key: cord-027713-8ohchx8p authors: abidine, m’hamed bilal; fergani, belkacem title: human activities recognition in android smartphone using wsvm-hmm classifier date: 2020-05-31 journal: the impact of digital technologies on public health in developed and developing countries doi: 10.1007/978-3-030-51517-1_35 sha: doc_id: 27713 cord_uid: 8ohchx8p being able to recognize human activities is essential for several applications such as health monitoring, fall detection, context-aware mobile applications. in this work, we perform the recognition of the human activity based on the combined weighted svm and hmm by taking advantage of the relative strengths of these two classification paradigms. one significant advantage in wsvms is that, they deal the problem of imbalanced data but his drawback is that, they are inherently static classifiers they do not implicitly model temporal evolution of data. hmms have the advantage of being able to handle dynamic data with certain assumptions about stationary and independence. the experiment results on real datasets show that the proposed method possess the better robustness and distinction. the advancement of technologies has facilitated the monitoring of human activities through the embedded sensors in a smartphone. recently, smart phones, equipped with a rich set of sensors, are explored as alternative platforms for human activity recognition (har) [1, 2] . har technology aims at recognizing the behavior and activities of users through a series of observations, which has wide application [3, 4] in different areas, such as healthcare and military monitoring. with smartphones becoming an integral part of daily human life [5] , they are being preferred as the most usable appliances that could recognize human activities due to its powerful in terms of mobility, user-friendly interface, network capability, strong cpu, memory, and battery. they contain a large number of hardware sensors such as accelerometer, gyroscope, temperature, humidity, light sensor, and gps receiver. the human sensor based activity recognition is a combination of sensor networks hand-in-hand with the data mining and machine learning techniques [6] . the smartphones provide enormous amount of sensor data for one to understand the daily activity patterns of an individual. the basic procedure for mobile activity recognition involves i) collection of labelled data, i.e., associated with a specific class or activity from users that perform sample activities to be recognized ii) classification model generation by using collected data to train and test classification algorithms iii) a model deployment stage where the learnt model is transferred to the mobile device for identifying new contiguous portions of sensor data streams that cover various activities of interest. sensor data can be processed in real-time or logged for offline analysis and evaluation. the model generation is usually performed offline on a server system and later deployed to the phone to recognize the activity performed. recently, several authors [7, 8] have proposed many applications related to activity recognition on multiple body positions. most of the work, like ahmad [9] , tran [10] , awan [11] , shoaib [12] , and abidine [13] , consider a single classifier approach to study activity recognition using smartphones. for the classification, svms are popular [8, 14] . it is also the case for hmms [15] which they commonly used for time-series activity recognition. however, there is very limited number of publications in the literature that investigate the application of the wsvm classifier for smartphone data, and no one is found about applying the latter one on smartphone data or even on har system's datasets. building a system with high precision to accurately identify these activities is a challenging task. in this work, we adopted a new method for physical activity recognition using mobile phones that uses labels outputting wsvm in hmm. wsvm investigated the effect of overweighting the minority class on svm modeling between the performed activities. hmm is a natural solution to address the activity complexity bycapturing and smoothing information during the transition between two activities (e.g. walking and standing). we also used the feature extraction approach that transforms the original high dimensional data to a lower dimensional feature space. the transformation can be linear or nonlinear. in this project, we employed the linear principal component analysis (pca) [16] to extract the feature vectors. figure 1 shows the architecture of the proposed activity recognition system. among the available labelled data, training and test subsets are chosen using the crossvalidation mechanism. the constructed pca space is then used for training and testing the weighted svm classifier. in the second step of the process is a pre-classification by 'wsvm', this phase is carried out by the 'cross-validation' will generate an estimate of the label vector. the principal component features concatenated with the wsvm estimated label vector are employed as a new training data to train hmm classifier. the final classification is performed with the 'viterbi' algorithm, by the use of a hmm model. an estimated label vector is generated by the 'viterbi' algorithm and the system will output the recognized activity (i.e., walking, running, and others). pca [16] is an orthogonal projection-based technique such that the variance of the projected data is maximized. in our case, a large number of features are extracted by prepossessing the raw signals generated from different sensors. it is a widely used technique for dimensionality reduction, feature extraction, and data visualization through the construction of uncorrelated principal components that are a linear combination of the original variables. the pca components can be counted by performing the eigenvector decomposition of the covariance matrix s: this problem leads to solve the eigenvalue equation with k is the eigenvalue of s and v is the eigenvector corresponding to the k: ð2þ is the n â n matrix containing n eigenvectors and k is an n â n diagonal matrix of eigenvalues of the covariance matrix. in eq. (2), each n dimensional eigenvector v i corresponds to the ith eigenvalue k i . osuna et al. [17] proposed an extension of the svm modeling, weighted svm algorithm to overcome the imbalance problem by introducing two different penalty parameter c à and c þ in the primal lagrangian (eq. 3) for the minority (y i = −1) and majority classes (y i = +1), as follow m þ (resp. m à ) the number of positive (resp. negative) instances in the initial database ( m à þ m þ ¼ m). solving the formulation dual of wsvm [17] gives a decision function for classifying a test point y 2 r p f ðxþ ¼ sgn we used the gaussian kernel as follows: kðx; yþ ¼ exp à x à y k k 2 =2r 2 . some authors [17] [18] [19] have proposed adjusting different cost parameters to solve the imbalanced problem. to extend weighted svm to the multi-class scenario in order to deal with n classes (daily activities), we have shown in [20] that the cost of misclassifying a point from the small class should be heavier than the cost for errors on the large class. they used different misclassification c i per class, use this conclusion can get a satisfactory result. by taking c − = c i and c + = c, with m þ and m i be the number of samples of majority classes and number of samples in the i th class, the main ratio cost value c i for each activity can be obtained by: hmm [21] comprises two parts: markov chain and stochastic process. markov chain, whose output is a sequence of state, can be described by the initial probability distribution for the states (p) and the state transition matrix (a), while stochastic process whose output is a sequence of observed values, is described by the observation probability matrix (b). thus, a hmm can be described as: with: i, j {1,2, …, n} o t : vector of observations a standard hmm is a generative probabilistic model, which generates hidden states y t from observable data x t at each discrete time instant. in our case the hidden variable is the activities that the subject was performing at a given time step and the observable variable is the vector of sensor readings. hmm model mainly works on two basic principles as follows: the observable variable at time t, namely x t , depends only on the hidden variable y t . the hidden variable at time t, namely y t , depends only on the previous hidden variable y t−1 . learning the parameters of these parameters corresponds to maximizing the joint probability p(x, y) between the sensor data and activities in the training data. the joint probability therefore factorizes as follows: the main aim of this model is to determine the best hidden state sequence from the observed output sequence that maximizes p(x, y). we validate our method on three public datasets whose information is summarized in hapt datasets provide a large extracted features extracted by prepossessing the raw signals generated from sensors. these algorithms are tested under matlab environment and the wsvm algorithm is tested with implementation libsvm [25] using gaussian kernel is used for all the datasets. each training dataset is normalized before classification within a range of [−1, 1]. we optimized the svm hyper-parameters (r, c) for all training sets in the range [0.1, 0.2, 0.5, 1] and {0.1, 1, 5, 10, 100}, respectively, to maximize the error rate of five fold-cross validation technique. the optimal parameters r opt = 0.9, 0.9, and 0.8 are found to be optimal the training dataset of har, hapt, and wisdm, respectively. we show in the table 2 that the fusion of principal component features with wsvm-hmm makes the model more robust, achieving better performance. one also notices for har dataset that the multi-class wsvm method improves the classification results over mc-svm, mc-hf-svm and hmm classifiers used alone. on the other hand, the results also show that wsvm outperforms hmm for recognizing activities for all datasets except for the hapt dataset. in terms of reducing the datasets, the feature reduction identifies the most relevant features for the learning process. we notice that pca features can improve the discrimination between different activities than the original features. for wisdm the performances of activity recognition are low than har and hapt datasets with 561 features. this is explained by the number of features (6) for wisdm is not sufficient when using pca algorithm. another reason to the lowest accuracy in wisdm dataset is attributed to the use only the accelerometer sensor comparatively to the har and hapt that use the both accelerometer and gyroscope sensors. to get a detailed knowledge of the performances on each class corresponding to current activity for the har dataset with six different activities. we calculate the confusion matrix of the proposed method in table 3 . from these tables, we see that the best performances were obtained for the proposed method for all classes, in particular for the static activities (sitting and standing). in the table 3 , 96.2% of 'w. upstairs' activity instances are correctly recognized, while 2.4% goes into 'w. downstairs' and 1.2% are confused with 'walking' activity. the similar classes such as 'walking', 'w. upstairs', and 'w. downstairs' show similar trend of sharing errors among each other. the reason is the similar status of smartphone when the user does these dynamic activities. we notice that the static activities share errors among each other. 12.2% of 'standing' activity instances are confused with 'sitting' activity and 7.4% of 'sitting' activity instances are confused with 'standing' activity. intuitively, this can be explained by the fact that the patterns in the acceleration data between these activities are somewhat similar. experimental results of the hybrid model presented demonstrate how it can be effectively employed for activity recognition of static and dynamic activities. it obtains a significant performance. specifically, we show how the hybrid system obtained by using the wsvm label output a new feature added to the reduced data for training and testing hmm outperforms other well known supervised pattern recognition approaches. we consider that wsvm approach has great potential to deal the imbalance class in this human activity recognition problem. however, it must be noticed that hybridizing these schemes implies a more complex system. fortunately, the training phase in a deployed activity recognizer is usually done offline, so we do not consider such growth of complexity a real problem in our domain. impact of smartphone's on society improving human activity recognition in smart homes healthcare in the pocket: mapping the space of mobile-phone health interventions an automatic data mining method to detect abnormal human behaviour using physical activity measurements a survey of online activity recognition using mobile phones human activity recognition and prediction smart phone based data mining for human activity recognition human activity recognition on smartphones using a multiclass hardware-friendly support vector machine sarm: salah activities recognition model based on smartphone human activities recognition in android smartphone using support vector machine subject-independent human activity recognition using smartphone accelerometer with cloud support towards physical activity recognition using smartphone sensors soft margin svm modeling for handling imbalanced human activity datasets in multiple homes pattern recognition of human activity based on smartphone data sensors using svm multiclass hmm machine learning and inference for activities of daily living recognition principal component analysis support vector machines: training and applications controlling the sensitivity of support vector machines weighted support vector machine for classification with uneven training class sizes the joint use of sequence features combination and modified weighted svm for improving daily activity recognition hmm machine learning and inference for activities of daily living recognition a practical guide to support vector classification activity recognition using cell phone accelerometers ), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license and indicate if changes were made. the images or other third party material in this chapter are included in the chapter's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the chapter's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use key: cord-258239-7xyqlz0u authors: gärtner, fabian; knippschild, uwe; burster, timo title: application of an activity-based probe to determine proteolytic activity of cell surface cathepsin g by mass cytometry data acquisition date: 2020-10-19 journal: acs omega doi: 10.1021/acsomega.0c04092 sha: doc_id: 258239 cord_uid: 7xyqlz0u [image: see text] during an immune response, cathepsin g (catg) takes on the role of adaptive and innate immunity and the outcome depends on the localization of catg. soluble, cell surface-bound, or intracellular catg is also responsible for pathophysiology conditions. we applied the activity-based probe mars116-bt to mass cytometry by time-of-flight to analyze catg activity on the cell surface of immune cells. the phosphonate warhead of mars116-bt binds covalently to the serine amino acid residue s195 of the catalytic center and thereby catg activity can be detected. this method contributes to observing the activation or inhibition status of cells during pathogenesis of diseases and enables accurate data acquisition from complex biological samples with a vast panel of cell subset markers in a single-cell resolution. hypertension, cardiac diseases, and diabetes are some of the consequences of cardiac dysfunction. elevated blood pressure is the result of vasoconstriction, which is triggered by angiotensin ii binding to the type 1 angiotensin receptor. additionally, angiotensin ii enhances inflammation and fibrosis. 1 how is angiotensin ii generated? angiotensinogen is cleaved by renin to angiotensin i and is further proteolytically digested by the angiotensin-converting enzyme (ace) to angiotensin ii. importantly, neutrophil-derived cathepsin g (catg), which can be soluble or bound to the cell surface of neutrophils, and mast cell-derived chymase are also able to convert angiotensin i to angiotensin ii. 2−5 ace2 processes angiotensin ii to angiotensin 1−7, which is, on the one hand, valuable in lowering blood pressure by vasodilation and provokes the kidneys to excrete sodium and water. on the other hand, it also exhibits an anti-inflammatory capacity. downregulation of ace2-expressing cells can cause activation of the renin angiotensin system, which regulates vascular and cardiac physiology and is central to common pathologic conditions such as hypertension and heart failure. 6, 7 neutrophils can be identified by a set of cell surface markers, among them are cd11b, cd16, and cd66b, which are reliably expressed across the neutrophilic population. in addition, these cell surface markers are independent from the location or activation status of neutrophils. 8 activated neutrophils, under conditions of both inflammation and homeostasis, express cell surface markers, such as cd62l (l-selectin), cd54 (intracellular adhesion molecule 1, icam-1), cd32 (fcγrii), and cd88 (c5a receptor). 9 activation of neutrophils can also be artificially induced using phorbol myristate acetate. 10 catg, a serine protease, together with neutrophil elastase, proteinase 3, and neutrophil serine protease 4 is secreted by activated neutrophils. cell surface catg on neutrophils is still proteolytically active, 11 although natural serine protease inhibitors, including α 1 -antitrypsin, are present at the site of inflammation but cannot inhibit cell surface catg. first, neutrophils release matrix metalloproteases in order to proteolytically inactivate α 1 -antitrypsin, 12 and second, the bulky natural serine protease inhibitor, α 1 -proteinase inhibitor might not be able to reach the catalytic center of cell surfacebound catg by steric hindrance. 11 catg possesses a catalytic triad, as with other proteases, containing histidine, aspartate, and serine amino acids within the active center (h57, d102, and s195). the active site cleft is perpendicularly oriented to the two β barrels where the hydroxyl group of s195 nucleophilically attacks the carbonyl carbon of the scissile peptide bond. 13, 14 human catg shows trypsin, chymotrypsin, metase, and lyase activity. 15, 16 additionally, it was demonstrated by performing protease profiling that catg has a preference at the p1 position holding an asparagine (n), at p2 proline (p), and at p3 glutamic acid (e) and at the alternate subsite p1′ isoleucine (i), alanine (a), and serine (s) as well as at the p2′ negatively charged amino acids (aspartic acid, d, and glutamic acid, e). 17, 18 in our previous work, we analyzed distinct cell populations in the peripheral blood of healthy donors for their cell surface catg activity by applying the activity-based probe mars116-bt in a flow cytometry approach. this method circumvents cell separation from a mixture of cells found in blood or tissue to detect catg activity by a western blot-based assay or by enzymatic kinetics. 19 mass cytometry by time-of-flight (cytof) is the next generation of flow cytometry to simultaneously analyze a complex panel of cell markers, which is not possible with the classical fluorescence flow cytometer. hence, a multiplexed profiling of up to 100 surface markers as well as intracellular signaling proteins is possible. 20, 21 until now, the detection of cell markers, including proteases, via application of cytof has been limited to analysis at the protein level. to this end, we established an approach to determine the proteolytic activity of catg on the cell surface of neutrophils, nk cells, b cells, and t cells in peripheral blood mononuclear cells (pbmcs) by combining the activity-based probe mars116-bt and the antibiotin-150 nd antibody with cytof analysis. the technique outlined here demonstrates that catg can be detected on cd16 + cd66b + neutrophils and nk cells using the cytof methodology. therefore, mars116-bt-anti-biotin-150 nd antibodies are useful to profile a vast panel of different cell subsets in order to evaluate catalytically active catg, its regulation, or inhibition on the cell surface. 2.1. detection of catg activity on the cell surface of neutrophils by cytof. activity-based probes are applied to detect cysteine and serine protease activity. 22−24 mars116-bt contains a biotin, spacer, amino acid sequence for specificity, and a warhead. mars116-bt, with an incorporated electrophilic phosphonic active site-directed warhead, binds covalently to the oxygen atom of s195 within the catalytic center of catg. 25 pbmcs were incubated with mars116-bt with a group of specific antibodies that were conjugated with different isotopes ( 147 sm-cd20, 154 sm-cd45, 155 gd-cd56, 160 gd-cd14, 162 dy-cd66b, 165 ho-cd16, 168 er-cd8, 170 er-cd3, 173 yb-hla-dr, and 174 yb-cd4) to determine immune cells and their respective subsets. furthermore, the groups were treated with different inhibitors to distinguish specificity. one control group was incubated without an inhibitor, two groups were preincubated with the reversible catg inhibitor (catginh.) in a final concentration of 12.5 or 50 μm, and one group was treated with the irreversible catg inhibitor suc-val-pro-phe p (oph) 2 (sucvpf) 26, 27 in a final concentration of 50 μm before adding mars116-bt. subsequently, antibiotin-150 nd antibodies were added to the samples and catg activity was monitored by cytof combined with the respective software (summarized in figure 1 ). after data acquisition, the fcs files were normalized based on the calibration beads (eq four element calibration beads) using the built-in normalizer of the helios. the beads were gated out of the analysis and the cells were gated for dna double-positive events ( 191 ir and 193 ir) to exclude doublets for accurate single-cell identification. cd45 was used as a marker for pbmcs. a further gating procedure was carried out to differentiate between cd4 + t cells (cd3 + , cd4 + , cd8 − , and cd20 − ), cd8 + t cells (cd3 + , cd4 − , cd8 + , and cd20 − ), b cells (cd20 + and hla-dr + ), monocytes (cd3 − , cd14 var , cd16 var , cd20 − , cd56 − , and hla-dr + ), nk cells (cd3 − , cd14 − , cd16 var , cd20 − , and cd56 var ), eosinophils (cd3 − , cd16 − , cd20 − , cd56 − , and cd66b + ), and neutrophils (cd3 − , cd16 + , cd20 − , cd56 − , and cd66b + ). nk cells were further subcategorized by gating for the cell surface expression of cd16 and cd56 based on previously established gating procedures of regular flow cytometry. 28 in the next step, the 150 nd channel was adjusted for catg activity selecting cd4 + t cells, cd8 + t cells, b cells, monocytes, nk cells, and their subsets, as well as eosinophils and neutrophils. in contrast to b cells or t cells, eosinophils and neutrophils showed a robust level of proteolytically active catg (figure 2 and supporting information s1). additionally, the nk cell subset cd16 + cd56 − (no. 5) harbors detectable catg activity, as demonstrated in figure 3 . the specificity of cell surface-active catg was determined by signal reduction when samples were preincubated with catg inhibitors (catginh. and sucvpf). thus, the methodology of cytof can be used for deep profiling of neutrophils, eosinophils, and nk cells as well as for http://pubs.acs.org/journal/acsodf article detection of proteolytically active catg by applying mars116-bt. furthermore, cytof is a valid method to detect not only the presence of specific antigens but also their proteolytic activity. the next-generation flow cytometry cytof setup in combination with mars116-bt-anti-biotin-150 nd antibodies allows us to analyze and gate distinct cell subsets and simultaneously determine the proteolytic activity of catg. this approach can be used to monitor neutrophils for the efficacy of inhibitors to arrest catg activity as well as disease progression where catg activity is upregulated. furthermore, mars116-bt-anti-biotin-150 nd antibodies can be added to a vast panel of activation markers in cytof analysis to characterize immune cells during inflammation, inducing a pathologic effect, in homeostasis, or when stimulated with different substances. additionally, different activity-based probes can be applied to cytof to detect further serine proteases or even cysteine proteases in a single-cell approach. in previous work, we detected catg activity in both nk cell subsets, cd3 − cd16 − cd56 dim and cd3 − cd16 dim cd56 − nk cells, 19 by common flow cytometry. compared to the cytof analysis demonstrated here, catg activity detection was limited to cd16 + cd56 -nk cells. this gives rise to speculation that a low amount of catg activity cannot be detected by cytof and is certainly a limitation of this technique. the use of mars116-bt-anti-biotin-150 nd antibodies for characterizing the catalytic activity of catg is not only practical for immune cells from the blood. we also suggest sputum from patients or tissue from animal models because application of the mars116-bt-anti-biotin-150 nd antibodies is not restricted to cells from humans. furthermore, treatment with protease inhibitors to regulate imbalanced proteolytic activity in several diseases is indicated. with respect to serine protease inhibitors, boswellic acids (bas) inhibit the catalytic activity of catg. 29 previously, it has been shown that ba interferes with the chemoinvasion of neutrophils, suppresses inflammation, and possesses potential cardioprotective properties. 29−31 inhibition of catg by ba could be monitored by mars116-bt-anti3.2. application of mars116-bt in cytof. pbmcs from one donor were thawed, washed twice with pbs, and used in a final concentration of 1.5 × 10 6 pbmcs/staining for both titration of antibodies and the experiments. the experiment was split into four groups, one control group treated with pbs, two groups that were preincubated with the reversible catg inhibitor (catginh., calbiochem, merck chemicals gmbh, schwalbach, germany, cat. no.: 219372) at two concentrations, 12.5 and 50 μm, and one group treated with 50 μm of the irreversible catg inhibitor, suc-val-pro-phe p (oph) 2 (sucvpf). 26 preincubation was performed for 15 min at room temperature (rt). afterward, cells were washed with pbs, centrifuged (300g for 8 min), and incubated with mars116-bt, which was synthesized as described previously, 25 in a final concentration of 2 μm for 30 min at rt, followed by three wash steps in pbs, and centrifugation at 300g for 8 min. cells were stained with the following mixture of antibodies, as depicted in table 1, in rpmi supplemented with 10% dmso until the day of acquisition. the samples were thawed followed by one washing step in pbs with 10% fcs, and three washing steps with milliq water (600g, 8 min). thereafter, cells were acquired at 300 events/second with a helios cytof system (fluidigm, markham, canada). calibration beads (eq four element calibration beads, fluidigm #201078, markham, canada) were used at a concentration of 10%. 3.3. data analysis. the data were analyzed using manual gating with flowjo x (flowjov10.6.2, flowjo llc, ashland, or, usa) as well as automated clustering approaches using spade (cytobank 7.3.0., implemented version, santa clara, ca, usa). statistical analysis was performed with the commercially available software, graphpad prism 6, inc., san diego, ca, usa. data were normalized to the specific untreated control population and expressed as normalized intensity in percent. the ± standard error of the mean (s.e.m.) is shown. significant differences were considered at p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), or p < 0.0001 (****). 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and cathepsin g as therapeutic targets in human diseases the 1.8 a crystal structure of human cathepsin g in complex with suc-val-pro-phep-(oph)2: a janusfaced proteinase with two opposite specificities how immune peptidases change specificity: cathepsin g gained tryptic function but lost efficiency during primate evolution new chromogenic substrates of human neutrophil cathepsin g containing non-natural aromatic amino acid residues in position p(1) selected by combinatorial chemistry methods extended cleavage specificity of human neutrophil cathepsin g: a low activity protease with dual chymase and tryptase-type specificities charge-synchronized" proteome-derived peptide libraries cell surface cathepsin g activity differs between human natural killer cell subsets application of mass cytometry (cytof) for functional and phenotypic analysis of natural killer cells coumarin as a structural component of substrates and probes for serine and cysteine proteases activity-based profiling of proteases recent advances in activity-based protein profiling of proteases application of a novel highly sensitive activity-based probe for detection of cathepsin g irreversible inhibition of serine proteases by peptide derivatives of (alpha-aminoalkyl)phosphonate diphenyl esters application of specific cell permeable cathepsin g inhibitors resulted in reduced antigen processing in primary dendritic cells cd56bright natural killer (nk) cells: an important nk cell subset identification of human cathepsin g as a functional target of boswellic acids from the anti-inflammatory remedy frankincense boswellia serrata: an overall assessment of in vitro, preclinical, pharmacokinetic and clinical data anti-inflammatory and anti-cancer activities of frankincense: targets, treatments and toxicities key: cord-016089-h122of8q authors: li, haixia; lu, chunbo title: lonicera japonica thunb 金银花 (jinyinhua, honey suckle) date: 2015-02-19 journal: dietary chinese herbs doi: 10.1007/978-3-211-99448-1_78 sha: doc_id: 16089 cord_uid: h122of8q jinyinhua, a sprawling and twining lianas in the family of caprifoliaceae, is a popular chinese herbal medicine used for the treatment of inflammatory diseases and as a well-known dietary supplement that has been used for many centuries. material can be obtained and marketed. during drying procedures, do not direcctly expose the flowers to strong sunlight because it will darken the flowers. other processing methods are further performed for some specific medicinal purposes, such as fried jinyinhua and charcoaled jinyinhua etc. in recent years, sulfurfumigation has been used to replace natural drying processing for efficiency and pest control [4] . organic acids, flavonoids and volatile oil are the three major classes of bioactive compounds found in jinyinhua [1] . in addition, shuangkangsu (shown in fig. 78 .2 (8) ), which has the marked anti-viral activity against influenza b virus, influenza a3 virus and respiratory syncytial virus, is an important chemical constituent with a novel skeleton structure of cyclic peroxide. it was found in 2008. organic acids are the main and effective components of l. japonica. chlorogenic acid (1), isochlorogenic acid (2), neochlorogenic acid (3) , and caffeic acid (4) (shown in fig. 78 .2) are representative compounds. among them, chlorogenic acid is received considerable attention for its part in the human diet with potential biological effects [5] , and is used as a standard compound for evaluation of the quality of jinyinhua and related pharmaceutical or natural health product containing the herb. according to the chinese pharmacopoeia, its content must be not less than because flavonids have a wide spectrum of biological activities, especially with antioxidative and anti-inflammatory properties, they play a part in the qualitative and quantitative analysis of jinyinhua. luteoloside (5) was added in chinese pharmacopoeia with chlorogenic acid to control the quality of medical material by hplc method. the content of luteoloside in the jinyinhua should be not less than 0.05 %. besides luteoloside, other flavonoids, such as luteolin (6) and lonicerin (7) (shown in fig. 78 .2), have exerted anti-inflammatory activity [6] . as one of the important compositions, volatile oil is significant in both the wide activity and utilization of jinyinhua. a total of about ninety compounds of volatile oil were identified, the main compound being linalool [7] . due to the differences in harvesting time and processing technics, the contents and components of volatile oil are different. existing research showed that the complete white and silver flower period are the preferable harvest times for volatile oil, which match with the best time to harvest for chlorogenic acid. low temperature and no-lighting were in favor of the volatile oil in the dry and extract processes [1] . as described previously, jinyinhua is one of widely used herbs in tcm, especially for almost all infectious diseases, due to its antimicrobial and anti-inflammatory activities. the two activities act synergistically to accelerate wound repair [8] . moreover, the modern pharmacological studies showed that jinyinhua and its active principles also possess the wide pharmacological actions, such as antiendotoxin, antipyretic, antihyperlipidemic, antithrombotic, anti-oxidative and anti-carcinogenic activities, and hepatoprotective etc. [1] . in addition, anti-lipase, insecticidal and acaricidal activities were also found in the crude extract of jinyinhua. in recent years new bioactivities, such as the potent anti-parkinsonism activity [9] and protecting neuronal cells against glutamate excitotoxicity via antioxidative activity [10] , inhibition of the allergic contact dermatitis [11] , and a possible use for antidiabetes, have been discovered and suggested to be in the compounds isolated from jinyinhua due to its potent inhibitor action for maltase [12] . researchers thought most of these effects may be related to the active compositions of volatile oil, chlorogenic acid, and flavones. chlorogenic acid and luteoloside, officially used as the indicator compound to characterize the quality of this herb and its related preparations, were shown to have beneficial effects in the aspects of anti-oxidation and antitumor [1, 13] . moreover, chlorogenic acid showed the antibacterial, antiviral, anti-inflammatory, hypoglycemic activities, and allergy-preventive properties [1, 14] . meanwhile, luteolin, another major flavonoid in jinyinhua, and volatile oil also showed significant anti-inflammatory activity. luteolin has significant bioactivities in the aspects of antifibrotic and anti-5-lipoxygenase activities [6, 15] . taken together, most of these activities matched to traditional usage. jinyinhua with heat-clearing and detoxifying effect has been called little fairyhood of herb store. in tcm clinical practice, jinyinhua is usually used to treat various infectious diseases. as the most famous herb of anti-inflammatory, it is constantly used for upper respiratory tract infections. 1500 years ago, jinyinhua had been used for the treatment of exopathogenic wind-heat, epidemic febrile diseases, carbuncles, sores, furuncles and infection diseases. also, it has also been made in preparations to treat chronic enteritis, pneumonia, acute tonsillitis, nephritis, acute mastitis, and leptospirosis in clinic. more than 500 prescriptions containing jinyinhua have been used to treat various diseases [1] . there are hundreds of manufacturers making these two products based on the same formula in china, these two products are two of the best-selling drugs on market. they are mainly used for the treatment of fever, cough, sore throat, acute and chronic tonsillitis, acute and chronic pharyngitis through its function clearing away the heat and toxic material, antibacterial, anti-inflammatory and antiviral effects. yinhuang buccal tablets and shuanghuanglian buccal tablets have the same compositions as oral liquid but are in a different form. the buccal table form is particularly suitable for swelling and pain of the throat caused by acute and chronic tonsillitis, pharyngitis and upper respiratory tract infections. yinhuang injection, shuanghuanglian injection, and compound acetaminophen jinyinhua injection are three common used preparations containing jinyinhua. compound acetaminophen jinyinhua injection is composed of the extract of jinyinhua, baicalin, and acetaminophen. these products have been used clinically for the treatment of upper respiratory tract infections, sore throat, tonsillitis, mumps and pneumonia, and compound acetaminophen jinyinhua injection has also been used to relieve moderate pain, such as arthralgia, headache, and toothache. adverse reactions of these products after intravenous injection were detected so these injections are better to be administered by intramuscular injection. jinyinhua granula prepared by itself is an extract in a convenient form that is used by being mixed with other herbs. recently, jinyinhua, as 'bouvardin', has been used extensively to prevent and treat some serious viral diseases of humans and animals, such as sars corona virus, h1n1 (swine) flu virus [16] . jinyinhua is a well-known dietary supplement due to its valuable bioactivies and because it's easy to obtain since it is planted in many areas as one of ornamental groundcover. it can be used in many ways historically. these include jinyinhua beverage, jinyinhua candy, and jinyinhua soup etc. the following dietary forms can be easily bought at a market or made at home. jinyinhua has been used to make healthy beverage through various technologies, such as jinyinhua tea, jinyinhua dew, jinyinhua nutritive dew, jinyinhua nutritive beverage, and jinyinhua yogurt etc. these beverages are employed to improve the body and prevent illnesses in china [1] . jinyinhua tea has a variety of practices, such as jinyinhua by itself, combined with tea, combined with honey, or combined with other herbs. all of them are popular ways to use jinyinhua. they are typically drunk in the hot season for clearing heat, detoxicating and strengthening the body's response against disease by improving the activity of the immune system. some examples are: jinyinhua dew composed of the distilled liquid of jinyinhua and water. jinyinhua tea can be drunk after boiling water of 150 ml to soak jinyinhua (5 g) and green tea (3 g) for 5-10 min. according to further needs, jinyinhua can be combined with other herbs with bioactivities for enhancing its effect. jinyinhua shanzha tea composed of jinyinhua (10 g), juhua (flowers of chrysanthemum morifolium, 10 g), and shanzha (fruits of crataegus pinnatifida, 10 g) for headache, fever and thirst caused by hotness. jinyinhua bohe tea composed of jinyinhua (15 g), bohe (aerial parts of mentha haplocalyx, 5 g), and gouqi (fruits of lycium chinense or l. barbarum, 15 g). jinyinhua itself or combined with different herbs can be used to make herbal wine for various specific needs of functions. some examples are: the extracting solution of jinyinhua (alcohol content 55 %) is added before fermentation, the following process is the same as used to brew wine [17] ; jinyinhua (45-55 g), gancao (roots and rhizomes of glycyrrhiza uralensis, g. inflate, or g. glabra, 5-10 g), gouqi (fruits of lycium chinense or l. barbarum, 25-30 g) and baizhi (roots of angelica dahurica, 5-10 g) are soaked in 500 ml wine of alcohol content between 55-60 % for more than 25-50 days, then it can be adjusted into different contents of alcohol according to needs. jinyinhua, the material which can be used as medicine and food, is often made into candy together with other herbs. some examples are: jinyinhua qingguo pipa candy composed of jinyinhua, qingguo (chinese olives, fruits of canarium album), pipa (fruits of eriobotrya japonica), jiegeng (roots of platycodon grandiflorum) and baimaogen (rhizomes of imperata cylindrica); jinyinhua cool candy is composed of jinyinhua, qingguo, luohanguo (fruits of siraitia grosvenorii), pangdahai (seeds of sterculia lychnophora) and bohe (aerial parts of mentha haplocalyx); jinyinhua juhua candy is composed of jinyinhua, bohe and juhua (flowers of chrysanthemum morifolium). white granulated sugar and liquid glucose are usually added to adjust the taste. you can buy the candy in the supermarket for the purpose of moistering and clearing the throat. jinyinhua can be used to make soups or porridge with rice, or mung bean etc. a typical way is to boil 100 g mung bean and a piece of ginger in 1 liter of water before adding 30 g jinyinhua, then continuously boil until the mung bean cracks and are fully cooked. other ingredients, such as wax gourd, lily bulbs, lotus root, almond, pears, and ham etc., can be boiled together with jinyinhua. beautiful white and yellow color, nutrient, and health-maintaining effect of jinyinhua can be employed simultaneously. the taste of jinyinhua-contained foods can be adjusted by adding honey, white granulated sugar or licorice. in addition, the oil and extracts from jinyinhua may be a potential source of preservatives for the food industries [1] . in qing dynasty of china, about 375 year ago, jinyinhua was used to moisturize the skin and for rejuvenation. recently, the extract of jinyinhua, as natural source of bioactive compounds, have been applied in cosmetics, extensively to exert its marked antibacterial and antisepticize activities, such as jinyinhua facial mask, jinyinhua facial cleanser, and jinyinhua shower gel. it could be made into toothpaste which could have the effects of preventing and treating the oral cavity's diseases [1] . in addition, the volatile oil isolated from jinyinhua would cover the smell from cigarettes. and chlorogenic acid and its analogues, which are beneficial to health, are rich in jinyinhua. it can be added into cigarettes to serve a useful role in improving the quality of cigarettes and preventing disease. jinyinhua is mostly used in combination with other herbs with heat-clearing and detoxifying effect, such as huangqin (roots of scutellaria baicalensis), lianqiao (fruits of forsythia suspensa) etc. more than 12 preparations, in which jinyinhua was the main and active compositions, were listed in chinese pharmacopoeia (2010 edition) and used to cure fever, cough and pharyngalgia and the swell of throat, constipation, conjunctival congestion, etc. except the aforementioned preparations: injection, oral solution, granular, or suppository of yinhuang, shuanghuanglian and yinzhihuang, simiaoyongan decoction, yinqiao jiedu tablets, and xiaoyin tablets etc. are the most commonly used preparations. there are large numbers of clinical related reports or observational studies published on the effects of jinyinhua and its related preparations for various diseases. clinical report showed shuanghuanglian oral solution's effect of antipyretic on wind and warm syndrome was 100 % within 72 h in 48 cases [18] ; and the preparation could effectively relief the cold symptom of cough, headache, nasal discharge, sore throat. it could also stop the cheek swelling of child with epidemic parotitis [19] . yinzhihuang oral solution, which is composed of four herbal components: jinyinhua, yinchen (aerial parts of artemisiae scopariae or a. capillaris), huangqin (roots of scutellaria baicalensis), and zhizi (fruits of gardenia jasminoides), may inhibit further increase in bilirubin levels, and reduced the phototherapy requirement in 1177 cases of neonatal indirect hyperbilirubinemia in term newborn infants [20] . simiao yongan (trade name: mailuoning) is used in treating ischemic cardiovascular and cerebrovascular diseases for many years in clinical and comprises jinyinhua, xuanshen (roots of scrophularia ningpoensis), danggui (roots of angelicae sinensis) and gancao (roots and rhizomes of glycyrrhiza uralensis, g. inflate or g. glabra), clinical studies have shown that it can inhibit the inflammatory response and antagonize the blood clotting process [21] . because jinyinhua is an edible herb and commonly used as raw material in health food, clinical reports on the toxicity or side effects were done to determine its safety at least 10 years ago. the extract of jinyinhua was found to be fairly nontoxic when oral taken by rats or mice. the detail was as follow. acute toxicity test showed its ld 50 was more than 15 g/kg body weight on mice orally. according to the classification standard of chemicals acute toxicity, it belongs to non-toxic level. micronucleus test of bone marrow cells up to 7.5 g/kg in mice orally and ames test/ mammals microsomal enzyme test showed it was safe without mutagenesis. meanwhile, sperm abnormalities and antifertility effect were undetected on male mice and sd female rats, respectively [22] . in addition, jinyinhua combined with the dried zhimu (rhizomes of anemarrhena asphodeloides) showed no signs of acute or chronic toxicity in terms of general behavior, gross appearance of the internal organs, blood chemistry, or mortality in male or female rats when orally administered a single dose of 5,000 mg/kg in acute toxicity test or 500, 1000 or 2,000 mg/kg daily for 13 weeks in chronic toxicity test. they didn't cause significant gastric mucosal damage after single or repeated doses, instead appearing to protect the mucosa from diclofenac-induced gastric damage [23] . to sum up, as a material of being used as medicine and food, jinyinhua is definitely a safe herbal medicine, and often used for the treatment of infectious diseases and health maintaining purpose. it can also be used for relieving cold and cleaning away poison. but the close attention must be paid when deciding to use this herb for cold because it is obvious that the cold treated by jinyinhua refers only to a pyretic cold rather than a frigid cold, and this herb is inapplicable for the hypofunction of person's constitute with cold manifestation consideration of its strong clearing heat activity. lonicera japonica thunb.: ethnopharmacology, phytochemistry and pharmacology of an important traditional chinese medicine combination of normal light and fluorescence microscopy for authentication of five lonicera species flower buds pharmacopoeia of people's republic of china profiling and characterization of volatile components from non-fumigated and sulfur-fumigated flos lonicerae japonicae using comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry coupled with chemical group separation an outlook on chlorogenic acids-occurrence, chemistry, technology, and biological activities luteolin isolated from the flowers of lonicera japonica suppresses inflammatory mediator release by blocking nf-kappab and mapks activation pathways in hmc-1 cells chemical composition of the essential oils from the flower, leaf and stem of lonicera japonica wound repair and anti-inflammatory potential of lonicera japonica in excision wound-induced rats lonicera japonica thunb. protects 6-hydroxydopamine-induced neurotoxicity by inhibiting activation of mapks, pi3 k/akt, and nf-kappab in sh-sy5y cells neuroprotective activity of the methanolic extract of lonicera japonica in glutamate-injured primary rat cortical cells characterization and anti-allergic effect of a polysaccharide from the flower buds of lonicera japonica α-glucosidase inhibitory activity by the flower buds of lonicera japonica thunb hplc-esi-ms/ms analysis and pharmacokinetics of luteoloside, a potential anticarcinogenic component isolated from lonicera japonica, in beagle dogs allergy-preventive effects of chlorogenic acid and iridoid derivatives from flower buds of lonicera japonica luteolin ameliorates experimental lung fibrosis both in vivo and in vitro: implications for therapy of lung fibrosis research and comprehensive utilization of honeysuckle studies on health food of honeysuckle research on antipyretic project of tcm emergency care for exogenous fever with analysis of 906 progress in clinical application of shuanghuanglian oral liqui clinical research collaborative group of yinzhihuang oral solution (2011) a multicenter randomized controlled study on the efficacy and safety of yinzhihuang oral solution for the treatment of neonatal indirect hyperbilirubinemia in term newborn infants effect of si-miao-yong-an on the stability of atherosclerotic plaque in a diet-induced rabbit model the toxicological assessment of lonicera japonica on food safety gastroprotective and safety effects of win-34b, a novel treatment for osteoarthritis, compared to nsaids key: cord-022779-himray6q authors: nan title: abstracts of oral presentations date: 2005-06-10 journal: biopolymers doi: 10.1002/bip.20321 sha: doc_id: 22779 cord_uid: himray6q nan s. tchertchian, f. oplinger, m. paolini, s. manganiello, s. raimondi, b. depresle, n. dafflon, h. gaertner, and p. botti geneprot inc., geneva branch, 2, pré de-la-fontaine, 1217 meyrin, switzerland the last decade has provided extensive demonstration of the key role played by native chemical ligation (ncl) for the preparation of small and medium size proteins [1] . yet the requirement for cysteine at the site of ligation in standard ncl has limited its flexibility. recently, different types of auxiliary groups [2, 3] have been developed to extend the application of ncl to other ligation sites. however, the generally slower ligation rates especially with large fragments and the additional step required to cleave the auxiliary post-ligation have reduced their utility. here we present a novel strategy to synthesize proteins through a chemical ligation using unprotected peptide segments. our scheme does not make use of auxiliary groups [2, 3] , instead originally exploits the features of some side chain removable functionalities. ligation rates are high, comparable to ncl and the residues available for ligation are more frequent than cysteine. furthermore the whole process is "one pot" and at the end a native polypeptide is obtained directly in the ligation mixture. the total chemical syntheses of c5a (1-74) using both ncl and our method will be presented and compared. [ during the biosynthesis of glycopeptide antibiotics of the vancomycin family, several oxidative phenol coupling reactions take place. the enzymes catalyzing these reactions are of interest from structural and mechanistic viewpoints. in this work [1, 2] , it is shown that the oxygenase oxyb from the vancomycin producer only catalyzes a phenol coupling reaction when the putative peptide substrate is linked as a thioester to a peptide carrier domain (pcd) derived from the nonribosomal peptide synthetase. an efficient access is described to representative free linear peptide substrates, which makes use of alloc-solid phase peptide chemistry, but largely avoids the use of amino acid side chain protecting groups. in this way, the target linear peptides can be released from the resin under very mild conditions, and then be activated as thioesters, prior to loading onto the pcd. [ we have recently discovered a new nonenzymatically-formed product from n-(3-oxododecanoyl)-l-homoserine lactone. interestingly, both the n-acylhomoserine and its novel tetramic acid degradation product 1 are potent antibacterial agents. bactericidal activity was observed against all tested gram-positive bacterial strains, while no toxicity was seen against gram-negative bacteria. we propose that p. aeruginosa utilizes this tetramic acid as an interference strategy to preclude encroachment by competing bacteria. additionally, we have discovered that this tetramic acid binds iron with comparable affinity to known bacterial siderophores, possibly providing an unrecognized mechanism for iron solubilization. using short portions (7-11 amino acid) rich in positively charged residues from either human lactoferricin or the marcks protein as templates, a panel of 70 peptides each possessing a specific chemical structure was synthesized. these included amino acid omissions, substitutions, and insertions in the aim to modify the peptides overall charge, hydrophobic core, and/or amphipathicity. the peptides antimicrobial activities against a large panel of bacteria were assessed using both conventional tests (mic, mbc) and non-conventional assays (mic quantified by an automated turbidimetry-based system and mbc measured on resting cells suspended in low-ionic strength medium-"survival assay"). furthermore, the membrane permeabilizing activity of the peptides on strains of several gram negative bacterial species was quantitated by measuring their ability both to decrease the mic of novobiocine and to promote the uptake of the hydrophobic fluorescent probe npn. while the mic determined by turbidimetry or by the conventional method did not significantly differ, bactericidal activity of the peptides measured by the survival assay was 1 to 2 orders of magnitude higher than that measured by the conventional mbc test. on the other hand, the two assays used to measure the permeabilizing activity of the peptides rendered similar results. interestingly, the most potent permeabilizers did not correspond with the peptides exhibiting the highest bactericidal activity thus indicating that these two activities have different structural bases. protein farnesyl transferase (pftase) catalyzes the attachment of farnesyl diphosphate (fpp) to proteins that contain a caax-box sequence at their c-termini [1] . several analogues of fpp that incorporate azide functional groups have been synthesized and shown to be incorporated into peptides using pftase as a catalyst. in particular, it has been shown that the prenyl azide moiety from 1 or related analogues can be transferred to the peptide substrate, n-dansyl-gcvia to yield the corresponding thioether-linked products. the resulting azide-containing peptides have been derivatized with a triphenylphosphine-based reagent to generate o -alkyl imidate-linked products rather than the amide-linked material expected via a staudinger reaction [2] . since caax-box sequences can be appended to the c-termini of many different proteins, these analogues provide a simple and general method for incorporating orthogonal azides into proteins at unique sites. subsequent functionalization of such azide groups via staudinger or "click" chemistry should provide a convenient method for linking proteins with a diverse array of probes, biomolecules, surfaces and other materials under mild conditions. chemoselective glycosylation, acylation, and alkylation of completely unprotected peptides can be accomplished by incorporating n-alkylaminooxy amino acids into the peptide sequence. the n-alkylaminooxy side chains react selectively with reducing sugars, activated alkyl halides, and various acylating agents in mildly-acidic aqueous buffers (ph 4) to furnish neoglyco-and neolipopeptides. a key feature of the approach is that a single parent peptide can be quickly reacted with a variety of agents to provide a large number of "post-translationally"modified peptides. the ability to easily synthesize arrays of modified peptides allows comprehensive studies of the effects that glycosylation and lipidation have on peptide structure and function. here we present an overview of the methodology and initial results on its application to studying problems of biological interest. this presentation describes cyclic peptides that fold into well-defined ␤-sheet structures in aqueous solution and can dimerize through ␤-sheet interactions. the cyclic peptides contain the unnatural amino acid hao, which mimics the hydrogen-bonding pattern of one edge of a peptide ␤-strand, and ␦-linked ornithine, which mimics a ␤-turn and provides enhanced water solubility or a linkage point for creating multivalent structures. institute for molecular bioscience, the university of queensland, queensland 4072, australia the human genome project and other major sequencing projects have rapidly provided a vast array of new protein/peptide sequences. in contrast, many other new proteins/peptides are also being uncovered from plant and animal sources whose genomes are yet to be tapped. in the post-genomic era, the physical form of many of these gene-encoded sequences will be vital for biomedical research and drug development. moreover, the advantages of peptide and protein chemical synthesis over recombinant-dna methods are increasingly being used to provide rapid structure-activity information of complex bioactive peptides, small proteins and functional receptor domains. in a program designed to exploit the potential of australian conus species we have isolated, characterised and chemically synthesised a wide range of novel conotoxins. these cysteine rich microproteins have well-defined tertiary structures with considerable rigidity and stability and contain many elements of protein secondary structure. of particular interest are the two disulfide bond containing conotoxins (examples below) which target transporters, ion channels and receptors at nanomolar potencies. in this presentation i will describe some of our research on controlling the shape and potency/selectivity of these microproteins through intramolecular native ligation chemical approaches. it appears that there is considerable scope to control the properties of these native sequences which in some cases may prove useful in the development of these molecules as therapeutic candidates. despite identical amino acid composition, differences in the properties of class a amphipathic helical peptides due to differences on the hydrophobic face results in substantial differences in anti-inflammatory properties. one of these peptides is an apolipoprotein a-i mimetic, d-4f. when given orally to mice and monkeys, d-4f caused the formation of pre-␤ hdl, improved hdl-mediated cholesterol efflux, reduced lipoprotein lipid hydroperoxides, increased paraoxonase activity and converted hdl from pro-inflammatory to anti-inflammatory. in apoe null mice d-4f increased reverse cholesterol transport from macrophages. oral d-4f reduced atherosclerosis in apoe null and ldl receptor null mice. in vitro d-4f caused the formation of pre-␤ hdl, reduced lipoprotein lipid hydroperoxides and converted hdl from pro-inflammatory to anti-inflammatory. physical properties and the ability of various class a amphipathic helical peptides to activate enzyme lcat in vitro did not predict biologic activity in vivo. in contrast, the use of cultured human artery wall cells in evaluating these peptides was more predictive of their efficacy in vivo. thus, anti-inflammatory properties of different class a amphipathic helical peptides depends on subtle differences in the configuration of the hydrophobic face of the peptides. physical-chemical properties provide an explanation for the mechanism of action of the active peptides. peptides to ameliorate atherosclerosis and other inflammatory diseases can be designed using this strategy. inflammatory diseases. this chemokine belongs to the family of cxc chemokines, its response is mediated through binding to seven transmembrane helical g-protein coupled receptors cxcr1 and cxcr2. in order to investigate the relevance of selected protein segments for biological activity we synthesized chemically modified and biologically active analogues of the 77-mer of hil-8 by expressed protein ligation (epl). for ligation naturally occurring cysteine at position 55 was chosen. c-terminal peptides carrying an n-terminal cysteine were synthesized by solid phase peptide synthesis (spps) applying the fmocstrategy and used to introduce modifications. ligation of the recombinantly produced thioester with synthetic peptides yielded in full length hil-8 that finally was correctly folded and stabilised by two disulfide bridges as in the native protein. in addition to fluorescent and photoactivatable analogues, we produced variants that contain a ␤-peptide helix instead of the naturally occurring ␣-helix. thus, for the first time, we received a protein containing a whole ␤-peptide segment and still showing high biological activity. depending on the linker between ␤-sheet and ␤-peptide helix of interleukin 8 we could discriminate between active and inactive proteins suggesting that the overall orientation of the c-terminal segment is highly relevant for the folding of the protein and subsequently for the signalling of interleukin 8. a peptide based on residues 109 -122 of the syrian hamster prion protein (h1) forms ␤-sheet aggregates in solution, which grow to form large fibers. isotope-edited infrared spectroscopy has shown that the initial antiparallel ␤-sheet formed by this peptide is disordered. a slow rearrangement occurs to form a structure in which the hydrophobic core of the strands (residues 112 -122) pack together, resulting in the alignment of residue 117 across the sheet. the kinetics of the realignment have been monitored for h1 and for peptides with mutations at residue 117 (a117i, a117l and a117b where b is aminobutyric acid). h1 and a117i align with non-exponential kinetics. at low concentrations h1 aligns via the repeated detachment and annealing of strands, whereas at higher concentrations a reptation mechanism is observed. a117b aligns instantaneously within the dead-time of our experiments. a117l does not align at residue 117 but some undefined reordering can still be observed as a shift of the 13 c band. these data are the first experimental probes of the types of intersheet rearrangements which are required for the nucleation of fibrous peptide aggregates, and the evidence for strand reptation within the ␤-sheet confirms observations in molecular dynamics simulations. [1, 2] . we have since established the microfluidic peptide chip as a miniaturizing platform. the challenging issues in making peptide chips a practical tool for understanding biology, drug discovery, and diagnostics are quality of synthesis, specificity in reported activities, and ability for quantitative measurements. we will present the results of the work which involves our intensive effort in: • development of the method for monitoring and analyzing quality of peptide chip synthesis • improvement in peptide chip synthesis • development of the methods for quantitative analysis of (a) the specific binding of antibodies/proteins to peptides on chip and (b) kinase enzymatic activities against substrate peptides on chip. our presentation should demonstrate novel applications of peptide chips that can be implemented as routine laboratorial processes. [1] gao, x., pellois, j. p., kim, k., na, y. , gulari, e., and zhou, x. to prototype this approach we developed a protein array consisting of the ras-binding domain of craf-1 (rbd) that was c-terminally modified with a 24mer oligonucleotide and immobilized via hybridization with the complementary oligonucleotide on a wafer [1] . the rbd-dna conjugate was generated by native chemical ligation using a recombinantly produced rbd-thioester and an oligonucleotide carrying a 5'-cystein residue [2] . incubation of the immobilized rbd with ras(gtp), the activated rbd-binding form, retains sufficient amounts of ras on the protein array to allow detection by mass spectrometry with high sensitivity. controls carried out with inactive ras(gdp) did not produce any signal, demonstrating a sufficient selectivity for biotechnological applications. protein microarrays in which proteins are immobilized to a solid surface are ideal reagents for high-throughput experiments that require very small amounts of analyte. such protein microarrays ('protein chips') can be used very efficiently to analyze all kind of protein interactions en masse. the present work describes a general method for the selective attachment of proteins to solid surfaces through its c-termini that can be used for the creation of protein chips. our method is based in the chemoselective reaction between a protein c-terminal ␣-thioester and a modified surface containing n-terminal cys residues. ␣-thioester proteins can be obtained using standard recombinant techniques by using expression vectors containing modified inteins. we also present an efficient solid-phase approach for the rapid synthesis of cys-containing linkers that can be used for the modification of au-and si-based surfaces. this new method was used to immobilize two fluorescent proteins and a functional sh3 domain. a series of glycopeptides based on the leu-enkephalin analogue yt-gfs*-conh 2 led to greatly enhanced stability in vivo and effective penetration of the bbb. transport through the bbb hinges on the biousian nature of the glycopeptides-the glycopeptides have two conflicting conformational manifolds, a h 2 o soluble state, and an amphipathic state at h 2 o-membrane phase boundaries. multiple lines of evidence suggest that the bbb transport mechanism is absorptive endocytosis. mixed /␦-agonists showed antinociceptive potencies greater than morphine, and lacked many of the side effects generally associated with classical -selective opiate analgesics. the biousian design was extended to larger glycopeptides (16 residues) related to ␤-endorphin, which also penetrated the bbb and produced antinociception in mice. plasmon waveguide resonance (pwr) studies showed that the amphipathic helices bound to membrane bilayers with m to low nm k d 's. the presence of diverse endogenous neuropeptide transmitters and neuromodulators in the human brain is potentially applicable to the treatment of a wide range of behavioral disorders. clemencia pinilla, 1 mireia sospedra, 2 yindong zhao, 3 we have recently demonstrated the feasibility of utilizing the ligase activity of inteins for the in vivo backbone cyclization of peptidic chains. this procedure -called siclopps for split intein circular ligation of peptides and proteins-provides a biosynthetic pathway for peptides that are metabolically stable, and can be produced with spatial and temporal control [1] . to screen for bacteriotoxic peptides, a sic-lopps library was introduced into an escherichia coli population, such that each bacterium encodes a different peptide sequence. siclopps library over-expression afforded six distinct bacteriostatic peptides that reduce cell growth. one of these peptides (ln05) also caused cell aggregation. an e. coli genomic library was introduced into cells encoding ln05. co-expression of the genomic library and ln05 peptide rescues growth only in cells expressing genomic fragments able to counteract peptide toxicity. genomic library and ln05 co-expression resulted in enrichment of a single genomic construct, a fragment of the narz gene. narz is part of a nitrate reductase complex and has a role in tuberculosis persistence [2] . ln05 production in mycobacterium smegmatis resulted in a slow-growth phenotype. [1] abel-santos, e., scott, c. p., and benkovic, s. j., methods in the special feature of proteins involved in alzheimer's or prion diseases is their ability to adopt at least two different (meta) stable conformations. thus, amyloid-forming proteins that mainly contain ␣-helical structures in their native conformation must undergo an ␣-helix3␤strand conversion before or during fibril formation. the conformational transition that shifts the equilibrium from the functional to the pathological isoform can happen sporadically. it can also be triggered by mutations in the primary structure, changes of the environmental conditions such as ph, ionic strength, metal ions, protein concentration, oxidative stress, free radicals, action of physiological, or pathological chaperones. alternatively, the introduction of a small quantity of protein polymer may act as a structural template and initiate the disease. therefore, the development of model systems which allow the investigation of the complex folding mechanisms that lead to ␤-sheet aggregation appears to be one of the main challenges in the detailed understanding of the pathways from incubation to mortality. in order to create an ␣-␤ switch system we designed ideal ␣-helical parallel coiled coil peptides, introduced trigger functions by mutations in the primary structure, and studied the consequences that these mutations have on the secondary structure properties of the resulting peptides under certain environmental conditions. based on these results we continued to change the primary structure of the coiled coil system subsequently by mutations in the heptad repeat untill we ended up with soluble ␤-sheet peptides. the most important feature of these new ␤-sheet peptides is that they still follow the characteristic hydrophobic heptad repeat of an ␣-helical coiled coil and that all of the positions which are part of the dimerization domains of the coiled coil remained untouched. thus, these new peptides bear all of the requirements which are necessary for formation of cooperatively interacting helical structures and, furthermore, contain domains for cooperative sheet aggregation as well. the folded structure will now strongly depend on the environment. this peptidic model system allows a systematic study of the subtle influences that environmental conditions may have on protein folding stepwise, which means changing these conditions one after one or in all of the possible combinations that nature applies in vivo. plasmon waveguide resonance (pwr) spectroscopy is a powerful new biophysical method which allows us to examine structural changes, kinetics and thermodynamics of anisotropic biological systems and processes such as proteolipid membranes. this method has probed the mechanisms of g-protein coupled receptor (gpcr) signal transduction, and has obtained new insights into specific signaling pathways of agonists and antagonists with gpcrs. we now have extended these studies to examine the effects of lipid microdomains (rafts) on the binding, signaling and transduction pathways. using a 1:1 mixture of palmitoyloleoylphosphatidyl-choline (popc) and sphingomyelin (sm), we have directly observed the formation of two lipid bilayer microdomains, and the preferred segregation of the delta opioid receptor (hdor) into sm lipid rafts when the agonist ligand was bound, but not for the unoccupied receptor which preferentially incorporated into the popc-rich domain. furthermore, we can demonstrate directly that, g-proteins bind much more strongly to the hdor receptor in the lipid raft (sm-rich) environment than in the fluid non-raft (popc-rich) domain of the lipid bilayer. the implications of these findings for novel design of drugs, and drug screening will be emphasized. † supported by grants from the u.s.p.h.s., national institute of drug abuse and national science foundation. their measurement in high resolution nmr requires partial molecular alignment. for proteins in aqueous solution a number of standard methods exist to achieve such small alignment. we found that swelling of cross-linked polymers inside the nmr tube results in anisotropic gels. peptides in such a gel phase exhibit well resolved spectra of the partially aligned molecules. this allows to scale the orientation depending on the cross-linking of the polymer, the thickness of the unswollen stick or the temperature. rdcs can now easily be measured in natural abundance in peptides 3] all common nmr solvents can be used. with the chiral gel gelatin it is possible to discriminate d-and l-alanine to determine enantiomeric purity. the procedure is demonstrated to refine the solution structures of peptides such as cyclosporin a, somatostatin analogues and others galia blum, 1 georges von degenfeld, 2 kinneret keren, 3 misregulation of cysteine protease activity is associated with numerous pathologies ranging from cancer to autoimmune disease. protease activity is controlled by a delicate balance of many factors such as levels of natural inhibitors and posttranslational modifications. thus developing a detection method for monitoring protease activity rather than abundance is desirable. here we describe the design and synthesis of a novel class of chemical tools, activity based probes (abps), that detect protease activity. these probes are composed of a fluorescent tag and its cognate quenching group, a peptide recognition scaffold, and a reactive "warhead". these fluorescently quenched "smart probes" covalently modify protease active sites in a fashion that is dependent on activity of the protease. this results in loss of the quenching group, producing a fluorescent signal. we report the production of selective, cell permeable activity based probes for the study of papain family cysteine proteases in cells and whole animals. these probes are used to monitor real time protease activity in living cells using fluorescence microscopy techniques as well as standard biochemical methods. key: cord-005337-3f6pwyy3 authors: yoon, hyun kyung; jung, sang taek; kim, jae-ho; yoo, tae hyeon title: recent development of highly sensitive protease assay methods: signal amplification through enzyme cascades date: 2013-01-04 journal: biotechnol bioprocess eng doi: 10.1007/s12257-012-0545-9 sha: doc_id: 5337 cord_uid: 3f6pwyy3 proteases are involved in almost all biological processes, and therefore, aberrant activity of many of these enzymes is an important indicator of disease. various methods have been developed to analyze protease activity, among which, protease assays based on resonance energy transfer are currently used most widely. however, quantitative methods with relatively higher sensitivity are needed, especially for disease diagnosis at early stages. one of the strategies to achieve higher sensitivity is to implement signal amplification of the protease activity. in this review, we briefly summarize the protease assay methods based on resonance energy transfer, and then elaborate the efforts to develop sensitive protease assays through signal amplification by using enzyme cascades. proteases (or proteolytic enzymes) hydrolyze the peptide bond of proteins by recognizing the side chains of specific amino acid sequences. approximately 2% of human genes encode proteases or their homologs, and proteases are involved in various biological processes, such as development, immunity, blood clotting, and wound healing [1, 2] . therefore, aberrant protease activity is associated with various diseases, including cancer, cardiovascular disease, alzheimer disease, inflammatory disease, and virus-related diseases [3] [4] [5] [6] [7] . to treat diseases resulting from hyperactivity of proteases, small molecule inhibitors have been developed. inhibitors against matrix metalloproteinase (mmp) and cathepsin, which play an important role in cancer metastasis, have been actively investigated [4, 8, 9] . protease inhibitors against the hiv protease are one of the most successful approaches to controlling the disease [7] . on the other hand, in diseases that are characterized by low or no protease activity, recombinant proteases are introduced into humans; for example, factor ix for hemophilia b and tissue-plasminogen activator (tpa) for breakdown of blood clots [10] . because of this relevance of proteases to disease states, the activity of specific proteases is an important indicator in the diagnosis of many diseases. for example, serine protease kallikrein 3, also known as prostate specific antigen, is a diagnostic marker for prostate cancer [11] . cathepsin, urokinase, and mmps are also known as markers of cancers [4, 8, 12, 13] . in addition, high activity of calpain is reported to be associated with altered calcium homeostasis, resulting in various pathologies [14] . a number of methods have been developed to assay proteases. immunoassays that rely on capturing proteases of interest by using specific antibodies have been developed to detect their abundance [15] . however, it should be noted that the protease activity, rather than its quantity, is indicative of disease states. consequently, the immunological methods have limitation, and are rarely applied in screening protease inhibitors. traditional biochemical methods, such as liquid chromatography and gel electrophoresis, have also been used to measure the activity of proteases [16] ; however, most of them are timeconsuming, and thus, cannot be easily adapted for highthroughput analysis of samples. recently, resonance energy transfer (ret)-based approaches have been actively investigated [17, 18] . two molecules, involved in ret, are linked by peptide substrates and cleavage of the substrate separates the two molecules and alters attributes of detectable signals. in the first part of this review, we summarize these methods briefly. early diagnosis of disease is closely associated with the likelihood of success in treatment of many diseases, and methods with high sensitivity and low noise are needed to facilitate early diagnosis. in the later parts, we introduce one of the strategies for developing sensitive protease assays, in which the signals generated by proteases are amplified through a process of enzymatic cascade. ret or fluorescence resonance energy transfer (fret) are processes in which a donor chromophore, in its excited state, transfers energy to an acceptor chromophore (typically, at a distance closer than 10 nm to the donor) via nonradiative dipole-dipole coupling. in the protease assays based on fret, the donor and acceptor molecules are linked together through short peptide substrates, and cleavage of the peptides results in reduction of the fret efficiency ( fig. 1) [17, 18] . two types of systems have been developed. in the first system (fig. 1a) , the donor is a fluorophore, and the acceptor is a quencher. the emission spectrum of the donor overlaps with the absorption spectrum of the acceptor, and the quencher reduces the intensity of fluorescence from the fluorophore. upon hydrolysis of the peptide substrate by protease, the donor and the acceptor move apart, and the emission of light from the fluorophore is restored. in the second configuration, a second fluorophore instead of the quencher can be linked to the donor fluorophore, (fig. 1b) , and the acceptor absorbs light from the donor and emits light of a different wavelength. cleavage of the peptide linker between the two fluorophores results in an increase in the fluorescence from the donor, and in the second system, a reduction or elimination of the acceptor fluorescence. fret methods have been widely used to assay protease activities because of its advantages, especially coming from the fact the signal is based on fluorescence [17, 18] . compared to the traditional biochemical methods, the signal-to-noise ratio is high, the signal can be measured without any additional purification step after reaction, and the assay cost is generally low. in particular, the platform can be easily developed into a high-throughput assay method, which makes it possible to analyze the samples in parallel for determining the substrate specificity of a protease or for finding protease inhibitors [19] [20] [21] [22] . the chemical compounds inhibiting proteases can then be developed further into therapeutics for diseases resulting from hyperactivity of proteases. in addition, these assays can also be used to reverse-engineer desired substrate specificity for a given protease. for instance, a highthroughput fret-based method for assaying ompt endopeptidase activity was used for engineering the substrate specificity of the enzyme [23, 24] . various organic dyes have been used as donors and acceptors [18, 25] . in particular, with the development of new chromophores with different absorption and emission wavelengths, various combinations are now possible, which enables multiplexed analyses of samples. despite advantages with using synthetic organic chromophores, their applications are limited because of some unfavorable inherent photophysical properties [17] . recently, much attention has been focused on synthesis of nanoparticles with enhanced properties, such as photostability, absorption capacity, quantum yield, and fluorescence lifetime, compared with those of organic dyes [17, 18] . with the discovery and engineering of new fluorescent proteins, protein chromophores have emerged as alternatives to synthetic probes [26] . fluorescent proteins are linked to synthetic compounds, such as organic dyes or nanoparticles, or two fluorescent proteins are connected to each other through a peptide linker. in the latter case, the entire protease sensor system can be expressed in a recombinant form in engineered cells, and the protein fret pairs can be used for monitoring protease activity inside the cells. however, synthetic chemical compounds generally have difficulty in penetrating the cellular membrane, thus, limiting their application in vivo. one limitation of fret-based methods that employ fluorescent chromophores as donors is the requirement for external luminescence to initiate fluorescence transfer. in addition, nanoparticles are not usually used as acceptors because they have broad absorption spectra, and thus, can be excited by external light that is used for exciting donor probes. in order to overcome these drawbacks, the luciferase enzyme has been investigated as a source for photon emission, in a process referred to as bioluminescence resonance energy transfer (bret) [17] . luciferase oxidizes luciferin into oxyluciferin, which relaxes back to the ground state by emitting a yellow-green light. because an external light source is not needed for excitation, bret methods have a lower background signal and usually have higher sensitivity than fret-based methods. detection of small amounts of marker proteins enables disease diagnosis at early stages, which often correlates with success in the treatment of these diseases. quantitative methods with high sensitivity and low noise are crucial for developing these diagnostic methods. even though fretbased protease assay methods have significantly improved with advancements in technologies for synthesizing fluorescent molecules and for light-detection instruments, there is still a need to develop new assay methods with higher sensitivity, while maintaining at least the same level of noise. one strategy for improving the assay sensitivity is to take advantage of a signal amplification processes via enzyme cascades ( fig. 2 and table 1 ). in these methods, a protease of interest activates another enzyme by removing the restrictions on the enzyme via a cleavage event, and the activated enzyme then generates detectable signals. these methods include signal amplification steps by reporter enzymes, and the degree of amplification is dependent on the catalytic activity of the activated enzymes. in the remaining part of this review, we will summarize recent research focused on developing methods of measuring protease activity utilizing the enzyme cascades. 3.1. pro-protease protease sensors a recent report described an assay method for enteropeptidase, which employs a naturally occurring enzymefig. 2 . signal amplification strategy using enzyme cascades. a protease activates a zymogen into an active one, which then generates a detectable signal. the activated enzyme can convert more than one substrate; thus, the protease activity signal is amplified, and the readout signal is theoretically higher than that generated directly by the protease activity, such as the signal generated in fig. 1 . autoinhibited split luciferase luciferin luminescence tev caspases [40, 41] autoinhibited split lactamase fluorocillin fluorescence tev [40] phospholipase a 2 nbd c 6 -hpc fluorescence sumo protease [34] cascade reaction. enteropeptidase is a serine protease and converts trypsinogen into active trypsin by cleaving the nterminal peptide (ddddk) of the zymogen [27] . in this assay, trypsinogen serves as the substrate for enteropeptidase, and the activated trypsin generated by the action of enteropeptidase cleaves a fret peptide substrate. the signal generated by enteropeptidase is amplified by the catalytic activity of trypsin. to develop the assay method, the authors first optimized fret peptide substrates of trypsin based on the fret efficiency and its cleavage efficiency/specificity, and further tested a range of enteropeptidase concentrations, using the optimized substrates and trypsinogen. the limit of detection (lod) was around 200 fm enteropeptidase in a complex biological mixture of escherichia coli lysate, as well as in a buffered solution. this is probably the lowest lod reported for any enteropeptidase assay, and the improved sensitivity is probably attributable to the signal amplification by the enzymatic cascade. an enteropeptidase assay is, perhaps, of limited clinical significance. however, the authors mentioned that the system can be adapted for the detection of any other protease. to apply this strategy for other proteases, the nterminal sequence of trypsinogen should be engineered in such that the protease of interest cleaves the modified sites; while the engineered trypsinogen remains inactive. one, and seemingly the only, approach is to insert a peptide sequence that the protease of interest recognizes and cleaves between the n-terminal peptide and trypsin. however, small changes in protein sequences can alter the function/structure of proteins, and additional studies, especially using proteases that are disease markers, are necessary to generalize the strategy employed in this study. verheijen et al. took a similar approach to develop an assay method for mmps, which are known to be involved in cancer metastasis [28] . pro-urokinase (pro-upa) is activated to urokinase (upa) through proteolytic cleavage by plasmin, and the activity of upa can be detected by using a chromogenic substrate (pyro-glu-gly-arg-p-nitroanilide). to engineer a form of pro-upa that can be activated by mmps instead of by plasmin, the authors replaced the sequence of amino acids in pro-upa that is recognized by plasmin (prfl ↓ iigg, where the arrow indicates the cleavage site) with one that can be hydrolyzed by many mmps (rplg ↓ iigg). using this system, the authors observed an increased mmp activity in synovial tissue extracts from patients with rheumatoid arthritis, compared to the activity of extracts from patients with osteorarthritis. the samples tested in this study might not have detectable upa activity, enabling detection of increased activity. however, blood and urine, which are most widely used for disease diagnosis, have significant activity of the reporter enzyme, upa, which definitely imposes a limitation on the clinical application of this assay method. firefly luciferase catalyzes the oxidation of firefly luciferin in the presence of mg·atp and oxygen into oxyluciferin, which emits a yellow-green light upon relaxation. luciferase provides high sensitivity and a wide dynamic range, and it has been widely used as both an in vivo and an in vitro bioluminescence reporter. in an attempt to exploit the beneficial properties of luciferase, fan et al. engineered luciferase asprotease sensors [29] . firefly luciferase has 2 domains, a large n-terminal domain and a small cterminal domain; both domains are connected through a hinge-like linker. the c-terminal domain rotates and translocates to the n-terminal domain upon substrate binding (fig. 3a) . based on these structural features, the authors engineered a circularly permuted construct: the n-and c-termini were connected through a linker that included sequence cleavable by a protease, and new c-and n-termini were created at the 233 and 234 positions, respectively (fig. 3b) . the linker restricts the conformational change induced by substrate binding, and the circularly permuted luciferase exhibits very little luminescence, several 1,000-fold lower than the wild-type enzyme. however, protease treatment increases the luminescence, in the range of 100 ~ 1,000-fold, depending on the specific protease and cleavable sequence used. laxmane et al. took another approach to engineer luciferase enzymes with attenuated activity, which can be activated by the action of caspase-3 [30] . the authors constructed caspase-3 reporters by fusing the estrogen receptor regulatory (er) domain to the c-terminus alone, or to both the n-and c-termini of firefly luciferase, with a caspase-3 cleavable sequence (devd) in the linker region between er and luciferase (fig. 3c) . the fusion of the er domain silences the activity of the reporter, and cleavage of the linkers by caspase-3 restores the luciferase activity. the mechanism by which the er domain inhibits luciferase has not been elucidated; however, the association of the er domain with heat shock proteins seems to play a role in the attenuation of luciferase activity [30] . in the case that both n-and c-termini of luciferase are fused to the er domain, the cleavage of the linkers resulted in approximately 10-fold increase in luminescence. in the study, the caspase-3 activity induced by tumor necrosis factor α-related apoptosis-inducing ligand (trail) was monitored real-time in tumors implanted in mice. despite the successful applications of the engineered luciferases, the systems have an important limitation. the enzymes are unstable in their purified forms, and the applications are restricted to in vivo bioluminescence or to in vitro analyses in an unpurified state, such as in cell lysate and in cell-free translation reactions. however, a system with a defined composition with purified proteins is needed in many cases to develop standardized diagnostic methods for disease detection. there are more stable luciferase enzymes from other organisms [31] , but they have to be engineered to have the desired properties, which can sometimes be extremely difficult. 3.3. sumo-pla 2 fusion-protein protease sensors sumolyation, wherein a small ubiquitin-like modifier (sumo) is covalently attached to the ε-nh 2 group of a lysine residue in proteins, through a series of enzymes (e1 activating enzyme, e2 conjugating enzyme, and e3 ligase), is a posttranslational modification and is known to be involved in many cellular processes, such as transport, transcription, apoptosis, and protein stability [32] . sentrinspecific proteases (senps) are isopeptidases that remove sumo moieties from proteins [32] . sumolyation is a dynamic process, and thus, the activity of sumo proteases is important in understanding sumo-related biological processes. phospholipase a 2 (pla 2 ) is an enzyme that releases fatty acids from the second carbon of glycerol and hydrolyzes fluorogenic substrates, such as 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-snglycero-3-phosphocholine (nbd c 6 -hpc). the enzyme requires a free amino terminus for catalytic activity [33] . by using these features of pla 2 , the researchers at progenra developed an assay platform for senps by constructing a fusion protein, sumo-pla 2 , which itself is inactive [34] . senps remove sumo from the fusion protein, and the activated pla 2 hydrolyses nbd c 6 -hpc and generates a fluorescence signal. leach et al. used this assay system to determine km values and to characterize inhibitors of sumo proteases [34] . split protein (or protein fragment) complement methods have been widely used to study biomolecular interactions [35, 37] . a protein is split into two fragments, and each fragment is fused to proteins of interest. when the two proteins interact with each other directly, or via another molecule, the split fragments are assembled into an active protein. autoinhibition is a naturally occurring mechanism for regulating protein activity: the protein has a polypeptide pseudosubstrate that blocks access to its active site, and the inhibitory region can be displaced through a conformational change or removed by a cleavage [38, 39] . shekhawat et al. employed these two mechanisms for regulation of protein activity, split protein complementation, and autoinhibition, to engineer protein reporters that can be activated by the action of protease (fig. 3d ) [40] . they used previously developed split protein complement systems: firefly luciferase and β-lactamase. one half of the reporter is linked to an antiparallel heterodimeric coiled coil (a-b), in which the a-b) is autoinhibited, and complementation of the two split fragments is thus prevented [40] . two coils are linked through a protease-cleavable linker, and the other half is linked to a coil (b'), which can dimerize with a. the heterodimeric coiled coil (a-b), which is autoinhibited, has a very low binding affinity to b', and the complementation of the 2 fragments is, therefore, unlikely. however, when a protease cleaves the linker connecting a and b, the interaction between a and b' can occur, and the split halves then assemble into an active reporter, such as luciferase or β-lactamase. using these systems, the authors developed and optimized protease sensors for the tev protease and caspase-3. the best luciferase sensor for tev provided a 1,000-fold increase of signal after protease treatment. in addition, logic gates were constructed by fusing autoinhibited heterodimer coiled-coils to both split halves. in a later study, the same group reported a panel of caspase sensors, which were used to investigate the substrate specificities of caspases and caspase activation pathways [41] . the protein sensors in this study were expressed using cell-free translation systems and then used to assess caspase activity in mammalian cytosolic extracts. however, it is plausible that the commercially available kits for in vitro translation would include proteases that activate the sensors. actually, when the caspase sensors were expressed using a rabbit reticulocyte lysate (rrl), significant luminescence signals were observed without the addition of a caspase, which was attributed to endogenous caspase activity in rrl. in addition, as mentioned above, the sensors must be expressed immediately before analysis, which can be an obstacle in developing standardized methods for analysis or diagnosis. cycling probe technology (cpt) is a technique originally developed for detecting specific dna sequences by using the unique property of rnase h, which hydrolyzes the 3'-o-p bond of rna in a dna/rna duplex [42] . kim and chung exploited the advantages of cpt to amplify the signal of a protease activity assay. this strategy differed from the ones that use the enzyme cascades that have been described so far, but the signal amplification method is worth introducing in this review [43] . the system used in this study included two kinds of gold nanoparticles (gnps), gnpa, in which the gnp is conjugated to a peptide-dna complex, and gnpb, in which the gnp is conjugated to a rna-fluorescein isothiocyanate (fitc) complex. gnpb particles were also pegylated to minimize heteroduplex formation between the gnpa dna and gnpb rna. thus, rnase h was prevented from cleaving the single-stranded rna of gnpb, and the fluorescence of fitc was quenched by gnps. when a protease of interest cleaves the peptide sequence linking the dna and gnpa, the dna oligomers are released from the nanoparticles and diffuse, eventually forming dna/rna duplexes with the rna in gnpb. duplex formation allows rnase h to hydrolyze the rna linked to gnpb, and the quenched fluorescence of fitc is thus recovered. dna oligomers, which are a product of the protease activity, are not consumed by the rnase h reaction, the cycle of duplex formation and rna hydrolysis can be continued, serving as the signal amplification step. the authors applied this system to the mmp2 protease, and the sensitivity of the protease was improved, to the detection of mmp2 at levels as low as 10 pm. proteases play important roles in diverse biological processes and are important markers of a number of diseases. various methods have been developed to analyze their activities, and the protease assay methods based on ret are, currently, the most common approaches. however, new tools with relatively higher sensitivity and at least the same level of noise, if not a further reduction in noise, are still needed. one solution to resolve this problem are the approaches that rely on amplifying the signal of protease activity by using enzyme cascades, and several specific strategies have been attempted so far. improved sensitivity has been demonstrated for these methods, but the optimization of these methods or development of new ones is required for application to diverse purposes. protease proteomics: revealing protease in vivo functions using systems biology approaches targeting proteases: successes, failures and future prospects dynamic imaging of protease activity with fluorescently quenched activity-based probes cancer. proteases--invasion and more protease-sensitive fluorescent nanofibers a genomic perspective on human proteases as drug targets discovery of next generation inhibitors of hiv protease matrix metalloproteinases: multifunctional contributors to tumor progression application of activitybased probes to the study of enzymes involved in cancer progression proteases as therapeutics the control of prostate-specific antigen expression and gene regulation by pharmacological agents cathepsin d in breast cancer: mechanisms and clinical applications, a 1999 overview fluorescent peptide probes for in vivo diagnostic imaging imaging calpain protease activity by multiphoton fret in living mice a one-step sandwich enzyme immunoassay for human matrix metalloproteinase 7 (matrilysin) using monoclonal antibodies application of in-gel protease assay in a biological sample: characterization and identification of urokinase-type plasminogen activator (upa) in secreted proteins from a prostate cancer cell line pc-3 analysis of protease activity using quantum dots and resonance energy transfer protease sensing with nanoparticle based platforms high throughput screening of potentially selective mmp-13 exosite inhibitors utilizing a triple-helical fret substrate activity-based fingerprinting of proteases activity based fingerprinting of proteases using fret peptides activity-based fingerprinting and inhibitor discovery of cysteine proteases in a microarray engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity highly active and selective endopeptidases with programmed substrate specificities using specificity to strategically target proteases using gfp in fret-based applications highly adaptable and sensitive protease assay based on fluorescence resonance energy transfer modified proenzymes as artificial substrates for proteolytic enzymes: colorimetric assay of bacterial collagenase and matrix metalloproteinase activity using modified pro-urokinase novel genetically encoded biosensors using firefly luciferase noninvasive real-time imaging of apoptosis redshifted renilla reniformis luciferase variants for imaging in living subjects protein modification by sumo active site and catalytic mechanism of phospholipase a2 detection and characterization of sumo protease activity using a sensitive enzyme-based reporter assay beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions split ubiquitin as a sensor of protein interactions in vivo kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals reprogramming control of an allosteric signaling switch through modular recombination autoinhibitory domains: modular effectors of cellular regulation an autoinhibited coiled-coil design strategy for split-protein protease sensors comprehensive panel of turn-on caspase biosensors for investigating caspase specificity and caspase activation pathways specific and sensitive detection of nucleic acids and rnases using gold nanoparticle-rna-fluorescent dye conjugates proteolytic fluorescent signal amplification on gold nanoparticles for a highly sensitive and rapid protease assay the authors acknowledge financial support from the new key: cord-027712-2o4svbms authors: urošević, vladimir; andrić, marina; pagán, josé a. title: baseline modelling and composite representation of unobtrusively (iot) sensed behaviour changes related to urban physical well-being date: 2020-05-31 journal: the impact of digital technologies on public health in developed and developing countries doi: 10.1007/978-3-030-51517-1_13 sha: doc_id: 27712 cord_uid: 2o4svbms we present the grounding approach, deployment and preliminary validation of the elementary devised model of physical well-being in urban environments, summarizing the heterogeneous personal big data (on physical activity/exercise, walking, cardio-respiratory fitness, quality of sleep and related lifestyle and health habits and status, continuously collected for over a year mainly through wearable iot devices and survey instruments in 7 global testbed cities) into 5 composite domain indicators/indexes convenient for interpretation and use in predictive public health and preventive interventions. the approach is based on systematized comprehensive domain knowledge implemented through range/threshold-based rules from institutional and study recommendations, combined with statistical methods, and will serve as a representative and performance benchmark for evolution and evaluation of more complex and advanced well-being models for the aimed predictive analytics (incorporating machine learning methods) in subsequent development underway. the urban public health, well-being monitoring, and prevention are recently being transformed from reactive to a predictive and eventually long-term risk mitigating systems, through a number of research initiatives and projects, such as the ongoing pulse project (participative urban living in sustainable environments, funded from the eu horizon 2020 programme) focusing on the chronic metabolic and respiratory diseases (such as type 2 diabetes and asthma) affected or exacerbated by the preventable or modifiable environmental and lifestyle factors, and well-being/resilience. a major challenge in the project is the modelling and assessment/prediction of citizen well-being from the collected and processed big data of unprecedented variety and from highly heterogeneous sources (health and vital activity personal data obtained through wearable devices and other sensing technologies, geo-located online surveys, open/public smart city datasets…), on individual and collective (population/cluster) levels. overall well-being and its main domains (vitality, supportive relationships, stress levels…) are all significant factors affecting the onset and exacerbation of the stated chronic diseases which are becoming more and more widespread and progressing in urban environments, and overall resilience of citizens and urban communities is increasingly important against other pertaining global and sustainability challenges, like climate change. the proposed and deployed elementary statistical model presented in this paper is to be the basis for interpretation and contextualization of changes to well-being, and a performance benchmark for evaluation and comparison of more complex and advanced well-being models of the aimed predictive analytics and final intelligent system (incorporating machine learning methods) in subsequent development, supporting the pulse phos (public health observatories established for the relevant policy making and execution in smart cities). the activity and vital/health parameters data measured mostly unobtrusively by wearable devices (wristbands, smartwatches) have particular significance for behaviour analysis and change recognition in pulse, as these are the input data streams with highest volume, acquisition "velocity", and temporal resolution/granularity of all the various data collected in the project, and therefore practically the most (and only) suitable data comprising the sufficiently continuous and non-sparse time series over months, to properly derive or construct the behavioural patterns and analyze behaviour changes. recent studies performed by the stated major wearable device manufacturers over billions of records of temporal measurements data [1, 2], as well as the experiences from projects like the just concluded city4age (www.city4ageproject.eu) [3, 4] , show the significance and general predictive ability of the measured main vital/health and activity parameters (walking, climbing stairs, physical activity/exercise, heart rate data, consumed calories…) for overall health and physiological/physical well-being assessment. the additional complementary socio-demographic, health, lifestyle/habits and environmental data in less frequent temporal resolution, ingested from the open/public datasets or manual "obtrusive" inputs, are combined to cross-check, adjust and improve integrity of the recognized behaviour changes derived from the main timeseries data acquired through the wearable devices. we adopt a combined knowledge-and data-driven approach in detection and characterization of relevant behaviours that denote significant variations in well-being, with multi-level hierarchical model topology and range/threshold based computational rules as basic primary formal knowledge structures, and statistical analytics as baseline (and performant) data-driven detection methods. the complexity of human behaviours is commonly represented through multi-level hierarchical structured models, decomposed to more granular "units" like activities and action events [5, 6] , with multiple variables from behavioural, physiological and environmental domains of well-being known to additionally increase complexity and dimensionality [7] . there are other contending approaches, like the monitoring and analysis of individual well-being or behavioural domain indicators or determinants independently in parallel, without hierarchical structuring and substantial synthesis into fewer higher-level composite factors or score(s) [8] . most, including the adopted and followed approach works (like [9] ), are comprehensively covered or referenced in the encyclopedia of quality of life and well-being research (springer 2014) that summarizes recent research works related to well-being and quality of life in spanned various research and policy-making/implementation fields. main advantages of a few composite synthetic indicators/factors over a battery of multiple separate indicators, namely: • ability to summarize complex and multi-dimensional real-life phenomena or domains (like well-being), • easier for interpretation and comparison among (socio-demographic, geospatial/regional…) groups or population clusters, • more effective for comprehending overall trends, particularly when a number of the underlying indicators denote opposing-trend changes, are of crucial significance in usage and context settings of the pulse project, with over 60 indicators formalized in the initial knowledge-based well-being model topology from the systematization of collected data, and with • visible set of indicators to various stakeholders (policy makers, researchers, general public) needing to be minimal without omitting important underlying information, • and collaboration, communication and comparison of complex dimensions by various stakeholders needing to be most straightforward, facilitated and effective. we therefore propose two complementary approaches for synthesis of the composite well-being indicators composed from underlying streamed iot-sourced timeseries data in the context of pulse. the indicators summarize multi-dimensional aspects of citizen well-being and enable the assessment of individual and synthesized collective urban well-being over time. the notion is illustrated through analysis within the scope of four representative and characteristical key summary indicators of citizen health and fitness, derived from activity and vital/health parameters measured, as stated, using wearable sensing devices: motility, physical activity, sleep quality and cardio-respiratory health/fitness (fig. 1) . in the first approach, daily and intra-daily underlying measurements (table 1 ) are used to estimate levels of adherence to rule-and range-based recommendations matured from institutional knowledge of relevant authorities and population-significant studies in the field, accumulated for over decades in the stated four example domains of motility, physical activity, sleep quality and cardio-respiratory fitness [8, 10, 11] . the complementary data-driven statistical approach is predicated on standard scores that denote the number of standard deviations that a given measurement deviates from the sample mean. this approach allows comparison of individual scores to the corresponding norm groups stratified by common socio-demographic parameters (age, gender…), when considered conditionally independent nodes in the complete model topology. it also allows to place a score for any individual and variable with respect to alternative descriptive statistic or measure of central tendency (variable median, geometric mean, standard deviation or error), so that more accurate or optimal comparison for specific variable distribution can be made. the data are collected by several types of health and fitness wearable tracker devices manufactured by fitbit, garmin and asus, monitoring physical and walking activity, sleep and heart/cardio parameters, for over 300 recruited citizens participating in the study in 7 global testbed cities (barcelona, birmingham, new york, paris, pavia(italy), singapore, and keelung/taipei), supplied with wearable tracker devices by the project. physical activity level as a single measure is mainly expressed in terms of time spent and calories burned while performing light/soft, moderate, and intense/vigorous physical activity. walking activity is captured with walked steps, distance, speed, and climbed stairs/floors measurements. heart rate measures capture time spent in different target heart rate zones (like peak, cardio, fat burn), resting and maximal heart rate (hr max ), and some still experimental measures like systolic and diastolic blood pressure, measured by the newest recently released devices such as asus vivowatch bp, but not yet acquired in significant volume sufficient for analysis. the peak heart rate zone by default definition ranges from 85 to 100% of person's maximum heart rate (hr max ), the cardio zone ranges from 70 to 84% of hr max , and the fat burn zone ranges from 50 to 69% of hr max . sleep quality/hygiene measures mainly capture time spent in defined phases of sleep. all the processed measures are listed in table 1 , collected or aggregated with a default daily periodicity, except the ones in gray-shaded rows which are acquired in higher intra-daily temporal resolution, mostly once in every 15 min or up to once in a minute, depending on the variable. in addition to stated behavioral time-series data, an extensive set of personal sociodemographic, profile (age, gender, ethnicity, educational and marital status, employment status and occupational environment…), as well as health state, risk factors and habits, lifestyle, neighbourhood and quality of life assessment, and other relevant behavioural data are manually input/submitted on each citizen participating in the study via online forms, composed from adapted relevant survey/assessment instruments for each specific field, like framingham, euroqol-5, ipaq-sf. these data are geolocalized to the residence location of each responding citizen for the purposes of analytics of collective/community well-being, and are collected from a greater number of recruited respondents, but just in rare cases in more than one iteration over time due to the high number and scope of covered variables, and therefore suitable for a broad but mainly static "snapshot" assessment of current well-being state rather than for behaviour change model and analytics. incorporating both these static and iot-sensed temporal data into a fully comprehensive predictive well-being model is an ongoing task in progress throughout the end of the project, with results to be presented in other upcoming publications. physical activity in our first approach stated above in sect. 2 can be discretized using several common baseline categorizations related or derived from the above mentioned relevant institutional/governmental and professional expert guidelines for the urban population groups. the example approach taken in the recent health survey of england from 2016 [12] compared well-being and mental health of adults in different sociodemographically stratified population groups by physical activity, among others. the activity level categories used in the analysis were the following: • meets aerobic guidelines: at least 150 min moderately intensive physical activity or 75 min vigorous activity per week or an equivalent combination of these • asserted activity: 60 to 149 min moderate activity or 30-74 min vigorous activity per week or an equivalent combination of these • low activity: 30 to 59 min moderate activity or 15 to 29 min vigorous activity per week or an equivalent combination of these • inactive: less than 30 min moderate activity or less than 15 min vigorous activity per week or an equivalent combination of these, and the corresponding linear scaled scoring function denotes "meets aerobic guidelines" with a score of 4, "certain activity" -3, "low activity" -2, and "inactive" with 1. this baseline scoring scale, besides sufficient granularity and robustness exhibited in referenced comprehensive studies, is also convenient for • mapping to the defined activity level categories used as input parameters for the consensus models for prediction of risk of type 2 diabetes (t2d) and asthma onset and exacerbation, developed for the pulse project [13, 17] • quantification of longer-term and/or periodic activity level behaviours directly from categorized daily or incidental activity level values as measured and acquired from the wearable tracker devices through relevant apis (light/soft, moderate, and vigorous/intense activity). similarly, the authors in [14] and [15] demonstrate the following referent threshold ranges of the number of daily walked steps to be used for classification of walking activity in healthy adults, and the corresponding scoring function linearly assigning the following 1-5 integer scores to the classification categories: highly active (12,500 or more steps/day) -5, active (10,000-12,499 steps/day) -4, somewhat active (7,500-9,999 steps/day) -3, lowly active (5,000-7,499 steps/day) -2, and sedentary (under 5,000 steps/day) -1. vo 2 max is the metric denoting the maximum amount of oxygen that an individual can use during intense exercise. it is widely and commonly used as an indicator of cardiorespiratory fitness. a simple generic estimate of vo 2 max of an individual can be obtained using their maximum and resting heart rates in the following formula, publised in [16] : vo 2 max % hr max hr rest ã 15:3 ml=ðkg ã minþ ð 1þ where hr max can be crudely estimated as 220age of the person. a relatively standard convenient and meaningful categorization of vo 2 max for western european and usa populations can be on a scale from 1 -very low, through 2 -low, 3 -fair, 4 -moderate, 5 -good, and 6 -very good, to maximal 7 -elite, depending of the individual's gender and age, with common categorization for males and females aged 6 to 75 published by shvartz & reibold in [11] . total average sleep duration in 24 h is a straightforward direct metric for assessing the quality of sleep in terms of longer-term stable behaviour across complete populations. the us national sleep foundation recently provided the following referent expert sleep duration recommendations (in terms of recommended (or not) threshold values for both oversleep and undersleep), categorized by precise granular age ranges [10] : these recommendations categorize possible output sleep duration times as either recommended, may be appropriate, or not recommended, and the optimal recommended duration is 7-9 h for majority of the populations. additionally, relevant recent findings like the extensive meta-analysis performed by the american diabetes association to assess the dose-response relationship between sleep duration and risk of type 2 diabetes [18] , have concluded that the lowest type 2 diabetes risk is for the average overnight sleep duration from 7 to under 9 h per day, and that both shorter and longer sleep durations than this optimum range denote up to 1.5 times increased risk (and up to 2 times increased cardiac conditions risk shown in the related studies [21] , also relevant in the project). we therefore slightly alter the may be appropriate category from the otherwise adopted recommendations from table 2 above to mildly risky, reflecting the importance of stated health risks in pulse, and the effect of common or periodically repeated behaviour patterns over months or years to the exasperation of the risks. this categorization will also consequently be communicated on the data visualizations and public health/prevention interventions and campaigns deployed and administered through relevant pulse system applications and modules (pho dashboards, pulsair gamified mobile app.) towards the citizens and urban communities, and the resulting function scores assigned to the categories are therefore: 1 for not recommended, 1.75 for mildly risky, and 2 for recommended, inversely proportional to the pesimistically estimated risks increase brought by shorter durations. complex eventual relations of detailed specific measured sleep parameters to well-being will be explored by more advanced methods in other subsequent work. from several existing elementary statistical approaches for aggregating the underlying dimensional indices and constructing the summary composite indicator value, we consider the weighted geometric mean of the four constituting dimensional indices as most adequate and appropriate for this specific well-being problematics: where summary dimensional indices denoted by the scoring function values are: i wwalking activity index, i pphysical activity/exercise index, i ssleep duration index, i ccardio-respiratory fitness index (through vo 2 max), and wt w , wt p , wt s , and wt c are respective weight factors, derived from expert assessments and rank data from relevant previous studies and experience, and assigned to adjust the relative importance and contribution of each of the indices to the resulting composite indicator value, per compositing methods outlined in [19] and [9] , or for derivation of composite un human development index (hdi). as all 4 constituting indices are directly proportional to the resulting composite indicator (i.e. the higher the activity levels or cardiorespiratory fitness scores, the higher the well-being), and low value of either of the four is significant for decreased overall composite (although there is some correlation between the indices -e.g. decrease in cardio-respiratory fitness in most cases causes decreased activity levels as well), the geometric mean is adequate for its sensitivity to low values of each individual constituting index, and ability to combine values on completely different scales without normalization required. initially assigned values of weight factors are 0.9 for i w , 1 for i p , and 1.05 for i s and i c , taking into account the importance of specific indices for respiratory disease and t2d risk, volatility of the collected data by now, and known overestimation of some measured variable values (like number of walked steps, vo 2 max estimate, or recognized sessions of cycling and some other exercise types) by the predominantly used wearable devices -fitbit charge 2 [20] . the weight factors are set as configuration parameters in the model, so they can be changed to fine-tune the composition according to the data insights acquired over time or the results of the validation described in sect. 5 below. time series of the values of the composite indicator are formed from weekly and monthly aggregations of underlying daily and intra-daily measurements into the 4 constituting index values. method for computing those values from the measured values of variables listed in table 1 above is as developed and introduced in [22] for synthesis of indicators and geriatric factors from the same source iot data, based in this case on univariate normalization of relative changes (quantified in standard scores, as stated above in the "approach" sect. 2) of acquired big temporal data during the complete study period, and then multivariate weighted linear aggregation of obtained normalized indicators and descriptive statistics into higher-level composite factors, to capture weekly and monthly behavioural patterns and trends, less susceptible to influence of outliers and ocassional notably deviating values. validation metric is the correlation with specific corresponding summary measure(s) of current well-being, self-reported by the respondent citizens through web and mobile app. questionnaires as mentioned above. they can be summarized from two relevant subset questionnaires: 1) european social survey (ess), and 2) euroqol-5d (eq5) survey instrument, both standardized (with minor adaptations) and common for measuring well-being in multiple continuous and/or repeated relevant europe-wide and national-level studies, and robust to some degree against extreme fully subjective bias. 15 statements of the ess questionnaire broadly cover social and most of the other aspects of personal and community well-being that the respondent rates on a 5-degree likert scale (strongly agree-4, agree-3, neither agree nor disagree-2, disagree-1, strongly disagree-0), the total possible questionnaire score thus ranging from 0, denoting the lowest/worst well-being, to 60 representing the optimum. eq5 instrument is focused on physical and mental health status and daily life activities measured in 5 dimensions (mobility, self-care, usual activities, pain/discomfort and anxiety/depression), also self-rated on a 5-degree scale from perceived worst to best like in ess. last question asks for assessment of the respondent's overall health state (hsa) on the current day, on the scale from 0 (worst) to 100 (best imaginable), also mapped to a number ranged from 1 to 5 by the formula 1 + 4 * hsa/100 for the purpose of this evaluation. total cumulative eq5 score thus ranges from 6 to 30. figure 2 below shows the correlation scatter plot of ess scores and composite well-being indices for 97 respondents of which 12 filled ees questionnaire twice, two filled it three times, and the rest only once during the observed period of 11 months. figure 3 shows the relationship between the composite well-being indices and the obtained eq5 scores of 107 respondents, 3 of which filled the questionnaire three times, 20 filled it twice, and the rest once during the observed data collection period. the analysis reveals a medium positive correlation of 0.424 (with the p-value of approx. 3.7 â 10 −7 ) between our constructed composite index and cumulative eq5 scores. the composite indicator and its constituting domain indices can therefore be considered promising for their intended purpose of basic represention of the urban physical well-being aspects modelled from a variety of heterogeneous underlying activity and health/vital parameters measured by iot wearable devices, summarized in 4 main dimensional and one overall derived score convenient for comparisons, interpretation and presentation to the end-users, particularly in the required shortest most concise manner and form, such as through a mobile app. ui or intervention messages. as almost half of the questions in eq5 are very remotely or not at all related to the physical well-being aspects summarized by the composite indicator, the correlation is expected to increase when the ongoing work in incorporating social and other wellbeung aspects fully in the model is completed. found small positive correlation of 0.287 (p-value 0.001) between the composite indicator and cumulative scores of ess questionnaire (in which most of the questions are not related to physical well-being) additionally points to the significance of this composite indicator to the overall well-being. the work also continues on the cleaning and pre-processing the data collected on the remaining monitored citizens, incorporation of machine learning methods in the model and exploration and modelling of the influence of detailed sleep and cardiac parameters, as well as of the sensed ambiental data, on the main well-being domains. the images or other third party material in this chapter are included in the chapter's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the chapter's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. ageing-friendly cities for assessing older adults' decline: iot-based system for continuous monitoring of frailty risks using smart city infrastructure a critical analysis of an iot-aware aal system for elderly monitoring semantic and fuzzy modelling for human behaviour recognition in smart spaces a scalable hybrid activity recognition approach for intelligent environments comparative impact study of the european social survey (ess) european research infrastructure consortium (eric) systematic scoping review of indicators of community wellbeing in the uk composite index construction national sleep foundation's sleep time duration recommendations: methodology and results summary aerobic fitness norms for males and females aged 6 to 75 years: a review health survey for england 2016: well-being and mental health. nhs digital health and social care information centre and the uk office for national statistics hapt2d: high accuracy of prediction of t2d with a model combining basic and advanced data depending on availability how many steps/day are enough? for adults estimation of vo2max from the ratio between hrmax and hrrest -the heart rate ratio method a bayesian network analysis of the probabilistic relations between risk factors in the predisposition to type 2 diabetes sleep duration and risk of type 2 diabetes: a meta-analysis of prospective studies indicators and methods for constructing a us human well-being index (hwbi) for ecosystem services research assessing the ability of the fitbit charge 2 to accurately predict vo 2 max sleep duration and risk of stroke events and stroke mortality: a systematic review and meta-analysis of prospective cohort studies data driven mci and frailty prevention: geriatric modelling in the city4age project empowering citizens through perceptual sensing of urban environmental and health data following a participative citizen science approach acknowledgment. this work has received funding from the european union's horizon 2020 research and innovation programme under the grant agreement no. 727816 (pulse). the performed research studies have all been granted ethical approval from the relevant irb authority in each pulse pilot testbed city (ethics committee of the parc de salut mar hospital in barcelona, through nhs health research authority iras (integrated research application system) in birmingham, new york academy of medicine irb in new york city, etc.), resulting from comprehensive multimonthly evaluation processes. the inclusion and exclusion methods and criteria for recruiting citizens have been specified in relevant previous publications of the project, such as in section 2.2.1. participation criteria in [23] . key: cord-260674-a0ejus6m authors: chopra, sakshi; ranjan, piyush; singh, vishwajeet; kumar, suraj; arora, mehak; hasan, mohamed shuaib; kasiraj, rhytha; suryansh; kaur, divjyot; vikram, naval k.; malhotra, anita; kumari, archana; klanidhi, kamal bandhu; baitha, upendra title: impact of covid-19 on lifestyle-related behavioursa cross-sectional audit of responses from nine hundred and ninety-five participants from india date: 2020-10-06 journal: diabetes metab syndr doi: 10.1016/j.dsx.2020.09.034 sha: doc_id: 260674 cord_uid: a0ejus6m background and aims: the impact of measures taken to contain covid-19 on lifestyle-related behaviour is undefined in indian population. the current study was undertaken to assess the impact of covid-19 on lifestyle-related behaviours: eating, physical activity and sleep behaviour. methods: the study is a cross-sectional web-based survey. a validated questionnaire to assess the changes in lifestyle-related behaviour was administered on adults across india using a google online survey platform. results: a total of 995 responses (58.5% male, mean age 33.3 years) were collected. an improvement in healthy meal consumption pattern and a restriction of unhealthy food items was observed, especially in the younger population (age <30 years). a reduction in physical activity coupled with an increase in daily screen time was found especially among men and in upper-socio-economic strata. quarantine induced stress and anxiety showed an increase by a unit in nearly one-fourth of the participants. conclusions: covid-19 marginally improved the eating behaviour, yet one-third of participants gained weight as physical activity declined significantly coupled with an increase in screen and sitting time. mental health was also adversely affected. a detailed understanding of these factors can help to develop interventions to mitigate the negative lifestyle behaviours that have manifested during covid-19. covid-19 is a global burden which continues to redefine daily lifestyle-related habits in a significant manner as the pandemic progresses through its different phases. public health recommendations and government measures taken to abate infection have indirectly impacted food availability, dietary quality, normal daily activities, access to recreational public settings, social activities, work and financial security [1] . these factors compound over time to radically change lifestyle-related behaviours, especially daily eating, activity and sleep behaviours that are known to be independent risk factors for metabolic complications such as obesity, diabetes and cardiovascular disorders [2, 3] . few preliminary studies from the west have highlighted a negative impact on various lifestylerelated behaviours as the potential implication of covid-19. however, these studies were done during the complete lockdown phase and suffer from methodological limitations like less representative sample and non-validated tools for data collection. moreover, the interplay of the severity of covid-19 infection with different social, economic and cultural constructs in determining the extent of changes in lifestyle-related behaviours might vary from country to country. there is a lack of evidence that evaluates the effect of covid-19 on lifestyle-related behaviours in india. it is important to investigate some key questions such as which lifestyle behaviours are most affected, how much is the impact of covid-19 on these behaviours, what are the reasons for these changes and which demographic section is the most impacted. considering these questions, we undertook this study to evaluate the overall impact of covid-19 on lifestyle changes experienced by individuals during the pandemic. the answers to these questions will establish a fundamental basis to develop appropriate recommendations for lifestyle modifications during this time. a web-based cross-sectional study was conducted on the general population to assess the impact of covid-19 on daily lifestyle-related practices such as dietary, activity and sleep pattern using a validated questionnaire. the study was approved by the institutional ethics committee this study was a rapid, large cross-sectional online survey conducted during the unlock phase (15 th august 2020 to 30 th august 2020) across various cities, towns and villages in india. the data was collected using google form web survey platform and telephonic interview. a standard study invitation message along with the link to the online survey was shared through personal and social contacts of the research group members via email, facebook, instagram, and whatsapp. we also asked the participants to share the study link to increase study participants, which allowed us to conduct a nationwide survey, especially during the pandemic situation. in cases, where participants had limited literacy levels or technical knowledge to fill the google form by themselves, the investigators conducted a telephonic interview and filled the form on their behalf. a brief description of the study, its objectives and the declaration of anonymity and confidentiality were given to the participants before the start of the questionnaire shared via google form. informed consent was taken from all the participants at the time of enrollment either by checking. participants were also requested to be honest in their responses. following this, the participants answered differential questions on the changes experienced in their lifestyle before and during the pandemic. during the survey, participants were able to stop study participation and leave the questionnaire at any stage before the submission process; if doing so, their responses would not be saved. responses were saved only by clicking on the "submit" button provided at the end of the questionnaire. the principle of maximum diversity was followed to recruit a representative sample for this study. quota sampling technique was used to identify the quotas based on different demographic variables such as age, gender, socio-economic strata and place of residence. the number of j o u r n a l p r e -p r o o f participants in each quota was compared with the prevalence of different categories in indian population and efforts were made to sustain maximum representativeness. a total of 1058 responses were collected using the google form link and telephonic survey, after excluding responses that met exclusion criteria such as younger participants (age <18 years), duplicates and invalid entries. the final data included 995 participants (as shown in supplementary figure 1 ). the electronic survey questionnaire was designed to assess changes in multiple lifestyle-related behaviors such as eating, physical activity, sleep and other health related behaviours during the covid-19 outbreak. the differential questionnaire used in this study was developed and validated as an extension of a short version lifestyle related practices questionnaire in indian adults [4] . the questionnaire has three sections assessing socio-demographic details, changes in lifestyle related behaviour and covid specific reasons for the changes in these behaviors. section a comprises questions relating to general information and demographic data, selfreported anthropometric data and one question of change in weight status during covid-19. section b consists of two parts with 24 items. part a (a1 to a24) assesses the baseline lifestylerelated behaviours and part b (b1 to b24) evaluates changes in different lifestyle related behaviors such as eating habits, physical activity and sleep pattern during the pandemic. the domain on eating behaviour consists of 12 items on meal pattern, portion size, frequency of meals, food group consumption pattern, emotional eating and intake of high fat, salt and sugar (hfss) foods and sugar-sweetened beverages (ssb) consumption. the domain on physical activity pattern has six items focusing on different components of activity such as aerobic exercise, involvement in household chores, leisure related activity, work-related sitting time and screen time. two items are for sleep patterns, one item for daily stress levels and two items for stress related addictive behaviours such as smoking and alcohol consumption. the five point likert-response choices are as follow: 'not routinely', 'one to two times a week', 'three to four times a week', 'five to six times a week' and 'almost daily'. the magnitude of the responses ranges from 5 (most acceptable behaviour) to 1 (least acceptable behaviour). section c has 6 items assessing the perceived covid-19 specific reasons for changes in lifestyle-related behaviours. j o u r n a l p r e -p r o o f descriptive statistics of the participants' baseline characteristics and responses were provided as frequency and percentage for categorical variables. continuous variables were reported as mean and standard deviation or median and range/interquartile range according to the distribution. the responses for before-covid-19 lifestyle scores and during-covid-19 lifestyle scores were assessed and these scores were subtracted for each item giving the mean difference scores which were associated with demographic variables. the association between the categorical variables was assessed using chi-squares test or fisher's exact test. the differences of continuous variables between two groups was assessed using t-test or wilcoxon test. while comparing more than two groups anova with bonferroni correction was done. for all analyses, p≤0.05 was considered as statistically significant. all statistical analyses were performed by using stata/se version 14.2 (statacorp lp, college station, tx, usa). the demographic details of the included participants (n=995) is shown in table 1 . the sample has slightly higher male participation (58.5%) with the mean age of 33.3 (14.5) (range, years. the representation from different socio-economic strata (according to kuppuswamy scale) and place of residence was fairly equal, with slightly greater number of participants from metropolitan cities (43.1%). the mean self-reported body mass index (bmi) was 24.8 ± 4.7 kg/m 2 . one-third participants reported a gain in weight during covid-19 pandemic. responses for differential items assessing the changes in lifestyle-related practice before and during covid-19 is given in table 2 . the habit of consuming meals routinely at regular intervals has slightly increased during covid-19 (42.5% vs 49.7%). the participants refraining from unhealthy eating behaviours such as consumption of fried food (64.1% vs 81.6%), junk food (44.3% vs 62.6%), fast food (53.2% vs 67.6%) and sugar-sweetened beverage (ssb) (50.1 vs 51.6%) also increased. participants reported marginal improvement in the frequency of j o u r n a l p r e -p r o o f consumption of different food groups such as fruits and vegetables (34 vs 37.8%), milk and its products (38% vs 40%) and pulses, meats and egg (18.2% vs 24%) during covid-19. in the physical activity domain, an increase in participants not routinely exercising for 30 minutes was observed (38.4% vs 50.4%). although, participants exercising more than three days a week (45.4% vs 45.1%) before the pandemic maintained the habit of exercising during the pandemic as well. participants refraining from routinely involvement in leisure-related physical activity also increased by more than double (29.4% vs 65.9%). one-third participants reported a daily screen time of 4-5 hours during covid-19 (13.7% vs 32.5%). participants reporting more than eight hours of sleep increased (10.2% vs 27.8%) but the overall quality of sleep marginally declined (50.7% vs 48.1%) and overall stress amongst participants increased (24.9% vs 38.2%). the comparison of mean scores of lifestyle related behaviours before and during covid-19 is shown in table 3 . there was a significant increase in routine consumption of meals at regular the frequency distribution of per unit difference in lifestyle scores before and during covid is shown in table 4 . the change in scores was calculated by subtracting during-covid lifestyle scores from before-covid lifestyle scores. in the eating behaviour domain, half of the participants experienced no change in regular meal pattern, whereas for 29.8% participants this habit was improved but for 18.56% this habit was worsened. the intake of high protein foods such as pulses, egg and meats increased during covid-19 (26.4%). the intake of unhealthy food items such as fast food (18.9%), junk food (1.98%), fried food (25.3%) and ssb (16.3%) improved by one unit. participants experienced a reduction in physical activity by 9.5% in comparison of the improved participants (22.05%) and leisure-related physical activity (45.63%) was three times higher than improved participants (15.68%). in addition to this, the daily sitting time increased for 33.07% participants. besides, the time spent daily on screen time increased by one unit in one-third of the sample (30.65%) and overall sleeping hours increased in one-fourth of the sample (25.53%). almost one-fourth (23.02%) participants reported an increase in stress level by one unit. although, overall social support from the family and friends (20.8%) during covid-19 improved as well. the reasons for change in lifestyle related behaviour is given in supplementary table 1 . factors such as fear of coronavirus infection (43.8%), preferring home-cooked food (25.2%) and less involvement in eating out and socializing (23.6%) were the prime reasons for improvement in healthy eating and decline in junk food consumption. although, participants involved in physical activity by walking (28.9%), at-home workout sessions (18.9%) and yoga (16%); adverse changes in physical activity levels were reported due to lack of motivation (24.5%), time availability (25.3%) and restricted access to parks, dance and fitness centre (28.6%). besides, participants' fear of getting infected by coronavirus (23.6%), worrying about their family (20.2%) followed by boredom and loneliness (18.2%) and financial loss (14.7%) were most commonly reported reasons for adverse changes in stress and anxiety levels during covid-19. j o u r n a l p r e -p r o o f the association of mean difference of during-covid domain-wise lifestyle scores from before-covid domain-wise lifestyle scores was studied with respect to different demographic groups as shown in table 5 . in the age category, a significant improvement in overall eating behaviour during covid-19 (2.44[6.49], p<0.001) was seen in the younger age group (≤30 years). besides, the overall physical activity worsen in all age groups (p<0.001). also, the overall physical activity worsened in both the genders, but men experienced a greater significant reduction in their activity status (-1. j o u r n a l p r e -p r o o f the outbreak of covid-19 and measures of its containment has evident impact on the lifestyle related behaviors in the population [5] . experts believe that lifestyle related predictors of weight gain and cardiometabolic risk are modifiable and should be screened and addressed during covid to prevent obesity and maintain general wellbeing [6] . the current study is a cross sectional web-based survey conducted to understand the impact of covid-19 on different lifestyle behaviors, severity of this impact across different demographic sections and covid-19 specific reasons for changes in lifestyle. we recruited a representative sample of 995 participants across india to complete a pre-validated questionnaire on lifestyle related behaviors using a webbased platform. the data collected was subjected to rigorous statistical analysis to generate robust inferences regarding the impact of covid-19 on lifestyle related behaviors in terms of both magnitude and direction. the key findings of the survey divulge certain trends in the eating habits and physical activity behaviour. firstly, a healthy eating trend was observed in terms of slight improvement in routine consumption of meals at regular intervals and consumption of protein-rich foods such as pulses, eggs and meat along with restricted intake of high fat, sugar, salt (hfss) food items, especially in the younger population (age <30 years). secondly, there was a significant reduction in moderate intensity aerobic exercises as well as leisure related activities coupled with an increase in daily screen and sitting time. overall, physical inactivity was comparatively higher in men and participants belonging to upper socio-economic groups. thirdly, quarantine induced stress and anxiety increased by a unit in almost one-fourth of the participants. the findings indicate that the participants improved slightly in terms of consuming meals at regular intervals in routine. regular meal pattern as a construct is often described as an individual's eating patterns at the level of a 'meal', such as a main meal (for example, breakfast, lunch or dinner) or a smaller-sized meal (for example, supper or snack) [7] . the consumption of nutritionally balanced small and frequent meals is associated with better dietary quality and is a common clinical recommendation for weight loss and reduction in metabolic comorbidities [8] . certain experts believe that a proportion of individuals may have marginally improved metabolism and other health outcomes during the covid-19 pandemic by adhering to the following dietary behaviors: (i) reducing meal frequency, (ii) consuming regular (i.e., breakfast j o u r n a l p r e -p r o o f (about 40% of daily total energy), lunch (30% of daily total energy) and dinner (30% of daily total energy) and having good quality meals (e.g., more fresh vegetables, good quality protein source, avoiding refined and high glycemic foods) [9] . the participants in our study also reported higher consumption of protein-rich foods such as pulses, eggs and meat. this is, however, contrary to the findings of another study conducted in the west, which found that daily consumption of regular meals had marginally lower contribution to the overall improvement in eating behavior during covid-19 (10) . a possible reason for this difference could be higher focus on home cooked food in the indian households. the results of this study highlight a significant but limited (mostly by one unit) improvement in quality of meals consumed by reduction in the consumption of calorie-dense fast food, fried food and ssbs. contrary to our findings, some studies suggest that confinement increased intake of hfss food items, which could be attributed to eating out of anxiety or boredom, a dip in motivation to maintain healthy eating or an increase in mood-driven eating [11, 12] . however, in our study that was conducted in india, the participants reported less socializing and eating out, preference to home cooked meals, time availability for meal preparation, incorporation of immunity-boosting foods to maintain health and better family support in maintenance of healthy eating pattern as prime reasons for reduction in unhealthy eating behaviors. it can also be noted that socializing and eating out practices mostly governed unhealthy food consumption in our sample. despite recommendations that covid-19 preventive measures should not hinder people from being physically active, present results show that there has been a decline in physical activity levels. a reduction in engagement in physical activity at all levels coupled with increase in daily sitting and screen time due to confinement was prominently found across the literature [10] . it is evident that government recommendations to limit outside movement and restrictions on social gathering (group sports and walking or exercise classes), availability (sports and gym facilities) and accessibility (public recreational spaces such as community centers parks and sports grounds) is linked to decrease in active participation in exercise and normal leisure related activity such as walking, grocery shopping, gardening etc. despite counteracting measures taken to increase overall activity at home by offering online "at home physical activity classes" through various social media platforms, present results indicate that it has not been possible for j o u r n a l p r e -p r o o f individuals to adequately maintain their normal activity patterns with suggested home activities [13] . the decline in time spent in engaging in physical activity was accompanied by increased screen and sitting time. a substantial increment in the number of hours (4-5 hours) spent in front of the screen for the recreational or work purpose was seen in our study as well. covid-19 has limited day-to-day social engagements such as workplace interactions, participation in recreational activities, socializing and eating out which might lead to an increase in mental health distress. we also found out that one-fourth of the participants reported an increase in stress and anxiety level by one unit due to fear of getting infected by coronavirus, boredom, loneliness and financial loss at work. similar findings were reported by a number of studies showing moderate levels of quarantine induced stress and anxiety in indian adults with more than 80% adults preoccupied with fearful thoughts of getting coronavirus infection [14, 15] . the study also highlights the association of demographic variables with changes in lifestyle related behaviors due to covid-19 pandemic. in our study, a significant improvement was observed in overall eating behavior in the younger age group (<30 years). similar findings were reported in an italian survey, where the highest compliance to a traditional mediterranean diet (high in antioxidants from fruits and vegetables and mufa from olive oil and fish) was exhibited by younger people (18-30 years) [12] . eating behavior also significantly improved in upper socio-economic status but conversely the physical activity decreased. despite reduction in food availability, the upper strata of society had the economic means and time to procure and maintain access to healthier ingredients (such as fresh fruits and vegetables, nuts, oilseed, milk and products) and transform them into healthy meals. it has been already seen in china that level of lockdown and socio-economic status defined level of physical activity [16] . the involvement of lower strata in delivering essential services to restore economic means for surviving the pandemic might have led to an increased daily overall activity level. we also found out that changes in these lifestyle related behaviors also led to weight gain in onethird of the sample. since individuals with obesity and associated metabolic comorbidities such as diabetes and cardiometabolic disease are more prone to getting covid-19 infection [17, 18] , the control on adaptation of negative lifestyle related behaviors becomes a crucial preventive step in containing the spread [19] . the findings also indicate that decline in physical activity and j o u r n a l p r e -p r o o f increase in stress outweighed marginal improvement in dietary behavior, which might have led to a positive calorie balance, further leading to weight gain in the sample. the results from this study are concurrent with western literature in establishing that adaptation of negative lifestyle related behaviors to abate coronavirus infection are one of the potential consequences of covid-19 pandemic. to our knowledge, this is the first study in india to understand the extent of changes in lifestyle related behaviors and its underlying covid specific reasons, in order to counteract these changes for maintenance of optimal health status at individual and community level. human behaviour is a product of a combination of environmental, cultural, economic and social variables, since all these variables are known to vary with changing situations during covid-19 pandemic, there is a need for further research to identify the correlated that has maximum impact on these behaviors to develop effective public health promotion strategies. the increase in usage of information and communication technology during pandemic should be used to our advantage in devising 'one stop lifestyle applications' to disseminate knowledge, change pandemic driven attitude and provide specific action points to manage healthy lifestyle habits amidst pandemic. the information on demographic variation in lifestyle practice during covid-19 can help to devise user-friendly behavioral support interventions using fitness applications, video streaming and motivation support. this is the first pan-india study which aimed to recruit a representative sample for collection of data using a pre-validated questionnaire to study the impact of covid-19 on lifestyle related behaviors. some limitations of the study are possibilities of reporting bias, due to e-survey and telephonic survey, validity of answers is a general problem of online surveys, differential approach described in the methods section. in conclusion, the results of the study indicate a mixed effect of the preventive measures enforced/adopted to control coronavirus on the lifestyle related behavior with a significant improvement in regular meal consumption pattern and healthy eating behavior and reduction in unhealthy food intake as positive indicators and significant reduction in physical activity and increase in sitting time, and screen time and stress as negative indicator. even though there were improvements in eating behaviors, but its affect was limited. the negative effect of lifestyle j o u r n a l p r e -p r o o f related behaviors would outweigh the positive effect of one unit correction in eating behavior, which would lead to higher incidents of weight gain and associated metabolic complications. these observations have potential implications that could aid the development of physical activity and nutritional recommendations to maintain health during the covid-19 pandemic. values are presented as mean ± standard deviation or number (frequency %). obesity risk during collective quarantine for the covid-19 epidemic dietary and lifestyle changes during covid-19 and the subsequent lockdowns among polish adults: a cross-sectional online survey plifecovid-19 study global physical activity levels:surveillance progress, pitfalls, and prospects a short questionnaire to assess changes in lifestyle-related behaviour during covid 19 pandemic impact of lockdown due to covid-19 outbreak : lifestyle changes and public health concerns in india covid-19 related home confinement in adults: weight gain risks and opportunities understanding meal patterns: definitions, methodology and impact on nutrient intake and diet quality effects of meal frequency on weight loss and body composition: a meta-analysis impact of sedentarism due to the covid-19 home confinement on neuromuscular, cardiovascular and metabolic health: physiological and pathophysiological implications and recommendations for physical and nutritional countermeasures effects of covid-19 home confinement on eating behaviour and physical activity: results of the eclb-covid19 international online survey food reward and food choice. an inquiry through the liking and wanting model eating habits and lifestyle changes during covid-19 lockdown: an italian survey traininginhome -home-based training during covid-19 (sars-cov2) pandemic: physical exercise and behavior-based approach depression, anxiety and stress among indians in times of covid-19 lockdown. community ment health j 2020 study of knowledge, attitude, anxiety & perceived mental healthcare need in indian population during covid-19 pandemic mental health outcomes of quarantine and isolation for infection prevention: a systematic umbrella review of the global evidence effects of nationwide lockdown during covid-19 epidemic on lifestyle and other medical issues of patients with type 2 diabetes in north india is excess weight a risk factor for the development of covid 19 infection? a preliminary report from india effects of covid-19 lockdown on lifestyle behaviors in children with obesity living in verona, italy: a longitudinal study key: cord-318528-yc0jw3s1 authors: romero-blanco, cristina; rodríguez-almagro, julián; onieva-zafra, maría dolores; parra-fernández, maría laura; prado-laguna, maría del carmen; hernández-martínez, antonio title: physical activity and sedentary lifestyle in university students: changes during confinement due to the covid-19 pandemic date: 2020-09-09 journal: int j environ res public health doi: 10.3390/ijerph17186567 sha: doc_id: 318528 cord_uid: yc0jw3s1 regular physical activity is related to many factors in a university student’s environment. the coronavirus pandemic and the resulting lockdown have restricted many elements of our environment. the aim of this study was to evaluate students’ physical activity and sedentary behaviour at two points in time: before and during the coronavirus lockdown. as a secondary aim, we also wanted to look at changes resulting from other factors (alcohol, tobacco, diet, stages of change, symptoms of anxiety/depression and sociodemographic characteristics). we conducted an observational, cross-sectional, pre-post study with two cut-off points. two hundred and thirteen students took part in the study. the main dependent variables were physical activity and sitting time, measured using the international physical activity questionnaire—short form (ipaq-sf). parametric and non-parametric tests were used for paired and unpaired data, as well as group-stratified analysis. during lockdown, both weekly physical activity (md: −159.87; ci: −100.44, −219.31) and weekly sitting time increased (md: −106.76; ci: −71.85, −141.67). in the group analysis, differences were observed in relation to gender, year of study, bmi, alcohol consumption, tobacco use, symptoms of anxiety/depression, mediterranean diet, living situation and stage of change. the results showed an increase in both physical activity and sitting time globally and by group. a healthy lifestyle should be promoted among all ages, but the earlier a habit is formed, the more likely it is to become rooted [1] . regular physical activity is one of the most effective ways of preventing premature death [2, 3] . the world health organization (who) recommends at least 150 min of moderate physical activity, 75 min of vigorous activity, or a combination of the two, per week [4] . independently of the physical activity carried out, it is important to assess sedentary behaviour (sb) as this is related to increased morbidity and cardiovascular risk factors [5] . by 2030, the who aims to reduce the prevalence of physical inactivity by 15% worldwide [6] . in spain, the amount of physical activity carried out by university students is low [7] and is in many cases linked to other healthy habits such as eating fruit and not smoking [8] . meanwhile, sedentary behaviour is a health problem in the child and youth population, which is aggravated with age [9] . in university students, sitting time can exceed 9 h a day [10] . it is known that individual factors such as age, sex and health status affect the physical activity that individuals do [11] . other factors associated with physical activity are motivation, lack of time and aspects related to body image or physical appearance [12] ; some of the beneficial effects of physical activity are reduced anxiety and depression [13, 14] . however, there are several factors that come into play throughout an individual's lifetime that can either facilitate or impede a behaviour, with the transition from secondary education to university being a decisive moment [15] . it is at this time that young adults form their behavioural habits, so the role of healthy universities and the healthy habits they acquire at this stage are fundamental in maintaining this behaviour in the years to come [16] . when it comes to making physical activity a regular habit, the elements that may be related have been studied in depth [17] . ecological models are considered one of the most significant theoretical approaches when it comes to analysing habit formation [18] . these models establish that in addition to individual factors, social and environmental factors are determinant in forming and maintaining physical activity habits [19] . the covid-19 pandemic led to the population being confined to their homes [20] . in spain, from march to april 2020, there was a prohibition on going outside to engage in sporting or social activities. during this period, elements of the built environment and other factors related to individuals' environments were restricted due to the state of alarm. this created a valuable opportunity to assess physical activity without taking these factors into account. experts' recommendations to prevent sedentary behaviour during lockdown included taking active breaks, getting up and walking around the house, and doing online workouts [21] . however, during the pandemic, an overall negative effect on physical activity intensity was observed, as well as a rise in the consumption of less healthy food and a 28.6% increase in sedentary behaviour [22] . a reduction in physical activity was also observed in university students [23] , along with increased levels of anxiety among 18-to 34-year-olds [24] . spanish university students had to continue attending classes online, and their social lives were limited due to the prohibition on going outside. during lockdown, physical activity could have been an opportunity to pass the time, or, conversely, sedentary behaviour could have increased. the other characteristics of each individual (gender, motivation, eating habits, mental state etc.) could have either facilitated or interfered with the decision to exercise. the hypothesis put forward was that students' sedentary behaviour would have increased during lockdown since they were confined to their homes, and that their physical activity would have decreased since they could not go outside to exercise. in this study, we aimed to analyse the physical activity university students did before and during lockdown. to broaden our approach, as a secondary aim, we also wanted to look at changes in physical activity and sedentary behaviour resulting from other factors such as alcohol and tobacco consumption, adherence to a mediterranean diet, motivation, symptoms of anxiety/depression and sociodemographic characteristics. we aimed to evaluate whether there were any differences when certain factors affecting individuals' environments were restricted. this was an observational, cross-sectional, pre-post study on health sciences students, with two cut-off points. the first cut-off point was between 15 and 30 january 2020, prior to the state of alarm being put in place, and the second sample point was between 1 and 15 april 2020. this study received the approval of the ethics and clinical research committee of ciudad real, in spain, with protocol number (c-291, 11/2019). this study was carried out within the context of another study that we conducted on healthy habits and lifestyles, with an estimated follow-up period of 9 months. due to the state of alarm and lockdown, recruitment of subjects was temporarily suspended and a decision was made to study the impact of lockdown on the population already participating. there were no exclusion criteria, other than failure to fully complete the questionnaire. to estimate the sample considering a bilateral hypothesis, the following criteria were used: variance in the pre-lockdown control group of 33,929.60, obtained using the total minutes of physical activity [25] , a beta risk of 20% (power = 80%), a confidence level of 95% and a clinically important difference of 60 min with respect to the control group. it was therefore estimated that a minimum of 148 study subjects would be needed. considering a missing values ratio of 20%, the resulting sample size would be 185 subjects. the students invited to take part were first-to fourth-year students who agreed to respond to the questionnaire at both time points. the questionnaires were administered during the second university semester. the first data collection point was two weeks after the end of the exam period, while the second data collection point was four weeks into lockdown. at the second data collection point, students could not leave their homes except for essential purposes such as buying food or going to hospital. outdoor exercise was prohibited across spain; anyone breaching the rules faced a 600 euro fine. during lockdown, university classes continued online with the same schedule as usual. the university provided internet access or technological devices to any students who requested them so that they could continue attending classes. online classes did not contain any recommendations for students to carry out physical activity. an ad hoc self-administered questionnaire was used, collecting sociodemographic information such as sex, age, weight, height, place of residence during the academic year, smoking habits (yes/no and number of cigarettes per day) and alcohol consumption (yes/no and number of drinks per week). for perceived health status and the existence of problems with anxiety/depression, the euroqol 5d (eq-5d) questionnaire was used [26] . to assess adherence to the mediterranean diet, the predimed questionnaire [27] was used, which uses 14 questions to assess the frequency of food consumption and eating habits. each question has a possible score of 0 or 1. the result allows classification into low adherence or high adherence. stages of change (soc) in physical activity were assessed using prochaska and diclemente's transtheoretical model (ttm) [28] . five stages of motivation for change were evaluated: pre-contemplation (i don't exercise and i don't intend to), contemplation (i don't exercise, but i'd like to), preparation (i exercise sometimes), action (i have been regularly exercising for less than 6 months) and maintenance (i have been regularly exercising for more than 6 months). physical activity was measured using the international physical activity questionnaire-short form (ipaq-sf), which contains 7 questions [29] . the questionnaire was used to obtain the total minutes of physical activity per week and sitting time per day. first, descriptive statistical analysis was performed using absolute and relative frequencies for categorical variables and mean with standard deviation (sd) for the quantitative variables. next, bivariate analysis was performed on the whole sample for paired data between weekly minutes of physical activity for the two sample points (pre-lockdown and lockdown). we used the kolmogorov-smirnov test to verify the normality of the quantitative variables. since there were variables that were not normally distributed, we then used the non-parametric wilcoxon signed-rank test. we also used the parametric student-fisher t-test to evaluate whether there were statistical differences in some comparisons and to obtain an approximation of the differences found. finally, the same analyses were performed again, but this time stratified for different sub-groups. mean differences (md) were obtained with a confidence interval of 95% (ci). all calculations were done using the program spss v24.0 (ibm corp, new york, ny, usa). two hundred and thirteen health sciences students participated in this study. the mean age was 20.5 years (sd = 4.56). of the participants, 80.8% (172) were women, 76.5% (163) were normal weight and 9.9% (21) were smokers. the rest of the demographic characteristics and health parameters are shown in table 1 . then, the results of the ipaq questionnaire were analysed: days and minutes of physical activity per week, as well as time spent sitting per week at both time points studied ( table 2) . we observed a significant increase in the number of days on which students engaged in physical activity, both vigorous we then analysed physical activity by group (table 3 ). when we looked at the differences in average minutes of physical activity, all groups analysed spent more time doing physical activity during lockdown (although not all of them significantly). groups that showed significant differences were women; first, second and third year of study; normal or low bmi; and those who did not eat a mediterranean diet. average physical activity time reduced during lockdown for participants in the pre-contemplation (md: 37.50; 95% ci: −115. 33, 190.33) and contemplation (md: 31.08; 95%ci: −15.87, 78.03) stages. in other words, they spent less time on physical activity, although this difference was not significant. conversely, for those in the preparation (md: −75.59; 95%ci: −0.92, −150.25) and action (md: 322.69; 95%ci: −214.84, −430.55) stages, significant differences (p < 0.05) were observed. in the rest of the groups analysed, statistically significant differences were observed between the two time points, except for men, final-year students, those that were overweight or obese and those that ate a mediterranean diet. finally, the analysis by group (table 4 ) showed significant differences (p < 0.05) in sitting time before and during lockdown in all groups except first-year students, those that were overweight or obese, smokers and those in the pre-contemplation stage. sitting time increased in all groups of the variables gender, alcohol, symptoms of anxiety/depression and mediterranean diet. it also increased in the following groups: second, third and fourth year of study; normal and underweight bmi; non-smokers; those living in a university residence, shared apartment or with family; and those in the contemplation, preparation, action and maintenance stages. this study aimed to evaluate physical activity and sedentary behaviour in health sciences students before and during the lockdown. at the first time point, students were in their normal study environment, while at the second, their social and environmental setting was limited due to lockdown. the results showed changes in physical activity and sedentary behaviour patterns both globally and by group. overall, students spent more time doing physical activity and spent more time sitting when their usual environment was limited. in the analysis by group, minutes of physical activity increased significantly during lockdown among the following groups: women; all years of study except final year; normal or low bmi; those who did not eat a mediterranean diet; and those in the preparation or action stage of change. sitting time increased in all groups of the variables gender, alcohol, symptoms of anxiety/depression and mediterranean diet. the groups that did not experience differences were: first year of study, overweight or obese, smokers and those in the pre-contemplation stage. these four groups spent the most time sitting at the first data collection point when compared with the rest of their cohort; in other words, sedentary behaviour was already high before lockdown and there were no significant differences at the second data collection point. some researchers believed that lockdown would cause inactivity and an increase in sedentary behaviour and that measures would need to be taken to prevent these effects [30] . in fact, during lockdown, people modified their lifestyles, with an increase in sitting time due to people spending more time at home, and there was also a reduction in the amount of time spent on physical activity [22] . in our study, the initial hypothesis was partially confirmed: there was an increase in sitting time, but unexpectedly, there was also an increase in both the amount of time spent doing physical activity and the number of days on which participants were active. we expected to find an increase in sitting time due to the restrictions on movement; however, we also thought that the increase in screen time would reduce physical activity time, since in previous studies conducted in the spanish university population, more screen time was associated with higher inactivity levels [31] . we do not know the exact reasons why physical activity increased, and we do not know if the effects on physical activity habits would have been maintained if the lockdown had gone on for longer. the environment in which students live affects their sedentary behaviour patterns [32] , and it seems that the characteristics of health sciences students' environments do not facilitate physical activity. rather than being an obstacle, restricted social relations and not having access to the built environment in their community increased the number of days and minutes students spent doing physical activity. in the case of health sciences students, another factor to consider is that their training in promoting healthy habits may have influenced their decision to exercise at home. no changes in physical activity were found in men. perhaps men and women had different motivations and the environment influences one gender more strongly. in previous studies on motives for physical activity by gender [33] , some variables that motivated men but not women were elements related to the environment, such as competition or social recognition, while weight control was the main motivation for women. in our study, women accounted for more than 80% of the sample, so the lack of results may also be due to the fact that there were fewer male participants. the effect of the built environment is yet to be determined for those with a high bmi [34] . the data in this study show that in overweight or obese students, there were no changes in time spent doing physical activity or sitting time. as we have seen, healthy habits that are ingrained in the population are not affected by the lockdown: this is the case of the mediterranean diet [35] . in this study, we observed that students that ate a mediterranean diet spent more time doing physical activity and that their physical activity patterns did not change significantly. this suggests that those that lead a healthy lifestyle pay attention to both diet and exercise and persist with their habits regardless of the environment. conversely, those with unhealthy habits stick to them and experience no changes during lockdown. this is the case for smoking and sedentary behaviour. grouping of healthy and non-healthy factors is habitual in university students [8, 25] : those that are more sedentary are also more likely to smoke or spend a lot of time watching screens, while those that exercise regularly tend to eat more fruit and vegetables and drink less alcohol. contrary to what we expected, smokers did spend more time doing physical activity during lockdown. it would be interesting to investigate the reasons for this. in our sample of the population, the percentage of smokers was very low, and the number of cigarettes smoked per day was also low, so we believe more research is needed in a sample with more smokers. in our results, we also found differences based on year of study. among final-year students, physical activity did not vary significantly. this group also spent the least time doing physical activity at both time points analysed. in their meta-analysis, keating et al. indicate that with regard to year of study, the majority of studies find no differences in physical activity, but that some studies suggest that higher years of study are less active [36] . as for sedentary behaviour, it was observed that first-year students spent more time sitting and that lockdown did not bring about any significant changes. some studies, contrary to the findings of our study, observed that students in higher years of study were more sedentary due to a higher workload [10] . in health sciences students, most of the theoretical workload is in the first year, while in their final year students spend most of their time on placement. another possible factor could be that first-year students might have practiced sport in secondary school and kept up the habit. it would have been interesting to ask students about their sports histories. in this study, we evaluated stages of change, one of the central concepts of the transtheoretical model of change. this model was initially used to treat tobacco and alcohol problems, but it was later adapted to other aspects of health such as physical activity and sedentary behaviour [37] [38] [39] . the analysis of the stages of change and how they affected the participants was very interesting. participants in the first two stages did not experience any changes, and neither did those in the last stage. the behaviour of participants that exercised as part of their routine remained practically the same, as did the behavior of those that did not do any exercise. however, for those that were motivated but had not yet made exercise a regular habit, lockdown was a good opportunity to increase their dedication. in line with these findings, di renzo et al. [35] observed in a recent study that lockdown increased activity among people that did sport occasionally because they had more time at home, but those that did not do any exercise did not use the situation as an opportunity to start. overall, the results show that minutes of physical activity increased, as did minutes of sitting time. although the results during lockdown are positive in terms of physical activity, it is necessary to recognise that this population might suffer from health issues in the future due to an increase in sedentary behaviour. it would be interesting to find out what the reasons were for students having this behaviour. perhaps they realised that their sitting time increased (they were not walking to class, walking to their car, going shopping, standing up, going to their jobs etc.) and compensated for this with some high-intensity exercise. another aspect that could have affected the results is that the students were involved in the health sciences field, so they may have been more prone to exercising during the pandemic than students in other majors such as engineering or literature. this is why we cannot exclusively consider the limitation of the environment during lockdown to be the cause of the changes in physical activity and sedentary behaviour. it would be interesting to continue studying the elements related to university students' physical activity/sedentary behaviour and their surroundings in order to plan strategies that promote an increase in physical activity levels in this group. our study has various limitations that should be considered. firstly, it is an observational study and all study subjects volunteered to participate in the questionnaire, so there may be a selection bias. secondly, we did not measure whether there was any risk of exposure to covid-19 infection, a factor that could have influenced our assessment of physical activity and sedentary behaviour. another limitation is the use of a self-administered questionnaire to evaluate physical activity and sedentary behaviour. it would have been more interesting to perform a real assessment of physical activity using accelerometry and also investigate their sports history. this could be a future line of research. finally, the lack of significance in some of the strata analysed could be due to a lack of statistical power because of the low number of subjects in some groups. furthermore, we do not know if these changes in physical activity would have been maintained if lockdown had gone on longer. as for the strengths, this is the first study to look at physical activity and sedentary behaviour in university students studying health sciences both before and during lockdown. in this study, we observed the behaviour of health sciences students when deprived of their usual social and community environment. participants spent more time doing physical activity and also spent more time sitting. university students' social environment may be a barrier to building an exercise habit, especially among women, and motivation seems to have a significant bearing on whether university students engage in physical activity. more efforts should be made to create strategies that motivate students to lead a healthy lifestyle in all aspects (diet, avoiding harmful substances, mental health etc.), with a particular emphasis on engaging in physical activity and reducing sitting time. programs and policies that promote positive youth development and prevent risky behaviors: an international perspective health benefits of physical activity: the evidence health benefits of physical activity: a systematic review of current systematic reviews world health organization. global recommendations on physical activity for health sedentary behavior and cardiovascular morbidity and mortality: a science advisory from the american heart association world health organization. global action plan on physical activity 2018-2030: more active people for a 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consumption, physical activity and diet quality the spanish version of euroqol: a description and its applications a 14-item mediterranean diet assessment tool and obesity indexes among high-risk subjects: the predimed trial stages and processes of selfchange of smoking:toward an integrative model of change validation of three short physical activity questionnaires with accelerometers among university students in spain how to deal with covid-19 epidemic-related lockdown physical inactivity and sedentary increase in youth? adaptation of anses' benchmarks cluster analysis of health-related lifestyles in university students neighborhood built environment and socioeconomic status are associated with active commuting and sedentary behavior, but not with leisure-time physical activity, in university students college students' motivation for physical activity: differentiating men's and women's motives for sport participation and exercise role of built environments in physical activity, obesity, and cardiovascular disease eating habits and lifestyle changes during covid-19 lockdown: an italian survey a meta-analysis of college students' physical activity behaviors role of counseling to promote adherence in healthy lifestyle medicine: strategies to improve exercise adherence and enhance physical activity application of the transtheoretical model to sedentary behaviors and its association with physical activity status levels of physical activity, motivation and barriers to participation in university students funding: this research received no external funding. the authors declare no conflict of interest. key: cord-016362-d3cpy37g authors: orhan, ilkay; özcelik, berrin; şener, bilge title: antiviral and antimicrobial evaluation of some heterocyclic compounds from turkish plants date: 2007-08-01 journal: bioactive heterocycles v doi: 10.1007/7081_2007_072 sha: doc_id: 16362 cord_uid: d3cpy37g antibiotic resistance has become a problem since the discovery of antibiotics. not long after the introduction of penicillin, staphylococcus aureus, which can be also transmitted to humans via milk and milk products, began developing penicillin-resistant strains. therefore, one approach that has been used for the discovery of new antimicrobial agents from natural sources is based on the evaluation of traditional plant extracts. natural products have played a pivotal role in antibiotic drug innovation and include aminoglycosides, cephalosporins, macrolides, cycloserine, novobiocin, and lipoproteins. however, only a few antiviral agents are available on the market. to this purpose, we have screened a great number of herbal extracts along with some pure natural substances and obtained interesting findings. this chapter covers the results of our rigorous search for new antiviral and antimicrobial alternative compounds from a number of turkish plants. since their discovery during the twentieth century, antimicrobial agents (antibiotics and related medicinal drugs) have substantially reduced the threat posed by infectious diseases. on the other hand, antibiotic resistance can be defined as the ability of any microorganism to withstand the effects of an antibiotic, which is a specific type of drug resistance and evolves naturally via natural selection through random mutation [1, 2] . the antibiotic action is an environmental pressure; those bacteria that have a mutation allowing them to survive will live on to reproduce. they will then pass this trait to their offspring, which will be a fully resistant generation [3, 4] . without doubt, antibiotic resistance is a global issue. because of cross-continental travel of both humans and goods, antibiotic-resistant bacteria are spread from one country to another. therefore, antibiotic resistance has been called one of the world's most pressing public health problems. much evidence supports the view that the total consumption of antimicrobials is the critical factor in selecting resistance. paradoxically, underuse through lack of access, inadequate dosing, poor adherence, and substandard antimicrobials may play as an important role as overuse [5, 6] . since misuse of antibiotics jeopardizes the usefulness of essential drugs, decreasing inappropriate antibiotic use is the ideal way to control resistance [7] . natural selection of penicillin-resistant strains in a bacterium known as staphylococcus aureus began soon after penicillin was introduced in the 1940s. recently more hospital-acquired infections are becoming resistant to the most powerful antibiotics available, such as vancomycin, which emerged in the united states in 2002, presenting physicians and patients with a serious problem [8] . a number of cases of community-associated methycillin-resistant s. aureus (mrsa) have also been reported, including cases in patients without established risk factors [9] . modern medical science and practice have something of an armory of effective tools, ranging from antiseptics and anesthetics to vaccines and antibiotics. however, one field has been weak in finding drugs to deal with viral infections, although viral diseases have affected humans many centuries. the emergence of antivirals is the product of a greatly expanded knowledge of the genetic and molecular function of organisms, allowing biomedical researchers to understand the structure and function of viruses. on the other hand, herbal medicine is the oldest form of healthcare known to mankind and has been used by all cultures throughout history. the world health organization (who) estimates that 4 billion people, 80% of the world population, presently use plants for some aspect of primary health care. who also notes that of 119 plant-derived pharmaceutical medicines, about 74% are used in modern medicine in ways that correlate directly with their traditional uses as plant medicines by native cultures. in our ongoing study on the plant extracts and their pure compounds from turkish medicinal plants, we have so far screened a number of plant extracts and pure components for their antimicrobial and antiviral activities. in this chapter, we intend to cover the recent results obtained from our antimicrobial and antiviral studies on heterocyclic compounds isolated from several turkish medicinal plants and marine organisms. although biodiversity in terrestrial environments is extraordinary, oceans covering more than 70% of our planet represent a greater diversity of life. a small number of marine plants, animals, and microbes have already yielded over 12 000 novel compounds [10] . among them, a great number of substances of marine origin have been reported to possess antimicrobial activity such as loloatins a-d, myticins a and b, psammaplin a, etc., which have been covered in several excellent reviews [10] [11] [12] . in our previous study, ten terpene-type compounds, which we isolated from the marine sponges ircinia spinulosa and spongia officinalis of the aegean sea collection, were assayed against the american-type culture collection (atcc) strains of bacillus subtilis (6633), s. aureus (6536), pseudomonas aeruginosa (9027), and escherichia coli (8739) by the disk diffusion method [13] . the results were evaluated by comparing the inhibition zones of the compounds, namely, furospinulosin-1, furospongin-1, 2-(hexaprenylmethyl)-2-methylchromenol, heptaprenyl-p-quinol, 12-epi-deoxoscalarin, 1,4,44-trihydroxy-2-octa-prenylbenzene, demethylfurospongin-4, 4-hydroxy-3-octaprenylbenzoic acid, 4-hydroxy-3-tetraprenylacetic acid, and 11-β-acetoxy-12-en-16-one. we found that furospinulosin-1, furospongin-1, 2-(hexaprenylmethyl)-2-methylchromenol, and heptaprenyl-p-quinol did not exhibit any inhibition against those bacterium strains, whereas 12-epi-deoxoscalarin (1) exerted a weak activity against b. subtilis and s. aureus, causing inhibition zones of 12 and 11 mm in diameter, respectively (fig. 1 ). demethylfurospongin-4 (2), 4-hydroxy-3-tetraprenylacetic acid (3), and 11-β-acetoxy-12-en-16-one (4) were also active against gram-positive bacteria (fig. 1 ). among them, 4-hydroxy-3-tetraprenylacetic acid (3) was the most effective causing 20 mm of inhibition zone (fig. 1) . none of the compounds tested had an ability to inhibit e. coli. owing to scarcity of the compounds, only one concentration (0.5 mg/disk) could be tested. in conclusion, we suggested that this assay should be better with higher yield, if repeated. in a similar study reported previously, 23 hydroquinone and quinone derivatives from the sponge ircinia spinulosa were tested for their antibacterial activity, and relevant structure-activity relationships (sar) were established [14] . as a consequence, sar studies indicated that the optimum length of side chain in the compounds for antibacterial activity should be 5-15 carbon atoms, which is in accordance with our most effective compound, 4-hydroxy-3-tetraprenylacetic acid (3). besides, it was reported that long-chain alcohols exert higher antimicrobial activity compared to the corresponding acids and aldehydes [15] . similarly, a relationship between antibacterial activity and the structure of aliphatic alcohols was described, which suggested that maximum activity against s. aureus might be depend on the number of carbon atoms in hydrophobic chain. this should be less than 12, but as close to 12 as possible [16] . however, according to the study of inoue et al., this finding did not support the anti-stapylococcus effect of the aliphatic terpene alcohols, farnesol, nerolidol, and plaunotol, which may result from configuration of functional groups and double bonds that affected activity [17] . therefore, in that study, it was concluded that anti-stapylococcus activity depends not only on aliphatic side chains, but also on the configurations of functional groups and double bonds. possibly, the difference observed in the antibacterial effect of our compounds might be due to this reason as well. flavonoids are a group of polyphenolic compounds ubiquitous in many plants, in which they occur as the free forms, glycosides, as well as as methylated derivatives. in addition to their diverse biological activities, there is an increasing interest in flavonoids due to their anti-infective properties [18] . for instance, the flavonoids quercetin and kaempferol, as well as the flavonoid glycosides rutin and isoquercitrin, were reported to have antibacterial and antifungal activities [19] . quercetin and kaempferol are known to be the most common flavonols present in many plants, and occur in different glycosidic forms. in many studies, they or their various glycosides have been proved to possess antimicrobial activity or, in other words, the antimicrobial activity of plant extracts (e.g., rubus ulmifolius, combretum erythrophyllum, morus alba, trollius chinensis, and propolis) has been attributed to quercetin and kaempferol [20] [21] [22] [23] [24] [25] . in one of our recent studies, we examined antimicrobial and antiviral activities of four flavonoid derivatives, namely, scandenone (5), tiliroside (6), quercetin-3,7-o-α-l-dirhamnoside (7), and kaempferol 3,7-o-α-ldirhamnoside (8) as shown in fig. 2 . these were tested against e. coli, p. aeruginosa, proteus mirabilis, klebsiella pneumoniae, acinetobacter baumannii, s. aureus, b. subtilis, and enterococcus faecalis, as well as the fungus candida albicans by the microdilution method [26] . in addition, both dna virus herpes simplex (hsv) and rna virus parainfluenza (pi-3) were employed for antiviral assessment of the compounds using madin-darby bovine kidney (mdbk) and vero cell lines. all four compounds were found to be most active against s. aureus and e. faecalis, with a minimum inhibitory concentration (mic) of 0.5 µg/ml, followed by e. coli (2 µg/ml), k. pneumoniae (4 µg/ml), a. baumannii, and b. subtilis (8 µg/ml). p. mirabilis and p. aeruginosa were the most resistant bacteria against the compounds (16 and 32 µg/ml, respectively). notably, antibacterial activity of the compounds was as potent as ampicillin (amp) and oflaxocin (ofx) towards s. aureus and e. faecalis. these compounds also possessed quite remarkable antifungal activity against c. albicans, as much as ketocanozole (ket) (1 µg/ml). as shown in table 1 , none of the compounds had the ability to inhibit hsv, while only quercetin-3,7-o-α-l-dirhamnoside had inhibitory activity against pi-3 in the range 8-32 µg/ml of maximum and minimum cytopathogenic effect (cpe) inhibitory concentrations, respectively. the inhibitory concentration range of this compound is on a vast scale, which resembles that of oseltamivir (32 to < 0.25 µg/ml). besides, its maximum non-toxic concentration (mntc) (64 µg/ml) was observed to be better than oseltamivir (32 µg/ml). one of the undisputed functions of flavonoids and related polyphenols is their role in protecting plants against microbial invasion, which accumulate phytoalexins in response to microbial attack plants. moreover, it is evident that a structure-activity relationship exists between the various flavonoids and their antimicrobial activity in most cases. a large number of antimicrobial flavonoids have been reviewed brilliantly and their sars discussed [27] [28] [29] [30] [31] [32] . the majority of antifungal flavonoids have been observed to have either isoflavonoid, flavan, or flavanone structures such as maackiain, mucronulatol, luteolin 7-(2 -sulfatoglucoside), etc., which is consistent with our data on flavonoids. accordingly, the presence of a phenolic group in the flavonoid structure suggests that it is necessary for antimicrobial activity. interestingly, increasing the number of hydroxyl, methoxyl, or glycosyl substituents resulted in the steady loss of antifungal effect of the flavonoids [33] . in the review of bylka et al. [34] , it was suggested that the antibacterial effect towards gram-negative bacteria is higher with flavones, while flavonoids containing two or three hydroxy groups in rings a and b are more active in inhibition of gram-positive bacteria. however, in our study, all four flavonoid derivatives, consisting of one prenylated isoflavone and three flavonol glycosides, exhibited an equal strength of antibacterial and antifungal activities, independent of their structural substitutions. recently, flavonoids have been investigated from the viewpoint of their antiviral effect, particularly against the human immunodeficiency virus (hiv), the causative agent of acquired immonodeficiency syndrome (aids). among them, quercetin has been shown to be effective against divergent virus types by many researchers, which supports our data on quercetin-3,7-oα-l-dirhamnoside [26] . in one of the earliest studies, oral application of quercetin in mice was found to display a protective effect towards intraperitoneal encephalomyocarditis, mengo m,l and mengo m virus infections, but not against intracerebral challenge with mengo m virus. it was not virucidal and did not interfere with mengo virus replication in l cells [35] . the potentiative interaction of quercetin with murine α/β interferon in mice against mengo virus infection [36] was also proved. moreover, quercetin was reported to greatly enhance the antiviral effect of tumor necrosis factor (tnf) that produces a dose-dependent inhibition of vesicular stomatitis virus, encephalomyocarditis virus, and hsv type-1 replication in wish cells [37] . in another study, the effect of different substituents of quercetin and luteolin on the ability to inhibit the hsv type-1 replication in rk-13 cells was studied [38] . in conclusion, our results demonstrated that scandenone, tiliroside, quercetin-3,7-o-α-l-dirhamnoside, and kaempferol-3,7-o-α-l-dirhamnoside possessed severe antibacterial and antifungal activities, whereas only quercetin-3,7-o-α-l-dirhamnoside exerted noticeable anti-pi-3 activity. the genus consolida, aconitum, and delphinium (ranunculaceae) are wellknown to be rich in diterpene alkaloids, which possess a diverse range of biological activities. these plants have also been the cause of poisonings, which primarily occur in cattle as well as human beings, due to toxicity of their alkaloids. in one of our recent studies, five diterpenoid-derivative alkaloids, lycoctonine (9), 18-o-methyllycoctonine (10), delcosine (11), 14acetyldelcosine (12), and 14-acetylbrowniine (13) (as shown in fig. 3) were screened for their antibacterial, antifungal, and antiviral activities [39] . once more, hsv and pi-3 were employed for antiviral assessment of the compounds using mdbk and vero cell lines. their mntc and cpe values were determined using acyclovir and oseltamivir as the references. besides antibacterial and antifungal activities, the alkaloids were tested against e. coli, p. aeruginosa, p. mirabilis, k. pneumoniae, a. baumannii, s. aureus, and b. subtilis, as well as the fungus c. albicans by the microdilution method as compared to the references amp, ofx, and ket. the results pointed out that these alkaloids possessed the highest antibacterial activity against k. pneumoniae and a. baumannii at 8 µg/ml concentration (table 2) , whereas they were moderately active to the rest of the bacteria. however, all the alkaloids tested were highly effective against c. albicans in a comparable manner to ket in the antifungal screening. conversely, a selective inhibition was observed towards pi-3 virus by these alkaloids, while they were entirely unsuccessful on inhibition of hsv (table 3) . pi-3 inhibitory activity of the alkaloids was fairly analogous to that of oseltamivir, ranging between 8-32 µg/ml as minimum and maximum inhibitory concentrations for the cpe. our results showed that the alkaloids possessed rather high antifungal activity against c. albicans and a compelling antibacterial effect only against k. pneumoniae and a. baumannii, while they exerted a strong inhibition against pi-3. even though much is already known about the toxicity of diterpene alkaloids that contribute to the toxicity of consolida, delphinium, and aconitium species, no antiviral study has been so far reported on this type of alkaloids. therefore, no sar studies have been encountered by us on the antiviral or antimicrobial activities of these alkaloids. however, a quantitative sar analysis performed on a number of diterpene alkaloids isolated from an aconitum sp. indicated that biological activity of these alkaloids may be related to their toxicity rather than to a specific pharmacological action [40] . in a current study on 43 norditerpenoid alkaloids from consolida, delphinium, and aconitum species against several tumor cell lines, lycoctonine and browniine were 312 i. orhan et al. found to be active among those screened [41] . in contrast to this data, lycoctonine and 14-acetyl derivative of browniine in our study showed a lesser amount of cytotoxicity on mdbk and vero cell lines at 64 µg/ml. gonzales-coloma et al. studied antifeedant activity and toxicity of some diterpene alkaloids (15-acetylcardiopetamine, cardiopetamine along with its amino alcohol, the β, γ unsaturated ketone, and the acetylated ketone derivatives) from delphinium sp. on the insects spodoptera littoralis and leptinotarsa decemlineata [42] . results of the study showed that the c13 and c15 hydroxy substituents are essential features of the active molecule, while the c11 benzoate group enhanced the biological effect on both insect species, where all of our alkaloids lacked those two substituents. in a taxonomic study done in 2002, lycoctonine-type alkaloids isolated from three delphinium species were classified into three groups according to the degree of their toxicity: n-(methylsuccinyl)-anthranoyllycoctonine (mal)-type with high toxicity, lycoctonine-type with moderate toxicity, and 7,8-methylene-dioxylycoctonine (mdl)-type with low toxicity [43] . in that paper, it was reported that the moiety attached to c14 is quite important for the toxicity of these alkaloids, which is also in accordance with our present data. furthermore, other functionalities on these molecules are also notable in terms of toxicity. it was noticed that the tertiary nitrogen, anthranilic acid substitution, and c1 moiety affect the toxicity degree within those alkaloids. for instance, when the methylsuccinyl group is removed from mal (which then converts to lycoctonine), lycoctonine becomes 93 times less toxic. briefly, our report was the first on antiviral, antibacterial, and antifungal activities of lycoctonine, 18-o-methyllycoctonine, delcosine, 14-acetyldelcosine, and 14-acetylbrowniine. furthermore, our data also suggest that all of the diterpene alkaloids are worthy of being evaluated for their antimicrobial and antiviral activities for future-promising results. in our recent work, we focused on investigation of antiviral activity of 33 isoquinoline alkaloids and seven derivatives of them, which are classified as protopine-type [protopine (14) and β-allocryptopine [44] , whose isolation procedures were described elsewhere [45] [46] [47] [48] [49] [50] (figs. 4 and 5) . moreover, the alkaloids were also tested against e. coli, p. aeruginosa, p. mirabilis, k. pneumoniae, a. baumannii, s. aureus, and b. subtilis, as well as the fungus c. albicans by the microdilution method, for their antibacterial and antifungal activities. according to our findings, all types of the alkaloids appeared to be more active to gram-negative bacteria than to gram-positive ones. most of the alkaloids, including protopine, β-allocryptopine, chelidimerine, fumarophycine, (±)-sibiricine, sibiricine acetate, (±)-dihydrosibiricine, parfumine acetate, α-hydrastine, (+)-bulbocapnine, berberine, (-)-canadine, (-)-ophiocarpine, ophiocarpine-n-oxide, corydalmine, oxosarcocapnidine, and corydaldine, showed significant inhibition on k. pneumoniae and a. baumannii, in particular, better than the rest of the gram-negatives, at 8 µg/ml concentration as compared to amp (2 µg/ml). all of the alkaloids, regardless of their structural differences, inhibited e. coli and p. mirabilis with mic of 32 µg/ml, while they inhibited s. aureus at 64 µg/ml. interestingly, the alkaloids that were found to inhibit k. pneumoniae and a. baumannii at 8 µg/ml also had remarkable inhibition on c. albicans (4 µg/ml), while a notable occurrence of antifungal activity was observed with the rest at 8 µg/ml concentration. the tested isoquinolines were observed to display a selective inhibition against pi-3 as seen in table 4 , except for (+)-isoboldine, (-)-stylopine, and (±)-corydalidzine, that were totally ineffective against both viruses. protopine, β-allocryptopine, chelidimerine, fumarophycine, α-hydrastine, (+)-bulbocapnine, (+)-isoboldine, (-)-sinactine, palmatine, dehydrocoryda-316 i. orhan et al. line, dehydrocavidine, (+)-cularicine, oxocularine, and oxosarcocapnine were completely inactive against hsv, whereas maximum cpe concentrations of the rest were the same as acyclovir (16 µg/ml). however, the alkaloids were revealed to be less cytotoxic than acyclovir on mdbk cells, (-)-canadine being the least cytotoxic alkaloid (128 µg/ml). the most active alkaloid with anti-pi-3 effect was shown to be protopine (1-32 µg/ml), followed by fumarophycine (2-32 µg/ml), chelidimerine, (+)-bulbocapnine, and (-)ophiocarpine (4-32 µg/ml), as well as β-allocriptopine and oxosarcocapnia number of antimicrobial, antiviral, antitumoral, antimalarial, and cytotoxicity studies have been so far reported on various derivatives of natural or synthetic isoquinoline alkaloids [51] [52] [53] [54] [55] [56] [57] . in one study, antimicrobial, cytotoxic, and anti-hiv activities of 26 simple isoquinolines and 21 benzylisoquinolines were investigated. the results showed that a quaternary nitrogen atom of isoquinoline or dyhydroisoquinoline type may enhance the potency of antimicrobial activity and cytotoxicity, whereas anti-hiv activity was higher with tetrahydroisoquinolines [58] . in the study of cui et al., simple isoquinolines, 15 of which were of 1-benzylisoquinoline-type and 19 of which were of protoberberine-derivatives, were screened against epstein-barr virus early antigen (ebv-ea) activation induced by 12-o-tetradecanoylphorbol-13-acetate (tpa), which is considered to be an indicator of antitumorpromoting activity. the study was carried out using raji cells and all 1benzylisoquinolines and 11 of the protoberberines exerted higher inhibitory activity than β-carotene [59] . regarding the structure-activity relationship, it was concluded that the inhibitory activity of 1-benzylisoquinolines increased as the number of hydroxyl groups on the aromatic ring increased and, additionally, the size of substituents at the c8 and c13, as well as type and position of the oxygenated substituents on a and d rings, influenced the virus inhibition. moreover, derivatives of the isoquinoline skeleton attached with carboxamide moiety were declared to be the potent and selective inhibitors of human cytolamegavirus [60] . in another study, the structure-activity relationships of berberine and its derivatives were examined for their antibacterial activity. among the 13-alkyl-substituted and the 13-unsubstituted protoberberinium salts, an increase in antibacterial activity was observed with the 13-ethyl-9-ethoxyl, the 13-ethyl, and the 13-methyl derivatives against s. aureus by eight-, four-, and twofold, respectively, over berberine, which suggested that steric effects played an noteworthy role in the antibacterial activity [61] . additionally, replacement of methoxyl groups at the c2 and the c3 of ring a by a methylenedioxy group caused a boost in activity. in this report, it was stated that the quaternary nitrogen atom (such as in protoberberinium salts) an alkylsubstituent at c13, and a methylenedioxy function at c2 and c3 are required for enhanced antibacterial activity. in a study by nakamoto et al., berberine was revealed to have a strong antifungal effect against c. albicans, c. tropicalis, and c. glabrata, respectively, which is in accordance with our data on berberine [62] . in a recent publication, a high occurrence of antibacterial activity in berberine was shown towards e. coli, k. pneumoniae, p. aeruginosa, p. fluorescens, s. aureus, salmonella typhi, enterococcus sp., and serratia marcescens, showing a better activity than streptomycin at 50 µg/ml by paper disc diffusion method. consequently, berberine was concluded to be responsible for the high antibacterial activity of coscinium fenestratum [63] . however, we found that berberine was only active against k. pneumoniae and a. baumannii by microdilution method, which might result from the application of two different methods. in another former study, berberine obtained from berberis heterophylla was tested against the atcc strains of s. aureus, enterecoccus faecalis, p. aeruginosa, e. coli, and c. albicans by agar diffusion method at 50, 100, and 200 µg/ml concentrations. the alkaloid was highly active against s. aureus at 100 and 200 µg/ml, whereas it did not possess any inhibitory effect against e. faecalis, p. aeruginosa, and e. coli [64] . this data was consistent with ours for berberine in the case of e. coli and p. aeruginosa. it was not active to s. aureus, which might again be the result of the use of two dissimilar methods. in the same work, antifungal activity screening was performed with berberine using the clinical strains of several candida sp. such as c. albicans, c. glabrata, c. haemulonii, c. lusitaniae, c. krusei, and c. parapsilosis. being the most active to c. krusei, followed by the rest in decreasing degrees of effectiveness, berberine was expressed as a novel antifungal agent. in one report, protopine and α-allocryptopine isolated from glaucium oxylobum were tested for their antifungal activity against microsporium canis, m. gypseum, tricophyton mentagrophytes, epidermophyton floccosum, c. albicans, aspergillus niger, and penicillium sp. [65] . among these fungi, protopine exerted low activity against m. canis and t. mentagrophytes, while α-allocryptopine had low activity towards m. gypseum and good inhibition of e. floccosum. in contrast, protopine was found to be inactive against c. albicans, whereas this alkaloid had a high inhibition against the same fungus in our study (4 µg/ml). α-allocryptopine was also inactive against c. albicans, whose β-counterpart exhibited a very good antifungal effect against c. albicans. this may reasonably be due to the αand β-conformation of the compound. in a previous study, the molluscicidal activity of argemone mexicana seeds were tested against the snail lymnaea acumi-nata, which led to isolation of protopine and sanguinarine as the active components [66] . from the structure-activity point of view, a few features about the isoquinoline alkaloids investigated herein can be pointed out. the quaternary nitrogen atom found on some of the isoquinolines such as dehydrocorydaline, dehydrocavidine, berberine, sanguinarine, and palmatine may have an effect on the decrease of antiviral activity. on the other hand, the synergistic interaction among the isoquinoline alkaloids isolated from f. vaillantii may be stated to contribute to the higher antiviral activity of this extract. protopinetype alkaloids seem to display a higher antiviral effect than the rest. from ancient to modern history, traditional plant-based medicines have played an important role in health care. many countries in africa, asia, and latin america still rely on traditionally used herbal medicines for primary health care needs. on the other hand, the complex nature of plant extracts and the high probability of competing or synergistic bioactivities within the same extract mean that these results represent the starting point for an activity-guided search for active plant metabolites. it is also evident that a structure-activity relationship exists between the various chemical structures and their antimicrobial activity in most cases. however, antiviral agents, unlike antibacterial drugs which may cover a wide range of pathogens, tend to be narrow in spectrum, and, unfortunately, have a limited efficacy. historically, the discovery of antiviral drugs has been largely fortuitous. spurred on by success with antibiotics, drug companies launched huge blind-screening programs with relatively little success. besides, lead compounds were modified by scientists in an attempt to improve bioactivity. however, there is still a great need to develop more effective antiviral and antimicrobial drug molecules. as a conclusion, these findings provide additional evidence for the supposition that the assays mentioned above play the part of useful primary screening in the survey of bioactive natural products. for the reasons outlined above, it is very important to focus on plants to discern novel antiviral/antimicrobial compounds. therefore, we truly hope that our studies, as well as similar reports by different researchers, may help find new antimicrobials/antivirals from herbal sources. the lancet (inf. diseases) key: cord-012623-bc9fj29h authors: pekmezaris, renee; kozikowski, andrzej; pascarelli, briana; handrakis, john p.; chory, ashley; griffin, doug; bloom, ona title: participant-reported priorities and preferences for developing a home-based physical activity telemonitoring program for persons with tetraplegia: a qualitative analysis date: 2019-05-16 journal: spinal cord ser cases doi: 10.1038/s41394-019-0188-6 sha: doc_id: 12623 cord_uid: bc9fj29h study design: focus group. objectives: the purpose of this qualitative study was to explore perceptions and priorities of persons with spinal cord injury (sci) for physical activity and to incorporate their feedback to inform future development of a physical activity program delivered via a telemonitoring platform. setting: new york. methods: qualitative data were collected from a purposive sample of adults with tetraplegia (n = 7). two investigators led an audio-recorded focus group using a moderator’s guide. data were analyzed using a six-phase thematic analysis approach. results: the discussion focused on two major areas, which resulted in multiple derived themes and subthemes. the first theme centered on the daily life of persons with tetraplegia, including changes after sci, gain of function prioritization, and identification of psychosocial support systems that facilitate community reintegration after injury. the second theme centered on participant perceptions and recommendations for a physical activity program delivered via a telemonitoring platform. desired design features included variations in schedule, diverse activities, or exercises included in each class, and optional two-way video to enable social interactions with classmates. conclusions: participants favorably viewed the concept of a physical activity program delivered via a telemonitoring platform and contributed program design ideas. although this was a small sample size, challenges to obtaining physical activity expressed by participants were consistent with those identified previously in larger studies of persons with tetraplegia. therefore, we expect these concepts and their recommendations to be relevant to the greater sci community. approximately 350,000 persons in the us are living with traumatic spinal cord injury (sci) [1, 2] . due to reduced mobility, persons with sci are at increased risk for developing obesity, muscle atrophy, osteoporosis, accelerated atherogenesis, type ii diabetes mellitus, and other medical consequences that increase the risk of stroke and coronary heart disease [1] [2] [3] . this reduced mobility often has deleterious psychosocial effects that impact quality of life, including increased social isolation, reduced social participation, reduced exercise self-efficacy, and depression [4, 5] . thus, there is a critical need for therapeutic strategies that reduce the risk of multiple medical and psychosocial consequences of sci. physical activity is a recommended therapeutic strategy to reduce risks of common medical consequences across diverse clinical populations [6, 7] . physical activity reduces risks of coronary heart disease and diabetes, increases immunity and blood circulation, and decreases inflammation, fat, anxiety, pain, and improves mood and sleep [8] [9] [10] [11] . the american college of sports medicine recommends that able-bodied adults perform 150 min of moderate-intensity aerobic exercise and participate in two or more days of muscle-strengthening exercise weekly [12] . the latest physical activity guidelines for adults with sci recommend, "at least 20 min of moderate to vigorousintensity aerobic exercise two times per week and three sets of strength exercise for each functioning muscle group, at moderate to vigorous intensity, two times per week" [13, 14] . for cardiometabolic health benefits, it is recommended that adults with sci engage in at least 30 min of moderate to vigorous-intensity aerobic exercise three times per week [13, 15] . persons with sci and other disabilities are less likely to engage in regular physical activity, due to many modifiable barriers. these include: lack of knowledge about existing programs/safe exercises, insufficient programming, lack of transportation, cost, and scheduling issues [16] . there are also other barriers, such as feeling too hot or cold outdoors or distance from an adaptive sports facility [17] . in the general population, telemonitoring approaches to delivering physical activity are part of a highly successful commercial fitness industry. consumers are offered the ability to choose a program to engage in at home, with recorded or live classes, that can be delivered to a tv, tablet, phone, or computer via a commercial internet provider. compared to a gym membership, telemonitoring is convenient, scalable, and relatively low cost. regardless of the modality, telemonitoring physical activity programs often require minimal exercise equipment and are delivered at home on a personalized schedule. in addition to the physical health benefits, such as increased muscle strength and improved cardiovascular fitness, many physical activity instructors also engage actively in motivational strategies, to promote adherence and increase exercise self-efficacy [18] . increasingly, telemonitoring enables a participant to experience self-efficacy in the following ways: (1) mastery of experiences, the strongest predictor of self-efficacy, relate to actual performance when successfully meeting a challenging task. participants performing daily health behaviors and seeing progress, experience mastery. (2) vicarious modeling (seeing others facing similar challenges and reaching their goals) will be achieved by viewing other participants of similar abilities attaining activity goals. (3) social persuasion (verbal encouragement) is provided by the instructor. (4) physiological factors, such as anxiety and distress, can be experienced by participants when they fail to meet activity goals; the instructor can interpret this as situational and not associated with overall success [19, 20] . home-based physical activity delivered via telemonitoring may be a particularly useful option for persons with sci as a way to modify common environmental barriers to achieve the benefits of regular physical activity [21] . to address these and other barriers, telehealth approaches are being increasingly studied in the context of sci [21] . sweet and colleagues are starting an rct of an 8-week tele-rehab program for persons with paraplegia to measure changes in psychosocial variables related to exercise participation and quality of life [22] . another study measured the effects of a home-based exercise program in persons with chronic sci, including outcome measures of metabolism, body composition, physical activity, energy intake, measures of health and wellbeing, resting metabolic rate, heart rate, and blood pressure, aerobic capacity, immune function, and adipose gene expression [23] . encouraging results using telemonitoring have been obtained across physical health measures (i.e., wound care), as well as psychological health [21] . there is a need to establish novel methods to facilitate regular physical activity for persons with sci [24] . here, we report the results of a qualitative study of priorities and preferences for developing a home-based physical activity telemonitoring program for persons with tetraplegia. we consider this to be a first step towards optimizing feasibility and acceptability in a physical activity program for persons with sci [13] . this is a qualitative study of adults with chronic (at least 1 year from injury) tetraplegia who were recruited from the ny metropolitan area. the rationale for including only persons with tetraplegia was because, in general, this group has fewer opportunities for achieving physical activity in their daily life, lower reference values of cardiovascular fitness (relative vo2 peak), higher risk factors for cardiovascular disease, and lower life expectancy than persons with paraplegia. a short demonstration video developed by the study team was presented to participants to show the concept of a telemonitoring physical activity program led by a physical therapist for persons with tetraplegia. moderators explained that they envisioned that participants would join the class via a tablet with a split screen that showed themselves, the instructor, and classmates conducting exercises. moderators described that an instructor would monitor vital sign data (heart rate and blood oxygenation) of participants in real time via a pulse oximeter. before engaging in exercise, participants would be trained on proper equipment use. for safety, participants would be asked by the instructor every 5 min during the intervention, to describe any symptoms of discomfort, including pain (musculoskeletal or other), fatigue, shortness of breath, or dizziness. frequency, duration, and type of proposed activities are based on the most recent guidelines on physical activity for persons with sci [24] . the intervention presented was proposed to be delivered three times/week for 45 min, with ≥30 min of activity. the circuit training program proposed was based on evidence of strength and cardiorespiratory benefit in persons with sci [25] . stretching, cardiovascular, and strengthening exercises would be tailored to participants' functional abilities. theraband, with open handgrips (loops), would be used to provide resistance for strength training [26] . moderators explained that the program would consist of three repetitions of: (a) warm-up: a series of active (nonresisted) movements: shoulder lateral raises, flies, shoulder rolls, wide biceps curls, shoulder shrugs, triceps extensions to rear; (b) circuit exercise program: resistance followed by aerobic (arm spinning) exercises with rest periods as needed (~15 s). resisted movements would include: set 1: seated rows, horizontal shoulder abduction, arm spinning/circles (aerobic exercise), set 2: shoulder internal rotation, shoulder external rotation, aerobic exercise, set 3: straight arm pulldowns, chest press, aerobic exercise [26] . a 3-h focus group was conducted in january 2018, led by two moderators previously unknown to participants. moderators used a moderator guide with open-ended questions and probes, related to a range of relevant topics including experiences with and priorities for benefits of physical activity before and after their injuries, technology use, and perceptions of important features that should be incorporated into a telemonitored physical activity program. the discussion was digitally recorded (using two recorders in case of technical failure), stored on an internal password protected server to ensure security, and transcribed professionally. transcripts were checked against the original recordings for accuracy. to optimize credibility, transferability, and dependability of results, we utilized analyst triangulation, peer debriefing, and conducted an audit trail of decisions made during the analysis and rationale. the transcript was analyzed by two researchers (a k and b p), to achieve triangulation to gain a more complex understanding of the data. a six-phase thematic analysis approach was utilized [27, 28] . in the first phase, transcripts were reviewed independently multiple times to become familiar with the data. researchers documented initial theoretical and reflective thoughts, and potential codes and themes. in the second phase, researchers focused on data patterns and generated a comprehensive set of codes through inductive and deductive coding. two researchers documented their reasoning for coding blocks of text from the transcript of the focus group to explain how the data were perceived and examined. the third phase consisted of searching for themes after coding and codes were collated. in the fourth phase, themes were reviewed and refined. criteria for retaining themes were that they needed to be specific enough to be concrete, while broad enough to capture ideas. themes with sparse data were eliminated and those with large amounts of data were further divided into separate themes. in the fifth phase, team members met and discussed the finalization of theme names. in the sixth phase, the report was generated. participants were persons with tetraplegia (n = 7: 5 males and 2 females) who were wheelchair users for community mobility. the discussion explored challenges of living with tetraplegia, gain of function prioritization, social networks, and design recommendations for a telemonitored physical activity program. participants were asked to rank their gain of function prioritization on a seven-point scale, with one being most, and seven being least important, in the following areas: arm/ hand function, upper body/trunk strength and balance, bladder/bowel function, lived experiences of sexual function, elimination of chronic pain, sensation and mobility ("mobility could be anything that gets your body around in space") [29] . most participants ranked either arm/hand function, sensation, or improvement of mobility as the most important. the next gain of function priorities ranked was upper body/trunk strength and balance, elimination of chronic pain, and sexual function. two major discussion themes emerged from a six-phase thematic analysis approach to the transcript. theme one: daily challenges pain several participants described challenges of performing activities of daily living (adls) while experiencing constant pain. the locations of pain symptoms varied by individual, including the back, neck, shoulders, and feet. multiple participants reported that pain symptoms were worse in the morning and did not resolve completely throughout the day. "right now, i feel like someone's kicking me in the back but that's normal for me, so it's just one of those things you kind of deal with…" "…i have chronic back pain that just will not go away. it's probablyif i say on a scale of one to ten, it's probably around a good eight most of the day…" "i'm in pain every day when i get home. i'm in bed by 7:00 because i can't even function." participants also discussed how pain impacted feelings of fatigue and strategies to cope with interruptions in sleep. "and you talk about getting exhausted during the day. i want to sleep every day by 12:00. but i'm at work, so i can go in my office for a little while just to try to rest for a minute." multiple participants reported being athletic and active prior to their sci, had careers in physically enduring professions or participation in active sports including swimming, motocross, running, cycling, and skiing. "yeah, motor cross. yeah. so i used to ride-a lot of cycling, a lot of swimming. i had a home gym that i worked out in all the time. running-i was a terrible runner because my knees weren't that great. so i would run a little bit, but not that much. mostly cycling. i loved cycling. anything with wheels, i was there." "i was very free spirited, i'd say. we'll put it that way. but yeah, i sort of was very spontaneous and enjoyed flying by the seat of my pants and all that. it's like losing a little piece of you." as expected, the intensity and type of physical activity changed for most participants after injury. most focus group participants were not engaged in regular physical activity, outside of the exercises prescribed during physical therapy. for participants who were active, post-injury activities include using a stationary bike, thera-bands for resistance training, and free weights. "when i first came home, i was doing them every day. and then little by little, you're slacking off. but like i said, every day, once i get into bed, that's when i do the most thera-bands or weights or anything because i'll put a wrist [adaptor] on my arm. i'll go on my side and i'll do the left arm. then i get turned the other way, i'll do the right arm." "...everything from thera-bands like you were talking, to cuff weights. i use cuff weights as well that-most people use them on their ankles when they're running or exercising, but they also work great for quads around your arm. it's like a velcro weight. the rickshaw [wheelchair rehab exercise machine]…. it's a great machine for people in wheelchairs. and they have another machine there which is called the upper tone…it's kind of a home gym-type looking machine that's specifically designed for people in wheelchairs and people with limited hand function." variation in physical therapy and interactions with physical therapy personnel were discussed and perceived as impacting the post-injury rehabilitation process. "i've been to great therapists and i've been to not-sogreat therapists, and what they did clinically was not that different from each other. the difference was the therapists' behavior, the interaction." "i mean, it felt like it's a total package there. you get a lot of focused attention. youand theystart on the dime and they give you every second of that hour." participants discussed the critical role of social networks (family and friends) in community reintegration after injury. in addition, participants were motivated and inspired by interacting with peers with sci who demonstrated resilience. "…when you see people getting better, it helps. it makes you believe you can do the same thing too." the importance of self-efficacy to obtaining functional gains was also discussed, including the importance of maintaining both physical and emotional health. the feelings of well-being obtained from exercise were reported to reinforce the desire to continue exercising. "when i'm in a good mood, i feel i can conquer the world. but when i'm in a lousy moodlike today is not a great mood for meis i don't feel good about anything, and i don't want to do anything because i'm miserable. but then tomorrow i'll feel great and say i can pretty much take on the world and do anything i want and just let me do what i got to do." "yeah, inspirational. yeah, it would raise inspiration, want me to build more muscle on my end to want to feel better and know that i'm healthier and to keep going for whatever reason, whether it's for walking or not." "and i can tell you personally that i should probably be further along physically than i am… i think i plateaued and then went the other direction because of my own inability to push those things out of my mind…like if your head's not there, like in anything in life, but especially with sci rehab, it's hard enough knowing that this happened." participants discussed the importance of recognizing that goals and priorities may vary by individual and that each person will begin the program looking for a different outcome. for some, success may be defined as an improvement in mobility, whereas for others success may be defined as increased social interactions, motivation, or health maintenance. "i think everybody's priorities and everybody's goals are different." "so i mean, so i don't want to lose those [functions] any worse than they've been getting over the years because it's like almost limited to what i can kind of do…" "well, i guess it depends on somebody's lifestyle and age has a lot to do with it. so i would say some people are just looking to maintain themselves and stay healthy to be able to continue to do the activities that they currently do." "yeah, and just feeling better as well in daily activities." interaction with other classmates a strong recommendation was made to foster potential interactions among classmates in order to motivate and inspire one another. an additional recommendation was to include a feature to extend class times to allow for social interactions among classmates before or after exercise. some participants suggested that two-way viewing among classmates should be optional, so as to not discourage those who might feel uncomfortable. "i think it would be key to interact with not only the therapist, but with other patients. so i see jack on the one screen and he's struggling, i'm like come on. do one more. do one more. and we're all telling himme, alex-jack, come on. do one more. and he pulls through, so it gives that mental back." "so you said a 45-minute designed program, but maybe its 60 min and we all log on 15 min before. we could all-oh, maria*, how's that going, or chris*, how's that? so the social muscle to it instead of just working on arms and then logging off, like good old talk." "but to have the ability to [see others] should certainly be an option… but they should at least have the option to turn it off if they want i think, right?" participants suggested that it would be helpful for someone to orient a class member, assist with equipment needs and demonstrate specific exercises included in the program prior to session initiation. participants suggested that including a variety of exercises within each class would be desirable, in order to meet personal preferences and to address varying physical abilities. multiple participants suggested that three times per week would be the preferred frequency of classes, held at a variety of days and times to accommodate different schedules (e.g., weekday, weekend, and evening sessions). the goal of this focus group was to discuss experiences with physical activity and gather input from persons with tetraplegia to inform future design of a physical activity program delivered via telemonitoring that would be feasible, acceptable, and consistent with exercise guidelines for those with sci. a minor aspect of the discussion revealed that, in general, priorities for improvement included: arm/hand function, sensation, and improvement of mobility as being most important. in addition, upper body/trunk strength and balance, elimination of chronic pain, and improving lived experiences of sexual function were also ranked as important. data demonstrate that different modalities of exercise and physical activity have indeed been shown to improve aspects of physical capacity, health, and abilities to perform activities of daily living (e.g., functional wheelchair maneuvers and transfers) in persons with sci [30] . participants perceived a home-based physical activity program as needed and important. intensity and type of physical activity performed before and after injury were discussed. participants identified family support, psychological state, and having a peer network (e.g., others with sci) as important factors for their overall recovery. participants regarded their input and feedback as critical for ensuring usability and feasibility, including the ability to make choices regarding whether a participant can be seen by classmates, types of exercises in a class, and timing of class delivery to suit multiple schedules. participants generally expressed enthusiasm for interacting with classmates, a desire for help from a caregiver or professional in initial set up, and comfort with a frequency of three times/week for classes and a duration of 45-60 min per class. participants perceived multiple potential benefits of a physical activity for persons with sci delivered via telemonitoring. participants had several practical suggestions to optimize design and delivery of such a program. clearly, a pilot study in this population testing this kind of intervention is needed. it is important that future studies incorporate feedback from participants on the design and implementation of a physical activity program. data generated during the focus group are not publicly available in order to protect privacy of participants. deidentified data can be made available upon request to the corresponding author. publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. annual statistical report for the spinal cord injury model systems public version traumatic spinal injury: global epidemiology and worldwide volume facts and figures at a glance facilitators and barriers to social and community participation following spinal cord injury social and community participation following spinal cord injury: a critical review position statement. part two: maintaining immune health position statement. part one: immune function and exercise reduction in trunk fat predicts cardiovascular exercise training-related reductions in c-reactive protein exercise and respiratory tract viral infections cardiovascular exercise training extends influenza vaccine seroprotection in sedentary older adults: the immune function intervention trial exercise, inflammation, and innate immunity american college of sports medicine position stand. quantity and quality of exercise for developing and maintaining cardiorespiratory, musculoskeletal, and neuromotor fitness in apparently healthy adults: guidance for prescribing exercise evidence-based scientific exercise guidelines for adults with spinal cord injury: an update and a new guideline development of scientific exercise guidelines for adults with spinal cord injury the development of evidence-informed physical activity guidelines for adults with spinal cord injury more than just a game: the public health impact of sport and physical activity for people with disabilities (the 2017 delisa lecture) functional and environmental factors are associated with sustained participation in adaptive sports self-efficacy: the exercise of control self-efficacy: the exercise of control self-efficacy mechanism in human agency correlates and determinants of physical activity in persons with spinal cord injury: a review using the international classification of functioning, disability and health as reference framework participation in physical activity in persons with spinal cord injury: a comprehensive perspective and insights into gender differences participation in sport in persons with spinal cord injury in switzerland telehealth for people with spinal cord injury: a narrative review circuit training provides cardiorespiratory and strength benefits in persons with paraplegia a comparison of 2 circuit exercise training techniques for eliciting matched metabolic responses in persons with paraplegia using thematic analysis in psychology causes of death during the first 12 years after spinal cord injury targeting recovery: priorities of the spinal cordinjured population exercise and health-related risks of physical deconditioning after spinal cord injury acknowledgements the authors appreciate the time, effort, and opinions of the focus group participants.funding a grant from the new york state spinal cord injury research board (to ob) and institutional funds supported this work. these funds were used to support sci-related research at our institution and did not influence the specific study in any way.authors' contributions rp, ak, jh, dg, and ob designed the study. rp and ak led development of the moderator guide, to which all authors made contributions. jh and dg developed the proposed physical activity program. rp, ak, jh, dg, and ob created the demo video of the proposed physical activity program. all the authors (rp, ak, jh, dg, ac, bp, and ob) were present for the focus group. rp and ak moderated the focus group. ak and bp analyzed the transcript and wrote the report. all the authors (rp, ak, jh, dg, ac, bp, and ob) contributed to interpreting the data and writing the manuscript. conflict of interest the authors declare that they have no conflict of interest.ethics study activities were deemed not human subject research by the local institutional irb research and therefore did not require irb review. key: cord-272943-q09i8fqu authors: dalhoff, a. title: antiviral, antifungal, and antiparasitic activities of fluoroquinolones optimized for treatment of bacterial infections: a puzzling paradox or a logical consequence of their mode of action? date: 2014-12-17 journal: eur j clin microbiol infect dis doi: 10.1007/s10096-014-2296-3 sha: doc_id: 272943 cord_uid: q09i8fqu this review summarizes evidence that commercially available fluoroquinolones used for the treatment of bacterial infections are active against other non-bacterial infectious agents as well. any of these fluoroquinolones exerts, in parallel to its antibacterial action, antiviral, antifungal, and antiparasitic actions at clinically achievable concentrations. this broad range of anti-infective activities is due to one common mode of action, i.e., the inhibition of type ii topoisomerases or inhibition of viral helicases, thus maintaining the selective toxicity of fluoroquinolones inhibiting microbial topoisomerases at low concentrations but mammalian topoisomerases at much higher concentrations. evidence suggests that standard doses of the fluoroquinolones studied are clinically effective against viral and parasitic infections, whereas higher doses administered topically were active against candida spp. causing ophthalmological infections. well-designed clinical studies should be performed to substantiate these findings. the history of quinolones began in 1962 with the isolation of a byproduct of chloroquine synthesis by george yohe lesher and colleagues [1] at the sterling-winthrop research institute in rensselaer, new york; this compound was found to be antibacterially active and was subsequently modified to yield nalidixic acid. nalidixic acid and chloroquine share structural features being essential for their antibacterial and antiparasitic activity, respectively. apart from its well-known antimalarial effects [2] [3] [4] , chloroquine exerts direct antiviral [5] [6] [7] [8] [9] [10] [11] [12] [13] , antifungal [13] [14] [15] [16] , and antibacterial effects [13, [17] [18] [19] [20] . furthermore, chloroquine exhibits immunomodulatory activity [21] [22] [23] [24] [25] and was found to reverse p-glycoprotein (p-gp)-mediated multidrug resistance, thereby increasing the cytotoxicity of some antineoplastic agents [26] [27] [28] [29] [30] . the antimalarial effects of chloroquine are due to its accumulation in acidic food vacuoles of intraerythrocytic trophozoites, thereby preventing hemoglobin degradation and inhibition of a haem polymerase enzyme [3, 4] . the antiviral, antifungal, and antibacterial activities of chloroquine are ph-dependent [10, 14, 16, 18 ]. this phenomenon is due to the fact that chloroquine is a weak base and, therefore, does not enter the cell if the extracellular fluid or the incubation medium is acidic. once chloroquine has entered cells, it intercalates into dna and prevents the introduction of topoisomerase ii-mediated dna breaks. the intercalation of chloroquine into dna protects cells against epipodophyllotoxins such as etoposide, acting as topoisomerase ii poison by hindering the dna cleavage reaction of this target enzyme [31, 32] . the use of chloroquine in the treatment of some autoimmune diseases and its antiinflammatory properties may be due to the inhibition of mhc class ii antigen presentation; the inhibition of t-cell response may be due to a direct interaction of chloroquine with the cell membrane [22] . furthermore, chloroquine was found to destabilize indirectly lysosomal and plasma membranes as a result of accumulation within the lysosome, followed by an increase in lysosomal volume; it also sequesters important cell membrane constituents in lysosomes [29] . chloroquine was found to adsorb to the plasma membrane of yeasts, inhibit competitively the binding of immunoglobulin g to the cell surface, altered phospholipid turnover, and influenced directly but non-specifically the membrane integrity and permeability of renal brush border vesicles, mast cell membranes, and fibroblasts [16, [33] [34] [35] . furthermore, chloroquine blocks the inward rectifier potassium channel kir2.1; it is bound at the center of the cytoplasmic domain of the channel [36, 37] . these data demonstrate that the congener of fluoroquinolones, i.e., chloroquine, exhibits, apart from its antimalarial activity, pleiotropic actions and interacts with multiple targets. as chloroquine and nalidixic acid share structural features being essential for their activity, it was not surprising that it has been recognized in the late 1980s that nalidixic acid and oxolinic acid derivatives exert trypanocidal and antitumor activities [38] ; in the early 1990s, it was described that fluoroquinolones used for the treatment of bacterial infections exert not only an antibacterial but also an antiprotozoal activity [39] and may find applications as antiparasitic, antifungal, or antiviral agents [40] . furthermore, and in analogy to chloroquine, the activity of antibacterially active fluoroquinolones is ph-dependent [41] , and they bind directly to bacterial dna, i.e., two molecules intercalate at the highly bent dna gate in the dna cleavage domain [42] [43] [44] [45] [46] . despite these phenotypic and molecular homologies between chloroquine and fluoroquinolones, the pharmaceuticals industry invested financial and human resources into focused research programs on the application of developmental fluoroquinolones as antibacterials only and into pre-and postmarketing studies supporting the use of fluoroquinolones in the oncegranted indications. studies on the function of an antibacterial agent exerting pleiotropic anti-infective actions have never been performed systematically. surprisingly, the use of fluoroquinolones in indications other than bacterial infections has never been exploited, although not only nalidixic acid and its congener chloroquine exerts pleiotropic actions but, e.g., β-lactams and aminoglycosides are characterized by a broad range of biological activities too [47, 48] , so that a multitude of antimicrobial effects would not have been unusual. this review summarizes the pleiotropic phenotypes of nonantibacterial actions of fluoroquinolones and addresses the question if the diversity of effects are due to one common mode of action of antibacterially active fluoroquinolones, i.e., inhibition of essential bacterial type ii topoisomerases, or if other mechanisms may mediate non-antibacterial activities. although the complexity and diversity of prokaryotic and eukaryotic topoisomerases is remarkable and little or no sequence homology of amino acids exists, type i and type ii topoisomerases share certain structural elements mediating identical functions like dna relaxation or dna transport in bacteria, dna viruses, yeasts, and parasites; the dna helicase coordinates the directionality of topoisomerase activity; rna helicases as present, e.g., in hepatitis c virus (hcv) directly interact with double-stranded dna or rna and assembles complexes with type ii topoisomerases [49] [50] [51] [52] [53] . as dna topoisomerases are ubiquitous enzymes controlling dna topology, it is conceivable that antibacterially active quinolones may not only inhibit the growth of bacteria at clinically relevant concentrations, but that of other prokaryotic and even eukaryotic organisms as well. ciprofloxacin, ofloxacin, levofloxacin, and gatifloxacin were found to be clinically effective in the treatment of the singlestranded rna hcv and the non-enveloped, encapsulated dna polyomavirus bk [54] [55] [56] [57] [58] [59] [60] . five and four patients with hcv-induced chronic hepatitis and compensated liver cirrhosis, respectively, were treated with 100 to 900 mg ofloxacin per day for one to eight weeks. in three patients with chronic hepatitis and one patient with compensated liver cirrhosis, hcv rna decreased at least by 1 log titer [54] . in another study, five patients with chronic hcv were treated with 500 mg ciprofloxacin twice daily (b.i.d.) for 30 days. serum hcv rna levels remained largely unchanged in these patients [55] . the latter study indicates that the anti-hcvefficacy of quinolones may be limited in patients with advanced liver cirrhosis. ciprofloxacin decreased bk peak viral load after hematopoietic stem cell transplantation [56] . a reduction of viremia was demonstrated two months after a 10-day course of gatifloxacin at 400 mg/d in 7 of 10 transplant recipients with active bk virus replication [57] . a retrospective analysis revealed that the use of either ciprofloxacin 250 mg b.i.d. or levofloxacin 250 mg once daily (q.d.) within the first month post transplantation and up to 3 months after transplantation was associated with significantly lower one-year rates of bk viremia [58] . a recent study in nine kidney transplant recipients with persistent bk infection revealed that, three months post ciprofloxacin treatment with 250 mg b.i.d for 30 days, the virus load was cleared completely in three patients and decreased by >50 % in another three patients [59] ; patients were not treated with anti-infectives other than fluoroquinolones. fluoroquinolones inhibit bk viral replication in vitro. ofloxacin and levofloxacin inhibited polyomavirus bk replication in primary human kidney cells in a dose-dependent manner, yielding a~90 % inhibition at 150 μg/ml. bk virus genome replication was reduced by 77 % at 48 h post infection of the kidney cells. at 72 h after inoculation of the kidney cells, the reduction in genome replication and protein expression was less pronounced. a dose-dependent cytostatic effect was noted. in infected cells, 150 mg/l ofloxacin led to a 26 % and 6 % inhibition of cellular dna replication and total metabolic activity, respectively, while 150 mg/l levofloxacin exhibited a slightly more marked cytostatic effect, particularly in uninfected cells [60] . ciprofloxacin, moxifloxacin, levofloxacin, ofloxacin, gatifloxacin, and norfloxacin inhibited bk virus replication to 50 % at concentrations ranging from 66.7 to 266.6 mg/l [61] . ciprofloxacin, ofloxacin, levofloxacin, gatifloxacin, and trovafloxacin inhibited viral replication of simian virus 40 (sv40), another member of the polyomaviridae, in permissive monkey cells, as well as plaque formation, dna replication, and helicase activity. ciprofloxacin, levofloxacin, and ofloxacin inhibited "significantly" helicase activity at 0.5, 1.0, and 2.0 mm, whereas trovafloxacin inhibited helicase activity at 50 μm [62, 63] . recently, it was demonstrated that norfloxacin, ofloxacin, flumequine, enrofloxacin, cinoxacin, enoxacin, fleroxacin, lomefloxacin, balofloxacin, and difloxacin inhibited hcv replication, in particular, hepatoma huh-7 and huh-8 cell lines, and hcv ns3 helicase activity. the concentrations inhibiting hcv rna replication to 50 % ranged from 3.3 to 8.2 μm and those inhibiting helicase activity ranged from 4.1 to 9.9 μm [64] . the clinical studies reviewed above and one recent report of a successful treatment of a kidney retransplant patient with ciprofloxacin (250 mg b.i.d. for 10 days) who needed an overall increase of immunosuppression due to acute rejection [65] suggest that fluoroquinolone treatment of polyomavirus bk infections in transplant patients may be beneficial. therefore, a study protocol for a randomized controlled clinical trial evaluating the prophylactic efficacy of fluoroquinolones has been designed and is registered at clinicaltrials.gov under nct01353339; levofloxacin at a dose of 500 mg q.d. will be administered for 3 months and will be compared to placebo [66] . another clinical study on the use of ciprofloxacin (250 mg q.d. for 3 months as compared to placebo) for the prevention of bk infections is registered under nct01789203 [67] . furthermore, it was demonstrated that ofloxacin [68] and levofloxacin [69] inhibited viral topoisomerase activity of vaccinia virus but not of herpes simplex virus and influenza virus [68] . in agreement with this finding, it was reported that 200 mg/l each of ciprofloxacin, lomefloxacin, ofloxacin, pefloxacin, and rufloxacin inhibited to 50 % the cytopathic effect of herpes simplex virus type 2 at concentrations being equivalent to the cytotoxic effect of the quinolones on the vero cells [70] . fluoroquinolones inhibit not only enzymic activity of viral topoisomerases/helicases, but inhibit in vitro human immunodeficiency virus (hiv) reverse transcriptase as well; complete inhibition was observed at concentrations of ciprofloxacin and ofloxacin of 3 μm and norfloxacin of 1 μm, respectively [71] [72] [73] . inhibition of rhinovirus (rv) infection by quinolones is due to the inhibition of cell functions required for viral replication. levofloxacin pretreatment of not yet infected human tracheal epithelial cells reduced the mrna level of intercellular adhesion molecule 1 (icam-1), a receptor for rv, in the cells and the concentration of the soluble form of icam-1 in the supernatant, so that rv infection of the tracheal epithelial cells was significantly reduced. levofloxacin pretreatment also decreased the number of the acidic endosomes from which rv rna enters the cytoplasm. furthermore, levofloxacin pretreatment inhibited the activation of nuclear factor κb proteins. these data suggest that levofloxacin inhibits rv infections first by reducing icam-1 expression levels and the number of acidic endosomes, and second by modulating airway inflammation [74] . fluoroquinolones other than levofloxacin have not been studied in this context. moxifloxacin and gatifloxacin inhibited, at a concentration of 0.5 % used for topical application in ophthalmology, candida spp. to >95 % [75] . gatifloxacin and sparfloxacin showed activity in a qualitative paper disk diffusion test against trichophyton rubrum, fusarium solani, and candida albicans, but not against saccharomyces cerevisiae [76] . ciprofloxacin, moxifloxacin, levofloxacin, trovafloxacin, and sitafloxacin enhanced the activities of antifungal agents against candida albicans and aspergillus fumigatus [77] [78] [79] [80] [81] [82] [83] [84] . furthermore, ciprofloxacin showed synergism with azoles against histoplasma capsulatum and coccidioides posadasii [85] , as well as in combination with amphotericin b against exophiala spinifera [86] . several but still rare reports of clinical and microbiological cure of fungal keratitis by quinolones have been published; recently, five additional cases of fungal keratitis treated successfully with topical moxifloxacin monotherapy were published [79] . the causative organisms curvularia spp., candida parapsilosis, paecilomyces lilacinum, and aspergillus fumigatus were treated with moxifloxacin 0.5 %, one drop every half-hour to every hour. all these cases of fungal keratitis were cured with topical moxifloxacin and the pathogens were eliminated [87] . these data demonstrate that topical administration of quinolones, thus generating high target site concentrations, are clinically effective in the treatment of fungal ophthalmological infections. topoisomerase ii has been identified as the primary target for quinolones in yeast [88, 89] , so that the antifungal activities of the fluoroquinolones tested are likely to be mediated by this enzyme. the dna topoisomerase ii isolated from candida albicans was more susceptible to quinolones than the calf thymus dna topoisomerase ii, despite the fact that both enzymes are of eukaryotic origin [80] . yeast dna topoisomerase ii selected for resistance to quinolones are characterized by amino acid mutations which are homologous to mutations in gyra of escherichia coli [90] [91] [92] . these differences between yeast and mammalian type ii topoisomerases may explain why fluoroquinolones exhibit an antifungal activity by maintaining in parallel a selective toxicity against prokaryotic topoisomerases. although antibacterially active fluoroquinolones were derived from the antimalaria agent chloroquine, the clinical efficacy of norfloxacin against plasmodium falciparum was discovered by chance when the agent was used for the treatment of typhoid fever in indian patients. norfloxacin was administered to nine hospitalized malaria patients orally with 400 mg norfloxacin b.i.d. for three days; treatment led to disappearance of splenomegaly [93] . later, another 15 patients with uncomplicated malaria were treated with norfloxacin (ten with 400 mg b.i.d. and five with 800 mg b.i.d.) for three days [94] . this study confirmed that norfloxacin is clinically effective in the treatment of falciparum malaria, but the efficacy of the lower dose was suboptimal. later, it was demonstrated that norfloxacin is inferior to chloroquine for falciparum malaria. a prospective, randomized trial revealed that the mean parasite clearance time as well as the mean defervescence time were shorter in the chloroquine group [95] . fluoroquinolones like ciprofloxacin, amifloxacin, enoxacin, norfloxacin, ofloxacin, pefloxacin, grepafloxacin, trovafloxacin, and 16 additional commercially available quinolones exhibit marked in vitro activity and in vivo efficacy against plasmodium spp. [96] [97] [98] [99] [100] [101] [102] [103] [104] [105] . nalidixic acid and several fluoroquinolones like ciprofloxacin, norfloxacin, enoxacin, ofloxacin, fleroxacin, clinafloxacin, pefloxacin, and sparfloxacin exerted an antitrypanosomal in vitro and in vivo effect at micromolar concentrations [38, [106] [107] [108] [109] [110] [111] [112] [113] [114] [115] [116] . in addition, nalidixic acid, norfloxacin, ofloxacin, moxifloxacin, gatifloxacin, lomefloxacin, and some more fluoroquinolones inhibited growth of the microsporidia encephalitozoon intestinalis and vittaforma corneae to 50 % at concentrations ranging from 0.9 to 98.4 μm [112] . furthermore, ciprofloxacin caused a 50 % growth inhibition of babesia microti, b. bigemina, b. caballi, b. equi, and b. bovis at concentrations of 2.5 to 15.8 μm [113] . fluoroquinolones exerted antitoxoplasma activities as well. moxifloxacin, gatifloxacin, trovafloxacin, and grepafloxacin were the most active agents, inhibiting growth of t. gondii to 50 % at concentrations ranging from 0.4 to 5.1 mg/l, while ciprofloxacin was poorly active, with a 50 % inhibitory concentration value of 79.4 mg/l [116] . the parasites of the phylum apicomplexa, i.e., plasmodium spp., toxoplasma spp., babesia spp., and leishmania spp. are characterized by the absence of organelles like mitochondria, but they have acquired a plastid by endosymbiosis of a green alga. the apicoplast is a nonphotosynthetic plastid in which several essential biosynthetic pathways are sequestered, so that interactions with these biosynthetic functions cause deleterious effects. elimination of the plastid or total inhibition of its function results in a "delayed death", i.e., the parasites grow and evade normally within and from the first host cell, but their replication is halted immediately after the invasion of a new host cell. the apicoplast harbors a circular dna and bacterial type dna gyrase. ciprofloxacin induced cleavage of apicoplast dna in p. falciparum, without targeting nuclear dna [117] [118] [119] . exposure of toxoplasma gondii to ciprofloxacin resulted in a decrease of the apicoplast genome copy number during replication [120] . although it was discussed that differences in the role of apicoplasts in toxoplasma and plasmodium may exist [121] , the apicoplast dna gyrases isolated from both species were inhibited by almost identical concentrations; the apicoplast dna gyrase isolated from plasmodium falciparum is inhibited by ciprofloxacin concentrations ranging from 7 to 38 μm and trovafloxacin inhibits apicoplast dna gyrase activity isolated from toxoplasma gondii and plasmodium falciparum, respectively, at 30 μm [102, [117] [118] [119] [120] [121] . consequently, prokaryotic type ii dna topoisomerase of apicomplexan protozoa are effectively targeted by fluoroquinolones. it has been summarized previously that fluoroquinolones are active in preclinical infection models against quinoloneresistant bacteria as well as candida albicans infections [122, 123] . furthermore, levofloxacin was active against rv infections [74] . these phenomena were found to be directly correlated to the immunomodulatory activities of fluoroquinolones [122, 123] . mechanisms underlying the various immunomodulatory effects of fluoroquinolones include an effect on intracellular cyclic adenosine-3,5-monophosphate and phosphodiesterases, as well as an effect on transcription factors and also a triggering effect on the eukaryotic equivalent of bacterial sos response with its ensuing intracellular events [124] . fluoroquinolones are routinely prescribed for the treatment of coronavirus-associated severe acute respiratory syndrome (sars) or opportunistic bacterial infections in hiv-positive patients. upon elimination of the bacterial pathogen or exclusion of bacterial pathogens, antibiotic therapy can be withdrawn. however, patients may benefit from the immunomodulatory activities of fluoroquinolones, but their effect on the course of sars or acquired immune deficiency syndrome (aids) is undetermined. although it is well documented that nalidixic acid and fluoroquinolones modulate immune responses by the modulation of intracellular signaling cascades, it is unknown which mechanism(s) may trigger signal transduction. it has been d e m o n s t r a t e d t h at , i n a n a l og y t o c hl o r o q ui n e , fluoroquinolones bind to and insert into pro-and eukaryotic membranes, respectively, thereby altering their fluidity [116] . changes in membrane fluidity may be sensed by the immunocompetent cells, so that gene expression may be controlled according to the signals triggered. furthermore, it can be hypothesized that fluoroquinolones exert direct antiinfective activities due to their physicochemical interactions with membranes, thus making the organisms leaky, followed by cell death. this latter aspect has never been addressed systematically. any fluoroquinolone used for the treatment of bacterial infections exerts, in parallel to its antibacterial action, antiviral, antifungal, and antiparasitic actions at clinically achievable concentrations. this broad range of anti-infective activities is due to one common mode of action, i.e., the inhibition of type ii topoisomerases, thus maintaining the selective toxicity of fluoroquinolones inhibiting microbial topoisomerases and eukaryotic topoisomerases of prokaryotic origin at low concentrations but mammalian topoisomerases at much higher concentrations. there is strong evidence that the broad range of anti-infective activities translates into the clinical arena. however, anti-infective activities other than antibacterial activities have never been evaluated systematically. this may be due to the strategy of both the pharmaceutical industry and regulatory authorities to develop an agent on the basis of its application, i.e., its use as an antibacterial agent. therefore, the antiviral or antifungal activities of fluoroquinolones have, so far, not been exploited systematically; two controlled studies evaluating the antiviral effects of fluoroquinolones have been initiated recently. the clinical evaluation of their antifungal and antiparasitic effects is justifiable and would be opportune. traditionally, clinical studies are designed on the basis of a monocausal microbe-outcome association, i.e., the presence of one bacterial species at the site of infection indicates pathogenicity. consequently, an anti-infective agent is considered to be effective if this single species is eradicated from the focus of infection. however, infections may be polymicrobial or chronically ill patients may suffer from opportunistic infections; hivpositive patients represent an extreme example for the acquisition of opportunistic infections caused in parallel by viruses, bacteria, and/or parasites. such patients could, in theory, benefit from treatment with agents which exert a broad range of anti-infective activities. a multifactorial analysis of the outcome of infectious diseases would be necessary. the corresponding outcome measures are quantifiable and can be linked to pharmacokinetics and overall clinical efficacy. in summary, based on one common mode of action, fluoroquinolones being commercially available as antibacterial agents are active against viruses, fungi, and parasites too, so this class of agents is probably representative of broad-spectrum anti-infectives in its true sense. brundage rp (1962) 1,8-naphthyridine derivatives. a new class of chemotherapeutic agents inhibition by chloroquine of a novel haem polymerase enzyme activity in malaria trophozoites chloroquine: mechanism of drug action and resistance in plasmodium falciparum inhibition of glutathione-dependent degradation of heme by 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effects of quinolones antibiotics and chemotherapy. basic research and clinical aspects of pseudomonas aeruginosa the author declares that he has no conflict of interest. key: cord-281137-j81fgnld authors: rahman, lubna; shinwari, zabta k.; iqrar, irum; rahman, lutfur; tanveer, faouzia title: an assessment on the role of endophytic microbes in the therapeutic potential of fagonia indica date: 2017-08-01 journal: ann clin microbiol antimicrob doi: 10.1186/s12941-017-0228-7 sha: doc_id: 281137 cord_uid: j81fgnld background: natural products of animals, plants and microbes are potential source of important chemical compounds, with diverse applications including therapeutics. endophytic bacteria that are especially associated with medicinal plants presents a reservoir of therapeutic compounds. fagonia indica has been recently investigated by numerous researchers because of its striking therapeutic potential especially in cancer. it is also reported that endophytes play a vital role in the biosynthesis of various metabolites; therefore we believe that endophytes associated with f. indica are of crucial importance in this regard. the present study aims successful isolation, molecular identification of endophytic bacteria and their screening for bioactive metabolites quantification and in vitro pharmacological activities. methods: 16s rrna gene sequencing was used for the identification of isolated endophytic bacteria. methanolic extracts were evaluated for total phenolic contents (tpc), total flavonoids contents (tfc), dpph free radical scavenging activity, reducing power and total anti-oxidant assays were performed. and also screened for antibacterial and antifungal activities by disc diffusion method and their mic were calculated by broth dilution method using microplate reader. further, standard protocols were followed for antileishmanial activity and protein kinase inhibition. analysis and statistics were performed using spss, table curve and origin 8.5 for graphs. results: bacterial strains belonging to various genera (bacillus, enterobacter, pantoea, erwinia and stenotrophomonas) were isolated and identified. total phenolic contents and total flavonoids contents varies among all the bacterial extracts respectively in which bacillus subtilis showed high phenolic contents 243 µg/mg of gallic acid equivalents (gae) and stenotrophomonas maltophilia showed high flavonoids contents 15.9 µg/mg quercitin equivalents (qa), total antioxidant capacity (tac) 37.6 µg/mg of extract, reducing power (rp) 206 µg/mg of extract and 2, 2-diphenyl-1-picrylhydrazyl (dpph) free radical scavenging activity with 98.7 μg/ml ic(50) value. although all the extracts tested were active to inhibit growth of selected pathogenic microbes (bacteria and fungi), but significant antibacterial activity was observed against klebsiella pneumonia and b. subtilis. an enterobacter cloaca was active against leishmania tropica with ic(50) value of 1.4 µg/mg extracts. b. subtilis and bacillus tequilensis correspondingly exhibit significant protein kinase inhibition of 47 ± 0.72 and 42 ± 1.21 mm bald zones, indicating anti-infective and antitumor potential. conclusions: our findings revealed that crude extracts of selected endophytic bacteria from f. indica possess excellent biological activities indicating their potential as an important source of antibiotics (antifungal, antibacterial) compounds. drug resistance has created new health challenges for humans. over the years, emergence of new infectious diseases such as ebola, swine flu (h1n1), dengue fever and severe acute respiratory syndrome (sars), have been added to this challenges [1] . there is a general need for chemotherapeutic agents, antibiotics and pharmaceutical drugs with high effectiveness and low cytotoxicity. new medicinal agents are also needed for nematode infections such as malaria and for the treatment of parasitic protozoan diseases such as leishmaniasis and trypanosomiasis [2] . natural resources are the potential source of novel bioactive molecules and can be used in the treatment of various infections. such sources may include various forms of life like plants, marine macro-organisms (sponge, corals and algae) and endophytes (fungi, bacteria and actinomycetes) [3] . plants which are used in traditional medicines are of significant importance and therefore considerable research has been carried out on medicinal plants for bioactive compounds however limited research has been performed on the associated microorganisms. endophytes are chemical synthesizer inside plants. they play a role as a selection system for microbes to produce pharmacologically active substances with low toxicity toward mammalians [4] . endophytes are sometimes responsible for the medicinal properties of their hosts. many endophytes synthesize bioactive compounds that can be used by plants for defense against pathogens and some of these compounds may be valuable as pharmaceutical drugs [5] . fagonia indica is a small spiny shrub that belongs to the family zygophyllaceae present in warm and arid regions of the world, especially asia and africa [6] . fagonia indica is of great ethno pharmacological importance with multiple therapeutic activities such as antimicrobial, antioxidant, antiseptic, anticancer, antidiabetic and antiinflammatory [7] . the plant is also used for the treatment of fever, thirst, vomiting, asthma, urinary discharge, dysentery, liver and stomach trouble, toothache, typhoid and skin diseases [8] . this study was designed to investigate the role of endophytic bacteria in the medicinal properties of f. indica. bacteria were isolated, identified and screened for the production of bioactive secondary metabolites. all the experiments were carried out in the molecular systematics and applied ethnobotany lab, department of biotechnology, quaid-i-azam university, islamabad. the plant samples were collected in sample bags from village mullazai, peshawar, khyber pakhtunkhwa, pakistan and brought to the laboratory at the same day for the isolation of endophytic bacteria. the plant material was taxonomically identified as f. indica at the department of plant sciences, quaid-i-azam university islamabad. to further confirm the taxonomic validity of the plant species, dna barcoding was executed using cdna markes such as matk, rbcl and trnh-psba. herbarium specimen (voucher no. mosel-320) was deposited in the herbarium of molecular systematics and applied ethnobotany lab at department of biotechnology; quaid-i-azam university, islamabad. the stem of the plant was cut into pieces of about 0.5-1 cm in length and surface sterilized with 70% ethanol for 2 min followed by washing with clorox (commercial bleach) for 5 min and finally rinsing at least three to five times with sterile distilled water. stem pieces (5) (6) were blotted on the blotting paper [9] and placed on selected tryptic soy agar (tsa) media for the isolation of endophytic bacteria. the plates were incubated at 30 °c for 24 h to obtain colonies of bacteria. bacterial genomic dna was isolated using alkaline lysis method and 16s rrna gene was amplified by performing colony pcr using universal bacterial primers 27f and 1492r, which produced a product of size 1465 base pairs [10] . purification of pcr product was done by using pure linktm quick pcr purification kit (invitrogen). sanger sequencing method was used to commercially sequence the purified pcr samples with 27f primer from macrogen (south korea). sequence results obtained were compared with nucleotide sequences available on ncbi database (www.ncbi.nlm.nih.gov/blast) and also confirmed from ez-taxon (http://www.ezbiocloud.net/ eztaxon). sequences of endophytic bacteria were submitted to the genebank (ncbi) and accession numbers were obtained. selected bacterial strains were inoculated in tryptic soye broth and incubated for 48 h in shaking incubator at 30 °c at 120 rpm. bacteria were then transferred into 50 ml falcon tube and centrifuged at 10,000 rpm for 10 min. the supernatant was separated in the tube and the pellet was processed further. pellet was dissolved in organic solvent (methanol) and incubated for 1 day. next day sonication was performed to rupture the cells for 30 min after every five min. after sonication the tubes were centrifuged for 10 min at 10,000 rpm. the organic supernatant was separated in the falcon tube (a) and in the remaining pellet, again organic solvent methanol was added and centrifuged for 10 min at 10,000 rpm. supernatant was separated in the falcon tube (b). the pellet was discarded and both the solvents (a) and (b) were mixed. the solvent was evaporated at room temperature to get crude extract of bioactive metabolites. the extract was dissolved in (dimethyl sulfoxide) dmso [11] . total flavonoid contents (tfc) aluminum trichloride (alcl 3 ) colorimetric method was used for total flavonoids content determination as described by quettire-dele et al. [12] with slight modifications as per system suitability. an aliquote of 20 μl of the test sample (4 mg/ml) along with negative and positive controls i.e. dmso and quercetin (1 mg/ml) respectively were taken in 96-well plate and incubated for 30 min at 37 °c. it was followed by the addition of 10 µl of aluminum chloride (10%), 10 µl potassium acetate (98.15 g/l) and final volume was raised up to 200 µl with distilled water. absorbance was measured at 405 nm through micro plate reader (elx800biotek) and triplicate results were analyzed as µg qe/mg extracts. total phenolic contents (tpc) folin-ciocalteu method as described by jagadish et al. [13] with slight modifications was followed for tpc determination. calculated volume of test sample 20 µl was taken form 4 mg/ml stock solutions and added to 96 well plates followed by addition of 90 µl of 10 times diluted folin-ciocalteu reagent incubated for 5 min. after incubation, 6% sodium chloride solution was added to each mixture and incubated for 90 min at room temperature. dmso and gallic acid (1 mg/ml) were taken as negative and positive controls, respectively. absorbance was measured at 630 nm wave length of triplicate analysis and results were expressed in term of mean ± sd. the method described by tai et al. [14] was used for the determination of free radicals scavenging activity with minor modification. the antioxidant potential of the crude extracts was gauged by monitoring its capacity to quench the stable 2, 2-diphenyl 1-picrylhydrazyl (dpph) free radical. activated crude extract samples of bacterial endophytes i.e. 100, 50, 25 and 12.5 µg/ml were taken in 96 well plates. dpph was added to the entire row of well containing samples to obtain the final concentration of 200 µl. dmso and ascorbic acid were taken as negative and positive controls, respectively. the absorbance was measured at 630 nm using microplate reader (elx800 biotek) after 1 h of incubation at room temperature. ic 50 values expressed as µg aae/mg of extracts. percent radical scavenging was calculated by using formula: total antioxidant assay was performed to evaluate the total antioxidant capacity of extracts by phosphomolybdenum method [15] . the method depends on the reduction of mo (vi) to mo (v) by the consequent formation of a green colored phosphate/mo (v) complex with a maximal absorption at 630 nm and through antioxidant mediators [16] . reaction mixture contains 180 µl of phosphomolybdenum reagent (0.6 m h2 so4, 28 mm nah2po4, 4 mm ammonium molydate) and 20 µl of test sample taken from stock solution (4 mg/ml) followed by incubated for 90 min at 95 °c in water bath. after incubation, samples had been cooled at room temperature and transferred to 96 well plates. dmso and ascorbic acid were taken as negative and positive controls, respectively and absorbance was measure at 630 using microplate reader (elx800 biotek). results were calculated as the number of µg equivalents of ascorbic acid per mg of extract (µg/mg). the experiment was performed in triplicate. the method described by oyaizu et al. [17] was followed for the estimation of total reducing power with minor modifications. a proper volume of test samples 40 µl from the stock solution (4 mg/ml) was taken in eppendrof tubes and incubated in water bath for 20 min at 50 °c, after the addition of reagents 0.2 m phosphate buffer (6.6 ph) and 1% potassium ferri cyanide [k3fe (cn)6] (1 g 100 ml −1 ). after incubation 10% trichloroacetic acid was added to all tubes and centrifuged at 2500 rpm for 10 min. 166.66 µl supernatant from each centrifuge mixture was taken and transferred into 96 well microplate followed by the addition of 33.3 µl ferric chloride (0.1%) solution to each well and mixed properly. absorbance was measured at 630 nm through micro plate reader (elx800 biotek). results were calculated as µg aae/mg extracts of triplicate analysis. dmso and ascorbic acid were used as negative and positive controls, respectively. triplicate analysis by disc diffusion method was used to evaluate the antifungal potential of test extracts described by lai et al. [18] with some modification. crude extracts were screened against four fungal pathogenic strains: mucor mycosis (fcbp (fungal culture bank of pakistan) -0041), aspergillus flavus (fcbp-0064), aspergillus fumigates (fcbp-1264) and aspergillus niger (fcbp-0198). dimethyl sulfoxide (dmso) disc was used as negative control whereas amphotericin b was used as positive control. plates were incubated at 37 °c for 24-48 h with periodic observations of inhibition zones. extracts were screened to determine minimum inhibitory concentration bacterial extract samples were tested at lower concentration with three-fold serial dilutions and relative optical density (od) was taken at 540 nm through micro plate reader (elx800biotek). ic 50 values were obtained from the dose-response curves generated by plotting percent growth versus drug concentration. percent viable cells were calculated by using the formula. (mic) producing an inhibition zone ≥10 mm in diameter at lower concentrations ranging from 100 to 12.5 μg/disc by standard disc diffusion method. in vitro antibacterial potential of the test extracts was evaluated using 96 well microplates method as described previously by gao et al. [19] with slight modifications. the extracts were tested against gram positive bacteria: bacillus subtilis (atcc-6633), staphylococcus aureus (atcc-6538), micrococcus luteus (attc-10240) and gram negative bacteria: escherichia coli (atcc-15224), salmonella typhi (atcc-14028) pseudomonas aeruginosa (atcc-9721) and klebsiella pneumonia (atcc-4619). bacterial strains were inoculated in 10 ml tsb and incubated for 24 h at 37 °c at 120 rpm in shaking incubator. for each bacterial strain, a standardized inoculum (1 × 10 8 cfu/ml) was prepared. the test samples were used in three fold dilutions i.e. 100, 33.3, 11.1 and 3.7 µg/ml. dmso was used as negative control and cefixime monohydrate (antibiotic) as positive control, respectively. reading was initially taken at zero hours and then again after 24 h incubation through microplate reader (elx800biotek) at 630 nm wave length [20] . the corresponding 50% inhibitory concentration (ic 50 ) of each sample was calculated. the process described by ma et al. [21] was used for in vitro antileishmanial activity with slight modifications against leishmania tropica in their promastigote stage. the parasite was sub cultured in the rpmi 1640 medium supplemented, 292 µg/ml l-glutamine, 4.5 mg/ml glucose and 10% fetal bovine serum (fbs). within these culture conditions, the stationary phase of parasite growth was obtained in 6 days. the culture was incubated at 25 °c and used within 2 weeks of cultivation. antileishmanial activity of extracts in comparison to amphotericin b drug was evaluated against parasite using mtt 3-(4, 5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide) based micro assay as a marker of cell viability. the the protein kinase inhibition assay was performed thrice with purified isolates of streptomyces 85e strain by observing hyphae formation using isp4 selective media [22] . the prepared media was subjected to autoclaving for 20 min at 121 °c and poured in the petri plates (autoclaved) under laminar flow cabinet to avoid contamination. after solidification of the media, streptomyces culture broth was spread on the surface of media with sterilized cotton swab. the test samples 20 mg/ml were tested on this media using the disc diffusion method. plates were incubated at 37 °c for 24-48 h. the zone formation was measured through vernier caliper. surfactin (antibiotic) was used as positive control while dmso as negative control, respectively. two types of zones were observed clear and bald in which the bald zones showed protein kinase inhibition activity [23] . all experiments were conducted in a completely randomized design at least three times. statistical analysis was carried out using spss 22.0 and statistic 8.1. the relationship between different parameters was assessed using pearson's correlation coefficient (r). one-way anova was used to check the significant mean difference with tukey's hsd for post hoc analysis. a p < 0.05 was used to define significant results. all the graphs were made using origin 8.1 and duncan's multiple-range test was used to find differences among treatments (p < 0.05). the isolated bacteria form (table 1) . the percent free radical scavenging activity (%rsa) of the bacterial crude extracts was evaluated by the discoloration of dpph reagent. the reaction was visible as a color change from reduction of the purple colored dpph to stable yellow-colored 2, 2-diphenyl-1-picrylhydrazyl molecule by hydrogen radical or accepting electron from donor antioxidant. results were evaluated by calculating the half maximal (50%) inhibitory concentration. it was found that s. maltophilia possess highest free radical scavenging activity with 98.7 µg/ml inhibitory concentration (ic 50 value), followed by p. dispersa and e. cloacae having 157.6 and 228.3 µg/ml ic 50 values. ascorbic acid was taken as standard and percent dpph free radical scavenging activity of each endophytic bacterial extracts are shown in table 2 . [fig. 2e (standard curve) ]. estimating total antioxidant capacity. it was found that s. maltophilia show maximum antioxidant activity among all the bacterial extracts with 37 µg/mg value followed by e. cloacae, p. cypripedi and b. subtilis with 35, 34 reducing ability was measured by change of fe 3+ to fe 2+ in reducing power assay. extracts were checked for their antioxidant reducing power. s. maltophilia and p. dispersa results indicated their electron donor ability for stabilizing free radicals and greater reductive potential with highest reducing power i.e. 206 and 175 µg ascorbic acid equivalents per mg of extract. activity of all bacterial extracts equivalent to ascorbic acid with respect to their absorbance values were shown in figs. 1d and 2c (standard curve). in antibacterial assay, all bacterial extracts show inhibition against two strains of pathogenic bacteria b. subtilis and k. pneumonia. against k. pneumonia, bacterial extracts showed ic 50 values ranging from 1 to 20 μg/ml with variable inhibition from 55 to 82 at a concentration of (100 µg/ml). while in case of b. subtilis the extracts show variable ic 50 ranging from 27 to 365 μg/ml with percent inhibition from 0 to 76. the inhibition value and effective ic 50 value of different endophytic bacterial extracts against k. pneumonia and b. subtilis were shown in (table 3) . antifungal activity of bacterial crude extracts was determined against four strains of filamentous pathogenic fungi namely; mucor mycosis, aspergillus flavus, aspergillus fumigatus and aspergillus niger through disc diffusion method. all the bacterial genera showed inhibitory activity against all the selected pathogenic fungi. almost all the bacterial extracts were active against a. niger but significant results observe are of stenotrophomonas maltophilia with 16 mm inhibitory zone and 12.5 μg/ml mic value (table 4 ; fig. 3 ). although best results showed by b. tequilensis extracts against m. mycosis with maximum zone of 12 mm and 50 μg/ml mic whereas all the extracts shows moderate antifungal activity against a. fumigatus with an average diameter of growth inhibition zone ranging from 7 to 10 mm. moreover, no inhibitory zone was observed for dmso which conform its inactivity against the selected fungal strains. although standard drug amphotericin b exhibited maximum, activity with 21.2 ± 0.985 mm zone. antileishmanial activity against promastigote was determined by mtt assay. variation was observed between bacterial extracts ( letter a-c represent; a highly significant, b slightly significant and c non-significant difference from control at p < 0.05 by one-way anova in the column * values are mean ± sd of triplicate in the current study, endophytic bacterial crude extracts were screened for the inhibition of protein kinases with streptomyces sp. the growth inhibition of streptomyces strain was used as parameter to check the cytotoxicity of endophytic crude extracts. clear zone indicates no bacterial growth having cytotoxicity while a bald zone with doted bacterial growth indicates protein kinase inhibition. in bald zone, only the hyphae formation capability of streptomyces was lost. in clear zones, complete cells were destroyed. bacterial extracts of genus bacillus show high inhibition value against streptomyces growth such as b. subtilis and b. tequilensis with 47 ± 0.72 and 42 ± 1.21 mm zone of inhibition, respectively with bald zones formation which show protein kinase inhibition activities dmso show no-toxic effect with no zone formation while positive control surfactin show the bald zone formation with 21 ± 1.02 mm ( table 6 ; fig. 3 ). a large number of bioactive metabolites with pharmacological properties have been isolated from medicinal plant's endophytes and structurally illustrated by employing various conventional as well as modern techniques. these metabolites have provided a base line for the researchers to do more work on development and formulation of bioactive compounds into useful therapies with tremendous applications in health care system and also in many other fields of human life. bacteria are common inhabitants of both internal and external tissues of most plants [12] . medicinal plants usually harbor endophytes related with their secondary metabolites and medicinal activities [5] . the present study was conducted on endophytic bacteria, isolated from f. indica with great ethno-botanical significance. it is reported that medical uses of a plant with medicinal history relates more to its endophytic population than its own biochemistry [5] . many biological assays were conducted to investigate whether bacteria associated with f. indica have potential medicinal properties. the isolated endophytic bacteria belong to diverse genera such as bacillus, enterobacter, pantoea, erwinia, stenotrophomonas as confirmed with 16s rrna gene sequence analysis. it was observed from the results that all the selected bacterial extracts exhibit various anti-bacteria, anti-fungal and protein kinase inhibition activities. antioxidants are the first line of defense against damage that may occur due to the generation of free radicals. antioxidants deactivate or stabilize free radicals before they attack the cells [24] . the free radical dpph scavenging activity model is a simple, rapid and classic method of assessing antioxidant activity [25] . methanolic extracts of endophytic isolates were able to reduce the stable radical dpph to a yellowcoloured diphenyl picryl hydrazine. among others, the culture extracts of s. maltophilia and p. dispersa showed the highest % scavenging activities which is considerable as compared to control (i.e. ascorbic acid equivalent). ic 50 value is a widely used parameter for the measurement of free radical scavenging activity. low ic 50 indicates significant activities as compared to high ic 50 value [26] . the current results are in agreement with the previous study reported by [27] , about endophytic bacteria associated with ethno-medicinal plants. oxidation in biological system is natural phenomenon resulting in the generation of highly reactive peroxyl and hydroxyl radical which may cause inadequate damage to dna, polyunsaturated fatty acid residues of cell membrane, phospholipids and proteins. it may also lead to pathological effects such as cancer and vascular diseases [28] . among the extracts total antioxidant activity of s. maltophilia was the highest followed by e. cloacae strain. reducing ability of a compound depends on the free radical scavenging capacity and electron donation [29] . endophytic bacteria isolated from centella asiatica, pantoea species such as pantoea agglomerans showed greater reductive potentials reported by rafat et al. [30] . all bacterial extracts were subjected for their reducing power but s. maltophilia and p. dispersa showed the highest values indicating their electron donating ability for table 6 protein kinase (pk) inhiation (i.e. zone's diameter in mm ± sd) during streptomyces hyphae formation of isolated bacterial extracts (bald zone indicates pk inhibition while clear zone shows cytotoxicity) surfactin stabilizing free radicals and showed the similar reductive potential as reported above. high antioxidant potential is usually related with higher proportion of the phenolic content and in the present study a significant correlation was also found in case with s. maltophili. the current results are in agreement with the previous study reported by swarnalatha et al. [31] . flavonoids have an important role in stabilizing lipid oxidation which is associated with antioxidant activity [32] . in present study highest flavonoid content was displayed by s. maltophilia culture extract which correlates with its highest radical scavenging and greatest reductive potential as discussed earlier. which are in agreement with the study reported by jalgaonwala et al. [33] about endophytic bacteria associated with host plant pongamia glabra. all isolated of endopytes extracts from f. indica showed varying degree to inhibited test organism growth but significant results were observe against k. pneumonia by each extracts. bacterial extracts of s. maltophilia, e. hormaechei, b. tequilensis and erwinia sp. also showed significant antibacterial activity against b. subtilis, with variable inhibition and ic 50 values. the crude extract of isolated b. subtilis strain showed no growth inhibition against the b. subtilis which show their similar metabolites productions and thus have no effect on test strain. moreover, extracts of all the endophytic bacteria inhibited the growth of nearly all the tested fungal pathogens. in current study the extracts producing a growth inhibitory zone ≥10 mm in agar disc diffusion assay were considered significant active. significant result was observed by s. maltophilia against a. niger with 16 mm inhibitory zone and 12.5 μg/ml mic value and b. tequilensis extracts against m. mycosis with maximum zone of 12 mm and 50 μg/ml mic. which is an agreement with the previous results reported by [34] non-toxic effect of dmso was confirmed through absence of growth inhibition zones while standard drug amphotericin b exhibited inhibitory zone of (21.2 ± 0.985). leishmaniasis, is a vector-borne disease caused by obligate intra-macrophage protozoa [35] . more investigations required to find anti-leishmanial effect by using leishmania amastigote through different in vitro assays and then to investigate in vivo activity in laboratorial infected animals which would help in obtaining a novel drug that could potentially be cost-effective against the leishmania parasite and less toxic [36] . most of the isolated endophytic bacterial crude extracts showed antileishmanial activities against leishmania tropica with ic 50 values ranging from 1.4 to 6.6 μg/ml. protein phosphorylation at threonine/serine and tyrosine residues by protein kinases is one of the major regulatory mechanisms in biological processes including cell proliferation, cell differentiation metabolism and apoptosis. deregulated phosphorylation at serine/threonine and tyrosine residues by protein kinases produced as a result of genetic alterations acquired early in tumorigenesis are often the cause of cancer. in this respect, inhibition of protein kinases has emerged as a promising target for cancer treatment [37] . using streptomyces 85e as an assay strain for kinase inhibitors appears to identify a wide range of eukaryotic kinase modulators, presumably because the streptomycete enzymes are evolutionary forerunners of their highly specific eukaryotic counterparts [38] . the growth inhibition of streptomyces strain was used as parameter to check the protein kinase inhibition of the crude extracts. bacterial extracts of genus bacillus showed high inhibition zone against streptomyces growth. b. subtilis and b. tequilensis showed bald zone formation (with 47 ± 0.72 and 42 ± 1.21 mm zone respectively), positive control surfactin also exhibit bald zone which indicate protein kinase inhibition activities. extracts were used in concentration of 100 µg/ ml while positive control with 25 µg/ml. b. subtilis is the source of surfactin antibiotic production as isolated and reported by [39] . protein kinase activity is critical for the aerial hyphae formation of streptomyces and this prerequisite has been exploited in the present study to bioprospect the extracts for their kinase inhibitory activity so that their anticancer potential could be assessed. from the preliminary study, it can be inferred that endophytes play a pivotal role in the medicinal activities of plants such as f. indica. we conclude excellent biological activities for the endophytic microorganisms associated with f. indica and postulate a viable role of the endophytic microbes in the medicinal potential of f. indica. we further recommend studies on the endophytic microbes associated with f. indica with state of the art spectroscopic and chromatographic techniques for the identification of targets and mechanism of their synthesis. hospital outbreak of middle east respiratory syndrome coronavirus thoughts and facts about antibiotics: where we are now and where we are heading fungal endophytes and bioprospecting endophytes-the chemical synthesizers inside plants bioprospecting for microbial endophytes and their natural products phytochemicals and biological activities of fagonia indica a novel steroidal saponin glycoside from fagonia indica induces cell-selective apoptosis or necrosis in cancer cells jodhpur: scientific publishers taxonomy of endophytic fungi of aerial plant tissues. microbiology of the phyllosphere selective isolation and characterization of agriculturally beneficial endophytic bacteria from wild hemp using canola endophytic fungal strains of fusarium solani, from apodytes dimidiata e. mey. ex arn (icacinaceae) produce camptothecin, 10-hydroxycamptothecin and 9-methoxycamptothecin phenolic compounds and antioxidant activities of buckwheat (fagopyrum esculentum moench) hulls and flour comparitive study on the antioxidant, anticancer and antimicrobial property of agaricus bisporus (je lange) imbach before and after boiling antioxidant activity and chemical constituents of edible flower of sophora viciifolia spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin e in vitro assessment of antioxidant potential and determination of polyphenolic compounds of hedera nepalensis k. koch studies on products of browning reaction-antioxidative activities of products of browning reaction prepared from glucosamine antioxidative, tyrosinase inhibiting and antibacterial activities of leaf extracts from medicinal ferns antimicrobial and antiprotozoal activities of secondary metabolites from the fungus eurotium repens an innovative microplate assay to facilitate the detection of antimicrobial activity in plant extracts antimicrobial and antileishmanial activities of hypocrellins a and b citrinin derivatives from the soil filamentous fungus penicillium sp. h9318 evaluation of actinomycete strains for key traits related with plant growth promotion and mycorrhiza helping activities from the editor: antioxidants and diseases of the brain determination of in vitro antioxidant and radical scavenging activities of propofol assessment of phenolic content and free radical-scavenging capacity of some thai indigenous plants investigation on the bioactivity of culturable endophytic and epiphytic bacteria associated with ethnomedicinal plants total phenolic contents and free radical scavenging activity of certain egyptian ficus species leaf samples free radical scavenging activity of an aqueous extract of potato peel a novel source of bioactive compounds: endophytic bacteria isolated from centella asiatica bioactive compound analysis and antioxidant activity of endophytic bacterial extract from adhathoda beddomei coumarins as antioxidants isolation and characterization of endophytic bacterial flora from some indian medicinal plants isolation, characterization, and antimicrobial activity of endophytic bacteria from polygonum cuspidatum the leishmaniases as model zoonoses vector control in cutaneous leishmaniasis of the old world: a review of literature extraction optimization of medicinally important metabolites from datura innoxia mill: an in vitro biological and phytochemical investigation identifying protein kinase inhibitors using an assay based on inhibition of aerial hyphae formation in streptomyces studies on the biosynthesis of surfactin, a lipopeptide antibiotic from bacillus subtilis atcc 21332 we acknowledge ikram ullah (ph.d. scholar), ali talha khalil (ph.d. scholar) and tariq khan (ph.d. scholar) from department of biotechnology, quaid-i-azam university islamabad for their help and valuable advices during research work. we acknowledge molecular systematics and applied ethnobotany lab, department of biotechnology, quaid-i-azam univeristy islamabad for providing the platform to perform the experiments. authors' contributions lr participated in the overall experimental design and wrote the manuscript. zks participated in providing lab facilities and management of the study. ii and ft participated in the coordination of the study and analyzing the data sequencing. lr participated in graphical and statistical data analysis. all authors read and approved the final manuscript. the authors declare that they have no competing interests. the nucleotide genome sequences of endophytic bacteria obtained in this work were deposited in genbank under accession numbers kt367786-kt367793. it is not applicable because the manuscript does not contain anyone's individual data. this research was approved by the research ethics committee of quaid-i-azam univeristy islamabad pakistan. the work was not funded from any source and further that the institution is not providing any funds for its publication. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. submit your next manuscript to biomed central and we will help you at every step: key: cord-318854-xaus3bma authors: sun, yi; kurokawa, masahiko; miura, motofumi; kakegawa, tomohito; motohashi, shigeyasu; yasukawa, ken title: chapter 4 bioactivity and synthesis of diarylheptanoids from alpinia officinarum date: 2016-12-31 journal: studies in natural products chemistry doi: 10.1016/b978-0-444-63601-0.00004-1 sha: doc_id: 318854 cord_uid: xaus3bma abstract many diarylheptanoids were isolated from the galangal (rhizome of alpinia officinarum). we are reported the antitumor-promoting, antiinflammatory, cytotoxic, antiinfluenza virus, and antiherpes simplex-1 virus activities of diarylheptanoids. these components in our dietary and herbal supplements are considered to be relatively nontoxic to human. in addition, we synthesized the new compounds. in this review, we discuss the antitumor-promoting, antiinflammatory, cytotoxic, antimicrobial, antiinfluenza, and antiherpes simplex-1 virus activities of diarylheptanoids isolated from galangal resulting from our own and other research groups' investigation. alpinia officinarum hance (zingiberaceae), known as lesser galangal, is a wellknown medicinal herb distributed in east asia whose rhizomes are used for stomachic, analgesic, and antiemetic treatment [1] . the plant grows several feet tall and has long leaves and reddish-white flowers. alpinia officinarum is widely cultivated throughout china, thailand, india, sri lanka, malaysia, indonesia, saudi arabia, and egypt. the species is native to asia, australia, and the pacific islands, where it occurs in tropical and subtropical climates. alpinia is the largest genus of about 230 species in zingiberaceae. historically, its rhizomes possess stimulant and digestive effects owing to their spicy flavor and aromatic scent, and as a result, it is widely used in curries and perfumes throughout asia. although it was previously used throughout europe, its use has declined in recent years, and it is now mainly used in eastern europe. homoeopaths use it as a stimulant. the rhizomes of a. officinarum have been used in traditional japanese herbal prescriptions (kampo medicine) mainly for dyspepsia, vomiting, flatulence, stomach trouble, and diarrhea. the plant is also used in traditional chinese medicine as an aphrodisiac, abortifacient, carminative, antipyretic, antiinflammatory, and emmenagogue, as well as to treat disorders of the heart and kidneys, bronchitis, chronic enteritis, renal calculus, diabetes, and rheumatism. furthermore, its rhizomes are used in various asian cuisines (for example, in thai and lao tom yum and tom kha gai soups, vietnamese hu cuisine and throughout indonesian cuisine). the rhizomes contain numerous active constituents, including essential oils [2] , resin [3] , flavonoids [4] , diarylheptanoids [5] , and phenylpropanoids [6, 7] . as the main components distributed in this genus, diarylheptanoids possess cytotoxic, antiemetic, antiinflammatory, antivirus, and antiproliferative activities. the rhizomes of a. officinarum are an important medicinal herb recorded in chinese, korean, and japanese pharmacopoeias [8] [9] [10] . in this chapter, we discuss the biological activity of natural diarylheptanoids isolated from a. officinarum, as well as synthetic diarylheptanoids. diarylheptanoids are a major class of bioactive constituents in a. officinarum that are categorized into linear, cyclic, and dimeric diarylheptanoids, or diarylheptanoids bearing special moieties ( fig. 4 .1, table 4 .1). diarylheptanoids isolated from a. officinarum are mainly linear diarylheptanoids. diarylheptanoids 2-11 mostly possess a common structural moiety of 5-ene and 3-oxo or 3,5-dioxo groups on the heptane skeleton [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] . the main types of linear diarylheptanoids (13-43) possess 5-hydroxy and 3-oxo groups in their structure [11] [12] [13] [14] [15] [16] [17] [18] [19] [22] [23] [24] [25] [26] [27] [28] [29] [30] . the other differences in structures lie in the pattern of substitution on aromatic rings. some diarylheptanoids (47) (48) (49) (50) (51) (52) (53) possess a common structural moiety of 3,5-dihydroxy on the heptane skeleton [12, 15, 17, 25, 29, 31 ]. in addition, compounds 1, 9, 12, and 44-46 are also linear diarylheptanoids that possess double bonds or ketone groups between c-1 and c-7 [15, 19, 32] . sun [14, [23] [24] [25] . several novel dimeric diarylheptanoids (58) (59) (60) (61) (62) (63) (64) were isolated from the rhizomes of a. officinarum [19, [32] [33] [34] . some dimeric diarylheptanoids were connected through cdc or cdodc bonds (58) (59) (60) (61) , and some were connected through the pyridine ring or six-numbered carbon ring (62) (63) (64) . both the pyridine and (5r)-5-(3,5,7-trihydroxyflavone)-7-(3-methoxy-4-hydroxyphenyl)-1-phenyl-3heptanone (officinin a) [35] six-numbered carbon rings are derived from the heptane unit of linear diarylheptanoids. liang et al. found that diarylheptanoid (65) has a novel skeleton bearing a flavonol moiety [35] . pharmacological research found that diarylheptanoids exhibited antiproliferative, cytotoxic, antiemetic, antiinflammatory, and antivirus activities. many related diarylheptanoids have been examined in order to study the structureactivity relationship (sar). several research groups have developed techniques for the detection, isolation, and identification of the components from a. officinarum. wong et al. isolated two diarylheptanoids (6 and 24) and two flavonoids using high-speed countercurrent chromatography (hsccc) from chloroform extracts [36] . ho et al. attempted to analyze the ethyl acetate extract directly by high-performance liquid chromatography (hplc) with photodiode array and electrochemical detection (hplc-ecd) techniques [37] . the hplc-edc method is also a very powerful tool for the detection of diarylheptanoid components at the nanogram level. furthermore, feng et al. discussed the separation and identification of diarylheptanoids in supercritical fluid extracts of a. officinarum using a uplc-q-tof-ms-ms system [38] . diarylheptanoids are widely distributed in nature. curcumin is the most common diarylheptanoid and possesses a range of bioactivities against human diseases, including antitumor, antiinflammatory, and antioxidant activities [39] . on the other hand, yashabushidiols were isolated from the male flowers of alnus sieboldiana (betulaceae) by hashimoto et al. [40] , and these compounds are also included in the diarylheptanoids from a. officinarum, such as compound (49) (fig. 4.2) . stereoselective synthesis of yashabushidiols and their derivatives has been reported by venkateswarlu's group, shinde's group, and yikang's group [41] [42] [43] . each group used a sugar or derivative as a starting material; for example, d-mannitol, d-glucose, and d-gluconolactone. after several steps, acetal compounds were obtained, and nucleophilic addition or wittig reactions then afforded yashabushidiols and their derivatives ( fig. 4.3) . we also synthesized yashabushidiols and their derivatives using kinetic resolution of sharpless asymmetric epoxidation reaction [44] (fig. 4.4) . first, 4-phenyl-1-butyne was treated with n-buli followed by addition of 3-phenylpropionaldehyde to a lithiated alkynyl solution. reduction of propargyl alcohol with red-al afforded the racemic allylic alcohol. sharpless epoxidation reaction of allylic alcohol gave enantio-enriched antiepoxy alcohol through kinetic resolution, followed by reduction using red-al to afford both natural and unnatural types of yashabushidiol b (49) . on the other hand, racemic allylic alcohol was treated with mcpba and preferentially generated syn-epoxy alcohol followed by reductions using cp 2 ticl 2 , zn, and zncl 2 to afford yashabushidiol a. compounds 35 and 32, and their enantiomers, were synthesized under almost the same conditions. compound 32 showed particularly strong antiviral activity against respiratory syncytial virus (rsv) in vitro and in vivo [44] . optically active compounds 35 and 32 were synthesized from racemic allylic alcohols according to the following procedure. first, kinetic resolution of optically active epoxy alcohol and chiral allylic alcohol was performed by sharpless asymmetric epoxidation (sharpless ae). chiral allylic alcohol was subjected to sharpless ae with l-dipt to afford the opposite configuration epoxy alcohol. these epoxy alcohols were then oxidized by dess-martin periodinane oxidation to afford optically active α-epoxy ketones. finally, compound 35 and its enantiomers were obtained by treating β-hydroxy ketone with meotf and 2,6-di-tert-butylpyridine; deprotection of β-hydroxy ketone by tbaf yielded compound 32 (fig. 4.5) . compound 50 was also isolated from a. officinarum. it resembled the structure of yashabushidiol b, which was synthesized by das et al. [45] . they also used kinetic resolution of sharpless ae reaction with the racemic allylic alcohol, followed by a ring opening reaction. the 3,5-dihydroxy compound was protected by 2,2-dimethoxypropane under acid conditions, followed by deprotection of the pmb group using ddq reagent. finally, the terminal hydroxyl group was oxidized by swern oxidation to afford aldehyde compound, and it was then subjected to wittig reaction followed by reduction using h 2 and pd on carbon to yield compound 50 ( fig. 4.6 ). alpinoids b (46) and c (45) were also isolated from a. officinarum by sun et al. [18] . both compounds have a unique moiety in the skeleton of the γ-hydroxy-α-enone carbon chain. generally, diarylheptanoids possess 3,5-diketo, 3-keto-5-hydroxy, or 3-keto-4-ene structures, but alpinoids b (46) and c (45) have a 3-keto-4-ene-5-hydroxy moiety. in addition, there is a chiral center at c-5, and its absolute configuration was determined as r by mosher's method. alpinoid c (45) and its analogues were synthesized by venkateswarlu's group and miura's group [46, 47] . the synthetic strategy of venkateswarlu's group is described below (fig. 4.7) . first, 4-phenyl-2-buten-1-ol was treated with (+) dipt, ti(oipr) 4 , and cumene hydroperoxide to afford chiral epoxy alcohol. after two further steps, epoxy alcohol formed chiral allylic alcohol followed by olefin metathesis coupling using grubb's second generation catalyst to afford alpinoid c (45) . on the other hand, miura et al. synthesized 45 using asymmetric 2,3-sigmatropic rearrangement of chiral α-sulfinyl enone ( fig. 4.8 ). chiral α-sulfinyl enone was readily synthesized from l-menthyl sulfinate [48] . chiral α-sulfinyl enone treated with catalytic amount of dbu and pph 3 , followed by oxidation with aqueous h 2 o 2 solution afforded target compound 45 in high enantiomeric excess. neuroblastoma is a common extracranial pediatric solid tumor, accounting for 10% of all tumors in the pediatric age group. the clinical presentation of neuroblastoma is variable and advanced cases are often found to be highly resistant to conventional treatment modalities based on surgery, chemotherapy, transplantation, and radiotherapy [49] . thus, development of new effective and safe therapeutic agents for the treatment of neuroblastoma is urgently needed. in a recent publication, we discussed the antitumor activity of naturally occurring compounds against neuroblastoma [50] . compounds 6, 24, and 30 exhibited the most potent activity against neuroblastoma imr-32 cells (table 4. 2), with ic 50 values of 0.11, 0.83, and 0.23 μm, respectively [17] , and were more potent than cisplatin (ic 50 : 0.85 μm). sun et al. found that diarylheptanoids containing the substituents of 3″-ome and 4″-oh on the benzene ring or only a carbonyl (c-3) and a double bond (c-4/5) at the aliphatic chain possessed potent cytotoxicity against the imr-32 cell line [17] . dose-dependent manner. they found that 5 induces s phase arrest and apoptosis via upregulation of activating transcription factor 3 (atf3) and stabilization of p53 in the shsy5y cell line [25] . compound 5 also exhibited potent cytotoxicity against hepg2, mcf-7, and sf-268 (atcc) human cancer cell lines (ic 50 : 6-10 μg/ml) [24] . furthermore, matsuda et al. also tested the inhibition of melanogenesis by 6, 21, 24, and 49 in theophylline-stimulated b16 melanoma 4a5 cells, and found ic 50 values of 10-48 μm. compound 6 showed the strongest activity among the four diarylheptanoids (ic 50 : 10 μm), and it also inhibited the mrna expression of tyrosinase, tyrosinase-related protein (trp)-1 and trp-2, as well as protein levels of microphthalmia-associated transcription factor (mitf) [52] . chronic inflammation may be a causative factor in a variety of cancers. the longer the inflammation persists, the higher the risk of cancer. in general, inflammatory leukocytes such as neutrophils, monocytes, macrophages, and eosinophils provide soluble factors that are thought to mediate the development of inflammation-associated cancer, including the cancer cells themselves, although other cells also participate. inflammatory mediators include metabolites of arachidonic acid, cytokines, chemokines, and free radicals. chronic exposure to these mediators leads to increased cell proliferation, mutagenesis, oncogene activation, and angiogenesis. emphasis will be placed on examining the role of the reactive oxygen (eg, o 2 − ) and nitrogen intermediates (eg, no), cytokines (eg, interferons, interleukins, tumor necrosis factor-α (tnf-α)), and prostaglandins (pgs). increased cancer incidence is associated with increased duration of inflammation. animal models have demonstrated experimentally that chronic inflammation can lead to the development of various forms of cancer, while providing further insights into possible mechanisms. skin tumors are induced by administration of carcinogens such as 7,12-dimethylbenz[a]anthracene (dmba), followed by repeated administration of tumor promoters such as 12-o-tetradecanoylphorbol-13-acetate (tpa) [53] . in recent publications, we discussed the inflammatory and tumor promotion, and its inhibitors from naturally occurring compounds [54, 55] . methanol extracts from the rhizomes of a. officinarum inhibited tumor promotion by tpa after initiation with dmba in icr mice [56] . fig. 4 .9a shows the percentage of tumor-bearing mice treated with dmba plus tpa was 80% at week 20, whereas that in the group treated with dmba plus tpa and methanol extract of a. officinarum was 20%. treatment with methanol extracts of the rhizomes of a. officinarum caused an 85% reduction in the average number of tumors per mouse at week 20 ( fig. 4 .9b). using bioassay-guided isolation, seven diarylheptanoids (2, 4, 6, 21, 24, 30, and 35) were isolated from active fractions of the methanol extracts of a. officinarum [56] . the inhibitory effects against tpa-induced inflammation closely paralleled those of the inhibition of tumor promotion in two-stage carcinogenesis initiated by dmba and tpa, a well-known tumor promoter, in a mouse skin model [57] . these diarylheptanoids inhibit tumor promotion in two-stage carcinogenesis in mouse skin. on the other hand, these diarylheptanoids inhibited tpa-induced inflammation in mice (table 4. 3). compounds 4 and 30 were similar in activity to indomethacin, an inflammatory drug [56] . the antiinflammatory mechanisms of diarylheptanoids have been reported by many researchers. matsuda et al. found that compounds 6, 21, 24, and 49 inhibited nitric oxide (no) production in lipopolysaccharide (lps)-activated mouse peritoneal macrophages on bioassay-guided isolation, and compound 6 showed particularly strong inhibitory activity with an ic 50 value of 33 μm [58] . bioassay-guided purification of ether extracts led to the isolation of a new diarylheptanoid (44) , as well as two known diarylheptanoids (2 and 17) . these compounds exhibited potent platelet-activating factor (paf) receptor binding inhibitory activity with ic 50 values of 1.3, 5.0, and 1.6 μm, respectively [16] . compound 15 also showed inhibitory and bactericidal activities against enteropathogenic escherichia coli (epec) clinical isolates and efficiently suppressed epec lps-induced inflammation in human peripheral blood mononuclear cells [59]. compound 6 exhibited antiinflammatory properties in a mouse macrophage cell line (raw 264.7) (6.25-25 μm) and suppressed lps-induced production of no, interleukin (il)-1β, and tnf-α by inhibiting nuclear factor-κb (nf-κb) activation and phosphorylation of p44/42 mitogen-activated protein kinases (mapks) [60] . compounds 2, 6, 15, 16, 47, and 48 were tested for their inhibitory effects on no production in the lps-activated macrophage cell line raw 264.7 [15] . compounds 6 and 2 showed potent inhibitory activities with ic 50 values of 0.6 and 6.8 μm, respectively. diarylheptanoid 3, isolated from the n-hexane extract, modulated nf-κb signaling involved in the inflammatory response, and inhibited lps-induced expression of tnf-α, il-1β, nitric oxide synthase (nos), and cyclooxygenase-2 (cox-2) at the gene level in raw 264.7 cells [21] . kiuchi et al. examined the effects of diarylheptanoids (6, 11, 13-15, 19, and 40) against pg and leukotriene. this suggested that compounds lacking the methoxy group adjacent to the phenol were less active than those possessing the methoxy group, presumably owing to the decrease in acidity from the phenol group [26, 28] . thus, the reports cited above suggest that diarylheptanoids possess inhibitory effects against inflammatory and tumor-promoting activities. diarylheptanoids exhibited antiviral activities against influenza virus [61] [62] [63] [64] [65] , rsv [44, 66] , poliovirus [44] , measles virus [44] , herpes simplex virus type 1 (hsv-1) [44] , human immunodeficiency virus (hiv) [67] , severe acute respiratory syndrome (sars) virus [68] , and epstein-barr virus in relation to carcinogenesis [69] . they are characterized as compounds possessing broad antiviral spectrum against dna and rna viruses. most diarylheptanoids possessing antiviral activities have been evaluated in vitro. antiviral activities in vivo have been documented for influenza virus and rsv using animal infection models [62, 65, 66] . sawamura et al. [61, 62] examined the antiinfluenza virus activity and cytotoxicity of 10 diarylheptanoids (2, 4, 6, 21, 24, 30, 32, 35, 36, and 49) isolated from a. officinarum by plaque reduction assay and mtt assay, respectively, using madin-darby canine kidney (mdck) cells (table 4 .4). in this study, influenza virus was more susceptible to 6 (ec 50 = 2.9 ± 0.3 μg/ml) and 32 (ec 50 = 0.7 ± 0.2 μg/ml) than to the others, and their therapeutic indexes (cc 50 / ed 50 ) were 11.7 and 114.3, respectively [61] . compound 32 has a 4-hydroxylphenyl moiety. platyphyllone and platyphyllone-5-xylopyranoside contain two 4-hydroxyphenyl moieties and have been reported to be active against influenza virus with therapeutic indexes of >10 [64] . thus, hydroxylation at the c-4 position of the phenyl moiety may be important for antiinfluenza virus activity in vitro. however, in a murine influenza virus infection model, 6 significantly reduced virus titers in bronchoalveolar lavage fluids of the lungs and prolonged survival times of the infected mice without toxicity, whereas 32 did not show this activity [62] . compound 6 possessed an unsaturated ketone and a methoxy group at the c-5 position of the 4-hydroxyphenyl moiety, but 32 did not. thus, the ketone and methoxy groups may be necessary for antiinfluenza activity in vivo. compound 6 exhibited antiviral activity against h1n1 virus, h3n2 virus, and b type virus, as well as oseltamivir-resistant h1n1 virus [62] . this indicates that the mode of antiinfluenza virus action of 6 was different from that of the known agents, such as oseltamivir, and suggests that it is a candidate antiviral compound against more virulent strains than the pandemic h1n1. in fact, 6 was shown to have no effect on virus adsorption or invasion into cells, instead suppressing the expression of viral messenger rna and antigens in infected mdck cells [62] . it is probable that 6 selectively suppressed influenza virus mrna synthesis in infected cells without cytotoxicity [62] . diarylheptanoids isolated from alpinia katsumadai such as katsumadain a and (e,e)-5-hydroxy-1,7-diphenyl-4,6-heptadien-3-one are reported to have inhibitory activity against the neuraminidase (na) of influenza virus (a/pr/8/34) in vitro [65] . some diarylheptanoids isolated from a. officinarum may therefore be effective in reducing na activity. konno et al. [34, 84] (table 4 .4). among these, 6 and 30 were not effective against the a2 strain of rsv [84] . the ec 50 values of 4 and 66 were 5.0 ± 0.0 and 7.0 ± 1.4 μg/ml (table 4 .4), respectively, and both compounds showed antiviral activity against the a2 strain of rsv with therapeutic indexes of 4.6 and 14.3, respectively, and were more potent than the other tested compounds [44, 66] . compound 4 was also active against influenza virus with an ec 50 value of <10 μg/ml [61] , which suggests that 4 is an effective diarylheptanoid against both rsv and influenza virus in vitro. compounds 22 and 22/32 were synthesized as an enantiomer and racemate, respectively, of 32, and compound 66 was synthesized as the enantiomer of 35 (fig. 4.10 ) [44] . the therapeutic indexes of stereoisomers (22, 32 , and 22/32) were similar (cc 50 /ec 50 : 1.3, 2.1, and 3.4, respectively) and their ec 50 values were 44.7 ± 1.5, 40.7 ± 3.5, and 24.3 ± 0.6 μg/ml (table 4 .4), respectively [44] . however, the therapeutic indexes of 35 and 66 (6.1 and 14.3) were higher than those of the three stereoisomers, with the ec 50 values of 16.3 ± 3.5 and 7.0 ± 1.4 μg/ml, respectively [44, 66] . the c-5 position of 35 and 66 is methoxylated with a saturated ketone at the c-3 position. compound 30 also contained a methoxy group with a saturated ketone and its ec 50 value was 13.3 ± 3.8 μg/ml [66] . thus, methoxylation in diarylheptanoids may contribute to anti-rsv activity in vitro. in a murine intranasal rsv infection model, compounds 22, 32, 22/32, and 66 were significantly effective in reducing virus titers, infiltration of lymphocytes and interferon-γ levels (marker of pneumonia severity) in the lungs of mice [44] . among these, 66 showed the strongest anti-rsv activity in mice, as shown by anti-rsv assay in vitro, and this may be a lead compound for the development of anti-rsv drugs in the future. six diarylheptanoids (2, 6, 21, 24, 30, and 35) were examined for their anti-rsv activity against poliovirus, measles virus, and hsv-1 by plaque reduction assay and trypan blue dye exclusion assay using vero cells (table 4 .4). among the six compounds, 6 and 30 did not exhibit any significant anti-rsv activity in hep-2 cells [84] . however, 30 exhibited relatively stronger antiviral activities against all three viruses (poliovirus, measles virus, and hsv-1), but 6 was only effective against measles virus. all the examined diarylheptanoids except 6 exhibited antipoliovirus activity with ec 50 values between 3.7 ± 0.6 and 44.3 ± 4.0 μg/ml, and all except 2 and 6 exhibited anti-hsv-1 activity with ec 50 values between 5.7 ± 0.6 and 58.7 ± 1.5 μg/ml (table 4 .4). all six examined compounds were significantly effective against measles virus with ec 50 values between 6.3 ± 0.6 and 47.0 ± 4.6 μg/ml. thus, diarylheptanoids appear to have a broad spectrum of antiviral activity. chareonkla et al. [67] reported that (3s,5s)-3,5-diacetoxy-1,7-bis(3,4,5-trimethoxyphenyl) heptane from zingiber mekongense exhibited anti-hiv activity in the antisyncytium assay using δtat/rev mc99 virus and 1a2 cell line system, but that it did not show activity on hiv-1 reverse transcriptase assay. hirsutenone isolated from alnus japonica has been shown to be a potent inhibitor of papain-like protease, which controls replication of the sars coronavirus. this compound is therefore thought to be a potential compound for the treatment of sars [68] . some cyclic diarylheptanoids isolated from acer nikoense and myrica rubra are reported to strongly inhibit epstein-barr virus early antigen activation in raji cells, and they also exhibit inhibitory activities on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test [69] . it is possible that some diarylheptanoids from a. officinarum possess antiviral activity against these viruses. obesity is a major health concern at present and is widely considered to be a global epidemic. conventional or allopathic medicines used to treat obesity have high abuse potential and frequently exhibit side effects. few botanicals included in natural weight loss products have been thoroughly researched on a basic or clinical level, and it is thus imperative to validate their widespread consumption for weight management. many natural weight loss products are sold and used globally with little or no proof of efficacy and quality, and concerns regarding safety have surfaced with good reason. orlistat inhibits pancreatic and gastric lipases that catalyze the hydrolysis of ingested fats, resulting in reduced dietary fat absorption and high fecal fat excretion [70] . it is certain that orlistat has a marked drug effect, as 35-45% of energy intake in western diets is attributed to dietary fat. however, malabsorption of fat leads to rather uncomfortable side effects, such as flatus with discharge, oil spotting from the rectum, fecal incontinence, fecal urgency, loose or liquid stools, and malabsorption of fat-soluble vitamins [70] . the water extract of rhizomes from a. officinarum showed the strongest inhibitory activity against pancreatic lipase in vitro. the extract was subsequently fractionated using organic solvents, and the ethyl acetate fraction had the strongest inhibitory activity. the most potent inhibitor in this fraction was identified as galangin-3-methyl ether (fig. 4.11) with an ic 50 value of 1.3 mg/ ml, as compared with the positive control (orlistat) with an ic 50 value of 0.8 mg/ml using triolein as the substrate. after oral administration of corn oil, triglyceride levels in mice decreased significantly to less than those in the control group by administration of h 2 o extract (1 g/kg) and the ethyl acetate fraction (0.5 g/kg). in triton wr-1339-induced hyperlipidemic mice, galanin-3-methyl ether significantly decreased triglyceride and cholesterol levels at a 20 mg/kg dose to 81.3% and 81.0%, respectively, while it increased high density lipoprotein (hdl) when compared with the control group. moreover, galanin-3-methyl ether did not show hypolipidemic activity in high cholesterol dietinduced hyperlipidemic mice [71] . compound 15 exhibited inhibitory activity against pancreatic lipase with an ic 50 value of 1.5 mg/ml using triolein as the substrate. the levels of serum triglyceride in corn oil fed mice were reduced significantly, while the levels of serum triglyceride and cholesterol were reduced in triton wr-1339-induced hyperlipidemic mice. hypolipidemic activity was not observed in the high-cholesterol diet-induced hyperlipidemic mice. inhibition of pancreatic lipase for both compounds when compared with the positive control (orlistat) was good in terms of other parameters, such as triglyceride, cholesterol and hdl levels. however, orlistat remained the most effective drug [72] . antiemetic drugs are effective against vomiting and nausea and may be used for severe cases of gastroenteritis, particularly if the patient is dehydrated. antiemetics can also be used for morning sickness, but there is little information about their effects on the fetus. some crude drugs inhibit vomiting among traditional japanese herbal prescriptions, while a. officinarum is used as an antiemetic in traditional chinese medicine. takahashi et al. reported that diarylheptanoids showed antiemetic activities in a copper sulfate (cuso 4 )-induced emesis assay in young chicks [12, 29] . the sar of diarylheptanoids was also investigated. among 14 diarylheptanoids, two types of essential functional structure showed inhibitory activities against emesis. diarylheptanoids 2, 4, 6, 21, 22, 24, 27 contained partial type a or b structures ( fig. 4.12) , and they therefore concluded that diarylheptanoids of types a and b might be the main antiemetic components of a. officinarum. 5α-reductase (reduced nicotinamide adenine dinucleotide phosphate: δ 4 -3-ketosteroid 5α-oxidoreductase), which is present as type 1 and type 2 isozymes in humans and rats, catalyze the reductive conversion of testosterone to 5α-dihydrotestosterone [73] . 5α-dihydroteststerone acts as a more active androgen than testosterone in many tissues, such as the prostate. therefore, inhibitors of this enzymatic conversion may be useful in the selective treatment of androgen-dependent diseases, such as benign prostate hyperplasia, male pattern baldness, and acne. most 5α-reductase inhibitors are steroids binding to the steroid receptors, and these steroids may produce various undesirable hormonal effects acting as agonists or antagonists. it has been reported that diarylheptanoids possess inhibitory activity against 5α-reductase. the sar of four diarylheptanoids (2, 22, 24, and 31) from a. officinarum was discussed [13] . it has been suggested that diarylheptanoids with unsaturated bonds, particularly the conjugated form in the alkyl structural moiety between the two aryl groups, had weak inhibitory activity, while diarylheptanoids with saturated counterpart bonds had potent inhibitory activity. thus, the alkyl parts of the diarylheptanoids appear to be important structural moieties for inhibitory activity against 5α-reductase. helicobacter pylori is a gram-negative, microaerophilic bacterium found in the stomach. it is also related to the development of duodenal ulcers and stomach cancer [74] . the eradication of h. pylori is effective in preventing duodenal ulcers and stomach cancer. lee et al. examined the inhibitory effects of compound 15 against h. pylori atcc 43504, atcc 700392, and atcc 700824 using the paper-disc diffusion and agar dilution methods [75] . rhizome-derived materials, particularly isolated diarylheptanoids, merit further study as potential antipylori functional food products or therapeutic products for preventing the diseases caused by h. pylori. zhang [19] . the ic 50 values of these compounds were 9-20 μg/ml and 25-47 μg/ml against hp-sydney and 1 hp-f44 strains, respectively. from bacteria to vertebrates, cells change their patterns of gene expression to respond to their microenvironment. in addition to classical dna microarray technology (transcriptome analysis), new analytical tools for high-throughput screening of whole transcriptome sequencing, polysome profiling (translatome analysis [76] ) or ribosome profiling (ribosome protection assay [77] ) have recently emerged and been applied to the analysis of gene expression (fig. 4 .13) [78] . in et al. reported that microarray global gene expression analysis showed 1′s-1′-acetoxyeugenol acetate (fig. 4.14) , a novel phenylpropanoid from alpinia conchigera, enhanced the apoptotic effects of paclitaxel in mcf-7 cells through nf-κb inactivation [79] . this suggests that the induction of tumor cell death through apoptosis is modulated through dysregulation of the nf-κb pathway. as genetic information transforms from dna to proteins, the cellular abundance of proteins is predominantly controlled at the translation level [80] . weak correlations between messenger rna (mrna) and protein levels [81] are observed because nontranslated mrnas may be present in rna granules, rna particles, processing bodies, stress granules, and mirna-risc (mirisc) complexes in cytosol (fig. 4.13) . analysis of the translatome can thus provide substantial and surprising new information [82] . whole free polysome and/or membrane-bound polysome analysis has also been applied. ribosome-associated mrnas (usually >3) are separated from mrnas associated with fewer ribosomes. these polysome-associated mrnas are applied to label probes on dna microarrays (translatome analysis) or are sequenced using next-generation sequencers (polysome profiling). these polysome-associated mrnas are then applied to ribosome-protection assay, and the resulting segments of rna are sequenced using next-generation sequencers (ribosome profiling). in these analyses, active and stalled ribosomes have been shown to cosediment during isolation of polysome complexes through sucrose gradients [83] , thus indicating that polysome profiling does not fully distinguish translationally active from repressed mrnas. diarylheptanoids 6, 24, and 30 inhibit proinflammatory mediators and exhibit cytotoxic and antiviral activities. however, the precise mechanisms of action and their effects on expression of specific genes are unknown. thus, we used translatome analysis to investigate the mechanisms and modes of action of these diarylheptanoids [84] . polysome-associated mrnas were prepared from diarylheptanoid-treated and control cells from a human b lymphoblastoid cell line; these mrna samples were then used for microarray analysis. the number of downregulated inflammatory-related transcripts was ranked as follows: 30 > 24 > 6. compound 6 showed greater influence on the translatome of bjab cells, while 24 showed less efficacy, except when upregulating the expression of genes related to rhodopsin-like gpcrs, mrna processing, and proteasomerelated proteins of wikipathways [85] . it is possible that the same host factors, such as splicing factors or hnrnps listed in mrna processing wp411 45374 (wp; wikipathways), affect virus structure and/or replication. sixteen transcripts were upregulated after treatment with 6, 24, or 30. among these, transcripts of heterogeneous nuclear ribonucleoprotein c (c1/c2), heterogeneous nuclear ribonucleoprotein k, non-pou domain containing, octamer-binding, and polypyrimidine tract-binding protein 1 were identified as internal ribosome entry site trans-acting factors. all of these studies have provided new insights into the mode of action of diarylheptanoids from a. officinarum with regard to its antiinflammatory, antitumor promotion, and antiviral effects. humans have used plants as foods and natural medicines since ancient times, and while they are crude drugs, are typically safer than synthetic drugs, and have been used as both spices and supplements. several active components have been isolated, and their chemical structures have been and continue to be determined. the diarylheptanoids of the rhizomes of a. officinarum are considered to be a particularly promising group of compounds. diarylheptanoids are minor but ubiquitous components in our diet and have the advantage of being nontoxic or relatively nontoxic to humans. natural diarylheptanoids have multiple physiological functions, including antiinflammatory, antitumor, cancer preventive, antiviral, antiemetic, and anti-pylori effects. challenges that must be overcome in order to find functionally useful compounds that can be applied clinically are further screening of natural diarylheptanoid compounds, examination of sars, elucidation of physiological action mechanisms, and the problems associated with supplying large quantities of compounds. in order to resolve these issues, collaboration between researchers in various fields will be necessary. activator protein-1 atf3 activating transcription factor 3 bjab the human b-lymphoma cell line encyclopedia of herbs & their uses studies in natural products chemistry chinese pharmacopoeia of the people's republic of china the korea food and drug administration notification, korean pharmacopoeia the japanese pharmacopoeia drug discovery research in pharmacognosy drug discovery and development -from molecules to medicine key: cord-011038-5t8az1hf authors: mor, satbir; sindhu, suchita title: synthesis, type ii diabetes inhibitory activity, antimicrobial evaluation and docking studies of indeno[1,2-c]pyrazol-4(1h)-ones date: 2019-10-26 journal: med chem res doi: 10.1007/s00044-019-02457-8 sha: doc_id: 11038 cord_uid: 5t8az1hf we report a convenient and efficient synthesis of indeno[1,2-c]pyrazol-4(1h)-ones (4a‒o) by the reaction of a variety of 2-acyl-(1h)-indene-1,3(2h)-diones (1) and 2-hydrazinylbenzo[d]thiazole/2-hydrazinyl-6-substitutedbenzo[d]thiazoles (2) in the presence of glacial acetic acid in good yields. the structure of the compounds thus prepared were confirmed by analytical and spectral (ft-ir, (1)h nmr, (13)c nmr, and hrms) techniques. all the synthesized indeno[1,2-c]pyrazol-4(1h)-ones (4a‒o) were assayed for their in vitro type ii diabetes inhibitory activity by using acarbose as standard drug and in vitro antimicrobial activity utilizing streptomycin and fluconazole as reference drugs. among the synthesized derivatives, 4e (ic(50) = 6.71 μg/ml) was found to be more potent against α-glucosidase enzyme as compared with the standard acarbose (ic(50) = 9.35 μg/ml) and 4i (ic(50) = 11.90 μg/ml) exhibited good inhibitory activity against α-amylase enzyme as compared with the standard acarbose (ic(50) = 22.87 μg/ml). also, all the titled compounds showed good antimicrobial activity. in addition, in vitro α-glucosidase and α-amylase inhibition were supported by docking studies performed on the derivatives 4e and 4o, respectively. [image: see text] diabetes mellitus is a metabolic disorder resulting from inadequate secretion of insulin characterized by chronic hyperglycemia caused by high calorie diets rich in fat, carbohydrates and proteins . the international diabetes foundation (idf) reports that there were 425 million diagnosed cases of diabetes globally in 2017 which is estimated to increase to 629 million by 2045. recently, there are more than 46 million diabetics in north america and the caribbean, 58 million in europe, 26 million in south and central america, 39 million in middle east and north africa, 16 million in africa and 82 million in south-east asia. there are 352 million people at the risk of developing type ii diabetes (idf diabetes atlas 2017). the emerging factors that contribute to the spread of type ii diabetes, comprising a progressively technological society, food habits with high calorie diets rich in fats and carbohydrates, and an increasingly inactive lifestyle (wagman et al. 2017) . type ii diabetes is associated with hypertension, dyslipidemia, obesity, cardiovascular disease, etc. it may also eventually cause tissue or vascular damage leading to severe diabetic complications such as retinopathy, neuropathy, and nephropathy (keri et al. 2015) . out of several enzymes known, α-amylase and α-glucosidase are the key enzymes in the lowering of postprandial hyperglycemia observed in case of type ii diabetes mellitus (t2dm) (patil et al. 2013) . α-amylase inhibits dietary starch from being absorbed into the body system and leads to lowering of blood glucose by the inhibition of salivary and pancreatic amylase (ajiboye et al. 2016) . α-glucosidase inhibitors have been reported to reduce postprandial hyperglycemia in diabetic mellitus resulting in the lowering of glucose absorption by carbohydrate digestion and increases digestion time (chaudhry et al. 2017) . likewise, the emergence of bacterial resistance of pathogenic microorganisms is rapidly becoming a major worldwide problem . therefore, the demand for new antimicrobial agents is necessary, but now days, it leads to a challenging task for chemists to synthesize new molecules with excellent activity (kim et al. 2012) . in the recent years, indeno-fused heterocycles are recognized as important frameworks with a broad spectrum of pharmacological properties. among them, indenopyrazoles have gained substantial attention due to their wide range of biological activities such as antitubercular (ahsan et al. 2011) , tyrosine kinase inhibitors , cns agents (lemke et al. 1978) , antioxidant activity , non-steroidal anti-inflammatory drugs (lemke et al. 1989) , anticancer (mor et al. 2016) , antimicrobial , anti-hiv and anticonvulsant activities (ahsan et al. 2012) , and cyclin-dependent kinase (cdk) inhibitors (singh et al. 2006) . moreover, methyl 3-((6-methoxy-1,4dihydroindeno [1,2-c] pyrazol-3-yl)amino)benzoate was the first indenopyrazole that was reported as a tubulin polymerization inhibitor (minegishi et al. 2015) . similarly, benzothiazole is a privileged bicyclic ring system associated with numerous pharmacological activities like antitumor (gabr et al. 2015) , anticonvulsant (amnerkar and bhusari 2010) , antimicrobial (chugunova et al. 2015; kamal et al. 2015) , antihelmintic (sarkar et al. 2013) , antileishmanial (keri et al. 2015) , antitubercular (patel et al. 2013) , anti-inflammatory (shafi et al. 2012) , antipsychotic (yevich et al. 1986 ), antioxidant (bhat and belagali 2016), antidiabetic (meltzer-mats et al. 2013; kamal et al. 2015) activities, etc. some of the important marketed drugs involving benzothiazole nucleus are riluzole, sibenadet hydrochloride (viozan), and pramipexole (scott and njardarson 2018) . zopolrestat is another significant drug containing benzothiazole core with antidiabetic effects (carvalho et al. 2006) . to the best of our knowledge as revealed by literature surveys , none of hetrocycles with indenopyrazole skeleton have been reported to exhibit antidiabetic effects. therefore, we thought of synthesizing some new benzothiazole tethered indenopyrazoles to see the additive effect of these moieties towards the preliminary examination of in vitro antidiabetic activity (doddaramappa et al. 2015) . in this perspective and in continuation of our interest in the synthesis of heterocycles containing nitrogen (zhou et al. 2017; huang and huang 2019) and sulfur as heteroatoms and their biological activities herein, we report the synthesis, characterization, α-amylase and α-glucosidase inhibition, antimicrobial evaluation and docking studies of several benzothiazole tethered indeno [1,2-c] pyrazol-4(1h)ones (4a-o). all reagents were used without any further purification. melting points were observed using electrothermal melting point apparatus, labco co., india and are not corrected. the ft-ir spectra were recorded in kbr on ir affinity-1 ftir (shimadzu) spectrophotometer, and results are reported in cm −1 . 1 h nmr and 13 c nmr spectra were recorded on bruker avance iii nmr spectrometer operating at 400 and 100 mhz, respectively, with cdcl 3 as the solvent and tetramethylsilane (tms) as the internal standard. chemical shifts (δ) are reported in parts per million (ppm), and coupling constants (j) are expressed in hertz (hz) . hrms were obtained from waters synapt g2-si qtof and sciex 5600 + qtof mass analyser by using the electrospray ionization (esi) method. the purity of synthesized compounds was checked by precoated tlc plates (sil g/uv 254 , alugram) using a mixture of hexane and ethyl acetate as eluent and visualization was achieved via uv light. general procedure for the synthesis of 2-acyl-(1h)indene-1,3(2h)-diones (1) 2-acyl-(1h)-indene-1,3(2h)-diones (1) needed for the purpose were prepared via claisen condensation of diethylphthlate and appropriate aliphatic ketones in presence of freshly prepared sodium methoxide following the procedure presented in literature (mor et al. 2016 thiazol-2-amines thus obtained was filtered through suction and washed with hexane. thereafter, the salt was dissolved in water upon warming and the product was precipitated by adding dil. naoh solution. the solid thus formed was filtered through suction and recrystallized from ethanol to afford the corresponding amines in high yields . to a solution of hydrazine hydrochloride in ethylene glycol was added the appropriate benzo[d]thiazol-2-amine/6-substitutedbenzo[d]thiazol-2-amines in portions with continuous stirring and the resulting mixture was heated to reflux on a heating mantle for 2 h. a fine crystalline solid was separated out on cooling which was filtered, washed with water and crystallized from rectified sprit to yield the corresponding 2-hydrazinylbenzo[d]thiazole/2-hydrazinyl-6-substitutedbenzo [d]thiazoles (2) in good yields ). general procedure for the synthesis of benzothiazolyl hydrazones (3) a solution of equimolar quantities of 2-acyl-(1h)-indene-1,3 (2h)-diones (1, 3 mmol) and hydrazines (2, 3 mmol) in dry methanol (15 ml) was heated on a water bath for 15 min in presence of catalytic amount of glacial acetic acid (4-5 drops). thereafter, reaction mixture was cooled at room temperature. the solid thus separated out was filtered through suction and recrystallized from ethyl acetate-ethanol to give the corresponding benzothiazolyl hydrazones (3a-o) as orange solids (sawhney and lemke 1983; . general procedure for the synthesis of indeno [1,2-c] pyrazol-4-ones (4a-o) benzothiazolyl hydrazones (3) were charged with glacial acetic acid (30 ml) and heated to reflux on a heating mantle for 7-9 h till the completion of reaction as indicated by tlc. the reaction mixture was cooled at room temperature and the solid thus obtained was filtered, and recrystallized from chloroform to furnish the target compounds 4a-o in good yields. the physical and spectral data of compounds 4a-o are as follows: 1-(benzo[d]thiazol-2-yl)-3-methylindeno [1,2-c] 122.83, 124.09, 124.29, 124.54, 125.45, 126.75, 130.69, 132.58, 133.32, 133.58, 140.10, 148.70, 150.95, 158.73, 184.34 (c-4) 121.49, 122.38, 123.97, 124.27, 124.54, 128.26, 130.64, 132.63, 133.45, 133.57, 135.73, 140.16, 143.67, 148.56, 148.94, 158.58, 184.38 (c-4) ; hrms: m/z (m + ) cacld. for c 19 h 13 n 3 os: 331.0779, found: 332.0839 [m+h] + . j = 7. 28 hz, ; 13 c nmr (100 mhz, cdcl 3 ): δ = 21.15 (-ch(ch 3 ) 2 ), 28.14 (-ch(ch 3 ) 2 ), 118.70, 123.11, 123.90, 124.23, 124.34, 130.22, 130.79, 132.63, 133.53, 134.99, 140.02, 144.69, 149.89, 159.39, 159.55, 159.83, 183.74 (c-4) 2 ), 27.79 (-ch 2 ch(ch 3 ) 2 ), 36.32 (-ch 2 ch(ch 3 ) 2 ), 121. 63, 122.79, 123.98, 124.24, 124.43, 125.39, 126.69, 130.62, 132.67, 133.34, 133.53, 140.13, 148.32, 150.93, 152.78, 158.81, 184 .14 (c-4); hrms: m/z (m + ) calcd 119.77, 120.95, 121.95, 123.88, 125.12, 127.58, 128.98, 130.64, 132.01, 139.18, 140.27, 148.66, 150.06, 151.58, 154.85, 159.83, 182.64 (c-4) 56, 115.82, 123.45, 123.83, 124.28, 124.41, 127.13, 130.61, 132.79, 133.55, 134.79, 136.96, 140.30, 145.23, 150.48, 152.66, 158.00, 184.23 (c-4) 120.97, 121.36, 121.49, 123.57, 124.37, 124.47, 127.28, 127.54, 130.78, 133.58, 140.09, 149.54, 151.28, 152.08, 153.03, 158.93, 185.16 (c-4) 123.85, 123.92, 124.24, 124.37, 124.47, 130.00, 130.46, 132.55, 133.57, 134.15, 140.09, 149.88, 150.21, 153.04, 159.80, 184.05 (c-4) in vitro α-glucosidase inhibition mccue's protocol was followed for evaluation of in vitro αglucosidase inhibitory activity, with some modifications (mccue et al. 2005) . the present activity was carried by using α-glucosidase enzyme (saccharomyces cereviciae). a solution of the enzyme was obtained by adding 20 μl αglucosidase (0.5 unit/ml) in 120 μl of 0.1 m phosphate buffer (ph 6.9). in microplate wells, the enzyme solution was mixed with 10 μl of each test samples which, in turn, were prepared by dissolving in dimethylsulphoxide (dmso) at various concentrations i.e. 12.5, 25, 50, 100 μg/ ml and incubated for 15 min at 37°c. thereafter, this was charged with 20 μl of substrate solution to 5 mm p-nitrophenyl-α-d-glucopyranoside in 0.1 m phosphate buffer (ph 6.9) and further incubated for 15 min. a solution of 0.2 m sodium carbonate (80 μl) was added to terminate the reaction, and absorbance was measured at λ = 405 nm on elisa microplate reader. the reaction system without test samples (4a-o) was used as control while the system without α-glucosidase was used as a blank, and acarbose was used as positive control. each experiment was performed in triplicate. the enzyme inhibitory rates of samples have been expressed as percentage (%) inhibition which is determined by eq. (1) as follows: the ic 50 values of compounds 4a-o were calculated. the protocol reported by xiao et al. and yoshikawa et al. with slight modifications was utilized for the evaluation of in vitro α-amylase inhibition activity (xiao et al. 2006; yoshikawa et al. 2001) . stock solutions of compounds 4a-o were prepared by dissolving the compound (5 mg) in dmso (5 ml) at room temperature. the α-amylase inhibitory activity was examined at different concentrations of each sample i.e., 12.5, 25, 50, and 100 μg/ml. the reagent solution without the test sample was used as the control and acarbose was used as standard reference. substrate solution was prepared by dissolving soluble starch (500 mg) in 0.4 m naoh (25 ml) and heated for 5 min at 100°c. after cooling in ice cold water, the ph of the solution was achieved to 7 by adding 2 m hcl, and water was added to make the volume to 100 ml. the sample (20 μl) and substrate (40 μl) solutions were mixed in a microplate well and the mixture in each case was preincubated at 37°c for 3 min. thereafter, 20 μl of α-amylase solution (50 μg/ml) was added to each well, and the microplate was incubated for 15 min. the reaction was terminated by adding 0.1 m hcl (80 μl). then 1 mm iodine solution (200 μl) was added to the reaction mixture and absorbance was measured at λ = 650 nm with elisa microplate reader. the enzyme inhibitory activity expressed as percentage (%) inhibition was calculated by eq. (2) as follows: where, abs1 = absorbance of incubated solution containing test sample, starch and amylase, abs2 = absorbance of incubated solution containing test sample and starch, abs3 = absorbance of incubated solution containing starch and amylase, and abs4 = absorbance of incubated solution containing starch. all the synthesized compounds 4a-o were screened for their antibacterial activity using agar well diffusion method (okeke et al. 2001) . the test microorganisms were enthused by inoculation in 25 ml of nutrient broth (peptone 5 g/l, sodium chloride 5 g/l, hm peptone 1.5 g/ l, yeast extract 1.5 g/l, ph = 7.4 ± 0.2). the media was solidified and the test bacterial strains were cultivated by pour plate method on nutrient agar plates. wells were bored in the seeded agar plates by using sterile cork borer of 8 mm diameter and these were loaded with a 100 μl of each synthesized compound reconstituted in dmso. all the plates were incubated at 37°c for 24 h. the diameter of inhibition zone of the test organisms was measured by using digital colony counter (lab and life instruments pvt. ltd., india) and reported in mm. sterile dmso was used as a negative control, whereas streptomycin was used as a positive control. the experiments were performed in triplicates and the mean values are reported. antifungal activity of the title compounds 4a-o was examined against a. niger by a quantitative microspectrophotometric assay (broekaert et al. 1990 ) in 96-well microplates. the growth inhibition was observed at 595 nm. initially, the fungus was grown on potato dextrose broth (pdb) (potatoes, infusion form = 200 g/l, dextrose = 20 g/ l, ph = 5.1 ± 0.2 at 25°c) at 27°c for 7 days. the spores of the fungus were collected from culture on broth plates. the sporangial suspension concentration was measured by hemocytometer and made to 1.7 × 10 5 spores/ml and the fungal spore suspension was stored at −40°c. the pdbfungal spore suspension solution was prepared by mixing pdb (25 ml) with 0.147 ml of the fungal spore suspension solution. experiment was performed with 100 μl of each of the compounds 4a-o to be assayed and 100 μl of pdbfungal spore suspension solution. dmso was used as a negative control and fluconazole was used as a positive control. after incubation at 27°c for 48 h, growth was observed by measuring absorbance at 595 nm on elisa plate reader. growth inhibition was reported by using eq. (3) given as follows: where a control is the absorbance of the control and a sample is the absorbance of the tested microculture. molecular docking analysis was performed using autodock vina in autodock tools (trott and olson 2010) . for αglucosidase (pdb id: 5nn5), the coordinates of center of grid box were center_x = −16.224, center_y = −35.668 and center_z = 95.439, the size of grid box was x_size = 24 å, y_size = 24 å and z_size = 24 å, and the exhaustiveness was equal to 40. for α-amylase (pdb id: 2qv4), the coordinates of center of grid box were cen-ter_x = 20.074, center_y = 46.866 and center_z = 29.866, the size of grid box was x_size = 28 å, y_size = 28 å and z_size = 28 å, and the exhaustiveness was equal to 40. structures of the newly synthesized compounds 4a-o were confirmed by their ft-ir, nmr ( 1 h and 13 c), and mass spectra. their ft-ir spectra exhibited strong absorption bands at 1585-1608 (c=n) and 1693-1716 (c=o) cm −1 . the main characteristic feature of 1 h nmr spectra of derivatives 4a-o is the resonance of a signal appeared as a doublet integrating for one proton in the range of δ 8.03-8.56 ppm, (j = 7.28-8.00 hz), which was safely assigned to 8-h. downfield shifting of this proton is probably due to anisotropic-diamagnetic effect of lone pair of electrons present on nitrogen/sulfur of benzothiazole moiety, which finds support from the results reported earlier (mor et al. 2016) . the significant feature of 13 c nmr spectra of compounds 4a-o demonstrated the downfield shifting of signal due to c-4 (carbonyl carbon) which was observed in the region at δ 182.64-185.16 ppm. however, the remaining protons in 1 h nmr and carbons in 13 c nmr spectra displayed signals in the expected regions. further, the hrms analysis results were found in consistent with their molecular formulae (vide experimental). in search of new antidiabetic agents, we recognized primarily various pyrazole derivatives as reported in the literature (wright et al. 1964) . to the best of our knowledge, this is the first report of antidiabetic activity possessed by synthesized indeno [1,2-c] pyrazol-4-ones (4a-o). all synthesized compounds 4a-o were assessed for their αglucosidase inhibitory activity against α-glucosidase enzyme (saccharomyces cerevisiae) at various concentrations ranging from 12.5 to 100 μg/ml following the developed earlier procedure (mccue et al. 2005 ) by using acarbose as the standard (table 1) . it is inferred from the data presented in table 1 that all the derivatives exhibited moderate to excellent % inhibition against α-glucosidase enzyme as compared with the standard. compound 4i was found to be more potent analogue of this series with 67. 02, 86.27, 90.47, and 94 .52% inhibition when explored at the concentrations of 12.5, 25, 50, and 100 μg/ml, respectively. similarly, compound 4e exhibited a rise in % inhibition from 76.41 to 82.76% on increasing the concentration from 12.5 to 50 μg/ml in comparison to the standard drug acarbose. compound 4l displayed 90.78% inhibition followed by 4a that exhibited 87.26% inhibition at concentration of 100 μg/ml. among the synthesized indeno [1,2-c] pyrazol-4-ones, 4e and 4i were found to be more active with ic 50 values 6.71 and 8.18 μg/ml, respectively (acarbose ic 50 = 9.35 μg/ml). however, the derivatives 4a and 4b exhibited good inhibitory activity with ic 50 values 9.87 and 10.59 μg/ ml, respectively. scheme 1 synthesis of indeno [1,2-c]pyrazol-4-ones (4a-o) in vitro α-amylase inhibitory activity the α-amylase inhibitory activity of the synthesized compounds 4a-o was screened following xiao's procedure (xiao et al. 2006; yoshikawa et al. 2001 ) by using acarbose as the standard reference ( table 2) . the results of the α-amylase inhibitory activity depicted in table 2 revealed that all the tested derivatives 4a-o displayed moderate to high % inhibition. compounds 4a, 4e, 4i, 4l, and 4o at concentration 12.5 μg/ml, 4e, 4f, 4i, 4l, and 4o at concentration 25 μg/ml, and 4a and 4o at concentration 50 μg/ml were found to be more potent than the standard. whereas compound 4i displayed inhibition equivalent to the standard drug acarbose at concentration 100 μg/ml. the analogues 4c and 4j at concentration 12.5 μg/ml, 4a and 4d at concentration 25 μg/ml, 4b, 4d, 4e, and 4i at concentration 50 μg/ml, and 4a and 4c at concentration table 1 in vitro α-glucosidase inhibitory activities of indeno [1,2-c] 100 μg/ml demonstrated comparable inhibitory activity as shown by the standard drug acarbose. furthermore, the remaining compounds were found to display lesser inhibitory activity as compared with the standard drug screened at different concentrations. among the synthesized derivatives, 4a (ic 50 = 21.23 μg/ml), 4e (ic 50 = 19.25 μg/ml), 4i (ic 50 = 11.90 μg/ ml), and 4o (ic 50 = 12.83 μg/ml) demonstrated higher activity than the standard (acarbose, ic 50 = 22.87 μg/ml). consequently, 4a, 4e, 4i, and 4o can be considered as a possible antidiabetic agent for further studies. structure activity relationship (sar) for antidiabetic activity of indeno [1,2-c] pyrazol-4-ones (4a-o) according to the presented data for antidiabetic activity of indeno [1,2-c] pyrazol-4-ones (4a-o), the following sars have been established: (1) results of antidiabetic activity indicated that the presence of r 1 = ch 3 and r 2 = h, br in the synthesized compounds 4a-o has led to increase the antidiabetic activity against α-glucosidase enzyme while the derivative 4e containing r 1 = ch 3 and r 2 = br has been found to exhibit improved inhibitory activity against α-amylase enzyme. (2) compound 4i containing r 1 = i-propyl and r 2 = cl has been found to enhance inhibitory activity against both α-glucosidase and α-amylase enzymes. (3) derivative 4l containing r 1 = i-butyl and r 2 = ch 3 has improved inhibitory activity against α-glucosidase, whereas presence of r 1 = i-butyl and r 2 = br in compound 4o had increased inhibitory activity against α-amylase enzyme. from these results, it is inferred that the compounds containing r 1 = ch 3 and r 2 = h, ch 3 , och 3 , cl, and br, are found to display more inhibitory activity against αglucosidase as compared with the remaining derivatives containing r 1 = i-propyl and i-butyl, and r 2 = h, ch 3 , och 3 , cl, and br, whereas no such trend was observed against α-amylase inhibitory activity. overall, we may conclude that there are different structural requirements for a compound to be effective against α-glucosidase and αamylase enzymes. however, no general trend for sar was established for both α-glucosidase and α-amylase enzymes. the above mentioned findings are summarized in fig. 1 . all synthesized indeno [1,2-c] pyrazol-4-ones (4a-o) were tested for their in vitro antibacterial activity against grampositive (b. subtilis, s. aureus) and gram-negative (e. coli, p. aeruginosa) bacteria by agar well diffusion method using streptomycin as the reference drug (table 3 ) (okeke et al. 2001) . it is revealed from the data presented in table 3 that compound 4l and 4g exhibited the highest activity against b. subtilis, while the derivative 4h demonstrated maximum inhibition zone against s. aureus. moreover, the analogues 4f and 4j displayed moderate activity against e. coli, while 4g demonstrated activity against p. aeruginosa. on the other hand, some compounds were inactive against the specific bacteria under study. overall, these results suggest that the synthesized compounds 4a-o exhibit lesser activity against the bacterial strains than standard drug streptomycin. all synthesized compounds 4a-o were evaluated for their in vitro antifungal activity against a. niger by the quantitative microspectrophotometric assay using fluconazole as the standard drug (broekaert et al. 1990) (table 4) . a perusal of accumulated data from table 4 reveals that all synthesized compounds 4a-o were found to inhibit fungal growth with inhibition ranging from 49.21 to 72. 25%, 54.71 to 76.96%, 59.42 to 80.89%, and 67.01 to 84.55% at concentration 125, 250, 500 , and 1000 μg/ml, respectively. derivative 4o was found to be more active with ic 50 value of 5.68 μg/ml than the standard (ic 50 = fig. 1 structure activity relationship (sar) for antidiabetic activity of synthesized indeno [1,2-c] pyrazol-4-ones (4a-o) table 3 in vitro antibacterial activity of indeno [1,2-c] 11.75 ± 1.50 sars for antimicrobial activity of indeno [1,2-c] pyrazol-4ones (4a-o) the sars approach to the synthesized derivatives 4a-o demonstrated good to moderate inhibitory potential against all the microbial strains under study. consequently, we may conclude that there are different structural requirements for a compound to be effective against different bacterial and fungal strains. moreover, no general trend has been established for the sars for antimicrobial activity. molecular docking analysis of some selected synthesized compounds with enzymes α-glucosidase and α-amylase was performed to find out the mechanism of action at the molecular level. crystal structure used for α-glucosidase was obtained from protein data bank and the pdb id for this structure is 5nn5 (roig-zamboni et al. 2017) . docking protocol was validated by docking the co-crystallized ligand. in vitro assay showed that compounds 4e and 4i exhibit best α-glucosidase inhibitory potential, hence molecular docking analysis was performed using these two compounds. compound 4e with a docking score of −7.3 showed three pi-pi interactions with amino acid residue phe-525, trp-376 (t shaped), and trp-481 (t shaped) as shown in fig. 2 and hydrophobic interactions with trp-481 and phe-649. compound 4i with docking score of −8.4 showed interactions very similar to that of 4e i.e., three pi-pi interactions with amino acid residue phe-525, trp-376 (t shaped), and trp-481 (t shaped) as shown in fig. 3 and further stabilizing interactions are provided by hydrophobic contacts with asp-282, ala-555, and phe-649. both the compounds were found to bind in similar orientation with a snug fit. in vitro results showed that most of the synthesized compounds 4a-o were stronger inhibitors of α-amylase compared with acarbose. compounds 4i and 4o showed superior inhibition as compared with the other synthesized compounds. therefore, 4i and 4o compounds were used for determining the binding pose and interactions responsible for the activity against human pancreatic α-amylase (pdb id: 2qv4) (maurus et al. 2008) . first docking protocol was validated by performing docking of co-crystallized ligand. compound 4o with docking score of −8.8 was found to be showing two hydrogen bond interactions with amino acid residues gln-63 and thr-163, three pi-pi interactions with trp-59 and hydrophobic contacts with tyr-62, leu-162, and leu-165. the binding pose of compound 4o is shown in fig. 4 . compound 4i with docking score of −9.3 binds in active site of α-amylase with orientation and interactions similar to that of 4o. it is showing one hydrogen bond with gln-63, one halogen bond with asp-197, three pi-pi interactions with trp-59 and hydrophobic contacts with trp-58 and tyr-62 as shown in fig. 5 . in conclusion, the present study describe the synthesis and characterization of heterocyclic frameworks i.e., indeno [1,2-c] pyrazoles (4), and their biological evaluation as inhibitor of α-glucosidase and α-amylase related to type ii diabetes, and antimicrobial activity. the chemistry for the synthesis of indeno [1,2-c] pyrazole (4) involved the reaction of 2-acyl-(1h)-indene-1,3(2h)-diones (1) (3), which upon subsequent refluxing in glacial acetic acid afforded the target compounds 4 in good yields. some of the synthesized compounds exhibited significant in vitro αglucosidase and α-amylase inhibitory activity viz. 4e was found to be more potent with ic 50 value 6.71 μg/ml against α-glucosidase enzyme and 4i showed good activity with ic 50 value 11.90 μg/ml against α-amylase enzyme as compared with reference drug acarbose (ic 50 = 22.87 μg/ml). moreover, some of the compounds exhibited convincing results for antimicrobial activity, however, with a degree of variation. the antidiabetic activity was found to be more prolific than antimicrobial activity. in vitro α-glucosidase and αamylase inhibition was further supported by docking studies of compounds 4e, 4i, and 4o. hopefully, these findings will prove helpful to medicinal chemists for the development of new inhibitors of enzymes related to type ii diabetes. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. c=n stretch), 1716 (c=o stretch), 2918, 2962 (aliphatic c-h stretch), 3086 (aromatic c-h stretch) cm −1 ; 1 h nmr (400 mhz c=n stretch), 1712 (c=o stretch), 2920, 2962 (aliphatic c-h stretch), 3089 (aromatic c-h stretch) cm −1 ; 1 h nmr (400 mhz 3089 (aromatic c-h stretch) cm −1 ; 1 h nmr (400 mhz -isopropylindeno[1,2-c]pyrazol-4 (1h)-one (4f): yellow solid 3057 (aromatic c-h stretch) cm −1 ; 1 h nmr (400 mhz 44 hz, 8-h); 13 c nmr (100 mhz -methylbenzo[d]thiazol-2-yl)indeno[1,2-c] pyrazol-4(1h)-one (4g): yellow solid; yield 67% cdcl 3 ): δ = 1.42 (6h, d, j = 6.92 hz, -ch(ch 3 ) 2 , 2.51 (3h, s, ch 3 ), 3.08-3.14 (1h, m, -ch(ch 3 ) 2 ) yellow solid; yield 65%; mp 212-214°c; ftir (kbr): ν max 759 (1h)-one (4i): yellow solid; yield 70%; mp 182-184°c; ftir (kbr): ν max 763, 1109, 1381, 1500, 1544, 1597 (c=n stretch), 1703 (c=o stretch), 2870, 2964 (aliphatic c-h stretch), 3086 (aromatic c-h stretch) cm −1 ; 1 h nmr (400 mhz ar-h), 8.46 (1h, d, j = 7.40 hz, 8-h) hrms: m/z (m + ) calcd -isopropylindeno[1,2-c] pyrazol-4(1h)-one (4j): yellow solid; yield 65%; mp 190-194°c; ftir (kbr): ν max 738 3062 (aromatic c-h 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evaluation and qsar studies of some 3-aryl-1-heteroarylindeno [1,2-c]pyrazol-4 (1h)-ones synthesis of indane-based 1,5-benzothiazepines derived from 3-phenyl-2,3-dihydro-1h-inden-1-one and antimicrobial studies thereof synthesis, biological evaluation, and molecular docking studies of some n-thiazolyl hydrazones and indenopyrazolones synthesis, type ii diabetes inhibitory activity, and antimicrobial tests of benzothiazole derivatives bridged with indenedione by methylenehydrazone evaluation of extracts of the root of landolphia owerrience for antibacterial activity synthesis of coumarin-based 1,3,4-oxadiazol-2ylthio-n-phenyl/benzothiazolyl acetamides as antimicrobial and antituberculosis agents synthesis, crystal structure and antidiabetic activity of substituted (e)-3-(benzo[d]thiazol-2-ylamino) phenylprop-2-en-1-one structure of human lysosomal acid α-glucosidase-a guide for the treatment of pompe disease synthesis of 1-[2(substituted phenyl)-4-oxothiazolidin-3-yl]-3-(6-fluro-7-chloro-1,3-benzothiazol-2-yl)-ureas as anthelmintic agent analysis of us fda-approved drugs containing sulfur atoms chemistry of β-triketones. 1. structure of schiff base intermediates of 2-acyl-1,3-indandiones synthesis of novel 2-mercaptobenzothiazole and 1,2,3-triazole based bis-heterocycles: their anti-inflammatory and anti-nociceptive activities design, synthesis, and antimicrobial evaluation of 1,4-dihydroindeno[1,2-c]pyrazole tethered carbohydrazide hybrids: exploring their in silico admet, ergosterol inhibition and ros inducing potential 3d-qsar comfa study on indenopyrazole derivatives as cyclin dependent kinase 4 (cdk4) and cyclin dependent kinase 2 (cdk2) inhibitors autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading synthesis, binding mode, and antihyperglycemic activity of potent and selective (5-imidazol-2-yl-4-phenylpyrimidin-2-yl)[2-(2-pyridylamino)ethyl] amine inhibitors of glycogen synthase kinase 3 the antidiabetic activity of 3,5-dimethylpyrazoles a quantitative starch-iodine method for measuring alpha-amylase and glucoamylase activities synthesis and biological evaluation of 1-(1,2-benzisothiazol-3-yl) and (1,2-benzisoxazol-3-yl) piperazine derivatives as potential antipsychotic agents polyphenol constituents from salacia species: quantitative analysis of mangiferin with α-glucosidase and aldose reductase inhibitory activities design, synthesis and biological activity of pyrazinamide derivatives for anti-mycobacterium tuberculosis conflict of interest the authors declare that they have no conflict of interest. key: cord-328834-yetnlb2j authors: mohsin, noor ul amin; irfan, muhammad; hassan, shams ul; saleem, usman title: current strategies in development of new chromone derivatives with diversified pharmacological activities: a review date: 2020-06-15 journal: pharm chem j doi: 10.1007/s11094-020-02187-x sha: doc_id: 328834 cord_uid: yetnlb2j chromone derivatives possess a spectrum of biological activities. chromone has been recognized as a privileged structure for new drug invention and development. substitution pattern of chromone scaffold determines different type of biological activities. the type, number and position of substituents connected to the chromone core play a vital role in determining pharmacological activities. in the present review, we have discussed new chromone derivatives as anticancer, anti-diabetic, antimicrobial, anti-inflammatory, antioxidant and as anti-alzheimer agents. this review deals with the chromone derivatives prepared by combining chromone molecule with various natural and synthetic pharmacophores and pharmacological activities presented by them. the main aim is to highlight the diversified pharmacological activities exhibited by chromone hybrid molecules during the last eight to ten years. chromone is a heterocyclic compound containing oxygen as heteroatom and has a benzo-g-pyrone skeleton (fig. 1, compound 1 ). chromone is a natural molecule present in the diet of human and animals and shows less toxicity to mammalian cells [1] . chromone containing natural and synthetic molecules displayed interesting biological activities [2] . medicinal properties exhibited by chromone derivatives are antibacterial, antifungal, antioxidant, antimalarial, neuroprotective and hiv inhibitory potential [3 -8] . chromone derivatives have also shown promising anticancer and antiviral potential [9 -13] . anti-inflammatory, antiallergenic and antiulcer are other properties displayed by chromone derived molecules [14 -16] . chromone is treated as an attractive source for the synthesis of new drugs due to its valuable activities and low toxicity [17] . chromone is considered as a single molecule which can combine with different types of receptors [18] . modifications of chromone scaffold have been performed at benzene or pyrone ring by attachment of different substituents. 3-formylchromone (fig. 1, compound 2) is a frequently used precursor for the synthesis of chromone derivatives and can be prepared easily by the vilsmeir -haack reaction [19] . chromone derived molecules have been discussed with respect to chemical structures, pharmacological activities and structure -activity relationship (sar) in following sections. cancer is a serious problem all around the world due to high mortality and there is demand to discover new leads as anticancer agents [20] . a number of anticancer drugs have been discovered from natural sources [21] . chromone derived molecules have displayed excellent anticancer activities. amin and co-workers synthesized new molecules by merging benzofuran and 5h-furo [3,2g] chrome-5-one and attached different heterocyclic rings through sulfonamide group. these compounds displayed in vitro activity against breast cancer cell line (mcf-7) ranging from 0.004 -0.87 mm. compound 3 (fig. 2) presented prominent in vitro activity (ic 50 = 0.056 ± 0.0027 mm) against mcf-7 cell line in comparison to standard drug (doxorubicin, ic 50 = 0.62 ± 0.0316 mm) and proved less toxicity to normal cell line (ic 50 = 23 ± 1.02 mm). these derivatives also showed p38a mapk (mitogen-activated protein kinase) inhibition activity. mapk controls many biological functions such as cell growth, differentiation and inflammation [22] . molecular docking studies showed that compound 3 formed four hydrogen bonds with k-53, m-109 and g-170 amino acids of mapk [23] . singh, et al. attached indole, pyrimidine, pyrazole with chromone to produce new derivatives. it was observed that introduction of 2,6-dichlorophenyl, 2,6-dichlorobenzoyl group along with indolinone produce notable activities. compound 4 manifested prominent in vitro antitumor activity with 50 -90% growth inhibition of all tumor cell lines and showed an average gi 50 value of 3.2 mm. compound 4 was more potent against leukemia (rpmi-8226 gi 50 [24] . synthesis of chromone and sulfonamide comprising molecules was carried out by awadallah, et al. in which two molecules were linked to each other by a large heterocyclic ring or by small linker groups such as methine amine or alkyl amine. compound separated by small linker group dispensed greater activity. upon in vi-tro evaluation, compound 5 emerged as the most active against breast (mcf-7, ic 50 = 0.72 mm) and lung (a-549, ic 50 = 0.50 mm) cancer cell lines as compared to doxorubicin (mcf-7 ic 50 = 33.13 ± 2.90 mm, a-549 ic 50 = 26.81 ± 2.50 mm). compound 5 presented selectivity for isoforms ix and xii of the human carbonic anhydrase (hca). this compound induced apoptosis in both types of cancer cell. it was also observed that compounds having free sulfonamide group presented higher activity. when the sulfonamide group was attached with heterocyclic scaffold such as pyridine, pyrimidine, and isoxazole, less active derivatives were obtained [25] . chen, et al. attached chromone molecule to 1-alkyl-1h-imidazole-2-yl via dienone as linker group. the nitrogen-containing heterocycles were used as bioisostere for phenols in the natural compound while the dienone linker was used as substitute for dienone in curcumin. compound 6 presented excellent activity against prostate cancer (pc-3, ic 50 = 1.8 ± 0.3 mm and lncap ic 50 = 1.0 ± 0.2 mm) cell lines. the nitrogen atom of imidazole carries ethyl group. replacement of ethyl group by longer chain has no significant influence on anticancer activity. therefore they are excellent molecules for future investigations [26] . dolatkhah, et al. used the three-component reaction involving chromone-3-carboxaldehyde, alkyl acetoacetate, urea or thiourea to produce 4h-chromone-1,2,3,4tetrahydropyrimdine-5-carboxylates using mcm-41-so 3 h nanoparticles as catalyst. the catalyst can be recycled and reused. compound 7 presented prominent activity against leukemia cell line upon evaluation by microculture tetrazolium test (mtt) assay. this compound showed no toxicity to normal cell line human foreskin fibroblast (hu02). compound 5 showed high affinity (binding energy = -10.10 kcal/mol) with ab1-kinase enzyme by autodock-4 program [27] . nam, et al. developed chromone derived analogues of lavendustin. upon anticancer evaluation, compounds 8 (ic 50 = 6.01 ± 2.7 mm) and 9 (ic 50 = 9.92 ± 3.6 mm) showed prominent activities against a-549 cell line. compound 8 (ic 50 = 6.89 ± 2.6 mm) and 9 (ic 50 = 7.86 ± 2.2 mm) also showed activity against hct-15 cell lines. in compound 8, replacement of 4-methoxybenzyl with benzyl or phenethyl decreased the activity. in compound 9, replacement of 4-nitrobenzyl with 4-methoxybenzyl or benzyl group produced less active compounds against hct-15 cell line [28] . bhatia and co-workers synthesized chalcone-chromenone derived molecules. upon in vitro evaluation, compound 10 ( fig. 3) showed prominent activity (87% growth inhibition) against colon cancer cell line (hct-116) as compared to fluorouracil (67% inhibition). compound 10 carries two halogen atoms each on the chromone core and chalcone fragment [29] . ozen, et al. conjugated chromone molecule with 5-membered heterocyclic scaffolds thiazolidindiones, imidazolidindiones and thiohydantoins to produce new hybrid molecules. compound 11 showed prominent activity against liver (huh-7 ic 50 = 5.2 mm) and breast cancer (mcf-7, ic 50 = 4.9 mm) cell lines. this compound contains unsubstituted chromone while ethyl group is attached with thiohydantoin. when the chromone ring is substituted with methyl group, resultant compound showed less activity. compound 11 did not show apoptosis but only reduced the replication of cells [30] . singh and co-workers synthesized chromenopyridine derivatives based on the activity of chromones and pyridones. derivatives bearing electron withdrawing groups at positions # 5, # 6 and # 7 of chromone scaffold showed better activity. compound 12 appeared as the most potent against pc-3 (ic 50 = 2.4 ± 3.4 mm), mcf-7 (ic 50 = 10.7 ± 2.5 mm) and hela (ic 50 = 7.0 ± 3.5 mm) cancer cell lines. this compound contains allyl group attached to pyridone and showed better activity as compared to unsubstituted compounds [31] . obreque-balbua and coworkers synthesized chromone derivatives as abcc1 modulators. an amino or carboxamide group was introduced at position # 2. abcc1 overexpression is detected in different types of cancers. compound 13 was the most effective (ic 50 = 11.3 ± 1.8 mm) in reducing the abcc1 mediated resistance in comparison to reversan (ic 50 = 4.3 ± 0.2 mm). derivative 13 showed selectivity for abcc1 over abcg2 and abcb1. it consists of chromone molecule connected to benzo [1, 2, 5] oxadiazole via piperazine linker group. insertion of carbonyl group between chromone and piperazine decreased the activity [32] . abdelhafez at al. synthesized benzofuran derivatives as vascular endothelial growth factor (vegf) inhibitor. bromovisnagin (14) was an intermediate product and consist of chromone core in its structure. bromovisnagin (14) showed prominent anticancer (ic 50 = 3.67´10 -7 -7.65´10 -13 mm) potential as compared to other products in this series. compound 14 was the most potent agent against mcf-7 (ic 50 = 3.67´10 -7 ) as compared to standard drug epirubicin (ic 50 = 2.22´10 -9 ). docking studies of compound 14 with vegfr2 (vascular endothelial growth factor receptor) kinase enzyme showed hydrogen bonding between furan oxygen and amino group of d1046. methoxy group showed interaction with nh moiety of k868 [33] . chand and colleagues synthesized substituted chromone, 4-oxo-4h-1-benzopyran derivatives as anticancer agents. compounds having acrylate group at position # 3 demonstrated prominent activity. derivatives having amide and acid group at this position proved less active. compound 15 was the most active (50 -60% inhibition) agent against colon (ht-29), breast (sk-ov-3) and ovarian (mda-468) cell lines as compared to doxorubicin (80% inhibition). compound 15 did not show in vitro src kinase inhibitory activity (ic 50 < 300 mm) as compared to staurosporin (ic 50 = 0.6 mm) [34] . han and colleagues synthesized succinimide derivatives by c-h functionalization of chromone, naphthoquinone and xanthone scaffolds with maleimide scaffold. chromone derivatives (16a, b, c) showed only minute activity (ic 50 < 50 mm) versus mcf cell line [35] . el-garah, et al. synthesized chromone carboxamide derivatives. compound 17 presented prominent activity (ic 50 = 0.9 mm) against mcf-7 cancer cell line in comparison to tamoxifen (ic 50 = 0.39 ± 0.01 mm). structure activity relationship (sar) studies highlighted that attachment of fluorine atom at position # 6 of chromone core produced more active derivatives. replacement of fluorine atom by chloro and methyl groups produced less active derivatives. the presence of propyl side chain at the amide group also increased the activity. compound 18 also presented in-vitro anti-inflammatory activity (79.9 ± 6.6% inhibition) as lipoxygenase (lox) inhibitor. derivatives having hydrophilic group showed better anti-inflammatory activity [36] . ali, et al. synthesized chromone annulated phosphorous heterocycles as anticancer agents. phosphorous reagents used in the synthesis were phosphorous halides, phosphorous sulfides and phosphonic acid. anticancer activity was evaluated by using crystal violet blue assay. compounds 19 and 20 exhibited prominent activities against hepg-2 (ic 50 = 1.61 mg/ml, ic 50 = 2.49 mg/ml) and hct-116 (ic 50 = 1.72 mg/ml, ic 50 = 1.56 mg/ml) as compared to doxorubicin (hep-g-2 ic 50 = 0.467 mg/ml, hct-116 ic 50 = 0.468 mg/ml). the thiophosphoryl (p=s) bond was described as major cause of cytotoxicity of these compounds as compared to phosphoryl group (p=o) [37] . sun, et al. carried out synthesis of thiopyrano [4,3-d] pyrimidne derivatives having chromone molecules. compound 21 displayed significant inhibition of mtor (ic 50 = 1.10 ± 0.10 mm) and p13ka (ic 50 = 0.92 ± 0.12 mm) kinases. compound 21 (fig. 4 ) displayed marvelous in vitro activity against mcf-7 (ic 50 = 11.8 ± 6.9 mm), hela (ic 50 = 10.3 ± 0.58 mm) and hepg2 (ic 50 = 8.77 ± 0.83 mm) cancer cell lines as evaluated by mtt assay. docking investigation with mtor and p13ka kinases highlighted that morpholine, chromone and hydrazinyl groups are essential for antitumor activities. substitution on the chromone nucleus manifested significant influence on activity. introduction of carboxylic group on the chromone core proved more active as compared to methyl, ethyl and hydroxyl group [38] . abu-aisheh, et al. synthesized amidrazone derivatives having flavone scaffold. upon evaluation as anticancer agents by mtt assay, compounds 22 (ic 50 = 1.42 ± 0.13 mm) and 23 (ic 50 = 2.92 ± 0.94 mm) showed prominent in vitro activity versus breast cancer cell line (t47d) as compared to doxorubicin (ic 50 = 0.33 ± 0.05 mm). replacement of piperazine ring by piperidine produced less active agents against breast cancer. derivatives having phenyl group at position # 2 presented higher activity as compared to methyl group. these compounds can be good candidates as anticancer agents [39] . singh, et al. synthesized chromone and isoxazolidine based compounds by regioselective and stereoselective 1,3-dipolar cycloaddition reaction. compound 24 (ic 50 = 0.7 mm) exhibited prominent in vitro activity against a549 cell line in comparison to paclitaxel (ic 50 = 0.4 mm). this compound bears disubstituted isoxazolidine scaffold. this compound also induced apoptosis in hl-60 cancer cell line [40] . satyajit singh and co-workers synthesized chromano piperidine fused isoxazolidine molecules. derivatives having electron withdrawing groups at the chromone core presented superior activity. compounds having aromatic ring attached with isoxazolidine presented better activity. compound 25 showed prominent in vitro cytotoxicity against colo (ic 50 = 12.6 mm) as compared to fluorouracil (ic 50 = 21 mm). compound 25 also exhibited moderate activity against imr-32 (ic 50 = 55.2 mm) as compared to adriamycin (ic 50 = 1.7 mm). compound 26 (ic 50 = 10.7 mm) presented prominent activity against neuroblastoma (imr-32) cell line [41] . zwergel and co-workers synthesized chromone and coumarin based benzofuran derivatives. biological evaluation on human leukemia (k562) cell line was carried out. compounds 27 and 28 exhibited prominent activities having 72% and 63% cell survival respectively by using 50 mm concentrations. compound 27 showed 24% apoptosis. substitution on chromone nucleus does not have significant influence on activity. replacement of benzofuran by naphthofuranone produced more active compound towards apoptosis [42] . zhu, et al. designed and synthesized thienopyrimidine and chromone derived compounds as mtor/p13ka inhibitors [43] . compound 29 (97.2 ± 0.4% inhibition) presented prominent activity against mtor/p13ka kinase. derivative 29 also manifested notable in vitro activity versus h460 (ic 50 = 1.2 ± 0.3 mm) and pc-3 (ic 50 = 0.85 ± 0.04 mm) cell lines in comparison to positive control (ic 50 = 9.52 ± 0.29 mm, ic 50 = 16.27 ± 0.54 mm against h-460 and pc-3). chromone molecule having carboxylic acid or nitro group was found to be optimum for the activity in this series. docking studies revealed that chromone molecule is vital for activity. carboxylic group of chromone interacted via hydrogen bonding with trp-2239 and arg-2348. morpholine group formed hydrogen bonding with val-2240 [44] . diabetes mellitus is caused by the elevated level of glucose in the blood due to improper production of insulin. it is a very common problem all around the world [45] . currently available anti-diabetic drugs are linked with various side effects [46] . anti-diabetic properties of chromone derivatives are described in this section. ceylan-unlusoy, et al. attached chromone scaffold with 2,4-imidazolidindione and 2,4-thiazolidindione. attachment of heterocyclic rings at position # 3 produced more active derivatives as compared to position # 2. compounds 30 (142.7 ± 17.60%) and 31 (155.4 ± 35.16%) (fig. 5 ) showed prominent in vitro insulinotropic potential with dose of 1 mg/ml as compared to standard drug glibenclamide (138 ± 13.99%). incorporation of methyl and ethyl group in the heterocyclic ring showed comparable activity to unsubstituted derivatives [47] . later on, ceylan-unlusoy, et al. synthesized chromone derivatives having imidazolidindione, 2,4-thiazolidindione and 2-thioxoimidazolidine-4-one heterocyclic cores. derivatives having substituted 2,4-thiazolidinione ring demonstrated better activity and the most active agent carries methyl group at heterocyclic nitrogen. therefore lipophilic groups at the heterocyclic nitrogen were found significant for increasing the activity. compound 32 (dose = 0.001 mg/ml) was the most prominent which increased the release of insulin (120.6 ± 13.53%) as compared to glibenclamide (145.7 ± 7.74%) [48] . wang, et al. linked chromone to the benzene sulfonamide through hydrazone linkage to produce new a-glucosidase inhibitors. the a-glucosidase enzyme is involved in the hydrolysis of carbohydrates and releases glucose. compound 33 showed excellent in vitro inhibition (ic 50 = 20.1 ± 0.19 mm) of this enzyme in comparison to acarbose (ic 50 = 817.38 ± 6.27 mm). compound 33 carries a free sulfonamide group attached to the phenylhydrazone. replacement of phenylhydrazone by benzohydrazide and benzene ring by thiophine ring produced less active molecules. compound 33 displayed non-competitive inhibition as determined by lineweaver-burk plot. in molecular docking studies, hydrogen bonding was observed between the drug molecule and asp-214 and arg-439 of enzyme [49] . one year later, wang, et al. synthesized hybrid molecules comprising chromone-isatin as a-glucosidase inhibitors. these molecules exhibited excellent in vitro blockage of this enzyme and compound 34 (ic 50 = 3.18 ± 0.12 mm) appeared as the most potent in this series. substitution on the chromone core was very important where introduction of hydroxy group significantly enhances the activity. molecular docking studies also showed interaction (binding energy = -8.8 kcal/mole) of this compound with the glucosidase enzyme. 4-bromophenyl moiety showed hydrophobic interactions with phe-157 and phe-177, ala-278 and phe-300. benzopyrone formed ð-ð interaction with phe-300. hydroxyl group formed hydrogen bonding with gln-350 [50] . alpha-amylase is an enzyme involved in the hydrolysis of carbohydrates. salar, et al. connected chromone to hydrazinyl thiazole to produce the new hybrid molecules. upon in vitro evaluation as the a-amylase inhibitors, compound 35 showed prominent inhibition (ic 50 = 2.826 ± 0.06 mm) of this enzyme in comparison to acarbose (ic 50 = 1.9 ± 0.07 mm). compound 35 showed a docking score of -7.1717 and p-p interactions with trp-59 of a-amylase. compound 35 also exhibited dpph (2,2-diphenyl-1-picrylhydrazyl) (ic 50 = 1.13 ± 0.15 mm) and abts (2,2 / -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ic 50 = 1.083 ± 0.15 mm) radical scavenging activities. compound 35 bears unsubstituted phenyl ring with thiazole. replacement of phenyl group with biphenyl core also produced more active compounds. incorporation of hydroxyl group at the aromatic ring decreased the activity. dichloro-substituted derivatives at the aromatic ring produced more active compounds as compared to mono-substituted compounds. attachment of methyl group with chromone core also produced less active derivatives [51] . parthiban, et al. synthesized quercetin based chromone derivatives as alpha-amylase inhibitors. various modifications in the chromone core of quercitin were carried out such as introduction of furan and indole ring. compounds 36 (ic 50 = 13 ± 0.16 mm) and 37 (ic 50 = 12 ± 0.14 mm) presented prominent in vitro activities as alpha amylase inhibitor as compared to quercitin (ic 50 = 22 ± 0.01 mm). in these derivatives chromone core is attached with indole ring. derivatives having aliphatic side chains at the indole nitrogen presented higher activity as compared to aromatic rings. molecular docking studies showed that chromone core forms hydrophobic interaction with trp58, tyr62 and leu165. diethylamino group of compound 37 interacted via hydrogen bonding as well as hydrophobic interaction with glu233 and ile235. these derivatives have the potential for further investigations as anti-diabetic agents [52] . parasites such as bacteria, viruses, fungi, protozoa, trypanosomes, etc. cause various diseases in human beings and are responsible for high mortality and morbidity [53] . ate activity (10 -12 mg/ml) against sensitive h37rv as compared to ethambutol (2 mg/ml) and streptomycin (2 mg/ml). it was assumed that these compounds act on the lipoprotein part of the cell wall of mycobacterium tuberculosis [62] . cano, et al. synthesized fluorine-containing chromone and tetrazole hybrid molecules by ugi-azide reaction [63] these derivatives displayed moderate antimicrobial activity as is evident by compound 51 (mic = 20 mg/ml) activity versus pseudomonas aeruginosa (p. aeruginosa) as compared to cefotaxime (mic = 0.5 mg/ml). compound 52 also displayed low antiamoebic activity (ic 50 = 61.7 mg/ml) as compared to metronidazole (ic 50 = 1.5 mg/ml). compound 53 exhibited antifungal activity against sporothrix schenckii (s. schenckii) at the concentration of 6.25 mg/ml. introduction of halogen atoms at the chromone core produced more active derivatives as compared to nonhalogenated derivatives [64] . reddy, et al. produced the fused hybrid molecules by reacting 2-amino chromone, benzaldehyde, 1,4-dimedone to produce chromeno-tetrahydroquinoline. antibacterial activity was tested against p. aeruginosa, s. aureus, e. coli and bacillus subtilis (b. subtilis). in these derivatives, tetrahydropyridine is embed-ded in dimedone and chromone scaffolds. agar well diffusion method was used for assessing antibacterial activity. compounds 54, 55 and 56 were most active derivatives (zoi = 22 -37 mm) as compared to streptomycin (zoi = 25 -40 mm) against these bacteria. derivatives bearing small alkyl chains demonstrated better activity. introduction of cyclic rings e.g. cyclopropyl, cyclobutyl and cyclopentyl at this position produced less active derivatives [65] . pouramiri, et al. showed excellent activity (100%) against meliodogyne incognita by using concentrations 10 mg/ml and 25 mg/ml. standard drug tioxazafen exhibited 100% inhibition at 25 mg/ml and 92.9% at 10 mg/ml. sar studies revealed that incorporation of methyl, ethyl and phenyl groups in the linker chain produced less active derivatives. attachment of trifluoromethyl group at position # 3 of pyrazole scaffold produced more active derivatives as compared to bromide, cyanide and methyl groups [68] . siddiqui, et al. synthesized prazolyl pyranopyridines derivatives by the reaction of 3-acetoacetyl benzopyranopyridone with different hydrazine derivatives. benzopyranopyridones contain condensed chromone ring and are the intermediate products in this series. among these derivatives, compound 65 (fig. 8 ) manifested prominent activity against salmonella typhi (s. typhi, zoi = 12 mm), salmonella dysenteriae (s. dysenteriae, zoi = 19 mm) and klebsiella pneumoniae (k. pneumoniae, zoi = 18 mm) as compared to chloramphenicol (zoi = 16 mm, 23 mm, 23 mm against these bacteria) [69] . batula, et al. synthesized aryl isoxazole and 6-fluorochromone carboxylate derivatives in which both these scaffolds were linked by ester linkage. the introduction of aryl groups at the isoxazole core enhanced the antibacterial activity. compound 68 showed activity against a. niger (mic = 18.75 mg/ml) and c. albicans (mic = 18.75 mg/ml) as compared to amphotericin b (mic = 1.562, 6.25 mg/ml respectively against these microbes). introduction of halogen-substituted phenyl rings significantly increased antifungal activity. compounds 66 and 67 were most active against b. subtilis having mic value of 9.375 mg/ml for each compound as compared to streptomycin (mic = 6.25 mg/ml) [70] . hessein and co-workers synthesized furochromone, furocoumarin and benzofuran derivatives having sulfonamide group. upon in vitro evaluation, compound 69 exhibited antimicrobial potential against e. coli (zoi = 20 mm) and aspergillus ochraceus (zoi = 19 mm). compound 70 (zoi = 19 mm) was most prominent against e.coli as compared to chloramphenicol (zoi = 38 mm). attachment of aminophenyl and biphenyl ring to the sulfonamide group significantly improved the antibacterial activity. addition of benzotriazole or biphenyl acetamide group did not improve the antimicrobial activity [71] . aggarwal, et al. synthesized 1,2,4-dithiazolyl derivatives as antimicrobial agents. compound 71 showed prominent activity against b. subtilis (mic = 0.78 mg/ml), s. cerevisiae (mic = 6.25 mg/ml) and c. albicans (mic = 3.12 mg/ml) as compared to fluconazole (mic = 1.9 mg/ml, 3.9 mg/ml versus s. cerevisiae, c. albicans). compound 72 presented excellent activity against e. coli (mic = 1.56 mg/ml) and b. subtilis (mic = 1.56 mg/ml) as compared to gentamicin (mic = 0.9 mg/ml and 0.75 mg/ml against e. coli and b. subtilis). sar investigation declared that substitution of the phenyl ring at 1,2,4-dithiazolyl ring is very important for antimicrobial activity and addition of the halogen atom at para-position significantly improves the activity. addition of electron-withdrawing group e.g. chlorine and bromine at position # 6 and # 7 of chromone scaffold also enhances activity while attachment of electron-donating group e.g. methyl lowers the activity [72] . gadhave, et al. synthesized fluorine containing pyrazolone derivatives having chromone molecule by a knoevenagel condensation reaction [73] . compound 75 (fig. 9) showed prominent activity against streptococcus pyogenes (mic = 62.5 mg/ml) in comparison to ampicillin (mic = 100 mg/ml). compounds 76 was most active against s. aureus having mic value of 62.5 mg/ml. compound 77 was most active against p. aeruginosa (mic = 62.5 mg/ml) in comparison to ampicillin (mic = 250 mg/ml). it was found that addition of propyl and trifluoromethyl group at the pyrazole ring produced potent derivatives against gram-positive bacteria [74] . knoevenagel-cope condensation reaction was also used by bari, et al . different concentrations were used for testing antibacterial activity and no direct relationship was observed between concentration and antibacterial activity [77] . badhade, et al. synthesized chromone and isoxazole conjugates without any linker group. upon in vitro evaluation as antibacterial agents, these derivatives exhibited excellent activity. compounds 82 and 83 (fig. 9 ) exhibited notable activities against b. subtilis and s. aureus presenting mic values of 10 mg/ml equivalent to standard drug. incorporation of halogen and alkyl groups in the chromone ring produced more active compounds [78] . chromone scaffold was attached with 1,2,3-triazole by dofe and co-workers to produce new derivatives. compounds 84 and 85 exhibited prominent antifungal activities (mic = 12.5 mg/ml) against c. albicans for both compounds as compared to miconazole (mic = 12.5 mg/ml). derivatives 84 and 85 showed prominent antibacterial activity (mic = 25 mg/ml) against b. noor ul amin mohsin et al. the process of inflammation is related to various diseases. nonsteroidal anti-inflammatory agents are frequently used as remedy of inflammation but are associated with the major adverse effect of gastrointestinal ulceration [82] . therefore the development of new anti-inflammatory drugs having good safety profiles is necessary. chromone derivatives have also presented anti-inflammatory activities. tnfa (87% inhibition) as compared to standard drug dexamethasone (71 and 84% inhibition of tnfa and il-6 respectively). in compound 97, piperazine and chromone scaffolds are separated by methylene group and piperazine is further attached to a pyrimidine. replacement of pyrimidine ring by pyridine, p-methoxyphenyl ring produced equipotent derivatives. substitution of pyrimidine ring by methyl, ethyl and acetyl groups produced less active derivatives [85] . alzheimer's disease (ad) is a neurological ailment mostly among the elder people and is indicated by memory loss and dementia [86] . chromone core has been attached with various other pharmacophores to produce new multifunctional agents. natural compounds have potential to inhibit some toxicities of alzheimer disease [87] . liu, et al. attached chromone-2-carboxamide with alkylbenzylamines as anti-alzheimer agents. compound 98 (fig. 11 ) displayed excellent in vitro acetylcholinesterase (ache) inhibition (ic 50 molecular modeling investigation of this derivative also showed interaction with the active site of ache. the presence of amino group at position # 2 of chromone ring forms additional hydrogen bonding with tyr-510 and gly-523 of ache. benzene ring of chroman fragment showed p-cation interaction with arg-522. the increased activity was also linked to the extra amino functionality in the chromone ring [18] . hybrid molecules of 4-oxo-4h-chromene and tacrine were synthesized by fernandez-bachiller and colleagues in which cholinesterase inhibitory property of tacrine and antioxidant property of chromone were combined. alkylene diamine was used as linker between these two pharmacophores and a chain of ten carbon atoms was found optimal for best activity. hybrid proved more potent than parent drug tacrine but showed inhibition of ache and buche enzymes. 2-amino-4-methyl phenol group occupied the hydrophobic pocket formed by leu-171, ile-198 and ile-199 [90] . mackhaeva, et al. synthesized 2-vinyl chromone derivatives as selective butyryl cholinesterase (buche) inhibitors. derivatives having n-benzyl and n-vinyl methoxy carbonyl substituted compounds presented more selectivity for buche. in this series, compound 104 appeared as the most potent inhibitor (ic 50 = 2.27 ± 0.18 mm) of buche in comparison to tacrine (ic 50 = 0.0290 ± 0.0002 mm). compound 104 carry bromine atom at position # 6 of chromone core. introduction of ethoxy group at position # 8 of chromone core produced less active derivatives. molecular docking studies showed that methoxy carbonyl group interacted with anionic site formed by gly116, gly117 and ala199. the n-benzyl group interacted with tyr440 and trp430 and showed more affinity (binding free energy = 1.5 kcal/mol) as compared to n-methyl substituent at this position [91] . cagide and co-workers synthesized chromone-2-phenyl carboxamide derived compounds as adenosine a1 (ha1) receptor ligands. different substituents were attached at the exocyclic phenyl ring. compound 105 presented prominent activity at ha1 (ki=0.219 mm) receptor and this compound also exhibited selectivity for this receptor as compared to ha3. it bears nitro group at ortho position of phenyl ring. shifting of nitro group at meta or para position produced less active derivatives. authors explained that receptor sites of ha1 (lined by asn170, glu170, glu172 and thr270) and ha3 (ser73, gln167, val169, leu264) are different from each other. these derivatives also follow lipinski rule of five and possess drug like properties [92] . ali abid, et al. synthesized sulfonyl hydrazone derivatives of 3-formyl chromones as monoamino oxidase (mao) inhibitors. these compounds showed capacity to inhibit mao-a and mao-b enzymes simultaneously. compound 106 was the most active (ic 50 = 0.33 ± 0.01 mm) in vitro mao-a inhibitor in comparison to clorgyline (ic 50 = 0.004 ± 0.0003 mm). it bears chlorine atom at position # 6 of chromone skeleton. replacement of chlorine by methyl group produced less active derivatives. compound 107 was the most active (ic 50 = 1.12 ± 0.02 mm) mao-b inhibitor as compared to deprenyl (ic 50 = 0.0196 ± 0.001 mm). it carries fluorine atom at position # 6 and replacement of fluorine atom by chlorine or bromine atom produced less active derivatives. molecular docking studies of compound 107 with mao-b showed that sulfonamide oxygen interacted by hydrogen bonding with tyr60. oxygen atoms of chromone ring and carbonyl group interacted through hydrogen bonding with gln206 and tyr435. derivatives in this series exhibited good pharmacokinetic properties such as oral bioavailability [93] . reactive oxygen species produce oxidative damage to various biomolecules. naturally occurring compounds have also displayed antioxidant activities [94] . chromone derivatives also play significant role as antioxidants and free radical scavengers [95] . berczynski, et al. (2013) evaluated chromone conjugated derivatives of 2,4-thiazolidinedione, 2,4-imidazolidinedione, 2-thioxo-imidazolidine-4-one as antioxidant agents. derivatives having 2-thioxo-imidazolidine scaffold exhibited prominent activity against oh·, dpph· free radicals. compound 108 (fig. 12 ) appeared as the most active in vitro free radical scavenger (ic 50 = 0.353 ± 0.04 mmol/l) in comparison to standard drug tiron (ic 50 = 0.451 ± 0.065 mmol/l). in compound 108, 2, 3-double of chromone is in conjugation with the 4-carbonyl group and this is the reason for increased antioxidant activity [96] . berczynskai, et al. (2013) evaluated chromone molecules having 2,4-thiazolidinedione and 2,4-imidazolidinedione heterocyclic rings for antioxidant activities. heterocyclic rings were further attached with substituted aromatic rings. in dpph (1,1-diphenyl-2-picryl-hydrazyl) radical scavenging assay, compounds 109 (ic 50 = 194 ± 3.5 mmol/l) and 110 (ic 50 = 364 ± 3.3 mmol/l) presented prominent activity as compared to vitamin c (ic 50 = 346 ± 28 mmol/l). by using the dmpo-oh (5,5-dimethyl-1-pyrroline-1-oxide) spin adduct method, 109 (41.7% inhibition) and 110 (35.1% inhibition) also exhibited free radical scavenging activity [97] . takao and colleagues synthesized 3-styryl chromone derivatives by konevenagel reaction as a-glucosidase inhibitor and as antioxidant agents. compounds 111 (ec 50 = 17 mm) and 112 (ec 50 = 23 mm) showed prominent in vitro activities as dpph scavenger as compared to ascorbic acid (ec 50 = 23 mm). compounds 111 (ic 50 = 16 mm) and 112 (ic 50 = 10 mm) also exhibited inhibition of a-glucosidase enzyme. both compounds contain catechol ring attached to the chromone scaffold. sar studies showed that incorporation of methyl group and halogen atoms on the ring b did not increase the activity but the incorporation of hydroxyl group produced active molecules. dihydroxy derivatives showed better activity as compared to monohydoxy derivatives. [98] . proenca, et al. attached chromone with 3,4-dihydroxy phenyl core via 1,3-diene system. compound 113 (ic 50 = 125 ± 13 mm) and 114 (ic 50 = 121 ± 9 mm) showed prominent activity as scavenger of hydrogen peroxide (h 2 o 2 ) as compared to standard drug quercetin (ic 50 = 1338 ± 42 mm). compound 114 also exhibited effective scavenging of hocl (ic 50 = 17 ± 3 mm), roo· and onoo -·(ic 50 = 0.29 ± 0.02 mm). incorporation of hydroxyl groups both at position # 5 and # 7 of chromone scaffold was important for the antioxidant activity. no prominent difference in the activity was observed for these hydroxyl groups [99] . soengas, et al. synthesized chromone-3-pyrazoly derivatives as free radical scavenger and alpha glucosidase inhibitor. compound 115 appeared as the most effective antioxidant agent (ic 50 = 23 mm) as compared to standard drug a-tocopherol (ic 50 = 23 mm). it possesses catechol group in its structure and it proved more active as compared to monohydroxyl derivatives. addition of hydroxyl group in the chromone ring had no significant influence on the activity [100] . rao, et al. synthesized styryl chromone derivatives in which styryl group was attached at position # 2 of chromone core. antioxidant activity was monitored as scavenger of superoxide free radical. antioxidant activity was dependent on the number of hydroxyl groups, and compound 116 emerged as the most active agent (ic 50 = 234 mm) in comparison to vitamin c (ic 50 = 852 mm). compound 116 also showed excellent antibacterial activity against xanthomonas campestris (zoi = 11.3 mm) and agarobacterium tumafaceins (zoi = 11 mm) as compared to streptomycin (zoi = 12 -15 mm). chromone derivatives having hydroxyl group also presented better antimicrobial activity. replacement of hydroxyl group with methoxy group decreased antibacterial activity and increased antifungal activity [101] . demetgül and co-workers linked chromone with chitosan to produce the chromone-chitosan schiff base. upon in vitro evaluation as antioxidant agent by dpph radical scavenging assay, compound 117 emerged as the most potent (ic 50 = 0.88 mg/ml) antioxidant agent as compared to chitosan alone. the increased antioxidant activity was related to the phenolic group present in chromone [102] . thus, chromone is a promising scaffold for the synthesis of new therapeutic agents, and hybrid molecules presented in this review can be used as lead for future drug discovery. actions of flavonoids and coumarins on lipoxygenase and cyclooxygenase el-edfawy, phosphorus, sulfur alzheimer's dis key: cord-000884-zq8kqf6h authors: shen, hsin-hui; lithgow, trevor; martin, lisandra l. title: reconstitution of membrane proteins into model membranes: seeking better ways to retain protein activities date: 2013-01-14 journal: int j mol sci doi: 10.3390/ijms14011589 sha: doc_id: 884 cord_uid: zq8kqf6h the function of any given biological membrane is determined largely by the specific set of integral membrane proteins embedded in it, and the peripheral membrane proteins attached to the membrane surface. the activity of these proteins, in turn, can be modulated by the phospholipid composition of the membrane. the reconstitution of membrane proteins into a model membrane allows investigation of individual features and activities of a given cell membrane component. however, the activity of membrane proteins is often difficult to sustain following reconstitution, since the composition of the model phospholipid bilayer differs from that of the native cell membrane. this review will discuss the reconstitution of membrane protein activities in four different types of model membrane—monolayers, supported lipid bilayers, liposomes and nanodiscs, comparing their advantages in membrane protein reconstitution. variation in the surrounding model environments for these four different types of membrane layer can affect the three-dimensional structure of reconstituted proteins and may possibly lead to loss of the proteins activity. we also discuss examples where the same membrane proteins have been successfully reconstituted into two or more model membrane systems with comparison of the observed activity in each system. understanding of the behavioral changes for proteins in model membrane systems after membrane reconstitution is often a prerequisite to protein research. it is essential to find better solutions for retaining membrane protein activities for measurement and characterization in vitro. the cell membrane separates intracellular components from the outside environment and is constituted by various phospholipids, cholesterol, glycolipids and proteins. integral membrane proteins have at least one polypeptide segment spanning the membrane bilayer whereas peripheral membrane proteins are temporarily attached to the lipid bilayer or to integral membrane proteins by various interactions such as hydrophobic, electrostatic and other types of non-covalent interactions. membrane proteins work as a selective filter to regulate molecules entering cells and also serve in communicating with the surrounding environment. thus, membrane proteins play an essential role in the physiological functions needed for cell survival. the functional activities of membrane proteins are modulated by the structure of the surrounding lipids molecules in the membrane [1, 2] ; thus the composition of the lipid bilayer can affect the inter-or intra-molecular interactions between the lipid bilayer and membrane proteins [3] . investigating membrane proteins in vivo is difficult because the membrane proteins are associated with a complex mixture of other proteins, and are prone to aggregation in solution [4] . it is still a major challenge at this stage to extract information needed in vivo to address specific questions in the function of the cell membrane. to simplify cell membrane systems, model membranes such as monolayers, bilayers, liposomes and nanodiscs have been developed, enabling detailed investigation of membrane protein structure in lipid membranes. model membrane environments more closely resemble the natural lipid bilayer than alternatives such as detergents. however, many features of phospholipid structure need to be considered and optimized in the creation of a suitable model membrane. for example, the hydrophobicity of the lipid chain defined by the lengths of the fatty acid chains, is an important parameter for retaining protein activity. other factors affecting the reconstituted membrane protein activity are the chemical properties of the lipid head groups which control membrane hydrophilicity. both parameters are crucial in stabilizing membrane protein structure. there are a number of approaches used to create a model membrane in order to mimic properties of the native cell membrane, and we will review these various approaches for reconstituting membrane proteins into different types of model membrane-monolayers [5] , supported planar lipid bilayer [6] and liposomes [7] as shown in figure 1a -c. we will also discuss the emerging technology of nanodiscs [8] ( figure 1d ). nanodiscs are a new class of model membrane, with attractive properties that address shortcomings of other approaches in the study of membrane proteins. the first section gives a brief summary of each method and a comparison of their strengths and weaknesses. in the following section, we describe four case studies and will compare the protein activity changes when the membrane proteins are reconstituted into different model membranes. in these case studies, we demonstrate how protein activities are modulated by the lipid environment and discuss how this environment helps to retain protein activities. and black in b represent water and a substrate respectively. nanodiscs contain membrane scaffold proteins, shown in green. one of the most common approaches to study the membrane protein structure and activity uses a langmuir monolayer at the air-water interface. this method has been extensively used for more than a century [9, 10] . reconstitution of membrane proteins helps obtain further information on their organization and structure in the langmuir membrane [11, 12] . it is a simple method to create a phospholipid monolayer at an air-water interface. basically, a desired amount of lipid or lipid mixtures are dissolved in organic solvents such as chloroform or chloroform/ethanol mixtures, followed by spreading the lipid/solvent mixtures on the water surface. by evaporating out the solvent, the phospholipid molecules self-assemble vertically as a monolayer film at the air-water interface, with their hydrophilic head groups immersed in the water and their hydrophobic tail pointed to the air as shown in figure 1a [13] . a major advantage of using the langmuir monolayer system is that parameters such as thickness, surface pressure, molecular area and subphase thickness can be well controlled [10] . more advanced characterization techniques, such as π-a isotherm uv-vis adsorption, x-ray reflectivity, ellipsometry and rheology, have been developed to gain detailed information on the binding of proteins onto the phospholipid monolayer and to monitor enzyme activities when binding to the monolayer [14] . however, a limitation of langmuir monolayers is that the lack of a layer comparing to the natural cell structure (bilayer) and the high surface tension of water that can cause protein denaturation. despite this limitation, there are several successful studies using this approach. two types of membrane proteins in monolayer model membrane system will be briefly described below: rhodopsin [15, 16] , bacteriorhodopsin [17, 18] and the aliphatic peptide gramicidin [19, 20] have been successfully reconstituted and studied in monolayers at the air-water interface. in order to obtain information on the secondary structure and orientation, the protein layer can be investigated in situ at the air-water interface by either polarization modulation infrared reflection absorption spectroscopy (pm-irras) or x-ray reflectivity in combination with surface pressure-area isotherms [21] . the study of gramicidin is an example of such an approach, and while gramicidin is unfolded at high molecular area (low pressure), it is refolded upon compression and retains its precise structure and orientation. likewise, for both rhodopsin and bacteriorhodopsin, the secondary structures measured in monolayers are indistinguishable from that in native membranes when appropriate conditions are used. while some experiments have suggested that spreading of rhodopsin in certain conditions (>5 m/n) leads to denaturation [21] , bacteriorhodopsin, in contrast, is very stable in most testing conditions (compression and temperature change). the different properties of the protein are probably due to the ability of baceriorhodopsin to form a stable two-dimensional crystalline structure at the air-water interface [21] . phospholipid monolayers are simple model membrane systems that are perfectly suited to study the binding of peripheral proteins onto a membrane surface. peripheral membrane proteins spontaneously bind onto phospholipid monolayers at the air-water interface by injecting themselves into the subphase underneath the lipid monolayer. in most cases, useful information can be obtained by measuring the binding of peripheral proteins onto the monolayer. for example, the kinetics and dynamics of adsorption of myristoylated and nonmyristoylated recoverin onto phospholipid monolayers have been investigated using surface pressure isotherm described in figure 2 [21] . the curve can be fitted with stretched exponential which can convert into the rate of adsorption of myristoylated and nonmyristoylated which is 0.028 s −1 and 0.0048 s −1 , respectively. this indicates that the adsorption of myristoylated recoverin is six times faster than nonmyristoylated recoverin. reconstituting enzymes into the langmuir monolayers at the air-water interface has been found to be a very useful approach to understand the hydrolysis of membrane phospholipids. for example, the interfacial recognition and adsorption of phospholipases a2 (pla2) and phospholipases c (plc) to the phospholipid membrane interface are poorly understood. by using this approach, it appears that both pla2 and plc are active at the monolayer model membrane, indicating that the kinetics of phospholipid hydrolysis at the air-water interface can be monitored by biophysical characterization techniques in situ such as pm-irras and infrared reflection adsorption spectroscopy [22] . moreover, it has been found that in the presence of calcium, phospholipid hydrolysis by pla2 resulted in the production of calcium-palmitate complexes. this suggests that calcium is necessary for pla2 secretion. the formation of a supported lipid bilayer on a solid substrate was reported by tamm and mcconnell in 1985 as a new model membrane system to study the physical properties of biological membranes and their constituent lipid and protein molecules [23, 24] . supported planar lipid bilayers are prepared by several methods [25, 26] . vesicle fusion is the simplest method for supported bilayer formation and the fusion mechanism on a hydrophilic support is well understood [27, 28] . essentially, the bilayer is prepared by the fusion of small unilamellar vesicles on solid supports such as sio 2 , glass and modified gold surface by van der waals, electrostatic, hydration and steric forces. the supported lipid bilayer has polar hydrophilic headgroups facing the aqueous surroundings and two hydrophobic tails that face the interior of the membrane which more closely resembles biological membranes than the langmuir monolayer. the supported lipid bilayer can confer many key functions to biological membranes. however, one side of the hydrophilic head group is still tightly attached to the solid support and this may, in some cases, affect the fluidity of the model membrane. this matters, since integral membrane proteins may not diffuse in the plane of the membrane. furthermore the orientation of membrane proteins cannot be controlled in the supported planer lipid bilayer. to alleviate some of these problems, a new tethered polymer-supported planar lipid bilayer system was developed to investigate the reconstitution of integral membrane proteins in a laterally mobile form into the supported lipid bilayer [29] . wagner and tamm [30] have successfully designed a supported lipid bilayer on a polyethyleneglycol cushion shown in figure 3 . the polymer cushion minimizes the interactions of the proteins with the substrate and the polymer. it also provides a soft support and, for increased stability, covalent linkage of the membranes to the supporting quartz or glass substrates. in low polyethyleneglycol concentration regimes, the bilayers were assembled with high lateral lipid diffusion coefficients (0.8-1.2 × 10 −8 cm 2 /s). cytochrome b5 and annexin v were used to test the polyethyleneglycol cushion system. two populations of laterally mobile proteins were observed in the polyethyleneglycol cushion-supported bilayers. approximately a quarter of cytochrome b5 diffused with a diffusion coefficient of 0.8-1.2 × 10 −8 cm 2 /s, and more than half of the cytochrome b5 diffused with a diffusion coefficient of ~2 × 10 −10 cm 2 /s. similar results were found in the annexin v system. annexin v diffused with two populations with diffusion coefficients of 3 × 10 −9 cm 2 /s and 4 × 10 −10 cm 2 /s. the new polymer-supported lipid bilayer system has increased the mobile fraction and retained the full lateral mobility of both cytochromes b5 and annexin when integrated or bound to the supported lipid. although polymer cushions allow for successful integration of small membrane proteins into bilayers, further challenges stem from studies with large transmembrane proteins. polymer cushions cannot provide large transmembrane proteins with good solvent accessibility, or enough space for the motion; required for the activity. while several types of polymer cushions have been developed, including polymethyl methacrylate diblock polymer cushions [31] , poly(ethylene imine) [32, 33] cushions and poly(ethylene glycol) tethered lipopolymers [30] , these cushions are mostly limited to a thickness of up to 10 nm. a recent development of a maleic anhydride copolymer thin film has film thickness up to 60 nm [34] . the hydrophilic polymer-cushioned supported lipid bilayers provide a higher mobility and homogeneous distribution of the incorporated beta-amyloid precursor protein cleaving enzyme (bace) on the bilayer surface, and enhances the enzymatic activity of bace (increased from 8% to 16%). even so, the activity of the incorporated bace remains significantly lower (16%) than that of the native enzyme (100%). another important classic category of membrane proteins are the transporters of ions and small molecules. studies of how ion channels regulate the transport of substrates [7] are important for fundamental biology. however, it is challenging to incorporate ion channels in supported lipid bilayers due to leakage or instability issues. detailed studies of ion channel conduction or gating require considerable period of time (possibly >1 h), and it is difficult to set up a stable and electrically quiet environment for the ion channel in planar lipid bilayer. a better alternative has proven to be reconstitution of ion channels into proteoliposomes. lipid vesicles, also known as liposomes, consist of a self-closed lipid bilayer. they have been widely used for more than 30 years to reconstitute the membrane proteins in unilamellar phospholipid vesicles. liposomes are relatively easy to construct by procedures such as extrusion method or ultrasonication, with reverse-phase evaporation. furthermore, giant vesicles of unilamellar or multilamellar nature can be "micro-manipulated" under an optical microscope. reconstitution of membrane proteins in liposomes usually requires detergents wherein purified membrane proteins are solubilized in detergent, then mixed with the desired phospholipid vesicles forming an isotropic solution of mixed phospholipid-protein-detergent micelles. the detergent can then be removed slowly by dialysis, gel filtration or biobead adsorption. when the detergent concentration reaches a critical level, the protein will spontaneously associate with the phospholipid membrane to form biologically active liposomes, called proteoliposomes. however, it has been a hard feat to control the final orientation of protein in the proteoliposomes [35] , as well as the amount of protein inserted due to the limited area available. in many cases, disorientation of the protein causes aggregation. despite these difficulties, there have been many successful cases of membrane proteins reconstituted in the proteoliposomes, and we describe two examples below. several integral membrane proteins have been successfully reconstituted into proteoliposomes such as rhodopsin [36] , g proteins [37, 38] , proapoptotic bcl-2 proteins and t-bid [39] , phosphocholine cytidylyltransferase (ct) [40] and p protein kinase c (pkc) [41] . however, these studies also found that the resulting protein activities are sensitive to the membrane curvature of the liposomes. this indicates that different phospholipids can cause considerable curvature stress changes in the liposomes [42] . specifically, the curvature stress has been suggested to modulate the free energy and folding of the integral membrane proteins [43] . sometimes the activity of different enzymes is modulated by the same driving force of the membrane curvature, but there may also be variation of activity through different mechanisms. for example, the activities of both ct [40] and pkc [41] are enhanced by increasing the negative curvature strain of the membrane. the activity of ct appears to be directly coupled with the membrane curvature, in contrast, the activity of pkc does not have a direct relationship with the curvature strain and enzymatic activity [41] . the activity of pkc is instead modulated by nonlamellar-forming lipids via a less direct mechanism. liposomes have been commonly used for reconstituting different types of transporters to allow for the free diffusion of solution or catalysis of obligatory co-transporters. a large number of functional membrane proteins have been successfully reconstituted into liposomes but only a few examples will be discussed here. the reconstitution of colicin ia and e1 in either soybean phospholipids or e. coli phospholipids show that there is channel formation in the liposomes but there are unspecific channels allowing passage of ions, such as rubidium, sodium, chlorine, potassium or phosphate but not of sugars [7, 44] . an example of the reconstitution of selective transport comes from the d-glucose transporter, purified from human erythrocytes and extracted from detergents followed by incorporation into proteoliposomes. with incorporation of the d-glucose transporter, the proteoliposomes become permeable to d-glucose but not to l-glucose. the transport was inhibited by cytochalasin b which is a potent inhibitor of d-glucose transporter [45, 46] . several types of atp-dependent ion transporters such as ca 2+ /mg 2+ -atpase, na + /k + -atpase, and h + /k + -atpase have been reconstituted into proteoliposomes [47] . upon addition of atp, ions are observed to be transported inwards and can form a complex. the single-channel property of channels incorporated into proteoliposomes can be investigated using the well-known patch-clamp method [48] . channel activity is monitored following excision of the patch from the proteoliposomes. ion-channel reconstitution makes possible the investigation of the influence of membrane lipid composition on channel function. the kinetic investigation of these channels under physiological conditions has been discussed elsewhere [47] . another up-to-date method is using organic solvent or oil mixed with water that creates water-in-oil (w/o) microdroplets coated by phospholipid. the hydrophilic head group immerses in the water and the hydrophobic tail locates in the oil/organic solvent phase. the application of the water-in-oil system could cover a wide range of applications from monolayer, planer lipid bilayer and liposomes. funakoshi et al. [49] and maglia et al. [50] used a planer lipid bilayer formed by two microdroplets driven to come in contact to reconstitute ion channels in the bilayer. this method is extremely simple and reproducible. recently, the water-in-oil microdroplets are extended to form liposomes by using droplet-transfer method invented by yoshikawa [51] . by using this approach, it is possible to modulate the lipid compositions of outer and inner leaflets and furthermore to orient a reconstituted membrane protein in liposomes [52] . nanodiscs offer a solution to some of the challenges described in the previous sections. the first attempt to reconstitute membrane proteins in the phospholipid bilayers using nanodisc technology was initiated by sligar's group a decade ago [8] . the nanodisc is a self-assembly of phospholipids and a membrane scaffold protein derived from human serum apolipoprotein a1. the detergent, cholate, can be used to solubilize phospholipids and membrane scaffold proteins into a micelle mixture. following detergent removal with dialysis or bio-beads adsorbent, a nanodisc self-assembles. the phospholipid associates as a bilayer domain while the membrane scaffold protein wraps around the edges of the discoidal structure in a belt-like configuration ( figure 1d ). it is possible to modify the diameter of the bilayer disc by genetically engineering the apolipoprotein a1 by changing the number of amphipathic helices. by this approach, the diameter of nanodiscs can be made anywhere from 9.8 to 17 nm, and therefore accommodate a range of membrane proteins. the ratio of phospholipid: membrane scaffold protein is precisely defined which helps engineer the different size of membrane proteins in the nanodiscs. detailed formation of different types of nanodiscs has been described elsewhere [53, 54] . the great advantage of using nanodiscs is keeping the membrane proteins in aqueous solution, in native-like phospholipid bilayer environment that is soluble, stable, monodisperse and detergent-free. most important, it isolates proteins or complexes as individual particles in monomeric or oligomeric states for analysis by techniques that range from activity assays to electron microscopy. since 2003, there have been more than 100 membrane proteins reconstituted into nanodiscs [54] , ranging from signaling receptors to transport machines. we will discuss the applications separately below. nanodiscs have been used to analyze the influence of binding substrate on monodisperse receptors which are isolated from the cell-surface membrane. those receptors include g protein-coupled receptors (gpcr) [55, 56] , cholera toxin receptor ganglioside g m1 , bacterial chemoreceptor [57] and epidermal growth factor receptor. introduced into nanodiscs, the receptors stay in monodispersed, controllable, predefined oligomeric states in which it is possible to characterize the oligomeric status. for example, two different gpcr proteins, the beta-adrenergic receptor (β 2 ar) and rhodopsin [58] have been extensively studied using nanodiscs. β 2 ar was one of the first receptors assembled into nanodiscs which was found to be functionally active (54% of starting activity recovered) and shown coupling to its g-protein. rhodopsin is a light-activated gpcr present in the photoreceptor cells of the retina and transducin is an important g-protein naturally expressed in retina rods and cones. assembly of functional rhodopsin into nanodiscs was found to activate transducin with high efficiency and to isolate the high affinity of transducin-metharhodopsin ii complex. this provides strong evidence that the monomeric state of rhodopsin can activate and interact with the transducin. a dimeric rhodopsin nanodisc was separated for monomeric forms using sucrose density gradients. even with two rhodopsins in the nanodiscs, interaction with a single transducin molecule was observed and found to activate the transductin with high efficiency [56] . numerous membrane associated enzymes have been incorporated into nanodiscs. cytochrome p450 (cyp) enzymes have been extensively studied, including cyp2b4 [59] , cyp6b1 [8] , cyp73a5 [60] and cyp19 [61, 62] . this system has provided a means for studying the extensive collection of membrane bound cytochromes p450 with the same biochemical and biophysical tools that have been previously limited to use with the soluble p450s. for example, the cytochrome p450 3a4 (cyp3a4) is a membrane-bound protein which is a human hepatic drug-metabolizing enzyme. most studies of the ligand binding by cyp34a are carried out in the presence of detergents below their critical micelle concentrations [63, 64] but are compared by the propensity of cyp34a to aggregate. even in studies attempting to use liposomes, cyp3a4 is unlikely to exist in its native state because the detergent concentrations are much higher than the phospholipid concentrations. as a result, the understanding of the structure and composition of cyp3a4 in the lipid phase was limited and the membrane effect on cyp3a4 ligand binding behavior is unclear. nanodiscs have been utilized to study cyp3a4 which displays monophasic reduction kinetics. with a high lipid-protein ratio, cyp3a4 is captured as a monomer. however, at lower lipid ratio, cy3a4 self-associates and heterogeneous behaviors are induced. the nanodiscs prohibit self-association in this case as there is only one cyp3a4 per nanodisc and show significant improvement in homogeneity and stability. this opens up new possibilities for detailed analysis of equilibrium and steady-state kinetic characteristics of catalytic mechanisms of cyp enzymes [63] . the sec translocon is a membrane-embedded protein assembly that drives protein translocation into or across membranes. the core translocon is formed from a trimeric arrangement of secy, sece and secg [65] . the secyeg promoter has 15 transmembrane helices sitting in the phospholipid membrane. the oligomerization of secyeg has been proposed to be necessary to proper function. researchers were successful in reconstituting sec into membrane vesicles in 1990 and have had great success in characterizing several partial reactions of secyeg functions [65] . reconstituting a single secyeg into a nanodisc with different types of lipids [66] suggests that the acidic lipids can stabilize the secyeg channel in the nanodisc bilayer and trigger dissociation of the seca dimer. a model has been proposed by alami et al. [66] , suggesting that the dissociation of the seca dimer provoked by the secyeg complex is followed by activation of the seca atpase. furthermore, dalal et al. [67] , using the nanodisc technology, have also shown that only the secy dimer together with acidic lipids supports the activation of the seca translocation atpase. recently, a high resolution single-particle cryo-em structure of single secyeg complexes in nanodiscs, bound to translating ribosomes was first solved at subnanometer resolution [68] . it allows the secyeg complex to be investigated in a natural lipid bilayer environment and identifies the ribosome-lipid interactions. wu et al. [69] also used surface plasmon resonance to investigate the competitive binding of ribosomes and seca. the data suggest that both ribosomes and seca can interact simultaneously with secyeg complex during membrane protein insertion, but seca competes with ribosome when it binds to the secyeg complex. in the previous section, we have shown that membrane proteins can be assembled into four different types of model membrane and the activities of some of the membrane proteins can be retained, allowing their physicochemical properties to be studied. but is there a model membrane system that is the best for membrane-protein reconstitution? the reconstitution of the same membrane protein into different model membranes has been compared and, in this section, we list four membrane proteins with varying activities in different model membranes. ganglioside g m1 is a naturally occurring native receptor that binds to cholera toxin via hydrogen bonds [70] . it is an excellent receptor for studying lipid-receptor interaction. several different approaches to reconstituting the glycolipid receptor g m1 in model membranes have enabled the measurement of binding of its interaction partner cholera toxin. in liposomes and supported lipid bilayer systems, the ganglioside g m1 is free to diffuse across long distances and exhibits a non-uniform lateral distribution, i.e., self-aggregation, even at low incorporation ratios. therefore the binding activity of ganglioside g m1 with cholera toxin b is restricted [71] . investigations of ganglioside g m1 incorporated into nanodiscs found reduced protein aggregation. bricarello et al. [72] found that the reconstitution of a low concentration of ganglioside g m1 in nanodiscs, shows binding of cholera toxin with a significantly higher affinity than in liposomes or supported lipid bilayers. this is due to the interaction of ganglioside g m1 with the headgroup region of the disc which reduces the oligomerization, thereby causing a potential effect on the affinity of toxin binding. thus, nanodisc technology restricts the ganglioside g m1 oligomerization by controlling the number of ganglioside g m1 monomer isolated by each nanodisc. borch et al. [73] have also used sensor chip-based surface plasmon resonance (spr) technology to measure the detailed kinetic binding of the interaction between soluble molecules and membrane receptors inserted in the bilayer of nanodiscs. the corresponding spr sensorgrams are displayed in figure 4 . overall, the change of the sensorgram indicates that the spr sensorchip is immobilized with histidine-modified nanodisc or the cholera toxin b bound to the nanodiscs. the sensorgrams in both figure 4a ,b shows the binding of nanodiscs (576 ru) on the antibody immobilization surface on the sensor chip. by injecting the cholera toxin b over two flow cells presented in figure 4a and 4b, the spr sensorgrams can detect the interaction of the cholera toxin b with nanodisc with or without the existence of g m1 . it has been revealed that the captured 2% g m1 -nanodiscs bound 238 ru of the cholera toxin b without binding to the capturing nanodiscs without g m1 ( figure 4b ). the measured kinetic values of the interaction are in agreement with those reported by previous studies on the interaction of the cholera toxin with the g m1 receptor embedded in different membrane systems. this, therefore, serves as a proof of concept that nanodiscs can be employed in kinetic spr studies. the nucleus envelope is composed of two bilayers (the outer nuclear membrane and inner nuclear membrane) and contains abundant ion channels, through which ions and small molecules diffuse between the cytoplasm, nucleoplasm and perinuclear (i.e., intermembrane) space. the nuclear ionic channels represent a ubiquitous structure in the nuclei in a wide range of cells, although little is known about its functional properties. to characterize nuclear ionic channels, guihard et al. [74] attempted to reconstitute nuclear envelope vesicles derived from the canine liver nuclei into a planar lipid bilayer and giant proteoliposomes. they found that the success rate of nuclear envelope fusion into planar lipid bilayers was extremely low although cardiac nuclear ionic channels were successfully incorporated into planar lipid bilayers. the detection of the nuclear ionic channels activity was not possible. such a low efficiency can be explained by the clustering of nuclear envelope vesicles, and the low density of single vesicles, as well as the presence of residual chromatin and/or nuclear proteins (histones or lamins) which would prevent fusion events with the bilayer. another approach is reconstituting nuclear envelope vesicles into giant proteolipsosmes and detecting the single ion channel by the patch-clamp technique [49] . large conductance, voltage-gated, k + and cl − selective nuclear ionic channels are characterized and plotted as a current-voltage relationship presented in figure 5a ,b respectively. it has been found that under asymmetrical 150/50 mm kcl conditions, the zero current potential for unitary currents is at 322 mv ( figure 5a ). calculated from the goldman-hodgkin-katz (ghk) flux equation, a p k +/p cl − ratio is 9.4. this value indicates the k + selectivity for this channel. in figure 5b , the cl − selective nuclear ionic channel yields a positive zero current potential of +27.3 mv, with a p cl −/p k + ratio of 80, indicative of a high cl − selectivity over k + . this suggests super fusion of the channel under asymmetrical (150/50 mm) kcl conditions. the current-voltage relationship curves indicate that the nuclear ion channels can be functionally characterized by incorporating the proteins into the giant proteoliposomes where it is possible to retain their channel activity. furthermore, the measured activities are consistent with those described for native nuclear ion channels. p-glycoprotein, the most extensively studied atp-binding cassette transporter, has been implicated in the phenomenon of multidrug-resistance in tumor cells and has been suggested to play a significant role in drug absorption and deposition. how p-glycoprotein interacts with its substrates is still unknown. functional studies are limited because of the difficulty of obtaining large quantities of stable p-glycoprotein. besides that, no atpase activity of p-glycoprotein solubilized in detergent could be detected. when p-glycoprotein is reconstituted into proteolipsomes, it has detectable atpase activity; however, the whole complex is very unstable. heikal et al. [75] have further found that p-glycoprotein reconstituted in the proteoliposomes has a half-life of less than one day. in 2009, ritchie et al. [76] performed a detailed study of drug-stimulated atpase kinase activity of p-glycoprotein using the nanodisc technology. the p-glycoprotein protein was reconstituted into both msp1e3d1 disc and liposomes in order to compare its atpase kinase activities. the results described in figure 6 demonstrate that p-glycoprotein is functionally active when reconstituted into the nanodiscs (close squares). comparing to the atpase kinase activity of p-glycoprotein reconstitution in lipsosomes (close circles), there is a twofold increase in the maximum atpase activity in the nanodiscs. this could be due to the uniform orientations of p-glycoprotein in the nanodiscs while there are two possible orientations in liposomes. these data not only show that p-glycoprotein is functionally active when reconstituted into the nanodiscs, but that it also exhibits higher specific activity than the current standard reconstitution system. figure 6 . comparsions of the atpase activity of p-glycoprotein in nanodiscs (square) and proteoliposomes (circle). open symbols: basal activity in the absence of drug; filled-in symbols: activity in the presence of nicardipine [76] . atp-binding cassette transporters utilize the energy of atp hydrolysis to transport a wide range of substrates across cellular membranes and for non-transport-related processes such as translation of rna and dna repair [77] . a member of the atp-binding cassette super family, the maltose transporter malfgk2 from e. coli, together with the substrate-binding protein male, is one of the best-characterized atp-binding cassette binding cassette transporters suitable for various reconstitution techniques. bao and fuong have reported the reconstitution of the maltose transporter in nanodiscs, in detergent and in proteoliposomes. the atpase activity of the malfgk2 complex in various environments is shown in figure 7 . the data presented in the first column of figure 7 show that the basal atpase activity for assembly in the nanodiscs and detergent (~700 nmol/min/mg) is 10-fold higher than in proteoliposomes because of the decrease in the activation energy barrier of the transporter [78] in detergent micelles and nanodiscs. however, in the presence of male, the rate of atp hydrolysis increases in all assembly conditions. this is because male captures maltose and delivers the sugar to the transporter. note that the basal atpase activity assembly in the nanodiscs dramatically increases from 700 to 2300 nmol/min/mg. the maltose alone has no effect on the basal atpase activity in the nanodiscs and detergent. however, in nanodisc and detergent, an inhibition of the atpase activity was observed in the presence of both maltose and male in the nanodiscs. this is because that maltose reduces the binding affinity of the male-malfgk 2 complex, which therefore has reduced the atpase activity. in proteoliposomes, the atpase activity (~40 nmol/min/mg) shows a further 10-fold increase in the presence of both maltose and mele in the figure. the author used another type of male mutant which binds maltose with higher affinity. this male mutant, in contrast, shows a reduction of the atpase activity in proteoliposomes which has the same effect as the nanodiscs and detergents. overall, proteoliposomes have shown a low basal atpase activity because the lipid stabilized the transporter. however, the nanodiscs have been shown to be a better medium than proteoliposomes for studying the atp hydrolysis ability of atp-binding cassette transporters. this review summarizes and compares the most up-to-date methods for reconstituting membrane proteins into model membranes. there is no superior method for reconstituting membrane proteins in the model membrane; instead two or more model membranes should be considered, depending on the particular needs of the system and the proteins of interest. in general, systems based on lipid bilayers supported on a solid substrate are still the most favored and well-developed of the methods to study membrane proteins in the bilayer. this approach allows detailed study of the fundamental properties of biological membranes and is practical to reproduce the bilayer system. on the other hand, the proteoliposome is more suitable for ion channel reconstitution in the bilayer, as well as for combination with the patch-clamp method to detect the ionic selectivity of the channel. finally the self-assembled nanodiscs system provides a robust and common means for rendering these targets soluble in aqueous media while providing a native-like bilayer environment that maintains functional activities. nanodisc technology offers another way to prepare monodisperse samples of membrane proteins in the bilayer environment, and it is emerging as the favored approach in studies concerning membrane protein complexes. biochemical and functional characterization of the membrane association and membrane permeabilizing activity of the severe acute respiratory syndrome coronavirus envelope protein lipid-protein interactions in human erythrocyte-membrane acetylcholinesterase. modulation of enzyme activity by lipids correlation between the effect of the anti-neoplastic ether lipid 1-o-octadecyl-2-o-methyl-glycero-3-phosphocholine on the membrane and the activity of protein kinase calpha weak dependence of mobility of membrane protein aggregates on aggregate size supports a viscous model of retardation of diffusion structure and phase transitions in langmuir monolayers allogeneic stimulation of cytotoxic t cells by supported planar membranes effect of colicins ia and e1 on ion permeability of liposomes direct solubilization of heterologously expressed membrane proteins by incorporation into nanoscale lipid bilayers surface enhanced raman scattering of a lipid langmuir monolayer at the air-water interface lipid monolayers: why use half a membrane to characterize protein-membrane interactions? langmuir monolayer of artificial pulmonary surfactant mixtures with an amphiphilic peptide at the air/water interface: comparison of new preparations with surfactant polymyxin b-lipid interactions in langmuir-blodgett monolayers of escherichia coli lipids: a thermodynamic and atomic force microscopy study langmuir balance investigation of superoxide dismutase interactions with mixed-lipid monolayers modern physicochemical research on langmuir monolayers structure of rhodopsin in monolayers at the air-water interface: a pm-irras and x-ray reflectivity study formation, structure, and spectrophotometry of air-water interface films containing rhodopsin proton transport by bacteriorhodopsin in planar membranes assembled from air-water interface films structural and spectroscopic characteristics of bacteriorhodopsin in air-water interface films spectroscopic and structural properties of valine gramicidin a in monolayers at the air-water interface effects of gramicidin-a on the adsorption of phospholipids to the air-water interface organization, structure and activity of proteins in monolayers monitoring of phospholipid monolayer hydrolysis by phospholipase a2 by use of polarization-modulated fourier transform infrared spectroscopy supported planar membranes in studies of cell-cell recognition in the immune system supported phospholipid bilayers formation of high-resistance supported lipid bilayer on the surface of a silicon substrate with microelectrodes supported lipid bilayer self-spreading on a nanostructured silicon surface simulations of temperature dependence of the formation of a supported lipid bilayer via vesicle adsorption simulations of lipid vesicle rupture induced by an adjacent supported lipid bilayer patch membrane lateral mobility obstructed by polymer-tethered lipids studied at the single molecule level tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins: silane-polyethyleneglycol-lipid as a cushion and covalent linker reversible activation of diblock copolymer monolayers at the interface by ph modulation, 1: lateral chain density and conformation polymer-cushioned bilayers. i. a structural study of various preparation methods using neutron reflectometry polymer-cushioned bilayers. ii. an investigation of interaction forces and fusion using the surface forces apparatus controlled enhancement of transmembrane enzyme activity in polymer cushioned supported bilayer membranes orientation and reactivity of nadh kinase in proteoliposomes modulation of rhodopsin function by properties of the membrane bilayer role of lipid polymorphism in g protein-membrane interactions: nonlamellar-prone phospholipids and peripheral protein binding to membranes influence of the membrane lipid structure on signal processing via g protein-coupled receptors the apoptotic protein tbid promotes leakage by altering membrane curvature modulation of ctp:phosphocholine cytidylyltransferase by membrane curvature elastic stress the role of membrane biophysical properties in the regulation of protein kinase c activity membrane lipid polymorphism: relationship to bilayer properties and protein function elastic coupling of integral membrane protein stability to lipid bilayer forces reconstitution of colicin e1 into dimyristoylphosphatidylcholine membrane vesicles the permeability of bilayer lipid membranes on the incorporation of erythrocyte membrane extracts and the identification of the monosaccharide transport proteins binding of cytochalasin b to human erythrocyte glucose transporter conformational dynamics of na+/k+-and h+/k+-atpase probed by voltage clamp fluorometry the extracellular patch clamp: a method for resolving currents through individual open channels in biological membranes lipid bilayer formation by contacting monolayers in a microfluidic device for membrane protein analysis droplet networks with incorporated protein diodes show collective properties cell-sized liposomes and droplets: real-world modeling of living cells oriented reconstitution of a membrane protein in a giant unilamellar vesicle: experimental verification with the potassium channel kcsa phospholipid phase transitions in homogeneous nanometer scale bilayer discs membrane protein assembly into nanodiscs functional reconstitution of beta2-adrenergic receptors utilizing self-assembling nanodisc technology transducin activation by nanoscale lipid bilayers containing one and two rhodopsins using nanodiscs to create water-soluble transmembrane chemoreceptors inserted in lipid bilayers atomic-force microscopy: rhodopsin dimers in native disc membranes single-molecule height measurements on microsomal cytochrome p450 in nanometer-scale phospholipid bilayer disks co-incorporation of heterologously expressed arabidopsis cytochrome p450 and p450 reductase into soluble nanoscale lipid bilayers the critical iron-oxygen intermediate in human aromatase the ferrous-oxy complex of human aromatase kinetics of dithionite-dependent reduction of cytochrome p450 3a4: heterogeneity of the enzyme caused by its oligomerization ligand binding to cytochrome p450 3a4 in phospholipid bilayer nanodiscs the effect of model membranes the atpase activity of seca is regulated by acidic phospholipids, secy, and the leader and mature domains of precursor proteins nanodiscs unravel the interaction between the secyeg channel and its cytosolic partner seca two copies of the secy channel and acidic lipids are necessary to activate the seca translocation atpase cryo-em structure of the ribosome-secye complex in the membrane environment competitive binding of the seca atpase and ribosomes to the secyeg translocon crystal structure of cholera toxin b-pentamer bound to receptor gm1 pentasaccharide self-aggregation-an intrinsic property of g(m1) in lipid bilayers ganglioside embedded in reconstituted lipoprotein binds cholera toxin with elevated affinity nanodiscs for immobilization of lipid bilayers and membrane receptors: kinetic analysis of cholera toxin binding to a glycolipid receptor patch-clamp study of liver nuclear ionic channels reconstituted into giant proteoliposomes the stabilisation of purified, reconstituted p-glycoprotein by freeze drying with disaccharides chapter 11-reconstitution of membrane proteins in phospholipid bilayer nanodiscs abc transporters, mechanisms and biology: an overview discovery of an auto-regulation mechanism for the maltose abc transporter malfgk2 the authors gratefully acknowledge financial support from the australian research council (arc). hhs is an arc super science fellow and tl is an arc federation fellow. tl and lm were awarded the arc super science fellowships and grant (fs110200015). we thank victoria hewitt for her critical reading of the manuscript. key: cord-287348-00yaxpkp authors: martinez, maria jose abad; olmo, luis miguel bedoya del; benito, paulina bermejo title: antiviral activities of polysaccharides from natural sources date: 2005-12-31 journal: studies in natural products chemistry doi: 10.1016/s1572-5995(05)80038-9 sha: doc_id: 287348 cord_uid: 00yaxpkp abstract the ever increasing resistance of human pathogens to current anti-infective agents is a serious medical problem, leading to the need to develop novel antibiotic prototype molecules. in the case of viruses, the search for antiviral agents involves additional difficulties, particularly due to the nature of the infectious viral agents. thus, many compounds that may cause the death of viruses are also very likely to injure the host cell that harbours them. natural products are increasingly appreciated as leads for drug discovery and development. screening studies have been carried out in order to find antiviral agents from natural sources, and the occurrence of antiviral activity in extracts of plants, marine organisms and fungi is frequent. the evidence indicates that there may be numerous potentially useful antiviral phytochemicals in nature, waiting to be evaluated and exploited. in addition, other plants, not previously utilized medicinally, may also reveal antivirals. among natural antiviral agents, recent investigations have reconsidered the interest of phyto-polysaccharides, which act as potent inhibitors of different viruses. this chapter will illustrate a variety of antiviral polysaccharides from natural sources since 1990, with the aim of making this matter more accessible to drug development infectious diseases account for one-third of all deaths worldwide. although the last decade has yielded significant advances in the treatment of infectious diseases, new therapies for viral, fungal, bacterial and parasitic infections are needed [1] . viruses are responsible for a large proportion of the morbidity and mortahty experienced worldwide. these infectious agents consist of a core genome of nucleic acid, dna or rna (nucleoid) contained in a protein shell (capsid) and sometimes surrounded by a lipoprotein membrane (envelope). some genera of viruses are known to cause human infections and include the following: adeno, pox and herpesviruses: herpes simplex virus type 1 and 2, varicella-zoster, cytomegalovirus, epstein-barr virus; papillomaviruses (papillomas, cancer); parvoviruses (erythema infectiosum); dna-viruses (e.g., hepatitis b); (+/-) rna viruses: reo-and rotavirus; (-) rna viruses: influenza, parainfluenza, measles, respiratory syncytial, vesicular stomatitis, rabies virus and togaviruses (rubella, yellow fever); retroviruses (e.g., human immunodeficiency virus, the causative agent of acquired immunodeficiency syndrome); and other several rna viruses such as arenaviruses (lymphocytic choriomeningitis), bunyaviruses (encephalitis), coronaviruses (upper respiratory infections) and picornaviruses (poliomyelitis, diarrhoea). most successful anti-infective agents inhibit essential steps in metabolic processes required by the pathogen but absent in the host. hov^ever, viruses cannot replicate independently. they must enter cells and use the cellular energy-generating, dna-or rnareplicating and protein-synthesizing pathways of the host to achieve viral replication. for this reason, in the case of viruses the search for antiviral agents is very hard since metabolic differences are not available: viruses utilize the host's protein synthesis leaving only some aspects of nucleic acid synthesis and macromolecule processing distinct from the host's metabolism. the identification of a retrovirus, human immunodeficiency virus, as the causative agent of acquired immunodeficiency syndrome, the steadily increasing incidence of various viral infections caused by viruses in immunodeficient patients, e,g., herpes simplex virus, varicella-zoster virus, cytomegalovirus and epstein-barr virus, the increasing evidence for the pathogenic role of a number of viruses, e.g., human papilloma virus in the pathogenesis of primary hepatocellular carcinoma, and the socioeconomic impact of viral infections of the respiratory (influenza, adeno-, corona-and rhinoviruses) and gastrointestinal tract (rotaviruses) have all been important factors in boosting the search for new antiviral agents and new modalities of antiviral chemotherapy. although many compounds with potent antiviral activity in cell cultures and in experimental animals have been detected, at present only several molecules and a-interferon have been approved by the health authorities for therapy of viral infections in humans. among these antiviral substances are several natural compounds isolated from plants used in traditional medicine, including polysaccharides [2] [3] [4] . polysaccharides (ps) constitute a stmcturally diverse class of biological macromolecules with a wide range of physicochemical properties, which are the basis for different applications in the broad field of pharmacy and medicine [5] [6] [7] [8] , several hundred natural ps are currently known and provide one of the richest and oldest reservoirs of structurally and functionally diverse biopolymers. ps constitute important members of the family of industrial water-soluble polymers. besides the classical applications of these biopolymers in industry and pharmaceutical practice, the relatively new field of pharmacologically active polymers will be discussed. ps from plants and other natural sources have long been known to exert antitumour, immunomodulatory, anticoagulant and other types of biological activity, including antiviral activity. the importance of the plant kingdom as a source of new antiviral substances will be illustrated by presenting a survey on natural-derived antiviral ps. in recent years, natural and synthetic polymers of a carbohydrate nature were proved to be selective inhibitors of several enveloped viruses, including such human pathogens as human immunodeficiency virus, herpes simplex virus, human cytomegalovirus, influenza virus and respiratory syncytial virus. recent efforts have been directed at the development of polysaccharidic molecules as selective inhibitors of different classes of viruses [9, 10] . in this search, the following molecular targets were envisaged: for dna viruses in general, the viral dna polymerase; for herpes simplex virus and varicella-zoster virus, the viral dna polymerase via a specific phosphorylation by the viral 2'deoxythymidine kinase; for (+/-) rna and (-) rna viruses, sadenosylhomocysteine hydrolase, a key enzyme in transmethylation reactions required for the maturation of viral mrna; for retroviruses, reverse transcriptase as an initiator of virus replication and/or cell transformation; and for several enveloped viruses (e.g., retro-, herpes-and rhabdoviruses), virus adsorption to the outer cell membrane. this review deals with antiviral ps compiled from several sources during the past decade. under the respective titles, we offer some brief descriptions of the most important global viral infections that are caused either by individual viruses or by groups of related viruses. they are restricted to those generally considered important in human and veterinary medicine and about which reports on active antiviral ps are recorded in the literature. acquired immunodeficiency syndrome (aids) is a pandemic immunosuppressive disease which results in life-threatening opportunistic infections and malignancies. in 1996, the number of aids infections was 23 million and 1.5 million people (half of them in africa) were killed by this disease worldwide. in order to combat human immunodeficiency virus (hiv), the causative agent of aids, enormous amounts of time and money have been dedicated to research into compounds which can be developed as therapeutic agents. hiv is a lentivirus which replicates within critical cells of the immune system, particularly cd4 t-cells and monocyte/macrophages, leading to a progressive loss of helper t-cells and profound immunosuppression. this condition is known as aids. without treatment, the cd4 t-lymphocytes of an infected patient are reduced markedly so that the patient is susceptible to a wide range of opportunistic infections, and ultimately dies. since this retrovirus, designated hiv, has been clearly identified as the primary cause of this disease, numerous compounds, also including plant-derived substances, have been evaluated for their inhibitory effects on hiv replication in vitro. furthermore, many plant products are being used by patients with aids in some countries without any scientific proof that they possess anti-hiv activity. several reviews on natural products for the chemotherapy of hiv infections have been recorded in the literature [11] [12] [13] [14] [15] [16] [17] [18] [19] . carbohydrates were among the natural molecules capable of treating hiv infection. various ps, including sulphated ps, have been found to be anti-hiv active substances of many antivirally active plant extracts, all of which are medicinal plants used in traditional medicine as anti-infectives. for example, a ps (mar-10) was isolated from the aqueous extract of the plant hyssop officinalis l., which was found to inhibit hiv-1 replication as demonstrated by the inhibition of hiv-1 p24 antigen and syncytia formation [20] . from aspalathos linearis burm., a popular medicinal plant in south africa, europe and japan, a ps with strong anti-hiv activity was isolated [21, 22] . this compound almost completely inhibited the binding of hiv-1 to mt-4 cells. the major sugar components of this purified ps were found to be glucose, galactose, mannose and xylose. premanathan et al [23, 24] investigated the ps extracted from rizophora apiculata blume and rizophora mucronata poir. in cell culture systems for their activity against human and simian immunodeficiency viruses (siv), both compounds inhibited hiv-1, hiv-2 or siv strains in various cell cultures and assay systems. it was found that these ps consisted mainly of neutral sugars and uronic acids. in a screening programe of thai plants for anti-hiv activity, it was found that 114 extracts of 81 species belonging to 32 families showed activity [25] . of the effective samples, the extracts of merremia peltata (l.) merrill were the most active. most of the anti-hiv agents were found to be anionic ps. furthermore, sulphated derivatives of paramylon, a (1-3)-p-d-glucan from euglena gracilis klebs. significantly inhibited the cytopathic effect of hiv-1 and hiv-2 [26] , while two naturally occurring ps isolated from the tropical climbing shrub ipomoea cairica (l.) sweet were found to strongly inhibit replication of hiv-1 in vitro [27] . according to de clercq [28] , the replicative cycle of hiv comprises several steps that could be considered adequate targets for chemotherapeutical intervention: virus entry, viral adsorption, virus-cell fusion, reverse (rna -> dna) transcription, proviral dna integration, viral (dna -> e^a) transcription (transactivation), viral (mrna -> protein) translation, virus release, viral assembly, budding and maturation. the most notable difference between hiv replication and other retroviruses is the intricate control of hiv gene expression by viral and cellular factors [29] . possible mechanisms by which hiv kills infected cells include the formulation of multinucleate syncytia, cytopathic components within the virions themselves, and interaction between viral envelope proteins and the cd4 molecule on the cell surface. three hiv enzymes are essential to the life cycle of the virus: hiv reverse transcriptase is crucial for viral replication; hiv protease processes viral polyproteins into fonctional enzymes and structural proteins, thereby facilitating maturation and infectivity of the virion particles; hiv integrase mediates hiv integration into the host chromosome. the chemotherapeutic strategies have therefore been focused on the development of inhibitors of these retroviral enzymes. recently, it has been reported that biological membranes arising from hiv-induced cell fusion, as well as syncytium formation between infected and non-infected cells are interesting approaches in research for a new hiv therapy [30] . natural sulphated ps such as dextran sulphate, pentosan sulphate, chondroitin sulphate and heparin have long been recognized as effective anti-hiv agents [31] [32] [33] [34] [35] . these compounds target the interaction between the viral envelope glycoprotein gpl20 and the cd4 receptor and, as a consequence, they inhibit not only vims adsorption to the cells but also virus-induced syncytium (giant cells) formation [36] [37] [38] [39] [40] [41] [42] . the inhibitory effect of dextran sulphate and its congeners on viral binding, viral replication and syncytium formation appears to be mediated by a specific interaction with the v3 region of gpl20. in addition, sulphated ps may also directly interfere with the binding of hiv particles to the heparin sulphate proteoglycan of the cell surface, whether or not this process occurs independently of, or cooperatively with, the viral envelope cd4 receptor interaction. this class of antiviral agents exhibits several unique properties that are not shared by other currently known antiviral agents: remarkable broadspectrum antiviral activity against hiv-1, hiv-2 and a series of other enveloped viruses; the ability to inhibit syncytium formation between hiv-infected and normal cd4 t-lymphocytes, a mechanism that drastically enhances hiv infectivity and a low induction of viral drugresistance [43] . it should be noted that sulphated ps owe their anti-hiv activity to the presence of the sulphate groups, which in turn are responsible for the inhibition of virus-cell binding. dextran is a highmolecular-weight ps consisting predominantly of a (1 -> 6) linked dglucose units. dextrans can differ in chain length and in degree of branching. the branching occurs via a (1 ^3) and a (1 -> 4) branch points. dextran fractions of different average molecular weight were obtained by partial hydrolysis. these different fractions were sulphated to obtain dextran sulphates of different molecular weight. busso and resnick [44] investigated the effects of three molecular weight ranges of dextran sulphate on five different hiv isolates. the higher molecular weight range of dextran sulphate showed the most potent activity as assessed by a quantitative syncytium formation assay. however, dextran sulphate is poorly adsorbed after oral administration and upon intravenous administration produces thrombocytopenia, so that there is little, if any, evidence for the in vivo efficacy of this compound and more generally of sulphated ps against hiv infection [45] . however, double-blind placebo-controlled clinical pilot trials in advanced hiv disease are being conducted with these compounds [46] . in summary, these compounds offer great promise as candidate drugs for the chemotherapy of hiv infections, and novel sulphated ps which have proved to be potent and selective inhibitors of hiv in vitro are being chemically synthesized [47] [48] [49] [50] [51] [52] . on the other hand, although several groups have reported that sulphated ps are potent and selective inhibitors of hiv, their therapeutic application is however limited by their anticoagulant activity. in view of possible improvements in therapeutic potential, a number of heparin derivatives with reduced anticoagulant activity were studied for their inhibitory activity in an hiv-dependent syncytium formation assay [53] . the most pronounced anti-hiv activity was observed with 30% succinylated standard heparin and 100% succinylated low molecular weight heparin. similarly, and in order to increase the ratio of anti-hiv activity to anticoagulant activity, selectively substituted sulphated ps with oh and/or cooh groups were chemically prepared [54, 55] . this group comprises a family of heterogeneous carbohydrates composed of long, unbranched ps modified, for example, by sulphations and acetylations. the acetylated derivatives are more active as inhibitors of hiv-induced giant-cell formation. furthermore, several soluble derivatized dextrans with different percentages of carbomethyl, benzylamide and sulphonate/sulphate groups were also evaluated for possible inhibitory effects on hiv-1 infection, and from the results obtained, their use as anti-hiv therapeutic agents can be proposed [56] . other natural sources of anti-hiv sulphated ps include algae and other marine organisms. sulphated ps have been found in a variety of marine animals, plants and microorganisms [57, 58] . aqueous extracts of many marine invertebrates have exhibited some activity in the national cancer institute's primary screen for anti-hiv cytopathicity [59] . carrageenans and fucoidan are sulphated ps extracted from red seaweed and brown algae, which have shown potent inhibitory activity against different viruses including hiv. for example, fucoidan, a complex sulphated ps from the alga fucus vesiculosus l. was found to inhibit hiv in vitro, presumably through a direct interaction of the ps with the hiv binding site of the target cells [60] . the aqueous-soluble extracts from this alga, a common littoral alga of the coast of the northern atlantic, the pacific ocean and the baltic sea, inhibited the activity of hiv reverse transcriptase enzyme as well as hiv-induced syncytium formation. beress et al [61] reported a new procedure for the isolation of the anti-hiv ps from this marine alga. a novel sulphated ps named calcium spirulan has been isolated as an antiviral principle from spirulina platensis (nordst.) geitl., a blue-green alga growing in some african and central and south american lakes rich in salts [62] [63] [64] [65] . chemical analyses of calcium spirulan suggest that it contains rhamnose, ribose, mannose, fructose, galactose, xylose, glucose, glucuronic acid and galacturonic acid. other sulphated ps from marine sources have demonstrated antiviral activities against hiv, blocking viral replication in cell culture without any toxicity to the host cells. examples include sulphated cell-wall ps from the mediterranean red alga asparagopsis armata harvey [66] , from the marine microalga cochlodinium polykrikoides blooms [67] , fiicose, the main component sugar of the ps isolated from the brown alga sargasum horneri (turner) c. agardh [68] and sargasum muticum (yendo) fensholt. [69] , sulphated ps from brazilian marine algae such as laminaria abyssalis ab joly & ec oliveira [70] , and from tropical marine algae of the codiaceae family [71] , and new sulphated p-galactans from the marine clam species meretrix petechialis lamarck [72] . other natural sources of anti-hiv sulphated ps include fiingi such as ganoderma lucidum (fr.) karst. [73] and lentinus edodes (lem.) mycelia [74] . inhibition of hiv-reverse transcriptases (hiv-rt) is currently considered a usefiil approach in the prophylaxis and intervention of aids and natural products have not as yet been extensively explored as inhibitors of this enzyme [75, 76] . furthermore, the mechanisms of action of sulphated ps may not only be due to the inhibition of virus cell adhesion, but also to the inhibition of hiv-rt [13, 15] . herpes infections are ubiquitous. approximately, 16-35%, 40-80% and >90% of the population are sero-positive for, or infected by herpes simplex virus type i (hsv-1), herpes simplex virus type ii (hsv-2) and varicella-zoster virus, respectively. more alarmingly, over the past decade, the incidence and severity of infections caused by hsv have increased due to the growth in number of immunocompromised patients produced by aggressive chemotherapy regimens, expanded organ transplantation and a greater occurrence of hiv infections. unfortunately, prolonged therapies with acyclovir, the most successful antiherpetic drug, have resulted in some undesirable complications and also induced the emergence of drug-resistant viruses. with this change in disease pattern, and the increase in drug use frequency, acyclovir-resistant hsv infections have emerged. infections with hsv range from simple cold sores and fever blisters to severe central nervous system disorders. development of effective antiviral medications has made prompt recognition important in primary care practice. appropiate therapy can significantly reduce both the medical and psychosocial ramifications of herpes infections and can greatly improve the quality of life of many patients [77] . thus, there is an urgent need for novel anti-hsv agents, especially those with a different mode of action from acyclovir. for the past decades, in addition to a variety of synthetic antiviral drugs with different molecular targets, a large number of phytochemicals have been recognized to control infections caused by viruses belonging to the herpesviridae family. ethnopharmacological screenings of medicinal plants from all over the world have led to the selection of several extracts which are active against herpes viruses. among natural antiviral agents, recent investigations have revived the interest in phyto-ps, which act as potent inhibitors of different enveloped viruses, including members of herpesviridae. for example, a neutral ps extracted from sclerotium glucanicum halleck., namely scleroglucan, revealed promising antiviral activity [78] . these ps are composed of a p-1,3-linked glucopyranose backbone with single p-l,6-linked glucopyranosyl branches every third subunit. the blockage on hsv infection takes place during the very early phases of the viral multiplication cycle, since the greatest inhibitory effect occurred when it was added during the attachment step. as a result of the intensive search for antiviral substances from medicinal plants, antiviral activity against hsv was found in extracts from cedrela tubiflora bertoni. leaves [79] , from prunella vulgaris l., a perennial plant commonly found in china, the british isles and europe [80] and from trichilia glabra l. leaves [81] . phytochemical studies indicate that these plants contain anionic ps as active constituents which may inhibit hsv by competing for cell receptors as well as by some unknown mechanisms after the virus has penetrated the cells. furthermore, the in vitro antiviral activity demonstrated by extracts of the medicinal plant achyrocline flaccida (weinm.) d.c. on hsv-1 is exerted early during viral replication, essentially during viral adsorption to host cells [82] . a bioguided purification process indicated that negatively charged ps were responsible for this antiviral activity. among natural antiviral agents of a carbohydrate nature, recent investigations have also revived interest in sulphated phyto-ps such as carrageenans and fucoidan from seaweed. sea algae represent a very interesting source of potential antiviral compounds, particularly the watersoluble sulphated ps which are abundant constituents of the cell wall. these compounds act as potent inhibitors of different enveloped viruses, including members of herpesviridae, and their activity is linked to the anionic features of the molecule which hinder the attachment of viral particles to host cells. for example, carrageenans are sulphated galactans that can be extracted from certain red seaweeds. they comprise a broad range of structures and can be divided into two families: the k-family, defined by the presence of a c4-sulphate group on the p-d-unit, and the x-family, characterized by the sulphate on c2 and constituted by all the variations of the ^.-structures [83] . investigations have detected antiviral activity in a significant number of algae from various marine environments such as california, united kingdom, the mediterranean, india and japan [84] . in the course of a screening study on the biological properties of natural marine products, diverse carrageenans isolated from the red seaweed gigartina skottsbergii setch. et gard. have proved to be potent inhibitors of hsv-1 and -2 in vitro [85] . mode of action studies are consistent with the carrageenans having a major effect on the attachment of virus to the cells. adsorption of hsv-1 is primarily mediated by the envelope glycoprotein gc, which binds to heparan sulphate residues present on the proteoglycans on the surface of target cells. antiherpetic activity was directly correlated to the amount of a-d-galactose 2,6-disulphate residues in the natural carrageenans. carrageenans were also isolated from chilean samples of stenogramme interrupta (c. ag.) mont., a red seaweed from california [86] . in the course of screening for antiviral properties in sulphated ps isolated from marine algae of the south american coast, carlucci et ah [87] reported the antiviral activity of mannans and xylogalactans isolated from the red seaweed nothogenia fastigiata turner (lam.), and a sulphated galactan from pterocladia capillacea (gmelim) barnet & thuset. hsv-1 and -2 were the most sensitive viruses of these compounds due to an inhibition of virus binding. structural analysis of two xylomannans extracted from nothogenia fastigiata was carried out [88, 89] . the results are consistent with the general pattern previously reported for other xylomannans of the same system, a(l -> 3)-linked dmannans 2-and 6-sulphated and with single stubs of p(l -> 2)-linked dxylose, but one of the new samples contains a significant amount of 2,6disulphated units. the presence of sulphate groups in the molecule was essential to the antiviral properties of these ps [90] . the antiviral activity of sulphated ps is known to increase with molecular weight or the degree of sulphation. however, sulphated ps are generally endowed with anticoagulant properties that may hamper their usefulness as antiviral drugs. duarte et al [91] reported the inhibitory effect of sulphated galactans from the marine alga bostrychia montagriei harvey. the results of the activated partial thromboplastin time assay and the thrombine time assay indicated that three natural sulphated ps have very low anticoagulant activity, confirming that there is no relation between the antiviral and anticoagulant properties. anti-hsv sulphated ps have also been isolated from other seaweeds such as rhamnan sulphate from monostroma latissimum wittrock [92] , and calcium spirulan from the blue-green alga spirulina platensis [62] . lee et al. [93] initiated studies into the structure-activity relationships of calcium spirulan. calcium ion binding with the anionic part of the molecule was replaced with various metal cations. replacement of the calcium ion with sodium and potassium ions maintained anti-hsv-1 activity while divalent and trivalent cations reduced the activity. the cell wall sulphated ps of the red microalga porphyridium spp. honey exhibited impressive antiviral activity against hsv-1 and -2 both in vitro (cell culture) and in vivo (rats and rabbits) [94] . it seems that this ps is able to inhibit viral infection by preventing adsorption of the virus into the host cells and/or by inhibiting the production of new viral particles inside the host cells [95] . novel fiican sulphates with anti-hsv activity were also isolated from the hot water extract of an edible brown alga sargassum horneri [96] and from the brown seaweed leathesia difformis (linnaeus) areschong [97] . other natural sources of anti-hsv ps include fimgi, e.g., various protein bound ps isolated from ganoderma lucidum, a basidiomycetous fungus used to treat human diseases in oriental folk medicine [98] . the possible mode of antiviral activity of these ps seems to be related to their binding with hsv-specific glycoproteins responsible for attachment and penetration, impeding the complex interactions of viruses with cell plasma membranes [99] [100] [101] . furthermore, different semisynthetic ps were evaluated for their inhibitory effect on in vitro replication of hsv-1 and -2 [102] [103] [104] . some neutral and negatively charged carbohydrates were able to inhibit viral infection by interfering mainly with the adsorption process. the in vitro and in vivo effects of sulphated ps were also investigated against the pseudorabies virus (suid hsv-1), the most notable of which is porcine hsv which has a well-documented history of epizootic infections, especially in europe [105] . in vitro experiments revealed that sulphated ps significantly reduced the number of lytic plaques, but in vivo only heparin protected mice against the pseudorabies virus. in terms of their biological and pathogenetic properties, the herpes viruses fall naturally into several subfamily groupings, including cytomegalovirus (cmv), although detailed classification is at present premature. nevertheless the cmv clearly constitute a group of their own with internal consistency. human cmv is, together with hsv-1, one of the agents responsible for opportunistic infections in hiv-infected people. as can be seen in the present review, sulphated ps show antiviral activitiy against enveloped viruses in vitro. it has been suggested that these antiviral mechanisms depend on their polyanionic properties, which interact with the positively charged amino acid sequence of viral envelope glycoproteins. natural sulphated ps can act as potent inhibitors of cmv. the cell wall mucilages of seaweeds are known to be rich in sulphated ps with potential anti-cmv activity. recently, blinkova^/a/. [106] reviewed information on spiridina platensis, a blue-green alga with diverse biological activity. preparations obtained from this alga have been found to be active against several enveloped viruses, including cmv. it was revealed that calcium spirulan, a sulphated ps isolated from spirulina platensis, selectively inhibited the penetration of the virus into host cells [62] . retention of molecular conformation by chelation of calcium ion with sulphate groups may be indispensable to its antiviral effect. the antiviral activity of a polysaccharidic fraction obtained from another red seaweed, nothogenia fastigiata, was also reported [107] . its mode of action against cmv could be ascribed to an inhibitory effect on virus adsorption. other natural sources of sulphated ps include brown algae. a sulphated polysaccharide with potent anti-cmv activity was isolated from the brown alga sargassum horneri [68] . time-of-addition experiments suggested that it inhibited not only the initial stages of viral infection, such as attachment to and penetration into host cells, but also later replication stages after virus penetration. fractions of fiicoidan, another polysaccharide isolated from the brown seaweed leathesia difformis were also found to be selective antiviral agents against human cmv [97] . these compounds offer great promise as candidate drugs for the chemotherapy of cmv infections, and novel sulphated ps are being chemically prepared [108] . ps from terrestrial plants have also been reported as anti-cmv agents. for example, ps from three plant species. astragalus brachycentrus d. c, astragalus echidnaeformis sirjaev and sterculia urens roxb., which are devoid of in vitro antiviral activity, were evaluated in mouse models of murine cmv infections [109] . treatment with the compounds needed to be started one day prior to virus inoculation for maximum protective benefit. treatments starting after virus inoculation were ineffective. the mannose-specific plant ps from the orchid species cymbidium hybrid cym., epipactis helleborine (l.) crantz. and listera ovata (l.) r.br. svenska are potent and selective inhibitors of human cmv in vitro [110] . they presumably interact at the level of virion fusion with the target cell. papillomaviruses are members of the papopavirus family and are distinguished from other members by virtue of their association with a variety of warts in different parts of the body. some types have been implicated in genital warts and cervical carcinomas, while others seem to be associated with other distinctive warts elsewhere. no generally effective control is available, although potentially dangerous lesions can be removed by cryosurgery or laser treatment. however, some medicinal plant preparations have been reported to be beneficial; conceivably these may work by promoting healing or stimulating immune responses, rather than directly inhibiting the virus. natural high-molecular weight sulphated or sulphonated ps, such as cellulose sulphate and dextran sulphate, may be useftil non-toxic microbicidal compounds that are active against a variety of sexuallytransmitted disease agents, including bovine papillomavirus type 1 and human papilloma virus type 11 and type 40 [111] . hepatitis b virus (hbv) is widespread throughout human populations, specially in asia and africa, and it has been estimated that over 200 million carriers exist, some of whom are eventually expected to develop liver carcinoma or cirrhosis. hbv shows a strict tropism for liver hepatocytes in which it displays a protected replication with resultant foci of liver necrosis. the virus is a member of the hepadnaviridae, along with several other species, and it replicates by a mechanism which appears to be unique to this family. in contrast, hepatitis a virus is a picornavirus and the hepatitis d agent appears to be a viroid-like rna enclosed within a hepatitis b capsid, and consequently depends upon its association with the hbv for its spread and survival. control may be effected by passive immunization (with hyperimmune globulin) or by various types of vaccines which are currently being developed and improved. specific chemotherapy has not been consistently successful, but in some countries (e.g., india and china), plant extracts have provided some success. among natural antiviral agents of carbohydrate nature, sulphated ps such as x and k carrageenans showed a potent inhibitory effect on the replication of hbv in the human hepatoma cell line plc/prf/5 [112] . the two types of carrageenans resulted in concentration-dependent reduction of hbv-antigen expression and hbv infectivity, emerging as promising candidates for chemotherapy of acute hepatitis. human rotavirus (hrv), a member of the reoviridae, is a non-enveloped virus which is the major etiologic agent of severe dehydrating gastroenteritis in children worldwide. for the treatment of rotavirus gastroenteritis, intravenous fluid administration has been used successfully for dehydration from diarrhoea. however, in the case of severe inpatients and immunocompromised hosts who are suffering from prolonged diarrhoea and fever, virus-specific treatment will be necessary if possible. several compounds, biomaterials and plant extracts have been found to be inhibitory for hrv of some species in vitro. for example, extracts of stevia rebaudiana bertoni, a family member of chrysanthemum originating from paraguay in south america, inhibited the replication of all four serotypes of hrv in vitro [113] . binding assays with radiolabeled purified viruses indicated that the inhibitory mechanism of this plant extracts is the blockage of virus binding. the inhibitory components of stevia rebaudiana were found to be heterogeneous anionic ps with different ion charges. influenza continues to have a significant impact on public health. annually, 20,000 deaths and 100,000 hospitalizations are attributed to "flu" in the united states alone. airborne transmission, facile viral mutation, vaccine shortages, and actual and perceived side effects and limitations of both vaccines and prophylactic drugs contribute to the drive for new therapies and preventive medicines for influenza. research during the last fifteen years has elucidated many of the mechanisms by which the influenza virus invades, captures and mobilizes the replication capabilities of a host cell, providing new targets in the search for antiviral treatments. for the past decades, besides a variety of synthetic antiviral drugs with different molecular targets, a large number of phytochemicals have been recognized to control infections caused by the influenza virus. serkedjieva et al [114] reported the antiviral activity from a lyophilized infusion from flowers of sambucus nigra l., aerial parts of hypericum perforatum l. and roots of saponaria officinalis l. the preparations contain ps which could be responsible for their antiviral properties. reports on the anti-influenza virus activity of natural ps are recorded in the literature. from a pine cone extract of pinus parviflora sieb. et zucc, an acidic polysaccharide was isolated [115, 116] . this compound showed significant inhibition of both the viral protein synthesis in infected cells and virion-associated rna-dependent rna polymerase activity. in animal models in vivo, most mice inoculated intranasally or intracerebrally with lethal doses of the influenza virus a/wsn/33 died within 12 days. however, the infectivity of the virus that had been preincubated with a polysaccharide prepared from cones of pinus parviflora was significantly reduced [117] . in 1991, sakagami et a/. [118] reviewed the potential antiviral efficacy of natural polysaccharidic-related materials isolated from the pine cone extracts. sulphated ps, potent antiviral agents, were also evaluated in vitro as inhibitors of influenza virus replication [119] . the fact that the sulphated ps are inhibitory to some myxoviruses and retroviruses but not to others seems to depend on the composition of the amino acid sequences of the viral envelope glycoproteins that are involved in virus-cell binding and fiision [120] . as can be seen in the present review, other natural sources of antiviral sulphated ps include the marine environment. sulphated and nonsulphated ps with anti-influenza activity have been isolated from marine sources such as the green marine alga ulva lactuca l. [121] , the japanese sea alga crenomytilus grayanus dunkei. [122, 123] , the blue-green alga spirulina platensis [106] , the red seaweed nothogenia fastigiata [107] and the marine microalga cochlodinium polykrikoides [67] . novel marine ps which have proved to be potent and selective inhibitors of the influenza virus in vitro are being chemically synthesized [124, 125] . the measles virus (mv) is a paramyxovirus which has been associated with several chronic diseases. since the advent of mass immunization campaigns, the incidence of measles has decreased dramatically in many developed countries. this is not the case elsewhere, however, where the virus still takes its toll in large numbers, especially in malnourished or immunocompromised individuals. secondary bacterial infections are prevalent in these populations. although most infections are relatively short-lived and innocuous in healthy individuals, encephalitis can develop in approximately 0.1% of cases; hence vaccination is desirable. following respiratory infection, the virus readily invades local lymphoid tissues and gains access to the blood, from where it disseminates widely in the body. there has not been much demand for chemotherapy; vaccination seems to be the choice. no really effective antiviral has been evaluated, although some plant compounds have been shown to inhibit mv replication effectively, such as ps from the blue-green alga spirulina platensis [62] . rabies has long been recognized as a scourge of livestock with occasional and often fatal intrusions into humans and their pets. initially the virus gains access to muscle, sensory organs or skin, and there replicates in local unmyelinated (sensory) nerve fibers. many layers of epithelial cells are susceptible to the virus but the principal target is the unprotected neuron. the brain is where most damage is done, resulting in the familiar psychomotor disturbances. in addition, the virus spreads into salivary glands (by axoplasmic flow again), from where it is secreted from the asinar mucous cells and transmitted to the victim. the objective of much research on the rabies virus has been the development of better vaccines and therapeutic measures, some of them from natural sources. for example, different natural polymeric carbohydrates inhibited rabies virus infection in chicken-embryo-related cells by interfering with the virus adsorption process [126] . the charge density and the polymeric backbone of the molecules seem to play a role in influencing the antiviral properties, whereas other features such as the sugar moieties do not appear to be relevant. rubella is the sole member of the rudivirus genus in the togavirus family. the virus is transmitted by the respiratory route, and multiplies in upper respiratory tissues from where it gains access to the blood, accompanied sometimes by a characteristic rash and other symptoms. the infection may be more severe in pregnant women. vaccination programmes are in place in many countries, and the belief is that the virus will eventually be brought under control. although there seems to be no apparent role or need for specific antiviral therapy, reports on the antirubella activity of natural products and extracts have been reported [57] . for example, the natural carbohydrate scleroglucan, three sulphate derivatives and dextran sulphate were investigated for their inhibitory effect on rubella virus infection on vero cells [127] . the results indicated that ps blocked a step in virus replication subsequent to virus attachment, such as internalisation and/or uncoating. bunyavirus sandfly fever sicilian virus (sfsv) is a phlebovirus, a member of the bunyaviridae family, which cause acute, incapaciting but self-limiting diseases in man, and which have had a major impact in europe, the middle east and africa. sfsv is a virus closely related to other viruses which are considerably more pathogenic both in humans and animals, such as the african swine fever virus (asfv) and the crimean-congo haemorrhagic fever virus (cchfv). the asfv is a highly contagious bunyavirus infection of pigs which is clinically indistinguishable from the unrelated hog cholera virus. since 1957, epidemics have been recorded intermittently in several european countries bordering the mediterranean, in the caribbean, and in brazil, and more recently in other european countries and parts of africa. although pigs appear to be the only species infected naturally, the virus can be adapted to grow in a variety of other cell types and experimentally infects goats and rabbits. in view of this, it seems worthwhile to consider the possibility of its natural persistence in animals other than swine and man. transmission of asfv usually occurs through the respiratory route. following inhalation, the virus invades and attacks local lymph nodes and the endothelium of blood vessels, with the resuh that high titers of virus circulate in the blood and lymph, and eventually in various secretory fluids. attempts have been made to produce vaccines, but without much success. in any case, the virus mutates frequently in the wild and crossprotection of animals is not complete. since the virion contains several enzyme activities, and the dna can probably code for more in the infected cell, the possibility of specific chemotherapy, along the lines of antiherpes chemicals, should be profitable. several studies have been undertaken to develop drugs which could be used for treatment of fever infections caused by viruses belonging to the bunyaviridae family, including natural ps [128, 129] . antiviral activity was estimated by the reduction of the cytophatic effect of sfsv on infected vero cells. several ps, such as carrageenans, fucoidan, dextran sulphate and pentosan polysulphate caused a concentration-dependent reduction in the virus yield. fabregas et al [130] screened several extracts from marine microalgae for in vitro inhibition of the replication of asfv. that this inhibition could be due to sulphated ps was suggested because the same pattern of viral inhibition was obtained by using exocellular extracts from microalgae enriched in these compounds and/or dextran sulphate of high molecular weight. other natural ps should be considered for the potential treatment of significant human infections caused by bunyaviruses. the mouse model of punta toro virus, a phlebovirus member of the bunyaviridae family, is a model for studying the treatment of rift valley fever infection. tragacanthin ps from astragalus brachycentrus and astragalus echidnaeformis plants caused reduction in mortality, liver infection scores, liver and spleen virus titers, and serum transaminases in treated mice [131] . poliomyelitis is not the scourge that it once was, thanks to improved hygienic practices which have effectively blocked the spread of wild-type polioviruses, especially in developed countries, and thanks to the widespread use of vaccines. the viruses, like other enteroviruses, are transmitted by the fecal-oral route; following ingestion, they replicate in various cell types in the pharynx and small intestine (they are quite stable to stomach acids) and gain access to the circulation. from there, they disseminate and occasionally gain access to the central nervous system, where they may produce the characteristic lesions leading to poliomyelitis. control of virus spread accompanied by vaccination continues to provide the best means of alleviating poliomyelitis. the use of antivirals seems to have less application than for many other viral infections, although the poliovirus continues to serve as a useful laboratory model for antiviral screening. the plant kingdom and seaweeds may be a source of potentially useful and interesting antiviral compounds against polioviruses. for example, anti-poliovirus activity was detected in a polysaccharidic-rich fraction from extracts of several korean seaweeds [132] , and from the leaves of the meliaceae tree trichilia glabra [81] . other compounds of carbohydrate nature with antiviral activity against other viruses have been isolated from natural sources. several compounds belonging to different classes of sulphated ps were evaluated for their inhibitory effects on the replication of the arenavirus junin and tacaribe in vero cells [133, 107] . very potent and selective inhibitors were the sulphated ps dextran sulphate, ^.-carrageenan, fucoidan, heparin and pentosan polysulphate. examples also include aloe polymannose, a high mannose purified from the aloe barbadensis miller plant, which showed activity in enhancing antibody titers against coxsackievirus b3-induced myocarditis in murine models of the disease [134] , and fungal ps which are active in vivo in sendai virus infection [135] . although relatively little work has been done on natural antivirals against plant viruses, several reports concerning antiviral activity against plant virus infection have been recorded; for example, yeast mannans with antiviral activity against the tobacco mosaic virus infection in tobacco plants [136] , and lichenan ps from iceland moss which exhibited antiviral activity against the potato viruses [8] . jacquir immune defic syndr hum retrovirol the technical assistance of ms. brooke-turner is gratefully acknowledged. key: cord-013176-6ckuya1w authors: ninfali, paolino; antonelli, antonella; magnani, mauro; scarpa, emanuele salvatore title: antiviral properties of flavonoids and delivery strategies date: 2020-08-21 journal: nutrients doi: 10.3390/nu12092534 sha: doc_id: 13176 cord_uid: 6ckuya1w this review summarizes the latest advancements in phytochemicals as functional antiviral agents. we focused on flavonoids, like apigenin, vitexin, quercetin, rutin and naringenin, which have shown a wide range of biological effects including antiviral activities. the molecular mechanisms of their antiviral effects mainly consist in the inhibition of viral neuraminidase, proteases and dna/rna polymerases, as well as in the modification of various viral proteins. mixtures of different flavonoids or combination of flavonoids with antiviral synthetic drugs provide an enhancement of their antiviral effects. recent strategies in drug delivery significantly contribute to overcoming the low bioavailability of flavonoids. frequent viral infections worldwide have led to the need for new effective antiviral agents, which can be identified among the various phytochemicals. in this light, screening the antiviral activities of a cocktail of flavonoids would be advantageous in order to prevent viral infections and improve current antiviral therapies. in the past two decades, studies conducted in our laboratory focused on the antioxidants present in vegetable foods and on their capacity to reduce the adverse physiological effects of the oxygen free radicals [1, 2] . the research was also aimed at the technologies of food transformation in order to preserve, as much as possible, the antioxidants in the final products [3] . antioxidants are mainly represented in nature by the liposoluble vitamins e and a, β-carotene, hydro-soluble vitamin c and a wide range of amphipathic molecules, broadly termed phenolic compounds [1] . these compounds are divided into several sub-classes, including phenolic acids and analogues, stilbenes, flavonoids and their analogues. flavonoids possess important health protective effects, including anti-inflammatory, anticancer and antiviral properties [4] [5] [6] [7] [8] [9] [10] . there are in nature more than 6000 flavonoids, which have been structurally identified and divided in classes: flavones (e.g., apigenin), flavanols (e.g., quercetin), flavins (e.g., epigallocatechin-3-gallate), isoflavones (e.g., genistein) and anthocyanidins (e.g., cyanidin). flavonoids occur in their free or conjugated form or are often esterified with one or two sugars with o-glycosidic or c-glycosidic bonds [11] . in the past decade we purified and studied the biological effects of a group of the flavonoid c-glycosides derived from apigenin, namely vitexin, vitexin-2-o-rhamnoside and vitexin-2-o-xyloside ( figure 1 ). the interest in the antiviral activity of natural flavonoids has increased in the last decade because of the frequency of viral infections, particularly influenza infections, which affect several million patients annually [12] . while vaccination is the primary strategy for influenza prevention, there are scenarios for which vaccination is not possible and antiviral molecules represent an important sanitary presidium. synthetic antiviral drugs often show limited efficacy and serious adverse effects [13] , whereas herbal extracts, known for their antiviral properties with no or mild side effects, may be a viable alternative for treating various viral diseases [14] . viruses consist of nucleic acid (dna or rna) enclosed in a protein structure, called capsid, which can be surrounded by a lipid membrane named the envelope. the infective unit of the virus is called the virion and this parasite can replicate only inside host cells, hijacking the molecular machinery and controlling dna replication, rna transcription and the protein translation processes. viruses attack the host cells through adsorption to receptors specific for each type of target cells, penetrating through the cell membrane, then the genetic material of the virus is liberated to replicate its own genome, produce new viral proteins and obtain new virions [15] . in 2017, the antiviral effects of various phytochemicals were reviewed by kapoor et al. [15] , taking into consideration many categories of compounds, ranging from flavonoids to saponins and lignans. in the same year, another group published a review focused specifically on the antiviral effects of the six classes of flavonoids [16] . in 2020, an interesting review regarding the methods for delivery of phytochemicals to increase their bioavailability in human tissues has been published [17] . in this review, we first summarize flavonoids along with their class and plant sources, with particular attention to apigenin, vitexin and its derivatives (table 1) . we then discuss the antiviral action mechanisms of flavonoids, their combinations with conventional antiviral drugs in multitarget cocktails, and the delivery strategies used to increase their bioavailability and antiviral efficacy. the interest in the antiviral activity of natural flavonoids has increased in the last decade because of the frequency of viral infections, particularly influenza infections, which affect several million patients annually [12] . while vaccination is the primary strategy for influenza prevention, there are scenarios for which vaccination is not possible and antiviral molecules represent an important sanitary presidium. synthetic antiviral drugs often show limited efficacy and serious adverse effects [13] , whereas herbal extracts, known for their antiviral properties with no or mild side effects, may be a viable alternative for treating various viral diseases [14] . viruses consist of nucleic acid (dna or rna) enclosed in a protein structure, called capsid, which can be surrounded by a lipid membrane named the envelope. the infective unit of the virus is called the virion and this parasite can replicate only inside host cells, hijacking the molecular machinery and controlling dna replication, rna transcription and the protein translation processes. viruses attack the host cells through adsorption to receptors specific for each type of target cells, penetrating through the cell membrane, then the genetic material of the virus is liberated to replicate its own genome, produce new viral proteins and obtain new virions [15] . in 2017, the antiviral effects of various phytochemicals were reviewed by kapoor et al. [15] , taking into consideration many categories of compounds, ranging from flavonoids to saponins and lignans. in the same year, another group published a review focused specifically on the antiviral effects of the six classes of flavonoids [16] . in 2020, an interesting review regarding the methods for delivery of phytochemicals to increase their bioavailability in human tissues has been published [17] . in this review, we first summarize flavonoids along with their class and plant sources, with particular attention to apigenin, vitexin and its derivatives (table 1) . we then discuss the antiviral action mechanisms of flavonoids, their combinations with conventional antiviral drugs in multi-target cocktails, and the delivery strategies used to increase their bioavailability and antiviral efficacy. the most common flow chart for studies regarding the antiviral activity of phytochemicals is focused on the immediate testing of the whole natural extract, by dividing the polar from the non-polar constituents. after that, fractions with remarkable activity in in vitro antiviral assays, such as cytophatic effect, neutralization assay, hemagglutination, viral enzyme inhibition, or virion number reduction assay, are further fractionated through chromatographic techniques in order to purify the active phytochemicals. effective compounds are then used on virus-infected animals or in human clinical trials in order to assess their effective antiviral properties [15] . the complete procedure, when interfaced with chemical libraries, represents the basis for high throughput screening assays [15] . the parameter used for assessing antiviral efficiency of drugs and phytochemicals is represented by 50% inhibitory concentrations (ic 50 ) or by 50% effective concentration (ec 50 ). table 2 shows the main flavonoids studied for antiviral activity along with their investigated mechanisms of action. in the first section, we point out the studies focused on apigenin, vitexin and their derivatives, which were found active against many viruses like human hepatitis c virus (hcv), herpes simplex virus-1 (hsv-1), human hepatitis a and b viruses (hav; hbv), rhesus rotavirus (rrv) and influenza virus. the natural extract of eclipta alba (asteraceae) was shown to be able to inhibit the hcv replicase in a cell culture system, which resulted in reduced hcv rna titer and translation level of viral proteins [19] . through bioassay-based fractionations of the natural extract, the authors identified two flavonoid compounds: apigenin and luteolin, which, tested individually, exhibited a dose-dependent inhibition of hcv replicase in vitro [19] . quercetin, extracted from embelia ribes (mirsinaceae), exhibited antiviral effects against hcv, exerted through activity inhibition of the viral protease non-structural protein 3 (ns3), leading to a decrease in hcv replication [36] . furthermore, the flavanol quercetagetin was found to inhibit hcv rna-dependent rna polymerase (rdrp) through inhibition of rna binding to the viral polymerase, a mechanism associated with broad genotypic activity against several hcv strains and a high barrier to resistance mechanisms [39] . luteolin and quercetin showed anti-hcv effects in hepatoma huh-7 cells transfected with non-structural protein 5b (ns5b) cloned gene vector, that codifies for the ns5b polymerase of hcv virus [40] . naringenin and quercetin possess antiviral activity against hcv, but naringenin showed stronger inhibition of virion assembly, whereas quercetin inhibited viral translation by blocking non-structural protein 5a (ns5a) and internal ribosome entry site (ires)-mediated translation, as well as heat shock protein 70 (hsp70) induction [41] . bioinformatics tools were also used to study the interaction of various phytochemicals with viral proteins that possess pivotal roles in the production of new virions and in the infection of the host cells. this approach may be a useful premise for deeper investigation regarding flavonoids which have provided interesting evidence of interactions with viral proteins. for instance, naringenin, diosmetin, apigenin and luteolin were all able to bind to the ns5b protein of hcv with higher affinity when compared with the antiviral drug sofosbuvir, inhibiting the activity of this viral enzyme [25] . epigallocatechin-3-gallate (egcg), the principal tea derived catechin, efficiently inhibited cell culture-derived hbv entry into hepatocellular cell lines, independent of the hbv genotypes, through a mechanism that involves the clathrin-dependent endocytosis of the hbv receptor sodium taurocholate co-transporting polypeptide (ntcp) from the plasma membrane, followed by its protein degradation [26] . the extract obtained from erythrina speciosa (fabaceae) exerted antiviral effects against hav-h10 viruses mainly due to vitexin which, isolated from the extract, exhibited an antiviral activity against this virus with ec 50 = 41.6 ± 7.6 µm [42] . the authors showed that the flavone vitexin can interact with the binding pocket of hav 3c proteinase, inhibiting this enzyme [42] . hcv virions inhibition of virion assembly vitexin, extracted from the plant flos trollii, (caryophyllaceae), exerted its anti-h1n1 influenza virus effects through partially down-regulating toll-like receptor 3 (tlr3) and toll-like receptor 7 (tlr7) pathways and up-regulating toll-like receptor 4 (tlr4) molecular pathway [38] . tlrs are transmembrane glycoproteins, which are privileged targets of several viruses and can be activated by endogenous molecules in the context of inflammation. tlr activation produces pro-inflammatory cytokines through nuclear factor kappa-light-chain-enhancer of activated b cells (nf-kb) signaling pathway or through interferon regulatory factor 3 (irf3) and interferon regulatory factor 7 (irf7). some viruses enter the host cells by binding tlr3, but after their entrance the viruses are able to inhibit cytokine production, thus impairing the immune response. phytochemicals able to decrease the binding between tlrs and virus particles can slow the infective process. interestingly, virus infection can lead to an induction of the inflammation process, characterized by excessive production of nitric oxide (no), interleukin-1 (il-1), interleukin-6 (il-6) and tumor necrosis factor-α (tnf-α). it was shown that various flavonoids enhance the production of interferon-β (ifn-β) in order to counteract the viral infections [38] . baicalin, baicalein, wogonin, chrysin and oroxylin a, isolated from scutellaria baicalensis, showed anti-h1n1 activities, with ic 50 values of 7.4, 7.5, 2.1, 7.7 and 12.8 µm, respectively, and were all more potent than the conventional antiviral drug oseltamivir phosphate, which had an ic 50 of 45.6 µm [22] . these flavonoids increased the transcriptional activity of nuclear factor erythroid 2-related factor 2 (nrf2), the master regulator of the antioxidant responses, whose activation is related to the antiviral effects of scutellaria baicalensis [22] . the natural extract of tetrastigma hemsleyanum (vitaceae) contains many flavonoids, including vitexin, vitexin-2-o-rhamnoside, isorhamnetin, rutin, kaempferol, astragalin, quercitrin, quercetin and iso-quercetin, which were shown to be able to exert anti-influenza virus activity, with different efficiency, through the reduction of the number of plaques induced by the influenza virus in infected madin-darby canine kidney (mdck) cells [21] . similarly, the flavonoid quercetin-3-rhamnoside, extracted from houttuynia cordata (saururaceae), exerted anti-influenza a/ws/33 activity reducing virus-mediated cytopathic effects, directly interacting with virus particles [37] . furthermore, the same authors showed that quercetin-3-rhamnoside exerted anti-influenza virus activity in mice, when used at 6.25 mg/kg/day for six days after influenza virus infection [45] . flavonoids sanggenon o, egcg and chamaejasmin were all potentially able to inhibit dengue virus replication by blocking the asn-130 glycosylation site of the viral protein non structural protein 1 (ns1) [23] . baicalin, a flavonoid derived from scutellaria baicalensis (lamiaceae), exhibited viricidal activity against dengue virus-2 (denv-2) extracellular virions with ic 50 = 19.6 ± 0.2 µm, exerted an anti-adsorption effect with ic 50 = 40.5 ± 0.4 µm and also inhibited virus replication with ic 50 = 30.2 ± 0.2 µm [44] . studies of the antiviral effects of the flavonoids fisetin, quercetagetin, and pinocembrin showed that fisetin can inhibit the replication molecular machineries of dengue virus and enterovirus a71 [35] . furthermore, other antiviral mechanisms of the pinocembrin consist in targeting the molecular machinery used by the zika virus to replicate its own genome inside the host cells [33] . this flavanone acts on the post-entry processes of zika virus replication cycle through the inhibition of both viral rna production and synthesis of envelope proteins [33] . interestingly, the plant moringa oleifera lam (moringaceae) provides a rich and rare combination of several phytochemicals, including the flavonoids quercetin and kaempferol, and its leaves extract can be applied as a prophylactic or therapeutic anti-hsv-1 medicine [29] . the extract obtained from moringa oleifera lam remarkably reduced the plaque formation induced by wt hsv, thymidine kinase-deficient hsv and phosphonoacetate-resistant hsv strains [29] . furthermore, the extract obtained from erythrina speciosa possessed antiviral properties against the hsv-1 virus, mainly due to vitexin which exhibited an antiviral activity with ec 50 = 80.9 ± 6.2 µm, exerted through the interaction of this flavone with the binding pocket of hsv-1 thymidine kinase [42] . vitexin and luteolin from aspalathus linearis (fabaceae) showed antiviral activity against rrv with ic 50 of 129 µm and 116 µm , respectively; interestingly, apigenin-7-o-glucoside from melissa officinalis (labiateae) inhibited viral growth, with an ic 50 of 150 µm, through the reduction of the number of rrv-induced plaques in infected ma104 cells [20] . tangeretin and nobiletin, two polymethoxyflavones extracted from citrus reticulate "chachi", possess anti-rsv properties. tangeretin exhibited a dose-dependent inhibition of rsv-induced plaque formation on hep-2 cells, through inhibition of rsv entry into host cells and viral replication. furthermore, tangeretin decreased the levels of rsv phosphoprotein (p protein), which is associated with the viral genome to form the holo-nucleocapsid. the extent of the antiviral effect of this phytochemical was comparable to the conventional antiviral drug ribavirin [32] . the knowledge of the molecular mechanisms of virus infection and phytochemical antiviral actions is fundamental in planning an effective therapeutic approach. the main mechanisms involved in the antiviral effects of phytochemicals are focused on the targeting of viral enzyme activities. many natural molecules target the catalytic activity of the influenza virus neuraminidase, also called sialidase. this enzyme is a glycoside hydrolase that cleaves the glycosidic linkages of sialic acids ( figure 2 ). various phytochemicals inhibit the activity of viral sialidases, hampering the release of new virions from the infected cells and preventing new infections [47] . another enzyme with a pivotal role in influenza a virus infection is rdrp, which is composed of three subunits: polymerase acidic subunit (pa), protein binding 1 subunit (pb1) and protein binding 2 subunit (pb2). the enzyme synthesizes viral mrnas using short capped primers derived from host cellular mrnas, which are cut by the viral endonuclease. the n-terminal domain of the pa subunit contains a typical endonuclease active site and harbors the rna/dna endonuclease activity, which is essential for viral growth [48] . the knowledge of the molecular mechanisms of virus infection and phytochemical antiviral actions is fundamental in planning an effective therapeutic approach. the main mechanisms involved in the antiviral effects of phytochemicals are focused on the targeting of viral enzyme activities. many natural molecules target the catalytic activity of the influenza virus neuraminidase, also called sialidase. this enzyme is a glycoside hydrolase that cleaves the glycosidic linkages of sialic acids (figure 2 ). various phytochemicals inhibit the activity of viral sialidases, hampering the release of new virions from the infected cells and preventing new infections [47] . another enzyme with a pivotal role in influenza a virus infection is rdrp, which is composed of three subunits: polymerase acidic subunit (pa), protein binding 1 subunit (pb1) and protein binding 2 subunit (pb2). the enzyme synthesizes viral mrnas using short capped primers derived from host cellular mrnas, which are cut by the viral endonuclease. the n-terminal domain of the pa subunit contains a typical endonuclease active site and harbors the rna/dna endonuclease activity, which is essential for viral growth [48] . enzymes involved in the hcv virus replication, like the protease ns3 and the polymerase ns5b can also be efficiently inhibited or modulated by phytochemicals ( figure 3 ). ns3 is a hcv nonstructural protein, which acts as a serine protease. its n-terminal domain can interact with the viral non structural protein 2 (ns2), while the c-terminal domain acts both as helicase and nucleoside triphosphatase. enzymes involved in the hcv virus replication, like the protease ns3 and the polymerase ns5b can also be efficiently inhibited or modulated by phytochemicals ( figure 3 ). ns3 is a hcv nonstructural protein, which acts as a serine protease. its n-terminal domain can interact with the viral non structural protein 2 (ns2), while the c-terminal domain acts both as helicase and nucleoside triphosphatase. the ns5b protein is rdrp with a pivotal role in replicating hcv's viral rna by using the viral ssrna+ as a template to catalyze the polymerization of ribo-nucleoside triphosphates during replication of the viral genome. interestingly, quercetin, apigenin and luteolin effectively inhibit ns5b polymerase activity [33] . when phytochemicals have been combined among them or with synthetic antiviral drugs, synergistic therapeutic effects were often evidenced. overall, when synergy occurred, it was often justified by the fact that the different molecules target different steps in the the ns5b protein is rdrp with a pivotal role in replicating hcv's viral rna by using the viral ssrna+ as a template to catalyze the polymerization of ribo-nucleoside triphosphates during replication of the viral genome. interestingly, quercetin, apigenin and luteolin effectively inhibit ns5b polymerase activity [33] . when phytochemicals have been combined among them or with synthetic antiviral drugs, synergistic therapeutic effects were often evidenced. overall, when synergy occurred, it was often justified by the fact that the different molecules target different steps in the molecular mechanisms of viral infection, and the final antiviral effect results therefore potentiated. naringenin is a flavanone, which exhibits anti-hcv activity by blocking the assembly of hcv particles [41] . the phytochemical quercetin exerted anti-hcv activity by reducing internal ribosome entry site-(ires-)mediated translation and also inhibiting the viral non-structural protein ns5a as well as the protease ns3 [41] . when naringenin and quercetin were used together they suppressed hcv rna replication and inhibited viral replication to a higher extent when compared with the phytochemicals used individually, thus demonstrating a synergistic effect [41] . ladanein, a flavone isolated from marrubium peregrinum l. (lamiaceae), exploited antiviral effects through the inhibition of the post-attachment entry step of hcv virions, with an ic 50 of 2.5 µm. ladanein, in combination with cyclosporine, showed a remarkable synergistic antiviral effect against various hcv genotypes [30] . the natural extracts from nymphaea alba l. (nymphaeaceae), containing iso-quercetin, hyperoside, quercetin, reynoutrin, apigenin and isokaempferide, showed anti-hcv activity, suppressing hcv ns3 gene expression in the transfected huh-7 cell line and inhibiting the viral genotype 1a replication. furthermore, the combination of nymphaea alba l. extract with the conventional drug boceprevir displayed synergistic effects for inhibition of hcv replication in a docking bioinformatics model [28] . an antiviral role of phytochemicals was also linked to the receptors recognized by viruses for their endocytosis, such as the membrane receptor ntcp. this protein has a mass of 56 kda and is involved in the transport of bile salt molecules, steroid hormones, thyroid hormones and xenobiotics. ntcp is required for the entry in hepatocytes of both hbv and human hepatitis d virus (hdv). in fact, the virus-receptor interaction leads to the clathrin-dependent endocytosis of hbv virions, which can replicate inside the host cells [26] . egcg, used at the dose of 50 µm, induced clathrin-dependent endocytosis of ntcp from the plasma membrane, leading to its degradation and inhibiting the entry of hbv virus into immortalized human primary hepatocytes (figure 3 ). two hiv enzymes address pivotal roles in virus replication and virion production: hiv reverse transcriptase and the homodimer of hiv protease (figure 4 ). hiv reverse transcriptase is an rna-dependent dna polymerase (rddp) and builds ssdna based on an rna template in its polymerase active site; the enzyme also cleaves the original rna template into pieces through its nuclease active site and finally it polymerizes a second dna strand to form the final dsdna, which is integrated in the host cell genome. interestingly, it was shown that egcg suppressed hiv-1 infection in hela-cd4-ltr-β-gal cells, with a ec 50 of 1.6 µm, by acting as a non-nucleoside hiv reverse transcriptase inhibitor [43] . furthermore, it was demonstrated that the flavonoids myricetin-3-rhamnoside and myricetin-3-(6-rhamnosylgalactoside) possessed antiviral activity in vitro against hiv with ec 50 of 120 µm and 45 µm, respectively [31] . this antiviral effect was exerted through the inhibition of hiv reverse transcriptase with ic 50 of 10.6 µm for myricetin-3-rhamnoside and of 13.8 µm for myricetin-3-(6-rhamnosylgalactoside) [31] . hiv protease is a retroviral aspartyl protease, which cleaves newly synthesized viral polyproteins (namely gag-pol) at nine cleavage sites to create the mature protein components of the virion. mature hiv-1 protease exists as a 22 kda homodimer, each one containing an asp25 amino acid, which acts as the catalytic residues are able to hydrolyze peptide bonds on the gag-pol polyproteins into fully functional viral proteins, like reverse transcriptase and integrase. kehinde et al. (2019) [27] showed that the phytochemicals kaempferol-7-o-glucoside and egcg were able to interact with hiv-1 protease, showing pronounced structural evidence as potential hiv-1 protease inhibitors ( figure 4) . interestingly, phytochemicals can also be used to reduce the extrusion of antiviral drugs from infected cells. in fact, apigenin, fisetin and luteolin were able to slow down the elimination of the antiviral drugs atazanavir, lopinavir, darunavir and saquinavir, which target the viral protease of hiv-1 [27] . dependent dna polymerase (rddp) and builds ssdna based on an rna template in its polymerase active site; the enzyme also cleaves the original rna template into pieces through its nuclease active site and finally it polymerizes a second dna strand to form the final dsdna, which is integrated in the host cell genome. interestingly, it was shown that egcg suppressed hiv-1 infection in hela-cd4-ltr-β-gal cells, with a ec50 of 1.6 µm, by acting as a non-nucleoside hiv reverse transcriptase inhibitor [43] . furthermore, it was demonstrated that the flavonoids myricetin-3-rhamnoside and myricetin-3-(6-rhamnosylgalactoside) possessed antiviral activity in vitro against hiv with ec50 of 120 µm and 45 µm, respectively [31] . this antiviral effect was exerted through the inhibition of hiv reverse transcriptase with ic50 of 10.6 µm for myricetin-3-rhamnoside and of 13.8 µm for myricetin-3-(6-rhamnosylgalactoside) [31] . hiv protease is a retroviral aspartyl protease, which cleaves newly synthesized viral polyproteins (namely gag-pol) at nine cleavage sites to create the mature protein components of the antiviral activity of flavonoids may be also due to the modulation of host cell enzymes used by the virus to take advantage for infection, such as the enzymes with a pivotal role in the production of radical oxygen species (ros), utilized to increase the number of virions. regarding hsv-1, which persists in the host in sensory neurons in latency, the enzyme nicotinamide adenine dinucleotide phosphate (nadph) oxidase (nox) family is a useful source of ros, which can be used to reactivate the viral infection under oxidative stress conditions [52, 53] . interestingly, delphinidin-3-rutinoside obtained from extracts of solanum melongena l. (solanaceae), inhibited hsv-1 replication through the reduction of nox4 protein levels, when added for 24 h after viral adsorption on vero cells [24] . the extract obtained from veronica persica poir (plantaginaceae) possessed a dose-dependent inhibitory activity against the herpes simplex virus strains hsv-1 and hsv-2 and a prominent synergistic activity in combination with acyclovir in anti-hsv therapy, exerted through a reduction of the percentage of plaque numbers of both hsv-1 and hsv-2 in the infected cero cells [54] . the natural extract of disticella elongata (bignoniaceae) exhibited antiviral effects against denv-2 virus and this effect was mainly due to pectolinarin and acacetin-7-o-rutinoside [18] . when the two flavonoids pectolinarin and acacetin-7-o-rutinoside were used in combination, the antiviral effect was eight times more potent against denv-2 virus (with ec 50 = 18.3 ± 2.6 µm) than the flavonoid pectolinarin used alone (with ec 50 = 138.8 ± 6.1 µm). the selectivity index of the combination (si = 45) was remarkably higher than the si of the isolated pectolinarin (si = 4.6), indicating that the phytochemical mixture specifically inhibited viral growth, with negligible effects on the vitality of the cells infected by denv-2 virus. the ethanol extract obtained from the leaves of disticella elongata showed antioxidant activities; therefore, it could detoxify cell damaging free radicals present in denv-2 viral infections [18] . the low water solubility and low bioavailability of natural flavonoids represent the major limit for their use in the nutraceutical sector. many delivery strategies have been used by researchers for increasing flavonoid bioavailability following oral consumption [16] , including: micelles, nanoparticles, microspheres, crystals, dendrimers, the self-micro-emulsifying drug delivery system (smdds) and the self-nanoemulsifying drug delivery systems (snedds), as recently reviewed [17, 55] . for instance, it was shown that the catechin and egcg-loaded chitosan nanoparticles led to a higher rate of intestinal absorption of the two phytochemicals [56] . interestingly, in chitosan particles the flavonoids maintain their antioxidant activity and can exploit their antioxidant effects in the blood stream [57] . the loading of myricetin into a cationic polymeric nanoparticle carrier with a cationic corona and hydrophobic core was investigated in order to improve the clinical relevance of this natural flavonoid by increasing its solubility [58] . smdds has been used to overcome the problem of low bioavailability of hydrophobic molecules as, in the intestinal lumen, the oil-in-water microemulsions containing phytochemicals may be efficiently formed with a consequent increase of the intestinal absorption of the flavonoid [59] . interestingly, puerarin, an isoflavone isolated from the root of pueraria lobata, exhibited 2.6 fold higher bioavailability when prepared using the smdds technique [34] . furthermore, the synthesis of silver nanoparticles linked with phytochemicals and their use for antimicrobial and antiviral treatments have been described, highlighting the various molecular mechanisms that lead to the phytochemical-mediated inhibition of viral replication [60] . poly (d,l-lactide) (pla) nanoparticles and polymeric micelles contributed to a more sustainable and efficient release of flavonoids characterized by a poor bioavailability, like quercetin and apigenin [61, 62] . quercetin was successfully encapsulated on the most uniformly distributed type of pla-4 nanoparticle, synthesized using lonicera japonica leaf extract, showing that these nanoparticles allowed a slow release of quercetin [63] . this nanoparticle approach paves the way for encapsulating drugs, small molecules, nutraceuticals and other bioactive ingredients to obtain safer cellular uptake, improved biodistribution, specific targeted delivery and enhancement of the therapeutic antiviral efficacy of encapsulated drugs and phytochemicals [64] . the increase in antiviral efficacy and bioavailability of flavonoids may be attained through their encapsulation into red blood cells (rbcs), as has occurred for other type of antiviral drugs and molecules, such as fludarabine (figure 4 ) [65] , vincristine and vinblastine [66, 67] . the idea of using rbcs as delivery system for flavonoids takes its advantage from the favorable characteristics of these cells. they have a long life-span of about 120 days and have a widespread circulation throughout the body, and hence can be used as drug reservoirs, enabling them to facilitate sustained drug release. moreover, rbcs protect encapsulated drugs and molecules from degradation; they are completely biodegradable and show no or only minor immunogenic responses. interestingly, the rbcs were used to determine cellular antioxidant activity of some flavonoids, specifically vitexin, vitexin-2-o-xyloside and vitexin-2-o-rhamnoside, with the aim of predicting their bioavailability [68] . moreover, it was demonstrated that flavonoids could have beneficial effects in preventing oxidative damage of erythrocyte membrane [69, 70] . some authors have also evidenced the possibility that human rbcs play a pivotal role in the distribution and bioavailability of circulating flavonoids such as quercetin [71] . furthermore, it was shown that drug-loaded rbcs can be modified in order to increase their macrophage-mediated phagocytosis by inducing band 3 clustering and opsonization through the complementary and autologous iggs [72] . in future perspective, this approach could be considered in order to possibly improve the antiviral activity of some flavonoids, like baicalin, that was able, like fludarabine [65] , to act against hiv-1 chronic infection of human monocytes and macrophages, inhibiting the fusion of hiv virus envelope proteins with these cells [73] . although polymeric nanoparticles, liposomes, dendrimers, micelles and inorganic nanoparticles have been widely accepted as drug delivery systems, they show toxicity and a short lifetime, thus making them relatively disadvantageous when compared with natural cell carriers, such as rbcs. for this reason, some authors in recent years have tried to mimic the erythrocyte cell membrane to produce biocompatible nanocarriers in order to decrease their toxicity and to prolong their survival in blood circulation [74] [75] [76] . rbcs, which are biodegradable and non-immunogenic, can be used as a valuable carrier system with a lifespan that is remarkably prolonged and controllable when compared to synthetic carriers. several approaches have been developed to load peptides, drugs and molecules into rbcs or to attach these agents onto rbcs' outer membrane surface by either chemical or physical methods [77] and the possibility of loading drugs into autologous rbcs prior to their transfusion into patients has been studied in small animal models and primates, as well as in clinical studies of human patients [78] [79] [80] [81] . the topic of phytochemical encapsulation in rbcs remains open for further studies, but we believe that flavonoid derivatives and nanoparticles able to bind flavonoids could be successfully considered for this application in the near future. diet and life-style play important roles in the defense against the attacks of viruses. the relationship between diet and the immune system involves the microbiota, i.e., the ecological community of commensals and potentially pathogenic microbes and symbionts that live in the gut [82] . the diet modulates the microbiota composition, leading to an increase or a decrease in immune defenses [82] . the mediterranean diet (and in general diets rich in fruits, vegetables and herbs) maintains gut microbiota homeostasis and provides protection against microbes and viruses [83] . the cross-talk between microbiota and immunity is supported by the aryl hydrocarbon receptor (ahr), which is ubiquitous, but mainly present in the cytoplasm of immune cells [84] . it was demonstrated that ahr binds different ligands, namely metabolites, pollutants and pathogenic molecules, and after this interaction it translocates into the nucleus, where it induces specific transcription programs and modulates the defensive functions of both t and b cells, dendritic cells and monocytes [84] . interestingly, it was shown that flavonoids can bind ahr [84] . on this basis, people eating vegetables, all of which contain flavonoids to a different extent, would strengthen their immune system [83] . this strengthening is a general effect that occurs with many nutrients, but there are more specific antiviral effects attributable to the flavonoids treated in this review. flavonoids, like apigenin, vitexin, quercetin, rutin and naringenin, show a wide range of antiviral effects (table 2) , because they are able to target different pathways of viral infections. it is therefore possible to increase the chances of blocking viral replication using mixtures of flavonoids with synergistic antiviral effects, because of the pleiotropic effects of their combination. an important question that arises is focused on the reasonable concentration range of flavonoids that should be used for an effective antiviral therapy, which is hard to be reached by diet alone. recent experiments performed in in vivo studies demonstrated the protective efficacy of various flavonoids, tested in the range 10-50 mg/kg body weight per day, in newborn mice challenged with a lethal dose of the enterovirus 71 [85] . interestingly, apigenin (50 mg/kg), luteolin (10 mg/kg) and quercetin (10 mg/kg) conferred survival protection of 88.9%, 91.7% and 50.1%, respectively, from the lethal enterovirus 71 infection; moreover, isorhamnetin provided the highest survival protection of 100% at a dose of 10 mg/kg. the authors hypothesized that the differences in concentrations are due to different times of absorption and renal clearance of these flavonoids [85] . the flavanol isorhamnetin was studied also by dayem et al. [86] , who showed that oral administration of this flavonoid at 1 mg/kg/day for five days in mice infected with the influenza a virus significantly decreased lung virus titer by two-fold, increased the survival rate (which ranged from 70% to 80%) and decreased mice body weight loss by 25%. these authors hypothesized that the methyl group of the b ring of isorhamnetin may contribute to its strong antiviral potency against influenza a virus [86] . guo et al. [87] focused their in vivo studies on the flavone wogonin, showing that, in human hbv-transgenic mice, this flavonoid administered once a day at 7, 14 and 28 mg/kg reduced plasma hbsag level and immunohistological staining of the liver confirming the hbsag reduction exerted by wogonin [87] . the potentiality of flavonoid bioactivity in vivo depends on the extent of their absorption after ingestion and their ability for distribution in various target tissues. in this light, liu et al. [88] developed a quercetin-loaded cationic nanostructure lipid carrier (q-cnlc), which increased the in vivo bioavailability of this flavonoid. this quercetin-nanostructure complex benefits from its strong interactions with negatively charged intestinal mucosa, which could increase its residence in the gastrointestinal tract. moreover, the authors showed an entrapment efficiency of quercetin of 89.3% and a slower release of this flavonoid from the q-clnc, when compared with the release profile of a simple quercetin solution, indicating that q-clnc exhibited a sustained and controlled release of this flavanol, in order to maintain its effective therapeutic concentration [88] . in addition, the same authors performed in vivo tissue distribution studies in c57bl/6j mice, comparing treatment with 25 mg/kg of orally administered quercetin solution with the administration of 25 mg/kg of q-cnlc, showing that the relative quercetin uptake from q-cnlc was 1.57 fold higher in lungs, 1.51 fold higher in liver and 1.68 fold higher in kidneys. on the contrary, the relative quercetin uptake from q-cnlc was lower in spleen, heart and brain, when compared with the quercetin solution [88] . these results indicate that the most suitable delivery strategy should be chosen in order to target a specific organ with a particular flavonoid-nanostructure complex for future clinical applications. furthermore, the safety of the used phytochemical should also be considered, as has already been done for hydroxytyrosol, which is quickly absorbed and eliminated by the kidneys in either free or conjugated forms. hydroxytyrosol has been considered safe at 50 mg/kg/day by the european food safety authority (efsa) panel [89] . in this light, we think that, for a 70 kg person, a flavonoid range between 0.5-3.0 g/day should be proposed. these values are close to the daily polyphenol intake in humans, calculated in various diet surveys [90] , such as the supplementation en vitamines et mineraux antioxydants (su.vi.max) study, which ranked the polyphenol intake at 1.19 ± 0.51 g/day [91] . accordingly, in another dietary intervention trial aimed at improving cognition in older adults, a total of 1.04 g/day of flavonoids was applied [92] . indeed, based on these results, we think that 0.5-3.0 g/day of flavonoids could be a reasonable concentration range in order to start preliminary experiments, focused on assessing the in vivo antiviral effects of flavonoids. concerning the combination of flavonoids in an antiviral cocktail, each phytochemical may be used initially at a concentration of about 0.5 g/day with the aim of reaching an intake of 3 g/day of antiviral flavonoids. in the case of antiviral synergistic effects [5, 6] or increase of the absorption of one specific flavonoid exerted by other phytochemicals [93] , the individual flavonoid concentrations can be modulated, according to the extent of the effect. it should also be emphasized that a significant antiviral effect is linked to the type of flavonoid mixture, the delivery system used, the pharmacokinetic pattern, the number of targets involved and the number of required daily doses. once these aspects have been defined, the more suitable regimen of administration consists of starting with the lowest effective concentration for a fixed time and proceeding with increasing doses of the antiviral flavonoids. monitoring the markers of antiviral efficacy and any side effects should also be considered in order to evaluate the risks-benefits pattern. overall, our review shows that many flavonoids exhibit antiviral activity and could offer a promising alternative for prevention of and therapy for viral infections. flavonoids are present in many vegetables and the first protection for the immune system resides in the ability to seek foods rich in bioactive nutrients. education programs for a healthy diet should be implemented during the outbreaks of viral infections [94] . in fact, a diet rich in vegetables activates the ahr in the gut for maintenance of microbiota homeostasis, which in turn regulates the immune system. in the critical periods of viral infections, oral dietary supplementation with nutraceutical preparations based on combinations of flavonoids can be useful in order to inhibit different steps of the viral infective cycle. molecular mechanisms underlying the antiviral effects of flavonoids, herein described, mainly focus on the inhibition of viral enzyme activities: neuraminidases, dna/rna polymerases and proteases. therefore, a cocktail of flavonoids, selected for their efficacy in the inhibition of different viral enzymes, could be associated with elevated immune response and offer a promising option for antiviral therapies. this option acquires great importance considering that the viral genome frequently mutates, due to the lack of proof-reading activity of most of the viral polymerases. these mutations could hamper the efficacy of antiviral synthetic drugs. on the contrary, antiviral flavonoids, as well as the combination of synthetic antiviral drugs with flavonoids, would enhance therapeutic strategies by targeting the multiple signaling pathways involved in the viral infections [95] . the active concentration of the flavonoids should be investigated, considering the pharmacokinetic studies available in the literature and the synergistic effects of the specific flavonoid combinations [15] [16] [17] . the scarce intestinal absorption and bioavailability of flavonoids, when given through food or in pills, may be enhanced by the use of new drug delivery strategies [96] . in fact, since flavonoids have some drawbacks after oral administration such as low stability, bioavailability and bio-efficacy, researchers are developing biocompatible nanomaterial synthesis as novel delivery systems (including nanospheres, nano-capsules, micro and nano-emulsions, micelles, solid lipid nanoparticles and capsules), for overcoming the delivery challenges of flavonoids in the biomedical sector. phytochemical-nanomaterial complexes can represent innovative drug delivery strategies (alongside those already known) for new antiviral therapies against the seven baltimore virus classes [97] . interestingly, three patents regarding the antiviral effects of flavonoids (us 7,998,937; ep1245230; us 6,399,654) have been already assigned to the korea research institute of bioscience and biotechnology and advanced life sciences inc. [35] . however, an important step that must be achieved is to obtain the authorization of novel food including flavonoids with antiviral properties, following regulation eu 2015/2283 [98] . nowadays, this authorization has been given only to hydroxytyrosol [89] and few other nutrients. if the antiviral efficacy and safety of flavonoids and their mixtures can be clearly demonstrated in vivo, it would be possible to obtain european food safety authority (efsa) authorization of novel foods, in order to provide new natural tools for preventing and facing outbreaks of viral infections. indeed, when flavonoids are administered through nano-sized delivery systems, they show increased stability and bioavailability with an enhanced and prolonged activity. however, the in vivo behavior and the antiviral actions of these nano-delivery systems are still under experimental evaluation. interferon regulatory factor 3 irf7 interferon polyphenols and antioxidant capacity of vegetables under fresh and frozen conditions antioxidant capacity of extra-virgin olive oils antioxidant capacity of vegetables, spices and dressings relevant to nutrition effect of flavonoids on human health: old subjects but new challenges vitexin 2-o-xyloside, raphasatin and (-) eepigallocatechin-3-gallate synergistically affect cell growth and apoptosis of colon cancer cells betalains increase vitexin-2-o-xyloside cytotoxicity in caco-2 cancer cells antiproliferative activity of vitexin-2-o-xyloside and avenanthramides on caco-2 and hepg2 cancer cells occurs through apoptosis induction and reduction of pro-survival mechanisms natural and synthetic avenathramides activate caspases 2,8,3 and downregulate htert, mdr1 and cox-2 genes in caco-2 and hep3b cancer cells a combination of moringin and avenanthramide 2f inhibits the proliferation of hep3b liver cancer cells inducing intrinsic and extrinsic apoptosis flavonoids from beta vulgaris cicla and betalains from beta vulgais rubra: antioxidant, anticancer, antiinflammatory activities-a review characterization and biological activity of the main flaonoids from swiss chard (beta vulgaris subspecies cycla) oseltamivir resistance-disabling our influenza defenses twenty years of research into medicinal plants: results and perspectives combination of western medicine and chinese traditional patent medicine in treating a family case of covid-19 in wuhan. front antiviral phytochemicals: an overview flavonoids: promising natural compounds against viral infections antiviral effects of phytochemicals from medicinal plants: applications and drug delivery strategies antiviral activity of disticella elongata (vahl) urb. 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french adults enhancing dentate gyrus function with dietary flavanols improves cognition in older adults enhancement of flavonoid ability to cross the blood-brain barrier of rats by co-administration with α-tocopherol the food systems in the era of the coronavirus (covid-19) pandemic crisis. foods viral mutation rates flavonoids loaded in nanocarriers: an opportunity to increase oral bioavailability and bioefficacy nanomaterials designed for antiviral drug delivery transport across biological barriers of the european parliament and of the council and repealing regulation (ec) no 258/97 of the european parliament and of the council and commission regulation (ec) no 1852 this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors want to thank university of urbino carlo bo for financial support. the authors declare no conflict of interest. the funders had no role in the writing of the manuscript or in the decision to publish this manuscript. key: cord-000445-2x7dfl1q authors: paliwal, sarvesh k.; verma, ankita narayan; paliwal, shailendra title: neglected disease – african sleeping sickness: recent synthetic and modeling advances date: 2011-05-10 journal: sci pharm doi: 10.3797/scipharm.1012-08 sha: doc_id: 445 cord_uid: 2x7dfl1q human african trypanosomiasis (hat) also called sleeping sickness is caused by subspecies of the parasitic hemoflagellate trypanosoma brucei that mostly occurs in sub-saharan africa. the current chemotherapy of the human trypanosomiases relies on only six drugs, five of which have been developed more than 30 years ago, have undesirable toxic side effects and most of them show drug-resistance. though development of new anti-trypanosomal drugs seems to be a priority area research in this area has lagged far behind. the given review mainly focus upon the recent synthetic and computer based approaches made by various research groups for the development of newer anti-trypanosomal analogues which may have improved efficacy and oral bioavailability than the present ones. the given paper also attempts to investigate the relationship between the various physiochemical parameters and anti-trypanosomal activity that may be helpful in development of potent anti-trypanosomal agents against sleeping sickness. african sleeping sickness remains one of the most neglected life threatening diseases that have been left untreated till date. two forms of human african trypanosomiasis (hat) have been identified that are parasite dependent. the first one trypanosoma brucei gambiense (t. b. gambiense) causes a human chronic infection, endemic in western and central africa while the other trypanosoma brucei rhodesiense (t. b. rhodesiense) has a vast animal reservoir and causes acute illness in people in eastern and southern african countries. hat has high occurance in the remote rural areas, where the surveillance is weak or nonexistent, with 50 to 70 thousand estimated cases. according to world health organization (who) estimation there are half-million cases of hat or "sleeping sickness" resulting from infections with t. brucei rhodesiense and t. brucei gambiense. who has attributed 50.000 deaths annually to the disease [1] . by more recent estimates, up to 25.000 new cases occur per year, and 50 million people are at risk [2, 3] . out of six clinically approved drugs for the treatment of hat, five (suramin, pentamidine, melarsoprol, eflornithine and nifurtimox) have had been discovered more than 30 years ago. because suramin and pentamidine are ionized at physiological ph, they are unable to cross the blood brain barrier in therapeutic concentrations and are thus used for the treatment of hemolymphatic early stage hat, caused by t. b. rhodesiense and t. b. gambiense infections, respectively. the treatment of the second or neurological stage, when the parasites invade the central nervous system (cns), relies on the organoarsenical drug melarsoprol and the more recently registered eflornithine. the latter is ineffective against t. b. rhodesiense sleeping sickness and is used primarily to control cns-involved hat caused by t. b. gambiense. all the existing anti-trypanosomal therapies suffer from unacceptable toxicity, poor efficacy, difficulties of administration, and increasing treatment failures due to the development of parasite resistance [4] [5] [6] [7] [8] [9] . in the last couple of decades, no new drug has been developed for treatment of early-stage hat, and only one drug has been developed for late-stage hat [1, 3, 5] . the need is great for new orally active drugs for the control and eradication of this disease. novel medicines are typically developed using a trial-and-error approach, which is timeconsuming and costly but yet has the potential to yield new drugs. the application of computer-assisted drug design (cadd) methodologies to this problem has the potential to greatly decrease the time and effort required to discover new medicines or improve current ones in term of their efficacy. this review focuses on the synthetic and computer-assisted drug design (cadd) approaches made by various research groups for the development of newer antitrypanosomal agents having improved efficacy and oral bioavailability. the current research also endeavors [10] to investigate the relationship between the various physiochemical parameters and anti-trypanosomal activity that may be helpful in development of potent anti-trypanosomal agents against sleeping sickness. safe, effective and affordable orally active therapies for trypanosomiasis capable of overcoming resistance are required, so the identification of new anti-trypanosomal drug candidates is an urgent priority. compared to the last 15 years there has been a revival of drug research and development regarding neglected parasitic diseases, and a number of drug development projects are currently ongoing [11] . in view of this, the researchers have endeavored to compile in the present section of the review, diverse series of compounds that have been recently synthesized by various research groups targeting dna minor groove and many other new targets. based on the mechanism of action this section has been divided into two sub-sections. the first sub-section includes the series of compounds acting as dna minor groove binders while in the second sub-section are included all the other anti-trypanosomal compounds and their respective targets. also are described herein prodrug approaches to provide oral bioavailability for the dication class. the wide range of antifungal and antiparasitic activities related to aromatic diamidines and its excellent results in preclinical and clinical phase of drug development has made aromatic diamidines an interesting class for the development of newer anti-trypanosomal drug therapy. the aromatic diamidines i.e. pentamidine ( figure 1) show their anti-parasitic action by binding strongly to at-rich sequences in the minor groove of dna. therefore dna minor groove has evolved as a productive target for designing new ant-parasitic drugs. in order to design newer analogues belonging to diamidine series, studies of the dna complexes with diamidine compounds have been conducted and a number of diamidines have been crystallized with the dna duplex d(cgcgaattcgcg)2 which provide valuable models for drug development in the diamidine series. structures of dna complexes of furamidine, berenil, and pentamidine, for example, reveal that they all bind in the dna minor groove at the central aatt sequence. these drugs penetrate deeply into the groove and fit snugly between the walls of the groove. their amidines form h-bonds with thymine-o2 and/or adenine-n3 acceptor groups on the edges of the bases at the floor of the groove. the amino group of g protrudes into the minor groove and prevents the compounds from assuming their preferred orientation deep in the minor groove. this binding to dna eventually leads to inhibition of one or more of the several dna-dependent enzymes (e.g., topoisomerases and nucleases) or direct inhibition of transcription. for more than 50 years aromatic diamidines and related dicationic molecules have been extensively used but so far only pentamidine [12] [13] [14] [15] has been widely employed as a drug in humans despite several adverse effects, such as hypotension, abdominal pain, vertigo, hypersalivation, hypoglycemia, nausea, and mild nephrotoxocity [5] [6] [7] [8] . being a highly flexible molecule that can assume an array of linked conformations related through torsional rotation, changes can be made in the nucleus to produce improved analogous against hat. in line to this with an aim to increase the efficacy and decrease the side effect related to pentamidine various research groups have made several changes in the pentamidine molecule [16] [17] [18] [19] [20] [21] [22] [23] large numbers of structurally related congeners of pentamidine were synthesized by tidwell rr et al [24] by introducing substitutions on the cationic groups, changing the position of dications from 4,4' to 3,3' position, changing the length of the aliphatic chain between the two aromatic rings, adding substituent on the aromatic rings at 2,2' and 3,3' position and replacing oxygen atoms in the alkyl linker with isosteric sulfur or nitrogen atoms (figure 2a-g) . on comparing the activities of the synthesized compounds with pentamidine and melarsoprol, it was found that few compounds showed activity in the range or better than that of pentamidine and/or melarsoprol. the unsubstituted diamidine compound 64 ( figure 3a ) with secondary amino group in place of oxygen atom exhibited subnanomolar activity (<0.001 µm) against t. b. rhodesiense and was nearly twice as selective against the pathogen as pentamidine. at the same time the diimidazoline compound 66 (figure 3b ) exhibited the second highest anti-trypanosomal activity in the series with the ic 50 value of 0.001 µm and also had the highest parasite selectivity (si t = 34500), being 63 times more selective against t. b. rhodesiense than pentamidine. the replacement of oxygen atom with sulphur atom as in compound 62 ( figure 3c ) also generated active congeners having ic 50 (0.005 µm) value better than that of pentamidine (0.007 µm). the compounds 20-31 ( figure 2c ) in which the cationic groups were present in the 3,3′-position of the aromatic rings and the length of the carbon linker was varied from 3-6 exhibited lower anti-trypanosomal activity compared to pentamidine whereas among the compounds 12-19 ( figure 2b ) of the series, in which the amidine groups were present at 4,4' position and the length of aliphatic chain was varied from three carbon atoms to six carbon atoms, compound 12 ( figure 3d , ic 50 =0.007 µm) with three methylene linker between the aromatic rings had same in vitro activity as that of pentamidine (0.007 µm). further introduction of 2,2′-dichloro substituent in compound 12 improved the antitrypanosomal properties as evident from the in vitro activity of compound 32 (figure 3e , ic 50 =0.004 µm). these compounds having excellent in vitro activity were evaluated in vivo in the stib900 mouse model of african trypanosomiasis. the screening was conducted using intraperitoneal dosing at 20 mg/kg daily for four days. the compounds 12, 62 and 64 showed very poor in vivo activity, whereas compound 32 and 66 exhibited excellent in vivo efficacies in the acute mouse model of trypanosomiasis, providing cures of all infected animals. thus, because of high selectivity, excellent in vitro and in vivo activity compounds 66 and 32 can serve as a novel lead for further pre-clinical and clinical trials, but its cytotoxcity profile needs to be monitored during its evaluation. also compound 64 which showed excellent in vitro, in vivo activity and high selectivity index against the t. b. rhodesiense merits further sar optimization in order to synthesized newer lead compounds with reduced cytotoxicity compared to pentamidine. pentamidine analogues with activity better than that of melarsoprol and/or pentamidine increased efficacies of pyridyl analogues of 2,5-bis(4-amidinophenoxy)furan (furamidine) [25] [26] [27] encouraged tidwell rr et al [28] to synthesize pentamidine analogues in which the phenyl rings of pentamidine were replaced with pyridyl fragments. this replacement resulted in series of 18 compounds (figure 4a ), most of which had lower cytotoxicity than pentamidine. sar study pointed out that the antiprotozoal properties of these compounds depended on the placement of cationic moieties on the pyridine rings as well as the nature of substituents on the amidine groups. the n-substituted congeners were lesser cytotoxic than the unsubstituted diamidines whereas the n-alkylation of cationic fragments reduces the activity of compounds against t. brucei rhodesiense compared to pentamidine. a same trend was observed in pentamidine analogues series. the 2, 6-substituted dications (compounds 11-13, figure 4a ) and 2,4-substituted dications (compounds 14-18, figure 4a ) displayed lower potencies against t. brucei rhodesiense than the corresponding 2,5-substituted isomers (compounds 1-10, figure 4a), while among the 2, 5-substituted dications, the compounds possessing cationic substituents adjacent to nitrogen atoms in pyridine rings displayed superior activities against parasites compared to pentamidine as evident from compound 6 (figure 4b) which showed promising anti-trypanosomal activity (0.001µm) and lower cytotoxicity (4.90µm) than pentamidine and melarsoprol, but had poor in vivo activity giving only 1/4 cures in the stib900 mouse model. however diamidoxime compound 9 (figure 4c ), an oral prodrug of diamidine compound 6 ( figure 4b ), exhibited excellent in vivo activity curing four out of four animals upon oral administration in stib900 mouse model. although compound 9 did not provide cure in the cns mouse model of infection but its bbb permeability could potentially be improved by developing prodrug using lipophillic substitutions in place of hydrophilic substitution as used in compound 9. this could provide higher concentration of compound 6 in cns. thus excellent in vitro activity of diamidine 6 and high in vivo efficacy of its prodrug diamidoxime 9 warrant further investigation of these dications as potential antitrypanosomal drug candidates with improved oral efficacies. the structure activity relationship of the two series indicates that in both the cases the unsubstituted diamidines were more active than the n-substituted diamidines. the replacement of oxygen atoms in alkyl chain with secondary amino group improves the activity against t. b. rhodesiense of pentamidine analogues while in case of pyridyl analogue the compounds with oxygen atoms in the alkyl chain have been the active one. the most potent compound of these two series are compound 6 ( figure 4b ) and compound 66 (figure 3b ), which have excellent in vitro activity (0.001µm) better than pentamidine and melarsoprol and also have high selective against the t. b. rhodesiense parasite as evident from their selectivity index (si of compound 6 = 4900 and compound 66 = 34500 µm). initially it has been believed that the oxygen atom in the aliphatic linker and the amidine groups are important for anti-trypanosomal activity of pentamidine as these groups were part of recognition motif for p2 amino purine transporter in trypanosoma species. thus the activity of compound 6 can be explained on this basis, but in case of compound 66 the amidine groups have been replaced by imidazoline and the oxygen atom has been replaced by secondary amino groups. despite these replacements, the compound has shown excellent in vitro and in vivo activity. thus this is in accordance to recently reported work [29] according to which there is no direct connection between the affinity for p2 carrier and anti-trypanosomal activity. in view of this, these two compounds can serve as a lead structure for the development of newer analogues of pentamidine with reduced side effect and improved pharmacokinetic profile. in the early 1970s dann o et al [30] [31] [32] fig. 6 . structure of two phenylbenzofuran dications with promising anti-trypanosomal activity tidwell rr et al found that the in vitro anti-trypanosomal activities of bisbenzofuran derivatives against trypanosoma brucei rhodesiense, and cytotoxicity against mammalian cells depended on the position and the type of cationic substituents as well as the length of the carbon linker between aromatic moieties. as observed in most of the dicationic molecules, the n-substituted congeners were lesser cytotoxic than the unsubstituted diamidines whereas the n-alkylation of cationic fragments reduced the activity of compounds against t. brucei rhodesiense compared to pentamidine. at same time the 5-substituted bisbenzofurans were generally less cytotoxic than compounds bearing substituents in the 4-or 6-positions where as the 4-substituted bisbenzofurans were significantly less active against t. b. rhodesiense than corresponding 5-and 6-substituted isomers. the in vitro activity of the bisbenzofuran series was not so promising and only lead compound 8, (figure 5b ) exhibited in vitro anti-trypanosomal activity (0.008 µm) comparable to that of pentamidine and melarsoprol. however some compounds from tidwell series (figure 7b ) showed reduced cytotoxicity profile compared to pentamidine (for example compound 43 has cytotoxicity of 1.85 µm), but had very poor selectivity profile. thus was proved that the selectivity of bisbenzofurans against t. b. rhodesiense decreases as number of methylene groups in the alkyl bridge increases. however, how the substitution on the amidine groups affects the anti-trypanosomal properties of bisbenzofurans has not been explained. thus in light of their reduced cytotoxicity compared to pentamidine these molecules require further investigation to study the influence of the type of cationic substituents and the distance between aromatic moieties on uptake and intracellular distribution of dicationic bisbenzofurans in order to understand better the mode of action and thus improve the efficacy of aromatic diamidines. thus promising in vitro, anti-trypanosomal activity, excellent potency in the acute mouse model of trypanosomiasis and the reduced cytotoxicity (1.9 µm) of compound 1 compared to pentamidine warrants further pre-clinical and clinical trials of this molecule. while compound 32 and 20 can serve as lead compounds for further sar optimization to derive congener with enhanced activity and lesser cytotoxicity. to combine the rigidity of 2-phenylbenzofurans with the flexibility of pentamidine congeners, tidwell rr et al [35] incorporated the benzofuran ring into molecules of pentamidine-related analogues. a series of 48 pentamidine congeners containing benzofuran fragments (figure 10a , b) were synthesized and tested in vitro against t. b. rhodesiense. most of the compounds in the series showed cytotoxicity less than that of pentamidine, but had lesser potency and selectivity index as compared to pentamidine. the properties and cytotoxicities of these dications depended on the nature of the cationic substituents, the placement of the benzofuran motif, and the length of the carbon linker. within the series cytotoxicity of the compounds decreased with the substitution on cationic groups and increased with the elongation of the carbon linker. dications with the benzofuran motif in the 4'-position (compounds 25-48, figure 10b ) and connected by the propylene linker are the most potent and more selective against t. b. rhodesiense among the series. thus on the basis of sar study further improvements can be made in order to produce newer anti-trypanosomal agents with improved pharmacological activity. the sar of the three series clearly indicates that the nature of cationic substituents and their position is important in deciding the anti-trypanosomal activity of benzofuran derivatives. the introduction of phenoxy fragment into a benzofuran structural motif may result into retention of affinity for the aminopurine p2 transporter, which could be the reason for anti-trypanospomal activity of benzofuran derivatives. however the precise mechanism by which these compounds show their anti-trypanosomal activity in not known. among the three series the 2-phenylbenzofuran derivatives are attractive molecules because of their high activity, low cytotoxicity and high selectivity against the hat parasite. the introduction of phenoxy fragment into the aromatic ring protects the phenylbenzofuran dervatives from metabolic deactivation. this structural modification retains affinity for the aminopurine p2 transporter. the diamidine and the hydroxy or methoxy group may be involved in binding to the dna minor groove. thus multiple modes of action may be the reason for promising anti-trypanosomal propertries of 2-phenylbenzofuran derivatives. thus these compounds require further assessment in order to develop as newer antitrypanosomal agents with promising pharmacokinetic profile. since a prodrug of furamidine, 2,5-bis[4-(n-methoxy)amidinophenyl]furan (pafuramidine), has shown promising results in clinical trials against hat [36] , therefore furamidine ( figure 11a ) has gained attention for further evaluation to produce newer antitrypanosomal analogous. two other analogues of furamidine (figure 11b &c) have been evaluated against t. b. rhodesiense mouse model and have shown promising results. one approach to enhance the activity of furamidine has been the replacement of the central furan ring with other heterocyclic systems, including thiophene, pyrrole, oxazole, oxadiazole, thiadiazole, pyridazine, methylpyrimidine, and triazine [37] [38] [39] [40] [41] . such structural modification in past has resulted into compound with good anti-parasitic activity. for example in 1977 boykin dw and das pb have reported the synthesis and antiprotozoal activity of eighteen substituted 2,5-bis(4-guanylphenyl)furans and related analogues, including "masked" amidines in which the guanyl function was incorporated into a heterocyclic ring. among these, six compounds have produced cures in mice at submilligram dosage levels and have been somewhat more active in this screen than stilbamidine, hydroxystilbamidine, and pentamidine. . 11 . structure of furamidine and its analogue that have shown promising antitrypanosomal properties tidwell rr et al [43] has recently synthesized a series of 3,5-diphenylisoxazole analogues ( figure 12 ), in which the central ring of furamidine was replaced by isoxazole, and their activities with furamidine and melarsoprol were compared. among 43 synthesized dications, the compounds with at least one p-amidine moiety (compound 22, figure 13a and compound 3, figure 13b ), displayed good ic 50 values (3.5 nm and 5.1 nm respectively) comparable to that of furamidine (4.3 nm) while loss in potency was observed in compounds with substituted diamidine at m-position (compound 32, ic 50 =29 nm, figure 13c ). in general, the introduction of nitro, chloro, or methoxy substituents on either aromatic ring resulted in decreased anti-trypanosomal activity. however, the introduction of methoxy group on the aromatic rings of compound 32 resulted into compound 41 ( figure 13b ) with comparable anti-trypanosomal activity (4.2 nm) as that of furamidine. hence these compounds of the isoxazole series that showed good in vitro antitrypanosomal activity and less cytotoxcity profile relative to furamidine, could be a candidate for further evaluation against animal models of the diseases. however in vivo evaluation of the present 3,5-diphenylisoxazole series has not been reported yet. [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] and also because 1,4-diphenyl-1,2,3-triazoles showed geometrical resemblance to furamidine. it was observed that the cytotoxicities of triazoles were as lower compared to that of pentamidine and were not affected by the alkylation on the amidine groups or the substitution on the aromatic rings. however, the placement of the cationic moiety in the 4-position increased the cytotoxicities of diamidines 46 ( figure 15 ) with respect to the pentamidine. except few, majority of unsubstituted diamidines exhibited higher in vitro activities against t. brucei rhodesiense than bis(n-isopropyl)amidines and diimidazolines. the compounds with cationic fragments in the 5,5'-position (compounds 1-15, figure 14a the replacement of central furan ring of furamidine either by isoxazole or 1,2,3-triazole resulted into compounds with better activity than that of furamidine. position and the nature of the cationic substituent governed the anti-trypanosomal activity of the two series. in general the para substituted diamidines in both the series were the most promising compounds with good activity. thus these compounds should be further evaluated to produce better analogue of furamidine the recent report on n,n'-bis(4-amidinophenyl)piperazine (figure 16) , which was shown to be very effective in vivo anti-trypanosomal agent has attracted the interest in this compound [56] . hence in search for new hat chemotherapy dardonville c et al [57] decided to carry out an in vitro screening of a total of 62 compounds against the parasite t. brucei rhodesiense taken from their in-house library. based on this they showed that bisguanidine and especially bis(2-aminoimidazoline)diphenyl compounds displayed potent anti-trypanosomal activity in vitro and vivo against t. b. rhodesiense, the causative agent of acute hat [58, 59] . among the 62 compounds screened, compounds1c, 28b, 32b and 41b ( figure 17 ) showed excellent in vitro activity (49nm, 69 nm, 22 nm and 118 nm respectively) as well as high selectivity (>5294, 3072, 29.5 and 881respectively) for the parasite. these studies revealed that compounds bearing 2-aminoimidazoline cations had higher selectivity for the parasite and similar activities with respect to their guanidine counterparts. in addition, a correlation between anti-trypanosomal activity and dna binding affinity was observed, suggesting a possible mechanism of action for these compounds. those molecules that showed an excellent in vitro activity as well as high selectivity for the parasite represent new anti-trypanosomal lead compounds. fig. 16. n,n'-bis(4-amidinophenyl) piperazine in light of these promising results, bis(2-aminoimidazoline) derivatives deserve more investigation as anti-trypanosomal agents and dna minor groove binders. the synthesis and study of new derivatives and prodrugs of these lead compounds is ongoing. progress has also been made on targets other than dna minor grove. in addition to newer targets some new lead compounds have also been identified with proper antitrypanosomal activity but uncertain mechanism of action. the enzyme trypanothione reductase (tr) which restores the oxidized trypanothione to reduced state has evolved as an effective drug target. many inhibitors of this enzyme have been developed but most of them combat with problems related to bioavailability, pharmacokinetics and metabolism. despite this quinolines have evolved as most important class of compounds against tr due to their broad spectrum of activity, excellent pharmacological and pharmacokinetic properties such as high plasma levels, high clearance, oral and parenteral applicability, chemical stability and rare side effect. in view of this gilbert ih [60] reported the screening of 62000 compound libraries against t. brucei. high through put screening (hts) which resulted into identification of two novel compound series active against the enzyme trypanothione reductase. series 1 was based on the quinoline scaffold (figure 18a ) in which compound 2 (figure 19a) with methylfuran group at r 3 position, br at 6 th position of quinoline ring and n-methylethanamine group at r 1 position was the most potent and significant tr inhibitor with tr inhibitory activity of 1.1 µm. according to sar study the replacement of methylfuran with other groups like furan, phenyl, pyridinyl, thiophene led to decrease in activity. also the replacement of br with h or f reduced the activity while cl group retained the activity. at 4 th position the nh and nme groups were equally active. the alkylamines at r 1 were active whereas the simple alkyl or aryl groups led to inactive compounds. the second series was based on the pyrimidopyridazine scaffold (figure 18b) , in which the compound 49 ( figure 19b ) showed promising inhibitory activity of 2.6 µm. in case of series 2 replacement of methyl group at r 1 position by h led to the decrease in activity. at r 2 position methyl and ethyl group showed activity while the h and chain extensions to propyl, butyl and cyclopentane led to decrease in activity. substituted phenyl and alkenylphenyl group at r 3 position gave the most active compound of the series. thus these quinoline compounds with promising activity could serve as lead compounds for further development of targeted drugs against african trypanosomiasis. in addition to this synthetic optimization study based on the lead anti-trypanosomal compound 1,2-dihydro-2,2,4-trimethylquinolin-6-yl 3,5-dimethoxybenzoate ( figure 20 ) was undertaken by werbovetz ka et al [61] in an attempt to discover new trypanocides with potent in vivo activity targeting tr enzyme. in course of this a total of 53 compounds were evaluated in vitro for their anti-trypanosomal activity and cytotoxicity. the compounds with oxygen atom at 6 th position were the most active compounds in the series, for example compound 9 ( figure 21 ) showed better anti-trypanosomal activity (0.007 µm) and low cytotoxicity (6.8 µm) than melarsoprol (ic 50 = 0.008µm and cytotoxicity = 7.9µm). whereas compounds lacking the 6-oxygen atom or bearing an oxygen atom at the 7-position rather than the 6-position were far less potent than those containing an alcohol or acyloxy group at the 6-position. compounds carrying aliphatic or a 2-phenylacetyl ester side chains were as potent and selective as their benzoylated or acetylated counterparts. however compound 9a was unstable due to auto-oxidation, so the unstable alcohols were esterified to generate prodrug 10a ( figure 21 ) having promising activity of 0.014µm against these parasites and a selectivity index of 1700. the in vivo evaluation of compound 10a in a murine model of african trypanosomiasis showed good results as the prodrug extended the lifespan of mice infected with t. b. brucei. thus compound 10a can serve as lead compound for further investigation of this class of molecules as potential candidates against hat. efforts are also be undertaken to further elucidate the metabolism, pharmacokinetics, and the anti-trypanosomal mechanism of action of this novel and promising class of compounds. dna topoisomerases have evolved as an effective drug target in prokaryotic and eukaryotic systems as these enzyme mediate mechanistic interactions such as supercoiling, relaxation, knotting or catenating of dna double helices. based on their mechanism of action, topoisomerases can be classified as type i enzymes, which break a single strand of the dna helix during the catalytic cycle, and type ii enzymes, which make double-stranded breaks. on the basis of primary sequence and reaction mechanism, type i topoisomerases are further subdivided into type ia and type ib. recently shapiro ta et al [62] evaluated the activity of indenoisoquinolines (figure 22) , originally known to have anti-cancer activity, against t. brucei and found that most of the compounds showed in vitro activity at submicromolar concentrations. the compound 12 ( figure 23 ) with propylamino group at r 6 position and methoxy group at r 2 , r 3 and r 9 was the most active one with in vitro anti-trypanosomal activity of 0.05 µm. the compound also showed good in vivo activity as it delayed parasitemia and extended survival in infected mice. according to structure-activity analysis the compounds with enhanced potency included alkylamino substitutions on n-6, methoxy groups on c-2 and c-3, and a methylenedioxy bridge between c-8 and c-9. testing of indenoisoquinolines with promising activity on l1210 mouse leukemia cells revealed all the compounds were more effective against trypanosomes than against mammalian cells. the indenoisoquinolines also showed appreciable water solubility indicating that these compounds have good quality for drug development. these compounds showed their anti-trypanosomal action by multiple mechanisms. the study indicated that they stabilize topoisomerase-dna complexes in situ and may also impede topoisomerase binding to dna. these agents markedly inhibited dna synthesis by interfering with topoisomerase and possibly other dna-metabolizing enzymes. concentrations in the range of 300 ng/ml up to 900 ng/ml. some of the dupont compounds, developed as anti-tumour drugs, were highly active but also showed high cytotoxicity on ht-29 cells. it was observed that the position r 1 and position r 2 in the quinolone core nucleus was not prerequisite for anti-trypanosomal activity and also substitution at r 8 position was not necessary for trypanocidal activity. thus, based on the result obtained from sar analysis special attention should be given to r 7 position and the tetracyclic derivatives. the in vivo results of these compounds were very poor, as none of the compounds evaluated produced cure of mice in dose escalation experiment up to 100mg/kg i.p. however no signs of toxicity were observed during the experiments. the in vivo ineffectiveness had not been explained and no drug level determination in the plasma of the treated mice was performed. polyamines are generally involved in growth and differentiation [64] [65] [66] [67] within the cell and their analogs are also used as anticancer agents, antiparasitic agents, antidiarrhoeals, anti-hiv agents, metal chelators, and gene delivery agents. since the inhibition of the initial polyamine biosynthesis enzyme, ornithine decarboxylase, by dl-α-difluoromethylornithine (dfmo) is toxic to african trypanosomes cells, [68, 69] polyamines can become a promising anti-trypanosomal compound. dfmo [70] [71] [72] is the most recently developed agent for late stage t. b. gambiense and t. b. brucei sleeping sickness, but has not been active against all strains of t. b. rhodesiense. the major drawbacks of dfmo are its cost, the duration of treatment and its availability. recent clinical studies have investigated that dmfo can be used in combination with clinically used trypanocides including suramin, nifurtimox and melarsoprol [73, 74] . these combinations result in significant reduction in dfmo dosage and time of administration. initial clinical study has shown that dfmo + nifurtimox are superior to dfmo + melarsoprol and melarsoprol + nifurtimox. the dfmo + nifurtimox regimen (nect regimen) allowed reduction in dfmo regimen from 14 to 7 days (56 versus 28 infusions) with a 94% cure rate and are associated with significantly reduced adverse side effects as compared to melarsoprol-based therapy. the success of the combined regimen has been most likely due to ability of dmfo to reduce trypanothione levels and resistance to oxidative stress and the ability of nifurtimox to generate oxidative stress. recently gilbert et al [76] designed, synthesized, and evaluated substituted polyamines, carrying 1,3,5-triazine units, as potential anti-trypanosomal drugs. preliminary results indicated that this route might be successful, and lead structure a (figure 26 ) was used as a starting point for the synthesis of two series of analogues. in the first series, the influence of structural changes of the central core unit was investigated while in the second series, the effect of additional methyl substituents on the 1,3,5-triazine was studied. the compounds were designed with the intention to selectively target the interior of t. brucei via the p2 amino-purine transporter. in the first series the compound containing the ndodecyl chain as core unit, showed weak activity against t. b. rhodesiense. the compound with n-nonyl chain was the most promising compound and its various analogues were designed by replacing nh 2 groups on the triazine ring with nhme and nme 2 groups. introduction of one or two methyl groups per triazine unit resulted in a 10fold increase in anti-trypanosomal activity. when four methyl groups per triazine unit were introduced, an 80-fold increase in activity was observed. similarly, replacement of nh 2 group in n-dodecyl chain led to 2-20-fold higher anti-trypanosomal activity for the methylated derivatives. monosubstituted compounds showed a slight increase in activity against t. b. rhodesiense as compared to the disubstituted compounds. the methylamino substituted triazines attached to the c9-(compound 8c, figure 27 ) or c12-(compound 8f, figure 27 ) polyamine precursor via an additional ch 2 linker resulted in most active trypanocidal compounds(ic 50 of 8c = 0.27µm and 8f = 0.18 µm). beside good activity, the compounds showed poor in vivo activity producing no cure to the infected mice and concentrations greater than 10 mg kg −1 induced severe acute toxicity. the actual mode of action for the reported triazine substituted polyamines remains unclear. so to understand and improve the activities of these compounds, further research has to verify intracellular drug targets and possible metabolic pathways. stanislaw fw et al [77] have reported the anti-trypanosomal activity of 5'-deoxy-5'-(e)-(iodomethylene)adenosine, which is a known inhibitor of adohcy hydrolase, [78, 79] the 5'-deoxy-5'-(e)-(iodomethylene)adenosine (eiddha) and its 6-n cyclopropyl analogue (figure 28a , b) have shown promising in vitro inhibitory activity (ic50 at 9 and 12 μg/ml) against t. brucei. the utilization of adenosine analogues as anti-parasitics should be explored as a therapeutic paradigm, as it has been shown previously that inhibitors of adohcy hydrolase are also very potent inhibitors against the growth of plasmodium falciparum [79] . this class of 6-n-cyclopropyl adenosine analogues modified at carbon 5', does not exhibit an inhibitory effect on human or parasite forms of the enzyme and displays only marginal antiviral activity in comparison to analogues which have been unmodified at 6-amino position (that are potent inhibitor of adohcy hydrolase). therefore these compounds require further structural modification in order to develop newer analogues with improved activity against t. brucei. the synthesis of 4-[5-(4-phenoxyphenyl)-2h-pyrazol-3-yl] morpholine derivatives by perozzo r et al [80] resulted in to the discovery of newer class of anti-trypanosomal compounds having stage specific action, as these compounds have shown moderate to very good activity against the blood stage of t. b. rhodesiense. the two compounds, 4-[3-(4-phenoxyphenyl)-1h-pyrazol-5-yl]morpholine (1.0µm) ( figure 29a ) and 1-[3-(4-phenoxyphenyl)-1h-pyrazol-5-yl]piperazine (1.1 µm) (figure 29b ) with a pyrazol ring, are the most potent anti-trypanosomals of the series and have same cytotoxicity (61.6 µm), indicating that the pyrazol ring is very important for anti-trypanosomal activity. the substitution of pyrazol ring with isoxazole derivative leads to a six fold reduction in activity as compared to the most potent compound. in addition, substitution with nitrophenyl or aminophenyl also results in strong reduction in activity. the phenoxy ring in the compounds is also important for activity as replacing it by an ethylene group results in nine fold reduction in efficacy. further substitution with a nitro group or an amino group reduces potency up to 4-fold or 18-fold respectively. the stage specific action of these compounds is unknown. further optimization of this class of compounds is required in order to lower the cytotoxicity profile of the compound. in vitro evaluation of a series of n-, s-, and cooh-blocked glutathione derivatives have been carried out by d'silva and daunes [81] against bloodstream form trypanosoma brucei trypomastigotes, to identify the determinants necessary for activity and for further development into an active lead structure. the results shows that n, s-blocked glutathione diesters are the most active inhibitors of t. brucei parasites and that n-acetyl-s-benzyloxycarbonylglutathione dimethyl ester (compound 5) and the n,s-benzyloxycarbonyl-s-2,4dinitrophenylglutathione diester derivatives (compounds 17-19 & 21) (figure 30 ) represent lead structures possessing minimal toxicity which potentially could be developed further to yield a therapeutically active agent for the treatment of trypanosomiasis. pentamidine, furamidine and its analogues lack oral bioavailability [82, 83] .in addition several analogues of furamidine show excellent activity on intravenous dosing but are ineffective on oral administration [83, 84] . generally, oral administration is the preferred dosing regime, and hence, prodrug strategies for diamidines that have the potential to overcome their limited oral bioavailability merit attention. the following works have been performed by boykin dw et al to develop prodrugs. boykin dw et al [85] syhthesized and evaluated five o-alkoxyamidine analogues of the prodrug 2,5-bis [4-methoxyamidinophenyl] furan against trypanosoma brucei rhodesiense in the stib900 mouse model by oral administration. it was observed that the size of the o-alkyl side-chain determined the metabolic stability of the prodrugs. the prodrugs with the o-methyl analogue were most susceptible to metabolism while the larger o-n-butyl and o-n-hexyl groups were least susceptible to metabolism. the in vivo studies in the stib900 mouse model for t. b. rhodesiense showed that compounds with an o-methoxy-amidine or o-ethoxyamidine group effectively cured all trypanosome-infected mice, whereas prodrugs with larger side-chains did not completely cure the mice. therefore the o-alkoxyamidine prodrugs, where the alkyl chain is less than three carbons, could effectively be used as prodrugs for amidines. in addition to above mentioned prodrug synthesis boykin dw et al [86] also reported that bis-amidoximes and bis-o-alkylamidoximes of a number of diamidine systems are effective prodrugs. in order to develop orally effective anti-trypanosomal agents, they synthesized these two types of potential prodrugs in the terphenyl series. it was found that compound 10b and 10d ( figure 32 ) showed good activity in the range of 2 nm. among these compounds 10b had lower cytotoxicity (6.4μm) profile but had very poor in vivo activity (cured none of the infected animal in stib900 mouse model). whereas compound 10d showed excellent in vivo activity, by curing all the infected animals upon oral administration in stib900 mouse model. to capitalize on the efficacy of these potent dications, other prodrugs that rely on different bioconversion pathways need to be developed. boykin dw et al [87] showed that some of the dicationic guanidine, n-alkylguanidine, and reversed amidine derivatives of fused ring systems have good in vitro activity against trypanosoma brucei rhodesiense [87, 88] . the dicationic n-isopropylguanidino-9hfluorene (12c, figure 33 ) showed promising in vivo biological results by giving 4/4 cures of the treated animals in the stib900 animal model for african trypanosomiasis. in addition the n-methyl analogue (12a, figure 33 ) also showed high activity giving 3/4 cures of the treated animals in the stib900 animal model for african trypanosomiasis. in order to enhance the oral bioavailability, two novel classes of potential guanidine prodrugs were prepared. the n-alkoxyguanidine derivatives 12d and 12e ( figure 33) were not effective as prodrugs. whereas the carbamate prodrugs (11c, figure 33 ), gave promising results with 4/4 cures on oral administration in the stib900 mouse model. the result showed that these compounds bind strongly to the dna minor groove, but despite strong bonding these compounds do not have high antiparasitic activity. as compared to the last 15 years, there has been a revival of drug research and development regarding neglected parasitic diseases, and a number of drug development projects are currently ongoing. however discovering lead compounds with anti-trypanosomal activity remains a crucial step to sustain the progress achieved till date. the use of computer-assisted drug design (cadd), since their start, has become increasingly helpful in understanding many aspects of chemical-biological interactions in drug and other scientific research. the latest technological advances (qsar, structure-based design, ligand-based design, cheminformatics & bioinformatics) are providing a much improved basis for the design of ligands and inhibitors with desired specificity. recently, computerassisted drug design approaches based on ligand-based and structure-based drug design have been successfully employed to develop new drugs for the treatment of cancer, aids and other diseases [89] [90] [91] [92] [93] [94] [95] [96] . qsar [97] has been widely used for years to provide quantitative analysis of structure and activity relationships of compounds. our core research group has also performed qsar analysis of dicationic diphenylisoxazoles [10] . in this study, attempt has been to investigate the relationship between the various physiochemical parameters and anti-trypanosomal activity of dicationic 3,5-diphenylisoxazoles that may be helpful in development of potent antitrypanosomal agents against sleeping sickness. several statistical expressions have been developed using stepwise multiple linear regression analysis (mlr) and partial least squares (pls). the best mlr model showed good correlative and predictive ability as shown in following equation 84 has also been obtained. the developed model has been validated by leave-one-out method of cross-validation and prediction of test set. the study indicates that the anti-trypanosomal activity is largely explained by cosmic energy, log p and total lipole descriptors. the qsar study has reported in present study provides important structural insights, related to anti-trypanosomal activity. authors have developed a validated and highly predictive model sharing important structural requirement for effective binding of anti-trypanosomal compounds to minor groove of t. b. rhodesiense dna. the model reported in the study should be helpful in development of new compounds with improved efficacy and oral bioavailability. in line to the developed model authors have also designed some molecules which showed good activity in silico. the further study of these compounds is in progress. the poor pharmacokinetic profile and toxicity produced by the drugs currently used to treat trypanosomiasis requires an immediate attention for the development of safe, economical and high affinity chemotherapeutic agents to meet the need for this class of drugs. the unacceptable truth is the lack of attention of the government and less interest of pharmaceutical companies in light of less monitory gain in the area of protozoal infection since most of the affected people belong to poorer country. surprisingly the endeavors of different research group discussed in this review belong to academic institutions. it is felt by the researchers that the treatment for hat requires a combined approach by government organization, pharmaceutical companies and academic institutions. the most promising fact is that in the last decade several synthetic approaches have been made in the field of development of anti-trypanosomal therapies. several compounds are being synthesized that yield new compounds for the treatment of hat, as a result of these synthetic approaches newer leads have been identified, which are under different phase of drug development. the promising in vitro and in vivo activities of dicationic molecules clearly indicate that aromatic diamidines are the most promising class of compounds for the development of newer drugs against hat. the most important one is the newer analogous of existing drugs pentamidine (compounds 32, figure 3e and 66, figure 3b ) which shows excellent in vivo and in vitro activity (0.004 and 0.001 μm respectively) and also has very good selectivity index against the parasite but is cytotoxic. in an attempt to synthesise newer compounds with reduced cytotoxicity than pentamidine molecules, several benzofuran derivatives have been synthesized. however the replacement of phenoxy fragement of pentamidine with a benzofuran motif has resulted in poor analogues with lower antitrypanosomal activity but improved cytotoxic profile. however the strong activity against t. b. rhodesiense isolates indicates that steps should be taken to initiate further studies of compound 66 and compound 32 which can be further evolved as new lead compound. the newer analogues of furamidine also show promising anti-trypanosomal activity along with lower cytotoxicity than furamidine. thus strong activity against t. b. rhodesiense and lower cytotoxicity of compound 3 (figure 13b ) indicates that these compounds should be further evaluated. thus the dna minor grove binders are still the most interesting and potential target for the development of newer anti-trypanosomal agents. apart from diamidines, polyamines and guanidine also have the potential to give antitrypanosomal compounds. the success of dmfo as polyamine inhibitors has attracted researchers for synthesis and development of agents targeting polyamine metabolism. recently polyamines carrying 1,3,5-triazine units have been synthesized and evaluated against t. brucei. these compound exhibited good activity but are cytotoxic. morpholine and dihydroquinolines have also shown promising in vivo anti-trypanosomal activity. in addition to this development of prodrugs, the existing compounds also promise to address the pharmacokinetic related problems, in near future. thus excellent in vitro and in vivo activities and high selectivity of aforementioned compounds merit further investigation in order to reduce the cytotoxicity that may result in development of newer anti-trypanosomal drug with reduced toxicity, improved efficacy and pharmacokinetic profile. as the drug discovery and development process is expensive in terms of time and money, the cross application of existing series of compounds with selective trypanocidal activity may be the best prospect to new anti-trypanosomal drugs in the short term. more emphasis has to be put in the field of cadd approaches for development of anti-trypanosomal agents as cadd study reduces the time and cost required for development of newer analogues. the trypanosomiases african trypanosomiasis (sleeping sickness) the fall and rise of sleeping sickness treatment of human african trypanosomiasis-present situation and needs for research and development chemotherapy of human african trypanosomiasis: current and future prospects human african trypanosomiasis. cook gc, zumla a, editors. in manson's tropical diseases chemotherapy of human african trypanosomiasis current chemotherapy of human african trypanosomiasis quantitative structure activity relationship analysis of dicationic diphenylisoxazole as potent anti-trypanosomal agents new approaches to the development of anti-protozoan drug candidates: a review of patents small molecule dna and rna binders; from synthesis to nucleic acid complexes treatment perspectives for human african trypanosomiasis challenges and new discoveries in the treatment of leishmaniasis treatment and control of human african trypanosomiasis the biochemical basis of arsenicaldiamidine crossresistance in african trypanosomes transporters in african trypanosomes: role in drug action and resistance adenosine transporters in bloodstream forms of trypanosoma brucei brucei: substrate recognition motifs and affinity for trypanocidal drugs a drug resistance determinant in trypanosoma brucei synthesis and trypanocidal activity of the bis-carba analogue of pentamidine structure-activity relationships of analogs of pentamidine against plasmodium falciparum and leishmania mexicana amazonensis analogues of 1,5-bis(4-amidinophenoxy)pentane (pentamidine) in the treatment of experimental pneumocystis carinii pneumonia trypanocidal activity of conformationally restricted pentamidine congeners structure-activity study of pentamidine analogues as antiprotozoal agents synthesis and antiprotozoal activity of 2,5-bis(4-guanylphenyl)furans synthesis and antiprotozoal activity of aza-analogues of furamidine accumulation and intracellular distribution of anti-trypanosomal diamidine compounds db75 and db820 in african trypanosomes synthesis and antiprotozoal activity of pyridyl analogues of pentamidine design and synthesis of a series of melamine-based nitroheterocycles with activity against trypanosomatid parasites trypanocide diamidine mit drei ringen in zwei isolierten ringsystemen trypanocide diamidine mit vier ringen in einem oder zwei ringsystemen synthesen biskationischer, trypanocider 1-benzofuran-verbindungen synthesis and in vitro antiprotozoal activity of bisbenzofuran cations synthesis and antiprotozoal activity of cationic 2-phenylbenzofurans synthesis and antiprotozoal properties of pentamidine congeners bearing the benzofuran motif db-289 immtech international synthesis and antiprotozoal activity of 2, 5-bis(4-guanylphenyl)furans synthesis and anti-trypanosomal activity of some bis(4-guanylphenyl) five-and six-membered ring heterocycles anti-pneumocystis carinii pneumonia activity of dicationic diaryl methylpyrimidines synthesis of dicationic diaryltriazines nucleic acid binding agents 4-diphenylfurandiamidines as novel anti-pneumocystis carinii pneumonia agents trypanocidal diamidines with three isolated ring systems synthesis and in vitro antiprotozoal activities of dicationic 3,5-diphenylisoxazoles superoxide dismutase-like activity of 1, 2, 3-triazole derivatives synthesis and muscarinic activities of quinuclidin-3-yltriazole and -tetrazole derivatives 3-triazoles: synthesis and evaluation of antiinflammatory and analgesic properties i 3-triazoles: synthesis and evaluation of antiinflammatory and analgesic properties ii bioisosteres of arecoline: 1,2,3,6-tetrahydro-5-pyridyl-substituted and 3-piperidyl-substituted derivatives of tetrazoles and 1,2,3-triazoles. synthesis and muscarinic activity synthesis and biological evaluation f novel 2-pyridinyl-[1,2,3]triazoles as inhibitors of transforming rowth factor beta 1 type 1 receptor rapid discovery and structure-activity profiling of novel inhibitors of human immunodeficiency irus type 1 protease enabled by the copper(i)-catalyzed synthesis of 1,2,3-triazoles and their further functionalization synthesis and biological evaluation of 4-aryl-5-cyano-2h-1, 2, 3-triazoles as inhibitor of her2 tyrosine kinase synthesis, hiv-rt inhibitory activity and sar of 1-benzyl-1h-1, 2, 3-triazole derivatives of carbohydrates 3-triazole derivatives as new cannabinoid cb1 receptor antagonists synthesis and cb1 cannabinoid receptor affinity of 4-alkoxycarbonyl-1,5-diaryl-1,2,3-triazoles trypanocidal activity of conformationally restricted pentamidine congeners new bis(2-aminoimidazoline) and bisguanidine dna minor groove binders with potent in vivo antitrypanosomal and antiplasmodial activity dna binding affinity of bisguanidine and bis(2-aminoimidazoline) derivatives with in vivo antitrypanosomal activity and polyamine derivatives as potent and selective chemotherapeutic agents against trypanosoma brucei rhodesiense. synthesis and in vitro evaluation investigation of tryoanothione reductase as a target in trypanosoma brucei activity of 1,2-dihydroquinolin-6-ols and their ester derivatives activity of indenoisoquinolines against african trypanosomes evaluation of quinolone derivatives for anti-trypanosomal activity biological activity and synthesis of polyamine analogues and conjugates synthesis of polyamines, their derivatives, analogues and conjugates polyamines as targets for therapeutic intervention trypanosoma brucei ornithine decarboxylase -enzyme purification, characterization, and expression in escherichia-coli polyamine metabolism: a potential therapeutic target in trypanosomes advances in sleeping sickness therapy safety and effectiveness of first line eflornithine for trypanosoma brucei gambiense sleeping sickness in sudan: cohort study three drug combinations for late-stage trypanosoma brucei gambiense sleeping sickness: a randomized clinical trial in uganda nifurtimox eflornithine combination therapy for second-stage trypanosoma brucei gambiense sleeping sickness: a randomized clinical trial in congo cure of trypanosoma brucei brucei and trypanosoma brucei rhodesiense infections in mice with an irreversible inhibitor of s-adenosylmethionine synthesis and biological evaluation of s-triazine substituted polyamines as potential new anti-trypanosomal drugs anti-trypanosomal activity of 5'-deoxy-5'-(iodomethylene) adenosine and related 6-n-cyclopropyladenosine analogues synthesis of 6' (e and z)-halohomovinyl derivatives of adenosine, inactivation of s-adenosyl-lhomocysteine hydrolase, and correlation of anticancer and antiviral potencies with enzyme inhibition structure, evolution, and inhibitor interaction of s-adenosyl-l-homocysteine hydrolase from plasmodium falciparum synthesis and evaluation of antiparasitic activities of new 4 structure-activity study on the in vitro antiprotozoal activity of glutathione derivatives dicationic diaryl furans as anti-pneumocystis carinii agents anti-pneumocystis activity of aromatic diamidoxime prodrugs 5-bis[4-(n-alkylamidino)phenyl]furans as pneumocystis carinii agents prodrugs of furamidine: in vitro transport and microsomal metabolism as indicators of in vivo efficacy in a mouse model of trypanosoma brucei rhodesiense infection dna affinity, and antiprotozoal activity of linear dications: terphenyl diamidines and analogues synthesis, dna affinity, and antiprotozoal activity of fused ring dicationic compounds and their prodrugs prodrugs for amidines: synthesis and anti-pneumocystis carinii activity of carbamates of 2,5-bis rational design of potent sialidase-based inhibitors of influenza virus replication bis tertiary amide inhibitors of the hiv-1 protease generated via protein structure-based iterative design structure-based inhibitor design by using protein models for the development of antiparasitic agents conformation-activity relationship study of 5-ht3 receptor antagonists and a definition of a model for this receptor site 3-hydroxy-3-methylglutaryl-coenzyme. a reductase: molecular modeling, three-dimensional structureactivity relationships, inhibitor design a 3d model of sars_cov 3cl proteinase and its inhibitors design by virtual screening a novel strategy for improving ligand selectivity in receptor-based drug design corona virus main proteinase (3clpro) structure: basis for design of anti-sars drugs correlation of biological activity of phenoxyacetic acids with hammett substituent constants and partition coefficients 3d qsar on a library of heterocyclic diamidine derivatives with antiparasitic activity activity of bisphosphonates against trypanosoma brucei rhodesiense qsar study on the contribution of log p and es to the in vitro antiprotozoal activity of glutathione derivatives authors pay sincere thanks to prof. aditya shastri, vice chancellor, banasthali university and dr. monali bhattacharga, dept. of english, banasthali university, rajasthan, india. the author declares no conflict of interest key: cord-296769-xnjmk4pm authors: valentová, kateřina title: cytoprotective activity of natural and synthetic antioxidants date: 2020-08-06 journal: antioxidants (basel) doi: 10.3390/antiox9080713 sha: doc_id: 296769 cord_uid: xnjmk4pm numerous chronic diseases including cancer, cardiovascular, chronic respiratory or neurodegenerative diseases, diabetes mellitus, retinal damage, and others are associated with oxidative stress [...]. mimetic, chelating agent, and intravenous magnetic resonance imaging agent mangafodipir showed preventive and protective effects against oxaliplatin-induced neurotoxicity in balb/c mice [14] . a growing number of studies deal with the mechanism of action of cytoprotection. the cytoprotective mechanism of natural anthocyanin delphinidin against oxidative stress induced by h 2 o 2 was investigated in human chondrocytes where it inhibited reactive oxygen species (ros)-induced apoptosis via activation of nrf2 and nuclear factor κb (nf-κb) and activated cytoprotective autophagy showing potential in the treatment of osteoarthritis [15] . embelin, a plant natural product from lysimachia punctata and embelia ribes fruit with quinone and hydroquinone functional groups plus a long hydrophobic tail completely abolished the superoxide radical generated in situ with hydrodynamic voltammetry. moreover, it exhibited cytoprotective activity in thp-1 human leukemic monocytes and bv-2 mice microglia probably thanks to its long alkyl tail enabling its insertion in cell membranes [16] . the mechanism of anti-inflammatory and antioxidant effects of aspirin on hyperoxia-induced acute lung injury was studied in nf-κb-luciferase transgenic mice. pretreatment with aspirin significantly reduced the protein levels of phosphorylated protein kinase b, nf-κb, and tumor necrosis factor α, indicating that aspirin reduces nf-κb activation [17] . a few publications were devoted to the effect of natural compounds in the form of complex but well-characterized extracts. thus, the essential oil from feijoa (acca sellowiana) fruit peal containing mostly sesquiterpenes showed strong antioxidant and free radical scavenging activity, cytoprotective activity on lymphocytes pre-treated with 100 µm tert-butylhydroperoxide (t-booh), as well as a decrease in intracellular ros, induced by t-booh on erythrocytes and antimicrobial and antifungal activities against staphylococcus aureus and candida albicans, respectively [18] . vitis vinifera var. fetească neagră tendrils and leaves extracts, intended to be used in oral hygiene products recommended in periodontal disease, displayed a cytoprotective effect against nicotine-induced cytotoxicity and anti-inflammatory activity in human gingival fibroblasts [19] . viburnum opulus berry phenolic extracts were able to decrease the uptake of glucose, free fatty acids, and accumulation of lipid droplets in caco-2 cells without affecting their viability, followed by a decrease in the cd36/fat gene expression, without influence on the glut2 and pparα levels. furthermore, the extracts revealed strong chemo-preventive activity against oxidative stress induced chemically by t-booh, as well as against dna damage through the induction of dna repair after cell exposition to methylnitronitrosoguanidine and h 2 o 2 [20] . in a subsequent study, v. opulus fruit fresh juice and a phenolic-rich fraction were able to decrease intracellular oxidative stress in mice insulinoma min6 cells, induced glucagon-like peptide-1 secretion in the presence of an elevated glucose concentration, and inhibited in vitro activity of the dipeptidyl peptidase-4 [21] . several publications in this special issue study flavonolignans from silymarin, an extract from the fruits of silybum marianum. twenty-six commercially available silymarin preparations, natural silymarin from sigma aldrich, and a model mixture of pure flavonoid/flavonolignans mimicking the silymarin composition, all analyzed by u-hplc-hrms/ms, were compared using biochemical (2,2 -azino-bis-(3-ethylbenzothiazoline-6 sulfonic acid) (abts), oxygen radical absorption capacity (orac), and 2,2-diphenyl-1-picrylhydrazyl (dpph)) and cellular (caa) antioxidant tests and significant differences in the antioxidant capacity of the supplements were observed [22] . antioxidant activities of pure stereomers a and b of silybin and 2,3-dehydrosilybin, their racemic mixtures, pure silychristin a, and its derivatives (anhydrosilychristin, isosilychristin, and 2,3-dehydrosilychristin a) were investigated by using orac and caa assays. moreover, their anti-inflammatory activity was studied in macrophages and multidrug resistance modulation as inhibition of p-glycoprotein and sensitization of doxorubicin-resistant ovarian carcinoma cells overproducing p-glycoprotein was detected and the effect on related gene expression revealed a distinct mechanism of action for the individual compounds [23, 24] . last but not least, new potential targets of silymarin constituents silybin and dehydrosilybin as dual inhibitors of braf and the hedgehog pathway receptor smoothened (smo), two major targets in anticancer therapy, were identified in silico and confirmed in vitro [25] . the content of the special issue clearly shows that cytoprotection by anti-and prooxidant active molecules is still a hot topic. however, for effects demonstrated in cell cultures other than those derived from the gastrointestinal tract, the bioavailability and metabolism of the active compounds often could not be taken into consideration. therefore, future studies should be directed towards the effects of the metabolites likely to be present in blood plasma. antioxidant and oxidative stress: a mutual interplay in age-related diseases the nrf2 system as a potential target for the development of indirect antioxidants the importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state direct and indirect antioxidant properties of inducers of cytoprotective proteins european contribution to the study of ros: a summary of the findings and prospects for the future from the cost action bm1203 (eu-ros) oxidative stress, aging, and diseases antioxidants: friend or foe? asian pac oxidative stress in cardiovascular diseases: still a therapeutic target? nutrients a bibliometric review of the keap1/nrf2 pathway and its related antioxidant compounds potential cytoprotective activity of ozone therapy in sars-cov-2/covid-19 aop1, a new live cell assay for the direct and quantitative measure of intracellular antioxidant effects the side effects of platinum-based chemotherapy drugs: a review for chemists quantification of berberine in berberis vulgaris l. root extract and its curative and prophylactic role in cisplatin-induced in vivo toxicity and in vitro cytotoxicity calmangafodipir reduces sensory alterations and prevents intraepidermal nerve fibers loss in a mouse model of oxaliplatin induced peripheral neurotoxicity cytoprotective effects of delphinidin for human chondrocytes against oxidative stress through activation of autophagy antioxidant properties of embelin in cell culture. electrochemistry and theoretical mechanism of scavenging. potential scavenging of superoxide radical through the cell membrane anti-inflammatory and reactive oxygen species suppression through aspirin pretreatment to treat hyperoxia-induced acute lung injury in nf-κb-luciferase inducible transgenic mice feijoa fruit peel: micro-morphological features, evaluation of phytochemical profile, and biological properties of its essential oil phytochemical profile and biological activities of tendrils and leaves extracts from a variety of vitis vinifera l viburnum opulus fruit phenolic compounds as cytoprotective agents able to decrease free fatty acids and glucose uptake by caco-2 cells evaluation of viburnum opulus l. fruit phenolics cytoprotective potential on insulinoma min6 cells relevant for diabetes mellitus and obesity complex evaluation of antioxidant capacity of milk thistle dietary supplements multidrug resistance modulation activity of silybin derivatives and their anti-inflammatory potential antioxidant, anti-inflammatory, and multidrug resistance modulation activity of silychristin derivatives dual smo/braf inhibition by flavonolignans from silybum marianum. antioxidants 2020 key: cord-253276-mqcwk2ow authors: desai, n. c.; bhatt, nayan; somani, hardik; trivedi, amit title: synthesis, antimicrobial and cytotoxic activities of some novel thiazole clubbed 1,3,4-oxadiazoles date: 2013-09-30 journal: european journal of medicinal chemistry doi: 10.1016/j.ejmech.2013.06.029 sha: doc_id: 253276 cord_uid: mqcwk2ow abstract a series of thiazole clubbed 1,3,4-oxadiazole derivatives (5a–l) have been synthesized and characterized by ir, 1h nmr, 13c nmr and mass spectral analysis. synthesized compounds were evaluated for their antimicrobial and cytotoxic activities. the results indicated that, compounds 5c and 5i exhibited the most potent antibacterial activity. compound 5f was found to be the most potent antifungal agent. the structure activity relationship revealed that the presence of electron withdrawing groups at para position of phenyl ring remarkably enhanced the antibacterial activity of synthesized compounds. further, the results of preliminary mtt cytotoxicity studies on hela cells suggested that potent antimicrobial activity of 5b, 5c, 5f, 5h and 5i is accompanied by low cytotoxicity. the treatment of infectious diseases still remain a challenging task because of combination of factors such as an alarming increase in number of multi-drug-resistant microbial pathogens and advent of newer infectious diseases such as severe acute respiratory syndrome (sars), and avian influenza. despite the availability of a large number of antibiotics and chemotherapeutics, the increasing clinical importance of drug-resistant microbial pathogens have lent additional urgency in microbiological and antifungal research [1e4] . a potential solution to the antibiotic resistance is to design and explore innovative heterocyclic agents with novel mode of actions. in this context, thiazole derivatives have been playing a crucial role in medicinal chemistry. thiazole nucleus constitutes an integral part of all the available penicillins, which have transformed the bacterial diseases therapy [5] . they display quite a broad spectrum of biological activities [6] , which have found applications in the treatment of allergies [7] , hypertension [8] , inflammation [9] , schizophrenia [10] , microbial infections [11, 12] , hiv infections [13] , hypnotics [14] and pain [15] . they are also used as new inhibitors of bacterial dna gyrase b [16] . further, thiazoles have emerged as new class of potent antimicrobial agents, which are reported to inhibit bacteria by blocking the biosynthesis of certain bacterial lipids and/ or by additional mechanisms [17, 18] . on the other hand, 1,3,4-oxadiazole heterocycles are very good bioisosteres of amides and esters, which can contribute substantially in enhancing pharmacological activity by participating in hydrogen bonding interactions with the receptors [19] . potent pharmacological activity of 1,3,4-oxadiazoles can be attributed to the presence of toxophoric en]ceoe linkage which may react with the nucleophilic centers of the microbial cell [20] . further, the widespread use of 1,3,4-oxadiazoles as a scaffold in medicinal chemistry established this moiety as a member of the privileged structures class and its derivatives have exhibited a wide range of biological activities such as antibacterial [21] , antitubercular [22] , vasodialatory [23] , antifungal [24] , cytotoxic [25] , anti-inflammatory and analgesic [26, 27] , hypolipidemic [28] , anticancer [29] and ulcerogenic [30] . oxadiazole derivatives have been found to possess broad spectrum antimicrobial activity and therefore are useful substructures for further molecular exploration [30] . the most prominent examples of prescribed agents featuring the 1,3,4-oxadiazoles nucleus include the antiretroviral raltegravir [31] , antihypertensive nesapidil [32] and the antibiotic furamizole [33] (fig. 1) . prompted by above-mentioned observations and in continuation of our search for new, potent, selective, and less toxic antimicrobial agents [34e39], we report herein the synthesis of some novel structural hybrids by combining thiazole and 1,3,4oxadiazole pharmacophores in single molecular framework in order to investigate their in vitro antimicrobial activity. in addition, cytotoxicity studies were also conducted in hela cell lines to evaluate the ability of these compounds to inhibit the cell growth. the substitution pattern of 1,3,4-oxadiazole ring was carefully selected in order to confer different electronic environment to the molecules. the reaction sequences employed for synthesis of the target compounds 5ael are outlined in scheme 1. ethyl 2-amino-4methylthiazole-5-carboxylate (1) was taken as starting material and reacted with acetic anhydride to afford ethyl 2-acetamido-4methylthiazole-5-carboxylate (2) , which on further reaction with hydrazine hydrate in absolute ethanol yielded intermediate n-(5-(hydrazine carbonyl)-4-methylthiazol-2-yl)acetamide (3) . the intermediate obtained thus, was refluxed with carbon disulfide in the presence of potassium hydroxide in ethanol (99.5%) to yield intermediate n-(4-methyl-5-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2yl)thiazol-2-yl)acetamide (4) . mannich condensation of intermediate 4 with 36% formaldehyde and an appropriately substituted aniline derivative in ethanol (99.5%) furnished the desired compounds 5ael. both analytical and spectral data of the synthesized compounds 5ael were fully in agreement with proposed structures. formation of titled compounds 5ael was confirmed by characteristic ir spectrum absorption bands in the range of 3330e 3350 cm à1 , 3200e3250 cm à1 , 2810e2840 cm à1 , 2750e2800 cm à1 and 1660e1690 cm à1 corresponding to enh stretching of secondary amine, enh stretching of amide, ech 3, ech 2 e and >c]o of amide respectively. singlets at around d 2.03e2.10, 2.40e2.50, 4.15e4.25, 4.35e4. 45 and 9 .10e9.20 ppm in 1 h nmr were due to e ch 3 in anilide group, hetech 3 , secondary amine, ech 2 e and enh amide group respectively. the aromatic ring protons were observed at d 6.20e8.05 ppm and j value were found to be in accordance with substitution pattern on phenyl ring. characteristic peaks at around d 168.5e168. 9 and 177.0e177.3 ppm in 13 c nmr confirmed the presence of >c]o and >c]s groups respectively. the mass spectrum of 5ael revealed that observed molecular ion peaks were in agreement with molecular weight of respective compound. all the synthesized compounds 5ael were evaluated for their in vitro antibacterial activity against staphylococcus aureus mtcc 96, streptococcus pyogenes mtcc 442 (gram-positive), escherichia coli mtcc 443 and pseudomonas aeruginosa mtcc 1688 (gramnegative) by conventional broth microdilution method using chloramphenicol as a control drug for antibacterial activity [40] . the results of the antimicrobial studies are presented in table 1 . in general, compounds 5ael demonstrated better antibacterial activity than antifungal activity. compounds 5c and 5i emerged as the most effective antibacterial agents with 2-to 4-fold higher mic (12.5e25 mg/ml) than the reference drug chloramphenicol. while, compounds 5b and 5h exhibited comparable antibacterial activity with the standard drug. from the results of the antimicrobial activity of the synthesized compounds 5ael, the following structure activity relationships can be derived: the antibacterial activity was considerably affected by substitution pattern on the phenyl ring and the most active compounds contain electron withdrawing substituent at para and meta positions of the phenyl ring (p > m > o). in contrast, the presence of electron releasing groups on the phenyl ring witnessed a substantial decrease in antibacterial activity for compounds 5def and 5kel. the role of electron withdrawing group in improving antimicrobial activity is very well supported by previous studies [41, 42] . compounds 5c and 5i, substituted with inductively electron withdrawing fluoro and nitro groups, respectively at para position showed the highest antibacterial activity (f > no 2 ). it is a very well-known fact that electron withdrawing substitution such as fluoro/nitro at the para position of the aromatic ring increases the lipophilicity of molecules. this property is directly related to antimicrobial activity as it facilitates a compound to diffuse through the biological membranes and reach to its site of action. the presence of lipophilic substituent at para position of phenyl ring provided a positive influence on antibacterial activity. on the other hand, presence of the same functional groups at meta position resulted in slight decrease in the antibacterial activity (5b and 5h) but, still produced significant inhibitory action compared to the standard drug chloramphenicol (f > no 2 ). while, substituting the phenyl ring with fluoro and nitro group at ortho position resulted in noticeable decrease in the antibacterial activity of compounds 5a and 5g respectively. on the basis of mic values, it may be concluded that electron withdrawing atom/group such as fluoro and nitro at the para position of phenyl ring induced a positive effect while electron donating groups such as methyl and methoxy induced a negative effect on antibacterial activity of compounds 5ael. the in vitro antifungal activity of synthesized compounds 5ael were determined against candida albicans mtcc 227, aspergillus niger mtcc 282 and aspergillus clavatus mtcc 1323 by conventional broth microdilution method. the results indicated that compound 5f substituted with methoxy group at para position of the phenyl ring was found to be the most promising agent against both c. albicans and a. niger having 2-fold higher mic (25 mg/ml) in comparison with control drug ketoconazole. the enhanced activity of compound 5f may be attributed to the presence of electron releasing group at para position. the contrasting nature of substitution pattern at para position of the phenyl ring of most active antibacterial and antifungal agents indicate that the structural requirements are different for binding of drug to bacterial or fungal targets, respectively [43] . all other compounds showed less inhibition against the tested microorganisms as compared to the standard drug. in vitro cytotoxicity of compounds 5b, 5c, 5f, 5h and 5i were evaluated against human cervical cancer cell line (hela) by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (mtt) colorimetric assay [44] , which measures the reduction of tetrazolium bromide salt into a formazan dye by mitochondrial dehydrogenases in treated versus untreated cells. the results are summarized in table 2 . it was observed that none of the tested compounds exhibited any significant cytotoxic effect on hela cells, suggesting a great potential for their in vivo use as antimicrobial agents. in conclusion, some new structural hybrids of thiazole and 1,3,4oxadiazole were synthesized and investigated for their antimicrobial property with an anticipation of generating new structural leads serving as potent antimicrobial agents. many of the synthesized motifs (5b, 5c, 5h and 5i), possessing electron withdrawing atom/group such as fluoro and nitro at para and meta positions were identified as the most potent antibacterial agents. albeit, it was observed that para position was more favorable for enhancing the antibacterial activity. while, compound 5f with electron releasing group at para position came out as the most promising antifungal agent. the potent antimicrobial activity of most active compounds 5b, 5c, 5f, 5h and 5i were accompanied with relatively low level of cytotoxicity. the results described here, merits further investigations in our laboratories using a forward chemical genetic approach for finding lead molecules as antimicrobial agents. all reactions except those in aqueous media were carried out by standard techniques for the exclusion of moisture. melting points were determined on an electrothermal melting point apparatus and were reported uncorrected. tlc on silica gel plates (merck, 60, f 254 ) was used for purity checking and reaction monitoring. column chromatography on silica gel (merck, 70e230 mesh and 230e400 mesh asth for flash chromatography) was applied when necessary to isolate and purify the reaction products. elemental analysis (% c, h, n) was carried out by a perkineelmer 2400 chn analyzer. ir spectra of all compounds were recorded on a perkine elmer ft-ir spectrophotometer in kbr. 1 h nmr spectra were recorded on varian gemini 300 mhz and 13 c nmr spectra on varian mercury-400 100 mhz in dmso-d 6 as a solvent and tetramethylsilane (tms) as an internal standard. mass spectra were scanned on a shimadzu lc-ms 2010 spectrometer. all reactions requiring anhydrous conditions were carried out under nitrogen atmosphere using oven-dried glassware. ethyl 2-amino-4-methylthiazole-5-carboxylate (0.01 mol) was taken in a round bottom flask and acetic anhydride (0.02 mol) was added. the reaction mixture was refluxed at 140e150 c for 1 h and then poured into cold water under constant stirring to get solid product. the mixture was heated to boiling to decompose excess of acetic anhydride and cooled. the solid obtained was filtered, washed with water and dried. the ethyl 2-(acetyl amino)-4methyl-1,3-thiazole-5-carboxylate was collected and recrystallized from ethanol to get pure white crystals. yield: 74%; m.p. 158 c; anal. obs. c, 47.08%; h, 5.48%; n, 12.44%. calcd. for c 9 h 12 n 2 o 3 s: c, 47.35%; h, 5.30%; n, 12.27%. intermediate (2) (0.01 mol) and 99% hydrazine hydrate (0.015 mol) were taken in a round bottom flask and mixture was refluxed for 10 min. alcohol was added till both the layers were miscible and refluxing was continued for 5 h. excess of alcohol and unreacted hydrazine hydrate was distilled out and the contents were poured into a beaker. the solid was recrystallized from ethanol to get pure crystalline product. dmso was used as diluents to get desired concentration of compounds to test upon standard bacterial strains. serial dilutions were prepared in primary and secondary screening. the control tube containing no antibiotic was immediately subcultured (before inoculation) by spreading a loopful evenly over a quarter of plate of medium suitable for the growth of the test organism and put for incubation at 37 c overnight. the tubes were then incubated overnight. the mic of the control organism was read to check the accuracy of the compound concentrations. the mic was defined as the lowest concentration of the antibiotic or test sample allowing no visible growth. all the tubes showing no visible growth (same as control tube) were subcultured and incubated overnight at 37 c. the amount of growth from the control tube before incubation (which represents the original inoculum) was compared. subcultures might show: similar number of colonies indicating bacteriostatic; a reduced number of colonies indicating a partial or slow bactericidal activity and no growth if the whole inoculum has been killed. the test must include a second set of the same dilutions inoculated with an organism of known sensitivity. each synthesized compound was diluted obtaining 2000 mg/ml concentration as a stock solution. in primary screening 500, 250 and 200 mg/ml concentrations of the synthesized compounds were taken. the active synthesized compounds found in this primary screening were further tested in a second set of dilution against all microorganisms. the compounds found active in primary screening were similarly diluted to obtain 100, 62.5, 50 and 25 mg/ml concentrations. the highest dilution showing at least 99% inhibition is taken as mic. in vitro cytotoxicity was determined using a standard mtt assay with protocol appropriate for the individual test system. test compounds were prepared prior to the experiment by dissolving in 0.1% dmso and diluted with medium. the cells were then exposed to different concentrations of the drugs (1e100 mm) in the volume of 100 mm/well. cells in the control wells received the same volume of medium containing 0.1% dmso. after 24 h, the medium was removed and cell cultures were incubated with 100 ml mtt reagent (1 mg/ml) for 5 h at 37 c. the suspension was placed on micro vibrator for 10 min and absorbance was recorded by the elisa reader. the experiment was performed in triplicate. human cancer cell lines, hela cells were cultured in mem medium supplemented with 10% fbs, 1% glutamine and 50 mm/ml gentamicin sulphate in a co 2 incubator in a humidified atmosphere of 5% co 2 and 95% air. ir (kbr): v/cm à1 3329 (secondary amine nh), 3225 (secondary amide nh), 3010 (aromatic ring ch -nitrophenyl)amino)methyl)-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)thiazol-2-yl)acetamide (5i) yield: 60%, m.p. 159e161 c; ir (kbr): v/cm à1 3331 (secondary amine nh), 3227 (secondary amide nh) (o-tolylamino)methyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl)thiazol-2-yl)acetamide (5j) yield: 62%, m.p. 142e144 c; ir (kbr): v/cm à1 3331 (secondary amine nh), 3220 (secondary amide nh (m-tolylamino)methyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl)thiazol-2-yl)acetamide (5k) yield: 57%, m.p. 151e153 c; ir (kbr): v/cm à1 3330 (secondary amine nh), 3229 (secondary amide nh anal. calcd. for c 16 h 17 n 5 o 2 s 2 c-51 p-tolylamino)methyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl)thiazol-2-yl)acetamide (5l) yield: 61%, m.p. 163e165 c; ir (kbr): v/cm à1 3327 (secondary amine nh) anal. calcd. for c 16 h 17 n 5 o 2 s 2 c-51 we would like to express our sincere gratitude to the department of chemistry, mahatma gandhi campus, maharaja krishnakumarsinhji bhavnagar university, bhavnagar for providing research and library facilities. key: cord-281597-x53ni6q1 authors: chiasson, melissa a; rollins, nathan j; stephany, jason j; sitko, katherine a; matreyek, kenneth a; verby, marta; sun, song; roth, frederick p; desloover, daniel; marks, debora s; rettie, allan e; fowler, douglas m title: multiplexed measurement of variant abundance and activity reveals vkor topology, active site and human variant impact date: 2020-09-01 journal: elife doi: 10.7554/elife.58026 sha: doc_id: 281597 cord_uid: x53ni6q1 vitamin k epoxide reductase (vkor) drives the vitamin k cycle, activating vitamin k-dependent blood clotting factors. vkor is also the target of the widely used anticoagulant drug, warfarin. despite vkor’s pivotal role in coagulation, its structure and active site remain poorly understood. in addition, vkor variants can cause vitamin k-dependent clotting factor deficiency or alter warfarin response. here, we used multiplexed, sequencing-based assays to measure the effects of 2,695 vkor missense variants on abundance and 697 variants on activity in cultured human cells. the large-scale functional data, along with an evolutionary coupling analysis, supports a four transmembrane domain topology, with variants in transmembrane domains exhibiting strongly deleterious effects on abundance and activity. functionally constrained regions of the protein define the active site, and we find that, of four conserved cysteines putatively critical for function, only three are absolutely required. finally, 25% of human vkor missense variants show reduced abundance or activity, possibly conferring warfarin sensitivity or causing disease. the enzyme vitamin k epoxide reductase (vkor) drives the vitamin k cycle, which activates blood coagulation factors. vkor, an endoplasmic reticulum (er) localized transmembrane protein encoded by the gene vkorc1, reduces vitamin k quinone and vitamin k epoxide to vitamin k hydroquinone (li et al., 2004; rost et al., 2004) . vitamin k hydroquinone is required to enable gamma-glutamyl carboxylase (ggcx) to carboxylate gla domains on vitamin k-dependent blood clotting factors. vkor is inhibited by the anticoagulant drug warfarin (czogalla et al., 2017; zimmermann and matschiner, 1974) , and vkorc1 polymorphisms contribute to an estimated~25% of warfarin dosing variability (owen et al., 2010) . for example, variation in vkorc1 noncoding and coding sequence can cause warfarin resistance (weekly warfarin dose >105 mg) or warfarin sensitivity (weekly warfarin dose <~10 mg) (osinbowale et al., 2009; yuan et al., 2005) . though 15 million prescriptions are written for warfarin each year (https://www.clincalc.com), fundamental questions remain regarding its target, vkor. for example, the structure of human vkor is unsolved, though a bacterial homolog has been crystallized . a homology model based on bacterial vkor has four transmembrane domains, but the quality of the homology model is unclear, as human vkor has only 12% sequence identity to bacterial vkor. moreover, experimental validation of vkor topology yielded mixed results: similar biochemical assays suggested either three-or four-transmembrane-domain topologies tie et al., 2012; shen et al., 2017; wu et al., 2018) . topology informs basic aspects of vkor function including where vitamin k and warfarin bind, so determining the correct topology and validating the homology model is critical. in particular, vkor has four functionally important, absolutely conserved cysteines at positions 43, 51, 132, and 135, the orientation of which differs between the two proposed topologies. in the four transmembrane domain topology, all four cysteines are located on the er lumenal side of the enzyme. in this topology, cysteines 43 and 51 are hypothesized to be 'loop cysteines' that pass electrons from an eranchored reductase, possibly transmembrane thioredoxin-related protein , to the active site (rishavy et al., 2011) . however, in the three transmembrane domain topology, these cysteines are located in the cytoplasm and other pathways would be required to convey electrons to the redox center. even for non-catalytic residues, topology plays an important role. for example, vitamin k presumably binds near the redox center, and topology dictates which residues make up the substrate binding site. to understand the effect of human variants and to define the vitamin k and warfarin binding sites, vkor variant activity has been extensively studied in cell-based assays (czogalla et al., 2017; shen et al., 2017; tie et al., 2013) . in addition to activity, vkor protein abundance has also been studied because abundance is an important driver of disease and warfarin response. for example, vkor r98w is a decreased-abundance variant that, in homozygous carriers, causes vitamin k-dependent clotting factor deficiency 2 . a 5' utr polymorphism reduces vkor abundance and can be used to predict warfarin sensitivity (gong et al., 2011) . however, so far, the activity and abundance of only a handful of vkor variants has been tested. here, we used multiplexed, sequencing-based assays (gasperini et al., 2016) to measure the effects of 2,695 vkor missense variants on abundance and 697 variants on activity. our analysis of the large-scale functional data supports a four transmembrane domain topology, which an orthogonal evolutionary coupling analysis confirmed. next, we identified distinct mutational tolerance groups, which are concordant with a four transmembrane homology model. combining this homology model with variant abundance and activity effects, we identified an active site that contains the catalytic residues c132 and c135 and shares six positions with a previously proposed vitamin k binding site (czogalla et al., 2017) . we found that of four conserved cysteines putatively critical for function, only three are absolutely required, and analyzed the mutational signatures of two putative er retention motifs. human vkorc1 variants present in genetic databases and contributed by a commercial genetic testing laboratory were each classified based on abundance and activity. while most variants show wild type-like activity, 25% show low abundance or activity, which could confer warfarin sensitivity or cause disease in a homozygous context. finally, we analyzed warfarin resistance variants and found that they span a range of abundances, indicating that increased abundance is an uncommon mechanism of warfarin resistance. to measure the abundance of vkor variants, we applied variant abundance by massively parallel sequencing (vamp-seq), an assay we recently developed (matreyek et al., 2018) . in vamp-seq, a protein variant is fused to egfp with a short amino acid linker. if the variant is stable and properly folded, then the egfp fusion will not be degraded, and cells will have high egfp fluorescence. in contrast, if the variant causes the protein to misfold, protein quality control machinery will detect and degrade the egfp fusion, leading to a decrease in egfp signal (figure 1a) . mcherry is also expressed from an internal ribosomal entry site (ires) to control for expression. differences in abundance are measured on a flow cytometer using the ratio of egfp to mcherry signal. to determine abundance score egfp:mcherry ratio figure 1 . multiplexed measurement of vkor variant abundance using vamp-seq. (a) to measure abundance, an egfp reporter is fused to vkor. egfp-tagged wt vkor is folded correctly, leading to high egfp fluorescence. however, a destabilized variant is degraded by protein quality control machinery, leading to low egfp fluorescence. (b) flow cytometry is used to bin cells based on their egfp:mcherry fluorescence intensity. density plots of vkor library expressing cells (grey, n = 12,109) relative to three controls: wt vkor (red, n = 4,756), vkor 98w (blue, n = 2,453), and vkor tmd1d (orange, n = 2,204) are shown. quartile bins for facs of the library are marked. (c) abundance score density plots of nonsense variants (dashed blue line, n = 88), synonymous variants (dashed red line, n = 127), and missense variants (filled, solid line, n = 2,695). the missense variant density is colored as a gradient between the lowest 10% of abundance scores (blue), the wt abundance score (white) and abundance scores above wt (red). (d) heatmap showing abundance scores for each substitution at every position within vkor. heatmap color indicates abundance scores scaled as a gradient between the lowest 10% of abundance scores (blue), the wt abundance score (white), and abundance scores above wt (red). grey bars figure 1 continued on next page whether vamp-seq could be applied to vkor, we fused egfp to vkor n-or c-terminally and found that both orientations had high egfp signal (figure 1-figure supplement 1) . we compared n-terminally tagged wild type (wt) vkor to r98w, a variant that ablates a putative er retention motif and reduces abundance (czogalla et al., 2014) , and to tmd1d, a deletion of residues 10-30 which comprise the putative first transmembrane domain (tmd1; figure 1b ). both reduced abundance variants exhibited much lower egfp:mcherry ratios than wt, demonstrating that vamp-seq could be applied to vkor. we constructed a barcoded site-saturation mutagenesis vkor library that covered 92.5% of all 3240 possible missense variants. to express this library in hek293t cells we used a bxb1 recombinase landing pad system we previously developed (matreyek et al., 2017) . in this system, each cell expresses a single vkor variant. recombined, vkor variant-expressing cells were then sorted into quartile bins based on their egfp:mcherry ratios. each bin was deeply sequenced, and abundance scores were calculated based on each variant's distribution across bins. raw abundance scores were normalized such that wt-like variants had a score of one and total loss of abundance variants had a score of zero ( figure 1c) . we performed seven replicates, which were well correlated (figure 1-figure supplement 1, mean pearson's r = 0.73; mean spearman's r = 0.7, supplementary file 1). abundance score means and confidence intervals for each variant were calculated from the replicates. the final dataset describes the effect of 2695 of the 3240 possible missense vkor variants on abundance (figure 1d and e). validation of 10 randomly selected variants spanning the abundance score range showed high concordance between individual egfp:mcherry ratios assessed by flow cytometry and vamp-seq derived abundance scores (figure 1f , pearson's r = 0.96, spearman's r = 0.97). western blots of these variants also showed high concordance with abundance scores (figure 1-figure supplement 2). we also measured vkor variant activity, adapting a hek293 cell assay based on vitamin k-dependent gamma-glutamyl carboxylation of a cell-surface reporter protein (haque et al., 2014) . in this assay, if vkor is active, a factor ix domain reporter is carboxylated, secreted and retained on the cell surface where it is detected with a carboxylation-specific, fluorophore-labeled antibody. however, if vkor is inactive, the reporter is not carboxylated and the antibody cannot bind (figure 2a) . we modified the hek293 activity reporter cell line to eliminate endogenous vkor activity by knocking out both vkorc1 and its paralog, vkorc1-like 1 (vkorc1l1) (tie et al., 2013; figure 2-figure supplement 1) . we also installed a bxb1 landing pad to facilitate expression of individual vkor variants or libraries (figure 2-figure supplement 1) . recombination of wt vkorc1 into the landing pad of the hek293 vkor activity reporter cell line yielded robust reporter activation, demonstrating that the reporter line could be used to assess the activity of a library of vkor variants (figure 2b ). we recombined a library of vkorc1 variants into the hek293 activity reporter cell line and sorted recombinant cells into quartile bins based on carboxylation-specific antibody binding. each bin was deeply sequenced and, as for vamp-seq, an activity score was computed for each variant. final activity scores and confidence intervals were computed from six replicates for a total of 697 comparing vamp-seq derived abundance scores to mean egfp:mcherry (n = 1 replicate) ratios measured individually by flow cytometry. variants were selected at random to span the abundance score range. error bars show standard error for abundance scores and standard error for egfp:mcherry ratio. the online version of this article includes the following source data and figure supplement(s) for figure 1: source data 1. vkor variant abundance and activity scores. source data 2. flow cytometry for monoclonal validation of variants. reporter is expressed inhek293 cells and consists of a prothrombin pre-pro-peptide which allows for processing and secretion, a factor ix gla domain, and proline rich gla protein 2 (prgp2) transmembrane and cytoplasmic domains. middle panel, cells expressing wt vkor carboxylate the reporter gla domain, which, upon trafficking to the cell surface, can be stained using a carboxylation-specific antibody conjugated to the fluorophore apc. right panel, vkor knockout cells do not carboxylate the reporter, so the fluorescent antibody does not bind. (b) density plots of hek293 activity reporter cells stained with apc-labeled carboxylation-specific antibody expressing no vkor (blue, n = 7,188), wt vkor (red, n = 4,107), or the vkor variant library (grey, n = 41,418). quartile bins for facs of the library are marked. (c) activity score density plots of nonsense variants (dashed blue line, n = 14), synonymous variants (dashed red line, n = 35), and missense variants (filled, solid line, n = 697). the missense variant density is colored as a gradient between the lowest 10% of activity scores (blue), the wt activity score (white) and activity scores above wt (red). the online version of this article includes the following figure supplement(s) for figure 2: two different domain models, one with three transmembrane domains and another with four, have been proposed for human vkor tie et al., 2012; figure 3a) . because charged amino acids occur infrequently in transmembrane domains and should be less tolerated, we reasoned we could discriminate between these two models using a sliding window average of the effect of charged substitutions on vkor abundance (elazar et al., 2016a; sharpe et al., 2010) . we found four clearly demarcated regions where charged substitutions profoundly reduced vkor abundance, relative to aliphatic substitutions ( figure 3b) . to exclude the possibility that the egfp tag used in our vamp-seq assay somehow affected topology, we also analyzed the activity score data. the activity data, derived using native, untagged vkor, revealed the same four minima as the abundance data ( figure 3c ). in addition to these four minima, we also observed an activity score minimum at position 57, corresponding to a conserved serine at this position. this serine occurs at the end of the lumenal half-helix hypothesized to shield the active site from non-specific oxidation, so it is likely this signal is the result of disruption of that half helix. together, these results strongly support the hypothesis that, like its distant bacterial homolog, human vkor has four transmembrane domains. to validate these findings, we performed evolutionary coupling analysis to infer the three-dimensional structure suggested by co-evolution. we aligned 6910 vkor sequences from both eukaryotes and prokaryotes (1118 sequences from eukaryotes, 5731 from bacteria, and 61 from environmental samples and viruses) and identified coupled residues using the evcouplings software (hopf et al., 2012; marks et al., 2011) . local patterns of evolutionary couplings (i.e. between nearby positions, i to i+4) supported a four-helix topology. the helices predicted by these local evolutionary couplings overlapped 70 of the 82 residues in alpha-helices of the bacterial structure (pdb 4nv5) (shen et al., 2017) and included in our alignment (hyper-geometric test p-value=3.26 à23 , figure 3d ). we identified non-local evolutionary coupling patterns characteristic of three-dimensional contacts, which also strongly supported the four transmembrane domain model. using these contacts, we computationally folded human vkor, yielding a modeled structure similar to the bacterial structure (rmsd = 2.58 å over 97/143 c alpha , figure 3 comparison of our abundance data to the energy required to insert different amino acids into the membrane yielded additional evidence for the four transmembrane domain model. the apparent change in free energy (ddg app ) of insertion relative to wild type for every amino acid has been determined experimentally using deep mutational scanning of bacterial membrane proteins (elazar et al., 2016a) . median abundance score and ddg app for each amino acid are correlated ( figure 3h ). in particular, the large energetic cost of insertion of transmembrane domains with charged amino acids is apparent, including within the second transmembrane domain tmd2. beyond insertion energies of individual amino acids, the overall hydrophobicity of transmembrane helices contributes to membrane protein insertion (elazar et al., 2016a) , as well as topology (elazar et al., 2016b) and degradation (guerriero et al., 2017) . to determine whether overall helix hydrophobicity was a large factor contributing to abundance scores, we calculated the free energy for insertion (dg helix ) of each helix in the four transmembrane domain model using the dg prediction server v1.0 (hessa et al., 2007) and topgraph (elazar et al., 2016b) . both predicted that transmembrane domain three had the most favorable dg helix for insertion (dg prediction server: tmd1: 0.435, tmd2: 1.551, tmd3: à1.749, and tmd4: 1.734; topgraph: tmd1: à6.3, tmd2: à5.5, tmd3: à12.6, tmd4: à4.3). interestingly, we observed that tmd3 has a high density of substitutions with wt-like scores (figure 3figure supplement 3a), suggesting that tmd3's favorable insertion energy might explain its mutational tolerance. in addition, the high concordance of hydrophobicity indices from bacterial and mammalian multiple sequence alignments further supports the conservation of a four transmembrane domain topology between bacteria and mammals ( having confirmed that human vkor has four transmembrane domains, we next explored the detailed pattern of mutational effects we observed in the context of a four transmembrane domain homology model. we generated a homology model of human vkor with i-tasser using the bacterial vkor structure (shen et al., 2017; yang et al., 2015, supplementary file 4) . we performed hierarchical clustering of positions based on abundance scores, which yielded four groups of positions with characteristic mutational patterns ( figure 4a ). in group 1, most substitutions were neutral or increased abundance; in group 2, charged amino acid and proline substitutions decreased abundance; in group 3, all substitutions decreased abundance; and in group 4, all substitutions decreased abundance profoundly. each group corresponded to a spatially distinct region of the homology model structure (figure 4b) . group one positions were located in or adjacent to cytoplasmic and er lumenal loops, which were more tolerant of substitutions than the transmembrane domains. at four group one positions, k30, r33, r35, and r37, almost every substitution increased abundance. these positively charged positions are positioned either at the edge of tmd1 (k30) or in the er lumen directly abutting the top of tmd1 (r33, r35, and r37). the 'positive inside rule ' (von heijne, 1989) , suggests that positive charges in membrane proteins generally reside in the cytoplasm, and this phenomenon is important for driving topology and membrane insertion (elazar et al., 2016b; nilsson and von heijne, 1990; von heijne, 1989) . k30, r33, r35, and r37 violate the positive inside rule, and substitutions at these positions may increase abundance by reducing charge inside the er, reducing topological frustration or increasing membrane insertion efficiency. compared to the other 12 arginine and lysine positions in wt vkor, k30, r33, r35, and r37 are the only ones where substitutions generally increased abundance (figure 4-figure supplement 1). our observations are consistent with a screen of rat vkor variants intended to improve protein expression in e. coli where deletion of positions 31 to 33 increased protein levels (hatahet et al., 2015) . in group two, charged amino acids or proline substitutions generally decreased abundance. group two consisted mostly of transmembrane positions that had side chains projecting into the lipid bilayer. such transmembrane positions usually have hydrophobic, nonpolar side chains (ulmschneider and sansom, 2001) . proline has poor helix forming propensity, explaining why proline substitutions decreased abundance at these positions. group 3 consisted of a mixture of cytoplasmic, er lumenal and transmembrane positions where most substitutions decreased abundance. the cytoplasmic positions in this group included the putative dilysine er localization motif at positions 159 and 161. also in this group were r98, part of another putative er retention motif at positions 98 and 100, and a glycine adjacent to tmd1 at position nine. the transmembrane positions had side chains projecting towards neighboring transmembrane helices, suggesting that, as for other membrane proteins (fleming and engelman, 2001; mravic et al., 2019) , intramolecular sidechain packing is important for abundance. finally, substitutions in group 4, consisting of positions g19, y88, i141, and l145, resulted in catastrophic loss of abundance. these positions are all in transmembrane domains with side chains projecting into the interior of the protein. on the basis of strict mutational intolerance of these positions, we hypothesized that their coordinated side chain packing comprises the core of the vkor four helix bundle. indeed, group four residues had dramatically lower relative solvent accessibility than groups 1-3 ( figure 4c) . the four transmembrane domain homology models also allowed us to explain vkor's unusual trimodal distribution of variant abundance scores. previous vamp-seq derived abundance score distributions for the cytosolic proteins tpmt and pten were bimodal (figure 4-figure supplement 2; matreyek et al., 2018) , and 15 of 16 deep mutational scans of other soluble proteins using a variety of other assays also exhibited bimodal functional score distributions (gray et al., 2017) . because vkor is an er resident, transmembrane protein, we hypothesized that its unusual trimodal abundance score distribution resulted from transmembrane domain substitutions. indeed, the lowest mode of the distribution was composed almost exclusively of deleterious transmembrane domain substitutions (figure 4d ). in contrast, the intermediate mode consisted of substitutions in the er lumen, cytoplasm, and transmembrane domains. similarly, substitutions that profoundly decreased activity occurred in transmembrane domains (figure 4e ). we reasoned that our activity and abundance data could reveal the location of functionally important positions in vkor, including the active site, since functionally important positions should have many loss-of-activity but few loss-of-abundance variants. thus, we calculated the specific activity for each variant by taking the ratio of its rescaled activity score and abundance score (see methods). we computed the median specific activity for each position; substitutions at positions with low median specific activity generally have low activity relative to their abundance. we set a specific activity threshold based on two absolutely conserved cysteines that form vkor's redox center, c132 and c135. using this threshold, positions with the lowest 12.5% of specific activity scores and with at least four variants scored for activity were deemed functionally constrained and mapped on the homology model of vkor ( , including f55, which is hypothesized to bind vitamin k. three functionally constrained positions, g60, r61, and a121, did not match any position in the predicted active site, but were immediate neighbors of w59 and l120, positions that are present in the predicted active site. besides c132 and c135, vkor has two additional absolutely conserved cysteines, c43 and c51. in the four transmembrane domain model, c43 and c51 are postulated to be loop cysteines that relay electrons to the c132/c135 redox center (liu et al., 2014) . we classified c43 as having low specific activity, but we only observed one variant at this position, so it was not included in our set of functionally constrained positions ( figure 5-figure supplement 2) . in contrast, substitutions at c51 resulted in only modest activity loss, a phenomenon that has been observed previously (shen et al., 2017) . interestingly, every substitution at c51 and 15 of 19 at c132 decreased vkor abundance ( figure 5-figure supplement 2) . inside cells, the majority of vkor molecules have a c51-c132 disulfide bond, and warfarin binds to this redox state of vkor (shen et al., 2017) . since disruption of this disulfide bond apparently impacts abundance as well as activity, this bond may be important for vkor folding and stability. vkor is thought to contain two sequences important for er localization. the first is a diarginine motif (rxr) at positions 98-100, and the second is a dilysine motif (kxkxx) at positions 159-163. while we did not directly measure localization, we found that only six of 19 r98 variants and seven of 14 r100 variants resulted in low abundance ( figure 5-figure supplement 3) . in contrast, nearly all variants at k159 (14 of 18) and k161 (17 of 19) resulted in low abundance ( figure 5-figure supplement 3) . a histidine substitution was tolerated at position 161, which mimics the kxhxx motif commonly found in coronaviruses and a small number of human proteins (ma and goldberg, 2013) . because protein localization and degradation are coupled (hessa et al., 2011) , we suggest that the reductions in abundance we observe are the result of degradation caused by mislocalization, and that the dilysine motif at positions 159-163 is essential for vkor er localization. overall, comparison of vkor variant activity and abundance revealed functionally important regions, refining our understanding of the active site, redox-active cysteines, and er retention motifs. (kabsch and sander, 1983; touw et al., 2015) and colored as in b. bold black line shows median, box shows 25th and 75th percentile. line shows 1.5 interquartile range above and below percentiles, and outliers are shown as black points. (d) histograms of abundance scores for missense variants in the cytoplasmic, er lumenal, or transmembrane domains. (e) histograms of activity scores for missense variants in the cytoplasmic, er lumenal, or transmembrane domains. the online version of this article includes the following figure supplement(s) for figure 4: variation in vkor is linked to both disease and warfarin response, but the overwhelming majority of vkor variants found in humans so far have unknown effects. thus, we curated a total of 215 variants that had either been previously reported in the literature as affecting warfarin response (supplementary file 5), were in clinvar (landrum et al., 2014) , were in gnomad v2 or v3 (karczewski et al., 2019) , or were present in individuals whose healthcare provider had ordered a multi-gene panel test from a commercial testing laboratory (color genomics) (supplementary file 6). of eight variants present in clinvar, we included only one (d36y) in our analysis as it was the only variant reviewed by an expert panel (kurnik et al., 2012) . 159 variants were present in gnomad, and all but one missense variant (d36y) had population frequencies less than 0.2%. 28 variants were literature-curated warfarin response variants, only 12 of which were in one of the databases surveyed. d36y was the only warfarin response variant present in all databases, clinvar, gnomad, and color ( figure 6-figure supplement 1) . we classified 193 of the 215 variants we curated according to their abundance (supplementary file 6). all synonymous variants with the exception of two were wt-like or possibly wt-like, while the three nonsense variants were scored as low abundance (figure 6a) . missense variants spanned all abundance categories, with 129 (60%) having wt-like or possibly wt-like abundance. 30 missense variants were low abundance, and 12 were high abundance. the single known pathogenic variant r98w was low abundance (figure 6b) . we also classified 54 variants according to their activity (supplementary file 6) . only one variant, a115v, exhibited low activity. it had wtlike abundance, indicating that the loss of activity is not due to loss of abundance. we examined warfarin response variants including w5x, the only variant observed so far linked to human warfarin sensitivity . as expected, w5x was low abundance, reinforcing that heterozygous loss of vkor is the cause of warfarin sensitivity in carriers of this variant. warfarin resistance variants, on the other hand, are predicted to abrogate warfarin binding , but it is unclear whether these variants have appreciable effects on abundance or activity. we found that warfarin resistance variants span a range of abundances and that the distribution of warfarin resistant variant abundance was not different from missense variants generally (figure 6c , twosided kolmogorov-smirnov test p=0.438). five warfarin-resistance variants had low abundance, suggesting that these variants must block drug binding or increase activity to confer resistance. one variant, a26t, had high abundance, a possible mechanism of warfarin resistance. the five warfarin resistance variants, r58g, w59l, v66m, g71a, and n77s, whose activity we scored, were all wtlike. thus, our abundance and activity data are consistent with warfarin resistance arising largely from variants that block warfarin binding. we conducted multiplexed assays to measure the effects of 2,695 vkor variants on abundance and 697 variants on activity. both abundance and activity data provided evidence for a four transmembrane topology, which was further supported by evolutionary couplings analysis. we evaluated a vkor homology model in the context of the patterns of variant effects on abundance we measured and found that the homology model could explain these patterns. low specific activity residues mapped onto this homology model identify, at least in part, the active site, which largely overlaps with the results of a vitamin k docking simulation (czogalla et al., 2017) . our active site is shallower than what the docking simulation predicts; this is the result of low abundance scores at some of the deeper, transmembrane positions predicted by docking to bind the isoprenoid chain of vitamin k (f87, y88), and poor coverage of activity scores for other positions (v112, s113). in light of the fact that substitutions at f87 and y88 resulted in low abundance, we note that the modeled vitamin k binding mode would disrupt packing of vkor core residues and require repacking of helices to maintain protein stability (merkle et al., 2018) . in addition to the active site, substitutions at the dilysine and, to a lesser extent, the diarginine er localization motifs caused abundance loss. we also used our large-scale functional data to analyze 215 vkor variants found in humans. 16% of these variants affect neither activity nor abundance; we identified 54 previously uncharacterized low abundance or low activity variants that could be pathogenic or alter warfarin response. we found that only one warfarin resistance variant had increased abundance, indicating that increased abundance is not a pervasive warfarin resistance mechanism. many of the other warfarin resistant variants have warfarin ic50s that are 10-to 100-fold higher than wildtype vkor in cell assays (shen et al., 2018) , and this high level of resistance probably cannot be gained through increased protein abundance alone. all five of the warfarin resistance variants whose activity we scored were wt-like. taken together these data support the notion that warfarin resistance generally involves alterations to warfarin binding rather than abundance or activity. we analyzed one known warfarin sensitivity variant, w5x, and found that it is low abundance, suggesting the possibility that any of the 53 other low abundance variants, if found in a person, might also confer warfarin sensitivity. while our vkor variant abundance and activity data illuminates various aspects of vkor's structure and function, the data have limitations. for example, neither assay captures variant effects on a b c 0.5 figure 6 continued on next page mrna splicing, which means we cannot determine the effect of human splice site variants on vkor activity or abundance. in addition, as a result of how these assays were engineered, both have limited dynamic ranges. thus, subtle effects on abundance or activity cannot be discerned, and it is difficult to translate the scores these assays generated to a more precise biochemical measure, like absolute vkor molecules present or enzymatic kinetics. in addition, both assays have inherent noise, largely arising from the limited number of cells we can sample due to the bottleneck of cell sorting. we account for this noise by filtering each dataset based on variant frequency and presenting a confidence interval for each abundance and activity score. reengineering the assay to be growth-based, instead of flow cytometry-based, would increase the number of cells sampled and would most likely improve library coverage and score estimation. in the future, we envision that the assays we used could be employed to better understand vkor's interaction with warfarin. here, we could measure warfarin's effect on both variant abundance and activity, mapping the warfarin binding site more finely. in addition, we could identify warfarin resistance mutations that have not yet been observed in the clinic and group variants by their putative resistance mechanism. overall, our work highlights the value of multiplexed assays of variant effect for better understanding protein structure, function and human variant effects. continued on next page source data 1. abundance and activity data for human variants found in clinvar, gnomad v2 and v3, and color genomics dataset. figure supplement 1 were cultured in dulbecco's modified eagle's medium supplemented with 10% fetal bovine serum, 100 u ml à1 penicillin, and 0.1 mg ml à1 streptomycin. cells were induced with 2.5 ug ml à1 doxycycline. cells were passaged by detachment with trypsin-edta 0.25%, and cells were prepared for sorting by detachment with versene. all cell lines tested negative for mycoplasma. cell line identity was not authenticated. because our activity assay is vitamin-k dependent, all activity assays were done with the same lot of fbs to ensure similar concentrations of vitamin k in each replicate. all synthetic oligonucleotides were obtained from idt and can be found in supplementary file 7. all non-library-related plasmid modifications were performed with gibson assembly (gibson et al., 2009 ). a gblock with a codon-optimized sequence for human vkor was ordered from idt. it was then cloned into the vector phsg298 (clontech). saturation mutagenesis primers were designed for each codon in vkor from positions 2 to 163 (jain and varadarajan, 2014) and ordered resuspended from idt. forward and reverse primers for each position were mixed at 2.5 mm, and used in a pcr reaction with 125 pg of phsg298-vkor, 5% dmso, and 5 ml of kapa hifi hotstart 2x readymix. pcr products were visualized on a 0.7% agarose gel to confirm amplification of the correct product. pcr products were then quantified using the quant-it picogreen dsdna assay kit (invitrogen) using dna control curves done in triplicate. to pool positions, a total amount of dna for each reaction was calculated that maximized the volume to be drawn from the lowest concentration pcr product. pooled pcr products were cleaned and concentrated using zymogen clean and concentrate kit and then gel extracted. the pooled library was phosphorylated with t4 pnk (neb), incubated at 37˚c for 30 min, 65˚c for 20 min, and then 4˚indefinitely. 8.5 ml of this phosphorylated product was combined with 1 ml of 10x t4 ligase buffer (neb) and 0.5 ml of t4 dna ligase (neb) to make a 10 ml overnight ligation reaction. this reaction was incubated at 16˚c overnight. the overnight ligation was then cleaned and concentrated (zymogen) and eluted in 6 ml of ddh2o. 1 ml of this ligation was then transformed into high efficiency e. coli using electroporation at 2 kv. each reaction contained 1 ml of ligation (or ligation control or puc19 10 pg/ul) and 25 ml of e. coli 975 ml of pre-warmed soc medium was added to each cuvette after electroporation, transferred to a culture tube, and recovered at 37˚c, shaking for 1 hr. at 1 hr, 1 and 10 ml samples from all cultures were taken and plated on appropriate medium (lb + kanamycin for ligation and ligation control; lb + ampicillin for puc19), the remaining 989 ml was used to inoculate a 50 ml culture (+ kanamycin). plates and 50 ml culture were incubated at 37˚c overnight (shaking for 50 ml culture). colonies on plates were then counted, and counts were used to calculate how many unique molecules were transformed to gauge coverage of the library. 50 ml culture was spun down and midiprepped. to transfer the library from phsg298 to the recombination vector, the phsg298 library and recombination vector were digested with xbai and aflii for 1 hr at 65˚c. the library and cut vector were then gel extracted. the library was then ligated with the cut vector at 5:1 using t4 ligase, overnight at 16˚c. the ligation was heat inactivated the next morning, clean and concentrated. another high efficiency transformation was performed the same as described above, except this ligation was plated on lb + ampicillin (antibiotic switching strategy). plates and 50 ml culture were incubated at 37˚c overnight (shaking for 50 ml culture). colonies on plates were then counted, and counts were used to calculate how many unique molecules had been transformed to gauge coverage of the library. a 50 ml culture was spun down and midiprepped. to barcode individual variants, plasmid library harvested from midiprep was digested with ecori-hf and ndei at 37˚c for 1 hr, 65˚c for 20 min. barcode oligos were ordered from idt, resuspended at 100 um, and then annealed by combining 1 ml each of primer with 4 ml cutsmart buffer and 34 ml ddh2o and running at 98˚c for 3 min followed by ramping down to 25˚c at à0.1˚c/second. after annealing, 0.8 ml of klenow polymerase (exonuclease negative, neb) and 1.35 ml of 1 mm dntps was then combined with the 40 ml of product to fill in the barcode oligo (cycling conditions: 25˚c for 15:00, 70˚c for 20:00, ramp down to 37˚c at à0.1˚c/s). digested vector and barcode oligo were then ligated overnight at 16˚c. the overnight ligation was then cleaned and concentrated and eluted in 6 ml of ddh2o. 1 ml of this ligation was then transformed into high efficiency e. coli using electroporation at 2 kv. each reaction contained 1 ml of ligation (or ligation control or puc19 10 pg/ul) and 25 ml of e. coli. 975 ml of pre-warmed soc medium was added to each cuvette after electroporation, transferred to a culture tube, and recovered at 37˚c, shaking for 1 hr. at 1 hr, 1 and 10 ml samples from water and puc19 cultures were taken and plated on lb supplemented with ampicillin. for ligation and ligation control, four flasks were prepared with 50 mls of lb and ampicillin, and then 500 ml, 250 ml, 125 ml, 62.5 ml was sample from the 1 ml of recovery and transferred into a corresponding flask. from those flasks, 1 ml, 10 ml, and 100 ml, were sampled and plated onto lb ampicillin plates. plates and 50 ml culture were incubated at 37˚c overnight. colonies on plates were then counted, and counts were used to calculate how many unique molecules were transformed to gauge number of barcodes. flask with the target number of barcodes was then spun down and midiprepped. cell line description vamp-seq assay cell line hek293t cells with a serine integrase landing pad integrated at the aavs1 locus were used (matreyek et al., 2017) . we used a previously published reporter cell line (haque et al., 2014) and inserted a recombinasebased landing pad at the aavs1 safe harbor locus using a previously published strategy (matreyek et al., 2017) . single cell clones were transfected with talens for aavs1 and the landing pad plasmid, and single cell clones were sorted. presence of one landing pad was confirmed by 1) barcode sequencing of the landing pad and 2) co-transfection experiment with gfp and mcherry. from this, we moved forward with one clone demonstrated to have only one landing pad present (clone 45). grnas to delete portions of the first exon of both vkorc1 and vkorc1l1 were ordered and cloned into pspcas9(bb)à2a-gfp (px458), which was a gift from feng zhang (addgene plasmid #48138; http://n2t.net/addgene:48138; rrid:addgene_48138). clone 45 was then transfected with these four plasmids, and single cells were sorted based on gfp positivity. disruption of vkorc1 and vkorc1l1 was confirmed by performing nested pcr, ta cloning, and then sequencing of products. we detected three alleles for both vkorc1 and vkorc1l1, indicating that these loci are triploid in hek293 cells. a western blot was also used to confirm absence of vkor protein product in our activity reporter cell line. protein lysates were harvested from~1 million cells using 100 ml np40 lysis buffer with freshly prepared protease inhibitor cocktail and 1 mm pmsf. protein lysates were qubited for concentration, and 20 ug of each protein lysate was loaded. 4-12% bistris nupage gel (thermo fisher) was used with mes buffer + 500 ml of antioxidant added to the inner chamber. the gel ran at 150v for 90 min. gel was then transferred to nitrocellulose using 1x transfer buffer 20% etoh at 24v for 1 hr on ice. the blot was washed for 5 min with 1x tbs-t 0.1% tween three times. blot was then blocked for overnight 1x tbs-t 0.1% tween + 5% milk. blot was then washed for 5 min with 1x tbs-t 0.1% tween three times. blot was then cut in half at the between the 25 kda and 35 kda molecular weight markers. the bottom blot was incubated with: avkor 1:1000 + 1x tbs-t 0.1% tween + 5% milk. the top blot was incubated with abeta-actin dhrp 1:1000+ 1x tbs-t 0.1% tween + 5% milk. both blots were incubated with their primary antibodies overnight at 4˚c. the avkor blot was washed for 5 min with 1x tbs-t 0.1% tween three times. the avkor blot was then incubated with 1:10,000 secondary anti-mouse-hrp (ge healthcare na931v) + 1x tbs-t 0.1% tween + 5% milk for one hour. the abeta-actin dhrp blot remained in primary antibody during this time, as no secondary antibody is needed for a direct hrp conjugate. both blots were then washed for 5 min with 1x tbs-t 0.1% tween three times. blots were then incubated with supersignal west dura extended duration substrate (thermo fisher). 500 ml of both substrates incubated on blot for 5 min. blots were then dried by kimwipe and exposed using the colorimetric and chemiluminescence functions on the biorad chemidoc mp (biorad). cells were transfected in six well plates, 250,000 cells per well (12-24 wells transfected total for each experiment). sequential transfections were performed. on day 1, 3 ug of pcag-nls-bxb1 was diluted in 250 ml of optimem and 6 ml of fugene (promega). on day 2, 3 ug of barcoded library was diluted in 250 ml of optimem and 6 ml of fugene6 and transfected. 48 hr after this second transfection, cells were induced with doxycycline at a final concentration of 2.5 ug/ml. cells were transfected in six-well plates, 500,000 cells per well (18-24 wells transfected total for each experiment). 272 ng of pcag-nls-bxb1 was diluted in 125 ul of optimem with 2.7 ug of barcoded library. 2.25 ul of lipofectamine 3000 (thermo fisher) was diluted in 125 ul of optimem in a separate tube. the dna mixture was then added to the lipofectamine 3000 mixture and incubated at room temperature for 15 min. transfection mixture was then added dropwise to one six-well plate. cells were induced with doxycycline 48 hr after transfection, with a final concentration of 2.5 ug/ml doxycycline. cells were washed once with pbs, then dissociated with versene. medium was added to dilute edta, and cells transferred to 15 ml conical and spun down at 300 x g for 4 min. medium was aspirated off, and cells were resuspended in pbs, then filtered through a 35 um nylon mesh filter. cells were sorted on a bd aria iii facs machine. mtagbfp2, expressed from the unrecombined landing pad, was excited with a 405 nm laser. recombined cells either expressed mcherry (abundance) or egfp (activity), and these were excited by a 561 nm laser and a 488 nm laser, respectively. samples were gated for live cells using fsc-a and ssc-a, then singlets using ssc-h vs. ssc-w, fsc-h vs. fsc-w. for activity assay reporter cell line, cells were then sorted for dsred positivity to ensure robust expression of reporter. cells that had successfully recombined a single vkor variant were gated on recombinant mtagbfp2 negativity and either mcherry positivity (abundance) or egfp positivity (activity) (see figure 2 -figure supplement 1b for gating example). recombined cells were sorted on 'yield' mode in the bd diva software and grown out for 3-5 days. recombined cells were run on a bd aria iii facs machine. cells were prepared for sorting as described above, and were then gated for live, recombined singlets. a ratio of egfp/mcherry was created using the bd diva software as a unique parameter, and the histogram of this ratio was divided into four equal bins. each quartile was sorted into a 5 ml tube on '4-way purity' mode. sorted cells were grown out for 2-4 days post sorting to ensure enough dna for sequencing. the details of replicate sorts for activity assay are in supplementary file 1. factor ix gla domain antibody specific for carboxylation was conjugated to apc following lynx rapid apc antibody conjugation kit instructions. antibody was resuspended at 1 mg/ml in nuclease-free water. 1 ml of modifier reagent was then added for every 10 ml of antibody and mixed by pipetting. that mixture was then pipetted directly onto the lynx lyophilized mix and gently mixed by pipetting up and down twice. the conjugation mixture was then capped and incubated in the dark at room temperature overnight. after overnight incubation, 1 ul of quencher reagent was added for every 10 ul of antibody used and left to incubate for 30 min. at that point, antibody was divided into 20 ul aliquots to be used for replicate experiments and stored at à20˚c. cells were plated in six-well plates at 500,000 cells per well with d10 medium with no doxycycline. all replicates were performed with 18-24 wells of cells total. after 24 hr, doxycycline was added to cells to induce expression of reporter and vkor variant. cells were then incubated with doxycycline for 48 hr. on day of cell sorting, each six well was washed with cold pbs, dissociated with 200 ml of versene, and then resuspended in 1 ml of phenol red-free dmem + 1% fbs and transferred to a 5 ml facs tube. cells were spun at 300 x g, then washed once with 1 ml of phenol red-free dmem + 1% fbs. cells were spun at 300 x g, and after aspirating supernatant, cell pellet was resuspended in 100 ul of antibody diluted 1:100 in phenol red-free dmem + 1% fbs. cells were incubated in antibody for 20 min at 4˚c in the dark, with vortexing at five minute intervals to ensure staining. after 20 min, 1 ml of staining buffer was added to each tube to dilute out antibody. cells were spun at 300 x g, washed twice more similarly with staining buffer, then resuspended in 200 ul. at this point, all tubes were pooled and filtered to remove clumps. cells were then sorted using a facsaria iii (bd biosciences) into bins based on their apc intensity. first, live, single, recombinant cells were selected as described above. a histogram of apc was created and gates dividing the library into four equally populated bins based on apc fluorescence intensity were drawn. the details of replicate sorts for activity assay are in supplementary file 2. western blotting for abundance score validation nine variants that spanned the abundance score range were chosen to validate abundance scores. cells were recombined using a selectable serine integrase landing pad (matreyek et al., 2020 ) using the transfection method described above for abundance experiments. recombinants were then selected with a small molecule, ap1903, at a concentration of 10 nm. protein lysates were harvested from~1 million cells using 100 ml np40 lysis buffer with freshly prepared protease inhibitor cocktail. 15 ml of each protein lysate was loaded onto a 4-12% bistris nupage gel (thermo fisher) with mes buffer + 500 ml of antioxidant added to the inner chamber. the gel ran at 200v for 35 min. gel was then transferred to nitrocellulose using 1x transfer buffer 10% metoh at 24v for 1 hr on ice. the blot was washed for 5 min with 1x tbs three times. blot was then blocked for one hour with 1x tbs-t + 5% milk and then washed for 5 min with 1x tbs-t 0.1% tween three times. the blot was incubated with avkor 1:1000 and acofilin 1:1000+ 1x tbs-t 0.1% tween + 5% milk overnight at 4c . the blot was washed for 5 min with 1x tbs-t 0.1% tween three times. the blot was then incubated with 1:10,000 goat anti-mouse igg (h+l) highly cross-adsorbed secondary antibody, alexa fluor plus 488 (thermofisher) and 1:10,000 goat anti-rabbit igg (h+l) cross-adsorbed secondary antibody, alexa fluor 647 (thermofisher)+ 1x tbs-t 0.1% tween + 5% milk for one hour. the blot was then washed for 5 min with 1x tbs-t 0.1% tween three times. the blot was then imaged using the alexafluor488 and alexafluor647 fluorescent channels on the chemidoc mp (biorad). cells were then collected, pelleted by centrifugation and stored at à20˚c. genomic dna was prepared using a dneasy kit, according to the manufacturer's instructions (qiagen), with the addition of a 30 min incubation at 37˚c with rnase in the re-suspension step. eight 50 ml first-round pcr reactions were each prepared with a final concentration of~50 ng mlàone input genomic dna, 1 â q5 high-fidelity master mix and 0.25 mm of the kam499/vkorampr 1.1 primers. the reaction conditions were 98˚c for 30 s, 98˚c for 10 s, 65˚c for 20 s, 72˚c for 60 s, repeat five times, 72˚c for 2 min, 4˚c hold. eight 50 ml reactions were combined, bound to ampure xp (beckman coulter), cleaned and eluted with 21 ml water. forty percent of the eluted volume was mixed with q5 high-fidelity master mix; vkor_indexf_1.1 and one of the indexed reverse primers, pten_seq_r1a through pten_seq_r2a, were added at 0.25 mm each. these reactions were run with sybr green i on a bio-rad miniopticon; reactions were denatured for 3 min at 95˚c and cycled 20 times at 95˚c for 15 s, 60˚c for 15 s, 72˚c for 15 s with a final 3 min extension at 72˚c. the indexed amplicons were mixed based in relative fluorescence units and run on a 1% agarose gel with sybr safe and gel extracted using a freeze and squeeze column (bio-rad). the product was quantified using kapa illumina quant kit. barcoded vkor library was subassembled using a miseq 600 kit (illumina). two amplicons were generated, one forward, one reverse. pcr reactions were each prepared with~500 ng input plasmid dna, 1 â kapa high-fidelity master mix and 0.25 mm of the vkor_sa_amp_f/vkor_sa_amp_r or vkor_sa_for_amp_r2.0/vkor_sa_rev_amp_f2.0 primers. pcr reactions were run at 95˚c for five minutes, then cycled 15 times at 98˚c for 0:20, 60˚c for 0:15, 72˚c for 0:30, with a final extension at 72˚c for 2:00. amplicons (741 bp) were gel extracted on a 1.0% gel run at 130v for 35 mins. the product was quantified using qubit and kapa illumina quant kit. read lengths were as follows: 289 bp forward read, 18 bp index1, 18 bp index 2 (index = barcode forward and reverse). all reads sharing a common barcode sequence were collapsed to form the consensus variant sequence, resulting in 175,052 barcodes after filtering. enrich2 was used to quantify barcodes from bin sequencing, using a minimum quality filter of 20 (rubin et al., 2017) . fastq files containing barcodes and the barcode map for vkor were used as input for enrich2. enrich2 configuration files for each experiment are available on the github repository. barcodes assigned to variants containing insertion, deletions, or multiple amino-acid alterations were removed from the analysis. scores and classifications were assigned using previously published analysis pipeline (matreyek et al., 2018) . briefly, for each protein variant, frequencies in each bin were calculated by dividing counts by total counts. from there, we filtered variants based on the number of experiments in which it was observed (f expt = 2) and their frequency (f freq = 10 à4 ), after noticing that low frequency variants introduced noise to the analysis. these frequencies were then each weighted by multiplying by 0.25, 0.5, 0.75, and one in a bin-wise fashion. we generated a replicate score for each variant by using min-max normalization: normalizing to the median weighted average of the nonsense distribution set at 0 and the median weighted average of the synonymous distribution set at 1. we then averaged those scores for a final, experiment-wide variant score. standard deviation and standard error were also calculated for each variant, and 95% confidence intervals were estimated using standard error, assuming a normal distribution. abundance and activity classifications were assigned by assessing variant score and confidence intervals in relation to synonymous variant distribution. to do this, we established a cut-off that separated the 5% of synonymous variants with the lowest abundance (or activity) scores from the 95% of synonymous variants with higher abundance (or activity) scores. variants with both scores and upper confidence intervals below this threshold were classified as 'low,' while those with scores below but upper confidence above were classified as 'possibly low.' variants with scores and lower confidence intervals above the threshold were classified as 'wt-like', while those with scores above lower confidence intervals below the threshold were classified as 'possibly wt-like.' finally, another threshold was set that separated the 5% of synonymous variants with the highest scores from the rest of the synonymous distribution. variants that had scores above this threshold, with lower confidence intervals above the lower threshold were classified as 'high'. windowed averages of abundance and activity scores were calculated using a window length of 10 positions with center alignment. scores were calculated for both charged amino acids (r, k, h, d, e,) and aliphatic amino acids (g, a, v, i, l). evcouplings extracts the constraints between pairs of residues, as evidenced in alignments of homologous sequences: first homologous sequences must be collected and aligned, and then a model of statistical energy costs and benefits between residues is fit to explain the sequence variation in the alignment. we collected an alignment of 2770 sequences using jackhammer (http:// hmmer.org/) to query the human vkor sequence against uniref100 (https://www.uniprot.org/uniref/), with a bitscore per residue cutoffs of 0.4 and 7 search iterations. we predicted secondary structures where the summed strength of couplings at would-be alpha helix and beta strand contacts scored above 1.5 for alpha helices and 0.75 respectively, for two or more consecutive residues. we extended the called helices and strands by one residue on each side for a minimum structure size of four residues. all methods used for building alignments, training the model, folding, and predicting secondary structure are part of the evcouplings software (https://evcouplings.org/) (hopf et al., 2019) . a homology model of human vkor was made by accessing i-tasser (yang et al., 2015) and using pdb structure 4nv5 as a template for threading. model1 from results was used for all figures in this paper. hierarchical clustering was performed on abundance score vectors for each position using the hclust function in r. dendrogram for hierarchically clustered heatmap was drawn using dendextend package (version 1.12.0). activity and abundance scores were rescaled so that the lowest score present in the dataset was set at 0, and the highest score at 1. a ratio of rescaled activity to rescaled abundance (specific activity) was then calculated for every variant. using variant specific activity scores, median specific activity was calculated for each position. threshold for classification as an active site position was drawn based on scores of known redox cysteines at positions 132 and 135, resulting in lowest 12.5% of median specific activity scores being classified as active site residues. we additionally required that any position within this group had been scored for at least four variants to eliminate noise from poor sampling. code for analysis is available at http://github.com/fowlerlab/vkor (chiasson, 2020a ; copy archived at https://github.com/elifesciences-publications/vkor). the code used to train the evolutionary couplings model is available at the evcouplings github repository (chiasson, 2020b ; https://github.com/debbiemarkslab/evcouplings; copy archived at https://github.com/elifesciences-publications/evcouplings). the data used to train the model is publically available at uniprot (https://uniprot.org). vkor abundance and activity score analysis. github. f80bb91 the arg98trp mutation in human vkorc1 causing vkcfd2 disrupts a di-arginine-based er retention motif warfarin and vitamin k compete for binding to phe55 in human vkor mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane interplay between hydrophobicity and the positive-inside rule in determining membrane-protein topology specificity in transmembrane helix-helix interactions can define a hierarchy of stability for sequence variants the power of multiplexed functional analysis of genetic variants enzymatic assembly of dna molecules up to several hundred kilobases clinical and genetic determinants of warfarin pharmacokinetics and pharmacodynamics during treatment initiation analysis of large-scale mutagenesis data to assess the impact of single amino acid substitutions transmembrane helix hydrophobicity is an energetic barrier during the retrotranslocation of integral membrane erad substrates r-vkorc1 expression in factor ix bhk cells increases the extent of factor ix carboxylation but is limited by saturation of another carboxylation component or by a shift in the rate-limiting step a cellular system for quantitation of vitamin k cycle activity: structure-activity effects on vitamin k antagonism by warfarin metabolites altered escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin k epoxide reductase recognition of transmembrane helices by the endoplasmic reticulum translocon molecular code for transmembrane-helix recognition by the sec61 translocon protein targeting and degradation are coupled for elimination of mislocalized proteins three-dimensional structures of membrane proteins from genomic sequencing the evcouplings python framework for coevolutionary sequence analysis a rapid, efficient, and economical inverse polymerase chain reaction-based method for generating a site saturation mutant library dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features variation across human exomes and genomes reveals the spectrum of loss-of-function intolerance across human protein-coding genes effect of the vkorc1 d36y variant on warfarin dose requirement and pharmacogenetic dose prediction clinvar: public archive of relationships among sequence variation and human phenotype identification of the gene for vitamin k epoxide reductase structure of a bacterial homologue of vitamin k epoxide reductase structures of an intramembrane vitamin k epoxide reductase homolog reveal control mechanisms for electron transfer rules for the recognition of dilysine retrieval motifs by coatomer protein 3d structure computed from evolutionary sequence variation a platform for functional assessment of large variant libraries in mammalian cells multiplex assessment of protein variant abundance by massively parallel sequencing an improved platform for functional assessment of large protein libraries in mammalian cells substratemodulated unwinding of transmembrane helices in the nss transporter leut packing of apolar side chains enables accurate design of highly stable membrane proteins fine-tuning the topology of a polytopic membrane protein: role of positively and negatively charged amino acids mutations in the vkorc1 gene cause warfarin resistance, warfarin sensitivity and combined deficiency of vitamin k dependent coagulation factors an algorithm for managing warfarin resistance vkorc1 pharmacogenomics summary novel insight into the mechanism of the vitamin k oxidoreductase (vkor): electron relay through cys43 and cys51 reduces vkor to allow vitamin k reduction and facilitation of vitamin k-dependent protein carboxylation mutations in vkorc1 cause warfarin resistance and multiple coagulation factor deficiency type 2 a statistical framework for analyzing deep mutational scanning data vitamin k epoxide reductase prefers er membrane-anchored thioredoxin-like redox partners a comprehensive comparison of transmembrane domains reveals organelle-specific properties warfarin traps human vitamin k epoxide reductase in an intermediate state during electron transfer stabilization of warfarin-binding pocket of vkorc1 and vkorl1 by a peripheral region determines their different sensitivity to warfarin inhibition alignme-a membrane protein sequence alignment web server human vitamin k epoxide reductase and its bacterial homologue have different membrane topologies and reaction mechanisms evaluation of warfarin resistance using transcription activator-like effector nucleases-mediated vitamin k epoxide reductase knockout hek293 cells structured states of disordered proteins from genomic sequences a series of pdb-related databanks for everyday needs amino acid distributions in integral membrane protein structures control of topology and mode of assembly of a polytopic membrane protein by positively charged residues warfarin and vitamin k epoxide reductase: a molecular accounting for observed inhibition the i-tasser suite: protein structure and function prediction a novel functional vkorc1 promoter polymorphism is associated with inter-individual and interethnic differences in warfarin sensitivity biochemical basis of hereditary resistance to warfarin in the rat we thank d nickerson and m dunham for helpful conversations in analyzing the data and writing the manuscript, a leith of the uw foege flow lab and d prunkard of the uw pathology flow cytometry core facility for assistance with cell sorting, and all members of the fowler lab for helpful feedback on figures. this work was supported by the nih (r24gm115277 p01gm116691 the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. melissa a chiasson, conceptualization, data curation, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, writing -original draft, writing -review and editing, carried out abundance and activity experiments and analyzed the data, wrote the manuscript with input from co-authors; nathan j rollins, debora s marks, conceptualization, formal analysis, supervision, validation, performed evolutionary couplings analysis; jason j stephany, methodology, prepared samples for next-generation sequencing; katherine a sitko, validation, cloned variants for abundance assay validation; kenneth a matreyek, conceptualization, provided abundance analysis framework; marta verby, song sun, frederick p roth, resources, provided a mutagenized vkor library; daniel desloover, data curation, writing -review and editing, provided human vkorc1 variants from color genomics; allan e rettie, conceptualization, resources, provided the parental activity reporter cell line; douglas m fowler, conceptualization, supervision, funding acquisition, writing -review and editing, wrote the manuscript with input from co-authors key: cord-016575-bn15006x authors: cox-georgian, destinney; ramadoss, niveditha; dona, chathu; basu, chhandak title: therapeutic and medicinal uses of terpenes date: 2019-11-12 journal: medicinal plants doi: 10.1007/978-3-030-31269-5_15 sha: doc_id: 16575 cord_uid: bn15006x terpenes, also known as terpenoids are the largest and most diverse group of naturally occurring compounds. based on the number of isoprene units they have, they are classified as mono, di, tri, tetra, and sesquiterpenes. they are mostly found in plants and form the major constituent of essential oils from plants. among the natural products that provide medical benefits for an organism, terpenes play a major and variety of roles. the common plant sources of terpenes are tea, thyme, cannabis, spanish sage, and citrus fruits (e.g., lemon, orange, mandarin). terpenes have a wide range of medicinal uses among which antiplasmodial activity is notable as its mechanism of action is similar to the popular antimalarial drug in use—chloroquine. monoterpenes specifically are widely studied for their antiviral property. with growing incidents of cancer and diabetes in modern world, terpenes also have the potential to serve as anticancer and antidiabetic reagents. along with these properties, terpenes also allow for flexibility in route of administration and suppression of side effects. certain terpenes were widely used in natural folk medicine. one such terpene is curcumin which holds anti-inflammatory, antioxidant, anticancer, antiseptic, antiplasmodial, astringent, digestive, diuretic, and many other properties. curcumin has also become a recent trend in healthy foods and open doors for several medical researches. this chapter summarizes the various terpenes, their sources, medicinal properties, mechanism of action, and the recent studies that are underway for designing terpenes as a lead molecule in the modern medicine. terpenes, also known as isoprenoids are the largest and most diverse group of naturally occurring compounds that are mostly found in plants but larger classes of terpenes such as sterols and squalene can be found in animals. they are responsible for the fragrance, taste, and pigment of plants. 1 terpenes are classified on the basis of organization and number of isoprene units it contains (see footnote 1). an isoprene unit is a building block of terpenes that is a gaseous hydrocarbon that contains the molecular formula c 5 h 8 (see footnote 1). terpenes and terpenoids are terms that are often used interchangeably but the two terms have slight differences; terpenes are an arrangement of isoprene units that are naturally occurring, volatile, unsaturated 5-carbon cyclic compounds that give off a scent or a taste to defend itself from organisms that feed off of certain types of plants (see footnote 1). terpenes have many functions in plants such as a thermoprotectant, signaling functions, and not limited to, pigments, flavoring, and solvents but also have various medicinal uses (yang et al. 2012) . table 15 .1 shows the different types of terpenes discussed in this chapter along with an example of that terpene. terpene is a natural compound with various medical properties and found in both plants and animals (gershenzon 2007) . among natural products that mediate antagonistic and beneficial interactions within the organism, terpene play a variety of roles (gershenzon 2007) . terpene protects many living organisms like microorganisms, animals and plants from abiotic and biotic stresses (gershenzon 2007) . terpene can ward off pathogens, predators, and competitors. living organisms use terpene for multiple reasons like medicinal purposes and communications about food, mates, or enemies (gershenzon 2007) . it is impressive how different organisms use terpene for common purposes even though terpene contain many forms and varieties (gershenzon 2007) . so far only a small percentage of terpene is investigated (franklin et al. 2001 ). cannabis is one of the most common sources for the medicinal terpene (franklin et al. 2001 ). this plant contains many medicinal properties like anticancer, antimicrobial, antifungal, antiviral, antihyperglycemic, analgesic, anti-inflammatory, and antiparasitic (franklin et al. 2001) . terpene is also used to enhance skin penetration, prevent inflammatory diseases (franklin et al. 2001) . nowadays modern medication use large scales of terpene for various treatment drugs (franklin et al. 2001) . there are commonly used plants like tea (melaleuca alternifolia), thyme, cannabis, salvia lavandulifolia (spanish sage), citrus fruits (lemon, orange, mandarin) etc. that provide wide range of medicinal values (perry et al. 2000) . tea tree oil has increased in popularity in recent years when it comes to alternative medicine (perry et al. 2000) . tea tree oil is a volatile essential oil and is famous for its antimicrobial properties, and acts as the active ingredient that is used to treat cutaneous infections (carson et al. 2006 ) apart from the flavor that gives to food, essential oil contain antimicrobial properties (bound et al. 2015) . thyme is one of plants that synthesize terpene alcohols and phenols which contain powerful antibacterial and antifungal properties (bound et al. 2015) . terpene synthesized from cannabis also long served as medicines (perry et al. 2000) . they also contain psychoactive properties and used against many infectious diseases (perry et al. 2000) . salvia lavandulifolia is famous for anti-dementia (current memory-enhancing) drugs by enhancing james and dubery (2009) cholinergic activity via inhibition of cholinesterase (perry et al. 2000) . in vitro examination method was used to study the effects of constituent terpenes on human erythrocyte acetylcholinesterase (perry et al. 2000) . some of the medicinal properties of terpenes are listed in table 15 .2. important properties associated with terpene are difficult to overstress (franklin et al. 2001 ). there are many important uses with terpene and these include antiinsect properties, antimicrobial properties and anti-herbivore properties (franklin et al. 2001) . terpene can be extracted through plants and thorough some insects (franklin et al. 2001) . without using harsh chemicals that could potentially contain side effects, terpene is a healthy alternative to ward off insects (franklin et al. 2001 ). there have been many pesticides made for killing domestic pests like lice, or mites (franklin et al. 2001) . in these cases, it is very important to make sure that these pesticides do not affect humans in harmful ways (franklin et al. 2001 ). there are many options like shampoo, sprays, lotions that were manufactured against pests that include one or more terpenes that are employed in the instant invention (franklin et al. 2001 ). these naturally occurring terpenes are generally not modified they were used in their raw form and the environment protection agency in the usa classified as "gras" which mean generally regards as safe (franklin et al. 2001 ). certain terpene is highly effective against both lice and lice eggs and there is a less than significant chance of resistance developing against this terpene based pesticides; reason for this is their observed modes of action (franklin et al. 2001 ). silva et al. (2008) unlike other types of pediculosis medication this terpene based instant inventions are not neurotoxins (franklin et al. 2001) terpenes are also used combined with terpene aldehyde called citral. citral derives from an essential oil that is extracted from lemongrass (cymbopogon citratus) (franklin et al. 2001) . citral possesses antibacterial and antifungal properties, while lemongrass possesses anti-insect properties (franklin et al. 2001) . a series of anti-insect formulation contain many terpenes (franklin et al. 2001 ) most of these pesticides are a mix of terpene and citral (franklin et al. 2001 ). table 15 .3 consists of what these terpenes include. antimicrobial properties or the ability to kill or stop growth of a microorganism in terpenes are commonly used in traditional and modern medicine (himejima et al. 1992 ). there are many terpenes with antimicrobial activities (himejima et al. 1992 ). the following plants produce terpenes which have antimicrobial properties: pinus ponderosa (pinaceae), spices (sage, rosemary, caraway, cumin, clove, and thyme), cretan propolis, helichrysum italicum, rosmarinus officinalis, and so on (himejima et al. 1992) . these antimicrobial terpenes can also be used against food borne pathogen like escherichia coli, staphylococcus aureus, and bacillus cereus (himejima et al. 1992) . pinus ponderosa cell extract contain wide-ranging antimicrobial activities (himejima et al. 1992) . after steaming and distillation from pinus ponderosa cell extract, a distillate and a residue are obtained (himejima et al. 1992) . the distillate consists of monoterpenes and some sesquiterpenes while the residue consists of four diterpene acids (himejima et al. 1992) . it was also reported that when a physical damage is caused to the pine tree or any other terpene containing tree from insect attacks, resin which contains terpene secret to protect the tree from further damage (himejima et al. 1992) . five different kinds of terpene can be isolated from cretan propolis, they are, the diterpenes, 14,15-dinor-13-oxo-8(17)-labden-19-oic acid and a mixture of labda-8(17),13e-dien-19-carboxy-15-yl oleate, palmitate and triterpene (popova et al. 2009 ). spectroscopic analysis and chemical evidence has been used to establish the structures of the different compounds (popova et al. 2009 ). these compounds that were isolated from terpene was tested for its antimicrobial activity against bacteria like gram positive and gram negative (popova et al. 2009 ). it was all tested for human pathogenic fungi which has broad-spectrum antimicrobial activity (popova et al. 2009 ). helichrysum italicum essential oil was analyzed using gas chromatography and mass spectrometry to fraction into terpene and terpenoid. fifty two compounds, including hydrocarbons of the oil; α-pinene (10.2%), α-cedrene (9.6%) aromadendrene (4.4%), β-caryophyllene (4.2%), and limonene (3.8%), neryl acetate (11.5%), 2-methylcyclohexyl pentanoate (8.3%), 2-methylcyclohexyl octanoate (4.8%), and geranyl acetate (4.7%) were identified (mastelic et al. 2017 ). the smallest of terpenes are monoterpenes. they contain the compound c 10 h 16 , come from different flowers, fruits and leaves and are known as the main component of essential oils, fragrances and many structural isomers (see footnote 1). monoterpenes are also the most fragrant of all the classes of terpenes (see footnote 1). examples for the types of monoterpenes found in natural scents are α-pinene, which imparts scent to pine trees, and limonene from citrus plants (see footnote 1). what is thought to be one of the main purposes of monoterpenes is to attract pollinators or to serve the purpose of repelling other organisms from feeding off of plants. they also may be related to the flowering process of the plants (loreto et al. 2002) . they are isolated from their plant sources by distillation with steam and have a boiling points in the range of 150 °c to 185 °c (see footnote 1). monoterpenes are purified using fractional distillation at pressures that are reduced or use another process in order to form a crystalline derivative (see footnote 1). many studies test the hypothesis of high emissions of monoterpenes under high temperatures using the leaves of quercus ilex, also known as evergreen oak (table 15 .1). the evergreen tree is native to the mediterranean area where it has to survive under hot and dry conditions and synthesis of these monoterpenes may have been an adaptive mechanism for the plants to survive under heat stress. 2 this tree does not emit isoprenes but it emits monoterpenes and is able to handle different environmental stresses such as drought, salt, and heat (see footnote 2). a particular study done by loreto et al. (2002) were conducted to visualize monoterpene production in response to high temperatures and to see if thermotolerance is increased with monoterpenes (loreto et al. 2002) . in this study, the leaves were exposed in 5 °c intervals ranging from the temperatures 30 °c to 55 °c and leaves were kept under conditions in which inhibited or allowed monoterpenes to synthesize (loreto et al. 2002) . the results that were found in this experience was a discovery of seven most abundant monoterpenes which was emitted at the maximum temperature of 35 °c and decreased its abundance over time as the temperatures increased and α-pinene had the greatest abundance of emittance at 35 °c as well as other terpenes but greatly reduced over higher temperatures (loreto et al. 2002) . at 55 °c the monoterpenes, myrcene and limonene had higher emission rates compared to temperatures around 35 °c (loreto et al. 2002) . photosynthesis was also decreased when the leaves were exposed to any temperature that was higher than 30 °c and at 55 °c showed a loss of co 2 and recovery occurred around 30 °c (loreto et al. 2002) . overall, the monoterpenes showed that their optimal temperature for emission was around 30-35 °c (loreto et al. 2002) . researchers prove that the emission of monoterpenes is under enzymatic control due to their optimal temperatures (loreto et al. 2002) . sesquiterpenes, containing the chemical formula c 15 h 24 , are much larger compounds than monoterpenes and are much more stable in comparison. 3 they are isolated by distillation with steam or by extraction and purified by methods such as vacuum fractional distillation or gas chromatography (see footnote 1). oxidation or rearrangement of isoprene units that are made to sesquiterpenes produce the corresponding sesquiterpenoids (see footnote 1). sesquiterpenes are naturally occurring and found in plants, fungi, and insects and act as a defensive mechanism or attract mates with pheromones in insects (see footnote 1). acyclic compounds of sesquiterpenes such as farnesans can be used as a natural pesticide for insects and also as pheromones for some insects and mammals such as elephants, to attract mates or to mark their territory (see footnote 1). sesquiterpenes have a vital role in plant growth hormones and signaling properties in response to its environment (giraudat 1995) . abscisic acid has a role in plants such as development, germination, cell division, and synthesis of protein storage and signalling (giraudat 1995) . it also plays a role in plants in response to various environmental stresses. it regulates the closure of the stoma by regulating ion channels and exchange of water across the plasma membrane (giraudat 1995) . cyclic adp-ribose signals abscisic acid in response to drought-stressing conditions from the environment (giraudat 1995) . abscisic acid is not unique to plants, it has shown to be present in the central nervous system of other organisms such as pigs and may play a role in humans as a pro-inflammatory cytokine and stimulator of insulin release in the human pancreas (chadwick et al. 2013) . gossypol is a sesquiterpene that is found in cotton plants. it has anticancer properties and can potentially inhibit fertility in male humans which is why it must be removed from essential oils and various other products before human use or consumption. avarol, a sesquiterpenoid that has shown to have antimicrobial and antifungal uses, is effective against the aids virus in humans (see footnote 3). 4 the medicinal properties of sesquiterpenes typically come from flowering plants that are included in the asteraceae family, which include, but not limited to sunflowers, marigolds, and daisies. this family of flowers is a significant resource for potent sesquiterpene lactones, which are usually found in the leaves and the flower portion of plants and are constantly being produced at high levels (chadwick et al. 2013) . the role of sesquiterpenes in these flowering plants are not solely made for human use but for the purpose of protecting the plant from predators and are produced de novo in response to microbial attack and ultraviolet ray protection (chadwick et al. 2013) . their bitter taste is a defense mechanism against herbivores from feeding on them but some have sweet tastes or tastes that are pleasant to certain organism for the purpose of spreading their seeds and being fertilized in different areas (chadwick et al. 2013) . sesquiterpenes have many uses in traditional, western medicine because they contain so many anticancer, antiplasmodial, and anti-inflammatory activities (chadwick et al. 2013) . sesquiterpenes lactones are able to reduce stomach ulcers in some people and are also present in powerful antimalarial drugs (chadwick et al. 2013 ). artemisinin, a metabolite produced from artemisia annua, which contains sesquiterpene lactone produced in the roots and shoots of the plants, is used in drugs to treat malaria (chadwick et al. 2013) . other uses of this family of flowers is for treatment of bacterial infections, migraines, and to improve skin (chadwick et al. 2013) . lettuce opium has been used for many years as a painkiller (chadwick et al. 2013 ). diterpenes are naturally occurring chemical compounds that contain the molecular formula, c 20 h 32 . diterpenes have physiologically active groups such as vitamin a activity well as plant growth hormones that regulate germination, flowering and switch reproductive cycles (from asexual to sexual reproduction) of plants (lee et al. 2015) . they can also be classified as a phytol, which is an oxygenated acyclic diterpene. over 650 diterpenoids have been isolated from euphorbia plants, which is a very diverse genus of flowering plants (popova et al. 2009 ). diterpenes have many therapeutic benefits such as antitumor, cytotoxic, and anti-inflammatory (vasas and hohmann 2014) . they are present in anticancer drugs such as taxol, and the tumor promoter, phorbol (vasas and hohmann 2014) . tanshinones are a class of diterpenes that are isolated from dried roots or rhizomes of an herb in traditional chinese medicine called salvia miltiorrhiza also known as danshen or tanshen (zhang et al. 2012) . tanshinones were first isolated in the 1930s, and since then, more than 90 chemicals have been identified and split up into two groups: 40 lipophilic and 50 hydrophilic compounds (zhang et al. 2012) . tanshinones have recently been extensively researched for their anticancer properties in vitro and in vivo (zhang et al. 2012 ). their potential use as an anticancer drug comes from their broad range of activities such as anti-proliferation and inhibiting adhesion, migration, and invasion (zhang et al. 2012) . analogues of tanshinone have been synthesized in many clinical trials because they have many anticancer attributes (lee et al. 2015) . this herb has been used in many asian countries for preventative and therapeutic solutions to many diseases such as heart disease, vascular diseases, and arthritis (zhang et al. 2012) . tanshinones may also reduce inflammation and increase immune responses (zhang et al. 2012) . cafestol and kahweol are diterpene alcohols that are found in the oil derived from coffee beans. these chemical structures are very similar but only differ by an extra double bond that is present in kahweol's chemical structure. 5 researchers have reported that coffee lowers the risk of depression in women, prostate cancer in men, stroke, diabetes, and some cancers (see footnote 5). it is thought that the antiinflammatory and antioxidant properties of these particular diterpenes are responsible for such events (see footnote 5). coffee benefits the liver as well by lowering liver enzymes that are in response to inflammation and damage and may offer some protection against liver cancer as well (see footnote 5). the adverse result of these diterpenes is that they raise cholesterol level, but it seems to be limited to coffee that has been unfiltered and has oily droplets of cafestol and kahweol (see footnote 5). filtered coffee may not have much impact on cholesterol levels (see footnote 5). triterpenes are composed of three or six isoprene units and have the chemical formula c 30 h 48 which includes steroids and sterols with squalene being the biological precursor of all triterpenes (see footnote 1). triterpenes are produced by animals, plants, and fungi. they play a role as precursors to steroids in animal and plant organisms, and are derived from mevalonic acid (see footnote 1). saponins come from the skins of many plants and have emulsion like properties that make them excellent detergents in the human digestive system (see footnote 1). chemical structures of steroid saponins are similar to hormones that are produced in the human body (see footnote 1). the medicinal uses of triterpenes are not quite as recognized as other different types of terpenes but their uses are being continuously investigated by researchers. their properties have been studied for anticancer, antioxidant, antiviral, and anti-atherosclerotic activities (nazaruk and borzym-kluczyk 2015) . some studies have shown that there is promising potential for the use of triterpenes for people with diabetes by aiming to reduce glucose levels and also by reducing sweetness inhibitors in sweet and high calorie foods (nazaruk and borzym-kluczyk 2015) . saponins have detoxification properties and act as a diuretic for the kidneys and wound healing properties (nazaruk and borzym-kluczyk 2015) . tetraterpenes are also known as carotenoids that have the molecular formula c 40 h 56 and can be in the category of terpenes because they are made from isoprene units. 6 most carotenoids are highly unsaturated and for this reason, they are extremely difficult to isolate and purify (see footnote 1). they are found in all different types of fungi, bacteria, and plants and mainly responsible for red, yellow, or orange fatsoluble plant and animal pigments (see footnote 6). one of the most crucial and common tetraterpene is beta-carotene that contributes to the yellow pigment in carrots. it is important to mammals especially because it is a precursor in producing vitamin a and other important terpenoids for vision (see footnote 1). higher order terpenes have been shown to increase thermotolerance (singsaas 2001) . the permeability of the thylakoid membranes increase at higher temperatures and this happens by an increase in cyclic photophosphorylation around photosystem ii (singsaas 2001) . when the temperature of the atmosphere continues to rise, the photophosphorylation system is not able to keep up with protons leaking, which causes the transmembrane gradient to drop and a reduction in atp synthesis occurs (singsaas 2001) . all these events can potentially cause lowering in the rubisco activation state due to an inhibition of rubp regeneration (singsaas 2001) . the mep pathway, also known as the non-mevalonate pathway or methylerythritol phosphate pathway, is a metabolic pathway for isoprenoid biosynthesis that creates the products isopentenyl pyrophosphate (ipp) and dimethylallyl pyrophosphate (dmapp). this pathway occurs in the chloroplasts and produce monoterpenes, specific sesquiterpenes, diterpenes, and carotenoids (zhang et al. 2012) . the vital application of this pathway is to develop antimicrobial agents to target diseases such as malaria and sexually transmitted diseases (hunter 2007) . since this pathway does not occur in humans, it is a valuable resource to develop antibacterial and antiparasitic drugs (seemann et al. 2009 ). the first steps of this pathway involve pyruvate and d-glyceraldehyde 3-phosphate to produce doxp which is catalyzed by 1-deoxy-d-xylulose-5-phosphate (dxs) (hunter 2007) . 1-deoxy-d-xylulose-5-phosphate reductoisomerase, otherwise known as ispc, coverts doxp to mep. from mep, it reacts with ctp to create 4-diphosphocytidyl-2c-methyl-d-erythritol (hunter 2007) . a phosphate is released in this reaction and then reacts with atp-dependent ispe to make 4-dipho sphocytidyl-2c-methyl-d-erythritol 2-phosphate and adp and then reacts with the enzyme ispf to create 2c-methyl-d-erythritol 2,4-cyclodophosphate (hunter 2007) . the enzyme requires metal cations. then finally, in the least understood step of the reaction, the two enzymes, ispg and isph make the two products, ipp and dmapp by using a two-electron reduction (hunter 2007) . the pathway is regulated by control of repression or activation of gene expression via feedback loops within the pathway or by effector molecules which target an enzyme or downstream activities (hunter 2007) . the mva pathway or mevalonic acid pathway occurs in the cytosol. it is responsible for the synthesis of sterols, specific sesquiterpenes, and also may play a role in the synthesis of transhinones (zhang et al. 2012) . in gram-positive bacteria, the genes in the metabolic pathways such as mva are organized into operons and are thought to be regulated by transcription (hunter 2007) . the use of cannabis is increasing for medicinal uses that commonly treat pain, the side effects of chemotherapy in cancer patients such as nausea, anxiety and depression, and its uses and benefits are continuously being researched by scientists (cathcart et al. 2015) . there are at least 80 compounds that come from the cannabis plant that are regarded as cannabinoids that cause psychotropic effects in the human brain due to cb 1 receptors (klein et al. 2011) . the main active ingredient, delta-9tetrahydrocannabinol, otherwise known as thc, is a psychoactive agent and is a focus for controversy in society because it binds to the human endocannabinoid receptors in areas of the brain such as the hippocampus and the frontal cortex, which are responsible for memory, cognition and attention. 7 how thc works is by taking the place of endocannabinoids, naturally occurring chemicals in the human body (see footnote 7). one of the most common and well known molecules that thc replaces in the human body is called anadamide (see footnote 7). to this day, scientists are researching to discover the exact role of this molecule in the human body. cannabidiol, or cbd is also a common ingredient in cannabis but compared to thc, it is a non-psychoactive and it can potentially reduce the effects of thc (klein et al. 2011 ). cbd does not bind to the same receptors as thc does in the human body and it works by inhibiting faah or the enzyme fatty acid amide hydroxyls (see footnote 7). this enzyme is responsible for degrading anadamide in the body and by inhibiting faah, cbd increases natural endocannabinoids already in the human system (klein et al. 2011) . cbd is thus an agent that works for depression, anxiety and neuroprotective effects (klein et al. 2011) . what are major components in cannabis are the monoterpenes that are responsible for many different medicinal properties. one of the main uses for thc is the potential for cancer treatment and can play a role in reducing size of tumors (see footnote 7). thc can also reduce inflammation caused by certain diseases in patients. other conditions that thc can help but are not limited to are adhd, arthritis, migraines, and glaucoma (see footnote 7). 8 it can also improve the symptoms in individuals that suffer from hiv by helping their appetite and thus causing weight again, improving their depression symptoms and their quality of life (lutge et al. 2013 ). terpenes have been shown to have a favorable antiplasmodial activity. with the rising malarial infections and drug resistance, terpenes have gained more attention towards it through antiplasmodial activity (nogueira and lopes 2011) . the interesting mechanism behind the terpene activity is that it binds to the hemin part of infected erythrocytes and kills the parasite just like the famous antimalarial drug chloroquine (orjih et al. 1981; kayembe et al. 2012) . hemin is made of iron which is necessary for the plasmodium development in the erythrocytes. though hemin breaking enzymes are not yet found in plasmodium, it could be one reason why hemin binding accounts for parasite lysis (ginsburg and demel 1984) . another study suggests that drug-hemin complex binds to phospholipid layers thereby disrupting the respective membrane structure and causing cell lysis (ginsburg and demel 1984) . moreover, it is also known that hemin can affect the carbohydrate metabolism of the parasites, which could lead to lysis of parasites (rodriguez and jungery 1986) . thus, terpenes can be designed to be promising drugs for malaria. different kinds of terpenes show different effects on the parasites. for instance, beta-myrcene the most common terpenes, is proven to have in vitro antiplasmodial activity (kpoviessi et al. 2014 ). beta-myrcene from cannabis sativa, the plant which is high in terpenes, does not show an anti plasmodial effect but extracts from stem, leaves, and seeds of clove basil showed a good antiplasmodial activity (small 2017; kpoviessi et al. 2014 ). additionally, it was also reported to have antitrypanosomal activity when tested against trypanosoma brucei brucei (habila et al. 2010) . this data leads to the fact that terpenes are effective against pathogenic protista. limonene regarded as the second most commonly found terpene, also possesses antiplasmodial activity against plasmodium falciparum. limonene achieves its goal by targeting the intermediates of the active isoprenoid pathway of the parasite. isoprenoid pathway plays a major role in parasite survival by mediating cell signaling, protein translation and several other biological processes (jordão et al. 2011) . specifically, the isoprenic products that are inhibited from being synthesized are dolichol and ubiquinone (goulart et al. 2004 ). the isoprenoid pathway of parasites is distinct from that found in mammals, which makes limonene a reliable constituent of antimalarial drug (goulart et al. 2004 ). thus, the host cell pathway will not be affected by the administration of the drug. pinene, commonly found monoterpene in pine trees is composed of two classesalpha-pinene and beta-pinene. both the classes of pinene were reported to be effective against the w2 strain of plasmodium falciparum, which is resistant to chloroquine (boyom et al. 2010) . of particular interest is the increase in antiplasmodial activity of pinene in cumin seed oil with increase in the distillation time. the study concluded that the optimal distillation time for increased antimalarial activity is 0-5 and 5-7.5 min (zheljazkov et al. 2015) . further investigation is needed to ascertain if distillation time is just increasing the yield of pinenes in the oil or improving the bioactivity of pinenes. the next most abundant terpene, caryophyllene has the ability to both prevent and cure malaria. caryophyllene is an active component of insect repellents especially for mosquitoes and other blood-feeding diptera (maia and sarah 2011) . recent studies ensured that silver nanoparticles synthesized from caryophyllene are highly effective against plasmodium falciparum (kamaraj et al. 2017 ). thus, terpenes could be a safer and a cost effective alternative for malarial treatment. the emerging viral diseases have necessitated the research for new effective antiviral agents such as terpenes. as a result, scientists evaluated various terpenes for their properties, among which monoterpenes showed a good result. monoterpenes are terpene classes that possess two isoprene units. they form a major constituent of essential oils in plants which indicates monoterpenes play a major role in defense for plants (grabmann 2005) . a 2005 study evaluated the in vitro antiviral activity of several essential oils extracted from south american plants (duschatzky et al. 2005) . the oil extracts were tested against three major human viruses-herpes simplex virus-1 (hsv1), dengue virus type 2, and junin virus. the oils that were proved to be virucidal were mainly composed of monoterpenes, namely, carvone, carveol limonene, alphaand beta-pinene, caryophylene, camphor, beta-ocimene, and one sesquiterpene which is germacrene (duschatzky et al. 2005) . a similar study in 2008 analyzed the essential oils of seven plants from lebanon for in vitro antiviral activity (loizzo et al. 2008 ). the viruses under investigation were hsv1 and severe acute respiratory syndrome corona virus (sars cov). the results were positive for antiviral effects, and the major constituents were alpha-and beta-pinene, beta-ocimene, and 1,8-cineole (loizzo et al. 2008) . following this, a 2009 study on salvia cedronella also had similar results which suggested 1,8-cineole, α-pinene, caryophyllene oxide, and sabinene to be the major components of virucidal oils (alim et al. 2009 ). functional data from these studies reveal that a few monoterpenes are shared by various plants for antiviral properties (alim et al. 2009 ). these shared monoterpenes could be of importance as they are present universally. of particular interest is the single main monoterpene that is contributing to the virucidal activity. this was studied by astani et al. (2009) using eucalyptus, tea tree, and thyme essential oil extracts (astani et al. 2009 ). they suggested that monoterpene hydrocarbons have a slightly higher virulent activity compared to the monoterpene alcohols against hsv-1. the monoterpenes with the highest virucidal activity were identified to be alpha-pinene and alpha-terpineol (astani et al. 2009 ). the mechanism behind the virucidal activity was suggested to be direct inactivation of free viral particles. however, the study concluded that more than isolated single monoterpenes, a mixture of monoterpenes are more effective and possessed lesser toxicity to host cells (astani et al. 2009 ). this was further bolstered by another study which evidenced the virucidal property of a combination of monoterpenes obtained from melaleuca alternifolia (zamora et al. 2016 ). the activity was tested against a human flavivirus west nile virus. the results were positive both in vivo and in vitro. the underlying mechanism was predicted to be induced cell cycle arrest at g0 or g1 phase. this indicates that a mixture of monoterpenes could act as a better antiviral agent rather than a single monoterpene (zamora et al. 2016) . recent studies have shown that triketone-terpene adducts also exert antiviral, antimicrobial and antitumor activity (chen et al. 2017 ). these adducts are obtained from myrtaceae as secondary metabolites in the form of sesquiterpenes called myrtucomvalones a, b, and c. the terpene adducts successfully inhibited the respiratory syncytial virus (rsv) (chen et al. 2017) . the bioactive terpenes present in various plants have shown various results for antiviral property. it would therefore be important to look for various plant source rather than various monoterpenes for therapeutic purposes. researchers are also focusing on synthesizing terpene hybrid from fungal sources as they are presumed to have antiviral and uv protective properties (yuan et al. 2017) . terpene synthesis from fungi can lead to cost effective and limited labor methods (yuan et al. 2017 ). the medicinal benefits of terpenes are not limited to pathogenic diseases. terpenes are widely acclaimed for their anticancer activity too. an early 1997 study concluded that a combination of monoterpenes, diterpenes and sesquiterpenes can be effectively used to treat cancers that occur in colon, brain, prostate gland, and bones. 9 it also claimed that administration of terpenes in humans inhibited the growth of prostate cancer cells and sensitized the tumor in such a way it becomes susceptible to radiotherapy (see footnote 9). the major advantage of this treatment was that, the drug can be administered through several routes among which oral and topical were most preferred (see footnote 9). among the different kinds of terpenes, limonene is well recognized as an anticancer agent. limonene is a bioactive food component found in citrus peels, orange peels, and several other citrus fruits (jirtle et al. 1993) . studies have reported limonene to exhibit strong cancer inhibition activity both in vitro and in vivo. the mechanism behind limonene activity is still under investigation. a study by jirtle et al. (1993) reported that limonene acts through induction of transforming growth factor b-1 and mannose-6-phosphate/insulin-like growth factor ii receptors (jirtle et al. 1993 ). in contrast a study by bishayee and rabi 2009) structural studies on limonene reported that they are lipophilic and have the tendency to be deposited in fatty tissues when administered orally. this indicates that limonene can act as an excellent chemopreventive drug for cancer as it can be deposited in the body (miller et al. 2010) . another study in 2013 concluded that limonene acts by suppressing the expression of breast tumor cyclin d1 (miller et al. 2013) . this lead to cell cycle arrest and mitigated proliferation of cancer cells in women with early stages of breast cancer (miller et al. 2013) . recent study showed that limonene from pinecones can kill lung cancer cells in vitro by apoptotic mechanism that is activated through caspase-3 pathway (lee et al. 2017) . these findings indicate a novel application of limonene towards fighting and preventing cancer. not just limonene, but also its metabolite perillyl alcohol is also said to exhibit antitumor activity in pancreatic cell lines through apoptotic mechanisms (sobral et al. 2014; dalessio et al. 2014) . apart from limonene, the terpene thymoquinone has all been widely studied for its chemoprotective and chemotherapeutic activity. thymoquinone is found to be an active constituent of the volatile oils of an annual herbaceous plant called nigella sativa (black cumin) (majdalawieh et al. 2017 ). the pathways affected by thymoquinone to exert its antitumor properties are p53, pparγ, mapk, nf-κb, pi3k/akt, and stat3 signaling pathways (majdalawieh et al. 2017) . thymoquinone has been proved to be anticancerous against several cancers such as breast cancer, skin cancer, non-small cell lung cancer, bile duct cancer, and brain cancer. the basic mechanisms underlying the cancer inhibition is apoptosis and cell cycle arrest (sobral et al. 2014; khader and eckl 2014) . most of the cancer related studies were performed using thermoquinone obtained from the n. sativa extracts. a 2012 study showed that thermoquinone can be obtained in larger amounts from the mint family, namely, monarda didyma and monarda media (taborsky et al. 2012) . thus, thermoquinone from alternative sources has to be tested for its precious potential in cancer therapy. other terpenes that have reported cytotoxic effects on cancer cells include alloocimene, camphor, beta-myrcene, pinene, alpha-and gamma-thujaplicin, terpinene, thymohydroquinone, carvone, camphene, and cymene (sobral et al. 2014) . terpenes being natural compounds are unlikely to affect the healthy cells or create a side effect, which attracts many researchers to exploit its capability in cancer treatment. diabetes is one of the widely prevalent diseases in the world. it is affecting both children and adults in both developing and developed nations (you and henneberg 2016; narayan et al. 2000) . the social and economic burden of diabetes continues to grow and it is expected to rise rapidly in developing countries (sarwar et al. 2010) . in usa, diabetes is one of the leading causes for visual impairment, limb amputation, renal diseases, heart diseases and death (saddinne et al. 1999) . diabetes can be of two types-type 1 (where the immune system of the body acts against the insulin-producing organs) and type 2 (where the insulin produced cannot be used by the body or insulin is produced in low amounts). 10 although there are several medications available, their use is limited due to their adverse effects. some of the commonly found side-effects include low blood sugar, vomiting, nausea, diarrhea, bloating, and weight gain. 11 this led to the research for natural products to be used as effective antidiabetic medication. phytochemicals from the medicinal plants have been recommended for treating type 2 diabetes, of which terpene forms a major constituent (jung et al. 2006) . medicinal plants of oriental morocco were studied for their antidiabetic property in rats. the report showed that terpenes, terpene diols, and terpene diol glucosides form major components of the extracts of plants under study (bnouham et al. 2010) . a similar study on medicinal plant and their natural products that were reported from 2001-2005 was conducted by jung et al. 2006 . this study was focused on non-insulin-dependent diabetes mellitus (type 2), and it proved that terpenes along with few other secondary metabolites such as alkaloids and flavonoids exhibit antidiabetic potential (jung et al. 2006) . the most promising terpene compound for treating diabetes is called andrographolide which is a diterpenoid lactone (brahmachari 2017) . this compound forms the major component of the leaves of the small herbaceous plant andrographis paniculata. a. paniculata is an asian plant that has already been reported to be used in traditional medicines for its therapeutic nature (brahmachari 2017) . the terpenoid acts by reducing the plasma glucose and increasing the utilization of glucose by the body in diabetes mellitus rats (gupta et al. 2008 ). the actual mechanism by how it does this is it activates the alpha-adrenoreceptors to increase the release of an opioid peptide beta-endomorphin (brahmachari 2017) which is reported to be secreted in low amounts in diabetic rats (forman et al. 1985) . this increased secretion in turn activates the opioid μ-receptors. these receptors can effectively curb the hepatic gluconeogenesis (glucose synthesis from non-carbohydrate precursors) and elevate the utilization of glucose by muscles. finally, this results in a reduced plasma glucose concentration (brahmachari 2017) . andrographolide is also observed to prevent the secondary complications of diabetes such as diabetic retinopathy, a condition that will lead to blindness (brahmachari 2017) . it significantly weakens the retinal angiogenesis and inflammation during the development of the disease (brahmachari 2017) . moreover, it can also fix the impaired or extended estrous cycle in diabetic rats (reyes et al. 2006) . andrographolide was orally administered in all the above studies. this indicates its efficiency for being used as a lead molecule in the future drugs designed for treating diabetes mellitus. another widely known terpene is curcumin obtained from curcuma longa which commonly called turmeric (nabavi et al. 2015) . it exhibits high antidiabetic property and acts by quashing the oxidative stress and inflammation. by regulating the polyol pathway, it can also reduce the plasma glucose and levels of glycosylated hemoglobin (nabavi et al. 2015) . moreover, curcumin is also reported to activate the enzymes present in the liver that are essential for glycolysis, gluconeogenesis, and lipid metabolism (zhang et al. 2013) . alike andrographolide, curcumin is also reported to reduce the complications of diabetes (nabavi et al. 2015) , for example, liver disorder which is a common manifestation of diabetes type 2 (zhang et al. 2013) . curcumin treats these disorders by reducing the liver weight and lipid peroxidation products. further, it is also reported to normalize the levels of fetuin-a in serum that contributes to insulin resistance and fatty liver in diabetic rats (zhang et al. 2013) . other complications that can be attenuated by curcumin include diabetes associated-retinopathy, microangiopathy, neuropathy, and nephropathy (zhang et al. 2013) . these findings confirm that curcumin is likely to be used in the future for diabetes treatment. depression has become a serious health concern by contributing to the emerging mental and emotional disorders throughout the world. it is hitting both the developed and developing countries. depression can pave way to various health issues from alcoholism to heart diseases (holden 2000) . it is also said to increase the rate of mortality significantly in breast cancer patients (hjerl et al. 2003) . moreover, depression immobilizes its victims thereby leading to economic loss (holden 2000) . by analyzing the social and economic burdens caused by depression, researchers have stepped out towards finding novel stress-relieving drugs. synthetic drugs have serious side-effects and unintended interactions with the body that negatively affects the treatment outcome (jawaid et al. 2011) . hence this necessitated the need for natural drugs. terpenes serves as one of the most relevant bioactive compound for treating depression and therefore can open doors for designing natural or synthetic antidepressant drugs (bahramsoltani et al. 2015) . twenty-five percentage of antidepressant drugs prescribed by doctors are obtained from herbs through various extracts (saki et al. 2014) . to estimate the important compounds contributing to the antidepressant effect, saki et al. (2014) performed an electronic database based study. the results revealed that terpenes formed a major part of the extracts of medicinal plants that exerted antidepressant effects (saki et al. 2014) . thus, scientists focused on identifying the active principles of plant extracts contributing to the antistress effects. different plant had different acting compounds. among the several terpenes, linalool and beta-pinene are commonly found to be active principles (both guzmángutiérrez et al. 2015; guzmán-gutiérrez et al. 2012) . they were discovered from the extracts of medicinal plants litsea glaucescens and tagetes lucida and flowers of lavender (appleton 2012; guzmán-gutiérrez et al. 2012; guadarrama-cruz et al. 2008 ). these monoterpenes act by interacting with the 5ht1a receptors of the serotonergic pathway. serotonins are important in the fact that their release and re-uptake levels can be altered to overcome stress (chaouloff 2000; guzmán-gutiérrez et al. 2012) . they also interact with adrenergic receptors of the body that play a major role in stress-induced behavioral changes (pandey et al. 1995; guzmán-gutiérrez et al. 2015) . another interesting finding is the interaction of beta-pinene with dopaminergic receptors namely d1 receptors. this is the mechanism followed by most of the antidepressant drugs available in the market (guzmángutiérrez et al. 2015) . a more interesting study would be to examine the beta-pinene and linalool efficiency through inhalation tests. this is because these monoterpenes are aromatic compounds that generally have an enhanced activity when inhaled as they can directly hit the central nervous system (guzmán gutiérrez et al. 2014) . apart from monoterpenes, sesquiterpenes also exhibit antidepressant effects. one striking example is beta-caryophyllene which was proved to ameliorate the depressive symptoms in mice (bahi et al. 2014) . the underlying mechanism of this compound is binding to a receptor called cb2 and activating it. cb2 is found in the brain and immune cells and plays a major role in regulating depressive-related disorders (bahi et al. 2014) . thus beta-caryophyllene curbs depression by acting as a cb2 receptor agonist (bahi et al. 2014) . other terpenes that have effective antidepressant properties include hyperforin which is present in the extracts of hypericum perforatum (subhan et al. 2010) . it has been shown that the extracts of h. perforatum differ in their antidepressant potential with the difference in concentration of hyperforin present in the extract (laakmann et al. 1999 ). similar to many other antidepressants hyperforin acts by inhibiting the neuronal uptake of mood regulators such as serotonin, dopamine and norepinephrine. in addition, it also has its own unique mechanism of controlling depression by inhibiting the neurotransmitters gaba and l-glutamate uptake (müller et al. 2001) . another fascinating antidepressant plant is valeriana wallichii, which is a short perennial herb. this plant not only reduces the stress and anxiety levels but also improves the symptoms of depression in humans (bhattacharyya et al. 2007 ). the major components of valeriana extracts are terpenoids called maaliol, patchouli alcohol, and 8-acetoxypatchouli alcohol (subhan et al. 2010) . the terpenoid-less extract of valeriana was found to be devoid of antidepressant activity which indicates that terpenes are the active components involved in reducing the depression (subhan et al. 2010 ). folk medicine has always been an eye-opener for designing novel drugs for diseases. to be more specific, almost three-fourths of the plant-based drugs were created based on the knowledge of folk medicine (table 15 .4) (efferth et al. 2008) . realizing this fact, western worlds are now turning back into old medicines and bioactive plant components to treat modern diseases (efferth et al. 2007 (efferth et al. , 2008 . this has boosted the export rates of chinese medicinal products (based on traditional chinese medicine) from china to other developed nations. plants used in traditional chinese medicine (tcm) are being extensively studied for their secondary metabolites and their therapeutic properties (efferth et al. 2007 ). one of the active principles of tcm products is terpenes (liu and jiang 2012) . due to their large availability and diversity, terpenes contribute the most to industrial and medicinal applications among all the secondary metabolites of plants (zwenger and basu 2008) . paclitaxel is one of the most successful terpenes available in the market today (efferth et al. 2008) . it is made out of yew trees which is a medicinal tree used in tcm. 12 raw material from yew contains taxol (brand name of paclitaxel) which is used in the treatment of cancers in breast, lung, ovary, pancreas, cervix, and blood (see footnote 12). 13,14 two variations of this drug are used now in chemotherapyconventional paclitaxel and albumin-bound paclitaxel (see footnote 13). the advantage of the latter is that concentration increases in tumor cells at a rate higher than that of the former (see footnote 13). the mechanism of anticancer activity is described as disruption of microtubules in the mitotic spindle, which will lead to incomplete chromosome separation thereby causing cell death (see footnote 13). in tcm and ayurveda (herbal medicinal science mainly developed in india), healers used the twigs and barks of the tree to make a special kind of tea that can be given to patients suffering from cancer. however due to the slow growing nature of yew tree, paclitaxel nowadays is produced by coalescing the products of endophytic fungus that grows under the tree and the bark of the tree 15,16 (heinig et al. 2013 one more common terpene present in the drugs used in tcm is pinene (wu et al. 2008) . pinene exhibits therapeutic properties such as anti-inflammatory, antiseptic, anticancer, and antibiotic properties. 17, 18 the source for pinene is eucalyptus and other related coniferous trees (see footnote 17, sartorelli et al. 2007) in olden days, the juice from the bark of eucalyptus was collected and mixed in water, milk or wine to be used as a drug (see footnote 17). currently, they are extracted in the form of oil and sold in the form of syrups and lozenges. 19 as eucalyptus oil contains several monoterpenes, a study analyzed the different constituents of eucalyptus oil for its effectiveness against bacteria. here it was concluded that alpha-pinene is the best monoterpene with the highest inhibitory activity (sartorelli et al. 2007) . recently scientists are studying another primary terpene in eucalyptus called cineole. cineole is reported to improve the memory power, cognitive performance and attenuate the symptoms of alzheimer's disease in humans (see footnote 19; moss and oliver 2012) . in addition, studies also showed that cineole is capable of improving the health of bronchitis patients by reducing their cough (fischer and dethlefsen 2013) . this is in agreement with the fact that eucalyptus oil was used as an expectorant in ayurvedic medicine. 20 it is also known that local brazilians used the eucalyptus leaves to treat several human diseases such as cancer (mathias et al. 2012) . further reports also suggest that eucalyptus oil has been involved in ancient indian ayurvedic and greco-european medicine systems (see footnote 19). ayurveda is a popular medicine system which originated about 3000 years ago in india. the ayurvedic medicines are based on medicinal herbs, minerals, and metals (see footnote 16) along with diet regimes such as vegetarianism (caldecott 2006) . this system of medicine has proven to cure chronic disorders that could not be treated by western medicine (sharma et al. 2007) . interestingly a lot of medicinal plants used by ayurvedic practitioners owe their therapeutic property to their terpene contents. one good example is turmeric, a family of ginger which is regarded as "golden goddess" by medical practitioners (see footnote 18). 21 it has numerous therapeutic properties that includes anti-inflammatory, antioxidant, anticancer, antiseptic, antiplasmodial, astringent, digestive, diuretic, and many more (see footnote 18). 22 recently, scientists discovered that most of the turmeric's properties are laid out by the yellow-colored terpene-curcumin (kocaadam and şanlier 2017) . studies are now trying to create curcumin analogues to improve the effects and activity of natural curcumin (kocaadam and şanlier 2017) . another popular example is clove which was used by both ayurveda and tcm as a painkiller in dental cases. it was applied topically on cavities to relieve toothache and abdomen to treat digestive problems (alqareer et al. 2006) . the essential oil of clove is mostly composed of eugenol, a bioactive terpene that is responsible for clove's aroma (alqareer et al. 2006) . eugenol by itself is said to enhance the blood circulation in the body and improve metabolism (see footnote 22). thus, based on the above data we can conclude that various terpenes have been in use even before their discoveries by modern science, due to their amazing medicinal properties. a schematic summary of different terpenes and their medicinal uses, that we discussed, is provided below in fig. 15 .1. 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valeriana wallichii extracts and antidepressant-like response profiles identification of potential sources of thymoquinone and related compounds in asteraceae, cupressaceae, lamiaceae, and ranunculaceae families euphorbia diterpenes: isolation, structure, biological activity, and synthesis volatility-dependent 2d ir correlation analysis of traditional chinese medicine 'red flower oil' preparation from different manufacturers enhancing production of bio-isoprene using hybrid mva pathway and isoprene synthase in e. coli type 1 diabetes prevalence increasing globally and regionally: the role of natural selection and life expectancy at birth polyketide-terpene hybrid metabolites from an endolichenic fungus pestalotiopsis sp the in vitro and in vivo antiviral properties of combined monoterpene alcohols against west nile virus infection the scientific contributions of m. judah folkman to cancer research novel norsesquiterpenoids from the roots of phyllanthus emblica tanshinones: sources, pharmacokinetics and anti-cancer activities curcumin and diabetes: a systematic review distillation time as tool for improved antimalarial activity and differential oil composition of cumin seed oil plant terpenoids: applications and potentials holy basil/ tulsi eugenol, β-elemene, β-caryophyllene and germacrenerestores functions of nervous system increases fertiltity used to treat asthma and cold key: cord-034066-fsp7e5x5 authors: di figlia-peck, stephanie; feinstein, ronald; fisher, martin title: treatment of children and adolescents who are overweight or obese date: 2020-10-21 journal: curr probl pediatr adolesc health care doi: 10.1016/j.cppeds.2020.100871 sha: doc_id: 34066 cord_uid: fsp7e5x5 nan m anaging the millions of children and adolescents who are either overweight or obese has become a major challenge for the healthcare community. in 1997, an expert committee was convened by the maternal and child health bureau of the health resources and services administration (hrsa), department of health and human services, (dhhs) to develop guidelines for healthcare providers. 1 in 2005, the american medical association, in cooperation with hrsa and the center for disease control and prevention, created an expert committee to update those initial guidelines. 2 and in 2008, the agency for healthcare research and quality of the hhs came out with an evidence-based/technology assessment entitled "the effectiveness of weight management programs in children and adolescents." 3 in addition to these government-sponsored guidelines, recommendations for management of overweight and obesity in this population have been issued by multiple other organizations. the one directive they all have in common is that a multicomponent program that focuses on physical activity, diet, and behavioral change should be the first line of treatment offered. this article highlights the evidence-based data, presents the various ways in which this multicomponent approach can be implemented, and includes the roles of school programs and bariatric surgery as weight management options. family-based group sessions coordinated by a registered dietitian (rd/rdn) are a crucial part of multicomponent interventions. the academy of nutrition and dietetics, which issued its pediatric weight management evidence-based guidelines in 2015, has reported positive weight status outcomes, both shorter-term (6 months) and longer-term (12 months), when group pediatric weight management sessions and family participation are coordinated. 4, 5 individual family and mixed-format (which includes some time with individual families and some group time) approaches have been found to be superior to group-only approaches as per the latest us preventative services task force (uspstf) recommendations. 6 however, including in addition to these governmentsponsored guidelines, recommendations for management of overweight and obesity in this population have been issued by multiple other organizations. the one directive they all have in common is that a multicomponent program that focuses on physical activity, diet, and behavioral change should be the first line of treatment offered. the academy of nutrition and dietetics, which issued its pediatric weight management evidence-based guidelines in 2015, has reported positive weight status outcomes, both shorter-term (6 months) and longer-term (12 months), when group pediatric weight management sessions and family participation are coordinated. 4, 5 some group sessions may offer the opportunity for social support and improve cost effectiveness. 7 the dose of treatment has a strong impact on success. multicomponent behavioral interventions of moderate (26à75 h of treatment contact per year) to high intensity (> 75 h) for obese children and adolescents, ages six and older, have been shown to yield short term improvements in up to 12 months. obtaining a qualitative assessment of a patient's diet with a particular focus on dietary patterns thought to be linked to excess energy intake and adiposity is recommended, as intervening with these patterns can significantly reduce intake and potentially improve nutritional status. 8 tailoring interventions by considering patient and family motivation, as well as readiness for change, is optimal. the family-based approach can be modified based on the age of the patient and the degree of parental involvement. it should be noted that family involvement has been shown to be less effective when the patients are older teens. 9 behavioral treatments at the heart of behavioral treatment for obesity is determining what behaviors are modifiable and what therapies to use to help patients achieve the needed modifications. motivational interviewing (mi), which is a patient-centered counseling style, has been shown to be effective in primary care settings. 10 a dietitian should be included, as the rdn's knowledge and skill base are critical in the ongoing process of addressing the diverse needs of clients and families. 4, 10 cognitive behavioral management and gradual stepwise change have been explored in depth for childhood and adolescent obesity treatment. individuals get acclimated to recommended changes over time by making adjustments in their dietary patterns and food environment and by learning to set limits on eating unhealthy food. short-term goals are established in order to lead to long-term habits that change the way individuals and their families think about food. cognitive behavioral therapy (cbt) focuses on breaking the negative cycle that is a part of weight-related difficulties in obesity, the "maladaptive daily patterns, cognition that is distorted, and problematic behaviors" cited by wilfley et al. 11 it allows for a restructuring of daily patterns. bloom et al. explores utilizing a form of cbt known as cbt-af to address appetite awareness and cues for eating. 12 caat is an adapted version used with children and adolescents to sensitize them to recognize and respond to internal appetite cues such as hunger and satiety in order to improve their self-regulation of energy intake. results of one study showed a significant reduction in body mass index (bmi) for children in a caat group compared to those in a control group. however, this impact was only studied short term. the researchers concluded that caat holds promise as a treatment modality since overweight and obese children are often less effective in regulating food intake compared to normal weight children. 13, 14 in the transtheoretical model of change, in which change occurs in stages, the readiness of parents for personal change, as well as their readiness to help their children make changes, becomes a pivotal factor for success in a weight management program. 15 tailored messages to parents may help modulate their "decisional balance," (the value of making behavioral changes versus the value of not making any changes) and contribute to the likelihood of treatment success for their children. yet influencing parents so as to influence their children in terms of weight management behaviors can be a challenge. weight loss is a complex behavior which encompasses two separate "domains" of changeà eating habits and physical activity. although these are often considered together, each carries unique challenges with respect to perceived confidence and readiness for change. 16, 17 in a cross-sectional study with a convenience sample of parents (or guardians) of children attending a tertiary care pediatric obesity clinic, parents completed surveys initially and again on follow up visits to assess their readiness for change. 16 those in the action/ cognitive behavioral management and gradual stepwise change have been explored in depth for childhood and adolescent obesity treatment. individuals get acclimated to recommended changes over time by making adjustments in their dietary patterns and food environment and by learning to set limits on eating unhealthy food. maintenance state of change were more likely to be actively making changes to multiple eating behav-iorsài.e. availability of sugar-sweetened beverages (ssbs) and salty snacks, and in physical activity patternsài.e. reaching recommended levels of increased activity and limiting screen time. their children were more likely to be more physically active and to consume less fast food and more fruits and vegetables than the children of parents in the other stages of change. 16 parents who believed their own weight was a health problem were less ready to make changes to their children's diet. 18 these authors suggest that maintaining both parent and patient motivation should be a focal point of treatment and that this may entail a variety of approaches, such as using texting or other electronic devices to assess the stage of change for readiness and decisional support. 18 use of mobile health technology as an adjunct to behavioral based weight management strategies is becoming more common. chen and researchers reported on a convenience sample of self-identified chinese-american adolescents with bmi 85th percentile who participated in a culturally focused intervention called smart start. 19 it provided general health education, wearable fitness trackers, online educational modules, and tailored biweekly text messages. a benefit in outcome occurred in both the control and intervention groups. however, over a six month period, the intervention group, as compared to the control group, had "statistically greater changes" in bmi that were associated with less fast food intake, a lower intake of ssbs, and an increase in physical activity levels and decreased sedentary behavior. 19 overall, mobile health use has shown mixed benefits for weight management in adolescents and young adults. 20 other mobile health initiatives have resulted in weight loss in the experimental groups that was not sustained 21, 22 or have displayed no further benefits above that of the standard care group. 23 researchers have thus noted limited evidence of efficacy of mobile health interventions as a stand-alone treatment modality. 24 the impact of combining the mobile health approach with components of behavior based interventions has been examined by cueto et al. 25 they evaluated the original kurbo app (circa 2014) before it became kurbo ww. 26 designed to promote behavior change and encourage healthy lifestyle choices, it used the evidence-based traffic light diet approach 27 and kurbo health coaching through the incorporation of behavior substitutions and habit formation. 28 although kurbo includes components of behaviorbased interventions proven successful in pediatric and adolescent weight management, it has come under fire for promoting behaviors that can be perceived as overly restrictive and potentially promoting eating disorder behaviors. 29 questions have been raised based on degree of weight loss in young subscribers and whether adequate monitors are in place to determine that degree. prior studies have warned about the potential for "growth velocity to be negatively impacted when caloric intake is restricted," and thus growth velocity must be followed carefully during and after weight loss in older children and younger adolescents, and medical supervision may be warranted. 30, 31 other combined interventions utilizing mobile health apps have yielded partial success. one 12month technology-based program for adolescents with type 2 diabetes "was not sufficient to produce weight loss with the combination of web intervention and group sessions and telephone follow up, but improvements in sedentary behavior and use of behavior change strategies expected to lead to behavior change was evidenced." 32 telemedicine, in theory, should be able to compensate for some of the barriers that prevent access to and utilization of family based comprehensive behavioral interventions for child and adolescent obesity. 33 these barriers include time, transportation, access, cost, scheduling challenges, stigmatization, language barriers and more. [34] [35] [36] [37] rural populations have been studied for feasibility and satisfaction with telemedicine treatment approaches, and results have been comparable to standard treatment outcomes. 38 urban populations can face similar barriers to attendance of programs held in hospitals or university medical use of mobile health technology as an adjunct to behavioral based weight management strategies is becoming more common. settingsà delays in acquiring care, fear of being judged based on native language or residency, and possible stigmatization. 39 consequently, there have been studies here too (even prior to the covid-19 pandemic) regarding the incorporation of telemedicine as a supplemental arm of treatment modalities involving group sessions and mixed formats with medical staff including physicians, nurse practitioners (nps), nurses, psychologists, family counselors, dietitians, physical therapists, exercise specialists, and social workers. 38, 39 with the dramatic increase in the use of telemedicine brought about by the covid 19 pandemic, this modality of treatment will certainly be utilized and studied considerably more in the upcoming months and years. mobile apps have proved an engaging way to involve children in health behavior changes, 40 allowing for delivery of health information in a portable, "entertaining" way. [41] [42] [43] these apps are capable of promoting some of the expert recommendations for healthy eating and physical activity, including setting goals/limits and reducing intake of ssbs, but they often do not go deeper into behavior change. one app, hyperant tm , utilized a set of "hyper activity cards tm " to give children ideas for health-promoting behaviors including physical activity, healthy eating, and sleep. 44 however, it only provides user messages without offering the opportunity for interaction. in a meta-analysis of mobile health technologies for selfmonitoring, darling et al. concluded that self-monitoring techniques using mobile health technologies have a small but significant effect on weight status in children and adolescents. 45 population health initiatives a more "macro approach" for educating and guiding children, adolescents, their families and guardians is called for to achieve greater success in maintaining better health and weight management. several such programs are described below. the choosemyplate teaching initiative from the u.s. department of agriculture (usda) came out of the need for a vehicle to effectively and "with maximum visibility" communicate the 2010 dietary guidelines for americans (dgas) in order to foster a healthier lifestyle. 46 using print and online resources to engage the public, it was translated into several languages, incorporated into health curriculum resources created for nutrition education for children and adults, and promoted to nutrition communicators, educators and the food industry, calling upon them to "get the message out." [47] [48] [49] its message: "americans can achieve a healthier weight by eating more of some foods," was thought to be one that consumers could embrace. when one's plate has a larger proportion of lower calorie vegetables, they, in essence sense, "crowd out' the more calorically dense other foods on the plate like refined grains and high fat proteins. thus, adding foods, rather than taking away foods, can result in a calorie deficit. designed to "impact behavior during meal planning" and "perception during meal consumption," this initiative aimed to be seen by individuals and groups as a positive way of collectively altering energy balance. choosemyplate calls for a shift in consumption patterns. it emphasizes less processed foods and more of whole grains, lower fat and non-fat dairy items over full-fat varieties, water in place of ssbs, and protein alternatives, including leaner meats. along with less saturated fat and added sugars, lower sodium options are promoted. central to this multimodal plan is the plate icon ( fig. 1 ) that replaces the food guide pyramid as both visual cue and accepted standard. 48, 49 the most current recommendations, as per myplate, my wins (see below), directs people to "find your healthy eating style and maintain it for a lifetime" by making half of the meal plate fruits and vegetables (varying the veggies and focusing on whole fruits), making a quarter of the plate grains (half of them whole grains), and making the remaining quarter of the plate proteins (varying the protein routine). individuals are advised to move to low-fat or fat-free milk or choosemyplate calls for a shift in consumption patterns. it emphasizes less processed foods and more of whole grains, lower fat and non-fat dairy items over full-fat varieties, water in place of ssbs, and protein alternatives, including leaner meats. along with less saturated fat and added sugars, lower sodium options are promoted. yogurt for dairy intake, which is depicted alongside on the right of the icon's plate. the "right mix" is based on variety, amount, and nutrition content. the original myplate teaching campaign was revamped to reflect changes in the updated dgas (2015à2020). myplate, my wins, launched in 2015, strongly focuses on food patterns. it added the concept of "a healthy eating style" which can be achieved with "small changes" to promote the goal of getting individuals to realize that "what you eat and drink over time matters and can help you be healthier now, and in the future," messaging that reflected the evolving emphasis of the dgas. the public was encouraged to be more engaged and active in their health, and was invited to virtually share personal experiences with my plate, my wins on social media using #myplatemywins. the present day choosemy-plate.gov website includes printable materials, images, and graphics available as downloadable pdfs, jpgs, and other files-all in the public domain so that no permission is required to print, reproduce, or use them. resources have grown to include a host of topics, from meal planning and food safety to physical activity and seasonal resources. information continues to be available in diverse formats like toolkits, quizzes, infographics, and videos. researchers out of the behavioral health and nutrition department at the university of delaware used myplate to test whether peer education improved selfefficacy, perceptions and attitudes toward healthy eating, and physical activity. 50, 51 they concluded that peer education could promote improved knowledge and attitudes about myplate among college students and increase their self-efficacy, helping them make healthier decisions with regard to food and food intake. the pilot first year experience course curriculum developed at the university became mandatory coursework for all incoming freshmen. 41 a florida study of elementary school children whose families qualified for federal assistance via the supplemental nutrition assistance program (snap), utilized the six-lesson youth understanding myplate (yum) curriculum to teach the students through grade specific activities. the children reported an increase in intake of fruits and vegetables, grains, low-fat/fat-free dairy, healthy snacks, eating breakfast, and physical activity, compared to baseline. 52 5-2-1-0 let's go! is another nationally recognized program that aims to create environments supporting healthy choices, healthy habits, and healthy living within a multi-setting model. [53] [54] [55] [56] developed in maine in 2006 by a group of professionals on a mission to tackle childhood obesity by using evidence-based tools and strategies, it has expanded and gained momentum through its strong, far-reaching program and campaign designed to reach out to families where they live, learn, work, and play. its premise is that if children and families are exposed to the same health message in multiple places across their community, and if those places have policies and environments that support healthy choices, then children and families will be more likely to adopt those behaviors and maintain them in their daily lives. the foundation for change as modeled in the 5-2-1-0 healthy habits message is based on the following daily measures: 5 or more fruits and vegetables, 2 h or less of recreational screen time (tv/ computers to be kept out of the bedroom and no screen time under the age of 2), 1 h or more of physical activity, and 0 the foundation for change as modeled in the 5-2-1-0 healthy habits message is based on the following daily measures: 5 or more fruits and vegetables, 2 h or less of recreational screen time (tv/computers to be kept out of the bedroom and no screen time under the age of 2), 1 h or more of physical activity, and 0 sugary drinks and more water intake (fig. 2) . sugary drinks and more water intake (fig. 2) . though this message has been found to increase awareness and healthy behaviors, it remains to be seen if that will translate to concrete behavioral changes. many pediatric and primary care offices across the country have started to implement 5-2-1-0 let's go! into their practices to potentially impact the health of their patients, as have hospital-based specialty programs. the power kids weight management program of cohen children's medical center at northwell health is the authors' multidisciplinary program for overweight and obese children and adolescents, 8 to 18 years of age. in advance of meeting with program staff or at the initial assessment by the program's registered dietitian nutritionist (rdn), prior to any interventions, the patient or the parent/guardian is asked to fill out a healthy habits questionnaire adapted from and directly correlated to the 5-2-1-0 let's go! program (fig. 3) . one version is for children up to 9 years of age, another for 10 to 18-year-olds, and both are available in spanish as well as english. the power kids questionnaire uses a modified food-frequency survey style to ask questions regarding food and beverages and includes other questions that address time allocation for activity and sedentary pursuits as well as family meal patterns and access to tv. what emerges are overall patterns, habits, and choices, ending with a glimpse as to what the child or adolescent is willing to change. answers to the questions help guide the direction of behavioral, nutritional, and exercise interventions. focusing on domains where program participants exhibit deficiencies, while reinforcing already established positive health-related behaviors, helps to pave the path to successful weight management. the goal is to use the 5-2-1-0 message to encourage the children and adolescents in the program to develop healthy habits that can positively impact what would otherwise be their trajectory for further excess weight gain and the associated comorbidities of obesity. let's move is the comprehensive initiative launched in 2010 by former first lady michelle obama the same day that president barak obama signed the memorandum creating the task force on childhood obesity. in partnership with the alliance for a healthier generation, it is dedicated to solving the problem of obesity "within a generation" so that "children born today will grow up healthier and be able to pursue their dreams." 57 the focus is on creating a healthy start for children, empowering parents and caregivers, providing healthy food in schools, improving access to healthy affordable foods, and increasing physical activity. one of its many ambitious goals is the commitment to giving children a voice and a presence. families are encouraged to recognize that children can create healthy lunches from their own kitchens and express their unique preferences as to what "healthy eating" translates into for them. the healthy lunchtime challenge has drawn representatives from every state and territory in the united states, and the accumulation let's move is the comprehensive initiative launched in 2010 by former first lady michelle obama the same day that president barak obama signed the memorandum creating the task force on childhood obesity. of recipes from the annual challenges is accessible online as "historical material." 57 the let's move! outside program, developed to bridge the growing disconnect between young people and the great outdoors, and to emphasize the need for active play, has been adopted by the ymca of the usa, through its youth development division, using programs and services shown to be instrumental in their diabetes prevention program (dpp) trials. 58 eligible children and adolescents, ages 5à17, representing a wide variety of socioeconomic backgrounds, were recruited for a randomized computer-assisted intervention that included their families, to assess whether eliminating financial barriers to ymca membership could encourage increased physical activity in the environment of a supportive family. 59 extensive resources were available to those who utilized the services. all participants and their parents and guardians were scheduled to attend 4 nutrition classes administered by a registered dietitian (rd) and to return for evaluation at 2, 4, 6, 9, and 12 months. children were randomized to nutrition class only (n = 39) or nutrition class and free family ymca membership (n = 44). nutrition classes did not differentiate between those in the control and treatment groups. of the 36 evaluable participants randomized to treatment, only 27 ever visited the ymca, with a median of 5 visits reported. overall attendance at scheduled study-related visits was poor. only 2 participants in each group attended all 6 scheduled visits. for nutrition classes, at least 1 class was attended by 67% of the treatment group, but only 30% of controls. attendance in the nutrition classes led to improvements in nutritional intake for both groups. four participants in the control group and 1 in the treatment group achieved the target reduction of 2 bmi percentile points. there was a positive, but very small, relationship for ymca attendees between the number of visits and the loss of either bmi or weight, which was not statistically significant. curr probl pediatr adolesc health care, & &&&& another major initiative promoting physical activity and healthy eating among children-(in this case, as young as kindergarten and through 12th grade) that has been studied and evaluated is the nfl play 60 fit-nessgram partnership project, led by teachers in school settings across 32 national football league franchise markets. (its two most popular programs are fuel up to play 60, in collaboration with the united states dairy association (usda), and the nfl play 60 challenge created in conjunction with american heart association (aha). the latter has its own app which originally allowed users to choose an avatar with which to complete a course through a virtual outdoor park while listening to health promoting messages like "make sure you drink enough water today"it no longer includes an "in the game" motion sensor but still gauges and delivers health concepts.) the longitudinal impact of nfl play 60 programming was measured using data based on students from 497 schools who completed fitnessgram assessments annually, starting in 2011 through 2015. for schools that participated in the program, annual improvements in aerobic capacity were significantly greater for both girls and boys, compared with non-programming schools. both girls and boys in participating schools showed annual improvement in bmi healthy fitness zone achievement. students in schools that implemented the program for the entire 4 years tended to have better improvements in aerobic capacity than those in schools enrolled for only 2 or 3 years. 60 it is fair to say that each of the national initiatives described in this section had some impact on nutrition and physical activity for many children and adolescents but that the impact was modest for most and minimal for many. going forward, it can prove useful to combine the messages of these multiple programs into one unified message that can be promoted throughout the country in a way that will strengthen their message and thereby yield a stronger effect on the nutritional and physical activity patterns for the youth of the nation. advances in technology have brought about the proliferation of electronic devices now available to children and adolescents who are spending long durations of time in sedentary activities involving handheld devices and video consuls. current guidelines call for limiting sedentary screen time to 2 h or less. 61 among the many concerns being addressed is that increased time on electronics/screen time becomes a potential source of additional energy intake. in a clever harnessing of this dynamic, health professionals are exploring the use of electronics and gaming for getting children to be more physically active. games like wii/wiiu, xbox connect, nintendo, and variations of them have offered small promise. active video games can acutely increase light to moderate physical activity. however, they are unlikely to impact increased habitual activity or significantly decrease sedentary behaviors. 62 rose et al. in their systematic review of digital interventions for improving diet and physical activity behaviors in adolescents, struggled with the heterogenicity of studies not being conducive to a meta-analysis and urged setting up future research initiatives in digital health as a cost-effective medium for health promotion. 63 a great deal of thought and programing is being directed to creating challenges and monitoring progress with physical activity. and sometimes the unexpected turns up with great outcomes. for a time in 2016, the pok emon go app set off a frenzy of interest in walking, sometimes long distances, to find and catch pok emon avatars. 64 an estimated 9à21 million people used the app and increased their daily step count, with some reaching as many as 15,000 steps a day. 65 step challenges have worked well in the adult population with competitions awarding badges, status recognition, and prizes for accumulating steps. in the early 2000s portable watches that were affordable and fashionable were introduced for use in tracking steps. prior to this, they had only been available at research grade. studies exploring step tracking have shown promising results in that a positive feedback loop is established, whereby accumulating steps reinforces continuation of the activity. efforts at encouraging step initiatives in children and adolescents hone in on impacting their motivation, which is often lacking. 66 research on how to encourage more physical activity among studies exploring step tracking have shown promising results in that a positive feedback loop is established, whereby accumulating steps reinforces continuation of the activity. children and adolescents yields findings on how to most effectively use pedometers in combination with other treatment modalities. organizations including the american medical association (ama) and the united states preventive services task force (uspstf), along with healthcare organizations and professionals abroad, have recommended counseling to promote increased physical activity. 67 pedometers, which are inexpensive and wearable devices, can provide children with objective ways to self-monitor their physical activity. several studies of weight management interventions have shown that children can successfully increase their step count from baseline as part of an intervention. 68 yet these studies fail to consistently demonstrate a significant change in bmi percentile from controlled conditions. 68,69 staiano et al. were able to demonstate weight loss in groups of children issued pedometers as part of a 10week, family-based weight management intervention which included physical activity, nutrition, and behavior modification (as well as money compensation). 68 those in the group issued pedometers and a step count goal increased their daily step count, as well as reduced their bmi and bmi z score significantly more than those issued a pedometer without a step goal count. both groups saw a reduction in bmi and an increase in step count from baseline. these same children issued pedometers (with or without a step count goal) had increased subjective health and increased health-related quality of life. ostendorf et al. examined what leads some people to be consistent exercisers and demonstrated that weight loss maintainers weren't using continuous calorie restriction to maintain their weight. 70 instead, the weight loss maintainers had a much higher energy burn from exercise despite eating approximately the same number of calories per day as the control participants with overweight/obesity. it takes a significant time commitment to achieve the level of activity observed in these weight-loss maintainers. in a commentary on the role of exercise, martin and church challenge researchers to identify the physiological, psychological, and environmental factors that help people maintain weight loss through large amounts of exercise so that strategies can be implemented for future weight loss maintenance success. the benefits of exercise cannot be argued. regular exercise can lower stress, moderate anxiety, and improve overall quality of life; however, there is great variation in these outcomes. 71 targeting the agent of change knowing that parents can be effective in modulating childhood obesity by serving as role models for children's eating and physical activity behavior, and knowing the positive impact parental involvement in childhood obesity efforts carries, golan and crowl compared targeting parents exclusively for treatment with a child-only intervention. 72 group sessions were utilized in this family-based health center intervention treatment, with parents attending 14 onehour support and educational sessions that started as weekly, became biweekly, and then took place once every six weeks with clinical dietitians delivering the topics. two similar groups were established, with 15 families participating in each, discussing such topics as limited responsibilities, nutrition education, eating and activity behavior modification, decreasing stimulus exposure, parental modeling, problem-solving, cognitive restructuring, and coping with resistance. parents were encouraged to practice an authoritative parenting style as opposed to an authoritarian style. 71 in authoritative parenting, "parents are both firm and supportive and then assume a leadership role in the environmental change with appropriate granting of child autonomy," whereas in the authoritarian style, child feeding practices are controlled by the adults 73à77 children in the child-only group were prescribed a 1500 calorie per day diet and participated in 30 one-hour group sessions led by a clinical dietitian. two similar groups were held with 15 children allocated to each. the first 7 sessions were conducted weekly and the remainder were held biweekly for the period of one year. at the end of the intervention, 35% of children in the parents-only group reached a non-obese status, compared to 14% in the child-only group. at the one-year follow-up, or one year after program termination, the weight loss in the children of the parent-only group was statistically significant compared with that of the child-only group. at the two-year follow-up, there was a mean reduction in overweight of 15% in children of the parent-only group and an increase of 2.9% in children of the child-only group. at the seven-year follow-up, both treatment conditions demonstrated substantial weight loss. however, the mean reduction of overweight status was 29% in children of the parent-only group and 20.2% in those of the child-only group; 60% of children of the parent-only group, compared with only 31% of children of the child-only group, were in a non-obese status. seven years after program termination, two (6.6%) of the girls from the child-only group reported eating disorder symptoms (both bingeing and purging); none of the children in the parent-only group reported any eating disorder symptoms. family-based programs require the family to be involved. with more families having both parents in the workforce, present-day parents are less available to their children, which makes it difficult for children and adolescents waiting for them to provide a source of physical activity, to engage them in physical activity, or to accompany them to physical activity. parents are less able to enroll in family-based weight management programs if their work schedules conflict with their ability to use free time to participate. 78 interventions targeting overweight and obese children and adolescents that require a large time commitment, a commitment from family members, travel to the intervention location, and potential cost may be poorly received and underutilized. solutions to some of these challenges could be reached with innovative restructuring, telehealth, or a mixed model that may evolve over time. accordingly, researchers collaborated to examine whether utilizing a school nurse delivered intervention for overweight and obese adolescents would be feasible and acceptable, and whether it would serve to improve common obesogenic behaviors (selected for intervention) while positively impacting bmi. 78 clearly there are potentially modifiable behaviors that are associated with improving overweight and obesity. these include decreasing fast food intake, the amount of screen time, on and off dieting, depressive symptoms, low self-esteem, and weight teasing, on the one hand, as well as increasing fruit and vegetable intake through home availability and having more family meals, plus participating in moderate to vigorous physical activity. 79 they are the behaviors most targeted in nutrition interventions using medical nutritional therapy (mnt) by an rd as part of a comprehensive weight management program. increased frequency of rd visits has been associated with improved bmi outcomes in obese youth participating in these programs: "the probability of success exceeded 78% with one rd visit per month versus 43% with minimal rd exposure." 80 both the choose myplate and the 5-2-1-0 education initiatives target these potentially modifiable behaviors. in conjunction with each other, they can have a synergistic effect. healthcare professionals can use these tools together to impact behavior change sessions and establish simple lifestyle goals. many adolescents engage in extreme weight control behaviors and that number has greatly increased over time, as innumerable studies have shown. one population-based survey of adolescents attending middle and high schools in 1998à99 and again in 2008à09 by project eating and activity among teens and young adults assessed personal, psychological, behavioral, and socio-environmental factors believed to play a role in obesity. it showed that informing adolescents and young adults that increased dieting is associated with the persistence of obesity may help motivate adolescents to use more healthful means of weight management. 81, 82 this study reemphasizes the crucial importance of promoting healthy eating, improving the quality of the home food environment, and increasing physical activity as a means of preventing unhealthy weight loss behaviors. the weight management and healthy living 2015 survey from the hartman group 83 found that consumers are more interested in lasting changes and lifelong healthy eating than in crash dieting. it demonstrated that a campaign like myplate, with its message that individuals can achieve a healthier weight by eating more of some foods and less of others, can have utility in helping consumers make lifestyle changes that prove formidable. studies on energy density by b. j. rolls suggest that decreasing energy density reduces energy intake, independent of the macro nutrient mix, because of effects on satiety. 84 the indication is that diets of low energy density, which are typically rich in vegetables, fruits, legumes, and minimally processed grain products, allow individuals to consume "satisfying portions of food," while simultaneously reducing their energy intake. 84 this concept has been used in her best-selling book series volumetrics and made into a diet plan. 85 another approach which has been used in many interventions is the "traffic light" or "stoplight diet," which groups foods based on their nutrient quality and calorie density such that "red foods" should be consumed rarely, "yellow foods" infrequently, and "green foods" most often. 27 it is predicated on the idea that children can learn to substitute lower energy-dense healthy foods for less healthy higher energy-dense foods and that parents can facilitate this transition via increasing access to healthy foods and decreasing access to less healthy foods by altering food purchasing and food storage habits for the family at large. 86 the vast number of children and adolescents in the united states attend public schools. health and wellness policies and programs have traditionally been an important part of the daily curriculum of the majority of these schools. during the 20th century, mandatory physical education classes and nutrition programs, including the national school lunch program (nslp) and the school breakfast program (sbp), were implemented to address problems including "food insecurity." the current obesity epidemic among children and adolescents in the united states has stimulated the further involvement of local, state, and federal agencies in an attempt to use public schools as a venue to combat this problem. in 2004, the u.s. federal government mandated that all school districts participating in the federal meal program create a school wellness program by establishing a committee that includes individuals impacted by this problem. legislation also required the development of nutrition standards for meals and snacks served in schools, as well as the setting up of goals for physical education. the healthy hunger-free act, passed in 2010, required school districts to measure policy implementation and make these results publicly available. what follows here is a look at the impact of some of these and other programs implemented by the schools. approximately 12.2 million public school students from low-income homes are provided a nutritious breakfast as part of the federal school breakfast program (sbp), which was established in 1966 and permanently authorized in 1975. studies have shown that this may be associated with improved academic performance and a reduction in the number of students affected by food insecurity. 87, 88 the number of students participating in the sbp is less than half of those participating in the national school lunch program (nslp). to increase participation in the sbp, the federal government allows school districts to serve breakfast in the classroom (bic). 89 in new york city, more than 70% of public-school students qualify for free or reduced-price meals. researchers reported in 2013 on the impact of bic on the percentage of children going without morning food, the number of locations where food was consumed, and the estimated calories each child consumed. comparisons were made between schools that offered bic and those that did not. results showed that students in bic schools were significantly more likely to eat more than once in the morning and, on average, ate an estimated 95 additional calories each morning. 89 a similar study in the philadelphia public school system, completed and another approach which has been used in many interventions is the "traffic light" or "stoplight diet," which groups foods based on their nutrient quality and calorie density such that "red foods" should be consumed rarely, "yellow foods" infrequently, and "green foods" most often. 27 approximately 12.2 million public school students from lowincome homes are provided a nutritious breakfast as part of the federal school breakfast program (sbp), which was established in 1966 and permanently authorized in 1975. studies have shown that this may be associated with improved academic performance and a reduction in the number of students affected by food insecurity. 87, 88 published in 2018, found that bic did not affect the combined incidence of overweight and obesity among public school students. 90 however, an increasing incidence and prevalence of obesity among the students was noted. in 2003, arkansas became one of the first states to pass legislation to specifically address the epidemic of obesity. it required annual body mass index (bmi) screenings for all public school students, elimination of elementary school students' access to vending machines, and creation of physical education and nutrition standards via district physical activity and nutrition committees along with input from a child health advisory committee. 91, 92 a study published in 2018 assessing the effectiveness of this policy concluded that it was very unlikely that the arkansas act was having an impact on preventing adolescent overweight and obesity. 93 california began bmi screening during the early part of the first decade of the 21st century. the state collected bmi data annually on fifth, seventh, and ninth grade students. parental notification of the results was optional. in 2001, bmi results were sent to 35% of parents or guardians, which rose to 52% in 2008. notification in fifth and/or seventh grade on subsequent bmi z scores, when compared to no notification, showed no significant difference in reducing the prevalence of obesity among this population of students. 94 one state that offered a program that achieved better success is massachusetts. in a pair-matched, cluster-randomized, and controlled school-based trial using a convenience sample of six public high schools, eligible 9th to 11th graders were recruited to participate in "lookin good feelin good," a school nurse-delivered counseling intervention with one-on-one sessions conducted over two months during the school day, during non-academic classes held in the privacy of the school nurse's office. 95 the 5-3-2-1-0 approach was used "to support making five behavioral changes" by utilizing cognitive behavioral techniques to facilitate changes in selfmanagement behaviors through health knowledge and the development of positive outcome expectations, self-control, and behavioral capacity skills and self-efficacy." targeted adolescents completed behavioral and physiological assessments at baseline, and at 2-month and 6-month follow-ups. at two months, compared to control participants, this intervention was able to impact both increased intake of breakfast, and decreased total sugar and added sugar intake. while these particular positive results were not maintained at further follow-up, other positive outcomes were noted at 6 months when the adolescents in the intervention were more likely to drink soda less than or equal to one time a day and eat at fast food restaurants less than or equal to one time per week compared to controls. total calorie intake and calories from fat did not change significantly between groups. screen time and time spent in moderate to vigorous physical activity were not statistically affected. although there was no statistically significant difference in bmi, students in the counseling intervention schools experienced favorable improvements in their bmi compared to students in the control schools. 95 there are clear factors standing in the way of more successful outcomes. an online survey of u.s. public school administrators completed in 2016 indicates that rarely are evidence-based obesity prevention programs being implemented. 92 many programs focus on students' weights rather than on healthy lifestyles. barriers to implementation include lack of funding, time, and training. the johns hopkins evidence-based practice center completed a study of 124 school-based interventions in 2013 and reported on two kinds of programs that demonstrated high evidence of effectiveness in preventing overweight and obesity in the schoolaged population. these are (1) school-based programs that combined physical activity and diet with a home-based component and (2) school-based physical activity and diet interventions that were combined with a home and community component. 96 medication is only recommended after an unsuccessful attempt at weight loss that includes the adoption of a healthy and age-appropriate diet and an increase in daily physical activity. presently, five medications are approved for adults in the united states for long-term management of obesity. 97, 98 weight loss associated with these ranges from approximately 3%à9%. side effects and adverse reactions are common with each. for adolescents greater or equal to twelve years of age, the only prescription medication approved by the united states food and drug administration (usfda) is orlistat. no medication is approved for use in children less than twelve years of age. [99] [100] [101] orlistat is a lipase inhibitor that blocks the absorption of fat. it is recommended to be taken with each meal. although it has been demonstrated to have a good safety profile, side effects can include cramping, excessive gas, oily spotting, fecal urgency, and abdominal pain. since these side effects occur not infrequently, it can be difficult to maintain compliance with this medication. studies have shown modest weight loss efficacy when orlistat is used along with a comprehensive weight loss program. in the largest study (n = 539) of orlistat use in combination with diet, exercise, and behavioral modification, a bmi reduction of approximately 2.4%, as compared to a placebo group, was seen over a treatment period of one year. 102 the only cardiometabolic benefit seen was a small reduction in diastolic blood pressure. at the present time there are no studies reporting long-term outcomes after cessation of orlistat use. phentermine, a norepinephrine reuptake inhibitor, has been approved by the usfda for short-term use for ages seventeen or older. no randomized clinical trials of phentermine have been conducted in individuals younger than seventeen years. common side effects observed in adults using this medication include rapid heart rate, high blood pressure, anxiety, insomnia, and headache. metformin, a biguanide used predominately for glycemic control in individuals with type 2 diabetes mellitus, has been studied for use in treatment of pediatric obesity along with lifestyle interventions. 103 it does not have usfda approval for this use in children and adolescents at the present time. one systematic review of the benefits and risks of using metformin in treating obesity in this population demonstrated a statistically significant, but very modest, reduction in bmi when combined with lifestyle interventions over the short term. no serious adverse events were reported to occur among individuals in the review. the authors concluded that metformin has not been shown to be clinically superior to other options for treating childhood obesity in the short term. bariatric surgery has become an optional treatment for adolescents who are severely obese. in 2004, an expert panel of pediatric surgeons and pediatricians made recommendations regarding selection criteria for bariatric surgery in individuals less than eighteen years of age. 104 selection criteria included: (1) failed >6 months of organized attempts at weight management, (2) has attained or nearly attained physiologic maturity, (3) >50 bmi, or >40 bmi with an associated severe co-morbidity (i.e. sleep apnea, diabetes, hypertension), (4) demonstrates commitment to comprehensive medical and psychological evaluations both before and after surgery, (5) agrees to avoid pregnancy for at least a year, (6) is capable of and willing to adhere to nutritional guidelines postoperatively, (7) provides informed consent, (8) demonstrates decisional capacity, (9) has a supportive family environment, and (10) surgery would be done in centers that have experience with bariatric surgery and a team of specialists trained to provide long-term follow-up care of the metabolic and psychosocial requirements of the patient and family. as an ancillary study of its observational study of adults undergoing bariatric surgery, the national institute of diabetes and digestive and kidney diseases (nddk) created teen-longitudinal assessment of bariatric surgery (teen-labs). funding was provided to five centers in the united states to enroll at least 200 adolescent bariatric surgical patients to serve as a prospective observational cohort study aimed at assessing the clinical, epidemiological, and behavioral parameters in a select population of adolescents undergoing bariatric surgery. 105 the majority of surgical procedures completed in the study were either gastric bypass (roux-en y), which creates a small gastric pouch that is connected directly to the jejunum, bypassing the upper portion of the small intestine, or the sleeve gastrectomy, which creates a narrow stomach pouch and removes the rest of the stomach. for adolescents greater or equal to twelve years of age, the only prescription medication approved by the united states food and drug administration (usfda) is orlistat. no medication is approved for use in children less than twelve years of age. [99] [100] [101] research published in 2018 has shown an increasing use of vertical sleeve gastrectomy compared to other surgical procedures. 106 multiple publications from the teen-labs study have documented that severely obese adolescents undergoing bariatric surgery, when compared to matched adolescents undergoing medical treatment alone, had better weight loss, improvement in cardiovascular risk markers and better glycemic control. the teen-labs researchers also reported identified risks including specific micronutrient deficiencies and the need for an acceptable rate (13%) of additional abdominal procedures. [107] [108] [109] [110] [111] overall similar findings were obtained by olbers in a prospective nationwide study of 81 swedish adolescents who were severely obese and underwent roux-en y gastric bypass. 112 a single study completed by alqalhtani in saudi arabia reviewed the effects of laparoscopic sleeve gastrectomy in 114 children younger than 14 years of age (mean § sd, 11.2 § 2.5 years). it was concluded that the procedure resulted in significant weight loss, improved growth, and a resolution of comorbidities, without mortality or significant morbidity. teen-labs researchers recently compared 5-year outcomes of gastric bypass in adolescents with those of adults. they reported that adolescents and adults who underwent gastric bypass surgery had similar significant weight loss 5 years after surgery, but adolescents had a higher rate of remission of hypertension and diabetes following gastric bypass than adults. they also found that abdominal operations and short-term nutritional deficiencies were more common among adolescents than adults following surgery. 113 data from another teen-lab study demonstrated that joint pain, physical function, and health-related quality of life improved after bariatric surgery. 114 in 2018, the american society for metabolic and bariatric surgery's (asmba) pediatric committee updated their recommendations for metabolic and bariatric surgery in children and adolescents following a comprehensive literature search. they proposed that metabolic and bariatric surgery is indicated for the following adolescents: (1) bmi >35 or 120% of the 95th percentile with clinically significant comorbidities (whichever is lower), and (2) bmi >40 or 140% of the 95th percentile (whichever is lower). in addition, the patient and family should demonstrate the ability and motivation to adhere to recommended pre-and postoperative treatment. the asmba's recommendations regarding contraindications for surgery included: (1) a medical correctable cause of obesity, (2) an ongoing substance abuse problem (within the preceding year), (3) inability to adhere to postoperative dietary and medication regimens as a result of a medical, psychiatric, psychosocial, or cognitive condition, and (4) current or planned pregnancy within 12à18 months of the procedure. at the same time, their guidelines stated that treatment should not be denied to those adolescents with cognitive disabilities, a history of mental illness, a history of eating disorders that are treated, immature bone growth or low tanner stage. their overall conclusion was that surgery was safe and effective in adolescents, and that early intervention can reduce the risk of persistent obesity as well as end organ damage from longstanding comorbidities. 115 multiple publications from the teen-labs study have documented that severely obese adolescents undergoing bariatric surgery, when compared to matched adolescents undergoing medical treatment alone, had better weight loss, improvement in cardiovascular risk markers and better glycemic control. in 2018, the american society for metabolic and bariatric surgery's (asmba) pediatric committee updated their recommendations for metabolic and bariatric surgery in children and adolescents following a comprehensive literature search. they proposed that metabolic and bariatric surgery is indicated for the following adolescents: (1) bmi >35 or 120% of the 95th percentile with clinically significant comorbidities (whichever is lower), and (2) bmi >40 or 140% of the 95th percentile (whichever is lower). the american academy of pediatrics, as well, has issued guidelines in a policy statement entitled "pediatric metabolic and bariatric surgery: evidence, barriers and best practices," published in 2019. 116 they recommended considering the following factors in deciding on surgery: (1) shared decision-making including patient, parents, medical and surgical providers, (2) bmi and comorbidity, (3) physiological, psychological, and developmental maturity, (4) ability to understand risks and benefits and be able to adhere to lifestyle modifications, (5) decision-making capacity, (6) robust family and social supports before and after the procedure. concluding that there was no evidence to support the application of age-based eligibility, the aap set forth the following indications for adolescent metabolic and bariatric surgery: (1) class 2 obesity: bmi i 35 or 120% of the 95th percentile for age and sex, whichever is lower, and with an associated clinically significant disease, including obstructive sleep apnea (ahi >5), t2dm, increased intracranial hypertension, nash, blount disease, scfe, gerd, and hypertension, and (2) class 3 obesity: bmi 40, or 140% of the 95th percentile for age and sex, whichever is lower without any associated comorbid conditions. multicomponent programs that focus on diet, behavior-change, and physical acitivity are recommended as the first line of treatment for children and adolescents who are overweight or obese. treatment should be guided by the patient's developmental, cognitive, and pubertal stage of development. the range of clinicians and environments providing these services is extensive with most services being provided through multidisciplinary tertiary care clinics and providers. these interventions have been proven to be beneficial in achieving small short-term reductions in bmi. presently, there is both a lack of long-term benefit and evidence that these interventions will reduce the incidence of obesity or the associated cardio-metabolic complications for children and adolescents (and adults) in the future. an almost universal consensus recommends a significant increase in research on all interventions including minority and special-needs populations with coordinated long-term follow-up. school-based programs, pharmacotherapy, and bariatric surgery are additional approaches that are increasingly being utilized for weight loss management; of these, 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surgery asmbs pediatric metabolic and bariatric surgery guidelines pediatric metabolic and bariatric surgery: evidence, barriers, and best practices key: cord-017131-rx1z4orm authors: patra, amlan kumar title: an overview of antimicrobial properties of different classes of phytochemicals date: 2012-02-18 journal: dietary phytochemicals and microbes doi: 10.1007/978-94-007-3926-0_1 sha: doc_id: 17131 cord_uid: rx1z4orm plants produce a great diversity of phytochemicals, the beneficial properties of which have been used by humans for centuries since the advent of human civilization. with the discovery of effective and potent antimicrobial compounds, these synthetic antimicrobial compounds are widely used to prevent and cure microbial diseases. however, the development of antibiotic resistant strains of bacteria, reduced efficacy and safety of antimicrobials and the search of new antimicrobials against emerging incurable diseases by conventional antimicrobial agents have revived to explore phytochemicals as an alternative to synthetic antimicrobial compounds. although numerous studies have been conducted in vitro and in vivo in the recent years on the efficacy of plant phytochemicals as antimicrobial agents, this chapter provides an overview of the antimicrobial properties of some major group of phytochemicals, namely, different phenolic compounds, alkaloids, saponins, iridoids and secoiridoids, polyacetylenes, glucosinolates, terpenoids, sulfinate, limonoids (tetranortepenoids) and anthranoids against pathogenic bacteria, fungi, viruses and commensal bacteria in the intestinal tracts of humans and animals. this chapter also discusses their antimicrobial mechanisms of action, the efficiency of different groups of phytochemicals against multiple-drug resistant bacteria, the effect of active dietary phytometabolites on the beneficial and pathogenic microbes of the gastrointestinal tracts and the outcomes of combination of phytofactors and drugs interactions. plants contain a wide array of phytochemicals , which have traditionally been utilized for centuries in folk medicines or ethnomedicines. the earliest information on the medicinal use of plants comes from china in 5000 bc (greathead 2003 ) , from india (in rigveda and atharvaveda) in 2000 bc (ramawat et al. 2008 ) , from mesopotamia in 2600 bc (newman et al. 2000 ) , and also from egypt in about 1550 bc (davidson and naidu 2000 ) . the natural medicines were widely used until the fi rst half of the twentieth century, when a shift towards synthetic medicines that were more effective, patentable and highly profi table, occurred (tyler 1999 ) . however, there have been increasing interests towards use of natural chemicals in medicinal purposes in recent years. these ethnomedicines are encouraging for both the public and national health care institutions as alternatives to synthetic drugs due to relatively lower incidences of adverse reactions compared to modern conventional pharmaceuticals along with their reduced cost (nair et al. 2005 ) . recently, the growing occurrences of multi-drug resistant strains of bacteria and the appearance of strains with decreased susceptibility to antibiotics have led to a resurgence of research interests in the discovery of novel antimicrobial agents from natural sources for therapeutic and preventive purposes against microbial diseases, food preservatives and feed additives in the animal industry. the ethnopharmacologists, botanists, microbiologists and natural-product chemists are constantly in search of medicinal effi cacy of plants and their phytochemicals , since the reported data so far available on plants are comparatively meager compared to the vast number of plant population. plants produce a great diversity of compounds. the structures of close to 50,000 compounds have already been elucidated and there are perhaps hundreds of thousands of such compounds in plants (pichersky and gang 2000 ) . only a few of these are part of 'primary' metabolic pathways (those common to all organisms). the rest are secondary metabolites or phytochemicals whose biosynthesis is restricted to selected plant groups (pichersky and gang 2000 ) . phytochemicals can be divided into many major classes depending upon the chemical structures, botanical origins, biosynthesis pathways or biological properties. the most phytochemical classifi cation scheme is based on chemical structures such as phenolics, alkaloids, saponins, terpenoids, limonoids, polyacetylenes and secoiridoids and so on. numerous studies have been conducted in vitro and in vivo in the recent years on the effi cacy of plant phytochemicals as antimicrobial agents. this paper presents the antimicrobial properties of some major group of phytochemicals against pathogenic bacteria, fungi and virus, and benefi cial microbes of the gastrointestinal tracts and their mechanism of action. phenolic compounds are a group of phytochemicals , which have a phenol structure, i.e. an aromatic benzene ring bearing at least one hydroxyl substituent (robbins 2003 ; vermerris and nicholson 2006 ) . phenolic compounds are commonly found 1 an overview of antimicrobial properties of different classes of phytochemicals throughout the plant kingdom, where they protect the plants from microbial infections, ultraviolet radiation and chemical stressors. this large and diverse group of phytochemicals is classifi ed into many subclasses depending upon chemical structures and occurrence in plants. the commonly categorized subclasses of phenolic compounds are simple phenolics (resorcinol and phloroglucinol ), phenolic acids and aldehydes , coumarins , fl avonoids , chalcones , aurones , benzophenones , xanthones , stilbenes , benzoquinones , naphthaquinones , anthraquinones , betacyanins , lignans , and polyphenols (proanthocyanidin , galloyl, hexahydroxydiphenyl ester , hydroxy cinnamic acid , and phloroglucinol derivatives) (vermerris and nicholson 2006 ; handique and baruah 2002 ) . the detailed structures and chemistry of these phenolic compounds are presented elsewhere (vermerris and nicholson 2006 ) . foods containing phenolics are becoming an important part of diets due to their potential anti-oxidative properties. besides, these compounds have also potent anti-microbial properties. the phenolic acid and aldehyde group of phenolic compounds is characterized by the presence of a carboxylic acid or aldehyde group substituted on a phenol (table 1. 1 ; vermerris and nicholson 2006 ) . the naturally occurring phenolic acids generally have two characteristic constitutive carbon frameworks: the hydroxycinnamic and hydroxybenzoic structures (robbins 2003 ) . majority of cinnamic and benzoic acid derivatives in plants are linked through ester, ether or acetal bonds to structural components, polyphenols , organic acids (quinic , maleic , tartaric and shikimic acid ), glucose and terpenes (robbins 2003 ) . chlorogenic acid is an ester of quinic acid and caffeic acid . some aldehyde analogues of phenols (e.g. vanillin ) are also grouped with phenolic acids (robbins 2003 ) . the numbers and positions of the hydroxyl and other groups on the aromatic ring can produce a large number of compounds in this subclass (robbins 2003 ; vermerris and nicholson 2006 ) . phenolic acids are present in a wide range of plants including in many common foods such as tea, coffee and berries. besides, phenolic acids and aldehydes could be formed by the intestinal microbial biotransformation of other phenolic compounds in the intestine, where they may infl uence intestinal microbiota. a number of simple phenols and phenolic acids possess antibacterial, antiviral and antifungal activities against a wide range of microbes, but at different concentrations. gallic acid and p -hydroxybenzoic acid reduced the viability of camplylobacter jejuni at concentrations as low as 1 mg/l (ganan et al. 2009 ) . synaptic acid , vanillic acid , and caffeic acid were microbicidal at concentrations starting at 10 mg/l. ferulic acid and cumaric acid were effective at a concentration of 100 mg/l (ganan et al. 2009 ) . ozçelik et al. ( 2011 ) recently tested some phenolic acids such as gallic acid, caffeic acid, chlorogenic acid , and quinic acid for their in vitro antiviral, antibacterial, and antifungal activities. all these phenolic acids were inhibitory to herpes simplex virus type 1 (hsv -1), whereas gallic acid, chlorogenic acid and quinic acid showed potent antiviral effect against parainfl uenza virus type 3 at the therapeutic range of 0.8-0.05 mg/l. in general, antibacterial activity of phenolic acids is stronger against gram-positive bacteria than gram-negative bacteria (merkl et al. 2010 ; cueva et al. 2010 ) . the outer membrane of gram-negative bacteria provides them with a hydrophobic surface structure that is able to exclude certain hydrophilic molecules, making them inherently resistant to many antimicrobial agents including phenolic acids (alakomi et al. 2007 ; cueva et al. 2010 ) . gram-positive bacteria are enclosed in a plasma membrane covered by a thick peptidoglycan wall and lack an outer membrane (alakomi et al. 2007 ; cueva et al. 2010 ) . although, phenolic acids are effective against gram-negative bacteria, their antimicrobial effect is strain dependent (e.g. different strains of escherichia coli ; cueva et al. 2010 ) . phenolic compounds are usually poorly absorbed in the small intestine, and thus most of the dietary phenolics accumulate in the colon (clifford 2004 ; van duynhoven et al. 2011 ) . therefore, higher concentrations of phenolic acids may reach in the intestine than the concentrations in diets. phenolics may selectively suppress or stimulate (tzounis et al. 2008 ) . chlorogenic, quinic and gallic acids stimulated growth of lactobacillus collinoides relative to control cultures (no additive) up to concentrations of 1 g/l of tomato broth media. in contrast, growth of lactobacillus brevis was little affected during early incubation, which has been suggested to be due to metabolism of these acids (stead 1994 ) . from structure-activity relationship , phenols having different alkyl chain length with hydroxyl groups could be important for antimicrobial actions (kubo et al. 1995 ) . p-hydroxybenzoic acid, protocatechuric , gentisic acid , vanillic acid , ferulic acid , caffeic acid and their methyl, ethyl, propyl and butyl esters were investigated for antibacterial action. it has been reported that the antimicrobial effect of phenolic acids derivatives increased with the increasing length of the alkyl chain (merkl et al. 2010 ) . the presence of hydroxyl groups on the phenol groups and oxidized status of phenol groups also determine the toxicity of microbes. the fl uidity of the cell membrane could be disturbed with increasing hydrophobic alkyl chains. the phenolic acids could enter the molecular structure of the membrane with the polar hydroxyl group oriented into the aqueous phase by hydrogen bonding and nonpolar carbon chain aligned into the lipid phase by dispersion forces (kubo et al. 1995 ) . thus, when the hydrophilic force exceeds hydrophobic one, the activity tends to disappear. also, the number and position of substitutions in the benzene ring of the phenolic acids and the saturated side-chain length infl uenced the bacteriocidal effects of phenolic acids against the different microorganisms, but in different ways against gram-positive and gram-negative bacteria (cueva et al. 2010 ) . for example, cueva et al. ( 2010 ) showed that for benzoic and phenylacetic acids, e. coli was inhibited in the following order of potency: non-substituted > 4-hydroxy-3-methoxy-> 3-hydroxy-> 4-hydroxy-> 3,4-dihydroxy-substituted acid. for phenylpropionic acids, the order differed slightly: nonsubstituted > 4-hydroxy-> 3-hydroxy->3,4-dihydroxysubstituted acid. however, the potency of phenolic acids was in different order for lactobacillus spp. for benzoic acids, the order of potency was: 4-hydroxy-> 3-hydroxy-> non-substituted > 4-hydroxy-3-methoxy-> 3,4-dihydroxy-substituted acids, except for lactobacillus coryniformis cect 5711 (4-hydroxy-> non-substituted > 3-hydroxy > 4-hydroxy-3methoxy-substituted acids). for phenylacetic acids, growth inhibition of lactobacilli was on the order of non-substituted > 3-hydroxy-> 4-hydroxy-> 3,4-dihydroxysubstituted acids. for phenylpropionic acids, growth inhibition was as follows: nonsubstituted > 4-hydroxy-> 3-hydroxy > 3,4-dihydroxy-substituted acids, except for lactobacillus fermentum cect 5716 (3-hydroxy > non-substituted > 4-hydroxyand 3,4-dihydroxy-substituted acids) and lactobacillus plantarum lch17 (nonsubstituted > 3-hydroxy-> 4-hydroxy-> 3,4-dihydroxy-substituted acids). coumarins are naturally found in many families of plants (apiaceae, asteraceae, fabiaceae, rosaceae, rubiaceae, rutaceae and solanaceae) and microorganisms, and approximately 1,000 coumarins have been isolated from these sources (weinmann 1997 ; smyth et al. 2009 ) . coumarins can be classifi ed into fi ve groups depending upon the structure, i.e. coumarins with substituents in benzene ring, coumarins with substituents in pyrone ring, furocoumarins, pyranocoumarins , and coumarin dimmers ( fig. 1.1 ; smyth et al. 2009 ) . coumarins exhibit a broad diversity for antimicrobial activity. o-acetylcolumbianetin, edultin, cniforin a, columbianadin and imperatorin isolated from the fruits of cnidium monnieri (l.) cuss exerted a little to no appreciable growth-inhibition of gram-positive and gram-negative bacteria (ng et al. 1996 ) . an amino-coumarin -7-amino-4-methylcoumarin showed broad-spectrum antibacterial and antifungal activities (liu et al. 2008 ) . melliou et al. ( 2005 ) studied the antibacterial activity of pyranocoumarins using an agar disc diffusion method. seselin, xanthyletin, 5-hydroxyseselin, and 7-hydroxyalloxanthyletin had no antibacterial effects. coumarin derivatives such as 5-methoxyseselin and its brominated derivatives, alloxanthoxyletine, the acetylated derivatives, and dipetalolactone were active against all the tested bacteria. a seselin derivative, 3-bromo-4-benzoyloxyseselin showed moderate activity, while three coumarins containing acetoxy groups in pyrano ring were only active against the two gram-positive bacteria. a new coumarin -cajanuslactone isolated from pigeon pea leaves showed anti-bacterial activity against staphylococcus aureus (atcc 6538), and the minimum inhibitory concentration (mic) and the minimum bactericidal concentration (mbc) were 0.031 and 0.125 mg/ml, respectively . some seselin derivatives, including derivatives of 5-methoxyseselin, were found to be potent against human immunodefi ciency virus (hiv ) (xie et al. 1999 ) . it has been suggested that the presence of oxygenated substituents in the ether or ester form usually enhances the antibacterial activity, while the presence of free hydroxyl group reduces the activity (melliou et al. 2005 ) . this fact could be at least partially attributed to the reduced lipophilicity of the hydroxyl derivatives, which hinders the penetration through the bacterial cell wall (melliou et al. 2005 flavonoids are one of the largest groups of secondary metabolites that are distributed in various plant species. they have signifi cant antioxidant properties, which are benefi cial for health. these polyphenolic compounds are constructed basically with an a and c ring of benzo-1-pyran-4-quinone and a b ring. the main classes of fl avonoids ( fig. 1.2 ) (having a hydroxyl group at the 3-position), e.g. kaempferol , quercetin , galangin , datiscetin , morin , robinetin , isorhamnetin , tamarixetin , quercetagetin and myricetin ; (3) fl avanones (2-3 bond saturated), e.g. hesperetin , taxifolin, eriodictyol and naringenin ; (4) flavan-3-ol, e.g. catechin and epicatechin; (5) isoflavone , e.g. genistein , daidzein and coumestrol ; (6) anthocyanidins : cyanidin , delphinidin , pelargonidin and peonidin (crozier et al. 2006 ) . the majority of fl avonoids commonly remain conjugated with sugars as glycosides . numerous fl avonoid derivatives showed antiviral activity against a wide range of viruses such as hsv , hiv , coxsackie b virus, coronavirus, cytomegalovirus, poliomyelitis virus, rhinovirus, rotavirus, poliovirus, sindbis virus, and rabies virus (de bruyne et al. 1999 ; evers et al. 2005 ; nowakowska 2007 ) . ozçelik et al. ( 2011 ) investigated the effects of quercitin, apigenin, genistein , naringin, silymarin and silibinin against hsv -1 and pi-3 virus. all fl avonoids inhibited hsv -1 activity, but only genistein inhibited parainfl uenza type-1 (pi-1) activity. of the three fl avonoids (baicalin, rutin and naringin) examined by ng et al. 1996 , baicalin was found to be the most potent in inhibiting the growth of s. aureus : 11 of the 16 strains tested were inhibited at 128 mg/l. however, no inhibitory activity of rutin and naringin against s. aureus was observed at 128 mg/l. at this concentration, naringin and baicalin inhibited two strains and rutin inhibited one strain of the eight p. aeruginosa strains tested. the fl avonoids compounds display different mode of antiviral action. for instance, baicalein probably block human cytomegalovirus infection at entry level while the primary mechanism of action for genistein may be to block immediateearly protein functioning off human cytomegalovirus (evers et al. 2005 ) . both these fl avoinoids did not inhibit the virus replication (evers et al. 2005 ) . puupponen-pimia et al. ( 2001 ) investigated 13 falovonoid compounds (apigenin, (+)-catechin , chlorogenic acid , cyanidin chloride, delphinidin chloride, isoquercitrin, kaempferol , cyanidin-3-glucoside (kuromanin), luteolin, myricetin , pelargonidin chloride, quercetin dehydrate and rutin trihydrate), and 4 phenolic acids (caffeic acid , 3-coumaric acid, ferulic acid , trans-cinnamic acid) on 7 gram-positive lactic acid bacteria of intestines, gram-negative e. coli cm 871 and salmonella . myrecetin strongly inhibited the growth of lactobacillus as well as e. coli , but did not affect salmonella . luteolin was weakly inhibitory to gram-positive lactic acid bacteria but not to gram-negative bacteria. the anthocyanidins pelargonidin, delphinidin and cyanidin, as well as cyanidin-3-glucoside, only inhibited growth of e. coli and had no effect on other bacterial strains (puupponen-pimia et al. 2001 ) . however, phenolic acids did not inhibit lactic acid bacteria, but inhibited gram-negative e. coli and salmonella sp. hatano et al. ( 2005 ) discussed that some prenylated fl avonoids such as licoricidin (an isofl avan) effectively suppressed the antibiotic resistance of methicillin-resistant s. aureus (mrsa ) compared to other fl avonoids. the addition of 4 m g/ml of licoricidin shifted the mic of oxacillin from 128-256 to 8-16 m g/ml, and 8 m g/ml of licoricidin reduced it to less than 0.5 m g/ml. the requirement for dimethylallyl or equivalent substituents suggests the importance of affi nity for the bacterial cell membrane. 1 an overview of antimicrobial properties of different classes of phytochemicals phenolic acids show greater antimicrobial potency than their corresponding fl avonoids precursors such as the monomers (+)-catechin and (−)-epicatechin (ganan et al. 2009 ; cueva et al. 2010 ) . therefore, microbial transformations of dietary fl avonoid compounds in the intestine could lead to more potent microbialinhibitory compounds (phenolic acids ) and could reach greater concentrations in the intestine. this may selectively infl uence intestinal bacteria species, and therefore could affect the diversity and metabolic activity of the intestinal microbiota, including the transformation of phenolics in the gut (cueva et al. 2010 ) . epigallocatechin gallate exerted strong antibacterial growth against gram-positive bacteria than against gram-negative bacteria (yoda et al. 2004 ; engels et al. 2009 ) . it has been stated that gram-positive bacteria absorb more epigallocatechin gallate into their peptidoglycan cell wall and aggregate its presence, while gram-negative bacteria do not aggregate and absorb less epigallocatechin gallate (ikigai et al. 1993 ; engels et al. 2009 ) because of the repulsive negative charge of lipopolysaccharides on the surfaces of gram-negative bacteria. the binding of epigallocatechin gallate to peptidoglycan disrupts its function in osmotic protection, cell division, and cell wall biosynthesis (yoda et al. 2004 ) . detailed information of antimicrobial activities of fl avonoids has been discussed elsewhere in this book (chap. 2 ). some phenolic acids (ellagic and gallic acids) or fl avonoids (fl avan-3-ol, fl avan-3-4-diol or fl avan-4-ol) in plants are esterifi ed or polymerized into dimeric, oligomeric or polymeric compounds. most abundantly present polyphenolic compounds in plants are tannins , which are usually of two types: hydrolysable tannins (ht) and condensed tannins (ct ). the ht are complex molecules with a polyol as a central core such as glucose, glucitol, quinic acids, quercitol and shikimic acid that is partially or totally esterifi ed with a phenolic group, i.e. gallic acid (3,4,5-trihydroxy benzoic acid ; gallotannins) or gallic acid dimmer hexahydroxydiphenic acid (ellagitannins) (haslam 1989 ) . the ct (proanthocyanidins) are mainly polymers of the fl avan-3-ols (epi)catechin and (epi)gallocatechin units, which are linked by c4-c8 and c4-c6 interfl avonoid linkages (ferreira et al. 1999 ; hagerman and butler 1989 ) . the polyphenols also exert a wide range of antibacterial and antifungal activities. ellagitannin extracts inhibited a range of pathogenic organisms including vibrio cholerae , shigella dysenteriae and campylobacter spp . (silva et al. 1997 ; puupponen-pimia et al. 2002 ) . puupponen-pimia et al. ( 2005 ) reported that berry extracts exhibit selective inhibitory properties against intestinal bacteria such as staphylococcus , salmonella , listeria and lactobacillus strains, and the selective inhibitory actions varied with berry extracts. in general, pathogenic staphylococcus and salmonella were sensitive to various berry extracts and ellagitannins fractions, while pathogenic listeria and benefi cial lactobacillus were not inhibited. rauha et al. ( 2000 ) studied antimicrobial effects of some berry extracts against food spoilage and poisoning bacteria. the widest antibacterial activity was present in berries belonging to the genus rubus (cloudberry and raspberry) that are rich in ellagitannins. ellagic acid has been reported to exhibit a dose-dependent inhibitory effect (ic50 = 1 mm) on helicobacter pylori isolated from peptic ulcer patients (chung 1998 ) . tannins isolated from dichrostachys cinerea roots exerted antimicrobial effects against s. aureus , e. coli , shiegella spp. and p. aeruginosa with mic of the tannins ranging between 4.0 and 5.5 mg/ml, while the mbc ranging between 4.5 and 6.0 mg/ml (banso and adeyemo 2007 ) . gallotannins extracted from the mango seed kernel inhibited the growth of gram-positive food spoilage bacteria and decreased the growth of gram-negative e. coli , but did not affect lactic acid bacteria (engels et al. 2009 ) . the antibacterial properties of cranberry juice with inhibition of e. coli adherence to mucosal surfaces by cranberry juice is reported to be associated with the presence of proanthocyanidins (howell et al. 1998 ) . many polyphenols have antiviral activities against different types of viruses (de bruyne et al. 1999 ; cheng et al. 2002 ) . it has been suggested that prodelphinidin b-2 3 ¢ -o -gallate (a proanthocyanidin gallate isolated from green tea leaf) showed anti-hsv -2 properties with the mechanism of inhibiting the attachment and penetration between cells and viruses possibly through the instability of viral glycoproteins (cheng et al. 2002 ) . the structure and functional groups of the polyphenol compounds may determine the effectiveness of the antiviral activities (de bruyne et al. 1999 ) . the content of small-molecular phenolic compounds have greater infl uence on the antibacterial activity of extracts than tannins (nazaruk et al. 2008 ) . thus, polyphenols could be cleaved by bacterial enzymes to form a number of phenolic acids in the intestine, where they may infl uence the microbial populations (bock and ternes 2010 ) . engels et al. ( 2009 ) recently studied the effects of gallotannins with different galloyl units from mango seed kernel on various gram-positive and gram-negative bacteria. gallotannins showed antibacterial activities with mics ranging from 0.1 g/l for s. aureus to 3.3 g/l for pediococcus acidilactici . they also observed that degree of galloylation did not affect the growth of bacteria. it has been suggested that the antibacterial activities of gallotannins are due to their strong affi nity for iron and the inactivation of membrane-bound proteins (engels et al. 2009 ) . it has also been shown that gallotannins changed the morphology of bacillus subtilis , which has been hypothesized due to inhibition of cell division by binding of gallotannins to the cell wall or inhibition of enzymes involved in cell separation (engels et al. 2009 ) . naphthoquinones are widely distributed in plants, fungi, and some animals. lapachol, plumbagone, juglone and lawsone are naturally occurring naphthoquinones 1 an overview of antimicrobial properties of different classes of phytochemicals of plant origin that have antimicrobial effects against various pathogenic bacteria and fungi. adeniyi et al. ( 2000 ) reported that two dimeric naphthoquinones, diospyrin and isodiospyrin , isolated from the root of diospyros piscatoria (gurke), a common ingredient in several folk medicines, exhibited a broad spectrum of antibacterial activity against s. pyogenes and s. pneumoniae (mics of diospyrin ranged from 1.56 to 50 m g/ml) salmonella choleraesuis serotype typhi ( s. typhi ) and mycobacterium chelonae (mics of diospyrin were between 25 and 100 m g/ml). isodiospyrin was more active than its racemic isomer diospyrin (mics against gram-positive bacteria ranged from 0.78 to 50 m g/ml, while those against pseudomonas aeruginosa and s. typhi ranged from 50 to 100 m g/ml). another naphthoquinones, lapachol and b -lapachone, found in species of tabebuia, had relevant effects against candida albicans, candida tropicalis, and cryptococcus neoformans, and were more active than the reference standard, ketoconazole. lapachone showed strong antimicrobial activity than lapachol against the fungi (guiraud et al. 1994 ) . methanol extract from the dried inner bark of tabebuia impetiginosa exhibited potent antibacterial activity against h. pylori which contained lapachol and anthraquinones (park et al. 2006 ) . alkaloids have been defi ned as n-heterocyclic basic metabolites, although the defi nition does not clearly separate from other n-containing compounds. alkaloids have been classifi ed in many ways depending upon biogenic precursors or carbon skeleton characteristics. they have a great structural diversity compared with other classes of phytochemicals . alkaloids are generally known according to their carbon skeleton structures. pyridine (e.g. piperine), piperidine , quinoline , indole , pyrrolidine , quinazoline , isoquinoline , glyoxaline , lupinane , tropan , phenanthridine , imidazoline , alkaloidal amines and terpenoid types of alkaloids are commonly found in plants (hegnauer 1988 ) . alkaloid fractions isolated from strychnos potatorum l.f. (loganiaceae) seeds, which were of indole type, were tested for their antimicrobial properties against some pathogenic gram-positive, gram-negative and acid-fast bacteria and fungi. these fractions had shown considerable antimicrobial activity against both bacteria and fungi at the tested concentrations (100 and 200 m g/ml). further, the growth of proteus vulgaris , s. aureus , salmonella typhimurium, vibrio cholerae , mycobacterium tuberculosis, aspergillus niger and c. albicans were signifi cantly inhibited (mallikharjuna and seetharam 2009 ) . similarly, two benzophenanthridine alkaloids , dihydrochelerythrine and dihydrosanguinarinealkaloid constituents of bocconia arborea showed considerable antimicrobial activity against gram-positive and gram-negative bacteria and c. albicans ( navarro and delgado 1999 ) . sensitivity of dna and rna viruses to alkaloids may differ. ozçelik et al. ( 2011 ) investigated various alkaloids namely yohimbine and vincamine (indole -type), scopolamine and atropine (tropane-type), colchicine (tropolone-type), allantoin (imidazolidine-type), trigonelline (pyridine-type) as well as octopamine, synephrine, and capsaicin (exocyclic amine-type) for their antiviral activities against dna virus herpes simplex (hsv -1) type 1 and rna virus parainfl uenza type-3 (pi-3). all the alkaloids were effective against hsv -1 at 0.05-1.6 mg/l, but atropin and octopamine showed potent antiviral activities against pi-3 at 0.05-0.8 mg/l (ozçelik et al. 2011 ) . antibacterial alkaloids from chelidonium majus linn, i.e. benzo [c] phenanthridine -type alkaloids, 8-hydroxydihydrosanguinarine, 8-hydroxydihydrochelerythrine were potently active against mrsa strains with mics/mbcs ranged from 0.49 to 15.63 and 1.95 to 62.50 m g/ml, respectively (zuo et al. 2008 ) . there are two rich sources of organosulphur compounds from plants; (1) alliaceae family containing alliin -alliinase system and (2) cruciferae ( brassicacae ) family e.g. brassica juncea , wasabia japonica (wasabi), armoracia rusticana (horseradish) and brassica oleracea (caulifl ower) containing glucosinolate-myrosinase (mithen 2006 ) . a number of sulphur-containing compounds can be derived from these plants through the action of myrosinase and alliinase enzymes. the primary sulphur-containing constituents in alliums spp. (e.g. a. sativum (garlic), a. cepa (onion), a. porrum (leek)) and brassica spp. (e.g. cabbage, kale, caulifl ower and turnip) are s -alk(en)yl-l-cysteine sulphoxides and g -glutamyl-s -alk(en)yl-lcysteine sulphoxides (block et al. 1992 ; ross and milner 2007 ; fig. 1.3 ) . the content of s-alk(en)yl-l-cysteine sulphoxides in garlic may range from 0.53% to 1.3% of fresh weight with s -allyl-l-cysteine sulphoxide (alliin ) being the largest contributor. by the action of alliinase enzyme present inside the cells, these compounds are converted into thiosulfi nate (a functional group consisting of the linkage r-s(=o)-s-r'), which are then spontaneously and enzymatically converted into a large array of volatile compounds, e.g. diallyl disulphide, diallyl trisulphide, allyl methyl disulphide and dipropyl and disulphide (mithen 2006 ) . antimicrobial activities of garlic and onion against a wide range of grampositive and gram-negative bacteria, virus and fungi are known for many years (ankri and mirelman 1999 ) . the antifungal activities of garlic oils appear to be more than the antibacterial activity (avato et al. 2000 ) . extracts of garlic exhibit the most potent antibacterial activity, followed by onion, and brassica including cabbage (kyung and lee 2001 ) . the principal antimicrobial compounds of allium and brassica are allicin ( s -allyl-l-propene thiosulfi nate ) and methyl methanethiosulfinate, respectively (kyung and lee 2001 ) . these compounds are derived from s -allyl and s -methyl derivatives of l-cysteine sulfoxide , respectively. avato et al. ( 2000 ) tested different mixtures of garlic distilled oils containing diallyl disulfi de (dds) and diallyl trisulfi de (dts), ranging from 1% to 51% and 88% to 38%, respectively, against yeasts ( c. albicans, c. tropicalis and b. capitatus ), gram-positive bacteria ( s. aureus and b. subtilis ) and gram-negative bacteria ( p. aeruginosa and e. coli ). incubation of garlic extracts made up of 1% dds and 88% dts did not show growth inhibition against all the tested microorganisms, whereas garlic oils with higher quantities of dds showed signifi cant inhibitory activity, increasing with the increase of dds amount, thus implicating the dds as the active antimicrobial agent (avato et al. 2000 ) . it has been reported that allicin (mic, 6 m g/ml; mbc, 6 m g/ml) was more potent than dds (mic range, 100-200 m g/ml; mbc range, 100-200 m g/ml), its corresponding sulfi de, but of a strength similar to that of diallyl tetrasulfide (mic range, 3-6 m g/ml; mbc range, 3-6 m g/ml) against h. pylori (o'gara et al. 2000 ) . kyung and fleming ( 1997 ) investigated the different s-compounds found in cabbages on the growth of 15 bacteria and 4 fungi. s -methyl-l-cysteine sulfoxide, sinigrin , and dimethyl sulfi de at 500 ppm did not inhibit the growth of any of the bacteria and yeasts. dimethyl disulfi de at 500 ppm retarded the growth of some bacteria, but was not bactericidal to any of the test microorganisms. dimethyl trisulfi de , methyl methanethiosulfi nate and methyl methanethiosulfonate had mics of 200 ppm, between 50 and 200 ppm, and between 20 and 100 ppm, respectively for bacteria, and 20 ppm, between 6 and 10 ppm and between 50 and 500 ppm for yeasts, respectively (kyung and fleming 1997 ) . there are numerous reports showing the effectiveness of garlic or allicin as antimicrobial agents in comparison to antibiotics (fujisawa et al. 2009 ; cai et al. 2007 ) . also, allicin with antibiotics may synergistically augment the antimicrobial actions (cai et al. 2007 ; an et al. 2009 ) . besides, thiosulfi nates and their derivatives show promising activity against multidrug resistant bacteria including mrsa (ankri and mirelman 1999 ; fujisawa et al. 2009 ) . the main mode of action of thiosulfi nate derivatives have been proposed to be due to its chemical reaction with the thiol groups of various enzymes (ankri and mirelman 1999 ) and thus antimicrobial properties of allicin may be abolished by cysteine, coenzyme a and glutathione (fujisawa et al. 2009 ) . antimicrobial activity of the diallyl sulfi des has been reported to increase with the number of sulfur atoms (o'gara et al. 2000 ) . glucosinolates are the sulphur-containing metabolites found in large number of edible plants. over 120 glucosinolates are present in 16 families of dicotyledonous angiosperms, most of which are clustered within the brassicaceae and capparaceae (fahey et al. 2001 ) . allyl (sinigrin ) and 3-butenyl (gluconapin) glucosinolate are found in brown mustard, p -hydroxybenzyl glucosinolate in white mustard, allyl and other glucosinolate in horseradish and wasabi, methylthiopropyl in cabbage and 2-hydroxy 3-butenyl glucosinolate in rapeseed ( fig. 1.4 ; fahey et al. 2001 ; mithen 2006 ) . the antibacterial and antifungal properties of glucosinolates are known for a long time (fahey et al. 2001 ) . intact glucosinolates do not show antimicrobial action, but the hydrolysis products of glucosinolates are active against various microorganisms (manici et al. 1997 ; tierens et al. 2001 ) . aires et al. ( 2009a ) observed that the in vitro growth inhibition and the sensitivities of the individual bacteria are infl uenced by the structure of glucosinolates and their hydrolysis products. the most effective glucosinolate hydrolysis products were the isothiocyanates ; sulforaphane and benzyl isothiocyanate were the strongest inhibitory against the growth of human pathogenic bacteria. regarding action of glucosinolates products on the type of bacteria, 4-methyl sulfi nyl butylisothiocyanate exhibited antibacterial activity against a larger range of bacteria. indole-3-carbinol had some inhibitory effects against the gram-positive bacteria, but had no effect against the gram-negative bacteria. indole-3-acetonitrile had some inhibitory activity against the gram-negative bacteria. glucosinolates, nitriles and amines were ineffective at the doses up to 3 m mol (aires et al. 2009b ) . saavedra et al. ( 2010 ) evaluated the in vitro antibacterial actions of different classes of common dietary phytochemicals , i.e. simple phenolics -tyrosol, gallic acid, caffeic acid , ferulic acid , and chlorogenic acid ; chalcone -phloridzin; fl avan-3-ol -(−) epicatechin; secoiridoid -oleuropein glucoside; glucosinolate hydrolysis products -allyl isothiocyanate, benzyl isothiocyanate and 2-phenylethyl isothiocyanate) against four pathogenic microbes. all of the isothiocyanates had signifi cant antimicrobial activities, while the phenolics were much less effi cient. no antimicrobial activity was observed with phloridzin. allyl isothiocyanate from cabbage had an mic between 50 and 500 ppm for bacteria and between 1 and 4 ppm for yeasts (kyung and fleming 1997 ) . iridoids is a group of cyclic monoterpenoids having iridane skeleton (cis-2-oxabicycle-(4.3.0)-nonane), which mostly remain as glycosides ( fig. 1.5 ; perez et al. 2005 ) . secoiridoids derive from iridoids by the elimination of the link 7-8 to yield the basic structure (perez et al. 2005 ) . this group of phytochemicals is found in a number of folk medicinal plants and many of them possess signifi cant biological and pharmacological activities (dinda et al. 2009 ) . a number of iridoids and secoiridoids (nepetalactones from serbian nepeta species , nestorović three iridoids, phloyoside1, phlomiol, and pulchelloside 1, isolated from the rhizomes of the iranian fl ora eremostachys laciniata ( lamiaceae ) had low to moderate levels of antibacterial activity (mic = 0.05-0.50 mg/ml) against fi ve bacterial strains, bacillus cereus , citrobactor freundii , proteus mirabilis , p. aeruginosa , s. aureus (modaressi et al. 2009 ) . out of these three compounds, pulchelloside 1 showed highest antibacterial activity against b. cereus , penicillin-resistant e. coli , p. mirabilis and s. aureus with an mic value of 0.05 mg/ml. nestorović et al. ( 2010 ) investigated the nepetalactones content in the methanol extracts of the shoot cultures of three endemic serbian nepeta species: nepeta rtanjensis , n. sibirica and n. nervosa , and evaluated the antimicrobial activity of these extracts against eight bacterial strains e. coli , p. aeruginosa , s. typhimurium , listeria monocytogenes , enterobacter cloacae (human isolate), b. cereus (clinical isolate), micrococcus fl avus and s. aureus , and eight fungal species: aspargillus fl avus , aspargillus fumigatus , aspargillus niger , fusarium sporotrichoides , fulvia fulvum, penicillium funiculosum, p. ochrochloron and trichoderma viride . trans, cisnepetalactone was present in shoots of n. rtanjensis , while cis,trans -nepetalactone stereoisomer was present in n. sibirica . no nepetalactone was observed in shoots of n. nervosa . all these extracts had signifi cant antibacterial and antifungal activities against all the tested species. n. rtanjensis extract showed the strongest antibacterial activity with mic of 50 m g/ml. n. nervosa and n. sibirica extracts showed antibacterial activities with mic of 50-100 and 100 m g/ml, respectively. similarly, n. rtanjensis, n. nervosa and n. sibirica extracts showed mic of 25-5, 50-100 and 25-100 m g/ml, respectively. the presence of trans-nepetalactone in n. rtanjensis extract was probably responsible for strongest activity against bacteria and fungi, while cis-nepetalactone in n. sibirica extract showed higher antibacterial and antifungal activity than that of n. nervosa extract. (geng et al. 2009a (geng et al. , b, 2011 . iridoid aglycone moieties, but not its glycosides , exhibit the antiviral activities. zhang et al. ( 2009 ) studied an anti-hepatitis c virus pseudoparticles (hcvpp) entry essay on both aqueous and methanol extracts of the fl owering tops of lamium album . iridoid glucoside lamalbid isolated from the methanol extract was inactive against hcvpp, whereas its aglycone, and epimers named lamiridosins a and b present as major constituents in the aqueous extract signifi cantly inhibited in vitro hcv entry (ic50 value of 2.31 m m). these were nontoxic to the hep g2 2.2 cells at a concentration of 50 m g/ml. they also demonstrated that the parent iridoid glycosides did not show anti-hcv entry activity, but the aglycones of shanzhiside methyl ester, loganin, loganic acid, verbenalin, eurostoside and picroside ii exhibited signifi cant anti-hcv entry and anti-infectivity activities. chemically, saponins are a group of high molecular-weight glycosides , in which saccharide chain units (1-8 residues) are linked to a triterpene (triterpene saponins ) or steroidal (steroid saponins) aglycone moiety, i.e. sapogenin (fig. 1.6 ) . they occur in a wide variety of plants with triterpene saponins (in soybean, alfalfa, quillaja, and guar), and are more widely distributed in nature than steroidal (in yucca, tomato, and oats) saponins ( hostettmann and marston 1995 ) . the steroidal saponins may possess furostanol or spirostanol (e.g. smilagenin and sarsapogenin) moiety. the saccharide chains are commonly attached at the c3 position (monodesmosidic), but some sapogenins contain two saccharide chains (bidesmosidic) attached at the c3 and c17 (via c28) position (vincken et al. 2007 ) . a large number of saponins could be possible depending upon the modifi cations of the ring structure of aglycone moieties and number of sugars added to it, and in turn producing different biological properties. many plant extracts containing saponins from various plants and purifi ed saponins show antimicrobial activities at different concentrations (sen et al. 1998 ; avato et al. 2006 ) . however, the types of saponins exhibit different spectra of antimicrobial effects. oleanolic acid isolated from the root bark of newbouldia laevis have broad-spectrum antimicrobial activity against 6 gram-positive, 12 gramnegative bacterial species and three candida species (kuete et al. 2007 ) . b -sitosterol-3-o -b -d-glucopyranoside isolated from this plant also showed antibacterial effects on three gram-positive, six gram-negative bacterial species and three candida species. a saponin fraction from the stem of y. schidigera exhibited potent growthinhibitory activity with mic ranging from 31.3 to 125 m g/ml against certain food-deteriorating yeasts ( c. albicans ), fi lm-forming yeasts ( debaryomyces hansenii, pichia nakazawae, zygosaccharomyces rouxii ), dermatophytic yeasts ( candida famata, hansenula anomala, pichia carsonii ), and against brewer's yeast ( saccharomyces cerevisiae ) (miyakoshi et al. 2000 ) . different saponins, i.e. tigogenin from tribulus terrestris , dioscin from the rhizomes of smilacina atropurpurea , minutosides from bulb of allium leucanthum were very active against different fungal strains such as c. albicans , c. glabrata and cryptococcus neoformans (zhang et al. 2006a, b ; barile et al. 2007 ) . saponins appear to have stronger activities against fungi, and act by disrupting the membrane integrity of fungal cells. different extraction procedures and storage may affect the antimicrobial action of saponins probably due to chemical transformation of saponins (guclu-ustundag and mazza 2007 ) . commercially produced quillaja ( quillaja saponaria ) and yucca ( yucca schidigera ) saponins showed different antibacterial activities against e. coli , suggesting that saponins from various commercial sources differ in their biological activities (sen et al. 1998 ) . in this study, commercial saponin-rich quillaja and yucca extracts exhibited antibacterial activity against s. aureus and e. coli at different concentrations. the antimicrobial activity of saponins may also be modifi ed by the ph of media. the tea saponins exhibited greater antimicrobial activities against gram-positive s. aureus (mic <0.006 vs. >0.2), gram-negative e. coli (mic 0.003 vs. >0.2) and c. albicans (mic <0.006 vs. >0.2) at low ph 4 than high ph 8.5 (li et al. 2009 ) . some saponins , in general, exhibit stronger antimicrobial activity against gram-positive bacteria than against gram-negative bacteria (avato et al. 2006 ) . saponins fraction from soapnut pericarps ( sapindus mukurossi , tanaka et al. ( 1996 ) and guar ( cyamopsis tetragonoloba , hassan et al. 2010a, b ) showed greater antibacterial activity against gram-positive bacteria than against gram-negative bacteria. conversely, saponins isolated from orchid tree ( bauhinia variegata l.) bark exhibited greater antibacterial activity for gram-negative bacteria than gram-positive bacteria at concentrations ranging from 2.5 to 10 mg/ml (morrissey and osbourn 1999 ) . the relationships between saponin structures and antimicrobial activity are strongly noted. the structure of sapogenin moiety, chain length and composition of sugars infl uences the antimicrobial activities. the y. schidigera saponin fraction possessing a trisaccharide chain without any oxygen functionalities at c-2 and/or c-12 of the aglycone exhibited potent anti-yeast activity, while saponins with 2b-oh or 12-keto groups showed very weak or no activity. low activity was observed for saponins with a disaccharide chain and no activity was observed for the aglycones obtained after acid hydrolysis (miyakoshi et al. 2000 ) . yang et al. ( 2006a ) noted that no activity was observed in the hecogenin saponins when its sugar moiety was less than four monosaccharide units. pentaglycoside was more active than tetraglycoside and shows extended antifungal spectrum against a. fumigatus . in the diosgenin saponin series, saponins with only triglycosides are active against c. albicans and c. glabrata , while the diosgenin saponins with monoglycoside and diglycoside did not show any activity. again, within the group of tigogenin saponins, their antifungal capacity was slightly infl uenced by the composition of the sugar moiety. the replacement of a glucosyl unit with a xylosyl unit showed enhanced activity against a. fumigatus . avato et al. ( 2006 ) suggested that the sugar moiety is not important for the antimicrobial effi cacy from their study since antibacterial activity increased from the saponin extracts to the sapogenin samples. terpenoid compounds derive from a basic structure of c5 isoprene units. they are classifi ed according to the number of isoprene unit involved for their synthesis, i.e. monoterpenoid (c10), sesquiterpenoids (c15), diterpenoids (c20), sesterterpenoids (c25) and triterpenoids (c30). they can be acyclic (myrcene and geraniol), monocyclic (cymene and carvacrol), bicyclic (pinene) and tricyclic with different groups (alcohol, phenol, and aldehyde). the most commonly occurring essential oils (eo) are included in two chemical groups (fig. 1.7 ) : terpenoids (monoterpenoids and sesquiterpenoids) and phenylpropanoids, which are synthesized through mevalonate and shikimic acid metabolic pathways, respectively (gershenzon and croteau 1991 ; calsamiglia et al. 2007 ) . among these two classes, terpenoids are the more diversifi ed group of plant bioactives abundantly found in many herbs and spices (gershenzon and croteau 1991 ) . within terpenoids, the most important components of eo of the majority of plants belong to the monoterpenoids and sesquiterpenoids (gershenzon and croteau 1991 ; calsamiglia et al. 2007 ) . phenylpropanoids have a side chain of three carbons bound to an aromatic ring of c6 (calsamiglia et al. 2007 ) . phenylpropanoids are less abundant compounds of eo compared with terpenoid family, but some plants contain in signifi cant proportions. the eo are a group of secondary plant metabolites obtained from volatile fractions of plants by steam distillation process (gershenzon and croteau 1991 ) . the eo are used traditionally by humans, for many centuries, which provide characteristic fl avor and aroma specifi c to many plants, and are used as antimicrobial agents and preservatives. the eo have diverse chemical composition, nature and biological properties. the eo can be obtained from fl owers, petals, leaves, stems, fruits, roots and barks and the concentrations of eo in these parts depends upon the stage of growth, environmental conditions (hart et al. 2008 ) . a number of eo are known for their strong anti-microbial activities against many pathogenic and non-pathogenic bacteria and fungi. curcumin and its derivatives, the phenylpropanoids, are the principal compounds in rhizome of curcuma longa (turmeric), which exhibit antibacterial properties against different bacteria and fungi. essential oil fractions of turmeric inhibited the growth of pathogenic gram-positive ( s. aureus and staphylococcus epidermidis ) and gram-negative ( e. coli , p. aeruginosa and s. typhimurium ) bacteria (singh et al. 2002 ) . the eo fraction was more effective against gram-positive compared to gram-negative strains, and was comparable to standard antibiotics gentamycin, ampicillin, doxycycline and erythromycin in these strains (singh et al. 2002 ) . a recent study by de et al. ( 2009 ) demonstrated that curcumin inhibited the growth of different clinical isolates of h. pylori with mics ranging from 5 to 50 m g/ml. the gingerols, another phynylpropanoids from zingiber offi cinalis (zinger), possess antifungal and antibacterial properties (park et al. 2008 ) . ginger extract containing gingerol inhibited the growth of h. pylori with mics ranging from 0.8 to 12.5 m g/ml (mahady et al. 2003 ) . constituents of eo differ in their antimicrobial activity against bacteria and fungi. investigating the antimicrobial properties (18 bacterial species and 12 fungi) of fi ve eo constituents (cineole, citral, geraniol, linalool and menthol), pattnaik et al. ( 1997 ) showed that linalool had the most antibacterial activity and inhibited 17 bacteria, followed by cineole, geraniol (each of which inhibited 16 bacteria), menthol and citral aromatic compounds, which inhibited 15 and 14 bacteria, respectively. however, the antifungal activities of these eo constituents did not follow the pattern of antibacterial activities. citral and geraniol oils were the most effective against fungi (inhibiting all 12 fungi), followed by linalool (inhibiting 10 fungi), cineole and menthol (each of which inhibited 7 fungi) compounds (pattnaik et al. 1997 ) . it has been suggested that the ph of eo in culture media may modify antimicrobial properties. for example, anise oil had higher antifungal activity at ph 4.8 than at 6.8, while the oil of cedrus deudorawas was most active at ph 9 (janssen et al. 1987 ) . the structure and stereochemistry of the essential oils have profound infl uences on the antimicrobial activities. alkenyl substituents incorporated into nonphenolic ring structures of essential oils such limonene showed increased antibacterial activities compared with alkyl substituents such as p -cymene with alkylation showing more inhibitory effect on gram-negative bacteria (dorman and deans 2000 ) . from stereochemistry of eo, it has been reported that a -isomers such as a -pinene are less active relative to b -isomers such as geraniol and nerol; cis -isomers are inactive contrary to trans -isomers; compounds with methyl-isopropyl cyclohexane rings are the most active; or unsaturation of the cyclohexane ring further increases the antibacterial activity, e.g. terpinolene, terpineol and terpineolene (hinou et al. 1989 ; dorman and deans 2000 ) . however, griffi n et al. ( 1999 ) reported that the specifi city and level of antimicrobial activity of terpenoids were not always characterized by the functional groups, but were associated with hydrogen-bonding parameters, and for gram-negative organisms a combination of hydrogen-bonding parameters and molecular size parameters. the antimicrobial properties of eo from different sources have been discussed in details elsewhere (chap. 5 ). chemically, limonoids are unique secondary metabolites, characterized by a tetranortriterpenoid skeleton with a furan ring ( fig. 1.8 ) . they are commonly isolated from citrous and maliaceae plants (hallur et al. 2002 ; rahman et al. 2009 ; vikram et al. 2010 ) . besides their health promoting effects, various limonoids have been shown to possess antibacterial, antifungal and antiviral effects (govindachari et al. 2000 ; battinelli et al. 2003 ; atawodi and atawodi 2009 ) . various limonoid compounds such as mahmoodin , azadirone , epoxyazadiradione, nimbin , gedunin, azadiradione , deacetylnimbin and 17-hydroxyazadiradione, isolated from various parts of azadirachta indica (meliaceae family) have been reported to have antimicrobial activities (siddiqui et al. 1992 ; govindachari et al. 2000 ; atawodi and atawodi 2009 ) . rahman et al. ( 2009 ) tested two limonoids isolated from the seeds of swietenia mahagoni ( meliaceae family), swietenolide and 2-hydroxy-3-o-tigloylswietenolide against various multiple-drug-resistant bacterial strains including gram-positive ( s. aureus , s. pneumoniae and haemophilus influenzae ) and gram-negative ( e. coli , klebsiella pneumoniae, salmonella typhi, and salmonella paratyphi ) strains. the most potent activity of swietenolide was observed against h. infl uenzae , s. typhi , and s. paratyphi , whereas 2-hydroxy-3-o-tigloylswietenolide was most active against s. pneumoniae, s. typhi, and s. paratyphi . the lowest activity was observed against k. pneumoniae for both compounds. the limonoids compounds may exhibit antibacterial properties against pathogenic bacteria by disrupting the quorum sensing system and biofi lm production. vikram et al. ( 2010 ) demonstrated limonin, nomilin, obacunone, deacetyl nomilin and limonin 17-o-b -d-glucopyranoside purifi ed from seeds of grapefruits to possess the anti-quorum sensing activity and inhibitory effect on biofi lm formation of pathogenic e. coli o157:h7 with obacunone exhibiting strong antagonistic activity. limonoids also have signifi cant antiviral activity. limonin and nomilin showed inhibitory effects on hiv -1 replication in peripheral blood mononuclear cells and monocytes/macrophages, which was not cytotoxic at the active concentrations (battinelli et al. 2003 ) . the antiviral activity was not much infl uenced by structural differences by limonin and nomilin in this study (battinelli et al. 2003 parida et al. ( 2002 ) demonstrated in an in vivo study that azadirachtin obtained from a. indica inhibited dengue virus type-2 replication as confi rmed by the absence of dengue-related clinical symptoms in sucking mice and absence of virus specifi c 511 bp amplicon. more than 700 polyacetylene compounds have been characterized from plants, which are mainly prominent in the asteraceae, apiaceae and campanulaceae including many medicinal plants from various parts of the world (hudson 1989 ) . food plants of the apiaceae plant family such as carrots, celery, parsley, fennel and parsnip contain a group of bioactive aliphatic c17-polyacetylenes including falcarinol, falcarindiol, panaxydiol, and polyacetylene 8-o-methylfalcarindiol (zidorn et al. 2005 ; christensen and brandt 2006 ) . avato et al. ( 1997 ) investigated the different polyacetylene compounds from the aerial organs of bellis perennis l. of the major constituents, methyl deca-4,6-diynoate and deca-4,6-diynoic acid, and their structural analogues, i.e. deca-4,6-diyne, dimethyl octa-3,5-diyne-1,8-dioate and deca-4,6-diyne-1,10-dioic acid, deca-4,6-diynoic acid and deca-4,6-diyne-1,10-dioic acid showed antimicrobial activity against gram-positive and gram-negative bacteria, respectively. polyacetylene carboxylic acids, 13( e ),17-octadecadiene-9,11-diynoic acid (13,14-dihydrooropheic acid, and the known 17-octadecene-9,11,13-triynoic acid (oropheic acid, isolated from the stem bark of mitrephora celebica demonstrated signifi cant activity against mrsa and mycobacterium smegmatis (zgoda et al. 2001 ) . similarly, pentayne diol, a polyacetylene which was isolated from bidens pilosa (a traditional medicinal herbs) showed highly potent and extensive inhibitory activities against several gram-positive and gram-negative pathogenic bacterial species, including mrsa , and vancomycin-resistant enterococcus faecalis and c. albicans (tobinaga et al. 2009 ) . in a recent fi nding, a polyacetylene compound from carlina acaulis , i.e. carlina oxide exhibited strong antibacterial activity against two mrsa strains, streptococcus pyogenes, p. aeruginosa, c. albicans , and c. glabrata with less toxicity to human hela cells (herrmann et al. 2011 ) . anthranoid compounds are widely distributed in various plants particularly in aloe , cassia, rheum , cassia and frangula , which are traditionally used in ethnomedicine for laxative and cathartic action (paneitz and westendorf 1999 ) . naturally occurring anthranoids can be chemically described as dihydroxyanthraquinones, -dianthrones and -anthrones, often present in plants as glycones (table 1. 2 ; paneitz and westendorf 1999 ) . different anthranoids such as aloe-emodin, rhein, emodin, physcion and chrysophanol occur in rheum species. anthranoids have shown antimicrobial properties in different studies. the anthranoid compounds from the rhizome of rheum emodi exhibited antibacterial and antifungal activities (babu et al. 2003 ) . the antimicrobial effects of the three anthraquinones on s . aureus found to be in the order of rhein > emodin > 1,8-dihydroxyanthraquinone (wu et al. 2006 ) . similarly, wang et al. ( 2010 ) demonstrated that the sequence of antimicrobial activity against bifi dobacterium adolescentis of the fi ve hydroxyanthraquinones was rhein > emodin > aloe-emodin > chrysophanol > physician. they also suggested the infl uence of substituent groups on phenyl ring in hydroxyanthraquinones against b. adolescentis activity might be related with the polarity and the sequence was carboxyl > hydroxyl > hydroxylmethyl > methyl and methoxyl. prenylated anthranoids from leaves of harungana madagascariensis have shown to inhibit bacillus megaterum (kouam et al. 2007 ) . additionally, the effect of emodin with antibiotics (ampicillin and oxacillin) was found to be synergistic or partially synergistic against mrsa, where emodin reduced the mics of the antibiotics (lee et al. 2010 ) . however, some of the anthranoids have potent mutagenic effect (paneitz and westendorf 1999 ) , which is required to consider when evaluating the antimicrobial properties of these compounds. there is considerable evidence that a number of phytochemicals have potential to become useful antimicrobial agents that could be employed as preventative or treatment therapies against microbial and viral diseases. although, there are some encouraging effects in vivo to inhibit pathogenic microbes without affecting benefi cial bacteria in the gastrointestinal tracts, more studies would be required for the safety and effi cacy of these phytochemicals to establish whether they could offer therapeutic benefi ts over conventional therapies. besides, the combination of some antimicrobial drugs and phytochemicals may act as better antimicrobial agents than antimicrobial drugs alone. for example, the application of dual combinations demonstrated synergy between streptomycin and gallic acid, ferulic acid , chlorogenic acid , allylisothiocyanate and 2-phenyle thylisothiocyanate against the gram-negative bacteria. moreover, they can act synergistically with less effi cient antibiotics to control bacterial growth (saavedra et al. 2010 ) . 3,4-dihydroxyphenylacetic acid and 3-hydroxyphenylacetic acid increased the susceptibility of s . enterica subsp. enterica serovar typhimurium strains for novobiocin. in addition, organic acids present in berries, such as malic acid, sorbic acid, and benzoic acid , were shown to be effi cient permeabilizers of salmonella as shown by an increase in the 1-n-phenylnaphthylamine uptake assay and by lipopolrsaccharide release (alakomi et al. 2007 ) . cinnamon essential oil and its major component (trans-cinnamaldehyde) enhanced the antibacterial activity of clindamycin against a toxicogenic strain of clostridium diffi cile ) . in addition, the enhancement activity of different essential oils ( mentha longifolia l. and mentha spicata l.) and different monoterpenes (piperitone, carvone and menthone) on the antibacterial activity of nitrofurantoin has been reported (rafi i and shahverdi 2007 ; shahverdi et al. 2004 ) . the antibacterial activity of cefi xime, cephotaxime, vancomycin and tetracycline was also increased by curcumin (moghaddam et al. 2009 ) . allicin has a synergistic effect with amphotericin b against c. albicans via enhancing the phospholipid peroxidation reaction in vitro and in vivo , which suggests that allicin could reduce the amphotericin b dose to lessen side effects ) . due to the growing use of phytochemicals and other dietary phytochemical-rich supplements, it is required to understand whether problems might arise from using these preparations in combination with conventional drugs. there is lack of comprehensive studies that can establish the consequences of phytochemicals-drug interactions. however, all these evidence also suggest that intake of phytochemicals rich foods could be considered in future research while antimicrobial agents are applied to the body. plant genomes contain 20,000-60,000 genes, and about 15-25% of these genes encode enzymes for secondary metabolism (bevan et al. 1998 ; somerville and somerville 1999 ) . the genome of a plant species encodes only a small fraction of all the enzymes that are required to synthesize the entire set of secondary metabolites found throughout the plant kingdom (pichersky and gang 2000 ) . identifi cation of particular genes for target phytochemicals and the genetic engineering techniques could allow expressing the biosynthetic pathways of some phytochemical synthesis in organisms such as e. coli , b. subtilis or s. cerevisiae . for example, miyahisa et al. ( 2006 ) reported that introduction of four genes for a phenylalanine ammonia-lyase, cinnamate/coumarate:coa ligase, chalcone synthase, and chalcone isomerase, in addition to the acetyl-coa carboxylase, in e. coli cells resulted in effi cient production of (2s)-naringenin from tyrosine and (2s)-pinocembrin from phenylalanine. finally, the possibility of using phytochemicals as antimicrobial compounds would be a paradigm shift towards the potential health benefi ts and safety overcoming the problem of microbial resistance to drugs. antibacterial activity of diospyrin, isodiospyrin and bisisodiospyrin from the root of diospyros piscatoria (gurke) (ebenaceae) initial in vitro evaluations of the antibacterial activities of glucosinolate enzymatic hydrolysis products against plant pathogenic bacteria the antimicrobial effects of glucosinolates and their respective enzymatic hydrolysis products on bacteria isolated from the human intestinal tract weakening of salmonella with selected 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toxicity of anthraquinones by microcalorimetric bioassay anti-aids agents. 37. synthesis and structureactivity relationships of (3 ¢ r,4 ¢ r)-(+)-cis-khellactone derivatives as novel potent anti-hiv agents antifungal activity of c-27 steroidal saponins five new iridoids from patrinia rupestris different susceptibilities of staphylococcus and gram-negative rods to epigallocatechin gallate polyacetylene carboxylic acids from mitrephora celebica antifungal activities and action mechanisms of compounds from tribulus terrestris l atropurosides a-g, new steroidal saponins from smilacina atropurpurea lamiridosins, hepatitis c virus entry inhibitors from lamium album polyacetylenes from the apiaceae vegetables carrot, celery, fennel, parsley, and parsnip and their cytotoxic activities antibacterial alkaloids from chelidonium majus linn (papaveraceae) against clinical isolates of methicillin-resistant staphylococcus aureus key: cord-102632-yazl9usb authors: lobet, guillaume; descamps, charlotte; leveau, lola; guillet, alain; rees, jean-françois title: quovidi: a open-source web application for the organisation of large scale biological treasure hunts date: 2020-07-01 journal: biorxiv doi: 10.1101/2020.06.30.177006 sha: doc_id: 102632 cord_uid: yazl9usb learning biology, and in particular systematics, requires learning a substantial amount of specific vocabulary, both for botanical and zoological studies. while crucial, the precise identification of structures serving as evolutionary traits and systematic criteria is not per se a highly motivating task for students. teaching this in a traditional teaching setting is quite challenging especially with a large crowd of students to be kept engaged. this is even more difficult if, as during the covid-19 crisis, students are not allowed to access laboratories for hands-on observation on fresh specimens and sometimes restricted to short-range movements outside their home. here we present quovidi, a new open-source web platform for the organisation of large scale treasure hunts. the platform works as follows: students, organised in teams, receive a list of quests that contain morphologic, ecologic or systematic terms. they have to first understand the meaning of the quests, then go and find them in the environment. once they find the organism corresponding to a quest, they upload a geotagged picture of their finding and submit this on the platform. the correctness of each submission is evaluated by the staff. during the covid-19 lockdown, previously validated pictures were also submitted for evaluation to students that were locked in low-biodiversity areas. from a research perspective, the system enables the creation of large image databases by the students, similar to citizen-science projects. beside the enhanced motivation of students to learn the vocabulary and perform observations on self-found specimens, this system allows faculties to remotely follow and assess the work performed by large numbers of students. the interface is freely available, open-source and customizable. it can be used in other disciplines with adapted quests and we expect it to be of interest in many classroom settings. teaching biology to first-year bachelor students is a challenge. as educators, our aim is usually twofold. first, we want the students to learn a new set of knowledge and integrate it. second, and this is for us equally important, we want the students to engage with the topic at hand. we want to transmit our passion and curiosity about the topic that we teach. third, we also want students to learn to observe the world around them. it is one thing to learn a topic from a textbook, it is another to observe it in real life. however, the main issue is that the classroom is, often by design, completely disconnected from the natural world. the challenge is therefore to find a way for students to learn and engage with biology, despite that given disconnection. last but not least, in the spring semester of 2020 (january to june) it was necessary for us to adapt the learning activities to the containment measures related to covid-19. the formal aim of our biology course -given in the bioengineering faculty, uclouvain, belgium -is to discover plant and animal structures, organs and their function at the individual scale. to achieve this, students need to learn specific vocabulary related to these structures. the classic way to present this vocabulary to a student audience is to review a series of slides illustrating these different characteristics. this vocabulary is usually very boring for teachers to describe (imagine the slides showing all the different shapes of leaves) and the content is not very interesting for students to listen to either. yet this vocabulary is an important prerequisite for describing any biological structure and for later systematic identification of taxons using dichotomous keys . its learning is essential. the question is therefore how to make this learning process motivating for the students and give them the opportunity to learn over time instead of memorising a list of words? the additional difficulty is that this learning activity must be able to be set up with more than 300 students and few teaching resources. to create this learning activity, we decided to draw inspiration from all the pedagogical techniques that aim to place the student at the centre of his learning. student-centred learning and active learning emerged as important pedagogical techniques during the last century [ref] . active learning is characterised by (i) involving the student in the construction of his or her learning, (ii) engaging the student in an in-depth treatment of the subject matter, (iii) constructing learning through interaction (with the teacher or other students), (iv) conceiving of learning as the evolution of knowledge and skills [1, 2] . studies have shown that the more cognitively and socially engaged the student is in a learning task, the more perennial the learning task becomes [1, 3] . active learning improves the performance of students and acts to reduce the gap achievement between advantaged and disadvantaged students [4] . in order to stimulate learning through interaction and create a collective emulation around this activity, the idea of creating a campus-wide biological treasure hunt finally emerged from the discussions. beyond simply being active through the manipulation of information, the student has to transform and produce new information that is not provided in the learning material. gamification is another recent technique to better engage the students in a learning activity. gamification is defined by [5] as "game-based mechanics, aesthetics, and game thinking to engage people, motivate action, promote learning, and solve problems". in many studies, students' levels of engagement increased significantly following the introduction of game elements, such as points, challenges, quests or progress bar [6] . the gamified environnement can afford intrinsic motivation and engagement, which are also targeted by active learning. to assemble these different elements -biological vocabulary, observation, active learning and gamification -in a comprehensive learning activity, we created a large scale biological treasure hunt for our students. in short, we provided students with a list of specific biological vocabulary. they had to understand the list and find the different elements outside of the classroom, in the natural world. external resources (books, selected websites, wiki pages) describing this vocabulary were available to them. complexity of understanding (some words are more difficult than others) as well as the difficulty of identification in the field were rewarded with different points. to manage the treasure hunt, we designed a new web-based platform, quovidi (which would loosely translate from latin as "where did you see"), for the organisation of large scale, decentralised, biological treasure hunts. quovidi is an open-source project available at www.quovidi.xyz . the objective of this publication is to describe the project, to show how we were able to adapt this learning activity to the covid-19 crisis, and finally, to show the results and success of the activity with the students. quovidi is a web application for the organisation and management of large scale biological treasure hunts. it was created to teach students to learn new biological terms (both in zoology and botany) and to teach them to observe the natural world surrounding them. first, educators have to prepare a list of quests to find in the natural world. these quests should be tailored and adapted for the target public. for instance, in our experience with first year biology students, the quests revolved around biological structures and families (tab. 1). each quest is given a specific reward (points) depending on its intrinsic difficulty and rareness. quests can be sorted in different groups (for instance "animal" and "plant") and subgroups (for instance "animal species" and "leaf shapes") to help students navigate them. find a siphonaptera animal animal groups 3 find an example of aposematism animal animal physical attributes 1 second, educators have to assign students to groups to perform the activity. students in the same group will be able to share pictures and collaborate on the data collection. when logging into the web interface, students will be able to see the collected pictures and rewards from their own group. they will also be able to see the total number of points of the competing groups. educators also have the possibility to define specific game parameters, such as specific geographic regions in which the game takes place or restriction on the number of submissions in each quest group (adding for instance a point penalty below a certain number of "animal" or "plant" submissions). once the list of quests, users and groups are defined, the activity can start. two main activities are available for the students : an in situ treasure hunt and an ex situ photo quiz activity. the main activity of the platform is the biological treasure hunt. students have to go outside (although some of the creatures may be also found in their home such as food parasites, e.g. lepisma sp. or flies) to find the different quests setup by the educators. once they find a specific quest, they have to take a picture of it with their smartphone. we ask the student to take unambiguous pictures, where the subject of the quest is clearly identified and visible. we also ask them to leave the natural environment intact, without killing any plant or animal in the process. they can then store the picture on the quovidi web interface. when stored, pictures are automatically resized (for efficiency) and added to the activity database. localisation information and date are extracted from the picture exif metadata. any other information is erased at this step. once pictures are stored on the web interface, students can assign them to a specific quest and submit it for evaluation. the web application allows users to follow their progress in detail (which picture was submitted for which quest, what is the evaluation status, etc.) as well as the global progress of the other groups (the total number of collected points). it is worth noting that in belgium -where the web application was first usedthe lockdown due to the covid-19 pandemic still allowed citizens to go outside for some walk and exercise, although at a limited range. as such, the treasure hunt could still be performed by the students, either in their own garden or in neighbouring areas. however, not everyone lives in the countryside or close to a natural environment, or had the opportunity to leave their home during the lockdown. this is why we created a second module in the interface, the photo quiz, which allowed students to learn from photos contributed by other students, without having to submit their own photos. the second module of the interface allows students to evaluate pictures submitted by other students (a modified version of peer evaluation). more precisely, in the photo quiz module, students are presented with pictures submitted by other groups and validated by the educators (see below "expert evaluation). they have to assess whether the picture corresponds to its assigned quests. their assessment is then compared to the assessment of the educators. if it matches, the students gain points that are added to their global group tally. when performing this activity for the first time, it is necessary to have a sufficient amount of submitted (and corrected pictures). without a database large enough, the activity loses some of its interest, as students might all review the same pictures. the third important module of the interface, central to the activity, is the expert evaluation. each submitted picture needs to be manually assessed by the educators. different feedback can be given for each submission, such as "correct", "correct and nice picture", "incorrect" , "not visible" (e.g. the object is not visible in the picture) or "out of rules" (e.g. picture of a houseplant, picture taken outside of the prescribed geographical zones). the interface was designed to easily navigate the different quests and quickly correct the submitted images. the web application was created using the r shiny framework , using the shinydashboard [7] , shiny [8] , shinywidgets [9] , shinybs [10] , miniui [11] packages for the user interface design. the data is stored in a sqlite database, hosted on the server. the database management is done using the dbi [12] and rsqlite [13] packages . pictures are transformed and managed using the magick [14] package. exif information is extracted using the exifr [15] package. data manipulation and visualisation is done using the tidyverse [16] , lubridate [17] , cowplot [18] , formattable [19] , dt [20] , plyr [21] , leaflet [22] packages. the text sentiment analysis was performed using the rfeel package [23] . in our exemple, the web application was deployed on the university server with the following specifications: quovidi is an open source project, released under an apache licence [24] . everyone is free to re-use and modify it, with attribution. the interface was created to be as much user-friendly as possible so that neither students nor staff need technical training . because it is web based, it can be used on any platform, whatever the operating system . it scales on mobile devices as well, allowing users to store and submit pictures directly from the field (if they have an internet connection). figure 1 shows the different panels of the web interface. figure 1a shows the "store" panel, where students can store pictures, before submitting them for evaluation. this allows students from the same group to share and visualise their pictures. at this step, students can already assign a quest to the picture, which can be changed later on. they can also assign a geographic region, if this is required by the educators. a default region will be automatically proposed, based on the metadata of the picture. figure 1b shows the "submit" panel. at this stage, students see all the pictures from their group. they can select a stored picture, assign it to a quest and submit it for evaluation. groups can only submit one picture for each quest. or not as well as their validation status. in the same panel, students can also see the global scores of each group taking part in the activity. this adds a strong gamification aspect to the activity. figure 1d shows the "quests" panel. in that panel students can navigate through the different quests proposed by the educators. they can sort them by groups, subgroups or rewards. in this panel, no explanation is given for the different quests. for instance if the quest is "find an achene", we do not define achene. this is done by design. we want students to look up the different biological terms by themselves. we do provide them with ressources to do so. when an educator logs into the web application, the "quests'' panel becomes the "admin" panel. in this panel, educators can follow the evolution of the activity ( fig. 2a) , change the activity parameters ( fig. 2b ) or correct the student submissions ( fig 2c) . depending on the number of participating students and allowed submissions, the number of corrections can quickly become quite large. therefore we designed the corrections interface to be fast and efficient. the educator first chooses one quest to correct. he·she will in spring 2020, we organised the activity with a rooster of 346 first year bachelor students from the bioengineering faculty of the uclouvain (belgium). students were spread in 346 groups (it was therefore set up as an individual activity). although students had to do the activity individually, we encouraged them to discuss the different quests and collect them together, as long as everyone took their own pictures. each group was allowed to submit a maximum of 50 pictures. 285 quests were created, divided in 175 plant quests and 110 animal quests. specific restrictions were added to the game. a minimal number of animal and plant quests had to be collected by each group. groups were also asked to the activity started on february 11. we had to pause the activity for 20 days at the beginning of the lockdown due to the covid19 crisis. during that pause, we implemented the peer-evaluation in the web interface (it was not part of the interface initially). the activity resumed on the 3d of april and finished on the 15th of may. for the second phase of the activity, during the lockdown, all restrictions (quests groups and zones) were lifted as many students had returned to their home far away from the campus. at the end of the activity, we sent an anonymous feedback form to the students and received 125 answers. a total of 6543 pictures were submitted by students during the 2020 activity. figure 3 shows the repartition of the submitted pictures by the students during the activity. figure 3a & b show the difference before and after the lockdown imposed during the covid-19 crisis. before the lockdown, as we asked students to take pictures around the university, most of them were taken in louvain-la-neuve. during the lockdown, almost no pictures were taken in louvain-la-neuve, as students went back home. the lockdown reduced the number of collected pictures, but did not stop it. this is due to several reasons. at the beginning of the activity, we encouraged students to look for quests in groups, to foster peer-learning between them. this was not possible anymore during the lockdown. the collection of biological data was also influenced by the direct surroundings of the students. students living in an urban area were potentially at a disadvantage compared to students in the countryside. however, because we included the photo quiz module at the beginning of the lockdown, every student could continue the activity. figure 4 shows, for every group, the proportion of points acquired either with the quests collection or the photo quiz. we can see that the dual system allowed students to choose different strategies, to adapt to their individual lockdown conditions. we also observed a strong trend toward the collection of plant-related quests by the students ( fig. 3c & d) . this is probably due to the fact that, in an urban setup, plants are easier to find that animals. for an inexperienced naturalist, it is also probably easier to take pictures of plants than animals that have a tendency to escape. all the pictures can be viewed interactively at the address http://2020.quovidi.xyz overall, we observed a high correctness in the students picture submissions ( fig 5a) . for the treasure hunt and the picture collection, only 10% and 14% of the quests (for the animal and plant, respectively) were assessed as incorrect by ourselves. one reason for such a high accuracy from the students might be the high level of engagement required by the activity. they have to learn the vocabulary and discuss with other students, and go outside often in groups to find what they have identified as appropriate for a quest submission. in the icap framework [3] , we believe this corresponds to the "interactive learning" level, enabling the highest learning capabilities. interestingly, we also observed a much lower accuracy for the photo quiz ( fig. 5b ). for that activity, 37% and 38% of the evaluations by the students (for the animal and plant, respectively) were incorrect. this can be due to several factors. first, contrary to the treasure hunt in itself, the evaluation activity requires a lesser level of engagement by the student. the activity is indeed "reduced" to click on a button in front of a computer screen. second, depending on the quality of the picture to evaluate, said evaluation could be challenging. we tried to keep only good pictures for that activity, but the quality remained nonetheless variable. overall, the activity was very well appreciated by the students. with a few exceptions, students like going outside to observe their surroundings and collect the quests. in a survey performed after the activity ( fig. 6 ), 125 students reported to like the activity and have the feeling to have learned during it. many students spontaneously expressed their enthusiasm for this activity (tab. 3). selected comments from the students "great activity to learn new concepts and look at our environment in a different way. " "i think the game is fun and interactive, it's a great way to learn by seeing things "in real life" and also to decipher the quests. " "very nice way to propose the course, it pushes the students to discover the surrounding nature in a playful way. " the quovidi platform was created for several reasons. we wanted students to learn and know specific plant and animal vocabulary, but we did not want to just give them a list of words to be memorized and repeated. we also wanted them to explore and learn to observe their direct environment. we wanted to show them that you do not need to go to a tropical forest to be able to see a great diversity of plant and animal forms and species. we wanted to spark a strong interest in their surrounding natural world. finally, we were also working with strong practical constraints. we needed to design an activity that was scalable for hundreds of students, without the need to increase the number of educators. this was possible, thanks to the current technologies (camera, mobile network and gps localisation) available in almost every mobile phone. with the creation of the web-platform for quovidi, we have met all those goals. the treasure hunt (and to a lesser extent the photo quiz) strongly motivates students to learn and remember the different technical terms used in the quests. then they have to apply these new terms directly in the field. the gamification process (quests, score points, personal progress panel and scoreboard between all the groups) is also a strong incentive to engage in the activity. the activity is also highly scalable. the number of participants is, from a technical point of view, only limited by the capacity of the server on which the platform is installed. the main limitation remains the expert correction step. as every single picture needs to be validated, the evaluation can quickly require a lot of time from the educator, even though we tried to make the process as efficient as possible. we hope in the future that the platform would benefit from advances in artificial intelligence algorithms to help correct the images (see below). finally, the activity is completely decentralised, which has been a great asset during the covid-19 crisis. students can collect quests at any time and place, making it easy to adapt to every individual situation. if they cannot go outside, or are not in a nature-rich environment, they can still participate in the activity via the peer evaluation module. from the educator point of view, all the management and corrections can be done from anywhere, as long as they have access to a computer and an internet connection. as such, the platform was a real asset during the lockdown period (13 march to 8 of june in belgium), as it enabled us to continue the activity almost seamlessly. similarly to citizen science projects, the use of our platform allows the collection of large numbers of geotagged, dated images of plant and animal structures. by helping create such a database over the years, the students are taking an active role in creating a valuable research ressource. this in itself is viewed by the students as a motivational element of the activity. such databases could be re-used in different ways. from an educational point of view, the images collected could be used to create a quiz to rehearse the vocabulary the following year. the student would therefore create their own teaching and rehearsal material. an example of a quiz created with the students pictures is visible here : http://quiz.quovidi.xyz . from a research point of view, an ever growing database of annotated plant and animal pictures (describing either organ, species or groups), on a limited and well defined area would be a valuable resource. as each record of the database has been validated by an expert (the educators), such a database could be used in research projects. another interesting valuation of the database would be to reuse it to train deep learning recognition algorithms. again, given the size and potential growth of the database, it will be an interesting resource to train machine learning models to recognise plant and animal structures. such models could, in turn, be integrated into the platform to help with the correction. so far, we use the quovidi framework within a single classroom (even if it was a very large one). since the activity is entirely centralised online, we could imagine collaboration between remote classrooms. students from different regions, countries or continents could participate in the same activity, hence increasing the degree of diversity of the observations. here we exemplified the use of our platform with a biological treasure hunt. students were asked to find, in the field, plant and animal structures. however, due to its flexibility, the platform could be used to organise large scale treasure hunts in any context. it could be used in architecture, design or geology classrooms, with quests related to different building structures, street art or rock, respectively. it could be used with children, with simplified quests, or with more advanced students, with more complex ones. in short, we expect the concept could be used in any context to deal with structures present in the "outside" world. we presented in this manuscript a new open-source web platform for the organisation for large tresor hunt, quovidi. during the spring 2020, in the midst of the covid-19 crisis, we successfully used the quovidi platform with more than 300 students, and allowed the collection of more than 6000 geotagged plant and animal pictures. the decentralised nature of the platform enabled us to ensure a continuity in our teaching, despite the nation-wide lockdown. we expect quovidi to be of interest for any teaching activity focused on the identification of real-world structures. quovidi is available at the address http://www.quovidi.xyz active learning increases student performance in science, engineering, and mathematics translating the icap theory of cognitive engagement into practice the icap framework: linking cognitive engagement to active learning outcomes increased structure and active learning reduce the achievement gap in introductory biology kapp bkm. games, gamification, and the quest for learner engagement. in: main [internet the effect of gamification on motivation and engagement create dashboards with shiny: web application framework for r shinywidgets: custom inputs widgets for shiny twitter bootstrap components for shiny shiny ui widgets for small screens dbi: r database interface sqlite" interface for r advanced graphics and image-processing in r exif image data in r easily install and load the "tidyverse dates and times made easy with lubridate streamlined plot theme and plot annotations for "ggplot2 formattable: create "formattable" data structures dt: a wrapper of the javascript library "datatables tools for splitting, applying and combining data leaflet: create interactive web maps with the javascript "leaflet" library feel: a french expanded emotion lexicon. language resources and evaluation apache license, version 2.0 | open source initiative quovidi, then called biogo, was one of the laureates of the "prix wernaers pour la vulgarisation scientifique" in 2020. original draft x x x x x key: cord-021897-yeih3tfo authors: page, stephen j. title: tourism today: why is it a global phenomenon embracing all our lives? date: 2011-10-28 journal: tourism management doi: 10.1016/b978-0-08-096932-9.10001-8 sha: doc_id: 21897 cord_uid: yeih3tfo nan the new millennium has witnessed the continued growth of interest in how people spend their spare time, especially their leisure time and nonwork time. some commentators have gone as far as to suggest that it is leisure time -how we use it and its meaning to individuals and familiesthat defines our lives, as a focus for non-work activity. this reflects a growing interest in what people consume in these non-work periods, particularly those times that are dedicated to travel and holidays which are more concentrated periods of leisure time. this interest is becoming an international phenomenon known as 'tourism': the use of this leisure time to visit different places, destinations and localities which often (but not exclusively) feature in the holidays and trips people take part in. the world travel and tourism council (wttc) estimate that travel and tourism as economic activities generates around us$6 billion, which is expected to grow to us$10 billion by 2015. at a global scale, travel and tourism today: why is it a global phenomenon embracing all our lives? 1 tourism supports around 235 million jobs: this is equivalent to 8 per cent of world employment and 9 per cent of world gdp. therefore, the growing international significance of tourism can be explained in many ways. in an introductory text such as this, it is important to stress at the outset the following types of factors and processes in order to illustrate the reasons why tourism assumes an important role not only in our lives but also globally: • tourism is a discretionary activity (people are not required to undertake it as a basic need to survive, unlike consuming food and water) • tourism is of growing economic significance at a global scale, with growth rates in excess of the rate of economic growth for many countries • many governments see tourism as offering new employment opportunities in a growing sector that is focused on service industries and may assist in developing and modernizing the economy • tourism is increasingly becoming associated with quality of life issues as it offers people the opportunity to take a break away from the complexities and stresses of everyday life and work -it provides the context for rest, relaxation and an opportunity to do something different • tourism is becoming seen as a basic right in the developed, westernized industrialized countries and it is enshrined in legislation regarding holiday entitlement -the result is many people associate holiday entitlement with the right to travel on holiday • in some less developed countries, tourism is being advocated as a possible solution to poverty (described as 'pro-poor' tourism) • holidays are a defining feature of non-work for many workers • global travel is becoming more accessible in the developed world for all classes of people with the rise of low-cost airlines and cut-price travel fuelling a new wave of demand for tourism in the new millennium. this is potentially replicating the demand in the 1960s and 1970s for new popular forms of mass tourism. much of that earlier growth was fuelled by access to cheap transport (i.e. the car and air travel) and this provided new leisure opportunities in the western world and more recently in the developing world and newly industrializing countries • consumer spending on discretionary items such as travel and tourism is being perceived as a less costly item in household budgets. it is also much easier to finance tourism with the rapid rise in credit card spending in developed countries, increasing access to travel opportunities and participation in tourism • technology such as the internet has made booking travel-related products easy and placed it within the reach of a new generation of computer-literate consumers who are willing to get rid of much of the traditional ritual of going to a travel agent to book the annual holiday. such technology now opens many possibilities for national and international travel at the click of a computer mouse and to check-in for a flight via a mobile phone. it is evident that tourism is also becoming a powerful process affecting all parts of the globe. it is not only embraced by various people as a new trend, a characteristic or defining feature of people's lives, but is also an activity in which the masses can now partake (subject to their access to discretionary forms of spending). this discretionary activity is part of wider post-war changes in the western society with the rise in disposable income and spending on consumer goods and services. yet tourism is not just a post-war phenomenon as it can be traced back through time as shown in chapter 2. this highlights how important tourism was in past societies as well as the historical processes of continuity and change which help us to understand tourism development throughout the book. the first major wave of growth in consumer spending was in home ownership, then in car ownership and, then, in accessing tourism and international travel. in fact international travel (and domestic travel, i.e. within a country) is a defining feature of the consumer society. whilst the car has given more people access to tourism and leisure opportunities within their own country, reductions in the price of aeroplane tickets have made international travel and tourism products and services more widely available. for example, the number of air travellers in the uk is expected to rise to 475 million by 2030. this is not without its environmental cost. there is a growing global concern about the ability of the earth's environment and resources to sustain the continued expansion of economic activity, including tourism. whilst scientists have pointed to these concerns since the 1960s, these environmental issues have only really begun to permeate government and people's thinking since the rise of global concerns over climate change and the international kyoto treaty seeking to address greenhouse gas emissions. tourism is the centre stage in these concerns because travel for leisure purposes is not a fundamental necessity, and it contributes to co 2 emissions through the consumption of fossil fuels used to transport people on holiday, at the destination and in the accommodation they use. transportation causes around 75 per cent of the co 2 emissions generated by tourism, with aviation responsible for around 40 per cent of these emissions. improving energy efficiency in transportation may be expected to generate a reduction of 32 per cent in the emissions per passenger kilometre between 2005 and 2035. however, the quantity of emissions varies depending on the mode of transport used, with long-haul travel the greatest contributor to highly emissionintense trips. the issue of tourist travel and its global environmental effect through pollution is a thorny issue since tourism is internationally significant and has an important role in society, as we have already seen. there is an almost unanimous reluctance among government policy-makers to directly limit or restrict tourist travel due to its economic effects on destination areas. consequently, many prefer to adopt the politically acceptable and palatable adaptation strategies -seeking to adapt human tourism today: why is it a global phenomenon embracing all our lives? 1 behaviour and destinations to the effects of climate change (see box 1.1). many people openly admit to being supportive of 'green' and 'sustainable' principles but are unwilling to sacrifice their annual or additional holiday to reduce carbon emissions: likewise, few are willing to sacrifice an overseas destination for a less carbon consumptive and polluting domestic holiday. this assumes a more interesting dimension when one sees some sections of the tourism industry responding to consumer interest in green issues, by offering more 'green' and 'sustainable' holidays, recognizing a business opportunity. critics have labelled this harnessing of green issues as one way of gaining a competitive edge without a complete commitment to implementing sustainability principles in their business practices as 'greenwash' (see table 1 .1). this reflects the fact that tourism in this respect is a phenomenon that is constantly evolving, developing and reformulating itself as a consumer activity. tourism, as a consumer activity, is constantly being developed by the tourism industry and individual businesses, as marketing is used to develop new ideas, products and services and destinations. the challenge for the tourism industry is in adopting new ideas developed in research, such as service dominant logic (see shaw et al., 2011 for more detail) which may assist, with the use of social marketing techniques, to adapt human behaviour so that they extend the daily activities which climate change has become a dominant theme in the analysis of the future for small island nations which are little more than a metre above sea level. this has become a major problem for governments when the scale of sea level change is set against natural changes in the land level, which is sinking at a rate of around less than a centimetre per year. however, this means that in less than 100 years some island states such as the maldives may be flooded and therefore uninhabitable. the maldives is a collection of 1200 small islands (198 of which are inhabited) and it is dependent upon tourism as its main source of external earnings, accounting for over 28 per cent of gdp and almost 60 per cent of foreign earnings' receipts. the dependence upon tourism has meant that the country's 600 000 international visitors each year are a key source of revenue for the country's economy and should climate change combine with sea level rises to accelerate the pace of change, the country's tourism industry could be completely eradicated. therefore in spite of the country's natural beauty and 80 tourist resorts located across 80 different atolls (i.e. small islands that are just above sea level) its competitiveness as a destination may well be threatened by natural environmental changes. to address these threats, the capital male has built a 3 m sea wall for just one island and other islands in the maldives suffer periodic flooding. despite these major challenges, the country's government is seeking to try and mitigate the worst impacts of climate change, as its resources are very limited and the scale of the problem is huge. it is a story that can be repeated across many similar island archipelagos across the south pacific where climate change may accelerate the pace of sea level rises putting the livelihoods and entire destination in peril for the future. embrace sustainability ideals (e.g. recycling, reuse and minimizing the use of natural resources) to their holiday-taking behaviour. of course, the cynic may argue that the most sustainable form of tourism is none at all if you are serious about your own footprint on the planet. for the tourism sector, they have embraced new ideas (including in some cases sustainability) and pursued strategies focused developing niche products reflecting the way tourism has developed a more specialist focus (see table 1 .2). tourism appeals to the human imagination. as an activity it knows no bounds: it is global and it affects the environment it occurs in, the people who host it, the economies it seeks to benefit and the tourists who consume it as an experience, product and an element of their lives. with tourism having this all-embracing role, it is no surprise that many commentators, researchers and governments have agreed on the need to manage it as a process and activity, especially since it has the potential to snowball and grow out of proportion if it is not managed. therein lies the basic proposition of this book -tourism needs managing if it is to be successful and beneficial rather than a modern-day scourge. tourism and its ability to be sustainable as an activity have been major growth areas of research since the 1990s. the guiding principles of sustainable tourism are based on the management of resources, the environment and economy and society/its culture for the long-term so they are not compromised or damaged by tourism development. a number of key studies exist which provide a very wide ranging overview of the subject's development: krippendorf, j. (1987) the holiday-makers. oxford: butterworth heinemann. this landmark study questioned the necessity of long-haul travel and the impact of tourism including the damage it caused to the environment. this is a complex but critical review of the sustainability debate which challenges current thinking and many of the conventional ideas that tourism can easily be translated into a sustainable activity, particularly in less developed countries. this report outlines many of the principles associated with setting out the principles which can be harnessed to try and make tourism sustainable. yet one of the fundamental problems in seeking to manage tourism is in trying to understand what it is: how it occurs, why it occurs, where it does, the people and environments that are affected by it and why it is a volatile activity that can cease as quick as it can start. these types of questions are what this book seeks to address. it will also look at why tourism as a consumer activity is built on dreams, images and what people like to do; this is notoriously difficult to understand as it involves entering the realms of psychology and the mind of the individual tourist. furthermore, these psychological elements are bound up in notions of enjoyment, feelings, emotions and seemingly intangible and unseen characteristics. the issue is further complicated by the way in which an individual's tastes and interests change throughout their life. in other words, being a tourist is based on the principle of non-work and enjoyment of one's free time in a different locality, and results in an experience, a treasured memory and something personal which develops through our life course. tourism is a dynamic phenomenon and a highly trend-driven activity in a post-modern society where travellers constantly seek new and diverse experiences. this has led the tourism sector to harness marketing techniques to create different products and experiences to very specific market segments based on consumers' interests and values. a range of some of the key trends and developments in recent years are listed below with a brief explanation of their underlying philosophy and examples. slow travel travel to a destination and savouring the journey by not flying, such as taking the train or bicycle so as the rush and stress is taken out of the travel experience so it is slowed down why study tourism? is it just about enjoyment and holidays? tourism and its analysis have become a relatively recent field of study among academics, researchers and commentators. some of the very early student textbooks on tourism (see table 1 .3) can be dated to the early 1970s (although there are examples of other reviews of tourism dating to the 1930s, 1940s and 1950s), with a second wave being produced in the 1980s and then a massive explosion in the late 1980s and 1990s as tourism education and training expanded worldwide. since the 1990s, a wide range of more specialist and niche books have been published on particular aspects of tourism research. there are a range of commonly recognized problems in studying tourism, a number of which are important to the way in which we understand whether it is just about enjoyment and holidaytaking: • tourism is a multidisciplinary subject which means that a wide range of other subjects, such as psychology, geography, economics, to name but a few, examine it and bring to it a range of ideas and methods of studying it. this means that there is no overarching academic agreement on how to approach the study of tourism -it really depends on how you are looking at tourism, and the perspective you adopt which determines the issues you are interested in studying • this has led to a lack of clarity and definition in how to study tourism, something that other researchers have defined as reductionism. what this means is that tourism is normally defined by reducing it (hence 'reductionism') to a simple range of activities or transactions (i.e. what types of holidays do people choose? or how do people purchase those holidays?) rather than by focusing on the framework needed to give a wider perspective or overview of tourism. these problems often compound the way people view tourism as a subject, emphasizing the holiday or enjoyment aspects of travelling (in one's spare time or on business) as the defining features or reference point of tourism. to the general public tourism is something everyone knows about -it is something many have engaged in and so have an opinion on what it is, its effects and widespread development. admittedly, tourism is about pleasure and enjoyment, but its global growth and expansion are now creating serious societal problems and issues; a fundamental understanding of tourism is required if we are to manage and control the impacts and problems it can cause. some critics argue that tourism epitomizes the extreme of post-modern consumption in a society that spends on travel and tourism because it can and not for an intrinsic need for holidays as access to travel is, in relative terms, very cheap and affordable for many. one way of beginning to understand that tourism is more than holidays and enjoyment is to think about why tourism is so important in modern society (i.e. its social, cultural and economic significance) by looking at an important process which has led to the demand for it -the rise of the leisure society. tourism is now widely acknowledged as a social phenomenon, as the nature of society in most advanced developed countries has now changed from one which has traditionally had an economy based on manufacturing and production, to one where the dominant form of employment is services and consumer industries (i.e. those based on producing consumer goods and services). at the same time, many countries have seen the amount of leisure time and paid holiday entitlement for their workers increase in the post-war period so that workers now have the opportunity to engage in the new forms of consumption such as tourism. these changes have been described as being part of what has been termed as the leisure society, a term coined in the 1970s by sociologists. they were examining the future of work and the way in which society was changing, as traditional forms of employment were disappearing and new service-related employment, increased leisure time and new working habits emerged (e.g. flexi-time and part-time work). some commentators described this as a 'leisure shock' in the 1980s since many workers were still not prepared for the rise in leisure time and how to use it. as society has passed from the stage of industrialization to one now described as post-industrial, where new technologies and ways of communicating and working have evolved, sociologists such as baudrillard (1998) in the consumer society: myths and structures, have argued that we have moved from a society where work and production have been replaced by one which leisure and consumption now dominate. this has been reflected in social changes, such as the rise of new middle classes in many developed and developing countries, and these middle classes have a defining feature, which is the concern with leisure lifestyles and the leisure society consumption. the new-found wealth among the growing middle class has been increasingly spent on leisure items and tourism is an element of this (e.g. in 1911, 1 per cent of the population had 70 per cent of wealth; this dropped to 40 per cent in 1960 and 23 per cent in 2002 in the uk). the international growth in holidaytaking is directly related to this new middle class. the increasing mobility of this group has been reflected in a massive growth globally in their propensity to travel and the growth of a society focused on leisure, of which tourism is prioritized as a key element of their household budgets and as a form of conspicuous consumption as the following statistics suggest: • factors promoting these changes include cheaper air fares and changing patterns of personal expenditure. this snapshot of the uk shows that tourism is a major element of the leisure spending of households and tourist travel to the uk is a major driver of the economy. the growing significance of travel and tourism in the household spending reflects what researchers have described as 'leisure lifestyles'. interest in tourism in europe, north america and other parts of the world has been given an added boost by the impact of new technology such as the internet and the worldwide web, which has rendered knowledge and awareness of tourism and the opportunities to travel worldwide more accessible. the worldwide web has been used as a medium to portray travel options and the product offerings of destinations, so that people can search and explore travel options at a global scale from the ease of a computer terminal. in europe, the impact of this new technology in the early years of the twenty-first century has generated a new tourism boom akin to the rise in international tourism in the 1970s, with new forms of technology and the supply of cheaper forms of travel (i.e. the low-cost airlines) fostering this demand. over 90 per cent of some low cost airline bookings are now made online which illustrates the power of the internet and its role in reaching a new customer base in the tourism sector. this has given rise to the rise of e-tourism, which is the digitization 1 of all elements in the tourism supply chain 1 , whereby the supply and demand for tourism can be met through new virtual forms of distribution such as the worldwide web, as opposed to conventional methods such as travel agents and paper brochures. this has certainly revolutionized tourism and the access to travel knowledge and information, hitherto largely within the confines of travel agents and travel organizers: now everyone can be their own travel agent if they have access to the technology. other commentators have also pointed to the changing sophistication of tourists as consumers, especially the middle classes with their pursuit of authentic and unique experiences. this is part of what pine and gilmore (1999) identified as the experience economy which is the next stage in the evolution of society from a service economy. they argue that businesses need to create experiences which create a sensation, can personalize the experience to build a relationship with the consumer and they suggest four areas of experience that we need to focus on: • entertainment • education • esthetic (i.e. an ability to immerse oneself in something) and • escapism in what is consumed. this has major implications for the types of tourism experience we develop now and in the future and it has gained momentum with the growth of the internet that now allows consumers to seek out these experiences globally. the internet e-tourism is only the first stage of the internet's impact upon tourism. the first wave of internet technology created an online travel community where tourism businesses were able to market and communicate with consumers through electronic media. this has been followed by a new wave of web-based communities known as web 2.0 (also described as computergenerated media or social media) where the online content is created by online users and made available to other users via the web 2.0 interactive technology. the importance of this technology is that it allows consumers to communicate about social themes such as holidays and travel. so the increasing use of the internet to make bookings and reservations for travel online has been combined with consumer ratings and reviews online through travel sites such as tripadvisor.com. therefore, many of the previous principles of travel planning, where the advice and knowledge of travel agents was seen as a key determinant of holiday decision-making have now been replaced by the technological power of the internet. access to and use of internet technology is increasing and one important feature which many studies confirm is that this technology is increasingly used to search out and peruse travel options as well as for making bookings. with these issues in mind, attention now turns to what is meant by the terms 'tourism', 'tourist' and 'travel'. attempts to define tourism are numerous and very often the terms 'travel' and 'tourism' are used interchangeably. according to the international organization responsible for tourism, the world tourism organization (un-wto): this seemingly straightforward definition has created a great deal of debate. in fact, controversy has surrounded the development of acceptable definitions since the league of nations' attempt to define a tourist in 1937 and subsequent attempts by the united nations conference in 1963 which considered definitions proposed by the then iuoto (now un-wto). there have also been attempts to clarify what is meant by the term 'visitor' as opposed to 'tourist' and the distinction between tourists who travel within their own country (domestic tourists) and those who travel to other countries (international tourists). what the debates on defining tourism at a technical level show is that it is far from an easy task in agreeing what constitutes a 'tourist'. for example, should we include someone who is a visitor staying in a second home?: they are technically away from their home, but are staying in another form of property they own. similarly, how far away from your home area must you travel before your activity is deemed tourism? a further problem is associated with the category of cruise ship passengers who dock at a port and visit briefly, not staying overnight, or cross-channel trippers who may cross an international boundary but then return within a day and do not stay overnight. to try and encompass many of these anomalies and problems, the un-wto produced guidelines and a useful categorization for defining a tourist, which is shown in figure 1 .1. what is increasingly obvious is that new forms of research on tourism are needed to understand how the phenomenon loosely defined as tourism is evolving as it is far from static. for example, research on tourism and migration has identified the short-term migration of the elderly who winter in warmer climatessuch as the uk pensioners who overwinter in the mediterranean -as a new type of tourist. these patterns of tourism migration incorporate owners of second homes, tourists and seasonal visitors who spend two to six months overseas in locations such as tuscany, malta and spain. for example, 328 000 people own a second home in the uk and 178 000 tourism is defined as the activities of persons travelling to and staying in places outside their usual environment for not more than one consecutive year for leisure, business and other purposes not related to the exercise of an activity remunerated from within the place visited. the use of this broad concept makes it possible to identify tourism between countries as well as tourism within a country. 'tourism' refers to all activities of visitors, including both 'tourists (overnight visitors)' and 'same-day visitors'. (www.world-tourism.org) 1 have purchased overseas properties. in the usa, estimates of domestic second-home ownership range between 3.6 million and 9.2 million properties, the majority of which are located in coastal or rural areas. this pattern of seasonal tourism and migration also generates flows of people known as 'visiting friends and relatives', and these are somewhat different to the conventional images of package holidaymakers destined for these locations in europe. in the usa, a long-established trend of a family vacation is the holiday home. some commentators also suggest that existing definitions of tourism are dated and are being challenged by new forms of tourism such as students engaging in a year abroad. therefore, the following definition of tourism might be useful where tourism is in the usa, there is a tendency still to use the term 'travel' when in fact 'tourism' is meant. what is clear is that tourism is associated with three specific issues: • 'the movement of people • a sector of the economy or an industry • a broad system of interacting relationships of people, their needs [sic] to travel outside their communities and services that attempt to respond to these needs by supplying products'. chadwick, 1994) the field of research on human and business activities associated with one or more aspects of the temporary movement of persons away from their immediate home communities and daily work environments for business, pleasure and personal reasons (chadwick 1994: 65) . from this initial starting point, one can begin to explore some of the complex issues in arriving at a working definition of the terms 'tourism' and 'tourist'. probably the most useful work to provide an introduction to tourism as a concept and the relationship with travel is burkart and medlik's (1981) seminal study tourism: past, present and future. this identified the following characteristics associated with tourism: • tourism arises from the movement of people to and their stay in various destinations • there are two elements in all tourism: the journey to the destination and the stay including activities at the destination • the journey and the stay take place outside the normal place of residence and work, so that tourism gives rise to activities that are distinct from those of the resident and working populations of the places through which tourists travel and in which they stay • the movement to destinations is of a temporary, short-term character, with intention to return within a few days, weeks or months • destinations are visited for purposes other than the taking up of permanent residence or of employment remunerated from within the places visited. source: burkart and medlik (1981: 42) all tourism includes some travel but not all travel is tourism, while the temporary and short-term nature of most tourist trips distinguishes it from migration. but how does tourism fit together -in other words how can we understand the disparate elements? one approach is to look at tourism as an integrated system, which means that one has to ask how tourism is organized and what the defining features are. the most widely used framework is that developed by leiper (1990 -see hall and page, 2010 for a posthumous review of his work) who identified a tourism system as comprising a tourist, a traveller-generating region, tourism destination regions, transit routes for tourists travelling between generating and destination areas, and the travel and tourism industry (e.g. accommodation, transport, the firms and organizations supplying services and products to tourists). this is illustrated in figure 1 .2 and shows that transport forms an integral part of the tourism system, connecting the tourist-generating and destination regions together. thus, a 'tourism system' is a framework which enables one to understand the overall process of tourist travel from both the supplier and purchaser's perspective (known respectively as 'supply' and 'demand') while identifying the organizations which influence and regulate tourism. it also allows one to understand where the links exist between different elements of tourism, from where the tourist interacts with the travel organizer (travel agent or retailer), the travel provider (airline, or mode of transport), the destination area and tourism sector within the destination. this approach is also helpful for understanding how many elements are assembled by 1 the tourism sector to create an experience of tourism. one major element in this experience of tourism is the tour, which is a feature of holidays and the use of leisure time. the tour, holidays, leisure time and the destination what is evident from leiper's model of the tourism system is that the tour -which is a trip or travel anywhere for pleasure, leisure or businessis a vital element. the tour is an underpinning feature of tourism, a prerequisite for tourism to occur -the consumer has to be brought to the product or experience, and has to travel, and it is a reciprocal event -the traveller travels out and back. transport and single or multiple locations are involved. the conventional definition of touring inevitably implies travel to one or more places, called 'destinations'. a destination typically comprises attractions (e.g. natural and man-made), need to be accessible, have available packages to attract visitors, provide ancillary services such as tour guides and have amenities such as accommodation and retailing. this notion of a destination is increasingly being used as a framework for tourism management by public sector organizations to understand how the visitor experience of a place can be developed and enhanced as well as how the synergies between businesses can be developed and the competitiveness of the destination can be improved. for the tourist, there are various forms of touring: the excursion by road or rail which may have a scenic element known as a touring route; some cruises, where the ship tours a range of destinations or ports of call. conversely, the excursion element may be something that the tourist undertakes at the destination on a day-trip basis or in the form of a more sustained trip, with a planned or unplanned itinerary. whilst the holiday is something which encompasses the entire experience or use of leisure time for a holiday, the tour is a distinct element of the holiday and has distinct travel patterns. these patterns contribute to the development of places as destinations which develop and grow through time. some researchers have attempted to explain the growth, stagnation and decline of tourist resorts such as spas in terms of a resort life cycle. the work of butler, published in 1980, suggested that resorts follow a specific cycle page, 1995 ; based on and modified from leiper, 1990) of growth. the initial exploration by tourists is followed by a period of involvement, often with patronage by a royal figure who started a trend towards visitation (e.g. king george iii visiting weymouth in england) or by its wider popularization as a resort for the elite to visit. this set the stage and created tourism tastes and fashions emulated by the visitors. the next stage of butler's model is development, followed by consolidation and then stagnation. at this point, the resort may decline or action may be taken by agents of development (i.e. an entrepreneur, the public sector or a combination of both) to rejuvenate the resort, and this rejuvenation is the last stage of the model. figure 1 .3 illustrates this pattern through time and shows the creation (i.e. birth) and decline (i.e. death) of resorts. although such models are highly generalized and simplify the reality of resort development, they are a starting point for the analyses of resorts such as spas through history. the model has also been used in recent years as a basis to try and understand what point specific destinations are in their life cycle, since the model follows the marketing concept of the product life cycle, where products may have definite or indefinite life courses. the same applies to tourist destinations which can decline when tourist tastes and patterns change and so fall out of favour and require a new focus or attraction to bring the visitors back. in view of these issues, which help to understand the nature of tourism as an entity, attention now turns to the scale, significance and importance of tourism as an international activity. once we agree a general definition of what tourism is, we can look for methods that add precision to the scale, volume and significance of tourism as a global activity. measuring tourism also helps to understand some of the problems which planners and decision-makers need to address in butler, 1980) tourism today: why is it a global phenomenon embracing all our lives? 1 planning for tourism and future growth scenarios. there are three basic considerations in trying to define tourism as an activity, which are: 1 what is the purpose of travel (e.g. business travel, holidaymaking, visits to friends and relatives)? 2 what time dimension is involved in the tourism visit, which requires a minimum and a maximum period of time spent away from the home area and the time spent at the destination? in most cases, this would involve a minimum stay of more than 24 hours away from home and less than a year as a maximum 3 what situations exist where some countries may or may not choose to include travellers, such as cruise passengers, travellers in transit at a particular point of embarkation/departure and excursionists who stay less than 24 hours at a destination, as tourists? there are five main reasons why measuring tourism is important: 1 to understand why and how significant it is for certain destinations, countries and regions in terms of the scale and value of the visitors 2 to understand how important it is for countries in terms of their balance of payments, as it is an invisible export that generates foreign currency and income 3 to assist the tourism industry and governments in planning for and anticipating the type of infrastructure which is required for tourism to grow and prosper 4 to assist in understanding what type of marketing is needed to reach the tourist as a consumer, and what factors will influence tourists to visit a country or destination 5 to help the tourism industry make decisions about what type of action is needed to develop tourism businesses. at a general level, measuring tourism through the collection, analysis and interpretation of statistics is essential to the measurement of the volume, scale, impact and value of tourism at different geographical scales from the global to the country level down to the individual destination. at the simplest level, this is shown in figure 1 .4, which demonstrates the trends in global tourism since 1950 and forecasts to 2020. part of this turbulence, as glaeßer (2006) notes, is the impact of natural catastrophes on tourism. for example, in the twentieth century there have been 50 000 natural disasters but between 1990 and 2005 there have been 500-700 such catastrophes each year. these events have periodically interrupted or at worst devastated the tourism industry (e.g. the earthquake that devastated haiti in 2009), contributing to the notion of turbulence in tourism activity. in other words, a range of factors impact upon visitor arrivals at an international level, because tourism is a very fickle activity (i.e. it is very vulnerable to the external factors mentioned above which act as deterrents to travel) and adverse events can act as shock waves which send ripples across the world and impact upon people's willingness to travel for pleasure reasons. this is because tourism needs relative stability for such activity to occur and the vulnerability to shock effects has been described as volatility in tourism demand which reacts very quickly to these crises or shock events such as wars, currency fluctuations and political instability. tourism also responds to very positive factors such as hosting the olympic games which may lead to a sudden change in the volume of visitors. one of the most recent shock events that have impacted on global tourism is the global credit crunch. whilst this has had different types of impacts on various tourism markets (the most substantial on business travel), its continued existence has led to a global decline in visitor arrivals internationally. in addition, in 2008, the effect of the credit crunch was compounded by the outbreak of a global pandemic associated with swine flu (see figure 1 .5) which initially developed in mexico and spread by travellers returning to their home areas or by visiting new areas so that a number of fatalities occurred in the affected countries as shown in figure 1 1,800,000 1 9 5 0 1 9 6 0 1 9 6 5 1 9 7 0 1 9 7 5 1 9 8 0 1 9 8 5 1 9 9 0 1 9 9 5 2 0 0 0 2 0 0 1 2 0 0 2 2 0 0 3 2 0 0 4 2 0 0 5 2 0 0 6 2 0 0 7 2 0 0 8 2 0 0 9 2 0 1 0 2 0 1 5 2 0 2 0 france, spain, the usa, china, italy, uk and germany • more established destinations in north-western europe and the usa have seen slower growth compared to emerging regions such as africa, n. e. asia, eastern europe, s. e. asia and the middle east. what these rates of growth mean for individual destinations can be seen in web case 1.1 which examines vietnam. but one of the enduring problems of tourism statistics are that they are an incomplete source of information because they are often only an estimate of the total pattern of tourism. in addition, such statistics are often dated when they are published because there is a significant time lag in their generation, analysis, presentation and dissemination. this is because many published tourism statistics are derived from sample surveys with the results being weighted or statistically manipulated to derive a measure which is supposedly representative of the real-world situation. hence, many tourism statistics at a country or regional level often state they are estimates of tourism for this reason. in reality, this often means that tourism statistics may be subject to significant errors depending on the size of the sample. the typical problems associated with measuring tourism are as follows: • tourists are a transient and highly mobile population making statistical sampling procedures difficult when trying to ensure statistical accuracy and rigour in methodological terms • interviewing mobile populations such as tourists is often undertaken in a strange environment, typically at ports or points of departure or arrival where there is background noise which may influence responses • other variables such as the weather may affect the responses. source: latham (1989) even where sampling and survey-related problems can be minimized, such tourism statistics have to be treated carefully as they may be influenced by how the tourist was measured and the type of approach used. the main ways of measuring tourists through surveys are as follows: • pre-travel studies of tourists intended travel habits and likely choice of destination (intentional studies) • studies of tourists in transit to provide information on their actual behaviour and plans for the remainder of their holiday or journey (actual and intended studies) • studies of tourists at the destination or at specific tourist attractions and sites, to provide information on their actual behaviour, levels of satisfaction, impacts and future intentions (actual and intended studies) • post-travel studies of tourists on their return journey from their destination or on-site experience, or once they have returned to their place of residence (post-travel measures). such studies can also be used to examine different facets of the tourist as the following three approaches suggest: • measurement of tourist volume, enumerating arrivals, departures and the number of visits and stays • expenditure-based surveys which quantify the value of tourist spending at the destination and during the journey • measurement of the characteristics and features of tourists to construct a profile of the different markets and segments visiting a destination. in the commercial world, tourism data are also collated by organizations that specialize in its collection and analysis including market research companies. tourism consultants may also be commissioned specifically to collect data for feasibility studies of tourism developments or new business opportunities and much of the information remains confidential to the client due to its commercial sensitivity. but in most cases, national governments collate tourism statistics through studies of domestic and international tourism which are then assembled by the un-wto. once we have an understanding of how tourism is measured and collated, then we can begin to think about what the patterns and trends in tourism mean at a global level and what the implications are, particularly in terms of the more critical issues of what forces are affecting tourism as a global activity. new forces affecting tourism -globalization, inequality and the developed and developing world when one looks at the patterns of tourism, and those areas which are growing in terms of international tourism, it is evident that the majority of outbound travellers are from the developed countries of europe and north america, australasia and the new middle class in many developing countries. in some cases, the tourists are travelling to developing countries where the standard of living often means the majority of the population lives at subsistence level or at a much lower standard than the visitor. the contrast in wealth between visitor and host is often very large and it highlights a clear inequality between those who have the disposable income to enjoy the luxury of international and domestic travel and the tourism employees who are working at low wage rates and in low-paid, unskilled jobs. this situation is made worse by the growing impact of globalization. globalization is a process associated with the growth of large international companies and corporations, which control various forms of economic development and production internationally from their host country, making goods and delivering services at a lower cost using low overheads and cheap labour in developing countries. tourism is no exception to this: large multinational hotel chains and tour operators use developing countries and destinations as the basis for their tourist product. in these situations, the economic linkages with the local community are limited, so that low-skill jobs and low economic benefits are traded off against the profits and economic benefits of tourism development being expropriated (i.e. returned) to the country of origin of the multinational firm. in many cases, the weakly developed nature of local economic linkages in developing countries' tourism economies means they are often trapped into such exploitative relationships because they do not have the indigenous capital or entrepreneurs to set up tourism businesses. a lack of education, know-how and power to negotiate with multinationals to new forces affecting tourism maximize the benefits for local people means that tourism can develop as a form of exploitation for such communities. this may mean that rather than importing foodstuffs, such as internationally recognizable brands, to meet the tastes of tourists, local products should be developed to nurture the linkages with the local economy, so local people may benefit. tourists bring their leisure lifestyles with them on holiday and these are increasingly consumptive and conspicuous. their spending power could be harnessed for the benefit of the local economy. a growing problem in many tourism destinations worldwide is that the growth of tourism and expropriation of its profits mean that the environmental resource base which is used to attract tourists (e.g. attractive beaches, wildlife and the cultural and built environment) is not invested in and may be spoilt. more and more, attention is turning to the extent to which tourism is a sustainable economic, social and environmentally based activity. that we should use the environment without conserving it for future generations is one of the central arguments in the sustainable tourism debate. this also raises the issue of inequalities related to tourism; for example, tourist use of local resources required by residents can destroy those resources and environmental quality. this means that local people, governments and international agencies have a responsibility to lobby and take action to ensure that tourism development which occurs in different countries and locations is not only sustainable but seeks to minimize negative impacts as far as possible. it should not marginalize vulnerable groups such as children and the local workforce: the international labour organization (ilo) has estimated that between 10 and 15 per cent of the tourism workforce worldwide comprised children who do not enjoy appropriate standards of labour and employment conditions (see the work of tourism concern at tourismconern.org.uk). among the common human rights abuses which tourism concern have highlighted are: the forced eviction of people to make way for tourism development; environmental damage resulting from tourism which impacts upon the resources people depend upon for their livelihoods; exploitation of tribal people as tourist attractions and poor levels of pay and poor working conditions for employees in the tourism sectors. tourism needs to be developed in an ethical manner so that exploitation is not its hallmark. this is a theme which will be returned to later in the book; at this point it is enough to emphasize that tourism development and activity not only needs to be socially and environmentally responsible, it must be sustainable and long-term rather than short term and exploitative (so that the goose that lays the golden egg is not killed off). the tourism industry needs to work with communities, local bodies and people to ensure that tourism is a win-win activity for everyone and is integrated into the local community rather than just exploiting its local assets. this may require a significant change in emphasis in the way tourism is developed and managed but it is an enduring theme, which is worth highlighting at different points in the book (see box 1.2 for more detail). tourists and tourism businesses have a greater responsibility to ensure that tourism is promoted as an activity which will not only enhance global understanding and interaction between people of different cultures and societies, but which will also promote dialogue, benefits and opportunities for the tourist, the host and the environment. so, in some situations, tourism may be a way of providing the stimulus and means for preserving and conserving endangered species and environments as well as providing benefits beyond those, which normally accrue to the tourism industry. tourism has to operate as a profitable activity, but for its long-term future, mutually beneficial relationships and links between the industry, people and the environment must exist to bring financial and sustainable benefits for all and enhance the reputation and image of tourism as a global phenomenon. this is the underlying basis of the pro-poor tourism lobby. in this way the welfare and benefits of tourism to tourists can also be extended to the host population and help to address many of the global inequalities which exist in the growing globalization of tourism activity as multinational enterprises seek to exercise greater control of the choice and nature of tourism being offered to consumers. although this book will not be able to address all of these issues, it is hoped that they will be at the forefront of the reader's mind so that they are aware of the implications of the tourism industry and its activities at a global, national and local level throughout the book. extreme poverty is a major problem for many developing countries who have a large proportion of their population living a subsistence lifestyle, often existing on less than $1 a day. at the same time, many of these countries have seen their tourism economies expand as tourists seek new destinations and governments embrace the expansion of this activity to generate foreign revenue. a considerable body of research from consultants and academics has arisen on how this expansion of tourism may be harnessed to address the development problems associated with poverty (see scheyvens, 2007 scheyvens, , 2011 mitchell and ashley, 2009 ). this new thinking has been described as pro-poor tourism which is designed to develop ways to maximize the benefits from tourism to raise local people out of poverty. this involves measures that will: encourage the employment of local people (as opposed to expatriate labour), to provide opportunities for local people to supply goods to tourists and tourism businesses and the creation of micro-enterprises so people can develop their own businesses. however, many obstacles have been identified in implementing pro-poor tourism strategies in less developed countries which include: a lack of awareness and understanding in poorer communities which limits their understanding of the opportunities available; a lack of skills and entrepreneurial talent to capitalize on the opportunities and access to finance to create new businesses focused on tourism as well as cultural concerns over how tourism may affect their way of life. where success stories of pro-poor tourism exist, these examples of best practice need to be shared so that tourism can be harnessed to address abject poverty through case studies of best practice which outline the principles and success factors associated with implementation of such an approach. this is vital if the benefits of tourism development are to be harnessed in the future to address poverty. tourism and management as a focus for the book a framework for the book the title of this book is tourism management and therefore it is useful to present an organizing framework for the book and what is meant by the term 'tourism management'. what is often seen and used as an ambiguous term is the word 'management'. therefore, in this section, the relationship of tourism with management and its meaning in the context of this book is examined. tourism and management as a focus for the book at a very general level, the word 'management' as applied to tourism refers to how tourism needs to be managed as a growing activity at a global, national and local level in order that its often contradictory forces (i.e. the pursuit of profit as a private sector activity and impact on the resource base it uses such as a beautiful coastline on a pacific island) are reconciled and balanced so that tourism develops and is pursued in a sustainable manner. this means there is a need to examine the basic principles associated with the term 'management' and how they can be integrated with tourism as an activity. the basic functions associated with management are: 1 planning, so that goals are set out and the means of achieving the goals are recognized 2 organizing, whereby the work functions are broken down into a series of tasks and linked to some form of structure. these tasks then have to be assigned to individuals 3 leading, which is the method of motivating and influencing staff so that they perform their tasks effectively. this is essential if organizational goals are to be achieved 4 controlling, which is the method by which information is gathered about what has to be done. each of these functions involves decision-making by managers, businesses, tourist destinations or organizations so that they can be harnessed to achieve the objectives and tasks associated with managing tourism. the word 'organization' is often used as an all-embracing term to refer to the type of tourism entity which is involved with tourism as a business or other level. these businesses are motivated by their involvement in tourism to make a profit and, therefore, the efficient organization and management of their activities are essential to ensure that company or organizational objectives are met. there is a school of management thought which argues that management only occurs when chaos occurs and that the function of management is to impose order and structure on that chaos. within organizations dealing with the tourism sector (e.g. travel agents, airlines, tour operators and associated businesses), resources are harnessed (e.g. employees, finance, capital, technology, equipment and knowledge) to provide an output, which in the case of tourism is normally a product or experience consumed by the tourist or service. this output is achieved through the management of the resources. managing tourism demand and supply: the perennial management challenge for tourism organizations one critical element of that management process is related to the way in which businesses have to address the following issues: • what should we produce as a business to meet a certain form of tourism demand? (i.e. should we produce an upmarket high-cost holiday package for ecotourists using tailor-made packages or aim for mass market, low-cost package holidays?) • how should it be produced? (i.e. should we contract in supplies to provide each element of the package product to reduce costs or should we produce each element to ensure quality control and consistency in product delivery?) • when, where and how should we produce the tourism product? (i.e. do we produce an all-year-round or seasonal tourism product?) • what destinations/places should be featured in the tourism experience? • what form of business or businesses do we need to produce the tourism services and products so that we meet demand? tourism businesses need to address these issues for their long-term viability and success or failure will depend upon the management of their organizations' resources to meet demand by consumers in an efficient and profitable manner. it is the concept of supply (i.e. what a business produces) which helps us to understand how the wide range of tourism businesses and organizations (and quite often businesses which do not see themselves as servicing tourists' needs such as taxi companies) combine to link the tourist with the services, experiences and products they seek in a destination. sessa (1983) categorized the supply of tourism services by businesses as follows: • tourism resources, comprising both the natural and human resources of an area • general and tourism infrastructure, which includes the transport and telecommunications infrastructure • receptive facilities, which receive visitors, including accommodation, food and beverage establishments and apartments/condominiums • entertainment and sports facilities, which provide a focus for tourists' activities • tourism reception services, including travel agencies, tourist offices, car hire companies, guides, interpreters and visitor managers. these 'elements of tourism' which combine at a destination highlight the scope of tourism supply, but a number of less tangible elements of supply (i.e. the destination image) also need to be considered. the business environment in which businesses operate can also have a major bearing on tourism supply. for example, in most countries tourism operates within a free market economy, and individual businesses operate in open competition. however, in some countries certain sectors of the tourism industry receive assistance from government through infrastructure provision, marketing and promotional support from tourist boards and other agencies. it is also apparent that when governments decide to promote inbound tourism to destinations (also see further web reading 1). the competitive environment which affects tourism businesses and their operation needs to be considered in relation to a number of underlying economic issues: • what competitive market conditions exist for a specific sector of tourism (i.e. the airline sector, hotel sector or attraction sector)? do conditions of monopoly, oligopoly (i.e. where a limited number of suppliers control supply) or other market conditions exist? • how many businesses are involved in these markets? what size are they? are they able to respond quickly to new competitive pressures, or are they characterized by complacency and an inability to redefine their operations in the light of aggressive competition? • do the businesses involved in tourism display patterns of market concentration, where a limited number of businesses dominate all aspects of production (i.e. from retailing through to supply of services and products in the destination such as in the uk tour operator market)? • what are the capital costs of entering a tourism market? are there high entry and exit barriers? for example, starting an airline has high entry and exit costs, requires a high level of technical know-how and large capital investment and ongoing finance to service the business. buying a guesthouse, on the other hand, has low entry costs and no barriers to entry in terms of technical competencies to be able to run and manage it and host visitors • what types of products already exist in the market? is there scope for innovation to develop new products without the risk of 'ambush marketing' by competitors who copy the idea and undercut the competition by loss-leaders to regain market share? aggressive marketing and a limited number of loss-leaders have characterized the low-cost airlines and privatized railways in the uk in an attempt by their owners to capture price-sensitive leisure travellers. in other words, is there scope for price discrimination in the market to differentiate a whole range of products? what these factors indicate is that the market conditions and business environment in which tourism operates are far from static. they are constantly changing, requiring businesses to adapt and to develop strategies to retain their market presence. for tourism businesses, recognizing these evolving patterns, new trends and the need for innovation (i.e. new ideas and products) to address market conditions re-emphasizes the importance of managerial skills in the supply of tourism products and services. this also highlights what mintzberg (1973) identified as the nature of managerial work in organizations -short-term coping, disparate activities and more concerned with brevity, variety and increasing fragmentation. tourism managers and businesses are no exception to this and mintzberg's research has an important bearing on how managers performed certain roles (see table 1 .4) labelled as interpersonal, informational and decisional roles. the ten managerial work roles which mintzberg identified illustrate the scope of activities 1 which operating and managing a tourism business require, as well as some of the complexities of how the individual business interacts with the wider body of interests conveniently labelled the 'tourism industry'. it also suggests how important prevailing market conditions are when they impact upon how a business operates, manages and responds to opportunities, threats and shortcomings in its own organization. yet to do this, a business needs also to understand its relationship to other tourism businesses. a convenient way to explain this is by using the tourism supply chain concept. as tourism is an amalgam of different interests, activities, stakeholders and businesses, the supply chain concept helps us to understand how different interests are functionally linked together to form a distinct method of service delivery. the supply chain concept originates in economics and has been used to explain how different businesses enter into contractual relationships to supply services, products and goods, and how these goods are assembled into products at different points in the supply chain. tourism is well suited to the concept of the supply chain because the product, service or experience that is consumed is assembled and comprises a wide range of suppliers. all too often our knowledge of the supply chain is quite restrictive, since a wide range of components are consumed in tourism including the use of bars, restaurants, handicrafts, food, infrastructure and related services. a schematic diagram of a typical tourism supply chain is shown in figure 1 .6. this shows that once the consumer has chosen a destination and product, the decision to purchase involves contacting a tourism retailer (e.g. a retail agent, a direct selling company or an internet-based seller such as www.expedia.co.uk). having chosen a booking medium and selected a package from a tour operator, the package is then assembled. the tour operator enters into contractual relationships with tourism suppliers such as airlines (although larger tour operators may also own their own charter or schedule airline), hotel operators and suppliers of associated services such as airport transfers. these suppliers, in turn, contract suppliers who service their business needs: in-flight caterers, airline leasing companies, airport terminal services (i.e. check-in services, baggage handling, flight controllers, customer service agents for visitors and those with special needs, such as the disabled). with so many organizations involved in the supply chain in relation to tourist spending and activity, it is clear that these are critical break or pressure points where the service provision could potentially fall down (see figure 1.7) . the business strategies, that travel companies can pursue to develop their supply of tourism services and products include: • focusing on core business (i.e. a holiday company focusing on selling holidays rather than being vertically integrated and operating its own airline and hotels) • seeking to diversify its products. the leading french holiday company club mediterranée (club med), which traditionally sold packages to its 120 holiday resorts, has used this strategy. since 1999 and its acquisition of jet tours (france's fourth ranked tour operator, which tourism today: why is it a global phenomenon embracing all our lives? 1 operated to 113 summer and 81 winter locations) it has diversified its operations to sell non-club med packages. rewe in germany has pursued a similar diversification strategy with its acquisition of a wider range of tour operating businesses in the long-and short-haul market • choosing to operate in all segments of the tourism market. tui has adopted this tactic and others such as kuoni are moving towards that goal • non-holiday companies may choose to enter the market: easyjet entered the cruise holiday business in 2005. to implement these business strategies, companies in the tourism industry have adopted marketing-related concepts such as branding to differentiate their products in an increasingly competitive marketplace. for example, club med relaunched its worldwide image to re-emphasize its famous name and association with consumers, and particularly its dominant position in the french market. thomas cook, now owned by the german company c&n touriste, has used its global image and historic association with pioneering tourism to continue its expansion throughout europe (see figures 1.8-1.10 ). there is also a debate among tourism researchers who argue that tourism is a unique sector in that it displays characteristics of partial industrialization which are explained more fully by leiper (1990: 25) where only certain organizations providing goods and services directly to tourists are in the tourism industry. the proportion of (a) goods and services stemming from that industry to (b) total goods and services used by tourists can be termed the index of industrialization, theoretically ranging from 100 per cent (wholly industrialized) to zero (tourists present and spending money, but no tourism industry). 1 what leiper's approach to the tourism sector shows is that managing the broad phenomenon called 'tourism' is complex for a number of reasons: • the tourism industry is not a homogenous sector or segment of the economy: it is made up of various organizations directly involved in tourism (i.e. those which directly service tourist needs) and those indirectly involved and so may be described as allied industries (i.e. food suppliers, retailers and other service providers) • some of the organizations directly involved in tourism are responsible for encouraging and promoting tourism development and marketing • the allied industries do not always see themselves as tourism-related enterprises • the destination or area which the tourists visit is not the sole responsibility of one business or group of businesses; usually the public sector intervenes to ensure that business objectives (i.e. profit and increasing 1 tourism numbers and revenue) are balanced with local needs and business interests (known as 'stakeholder interests') in relation to the resource base which tourism utilizes (i.e. beaches, attractions, the infrastructure and overall environment) • the public sector is responsible for trying to liaise, plan and manage these diverse group of interests that are associated with tourism as a phenomenon as well as having an underlying responsibility in many cases for the marketing and promotion of the destination. therefore, one can see how complex the management of tourism is when the interests and variety of organizations involved in tourism are considered and then the concept of partial industrialization is introduced. from this discussion, who is responsible for tourism management can be examined at a number of levels, although this is not an exclusive list but a range of illustrations: • at the individual business level, the manager(s) is (are) involved with the functioning and running of the enterprise • at the destination level, responsibility often lies with a public sector led agency such as a tourism department (either as a stand-alone body or as part of a local authority department). in extreme situations where a destination is deluged with tourists due to its popularity, the public sector may lead with a public-private sector partnership involving business interests to manage the visitors on the ground • at the country level, it is the national tourism organizations, funded by the public sector through taxes and sometimes with private sector members, who promote and market the country as a place to visit and attempt to manage the diverse interests involved in tourism • at each level, be it the individual business, destination or country, a complex web of interactions and interrelationships exist which need to be taken into account in the decisions, interests and actions taken to manage tourism. in each of these illustrations, the functions of management are harnessed. tourism management as a pursuit, however, is further complicated in that there is a great debate as to what tourism is, what needs to be managed and who should be responsible. the fact that tourism can be seen as an experience based on the pursuit of pleasure and profit raises many complex issues such as whether the tourist is consuming a product, experience or service, and it leads to many debates on what to manage and how far management controls should be exercised by the tourism industry and public sector. so how does this book address these questions? one way is to view the managerial process of tourism as a multilayered process, in which the various organizations and stakeholders involved in tourism engage at different levels through time. figure 1 .11 demonstrates this. the focus begins with the individual business and the management processes (controlling, planning, leading and organizing) are continuous through the interconnected stakeholder groups from the individual business through to the various interests known as the tourism industry. these interests and the connections between management at different levels and between groups mean that, in reality, these groups also have to be aware of external factors that will impact upon management such as the visitor, the business environment, consumer trends, the growth of the leisure society and political processes affecting tourism at government level. the book is organized in such a way that these issues are explained in a manner where the links between different elements of the tourism sector are addressed through examples and case studies. each chapter builds upon the one preceding to develop the knowledge and understanding of what the tourism industry is, the management challenges facing each sector and how tourism affects changes in different contexts. accommodating, anticipating and responding to that level of change is among the major challenges for tourism management in the new millennium. englishmen at rest and play the tourist movement the englishman's holiday the travel trade tourism, principles and practice tourism volumes 1-6: sage library of tourism and hospitality management the consumer society: myths and structures tourism, past present and future the concept of the tourist area cycle of evolution: implications for the evolution of resources (eds) travel, tourism and hospitality research: a handbook for managers and researchers crisis management in the tourism industry the contribution of neil leiper to tourism studies the statistical measurement of tourism tourism systems: an interdisciplinary perspective the nature of managerial work tourism and poverty reduction: pathways to prosperity the experience economy exploring the tourism-poverty nexus tourism and poverty elements of tourism. rome: catal aspects of service dominant logic and its implications for tourism management further reading questions 1 why is tourism such an important activity in the twenty-first century? 2 how would you classify tourists? 3 why is tourism management important for a business operating in the tourism sector? 4 how stable is tourism as an economic activity? key: cord-257749-eyhsc8q8 authors: koul, bhupendra; taak, pooja; kumar, arvind; kumar, anil; sanyal, indraneel title: genus psoralea: a review of the traditional and modern uses, phytochemistry and pharmacology date: 2019-03-25 journal: j ethnopharmacol doi: 10.1016/j.jep.2018.11.036 sha: doc_id: 257749 cord_uid: eyhsc8q8 ethnopharmacological relevance: the genus psoralea (fabaceae) harbours 105 accepted species that are extensively used by local peoples and medicinal practitioners of china, india, and other countries for treatment of tooth decay, psoriasis, leucoderma, leprosy, kidney problems, tuberculosis, indigestion, constipation and impotence. presently, pharmacological research reports are available on only few species namely bituminaria bituminosa (syn: p. bituminosa), p. canescens, p. corylifolia, p. esculenta, p. plicata and p. glandulosa which are valued for their chemical constituents and traditional uses. aim of the review: this review article provides explicit information on traditional uses, phytochemistry, and pharmacological activities of selected psoralea species. the possible trends and perspectives for future research on these plants are also discussed. materials and methods: an extensive and systematic review of the extant literature was carried out, and the data under various sections were identified using a computerized bibliographic search via the pubmed, web of science and google scholar, cab abstracts, medline, embase, inmedplan, natts as well as several websites. key findings: a total of 291 bioactive compounds from 06 species of genus psoralea have been isolated and characterized. however, p. bituminosa alone possess nearly 150 compounds. these bioactive compounds belong to different chemical classes, including flavonoids, coumarins, furanocoumarins, chalcones, quinines, terpenoids and some others due to which these species exhibit significant anti-oxidant, anti-bacterial, anti-fungal, anti-viral, anti-helmintic, anti-diabetic, diuretic, hepatoprotective, anti-cancer and anti-tumor activities. p. corylifolia l. (babchi), a chinese traditional medicinal plant has been used in traditional medicine for many decades for its healing properties against numerous skin diseases such as leprosy, psoriasis and leucoderma. conclusions: the in vitro studies and in vivo models have provided a simple bio-scientific justification for various ethnopharmacological uses of psoralea species. from the toxicological perspective, the root, leaf, and seed extracts and their preparations have been proven to be safe when consumed in the recommended doses. but, meticulous studies on the pharmaceutical standardization, mode of action of the active constituents, and sustainable conservation of psoralea species are needed, to meet the growing demands of the pharmaceutical industries, and to fully exploit their preventive and therapeutic potentials. the medicinal herbs have been a major source of biodynamic compounds of therapeutic value in ayurveda, unani, homeopathy, traditional chinese medicine (tcm) and other traditional system of medicines. several dreadful diseases including cancer, aids, kidney psoralea species have been used in folklore and indigenous system of medicine for a long time. several psoralea products are successfully commercialized and available in the markets (supplemental information 2, table s2 ). the roots of p. argophylla pursh (silver leaf scurf pea, silver leaf indian breadroot) are eaten raw or cooked (tanaka, 1976; yanovsky, 1936) . the dried roots may be ground into a powder and used an ingredient of soups and bread (yanovsky, 1936) . a tea prepared from the leaf and stem powder possess anti-pyretic properties (weiner, 1990) . a decoction of the plant is used as a wash for wounds (moerman, 1998) . the root extract is used as a remedy for chronic constipation (moerman, 1998) . bituminaria bituminosa (l.) c.h.stirt. (arabian pea or pitch trefoil) is used as a forage crop. psoralea canescens michx. (buckroot) roots are eaten raw or cooked (hedrick, 1972; tanaka, 1976; yanovsky, 1936) . the powdered roots are used in preparation of soups or breads (yanovsky, 1936) . as the plant has analgesic properties, a poultice prepared from roots is applied on painful areas of the body (moerman, 1998 ). an infusion of the roots and its steam is used for the treatment of cold, cough, headache and sore throat (moerman, 1998) . psoralea castorea s. watson (beaver indian breadroot) roots are also eaten raw or cooked and the root-powder can be used in soups or breads (hedrick, 1972; tanaka, 1976; uphof, 1968; yanovsky, 1936) . psoralea corylifolia l. (bu gu zhi) is revered in chinese traditional medicine as a tonic to improve general vitality. the name 'buguzhi' (fructus psoraleae) actually comprises of three chinese words: 'bu' means 'to invigorate'; 'gu' means 'bone' and the third word 'zhi' means 'fat'. the chinese name of the herb suggests the function of the herb to provide fat for the invigorating bones. one of the most important features of p. corylifolia is that each and every part of the plant is beneficial which includes roots, stems, leaves, seeds and even blooms (hodges, 2015) . since p. corylifolia is a leprosy destroyer it is revered to as "kushtanashini" in sanskrit. moreove, it is an ancient remedy for leucoderma among the traditional system of medicines in india and china and also among the people in the west (chopra and chopra, 1958 ). in unani system, the plant has been effective against fever, skin diseases and internal ulcers (chopra and chatterjee, 1927) . it is also found to be an effective antihelmintic and sedative (nadkarni, 1976) . for treating leprosy and leucoderma the leaves are consumed as powder and also apllied on skin in the form of paste (anon, 1998; nadkarni, 1976; panda, 2000) . however, some precaution should be taken when applying the herbalpaste externally, since it can cause a skin-allergic reaction when exposed to sunlight (anderson and voorhees, 1980) . the leaves are also used to treat dermatitis, inflammation, mucomembranous disorders, oedematous conditions of the skin and to alleviate diarrhea (rajpal, 2005; sharma et al., 2001; krishnamurthi et al., 1969) . the plant possesses blood purifying properties and therefore used to treat boils, itching eruptions or red papules, ringworm-infection, extensive eczema, rough and discolored dermatosis with fissures, and scabies (khare, 2004) . the essential oil from plant is reported to have a strong effect on streptococcal infection of the skin (rajpal, 2005) . moreover, it is known to improve the color of skin, hair and nails. seeds are sweet, bitter, acrid, and astringent. the seeds are anti-pyretic and also possess alexiteric properties and therefore are given in scorpion-sting or snake bite and in bilious disorders (agharkar, 1991; kapoor and boca, 2001; nadkarni, 1976; panda, 2000) . both the seeds and fruits contain psoralen (furocoumarin), known to regulate pigmentation (rashid and agarwala, 1965; sebastian, 2006) . p. corylifolia seed extracts have been reported to possess anti-hyperglycemic, antidepressant, anti-tumor, anti-bacterial and anti-oxidant property (steven and russell, 2007; wang et al., 1990) . seed extract and powder are beneficial as anti-helmintic, laxative, diuretic, and for healing wounds (rajpal, 2005) . they are used as stomachic, diaphoretic and aphrodisiac . the major components psoralen (92) and isopsoralen (2) are known to possess anti-bacterial, anti-viral and antitumor properties (liu et al., 2004) . therefore, the seeds are used for curing various disorders such as cough, asthma, nephritis, alopecia areata, menstruation, uterine disorders and haemorrhages (qiao et al., 2007; ruan et al., 2007) . the crude extracts of seeds are used in the treatment of febrile diseases, impotence, spermatorrhea, premature ejaculation, lower back pains, incontinence, enuresis, pollakiuria, and cold symptoms in the waist and knees (chopra et al., 1956; lin et al., 2007; zhao et al., 2005a zhao et al., , 2005b . it also possesses coronary vasodilatory activity . the seeds act as deobstruent and heal ulcers, heart troubles, cure blood disorders and elephantiasis (anonymous, 1969; drury, 1873) . the extracts also possess cytotoxic, anti-mutagenic and anti-repellant properties . the seeds are also used to make perfumed oil (gupta et al., 1979; nadkarni, 1976) . in japan, the ethanol extract of the seeds has been used as a preservative for pickles and some processed foods (qiao et al., 2007) . the seed cake is rich in nitrogen and minerals and is used as cattle feed or manure . the fruits of the p. corylifolia also have valuable medicinal uses. the seeds or the seed with the seed pod, possess high aphrodisiac properties allo-aromadendrene (1) u (azzouzi et al., 2014a (azzouzi et al., , 2014b bertoli et al., 2004; innocenti et al., 1998; khatune et al., 2004; pazos-navarro et al., 2011; pistelli et al., 2003; tava et al., 2007) [ reference(s) psoralea canescensmichx. angelicin (isopsoralen) (2) n (innocenti et al., 1997) [ and bhakuni, 2010; wang et al., 2014; won et al., 2015; wu et al., 2007 a, b; wu et al., 2008; xiao et al., 2012; xu et al., 2012; yadava and verma, 2003; yadava and verma, 2005; yang et al., 2006; yang et al., 2009; yin et al., 2004; yu et al., 2005; zhang et al., 2016; zhao et al., 2005a) bakuchicin ( xanthoangelol ( (christensen et al., 2008; kaye and moodie, 1978; perara, reese, 2002; stahnke et al., 2008) [ leaf, flower, seed angelicin (isopsoralen) (2) n (arfaoui et al., 2013; cheikh et al., 2015; el-abgy et al., 2012; hamed et al., 1997 hamed et al., , 1999 khatune et al., 2004; menon et al., 1999; rasool et al., 1989 rasool et al., , 1990 1991; youssef et al., 2013) [ (backhouse et al., 2001; labbe et al., 1996; li et al., 2016; madrid et al., 2012 madrid et al., , 2013 madrid et al., , 2015a madrid et al., , 2015b bakuchiol ( psoralen ( and are used as a tonic to strengthen the genital organs (ven, 1987) . the fruits are bitter in taste, can prevent vomiting, cure difficulty in micturition, cure piles, bronchitis and anaemia and are known to improve complexion (joshi, 2000) . moreover, fruit extract inhibits the growth of mycobacterium tuberculosis (yeung, 1985) . the roots and seeds of p. corylifolia are used for preventing tooth decay (duke, 2009 ). they also promote bone calcification, and hence are beneficial for treating bone fractures and osteoporosis (joshi, 2000; krishnamurthi et al., 1969; rabie, 2010a, 2010b) . psoralea esculenta pursh. (breadroot, large indian breadroot) roots are starchy (70% starch), glutinous (9% protein) with a sweetish turniplike taste (5% sugars) and are eaten raw or cooked (hedrick, 1972; saunders, 2011; tanaka, 1976; uphof, 1968; yanovsky, 1936) . the dried roots are pulverized and used with cereals in making cakes and porridge (facciola, 1983) . a poultice prepared from the crushed roots is applied to sprains and fractures. an infusion of the dried roots is used in the treatment of sore throats, gastro-enteritis and chest problems. the roots are chewed by children as a treatment for bowel complaints (moerman, 1998) . psoralea glandulosa l. is cultivated in chile for its leaves and young shoots, which are used to make a refreshing cold drink. the leaves are anti-helmintic. psoralea hypogaea torr. & a. gray (small indian breadroot) roots are eaten raw or cooked (elias and dykeman, 2009; harrington, 1974; hedrick, 1972; moerman, 1998; yanovsky, 1936) . the roots are rich in starch and can be ground into a powder and used in soups or with cereals for making bread (yanovsky, 1936) . the roots were used an important source of food for the native north american indians (diggs et al., 1999) . psoralea macrostachya dc. (large leather root) roots are eaten raw or cooked and may be dried for winter use. moreover, the plant has been used in the treatment of ulcers and sores (moerman, 1998) . a strong fibre is obtained from the inner bark of the stem (saunders, 2011; usher, 1974) . a fibre is also obtained from the roots, which is used to make ropes and bags (moerman, 1998; uphof, 1968) . roots are aromatic and the perfume persists for several months (saunders, 2011) . a yellow dye is also obtained from the roots (moerman, 1998) . psoralea orbicularis lindl. (roundleaf leather root) leaves are cooked and eaten (moerman, 1998; tanaka, 1976; yanovsky, 1936) . a decoction of the root is used to purify blood and to treat prexia (moerman, 1998) . the plant is a good soil stabilizer (huxley, 1992) . psoralea pedunculata (mill.) vail (sampson's snakeroot) is used as a bitter tonic. psoralea species have been investigated since 1890s (dymock et al., 1893) . leaf, rhizome, root, seeds, fruit and resinous extracts of psoralea species have been subjected to hplc and hptlc followed by pharmacological analyses (pandey et al., 2012; shailajan et al., 2012; uikey et al., 2010; won et al., 2015; yin et al., 2015; zhao et al., 2005a) . detailed and extensive chemical investigation of six psoralea species viz: p. bituminosa l. (syn: bituminaria bituminosa), p. canescens michx., p. corylifolia l., p. esculenta pursh, p. plicata delile and p. glandulosa l. led to the characterization of a large number of bioactive constituents. review of literature reveals the presence of altogether 291 chemical constituents including volatile compounds in the aforementioned species (table 1) . these chemical compounds have been categorized into coumarins, furanocoumarins, flavonoids (polyphenols), isoflavones, meroterpenes, chalcones, phenols, phenolic cinnamates, phenylpropene, sterols, terpenes, tocopherols, benzofurans, sesquiterpenes, acids, fatty acids, alkyl aldehydes, alcohols and esters (agarwal et al., 2006; bertoli et al., 2004; hsu et al., 2001; qiao et al., 2006; ruan et al., 2007; tava et al., 2007; yin et al., 2006 yin et al., , 2007 yu et al., 2005) . the chemical structures of 1-291 compounds are shown in the supplemental information (supplemental information 3, fig. s1 ). their chemical names, chemical class and the corresponding plant sources are compiled in table 1 . the structures of those bioactive compounds which are representative of the genus and with reported pharmacological activities are presented in the main text (fig. 2) . recently, an isoflavone synthase (ifs) gene has been isolated and functionally characterized from p. corylifolia (misra et al., 2010) . the active principle found in p. corylifolia is bakuchiol (155) and psoralen (92) (dev, 1999; wang et al., 2009) . psoralen is linear in structure and may be called as a derivative of umbelliferone (jois et al., 1933; rangari and agrawal, 1992) . the production of psoralen enhances when the plant is exposed low dose of gamma radiation (jan et al., 2011) . isopsoralen (2) is a structural isomer of psoralen (92), and it was found to be identical to angelicin (2) (jois et al., 1933; jois and manjunath, 1934; seshadri and venkata rao, 1937) . angelicin (isopsoralen) (2) is a photosensitizing agent and used for determination of dna and rna structures in cells and microorganisms (kittler et al., 1980) . angelicin (2) and its derivatives occur in a number of plants belonging to the family umbelliferae. the active compound, bakuchiol (155) is a monoterpene phenol, has been obtained in a pure state and named after sanskrit name of the plant (mehta et al., 1973) and possess the potent anti-bacterial property (satyavati et al., 1987) . the seed contains volatile oils, monoterpenes, flavones, coumarins, stigmasteroids, resins, lipid compounds and phenols. the volatile oils include limonene (64), linalool (65), β-caryophyllene (126), geranyl acetate (199) and terpinen-4-ol (97), coumarin derivatives include psoralen (92), isopsoralen (2), isopsoralidin (209), corylidin (179) (211). lipids include triglycerides, diglycerides and monoglycerides. monoterpene phenol includes bakuchiol (155). others include free fatty acids, stigmasterol (232), triacontane (233), daucosterol (192), glucose and saponin. the fatty acids obtained from oil were found to be primarily palmitic acid (86) and linoleic acid (66) together with small fraction of linolenic acid (67). the pharmacologically active oil is identical with the unsaponifiable oil isolated by earlier workers (gaind et al., 1965; gupta et al., 1962) . more that 188 chemical constituents (belonging to furanocoumarins, coumestrol group, chalcones and flavones) have been reported from the seeds chakrovarti et al., 1948; chen et al., 2005; satyavati et al., 1987; tsai et al., 1996) . psoralea species have received tremendous attention because of their bioactive principles possessing remarkable pharmaceutical properties ( fig. 3 ; supplemental information 4, table s3 ). several protocols have been followed to analyse the anti-oxidant property of seeds and leaf extracts of p. bituminosa, p. glandulosa, p. corylifolia, p. plicata, p. esculenta and p. glandulosa. the phenolic compounds obtained from different extracts were found to protect the biological membranes from oxidative stresses (haraguchi et al., 2002; guo et al., 2005; borchardt et al., 2008; kim et al., 2013; madrid et al., 2013; huang et al., 2014) . the antioxidant activity of fruit extracts of psoralea plicata was evaluated by dpph assay. the study indicated that the methanol extract shows an anti-oxidant activity (288.32 micromol trolox equivalent/100 g dry material) which is slightly higher as compare to aqueous extract (258,65μmol trolox equivalent/100 g dry material) (cheikh et al., 2015) . in a study on p. corylifolia, several bioactive compounds such as bakuchiol (155), psoralen (92), isopsoralen (2), corylin (187), corylifolin (185) and psoralidin (228) were screened for their anti-oxidant potential. their antioxidant activities were investigated individually and compared with butylated hydroxytoluene (bht) and α-tocopherol by the oxidative stability instrument (osi) at 100ºc among these compounds psoralidin exhibited highest anti-oxidant activity (5.23) than that of standard compounds (psoralidin > bht > α-tocopherol > bakuchiol > corylifolin > corylin > isopsoralen~psoralen) (jiangning et al., 2005) . however, antioxidant activity alone cannot be of any pharmacological significance. it is accompanied by other specific pharmacological activities to claim any therapeutic benefit. there are several reports on the anti-bacterial activitiy of p. bituminosa and p. corylifolia. the ethanol and methanol extracts obtained from the aerial parts of p. bituminosa contain flavones and isoflavones, while the seed extracts of p. corylifolia contain psoralidin (228), bakuchicin (154), psoralen (92) and angelicin (2), which have shown significant anti-bacterial activities. techniques such as disc-diffusion method, uv (ultraviolet), h nmr (proton nuclear magnetic resonance), c nmr (carbon nuclear magnetic resonance), anti-bacterial assay, broth-dilution method, column chromatography, hplc (high performance liquid chromatography) and tlc (thin layer chromatography) were used to quantitate and analyse the anti-bacterial property of these natural compounds (azzouzi et al., 2014a (azzouzi et al., , 2014b bhawna et al., 2013; cui et al., 2015) . bakuchiol obtained from seed extract of p. corylifolia has been reported to inhibit the growth of staphylococcus mutans and actinomycess viscosus and hence possess strong anti-bacterial activity and mic value of bakuchiol was found to be 9.76-19.5 µg/ml (khushboo et al., 2010; rao et al., 2011) . in a study, the compounds psoralidin (228), bakuchicin (154), psoralen (92) and angelicin (2) obtained from different extracts of the plant were found to show antibacterial activity against different gram-positive and gram-negative bacteria. among them, psoralidin (228) showed highest anti-bacterial activity against shigella sonnei and s. flexneri as depicted by disc diffusion assay . 6.3. anti-fungal activity p. corylifolia and p. glandulosa are known to possess significant antifungal activity. aqueous and methanol extract of seeds and petroleum ether extract of the aerial parts p. corylifolia have been tested against various seed borne fungi such as (alternaria, cladosporium, dreschslera and rhizophus spp.) of maize. in aqueous extract, maximum inhibition (95.4% inhibition at 50% concentration) was observed against a. alternata followed by c. lunata (86.0%), rhizopus sp. (82.3%), d. halodes (68.0%) and c. cladosporioides (57.7%). among the solvents used for the preparation of extracts, maximum inhibition was observed with petroleum ether extract and moderate activity was observed with methanol extract (kiran et al., 2011) . in an anti-fungal assay, the essential oil extracted from p. corylifolia, was studied against three dermatophytic fungi microsporum canis, trichophyton rubrum and trichophyton mentagrophytes. the zone of inhibition (by disc-diffusion assay) for m. canis, t. rubrum and t. mentagrophytes was found to be 20, 35 and 37 mm respectively, while the minimum inhibitory concentration was reported to be 1.4, 0.4 and 0.5 μl/ml, respectively (sharma and tiwari, 2012) . in another study, methanol extract of p. corylifolia seeds was found to be most effective against tomato late blight (phytophthora infestans) and wheat leaf rust (puccinia recondite) (shim et al., 2009) . the crude extract of p. corylifolia exhibited significant antifungal activity against candida albicans . in a study by borate et al. (2014) , methanol extract of p. corylifolia seeds was reported to exhibit a maximum zone of inhibition against c. albicans the crude ethanol extract of the seeds of p. corylifolia exhibited significant anti-viral activity against the severe acute respiratory syndrome corona virus (sars-cov) papain-like protease (plpro). the ic50 value for the same was 15 μg/ml. sars-cov-plpro is the enzyme that is crucial for replication of sars virus . in a recent study, it was reported that bakuchiol (155) present in seeds of p. corylifolia inhibited the influenza a viral infection and growth and activated the nuclear factor erythroid 2-related factor (nrf2) pathway. this pathway is responsible for cellular defense against electrophilic or oxidative stress (shoji et al., 2015) . bakuchiol (155) extracted (petroleum ether extract, dichloromethane extract, and methanol extract) from the aerial parts of p. glandulosa inhibits degranulation in human neutrophils and decreases the cell migration, eicosanoid levels and myeloperoxidase activity in mice thus, confirming its anti-inflammatory potential (ferrándiz et al., 1996; backhouse et al., 2001) . in another study, bakuchiol (155) from p. corylifolia was reported to inhibit the expression of inducible nitric oxide synthase (nos) gene through the inactivation of nuclear transcription factor-b in raw 264.7 macrophages (pae et al., 2001) . the bioactive compounds obtained from leaves, fruits and seeds of p. corylifolia inhibit the functioning of tumor necrosis factor-alpha (tnf-α) and exhibt anti-inflammatory activity (mueller et al., 2010) . the production of inflammatory mediators such as, reactive oxygen species (ros), reactive nitrogen species (rns), and cytokines such as il-1β, il-6 and tnf-α (tumor necrosis factor) in pma (phorbol 12-myristate 13acetate)/lps (lipopolysaccharide), in ifn-stimulated murine peritoneal macrophage cell line (raw 264.7) was inhibited by neobavaisoflavone (212) (szliszka et al., 2011a) . cytotoxicity of neobavaisoflavone (212) was tested by using ldh assay (lactate dehydrogenase assay). psoralidin (228) suppresses the activity of pro-inflammatory cytokines which regulate pulmonary inflammation in human lung-fibroblasts and in mice, by ionizing radiation, has been reported (yang et al., 2011) . in a recent study the activity of il-6-induced stat3 (signal transducer and activator of transcription 3) was found to be inhibited by bavachin (159), bakuchiol, bavachinin (160), corylin (187), corylifol a (180), neobavaisoflavone (212), and isobavachalcone (203). hence, these bioactive compounds hold promise to cure inflammatory diseases . in a study by newton et al. (2002) , forty-three plant species were screened for their anti-mycobacterial activities. among them, bakuchiol (155) extracted from p. corylifolia hexane seed-extract exhibited significant anti-bacterial activity (mic = 31.25 g/ml) against mycobacterium aurum and m. smegmatis thus, confirming its potential for treatment of leprosy. however, these studies further require in vitro and in vivo analyses for their large-scale utilization . in a study by dwarampudi et al. (2012) , the ethanolic seed extract of p. corylifolia showed an ic50 value of 255 μg/ml and a considerable anti-psoriatic activity (75.87%), using the mouse tail model. the seed extract converted parakeratosis stage (keratinization) to orthokeratosis (formation of anuclear keratin layer) stage of the cell, thus confirming its anti-psoriatic potential (dwarampudi et al., 2012) . in a recent report, different micro-emulsions containing single and both commiphora mukul powder and babchi-oil from p. corylifolia were used to assess the anti-psoriatic efficacy on diseased rat paw. the synergistic effect of both the natural products gave better results, hence this herbal combination could be a cheap and effective source of anti-psoriatic agent (marwaha, 2013) . in a study involving human subjects, p. corylifolia hexane-seeds extract was prepared into a cream using stearic acid followed by an open clinical trial on thirty patients suffering from eczema, for a period of one month. the placebo preparation, for this experiment contained all the ingredients except the seed extract. the parameters studied were length of the lesion, exudation rate and rate of itching. the symptoms score reduced after two weeks of cream application. finally, the length of the lesion reduced from 6.367 ± 1.098-0.333 ± .279, exudation rate reduced from 1.333 ± .994-0.165 ± .087 and the rate of itching reduced from 2.567 ± .504-0.165 ± .132. this study concluded that p. corylifolia seed extracts could be used effectively used for the treatment of eczema (gidwani et al., 2010a). the compounds obtained from the p. corylifolia extract have played an influential role for the treatment of vitiligo. the furocoumarins, psoralen (92) and isopsoralen (2) present in psoralea species assists the skin to produce new pigment. they initiate the transformation of dihydroxyphenylalanine (dopa) to melanin when exposed to sunlight and are therefore being used for treating vitiligo, psoriasis and leprosy (fitzpatrick and pathak, 1959; hussain et al., 2016; song et al., 2004; yin et al., 2004) . psoralen (92) alone can effectively treat psoriasis and alopecia areata (ji and xu, 1995; wang and wang, 2007) . in a report by abu tahir et al. (2010), human melanocytes were used to test the antivitiligo activity of the oleo-resinous extracts of the seeds. the seed extract acted as an effective anti-vitiligo agent by restoring the melanocytes of the affected area (abu tahir et al., 2010). ethanol seed extract of p. corylifolia when administrated orally to streptozotocin-nicotinamide (stz) induced diabetic rats lead to an increase in glycogen content of liver and insulin level in plasma with a decrease in plasma cholesterol and blood glucose level (kamboj et al., 2011) . in a report by bera et al. (2013) , the aqueous seed extract of p. corylifolia was found to regulate the functioning of insulin sensitive enzyme activities in wistar strain male albino rats. the seed extract tends to increase the activity of liver hexokinase, glucose-6-phosphate and decrease the level of glucose-6-phosphatase (bera et al., 2013) . the furanocoumarins psoralen (92) and isopsoralen (2) obtained from the seed extract of p. corylifolia have showed anti-depressant activity in mice by hindering the mao (monoamine oxidase) activity, hypothalamic-pituitary-adrenal axis action and oxidative stress . another compound, psoralidin (228) inhibits the transcription of crf (corticotrophin releasing factor) gene, which is responsible for stress response . the anti-depressant activity of psoralidin (228) was evaluated by applying the forced swimming test on rats . psoralen was reported to successfully change the level of 5-hydroxyindoleacetic acid (5-hiaa) and serotonin (5-ht) in hippocampus and frontal cortex of mice . bakuchiol (155) decreases the immobilization time in behavioral despair mouse and plasma epinephrine and norepinephrine (neurotransmitter) levels in bovine adrenal medullary cells hence, exhibits anti-depressant activity . these results depict the anti-depressant activity of psoralen and bakuchiol. psoralea species possess unique bioactive compounds with anticancer properties. p. corylifolia leaves contain remarkably high concentrations (more than 2 g per kg dry weight) of genistein (198), the anti-cancer metabolite. bioactivity of two furocoumarins psoralen (92) and isopsoralen (2) was determined for their cytotoxicity on carcinoma lines kb, kbv200 (vincristine resistance subline of kb), human erythroleukemia cell k562 and k562/adm (doxorubicin resistance subline of k562). both the compounds induced apoptosis in these cells thus, confirming their anti-cancer potential. the ic50 values of psoralen were 88.1, 86.6, 24.4 and 62.6, which of isopsoralen were 61.9, 49.4, 49.6 and 72.0, respectively (wang et al., 2011) . psolaren (92) when subjected to human hepatocarcinoma cells, showed its inhibitory activity by inducing the mechanism of apoptosis. psoralen (92) was able to inhibit the growth of smmc-7721 cells in a dose-and timedependent manner and had a strong proapoptotic effect on these cells (jiang and xiong, 2014; khan et al., 2015) . the bioactive compounds 7,2 ' ,4 ' -trihydroxy-3-arylcoumarin (263), psoracoumestan (224) and corylifol c (182) from p. corylifolia showed strong anti-cancer potential by inhibiting the enzyme mapk/erk kinase phosphorylation and inducing aptotic cell death . in a study, psoralidin (228) showed its activity in mcf-7 cancer cells (isolated from human breast) by induction of ps2 gene activity, with an ec50 value of ere-reporter gene transcription activity of 1.85 μm . the seed extract of p. corylifolia induced apoptosis in the human breast cancer (mcf-7) cells followed by mitochondrial cell death (rajan et al., 2014) . in another study, psoralidin (228) was reported to generate reactive oxygen species and also inhibited a549 cell proliferation. the method adopted was mtt assay and the ic50 values obtained after 24-, 48-and 72-h treatment were 19.2, 15.4, and 11.8 μm, respectively . psoralidin (228) and neobavaisoflavone (212) in combination with trail (tumor necrosis factor-related apoptosis-inducing ligand) have showed their anti-cancer property by inducing apoptosis in lncap (human adinocarcinoma prostate cancer cells) (szliszka et al., 2011a) . in another report, psoralidin (228) in combination with trail influenced apoptosis in hela cells by increasing the expression of death receptor (trail-r2) (bronikowska et al., 2012) . in a similar report, psoralidin (228) compound obtained from different extracts of p. corylifolia exhibited the anti-cancer activitiy against human lung cancer (a549) cells. psoralidin dramatically decreased the cell viabilities in dose and time-dependent manner . psoralea fructus suppressed the proliferation of human colorectal cancer cell lines, such as lovo (ic50: 23.3 ± 1.9 μg/ml), sw480 (ic50: 37.9 ± 1.6 μg/ml), ht-29 (ic50 value: 40.7 ± 1.5 μg/ml), and hct116 (ic50: 45.3 ± 1.2 μg/ml) by decreasing the protein expression of cyclin d1 and cdk4. succeeding experiments with many kinase inhibitors revealed that p. fructus-mediated degradation of cyclin d1 and cdk4 is dependent on gsk3β and/or erk1/2 (park et al., 2016) . psoralen (92), isoporalen (2) and bakuchiol (155) identified in p. corylifolia extracts (chloroform and ethanol) have shown their cytotoxic property against hep-2 cell line (whelan and ryan, 2003) , bgc-823 cancer cell (guo et al., 2003) and cultured human cancer cells (sk-ov-3, a549, xf498 and sk-mel-2hct15) (ryu et al., 1992) . bakuchiol (155) is one of the active ingredients of the dried ripe fruit of p. corylifolia. in a study by miao et al., 2013, bakuchiol (155) suppressed the testosterone induced cell proliferation and gene expression in androgen-dependent prostate cancer (pca) cell line (lncap). the ic50 of bakuchiol (155) to androgen receptor was 8.87 × 10 4 , which was similar to the standard flutamide (10.00 × 10 4 ). bakuchiol (155) has also showed a strong anti-cancer action against human lung adenocarcinoma cell line a549 and showed better results than its analogue resveratrol. ic50 of bakuchiol (155) at 72 h was 9.58 ± 1.12 μmol/l, much lower than that of resveratrol (33.02 ± 2.35 μmol/l). compared to resveratrol, bakuchiol (155) triggered the process of apoptosis to a higher level. it was also observed that oxygen species mediated apoptosis contributes to the cytotoxic properties of bakuchiol (155) and can therefore be used against non-small-cell lung cancer . invitro experiments with a2058 melanoma cells using p. glandulosa resinous exudates revealed that it can inhibit the growth of cancer cells after 48 h of treatment. these experiments confirmed the anti-cancer potential of p. glandulosa which can be attributed to the presence of bakuchiol, 3-hydroxy-bakuchiol and 12-hydroxy-isobakuchiol (madrid et al., 2015b) . ethanol, methanol, chloroform and aqueous seed extracts of p. corylifolia were tested against the tumor cells of mice and were found to stimulate the antibody complement-mediated cytotoxicity during tumor development (latha et al., 2000) . anti-tumor property of bakuchiol (155) was also compared with resveratrol and it was observed that bakuchiol was more efficient in inhibiting the growth of tumor cells, when tested against human lung adeno-carcinoma a549 cell line . in a similar study on tumor cells of murine origin, radioiodinated bakuchiol showed greater cytotoxic effect than bakuchiol (bapat et al., 2005) . bakuchiol (155) also induced erβ expression and suppressed the erα expression in mcf-7 cells (breast cancer cells). it also caused the arrest of s-phase in mcf-7 and mda-mb 231 cells and showed a stronger anti-proliferative effect than resveratrol. additionally, bakuchiol (155) caused the apoptotic cell induction and interrupted membrane potential in mitochondria of mcf-7 cells via an intrinsic apoptotic pathway. the same compound when tested for in vivo anti-breast cancer effect in zebrafish xenografts showed a significant reduction in mcf-7 cell mass (li et al., 2017) . similarly, two more compounds from the same species identified as isobavachalcone (203) and bavachinin (160) attenuate aβ42-induced cell toxicity. the investigation was carried out on yeast two-hybrid system. during this study, eight compounds were tested, and among them ibc (3 μm) and bcn (30 μm) proved to be active at non-toxic concentrations . this is a significant property which is associated with the p. corylifolia fruit extract, the osteoblastic proliferation in cultured umr106 (osteosarcoma) cell line and enhancing the formation of bones . in several studies, p. corylifolia extracts showed significant inhibitory effect on osteoclasts (yang et al., 2007; zhang et al., 1995) . corylin (187) and bavachin/coryfilolin (159) were reported to promote the proliferation of osteoblasts and inhibit bone resorption . bakuchiol (155) compound in the extracts showed inhibitory effect on osteoporosis, mediated through estrogen deficiency (lim et al., 2009; tsai et al., 2007) . in a report by tsai et al. (2007) , it was observed that p. corylifolia extract lead to a decrease in calcium and osteocalcin through urinary excretion in ovariectomized (ovx) rat model and thus, maintained the bone density (tsai et al., 2007) . in a similar report, ethanolic bakuchiol present in seed extract of p. corylifolia reduced the bone loss in ovx rat model thus, showed promising anti-osteoporosis activity (lim et al., 2009 ). the ethanolic fruit extract of p. corylifolia showed estrogenic activity, which was demonstrated by transcription of lacz in recombinant yeast system . in another report by zhao et al. (2007) , proliferation rates of mcf-7 cells increased significantly when treated with the p. corylifolia extract (zhao et al., 2007) . in a study by lim et al. (2011) bakuchiol (155) showed a higher estrogenic activity and estrogen receptor (er) binding affinity than genistein, both in vitro and in vivo (lim et al., 2009 . among the seven bioactive components of p. corylifolia extract exhibited isobavachalcone (203), bavachin (159), corylifol a (180), neobavaisoflavone (212), bakuchiol (155), and two coumarins psoralen (92) and isopsoralen (2) compounds selectively activated er-α while the other compounds activated both er-α and er-β (xin et al., 2010) . park et al. (2012) , reported that bavachin (159) could bind and activate the estrogen receptor . in addition to it, psoralidin (228) was reported as aganist for both er-α and er-β . in a similar study by xin et al. (2010) , seed extract of p. corylifolia containing psoralen (92) and isopsoralen (2) enhanced the mcf7 cell prolification by enhancing the activity of erα (estrogen receptors), hence showed significant estrogenic activity. 6.14. hepatoprotective activity p. corylifolia is revered for its hepatoprotective potential (dai et al., 2009; ye et al., 1999) . three compounds psoralen (92), bakuchicin (153) and bakuchiol (155) (ec50 values of 1.0, 47.0, and 50.0 μg/ml, respectively) have shown hepatoprotective activity towards the liver cells (hep g2 cells) in which the cytotoxicity was induced by tacrine (cholinesterase inhibitor) . in a similar study, bakuchiol (155) reduced the toxic activity of carbon tetrachloride (ccl 4 ), d-galactosamine (d-gain), and tert-butylhydroperoxide (tbh) in primary rat hepatocytes (park et al., 2005) . the activity of hepatic stellate cells (involved in liver fibrosis) was reduced by bakuchiol in liver injured rats thus, confirming its hepatoprotective activity (park et al., 2007) . in a recent report, bakuchiol (155) obtained from the seed extract of the p. corylifolia exhibited hepatoprotective activity by inhibiting the production of reactive oxygen species (ros) and malfunctioning of the mitochondria in human diploid fibroblast (hdf) (seo et al., 2013) . in a study, the cultured rat pheochromocytoma (pc12) cells pretreated with p. corylifolia l. seed extract significantly attenuated 3-np induced cell death, reduced atp levels, and lowered the mitochondrial membrane potential. thus, p. corylifolia seed extract has the potential to treat neurodegenerative diseases (im et al., 2014) . in another study, it was revealed that isobavachalcone, a flavonoid from p. corylifolia, has the ability to ameliorate the neuronal injury in brain diseases related to inflammation, and this was accomplished through inhibition of the expression of lipopolysaccharide induced intercellular adhesion molecule-1 and leukocyte adhesion to brain endothelial cell, by blocking toll-like receptor 4 signaling . 6.16. immunomodulatory activity p. corylifolia extract effectively increased the proliferation rate of diploid fibroblasts (in mice) and increased the ability of non-specific immunity (wang et al., 1990) . in the study, a mixture of fruit extracts of p. corylifolia and brucea javanica improved the immunological regulation in rats that were infected with pneumocystis carinii pneumonia. as a result of the treatment, gain in body weight, decline in the number of cysts produced in the lungs and the level of t cells (cd4 + and cd8 + ) and tnf-alpha in the serum also increased considerably in the immunosuppressed rats (qin et al., 2006) . in another study, the ethanol seed-extracts of p. corylifolia stimulated the immune system in mice by increasing both cell-mediated and humoral immune responses in mice (latha et al., 2000) . 6.17. anti-asthma activity p. corylifolia has long been used for its anti-asthmatic properties. experiments have shown that coumarins isolated from p. corylifolia exhibits anti-asthmatic activity by markedly increasing the level of serum camp (deng et al., 2001; yu et al., 2006) . in another study, a chinese herbal decoction, which contained six herbs, along with p. corylifolia seeds, could prompt treatment for asthma in the convalescent stage, to prevent emphysema (fu, 1989) . in a study by hongfen (2007) , the preparations obtained from the p. corylifolia showed high antiasthma activity by stabilization of mast cells and inhibition of histamine release. thus, the natural compounds obtained from p. corylifolia can act as promising molecules to cure asthma. several studies on animal models showed that a trihydroxyflavone, genistein (198) has the potential to decrease body weight by decreasing the food intake. one such study was conducted on ovariectomised mice wherein, it reduced the fat pad weight and enhanced the apoptosis of adipose tissues. genistein (198) isolated from p. corylifolia, exhibited a potential anti-obesity and ant-diabetic activity through multiple mechanisms and cell signaling pathways by the action on adipocyte life cycle, obesity-related low-grade inflammation, and oxidative stress . in a report, the aqueous and alcohol extracts of the leaves and seeds of p. corylifolia showed significant anti-filarial activity against setaria cervi. alcohol extracts of both leaves and seeds caused death of microfilariae in vitro. the lc50 and lc90 for alcohol extract of leaves and seed was 15, 25 ng/ml and 12, 18 ng/ml respectively (qamaruddin et al., 2002) . the extracts also caused the inhibition of spontaneous movements of the whole worm and the nerve muscle preparation of s. cervi (qamaruddin et al., 2002) . the methanolic p. corylifolia seed extracts was observed to inhibit the aggregation of rabbit platelets induced by arachidonic acid, collagen, and platelet activating factors. the anti-platelet aggregation activity of isobavachalcone was found to be most effective against aggregation induced by arachidonic acid, with a 50% inhibitory concentration (ic50) of about 0.5 µm (tsai et al., 1996) . using rabbit as an animal model, and escherichia coli endotoxin (13 ng/kg) as a pyrogen, the petroleum ether, dichloromethane, and methanol extracts of the aerial parts of p. glandulosa have been reported to possess anti-pyretic activity. the anti-pyretic effect (21.7%) of the petroleum ether extract was reported because of bakuchiol compound present in the extract. maximum anti-pyretic effect (68%) was observed at the dose of 17 mg/kg (backhouse et al., 2001) . isobavachalcone (203) and bavachinin (160) from psoralea modulate amyloid β (aβ) peptides, especially the peptides with 40 (aβ40) or 42 (aβ42) residues, which are believed to be responsible for the development of amyloid plaques in alzheimer's disease. isobavachalcone significantly inhibits both oligomerization and fibrillarization of aβ42, whereas, bavachinin converts aβ42 into large unstructured aggregates in neuroblastoma cells . psoralen (92) isolated from p. corylifolia fruits was investigated as an inhibitor of ache enzyme in an attempt to explore its potential for the management of alzheimer's disease. the concentration of psoralen used was 25-400 μg/ml. it inhibited the ache in a dose-dependent manner in animal models. adult male wistar rats, weighing 180-250 g, were used in the study. while molecular docking study was also carried out, which showed that psoralen (92) binds well within the binding site of the enzyme showing interactions such as hydrogen bonding and π-π stacking . these findings may provide valuable information for the synthesis of new drugs or for the treatment of alzheimer's disease. although having excellent bioactivities, excess intake or use of psoralea is not free from side-effects. p. fascicularis (synonym: psoralea tenuifolia thunb.) has been reported to be toxic to horses, cattle and therefore is not recommended as a fodder. there have been reports on skin allergic reactions after the oral and injected preparations of psoralea (bensky et al., 2004) . in over-dose, psoralea has been associated with dizziness, general weakness, blurred vision, rapid breathing, and vomiting. severe cases of overdose have been associated with vomiting of blood, loss of consciousness, and coma (bensky et al., 2004; chen and chen, 2004) . psoralen plus uva (puva) therapy has been used to treat skin diseases, such as vitiligo and psoriasis. the reported side-effects include dermatitis, blistering, edema, renal complications, loose-motions, biliousness, malaise, sleeplessness, annoyance and mental depression. prolonged therapy has reported to affect liver, eyes, and the immune system (rajpal, 2005) . the psoralen (92) treatment along with uva can cumulatively cause extensive chromosome damage to mammalian cells and could lead to malignancy. a mixture of psoralen (92), isopsoralen (2), and imperatorin caused hypertrophy of liver, kidney, and spleen in rats at a daily dose of 2.5 mg/75 g for 60 days (sharma, 2001) . in a similar study, different concentrations of p. corylifolia extracts were administered to the rats for 90 days. the treatment decreased the body weight and gonad weight (testes and ovaries) thus indicating psoraleninduced reproductive toxicity (takizawa et al., 2002) . there are several reports on hepatotoxicity symptoms in mice and rats after long-term usage of psoralen or isopsoralen (khushboo et al., 2010; totonchy and chiu, 2014; wang et al., 2012) . in an investigation, p. corylifolia and its natural compounds (bavachin, corylifol a, neobavaisoflavone, ibc, and bcn) were evaluated for their potential toxicity, and the results showed it had a potent inhibitory effect against human udp-glucuronosyltransferase 1a1 (ugt1a1) which is considered a stimulant for p. corylifolia related toxicity, including hepatic injury and raised bilirubin levels . in another study, p. corylifolia extract and fractionated compounds such as psoralen (92) and isopsoralen (2) were incubated with the recombinant cyp3a4 enzyme or differentiated huh-7 and heparg cells. p. corylifolia extract, psoralen, and isopsoralen caused the inhibition of concentration cyp3a4 activity in a dose dependent manner with different potency in vitro. it was also noted that none of the sample tested showed any toxicity (liu and flynn, 2015) . three cases of liver injury have been reported in chinese population associated with consumption of p. corylifolia dried seeds (fructus psoraleae) (cheung et al., 2009) . pregnant women are discouraged from consuming p. corylifolia (bensky et al., 2004; chen et al., 2007) . in a case study, a 44-year-old female ingested p. corylifolia seeds every 1 h for seven weeks for treating osteoporosis but, developed acute cholestatic hepatitis (nam et al., 2005) . in another case study, a 64year-old female developed severe hepatotoxicity after administration for nine months of three kinds of herbal tablets and herbal tea for treating vitiligo. tablets made from p. corylifolia leaves were identified as the most probable cause of the hepatotoxicity (teschke and bahre, 2009; teschke et al., 2014) . however, the underlying hepatotoxic mechanisms of p. corylifolia and its major components are not well elucidated. a text on traditional chinese medicine reports that no adverse effects of psoralea occur when a normal dose range (decoction of 4.5-9.0 g) is consumed (bensky et al., 2004) . in all the case-studies/ experiments on psoralea induced toxicity one thing was found common and that is purposeful/deliberate and continuous/prolonged intake/ application of a high dose. going through the available literature we can conclude that there are mixed opinions about the psoralea induced toxicity and therefore there is still a research gap with regard to its mode of action and its pharmaceutical standardization. as already mentioned the genus psoralea has immense potential to cure various diseases and the most important among these are psoriasis, leprosy and vitiligo. although pre-clinical studies have shown promising results, further studies are required to explore the in-depth molecular mechanisms responsible for the afore-mentioned pharmacological activities and to test the efficacy of isolated compounds, in properly designed experiments. in addition, long term toxicity studies and data on interaction with other drugs are also required to establish the safety profile of extracts before the commencement of clinical trials. the efficiency and efficacy of the bioactive compounds present in the psoralea species has been evaluated from time to time. now, since there is awareness complemented with scientific proof regarding the medicinal benefits, their mass cultivation/propagation and extraction of bioactive compounds should also be the objective of further research. the commercialization of pharmaceutical drugs containing psoralea as a sole or a part of the ingredients shall bring relief to millions of people suffering from psoriasis, leprosy and vitiligo, in a natural way. looking into the available literature on the genus psoralea, including the discovery of the number of bioactive compounds from each species (as mentioned in table 1 ) it is very clear that p. corylifolia is an important member of the genus from ethnobotanical, ethnopharmacological, biological, and phytochemical point of view. several bioactive compounds isolated from psoralea are commercially available. the compounds such as daidzin (cas 552-66-9) is a potent inhibitor of human mitochondrial aldehyde dehydrogenase that indicates it chemopreventive property (keung and vallee, 1993) ; angelicin (cas 523-50-2) is an antifungal compound (sardari et al., 2000) ; 8-methoxypsoralen (cas 298-81-7) has been known as a potent suicide inhibitor of cytochrome p-450 (tinel et al., 1987) ; corylifol c and xanthoangelol are potent protein kinase inhibitors and induce apoptotic cell death therefore, possess anti-cancer property ; bakuchiol (cas 10309-37-2) is a ptp1b and dna polymerase inhibitor (choi et al., 2015) ; genistein (cas 446-72-0) is a highly specific inhibitor of protein tyrosine kinase (zarmouh et al., 2016) ; psoralen has been used as photochemical probe in dna mutation and repair studies (gasparro, 1988) ; and bavachin (cas 19879-32-4) stimulates bone formation (anti-osteoporotic activity) xin et al., 2010) . these examples indicate the utility of bioactive compounds isolated from psoralea species. psoralea species are difficult to propagate because of poor seed-germination and high seedling-mortality (mitter et al., 1993; saxena et al., 1997; siva et al., 2014) . to ensure sustained production and benefits of psoralea products we must aim at its mass cultivation through conventional approaches as well as micropropagation (koul and chase, 2015) . psoralea guenzii harv. has become extinct while species like p. asarina (p.j.bergius) t.m.salter, p. fascicularis dc. and p. glaucina harv. are vulnerable to extinction. their bioactive principles and mode of action has not been explored yet. their conservation would also ensure an alternative source of continuous and sustainable supply of elite bioactive compounds such as bakuchiol, psoralen, angelicin and psoralidin, for the pharmaceutical industry (supplemental information 5, table s4 ). although, reproducible or rapid regeneration protocols for all the psoralea species are not yet developed, but the same can ensure the enhancement of bioactive compound(s) production in near future, through suspension culture or transgenic technology (arya and gothalwal, 2015) . these techniques may also streamline the path for further research on the bioactive principles and for the discovery of new compounds with novel properties in future. this is the first report wherein a detailed botanical description, traditional uses, phytochemistry and conservation of psoralea species has been discussed. to conclude, psoralea species have immense potential to act as panacea to several health-related maladies and so its conservation before excessive exploitation should be a prerequisite. current remedies for vitiligo isolation of chalcones from the seeds of psoralea corylifolia linn medicinal plants of bombay presidency psoralea corylifolia l: ethnobotanical, biological, and chemical aspects: a review antipsoriatic microemulsion gel formulations for topical drug delivery of babchi oil (psoralea corylifolia) psoralen photochemotherapy of cutaneous disorders the wealth of india, a dictionary of indian raw materials and industrial products. csir phytopharmacological assessment of medicinal properties of psoralea corylifolia. afr experimental and density functional theory study of a new dimer with tetra-substituted cyclobutane ring system isolated from psoralea plicata seeds phytochemical and biological activities of bituminaria bituminosa l (fabaceae). asian pac anti-oxidant potential of n-buoh extract was evaluated through two methods, dpph and cupric ion reducing anti-oxidant capacity assay. asian pac active constituents isolated from psoralea glandulosa l. with anti-inflammatory and anti-pyretic activities a new chromenochalcone bavachromene from the seeds of psoralea corylifolia preparation and in vitro evaluation of radio iodinated bakuchiol as an anti-tumor agent genistein: a promising therapeutic agent for obesity and diabetes treatment chinese herbal medicine: materia medica protective effect of aqueous extract of seed of psoralea corylifolia (somraji) and seed of trigonella foenum-graecum l (methi) in streptozotocin-induced diabetic rat, comparative evaluation volatile constituents of different organs of psoralea bituminosa l some new flavonoids from psoralea corylifolia antimicrobial activity of psoralea corylifolia linn. 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seeds of psoralea corylifolia l. against seed borne fungi of maize cross-link formation of phage lambda dna in situ photochemically induced by the furocoumarin derivative angelicin determination and comparison of mineral elements in traditional chienese herbal formulae at different decoction times used to improve kidney function -chemometric approach bakuchicin a new simple furanocoumarin from psoralea corylifolia moringa oleifera lam, panacea to several maladies the wealth of india bakuchiol derivatives from the leaves of psoralea glandulosa two antifungal components isolated from fructus psoraleae and folium eucalypti globuli by bioassay--guided purification anti-dermatophytic activity of bakuchiol: in vitro mechanistic studies and in vivo tinea pedis-inhibiting activity in a guinea pig model immunomodulatory and anti-tumour properties of psoralea corylifolia seeds biological effects of the herbal plant-derived phytoestrogen bavachin in primary rat chondrocytes phenolic compounds isolated from psoralea corylifolia inhibits il-6-induced stat3 activation osteoblasts proliferation and differentiation stimulating activities of the main components of fructus psoraleae corylifoliae fructus psoraleae contains natural compounds with potent inhibitory effects towards human carboxylesterase 2 mechanistic study of bakuchiol-induced anti-breast cancer stem cell and in vivo antimetastasis effects ethanol extract of psoralea corylifolia l. and its main constituent bakuchiol reduce bone loss in ovariectomised sprague-dawley rats estrogenic activities of psoralea corylifolia l. seed extracts and main constituents compounds isolated from psoralea corylifolia seeds inhibit protein kinase activity and induce apoptotic cell death in mammalian cells analysis of bakuchiol psoralen and angelicin in crude drugs and commercial concentrated products of fructus psoraleae two new benzofuran derivatives corylifonol and isocorylifonol from the seeds of psoralea corylifolia preparative isolation and purification from psoralea corylifolia by high speed counter current chromatography psoralidin, a coumestan analogue, as a novel potent estrogen receptor signaling molecule isolated from psoralea corylifolia cyp3a4 inhibition by psoralea corylifolia and its major components in human recombinant enzyme differentiated human hepatoma huh-7 and heparg cells phytoestrogen bakuchiol exhibits in vitro and in vivo anti-breast cancer effects by inducing s phase arrest and apoptosis anti-fungal study of the resinous exudates and of meroterpenoids isolated from psoralea glandulosa (fabaceae) study of the chemical composition of the resinous exudate isolated from psoralea glandulosa and evaluation of the antioxidant properties of the terpenoids and the resin antiphytopathogenic activity of psoralea glandulosa (fabaceae) against botrytis cinerea and phytophthora cinnamomi psoralea glandulosa as a potential source of anticancer agents for melanoma treatment extraction modelling and characterization of bioactive components from psoralea corylifolia l. obtained by supercritical carbon dioxide cytotoxic constituents of psoralea corylifolia formulation design and evaluation of herbal anti-psoriatic emulgel bioactive constituents from chinese natural medicines xx inhibitors of antigen-induced degranulation in rbl-2h3 cells from the seeds of psoralea corylifolia meroterpenoids-1 psoralea corylifolia linn-1 bakuchiol a novel monoterpene phenol structure-anti-mutagenic activity relationship study of plicatin b characterization of isoflavone synthase gene from psoralea corylifolia, a medicinal plant overcoming hard seededness on psoralea corylifolia native american ethnobotany a case of acute cholestatic hepatitis associated with the seeds of psoralea corylifolia the evaluation of forty-three plant species for in vitro antimycobacterial activities; isolation of active constituents from psoralea corylifolia and sanguinaria canadensis assessment of antifungal activity of bakuchiol on oral-associated candida spp traditional medicinal knowledge about herb bemchi (psoralea corylifolia) in chhatisgarh herbs cultivation and medicinal uses simultaneous separation and quanti-fication of targeted group of compounds in psoralea corylifolia l. using hplc-pda-ms-ms activation of estrogen receptor by bavachin from psoralea corylifolia anti-cancer activity of psoralea fructus through the downregulation of cyclin d1 and cdk4 in human colorectal cancer cells bakuchiol from psoralea corylifolia inhibits the expression of inducible nitric oxide synthase gene via the inactivation of nuclear transcription factor-κb in raw264.7 macrophages micropropagation from apical and nodal segments of bituminaria bituminosa and the furanocoumarin content of propagated plants neo-psoralen isolated from psoralea corylifolia linn nutritional value of psoralea esculenta pterocarpans from bituminaria morisiana and bituminaria bituminosa meroterpenoids ii psoralea corylifolia linn 2 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plicata a benzoquinone and a coumestan from psoralea plicata lucknow and niscir studies on the chemical constituents of psoralea corylifolia l anti-tumor activity of psoralea corylifolia isolation and identification of furocoumarins from the seeds of psoralea corylifolia l estimation of psoralen content by hplc method in different organs of psoralea corylifolia l. as influenced by sulphur and nitrogen nutrition synthesis and anti-fungal activity of coumarins and angular furanocoumarins antifungal activity of diplotaenia damavandica medicinal plants of india edible and useful wild plants of the united states and canada plant regeneration from callus cultures of psoralea corylifolia linn ayurvedic medicine: the principles of traditional practice, part 2 development of an efficient in vitro regeneration protocol for rapid multiplication and genetic improvement of an important endangered medicinal plant psoralea corylifolia protective role of psoralea corylifolia l. seed extract against 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effect of psoralen cooperated with substrates on tyrosinase activation screening anti-tumor compounds psoralen and isopsoralen from psoralea corylifolia l. seeds. evid. based complement a uplc-ms/ms method for in vivo and in vitro pharmacokinetic studies of psoralenoside isopsoralenoside psoralen and isopsoralen from psoralea corylifolia extract a rapid method for the analysis of ten compounds in psoralea corylifolia l. by uplc earth medicine, earth food ethanolic extracts of euphorbia and other ethnobotanical species as inhibitors of tumor cell growth bioactive metabolites from the fruits of psoralea corylifolia effect of buguzhi (psoralea corylifolia fruit) extract on bone formation systemic effect of fructus psoraleae extract on bone in mice phototherapy in psoriasis. a review of mechanisms of action psoralen and isopsoralen two coumarins of psoraleae fructus can alleviate scopolamine-induced amnesia in rats bisbakuchiols a and b novel dimeric meroterpenoids from psoralea corylifolia hypoxiainducible factor-1 and nuclear factor-κb inhibitory meroterpene analogues of bakuchiol a constituent of the seeds of psoralea corylifolia isolation of a new meroterpene and inhibitors of nitric oxide production from psoralea corylifolia fruits guided by tlc bioautography phytoestrogens from psoralea corylifolia reveal estrogen receptor subtype selectivity simultaneous characterization of prenylated flavonoids and isoflavonoids in psoralea corylifolia l. by liquid chromatography with diode-array detection and quadrupole time-of-flight mass spectrometry anti-depressant-like effects of psoralen isolated from the seeds of psoralea corylifolia in the mouse forced swimming test a new biologically active flavonol glycoside from psoralea corylifolia (linn) effect of psoralea corylifolia l. on the proliferation and differentiation of osteoblast isolated from neonatal rat calvarium in vitro two new compounds from psoralea corylifolia l psc-afp an anti-fungal protein with trypsin inhibitor activity from psoralea corylifolia seeds food plants of the north american indians. united states department of agriculture pharmacokinetics of fructus psoraleae in bile elimination. traditional chinese handbook of chinese herbal formulas anti-depressant-like effects of psoralidin isolated from the seeds of psoralea corylifolia in the forced swimming test in mice quality assessment of psoralea fructus by hplc fingerprint coupled with multi-components analysis psoracorylifols a-e, five novel compounds with activity against helicobacter pylori from seeds of psoralea corylifolia. state key lab anti-bacterial prenylflavone derivatives from psoralea corylifolia, and their structure-activity relationship study cyclobakuchiol c, a new bakuchiol derivative from psoralea coryllfolia experimental and density functional theory study of a new dimer with tetrasubstituted cyclobutane ring system isolated from psoralea plicata seeds chalcones from the seeds of psoralea corylifolia effects of total coumarin on cgmp/cgmp of asthmatic rats evaluation of the isoflavone genistein as reversible human monoamine oxidase-a and-b inhibitor in vitro estrogenic activities of chinese medicinal plants traditionally used for the management of menopausal symptoms the effect of psoralea corylifolia on isolated osteoclasts cells the chemical constituents and bioactivities of psoralea corylifolia linn. a review fingerprint analysis of psoralea corylifolia l.by hplc and lc-ms analysis of psoralea corylifolia l. fruits in different regions evaluation on phytoestrogen effects of ten kinds of chinese medicine including flos carthami the authors are thankful to lovely professional university (lpu), punjab, india for the infrastructural support. the authors are also thankful to the center for chromatography and mass spectrometry, industrial university of santander, columbia for awarding the postkey: cord-007084-4niom5mw authors: popplewell, philip y.; butte, john; azhar, salman title: the influence of age on steroidogenic enzyme activities of the rat adrenal gland: enhanced expression of cholesterol side-chain cleavage activity(*) date: 1987-06-01 journal: endocrinology doi: 10.1210/endo-120-6-2521 sha: doc_id: 7084 cord_uid: 4niom5mw the ability of isolated adrenocortical cells to secrete corticosterone in response to acth challenge declines as rats age, but the site or mechanism(s) of this impairment is still unknown. to test the functionality of steroidogenic capacity per se, we measured the key enzyme activities involved in corticosterone biosynthesis. we also measured the mitochondrial cytochrome p-450 content and nonsteroidogenic enzymes specific for subcellular fractions. mitochondria and microsomal fractions were isolated from the adrenals of 2-, 12-, and 18month-old animals and used for various enzyme measurements. mitochondrial side-chain cleavage enzyme activity (nanomoles per min mg protein(-1)) increased from a mean of 0.43 ± 0.06 in 2-month-old rats to 1.26 ± 0.11 and 1.51 ± 0.06 in 12and 18-month old rats, respectively. after incubation with 5cholesten-3j8,25-diol (25-hydroxycholesterol; 25 μg/ml) sidechain cleave activity rose to 5.0 ± 0.6, 12.4 ± 1.2, and 16 ± 1.4 nmol min(-1) mg protein(-1) in adrenal mitochondrial fractions from 2-, 12-, and 18-month-old rats, respectively. in contrast, mitochondrial cytochrome p-450 content did not vary with advancing age. microsomal δ(5)-3β-hyroxysteroid dehydrogenase-δ(5)-δ(4)isomerase activities were similar in 2and 12-month-old rats, but 21-hydroxylase (nanomoles per min mg protein(-1)) activity was significantly increased in 12-month-old rats (2-month-old, 5.2 ± 0.2; 12-month-old, 7.7 ± 0.5). finally, mitochondrial 11βhydroxylase was comparable in both age groups. in addition, activities of mitochondrial nonsteroidogenic enzymes, such as monoamine oxidase, amytal insensitive nadh cytochrome c reductase, cytochrome c oxidase, succinate dehydrogenase, and malate dehydrogenase, did not change with age. it appears from the evidence presented that the activities of the steroidogenic enzymes are not responsible for the diminished capacity in corticosterone production seen with aging in the rat. (endocrinology120: 2521–2528,1987) i t has been reported previously from this laboratory that the steroidogenic capacity of adrenocortical cells declines as rats age (1) . furthermore, this defect seems to be due to biochemical events distal to binding of acth to its receptor and stimulation of camp production (1) . although the exact mechanism of this defect is yet to be defined, the possibility that this phenomenon may represent a direct effect on the steroidogenic process per se is quite appealing. in rat adrenals, steroid (corticosterone) biosynthesis involves the active participation of cholesterol side-chain cleavage, a 5 -3/?-hydroxysteroid dehydrogenase-a 5 " 4isomerase (3/3-hsd), 21-hydroxylase, and 11/3-hydroxylase activities (2) (3) (4) (5) (6) (7) (8) . among these, cholesterol side-chain cleavage (cytochrome p-450 scc ) catalyzes the first step in corticosterone biosynthesis (i.e. the conversion of cholesterol to pregnenolone) and is generally considered to be the rate-limiting step in steroidogenesis (2) (3) (4) (5) (6) (7) (8) . furthermore, the levels of various steroidogenic enzyme activities have been shown to be influenced and/or regulated by acth (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) . therefore, the age-related decline in corticosterone secretion would be expected to have a profound effect on the activity of some or all steroidogenic enzymes. moreover, this age-related defect may, in fact, result from alterations in the key enzyme(s) involved in corticosterone biosynthesis. the present studies were conducted, therefore, to examine the effects of aging on steroidogenic enzymes responsible for the production of corticosterone. additionally, changes in mitochondrial cytochrome p-450 were compared with the activity of cholesterol side-chain cleavage. finally, to assess nonspecific age-related effects, the activities of several unrelated enzymes associated with specific compartments of mitochondria were also measured. [7-14 c] benzylamine hydrochloride (sa, 50-60 mci/mmol) and [l,2-n3 h]deoxycorticosterone (sa, 35-60 ci/mmol) were obtained from amersham corp. (arlington heights, il). fatty acid-free bsa, glucose-6phosphate, adenine nucleotides, sodium succinate, and cytochrome c were the products of sigma chemical co. (st. louis, mo). all unlabeled steroids were supplied by research plus laboratories (denville, nj). 5-cholesten-3j8,25-diol (25-hydroxycholesterol) was purchased from steraloids, inc. (wilton, ny). all other reagents used were of analytical grade. male retired breeder sprague-dawley rats [crl:cobs cd (sd)br], 9-10 months of age, were obtained from charles rivers breeding laboratories (wilmington, ma) and allowed to age to 12 and 18 months in our facility for aging animals, which is isolated from the general animal quarters in this v.a. center. two-month-old rats of the same strain were obtained from the same source. the rats were housed under conditions of controlled light (constant light from 0600-1800 h each day) and temperature (22-24 c) and given free access to purina laboratory chow (no. 5012, ralston-purina, st. louis, mo) and water. the animals in this colony are routinely monitored for the following viruses and infectious agents: pneumonia virus of mice (pvm), rev-3 virus, encephalomyelitis virus (gd-v11), sendai virus, kilham rat virus, rat corona virus/sialodacryoadenitus (rcv/sda), and mycoplasma pulmonis. occasional animals tested positively for pvm, h-1 virus and rev/sda, but all animals were negative for the remaining agents. routine diagnostic necropsies were not performed. in addition, animals were checked for gross pathology before use. those with visible tumors or apparent defects were discarded. preparation of mitochondria. rats were killed by cervical dislocation, and the adrenals were rapidly removed and trimmed of excess fat and external connective tissue. adrenal mitochondria were isolated by a slightly modified procedure of toaff et al. (16) , as described previously (17, 18) . briefly, adrenals from four to six rats were homogenized in 5 times their wet weight of ice-cold buffer containing 0.25 m sucrose, 1 mm edta, 25 mm hepes (ph 7.0) and 10 mg/ml fatty acid-free bsa (buffer a). the homogenate was then centrifuged at 600 x g (ja 20 rotor, beckman j21, palo alto, ca) for 10 min to remove intact cells, nuclei, and debris. the supernatant was then subjected to centrifugation at 8700 x g for 10 min to sediment mitochondria. the pellet (sedimented mitochondria) was resuspended in buffer a and centrifuged at 600 x g for 10 min, and mitochondria were resedimented at 8700 x g for a further 10 min. the resulting pellet was resuspended in buffer b (0.2 m sucrose, 20 mm kc1,5 mm mgcl 2 , and 25 mm tris-hcl ph 7.4) to a desired protein concentration and immediately used for enzyme measurements. preparation of microsomal fraction. the 8,700 x g supernatant was centrifuged at 15,000 x g for 15 min, and the pellet was discarded. the supernatant fraction was next centrifuged at 105,000 x g for 60 min (60-ti rotor, beckman lm 8 ultracentrifuge) to sediment microsomal fraction. the pellet was washed in buffer a (without bsa) and again collected at 105,000 x g for 60 min. the sediment was resuspended in buffer c (0.25 m sucrose, 1 mm edta, and 10 mm potassium phosphate, ph 7.0) and immediately used for enzyme assays. assay for cholesterol side-chain cleavage activity. the enzyme activity was measured by a slight modification of the procedure of toaff et al. (16) , as described by mori and marsh (17) . the incubation medium in a final volume of 1 ml contained 0.2 m sucrose, 10 mm kc1, 5 mm mgcl 2 , 0.2 mm edta, 10 mm potassium phosphate, 25 mm tris-hcl (ph 7.4), 1 mg/ml bsa (essentially fatty acid free), 3 mm sodium succinate, 6 ftm cyanoketone, and a suitable aliquot of mitochondrial preparation (50-100 ^tg protein/ml) in the presence or absence of 25hydroxycholesterol (25 /ng/ml). after incubation at 37 c for 5-10 min, the reaction was terminated by quick freezing at -60 c. the reaction product, pregnenolone, was extracted from the incubation mixture with hexane (three times, 2 ml each) and assayed by specific ria (19) . cyanoketone was included to inhibit further metabolism of pregnenolone to progesterone. all assays were run in triplicate. the cholesterol side-chain cleavage activity is expressed as nanomoles of pregnenolone formed per min mg protein" 1 . assay for 3/3-hsd. 3/3-hsd activity was determined by measuring the conversion of [ 3 h]pregnenolone to [ 3 h]progesterone as described by shaw et al. (20) . briefly, incubation medium in a final volume of 1.0 ml contained 50 mm potassium phosphate buffer (ph 7.4), 50 nm [ 3 h]pregnenolone (400 dpm/pmol), 0.5 mm nad + , and a suitable aliquot of microsomal fraction. the reaction, maintained at 37 c for 15-30 min, was initiated by the addition of [ 3 h]pregnenolone (dissolved in 30 n\ dimethylsulfoxide). the reaction was stopped by the addition of 0.1 ml 1 n naoh, and unlabeled pregnenolone (50 ng) and progesterone (50 fig) were added in addition to [ 14 c]progesterone (5000 dpm) to monitor recovery. the steroids were extracted two times with 5 ml diethyl ether, and pooled diethyl ether extracts were dried under n 2 . pregnenolone and progesterone were separated by ascending tlc on silica gel h using chloroformdiethyl ether (5:1, vol/vol) as a solvent system. the radioactivity associated with progesterone was measured by liquid scintillation spectrometry (beckman ls3801 scintillation counter). the purity of the [ 3 h]progesterone was determined by repeated recrystallization. all enzyme assays were carried out in triplicate. 3/3-hsd activity is expressed as nanomoles of progesterone produced per min mg protein" 1 . assay for 21-hydroxylase activity. enzyme activity was determined by measuring the formation of [ 14 c]deoxycorticosterone from [ 14 c]progesterone according to the procedure of menard et al. (21) . all incubations were performed at 37 c for 15-20 min. the : incubation medium in a final volume of 0.4 ml contained 50 mm tris-hcl (ph 7.4), 0.6 mm nadph, 10 mm glucose-6-phosphate, 1 u/ml glucose-6-phosphate dehydrogenase, 5 mm mgcl 2 , 1 mm [ 14 c]progesterone (10,000 dpm/nmol in 2.5% propylene glycol), and a suitable aliquot of the micro-somal fraction. the reaction was initiated by the addition of microsomal fraction and stopped by the addition of 0.04 ml 1 n naoh. fifty micrograms each of progesterone and deoxycorticosterone (containing 5000 dpm [ 3 h]deoxycorticosterone to monitor recovery) were added to each tube, and steroids were extracted with dichloromethane. steroids were separated by tlc (silica gel f254 plates) using dichloromethanebutyl alcohol (1:1, vol/vol) as a mobile phase. the radioactivity associated with deoxycorticosterone was measured by liquid scintillation spectrometry, as described above. all assays were carried out in triplicate. the enzyme activity is expressed as nanomoles of deoxycorticosterone formed per min mg protein" 1 . assay for llfi-hydroxylase. the enzyme activity was measured by following the conversion of [ 3 h]deoxycorticosterone to [ 3 h] corticosterone, as described by churchill et al. (22) . briefly, incubation medium in a final volume of 0.5 ml contained 50 mm tris-hcl (ph 7.4), 1 mm mgcl 2 , 1 mm [ 3 h]deoxycorticosterone (20,000 dpm/nmol in 2.5% propylene glycol), 0.25 m sucrose, 4 mm sodium succinate, and a suitable aliquot of mitochondrial preparation. all incubations were carried out at 37 c for 15-20 min. the reaction was initiated by the addition of mitochondrial suspension and terminated by the addition of 0.05 ml 1 n naoh. fifty micrograms each of deoxycorticosterone and corticosterone were added to each tube, and steroids were extracted with dichloromethane. steroid were separated by tlc using silica gel f 254 plates and dichloromethane-butyl alcohol (1:1, vol/vol) as a solvent system. the plates were run three times in same solvent system, and the corticosteronecontaining areas were isolated, eluted, and counted for radioactivity by liquid scintillation spectrometry, as described above. all enzyme assays were carried out in triplicate. the enzyme activity is expressed as nanomoles of corticosterone formed per min mg protein" 1 . measurement of cytochrome p-450. mitochondrial cytochrome p-450 was measured according to the method of omura and sato (23) . the extinction coefficient of 91 cm" 1 mm" 1 was used to calculate the cytochrome p-450 concentration from the absorbancy difference between 450 and 490 nm. miscellaneous enzyme assays. succinate dehydrogenase activity was measured according to the method of pennington (24) . the activity is expressed as nanomoles of 2-(p-iodophenyl)3-(pnitrophenyl)5-phenyltetrazolium reduced per min mg protein" 1 . malate dehydrogenase was assayed by a modification of the procedure of ochoa (25) , as described by vardanis (26) , with activity expressed as nanomoles of nadh oxidized per min mg protein" 1 . the radiochemical procedure of mccaman et al. (27) was used to quantitate monoamine oxidase, with activity expressed as nanomoles of benzaldehyde produced per min mg protein" 1 . amytal-insensitive nadh cytochrome c reductase was measured by a combination of the procedures of sottocasa et al. (28) and privalle et al. (29) . the enzyme activity is expressed as nanomoles of cytochrome c reduced per min mg protein" 1 . cytochrome oxidase activity was measured according to the methods of cooperstein and lazarow (30) , with activity expressed as nanomoles of cytochrome c oxidized per min mg protein" 1 . the protein content of all fractions was measured by the modified procedure of lowry et al. (31) , as described by markwell et al. (32) . statistical analyses. the results are expressed as the mean ± se. the results were analyzed by analysis of variance. the analysis used the general linear models procedures of sas (sas institute, cary, nc) when giving a significant f value; scheffe's posttests were used to determine differences between any two age groups. p < 0.05 was considered statistically significant. cholesterol side-chain cleavage enzyme activity, the rate-limiting enzyme in corticosterone biosynthesis, was measured in adrenal mitochondria isolated from 2-, 12-, and 18-month-old male sprague-dawley rats. all assays were carried out in the presence and absence of 25hydroxycholesterol (25 /ug/ml). the enzyme-catalyzed reaction product, pregnenolone, was quantitated by a specific ria. results presented in figs. 1 and 2 show incubation time-and enzyme concentration-dependent pregnenolone formation by the adrenal mitochondria isolated from 2-and 12-month-old rats. under basal conditions, the rate of pregnenolone formation was low and exhibited a biphasic response (fig. 1, inset) . in contrast, addition of oxygenated sterol (25-hydroxycholesterol) greatly enhanced pregnenolone production, and again this effect was biphasic. furthermore, at each time point studied, mitochondria from senescent rats synthesized 2-3 times more pregnenolone compared to corresponding values from young control groups. similarly, the rate of pregnenolone formation was linear with protein concentration up to 100 /kg/ml in both age groups examined (fig. 2) . like time-course studies, enzyme concentration studies also revealed enhanced steroidogenic capacity of mitochondria from 12-month-old rats, and this effect was potentiated in the presence of 25-hydroxycholesterol. we next examined the side-chain cleavage enzyme activity in mitochondria isolated from rats 2, 12, and 18 months of age, and the results are presented in fig. 3 . under basal conditions (without 25-hydroxycholesterol), cholesterol side-chain cleavage activity (expressed as nanomoles of pregnenolone produced per min mg protein" 1 ) increased (p < 0.001) from a mean (±se) of 0.43 ± 0.06 in 2-month-old rats to 1.26 ± 0.11 and 1.51 ± 0.06 in the 12-and 18-month-old rats, respectively. similarly, mitochondria isolated from 12-and 18-month-old rats showed significantly higher activity in the presence of more polar exogenous substrate (25-hydroxycholesterol) compared to the 2-month-old groups (18-month-old, 16 ± 1.4; 12-month-old, 12.4 ± 1.2; 2-month-old, 5.0 ± 0.6). all of these enzyme assays were carried out under a nearlinear range of time and enzyme protein concentration. thus mitochondria isolated from the 12-or 18-monthold rats exhibited an enhanced capacity to synthesize pregnenolone compared to mitochondria from control groups. moreover, addition of 25-hydroxycholesterol produced a further substantial increase in pregnenolone formation in mitochondria isolated from older animals, and levels were significantly higher than those in mitochondria from control groups. in contrast to progressive changes in side-chain cleavage activity, mitochondrial cytochrome-p450 contents did not change with age (table 1). a comparison of the activities of other steroidogenic enzymes, 3/3-hsd 21-hydroxylase, and 11/miydroxylase, in microsomal and mitochondrial fractions of adrenals from 2-and 12-month-old animals is shown in table 2 . no significant age-related changes were noted for 3/3-hsd (29.8 ± 1.2 nmol min" 1 mg protein" 1 in 2-monthold rats us. 29.5 ± 2.2 nmol min" 1 mg protein" 1 in 12month-old rats; p = ns). similarly, mitochondrial 110hydroxylase activity was comparable in both age groups examined (1.21 ± 0.15 nmol min" 1 mg protein" 1 in 2month-olds us. 1.19 ± 0.09 nmol min" 1 mg protein" 1 in 12-month-olds; p = ns). in contrast microsomal 21hydroxylase activity was slightly but significantly (p < 0.001) increased in 12-month-old rats (7.7 ± 0.5 nmol min" 1 mg protein" 1 ) compared to that in 2-month-old animals (5.2 ± 0.2 nmol min" 1 mg protein" 1 ). mean activities (±se) of monoamine oxidase, amytalinsensitive nadh cytochrome c reductase, succinate dehydrogenase, cytochrome oxidase, and malate dehydrogenase are presented in table 3 . monoamine oxidase (33, 34) and amytal-insensitive results are the mean ± se. the number of experiments is given in parentheses. "expressed as nanomoles of progesterone produced per min mg protein" 1 . 6 expressed as nanomoles of deoxycorticosterone produced per min mg protein" 1 . c expressed as nanomoles of corticosterone produced per min mg protein" 1 . results are the mean ± se of eight separate experiments. 0 expressed as nanomoles of benzaldehyde produced per min mg protein" 1 . 6 expressed as nanomoles of cytochrome c reduced per min mg protein" 1 . c expressed as nanomoles of iodonitrotetrazolium reduced per min mg protein" 1 . d expressed as nanomoles of cytochrome c oxidized per min mg protein" 1 . e expressed as nanomoles of nadh oxidized per min mg protein" 1 . nadh cytochrome c reductase (18, 28, 29, 35) are located on the outer mitochondrial membrane and were chosen for study to serve as a control for the increase in sidechain cleavage activity associated with preparation of mitochondrial fractions. succinate dehydrogenase and cytochrome oxidase are conventional markers of the inner mitochondrial membrane (26) and were chosen as a control for the possibility that age leads to a generalized change in the activity of adrenal enzymes. malate jdehydrogenase is a classical marker for mitochondrial matrix (26, 29) and was employed to serve as a control for mitochondrial integrity. the fact that the activity of none of these enzymes changed as rats grew older provides support for the relative specificity of the age-related increase in cholesterol side-chain cleavage activity. the ability of isolated adrenocortical cells to respond to acth declines with advancing age (1, 36) . we have shown previously that this aging defect lies distal to both binding of acth to its receptor and the consequent production of its secondary messenger camp (1) . in the present studies the possible relationship between steroidogenic enzyme activities and steroidogenesis in rat adrenals during aging was explored. accordingly, we measured cholesterol side-chain cleavage, 3/3-hsd, 21-hydroxylase, 11/3-hydroxylase activities, and cytochrome p450 contents in appropriate mitochondrial and microsomal fractions of adrenals from 2-, 12-, and 18-month-old rats. an unexpected finding was that cholesterol side-chain cleavage activity showed a progressive increase with advancing age. a slight but significant increase in the activity of 21-hydroxylase was also noted in adrenal microsomal fractions from senescent rats. in contrast, activities of 3/3-hsd and 110hydroxylase were insensitive to the aging process. interestingly, the current results have clearly indicated that adrenal side-chain cleavage enzyme activity increased at a time when acth-induced cellular steroidogenesis has been more than 60% depressed (1, 36) . several mechanisms might account for the stimulatory effect of aging on cholesterol side-chain cleavage activity. one possibility is an augmentation of enzyme synthesis. in addition, if degradation of cholesterol side-chain cleavage enzyme protein were to proceed at a lower rate than in young rats, the levels of this enzyme would be expected to be higher, since the steady state level of any given protein is achieved by its relative rates of synthesis and degradation. a second possibility may be that an increased activity represents cellular adaptive changes to deal with the diminished capacity of the adrenal gland to mobilize and use cholesterol esters for steroidogenesis. indeed, we have recently shown that cholesterol ester content increases with age and that this defect was related to specific changes in neutral cholesterol esterase (popplewell, p. y., and s. azhar, submitted). thus, an increase in cholesterol side-chain cleavage activity can be viewed as a compensation on the part of the enzyme to the elevated cholesterol esters levels. that is, mitochondrial side-chain cleavage enzymes increase in activity in order to effectively use that cholesterol which becomes available from the stored cholesterol ester. however, the fact that the adrenal steroidogenic response is still blunted in senescent rats demonstrates this to be a partially successful compensation. another possibility of equal importance is that the observed increase in cholesterol side-chain activity may be in response to vastly elevated levels of plasma acth, previously reported in senescent rats (37) . this hyperstimulation hypothesis is supported by the observations that the cholesterol side-chain cleavage step is the rate-limiting step in steroidogenesis and, as such, activated/regulated by acth (2, 4, 5) . finally, evidence has recently been presented to support the idea that adrenal mitochondria contain more than one cholesterol side-chain cleavage system (38) (39) (40) . thus, it is conceivable that elevated levels represent stimulation of only one isoenzymic form which may or may not actively participate in steroidogenesis under in vivo situations. obviously more rigorous experimental approaches are needed to sort out these various possibilities. such investigations are currently in progress in our laboratory. we were unable to document any age-related changes in mitochondrial cytochrome p450 levels. these results point to a dissociation between cytochrome p450 levels and cholesterol side-chain cleavage enzyme activity. this discrepancy could be attributed to a variation in the relative compositions of various forms of cytochrome p450 in total mitochondrial preparations from young and senescent rat adrenals (41, 42) . in addition, a pool of cytochrome p450 not associated with steroidogenic enzymes (i.e. cytochrome p450 8cc or cytochrome p450 u/ 3) may exist (12) , which, in turn, could mask a real age effect on specific cytochrome p450 8cc . the above hypothesis is of interest in view of the previous finding demonstrating a dissociation in half-lives of 21-hydroxylase and cytochrome p450 (4.5 vs. 2.9 days) in rat adrenals after hypophysectomy (12) . additional evidence for this hypothesis is the recent finding of naumoff and stevenson (43) , who observed a delayed developmental maturation pattern for cholesterol side-chain activity compared to cytochrome p450 in rat ovaries after gonadotropin treatment. in view of these observations, it is not unreasonable to speculate that levels of cytochrome p450 and side-chain cleavage are modulated differentially as rats grow old. finally, demonstration of any specific age effect on cytochrome p450 8cc will require the purification of a side-chain cleavage enzyme system from adrenals of young and old rats. results of the present study also suggest that the decline in steroidogenesis observed in adrenocortical cells isolated from senescent rats is not related to a reduction in cholesterol side-chain cleavage activity or other steroidogenic responses, as measured in vitro in isolated subcellular components. these in vitro results do hot exclude the possibility, however, that side-chain cleavage activity is reduced (or blunted) in intact cells or in vivo. furthermore, the possibility that the aging process directly affects the performance (in vivo) of recently identified regulator (29, (44) (45) (46) (47) (48) (49) (50) (51) (52) (53) (54) (55) of the side-chain cleavage enzyme activity should also be considered. previous reports indicate that the aging process in mouse and rat adrenals is associated with changes in 3/3-hsd (55, 56) . we realize that our inability to demonstrate an age effect on this enzyme are in conflict with earlier studies (55, 56) . several technical/physiological differences in the experimental approach to the problem may account for the observed incongruent findings. for instance, the earlier studies were carried out with female rats or mice, as opposed to male rats employed in the present studies. additionally, variations in the assay mixture and other experimental conditions may account for the observed discrepancy. likewise, alterations in nutritional status, animal strain, or other physiological factors could also contribute significantly. finally, since this enzyme is not rate limiting, even a modest decline in its activity should have very little adverse effect on the overall steroidogenic capacity of the adrenal gland. significant changes in some steroidogenic enzymes in other steroid-producing tissues have been demonstrated in senescent rats. for example, testicular 30-hsd activities have been shown to decline with advancing age (57) (58) (59) . similarly, other researchers have reported significant age-related loss of this enzyme activity in rat ovary (60) (61) (62) . likewise, other researchers have presented evidence that activities of 17a-hydroxylase (57, 59, 63) , c17-20 lyase (63), as well as microsomal cytochrome p-450 (63) are depressed in testes from aged rats. however, comparison of data from different steroidogenic tissues suggests that changes in enzyme activities that occur with aging in one steroid-producing tissue may not necessarily be applicable to other steroid-producing tissues. in conclusion, the results of the present study suggest that the impairment of corticosterone secretion in adrenocortical cells isolated from senescent rats is probably not related to a reduction in the activity of steroidogenic enzymes, as measured in vitro. however, since the transport of cholesterol to cytochrome p450 scc rather than the activity of cytochrome p450 scc per se limits the rate of steroidogenesis, it is possible at this time to at least speculate that the age-related decline in steroidogenesis is related to a decrease in the transport of cholesterol to the mitochondria. the roles of microtubules (44), microfilaments (44) , phospholipids (40, (45) (46) (47) 52) , sterol carrier proteins (48) (49) (50) (51) , and other factors (28, 53, 54) have all been described as playing a part in cholesterol transport. failure of or changes in any of these mechanisms with age would reduce the amount of cholesterol substrate available for steroidogenesis within the mitochondria, and we are presently studying the possibility that cholesterol transport is also altered with age. differential effects of aging on acth receptors, adenosine 3',5' cyclic monophosphate response and corticosterone secretion in adrenocortical cells from sprague-dawley rats on the mechanism of action of acth adrenal gland cholesterol side-chain cleavage, cytochrome p450, and the control of steroidogenesis cholesterol metabolism in the adrenal cortex regulation of the adrenal and gonadal microsomal mixed function oxygenases of steroid hormone biosynthesis the roles of cytochrome p450 in the synthesis and metabolism of steroids a heuristic proposal for understanding steroidogenic processes the mechanism of action of adrenocorticotropic hormone: the role of mitochondrial cholesterol accumulation in the regulation of steroidogenesis evidence that the cyclohexamide-sensitive site of adrenocorticotropic hormone action is in the mitochondrion: changes in pregnenolone formation, cholesterol content, and the electron paramagnetic resonance spectra of cytochrome p-450 regulation of cytochrome p-450 supported 11-/3-hydroxylation of deoxycortisol by steroids, oxygen and antioxidants in adrenocortical cell cultures life time of adrenal cytochrome p450 as influenced by acth mechanism of induction of a 5 -3/miydroxysteroid dehydrogenase-isomerase activity in rat adrenocortical cells by corticotropin steroidogenic enzyme activities in cultured human definitive zone adrenocortical cells: comparison with bovine adrenocortical cells and resultant differences in adrenal androgen synthesis in vitro spontaneous and adrenocorticotropin dependent maturation of the steroidogenic pathway of ovine fetal adrenal cells relationship of cholesterol supply to luteal mitochondrial steroid synthesis the site of luteinizing hormoe stimulation of steroidogenesis in mitochondria of the rat corpus luteum oxygen dependence of adrenal cortex cholesterol side chain cleavage': implications in the rate-limiting steps in steroidogenesis radioimm'unoassay of serum and ovarian pregnenolone in immature rats synergistic effect of follicle-stimulating hormone and luteinizing hormone on testicular a 5 -3/3-hydroxysteroid dehydrogenase-isomerase: application of a new method for the separation of testicular compartments spironolactone and cytochrome p-450: impairment of steroid 21-hydroxylation in the adrenal cortex topological studies of the steroid hydroxylase complexes in bovine adrenocortical mitochondria the carbon-monoxide binding pigment of liver microsomes. i. evidence for its homoprotein nature biochemistry of dystrophic muscle: mitochondrial succiniate tetrazolium reductase and adenosine triphosphatase malic dehydrogenase from pig heart protein kinase activity at the inner membrane of mammalian mitochondria microdetermination of monoamine oxidase and 5-hydroxytryptophan decarboxylase activities in nervous tissues an electron-transport system associated with the outer membrane of liver mitochondria: a biochemical and morphological study regulation of intramitochondrial cholesterol transfer to side-chain cleavage cytochrome p-450 in rat adrenal gland a microspectrophotometric method for the determination of cytochrome oxidase protein measurement with the folin phenol reagent a modification of the lowry procedure to simplify protein determination in membrane and lipoprotein samples the submitochondrial localization of monoamine oxidase-enzymatic marker for the outer membrane of rat liver mitochondria utilization of intramitochondrial membrane cholesterol by cytochrome p-450 dependent cholesterol side-chain cleavage reaction in bovine adrenocortical mitochondria: steroidogenic and non-steroidogenic pools of cholesterol in the mitochondrial inner membranes localization of l-lactate dehydrogenase in mitochondria aging of the rat adrenocortical cells: response to acth and cyclic amp in vitro the neuroendocrinology of stress and aging: the glucocorticoid cascade hypothesis cytochrome p-450 of adrenal mitochondria: steroid binding sites of two distinguishable forms of rat adrenal mitochondrial cytochrome p-4508c c evidence suggesting that more than one sterol side chain cleavage system exists in mitochondria from bovine adrenal cortex effects of phospholipid and detergent on the substrate specificity of adrenal cytochrome p-450scc: substrate binding and kinetics of cholesterol side-chain oxidation purification and characterization of mitochondrial cytochrome p-450 associated with cholesterol side-chain cleavage from bovine corpus luteum inhibition of cholesterol side chain cleavage by active site directed antibody to corpus luteum cytochrome p450 the differential development of mitochondrial cytochrome p-450 and the respiratory cytochromes in rat ovary intracellular movement of cholesterol in rat adrenal cells: kinetics and effects of inhibitors on the mechanism whereby acth and cyclic amp increase adrenal polyphosphoinositides: rapid stimulation of the synthesis of phosphatidic acid and derivatives of cdp ~ diacylglycerol phosphoinositide metabolism and hormone action adrenocorticotropic hormone-mediated changes in rat adrenal mitochondrial phospholipids sterol carrier protein 2 : delivery of cholesterol from adrenal lipid droplets to mitochondria for pregnenolone synthesis sterol carrier protein 2 : identification of adrenal sterol carrier protein 2 and site of action for mitochondrial cholesterol utilization intramitochondrial movement of adrenal sterol carrier protein with cholesterol in response to corticotropin scallen tj 1985 phospholipid, sterol carrier protein 2 and adrenal steroidogenesis polyphosphoinositide activation of cholesterol side chain cleavage with purified cytochrome p-4508cc brownie ac i983 cholesterol side-chain cleavage in the rat adrenal cortex: isolation of a cycloheximide-sensitive activator peptide modulation of the kinetics of cholesterol side-chain cleavage by an activator and by an inhibitor isolated from the cytosol of the cortex of bovine . adrenals aging and adrenal a 5 -3-/3-hydroxysteroid dehydrogenase in female mice aging and adrenal a 6 -3/3-hydroxysteroid dehydrogenase in female rats the effect of vasectomy on steroid metabolism by the seminiferous tubules and interstitial tissue of the rat testis: a comparison with the effects of aging effect of age on testis a 6 -3j8-hydroxysteroid dehydrogenase in the rat testicular function and sexual activity in senescent mice ovarian a 5 -3/3-hydroxysteroid dehydrogenase in aging rats aging and ovarian a 5 -3j8 hydroxysteroid dehydrogenase in rats aging and ovarian a 5 -3/3-hydroxysteroid dehydrogenase in the pregnant mouse an analysis of the age-related decline in testicular steroidogenesis in the rat key: cord-270037-tq82srhn authors: kramer, holger b.; nicholson, benjamin; kessler, benedikt m.; altun, mikael title: detection of ubiquitin–proteasome enzymatic activities in cells: application of activity-based probes to inhibitor development() date: 2012-05-19 journal: biochim biophys acta mol cell res doi: 10.1016/j.bbamcr.2012.05.014 sha: doc_id: 270037 cord_uid: tq82srhn background: synthetic probes that mimic natural substrates can enable the detection of enzymatic activities in a cellular environment. one area where such activity-based probes have been applied is the ubiquitin–proteasome pathway, which is emerging as an important therapeutic target. a family of reagents has been developed that specifically label deubiquitylating enzymes (dubs) and facilitate characterization of their inhibitors. scope of review: here we focus on the application of probes for intracellular dubs, a group of specific proteases involved in the ubiquitin proteasome system. in particular, the functional characterization of the active subunits of this family of proteases that specifically recognize ubiquitin and ubiquitin-like proteins will be discussed. in addition we present the potential and design of activity-based probes targeting kinases and phosphatases to study phosphorylation. major conclusions: synthetic molecular probes have increased our understanding of the functional role of dubs in living cells. in addition to the detection of enzymatic activities of known members, activity-based probes have contributed to a number of functional assignments of previously uncharacterized enzymes. this method enables cellular validation of the specificity of small molecule dub inhibitors. general significance: molecular probes combined with mass spectrometry-based proteomics and cellular assays represent a powerful approach for discovery and functional validation, a concept that can be expanded to other enzyme classes. this addresses a need for more informative cell-based assays that are required to accelerate the drug development process. this article is part of a special issue entitled: ubiquitin drug discovery and diagnostics. the last two decades have witnessed the development and application of activity based probes for the detection of enzymatic activities related to a wide range of protein post-translational modifications (ptms). these tools have allowed for the detection of many enzymatic activities in relation to conjugation and deconjugation of ptms. the greatest success and widest application of this approach have probably been found within the ubiquitin proteasome pathway [1] . the ubiquitin proteasome system (ups) consists of three different elements with partially opposing, but complementary roles; the ubiquitin conjugating cascade, the deubiquitylating enzymes and the proteasome complex (fig. 1) . the ubiquitin conjugating cascade of enzymes (e1, e2, e3 and e4) is responsible for conjugating ubiquitin (ub)/ubiquitin-like proteins (ubls) to other proteins as signaling molecules in several biological processes [2, 3] . the deubiquitylating enzymes (dubs) are responsible for removing, trimming or editing the ub/ubl modifications from proteins [2] . the proteasome complex, composed of at least three different proteolytically active subunits β1, β2 and β5, degrades the majority of soluble proteins within the cell following polyubiquitylation [4] . the ups not only enables the targeted destruction of proteins no longer required by the cell, but also enables the selective deactivation of signaling molecules, removal of pathogenic proteins and production of peptides for presentation to the immune system. the entire pathway comprises close to 1000 genes, is vital for many normal cellular functions and is frequently disrupted in cellular pathologies [5, 6] . a likely explanation for the remarkable success of development of activity-based probes in the ups when compared to small molecule probes related to other ptms, is the ability to target cysteine and threonine proteases successfully by covalent inhibition. these represent the major enzymatic activities of dubs and the proteasome respectively. improved recognition of the protein-based ubiquitin and peptide-derived proteasome probes as compared with small molecule based probes may also contribute. the generation of activity based probes for the ubiquitin pathway as important tools to obtain insights into the ubiquitin proteasome system has been reviewed previously [7] [8] [9] [10] . the focus of this review is the application of activity-based probes in the context of inhibitor development and characterization. this process represents a critical step in arriving at a better understanding of fundamental cellular processes and enables the development of novel targeted therapeutic agents. activity-based probes (abps) are defined by their ability to target subsets of a complex proteome based on labeling of specific enzyme classes in a covalent fashion. the incorporation of specific tags for detection and enrichment allows for visualization and further analysis for example by tandem mass spectrometry. the abp approach is highly complementary to mass spectrometry-based proteomics due to the ability to enrich for specifically captured substrates with the aid of an affinity tag. this also distinguishes the method from the use of radiolabeled covalent inhibitors that support detection but not affinity enrichment. a critical parameter is also the choice of reactive group for covalent labeling of the target enzyme at or close to the enzyme active site. depending on the mechanism of enzymatic catalysis different reactive groups can be chosen for covalent capture. the alkylation of nucleophilic residues (cys, ser, thr) present in protease active sites by reactive electrophiles has been a very successful realization of this method. the utility of abps is particularly evident in the study of enzyme systems involved in the attachment and removal of ptms. due to the enzyme active site chemistries involved abp development has been generally more fruitful for the targeting of deconjugating enzymes that usually contain strong nucleophiles at their active sites when compared to the respective ligases/transferases. the discovery and development of proteasome inhibitors have been previously covered by comprehensive reviews [10, 11] . here, we will therefore only give a brief introduction to the proteasome complex and proteasome active site probes. the proteolytically active sites of the proteasome complex are located on distinct subunits (for example β1, β2 and β5 in the constitutive proteasome) and this is reflected by the labeling pattern obtained by active site-directed proteasome probes [12] . the three subunits responsible for proteolysis also correspond to different activity profiles with respect to substrate specificity which were named after prototypic proteases: chymotrypsin-like activity for β5 (ct-l), trypsinlike activity for β2 (t-l) and petidylglutamyl peptide hydrolyzinglike (pgph-l) activity (also referred to as caspase-like) for β1 [13] . exchange of the active subunits with close homologues can be induced in immune cells [14] and the thymus [15] . this allows for the tuning of the peptide repertoire produced by proteasomal proteolysis, which underscores the importance of presentation of these peptides (after further processing steps) in immune surveillance. proteolytically active subunits of the proteasome have been targeted by probes based on peptide scaffolds bearing a c-terminal electrophile (fig. 2) . the most popular designs have been based on the trileucine vinylsulfone (l 3 vs) sub-structure and on functionalized epoxomicin derivatives. profiling with these probes is enabled by detection with dansyl [16] and biotin [17, 18] tags and with strong fluorophores [19, 20] as well as by radiolabels [12, 21] . alternatively, a two step protocol can be applied whereby the tag is attached in a bioorthogonal ligation step following labeling of active proteasome subunits in a cell lysate [22] . this selective ligation is possible through application of the staudinger ligation [23] or 1,4-triazole formation also known as "click" chemistry [24, 25] . a more recent addition to the arsenal of proteasome-directed probes has been the belacostin a derived dansyl-kf33955 probe [26] . this β-lactone containing probe was used successfully in the labeling of β1, β2 and β5 subunits of the 26s proteasome in hela cells as well as the 20s proteasome from erythrocytes. ubiquitin derived abps [27, 28] have been used for the profiling of the active dub population in cell lines, primary cells and tissues [29] . subsequently this concept has been extended to the study of proteases specific for ubiquitin-like proteins (ubls) [30] . a complex proteome such as a cell or tissue lysate is labeled by incubation with ubiquitin probe, followed by sds-page separation and immunoblotting against the epitope tag genetically encoded in the probe construct. thereby a read-out, which is dependent on dub abundance and activity, is obtained. this approach was also extended to the investigation of cancer cell lines [29, 31] and patient biopsies [31] . particularly fruitful was the study of in vitro infection models of viral [32] [33] [34] [35] , parasitic [36, 37] and bacterial [38, 39] pathogens. in this way previously unknown dubs encoded by pathogens were identified and other interactions of pathogen encoded factors with the host ubiquitin proteasome system were elucidated. dub related pathogen-host interactions have been reviewed in detail elsewhere [40] [41] [42] [43] . ubiquitin probes have further enabled structural studies on dub-ubiquitin complexes [44] . in these investigations the crystallization and x-ray diffraction of covalent adducts formed between enzyme and the ubiquitin probe allowed insights into some of the molecular determinants of interactions of dubs with ubiquitylated substrates. these types of studies have also been successfully carried out with complexes involving ubiquitin aldehyde [45] [46] [47] or ubiquitin probes devoid of an epitope tag [48] [49] [50] . in addition to the utility of ubiquitin abps targeting cys protease dubs, ms-based proteomics experiments of ubiquitin probes have also been shown to retrieve other regulators of ubiquitylation, for example metalloprotease dubs were retrieved under native pull-down conditions [51] . this finding might be explained by the formation of non-covalent complexes which can be enriched in immunoprecipitations (ips) under non-denaturing conditions. the targeting and retrieval of ubiquitin ligases by ubiquitin probes have also been observed [52] . usually the reduced nucleophilicity of cys residues in this enzyme class (when compared to cys as part of the catalytic triad in dub proteases) results in strongly reduced capture. this can apparently be compensated for to a certain extend by strong c-terminal electrophiles conjugated to the ub probes [52] . in addition to the above-mentioned applications activity-based proteomics probes have also enabled the development of pharmacologically active enzyme inhibitors. in one format, competition assays between an inhibitor candidate compound and the activitybased probe can be performed in cell lysates. in this type of assay format the competition between the small molecule inhibitor and the probe leads to a reduced labeling profile for the activity-based probe. the loss of signal for labeling of specific target enzymes allows assessment of the specificity of inhibition, while titration of concentration ranges provides a measure of ic 50 values for the respective inhibitor. several features make this type of assay format particularly attractive: the approach represents a cell-based assay in which treatment by the inhibitor candidate can be performed on intact cells. in contrast to the majority of in vitro enzyme assays, a range of active cellular enzymes can be assayed simultaneously. this is only limited by the number of enzymes successfully labeled by the activity-based probe and the representation of active enzymes in the cellular proteome. quantifiable results can be obtained using various readout formats including immunoblotting, in-gel fluorescence detection or ip followed by quantitative msbased proteomics. such an approach has been employed successfully for a number of abps including in the identification and characterization of selective inhibitors of the proteasome [53, 54] , of cysteine proteases in the dub enzyme family [51] and serine hydrolases in the fatty acid amide hydrolase class [55] . promiscuous inhibitors can be tested in this way in order to demonstrate their broad inhibition profile [56] and competition assays can be applied to the characterization of the specificity of known drugs. the development of novel cancer therapeutics is a prime example where the success of bortezomib [16, 57] for treatment of multiple myeloma [58] has provided proof of concept for the value of interventions targeting the ubiquitin proteasome system. the effects of bortezomib (ps-341) on the activity and composition of proteasomes in multiple myeloma cells were investigated by labeling with proteasome probe ada-[ 125 i]-y-ahx 3 l 3 vs demonstrating that the inhibitor selectively targets β5 and β1 subunits (as well as immunoproteasome subunits β5i and β1i) [57] . the corresponding experiments were carried out in the presence or absence of cytokines, which induce the formation of immunoproteasomes under conditions that may be encountered in the bone marrow. a similar characterization of the subunit specificity of proteasome inhibition was also carried out for the orally active inhibitor npi-0052 (salinosporamide a) in multiple myeloma cells [59] . the results of competitive labeling with the proteasome probe dansyl-ahx 3 l 3 vs indicated that the inhibitor targets the β1, β2 and β5 subunits. this result was consistent with the finding that cleavage of fluorogenic substrates showed inhibition of the ct-l, t-l and pgph-l activities of the proteasome. in a comparable study the specificity of the three proteasome inhibitors bzlllcocho, bortezomib (ps-341) and mg132 was assessed by labeling with the cell permeable dansyl-ahx 3 l 3 vs probe following treatment with individual inhibitors for 24 h [60] . this demonstrated clearly distinct labeling pattern of the proteolytically active proteasome subunits β1, β2 and β5 for the three inhibitors. the observed labeling patterns were positively correlated with the inhibition of the ct-l, t-l and pgph-l activities of the proteasome by hydrolysis of suitable fluorogenic substrates in the presence of inhibitors. the selective inhibition of the immunoproteasome subunit β1i (lmp2) by synthetic dihydroeponemycin analogs was demonstrated by competitive labeling experiments in el4 cells using biotinepoxomicin and biotin-eponemycin as a proteasome active site probes [61] . these examples highlight the value of utilizing active site-directed probes in the development and characterization of proteasome inhibitors. this is enabled by their ability to provide a rapid assay of the corresponding proteolytically active subunits in different cell types and in the presence or absence of added competitive inhibitors the specificities of which can thereby be assessed. this approach has been informative to basic science as well as to the development of lead compounds for novel therapeutics. due to the large number of cellular dubs there is a need for innovative assay designs that allow assessment of dub activity in a cellular context. competitive labeling with ub probes provides characterization of lead compounds in a cell culture model thereby providing valuable data for assessment of both compound potency and selectivity of different dubs relevant to a given cell type. a number of dub inhibitors have been characterized and developed into lead compounds for medicinal chemistry programs. generally the initial hit has been obtained by high throughput screening (hts) of compound libraries followed by further optimization using structureactivity relationships. it is noteworthy that many of the identified lead compounds and inhibitors have the ability to covalently label the active site cysteine residue present in the majority of dubs (fig. 3 ). this may occur by conjugate addition, nucleophilic aromatic substitution or disulfide bond formation depending on the chemistry of the small molecule inhibitor. one example of ub probe labeling in the presence of a small molecule inhibitor has been the characterization of wp1130 as a partially selective dub inhibitor which induces aggresome formation and tumor cell apoptosis by kapuria et al. [62] . the selectivity of inhibition for different members of the dub family of enzymes was also investigated by in vitro inhibition assays using ubamc as a substrate in combination with recombinant purified dubs. the total cellular dub activity in lysates treated with inhibitors was tested by ubamc hydrolysis. labeling of treated versus untreated cell lysates with the activity-based probe haubvs was performed in order to assess the dub inhibitory profile of wp1130. in this assay format reduced labeling by the haubvs probe as detected in anti-ha immunoblotting of lysates from cells treated with wp1130 was used as readout of selectivity of dub inhibition. a further focus of this work was the observation of accumulation of polyubiquitylated protein material upon treatment with wp1130 despite a lack of inhibition of proteasome activity. altun et al. described a more extensive application of activitybased probes to the characterization and development of novel dub inhibitors [51] . in this study two dub inhibitors, pr-619 and p22077, were characterized by in vitro enzyme inhibition assays and in cell culture models. labeling of hek293t cell lysates treated with inhibitors at varying concentrations was performed with the ubiquitin probes haubvme and haubbr2 to investigate dub inhibition profiles. as a read-out of these assays immunoblotting against ha-tag or specific dubs of interest was carried out enabling a rapid assessment of inhibitor specificity. in order to obtain an alternative quantitative read-out format, anti-ha immunoprecipitation (ip) was combined with identification and label-free quantification by mass spectrometry based proteomics (fig. 4) . using this approach, quantitative data illustrating the degree of inhibition of 25 cellular dubs was obtained. these data were consistent with the in vitro enzyme characterization data, demonstrating that pr-619 exhibits a broad inhibition profile whereas p22077 is a selective inhibitor of usp7 and usp47. both usp7 and usp47 are considered oncology targets and p22077 and related analogs are the subject of further development as anti-cancer therapeutics [63, 64] . another application of ub probe labeling for the development of usp7 inhibitors was recently demonstrated by reverdy et al. [65] . the screening hit hbx 19,818, which had been identified by high throughput screening (hts) of a compound library employing the ubamc assay, was characterized by haubvs labeling of hct116 cells treated with increasing concentrations of the inhibitor. the readout of the assay was performed by anti-ha immunoblotting and showed reduced probe labeling of usp7 while leaving the competitive labeling of other cellular dubs unaffected. this result was consistent with the in vitro enzyme inhibition data that also indicated a lack of inhibition of other tested cysteine proteases, dubs and ubl specific proteases, but the activity of hbx 19,818 towards the closely related dub usp47 was not discussed. the mechanism of inhibition was investigated by the authors and covalent labeling of the active site cys223 in usp7 was demonstrated, indicating that alkylation of this residue leads to irreversible inactivation of the dub. furthermore the cellular effects of the inhibitor were characterized as reduction in cell proliferation, induction of apoptosis and g1 arrest in a dosedependent fashion. a novel dub inhibitor, b-ap15 was recently described by d'arcy et al. [66] . dub profiling was carried out in the presence of ubiquitin probe haubvs in hct-116 cells after 3 h treatment with 1.0 μm inhibitor; demonstrating that usp14 and to a lesser extend uchl5 were inhibited at this concentration. treatment of cells with b-ap15 led to tumor cell apoptosis, which was independent of tp53 status. additionally an accumulation of high molecular weight ub conjugates and reduced levels of free ubiquitin were observed and ascribed to the inhibition of the 19s associated dubs usp14 and uchl5. the authors concluded that the compound does not act as a broad spectrum dub inhibitor at the concentration employed, rather it inhibits the proteasome associated dubs usp14 and uch-l5. thus, b-ap15 effectively inhibits proteasomal activity by inhibiting the dubs that process polyubiquitylated proteins prior to their engagement by the proteasome. this approach has utility for viral dubs as well. the ubiquitin probe haubvs has been successfully applied to demonstrate the selectivity of inhibitor grl0617 for the sars coronavirus protease and isopeptidase papain-like protease (plpro) in vero e6 cell lysate [67] . no change in labeling profile was observed for cellular dubs at inhibitor concentrations of up to 40 μm. in contrast, the labeling of added plpro was almost completely abolished at the same inhibitor concentration. the usefulness of activity based probes (abps) in the development and characterization of enzyme inhibitors has also been recognized in fields outside the ubiquitin proteasome system. below we describe the advances made in the application of this concept to kinases and phosphatases involved in protein phosphorylation. abps for the targeting of kinases and phosphatases have received considerable attention due to the central role of protein phosphorylation in cellular signaling, protein translocation and ultimately its aberrations in disease. initial kinase probes have utilized atp and adp analogs as molecular scaffolds thereby targeting the essential kinase co-factor in probe design. later approaches were built on modification of known kinase inhibitors of natural product origin or related to licensed drugs. kinase probes based on acyl phosphate atp and adp analogs which lead to the transfer of a biotin to amino acid residues on the interacting protein resulted in the labeling and identification of a range of kinases, atpases, nucleotide biosynthesis-related and other proteins [68] . the lack of selectivity for kinases in a cellular environment is understandable given the wide range of proteins that utilize atp as a cofactor. a tetramethylrhodamine-labeled wortmannin analog ax7503 allowed labeling in mammalian cell lysates and readout of the assay by in gel fluorescence [69] . this fluorescent kinase probe labeled dna dependent protein kinase (dna-pk), phosphoinositide 3 kinase and mammalian polo-like kinase 1 (plk1). previously it had not been appreciated that plk1 is inhibited by wortmannin at relevant concentrations. further wortmannin derivatives labeled with borondipyrromethene (bodipy), tetramethylrhodamine or biotin were prepared successfully [70] . it was shown that the bodipy derivative is cell-permeable thereby enabled labeling of intact cells while the biotin derivative allows isolation of labeled protein material from cell lysates. in a follow-up study to this work liu et al. also demonstrated that polo-like kinase 3 (plk3) is a further molecular target of wortmannin as shown by competitive labeling between ax7503 and wortmannin [71] . the labeling sites of ax7503 for both plk1 and plk3 were determined by tandem mass spectrometry and were found to represent conserved lysine residues in the atp binding site of each of the kinases. a bisindolylmaleimide motif was chosen as the scaffold for the design of a further kinase probe ax4697 [72] . this recognition motif with a high affinity for protein kinase c (pkc) was combined with a chloroacetamide electrophile and a tetramethylrhodamine fluorophore. successful labeling of α and β pkc in jurkat cells was demonstrated as well as loss of signal in staurosporine-pretreated cells. a recent design of a probe for the abelson tyr kinase (abl) was based on the anti-cancer drug imatinib [73] . the reversible mode of enzyme inhibition necessitated the incorporation of a photoreactive benzophenone moiety or a dialdehyde motif for covalent labeling. detection was enabled by the incorporation of a terminal alkyne into the structure allowing attachment of a rhodamine azide dye by cu(i) catalyzed triazole formation (click chemistry) [24, 25] . targeting and detection of abl kinase in the presence of added cell lysate occurred, but the labeling efficiency diminished with increasing amounts of lysate and the approach did not extend to labeling and detection at endogenous levels. the reversible and non-selective kinase inhibitor staurosporine has also been utilized as the recognition motif in probe design by several groups. in these probe designs covalent labeling is enabled by incorporation of photoreactive phenylazide [74] or diazirine [75] groups and irradiation by uv light. enrichment of the trapped proteins is performed by a further incorporated biotin moiety [74] or a terminal alkyne for attachment of either a biotin group or a fluorophore for detection following the covalent capture by click chemistry [75] . activity based probes for protein tyrosine phosphatases (ptps) were developed on several scaffolds that allow mimicry of phosphotyrosine motifs while incorporating reactive electrophiles for the covalent capture of active phosphatases (fig. 5) . several groups exploited the generation of quinone methides for the generation of reactive electrophiles for trapping of ptps [76] [77] [78] [79] . these are generated from substituted phenyl phosphates with benzylic fluoride substituents in ortho or para position. in a recent publication this concept was extended to the design of the unnatural amino acid 2fluoromethyl phosphotyrosine which was incorporated into peptides by solid phase peptide synthesis (spps) [80] . in this way peptidic activity based probes were produced based on known ptp target sequences. in earlier work kumar et al. combined a minimal motif incorporating a bromomethyl phosphonate-substituted phenyl derivative attached to either a fluorophore or biotin for detection [81] . successful labeling of ptps was demonstrated using both isolated enzymes in vitro and in cell lysates of different cancer cell lines. a further alternative approach exploited conjugate addition to substituted aryl vinyl sulfonates and sulfones for the covalent labeling of active ptps [82] . this methodology was then applied to a structural study of a covalent complex between the bacterial ptp yoph and its mechanism based inhibitor. recently a novel probe design was implemented that enables microscopy-based read out through the use of quenched activity based probes [83] . in this strategy the quencher is released upon quinone methide formation and reaction with the target enzyme. the methodology has also been applied to one-and two-photon imaging experiments. in comparison, the targeting of ser/thr phosphatases by activitybased probes has been less frequently realized. a successful example is that of a microcystin derived probe which was prepared and applied to the labeling and isolation of endogenous levels of ser/thr protein phosphatases from jurkat cell lysate [84] . acylphosphate kinase probes based on the atp cofactor [68] were used recently in an extensive study evaluating the specificity profiles of a range of kinase inhibitors [85] . in this 'in situ native kinase profiling' (kinativ) approach, labeling of mammalian cell lysates with the kinase probes was conducted in the presence or absence of the respective inhibitor. the complex samples were then digested with trypsin and biotinylated peptides enriched with streptavidin resin. following elution the captured peptides were analyzed by quantitative proteomics experiments using a targeted lc-ms 2 approach. this detection method significantly improved the signal to noise ratios and allows detection of lower amounts of analyte. quantitative inhibition data for over 200 kinases with six kinase inhibitors was obtained by this method. this approach provides access to quantitative inhibition profiles that are not easily obtained by other experimental approaches and exploits the strengths of both activity-based probe profiling and targeted mass spectrometry-based proteomics. the introduction of activity-based probes into the development of enzyme inhibitors enables the assessment of compound selectivity in a cellular context. this ideally complements more conventional in vitro enzyme assays and allows implementation of this assay format at an early stage in drug development. furthermore competitive labeling experiments have greatly aided the definition of specificities of inhibitors used in cellular research. new therapies in the ups will employ the manipulation of dub activity in a highly selective manner to achieve the desired outcomes. therefore inhibition of selected intracellular dub activities will receive increasing attention as a strategy for pharmacological intervention in disease. dubs with a validated role in oncology are expected to be the first in line for future dub inhibitors. provided that challenges of developing specific inhibitors with desirable compound profiles can be overcome, it is expected that this will enable successful intervention in other pathologies such as inflammatory diseases. activity-based probe designs have provided insights into multiple cellular pathways and have proven extremely valuable as tools for the study of ptm conjugating and deconjugating enzymes. the investigation of the ubiquitin proteasome system has benefited from the development of probes for proteasomal proteolysis as well as deubiquitylating enzymes. specificities and overlapping reactivities of ubiquitin-like proteases were investigated by analogous ubl probes. inhibitor development has been facilitated through competitive labeling assays enabling elucidation of inhibition profiles in a cellular environment. in addition, natural product derived kinase probes have demonstrated the protein targets of these covalent inhibitors, indicating previously unknown molecular targets and providing information in situations where specificities of the respective compounds are not always fully elucidated. the importance of access to selective inhibitors for basic cell biology and drug development makes this area highly suited for successful industry-academia collaborations. the ubiquitin-proteasome pathway: on protein death and cell life mechanism and function of deubiquitinating enzymes the ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on mhc class i molecules targeting proteins for destruction by the ubiquitin system: implications for human pathobiology protein degradation by the ubiquitinproteasome pathway in normal and disease states chemistry-based functional proteomics: mechanism-based activity-profiling tools for ubiquitin and ubiquitin-like specific proteases putting proteomics on target: activity-based profiling of ubiquitin and ubiquitin-like processing enzymes active-site directed probes to report enzymatic action in the ubiquitin proteasome system chemical tools to study the proteasome proteasome inhibitors: dozens of molecules and still counting substrate binding and sequence preference of the proteasome revealed by active-site-directed affinity probes the active sites of the eukaryotic 20s proteasome and their involvement in subunit precursor processing immunoproteasome assembly: cooperative incorporation of interferon γ (ifn-γ)-inducible subunits regulation of cd8+ t cell development by thymus-specific proteasomes activity probe for in vivo profiling of the specificity of proteasome inhibitor bortezomib total synthesis of the potent proteasome inhibitor epoxomicin: a useful tool for understanding proteasome biology extended peptide-based inhibitors efficiently target the proteasome and reveal overlapping specificities of the catalytic β-subunits profiling proteasome activity in tissue with fluorescent probes a fluorescent broad-spectrum proteasome inhibitor for labeling proteasomes in vitro and in vivo epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools chemistry in living cells: detection of active proteasomes by a two-step labeling strategy cell surface engineering by a modified staudinger reaction peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides a stepwise huisgen cycloaddition process: copper(i)-catalyzed regioselective "ligation" of azides and terminal alkynes affinity labeling of the proteasome by a belactosin a derived inhibitor chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family a novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of usp14 activity-based ubiquitin-specific protease (usp), profiling of virus-infected and malignant human cells specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins activity profiling of deubiquitinating enzymes in cervical carcinoma biopsies and cell lines a functional ubiquitin-specific protease embedded in the large tegument protein (orf64) of murine gammaherpesvirus 68 is active during the course of infection a gammaherpesvirus ubiquitin-specific protease is involved in the establishment of murine gammaherpesvirus 68 infection a deubiquitinating enzyme encoded by hsv-1 belongs to a family of cysteine proteases that is conserved across the family herpesviridae high-molecular-weight protein (pul48) of human cytomegalovirus is a competent deubiquitinating protease: mutant viruses altered in its active-site cysteine or histidine are viable artavanis-tsakonas, characterisation of the trichinella spiralis deubiquitinating enzyme, tsuch37, an evolutionarily conserved proteasome interaction partner identification by functional proteomics of a deubiquitinating/deneddylating enzyme in plasmodium falciparum chlamydia trachomatis-derived deubiquitinating enzymes in mammalian cells during infection ubiquitin and ubiquitin-like specific proteases targeted by infectious pathogens: emerging patterns and molecular principles ubiquitination, ubiquitin-like modifiers, and deubiquitination in viral infection bacterial interference of ubiquitination and deubiquitination viral avoidance and exploitation of the ubiquitin system structure of the ubiquitin hydrolase uch-l3 complexed with a suicide substrate crystal 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a tool for enzyme family subclassification, target identification, and inhibitor design discovering potent and selective reversible inhibitors of enzymes in complex proteomes protein cross-linking as a novel mechanism of action of a ubiquitin-activating enzyme inhibitor with anti-tumor activity effects of ps-341 on the activity and composition of proteasomes in multiple myeloma cells a phase 2 study of bortezomib in relapsed, refractory myeloma a novel orally active proteasome inhibitor induces apoptosis in multiple myeloma cells with mechanisms distinct from bortezomib comparative selectivity and specificity of the proteasome inhibitors bzlllcocho, ps-341, and mg-132 lmp2-specific inhibitors: chemical genetic tools for proteasome biology deubiquitinase inhibition by small-molecule wp1130 triggers aggresome formation and tumor cell apoptosis the multifaceted roles of usp7: new therapeutic opportunities usp47 is a deubiquitylating enzyme that regulates base excision repair by controlling steady-state levels of dna polymerase beta discovery of specific inhibitors of human usp7/hausp deubiquitinating enzyme inhibition of proteasome deubiquitinating activity as a new cancer therapy a noncovalent class of papain-like protease/deubiquitinase inhibitors blocks sars virus replication functional interrogation of the kinome using nucleotide acyl phosphates wortmannin, a widely used phosphoinositide 3-kinase inhibitor, also potently inhibits mammalian polo-like kinase a cell-permeable, activity-based probe for protein and lipid kinases polo-like kinases inhibited by wortmannin: labeling site and downstream effects design and synthesis of ax4697, a bisindolylmaleimide exo-affinity probe that labels protein kinase c alpha and beta small molecule probes that target abl kinase comprehensive identification of staurosporine-binding kinases in the hepatocyte cell line hepg2 using capture compound mass spectrometry (ccms) proteome profiling reveals potential cellular targets of staurosporine using a clickable cell-permeable probe study of the preferred modification sites of the quinone methide intermediate resulting from the latent trapping device of the activity probes for hydrolases design and synthesis of class-selective activity probes for protein tyrosine phosphatases activity-based fluorescent probes that target phosphatases activity-based probes for protein tyrosine phosphatases peptide-based activity-based probes (abps) for target-specific profiling of protein tyrosine phosphatases (ptps) global analysis of protein tyrosine phosphatase activity with ultra-sensitive fluorescent probes aryl vinyl sulfonates and sulfones as active site-directed and mechanism-based probes for protein tyrosine phosphatases multicolor, one-and two-photon imaging of enzymatic activities in live cells with fluorescently quenched activity-based probes (qabps) design and synthesis of ax7574: a microcystinderived, fluorescent probe for serine/threonine phosphatases situ kinase profiling reveals functionally relevant properties of native kinases key: cord-007682-01iom9al authors: harrigan, jeanine; jacq, xavier title: monitoring target engagement of deubiquitylating enzymes using activity probes: past, present, and future date: 2016-09-10 journal: proteostasis doi: 10.1007/978-1-4939-3756-1_26 sha: doc_id: 7682 cord_uid: 01iom9al deubiquitylating enzymes or dubs are a class of enzymes that selectively remove the polypeptide posttranslational modification ubiquitin from a number of substrates. approximately 100 dubs exist in human cells and are involved in key regulatory cellular processes, which drive many disease states, making them attractive therapeutic targets. several aspects of dub biology have been studied through genetic knock-out or knock-down, genomic, or proteomic studies. however, investigation of enzyme activation and regulation requires additional tools to monitor cellular and physiological dynamics. a comparison between genetic ablation and dominant-negative target validation with pharmacological inhibition often leads to striking discrepancies. activity probes have been used to profile classes of enzymes, including dubs, and allow functional and dynamic properties to be assigned to individual proteins. the ability to directly monitor dub activity within a native biological system is essential for understanding the physiological and pathological role of individual dubs. we will discuss the evolution of dub activity probes, from in vitro assay development to their use in monitoring dub activity in cells and in animal tissues, as well as recent progress and prospects for assessing dub inhibition in vivo. otherwise distinct. the ubiquitin-proteasome system has multiple essential biological roles, and thus its function and dysfunction, are important factors in various human diseases, including cancer, infection, infl ammation , and neurodegeneration [ 1 -4 ] . ubiquitylation of substrate proteins fi rst involves an atpdependant activation of the ubiquitin polypeptide by the activating enzyme e1 . activation involves covalent linkage between the carboxy terminus of ubiquitin and a cysteine residue present on the e1, forming a thioester bond. the activated ubiquitin is then transferred to an e2 ubiquitin-conjugating enzyme forming a thioester linkage. in the fi nal step, an e3 ligase transfers the ubiquitin from the e2 to the substrate protein. the majority of e3 ligases are classifi ed as ring fi nger e3s and act by bringing the substrate and e2 enzyme in close proximity. the ring fi nger e3s directly transfer ubiquitin from the e2 to the substrate. the hect domain e3s act by forming an intermediate thioester linkage with ubiquitin before transfer to the substrate (reviewed in [ 5 ] ). more recently, a third class of e3 ligases with an intermediate mechanism of action has been identifi ed. the ring-in-between-ring (rbr) e3s are an unusual family of ubiquitin e3-ligases composed of a dozen proteins. their activities are autoinhibited, causing a requirement for activation by protein-protein interactions or posttranslational modifi cations . they catalyze ubiquitin conjugation by a concerted ring/hect-like mechanism in which the ring1 domain facilitates e2-discharge to directly form a thioester intermediate with a cysteine in ring2. this short-lived, hect-like intermediate then modifi es the target [ 6 , 7 ] . following monoubiquitylation of a substrate, the process can either stop, forming monoadducts of ubiquitin, or be repeated forming an elongated chain of ubiquitin residues. polyubiquitin chains can be formed using the n-terminus (linear) or any of the seven internal lysine residues found in ubiquitin, and these various chain topologies lead to different functional outcomes. of the most well studied linkages, k63-linked polyubiquitin chains are often involved in nonproteolytic signal transduction while k48linked chains generally target substrates for proteasomal degradation. a number of additional linkages such as met1, k6, k11, k27, k29, and k33 have been identifi ed and their nondegradative cellular signaling roles are still subject to a number of investigations. the complexity of ubiquitin chain signaling is further enhanced by the existence of mixed-lineage chains [ 8 , 9 ] . proteins destined for degradation via the ubiquitin-proteasome system include proteins that are damaged, improperly folded, or that have short half-lives [ 10 ] . proteins that have been appropriately polyubiquitylated are recognized and degraded by the 26s macromolecular proteasome complex [ 11 ] . the 26s complex consists of a 20s catalytic core particle that is capped at both ends by 19s regulatory particles . the 19s regulatory particle can be further subdivided into lid and base components. following recruitment to the proteasome, polyubiquitylated proteins undergo deubiquitylation and unfolding. the removal of ubiquitin is accomplished by deubiquitylating enzymes (dubs) associated with the 19s lid. ubiquitin polypeptides that are removed from substrate proteins can be directly recycled by the cell. the 19s base component plays a key role in the unfolding of the substrate protein and delivery of the deubiquitylated, unfolded protein into the 20s catalytic core particle. the 20s consists of four layers of ring-like structures [ 12 ] . the outer rings are composed of seven α subunits with the inner rings composed of seven β subunits. the β1 subunits exhibit caspase-like activity, the β2 subunits trypsin-like activity, and β5 subunits chymotrypsin-like activity, collectively degrading proteins into short oligopeptides as well as recycling amino acids [ 5 , 13 ] . ubiquitin is covalently linked to many cellular proteins and regulates their activity, stability, localization, or interactions. ubiquitylation is a reversible process carried out by the opposing activities of ubiquitin ligases and dubs. the human genome encodes approximately 100 dubs [ 14 -16 ] . of the fi ve families of dubs, four ( uch , usp, mjd , and otu ) belong to the cysteine peptidase class, while one (jamm) belongs to the metallopeptidase class. as dubs have been shown to play critical roles in many pathological processes, particularly cancer, infectious disease, and neurodegeneration, they have begun to attract signifi cant attention from the pharmaceutical industry [ 17 -20 ] . unlike most posttranslational modifi cations , ubiquitin is able to form polymeric chains [ 21 ] : the ubiquitin linkage in the chain as well as the length of the chain will impact on the fate of the protein modifi ed by the polymer of ubiquitin [ 9 , 22 ] . pharmacological modulation of dubs using a multitude of approaches in the last decade has seen limited success to date; however, recent progress is beginning to identify dub inhibitors with the potential for drug development [ 23 -27 ] . a number of conceptual and technological obstacles need to be overcome in order to progress genuine dub therapies. a major challenge in characterization of dub inhibitors is the development of high throughput assays monitoring "on-target" inhibition in cells and in vivo. monitoring dub target engagement by small molecule inhibitors in vivo has a number of implications. firstly, as a biomarker readout of inhibition and for understanding the physiological implication of inhibiting a class of enzymes for which there is usually no known unique ubiquitylated substrate. secondly, for assessment of the selectivity of compounds as well as understanding the mechanism of action of the inhibition, including duration, reversibility, and pharmacodynamic parameters. activity-based probes (abps) rely on the design of chemical warheads which selectively react with the active site of an enzyme. abps are usually composed of a reactive electrophile, to covalently modify an active-site residue, and a reporter group to allow detection of the labeled enzyme [ 28 ] , see fig. 1a , b . activity probes have been designed for a number of enzyme classes such as serine hydrolases [ 29 ] , metalloproteases [ 30 , 31 ] , proteasomes [ 32 ] , and oxidoreductases [ 33 ] . epitope-tagged ubiquitin and ubiquitin-like derivatives have been utilized in a variety of assays to identify or monitor active dubs in biological samples [ 34 , 35 ] (fig. 1c ) . ubiquitin abps have been instrumental in the identifi cation of a number of new dubs [ 36 ] including a novel class of dubs: otus [ 37 ] . unlike other proteolytic enzymes, for optimal recognition, dubs require not only an electrophilic trap but also a very large portion of ubiquitin or chains of ubiquitin for binding and recognition in the enzyme active site: truncated portions of ubiquitin are usually not suffi cient to trap dubs. in addition, the isopeptide nature of the covalent linkage of ubiquitin to the target protein monitoring the activity of endogenous enzymes such as dubs in their native, full-length status as well as under all possible naturally occurring posttranslational modifi cations or interference/allosteric regulation from binding partners is a major advantage of abps . the irreversible covalent nature of abps toward their enzyme targets has a number of advantages when compared to many other analytical technologies that rely on weak, naturally transient and diffi cult to capture interactions between an enzyme and its substrate. various warheads (fig. 2 ) have been employed including alkyl halides (chloroethyl, bromoethyl, bromopropyl), michael acceptors ( vinyl methyl ester (vme) , vinyl methyl sulfone (vms) , vinyl phenyl sulfone, vinyl cyanide) and more recently propargyl (pa) [ 36 , 38 , 39 ] . the fi rst attempt at generating activity probes to label dubs on their catalytic site thiol group was described by hidde ploegh and colleagues [ 35 ] . using a trypsin catalyzed transpeptidation to modify ubiquitin at its carboxy terminus with a vinyl sulfone group, they were able to demonstrate that ubiquitin vinyl sulfone labeled not only recombinant purifi ed dubs but also a number of yeast dubs in a crude lysate. the identity of each labeled band was verifi ed using individual yeast dub mutant strains. the initial version of the ubiquitin vinyl sulfone probe was labeled with iodine 125 and allowed for detection of a number of dubs in mouse tissues as well as in mouse cell lysates. in the same study, borodovsky et al. described the use of unlabeled ubiquitin vinyl sulfone to detect a specifi c dub by monitoring a shift in the apparent molecular weight in sds-page followed by immunoblotting: usp7 was labeled effi ciently in mammalian cell lysates. finally, the authors were also able to identify usp14 as a novel dub associated with the proteasome thanks to the use of ubiquitin vinyl sulfone in fractionation and immune-purifi cation assays. in a second generation of activity probes, the thiol-reactive group was added to ubiquitin using an intein-based chemical ligation method [ 36 ] . the reactivity of the dubs depends on the type of electrophilic warhead fused to ubiquitin. the second generation of probes were additionally used for the identifi cation of bound dubs by affi nity purifi cation/ mass spectrometry [ 34 ] . more recently, abps using a fl uorescent reporter tag have been generated to replace the initial tags (e.g., ha) to allow replacement of the immunoblot procedure with fl uorescent imaging [ 39 -41 ] . while the historical production of ubiquitin abps was based on a trypsin catalyzed transpeptidation to modify ubiquitin at its carboxy terminus with a vinyl sulfone group or based on the addition of the electrophilic warhead via intein-based chemical ligation methods, recent approaches have moved toward the full-chemical synthesis of ubiquitin abps [ 41 , 42 ] . this latest improvement has the added advantage of allowing the incorporation of modifi ed amino acid residues at any position in the abps, whether natural or not. in addition their major role in monitoring or identifying active dubs in biological samples, ubiquitin-based probes are useful tools for structural analysis of dubs . a number of cocrystals of dubs with ubiquitin have been solved [ 34 , 39 ] and in some cases, the structure of the apo-dub was not achieved in the absence of the modifi ed ubiquitin abp [ 43 ] . abps are sometimes the only option available for cocrystallizing dubs with ubiquitin substrates or ubiquitin chains . a limited number of studies have demonstrated the utility of activity probes for monitoring dub activity in normal or diseased animal tissues . in earlier dub activity probe publications, mouse tissues were examined and signifi cant differences in the profi le of active dubs in tissues was observed [ 35 ] . more recently, in a very detailed study, altun et al. investigated the activity of dubs in models of aging and dietary restriction [ 44 ] . dramatic differences in the levels of active dubs in cell lines derived from various tissues as well as in primary tissues has been observed [ 45 ] . for a small number of dubs , the activity as monitored by activity probe binding can be correlated with the malignant status of the cell line or tissue, suggesting a possible therapeutic window for dub inhibitors [ 45 ] . given the sensitivity and signifi cance of such techniques, we can expect an increase in the number of studies taking full advantage of ubiquitin abps to monitor differential dub activity in pathological versus normal conditions in the near future. abps have been used to identify and monitor the activity of bacterial, viral, or parasitic dubs including herpes viridae , chlamydia trachomatis , toxoplasma gondii , and plasmodium falciparum : abps are invaluable tools to identify functionally active dubs in complex in sometimes relatively poor or diffi cult to annotate organisms [ 46 -50 ] . while viruses or bacteria do not encode a full complement of ubiquitin proteasome enzyme systems, they express dubs to evade the detection of their proteins by the immune system or otherwise enhance virulence [ 51 ] . in addition, since dubs are essential for viral proliferation, viral dubs have been considered as possible therapeutic strategies for the treatment of certain viral infections such as sars or mers [ 52 ] . mass spectrometry has emerged as an important tool for characterizing the various forms of ubiquitin. initial global characterization of the ubiquitin-modifi ed proteome has been made possible in proteomic studies taking advantage of a monoclonal antibody that recognizes (di-gly)-containing isopeptides following trypsin digestion of complex cellular lysates [ 53 , 54 ] . in the ubiquitin-aqua approach, synthetic isotopically labeled internal standard peptides are used to quantify branched peptides and the branched -gg signature peptides generated by trypsin digestion of ubiquitin signals [ 55 ] . proteomic studies looking at dub interaction partners have also generated a great deal of information about their substrates, regulation, and function [ 56 ] . additional studies have evaluated the functional role of dubs using rnai libraries [ 57 , 58 ] or gfp-dub fusions [ 59 , 60 ] , and have linked dubs to specifi c cellular pathways. while such studies are very informative and have generated a wealth of data on the biological roles of dubs, they provide only limited information regarding the dynamic activity profi le of dubs, and are not able to distinguish the catalytic state (active versus inactive) of dubs. as the cellular activity of dubs can be controlled by multiple factors including protein interactions [ 61 ] , stoichiometric changes to the structure of the protein [ 62 , 63 ] , and posttranslational modifi cations [ 64 , 65 ] , the advantage of activity probes is their specifi c reactivity with catalytically active dubs . one of the benefi ts of abps for the characterization of dub inhibitors is the ability to monitor compound selectivity. a chemical activity-based proteomic approach using ha-tagged ubiquitin labeled with electrophilic warheads (ha-ubbr2 or ha-ub-vme) was undertaken to characterize the selectivity of two usp7 inhibitors either in immunoblots or by quantitative mass spectrometry following treatment of cells or cell lysates with compounds [ 66 ] . an independent study using another usp7 inhibitor displaying selectivity in a panel of biochemical dub assays, was also subjected to cellular selectivity profi ling using ha-ub-vms followed by immunoblotting [ 24 ] . in a more targeted approach, an active-site ubiquitin probe (ha-ub-vms) has been used to demonstrate that usp14/uchl5 inhibition by a small molecule (b-ap15) inhibits the 19s proteasome in a reconstituted biochemical assay. a similar probe approach was also used to demonstrate that b-ap15 is not a general inhibitor of dubs in a cell lysate probed with an anti-ha antibody detecting the conjugated ubiquitin species [ 67 ] . while the studies mentioned above are paving the way for elucidating dub selectivity profi les in a cellular context, coverage of the " dubome " is still limited. technological improvements are still required to increase sensitivity and accurately monitor dubs in a given cell or tissue experiment. while dub proteomic studies using activity probes have mainly been used for monitoring the selectivity of fi rst generation dub inhibitors , the potential for ubiquitin abps is much broader. indeed, it is possible to determine the dynamic nature of dub inhibitors by using abps to monitor the reversibility or the duration of dub inhibition. furthermore, most of the work so far on dubs using abps has been restricted to cellular studies. recent progress in developing dub inhibitors with in vivo preclinical potential is currently driving the tools for pharmacodynamic as well as mode-of-action understanding of dub inhibitors in vivo. activity probes based on selective inhibitors of peptidases have already been developed such as probes targeting proteasomes [ 68 ] , cathepsins [ 69 ] or caspases [ 70 ] and are proving their usefulness for in vivo imaging studies as well as for diagnostic purposes [ 71 ] . the utility of ubiquitin activity probes to identify and characterize dubs in a number of conditions is not limited to ubiquitin. indeed, probes for enzymes that remove ubls have been generated. the exquisite selectivity of dubs for their cognate substrates suggested that specifi c probes are also required for ubl peptidases. an initial approach based on the synthesis of peptide vinyl sulfones harboring various portions of the ubiquitin-like carboxy terminus has suggested that truncated ubls are able to bind ubl-specifi c proteases in a manner similar to the ubiquitin-based vinyl sulfone polypeptides [ 72 ] . ubl-based probes for nedd8 , sumo-1, isg15, gate-16, map1-lc3, gabarap, and apg8l have been successfully synthesized [ 73 -75 ] . an alternative to classical activity probes containing a full ubiquitin or ubiquitin-like polypeptide is based on the use of small molecule inhibitors to label the catalytic site of desumoylating enzymes (sentrin-specifi c proteases, senps ). a peptide acyloxymethyl ketone (aomk) containing a large aromatic o-acyl group are selective covalent inhibitors of senps and can be modifi ed using fl uorescent labels to detect senps activity in biological samples [ 76 ] . a similar approach has been described using a different family of proteins: glycine fl uoromethylketones, which serve as probes to selectively target senps [ 77 ] . a more conventional derivatization of the carboxy-terminal end of ubls with electrophilic warheads has also been pursued and a general derivatization procedure to produce any ubl domain chemically activated at its c-terminus by formation of a thiol ester. reaction of the thiol with a nucleophile produces the desired derivatives taking advantage of the intein fusion technology [ 78 ] . there is no technical challenges preventing the development of fully synthetic ubl abps and indeed, a number of such reagents are already commercially available from various sources. as the mechanism for the removal of ubls by specifi c enzymes has not yet been fully characterized, abps will certainly play a key role in the elucidation of such understanding. similarly, the biological or mechanistic functions of a number of dubs or senps remains poorly understood, and existing abps or novel more selective abps can serve as tools for extending our knowledge. in parallel with the development of monoubiquitin abps , a number of groups have also achieved total (semi)-synthesis of di-ubiquitin [ 42 , 79 -81 ] or even tetra-ubiquitin chains [ 82 , 83 ] . however, incorporation of electrophilic warheads into polyubiquitin chains remains problematic. an intermediate approach to the generation of polyubiquitin abps was elaborated on the basis of the synthesis of branched-peptides incorporating an isopeptide-linked ubiquitin and an electrophilic warhead [ 84 ] . in addition, the synthesis and characterization of k48or k63-linked di-ubiquitin probes bearing dehydroalanine as a warhead near the isopeptide bond has been described [ 85 ] . finally, abps engineered for di-ubiquitin chains incorporating the 8 known ubiquitin linkages have been successful and now allow dub ubiquitinlinkage specifi city in a cellular context to be addressed [ 86 -88 ] . structural studies of dubs with di-ubiquitin have demonstrated that in addition to the peptide fl anking the ubiquitylated residues, more extensive interactions between dubs and the proximal ubiquitin in the chain also contribute to the recognition by dubs. probing dub selectivity with the latest generation of probes not only generates a distinct pattern from that obtained using mono-ubiquitin abps, but also suggests that the promiscuity of some dubs for their substrates is probably much less pronounced than initially anticipated. in the last couple of years, posttranslational modifi cations of ubiquitin, especially phosphorylation of ubiquitin at specifi c residues (e.g., ser57 and ser65) have been shown to play important roles in a number of cellular processes [ 89 , 90 ] . ubiquitin abps bearing the phosphorylated variants of ubiquitin have been generated and used to probe the selectivity of the modifi cations for conjugating and deconjugating enzymes. e1 and e2 enzymes are usually able to tolerate phosphorylated ubiquitin, however, a number of dubs have diffi culty recognizing the modifi ed substrates [ 91 , 92 ] . studies evaluating additional posttranslational modifi cations of ubiquitin such as methylation, acetylation, hydroxylation, or other phosphorylation will certainly be unraveled in the near future: the corresponding abps will again serve as useful tools to understand the mechanistic and physiological role of novel variants of ubiquitin. a key issue facing researchers involved in deciphering the roles of dubs in a cellular context is the lack of understanding of the most direct or relevant substrate of specifi c dubs in a given cellular pathway. some dubs have very well characterized substrates (e.g., usp1 or usp7) [ 15 ] that are clearly linked to the function of the dubs, however, the known substrate specifi city is still relatively poor or partial at best for most dubs. in certain cases, it is quite clear that unique substrates do not exist: e.g., usp14 or uchl5 are dubs that indiscriminately recognize any ubiquitylated substrates which is targeted to the proteasome [ 93 ] . ubiquitin abps can play a critical role as tools to monitor the dynamics of the activation or inhibition of dubs under specifi c physiological or pharmacological pathway alterations. the problem is especially acute for the monitoring of dub activity upon inhibition with specifi c inhibitors: the pharmaceutical development of dub inhibitors requires a good understanding of the pharmacokinetic modulation of the target upon treatment with compounds. the development of abps for proteomic evaluation of target engagement is currently being investigated by a number of groups. in addition, higher throughput abp-based strategies are also under development for the determination of dub target engagement in cellular contexts as well as in tissues or eventually for clinical sample evaluation (fig. 3 ) . the development of activity probes for dubs has lagged behind the development of probes for more classical proteases. indeed, the complexity of the recognition site of dubs, which requires the binding of full-length ubiquitin in the catalytic site as well as the challenges in the characterization of potent and selective dub inhibitors , has hindered production of abps for dubs. however, following on from the ground-breaking evolution of cell-permeable and in vivo-compatible activity-based imaging probes developed for other proteases such as caspases or cathepsins [ 69 , 70 ] , the next generation of probes for dubs will certainly be agents that enable direct visualization and quantifi cation of dub activity in vivo. such noninvasive agents have great potential for early diagnosis as well as pharmacodynamic evaluation of dub inhibition in preclinical as well as clinical settings. one attractive avenue to explore for the development of selective dub activity probes is based on the design of copper-catalyzed click-labeled dub inhibitors with quenchable or nonfl uorescent labels [ 94 ] . click-labeled abps allows for selective labeling, visualization, and enrichment of active enzymes in a complex proteome. another approach will likely be based on the generation of noninvasive substrate probes that do not bind covalently to the enzyme. the advantage of this approach is based on the theoretically higher signal that can be generated, in contrast to covalent activity probes which are limited by the stoichiometric labeling of the enzyme (the signal being proportional to the amount of enzyme in various tissues). so far a very limited number of reporter substrates are available, none being cell permeable, or suitable for in vivo applications. again, noninvasive permeable substrates will likely be derived from selective inhibitors of individual dubs or knowledge around selectively ubiquitylated sites on dub substrates. probably one of the most promising avenues for developing cell-and tissue-permeable selective ubiquitin abps for dubs will rely on the modifi cation of selective small molecule inhibitors of dubs . similar approaches have already achieved some preliminary success for other enzymes of the ups such as e1 enzymes [ 95 ] and proteasome probes [ 96 ] . the limiting step in developing such probes for dubs is currently a lack of potent, specifi c and selective dub inhibitors available, however, the community is successfully designing novel generations of selective dub inhibitors. while a number of abps have been successfully designed for monitoring the activity of cysteine peptidase dubs , there is still a gap in the development of abps for dubs of the metalloenzyme class (mpn+/jamms) . a number of approaches are currently being investigated for the design of abps for metallo-dubs and will certainly aid the characterization of inhibitors for that class of enzymes which is showing great promise as therapeutic targets [ 97 -99 ] . protein ubiquitylation is critical for the control of protein half-life, localization, and function. deregulation of this process is a causative factor of many diseases. the development of abps has allowed for major advancement in the identifi cation and characterization of cysteine dubs. signifi cant progress has been made in terms of probe design and preparation. for example, fi ve papers have been published in the past 3 years describing di-ubiquitin abps, underscoring the importance of these tools for dub research. the jamm family remains diffi cult to target using abps due to the catalytic mechanism which does not involve a covalent dubsubstrate intermediate. hopefully new approaches and novel probe designs will yield better tools to investigate this class of metalloproteases. abps will ultimately shed light on the function and relevance of dubs involved in various chain-specifi c ubiquitin signaling, and will continue to advance our knowledge of dub regulation and function in a cellular context. furthermore, abps will aid the development and characterization of dub inhibitors , allowing the monitoring of target engagement as well as selectivity in vivo. finally, while ubiquitin abps have not yet been as broadly used as one might expect to monitor dubs in developmental or pathological evaluations, they can provide a unique dynamic assessment of the activity of dubs, and will undoubtedly become a more familiar option for many researchers. ubiquitin-like protein conjugation and the ubiquitin-proteasome system as drug targets mdc1 is required for the intra-s-phase dna damage checkpoint diversity of 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understanding ubiquitin-modifying enzymes: from pharmacological targeting to proteomics deubiquitylating enzymes and dna damage response pathways ubiquitin chain conformation regulates recognition and activity of interacting proteins dubs, the regulation of cell identity and disease small-molecule inhibitor of usp7/hausp ubiquitin protease stabilizes and activates p53 in cells discovery of specifi c inhibitors of human usp7/hausp deubiquitinating enzyme a small molecule inhibitor of ubiquitin-specifi c protease-7 induces apoptosis in multiple myeloma cells and overcomes bortezomib resistance a novel small molecule inhibitor of deubiquitylating enzyme usp14 and uchl5 induces apoptosis in multiple myeloma and overcomes bortezomib resistance targeting deubiquitinase activity with a novel small molecule inhibitor as therapy for b-cell malignancies activity-based protein profi ling: from enzyme chemistry to proteomic chemistry activity-based protein profi ling: the serine hydrolases developing photoactive affi nity probes for proteomic profi ling: hydroxamate-based probes for metalloproteases activity-based probes for the proteomic profi ling of metalloproteases activity probe for in vivo profi ling of the specifi city of proteasome inhibitor bortezomib proteomic profi ling of mechanistically distinct enzyme classes using a common chemotype mechanism-based proteomics tools based on ubiquitin and ubiquitin-like proteins: crystallography, activity profi ling, and protease identifi cation a novel active sitedirected probe specifi c for deubiquitylating enzymes reveals proteasome association of usp14 chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family otubains: a new family of cysteine proteases in the ubiquitin pathway chemistry-based functional proteomics: mechanism-based activityprofi ling tools for ubiquitin and ubiquitin-like specifi c proteases on terminal alkynes that can react with active-site cysteine nucleophiles in proteases fluorescence-based active site probes for profi ling deubiquitinating enzymes ubiquitin-based probes prepared by total synthesis to profi le the activity of deubiquitinating enzymes chemical synthesis of ubiquitin, ubiquitin-based probes, and diubiquitin structural basis of ubiquitin recognition by the deubiquitinating protease usp2 muscle wasting in aged, sarcopenic rats is associated with enhanced activity of the ubiquitin proteasome pathway activity-based ubiquitinspecifi c protease (usp) profi ling of virusinfected and malignant human cells a deubiquitinating enzyme encoded by hsv-1 belongs to a family of cysteine proteases that is conserved across the family herpesviridae structure of a herpesvirus-encoded cysteine protease reveals a unique class of deubiquitinating enzymes chlamydia trachomatisderived deubiquitinating enzymes in mammalian cells during infection apicomplexan uchl3 retains dual specifi city for ubiquitin and nedd8 throughout evolution identifi cation by functional proteomics of a deubiquitinating/deneddylating enzyme in plasmodium falciparum chladub1 of chlamydia trachomatis suppresses nf-kappab activation and inhibits ikappabalpha ubiquitination and degradation the sars-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds systematic and quantitative assessment of the ubiquitin-modifi ed proteome improved quantitative mass spectrometry methods for characterizing complex ubiquitin signals dynamics of cullin-ring ubiquitin ligase network revealed by systematic quantitative proteomics defi ning the human deubiquitinating enzyme interaction landscape functional annotation of deubiquitinating enzymes using rna interference deubiquitinase activities required for hepatocyte growth factor-induced scattering of epithelial cells systematic survey of deubiquitinase localization identifi es usp21 as a regulator of centrosome-and microtubuleassociated functions systematic characterization of 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sensitivities of e1-e2 enzymes and deubiquitinases the proteasome under the microscope: the regulatory particle in focus applications of copper-catalyzed click chemistry in activity-based protein profi ling development of activity-based probes for ubiquitin and ubiquitin-like protein signaling pathways activity-based imaging probes of the proteasome endocytosis: the dub version jamm: a metalloprotease-like zinc site in the proteasome and signalosome cop9 signalosome function in the ddr the authors would like to acknowledge networking support by the proteostasis cost action (bm1307) . key: cord-307983-gjdza9bh authors: hawdon, james; parti, katalin; dearden, thomas e. title: cybercrime in america amid covid-19: the initial results from a natural experiment date: 2020-06-10 journal: am j crim justice doi: 10.1007/s12103-020-09534-4 sha: doc_id: 307983 cord_uid: gjdza9bh the covid-19 pandemic has radically altered life, killing hundreds of thousands of people and leading many countries to issue “stay-at-home” orders to contain the virus’s spread. based on insights from routine activity theory (cohen & felson 1979), it is likely that covid-19 will influence victimization rates as people alter their routines and spend more time at home and less time in public. yet, the pandemic may affect victimization differently depending on the type of crime as street crimes appear to be decreasing while domestic crimes may be increasing. we consider a third type of crime: cybercrime. treating the pandemic as a natural experiment, we investigate how the pandemic has affected rates of cybervictimization. we compare pre-pandemic rates of victimization with post-pandemic rates of victimization using datasets designed to track cybercrime. after considering how the pandemic may alter routines and affect cybervictimization, we find that the pandemic has not radically altered cyberroutines nor changed cybervictimization rates. however, a model using routine activity theory to predict cybervictimization offers clear support for the theory’s efficacy both before and after the pandemic. we conclude by considering plausible explanations for our findings. presence of a suitable target, and (3) the absence of a capable guardian. routine activity theory proposes that victimization stems from the "recurrent and prevalent activities" that individuals are involved in, which in turn influence the likelihood that the three necessary factors are present (cohen & felson, 1979) . therefore, routines influence an individual's risk of being victimized. while rat cannot be directly applied to the online world (see yar, 2005; yar, 2013; tillyer & eck, 2009) , the cyberlifestyle-routine activities perspective (see reyns, henson, & fisher, 2011; eck & clarke, 2003) overcomes the primary issue limiting the theory's applicability. most notably, while offline victimization requires a convergence in time and space of offenders and victims, cybervictims and offenders can come into virtual contact through their networked devices, and this contact can happen asynchronously (leukfeldt & yar, 2016; reyns et al., 2011; vakhitova, reynald, & townsley, 2015) . with this modification in mind, online routine activities can increase the likelihood of victimization by bringing potential targets into virtual contact with offenders in environments lacking guardians (see eck & clarke, 2003; reyns et al., 2011) . researchers have now applied rat to a variety of types of cybervictimization, ranging from fraud and identity theft to harassment and other forms of cyberviolence (e.g., bossler & holt, 2009; bossler, holt, & may, 2012; costello, hawdon, ratliff, & grantham, 2016; hawdon, bernatzky, & costello, 2019; hawdon, oksanen, & räsänen, 2015; hawdon, oksanen, & räsänen, 2017; holt & bossler, 2008; holt & bossler, 2013; marcum, higgins, & ricketts, 2010; navarro & jasinski, 2012; navarro & jasinski, 2013; pratt, holtfreter, & reisig, 2010; reyns, 2013; reyns & henson, 2015; van wilsem, 2011) . studies using rat to predict cybervictimization generally find that engaging in risky online behaviors such as downloading games and music from unknown websites, using file-sharing programs, instant messaging, opening unknown email attachments, and clicking on pop-up messages increases cyberharassment (hinduja & patchin, 2009; marcum, 2009; marcum et al., 2010; navarro & jasinski, 2012) . similarly, general computer use, anonymously confiding in others online, playing video games, spending time in chatrooms, online shopping, and the use of social networking sites, and adding strangers as friends to the social networking accounts have been reported to increase the likelihood of being a victim of cyberviolence (e.g. bossler & holt, 2009; bossler et al., 2012; costello et al., 2016; hawdon, oksanen, & räsänen, 2014; holt & bossler, 2008; leukfeldt & yar, 2016; navarro & jasinski, 2012; reyns et al., 2011; reyns, henson, & fisher, 2016; van wilsem, 2011) . the use of target-hardening devices such as antivirus programs, firewalls, and filtering and blocking software can potentially reduce cybervictimization, although the effect may only apply to economic victimization (e.g., leukfeldt, 2014; marcum, 2008; marcum et al., 2010) . as mentioned above, the stay-at-home orders enacted to combat the spread of covid-19 have radically altered the daily routines or millions of americans. with decreased mobility due to shelter-in-place orders, people are increasingly teleworking. according to one study, 88% of organizations have encouraged or required their employees to work from home because of the pandemic (gartner, 2020) . in addition to more people relying on technology to telework, the use of social media sites such as tiktok, twitter, facebook, and instagram are also spiking (yitzhak, 2020) . as we spend more time online, our cyber-routines change, and we would anticipate these changes would alter cybervictimization rates. but how specifically would the covid-19 pandemic likely alter our proximity to motivated offenders, suitability as a potential target, and online guardianship? let us speculate on each of these. covid-19 and virtual proximity to motivated offenders first, the unemployment rate has surged above 20% and is expected to reach levels not seen since the great depression, millions have lost their jobs, had their hours reduced, or have been furloughed, and the nation's small business owners are struggling to remain in business (bartash, 2020; lambert, 2020; wolfer, 2020) . as a revised version of rat argues (see bryant & miller, 1997) areas with large secondary labor markets have high crime rates in part because workers in the secondary sector frequently experience unemployment, which may compel them to find alternative means of support. thus, the radical shift in the employment structure of the nation to which these dire economic numbers attest has likely led to heightened economic need and increased motivations to steal. combined with the increase in the number of people going online, we would anticipate an increase in the virtual presence of motivated offenders in cyberspace. the increased presence of motivated offenders in cyberspace during the pandemic, assuming it is indeed the case, should increase overall rates of cybervictimization. while this proposition is likely to be true, motivated offenders are a necessary but not sufficient condition for victimization. indeed, cohen and felson (1979) assumed such offenders were omnipresent, and this truism is probably even more enhanced in cyberspace because its asynchronous nature allows offenders to be "virtually present" even when they are not personally online. thus, while more motivated offenders being online is likely to elevate rates of cybervictimization, the overall patterns are likely more affected by changes in target suitability and guardianship that result from the pandemic. covid-19 and target suitability independent of the number of offenders prowling virtual space, the online routines of potential victims also shapes their likelihood of being victimized by determining a target's suitability. suitable targets include any person or object that can fulfill the needs or wants of a motivated offender (cohen & felson, 1979) , and target suitability is a function of viva: the target's value, inertia, visibility, and access (felson & clarke, 1998) . value is the worth a person or object has in the eyes of a potential offender, inertia is the target's ability to avoid the offender, access is the opportunity for an offender to commit the illegal act, and visibility is the extent to which offenders can see a possible victim. these factors are interrelated and also related to the extent of contact targets have with motivated offenders. it is likely that the pandemic would influence target suitability in several ways. as noted above, the pandemic has resulted in people spending more time online. all things being equal, spending more time online would increase the potential victim's visibility to likely offenders. indeed, research indicates that the proportion of users who access the internet only from home is positively related to cybertheft victimization (song, lynch, & cochran, 2016) . however, simply spending more time online may not necessarily result in a greatly enhanced probability of being victimized because overall time spent online is likely less important than the specific online activities in which one engages. for example, spending 8 hours online teleworking is probably not likely to bring one into a virtual space where motivated offenders lurk. in contrast, spending even 1 hour surfing the dark web very well might increase one's exposure to motivated offenders. thus, online activities that lead one to visit "dangerous virtual spaces" will increase a potential target's visibility and the offender's access more so than those activities that occur in more secure online spaces (see costello, barret-fox, bernatzky, hawdon, & mendes, 2018; räsänen et al., 2016) . how the covid-19 pandemic and resulting stay-at-home orders will likely affect target suitability is undoubtedly complex. for example, as previously mentioned, the limited available evidence suggests that some activities known to be related to victimization because they may lead users into dangerous virtual spaces have undoubtedly increased (yitzhak, 2020) . these "dangerous" online routines would include surfing the dark web, playing online video games, online shopping, and visiting social media sites as all of these activities have been reported to increase cybervictimization (bossler & holt, 2009; bossler et al., 2012; costello et al., 2016; hawdon et al., 2014; leukfeldt & yar, 2016; navarro & jasinski, 2012; reyns et al., 2011; van wilsem, 2011) . all of these activities would increase the target's visibility and the offender's access, and we would anticipate that increases in these behaviors would result in higher rates of cybervictimization. however, time spent performing other online routines, such as working online or reading the news, may have also increased due to the pandemic, but these activities are unlikely to affect cybervictimization since they would not bring one into "dangerous" virtual spaces. another factor that can influence target suitability by decreasing an offender's access and possibly increase the target's ability to avoid an attack (i.e. decrease the target's inertia) is the use of target-hardening devices. some evidence suggests that the use of antivirus programs, firewalls, and filtering and blocking software can reduce the likelihood of becoming a victim of an economic cybervictimization; however, there is little evidence such devices can protect one from violent cybercrimes (see holt & bossler, 2008; leukfeldt, 2014; marcum et al., 2010) . how the pandemic would influence the use of target-hardening devices is difficult to predict. while one would hope people would be more vigilant in terms of updating their anti-virus software and making sure their firewalls are set, the pandemic has probably not influenced the overall use of computer technology for those with high levels of computer skills since these people were probably frequent users prior to the pandemic. instead, the pandemic has probably led to more inexperienced and unsophisticated computer users spending more time online. if this is the case, we would predict that overall rates of economic cybervictimization should increase. we note here that we would not expect violent cybercrimes to increase since these are reportedly unaffected by target-hardening devices. finally, another factor that patterns victimization is guardianship. guardianship is "the presence of a human element which acts-whether intentionally or not-to deter the would-be offender from committing a crime against an available target" (hollis, felson, & welsh, 2013: 76) . the findings with respect to guardianship and cybercrime are inconsistent (e.g., bossler & holt, 2009; leukfeldt & yar, 2016; reyns, 2015) , in part due to conceptual uncertainty across both study design and types of victimization (vakhitova & reynald, 2014 ). yet, as argued by costello, hawdon, and ratliff (2017) , the virtual world is a truly socially disorganized community. in cyberspace, actors are truly transient as they come and go regularly, and they do so anonymously. moreover, many online spaces have no way for anyone even trying to monitor the activity to intervene, and most offenders likely know this. even sites that closely monitor activity and have policies to censor or delete material struggle to keep pace with the amount of activity that must be monitored. moreover, online norms that would stimulate intervention on one's behalf tend to be weak and underdeveloped (see costello et al., 2017) . thus, overall guardianship is always low online and the pandemic is unlikely to have changed that. as such, we would not anticipate rates of cybervictimization to have changed due to any influence the pandemic would have had on cyber guardianship. taking all of these factors together, we would anticipate an increase in cybervictimization amid the covid-19 pandemic due to more motivated offenders, a change in some "dangerous" online routines, and perhaps less target-hardening. however, given that many online routines that have likely increased would not necessarily result in increased victimization and the fact that guardianship is likely unchanged by the pandemic (because it is always lacking online), any observed increase is expected to be modest. the above discussion gives rise to the following hypotheses that will be tested using samples collected pre (november 2019) and post (april 2020) pandemic. first, as stated above, given the anticipated changes in online routines, we hypothesize (h1) rates of cybervictimization will be modestly higher among post-covid-19 respondents than they are among pre-covid-19 respondents. next, as explained above, we anticipate the increase in victimization because people were forced to shift their daily activities online and radically enlarge their digital footprint. thus, we hypothesize that (h2) the extent to which respondents engage in online activities will be higher in the post-covid-19 sample than in the pre-covid-19 sample. we now turn to our analysis. data were collected using online panels from dynata. dynata uses random digit dialing, banner ads, and other permission-based techniques to recruit respondents. from this database dynata randomly invites panel members to participate in the survey. the sample was balanced based on us census data to represent sex, ethnicity, and race. online proportional sampling has been found to yield similar results as random probability-based samples due to several strategies (weinberg, freese, & mcelhattan, 2014; simmons & bobo, 2015; contrast macinnis, krosnick, ho, & cho, 2018) . first, both repeat participants and individuals who speed through the survey are eliminated to increase sample validity (wansink, 2001; evans & mathur, 2005) . in addition, the rewards offered by the panel company have been shown to increase validity of the overall data (see wansink, 2001 ). the first survey was fielded between november 24 and november 30, 2019 (pre-covid-19 sample), and the second was fielded between april 14 and april 17, 2020 (post-covid-19 sample). overall, 1315 respondents began the pre-covid-19 survey, but 81 respondents completed the survey in less than 3 min and were considered "speeders." they were removed from the sample. in addition, 125 participants did not complete the survey and were eliminated from the analysis. in total, 1109 respondents had usable data and were included in our analysis. in the post-covid-19 sample, 1120 respondents began the survey, with 58 "speeders" who were dropped from the analysis. dropping these respondents resulted in a sample of 1021 participants in the post-covid-19 sample. a comparison between the pre-covid-19 and post-covid-19 sample in terms of demographics can be found in table 1 . of note was an expected increase in unemployment in the post-covid-19 sample. in addition, the samples differed in average age and education, but did not differ in terms of racial/ethnic composition or gender. to examine the first hypothesis, we investigate rates of cybervictimization in the two samples. to measure cybervictimization, respondents were asked if they had been a victim of seven different types of cybercrime (see table 4 for the types of crimes and survey items used to measure them). we created a summated variable of all victimization behaviors. this count variable reflected the number of different victimization experiences the participants had experienced in the past 12 months. even upon visual inspection we noticed that, if anything, victimization was slightly higher in the pre-covid-19 sample. we examined victimization in relation to the samples using a negative binomial regression because our data are over-dispersed count data. we examined the differences between the samples by regressing victimization on a covid-19 indicator variable (0 = pre-covid-19; 1 = post-covid-19). the covid-19 indicator variable failed to reach standard levels of statistical significance, and the overall model had extremely low predictive power, with a pseudo r 2 of <.001. as such, our first hypothesis is not supported, at least when we consider overall cybervictimization. given that certain types of cybercrimes may have increased while others decreased, we also examined specific victimization and self-protection behaviors between the pre and post-covid-19 sample to help understand why we were not seeing a difference between the samples. we utilized χ 2 tests to consider differences between the samples. types of victimizations tested included scams, identity theft, unknown transactions, notification from organizations about data theft, online bullying, online sexual harassment, and malware/viruses. only one significant difference was found. the post-covid-19 sample reported fewer notifications by companies that their data had been stolen (χ2 = 7.97(1), p = .005). in the pre-covid-19 sample 21% of respondents indicated they had been notified by a company about data loss whereas in the post-covid-19 sample only 16% indicated they had been notified by a company about data loss. we also examined differences in self-protection measure use in the pre/post-covid-19 samples using similar χ 2 tests. only one significant difference was found. while 70% of the post-covid-19 sample indicated that they used virus software or firewalls, only 66% of the pre-covid-19 reported that they did (χ 2 = 3.97(1), p = .046). we advise caution in interpreting this result as the p value was close to .05 and we ran a total of 11 χ 2 tests, increasing the likelihood of a false positive. all victimization and self-protection difference tests can be found in table 2 . to test our second hypothesis regarding differences in computer behaviors between the samples, we compared pre-covid-19 and post-covid-19 computer-related activities. these activities include playing online games, reading news or other articles online, browsing social media, using a computer while working, and shopping online. as seen in table 3 , only one activity, reading news or other online articles was significantly higher in the post-covid-19 sample (t = −4.4(2093), p < .001). therefore, our second hypotheses is also not supported. given the failure of either of our hypotheses to be supported, we investigate if the rat model still applies to cybervictimization in the post-covid-19 world. given the reported fourfold increase in cybercrimes during the pandemic (cimpanu, 2020; england, 2020) yet our data not reflecting such an increase, we need to consider if to do this, we turn to a test of rat using our two samples. to examine if rat is an adequate predictor of cybervictimization, we included several variables in a negative binomial regression. the model included an index variable of pre/post-covid-19 sample, time spent in the various online activities mentioned above, computer self-protection behaviors, and demographic variables. table 4 reports the results of the analysis. overall the model was significant (p < .001). factors related to increased risk of victimization included dark web use per week (irr = 1.14; p < .001), time reading online news/articles (irr = 1.08; p < .001), time browsing social media (irr = 1.04; p < .05) and age (irr = 1.02; p < .001). age was the only demographic factor to achieve statistical significance in the model. factors significantly related to lower risk of victimization included time working on a computer (irr = 0.95; p < .005) and all protective behaviors including covering a webcam (irr = 0.70; p < .001), having identity theft protection (irr = 0.78; while the covid-19 indicator was again not significant, almost all of the ratspecified variables were significant predictors in the model, and all of the relationships between these variables and cybervictimization were in the direction rat would predict. it is also worth noting that age was the only demographic variable that was significantly related to cybervictimization. this finding is also supportive of rat's argument that one's routines determine victimization. finally, we tested to see if any interaction between the covid-19 indicator variable and the rat variables were significant to be certain that rat applied equally well in the pre and post covid-19 world. results (not shown here) indicated that no interactions were significant, suggesting that indeed rat still performs well as an explanation of cybervictimization even during the pandemic. to our knowledge, our study is the first empirical evidence concerning how the covid-19 pandemic influenced cyber-routines and cybervictimization. we assumed that the pandemic and the results of stay-at-home orders would result in increased online presence, an increased level of routine activities online, and, as such, enhanced levels of target suitability and target proximity to motivated offenders. consequently, we expected that rates of cybervictimization would be higher in the post-pandemic sample than what was observed in the pre-pandemic sample. the results show that we were clearly wrong. based on our results, the stay-at-home orders did not radically alter our cyber-routines, and cybervictimization did not increase either. instead, global levels of cybervictimization were nearly identical pre and post-pandemic, and only one type of victimization (being informed that your identity or private information had been stolen) changed. moreover, this victimization decreased in the post-covid-19 sample. among the indicators of cyber-routine activities, including playing online games, a scale is nonlinear as hours were represented in increasing increments, 0, <1, 1-2, 2-4, 4-6, 7-8, 8-10, 10 or more *p < .05, **p < .01, ***p < .001 reading news or other articles online, browsing social media, using a computer while working, and shopping online, only reading news or other online articles increased. one online activity, online shopping, even decreased in the post-covid-19 sample. we also wanted to see if specific types of victimization and protection behaviors changed after the pandemic. among all the specific victimization variables, only one showed a significant difference: there was less notification from companies concerning data theft in the post-covid-19 sample. in terms of target-hardening behaviors, participants reported using more self-protection (i.e. virus software and a firewall) in the post-covid-19 sample. thus, while there were minor differences between the samples, contrary to our expectations and fbi reports (cimpanu, 2020; england, 2020) , our data show that the pandemic has not radically altered our cyber-routines or levels of cybervictimization. fearing rat may not apply in the post-pandemic virtual world, we tested it with a negative binominal regression. our model showed dark web use, time spent online reading newspapers and other articles, and time using social media significantly increased the likelihood of being a cyber victim. time spent working on a computer, protective behaviors such as covering the webcam, having identify theft protection, freezing credit, and having virus protection were all inversely related to the likelihood of victimization. these results clearly support rat, and the insignificance of the covid index variable or any interactions between it and the various cyber-routines indicate that the theory applies equally well in the pre and post covid-19 world. although cybervictimization has not changed substantially, our binominal regression model indicates that rat can account for patterns of cybervictimization in both pre and post-pandemic samples. so why were our expectations so wrong? first, proximity to motivated offenders may have increased as people went online to work, study, and network, but target suitability did not increase as, according to our results, people likely used online platforms similarly to how they had before the pandemic. most of their online behaviors did not put them at more than average risk of victimization. there was no evidence that dangerous online behaviors such as dark web use, online shopping, or visiting social media platforms changed significantly after the pandemic. indeed, our data suggest users abandoned online activities such as online shopping that would pose risk to their bank accounts. instead, it seems that people kept their cyber-routines concentrating on less dangerous routine activities, such as working online and reading news articles. the online routine activity that significantly increased was reading online news, but that activity would not heighten victimization as most traditional online news sites are reputable and do not increase their readers' target suitability. we also expected that the overall rates of cybervictimization, and especially economic cybervictimization, would increase because of the nation's swift shift to the virtual world likely did not leave time for users to upgrade their computer security measures (e.g. firewalls, anti-virus software, etc.). while more computer savvy users likely have security measures already installed, those with fewer computer skills could be more vulnerable now that they are spending more time online. however, our data indicated that more people engaged in target-hardening measures and cybervictimization was unrelated to computer familiarity. a plausible explanation for this is that workers' relatively unfamiliar with computers were moved online by their companies who provided sufficient it support. we cannot say this happened, but it is likely that corporations were keenly aware that some of their less-thantech-savvy employees who were now teleworking needed support and failing to do so could put their company at heightened risk. indeed, there is reason to suspect there was heightened concern about cybercrime and possibly greater vigilance practiced to protect oneself from it. for example, the fbi noted how cybercriminals would likely target both companies and individuals working from home via teleworking software, education technology, and business email platforms. on april 15, the us departments of state, homeland security, and treasury, and the fbi issued an advisory to raise awareness of the cyber threat posed by north korea's malicious cyber activities, a significant threat to the integrity and stability of the international financial system (us cert, 2020). on april 20, the fbi charlotte warned (fbi charlotte, 2020) social media users to pay attention to trending social media topics (e.g. high school support photo trend, posting a picture of your first car, answering questions about your best friend, providing the name of your first pet, identifying your first concert, etc.), which might collect users' personal information, including passwords to reset accounts and gain access to protected data. thus, it is possible that these warnings worked. while we lack the data to adequately test if companies' policies attempted to protect their users or the government warnings worked, this possibility should be further explored by future research. our data allows us to say that the relative unchanged nature of our respondents' "dangerous" cyber-routines combined with their use of stronger security measures such as antivirus software and firewalls likely kept cybervictimization low, even if there was elevated motivation among offenders due to souring rates of unemployment and economic struggles. we can also say that our findings contradict recent reports about heightened levels of cybercrime being reported to the fbi (cimpanu, 2020; england, 2020; ic3, 2020) . like us, the fbi anticipated that virtual environments will be increasingly affected adversely by cybercriminals, and as of march 30, 2020 (ic3, 2020), their data gives credence to their fears. the fbi internet crime complaint center (ic3) reports receiving more than 1200 complaints related to covid-19 scams, including phishing campaigns against first responders, ddos attacks against government agencies, ransomware against medical facilities, and fake covid-19 websites downloading malware to users' computers. in total, the fbi reports cybercrimes have quadrupled during the covid-19 pandemic (cimpanu, 2020; england, 2020) . how do our results showing decreased cybervictimization make sense when cybercrime has an upward trend in the news and in fbi reports? it might just show the usual discrepancies between official crime statistics relying on reporting and victimization surveys. it is well documented how official statistics seriously underestimate crime rates. what we might be seeing is that while rates of crime are actually unchanged, rates of reporting crimes is increasing. the repeated warnings of federal and state law enforcement and international policing agencies about the expected increase in cyber-offending may alert people and lead them to the dangers of cybercrime. this heightened awareness would then lead them to notice and report these crimes more than they did prior to the pandemic. another possibility is that the increased rates of reporting to the fbi are more due to attacks on companies than on individual users. our data focused on individual, not corporate, victimization. thus, because our survey only included individual level cybervictimization while ignoring attacks on companies and critical infrastructures, we may not be detecting the increase in cybercrimes through our victimization surveys. this possibility should also be investigated by future researchers. our lives have been radically altered by a pandemic that is considered to be among the most widespread in history. the change in our daily routines appears to have resulted in an abrupt drop in street crimes. however, the shift to the digital world undoubtedly creates new opportunities and platforms for motivated offenders to engage in various illegal activities. this shift should increase the number of suitable targets, as millions of people are confined to their homes and forced to work, study, and socialize online. the current shift was swift, but, at least according to our data, this shift apparently did not result in people being more affected by cybercrime. they may be reporting more of it, but it is also possible that the pandemic has led to a decrease in most street crimes, an increase in domestic crimes, and no change in cybercrimes. the reasons of the crime drop experienced all over the world in the 1990s (pope & pope, 2012) is still debated (rosenfeld & messner, 2012) and is indeed a complex issue. it is likely the current crime trends will be studied and debated for some time too. our aim here was simply to provide one piece of evidence that will hopefully significantly contribute to that future analysis and debate. we live in unprecedented times. we did not, nor can we, measure every possible underlying reason why cybervictimization occurs. we cannot predict if cybervictimization will remain low as social distancing continues. however, we can say that routine activities theory appears to still apply. we live in unprecedented times, but we still have theories to help us make sense of them. thomas dearden : is assistant professor of sociology at virginia tech. dr. dearden specializes in research technology and crime, and corporate crime. he has conducted research for organizations across the globe, including the polynesian cultural center in hawaii, food for life vrindavan in uttar pradesh, india, and pay tel in north carolina. he has published his research in peer-reviewed journals including the journal of financial crime and the journal of investigative 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pursued online applying cyberlifestyle-routine activities theory to cyberstalking victimization guardians of the cyber galaxy: an empirical and theoretical analysis of the guardianship concept from routine activity theory as it applies to online forms of victimization the crime drop in comparative perspective: the impact of the economy and imprisonment on american and european burglary rates the international crime drop. crime prevention and security management staying home saves lives can non-full-probability internet surveys yield useful data? a comparison with full-probability face-to-face surveys in the domain of race and social inequality attitudes a macro-social exploratory analysis of the rate of interstate cyber-victimization routine activities north-korean cyber threat cyberguardians: an empirical study of guardianship against cyber abuse toward the adaptation of routine activity and lifestyle exposure theories to account for cyber abuse victimization worlds tied together? online and non-domestic routine activities and their impact on digital and traditional threat victimization editorial: the power of panels comparing data characteristics and results of an online factorial survey between a population-based and a crowdsource-recruited sample the unemployment rate is probably around 13 percent. the new york times accessed 3 the novelty of 'cybercrime': an assessment in light of routine activity theory cybercrime and society social media interest is spiking worldwide-except for linkedin. the next web the great american crime decline publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations james hawdon : is a professor of sociology and director of the center for peace studies and violence prevention at virginia tech. he researches how communities influence the causes and consequences of violence. most recently, he has focused on how online communities influence political polarization, online hate, extremism, and cybercrime. he has been funded by the national science foundation, the national institute of justice, the national consortium on violence prevention, and several other agencies she evaluated cyberbullying programs of the massachusetts aggression reduction center as a fulbright fellow, and was awarded the european safety and prevention award for channeling academic research results to schools. she has published in peer-reviewed journals such as the pediatrics, the acknowledgements this research was funded by the center for peace studies and violence prevention at virginia tech (grant number 025-19), the institute for culture, society, and environment at virginia tech, and the integrated security destination area at virginia tech. key: cord-256992-rwy0n01l authors: taheri, yasaman; suleria, hafiz ansar rasul; martins, natália; sytar, oksana; beyatli, ahmet; yeskaliyeva, balakyz; seitimova, gulnaz; salehi, bahare; semwal, prabhakar; painuli, sakshi; kumar, anuj; azzini, elena; martorell, miquel; setzer, william n.; maroyi, alfred; sharifi-rad, javad title: myricetin bioactive effects: moving from preclinical evidence to potential clinical applications date: 2020-08-01 journal: bmc complement med ther doi: 10.1186/s12906-020-03033-z sha: doc_id: 256992 cord_uid: rwy0n01l several flavonoids have been recognized as nutraceuticals, and myricetin is a good example. myricetin is commonly found in plants and their antimicrobial and antioxidant activities is well demonstrated. one of its beneficial biological effects is the neuroprotective activity, showing preclinical activities on alzheimer, parkinson, and huntington diseases, and even in amyotrophic lateral sclerosis. also, myricetin has revealed other biological activities, among them as antidiabetic, anticancer, immunomodulatory, cardiovascular, analgesic and antihypertensive. however, few clinical trials have been performed using myricetin as nutraceutical. thus, this review provides new insights on myricetin preclinical pharmacological activities, and role in selected clinical trials. polyphenols are a wide group of plant-derived molecules resulting from secondary metabolism, ubiquitously distributed in vegetable kingdom where they display different activities such as protective effect against uv rays, bacteria, virus and fungi infections, modulation of plant hormones, enzyme inhibition and pollinator attraction [1] . in nature, there are a plethora of different polyphenols that can be classified in the following main classes: simple phenolic acids (e.g. gallic, vanillic, syringic, p-hydroxybenzoic), hydroxycinnamic acid derivatives (such as caffeic acid, p-coumaric, ferulic, sinapic), flavonoids, stilbenes and lignans. the largest common class of polyphenols present in human diet is represented by flavonoids [2, 3] . chemically flavonoids are classified in flavans, flavones, flavonols, and anthocyanidins [4] . among the flavonols, myricetin, a 3,3′,4′,5,5′,7-hexahydroxyflavone, possess one of the most hydroxylated structures (fig. 1) . the solubility of myricetin in water is poor (16.6 μg/ml) but increases when deprotonated in basic aqueous media and in some organic solvents (dimethylformamide, dimethylacetamide, tetrahydrofuran and acetone) [5] . the chemical stability of myricetin is ph and temperature dependent [6] . depending on the environment conditions, myricetin can exert, in vitro, both a potent antioxidant and a pro-oxidant effect. buchter et al. [7] attributed its direct antioxidant action to several structural elements. on the other hand, chobot and hadacek [8] demonstrated the pro-oxidative properties of myricetin to molecular oxygen reduction to reactive oxygen species (ros) and iron (iii) to iron (ii) and also highlighted the ability of myricetin to serve as a substitute for ascorbic acid, albeit less efficiently. myricetin is mainly present in the glycoside form (o-glycosides), in vegetables, fruits, nuts, berries, herbs, plants together with beverages, such as tea, wine, fruit and medicinal plants [9] [10] [11] [12] [13] [14] [15] . there are numerous factors that can influence myricetin levels in plant foods such as genetic and environmental factors, germination, and ripeness degree, variety, seasonal variation, and storage, processing and cooking. the estimate of total flavonoid intake is difficult to calculate, as appropriate tables of food composition are not yet available. however, reliable data on daily flavonoid intake in a population are needed to develop proper dietary recommendations and even for correct data interpretation from intervention studies. the flemish dietetic association database determined an average daily intake of myricetin of 2.2 ± 2.5 mg mullie et al. [16] . in a korean adult population, jun et al. [17] estimated an average intake of 0.8 mg/day representing about 1-2% of flavonol subclass, while a mean intake of myricetin 2 mg/day ranged from 1 to 4 mg/day in adults (18 to 64 years) in the european union was reported by vogiatzoglou et al. [18] . the knowledge on habitual flavonoids consumption is also crucial to determine their possible impact on human health. myricetin exhibited antioxidant properties and free radical-scavenging effects [19] . these activities seem to support a wide range of beneficial outcomes including, anti-platelet aggregation, antihypertensive, immunomodulatory, anti-inflammatory, anti-allergic, analgesic, anticancer actions and so on [6, [20] [21] [22] [23] [24] [25] . the main goal of the present review is to provide new insights on myricetin preclinical pharmacological activities, and its role in selected clinical trials. [27] . the first time myricetin was identified was in plants of the myricaceae, comptonia peregrina (l.) coult. and later morella cerifera (l.) small [28, 29] . the myricetin concentration in the plants such as rosa canina l. (rosa hip), urtica dioica l. (nettle), and portulaca oleracea l. (purslane) found between 3 and 58 mg/kg [13] . myricetin was isolated from polygonum bellardii all. (polygonaceae) as yellow needles (50 mg) from aerial parts using meoh extract [30] . previously, a prescreening of leaves of 28 polygonaceous plants was estimated that myricetin glycosides were relatively rare consituents [31] . trigonella foenum-graecum l. gemmo-modified extract had the richest content in myricetin (830 mg/kg), followed by euphorbia tirucalli l. (821 mg/kg), rhizomes of cyperus rotundus l. (702 mg/kg) and seed extract of t. foenum-graecum (547 mg/kg). c. rotundus gemmomodified extracts contained 104 mg/kg myricetin [10] . the highest level of myricetin content has been identified in the strawberry and spinach [9] . species of anacardium and mangifera (anacardiaceae) found to have high levels of hydroxylated compounds like myricetin, gallic acid, proanthocyanidins and flavonols. in marantodes pumilum (blume) kuntze (primulaceae) were identified quercetin, myricetin, kaempferol, catechin and epigallocatechin [32] . the most common sources of myricetin are vegetables, fruits, nuts, berries and tea [33] . myricetin-rich foods are listed in table 1 based on the usda food database (compiled data from all fruits and vegetables that contain information on myricetin concentration) [34] . in black fruits the quantities varied between 14 and 142 mg/kg [12] . myricetin is the most abundant flavonol of black currant, and its quantity varied significantly among black currant cultivars [35] . at the same time, honey is also a source of flavonoids, especially myricetin. the hplc analyses of honeys from australian eucalyptus have shown that the flavonoids myricetin, quercetin, tricetin, kaempferol and luteolin exist in all honeys. myricetin was found in range from 29.2-289.0 μg/100 g honey [36] . in grapes, flavonol glycosides from the following aglycons have been identified: myricetin (3′,4′, 5′-trioh), laricitrin (3′-meo analog of myricetin) and syringetin (3′,5′-dimeo analog of myricetin), quercetin and kaempferol [37] . the simultaneous presence of these aglycons was detected in different types of red wine vitis vinifera l. grapes [38] , while in white wine, only quercetin, kaempferol and isorhamnetin were detected [37] . myricetin displays multiple preclinical biological effects [19] . thus, in the following subsections, the antimicrobial, antioxidant, neuroprotective, antidiabetic, anticancer, immunomodulatory, cardioprotective, analgesic, anti-hypertensive and wound healing potential of myricetin are briefly discussed and summarized. antimicrobial mechanism of flavonoids may involve membrane disruption, inhibition of cell envelope synthesis, inhibition of nucleic acid synthesis, inhibition of bacterial virulence and quorum sensing, which impairs their ability to form biofilms, inhibition of efflux pumps, and inhibition of nadh-cytochrome c reductase activity and atp synthase [39, 40] . myricetin inhibited escherichia coli dna gyrase (ic 50 1.18 mg/dl) [41] , and dnab helicase (ic 50 11.3 μm) [42] , and cellular dna and rna polymerases [43] . myricetin showed a significant antimicrobial activity against foodborne pathogens in terms of minimum inhibitory concentration (mic, mg/ml) <15.0, <15.0, < 20.0, <10.0 at 24 h and <20.0, <20.0, <15.0, <5.0 at 60 h incubation for escherichia coli, salmonella paratyphi, salmonella cholerasuis, and salmonella enteritidis, respectively [44] . the compound myricetin revealed curlidependent e. coli biofilm formation inhibition (ic 50 = 46.2 μm), curli contributes to the robustness of e. coli biofilms [45] . yadav et al. [47] demonstrated the anti-tubercular activity of 15 selected flavonoids including myricetin and their structure-activity relationships were evaluated against mycobacterium tuberculosis h37rv strain radiometrically. myricetin was found to be active against m. tuberculosis, with a mic of 50 μg/ml, and structure-activity relationships authenticated their anti-tubercular potential due to the presence of hydroxy groups in their structure. the inhibitory activity of the compounds were evaluated against dna gyrase from e. coli by dna supercoiling. mean antibacterial activity in terms of mic and ic 50 were 142 μg/ml and 1.18 mg/ml respectively. the structureactivity relationship analysis suggests that, the presence of hydroxyl and substitution in the ring a and b position are essential for the best inhibitory effects [41] . the inhibitory effect of myricetin on severe acute respiratory syndrome-coronavirus (sars-cov) helicase, nsp13, and hepatitis c virus (hcv) helicase, ns3h was also assessed [48] . myricetin was found to inhibit sars-cov helicase protein by affecting the atpase activity (ic 50 2.71 μm), however, it failed to affect the atpase activity of the hcv ns3 helicase. desouza and wahidullah [49] reported the antimicrobial activity on e. coli, klebsiella pneumoniae, proteus mirabilis, pseudomonas aeruginosa, salmonella typhi, shigella flexneri, staphylococcus aureus, vibrio cholerae and myricetin showed the best activity against p. aeruginosa (mic 1.5 μg/ml). gendaram et al. [50] reported the myricetin antibacterial effect against s. aureus by the disc diffusion method (300 μg/disc, inhibition zone 9 mm) but reported no antibacterial activities against p. aeruginosa, e. coli, enterococcus faecalis, or micrococcus luteus. however, at 100 μm concentration, myricetin did not exhibit antimicrobial activity on gram-positive bacteria but showed inhibitory activity against sortase a (srta) from s. aureus (92%; ic 50 4.63 μm) [51] . in vitro antimicrobial activity of six natural phytochemicals including myricetin (alone and with combination) were evaluated against five strains of p. aeruginosa by using a time-kill assay. the compound showed the mic as 500 μg/ml against all five strains of p. aeruginosa [52] . other reports of the compound based on antimicrobial and antiviral studies are presented in table 2 . plant-based compounds considered as natural antioxidants have attracted a large number of communities of scientist, researchers, industries and traditional healers for their health-promoting characteristics. the antioxidant table 1 myricetin (mg/100 g) rich foods [34] cranberry 6600 dock 5700 sweet potato leaves 4400 chard, swiss 3100 broadbeans, immature seeds 2600 rutabagas 2100 garlic 1600 blueberry 1300 peppers, hot chili, green 1200 blackberry 700 lotus root 600 lemon 500 source: usda food database (compiled data from all fruits and vegetables that contain information on myricetin concentration) potential of myricetin has been reported by several authors in the last few decades. hou et al. [61] studied the antioxidant effect of hs15-myr micelles and independent myricetin by using frap (ferric reducing antioxidant power) and abts (2,2′azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) assays. the abts assay displayed an improved value from 22.20 to 41.77% in hs15-myr micelles and 0 to 6.12% in independent myricetin at two different concentrations and incubation periods. the frap assay also presented an improved value from 1.27 to 8.94 mm fe 2+ /g in hs15-myr micelles and 13.63 to 16.33 mm fe 2+ /g in independent myricetin at two different concentrations and incubation periods. myricetin in hs15-myr micelles exhibited in both assays stronger antioxidant effects when compared to independent myricetin. barzegar [62] reported the ros-protection efficiency of the compound myricetin in a cell-free and cell-based system. a low concentration of compound significantly inhibited intracellular ros production and also protected cells against toxicity induced by peroxide compounds. guitard et al. [63] reported that, myricetin is more efficient than α-tocopherol and synthetic antioxidants on preservation of omega-3 oils. other studies on antioxidant potential of the compound are presented in table 3 . natural flavonoids have exerted positive impacts on body through affecting multiple cell systems and modulating the activity of various pathways to reduce cognitive decline and neuronal dysfunction [79] . myricetin is one of such flavonoids, and multiple studies have been conducted to assess the neuroprotective effects of this compound and its interaction with brain receptors ( table 4 ). the main mechanisms are shown in fig. 2 . myricetin antidiabetic activity has been reported by several authors in the last few years and limited reports are also available on its anti-obesity activity but in this review, we focused on only its antidiabetic potential. karunakaran et al. [101] reported the in vitro effect of myricetin on high glucose-induced β-cell apoptosis, possibly via cyclin-dependent kinase 5 (cdk5) inhibition. data revealed that myricetin (20 μm) significantly protect β-cells reducing apoptosis in ins-1 cells and rat islets that were incubated with glucose at the concentration of 30 mm for 24 and 48 h, respectively. docking studies predicted myricetin inhibited activation of cdk5. the effect of myricetin was evaluated in diabetes mellitus-associated kidney injuries and dysfunction in an experimental mouse model with diabetes mellitus induced by 5 consecutive injections of low-dose streptozotocin (stz) [20] . the data revealed that myricetin (orally twice a day, 100 mg/kg/day, for 6 moths) inhibited the iκbα/nf-κb pathway, with this pathway being independent of nuclear factor erythroid 2related factor (nrf2) regulation. it was also reported that myricetin activates glucagon-like peptide 1 receptor (glp-1r) and its long-term oral administration (200 mg/kg, for 40 days) validates its glucoregulatory effects [102] . insulin's metabolic action is mediated via the activation of phosphatidylinositol 3-kinase (pi3k) and its downstream effectors, the protein kinase b (pkb/akt) kinases [103] . in contrast, amp-activated protein kinase (ampk) signal pathway is likely to mediate the effect of insulin-independent stimuli for glucose uptake in muscle [104] . in an in vitro study, myricetin enhanced akt and ampk protein activity, encouraged glucose uptake and reduced insulin resistance [105] . the mechanisms of myricetin for improving insulin-sensitive tissue might be the amelioration of impaired signaling intermediates downstream of insulin receptors through enhancing the secretion of β-endorphin, which in turn led to the activation of peripheral μ-opioid receptors [106, 107] . then, myricetin affects insulin receptor phosphorylation, insulin receptor substrate-1 (irs-1), the p85 regulatory subunit of pi3k, akt and akt substrate of 160 kd, with subsequent effects on glucose transporter 4 (glut4) translocation [108] . other previous studies on antidiabetic potential of the compound are shown in table 5 . cancer is responsible for second highest cause of death across the globe [124, 125] . it has been reported that number of death due to this devastating disease would expand to over 13 million by 2030 [126, 127] . laboratory and clinical studies have reported that myricetin from natural sources exerts promising effects against various types of cancer [19, 21] . the dietary compound myricetin also has the potential to inhibit key enzymes involved in cancer initiation and growth. myricetin has presented cytotoxic activity in human colon cancer cells. kim et al. [21] demonstrated that myricetin significantly induces the bcl2-associated x dose-dependent reduction in lithium-induced head twitches and anxiolytic activity by altering 5-hydroxytryptamine transmission. [80] in vitro pro-oxidant agent and reduced the formation of ordered amyloid beta (aβ)42 aggregation. [81] in silico destabilizes the β-sheet ordered amyloid oligomers formed by the undecapeptide aβ (25-35) model. [82] in vitro marked modulation of metal-induced aβ aggregation, more than metal-free aβ aggregation. increase cell survival rate of aβ (with metal ions). [83] in vitro increases α-secretase (adam10) enzyme activity and decreases of β-secretase (bace-1). it also exerts neuroprotective activity against aβ (1-42) with multifunctional role in counteracting ad progress. [84] in vitro dose-dependent inhibition of α-synuclein fibrils formation and destabilization (ec 50 = 0.21-1.8 μm). [85] in vitro dose-dependent inhibition of aβ fibrils formation from fresh aβ (1-40) and aβ . the ec 50 value for formation, extension and destabilization aβ fibrils ranges from 0.13-1.8 μm. [86] in vivo increases the number of hippocampal ca3 pyramidal neurons and survival in a rat model (10 mg/kg). improved learning and memory in a rat model with ad. [87] reduces the aggregation of different abnormal proteins and eliminates various toxic proteins related to neurodegenerative diseases. improves physiological functions of hsp70 molecular chaperone and reduces mis-folded proteins. [ 88] in vitro and in vivo increases gaba receptor activity via calcium channel/ camk-ii dependent mechanism, which is distinctively different from that of most existing benzodiazepine binding site agonists of gaba receptor. [89] in vivo increases mrna for brain-derived neurotrophic factor (bdnf) in the hippocampus of male c57bl/6 mice at 10 and 20 mg/ kg (7 days). [90] in vivo increases bdnf concentrations in the hippocampus of male c57bl/6 mice at 50 mg/kg (21 days). [91] in vivo enhances expression and activity of erk1/2-creb pathway and na + , k + -atpase while reduces oxidative stress level in hippocampus. improves learning and memory when compared with d-galactose. [92] in vivo reduces seizure severity and mortality rates in mouse models and signaling pathways (bdnf-trkb) and regulates gad65/ gaba with mmp-9 expression. [93] in vivo interacts with rna, especially cag motif, and decreases the huntingtin protein translation and sequestration. reduces cytotoxicity in hd and other polyq disease models. [94] in vitro suppresses intracellular ros production, re-establishes mitochondrial trans-membrane potential, and inhibits mkk4 and jnk activation. [ 95] in vitro and in vivo inhibits activation of microglia (neuroinflammation), expression of pro-inflammatory mediators and reduces the number of dopaminergic neurons. [96] in vivo dose-dependent delay in climbing ability loss, but increases the life span of flies expressing human α-synuclein in brain. [97] in vivo prevents the loss of dopaminergic neurons and dopamine content in brain of parkinson flies. [98] in vivo dose-dependent inhibitory activity on α-synuclein aggregation. [99] in vivo diminishes dopamine neuron degeneration, which is induced by 6-hydroxydopamine and 1-methyl-4-phenyl-pyridinium in substantia nigra-striatum. [100] aβ amyloid beta, cns central nervous system, bdnf brain-derived neurotrophic factor taheri this study suggested that myricetin can be utilized for the design of therapeutic agents against human colon cancer. myricetin also acts as a potent inhibitor of human flap endonuclease 1 (hfen1) protein (ic 50 690 nm), based on inhibitory mechanisms, molecular docking, and cancer cell-based assays [128] . the hfen1 protein is a functional member of the 5′-nuclease superfamily. by chemical nature, hfen1 is a metal iondependent and structure-specific nuclease and also instrumental in dna replication and repairing processes. molecular docking studies revealed that ring a of myricetin compound, including 4-keto and 5-oh, was found stretched towards the two divalent metal ions. both metal ions are critical as they seem to interact with arg100 and lys93 amino acids through hydrogen bonds. these interacted residues are well known for their critical interplay in hfen1's activity during human colon cancer. myricetin has also been shown to protect against ovarian cancer through suppressing ovarian cancer cell angiogenesis [129] . anti-angiogenic effects of myricetin (5 to 20 μm) assessed through in vitro (huvec) and in vivo (cam) models revealed that this compound significantly inhibits angiogenesis induced by ovcar-3 cells. in skov3 human ovarian cancer cells, myricetin inhibited viability and induced apoptosis (40 μg/ml, time-dependent manner) through endoplasmic reticulum stress and dna double-strand breaks [130] . zheng et al. [131] stated that in a2780 and ovcar3 ovarian cancer cells, the dietary flavonoid myricetin induced significant cytotoxicity (ic 50 = 25 μm). in a recent study, tavsan and kayali [132] reported that myricetin suppressed ovarian cancer cell growth, induced apoptosis, arrested cell cycle and also had the potential to inhibit cell invasion in a significant manner (ic 50 = 184 μm a2780, 32 μm ovcar-3, 3.3 μm skov3, and > 500 μm osf). thus, it can be concluded that myricetin has enough potential to cope with ovarian cancer in a significant manner. myricetin has potent anticancer-promoting activity against skin cancer. it was found capable of inhibiting neoplastic cell transformation and mitogen-activated protein kinase 1 (mek1) activity (myricetin 1 or 5 μm) [133] . molecular interaction between myricetin and mek1 suppressed mek1 activity leading to downstream signaling to the erk/p90rsk/ap-1 pathway. in another study, myricetin has been presented as a potent chemoprotective agent against skin cancer [134] . myricetin can bind directly to central kinases including pi3-k, akt, jak1, raf1, mek1, mkk4, and fyn, which regulate multiple cell signaling pathways in cancer cells. myricetin inhibited 12-o-tetradecanoylphorbol-13-acetate (tpa)and epidermal growth factor (egf)-induced cell transformation by 76 and 72%, respectively at 10 μm concentration. sun et al. [135] recently reported that myricetin has anticancer activity against skin cancer a431 cell lines, by inducing apoptosis and cell cycle arrest and exhibited low toxicity. an earlier in vitro study demonstrated the antimetastatic effect of myricetin in human lung adenocarcinoma a549 cells [136] . this study revealed that myricetin (5 to 20 μm) suppresses adenocarcinoma a549 cell invasion and migration through inhibition of the erk pathway in a time-dependent manner. along with a combination of radiotherapy, myricetin was found responsible to enhance the tumor radio-sensitivity of lung cancer a549 and h1299 cells through significant suppression of cell-surviving fraction and proliferation [137] . wang et al. [138] found that the combination of myricetin with 5-fluorouracil chemotherapy has the potential to enhance tumor chemo-sensitivity of esophageal cancer ec9706 cells. sun et al. [139] investigated the function of myricetin phytochemical against human t24 bladder cancer in a dose-and time-dependent fashion, and stated that myricetin significantly inhibits both t24 cancer cells viability and proliferation (ic 50 = 85 μm). the preclinical immunomodulatory effects of myricetin have also been increasingly reported. ghassemi-rad et al. [140] concluded that myricetin has the potential to inhibit t-lymphocyte activation in a mouse model through bead-immobilized anti-cd3 and anti-cd28 monoclonal antibodies. this study clarified the mechanism of action and reported the suppressive effect of myricetin on t lymphocytes mediated through extracellular h 2 o 2 generation. in mouse primary macrophages and raw264.7 monocytic cell-line, this phenolic compound was found to inhibit the lipopolysaccharide (lps)-induced interleukin (il)-12 production in a significant manner through down-regulation of nf-κb binding activity [22] . in isolated rat aortic rings, myricetin induced endothelium-dependent contractile responses at 50 μm. earlier, jiménez et al. [141] reported that, in cultured bovine endothelial cells, this compound is responsible for stimulating the production of cytosolic free calcium. in a myricetin in vivo enhanced enzymatic and non-enzymatic antioxidant defense system and showed protective effects against oxidative damage in liver and kidney of streptozotocin-cadmium-induced diabetic model. [109] myricetin in vivo inhibitory activity against α-glucosidase (ic 50 = 414 μm) in dose dependent manner. [110] myricetin in vivo anti-hyperglycemic and renoprotective effects at 1.0 mg/kg. [111] myricetin in vivo improved and re-established renal functions and activities of the glutathione peroxidase and xanthine oxidase enzymes in diabetic rat model. [112] myricetin in vivo antidiabetic activity against t-bhp-induced oxidative stress. [113] myricetin in vivo reduced glycemia in diabetic rats up to 50% after 2 days of treatment at 3 mg/12 h. [114] myricetin in vivo stimulated lipogenesis in rat adipocytes and enhanced the stimulatory effect of insulin (ec 50 = 65 μm). [115] myricetin in vitro inhibited intestinal α-glucosidase (29%) and porcine α-amylase (64%) with ic 50 vale of 0.38 mm. [116] abelmoschus moschatus medik. (aerial part) in vivo improved insulin sensitivity in rats. [117] ampelopsis grossedentata (hand.-mazz.) w.t. wang (leaves) in vivo inhibitory activity against α-glucosidase (ic 50 = 319.3 μm). [118] azadirachta indica a.juss. (leaves) in vivo enhanced insulin signaling pathway and glucose utilization in skeletal muscle. [119] hovenia dulcis thunb. (seeds) in vitro inhibited intestinal α-glucosidase with ic 50 = 3 μg/ml and α-amylase with ic 50 = 662 μg/ml. [120] myrtus communis l. (leaves) in vivo significant antidiabetic activity in diabetic models. [121] syzygium cumini (l.) skeels (seeds) in vitro inhibitory activity against α-glucosidase (ic 50 = 1.7 μg/ml) and α-amylase (ic 50 = 7.62 μg/ml). dose-dependent manner, myricetin inhibited the secretion of a potent t cell growth factor, namely il-2 protein from mouse el-4 t cells, activated with phorbol 12myristate 13-acetate (pma) plus ionomycin [142] . in vitro evidence demonstrated that at 5-100 μm, myricetin inhibits cd69 expression and lymphocytes proliferation in a mouse model. moreover, an in vitro investigation revealed that myricetin significantly effects il-2 expression. however, further in vitro and in vivo investigations are required to explore myricetin as an immunomodulatory agent. previous studies have demonstrated that myricetin also has beneficial effects on the human vascular system [23] . in human umbilical vein endothelial cells, myricetin (100 μm), revealed vasculoprotective effects through changes at the transcriptional level [143] . myricetin has been presented as a functional agent towards preventing atherosclerosis through inhibition of cd36 cell surface protein and mrna expression in a significant manner [144] . in isolated and langendorff-perfused rat hearts, without affecting contractility and relaxation, myricetin elicited coronary dilation [145] . in triton-treated hyperlipidemic rats, evidence from an in vivo investigation demonstrated that myricetin exerts lipid-lowering activity and suggests that myricetin can be utilized in the treatment of hyperlipidemia and cardiovascular diseases (cvd) [146] . in wistar rats, myricetin significantly inhibited the effects of histopathological changes of isoproterenol on heart rate, the levels of different cardiac marker enzymes, including lactate dehydrogenase (ldh), creatine kinase (ck), aspartate aminotransferase (ast), superoxide dismutase (sod) and catalase (cat), as well changes in vascular reactivity and electrocardiographic patterns [147] . a mechanism-based study by scarabelli et al. [148] demonstrated that myricetin exerts strong inhibitory activity against signal transducer and activator of transcription 1 (stat1) activation, and also protects the heart from ischemia/reperfusion-injury. the available genomic and genetics data from preclinical experiments have shown that myricetin is likely to confer the first line of defense against cardiovascular and other associated diseases. in acetic acid-induced writhing response, formalininduced paw licking, sedative activity and hot plate test models, myricetin revealed potent analgesic effects, closely related with peripheral analgesia, but not with the opioid system [24] . the compound also produced a significant analgesic effects in a rat model of neuropathic pain, by decreasing spinal nerve ligation-induced mechanical allodynia and thermal hyperalgesia lasting for several hours (0.1-10 mg/kg i.p.) [149] . the antihypertensive effects of myricetin were evaluated in the deoxycorticosterone acetate (doca)-salt-hypertensive rat model. myricetin reduced systolic blood pressure, vascular reactivity changes and reversed the increased heart rate induced by doca. at oral doses of 100 and 300 mg myricetin/kg b.w., the compound displayed antihypertensive propertie in the doca rat model of hypertension [25] . in another study, the compound lowered the high blood pressure that was induced by fructose doses of 100 and 300 mg/kg p.o. in rats and reversed sugar-triggered metabolic changes [150] . the wound-healing effects of myricetin-3-o-β-rhamnoside were investigated on three different types of cells, keratinocytes, fibroblasts, and endothelial cells. the compound exhibited significant wound healing activity at 10 μg/ml [151] . although the number of clinical studies reporting myricetin health benefits in ailments and disorders is low, the increasing data from preclinical studies have supported its beneficial effects [152, 153] . in a 4-week randomized placebo-controlled clinical trial the effect of 300 mg blueberin (250 mg blueberry leaves, vaccinium arctostaphylos l., and 50 mg myricetin, three times per day) on fasting plasma glucose and some other biochemical parameters has been investigated in 42 female volunteers (46 ± 15 years; body mass index, bmi, 25 ± 3 kg/m 2 ) with diabetes type 2. the blueberin treatment significantly reduced fasting plasma glucose from 143 ± 5.2 mg/l to 104 ± 5.7 mg/l. in addition to antidiabetic effects, results showed that blueberin also possessed pharmacologically relevant antiinflammatory properties, reduced plasma enzyme levels of alanine aminotransferases (alt), ast, glutamyltransferase (ggt), and reduced serum c-reactive proteins (crp) [154] . emulin™ (250 mg of patented blend of chlorogenic acid, myricetin, and quercetin), when regularly consumed, was able not only to lower the acute glycemic impact of foods, but also to chronically decrease blood glucose levels in type 2 diabetic humans (reductions between 1 and 5%) [155] . this study was performed in 40 male and female with fasting glucose range between 126 to 249 mg/ml and a bmi ≥ 30 kg/m 2 . data from different studies also indicate the importance of myricetin as a chemopreventive agent, acting on cell proliferation, signaling mechanisms, apoptosis, angiogenesis, and tumor metastasis [156] . through the analysis of habitual food consumption of 10,054 participants of finnish mobile clinic health examination survey developed during 1966-1972, knekt et al. [157] estimated that higher myricetin intakes in men led to lower prostate cancer risk. in a prospective study, gates et al. [158] analyzed the association between the 5 common dietary flavonoids (myricetin, kaempferol, quercetin, luteolin and apigenin) intake and epithelial ovarian cancer incidence in 66 [158] . the association between flavonoids and flavonoid-rich foods intake and exocrine pancreatic cancer development within the α-tocopherol, β-carotene cancer prevention study cohort were also examined [159] . of the 27,111 male smokers with 306 pancreatic cancers, the data obtained suggests that a flavonoid-rich diet may decrease pancreatic cancer risk in male smokers not consuming supplemental α-tocopherol and/ or β-carotene. tang et al. [160] showed that high/increased flavonoids (e.g., myricetin) intake is associated with lower lung cancer risk in their studied population (meta-analysis of 8 prospective studies and 4 casecontrol studies involving 5073 lung cancer cases and 237,981 non-cases). the intake of 36 g lyophilized grape powder (rich in flavans, anthocyanins, quercetin, myricetin, kaempferol, and resveratrol) also had a great impact in key risk factors for coronary heart disease (lowered levels of triglyceride, lowdensity lipoproteins, apolipoproteins b and e) in both preand post-menopausal women [161] . the study was performed on 24 pre-and 20 post-menopausal women for 4 weeks. however, wide ranges of clinical studies are still needed on the potential activities of myricetin which have been already indicated through in vitro and in vivo experiments. myricetin is a flavonoid present in many foods that has shown biological activities in numerous studies and has a potential use as a nutraceutical. its antimicrobial and antioxidant role is widely studied, and numerous studies have shown neurobiological activities and a potential beneficial impact on ad, pd, hd and als. also, preclinical studies have revealed antidiabetic, anticancer, immunomodulatory, anti-cardiovascular, analgesic and antihypertensive activities. these studies investigated the effect of myricetin, pure compound or plant extract rich in this compound. in plant studies, the extracts rich in myricetin always have other flavonoids that have also shown antioxidant activity alone. nevertheless, new well-designed studies have to be performed to study all of the biological effects described before, as well as preclinical studies comparing the effect of myricetin compared to other flavonoids and phytochemicals. in the case of neurological diseases, more in-depth studies have to be designed to show the pre-clinical results. chapter 10 -metabolic responses of plants upon different plant-pathogen interactions kaempferol: a key emphasis to its anticancer potential in vitro and in vivo assessment of free radical scavenging and antioxidant activities of veronica persica poir nomenclature of flavonoids (iupac recommendations 2017) preformulation studies of myricetin: a natural antioxidant flavonoid myricetin: biological activity related to human health myricetin-mediated lifespan extension in caenorhabditis elegans is modulated by daf-16 exploration of pro-oxidant and antioxidant activities of the flavonoid myricetin flavonols (kaempeferol, quercetin, myricetin) contents of selected fruits, vegetables and medicinal plants phenolic acid and flavonol contents of gemmo-modified and native extracts of some indigenous medicinal plants total phenolic compounds, flavonoids, and radical scavenging activity of 21 selected tropical plants content of the flavonols quercetin, myricetin, and kaempferol in 25 edible berries determination of myricetin in medicinal plants by highperformance liquid chromatography antioxidant constituents of three selected red and green color amaranthus leafy vegetable characterization of bioactive compounds and antioxidant activity of fruit beers estimation of daily human intake of food flavonoids estimation of dietary flavonoid intake and major food sources of korean adults flavonoid intake in european adults (18 to 64 years) myricetin: a dietary molecule with diverse biological activities myricetin attenuated diabetes-associated kidney injuries and dysfunction via regulating nuclear factor (erythroid derived 2)-like 2 and nuclear factor-κb signaling myricetin induces cell death of human colon cancer cells via bax/bcl2-dependent pathway inhibition of interleukin-12 production in mouse macrophagesvia decreased nuclear factor-κb dna binding activity by myricetin, a naturally occurring flavonoid polyphenols: potential use in the prevention and treatment of cardiovascular diseases analgesic activity of myricetin isolated from myrica rubra sieb effect of myricetin on deoxycorticosterone acetate (doca)-salt-hypertensive rats antiproliferative activity of pteleopsis suberosa leaf extract and its flavonoid components in human prostate carcinoma cells two new flavonoid glycosides from the halophyte limonium franchetii flavonoids from comptonia peregrina the diarylheptanoid (+)-ar,11s-myricanol and two flavones from bayberry (myrica cerifera) destabilize the microtubule-associated protein tau polyphenols from aerial parts of polygonum bellardii and their biological activities flavonoids in the leaves of twentyeight polygonaceous plants flavonoids and phenolic acids from labisia pumila (kacip fatimah) dietary flavonoids: bioavailability, metabolic effects, and safety department of agriculture (usda) food database flavonol content varies among black currant cultivars flavonoids in monospecific eucalyptus honeys from australia wine and grape polyphenols -a chemical perspective syringetin, a flavonoid derivative in grape and wine, induces human osteoblast differentiation through bone morphogenetic protein-2/extracellular signal-regulated kinase 1/2 pathway comprehensive review of antimicrobial activities of plant flavonoids phytochemicals in helicobacter pylori infections: what are we doing now? structure-activity relationship of flavonoids on their anti-escherichia coli activity and inhibition of dna gyrase myricetin inhibits escherichia coli dnab helicase but not primase differential inhibitory effects of various flavonoids on the activities of reverse transcriptase and cellular dna and rna polymerases antimicrobial efficacy of plant phenolic compounds against salmonella and escherichia coli novel strategy for biofilm inhibition by using small molecules targeting molecular chaperone dnak anti-hiv-1 activity of flavonoid myricetin on hiv-1 infection in a dual-chamber in vitro model screening of flavonoids for antitubercular activity and their structure-activity relationships identification of myricetin and scutellarein as novel chemical inhibitors of the sars coronavirus helicase, nsp13 antibacterial phenolics from the mangrove lumnitzera racemosa anti-oxidative and antibacterial constituents from sedum hybridum molecular docking and screening studies of new natural sortase a inhibitors activity and interactions of antibiotic and phytochemical combinations against pseudomonas aeruginosa in vitro inhibitory effects of flavonoids on moloney murine leukemia virus reverse transcriptase activity compounds from syzygium aromaticum possessing growth inhibitory activity against oral pathogens activity of plant flavonoids against antibiotic-resistant bacteria antibacterial activity directed isolation of compounds from punica granatum flavonols inhibit sortases and sortasemediated staphylococcus aureus clumping to fibrinogen structure elucidation, conformational analysis and thermal effects on membrane bilayers of an antimicrobial myricetin ether derivative antimicrobial activity of antibiotics in combination with natural flavonoids against clinical extended-spectrum β-lactamase (esbl)-producing klebsiella pneumoniae inhibitory effect of dietary phenolic compounds on chlamydia pneumoniae in cell cultures ultra-small micelles based on polyoxyl 15 hydroxystearate for ocular delivery of myricetin: optimization, in vitro, and in vivo evaluation antioxidant activity of polyphenolic myricetin in vitro cell-free and cell-based systems rosmarinic and carnosic acids as superior natural antioxidant alternatives to α-tocopherol for the preservation of omega-3 oils antioxidant behavior of mearnsetin and myricetin flavonoid compounds-a dft study microarray and pathway analysis highlight nrf2/are-mediated expression profiling by polyphenolic myricetin development of a myricetin/ hydroxypropyl-β-cyclodextrin inclusion complex: preparation, characterization, and evaluation antiinflammatory and antioxidant activities of some extracts and pure natural products isolated from rhus tripartitum (ucria) ferric reducing and radical scavenging activities of selected important polyphenols present in foods antioxidant capacity and vasodilatory properties of mediterranean food: the case of cannonau wine, myrtle berries liqueur and strawberry-tree honey structural elucidation and antioxidant activities of proanthocyanidins from chinese bayberry (myrica rubra sieb. et zucc.) leaves activity-guided isolation of antioxidant principles from limoniastrum feei (girard) batt effect of myricetin, pyrogallol, and phloroglucinol on yeast resistance to oxidative stress myricetin suppresses oxidative stress-induced cell damage via both direct and indirect antioxidant action nitric oxide scavenging rates of solubilized resveratrol and flavonoids myricetin affords protection against peroxynitrite-mediated dna damage and hydroxyl radical formation comparative study on antioxidant capacity of flavonoids and their inhibitory effects on oleic acid-induced hepatic steatosis in vitro myricetin protects cells against oxidative stress-induced apoptosis via regulation of pi3k/akt and mapk signaling pathways protective effect of flavonoids against reactive oxygen species production in sickle cell anemia patients treated with hydroxyurea the neuroprotective potential of flavonoids: a multiplicity of effects effect of myricetin on behavioral paradigms of anxiety disclosure of a fundamental clue for the elucidation of the myricetin mechanism of action as amyloid aggregation inhibitor by mass spectrometry amyloid beta-peptide 25-35 selfassembly and its inhibition: a model undecapeptide system to gain atomistic and secondary structure details of the alzheimer's disease process and treatment myricetin: a naturally occurring regulator of metal-induced amyloid-β aggregation and neurotoxicity multifunction of myricetin on aβ: neuroprotection via a conformational change of aβ and reduction of aβ via the interference of secretases antioxidant compounds have potent anti-fibrillogenic and fibril-destabilizing effects for α-synuclein fibrils in vitro potent anti-amyloidogenic and fibril-destabilizing effects of polyphenols in vitro: implications for the prevention and therapeutics of alzheimer's disease myricetin protects hippocampal ca3 pyramidal neurons and improves learning and memory impairments in rats with alzheimer's disease polyphenolic flavonoid (myricetin) upregulated proteasomal degradation mechanisms: eliminates neurodegenerative proteins aggregation flavonoid myricetin modulates receptor activity through activation of channels and camk-ii pathway dihydromyricetin exerts a rapid antidepressant-like effect in association with enhancement of bdnf expression and inhibition of neuroinflammation myricetin attenuates depressant-like behavior in mice subjected to repeated restraint stress in vivo investigation on the potential of galangin, kaempferol and myricetin for protection of d-galactose-induced cognitive impairment myricetin attenuates the severity of seizures and neuroapoptosis in pentylenetetrazole kindled mice by regulating the of bdnf-trkb signaling pathway and modulating matrix metalloproteinase-9 and gabaa myricetin reduces toxic level of cag repeats rna in huntington's disease (hd) and spino cerebellar ataxia (scas) myricetin attenuated mpp+-induced cytotoxicity by anti-oxidation and inhibition of mkk4 and jnk activation in mes23. 5 cells myricetin prevents dopaminergic neurons from undergoing neuroinflammation-mediated degeneration in a lipopolysaccharide-induced parkinson's disease model effect of myricetin on the transgenic drosophila model of parkinson's disease effect of myricetin on the loss of dopaminergic neurons in the transgenic drosophila model of parkinson's disease inhibition and disaggregation of α-synuclein oligomers by natural polyphenolic compounds myricetin reduces 6-hydroxydopamine-induced dopamine neuron degeneration in rats myricetin protects against high glucose-induced β-cell apoptosis by attenuating endoplasmic reticulum stress via inactivation of cyclin-dependent kinase 5 myricetin: a potent approach for the treatment of type 2 diabetes as a natural class b gpcr agonist insulin signalling and the regulation of glucose and lipid metabolism ampactivated protein kinase and muscle insulin resistance. front biosci (landmark ed) myricetin attenuates hyperinsulinemia-induced insulin resistance in skeletal muscle cells myricetin ameliorates defective post-receptor insulin signaling via β-endorphin signaling in the skeletal muscles of fructose-fed rats minireview: therapeutic potential of myricetin in diabetes mellitus myricetin ameliorates defective post-receptor insulin signaling via beta-endorphin signaling in the skeletal muscles of fructose-fed rats myricetin modulates streptozotocincadmium induced oxidative stress in long term experimental diabetic nephrotoxic rats α-glucosidase inhibitory activities of myricetin in animal models of diabetes mellitus myricetin, a natural flavonoid, normalizes hyperglycemia in streptozotocin-cadmium-induced experimental diabetic nephrotoxic rats beneficial effect of myricetin on renal functions in streptozotocin-induced diabetes myricetin may provide protection against oxidative stress in type 2 diabetic erythrocytes effects of myricetin on glycemia and glycogen metabolism in diabetic rats insulinomimetic effects of myricetin on lipogenesis and glucose transport in rat adipocytes but not glucose transporter translocation inhibition of α-glucosidase and α-amylase by flavonoids improvement of insulin sensitivity in obese zucker rats by myricetin extracted from abelmoschus moschatus α-glucosidase inhibition and antihyperglycemic activity of flavonoids from ampelopsis grossedentata and the flavonoid derivatives molecular approach to identify antidiabetic potential of azadirachta indica evaluation of total flavonoids, myricetin, and quercetin from hovenia dulcis thunb. as inhibitors of α-amylase and α-glucosidase biochemical studies on the effect of phenolic compounds extracted from myrtus communis in diabetic rats syzygium cumini seed exhibits antidiabetic potential via multiple pathways involving inhibition of αglucosidase, dpp-iv, glycation, and ameliorating glucose uptake in l6 cell lines potential antihyperglycaemic effect of myricetin derivatives from syzygium malaccense assessment, origin, and implementation of breath volatile cancer markers vocc: a database of volatile organic compounds in cancer age and cancer risk: a potentially modifiable relationship programmed cell death, from a cancer perspective: an overview discovery of myricetin as a potent inhibitor of human flap endonuclease 1, which potentially can be used as sensitizing agent against ht-29 human colon cancer cells myricetin inhibits proliferation of cisplatin-resistant cancer cells through a p53-dependent apoptotic pathway myricetin induces apoptosis via endoplasmic reticulum stress and dna double-strand breaks in human ovarian cancer cells myricetin induces apoptosis and enhances chemosensitivity in ovarian cancer cells flavonoids showed anticancer effects on the ovarian cancer cells: involvement of reactive oxygen species, apoptosis, cell cycle and invasion myricetin is a novel natural inhibitor of neoplastic cell transformation and mek1 myricetin is a potent chemopreventive phytochemical in skin carcinogenesis myricetin exerts potent anticancer effects on human skin tumor cells myricetin suppresses invasion and migration of human lung adenocarcinoma a549 cells: possible mediation by blocking the erk signaling pathway enhancement of recombinant myricetin on the radiosensitivity of lung cancer a549 and h1299 cells myricetin enhance chemosensitivity of 5-fluorouracil on esophageal carcinoma in vitro and in vivo potential anticancer activity of myricetin in human t24 bladder cancer cells both in vitro and in vivo myricetin-induced oxidative stress suppresses murine t lymphocyte activation involvement of thromboxane a2 in the endothelium-dependent contractions induced by myricetin in rat isolated aorta inhibition of interleukin-2 production by myricetin in mouse el-4 t cells gene expression profiling of human umbilical vein endothelial cells exposed to myricetin morin and myricetin attenuate cd36 expression and oxldl uptake in u937-derived macrophages distinct signalling mechanisms are involved in the dissimilar myocardial and coronary effects elicited by quercetin and myricetin, two red wine flavonols lipid lowering and antioxidant activity of flavones in triton treated hyperlipidemic rats cardioprotective potential of myricetin in isoproterenol-induced myocardial infarction in wistar rats targeting stat1 by myricetin and delphinidin provides efficient protection of the heart from ischemia/reperfusion-induced injury antiallodynic effect of the flavonoid myricetin in a rat model of neuropathic pain: involvement of p38 and protein kinase c mediated modulation of ca2+ channels effect of myricetin on blood pressure and metabolic alterations in fructose hypertensive rats wound healing potential of chlorogenic acid and myricetin-3-o-β-rhamnoside isolated from parrotia persica protective functions of myricetin in lps-induced cardiomyocytes h9c2 cells injury by regulation of malat1 development of m10, myricetin-3-o-beta-d-lactose sodium salt, a derivative of myricetin as a potent agent of anti-chronic colonic inflammation effect of blueberin on fasting glucose, c-reactive protein and plasma aminotransferases, in female volunteers with diabetes type 2: double-blind, placebo controlled clinical study effect of emulin on blood glucose in type 2 diabetics molecular mechanisms underlying anticancer effects of myricetin flavonoid intake and risk of chronic diseases a prospective study of dietary flavonoid intake and incidence of epithelial ovarian cancer flavonoid intake and risk of pancreatic cancer in male smokers (finland) flavonoids intake and risk of lung cancer: a meta-analysis grape polyphenols exert a cardioprotective effect in preand postmenopausal women by lowering plasma lipids and reducing oxidative stress publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-269943-g77qe5ml authors: di sotto, antonella; vitalone, annabella; di giacomo, silvia title: plant-derived nutraceuticals and immune system modulation: an evidence-based overview date: 2020-08-22 journal: vaccines (basel) doi: 10.3390/vaccines8030468 sha: doc_id: 269943 cord_uid: g77qe5ml immunomodulators are agents able to affect the immune system, by boosting the immune defences to improve the body reaction against infectious or exogenous injuries, or suppressing the abnormal immune response occurring in immune disorders. moreover, immunoadjuvants can support immune system acting on nonimmune targets, thus improving the immune response. the modulation of inflammatory pathways and microbiome can also contribute to control the immune function. some plant-based nutraceuticals have been studied as possible immunomodulating agents due to their multiple and pleiotropic effects. being usually more tolerable than pharmacological treatments, their adjuvant contribution is approached as a desirable nutraceutical strategy. in the present review, the up to date knowledge about the immunomodulating properties of polysaccharides, fatty acids and labdane diterpenes have been analyzed, in order to give scientific basic and clinical evidence to support their practical use. since promising evidence in preclinical studies, limited and sometimes confusing results have been highlighted in clinical trials, likely due to low methodological quality and lacking standardization. more investigations of high quality and specificity are required to describe in depth the usefulness of these plant-derived nutraceuticals in the immune system modulation, for health promoting and disease preventing purposes. immunomodulators are defined as agents able to affect the immune response, which represents the set of reactions activated to protect the organism against infective agents, environmental injuries and illness; moreover, immune response can counteract the invasion of harmful native cells, such as precancerous and cancerous ones [1] . immune response is mediated by a first line of defence, namely innate immunity (figure 1 ), which is characterized by physical and biochemical barriers, alongside a non-specific cell-mediated immune response, including granulocytes, macrophages, natural killer cells and humoral elements, which cooperate to counteract pathogen infection and malignant transformation [2] . moreover, an adaptive immunity is activated as a second defense line after a macrophage-mediated presentation of antigens to b lymphocytes, with the help of t lymphocytes; then, b cells can mediate the humoral immunity through the production of high-affinity antibodies and establish immunological memory [3] . moreover, t lymphocytes can mediate cellular immunity after activation by cytokines released from helper t cells [2] . responses involved in innate and adaptive immunity. fast and nonspecific responses, occurring against all factor identified as nonself, are involved in innate immunity; conversely, adaptive immunity is a highly specific, complex and slow response mediated by t and b lymphocytes, which release antigen-specific antibodies and cytokines. immunomodulators can directly affect innate and adaptive response or the factors involved, thus leading to immunostimulant or immunosuppressive effects. a further group of immunomodulators is represented by immunoadjuvants, able to enhance immune response to vaccines without producing specific antigenic effects, and more recently approached as adjuvant pharmacological treatments, especially for viral infections and cancers [8] [9] [10] [11] [12] . immune-associated disorders, including autoimmune diseases, viral or bacterial infections, and chronic diseases, are usually associated with acute inflammation, which represents a key component for the activation of immune response [13] . on the other hand, the chronicity of inflammatory response can negatively influence the immune function, affecting both innate immune cells and t and b lymphocytes, thus suggesting a possible usefulness of anti-inflammatory immunomodulators [13] . accordingly, immunomodulatory agents, with antioxidant and anti-inflammatory activity, have attracted great attention as possible chemopreventive agents, due to their ability to counteract chronic inflammation, which provides favorable conditions for the transition from normal to cancer cell [14] . responses involved in innate and adaptive immunity. fast and nonspecific responses, occurring against all factor identified as nonself, are involved in innate immunity; conversely, adaptive immunity is a highly specific, complex and slow response mediated by t and b lymphocytes, which release antigen-specific antibodies and cytokines. immunomodulators can directly affect innate and adaptive response or the factors involved, thus leading to immunostimulant or immunosuppressive effects. immunomodulating agents can affect immunity in a negative or positive manner, thus being categorized as suppressing or stimulant [4] . particularly, immunosuppressors inhibit the activation of immune response or decrease the activity of its components, thus restoring normalcy. they are of interest in organ transplantations and in autoimmune disorders, wherein the immune system mistakenly activates an immune response against the own body tissues, leading to their destruction [4] . for instance, vitamin d has been shown to counteract the aberrant immune responses of systemic lupus erythematosus, without compromising the physiological defences and to produce benefits in atopic dermatitis too [5, 6] . conversely, immunostimulants boost the endogenous immune defences, thus allowing one to restore or maintain the body homeostasis [4] . they can be usefully exploited as immunotherapeutic agents by individuals with immunocompromised conditions; however, they can represent suitable prophylactic strategies for healthy individuals or more susceptible subjects against viral infections [4] . in support, during the current sars-cov-2 pandemic, the trained immunity by vaccines, which induce heterologous protection, have been proposed as a rational strategy to boost antiviral defences and reduce susceptibility to infection [7] . a further group of immunomodulators is represented by immunoadjuvants, able to enhance immune response to vaccines without producing specific antigenic effects, and more recently approached as adjuvant pharmacological treatments, especially for viral infections and cancers [8] [9] [10] [11] [12] . immune-associated disorders, including autoimmune diseases, viral or bacterial infections, and chronic diseases, are usually associated with acute inflammation, which represents a key component for the activation of immune response [13] . on the other hand, the chronicity of inflammatory response can negatively influence the immune function, affecting both innate immune cells and t and b lymphocytes, thus suggesting a possible usefulness of anti-inflammatory immunomodulators [13] . accordingly, immunomodulatory agents, with antioxidant and anti-inflammatory activity, have attracted great attention as possible chemopreventive agents, due to their ability to counteract chronic inflammation, which provides favorable conditions for the transition from normal to cancer cell [14] . furthermore, immunostimulants can act as adjuvant anticancer treatments, to counteract their immunosuppressive side-effects [14] . particularly, aristolochic acid, an alkaloid from aristolochia clematitis l., showed immunostimulatory properties, by enhancing the phagocytic activity of peritoneal macrophages and leukocytes; however, its potential cannot be exploited, because of its carcinogenic risk [50] . likewise, vincristine and staurosporine act as immunostimulants at low doses, while as immunosuppressors particularly, aristolochic acid, an alkaloid from aristolochia clematitis l., showed immunostimulatory properties, by enhancing the phagocytic activity of peritoneal macrophages and leukocytes; however, its potential cannot be exploited, because of its carcinogenic risk [50] . likewise, vincristine and staurosporine act as immunostimulants at low doses, while as immunosuppressors at higher doses [51] . among polyphenols, resveratrol stimulated both cellular and humoral immunity in preclinical models, thus preventing pathogen replication and inflammation, and promoted antitumor immune response too [52] . furthermore, cichoric acid from echinacea promoted phagocytic activity, both in vitro and in vivo [53] . anti-inflammatory and immune-modulatory effects has been highlighted for curcumin too, although the poor bioavailability limits its clinical application [54] . in the present review, up to date knowledge on the scientific basis for the immunomodulatory activity and clinical relevance of some emerging classes of plant-derived nutraceuticals, including polysaccharides, fatty acids and labdane diterpenes, has been reported. a comprehensive search was made using pubmed and scopus electronic databases and selecting english as the preferred language, although no language limitations nor filters were applied. for more specific requirements, google scholar and clinicaltrials.gov were considered too. the following searching keywords and their combinations through the boolean logical operators were used: "herbal immunomodulators", "phytochemicals", "immune system", "nutraceuticals", "medicinal plants", "immunomodulation", "immune system boosters", "immunosuppressors", "immunoadjuvants", "gut microbiome", "natural occurrence", "chemical features", "preclinical studies", "clinical trials", "polysaccharides", "echinacea", "astragalus", "β-glucan", "fatty acids", "pufa", "oleic acid", "punicic acid", "γ-linolenic acid", "linoleic acid", "evening primrose oil", "borage oil", "flaxseed oils", "labdane diterpenes" and "andrographolide". this overview allows one to identify novel immune system modulators to be usefully exploited for health promoting and disease preventing purposes. polysaccharides are carbohydrate macromolecules containing at least 10 monosaccharide units, joined by glycosidic linkages to form long-chain molecules, which can be both linear and highly branched. they are called homopolysaccharides when constituted of the same monosaccharide unit, while heteropolysaccharides if different units are present. some of them are also referred to as dietary fibres, meaning that these macromolecules are neither digested nor absorbed in the human small intestine [55] . several polysaccharides have been found to modulate both innate and adaptive immune responses, among which, glucans, mannans, pectins, fucoidans, galactans, fructans, and xylans are the most studied ( figure 3 ) [56] . chemical structure, molecular weight, conformation, the presence of functional groups (i.e., acetyl and sulfate groups), and branching have been identified as structural features for the immunostimulatory properties of polysaccharides. the chemical structures of the polysaccharides associated with immunomodulatory properties are displayed in figure 4 . glucans are based on the d-glucopyranosyl unit (homoglucans); the different glycosidic bonds, namely (β1→4), (β1→3), and (β 1→6) or (α1→3), (α 1→4), and (α 1→6), allow the production of linear and branched glucans. it seems that (β1→3)-d-glucan moiety, triple helix conformations, sulfation and carboxymethylation of (β 1→3)-d-glucans, and chain acetylation are involved in glucan immunostimulatory activity. regarding (α1→6) (α1→4)-d-glucans, their structure activity relationship is less characterized [56] . while heteropolysaccharides if different units are present. some of them are also referred to as dietary fibres, meaning that these macromolecules are neither digested nor absorbed in the human small intestine [55] . several polysaccharides have been found to modulate both innate and adaptive immune responses, among which, glucans, mannans, pectins, fucoidans, galactans, fructans, and xylans are the most studied ( figure 3 ) [56] . chemical structure, molecular weight, conformation, the presence of functional groups (i.e., acetyl and sulfate groups), and branching have been identified as structural features for the immunostimulatory properties of polysaccharides. the chemical structures of the polysaccharides associated with immunomodulatory properties are displayed in figure 4 . glucans are based on the d-glucopyranosyl unit (homoglucans); the different glycosidic bonds, namely (β1→4), (β1→3), and (β 1→6) or (α1→3), (α 1→4), and (α 1→6), allow the production of linear and branched glucans. it seems that (β1→3)-d-glucan moiety, triple helix conformations, sulfation and carboxymethylation of (β 1 → 3)-d-glucans, and chain acetylation are involved in glucan immunostimulatory activity. regarding (α1 → 6) (α1 → 4)-d-glucans, their structure activity relationship is less characterized [56] . mannans consist of a d-mannose backbone, linked mainly by β1→4 bonds, which can be ramified with other monosaccharide, so originating glucomannan, galactomannan, and galactoglucomannan [57] . furthermore, (β1→3)-or (α1→3)-, (β1→2)-, and (β1→6)-or (α1→6)-dmannosidic bonds are reported [56] . the (β1→6)-d-mannan moiety (e.g., galctoglucomannans), acetyl and sulfate group presence, and this kind of branching seems to confer a high immunostimulatory activity [58] [59] [60] . pectins are complex polysaccharides which contain a common galactopyranosyluronic acid. homogalacturonans, xylogalacturonan, apiogalacturonan, rhamnogalacturonan, type i and ii arabinogalactans belong to this class. particularly, type i arabinogalactans (ag-i) possess an α-1arabinofuranosyl and β-d-galactopyranosyl units linked via position 3 at the main chain, while type ii arabinogalactans (ag-ii) comprise highly branched polysaccharides with ramified chains of (β1→ 3)-and (β1→6)-d-galactopyranosyl units [61, 62] , to which the arabinosyl units might be attached. the degree of branching, methyl esterification, acetylation, and the type of branched chains and mannans consist of a d-mannose backbone, linked mainly by β1→4 bonds, which can be ramified with other monosaccharide, so originating glucomannan, galactomannan, and galactoglucomannan [57] . furthermore, (β1→3)-or (α1→3)-, (β1→2)-, and (β1→6)-or (α1→6)-d-mannosidic bonds are reported [56] . the (β1→6)-d-mannan moiety (e.g., galctoglucomannans), acetyl and sulfate group presence, and this kind of branching seems to confer a high immunostimulatory activity [58] [59] [60] . pectins are complex polysaccharides which contain a common galactopyranosyluronic acid. homogalacturonans, xylogalacturonan, apiogalacturonan, rhamnogalacturonan, type i and ii arabinogalactans belong to this class. particularly, type i arabinogalactans (ag-i) possess an α-1-arabinofuranosyl and β-d-galactopyranosyl units linked via position 3 at the main chain, while type ii arabinogalactans (ag-ii) comprise highly branched polysaccharides with ramified chains of (β1→3)-and (β1→6)-d-galactopyranosyl units [61, 62] , to which the arabinosyl units might be attached. the degree of branching, methyl esterification, acetylation, and the type of branched chains and molecular weight determine the structural diversity [63] . moreover, flexible chain conformation and branched regions are the main ones responsible for the immunomodulatory properties [64, 65] . galactans are polysaccharides rich in galactose and include, beside type i and ii arabinogalactans, carrageenans, chemically characterized by repeating disaccharide units of sulfated or unsulfated d-galactose, that are linked by (β1→4)-and (α1→3)-bonds. low molecular weight (<20 kda) and a high degree of sulfation have been reported as features that high influence their immunomodulatory properties [66, 67] . fucoidans are heteropolysaccharides rich in l-fucopyranosyl sulfated units linked by (α1→2), (α1→3) or (α1→4) bonds. other monosaccharides can be present, such as galactopyranosyl, mannopyranosyl, xylosepyranosyl and uronic acids [68] . the naturally higher content of sulfate groups and the presence of acetyl groups are associated with a higher stimulatory activity [56] . fructans are polysaccharides which constitute up to 70 fructose units with a sucrolose terminal molecule. they are classified in inulin with a (β2→1)-d-fructofuranosyl, levan with a (β2→6)-d-fructofuranosyl, and mixed type, with both (β2→1)-and (β2→6)-linked d-fructofuranosyl moieties. a helical conformation has been associated with the modulatory activity on the immune system [69, 70] . at last, xylans are polysaccharides containing predominantly a backbone of (β1→4)-dxylosepyranosyl units. other monomers attached to their backbone include α-dglucopyranosyl a units (glucuronoxylans) and α-l-arabinofuranosyl units (arabinoxylans). a correlation between their structure and activity has not been elucidated yet [71, 72] . polysaccharides are naturally occuring in animal body fluids, cell walls, bacteria, yeast and fungi, extra cellular fluids, and in plant seeds, stems and leaves, which represent the focus of the present review. the main advantage of plant polysaccharides seems to be the low toxicity with respect to immunomodulatory bacterial polysaccharides and synthetic compounds [73] . thus, they represent an ideal alternative for immune modulation. a variety of polysaccharides with immunomodulatory properties have been discovered in different species of plants (table 2 ). among the most studied, there are type i and ii arabinogalactans from astragalus membranaceus (fisch.) bge., fructans from allium sativum l. [56] , fucogalactoxyloglucan and type ii acidic arabinogalactan from echinacea purpurea l. (moench), ginsan and panaxanes from panax ginseng c.a. meyer, acemannan and aloeride from aloe vera l. [74] , and glucomannan from amorphallus konjac koch [75] . several studies have shown that polysaccharides from plants can modulate both innate and acquired intestinal immunity, by direct and indirect mechanisms. the former include the activation of immune cells (e.g., macrophages, dendritic cells, natural killer cells, t cells, b lymphocytes), while the latter the short-chain fatty acid (scfa) formation ( figure 5 ). of immune cells (e.g., macrophages, dendritic cells, natural killer cells, t cells, b lymphocytes), while the latter the short-chain fatty acid (scfa) formation ( figure 5 ). the immunomodulatory effects of plant polysaccharides on macrophages are mainly achieved through the generation of reactive oxygen and nitrogen species (ros and nos), and the stimulation of cytokines secretion, cell proliferation, and macrophage phagocytic activity [101] . for example, a. membranaceus polysaccharides have been shown to promote nitric oxide (no) synthesis in macrophages, by inducing the gene expression of inducible nitric oxide synthase (inos), through the activation of nuclear factor kappa-b (nf-κb)/rel [102, 103] . moreover, they were also able to increase the macrophage phagocytic activity, by enhancing their secretion of release factor and intracellular ca 2+ concentration [104, 105] . pectic polysaccharides from citrus unshiu marc. have been shown to simultaneously regulate the expression of pro-and anti-inflammatory cytokines. particularly, they increased the production of the pro-inflammatory cytokines tumor necrosis factor (tnf)-α and interleukin (il)-6 and the antiinflammatory cytokine il-12 in macrophage raw264.7, so showing a regulatory mechanism to maintain an equilibrium state [106] . arabinogalactan from e. purpurea has been reported to increase macrophages activation and il-1, tnf-α and interferon (ifn)-β production [86] . the activation of macrophages by plant polysaccharides seems to be due to specific receptors present on their surface, which initiates the immune response, and exerts an immunomodulatory effect. these receptors are called pattern recognition molecules, and include: toll-like receptor 4 (tlr4), cd14, complement receptor 3 (cr3), scavenger receptor (sr), mannose receptor (mr), and dectin-1. their activation determines a series of intracellular signaling cascades, leading to the transcriptional activation and production of inflammation-related cytokines [101] . immunity modulation by plant polysaccharides can be achieved also by modulating the cytokine release from intestinal dendritic cells. indeed, pectin has been shown to reduce il-6 and il-10 release induced by the synthetic lipopeptide p3csk4 [107] . moreover, inulin, pectin, arabinoxylan and β-glucan have been found to elevate il-10/il-12 ratio and to reduce the release of ifn-γ, il-12, il-1, il-6, il-8, monocyte chemoattractant protein (mcp)-1, macrophage inflammatory proteins the immunomodulatory effects of plant polysaccharides on macrophages are mainly achieved through the generation of reactive oxygen and nitrogen species (ros and nos), and the stimulation of cytokines secretion, cell proliferation, and macrophage phagocytic activity [101] . for example, a. membranaceus polysaccharides have been shown to promote nitric oxide (no) synthesis in macrophages, by inducing the gene expression of inducible nitric oxide synthase (inos), through the activation of nuclear factor kappa-b (nf-κb)/rel [102, 103] . moreover, they were also able to increase the macrophage phagocytic activity, by enhancing their secretion of release factor and intracellular ca 2+ concentration [104, 105] . pectic polysaccharides from citrus unshiu marc. have been shown to simultaneously regulate the expression of pro-and anti-inflammatory cytokines. particularly, they increased the production of the pro-inflammatory cytokines tumor necrosis factor (tnf)-α and interleukin (il)-6 and the anti-inflammatory cytokine il-12 in macrophage raw264.7, so showing a regulatory mechanism to maintain an equilibrium state [106] . arabinogalactan from e. purpurea has been reported to increase macrophages activation and il-1, tnf-α and interferon (ifn)-β production [86] . the activation of macrophages by plant polysaccharides seems to be due to specific receptors present on their surface, which initiates the immune response, and exerts an immunomodulatory effect. these receptors are called pattern recognition molecules, and include: toll-like receptor 4 (tlr4), cd14, complement receptor 3 (cr3), scavenger receptor (sr), mannose receptor (mr), and dectin-1. their activation determines a series of intracellular signaling cascades, leading to the transcriptional activation and production of inflammation-related cytokines [101] . immunity modulation by plant polysaccharides can be achieved also by modulating the cytokine release from intestinal dendritic cells. indeed, pectin has been shown to reduce il-6 and il-10 release induced by the synthetic lipopeptide p3csk4 [107] . moreover, inulin, pectin, arabinoxylan and β-glucan have been found to elevate il-10/il-12 ratio and to reduce the release of ifn-γ, il-12, il-1, il-6, il-8, monocyte chemoattractant protein (mcp)-1, macrophage inflammatory proteins (mip)-1α, rantes and tnf-α by dendritic cells [108] . polysaccharide enriched extracts of e. purpurea have been found to promote the phenotypic and functional maturation of dendritic cells by modulating c-jun n-terminal kinase (jnk), p38 mitogen-activated protein kinase (mapk) and nf-κb pathways [109] . the activation of natural killer (nk) cells also contributes to the immunity modulation by polysaccharides. indeed, it has been shown that a. membranaceus polysaccharides can enhance the activity and killing effects of nk cells and promote their proliferation in rats with gastric cancer [110] . moreover, they were able to increase cd3-cd4-cd8+ nks in peripheral blood lymphocytes [111] . nks activation is probably due to the polysaccharides interaction with the killer cell lectin-like receptor k1(klrk1) of nks [112] . arabinoxylans extracted from wheat bran have been shown to inhibit the growth of transplantable tumors, and to promote the nk cell activity in s180 tumor-bearing mice [113] . moreover, the mgn-3 rice bran arabinoxylan showed to enhance natural killer (nk) cell activity in aged c57bl/6 and c3h mice upon its intraperitoneal injection [114] . adaptive immunity is also modulated by plant polysaccharides. particularly, fan et al. have shown that polysaccharides from a. membranaceus significantly up-regulated the proliferation of b lymphocytes, probably through the interaction with immunoglobulin on the surface of b cells [115] [116] [117] . a. membranaceus polysaccharides were also able to increase the number of cd3+cd4+cd8+ memory t helper (th) cells and cd3+cd4-cd8+ cytotoxic t cells [111] . moreover, they also enhance the cd4+/cd8+ t cell ratio [118] . furthermore, arabinoxylan were found to increase the activation of tand b-cells and humoral and cell-mediated immunity in tumor bearing mice [71] . at last, β-glucan microparticles enhanced t-cell activation and proliferation in vitro [119] . their ability to affect the immune system by inducing th1 and/or th2 type immune response makes polysaccharides suitable adjuvants of the vaccine. among them, inulin, chitosan, glucans and mannans have been most extensively studied. particularly, the gamma and delta forms of inulin fructan have shown adjuvant activity against infectious pathogens by stimulating both th1 and th2 responses without inducing immunoglobulin e (ige) production [120] . moreover, advax, a polysaccharide derived from delta inulin, has demonstrated to increase the immunization derived from influenza vaccine in mice. particularly, an induction of neutralizing antibody and memory b-cell against influenza, an increase in cd4 and cd8 t-cell proliferation, and enhanced levels of il-2, ifn-γ, il-5, il-6 were highlighted [121] . advax also enhanced the immunogenicity of hepatitis b surface antigen (hbs) in mice and guinea pigs, by increasing both anti-hbs antibody titers and anti-hbs cd4 and cd8 t-cells. th1, th2 and th17 responses were increased too [122] . astragalus polysaccharides were also used as adjuvants of hepatitis b virus dna vaccine in a mice model, showing increased hbsag-specific antibody levels, higher activity of t cells, the production of il-4, il-2 and ifn-γ by cd4+ t cells, and ifn-γ expression of cd8+ t cells. moreover, a stimulation of cytotoxic lymphocytes and dendritic cells maturation, and a reduction in the frequency of regulatory t cells were observed [123] . mannans and fructooligosaccharide have also been shown to possess adjuvanticity [124, 125] . furthermore, indirect effects are involved in the immunomodulatory properties of polysaccharides. in particular, dietary fibers (e.g., inulin, mannan, β-glucan, pectin) are metabolized by intestinal bacteria in the anaerobic environment of the cecum and colon, so generating scfa, such as acetate, propionate and butyrate [126] . these molecules are able to cross the gut epithelium and interact with surface receptors on the immune cells, such as the g-protein coupled receptors (gprs) 41 and 43 [127] . the activation of gprs by scfa modulates inflammatory signalling pathways, such as nf-κb, erk and p38 mapk [128, 129] . moreover, it has been highlighted that scfa can reach t lymphocyte nucleus, so modulating several functions through a histone deacetylase (hdac) inhibition. recently, scfa have been reported able to induce t cells metabolic alterations by enhancing the mtor complex activity. particularly, after absorption into t cells, scfa can stimulate the activity of mtor complex, so increasing the conversion of pyruvate into acetyl-coa. moreover, the acetyl groups from scfa can be link to coa and enter the tricarboxylic acid cycle. the increased levels of citrate are exported from mitochondria into the cytoplasm, where the enzyme atp citrate lyase converts it into acetyl-coa, then used by histone acetyltransferases (hats) for histone acetylation and the regulation of cytokine gene expression [126] . some clinical studies have been carried out on the potential immunomodulatory properties of polysaccharides, and a. membranaceus, e. purpurea and β-glucan have been most investigated. in a clinical trial on a. membranaceus by jiang et al. [130] , twenthy-eight stable continuous ambulatory peritoneal dialysis patients were treated with peritoneal dialysis fluid containing astragalus (20 ml/2 l) for one week. an increase in the macrophage phagocytic capacity, no and tnf-α contents were observed in patients compared to those before the treatment [130] . furthermore, ji et al. [131] investigated the effect of astragalus pre-operative treatment of colorectal cancer patients (n = 128) on immune function. results showed that astragalus pre-operative treatment promoted the nk cell activity in postoperative patients. in addition, the possible immunomodulatory activity of astragalus in patients with acute exacerbations of bronchial asthma (n = 72) has been investigated [132] . particularly, it was observed that the combination of conventional therapy with astragalus injection for 14 days improved the effects of routine treatment, by enhancing t lymphocyte and nk-cells immune function. results of clinical trials on the immune system modulation by e. purpurea are controversial. particularly, a randomized blinded trial carried out on 108 patients revealed that there was no significant difference in the incidence and severity of colds and respiratory infection between echinacea treatment (8 weeks) and placebo groups. however, a small decrease of total lymphocyte counts was observed [133] . another randomized, placebo controlled, double-blind clinical trial investigated the effect of different echinacea preparations, namely echinaforce ® (e. purpurea preparation from 95% herba and 5% radix), e. purpurea concentrate (same preparation at 7 times higher concentration), special e. purpurea radix preparation (totally different from that of echinaforce ® ) on the reduction of the complaint index, defined by 12 symptoms in healthy, adult volunteers who caught a common cold. the treatment continued until the enrolled patients felt healthy again, but not longer than 7 days. the supplementation with echinaforce ® and its concentrated preparation showed to be significantly more effective than the special echinacea extract or placebo. moreover, all treatments were well tolerated [134] . furthermore, prevention trials have been carried out, showing that echinacea products slightly reduce the risk of getting a cold in healthy individuals [135] . however, the heterogeneity (e.g., different species and part used) of preparations used in the trials makes the conclusions on the potential immunomodulatory properties of echinacea difficult. clinical trials concerning the β-glucan immunomodulatory properties have also been carried out, although in some cases, yeast-derived-glucan were used. particularly, three randomized, double-blind, placebo-controlled studies have evaluated the effects of short-term β-glucan supplementation on children with chronic respiratory problems. after 30 days' treatment, significant improvements in immunoglobulin, lysozyme, exhaled nitric oxide, and calprotectin production were found [136] [137] [138] . furthermore, the combination of resveratrol plus carboxymethyl-β-glucan as a solution for aerosol has been tested in clinical trials. particularly, the ability of the combination to prevent or treat recurrent respiratory infections in children was studied [139, 140] . in both cases, resveratrol plus carboxymethyl-β-glucan had a positive impact on children clinical conditions. indeed, nasal obstruction, rhinorrhea, sneezing, cough, fever, medication use, medical visits, and school absence were significantly reduced. moreover, resveratrol plus carboxymethyl-β-glucan have also been shown to relief nasal symptoms in children with allergic rhinitis, due to pollen allergy [141] . at last, mannans should be mentioned. they have been reported to possess adjuvant-vaccine properties in clinical studies, probably mediated by its interaction with mannose receptors. particularly, it has been shown that oxidized mannan-mucin 1 can be useful as an adjuvant in the breast cancer immunotherapy. indeed, a 12-15 years follow-up has highlighted that it decreases the cancer recurrence rate and prolongs recurrence time, without inducing toxicity or adverse reactions [124] . fatty acids (fa) are a large group of lipids, characterized by a different number of carbons, arranged in a linear carbon chain skeleton of variable length with a terminal carboxylic group [142] . based on the number of carbons in the chain, fatty acids can be classified as shortchain fatty acids (scfa; aliphatic tails up to a maximum of six carbons), medium-chain fatty acids (mcfa; aliphatic tails of 7-12 carbons), long-chain fatty acids (lcfa; aliphatic tails of 13 to 21 carbons) and very long-chain fatty acids (vlcfa; aliphatic tails of 22 and more carbons) [143] . among them, scfa, such as acetate, propionate, and butyrate, are produced by gut microbiota enzymes (i.e., propionate-coa transferase and propionaldehyde dehydratase) during the metabolism of carbohydrates and peptides containing branched-chain amino acids [144] . bacteroidetes are reported to be mainly responsible for the production of acetate and propionate, while firmicutes are the primary contributors of butyrate; however, other bacteria such as lactobacillus and bifidobacterium spp. are involved too [144] . based on the presence of different double bonds in this structure, fatty acids can be distinguished in saturated fatty acids (sfa), lacking double bonds in their carbon backbone, and unsaturated fa (ufa), which may contain one or more double bonds, thus leading to monounsaturated (mufa) and polyunsaturated fa (pufa) [143] . sfa include palmitic acid (c16:0), lauric acid (c12:0), myristic acid (c14:0), and stearic acid (c18:0), whereas n-9 oleic acid (c18:1) is an example of mufa. furthermore, pufa class includes fatty acids such as α-linolenic acid (ala; c18:3), linoleic acid (la; c18:2) and further long-chain metabolites [143] . the number of carbon atoms and unsaturated bond position are used for the systematic nomenclature of fa. moreover, the greek letters omega (ω) and delta (∆) are included, to indicate how far a double bond is from the terminal methyl carbon and the presence and position of one or more double or triple bonds in the carbon backbone, respectively [143] . a further "ω" or "n" classification designates the position of the first double bond in the skeleton from the end opposite to the carboxy group. accordingly, oleic acid is classified as a ω-9 (or n-9) fatty acid, while linoleic acid and α-linolenic acid are ω-6 (or n-6) and ω-3 (or n-3) fatty acids, as they contain the double bond nine, six and three carbons from the methyl end [143] . nomenclature of the major representative fatty acids in the different fa classes is displayed in table 3 . unsaturated fatty acids can be characterized on the basis of the cisor transorientation of the double bonds. usually, natural fatty acids carry a cisconfiguration, although some trans-fatty acids can also occur in foods as a consequence of the hydrogenation process, which can move double bonds from their naturally occurring position to a trans-configuration [143] . trans-fatty acids are considered undesirable compounds in foods, as their intake is associated with an increased risk of cardiovascular and metabolic diseases [145] . pufa can be further classified depending on the relative positions of the double bonds, as conjugated (double-bonded carbon atoms alternate with single bonds) and unconjugated (double bonds separated by one or more single bonds) [143] . unconjugated pufa, especially ω-3, ω-6, and ω-9 series, are the most occurring in nature. the most common conjugated pufa are trienes, such as octadecatrienoic acids (e.g., punic acid, calendic acid). fatty acids within the series are biosynthetically related, being synthesized through enzymatic processes of desaturation, chain elongation, and chain shortening [146] . particularly, the biosynthesis of ω-3 and ω-6 pufa starts from α-linolenic acid (ala or linolenate; 9,12,15-18:2) and linoleic acid (la or linoleate; 9,12-18:2), respectively ( figure 6 ). these precursors cannot be synthetized by mammals, which lack the ∆12 and ∆15 desaturases responsible for the convertion of 18:1 ω-9 fa to 18:2 ω-6 and 18:3 ω-3 pufa, and must be supplied by the diet, thus being considered as essential fatty acids. the initial rate-limiting step for the biosynthesis of ω3 and ω6 fatty acids is the insertion of a further double bond at the ∆6 carbon into the carbon chain of ala and la, through the help of a ∆6 desaturase enzyme: stearidonic acid (sa; 6,9,12,15-18:4) and γ-linolenic acid (gla; 6,9,12-18:3) are formed, respectively. these compounds are converted to eicosatetraenoic acid (eta; 2,4,6,8-20:4) and dihomo γ-linolenic acid (dgla; 8,11,14-20:3) by the elongase 5, being further converted to eicosapentaenoic acid (epa; 2,4,6,8,10-20:5) and arachidonic acid (aa; 5,8,11,14-20:4) , by the addition of a double bond at the ∆5 position, through a ∆5 desaturase. further elongations convert epa and aa to docosapentaenoic acid (2,4,6,8,10-22:5) and adrenic acid (7,10,13,16-22:4) ; then, a desaturation by ∆6 desaturase generates docosahexaenoic acid (dha; 4,7,10,13,16,19-22:6) [146] . both series of fatty acids can be further metabolized by cyclooxygenase and lipoxygenase enzymes, to obtain eicosanoids, including prostaglandins, thromboxanes and leukotrienes, acting as central modulators of the inflammatory process [146] . the byosynthetic pathways of ω3 and ω6 fatty acids are interconnected; indeed, it is known that long-chain derivatives from linolenic acid are accumulated in tissue only slightly when competing ω6 analogues exceed their amounts. therefore, suitable levels can be reasonably obtained through diet and when an optimum ratio of ω3 and ω6 series is maintained. both series of fatty acids can be further metabolized by cyclooxygenase and lipoxygenase enzymes, to obtain eicosanoids, including prostaglandins, thromboxanes and leukotrienes, acting as central modulators of the inflammatory process [146] . the byosynthetic pathways of ω3 and ω6 fatty acids are interconnected; indeed, it is known that long-chain derivatives from linolenic acid are accumulated in tissue only slightly when competing ω6 analogues exceed their amounts. therefore, suitable levels can be reasonably obtained through diet and when an optimum ratio of ω3 and ω6 series is maintained. figure 6 . biosynthetic pathways of ω-3 and ω-6 fatty acids. fatty acids occur widely in nature, being identified in both animal tissue and plants. particularly, short-chain saturated acids are components of milk fats: in bovine milk, butanoic acid along with other scfa and mcfa have been reported [147] . likewise, the mcfa lauric acid and myristic acid are the major components of the oils obtained from some lauraceae and myristiceae species [148] . moreover, palmitic acid is the most representative sfa in vegetable oils, such as palm oil [149] . fatty acids with immune modulating properties mainly belong to the long-chain classes; among them, punicic acid is a peculiar conjugated triene, found to be a unique component of pomegranate seed oil [150] , while oleic acid is one of the most widely distributed fatty acids: it represents 49% to 83% of total fa in olive oil, although it does occur in high amounts in other oils, such as those from grape seeds, canola and sufflower [151, 152] . moreover, ω3 and ω6 fatty acids have been highlighted in several natural sources, wherein both series co-occur (table 4) , although in different amounts [153] . table 4 . major natural sources of long-chain monounsaturated (mufa) and polyunsaturated fatty acids (pufa) associated with immune system modulating activities and relative amounts. some vegetable oils, including rapseed, hemp seed, and sunflower oils, contain higher levels of la (essential ω6 pufa), while ala (essential ω3 pufa) is in lower proportion; a similar trend has also been reported for soybean, corn, and for dried black walnuts and brazilnuts; conversely, higher amounts of ala respect to la are reported in flaxseed oil and in the seeds of chia and perilla [153] . likewise, green leafy vegetables seem to be an interesting source of ala [163] . fish oils are also sources of both epa and dha (ω3 pufa), with lower amounts of dpa (ω6 pufa) [153] . wild marine species showed to contain higher ω3 pufa levels compared to farmed ones, likely due to the feed composition [153] . some vegetables can supply both the essential pufa and some derivative fatty acids. particularly, the oils obtained from the seeds of borago spp., echium spp., ranunculus spp. and oenothera biennis l. have been reported to contain high levels of both la and γ-linolenic acid (gla) [159, 160] . fatty acids play energetic, metabolic, and structural functions, being the main component of phospholipids, triglycerides, diglycerides and monoglycerides. a separate category is represented by scfa, which act as metabolites of carbohydrates, produced by gut microbiota: their role in the modulation of immune function is described in section 2.3. long-chain fatty acids have been found involved in immune modulation, being able to affect both innate and adaptive response. although specific profiles characterize each class of fatty acids, these effects are mainly ascribed to their ability to target the cell membrane, where they can be incorporated, thus changing membrane composition and fluidity and modulating membrane-protein interaction and signal transduction. furthermore, a role in the control of inflammation has been reported. epithelial growth factor receptors (a critical crossroad of multiple receptor pathways which is potentially implicated in the regulation of proliferation and possibly involved in atherogenesis) are considered possible targets for unsaturated fatty acids [164] . mufa, especially oleic acid, have attracted great attention in the years as possible immunomodulating nutrients. preclinical studies demonstrated the ability of oleic acid to modulate the immune system, through affecting both innate and adaptive immunity response [164] . indeed, it diminished nk cell activity [165] and the expression of the leucocyte adhesion molecules, which have shown to be implicated in some pathophysiological conditions, such as rheumatoid arthritis [165] . furthermore, it enhanced neutrophil aggregation and neutrophil-endothelial cell attachment, phagocytic and candidacidal capacities [166, 167] . in regard to adaptive response, it inhibited the proliferation of immune cells, such as jurkat t cells and lymphocytes, likely through the regulation of the cell cycle, although the true mechanisms remain to be clarified [164] . similar suppressive effects were also highlighted for its synthetic analogue minerval and confirmed in animal models [164, 168] . furthermore, the treatment with oleic acid and minerval induced proapoptotic effects in jurkat (t lymphocyte) and raji (b lymphocyte) cells, likely due to mitochondrial depolarization and ros production [168] [169] [170] . recently, oleate has been reported to be able to protect macrophages from palmitate-induced lipotoxicity; moreover, it has been associated with an increase in the regulatory phenotype of the myeloid msc-2 suppressor cells and suppression of activated t cells [171, 172] . in the skin, oleic acid, along with other unsaturated fa, seems to be incorporated into the lipid moiety of staphylococcus aureus lpp, inducing an immune response against the pathogen [173] . regarding conjugated pufa, punicic acid has been shown to improve the immune system development, stimulate the cd4+ and cd8+ lymphocyte-mediated immunity and increase the immune response against viruses [174] . these immune boosting effects are due to nuclear peroxisome proliferator-activated receptor (ppar)γ-and δ-dependent mechanisms, as punicic acid is able to act as an agonist of these receptors; in support, the loss of pparγ in immune cells impaired its effects [174, 175] . moreover, punicic acid inhibited the tnf-α-induced priming of ros production by inhibiting the ser345-p47phox phosphorylation and upstreaming kinase p38mapk; likewise, it blocked the tnf-α-induced release of myeloperoxidase from neutrophils, and decreased neutrophil-activation and ros/mpo-mediated tissue damage in vivo [176] . antinflammatory properties were found to be related to the activation of pparγ and the suppressed expression of inflammatory genes (encoding cytokines, chemokines, cyclooxygenase, no synthase, and metalloproteinases) [150] . immunomodulatory properties of ω-6 and ω-3 pufa have been highlighted in different preclinical models and have been associated with their ability to modulate the inflammatory process [177] . these fatty acids share common biosynthetic enzymes which mediate the production of different series of eicosanoids, starting from typical precursors, including dihomo-γ-linoleic acid (dgla), arachidonic acid (aa) and eicosapentaenoic acid (epa). among prostanoids, three types of prostaglandins (pg), including pg1, pg2 and pg3, can be obtained. pg1 is associated with beneficial effects and lower inflammation, thus being considered as an antinflammatory prostanoid; conversely, pg2 has opposite behaviour, increasing inflammation, vasoconstriction and blood clotting. pg3 acts through a mixture of functions and is able to reduce the pg2-mediated inflammation [177] . starting from dgla, both anti-inflammatory pg1 and pro-inflammatory pg2, through the conversion into arachidonic acid, can be produced (figure 7) . effects and lower inflammation, thus being considered as an antinflammatory prostanoid; conversely, pg2 has opposite behaviour, increasing inflammation, vasoconstriction and blood clotting. pg3 acts through a mixture of functions and is able to reduce the pg2-mediated inflammation [177] . starting from dgla, both anti-inflammatory pg1 and pro-inflammatory pg2, through the conversion into arachidonic acid, can be produced (figure 7) . this synthesis is controlled by the activity of δ5-desaturase and δ6-desaturase enzymes, which are often compromised during inflammatory conditions and diseases. it has been found that diets enriched in ω-3 fatty acids are able to activate the conversion of dgla into pg1, whereas low ω-3 intake induces the conversion in aa with the synthesis of proinflammatory prostanoids [178] . aa can also be released by the cell membranes through the action of phospholipase a2 during cell injuries or changes in biomembrane composition, thus representing a physiological activator of inflammation as a defence response. aa and eicosapentaenoic acid (epa) compete for the synthesis of different series of pg, mediated by cyclooxigenase, while 5-lipooxygenase (lox) is involved in their conversion into thromboxanes and leukotrienes. particularly, tromboxane a2 and leukotriene b4 are produced from aa, while tromboxane a3 and leukotriene b5 from epa. epa and dha are also precursors of lipoxins, resolvins and protectins, which produced anti-inflammatory effects and regulate vascular tone and blood pressure [178] . like punicic acid, the anti-inflammatory effects of epa and dha are mediated by the activation of the pparα/γ [179] . despite the antinflammatory role of ω-3 and ω-6-based diets, aa increases the plasmatic levels of proinflammatory eicosanoids, associated with an increased incidence of allergic and inflammatory disorders and with excessive cell proliferation. anyhow, it is not clear the usefulness to select ω-3 with respect to ω-6 in the diet: a balance between ω-3 and ω-6 pufa seems to be essential for mantaining ahealth status. the ability of fatty acids to be incorporated in the cell membrane seems to represent a key mechanism accounting for the immunomodulating properties of ω-3 and ω-6 pufa. indeed, immune cells (i.e., t cells and neutrophils) can incorporate exogenous fatty acids into membrane with a lateration in the function of cell surface pattern recognition receptors [180] . dietary ω-3 pufa has been shown able to modulate the macrophage function, through the activation of g protein coupled receptors (gpr) and to induce a shift to an anti-inflammatory phenotype [49] . modulating signalings through the gpr receptor activation can also affect leukocyte function. likewise, an inhibition of the pro-inflammatory phenotype of dendritic cells and of the t cell responses has been reported [49] . they are also able to inhibit neutrophil and monocyte adhesion, depending on the activation of ppar-α [49] . conversely, ω-6 pufas seem to promote inflammation, associated with incresead ros levels, in neutrophils [49] . particularly, linoleic acid increased the marginated pool of neutrophils in tissues by the induced expression of adhesion molecules;it also complexed with the anti-inflammatory this synthesis is controlled by the activity of ∆5-desaturase and ∆6-desaturase enzymes, which are often compromised during inflammatory conditions and diseases. it has been found that diets enriched in ω-3 fatty acids are able to activate the conversion of dgla into pg1, whereas low ω-3 intake induces the conversion in aa with the synthesis of proinflammatory prostanoids [178] . aa can also be released by the cell membranes through the action of phospholipase a2 during cell injuries or changes in biomembrane composition, thus representing a physiological activator of inflammation as a defence response. aa and eicosapentaenoic acid (epa) compete for the synthesis of different series of pg, mediated by cyclooxigenase, while 5-lipooxygenase (lox) is involved in their conversion into thromboxanes and leukotrienes. particularly, tromboxane a2 and leukotriene b4 are produced from aa, while tromboxane a3 and leukotriene b5 from epa. epa and dha are also precursors of lipoxins, resolvins and protectins, which produced anti-inflammatory effects and regulate vascular tone and blood pressure [178] . like punicic acid, the anti-inflammatory effects of epa and dha are mediated by the activation of the pparα/γ [179] . despite the antinflammatory role of ω-3 and ω-6-based diets, aa increases the plasmatic levels of proinflammatory eicosanoids, associated with an increased incidence of allergic and inflammatory disorders and with excessive cell proliferation. anyhow, it is not clear the usefulness to select ω-3 with respect to ω-6 in the diet: a balance between ω-3 and ω-6 pufa seems to be essential for mantaining ahealth status. the ability of fatty acids to be incorporated in the cell membrane seems to represent a key mechanism accounting for the immunomodulating properties of ω-3 and ω-6 pufa. indeed, immune cells (i.e., t cells and neutrophils) can incorporate exogenous fatty acids into membrane with a lateration in the function of cell surface pattern recognition receptors [180] . dietary ω-3 pufa has been shown able to modulate the macrophage function, through the activation of g protein coupled receptors (gpr) and to induce a shift to an anti-inflammatory phenotype [49] . modulating signalings through the gpr receptor activation can also affect leukocyte function. likewise, an inhibition of the pro-inflammatory phenotype of dendritic cells and of the t cell responses has been reported [49] . they are also able to inhibit neutrophil and monocyte adhesion, depending on the activation of ppar-α [49] . conversely, ω-6 pufas seem to promote inflammation, associated with incresead ros levels, in neutrophils [49] . particularly, linoleic acid increased the marginated pool of neutrophils in tissues by the induced expression of adhesion molecules; it also complexed with the anti-inflammatory molecule 1-antitrypsin, thus reducing lps-induced il-1 secretion in neutrophils [49] . on the whole, preclinical evidence highlighted that these fatty acids could increase neutrophil function, thus promoting innate immunity. regarding adaptive immunity, ω-3 pufa have been reported able to improve the mitogen-mediated activation of immune cells and to promote the development of a th2-type immune response [180] . moreover, an increased production of associated anti-inflammatory cytokines like il-4, in spite of a reduction of pro-inflammatory tnf-α, was found [180] . similar effects were highlighted with both fish oil-enriched diets and the purified epa and dha [49] . the beneficial influence of ω-3 pufa has been highlighted also on epithelial cells during inflammation, being able to restore impaired barrier function and reduce the production of pro-inflammatory mediators [49] . moreover, a strictly interplay between omega-3 fatty acids, immunity and gut microbiota has been reported and seems to be an essential factor to maintain the intestinal wall integrity. these effects have been ascribed to the ability of ω-3 pufa to positively affect the microbiota composition and increase the production of anti-inflammatory compounds, like short-chain fatty acids [181] . although a major interest over the years has been focused on marine sources of ω-3-enriched oils or on pure compounds, some plant species have been studied for their immunomodulating and anti-inflammatory properties, likely ascribable to ω-3 and/or ω-6 pufa, although the major evidence has been highlighted for linum usitatissimun l., oenothera biennis l. and borago officinalis l. [160, 182, 183] . the seed oil from l. usitatissimum, also known as flaxseed oil, has been reported to induce immunomodulating effects, likely through suppressing cell mediated immunity, without the involvement of humoral immunity. being a rich source of ala, its effects are mainly ascribed to this compound, although further studies suggested a possible contribution of bioactive phenolics [184] . flaxseed oil was found to be effective in reducing skin inflammatory responses, although with a lower immunosuppressive power with respect to fish oil [185] . moreover, it improved systemic and gut immunity, in a piglet model with intrauterine growth retardation: increased plasma concentration of immunoglobulin g, decreased cd3+cd8+ t lymphocytes, and the downregulation of genes expression for proinflammatory factors have been reported [186] . regarding o. biennis, the administration of the seed oil (namely, evening primrose oil) in animal models enhanced pge1 synthesis in peritoneal macrophages, decreased pge2 amounts in granulocytes, and suppressed the natural killer (nk) cell activity and lymphocyte proliferation; moreover, it decreased the serum levels of interferon γ (ifn-γ) and mcp-1, while stimulating tnf-α [187] [188] [189] [190] [191] . furthermore, anti-inflammatory effects have been found to be involved in the immunomodulation by evening primrose oil [160] . these effects were ascribed to the content of gla, whose t-regulatory cell activity in autoimmune disease models was highlighted [192] . however, a contribution of la to the antinflammatory effects seems to be likely; indeed, la can itself modulate inflammation as it is metabolized by lox to hydroxyoctadecadienoic acids (hodes) and oxo-hodes, characterized by antinflammatory properties [193] . similarly, the seed oil from b. officinalis seeds produced immonomodulating and antinflammatory effects, likely through its gla content [183] . a chemotactic migration of monocytes to necrotic site that differentiate into macrophages is associated with the administration of this product. moreover, it is known to reduce the levels of proinflammatory cytokines, such as tnf-α, and to promote pge1 generation; a reduced expression of inflammatory genes, especially those of macrophages involved in atherosclerosis, has been reported too [183] [184] [185] [186] [187] [188] [189] [190] [191] [192] [193] [194] . clinical studies mainly focused on the effects of fatty acid-enriched diet on inflammation, although specific immune-based pathological conditions associated with inflammation were assessed too. regarding mufa, few studies are available, and results differ from those in animal models. indeed, a mufa-rich diet (with highly refined olive oil for 8 weeks) did not alter the immune function in healthy subjects; such effects could be due to the high amounts administered in animal models [164] . conversely, clinical evidence about the immunomodulatory power of punicic acid in healthy or sick subjects is lacking [150] . the ω-3 pufa series and the relative enriched fish oils have been mainly evaluated for their immunomodulating and antinflammatory effects in humans. although preclinical evidence highlighted their ability to influence both innate and adaptive immunity, the clinical relevance of these results remains to be clarified, due to lacking or inconclusive data [49] . inadequacy of clinical results should be due to the different doses used in preclinical studies, wherein often high fatty acids levels were administered; moreover, other factors such as genetic and epigenetic heterogeneity of the recruited subjects, diet diversity, nutritional habits and microbiome can be considered as additional confounding factors [49] . although limitations of clinical studies require further confirmation, ω-3 pufa intake produced significant clinical benefits and reduction of the symptoms in patients with autoimmune disorders, especially rheumatic diseases and systemic lupus erythematosus [195] [196] [197] . in support, low levels of pufas have been found in the serum of patients with rheumatic diseases [198] . conversely, inconsistent results are reported for multiple sclerosis, thus the possible usefulness of these fatty acids as supportive therapy requires more clinical trials [199] . regarding ω-6 pufa, although they are associated with possible increased inflammatory conditions, being arachidonic acid a presursor of proinflammatory prostanoids, such a risk is not confirmed by clinical evidence [200] . indeed, studies in healthy human adults highlighted that an increased intake of these fatty acids did not induce inflammation; conversely, epidemiological evidence reported reduced inflammatory conditions [200] . the antinflammatory effects of la have been reported too [193] . similarly, increased la intake was found to be not related to increased amounts of ara and proinflammatory factors; however, an inverse correlation with epa and dha was reported [201] . this suggests that the interaction between ω-e and ω-6 series is regulated by complex mechanisms that requires further clarifications. major clinical studies have been performed using evening primrose oil (from the seeds of o. biennis), as a source of la and gla, in inflammatory diseases associated with immune system disorders, including atopic dermatitis, psoriasis, multiple sclerosis and rheumatoid arthritis. standardized oils for the content in la and ala (for instance, efamol is titred to contain 72% la and 9% gla) were usually used [202] . the treatment with evening primrose oil (4 and 7 weeks) produced clinical improvements in patients with atopic dermatitis, as revealed by measuring the scoring atopic dermatitis [203] . some beneficial effects were also reported in multiple schlerosis patients, although the few available studies limited the evidence in this disorder. conversely, evening primrose oil in combination with fish oil and vitamin e (efamol marine) failed to improve the symptoms of psoriasiac patients but produced antinflammatory effects [204] . similarly, in association with ω-3 fatty acids, it did not induce improvements in patients with rheumatoid arthritis [205] . a cochrane revision highlighted moderate evidence for oils containing gla (i.e., evening primrose, borage, or blackcurrant seed oil) to produce benefit in rheumatoid arthritis [205, 206] . evening primrose oil along with borage oil were not effective to treat eczema too [207] . highly variable results were also obtained for borage oil in the treatment of atopic dermatitis, although, in all the studies, a moderate efficacy degree was displayed [208] . regarding flaxseed oils, some clinical trials higlighted a significant improvement of inflammatory parameters in subjects with cardiovascular diseases non-associated with the immune system [209] . reported studies, although performed in pathological conditions associated with immune system disfunction, did not give a direct measure of the immunomodulatory effects of the treatments. furthermore, specific and high-quality studies are required for better characterizing the possible usefuleness of these pufa-enriched oils as anti-inflammatory and immunomodulating treatments. labdane compounds have a molecular formula c 20 h 38 with an average mass of 278.516 da ( figure 8 ). labdane-related molecules have a hydrocarbon skeleton, originated from dual biosynthetic cyclization and/or rearrangement reactions, produced through the biosynthetic pathway of gibberellin phytohormones by the diterpene cyclases. the labane diterpenoids belong to a superfamily of natural products, in which the hydrocarbon skeleton might serve as privileged scaffolds for their biological activity [210] . labdane compounds have a molecular formula c20h38 with an average mass of 278.516 da ( figure 8 ). labdane-related molecules have a hydrocarbon skeleton, originated from dual biosynthetic cyclization and/or rearrangement reactions, produced through the biosynthetic pathway of gibberellin phytohormones by the diterpene cyclases. the labane diterpenoids belong to a superfamily of natural products, in which the hydrocarbon skeleton might serve as privileged scaffolds for their biological activity [210] . labdane diterpenes have been found in the various matrix of vegetal origin (leaves, rizomes, fruits, etc.) of different plants. in table 1 , some of them (where diterpenes have been found), their botanical family (in parentheses) and the part of plant of biological interest are reported. some labdane diterpenoids, isolated from plant matrix, include the following: andrographolide ( figure 9 ) (from andrographis paniculata (burm.f.) nees) [211] , labda-8(17), 12-diene-15, 16-dial (from curcuma amada roxb) [212] , podoimbricatin c (a 12,17-cyclo-labdane diterpenoid from dacrycarpus imbricatus (blume) de laub) [213] chapecoderins a-c (from echinodorus macrophyllus (kunth) micheli), [214] , (4r,5s,9s,10r)-13-des-ethyl-13-oxolabda-8(17),11e-dien-19-oic acid (from juniperus oblonga m. bieb) [215] , leoheteronin d and leojaponin a (from leonurus japonicus houtt) [216] , marrubasch a-f and marrubenol (from marrubium aschersonii p.magnus), marrulibanoside (from marrubium globosum boiss. and balansa) [217] , vitexlimolides a-c (from vitex limonifolia wall. ex c.b. clarke) [218] . labdane diterpenes have been found in the various matrix of vegetal origin (leaves, rizomes, fruits, etc.) of different plants. in table 1 , some of them (where diterpenes have been found), their botanical family (in parentheses) and the part of plant of biological interest are reported. some labdane diterpenoids, isolated from plant matrix, include the following: andrographolide ( figure 9 ) (from andrographis paniculata (burm.f.) nees) [211] , labda-8(17), 12-diene-15, 16-dial (from curcuma amada roxb) [212] , podoimbricatin c (a 12,17-cyclo-labdane diterpenoid from dacrycarpus imbricatus (blume) de laub) [213] chapecoderins a-c (from echinodorus macrophyllus (kunth) micheli), [214] , (4r,5s,9s,10r)-13-des-ethyl-13-oxolabda-8(17),11e-dien-19-oic acid (from juniperus oblonga m. bieb) [215] , leoheteronin d and leojaponin a (from leonurus japonicus houtt) [216] , marrubasch a-f and marrubenol (from marrubium aschersonii p.magnus), marrulibanoside (from marrubium globosum boiss. and balansa) [217] , vitexlimolides a-c (from vitex limonifolia wall. ex c.b. clarke) [218] . imbricatus (blume) de laub) [213] chapecoderins a-c (from echinodorus macrophyllus (kunth) micheli), [214] , (4r,5s,9s,10r)-13-des-ethyl-13-oxolabda-8(17),11e-dien-19-oic acid (from juniperus oblonga m. bieb) [215] , leoheteronin d and leojaponin a (from leonurus japonicus houtt) [216] , marrubasch a-f and marrubenol (from marrubium aschersonii p.magnus), marrulibanoside (from marrubium globosum boiss. and balansa) [217] , vitexlimolides a-c (from vitex limonifolia wall. ex c.b. clarke) [218] . figure 9 . chemical structure of andrographolide. this has been obtained using chemspider ® chemical structure database. the body's defense responses can be improved through various properties induced by plants. some of them, referring to the plants in table 5 , are shown below. for each plants and plant-derived nutraceutical, only properties potentially attributable to the labdane skeleton and useful to improve the immune system are reported. as both inflammation (biological response of body tissues to harmful stimuli) and oxidative stress (imbalance between reactive oxygen species and a biological system's ability to detoxify/repair the resulting damage of the reactive intermediates) are the main self-defend methods to eliminate pathogens and protect living bodies, plants with antinflammatory and/or radical scavenger properties are considered too [211] . indeed, labdane diterpenoids have recently gained greater attention from the scientific point of view, due to a wide range of biological activities, including the anti-inflammatory modulation of immune cell functions [217] . a. paniculata exhibited, in vitro and in vivo, various pharmacological activities, including antihyperglycemic, antiplatelet aggregation, anti-microbial, anti-inflammatory, anti-hiv, anti-cancer, anti-nociceptive activity, etc. it has also been used for autoimmune encephalomyelitis and, in indian and chinese medicine, for respiratory tract infections [217, 219] . more recently, a. paniculata has been used to stimulate the immune system and treat myocardial ischemia [211] . a. paniculata inhibited interleukin (il)-6, tnf-α mrna, lps-induced expression, and suppressed levels of tnf-α, il-1β, jnk, c-reactive protein, and nf-κb [211] . many labdane diterpenoids compounds have been found to act on the latter. the activation of the nf-κb pathway leads to several physiological responses, including inflammatory or innate immune response [217] . in vitro, andrographolide (the main phytoconstituent of a. paniculata) can inhibit inflammation, by regulating protein expression (cytokines, chemokines) and by reducing immune cell infiltration. andrographolide was shown to inhibit also oxidative stress by binding to adenosine a2a receptor, by inducing nuclear factor (erthroid-derived 2)-like 2 (nrf2) translocation, and by increasing the expression of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase-2 [211] . these effects can contribute to the immunoregulatory activity of this plant-derived nutraceutical, as it can modulate the innate and adaptive immune responses by regulating macrophage phenotypic polarization and antibody productions [25] . moreover, it was found to exert cytotoxic/anticancer effects on almost all types of tumour cell lines (human leukemia, renal tubular epithelial cells, breast cancer cells, etc.), mainly by cell cycle arrest, autophagy, cell death, anti-inflammatory and immune system mediated effects [220] . other preclinical studies have highlighted pharmacological properties of labdane diterpenoidscontaining plants. data are limited, and consequently also their preclinical evidence. some examples are reported below. c. amada, also known as mango ginger, and its labdane-diterpenoids have shown antiflammatory, antibacterial, insecticidal, antifungal, antipyretic, antioxidant, anticancer, and antitubercolar properties in preclinical trials [212] . d. imbricatus displayed cytotoxic and anti-neuroinflammatory activities, but it had no cytotoxic activity against human tumour cell lines [213] . e. macrophyllus, brazilian plant, also known as "leather hat", is used as a methanolic (which contains mainly labane diterpenoids, steroids, alcaloids, etc.) or aqueous extract (rich in flavonoids) of the aerial parts, leaves in particular. in folk medicine, e. macrophyllus is used for various illnesses (respiratoy and urinary diseases, rheumatoid arthritis, atherosclerosis, etc.), as it has been shown to possess tissues protective activity and immunosuppressive effects (impaired secretion and function of b and/or t cells), on humoral or cellular immune responses and on autoimmune rheumatic diseases [221] . in in vitro/vivo studies, the aqueous extract of e. macrophyllus exhibited strong antinflammatory activities by decreasing rats paw edema, inflammatory exudates, infiltrate tissues, no production, ltb4 release, and neutrophil migration [222] . in preclinical studies, the methanolic extract of e. macrophyllus was not cytotoxic, genotoxic, mutagenic, and no acute toxicity (up to the maximum dose of 2000 mg/kg b.w.) has been observed in tested animals [221] . however, the extrapolation of animal experiments to clinical practice must be done with caution [223] . compounds from j. oblonga have shown anti-tumor effects, through moderate cytotoxicity against human tumor cell lines obtained from various human tissues, including: hepatocellular carcinoma (hepg2), breast cancer (mcf-7), and cervical carcinoma cancer (hela) [215] . the berries from j. oblonga also have antimicrobial activity and anti-inflammatory effects. labdane diterpenoid (e.g., leonurine), extracted from l. japonicus, exhibited cytotoxicity and cell cicle arrest against cancer cell lines and presented immunomodulatory and antinflammatory activities (suppresses tnf-α, nf-κb, and down-regulated expression of inos, cox2, and conseguently peg2 and no levels) [216] . marrubium spp. (aschersonii, globosum, etc.) have multiple actions, including antimicrobial and anti-inflammatory activities. marrubasch a-f and marrubenol, isolated from the ethanolic extract of m. aschersonii, exhibited weak reduction in inos activity and, consequentely, no production [217] . marrulibanoside, obtained from the aerial parts of m. globosum, inhibited catalytic activity of inos and cox-2enzymes, and consequentely, the peg2 and no production. v. limonifolia, in preclinical trial, have shown a strong antiviral activity against coxsackievirus b3, human rhinovirus 1b, and enterovirus 71 (ev71). all of them could be responsible for various illnesses, ranging from common cold, hand, foot, and mouth diseases, to acute flaccid paralysis [218] . the clinical efficacy of the medicinal plants, and plant-derived nutraceuticals discussed above are almost totally lacking. only for a. paniculata there are some evidence in humans. andrographis extract (various and not standardized), andrographolide, and its derivatives have been studied in the treatment of various disease (multiple sclerosis, infection disease, gastro-intestinal upsets, respiratory ailments, pain), and in the maintenance of immune function. in this last context, it seemed to improve the response to cough and sore throat, shortening the sick leave/time to resolution [219] . the most interesting activity is the increase of cd4+ lymphocyte levels, in hiv-positive patients [211] . the increase in these lymphocytes testifies an improvement in the state of the immune system. moreover, a chinese product containing andrographolide improved the efficacy of glucocorticoids and immunoglobulin in patients with severe hand, food, and mouth disease. however, andrographolide is considered a hazard, as it is irritating, and its injectable use is limited because it could induce allergic reactions (erythema, pruritus, etc.), which are sometimes life-threatening [211] . preclinical data suggested that andrographolide could be responsible of pharmacokinetics interactions, as it induced cyp1a2 [219] . the european medicines agency (ema) reports a possibility of causing reproductive toxicity of andrographis extracts (decreases in sperm motility and counts) [219] . on the other hand, no major adverse effects have been reported for a. paniculata; only minor side effects, mainly gastrointestinal, are known [219] . notably, even if a. paniculata presents numerous pharmacological properties, andrographolide possess poor solubility (principally in dmso), which severely limits the possibility of achieving a therapeutic effect (if not properly formulated). its better absorption could be achieved by nano-formulations (e.g., nano-emulsion, nano-capsules). immunomodulation by plant-based nutraceuticals represents an interesting tool to be exploited for the treatment and preventing purposes of immune system disorders, due to their multiple bioactivities, well tolerability and good patient compliance. however, as often reported for several herbal medicinal products, some points require being underlined to improve the research in the field and provide solid evidence to support their rational use. according to previous stated critical issues [224, 225] , herbal products under study must be characterized for the phytochemical composition, using validated analytical methodologies, and for the extraction procedures; moreover, the starting material should be fully defined in terms of origin (country and region), cultivation conditions, botanical identity and plant part. the content of specific compounds, used as analytical or active markers, should be determined too. these requirements are needed to ensure reproducible pharmacological/clinical activity and to compare different studies. indeed, using nonstandardized phytocomplexes increases variability of the biological response, thus limiting the reliability and validity of the studies. furthermore, to assess the pharmacological activity of specific compounds, purity (at least 95%) and identity should be characterized. indeed, when assessed as mixtures, the subtle interactions which can be established among phytochemicals make it difficult to understand whether the observed benefits are attributable to a specific class or to the whole phytocomplex. for instance, both fatty acids and polyphenols can be involved in the immunomodulating effects of pufa-enriched plant oils. moreover, as found for both polysaccharides and fatty acids, among the same class, different subclasses can co-occur, thus contributing to the whole effects. regarding preclinical studies, detailed methodologies, including information about specific extraction process, the choice of the tested concentrations and experimental procedures, vehicle effects, and comparison with standard effective compounds (positive controls) should be reported. in order to validate the "goodness" of the treatment, promising results in preclinical studies should be confirmed by clinical evidence of efficacy and lack of toxicological concerns for both the isolated compounds and the whole phytocomplex. at last, possible interactions with diet constituents or possible pharmacological treatments, as reported for andrographolide, which is a cyp1a2 inducer, should be considered. as highlighted for a number of natural products, clinical evidence is a major challenge for plant-based immunomodulating nutraceuticals too, due to limited specific studies. moreover, methodological quality of the available trials was overall poor, the studies often being not blinded, protocol unavailable and lacking the standardization of tested products, thus making the claimed effect difficult to be reproduced. at last, standardized methodologies for systematic reviews and meta-analyses, such as the prisma guidelines [226] , would allow a rational interpretation of the results and suggestions for future research. medicinal plants are rich sources of bioactive phytochemicals, characterized by multiple and often pleiotropic activities, which can be exploited both therapeutically and as nutraceutical strategies for preventive purposes. among plant-based nutraceuticals, immunomodulators have been highlighted to be of interest as boosters of the immune system, to counteract infectious or exogenous injuries, immunosuppressor, to control the abnormal immune response occurring during autoimmune diseases, or as adjuvants, which contribute by modulating nonimmune targets. in this review, we highlighted the scientific evidence about the immunomodulating properties of three emerging 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reproducibility of natural product research preferred reporting items for systematic reviews and meta-analyses: the prisma statement this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license a.d.s. and s.d.g. fellowships were funded by grants from sapienza university (ateneo 2019) and regione lazio. the authors thank "enrico and enrica sovena" foundation (italy) for supporting the study. the authors declare no conflict of interest. key: cord-295335-oa4twg2z authors: pastorino, giulia; cornara, laura; soares, sónia; rodrigues, francisca; oliveira, m. beatriz p.p. title: liquorice (glycyrrhiza glabra): a phytochemical and pharmacological review date: 2018-08-17 journal: phytother res doi: 10.1002/ptr.6178 sha: doc_id: 295335 cord_uid: oa4twg2z in the last years, consumers are paying much more attention to natural medicines and principles, mainly due to the general sense that natural compounds are safe. on the other hand, there is a growing demand by industry for plants used in traditional medicine that could be incorporated in foods, nutraceuticals, cosmetics, or even pharmaceuticals. glycyrrhiza glabra linn. belongs to the fabaceae family and has been recognized since ancient times for its ethnopharmacological values. this plant contains different phytocompounds, such as glycyrrhizin, 18β‐glycyrrhetinic acid, glabrin a and b, and isoflavones, that have demonstrated various pharmacological activities. pharmacological experiments have demonstrated that different extracts and pure compounds from this species exhibit a broad range of biological properties, including antibacterial, anti‐inflammatory, antiviral, antioxidant, and antidiabetic activities. a few toxicological studies have reported some concerns. this review addresses all those issues and focuses on the pharmacological activities reported for g. glabra. therefore, an updated, critical, and extensive overview on the current knowledge of g. glabra composition and biological activities is provided here in order to explore its therapeutic potential and future challenges to be utilized for the formulation of new products that will contribute to human well‐being. prepare a tea that is an excellent thirst quencher. the dried root has been described as a tooth cleanser (armanini et al., 2002) . actually, the most important industrial use of g. glabra is the production of food additives, such as flavours and sweetening agents (mukhopadhyay & panja, 2008) . in particular, the root is used as a flavouring agent for american-type tobacco, chewing gum, candies, baked goods, ice cream, and soft drinks (rizzato, scalabrin, radaelli, capodaglio, & piccolo, 2017) . in beers and fire extinguishers, the root extracts are used as foaming agents, whereas the root fibbers are used in insulation, wallboard, and boxboard materials, after removal of the medicinal and flavouring constituents. in the cosmetic field, g. glabra is described as a skin depigmentation agent and is being incorporated in topical products for that purpose. with regard to government approval, liquorice extract and glycyrrhizin have been allowed for use in foods by the united states food and drug administration, the council of europe, and the joint fao/who expert committee on food additives (fao, 2005) . indeed, the u.s. flavor and extract manufacturers association has recognized it as generally safe. to the best of our knowledge, a limited number of reviews have been published on this plant, particularly in what concerns to pharmacological aspects (asl & hosseinzadeh, 2008; fiore, eisenhut, ragazzi, zanchin, & armanini, 2005) . the objective of this review was to examine the bioactive compounds of g. glabra and the biological activities associated with these compounds. g. glabra is a typical herbaceous perennial, growing to 1 m in height, presenting pinnate leaves with a length of 7 to 15 cm. the flowers are purple to pale whitish blue, being arranged in a hermaphrodite inflorescence, whereas the fruit is an oblong legume with 2 to 3 cm of length and containing several seeds. the genus glycyrrhiza (fabaceae) consists of about 30 species, such as g. glabra, g. uralensis, g. inflata, g. aspera, g. korshinskyi, or g. eurycarpa. like the other plants of fabaceae, g. glabra is able to fix nitrogen, due to symbiosis with bacteria of the genus rhizobium, at the root level, being suitable for sandy and clay soils, though preferring humid soils. since the egyptian age, the therapeutic properties of g. glabra are well documented (fiore et al., 2005) . the roots are the most used parts whereas leaves are considered an agrochemical waste. however, in the last years, different authors studied the phytochemical composition of g. glabra leaves, demonstrating that certain compounds present in the roots are also identified in leaves, although in smaller quantities (hayashi & sudo, 2009; siracusa et al., 2011) . in the last years, the chemical constituents of liquorice have been extensively investigated by different authors siracusa et al., 2011) . nevertheless, few studies were carried out on the nutritional composition of g. glabra. nutritionally, liquorice is a source of proteins, amino acids, polysaccharides and simple sugars, mineral salts (such as calcium, phosphorus, sodium, potassium, iron, magnesium, silicon, selenium, manganese, zinc, and copper), pectins, resins, starches, sterols, and gums (q. . oestrogens, tannins, phytosterols (sitosterol and stigmasterol), coumarins, vitamins (b1, b2, b3, b5, e, and c), and glycosides have been reported (q. wang, qian, et al., 2015) . a large number of biological compounds have also been isolated, mostly triterpenes, saponins (responsible for the sweet taste), and flavonoids (rizzato et al., 2017; q. wang, qian, et al., 2015) . the liquorice saponins are present as glucuronides, whereas the aglycones are present as oleananes. the triterpene saponins are the major characteristic constituents of liquorice, being responsible for the sweet taste. the contents of these compounds may vary significantly due to geographic sources, harvesting, and processing, affecting the therapeutic effects of liquorice. the main constituent of roots is glycyrrhizin, a triterpenoid saponin that is almost 50 times sweeter than sucrose, being the primary active ingredient (j. y. yu et al., 2015) . glycyrrhizin represents about 10% of the liquorice root dry weight, being a mixture of potassium, calcium, and magnesium salts of glycyrrhizic acid that varies between 2% and 25% (rizzato et al., 2017) . after oral administration, glycyrrhizin is metabolized to 18-glycyrrhetic acid 3omonoglucuronide and glycyrrhetic acid by intestinal bacteria (albermann, musshoff, hagemeier, & madea, 2010) . the yellow colour of liquorice is due to the flavonoid content. the flavonoids identified belong to different classes, including flavanones, flavones, flavanonols, chalcones, isoflavans, isoflavenes, isoflavones, and isoflavanones. the major flavonoids are glycosides of liquiritigenin (4′,7-dihydroxyflavanone) and isoliquiritigenin (2′,4,4′trihydroxychalcone), such as liquiritin, isoliquiritin, liquiritin apioside, and licuraside (rizzato et al., 2017) . five new flavonoids have been isolated from dried roots: glucoliquiritin apioside, shinflavanone, shinpterocarpin, prenyllicoflavone a, and 1-methoxyphaseolin. pinocembrin and licoflavanone were also isolated from the leaves (fukui, goto, & tabata, 1988) . glabridin is the principal isoflavone identified, ranging between 0.08% and 0.35% of roots' dry weight (simmler, pauli, & chen, 2013 liquorice is one of the oldest and most popular herbal medicines in the world. many of the liquorice historical uses are still practised the antioxidant activity of g. glabra is one of the major reasons for its uses. the phenolic content is probably responsible for the powerful antioxidant activity observed (rackova et al., 2007) . varsha and sonam (2013) attributed this activity to flavonoids, whereas singh et al. (2015) reported that mostly isoflavones, such as glabridin, hispaglabridin a, and 30-hydroxy-4-o-methylglabridin, are the responsible compounds. biondi, rocco, and ruberto (2003) reported a huge antioxidant activity of the dihydrostilbene derivates present in g. glabra leaves. also, licochalcones b and d are present in g. glabra, showing a strong scavenging activity on dpph radical and the ability to inhibit the microsomal lipid peroxidation (biondi et al., 2003; v. sharma, katiyar, & agrawal, 2016) . these phenolic compounds are effective in the protection of biological systems against oxidative stress, being able to inhibit the onset of skin damages (haraguchi et al., 1998) . according to castangia et al. (2015) , the topical application of liquorice extract formulations may be of value in innovative dermal and cosmetic products as it counteracts oxidative stress damage, maintaining the skin homeostasis due to its high antioxidant content. table 1 summarizes the most important studies of antioxidant activity. the anti-inflammatory activity of g. glabra and its use in the treatment of inflammatory diseases have been documented since ancient times (r. yang, yuan, ma, zhou, & liu, 2017) . shalaby, ibrahim, mahmoud, and mahmoud (2004) evaluated the anti-inflammatory activity of g. glabra in male rats after 4 weeks of food intake. the authors observed a significant decrease in the total cholesterol and triglyceride levels as well as in the levels of serum liver enzymes. harwansh, patra, pareta, singh, and biswas (2011) reviewed the positive effects of g. glabra on the treatment of the upper respiratory tract and gastric system diseases. these pharmacological effects were due to an increase in the secretion of serotonin and prostaglandins in the stomach that led to a decrease of gastric inflammation (bahmani et al., 2014) . different authors described that the anti-inflammatory action is primary mediated by glycyrrhizin, which in vitro could inhibit factors responsible for inflammation as well as promote the healing of stomach and mouth ulcers (rackova et al., 2007; yin et al., 2017) . in fact, the anti-inflammatory effects of glycyrrhizin were described as similar to those of glucocorticoids and mineralocorticoids (kageyama, suzuki, & saruta, 1994) . furthermore, g. glabra is used in renal and liver complications on the basis of its strong antiinflammatory effects (y. xiao et al., 2010) . y. xiao et al. (2010) reported the inhibition of liver granuloma formation and the inflammatory cytokine production by glycyrrhizin, whereas x. r. wang, hao, and chu (2017) described the anti-inflammatory effects on endometriosis. moreover, liu et al. (2017) proved the anti-inflammatory activity of glabridin on raw cells. the antitussic and expectorant effects of liquorice have been reported by different authors, particularly its useful effects on the treatment of sore throat, cough, and bronchial catarrh (damle, 2014; fiore et al., (hasanein, 2011) glycyrrhiza glabra extract 75-300 mg/kg, 7 days in vivo-oral administration to swiss young male albino mice production of antidepressant-like effect in mice in forced swim test and tail suspension test, probably by interaction with adrenergic and dopaminergic system (dhingra & sharma, 2006) g. glabra aqueous extract 75-300 mg/kg, 7 days in vivo-oral administration to mice dose of 150 mg/kg significantly improved learning and memory of mice (parle, dhingra, & kulkarni, 2004) sedative activity (cho et al., 2010) antidepressive activity g. glabra aqueous extract 75-300 mg/kg in vivo-forced swim test and tail suspension test applied to mice antidepressant-like effect of liquorice extract seems to be mediated by increase of brain norepinephrine and dopamine, but not by increase of serotonin (dhingra & sharma, 2006) oestrogenic activity female wistar rats stimulation of creatine kinase specific activity (tamir, eizenberg, somjen, izrael, & vaya, 2001) liquorice root extract 25 μg/day, 2 weeks in vivo-oral administration to female rats increase in creatine kinase activity (tamir et al., 2000) liquiritigenin 2-10 μg/ml in vitro-mcf-7 and t47d cells induction of oestrogen responsive alkaline phosphatase activity in endometrial cancer cells, oestrogen responsive element luciferase in mcf-7 cells and tff1 mrna in t47d cells (somjen et al., 2004) isoliquiritigenin 0-0.04 mg/ml in vivo-intraperitoneal injection of female icr mice improvement of ivf rate (tung, shoyama, wada, & tanaka, 2014) skin effects glycyrrhizinic acid in vivo-double-blind clinical trial in human patients reduction of erythema, oedema, and itching scores (halder & richards, 2004) -in vitro-topical treatments in human patients during 4 weeks lighten hand solar lentigines (nerya et al., 2003) glycyrrhetinic acid; glabridin 0-120 μm in vitro-human keratinocyte culture prevention of oxidative dna fragmentation and activation of apoptosis-associated proteins in human keratinocyte (grippaudo & di russo, 2016) (continues) in vitro-vero cells in vivo-ducks stimulation of immune and antiviral effect against dhv (soufy et al., 2012) 0.1 μg/ml (extract) in vitro-human foreskin cell line protection of host cells against ev71 infection (kuo, chang, wang, & chiang, 2009) in vitro-vero cells protection against coronavirus (cinatl et al., 2003) 100 μg/ml (compound) in vitro-peripheral blood mononuclear cells inhibition of nonsyncytium-inducing variant of hiv replication (sasaki, takei, kobayashi, pollard, & suzuki, 2002) 400-1,600 mg/day (compound) in vitro-human immunodeficiency virus type 1 (hiv-1) p24 antigen inhibition of hiv-1 replication (hattori et al., 1989) 60 mg in vivo-oral administration to humans (ldl isolation) (carmeli & fogelman, 2009) licochalcone 2-20 μg/ml dpph, superoxide anion, lipid peroxidation, red blood cells inhibition of the microsomal lipid peroxidation (haraguchi, ishikawa, mizutani, tamura, & kinoshita, 1998) hepatoprotective activity liquorice aqueous extract 100-300 mg/kg 15 days in vivo-oral administration to wistar rats stimulation of the antioxidant enzymes and arrest of inflammatory cytokine production (huo, wang, liang, bao, & gu, 2011) g. glabra aqueous root extract 2 g/day, 2 months in vivo-humans alt and ast decrease (hajiaghamohammadi, ziaee, & samimi, 2012) 10, 30, 100 mg/kg 2005). these effects are associated with the presence of glycyrrhizin that helps to expel congestion in the upper respiratory tract and accelerates tracheal mucus secretion (v. sharma et al., 2016) . likewise, liquiritin apioside, an active compound reported in the methanolic extract of liquorice, has the ability to inhibit capsaicin, a compound that induces cough (kamei, nakamura, ichiki, & kubo, 2003) . the effect on sore throat has been compared with that of carbenoxolone, a glycyrrhetinic acid derivative with a steroid-like structure, which stimulates gastric mucus secretion (damle, 2014) . the use of g. glabra extract as antiulcerative is widely known. for the gastrointestinal system, it is used in gastric and duodenal ulcers (bardhan, cumberland, dixon, & holdsworth, 1978) , whereas for the treatment of spasmodic pains of chronic gastritis, it is employed as an adjuvant (armanini et al., 2002) . the benefits of g. glabra in the treatment of duodenal and peptic ulcers have been reported since the 1970s, and this traditional use is related to the presence of antiinflammatory saponins (krausse et al., 2004) . the major compound responsible for this activity is glycyrrhizin, which can raise the concentration of prostaglandins in the digestive tract, promoting stomach mucus secretion (jafarian & ghazvini, 2007) . in addition, liquorice prolongs the lifespan of stomach surface cells, demonstrating an antipepsin effect (ram, lachake, kaushik, & shreedhara, 2010) . furthermore, deglycyrrhizinated liquorice has shown some effects in the treatment of gastrointestinal ulcers, suggesting the presence of other active ingredients (zadeh, kor, & goftar, 2013) . indeed, carbenoxolone, a glycyrrhetinic acid analogue, is reported to inhibit two important enzymes for the metabolism of prostaglandin, 15-hydroxyprostaglandin dehydrogenase and δ 13 prostaglandin reductase, raising prostaglandin levels and leading to positive effects in clinical trials for gastric and duodenal ulcers (damle, 2014) . in fact, prostaglandins stimulate mucous secretion and cell proliferation leading to ulcer healing. nevertheless, the glycyrrhetic acid derivative carbenoxolone presents secondary effects such as the potential development of pseudo aldosteronism, which limits its use. in a clinical trial, bardhan et al. (1978) studied the effect of liquorice by oral administration in 96 patients with gastric ulcer. the patients were randomly allocated to the treatment either with deglycyrrhizinated liquorice or with placebo. however, after 4 weeks, no differences were observed between groups in the percentage of ulcer area reduction or clinical improvements (bardhan et al., 1978) . particular, glabridin, glabrol, glabrene, hispaglabridin a, hispaglabridin b, 40-methylglabridin, and 3-hydroxyglabrol, isolated from g. glabra, are responsible for this activity (l. . the mechanism behind this could be the decrease of bacterial gene expression, the inhibition of bacterial growth, and the reduction of bacterial toxin production (gupta et al., 2008; l. wang, yang, et al., 2015) . in 2014, s.-j. ahn, song, mah, cho, and kook (2014) (fukai et al., 2002a (fukai et al., , 2002b l. wang, yang, et al., 2015) . on the other hand, the ability to inactivate methicillin-resistant s. aureus (mrsa) by decreasing the expression of saer and hla, the key virulence genes of mrsa, have also been reported by different authors (fukai et al., 2002a (fukai et al., , 2002b l. wang, yang, et al., 2015) . it is also suggested that licochalcone e could be used for chemical synthesis of novel anti-s. aureus compounds, reducing the production of α-toxin in both methicillin-sensitive s. aureus and mrsa (l. . besides, α-haemolysin is an important exotoxin in the pathogenesis of s. aureus infections (berube & bubeck wardenburg, 2013) . such infections are associated with a broad spectrum of diseases, ranging from endocarditis to minor skin infections, toxinoses, and lethal pneumonia. liquiritigenin, one of the most active compounds of liquorice, demonstrated the capacity to prevent human lung cells (a549) from α-haemolysin-mediated injuries, by decreasing αhaemolysin production (l. . similarly, glabrin and glycyrrhetinic acid have shown antibacterial activity against s. aureus . different authors reported the antibacterial action of g. glabra against mycobacterium tuberculosis (gupta et al., 2008) , demonstrating that glabridin is the responsible compound for this activity, instead of hispaglabridin b . the antitubercular phenolic compounds were previously identified as licoisoflavone and licochalcone a (chakotiya, tanwar, srivastava, narula, & sharma, 2017) . in a mice lung infection model, g. glabra was therapeutically active against multidrug-resistant strain of p. aeruginosa (chakotiya et al., 2016) , and the hydro-alcoholic extract led to a reduction of the microbial load in the blood, mainly due to the presence of stigmasterol, ergosterol, licochalcone, and glabridin (chakotiya et al., 2017) . the activity of g. glabra against helicobacter pylori has been also reported, as mentioned in the previous subsection (krausse et al., 2004) . according to krausse et al. (2004) , the compounds responsible for this activity are glabridin and glabrene. cao et al. (2016) also reported that 18β-glycyrrhetinic acid significantly attenuated the gastritis infection caused by h. pylori. asha et al. (2013) found that the flavonoid glabridin exhibits activity against h. pylori whereas glycyrrhizin did not present activity even at a concentration of 250 μg/ml. these flavonoids also showed activity against h. pylori strains resistant to clarithromycin and amoxicillin (fukai et al., 2002a (fukai et al., , 2002b . the probable mechanism behind these action is the inhibition of the protein synthesis, dna gyrase, and dihydrofolate reductase (fukai et al., 2002a (fukai et al., , 2002b . moreover, the liquorice polysaccharides also present activity against porphyromonas gingivalis adhesion, which is of huge importance because no specific adhesion inhibitors have been described (chinsembu, 2016) . the antifungal activity of g. glabra is also detailed (sato, goto, nanjo, kawai, & murata, 2000) . sato et al. (2000) reported that the methanolic extract of liquorice presents fungicidal activity against arthrinium sacchari and chaetomium funicola, whereas glabridin was found to be the active compound responsible for the observed effects (sato et al., 2000) . in fact, isoflavonoids, such as glabridin, glabrol, and their derivatives, are responsible for the in vivo inhibition of mycobacterium smegmatis, shigella, salmonella, e. coli, s. mutans, and lactobacillus acidophilus (ajagannanavar et al., 2014) . recently, chandra and gunasekaran (2017) also proved the antifungal activity of crude methanolic extract of g. glabra against aspergillus niger. different authors reported that c. albicans is susceptible to liquorice extracts due to their richness in liquiritigenin, liquiritin, licochalcone a, and glabridin (chandra & gunasekaran, 2017 ; j. y. lee et al., 2009; singh et al., 2015) . nevertheless, according to karahan, avsar, ozyigit, and berber (2016) , the antimicrobial activity could be influenced by the environmental conditions that may affect the chemical compound contents and the biological activity. according to messier et al., licochalcone a and glabridin present a therapeutic potential against c. albicans oral infections, whereas glycyrrhizic acid had no effect (messier & grenier, 2011; moazeni et al., 2017) . fukui et al. (1988) isolated licoflavanone from the leaves of g. glabra, demonstrating its antimicrobial activity. indeed, chen et al. (1993) reported that licochalcone a is an antiparasitic compound with potential activity against human pathogenic protozoan leishmania species. the antiviral activity of g. glabra extracts against different viruses has been reported, including herpes simplex, varicella zoster, japanese encephalitis, influenza, and vesicular stomatitis virus (l. . different studies have demonstrated that two triterpenoids are responsible for the antiviral activity reported: glycyrrhizin and 18β-glycyrrhetinic acid (l. . these compounds have the ability to inhibit virus gene expression and replication, decreasing the adhesion force and stress and reducing hmgb1 binding to dna (l. . also, they can herpes simplex virus (hsv) is one of the most common viruses infecting humans and animals. during hsv infection, the cellular adhesion is increased, playing a key role in inflammatory response. w. huang et al. (2012) reported that the adhesion force and stress between the cerebral capillary vessel endothelial cells and the polymorphic nuclear leukocytes were amplified during hsv infection. glycyrrhizin stimulates the mouse defence system against hsv-1 infection (sekizawa, yanagi, & itoyama, 2001) . furthermore, glycyrrhizic acid was found to have a distinctive effect against kaposi sarcoma-associated herpes virus (kshv). it was proved that glycyrrhizic acid could terminate the latent infection of kshv when all current drugs are ineffective (damle, 2014) . also, glycyrrhizic acid down-regulates the expression of latency-associated nuclear antigen in b lymphocytes leading to natural cell death (apoptosis) of the kshv-infected cells (damle, 2014) . recently, the antiviral activity of glycyrrhizin against severe acute respiratory syndrome virus was evaluated (cinatl et al., 2003) . glycyrrhizin affects the cellular signalling pathways such as protein kinase c, casein kinase ii, and transcription factors, namely, activator protein 1 and nuclear factor κb (cinatl et al., 2003) . furthermore, glycyrrhizin and its aglycone, 18β the hepatoprotective activity of glycyrrhizin and 18β-glycyrrhetic acid by inhibition of free-radical generation and lipid peroxidation has been extensively reported (huo et al., 2011) . one of these studies indicated that the hydromethanolic root extract of g. glabra exhibits a significant protection against hepatotoxicity induced by carbon tetrachloride in the liver tissue of mice (v. sharma & agrawal, 2017) . the effects of liquorice on nonalcoholic fatty liver disease have also been investigated (hajiaghamohammadi et al., 2012) . according to rizzato et al. (2017) , glycyrrhizin and glycyrrhetinic acids prevent drug-induced liver injury and ensure the disruption of bile acid metabolism in humans. indeed glycyrrhetinic acid has been reported as anti-inflammatory and hepatoprotective compound (yin et al., 2017) , whereas glycyrrhizin, when compared with the placebo, induced a significant reduction in the serum aminotransferases and improved the liver histology (van rossum et al., 1998) . it has also been reported that the long-term use of glycyrrhizin prevents the development of hepatocellular carcinoma in chronic hepatitis c (van rossum et al., 1998) . in vitro studies have shown that glycyrrhizin modifies the intracellular transport and suppresses the hepatitis b virus surface antigen (sato et al., 1996) . in addition, it prevents the oxidative and hepatic damage caused by aflatoxins through increasing cyp1a1 and glutathione-stransferase activity, contributing to the anticarcinogenic activity by metabolic deactivation of the hepatotoxin (y. . mahmoud, hussein, hozayen, and abd el-twab (2017) reported that the treatment with 18β-glycyrrhetinic acid significantly reduced the serum enzymes, bilirubin, and proinflammatory cytokines in the liver, decreasing the expression of p450 e1. different studies suggest that the extract of g. glabra may be a potential supplemental source for different cancer treatments (c. s. lee, kim, lee, han, & lee, 2008; ohtsuki, oh-ishi, & nagata, 1992) . this activity is due to the 18β-glycyrrhetinic and glycyrrhizic acids that x. y. xiao et al., 2011) . the results indicated that licochalcone a inhibits gastric cancer cells growth in a dose-dependent way, by blocking cell cycle progression at the g2/m transition, inducing apoptosis. in addition, licochalcone a induced apoptosis by its effects on the expression of parp, caspase-3, bcl-2, and bax (x. y. xiao et al., 2011) . kanazawa et al. (2003) and jung et al. (2006) showed that isoliquiritigenin inhibits the cell growth by g2/m cell cycle arrest in breast and prostate tumour cells. different studies demonstrated that isoliquiritigenin suppresses pulmonary metastasis in mice (yamazaki et al., 2002) and human hepatoma cells (hsu, kuo, & lin, 2005) . apoptosis was primarily mediated through mitochondrial death cascade, as shown by loss of mitochondrial membrane potential, release of cytochrome c, and activation of caspase-9. a possible explanation is that cell growth was arrested through up-regulation of p53 and p21 and down-regulation of cdk2, cyclin e, and e2f-1 while apoptosis was induced by increasing bax protein expression and activating caspase-7 (g. sharma et al., 2012) . glabridin exhibited antitumour properties in various human cancer cells . the results revealed that glabridin induced apoptosis in dose dependently in huh7 cells through caspase-3, caspase-8, and caspase-9 activation and parp cleavage (hsieh et al., 2016) . the effects of g. glabra on learning and memory were investigated in mice (dhingra & sharma, 2006; parle et al., 2004) . in 2004, parle et al. (2004) administered the extract of g. glabra orally to mice during 7 days at different concentrations (75-300 mg/kg). chakravarthi and avadhani (2013) and dhingra and sharma (2006) studied the effects of g. glabra root aqueous extract on the learning and memory of 1month-old male wistar albino mice at doses between 75 and 300 mg/kg, orally administered during six successive weeks. both studies demonstrated a significant improvement of learning and memory in mice, but the exact mechanism behind this action remains unknown (chakravarthi & avadhani, 2013; dhingra & sharma, 2006 ). these findings suggest a possible neuroprotective role of liquorice in the prevention of diseases such as alzheimer. the basis of alzheimer is the chronic inflammation of certain brain regions. thus, the antiinflammatory activity of liquorice might contribute to the observed memory-enhancing effects (yokota, nishio, kubota, & mizoguchi, 1998) . also, oxygen free radicals are implicated in the process of aging and could be responsible for the development of alzheimer's disease in elderly persons. the protective role of liquorice extract may be attributed to its antioxidant properties, resulting in reduced brain damage and improvement of neuronal function and memory. the combination of anti-inflammatory and antioxidant activities with neuroprotective role could lead to memory-enhancing effects. hasanein (2011) gamma-aminobutyric acid (gaba) is the major inhibitory neurotransmitter in the central nervous system, being gaba a receptors a target for anaesthetics and neuroleptic, anxiolytic, and anticonvulsant compounds . g. glabra acts as a modulator of gaba a receptors (hoffmann, beltrán, ziemba, hatt, & gisselmann, 2016) , being able to induce sedative and anxiolytic effects. glabridin was evaluated by examining gaba responses in acutely isolated dorsal raphe neurons of a rat . according to the authors, glabridin potentiated gaba-induced responses by positive modulation of gaba a receptors, exhibiting sedative and hypnotic effects . glabridin potentiation is not sensitive to flumazenil and uses a similar mechanism of the general anaesthetics involving the amino acids n265 and m286, which are located in the second and third transmembrane domains on the β-subunit of gaba a receptors (hanrahan, chebib, & johnston, 2011) . also, glabridin could contribute to the hypnotic effect, as it is able to cross the blood-brain barrier . liquorice extract may have potential therapeutic value for the treatment of depressive disorders. recent studies have shown that liquorice extract produces significant antidepressant effects in mice during forced swim test (fst) and tail suspension test (tst; dhingra & sharma, 2006) . in the fst model, mice were forced to swim in a restricted space and induced to a characteristic behaviour of immobility. this situation reflects a state of depression. the tst model also induces a state of immobility that is claimed to reproduce a condition similar to human depression. both models are widely used to screen antidepressant drugs. the precise mechanisms by which liquorice extract produced this effect are not completely understood. however, it is suggested that the extract may interact with α1-adrenoceptors and dopamine d2 receptors, increasing the levels of norepinephrine and dopamine in the mice brain (dhingra & sharma, 2006) . besides, p-cpa (a serotonin synthesis inhibitor) did not attenuate the antidepressant effect of liquorice extract, suggesting that this is not mediated by the serotonergic system. on the other hand, p-cpa reversed the antidepressant effect of fluoxetine, suggesting that fluoxetine acts in the serotonergic system. reserpine produces a significant depression by depleting biogenic amines in the brain of rodents. as liquorice extract reversed reserpine-induced depression, its antidepressant effect can be associated with the restoration of brain monoamines, such as norepinephrine and dopamine. since ancient times, the influences of liquorice on the action of cortisol, reduction of testosterone synthesis, and the influence on oestrogen activity are well known (armanini et al., 2002) . s. h. kim and park (2012) reported that isoflavones can influence sexual development and impair oestrous cycling and ovarian and hypothalamus and pituitary glands function (s. h. kim & park, 2012) . the oestrogenic effect of liquorice ethanolic extract could be explained by its agonist activity on mcf-7 breast cancer cells, being this action mediated by 18β-glycyrrhetinic acid (g. sharma et al., 2012) . glabridin is a common component of herbal remedies used for the treatment of menopausal symptoms, resulting in favourable outcomes similar to those of 17βoestradiol (su wei poh et al., 2015) . in concentrations between 2.5 and 25 μg per animal, glabridin induces similar effects to the administration of oestradiol in a concentration of 5 μg per animal. glabridin was found to be three to four times more active than 2′-omethylglabridin and 4′-o-methylglabridin (tamir et al., 2000) . moreover, according to tamir et al. (2001) , glabrene has a considerable oestrogenic activity. glabridin and glabrene are similar to 17βoestradiol in the stimulation of the specific activity of creatine kinase, although at higher concentrations, differing in the extension rate of action as well as in the interaction with other drugs. in human premenopausal bone cells, the response to 17β-oestradiol and glabridin (at a higher concentration) was higher than in postmenopausal cells, whereas glabrene (at a higher concentration) was more effective in postmenopausal cells (somjen et al., 2004) . isoliquiritigenin has a strong oestrogen-like activity, suggesting that this compound may be cyclized to liquiritigenin, which is an active flavonoid under physiological conditions (hajirahimkhan et al., 2013) . in vivo, the stimulatory effects of glabrene are similar to those of oestradiol (powers & setzer, 2015) . it is also interesting to observe that isoliquiritigenin and formononetin stimulate sperm during fertilization (tung et al., 2014) . this reveals that both phytoestrogens may be useful therapeutic agents for infertility treatments (tung, shoyama, wada, & tanaka, 2015) . zamansoltani, nassiri-asl, sarookhani, jahani-hashemi, and zangivand (2009) reported that the alcoholic extract of g. glabra has antiandrogenic effects probably by increasing the testosterone metabolism, down-regulating androgen receptors, or activating oestrogenic receptors. the main skin benefits reported for g. glabra are based on the antioxidant and anti-inflammatory activities as well as on the ultraviolet (uv) protection (halder & richards, 2004) . saeedi, morteza-semnani, and ghoreishi (2003) reported the use of liquorice mainly for skin eruptions, including dermatitis, eczema, pruritus, and cysts. in particular, the g. glabra flavonoids present depigmenting capabilities and tyrosinase inhibition effects (solano, briganti, picardo, & ghanem, 2006) . the presence of an α-keto group in flavonoids is responsible for this activity (y. j. kim & uyama, 2005; parvez et al., 2007) . castangia et al. (2015) have reported the skin protective effects of liquorice against damage from oxidative stress. according to the authors, liquorice extract can scavenge dpph free radicals with an inhibition of 80% and protect fibroblasts against oxidative stress (castangia et al., 2015) . nevertheless, when evaluated in the isolated form, glycyrrhizin showed a poor antioxidant activity, being not able to efficiently counteract the oxidative effect (castangia et al., 2015) . tyrosinase is essential for skin pigmentation due to its role in melanin biosynthesis (solano et al., 2006) . the use of tyrosinase inhibitors is important in the cosmetic and medicinal industries, due to their preventive effect on pigmentation disorders such as melasma, age spots, and sites of actinic damage (nerya et al., 2003) . alternatively, tyrosinase inhibitors may be targets for developing medicines to treat hypopigmentation-related problems, such as albinism and piebaldism (y. j. kim & uyama, 2005) . in particular, glabridin, glabrene, isoliquiritigenin, licochalcone a, and liquiritin have been reported as g. glabra compounds able to inhibit the tyrosinase activity (ebanks, wickett, & boissy, 2009; nerya et al., 2003) . recently, grippaudo and di russo (2016) described the effects of the topical application of glycyrrhetinic acid combined with fractional carbon dioxide laser for the benign treatment of hand hyperpigmentation during 4 weeks. likewise, the treatment of human keratinocytes with 18βglycyrrhetinic acid and glabridin was documented to directly and indirectly prevent dna damage, avoiding the apoptosis activation caused by uv b radiation (veratti et al., 2011) . indeed, yokota et al. (1998) described that glabridin inhibits tyrosinase activity, melanogenesis, and skin inflammation. besides, glabrene acts as a tyrosinase inhibitor, preventing the formation of melanin in melanocytes, probably acting as skin-lightening agent. saeedi et al. (2003) liquorice has been traditionally used as a sweetener due to its taste, (tian, liu, zhen, & tong, 2013; tong, xie, rong, zhou, & meng, 2015) . according to bahmani et al. (2014) , liquorice can reduce diabetes symptoms, such as polydipsia and frequent urination, but cannot reduce blood glucose. takii et al. (2001) suggested that glycyrrhizin has an antidiabetic effect in noninsulin-dependent diabetes mice model, reducing the postprandial blood glucose rise. licochalcone a is the most promising one, inhibiting in vitro the growth of chloroquine-susceptible and chloroquine-resistant plasmodium falciparum strains (chen et al., 1994) . glabridin also showed in vitro activity against this parasite, probably by an induction of oxidative stress, mainly through the generation of reactive oxygen and nitrogen species that lead to apoptosis (cheema et al., 2014) . it is well known that myocardial ischaemia is one of the principal diseases in the western world. this disease occurs through occlusion or blockage of coronary arteries, resulting in myocardial cell death. however, the reperfusion produces the salvage of ischaemic tissue but also contributes to the myocardial cellular injury. the pretreatment with g. glabra significantly attenuates the ischaemic reperfusion, through an improvement of the heart antioxidant status, a positive modulation of the perturbed haemodynamic, and a recovery of left ventricular contractile function, along with histological salvage (di paola et al., 2009; ojha, golechha, kumari, bhatia, & arya, 2013) . in particular, glycyrrhizic acid induced protection against myocardial ischaemia in rats, probably due to its antioxidant potential (ojha et al., 2013) . similarly, nakagawa, kishida, arai, nishiyama, and mae (2004) demonstrated that g. glabra is safe for cardiomyocytes in a long-term administration. the extract of g. glabra has been used in the treatment of low bone mass, osteoporosis, fractures, bone defects, osteomalacia, osteogenesis imperfecta, bone disease, and periodontal diseases . rajesh (2004) described the inhibitory effect on bone reabsorption of g. glabra, whereas choi (2011) reported that glabridin is responsible for this activity. mitochondrial dysfunction, especially respiratory chain disruption, is responsible for aging-related bone diseases. hence, the target of glabridin is the reduction of mitochondrial dysfunction induced during aging and the prevention of osteoblast damage in osteoporotic patients (choi, 2011) . study on mice demonstrated that g. glabra, particularly glabridin, when integrated in a dietary supplement, could reduce the susceptibility of low-density lipoprotein (ldl) to oxidation and the atherosclerotic lesion area (fuhrman & aviram, 2001; grassi, desideri, & ferri, 2010) . these results could be related to the absorption and binding of glabridin to the ldl and a subsequent protection of the ldl from oxidation (fuhrman et al., 1997) . the methanolic extract of g. glabra rhizomes, at a dose of 150 mg/kg, has antiarthritic activity in male rats by inhibition of leukocyte migration, autoantigen production, and exhibition of antiproteinase activity (choudhary, kumar, malhotra, & singh, 2015) . also, mishra, bstia, mishra, chowdary, and patra (2001) reported that a combined formulation of g. glabra and boswellia serrata (1:1) had a significant synergistic action on arthritis. shin et al. (2007) studied the antiallergic effects, namely, the antiscratching behaviour and the ige production inhibitory activity, of glycyrrhizin, 18β-glycyrrhetinic acid, isoliquiritin, and liquiritigenin in dermatitis and asthma (shin et al., 2007) . in particular, 18β different adverse side effects were reported for high doses of g. glabra such as hypertension, hypokalaemia, or fluid retention (omar et al., 2012) . the exposure to high levels of glycyrrhizin can produce hypermineralocorticoid-like effects. glycyrrhetic acid and liquorice saponins can inhibit 11-β-hydroxysteroid dehydrogenase enzyme, leading to a cortisol-induced mineralocorticoid effect and a consequent tendency to the elevation of sodium and reduction of potassium levels (isbrucker & burdock, 2006) . for example, in 2010, a 34-yearold woman was suspected to have suffered a lethal acute intoxication from eating liquorice over a period of several months (albermann et al., 2010) . albermann et al. associated the effects with the potential mineralocorticoid action of glycyrrhizin and its metabolite, glycyrrhetic acid, and quantified by liquid chromatography-tandem mass spectrometry these compounds in the blood. nevertheless, only traces of glycyrrhetic acid had been found in the blood and stomach content of the deceased woman, which means that the possibility of acute lethal glycyrrhetic acid intoxication could be eliminated (albermann et al., 2010) . based on in vivo assays and clinical evidence, the amount of liquorice ingested daily by patients with mineralocorticoid excess syndromes appears to vary over a wide range (1.5-250 g/day ; isbrucker & burdock, 2006) . in addition to hypertension, patients may experithus, side effects remain an area of potential future study. this review not only details and explains the traditional use of this plant but also highlights its potential uses for other industries, such as cosmetic or pharmaceutical ones. more clinical trials should be performed to provide scientific basis for new uses. in conclusion, although evidence has grown in the past decade, there is still a need to conduct further robust double-blind randomized controlled trial about g. glabra. there is also an immense scope to explore different combinations of liquorice preparations in a wide range of disorders. anti-obesity effects of glabridin-rich supercritical carbon dioxide extract of licorice in highfat-fed obese mice determination of optimal concentration of deglycyrrhizinated licorice root extract for preventing dental caries using a bacterial model system effect of aqueous and alcoholic licorice (glycyrrhiza glabra) root extract against streptococcus mutans and lactobacillus acidophilus in comparison to chlorhexidine: an in vitro study determination of glycyrrhetic acid after consumption of liquorice and application to a fatality history of the endocrine effects of licorice in vitro anti-helicobacter pylori activity of a flavonoid rich extract of glycyrrhiza glabra and 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aquo-alchoholic extract of glycyrrhiza glabra against pseudomonas aeruginosa in mice lung infection model beneficial effect of aqueous root extract of glycyrrhiza glabra on learning and memory using different behavioral models: an experimental study screening of the phytochemical, antimicrobial and antioxidant activity of glycyrrhiza glabra root extract glabridin induces oxidative stress mediated apoptosis like cell death of malaria parasite plasmodium falciparum licochalcone a, a novel antiparasitic agent with potent activity against human pathogenic protozoan species of leishmania licochalcone a, a new antimalarial agent, inhibits in vitro growth of the human malaria parasite plasmodium falciparum and protects mice from p. yoelii infection plants and other natural products used in the management of oral infections and improvement of oral health hypnotic effects and binding studies for gabaa and 5-ht2c receptors of traditional medicinal plants used in asia for insomnia glabridin protects osteoblastic mc3t3-e1 cells against antimycin a induced cytotoxicity medicinal plants with potential anti-arthritic activity chemical composition and antimicrobial activities of the essential oil from glycyrrhiza glabra leaves glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus glycyrrhiza glabra (liquorice)-a potent medicinal herb effects of glycyrrhizin in a mouse model of lung adenocarcinoma antidepressant-like activity of glycyrrhiza glabra l. in mouse models of immobility tests. progress in neuro-psychopharmacology and biological psychiatry protective effects of glycyrrhizin in a gut hypoxia (ischemia)-reoxygenation (reperfusion) model. intensive care medicine mechanisms regulating skin pigmentation: the rise and fall of complexion coloration evaluation of certain food additives a history of the therapeutic use of liquorice in europe flavonoids protect ldl from oxidation and attenuate atherosclerosis licorice extract and its major polyphenol glabridin protect low-density lipoprotein against lipid peroxidation: in vitro and ex vivo studies in humans and in atherosclerotic apolipoprotein e-deficient mice anti-helicobacter pylori flavonoids from licorice extract antimicrobial activity of licorice flavonoids against methicillin-resistant staphylococcus aureus two antimicrobial flavanones from the leaves of glycyrrhiza glabra flavonoids: antioxidants against atherosclerosis effects of topical application of β-resorcinol and glycyrrhetinic acid monotherapy and in combination with fractional co 2 laser treatment for benign hand hyperpigmentation treatment antimicrobial potential of glycyrrhiza glabra roots the efficacy of licorice root extract in decreasing transaminase activities in non-alcoholic fatty liver disease: a randomized controlled clinical trial evaluation of estrogenic activity of licorice species in comparison with hops used in botanicals for menopausal symptoms topical agents used in the management of hyperpigmentation flavonoid modulation of gaba (a) receptors antioxidative and superoxide scavenging activities of retrochalcones in glycyrrhiza inflata pharmacological studies of glycyrrhiza glabra: a review chemopreventive effect of 18β-glycyrrhetinic acid via modulation of inflammatory markers and induction of apoptosis in human hepatoma cell line (hepg2) glabridin as a major active isoflavan from glycyrrhiza glabra (licorice) reverses learning and memory deficits in diabetic rats preliminary evidence for inhibitory effect of glycyrrhizin on hiv replication in patients with aids economic importance of licorice characterization of g. glabra and g. bucharica collected in tajikistan estimation of dietary intake of ochratoxin a from liquorice confectionery potentiating effect of glabridin from glycyrrhiza glabra on gaba a receptors glabridin induces apoptosis and autophagy through jnk1/2 pathway in human hepatoma cells isoliquiritigenin induces apoptosis and cell cycle arrest through p53-dependent pathway in hep g2 cells glycyrrhizin inhibits porcine epidemic diarrhea virus infection and attenuates the proinflammatory responses by inhibition of high mobility group box-1 protein inhibition of intercellular adhesion in herpes simplex virus infection by glycyrrhizin 18α-glycyrrhetinic acid induces apoptosis of hl-60 human leukemia cells through caspases-and mitochondria-dependent signaling pathways hepatoprotective and antioxidant effects of licorice extract against ccl(4)-induced oxidative damage in rats risk and safety assessment on the consumption of licorice root (glycyrrhiza sp.), its extract and powder as a food ingredient, with emphasis on the pharmacology and toxicology of glycyrrhizin in vitro susceptibility of helicobacter pylori to licorice extract glabridin inhibits cancer stem cell-like properties of human breast cancer cells: an epigenetic regulation of mir-148a/smad2 signaling. molecular carcinogenesis potentiating effect of glabridin on gabaa receptor-mediated responses in dorsal raphe neurons isoliquiritigenin induces apoptosis by depolarizing mitochondrial membranes in prostate cancer cells role of glucocorticoid in the development of glycyrrhizin-induced hypertension. clinical and experimental hypertension antitussive principles of glycyrrhizae radix, a main component of the kampo preparations bakumondo-to (mai-men-dong-tang) isoliquiritigenin inhibits the growth of prostate cancer antimicrobial and antioxidant activities of medicinal plant glycyrrhiza glabra var. glandulifera from different habitats 18β-glycyrrhetinic acid, the major bioactive component of glycyrrhizae radix, attenuates airway inflammation by modulating th2 cytokines, gata-3, stat6, and foxp3 transcription factors in an asthmatic mouse model effects of phytoestrogen on sexual development tyrosinase inhibitors from natural and synthetic sources: structure, inhibition mechanism and perspective for the future intestinal absorption and biliary elimination of glycyrrhizic acid diethyl ester in rats. drug design in vitro anti-helicobacter pylori activity of extractum liquiritiae, glycyrrhizin and its metabolites biphasic calcium phosphate-casein bone graft fortified with cassia occidentalis for bone tissue engineering and regeneration water extract of glycyrrhiza uralensis inhibited enterovirus 71 in a human foreskin fibroblast cell line 18β-glycyrrhetinic acid induces apoptotic cell death in siha cells and exhibits a synergistic effect against antibiotic anti-cancer drug toxicity liquiritigenin, a licorice flavonoid, helps mice resist disseminated candidiasis due to candida albicans by th1 immune response, whereas liquiritin, its glycoside form, does not synthesis and anticancer activities of glycyrrhetinic acid derivatives metabolomics analysis to evaluate the anti-inflammatory effects of polyphenols: glabridin reversed metabolism change caused by lps in raw 264.7 cells methotrexate hepatotoxicity is associated with oxidative stress, and down-regulation of ppargamma and nrf2: protective effect of 18β-glycyrrhetinic acid antithrombotic effect of glycyrrhizin, a plantderived thrombin inhibitor effect of licorice compounds licochalcone a, glabridin and glycyrrhizic acid on growth and virulence properties of candida albicans glycyrrhizin exerts antioxidative effects in h5n1 influenza a virus-infected cells and inhibits virus replication and proinflammatory gene expression antiarthritic activity of glycyrrhiza glabra, boswellia serrata and their synergistic activity in combined formulation studied in freund's adjuvant induced arthritic rats evaluation of immunomodulatory activity of glycyrhiza glabra l. roots in combination with zing glabridin triggers over-expression of mca1 and nuc1 genes in candida glabrata: is it an apoptosis inducer a novel process for extraction of natural sweetener from licorice (glycyrrhiza glabra) roots. separation and purification technology licorice flavonoids suppress abdominal fat accumulation and increase in blood glucose level in obese diabetic kk-a(y) mice glabrene and isoliquiritigenin as tyrosinase inhibitors from licorice roots the stimulatory and inhibitory effects of glycyrrhizin and a glycyrrhetinic acid derivative on phosphorylation of lipocortin i by a-kinase in vitro glycyrrhiza glabra protects from myocardial ischemia-reperfusion injury by improving hemodynamic, biochemical, histopathological and ventricular function licorice abuse: time to send a warning message antibacterial effects of glycyrrhetinic acid and its derivatives on staphylococcus aureus licochalcone-a induces intrinsic and extrinsic apoptosis via erk1/2 and p38 phosphorylation-mediated trail expression in head and neck squamous carcinoma fadu cells memory-strengthening activity of glycyrrhiza glabra in exteroceptive and interoceptive behavioral models naturally occurring tyrosinase inhibitors: mechanism and applications in skin health, cosmetics and agriculture industries the pharmacokinetics of glycyrrhizic acid evaluated by physiologically based pharmacokinetic modeling a molecular docking study of phytochemical estrogen mimics from dietary herbal supplements mechanism of anti-inflammatory action of liquorice extract and glycyrrhizin protective activity of glycyrrhiza glabra linn. on carbon tetrachloride-induced peroxidative damage formulation and evaluation of floating tablets of liquorice extract a new exploration of licorice metabolome anti-hiv activity of indian medicinal plants the treatment of atopic dermatitis with licorice gel effect of glycyrrhizin, an active component of licorice roots, on hiv replication in cultures of peripheral blood mononuclear cells from hiv-seropositive patients therapeutic basis of glycyrrhizin on chronic hepatitis b antifungal activity of plant extracts against arthrinium sacchari and chaetomium funicola hair growth stimulating effect and phytochemical evaluation of hydro-alcoholic extract of glycyrrhiza glabra glycyrrhizin increases survival of mice with herpes simplex encephalitis some effects of glycyrrhiza glabra (liquorice) roots extract on male rats 18β-glycyrrhetinic acid induces apoptosis through modulation of akt/foxo3a/bim pathway in human breast cancer mcf-7 cells in vivo antioxidant and hepatoprotective potential of glycyrrhiza glabra extract on carbon tetra chloride (ccl 4 ) induced oxidative-stress mediated hepatotoxicity assessment of median lethal dose and anti-mutagenic effects of glycyrrhiza glabra root extract against chemically induced micronucleus formation in swiss albino mice glycyrrhiza glabra: chemistry and pharmacological activity in vitro and in vivo antiallergic effects of glycyrrhiza glabra and its components phytochemistry and biological properties of glabridin a polyphenolic flavonoid glabridin: oxidative stress response in multidrug-resistant staphylococcus aureus phytocomplexes from liquorice (glycyrrhiza glabra l.) leaves-chemical characterization and evaluation of their antioxidant, anti-genotoxic and anti-inflammatory activity hypopigmenting agents: an updated review on biological, chemical and clinical aspects estrogenic activity of glabridin and glabrene from licorice roots on human osteoblasts and prepubertal rat skeletal tissues antiviral and immune stimulant activities of glycyrrhizin against duck hepatitis virus estrogenicity of glabridin in ishikawa cells antidiabetic effect of glycyrrhizin in genetically diabetic kk-ay mice estrogen-like activity of glabrene and other constituents isolated from licorice root estrogenic and antiproliferative properties of glabridin from licorice in human breast cancer cells successful treatment of latent autoimmune diabetes in adults with traditional chinese medicine: a case report detection of consensuses and treatment principles of diabetic nephropathy in traditional chinese medicine: a new approach improved in vitro fertilization ability of mouse sperm caused by the addition of licorice extract to the preincubation medium two activators of in vitro fertilization in mice from licorice review article: glycyrrhizin as a potential treatment for chronic hepatitis c pharmacokinetics of intravenous glycyrrhizin after single and multiple doses in patients with chronic hepatitis c infection phytochemical screening and determination of anti-bacterial and anti-oxidant potential of glycyrrhiza glabra root extracts 18β-glycyrrhetinic acid and glabridin prevent oxidative dna fragmentation in uvb-irradiated human keratinocyte cultures the antiviral and antimicrobial activities of licorice, a widely-used chinese herb metabolites identification of bioactive licorice compounds in rats 18β-glycyrrhetinic acid exhibits potent antitumor effects against colorectal cancer via inhibition of cell proliferation and migration liquorice, a unique "guide drug" of traditional chinese medicine: a review of its role in drug interactions glycyrrhizin inhibits lps-induced inflammatory mediator production in endometrial epithelial cells hypoglycemic effects of glabridin, a polyphenolic flavonoid from licorice, in an animal model of diabetes mellitus licochalcone a inhibits growth of gastric cancer cells by arresting cell cycle progression and inducing apoptosis 18β-glycyrrhetinic acid ameliorates acute propionibacterium acnes-induced liver injury through inhibition of macrophage inflammatory protein-1α isoliquiritigenin suppresses pulmonary metastasis of mouse renal cell carcinoma the anti-inflammatory activity of licorice, a widely used chinese herb protective effects of hepatocyte-specific glycyrrhetic derivatives against carbon tetrachloride-induced liver damage in mice efficacy of intravenous glycyrrhizin in the early stage of acute onset autoimmune hepatitis glycyrrhetinic acid attenuates lipopolysaccharide-induced fulminant hepatic failure in d-galactosamine-sensitized mice by up-regulating expression of interleukin-1 receptor the inhibitory effect of glabridin from licorice extracts on melanogenesis and inflammation antiinflammatory activities of licorice extract and its active compounds, glycyrrhizic acid, liquiritin and liquiritigenin, in bv2 cells and mice liver licochalcone-e induces caspase-dependent death of human pharyngeal squamous carcinoma cells through the extrinsic and intrinsic apoptotic signaling pathways licorice (glycyrrhiza glabra linn) as a valuable medicinal plant antiandrogenic activities of glycyrrhiza glabra in male rats glycyrrhizin administration ameliorates coxsackievirus b3-induced myocarditis in mice liquorice (glycyrrhiza glabra): a phytochemical and pharmacological review the authors declare that they have no conflict of interest. key: cord-303879-3hug5hj3 authors: cushnie, t.p. tim; lamb, andrew j. title: antimicrobial activity of flavonoids date: 2005-10-19 journal: int j antimicrob agents doi: 10.1016/j.ijantimicag.2005.09.002 sha: doc_id: 303879 cord_uid: 3hug5hj3 flavonoids are ubiquitous in photosynthesising cells and are commonly found in fruit, vegetables, nuts, seeds, stems, flowers, tea, wine, propolis and honey. for centuries, preparations containing these compounds as the principal physiologically active constituents have been used to treat human diseases. increasingly, this class of natural products is becoming the subject of anti-infective research, and many groups have isolated and identified the structures of flavonoids possessing antifungal, antiviral and antibacterial activity. moreover, several groups have demonstrated synergy between active flavonoids as well as between flavonoids and existing chemotherapeutics. reports of activity in the field of antibacterial flavonoid research are widely conflicting, probably owing to interand intra-assay variation in susceptibility testing. however, several high-quality investigations have examined the relationship between flavonoid structure and antibacterial activity and these are in close agreement. in addition, numerous research groups have sought to elucidate the antibacterial mechanisms of action of selected flavonoids. the activity of quercetin, for example, has been at least partially attributed to inhibition of dna gyrase. it has also been proposed that sophoraflavone g and (−)-epigallocatechin gallate inhibit cytoplasmic membrane function, and that licochalcones a and c inhibit energy metabolism. other flavonoids whose mechanisms of action have been investigated include robinetin, myricetin, apigenin, rutin, galangin, 2,4,2′-trihydroxy-5′-methylchalcone and lonchocarpol a. these compounds represent novel leads, and future studies may allow the development of a pharmacologically acceptable antimicrobial agent or class of agents. resistance to antimicrobial agents has become an increasingly important and pressing global problem. of the 2 million people who acquire bacterial infections in us hospitals each year, 70% of cases now involve strains that are resistant to at least one drug [1] . a major cause for concern in the uk is methicillin-resistant staphylococcus aureus (mrsa), which was at low levels a decade ago but now accounts for ca. 50% of all s. aureus isolates [2] . substantial investment and research in the field of anti-infectives are now desperately needed if a public health crisis is to be averted. structural modification of antimicrobial drugs to which resistance has developed has proven to be an effective means of extending the lifespan of antifungal agents such as the azoles [3] , antiviral agents such as the non-nucleoside reverse transcriptase inhibitors [4] , and various antibacterial agents including ␤-lactams and quinolones [5] . it is not surprising then, that in response to antimicrobial resistance, major pharmaceutical companies have tended to concentrate their efforts on improving antimicrobial agents in established classes [6] . however, with the portfolio of chemotherapeutics currently available, it has been acknowledged that researchers are getting close to the end game in terms of parent structure alterations. a call has therefore been made for the development of new classes of drug that work on different target sites to those in current use [7, 8] . rational drug design does not always yield effective antimicrobials. in the past, potent enzyme inhibitors have been successfully designed and synthesised but they had only modest antibacterial activity, probably owing to the complex issue of drug uptake by cells. broad empirical screening of chemical entities for antimicrobial activity represents an alternative strategy for the development of novel drugs. natural products have been a particularly rich source of anti-infective agents, yielding, for example, the penicillins in 1940, the tetracyclines in 1948 and the glycopeptides in 1955 [9] . the following review will examine the antimicrobial activity of flavonoids, a class of natural products possessing a diverse range of pharmacological properties. compounds with antifungal, antiviral and antibacterial activity will each be discussed in turn, with particular emphasis on those flavonoids with antibacterial activity. flavonoids are ubiquitous in photosynthesising cells and therefore occur widely in the plant kingdom [10] . they are found in fruit, vegetables, nuts, seeds, stems and flowers as well as tea, wine [11] , propolis and honey [12] , and represent a common constituent of the human diet [13] . in the us, the daily dietary intake of mixed flavonoids is estimated to be in the range 500-1000 mg, but this figure can be as high as several grams for people supplementing their diets with flavonoids or flavonoid-containing herbal preparations [14] . the function of flavonoids in flowers is to provide colours attractive to plant pollinators [11, 15] . in leaves, these compounds are increasingly believed to promote physiological survival of the plant, protecting it from, for example, fungal pathogens and uv-b radiation [13, 15] . in addition, flavonoids are involved in photosensitisation, energy transfer, the actions of plant growth hormones and growth regulators, control of respiration and photosynthesis, morphogenesis and sex determination [11, 13] . the basic structural feature of flavonoid compounds is the 2-phenyl-benzo[␣]pyrane or flavane nucleus, which consists of two benzene rings (a and b) linked through a heterocyclic pyrane ring (c) (fig. 1 ) [16] . flavonoids can be classified according to biosynthetic origin. some classes, for example chalcones, flavanones, flavan-3-ols and flavan-3,4-diols, are both intermediates in biosynthesis as well as end products that can accumulate in plant tissues. other classes are only known as end products of biosynthesis, for example anthocyanidins, proanthocyanidins, flavones and flavonols. two additional classes of flavonoid are those in which the 2-phenyl side chain of flavanone isomerises to the 3 position, giving rise to isoflavones and related isoflavonoids. the neoflavonoid is formed through further isomerisation to the 4 position [13] . structures of the major classes of flavonoids are given in fig. 2 . the structures of specific compounds within these classes that possess antimicrobial activity and that are discussed in the present review are summarised in table 1 . individual flavonoids may be assigned names in three different ways. trivial names are employed extensively and sometimes indicate flavonoid class or plant source. for example, names ending in 'inidin' can denote an anthocyanidin, names ending in 'etin' generally denote a flavonol, and compounds tricin and hypolaetin have been extracted from plants belonging to the genera triticum and hypolaena. flavonoids may also be named in a semi-systematic manner based on trivial names such as flavone or chalcone as the parent structure, e.g. 3,5,7,3 4 -pentahydroxyflavone or 3,3 ,4 ,5,7-pentahydroxyflavone. lastly, flavonoids may be given systematic chemical names, e.g. 3,4-dihydro-2-phenyl-2h-1-benzopyran for flavan, but this method is cumbersome and rarely used [13] . in the present review, trivial names will be used wherever possible. increasingly, flavonoids are becoming the subject of medical research. they have been reported to possess many useful properties, including anti-inflammatory activity, oestrogenic activity, enzyme inhibition, antimicrobial activity [10, 13] , antiallergic activity, antioxidant activity [11] , vascular activity and cytotoxic antitumour activity [15] . for a group of compounds of relatively homogeneous structure, the flavonoids inhibit a perplexing number and variety of eukaryotic enzymes and have a tremendously wide range of activities. in the case of enzyme inhibition, this has been postulated to be due to the interaction of enzymes with different parts of the flavonoid molecule, e.g. carbohydrate, phenyl ring, phenol and benzopyrone ring [10] . several reviews have been written on the interaction between flavonoids and mammalian cells, including comprehensive articles by harborne and williams [15] and middleton et al. [20] . an extensive review on the biochemistry and medical significance of flavonoids has also recently been produced by havsteen [21] . for centuries, preparations that contain flavonoids as the principal physiologically active constituents have been used by physicians and lay healers in attempts to treat human diseases [10] . for example, the plant tagetes minuta (containing quercetagetin-7-arabinosyl-galactoside) has been used extensively in argentine folk medicine to treat infectious disease [22] . the healing properties of propolis (or 'tzori' in hebrew) are referred to throughout the old testament [23] , and this balm was prescribed by hippocrates (460-377 bc) in ancient greece for the treatment of sores and ulcers [24] . [17, 18] , isoflavones [10] , chalcones [13, 19] , flavanones [10, 13] , flavones [10] , flavonols [10] , flavanon-3-ols [13] , anthocyanidins [13, 20] , flavan-3-ols [10, 13] , proanthocyanidins (occur as dimers, trimers, tetramers and pentamers; r = 0, 1, 2 or 3 flavan-3-ol structures) [13] , flavans [13] , flavan-3,4-diols [13] and dihydrochalcones [13] . table 1 a summary of the structures of antimicrobial flavonoids discussed within the present review article (compiled from the handbook of natural flavonoids [13] and individual research papers) substituents at carbon position: 2 3 4 5 6 7 8 2 3 4 5 6 flavones and their glycosides acacetin galangin flavan-3,4-diols and anthocyanidins leucocyanidin the antimicrobial properties of propolis have been attributed to its high flavonoid content and in particular the presence of the flavonoids galangin and pinocembrin [12, [25] [26] [27] . huangchin (scutellaria baicalensis) is yet another example. this herbal medicine has been used systemically and topically for thousands of years in china for the treatment of periodontal abscesses and infected oral wounds. the flavone baicalein is reported to be largely responsible for this plant's antimicrobial effects [28] . a oh r3 oh r3 oh naringenin oh oh oh naringin oh or7 oh pinocembrin oh oh ponciretin oh oh och 3 sophoraflavanone g oh oh r10 oh oh 3-methyleneflavanone ch 2 5,7,4 -trihydroxy-8-methyl-6-(3-methyl-[2butenyl])-(2s)-flavanone o h r 3 o h c h 3 o h chalcones licochalcone a r11 oh och 3 o h licochalcone c oh r3 och 3 o h 2,4,2 -trihydroxychalcone oh oh o h 2,4,2 -trihydroxy-5 -methylchalcone oh oh ch 3 o h -oh oh oh oh oh oh pelargonidin chloride cl oh oh oh flavans 6,4 -dichloroflavan c l c l 7-hydroxy-3 ,4 -(methylenedioxy)flavan o h # # r1 it has been suggested that because flavonoids are widely distributed in edible plants and beverages and have previously been used in traditional medicine, they are likely to have minimal toxicity. however, this family of compounds has a diverse range of activities in mammalian cells [14, 20] and in vivo confirmation of their side effects would be necessary for a full evaluation of their practical usefulness in the field of modern medicine [29] . given that the selectivity of flavonoids for eukaryotic enzymes appears to vary from compound to compound [15, 20] , such a study would need to assess the toxicity of these phytochemicals on an individual basis. owing to the widespread ability of flavonoids to inhibit spore germination of plant pathogens, they have been proposed for use against fungal pathogens of man [15] . a new prenylated flavanone recently isolated from the shrub eysenhardtia texana has been identified as 5,7,4 -trihydroxy-8methyl-6-(3-methyl-[2-butenyl])-(2s)-flavanone and shown to possess activity against the opportunistic pathogen candida albicans [30] . the flavonoid 7-hydroxy-3 ,4 -(methylenedioxy)flavan, isolated from terminalia bellerica fruit rind, has also been shown to possess activity against c. albicans [31] . two new flavones from artemisia giraldi, identified as 6,7,4 -trihydroxy-3 ,5 -dimethoxyflavone and 5,5dihydroxy-8,2 ,4 -trimethoxyflavone, together with 5,7,4 -trihydroxy-3 ,5 -dimethoxyflavone have been reported to exhibit activity against aspergillus flavus [32] , a species of fungi that causes invasive disease in immunosuppressed patients [33] . the activity of propolis against dermatophytes and candida spp. has been attributed at least partially to its high flavonoid content [34] . galangin, a flavonol commonly found in propolis samples [24] , has been shown to have inhibitory activity against aspergillus tamarii, a. flavus, cladosporium sphaerospermum, penicillium digitatum and penicillium italicum [35] . a recent area of research that is of particular interest is the apparent inhibitory activity of some flavonoids against human immunodeficiency virus (hiv). to date, most if not all investigations have involved work with the pandemic hiv-1 strain and its enzymes. in vitro studies have shown that baicalin inhibits hiv-1 infection and replication. inhibition of hiv-1 entry into cells expressing cd4 and chemokine co-receptors [36] , and antagonism of hiv-1 reverse transcriptase [37] by the flavone o-glycoside have been demonstrated by li and colleagues. baicalein [38] , robustaflavone and hinokiflavone [39] have also been shown to inhibit hiv-1 reverse transcriptase, as have several catechins, but catechins inhibit other dna polymerases and their interaction with the hiv-1 enzyme is therefore thought to be non-specific in nature [40] . in addition, it has been demonstrated that several flavonoids, including demethylated gardenin a and 3,2 -dihydroxyflavone, inhibit hiv-1 proteinase [41] . robinetin, myricetin, baicalein, quercetagetin [42] and quercetin 3-o-(2 -galloyl)-␣-l-arabinopyranoside [43] inhibit hiv-1 integrase, although there are concerns that hiv enzyme inhibition by quercetagetin and myricetin is non-specific [44] . it has also been reported that the flavonoids chrysin, acacetin and apigenin prevent hiv-1 activation via a novel mechanism that probably involves inhibition of viral transcription [45] . interestingly, in a study by hu and colleagues, chrysin was reported to have the highest therapeutic index of 21 natural and 13 synthetic flavonoids against hiv-1 [46] . several research groups have investigated the relationship between flavonoid structure and inhibitory activity against hiv-1 and its enzymes [39, 41, 42, 44, 46] . furthermore, at least two groups have proposed mechanisms of action for hiv-1 enzyme inhibition [41, 42] . flavonoids also have inhibitory activity against a variety of other viruses. for example, selway reports that quercetin, morin, rutin, dihydroquercetin, dihydrofisetin, leucocyanidin, pelargonidin chloride and catechin possess activity against up to seven types of virus, including herpes simplex virus (hsv), respiratory syncytial virus, poliovirus and sindbis virus [11, 47] . proposed antiviral mechanisms of action include inhibition of viral polymerase and binding of viral nucleic acid or viral capsid proteins [47] . in addition to the flavonoids mentioned above, three proanthocyanidins from pavetta owariensis (with structural similarity to proanthocyanidin a2 and cinnamtannin b1 and b2) have been shown to have activity against hsv and coxsackie b virus [48, 49] . it has also been demonstrated that two of the flavonoids found in propolis, chrysin and kaempferol, inhibit viral replication of hsv, human coronavirus and rotavirus [50] . more recently, the flavonol galangin has been reported to have significant antiviral activity against hsv and coxsackie b virus [51] . although naturally occurring flavonoids with antiviral activity have been recognised since the 1940s, it is only in the last 25 years that attempts have been made to synthetically modify flavonoids for improved antiviral activity. one such synthesised compound is 6,4 -dichloroflavan. however, despite showing strong in vitro activity, this compound proved unsuccessful in clinical trials [11] . synergism has been demonstrated between various combinations of flavones and flavonols. for example, kaempferol and luteolin show synergy against hsv. it has been suggested that this is why propolis is more active than its individual component compounds [52] . synergism has also been reported between flavonoids and other antiviral agents. quercetin, for example, potentiates the effects of 5-ethyl-2 -dioxyuridine [11] and acyclovir [53] against hsv and pseudorabies infection. apigenin also enhances the antiviral activity of acyclovir against these viruses [53] . the antibacterial activity of flavonoids is being increasingly documented. crude extracts from plants with a history of use in folk medicine have been screened in vitro for antibacterial activity by many research groups. flavonoidrich plant extracts from species of hypericum [54] , capsella [55] and chromolaena [55] have been reported to possess antibacterial activity. many other phytochemical preparations with high flavonoid content have also been reported to exhibit antibacterial activity [22, [56] [57] [58] [59] [60] [61] [62] [63] . propolis has been analysed on many occasions too, and samples containing high concentrations of flavonoids are frequently reported to show antibacterial activity [12, [25] [26] [27] 50, 64] . many research groups have gone one step further and either isolated and identified the structure of flavonoids that possess antibacterial activity, or quantified the activity of commercially available flavonoids. examples of such flavonoids are apigenin [65] [66] [67] [68] [69] [70] [71] [72] [73] , galangin [35, [74] [75] [76] [77] , pinocembrin [78, 79] , ponciretin [80, 81] , genkwanin [66, 82] , sophoraflavanone g and its derivatives [29, [83] [84] [85] , naringin and naringenin [29, 60, 86, 87] , epigallocatechin gallate and its derivatives [74, [88] [89] [90] [91] [92] [93] [94] [95] , luteolin and luteolin 7glucoside [69, 73, 96] , quercetin, 3-o-methylquercetin and various quercetin glycosides [60, 65, 72, 87, [97] [98] [99] [100] [101] [102] and kaempferol and its derivatives [60, 65, 74, 76, 87, 98, 100, 103] . other flavones [32, 60, 74, [104] [105] [106] [107] , flavone glycosides [86, 108, 109] , isoflavones [110, 111] , flavanones [29, 30, 78, 79, 104, [111] [112] [113] [114] , isoflavanones [115] , isoflavans [116] , flavonols [74, 114, 117] , flavonol glycosides [86, [118] [119] [120] and chalcones [79, 104, 111, 121] with antibacterial activity have also been identified. some researchers have reported synergy between naturally occurring flavonoids and other antibacterial agents against resistant strains of bacteria. examples of these include epicatechin gallate [122] [123] [124] [125] and sophoraflavanone g [83, 84] . at least one group has demonstrated synergy between flavonoids with antibacterial activity [126] . others have synthetically modified natural flavones and analysed them for antibacterial activity [94, [127] [128] [129] [130] [131] . for example, wang and colleagues have complexed 5-hydroxy-7,4 -dimethoxyflavone with a number of transition metals and shown that this process increases antibacterial activity [130] . another group reported increased antibacterial activity of 3-methyleneflavanones when the b ring contained bromine or chlorine substituents [131] . two research groups have described the use of flavonoids in vivo. in a study by vijaya and ananthan, oral administration of either 142.9 mg/kg quercetin or 214.3 mg/kg quercetrin protected guinea pigs against an induced shigella infection that killed untreated control animals [132] . more recently, dastidar and co-workers reported that intraperitoneal injection of either 1.58 mg/kg sophoraisoflavone a or 3.16 mg/kg 6,8diprenylgenistein gave significant protection to mice challenged with ∼9.5 × 10 8 colony-forming units (cfus) of salmonella typhimurium [110] . when reports of the antibacterial activity of flavonoids are compared, the results appear widely conflicting (table 2) . for example, it was published that apigenin had no activity against s. aureus at concentrations up to 128 g/ml [72] ; a separate study in the same year reported that the flavone inhibited the growth of 15 strains of mrsa and 5 sensitive strains of s. aureus at concentrations between 3.9 g/ml table 2 the inhibitory activity of apigenin against numerous species of gram-positive and + + dd, disk diffusion assay; bmad, broth macrodilution assay; bmid, broth microdilution assay; ns, assay type not stated in report; awd, agar well diffusion assay; ad, agar dilution assay; +, antibacterial activity detected; −, no antibacterial activity detected. and 15.6 g/ml [73] . from table 2 it can be seen that such discrepancies could perhaps be attributed on occasion to different assays being used (e.g. [65, 70] and [72, 73] ). many different assays are employed in flavonoid research, including the agar dilution technique [29] , the paper disk diffusion assay [115] , the hole-plate diffusion method [22] , the cylinder diffusion method [60] , the broth macrodilution technique [71] and the broth microdilution technique [134] . in particular, assays relying on diffusion of test flavonoids may not give a reliable quantitative measure of antibacterial activity because a potent antibacterial flavonoid may have a low rate of diffusion [32] . however, it is clear from table 2 that additional factors are involved in causing these discrepancies because even groups using the same assay are obtaining conflicting results (e.g. [67, 96] and [67, 72] ). such inconsistencies may be due to variations within each assay. for example, different groups using the agar dilution technique have used different sizes of bacterial inoculum [81, 86] . in a report by the national committee for clinical laboratory standards (nccls), inoculum size was considered the single most important variable in susceptibility testing [135] . it should be noted that many groups assaying flavonoid antibacterial activity have not quantified the test bacterial suspension [60, 115] and others have not even standardised the size of their unenumerated inocula [35, 56, 76, 90, 97] . from the published work it is clear that, in addition to inoculum size, there are many other variable factors for each type of assay. these include volume of broth or agar [90, 116] , type of broth or agar [86, 92] , size of wells [56, 60] , size of paper disks [57, 65] , strains of a particular bacterial species used [69, 72] and incubation period [90, 116] . recently, a set of guidelines was published for standard agar dilution, broth macrodilution and broth microdilution methods [136] . this may help to reduce the number of conflicting reports of flavonoid antibacterial activity in the future. however, it will remain necessary to consider carefully additional variables such as the solvent used to dissolve test flavonoids [116, 118] . it has previously been shown that precipitation occurs when selected flavonoids are dissolved in organic solvents and diluted with neutral polar solutions [75] . precipitation of flavonoids in a minimum inhibitory concentration (mic) assay is likely to cause diminished contact between bacterial cells and flavonoid molecules and may lead to false negative reports of antibacterial activity. also, in improperly controlled experiments, flavonoid precipitation could be misinterpreted as bacterial growth and further false negative results may be recorded as a consequence. the structural alteration of flavonoids such as galangin in alkaline solvents is another matter for consideration [75] . if flavonoid salts are formed and these have increased or decreased potency compared with the parent structure, this may lead to false positive/negative reports of antibacterial activity. other variables worth noting include whether the test flavonoids are obtained from a commercial or natural source [35, 74] and which companies [74, 75] /natural products [71, 72] the compounds are from. the diverse range of cell functions affected by flavonoids in eukaryotic systems is well documented [10, 20] . although there have been comparatively few studies into the mechanisms underlying flavonoid antibacterial activity, information from published literature indicates that different compounds within this class of phytochemicals may target different components and functions of the bacterial cell [137] [138] [139] . if this is the case, it is surprising that the small number of groups which have investigated the relationship between flavonoid structure and antibacterial activity (summarised below) have been able to identify common structural features among active compounds. however, it may be that individual antibacterial flavonoids have multiple cellular targets, rather than one specific site of action. alternatively, these common structural features may simply be necessary for flavonoids to gain proximity to or uptake into the bacterial cell. tsuchiya and colleagues sought to establish a structureactivity relationship for flavanones by isolating a number of differently substituted compounds and determining their mics against mrsa [29] . their study indicated that 2 ,4 -or 2 ,6 -dihydroxylation of the b ring and 5,7-dihydroxylation of the a ring in the flavanone structure was important for anti-mrsa activity. substitution at the 6 or 8 position with a long chain aliphatic group such as lavandulyl (5-methyl-2isopropenyl-hex-4-enyl) or geranyl (trans-3,7-dimethyl-2,6octadienyl) also enhanced activity [29] . interestingly, a recent report by stapleton and colleagues demonstrated that substitution with c 8 and c 10 chains also enhanced the antistaphylococcal activity of flavonoids belonging to the flavan-3-ol class [94] . osawa et al. assessed the activity of a number of structurally different flavonoids including flavones, flavanones, isoflavones and isoflavanones based on the paper disk agar diffusion assay [115] . it was shown that 5-hydroxyflavanones and 5-hydroxyisoflavanones with one, two or three additional hydroxyl groups at the 7, 2 and 4 positions inhibited the growth of streptococcus mutans and streptococcus sobrinus. these results correlate well with those of tsuchiya and colleagues [29] . it was also reported by osawa and colleagues that 5-hydroxyflavones and 5-hydroxyisoflavones with additional hydroxyl groups at the 7 and 4 positions did not exhibit this inhibitory activity [115] . however, when sato et al. examined two isoflavones with hydroxyl groups at the 5, 2 and 4 positions using an agar dilution assay, intensive inhibitory activity was detected against a wide range of streptococcal species [107] . this may suggest that hydroxylation at position 2 is important for activity. alternatively, the lack of activity detected by osawa et al. may simply be due to the poor diffusion of flavones and isoflavones (compared with flavanones and isoflavanones) through the medium. a more recent paper [104] also reports the importance of a hydroxyl group at position 5 of flavanones and flavones for activity against mrsa, supporting the earlier findings of tsuchiya et al. [29] . it further states that chalcones are more effective against mrsa than flavanones or flavones, and that hydroxyl groups at the 2 position are important for the antistaphylococcal activity of these compounds. methoxy groups were reported to drastically decrease the antibacterial activity of flavonoids [104] . the importance of hydroxylation at the 2 position for antibacterial activity of chalcones is supported by earlier work from sato and colleagues, who found that 2,4,2 -trihydroxy-5 -methylchalcone and 2,4,2trihydroxychalcone inhibited the growth of 15 strains of cariogenic streptococci [140] . as mentioned previously, ward and colleagues synthesised a number of halogenated derivatives of 3methyleneflavanone [131] . substitution of the b ring was found to enhance antibacterial activity, with 3 -chloro, 4chloro and 4 -bromo analogues each being approximately twice as effective as their parent compound against s. aureus, and four times more active against enterococcus faecalis. also, the 2 ,4 -dichloro derivative exhibited a four-to eightfold improvement in activity against s. aureus and a twoto four-fold improvement against e. faecalis. by contrast, 3-methylene-6-bromoflavanone was less potent than the parent compound and the authors suggested that halogenation of the a ring may diminish activity [131] . clearly, however, it would be necessary to prepare analogues with substitution at other a-ring positions before this could be said with any certainty. in chalcones, neither fluorination nor chlorination at position 4 of the b ring is reported to affect antibacterial potency significantly [104] . again, however, other structural analogues of this class of flavonoids would need to be synthesised and examined before the effect of halogenation upon antibacterial activity could be properly assessed. several research groups have attempted to determine whether flavonoid activity is bacteriostatic or bactericidal by conducting time-kill studies. in such experiments, epigallocatechin gallate [89] , galangin [75] and 3-o-octanoyl-(+)-catechin [94] have been shown to cause a reduction of 1000-fold or more in viable counts of mrsa-yk, s. aureus nctc 6571 and emrsa-16, respectively. this would immediately appear to suggest that flavonoids are capable of bactericidal activity. however, it has recently been demonstrated that 3-o-octanoyl-(−)-epicatechin induces the formation of pseudomulticellular aggregates both in antibiotic-sensitive and antibiotic-resistant strains of s. aureus [94] . if this phenomenon is induced by other compounds within the flavonoid class (and liposomal studies suggest that this is the case for epigallocatechin gallate [88] ), questions are raised regarding the interpretation of results from time-kill studies. it may be that flavonoids are not killing bacterial cells but merely inducing the formation of bacterial aggregates and thereby reducing the number of cfus in viable counts. in a study using radioactive precursors, mori and colleagues showed that dna synthesis was strongly inhibited by flavonoids in proteus vulgaris, whilst rna synthesis was most affected in s. aureus [138] . flavonoids exhibiting this activity were robinetin, myricetin and (−)-epigallocatechin. protein and lipid synthesis were also affected but to a lesser extent. the authors suggested that the b ring of the flavonoids may play a role in intercalation or hydrogen bonding with the stacking of nucleic acid bases and that this may explain the inhibitory action on dna and rna synthesis [138] . ohemeng et al. screened 14 flavonoids of varying structure for inhibitory activity against escherichia coli dna gyrase, and for antibacterial activity against staphylococcus epidermidis, s. aureus, e. coli, s. typhimurium and stenotrophomonas maltophilia [68] . it was found that e. coli dna gyrase was inhibited to different extents by seven of the compounds, including quercetin, apigenin and 3,6,7,3 ,4pentahydroxyflavone. interestingly, with the exception of 7,8-dihydroxyflavone, enzyme inhibition was limited to those compounds with b-ring hydroxylation [68, 141] . the authors proposed that the observed antibacterial activity of the seven flavonoids was due in part to their inhibition of dna gyrase. however, since the level of antibacterial activity and enzyme inhibition did not always correlate, they also suggested that other mechanisms were involved [68] . more recently, plaper and colleagues reported that quercetin binds to the gyrb subunit of e. coli dna gyrase and inhibits the enzyme's atpase activity [142] . enzyme binding was demonstrated by isolating e. coli dna gyrase and measuring quercetin fluorescence in the presence and absence of the gyrase subunits. the flavonoid-binding site was postulated to overlap with those of atp and novobiocin, since addition of these compounds interfered with quercetin fluorescence. inhibition of gyrb atpase activity by quercetin was also demonstrated in a coupled atpase assay. this research is in agreement with the earlier findings of ohemeng et al. [68] and supports the suggestion that quercetin's antibacterial activity against e. coli may be at least partially attributable to inhibition of dna gyrase. when screening natural products for type ii topoisomerase inhibitors, bernard and co-workers found that the glycosylated flavonol rutin was very effective [143] . this compound exhibited antibacterial activity against a permeable e. coli strain (a strain into which the enva1 allele had been incorporated [144, 145] ). using enzyme assays and a technique known as the sos chromotest, it was shown that rutin selectively promoted e. coli topoisomerase iv-dependent dna cleavage, inhibited topoisomerase iv-dependent decatenation activity and induced the sos response of the e. coli strain. the group suggested that since topoisomerase iv is essential for cell survival, the rutin-induced topoisomerase iv-mediated dna cleavage leads to an sos response and growth inhibition of e. coli cells [143] . within our own laboratory, a 4-quinolone-resistant s. aureus strain was shown to have increased susceptibility to the flavonol galangin compared with other 4-quinolonesensitive and -resistant strains [146] . interestingly, this strain possesses a distinct amino acid substitution (serine to proline) at position 410 of the grlb subunit. this may suggest that topoisomerase iv and the relatively homologous gyrase enzyme are involved in the antibacterial mechanism of action of galangin. clearly, however, further work with mutant strains and purified enzymes would be necessary before this could be verified. a research team that had previously found sophoraflavanone g to have intensive antibacterial activity against mrsa and streptococci [83] [84] [85] recently reported attempts to elucidate the mechanism of action of this flavanone [139] . the effect of sophoraflavanone g on membrane fluidity was studied using liposomal model membranes and compared with the less active flavanone naringenin, which lacks 8-lavandulyl and 2 -hydroxyl groups. at concentrations corresponding to the mic values, sophoraflavanone g was shown to increase fluorescence polarisation of the liposomes significantly. these increases indicated an alteration of membrane fluidity in hydrophilic and hydrophobic regions, suggesting that sophoraflavanone g reduced the fluidity of outer and inner layers of membranes. naringenin also exhibited a membrane effect but at much higher concentrations. this correlation between antibacterial activity and membrane interference was suggested to support the theory that sophoraflavanone g demonstrates antibacterial activity by reducing membrane fluidity of bacterial cells [139] . another group, ikigai and colleagues, carried out research on (−)-epigallocatechin gallate, a strongly antibacterial catechin found in green tea. catechins are a group of flavonoids that appear to have greater activity against gram-positive than gram-negative bacteria [88] . in this study, liposomes were again used as model bacterial membranes, and it was shown that epigallocatechin gallate induced leakage of small molecules from the intraliposomal space. aggregation was also noted in liposomes treated with the compound. the group therefore concluded that catechins primarily act on and damage bacterial membranes. it was not known how this damage occurred but two theories were put forward. first, catechins may perturb the lipid bilayers by directly penetrating them and disrupting the barrier function. alternatively, catechins may cause membrane fusion, a process that results in leakage of intramembranous materials and aggregation. interestingly, the group also demonstrated that leakage induced by epigallocatechin gallate was significantly lower when liposome membranes were prepared containing negatively charged lipids. it was therefore suggested that the low catechin susceptibility of gram-negative bacteria may be at least partially attributable to the presence of lipopolysaccharide acting as a barrier [88] . as mentioned previously, stapleton and colleagues found that substitution with c 8 and c 10 chains increased the antibacterial activity of selected flavan-3-ols (catechins). the group went on to show that cells of an mrsa clinical isolate treated with (−)-epicatechin gallate and 3-o-octanoyl-(+)-catechin, respectively, exhibited moderately and highly increased levels of labelling with the selectively permeable fluorescent stain propidium iodide. in addition, when s. aureus cells were grown in the presence of either (−)-epicatechin gallate or 3-o-octanoyl-(−)-epicatechin and examined by transmission electron microscopy, they were shown to form pseudomulticellular aggregates [94] . this work constitutes a substantial advance in the development of catechins as antibacterial agents and lends support to ikigai's argument that catechins act on and damage bacterial membranes. it has also been demonstrated by sato and colleagues that the chalcone 2,4,2 -trihydroxy-5 -methylchalcone induces leakage of 260 nm absorbing substances from s. mutans. this observation generally indicates leakage of intracellular material such as nucleotide, and the authors suggested that 2,4,2 -trihydroxy-5 -methylchalcone exerts its antibacterial effect by changing the permeability of the cellular membrane and damaging membrane function [140] . in addition, the effect of galangin upon cytoplasmic integrity in s. aureus has been investigated by measuring loss of internal potassium [147] . when high cell densities of s. aureus were incubated for 12 h in media containing 50 g/ml of the flavonol, a 60-fold decrease in the number of cfus was noted and cells lost ca. 20% more potassium than untreated control bacteria. these data strongly suggest that galangin induces cytoplasmic membrane damage and potassium leakage. whether galangin damages the membrane directly, or indirectly as a result of autolysis or cell wall damage and osmotic lysis, remains to be established however [147] . in an investigation into the antimicrobial action of propolis, mirzoeva and colleagues showed that one of its constituent flavonoids, quercetin, caused an increase in permeability of the inner bacterial membrane and a dissipation of the membrane potential [148] . the electrochemical gradient of protons across the membrane is essential for bacteria to maintain capacity for atp synthesis, membrane transport and motility. mirzoeva et al. suggested that the effect of propolis on membrane permeability and membrane potential may contribute enormously to its overall antibacterial activity and may decrease the resistance of cells to other antibacterial agents. it was thought that this might explain the synergistic effect that occurs between propolis and other antibiotics such as tetracycline [148] and ampicillin [149] . the group also demonstrated that the flavonoids quercetin and naringenin significantly inhibited bacterial motility, providing further evidence that the proton motive force is disrupted. bacterial motility and chemotaxis are thought to be important in virulence as they guide bacteria to their sites of adherence and invasion. mirzoeva et al. suggested that the antimotility action of propolis components may have an important role in inhibition of bacterial pathogenesis and the development of infection [148] . the cytoplasmic membrane activity detected for quercetin by mirzoeva and co-workers may represent one of the additional mechanisms of antibacterial action that was suspected to be present among the seven dna gyrase-inhibiting flavonoid compounds tested by ohemeng and colleagues [68] . haraguchi and colleagues recently carried out an investigation into the antibacterial mode of action of two retrochalcones (licochalcone a and c) from the roots of glycyrrhiza inflata [137] . these flavonoids demonstrated inhibitory activity against s. aureus and micrococcus luteus but not against e. coli, and in preliminary tests licochalcone a inhibited incorporation of radioactive precursors into macromolecules (dna, rna and protein). the group hypothesised that the licochalcones may be interfering with energy metabolism in a similar way to respiratory-inhibiting antibiotics, since energy is required for active uptake of various metabolites and for biosynthesis of macromolecules [137] . interestingly, the licochalcones were found to inhibit strongly oxygen consumption in m. luteus and s. aureus but not in e. coli, which correlated well with the observed spectrum of antibacterial activity. the group further demonstrated that licochalcones a and c effectively inhibited nadh-cytochrome c reductase, but not cytochrome c oxidase or nadh-coq reductase. it was therefore suggested that the inhibition site of these retrochalcones was between coq and cytochrome c in the bacterial respiratory electron transport chain [137] . merck research laboratories recently reported that the flavanone lonchocarpol a inhibits macromolecular synthesis in bacillus megaterium. using radioactive precursors, it was demonstrated that rna, dna, cell wall and protein synthesis were all inhibited at concentrations similar to the mic value [150] . this may represent another example of a flavonoid that interferes with energy metabolism. with regard to natural products, it is generally accepted that phytochemicals are less potent anti-infectives than agents of microbial origin, i.e. antibiotics [48] . however, new classes of antimicrobial drug are urgently required and the flavonoids represent a novel set of leads. future optimisation of these compounds through structural alteration may allow the development of a pharmacologically acceptable antimicrobial agent or group of agents. existing structure-activity data suggest that it might be possible, for example, to prepare a potent antibacterial flavanone by synthesising a compound with halogenation of the b ring as well as lavandulyl or geranyl substitution of the a ring. also, it is worth noting that the rapid progress which is being made toward elucidation of flavonoid biosynthetic pathways [151] may soon allow the production of structural analogues of active flavonoids through genetic manipulation. screening of these analogues might lead to the identification of compounds that are sufficiently potent to be useful as antifungal, antiviral or antibacterial chemotherapeutics. in addition to the structural alteration of weak and moderately active antimicrobial flavonoids, investigation into the mechanisms of action of these compounds is likely to be a productive area of research. such information may assist in the optimisation of a lead compound's activity, provide a focus for toxicological attention and aid in the anticipation of resistance. also, characterisation of the interaction between antimicrobial flavonoids and their target sites could potentially allow the design of secondgeneration inhibitors. infectious diseases society of america. statement of the idsa concerning 'bioshield ii: 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in staphylococcus aureus by catechins and gallates rutin-enhanced antibacterial activities of flavonoids against bacillus cereus and salmonella enteritidis synthesis and antimicrobial activity of flavone-6-carboxaldehyde oxime ether derivatives synthesis and antimicrobial activity of flavone-3 -carboxaldehyde oxime ether derivatives synthesis and antimicrobial activity of some new flavonyl oxime ether derivatives synthesis, characterization, and antibacterial activity of transition metal complexes with 5-hydroxy-7,4 -dimethoxyflavone antimicrobial 3-methyleneflavanones therapeutic efficacy of medicinal plants against experimentally induced shigellosis in guinea pigs isolation of the antimicrobial alkaloid stemmadenine from iraqi rhazya stricta baicalin synergy with beta-lactam antibiotics against methicillin-resistant staphylococcus aureus and other beta-lactam-resistant strains of s. aureus methods for determining bactericidal activity of antimicrobial agents methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically mode of antibacterial action of retrochalcones from glycyrrhiza inflata antibacterial activity and mode of action of plant flavonoids against proteus vulgaris and staphylococcus aureus reduction of membrane fluidity by antibacterial sophoraflavanone g isolated from sophora exigua growth inhibition of oral bacteria related to denture stomatitis by anticandidal chalcones a comparison of active site binding of 4-quinolones and novel flavone gyrase inhibitors to dna gyrase characterization of quercetin binding site on dna gyrase glycosylated flavones as selective inhibitors of topoisomerase iv transduction and dominance studies of the enva gene present in a chain-forming mutant of escherichia coli k12 mutant of escherichia coli with anomalous cell division and ability to decrease episomally and chromosomally mediated resistance to ampicillin and several other antibiotics assessment of the antibacterial activity of galangin against 4-quinolone resistant strains of staphylococcus aureus detection of galangin-induced cytoplasmic membrane damage in staphylococcus aureus by measuring potassium loss antimicrobial action of propolis and some of its components: the effects on growth, membrane potential and motility of bacteria svabic-vlahovic m. in vitro antimicrobial activity of propolis and synergism between propolis and antimicrobial drugs antibacterial activity of lonchocarpol a prospects for the metabolic engineering of bioactive flavonoids and related phenylpropanoid compounds the authors are very grateful to dr paul kong and dr satyajit sarker for critiquing preliminary drafts of the manuscript and for advice on flavonoid classification and structure. thanks are extended to dr peter taylor for insightful comments regarding interpretation of data from time-kill studies with flavonoids. thanks also to dr derek chapman, miss vivienne hamilton, dr bruce thomson and mrs amina al-mossawi for their kind support and encouragement. key: cord-270424-8yhsjbmi authors: kang, dongwei; zhang, heng; zhou, zhongxia; huang, boshi; naesens, lieve; zhan, peng; liu, xinyong title: first discovery of novel 3-hydroxy-quinazoline-2,4(1h,3h)-diones as specific anti-vaccinia and adenovirus agents via ‘privileged scaffold’ refining approach date: 2016-11-01 journal: bioorg med chem lett doi: 10.1016/j.bmcl.2016.09.071 sha: doc_id: 270424 cord_uid: 8yhsjbmi a series of 1,2,3-triazolyl 3-hydroxy-quinazoline-2,4(1h,3h)-diones was constructed utilizing cu(i)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (cuaac) method. the biological significance of the novel synthesized quinazolines was highlighted by evaluating them in vitro for antiviral activity, wherein several compounds exhibited excellent activity specifically against vaccinia and adenovirus. especially, 24b11 displayed the most potent inhibitory activity against vaccinia with an ec(50) value of 1.7 μm, which was 15 fold than that of the reference drug cidofovir (ec(50) = 25 μm). 24b13 was the most potent compound against adenovirus-2 with an ec(50) value of 6.2 μm, which proved lower than all the reference drugs. preliminary structure–activity relationships were also discussed. to the best of our knowledge, no data are present in the literature on antiviral activity of 3-hydroxy-quinazoline-2,4(1h,3h)-diones against dna-viruses. thus, these findings warrant further investigations (library expansion and compound refinement) on this novel class of antiviral agents. poxvirus-associated diseases are a major threat for human health. 1 smallpox, a highly transmissible and infectious disease with high morbidity and mortality, was the most dangerous human pathogen of poxviruses group. 2 although smallpox was declared eradicated in 1980 by the world health organization (who) after an intensive immunization campaign with the vaccinia virus vaccine of the global, there are stocks of varv were kept in two who-approved laboratories: the u.s. center for disease control and prevention (cdc) in atlanta and the russian state research center of virology and biotechnology in novosibirsk. 3, 4 by the fact that the vaccinia virus vaccines have substantial side effects, 5 the vaccination programs have been terminated since the last century, which led to the human population today more susceptible to a smallpox disaster. in addition, the emergence of zoonotic poxvirus infections such as the monkeypox virus in both the us and western africa in human populations aggravated the people's panic. 6 for all of these reasons, special attention has been paid to establish efficient safe therapies for dealing with poxvirus infections. in spite of number of potential antipoxviral agents have been reported recently, there have no approved drugs by us food and drug administration (fda) for the prevention and treatment of smallpox infections available on the market currently. cidofovir (cdv, 1) is a potent and selective anti-dna virus agent and can inhibit viral dna replication (fig. 1) , so it has a broad-spectrum activities and has been approved for the treatment of smallpox virus. but the low oral bioavailability of cdv and potential nephrotoxicity accompany with its intravenous administration limited the clinical application of the cdv. recently, the lipophilic prodrug of cdv, hexadecyloxypropyl ester (hdp-cdv, 2), was demonstrated that have improved bioavailability and equivalent effectiveness against orthopoxvirus infections and is in phase i/ii clinical studies currently. 7 moreover, tecovirimat (st-246, 3), an orally bioavailable compound that targets the f13l protein of the virus, which inhibit the growth of multiple orthopoxviruses and has significant antiviral activity in various poxvirus disease animal models, was demonstrated favorable safety, tolerability, and pharmacokinetics in phase i clinic trial. 8, 9 in 2010, tecovirimat was received both orphan drug designation and fast-track status from the us fda and with the hope that it can be approved for the prevention and treatment of smallpox infections. adenoviruses (advs) are double-stranded dna viruses (about 60-100 nm in size) with a nonenveloped icosahedral capsid and a genome of 26-45 kb. 9 advs comprise more than 50 human ad serotypes, which have been identified and classified into six species (a-f) in terms of their biological, physiochemical and genetic properties. 10 advs are opportunistic pathogens and associated with a wide variety of severe clinical symptoms in healthy individuals, such as respiratory illness, renal disease, gastroenteritis, hemorrhagic cystitis, and so on. 11-13 however, they are generally not considered to be highly pathogenic viruses for the reason that the adenovirus infections are most often self-limited in immunocompetent individuals. but an adenovirus infection might led to severe and life-threatening disease (multiple organ failure) in the immunocompromised individuals. 14 during the last two decades, a number of adenovirus serotypes that largely from species a, b, and c were isolated from immunocompromised patients successfully. ad2, a species a serotype that has been most detailed studied of adenovirus so far, are often associated with respiratory illness with a lethal outcome occasionally. currently, there have no available drugs for treatment of advs infections. cidofovir was proved to be the most promising anti-adenoviral agent of those currently used in clinical settings, but the outcome in the hematopoietic stem cell recipients has been found to be poor adenovirus infections. 15 therefore, there is compelling need for the discovery of new antiviral drugs of vaccinia and adenovirus that possess an improved safety property and oral bioavailability. over the past few decades, serendipitous or high-throughput screen (hts) campaign of heterocyclic compound collections continues to remain a major paradigm for antiviral hits or leads discovery. 16, 17 due to their chemical and biological importance, hydroxy-(iso)quinazoline-2,4(1,3)-dione and its analogues are attractive 'privileged structures' in antiviral medicinal chemistry. recently, 2-hydroxyisoquinoline-1,3(2h,4h)-dione and 3-hydroxypyrimidine-2,4-dione derivatives were reported as miscellaneous inhibitors targeting bridged dinuclear metalloenzymes, such as hiv rnase h and integrase (in) dual inhibitors 4-7 18 , 8, 9 19 , 10 20 , hiv rnase h active-site inhibitors mb-76 (11) 21-23 and 12 24 , as well as hepatitis c virus (hcv) ns5b polymerase inhibitor 13 25 (fig. 2) , suggesting that these divalent metal ion chelators could be useful inhibitor scaffolds with a broad range of biological activity via various modifications. 26 evolved from the concept of drug repositioning, 'privileged structure'-guided scaffold refining is a very effective strategy to exploit undescribed bioactivites by making full use of readily derivatized scaffolds with well-established synthetic methods. 27 in this context, in view of the above fact and to discover completely new anti-vaccinia and adenovirus agents with a novel skeleton and unique mode of action, a relatively small library of 6-(1-benzyl-1h-1,2,3-triazol-4-yl)-3-hydroxyquinazoline-2,4(1h,3h)-dione compounds (the general formula in scheme 1) was constructed via the copper(i)-catalyzed azidealkyne cycloaddition (cuaac) reaction 28 and the biological significance of the novel synthesized quinazolines was highlighted by evaluating them in cell culturebased antiviral high-throughput screening (hts) assays against a broad panel of dna viruses, retroviruses and several rna viruses. the library of 3-hydroxyquinolin-2(1h)-one compounds was constructed by the following general synthetic route, which was straightforward and depicted in scheme 1. the starting material 2-hydroxyisoindoline-1,3-dione (14) was firstly reacted with benzyl bromide to obtain 2-(benzyloxy)isoindoline-1,3-dione (15) . then 15 was treated with hydrochloric acid and acetic acid via an acidulation reaction to form the key intermediate o-benzylhydroxylamine hydrochloride (16) . 29 meanwhile, the commercially available material 2-amino-5-iodobenzoic acid (17) (19) was obtained by ring closure of 18 with carbonyldimidazole (cdi) and 16 under the condition of sodium hydroxide. 30, 31 then this heterocyclic scaffold was alkylated with iodomethane and iodoethane in dmf afforded the n 1methylation product 20a and n 1 -ethylation product 20b respectively. the key alkyne building block 22a or 22b was prepared from 20a or 20b via the sonogashira cross-coupling reaction and trimethylsilyl-removal reaction successfully. copper(i) catalyzed click reaction (cuaac) of the alkyne key intermediate 22a or 22b with different azido substituent groups generated the corresponding key 1,2,3-triazole intermediates (23a01-23a13 and 23b01-23b13), which were deprotected under basic condition affording two series of target compounds 1,2,3-triazole-substituted 3hydroxy-quinazoline-2,4(1h,3h)-diones 24a01-24a13 and 24b01-24b13. their structures were determined by their 1 h nmr, 13 c nmr, and ms (esi) spectra. notably, the 1 h nmr spectrum showed a singlet at 8.50 corresponding to the triazolyl proton while the 13 c nmr spectrum showed peaks at 125.73-125.90 and 149.12-149.65 corresponding to ch and qc characteristic to the triazole core unit. the newly synthesized 1,2,3-triazole-linked 3-hydroxy-quinazoline-2,4 (1h,3h)-diones were performed to evaluate against their antiviral activity against a broad panel of dna virus, including herpes simplex virus-1 (kos), herpes simplex virus-2 (g), herpes simplex virus-1 tkkos acvr, vaccinia virus and adeno virus-2 (evaluated in infected human embryonic lung fibroblast (hel) cells). in addition, all the compounds were also examined against retroviruses [i.e., human immunodeficiency virus type 1 (hiv-1) and type 2 (hiv-2)] and several rna viruses [i.e., human coronavirus and influenza virus]. the results were expressed as ec 50 and mcc (minimum cytotoxic concentration) ( table 1) . as shown in table 1 hdp-cdv (2) on vaccinia and adenovirus and this study can help to relate the structural characteristics of this complexes to their antiviral activity. preliminary investigation of the structure-activity relationships (sars) revealed that the nature of the n 1 -r substituent and the aryl group which connected to the triazole influenced the antiviral activity remarkably. for instance, the result revealed that the antiviral activity of n 1 -ethyl substituted analogues are more potent than that of the corresponding n 1 -methyl substituted counterparts (ec 50 : 24b07 > 24a07, 24b11 > 24a11 and 24b13 > 24a13). especially, most of n 1 -ethyl substituted analogues showed favorable activity against adenovirus, but nearly all the compounds lost their activity when the compounds with n 1 -methyl substituted counterparts with an exception of compound 24a12. introduction of an electron-withdrawing group at the para substituents of the aryl can remarkable improve the anti-vaccinia activities (no 2 > -cn > f > ch 3 ). to the contrary, a strong electron-withdrawing group can result in the compound lost their adenovirus activities, but the compounds with electrondrawing group (24b13 and 24a13) exhibited the most potent antiviral activity against adenovirus. when comparing compound 24b01 with 24b02, 24b03, 24b06, 24b07 and 24b08, only compound with fluorine substituted in the meta and para position of the aryl simultaneous can display potent inhibitory activity toward vaccinia (24b07, ec 50 = 1.9 lm). but for the adenovirus, compounds with ortho or meta substituted showed moderate inhibitory activity (24b01, ec 50 = 6.5 lm; 24b03, ec 50 = 13 lm), and para-substituted counterpart was inactive (24b02, ec 50 > 100 lm); moreover, changing the pattern of substitution on the aryl to disubstituted resulted in reduced activity (24b06, ec 50 = 8 lm), even completely lost inhibitory activity (24b08 and 24b09, ec 50 > 100 lm). in conclusion, a series of novel 1,2,3-triazole-containing 3hydroxy-quinazoline-2,4(1h,3h)-diones has been synthesized using cuaac reaction, and was firstly identified as potent and specific vaccinia and adenovirus inhibitors in vitro. among them, 24b11 and 24b13 was demonstrated with the most potent vaccinia and adenovirus inhibitory activity respectively, which were promising compounds for further exploration as drug candidates for anti-poxvirus or adenovirus therapy. preliminary sars were discussed with the hope to provide a helpful guidance for the design of next-generation of quinolinone analogues. obviously, the mechanism of action and precise viral target of these 1,2,3-triazolyl 3-hydroxy-quinazoline-2,4(1h,3h)-diones derivatives presented here remain to be identified. consequently, a detailed study on further elaboration of these 3-hydroxy-quinazoline-2,4 (1h,3h)-diones and investigation of their prospective mechanism of action is currently underway in our lab and would be disclosed in due course. the financial support from the national natural science molecular epidemiology of human adenoviruses key: cord-265764-h4zg0q8x authors: singh, kamaljit; kaur, hardeep; chibale, kelly; balzarini, jan title: synthesis of 4-aminoquinoline–pyrimidine hybrids as potent antimalarials and their mode of action studies date: 2013-06-10 journal: eur j med chem doi: 10.1016/j.ejmech.2013.05.046 sha: doc_id: 265764 cord_uid: h4zg0q8x one of the most viable options to tackle the growing resistance to the antimalarial drugs such as artemisinin is to resort to synthetic drugs. the multi-target strategy involving the use of hybrid drugs has shown promise. in line with this, new hybrids of quinoline with pyrimidine have been synthesized and evaluated for their antiplasmodial activity against both cq(s) and cq(r) strains of plasmodium falciparum. these depicted activity in nanomolar range and were found to bind to heme as well as at rich puc18 dna. malaria is one of the most widespread diseases besides tuberculosis and aids which affects more than 500 million people worldwide and results in around 1e3 million causalities every year [1] . in africa alone, around 20% childhood deaths are due to malaria and a child dies every 30 s [2] and it is estimated that an african child has on an average 1.6e5.4 episodes of malaria fever each year. of the four typically recognized plasmodium species causing disease in humans, plasmodium falciparum is most deadly to children below the age of five leading to mortality while plasmodium vivax is most morbidity prone, and is responsible for latent infection that hampers current control and future elimination efforts [3] . the development of drug resistance for the common antimalarials such as 4-/ 8-aminoquinolines, 4-methanol quinolines, antifolate drugs, sesquiterpene lactones etc. (fig. 1) is a rather serious issue which has stimulated considerable research efforts in the development of new drugs using different approaches [4, 5] of which the molecular hybridization approach [6, 7] is quite an attractive strategy which involves design of new chemical entities by covalent fusion of two drugs, both active compounds and/or pharmacophoric units derived from known bioactive molecules with complimentary activities and multiple pharmacological targets. the multiple target strategy led to the design of hybrid of 4-aminoquinoline with species such as triazine [8, 9] , ferrocene [10] , rhodanine [11] , thiazolidine-4-one [12] , chalcone [13] , trioxane [14] , isatin [15] and recently, pyrimidines [16e18] (fig. 2) . quinoline containing drugs (chloroquine and primaquine, fig. 1 ) are known to affect parasite metabolism and cause parasite death by blocking the polymerization of the toxic heme, into an insoluble and non-toxic pigment, hemozoin, resulting in cell lysis and parasite cell auto digestion [19e21] . on the other hand, pyrimidine-based compounds are well known for their wide range of promising antiviral [22] , antitubercular [23] , anti-aids [24] , antinociceptive [25] , antifungal [26] , antitumor [27] and antimalarial activities [28] apart from their role in the nucleic acid synthesis. thus, linking of the quinoline unit with pyrimidine might furnish conjugate hybrids that are capable of showing useful antimalarial activity. recently, antimalarial activities of some quinolineepyrimidine hybrids with activities in the micromolar to nanomolar range have been reported (fig. 3) [16e18 ,29] . in yet another report on the evaluation of quinolineepyrimidine, the activity (in micromolar range) was also reported for fixed combinations of the chloroquine and pyrimethamine. in all these reports, the pyrimidines were linked to the quinoline unit through 2-, 4-and 6-positions. we have employed rather conformationally flexible pyrimidine-5-carboxylates linked covalently to 4-aminoquinoline core. these novel pyrimidine carboxylate hybrids interact with the iron center of free heme within the physiological environment (ph 5.6), a key step in the accumulation of heme which is selectively toxic to the parasite. to enhance the possibility to accumulate within the digestive vacuole via weak-base trapping (the mechanism by which cq and other quinoline antimalarials attain high concentrations inside this compartment), we developed a novel class of antimalarials using a pharmacophore hybridization approach in which the pyrimidine-5-carboxylate motif was hybridized with an iron-complexing, 4-aminoquinoline moiety through c-2 position [29] . to further elaborate the structureeactivity profile, here, we present additional new 4-aminoquinolineepyrimidine carboxylate hybrids. we also report on their antimalarial activity against both cq sensitive (cq s dd2) as well as cq resistant (cq r d10) strains. finally, the mechanism of action studies with the representative compounds has also been performed. the 4-aminoquinolineepyrimidine-5-carboxylate hybrids were synthesized in economical way using synthetic approach outlined in scheme 1. the key starting compound, 3,4-dihydropyrimidin-2(1h)-one 1 was prepared through nh 4 cl/tfa [30, 31] catalyzed three-component biginelli condensation of an alkyl acetoacetate, urea and appropriate aldehyde or its formyl equivalent: 1,3oxazinane derivative, in acetonitrile or under solvent-free reaction conditions, in some cases. subsequent oxidation of 1 using 60% nitric acid readily furnished pyrimidinones 2 which upon chlorination with pocl 3 yielded 3 [32] . the nucleophilic substitution reaction of 3 with appropriate 4-amino-7-chloroquinoline 4 which in turn was prepared from the commercially available 4,7dichloroquinoline and diaminoalkanes [33] , gave 5aeg in good yields ( table 1) . structures of 1e5 were unambiguously established on the basis of spectral ( 1 h nmr, 13 c nmr, ms, ft ir) as well as microanalytical analysis. we have previously established that the 4-aminoquinolinee pyrimidine hybrids 6aec intercepted by a diaminoalkyl spacer showed optimum potency (table 1) , when the flexible spacer consisted of three or four carbon atoms [29] . further, the introduction of nitro substituent at ortho position of the phenyl ring at the c-4 of the pyrimidine core furnished the most potent compound 6c with antimalarial activity superior to the standard cq and close to artesunate [29] . keeping these observations in mind, we planned to further refine the activity of these persuasive 4aminoquinolineepyrimidine by incorporating electron withdrawing substituents at c-4 phenyl of pyrimidine core, as well as by varying the c-5 ester substituent and also by altering the basicity of diaminoalkyl spacer. the in vitro antimalarial screening of the new synthesized compounds 5aeg revealed good to moderate activities in nm range against both the tested dd2 (cq s ) and d10 strains (cq r ) of p. falciparum (table 1) . although the tested hybrids were not as active as the standard drugs viz. cq and asn, interesting sars have been drawn. analysis of the activity of the compounds recorded in table 1 reveals that replacing c-5 ethyl ester of the previously reported [29] decrease in antimalarial activity against both the chloroquine sensitive and chloroquine resistant strains of p. falciparum. however, comparison of hybrids 5b, 5c with 6b, 6c having an identical butyl spacer showed that incorporation of isopropyl ester at c-5 of pyrimidine motif (5b and 6b) increased the antimalarial activity against the cq s strain whereas considerable decrease in activity was observed for cq r strain of p. falciparum. also, the most potent compound 5b of the series displayed 2-fold increase in antimalarial activity than the standard cq against cq r strain of p. falciparum. when the diaminoalkyl linker of compound 6a was replaced with relatively less basic alkoxy amino linker 5d considerable decrease in antimalarial activity was observed which in turn linked to the decreased accumulation of compound via ph trapping into the digestive vacuole. it was not unexpected since the basicity of alkyl chain linker plays crucial role in determining the antimalarial activity of this class of compounds. furthermore, the introduction of a nitro substituent on the phenyl ring at the c-4 position of the pyrimidine core to create 5c resulted in a significant increase in antiplasmodial activity in comparison to the p-chloro/p-fluoro substituents (5e and 5f). moreover, the hybrid 5g lacking a c-4 substituent on the pyrimidine motif led to an increase in antimalarial activity against both the cq s as well as cq r strains of p. falciparum. however, although the antimalarial activity of 5g was superior to 5c, 5e & 5f, it was less than the corresponding c-4 phenyl substituted analogs 5b as well as 6b. thus, the sar study suggested that both the substitution of the c-4 phenyl group with electron withdrawing groups and alterations in basicity of linker leads to better antimalarial activity. unfortunately, these compounds suffer from high clogp values which are in the range 5e8 (table 1) , which are suggestive of the fact that these possess limited aqueous solubility. however, it is not a serious limitation in view of recent advancements in formulation methods. compounds 5aeg were evaluated for their toxicity against various (hela, vero, crfk, hel and mdck) cell cultures (table 1 & si table s1 ). toxicity data revealed that these compounds exhibit high toxicity (low cc 50 ) against mdck cell cultures while cc 50 values are quiet high for other cell cultures. the cc 50 values for inhibition of mdck cells summarized in table 1 indicate that the strongest antimalarial compound 5b was mildly cytotoxic (table 1) . further, the ratio of the cytotoxicity (cc 50 in mm) and in vitro antimalarial activity (ic 50 in nm for dd2 strain) enabled the determination of selectivity index (si) for these compounds. compound 5d with alkoxy amino linker and compound 5g bearing c-4 unsubstituted pyrimidine motif exhibited high cc 50 values and thus led to fairly safe selectivity index values (table 1) . compound 5d having less basic alkyl spacer, displayed highest si (43.6) whereas the most potent compound 5b exhibit relatively low si value (3.92 table s1 ) and (vii) feline corona virus (fipv) and feline herpes virus activity in crfk cell cultures ( table 2 ). the anti-viral activity of most of the compounds was not impressive except compounds 5a and 5c which exhibited relatively low ec 50 's only against the feline corona virus (fipv) and feline herpes virus in crfk cell cultures ( table 2) . quinoline antimalarials (e.g., cq, amodiaquine and quinine) act principally by forming adducts with ferriprotoporphyrin ix, thus blocking haemozoin formation [38] . in this study, we have evaluated the mechanism of antimalarial activity of the most potent compound 5b of the series by studying its binding with heme (fig. 4) . the titration of monomeric heme was also performed at the plasmodial food vacuole ph 5.6 using mes buffer instead of hepes to ensure that the compound 5b binds with heme even at acidic ph (s1). a 1:1 stoichiometry of the most stable complex of 5b with monomeric heme at ph 7.4 and 5.6 was established from the job's plot (si figure s1 ). the association constants (table 3) were calculated by analyzing the titration curves obtained at ph 7.4 using hypspecea non-linear least square fitting programme [39] . the binding of cq with heme under identical conditions was also determined in the similar manner and the results are presented in table 3 for comparison. table 3 shows that the association constants for the complexes formed between monomeric heme and 5b (log k 4.96) are comparable with those of standard antimalarial drug, cq (log k 5.15). furthermore, the decrease of apparent ph from 7.4 to 5.6 ( table 3) has little effect on the binding constants indicating binding is stronger even at acidic ph. to further establish the binding of 5b with monomeric heme, 1 h nmr titrations were performed and shifts in the peaks as well as peak intensity noted. the addition of 30 mol% of heme dissolved in 40% dmso to a solution of 5b in 40% dmso:d 2 o\d 2 so 4 (10 ml) caused a shift in the aromatic proton signals (fig. 5) , indicating binding of 5b with heme but further addition of heme led to broadening of the peaks. an equimolar (3.9 mmol) solution of hemin chloride and 5b when analyzed in mass spectrometer depicted an intense molecular ion peak at 1119.3769 da (fig. 6a) , corresponding to the molecular formula c 62 h 62 clfen 9 o 6 , suggesting the formation of 1:1 complex. thus, we propose that 5b interacts with heme by replacing chloride atom of hemin chloride and coordinating the iron atom with its endocyclic quinoline nitrogen as proposed in fig. 6b . similar titration of dimers of m-oxo type (10 mm) at ph 5.8 using standard procedure [29] with increasing concentration of compound 5b (0e14 mm), resulted in decrease in intensity of broad peak at 362 nm (fig. 7a, s1) . further, job's plot calculations indicated a 1:1 stoichiometry for the most stable m-oxo: 5b complex (fig. 7b) . in table 3 the association constants of 5b (log k 5.72) are compared to that of standard cq (log k 5.58) and also suggests that the binding of 5b is stronger with m-oxo heme (log k 5.72) than monomeric heme (log k 4.96). thus, the compound 5b inhibits hemozoin formation by blocking the growing face of heme resulting in the observed antimalarial activity. furthermore, the b-hematin inhibition assay (si table s2 ) shows that there is no correlation between antimalarial activity and b-hematin inhibition and also, all the compounds inhibit b-hematin formation although less than that of standard cq. the mechanism of many antimalarial drugs such as cq, quinacrine and quinine relies upon the interaction with dna presumably through ionic interactions between phosphate groups of dna and protonated amine in addition to the interactions between aromatic nuclei of the drug with nucleotide bases [40, 41] . therefore, the dna binding properties of 4-aminoquinolineepyrimidine hybrids have been evaluated using both the uvevisible spectrophotometer and fluorescence spectrophotometry in order to probe interaction of these compounds with dna. the addition of ct-dna (4e200 mm) to the buffered methanolic solution of 5b (30 mm) induced hyperchromic shift of 112% in absorption band at 255 nm whereas hypochromic shift of 37% in the characteristic quinoline ring absorption at 330 nm (fig. 8) . also, the bathochromic shift of w3 nm was observed for both the absorptions. the observed hyperchromic as well as hypochromic shifts in absorption bands of 5b upon addition of dna results from the intercalation of 5b with ct dna as suggested in the literature [42] . the intercalative nature of interaction of compound 5b with ct dna was additionally supported by thermal denaturation experiment. intercalation of molecules into the double helix is known to stabilize the dna against thermal strand separation and thus increases thermal melting temperature (t m ) [43, 44] . the derivative melting curve presented in fig. 9 shows an increase of 7.5 c in thermal melting temperature of ct dna upon addition of 5b which is less than that observed for the cq (table s3) . thus, both the uvevisible titrations and thermal denaturation experiment advocate partial intercalative nature of interactions between compound 5b and ct dna. further, to visualize the effect of dna base composition, the fluorescence titrations of 5b were performed with both gc-rich ct dna and at-rich puc18 dna in buffered methanol. fig. 10 shows decrease in the intensity of the emission band of 5b at 380 nm, upon addition of increasing concentration of both the dnas. a shift of 80 nm in emission band at 380 nm was observed upon addition of ct dna but no such shift in emission band was observed for puc18 dna. comparison of binding constant of 5b with ct dna (log k 5.76) and puc18 dna (log k 5.73) calculated from titration data using hypspec [39] , suggest that 5b does not discriminate between gc rich dna and at rich dna. a series of potent 4-aminoquinolineepyrimidine hybrids with antimalarial activity in nanomolar range were reported. the compound 5b exhibits lowest ic 50 value within the series against both cq s and cq r strains of p. falciparum. these hybrids displayed mild toxicity against mdck cell cultures. the antiviral activity profiles of these hybrids indicate that the compound 5a and 5c effectively inhibit feline corona virus and feline herpes virus. further, the mechanism of observed antimalarial activity was established in terms of binding with heme as well as dna. all liquid reagents were dried/purified following recommended drying agents and/or distilled over 4 a molecular sieves. thf was dried (na-benzophenone ketyl) under nitrogen. 1 h nmr (300 mhz) and 13 c (75 mhz) nmr spectra were recorded in cdcl 3 on a multinuclear jeol ft-al-300 spectrometer with chemical shifts being reported in parts per million (d) relative to internal tetramethylsilane (tms, d 0.0, 1 h nmr) or chloroform (cdcl 3 , d77.0, 13 c nmr). mass spectra were recorded at department of chemistry, guru nanak dev university, amritsar on a bruker lc-ms microtof ii spectrometer. elemental analysis was performed on flash ea 112 (thermo electron corporation) analyzer at department of chemistry, guru nanak dev university, amritsar and the results are quoted in %. ir spectra were recorded on perkin elmer ftir-c92035 fourier transform spectrometer in the range 400e4000 cm à1 using kbr pellets. melting points were determined in open capillaries and are uncorrected. for monitoring the progress of a reaction and for comparison purpose, thin layer chromatography (tlc) was performed on pre-coated aluminum sheets of merck (60f 254 , 0.2 mm) using an appropriate solvent system. the chromatograms were visualized under uv light. for column chromatography silica gel (60e120 mesh) was employed and eluents were ethyl acetate/ hexane or ethyl acetate/methanol mixtures. the steady state fluorescence experiments were carried out on perkin elmer ls55 fluorescence spectrometer at ambient temperature. uvevisible spectral studies were conducted on shimadzu 1601 pc spectrophotometer with a quartz cuvette (path length, 1 cm). the absorption spectra have been recorded between 1100 and 200 nm. the cell holder of the spectrophotometer was thermostated at 25 c for consistency in the recordings. to the stirred solution of 3 (2 mmol) and potassium carbonate (5 mmol) in dry thf (30 ml), a solution of appropriate 4aminoquinoline 4 (1.0 mmol) in dry thf (50 ml) was added. the reaction mixture was stirred for 48 h at room temperature. the reaction mixture was filtered and thf was removed under vacuum. the residue was purified by column chromatography using meoh/ ethyl acetate as eluent to obtain corresponding 5, which was recrystallized from dcm/hexane. using this procedure the following compounds were isolated. (for optimized structure of 5b, see figure s2 ). to the stirred solution of appropriate 4-aminoquinoline 4 in dry acetonitrile (50 ml) mixture of 3 (in a 1:2 molar ratio) and potassium carbonate in dry acetonitrile was added. the reaction mixture was refluxed for 24 h and then filtered. acetonitrile was removed under vacuum and the residue was purified by column chromatography using meoh/ethyl acetate as eluent to give 5 which is recrystallized from dcm/hexane. 6. material and methods the test samples were tested in triplicate on one or two separate occasions against chloroquine sensitive (cq s ) strain of p. falciparum (d10). continuous in vitro cultures of asexual erythrocyte stages of p. falciparum were maintained using a modified method of trager and jensen [45] . quantitative assessment of antiplasmodial activity in vitro was determined via the parasite lactate dehydrogenase assay using a modified method described by makler et al. [46] . the test samples were prepared to a 20 mg/ml stock solution in 100% dmso and sonicated to enhance solubility. samples were tested as a suspension if not completely dissolved. stock solutions were stored at à20 c. further dilutions were prepared on the day of the experiment. chloroquine (cq) was used as the reference drug in all experiments. test samples were initially tested at three concentrations (10 mg/ml, 5 mg/ml and 2.5 mg/ml) to determine the starting concentration for the full doseeresponse assay. cq was tested at three concentrations namely 30 ng/ml, 15 ng/ml and 7.5 ng/ml. a full doseeresponse was performed for all compounds to determine the concentration inhibiting 50% of parasite growth (ic 50 -value). test samples were tested at a starting concentration of 10 mg/ml, which was then serially diluted 2-fold in complete medium to give 10 concentrations; with the lowest concentration being 0.02 mg/ml. the same dilution technique was used for all samples. cq was tested at a starting concentration of 1000 ng/ml. several compounds were tested at a starting concentration of 1000 ng/ml. the highest concentration of solvent to which the parasites were exposed to had no measurable effect on the parasite viability (data not shown). the ic 50 -values were obtained using a non-linear doseeresponse curve fitting analysis via graph pad prism v.4.0 software. cytotoxicity was determined by exposing different concentrations of samples to vero, hel, hela and mdck cells [29] . the antiviral assays were based on inhibition of virus-induced cytopathicity in hel [herpes simplex virus type 1 (hsv-1), hsv-2 (g), vaccinia virus, and vesicular stomatitis virus], vero (parainfluenza-3, reovirus-1, coxsackie b4, and punta toro virus), hela (vesicular stomatitis virus, coxsackie virus b4, and respiratory syncytial virus) and mdck (influenza a (h1n1; h3n2) and b virus) cell cultures. confluent cell cultures in microtiter 96-well plates were inoculated with 100 cell culture inhibitory dose-50 (ccid 50 ) of virus (1 ccid 50 being the virus dose to infect 50% of the cell cultures) in the presence of varying concentrations of the test compounds. viral cytopathicity was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds [29] . shrinking the malaria map: progress and prospects thousands of chemical starting points for antimalarial lead identification artemisinin resistance in plasmodium falciparum malaria malaria chemotherapeutics, part i: history of antimalarial drug development, currently used therapeutics, and drugs in clinical development hybrid molecules with a dual mode of action: dream or reality? next-generation antimalarial drugs: hybrid molecules as a new strategy in drug design synthesis, antimalarial activity and cytotoxicity of 4-aminoquinolineetriazine conjugates synthesis of 4-aminoquinoline-1,2,3-triazole and 4-aminoquinoline-1,2,3-triazole-1,3,5-triazine hybrids as potential antimalarial agents synthesis and antimalarial activity in vitro and in vivo of a new ferroceneechloroquine analogue synthesis and biological evaluation of a new class of 4-aminoquinolineerhodanine hybrid as potent anti-infective agents synthesis and antimalarial activity of side chain modified 4-aminoquinoline derivatives enone-and chalconeechloroquinoline hybrid analogues: in silico guided design, synthesis, antiplasmodial activity, in vitro metabolism, and mechanistic studies preparation and antimalarial activities of trioxaquines, new modular molecules with a trioxane skeleton linked to a 4-aminoquinoline design, synthesis and anti-plasmodial evaluation in vitro of new 4-aminoquinoline isatin derivatives novel 4-aminoquinolineepyrimidine based hybrids with improved in vitro and in vivo antimalarial activity synthesis, characterization and antimalarial activity of quinolineepyrimidine hybrids substituted quinolinyl chalcones and quinolinyl pyrimidines as a new class of anti-infective agents falcipain-2 inhibitors quinoline antimalarial: mechanisms of action and resistance discovery of dual function acridones as a new antimalarial chemotype synthesis and antiviral activity of the alpha-analogues of 1,5-anhydrohexitol nucleosides facile transformation of 3,4-dihydropyrimidin-2(1h)-ones to pyrimidines in vitro evaluation as inhibitor of mycobacterium tuberculosis and modulators of cytostatic activity optimization of an anti-hiv hairpin ribozyme by in vitro selection adenosine kinase inhibitors. synthesis, water solubility, and antinociceptive activity of 5-phenyl-7-(5-deoxybeta-d-ribofuranosyl) pyrrolo[2,3-d]pyrimidines substituted at c4 with glycinamides and related compounds the synthesis of substituted pyridylpyrimidine fungicides using palladium catalysed crosscoupling reactions nucleoside analogues and nucleobases in cancer treatment synthesis and antiplasmodial activity of novel 2,4-diaminopyrimidines 2-aminopyrimidine based 4-aminoquinoline anti-plasmodial agents. synthesis, biological activity, structureeactivity relationship and mode of action studies ammonium chloride-catalyzed one-pot synthesis of 3,4-dihydropyrimidin-2-(1h)-ones under solvent-free conditions an expedient protocol of the biginelli dihydropyrimidine synthesis using carbonyl equivalents on the reaction of 3,4-dihydropyrimidones with nitric acid. preparation and x-ray structure analysis of a stable nitrolic acid chloroquine analogues: influence of side chain length and quinolyl nitrogen pk a on activity vs chloroquine resistant malaria treatment of chronic active hepatitis b (cah b) with chloroquine: a preliminary report inhibition of human coronavirus 229e infection in human epithelial lung cells (l132) by chloroquine: involvement of p38 mapk and erk inhibition of human immunodeficiency virus infectivity by chloroquine mechanism of enhancement of the antiviral action of interferon against herpes simplex virus-1 by chloroquine malarial haemozoin/betahaematin supports haem polymerization in the absence of protein investigation of equilibria in solution. determination of equilibrium constants with the hyperquad suite of programmes chloroquine as intercalator: a hypothesis revived intercalating drugs: dna binding and molecular pharmacology cytotoxic activity, cell imaging and photocleavage of dna induced by a pt(ii) cyclophane bearing 1,2 diamino ethane as a terminal ligand spectrophotometric studies of the interaction of chloroquine with deoxyribonucleic acid spectroscopic studies on the thermodynamic and thermal denaturation of the ct-dna binding of methylene blue human malaria parasite in continuous culture parasite lactate dehydrogenase as an assay for plasmodium falciparum drug sensitivity we gratefully acknowledge financial assistance from csir, new delhi (project 01(2364)/10/emr-ii) and ugc, new delhi for special assistance programme (sap). h.k. thanks csir, new delhi for senior research fellowship. jb thanks ku leuven for financial support (goa 10/14). supplementary material associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ejmech. 2013.05.046. key: cord-321985-w84wwkgi authors: xing, shihua; wang, mengyue; peng, ying; chen, daofeng; li, xiaobo title: simulated gastrointestinal tract metabolism and pharmacological activities of water extract of scutellaria baicalensis roots date: 2014-02-27 journal: j ethnopharmacol doi: 10.1016/j.jep.2013.12.056 sha: doc_id: 321985 cord_uid: w84wwkgi ethnopharmacological relevance: . scutellaria baicalensis: georgi (labiatae) is a well-known traditional chinese medicine to treat inflammation, cardiovascular diseases, respiratory and gastrointestinal infections, etc. the present study was to understand the metabolism of the root of scutellaria baicalensis (a.k.a. huangqin in chinese) in the gastrointestinal tract and the correlation between the metabolites and their respective pharmacological activities. materials and methods: the water extract of the root of scutellaria baicalensis (wesb) was incubated with simulated gastric and intestinal juices, and human fecal microflora for 24 h at 37 °c. the hplc–dad analysis was used to monitor the in vitro metabolic process and identify its metabolites by comparing their absorption spectrum and retention time with those of chemical references. the in vitro anticomplementary and antimicrobial activity was evaluated with hemolysis assay, agar disc-diffusion method and mic value, respectively. results: main constituents of wesb remain unchanged during the incubation with simulated gastric juice (ph=1.5) and intestinal juice (ph=6.8), whereas four flavones, baicalin, wogonoside, oroxyloside and norwogonoside were metabolized into their respective aglycons by human intestinal bacteria. all four metabolites were demonstrated to have higher anticomplementary and antimicrobial activity than those of wesb. the anticomplementary active metabolites were identified to be baicalein, oroxylin a and norwogonin, among them, norwogonin is the most active compound. conclusion: the presence of intestinal bacteria is demonstrated to play an important role in the gastrointestinal metabolism of wesb, and the pharmacological effects of scutellaria baicalensis may be dependent on the intestinal bacteria metabolism. the root of scutellaria baicalensis georgi (labiatae), also known as huangqin, is a widely used herb in traditional chinese medicine (tcm) with anticancer, antiviral, antibacterial and antiinflammatory properties (yoon et al., 2009) . traditionally, huangqin has been prescribed as a diuretic, laxative, febrifuge, an antipyretic, and for hemoptysis, bloody stool, and nasal haemorrhage when prescribed in a compound recipe (the pharmacopoeia commission of prc, 2010). remarkably, huangqin was recommended for the treatment and prevention of severe acute respiratory syndrome (sars) by the state tcm administration of the people 0 s republic of china (miller et al., 2005; zhang and chen, 2008) . huangqin was found to exert anti-inflammatory, antioxidant, anti-hepatitis b virus, anti-tumor, anti-allergic and anxiolytic properties (li et al., 2009; tong et al., 2012) . the main active constituents of huangqin are flavonoids, such as baicalin (baicalein-7-glucuronide), wogonoside (wogonin-7-glucuronide), baicalein, wogonin, oroxylin a and oroxyloside (oroxylin a-7glucuronide) (fig. 1 ). among these flavonoids, baicalin is regarded as the most important determinants of the quality of huangqin (yuan et al., 2011) . due to its orally administered as decoctions in tcm, the metabolism of the constituents often occurs in the gastrointestinal tract caused by the low gastric ph conditions, as well as the presence of digestive enzymes or intestinal bacteria (akao et al., 1994) . it was found that baicalin was initially hydrolyzed to baicalein prior to the absorption, while baicalein could be readily absorbed with a fast and extensive first pass metabolism (akao et al., 2000; lu et al., 2007) . wogonoside was found to be metabolized to wogonin by human fecal microflora (trinh et al., 2009 ). in addition, pharmacokinetic study of huangqin-tang decoction indicated that the constituents of wogonoside and oroxyloside had double-site absorption kinetics in rats which may due to the enteric circulation and enterohepatic circulation after oral dosing (zuo et al., 2003) . so far, there is no report on the metabolism of huangqin in gastrointestinal tract. in addition, most studies on bioactivity of huangqin were focused on antibacterial and anti-endotoxin activities but not anticomplementary activity. in this study, the water extract of huangqin, the root of scutellaria baicalensis (wesb) was incubated with simulated gastric and intestinal juices, and human fecal microflora, their metabolites were identified and the in vitro anticomplementary activity was evaluated. the inhibitory activity of main compounds in huangqin as well as their metabolites against the standard strains of methicillin-resistant staphylococcus aureus (mrsa), methicillin sensitive staphylococcus aureus (mssa), enterococcus faecalis (gram-positive bacteria), escherichia coli, pseudomonas aeuroginosa (gram-negative bacteria) was determined to understand the role of gastrointestinal tract conditions on pharmacological effects of huangqin. the roots of scutellaria baicalensis (huangqin) were purchased from leiyunshang drugstore (shanghai) and authenticated by one of the authors mengyue wang. voucher specimen (hn001) has been deposited at herbarium of school of pharmacy, shanghai jiao tong university, shanghai, china. pepsin (1: 250) and pancreatin were purchased from sangon biotech co., ltd (shanghai, china). general anaerobic medium broth (gam broth) was purchased from shanghai kayon biological technology co., ltd (shanghai, china). sheep erythrocytes, rabbit erythrocytes and anti-sheep erythrocyte antibody were purchased from shanghai fortune biological technological co., ltd (shanghai, china). heparin (sodium salt, 160 iu/mg) and macroreticular resin (hz-801) were purchased from china national medicines co., ltd (shanghai, china). normal human serum was obtained from healthy adult donors. guinea pigs serum was obtained from healthy guinea pigs from laboratory animal research center of fudan university. mrsa (atcc33591), mssa (atcc25923), enterococcus faecalis (atcc29212), escherichia coli (atcc8739), and pseudomonas aeuroginosa (atcc27853) provided by dr. xiuping qian 0 s lab. reference substances: baicalein, baicalin, wogonin, wogonoside, oroxylin a, oroxyloside were purchased from shanghai winherb medical technology co., ltd.; norwogonin was purchased from j&k chemical ltd.; norwogonoside was isolated from huangqin in our laboratory. all lab-made compounds were characterized by their spectra data, which were in accordance with references. all other chemicals and reagents were of analytical grade. dried huangqin (30 g) was decocted twice with a ten-fold mass of water to boil 1 h. after filtrated, the two filtrates were combined and then concentrated to 30 ml (equivalent to 1 g (crude drug)/ml). the hplc-dad analysis of wesb was conducted (fig. 2) . peaks 1-8 were confirmed by comparing the retention times and uv spectra with reference standards, and attributed to baicalin, norwogonoside, oroxyloside, wogonoside, norwogonin, baicalein, wogonin and oroxylin a, respectively. noted that norwogonoside was isolated from huangqin for the first time. a 0.4 ml wesb was placed in 5 ml simulated gastric juice (0.08 m hcl containing 50 mg pepsin, ph 1.5) and incubated at 37 1c for 0, 1, 2, 4 h, and the reactions were stopped by water saturated n-butanol immediately. the mixture was centrifuged at 4800 rpm for 20 min, and then the supernatant was evaporated. the residue was dissolved in 0.5 ml methanol, filtered through a 0.22 μm membrane and analyzed by hplc. repeat above all steps of experiment once more. a 0.4 ml wesb was placed in 5 ml simulated intestinal juice (0.05 m kh 2 po 4 containing 50 mg pancreatin, ph 6.8) and incubated at 37 1c for 0, 2, 4, 6 h. the treatment of samples was same to the simulated gastric juice. fresh fecal samples, obtained from five healthy subjects, were immediately homogenized with 25 volumes of gam broth. the sediments were removed by filtration through three pieces of gauze. the suspension was incubated at 37 1c in an anaerobic incubator in which the air had been replaced with a gas mixture (h 2 5%, co 2 10%, n 2 85%). a 1 ml wesb was added to human fecal suspension (100 ml) and the mixture was incubated at 37 1c in an anaerobic incubator for 24 h. the cultured mixture was respectively taken out and extracted with water saturated n-butanol. the extract was evaporated, and then the residue was dissolved in methanol (0.5 ml) and analyzed with hplc-dad. the hplc system consisted of a multi-solvent delivery pump (agilent 1200, usa), equipped with a quaternary solvent delivery system, an auto sampler and a dad detector. waters xterra reversed-phase column (particle size 5 μm, 4.6 mm â 250 mm i.d., agilent ltd., usa) were used. the purity of compounds and metabolic samples were analyzed under chromatographic condition as follows: acetonitrile (a)-water-phosphoric acid (100: appropriately. the addition of acid into the mobile phase was carried out in order to improve the peak shapes of analysis. uv absorption was monitored at 280 nm. the column temperature was set at 35 1c. the flow rate was 1.0 ml/min and sample injection volume was 10 μl. the dried huangqin (2.0 kg) were extracted for three times with 75% ethanol by heating reflux for 1.5 h each time, with the solvent removed under reduced pressure. the 75% ethanolic extract was suspended in water, and then purified with a column of macroreticular resin (column, 7.0 cm â 65 cm) washed with distilled water, 30% ethanol, 60% ethanol and 90% ethanol. the 60% ethanol soluble fraction (50 g) were firstly separated by silica gel column chromatography (12.0 cm â 70 cm, eluted with a gradient system of methanol and chloroform), and then by sephadex lh-20 column (2.5 cm â 70 cm, eluted with methanol) to obtain norwogonoside. 1 h and 13 c spectra data agree with those reported in the literature (shibata, 1988) . norwogonin-7-o-glucuronide yellow powder, the absorbance maximum (λ max ) of uv absorption is at 246, 285 and 357 nm in methanol, suggesting the compound to be a flavonoid. 1 h nmr (500 mhz, dmso-d6) δ: 12.23 (1h, s, 5-oh), 8.10-8.15 (2h, m, h2 0 , 6 0 ), 7.58-7.64 (3h, m, h3 0 , 5 0 ), 7.02 (1h, s, h-3), 6.65 (1h, s, h-6); 13 c nmr (125 mhz, dmso-d6) δ: 182.64 (c-4), 163.58 (c-2), 158.06 (c-7), 152.21 (c-9), 151.47 (c-5), 132.32 (c-4 0 ), 130.58 (c-1 0 ), 127.76 (c-8), 129.12 (c3 0 , 5 0 ), 126.56 (c-2 0 , 6 0 ), 105.13 (c-3), 105.56 (c-10), 99.36 (c-6). based on mayer's (1961) modified method, sensitized erythrocytes (ea) were prepared by incubation of sheep erythrocytes with rabbit anti-sheep erythrocyte antibody in gvb 2 þ (veronal buffer saline containing 0.1% gelatin, 0.5 mm mg 2 þ and 0.15 mm ca 2 þ ). the compounds and heparin sodium, used as reference, were dissolved in gvb 2 þ . guinea pigs serum was used as the complement source. the 1:40 diluted guinea pigs serum was chosen to give sub maximal lysis in the absence of complement inhibitors. first, various dilutions of tested samples (100 μl) were preincubated with 100 μl guinea pigs serum and 200 μl gvb 2 þ at 37 1c for 10 min. then, 200 μl ea was added and the mixture was incubated at 37 1c for 30 min. the different assay controls were incubated in the same conditions: (1) vehicle control, 200 μl ea in 400 μl gvb 2 þ ; (2) 100% lysis control, 200 μl ea in 400 μl water; (3) sample control, 100 μl dilution of each sample in 500 μl gvb 2 þ . the reaction mixture was centrifuged immediately. optical density of the supernatant was measured at 405 nm with a spectrophotometer (thermo, varioskan flash). the absorbance of sample (a 1 ), sample control (a 0 ) and 100% lysis control (a) were recorded. according to the method of klerx et al. (1983) , each sample was dissolved in gvb-mg-egta (veronal buffer saline containing 0.1% gelatin, 2.5 mm mg 2 þ and 8 mm ethylene glycol tetraacetic acid), and various dilutions were made. after pre-incubation of dilutions of each sample (150 μl) with 1:10 diluted normal human serum (150 μl) in enzyme label plate at 37 1c for 10 min, 200 μl rabbit erythrocytes (er) were added, followed a second incubation step at 37 1c for 30 min. after incubation, the resulting mixture was centrifuged immediately, and the optical density of the supernatant was measured at 405 nm. the inhibition rate of haemolysis was calculated by the following formula: a àða 1 à a 0 þ=a â 100%, where a, a 1 and a 0 represented the absorbance of 100% lysis control, the sample and the sample control, respectively. the ch 50 (concentrations resulted in 50% inhibition of sheep erythrocytes) or ap 50 (concentrations that resulted in 50% hemolysis inhibition of rabbit erythrocytes) value was obtained by plotting the hemolysis inhibition percentage against the logarithm of sample concentrations (lg c). heparin sodium salt was used as the positive control. 2.9. antibacterial activity by the agar disc-diffusion method and determination of minimal inhibitory concentration (mic) the antibacterial activity was carried out on wesb, metabolic sample incubated 24 h, the blank of intestinal bacterial liquid and eight flavonoids from huangqin with five test bacterial strains, and all of samples were three replicates. the positive controls were ampicillin for gram-positive bacteria, gentamycin for gramnegative bacteria and negative control was sterile distilled water. the antimicrobial effect of all samples was in petri dishes with 20 ml of nutrient agar plus 0.2 ml of microorganism suspension (10 8 cfu/ml, o.d 0.1, λ¼590 nm). once the agar had solidified, 200 ml of each sample and sterile distilled water (negative control) was added to wells of 3 mm diameter. the plates were incubated at 37 1c for 24 h, and the inhibited halos were evaluated (mm). the antibacterial effect was determined by measure of inhibited halos formed around the wells (murray et al., 1995; boussaada et al., 2008) . the mic value was determined by the dilution method according to national committee for clinical laboratory standards (1985) . the active samples and positive controls were dissolved in dimethylsulphoxide at a concentration of 2000 mg/ml. two fold serial dilutions of the solution were then prepared (2000, 1000, 500, …, 3.9 mg/ml). inoculated nutrient broth with 1% test bacteria strains (10 8 cfu/ml, o.d 0.1, λ¼590 nm) was prepared. ten concentrations of each sample â three test bacterial strains â three wells as repetitions were carried out in the sterile round-bottom 96-well plates by comparison of the sample with the non-inoculated nutrient broth. in each well, 0.9 ml inoculated nutrient broth plus 0.1 ml sample. plates were incubated at 37 1c for 24 h. the mic values were determined as the lowest concentration that inhibited visible growth of the bacterial as detected by unaided eye. wesb was found to be stable in simulated gastric juice (ph¼1.5) and intestinal juice (ph ¼6.8). after incubated with artificial juices, main constituents of wesb remained unchanged based on the comparison of peak areas of individual constituents during the course of incubation (rsd o3.0%). wesb was anaerobically incubated for 24 h with a bacterial mixture from human feces. the content changes of eight ingredients in wesb were analyzed at incubation time points of 0, 12 and 24 h. (fig. 3) . according to the content changes as well as the chemical structure of the eight ingredients in wesb, we concluded that four flavone glycosides, baicalin, wogonoside, oroxyloside and norwogonoside were converted to their respective aglycons, i.e. baicalein, wogonin, oroxylin a and norwogonin by intestinal bacteria. the effect on activation of guinea pig complement inhibition through the classical pathway was examined in 1:40 diluted guinea pig serum and heparin sodium was used as the positive control. the percent of activation that 1:40 diluted guinea serum occurred in the classical pathway was 98.34 75.23% in the complement control group. the percentage of activation that 1:10 diluted normal human serum occurred in the alternative pathway was 97.26 77.85% in the complement control group. wesb showed no anticomplementary effect through the classical pathway or alternative pathway. the ch 50 and ap 50 values of the metabolic samples at 24 h were both smaller than those of the blank of intestinal bacterial liquid as shown in fig. 4 . it indicated that the metabolic sample is more effective than the blank of intestinal bacterial liquid in complement inhibition. these results suggested that anticomplementary metabolites may be generated during the metabolic process. none of the flavonoid glycosides, baicalin, wogonoside, oroxyloside and norwogonoside, showed anticomplementary activities through the classical or alternative pathway, however, their respective metabolites, baicalein (ch 50 ¼0.18270.008 mg/ml; ap 50 ¼0.2437 0.013 mg/ml), oroxylin a (ch 50 ¼0.53670.032 mg/ml; ap 50 ¼ 0.83570.043 mg/ml) and norwogonin (ch 50 ¼0.13870.004 mg/ml; ap 50 ¼ 0.14770.008 mg/ml) did display an interesting anticomplementary activity through both classical and alternative pathways, while wogonin had no anticomplementary activity. remarkably, norwogonin demonstrated significant activity comparable to the positive inhibitor (heparin sodium, ch 50 ¼0.05670.005 mg/ml; ap 50 ¼0.07570.011 mg/ml). the primary antimicrobial screening was carried out using the agar disc-diffusion. as showed in fig. 5 , the wesb (1.25 mg/ml) had no inhibitory effect against any bacteria tested. the metabolic samples incubated for 24 h exhibited better antibacterial activity than the blank of intestinal bacterial liquid. specifically, baicalin, baicalein, wogonin and norwogonin revealed inhibitory effects against all bacteria studied, whereas oroxylin a was limited to mrsa, mssa and pseudomonas aeuroginosa, while wogonoside, oroxyloside and norwogonoside had no antimicrobial effect. the mic values of baicalin, baicalein, wogonin, oroxylin a and norwogonin against the bacteria were summarized in table 2 . norwogonin displayed an overall highest antimicrobial activity, followed by baicalein, oroxylin a and wogonin, and then baicalin. in order to identify the effective substances of huangqin by oral administration in human body, the metabolism in the gastrointestinal tract was investigated in vitro. although no change was observed in artificial gastric or intestinal juice, the main constituents, baicalin, wogonoside and oroxyloside, were found to be metabolized into their respective aglycons by intestinal bacteria. these findings are consistent with the reports that monomeric compounds baicalin, wogonoside and oroxyloside could be converted to metabolites baicalein, wogonin and oroxylin a by intestinal bacteria, respectively (akao et al., 2000; trinh et al., 2009; zuo et al., 2002) . the biotransformation of individual compound was the same as that in whole herb decoctions. moreover, a low content norwogonoside was isolated and identified from huangqin for the first time, and confirmed to be metabolized to norwogonin. baicalein, wogonin and oroxylin a were detected in rat plasma and urine samples after oral administration of gegen-qinlian-wan (gqw, including huangqin) by uhplc/dad/ qtof-ms, but not norwogonin which was present in our metabolic study in vitro (miao et al., 2013) . it is probably due to the difference between human and rat or the low content in specimens. nevertheless, both results suggest that flavonoid o-glycosides from huangqin could be easily transformed into their aglycones. the human complement system plays an important role in the host defense foreign invasive organisms. its effects are normally beneficial to the host (min et al., 2003) , but over-activation of the system may lead to pathological reaction in a variety of inflammatory and degenerative diseases such as rheumatoid arthritis, type i diabetes mellitus, systemic lupus erythematosus, sars, vasculitis and many others (valeriya et al., 2011) . numerous naturally occurring agents have been reported to effectively block the complement activation, which provide the prospect of novel anticomplementary drugs in non-toxicity based on abundant herb resources. the best described anticomplementary agents are polysaccharides, flavones and triterpenes (xu et al., 2007) . in the present study, wesb (1.25 mg/ml) showed no anticomplementary activity through the classical pathway or the alternative pathway, even though wesb was abundant in flavonoids. our studies on main flavonoid glycosides from wesb, baicalin, norwogonoside, oroxyloside and wogonoside generated the similar results (table 1) . however, their metabolites, especially norwogonin, showed obvious anticomplementary activity in inhibiting the classical pathway and the alternative pathway, suggesting a correction between 5,7-dihydroxyflavone and the anticomplementary effect. specifically, the inhibitory potencies against complement system were enhanced by the number of free hydroxyls on a ring of 5,7-dihydroxyflavone (xi et al., 2012) . this finding also agrees with the anticomplementary properties in some acylated flavonol glycosides obtained from magnolia fargesii (jung et al., 1998) and persicaria lapathifolia (park et al., 1999) . on the other hand, glycosylation on a ring of flavones might play an important role in its anticomplementary effect that most of the effect even disappeared (yao et al., 2013) . these results were also coincident with the previous suggestion that the intestinal flora might regulate the metabolism of compounds administered orally or excreted into bile (zuo et al., 2002) , and the herbal components should be transformed to bioactive compounds by the intestinal bacteria prior to their pharmacological actions (lee et al., 2002a; sousa et al., 2008) . further study should be carried out to elucidate their anticomplementary activity in vivo. published literature strongly support that the antimicrobial activities of some plant extracts are likely to be due to their high flavonoid content (cushnie and lamb, 2005; zhang et al., 2011) . in our studies, baicalin, baicalein, oroxylin a and norwogonin, four typical flavones possessed different degree of antimicrobial activities. a small number of research groups have investigated the relationship between structure and antibacterial activity of flavonoid to identify common structural features among active compounds. tsuchiya 0 s study indicated that 5,7-dihydroxylation of the a ring in the flavone structure was important for anti-mrsa activity (tsuchia et al., 1996) . it is consistent that baicalein, wogonin, oroxylin a and norwogonin, all featured such structures (shown in fig. 1) , and display better effective antimicrobial properties than other compounds. interestingly, a report by stapleton demonstrated that substitution with c-8 chain also enhanced the antistaphylococcal activity of flavonoids (stapleton et al., 2004) . independently, our data also revealed that norwogonin, 5,7,8-trihydroxyflavone has the highest antibacterial activity. these results suggest that the overall antimicrobial effect of huangqin may be dependent on its metabolism by intestinal bacteria. moreover, gram-negative bacteria escherichia coli and pseudomonas aeuroginosa have been inhibited to a less extent as compared to the gram-positive bacteria mrsa, mssa, and enterococcus faecalis as shown in table 2 . this result is in agreement with that flavonoids of the plant origin appear to have greater activity against gram-positive than gram-negative bacteria (ikigai et al., 1993) and could selectively inhibit gram-positive bacteria (wyman and van-etten, 1978) . further characterization of the interaction between these antimicrobial flavonoids and their target sites are needed, however. in the present study, norwogonin, one of the metabolites of wesb, presented most significant anticomplementary and antimicrobial activities. specifically, norwogonin has the most potent activity against multidrug-resistant acinetobacter baumannii strains (mic ¼0.256 mg/ml) among all identified antimicrobial compounds (miyasaki et al., 2013) , and exhibited more potent cytotoxicity than wogonin according to the mtt and ldh release assays in leukemia hl-60 cells (chow et al., 2008) . norwogonin also exhibited potent inhibitory activity toward vhr (ic 50 ¼ 1.1 μm), but had no inhibitory activity against t-cell protein tyrosine phosphatase or serine/threonine protein phosphatase 1 (lee et al., 2002b) . norwogonin possessed significant antioxidant activity in aaph (2,2 0 -azobis (2-amidinopropane hydrochloride)induced haemolysis (liu et al., 2004) . in addition, it is reported that norwogonin was determined in test strains differing in excision-repair capability and in the presence or absence of plasmid pkm101 (macgregor and wilson, 1988) . although some biological effects of norwogonin have been observed, the mechanism of action still remains unclear and requires further investigation. our results demonstrated that the presence of intestinal bacteria played an important role in the gastrointestinal metabolism of wesb. the process of metabolism by intestinal microbiota is involved in hydrolytic reactions generating non-polar low molecular weight byproducts. active metabolites were generated during the metabolic process. the results indicated that pharmacological effects of huangqin may be dependent on its metabolism by intestinal bacteria, consistent with the fact that wesb has no anticomplementary and antimicrobial activities in vitro. intestinal bacterial hydrolysis is indispensable to absorption of 18 beta-glycyrrhetic acid after oral administration of glycyrrhizin in rats baicalin, the predominant flavone glucuronide of scutellariae radix, is absorbed from the rat gastrointestinal tract as the aglycone and restored to its original form antimicrobial and antioxidant activities of methanol extracts of evax pygmaea (asteraceae) growing wild in tunisia differential apoptotic effect of wogonin and nor-wogonin via stimulation of ros production in human leukemia cells antimicrobial activity of flavonoids bactericidal catechins damage the lipid bilayer anticomplement activity of tiliroside from the flower buds of magnolia fargesi microassay for colorimetric estimation of 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phytochemical flavanones against methicillin-resistant staphylococcus aureus anti-inflammatory activity of devil 0 s claw in vitro systems and their active constituents antibacterial activity of selected isoflavonoids anti-complementary activity of flavonoids from gnaphalium affine d. don isolation and characterization of an anti-complementary polysaccharide d3-s1 from the roots of bupleurum smithii chemical constituents of rabdosia japonica var. glaucocalyx and their anti-complementary activity anti-inflammatory effects of scutellaria baicalensis water extract on lps-activated raw 264.7 macrophages high temperature effects on flavones accumulation and antioxidant system in scutellaria baicalensis georgi cells antioxidant and antiinflammatory activities of selected medicinal plants containing phenolic and flavonoid compounds anticomplementary principles of a chinese multiherb remedy for the treatment and prevention of sars metabolism of constituents in huangqin-tang, a prescription in traditional chinese medicine, by human intestinal flora pharmacokinetic study on the multi-constituents of huangqin-tang decoction in rats this work was financially supported by the national project in significant creation of new drugs during the eleventh five-year plan period (2009zx09502-013). the authors are grateful to dr. xiuping qian 0 s lab to provide all the bacteria strains and technical support of antimicrobial test, and dr. james l. fu for the careful english revision. supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2013.12.056. key: cord-267284-3uz0v29k authors: schneiderová, kristýna; šmejkal, karel title: phytochemical profile of paulownia tomentosa (thunb). steud. date: 2014-08-29 journal: phytochem rev doi: 10.1007/s11101-014-9376-y sha: doc_id: 267284 cord_uid: 3uz0v29k paulownia tomentosa, a member of the plant family paulowniaceae and a rich source of biologically active secondary metabolites, is traditionally used in chinese herbal medicine. flavonoids, lignans, phenolic glycosides, quinones, terpenoids, glycerides, phenolic acids, and miscellaneous other compounds have been isolated from different parts of p. tomentosa plant. recent interest in this species has focused on isolating and identifying of prenylated flavonoids, that exhibit potent antioxidant, antibacterial, and antiphlogistic activities and inhibit severe acute respiratory syndrome coronavirus papain-like protease. they show cytotoxic activity against various human cancer cell lines and inhibit the effects of human cholinesterase, butyrylcholinesterase, and bacterial neuraminidases. most of the compounds considered here have never been isolated from any other species of plant. this review summarizes the information about the isolated compounds that are active, their bioactivities, and the structure–activity relationships that have been worked out for them. the plant paulownia tomentosa (thunb.) siebold & zucc. ex steud. is a very adaptable and extremely fastgrowing timber tree native to central and western china traditionally used in chinese medicine. this deciduous tree is now grown in many areas worldwide, mostly as a decorative ornamental tree (erbar and g} ulden 2011; zhu et al. 1986) . two related varieties of p. tomentosa have been described-var. tsinlingensis has a round to shallowly cordate leaf base and a glabrous or sparsely hairy lower leaf surface, whereas var. tomentosa is characterized by a cordate leaf blade base and an abaxial surface that is densely hairy when mature (hong et al. 1998) . paulownia was named paulownia in honour of anna paulowna (1795 paulowna ( -1865 , queen consort of the netherlands and a daughter of tsar paul i of russia. nowadays, it is commonly known under its synonym bignonia tomentosa (zhu et al. 1986) or as the princess-tree, empress-tree, foxglove tree, royal paulownia, kiri, or mao pao tong (yuan bian zhong) (erbar and g} ulden 2011; hong et al. 1998) . the genus paulownia was first assigned to family bignoniaceae by swiss botanist thunberg (1781). it was then transferred to the scrophulariaceae family by the dutch scholars zuccarini and siebold (1835) (zhu et al. 1986 ). at last, paulownia has been categorized as a family of its own, paulowniaceae, based on data from the latest molecular phylogenetic studies. this table 1 non-prenylated flavonoid aglycones isolated from p. tomentosa leaves (zhu et al. 1986 ) weak cytotoxic activity in vitro against human leukaemia (hl-60) and human hepatoma (smmc-7721) cell lines ; no a-glucosidase inhibitory effect (zhao et al. 2009 ); weak aldose-reductase inhibitory activity (kadota et al. 1994) ; moderate inhibitory effect on human immunodeficiency virus-1 protease (lee et al. 2008) ; no activity against lipopolysaccharide (lps)induced no production in raw 264.7 macrophages (li et (2) leaves , flowers jiang et al. 2004 ) antioxidant (prince vijeya singh et al. 2004 ); antibacterial, anti-inflammatory, antispasmodic, antidiarrhoic, antiproliferative, vasorelaxant (jiang et al. 2004 ); neuroprotective (losi et al. 2004 ); cardioprotective (psotová et al. 2004 ); chemopreventive activity against skin cancer ); activity reviewed by shukla and gupta (2009) and patel et al. (2007) h h oh h oh kaempferol (3) leaves (si et al. 2008a ) cytotoxic, pro-apoptic (suppresses cell metastasis via inhibition of the erk-p38-jnk and ap-1 signaling pathways in u-2 os human osteosarcoma cells) ; antioxidant (e.g., attenuates bladder hyperactivity caused by potassium chloride after protamine sulphate-induced bladder injury) (huang et al. 2014) ; impact on human health and cancer chemoprevention reviewed by , luteolin (4) leaves (si et al. 2008a; li 2011) memory-improving (yoo et al. 2013 ); inhibition of aamylase activity (funke and melzig 2005) ; cytotoxic (bgc-823 gastric carcinoma xenografts in nude mice) ; vascular protective (si et al. 2013 (5) bark (si et al. 2011b) , leaves (si et al. 2008a ) antifibrotic (yoon et al. 2012) ; antioxidant, antiproliferative, anti-inflammatory, cardioprotective ; neuroprotective (ghosh et al. 2013 ); anti-diabetic [dose-dependent inhibition of both na ? atpase and sodium hydrogen exchanger in type 2 diabetic erythrocytes (mishra and rizvi 2012) ; inhibition of pi3k (koch et al. 2013) ]; antiviral [anti hcv (khachatoorian et al. 2012) ; hcmv (cotin et al. 2012) ]; no anti-hiv activity in vitro tested on h9 cells in the absence of toxicity (tang et al. 1994 ); review of bioactivities by russo et al. (2012) oh h oh oh oh 7,3 0 -dimethylquercetin (syn. rhamnazin) leaves, green immature fruit (wollenweber et al. 2008 ) antimicrobial activity, poor antifungal effect (omosa et al. 2014 ); low antimicrobial activity, low toxicity against human lymphocytes and monocytes, antioxidant/antiinflammatory activity (martini et al. 2004; pelzer et al. 1998 ); low affinity to acetylcholinesterase (remya et al. 2012) ; low inhibitory effect on no production in raw264.7 cells (sudsai et al. 2013) ; no activity against hsv-1, low toxicity against vero cells (tian et al. 2009 ); no antioxidant activity (takamatsu et al. 2003) ; no trypanocidal activity (grael et al. 2000) ; activity against lipid peroxidation in rat liver microsomes (yun et al. 2000) ; cytotoxicity against tk-10, mcf-7 and uacc-62 cells (lopez-lazaro et al. 2000) ; no inhibition of glycolysis in various tumor cells (suolinna et al. 1975 ,4 0 -trimethylquercetin (7) thrombin inhibition (shi et al. 2012 ); inhibition of il-4 synthesis in basophils (hirano et al. 2006) ; weak trypanocidal activity (jordao et al. 2004 ); weak inhibition of no production in lps-activated mouse peritoneal macrophages (matsuda et al. 2003) ; weak inhibition of degranulation of rbl-2h3 cells (mastuda et al. 2002) ; inhibition of pgp activity (scambia et al. 1994) ; no inhibition of glycolysis in a variety of tumor cells (suolinna et al. 1975 flowers jiang et al. 2004; kim et al. 2010a , b) inactive against glutamate-induced neurotoxicity studied in primary-cultured rat cortical cells (kim et al. 2010a, b) ; no trypanocidal activity (grael et al. 2000) h h oh ome ome 5-hydroxy-7,3 0 flowers (kim et al. 2010a , b) inactive against glutamate-induced neurotoxicity studied in primary-cultured rat cortical cells (kim et al. 2010a, b) ; inhibition of inflammation by induction of synovial apoptosis of fibroblast-like synoviocytes through caspase 3 activation in rats with adjuvant arthritis (li et al. 2010 ; inhibition of jak2-stat3 signal pathway in rats ); inhibition of phosphodiesterase 4 (yang et al. 2011) ; low toxicity on b16f10 and sk-mel-1 melanoma cells (rodriguez et al. 2002) ; antimutagenic activity (miyazawa et al. 2000) h family includes only one genus and between six and ten species. it was originally introduced in 1949 by the research of nakai (erbar and g} ulden 2011) . biological activity and traditional uses of p. tomentosa extracts according to both legends and records, people were already using paulownia for various purposes about 2,600 years ago. chinese herbal medicine has used p. tomentosa traditionally to relieve bronchitis, especially by reducing coughing, asthma, and phlegm (zhu et al. 1986 ). it has also been used to treat conjunctivitis, dysentery, enteritis, erysipelas, gonorrhea, hemorrhoids, parotitis, traumatic bleeding, and tonsillitis (jiang et al. 2004; si et al. 2009 ). solutions prepared from the leaves and fruit extracted in water have been used in daily applications to promote the healthy growth of hair and turn grey hair dark. an extract prepared from the wood and leaves may relieve swollen feet. pharmacological experiments have shown that extracts from the fruit can reduce blood pressure. nowadays, injections and tablets prepared from paulownia flowers and fruit are used for the herbal treatment of chronic bronchitis and other kinds of inflammation (zhu et al. 1986 ). more than 130 physiologically active constituents have been isolated from different parts of the paulownia plant. their biological activity has been tested using both the isolated compounds and different types of extracts. for example, n-butanol, etoac, and meoh extracts obtained from the fruit have displayed antiradical activity in anti-dpph and peroxynitrite assays, due to mainly the presence of flavonoids and phenolic glycosides, but not of all compounds present in these extracts have been identified) (š mejkal et al. 2007b ). an meoh extract obtained from the fruit inhibited hache (ic 50 = 1.44 mg/ml) and bche (ic 50 = 0.97 mg/ml) significantly more strongly than chcl 3 and water extracts (possibly due to the content of phenolic glycosides and c-prenylated dihydroflavonols and flavanones) (cho et al. 2012 ). significant concentration-dependent anti-inflammatory properties of etoh extracts of the bark of the tree have also been observed recently using a lipopolysaccharide (lps)induced nitric oxide production inhibition model in the murine macrophages cell line raw264.7 (si et al. 2011a ). kim et al. (2010a, b) showed potential of aqueous extract of p. tomentosa to suppress glutamate induced toxicity in primary cultured rat cortical cells (with possible sesquiterpene lactone as active substance). the bio-activities of individual compounds and, where possible, the structure-activity relationships are in tables 1, 2, 3 and 4 and discussed in separate chapters. flavonoids represent the most numerous group of secondary metabolites isolated from p. tomentosa. they can be divided into simple non-prenylated flavonoids 1-18 (table 1) , c-prenylated and c-geranylated flavonoids 19-59 (tables 2, 3 , respectively), and flavonoid glycosides 60-65. compounds 16, 17, 19, 20, [25] [26] [27] [28] [29] [30] [31] [33] [34] [35] [36] have never been isolated from any other species. most of the isolated flavanones are characterized by a 2s configuration in contrast to the dihydroflavonols, for which 2r, 3r isomer is often observed. some of the flavonoid compounds found in p. tomentosa have been categorized as dietary flavonoids. consuming these compounds is believed to deliver health benefits. the activities of these flavonoids are frequently been reviewed, for example in the papers of romano et al. (2013) , marzocchella et al. (2011) , ross and kasum (2002) , and havsteen (2002) . some simple flavonoid substances isolated from fruit of p. tomentosa are commonly observed as the lipophilic components of different plant exudates. flavonoids 1-18 show broad-spectrum pharmacological activities (table 1) . many papers report their potential role as anticancer compounds for use against human cervical and breast (bulzomi et al. 2010 (bulzomi et al. , 2012 , hepatoma, leukemic , osteosarcoma , gastric carcinoma , colon adenocarcinoma (sánchez-tena et al. 2013) , or prostatic (zhang and coultas 2013) cancer cell lines. their antioxidant properties could explain their promising cardioprotective (paneerselvam et al. 2010; psotová et al. 2004 ) and neuroprotective effects (losi et al. 2004 ). antimicrobial (betts et al. 2013; jiang et al. 2004; mankovskaia et al. 2013 ) and antiviral (khachatoorian et al. 2012) bio-activities against different pathogens have also been discovered. in some cases, no specific biological activity has been observed (kim et al. 2010a (kim et al. , 2010b tang et al. 1994) . paulownia tomentosa is also a rich source of prenylated and geranylated flavonoids (19-59), whose occurrence is limited to relatively a few plant families (yazaki et al. 2009 ). there are sometimes misunderstandings in the naming of these compounds. a compound containing both a phenolic skeleton and a terpenoid side chain is often designated as a ''prenylated'' phenolic substance, even though it contains not a prenyl, but rather a geranyl or other type of terpenoid side-chain. prenylated flavonoids are biosynthesized by a combination of several pathways: the acetate, shikimate, and mevalonate ( fig. 1) . it is now known that the prenyl and geranyl moieties are biosynthesized via mevalonate or deoxyxylulose pathway. the connection of terpenoid and flavonoid biosynthetic antioxidant ; cytotoxic (a2780 human ovarian cancer cell line) (murphy et al. 2005 (murphy et al. , 2006 oh bark (sticher and lahloub 1982; zhu et al. 1986 ) antiphlogistic (choi et al. 2004 ), low cox-2/1 and 5-lox inhibition (wang et al. 2013; diaz lanza et al. 2001 kuzuyama et al. 2005) . the side chain can later be converted into different moieties by several reactions. it is unclear, whether the described modifications of the prenyl side-chain are natural-sunlight, the presence of oxygen and an elevated temperature can all affect the terpenoid metabolism-or are artefacts of isolation. some of the changes in the prenyl moiety have been observed after treatment of an extract, or during the separation of a mixture in the acidic environment of silica gel (navrátilová et al. 2013 ; š mejkal 2014) (fig. 2) . most of the prenylated flavonoids isolated from p. tomentosa belong to c-geranylated group of flavonoids (21-52). some of them have their sidechain further modified by hydroxylation (35-44, 50, 51, 53-59), methoxylation (45-47, 49, 50), oxidation (47, 48, 52), cyclization (51, 53-59), or reduction (19, 20, 47-52) . interestingly, these compounds have been isolated from the leaves, flowers and fruit, but they are most commonly isolated from the roots, root bark, or bark of different plants (botta et al. 2005) . compounds 23, 24, and 28 have been found in the yellow dendritic trichomes on the adaxial side of the p. tomentosa leaves. on the other hand, no significant detected concentration of secondary metabolites has been detected in the white dendritic trichomes on the adaxial side of the leaves or the brown dendritic trichomes on the flower buds ). glandular hairs found on the young reproductive organs of p. tomentosa are rich in flavonoids, with concentrations over 1,000 times greater than those on the surfaces of the young leaves . some seasonal variations in the appropriate time for harvesting the fruit have been discovered. autumn is the best time to obtain high concentrations of flavonoids whereas early summer is better for phenylpropanoid glycosides (holubová and š mejkal 2011) . the antioxidant š mejkal et al. 2007a, b; zima et al. 2010) , antibacterial (navrátilová et al. 2013; š mejkal et al. 2008b) , antiphlogistic , cytotoxic (kollár et al. 2011; š mejkal et al. 2007a , and severe acute respiratory syndrome coronavirus papain-like protease (sars-cov plpro) activities (cho et al. 2013 ) of isolated geranylated flavonoids have been described recently, together with their inhibitory effect on both human acetylcholinesterase and butyrylcholinesterase (cho et al. 2012) . the great ability of several geranylated flavanones to interact with bacterial sialidase isolated from clostridum perfringens (cp-nani), a bacterium causing various gastrointestinal diseases, has recently been reported (lee et al. 2014) . possible relationships between structure and activity have been proposed for each of these biological activities. nevertheless, it is difficult to evaluate real structure-activity relationships only by comparing the published studies, because the study conditions and the assays employed are often different. generally, the addition of an isoprenoid chain renders the derivate molecule more pharmacologically effective than the parent compound, probably because the prenyl group increases the lipophilicity and confers a strong affinity for biological membranes (botta et al. 2005; epifano et al. 2007; chen et al. 2014) . interestingly, neither the geranyl side-chain nor its substitution affects the antioxidant activity of flavonoids. the spatial arrangement of the substitution of the flavanone skeleton is more important. for example, the antiradical activity is increased by 2 0 -hydroxyl substitution of the ring b, whereas 4 0 -methoxyl substitution diminishes it. the general rules postulated for the antioxidant activity of flavonoids in vitro are applicable (havsteen 2002; chen et al. 2002) , there are many review publications that touch on this topic, and it is not aim of this paper to delve deeply into this (plaza et al. 2014) . the type of antioxidant assays used for the experiment could also be a factor that significantly affects this activity (zima et al. 2010) . numerous reports about structure related antimicrobial activity have been published, but comparison shows the results to be widely conflicting (cushnie and lamb 2005) . based on several studies, it has been postulated that hydroxylation at position c-5 or c-7 of ring a and positions c-2 0 or c-4 0 of ring b increases the antibacterial activity (š mejkal et al. 2008b; navrátilová et al. 2013) . however, contrary to this assumption, 45 and 50 had no significant antibacterial activity (navrátilová et al. 2013) . interestingly, some c-geranylated flavonoids do not able inhibit the growth of gram-negative bacteria (21, 22, 25-27, 35, 38, 45, 47-48, 50, 53, and 54) . resistance to these compounds is probably due to the more complex structure and hydrophilic nature of g-cell walls (navrátilová et al. 2013; š mejkal et al. 2008b) . furthermore, substitution of the geranyl side-chain with carbonyl, hydroxyl or methoxyl groups diminishes the antibacterial activity in a manner similar to what is seen when the geranyl substituent at c-6 forms a ring by reacting with the hydroxyl group at c-7. compounds 45, 47, 48, 50, 53 , and 54 exhibit some degree of activity in the range of the concentrations tested on the gram-positive bacteria staphyloccocus aureus and various methicillin resistant strains of s. aureus. the level of activity varied in depending on both the structure and the bacterial strain used in the assay. flavonoid structures like 24 are more effective in protecting plants from water loss because of their reduced polarity navrátilová et al. 2013). compounds 45, 47, 48, 50 , and 53 were also tested for their ability to affect the initiation of the eukaryotic translation via dual-luciferase reported assay (firefly and renilla), but only 45 showed a modest activity in comparison with anisomycin (navrátilová et al. 2013) . the cytotoxicity of the prenylated flavonoids obtained from p. tomentosa has been tested in more than 20 different cell lines. the type of prenylation strongly affects the cytotoxicity of a flavonoid in the traditional p-388 murine leukemia model. the unmodified 3-prenyl group and the presence of corresponding ortho-dihydroxy or trihydroxy substitution of the flavonoid ring b are crucial to its activity against p-388 as compared with other prenyl substituents (hakim et al. 1999 (hakim et al. , 2002 (hakim et al. , 2006 . similar findings have been observed for modified c-6 geranyl groups and the substitution of the ring b for other cell lines tested (š mejkal et al. 2010) . however, replacing the parahydroxy group of the ring b of a c-8 prenylated flavanone with several different acyl substituents resulted in greater cytotoxicity (aniol et al. 2012; š mejkal 2014) . it has also been found that cytotoxic activity is diminished by the presence of a c-3 hydroxyl substituent on the ring c, 4 0 -methoxy substitution of the ring b or a para-hydroxy substituted ring b (š mejkal et al. 2008a) . for this reason, it is important to emphasize that the relative importance of the substitution of ring b can differ according to the cell line used. modification of a prenyl or geranyl sidechain can not only change the direct cytotoxicity, but it may also affect the proliferation cells or trigger apoptosis (kollár et al. 2011; šmejkal et al. 2007a š mejkal 2014) . prenylated flavonoids 55-59, modified with an unusual 3,4-dihydro-2h-pyran moiety, have been found to inhibit the severe acute respiratory syndrome corona virus plpro enzyme more effectively than their parent compounds, the precursors from which were they derived (cho et al. 2013) . the presence of a geranyl group at the c-6 position (21, 22, 24-26, 30, 31, 33 , and 34) seems to be crucial for the hache and bche inhibitory effects. the most effective inhibitor was 22. all of the geranylated flavonoids, apart from 26, inhibited hache dosedependently. it appears that greater inhibition is observed when ring b of the flavanone bears free hydroxyl groups (cho et al. 2012 ). phytochem rev (2015 the crystal structure of the bacterial sialidase cp-nani catalytic domain in a complex with the geranylated flavonoid diplacone (22) provides structural insights into the binding mode of natural flavonoidbased inhibitors at atomic resolution. it shows how the geranyl and c-3 0 hydroxyl groups of 22 contribute to the stability of the enzyme-inhibitor complex. time-dependent competitive inhibition patterns have been observed. structural comparison of the human sialidases neu1-neu4 with the cp-nani-diplacone (22) complex suggests that the interaction between human sialidases and 22 is likely to be unfavourable because of polar or ionic repulsion (lee et al. 2014) . six flavonoid glycosides have been extracted from the stem bark 60-62 (si et al. 2009 ) and flowers 63-66 (scogin 1980) of p. tomentosa. apigenin-7-o-b-d-glucopyranoside (synonym: cosmosiin) (kurkina et al. 2011) (60) shows anti-hiv activity in vitro on h9 cells (tang et al. 1994 ) and enhances the secretion of adiponectin, the phosphorylation of the tyrosine residue of insulin receptor-b, and the translocation of glut5 (rao et al. 2011) . compound 60 shows no significant hepatic glc-6-phosphatase inhibitory activity in vitro (kumar et al. 2010 ). apigenin-7-o-b-d-glucuronopyranoside (61) was more potent than apigenin (2) and omeprazole in an assay analysing the inhibition of reflux esophagitis and gastritis promoted surgically and by application of indomethacine in rats (min et al. 2005) . no information about the biological activity of apigenin-7-o-[b-d-glucuronopyranosyl (1 ? 2)-o-b-d-glucuronopyranoside] (62) has been found in the literature. delphinidin-3-o-glucoside (synonym: myrtillin) (63) protects microglia from inflammationinduced stress signaling (carey et al. 2013) , is cytotoxic against the mcf-7 (breast) cancer cell line (vareed et al. 2006) , and has an the affinity for the estrogenic erb receptor (hidalgo et al. 2012 ). delphinidin-3,5-di-o-glucoside (64) and cyanidin-3,5-di-o-glucoside (65) are potent antioxidants tanaka et al. 2013) . compounds 63 and 65 are potent ace inhibitors in vitro (hidalgo et al. 2012; persson et al. 2009 ). only three lignans: (?)-paulownin (66), (?)-sesamin (67), (?)-piperitol (68) have been isolated from the wood of p. tomentosa (ina et al. 1987; takahashi and nakagawa 1966; zhu et al. 1986) (fig. 3 ). several reports have described their pharmacological effects pan et al. 2009 ). compound 66 is a promising antifungal agent that acts against the basidiomycetes fomitopsis palustris and trametes versicolor (kawamura et al. 2004) . sesamin (67) has been previously isolated from the wood of p. tomentosa, and p. kawakamii and the hybrid of p. elongata 9 p. fortunei (takahashi and nakagawa 1966; zhu et al. 1986 ). compound 67 is the major lignan in sesame, and has various biological activities, such as antioxidant, angiogenic (chung et al. 2010) , antiphlogistic (chatrattanakunchai et al. 2000) , antihypertensive (nakano et al. 2006) , insecticidal (nascimento et al. 2004) , neuroprotective , and anticarcinogenic ( . however, this compound showed no significant ability to reduce the formation of atherosclerotic lesions in the apoe (-/-) gene-knockout mouse, whereas specific dietary polyphenols, especially quercetin and theaflavin were more active (loke et al. 2010) . it has been suggested that some of the biological effects attributed to sezamine (67) may, in fact, be caused by its metabolites and that 67 may be acting as a proactive substance in the body (yasuda and sakaki 2012) . the metabolism of sesamim (67) by cytochrome p450 and udp-glucuronosyltransferase has been found remarkably different in humans as compared to other animals. further investigation into the safety of taking sesamin (67) with therapeutic drugs that are metabolised by cyp2c9 is also needed (yasuda and sakaki 2012) . a total of thirteen phenylpropanoid glycosides (69-81) with multifarious bioactivities, natural compounds derived biosynthetically from the amino acid phenylalanine, have been isolated from different parts of the p. tomentosa plant (table 4 ) (damtoft and jensen 1993; kang et al. 1994; ota et al. 1993; schilling et al. 1982; si et al. 2008b , d, si et al. 2011b sticher and lahloub 1982; šmejkal et al. 2007b; zhu et al. 1986 ). phenylpropanoids are of great interest for fabricating effective tonic, anticancer, hepatoprotective, immunostimulating, antimicrobial, and anti-inflammatory phytopreparations (kurkin 2003; galvez et al. 2006; panossian and wagner 2005; fu et al. 2008; pan et al. 2003) . a new furanoquinone, methyl-5-hydroxy-dinaphthol [1,2-2 0 ,3 0 ]furan-7,12-dione-6-carboxylate (mhddc) (82) (fig. 4) identified in p. tomentosa stem bark (meoh extract) has been shown to possess antiviral activity against poliovirus types 1 and 3, using hela cells in vitro (kang et al. 1999 ). its cathepsin k and l inhibitory activities in vitro have recently been discovered. the 5-oh functional group may have a favourable effect on this reduction potential which prevents the degradation of bone the matrix carried out by osteoclasts (park et al. 2009 ). the naphtoquinone plumbagin (83) has been detected in the leaves and fruit of p. tomentosa (babula et al. 2006) . it has been used in traditional systems of medicine since ancient times (pile et al. 2013) . it has exhibited promising antimalarial activity in vitro with ic 50 of 580 (270-640) nm and 370 (270-490) nm, respectively, against 3d7 chloroquine-sensitive p. falciparum and k1 chloroquineresistant p. falciparum clones, using an assay based on sybr green i. toxicity testing indicated relatively low toxicity at dose levels up to 100 mg/kg body weight (for a single oral dose) and 25 mg/kg (for daily dose given for consecutive 14 days) for acute and subacute toxicity, respectively. based on the results of in vivo antimalarial testing, plumbagin administered at a the dose of 25 mg/kg of body weight for 4 consecutive days exhibited moderate to weak antimalarial activity with regard to its ability to reduce parasitaemia and prolong survival time (sumsakul et al. 2014) . other published studies of plumbagin (83) described effects such as antifungal, antibacterial, cytotoxic (krishnaswamy and purushothaman 1980) , anticoagulant (santhakumari et al. 1978) , anthelmintic (antischistosomal) (zhang and coultas 2013) and an anti-inflammatory effect on the amelioration of experimental ulcerative colitis in mice at a dose of 8-10 mg/ kg (pile et al. 2013) . six iridoids: 7-b-hydroxyharpagide (84), paulownioside (85), catalpol (86), aucubin (87), tomentoside (88) and 7-hydroxytomentoside (89) have been isolated from the leaves of p. tomentosa (84-87) (adriani et al. 1981; franzyk et al. 1999) , the bark of the trunk and roots (86) (plouvier 1971 ) and parts of the young plant (85, 86, 88, and 89) (damtoft and jensen 1993) (fig. 5) . compound 86 shows neuroprotective activity (li et al. 2004 ), a cardioprotective effect against myocardial infarction-specifically reperfusion damage (huang et al. 2013a, b) , and radioprotective effects . it also increases glucose utilization by increasing the secretion of b-endorphin from the adrenal gland (shieh et al. 2011) . compounds 86 and 87 possess antispasmodic activity (deurbina et al. 1994) . aucubin (87) had antioxidant and pancreasprotective effects on streptozotocin-induced diabetes in rats (jin et al. 2008) and it may improve obesityinduced atherosclerosis by attenuating the tnf-ainduced inflammatory response (park 2013) . a weak antibacterial effect (against streptococcus pneumoniae and mg-hemolytic streptococcus, mic 28.946 mg/ml) (zheng et al. 2012 ) and neuroprotective effects of 87 on diabetes and diabetic encephalopathy (xue et al. 2012) have also been observed. the sesquiterpenic lacton isoatriplicolide tiglate (90) has been isolated from p. tomentosa flowers. it has neuroprotective effects against glutamate-induced neurotoxicity (kim et al. 2010a (kim et al. , 2010b and cytotoxic activity against several cancer cell lines: a549 lung carcinoma; sk-ov-3 adenocarcinoma; sk-mel-2 malignant melanoma; xf498 central nervous system tumors; hct15 colon adenocarcinoma, against which its effect is comparable to that of reference substance adriamycin (moon and zee 2001) ; human breast cancers mda-mb-231, mcf7, hs578t, and t47d; and the hela, siha and c33a cervical cancer cell lines (jung et al. 2012) . seven phytosterols have been isolated from p. tomentosa leaves: ursolic acid (91) (zhu et al. 1986; zhang and li 2011) , 3-epiursolic acid (92), pomolic acid (93), corosolic acid (94), maslinic acid (95), b-sitosterol (96), and daucosterol (97) (zhang and li 2011) . most of these show various biological activities, e.g., 91 is a potentially useful for treating alzheimer's disease (because of its ability to block the interactions of the amyloid b-cd36) (wilkinson et al. 2011) . it can prevent the recruitment of the monocytes that accelerate atherosclerosis, a major complication of diabetes, in mice (ullevig et al. 2011) , and it also possesses antibacterial (wong et al. 2012) , anti-trypanosomal, and anti-leishmanial properties (bero et al. 2011) . compound 91 has also shown cytotoxic effects against k562 and k562/adr human chronic myelogenous leukaemia, hl60 and hl60/adr human acute myelocytic leukemia cancer cells, and the human colon cancer cell lines sw480 and sw620 (shan et al. 2011) . compounds 93-96 also show promising cytotoxic potential, e.g., 93 is effective against the human chronic myelogenous leukaemia cell line 562 and also against cells derived from chronic myeloid leukaemia patients (vasconcelos et al. 2007 ). compound 94 shows cytotoxic effects against osteosarcoma mg-63 cells (cai et al. 2011) and immunosuppressive activity on myeloid-derived suppressor cells in the murine sarcoma model (horland et al. 2013 (deepak and handa 2000) , and 93 (schinella et al. 2008 ). an immunomodulatory effect of 97 in protecting mice against candidiasis disseminated by the cd4? th1 immune response has been seen (lee et al. 2007 ). other recently discovered pharmacological effects include antimalarial (95) (moneriz et al. 2011) , hypotensive and endothelium-dependent vasorelaxant effects (93) (estrada et al. 2011 ). compound 95 is potentially useful for treating cerebral ischemic injuries (guan et al. 2011) , and 94 has promising antidiabetic effects (sivakumar et al. 2009 ). (127). the relatively most abundant constituents were 111 (20 % of the total glycerides), 126 (14 %), and 120 and 127 (12 %) (asai et al. 2009 ). another analysis of the secretions of the glandular hairs on the leaves of both bud flushes and adult trees as well as the flowers of adult trees showed that these secretions contain ten glycerides (106, 108-110, 118, 122, 124-127) . these compounds showed sticky character, but they were not toxic to several insects. therefore, the glandular hairs on the leaves and flowers may serve only to physically deter herbivores (fig. 6) . six known phenolic acids, namely p-hydroxybenzoic acid (128), vanillic acid (129), gallic acid (130), cinnamic acid (131), p-coumaric acid (132), and caffeic acid (133) have been identified in the leaves of p. tomentosa (128-133), bark (130 and 131) and wood (133) (ota et al. 1993; si et al. 2008c si et al. , 2011b , and p-ethoxybenzaldehyde (134) is present in flowers (yuan et al. 2009 ). the 5,7-dihydroxy-6-geranylchromone (135) isolated from fruits shows only moderate cytotoxic activity against a suspension culture of nicotiana tabacum cv. bright yellow (by-2), which confirms that ring b of the flavonoid skeleton is important for this activity (š mejkal et al. 2008a) . a large group of essential oil substances found in the flowers of p. tomentosa have also been identified (oprea et al. 2004; ibrahim et al. 2013) (fig. 7) . plants are still highly esteemed all over the world as rich sources of therapeutic agents. in this context, traditional chinese herbal medicines, such as paulownia continue to influence a modern healthcare. it has been estimated that approximately 420,000 plant species exist on earth, but little is known about the phytochemical or therapeutic qualities of most of them. thanks to the development of the spectral and other analytical methods used in modern phytochemistry, many of the principal physiologically active secondary metabolites have been identified and researched in detail. the accumulated knowledge of traditional medicine is therefore, playing an important role in enhancing the success of drug discovery in herbal medicine. approximately 80 % of the currently known antimicrobial, cardiovascular, immunosuppressive, and anticancer drugs are of plant origin (pan et al. 2013) . as mentioned in this review, p. tomentosa is a rich source of multifarious secondary metabolites, mainly prenylated flavonoids. as of today 135 compounds, including flavonoids, lignans, phenolic glycosides, quinones, terpenoids, glycerides, phenolic acids, and other miscellaneous compounds have been isolated from various extracts of this plant. of increasing interest are the isolation and identification of p. tomentosa prenylated flavonoids, as they have shown promising pharmacological effects. in the first experiments, their antioxidant, antibacterial, antiphlogistic, cytotoxic, and sars-cov pl activities have been discovered along with inhibitory effects on human acetylcholinesterase, butyrylcholinesterase, and bacterial neuraminidases. more than 40 compounds with modified prenyl or geranyl side-chains attached at c-6 of the flavonoid skeleton have been isolated from the flowers, fruit, and leaves of p. tomentosa. only two of these compounds have shown the presence of a five-carbon side-chain; for the others a ten-carbon side-chain is typical. further, only a few compounds have shown a geranyl moiety modified by hydroxylation at c-6 00 or c-7 00 . some compounds have a geranyl group modified by formating a heterocyclic moiety, which is also unusual. most of them have never been isolated from any other plant species. however, further in vivo pharmacology studies are needed to precisely elucidate biological mechanism of action, efficacy, and toxicity of these promising therapeutic agents. elucidation of the structure-activity relationships is also crucial for their further total syntheses and introduction into medical practice. for these reasons, the study of p. tomentosa as a source of biologically active metabolites is significant and future interest in this plant is ensured. isolation and characterization of paulownioside, a new highly oxygenated iridoid glucoside from paulownia tomentosa acteoside: a new antihypertensive drug in vitro immunomodulatory activity of verbascoside from nepeta ucrainica l antiproliferative activity and synthesis of 8-prenylnaringenin derivatives by demethylation of 7-o-and 4 0 -o-substituted isoxanthohumols effects of taxifolin on the activity of angiotensin-converting enzyme and reactive oxygen and nitrogen species in the aorta of aging rats and rats treated with nitric oxide synthase inhibitor and dexamethasone geranylated flavanones from the secretion on the surface of the immature fruits of paulownia tomentosa acylglycerols (=gly-cerides) from the glandular trichome exudate on the leaves of paulownia tomentosa chromatografické stanovení naftochinonů v rostlinách (chromatographic evaluation of naphtochinones in plants) active components from siberian ginseng (eleutherococcus senticosus) for protection of amyloid b(25-35)-induced neuritic atrophy in cultured rat cortical neurons catechin prodrugs and analogs: a new array of chemical entities with improved pharmacological and pharmacokinetic properties in vitro antitrypanosomal and antileishmanial activity of plants used in benin in traditional medicine and bio-guided fractionation of the most active extract antifungal synergy of theaflavin and epicatechin combinations against candida albicans prenylated flavonoids: pharmacology and biotechnology antiproliferative effect of catechin in grx cells naringenin and 17b-estradiol coadministration prevents hormone-induced human cancer cell growth the naringenininduced proapoptotic effect in breast cancer cell lines holds out against a high bisphenol a background corosolic acid triggers mitochondria and caspase-dependent apoptotic cell death in osteosarcoma mg-63 cells stilbenes and anthocyanins reduce stress signaling in bv-2 mouse microglia investigation of two flacourtiaceae plants: bennettiodendron leprosipes and flacourtia ramontchi cardiovascular protective flavonolignans and flavonoids from calamus quiquesetinervius sesamin inhibits lysophosphatidylcholine acyltransferase in mortierella alpine a review of the dietary flavonoid, kaempferol on human health and cancer chemoprevention structure-activity relationship of natural flavonoids in hydroxyl radicalscavenging effects propolin c from propolis induces apoptosis through activating caspases, bid and cytochrome c release in human melanoma cells determination of flavonoids in the flowers of paulownia tomentosa by high-performance liquid chromatography chemical modification and anticancer effect of prenylated flavanones from taiwanese propolis radio-protective effect of catalpol in cultured cells and mice a systematic review on biological activities of prenylated flavonoids cholinesterase inhibitory effects of geranylated flavonoids from paulownia tomentosa fruits geranylated flavonoids displaying sars-cov papain-like protease inhibition from the fruits of paulownia tomentosa anti-inflammatory and antinociceptive effects of sinapyl alcohol and its glucoside syringin angiogenic activity of sesamin through the activation of multiple signal pathways eight flavonoids and their potential as inhibitors of human cytomegalovirus replication antimicrobial activity of flavonoids tomentoside and 7-hydroxytomentoside, two new iridoid glucosides from paulownia tomentosa antimicrobial activity of brazilian propolis extracts against rumen bacteria in vitro antiinflammatory activity and chemical composition of extracts of verbena officinalis studies on the vascular effects of the fractions and phenolic compounds isolated from viscum album ssp. album in vitro antispasmodic activity of peracetylated penstemoniside, aucubin and catalpol lignan and phenylpropanoid glycosides from phillyrea latifolia and their in vitro anti-inflammatory activity phenolic compounds from artemisia iwayomogi and their effects on osteoblastic mc3t3-e1 cells chemistry and pharmacology of oxyprenylated secondary plant metabolites ontogeny of the flowers in paulownia tomentosa-a contribution to the recognition of the resurrected monogeneric family paulowniaceae pomolic acid of licania pittieri elicits endotheliumdependent relaxation in rat aortic rings dietary flavonoids: role of (-)-epicatechin and related procyanidins in cell signaling halohydrins of antirrhinoside-the correct structures of muralioside and epimuralioside naturally occurring phenylethanoid glycosides: potential leads for new therapeutics dietary flavonol epicatechin prevents the onset of type 1 diabetes in nonobese diabetic mice effect of different phenolic compounds on a-amylase activity: screening by microplatereader based kinetic assay pharmacological activities of phenylpropanoids glycosides neuroprotective role of nanoencapsulated quercetin in combating ischemiareperfusion induced neuronal damage in young and aged rats hepatoprotective effects of syringin on fulminant hepatic failure induced by d-galactosamine and lipopolysaccharide in mice a study of the trypanocidal and analgesic properties [of substances] from lychnophora granmongolense (duarte) semir & leitao filho maslinic acid, a natural inhibitor of glycogen phosphorylase, reduces cerebral ischemic injury in hyperglycaemic rats by glt-1 up-regulation studies on b-sitosterol and ceramideinduced alterations in the properties of cholesterol/sphingomyelin/ganglioside monolayers artoindonesianins a and b, two new prenylated flavones from the root of artocarpus champeden artoindonesianin p, a new prenylated flavone with cytotoxic activity from artocarpus lanceifolius prenylated flavonoids and related compounds of the indonesian artocarpus (moraceae) the biochemistry and medical significance of the flavonoids advanced research on acteoside for chemistry and bioactivities potential anti-inflammatory, anti-adhesive, anti/estrogenic, and angiotensin-converting enzyme inhibitory activities of anthocyanins and their gut metabolites luteolin, a flavonoid, inhibits ap-1 activation by basophils changes in the level of bioactive compounds in paulownia tomentosa fruits corosolic acid impairs tumor development and lung metastasis by inhibiting the immunosuppressive activity of myeloid-derived suppressor cells effect of diplacone on lps-induced inflammatory gene expression in macrophages prenylated and geranylated flavonoids increase production of reactive oxygen species in mouse macrophages but inhibit the inflammatory response catalpol decreases peroxynitrite formation and consequently exerts cardioprotective effects against ischemia/reperfusion insult eleutheroside b or e enhances learning and memory in experimentally aged rats anti-oxidant activity and attenuation of bladder hyperactivity by the flavonoid compound kaempferol chemical composition, antimicrobial activity of the essential oil of the flowers of paulownia tomentosa (thunb.) steud. growing in egypt antiplatelet aggregatory effects of the constituents isolated from the flower of carthamus tinctorius piperitol from paulownia tomentosa antiinflammatory role of naringenin in rats with ethanol induced liver injury determination of flavonoids from paulownia tomentosa (thunb) steud. by micellar electrokinetic capillary electrophoresis phenylethanoid glycosides in plants: structure and biological activity antioxidant and pancreasprotective effect of aucubin on rats with streptozotocininduced diabetes trypanocidal activity of chemical constituents from lychnophora salicifolia mart inhibitory effect and mechanism on antiproliferation of isoatriplicolide tiglate (pcac) from paulownia coreana matteuorienate a and b, two new and potent aldose reductase inhibitors from matteuccia orientalis (hook.) trev antibacterial phenylpropanoid 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antidepressant activity of some phytopharmaceuticals and phenylpropanoids flavonoids from tanacetum vulgare flowers structural basis for the promiscuous biosynthetic prenylation of aromatic natural products immunoregulatory activity of daucosterol, a beta-sitosterol glycoside, induces protective th1 immune response against disseminated candidiasis in mice two new triterpenes from the rhizome of dryopteris crassirhizoma, and inhibitory activities of its constituents on human immunodeficiency virus-1 protease anti-inflammatory effect and hplc analysis of extract from edible cirsium setidens anthocyanin compositions and biological activities from the red petals of korean edible rose (rosa hybrid cv. noblered) structural basis of sialidase in complex with geranylated flavonoids as potent natural inhibitors neuroprotection of catalpol in transient global ischemia in gerbils 0 -dimethoxy hesperetin induces apoptosis of fibroblast-like synoviocytes in rats with adjuvant arthritis through caspase 3 activation phenolic compounds of abies nephrolepis and their no production inhibitory activities therapeutic effect of 7,3 0 -dimethoxy hesperetin on adjuvant arthritis in rats through inhibiting jak2-stat3 signal pathway 0 -dimethoxy hesperetin inhibits inflammation by inducing synovial apoptosis in rats with adjuvant-induced arthritis the inhibitory effect of phenylpropanoid glycosides and iridoid glucosides on free radical production and b2-integrin expression in human leucocytes anti-inflammatory effect of the 5,7,4 0 -trihydroxy-6-geranylflavanone isolated from the fruit of artocarpus communis in s100b-induced human monocytes release of acetylcholine by syringin, an active principle of eleutherococcus senticosus, to raise insulin secretion in wistar rats specific dietary polyphenols attenuate atherosclerosis in apolipoprotein e-knockout mice by alleviating inflammation and endothelial dysfunction distribution and biological activities of the flavonoid luteolin cytotoxic activity of flavonoids and extracts from retama sphaerocarpa boissier apigenin modulates gabaergic and glutamatergic transmission in cultured cortical neurons inhibitory effects of luteolin on human gastric carcinoma xenografts in nude mice and its mechanism catechinincorporated dental copolymers inhibit growth of streptococcus mutans biological activity of five antibacterial flavonoids from erythrophyllum (combretaceae) dietary flavonoids: molecular mechanisms of action as anti-inflammatory agents structural requirements of flavonoids for inhibition of antigeninduced degranulation, tnf-a and il-4 production from rbl-2h3 cells studies on physiologically active substances in citrus fruit peel. part xx. structure and physiological activity of phenyl propanoid glycosides in lemon (citrus limon burm. f.) peel structural requirements of flavonoids for nitric oxide production inhibitory activity and mechanism of action the endocrine activities of 8-prenylnaringenin and related hop (humulus 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b-endorphin secretion by syringin, an active principle of eleutherococcus senticosus, to produce antihyperglycemic action in type 1-like diabetic rats role of sympathetic tone in the loss of syringin-induced plasma glucose lowering action in conscious wistar rats hypoglycemic effect of syringin from eleutherococcus senticosus in streptozotocin-induced diabetic rats antileishmanial phytochemical phenolics: molecular docking to potential protein targets antimicrobial flavonoids and diterpenoids from dodonaea angustifolia the analysis of the volatile and semi-volatile compounds of the paulownia tomentosa flowers by gas chromatography coupled with mass spectrometry the chemistry of color changes in kiri wood (paulownia tomentosa steud.) iii. a new caffeic acid sugar ester from kiri wood pharmacological activities and mechanisms of natural phenylpropanoid glycosides an update on lignans: natural products and synthesis bioactive constituents from chinese natural medicines. xxxvi. four new 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the chemical constituents from the leave of paulownia tomentosa antitumor activities of extracts and compounds from the roots of daphne tangutica maxim new lignans and their biological activities glucosidase inhibitory constituents from toona sinensis isolation, modification and cytotoxic evaluation of flavonoids from rhododendron hainanense enzymatic extraction and antibacterial activity from eucommia ulmoides leaves paulownia in china: cultivation and utilization. asian network for biological science and international development research centre, chinese academy of forestry antiradical and cytoprotective activities of several c-geranyl-substituted flavanones from paulownia tomentosa fruit key: cord-300522-okbupw61 authors: sansone, clementina; brunet, christophe; noonan, douglas m.; albini, adriana title: marine algal antioxidants as potential vectors for controlling viral diseases date: 2020-05-07 journal: antioxidants (basel) doi: 10.3390/antiox9050392 sha: doc_id: 300522 cord_uid: okbupw61 as the covid-19 epidemic expands in the world, and with the previous sars epidemic, avian flu, ebola and aids serving as a warning, biomedical and biotechnological research has the task to find solutions to counteract viral entry and pathogenesis. a novel approach can come from marine chemodiversity, recognized as a relevant source for developing a future natural “antiviral pharmacy”. activities of antioxidants against viruses can be exploited to cope with human viral infection, from single individual infections to protection of populations. there is a potentially rich and fruitful reservoir of such compounds thanks to the plethora of bioactive molecules and families present in marine microorganisms. the aim of this communication is to present the state-of-play of what is known on the antiviral activities recognized in (micro)algae, highlighting the different molecules from various algae and their mechanisms of actions, when known. given the ability of various algal molecules—mainly sulfated polysaccharides—to inhibit viral infection at stage i (adsorption and invasion of cells), we envisage a need to further investigate the antiviral ability of algae, and their mechanisms of action. given the advantages of microalgal production compared to other organisms, the opportunity might become reality in a short period of time. the ongoing global emergency linked to the covid-19 pandemic [1] teaches us that viral diseases can be dramatic all over the world in the present period of climate, political, social and globalization change. scientific solutions were not available and society was unprepared, claiming that research activities for the discovery of compounds for the prevention and treatment of severe and acute viral infections are nowadays a priority. oxidative stress-a loss in the balance between the production of free radicals including reactive oxygen species (ros) and antioxidant cell signaling pathways [2] -can be a key factor of the pathogenesis in many acute or chronical human diseases [3] . dietary intake of exogenous antioxidants in humans has a well-known role in preventing and protecting cellular oxidative stress. they operate diverse mechanisms of action and possess different therapeutic effects. as a result, modern medicine tends to use them in the prevention and treatment of various oxidative-stress-associated diseases [4] . viral infections are promoted by oxidative processes, the latter acting on replication in infected cells, cell proliferation inhibition and cell apoptosis induction [5] . in patients affected by herpes simplex [6] , it was observed that an increase of the ros-induced membrane phospholipids peroxidation caused dysfunction of vital cellular processes such as membrane transport and mitochondrial respiration [7] . epstein-barr virus (ebv) infections cause an increase in dna damage and significant ros accumulation, and, interestingly, treatment with ros scavengers was able to lower dna damage in both mitogen-stimulated and ebv-infected cells [8] . in this framework, discovery and screening of antioxidant compounds with antiviral properties is promising since the treatment of viral diseases requires the suppression of viral replication and cell survival promotion. the most known ros scavenger compounds belong to different chemical families, such as polyphenols (phenolic acids, flavonoids, anthocyanins, lignans and stilbenes), carotenoids (xanthophylls and carotenes), sterols, or vitamins (vitamins b, d, e, and c) [9] . for instance, there is a lot of evidence of the capability of natural antioxidants such as vitamins c and e (ascorbic acid and tocopherol, respectively) or polyphenols (e.g., curcumin) scavenging ros levels in infected cells, inhibiting proapoptotic factors and thus reestablishing the intracellular equilibrium between the stress-related proteins (such as c-jun n-terminal kinases-jnk) and promitotic (mapk) and transcription factors (nf-kb) [10] [11] [12] [13] . the most effective antioxidants are mainly synthetized by photosynthetic organisms, sharing these precious compounds with herbivorous animals through diet, and bioaccumulating along the trophic web [14] , as occurs in marine systems [15] . indeed, marine organisms represent a rich source of antioxidants, in terms of quantity and/or diversity. vitamins b 12 , c, d, e, peptides, amino acids, chitooligosaccharide derivatives, carotenoids, sulphated polysaccharides, sterols, phlorotannins, phenolic compounds and flavones are examples of marine antioxidant richness [16] . it is not surprising, therefore, that marine pharmacology is increasingly growing. recently, mayer et al. [17] reported that 21 studies were published in 2014/2015, focusing on marine antiviral drugs acting against human enterovirus 71, human cytomegalovirus, human immunodeficiency virus type-1 (hiv-1), human herpes simplex virus (hsv), influenza virus, hepatitis b virus, murine norovirus, respiratory syncytial virus (rsv) or sindbis virus. the mechanism of action of these compounds is known for five of them, although for the others it is still undetermined [17] . the aim of the present study is to show the state of the art, exploring the potential antiviral activity of known marine antioxidant compounds. for that, we explore the relationship between oxidative stress and viral infections, looking for solutions through the deciphering of cell signaling pathways that can inhibit virus replication and infections, and the mechanisms of action of potential antiviral molecules. the global public health emergency of international concern by 30 january 2020 regards the novel coronavirus [18] , which is the direct cause of the severe pneumonia (covid-19), with a high rate of transmission and infectivity. the 85% of critically ill patients with covid-19 showed leukocytosis, high levels of monocytes and neutrophils, and lymphopenia [19] , which is a lack of lymphocytes, with patients dying with comorbidity and high levels of plasma cytokines [20] . lymphopenia and hypercytokinemia are directly correlated with increased severity, mortality and a dysregulated immunological response [21] . first epidemiological indications reveal that the covid-19 patients requiring intensive care are older and are more likely to have hypertension, diabetes, cardiovascular or cerebrovascular pathologies [22] . aging and chronic diseases induce chronic endothelial dysfunction that provokes disassembly of intercellular junctions, endothelial cell death and blood-tissue barrier disruption, along with enhanced leukocyte adhesion and extravasation, which could contribute to explaining the lymphopenia observed in severe covid-19 patients [23] . endothelial dysfunction increases oxidative stress and systemic inflammation, glycocalyx degradation and inducing a procoagulant and antifibrinolytic state [24] . in some, especially old patients, with covid19 infection together with other persistent comorbidity chronic states, these pathways could induce severe respiratory failure [25] . covid-19 infection enters into the elective targets for viral diseases, which are, more generally, epithelial tissues, lung (influenza virus, coronavirus [26] ), liver (hcv and hcv [27] ) and cervix (hpv [28] ), all of which are strongly sensitive to oxidative stress and damages [29] . ros play a fundamental role in the normal functioning of the immune system and in the induction of proliferation of t cells and immunological defense [30] . oxidative stress represents a key factor in inflammation cell signaling for the regulation of cytokines and growth factors, as well as for immunomodulation and apoptosis [31] . recent observations have drawn attention to the possibility that interactions between ros and the human immunodeficiency virus (hiv) may also play a role in the pathogenesis of many other viruses as well [32] . indeed, the role of oxidative stress has been demonstrated in a number of viral infection diseases [33] , where cell pro-oxidative signaling co-occurred with viral infections [34] . viral infection is generally divided into three stages: • stage i, which consists of virus adhesion, adsorption, entry and invasion of cells; • stage ii, or eclipse phase, during which the cell is forced to replicate multiple copies of virus genome; • stage iii, or maturity and release of virus particles (virions). viruses enter the cell through specific receptors and coreceptors using the phagocytosis mechanism. the virus must then break out of the vesicle by which it was taken up in order to gain access to the cytoplasm [35] . the activation of phagocytes induces the release of pro-oxidant cytokines, such as tumor necrosis factor alpha (tnfa), or interleukin-1 and 6, paralleled with a significant iron uptake by the reticuloendothelial system [36] . in turn, the release of tnfa from activated phagocytes induces different intracellular responses. first, it acts on cell mitochondria inducing a pro-oxidant effect through the inhibition of the site of superoxide production (site ii). second, tnfa induces the release of nuclear transcription factor kappa b(nf-k-b) from the cytoplasmic inhibitor protein ikb, activating at a genomic level the transcription of cellular and/or viral genes. many viruses induce host cell death, by apoptosis or pyroptosis among others [37] , probably to enhance replication and expansion into the entire organism [38] . for example, hiv-1 activates pathways causing cd4 t-cell death in infected hosts due to caspase-1-mediated pyroptosis triggered by abortive viral infection. pyroptosis corresponds to an intensely inflammatory form of programmed cell death in which cytoplasmic contents and proinflammatory cytokines, including il-1β, are released. this death pathway relies on the two signature events in hiv infection-cd4 t-cell depletion and chronic inflammation-and creates a pathogenic vicious cycle in which dying cd4 t cells release inflammatory signals inducing enlarged cell death [39] . considering this evidence, antioxidant compounds are able to inhibit immune system cells (lymphocytes) through apoptosis and then release proinflammatory cytokines (il-1, il-6 and tnfa) that are likely to prevent lymphopenia, hypoferremia and, thus lower the viral replication in patients suffering severe viral diseases [40] . antioxidants such as vitamin e [41] are able to prevent the inhibition of the site of superoxide production (site ii), while the nf-kb-induced gene transcription can be inhibited by thiol groups [42] belonging to molecules that strongly interact with the antioxidant system [43, 44] . antioxidant therapy might further be related to the inhibition of virally induced cell death by antioxidants that scavenge peroxides, such as n-acetylcysteine and glutathione peroxidase [45] . it was observed that the antioxidant n-acetylcysteine blocks viral production in monocyte cell lines, which are the major reservoir for hiv in infected patients [46] . the epigallocatechin-3-gallate from green tea blocks hiv entry [47] due to its antioxidant properties. therapeutic treatments to inhibit or reduce virus replication in human cells are unfortunately restricted. known examples are mostly anti-hiv drugs, such as ritonavir, sequinavir, or antiflu, such as lopinavir, abifìgavan, or anti-ebola reagents. they all can have short-term or long-term adverse effects [48] , which makes it necessary to explore new molecular moieties in the perspective of producing new pharmaceutical tools [49] . many antiviral drugs are synthetic organic chemicals or natural derived products, for example, from secondary metabolites of plants [50] . in the emergent "blue" technology era, there is a growing interest in marine-derived antiviral compounds. marine organisms represent a rich source of many antioxidants that are promising for the development of drugs for the prevention and treatment of various chronic and acute human diseases [51] . thus, thousands of compounds from various marine organisms such as algae, bacteria, fungi, marine invertebrates or sponges have been screened and 21 of them have demonstrated antiviral activities [17] . in 2018, fedoreyev et al. [2] reported that an antioxidant "soup", containing echinochrome a (pigment of sea urchins), ascorbic acid (vitamin c) and α-tocopherol (vitamin e) possess in vitro antiviral activity against rna-containing tick-borne encephalitis virus and dna-containing herpes simplex virus type 1 [2] . this study also demonstrated that a synergistic effect of antioxidant molecules can be effective against virus infections. while the research and development of antiviral compounds are mainly focused on macroalgae [52] , the interest in microalgae and cyanobacteria has increased since they are known to produce many antioxidant molecules, which also possess antimicrobial, anticancer and antiviral activities [53] . moreover, cyanobacteria and microalgae in general are promising since the relatively low-compared to higher plants-requirements for growth make them good candidates for the production of antiviral agents at an industrial scale [54] . the compounds mainly capable of antiviral activities comprise sulfated polysaccharides, phenolic compounds, and organic acids [55] . in recent years, it has been shown that marine sulfated polysaccharides possess a wide range of bioactivities, such as anticoagulant, antioxidant, anti-inflammatory, antiviral, antibacterial, antiproliferative, antitumor, anticomplementary and antiadhesive activities [56] . interestingly, the antiviral activity exhibited by these compounds is often related to their immunomodulation and anticancer activities [57] . the double effect, anticancer and antiviral, of these natural compounds is mainly related to their capability to inhibit cell proliferation and to activate ifn signaling pathways that inhibit kidney and liver cancer progression [58] . in general, antiviral compounds extracted from (micro)algae are mainly polysaccharides that have been screened against pathogenic human viruses ( table 1 ). the mechanisms of action of these compounds against viruses are not fully clarified, but the activity seems to be related to their antioxidant power inhibiting the different stages of the viral infection, and interfering with its adhesion, penetration or replication [59] . for example, the sulfated polysaccharide isolated from the cyanobacteria spirulina platensis, named spirulan, has exhibited potent antiviral activity against both the herpes simplex virus type 1 (hsv-1) and the human immunodeficiency virus type 1 (hiv-1) [87] . the sulfated polysaccharides (sps) carrageenans from red algae (rhodophyta) display antiviral effects on several viral agents [60] , and are significantly active against hpv [61] . they predominantly act by inhibiting the binding or the internalization of virus into the host cells [62] . the carrageenan isolated from gigartina skottsbergii exert promising antiviral activities towards diverse strains of hsv-1 and hsv-2 during virus attachment stage [63] and against human rhinovirus (hrv) proliferation by preventing the primary phases of virus replication [64] . a recent in vivo study in mice indicated that low molecular weight carrageenans (3, 5, and 10 kda), as well as acetylated and sulfated derivatives, were active against influenza virus and reduced the hiv activity by depolymerization and sulfation processes [65] . another class of sulfated polysaccharides is represented by galactans, present in the external membranes of red algae [66] . the various structural types of this class of polysaccharides display relevant antiviral activity against several enveloped viruses, such as hsv-1 and hsv-2, denv, hiv-1 and hiv-2, and hepatitis a virus [67] . the galactan sulfate (gs), isolated from the red alga agardhiella tenera, displays an effective control against hiv-1 and hiv-2, blocking the virus adhesion to the cell as well as the attachment of gp120 on cd4+ t cell receptor to hiv-1 gp120 [68] . another sulfated galactan isolated from the red seaweed, schizymenia binderi, presented highly selective antiviral effects against hsv types 1 and 2 by the inhibition of the attachment of virus to host cells [69] . the d,l-galactan hybrid c2s-3, extracted from the marine red alga cryptonemia crenulata, blocks the multiplication of denv-2 in vero cell line [70] . alginates are polysaccharides widely distributed in brown algae (phaeophyceae), which have also been particularly attractive for their antiviral activities [71] . in particular, an alginate named 911 exhibited promising activity against hiv-1 decrementing the activity of reverse transcriptase (rtase), discontinuing the virus adsorption, and immunostimulating the host cells [72] . alternative inhibitory results were also reported against the hepatitis b virus (hbv), where 911 alginate could inhibit the virus replication by suppressing the activity of dna polymerase [73] . furthermore, the sulfated polymannuroguluronate (spmg) is a promising anti-aids drug candidate, inhibiting the robust attachment of hiv-1 gp120 protein with cd4 molecules on the surface of t cells [74] . fucans are high-molecular-weight sulfated polysaccharides, present in the intercellular tissues or mucilaginous matrix of brown algae [75] . beside many bioactivities, such as antiproliferative, antiadhesive properties can protect the cells from viral infections [76] . for instance, the sulfated fucans from the brown seaweeds, dictyota mertensii, lobophora variegata, fucus vesiculosus, and spatoglossum schroederi, could prevent hiv infection via the blocking effect of the activity of reverse transcriptase [77] . this study highlighted the necessity of sulfate and carboxyl groups in the inhibitory viral activity of the polysaccharides. the compound named mc26, a new type of fucose polysaccharide isolated from the marine brown alga, sargassum piluliferum exhibited a strong anti-influenza virus activity [78] . furthermore, the sulfated fucans extracted from the brown seaweed cystoseira indica showed a promising activity against hsv-1 and hsv-2 [79] . it was suggested that these compounds might act against viral infection through the inhibition of virus adsorption [80] . the polysaccharides fucoidan, based on the sulfated l-fucose, possesses various biological activities, among them activities against many rna and dna viruses such as hiv, hsv1-2, dengue virus, and cytomegalovirus [81] . fucoidans exert their antiviral activities by blocking the interaction of viruses with the cells, thus inhibiting viral-induced syncytium formation [82] . this property led to attachment of fucoidan to the f0 protein resulting in a great antiviral potency of fucoidan, e.g., higher than the antiviral drug ribavirin [83] . last but not least, fucoidans also display bioactivity on the immune system at cellular and humoral level by increasing macrophage phagocytosis [84] . the glucan laminaran, one of the most common polysaccharides in brown algae, exhibits a great antiviral activity and low toxicity in vivo [78] . laminaran polysaccharides extracted from brown algae are proficient to prevent the activity of hiv by preventing its adsorption on human-derived lymphocytes and by blocking the ability of hiv reverse transcriptase, thus the virus' proliferation [72] . three polysaccharides extracted from marine microalgae, naviculan from the diatom navicula directa, and two others (named a1 and a2) from the dinoflagellate cochlodinium polykrikoides also displayed antiviral activities against several enveloped viruses, such as hiv-1, hsv-1 or influenza virus type a (ifv-a) [71] . the sulfated polysaccharide p-kg03 extracted from another dinoflagellate gyrodinium impudicum has been also reported active against the encephalomyocarditis rnavirus (emcv) [85] , and against several strains of influenza viruses, with efficiency comparable to some existing drugs [86] . the common characteristic between the above-described examples of antiviral activity from algal polysaccharide is the presence of sulfated groups in their chemical structure [96] . although these compounds might act on stage iii of viral infection, selectively inhibiting reverse transcriptase in the case of hiv, thus hampering production of new viral particles after infection [97] , the inhibitory effect generally might refer to stage i of viral infection. this is a crucial aspect to investigate since the antiviral property starts with the interaction between the molecule and the positive charges of the virus or on the cell surface, preventing penetration of the former into the host cells [98] . indeed, sulfated exopolysaccharides from some marine microalgae have been hypothesized to interfere with stage i of viral infection [99] . therefore, small molecules, such as sulfated polysaccharides, represent a good challenge in antiviral drug discovery studies, since the actual antiviral pharmaceutics are proteins and act at the stage ii of the infection. considering the covid19 disease [100] , this virus is very infective due to the high adhesion capacity on the oral cell surface [101] and for the easy entrance ability through the ace2 receptor on the lung cell surface (stage i of the viral infection) [102] . less investigated, but with a high antioxidant activity, and therefore as potential antiviral, are the polyphenolic compounds: antioxidants produced by marine algae, such as flavonoids, cinnamic acid, benzoic acid, gallic acid, quercetin [91] and phlorotannins, the latter being found in brown macroalgae [92] , or diatoms [93] . phlorotannins are tannin derivatives exhibiting numerous bioactivities, such as antioxidant, anti-inflammatory [94] antibacterial, and antimatrix metallopeptidases (mmp) activities [95] . ahn et al. [93] reported that phloroglucinol derivative 8,8 -bieckol inhibited the activity of recombinant rt and protease of hiv-1 in vitro. another peculiar polyphenol discovered in some diatoms, (haslea sp.) marennine, presented relevant bioactivity and antiviral properties [95] . viral infections are often promoted by oxidative processes, favoring replication in infected cells, induction and inhibition of cell proliferation. antiviral activities of antioxidants acting in the antiviral infection task can be exploited as the sea is a fruitful reservoir of such compounds. many algal sulfated polysaccharides-several of them being exclusively marine-present strong antiviral activities. furthermore, tannins, e.g., phlorotannin-exclusive in brown algae-as well as some phycobiliproteins-exclusive in marine algae-exert antiviral activities. to implement natural antiviral tools, there is a priority of: (i) investigating the antiviral activities and mechanisms of action, and (ii) focusing on microalgae, as fast dividing and easily exploitable organisms. the antiviral activity of various microalgae has been demonstrated, although there is a lack of information on many classes. since microalgae are known to be rich in bioactive molecules-the so-called biofactory cells-and present a high diversity and complementarity of antioxidant compounds, they are the ideal reservoir of a sea-derived antiviral pharmacy. this is a fundamental aspect since synergy between molecules, and combinations of diverse backbones, can be further effective against virus infections, as demonstrated in the development and success of the haart combination therapy for hiv. the promising results reviewed in this communication suggest and urge for investment and advanced research into the development of knowledge on marine antiviral drugs and to develop a pipeline reaching into the biodiversity and chemodiversity of (micro)algae screening to screen them with an antiviral focus. author contributions: all authors have 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blue pigment sulfated seaweed polysaccharides as multifunctional materials in drug delivery applications molecular strategies to inhibit hiv-1 replication cell entry mechanisms of hsv: what we have learned in recent years antimicrobial activities of microalgae: an invited review. in science against microbial pathogens: communicating current research and technological advances; mendez-vilas, a., ed.; formatex microbiology book series coronavirus disease 2019 (covid-19) in italy mechanisms of coronavirus cell entry mediated by the viral spike protein ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia acknowledgments: the authors acknowledge the two reviewers for their comments on the previous version of the manuscript. the authors declare no conflict of interest. key: cord-261170-arnwk287 authors: gallimore, w. title: chapter 18 marine metabolites oceans of opportunity date: 2017-12-31 journal: pharmacognosy doi: 10.1016/b978-0-12-802104-0.00018-4 sha: doc_id: 261170 cord_uid: arnwk287 abstract the marine environment provides an array of compounds often with unique molecular architectures boasting an equally wide array of bioactivities including anticancer, antiinflammatory, and antimicrobial activity. typically without the benefit of folklore therapeutic knowledge, marine organisms are collected, extracted, and fractionated to afford compounds that undergo evaluation with in vivo and in vitro assays en route to clinical applications. the pharmaceutical industry has benefited from research into marine metabolites with the development of marine-derived drugs including cytarabine, vidarabine, and ziconotide along with the more recently developed formulation carragelose, an antiviral spray. cosmetic applications incorporating marine extracts include abyssine and refirmar. research with macroinvertebrates, macroalgae, and microorganisms continue in the hope that drugs of the future will be culled from the oceans of the world. while obtaining a consistent and adequate supply of the bioactive compounds remains a challenge to be overcome, synthetic methods are being employed along with the application of biotechnological techniques to ensure that the drugs, when developed, will be in sufficient quantities for distribution to those who are in need. to gain an understanding of the importance of marine natural products chemistry in drug development g to be able to map the process involved in drug development from marine natural products g to gain an appreciation of the range of biological activities associated with compounds isolated from micro-and macroorganisms g to identify the marine-derived drugs which are undergoing clinical evaluation over 75% of the earth's surface is covered by vast expanses of ocean. its inhabitants are diverse with 15 of the 34 phyla occurring exclusively in the oceans with only one phylum (onychophora) being reported as present on land only [1] . the marine environment provides an array of structurally unique and diverse constituents produced by an equally diverse consortium of marine organisms living on our coral reefs and in benthic communities. the marine organisms are highly variable in species, color, and morphology and belong to several phyla including porifera (sponges), ascidiacea (sea squirts), and octacorallia (soft corals). the metabolites of marine origin emanate from a variety of parts of the plants and animals and are thought to be produced as a form of chemical communication, defense, or to ward off potential predators [2à12] (figs. 18.1à18.3). the potential for a range of applications including anticancer, antibacterial, antiviral, antiinflammatory, antimalarial, antituberculosis activity, as well as pharmacological and industrial applications. the classes of compounds manufactured by marine organisms include alkaloids, terpenoids, shikimates, peptides, and polyketides [2à15] . temperature, salinity, ph, and dissolved oxygen concentrations in the water, thereby providing useful information to facilitate environmental studies [20, 21] . caution should always be exercised in the collection of marine species. gloves should be worn in the collection and subsequent handling of specimens. scuba divers should be clad in wet suits to protect against the possible deleterious effects of chemicals being exuded into the water by the organisms being collected. the personal unfortunate experience (author's) of hours of severe discomfort and rashes as a result of collecting the sponge neofibularia nolitangere from a reef in discovery bay, jamaica, provides clear evidence regarding the level of respect which should be accorded to figure 18 .5 student snorkeling to collect marine specimens. marine organisms whose chemistry is yet to be investigated. records are made of the depth, habitat, global positioning system coordinates (latitude and longitude), color, morphology, and associated organisms. an appropriate coding system should be employed to distinguish specimens. where possible, the specimens are photographed in situ as well as by the dockside (figs. 18.7 and 18.8) . a voucher specimen of each organism is usually preserved in 70% aqueous ethanol for the purpose of taxonomic identification. ascidians are usually preserved in seawater containing menthol crystals with more long-term storage in 10% formalin solution [22] . it should be noted that the recollection of organisms has proved to be a challenge in some instances. an ascidian species, e.g., found to be thriving on the mangrove in the summer of one year could all but disappear from the ecological landscape 6 or 12 months later, while a healthy bed of algae may be short-lived if there are dynamic factors involved in their growth. for example, the occasional nutrient runoff or groundwater seepage event could provide the ideal environment for the growth of selected algal species. environmental factors are key in the marine landscape and often provide a source of frustration to the specimen collector. prior to extraction of the collected organism, the specimens may be frozen, air-dried, freeze-dried, or could be retained in the fresh state. the majority of the marine organisms are extracted fresh or frozen while the remaining specimens are lyophilized or dried in air before extraction [22] . in some instances, dried algal species are ground to a powder prior to extraction as described by sansom and coworkers who isolated an antiproliferative bis-prenylated quinone from the alga perithalia capillaris [23] . the extraction of marine organisms may be carried out using a range of organic solvents including hexanes, dichloromethane, acetone, ethyl acetate, as well as more polar solvents such as ethanol and methanol. in many instances, a mixture of polar and medium polarity or nonpolar solvents is utilized in the extraction protocol. for example, the extraction of the madagascar sponge monanchora dianchora was achieved in ch 3 cl:meoh (1:1) to yield two polycyclic guanidine alkaloids [24] . extractions are usually exhaustively performed over several days with at least three aliquots of the solvent being used. the solvent is then removed in vacuo by rotary evaporation. solvent partitioning is another strategy employed in the extraction of the organisms. this involves single one-step or two-step partitioning systems usually involving an aqueous phase portioned with a solvent immiscible with that phase. the kupchan and modified kupchan procedures are often employed in natural products as was described in the isolation of a diterpene from an axinella species [25] . in this procedure, the concentration of the aqueous layer is progressively adjusted to afford three or four different fractions. complex partitioning procedures are also employed, albeit rarely so. simple partitioning has been most commonly employed with kupchan schemes being utilized with less frequency [22] . chromatographic methods of separation include gravity column chromatography, flash column chromatography, and vacuum liquid column chromatography utilizing silica gel as the packing material. with silica gel, the components of the marine extract are separated on the basis of polarity of the compounds. as the polarity of the eluting solvent increases compounds of increasing polarity are eluted from the column with hydrocarbons, e.g., eluting before alcohols. the elution of the components of a column is monitored by using thin layer chromatography (tlc) plates which are spotted to show the sequence of elution of the compounds (figs. 18.9 and 18.10). bonded reverse phase silica is employed in instances where the constituents of the marine extract include polar metabolites. bonded phases include ods (c 18 ), c 8 , cyano, and diol columns. separation of constituents may also be effected using gel permeation chromatography which effects separation of constituents on the basis of the size of the compounds. in this regard, sephadex lh-20 is commonly utilized in marine natural products isolation work [26] . resins such as biobeads, amberlite, xad-2, and xad-4 are also utilized in separating components of relatively high polarity. the use of xad-2 in the separation of antiviral trisulfated triterpene glycosides from the sea cucumber staurocucumis liouvillei is one such example in marine natural products isolation work [27] . the use of hplc employing a reversed phase stationary phase system is commonplace in marine natural products isolation work with c 18 and c 8 semipreparative and preparative columns being used. mplc and recycling hplc techniques are related techniques for purification of a range of metabolites including alkaloids, peptides, and terpenoids. tandem systems such as liquid chromatography-mass spectrometry systems are also employed to assist with dereplication efforts. unusual ms peaks in the profile suggest that novel components are present in the fraction or extract being evaluated. those fractions with unusual constituents may then become the focus of the research efforts. solidphase extraction methods are also employed in separating compounds. the structural identification of compounds isolated from the range of marine sources is facilitated by the use of spectroscopic techniques such as 1d and 2d nuclear magnetic resonance (nmr) spectroscopy and infrared (ir) spectroscopy. x-ray crystallographic techniques are also important in aiding in the determination of the stereochemistry of the compound. the identification of nanogram quantities of a novel compound is becoming increasingly more facile with the use of the cryoprobe, capillary probe, and mans probe [15] . in vitro activities of marine metabolites have been investigated for a diverse range of cell systems including antiinflammatory, antimicrobial, and anticancer activities. crude extracts, fractions from crude extracts, as well as pure compounds are typically evaluated for biological activity. the in vitro biological evaluation of the isolated compound may be performed using cell lines from human subjects or animals. brine shrimp, fish, and sea urchin are among the organisms employed in the evaluation of compounds or extracts for ecological and therapeutic importance (figs. 18.11 and 18.12) . a summary of the biological activity of some of the organisms discussed in this section is presented in preclinical trials are an essential component of the process of evaluation of the therapeutic potential of a compound. these trials often include animal models such as rats, dogs and monkeys. the major sources of biologically relevant compounds have been found to be from sponges, coelenterates, algae, echinoderms, ascidians, molluscs and microorganisms [14] . macroinvertebrates include sponges, ascidians, and soft coral. it has been found that the vast majority (75%) of novel compounds obtained from the marine environment have been sourced from the porifera and coelenterata (cnidaria) phyla [15] . scheme 18.1 shows representative structures of compounds isolated from macroinvertebrates. macroinvertebrates include: 1. sponges 2. ascidians 3. soft coral sponges (porifera) are sedentary, filter feeding metazoans which utilize a single layer of flagellated cells (choanocytes) to pump water current through their bodies in a unidirectional manner. there are over 5000 species of sponges accounting for much of the epifaunal biomass. extracted fresh or freeze-dried, sponge extracts are an important source of biologically active compounds. these isolates exhibit an impressive array of biological activities, some of which are described here. one sponge which has gained a place in history due to the promising biological activity being displayed is halichondria okadai, the producer of halichondrin b, which underwent evaluation as an anticancer agent. okadaic acid, also from h. okadai, exhibited inhibitory activity against phosphatase-1 and phosphatase-2a [28] (fig. 18 .13). agelaspin, an antitumor glycosphingolipid obtained from the marine sponge agelas mauritianus, demonstrated antitumor activity in vivo against murine b16 melanoma. this compound was also found to stimulate the immune system. a derivative of agelaspin, krn-7000, underwent clinical investigations for cancer immunotherapy [28] . more recently, the extracts of another agelas sp., a. nakamurai, contained the compound agelasine d which exhibited high antibacterial activity [29] . the deep water sponge discodermia dissoluta produced discodermolide, a polyhydroxylated lactone which exhibited anticancer activity, as well as immunosuppressive activity. it was found to stabilize microtubules in a manner similar to the drug taxol and underwent evaluation for use in tumors resistant to taxol [30, 31] . dysidea arenaria was found to contain arenastatin a which showed potent activity against kb cell lines (ic 50 5 5 pg/ml) [32] . girolline is a substituted imidazole isolated from the sponge pseudaxinyssa cantharella which functions by inhibiting the termination step in eukaryotic protein synthesis. having entered phase 1 clinical trials, it was withdrawn due to its adverse hypertensive effects seen in treated patients [28] . mycalamides a and b are protein synthesis inhibitors isolated from the new zealand sponge mycale sp. in vivo activity against a59 coronavirus was observed in mice when treated with a 2% mycalamide mixture at a dosage of 0.2 μg/kg daily with 100% survival over a two-week period. pure mycalamide a inhibited the herpes simplex virus 1 and polio virus type 1 at a concentration of 0.005 μg/disk. mycalamide b was found to exhibit more potent antiviral activity and cytotoxicity than mycalamide a [28] . the baculiferins i, j, l, and m from the marine sponge iotrochota baculifera have been found to inhibit human immunodeficiency virus-1 (hiv-1) with ic 50 values between 0.2 and 7.0 μm [33] . jasplakinolide, the first example of a cyclodepsipeptide isolated from a sponge, is a 19-membered macrocyclic depsipeptide from the jaspis sp. exhibiting in vitro antimicrobial activity at a minimum inhibitory concentration of melinacidins and gencidin antibacterial marinospora sp. lynamicins a-e antibacterial 25 μg/ml against candida albicans. with a topical administration of 2% jasplakinolide solution, an effect similar to that of miconazole nitrate was achieved in vivo [28] . discorhabdin r is a novel pyrroloiminoquinone isolated from the southern australian sponge negombata sp. and antarctic latruncula sp. which was found to display antibacterial activity against both gram-positive (staphylococcus aureus and micrococcus luteus) and gram-negative bacteria (serratia marcescens and escherichia coli), respectively [34] . antibacterial activity against a strain of the bacterial parasite plasmodium falciparum was reportedly identified in monanchora arbuscula with the active agents being the batzellidine alkaloids (ic 50 5 0.2à0.9 μm) [35] . an important isolate from a spongia sp. is the polyhydroxylated steroid, agosterol a, which functions by reversing multidrug resistance caused by the overexpression of two kinds of membrane glycoprotein in cancer cells [36] . from the phylum cnidaria the genera sinularia and briareum have proven to be prolific sources of novel compounds. cembranoids, 5,8-epidoxysteroids, sinulaflexiolides, and africanenes have been isolated from sinularia species [1] (fig. 18.14) . examples of other species of soft corals include the taiwanese soft coral cespitularia taeniata which was extracted with ethanol to yield a group of verticillene diterpenoids including cespitulactam k. the compounds were evaluated against human epidermal carcinoma and murine l1210 leukemia cell lines. cespitulactam k exhibited activity against the cancer cell lines (3.7à5.1 μg/ml) and also showed marked antimicrobial activity against m. luteus and cryptococcus neoformans [37] . the methanol extract of the octocoral muricea austera showed in vitro activity against chloroquine-resistant p. falciparum and was found to contain a range of different classes of compounds including tyramine derivatives, steroidal pregnane glycosides, and sesquiterpenoids [38] . cytotoxic dolabellane diterpenes were isolated from the formosan soft coral clavularia inflata var luzoniana and bioactivity against p388 cell lines with ed 50 values between 0.5 and 3.6 μg/ml was observed [39] . tunicates, sea squirts, or ascidians belong to the subphylum of tunicata (urochordata). they are so named because of their cellulose-containing protective tunic surrounding the organism. tunicates attach to a substratum, usually a marine solid surface such as a mangrove root, rocks, jetties, or even algal species (fig. 18.15 ). much like sponges and soft corals, ascidians have also been found to be a good source of bioactive agents. didemnin b, isolated from the tunicate trididemnum solidum, is one such bioactive compound, showing remarkable antiviral and cytotoxic activity. didemnin b demonstrated activity against p388 and l1210 murine leukemia cell lines. it was advanced into preclinical and clinical trials 1 and 2, but had to be withdrawn due to its harsh toxicity [30] . aplidine, formally known as dehydrodidemnin, an isolate from the mediterranean tunicate aplidium albicans, is one such bioactive compound. being structurally related to didemnin b, aplidine was found to be up to 10 3 more active and less toxic than didemnin b. it entered into phase 1 clinical trials in 1999 under investigation for the treatment of solid tumors and non-hodgkin's lymphoma. broad spectrum activity was displayed in vitro and in vivo against leukemia, melanoma, breast, ovarian, colon, and lung (nonsmall cell) cancer. having advanced to phase 2 clinical trials, aplidine affects protein synthesis through gtp-dependent inhibition of elongation factor 1-α [30] . the extract of the palauan ascidian didemnum guttatum afforded the sulfonated serinolipid cyclodidemniserinol trisulfate which exhibits an antiviral effect by inhibiting hiv-1 integrase, an attractive target for antiretroviral chemotherapy [30] . macroalgae belong to three main phyla: rhodophyta (red algae), chlorophyta (green algae), and phaeophyta (brown algae). biological activities identified in extracts and metabolites of algal origin include anticancer, antiobesity, neuroprotective, and antioxidant activity and scheme 18.2 shows chemical structures of representative bioactive compounds isolated from the macroalgae. a wide range of algal species are utilized in fresh or dried forms as food particularly in asian countries where folklore traditions govern their industrial and medicinal usage [40] . macroalgae are the source of agar, carrageenan, and alginate, which are all of importance in the food industry. the range of compounds isolated from algal sources has been variable. representative examples of bioactive constituents from macroalgae are mentioned below. cytotoxic activity has been identified in 8α,11-dihydroxypachydictyol a, a diterpenoid compound from a dictyota sp. collected on bangsaen beach in thailand. antimalarial activity was also found in the diterpene isolated from this extract when the compound was tested with malarial parasites [41] . stypolactone, an isolate from the brown alga stypopodium zonale, was found to exhibit weak cytotoxic activity in vitro when evaluated with a-549 and h-116 cell lines [42] . zonaquinone acetate, obtained from jamaican populations of s. zonale, displayed in vitro activity against breast and colon cancer cell lines [43] . specimens of taonia atomaria produced atomarianones a and b which were reportedly found to be cytotoxic against nsclc-n6 and a-549 cell lines [44] (fig. 18.16) . crude extracts of algal species have been found to exhibit a range of biological activities. for example, aqueous extracts of gracilaria corticata and sargassum oligocystum exhibit bioactivity against cancerous human leukemia cells [45, 46] while a methanol extract of plocamium telfairiae was observed to display bioactivity against ht-29 colon cancer cells [47] . antiinflammatory activity was found in the green alga from which 2-(2,4 0 -dibromophenoxy)-4,6-dibromoanisol was isolated. this activity was identified using a snake toxin-induced mouse limb model [48] . also exhibiting antiinflammatory activity is a mixture of phytosterols obtained from dunaliella tertiolecta. when administered in a sheep model of inflammation-induced cytokine production, an inhibitory effect was observed [49] . polyphenolic extracts from the red alga laurencia undulatea displayed antiinflammatory activity in vivo. these extracts served to inhibit asthmatic reactions in mice sensitized and challenged with ovalbumin which was used to induce murine allergic reactions in test subjects [50] . antiinflamatory agents floridoside and d-isofloridoside from the south korean alga l. undulatea were found to inhibit free radical oxidative stress at ic 50 values between 22 and 43 μm [51] . biologically active compounds have been isolated from the brown seaweed dictyota cervicornis from which was obtained sulfated polysaccharides with powerful anticoagulant activity [52] . antioxidant activity, evaluated using the dpph method, was reported in phenolic isolates of halimeda monile when liver injury was induced in a rat model. the phenolic fraction was administered over a 20-day period and led to protective effects against chemicals harmful to the liver [53] . with ic 50 values between 0.5 and 2.9 μm, potent antimalarial activity against the human malarial parasite p. falciparum was identified in new macrolides bromophycolides j, m, n, o, p, and q from the red algae callophycus serratus [54] collected in fiji. the marine alga halimeda tuna was studied by koehn and coworkers, leading to the isolation of halitunal, a diterpene displaying in vitro antiviral activity against murine coronavirus a59 [55] . ecologically important roles are played by some compounds from alga sources. for example, halimedatrial, a diterpene isolated from halimeda lamouroux, exhibited toxicity toward reef fishes and appeared to be a feeding deterrent. antimicrobial activity was also reported from this compound [56] . almost 20% of all bioactive marine compounds currently being studied are obtained from marine microorganisms [15] . these microbes are found in swabs from the surfaces of marine plants and animals, suspended in the water from geothermal vents and deep water environments, or on sediment surfaces. they thrive in a variety of environments including locales characterized by high pressures of up to 600 atmospheres, high temperatures, and high salinities. efforts at culturing some of the microorganisms have met with varying degrees of success. the ability to propagate these microorganisms in an economically feasible way will be of great significance as potent bioactive metabolites are discovered [57] (figs. 18.17à18.19 ). marine microorganisms are found 1. on the surface of marine plants and animals 2. suspended in water 3. on sediment surfaces historically, terrestrial microbes have been a potent source of pharmaceutical agents with the seminal discovery of penicillin. the discovery of new antibacterial agents is a serious priority because of the development of potent resistance to current antibiotics on the market. marine bacteria produce a wide variety of secondary metabolites for the purpose of defending themselves against other microbes. scheme 18.3 shows structures of representative compounds from microorganisms associated with marine specimens. marine bacteria which produce compounds of biological significance include pseudoalteromonas species which was found to produce 3,3 0 , 5,5 0 -tetrabromo-2,2 0 -diphenyl diol, an inhibitor of methicillin-resistant s. aureus. the class of 4-methoxypyrrole-containing compounds, the tambjamines, isolated from p. tunicata, was found to be active antifungal, immunosuppressive, and antimicrobial agents. biologically active compounds from marine bacteria also include streptomyces species from sediment and fish gut from which anticancer (e.g., halichomycin and δ-indomycinone) and antibacterial agents (e.g., phenazines) have been obtained [58à60]. vibrio species obtained from sponge specimens have produced phenolic and trisindole compounds with antibacterial activity [61, 62] . a micromonospora sp. obtained from a soft coral produced thiocoraline, a compound exhibiting anticancer activity [63] . marine fungi have also been known to produce compounds with a range of bioactivities including antiviral, antifungal, enzyme inhibition, and anticancer and antibacterial activities. the isolation and cultivation of fungi from the marine environment is of critical importance for propagation of the microbes from which biologically relevant compounds may be obtained. protocols have been established for this work [64] . fungal species which have produced antibacterial compounds include corallospora pulchella isolated from sand. this species produced melinacidins and gencidin [65] . anticancer activity has been reported from metabolites of aspergillus sp. (including the aspergillamides and fumiquinazolines) and penicillium sp. sourced from a marine alga which was found to contain pentostatins and communesins among other compounds [66, 67] . antiviral activity, attributable to the presence of halovirs, was identified in a scytalidium sp. collected from a seagrass species. potent antiviral activity against h. simplex virus (type 1) was observed and may be acting by binding directly to the virus [68] . actinomycetes have been the source of a wide range of antimicrobial agents, the most common of which include tetracycline and streptomycin. other bioactive compounds originating from actinomycetes include antitumor and antimicrobial agents. a marinospora sp. produced a group of bisindole pyrroles, lynamicins aàe, which exhibited biological activity against gram-positive and gram-negative species. importantly, activity was also shown against drug-resistant pathogens including methicillin-resistant s. aureus [69] . anticancer activity against lung, colon, and breast cancer cell lines was exhibited by isolates from the fermentation of a streptomyces sp. (mbg-04-17-069). tartrolon d was found to be the bioactive agent [70] . microalgae are found in seven phyla. these include chlorophyta, phaeophyta, rhodophyta, crystophyta, cryptophyta, eugelophyta, and pyrrhophyta. the blue-green algae, cyanophyta, are cyanobacteria which have been found to share characteristics with eukaryotic algae. these microalgae produce compounds with a high degree of structural diversity and species, such as lyngbya majuscula, have produced a vast array of biologically active compounds [71] . curacin a, e.g., isolated by gerwick and coworkers in 1994 [72] , was found to function by disturbing microtubule assembly, thereby functioning as a lead compound in chemotherapy. microcystis aeruginosa is the source of potent protein phosphatase-1 and phosphatase-2a inhibitors identified in microcystins [73] . other microalgal species under examination include dinoflagellates which produce an array of bioactive toxins including saxitoxin and maitotoxin which function by blocking or activating sodium/calcium channels. challenges exist with respect to the culturing of these organisms due to relatively low proliferation rates and the large quantities of culture required to obtain small amounts of bioactive compounds. diatoms, microscopic unicellular colonial algae, grow at a faster rate and are amenable to culturing but few bioactive metabolites have been identified from these microalgae [28] . some marine compounds sourced from microbes are of clinical significance, undergoing evaluation as potential pharmaceutical agents. the marine-derived drug pipeline, almost nonexistent in decades gone by, now has a range of candidates at various stages of development as shown in table 18 drugs in phase three clinical trials include tetrodotoxin, a guanidinium alkaloid under the trademark name tectin obtained from the pufferfish [34] . affecting the sodium channels, this drug is being investigated for the treatment of chronic pains (scheme 18.5). a depsipeptide from a tunicate, plitidepsin, is being tested by pharmamar in the treatment of a variety of cancers, namely leukemia, multiple myeloma, and lymphoma. another drug under evaluation by pharmamar for cytotoxic activity is zalypsis (pm00104) sourced from a mollusc which targets the dna-binding capacity of diseased uterine, lymphoma, cervical, and endometrial cancer cells. the alkaloid-derived compound pm01183 is another drug candidate from pharmamar being evaluated for its efficacy against a range of cancers including ovarian, breast, lung, acute leukemia, and endometrial cancer [74, 75] . bryostatin i, from the bryozoan bugula neritina has been involved in a battery of clinical trials being investigated for its potency against cancer. it is currently under phase i evaluation as a treatment for alzheimer's [74] . in the early years, the challenge associated with the supply of the drug was underscored by the fact that, in order to obtain 18 g of a cgmp quality bryostatin i, 13 tonnes of b. neritina had to be collected in californian waters [13, 76] . the gene cluster of the uncultivated microbial symbiont of b. neritina, candidatus endobugula sertula has been successfully identified, thereby opening the potential for the supply of the compounds [77] . kahalalide f, a cyclic depsipeptide, was found in the mollusc elysia rufescens as well as the green algae bryopsis sp. on which it feeds. this compound is currently in phase i/ii trials as a treatment against prostate cancer [74] (fig. 18.20) . dmxba [(3-(2,4-dimethylxybenzylidene)]-anabaseine is a derivative of anabeseine, an alkaloid found in marine worms. found to improve cognition in animal models, dmxba and other related compounds have demonstrated neuroprotective activity in both in vitro and in vivo screens. thought to have an effect on macrophage 7 receptors, antiinflamatory activity was also observed in animal models. phase i evaluation of healthy males and schizophrenics have shown that dmxba has led to marked improvements in cognitive function [74] . there are several marine compounds sourced from microbes which are of clinical significance. clinical trials are being conducted on plinabulin (npi-2358), a vascular disrupting agent obtained from a marine fungal extract with potential for activity against multidrug resistant tumor cells. marizomib (salinosporamide a, npi-0052), an isolate from a marine bacterium salinospora tropica, is a novel proteasome inhibitor which is currently under investigation for its efficacy against solid tumor models. the compound exhibits low cytotoxicity to normal cells and has significant potential for oral and intravenous administration [74] . the ultimate goal of many marine natural products and synthetic chemists is that the isolated or synthesized molecule possesses therapeutic applications. there are several food and drug association (fda)-approved drugs of marine origin obtained from sponges, a fish, a cone snail, a mollusc, and cyanobacterium species, while yondelis (trabectidin) obtained from the ascidian ecteinascidia turbinata, has been approved in the european union. the antitumor effects of aqueous ethanol extracts of e. turbinata were observed from 1969. in vitro trials had been carried out on a 60 human cancer cell panel by the company developing the drug, pharmamar, and the national cancer institute. aquaculture of the ascidian proved to be the initial strategy used to obtain sufficient quantities for evaluation of the efficacy of the compound. semisynthetic procedures involving the fermentation of pseudomonas florescens are now currently employed in the pharmaceutical preparation of the drug which is sold in over 80 countries, including south korea and russia, under the trade name yondelis. yondelis is also used in patients with relapsed platinum-sensitive ovarian cancer. this drug is currently under evaluation in phase ii for breast, prostate, lung, and pediatric cancers. the sponge tethya crypta (cryptotethia crypta) was the original source from which the drug cytarabine was developed. cytarabine is a synthetic analogue of the nucleoside which was originally isolated from the sponge. sold under the trade name cytosar-u, this cytotoxic agent inhibits deoxyribonucleic acid (dna) polymerase and dna synthesis. acute lymphocytic leukemia, non-hodgkin's lymphoma, and acute myelocytic leukemia are among the conditions being treated by this drug approved by the fda in 1969 [74] . produced by fermentation of streptomyces griseus, cytarabine has limited bioavailability but improvements in the delivery system have been made [78] . a slow-release liposomal form of cytarabine (depo cyle) has been approved in the united states and europe for the prolonged administration/exposure in cerebrospinal fluid. a related drug, vidarabine (vira-a), was developed from spongouridine and found use as an antiviral treatment for epithelial and superficial keratitis caused by the h. simplex virus types 1 and 2. viral dna polymerase and dna synthesis of herpes are inhibited by this drug which was discontinued over 10 years ago. this drug is still in use in europe for ophthalmological challenges. prialt (ziconotide) was obtained from a peptide ω-conotoxin mviia isolated from the cone snail conus magus. with a unique mode of action, this drug acts by reversibly blocking n-type calcium channels in some specific nerves in superficial layers of the spinal cord. this drug is used for the management of severe and chronic pains in patients suffering from cancer and acquired immunodeficiency syndrome who are unable to use or are unresponsive to other drugs such as morphine. ziconotide had to be synthesized using solid-phase peptide synthesis due to the insufficient quantities supplied by the cone snail, c. magus [79] . the blockage of the spinal cord induced by this drug prevents the release of neurotransmitters responsible for pain from specific neurons. related conus peptides are undergoing evaluation in human clinical trials [80] . brentuximab vedotin (sgn-35) is being marketed under the trade name adcetris by seattle genetics and has gained repute for the treatment of hodgkin and systemic anaplastic large cell lymphoma [81] . this drug is an analogue of dolastatin 10, a compound isolated from the sea hare dolobella auricularia, which was later found to be produced by diet-associated cyanobacteria symploca hydnoides and l. majuscula. preliminary phase i and ii clinical trials of dolastatin 10 and a related analogue were largely unsuccessful. antibody-drug conjugates function by selectively delivering the drug to the cancer cell by linking the dolastatin 10, e.g., to an antibody that targets a cell membrane protein on the surface of hodgkin's lymphoma cells. this technology has proven to be a seminal development. omega-3 fatty acids from fish oils are being marketed under the trade name lovaza by glaxosmithkline. used in the treatment of hypotriglyceridemia, the drug controls ethyl esters of eicosapentaenoic acid and docosahexaenoic acid and functions by lowering triglyceride levels. [81] . eribulin mesylate (e 7389), with the trade name halaven was formulated from the macrolide halichondrin b sourced from the sponge h. okadai. studies related to the anticancer activity of simpler analogues of halichondrin b showed that the efficiency is retained leading to the development of eribulin mesylate which is more water soluble than the parent macrolide. now approved for use, potent and irreversible inhibition in cancer cells medicated by this drug resulted in the death of the cells by apoptosis. in the absence of tubulin, cell growth grinds to a halt. related compounds are currently being evaluated in phase ii trials [81] . one of the more recent formulations on the market is carragelose, an antiviral nasal spray which functions by creating a physical antiviral barrier in the nasal cavity. the company marinomed biotechnologie gmbh, utilized iotacarrageenan, sulfated polysaccharides found in the rhodophyceae seaweed as well as other seaweeds. the product is effective against the early symptoms of the common cold [81] . it should be noted that, in addition to the pharmaceutical applications of marine-sourced therapies, a range of cosmetic applications also exist and are thriving industries. the foray into cosmetic applications was led by estee lauder with the antiaging skin care remedy resilience which contains an extract from the caribbean sea whip pseudopterogorgia elisabethae. the active antiinflammatory and analgesic agents are the pseudopterosins, tricyclic diterpene glycosides, which have been found to inhibit pla2 and 5-lipoxygenase. derivatives of the pseudopterosins underwent phase i and ii trials to examine wound healing efficiency but the lipophilic and insoluble nature of the compounds have served to limit its potential as an effective drug. compounds from this group of tricyclic diterpene glycosides also underwent preclinical evaluation as antiinflammatory drugs [81] . abyssine is marketed as a product used to soothe and reduce irritation in skin sensitive to ultraviolet b light as well as chemical and mechanical attack. it consists of an extract from an alteromonas species and contains a high molecular weight polymer with two different oligosaccharides (exopolysaccharide), while seacode represents another exopolysaccharide which occurs as a mixture of extracellular glycoproteins and other glucidic exopolymers produced by fermentation of a pseudoalteromonas sp. this product has been found to improve skin roughness after up to four weeks of administration. refirmar, a recent product to be introduced, was obtained from an intracellular extract from a fermentation of a new pseudoalteromonas sp. isolated from a deep (2300 m) hydrothermal vent in portugal's exclusive economic zone, extraction of the cultured biomass afforded a mixture of macromolecules which inhibit muscle contraction. the hydrating and antiaging potential of the product has been evaluated in vivo and in topically applied formulations [81] . the area of marine natural products chemistry has clearly developed leaps and bounds as evidenced by the relatively large number of marine-derived drugs undergoing evaluation as potential therapeutic agents. buoyed by the potential for the development of natural products from the sea, research work continues to advance with the discovery of new bioactive compounds and new applications for previously isolated molecules [2à15]. the supply issue, however, remains one of critical importance as it relates to the development of drugs from a marine organism. for example, (1) spongistatin 1 has been reported to be highly cytotoxic. it has been deemed to be the most active of all natural and synthetic compounds investigated by the national institute of cancer (usa). three tonnes of the sponge yielded 0.8 mg of the compound. another collection and processing of 400 kg of the sponge afforded 10 mg of the compound. this isolation work facilitated structure elucidation work. the ic 50 value for this compound was evaluated at 10 26 m in colon cancer cells and 10 212 m for breast cancer cell lines [82] . synthetic approaches to the compound have been presented by research groups including petit and coworkers [83, 84] . total synthesis of biologically active marine compounds is often fraught with its attendant challenges due to the length of multistep synthetic procedures and the general complexity of the structural motifs which must take into account stereochemical considerations. propagation through mariculture and aquaculture are also being studied to determine the viability of using these approaches to deal with the challenges associated with procuring sufficient quantities for clinical trials and subsequent formulation into drugs [85] . the timeline from discovering the drug, leading to the entry into the market typically spans a 20-to 30-year period during which time the capital injection is considerable, often necessitating support from the large pharmaceutical entities which are sometimes hesitant about making investments which may not yield significant financial rewards [86] . the caribbean region, being an important source of marine species with which much research work has been carried out, is not likely to become the recipient of the potential benefits to be derived from the development unless more research work in this area is undertaken in the region with support from the appropriate collaborators. in the future, it is expected that new strategies will be employed to ensure the supply of large quantities of the target compounds. these include optimization or fermentation techniques for propagation of microbes, including mixed fermentation methods. biotechnological approaches are likely to include whole genome sequencing, genome mining, genetic engineering, chemoenzymatic synthesis, and in vitro enzymatic synthesis in the hope that new therapeutic drugs will come from our seas [87] . 1. if you were required to evaluate an extract for its potential as a drug, what approach would you adopt? 2. silica gel chromatography is essential for the purification of organic compounds. identify three methods of chromatography. 3. design a form which could be used to document information when collecting a specimen. trends in the discovery of new marine natural products from invertebrates over the last two decades à where and what are we bioprospecting? marine natural products: metabolites of marine algae and herbivorous marine molluscs marine natural products: metabolites of marine invertebrates marine natural products marine natural products marine natural products marine natural products marine natural products marine natural products marine natural products marine natural products marine natural products drugs and cosmetics from the sea marine natural products and their potential applications as anti-infective agents biogeography of sponge chemical ecology: comparisons of tropical and temperate defenses temperature and spatiotemporal variability of salicylihalamide a in the sponge haliclona sp sources of secondary metabolite variation in dysidea avara (porifera: demospongiae): the importance of having good neighbors chemical mediation of interactions among marine organisms marine advanced technology education-marinetech.org. date accessed a survey of deep-water coral and sponge habitats along the west coast of the us using a remotely operated vehicle. noaa technical memorandum nos nccos 138 isolation of marine natural products an antiproliferative bis-prenylated quinone from the new zealand brown alga perithalia capillaris ptilomycalin d, a polycyclic guanidine alkaloid from the marine sponge monanchora dianchora a new cycloamphilectene metabolite from the vanuatu sponge axinella sp bastadin 20 and bastadin o-sulfate esters from ianthella basta: novel modulators of the ry 1 r fkbp12 receptor complex two new cytotoxic and virucidal trisulfated triterpene glycosides from the antarctic sea cucumber staurocucumis liouvillei drugs from the sea from anti-fouling to biofilm inhibition: new cytotoxic secondary metabolites from two indonesian agelas sponges marine natural products and related compounds in clinical and advanced pre-clinical trials discodermolide, a new bioactive polyhydroxylated lactone from discodermia dissolute arenastatin a, a potent cytotoxic depsipeptide from the okinawan marine sponge dysidea arenaria baculiferins aào, o-sulfated pyrrole alkaloids with anti-hiv-1 activity, from the chinese marine sponge iotrochota baculifera marine pharmocolgy in 2000: marine compounds with antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiplatelet, antitubercolosis, and antiviral activities; affecting the cardiovascular, immune, and nervous systems and other miscellaneous mechanisms of action bioactive guanidine alkaloids from two caribbean marine sponges agosterol a, a novel polyhydroxylated sterol acetate reversing multidrug resistance from a marine sponge of spongia sp nitrogen-containing verticillene diterpenoids from the taiwanese soft coral cespitularia taeniata antiplasmodial metabolites isolated from the marine octocoral muricea austera cytotoxic constituents from the formosan soft coral clavularia inflata var. luzoniana a guide to the common edible and medicinal sea plants of the pacific islands novel diterpenes with cytotoxic, anti-malarial and anti-tuberculosis activities from a brown alga dictyota sp an interesting diterpenoid from the brown alga stypopodium zonale antiproliferative activity and absolute configuration of zonaquinone acetate from the jamaican alga stypopodium zonale atomarianones a and b: two cytotoxic meroditerpenes from the brown alga taonia atomaria in vitro antitumor activity of gracilaria corticata (a red alga) against jurkat and molt-4 human cancer cell lines anticancer activity of sargassum oligocystum water extract against human cancer cell lines methanolic extracts of plocamium telfairiae induce cytotoxicity and caspase-dependent apoptosis in ht-29 human colon carcinoma cells biological importance of marine algae a mixture of phytosterols from dunaliella tertiolecta affects proliferation of peripheral blood mononuclear cells and cytokine production in sheep anti-asthmatic effect of marine red alga (laurencia undulata) polyphenolic extracts in a murine model of asthma inhibitors of oxidation and matrix metalloproteinases, floridoside, and d-isofloridoside from marine red alga laurencia undulata biological activities of sulfated polysaccharides from tropical seaweeds free phenolic acids from the seaweed halimeda monile with antioxidant effect protecting against liver injury antimalarial bromophycolides jàq from the fijian red alga callophycus serratus halitunal, an unusual diterpene aldehyde from the marine alga halimeda tuna isolation of halimedatrial: chemical defense adaptation in the calcareous reef-building alga halimeda screening for new metabolites from marine microorganisms δ-indomycinone: a new member of pluramycin class of antibiotics isolated from marine streptomyces sp rare phenazine l-quinovose esters from a marine actinomycete a novel antimicrobial substance from a strain of the bacterium vibrio sp marine natural products. 34. trisindoline, a new antibiotic indole trimer, produced by a bacterium of vibrio sp. separated from the marine sponge hyrtios altum thiocoraline, a new dipsipeptide with antitumor activity produced by a marine micromonospora. 1. taxonomy, fermentation, isolation, and biological activities methods for isolation of marine-derived endophytic fungi and their bioactive secondary products corollospora pulchella, a marine fungus producing antibiotics, melinachidins iii, iv and gancidin w new cytotoxic sequiterpenoid nitrobenzoyl esters from a marine isolate of the fungus aspergillus halovirs a-e, new antiviral agents from a marine-derived fungus of the genus scytalidium lynamicins a-e, chlorinated bisindole pyrrole antibiotics from a novel marine actinomycete tartrolon d, a cytotoxic macrodiolide from the marine-derived actinomycete streptomyces sp. mdg-04-17-069 continuing studies on the cyanobacterium lyngbya sp.: isolation and structure determination of 15-norlyngbyapeptin a and lyngbyabellin d structure of curacin a, a novel antimitotic, antiproliferative, and brine shrimp toxic natural products from the marine cyanobacterium lyngbya majuscula structure and biosynthesis of toxins from blue-green algae (cyanobacteria) the odyssey of marine pharmaceuticals: a current pipeline perspective the bryostatins identification of the putative bryostatin polyketide synthase gene cluster from "candidatus endobugula sertula", the uncultivated microbial symbiont of the marine bryozoan bugula neritina development of cytarabine prodrugs and delivery systems for leukemia treatment industrial natural product chemistry for drug discovery and development conus peptides: biodiversity-based discovery and exogenomics marketed marine natural products in the pharmaceutical and cosmeceutical industries: tips for success marine natural products: a way to new drugs towards a more step-economical and scalable synthesis of spongistatin 1 to facilitate cancer drug development efforts antineoplastic agents. 257 isolation and structure of spongistatin 1 aquaculture of three phyla of marine invertebrates to yield bioactive metabolites: process development and economics mariculture trials with mediterranean sponge species. the exploitation of an old natural resource with sustainable and novel methods marine natural products: a new wave of drugs? key: cord-252108-04xr5xdl authors: havrylyuk, dmytro; zimenkovsky, borys; vasylenko, olexandr; day, craig w.; smee, donald f.; grellier, philippe; lesyk, roman title: synthesis and biological activity evaluation of 5-pyrazoline substituted 4-thiazolidinones date: 2013-06-06 journal: eur j med chem doi: 10.1016/j.ejmech.2013.05.044 sha: doc_id: 252108 cord_uid: 04xr5xdl a series of novel 5-pyrazoline substituted 4-thiazolidinones have been synthesized. target compounds were evaluated for their anticancer activity in vitro within dtp nci protocol. among the tested compounds, the derivatives 4d and 4f were found to be the most active, which demonstrated certain sensitivity profile toward the leukemia subpanel cell lines with gi(50) value ranges of 2.12–4.58 μm (4d) and 1.64–3.20 μm (4f). the screening of antitrypanosomal and antiviral activities of 5-(3-naphthalen-2-yl-5-aryl-4,5-dihydropyrazol-1-yl)-thiazolidine-2,4-diones was carried out with the promising influence of the mentioned compounds on trypanosoma brucei, but minimal effect on sars coronavirus and influenza types a and b viruses. the non-condensed heterocyclic systems with thiazolidinone [1] and pyrazoline [2, 3] moieties have emerged as powerful scaffolds in drug design. among diazole-substituted 4-thiazolidinones highly active anticancer agents have been identified including inhibitors of necroptosis [4] , tumor necrosis factor a [5] and tyrosine phosphatase [6] . our previous study, based on a hybrid pharmacophore approach, allowed to establish a number of patterns in the structureeactivity relationship (sar) context for 4-thiazolidinones with a pyrazoline fragment in 2, 3 and 4 positions of the thiazolidone cycle, which possessed antitumor activity [7, 8] . on the other hand, thiazolidinones and pyrazolines have occupied a unique position in the design and synthesis of novel biologically active agents that exert trypanocidal activity [9e13]. the 2-thioxo-4thiazolidinone-3-acetic acid derivatives were identified as inhibitors of trypanosoma brucei dolicholphosphate mannose synthase [11] . the 2-hydrazolyl-4-thiazolidinone-5-carboxylic acid derivatives have shown promising activity on the cruzipain protease [12] . the most promising compound in series of aryl-4-oxothiazolylhydrazones was shown to be very active at non-cytotoxic concentrations in in vitro assays against trypanosoma cruzi cell cultures and exhibited potency comparable with the reference compounds (ic 50 (y strain) ¼ 0.3 mm) [9] . among pyrazoline derivatives, some novel compounds have been identified as inhibitors of the trypanosomal cysteine protease cruzain with ic 50 of 40e230 nm [13] . the antiviral activity of heteryl substituted 4-thiazolidinones is promising. among thiazoleethiazolidine conjugates [14] and noncondensed derivatives with thiazolidinone and pyridine [15e17] or pyrimidine [18e20] cycles, anti-hiv agents were identified. in addition, this group of compounds was active against hepatitis c virus [21] , tobacco mosaic virus [22] and vesicular stomatitis virus [23] . previously we also demonstrated the efficiency of certain pyrazolineethiazolidinone conjugates on influenza viruses and sars coronavirus [24] . the present work is an extension of our ongoing efforts toward developing promising biologically active agents using a hybrid pharmacophore approach. we made the design (fig. 1 ) and synthesized hybrid compounds by linking the main structural unit of the 4-thiazolidinone ring system with the pyrazoline, and examined their antitumor, trypanocidal and antiviral activities in vitro. we have found two compounds from 5-pyrazoline substituted 4-thiazolidinones, which possessed a commensurate antitumor activity compared to the pyrazolineethiazolidinone analogous compounds reported previously [7, 8] and evaluated anti-trypanosomal activity and antiviral activity of the synthesized compounds. the general methods for synthesis of target thiazolidinonee pyrazoline conjugates are depicted in schemes 1 and 2. the starting 3,5-diaryl-4,5-dihydropyrazoles synthesized using known methods from appropriate chalcones [25] easily reacted with 5-bromothiazolidine-2,4-dione [26] yielding 5-(3-naphthalen-2-yl-5-aryl-4,5-dihydropyrazol-1-yl)-thiazolidine-2,4-dione 1a and 1b. it is known that chemical modification of the n3 position of thiazolidinone cycle has an essential influence on the antitumor activity [27, 28] . relying on these observations we utilized potassium salt of 5-(3-naphthalen-2-yl-5-aryl-4,5-dihydropyrazol-1-yl)-thiazolidine-2,4-dione, generated in situ, in the reactions with 2-chloro-n-arylacetamides. following the mentioned reaction the new n3substituted non-condensed thiazolidinoneepyrazoline conjugates 2ae2c were synthesized. based on the mannich reaction of 1a and 1b with secondary amines the thiazolidinone analogs 3ae3g were obtained (scheme 1). aiming at the detailed elaboration of sar, especially the influence of the linking group of thiazolidinoneepyrazoline conjugates on the anticancer activity, 5-[2-(3,5-diaryl-4,5-dihydropyrazol-1-yl)-2oxoethylidene]-thiazolidine-2,4-diones (4ae4f) were synthesized by the method, described previously [29] . reaction of 3,5-diaryl-4,5-dihydropyrazoles with (2,4-dioxothiazolidine-5-ylidene)-acetyl chloride [27] afforded in excellent yield and purity the compounds 4ae4f. following the reaction of generated in situ potassium salts of 4b and 4e with 2-chloro-n-arylacetamides the group of n3substituted 4-thiazolidinones 5ae5e were synthesized (scheme 2). the data characterization of synthesized novel heterocyclic substituted thiazolidones are presented in experimental part. analytical and spectral data ( 1 h nmr, 13 c nmr) confirmed the structure of the synthesized compounds. protons ch 2 ech of pyrazoline fragment in the 1 h nmr spectra of synthesized compounds showed characteristic patterns of an amx system. the proton (ch) of thiazolidinone core of 1ae1b, 2ae2c and 3ae3g showed the broad singlet at d w5.59e5.99 and the protons of the methylene group (ch 2 co) of 2ae2c and 5ae5e appeared as a broad singlet at d w4.44e4.49 ppm. in the 1 h nmr spectra of the 1ae1b and 4ae4f nh proton of thiazolidinone cycle the broad singlet at dw12.20e12.72 was found. synthesized derivatives 1a, 1b, 2a, 3ae3d, 3f, 4a, 4d, 4e, 4f and 5d were selected by national cancer institute (nci, bethesda usa) developmental therapeutic program (dtp) and evaluated at the concentration of 10 à5 m toward a panel of approximately sixty cancer cell lines (http://dtp.nci.nih.gov). the human tumor cell lines were derived from nine different cancer types: leukemia, melanoma, lung, colon, central nervous system, ovarian, renal, prostate and breast cancers. primary anticancer assays were performed according to the nci protocol as described elsewhere [30e33]. the compounds were added at a single concentration and the cell cultures were incubated for 48 h. the end point determinations were made with a protein binding dye, sulforhodamine b (srb). the results for each compound are reported as the percent growth (gp%) of treated cells when compared to untreated control cells ( table 1 ). the range of percent growth shows the lowest and the highest percent growth found among the different cancer cell lines. the most active compounds 4d and 4f were found to be effective against 12 and 18 cell lines, respectively, compound 4a was found to be moderately effective against few cell lines, while the other compounds (1a, 1b, 2a, 3ae3d, 3f, 4e, 5d) did not show any activity (table 1) . finally, compounds 4d and 4f were selected for an advanced assay against a panel of approximately sixty tumor cell lines at 10fold dilutions of five concentrations (100 mm, 10 mm, 1 mm, 0.1 mm and 0.01 mm) [30e33] . the percentage of growth was evaluated spectrophotometrically versus controls not treated with test agents after 48-h exposure and using srb protein assay to estimate cell viability or growth. doseeresponse parameters were calculated for each cell line: gi 50 e molar concentration of the compound that inhibits 50% net cell growth; and tgi e molar concentration of the compound leading to the total inhibition. furthermore, a mean graph midpoints (mg_mid) were calculated for each of the parameters, giving an average activity parameter over all cell lines for the tested compound. for the mg_mid calculation, insensitive cell lines were included with the highest concentration tested ( table 2 ). the tested compounds showed inhibition activity (gi 50 < 10 mm) against 47 from 55 (4d) and 56 from 59 (4f) human tumor cells with average gi 50 /tgi values of 7.02 mm/38.07 mm (4d) and 4.38 mm/ 50.99 mm (4f) ( table 2) . with regard to the sensitivity against some individual cell lines among several subpanel, the compounds 4d and 4f demonstrated a certain sensitivity profile toward the leukemia subpanel tumor cell lines with gi 50 values range of 2.12e4.58 mm (4d) and 1.64e3.20 mm (4f) ( table 3 ). the sar study revealed that: (1) the level of antitumor activity of active thiazolidinones with pyrazoline fragment in 5 position (4d and 4f) is compatible with effectivity levels of heteryl substituted thiazolidones, described previously [34e42]; (2) conjugation of pyrazoline and thiazolidinone cycles using oxomethylidene linking group (4f) allowed us to increase the activity, in comparison with the structurally related conjugate representative 5-(3-naphthalen-2-yl-5-aryl-4,5-dihydropyrazol-1-yl)-thiazolidine-2,4-dione 1b; (3) introduction of the substituents in 3n-position of thiazolidine fragment did not have significant influence on the antitumor activity. nci's compare algorithm [30e33] allows to assume biochemical mechanisms of action of the novel compounds on the basis of their in vitro activity profiles when comparing with those of standard agents. we performed compare computations for the compounds 4d and 4f against the nci "standard agents" database at the gi 50 and tgi levels (table 4 ). however, obtained pearson correlation coefficients (pcc) did not allow to distinguish cytotoxicity mechanism of tested compounds with high probability. the compound 4d showed the highest correlation at the gi 50 level with dihydroorotate dehydrogenase inhibitor brequinar (pcc ¼ 0.651) and compound 4f e with maytansine (rna/dna antimetabolite, pcc ¼ 0.636). antiviral activity of 1a, 1b, 2ae2c, 3d and 3e was determined against sars coronavirus (sars cov) and influenza types a and b viruses (flu a, flu b). the obtained results are summarized in table 5 . although antiviral activity was evident, virus inhibition occurred at or near the cytotoxic concentration. the compounds showed insignificant activities against the four strains of influenza virus with the range levels of selectivity index from 1.0 to 2.1. compound 2a had moderate activity against the duck strain of influenza a with a 50% effective concentration (ec 50 ) of 21.78 mm and selective index (si) of >16.3; but did not have significant activity against other influenza strains. the majority of the compounds showed no activity against sars cov. the positive control compounds ribavirin, oseltamivir carboxylate, and m128533 were active as expected in the assays. the compounds 1a, 2b and 2c were selected in advanced in vitro assay against trypanosoma brucei brucei (tbb) and trypanosoma brucei gambiense (tbg). the doseeresponse curves with drug concentrations ranging from 10 mg/ml to 0.625 mg/ml are depicted on the results showed a moderated activity of compounds (table 6) on both parasite strains, namely ic 50 (tbb) ¼ 5.43e13.87 mm and ic 50 (tbg) ¼ 2.53e6.66 mm. in the present paper new 4-thiazolidinone based conjugates with pyrazoline moiety at 5 position are described. antitumor activity assay of thirteen synthesized compounds allowed us to identify highly active thiazolidinoneepyrazoline hybrids 4d and 4f, which demonstrated certain sensitivity profile toward the leukemia subpanel tumor cell lines with gi 50 values range of 2.12e4.58 mm (4d) the starting 3,5-diaryl-4,5-dihydro-1h-pyrazole [25] , 5-bromothiazolidine-2,4-dione [26] , and (2,4-dioxothiazolidine-5-ylidene)-acetyl chloride [27] were obtained according to the table 1 anticancer screening data at the concentration of 10 mm. methods described previously. preparation of compounds 4ae4d and 4f was described in our previous report [29] . a suspension of compound 1a or 1b (3 mmol) and potassium hydroxide (3 mmol) was stirred at r.t. during 5 min, later appropriate 2-chloro-n-arylacetamide (3.3 mmol) was added and the mixture was refluxed for 5 h in etoh (10 ml). obtained powders were filtered off, washed with ethanol and recrystallized with dmf:ethanol (1:2) mixtures. (2b). yield 68%, mp 220e222 c. 1 anti-trypanosomal activity of 5-(3-naphthalen-2-yl-5-aryl-4,5-dihydropyrazol-1yl)-thiazolidine-2,4-dione derivatives (1a, 2b, 2c). 137.9, 133.9, 133.3, 133.2, 130.1, 129.3, 128.9, 128.7, 128.6, 128.2, 127.6, 127.2, 127.1, 123.6 a solution of (2,4-dioxothiazolidin-5-ylidene)-acetyl chloride (3 mmol) in 5 ml of dioxane was added to a mixture of appropriate 3,5-diaryl-4,5-dihydro-1h-pyrazole (3 mmol) and triethylamine (3 mmol) in 5 ml of dioxane and later was heated to 70e80 c during 15 min, cooled and poured water (50 ml). obtained powder was filtered off, washed with water and recrystallized with dmf:ethanol (1:2) mixtures. results for each tested compound were reported as the percent of growth of the treated cells when compared to the untreated control cells. the percentage growth was evaluated spectrophotometrically versus controls not treated with test agents. the cytotoxic and/or growth inhibitory effects of the most active selected compounds were tested in vitro against the full panel of human tumor cell lines at concentrations ranging from 10 à4 to 10 à8 m. 48-h continuous drug exposure protocol was followed and an srb protein assay was used to estimate cell viability or growth. using absorbance measurements [time zero (tz), control growth in the absence of drug (c), and test growth in the presence of drug (ti)], the percentage growth was calculated for each drug concentration. percentage growth inhibition was calculated as: ½ðti à tzþ=ðc à tzþ â 100 for concentrations for which ti ! tz; ½ðti à tzþ=tz â 100 for concentrations for which ti < tz: dose response parameters (gi 50 , tgi) were calculated for each compound. growth inhibition of 50% (gi 50 ) was calculated from [(ti à tz)/(c à tz)] â 100 ¼ 50, which is the drug concentration resulting in a 50% lower net protein increase in the treated cells (measured by srb staining) as compared to the net protein increase seen in the control cells. the drug concentration resulting in total growth inhibition (tgi) was calculated from ti ¼ tz. values were calculated for each of these parameters if the level of activity was reached; however, if the effect was not reached or was excessive, the value for that parameter was expressed as more or less than the maximum or minimum concentration tested. the lowest values were obtained with the most sensitive cell lines. compounds having gi 50 values 100 mm were declared to be active. primary antiviral assay was performed on a respiratory viruses panel (flu a (h1n1), flu a (h3n2), flu a (h5n1), flu b, sars cov) [43] . compounds were diluted to 20 mg/ml in dmso then eight half-log dilutions were prepared in mem solution with 50 mg/ml gentamicin. each dilution was added to 5 wells of a 96-well plate with 80e100% confluent cells, and three wells of each dilution were then infected with the test virus using a multiplicity of infection of <0.006 ccid50 per cell for each virus. two wells remained uninfected as toxicity controls. a known active compound was run in parallel as a control. after cytopathic effect (cpe) was observed microscopically, plates were stained with neutral red dye for approximately 2 h, then supernatant dye was removed from the wells and the incorporated dye was extracted in 50:50 sorensen citrate buffer/ethanol and read on a spectrophotometer at 540 nm. the optical density of test wells was converted to percent of cell and virus controls, then the concentration of test compound required to inhibit viral cpe by 50% (ec 50 ) was calculated by regression analysis. the concentration of compound that would cause 50% cytotoxicity in the uninfected cells was similarly calculated (cc 50 ). ec 50 and cc 50 were presented in mm. the selective index (si) is the cc 50 divided by ec 50 . bloodstream forms of t. brucei brucei strain 90-13 and t. brucei gambiense feo strain were cultured in hmi9 medium supplemented with 10% fcs at 37 c under an atmosphere of 5% co 2 [44] . in all experiments, log-phase parasite cultures were harvested by centrifugation at 3000âg and immediately used. drug assays were based on the conversion of a redox-sensitive dye (resazurin) to a fluorescent product by viable cells as previously described [45] . drug stock solutions were prepared in pure dmso. t. brucei bloodstream forms (10 5 cells/ml) were cultured in 96-well plates either in the absence or in the presence of different concentrations of inhibitors in a final volume of 200 ml. after a 72-h incubation, resazurin solution was added in each well at the final concentration of 45 mm and fluorescence was measured at 530 nm and 590 nm absorbance after a further 4-h incubation. the percentage of inhibition of parasite growth rate was calculated by comparing the fluorescence of parasites maintained in the presence of drug to that of in the absence of drug. dmso was used as control. concentration inhibiting 50% of parasite growth (ic 50 ) was determined from the doseeresponse curve with a drug concentrations ranging from 10 mg/ml to 0.625 mg/ml and presented in mm. ic 50 value is the mean þ/à the standard deviation of three independent experiments. yield 82%, mp 268e270 c. 1 h nmr (400 mhz, dmso-d 6 þ ccl 4 ): d 12.71 (br. s, 1h, nh), 8.27 (s, 1h, arom), 7.95e8.11 (m, 4h, arom), 7.88 (s, 1h, ch), 7.56e7.60 (m, 2h, arom) 5-diaryl-4,5-dihydropyrazol-1-yl)-2-oxoethylidene]-2,4-dioxothiazolidin-3-yl}-n-arylacetamides (5ae5e) a suspension of compound 4b or 4f (3 mmol) and potassium hydroxide (3 mmol) was stirred at r.t. during 5 min, later appropriate 2-chloro-n-arylacetamide (3.3 mmol) was added and the mixture was refluxed for 5 h in etoh (10 ml). obtained powders were filtered off 3-phenyl-4,5-dihydropyrazol-1-yl]-2-oxoethylidene}-2,4-dioxothiazolidin-3-yl)-n-p-tolylacetamide (5a) 04 (d, 2h, j ¼ 7.9 hz, arom), 5.70 (dd, 1h, ch 2 ch dmso-d 6 þ ccl 4 ): d 10.04 (s, 1h, nh), 8.02 (s 4 -d ioxoth iazolidin-3-yl )-n-(4-chlorophenyl)-acetamide (5c). yield 79%, mp 289e290 c. 1 h nmr (400 mhz, dmso-d 6 þ ccl 4 ): d 10.51 (s, 1h, nh), 7.98 (s, 1h, coch), 7.83e7.86 (m, 2h, arom), 7.48e7.58 (m, 5h, arom), 7.34e7.42 (m, 3h, arom) -{2-[5-(4-methoxyphenyl)-3-naphthalen-2-yl-4,5-dihydropyrazol-1-yl]-2-oxoethylidene}-2,4-dioxothiazolidin-3-yl)-acetamide (5d). yield 86%, mp 268e269 c 46 (d, 2h, j ¼ 6.4 hz, arom), 7.21 (d, 2h, j ¼ 6.4 hz, arom), 6.90 (br. s, 3h, arom), 5.68 (dd, 1h, ch 2 ch 4-thiazolidones: centenarian history, current status and perspectives for modern organic and medicinal chemistry biological activities of pyrazoline derivatives e a recent development recent advances in the therapeutic applications of pyrazolines structureeactivity relationship study of a novel necroptosis inhibitor photochemically enhanced binding of small molecules to the tumor necrosis factor receptor-1 inhibits the binding of tnf-a 2-thiazolylimino/heteroarylimino-5-arylidene-4-thiazolidinones as new agents with shp-2 inhibitory action synthesis of novel thiazolone-based compounds containing pyrazoline moiety and evaluation of their anticancer activity synthesis of new 4-azolidinones with 3,5-diaryl-4,5-dihydropyrazole moiety and evaluation of their antitumor activity in vitro synthesis, cruzain docking, and in vitro studies of aryl-4-oxothiazolylhydrazones against trypanosoma cruzi synthesis and structureeactivity relationships of parasiticidal thiosemicarbazone cysteine protease inhibitors against plasmodium falciparum, trypanosoma brucei, and trypanosoma cruzi first small molecular inhibitors of t. brucei dolicholphosphate mannose synthase (dpms), a validated drug target in african sleeping sickness synthesis of 2-hydrazolyl-4-thiazolidinones based on multicomponent reactions and biological evaluation against t. cruzi synthesis and structureeactivity relationship study of potent trypanocidal thio semicarbazone inhibitors of the trypanosomal cysteine protease cruzain design, synthesis, and evaluation of 2-aryl-3-heteroaryl-1,3-thiazolidin-4-ones as anti-hiv agents synthesis and anti-hiv studies of 2-adamantyl-substituted thiazolidin-4-ones discovery of 2,3-diaryl-1,3-thiazolidin-4-ones as potent anti-hiv-1 agents design and synthesis of 2-(2,6-dibromophenyl)-3-heteroaryl-1,3-thiazolidin-4-ones as anti-hiv agents synthesis and evaluation of 2-(2,6-dihalophenyl)-3-pyrimidinyl-1,3-thiazolidin-4-one analogues as anti-hiv-1 agents predicting anti-hiv activity of 1,3,4-thiazolidinone derivatives: 3d-qsar approach design, microwave-assisted synthesis and hiv-rt inhibitory activity of 2-(2,6-dihalophenyl)-3-(4,6-dimethyl-5-(un)substituted-pyrimidin-2-yl)thiazolidin-4-ones non-nucleoside inhibitors of the hepatitis c virus ns5b rna-dependant rna polymerase: 2-aryl-3-heteroaryl-1,3-thiazolidin-4-one derivatives synthesis and biological activity of 4-(3-aryl-4-oxo-2-thioxothiazolidin-5-ylimino)-3-methyl-1-(n,n-disubstituted aminomethyl) pyrazolin-5-ones synthesis and antiviral activity of new pyrazole and thiazole derivatives synthesis and anticancer and antiviral activities of new 2-pyrazoline-substituted 4-thiazolidinones synthesis and antidepressant activities of some 3,5-diphenyl-2-pyrazolines synthesis and antihyperglycemic activity of novel 5-(naphthalenylsulfonyl)-2,4-thiazolidinediones synthesis and in vitro anticancer activity of 2,4-azolidinedione-acetic acids derivatives structureeanticancer activity relationships among 4-azolidinone-3-carboxylic acids derivatives synthesis and evaluation of antitumor activity of 5-[2-(3,5-diaryl-4,5-dihydropyrazol-1-yl)-2-oxoethylidene feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines some practical considerations and applications of the national cancer institute in vitro anticancer drug discovery screen cancer drug discovery and development the nci60 human tumour cell line anticancer drug screen synthesis and anticancer activity of novel nonfused bicyclic thiazolidinone derivatives, phosphorus new 5-arylidene-4-thiazolidinones and their anticancer activity a facile synthesis and anticancer activity evaluation of spiro thiazolidinone motif in anticancer drug discovery. experience of dh lnmu medicinal chemistry scientific group synthesis and anticancer activity evaluation of 4-thiazolidinones containing benzothiazole moiety synthesis and anticancer activity of isatin-based pyrazolines and thiazolidines conjugates synthesis and antitumor activity of novel 2-thioxo-4-thiazolidinones with benzothiazole moieties synthesis of new 4ethiazolidinone-, pyrazoline-, and isatin-based conjugates with promising antitumor activity study of molecular mechanisms of proapoptotic action of novel heterocyclic 4-thiazolidone derivatives in vitro and in vivo assay systems for study of influenza virus inhibitors prolyl oligopeptidase of trypanosoma brucei hydrolyzes native collagen, peptide hormones and is active in the plasma of infected mice new protein farnesyltransferase inhibitors in the 3-arylthiophene 2-carboxylic acid series: diversification of the aryl moiety by solid-phase synthesis we are grateful to dr. v.l. narayanan from drug synthesis and chemistry branch, national cancer institute, bethesda, md, usa, for in vitro evaluation of anticancer activity. evaluations of compounds for antiviral activity were supported by funds from contract n01-ai-15435 from the virology branch, division of microbiology and infectious diseases, national institute of allergy and infectious diseases, national institutes of health, bethesda, md, usa. key: cord-277077-kwaiorp8 authors: tița, ovidiu; constantinescu, maria adelina; tița, mihaela adriana; georgescu, cecilia title: use of yoghurt enhanced with volatile plant oils encapsulated in sodium alginate to increase the human body’s immunity in the present fight against stress date: 2020-10-19 journal: int j environ res public health doi: 10.3390/ijerph17207588 sha: doc_id: 277077 cord_uid: kwaiorp8 (1) background: the covid–19 pandemic and the imposition of strict but necessary measures to prevent the spread of the new coronavirus have been, and still are, major stress factors for adults, children, and adolescents. stress harms human health as it creates free radicals in the human body. according to various recent studies, volatile oils from various aromatic plants have a high content of antioxidants and antimicrobial compounds. an external supply of antioxidants is required to destroy these free radicals. the main purpose of this paper is to create a yoghurt with high antioxidant capacity, using only raw materials from romania; (2) methods: the bioactive components used to enrich the cow milk yoghurt were extracted as volatile oils out of four aromatic plants: basil, mint, lavender and fennel. initially, the compounds were extracted to determine the antioxidant capacity, and subsequently, the antioxidant activity of the yoghurt was determined. the 2,2-diphenyl-1-picrylhy-drazyl (dpph) method was used to determine the antioxidant activity; (3) results: the results show that cow milk yoghurt enhanced with volatile oils of basil, lavender, mint and fennel, encapsulated in sodium alginate has an antioxidant and antimicrobial effect as a staple food with multiple effects in increasing the body’s immunity. the antioxidant activity proved to be considerably higher than the control sample. the highest antioxidant activity was obtained on the first day of the analysis, decreasing onwards to measurements taken on days 10 and 20. the cow milk yoghurt enriched with volatile basil oil obtained the best results; (4) conclusions: the paper shows that yoghurts with a high antioxidant capacity were obtained, using only raw materials from romania. a healthy diet, compliance with safety conditions and finding appropriate and safe methods to increase the body’s immunity is a good alternative to a major transition through harder times, such as pandemics. the creation of food products that include natural antioxidant compounds combines both the current great possibility of developing food production in romania and the prevention and reduction of the effects caused by pandemic stress in the human body. according to the food and agriculture organization (fao) of the united states, agri-food production will increase by about 70% in the coming decades [1] . in 2019, romania exported food and animals worth 4.77 billion euros, while imports were over 6.7 billion euros, according to data from the national institute of statistics. the crisis generated by the coronavirus could after several types of research, it was discovered that the volatile oils extracted from different plants bring an extraordinary benefit to the health of consumers. these oils have antiseptic action especially on pathogenic bacteria such as listeria monocytogenes, listeria innocua, salmonella typhimurium. antioxidant activity is another benefit of volatile oils. free radicals cause oxidation of biomolecules, including proteins, amino acids, deoxyribonucleic acid (dna), etc., and eventually cause molecular changes related to ageing, arteriosclerosis and cancer [4] . fennel (foeniculum vulgare) is an aromatic plant that belongs to the apiaceae family and is considered one of the oldest plants cultivated in the world. this is an annual, biennial or perennial plant native to the mediterranean area grown for its aromatic fruits, which are used as culinary spices. it grows especially well in coastal climates and riverbanks [19, 20] . fennel essential oils are used as a flavoring agent in foods such as beverages, bread, pickles, pastries and cheese. it is also used as a component in cosmetics and pharmaceuticals. fennel medicines and fennel essential oils have hepatoprotective effects as well as antispasmodic effects. additionally, volatile fennel oil is known for its diuretic, anti-inflammatory, analgesic and antioxidant activity [21] . the antioxidant and antimicrobial activity of volatile fennel oil is offered by the high content of trans-anethole (63.30%), pinene (11.11%) and fenchone (8.32%) [20] [21] [22] [23] [24] [25] basil (ocimum basilicum) is an aromatic plant that belongs to the lamiaceae family and is considered a rich source of essential oils. the basil plant is native to asia, africa, south and central america [26] . volatile basil oil has antimicrobial, antihistaminic, anti-inflammatory, anthelmintic, antioxidant properties, has an immunomodulatory effect, it is an antidepressant, antidiabetic and anti-hyperlipidemiac, has a hepatoprotective, neuroprotective and cardioprotective effect and it linked to anticancer activity [27] . due to the content of estragole (41.40%), 1,6-octadien-3-ol, 3,7-dimethyl (29.49%), trans-alpha-bergamotene (5.32%), eucalyptol (3.51), citral (3.31%), n-cyano-3-methylbut-2-enamine (3.08%), cis-alpha-bisabolene (1.92%), levomenthol (1.81%), and beta-myrcene (1.11%), volatile oil basil has a good antimicrobial and antioxidant activity [28] [29] [30] [31] [32] [33] . mint (mentha piperita) is an aromatic plant that is part of the lamiaceae family and is widely grown in europe, asia, egypt, south africa and arabia [34] . mint leaves are traditionally used as a tea in the treatment of headaches, fever, digestive disorders and various minor conditions. in modern medicine, mint is widely used in the treatment of gastrointestinal disorders [35] . many studies that have evaluated the antioxidant activities of volatile mint oil have focused exclusively on chemical tests, while the effectiveness of volatile mint oil in preventing oxidative stress at the cellular level or in a living organism has not been characterized [36] . the main components of the mint volatile oil are menthol, menthone, menthofuran, isomenthone, (e)-caryophyllene, 1,8-cineole, linalool, limonene, carvone, pulegone and α-terpineol. they give the volatile mint oil an antioxidant and antimicrobial capacity [37] [38] [39] . lavender (lavandula angustifolia) is an aromatic plant that belongs to the lamiaceae family. lavender is a herbaceous plant native to the mediterranean area and is widely cultivated. the smell of lavender improves mood, reduces mental stress and anxiety, and improves sleep [40] . lavender essential oil is commonly used in aromatherapy and various complementary medicines and cosmetics [41] . numerous studies have shown that volatile lavender oil has antioxidant, antimicrobial and antifungal properties [42] . the antioxidant and antimicrobial capacity of lavender volatile oil is offered by compounds such as the monoterpenoids, linalool, linalyl acetate, 1,8-cineole, β-ocimene, terpinen-4-ol, and camphor [43] [44] [45] [46] . in recent years, romanian entrepreneurs have focused on cultivating aromatic and medicinal plants. they focused especially on plants that are found in the mediterranean area. if until a few years ago we met many crops of vegetables or plants specific to the romanian area, in recent years many crops of lavender, fennel, mint or basil have appeared. the lavender culture is found in the plain and depression area of romania because the plant needs a warm and moderately dry climate. the fennel culture is most often found in the southern part of romania, where it is mostly plain and there is a warm climate. the mint culture is the most common in the depression area of romania because it is a plant that has a moderate tolerance to drought, requiring numerous irrigations. basil crops are most often found in southern romania, more precisely in the plains because the plant prefers a warm climate. basil is in first place at the top of romanians' preferences in terms of cultivating aromatic and medicinal plants [47] [48] [49] [50] [51] . in the study by kokina et al. in 2019 lavender and peppermint volatile oils have been shown to have the highest antioxidant capacity. they combined two methods, dpph and 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (abts), to increase the efficiency of the evaluation of the antioxidant activity of the volatile oils studied. volatile oils were stored for 12 months and a significant decrease in antioxidant activity was observed [52] . in the study conducted by köksal and gülçin in 2008 and 2010, they demonstrated that cauliflower and lintite extracts have strong antioxidant activity [53, 54] . in 2016, aslam et al. conducted a study that demonstrated the antioxidant capacity of spinach leaves [55] . numerous studies conducted in recent years have shown that regular consumption of dairy products can have a protective effect against the development of obesity and cardiovascular disease [5] . yoghurt consumption has increased worldwide due to its nutritional value, therapeutic effects and functional properties [20] . it is very important to know the morphology of the plant and its active substances. from this point of view, the selective use of useful compounds from the plant that can be directed exactly to obtain the expected effect is preferred, and less of the plants that have been proven to produce unwanted and sometimes toxic interactions [56] . the oils obtained, once analyzed and the useful components identified, offer the possibility of their exact dosage in yoghurt so that the antioxidant effect can be ensured without substantially changing the taste and smell of the product [57, 58] . stress in the environment, especially heavy metal pollution, leads to the production of oxidative stress in plants. the population is constantly growing and the problems related to the supply of food are becoming more and more pressing. finding viable solutions for the realization of basic food products, with the widest possible destination, which in addition to a longer life cycle can ensure at the same time a healthy lifestyle, is of utmost importance. plants develop numerous enzymatic and non-enzymatic antioxidant mechanisms for detoxification. aromatic plants are especially rich in antioxidant phenolic compounds. their antioxidant activity is due to the redox properties and chemical structure, which play an important role in neutralizing free radicals and peroxides [20] . in a study conducted in 2019 on a sample of 1000 people in romania, it was shown that 98% of them suffer from diseases caused by stress. work related issues and the insecurity of tomorrow are the main reasons for the respondents' anxiety [59] . as last year in romania, the uncertainty of tomorrow and employment were the most widespread causes of stress, this year the world situation impacted by the covid-19 crisis will only deepen this even more. the large numbers of illnesses reported in the country at the moment, as well as the severely affected economic situation, are the most important causes of stress. social distancing, isolation and lack of certainty at work affects many people, who even reach states of anxiety and depression. stress development is associated with an increase in the number of free radicals, a decrease in the levels of antioxidant enzymes and an increase in oxidative lipids in the brain tissues. this free radical activity is associated with impaired cognitive function. major stress for a single period of eight hours increases the level of oxidative stress and the attack of free radicals on the brain, being accompanied by the weakening of memory and cognitive function. antioxidant nutrients have been shown to alleviate these effects when administered before or after stress-induced circumstances [60] . antioxidants are widely used as food additives to avoid food degradation. antioxidants also play an important role in preventing a variety of lifestyle disorders and ageing conditions, as they are closely linked to active oxygen and lipid peroxidation [54] . vegetable foods contain more antioxidants than those of animal origin, so the world health organization (who, geneva, switzerland) recommends about 400-600 g of vegetables and fruits daily to reduce the risk of cardiovascular disease, cancer, cognitive impairment and other eating disorders [61] . according to studies, it has been shown that the intake of antioxidants in the form of artificial supplements has not always brought the desired effect, so replacing them with natural antioxidants from plants can improve the desired effect. the intake of antioxidants, especially in this period when stress is present at maximum levels, is essential. the creation of food products that include natural antioxidant compounds combines both the current great possibility of developing food production in romania and the prevention and reduction of the effects caused by pandemic stress in the human body. to determine the antioxidant capacity, we used the dpph method. the dpph method measures the radical scavenging activity of antioxidants against free radicals, such as the dpph radical [62] . the main purpose of this work is to create yoghurts with high antioxidant and antimicrobial capacity using only raw materials from romania. external intake of antioxidants is essential to reduce the effects of daily stress. volatile oils are an excellent source of antioxidants, and their use in food production can be a great direction for the current situation. additionally, by using volatile oils, we aim to eliminate artificial preservatives added to yoghurts. according to studies in recent years, food preservatives harm consumer health, so using volatile oils with antimicrobial activity avoids the use of synthetic ones. to achieve this objective, we decided to enrich cow milk yoghurt with volatile oils encapsulated in sodium alginate. we used volatile oils from four aromatic plants: lavender, fennel, mint and basil. to achieve our purpose, we used the dpph method to determinate antioxidant capacity, and the measurements were made on the first day after making yoghurt from cow's milk, after 10 days and after 20 days. for each determination, we had two samples, a test sample (yoghurt with volatile oils) and a control sample (yoghurt without volatile oils). the yoghurt samples were packed in 150g plastic cups and stored in the refrigerator at a temperature between 0-4 • c. during the entire storage period, the glasses were covered with aluminum foil. we used five samples, first called control sample (yoghurt in which no alginate capsules were added with volatile oil), the second one called the yoghurt sample with the addition of volatile mint oil, the third one called the yoghurt sample with volatile basil oil, the fourth one called the yoghurt sample with volatile fennel oil and the fifth one called the yoghurt sample with volatile lavender oil. we used the dpph method to determinate antioxidant capacity, and the measurements were made on the first day after making yoghurt from cow milk, after 10 days and after 20 days. the cow's milk was taken from a farm in the sibiu area. lavender, mint and basil were taken from a culture located in the mures , area, and fennel was taken from a culture in the ialomit , a area. in 2019-before deciding to make a food product with a high content of antioxidants-we conducted a market study and a sensory analysis for these types of yoghurts so that we can conclude whether the products made by us are to the liking of consumers. following the sensory analysis, the tasters stated that the volatile oil added to the yoghurt only slightly influences its taste. the specific taste of yoghurt is predominant, only at the end, stimulating a slight taste of the plant from which the volatile oil is extracted. in addition to these two methods, we determined the ph and the lactic acid content to determine the antimicrobial activity of the yoghurt samples. to verify the antimicrobial activity of volatile oils, we created a mixture of the four oils studied and tested them on different cultures of enterobacteria, yeasts and molds. for the mixture of volatile oils, we used 25% volatile mint oil, 25% volatile lavender oil, 25% volatile basil oil and 25% volatile fennel oil. after obtaining the dilutions, the sowing took place on different culture mediums. the volatile oil mixture had a strong effect on colonies of enterobacteria, yeasts and molds: zero colony-forming units (cfu). no colony developed compared to the comparison plates (standard) [63] . to make the yoghurt enriched with bioactive components extracted from aromatic plants we used raw cow's milk with a physico-chemical composition representative of the lactation period, which we pasteurized, then cooled it and added lactic crops. for the inoculation operation, we used a starter culture from hansen (product name: f-dvs yc-x11 yo-flex). this is a thermophilic culture formed from lactobacillus delbrueckii subsp. bulgaricus and streptococcus thermophillus. the volatile oils did not influence the process of obtaining yoghurt. volatile oil compounds are gradually released into yoghurt due to its encapsulation in sodium alginate. the gradual release of antimicrobial and antioxidant action of volatile oils ensures a longer time of action and an avoidance of losses. for the extraction of volatile oils, we used mint, basil, lavender-dried and crushed aerial parts-and fennel seeds. the volatile oils were extracted by steam entrainment using the neo-clevenger apparatus modified by moritz. the extraction time was five hours for each sample, and for efficient extraction, the plants were soaked the day before. at the end of the extraction, the volatile oil obtained was measured and 1 ml of benzene is added over it. it was then placed in a glass vial containing sodium sulfate anhydrous to remove any traces of water. using a pasteur pipette, we extracted the volatile oil from the glass vial and the benzene evaporated. to preserve the characteristics of the volatile oil until analysis, it was sealed in a dark ampoule and refrigerated. the 4 samples of alginate capsules were obtained from 25 g 2% sodium alginate solution and 30 µl volatile basil, mint, fennel and lavender oil. the alginate solution was added gradually to the calcium chloride solution under centrifugation, thus obtaining the alginate capsules which were then washed with distilled water [63] . for the extraction of the compounds to determine the antioxidant capacity of the yoghurt samples with volatile oils, we used the extraction method adapted after patel et al. (2016) . we weighed 0.5 g of the sample to be analyzed and then extracted it with 10 ml of the mixture methanol:water:hydrochloric acid 0.12 m = 70:29:1 (v/v/v), at room temperature, for 24 h. the mixture was then kept on the ultrasonic water bath for 30 min at a temperature of 25 • c. after the time ran out, the supernatant was collected and centrifuged at 8000 rpm for 10 min. the residue was suspended in 10 ml of solvent to perform a second extraction for 15 min, on the bath of water at 25 • c. the resulting supernatant was centrifuged under the same conditions as the first. the total amount of supernatant was evaporated to the rotary evaporator and the residue was taken up with 10 ml of methanol. we filtered the mixture of supernatant and methanol and filled it to a volume of 10 ml with the same solvent [64] . to determine the antioxidant activity of the yoghurt samples with the addition of volatile oils encapsulated in sodium alginate, we used a method adapted according to the method applied by tylkowski et al. (2011) for the ethanolic extracts of sideritis ssp. l. we prepared a 25 µg/ml dpph solution by solubilizing a quantity of dpph in absolute methanol -stock solution.this mixture needed to be prepared in advance-at least 1-2 h-for complete solubilization. a volume of 970 µl dpph solution 25 µg/ml is measured and added over 30 µl methanol extract from the samples to be analyzed, which we obtained using the extraction shown above. to interpret the results, we measured the absorbance at 515 nm for each sample using the cecil 1021 uv-vis spectrophotometer and the concentration is determined according to the standard curve obtained from different concentrations of the stock solution [65] . the determination of antioxidant activity was performed for each sample of yoghurt. for all these samples, there were 10 spectrophotometer readings, because from each type of yoghurt we made five samples (five containers of 100 g each). after extracting the necessary compounds to determine the antioxidant activity of each extracted container we took two readings. we wanted to eliminate all errors related to equipment, human error and differences in temperature or humidity. all readings were made on the same day of the analysis, to be more exact on the first day, on the 10th day and on the 20th day after the yoghurt samples were made. the results obtained in this research are presented as follows. figure 1a shows the antioxidant activity of the control sample on the first day, the 10th day and the 20th day. on the first day of the analysis, the highest values of the antioxidant activity of the control sample were obtained. on this day, the maximum value obtained was 0.23%, and the minimum value was 0.16%. the value of the antioxidant activity for the control sample decreased on the 10th day compared to the first day. on day 10 of the analysis, the highest value obtained was 0.16%, and the lowest value was 0.10%. the lowest values of antioxidant activity for the control sample were obtained on day 20 of the analysis. on this day, the highest measured value was 0.11%, and the lowest was 0.06%. the average value for each day was 0.20% for the first day, 0.13% for the 10th day and 0.08% for the 20th day. figure 1a shows the antioxidant activity of the control sample on the first day, the 10th day and the 20th day. on the first day of the analysis, the highest values of the antioxidant activity of the control sample were obtained. on this day, the maximum value obtained was 0.23%, and the minimum value was 0.16%. the value of the antioxidant activity for the control sample decreased on the 10th day compared to the first day. on day 10 of the analysis, the highest value obtained was 0.16%, and the lowest value was 0.10%. the lowest values of antioxidant activity for the control sample were obtained on day 20 of the analysis. on this day, the highest measured value was 0.11%, and the lowest was 0.06%. the average value for each day was 0.20% for the first day, 0.13% for the 10th day and 0.08% for the 20th day. in figure 1a , the decline is calculated on day 10 and day 20 compared to the first day for the control sample. the decline on day 10 compared to the first day is smaller than the decline on day 20 compared to the first day. the results obtained on day 20 are lower compared to day 10. the decline from day 10 compared to the first day is between 52-88%. the decline from day 20 compared to the first day is between 27-58%. in figure 1a , the decline is calculated on day 10 and day 20 compared to the first day for the control sample. the decline on day 10 compared to the first day is smaller than the decline on day 20 compared to the first day. the results obtained on day 20 are lower compared to day 10. the decline from day 10 compared to the first day is between 52-88%. the decline from day 20 compared to the first day is between 27-58%. figure 2a shows the antioxidant activity of the yoghurt sample from cow milk with the addition of volatile mint oil encapsulated in sodium alginates on the first day, the 10th day and the 20th day. the strongest antioxidant activity of the yoghurt sample with volatile mint oil was on the first day. the lowest value of this day was 1.57%, and the highest was 1.62%. the value of the antioxidant activity decreased on the 10th day compared to the first day. on day 10 the highest value was 1.23%, and the lowest at 1.17%. for this yoghurt sample, the lowest antioxidant activity was recorded on day 20. the highest value on day 20 was 1.06%, and the lowest was 0.95%. the average value for each day is 1.60% for the first day, 1.20% for the 10th day and 1.00% for the 20th day. figure 2a shows the antioxidant activity of the yoghurt sample from cow milk with the addition of volatile mint oil encapsulated in sodium alginates on the first day, the 10th day and the 20th day. the strongest antioxidant activity of the yoghurt sample with volatile mint oil was on the first day. the lowest value of this day was 1.57%, and the highest was 1.62%. the value of the antioxidant activity decreased on the 10th day compared to the first day. on day 10 the highest value was 1.23%, and the lowest at 1.17%. for this yoghurt sample, the lowest antioxidant activity was recorded on day 20. the highest value on day 20 was 1.06%, and the lowest was 0.95%. the average value for each day is 1.60% for the first day, 1.20% for the 10th day and 1.00% for the 20th day. in figure 2b , the decline is calculated on day 10 and day 20 compared to the first day for the yoghurt sample from cow milk with the addition of volatile mint oil encapsulated in sodium alginates. the decline on day 10 compared to the first day is smaller than the decline on day 20 compared to the first day. the results obtained on day 20 are lower compared to day 10. the decline from day 10 compared to the first day is between 73-78%. the decline from day 20 compared to the first day is between 61-66%. figure 3a shows the antioxidant activity of the yoghurt sample from cow's milk with the addition of volatile basil oil encapsulated in sodium alginates on the first day, the 10th day and the 20th day. on the first day, the yoghurt sample with volatile basil oil showed the highest antioxidant activity, and on the 20th the lowest. the highest value from the first day was 9.65% and the lowest at 9.56%. on day 10 the highest value was 9.33%, and the lowest at 9.26%. on day 10 the antioxidant activity was lower than on the first day, but it was higher than on day 20. day 20 showed the lowest antioxidant activity, and the lowest value was 8.65%. the average value for each day is 9.60% for the first day, 9.30% for the 10th day and 8.70% for the 20th day. in figure 2b , the decline is calculated on day 10 and day 20 compared to the first day for the yoghurt sample from cow milk with the addition of volatile mint oil encapsulated in sodium alginates. the decline on day 10 compared to the first day is smaller than the decline on day 20 compared to the first day. the results obtained on day 20 are lower compared to day 10. the decline from day 10 compared to the first day is between 73-78%. the decline from day 20 compared to the first day is between 61-66%. figure 3a shows the antioxidant activity of the yoghurt sample from cow's milk with the addition of volatile basil oil encapsulated in sodium alginates on the first day, the 10th day and the 20th day. on the first day, the yoghurt sample with volatile basil oil showed the highest antioxidant activity, and on the 20th the lowest. the highest value from the first day was 9.65% and the lowest at 9.56%. on day 10 the highest value was 9.33%, and the lowest at 9.26%. on day 10 the antioxidant activity was lower than on the first day, but it was higher than on day 20. day 20 showed the lowest antioxidant activity, and the lowest value was 8.65%. the average value for each day is 9.60% for the first day, 9.30% for the 10th day and 8.70% for the 20th day. in figure 2b , the decline is calculated on day 10 and day 20 compared to the first day for the yoghurt sample from cow milk with the addition of volatile mint oil encapsulated in sodium alginates. the decline on day 10 compared to the first day is smaller than the decline on day 20 compared to the first day. the results obtained on day 20 are lower compared to day 10. the decline from day 10 compared to the first day is between 73-78%. the decline from day 20 compared to the first day is between 61-66%. figure 3a shows the antioxidant activity of the yoghurt sample from cow's milk with the addition of volatile basil oil encapsulated in sodium alginates on the first day, the 10th day and the 20th day. on the first day, the yoghurt sample with volatile basil oil showed the highest antioxidant activity, and on the 20th the lowest. the highest value from the first day was 9.65% and the lowest at 9.56%. on day 10 the highest value was 9.33%, and the lowest at 9.26%. on day 10 the antioxidant activity was lower than on the first day, but it was higher than on day 20. day 20 showed the lowest antioxidant activity, and the lowest value was 8.65%. the average value for each day is 9.60% for the first day, 9.30% for the 10th day and 8.70% for the 20th day. in figure 3b , the decline is calculated on day 10 and day 20 compared to the first day for the yoghurt sample from cow milk with the addition of volatile basil oil encapsulated in sodium alginates. the decline on day 10 compared to the first day is smaller than the decline on day 20 compared to the first day. the results obtained on day 20 are lower compared to day 10. the decline from day 10 compared to the first day is between 96-97%. the decline from day 20 compared to the first day is between 90-91%. figure 4a shows the antioxidant activity of the yoghurt sample from cow milk with the addition of volatile fennel oil encapsulated in sodium alginates on the first day, the 10th day and the 20th day. the highest antioxidant activity was on the first day and the lowest on the 20th day. on the first day, the highest value was 6.43%, and the lowest value was 6.37%. the antioxidant activity on day 10 is lower than on the first day. the highest value on the 10th day was 6.18%, and the lowest was 6.22%. on day 20, the yoghurt sample with volatile fennel oil had the lowest antioxidant activity. the highest value recorded was 6.04%, and the lowest was 5.95%. the average value for each day was 6.40% for the first day, 6.20% for the 10th day and 6.00% for the 20th day. figure 3 . (a) antioxidant activity of the yoghurt sample from cow milk with the addition of volatile basil oil encapsulated in sodium alginates on the first day, the 10th day and the 20th day; (b) decline on day 10 and day 20 for the yoghurt sample from cow milk with the addition of volatile basil oil encapsulated in sodium alginates compared to day one. in figure 3b , the decline is calculated on day 10 and day 20 compared to the first day for the yoghurt sample from cow milk with the addition of volatile basil oil encapsulated in sodium alginates. the decline on day 10 compared to the first day is smaller than the decline on day 20 compared to the first day. the results obtained on day 20 are lower compared to day 10. the decline from day 10 compared to the first day is between 96-97%. the decline from day 20 compared to the first day is between 90-91%. figure 4a shows the antioxidant activity of the yoghurt sample from cow milk with the addition of volatile fennel oil encapsulated in sodium alginates on the first day, the 10th day and the 20th day. the highest antioxidant activity was on the first day and the lowest on the 20th day. on the first day, the highest value was 6.43%, and the lowest value was 6.37%. the antioxidant activity on day 10 is lower than on the first day. the highest value on the 10th day was 6.18%, and the lowest was 6.22%. on day 20, the yoghurt sample with volatile fennel oil had the lowest antioxidant activity. the highest value recorded was 6.04%, and the lowest was 5.95%. the average value for each day was 6.40% for the first day, 6.20% for the 10th day and 6.00% for the 20th day. in figure 4b , the decline is calculated on day 10 and day 20 compared to the first day for the yoghurt sample from cow milk with the addition of volatile fennel oil encapsulated in sodium alginates. the decline on day 10 compared to the first day is smaller than the decline on day 20 compared to the first day. the results obtained on day 20 are lower compared to day 10. the decline from day 10 compared to the first day was between 96-97%. the decline from day 20 compared to the first day was between 93-95%. figure 5a shows the antioxidant activity of the yoghurt sample from cow milk with the addition of volatile lavender oil encapsulated in sodium alginates on the first day, the 10th day and the 20th day. the highest antioxidant activity of the yoghurt sample with volatile lavender oil was on the first day, and the lowest was on the 20th day. the highest value on the first day was 5.23%, and the lowest was 5.17%. the antioxidant activity on the 10th day was lower than on the first day, but it was higher than on the 20th day. the highest value on day 10 was 5.13%, and the lowest was 5.07%. on day 20, the lowest antioxidant activity was recorded, and the lowest value was 4.67%. the average value for each day was 5.20% for the first day, 5.10% for the 10th day and 4.70% for the 20th day. figure 4 . (a) antioxidant activity of the yoghurt sample from cow milk with the addition of volatile fennel oil encapsulated in sodium alginates on the first day, the 10th day and the 20th day; (b) decline on day 10 and day 20 for the yoghurt sample from cow milk with the addition of volatile fennel oil encapsulated in sodium alginates compared to day one. in figure 4b , the decline is calculated on day 10 and day 20 compared to the first day for the yoghurt sample from cow milk with the addition of volatile fennel oil encapsulated in sodium alginates. the decline on day 10 compared to the first day is smaller than the decline on day 20 compared to the first day. the results obtained on day 20 are lower compared to day 10. the decline from day 10 compared to the first day was between 96-97%. the decline from day 20 compared to the first day was between 93-95%. figure 5a shows the antioxidant activity of the yoghurt sample from cow milk with the addition of volatile lavender oil encapsulated in sodium alginates on the first day, the 10th day and the 20th day. the highest antioxidant activity of the yoghurt sample with volatile lavender oil was on the first day, and the lowest was on the 20th day. the highest value on the first day was 5.23%, and the lowest was 5.17%. the antioxidant activity on the 10th day was lower than on the first day, but it was higher than on the 20th day. the highest value on day 10 was 5.13%, and the lowest was 5.07%. on day 20, the lowest antioxidant activity was recorded, and the lowest value was 4.67%. the average value for each day was 5.20% for the first day, 5.10% for the 10th day and 4.70% for the 20th day. in figure 5b , the decline is calculated on day 10 and day 20 compared to the first day for the yoghurt sample from cow milk with the addition of volatile lavender oil encapsulated in sodium alginates. the decline on day 10 compared to the first day is smaller than the decline on day 20 compared to the first day. the results obtained on day 20 are lower compared to day 10. the decline from day 10 compared to the first day was between 97-99%. the decline from day 20 compared to the first day was between 89-91%. the determination of the antioxidant activity was performed for each sample of yoghurt. for all samples, 10 spectrophotometer readings were performed to eliminate all errors related to equipment, human error and differences in temperature or humidity. all readings were made on the same day of the analysis, to be more exact on the first day, on the 10th day and on the 20th day after the yoghurt samples were made. according to figure 6 , the analytical results obtained show that the highest antioxidant activity was shown by the sample of yoghurt with basil volatile oil encapsulated in sodium alginate, followed by the sample of yoghurt with fennel volatile oil encapsulated in sodium alginate, then the sample of yoghurt with lavender volatile oil encapsulated in the sodium alginate and yoghurt sample with mint volatile oil encapsulated in sodium alginate. the antioxidant activity proved to be considerably higher than the control sample. decline on day 10 and day 20 for the yoghurt sample from cow milk with the addition of volatile lavender oil encapsulated in sodium alginates compared to day one. in figure 5b , the decline is calculated on day 10 and day 20 compared to the first day for the yoghurt sample from cow milk with the addition of volatile lavender oil encapsulated in sodium alginates. the decline on day 10 compared to the first day is smaller than the decline on day 20 compared to the first day. the results obtained on day 20 are lower compared to day 10. the decline from day 10 compared to the first day was between 97-99%. the decline from day 20 compared to the first day was between 89-91%. the determination of the antioxidant activity was performed for each sample of yoghurt. for all samples, 10 spectrophotometer readings were performed to eliminate all errors related to equipment, human error and differences in temperature or humidity. all readings were made on the same day of the analysis, to be more exact on the first day, on the 10th day and on the 20th day after the yoghurt samples were made. according to figure 6 , the analytical results obtained show that the highest antioxidant activity was shown by the sample of yoghurt with basil volatile oil encapsulated in sodium alginate, followed by the sample of yoghurt with fennel volatile oil encapsulated in sodium alginate, then the sample of yoghurt with lavender volatile oil encapsulated in the sodium alginate and yoghurt sample with mint volatile oil encapsulated in sodium alginate. the antioxidant activity proved to be considerably higher than the control sample. the highest antioxidant activity was obtained on the first day of the analysis, before decreasing on day 10 and day 20. in the case of yoghurt samples with volatile basil, fennel and lavender oil, the greatest decreases in antioxidant activity were recorded between the 10th and the 20th day. in the case of the yoghurt sample with volatile mint oil, the greatest decrease in antioxidant activity was recorded between the first and the 10th day. these measurements show us that volatile basil, fennel and lavender oils have a higher antioxidant activity and are more stable during the first 10 days of preservation. in the case of volatile mint oil, it has a lower antioxidant activity and begins to stabilize after 10 days of preservation. all the data obtained show us that the chosen product has a significant antioxidant capacity and can be used as an external source of antioxidants. it was thus demonstrated that there exists a way of the highest antioxidant activity was obtained on the first day of the analysis, before decreasing on day 10 and day 20. in the case of yoghurt samples with volatile basil, fennel and lavender oil, the greatest decreases in antioxidant activity were recorded between the 10th and the 20th day. in the case of the yoghurt sample with volatile mint oil, the greatest decrease in antioxidant activity was recorded between the first and the 10th day. these measurements show us that volatile basil, fennel and lavender oils have a higher antioxidant activity and are more stable during the first 10 days of preservation. in the case of volatile mint oil, it has a lower antioxidant activity and begins to stabilize after 10 days of preservation. all the data obtained show us that the chosen product has a significant antioxidant capacity and can be used as an external source of antioxidants. it was thus demonstrated that there exists a way of increasing both the food and nutritional quality of the yoghurt but also the validation of the method of increasing the shelf life of the yoghurt. acid dairy products are appreciated worldwide because of the benefits they bring to the consumer's health, as well as the possibility of consuming them from an early age [5] . in this case, we used yoghurt as a representative product for consumers, both in terms of favorable intake for the harmonious development of the body at different stages of life, but also in terms of frequency of consumption. enriching it with bioactive components extracted from native herbs mint, basil, fennel and lavender, respectively, by adding volatile oils extracted from these plants and encapsulated using 2% sodium alginate, proved to be a beneficial option to increase the value of the product. the use of sodium alginate capsules also solved the problem of ensuring the stability of the volatile oils added to the food. the statistically processed results demonstrate the validity of the method of obtaining valuable food products, enriched in bioactive components, respectively, of using volatile oils with antioxidant and antimicrobial activity. the amount of volatile oils extracted from mint, basil, fennel and lavender is dependent on the growing conditions, soil and climatic conditions, the extraction method used, but the average values obtained support the potential for their use, especially in terms of the benefits for consumers' health. the creation of foods containing natural antioxidant and antimicrobial compounds must take precedence in food management. the problem of daily stress has become a major health problem in recent years, and the global situation impacted by the covid-19 crisis deepens this further. it is a reality nowadays that social distancing, isolation and the lack of certainty at work affects many people, even reaching states of anxiety and depression. making such foods to increase the body's immunity to viruses or to alleviate many chronic health problems is an effective and safe alternative to ensuring physical and mental health. to get over this period more easily and to reduce-as much as possible the effects-caused by the stress resulted from the pandemic, doctors recommend adopting a healthier lifestyle. therefore, a healthy diet, compliance with safety conditions and especially finding appropriate and safe methods to increase the body's immunity are safe alternatives to an easier passage through harder periods such as the pandemic. the use of volatile oils also ensures the complete elimination of artificial preservatives added to dairy products in this case of yoghurts. food preservatives harm consumer health, often causing food poisoning, so using volatile oils with antiseptic and antioxidant activities ensures an increase in the shelf life of food and attention to toxicology and public health. crossref] 2. #cumreînviembusinessul: industria alimentară merge înainte. producţia de alimente, o piaţă de 50 de miliarde de lei anual, s-ar putea dubla dacă folosim materie primă locală physicochemical, functional, and sensory properties of yogurts containing persimmon antioxidant and antiseptic properties of volatile oils from different medicinal plants: a review dairy consumption at snack meal occasions and the overall quality of diet during childhood. 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random house canada therios, i. boron and maturity effects on biochemical parameters and antioxidant activity of pepper (capsicum annuum l.) cultivars comparative analysis of yoghurts obtained with bioactive compounds non-targeted metabolite profiling and scavenging activity unveil the nutraceutical potential of psyllium (plantago ovata forsk). front concentration of biologically active compounds extracted from sideritis ssp. l. by nanofiltration. food bioprod. process we would like to express our sincere gratitude to the research center in biotechnology and food engineering (c.c.b.i.a.), lucian blaga university of sibiu for the entire support granted throughout the research period. we also appreciate the editor and the anonymous reviewers for their constructive comments and insightful suggestions on the manuscript. the authors declare no conflict of interest.int. j. environ. res. public health 2020, 17, 7588 key: cord-324217-secnz2ta authors: pasdaran, a.; pasdaran, a.; sheikhi, d. title: volatile oils: potential agents for the treatment of respiratory infections date: 2016-08-05 journal: the microbiology of respiratory system infections doi: 10.1016/b978-0-12-804543-5.00016-6 sha: doc_id: 324217 cord_uid: secnz2ta due to presence of secondary bioactive metabolites, natural compounds are considered a major source of new active molecules that can be developed as new drugs. infectious diseases, and mainly the common respiratory infections, are major challenges to the current chemotherapy systems and, therefore, there is a requirement to find new compounds with therapeutic potential. the volatile natural compounds and essential oils are the main treasure agents in the natural compounds with antibiotic potential. the present chapter reviews natural traditional remedies used in the treatment of respiratory infections with the emphasis on antibacterial, antiviral, and antiinflammation activities of the volatile natural compounds (essential oils, etc.), and provides a brief view in some of structural activity relationships between antibacterial potencies and chemical structures of the essential oil’s constituents. referring to infectious disease, respiratory tract infections engage with all surfaces in the respiratory tract. based on the infected zone, respiratory infections can be categorized into upper tract infection (uri or urti) and lower tract infection (lri or lrti). each involves different parts of the respiratory tract infections, which vary in type and severity of microorganisms. although there are different types of respiratory tract infections, the acute form in the upper respiratory tract infection predominates and includes several complications, such as sinusitis, pharyngitis, epiglottitis, laryngitis, and tracheitis. on the other hand, lower respiratory tract infection (lrti) includes both acute and chorionic types, such as pneumonia and bronchitis. based on pathogenicity, bacterial and viral pathogens are the most common microorganisms in both types (ie, lrti and urti). moreover, infection distribution leads to varieties based on the patient's age; for example, acute respiratory infections pose severe problem in childhood, which mainly occur in upper respiratory tract. although the bacterial pathogens play a significant role in intensifying lrtis, the major acute respiratory infections occur in upper respiratory tract, in these cases viral pathogens are the primary common pathogens, including influenza a and b, parainfluenza (type 1 and 3), adenovirus, and respiratory syncytial virus. some of the common pathogens of the respiratory tract are listed in table 16 .1. pathogen biodiversity, complexity, and mixed infections in many cases of respiratory tract infection have generated several problems for the treatment of respiratory infections. for example, various bacterial pathogens are encountered in several cases of viral infections. therefore, the treatment of respiratory infections is a complex therapy which consists of several chemotherapy strategies. 1-3 antiviral (the same as antibacterial medication) is used to control the treatment and prevention of respiratory infections. there are several restrictive factors, such as medication resistance, recurrency, and inflammation, which will guide researchers to find new effective compounds. this will be an important field in drug development for respiratory infections. natural compounds are considered to be one of the main sources in new drug development. historically, numerous plants have been utilized as traditional medicines by people in many nations. 4 many of these plants have been investigated for their antimicrobial and antiviral properties. [5] [6] [7] [8] [9] with regard to massive variation among natural products, chemical structure diversity causes different antimicrobial potential in natural compounds. 10 besides the antimicrobial activity of the essential oils in natural products, other characteristics such as high vapor pressure, low toxicity, and antiinflammatory potential create a worthwhile theme for using of these natural compounds for new drug development in respiratory infections. parallel to the roles of the microorganisms in the pathology of respiratory infections diseases, inflammatory process also have a considerable role in the persistence and recurrence of respiratory infectious diseases. this chapter reviews the antibacterial, antiviral, and antiinflammation effects of essential oils as effective natural compounds. it will also discuss the use of these natural compounds as traditional remedies in treatment of respiratory infections. traditional medicines utilize natural sources for the treatment of the many diseases. [11] [12] [13] historically, infectious diseases have been the major human ailment. natural sources are used in a variety of forms, including water extracts, tincture or alcoholic extract and incense. 14 based on the historical uses and effective treatments that have been based on many of these traditional remedies, extensive pharmacological research of their antibacterial, antiviral, and antiinflammation activity have been performed. [15] [16] [17] abundant information about plants and active compounds in infectious diseases and inflammation related process is available. [18] [19] [20] [21] aromatic and fragrant plants are a major part of traditional therapeutic remedies, and they have shown remarkable antibacterial and antiviral activity. furthermore, many of them also have a significant antiinflammatory activity and are used as adjuvant remedies in the treatment of infection (table 16 .2 and fig. 16.1) . some of the most active extracts of traditional herbs which have been used as antibacterial and antiviral in the treatment of respiratory infections are summarized in table 16 . 3 . the use of aromatic extracts or burning plants is a common process in traditional medicine. the resultant smoke or fragrance is inhaled to treat respiratory complaints, including cough, cold, infections, and asthma. 22, 23 inhalation administration goes back to the ancient cultures and its techniques may be considered as a progressive point in respiratory complaints treatment. the direct effect of such fragrance on the respiratory tract is an advantage of this form of treatment. inhalation therapy often involves the aromatic extracts or burning of plant material, and the volatile fraction liberated during the process is inhaled to aid in the healing process. inhalation of the volatile fraction from aromatic extracts or burning plant matter is a unique method of administration and has been used traditionally to treat respiratory conditions, such as, asthma, bronchitis, and other respiratory infections including the common cold. 24 in addition, aerosol delivery of such remedies is well practiced in allopathic medicine and has the advantage of being site specific, thus enhancing the therapeutic ratio for respiratory ailments. 25 the antimicrobial effects of plants and their extracts have been recognized for a long time. essential oil is one of the most important and wide spread secondary metabolite in plants and this class of phytochemical compounds and their activities needs attention. these phytochemicals are generally isolated from plant material by distillation methods, such as, hydrodistillation and steam distillation. they contain variable mixtures of several chemical classes, such as terpenoids, specifically monoterpenes and simple phenolic compounds. some of the higher molecular structures with high molecular weight, such as sesquiterpenes and diterpenes, may be present. a variety of low molecular weight aliphatic hydrocarbons, acids, alcohols, esters or lactones, sulfur-containing compounds and other chemical groups may also be observed. among the phytochemical compounds, terpenes are responsible for many therapeutic effects in medicinal plants. [26] [27] [28] [29] [30] most terpenes are derived from the condensation of isoprene units and are categorized according to the number of these units present in the carbon skeleton. these compounds are responsible for aromaticity and fragrance in many of the plants. the antibacterial activity of volatile oils has been assessed by many researchers. [31] [32] [33] [34] this potential of essential oils has been used in many pharmaceutical, cosmeceutical, and nutraceutical applications and industrials. there are many differences between the antimicrobial effects of different essential oils. essential oils and their constituents are an attractive source in new antimicrobial compounds evaluation. 35 many of the essential oils have been tested for bactericidal and bacteriostatic effects against a wide range of microorganisms including food spoiling organisms, pathogenic bacteria, yeasts, fungi, and many others. the major differences in antimicrobial activity have been yielded of several distinctive parameters which identify antibacterial characters of the essential oils, some of the major parameters include: (1) bacterial membrane permeability, (2) the hydrophobicity/hydrophilicity of the bacterial membrane, (3) the metabolic characteristics of the microorganism, and (4) their gram-positive or negative pattern. although susceptibility of the bacteria to the essential oils is not exactly predictable, many researchers have tried to determine the relationship between the origin of the essential oils and their compounds with their antimicrobial activity. furthermore, the delivery of medications to the respiratory tract has become an increasingly important method for respiratory disease treatment. the use of inhaler medications has become an invaluable therapeutic in the treatment of different pulmonary disorders, including bronchitis, pneumonia, and others complications. 37 several studies have reported the clinical efficacy of inhalation therapy for the treatment of lung disorders. 38, 39 through the effective delivery of medication to the action site, the active compounds are delivered directly into the lungs and this can result in respiratory tract local treatment. this method achieves maximum therapeutic biebersteinii. 3 screening of the antibacterial effects of essential oils effect, small dose usage, and has fewer side-effect risks compared with those associated with larger doses. inhalation is a unique treatment with direct effects on respiratory disorder site and is based on the volatility potential of essential oils. furthermore, there is a need to develop new therapeutic agents for respiratory infections. [40] [41] [42] research has been carried out on the wide spectrum of edible plants essential oils to determine the antibacterial potential of their essential oils. the role of these plants as therapeutic agents is remarkable in many cultures. investigations have reported that thyme and oregano essential oils, based on the phenolic components [such as carvacrol (1) and thymol (2) (fig. 16. 3)] have shown a strong correlation with the inhibition of some of the pathogenic bacterial strains (eg, in escherichia coli). the correlation between the antibacterial effect of the volatile oils and their chemical compounds, including high amount of the phenolic components such as carvacrol (1) or eugenol (3), has also been confirmed. 43 other essential oils such as oregano, savory, clove, and nutmeg with high concentrations of volatile phenolic compounds inhibit gram-positive more than gram-negative pathogenic bacteria. 44 however, in some essential oils such as achillea spp. (yarrow) strong antibacterial activity was observed against the gram-negative respiratory pathogens (haemophilus influenzae, pseudomonas aeruginosa) while streptococcus pyogenes was the most resistant to the this oil. 45 the other essential oils such as peppermint and spearmint inhibit the methicillin-resistant type of staphylococcus aureus. previous reports have clarified that the essential oils containing aldehyde or phenol as a major component represent the highest antibacterial activity. these antibacterial potencies are lower in the essential oils that contain high amounts of terpene alcohols compared to the essential oils containing aldehyde or phenol as a major component. other essential oils containing terpene ketone, or ether showed much weaker activity, and oil containing terpene hydrocarbon was relatively inactive. based on these findings, essential oils such as thyme, cinnamon, lemongrass, perilla, and peppermint have demonstrated suitable effects on respiratory tract infection. 46 the tolerance of gram-negative bacteria to essential oils has been attributed to the presence of a hydrophilic outer membrane that blocked the penetration of hydrophobic essential oils to the target cell membrane because the gram-positive bacteria were more exposed to the essential oils than gram-negative bacteria, which has been reported several times. [47] [48] [49] [50] lipids are one of the principal constituents for normal cell membrane function and these compounds supply many operations, such as barrier function in the bacterial cell membrane. the external capsule of some gram-negative bacteria limits or prevents the penetration of the essential oils into the microbial cell. one of the pronounced examples of the hydrophobicity/hydrophilicity role in bacterial sensitivity to antibacterial compound is h. influenzae. it should be pointed out that the outer membrane of h. influenzae (which forms rough colonies) was more hydrophobic. hydrophobic antibiotics, such as macrolides, are more active against h. influenzae than e. coli through their shorter oligosaccharide chains than those in e. coli. the effects of the cytoplasmic membrane and/or the embedded enzymes in it have demonstrated lipophilic biocide actions. 51 it is generally recognized that the antimicrobial action of essential oils depends on their hydrophilic or lipophilic character. based on these observations, investigators are trying to indicate the relationship between structural activity of the essential oils compounds and their antibacterial activity. certain components of the essential oils can act as uncouplers, which interfere with proton translocation over a membrane vesicle and subsequently interrupt adp phosphorylation pathways (primary energy metabolism). as a member of the phytochemicals, terpenoids have been observed as a model of lipid soluble agents, which have an impact on the activities of membrane catalyzed enzymes; for 3 screening of the antibacterial effects of essential oils example, enzymes involved in respiratory pathways. particular terpenoids with functional groups, such as phenolic alcohols or aldehydes, also interfere with membrane-integrated or associated enzyme proteins, inhibiting their production or activity. a good deal of antimicrobial compounds which act on the bacterial cytoplasmic membrane cause the loss of 260 nm absorbing material. this causes an increased susceptibility to nacl, the lysosomes formation and loss of potassium ions, which results in inhibiting respiration and the loss of cytoplasmic material. investigations about the cytoplasmic membrane effects of α-pinene (4), β-pinene (5), 1,8-cineole (6) , and electron microscopy studies have shown that the essential oils containing these compounds triggered such cytoplasmic material with losing in treated bacterial cells. 31, 32 the perturbation of the lipid fraction in the plasma membrane causes antimicrobial activity of some of the phytochemicals such as α,β-unsaturated aldehydes and some of monoterpenes. although these aldehyde compounds can elicit antibacterial effects by acting on membrane functional proteins, such antibacterial effect would be achieved with modifications of membrane permeability and intracellular materials leakage. [52] [53] [54] the membrane damage leading to whole-cell lysis has been reported by oregano and rosewood essential oils which contains major components as: carvacrol (1), citronellol (7), and geraniol (8) . 26, 55 phenols such as carvacrol (1), thymol (2), eugenol (3), and other oxygenated aromatic essential oil compounds including phenol ethers [trans-anethole (9), methyl chavicol (10)] and aromatic aldehydes [cinnamaldehyde (11), cuminaldehyde (12) ] have been reported to exert both antibacterial and antifungal activity. however, this chemical class-based on the concentration used-are known as either bactericidal or bacteriostatic agents, 56 but the phenolic component's high activity may be further explained in terms of the alkyl substitution into the phenol nucleus, which is known to increase the antimicrobial activity of phenols. the alkylation has been known to change the distribution ratio between the hydrophilic and the hydrophobic phases (including bacterial phases) by the surface tension reduction or the species selectivity mutate based on the bacteria cell wall characters. 57 this does not happen with etherified or esterified isomeric molecules, it is possible by describing their relative lack of activity. 58 as a member of these compounds carvacrol (1) is one of the few components that has a break apart from effect on the outer membrane of gram-negative bacteria and causes release of lipopolysaccharide and alters cytoplasmic membrane ions transportation, similar to carvacrol (1), thymol (2) antimicrobial activity results in structural and functional alterations in the cytoplasmic membrane. 59 interestingly, eugenol (3) and isoeugenol (13) exhibit higher activity against gram-negative bacteria than gram-positive bacteria, and when cinnamaldehyde (11) is used against e. coli, its activity is similar to carvacrol (1) and thymol (2) (fig. 16.3) . these compounds alter the membrane, affect the transport of ions and atp, and change the fatty acid profile of different bacteria. 60 although in some cases alcoholic form shows better potencies compared to acetate form, the presence of an acetate moiety in the structure appeared to increase the activity of the parent compound. in the case of geraniol (8), the geranyl acetate (14) demonstrated an increase in activity against the test microorganisms. 48, 61, 62 a similar effect was also observed in the case of borneol (15) , bornyl acetate (16) , linalool (17) , and linalyl acetate (18) (fig. 16.3 ). in addition, the effectiveness of alcoholic compounds very closely depended on the bacterial cell wall, which showed different permeability to alcohol based on chain length. 44, 63 it has been suggested that an aldehyde group conjugated to a carbon double bond such as citral (19) is an extremely electronegative order, which may explain their activity, and an increase in electronegativity can raise up the antibacterial activity. 64 in addition, the under research of aldehydes potency seems to depend not only on the existence of the α,β-double bond but also on the chain length from the renal group and on microorganism tested. it seems that some electronegative compounds, mainly from the cell surface, are responsible for the inhibited growth of the microorganisms, which may interfere in biological processes involving electron transfer and respond with vital nitrogen components and alteration in the operation of membrane-associated proteins. actually, a greater electronegativity of the molecule would cause a greater encounter of intermolecular hydrogen bond formation with membrane nucleophilic groups and thus a significant irregularity in the lipidic bilayer. some studies have recommend that carbon tail length also affects the electronegativity of the aldehyde oxygen atom and thus its interaction with the nucleophilic groups of the cell membrane. 65 comparably, the similar antimicrobial activity was detected in the series of the long-chain alcohols which is demonstrated to be resulted from the alkyl chain length. 66, 67 this structural activity relationship is notable between farnesol (20) , nerolidol (21), plaunotol (22) , geranylgeraniol (23), phytol (24), geraniol (8) , and linalool (17) which act on s. aureus with damages of the cell membranes and losing of k+ ions, while similar mode of actions can be detected by the aminoglycosides such as kanamycin and streptomycin. farnesol (20) was able to damage cell membranes most effectively than other terpene alcohols. the activities of farnesol (20) , nerolidol (21) (sesquiterpenes compounds) on s. aureus were higher than that of plaunotol (22) (diterpene). the effectiveness against s. aureus are in order as follows: farnesol (19) > nerolidol (20)> plaunotol (22) > geranylgeraniol (23), phytol (24) > geraniol (8) and linalool (17) (fig. 16.3 ). it has been suggested that maximum activity against s. aureus might depend on the number of carbon atoms in the hydrophobic chain from hydrophilic hydroxyl group, which should be less than 12 but as close to 12 as possible. neither a shorter nor a longer aliphatic carbon chain, could increase such activity. 68, 69 the increased effectiveness of sesquiterpenes as enhancers of membrane permeability may stem from their structural resemblance to membrane lipids (eg, linear molecules with internal lipophilic character and a more polar terminus). 70 the bacteriostatic potential of the terpenoids was also increased when the carbonyl groups increased in structure. 63 the type of alkyl substituent incorporated into a nonphenolic ring structure is responsible for enhancement of antibacterial activity. such as an alkenyl substituent (1-methylethenyl) makes an increase in antibacterial activity, as seen in limonene [1-methyl-4-(1-methylethenyl)-cyclohexene] (25), compared to an alkyl (1-methylethyl) substituent as in p-cymene [1-methyl-4-(1-methylethyl)-benzene] (26) . furthermore, principally gram-negative were the sensitive organisms that propose alkylation control of the gram reaction sensitivity of the bacteria. an allylic side-chain seems to raise the inhibitory role of the simple phenols mainly against gram-negative organisms. this was suggested due to the majority of the antimicrobial activity of alkylated phenols in relation to phenol which has been earlier reported (fig. 16.3) . 56 it was observed that α-isomers are inactive relative to β-isomers in many compounds in stereochemistry, which is also effective in antibacterial activity observed from essential oils. 44 the (e,e)-2,4-decadienal (26) appears to be more toxic to bacterial cells than the correspondent monounsaturated aldehyde (e)-2-decenal (27) as another example of stereochemistry effectiveness in activity potential observed in the unsaturated aldehyde, but it is noticeable that two double bonds in the cis configuration in the side-chain of 2,4-decadienal (26) produce more bends and shorten the length of the carbon tail, such α,β-unsaturated aldehydes might be a good choice compared to other highly toxic sterilizers (fig. 16.3) . 53 in summary, the antimicrobial activity of essential oils depends on different amounts of specific compounds. as an example, antimicrobial properties of the essential oils with high concentrations of eugenol (3), cinnamaldehyde (11) , or citral (19) , is predictable. remarkable antimicrobial, antifungal, 3 screening of the antibacterial effects of essential oils and antiviral activity of the monoterpenes and phenols relieve from essential oils present in thyme, sage, and rosemary. due to the formation of polysaccharides that increase the resistance to essential oils there are some other essential oils, such as basil, sage, hyssop, rosemary, and oregano, which are active against e. coli, s. aureus but are less effective against pseudomonas spp. [71] [72] [73] the typical characteristic of the essential oils is hydrophobicity, which is responsible for the disruption of bacterial structures and makes an increase in permeability due to a weakness to pull apart the essential oils from the bacterial cell membrane. many cellular functions, including maintaining the energy status of the cell, membrane-coupled energy transducing processes, solute transport, and metabolic regulation result from the cell membrane permeability barrier. actually, they are responsible for leakage of the cell contents, reducing the proton motive force, reducing the intracellular atp pool via decreased atp synthesis and augmented hydrolysis are the mechanisms of action of the essential oils including the degradation of the cell wall, damaging the cytoplasmic membrane, cytoplasm coagulation, damaging the membrane proteins, increased permeability that is different from the increased membrane permeability and reducing the membrane potential via increased membrane permeability. 33, 74, 75 unfortunately, scientific investigations on the antimicrobial activity of essential oils have been retarded by the lack of appropriate susceptibility testing methods for the essential oils and because no generally approved assay method has been established for the assessment of their antimicrobial activity. many researchers have employed the disk assay method. however, the results of this method were not always in parallel with those of dilution assay methods. the differences were caused not only by differences in the solubility of the oils but also by interactions of the components in the concentrated solution used in the disk assay. 48 the dilution method could be more reliable than the disk method with regard to reproducibility and clinical relevance. when testing nonwater-soluble and highly volatile essential oils by the dilution method, it is necessary to obtain a homogeneous dispersion of the oils in the medium. chemical emulsifiers such as tween 80, tween 20 and other have been used frequently for this homogenization, but it has been reported that emulsifiers reduced the bioactivity of the oils, probably because of the formation of micelles, which inhibit adequate contact between the oil and the test organism. 48 evidence also shows that the minimum inhibitory concentration (mic) values of the essential oils under open conditions of incubation caused a two-to eight-fold rise in the mics of highly volatile oils, as compared with values obtained under sealed conditions. sealed conditions are used to examine whether essential oils showed antibacterial activity against major respiratory tract pathogens (this method was authorized by the japan society of chemotherapy to adjust for the physico-chemical properties of essential oils). the scientific information concerning the antimicrobial effectiveness of the essential oils in the vapor phase compared with direct contact case show that potential of this form of the essential oils is ambiguous, although some degree of inhibition by volatile components of the essential oils has been demonstrated in the vapor phase. in fact, more investigation into the antibacterial effectiveness of the vapor phase is required. it seems that the composition of the atmosphere generated by the essential oils is also potentially correlated with their antimicrobial behavior. this part mentioned the main antibacterial activity laboratory assay methods, as already noted. these methods do not show coordinated results in some case and this appearance is reasoned to the necessity of simultaneously running two or more methods for antibacterial activity determination of essential oils. in this method, a petri dish is commonly used as an assay chamber (5-12 cm diameter and filled with 10-20 ml of agar broth). the solidified medium in the petri dish was inoculated with an appropriate colony of the microorganisms. this inoculation was accomplished by using a solution such as physiological saline solution containing adequate colony unit (commonly 100-200 (cfu)/ml). the essential oil is incorporated into the mediums in two ways: on a paper disc or into a well (hole) which is made in the agar medium. diluted or undiluted essential oils based on the goals were added to a sterile blank filter disk (5 mm diameter) and placed on top of the cultured media in a petri dish or added to the holes. after incubation under optimal conditions such as temperature and time, two different zones were considered in view of the average diameter of changes: (1) the zone where there is no growth of the microorganism, called total inhibition; and (2), the zone where growth of the microorganisms was significantly reduced in terms of amount of colonies, compared to blank assays. the growth of the microorganisms is recognizable with instruments or can be seen visually by one of the commonly used techniques, called turbidimetry, in which the optical density changes in the growing culture (od) are measured. some commercial systems have been produced for monitoring of the microorganisms growth as well as other methods were proposed (eg, bioautography). 76 an appropriate volume (100 µl physiological saline solution) of the microorganism's colony (10-20 cfu/ml) will be inoculated to the solidified medium. each essential oil sample was diluted in suitable solvent such as ethyl ether to obtain serial dilutions (v/v). the required volume (10 µl) of each dilution was then added to sterile blank filter disks or cups and placed on the medium free cover of each petri dish. experiments were designed in two forms of petri dish positions, including direct and invert placement. the petri dishes were then sealed using sterile adhesive tape. blanks were prepared by adding the same volume (10 µl) of samples of the solvent to the filter disks or cups, and this had no effect on the viability of any of the tested organisms. after the incubation period, the minimal inhibitory concentration (mic), expressed as microliters of the essential oil per volume unit of atmosphere above the organism growing on the agar surface, that caused inhibition by comparison with control tests was measured. 77 although the modification with liquid broth is mostly practical for fungi, the serial dilution agar method is also common for bacteria and fungi. the difference is that agar broth cultures are grown in petri dishes or tubes, but liquid broth cultures are cultivated in conical flasks filled with 100 ml medium or test tubes with 2.5-5 ml medium (bacteria and moulds). the inhibitory growth index is determined for the liquid broth in conical flasks (percent changes in mould's biomass comparing to the control culture). there is an inhibitory effect of essential oil which appears in the test tube cultures and is measured turbidimetrically or with the plate count method. the estimation of essential oil activity both in agar and liquid broth would be simplified through counting the tested microorganisms that can remain in the membrane. the lowest essential oil concentration in the broth that results in the lack of visible microorganism growth changes is known as the mic. the microorganisms are then transferred from the lowest essential oil concentration medium (with no visible microorganism growth) into a new broth medium and incubation to determine the lethal activity of essential oil. 78 viruses as invasive microorganisms may cause serious respiratory illness and in some cases lifethreatening conditions, such as acute pneumonia. although acute respiratory infection rates are not very high, this condition has been steadily increasing in children and persons over 60 years of age. the rates of hospitalization and death increase substantially in these cases. multiple factors, such as decline in respiratory and immune function, likely contribute to increased morbidity. natural products in all forms including pure compounds or extracts provide massive opportunities for new antiviral-lead compounds. 4, 80 at the moment, only a few effective antiviral drugs are available for the treatment of viral diseases, especially in respiratory viral infections. therefore, finding new substances with antiviral properties is a required for medical systems. much evidence has been reported about antiviral potential of various essential oils and their constitutions on several genera of viruses. 9, 81 in some investigations the essential oils with high hydrocarbons long-chain contents such as (e,e)-2,4-decadienal (26) , showed activities against influenza virus. 82,83 on other spectrum of antiinfluenza virus compounds, sesquiterpenes and sesquiterpenes-rich essential oils showed clear effects as well as aromatic rich essential oils. [84] [85] [86] among the terpenoids compounds, terpinen-4-ol (28), terpinolene (29) , and α-terpineol (30), show inhibitory effect on influenza a/pr/8pr virus subtype h1n1. also indicated that some sesquiterpenes such as β-sesquiphellandrene (31), and tetrahydronaphthalenol (32) , showed potent antirhinoviral activity in a plaque reduction test. 87 in other researches that conducted about the antiviral essential oils and their active constitutions indicated that laurus nobilis essential oil which was characterized by the presence of β-ocimene (32), 1,8-cineole (6), α-pinene (4), and β-pinene (5), as the main constituents, showed interesting activity against sars-cov. 88 this overview caused the development of commercial therapeutics based on monoterpens compounds for viral infections especially in respiratory viral infections (fig. 16.3) . 89, 90 although, antiviral potential is an important factor for bioactivity of natural compounds, cytotoxicity effects that probably triggered host cell damaged cycles are important. therefore, this is a limiting factor for presentation of the new useful compound in treatment or control of the viral infections. viruses as invasive parasites are capable of using host cell organelles and mechanisms for reproduction, such perspicaciously reproducing process caused a creation of tenacious shield against many potent natural molecules used. therefore selective activity is an important point, as are antiviral potencies. the immune response to respiratory tract infection is a double-edged sword because many of the symptoms that accompany these infections are largely due to the microorganism's induction of cytokines and chemokines, which may result in protracted inflammatory responses. phagocytic clearance of an infecting organism by inflammatory cells is an appropriate and necessary component of the host defense system. 91 at the same time, much of the evidence points to destructive effects spectrum made by products of these inflammatory cells. 92 these products can increase mucus secretion and impair ciliary clearance, thereby setting the stage for exacerbations and recurrences of infection. primary inflammatory cell products can amplify the other steps of the inflammatory cycles and can paradoxically even impair the immune response. these observations suggest that modulating the inflammatory response may be an important aspect of definitive therapy for respiratory tract infection. the primary defense systems are the lung secretions and the mucociliary escalator, which entrap organisms and sweep them away. at the same time, the lung secretions also contain a variety of proteins that inhibit microorganisms, especially bacterial adherence for example immunoglobulin a (iga) systems, which prevents bacterial adherence to epithelial cells, inhibit bacterial growth, and attempt to neutralize bacteria. the macrophages are a secondary defense system, which not only phagocytose microorganisms but also release biochemotactic factors that recruit other defense cells such as monocytes and neutrophils that are necessary for further phagocytosis process. 93 the inflammatory response form a key part of the secondary defense system that accompanies additional proteins, such as immunoglobulins and complement factors. although neutrophil infestation is part of the natural defenses against an invading organism, it may have destructive effects on the pulmonary systems. one of the destructive factors is the proteolytic enzyme neutrophil elastase, which is preformed and stored within the neutrophil. this proteinase is released after the neutrophil is activated and migrates into the tissues and phagocytosis invasive microorganisms. it produces a condition that histologically simulates chronic bronchitis. the potential role of neutrophil elastase in the pathogenesis of pulmonary diseases is characterized by neutrophil infiltration of the airways and increased mucus secretion. 94 it has also been reported that "secretions from patients with acute bronchial infection cause a significant reduction in ciliary beat frequency and that the addition of a neutrophil elastase inhibitor can reverse this effect." 95 the increase in the mucus secretion and the decrease in ciliary beat have been found as an important feature of chronic lung disease frequently. besides the phagocytic cell immediate responses, stimulated epithelial cells and macrophages produce the potent neutrophil chemoattractant, such as interleukin-8 (il-8), which was found to be present in high concentrations in the sputum of patients with chronic inflammatory airway disease. 96 at the same time, elastase released by activated neutrophils also stimulates il-8 production by the epithelial cells. 97 therefore, when these events are initiated, they become a self-amplifying cycle. this phenomenon is observed in patients with acute pneumonia that has apparently been cured by appropriate antibiotic therapy. natural products as inflammation inhibitors have for a long time played a key role in many traditional treatment systems. several mechanisms including interaction with prostaglandin biosynthesis, interaction with other inflammatory mediators, and corticosteroid-like effects are involved in antiinflammatory action of natural products. 98 among the natural chemical compounds, significant antiinflammatory activities of plant-based essential oil have been reported by many researchers, which showed the basis for folk and traditional uses of these herbs for treatment of inflammatory diseases. the essential oils and aromatic plants have had a significant place in inflammation control in many folklore medications. this evidence supports the view that appropriate investigations about the potential of essential oils should be made and their active constituents should also be evaluated for inflammation control. monoterpene alcohols such as linalool (17) and linalyl acetate (18) and other corresponding esters which are reported as one of the major volatile components of many aromatic plants essential oil were evaluated for antiinflammatory activity. these compounds have shown promising antiinflammatory potency among the many essential oil compounds. 99 for example, 1,8-cineole (6) the major constituent of eucalyptus oil is another active essential oil constituent that is well tolerated in inhalation administrations. this compound can also be effective for airways inflammation in clinical trials, based on such finding 1.8-cineole (6) is registered as a licensed medicinal product and has been available for airways inflammation for many years. 100 similar effects were also observed in other monoterpenes especially in carvacrol (1) and thymol (2) , which are main constituents in thymus, oregano, savory, clove, and nutmeg essential oils. in other reports, similar observations have confirmed that many of the other essential oils and their constitutions can play an important role in inflammation control. emphasizing the structure-activity relationships is necessary to define different potential of natural compounds such as essential oils compositions. for instance, aliphatic aldehydes and aromatic aldehydes are said to predominantly have antiinflammatory and antimicrobial potentials. 101 the terpenes and sesquiterpenes evaluated appeared to be good inhibitors of 5-lox in vitro. there is a good correlation observed between antiinflammatory activity of the limonene (25) and the essential oils rich in limonene (25) like grapefruit, lime, and celery. other similar activity has been observed on the various inflammations pathways such as cyclooxygenase (cox) and lipoxygenases (lox) between sesquiterpenic alcohols, aliphatic aldehydes, and some phenolic esters that caused these phytochemicals considered for more investigations in respiratory inflammations during the air ways infections (fig. 16.3) . 101, 102 it has been evaluated that activity of some of the essential oils such as western red cedar (thuja plicata) to inactivate several viruses implicated in respiratory infections, and to inhibit the influenza virus-induced secretion of cytokine (il-6) in cultured human lung cells. however, the liquid essential oil phases are generally a higher irritant and possibly toxic for nasopharyngeal or oral applications, although a few reports have indicated that the vapors of some essential oils might be useful for application in inhaled vapors for respiratory infections in low concentrations. 103 because of the noteworthy role of inflammation in respiratory infectious disease persistence and recurrence, many laboratory models have been developed for inflammation and inflammation/infection-mixed respiratory complications research. some of these models, such as ovalbumin-induced respiratory allergic eosinophilia arise especially for inflammation process which responds to them, including eosinophils, neutrophils, and other immune cell infiltration to the inflammation sites. 104 in other models, in addition to this cellular pathogenesis, more attention is considered on the infection's correlation with inflammation mechanisms. 105 table 16 .5 shows some of the attributes of inflammation and inflammation/infection major models that are used in screening of the new antiinflammation compounds. respiratory infections are one of the most prevalent human health problems and they cause major difficulties for all age ranges. 106 research to find new and effective therapeutic compounds for the control and treatment of these ailments is a most attractive field in natural product screening. the massive biodiversity of natural sources, such as plants, means that the selection of the suitable starting source point is a critical step for achieving the best screening results. in particular, paying attention to the main chemical constitutions of these natural sources will help to clarify the results of this research. analysis of the relationship between the chemical structures of natural compounds has led to a better overview of the rational uses of natural compounds and natural remedies for the control and treatment of disease, and has led to the development of therapeutics. the broad diversity of the chemical makeup of essential oils has caused some uncertainty over their best selection in complementary or investigative research. therefore, a correct understanding of the relation between the potential effects and the plant's family is a useful guide for physicians and researchers. according to this finding, the volatile oils of plant families such as pinaceae have good potential for use in infectious and inflammatory respiratory diseases, in both research and treatment. these plant based essential oils have a good feasibility for presentation as therapeutics in many of the respiratory infections and inflammatory diseases. although these natural compounds have been used as complementary therapeutics, scientists must still give attention to their safety and 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pseudomonas aeruginosa in a rat model of chronic pulmonary infection respiratory viral infections in the elderly key: cord-328176-fck2ktxi authors: mahapatra, manojkumar; kulandaivelu, umasankar; saiko, philipp; graser, geraldine; szekeres, thomas; andrei, graciela; snoeck, robert; balzarini, jan; jayaprakash, venkatesan title: methyl-2-arylidene hydrazinecarbodithioates: synthesis and biological activity date: 2013-02-19 journal: chem zvesti doi: 10.2478/s11696-013-0346-4 sha: doc_id: 328176 cord_uid: fck2ktxi methyl-2-arylidene hydrazine-carbodithioate has not been of particular interest to researchers even though its metal complexes are extensively reported on due to their biological activity. this study examined the cytostatic and antiviral activity of twelve methyl-2-arylidene hydrazinecarbodithioates reported by many researchers as intermediates for the synthesis of thiosemicarbazides and the preparation of their metal complexes. compounds iic, iii, and iil with tridentate ligand features were found to have the lowest ic(50) value (6.5 μm, ≈ 1 μm, and 0.8 μm, respectively) against hl60 human promyelocytic leukemia cells. they were also most inhibitory to human embryonic lung (hel) fibroblast proliferation (5.3 μm, 17 μm, and 2.6 μm). compound iic and iil show antiviral activity against wild-type herpes simplex virus (hsv), varicella zoster virus (vzv), and acyclovirresistant hsv; however, these activities were observed at concentrations at which the compounds also markedly inhibit hl60 and hel cell proliferation. many n-substituted thiosemicarbazones were prepared by the condensation of schiff's bases of methyl-2-arylidene hydrazinecarbodithioate (mahcd) with amines (casero et al., 1980; collins et al., 1982; klayman et al., 1979 klayman et al., , 1991 scovill et al., 1982; shipman et al., 1981) . while thiosemicarbazones and their metal complexes were studied for their biological activity (beraldo & gambinob, 2004; ettari et al., 2010; jiang et al., 2006; katz, 1987; liberta & west, 1992; matesanz & souza, 2009; pandeya & dimmock, 1993; wnuk & robins, 2006; yu et al., 2009) , the intermediate mahcd was rarely studied for its metal complexation behaviour and the complexes for their biological activity (ali et al., 2002; kanwar et al., *corresponding author, e-mail: venkatesanj@bitmesra.ac.in 2008; neelam et al., 2000; saxena & tandon, 1983; singh et al., 1997) . very few investigators have reported biological activity of compounds with a dithiocarbamate side chain (cao et al., 2005; huang et al., 2009; kumar et al., 2010) . it is interesting to note that mahcd shares structural similarity with schiff's bases of n-hydroxy semicarbazides (snhs) and nhydroxy aminoguanidines (snhag) (fig. 1) . both snhs and snhag were rigorously documented for their anticancer activity and antiviral properties. it was proposed that these compounds act by inhibiting ribonucleotide reductase (rr) through metal chelation (das et al., 1999; ren et al., 2002; t'ang et al., 1985) . with this background, twelve mahcd derivatives were synthesised and evaluated for anticancer activity against hl60 human promyelocytic leukemia cells and antiviral activity against a panel of viral cell lines. synthesis of methyl hydrazinecarbodithioate (i) and substituted benzaldehyde hydrazones of methyl hydrazine carbodithioate (ii) to an ice cooled solution (< 10 • c) of 19.8 g (0.300 mol) of potassium hydroxide in 24 ml of water and 20 ml of propan-2-ol, 17.1 ml of 80 % pure hydrazine hydrate were added and constantly stirred. the amount of 18.2 ml (0.300 mol) of ice cooled carbon disulfide was added drop wise to the stirred solution, maintained at < 10 • c for over about 1-1.5 h. the bright yellow mixture formed was stirred for an additional 1 h after which ice cooled iodomethane (18.7 ml, 0.300 mol) was added drop wise over a 2 h period. stirring was continued for an additional 1 h, and the white precipitate obtained was filtered and washed with ice-cold water. the crude product was recrystallised from dichloromethane: yield of 43 %; audrieth et al. (1954) ; 81-83 • c, klayman et al. (1979) ). methyl hydrazinecarbodithioate (20.0 mmol) was dissolved in 20 ml of methanol and then, equimolar amount of aromatic/heteroaromatic aldehyde was added. the mixture was refluxed for 6-12 h and the reaction was monitored by tlc. the hot solution was poured onto crushed ice and the precipitate obtained was filtered (klayman et al., 1979; scovill et al., 1982) : yield of 60-80 %. the hl-60 human promyelocytic leukemia cell line was purchased from atcc (american type culture collection, manassas, va, usa). cells were grown in a rpmi 1640 medium supplemented with a 10 % heat inactivated fetal calf serum (fcs), 1 % l-glutamine and 1 % penicillin-streptomycin in a humidified atmosphere containing 5 % of co 2 . all media and supplements were obtained from life technologies (paisley, scotland, uk). cell counts were determined using a micro cell counter cc-108 (sysmex, kobe, japan). cells growing in the logarithmic phase of growth were used in all experiments described below. hl-60 cells (10 5 per ml) were seeded in 25 cm 2 nunc tissue culture flasks and incubated with increasing concentrations of drugs (iia-iil) at 37 • c under cell culture conditions. cell counts and ic 50 values were determined after 24 h, 48 h, and 72 h using the micro cell counter cc-108. viability of the cells was determined by trypan blue exclusion. results were calculated as the number of viable cells. antiviral assays (except anti-human immunodeficiency virus (hiv) assays) were based on the inhibition of virus-induced cytopathic effect in hel for the hcmv assays, confluent hel fibroblasts were grown in 96-well microtiter plates and infected with the human cytomegalovirus strains davis and ad-169 at 100 pfu per well. after a 2 h incubation period, the residual virus was removed and the infected cells were further incubated with a medium containing different concentrations of the test compounds (in duplicate). after incubation for 7-days at 37 • c, the virus-induced cytopathic effect was monitored microscopically after ethanol fixation and staining with giemsa dye. antiviral activity was expressed as the ec 50 or compound concentration required for the reduction of virus-induced cytopathogenicity by 50 %. the varicella zoster virus (vzv) drug susceptibility tests were performed on confluent hel cells in 96well microtiter plates by the plaque reduction assay. monolayers were infected with 20 plaque forming units (pfu) of the cell-associated virus per well. for each assay, virus controls (infected-untreated cells) were included. after a 2 h incubation period, the virus inoculum was removed and the media were replaced by different dilutions (in duplicate) of the tested molecules. serial dilutions of test compounds were incubated with the infected monolayers for five days. after a five day incubation period, the cells were fixed and stained with giemsa, and the level of the virus-induced cytopathic effect was determined by counting the number of plaques for each dilution. activity was expressed as ec 50 (effective compound concentration required to reduce virus plaque formation by 50 %) compared to the untreated control. the anti-hiv activity of the compounds was evaluated against the wild type hiv-1 strain iiib in mt-4 cell cultures using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (mtt) method. briefly, virus stocks were titrated in mt-4 cells and expressed as the 50 % cell culture infective dose (ccid50). mt-4 cells were suspended in culture medium at 10 5 cells per ml and infected with hiv at the multiplicity of infection of 0.02. immediately after viral infection, 100 µl of the cell suspension were placed in each well of a flat-bottomed microtiter tray containing various concentrations of the test compounds. the test compounds were dissolved in dmso at 50 mm or more as stock solutions. then, serial dilutions of the test compounds were made. the highest concentration tested was 100 µm of the test compound containing 0.2 % of dmso, which is by itself not toxic to cell proliferation. after four days of incubation at 37 • c, the number of viable cells was determined using the mtt method. compounds iia-iil were synthesised following the reaction sequence outlined in fig. 2 . methyl hydrazinecarbodithioate (i ) was prepared by the reaction of hydrazine hydrate (85 mass %) with carbon disulfide in the presence of potassium hydroxide at a temperature below 10 • c; followed by the addition of methyl iodide (audrieth et al., 1954) . condensation of i with aromatic aldehydes/ketones in methanol provided compounds iia-iil (klayman et al., 1991; scovill etal., 1982) . all compounds were characterised by their 1 h-nmr and fab-ms data. physicochemical characterisation data are presented table 1 and spectral data of compounds iia-iil are presented in table 2 . all compounds were tested for their anticancer activity against the hl-60 human promyelocytic leukemia cell line and the results are presented in table 3 . the compounds were also tested for their antiviral activity against herpes simplex virus-1 (kos), herpes simplex virus-2 (g), herpes simplex virus-1 tk − (kos acv r ), varicella-zoster virus (oka and 07/1), cytomegalovirus (davis and ad169), vaccinia virus, vesicular stomatitis virus, feline corona virus, feline herpes virus, human immunodeficiency virus (hiv) type 1 (iii b ) and type 2 (rod), coxsackie virus b4, respiratory syncytial virus, parainfluenza 3 virus, retable 4 . compound iil, iii, and iic showed ic 50 values against hl60 cells at the concentration of 0.8 µm, ≈ 1 µm, and 6.5 µm, respectively, after 72 h. it is interesting to note that preferably compounds with tridentate ligand characteristics (iil, iii, and iic) showed potent anti-proliferative activity against the hl60 cells (table 3 ). the activity of compound iii (2 -hydroxy acetophenone derivative) was higher than that of iic (2-hydroxy benzaldehyde derivative) and almost equally potent to iil (pyridine 2carboxaldehyde derivative). all the other compounds (iia, iib, iid-iih, and iik) were inhibitory at concentrations between 55 µm and 63 µm after 72 h, except for iij, irrespective of the nature and position of the substitution. compound iij did not exert any inhibitory activity on the hl60 cells at the maximum concentration studied (100 µm, after 72 h). all the compounds exhibited a cytotoxic concentration (cc 50 ) of ≥ 100 µm in the hela cell cultures except for iil (36 µm), (table 3) . compounds iii and iil showed the best selectivity towards the hl60 cells and therefore, further investigations are required. compounds iic and iii resemble the iron chelator 311 (n -[(2-hydroxynaphthalen-1-yl)-methylidene]pyridine-4-carbohydrazide) and iil resembles triapine (3-aminopyridine-2-carbaldehyde thiosemicarbazone). compound iii having a ketonic methyl group showed activity comparable to that of iron chelator 311 at 48 h. compound iil also exhibited an anti-proliferative activity comparable to that of triapine. the analogue of iil with a ketonic methyl group has to be considered for further improvement of its activity. it is interesting to observe that only compounds with tridentate ligand characteristics (iic, iii, and iil) have shown antiviral activity against a few viruses and are presented in table 4 . compound iil displayed pronounced antiviral activity against hsv-1 (8-42 µm), hsv-2 (9-11 µm ), vzv (6.5-42 µm), and vaccinia virus (15-45 µm). it should be noted that iil inhibits the wild-type as well as the acyclovir-resistant hsv at comparable compound concentrations (8-20 µm, table 4). compound iic with a 2-hydroxy substitution and compound iii, an acetophenone analogue of iic, show antiviral activity at somewhat higher concentrations than iil. compound iid that differs from iic by a hydroxy functional group at the para position did not show any appreciable antiviral activity. additional analogues of iic and iil are currently under further investigation. it should, however, be noted that iic, iii, and iil are poorly cytotoxic but they show pronounced cytostatic activity in proliferating hel (and hl60) cell cultures. therefore, the observed antiviral activity might not be due to direct antiviral activity but rather due to indirect inhibitory action caused by the underlying cytostatic potential of these compounds. the present investigation revealed that: i) compounds with a metal chelating functional group (iic, iii, and iil ) at their aryl end were found to be active and ii) the methyl dithioate (-cssch 3 ) group is not an bioisosteric equivalent of hydroxamate (-conhoh) or amidoxime (-cnhnhoh) (fig 1.) . the compounds investigated are known as intermediates in the synthesis of many thiosemicarbazones but their usefulness as medicinally active agents has not yet been studied. the study suggests a possibility of intermediates with specific pharmacophoric features for the intended activities that can provide a new scaffold for these activities. hydrazine derivatives of the carbonic and thiocarbonic acids. i. the preparation and properties of thiocarbohydrazide preparation, characterization and antifungal properties of nickel(ii) complexes of tridentate ons ligands derived from n-methyl-s-methyldithiocarbazate and the x-ray crystal structure of the [ni(onmes)cn]·h 2 o complex the wide pharmacological versatility of semicarbazones, thiosemicarbazones, and their metal complexes. mini-reviews in medicinal chemistry synthesis and in vitro antitumor activity of 4(3h)-quinazolinone derivatives with dithiocarbamate side chains activity of 2-acetylpyridine thiosemicarbazones against trypanosoma rhodesiense in vitro activity of 2-acetylpyridine and 2-acetylquinoline thiosemicarbazones tested in vitro in combination with other antituberculous drugs inhibition of herpes simplex virus type 1 and adenovirus type 5 by heterocyclic schiff bases of aminohydroxyguanidine tosylate falcipain-2 inhibitors synthesis and antitumor activity of novel dithiocarbamate substituted chromones neuroprotective activity of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (pan-811), a cancer therapeutic agent synthesis and mycobactericidal properties of metal complexes of isonicotinoyldithiocarbazic acid thiosemicarbazones: inhibition of the growth of pox viruses and requirement for the growth of an isatinβ-thiosemicarbazone dependent mutant 2-acetylpyridine thiosemicarbazones. 1. a new class of potential antimalarial agents 2-acetylpyridine thiosemicarbazones. 13. derivatives with antifilarial activity impact of metal coordination on cytotoxicity of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (triapine) and novel insights into terminal dimethylation imidazole derivatives as possible microbicides with dual protection antifungal and antitumor activity of heterocyclic thiosemicarbazones and their metal complexes: current status α-n-heterocyclic thiosemicarbazone derivatives as potential antitumor agents: a structure-activity relationships approach. mini-reviews in medicinal chemistry palladium(ii) complexes of ns donor ligandsderived from s-methyl-dithiocarbazate, s-benzyldithiocarbazate and thiosemicarbazide as antiamoebic agents recent evaluations of thiosemicarbazones and semicarbazones and related compounds for antineoplastic and anticonvulsant activities synthesis, biological evaluation, and quantitative structure-activity relationship analysis of new schiff bases of hydroxysemicarbazide as potential antitumor agents the potential of iron chelators of the pyridoxal isonicotinoyl hydrazone class as effective antiproliferative agents ii: the mechanism of action of ligands derived from salicylaldehyde benzoyl hydrazone and 2-hydroxy-1-naphthylaldehyde benzoyl hydrazone antitumor activity of some diorganotin and tin(iv) complexes of schiff bases 2-acetylpyridine thiosemicarbazones. 4. complexes with transition metals as antimalarial and antileukemic agents antiviral activity of 2-acetylpyridine thiosemicarbazones against herpes simplex virus antitumor activity studies of newly synthesized n-salicyloyl-n -(p-hydroxybenzthioyl)hydrazine and its copper(ii) complex both in vivo and in vitro optimization of the schiff bases of n-hydroxy-n -aminoguanidine as anticancer and antiviral agents ribonucleotide reductase inhibitors as anti-herpes agents thiosemicarbazones from the old to new: iron chelators that are more than just ribonucleotide reductase inhibitors key: cord-293867-c4wnr5xe authors: gürsoy, elif; dincel, efe doğukan; naesens, lieve; ulusoy güzeldemirci, nuray title: design and synthesis of novel imidazo[2,1-b]thiazole derivatives as potent antiviral and antimycobacterial agents date: 2019-12-06 journal: bioorg chem doi: 10.1016/j.bioorg.2019.103496 sha: doc_id: 293867 cord_uid: c4wnr5xe a series of novel acyl-hydrazone (4a-d) and spirothiazolidinone (5a-d, 6a-d) derivatives of imidazo[2,1-b]thiazole were synthesized and evaluated for their antiviral and antimycobacterial activity. the antituberculosis activity was evaluated by using the microplate alamar blue assay and the antiviral activity was evaluated against diverse viruses in mammalian cell cultures. according to the biological activity studies of the compounds, 5a-c displayed hope promising antitubercular activity, 6d was found as potent for coxsackie b4 virus, 5d was found as effective against feline corona and feline herpes viruses. consequently, the obtained results displayed that, 5a-d and 6d present a leading structure for future drug development due to its straightforward synthesis and relevant bioactivity. lately, much interest has been focused on the chemistry and the biological activity of fused heterocyclic systems because of their broad spectrum of physiological activities [1, 2] . among these heterocyclic compounds carrying nitrogen atom, imidazo [2,1-b] thiazole derivatives possess specific importance because of their diverse pharmacological activities. the imidazo [2,1-b] thiazole derivatives have been reported in the literature as antibacterial [3, 4] , antitubercular [5] , antifungal [6] , antitumoral [7] , antiviral [8, 9] , antihelmintic [10] , analgesic [11] , anti-inflammatory [12] , antihypertensive [13] , cardiotonic [14, 15] , diuretic [16] , herbicide [17] and insecticide [18] agents. levamisole [(6s)-2,3,5,6-tetrahydro-6-phenylimidazo[2,1-b]thiazole] (fig. 1) , which is the levogyre isomer of antihelminthic tetramisol and contains imidazo [2,1-b] thiazole moiety, is a drug that has significant immunomodulatory properties [19] in addition to its antihelminthic activity [20] . besides the wide biological activity spectrum of imidazo[2,1-b] thiazole derivatives, also the compounds bearing hydrazide, acyl-hydrazone and spirothiazolidinone moiety, have been reported in the literature with their various effects such as antibacterial [21] , antifungal [22] , antitubercular [23] , antiviral [24] , anticonvulsant [25] and antidepressant [26] . on the other hand, the two compounds bmy-27709 and bms-199945, which are known in the literature for their antiviral activity and especially for their effects on influenza virus, have attracted the attention of researchers as compounds, that has a specific chemical character by an amide structure which connects an aliphatic cyclic system with an aromatic system [27, 28] . in this study, we further explored the scaffold containing the imidazo [2,1-b] thiazole ring as the aromatic moiety, that is linked by an amide to a spirothiazolidinone ring system as the aliphatic cyclic moiety and from this point forward, novel derivatives were synthesized (table 1) , and broadly evaluated for their antiviral and antimycobacterial activity (fig. 2) . two hit compounds were found to inhibit feline coronavirus or coxsackie b4 virus. besides, we observed some activity against mycobacterium tuberculosis, suggesting the versatility and relevance of this compound class to achieved anti-infective agents. compound 1 was obtained according to the procedure described by robert et al [29] . compound 2 was obtained according to the procedure described by robert et al [29] . compound 3 was obtained according to the procedure described by harraga et al [30] . general procedure for the synthesis of 6-(4-bromophenyl)-n 2 -(substituted/non-substituted cycloalkylidene)imidazo[2,1-b]thiazole-3-acetohydrazides (4a-d) 0,005 mol of 3 was boiled in a water bath under reflux with 30 ml of ethanol until a clear solution was obtained. 0.01 mol of cyclic ketone was added and heated for 6 h. after cooling the mixture to room temperature, it was filtered and purified by crystallization with warm ethanol or washing. [2,1-b] to a suspension of 0.005 mol of 4a-d in 30 ml of anhydrous benzene is added 2 ml of mercaptoacetic acid / 2-mercaptopropionic acid. the reaction mixture is heated under a reflux condenser using a dean-stark trap in a water bath for 6 h. it is concentrated under reduced pressure and the excess acid is neutralized with nahco 3 solution. the resulting product is kept in the refrigerator until solidified. the crude product is filtered, washed with water, dried and purified by crystallization or elution with ethanol. white solid, mp 162-163°c, yield: 54.4%. anal. calcd. for c 21 white solid, mp, 178-180°c, yield: 66.4%. anal. calcd. for c 27 white [4.4] [4, 5] the antimycobacterial activity studies of the compounds were performed by the taacf (tuberculosis antimicrobial acquisition and coordinating facility by the national institute of health of the us government). primary screening was conducted at 6.25 µg/ml against mycobacterium tuberculosis h37rv in bactec 12b medium using a broth microdilution assay the microplate alamar blue assay (maba) [31] . compounds exhibiting fluorescence were tested in the bactec 460 radiometric system. compounds affecting less than 90% inhibition in the primary screen were not generally evaluated further. compounds demonstrating at least 90% inhibition in the primary screen were retested at lower concentrations against m. tuberculosis h37rv in order to determine the actual minimum inhibitory concentration (mic) using maba. rifampin was utilized as the standard compound in the assays and each assay was replicated four times. the mic was defined as the lowest concentration affecting a reduction in fluorescence of 90% relative to controls. concurrently with the determination of mics, compounds were tested for cytotoxicity (ic 50 ) in vero cells at concentrations ≤6.25 µg/ml or 10 times the mic for m. tuberculosis h37rv (solubility in media permitting). after 72 h exposure, viability was assessed on the basis of cellular conversion of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (mtt) into a formazan product using the promega celltiter 96 non-radioactive cell proliferation assay. compounds for which the selectivity index ic 50 :mic ratio) si > 10 were assumed to possess in vitro activity confirmed in the bactec 460 at 6.25 µg/ml. alamar blue susceptibility assay (maba). antimicrobial susceptibility testing was performed in black, clear-bottomed, 96-well microplates (black view plates; packard instrument, meriden, connecticut, usa) in order to minimize background fluorescence. outer perimeter wells were filled with sterile water to prevent dehydration in experimental wells. initial drug dilutions were prepared in either dmso or distilled deionized water, and subsequent twofold dilutions were performed in 0.1 cm 3 of 7h9gc (no tween 80) in the microplates. bactec 12b-passaged inocula were initially diluted 1:2 7h9gc, and 0.1 cm 3 was added to wells. subsequent determination of bacterial titers yielded 1 × 10 6 , 2.5 × 10 6 and 3.25 × 10 5 cfu cm −3 in plate wells for m. tuberculosis h37rv. frozen inocula were initially diluted 1:20 in bactec 12b medium followed by a 1:50 dilution in 7h9gc. addition of 0.1 cm 3 to wells resulted in final bacterial titers of 2.0x10 5 and 5x10 5 cfu cm −3 for h37rv. wells containing drugs only were used to detect autofluorescence of compounds. additional control wells consisted of bacteria only (b) and medium only (m). plates were incubated at 37°c. starting at day 4 of incubation, 20 mm 3 of 10x alamar blue solution (alamar biosciences/accumed, westlake, ohio, usa) and 12.5 mm 3 of 20% tween 80 were added to one b well and one m well, and plates were reincubated 37°c. wells were observed at 12 and 24 h for a color change from blue to pink and for a reading of ≥50,000 fluorescence units (fu). fluorescence was measured in a cytofluor ii microplate fluorometer (perseptive biosystems, framingham, massachusetts, usa) in bottom-reading mode with excitation at 530 nm and emission at 590 nm. if the b wells became pink by 24 h, the reagent was added to the entire plate. if the well remained blue or 50,000 ≤fu was twofold drug dilutions were prepared in either dmso (sigma) or distilled deionized water and delivered via a 0.5-cm 3 insulin syringe in a 50-mm 3 volume. drug-free control vials consisted of solvent with bacterial inoculum and solvent with a 1:100 dilution of bacterial inoculum (1:100 controls). vials were incubated at 37°c, and the gi was determined in a bactec 460 instrument (becton dickinson) until the gi of the 1:100 controls reach at least 30. all vials were read the following day, and the gi and daily changes in gi (δgi) were recorded for each drug dilution. the mic was defined as the lowest concentration for which the δgi was less than the δgi of the 1:100 control. if the gi of the test sample was greater than 100, the sample was scored as resistant even if the δgi was less than the δgi of the 1:100 control. the synthesized compounds (4a-d, 5a-d and 6a-d) were evaluated against diverse rna and dna viruses, using the following cellto perform the antiviral assays, the virus was added to subconfluent cell cultures in 96-well plates, and at the same time, the test compounds were added at serial dilutions. appropriate reference compounds were included such as the virus entry inhibitors urtica dioica agglutinin lectin and dextran sulfate; broad virus inhibitors ribavirin and mycophenolic acid; antiherpetic drugs ganciclovir and cidofovir; and hiv inhibitor azidothymidine and nevirapine. after 3-6 days incubation at 37°c (or the target compounds 4a-d, 5a-d and 6a-d were synthesized from 2-(6-(4-bromophenyl)imidazo [2,1-b] thiazol-3-yl)acetohydrazide by a four and five-step synthesis through the pathways shown in fig. 3 . 4-bromophenacyl bromide and ethyl 2-aminothiazole-4-acetate were dissolved in acetone and mixed in room temperature. after standing for 3 days, 2amino-3-[(4-bromobenzoyl)methyl]-4-(ethoxycarbonylmethyl)thiazolium bromide (1) was obtained [29] . thereafter, compound 1 was boiled under reflux in absolute ethanol and via the ring closure of compound 1, ethyl [6-(4-bromophenyl)imidazo[2,1-b]thiazole-3-yl]acetate hydrobromide (2) was obtained [29] . by heating the compound 2 and hydrazine hydrate in ethanol, 2-[6-(4-bromophenyl)imidazo[2,1-b]thiazole-3-yl]acetohydrazide (3) was obtained [30] . the compound 3 and cyclic ketones were heated under reflux in ethanol to yield 6-(4-bromophenyl)-n 2 -(substituted/non-substituted cycloalkylidene)imidazo [2,1-b]thiazole-3-acetohydrazides (4a-d). the reaction yields of compounds 4a-d were between the interval of 95%-72.5%. 4a-d were then reacted with mercaptoacetic acid / 2-mercaptopropionic acid in anhydrous benzene under reflux by using a dean-stark water separator. the obtained reaction mixture was eventually neutralized with nahco 3 solution and by that way 2-[6-(4-bromophenyl)imidazo[2,1-b]thiazol-3yl]-n-(2-nonsubstituted/methyl-6,7,8-nonsubstituted/alkyl/aryl-3-oxo-1-thia-4-azaspiro [4.4] non/ [4.5] dec-4-yl]acetamides (5a-d, 6a-d) were obtained. the reaction yields of the compounds 5a-d and 6a-d were between the interval of 87.8%-54.4% and 95%-54%, respectively. when the chemical structures of the synthesized compounds were examined, it was observed that the carbonyl group was not present in the compounds 4a-d whereas the same group was present in the spirothiazolidinone ring of the compounds 5a-d and 6a-d. also according to the ir spectroscopy results, the stretching band belonging to the carbonyl group was not visible in the spectrum of the compounds 4a-d whereas the mentioned band was able to be observed in the range of 1724-1707 cm −1 for 5a-d and 6a-d. the ir values belonging to the carbonyl groups of spirothiazolidinone ring of 5a-d and 6a-d were in agreement with the literature [33, 34] . in the 1 h nmr analysis, the nh 2 protons of compound 3, which were observed as a broad singlet peak (2h) at 4.38 ppm, was disappeared in compounds 4a-d, whereas the peaks of aliphatic ch 2 groups belonging to cyclic ketone arose as multiplet in the range of 3.15-1.44 ppm. when the results obtained from compounds 5a-d examined, it was determined that the peaks of s-ch 2 protons were added to the spectrum in the range of 3.68-3.60 ppm as a distinctive indicator related to the formation of the mentioned compound [33, 35, 36] . also from the 1 h nmr analysis data obtained from compounds 6a-d, it was determined that the s-ch and ch-ch 3 peaks of the spirothiazolidinone ring were in the range of 3.95-3.90 and 1.43-1.40 ppm, respectively and the values were in compliance with the literature [37, 38] . the ir, 1 h nmr, 13 c nmr and ms spectra of the novel compounds are in agreement with the assigned structures. no unacceptable side reactions were observed, and products were obtained in moderate to good yields. the broad antiviral evaluation demonstrated that compound 5d was quite effective against feline coronavirus in crfk cells (table 2) , with an antiviral ec 50 value of 4.8 µg/ml and a superior selectivity index (ratio of cytotoxic to antiviral concentration) higher than 20. 5d was four-fold less active against feline herpesvirus. similarly, two compounds (4c and 4d) had weak anti-dna virus activity in hel cells, with antiviral ec 50 values about 45 µg/ml ( table 3) . the most effective compound against feline coronavirus was 5d. when the sar analysis of 5d is evaluated by comparison with the other compounds, it can be understood that the existence of the structure of the spirothiazolidinone moiety is significant in the activity against the mentioned virus. it can also be observed that the spirothiazolidinone structure of 5d is separated from 5b-c by the 4-hydroxyphenyl substituent at the 8-position, and thus it can be concluded that the corresponding substituent is significant in the activity of the compound. additionally, the 2-position of the spirothiazolidinone structure separates 5d from 6b-d, indicating that the nonsubstitution of position 2 is crucial for the antiviral activity against the feline coronavirus. the results also displayed that, the compounds bearing spirothiazolidinone structure did not show activity against dna viruses, whereas compound 4c and 4d, which are carrying acyl-hydrazone moiety, showed activity against dna viruses. the obtained result displayed the importance of the existence of the acyl-hydrazone residue for the activity against dna viruses. also, 4-phenylcyclohexyl and 4-(4hydroxyphenyl)cyclohexyl derivatives of acyl-hydrazones have been found to further increase the activity against dna viruses. another hit compound, 6d, was somewhat active against coxsackie b4 virus (ec 50 : 10 µg/ml and selectivity index ≥2 (table 4) ). the antimycobacterial evaluation of compounds (4a-c, 5a-c) was performed. the results belonging in vitro primer analyses, that were performed against mycobacterium tuberculosis h37rv strain by using maba in bactec 12b media, were given in table 5 . according to the in vitro primer analyses, the compounds possessing any value where the mic value was equal to less than 10 µg/ml were considered as active for antimicrobial activity and further analysis of the mentioned compounds were conducted. from this point forward compounds 4a-c were not detected as active and no further antimycobacterial activity was performed for them. in contrast to compounds 4a-c, compounds 5a-c were detected as active and further antimycobacterial activity of these compounds was conducted and the results were displayed in table 6 . the obtained results displayed that, 4a-c, the acyl-hydrazone moiety containing imidazo[2,1-b]thiazole derivatives, showed no or weak antitubercular activity and the ring closure raised the antitubercular activity of the compounds as can be seen from the results related to the compounds 5a, 5b, and 5c. the fact that the conversion of acyl-hydrazone derivatives to spirothiazolidinone derivatives by ring closure increased the activity, indicates the importance of the existence of spirothiazolidinone structure for antitubercular activity. antibiotic resistance is rising to high levels sharply all over the world and new kinds of resistance mechanisms are emerging worldwide. infections such as pneumonia, tuberculosis, gonorrhea, blood poisoning are becoming harder to treat and current antibacterials are losing their effectivity day by day. designing of new effectual compounds to deal with these resistant bacterias has become one of the most important issues today. similarly, mycobacterium tuberculosis remains a leading infectious cause of death worldwide today. especially the evolution of multi-drug-resistant (mdr) strains of mycobacterium tuberculosis, is the main reason for the increased incidence of tuberculosis, therefore, the development of new antimycobacterial agents has become an obligation. another important is that needs new drug candidates are antiviral therapy and it is definitely necessary to design and develop new effective antiviral agents. in the present study, in an attempt to find novel antiviral and antitubercular agents, 12 diverse derivatives of imidazo[2,1-b]thiazole were designed and synthesized. the compounds were screened for their antiviral and antitubercular activity. the antimycobacterial activities of the compounds were tested against the mycobacterium tuberculosis h37rv strain and in the test method, each value, in which the mic value was equal to less than 10 µg/ml, was actively accepted for antimycobacterial activity. the mic values of the compounds 5a, 5b, and 5c were determined as 8.453 µg/ml, 1.566 µg/ml and 0.854 µg/ml and the molecules were found as active. also, compound 6d was found as effective for coxsackie b4 virus. the antiviral activity and cytotoxicity of the compounds against feline corona and feline herpes viruses were investigated in crfk cell cultures and in comparison with hha, uda and ganciclovir references, compound 5d was found as highly effective. the findings revealed the promising antiviral and antitubercular activity of acyl-hydrazone, spirothiazolidinone and 2-methyl-substituted spirothiazolidinone derivatives of imidazo [2,1-b] thiazole and these derivatives could be an interesting starting point for further structural optimization to obtain new promising and more potent antiviral and antitubercular agents. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. the levamisole story the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discusses in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.no writing assistance was utilized in the production of this manuscript. the authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the declaration of helsinki for all human or animal experimental investigations. supplementary data to this article can be found online at https:// doi.org/10.1016/j.bioorg.2019.103496. key: cord-270602-599vweqe authors: donia, marwa; hamann, mark t title: marine natural products and their potential applications as anti-infective agents date: 2003-05-22 journal: lancet infect dis doi: 10.1016/s1473-3099(03)00655-8 sha: doc_id: 270602 cord_uid: 599vweqe the oceans are a unique resource that provide a diverse array of natural products, primarily from invertebrates such as sponges, tunicates, bryozoans, and molluscs, and from marine bacteria and cyanobacteria. as infectious diseases evolve and develop resistance to existing pharmaceuticals, the marine environment provides novel leads against fungal, parasitic, bacterial, and viral diseases. many marine natural products have successfully advanced to the late stages of clinical trials, including dolastatin 10, ecteinascidin-743, kahalalide f, and aplidine, and a growing number of candidates have been selected as promising leads for extended preclinical assessment. although many marine-product clinical trials are for cancer chemotherapy, drug resistance, emerging infectious diseases, and the threat of bioterrorism have all contributed to the interest in assessing natural ocean products in the treatment of infectious organisms. in this review, we focus on the pharmacologically tested marine leads that have shown in-vivo efficacy or potent in-vitro activity against infectious and parasitic diseases. the oceans are a unique resource that provide a diverse array of natural products, primarily from invertebrates such as sponges, tunicates, bryozoans, and molluscs, and from marine bacteria and cyanobacteria. as infectious diseases evolve and develop resistance to existing pharmaceuticals, the marine environment provides novel leads against fungal, parasitic, bacterial, and viral diseases. many marine natural products have successfully advanced to the late stages of clinical trials, including dolastatin 10, ecteinascidin-743, kahalalide f, and aplidine, and a growing number of candidates have been selected as promising leads for extended preclinical assessment. although many marineproduct clinical trials are for cancer chemotherapy, drug resistance, emerging infectious diseases, and the threat of bioterrorism have all contributed to the interest in assessing natural ocean products in the treatment of infectious organisms. in this review, we focus on the pharmacologically tested marine leads that have shown in-vivo efficacy or potent in-vitro activity against infectious and parasitic diseases. although the diversity of life in the terrestrial environment is extraordinary, the greatest biodiversity is in the world's oceans, with 34 of the 36 phyla of life represented. the oceans cover more than 70% of the earth's surface and contains more than 300 000 described species of plants and animals. 1, 2 macroscopic plants and animals have adapted to all regions of the oceans, including polar, temperate, and tropical areas. the diversity in species is extraordinarily rich on coral reefs, where there are around 1000 species per m 2 in some areas, and the indo-pacific ocean has the world's greatest tropical marine biodiversity. 2 the marine environment represents a treasure of useful products awaiting discovery for the treatment of infectious diseases (figure 1). ecological pressures, including competition for space, the fouling of the surface, predation, and successfully reproducing have led to the evolution of unique secondary metabolites with various biological activities. 3 the importance that these secondary metabolites play in the control of infectious and parasitic organisms was for many years largely overlooked. in the past 30-40 years, marine plants and animals have been the focus of a worldwide effort to define the natural products of the marine environment. a small number of marine plants, animals, and microbes have already yielded more than 12 000 novel chemicals, with hundreds of new compounds still being discovered every year. these discovery efforts have yielded several bioactive metabolites that have been successfully developed by the pharmaceutical industry. 4, 5 several clinically useful drugs, investigational drug candidates, and pharmacological tools have already resulted from these marine-product discovery programmes. 4, 5 one of the marine natural products that is currently under clinical investigation as a potential new anticancer agents is the marine alkaloid ecteinascidin-743, isolated from the tunicate ecteinascidia turbinate. 6 ecteinascidin is a broad-spectrum antitumour agent and is among the most advanced compounds with clinical applications. 6 in this review, we focus on the pharmacologically tested marine leads that have shown in-vivo efficacy or potent invitro activity against infectious and parasitic diseases, including malaria, toxoplasmosis, trypanosomiasis, and viral, bacterial, and fungal infections. our objective was to highlight the most promising compounds that have the greatest potential to lead to clinically useful treatments. there was not room to review treatments for specific infectious diseases, but there are several helpful reviews for antiparasitic drugs, 7 antinematodal drugs, 8 antituberculosis agents, 9 antiviral leads, 10 and antifungal agents. 11 furthermore, there are some excellent general and extensive reviews focusing on marine natural products, such as those by capon, 12 newman and colleagues, 13 and faulkner. 14 the antifungal screening of marine samples has led to the characterisation of many unprecedented natural products in regard to antifungal activity and chemical structures. the frequency of invasive fungal infection has risen substantially with the increasing numbers of immunocompromised patients, such as those infected with hiv, receiving cancer chemotherapy, immunosuppressive therapy, or treatment with broad-spectrum antibiotics. 11 evidence is mounting that fungi display highly specific adaptations in the marine environment that include the production of unique secondary metabolites. 11 the fact that marine organisms contain secondary metabolites different from their terrestrial counterparts in structure and biological activity has led to the hypothesis that marine organisms may contain efficient antifungal compounds with different modes of action and selective antifungal activity compared with human cells. 11 jasplakinolide is the first example of a cyclodepsipeptide isolated from a sponge (figure 1) and was identified from a jaspis sp collected in fiji. 15 jasplakinolide, also named jaspamide, is a 19-membered macrocyclic depsipeptide with selective in-vitro antimicrobial activity and a minimum inhibitory concentration (mic) of 25 g/ml against candida albicans. the in-vivo topical activity of a 2% solution of jasplakinolide against a candida vaginal infection in mice is similar to that of miconazole nitrate. 15 gambieric acids are extremely potent antifungal metabolites isolated from a strain of the epiphytic marine dinoflagellate gambierdiscus toxicus. these metabolites are the first antifungal representatives of the brevetoxin-type (fused polyether rings) structures, consisting of nine contiguous ether rings and one isolated tetrahydrofuran (figure 2). gambieric acid a inhibits the growth of aspergillus niger at a concentration of 10 ng/disk. the potency of gambieric acids exceeds that of amphotericin b by 2000-fold. 16 toxic effects in mice or cultured mammalian cells are moderate for a dose of 1 mg/kg given by intraperitoneal injection. additional marine natural products with antifungal activity are listed in table 1. most antifungal compounds from marine origin are cytotoxic. consequently, they have not generally been considered promising antifungal agents for clinical applications. we have reviewed marine natural products that show potent antifungal activity, but evidence of cytotoxicity was not available for all. 11 in many cases, the assessment of whether antifungal activity outweighs cytotoxic effects will be required, followed by rational modifications to improve the therapeutic index for these molecules. the search for new antituberculosis agents has become increasingly important with the emergence of multidrugresistant strains of mycobacterium tuberculosis. despite the small number of investigators looking at marine products as potential leads against m tuberculosis, the research in this area has been productive (figure 3). anti-infectives from the sea the alkaloid (+)-8-hydroxymanzamine a is characterised by a complex heterocyclic ring system attached to a ␤-carboline moiety. (+)-8-hydroxymanzamine a was first isolated from a sponge pachypellina sp and later from an undescribed petrosiidae genus. 26 this alkaloid exhibits potent inhibitory activity against m tuberculosis h37rv, with an mic of 0·91 g/ml. 27 the likely biogenetic precursor ircinol a does not have the ␤-carboline moiety but still has an mic of 1·93 g/ml. ircinol a represents a useful candidate for assessment in vivo against m tuberculosis, since it shows low cytotoxicity and structural complexity compared with the other manzamine-type alkaloids. 27 in addition, manzamine a inhibits m tuberculosis h37rv, with an mic of 1·56 g/ml. 27 axisonitrile-3 is a cyanosesquiterpene isolated from the sponge acanthella klethra and shows potent inhibitory activity against m tuberculosis, with an mic of 2·0 g/ml. 28 pseudopteroxazole, a benzoxazole diterpene alkaloid isolated from the west indian gorgonian pseudopterogorgia elisabethae, induces 97% growth inhibition for m tuberculosis h37rv at a concentration of 12·5 g/ml without substantial toxic effects. 29 ergorgiaene, a serrulatane-based diterpene (also known as biflorane) was isolated from the hexane extract of the same west indian gorgonian, and induced 96% growth inhibition for m tuberculosis h37rv at a concentration of 12·5 g/ml. 30 litosterol is a c-19 hydroxysteroid isolated from an okinawan soft coral litophyton viridis. it inhibited 90% of the growth of m tuberculosis with an mic of 3·13 g/ml. the poor solubility of litosterol in the aqueous tissue culture media obscured assessment of cytotoxic effects. 9, 31 puupehenone induced 99% inhibition of m tuberculosis h37rv growth, with an mic of 12·5 g/ml and a 50% inhibitory concentration (ic 50 ) of 2·0 g/ml. the puupehenones are shikimate-sesquiterpene derived metabolites isolated from sponges of the order verongida and dictyoceratida, collected from the hawaiian islands. 9, 32 anthelmintic activity anthelmintics are drugs used to rid host organisms of helminth parasites. parasitism by nematodes (unsegmented worms that constitute the phylum nematoda) represents a major issue in the commercial livestock industry, and contributes substantially to malnutrition and disease in human beings. particularly difficult to eradicate is ascaris lumbricoides, the large gut worm, which causes malnutrition and obstructive bowel disease, and the soiltransmitted blood-sucking hookworms ancyclostoma duodenale and necator americanus, which lead to severe blood loss and iron-deficient anaemia, decreased food intake, impaired digestion, malabsorption, and poor growth rate. 33 despite the availability of excellent commercial anthelmintics, growing resistance to key structural classes (benzimidazoles and macrolides) necessitates the search for new bioactive agents. 33 jasplakinolide represents a potent antiparasitic as well as antifungal agent. it exhibited an in-vitro 50% effective dose of less than 1 g/ml against the nematode nippostrongylus braziliensis. 15 respectively. 34 this tetrahydrofuran inhibits the development of eggs for the infective free-living third stage of these two species. this degree of nematocidal activity is similar to that of the commercially available nematocides levamisole and closantel. unfortunately, attempts to translate this in-vitro nematocidal activity to in vivo proved unsuccessful, but is not surprising given the hydrophobic properties of this drug. 34 however, understanding of the in-vitro mode of action of these compounds may yet contribute to the discovery of new and improved anthelmintics. the amphilactams, isolated from a sponge of amphimedon sp collected in the great australian bight, feature an unusual carbon skeleton and an enamino lactone or lactam moiety (figure 4). amphilactam d has an in-vitro nematocidal activity against the free-living stages of the parasitic nematode h contortus, with an ld 99 of 0·39 g/ml. this substance inhibits larval development at the l1 stage, but has little or no activity against nematode eggs. this degree of in-vitro activity is similar to that of existing commercial anthelmintics, such as levamisole and closantel, and is thought to merit in-vivo assessment. 35 a sponge, geodia sp, collected from southern australia has yielded a potent nematocidal agent, geodin a magnesium salt, which is a macrocyclic polyketide lactam tetramic acid showing an ld 99 value of 1·0 g/ml. 36 diseases caused by protozoan parasites lead to high rates of mortality and morbidity worldwide. we have focused on the marine-derived natural products showing promising efficacy against the protozoal infections caused by leishmania sp, trypanosoma sp, toxoplasma sp, and plasmodium sp ( figure 5 ). leishmaniasis is a disease caused by an obligate intracellular parasite of the genus leishmania. types of disease range from self-healing ulcers (cutaneous leishmaniasis) to progressive nasopharyngeal infections (mucocutaneous leishmaniasis) to disseminating visceral leishmaniasis, which is generally fatal if left untreated. 37 in mediterranean countries adult visceral leishmaniasis is recognised as an aids-related opportunistic disease, largely due to reactivation of latent infections by immunosuppression. the most common drugs for the treatment of leishmaniasis contain pentavalent antimonials, such as sodium stibogluconate and meglumine antimonite, that have cardiotoxic effects at the recommended doses and consequently signify the urgent need for alternative treatments. 38 sponges of the genus plakortis are well known for their production of cyclic peroxides. two peroxides produced by the palauan sponge plakortis aff angulospiculatus are active against leishmania mexicana. the most active cyclic peroxide (ld 50 0·29 g/ml) causes lysis of the cell membrane after 24 h at a concentration of 1·0 g/ml, and a striking decrease in motility after 30 min. this peroxide is, however, less effective than ketoconazole (ld 50 0·06 g/ml). 39 toxoplasmosis can be transmitted to human beings from cats infected with the parasite toxoplasma gondii. the severity ranges from a self-limiting adenopathy to fatal encephalitis. this infection is particularly dangerous for pregnant women and immunocompromised individuals; congenital infection of infants is fatal in most cases. 40 new drugs or drug combinations that can eradicate the tissue cysts that cause relapses are urgently needed, particularly for patients intolerant to folate inhibitors (pyrimethamine, trimethoprim), which frequently cause undesirable side-effects. the remaining group of drugs used for the treatment of toxoplasmosis include the macrolides, which act only against the tachyzoite, leaving the cysts unaffected. 41 the alkaloid manzamine a displays 70% inhibition of the t gondii parasite, at 0·054 g/ml concentration, without cell toxic effects. the activity increases notably at concentrations of 0·54 g/ml and 5·40 g/ml, even though it is accompanied by an increase in the cytotoxic effects for the host cells, and was therefore selected for in-vivo assessment. a daily intraperitoneal dose of 8 mg/kg of manzamine a for 8 consecutive days, started on day 1 after infection, prolonged the survival of swiss webster mice to 20 days, compared with 16 days for untreated controls. exploration of new manzamines, structure-activity relation studies, and studies of optimum dose will be important to improve the in-vivo efficacy of the manzamines against t gondii. 42 sigmosceptrellin-b isolated from the red sea sponge diacarnus erythraeanus, exhibits potent in-vitro activity against t gondii at a concentration of 0·039 g/ml without severe toxic effects. sigmosceptrellin-b is active (84-99% inhibition) against the parasite in human diploid fibroblast cells. 43 a cyclic peroxylactone, plakortide, was isolated from the jamaican sponge plakinastrella onkodes and has potent in-vitro activity against t gondii, with an ic 50 of 0·023 g/ml and no toxic effects for the host cells up to a concentration 0·12 g/ml. 44 the flagellated protozoa trypanosoma cruzi and trypanosoma brucei are, respectively, the causal agents of south american trypanosomiasis (chagas disease) and human african trypanosomiasis (sleeping sickness). the drugs used to treat these diseases, including pentamidine and suramin, require parenteral administration and are effective only against the early haemolymphatic stage of the disease. the arsenical drug melarsoprol is available for late-stage central nervous system infection, but it causes reactive encephalopathy and is difficult to administer. the nitroheterocyclic drug benznidazole, used to treat acute stages of chagas disease, is not effective against the chronic phases of the disease and is poorly tolerated, which clearly indicates the need for new drugs with structures and mechanisms of action different from those that are presently used. 45 from the green alga ulva sp the endophytic and obligate marine fungus ascochyta salicorniae was isolated and cultivated on a preparative scale, producing a structurally unusual tetramic acid metabolite ascosalipyrrolidinone a, which shows activity against t cruzi with an mic of 1·1 g/ml, whereas the control (benznidazole) has an mic of 30·0 g/ml. the main limitation for further development of this compound is its cytotoxic effects at 3·7 g/ml tested against rat skeletal muscle myoblast cells. 46 malaria is a particularly serious disease in sub-sahran africa, 47 but it is also a serious public-health issue in certain regions of southeast asia and south america. most malaria cases and deaths are caused by the parasite plasmodium falciparum. 47 since removal of the vector of transmission (the anopheles mosquito) is almost impossible, new antimalarial agents providing novel mechanisms of action will always be needed to combat resistance to drugs such as chloroquine, mefloquine, quinine, and sulfadoxine-pyrimethamine. manzamine a exhibits potent in-vitro activity against p falciparum (d6 clone), with an mic of 0·0045 g/ml, compared with control-drug (chloroquine and artemisinin) mics of 0·0155 g/ml and 0·010 g/ml, respectively. 48 manzamine a inhibits the growth of the rodent malaria parasite plasmodium berghei in vivo, with more than 90% of the asexual erythrocytic stages of p berghei inhibited after one intraperitoneal injection of 50 or 100 mol/kg manzamine a into infected mice. this treatment prolongs the survival of highly parasitaemic mice to more than 10 days compared with 2-3 days among untreated controls, 2 days among mice treated with artemisinin, and 6 days among mice treated with chloroquine; it has a 40% recovery rate 60 days after one injection. oral administration of an oil suspension of manzamine a also substantially reduces parasitaemia. interestingly, the manzamine a hydroxyl derivative (-)-8-hydroxymanzamine a, as well as the dimer neo-kauluamine, extend the lives of p berghei-infected mice far more than the two most important human therapeutic antimalarial drugs when administered as one intraperitoneal dose of 100 mol/kg. manzamine a and selected derivatives have a rapid onset of action because of high bioavailability and sustained antiparasitic activity with no apparent toxic effects. 48 manzamine a has a similar therapeutic index to chloroquine, whose toxic dose at 500 mol/kg is ten times higher than the dose required (50 mol/kg) to clear the parasite if administered three times with 2-day intervals. 48 the effectiveness of manzamine a and selected analogues against malaria makes them one of the most promising antiinfective leads to be discovered from the oceans, and understanding the structure-activity relation of this unique class of alkaloids is certain to lead to more effective and safer manzamine-related antimalarial drug leads. the previously mentioned antifungal macrolide halichondramide also has notable activity against p falciparum (d6 clone), with an ic 50 of 0·002 g/ml, which is approaching the value for the most active clinically used compounds (mefloquine 0·0003 g/ml). although halichondramide has a much lower selectivity index than currently used drugs, it represents a potential new class of compounds in the development of alternative chemotherapy for the treatment of malaria after further structure-activity relation investigations. 49 an example of a marine secondary metabolite containing the isonitrile group and eliciting significant antimalarial activity is di-isocyanoadociane, a tetracyclic diterpene with an isocycloamphilectane skeleton. di-isocyanoadociane has been isolated from the sponge cymbastela hooperi (axinellidae, halichondrida) and displayed antiplasmodial potency, with ic 50 values of 0·005 g/ml, and selectivity that rivals the invitro results obtained from clinically used antimalarial drugs (chloroquine and artemisinin). 50 the kalihinane diterpenoids exhibit several types of biological activities, of which the antimalarial activity of kalihinol a is the most noteworthy. kalihinol a was isolated from an okinawan marine sponge, acanthella sp, and it possesses notable in-vitro antimalarial activity (ec 50 0·0005 g/ml) and selectivity index (si 317) similar to those for mefloquine. 51 the development of resistance to current antibacterials continues to be a serious difficulty in the treatment of infectious diseases, and therefore the discovery and development of new antibiotics has become a high priority review anti-infectives from the sea in biomedical research. in the continuing effort by the marine natural products community, many antibacterial agents have been identified; we have focused only on those that seem to possess the greatest potential ( figure 6) . squalamine is the first aminosterol isolated from the dogfish shark squalus acanthias (squalidae). it has potent antimicrobial activity, with an mic of 1·0 g/ml against staphylococcus aureus, and antiangiogenic and antitumour properties. it is currently being tested in clinical trials for the treatment of advanced non-small-cell lung cancer. 52 cribrostatins were isolated from a blue sponge cribrochalina sp, and showed potent antineoplastic and antimicrobial activities. cribrostatin 3 has potent inhibitory activity against neisseria gonorrheae, with an mic of 0·09 g/ml. cribrostatin 3 is also active against penicillinresistant n gonorrheae (clinical isolate), with an mic of 0·39 g/ml. 53 sphaerococcus coronopifolius, a cosmopolitan red alga collected along the atlantic coast of morocco, contains the potent antibacterial diterpene, bromosphaerone. it shows antibacterial activity against s aureus, with an mic of 0·047 g/ml. 54 a marine fungus isolated from the surface of the brown alga rosenvingea sp, of the genus pestalotia, collected in the bahamas, was cocultured with a unicellular marine bacterium to yield pestalone. pestalone has potent antibiotic activity against meticillin-resistant s aureus, with an mic of 0·037 g/ml, and vancomycin-resistant enterococcus faecium, with an mic of 0·078 g/ml. these results represent an important achievement in showing that mixed fermentation can induce the biosynthesis of novel antibiotics, suggesting that this method may have use for drug discovery in the future. in addition, the potency of this agent toward drug-resistant pathogens suggests that pestalone should be assessed in more advanced animal models against infectious diseases. 55 jorumycin is a dimeric isoquinoline alkaloid that was isolated from the mantle and mucus of the pacific nudibranch jorunna funebris. it inhibits the growth of various grampositive bacteria-eg, bacillus subtilis, s aureus-at a concentration of 0·050 g/ml, with an inhibition zone of 16 mm. despite this high potency, there are several limitations for further investigation, which include its cytotoxic effects at an ic 50 of 0·012 g/ml and the difficulty in obtaining a pure stable preparation. 56 viruses have remained resistant to treatment or prophylaxis longer than any other infectious organism. the search for viral chemotherapeutic agents from marine sources has yielded several promising therapeutic leads reported to display notable antiviral activity (figure 7). perhaps the most important antiviral lead of marine origin reported thus far is the nucleoside ara-a. ara-a is a semisynthetic compound based on the arabinosyl nucleosides isolated from the sponge cryptotethia crypta. 57 once it was realised that biological systems would recognise the nucleoside base after modifications of the sugar moiety, chemists began to substitute the typical pentoses with acyclic entities or with substituted sugars, leading to the drug azidothymidine (zidovudine). ara-a (vidarabine), ara-c (1-␤-d-arabinosylcytosine; cytarabine), acyclovir, and azidothymidine are in clinical use and are all examples of products of semisynthetic modifications of the arabinosyl nucleosides. 58 the didemnins are a family of closely related cyclic depsipeptides obtained from trididemnum solidum, a caribbean tunicate, or sea squirt, of the family didemnidae. 59 in addition to their antitumour activity, they exhibit significant in-vitro and in-vivo antiviral properties. in 1982, canonico and colleagues 60 reported that didemnin b has in-vitro inhibitory effects against the viruses causing rift valley fever (ld 50 0·04 g/ml), venezuelan equine encephalomyelitis (ld 50 0·08 g/ml), and yellow fever (ld 50 0·08 g/ml). in-vivo didemnin b at a dose of 0·25 mg/kg daily in mice infected with rift valley fever virus gave a 90% survival rate. despite its important antiviral activity, didemnin b is cytotoxic and inhibits cellular dna, rna, and protein synthesis at concentrations close to those at which viral growth was inhibited, and hence it has low antiviral selectivity and therapeutic index. the didemnins might, however, be modifiable or useful in combination with other antiviral agents to combat such difficult diseases. 61 didemnin b interferes with mitogenic signal transmission such as inhibiting kinases, phosphatases, and elongation factors. since 1986, didemnin b has been subjected to phase 1 and 2 clinical trials for cancer by different groups, and the consensus is that with the dose schedules used, the drugs provided little efficacy and substantial toxic effects. 61 eudistomins represent a large family of ␤-carboline alkaloids that have been isolated from shallowwater tunicates from the genus eudistoma. there are four types of eudistomins, including: unsubstituted, pyrrolyl-substituted, pyrrolinyl-substituted, and tetrahydro-␤-carbolines containing a uniquely condensed oxathiazepine ring system. the in-vitro antiviral potency of the eudistomins against herpes simplex viruses 1 and 2 ranges from 5 ng/disk to 500 ng/disk and follows a trend in which the oxathiazepino-tetrahydro-␤-carbolines show greater potency than 1-pyrrolinyl-substituted, which in turn have better antiviral activity than the 1-pyrrolyl-substituted compounds. 62 perry and colleagues 63 reported isolating and investigating the structure of mycalamide a from a new zealand sponge mycale sp. they found that a material consisting of 2% mycalamide a was effective against a59 coronavirus in vivo in mice at 0·2 g/kg daily, with 100% survival after 14 days. when pure mycalamide a has been obtained, it inhibited herpes simplex virus 1 and poliovirus type 1 at 0·005 g/disk. 63 when mycalamide b was isolated, it showed greater antiviral activity and cytotoxicity than mycalamide a. in-vitro antiviral testing showed a minimum dose of inhibition of 0·001-0·002 g/disk for mycalamide b. mycalamide a and b are protein synthesis inhibitors. 64 further antiviral marine natural products are presented in table 2. the unprecedented diversity of marine natural products combined with an improved global awareness about the need for new anti-infective treatments is certain to result in an increased effort to move the product of the marine environment into clinical applications. although marineproduct leads have historically been confronted with many obstacles, including a sustainable supply-ie, the compounds may account for less than 10 -6 % of the wet weight 73 -there have been substantial advances, which suggest that sustainable sourcing will be achievable. we have learned from terrestrial drug leads that continuous and exhaustive harvesting from nature is not reliable and puts the respective species under risk of extinction. consequently, the large-scale production of metabolites from marine natural products for clinical applications is a real challenge and alternative strategies for environmentally sound and economically feasible supplies are clearly needed. traditionally, among the first options to be explored for the supply of marine-derived small molecules is chemical synthesis. unfortunately, the structural complexity of marine molecules, which suggests novel mechanisms of action and high selectivity, has also resulted in few economically feasible strategies for total chemical synthesis. one successful example of the synthetic production of a marine-product drug in unlimited quantities is the conus toxin ziconotide, because of its peptide nature. 74 a second but also fairly labour-intensive strategy is to do studies of the biological roles of marine natural product pharmacophores, with a clearly defined structural moiety, and attempt to define whether the critical pharmacophore via synthesis, chemical degradation, modification, or a combination of these, can result in more practical drugs based on a marine prototype. the aquaculture of the source organisms, including sponges, tunicates, and bryozoans, with the aim of securing a steady supply of drug product, has progressed notably in cancer applications, although in most cases the biomass currently generated is still far short of those required should a marine-based drug finally enter the market. 75 furthermore, the cultivation of invertebrates in their natural environment is subject to several uncertainties, such as destruction by storms or disease. an intriguing strategy has been to identify the true producers of bioactive compounds to find out whether or not they are of microbial origin, based on the fact that most if not all of the marine invertebrates harbour large communities of microorganisms, including bacteria, cyanobacteria, and fungi within their tissues. 76 many studies have successfully provided data to support the involvement of microorganisms in the biosynthesis of natural products isolated from invertebrates. the major breakthrough in the characterisation of the microbial communities associated with sponges involve the application of molecular methods such as fluorescence in-situ hybridisation, with the use of group-specific 16 s rrna-targeted oligonucleotide probes. this approach has led to the identification of the bacterial communities associated with many marine invertebrates. 77 review anti-infectives from the sea if bacteria or other associated microorganisms do produce these compounds of interest, the careful design of special media for culture would be useful for large-scale fermentation. currently, only an estimated 5% or less of the bacteria seen in marine samples by microscopic methods can be cultivated under standard conditions. 78 as a result, molecular approaches offer particularly promising alternatives through the transfer of biosynthetic gene clusters to a vector suitable for large-scale fermentation, thereby avoiding the difficulties in culturing symbiotic bacteria. clearly, the world's oceans will play an important part in the future control of the global infectious-disease burden. although substantial progress has been made in identifying novel drug leads from the ocean's resources, great efforts are still needed to advance to clinical applications. none declared. a 56-year-old man presented to the emergency department with a 4-week history of a painful left-sided preparotid swelling. computed tomography suggested that the lesion was most likely a tuberculous abscess (figure). samples for a clinical armamentarium of marinederived anti-cancer compounds the bioprocess-technological potential of the sea marine and freshwater products handbook biological activities of selected marine natural products marine natural products et-743 natural products as potential antiparasitic drugs secondary metabolites with antinematodal activity marine natural products as antituberculosis agents natural products as antiviral agents antifungal metabolites from marine sponges marine bioprospecting: trawling for treasure and pleasure the influence of natural products upon drug discovery marine natural products jasplakinolide a cyclodepsipeptide from the marine sponge jaspis sp gambieric acids: new potent antifungal substances with unprecedented polyether structures from a marine dinoflagellate gambierdiscus toxicus aurantosides d e and f: new antifungal tetramic acid glycosides from the marine sponge siliquariaspongia japonica phorboxazoles a and b: potent cytostatic macrolides from marine sponge phorbas sp halishigamides a-d new cytotoxic oxazolecontaining metabolites from okinawan sponge halichondria sp macrocyclic antifungal metabolites from the spanish dancer nudibranch hexabranchus sanguineus and sponges of the genus halichondria fascaplysin: an unusual antimicrobial pigment from the marine sponge fascaplysinopsis sp antifungal activity of meridine a natural product from the marine sponge corticium sp bengazoles c-g from the sponge jaspis sp synthesis of the side chain and determination of absolute configuration ptilomycalin a: a novel polycyclic guanidine alkaloid of marine origin haliclonadiamine: an antimicrobial alkaloid from the sponge haliclona sp 8-hydroxymanzamine a a ␤-carboline alkaloid from a sponge pachypellina sp 1234-oxamanzamines novel biocatalytic and natural products from manzamine producing indo-pacific sponges assessment of antimycobacterial activity of a series of mainly marine derived natural products novel antimycobacterial benzoxazole alkaloids from the west indian sea whip pseudopterogorgia elisabethae serrulatane diterpenes with antimycobacterial activity isolated from the west indian sea whip pseudopterogorgia elisabethae novel 19-oxygenated sterols from the okinawan soft coral litophyton viridis puupehenone-related metabolites from two hawaiian sponges hyrtios spp nutritional impact of intestinal helminthiasis during the human life cycle marine nematodes: tetrahydrofurans from a southern australian brown alga notheia anomala amphilactams a-d: novel nematocides from southern australian marine sponges of the genus amphimedon geodin a magnesium salt: a novel nematocide from a southern australian marine sponge chemotherapy of leishmaniasis: recent advances in the treatment of visceral disease leishmania and human immunodeficiency virus coinfection: the first 10 years antileishmanial cyclic peroxides from the palauan sponge plakortis aff angulospiculatus toxoplasmosis in aids patients anti-toxoplasmosis drugs new manzamine alkaloids with potent activity against infectious diseases antimalarial antiviral and antitoxoplasmosis norsesterterpene peroxide acids from the red sea sponge diacarnus erythraeanus new peroxylactones from the jamaican sponge plakinastrella onkodes with inhibitory activity against the aids opportunistic parasitic infection toxoplasma gondii recent advances in identifying and validating drug targets in trypanosomes and leishmanias ascosalipyrrolidinone a: an antimicrobial alkaloid from the obligate marine fungus ascochyta salicorniae survey of malaria treatment and deaths in vivo antimalarial activity of the betacarboline alkaloid manzamine a the marine environment: a resource for prototype antimalarial agents novel potent antimalarial diterpene isocyanates isothiocyanates and isonitriles from the tropical marine sponge cymbastela hooperi antimalarial activity of kalihinol a and new relative diterpenoids from the okinawan sponge acanthella sp aminosterols from the dogfish shark squalus acanthias antineoplastic agents 430 isolation and structure of cribrostatins 3 4 and 5 from the republic of maldives cribrochalina sp marine lit, and references from relevant articles. search terms were "antifungal marine new bromoditerpenes from the red alga sphaerococcus coronopifolius pestalone a new antibiotic produced by a marine fungus in response to bacterial challenge a new antitumor isoquinoline alkaloid from the marine nudibranch jorunna funebris contributions to the study of marine products: the nucleosides of sponges new anti-hiv agents and targets structures of the didemnins antiviral and cytotoxic depsipeptides from a caribbean tunicate inhibition of rna viruses in vitro and in rift valley fever-infected mice by didemnins a and b natural products as probes of cell biology: 20 years of didemnin research eudistomins c e k and l potent antiviral compounds containing a novel oxathiazepine ring from the caribbean tunicate eudistoma olivaceum mycalamide a an antiviral compound from a new zealand sponge of the genus mycale antiviral and antitumor agents from a new zealand sponge mycale sp 2: structures and solution conformations of mycalamides a and b papuamides a-d hiv-inhibitory and cytotoxic depsipeptides from the sponges theonella mirabilis and theonella swinhoei collected in papua new guinea inhibition of replication of the etiologic agent of acquired immune deficiency syndrome (human t-lymphotropic retrovirus/ lymphadenopathy-associated virus) by avarol and avarone debitus c: in vitro antiviral activity on dengue virus of marine natural products microspinosamide a new hiv-inhibitory cyclic depsipeptide from the marine sponge sidonops microspinosa solenolides new antiinflammatory and antiviral diterpenoids from a marine octocoral of the genus solenopodium hennoxazoles bioactive bisoxazoles from a marine sponge venustatriol a new anti-viral triterpenes tetracyclic ether from laurencia venusta antitumor and antiviral furanoditerpenoids from a marine sponge drugs from the seas: current status and microbiological implications mviia: from marine snail venom to analgesic drug aquacultural production of bryostatin 1 and ecteinascidin 743 electron microscope study of the association between some sponges and bacteria microbial diversity in the marine sponge aplysina cavernicola analyzed by fluorescence in situ hybridization (fish) chemical studies of marine bacteria: developing a new resource review anti-infectives from the sea the preparation of this review was supported by nih grants r01ai 36596 and ko2ai01502 from the national institute of allergy and infectious diseases, and the egyptian government (predoctoral fellowship for md). we thank charles stanley for his assistance in the preparation of this review. anti-infectives from the sea rabbit's revenge analysis were obtained from incision and drainage of the abscess; however, no acid-fast bacilli could be detected and culture media for bacteria, fungi, and mycobacteria remained without growth. a few days after his admission, the patient's wife joined her husband with a painful cervical lymph node. on further investigation it emerged that the couple had eaten a medium roasted wild rabbit in a berlin restaurant 2 months earlier. 1 week after the meal they both developed an exudative pharyngitis. oropharyngeal tularaemia was suspected and confirmed for both patients by positive serology. they both received a 2-week course of streptomycin and recovered uneventfully.tularaemia is caused by the gram-negative rod francisella tularensis. natural reservoirs and vectors include several small mammals, as well as insects like ticks and mosquitoes. transmission to people is through direct contact with infected animals, inhalation of contaminated fomites, insect bites, and ingestion of contaminated food. the primary clinical presentation of tularaemia depends on the site of entry of the bacterium into the body. oropharyngeal tularaemia results from oral exposure from contaminated fluids or foods. key: cord-315918-12rbbe8c authors: mukherjee, pulok k. title: antiviral evaluation of herbal drugs date: 2019-06-21 journal: quality control and evaluation of herbal drugs doi: 10.1016/b978-0-12-813374-3.00016-8 sha: doc_id: 315918 cord_uid: 12rbbe8c the viral infection and resistance to the existing antiviral drugs are alarming, which is a serious public health concern. medicinal plants are valuable resources for treatment of viral infections and can be used for the management of infections like herpes simplex virus (hsv), human immunodeficiency virus (hiv), influenza, etc. the antiviral screening of plant extracts should be highly selective, specific, and sensitive for bioactivity guided isolation of the active compounds from the plant extracts. the antiviral screening system should be validated for accuracy, reproducibility, simplicity, and cost effectiveness. this chapter highlights on various aspects for screening and evaluation of antiviral natural components including factors affecting antiviral in vivo studies, host cells, organisms, and culture media followed by different virus-specific assays for antiviral screening of natural products. although vaccines have been very successful in controlling many viral diseases, some diseases are likely to be controlled only by antiviral chemotherapy. the concept of antiviral drugs has only been accepted slowly, partly because of the toxicity of many of the earlier antiviral agents. in contrast to the development of antibiotics, attempts to develop antiviral drugs have indeed met a variety of problems. being strictly dependent on cellular metabolic processes, viruses possess only limited intrinsic enzyme systems and building blocks that may serve as specific targets for a drug. moreover, contrary to a bacteriostatic compound, an effective antiviral drug should not only display considerable specificity in its antiviral action, but should also irreversibly block viral synthesis in order to stop cell suicide due to the viral infection and restore normal cell synthesis (vanden berghe et al., 1986) . in addition to this inhibition, the antiviral agent must have a broad spectrum of activity, favorable pharmacodynamic properties, and not be immunosuppressive. in the ideal situation, the antiviral drug checks the infection while the immune system prepares to destroy the last virus particles (munro et al., 1987) . this point is critical for those immune-compromised by illness (aids, cancer) or drug therapy (transplants, cancer). a frequent cause of death in these instances is from viral infections, so that adjuvant antiviral chemotherapy is vital in these circumstances (shannon and schabel, 1980) . many viral infectious diseases still cause high mortality. although antiviral chemotherapy has shown outstanding progress, antiviral agents are still required. the emergence of drug-resistant viruses during treatment raises a potential problem for effective therapy. furthermore, new viral pathogens may be discovered. biologically active substances of plant origin have long been known as viral inhibitors. these antiviral compounds may be extracted from sources, such as higher plants, which have, for various reasons, been explored considerably less than the traditional ones. the first clinically useful antiviral drug was methylisatin-thiosemicarbazone (methisazone), which was active against pox viruses, especially smallpox virus. methisazone (25 mg/kg) was found to inhibit variola virus in mice and was later used successfully to treat cases of eczema vaccinatum and to treat and prevent smallpox. kaufman et al. (1962) , an ophthalmologist, successfully treated a herpes eye infection with an antineoplastic drug, idoxuridine. at the same time, a group of chemists at the du pont chemical company in the united states investigated the antiviral activity of a molecule called amantadine. it was active against influenza virus type a. in rapid succession, further nucleoside analogs, cytosine arabinoside, trifluorothymidine, and adenine arabinoside, were found to inhibit herpes virus. in the 1970s, the antiherpes activity of a new compound, 9-(2-hydroxyethoxymethyl) guanine (acyclovir), was detected by j. bauer at the wellcome research labs. within a decade, the same group was to discover azidothymidine (zidovudine), the first effective inhibitor of the newly emerged hiv. the alkaloid from the australian horse chestnut (castanospermine australe), isolated in the 1980s, was also found to be active against hiv. a research program to detect and isolate antiviral compounds from higher plants is best carried out by a multidisciplinary team, consisting of at least a pharmacognosist and a virologist. the antiviral screening system should meet all requirements of any good assay, including validity, lack of ambiguity, accuracy, reproducibility, simplicity, and reasonable cost. moreover, because we are dealing with plant extracts, the antiviral screen should be highly selective, specific, and sensitive; it is advisable to discriminate a true antiviral activity from a viricidal one at this stage. because most of the aforementioned requirements are better met by in vitro testing, we not only prefer in vitro screening of the plant extracts, but also the use of the same bioassay to guide the isolation of the antivirally active compounds from the plant extracts. the antiviral activity of the pure compounds then has to be confirmed in a later stage by in vivo assays (leven et al., 1982; vanden berghe et al., 1986) . one of the possible strategies for finding new antiinfective drugs may involve the search for compounds with chemotherapeutic activities supplementary to, and structures widely different from, those in current use. these compounds could be extracted from sources that have, for various reasons, been explored considerably less than the traditional microorganisms, including, among others, higher plants (mitscher and rao, 1984) . considering the enormous number and the amazing structural diversity of the currently available antimicrobially and antivirally active plant constituents, one might hope that promising systemic and/or locally acting antiinfective agents might be discovered in the plant kingdom (vanden berghe et al., 1986; vlientinck et al., 1988) . the increase of drug-resistant viruses in treatment raises an important issue for effective treatment. moreover, new viral pathogens might be found. along these lines, there is a strong requirement for promptly accessible antiviral medications at moderate costs with minimal side effects. henceforth, traditional medicines ought to be investigated as novel antiviral agents, as many of these ancient medicaments, containing different plant metabolites, have potent antiviral activities (chattopadhyay and khan, 2008) . the design of new antiviral drugs potentially focuses on the structural dynamics and replication cycles of the various infections causing viruses. a suitable example is the invention of acyclovir, which hinders certain proteins of herpes infections responsible for the triggering of disease. ethnomedicines are a vast repository of structural diversities and extensive bioactivities that can serve as a huge source of potential antiviral drugs. a significant number of medicinal plants from ayurveda and the traditional chinese system of medicine serve as potential remedies to decrease the severity of illness caused by viruses (chattopadhyay et al., 2009; khan et al., 2005; jadhav et al., 2012) . research on the antiviral potentials of plants was first started in 1952, and 12 out of 288 plants were found to be effective against influenza (chattopadhyay and naik, 2007) . numerous screening studies have been conducted in the last few years to determine the antiviral efficacy of medicinal plants using in vitro and in vivo assays. a few out of a 100 british colombian medicinal plants showed antiviral efficacy against respiratory syncytial virus (rsv), corona virus, influenza virus, and herpes simplex virus (hsv) (mccutcheon et al., 1995) , while the marine algae spirulina showed antimutagenic, immunomodulatory, and antiviral activities (chamorro et al., 1996) . interestingly, cyanovirin n, an 11-kda protein of blue green algae, inactivates hiv-1 by binding with its glycoprotein120 (clercq , while sulfated polysaccharides of seaweeds and algae showed anti-hiv and anti-hsv activities (schaeffer and krylov, 2000) . the plant kingdom is highly diverse and ranges from unicellular microscopic plants to long lived, huge trees. to screen each and every plant or their individual parts for the identification of antiviral components is a huge task. several examples of plants having antiviral properties and newly identified active compounds from them are reported in various journals. one of the examples that can be cited here is cyanovirin n (cv-n), an 11-kda protein isolated from the cyanobacterium nostoc ellipsosporum, which exhibits the property of inhibiting hiv-1 infection and also possesses broad-spectrum activity. the phytochemical, podophyllotoxin, isolated from the aqueous extract of podophyllum peltatum l., inhibited hsv type 1 (hsv-1) (bedows and hatfield, 1982) . the acetone extract of another plant, phyllanthus urinaria, also suppressed hsv-2 and hsv-1 (yang et al., 2007) . bessong et al. (2006) reported a comparison of various plants and their individual parts (stem, leaves, roots, and so forth.) in repressing viral reverse transcriptase (rt) and integrase, the two basic enzymes in hiv disease. after comparing all the extracts and fractions of the various plants, it was found that the n-butanol fraction of bridelia micrantha (hochst) exhibited the highest anti-rt activity. it has also been reported that the aqueous extract from the roots of carissa edulis (forssk.) vahl, a plant grown in kenya, displayed noteworthy activity against hsv for both wild type and resistant strains (tolo et al., 2006) . polyphenol-rich extract from the medicinal plant geranium sanguineum l. has been reported to show a strong antiinfluenza virus activity, as well as antioxidant and radical scavenging capacities (sokmen et al., 2005) hepatitis a, b, c, d, and e viruses are the leading causes for the prevalence of viral hepatitis and liver inflammation. despite the fact that presentation to any of these infections prompts intense disease, in any case, types b, c, and d are unique in causing chronic infection. plants belonging to the genus phyllanthus of the euphorbiaceae family were extensively used as a traditional remedy against these infections. clinical investigations were additionally intended to look at the inhibitory effects of different species of phyllanthus, that is, p. amarus (l.), p. niruri (l.), and p. urinaria (l.) (wang et al., 1995) . the screening of 56 different chinese medicinal herbs led to the identification of two potent plant extracts against duck hepatitis b virus, namely, ardisia chinensis and pithecellobium clypearia . similarly, this also led to the identification of oenanthe javanica (blume) dc flavones (ojf). they acted as a strong inhibitor of hbsag and hbeag secretion (involved in viral pathogenesis) in 2.2.15 cells and also reduced dhbv-dna levels in the hbv-infected duck model (wang et al., 2005) . because of the strong prevalence of hcv infection in poor countries, screening for the identification of anti-hcv potentials from medicinal plants are still ongoing. according to hussein et al. (2000) various plant extracts, such as methanol extracts acacia nilotica l. willd ex delile, boswellia carterii, embelia schimperi, quercus infectoria, trachyspermum ammi l., and aqueous extracts of piper cubeba l., q. infectoria, and syzygium aromaticum l., were found to possess significant inhibitory activity against hsv. combination therapy for treating diseases is an age-old practice of traditional medicine in which several plants are mixed together to develop an effective formulation for a particular disease. such combination therapies have also been tried for the inhibition of viral hepatitis. as an example, a chinese herbal medicine, prepared by liquid fermentation broth of ganoderma lucidum supplemented with an aqueous extract of radix sophorae flavescentis, was potent against hepatitis b virus activity in vitro and in vivo. viral infections are a matter of great concern for this planet. plants having broad-spectrum activity against many viruses could be evaluated for isolation and identification of molecules for treating viral infections. as an example, glycyrrhizin, a bioactive component of licorice (glycyrrhiza uralensis fisch), and lycorine, isolated from lycoris radiata l., showed strong anti-sars-cov activity, and was initially used for treating other indications (li et al., 2005a, b) . a variety of herbal preparations have shown potentials for inhibiting viruses that cause serious infections among humans, such as measles viruses (olila et al., 2002) , human rotaviruses (hrv) (husson et al., 1994; takahashi et al., 2001) , respiratory syncytial virus (rsv), human rhinoviruses (glatthaar-saalmuller et al., 2001) , the coxsackie group of viruses (evstropov et al., 2004; su et al., 2006) , neurotropic sindbis virus (nsv) (paredes et al., 2001) , and various strains of polio virus (andrighetti-frohner et al., 2005; melo et al., 2008) . one such illustration is the atomic investigation of the heated water concentrates of stevia rebaudiana l., which blocked a section of different irresistible serotypes of hrv into permissive cells by an anionic polysaccharide having a molecular weight of 9800 with uronic acid as a noteworthy sugar constituent (takahashi et al., 2001) . thus, an alkaloid concentrate of haemanthus albiflos globules repressed rna amalgamation of hrv spread in ma-104 cells (husson et al., 1994) . in contrast to the many publications on antibacterial and antifungal screening of plant extracts that have appeared in the last decades, far fewer antiviral screening studies of plant extracts have been reported. this is due chiefly to the complexity of the different techniques involved in such research, which consequently requires the know-how and dedication of a multidisciplinary team. nevertheless, many antiviral agents have been isolated from natural sources and partly or completely characterized. from these studies, several substances have emerged as true antivirals having a good chemotherapeutic index based on the viability of infected cells and on in vivo tests. thus, different 3-methoxy flavones and synthetic derivatives have shown to be promising leads for the development of antirhinovirus drugs (van hoof et al., 1984; vlientinck et al., 1988) , whereas the spanonin, glycyrrhizic acid, was found to be highly active in vitro against herpes simplex (pompei et al., 1979) , varizella-zoster (baba and shijeta, 1987) , and human immunodeficiency viruses (ito et al., 1987) . whether these compounds have any clinical potential, that is, in the therapy of the corresponding viral diseases, remains to be determined. there, in vivo bioavailability and other pharmacokinetic parameters are subjects of future study. because of the problems of drug resistance stated earlier and of side effects, the pharmaceutical industry is looking forward toward natural products (mainly medicinal plant extracts) as a source of possible antiviral agents. approximately 2500 medicinal plant species have been recorded globally to treat a myriad of inflictions and diseases. polyphenols, alkaloids, flavonoids, saponins, quinones, terpenes, proanthocyanidins, lignins, tannins, polysaccharides, steroids, thiosulfonates, and coumarins are prominent bioactive phytochemicals that have been observed to combat viral infections, as they are harmless to the systems of the human body. some selected anthraquinones and anthraquinone derivatives are noted for their dammarane saponins, ginsenoside rb1 (grb1), and chikusetsusaponin (chi-iii) have been found to possess antiviral activity against hsv-i using an in vitro plaque elimination assay. chi-iii prevented plaque formation at half the concentration of grb1 (fukushima et al., 1995 (hudson and towers, 1995) . the inhibitory effect of ferulic acid and isoferulic acid on murine interleukin-8 production in response to influenza virus infections in vitro has been reported (hirabayashi et al., 1995) and the effect of isoferulic acid was found to be greater than that of ferulic acid. hayashi et al. (1995) found a direct inhibitory activity of the steam distillate prepared from fresh plants of houttuynia cordata (saururaceae) against hsv-1, influenza virus, and hiv-1, without showing cytotoxicity, but not against poliovirus and coxsackie virus. montanha et al. (1995a) evaluated the action of a series of 19 aporphine alkaloids against hsv-1 in cell cultures. on the basis of viral titer reduction, six alkaloids were found to be active. ellagitannins isolated from phyllanthus myrtifolius and p. urinaria (euphorbiaceae) showed activity against epstein-barr virus dna polymerase. flavonoidal constituents, such as biflavonoids and robustaflavones, exhibited strong inhibitory effects against influenza a and b viruses. the antiviral potential of the flavonoids of chamaesyce thymifolia has been reported; they showed high cytotoxicity on hep-2 cells and moderate inhibitory activity against hsv-1 and bovine viral diarrhea virus (amaral et al., 1999) . sotanaphun et al. (1999) isolated sclerocarpic acid from the stem bark of glyptopetalum sclerocarpum, which exhibited antiviral activity against herpes simplex virus types 1 and 2. rhamnan sulfate, a natural sulfated polysaccharide isolated from monostroma latissimum, showed potent inhibitory effects on the virus replication of hsv-1, hcmv, and hiv-1 in vitro . matsuse et al. (1999) tested aqueous and methanolic extracts of 39 panamanian medicinal plants for anti-hiv effects. seven of these were found to be moderate inhibitors of hiv-protease enzyme. serkedjieva and ivancheva (1999) investigated the inhibitory effect of five extracts from the bulgarian medicinal plant g. sanguineum on herpes simplex virus types 1 and 2. yoosook et al. (1999) evaluated the anti-hsv-2 activities of barleria lupulina and clinacanthus nutans. the results suggested a therapeutic potential against hsv-2 for b. lupulina, but not for c. nutans. the antiviral activities of various water and methanol soluble substances isolated from g. lucidum against hsv types 1 and 2, influenza a virus, and vesicular stomatitis virus were studied using cytopathic effect inhibition assay and plaque reduction assay (eo et al., 1999) . kudi and myint (1999) investigated the antiviral activity of medicinal plant extracts against poliovirus, astrovirus, hsv, and parvovirus. most of the extracts showed activity against more than one virus at a dose rate of between 100 and 400 î¼g/100 î¼l (eo et al., 1999) . bourne et al. (1999) assessed 19 plant products in vitro by plaque reduction assay to determine their activity against a commonly transmitted pathogen, herpes simplex virus type 2. docherty et al. (1999) found that resveratrol, a phytoalexin, inhibited hsv-1 and hsv-2 replication in a dose-dependent reversible manner. anani et al. (2000) prepared methanol extracts from 19 medicinal plants of togo and analyzed them for antiviral and antibiotic activities. ten of the 19 showed significant antiviral activity against one or another of the test viruses (herpes simplex, sindbis, and poliovirus). hudson et al. (2000a, b) evaluated ethanolic extracts of 11 plants, endemic to madagascar, in order to determine the potential of malagasy plants as sources of antiviral activities. nine of the extracts had significant activity against hsv, whereas only four were active against the sindbis virus. a bioactive flavonoid, "baicalein," isolated from the chinese medicinal plant scutellaria baicalensis georgi showed antiviral properties using the high-speed countercurrent chromatography (hsccc) technique (li and chen, 2005) . many other substances, including flavonoids, phenolics, tannins, triterpenes, and alkaloids, interfere with host cell replication at their antivirally active concentrations or only exhibit extracellular viricidal activities. some of these viricides, including flavonoids and tannins present in foodstuffs, might be important, because they can inhibit virus replication of picorna-, rota-, and arena viruses in the gastrointestinal tract of humans and animals. artemisia capillaris was found to possess an active 6,7-dimethylesculetine having potent cytotoxic potential. in the fruits of schisandra chinensis (schizandraceae) used in oriental medicine, 22 lignans were identified, some of which, such as wuweizisu c and gomisine n, are very active. the same method threw light on the mechanism of the antihepatotoxic action of such well-known compounds as glycyrrhizin and its intestinal metabolites, which are protective against the first stages preceding hepatic lesions. other tests of this type are used to track down active substances. from taxus baccata, potier's group isolated new analogs of taxol, a terpenic compound displaying very good antileukemic and antitumor activities. taxol promotes the polymerization of tubulin into microtubules and inhibits their depolymerization. the choice among various fractions obtained by extraction was guided by the antitubulin activity in an in vitro test. many substances that are present in only trace quantities and are difficult to purify have been studied chemically; for example, the demonstration of xylose-bearing derivatives is new in this series of compounds. regarding structure activity relationships, in vitro cytotoxicity tests have shown that the activity is mainly related to the presence of a complex ester function in the compound. a list of plant extracts and their phytoconstituents that have antiviral potentials are listed in table 16 .1. in recent years, a lot of development has taken place regarding the identification of antiviral molecules from plant sources and the molecular mechanistic approach. compounds, such as spiroketalenol ether derivatives isolated from rhizome extract of tanacetum vulgare, have been reported to block virus entry and also arrest the synthesis of hsv-1 gc and hsv-2 gg glycoproteins (fernandes et al., 2012) . samarangenin b from the roots of limonium sinense exhibited inhibition of hsv-1î± gene expression (kuo et al., 2002) , whereas oxyresveratrol from artocarpus lakoocha plant was found to inhibit the early and late phases of viral replication of hsv-1 and hsv-2, respectively (chuanasa et al., 2008) . also, pterocarnin a compound isolated from pterocarya stenoptera inhibited hsv-2 from binding and penetrating to the host cells (cos et al., 2003) . the structures of some of the potential phytoconstituents having significant antiviral activity are depicted in fig. 16 .1. many of the antiviral drugs now known have been discovered by random search in the laboratory. most labs use a biological test system in which new molecules are added to tissue culture cells at a range of concentrations (e.g., 100-1000 î¼g/ ml); the drug-treated cells (and untreated cells as control) are then infected with a known multiplicity of infective virus particles. thousands of compounds can inhibit viral replication in a cell culture. in general, the more complex the regulatory mechanisms of a virus, the easier it is to find molecules that inhibit it. a broad estimate of the ratios of the activity of antiviral compounds in a cell culture, animal models, and humans is 1000:10:1. during the evaluation of antiviral agents, many test conditions, such as the cell culture system, virus strain, virus challenge dose, virus input multiplicity of infection, and time of harvesting, can affect or even alter the test results. to test the inhibitory activity of a new antiviral agent, it is first necessary to select the host cell system(s) in which the virus replication can be measured. viruses vary considerably in their ability to replicate in cultured cells. many viruses can cause cpe while some of them can form plaques. others can produce some specialized functions, such as hemagglutination, hemadsorption, or cell transformation. virus replication in cell cultures can also be detected by the presence of viral products, namely, viral dna, rna, or polypeptides. the antiviral tests selected may be based on: (a) inhibition of the virus-induced cytopathic effect (cpe) in which the 50% effective dose (ed 50 ) of the antiviral agent is expressed as the concentration that inhibits cpe in half of the quadruplicate cultures. (b) plaque reduction assay in which the dose of the drug required to reduce the plaque number by 50%, that is, ed 50 is calculated. (c) virus yield reduction assay in which the drug concentration required to reduce 90% (1 log 10 reduction), or 99% (2 log 10 reduction) of the virus yield as compared with the virus control (infected cultures without drug) are determined from the dose-response curves and are expressed as ed 90 or ed 99 of the antiviral agent. (d) assay systems based on the measurement of specialized functions and viral products; a number of viruses do not produce plaques nor do they cause cpe readily, but they may be quantified by certain specialized functions based on their unique properties, for example, hemagglutination and hemadsorption tests used to study the antiviral activity against myxoviruses and elisa, used to determine the extent of virus replication and, thus, obtain a measure of the inhibitory effect of various antiviral agents on virus replication, etc. (hu and hsiung, 1989 ). colorimetric assays quantify cell viability through enzyme-mediated biochemical reactions owing to ingress of certain dyes inside living cells. mosmann, borenfreund, and puerner first advocated the application of tetrazolium (mtt) and neutral red (nr) assay, respectively, to quantitate cell viability and the cytotoxicity to cells in vitro. parida et al. (1999) compared the efficacy of two colorimetric assays (mtt and neutral red) to determine viral infectivity in microculture virus titration employing polio virus type-3 and dengue virus type-4. mtt assay, also known as tetrazolium assay, has been exploited extensively to reveal the protective efficacy of therapeutic agents and plant extracts against cancer, hiv-1, hsv, polio virus type 3, den-4 virus, and to the determination of neutralizing antibody levels to hiv and respiratory syncytial virus. mtt assay using microculture virus titration (mcvt) was applied for the determination of infectivity titers of influenza viruses and was found to be compatible with the well-established procedure of egg infectivity assay. unlike mtt, neutral red dye uptake assay has not been substantially exploited in virologic research. nr dye assay was earlier performed for the study of the antiviral efficacy of basil leaves extract against polio virus type 3. mtt, a tetrazolium salt, is a yellow-colored dye [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] which gets cleared by mitochondrial succinate dehydrogenase enzyme into a blue-colored formazan in active cells. this product does not form crystals when it interacts with isopropyl alcohol and thus can be accurately measured. there is no need to harvest the viable cells, wherein the cell viability can be directly measured by a spectrophotometer (parida et al., 1999) . the assay procedure has been discussed in section 16.6.7. a colorimetric assay based on the cleavage of the tetrazolium salt wst-1 has been developed for human cytomegalovirus (hcmv) antiviral susceptibility testing and adapted to a microtiter plate format. bedard et al. (1999) developed a colorimetric assay based on the cleavage of the tetrazolium salt wst-1 for human cytomegalovirus antiviral susceptibility testing. the response of different cell cultures to a given antiviral agent, including the drug metabolism and toxicity, among other factors, may vary greatly. to perform antiviral testing against a particular virus infection, it is necessary to obtain a suitable host cell system for that virus infection. the same antiviral agent may behave very differently in different cell culture systems, although the same virus strain is used. (ii) virus strain and passage history the variability of sensitivity to a given antiviral agent has been noted among different strains of hsv and cmv. drugresistant strains have emerged to some antiviral agents, especially in the herpes virus group. the passage history of a virus strain may also affect its sensitivity to some antiviral agents. moi can influence substantially the evaluation of antiviral activity by the plaque reduction method or the virus yield reduction assay. high moi will decrease the sensitivity of the virus to an antiviral agent. this may also contribute to the disparity among antiviral evaluation results, even when the virus moi is kept constant (hu and hsiung, 1989) . ( the activities of several known antiviral phytochemicals are profoundly affected by the presence of serum components. for example, the terthiophene, î±-terthienyl (î±-t) was inhibited in a concentration-dependent manner by serum. in the case of the carboxylic acid derivative of î±-t, the compound appeared to have no antiviral activity at all in the presence of serum, yet in its absence this compound was as effective as î±-t. in contrast, the complex anthraquinone hypericin required a small amount of serum for maximal antiviral activity. the reactions were also strongly affected by the order of incubation of the components: virus, compound, serum, and light (hudson and towers, 1995) . the viruses to be selected for initial evaluation of plant extracts are obviously of major importance. they must be chosen to represent the different groups of viruses according to their morphology and various multiplication mechanisms and a range of virus diseases for which chemical control would be useful. besides the need for control, also the prevalence of the viral diseases and the resulting projection of the market potential, which are determined by the antigenic abundance of the causative viruses and the problems this represents for vaccine control, are important selection criteria (grunert, 1979) . in vitro methods are therefore more appropriate, because they allow simultaneous screening of a battery of viruses. in vivo screening of extracts against a broad array of viruses, in contrast, is not only very expensive but also extremely laborious. in vitro antiviral bioassays utilize thinly confluent monolayers of tissue culture cells with sufficient susceptibility to the infecting viruses that a visibly cytopathogenic effect (cpe) occurs, for example, rounding up. shrinking or detaching of cells from the monolayer can be produced and readily observed microscopically. a monolayer of cells consists of animal or human cells, such as chick embryo, rabbit, or green monkey kidney cells (vero cells), or human skin fibroblasts and carcinoma cells (hela cells), grown in a culture medium. such continuous cell lines used in virology are mostly "transformed" cells that can be maintained for an indefinite number of generations. the host cells require an appropriate tissue culture medium in which they can survive for at least 1 week without having to renew the medium. renewal of the medium causes changes in intra-and extracellular products and alters the virus concentration. the medium must have a stable ph during the whole incubation time and may contain only a small amount of serum, because blood products tend to absorb many compounds. mostly, a defined synthetic medium, supplemented by some type of serum (such as fetal bovine, calf, or horse), a buffer on sometimes bacterial and fungal inhibitory antibiotics, is used. according to experience, vero cells, which allow the growth of many human viruses with visible cpe, grown in the medium described by hronovosky et al. (1975) , and slightly modified by lowering the pyruvate concentration, are most suitable for antiviral screening of plant extracts (van den berghe et al., 1978) . many combinations of test viruses are possible, but a battery of six viruses seems to be quite acceptable. virus types and strains may vary in sensitivity, but have to be selected as a function of their ability to multiply in the same tissue culture when cell culture models are used as screening systems. in this way, an objective comparison of antiviral activities is possible, whereas toxicity tests are minimized. moreover, virus multiplication must cause a visible cpe within a reasonable period of time, preferably within a week after infection. which are expressed as the number of infectious units per volume. an infectious unit is defined as the smallest amount of virus capable of producing a reaction after virus inoculation and can be carried out by two generally applied methods, namely, the plaque test (pt) and the 50% endpoint titration technique (eptt). in the plaque test, monolayers of cells grown in plastic or glass petri dishes are inoculated with dilutions of a viral suspension. after adsorption of infectious virus particles to the host cell, the monolayers are overlaid with an agarosecontaining medium so that the newly formed virus particles are localized by the solid agar over layer. these newly formed infectious particles spread from the initially infected cell to the adjacent cells and develop well-circumscribed foci of cellular degeneration. these areas of dead cells are called "plaques" and are visualized by staining the cell monolayer with a vital dye, such as neutral red. the plaques may also be detected microscopically by determining the multinucleated cells (e.g., measles) or by immunofluorescence. the concentrations of viral suspensions measured by counting the plaques are expressed as plaque-forming units per ml (pfu ml â��1 ). in the endpoint titration technique, the concentration of infectivity is measured by determining the highest dilution of the suspension, which produces cpe in 50% of the cell cultures inoculated. this dilution is called the 50% tissue culture dose endpoint (tcd 50 ). dilutions are therefore made in a maintenance medium and a certain volume of each of them (0.05-0.1 ml) is added to four or more tube cultures. the proportion of positive cultures is registered for each dilution and the precise dilution at which 50% of the inoculated tube cultures are infected is calculated. at this dilution, the inoculum contains, on average, one tcd 50 or one tissue culture (infectious) dose for 50% of the tissue culture tubes. the influence of a plant extract on virus multiplication can be determined as a viricidal or an antiviral activity. the viricidal activity is measured by titration of the residual infectious virus after incubation of the plant extract with a virus suspension of at least 10 6 tcd 50 ml â��1 . on the other hand, the antiviral activity is determined by comparing the virus titers of infected cells, which have been cultured with a maintenance medium containing plant extracts or test substances and a maintenance medium without test material (colegate and molyneux, 1993) . in contrast with antibacterial screening, no solvents, other than physiological buffer solutions, should ideally be used in the in vitro antiviral screening of plant extracts because the samples have to be added to tissue culture cells. it has been observed that many nonpolar plant extracts, prepared and evaporated, are reasonably soluble in dimethyl sulfoxide (dmso), especially if little or no water is present in the sample and the dissolving sample is heated on a water bath. viricidal and antiviral determinations may then be carried out on test solutions containing not more than 10% and 1% dmso, respectively. therefore, dissolved samples of nonpolar plant extracts in dmso are added dropwise to the maintenance medium in a ratio of 1:10 or 1:100 under stirring. as already mentioned, the maintenance medium may contain antibiotics, such as penicillin g (20 î¼g ml â��1 ), neomycin (1 î¼g ml â��1 ), and amphotericin b (1 î¼g ml â��1 ), in order to avoid sterilization of the test solutions. any contamination by bacteria or fungi would indeed ruin the in vitro antiviral bioassay (colegate and molyneux, 1993) . there are various methods for validation of antiviral activity. the major techniques have been highlighted in the preceding sections and in fig. 16.2. most currently used antiseptics and disinfectants kill pathogenic bacteria and fungi at 25â°c within 5 min when present in a concentration of about 0.5% (3-log titer reduction). because it has been noticed that most of these preparations fail to kill all pathogenic viruses under these circumstances, a method has been developed for testing the in vitro viricidal effect of plant extracts, as will be described in the following section. thoroughly mix the preincubated (25â°c) plant extracts, dissolved in a physiological buffer, or their twofold dilutions (e.g., 1/2 to 1/16), with the same volume of a preincubated (25â°c) virus suspension of, for example, 10 6 pfu ml â��1 or tcd 50 ml â��1 in physiological buffer. incubate the mixture at 25â°c for 5 min. stop the incubation by the addition of a 10fold volume of ice-cold maintenance medium and filter the mixture immediately through a 0.22 î¼m filter to eliminate all possible precipitate. then, filter the ice-cold filtrate through a 0.01 î¼m filter to concentrate residual virus on the filter and separate the virus from possibly cytotoxic plant components, which pass the filter. remove the residual virus from the filter with maintenance medium, supplement with 5% serum (1 ml), sonicate in an ice-bath for 30 s, and titrate in 10-fold dilutions at 37â°c by plaque formation or in microtiter plates according to the eptt. carry out a virus control in a physiological buffer containing no plant extract simultaneously. an essential step of this methodology is the separation of all cytotoxic plant components from the residual virus, which has to be measured at 37â°c. cytotoxic substances have a greater influence on the activity of an extracellular virus at 37â°c than at 25â°c. this step, however, can be omitted when the plant extracts to be tested are not toxic to the host cells under the conditions of the evaluation procedure (colegate and molyneux, 1993) . the eptt technique is performed on preemptied confluent monolayers of vero or other cells, grown in the holes of microtiter plates, which are infected with serial 10-fold dilutions of a virus suspension (100 î¼l). starting with monolayers containing 10 4 cells per hole and a virus suspension of, for example, 10 7 tcid 50 ml â��1 or pfu ml â��1 , infect the first monolayers of cells with a multiplicity of infection (moi). the antiviral activity is expressed as the virus titer reduction at the maximal nontoxic dose (mntd) of the test substance, that is, the highest concentration (î¼g ml â��1 ) that does not affect the monolayers under the antiviral test condition. viral titer reduction factors, that is, the ratio of the viral titer reduction in the absence (virus control) and presence of the mntd of the test sample of 1 ã� 10 3 to 1 ã� 10 4 indicate a pronounced antiviral activity and are suitable as selection criteria for further investigation of plant extracts (colegate and molyneux, 1993) . the eptt is more suitable for testing complex samples, such as plant extracts, for many reasons. (i) first, the concentration of many compounds in the extract remains constant, and consequently the proportion of toxic versus active compounds does not change. (ii) second, the exact duration of the antiviral action can be determined by using the eptt, because the action starts from the moment the extract is added to the cells. (iii) third, the eptt using serial diluted extracts deals with a dynamic process, because the cells are subsequently infected with different moi. (iv) this system allows the correlation of all possible moi values in the same microtiter plate with decreasing amounts of plant extracts, so that the noncytotoxic concentration of plant extracts can be determined. at the same time, a correlation between extract toxicity and antiviral activity according to the corresponding moi can be determined in the same microtiter plate. (v) it can be stated as a general rule that the detected antiviral activity should be stable in at least two subsequent dilutions of nontoxic concentration of the extract; otherwise the activity is directly correlated with the toxicity of the extract or is only viricidal. moreover, a true antiviral product has to protect the cells, which have been infected with low virus dilutions (starting from 0.1 pfu per cell onward). (vi) finally, it should be stressed that all possible steps of virus manipulation are to be completed before the plant extracts are added. this means that an antiviral product tcd 50 ml â��1 ), under nontoxic conditions, must act on virus replication steps after uncoating. when the cells are completely protected only in the lower moi (0.1 tcd 50 ml â��1 ), replication processes before uncoating may be involved. an important aspect of the inhibition of viral cytopathic effect (cpe) is the determination of tcid 50 (50% tissue culture infective dose). after harvesting, dilute the virus stock 10-fold. add 0.1 ml of each dilution in 10 wells each of a 96-well microtiter plate. add 0.1 ml of cell suspension of 10,000 cells/well, incubate at 37â°c with 5% co 2 atmosphere, and observe for vial cpe on alternate days. take a final reading on the fifth day and calculate tcid 50 as per the method of reed and muench (1938) , from which tcid 50 can be further calculated from the log value. an antiviral drug will inhibit the cpe of a virus. therefore, for detecting an antiviral agent, the amount of inhibition of cpe of a virus can be observed microscopically (kenny et al., 1985) . for this purpose, trypsinise the monolayer and allow to seed in 96-well microtiter plates. after a 24-h incubation, wash the monolayer in each well and add different test drug dilutions and incubate. after 24 h, wash the cell culture and inoculate with 0.1 ml of 10 tcid 50 , 50 tcid 50 , and 100 tcid 50 of the virus suspensions in different wells. incubate them for 1 h at 37â°c in an incubator for the adsorption of the virus onto the cells. after incubation, remove excess virus suspension by washing with rpmi. add 0.1 ml of selected concentration of the test compound and keep both virus and cell control wells with 0.1 ml of rpmi containing 2% sheep serum. observe the plates under a microscope every 24 h until the virus control shows 100% cpe and tabulate the results (hu and hsiung, 1989) . trypsinize the cell monolayer, allow to seed in a 96-well microtiter plate and incubate for 24 h at 37â°c with 5% co 2 atmosphere. remove the medium, wash the cell monolayer with rpmi without serum, and add 0.1 ml of different virus suspensions in different wells containing the cell layer and incubate for 1 h for virus adsorption; wash off the excess virus suspension after adsorption. to each well, add the selected concentration of the test drug diluted in rpmi containing 2% sheep serum. to the virus control and cell control, rpmi is added (2% serum) and incubated for 24 h. freeze the plates at â��70â°c and thaw at room temperature a couple of times to liberate the associated virus. determine the virus titer by the end point dilution method and express as tcid 50 (cinatl et al., 1997) . trypsinize the monolayer culture and allow to seed in a 96-well microtiter plate. after a 24-h incubation, wash the monolayer in each well and add different test drug dilutions and incubate. after 24 h, wash the cell culture and inoculate with 0.1 ml of 10 tcid 50 , 50 tcid 50 , and 100 tcid 50 of the virus suspensions in different wells. incubate for 1 h at 37â°c in a co 2 incubator for adsorption of the virus onto the cell. after incubation, remove excess virus suspension by washing with rpmi without serum. add 0.1 ml of the selected concentrations of the test compound and keep both the virus and cell control wells with 0.1 ml of rpmi containing 2% sheep serum. incubate the plates at 37â°c for 72 h. after 72 h, discard the old media from the cell cultures and add 50 î¼l of 2 mg/ml of mtt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to each well and incubate for 3 h. after 3 h of incubation, discard the mtt and add 100 î¼l of isopropyl alcohol to each culture and set aside for 10 min. record the absorbance using an elisa plate reader at 540 nm. in this experiment, the effect of the test drug on mitochondrial synthesis due to viral infection is studied. the "plaque" is a confined region of contaminated cells formed by multiplying virus particles. the plaques are revealed either as regions of dead/decimated cells recognized by cell stains or as zones of polluted cells by immunostaining. the blended cell monolayer is infected with a log10 dilution of viral plaque-forming unit (pfu) in the absence or presence of the test drug and permitted to adsorb (1 h at 37â°c in 5% co 2 ); afterward, the cells should be washed twice with prewarmed tcid cpe at dilution next above cpe at dilution nex 50 50 50 % % % t t above cpe at dilution next below 50 50 % % % ⧠⩠ⷠminimum essential medium (mem). the overlaid drug dilutions are arranged in the overlay medium on the contaminated culture, without the test drug. then, 45 ml of carboxy methyl cellulose is added to 9 ml of the medium; the plates are incubated for 3-5 days, then settled with 10% formalin or 4% formaldehyde for 30 min. the cells are stained with methylene blue (1 ml/well) or 1% gem violet (w/v), washed, and dried to check the plaques (dark areas) by low-power amplification of a binocular microscope. the antiviral impact should be measured as the percentage inhibition of plaque formation: the concentration of the test drug required to exert 50% of virus inhibition (ic 50 or ec 50 ), as compared with the infection control, is evaluated from the graphical plot as dose-response curves by regression analysis . viruses, for example, influenza, can agglutinate rbcs due to surface ha proteins, which can be analyzed visually by blending viral dilutions with rbc. this can be utilized to inspect the inhibitory impact of any medication on ha. the 10-overlay serially diluted (1-1000x) test drug is used alongside the diluted viral stocks (1:4 to 1:128). this dilution (50 ml/well) is added to drug-containing wells. it should be preincubated for 45 min and mixed with rbc (1/20 in pbs) sample solution. here, up to a specific dilution, the viral particles possibly lose their capacity to agglutinate rbcs, which shows a linkage of the drug with the viral ha. known quantities of virus (moi 5-10) are used for infecting both untreated or drug-treated cells and allowed to adsorb for 45-60 min. the unabsorbed virus particles are washed and fresh media is added to incubate for 24-36 h. then, cells are washed with pbs, fixed with paraformaldehyde (3%-4%), and permeabilized with acetone or triton x-100. the cells are again washed and blocked with 1% bovine serum albumin (bsa) in pbs for 30 min. then, the cells are incubated with mouse or rabbit antibody against a specific viral protein for 1-4 h at 37â°c. after that, the cells are subjected to repeated washing and incubated with a fluorescent-tagged secondary antibody for 1 h and washed again. after washing, the cells are visualized under a fluorescent microscope and compared with the fluorescence of untreated and drug-treated cells. on the other hand, for quantitation, the cells are trypsinized after treatment and fixed with 4% paraformaldehyde. the cells are then washed, permeabilized, and labeled with a fluorescent-tagged antibody, followed by propidium iodide (pi: 50 mg/ml in pbs). the cells are then counted in a fluorescent-activated cell counter to quantitate the fluorescence percentage. known quantities of virus (moi 5-10) are used for infecting both untreated and drug-treated cells, adsorbed for 1 h, washed, and incubated (2-4 days) for evaluation of the inhibition of the virus-induced cytopathic effect (cpe). the virus stock is centrifuged after freeze thawing and diluted for elisa. each well of a plate coated with a virus-specific antibody is mixed with 100 ml of controls or test drug and incubated at room temperature (1 h) with horseradish peroxidase conjugate, alkaline phosphatase, or b-d-galactosidase-labeled virus-specific antibody. the wells are washed five times and the substrate (100 ml) is added and incubated in the dark for 10 min. the reaction is stopped by adding 5% h 2 so 4 solution and the absorbance is read at 450 nm. the 96-well plates are seeded with a quadruplicate cell monolayer having a log 10 dilution of the test drug followed by infection with the virus. after 16-20 h of incubation at 37â°c, the monolayers are fixed with 0.05% glutaraldehyde and examined for virus-specific protein(s) on the cell surface. the elisa should be performed with a monoclonal antibody to the specific protein of the corresponding virus and protein and the od (optical density) is measured at 450 nm. the concentration is calculated by 50% reduction in od values (ec 50 ) from extrapolating graphical plots followed by the determination of si (ratio of cc 50 :ec 50 ) in which the results are expressed as a percentage of virusinfected cells (virus control) . the test drug and the virus (10 4 pfu/ml) are mixed to incubate for 1 h at 37â°c. then, immediately dilute the virus-drug mixture to 100-fold (final inoculums 50 pfu/well) with media containing 2% fbs to get a subtherapeutic concentration of mean number of plaques in control mean number of plaques in sample m ean number of plaque in control the test drug. following that, mix the monolayer, with the virus inoculums seeded using a 12-well plate. alternately, add the virus-test drug mix diluted to 100-fold, with no incubation period, with the respective cells for infection. allow to adsorb for 1 h at 37â°c, discard the diluted inoculums, and wash the cells with pbs. pour an overlay medium (with 2% fbs), and incubate at 37â°c for 72 h, followed by plaque reduction assay. count the viral plaque numbers obtained from infections set in the presence of the test drug and compare it with the control. viral attachment to the cell surface is carried out at 4â°c, as it permits binding, but stops viral entry, by elisa. briefly, incubate susceptible cells (2 ã� 10 4 cells/well) in 96-well plates and allow growth overnight. the cell monolayers are cooled at 4â°c. the cells are infected with the virus (moi 5) using heparin in presence of the test drug for 3 h at 4â°c as a control. after washing the wells with ice-cold pbs, fix with prechilled 4% paraformaldehyde in pbs for 1 h on ice, blocked with 5% bsa at 4â°c. the samples are incubated at 37â°c for 1 h with a primary antibody in pbst pbs with 0.05% tween 20 along with 5% bsa. the wells are washed twice with pbst, 5% bsa, and again only with pbst twice, at 5-min intervals on a shaker. this is mixed with secondary antibody in pbst with 5% bsa and incubated at 37â°c for 1 h. the reaction is developed with a 3,3â�²,5,5â�²-tetramethyl-benzidine two component microwell peroxidase substrate for 20 min; the reaction is stopped with 1 m phosphoric acid. the absorbance is measured at 450 nm, and the values are expressed as the fold change of absorbance relative to the mock infection control (lin et al., 2011) . the cell monolayers are cooled and grown in 12-well plates at 4â°c for 1 h. subsequently, infect the prechilled cells with hsv-1 (100 pfu/well) and incubate for 3 h at 4â°c to allow for viral adsorption. incubate the infected cell monolayers in the presence of test drug or heparin (100 mg/ml) for an additional 20 min at 37â°c to facilitate hsv-1 penetration. the extracellular virus is inactivated by citrate buffer (ph 3.0) for 1 min, and the cells are washed with pbs followed by overlay with dmem containing 2% fbs. after 48 h of incubation at 37â°c, the viral plaques are stained and counted (lin et al., 2011) . add the plated cells (0.8 ã� 10 5 cells/well for a 12-well plate) grown overnight at 30% confluence (300 ml) with virus dilution and deae dextran at a final concentration of 20 mg/ml. after adsorption (2 h at 37â°c in co 2 incubator), place the plates in a rocker to prevent the cells from drying and add fresh medium (1-2 ml) containing the test drug to each well and incubate for 40-48 h in 5% co 2 at 37â°c for subconfluent growth. after removing the media, add fixing solution (1-2 ml) to each well and incubate for 5 min at room temperature (î²-galactosidase activity decreases dramatically if the fixing solution is left for >10 min). then, discard the fixing solution, wash the cells twice with pbs, stain, and incubate at 37â°c for 50 min. finally, stain the plates to count the number of blue syncytia and express the titration values as the number of stained cells multiplied by the viral dilution . this assay is applicable for hiv-1, simian immunodeficiency virus (siv), and simian-hiv and is carried out in tzm-bl cells as it reveal the reduction in tat-induced luciferase (luc) reporter gene expression after a single round of virus infection. place the tzm-bl cells (4 ã� 10 4 /well) in a 24-well plate and incubate overnight. in a separate vial, mix the hiv-1nl4.3 (moi 0.05) virion with the test drug or vehicle for 1 h at 37â°c, then add to tzm-bl cells and incubate for 4 h. wash the cells (with cold pbs), and add fresh media with the test drug to culture for 48 h, using untreated hiv-1-infected cells (negative) and azidothymidine (azt)-treated cells (positive) as controls. wash the cells twice with pbs, lyse with 1x lysis buffer, and add the supernatant with the substrate, which should then be analyzed for luciferase activity in an optiplate using a fluorimeter. the results are expressed as percentage inhibition: and the percent inhibition is calculated by subtracting the above value from 100 (wan et al., 2012) . luminescence in the experimental group test drug or azt lu / m minescence of infected cells without the drug u100 (b) cem-green fluorescent protein (gpt) cell-based assay cem-gfp is a stable t-cell line-containing a plasmid encoding gfp and is suitable for hiv-1nl4.3 (moi 0.05) culture. for postinfection, incubate the cells (2.0 ã� 10 5 /well) with the test drug up to 8 days, using azt and solvents (used to prepare the test drug) as control(s). lyse the virus-infected cells with 1x promega cell lysis buffer (150 ml), and transfer to a culture plate to read the absorbance at 485 nm (excitation) and 520 nm (emission) by a fluorimeter. the results can be expressed as percentage inhibition: with the he percent inhibition calculated by subtracting the above value from 100 . place hep ad38 cell suspension (6 ã� 10 5 viable cells/ml of hep ad38 seeding medium) in a 96-well microtiter plate, and incubate at 37â°c for 3 days. discard the medium and wash the cell monolayers with warm (37â°c) dpbs. to the proper wells, add 100 î¼l of hepad38 assay medium that contains either test or control compounds at the desired concentrations. also include wells with hep ad38 assay medium alone as "virus only" controls. incubate at 37â°c for 3 days. on day 3, wash the cells once with warm dpbs and add fresh medium containing the appropriate compound to the wells. after 24 h, transfer the supernatants to v-bottomed 96-well plates and remove cellular debris by centrifugation (15 min, 2500 rpm at 4â°c). transfer 90 î¼l of the clarified supernatants to new v-bottomed plates and store at â��70â°c for quantification of hbv dna. thaw the supernatants that were collected and add 90 î¼l of 2x denaturation solution to each well and mix. incubate at 37â°c for 20 min. cut the nylon membrane to size and prepare it for blotting by wetting it first with distilled water and then 20x hybridization buffer, ssc (saline sodium-citrate). dot-blot the denatured supernatants on to the nylon membrane as directed by the manufacturer. wash the blot with 200 î¼l of neutralization solution followed by 200 î¼l of 20x ssc. remove the blot, rinse it briefly in 2x ssc, and then crosslink the dna to the nylon filter by uv irradiation. prehybridize the blot at 42â°c for 1 h in 20 î¼l of hybridization solution. prepare a 32p-labeled probe by random priming using a portion of the hbv genome as a template. purify the probe through a clontech chroma spin column. denature the probe by boiling for 5 min and add it immediately to the hybridization solution. hybridize the nylon filter overnight at 42â°c. wash the nylon filters twice with 50 ml of 2x ssc, 0.1% sds (sodium dodecyl sulfate) at room temperature for 20 min and twice with 50 ml of 0.2x ssc, 0.1% sds at 65â°c for 20 min. expose the nylon filters to a molecular imager screen for 4 h and scan on a phosphor imager to obtain the data. to determine the percent inhibition of hbv replication, subtract the background value (counts of radiation detected from the nylon filter itself) from all control and experimental values. divide the average values of the experimental wells (cells treated with test compounds) by the average value for the "virus only" control (cells not treated with compound or tetracycline during the experiment) and multiply this number by 100 (king and ladner, 2000) . the above mentioned in vitro studies are listed in table 16 .2. drug development programs include preclinical screening of immense quantities of chemicals for specific and nonspecific cytotoxicity against numerous sorts of cells, which is imperative to show the potential therapeutic target and safety evaluation. the screening of plant extracts or pure compounds for investigating their antiviral properties can be more significant with cytotoxicity measures (meyer et al., 1982) . it is essential for an investigational item to establish the antiviral activity at concentrations that can be accomplished in vivo without inducing toxicity to cells. moreover, in a cell culture display, antiviral activity of an investigational item can be the aftereffect of host cell death after exposure to the item. cytotoxicity tests utilize a series of increasing concentrations of the antiviral product to determine what concentration results in the death of 50% of the host cells. this value is referred to as the median cellular cytotoxicity concentration (cc50 or ctc50 or ccic50). the relative effectiveness of the investigational product in inhibiting viral replication compared with inducing cell death is defined as the therapeutic or selectivity index (cc50 value/ec 50 value). it is desirable to have a high therapeutic index giving maximum antiviral activity with minimal cell toxicity. according to us fda guidelines, it is recommended to determine cc50 values in both stationary and dividing cells from multiple relevant human cell types and tissues to establish the potential for cell cycle, species, or tissue-specific toxicities. studies determining cytotoxicity and therapeutic indexes should be conducted before the initiation of phase 1 clinical studies. there are a number of advantages for in vitro testing using cell cultures, which include gfp fluorescence in the experimental group fluorescence in / i infected cells without the test drug ã�100 analysis of species specificity, feasibility of using only small amounts of test substances, and facility to do mechanistic studies (guidance for industry, 2006). after confirming the cytotoxic concentration, the drug concentrations are selected for antiviral studies based on the percentage viability of cells and are used to study the antiviral activity by cpe inhibition assay, virus yield assay, followed by mtt assay. any compound that is cytotoxic to cells inhibits the cell proliferation and kills the cells. trypan blue is a dye that is capable of penetrating dead cells; therefore, the dead cells take up the blue stain whereas the viable cells do not. this method gives an exact number of dead and viable cells (strober, 2001) . protein content is widely used for estimating total cellular material and can be used in growth experiments. the colorimetric method of estimating protein is more sensitive. the cell pellets are treated with 11% cold trichloroacetic acid to remove amino acid pools and dissolved in alkaline cupric sulfate and folin ciocalteau phenolic reagent. folin's reagent and cupric sulfate together react with amino acid to give a blue color and this color intensity is proportional to the protein concentration, which can be measured colorimetrically (maya et al., 1995) . to proceed with the same technique, the cells from the wells were trypsinized using 100 î¼l trypsin and transferred into eppendorf tubes and centrifuged at 5000 rpm for 10 min to obtain pellets. the cell pellets are dissolved in naoh and diluted , tzm-bl cell-based assay (hiv), cem-green fluorescent protein cell-based assay (hiv), hep ad38 assay (hbv), immunofluorescence assay, enzyme-linked immunosorbent assay (elisa) â�¢ reduction or inhibition of virus-specific polypeptides synthesis in infected cell cultures, e.g., viral nucleic acids, determination of the uptake of radioactive isotope labeled precursors or viral genome copy numbers other assays for validation of antiviral activity â�¢ virus inactivation assay, virus adsorption assay, virus attachment, and penetration assay to 0.1 n. the test drug is to be added to 200 î¼l of protein sample, mixed, and left for 10 min. to this, 100 î¼l of test reagent is added with constant mixing and left for 40 min in incubator. the absorbance was read at 655 nm using an elisa reader (bio-rad). the color development was correlated with the cell number as follows: the cytotoxic concentration found by dye exclusion techniques gives superficial data. the selected concentrations from a trypan blue dye exclusion study are used further for estimating proteins. mtt (3-(4,5-dimethylthiazol-2yl)-2,5 diphenyl tetrazolium bromide) in live cells enters the cells and enzyme succinate dehydrogenase present in mitochondria reduces it to formazan blue product. the color intensity is directly proportional to the number of live cells. to perform this process, the plates were seeded with hep-2 cells at 10,000 cells/well. they are incubated for 24 h. after 24 h, the medium is discarded and drug concentrations were added and incubated for 72 h. then, 50 î¼l of 2 mg/ml of mtt is to be added and incubated for 3 h and then 100 î¼l of isopropyl alcohol is added and absorbance is read at 540 nm in an elisa plate reader (bio-rad). the results are tabulated and percentage growth inhibition is calculated using the following formula: the concentrations of the test drug used in the previous experiments can be further confirmed by studying the mitochondrial synthesis by mtt assay. the formazan blue color formation is directly proportional to the number of viable cells and therefore the absorbance is to be read at 540 nm. in this test, brine shrimp (artemia salina) eggs are hatched in artificial sea water (38 g/l of sea salt). the brine shrimp test (bst) bioassay experiment is performed according to the procedure described by meyer et al. (1982) . generally, samples of the test drugs for the experiment are prepared in methanol solution, which acts as control vehicle. after 48 h of incubation, 10 brine shrimps are transferred to each sample vial using a pasteur pipette and artificial sea water is added to make 5 ml. sample vials are previously prepared by dissolving specific concentrations of test drugs with different dilutions. the solvent is then evaporated overnight. survivors are counted after 24 h and the lc 50 values, with 95% confidence intervals are determined using probit analysis, as described by finney (1971) . control vials are prepared using methanol only. three replicates are prepared for each concentration of the test drugs. control disks are prepared using only methanol. replicates are prepared for each dose level. to begin the bioassay, brine shrimp eggs are hatched in a shallow rectangular dish (22 ã� 32 cm 2 ) under the same conditions described in the literature except that natural instead of artificial seawater is used. ten shrimps are selected and transferred into each sample vial by means of a 23-cm disposable pasteur pipette and the final volume in each vial is adjusted to 5 ml using natural seawater. a drop of dry yeast suspension (3 mg in 5 ml seawater) is added as food to each vial. the vials are maintained under illumination. survivors are counted with the aid of a stereomicroscope, after 6, 24, and 48 h, and the deaths at each dose level and control are determined. no deaths are usually observed to occur in the control after 48 h. the brine shrimp test (bst) represents a rapid, inexpensive, and simple bioassay for testing plant extract lethality, which, in most cases, correlates reasonably well with cytotoxic properties (mclaughlin, 1991) . following the procedure of meyer et al. (1982) , the lethality of the test drugs/plant extracts to brine shrimp is determined. the lethal concentrations of test drugs/plant extract resulting in 50% mortality of the brine shrimp (lc 50 ) and 95% confidence intervals are determined from the 24 and 48-h counts and the dose-response data are transformed into a straight line by means of a trend line fit linear regression analysis; the lc 50 is derived from the best-fit line obtained. caffeine (lc 50 = 306 î¼g/ml) (meyer et al., 1982) is used as a positive control and methanol (500 î¼l) as a solvent and a negative control in the bioassay experiments. the current scenario of viral diseases is lethal and there is an upsurge in new viral diseases and resistance to existing viral infections worldwide. the currently accessible antivirals, however effective, are exorbitant and past the reach of a majority of individuals. along these lines, the advancement of safe, effective, and low-cost antiviral medications, for example, rt inhibitors, is among the top priorities, as many viral infections are not yet treatable and have high death rates. for the past few years, substantial work has been carried out regarding the effectiveness of medicinal plants on hiv infection (premanathan et al., 1999; calabrese et al., 2000; asres et al., 2001) and an increasing popularity of over-the-counter plant products containing orthodox drugs has been observed. the main focus is to lower the adverse effects associated with viral infections and an inclination toward synergistic interactions of multiple molecules present in plant extracts. be that as it may, because mostly pharmacological mechanisms of the combinations are not studied, antagonistic impacts or remedial disappointments have been seen (chan et al., 2000) . a prerequisite that should be considered significant for medicinal plants is to identify and standardize the method of extract preparation, the suitable season for collecting plant material, and the details of its administration (chattopadhyay et al., 2006) . as a lot of plant extracts and subsequent formulations have shown significant outcomes, it seems to be rational to endorse the idea of the study of medicinal plants as a quest to find potential antivirals. antiviral investigation on the flavonoids of chamaesyce thymifolia investigation of medicinal plants of togo for antiviral and antimicrobial activities in vitro virucidal activity of selected anthraquinones and anthraquinone derivatives antiviral evaluation of plants from brazilian atlantic tropical forest antiviral activity against human immunodeficiency virus type 1 (hiv-1) and type 2 (hiv-2) of ethnobotanically selected ethiopian medicinal plants suppression of hepatitis c virus by the flavonoid quercetin is mediated by inhibition of ns3 protease activity anti-herpes virus activities of bioactive fraction and isolated pure constituent of mallotus peltatus: an ethnomedicine from andaman islands the mannose-specific plant lectins from cymbidium hybrid and epipactis helleborine and the (n-acetylglucosamine) n -specific plant lactin from urtica dioica are potent and selective inhibitors of human immunodeficiency virus and cytomegalovirus replication in vitro 1-cinnamoyl-3,11-dihydroxymeliacarpin is a natural bioactive compound with antiviral and nuclear factor-kappa b modulating properties a high throughput colorimetric cell proliferation assay for the identification of human cytomegalovirus inhibitors an investigation of the antiviral activity of podophyllum peltatum in vitro anti-hiv-1 activities of kaempferol and kaempferol-7-o-glucoside isolated from securigera securidaca further screening of venda medicinal plants for activity against hiv type 1 reverse transcriptase and integrase the natural compound silvestrolis a potent inhibitor of ebola virus replication plant products as topical microbicide candidates: assessment of in vitro and in vivo activity against herpes simplex virus type 2 a phase i trial of andrographolide in hiv positive patients and normal volunteers comparative study of propofol versus midazolam in the sedation of critically ill patients: results of a prospective, randomized, multicenter trial survey for the presence and distribution of human herpesvirus 8 in healthy brain ethnomedicines and ethnomedicinal phytophores against herpes viruses antivirals of ethnomedicinal origin: structure-activity relationship and scope dose dependent therapeutic antiinfectives from ethnomedicines of bay islands recent advancements for the evaluation of anti-viral activities of natural products validation of antiviral potential of herbal ethnomedicine inhibitory effects of quercetin 3-rhamnoside on influenza a virus replication anti-herpes simplex virus (hsv-1) activity of oxyresveratrol derived from thai medicinal plant: mechanism of action and therapeutic efficacy on cutaneous hsv-1 infection in mice chemistry, biological activity, and chemotherapeutic potential of betulinic acid for the prevention and treatment of cancer and hiv infection antiviral effects of 6-diazo-5-oxo-1-nor-leucine on replication of herpes simplex type-1 novel compounds in preclinical/early clinical development for the treatment of hiv infections molyneux bioactive natural products plant substances as antiviral agents: an update potential antiviral lignans from the roots of saururus chinensis with activity against epstein-barr virus lytic replication drug screening for autophagy inhibitors based on the dissociation of beclin1-bcl2 complex using bifc technique and mechanism of eugenol on anti-influenza a virus activity structure and in vitro antiviral activity of triterpenoid saponins from calendula arvensis antiviral activity of chlorogenic acid against influenza a (h1n1/h3n2) virus and its inhibition of neuraminidase resveratrol inhibition of herpes simplex virus replication antiviral activities of various water and methanol soluble substances isolated from ganoderma lucidum sennoside a, derived from the traditional chinese medicine plant rheum l., is a new dual hiv-1 inhibitor effective on hiv-1 replication anti-enterovirus and immunostimulating activity of the polyphenol complex extracted from pethaphylloides fruticosa (l.) o. schwarz honokiol, a lignanbiphenol derived from the magnolia tree, inhibits dengue virus type 2 infection screening of brazilian plants for antiviral activity against animal herpes viruses probit analysis antiviral effects of glycyrrhiza species biotransformation of ursolic acid by syncephalastrum racemosum cgmcc 3.2500 and anti-hcv activity anti-aids agents 11. betulinic acid and platanic acid as anti-hiv principles from syzygium claviflorum, and the anti-hiv activity of structurally related triterpenoids antiviral activity of dammarane saponins against herpes simplex virus type 1 activity of melaleuca alternifolia (tea tree) oil on influenza virus a/pr8: study on the mechanism of action antiviral activity of an extract derived from roots of eleutherococcus senticosus search for antiviral agents antiviral product development-conducting and submitting virology studies to the agency. u.s. department of health and human services, food and drug administration michellamines d-f, new hiv-inhibitory dimeric naphthylisoquinoline alkaloids, and korupensamine e, a new antimalarial monomer, from ancistrocladus korupensis virucidal effects of the steam distillate from houttuynia cordata and its components on hsv-1, influenza virus, and hiv inhibitory effect of ferulic acid and isoferalic acid on murine interleukin-8 production in response to influenza virus infections in vitro and in vivo a modified plaque method for arboviruses on plastic panels evaluation of new antiviral agents: i, in vitro perspectives anti-viral effect of a compound isolated from liriope platyphylla against hepatitis b virus in vitro isolation of the anthropogenic compound fluoranthene in a screening of chinese medicinal plants for antiviral compounds further investigations on the antiviral activities of medicinal plants of togo antiviral activities in plants endemic to madagascar inhibitory effects of sudanese medicinal plant extracts on hepatitis c virus (hcv) protease study of antiviral action of total alkaloids from haemanthus albiflos inhibitory effect of glycyrrhizin on the in vitro infectivity and cytopathic activity of the human immunodeficiency virus antiviral potential of selected indian medicinal (ayurvedic) plants against herpes simplex virus 1 and 2. n anti-hbv active constituents from piper longum the flavonoid ellagic acid from a medicinal herb inhibits host immune tolerance induced by the hepatitis b virus-e antigen comparison of specific antiviral agents in herpes simplex keratitis in vitro and in vivo anti picorna virus activity of some phenoxypyridine carbonitriles extracts and molecules from medicinal plants against herpes simplex viruses hep ad38 assay a high-throughput, cell-based screen for the evaluation of compounds against hepatitis b virus samarangenin b from limonium sinense suppress herpes simplex virus type 1 replication in vero cells by regulation of viral macromolecular synthesis antiviral activities against hsv-1, hcmv, and hiv-1 of rhamnan sulfate from monostroma latissimum antiviral effects of black raspberry (rubus coreanus) seed and its gallic acid against influenza virus infection in vitro antiviral activities of chinese medicinal herbs against duck hepatitis b virus plant antiviral agents iii. isolation of alkaloids from clivia miniata regel (amaryllidaceae) isolation and purification of baicalein, wogonin and oroxylin a from the medicinal plant scutellaria baicalensis by high-speed counter-current chromatography antiviral activity and mode of action of caffeoylquinic acids from schefflera heptaphylla (l) identification of natural compounds with antiviral activities against sarsassociated coronavirus mechanism of action of glycyrrhizic acid in inhibition of epstein-barr virus replication in vitro hydrolyzable tannins (chebulagic acid and punicalagin) target viral glycoproteine glycosaminoglycan interactions to inhibit herpes simplex virus 1 entry and cell-to-cell spread saikosaponin b2 is a naturally occurring terpenoid that efficiently inhibits hepatitis c virus entry ethnopharmacology in overdrive: the remarkable anti-hiv activity of artemisia annua the alkaloids. vol antiviral potential of curcumin a search for anti-viral properties in panamian medicinal plants, the effects on hiv and its essential enzymes interaction of filacial proteins on growth regulation of normal lung epithelial cells in vitro antiviral screening of british columbian medicinal plants crown-gall tumors in potato discs and brine shrimp lethality: two simple bioassays for higher plant screening and fractionation the in vitro antivirial activity of an aliphatic nitro compound from heteropteris aphrodisiaca brine shrimp: a convenient general bioassay for active plant constituents natural products and drug development. munksgaard anti-herpes virus activity of apporphine alkaloids anti-herpes virus activities of achyranthes aspera: an indian ethnomedicine, and its triterpene acid bio organic marine chemistry pedilanthus tithymaloides inhibits hsv infection by modulating nf-îºb signalling screening extracts of zanthoxylum chalybeum and warburgia ugandensis for activity against measles virus (swartz and edmonston strains) in vitro biological activity of guatteria cardoniana fractions comparison of two colorimetric assays to determine viral infectivity in microculture virus titration characteristic of alkylated chalcones from angelica keiskei on influenza virus neuraminidase inhibition glycyrrhizic acid inhibits virus growth and inactivates virus particles in vitro anti-human immunodeficiency virus activity of polysaccharide from rhizophora mucronata poir maslinic acid, a natural triterpenoid compound from olea europaea, protects cortical neurons against oxygen-glucose deprivation induced injury in vitro study of the antiviral activity of some b-carboline alkaloids a simple method of estimating fifty percent endpoints anti-hiv activity of extracts and compounds from algae and cyanobacteria antiviral flavonoid from pterocaulon sphacelatum, an australian aboriginal medicine in vitro antiviral activity of the anthraquinone chrysophanic acid against poliovirus antiherpes virus activity of extracts from the medicinal plant geranium sanguineum l antiviral agents as adjuncts in cancer chemotherapy effects of phyllanthus plant extracts on duck hepatitis b virus in vitro and in vivo antiviral activity of carnosic acid against respiratory syncytial virus in vitro antioxidant activity of polyphenol extracts with antiviral properties from geranium sanguineum l a new antiviral and antimicrobial sesquiterpene from glyptopetalum sclerocarpum trypan blue exclusion test of cell viability antiviral activity and constituent of ardisia chinensis benth against coxsackie b3 virus analysis of anti-rotavirus activity of extract from stevia rebaudiana proanthocyanidin from blueberry leaves suppresses expression of subgenomic hepatitis c virus rna anti-viral activity of the extracts of a kenyan medicinal plant carissa edulis against herpes simplex virus screening of higher plants for biological activities. ii. antiviral activity plant products as potential antiviral agents plant antiviral agents: v. 3-methoxyflavones as potent inhibitors of viral-induced block of cell synthesis in vitro antiviral activity of plant extracts from asteraceae medicinal plants current organic chemistry, natural product chemistry issue plant flavonoids in biology and medicine ii: biochemical, cellular and medicinal properties inhibition of hepatitis c virus replication by chalepin and pseudane ix isolated from ruta angustifolia leaves fangchinoline inhibits human immunodeficiency virus type 1 replication by interfering with gp160 proteolytic processing herbs of the genus phyllanthus in the treatment of chronic hepatitis b: observations with three preparations from different geographic sites effect of oenanthe javanica flavone on human and duck hepatitis b virus infection antiviral effect of cimicifugin from cimicifuga foetida against human respiratory syncytial virus three new secoiridoids, swermacrolactones a-c and anti-hepatitis b virus activity from swertia macrosperma lignans with antihepatitis b virus activities from phyllanthus niruri l antiviral activity of polymethoxylated flavones from guangchenpi, the edible and medicinal pericarps of citrus reticulata 'chachi the in vitro activity of geraniin and 1,3,4,6-tetra-o-galloyl-beta-d-glucose isolated from phyllanthus urinaria against herpes simplex virus type 1 and type 2 infection the protective effect of 3-deoxysappanchalcone on in vitro influenza virusinduced apoptosis and inflammation mechanism of inhibition of hiv-1 infection in vitro by purified extracts of prunella vulgaris evaluation of anti-hsv-2 activities of barleria lupulina and clinacanthus nutans novel antiviral activity of baicalein against dengue virus a dihydro-pyrido-indole potently inhibits hsv-1 infection by interfering the viral immediate early transcriptional events comparative inhibitory effects of suramin and other selected compounds on the infectivity and replication of human t-cell lymphotropic virus (htlv-iii)/lymphadenopathy-associated virus (lav) discovery of cyanovirin-n, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development emerging anti-hiv drugs agar diffusion method for detection and bioassay of antiviral antibiotics anti-herpes virus activity of aporphine alkaloids antiviral potentials of medicinal plants evaluation of anti-infective potential of a tribal folklore odina wodier roxb against some selected microbes and herpes simplex virus associated with skin infection sensitive and rapid assay on mt-4 cells for the detection of antiviral compounds against the aids virus a rapid and simple colorimetric test for the study of anti-hiv agents plant-derived leading compounds for chemotherapy of human immunodeficiency virus (hiv) infection resistance of human immunodeficiency virus type 1 to the high-mannose binding agents cyanovirin n and concanavalin a key: cord-024652-4i6kktl0 authors: santra, hiran kanti; banerjee, debdulal title: natural products as fungicide and their role in crop protection date: 2020-05-12 journal: natural bioactive products in sustainable agriculture doi: 10.1007/978-981-15-3024-1_9 sha: doc_id: 24652 cord_uid: 4i6kktl0 seeking solutions from nature for solving one and all problems is the age-old practice for mankind, and natural products are proved to be the most effective one for keeping up the balance of development as well as the “healthy, wealthy, and well” condition of mother nature. fungal pathogens are proved to be a common and popular contaminant of agroecosystem that approximately causes 70–80% of total microbial crop loss. to meet the proper global increasing need of food products as a result of population explosion, managing agricultural system in an eco-friendly and profitable manner is the prime target; thus the word “sustainable agriculture” plays it part, and this package is highly effective when coupled with nature-derived fungicidal products that can minimize the event of fungal infections in agrarian ecosystem. present study enlists the most common and effective natural products that might be of plant or microbial origin, their mode of action, day-by-day development of phytopathogenic resistance against the prevailing fungicides, and also their role in maintenance of sustainability of agricultural practices with special emphasis on their acceptance over the synthetic or chemical one. a large number of bioactive compounds ranging from direct plant (both cryptogams algae and moss and phanerogams)-derived natural extracts, essential oil of aromatic plants, and low-molecular-weight antimicrobial compounds known as phytoalexins to secondary metabolites that are both volatile and nonvolatile organic compounds of microbes (fungal and actinobacterial members) residing inside the host tissue, called endophyte, are widely used as agricultural bioweapons. the rhizospheric partners of plant, mycorrhizae, are also a prime agent of this chemical warfare and protect their green partners from fungal invaders and emphasize the concept of “sustainable agriculture.” natural products are the best weapon for the survival of any type of problems regarding infection, pathogenesis, or protection from diseases. due to their degradability in nature, they are the first options to be used by agriculturalists and plant biologist for combating fungal pathogenesis. the eukaryotic organism fungi have a separate kingdom in whittaker's five kingdom classification and are prime member of this ecosystem as a potent decomposer. in spite of their heavy and multidimensional applications in agricultural, medicinal, and industrial field ranging from the production of life-saving medicines to food supplements, they are the cause of huge global crop loss each year and lead to economic exhaustion. macro-and microscopic fungi-producing fruit bodies on different portions of a plant body (stem, leaf, fruit, root) lead to the death and decline of the crop species. several methods have already been tried since the start of civilization for crop protection but because of plant and fungal coevolution, fungi have dominated on the green eukaryotes and caused significant reduction in the crop yield. particularly in a country like india where the central gdp largely depends on agricultural output, the fungal pathogenesis has been a matter of grave concern for the agriculture department and policy makers. huge amount of money and manpower is invested to fight against the fungal diseases for ensuring higher and qualitative yield, but still it has been a burning problem of today's conditions. the problem with chemical and synthetic tools for combating the parasitic infections includes their toxicity leading to quality deterioration and environmental pollution accompanied with side effects on human health. in a case study, it has been reported that the extreme use of antibiotics in agricultural field and their direct consumption by humans through their daily meal have led to resistance of those antibiotics in human fungal pathogens. so we are in search of bioactive agents that will be of biological origin, selective to their host, and produce no secondary symptoms with least negative impact. the problem with fungi is their secretion of various types of mycotoxins (aflatoxins, ochratoxins, patulin, fumonisin, zearalenone, deoxynivalenol, etc.) in the stored food products, causing postharvest loss of cereals, pulses, dry fruits, and spices. mycotoxins are not only food spoilers but also potent disease-causing agents in humans leading to cancer, liver damage, kidney failure, and paralysis (miller 1995) . the severe effects of fungal crop loss are visible largely in tropical or subtropical regions where the temperature is moderately higher than the other parts of the world. fungal devastation occurs in two phases: firstly, when the crops are growing on the field and, secondly, when they are stored for further transportation, postharvest loss. the third type of contamination occurs when the microscopic airborne pathogens like molds grow on cooked foods and lead to food spoilage. at each and every level, scientists have developed techniques to minimize fungal food loss. on a gross annual estimate, almost 25% of agricultural food items are of no use due to fungal contamination (pittet 1998) . the major issues with fungi-related crop loss are deterioration as a result of increase of fatty acid conditions, change of color and texture of food items, poor nutritional conditions, and poor germinability of stored seeds (dhingra et al. 2001) . reports from asia and africa include death of humans and animals due to consumption of mycotoxin-contaminated foods (reddy and raghavender 2007) . fungal pathogens are sometimes dependent on more than one host for their successful completion of life cycle and disease development (puccinia graminis var. tritici, causal agent of black stem rust of wheat that requires berberis aristata for successful infection other than there main target wheat plant). so physical controls like eradication of secondary or collateral host and burning of the old livestocks and remnants of the field are the primary measures adopted by the farmers for disease-free crop production. so maintaining the sustainability along with less pathogenic infection is the deep ecological movement for crop maintenance. there are reports of resistance developed against the common and widely used antibiotics of agricultural importance. blasticidin s, an antibiotic obtained from streptomyces sp. (a type of actinobacteria predominantly present in soil samples), interacts with the protein synthesis and causes the death of the rice blast pathogens. development of resistance of this antibiotic is reported to be present in some fungal pathogens that detoxify it by deamination (dayan et al. 2009 ). compounds of bacterial and fungal origin from both soil and endophytic sources are used as an alternative source over the chemical ones. plant extracts especially essential oils from plant taxa of lamiaceae family are of immense importance and are used as fungicidal or fungistatic. most of the active ingredients act upon the fungal cell wall by either blocking the cellular processes like respiration, cell wall and cell membrane synthesis, ergosterol biosynthesis, protein synthesis, or dna replication. not only the secondary metabolites of plant and microbial origin but also the direct application of microorganisms in terms of biocontrol agent could be used as potent antifungals. other than these, plants' own defense molecules, known as the phytoalexins, could provide a strong line of defense against mycorrhizae; the root symbionts of higher plants can physically, biologically, and biochemically protect the plant root from pathogenic invasion and provide an enhanced resistance conditions to their hosts. this study includes the role of these compounds as natural agents of antifungal property and their role in disease prevention. mycorrhizae as a biocontrol agent mycorrhiza being the perfect example of symbiosis is known to be the oldest association between higher plant (both angiosperm and gymnosperm, monocot and dicot plants) and fungi and is an astonishing phenomenon of nature. the mycorrhizal association is one of nature's privileges for maintaining the sustainability of agriculture. in present day's changing environment, haphazard use of pesticides (fungicides) and chemicals poses a great risk to the existence and survival of mycorrhizal species in its complete biologically active form. there is a need to increase awareness in order to save mycorrhizal fungi from extinction. plants form beneficial association with other variants of life forms (animals, bacteria, or fungi) to complete their life processes, to fight against pathogenic microorganisms, and most importantly to thrive in adverse environmental situations. the plant root and its associated living microbial flora are together called "rhizosphere," particularly the area of mycorrhizal occurrence. the term mycorrhiza is derived from two greek words: mycos which means fungus and rhiza which means roots. in nature, more than 80% of angiosperms and almost all of gymnosperms are known to have mycorrhizal associations. the common two types of mycorrhizal associations that exist in nature are endomycorrhizae, also called arbuscular mycorrhizae (am), for example, endogone sp. and rhizophagus sp., and ectomycorrhizae (em), for instance amanita muscaria and laccaria bicolor. mycorrhizal associations support its host plants to survive in untimely soil conditions and drought situations by increasing the surface area of root and efficiency of mineral uptake. environmental threats including problems of temperature increase, climate changing, drought, and infertility of soil are some of the major challenges in agriculture and have to be mitigated to ensure global food security. in this context, mycorrhiza-based crop production is one of the key components of sustainable agriculture practices. in most of the cases, am fungi-mediated suppression of root pathogenic fungi is achieved by either morphological, physiological, or biochemical alterations of the host. several experiments on fungistatic activity of the mycorrhizal species have been done, and fruitful results are found against pathogenic fungi such as aphanomyces spp., botrytis fabae, chalara (thielaviopsis) basicola, dothiorella gregaria, fusarium oxysporum, gaeumannomyces graminis var. tritici, ganoderma pseudoferreum, pythium ultimum, p. splendens, phytophthora parasitica, p. cactorum, p. vignae, rhizoctonia solani, r. bataticola, and sclerotium rolfsii (lioussanne et al. 2009; bagyaraj 2006; bagyaraj and chawla 2012) . the most common outcome of am fungal colonization is seen as an increase in number of branches, resulting in a relatively larger proportion of higher-order roots in the root system. thickening of the cell walls due to lignification and production of polysaccharides in mycorrhizal plants are the common mode of prevention of penetration and growth of pathogens like fusarium oxysporum and phoma terrestris. a huge percentage of am-root pathogen interaction studies have been conducted in crop plants of agricultural and horticultural importance. but the information available on forest tree species is scanty. mycorrhizal technology can thus play an important role in production of low-cost quality seedlings and provide plant protection. like other methods of biological control, am fungi are not able to offer complete immunity against the infection caused by plant pathogens. they could only impart a degree of resistance against soilborne plant pathogens. however, the possibility of biologically controlling soilborne plant pathogens looks promising. am fungi play a protective role for plants by activating the defense mechanisms for the better resistance of crop plants and thus may protect the host plant from further fungal pathogenic attack, thus working as a potent biocontrol agent. researchers have proved that am symbiosis triggers the activation of several defense-related genes and also expression of pathogenesis-related proteins. evidences are drawn from modern techniques like molecular biology methods and immunological and histochemical analysis that strongly supports this concept. am fungi first act as a biotrophic agent, and before entering the host plant's root cell, they cause a sharp change in endogenous salicylic acid that is reflected in quick accumulation of reactive oxygen species (ros), a wide range of hydrolytic enzymes, and also the activation of phenylpropanoid biosynthetic pathway (güimil et al. 2005 , paszkowski 2006 , roman et al. 2011 . research findings have proved that the amount of defense-related compounds (essential enzymes like pal, phenylalanine ammonialyase, a product of phenylpropanoid pathway, enzymes needed for flavonoid or isoflavonoid biosynthesis like chalcone isomerase) that act for the protection of plant from fungal and bacterial pathogen is higher in the case of mycorrhiza-inoculated plant than in the uninoculated ones (volpin et al. 1994 (volpin et al. , 1995 . host's physiological and biochemical processes are greatly influenced by the mycorrhizal association in terms of decreased root exudation, higher concentration of phenylalanine and serine contents, ortho-dihydroxy phenols, increased membrane phospholipid content, etc. (smith et al. 1994) . when the phospholipid contents are high, it reduces the chances of root pathogenic attack, and higher concentrations of ortho-dihydroxy phenols show inhibitory activity against root rot pathogen sclerotium rolfsii (causal agent of southern blight), whereas the non-mycorrhizal plants are affected by the southern blight disease. tomato plants when inoculated with g. fasciculatum show inhibitory activity against root knot nematodes. host-am association leads to the formation of defense-related compounds like phytoalexins, chitinases (chi), β-1,3-glucanase (glu) (enzyme related to hydrolysis of fungal cell wall), peroxidases (pox), hydroxyproline-rich glycoproteins, and phenolics (st arnaud and vujanovic 2007) . synergistic effect of pgprs along with am fungi is proved to be a system of better protection than the use of am fungi alone (linderman 1994; bagyaraj and chawla 2012) . fungal wilt of common medicinal plant indian coleus (coleus forskohlii) caused by fusarium chlamydosporium could be minimized by the joint action of am fungus and trichoderma viride and cause a sharp increase in root yield and root forskolin concentration and may also reduce the severe disease conditions (singh et al. 2012 ). am causes a drastic change in the rhizospheric microbiota and intentionally either removes directly the pathogenic microorganisms or stimulates the accumulation of potent microbial partners especially fungus that are heavily antagonistic to the plant pathogenic ones. plants with mycorrhizal association harbor higher population of rhizospheric microorganisms, thus making it impossible for the pathogen to compete and invade the root. in the case of phytophthora cinnamomi, the numbers of sporangia and zoospores are found to be reduced when rhizospheric soil extracts of am plants are applied; it means the am fungi are able to alter the microbial population and particular functional groups of rhizospheric microorganisms (meyer and linderman 1986; larsen and bodker 2003) . they cause qualitative and quantitative changes in the fungal community by several factors like changed exudation patterns; altered root size and architecture; different physiological and biochemical parameters like sugar, organic acids, and amino acids; and also putative direct am fungal effects (toljander et al. 2007; ahmed et al. 2013; vigo et al. 2000) . fungistatic siderophore (low-molecular-weight chelating agents having higher affinity for ferric ion)-producing microorganisms are found to be crowded in mycorrhiza-infected roots and rhizospheric regions. mycorrhizal plants are to be reported with more actinomycetes and bacterial (gram-positive paenibacillus sp. against phytophthora parasitica) flora antagonistic to soilborne root pathogens (azcon-aguilar and barea 1996; budi et al. 1999 ). apart from providing biochemical and physiological defense strategies, arbuscular mycorrhizal species also exhibit physical barrier of defense by changing the root anatomy, morphology, and even architecture in terms of increased nutrient uptake, meristematic and nuclear activities of root cell, higher rate of growths, and branching patterns (atkinson et al. 1994; gamalero et al. 2010; gutjahr and paszkowski 2013) . thus responses of root morphology as a result from afm colonization seem to depend on plant characters, tap root system, etc. more benefits are seen in tap root system than fibrous root system in terms of gained biomass and nutrient acquisition. though there is a gap of knowledge in how increased root branching caused by mycorrhizal infection help the plant to defend fungal pathogenesis, synergism is seen as something that can balance the suppressed root growth caused by several root pathogens and restore the root health. mycorrhiza-mediated strengthening of the vascular system allows the higher rate of flow of nutrients, increased mechanical strength, and also inactivation of vascular pathogens. in conditions of limited resource such as carbon requirement and space for inoculation, a competition between the symbiotic partner (mycorrhizae) and pathogenic fungi is very common and expected (vos et al. 2014 ). in the direct warfare, mycorrhizae win over the pathogenic one and thus obtain higher amount of nutrients (almost 4-20% of total assimilated carbon by host plant) and occupy large areas of available root cortical cells (jung et al. 2012; vierheilig et al. 2008) . defeating the pathogenic fungi in terms of nutrient uptake and providing a little or no room for infection are probably the mightiest cause of biocontrol ability of am fungus (hammer et al. 2011) . output of am and phytophthora interaction indicates that the pathogen does not penetrate cortical arbuscular cells, suggesting that localized competition for infection site does occur between the pathogenic fungi and the am fungus. not only fungi but also plant-invading nematodes are in the queue for colonization and nutrient uptake (smith 1988) . the infection of southern root-knot nematode (meloidogyne incognita, m. exigua) is reduced when the roots are priorly inoculated with symbiotic partners like in the case of coffee plants also (alban et al. 2013; dos et al. 2010) . reports have suggested that the number of infected sites is reduced within mycorrhizal root system than in the uninoculated one and thus strongly supports the mycorrhizal role as a biofungicide (vigo et al. 2000) . am fungi can help the plant uptake of nutrients like phosphate, nitrogen, minerals, microelements (zinc), and water at a higher rate than the uninfected one (baum et al. 2015; parniske 2008) , and as a result, they are provided with photosynthetic carbon (smith and smith 2011) . the plants capable of absorbing higher amount of nutrients in terms of am fungal association have the potential to tolerate pathogenic infections (karagiannidis et al. 2002) . though the improved nutrition and increased tolerance are not involved in a cause-effect relationship, proofs are there that higher uptake of phosphate results in remarkable reduction in pathogenic infection in mycorrhizal plant but not in non-mycorrhizal plant (bodker et al. 1998) . tomato plants already infected with rhizophagus irregularis are not colonized by the pathogen a. solani, whereas non-mycorrhizal plant is affected by the pathogen (fritz et al. 2006) . mixed action of arbuscular mycorrhizal fungi (amf) glomus intraradices and trichoderma harzianum as a biocontrol agent significantly reduces the damping off disease caused by rhizoctonia solani in the case of tomato seedlings (amer and seud 2008) . in order to combat parasitic (fungal, bacterial, viral, nematoidal, and insectal) infection like mammalian cells, plant cells also develop defense systems that mediate the release of low-molecular-weight and short-lived (generally 72-96 h of existence) antimicrobial compounds or molecules known as the phytoalexins (braga 1991; echiverri et al. 2010; paxton 1980) . these secondary metabolites help the plant to withstand biotic and abiotic stress (grayer and kukubun 2001) . most of them being lipophilic compounds can cross the plasma membrane and act inside the fungal cell causing cytoplasmic granulation of the infecting fungal cells, disorganization of the cellular components, rupture of the plasma membrane, and inhibition of the fungal enzymes and mycelial growth (cavalcanti 2005) . mode of action of phytoalexins against fungal pathogenesis varies from species to species (table 9 .1). metabolism of phytoalexin mediated by fungus involves the tendency for its increased polarity by addition of hydroxyl group (oxygenation), removal of methyl group (demethylation), etc. (jeandet et al. 2014) . muller and borger first enlightened the concept of phytoalexins almost 70 year ago (muller and borger 1940). the first reported case analyzed with the concept of phytoalexin was potato tuber infection by the different strains of causal organism of "late blight of potato," phytophthora infestans. this pathogenic fungus initiated the hypersensitive reactions that lead to the formation of some "plant secondary metabolite" that inhibited further infection of the same plant when infected with another strain of the same genus of phytophthora. muller and his coworker named this "principle" as "phytoalexins" that have protected the plant from secondary infection (deverall 1982) . accumulation of phytoalexins in the green plant tissue clearly indicates the presence of remarkable amount of fungal and bacterial infections in the host tissue (stoessl 1980) . phytoalexins are naturally occurring products secreted and accumulated temporarily by plants in response to pathogenic attack or abiotic stress and agents like heavy metal toxicity, uv radiation, and wounds on tissue (naoumkina et al. 2007 ). the inducer agent may be of two types, elicitor and elicitin. the elicitors are commonly the oligosaccharides from fungal cell origin (like hepatosaccharide from soja cell wall) (sharp et al. pezet and pont (1990) , adrian et al. (1997) , adrian and jeandet (2012) camalexin induction of the fungal programmed cell death (pcd) by apoptotic mechanisms et al. (2011) 1984). the elicitin types of molecules are generally a type of glycoproteins secreted by the fungal cells (cordelier et al. 2003) . reports on detailed investigations about phytoalexins have covered a very few families (leguminosae and solanaceae) of the green world (ingham 1982; kuc 1982) . though investigations on some selected number of species and genera are made from plant families including both monocotyledonous (amaryllidaceae, orchidaceae, poaceae) and dicotyledonous plants (apiaceae, asteraceae, convolvulaceae, chenopodiaceae, euphorbiaceae, linaceae, moraceae, piperaceae, rosaceae, rutaceae) and even gymnospermic taxa (ginkgoaceae) (coxon 1982) , cash crops like members of poaceae (focusing on maize and rice), vitaceae, and malvaceae (cotton) have been studied for their phytoalexin production (schmelz et al. 2014; langcake and pryce 1976; jeandet et al. 2010; sunilkumar et al. 2006) . though till date a lot of researches have already been performed regarding phytoalexins, a natural weapon against mycopathogens, but still to increase the fungitoxic effectivity of these stress metabolites, further advancement in design and genetic control is needed ). phytoalexin synthesis not only is dependent on pathogenic attack but also could be influenced by various abiotic factors such as temperature, humidity, and water availability ( fig. 9.1 ). there are evidences that different parts of the plant like leaves, flowers, stems, seeds, and root tubers are site of phytoalexin biosynthesis (mikkelsen et al. 2003) . different biochemical pathways are used for producing various types of phytoalexins. the three most common pathways include (i) the phenylpropanoic-polymalonic acid pathway, (ii) the methylerythritol phosphate (mep) and geranylgeranyl diphosphate (ggdp) route, and (iii) the indole phytoalexin (ip) pathway (jeandet et al. 2014 ). it is not always obvious that phytoalexins could be categorized not only by their chemical structure or biosynthetic pathway but also by their function and tissue specificity. examples include the occurrence of momilactone a on different plant parts of rice plant (lee et al. 1999; cartwright 1981) . momilactone a is known to be residing in rice husks and rice stems constitutively, but they are also a phytoalexin of rice leaves. further studies by toyomasu and his coworkers conclude that momilactone a is constitutively synthesized and oozed out from root of rice plants. still there is no sufficient data available to consider phytoalexins as ubiquitous throughout the whole plant kingdom. a lot of studies have revealed their complex biochemical synthetic machinery that involves their de novo synthesis, regulation, and mode of action (jeandet et al. 2013 ahuja et al. 2012 . regulatory mechanisms involve defense-related marker genes, calcium sensors, hormone signaling, phosphorylation cascades, and also their antipathogenic activity. there are reports on genetic engineering-mediated manipulation of phytoalexin production and increased disease resistance in the case of plants (delaunois et al. 2009; jeandet et al. 2012 jeandet et al. , 2013 . phytoalexins are secondary or stress metabolites that are produced when the host plant is infected with pathogenic fungus. phytoalexin-mediated defense response includes the expression of lytic enzymes such as chitinases and glucanases and a number of pathogenesis-related (pr) proteins, oxidizing agents, and lignification of cell walls (dixon and lamb 1999) . mode of action of phytoalexin involves the coordinated synergism between several defense factors for the effective inhibition of the fungal pathogen (purkayastha 2017; mansfield 1999) . in the case of sorghum plant, significant infection caused by fusarium proliferatum and fusarium thapsinum stimulates the production of 3-deoxyanthocyanidin, apigeninidin, and luteolinidin and also the concentration of defense-related proteins like peroxidases, β-1,3 glucanases, and chitinases that help to fight the pathogenic infection (huang and backhouse 2004 (koga et al. 1997; fukuta et al. 2007 ). there are several ways of blocking the fungal infection in host plant tissues by phytoalexin-mediated response. that includes inhibition of fungal spore on the leaf surface and inhibition during and after penetration to host cell (usman et al. 2018) . the occurrence of fungal germ tube on the leaf surface and diffusion of fungal metabolites through the leaf cells cause the accumulation of phytoalexins by the underlying cells and provide the first line of induced chemical defense (vanwees et al. 2003) . phytoalexins may be located on papillae or cell walls, thereby producing a localized, fungitoxic barrier to penetration (friend 2016) . examples include occurrence of fungitoxic (against erysiphe graminis) flavonoid (thought to be phytoalexin) on papillae of resistant barley leaves. phytoalexins are known to be solely produced as a result of induction or stimulus by external agents. fruitful evidences could be drawn regarding this fact. induction of disease resistance in plants is developed through the direct and indirect involvement of elicitors. extracts of fungal basidiocarp, essential oils of aromatic plants (walters et al. 2013) , and also synthetic chemicals like aminobutyric acid, salicylic acid, jasmonic acid, and acibenzolar-s-methyl ( (matiello and bonaldo 2013) , hymenolobium petraeum, qualea albiflora, and corymbia citriodora (matiello et al. 2016 ) that act as the elicitors of deoxyanthocyanidins and glyceolin production. homeopathic preparations of species of calcarea (c. citriodora and calcarea carbonica), essential oils of eucalyptus globulus (telaxka et al. 2014; oliveira et al. 2014) , and mild concentrations of salicylic acid (durango et al. 2013 ) are major elicitins of pistain production and accumulation in cotyledons of common bean (phaseolus vulgaris). silicon-mediated enhancement of disease resistance by peroxidase (pox), polyphenol oxidase (ppo), chitinases (chi), β-1,3-glucanases (glu), and phenylalanine ammonia-lyase (pal) is found in the case of leaf spot of cotton plant caused by ramularia areola (curvêlo et al. 2013 ). southern amazonian amphibian family bufonidae represents the true toads, and their cutaneous secretions are of diverse source of bioactive compounds which can be fruitful as new chemical weapons for agrochemical development. use of elicitors in the case of crop protection nowadays is becoming a very popular method of inducing response which are proved to be durable and broad-spectrum disease control mechanism where the plant's own resistance is used. a group of seven brazilian scientists (deice et al. 2019) evaluated the possibilities of methanolic extracts of cutaneous secretions of two species of bufonidae, rhaebo guttatus and rhinella marina, on synthesis of phytoalexins named glyceolin (soybean plant), deoxyanthocyanidins (sorghum plants), and phaseolin (mung plant) in soybean cotyledons, sorghum mesocotyls, and bean hypocotyls, respectively. there is a direct relationship between the phytoalexins production and defense ability of the host plant against the fungal pathogenesis. studies reveal that when the phytoalexin glyceolin is produced in higher amount in the soybean plant (cultivar tmg 132 rr) as a result of methanolic extracts of amphibian's (r. guttatus) cutaneous secretion (at a concentration of 0.2 mg/ml), stimulates the enzyme β-1,3-glucanase that can cause the hydrolysis of the fungal cell wall along with other defense-related enzymes (chitinase) is also produced in higher amount, but when suppression of glyceolin occurs, that particular enzyme is also not produced. there are evidences in the case of glycine max that the effectivity of phytoalexins varies from cultivar to cultivar. application of r. marina (amphibian) methanolic extracts induced glyceolin production in tmg 132 rr and monsoy 8372 cultivars ipro but did not induce tmg 132 rr cultivars to synthesize these defense-related compounds. less toxicity of phytoalexins than chemical fungicides is the reason for their universal acceptance. for over 75 years, phytoalexins have been a detailed area of study for its antimicrobial activity, especially antifungal properties. several investigations include the in vivo bioeffectivity of the phytoalexins against serious plant pathogenic fungi (table 9 .2). phytoalexin synthesizing genes have also been genetically modified to cope up with the pathogenic evolution. still reports are there that include examples of cruciferous phytoalexins detoxification by fungal enzymes (pedras and abdoli 2017) . modification of pathogen to overcome phytoalexinmediated damage includes curved germ tubes as a result of asymmetric growth of the germ tube. phytoalexins are natural products of diverse chemical nature, for example, alkaloids, coumarins, isoflavonoid (coumestans, isoflavans, isoflavones, isoflavanones, pterocarpans, pterocarpenes), lignans, polyacetylenes, pterocarpons (pisatin, phaseolin, glyceollin, medicarpin, and maackiain), terpenes, and non-isoflavonoid compounds (furanoacetylenes and stilbenes) ( fig. 9 .2) (grayer and kokubun 2001) . both in vitro and in vivo fungicidal activity are shown by sakuranetin (rice phytoalexins) against the blast fungus (hasegawa et al. 2014 ). reduction of green mold (caused by penicillium digitatum) infections is achieved by the action of coumarin type of phytoalexin (scopoletin) of orange (sanzani et al. 2014 ). the loss of apples production caused by penicillium expansum and accumulation of patulin is minimized by the action of phenolic phytoalexins like resveratrol, scopoletin, scoparone, and umbelliferone (sanzani et al. 2009 ). in the case of medicago sativa (alfalfa), the isoflavonoid 7-o-methyltransferase provides increased resistance against phoma medicaginis by synthesizing maiackiain (he and dixon 2000) . for soybean plants, transformation of resveratrol to pterostilbene 7, langcake and pryce (1976) , coxon (1980) , and harding and heale (1981) (continued) (zernova et al. 2014) . scientists have proven that not only fungal infection acts as the stimulus for phytoalexin synthesis but also the hormone levels; phosphorylation cascades play a major role in this purpose. cytokinin overexpression in nicotiana tabacum is directly associated with its resistance against p. syringe by higher concentration of capsidiol and scopoletin (grosskinsky et al. 2011 .) the fungitoxicity of the phytoalexin could be enhanced by methylation or presence of electron-attracting groups on aromatic rings that is directly involved in affinity with membrane proteins being an uncoupler of ets system. endophytes are a type of hidden beneficial microorganisms that reside within the host plant causing no visible disease symptoms and syndrome and promote the plant to maintain its existence in typical harsh conditions. sometimes they could be latent pathogens at a very distant path of the host's life cycle but are simply a unique area of research where plant science and their microbial association get new definition. endophytes have been a constant and reliable source of exploration of bioactive compounds, but extensive search has not been performed till date, and that has given the endophyte biologists a great opportunity to search endophytic fungal and actinobacterial flora for the establishment of novel bioactive compounds. selection of plant for endophytic isolation is the most vital part of this study. exploitation of the proper isolates accelerates this search and opens up new angle of research. the search for uncommon products of agrochemical importance is a common demand of todays' world. the safer the antifungal agents become, the more it is well accepted in the scientific community as well as agricultural market. in general, the screening of thousands of natural products ends up giving only one commercial product. so indeed it's a tough job to end the search of new antibiotics with a hopeful result. a total of 6 out of 20 of the popular prescribed medicines are of fungal origin, and it is a fact that 5% of the fungi have been described till date (hawksworth 1991 (hawksworth , 2001 . so fungi serve as a continuous dependable source of new natural products. the intelligent screening procedure includes the selection of fungal flora of endophytic sources to open up the untapped potential of secondary metabolites synthesized by fungi. microorganisms grown in the petri plates or culture broth constitute minimal growth medium needed for their survival. any kind of stress or transfer of microorganisms on selective media acts as a stimulation for production of their secondary bioactive compounds. these secondary metabolites are produced for their survival in odd environments and strictly act as the selection force for the expression of their antimicrobial-producing genes. these crude by-products of microbial cultures are filtered and purified for their industrial, medicinal, and agricultural exploitation. soil microorganisms have been exploited for a long time for production of antibiotics, but microorganisms inhabiting plants are a new source in that respect. plants are selected usually with potent medicinal applications. here the knowledge of ethnobotany and folk taxonomy contributes a lot in this selection procedure. a strong literature survey supports the plant selection. the plants are surface sterilized and plated in nutrient-less solid plates. the fungi emerge out from different explants using the decaying plant parts as their primary growth substance. the isolates are identified by microscopic structures focusing on their conidial morphology, spore sculpturing, and colony characters. confirmatory identification includes 18s rrna analysis. endophytic fungi are tested for their antifungal activity against phytopathogenic fungi by one-to-one inhibition assay or antagonistic test ( fig. 9 .3). two agar portions containing fungal hypha of endophyte and pathogen are placed on opposite sides of the plate. if the growth of pathogenic one is arrested partially or completely, that endophytic isolate is marked as antifungal agent and selected for further studies. another way of screening includes separating the agar plate into two equal halves, and two fungi are placed on two separate sides of the discontinuous plate. this test aims to screen the endophytes that produce volatile antifungals. if the isolate is potent enough to produce volatile organic compounds with fungi static or cidal activity, this will cease the growth of the pathogenic strain. then that isolate would be qualitatively and quantitatively measured for their volatile emissions using gc-ms as the master equipment ( fig. 9 .4). liquid extracts of endophytic fungi are also tested for antifungal potentials by agar well-diffusion method. the fungal extract having antibiotic property shows clear zone of inhibition of growth of the pathogenic fungus surrounding the area of application of that fungal liquid. the potent isolate will be mass cultured, and the bioactive molecules will be extracted using organic solvents like ethyl acetate, ethyl ether, and n-hexane. steps include purification of that fungal extract by column chromatography, detection of the compound by thin-layer chromatography, and analysis of compounds by hplc and mass chromatography. field experiment includes the synergistic effect of a pure compound with mixture of natural compounds, and the effectivity of a newly applied antifungal agent strictly depends on the host plant and pathogenic microorganism's interaction, environmental condition, and development of drug resistance by that organism. application of pellets soaked in fungal extracts also is a method of determination of antifungal activity. secondary metabolites are itself diverse in nature. a variety of bioactive secondary metabolites are produced at significant concentrations by the endophytic microbial flora. the major components include quinones, phenols, phenolic esters, steroids, terpenoids, cytochalasins, benzopyranone, alkaloids, isocoumarins, and chromones. till date, a large number of plants have been studied for their endophytic flora as antifungal agents (table 9 .3). alkaloid was the first ever reported insecticidal bioactive product. cryptocin was isolated from endophyte of tripterygium wilfordii, a plant of celastraceae family. the inner barks of the stem were used as explant, and cryptosporiopsis cf. quercina was isolated as a potent endophyte active against pyricularia oryzae and some other phytopathogenic fungi (li et al. 2000) . colletotrichum sp. produces 6-isoprenylindole-3-corboxylic acid having inhibitory action against phytophthora capsici, a pathogen of cucurbitaceae, fabaceae, and solanaceae, and also other phytopathogens rhizoctonia cerealis and gaeumannomyces graminis var. tritici, a common pathogen of poaceae family . epoxycytochalasin h and cytochalasins n and h were isolated as chloroform and methanolic extracts of (fu et al. 2011) . a lot of endophytes have been explored for their antifungal production, but only a few of them were positive for antifungal metabolites categorizing in alkaloids. the common alkaloids acting as the antifungal agents of endophytic fungal origin are gliotoxin, cryptocanadin, tyrocidine a, fumigaclavine c, fumitremorgin c, 1-n-methyl albonoursin, and phomapsichalasin. the terpenoids, usually called isoprenoids, are large and diverse group of naturally occurring organic compounds derived from terpenes that are multicyclic. sixty percent of all the known natural products are terpenoids in nature. some endophytic fungicidal products are of terpenes by their native chemical structure. endophytic isolates (hormonema sp.) of gymnospermous plant juniperus communis were reported to be antifungal producers of a triterpene glycoside enfumafungin (pelaez et al. 2000) . known antifungal sterols of endophytic origin are 3β-hydroxyergosta-5-ene, 3-oxoergosta-4,6,8,22-tetraene, etc. the sterols are strong inhibitors of helminthosporium sativum (present name: bipolaris sorokiniana), the asexual stage of cochliobolus sativus, a common root rot pathogen of wheat and barley crops which also infects leaf and stems of poaceae plants . sesquiterpenes are reported to be the growth inhibitors of cladosporium phlei (causal organism of leaf spot disease of timothy grass, phleum pratense). this is a unique example where the host plant (phleum pratense) itself harbors the endophyte (epichloe typhina) that inhibits the growth of its leaf spot pathogen (cladosporium phlei). from the point of view of organic chemistry, isocoumarins are defined as the isomer of coumarin where the orientation of the lactone is reversely arranged. zhang and his coworkers in the year 2008 isolated an endophytic fungus named microdochium bolleyi from fagonia cretica (also known as virgin's mantle of zygophyllaceae family), a herb of semiarid regions of gomera. isocoumarins were identified as the active compounds having antifungal activity against microbotryum violaceum (previously known as ustilago violacea), an obligate parasite of basidiomycete group and a common infectant of members of caryophyllaceae causing smut of anther. the four isolated and identified isocoumarins are monocerin, 12-oxo epimers of monocerin, and open-ring derivative compounds of monocerin. the compounds are obtained as mixtures by column chromatography followed by sephadex lh-20 chromatography techniques. preparative tlc further differentiated the four compounds. monocerin and its analogues were previously reported as antifungal compounds from fungal sources of drechslera monoceras, exserohilum monoceras, helminthosporium monoceras, exserohilum turcum, and fusarium larvarum (aldridge and turner 1970; robeson and strobel 1982; grove and pople 1979; claydon et al. 1979) . these secondary metabolites act on pathogens by interfering stages of divisional phases of cell cycle. the second isocoumarin was colorless oil. the third and fourth one are represented by the empirical formula of c 16 h 20 o 7 and c 16 h 22 o 7 . the fourth one is structurally correlated to fusarentin 6,7-dimethyl ether. both the compounds are of heptaketide in their origin, and it is revealed that fusarentins are the probable precursors of the active compounds monocerins (scott et al. 1984; axford et al. 2004 ). dihydroisocoumarins, mellein (an isocoumarin derivative), (r)-7-hydroxymellein, and fonsecinone were reported from species of xylaria (endophyte of piper aduncum), pezicula, penicillium (alibertia macrophylla), and aspergillus (cynodon dactylon), respectively (oliveira et al. 2011; schulz et al. 1995; song et al. 2004 ). phenols (popularly known as phenolics) represent a class of chemical compounds characterized with a hydroxyl group attached to an aromatic hydrocarbon group. phenol (or carbolic acid) is a colorless crystalline solid, aromatic compound having benzene rings. they are predominantly found in the plant kingdom as a response to stress and are of utmost importance. endophytic culture extracts are also known to be rich sources of phenolics; usually they are directly proportional to the antioxidative property of any fungal isolate, but in some particular cases, they are characterized with their antifungal potentials against phytopathogenic fungus. usually the liquid culture extracts of the endophytic isolates are subjected to solvent extraction using ethyl acetate, n-hexane, ethyl ether, etc. those organic solvents are believed to extract the phenolics from the water-based culture broth. those extracted compounds are further screened for their antifungal efficiency. ethyl acetate extracts of endophytic phoma sp. are reported to contain tetralone metabolites (derivatives of α-tetralone, 3,6,7-trihydroxy-α-tetralone) inhibiting the growth of two common broad phytopathogenic fungus fusarium oxysporum and rhizoctonia solani. griseofulvin is known to be the first antifungal compound isolated from penicillium griseofulvum. later it is isolated from several species of fungi including endophytic penicillium canescens and xylaria sp. (member of xylariaceae family). griseofulvin from endophytic p. canescens of popular chinese medicinal plant polygonatum cyrtonema (polygonaceae) showed strong inhibitory effectivity against phytopathogenic botrytis cinerea, sclerotinia sclerotiorum, colletotrichum orbiculare, and didymella bryoniae . other than penicillium, endophytic xylaria sp. isolated as an endophyte of abies holophylla yields griseofulvin and dechlorogriseofulvin for in vitro and in vivo effectivity against pathogenic magnaporthe grisea, corticium sasakii, blumeria graminis (park et al. 2005) . the ascomycete fungus pestalotiopsis is known to be a common plant pathogen but also has been reported many times because of their endophytic existence in the host plants. the two common species pestalotiopsis microspora (host: tropical plant terminalia morobensis) and p. fici are reported to be producing antifungal metabolites isopestacin and pestalofones d-e (harper et al. 2003; liu et al. 2009a, b) . chlorogenic acid and colletotric acids are antifungal phenolics of colletotrichum gloeosporioides and sordariomycetes sp., respectively zou et al. 2000) . they were isolated from medicinal plants of china (artemisia mongolica and eucommia ulmoides) and effective against fungi imperfecti helminthosporium sativum. orcinol is used for the production of a dye called orcein used randomly for the staining of cells and chromosomes. orcinol is popularly known for its antifungal activity too and has been isolated as a product of endophytic origin of penicillium sp. from alibertia macrophylla (a plant of rubiaceae) showing bioactivity against cladosporium cladosporioides and cladosporium sphaerospermum (oliveira et al. 2014) . endophytic phomopsis sp., dothiorella sp., and diaporthe sp. have also been tested for their antifungal production and antifungal compounds that were detected (brady et al. 2000; xu et al. 2004; huang et al. 2008 ). volatile organic compounds (vocs) are said to be a type of organic low-molecularweight carbon-containing small compounds (up to c20) that have a high vapor pressure with low molecular mass (100-500 daltons) at room temperature. the high vapor pressure results from a low boiling point of that chemical compound, which causes a huge quantity of molecules to evaporate from the liquid, solid, or semisolid form of the compound and gets released into the surrounding environment. the endophytes are unique in their volatile emissions. the term mycofumigation that is very much popular with the treatment of agricultural phytopathogens is actually the output of vocs that originated from endophytic isolates. the first ever reported volatile antibiotic producer was muscodor albus (xylariaceae family), an endophyte of guazuma ulmifolia (a plant of sterculiaceae family collected from tropical forest of sw ecuador), isolated by gary strobel and his co-workers ). the major compounds isolated by gcms are known to be involved in antifungal, antibacterial activity. compounds include butanoic acid, 2-methyl-; butanoic acid, 3-methyl-; 2-butenal, 2-methyl-; butanoic acid, 3-methylbutyl ester; 3-buten-1-ol, 3-methyl; guaiol; 1-octene, 3-ethyl-; formamide, n-(1-methylpropyl); azulene and naphthalene derivatives; caryophyllene; phenylethyl alcohol; acetic acid, 2-phenylethyl ester; bulnesene; and various propanoic acid, 2-methyl-derivatives. these compounds were tested against a number of phytopathogenic fungi (botrytis cinerea, mycosphaerella fijiensis, pythium ultimum, phytophthora cinnamomi) showing partial or complete death or growth inhibition of those pathogens after 2 or 4 days of incubation. muscodor albus was reported from a diverse type of host plants, i.e., myristica fragrans, terminalia prostrata, cinnamomum zeylanicum, and ginkgo biloba, by several workers (worapong et al. 2001; sopalun et al. 2003; ezra and strobel 2003; mercier et al. 2004; ezra et al. 2004a, b; atmosukarto et al. 2005; lacey and naven 2006; lacey et al. 2009; strobel et al. 2007; banerjee et al. 2010a, b; corcuff et al. 2011; alpha et al. 2015) . the mycofumigants are effective against pathogen fusarium culmorum, causal agent of seedling blight, foot rot, ear blight, stalk rot, and common rot of cereals. sexual stage (teleomorph) of glomerella cingulata, a fungus of glomerellaceae, is a potent pathogen causing anthracnose-like symptoms of water-soaked, sunken spots and necrotic lesions on fruits of forest trees. this phytopathogen is strictly inhibited by the volatile emissions of this novel endophyte. banerjee et al. (2010a, b) first reported muscodor albus strain gba from the usa as an isolate of ginkgo biloba (first isolate of m. albus from g. biloba) and tested the biological efficacy of its volatile mixtures against agricultural pathogens and also evaluated its promises to be used as a commercial mycofumigant agent for controlling the fungal diseases in storage fruits and vegetables, that is, agricultural productions and during food transportation. the strain gba in comparison to other strains of muscodor e6 and cz620 completely inhibits and potentially kills the member of phycomycetes, pythium ultimum after 2 days of exposure of the mixture of volatiles. the organic compounds include alcohols, acids, esters, ketones, and lipids as their active components. 1-butanol, 3-methyl-, acetate was found in significant quantities. vitrine, a terpenoid, was first isolated from muscodor albus strain gab. the volatile mixture is artificially produced by the mixture of the pure compounds, and that mixture is again checked for antifungal activity. a positive mycocidal or mycostatic effect similar to the effect of endophyte's volatile emission will confirm establishment of the endophyte and its mixture as the biocontrol or antifungal agent. myrothecium inundatum, an endophyte of herbaceous acalypha indica (euphorbiacea member collected from northeastern part of india), produces unique mixture of volatile components having 3-octanone, 3-octanol, 7-octen-4-ol, sesquiterpenes, organic acids, methyl esters, naphthalene, 2-octanoic acid, heptanoic acid, etc. this endophyte produces foam in its liquid culture predominant with long-chain carbon compounds like octane, 1,4-cyclohexadiene, 1-methyl-and cyclohexane, and 1-ethylpropyl. (meshram et al. 2013 (meshram et al. , 2017 suwannarach et al. 2015; saxena et al. 2015; suwannarach et al. 2010 suwannarach et al. , 2012 suwannarach et al. , 2013 kudalkar et al. 2012; mitchell et al. 2010; worapong et al. 2002; daisy et al. 2002; siri-udom et al. 2016 postharvest fungal disease is one of the prime causes of agricultural loss of crops. use of biological agent to minimize this loss is one of the vital targets of agriculturalists, horticulturalists, and plant biologists. several chemical agents have been already tested for practical applications, but endophytes are less explored organisms in this arena. volatiles from endophytic source open up new scope of utilization of unique mixtures of chemicals to be used as mycofumigant agents. a large number of endophytes have already been screened for their postharvest disease management ability (table 9 .4). the volatiles of the endophyte could be considered as the natural fungicides. muscodor vitigenus, an endophyte of hevea brasiliensis, was analyzed in gc-ms for their volatile production. the isolates produce a unique mixture of myroxylon balsamum 1, 4-cyclohexadiene, 1-methyl-; 1,4-pentadiene and cyclohexene, 1-methyl-4-(1methylethenyl)-; alkyl alcohols starting with 1-butanol-3-methyl, 1-propanol-2-methyl, cinnamomum bejolghota. this isolate was known to produce azulene, a new compound detected first from any muscodor species. this species was tested in vitro and in vivo for antifungal activity against a common worldwide devastating pathogen rhizoctonia solani (causal agent of damping off). the vocs produced by this fungi include (s)-(+)-5-methyl-1-heptanol; ethyl acetate; propanoic acid, 2-methyl-, methyl ester; cis-2,4-dimethylthiane; s,s-dioxide; cyclopentane; butanoic acid, 2-methyl-, methyl ester; 1-butanol, 3-methyl-, acetate; β-humulene; azulene, 1,2,3,5,6,7,8,8a-octahydro-1,4-dimethyl-7-(1-methylethenyl)-; 1s-(1.α., 7.α., 8a.β); and eudosma-4(14),11-diene 1,1,1,5,7,7,7-heptamethyl-3,3-bis(trimethylsiloxy) tetrasiloxane. rhizoctonia solani-infected seedlings were treated with volatile mixtures to assess the mycofumigation property. in vivo experiment was conducted on four seedlings of bird pepper, bush bean, garden pea, and tomato. it was concluded that 30 gm of muscodor cinnamomi prepared on rye grain solid media is the minimum dose required for inhibition of rhizoctonia infection and total control and elimination of damping off symptoms. muscodor cinnamomi-infected soil does not show any seed germination inhibition in comparison to rhizoctonia solani-infected soil. so it is a type of pioneer study of using endophytic species as potent agents of fumigation and biocontrol. candida intermedia strain c410 (saccharomycetaceae) was isolated as an endophyte of strawberry (fragaria ananassa), and the volatile emission was known to be a mixture of 49 organic compounds including esters, alcohols, alkenes, alkanes, alkynes, organic acids, ketones, and aldehydes of which 1, 3, 5, 7-cyclooctatetraene and 3-methyl-1-butanol were the most dominant (huang et al. 2011 ). volatiles of strawberry endophyte were itself useful as postharvest control agent for the host plant against botrytis fruit rot. other compounds include 1,3,5,7-cyclooctatetraene; 3-methyl-1-butanol; 2-nonanone; pentanoic acid, 4-methyl-, ethyl ester; 3-methyl-1-butanol, acetate; acetic acid, pentyl ester; and hexanoic acid, ethyl ester that were found to be extremely inhibitory to conidial germination (reproductive growth) and also vegetative (mycelial) proliferation of b. cinerea. when the fruits are exposed to c. intermedia synthetic volatiles or itself to the fungus, the incidence of botrytis fruit rot reduces significantly. strawberry fruits inoculated directly with the endophyte also remain disease-free. so mixtures of candida intermedia c410, the unique natural products, are useful as mycofumigation technique or for postharvest disease management by biological control policies. tangerine fruit (citrus tangerine), the commercial citrus crop of northern thailand, faces huge postharvest losses due to pathogenesis of green mold (penicillium digitatum). the pathogen is the prime cause of worldwide deterioration of tangerine fruits by mycopathogenesis. out of 32 detected compounds, the most predominant were 2-methylpropanoic acid and 3-methylbutan-1-ol. other compounds include carbitol, octanoyl chloride, azulene, 3-methylhexane, 2-methylpropan-1-ol, 2,3-butanediol, caryophyllene, 2-methylbutyric acid, ethyl 2-hydroxyproponate, etc. the pathogen was treated both in vitro and in vivo for their inhibition by endophytic volatile components. in both cases, pathogen growth was restricted. during transportation of the fruits, fungus causes huge crop loss by infecting the fruits; when fruits were inoculated with 30 gm of rye grain culture of m. suthepensis (1 month old), the disease development is ceased. so it is a classic example of mycofumigation by the biocontrol agent of tangerine fruit for the control of rot lesions caused by p. digitatum infection. the in vivo application requires the proper surface sterilization (using sodium hypochlorite) of the targeted parts where the inoculation is going to be done, for example, fruit, stem, root, and leaf. usually infection on fruits for assessing the biocontrol potential is the most common and popular method. the seeds will be washed in distilled water, and using sterile needle, uniformly the whole area would be done, and the whole area would be infected or inoculated with the endophytic liquid extracts containing spore suspensions. muscodor albus vocs are potent enough to cause a significant reduction of in vitro spore germination of the tilletia species t. horrida, t. indica, and t. tritici. endophytic nodulisporium spp., trichoderma spp., phomopsis spp., and oxyporus latemarginatus are reported to produce vocs that inhibit mycelial growth of phytopathogenic fungi (lee et al. 2009; park et al. 2010; ajith and lakhsmidevi 2010; amin et al. 2010) . black sigatoka disease (also known as leaf spot or black leaf streak disease) of banana (musa paradisiaca) is caused by mycosphaerella fijiensis (ascomycete fungus). this phytopathogen is inhibited by the volatile emissions of muscodor sutura, an endophytic isolate of prestonia trifidi. the volatiles are effective also against ceratocystis ulmi, the causal agent of dutch elm disease of american elm (ulmus americana). the volatile mixtures include thujopsene, chamigrene, isocaryophyllene, and butanoic acid, 2-methyl-that are potent inhibitors of the common anthracnose pathogen of cucumber, muskmelon, and watermelon (members of cucurbits), colletotrichum lagenarium. so this unique endophyte and its chemical mixtures are potent mycofumigants and ensure crop protections against destructive pathogens like c. ulmi and c. lagenarium, sclerotinia sclerotiorum (causing white mold, cottony rot, water soft rot, stem rot, drop, crown rot, and blossom blight diseases of the host), and also phytophthora palmivora (oomycete fungi), the causal agent of bud root of palms and areca nut predominantly occurring in regions of south india (kudalkar et al. 2012) . liarzi and his coworkers tested the biological control efficacy of the endophytic daldinia cf. concentrica, isolated from olive tree (olea europaea l.) of israel against 18 phytopathogens, and the unique mixtures of 27 volatile were effective against the phytopathogenic mycelial growths. the mixtures include a variety of organic compounds: 3-methyl-1-butanol, 2-methyl-1-butanol, 1-methyl-1,3-cyclohexadiene, 1-methyl-1,4-cyclohexadiene, 4-heptanone, isoamyl acetate, 4-heptyn-2-ol, 2-octenal, octanal, β-elemene, α-guaiene, β-selinene, α-selinene, α-bulnesene, germacrene a, etc. the unique mixtures having broad-spectrum antifungal property could be used for fumigation for eliminating the pathogenic infections of aspergillus niger (mold-causing organism on fruits of economic importance). so the endophytic d. cf. concentrica opens up opportunities for fungal disease control in food and agricultural industries (liarzi et al. 2016) . nodulisporium sp. strain gs4d2ii1 (hypoxylon anthochroum) and hypoxylon anthochroum strain blaci are potent enough to be used as biopesticide against fusarium oxysporum, a common contaminant of solanum lycopersicum var. cerasiforme (cherry tomato) causing a great percentage of crop loss globally. six vocs of alcohols' mixture, phenylethyl alcohol, 2-methyl-1-butanol, 3-methyl-1-butanol, eucalyptol, ocimene, and terpinolene, were detected and applied together with synergistic effect and individually both in vitro and in vivo. inoculation of pathogen on the cherry tomato fruits yields significant reduction in fusarium contamination. both agar dilution techniques and gas test were done to assess the in vitro antifungal activity, and the endophytic volatile mixtures were effective in both the cases. volatiles kill the pathogens probably by interfering cell membrane permeability, hyphal morphology, and respiratory activity of the pathogenic fusarium oxysporum. so it is a great opportunity to use the unique mixture of volatile organic compounds of the endophytic isolate to reduce the crop loss caused by the pathogenic infection on the commercially valuable plant of cherry tomato worldwide. endophytic phoma sp. (didymellaceae) and phomopsis sp. (valsaceae) were isolated from larrea tridentata and odontoglossum sp. singh et al. 2011) . the volatiles detected are effective against phytopathogens verticillum sp., ceratocystis sp., cercospora sp., sclerotinia sp. sclerotinia sp., and botrytis sp. algae are diverse group of autotrophs and the leading producers of o 2 in the ecosystem. they range from prokaryotic unicellular to eukaryotic complex multicellular forms involved in the marine and terrestrial food chain. antifungal activity of the seaweed (members of phaeophyceae and rhodophyceae) is a major weapon for natural fungicides along with their antibacterial, anti-protozoan, and antiviral activities. algal seaweeds are potent holders of large number of secondary metabolites including phenolics, terpenes, alkaloids, and lectins which are not directly involved in photosynthesis and reproduction and thus fall under the category of secondary metabolites. they are common antimicrobial of algal origin that act on the target organisms by altering the microbial cell permeability accompanied with the loss of internal macromolecules or sometimes interfere with the membrane function causing cellular disintegrity ultimately leading to cell death (abu-ghannam and rajauria 2013). several studies include antifungal activity of algal members against human pathogens; a very few studies include their efficacy against plant pathogens (cheung et al. 2014; singh et al. 2007; stirk et al. 2007; padmakumar and ayyakkannu 1997; ismail et al. 2014; genovese et al. 2013; lopes et al. 2015 ). padmakumar and ayyakkannu tested 80 species of algae against a variety of bacterial and fungal pathogens. out of the all screened organisms, 70% exhibited antibacterial efficiency, and only 27.5% inhibited fungal growth. polysaccharides found in the cell wall and deposited in terms of storage food from red and brown algal sources include ulvans (obtained from ulva sp.), alginates and fucans (from fucus sp.), laminarin (laminaria sp.), and carrageenans that can induce defense responses in plants against phytopathogens by pathogen-associated molecular patterns (maps) and are capable of inducing plant resistance (vera et al. 2011) . polysaccharides stimulate regular cellular changes associated with pathogen perception and defense activation by change in ca 2+ concentration and burst due to oxidative stress activation of salicylate, ethylene, and jasmonate biosynthetic pathways and by activating pathogenesis-related proteins (prps) (jaulneau et al. 2010; zhao et al. 2012) . as a result of the depolymerization of the polysaccharides, the obtained oligosaccharides induce protection against a variety of fungal, viral, and bacterial diseases by accumulation of the antimicrobial compounds in the cell. algal polysaccharides as an alternative weapon over the synthetic agricultural drugs for controlling plant disease have been widely studied (stadnik and freitas 2014; hahn et al. 2008 ). brown algae laminaria digitata, a genus of phaeophyceae, is commonly called seaweeds and known to be the potent producers of kelp, an iodine-rich substance needed for the normal functioning of thyroid gland. laminaria produces laminarin, glucan polysaccharide-containing 1,3-linked β-d-glucose moiety, a reserve food material found on the vacuoles of the vegetative cells of this genus. β-glucans are involved as a major part of daily diet and obtained from the brands of common cereals. they are involved in the defense responses of agricultural crops like tomato (lycopersicon esculentum), eggplant (solanum melongena), pepper (piper nigrum), watermelon (citrullus lanatus), grape (vitis vinifera), apple (malus sp.), and pear (pyrus communis). elicitation of defense response by laminarin against causal agents of gray mold (botrytis cinerea) and downy mildew (plasmopara viticola) in grapevine plants remarkably suppresses their infection up to 55% and 75%, respectively (copping et al. 2004 ). so, natural product from brown algae laminaria sp. known as laminarin or laminaran can act as the biofungicide or biocontrol compounds. use of laminarin significantly reduces the mycelial growth and aflatoxin production in aspergillus flavus and ensures its use as a fungicide (liangbin et al. 2012) . the advantage of using laminarin over other products is that as it breaks down finally to glucose molecules, it has no maximum residue limit (mrl) on the plant treated with this product. so, there is no need of preharvest interval constraint. this has been a prime cause why laminarin has substituted five popular fungicides involved in the treatment of apple scab (venturia inaequalis) in france (mery et al. 2013 ). this phyto-pharmaceutical is used widely in france and some countries of europe in the name of vacciplant (major active constituent is laminarin). laminarin has broad-spectrum applicability on fire blight of apples and pears in greece, france, belgium, switzerland, portugal, and also morocco. it is effective for apple scab disease in france and belgium and for curing storage diseases of apples caused by gloeosporium sp. in belgium. laminarin comes out as a fungicide of natural origin after being eligible in 33 tests between 2001 and 2011 in several parts of europe, for example, france, belgium, italy, and poland, on natural contamination of orchards on several sensitive strains of scab fungus including golden delicious, golden smoothie, read cheaf, galaxy, gala, and pink lady. laminarin is applied widely against secondary scab (to minimize secondary scab during summer and up to harvest) as a result of its uniqueness in its mode of action. it does not involve cell death of the host plant or hypersensitivity induction in the host organism but rather stimulates plants' natural resistance (klarzynski et al. 2000) . aziz et al. (2003) reported its effectiveness in tobacco plants, wheat, strawberries, apples, and vines. the application of laminarin and alginate reduced the development of wilt symptoms caused by verticillium dahliae on olive twigs, stimulating its phenolic metabolism (salah et al. 2018) . moreover, alginates reduced pathogen growth in vitro. laminarin induces the release of h 2 o 2 in cells of tobacco plants and leads to the increase in pal activity (phenylalanine ammonia-lyase) and causes the accumulation of pr-1, pr-2 (glucanase), pr-3 (chitinase), and pr-5. concerning red algae polysaccharides, carrageenans induced protection against a broad range of pathogens such as tobacco mosaic virus (tmv), b. cinerea, and e. carotovora on tobacco (vera et al. 2011) . again on tobacco, mercier et al. (2001) showed that carrageenan infiltrated the leaves and increased the expression of genes coding for a sesquiterpene cyclase involved in the synthesis of the antimicrobial terpenoid capsidiol, pr-3 proteins (basic chitinases), and proteinase inhibitor with antipathogenic activity. an adequate percentage of growth and spore germination inhibition of botrytis cinerea was mediated by the hexane extracts of laminaria digitata and undaria pinnatifida. porphyra umbilicalis, laverbread, is an edible seaweed (corato et al. 2017) . other than b-glucan polysaccharides (laminarin) of laminaria, other secondary metabolites (phenols, terpenes) of phaeophycean algae (sargassum sp.) showed effectivity against common pathogens fusarium solani, rhizoctonia solani, aspergillus spp., fusarium oxysporum, penicillium spp., and botrytis cinerea (khallil et al. 2015; ibraheem et al. 2017; mabrouk et al. 1985; liu et al. 2014 ). cyanophycean blue-green algae are abundant all over the world and ranging from pond ecosystem to oceanic system. though they have been reported to produce a large number of toxins and involved in death and disease of cattle and human being, they are of serious interest from the point of view of natural fungicidal products. drawing the similarities with bacteria, they are characterized with a mucilaginous or gelatinous sheath composed of polysaccharides which are the weapon against fungal pathogenesis. cyanobacterial polysaccharides (pol) show higher disease resistance against b. cinerea when they are applied on the intact fruit (preharvest conditions when fruit is attached to the plant) rather than the fruit detached (postharvest conditions) from the plant (zheng et al. 2011; feliziani et al. 2015; yao and tian 2005) . polysaccharides are involved in elicitation as elicitors for development of local and systemic disease resistance and expression of defense enzyme synthesis, for example, chitinases and glucanases that are involved directly in antifungal responses (paulert et al. 2009; reymond and farmer 1998; sharma et al. 2014) . water extracts of common bga anabaena sp., ecklonia sp. (common edible marine algae of japan and korea), and corallina sp. (hard seaweed of corallinaceae family) exhibit antifungal activity against podosphaera xanthii (causal agent of powdery mildew of cucurbits) on zucchini plant, cucurbita pepo, of cucurbitaceae (roberti et al. 2015 (roberti et al. , 2016 . in the recent past, fungi inhibitory ability of algal members has been reported by several workers (righini et al. 2018; corato et al. 2017; khallil et al. 2015; ibraheem et al. 2017) . in vitro growth inhibition of aspergillus oryzae and penicillium notatum has been seen by cyanophycean anabaena laxa (frankmölle et al. 1992) . devastating plant pathogens pythium sp., fusarium sp., and rhizoctonia sp. were restricted by extracts of anabaena sp. (moon et al. 1992; manjunath et al. 2010) . the use of bga extract as the growth inhibitor of pathogenic chaetomium globosum, cunninghamella blakesleeana, aspergillus oryzae, rhizoctonia solani, fusarium sp., pythium sp., and sclerotinia sclerotiorum is reported. the extracts of phormidium fragile and nostoc muscorum (rizk 2006 bryophytes, the simplest member of the broad umbrella of embryophyta, are situated between algae and pteridophytes, are known to be plant amphibians growing in the marshy or shady habitat, and require water for their fertilization and for the perfect swimming motility of their sperms. they have been evaluated for their antimicrobial activity for a long time. it has been proved that these cryptograms are rich source of bioactive secondary metabolites and can easily be exploited as an alternative source of fungicidal compounds. as they grow in marshy habitats and can protect themselves from biotic (ultraviolet rays, heat stress, and predation) and abiotic stress (fungal or bacterial attack), they are store house of diverse bioactive chemicals (xie and lou 2008) . members of hepaticopsida and mosses (the evolved members of bryophytes) are known to possess antifungal activity and are rich source of flavonoids, terpenoids, bibenzyls, and fatty acids of therapeutic importance (krzaczkowski et al. 2008) . bryophytes are known to possess antibiotic property (banerjee and sen 1979; banerjee 2000; singh et al. 2007; shirzadian et al. 2009; savaroglu et al. 2011) . their antibiosis has been evaluated against a large number of plant and human pathogenic fungus (mekuria et al. 2005) . antimicrobial compounds from bryophyte can cure the problems of conventional antibiotic resistance (vanden bossche et al. 1998) . the antifungal efficacy is tested by disc diffusion assay and microdilution method ( fig. 9 .5). different concentrations of the extracts are prepared and checked for their antifungal efficacy against phytopathogenic fungi. they may be fungicidal or fungistatic in nature, interfering at cellular, genetic level and creating blockage at metabolic pathways. extracts are made on several organic solvents or water extractions and also mixture of one or two organic solvents. the solvents popularly used are ethanol, methanol, chloroform, ether, dimethyl sulfoxide (dmso), acetone, chloroform, and hexane (table 9 .5). sporophytes and gametophytes of different bryophytes at different stages of growth and at a different amount are first surface sterilized and then crushed on the organic solvents and used as antifungals in vitro against the fungal pathogens (wolters 1964 (sabovljevic et al. 2011; pejin et al. 2012; veljic et al. 2009; gahotri and chaturvedi 2011; alam et al. 2011; deora and jain 2008; dey and de 2011; deora and suhalka 2017) . actinobacteria are a group of gram-positive filamentous bacteria that are called as the branched bacteria or ray fungi (from greek actis, ray beam, and mykes, fungus) and are characterized with the high g + c content occurring in mostly aerobic conditions but occasionally being anaerobes (ludwig and klenk 2005; olanrewaju and babalola 2019). their morphology varies from forming branching filaments or mycelial growth to external spores. they are ubiquitous in nature ranging their distribution from soil and human microbiota to plant and even animal kingdom. they are predominant in aquatic as well as terrestrial ecosystem playing a major part in mineralization and recycling of organic matters leading to soil formation (sharma et al. 2014 ). they are not only free-living members of the ecosystem but also a plant symbiont or endophyte, contributing to the plants' survival in extreme conditions and pursuing several bioactivities in vivo and in vitro. actinomycetes produce a diverse range of secondary metabolites, for example, antibiotics, antitumor, insectrepellent, and immunosuppressive agents, and plant growth-promoting regulators (pgprs) that are of immense pharmaceutical and agricultural importance. they are the prime producers of diverse antibiotics after the landmark discovery of penicillin in the year 1928. the single genus of streptomyces sp. itself produces 76% of the total known bioactive (10,000 are produced by actinobacteria out of 23,000 produced by microorganisms, almost 45%) compounds from actinobacterial and riccia gangetica curvularia lunata deora and suhalka (2017) , guhil (2015, 2016) bacterial source (berdy 2012) and is known to be the prime organism in the pharmaceutical world. they are equally profitable when isolated from plant source and designated as endophytic actinomycetes. so exploitation of the actinobacterial novel bioactive compounds both from endophytic and non-endophytic source is the ultimate way to fight against human and plant diseases. here we focus only on actinobacterial compounds' antifungal activity and role in plant protection from deadly diseases caused by severe phytopathogens leading to irreparable crop loss and economic breakdown of agricultural sectors. actinomycetes from soil source are selected based on the enrichment culture technique and are plated on selective media for isolation. antifungal agents, for example, nystatin and cycloheximide, are supplemented for the inhibition of fungal contamination. for isolation of endophytic actinobacteria from plant source, plants are first selected and surface sterilized for the elimination of the epiphytic contaminants and finally plated on the selective growth media like starch casein nitrate agar (scna), chitin-vitamin b, tap water yeast extract agar (twya), soybean, humic acid-vitamin b (hv), yeast extract casamino acid (yeca), modified gausse, and glycine-glycerol (ivantiskaya et al. 1978; küster 1959; küster and williams 1964; williams and davies 1965; hayakawa and nonomura 1987; crawford et al. 1993) . international streptomyces project (isp) medium is also popular media used for isolation, and they are supplemented with amino acids (l-asparagine for isp 5, tryptone for isp 1), inorganic trace salts, starch or carbohydrate sources (malt extract for isp 4), and agar as solidifying agent. ph set at near to optimum or slightly basic is mandatory for proper isolation techniques using isp medium. the actinomycete isolates are grown in solid or liquid medium for their antifungal bioactivity detection. antagonistic activities of the potent isolates are tested by growing them on both sides of the fungal hyphae, and isolate having anti-phytopathogenic activity will inhibit the growth of the pathogens. actinobacterial aqueous-or solvent-based extracts will be evaluated for either fungistatic or fungicidal activity by agar welldiffusion techniques. soluble bioactive compounds of antifungal importance will be extracted using wide range of organic solvents followed by purification by column and thin-layer chromatographic techniques. hplc analysis will be the most useful method for the detection of the purity of the compound, and further nmr studies are needed for the proper identification of the bioactive compound. cell line studies are made with the coupling of bioinformatics tools for the proper knowledge about their mode of action. actinobacteria can be a part of plant as endophyte, rhizospheric soil as symbiont for plant growth-promoting substance producer, and organisms' normal microbial flora as gut microorganism. so they are ubiquitous in their distribution. out of several biologically potent compound produced from the actinobacterial source, antibiotics are the major contribution of these microorganisms toward human civilization. all the known antibiotics (blasticidin, mildiomycin, natamycin, validamycin, kasugamycin) are of actinobacterial (most of them are the streptomyces sp.) source showing protective activity for the plants against agricultural fungal pathogens (tables 9.6 and 9.7). as human are dependent completely on nature and more particularly natural components of agricultural origin and importance, dependence on agricultural crops is of a known fact. but the problem arises when the crops are affected most by the fungal pathogens leading to huge crop loss, and thus the search for novel antibiotics is on, and the search has shifted to actinobacterial source, and endophyte plays an important role in this respect. there are significant reports of antifungal compounds from bacterial origin, but now the focus has shifted to microbes of endophytic origin (table 9 .8). till date, a huge number of antibiotics are already reported and have minimized the crop loss to a notable amount ( fig. 9.6 ). antibiotics and other antifungal compounds include munumbicins a, b, c, d, e-4, and e-5, vanillin, saadamycin, 5,7-dimethoxy-4-p-methoxyphenyl coumarin, coronamycin, and fistupyrone isolated from different strains of streptomyces (shan et al. 2018; costa et al. 2013; igarashi et al. 2002; tian et al. 2004; zin et al. 2007 ) and are protecting a large number of cereals and other important cash crops from being affected by these common contaminants. endophytic actinobacteria directly counteract with fungal plant pathogens not only by producing bioactive compounds but also by enhancing the plant's growth through the production of plant growth promoters and making the plant less susceptible to pathogenic invasion. they are efficient agent of reducing the symptoms that arise due to exposure to environmental stress (shimizu 2011) . enhanced production of indole acetic acid (iaa) was mediated by streptomyces sp. (isolated from centella asiatica) and nocardiopsis sp. (dochhil et al. 2013; shutsrirung et al. 2014; gangwar et al. 2014) . experimental trials on cucumber indicate positive result as the isolates actinoplanes campanulatus, micromonospora chalcea, and streptomyces spiralis enhanced plant growth and improved yield conditions (el-tarabily et al. 2010) . other than auxin, auxin-like similarly functioning molecules named as pteridic acids a and b are found to be inducers of adventitious root proliferation in kidney bean plants at very minute concentrations of 1 mm (igarashi et al. 2002) . chitin is a major fungal cell wall polysaccharide (the second most abundant polysaccharide in nature after cellulose) component and is the first line of defense of fungal cells. actinobacteria antagonize the fungal cell by producing chitinases (an enzyme capable of hydrolyzing fungal cell wall) and break the glycosidic bonds in chitin and lead to the death of the pathogenic cell. endophytic kitasatosporia sp. (isolate of catharanthus roseus) and kibdelosporangium sp. (isolate of achillea fragrantissima) are reported to be chitinase producers (el-shatoury et al. 2009; mini priya 2012) . actinoplanes missouriensis isolated from lupinus sp., a member of fabaceae family, produces chitinase causing hyphal cell lysis and reducing the conidial germination rate and protects the plant from pathogenic attack of plectosporium tabacinum, the causal agent of lupin root rot in egypt (el-tarabily 2003; el-tarabily and sivasithamparam 2006) . siderophores are soluble, small, high-affinity iron carriers produced by bacterial or fungal members and are involved in the transportation of iron (fe 3+ ) across the cell membrane. they have caught sudden attention due to their involvement in plant growth promotion as well as antagonistic ability against phytopathogens (cao et al. 2005; tan et al. 2006; rungin et al. 2012) . endophytic actinobacteria from aloe vera, mentha arvensis, and ocimum sanctum are known to be producers of hydroxymate type and catechol type of siderophores, and the isolate saccharopolyspora o9 is known to be the potent inhibitor howell and stipanovic (1980) , homma et al. (1989) , thomashow et al. (2002) and smith et al. (1993) harpin proteins (erwinia amylovora), trade name: harpin αβ (proact) induction of systemic acquired resistance (sar) and less susceptibility to fungal and bacterial disease wei et al. (1992) strobilurin and oudemansin (members of basidiomycete grows on dead wood) commercial synthetic analogues: azoxystrobin and kresoxim-methyl of alternaria brassicicola, botrytis cinerea, and fusarium oxysporum (gangwar et al. 2014; el-shatoury et al. 2009 ). endophytic isolates of cucumis sativus (cucumber), identified as actinoplanes campanulatus, micromonospora chalcae, and streptomyces spiralis, are reported to control the growth and development of damping off, crown rot, and root rot pathogen pythium aphanidermatum. they are known to promote plant growth and to protect seedlings and mature plants. a novel bioactive compound identified as 6-prenylindole was isolated from endophytic streptomyces sp. showing strong antifungal activity against a broad range of phytopathogens: alternaria brassicicola and fusarium oxysporum (igarashi 2004) . another new prenylated indole derivative from endophytic actinobacterial source inhibited the growth of colletotrichum orbiculare, phytophthora capsici, corynespora cassiicola, and fusarium oxysporum (zhang et al. 2014) . naphthomycins a and k isolated from streptomyces sp. cs have antifungal activity against penicillium avellaneum shen 2003, 2007) . biocontrol ability of fistupyrone has made it a useful tool to minimize the crop loss of brassica due to black leaf spot disease caused by alternaria brassicicola (igarashi 2004) . interest on actinomycetes of endophytic origin as an alternative tool for antifungal agent is increasing day by day (table 9 .9). since the beginning of human civilization, whenever human race has faced any turbulence in its path of existence, they have rushed to their green friends, trees, for the ultimate solution. search for bioactive products of medical importance has been a thirst area from time immemorial. whether it is a concern of human or plant health, trees have given answers in all aspects. in the recent past, phytopathogenic infection has pushed the agricultural productive parameters to a real challenge, and plant extracts in its crude and purified form are applied as biocontrol methods (table 9 .10). the existing synthetic chemicals are facing problem of immediate or delayed drug resistance and also issues of nephrotoxicity (the gold standard; amphotericin b), biomagnification, or quality assurance of the food products and thus are inconsistent in their business (goa and barradell 1995; cuenca-estrella et al. 2000) . so green plant extracts are the novel, safest, and the best effective treatment tool in this arena. plants are mysterious in their chemical nature and in respect to their secondary metabolite production. the faith is consistent on green plants due to the fact that plants protect themselves from fungal or bacterial diseases specially for the taxa that occur in marshy shady or water-logged or stress conditions (gurgel et al. 2005) . so the search is primarily made on the wild native taxa or invasive species that have higher potential of antimicrobial production. the knowledge of ethnobotany comes in this context, and tribal people are imitated for the gathering of crude knowledge. the problem of fungal pathogenesis is mainly faced by plants of economic importance, that is, cash crops. a single event of pathogenic attack can affect seriously the demand and supply ratio; thus the sustainability is lost, and restoring the good health of crops is a basic need of agricultural sectors but in an efficient way not hampering the soil health, ecosystem characters, and human health and also should be budget friendly. the search is strictly focused on plants of ethnomedicinal importance as history indicates the ability of medicinal plant extracts in human and animal mycoses and antifungal ability (mathias-mundy and mccorkle secondary metabolites are plants' best weapon against phytopathogenic invasion. several plant extracts have been assessed for their antifungal activity against a variety of phytopathogens of serious agricultural threats (table 9 .11). the metabolites are divided into terpenoids, saponins, phenolic compounds, flavones, flavonoids, flavonols, alkaloids, and coumarins (table 9 .12). plant extracts are primarily tested for antifungal efficacy and further are purified by solvent extraction and chromatographic procedures leading to discovery of new antifungal agents. terpenoids, also called as isoprenoids (under the chemical subclass of prenyllipids), are known to be the oldest group of widespread molecular compounds produced by plants. scher et al. (2004) reported a variety of six sesquiterpenes of antifungal importance against the causal organisms of bunch rot (botrytis cinerea) on grapes, scab of cucurbits (cladosporium cucumerinum), potato blight (phytophthora infestans), rice blast (pyricularia oryzae), and blotch of wheat (septoria tritici). sesquiterpene isolated from polygonum punctatum (dotted knotweed of knotweed family polygonaceae) named after the chemical polygodial is an effective control agent of zygosaccharomyces bailii (a common food spoilage yeast). scab of cucurbits is a common and devastating fungal pathogenic disease in agricultural fields, and this disease is to some extent prevented by the use of clerodane diterpenes extracted from detarium microcarpum, a plant of leguminosae family (cavin et al. 2006) . skaltsa (2000) reported fungi inhibitory (cunninghamella echinulata) activity of costunolide and eudesmane derivatives isolated from centaurea plants. other than terpenes, saponins (triterpene ad steroidal saponins) are also effective antifungals reported from plant sources. tea is one of the most vital cash crops in terms of foreign money earning and the most popular beverage having antioxidative properties. pathogenic infection by pestalotia longiseta causes a huge loss of tea production. nagata et al. in the year 1985 isolated triterpenoid saponins camelids i and ii from the leaves of camellia japonica (japanese camellia) that inhibited the tea pathogen p. longiseta. cucurbitacins i, a, b, q, and e isolated from cucurbitacins (ecballium elaterium) have antifungal activity against botrytis cinerea (har-nun and meyer 1990) . phenolics are odorous compounds having antifungal compounds and are also responsible for the plant pigment production. phenolics cover a large number of chemical compounds, for example, alkylated phenols, anthraquinones, coumarins, phenolic acid, phenols, phenylpropanoids, quinines, xanthones, hydroxycinnamic acid, p-coumaric acid, ferulic acid, and chlorogenic acid. phenol derivatives like crassinervic acid (p. crassinervium), aduncumene (p. aduncum), hostmaniane (p. hostamannianum), and gaudichaudanic acid (p. gaudichaudianum) are effective against strawberry blossom blight pathogen cladosporium cladosporioides (lago et al. 2004 ). 3-acetyl-4-acetoxyacetophenone showed antifungal activity against spendley et al. (1982) , potterat et al. (1987) , martson et al. (1988) , marston et al. (1993) , viturro et al. (2004) , cavin et al. (2006a) , and dhatwalia et al. (2009) pinocembrin from leaves of populus deltoides (salicaceae) shain and miller (1982) , hoof et al. (2008) cladosporium fruit and leaf rot and bitter root (cladosporium gloeosporioides) long-chain alcohol from peels of young fruit of persea americana from lauraceae, methylripariochromene a from roots of eupatorium riparium (asteraceae) prusky et al. (1983) , ratnayake bandara et al. (1992) pine needle pathogen (dothistroma pini) stearic acid from needles of pinus radiata (pinaceae) franich et al. (1983) pathogen of corn, sorghum, apple (helminthosporium carbonum) luteone and wighteone from leaf surface of lupinus albus (leguminosae) ingham et al. (1983) black and brown spot of banana (colletotrichum musae) dopamine from unripe banana fruit (musa sp.) muirhead and deverall (1984) powdery mildew of grains (erysiphe graminis) gramine from leaves of hordeum vulgare (poaceae) wippich and wink (1985) leaf spot, rots, and blights (alternaria alternata), disease of cereal (penicillium verrucosum) alizarin and emodin from root of rubia tinctorum of rubiaceae, alkylated phenols of peel and pulp of mangifera indica (anacardiaceae) cojocaru et al. (1986) , manojlovic et al. (2005) maize rot (fusarium moniliforme), epidemic outbreak of glume and kernel discoloration (curvularia lunata) flavan-4-ols of root bark of sorghum cultivars of poaceae jambunathan et al. (1986) (continued) kobayashi et al. (1987) , endo et al. (1990) , cho et al. (1998) blue mold of tobacco (peronospora tabacina) diterpenoids from nicotiana tabacum of solanaceae reuveni et al. (1987) leaf and fruit pathogen (cladosporium cladosporioides) canaliculatol from bark of stemonoporus canaliculatus and long-chain alcohol from persea americana, phenylethanone from euodia lunuankenda, sinharine and methylsinharine from glycosmis cyanocarpa, illukumbin from glycosmis mauritiana (rutaceae), phenylethanone from euodia lunuankenda (lauraceae), benzoquinone from croton lacciferus (euphorbiaceae) bokel et al. (1988) , ratnayake bandara and wimalasiri (1988) , kumar et al. (1990) , greger et al. (1992) , pacher et al. (2001) , springob and kutchan (2009) miles et al. (1991) , miles et al. (1993) , lee et al. (2003) , deng and nicholson (2005) and yoganandam et al. (2009) (continued) sclerotinia sp. phenolic structures when contain a carbonyl group are known to be flavones, and the addition of an extra 3-hydroxyl group indicates flavonol. flavonoids are also known to hydroxylated phenolics but occurring as a c6-c3 unit linked to aromatic ring. not only plant samples directly but also plant derivatives like porpolis (galangin isolated from the bee glue or resinous mixture produced as a result of the mixture of tree buds, sap, botanical extracts, and bee exudates) are shown to be antifungal against green rot or mold of tangerine (pathogens penicillium digitatum, p. italicum) and also control postharvest disease of cereal grains, legumes, and tree nuts caused by a. flavus (afolayan and meyer 1997) . flavones (6,7,4′-trihydroxy-3,5′dimethoxyflavone, 5,5′-dihydroxy-8,2′,4′-trimethoxyflavone) from artemisia giraldi are effective against a. flavus infections (cowan 1999) . leaf wax of arrabidaea brachypoda (brazilian medicinal plant from bignoniaceae) contains herger et al. (1988) and abdu-allah and elyousr (2017) cassia tora (dealcoholized extract of leaves) mukherjee et al. (1996) thymol, carvacrol, citronellol, geraniol, citral, perillyl, menthol, eugenol, 1,8cirsiliol, cirsimaritin, and hispidulin and is showed to be effective against cladosporium sphaerospermum (alcerito et al. 2002) . galeotti et al. (2008) against fusarium oxysporum f. sp. dianthi (galeotti et al. 2008) . fusarium culmorum, a serous pathogen of seedling blight, foot rot, ear blight, stalk rot, and common rot of cereals and grasses, is found to be inhibited by six commercial coumarins: bergapten, herniarin, umbelliferone, xanthotoxin, and scopoletin. tithonia diversifolia, the source of tithoniamarin, is effective against the anther smut fungus microbotryum violaceum, earlier known as ustilago violacea (yemele-bouberte et al. 2006) . berberine and jatrorrhizine (alkaloids) are isolated from mahonia aquifolium (a plant of berberidaceae family commonly called as oregon grape and native to western north america) and are effective against human pathogenic candida species. pathogens of mango (c. gloeosporioides), anthracnose of lupin species, postbloom fruit drop of citrus, valencia and navel oranges in florida (caused by c. acutatum), and strawberry (caused by colletotrichum fragariae) are inhibited by findersine, anhydroevoxine, and haplamine (cantrell et al. 2005) . roots of cyathobasis fruticulosa are source of beta-carboline, tryptamine, and phenylethylamine-derived alkaloids and are antifungal in nature (bahceevli et al. 2005 ). essential oils (eos) of aromatic and medicinal plant origin are reported to possess antifungal properties and are of wide spectrum in their application for the control of agricultural pathogen (table 9 .13). eos are mainly categorized under the plants' secondary metabolites and may fall under the category of terpenes, ketones, esters, aromatic phenols, ethers, alcohols, oxides, etc. (fig. 9.7) . they act by inhibiting the fungal hyphal growth either by accumulating in the fungal cell membrane or by crossing the cell membrane and entering into the eukaryotic cell. being lipophilic in their chemical nature, they can easily cross the cell and interrupt in sterol biosynthesis leading to growth retardation and finally cell death. as sterols are the maintenance, compounds of cellular integrity treatment with eo cause fungal cell death. metabolic processes like respiration, replication, transcription, and translation are inhibited. membrane permeability is drastically changed as they cause swelling and disruption of protein-lipid-protein membrane. leakage of useful ions like ca 2+ and k + causes cell death. thymol, carvacrol, eugenol, and related phenolic compounds cause h + and k + leakage and water imbalance and deplete intracellular high-energy molecule (atp). essential oils are extracted from almost every parts of a plant, for example, roots, fruits, barks, twigs, leaves, seeds, and flowers, by several extraction procedures that include hydro and steam distillation, cold pressing, and zataria multiflora lamiaceae fermentation. the antifungal efficacy is checked by direct contact of the essential oil components and fungal hypha and poison food method, following micro or broth dilution techniques, or in vivo fumigation assay is also performed in case of field trials. essential oils from leaves of chenopodium ambrosioides, a member of amaranthaceae family, are effective against storage fungi aspergillus flavus, a. glaucus, a. niger, a. oryzae, colletotrichum gloeosporioides, c. musae, fusarium oxysporum, and fusarium semitectum (jardim et al. 2008) . lemongrass oil from cymbopogon citratus and cymbopogon martini are potent inhibitors of botrytis cinerea, rhizoctonia solani, aspergillus tamari, a. fumigatus, and a. conicus (tzortzakis and economakis 2007; mishra et al. 2015) . the members of lamiaceae family are well known for their pungent odor and are tested for their antifungal activity by agar and broth dilution methods (roby et al. 2013; omidbeygi et al. 2007 ). essential oils extracted from laurus nobilis, syzygium aromaticum, and origanum vulgare are effective antifungal compounds against two pathogens of rice, fusarium culmorum and fusarium verticillioides (rosello et al. 2015) . essential oils from cymbopogon exhibited antifungal activities against rot molds (soundharrajan et al. 2003) . antifungal activities of peppermint and sweet basil were tested against plant pathogenic fungi s. sclerotiorum, rhizopus stolonifer, and mucor sp. (edris and farrag 2003) . antifungal activity of β-dolabrin, γ-thujaplicin, and 4-acetyltropolone was tested against pythium aphanidermatum ifo 32440 (morita et al. 2004) . boyraz and ozcan (2006) tested the antifungal activity of the essential oils isolated from wild turkish summer savory (satureja hortensis). essential oils (carvacrol, thymol, p-cymene) extracted from origanum acutidens are effective against phytopathogens. growth of a. humicola, colletotrichum gloeosporioides, rhizoctonia solani, and phytophthora cactorum was inhibited by the essential oil of asarum heterotropoides var. mandshuricum (dan et al. 2010) . though there are several reports of essential oils being potent anti-phytopathogenic (penicillium purpurogenum, rhizopus stolonifer, spondylocladium austral, penicillium digitatum, penicillium luteum, monilinia laxa, curvularia lunata, etc.) in nature, still there are some problems regarding their maximum use and optimum effectivity. that includes their volatile natures, requirement of close systems, and degradation of eos by oxidation due to presence of extreme amount of hydrogenated compounds (kim et al. 2003 ). we are nourished by mother nature. so it is our prime duty to keep up the normal equilibrium of natural parameters. but in a way to seek solutions, some steps taken toward success may have negative impact on our environment. to fight against the fungal pathogens for the ensuring of better crop productivity, use of chemical fungicide is just another example of that fact. but we must emphasize on products from direct natural origin over the chemically synthesized one. natural products are the best weapon to fight fungal pathogenic diseases on economically important crop species. they are less toxic, stable, and of no side effects when used in crop fields. the crying need of modern era is obtaining pathogen-free crop species in one hand and assurance of environmental sustainability on the other. fungal and bacterial products are already used in large scales followed by the plants' secondary metabolites. phytoalexins as internal molecules are the plants' own defense system. the detailed biochemical analysis of the phytoalexins and study of their regulatory mechanisms are opening up new horizons for universal use of phytoalexin inducing elicitors as plant defense enhancers. mycorrhizae provide the basic line of physical barrier against pathogenic invasion, and reports include their ability to enhance plant growth, thus making the plant nonsusceptible to fungal attack. endophyte on the other hand can enhance the plants' defense system by direct incorporation and open up popular angles of green immunization or plant vaccination. researches on these fields are still scanty, but in the near future, they could lead to the ultimate solution of fungal pathogenic crop loss. effect of certain plant extracts and fungicides against powdery mildew disease of grapevines in upper egypt antimicrobial activity of compounds isolated from algae flavonoid and other constituents of bauhinia manca effects of resveratrol on the ultrastructure of botrytis cinerea conidia and biological significance in plant/pathogen interactions biological activity of resveratrol, a stilbenic compound from grapevines, against botrytis cinerea, the causal agent for gray mold the antimicrobial activity of 3, 5, 7-trihydroxyflavone isolated from the shoots of helichrysum aureonitens extraction and identification of bioactive compounds (eicosane and dibutyl phthalate) produced by streptomyces strain kx852460 for the biological control of rhizoctonia solani ag-3 strain kx852461 to control target spot disease in tobacco leaf 2-9 phytoalexins in defense against pathogens effect of volatile and non-volatile compounds from trichoderma spp. against colletotrichum capsici incitant of anthracnose on bell peppers in vitro antifungal efficacies of aqueous extract of dumortiera hirsuta (swaegr.) nees against sporulation and growth of postharvest phytopathogenic fungi evaluation of streptomyces griseorubens e44g for the biocontrol of fusarium oxysporum f. sp. lycopersici: ultrastructural and cytochemical investigations interactions between a root-knot nematode (meloidogyne exigua) and arbuscular mycorrhizae in coffee plant development (coffea arabica) foliar epicuticular wax of arrabidaea brachypoda: flavonoids and antifungal activity metabolites of helminthosporium monoceras: structures of monocerin and related benzopyrans trans-trans-3, 11-tridecadiene5, 7, 9-triyne-1,2-diol, an antifungal polyacetylene from diseased safflower (carthamus tinctorius) mycofumigation by the volatile organic compound-producing fungus muscodor albus induces bacterial cell death through dna damage mycorrhizal fungi and trichoderma harzianum as biocontrol agents for suppression of rhizoctonia solani damping off disease of tomato effect of volatile metabolites of trichoderma species against seven fungal plant pathogens in vitro production of gliotoxin on natural substrates by trichoderma virens studies on antagonistic effect against plant pathogenic fungi from endophytic fungi isolated from houttuynia cordata thunb. and screening for siderophore and indole-3-acetic acid production effect of mushroom extracts in the induction of phytoalexins and in the control of soy oidium in a greenhouse impact of mycorrhizal colonisation on root architecture, root longevity and the formation of growth regulators isolation and characterization of muscodor albus i-41.3s, a volatile antibiotic producing fungus influence of seed priming on the development of pearl millet downy mildew (sclerospora graminicola) synthesis and incorporation of the first polyketide synthase free intermediate in monocerin biosynthesis laminarin elicits defense responses in grapevine and induces protection against botrytis cinerea and plasmopara viticola streptomyces sanglieri which colonised and enhanced the growth of elaeis guineensis jacq. seedlings was antagonistic to ganoderma boninense in in vitro studies current status of biological control of plant diseases using antagonistic organisms in india status and prospects for enhancing the uptake of antagonistic organisms for nematode management in india alkaloids and aromatics of cyathobasis fruticulosa (bunge) aellen antimicrobial activities of bryophytes a review antibiotic activity of bryophytes an endophytic myrothecium inundatum producing volatile organic compounds muscodor albus strain gba, an endophytic fungus of ginkgo biloba from united states of america, produces volatile antimicrobials increasing the productivity and product quality of vegetable crops using arbuscular mycorrhizal fungi: a review seaweed polysaccharides as bio-elicitors of natural defenses in olive trees against verticillium wilt of olive thoughts and facts about antibiotics: where we are now and where we are heading in vitro screening of bryophytes for antimicrobial activity effect of phosphate and the arbuscular mycorrhizal fungus glomus intraradices on disease severity of root rot of peas (pisum sativum) caused by aphanomyces euteiches canaliculatol, an antifungal resveratrol trimer from stemonoporous canaliculatus induction and identification of sativan and vestitol as two phytoalexins from lotus corniculatus the cytosporones, new octaketide antibiotics isolated from an endophytic fungus phytoalexins induction in rubiaceae the camalexins: new phytoalexins produced in the leaves of camelina sativa (cruciferae) anti-fungal effects of cocoa tannin on the witches' broom pathogen crinipellis pernicious applied and environmental microbiology the chemical composition, antifungal, antioxidant and antimutagenicity properties of bioactive compounds from fungal endophytes associated with thai orchids isolation and identification of antifungal and antialgal alkaloids from haplophyllum sieversii isolation and characterization of endophytic streptomyces antagonists of fusarium wilt pathogen from surface sterilized banana roots isolation and characterization of two phytoalexins from rice as momilactones a and b munumbicins, wide-spectrum antibiotics produced by streptomyces nrrl 30562, endophytic on kennedia nigriscans munumbicins e-4 and e-5: novel broad-spectrum antibiotics from streptomyces nrrl 3052 aspectos bioquímicos e moleculares da resistência induzida bioactive diterpenes from the fruits of detarium microcarpum in vitro evaluation of fungicides, plant extracts and biocontrol agents against brown leaf spot of paddy bioactive diterpenes from the fruits of detarium microcarpum crop diseases and their management. phi learning private limited antifungal activity and action mechanism of ginger oleoresin against pestalotiopsis microspora isolated from chinese olive fruits studies on a chlorogenic acid-producing endophytic fungi isolated from eucommia ulmoides oliver antifungal activity of cinnamaldehyde and eugenol congeners against wood-rot fungi antifungal and antiviral products of marine organisms cytotoxic and antifungal triterpene glycosides from the patagonian sea cucumber hemoiedema spectabilis antimicrobial activity of 4-hydroxybenzoic acid and trans 4-hydroxycinnamic acid isolated and identified from rice hull diversity and antifungal activity of fungal endophytes of asparagus racemosus willd insecticidal secondary metabolic products from the entomogenous fungus fusarium larvarum 12-heptadecenyl)-resorcinol, the major component of the antifungal activity in the peel of mango fruit antifungal activity of neo-clerodane diterpenoids from scutellaria the manual of biocontrol agents effect of water activity on the production of volatile organic compounds by muscodor albus and their effect on three pathogens in stored potato biological and molecular comparison between localized and systemic acquired resistance induced in tobacco by phytophthora megasperma glycoprotein elicitin biological control of phytopathogenic fungi by endophytic actinomycetes isolated from maize (zea mays l) plant products as antimicrobial agents identification of three hydroxyflavan phytoalexins from daffodil bulbs phytoalexins from other plant families isolation and characterization of actinomycete antagonists of a fungal root pathogen susceptibility of fluconazole-resistant clinical isolates of candida spp. to echinocandin ly303366, itraconazole and amphotericin b biochemical defense mechanisms in cotton plants against ramularia leaf spot mediated by silicon in vitro antifungal activity of 2-(3,4-dimethyl-2,5-dihydro-1h-pyrrol-2-yl)-1-methylethyl pentanoate, a dihydro -pyrrole derivative phytoalexin accumulation in tissues of brassica napus inoculated with leptosphaeria maculans naphthalene, an insect repellent, is produced by muscodor vitigenus, a novel endophytic fungus activities of essential oils from asarum heterotropoides var. mandshuricum against five phytopathogens antagonist actinomycetes metabolites against plant pathogens fungi of agricultural importance induction of phytoalexins and proteins related to pathogenesis in plants treated with extracts of cutaneous secretions of southern amazonian bufonidae amphibians natural products in crop protection bioassay-guided isolation of allelochemicals from avena sativa l.: allelopathic potential of flavone c-glycosides antifungal activity of crude extracts from brown and red seaweeds by a supercritical carbon dioxide technique against fruit postharvest fungal diseases rhizospheric streptomycetes as potential biocontrol agents of fusarium and armillaria pine rot and as pgpr for pinus taeda geldanamycin, a new antibiotic induction of fusarium solani mutants insensitive to tomatine, their pathogenicity and aggressiveness to tomato fruits and pea plants molecular engineering of resveratrol in plants antifungal properties of surangin b, a coumarin from mammea longifolia phytochemical analysis and antifungal activity of moss bryum cellulare against some phytopathological fungi studies on antifungal potential of bryum cellulare against spore germination of fungus curvularia lunata in vitro antifungal activity of plagiochasma appendiculatum against alternaria solani evaluation of bryophyte for green fungicides as alternative treatment to control plant pathogen oxidative ring contraction of the phytoalexin cyclobrassinin: a way to brassilexin brassilexin, a novel sulphur-containing phytoalexin from brassica juncea l., (cruciferae) secondary metabolites production by actinomycetes and their antifungal activity antifungal bryophytes: a possible role against human pathogens and in plant protection isolation, characterization and antimicrobial activity at diverse dilution of wheat puroindoline protein free fatty acid accumulation and quality loss of stored soybean seeds invaded by aspergillus ruber molecular communication in interactions between plants and microbial pathogens seed germination enhancing activity of endophytic streptomyces isolated from indigenous ethno-medicinal plant centella asiatica interactions between an arbuscular mycorrhizal fungus (scutellospora heterogama) and the root-knot nematode (meloidogyne incognita) on sweet passion fruit (passiflora alata) application of plant extracts as inducers to challenge leaf rust of wheat inhibitory effect and mechanism of tagetes erecta l. fungicide on fusarium oxysporum f evaluating novel microbe amended composts as biocontrol agents in tomato biological activity summary for cocoa (theobroma cacao l.) effect of salicylic acid and structurally related compounds in the accumulation of phytoalexins in cotyledons of common bean phenylphenalenone phytoalexins, will they be a new type of fungicide? antifungal activity of peppermint and sweet basil essential oils and their major aroma constituents on some plant pathogenic fungi from the vapor phase production and genetic improvement of a novel antimycotic agent, saadamycin, against dermatophytes and other clinical fungi from endophytic streptomyces sp. hedaya48 antimicrobial activities of actinomycetes inhabiting achillea fragrantissima (family: compositae) an endophytic chitinase-producing isolate of actinoplanes missouriensis, with potential for biological control of root rot of lupine caused by plectosporium tabacinum performance of three endophytic actinomycetes in relation to plant growth promotion and biological control of pythium aphanidermatum, a pathogen of cucumber under commercial field production conditions in the united arab emirates nonstreptomycete actinomycetes as biocontrol agents of soil-borne fungal plant pathogens and as plant growth promoters structures of antifungal diarylheptenones, gingerenones a, b, c and isogingerenone b, isolated from the rhizomes of zingiber officinale coronamycins, peptide antibiotics produced by a verticillate streptomyces sp. (msu-2110) endophytic on monstera sp antifungal, anti-oomycete and phytotoxic effects of volatile organic compounds from the endophytic fungus xylaria sp. strain pb3f3 isolated from haematoxylum brasiletto new endophytic isolates of muscodor albus, a volatileantibiotic-producing fungus effect of substrate on the bioactivity of volatile antimicrobials produced by muscodor albus antifungal volatile organic compounds from the endophyte nodulisporium sp. strain gs4d2ii1a: a qualitative change in the intraspecific and interspecific interactions with pythium aphanidermatum preharvest treatments with chitosan and other alternatives to conventional fungicides to control postharvest decay of strawberry fungistatic effects of pinus radiata needle epicuticular fatty and resin acids on dothistroma pini blue-green alga anabaena laxa. i isolation and biological properties plant phenolics, lignification arbuscular mycorrhiza reduces susceptibility of tomato to alternaria solani antifungal metabolites from phomopsis sp. by254, an endophytic fungus in gossypium hirsutum comparative efficacies in vitro of antibacterial, fungicidal, antioxidant, and herbicidal activities of momilactones a and b antifungal and antibacterial potential of methanol and chloroform extracts of marchantia polymorpha l flavonoids from carnation (dianthus caryophyllus) and their antifungal activity interactions between a fluorescent pseudomonad, an arbuscular mycorrhizal fungus and a hypo virulent isolate of rhizoctonia solani affect plant growth and root architecture of tomato plants diversity and biopotential of endophytic actinomycetes from three medicinal plants in india the diversity, plant growth promoting and antimicrobial activities of endophytic actinomycetes isolated from emblica officinalis gaertn agriculture and bioactives: achieving both crop yield and phytochemicals isolation and characterization of endophytic actinomycetes from mangrove plant for antimicrobial activity two phytoalexins from sugar beet (beta vulgaris) leaves lass-florl c (2013) the mediterranean red alga asparagopsis taxiformis has antifungal activity against aspergillus species introduction of some new endophytic bacteria from bacillus and streptomyces genera as successful biocontrol agents against sclerotium rolfsii fluconazole: an update of its pharmacodynamic and pharmacokinetic properties and therapeutic use in major superficial and systemic mycoses in immunocompromised patients plant-fungal interactions: the search for phytoalexins and other antifungal compounds from higher plants sulfur containing cinnamides with antifungal activity from glycosmis cyanocarpa phytoalexin emit indolstruktur aus kohlrabi (brassica oleracea var. gongylodes) cytokinins mediate resistance against pseudomonas syringae in tobacco through increased antimicrobial phytoalexin synthesis independent of salicylic acid signaling metabolic products of fusarium larvarum fuckel. the fusarentins and the absolute configuration of monocerin potential of horsetail (equisetum sp.) preparations in the synthesis of defense metabolites in soy (glycine max l.) cotyledons and the effect on the growth of rhizoctonia solani kuhn, in vitro comparative transcriptomics of rice reveals an ancient pattern of response to microbial colonization chemical composition, antifungal and antitumor properties of ether extracts of scapania verrucosa heeg. and its endophytic fungus chaetomium fusiformis in vitro antifungal activity of dragon's blood from croton urucurana against dermatophytes multiple control levels of root system remodelling in arbuscular mycorrhizal symbiosis antifungal effect of five aqueous plant extracts on mycelial growth of penicillium expansum isolated from rotted yam tubers in storage alterations in root exudation of intercropped tomato mediated by the arbuscular mycorrhizal fungus glomus mosseae and the soil borne pathogen fusarium oxysporum f.sp. lycopersici host-pathogen interactions: xix. the endogenous elicitor, a fragment of a plant cell wall polysaccharide that elicits phytoalexin accumulation in soybeans tit for tat? a mycorrhizal fungus accumulates phosphorus under low plant carbon availability the accumulation of inhibitory compounds in the induced resistance response of carrot root slices to botrytis cinerea cucurbitacins protect cucumber tissue against infection by botrytis cinerea pestacin: a 1,3-dihydro isobenzofuran from pestalotiopsis microspora possessing antioxidant and antimycotic activities the effect of post-infectional potato tuber metabolites and surfactants on zoospores of oomycetes the isolation of xanthoxylin from the bark of phytophthora and hendersonula-infected citrus lemon and its fungitoxic effect analysis on blast fungus-responsive characters of a flavonoid phytoalexin sakuranetin; accumulation in infected rice leaves, antifungal activity and detoxification by fungus the fungal dimension of biodiversity: magnitude, significance, and conservation the magnitude of fungal diversity: the 1±5 million species estimate revisited efficacy of artificial humic acid is a selective nutrient in hv agar used for the isolation of actinomycetes genetic manipulation of isoflavone 7-o-methyltransferase enhances biosynthesis of 4′-o-methylated isoflavonoid phytoalexins and disease resistance in alfalfa die wirkung von auszigen aus dem sachalin-staudenknoterich reynoutria sachalinensis (f. schmidt) nakai gegen plizkrankheiten, insbesondere echte mehltauplize production of antibiotics by pseudomonas cepacia as an agent for biological control of soilborne plant pathogens screening of poplar trees for antibacterial, antifungal and antiviral activity suppression of pythium ultimum induced damping -off of cotton seedlings by pseudomonas fluorescens and its antibiotic, pyoluterin effects of fusarium species on defence mechanisms in sorghum seedlings control of post harvest botrytis fruit rot of strawberry by volatile organic compounds of candida intermedia biodiversity of endophytic fungi associated with 29 traditional chinese medicinal plants novel acidic sesquiterpenoids constitute a dominant class of pathogeninduced phytoalexins in maize antimicrobial activities of some brown macroalgae against some soil borne plant pathogens and in vivo management of solanum melongena root diseases antifungal and antiproliferative activities of endophytic fungi isolated from the leaves of markhamia tomentosa screening of novel bioactive compounds from plant-associated actinomycetes isolation of actinomycetes from live plants and evaluation of anti phytopathogenic activity of their metabolites isolation and identification of endophytic actinomycetes and their antifungal activity phytoalexins from the leguminosae fungitoxic isoflavones from lupinus albus and other lupinus species the polyoxins: pyrimidine nucleoside peptide antibiotics inhibiting fungal cell wall biosynthesis validoxylamines as trehalase inhibitors suppression of damping-off disease in host plants by the rhizoplane bacterium lysobacter sp. strain sb-k88 is linked to plant colonization and antibiosis against soilborne peronosporomycetes antibacterial and antifungal activities of brown alga zonaria tournefortii (jv lamouroux) direct isolation of micromonospora on selective media with gentamicin polyphenol concentrations in grain, leaf and callus tissues of mold-susceptible and mold-resistant sorghum cultivars composition and antifungal activity of the essential oil of the brazilian chenopodium ambrosioides l ulvan, a sulfated polysaccharide from green algae, activates plant immunity through the jasmonic acid signaling pathway modulation of phytoalexin biosynthesis in engineered plants for disease resistance metabolic engineering of yeast and plants for the production of the biologically active hydroxystilbene, resveratrol biosynthesis, metabolism, molecular engineering and biological functions of stilbene phytoalexins in plants deciphering the role of phytoalexins in plant-microorganism interactions and human health phytoalexins produced in the leaves of capsella bursapastoris (shepherd's purse) xanthotoxin: a phytoalexin of pastinaca sativa root mycorrhiza-induced resistance and priming of plant defenses isolation of endophytic actinomycetes from catharanthus roseus (l.) g. don leaves and their antimicrobial activity. iranian effect of verticillium wilt (verticillium dahliae kleb.) and mycorrhiza (glomus mosseae) on root colonization, growth and nutrient uptake in tomato and eggplant seedlings the possible association of phytoalexins with resistant gene expression in flax to melampsora lini antifungal potential in crude extracts of five selected brown seaweeds collected from the western libya coast in vitro antifungal, anti-elastase and anti-keratinase activity of essential oils of cinnamomum-, syzygium-and cymbopogon-species against aspergillus fumigatus and trichophyton rubrum insecticidal activities of aromatic plant extracts and essential oils against sitophilus oryzae and callosobruchus chinensis anthraquinones isolated from cassia tora (leguminosae) seed show an antifungal property against phytopathogenic fungi recent development in the use of blasticidin s, a microbial fungicide, as a useful reagent in molecular biology linear b-1,3 glucans are elicitors of defense responses in tobacco-france induce systemic resistance and promotion of plant growth by bacillus spp antifungal activity of pisiferic acid derivatives against the rice blast fungus functional moiety for the antifungal activity of phytocassane e, a diterpene phytoalexin from rice phytoalexin induction in the sapwood of plants of the maloideae (rosaceae): biphenyls or dibenzofurans structural and functional characterization of gene clusters directing non-ribosomal synthesis of bioactive lipopeptides in bacillus amyloliquefaciens strain fzb42 evaluation of essential oils and their components for broad-spectrum antifungal activity and control of late leaf spot and crown rot diseases in peanut bryophytes, a potent source of drugs for tomorrow's medicine? a plant extract acts both as a resistance inducer and an oomycide against grapevine downy mildew outline of a comparative study of criteria used in characterization of the actinomycetes selection of media for isolation of streptomycetes phytoalexins from the solanaceae muscodor sutura, a novel endophytic fungus with volatile antibiotic activities endophytic fungi isolated from oil-seed crop jatropha curcas produces oil and exhibit antifungal activity identification of antifungal principle in the solvent extract of an endophytic fungus chaetomium globosum from withania somnifera isolation, characterization, and bioactivity of endophytic fungi of tylophora indica an endophytic nodulisporium sp. producing volatile organic compounds having bioactivity and fuel potential two fungicidal phenylethanones from euodia lunu-ankenda root bark antifungal and insect antifeedant 2-phenylethanol esters from the liverwort balantiopsis cancellata from chile efficacy of the biofumigant fungus muscodor albus (ascomycota: xylariales) for control of codling moth (lepidoptera: tortricidae) in simulated storage conditions the potential of the fungus, muscodor albus, as a microbial control agent of potato tuber moth (lepidoptera: gelechiidae) in stored potatoes benzoic acid derivatives from piper species and their fungitoxic activity against cladosporium cladosporioides and c. sphaerospermum the production of resveratrol by vitis vinifera and other members of the vitaceae as a response to infection or injury interactions between pea root-inhabiting fungi examined using signature fatty acids mycosubtilin overproduction by bacillus subtilis bbg100 enhances the organism's antagonistic and biocontrol activities momilactones a and b in rice straw harvested at different growth stages antibacterial activity of oriental medicinal plant extracts toward helicobacter pylori mycofumigation with oxyporus latemarginatus ef069 for control of postharvest apple decay and rhizoctonia root rot on moth orchid screening for endophytic fungi with antitumour and antifungal activities from chinese medicinal plants cryptocin, a potent tetramic acid antimycotic from the endophytic fungus cryptosporiopsis cf. quercina antifungal activity of camptothecin, trifolin, and hyperoside isolated from camptotheca acuminata effect of laminarin on aspergillus flavus growth and aflatoxin production use of the endophytic fungus daldinia cf. concentrica and its volatiles as bio-control agents mycorrhizae and plan health isoquinoline alkaloids from macleaya cordata active against plant microbial pathogens pestalofones a-e, bioactive cyclohexanone derivatives from the plant endophytic fungus pestalotiopsis fici bis(2,3-dibromo-4,5-dihydroxybenzyl) ether, a marine algae derived bromophenol, inhibits the growth of botrytis cinerea and interacts with dna molecules screening of a marine algal extract for antifungal activities a new macrolide antibiotic with antitumor activity produced by streptomyces sp. cs, a commensal microbe of maytenus hookeri a novel ansamycin, naphthomycin k from streptomyces sp new bioactive metabolites produced by colletotrichum sp., an endophytic fungus in artemisia annua overview: a phylogenetic backbone and taxonomic framework for prokaryotic systematics inhibitory activities of some marine algae on aflatoxin accumulation naphthoquinone spiroketal with allelochemical activity from the newly discovered endophytic fungus edenia gomezpompae plant disease control: understanding the roles of toxins and phytoalexins in host-pathogen interaction plant extracts, bau-biofungicide and fungicides in controlling some important diseases of rice cv. brri dhan40 biocontrol potential of cyanobacterial metabolites against damping off disease caused by pythium aphanidermatum in solanaceous vegetables bioprospecting for endophytes from australian flora with mycofumigation potential antifungal activity of rubia tinctorum, rhamnus frangula and caloplaca cerina antimicrobial compounds and resistance: the role of phytoalexins and antianticipins fungicidal and molluscicidal saponins from dolichos kilimandscharicus xanthones from polygala nyikensis ethnoveterinary medicine and development: a review of the literature elicitor activity of phytoalexins in soy and sorghum by extracts and tinctures of medicinal plant species synthesis of phytoalexins in soy and sorghum by extracts and tinctures from three forest species structures of ent-herbertane sesquiterpenoids displaying antifungal properties from the liverwort herberta adunca endophytic fungi associated with monarda citriodora, an aromatic and medicinal plant and their biocontrol potential bioactivity of bryophyte extracts against botrytis cinerea, alternaria solani and phytophthora infestans control of fungal decay of apples and peaches by the biofumigant fungus muscodor albus the algal polysaccharide carrageenans can act as an elicitor of plant defense laboratoires goëmar. parc technopolitain atalante muscodor ghoomensis and muscodor indica: new endophytic species based on morphological features, molecular and volatile organic analysis from northeast india muscodor camphora, a new record from cinnamomum camphora muscodor kashayum sp. nov. -a new volatile antimicrobial producing endophytic fungus muscodor strobelii, a new endophytic species from south india response of subterranean clover to dual inoculation with vesicular-arbuscular mycorrhizal fungi and a plant growth-promoting bacterium modulation oh cyp79 genes and glucosilate profiles in arabidopsis by defense pathways potential agrochemicals from leaves of wedelia biflora 60-trihydroxydihydrochalcone from psidium acutangulum fungi and mycotoxins in grain: implications for stored product research endophytic actinomycetes from indian medicinal plants as antagonists to some phytopathogenic fungi chemically characterized cymbopogon martinii essential oil for shelf life enhancer of herbal raw materials based on antifungal, antiaflatoxigenic, antioxidant activity and favorable safety profile volatile antimicrobials from muscodor crispans, a novel endophytic fungus volatile plant metabolites for postharvest crop protection 4-methoxybrassinin, a sulphur-containing phytoalexin from brassica oleracea brassicanal c and two dioxindoles from cabbage brassicanal a and b, novel sulfur-containing phytoalexins from the chinese cabbage brassica campestris l. ssp pekinensis dehydro-4-methoxycyclobrassinin, a sulfur-containing phytoalexin isolated from turnip brassica campestris l. ssp. rapa calophycin, a fungicidal cyclic decapeptide from the terrestrial blue-green alga calothrix fusca biological activity of β-dolabrin, γ-thujaplicin, and 4-acetyltropolone, hinokitiol-related compounds bacillomycin d: an iturin with antifungal activity against aspergillus flavus chemical composition and fungitoxic properties to phytopathogenic fungi of essential oils of selected aromatic plants growing wild in turkey evaluation of 3, 4-dihydroxybenzaldehyde, dopamine and its oxidation products as inhibitors of colletotrichum musae (berk. and curt.) arx in green banana fruits antifungal activities of the leaf extract of cassia tora linn experimentelle untersuchungen über die phytophthora resistenz der kartoffel camellidins, antifungal saponins isolated from camellia japonica different mechanisms for phytoalexin induction by pathogen and wound signals in medicago truncatula glyceollin, a soybean phytoalexin with medicinal properties an endophytic actinomycete, streptomyces sp. aok-30, isolated from mountain laurel and its antifungal activity antimicrobial activities of vernonia tenoreana streptomyces: implications and interactions in plant growth promotion dihydro isocoumarins produced by xylaria sp. and penicillium sp., endophytic fungi associated with piper aduncum and alibertia macrophylla activation of biochemical defense mechanisms in bean plants for homeopathic preparations inhibition of protein biosynthesis by mildiomycin, an antimildew substance antifungal activity of thyme, summer savory and clove essential oils against aspergillus flavus in liquid medium and tomato paste activity of fungal endophytes against four maize wilt pathogens efficacy of some agricultural wastes in controlling root rot of glycine max l. induced by rhizoctonia solani effect of seed inoculation with bacillus subtilis and streptomyces griseus on the growth of cereals and carrots stress induced carbazole phytoalexins in glycosmis species seasonal variation of antibacterial and antifungal activities of the extracts of marine algae from southern coasts of india griseofulvin from xylaria sp. strain f0010, and endophytic fungus of abies holophylla and its antifungal activity against plant pathogenic fungi arbuscular mycorrhiza: the mother of plant root endosymbiosis potential of the volatile producing fungus nodulisporium sp. cf016 for the control of postharvest diseases of apple isolation, abundance and phylogenetic affiliation of endophytic actinomycetes associated with medicinal plants and screening for their in vitro antimicrobial biosynthetic potential distribution and identification of endophytic streptomyces species from schima wallichii as potential biocontrol agents against fungal plant pathogens mutualism and parasitism: the yin and yang of plant symbioses effects of sulfated polysaccharide and alcoholic extracts from green seaweed ulva fasciata on anthracnose severity and growth of common bean (phaseolus vulgaris l.) biological control in greenhouse systems a new working definition of the term "phytoalexin biotransformation of the brassica phytoalexin brassicanal a by blackleg fungus phytoalexins from brassicas: overcoming plants' defenses phytoalexin accumulation and antifungal compounds from the crucifer wasabi pathogen inactivation of cruciferous phytoalexins: detoxification reactions, enzymes and inhibitors antimicrobial activity of rhodobryum ontariense. hemijska industrija the discovery of enfumafungin, a novel antifungal compound produced by an endophytic hormonema species biological activity and taxonomy of the producing organisms ultrastructural observations of pterostilbene fungitoxicity in dormant conidia of botrytis cinerea pers natural occurrence of mycotoxins in foods and feeds -an update review relation between the chemical structure and biological activity of hydroxystilbenes against botrytis cinerea two new antifungal naphthoxirene derivatives and their glucosides from sesamum angolense welw potential of plant extracts and fungicides for managing fusarium oxysporum f. sp lycopersici further evidence for the involvement of a pre-formed antifungal compound in the latency of colletotrichum gloeosporioides on unripe avocado fruits progress in phytoalexin research during the past 50 years antifungal potential and defense gene induction in maize against rhizoctonia root rot by seed extract of ammi visnaga (l.) lam diterpene alcohols from croton lacciferus an antifungal chromene from eupatorium riparium outbreaks of aflatoxicoses in india removal of duvatrienediols from the surface of tobacco leaves increases their susceptibility to blue mold jasmonate and salicylate as global signals for defense gene expression use of algae in strawberry management antimicrobial activity of essential oils and ethanol natural products from plants and fungi as fungicides 227 extract of phlomis fruticosa l. (lamiaceae) growth activities of the sugar beet pathogens sclerotium rolfsii sacc. rhizoctonia solani kühn. and fusarium verticillioides sacc. under cyanobacterial filtrates stress induction of defense responses in zucchini (cucurbita pepo) by anabaena sp. water extract activity of seaweed and cyanobacteria water extracts against podosphaera xanthii on zucchini antioxidant and antimicrobial activities of essential oil and extracts of fennel (foeniculum vulgare l.) and chamomile elicitation of foliar resistance mechanisms transiently impairs root association with arbuscular mycorrhizal fungi antifungal activity and potential use of essential oils against fusarium culmorum and fusarium verticillioides plant growth enhancing effects by a siderophore producing endophytic streptomycete isolated from a thai jasmine rice plant (oryza sativa l. cv. kdml105) bioactivities of extracts from some axenically farmed and naturally grown bryophytes antimicrobial activity of bryum argenteum screening of antimicrobial and antioxidant secondary metabolites from endophytic fungi isolated from wheat (triticum durum) structure biological activity relationships in triterpenic saponins: the relative activity of protobassic acid and its derivatives against plant pathogenic fungi pantoea agglomerans strain eh318 produces two antibiotics that inhibit erwinia amylovora in vitro effectiveness of phenolic compounds against citrus green mould control of penicillium expansum and patulin accumulation on apples by quercetin and umbelliferone determination of antimicrobial and antiproliferative activities of the aquatic moss fontinalis antipyretica hedw muscodor tigerii sp. nov.-volatile antibiotic producing endophytic fungus from the northeastern himalayas muscodor darjeelingensis, a new endophytic fungus of cinnamomum camphora collected from northeastern himalayas bioactivity guided isolation of antifungal compounds from the liverwort bazzania trilobata biosynthesis, elicitation and roles of monocot terpenoid phytoalexins biologically active secondary metabolites of endophytic pezicula sp pinocembrin: an antifungal compound secreted by leaf glands of eastern cottonwood endophytic actinomycetes from tea plants (camellia sinensis): isolation, abundance, antimicrobial, and plant-growth-promoting activities isolation of 2,4-diacetylphloroglucinol from a fluorescent pseudomonad and investigation of physiological parameters influencing its production plant bio-stimulants: a review on the processing of macroalgae and use of extracts for crop management to reduce abiotic and biotic stresses purification and partial characterization of a b-glucan fragment that elicits phytoalexin accumulation in soybean diversity and antimicrobial activity of culturable endophytic fungi isolated from moso bamboo seeds in: maheshwari dk (ed) bacteria in agrobiology: plant growth responses studies on endophytic actinomycetes (i) streptomyces sp. isolated from rhododendron and its antifungal activity introduction study of antifungal activities of bryophyte extracts anti-apoptotic machinery protects the necrotrophic fungus botrytis cinerea from host-induced apoptotic-like cell death during plant infection diversity of endophytic actinomycetes in mandarin grown in northern thailand, their phytohormone production potential and plant growth promoting activity diversity and antifungal activity of the endophytic fungi associated with the native medicinal cactus opuntia humifusa (cactaceae) from the united states antifungal activity of securinine against some plant pathogenic fungi antimicrobial activity of some indian mosses an endophytic phomopsis sp. possessing bioactivity and fuel potential with its volatile organic compounds existence of muscodor vitigenus, m. equiseti and m. heveae sp. nov. in leaves of the rubber tree (hevea brasiliensis müll. arg.), and their biocontrol potential casbene: an antifungal diterpene produced in cell-free extracts of ricinus communis seedlings sesquiterpene lactones from centaurea thessala and centaurea attica: antifungal activity mechanisms of resistance to plant diseases what is the significance of the arbuscular mycorrhizal colonisation of many economically important crop plants? the role of phosphorous nutrition in interactions of vesicular arbuscular mycorrhizal fungi with soil borne nematodes and fungi suppression of cottony leak of cucumber with bacillus cereus strain uw85 management of mycorrhiza in agriculture, horticulture and forestry endophytic naphthopyrone metabolites are co-inhibitors of xanthine oxidase, sw1116 cell and some microbial growths a record of muscodor albus, an endophyte from myristica fragrans in thailand antifungal activity of some essential oils two novel antifungal alka-2,4-dienals from triticum aestivum plant-derived natural products synthesis, function, and application streptomyces sp. 9p as effective biocontrol against chilli soilborne fungal phytopathogens algal polysaccharides as source 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experimental establishment in the host plant antifungal activities of a steroid from pallavicinia lyellii, a liverwort engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol muscodor cinnamomi, a new endophytic species from cinnamomum bejolghota evaluation of muscodor suthepensis strain cmu-cib462 as a postharvest biofumigant for tangerine fruit rot caused by penicillium digitatum molecular and morphological evidence support four new species in the genus muscodor from northern thailand biocontrol of rhizoctonia solani ag-2, the causal agent of damping-off by muscodor cinnamomi cmu-cib 461 antitumor activity of 4-arylcoumarins from endophytic streptomyces aureofaciens cmuac130 identification of streptomyces sp. tc022, an endophyte in alpinia galanga, and the isolation of actinomycin d structures of moracins e, f, g and h, new phytoalexins from diseased mulberry isolation of three novel sulphur-containing phytoalexins from the chinese cabbage brassica campestris l. ssp. pekinensis (cruciferae) novel sulfur-containing phytoalexins from the chinese cabbage brassica campestris l. ssp. pekinensis (cruciferae) mechanism of kasugamycin action on polypeptide synthesis isolation of endophytic actinomycetes from different cultivars of tomato and their activities against ralstonia solanacearum in vitro isolation, purification and characterization of trichothecinol-a produced by endophytic fungus trichothecium sp. and its antifungal, anticancer and antimetastatic activities antibacterial activity and induction of phytoalexins in bean plants by homeopathic preparations of essential oil of eucalyptus globulus antibiotic production by soil and rhizosphere microbes in situ study on the communities of endophytic fungi and endophytic actinomycetes from rice and their antipathogenic activities in vitro identification of volatile metabolites from fungal endophytes with biocontrol potential towards fusarium oxysporum f. sp. cubense race 4 influence of arbuscular mycorrhizal mycelial exudates on soil bacterial growth and community structure antifungal activity of lipopeptides from bacillus xt1 cect 8661 against botrytis cinerea hypoxylon sp., an endophyte of persea indica, producing 1,8-cineole and other bioactive volatiles with fuel potential diterpene phytoalexins are biosynthesized in and exuded from the roots of rice seedlings antifungal activity of lemongrass (cymbopogon citratus l.) essential oil against key postharvest pathogens antifungal drug resistance in pathogenic fungi phytotoxic and antimicrobial activity of volatile and semi-volatile organic compounds from the endophyte hypoxylon anthochroum strain blaci isolated from bursera lancifolia (burseraceae) volatile organic compounds from endophytic fungi as innovative postharvest control of fusarium oxysporum in cherry tomato fruits studies on the mode of action of the phytoalexin phaseolin characterization of the early response of arabidopsis to alternaria brassicicola infection using expression profiling antimicrobial activity of methanol extracts of fontinalis antipyretica, hypnum cupressiforme and ctenidium molluscum sea weed polysaccharides and derived oligosaccharides stimulate defense responses and protection against pathogens in plants endophytic actinomycetes from azadirachta indica a. juss.: isolation, diversity, and anti-microbial activity the biocontrol effect of mycorrhization on soil borne fungal pathogens and the autoregulation of the am symbiosis: one mechanism, two effects? biocontrol of the pathogen phytophthora parasitica by arbuscular mycorrhizal fungi is a consequence of effects on infection loci 5-methylcoumaranones from mutisia friesiana and their bioactivity a vesicular arbuscular mycorrhizal fungus (glomus intraradix) induces a defense response in alfalfa roots suppression of an isoflavonoid phytoalexin defense response in mycorrhizal alfalfa roots fungal (−like) biocontrol organisms in tomato disease control controlling crop diseases using induced resistance: challenges for the future antifungal activities of essential oils and their constituents from indigenous cinnamon (cinnamomum osmophloeum) leaves against wood decay fungi antifungal activity screening of soil actinobacteria isolated from inner mongolia isolation and identification of an endophytic fungus of polygonatum cyrtonema and its antifungal metabolites loroglossol: an orchid phytoalexin evaluation of fungicides, bio-agents and plant extracts against pyricularia oryzae temporal synthesis and radiolabelling of the sorghum 3-deoxyanthocyanidin phytoalexins and the anthocyanin, cyanidin 3-dimalonyl glucoside peptide synthetase gene in trichoderma virens use of antibiotics for selective isolation and enumeration of actinomycetes in soil biological properties of alkaloids. influence of quinolizidine alkaloids and gramine on the germination and development of powderly mildew, erysiphe graminis f. sp. hordei die verbreitung antifungaler eigenschaften bei moosen the role of stilbenes in resistance of sitka spruce (picea sitchensis (bong) carr) to entry of fungal pathogens muscodor roseus anam. sp. nov., an endophyte from grevillea pteridifolia muscodor albus anam. sp. nov., an endophyte from cinnamomum zeylanicum chemical constituents from the chinese bryophytes and their reversal of fungal resistance metabolites from mangrove endophytic fungus dothiorella sp tetran or triterpenoids from chisocheton paniculatus effects of pre-and post-harvest application of salicylic acid or methyl jasmonate on inducing disease resistance of sweet cherry fruit in storage endophytic fungi harbored in the root of sophora tonkinensis gapnep: diversity and biocontrol potential against phytopathogens tithoniamarin and tithoniamide: a structurally unique isocoumarin dimer and a new ceramide from tithonia diversifolia evaluation of wedelia biflora (linn) d.c for anthelmintic and antimicrobial activity recent trends in studies on botanical fungicides in agriculture effect of polyacetylenic acids from prunella vulgaris on various plant pathogens potent in vivo antifungal activity against powdery mildews of pregnane glycosides from the roots of cynanchum wilfordii fungitoxic non-glycosidic iridoids from alibertia macrophylla diversity and antifungal activity of endophytic fungi associated with camellia oleifera potential of endophytic fungi isolated from cotton roots for biological control against verticillium wilt disease natural plant products as eco-friendly fungicides for plant diseases control-a review regulation of plant immunity through modulation of phytoalexin synthesis a new prenylated indole derivative from endophytic actinobacteria streptomyces sp. neau-d50 studies on chemical constituents in root tuber of cynanchum auriculatum muscodor fengyangensis sp. nov. from southeast china: morphology, physiology and production of volatile compounds bioactive isocoumarins isolated from the endophytic fungus microdochium bolleyi effects of yeast polysaccharide on growth and flavonoid accumulation in fagopyrum tataricum sprout cultures actinobacteria associated with chinaberry tree are diverse and show antimicrobial activity the diversity and anti-microbial activity of endophytic actinomycetes isolated from medicinal plants in panxi plateau china preharvest l -arginine treatment induced postharvest disease resistance to botrytis cinerea in tomato fruits neoverataline a and b, two antifungal alkaloids with a novel carbon skeleton from veratrum taliense bioactive endophytic streptomycetes from the malay peninsula camalexin accumulation in arabis lyrata metabolites of colletotrichum gloeosporioides, an endophytic fungus in artemisia mongolica key: cord-274101-vm9nh8lc authors: perez espitia, paula judith; de fátima ferreira soares, nilda; dos reis coimbra, jane sélia; de andrade, nélio josé; souza cruz, renato; alves medeiros, eber antonio title: bioactive peptides: synthesis, properties, and applications in the packaging and preservation of food date: 2012-02-29 journal: compr rev food sci food saf doi: 10.1111/j.1541-4337.2011.00179.x sha: doc_id: 274101 cord_uid: vm9nh8lc abstract: bioactive peptides are protein fragments which have a positive impact on the functions and conditions of living beings. peptides have shown several useful properties for human health, including antimicrobial, antifungal, antiviral, and antitumor activities. these compounds are produced by almost all species of life. however, they are produced in limited quantities in nature. as a result, researchers have tried to synthesize bioactive peptides to study their properties and applications in various areas. among their applications in food preservation, peptides have been incorporated into packaging materials. this review begins with a brief description of the methods used for the synthesis, purification, and characterization of peptides. also, the main bioproperties and mechanisms of action of peptides are discussed. finally, some applications of peptides are presented, especially their use in active packaging, their effects on the polymeric matrix, and peptide migration. food safety is a growing concern of great importance worldwide. recently, the estimated costs of diseases caused by foodborne pathogens was about $152 billion in the united states (scharff 2010) , and it is estimated that in the united states alone about 47.8 million illness cases, 128000 hospitalizations and 3000 deaths will be caused by foodborne pathogens in 2011. the consumption of processed foods with chemical preservatives has led to increased consumer concern and the demand for more natural and minimally processed foods. as a result, researchers have shown a growing interest in natural antimicrobial agents such as certain peptides. bioactive peptides are defined as specific protein fragments that have a positive impact on the functioning or conditions of living beings, thereby improving their health (korhonen and pihlanto 2006) . the beneficial effects are attributed to different properties found in peptides such as antimicrobial (reddy and others 2004; rajanbabu and chen 2011) , antioxidant (sarmadi and ismail 2010) , antithrombotic (wang and ng 1999) , anti-hypertensive (erdmann and others 2008) , and immunomodulatory activities (st georgiev 1990; gauthier and others 2006) , among others. ms 20111071 submitted 9/7/2011, accepted 11/29/2011. authors espitia, soares, coimbra, de andrade, and medeiros are with food technology dept., federal univ. of viçosa, av. p. h. rolfs, s/n, campus univ., 36570-000. viçosa, minas gerais, brazil. author cruz is with food technology dept., state univ. of feira de santana, av. transnordestina, s/n, campus univ., 44036-900. feira de santana, bahía, brazil. direct inquiries to author soares (e-mail: nfsoares10@gmail.com) . peptides with antimicrobial properties are used as the first chemical barrier against microbial attack, being synthesized in response to bacterial infections. they are produced by almost all species of life, from microorganisms, plants and animals, to humans (st georgiev 1990; hancock and diamond 2000) . in animals, antimicrobial peptides are produced mainly in those tissues exposed to adverse conditions such as skin, eyes, and lungs, which are more likely to be in contact with microorganisms (zasloff 2002; papo and shai 2003) . more than 700 antimicrobial peptides have been reported, showing significant variations with respect to their sequence, length, and structure (papo and shai 2003) . antimicrobial peptides have found many applications, including those in biomedical devices, food processing equipment, and food preservation. in food preservation, peptides can be incorporated into materials to create antimicrobial packaging (appendini and hotchkiss 2002) . in this way, antimicrobial packaging plays an important role in maintaining the safety and quality of food, since the aim is to prolong food shelf life and to reduce bacterial growth on the product surface (soares and others 2009a) . this type of active packaging interacts with the product and/or the headspace inside to reduce, inhibit, or retard the growth of microorganisms that may be present (soares and others 2009b) . this review highlights the main methods of peptide synthesis and noteworthy peptide bioproperties. also, specific peptide applications in food preservation are reviewed, focusing on their incorporation in polymeric matrices. finally, the effects of peptide incorporation on packaging characteristics as well as their migration into food are discussed. borgia and fields (2000) . copyright (2000) , elsevier. peptides are biomolecules that contain between 1 to several dozen of amino acid residues joined by peptide bonds. the discovery of the different peptide activities has generated enormous interest in this class of compounds and in the methods of isolation, analysis, purification, identification, and quantification. these methods have been systematically studied and improved. however, most sources of natural peptides are poor in these compounds, thus preventing their isolation in sufficient quantities for research. as a result, there was a growing need to synthesize peptides for application in physiological, chemical, physical, pharmacological, biochemical, and clinical studies. total of 3 methods of peptide synthesis have been developed and improved: chemical synthesis, which uses chemical reagents to mediate peptide bond formation (andreu and rivas 2002) , enzymatic synthesis, in which the peptide bond formation is catalyzed by enzymes (bongers and heimer 1994; boeriu and others 2010) , and the dna recombinant technology synthesis, based on the use of cloning and ribosomal techniques from biological systems for peptide formation (sewald and jakubke 2002) . research on this synthesis method was first initiated more than 30 y ago. however, the construction of peptides has recently become more accessible due to advances in process efficiency, including the development and use of fast coupling reagents, as well as the minimization of side reactions (borgia and fields 2000) . the main aspects of chemical synthesis are protection and activation. protection strategies are intended to provide chemical selectivity necessary for the construction of a particular peptide sequence. activation refers to the chemical coupling necessary to ensure quantitative formation of each peptide bond in the sequence (andreu and rivas 2002) . in chemical synthesis, chemical reagents are used to activate the carboxylic acid (rcooh) of the amino acid, which will donate the acyl group (r-co-) to form the peptide bond. the peptide bond presents a nucleophilic attack of the α-amino group by another amino acid (h 2 n-r). in this synthesis, the reactive functional groups that are not directly involved in peptide bond formation receive prior protection (machado and others 2004) . there are 2 types of chemical peptide synthesis, synthesis in solution (classical synthesis) and solid-phase synthesis. chemical synthesis in solution is performed with all reagents and reaction products dissolved in the medium (kent 1988) . in comparison, solid-phase synthesis (sps) is a simple procedure to produce peptides in large quantities on a solid support which remains insoluble in the reaction medium (shigeri and others 2001) . the solid support is a polymeric resin that has a functional group on its surface (linker) that allows it to form stable bonds in the peptide sequence to the reagent used for the de-protection of the n-amino group. peptide synthesis in the solid phase generally consists on the acylation of an amino acid to be linked to an insoluble support (resin) via a linker ( figure 1 ). after that, the protecting group of the n-terminal is removed (the unprotecting step) to allow the next amino acid of the sequence to be attached to the complex "peptide-linker-resin." the unprotecting-coupling cycle is repeated until the desired sequence is complete. finally, the cleavage reagent is used to separate the complex "peptide-linker-resin." this reagent should also remove the protecting groups of side chains that are stable to unprotecting conditions of the n-terminal group (borgia and fields 2000) . peptide chemical synthesis can use 2 protocols, boc (tertbutyloxycarbonyl) and fmoc (9-fluorenylmethyloxycarbonyl), named according to the type of protector of the reactive group of the amino acids (n-terminal) involved in the synthesis. the first protocol employs the tert-butyloxycarbonyl (boc) group for n-amino protection. this protocol is based on gradual differences in their sensitivity to acids. thus, the boc group is typically removed with trifluoroacetic acid (tfa), while the protecting groups of the lateral chains (ester, ether, and urethane derivatives based on benzyl alcohol) are specifically designed to be stable to repeated cycles of boc removal and are removed only with a specific reagent, a relatively stronger acid, usually hydrofluoric acid (borgia and fields 2000) . the second protocol uses a 9-fluorenylmethyloxycarbonyl (fmoc) as the n-amino protecting group. this protocol provides a greater degree of chemoselectivity than the boc protocol, since the fmoc group is removed under basic conditions (piperidine in n, n-methylpyrrolidone or dimethylformamide), without alteration of the acid-sensitive lateral chains (andreu and rivas 2002) . protection groups of lateral chains are compatible with the fmoc protection group; these are mainly ether, ester, and urethane derivatives based on t-butanol. protection groups of lateral chains are removed by the end of the synthesis using tfa (borgia and fields 2000) . in this method, the peptide bond formation is mediated by an enzyme (protease) in free or immobilized form. the enzymatic method is especially useful in the synthesis of very short peptides (2-5 oligomers) and in the condensation of large peptide fragments (so and others 1998) . proteolytic enzymes such as chymotrypsin, papain, pepsin, subtilisin, termolisin, trypsin, among others, have been used in the presence of organic solvents as catalysts for the synthesis of peptide bonds (ogino and others 1999) . the enzymatic synthesis of peptides has several advantages over chemical methods, including good stereoselectivity and regioselectivity. however, it has certain shortcomings, such as peptide synthesis being thermodynamically unfavorable in water, as well as the secondary hydrolysis of synthesized peptide chains, which hinders their use in peptide synthesis with long sequences (so and others 1998) . thus, the main practical obstacle to employment of a protease for peptide bond formation is finding suitable conditions to allow bond formation without mediating secondary hydrolysis of the peptide or peptide fragments used as reagents (bongers and heimer 1994) . the formation of a peptide bond by enzyme catalysis can occur through several mechanisms, including the reverse hydrolysis reaction of amides and transpeptidation (machado and others 2004; boeriu and others 2010) . the mechanism of the reverse hydrolysis reaction is based on the microscopic reversibility principle. this indicates that the peptide bond formation and hydrolysis reaction come from the same intermediate ( figure 2 ). thus, the reaction conditions are manipulated to shift the equilibrium towards peptide bond formation. the transpeptidation mechanism occurs as a result of the break of a peptide bond, with the formation of an active acyl-enzyme intermediate ( figure 3 ). this intermediate is attacked in the presence of a nucleophile (peptide or amino acid blocked in the α-carboxyl group) and consequently causes the formation of a new peptide bond. for both mechanisms, the equilibrium should shift to the synthesis reaction direction, requiring the use of protective groups of α-amino and carboxyl substrates, the addition of organic solvents to the media reaction, excess substrates, and the removal of products from the reaction medium (machado and others 2004) . this synthesis uses modern methods of cloning and gene expression in microorganisms, allowing the production of a recombinant peptide or several peptides simultaneously. bacteria are the expression system generally used, with e. coli being the most widely used host. since antimicrobial peptides present a natural destructive activity against the host and relative sensitivity to proteolytic degradation, peptides are often expressed as fusion proteins to neutralize their innate toxic activity and increase their expression levels (wang and others 2011) . compared with isolation from natural sources and the other synthesis methods, the recombinant approach offers the most costeffective alternative for large-scale peptide production (li 2011) . peptides are increasingly being produced for various purposes, and these may contain closely related impurities resulting from incomplete reactions or from several side reactions. peptides synthesized for therapeutic and clinical research, as well as for biological and structural studies to explore the structure-activity relationships must have 95% purity or greater (ridge and hettiarachchi 1998) . however, there are other applications where low values of purity, between 70% and 95%, are tolerable (table 1) . peptide purification depends on a series of separation techniques. peptides made on a preparative scale (in gram amounts) can be obtained from a separation process, to isolate one or more individual components from a peptide mixture for future research, or on an analytical scale (about 1 mg of peptide) to identify and determine the relative amounts of some or all components in the mixture. studies on an analytical scale are the first steps for improving separation conditions, which are developed prior to the execution of any preparative separation process (sewald and jakubke 2002) . after the synthesis process, peptides are submitted to a separation procedure consisting of centrifugation and washing to remove residues of the reagents used, as well as products of side reactions. subsequently, peptides are cleaved and subjected to filtration, as well as lyophilization (dagan and others 2002) . the most widely used methods used for the purification of peptides are reverse-phase high-performance liquid chromatography (rp-hplc), ion-exchange chromatography, size exclusion chromatography, affinity chromatography, and capillary electrophoresis ( table 2) . the purity of a peptide must be verified by a method different from that used for purification, since the results of homogeneity derived from such a system can lead to misinformation and be misleading (ridge and hettiarachchi 1998) . thus, the characterization should be analyzed by different methods of mass spectrometry. mass spectrometry has different ionization methods, such as electrospray ionization mass spectrometry (esi-ms), fast atom bombardment mass spectrometry (fab-ms) or matrix-assisted laser desorption/ionization mass spectrome-try (maldi-ms) that can be used for peptide characterization ( table 3) . the different mass spectrometry techniques are based on the accurate determination of the molecular mass-charge ratio of the peptide, as well as on a chemical structure determination, with high sensitivity and resolution (sewald and jakubke 2002) . the growing resistance of pathogens against many commonly used antibiotics has led to research of new compounds with the same functions. an interesting approach is the study of molecules of natural origin to replace antibiotics (bechinger and lohner 2006) . several studies in recent decades have shown that peptides have certain bioactive properties (agyei and danquah 2011). short peptides (1-50 amino acids) with cationic and hydrophobic properties are known to be potent defenses of the host organism, providing activity against a wide variety of pathogenic microorganisms such as gram-negative and gram-positive bacteria, fungi, viruses, and parasites (hancock and sahl 2006) . studies have shown remarkable results of peptide antitumor activity, observed mainly in cancer therapy (korhonen and pihlanto 2006) . although several peptides have biological activity, antimicrobial activity is one of the most studied. one of the most-used analytical techniques to determine peptide antimicrobial activity is the broth microdilution test. in this test, the microorganisms are cultured in titration microplates and the peptide to be tested is added to each well at different concentrations. the microorganism growth causes turbidity in the wells. however, when a certain concentration of the peptide tested inhibits bacterial growth no turbidity is observed. turbidity is usually read by spectrophotometry, with the greatest frequency at 600 nm, but it can also be seen through visual inspection of the wells (otvos and cudic 2007) . the standard methods developed by the clinical and laboratory standards inst. (clsi) have been used to test the activity of antimicrobial peptides. among them are the standards for antimicrobial disk susceptibility tests (clsi 2003) , the method for dilution antimicrobial susceptibility tests for bacteria that grow aerobically m7-a6 (clsi 2006) , and the method for broth dilution antifungal susceptibility testing of yeast m27-a2 (clsi 2002) , all of which have been widely used (jang and others 2006; rubinchik and others 2009; hwang and others 2010) . the most studied peptides are those with antimicrobial activity, characterized by their interaction with the cytoplasmic membrane of the microorganism regardless of the final target (powers and hancock 2003) . factors influencing the antibacterial activity are the electrostatic interactions between the peptide and positively charged and anionic lipids on the surface of the target microorganism. also, the hydrophobicity of the peptide (factor required for insertion into the membrane) and peptide flexibility allow peptide interaction with the microbial membrane (jenssen and others 2006) . although these characteristics are variable according to each peptide, all of them are essential to the function of peptides as antimicrobials. the exact mechanism of action of antibacterial peptides is not yet fully understood. however, there is a consensus among researchers regarding the first step in the initial interaction between peptide and the target cell (reddy and others 2004) . exclusion liquid chromatography based on separation process according to the size of the peptide relative to pore sizes in the stationary phase. used primarily in the early stages of purification of the peptide, when performed in multiple steps used to separate low-molecular-weight impurities from a mixture of peptides. however, the separation of the peptide of interest with other closely related peptides is virtually impossible affinity chromatography based on the biological specificity of the peptide. consists of a ligand (small specific biomolecule such as an antibody) that is immobilized in the column. the separation occurs because of highly specific biochemical interactions between the peptide and the ligand used when a high degree of specificity is required, for example, isolation of a target protein present in low concentration in a biological fluid or a cell extract capillary electrophoresis based on the migration of the peptide according to its charge in solution, depending on the application of an electric field. complementary technique to reversed-phase chromatography used for peptides and proteins table 3 -ionization methods used in mass spectrometry. fundamental principle the ions are produced from a peptide contained in a solvent (for example, an organic compound such as methanol or acetonitrile) that is scattered in a fine aerosol fab-ms the peptide analyzed is mixed with a matrix, which is a non volatile reagent of protection (glycerol, diethanolamine, and triethanolamine, among others), and is bombarded with a beam of high-energy atoms (4000 to 10000 ev) in a vacuum. atoms are of an inert gas such as argon or xenon maldi-ms the peptide analyzed is bombarded by a laser beam (nitrogen), while a matrix (sinapinic acid) is used to protect the peptide. the matrix allows avoiding direct contact of the peptide with the beam, facilitating its vaporization, and ionization the initial attraction between the peptide and the target cell occurs via electrostatic binding between the cationic peptide and the components of the negatively-charged outer cell membrane, such as lipopolysaccharides in gram-negative bacteria or lipoteichoic acid on the surface of gram-positive bacteria (jenssen and others 2006) . this electrostatic interaction removes the native divalent cations (mg 2+ , ca 2+ ) from the cell surface, thus destabilizing the outer membrane and facilitating the entry of the peptide and subsequent peptide contact with the cytoplasmic membrane, a process known as autopromoted uptake (powers and hancock 2003) . after the peptide is bound to the target cell, an arrangement of the peptide occurs on the surface of the cytoplasmic membrane. this fact is of considerable debate, since several arrangement models have been proposed, such as the barrel-stave or the carpet model among others. depending on the model, the peptide can permeabilize the cytoplasmic membrane and/or translocate through it. thus, antimicrobial peptides can be classified into 2 major groups, the first consisting of those peptides which act on the cytoplasmic membrane, and the second consisting of those which have no action on the cytoplasmic membrane of the target microorganism. this means that the peptide just moves into the cell without causing major disturbances in the membrane (powers and hancock 2003; jenssen and others 2006) . peptides acting on the bacterial membrane. several models have been proposed to explain how, after initial attachment, antibacterial peptides are distributed on the surface of the bacterial cytoplasmic membrane to form pores. pore formation results in membrane permeabilization, thereby affecting cellular respiration. it also deprives the microorganisms of their source of energy by interrupting the electrochemical gradient and causing an increase in the flow of water and ions across the membrane, thus leading to cell swelling followed by cellular lysis (bechinger and lohner 2006) . to explain the formation of pores, the aggregate toroidal pore, barrel-stave, and the carpet models have been proposed. the last 2 models, the barrel-stave and carpet model, have been the most widely studied. barrel-stave model. this model describes the formation of a transmembrane channel (pore) through the binding of amphipathic α-helices. the hydrophobic surface of the peptide interacts with the lipid core of the membrane, while the hydrophilic surface of the peptide is oriented inside, producing an aqueous pore (figure 4) . the progressive recruitment of additional peptides to the membrane surface increases the size of the pores, causing the loss of cell content and thus cell death (reddy and others 2004) . this model has been proposed to explain the activity of antimicrobial peptides, such as magainins (matsuzaki and others 1998) . carpet model. in this model, the peptide in high concentration is in contact with phospholipids located on the outer surface of the bacterial membrane, a fact that allows the peptide to permeate the membrane ( figure 5 ). the peptides bind to the surface of the target membrane and cover it like a carpet. according to this model, the peptides exhibit a preferential binding for the phospholipid groups. the binding step is followed by the alignment of the peptide on the membrane surface so the hydrophilic surface is in contact with phospholipid or water molecules, causing a reorientation of hydrophilic residues and creating a hydrophobic core. finally, the peptide disintegrates the membrane by deformation of the membrane curvature (reddy and others 2004) . this model has been proposed to explain the action mechanism of dermaseptins (dagan and others 2002) . toroidal pore model. this model is considered a variant of the barrel-stave model. it is suggested that a perpendicular inclusion of the peptides to the membrane with their hydrophilic regions is associated with phospholipids, whereas their hydrophobic regions are associated with the lipid core. in this process, the membrane is bent inward so the pores are formed (jenssen and others 2006) . the main difference between this model and the barrel-stave model is the intercalation of the peptide with phospholipids to form the pore ( figure 6 ). this model has been used to explain the mechanism of action of the peptide melittin (yang and others 2001) . aggregate model. this model, proposed by wu and others (1999) , has some similarity to the toroidal pore model. this model consists mainly of the arrangement of the peptide in the membrane forming an extension and developing micelle-like aggregate of peptides and lipids, but without adopting a particular orientation (jenssen and others 2006) . peptides with no activity on the bacterial membrane. these antimicrobial peptides have the ability to translocate into bacterial cells without causing membrane permeabilization. the peptide is accumulated within the cell where it reaches a variety of essential cellular processes that result in bacterial cell death (jenssen and others 2006) . the target process includes inhibitions of nucleic acid synthesis, protein synthesis, enzyme activity, and cell wall synthesis. peptides such as buforin ii and pleurocidin have shown this mechanism of action (park and others 1998; patrzykat and others 2002) . there is a growing need for new antifungal agents due to the increased resistance of molds to therapies with regularly used compounds (de lucca and walsh 1999) . peptides have emerged as alternative antifungal agents. initially, the antifungal mechanism of action was described as a result of fungal cell lysis or as a result of interferences in fungal cell wall synthesis. however, the discovery of new antifungal peptides in the last decade has led to the identification of new mechanisms of action, including membrane permeabilization, binding to ergosterol/cholesterol in the fungal membrane, the attack of mitochondria or other intracellular organelles, and the deformation of cell membrane structure (jenssen and others 2006) . according to de lucca and walsh (1999) , fungal peptides can be classified with respect to their mode of action into 3 groups: peptides that act through cellular lysis; peptides that cross the fungal membrane and interact with the intracellular target; and peptides that act by forming pores. peptides acting by cellular lysis. these peptides are characterized by their amphipathic nature, being molecules with 2 faces, one positively charged and the other neutral and hydrophobic. some of these peptides bind only to the membrane surface, damaging the membrane structure, and they may or may not pass through it. peptide smap-29 (a synthetic peptide derived from the sequence of cathelicidins) has shown antimicrobial activity against the fungus trichosporon beigelii by interaction, penetration, and subsequent damage to the cell membrane (lee and others 2002) . this result suggests that the main target of smap-29 peptide is the fungal plasmatic membrane. a similar mechanism was observed for the synthetic peptide ib-amps (an analogue sequence to peptides isolated from seeds of impatiens balsamina), showing antifungal activity by bonding the peptide to the fungal cell membrane and subsequent penetration (thevissen and others 2005) . peptides that pass into the membrane and interact with intracellular targets. these peptides interfere with cell wall synthesis or the synthesis of essential cellular components such as chitin or glucan. as such, the synthetic peptide omiganan (an indolicin analogue peptide, isolated from bovine neutrophils) has shown antifungal activity against candida albicans, and the main mechanism of action of this peptide is related to its activity in the cytoplasmic membrane, resulting in macromolecules synthesis inhibition of macromolecules and finally cell death (rubinchik and others 2009) . pore-forming peptides. these peptides are aggregated in a selective way to form pores of varying sizes, which then allow the passage of ions and other solutes. the synthetic peptide di-k19hc (a halocidin analogue peptide, isolated from the invertebrate marine animal halocynthia aurantium known as sea peach), has shown antifungal activity against several strains of aspergillus and candida (jang and others 2006) . the activity of di-k19hc results in the formation of pores on the surface of fungal membranes. moreover, these researchers pointed out the specific binding of di-k19hc with b-1,3-glucan, a component of the cell wall of fungi. this mechanism has also been observed with the antifungal peptide psacotheasin, isolated from the yellow-spotted long-horned beetle (psacothea hilaris), which has shown activity against c. albicans (hwang and others 2010) . the researchers indicated that there was damage to the cell wall, membrane depolarization with the formation of pores (2.3-3.3 nm), as well as an increase in membrane permeability, all being responsible for the antifungal activity of this peptide. several studies have shown the ability of cationic peptides to inhibit viral infections. the peptide cecropin a has shown antiviral activity against junin virus (jv-which causes argentine hemorrhagic fever). the peptide melittin inhibited jv and herpes simplex virus 1 (hsv-1) multiplication, as well as magainin i and ii, and has shown inhibitory activity against hsv-1 and hsv-2 (albiol-matanic and castilla 2004). antimicrobial peptides isolated from fish, such as tilapia hepcidin 1-5, have shown activity against the nervous necrosis virus (nn virus), an infectious agent that causes mass mortality of several species of marine fish in the larval stage (chia and others 2010) . in addition, synthetic peptides consisting of arginine and tryptophan repetitions have shown activity against vaccinia virus (the cause of cowpox) (mohan and others 2010). the antiviral activity of peptides is often related to virus adsorption and its entry into the host cell or, in other cases, is the result of a direct effect on the viral envelope. thus, the antiviral activity of peptides may result from multiple mechanisms of action, the most important being blocking virus entry through interaction with the host cell and blocking viral entry through interaction with the virus. blocking viral entry through interaction with the host cell. peptides can interact directly with specific viral receptors on the host cell, thus preventing the virus from binding to the cell membrane or binding intracellularly (jenssen and others 2006) . proteoglycans are proteins found in all types of tissue, in intracellular granule secretions as well as in the extracellular matrix and cell surface. proteoglycans are covalently linked to one or more chains of glycosaminoglycans (gag), long polysaccharide unbranched structures, which have a sugar that contains nitrogen and are usually sulfated. gag chains are present on the surface of mammalian cells and their degree of sulfation makes these compounds more anionic. this network of strong negative charges allows gag to attract and bind to small cations, such as enzymes and proteins, and also pathogens such as viruses (spillmann 2001) . heparan sulfate, one type of gag chain, is one of the most important molecules related to viral binding (spillmann 2001) . thus, by blocking heparan sulfate molecules can be inhibited viral infection. jenssen and others (2006) have suggested that antimicrobial peptides which interact with heparan sulfate have the ability to block a number of viral infections. due to the large number of amino acid residues positively charged peptides can interact electrostatically with negatively charged heparan sulfate molecules on the cell surface. studies on lactoferrin (lf) have shown that this peptide prevents infection of the host cell rather than inhibiting virus replication after infection of the target cell. the interaction of lf with heparan c 2012 institute of food technologists ® vol. 11, 2012 r comprehensive reviews in food science and food safety 193 sulfate molecules has been proposed as the mechanism responsible for lf antiviral activity (van der strate and others 2001). similarly, jenssen and others (2006) showed the antiviral activity of synthetic peptides (consisting of arginine and lysine residues) against herpes simplex virus 1 and 2 (hsv-1 and hsv-2). the peptides presented higher affinity in binding to heparan sulfate with an increasing number of cationic residues, thereby blocking the entry of hsv (-1 or -2). in addition, luganini and others (2010) reported the inhibition of cytomegalovirus by binding synthetic peptide dendrimers with molecules of heparan sulfate on the surface of fibroblasts and endothelial cells. thus, cytomegalovirus infection was blocked by the interaction of synthetic peptide binding sites with heparan sulfate. blocking viral entry through interaction with the virus. the interactions of peptides with the glycoproteins (gp) in the viral envelope have been proposed as another mechanism that influences the process of viral entry and virus inactivation. in this way, peptides generated from chemical modification of milk proteins, such as α-lactalbumin, β-lactoglobulin, and lysozyme with 3-hydroxyphthalic anhydride (3-hp) inhibited infection of vero cells with hsv-1 (oevermann and others 2003) . according to those researchers, the antiviral activity of these peptides is based on their direct interaction with viral glycoproteins (gb, gc, gd), which are responsible for adsorption and penetration of the virus into the host cell. similarly, lf has shown the ability to bind to the gp120 glycoprotein (a protein present in the outermost layer of the hiv virus) with antiviral effects, since the gp120 glycoprotein plays an important role in the adsorption and entry of hiv into target cells (van der strate and others 2001; pan and others 2006) . on the other hand, other peptides, such as magainins, have shown antiviral effects through direct interaction with virus cells. egal and others (1999) have indicated that the effect of magainins is the result of the peptide acting on the viral envelope. a similar mechanism was suggested for the activity of mucroporin-m1, a defense cationic peptide present in scorpion venom, which has shown activity against the measles virus, the coronavirus that causes severe accurate respiratory syndrome (sars), and flu virus h5n1 (better known as the bird flu virus) (li and others 2011) . the researchers have suggested that the antiviral activity of the peptide is the result of direct interaction with the virus envelope, thereby reducing viral activity in the host cell. cancer, also known as malignant neoplasm, is a general term that refers to more than 100 different diseases affecting various tissues and different types of cells. all forms of cancer are characterized by abnormal cell growth, that is, they lack the mechanisms that control normal cell division. this lack of regulatory mechanisms is the result of a multistep process involving genetic mutations induced by inheritance or environmental changes (hütter and sinha 2001) . despite major advances in cancer therapy, there is considerable interest in the development of antitumor agents with a novel mode of action, since the cells have shown carcinogenic development of resistance to current chemotherapy (hoskin and ramamoorthy 2008) . carcinogenic cells often become resistant to chemotherapy. this mainly occurs as a result of increased expression of intracellular enzymes for the detoxification of antitumor agents, the correction of dna damage, generation of intracellular organelles with the ability to eliminate and/or transport the drugs out of the tumor, and irreversible defects in the cellular machinery that mediates apoptosis (hütter and sinha 2001) . thus, recent studies have shown peptides as an alternative to conventional cancer treatments. however, not all peptides have selective activity against carcinogenic cells. according to hoskin and ramamoorthy (2008) peptides that have antitumor activity can be classified into 2 major groups: peptides with selective activity, and peptides with non selective activity, that is, those that have activity against bacteria, carcinogenic cells, and healthy cells. peptides with selective activity toward carcinogenic cells. these peptides have activity against bacteria and carcinogenic cells, but not against normal cells. several peptides, such as the cecropins, buforins, and magainins have shown antitumor activity without affecting normal eukaryotic cells (cruciani and others 1991; cho and others 2009) . studies with magainin ii have shown to inhibit the proliferation of carcinogenic cells (in bladder cancer) without any effect on normal cells (lehmann and others 2006) . similar results were observed by chen and others (2009) in the study of the synthetic peptide th2-3 (isolated from tilapia and analogous to the peptide hepcidin), with antitumor activity shown primarily by direct interaction and lysis of target carcinogenic cells (human fibrosarcoma cells). these researchers indicated that the lytic activity of the peptide and proliferative cells were restricted mainly to carcinogenic cells, since normal cells showed no significant effects. likewise, the synthetic peptide th1-5 (isolated from tilapia and an analogue to the peptide hepcidin) has shown antitumor activity against carcinogenic cells, due to interaction with and penetration of the membrane. this peptide has less toxicity toward normal cells supposedly because it can discriminate between healthy cells and carcinogenic ones (chang and others 2011) . researchers have also indicated that the interaction with the cell membrane and its subsequent damage is caused by the formation of pores on its surface. it has been suggested that the internalization of the peptide and the subsequent damage to the mitochondrial membrane activates apoptotic pathways (chang and others 2011) . according to hoskin and ramamoorthy (2008) there are fundamental differences between the membranes of malignant cells and normal cells which allow the selectivity of certain peptides to attack carcinogenic cells without affecting healthy cells. electrostatic interactions between cationic peptides and anionic components of the cell membrane have also been considered an important factor. carcinogenic cells typically have a negative charge due to a higher expression than normal of anionic molecules such as phosphatidylserine (ps) and mucin (glycoprotein) (oren and shai 1997) . however, normal cells are not affected, since these cells have a neutral surface charge, conferred by the zwitterionic nature of most membrane components such as phosphatidylethanolamine (also known as cephalin), phosphatidylcholine, and sphingomyelin (sok and others 1999) . membrane fluidity and the surface area of the cell are also considered factors that contribute to the selectivity of peptides for carcinogenic cells. the fluidity of carcinogenic cells is greater than that of normal cells, which may increase the activity of lytic peptides through the easy destabilization of the membrane. in addition, the carcinogenic cells have a higher surface area than healthy cells due to the presence of greater numbers of microvilli, which are small projections of the cell membrane, irregular in size and shape. the microvilli may allow the bonding between peptide and carcinogenic cells (hoskin and ramamoorthy 2008) . peptides with nonselective activity. this group is comprised of peptides with activity against bacteria, carcinogenic cells, and against normal eukaryotic cells (hoskin and ramamoorthy 2008) . according to papo and shai (2003) non selective activity of these antimicrobial peptides results from their ability to interact with and cause damage to negatively charged membranes and those of a zwitterionic nature. dathe and others (1997) have indicated that the hydrophobic moment of antimicrobial peptides exerts a substantial influence on the neutral lipidic membranes, although it has a small role in the permeabilization of highly charged lipid membranes. peptides of this group include melittin, isolated from bee venom; taquiplesin ii, isolated from the horseshoe crab; defensins, isolated from insects; and plantaricin, a bacteriocin isolated from lactobacillus plantarum (schweizer 2009 ). plantaricin has shown activity against carcinogenic cells and against normal lymphocytes and neuronal cells (sand and others 2010) . the mechanism of action of antitumor peptides consists of permeabilization of the cell membrane mediated by electrostatic interaction. the electrostatic interaction is generated by the negatively charged phospholipids in the cell membrane and the positively charged peptide (schweizer 2009 ). unlike carcinogenic cells, eukaryotic cells have most of their negatively charged phospholipids, particularly ps, in the inner membrane, while neutral lipids are positioned on the outside (zhao and others 2006) . however, the result obtained by sand and others (2010) suggests that in addition to the mechanism of action related to the electrostatic interaction, there is another mechanism which explains the sensitivity of normal eukaryotic cells to plactaricin. probably another negatively charged macromolecule present on the membrane surface of healthy cells is also involved in plantaricin activity. similar results were observed by nan and others (2010) in the study of synthetic peptides consisting of lysine or arginine enriched with tryptophan. the peptide with arginine residues showed higher toxicity against human erythrocytes and mammalian cells. the hydrophobicity of the peptides has been suggested as an important factor in the increase of hemolytic activity and cytotoxicity in mammalian cells, as hydrophobic regions are required for direct interaction between peptide with membrane lipid components. the peptide with arginine residues was slightly more hydrophobic than the peptide with lysine residues. thus, these researchers suggested that small differences in hydrophobicity of these peptides may be responsible for the cytotoxic activity of this peptide in mammalian cells. the growing problem of microorganism resistance to conventional antibiotics, as well as the need for new agents with antibiotic properties has stimulated interest in developing antimicrobial peptides aiming for their application in the medical field (zasloff 2002) . most of the studies are devoted to the development of topical agents with antibacterial and antifungal activities. also, due to their antiviral activity, antimicrobial peptides have also been proposed as chemical preservatives. in the food industry, antimicrobial peptides, especially those produced by bacteria, have been widely researched in recent years due to their potential use as natural preservatives (papagianni 2003; coma 2008; settanni and corsetti 2008) . the direct application of antimicrobial peptides in food preservation can be achieved by 2 methods: the direct addition of peptide to the food matrix, or the inoculation of the food matrix with the bacteriocin producer strain under the conditions favorable for the in situ production of the antimicrobial peptide. bacteriocins can be obtained ex situ by the cultivation of the producer strain at an industrial scale in a food-grade substrate, followed by a series of separation and purification techniques. these ex situ bacteriocins are commercially available in concentrated form, such as alta tm 2341 or microgard tm , and can be added directly to the food matrix. the production of bacteriocins in the food matrix offers several legal and cost advantages. the use of bacteriocin producer strain requires careful selection depending on the particular food intended for inoculation to ensure the producer strains will produce bacteriocins in the necessary amounts to inhibit the target microorganism. in addition to the peptides being studied as antimicrobial agents for direct addition to foods, they also have shown potential for being incorporated into food preparation surfaces (such as cutting surfaces) and processing equipment, as well as in food packaging (appendini and hotchkiss 2002) . active packaging includes the incorporation of antimicrobial agents in the packaging material to control and extend the shelflife of food (soares and others 2009a). these types of packaging are considered an innovative technology in food preservation, since they allow better antimicrobial efficiency on food surfaces, thus improving stability. the development of active packaging by incorporating antimicrobial peptides in food packaging material can be done either to prolong the life of the product or to reduce the microbial load of the packing before use (steven and hotchkiss 2008) . the development of active packaging with antimicrobial peptides can be accomplished by 3 main methods of incorporation: direct peptide incorporation in the polymer; peptide coating on the polymeric surface; and peptide immobilization in the polymer. numerous studies have reported the incorporation of antimicrobial peptides directly in the polymeric material, especially bacteriocins. the peptides are relatively resistant to heat (appendini and hotchkiss 2001) . however, their antimicrobial activity may be greater when heat is not used in the incorporation process. moreover, bioactive peptides incorporated in polymer films must be able to diffuse to the package surface over time to be effective. thus, polymers such as cellulose acetate, alginate, chitosan, and soy protein, among others, have been widely used to develop films with direct incorporation of these antimicrobials (marcos and others 2008; pires and others 2008; sivarooban and others 2008; santiago-silva and others 2009). researchers have studied the antimicrobial activity of bacteriocins incorporated into polymeric materials in synergy with other antimicrobial agents. synergistic activity against staphylococcus aureus, listeria monocytogenes, and bacillus cereus has been observed for nisin with potassium sorbate and garlic oil when incorporated into chitosan films (pranoto and others 2005) . in addition, soy protein films incorporated with nisin, grape seed extract, and ethylenediaminetetraacetic acid (edta) have shown inhibitory synergistic activity against pathogenic microorganisms such as l. monocytogenes, e. coli o157: h7, and salmonella typhimurium (sivarooban and others 2008) . the activity of bacteriocins incorporated into polymeric materials in synergy with other conservation technologies has also been reported. films incorporated with enterocins a and b (bacteriocins produced by enterococcus faecium) have shown synergistic activity when used together with high-pressure processing. thus, the use of antimicrobial packaging developed in conjunction with the high-pressure process allowed the control of l. monocytogenes at below detectable levels after 90 d of storage at 6 • c (marcos and others 2008) . this is an alternative method when the polymer requires extreme processing conditions during packaging material manufacture, such as high pressure and temperature, which can result in inactivation of the antimicrobial agent (appendini and hotchkiss 2002) . in some cases, the antimicrobial coating is done by contacting the film with or immersing it in the peptide solution. in this way, linear low-density polyethylene (lldpe) has been coated with lactocin 705 and lactocin al705 (both bacteriocins produced by lactobacillus curvatus crl705), by direct contact of the film with a bacteriocin solution, showing antimicrobial activity in vitro against lactobacillus plantarum crl691 and listeria innocua 7 (massani and others 2008) . similarly, scannell and others (2000) used alternatively lacticin 3147 and nisin adsorbed on the surface of plastic bags (polyethylene/polyamide) through direct contact of the polymeric material with bacteriocin solution. the film coated with nisin showed inhibitory activity against l. innocua and s. aureus, maintaining its activity for 3 mo either at room temperature or under refrigeration. however, the film coated with lacticin 3147 did not show antimicrobial activity. the researchers suggested that lacticin 3147 was not retained by the polymer (scannell and others 2000) . proper handling of solvents and polymeric structures has been suggested to increase the adsorption of the peptide into the polymer matrix (appendini and hotchkiss 2002) . for example, the polymeric surface can be coated by applying a filmogenic solution that can be deposited on the film surface by the casting method. accordingly, chollet and others (2009) developed a laminated film of polyethylene (pe) and polyamide (pa), with the structure pe/pa/pe, coated with a filmogenic solution of hydroxypropyl methyl cellulose (hpmc) and adsorbed with nisin. the developed film presented antimicrobial activity in vitro against kocuria rhizophila (chollet and others 2009) . peptides can be immobilized or attached to solid supports by physical methods, such as layer-by-layer assembly, or by chemical methods, such as covalent bonding (onaizi and leong 2011) . layer-by-layer assembly. in this process, the peptide is sandwiched between 2 polyionic polymers and the number of peptides and polymers is flexible (figure 7) . the effectiveness of the peptide depends on its relative mobility. the advantage of this method is that it allows the slow release of the peptide embedded in the surface of the polymer. however, a key drawback of this method is that the peptide immobilized in the layers closest to the solid support will not be in direct contact with the target surface, thus reducing peptide activity. this peptide must be able to diffuse through the different layers of the assembly to the interface (onaizi and leong 2011) , to ensure efficient release and consequent bioactivity. the diffusion process of the peptide in the different layers is more complex than its diffusion in solution, since additional factors such as tortuosity of the diffusion path, the number of layers, and the polymer-peptide interactions can affect the diffusion process (appendini and hotchkiss 2002; sukhishvili 2005) . covalent bonding. in this process, the antimicrobial peptide will react chemically with a given surface to form a stable bond, which results in the formation of an antimicrobial coating on the polymeric surface (haynie and others 1995) . covalent bonding offers several advantages, including a more stable attachment between the peptide and the polymer surface (goddard and hotchkiss 2007) . covalent bonding reduces attached peptide ability to destabilize and improves its bioactivity, by protecting it from denaturation. due to the inert nature of most polymers, they must be subjected to a functionalization process on the surface before bonding with the peptide. the polymers can be functionalized with different spacers, which are reactive functional groups that allow peptide attachment on the spacer surface (humblot and others 2009) . the quantity of reactive functional groups generated in the functionalization process results in a restricted number of covalent bondings, which limits the amount of peptide that can be attached to the packaging. according to goddard and hotchkiss (2007) , direct bioactive compound applications require small amounts to be effective. however, for the applications of peptides in polymeric matrixes it is necessary to maximize the amount of peptides per unit area. to accomplish this, the functionalization technique must be optimized with the objective of linking the desired type and quantity of reactive functional groups. stiff or flexible spacers have been used as reactive groups for the functionalization of polymers (figure 8 ). stiff spacers, such as polymethyl-methacrylate (pmma) and polyvinyl chloride (pvc), restrict the lateral mobility of the peptide bond, keeping the peptide firmly in a specific orientation. on the other hand, flexible spacers, such as polyethylene glycol (sand and others 2010) allow lateral mobility of the peptide bound, which can result in different orientations of the peptide molecules at the interface (onaizi and leong 2011) . among the different types of spacers, polyethylene glycol (peg) is widely used in the immobilization of peptides. the use of peg has several advantages, such as rapid and free peptide orientation, promoting peptide-bacteria interactions (costa and others 2011) . although the potential penetration and translocation of peptides through the microorganism cytoplasmic membrane is low due to the covalent bond that attaches the peptide to the polymer, it has been reported that peptides have sustained their bioactivity after attachment to polymeric matrixes. the synthetic peptide 6k8l (a peptide sequence derived from magainin) was covalently bound to polystyrene (ps) resin by functionalization with peg and showed antimicrobial activity against foodborne pathogenic microorganisms, including e. coli o157: h7, l. monocytogenes, and pseudomonas fluorescens (appendini and hotchkiss 2001) . similarly, the synthetic peptide e14lkk was covalently immobilized in ldpe film after chromium oxidation and functionalization with peg. this active packaging had antimicrobial activity against e. coli showing a reduction of 3 log cycles when compared with the control (steven and hotchkiss 2008) . the sustained bioactivity of attached peptides is caused by the presence of spacers which allow sufficient freedom of motion for the active portion of the peptide to contact microorganisms on the food surface (appendini and hotchkiss 2002) . haynie and others (1995) have previously demonstrated that peptide-bacteria interactions are sufficient for peptide bioactivity. moreover, costa and others (2011) have indicated that the efficacy of attached peptides could possibly result from a higher peptide-relative surface availability, contrary to the other methods of peptide applications in which peptide aggregation can occur, producing uneven distribution. on the other hand, the diffusion of attached peptides into the food surface is restricted due to the covalent bonding. however, diffusion to the food product can occur in extreme conditions, such as high temperatures, which can promote hydrolysis reactions. the characterization of active packaging involves 2 processes: structural analysis and measurement of their properties (table 4) . according to goddard and hotchkiss (2007) , the type of analytical tool used in the structural characterization of polymers depends on the kind of modification, the specificity required, and available resources. some of the techniques used in structural analysis of active packaging with antimicrobial peptides are the contact angle, x-ray photoelectron spectroscopy (xps), fourier transform infrared spectroscopy (ftir), and scanning electron microscopy (sem). steven and hotchkiss (2008) used the techniques of contact angle and xps to assess changes in the surface of ldpe films after treatment with chromic oxide and functionalization with peg as spacer, subsequently covalently binding a synthetic peptide. the contact angle for the film before being subjected to any process of functionalization showed values of 101 • . however, after chromic oxidation and peg bonding, films presented values of 61 • and 45 • , respectively; which indicated that the film surface became hydrophilic. these researchers concluded that the decrease in the contact angle is the result of an increased ionization at the film surface after oxidation, due to the presence of functional groups, such as carboxylic acid (cooh); and a value even lower after functionalization was observed due to solubility of peg. the xps technique showed changes in chemical composition on the film surface, resulting in the detection of nitrogen (2.6%) and an increased percentage of oxygen (initially from 6.7% to 13.3% after oxidation and 19.4% after functionalization). the oxygen increase was due to the presence of carboxylic acid after chromic oxidation. also, this increase was the result of functionalization with peg due to the main presence of o 2 in the peg chain backbone. in addition, the functionalization with peg also introduced nitrogen originating from its amino-terminal functions (nh 2 -peg-nh 2 ). the ftir spectroscopic technique has been used by pranoto and others (2005) to study the interactions between chitosan films and nisin. they observed an increase in the band of the amide i corresponding to the wave number 1638 cm −1 related to the increased concentration of nisin incorporated in the film. according to the researchers, this is probably due to the interaction between the amine functional groups of chitosan and functional groups of nisin, which resulted in covalent bonds, and consequently in a larger peak. microscopic techniques have also been widely used to evaluate morphological changes in the surface of films that have been incorporated with antimicrobial peptides. pires and others (2008) used sem and observed that cellulose-based films incorporated with nisin, or a mixture of nisin and nantamicin, showed crystals deposited on the surface. these results indicated a heterogeneous distribution of the peptide in cellulosic films, while the control film presented a homogeneous structure. similarly, santiago-silva and others (2009) used sem to observe changes in surface morphology of cellulose acetate film incorporated with pediocin. when the concentration of peptide was increased, the films incorporated with pediocin had a rough surface c 2012 institute of food technologists ® vol. 11, 2012 r comprehensive reviews in food science and food safety 197 table 4 -characterization techniques of packaging incorporated with antimicrobial peptides. technique factor studied structural analysis contact angle quantifies surface hydrophobicity by measuring how far a droplet of water spreads on a surface x-ray photoelectron spectroscopy (xps) determines the atomic composition of the top several nanometers of a solid. this technique can be used to quantify the percent atomic composition and stoichiometric ratios fourier transform infrared spectroscopy (ftir) detects and identifies the chemical functional groups present in the polymer scanning electron microscopy (sem) allows the characterization of the polymer surface morphology and the observation of the dispersion quality of the peptide in the polymeric matrix property measurements mechanical properties measurement of the mechanical performance of the polymer. generally according to the standard method astm d882 (astm 2010a) barrier properties measurement of water vapor permeability. generally according to the standard method astm e96/e96m (astm 2010b); astm f1249 (astm 2006) due to large amounts of pediocin granules dispersed in the matrix. this resulted from the lack of peptide solubility. on the other hand, the control film showed a homogeneous and transparent surface. in addition to the analysis of structural changes and interactions between the peptide and the polymeric matrix, the study of packaging properties is important as well. these properties show the performance of the developed material and how it will relate to the primary functions of food packaging, such as physical integrity. thus, mechanical and barrier properties have become increasingly relevant and are more frequently studied. the mechanical properties of films incorporated with antimicrobial peptides serve as the basis for assessing the effects on the mechanical performance resulting from the modification made to the polymer (table 5) . guiga and others (2009) investigated the effect of nisaplin ® (2.5% purity of nisin) coating on the mechanical properties of laminated films (pe/pa/pe), studying the mechanical properties of elongation at break and young's modulus. their results showed a significant difference in mechanical properties between the films incorporated with the peptide and the control treatment; peptideincorporated films showed an increase of young's modulus and a decrease in elongation at break. the polymer-based coating (hpmc) applied in laminated film, as well as the interaction between proteins and salts present in nisaplin, may have modified the mechanical behavior of the manufactured packaging, thereby increasing the rigidity of the film with the consequent decrease in its elongation. a similar result was observed by santiago-silva and others (2009) in their study of cellulosic films incorporated with pediocin. the researchers indicated that the addition of 25% of the peptide increased the maximum load required for film rupture when compared to the control. the researchers pointed out that a possible interaction between the pediocin and the polymeric matrix allowed the development of a more resistant film. however, a significant drop in the maximum load value was observed at a 50% concentration. according to the researchers, there was an excessive amount of the peptide incorporated, which weakened the cellulose chains of the film and resulted in a reduction of film resistance. a decrease in the mechanical strength of films incorporated with antimicrobial peptides has also been observed. sivarooban and others (2008) reported a decrease in puncture resistance and tensile strength values of soy protein films incorporated with nisin. similarly, pires and others (2008) indicated that nisin incorporated into cellulose-based films affected the film structure, reducing maximum load and elongation values. these resulted from the heterogeneous distribution of the peptide in the polymeric matrix, which consequently lead to the formation of stress points and reduced film resistance. although several studies have indicated changes in film properties, in some cases peptide incorporation into polymeric matrices had no significant effects. massani and others (2008) reported no significant difference in tensile strength, elongation, and water vapor permeability of ldpe films coated with lac-tocin705 and lactocin al705. similar results were observed by chollet and others (2009) who indicated that the incorporation of nisin into pe/pa/pe laminated films, by coating with hpmc, showed neither changes in tensile strength at break nor in water vapor permeability. similarly, guiga and others (2010) reported that the direct incorporation of nisin into multilayer films of ethyl cellulose (ec) and hpmc (ec/hpmc/ec) did not alter the properties of tensile strength, young's modulus, or elongation at break. barrier properties of packaging include resistance to water vapor or gases (o 2 and co 2 ). the water vapor barrier property of the packaging can be determined by calculating the water vapor permeability (wvp) or the water vapor transmission rate (wvtr). both parameters cover the determination of the passage of water vapor through a polymeric material. however, the wvp considers vapor pressure difference between 2 specific surfaces (internal and external) of the analyzed packaging (astm 2010b). both parameters, wvp as well as wvtr, have been used to indicate that the addition of nisin in different polymeric matrixes, such as ldpe film coated with a cellulose-based solution (grower and others 2004; massani and others 2008) , sodium caseinate films (kristo and others 2008) and pe-film coated with hpmc (chollet and others 2009), causes no significant changes in the water vapor barrier property. on the other hand, studies regarding gas permeability in active packaging have been limited due to their applications as films or coatings for food products. during the evaluation and characterization of antimicrobial packaging it is important to research the transference of antimicrobial substances from the packaging material into the food, since this information allows the determination of how the antimicrobial agent is released from the active packaging. the mass transfer can occur by the diffusion mass transfer mechanism or by the convective mass transfer mechanism. the convective mass transfer mechanism occurs in a moving fluid, known as natural convection, if the movement is caused by differences in the density, or as forced convection, if the movement is caused by external agents or when a fluid is flowing on the solid surface by forced movement. on the other hand, diffusion mass transfer consists of a random motion of individual molecules as a result of a concentration gradient (crank 1975; geankoplis 1993) . the migration of substances from packaging materials takes place through the diffusion mass transfer mechanism, since the active packaging and the food contain a concentration gradient for the antimicrobial agent incorporated in the packaging. unlike the research on the release of active substances from drugs or the release of solvents from polymers, the study of antimicrobial release from active packaging is still limited (buonocore and others 2003; bastarrachea and others 2010) . the knowledge of diffusion parameters allows an efficient design of active packaging. several factors must be considered when studying the migration from antimicrobial packaging, including the release rate of the antimicrobial molecules from the packaging. if this rate is high, the active packaging would release the antimicrobial rapidly, resulting in a large concentration at a determined time. however, the large concentration would not be maintained over time, depending on the solubility of the antimicrobial in the selected food. if the solubility is very high, the antimicrobial will migrate rapidly to the food matrix, and therefore result in a decreased concentration of the antimicrobial on the food's surface along time. on the other hand, if the release rate is low, the antimicrobial agent will be slowly released in a desired concentration and if it presents a low solubility in the selected food, the antimicrobial can accumulate on the food surface and slowly migrate into the food matrix. in this situation the release rate should not be slower than the microbial growth (bastarrachea and others 2011) . in either case, the release of the antimicrobial agent from the packing material is indicated by the diffusion coefficient (d). thus, the diffusion characteristics of the antimicrobial agent can be used to determine the amount needed to maintain the proper concentration on the food surface (buonocore and others 2003) . the literature reports a few migration studies of antimicrobial peptides incorporated in active packaging. most evaluate the migration of nisin, probably due to the fact that this is the only antimicrobial peptide substance indicated as generally recognized as safe (gras) for direct contact with food in the united states (fda 2011). nisin is also widely accepted as a food preservative in the european community where it is classified as a safe preservative for food contact, coded as e 234 (fsa 2010), as well as in brazil where the use of nisin is permitted by brazilian law as a natural preservative for biological products (anvisa 1996) . diffusion of several antimicrobial agents, such as potassium sorbate or lysozyme incorporated in active packaging, has been explained by fick's second law (han and floros 1998; gemili and others 2009) : where, c is the diffusing substance concentration; d is the apparent diffusion coefficient; t is the diffusion time; and x the distance. depending on the conditions of the migration test, different analytical solutions have been applied to solve fick's second law and to calculate the d-value in the migration of the antimicrobial peptide nisin incorporated in different types of packaging materials (table 6) . different analytical solutions of fick's second law have been used in previous studies to calculate the d-value of nisin at a specific temperature ( table 7) . some of these studies were conducted at different temperatures to characterize the d-value as a function of this parameter. protein films (corn zein or wheat gluten) and poly(butylene adipate-coterephthalate) (pbta) films incorporated with nisin showed an increase in d-value with increasing temperature, indicating that the peptide concentration was higher in the simulant at equilibrium state with increasing temperature (teerakarn and others 2002; bastarrachea and others 2010) . similarly, at low temperatures, lower d-values of nisin diffusivity indicate that the film retains larger amounts of the peptide in the polymer matrix while in contact with the simulant. the arrhenius activation energy model (eq. 2) has been shown to confirm the dependence of the diffusivity with respect to temperature. where, d 0 is a constant; e a is the activation energy for the diffusion process (j/mol); r is the universal gas constant (8.314 j/mol k); and t abs is the absolute temperature (k (−1) n ierfc nh 2 √ d t m 0 , is the initial amount of nisin in the film; m t , released amount of nisin at time t; h, film thickness; d, diffusion coefficient; ierfc, associated function of the mathematical error function teerakarn and others (2002) paper coated with acrylic polymer and ethylene-vinyl acetate co-polymer (eva) is the amount of nisin released at time t; m ∞ , is the migration in a state of equilibrium; l p coating layer thickness, d, diffusion coefficient kim and others (2002) hydroxypropyl methyl cellulose (hpmc) films is the amount of nisin in the simulant at time t; m f,0 , amount of nisin in the film when t = 0; α, mass ratio between the amount of nisin in the simulant and in the film at equilibrium; q n , is the ''n" root of tanq n = −αq n ; l, is a half of the film's thickness bastarrachea and others (2010) teerakarn and others (2002) indicated e a values of 85.8 and 53.1 kj/mol for corn zein and wheat gluten films, respectively, and bastarrachea and others (2010) obtained an e a value of 38.3 kj/mole for pbta film incorporated with nisin. the value of e a represents the degree of molecular interactions between the antimicrobial substance incorporated and the polymeric matrix. thus, higher e a values represent stronger antimicrobial-polymer interactions, which is reflected in a lower d-value due to the greater energy level required for antimicrobial release (bastarrachea and others 2010) . the relationship between temperature and diffusivity of the antimicrobial agent is the result of structural changes in the polymer matrix, since above the glass transition temperature (t g ) the molecular mobility in the system increases along with temperature, which leads to an increase in the ability of the packaging material to transport substances through its polymeric matrix (teerakarn and others 2002) . in addition to the interactions between polymer and antimicrobial agent, the d-value is also influenced by interactions between the antimicrobial and the food matrix. thus, the food nh2-r-w-c-f-r-v-c-y-r-g-i-c-y-r-k-c-r-conh2 miyata and others (1989) * x = means that the specific identity of an amino acid cannot be determined unambiguously. * * dhb = (z)-2,3-didehydrobutyrine. * * * abu = 2-aminobutyric acid. composition, as well as the solubility of the antimicrobial in these components also affects the d coefficient. in the study of paper coated with eva incorporated with nisin, the d-values varied according to the composition of the food in contact with the active packaging (kim and others 2002) . the highest d-value was observed when the film was in contact with a 2% citric acid solution, and the lowest value was observed when in contact with a 2% nacl solution. characteristic parameters of each solution, such as ph and ionic strength, have been shown to influence nisin solubility. nisin has a high solubility (up to 40 mg·ml −1 at ph 2) at low ph, but at high concentrations of nacl (above 1 m) nisin solubility dependence on ph almost disappears and the solubility decreases to values below 1 mg·ml −1 at any ph (rollema and others 1995) . chollet and others (2009) also investigated the influence of food composition on the migration of nisin incorporated in pe/pa/pe films coated with hpmc by changing the fat percentage. they found that increasing the fat content in the food resulted in an increased d-value and, therefore, in a greater diffusion of incorporated nisin. in their experiment, nisin diffusion mechanism was governed by the fat content. the increase in fat content resulted in microstructural changes, such as enlargement of pore size in the food matrix, which favored nisin diffusion into it. consumer demand for minimally processed foods and additivefree products has led to the development of antimicrobial packaging. peptides have shown various bioproperties, among them antimicrobial activity, leading to the application of these compounds in the food preservation area by either direct addition or incorporation into packaging materials (table 8) . active packaging materials incorporated with antimicrobial peptides have shown effectiveness in inhibiting pathogenic microorganisms, an improvement in food safety. moreover, antimicrobial peptides incorporated into the polymeric matrix may af-fect the engineering characteristics of the packaging material, and lead to differentiated diffusion performance. this review highlights the characteristics of pure peptides, as well as their incorporation into polymeric matrices. several studies have indicated significant changes in mechanical properties and surface morphology of the films incorporated with antimicrobial peptides. however, research related to the study of barrier properties to gases and water vapor is still limited. more studies on the release of other peptides, different from nisin, from food packaging materials are needed to better understand the mechanism of dissemination of antimicrobial agents. finally, in the years ahead, the advent of nanotechnology will lead to research on the synergistic effects of antimicrobial peptides and nanoparticles, such as metals, metal oxides, and nanoclays, with the objective being to improve the mechanical and barrier properties of antimicrobial packaging. bioactive peptides: synthesis, properties, and applications shortened cecropin a-melittin hybrids significant size reduction retains potent antibiotic activity uso da nisina com a função de conservador para queijos pasteurizados. portaria deten/ms n • 29, de 22 de janeiro de 1996 surface modification of poly(styrene) by the attachment of an antimicrobial peptide review of antimicrobial food packaging in: standard test method for water vapor transmission rate through plastic film and sheeting using a modulated infrared sensor astm d882 in: standard test method for tensile properties of thin plastic sheeting astm e96/e96m in: standard test methods for water vapor transmission of materials biochemical and genetic characterization of enterocin a from enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins engineering properties of polymeric-based antimicrobial films for food packaging: a review release kinetics of nisin from biodegradable poly(butylene adipate-co-terephthalate) films into water detergent-like actions of linear amphipathic cationic antimicrobial peptides optimized enzymatic synthesis of c-terminal peptide amides using subtilisin a from bacillus licheniformis recent applications of enzymatic peptide synthesis chemical synthesis of proteins a general approach to describe the antimicrobial agent release from highly swellable films intended for food packaging applications tilapia (oreochromis mossambicus) antimicrobial peptide, hepcidin 1-5, shows antitumor activity in cancer cells a fish antimicrobial peptide, tilapia hepcidin th2-3, shows potent antitumor activity against human fibrosarcoma cells antimicrobial peptides (amp) with antiviral activity against fish nodavirus buforins: histone h2a-derived antimicrobial peptides from toad stomach monitoring nisin desorption from a multi-layer polyethylene-based film coated with nisin-loaded hpmc film and diffusion in agarose gel by an immunoassay (elisa) method and a numerical modeling reference method for broth dilution antifungal susceptibility testing of yeast performance standards for antimicrobial disk susceptibility tests methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically bioactive packaging technologies for extended shelf life of meat-based products covalent immobilization of antimicrobial peptides (amps) onto biomaterial surfaces the mathematics of diffusion antibiotic magainins exert cytolytic activity against transformed cell lines through channel formation in vitro antiplasmodium effects of dermaseptin s4 derivatives hydrophobicity, hydrophobic moment and angle subtended by charged residues modulate antibacterial and haemolytic activity of amphipathic helical peptides antifungal peptides: novel therapeutic compounds against emerging pathogens antiviral effects of synthetic membrane-active peptides on herpes simplex virus, type 1 the possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease direct food substances affirmed as gras -electronic code of the bactericidal activity of pediocin pa-1 is specifically inhibited by a 15-mer fragment that spans the bacteriocin from the center toward the c terminus food standards agency. current eu approved additives and their e numbers defensins: antimicrobial peptides of innate immunity immunomodulatory peptides obtained by the enzymatic hydrolysis of whey proteins principles of mass transfer development of cellulose acetate-based antimicrobial food packaging materials for controlled release of lysozyme polymer surface modification for the attachment of bioactive compounds development and characterization of an antimicrobial packaging film coating containing nisin for inhibition of listeria monocytogenes antimicrobial plastic film: physico-chemical characterization and nisin desorption modeling innovative multilayer antimicrobial films made with nisaplin ® or nisin and cellulosic ethers: physico-chemical characterization, bioactivity and nisin desorption kinetics simulating diffusion model and determining diffusivity of potassium sorbate through plastics to develop antimicrobial packaging films the role of cationic antimicrobial peptides in innate host defences antimicrobial and host-defense peptides as new anti-infective therapeutic strategies antimicrobial activities of amphiphilic peptides covalently bonded to a water-insoluble resin a coating for use as an antimicrobial and antioxidative packaging material incorporating nisin and [alpha]-tocopherol studies on anticancer activities of antimicrobial peptides the antibacterial activity of magainin i immobilized onto mixed thiols self-assembled monolayers proteomics for studying cancer cells and the development of chemoresistance antifungal properties and mode of action of psacotheasin, a novel knottin-type peptide derived from psacothea hilaris antifungal activity of synthetic peptide derived from halocidin, antimicrobial peptide from the tunicate, halocynthia aurantium peptide antimicrobial agents chemical synthesis of peptides and proteins properties of nisin-incorporated polymer coatings as antimicrobial packaging materials bioactive peptides: production and functionality thermal, mechanical and water vapor barrier properties of sodium caseinate films containing antimicrobials and their inhibitory action on listeria monocytogenes antifungal mechanism of smap-29 (1-18) isolated from sheep myeloid mrna against trichosporon beigelii antitumor activity of the antimicrobial peptide magainin ii against bladder cancer cell lines virucidal activity of a scorpion venom peptide variant mucroporin-m1 against measles, sars-cov and influenza h5n1 viruses recombinant production of antimicrobial peptides in escherichia coli: a review peptide-derivatized dendrimers inhibit human cytomegalovirus infection by blocking virus binding to cell surface heparan sulfate sínteses química e enzimática de peptídeos: princípios básicos e aplicações high-pressure processing and antimicrobial biodegradable packaging to control listeria monocytogenes during storage of cooked ham development and characterization of an active polyethylene film containing lactobacillus curvatus crl705 bacteriocins relationship of membrane curvature to the formation of pores by magainin 2 antimicrobial peptides, isolated from horseshoe crab hemocytes, tachyplesin ii, and polyphemusins i and ii: chemical structures and biological activity antiviral activity of selected antimicrobial peptides against vaccinia virus mammalian cell toxicity and candidacidal mechanism of arg-or lys-containing trp-rich model antimicrobial peptides and their d-enantiomeric peptides purification and characterization of plantaricin a, a lactobacillus plantarum bacteriocin whose activity depends on the action of two peptides the antiviral activity of naturally occurring proteins and their peptide fragments after chemical modification peptide synthesis catalyzed by organic solvent-stable protease from pseudomonas aeruginosa pst-01 in monophasic aqueous-organic solvent systems tethering antimicrobial peptides: current status and potential challenges selective lysis of bacteria but not mammalian cells by diastereomers of melittin: structure-function study broth microdilution antibacterial 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sorbate and nisin applications of antimicrobial peptides from fish and perspectives for the future antimicrobial peptides: premises and promises peptide purity and counter ion determination of bradykinin by high-performance liquid chromatography and capillary electrophoresis improvement of solubility and stability of the antimicrobial peptide nisin by protein engineering antimicrobial and antifungal activities of a novel cationic antimicrobial peptide, omiganan, in experimental skin colonisation models plantaricin a, a peptide pheromone produced by lactobacillus plantarum, permeabilizes the cell membrane of both normal and cancerous lymphocytes and neuronal cells antimicrobial efficiency of film incorporated with pediocin (alta ® 2351) on preservation of sliced ham antioxidative peptides from food proteins: a review development of bioactive food packaging materials using immobilised bacteriocins lacticin 3147 and nisaplin ® bioactive peptides: synthesis, properties, and applications health-related costs: from foodborne illness in the united states. produce safety project. available from: www.producesafetyproject.org. accessed cationic amphiphilic peptides with cancer-selective toxicity controlled diffusion of an antimicrobial peptide from a biopolymer film application of bacteriocins in vegetable food biopreservation synthesis and application of caged peptides and proteins physical and antimicrobial properties of grape seed extract, nisin, and edta incorporated soy protein edible films lipase-catalyzed synthesis of peptides containing d-amino acid: facts and artifacts recent patents on active packaging for food application active and intelligent packaging for milk and milk products membrane fluidity characteristics of human lung cancer heparan sulfate: anchor for viral intruders? immunomodulatory activity of small peptides covalent immobilization of an antimicrobial peptide on poly(ethylene) film responsive polymer films and capsules via layer-by-layer assembly nisin diffusion in protein films: effects of film type and temperature influence of amino acid substitutions in the nisin leader peptide on biosynthesis and secretion of nisin by lactococcus lactis antiviral activities of lactoferrin natural products with hypoglycemic, hypotensive, hypocholesterolemic, antiatherosclerotic and antithrombotic activities expression and purification of antimicrobial peptide buforin iib in escherichia coli lantibiotics: peptides of diverse structure and function mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of escherichia coli barrel-stave model or toroidal model? a case study on melittin pores antimicrobial peptides of multicellular organisms interaction of the antimicrobial peptide pheromone plantaricin a with model membranes: implications for a novel mechanism of action the authors would like to thank to nicholas j. walker for providing language help and writing assistance. financial support for this research was provided by coordenação de aperfeiçoamento de pessoal de nível superior (capes) and the conselho nacional de desenvolvimento científico e tecnológico (cnpq). key: cord-293974-jcpkla55 authors: simões, marta filipa; ottoni, cristiane angélica; antunes, andré title: mycogenic metal nanoparticles for the treatment of mycobacterioses date: 2020-09-02 journal: antibiotics (basel) doi: 10.3390/antibiotics9090569 sha: doc_id: 293974 cord_uid: jcpkla55 mycobacterial infections are a resurgent and increasingly relevant problem. within these, tuberculosis (tb) is particularly worrying as it is one of the top ten causes of death in the world and is the infectious disease that causes the highest number of deaths. a further concern is the on-going emergence of antimicrobial resistance, which seriously limits treatment. the covid-19 pandemic has worsened current circumstances and future infections will be more incident. it is urgent to plan, draw solutions, and act to mitigate these issues, namely by exploring new approaches. the aims of this review are to showcase the extensive research and application of silver nanoparticles (agnps) and other metal nanoparticles (mnps) as antimicrobial agents. we highlight the advantages of mycogenic synthesis, and report on their underexplored potential as agents in the fight against all mycobacterioses (non-tuberculous mycobacterial infections as well as tb). we propose further exploration of this field. currently, there are almost 200 described species of the ubiquitous acid-fast bacteria of the genus mycobacterium [1] . mycobacteria can cause many different mycobacterioses, being the most worrying tuberculosis (tb) [1] , while common mycobacterioses can be caused by non-tuberculous mycobacteria (ntm). some ntm such as mycobacterium abscessus, m. avium, m. kansasii, m. malmoense, and m. xenopi can cause pulmonary diseases; others like m. chelonae, and m. haemophilum are able to cause disseminated diseases; furthermore, m. fortuitum, m. marinum, and m. ulcerans are able to cause skin, soft tissue, and bone diseases [2] . even though there are some pathogenic species, ntm are opportunistic and considered as nontransmissible [3, 4] . the distinction between pathogenic and non-pathogenic species is not always trivial as many of them share the same phenotypic and genotypic characteristics and have very limited differences [3, 5] . despite their similarities, ntm have lower human pathogenicity than mycobacteria from the mycobacterium tuberculosis (mtb) complex [6, 7] (species with 85-100% dna homology with mtb, which include m. africanum, m. bovis, m. caprae, m. canetti, m. pinnipedii, m. tuberculosis, m. microti, or m. mungi, which are all pathogenic [3, 7, 8] ). the fact that ntm share many infectious traits with the causing agents of tuberculosis (tb) allows them to be used in many research studies as model organisms of infection for this disease, with the advantage of being less pathogenic and faster growing species [7, 9] . relation with similar clinical symptoms. therefore, aspergillosis has long been reported to lead to tb misdiagnosis [23] [24] [25] . when there are co-infections or direct links between tb and inflammation of cells due to other diseases, further challenges become evident: therapeutic limitations, acquired resistance, toxic side-effects, and drug-drug interactions [26, 27] . a disease for which tb has been referred as a risk factor is lung cancer, since it can cause alterations in the lungs, which might become a cause for later malignant cell changes [27] . the coronavirus disease 2019 (covid-19) pandemic is having a huge social impact. outcomes of this pandemic include the reduction or suppression of certain healthcare infrastructures and their access, mainly due to lockdown and other control measures (quarantine of suspected cases, isolation of infected patients, and contact tracing). as a result, less cases of tb will be detected, and its infection rate will increase [28] . the lockdown also affects the production and transport of drugs and supplies, and limits the access to healthcare services, causing the disruption of treatments of certain diseases, which will be particularly negative for people with drug-resistant tb [29] . this will certainly worsen the problem of resistance in tb and other infections. confinement also facilitates the contact of infected and non-infected members that share the same house, thus increasing household transmission of tb [30] . both tb and covid-19 present similar and non-specific clinical features-fever, cough, and dyspnea, or breathlessness [15, 31, 32] . when covid-19 testing is not available, their similarities can easily lead to misdiagnosis and ineffective treatments [30, [32] [33] [34] . another potential challenge is the stigmatization of tb patients (for coughing), due to the fear of covid-19 [30, 31] . these patients become afraid of visiting healthcare services, and many (infected with tb) end-up not being properly diagnosed within a timeline that would contribute to control the infection [31] . cases of patients simultaneously infected with tb and covid-19 present other risks: when radiology data is not available, tb might not be diagnosed; covid-19 therapy can reactivate latent tb; pre-existing tb, especially if active, will worsen the clinical state of covid infection; and the simultaneous therapy for both infections can lead to drug-drug interactions and added hepatotoxicity [32] . the who has recognized the impact of covid-19 on tb and has issued a note on how to tackle this serious issue [15] . nevertheless, some of these negative consequences will be unavoidable and long-lasting [16, 35] . as a result of the pandemic, the next five years are expected to reverse the trend of the past decades [29] and will lead to an increase in tb incidence and mortality [36] . the common treatment for tb is a multidrug combination of first-line drugs, consisting of two months of rifampicin (rif), isoniazid (inh), pyrazinamide (pza) and ethambutol (emb), followed by four months of rif and inh [6, 10, 37] . these standard combinations are reasonably low-priced and effective against sensitive mycobacterial strains [9] . however, due to the long duration and toxic side-effects of the therapy (especially when treating drug-resistant cases), many patients end-up not following the complete treatment, thus increasing the chance of reemergence of the disease and development of resistance [10, 14, 38] . unfortunately, the six-month treatment is not effective against mdr nor xdr tb [39] . mdr tb implies simultaneous resistance to rif and inh, whereas xdr tb implies an additional resistance to a quinolone and at least one injectable drug [17] . the increasing number of cases of mdr and xdr tb lead to the use of second-line drugs which are more toxic and less effective than first-line ones [17] . the treatment of mdr tb is more expensive, longer (can go up to 24 months), and uses a combination of at least five drugs with many harmful secondary effects (such as hepatotoxicity) [40] . failure of treatment hardens the challenge of fighting tb because it increases the infection rate and raises mortality [39] ; 70% of xdr tb patients have been estimated to die within a month of diagnosis [14] . therefore, all options that can contribute to improve efficiency of antimycobacterial activity and reduce toxicity should be fully explored. carrier or delivery systems, such as liposomes and microspheres, have been developed for the sustained delivery of anti-tb drugs and have shown better chemotherapeutic efficacy [17, 41] . conjugating existing drugs with nps is another strategy that has great potential for the treatment of mdr tb. this is the case of mesoporous silicon nps conjugated with ethionamide (eth), a second-line drug, that has increased activity against mtb when compared to eth alone [42] . hakkimane and colleagues synthesized nps with poly lactic-co-glycolic acid polymers encapsulating rif or inh, and found both formulations to be more effective than rif or inh against mtb, having a higher activity and requiring a lower drug concentration [39] . chemical synthesis of new drugs, such as prodrugs derived from first-line agents, is another strategy also studied as an alternative solution to fight tb where different chemical structures might circumvent previous resistance [43] . some peptides, part of the host first-line of defense and produced by the innate immune response, have been identified as having antimicrobial activity, presenting good biocompatibility and low probability of leading to microbial resistance [44] . these have been described as having a direct action against microorganisms, by creating cytotoxic pores on their cell walls, and an indirect one, by modulating the host immune system through upregulated secretion of pro-inflammatory cytokines and chemokines and contributing to contain infection [44] [45] [46] . therapies using these antimicrobial peptides have been investigated, and synthetic peptides have been shown to have great potential and activity against pathogens, combined with decreased toxicity [46, 47] . however, despite all this research and promising leads, their application to tb therapy remains underwhelming and a completely effective tb vaccine has also not yet been developed [14] . the current research was developed according to the prisma protocol. a literature search was performed on three databases (research gate (www.researchgate.net), google scholar (https://scholar.google.com), academia (www.academia.edu), and further cross-checked and complemented with searches on web of science (www.webofknowledge.com), scopus (www.scopus. com/home.uri), and pubmed (https://pubmed.ncbi.nlm.nih.gov). the search criteria were based on the following key terms: nanoparticle, mycogenic, biologic, mycobacterium, and agnps. searches with all possible combinations of the referred terms were performed. selected articles included all of those mentioning the use of mycogenic metallic nanoparticles against mycobacteria, as well as the most recent (≈last decade) manuscripts on agnps. the last date for these searches was 4 august 2020. any article matching the searching criteria was checked and used in our review. the rise of microbial resistance against antimicrobial drugs has encouraged and promoted nanotechnology research as a potential viable source of solutions [48] . np-based systems are able to circumvent many of the challenges related to mycobacterial infections since they can target the infected cells and act directly on the cell wall of intracellular pathogens, as is the case of mtb when it infects macrophages in pulmonary tb [26] . it is common knowledge that metal nanoparticles (mnps) have antimicrobial activity against a multitude of microorganisms [40] . mnps represent promising potential solutions against many infections and resistance to traditional drugs. they use different mechanisms of action from those identified for common drugs, exhibit activity against many microbial resistant species and strains, and target several biomolecules interfering in the development of microbial resistance [49] . the mechanisms that have been reported to explain the effects of mnps on microbial cells include: dna damage, protein damage, mitochondrial damage, attachment to 30s ribosome subunit, oxidation of cellular components, release of metal ions, damage to the proton efflux pump, disruption, or prevention of biofilm formation, disruption of cell membrane, disruption of transmembranar electron transport, and production of reactive oxygen species (ros) [17, 49] . some of these mechanisms vary depending on the target species and the specific characteristics of the nps [17] . silver (ag) has the highest reflectivity of all metals [50] . it has been used for centuries as an antimicrobial agent [40] . throughout history, civilizations incorporated silver into daily life objects to avoid spreading diseases, for example, in ancient times, silver containers were used to keep water potable and prevent wine spoilage. however, once antibiotics were discovered, the use of silver for its antimicrobial activity decreased [50] . nowadays, nanotechnology is responsible for a resurgence in the exploration of silver for these purposes. agnps are the most widely used mnps due to their potential as therapeutic agents and antimicrobial agents, showing activity against almost 700 pathogens [19, 51] . agnps are used in a vast number of different products and applications (namely textiles, cosmetics, food packaging, medical appliances, pharmaceutical ointments among many others) [40, 50, 52] . they have a significant impact on respiratory medicine and can be applied against a broad range of microbial infections [27, 52] . agnps are an alternative way to overcome drug resistance [53] , mostly due to their particular characteristics: small size, even morphology, and capacity to interact with biomolecules [19] . furthermore, agnps have been reported as having good conductivity, chemical stability, catalytic activity, cytotoxic effect on cancer cells, and antimicrobial activity [49, 54] . agnps have proven antimycobacterial activity, but this activity is highly variable, dependent on several parameters [52] , and also on target species [26, 53, 55] . smaller agnps tend to have higher activity due to their larger surface/area, which allows them to release higher amounts of silver ions and inhibit microbial growth [26, 56] . in addition, higher concentrations lead to higher activity [55, 57] . some studies also point to the potential relevance of agnps shape, although this has only been rarely analyzed. triangular-shaped agnps seem to be more effective against escherichia coli, likely due to an increase in positive charges and more active facets induced by this morphology [58, 59] . although agnps' antimicrobial mechanism of action is not fully clear, we know that they kill by contact and ion release [19] . it was also detected that the antimycobacterial activity of agnps is higher than other metallic nps, making them a favorite focus on this research field [40, 56, 60] . furthermore, in addition to antimycobacterial properties in vitro-when applied directly on mycobacteria, agnps have also shown ex vivo activity by suppressing innate responses of infected macrophages, induced by mycobacteria [40] . mycogenic agnps have also been reported to have anti-inflammatory activity [54] . all of the characteristics of agnps make them an undoubtedly easy focus of nanotechnology research. an overview of recent research on the use of agnps against mycobacterial species, not including mtb, is presented below ( table 1 ). this overview excludes mtb which will be analyzed afterwards due to its epidemiological relevance. we can note that the majority of these studies relied on chemically synthesized agnps. furthermore, they have focused on a reduced number of species, the most common being m. smegmatis and m. bovis. the reasoning for this focus is the fact that m. smegmatis is the most used model organism for the study of mycobacteriosis and tb, as they are safe and non-pathogenic mycobacteria as well as fast-growing and easy to manipulate genetically [61] . m. bovis (strain bcg) is a slow-growing mycobacterium, whose choice as a model is mostly due to its placement within the mtb complex, being an attenuated strain and a biohazard level 2 microorganism (versus biohazard level 3 for mtb) [62] . only a few studies have looked specifically into the production of agnps against mtb (table 2 ). these include agnps produced via physical-chemical methods, as well as biological ones (mostly using parts of plants), and consist of a wide-range of np sizes and tested strains (avirulent, virulent, isolated from clinical samples, drug-sensitive and drug-resistant-mdr and xdr). the research made so far on the use of agnps against mtb has proved that these are effective. the lack of standardization among all research studies makes it more difficult for us to compare them across, although they provide some relevant insights into the use of this type of nps against mtb. the general consensus that smaller sized agnps are more active against bacteria is also valid for mycobacteria [56, 71] . this is the case, for example, of the smaller bsa-agnps ( table 2 ) that showed higher activity than the produced pvp-agnps [64] . furthermore, these studies present promising results with cases of activity reported against mdr and xdr strains [19, 26, 71, 72, 78] , as well as activity against intracellular mtb [56, 73] . this is especially relevant for latent tb where mtb remain inside granulomas [79] , where they adapt and thrive under adverse conditions, such as nutrient deprivation and hypoxia [3] . mycogenic processes are biological processes developed by fungi, mostly filamentous fungi (molds)-fungi that form mycelia. these organisms have the capacity to accumulate metals by sequential action of reductase enzymes (such as nadph-dependent nitrate reductase), leading to the reduction of metal salts and final production of metal nanoparticles (mnps) [58] . as such, they can be affected by several parameters (table 3) . resultant morphology (size). • smaller concentrations tend to lead to smaller mnps and increased dispersion. higher concentrations can generate increased toxicity. culture media quantity of mnps. • media containing enzymes-specific substrates increases enzyme production, which can generate more mnps. quantity of mnps. • more biomass leads to increased enzyme release, facilitating the mycosynthesis of mnps and increasing their production. resultant morphology (size), quantity of mnps, and synthesis rate. • little or no agitation decreases the synthesis rate and might lead to agglomeration, increased sizes, and reduced mnp production. light intensity quantity of mnps and synthesis rate. • light stimulates fungal growth and metabolite production. higher metabolite concentration results in faster synthesis and increased mnp production. these parameters can be adjusted and optimized. they are variable for some processes and for the species used for the production of mnps [52] . interestingly, the mnps used in in vitro assays don't have their antimicrobial activity affected by temperature or ph [17] . however, the full extent of the effects of these parameters (table 3) is not yet completely understood and requires further research. even though mycogenic mnps can be produced intra-or extracellularly, the latter production process is more appealing. extracellular production involves fewer steps and does not require cell disruption to release the mnps or complex washing steps to recover and purify them [17] . many studies have reported and listed the capacity of different fungal species to synthesize mnps, from ubiquitous groups, to extremophiles, and ranging from yeasts, to filamentous fungi and mushrooms [18, 54, 58, [80] [81] [82] [83] [84] [85] [86] . despite this wide diversity, most mycogenic processes rely on filamentous fungi and follow a four-main steps method ( figure 1 ). generally, the process includes the growth of fungal biomass (steps i and ii in figure 1 ), followed by a sequential shorter incubation of that biomass in water (step iii in figure 1) , and then the mixture of this supernatant with a chemical precursor (step iv in figure 1 ). the fungal metabolites act as reducing and stabilizing agents, and lead to the reduction of metal ions and agglomeration of metal atoms, which result in mnps. the mnp synthesis is noticeable by the change in the color of the suspension and can be further confirmed by uv-visible spectroscopy, which detects changes in the optical properties of the mnps, by reading the absorbance of the surface plasmon resonance bands (usually localized at a wavelength of 400-450 nm). the mnp suspension should then be cleaned and purified either by filtration, dialysis, or ultracentrifugation. the last few years have shown a considerable amount of research and development in the use of mycosynthesis of the most studied mnps-agnps (supplementary table s1 ). these further highlight their wide applicability and support the use of mycogenic agnps as antimicrobial agents as well as for all other applications of general mnps [48, 55, [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] [104] [105] [106] . most mycogenic agnps studied are spherical although some variations, mostly due to the formation of clusters, can also be found [41] . in most mycogenic processes (those following the method shown in figure 1 ), the common concentration of the precursor-silver nitrate (agno 3 )-is 1 mm [48, 89, 103, 106] . within the size variation described, mycosynthesized agnps when produced with enzymatic stimulation (from co-cultures) form smaller and more active nps [106] . mycogenic synthesis of agnps combines the best of both worlds: the most effective mnps that nanotechnology has developed together with the green production process mediated by fungi [80] . these mycogenic processes allow us to obtain smaller sized nps than those produced as a result of chemical or physical synthesis, which in turn allow a better control over production size, being less size-variable [83] . furthermore, synthesis parameters can be altered in order to change their size and therefore activity [52, 54] . so far, little has been explored combining the use of mycogenic agnps and the fight against tb and other mycobacterioses, but the efforts presented in table 4 show the potential to exert some control over this infection and its increasing resistance. in general, mycogenic mnps are mostly monodispersed, with well-defined size and shape (which is most commonly spherical) [85] . moreover, the mycogenic agnps reported to be successful against mycobacteria were obtained from a diverse range of fungi, which included yeasts and species within different families of filamentous fungi (table 4 ). this supports the assumption that fungi constitute a potential source of mnps relevant for fighting tb and other mycobacterioses. biological agnps are considered relatively safe and less toxic (less cyto/genotoxic in vivo) than chemically synthesized agnps [110, 111] . their toxicity depends on the concentration used [112] . in addition, generally, all agnps show increased toxicity once dissolved and after losing their spherical structure [49] . transition metals (the 38 elements in groups 3 through 12 of the periodic table) are recognized as the most suitable elements for the synthesis of mnps [49] . within these, nps incorporating silver are the most widely studied and used. the last few years have seen an increase in testing some of the other transition metals and others as a way of uncovering new mnps and exploring their potential use as antimicrobial compounds ( table 5 ). the study of mnps with alternative metals against mycobacteria is arguably less dynamic. nonetheless, a few researchers have investigated the use of elements such as copper, gallium, selenium, titanium, zinc, or even bimetallic alternatives. within these, copper and zinc seem to be the two transition metals most commonly explored, with gallium (a post-transition metal) also being frequently studied. exploring these alternatives can be relevant to better understand which mnps have lower probability of developing toxicity. many studies have focused on the use of mnps complemented with other substances or different mnps (table 6 ). such combined uses of mnps have often proved to be synergetic, increasing the antimicrobial activity of its individual components. both citrate-aunps and pah-aunps have activity against m. bovis (strain bcg), lower than tested agnps [66] copper (cunps) nr biologically synthesized from leaves of psidium guajava l. activity against mtb, m. smegmatis, and m. pheli, but lower than other mnps [57] gallium (ganps) 305 nm sized and cylindrical chemically synthesized by double emulsification and sonication polydispersed, with prolonged activity against intracellular m. smegmatis [113] copper oxide and zinc oxide (cu(ii)onps and znonps) spherical chemically synthesized activity against m. avium subsp. paratuberculosis [69] 10-70 nm sized and polydispersed phyto-synthesized from barleria prionitis 90 nm sized and hexagonal phyto-synthesized from plumbago zeylanica 10-20 nm sized and spherical phyto-synthesized from syzygium cumini 16 µg/ml) higher activity than aunps or agnps (mic ≈ 2.5 µg/ml), and more specific for mycobacteria with a higher selectivity index in addition, the smaller mnps (from s. cumini) are more effective [60] zinc oxide (znonps) 12-53 nm sized and spherical biologically synthesized from leaves of limonia acidissima linn. also known as feronia elephantum correa or wood apple activity against mtb (strain h37rv) [114] ganps ≈300 nm sized and cylindrical chemically synthesized by double emulsification and sonication activity against intracellular mtb (strain h37ra) in monocyte-derived macrophage (mdms) and thp-1 macrophages (up to 70% mtb growth inhibition) [115] zinc (znnps) ≈60 nm sized and variable shapes, mostly spherical biologically synthesized from pseudomonas hibiscicola combination therapy for mycobacterial infections can increase the potential activity of mnps, contribute to decrease the effective dose of antibiotics potentiating them; reduce side effects, drug toxicity, and mnps toxicity; enhance bioavailability; and enhance solubility and retention time [103, 124] . the exact interaction mechanisms aren't always fully understood. generally, negatively charged microbial surfaces attract positively charged mnps due to electrostatic interactions. the mnps then establish bonds with the cellular membranes, disrupting the cell walls and making them more permeable. as a consequence, microorganisms become more sensitive to drugs [49] . the increased activity from the conjugation of mnps with antimicrobial peptides, for example, might be due to the higher membrane permeability of the peptides, which then help deliver mnps into the microbial cells [44] . another example of increased antimycobacterial activity is the combination of agnps with chloroform, which is due to chloroform's ability to remove lipids and rupture the mycobacterial cell wall [41] . one should also note that the conjunction of biomolecules such as peptides or chitosan results in increased antimycobacterial activity, but this effect is limited after mycobacteria are internalized by macrophages [7] . more effective strategies rely on combining nps with classical anti-tb therapeutics that ensure both extra-and intracellular activity, although only a few in vivo studies have explored this option [7] . the increased applications of mnps in the medical field demand more biocompatible, safe, and effective nanostructures with less hazardous byproducts of synthesis reactions [83] . mycogenic mnps (as well other biological mnps) are regarded as safe, less toxic, biocompatible, eco-friendly, and cheaper alternatives, with lower consumption of energy and higher yields when compared with physical-chemical synthesis [56, 106] . fungi are more efficient than most microorganisms when it comes to the biological production of mnps [51] . this is due to the fungal capacity of producing a high number of bioactive metabolites, accumulating metals and having enhanced processes [54, 125] . as additional advantages, fungi are easy to manipulate, easy to grow, do not require complex nutrients, have high production of biomass and metabolites, and have high wall-binding capability and high metal uptake [52, 80, 83, 85, 89, 106, 126] . many of the fungal metabolites involved in the mycogenic synthesis also cap the mnps, conferring a higher control of size and stability. in addition, because most mycogenic processes are extracellular, there is no requirement for additional steps, or downstream processing, to release the mnps for further processing or use [49, 80] . moreover, extracellular mycogenic processes facilitate handling and scale-up, and mycelia from filamentous fungi are more resistant to agitation and pressure making them more suitable for large-scale synthesis in bioreactors and chambers [54, 106] . looking specifically at mycogenic agnps, a recent study observed that they were more active against pathogenic bacteria than chemically synthesized agnps [55] , further highlighting the advantages of their use. a wide variety of fungal genera are recognized as being able to precipitate agnps (namely aspergillus, fusarium, penicillium and verticillium) [126] . however, there is a much wider fungal diversity that remains completely unexplored. current estimates point to the existence of 1.5-3.8 million fungal species on earth with only 120,000 (3-8% of the total) being validly described, leaving much to be discovered, isolated, and characterized [127, 128] . the fungal kingdom is therefore underexplored with only a small percentage of total species already surveyed regarding their capacity to mycosynthesize mnps. produced mnps can have a very diverse range of characteristics and biological activities which are dependent on the formation process and the enzymatic profile of each fungal species [90] . despite the well-recognized advantages of mnps as one of the best alternatives against antimicrobial-resistant strains of mycobacteria and other taxa, several challenges and opportunities are ahead of us. given that the effects of nps result from a combination of multiple, synergistic mechanisms, the potential development of resistance against them is more arduous and less likely [124] . one should note, however, that nps are unlikely to offer a full, definitive solution, and their misuse should be avoided, as it can lead to further issues. a recent report pointed to a case of an agnps and agno 3 resistant mutant strain of m. smegmatis, developed after one single exposure and associated with increased mic for inh [129] . this unexpected case seems to be a rare event but further highlights our need to study and understand mnps. the focus of such future studies should be on testing new strains, discovering new nps, and clarifying their synthesis and mode of action as antimicrobial agents. lines for potential novel discoveries on mycogenic mnps include the study of endophytic and extremophilic strains. while the former has increased its relevance within the last years (table 2) , the latter is still restricted to a very small number of studies and is focused on other applications rather than their use as antimicrobial agents [130] . the application of new approaches such as synthesis optimization via statistical methods (such as central composite design and response surface methodology) has been proposed in a few studies and showed some promise but remains mostly unexplored [98, 101] . such approaches are expected to significantly reduce the number of different lab tests required and lead to a quicker optimization of mnp production, thus warranting a closer look. like with any other drug, microorganisms might develop resistance to mnps [40, 131] , so it is essential to thoroughly investigate all aspects related to their application. this will allow us to understand and manipulate all caveats regarding their synthesis and antimicrobial mode of action, and standardize synthesis methodologies to attain best scale-up yields. furthermore, as mentioned by tăbăran et al. [7] in relation to agnps, there are a few other therapeutic obstacles to overcome: "poor delivery, variable intramacrophagic antimycobacterial efficiency, and residual toxicity". the same applies to all other mnps effective against mycobacteria. investing in exploring the capacity of new fungal species to mycosynthesize mnps is a potential source of therapeutic alternatives. this requires the investment in isolation and bioprospection of uncommon and unexplored or under-explored environments. the great resource of new fungal species to be discovered might bring to light species with even more effective and better capabilities. there is much work to be done and much to be explored, but the prospects of mycogenic mnps are very promising. mycobacterioses are an increasing public health threat and it is imperative to develop new and better solutions taking into consideration all options to control them. the scenario is particularly bleak for tb. innumerous actions and campaigns over the recent decades have contributed to a tendency to control the infection, but tb is still neglected by many sectors. worrying recent estimates [132] ) predict that covid-19 containment measures will exact a heavy toll on health services and therefore an increase on infections such as mycobacteriosis and tb cases. mnps constitute a viable option to aid us to face this threat. this is supported by the many studies done on the different mnps synthesis processes and their vast array of applications. furthermore, mycogenic processes of mnps production, namely those relying on the use of filamentous fungi, are particularly promising. among its many advantages, the use of this production method is simple, quick, low-cost, and eco-friendly. this nanotechnology could help in killing mycobacteria, lowering drug doses and therapy periods, which in turn would help control infections. despite their benefits, mycogenic mnps remain an underexplored weapon to fight tb and other mycobacterial infections. furthermore, mycobacteria and mtb in particular have a complex pathogenesis that is not yet fully understood. considering that, according to sarkar et al. 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distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-278654-dxx6z2fj authors: wysocki, jan; batlle, daniel title: reduced plasma ace2 activity in dialysis patients: another piece in the conundrum of factors involved in hypertension and cardiovascular morbidity? date: 2013-06-19 journal: nephrology dialysis transplantation doi: 10.1093/ndt/gft240 sha: doc_id: 278654 cord_uid: dxx6z2fj the reduction in plasma ace2 activity reported in esrd patients treated by dialysis, particularly in female subjects, could limit ang ii degradation leading to increased levels of this peptide, which could contribute to the high prevalence of hypertension and cardiovascular morbidity that afflicts the dialysis population predialysis patients with ckd or renal transplant patients. when compared with historic samples from healthy subjects, however, all ckd groups examined, i.e. predialysis, transplant patients and even subjects on dialysis, seemed to have increased levels of plasma ace2 activity. statistical comparisons with healthy controls, however, could not be done because samples from healthy individuals were not assayed concurrently with those in the present study. what the study shows, in our opinion, is that while plasma ace2 activity tends to increase in ckd patients, perhaps as a compensatory mechanism to attenuate ang ii overactivity, at the time that esrd is reached and dialysis initiated a relative deficiency in plasma ace2 activity ensues. what is then the significance of reduced plasma ace2 activity in dialysis patients? as ace2 is mainly involved with the degradation of ang ii, one could readily speculate that the levels of this peptide could be augmented thereby predisposing to hypertension and other cardiovascular morbidity. as the levels of ang ii or ang (1-7) were not measured in this study, one can only consider this as a predictable consequence of ace2 deficiency that, however, still needs to be demonstrated. one also wonders about the significance of plasma ace2 activity since ace2 is mainly a tissue enzyme and its levels in the circulation, unlike the levels of ace, are relatively low. initial attempts to measure ace2 directly in plasma from healthy individuals were unsuccessful [17, 18] . moreover, circulating ace2 enzymatic activity has also been shown to be low or even undetectable in animals under physiological conditions [19] [20] [21] [22] . interestingly, in pathological states in humans, such as ischemic heart disease [23] , heart failure [24] and diabetes accompanied by vascular complications [25] as well as in rodent models of diabetes [19, 26] circulating ace2 activity is augmented. other studies in patients with connective tissue diseases, by contrast, have reported antibodies against plasma ace2 that reduce enzymatic activity [27] . the intriguing observation that human plasma itself may inhibit ace2 enzymatic activity was made based on incubation of purified recombinant ace2 with human plasma [18] . these findings suggested the presence of a small molecular endogenous inhibitor of ace2 in human plasma. consistent with this, roberts et al. now show that in a small subset of patients from each ckd subgroup, ace2 activity in unprocessed plasma was markedly lower than in plasma samples that had undergone the extraction process to remove the endogenous inhibitor of ace2 [16] . one wonders whether the process of hemodialysis itself could alter the levels of the ace2 inhibitor in plasma owing to its small molecular size. studies before and after the dialysis procedure could be informative in this regard. in the study by roberts et al. [16] , samples from patients on hemodialysis were collected prior to commencing dialysis, and on the middle dialysis day of the week. removal of the inhibitor during the dialysis procedure, however, could only increase plasma ace2 activity which is the opposite of what was observed in dialysis patients. the results of this study can therefore be interpreted to signify that the extraction of the endogenous inhibitor by dialysis is not a cause of reduced plasma ace2 activity. a possibility that the small-molecular inhibitor of ace2 might form a complex with ace2 protein thereby evading extraction during dialysis needs to be considered. if the plasma ace2 inhibitor indeed does not play a role, the question that remains is what causes the observed reduction in plasma ace2 activity in patients with esrd undergoing dialysis. the source of circulating ace2 in healthy individuals and ckd patients is not clear but release from the kidneys is a possibility. the levels of ace2 activity have been found to be 10-to 30-fold higher in mouse kidney cortex than in the heart [22] and urinary ace2 activity is about 10-fold higher in urine than in plasma [28] . the mechanism of how ace2 reaches the circulation and is then released into plasma is not well understood and requires further examination. it has been proposed that soluble ace2, which lacks its cytosolic and transmembrane domains, arises from proteolytic 'shedding' of the membrane-bound enzyme [15] . it is, nevertheless, conceivable that the full-length, membrane-bound ace2 can also be released into plasma, especially in disease states associated with tissue damage, such as myocardial infarction. in cell culture experiments, shedding of soluble ace2 is stimulated by a disintegrin and metalloproteinase, adam17, [15] and inhibited upon interaction of ace2 with calmodulin [29] . unlike ace, which is an endothelial enzyme, ace2 is normally not expressed or only minimally expressed in the endothelial layer [30] . it is possible, however, that under pathologic conditions, there may be an aberrant neoexpression of ace2 in endothelial cells [31] . this abnormally expressed endothelial protein could be shed into the circulation and could be the source of increased plasma ace2 activity in certain conditions such as myocardial infarction, kidney disease or diabetes. there have been no studies, to our knowledge, that examined plasma ace2 and the possible pathways such as adam17 or calmodulin that might be involved in modulating the release of this enzyme into the circulation. as the ace2 gene is located on chromosome x, possible differences in plasma ace2 activity between the sexes were examined by roberts et al. [16] in a multivariate analysis for males and females separately. the predictors of plasma ace2 activity in patients undergoing dialysis appeared to be different in both sexes. while in males plasma ace2 activity was strongly associated with bnp, which is increased in left ventricular hypertrophy and systolic dysfunction, female patients undergoing hemodialysis showed significant associations with diabetes and postdialysis systolic blood pressure. whether these associations have pathogenic or therapeutic implications awaits further examination. moreover, the authors found that female hemodialysis patients compared with male counterparts had lower plasma ace2 activity. analogous trend of lower plasma ace2 activity in the female group was observed for kidney transplant patients, which is similar to the recent findings of soler et al. [23] who found plasma ace2 activity significantly lower in female transplant patients when compared with males. in summary, the article by roberts et al. suggests that, in patients with ckd, plasma ace2 activity is increased whereas in patients with esrd undergoing dialysis, by contrast, plasma ace2 activity is reduced when compared with predialysis ckd patients. whether ace2 in plasma is indeed altered in ckd patients needs to be confirmed in further studies that include contemporary measurements from healthy control subjects. moreover, a longitudinal follow-up of a ckd cohort would be ideal to monitor ace2 activity as renal function declines over time. several other questions remain unanswered, such as the mechanism driving ace2 into circulation in disease states. regardless of the mechanism, the reduction in plasma ace2 activity reported in esrd patients treated by dialysis, particularly in female subjects, could limit ang ii degradation leading to increased levels of this peptide 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insights into the endogenous counter-regulatory pathway of the renin-angiotensin-aldosterone system circulating ace2 activity is increased in patients with type 1 diabetes and vascular complications ace2 deficiency modifies renoprotection afforded by ace inhibition in experimental diabetes autoantibodies to angiotensin-converting enzyme 2 in patients with connective tissue diseases urinary excretion of ace2-a biomarker of diabetic kidney disease? calmodulin interacts with angiotensin-converting enzyme-2 (ace2) and inhibits shedding of its ectodomain glomerular localization and expression of angiotensin-converting enzyme 2 and angiotensin-converting enzyme: implications for albuminuria in diabetes renal ace2 expression in human kidney disease prevalence, treatment, and control of hypertension in chronic hemodialysis patients in the united states baseline characteristics of patients with chronic kidney disease stage 3 and stage 4 in spain: the merena observational cohort study chronic kidney disease and the risks of death, cardiovascular events, and hospitalization key: cord-278513-ajfpghze authors: boyle, john t.; celano, paul; koldovský, otakar title: demonstration of a difference in expression of maximal lactase and sucrase activity along the villus in the adult rat jejunum date: 1980-09-30 journal: gastroenterology doi: 10.1016/0016-5085(80)90375-3 sha: doc_id: 278513 cord_uid: ajfpghze abstract lactase and sucrase are two disaccharidases that differ not only in their substrate specificity and developmental patterns, but also in their resistance to mucosal insult. in this experiment, we tested the hypothesis that there might be a dichotomy in expression of enzyme activity along the jejunal villuscrypt unit. sectioning of the villus-crypt unit in a cryostat enabled direct comparison of the distribution of lactase and sucrase enzyme activities in the adult rat. there is a stepwise increase in mean lactase/sucrase ratio going from crypt to villus. the data indicate that unlike sucrase activity, which is expressed maximally in enterocytes along the entire villus, maximal lactase activity is not attained until midvillus. the delay in expression of maximal lactase activity might help to explain the vulnerability of this enzyme to acute mucosal insult such as occurs in viral gastroenteritis. lactase and sucrase are two disaccharidases that differ not only in their substrate specificity and developmental patterns, but also in their resistance to mucosai insult. in this experiment, we tested the hypothesis that there might be a dichotomy in expression of enzyme activity along the jejunal villuscrypt unit. sectioning of the villus-crypt unit in a cryostat enabled direct comparison of the distribution of lactase and sucrase enzyme activities in the adult rat. there is a stepwise increase in mean lactase/sucrase ratio going from crypt to villus. the data indicate that unlike sucrase activity, which is expressed maximally in enterocytes along the entire villus, maximal lactase activity is not attained until midvillus. the delay in expression of maximal lactase activity might help to explain the vulnerability of this enzyme to ncute mucosal insult such as occurs in viral gastroenteritis. since the work of leblond and stevens,' it has been recognized that the enterocytes mature both morphologically and biochemically while migrating from the crypts to become digestive-absorptive cells of the villus. using various techniques to isolate crypt from villus cells, several different laboratories have shown in the rat that activity of enzymes asso-ciated with dna synthesis decrease,*.3 activity of lysosomal enzymes remain unchanged,z.4.6 and activity of microvillus enzymes such as alkaline phosphatase and disaccharidases increase'-' as cells migrate along the heights of the villus. early in this century, finkelstein and meyer recognized that infants with acute gastroenteritis could not tolerate "complex sugars."" subsequent studies have shown that this is usually a manifestation of transient lactose intolerance,'*'* whereas an ability to absorb sucrose is usually preserved.13-'5 thus, these two disaccharidases differ not only in their substrate specificity and developmental patterns,18"' but also in their resistance to mucosal insult. since the absolute activity of lactase is low compared to sucrase both in the human" and rat," a comparable loss of both enzymes after mucosal injury may reduce lactase activity below a critical threshold to allow for physiologic hydrolysis of dietary lactose. however, the decrease in the ratio of lactase to suerase activity in small bowel biopsies of patients who are lactose intolerant after acute mucosal injury indicates a higher vulnerability of lactase compared to sucrase.13-'5 by studying incorporation and turnover of radiolabeled amino acids, james et al. have shown that microvillus proteins do not have a uniform rate of turnover.20 alpers showed that the largest molecular weight proteins (including the disaccharidases) have the fastest rate of turnover.2l therefore, both synthesis and degradation of disaccharidases are taking place along the entire length of the villus crypt! differences in location where synthesis begins, ot differences in the rate of turnover of lactase and suerase might lead to a difference in enzyme distribution along the villus-crypt unit. differences in localization of enzyme activity in turn might help explain the decrease in the ratio of lactase to sucrase (0), and acid &alactosidase (a) along the villus-crypt unit. abcissa depicts "idealized villus-crypt unit" as explained in the methods section, with 100% representing the top-most part of the villus and 0% the bottom-most part of the crypt. histologic examination of sections revealed villi to make up 65%; mixed villus crypt, 20%; and crypt, 15% of the total villus-crypt height. because of very low protein in homogenates from apical 10% of villus crypt, the amount of absorbance measured by spectrophotometer was very low both for lactase and sucrase, and. therefore, enzyme activity was not considered accurate for presentation. short vertical lines denote 1 sem. absence of vertical line indicates that value of sem is smaller than the symbols used. all figures in the text represent data from eight rats; each point on graph represents mean data from 6 to 8 homogenates. activity in small bowel biopsies after intestinal mucosal insult. although different investigators have described an apical localization of one or the other enzyme along the villus-crypt unit, no one to date has systematically compared the localization of lactase and sucrase activities on the same villus-crypt unit. in the present study, we compare localization of lactase and sucrase activity on the same villus-crypt unit after sequential sectioning in a cryostat by using the technique of van genderen and engel" as modified by nordstrom et al.' the data indicate that, unlike sucrase activity, which is maximally expressed in enterocytes along the entire villus, maximal lactase activity is not attained until midvillus. male rats of charles river strain cd (wilmington, mass.) were shipped at 2 mo old and were kept 4-6 wk in our animal house before being used in experiments. animals were fed a standard rat chow (purina co. no. 5001) ad libitum, and weighed between 300 and 350 g at time of gastroenterology vol. 79, no. 3 study. fed rats were killed by decapitation at 9:oo am, and a 5 x 5-mm segment of jejunum was sectioned within a cryostat at -18% as previously described.' horizontal sections were cut 10 pm thick (no. 5 setting on iec microtome, needham hts., mass.). at various depths into the villus-crypt unit, a section was attached to the microscope slide for immediate inspection of histology under a phase-contrast microscope. the tissue blocks were sectioned through the submucosa to the proximal muscular layer. commencing with counting of sections, every six consecutive sections were combined and homogenized in 0.5 cm3 distilled water by vortex shaking and sonification x 30 set (sonifier cell disrupter, model v185, heat systems-ultrasonics, inc., plainview, n.j.). assays of lactase and sucrase activities were performed on each homogenate according to the method of dahlqvist.23 the lactase assay mixture contained p-chloromercuriobenzoate (pcmb) (aldrich chemical co., milwaukee, wis.) in order to inhibit any residual lysosomal acid /3-galactosidase activity." all enzyme determinations were made under conditions of linear activity with time and concentration of the enzyme. protein was determined according to the method of lowry et a1.25 all chemicals used were of reagent grade. three possible problems associated with the lactase assay were investigated (data not shown): (a) to test for the presence of an inhibitor of the glucose oxidase reaction, homogenates obtained from different heights of the villuscrypt unit were boiled and the same protein concentration as used in the enzyme assays were added to the glucose standards.zb the resulting curves showed no significant inhibition. (b) the possibility of an inhibitor of enzyme activity in lower sections of the villus crypt was studied by combining homogenates of equal protein concentration from apical villus and lower villus-mixed crypt sections. the resulting activity for both lactase and sucrase approximated the arithmetic mean activity calculated to result from such a mixing. (c) the dependence of lactase activity on ph was determined in homogenates from apical, mid-, and lower villus-mixed villus-crypt areas of the villuscrypt unit. the ph optimum for lactase activity at all heights was between 5.0 and 6.0. in the presence of pcmb, all activity at ph 3.5 was abolished. since the ph optimum of acid /3-galactosidase is 3.5, this verifies that in homogenates of cryostat sections, pcmb totally inhibits the acid /%galactosidase activity as has also been shown in whole intestinal homogenates, and partially purified fractions of this lysosomal enzyme.z4*2'~28 since there was some variation in the number of sections obtained from different animals, the activities of various enzymes in serial homogenates were related in an "idealized villus-crypt unit."%' this maneuver allowed data from a number of animals to be easily compared. the percent distance each homogenate represented of the total villus-crypt unit was determined in any given tissue block. thus, enzyme activity in the sixth of 16 homogenates was considered to represent enzyme activity at a point between 35% and 40% of the distance between tip and base of an "idealized villus-crypt unit." table 1 . the enzyme activity of each homogenate was calculated both as specific activity (micromoles of substrate hydrolyzed per hour per milligram protein) and as total activity (micromoles of substrate hydrolyzed per hour per homogenate). since both lactase and sucrase activities were performed on the same homogenates, the lactase/sucrase ratio is independent of how the individual activities are expressed. however, to allow for statistical comparison between individual lactase and sucrase activities at any point along the villus-crypt unit, two alternative modes of expression of data were utilized: (a) the highest specific activity of each enzyme along the villus-crypt unit in any given rat was set at 100% and all other activities on the same unit expressed as a percent of that highest activity. (b) the total activity of each tissue block was determined by summation of total activities of consecutive homogenates. the total activity of each enzyme in each homogenate was then expressed as a percent of the total activity of the tissue block. the data from each of eight rats were plotted along the idealized villus-crypt unit as explained above. average values for each 5% of distance were calculated. when comparing lactase to sucrase activity at any given height of the villus-crypt, student's t-test was used.2q one-way analysis of variance30 was used to determine if there was significant variation of mean lactose/sucrose (l/s) ratios at different heights of the villus crypt. figure 1 shows the distribution of specific lactase, sucrase, and acid /&galactosidase activity along the villus-crypt unit. the general pattern of distribution of activity agrees with previous reports.'-' the mean l/s ratios at different heights of the villus crypt are plotted in figure 2 . since both enzyme assays were performed on the same homogenate, the l/s ratio is independent of how enzyme activity is expressed. the results show that there is a definite gradient of increasing l/s ratio in going from crypt to apical villus. using one-way analysis of variance, the ratio in lower villus is significantly less than in apical and midvillus ( table 1 ). the reason for this increase in l/s ratio can be seen if the specific activity of each enzyme is expressed as a percent of the maximal specific activity along a particular villuscrypt unit (figure 3 ). sucrase reaches maximal activity in enterocytes along the upper 70%-75% of the villus-crypt height, whereas lactase does not peak until the top 45%-50%. if the total activity of each enzyme in each homogenate is expressed as a percent of the total activity of the tissue block, the lag in expression of maximal lactase activity is also seen (data not shown). total activity of both disaccharidases decreases sharply in the apical portions of the villus-crypt unit because of small amounts of protein in apical homogenates. the net result of the different patterns of distribution is that 63% + 3% (mean +-sem) of total lactase activity along the villus-crypt unit is present on the top 50% of the unit as compared with 51% + 3% of sucrase activity (p < 0.02) this is the first report that directly compares distribution of lactase and sucrase activities along the crypt-villus unit of the adult rat. the technique x, y x = p < 0.05; y = p < 0.01. for statistical purposes, individual data for each 10% of height of the villus crypt were pooled. one-way analysis of variance confirmed that population means differ substantially, f = 18.9. least significant difference (lsd) was used to compare individual means. figure 3 . relative activity expressed as mean percent maximal specific activity of sucrase (0) and lactase (0) along the villus-crypt unit. the highest specific activity in each tissue block was set at 100% and activities in all other homogenates expressed as a percent of that highest activity. letters denote level of statistical significance between lactase and sucrase activities at any point along the villus-crypt unit. (a. p < 0.05; b. p < 0.02; c. p c: 0.01; d. p < 0.001.) absence of letter indicates no significant differences between maximal activities at a given height. of cryostat sectioning of frozen intestine was chosen because it provides precise sequential separation of cells along the villus-crypt unit after minimal manipulation of bowel. the other accepted method of cell separation is the weiser "washing" technique.3 while this is an excellent method for obtaining isolated enterocytes from apical villus, lower villus, and crypt, we were concerned with the specificity of "washing" cells sequentially down the villus in order to determine a true gradient of enzyme activity. in addition, the method does not control for the possibility of differential binding of the two enzymes onto the microvillus. selective solubilization of lactase in early washings from cells still adherent to the villus-crypt unit may result in erroneously low levels in lower portions of the villus. in our present study, the general pattern of distribution of both lactase and sucrase along the villus crypt agrees with previous reports,z-7 i.e., localization of activity to the villi and absence of activity in the crypts. however, by directly comparing enzyme activity in the same homogenate at each height of the villus crypt, we found that, unlike sucrase, lactase fails to achieve maximal activity until midvillus. this observation is based on the stepwise increase in mean l/s ratio going from crypt to midvillus. since both lactase and sucrase enzyme assays were performed on the same homogenate, the l/s ratio is independent of how enzyme activity is expressed. the more apical localization of lactase activity compared to sucrase activity on the crypt-villus unit may help to explain the vulnerability of this enzyme to acute mucosal injury (e.g., acute gastroenteritis). histologic studies in acute viral gastroenteritis have shown blunting of villus height and disorganization of surface epithelial cells.3*~32 in the event of apical destruction of villus cells carrying lactase activity, the remaining activity in cells in the mid to lower levels of the villus may be below critical threshold to allow for physiologic hydrolysis of lactose. it is important to remember that total jejunal lactase activity is between one-fifth (unpublished from our laboratory) and one-tenth" that of sucrase activity in the adult rat. the more even distribution of sucrase along the crypt-villus unit would then explain the usual normal ability to absorb sucrose in acute gastroenteritis. recent data suggest that invasion and damage of epithelial cells by the virus itself may not be the only factor in the pathogenesis of diarrhea in this disease. study of transmissible gastroenteritis in piglets (corona virus) has suggested that intestinal epithelial cell migration rates increase in response to viral invasion.33.34 while this leads to rapid desquamation of infected cells, the villi become populated by relatively immature cryptlike cells. based on results of the present experiment, lactase activity, which is more dependent on cell maturity than sucrase, would be primarily affected by reparative events occurring after infection. nordstrom et a1.35 have described the distribution of disaccharidase activity along the crypt-villus unit in 1 human patient using the same technique as in our report. difficulties in mounting and slicing human tissue restricted statistical comparison between a number of patients. although it is not stated that the activities of all enzymes were assayed on the same villus-crypt unit, the authors report a more apical distribution of jejunal lactase activity when compared to sucrase and maltase. this observation lends further clinical credence to the significance of the present studies in the rat. further studies are needed to determine the mechanism responsible for the differences in distribution of lactase and sucrase activities. differences in location where synthesis begins, or differences in the rate turnover of lactase and sucrase might lead to a difference in enzyme distribution along the villuscrypt unit. in view of the similarities of the lactase and sucrase distribution pattern between rat and human, the rat would be a suitable model for these studies. studies of small intestine. iii. infantile diarrhea associated with intolerance to disaccharides carbohydrate intolerance in infants with diarrhea jejunal disaccharidase activities in acute diarrhea and convalescence (abstr) gastroenterology 54:1244 evaluation of a sucrose/electrolyte solution for oral rehydration of acute infantile diarrhea prospective comparison of indirect methods for detecting lactase deficiency effect of cortisone on the developmental pattern of the neutral and the acid fi-galactosidase of the small intestine in the rat the constant renewal of the intestinal epithelium of the albino rat intestinal enzymes: indicators of proliferation and differentiation in the jejunum intestinal epithelial cell surface membrane glycoprotein synthesis dahlqvist a: localization of /?-palactosidases and acid phosphatase in the small intestine wall. comparison of adult and suckling rat characteristics and postnatal development of acid lipase activity of the rat small intestine protein synthesis in small intestine: localization and correlation with dna synthesis and sucrase activity quantitative determination of enzymes in different parts of the villi and crypts of rat small intestine the digestive function of the epithelium of the small intestine. ii. localization of disaccharide hydrolysis in the isolated brush border portion of intestinal epithelial cells zur technik und indikation der ernahrung mit eiweissmilch acquired milk intolerance in adult caused by lactose malabsorption due to a selective deficiency of intestinal lactase activity koldovsky 0: cell migration and cortisone induction of sucrase activity in jejunum and ileum isolated intestinal lactase deficiency in the adult intestinal disaccharidase activities in adult and suckling rats the turnover of disaccharides and brush border proteins in rat intestine the relation of size to the relative rates of degradation of intestinal brush border proteins on the distribution of some enzymes in the duodenum and ileum of the rat method for assay of intestinal disaccharidases a method for the separate assay of "neutral" and "acid" p-galactosidase in homogenates of rat small intestine mucosa protein measurement with folin phenol reagent use of the glucose oxidase method for assay of disaccharidase activities in the small intestine-a limitation rat small intestinal p-galactosidases separation and isolation of rat and human intestinal /3-galactosidases the principles and practice of statistics in biological research acute infectious nonbacterial gastroenteritis: intestinal histopathology recent advances in viral gastroenteritis transmissible gastroenteritis of swine: virus-intestinal cell interactions transmissible gastroenteritis: sodium transport and the intestinal epithelium during the course of viral enteritis quantitative distribution of some enzymes along villi and crypts of human small intestine key: cord-320693-de1lmzl1 authors: hu, han; guo, nan; chen, shuhua; guo, xiaozhen; liu, xiaoli; ye, shiyi; chai, qingqing; wang, yang; liu, binlei; he, qigai title: antiviral activity of piscidin 1 against pseudorabies virus both in vitro and in vivo date: 2019-07-31 journal: virol j doi: 10.1186/s12985-019-1199-4 sha: doc_id: 320693 cord_uid: de1lmzl1 background: swine-origin virus infection spreading widely could cause significant economic loss to porcine industry. novel antiviral agents need to be developed to control this situation. methods: in this study, we evaluated the activities of five broad-spectrum antimicrobial peptides (amps) against several important swine-origin pathogenic viruses by tcid(50) assay. plaque reduction assay and cell apoptosis assay were also used to test the activity of the peptides. protection effect of piscidin against pseudorabies virus (prv) was also examined in mouse model. results: piscidin (piscidin 1), caerin (caerin 1.1) and maculatin (maculatin 1.1) could inhibit prv by direct interaction with the virus particles in a dose-dependent manner and they could also protect the cells from prv-induced apoptosis. among the peptides tested, piscidin showed the strongest activity against prv. moreover, in vivo assay showed that piscidin can reduce the mortality of mice infected with prv. conclusion: in vitro and in vivo experiments indicate that piscidin has antiviral activity against prv. swine-origin virus infection is one bottleneck for the development of the porcine industry worldwide. the pathogenic viruses isolated from pigs including pseudorabies virus (prv), porcine reproductive and respiratory syndrome virus (prrsv), porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev), and rotavirus (rv) are commonly observed in china. the viral infection is also responsible for the secondary infection by bacteria which has greatly increased the application of antibiotics [1, 2] . in the past decades, the efforts to alleviate pig viral diseases have focused on the development of vaccines to enhance the adaptive immunity of the hosts [3, 4] . however, some of the evolving viruses could escape from the host immunity through different kinds of strategies. several viral diseases have broken out in recent years among pig herds, such as the reemergence of swine-origin influenza in 2009 [5] , ped in late 2010 [6] and pseudorabies in 2012 [7] . thus, there is an urgent need to develop novel potential agents to kill these viruses or block their infection. amps are common host defense molecules in nearly all forms of life. ever since their discovery, amps have gained worldwide attention as important alternatives in the field of disease prevention and immune modulation [8, 9] . previous studies mostly focused on the protection effect of amps against bacterial and fungal infection [10] . later on, amps were also reported to be effective against viral infection [11] . it has been reported that some alpha-helical peptides can act as virucidal agents. for example, caerins and maculatins isolated from amphibian skin could completely inhibit human immunodeficiency virus type 1 (hiv-1) infection after virus is exposed to peptides within minutes [12] . piscidins, discovered in the mast cells of fish, could reduce the infectivity of several important fish-origin viruses [13] . indolicidin, a natural 13-amino acid antimicrobial peptide isolated from bovine neutrophils, could directly kill hiv-1 [14] . bovine lactoferricin derived from bovine lactoferrin has been reported to exert antiviral activity to fight against human cytomegalovirus (hcmv), herpes simplex virus type 1 (hsv-1), hsv-2, and adenovirus [15] . other antimicrobial agents, such as human defensins which is a group of beta-sheet peptides, can inhibit enveloped viruses such as hsv-1 and hsv-2, hiv-1, vesicular stomatitis virus (vsv), influenza virus, and hcmv by directly inactivating viruses [16] . therefore, amps might be promising agents against viral infections. prv, a large enveloped dna virus, is a swine neurotropic herpesvirus [17] . although the pigs are the natural reservoir for the virus, most mammals and some avian species are also susceptible to prv. prv infected animals may die from central nervous system disorders [18] . prv infection poses a severe threat to pig industry and either attenuated live or inactivated vaccines are usually used to control the disease [19] . although vaccination can suppress development of the disease, vaccines cannot eliminate virus infection. mutant isolates emerged and caused pseudorabies outbreak in 2012 [7] . thus, novel antiviral agents should be developed as a complementary to vaccination. in one of our recently published papers, we described the antiviral activity of caerin against porcine epidemic diarrhea virus (pedv) in vitro [20] . in this study, we investigated the activity of five amps including piscidin, caerin, maculatin, lactoferricin b, and indolicidin against several porcine-origin viruses. more inhibitory activity of piscidin, caerin, and maculatin against prv was also explored. the in vivo protection effect of piscidin against prv infection was further investigated. chen et al found that piscidin has the anti-inflammatory and anti-nociceptive properties in inflammatory animal models [21] . kumar et al reported the obvious anti-endotoxin and anti-bacteria properties of piscidin-1 analogues both in vitro and in vivo [22] . the peptides (table 1 ) used in this study were synthesized on an automated solid-phase peptide synthesizer by neweast biosiences inc. (wuhan, china) [23] . the crude peptides were purified via a reverse-phase highpressure liquid chromatography (rp-hplc) using a c 18 column (waters xbridge). the elution was conducted using a water-acetonitrile linear gradient (0-80% of acetonitrile) containing 0.1% (v/v) trifluoroacetic aicd (tfa). finally, the purity and accurate masses of the product peptides were determined using hplc and mass spectrometry, respectively. the prv strains of ea, hnxx, 152, and tgev wh-1 were propagated in pk-15 cells based on the method reported in previous studies [24] . rv tm-a was propagated in rhesus monkey kidney cell line (ma104) [25] . prrsv ya was cultured in marc-145 cells [26] . pedv strain ch/ynkm-8/ 2013 was cultured in vero cells as described before [27] . the virus suspension was stored at − 80°c until used. in our initial topical screening assays, the viruses were pre-incubated with peptides (50 μg/ml) for 1 h at 37°c before they were added to the target cells. peptide-free controls (virus plus cell culture medium only) were incubated in parallel. after incubation, the tcid 50 of peptidetreated viruses and peptide-free viruses was measured. residual virus was calculated according to the following formula, residual infectivity = b/a × 100% ("a" represent tcid50 of residual virus non-treated by the amps (control), "b" represent tcid50 of residual virus after treated by amps). a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (mtt) cell proliferation assay was used to assess cell viability as described previously [28] . briefly, pk-15 cells were added to 96-well tissue-culture plates (coster, usa) and incubated overnight at 37°c. serial two fold dilutions of amps ranging from 200 to 3.12 μg/ml were added into the plate. the cells were incubated with amps at 37°c for 48 h, and the morphology of the cells was evaluated under light microscope. then, the cells were further incubated with mtt solution for 4 h and the cell pellets were collected for measurement of absorbance at 490 nm by an elisa reader. the antiviral activity was evaluated by plaque reduction assay [29] at different infection stages: pre-infection and post-infection, as well as direct inactivation of the viruses. briefly, the direct effect of peptides on virus per se was analyzed by the following procedures. monolayer of to analyze the post-infection effect of the peptides on virus growth, cells were infected with the same amount of virus mentioned above at 37°c for 1 h. subsequently, the cells were washed 3 times with dmem and treated by the peptides at different concentrations for 1 h at 37°c. the peptides were removed from the cell culture and replaced with the overlaid medium. the resulting pfu titer was determined as described above. to understand the pre-infection effect of the peptide on virus, the cells in 12-well tissue culture plates were treated with peptides at serial concentrations at 37°c for 1 h. then, cells were washed with dmem for 3 times, followed by infection with viruses. the cells were covered with the overlaid medium for plaque assays. cell apoptosis was analyzed with annexin v-fitc kit (nanjing keygen biotech. co., ltd). briefly, fitc-conjugated annexin v (50 μl/well) and propidium iodide (pi, 50 μl/well) were added to the cells infected by amptreated viruses. then, the obtained mixture was incubated at room temperature for 15 min in the dark prior to fluorescence observation. cell apoptosis was determined on basis of the observation soon after initiating apoptosis. cells translocated the membrane phosphatidylserine (ps) from the inner surface of the plasma membrane to the cell surface. thereafter, ps can be easily detected by staining with a fluorescent conjugate of annexin v (green), a protein that has a high affinity to ps. cellular late apoptosis was determined by cell staining with pi (red). the results were analyzed by fluorescence microscope (nikon, japan) at 36 hpi. in vivo prv challenge assay the 6 to 8 week-old specific-pathogen-free balb/c mice were purchased from the experimental animal center of zhongnan hospital of wuhan university (china) and randomly divided into six groups consisting of 10 mice each. individuals in each group of mice were anesthetized with 1-3% isoflurane gas and challenged by the intra-footpad injection with 50 μl of dmem containing 5 × 10 3 tcid 50 of prv-ea in the presence or absence of 10, 5, 1, 0.2 μg/ml piscidin. after 14 days, the surviving mice were challenged with 5 × 10 3 tcid 50 of prv again. mice viability and behaviors were monitored on a daily basis. in the first 2 days after the challenge, the mice commonly did not exhibit severe symptoms. however, mice gradually showed neurological symptoms in the subsequent days during the monitoring period and the post-challenged mice were monitored since day 3 post prv infection once every 6-8 h for 8 days to obtain the survival information of the mice. the level of prv infection symptoms was scored for every mouse by the following system: 0 = posture normal, absence of neurological symptoms, 1 = mild neurological symptoms: excitation, unrest, occasional itching, and foot swollen; 2 = severe neurological symptoms: ataxia, severe pruritus, and self-mutilation, biting and bleeding of the footpad. mice were euthanized in the chamber with co2 gradually filled when the score reached 2. on day 6, three mice from control group and noncontrol groups were euthanized and brain tissues were collected and transferred to 4% paraformaldehyde. the tissues were dehydrated in ascending grades of ethyl alcohol i.e. 70, 90 and 100% ethanol. afterwards, the tissues were cleared with xylene. slides were stained with haematoxylin and eosin (h&e) stain and analyzed through a light microscope. all of the animal experimental protocols were approved by the ethics committee of huazhong agricultural university according to hubei province laboratory animal management regulations (hzaumo2015-0015). during the experiments, mice were offered ad libitum access to water and food in a controlled environment of a 12 h light/dark cycle. all efforts have been made to reduce suffering of animals. the antiviral effects of the peptides (maculatin, caerin, piscidin, lactoferricin b, indolicidin) were investigated in vitro against several viral pathogens that severely threaten the porcine industry. four enveloped viruses were used in this assay including the prv ea strain, pedv ynkm strain, tgev wh-1 strain, and prrsv ya isolate, as well as one non-enveloped rv tm-a strain. the peptides exhibited different inhibitory activities against these viruses (fig. 1) . the relative potency of the peptides against prv was: piscidin≈caerin>ma-cualtin>indolicidin> lactoferricin b. piscidin and caerin showed strong virucidal activity with the residual infectivity being around 2.3% while indolicidin and lactoferricin b exhibited mild antiviral activity. in descending order of potency, the peptides against pedv ranked as follows: caerin>piscidin> maculatin>lactoferricin b > indolicidin. caerin had the most potent activity with the residual infectivity being 0.2%. the assay results indicated, that the order of potency versus prrsv was: caerin>lactoferricin b > piscidin>maculatin>indolicidin. caerin exhibited the best inhibitory activity with the residual infectivity being 41.1%, separately. the assay results of antivirus activity against tgev indicated the activity order as follows: piscidin>lactoferricin b > indolicidin>maculatin>caerin. piscidin was the most potent peptide versus tgev with the residual infectivity being 41.5%. the assay result also indicated that prrsv and tgev were less sensitive to the tested peptides, compared with other viruses (fig. 1) . when it comes to rv, the relative potency order was: piscidin>caerin>maculatin>lactoferricin b > indolicidin. piscidin displayed the most potent activity against rv with residual infectivity being 7.4%. piscidin and caerin exhibited activity against almost all the viruses with the residual infectivity being lower than 50% with exception of piscidin versus prrsv and caerin versus tgev. caerin showed the greatest inhibitory activity against pedv with the residual infectivity being 0.2%, which was the lowest value among all the viruses tested. maculatin exhibited strong activity against prv (10.1%) and mild activity against rv (43.4%). however, it showed limited activity against pedv, prrsv, and tgev. lactoferricin b and indolicidin were not as potent as other peptides to fight against viruses. lactoferricin b showed stronger inhibitory activity against tgev than against other tested viruses with residual infectivity being 54.1%, and residual infectivity of indolicidin versus prv was 34.6%. we examined the effects of amps on cell viability after incubating pk-15 cells with different concentrations of each peptide. cytotoxicity was evaluated using mtt method (fig. 2) . the non-linear regression analysis result indicated that the 50% toxic concentration (tc 50 ) of caerin, maculatin, piscidin, and indolicidin were 67.8, 76.6, 65.2, and 110.2 μg/ml, respectively. indolicidin was fig. 1 spectrum of antiviral activity of the peptides (tcid 50 assay). peptides (50 μg/ml) and viruses were incubated at 37°c for 1 h before they were added to the target cell monolayers. after incubation for 48-72 h, tcid 50 of the virus was recorded. the bars represent ± se. three separate assays were conducted (n = 3). the dashed line means 50% residual infectivity fig. 2 cytotoxic properties of the peptides. pk-15 cell monolayers were incubated with peptides. the cytotoxicity was measured by mtt assay (n = 3). the cell survival rates at different peptide concentrations were plotted and the dashed line means 50% cell survival less cytotoxic than piscidin, caerin and maculatin. lactoferricin b exhibited the weakest cytotoxic activity and it did not exhibit obvious cytotoxic activity even at maximum concentration of 200 μg/ml. to determine whether the inhibitory activity against prv by maculatin, caerin, piscidin was strain-specific or not, we tested the activity of these peptides against different prv isolates including prv-hnxx (a newly prv isolate in china in 2012) and prv-152 (modified bartha strain). we found that all the three peptides displayed inhibitory activities against all the tested strains (prv hnxx, prv 152, and prv-ea) (fig. 3a) . this finding indicated that the activity of the peptides versus prv was not strain-specific. when the concentration of the peptides increased from 50 to 100 μg/ml, the residual infectivity of prv decreased (fig. 3b) . this trend was the most obvious for maculatin with the residual infectivity decreasing from 10.11 to 0.0017%, while the groups treated with piscidin and caerin exhibited a decrease by more than 30 folds in the residual infectivity. these data indicated that these peptides inhibited the virus in a dose-dependent manner. in order to better understand the inhibitory effect of amps on the propagation of prv-ea, we examined a b fig. 3 prv inhibitory activity displayed by maculatin, piscidin and caerin (tcid 50 assay). a prv of ea, hnxx strain and 152 isolate were treated with the peptides (50 μg/ml) for 1 h before they were added to the cell monolayers. the bars represent means ± standard errors of the means of three separate experiments (n = 3). *, statistically significant difference by one-way anova with tukey post hoc test (p < 0.05), ns means not significant. b prv ea was treated with peptides (100 μg/ml) for 1 h before added to the cell monolayers. the effects of the peptides at 50 μg/ml and at 100 μg/ml against prv ea were compared whether amps directly damaged virus particles or indirectly interacted with the host cells pre-or post-infection. plaque-reduction assay was also performed to confirm the peptides' antiviral activity. the viruses were incubated with the peptides prior to infection. the result showed that all the three peptides inhibited the virus infection in a dose-dependent manner (fig. 4) . at the concentration of 25 μg/ml, pisicidin, caerin and maculatin were found to inhibit most of the prv particles. pisicidin exhibited the strongest activity and obvious inhibitory ability at concentrations above 5 μg/ml. the 50% effective concentration (ec 50 ) values of the peptides were calculated using probit analysis by ibm spss (version 21, new york, usa). the analysis revealed that ec 50 values of caerin and maculatin were 2.63 μg/ml and 1.09 μg/ml, respectively. ec 50 of piscidin was the lowest (0.23 μg/ml) among the peptides tested, which indicated that piscidin was the most potent antiviral peptide against prv in this study. subsequently, the experiments were conducted to further determine whether the peptides could function at post-(curative) or pre-infection (prophylactic) stage. the cell monolayers were treated with piscidin, caerin, and maculatin before (prophylactic) or after (curative) the virus binding stage, separately. no inhibitory effect of the three peptides on prv could be detected (the obtained data not shown). this implied that these peptides probably inhibited prv infection by directly interacting with the virus particles. the cell apoptosis assay showed that prv could induce both early and late apoptosis of pk-15 cells (fig. 5) . the late apoptosis rate of cells infected by the peptides-treated viruses decreased as the concentration of the peptide increased (fig. 5b) . the inhibition of late cellular apoptosis was obvious at the concentration of 25 μg/ml (fig. 5a) . in the control group of the in vivo assay, mice (n = 10) were injected with 50 μl of piscidin (200 μg/ml) by the intra-footpad route. all mice survived and behaved normally. prv was injected into mice with or without piscidin at various dosages (0.5, 2.5, 5, 10 μg/ml). and the surviving mice were re-challenged with prv on day 14 (fig. 6) . the mice survival rate was recorded for 28 days. protected against prv infection (fig. 6) . however, one mouse from the 2.5 μg/ml peptide-treated group and nine mice from the 0.5 μg/ml group died within 10 days. to further characterize the prevention effect of piscidin against brain damage induced by prv, the brain sections of mice from the control group, prv-infected, and co-injection group were collected respectively and subjected to pathological examination. the blood stasis was observed in the brain of mice from prv-infected group (fig. 6a) . the brain of mice from co-injection group showed no specific symptom. after 14 days, the surviving mice were re-challenged with prv at 5 × 10 3 tcid 50 . the groups co-injected with piscidin and prv failed to provide protection with the survival rates dropping to 30% at the end of the experiment and the mice in the 5 μg/ml peptide treated group all died. we previously reported the proof-of-concept usage of amps as the bactericidal agents against pathogenic bacteria isolated from pig herds [23] . the five antimicrobial peptides used in this study have been reported to inhibit the growth of various kinds of viruses including hiv, hsv-1, hsv-2, etc. [12] [13] [14] [15] [20] [21] [22] . however there has been limited information about the inhibitory activity of these peptides against the swine-origin viruses. considering this, this study evaluated the effect of these peptides against several porcine viral pathogens. three peptides (caerin, piscidin, maculatin) exhibited inhibitory activity against prv, pedv, tgev, prrsv, and rotavirus. plaque reduction assay showed that the prv infection could be inhibited in a dose-dependent manner by direct treatment of the peptides. a previous study indicated that caerin and maculatin could inhibit hiv infection without affecting t-cell viability and that the activity of caerin was more potent than that of maculatin [12] . in our research, we further tested the activity of caerin and maculatin against prv, pedv and prrsv. compared with maculatin, caerin showed better activity against most of the tested viruses except tgev (fig. 1) . the fact that caerin has a longer α-helix than maculatin, which enables caerin to disintegrate the membrane more potently, might explain the better activity of caerin. usually, the bacterial membrane and viral envelope were reported to be the main targets of the amps. for example, hiv envelope integrity was directly disrupted by caerin and maculatin [12] . however in this study, tgev and prv which both acquired their envelopes from pk-15, exhibited different levels of sensitivity to the tested peptides. in addtion to its envelope, physical characteristics of the virus such as viral size, surface area, and morphology might affect its sensitivity. hnp-1 also demonstrated a 1000-fold difference in activity against different enveloped viruses [16] . it should also be noted that the antiviral activity of the amps tested in this study is only observed at concentrations that are already somewhat cytotoxic at 48 h and could be even more so at 72 h or longer. to our surprise, piscidin, caerin, and maculatin a b fig. 6 piscidin protects mice from prv-induced death. a. processed brain sections from control, co-treated, and prv infected groups were subjected to haematoxylin and eosin (h&e). the representative h&e results were shown. b. the mice injected with piscidin or prv, co-treated with piscidin and prv were divided into different groups. surviving mice were challenged with prv on day 14 again. survival status of control and experiment groups were monitored on a daily basis for 28 days (n = 10). in the group of piscidin (10 μg/ml), 2 mice were heavily infected and died on day 19 and 20. and another 5 mice with severe neurological symptoms were euthanized for animal welfare reasons; in the group of piscidin (0 μg/ml) + prv, 2 mice were heavily infected and died on day 5. and another 7 mice with severe neurological symptoms were euthanized for animal welfare reasons; in the group of piscidin (10 μg/ml) + prv, 1 mouse was heavily infected and died on day 19. and another 6 mice with severe neurological symptoms were euthanized for animal welfare reasons; in the group of piscidin (5 μg/ml) + prv, 3 mice were heavily infected and died on day 19 and 20. and another 7 mice with severe neurological symptoms were euthanized for animal welfare reasons; in the group of piscidin (2.5 μg/ml) + prv, 1 mice were heavily infected and died on day 20. and another 6 mice with severe neurological symptoms were euthanized respectively for animal welfare reasons; in the group of piscidin (0.5 μg/ml) + prv, 3 mice were heavily infected and died on day 5 and 7. and another 6 mice with severe neurological symptoms were euthanized for animal welfare reasons; a total of 11 mice from all groups survived and were euthanized by the end of the experiment could consistently inhibit the growth of rv, a non-enveloped rna virus. among these three peptides, piscidin showed the strongest inhibitory activity. it has been reported that piscidin possessed significant antiviral activity to fight against both frog virus 3 and channel catfish virus [30] . the reduction of fv3 infectivity resulting from the application of piscidin is due to its capacity to interact with the essential lipid membrane of fv3 [13] . based on these results, it could be speculated that there might be other targets for these peptides in addition to the viral envelope, which need further exploration. lactoferricin b and indolicidin showed weak or no activity against the five tested viruses compared with caerin, maculatin, and piscidin. robinson et al reported that the ic 50 of indolicidin against hiv-1 ranged from 67 μg/ml to 100 μg/ml [31] . the weak activity of indolicidin against the tested viruses in this study was probably due to the low concentration we used (50 μg/ml). it has been reported that amps could inhibit both extracellular and intracellular viruses [32] [33] [34] . our plaque reduction assay result indicated that piscidin, caerin, and maculatin could inhibit prv by directly interacting with the virus. addition of the peptides pre-or post-infection could not help inhibit the prv infection. vancompernolle et al reported that caerin inhibited hiv infection by disrupting the virion envelope [12] . chinchar et al also reported that amphibian-origin esculentin-2p (e2p) and ranatuerin-2p (r2p) could inactivate the channel catfish virus (ccv) by directly interacting with the virions [35] . the re-emergence of pseudorabies in china since 2011 has caused a huge economic loss to the pig farms. three prv strains, i.e. ea, 152 and hnxx, were chosen for further evaluation of peptide's antiviral activity. the peptides inhibited replication of the three prv strains. this is of great importance in fighting against the mutant viruses resulting from natural selection or antibodyinduced events. on the other hand, these peptides had some cytotoxicity on pk-15 cells (fig. 2) . piscidin has been reported to have antiviral and antibacterial properties in vitro [13, 36] . however, this had not been tested in vivo. in this study, the data showed that coinjection of prv with piscidin at the concentration of above 5 μg/ml could offer protection against prv infection. even when the concentration of piscidin decreased to 2.5 μg/ml, the survival rate of mice could reach 90%. huang et al previously reported that th1-5, as an amp isolated from tilapia, could offer 80% protection against jev infection at 100 μg/ml [37] . compared with th1-5, piscidin was more effective even at low concentration. as huang et al noted, th1-5 treated mice surviving from the first jev infection remained alive after second challenge with jev at day14 [37] . however, our in vivo studies exhibited a different result. the possible reason needs further exploration. this study indicated that piscidin, maculatin and caerin could inhibit the infection of prv, prrsv, pedv, tgev and rotavirus. among the peptides examined in this study, piscidin showed the strongest antiviral activity against prv both in vitro and in vivo. and it could block the prv-induced cell apoptosis as well. abbreviations amp: antimicrobial peptide; prv: pseudorabies virus; tcid 50 : tissue culture infective dose assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: current status and future direction s1 domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen pathogenesis and transmission of swine-origin 2009 a(h1n1) influenza virus in ferrets new variants of porcine epidemic diarrhea virus, china antimicrobial peptides: do they have a future as therapeutics? in: antimicrobial peptides defensins: antimicrobial peptides of innate immunity antimicrobial peptides: promising alternatives in the post feeding antibiotic era the immunology of host defence peptides: beyond antimicrobial activity inhibition of hiv infection by caerin 1 antimicrobial peptides the chemistry and biological activities of peptides from amphibian skin secretions generation of a dual-target, safe, inexpensive microbicide that protects against hiv-1 and hsv-2 disease a review of the design and modification of lactoferricins and their derivatives defensins at the mucosal surface: latest insights into defensin-virus interactions generation and characterization of ul41 null pseudorabies virus variant in vitro and in vivo role of the pseudorabies virus gi cytoplasmic domain in neuroinvasion, virulence, and posttranslational n-linked glycosylation vaccines against pseudorabies virus (prv) caerin1.1 suppresses the growth of porcine epidemic diarrhea virus in vitro via direct binding to the virus the use of the antimicrobial peptide piscidin (pcd)-1 as a novel anti-nociceptive agent single amino acid substitutions at specific positions of the heptad repeat sequence of piscidin-1 yielded novel analogs that show low cytotoxicity as well as in vitro and in vivo anti-endotoxin activity broad activity against porcine bacterial pathogens displayed by two insect antimicrobial peptides moricin and cecropin b a crispr/cas9 and cre/lox system-based express vaccine development strategy against re-emerging pseudorabies virus establishment and clinical application of a multiplex reverse transcription pcr for detection of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine group a rotavirus porcine reproductive and respiratory syndrome virus (prrsv) suppresses interferonbeta production by interfering with the rig-i signaling pathway porcine epidemic diarrhea virus orf3 gene prolongs s-phase, facilitates formation of vesicles and promotes the proliferation of attenuated pedv the ubiquitin-proteasome system is required for the early stages of porcine circovirus type 2 replication antiviral effect of diammonium glycyrrhizinate and lithium chloride on cell infection by pseudorabies herpesvirus inactivation of viruses infecting ectothermic animals by amphibian and piscine antimicrobial peptides anti-hiv-1 activity of indolicidin, an antimicrobial peptide from neutrophils antiviral peptides targeting the west nile virus envelope protein novel influenza virus ns1 antagonists block replication and restore innate immune function fusion core structure of the severe acute respiratory syndrome coronavirus (sars-cov): in search of potent sars-cov entry inhibitors inactivation of frog virus 3 and channel catfish virus by esculentin-2p and ranatuerin-2p, two antimicrobial peptides isolated from frog skin piscidin: antimicrobial peptide of rock bream modulation of the immune-related gene responses to protect mice against japanese encephalitis virus using the antimicrobial peptide, tilapia hepcidin 1-5 publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. availability of data and materials data and materials are available upon request by the corresponding author.ethics approval and consent to participate all of the animal experimental protocols were reviewed and approved by the ethics committee of huazhong agricultural university. not applicable. the authors declare that they have no competing interests. key: cord-280795-wtrt13ij authors: han, yu-tsung; tsai, chia-sheng; chen, ya-chio; lin, ming-kuem; hsu, yau-heiu; meng, menghsiao title: mutational analysis of a helicase motif-based rna 5′-triphosphatase/ntpase from bamboo mosaic virus date: 2007-10-10 journal: virology doi: 10.1016/j.virol.2007.05.013 sha: doc_id: 280795 cord_uid: wtrt13ij the helicase-like domain of bamv replicase possesses ntpase and rna 5′-triphosphatase activities. in this study, mutational effects of the helicase signature motifs and residue l543 on the two activities were investigated. either activity was inactivated by k643a-s644a, d702a, d730a, r855a, or l543p mutations. on the other hand, q826a, d858a and l543a had activities, in terms of k(cat)/k(m), reduced by 5to 15-fold. amppnp, a nonhydrolyzable atp analogue, competitively inhibited rna 5′-triphosphatase activity. analogies of mutational effects on the two activities and approximation of k(i(amppnp)) and k(m(atp)) suggest that the catalytic sites of the activities are overlapped. mutational effects on the viral accumulation in chenopodium quinoa indicated that the activities manifested by the domain are required for bamv survival. results also suggest that q826 in motif v plays an additional role in preventing tight binding to atp, which would otherwise decrease further rna 5′-triphosphatase, leading to demise of the virus in plant. bamboo mosaic virus (bamv), a member of potexvirus genus belonging to the alphavirus-like superfamily, has a positive-strand rna genome (∼ 6.4 kb) with a 5′ m 7 gpppg cap structure and a 3′ poly(a) tail . open reading frame 1 of the viral genome encodes a 155-kda replicase consisting of mrna capping enzyme, helicase-like and rnadependent rna polymerase (rdrp) domains sequentially from n to c termini. a disordered region of more than 100 amino acid residues and a proline-rich segment separate respectively the neighboring functional domains. these two flexible regions may allow one domain to interact with others dynamically during the viral replication process. the capping enzyme domain, expressed in membrane fractions of yeast, exhibited an s-adenosylmethionine-dependent guanylyltransferase activity by which gtp is methylated to form m 7 gtp before the m 7 gmp moiety is further transferred to the 5′-diphosphate end of mrna to form the m 7 gpppn cap structure (huang et al., 2005; li et al., 2001a) . such distinctive capping activity has also been demonstrated in semliki forest virus (sfv) (ahola and kääriäinen, 1995) , brome mosaic virus (ahola and ahlquist, 1999; kong et al., 1999) , tobacco mosaic virus (tmv) (merits et al., 1999) and hepatitis e virus (magden et al., 2001) ; thus, it is probably a common feature of the capping enzymes from the alphavirus-like superfamily. the e. coli-expressed helicase-like domain possessed both rna 5′-triphosphatase and ntp hydrolase activities (li et al., 2001b) . with the rna 5′triphosphatase and the aforementioned capping enzyme activities, a cap structure could be formed at the 5′ end of bamv mrna in vitro. the recombinant rdrp domain showed a template-dependent rna polymerase activity and had preferential binding activity to the 3′ noncoding region of the viral rna (huang et al., 2001; li et al., 1998) . rna 5′-triphosphatase specifically cleaves the 5′ γ-phosphate out of the nascent mrna in the first reaction step toward the cap formation. the documented rna 5′-triphosphatases can be grouped into three classes according to their primary structures and the catalytic mechanism employed. the meta-zoan and plant enzymes are of the metal-independent cysteine phosphatase type and distinguish themselves by possessing an hc(x) 5 rs/t motif, in which the cysteine residue acts as a catalytic nucleophile within the catalytic pathway (changela et al., 2001; takagi et al., 1997) . the enzymes from fungi, protozoa, and some dna viruses constitute a metal-dependent phosphohydrolases class with characteristic of possessing two essential glutamate-containing motifs (ho et al., 1998; martins and shuman, 2003) . rna triphosphatases, derived from rna helicases or helicase-like proteins, constitute another metaldependent phosphohydrolases class. this class includes the enzymes identified from rna viruses such as reovirus , alphavirus (vasiljeva et al., 2000) , dengue virus (matusan et al., 2001) , coronavirus , and bamv (li et al., 2001b) . both classes of the metal-dependent rna triphosphatases are also capable of hydrolyzing nucleoside triphosphates to nucleoside diphosphates and inorganic phosphate. according to motif-based classification, helicase-like proteins from rna viruses have been classified into three superfamilies (sf) (kadaré and haenni, 1997) . members of sf1 share similarity to nsp2 of alphavirus, while ns3-like proteins of potyvirus, flavivirus and pestivirus, and 2c-like proteins of picornavirus are representatives of sf2 and sf3, respectively. across the three sf, gks/t sequence of motif i (walker a site) is conserved, whereas the consensus sequences of motif ii (walker b site) are varied, with de/d sequences in sf1 and sf3 and dexh in sf2. based on the classification principle, the helicase-like domain of bamv replicase can be grouped into sf1. fig. 1 shows comparison of partial amino acid sequences of the bamv domain and some of the sf1 members. in relation to enzymatic activities, all three sf proteins have atpase activity. an rna helicase activity has also been broadly corroborated on proteins of sf2; however, the activity was so far demonstrated only on a couple of sf1 proteins, e.g., nsp2 of sfv (gomez de cedron et al., 1999) and the helicase domain of tmv replicase (goregaoker and culver, 2003) . like the helicase-like domain of bamv replicase, nsp2 of sfv has also been reported to have rna 5′-triphosphatase activity (vasiljeva et al., 2000) . ntp binding and hydrolysis functions of motif i and ii of helicases have been suggested by numerous mutational studies and confirmed by crystal structures of helicases such as pcra of bacillus stearothermophilus (subramanya et al., 1996; velankar et al., 1999) , rep of escherichia coli (korolev et al., 1997) and ns3 of hepatitis c virus (yao et al., 1997) . the lysine residue of motif i interacts with the phosphates of mgatp/mgadp and the threonine or serine ligates the mg 2+ ion. the first aspartic acid of motif ii also coordinates mg 2+ ion and the following glutamate or aspartate residue may act as catalytic base in atp hydrolysis. by contrast, functions of other signature motifs, particularly those of sf1, are relatively less addressed. as a step toward better understanding the relationship between activities and structures of the helicase-like domain of bamv replicase, we set out to investigate the importance of each signature motif to its ntpase and rna 5′-triphosphatase activities by mutational and kinetic analyses. results of this study strongly sug-gest a common catalytic site for the removal of 5′γ-phosphate from ntp and rna. the relation between the enzymatic activities in vitro and the viral replication in vivo is also discussed in this study. previous studies demonstrated that the helicase-like domain of bamv replicase exhibits an mg 2+ /mn 2+ -dependent γphosphohydrolase activity toward both nucleoside triphosphate and rna (li et al., 2001b) . the former (ntpase) could be a prerequisite for the putative rna helicase activity, and the latter (rna 5′-triphosphatase) could be involved in the cap structure formation. substitution of gaa for gks in motif i abolished ntpase as well as rna 5′-triphosphatase activities. in the present study, we examined the mutational effects of other conserved motifs of sf1 helicases with respect to these two activities. leucine at position 543 was also included in the mutation list because the accumulation level of the viral coat protein decreased significantly in protoplast of nicotiana benthamiana as leu543 to proline mutation was introduced into the viral replicase . in addition, a deih sequence immediately downstream motif vi was mutated because it is analogous to dexh box of motif ii of sf2. each of the targeted residues, as indicated in fig. 1 , was replaced by alanine except leu543 that was also replaced by proline. all the proteins were expressed in e. coli and purified by immobilized metal affinity and anionic exchange chromatography as described in materials and methods. the purification results are shown in fig. 2 . the importance of the mutated residues to atp hydrolysis activity was first analyzed qualitatively by tlc assay (fig. 3) . alanine substitution at k643-s644 or at d702 reduced the activity to extents similar to the background level. mutation of d730 or r855 to alanine or l543 to proline also resulted in severe damage to atp hydrolysis activity. in contrast, q826a and d858a remained active but with diminished activities. atpase activity manifested by helicases of sf2, e.g., ns3 proteins of yellow fever virus , hepatitis c virus (suzich et al., 1993) and bovine viral diarrhea pestivirus , is usually stimulated by single stranded rna. effect of rna on atpase activity of the bamv helicaselike domain was also examined in this study. inclusion of an rna transcript, corresponding to the first 200 nucleotides of bamv genome, in the reactions did not cause apparent changes in the atpase activity of any of the tested enzymes. to better characterize the ntpase activity, steady-state rates of the reaction were measured by enzyme-coupled assay, in which ntp hydrolysis was coupled to nadh oxidation. apparent k m and k cat of the wild-type enzyme toward four different mononucleotides were subsequently calculated based on ntp concentration dependences of rate (table 1 ). all four mononucleotides could be hydrolyzed by the wild-type enzyme at rates with apparent k m value ranging from 0.15 to 0.33 mm and apparent k cat from 24 to 67 s − 1 . the specificity constants (k cat /k m ) of the reactions are comparable to that of λ1 of reovirus and nsp13 of sars coronavirus and larger than that of nsp2 of sfv and ns3 of dengue virus (matusan et al., 2001; vasiljeva et al., 2000) . mutational effects of the targeted amino acid residues on the apparent kinetic parameters of atpase were subsequently analyzed by enzyme-coupled assays. consistent with tlc results, proteins with specified mutation in motif i, ii, iii or vi did not show atpase activity to any appreciable extent (not shown). l543p mutant was also inactive; however, l543a was active with an unchanged k m but a 14-fold reduced k cat (table 2) . d858a had k m and k cat reduced by factors of 2 and 10, respectively. q826a had 17-fold and 90-fold decreases in values of k m and k cat , respectively. it is worth noting that the specificity constant of q826a decreased only by a factor of 5.3; this and overtime incubation may account for the apparent atpase activity of q826a shown in the previous tlc assay. the significantly enhanced affinity to atp should account for, to some extent, the dramatic decrease of k cat in q826a. besides ntpase activity, the helicase-like domain of bamv replicase also catalyzes the removal of 5′ γ-phosphate from triphosphate-terminated rna. results of typical experiments with wild-type enzyme are shown in fig. 4 . phosphate increased steadily with time in the first 5 min under the reaction condition fig. 1 . alignment of partial amino acid sequences of the helicase-like domains of some rna viral replicases. the consensus residues within each signature motif of sf1 helicases are shown in bold. residues mutated in this study on bamv protein are indicated by asterisk. protein secondary structure was predicted at the psipred protein structure prediction server (http://bioinf.cs.ucl.ac.uk/psipred/). the rectangle and arrow symbolize the α-helix and β-strand, respectively. bamv, pvx, tmv, tymv, and hev represent bamboo mosaic virus, potato virus x, tobacco mosaic virus, turnip yellow mosaic virus and hepatitis e virus, respectively. (panel a). in addition, phosphates released were roughly proportional to the applied amounts of enzyme within a short period of reaction time (panel b). to define the mutational effects on the activity, reactions catalyzed by wild-type and various mutants were allowed to occur under conditions of enzyme excess (fig. 5) . the activities of k643a-s644a and d702a were nearly abolished. likewise, l543p, d730a and r855a had activities hardly to be observed. the apparent activity of l543a was comparable to that of wild type, while that of q826a and d858a was reduced noticeably. to characterize the effects in detail, reaction rates of l543a, q826a, d858a and the wild-type enzyme were determined at different rna concentrations, and the kinetic parameters were subsequently calculated (table 2) . considering the limitations, such as suboptimal substrate concentration, in data processing, the determined values of k m and k cat were apparent. still, they should be helpful to evaluate the mutational effects. comparing with mononucleotides, rna was hydrolyzed at much slower rates. alanine substitution at l543, q826 or d858 had unfavorable effects on both rna binding and catalysis as evidenced by the increase of apparent k m and the decrease of apparent k cat , respectively. in general, the specificity constants of the three mutants for rna hydrolysis reduced approximately 5-to 14-fold. overall, effects of the investigated mutations on atpase and rna 5′-triphosphatase were parallel, implying that the catalytic sites of the two activities are overlapped. to support the notion, effects of amppnp, a nonhydrolyzable analogue of atp, and amp on rna 5′-triphosphatase activity were examined. amppnp exerted an inhibitory effect on the activity (fig. 6a ). the higher concentration of amppnp, the greater extent of inhibition was observed. by contrast, amp did not significantly affect rna 5′-triphosphatase activity even as its concentration was up to 2 mm (fig. 6b) , suggesting that the 5′terminal γand β-phosphate groups of substrates are the major determinants for the competition. to know the inhibition mode of amppnp, the dependence of rate on rna substrate concentration was determined under different amppnp concentration (fig. 7a ). double-reciprocal plot of the data showed an approximately unchanged v max (fig. 7b) , suggesting that amppnp acted as a competitive inhibitor. the apparent k i value of amppnp in inhibiting rna 5′-triphosphatase activity the enzymatic activity was determined at 20°c by enzyme-coupled assay in 1 ml solution that contained 10 pmol enzyme, 0.1 to 3 mm ntp and other buffer components as described under materials and methods except that the amounts of pyruvate kinase were increased up to 50, 75 and 300 u for gtpase, utpase and ctpase assay, respectively, to assure the rate of ntp hydrolysis being the limiting step within the coupling reaction. was calculated to be 93 μm, which is comparable to the k m value of atp. probably, the catalytic sites for hydrolyzing 5′ γphosphate from rna and mononucleotide are identical or extensively overlapped. the competence of bamv to multiply in vivo was investigated by inoculating plasmid pcbg into leaves of c. quinoa, in which the viral replication and cell-to-cell movement could b atpase activity was determined at 20°c by enzyme-coupled assay in 1-ml solution that contained 0.1 to 3 mm atp, 10 to 600 pmol enzyme and other buffer components as described under materials and methods. data are averages of at least two independent experiments. c rna 5′-triphosphatase activity was determined by tlc analysis. reactions were carried out at 20°c in 3-μl solution that contained 1 pmol enzyme, 10 to 80 μm 5′-[γ-32 p]rna, and other buffer components as described under materials and methods. data are averages of two independent experiments. be monitored by the appearance of green fluorescence because the expression of the introduced gfp depends on the fulfillment of bamv replication (lin et al., 2004) . to see the mutational effects, each of the selected mutations was introduced into pcbg, and the resulting mutant plasmid was mechanically inoculated into plant cells. as shown in fig. 8 , spots with green fluorescence began to appear on leaves of plants infected with wild type, l543a, or d858a virus a week later after inoculation. the respective frequency and average diameter of fluorescent loci appeared on leaves were similar, suggesting that the three variants replicated equally well in c. quinoa. local lesions formed later on leaves that had shown green fluorescence spots earlier (fig. 9) . no signs of green fluorescence or disease symptoms were noted on leaves of plants infected with other mutant viruses, including that carries q826a mutation. repeated experiments with wild type, l543a, q826a and d858a confirmed the negative result of q826a mutation. disability on rna 5′-triphosphatase alone is sufficient to account for the replication incompetence of those carrying specified mutation in motif i, ii, iii, and vi and l543p since formation of 5′ cap at viral mrna is essential for substantial viral protein translation. however, the reasons for the opposite effects of the d858a and q826a mutations in plant are uncertain because the two mutations decreased rna 5′-triphosphatase activity to similar extents. would it be possible that alanine substitution at q826 affects not only the enzymatic activity but also the protein stability? protein stabilities of wild type, l543a, q826a and d858a were, therefore, investigated under 25°c by measuring their residual enzymatic activity as a function of time. the inactivation of the proteins followed first-order kinetics. the half lives (t 1/2 ) of wild type, l543a and q826a were approximately 2 h, whereas t 1/2 of d858a was 40 min (data not shown), indicating that protein stability would not be the cause. alternatively, the distinct feature of q826a on atpase activity might underlie the failure of the mutant virus to replicate in vivo. the mutant protein might not be capable of providing sufficient energy by hydrolyzing atp to the putative helicase activity, or the tight binding of atp deteriorated further the weakened rna 5′-triphosphatase activity. activity of rna 5′-triphosphatase was assayed in the presence of 0.3 mm atp, which is within physiological concentration range (hampp et al., 1982; usuda, 1988) , to explore the latter possibility (fig. 10) . under the competition conditions, rna 5′-triphosphatase activities of wild type and d858a remained approximately 30%, whereas little was left in q826a mutant. the results suggest that rna and mononucleoside triphosphate could have reciprocal inhibitory effects in vivo; and the deterioration of rna 5′-triphosphatase activity might be simply enough to disable the replication function of q826a mutant virus. helicases are defined as proteins that catalyze the separation of duplex nucleic acids into single strands in an ntp-dependent reaction. sequence comparison of the helicase-like proteins from rna viruses has disclosed the conservation of several signature motifs and grouped them into three sf (kadaré and haenni, 1997) . functions of helicase motifs have been addressed through mutational and structural analyses owing to the involvement of helicases in various important biological processes. as regards sf1, crystal structures of dna helicases pcra (subramanya et al., 1996; velankar et al., 1999) and rep (korolev et al., 1997) could provide us insights into the relationships between structures and functions of the proteins. in brief, residues on both motif i and ii are involved in the binding of atp through phosphate group recognition and mg 2+ coordination, while the second conserved acidic residue of motif ii acts as catalytic base in atp hydrolysis. other residues such as the glutamine of motif iii and the arginine of motif vi also participate in accommodating the γ-phosphate group of atp. as with other potexvirus, bamv replicase contains a helicase-like domain which can be classified into sf1. in this study, contributions of the conserved motifs of the helicase-like domain to ntpase and rna 5′-triphosphatase were investigated by mutagenesis. with respect to atpase activity, motifs i and ii are essential, consistent with the suggestions by crystal structures and mutational results reported in numerous literatures. mutations at the aspartate of motif iii and the arginine of motif vi brought on severe damages to the activity, also consistent with their involvement in atp binding. function of deih sequence immediately downstream motif vi is not analogous to that of dexh box (motif ii) of sf2 helicases because mutation of the sequence merely caused about 10-fold decrease in k cat value. q826 in motif v seems to avoid the tight binding of the protein to ground-state atp since alanine substitution at the residue reduced value of k m(atp) notably. the otherwise enhanced affinity to atp in q826a not only had adverse effect on atp hydrolysis but also caused significant interference with rna 5′-triphosphatase activity. l543, located in a predicted short β-strand outside the helicase motifs, might have a structural role supporting the active-site architecture for catalyzing atp hydrolysis. for one thing, l543 bears a hydrophobic side chain; for another, the destructive effect caused by proline substitution could be reversed to a certain extent by alanine substitution. nonetheless, this proposition needs to be verified by further structural analysis. as for rna 5′-triphosphatase activity, the effects caused by the various mutations were similar to those on atpase activity, except for q826a: the alanine substitution increased k m(rna) slightly for the rna 5′-triphosphatase activity, while decreasing k m(atp) for the ntpase activity more than 17-fold. the importance of the mutated residues in the viral accumulation in plant was also investigated. mutations of residues within the conserved motifs would disable the viral replication in vivo. by contrast, the two mutations outside the motifs, l543a and d858a, had insignificant consequence. the helicase motifs in 1a protein of brome mosaic virus have also been shown to play essential roles in rna replication in vivo by presumably being involved in viral rnas import (wang et al., 2005) . another aim of this study is to know whether the two enzymatic activities occur at a single or two independent catalytic sites. the generally parallel effects of the mutations on the two activities suggest that the protein employs the same amino acid constellation for the removal of 5′ γ-phosphate from rna and ntp. approximation between k i(amppnp) in rna 5′-triphosphatase competition assays and k m(atp) in atpase assays supports the same idea. still, we cannot rule out another possibility that the substrates might bind to two closely related sites with the atpase activity driving a conformational change that activates the rna 5′-triphosphatase activity, considering the opposite mutational effects on k m(atp) and k m(rna) of q826a. further structural evidence is needed to clarify the argument. nonetheless, employment of a common catalytic site for ntpase and rna 5′-triphosphatase activities has also been suggested on several helicase motif-base proteins such as sfv nsp2 (balistreri et al., 2007) , reovirus λ1 , sars-coronavirus nsp13 and dengue virus nsp3 proteins (bartelma and padmanabhan, 2002) . this raises a question as whether rna 5′-triphosphatase activity is an attendant function of helicases, or it is an adaptive outcome in the course of viral evolution. bamv genome contains two copies of helicase-like domains. one is the domain within viral replicase, the other is the p28 movement protein encoded by triple gene block. with the essential role to the movement of virus in the infected plant, p28 protein also possesses ntpase and rna-binding activities (wung et al., 1999) ; however, it did not show rna 5′-triphosphatase activity (li et al., 2001b) . taken together with the fact that only a few helicases (or helicase-like proteins) have been reported to have rna 5′-triphosphatase activity, we speculate that the 5′ γphosphohydrolase activity for rna molecule has evolved from ntpase activity driven by the demand for functional capping machinery, by which the mrna of rna viruses can be capped within cytoplasm of the infected cells. in spite of lacking explicit biochemical data, the helicase-like domain of bamv replicase has been thought to have rna helicase activity, which may be required for resolving intramolecular base pairing in the template rna and/or preventing the formation of extensive base pairing between template and the nascent complementary strand during rna replication process. is it possible that a protein domain can participate in rna replication while it is also responsible for the removal of 5′ γ-phosphate from the nascent rna to allow cap structure formation? with the help of the flanking flexible hinges, the helicase-like domain may be able to work with the rdrp or the capping domains at different steps during replication process. yet, we cannot rule out another possibility at this moment that the function of the helicase-like domain is for the formation of cap structure while the helicase activity required for bamv replication is provided by hosts. deferring the uncertainty of helicase activity, rna 5′-triphosphatase itself is definitely sufficient to dictate the survival of bamv in plant. mutations that abolished the enzymatic activity in vitro could result in the demise of the virus in c. quinoa. nonetheless, ∼7% of the wild-type activity, in terms of k cat /k m , seemed to be enough for the need since bamv bearing d858a mutation survived. the failure of q826a mutant in c. quinoa is intriguing because it has even higher rna 5′-triphosphatase function than d858a. further inhibition of the weaken rna 5′triphosphatase activity by tight binding of atp in vivo might account for the fatal outcome, indicating the need of a multiplefunction enzyme to coordinate all its activities for the survival of the organism. plasmid phwt, a pet32 derived-vector containing a cdna fragment encoding amino acids 514-892 of bamv replicase, was used for protein expression in e. coli as described previously (li et al., 2001b) . plasmid pcbg is an infectious clone of recombinant bamv, in which a green fluorescence protein (gfp) gene preceded by a duplicated coat protein promoter is situated between triple gene block and coat protein-coding region as described previously (lin et al., 2004) . initial transcription of the recombinant bamv genome from pcbg in plant is driven by 35s promoter of cauliflower mosaic virus. plasmid puhel is a puc18-based vector that contains an sphidigested fragment (4168 nt) isolated from pcbg. site-directed mutagenesis was done on phwt and puhel based on the protocol of quikchange site-directed mutagenesis kit (stratagene). after confirming the mutations with abi prism 3100 auto sequencer (perkinelmer), the sphi-digested fragment from the mutated puhel was put back into pcbg. the helicase-like domain of bamv replicase, fused with a thioredoxin, a poly-histidine tag, and an s-tag at the n terminus, was expressed in e. coli novablue cells (novagen) and purified as described previously with minor modifications (li et al., 2001b) . briefly, the recombinant protein, expressed as inclusion bodies, was first dissolved in urea-containing lysis buffer (50 mm tris [ph 7.5], 150 mm kcl, 0.1% brij-35, 10% glycerol, 10 mm β-mercaptoethanol, 1 mm pmsf and 8 m urea). refolding of the protein was done at 4°c by dropping 1 ml denatured protein solution (∼ 15 mg/ml) into 25 ml stirred lysis buffer. after centrifugation, the refolded protein in the supernatant was then purified by ni 2+ -nitriloacetic acid resin (qiagen) followed by q sepharose (pharmacia) using a linear gradient of nacl (20-500 mm) in equilibrium buffer (50 mm tris [ph 8.0], 0.1% brij-35, 20% glycerol, 10 mm β-mercaptoethanol, and 5 mm egta). the eluted protein was finally subjected to dialysis against equilibrium buffer containing additional 20 mm kcl. rna transcript consisting of the first 50 nucleotides of the plus-strand rna of bamv was used as substrate for rna 5′triphosphatase assay. the corresponding cdna fragment preceded by t7 promoter was first amplified from a cdna clone of bamv by pcr and purified through steps of page (10%), gel extraction and ethanol precipitation. the amplified cdna fragment was then used as template to produce 5′-[γ-32 p]rna in a 20-μl in vitro transcription reaction that contained 0.3 μg template dna, 30 mm ntp (7.5 mm each), 0.05 mci [γ-32 p] gtp (6000 ci/mmol, perkinelmer), 2 μl t7-megashortscript enzyme mix (ambion) and 1× t7 transcription buffer. after 2 h of incubation at 37°c, the reaction product was treated with 2 units of rnase-free dnase i (ambion) at 37°c for 15 min and the 5′-labeled rna was purified through 8 m urea-page (10%), gel extraction and ethanol precipitation. rna concentration was determined according to its optical density at 260 nm measured with nanodrop spectrophotometer. a 200nucleotide 5′-terminal fragment of the plus-strand rna of bamv was also prepared based on the above procedure and described before (li et al., 2001b) . ntpase activity was analyzed by detecting the liberated [α-32 p]ndp from [α-32 p]ntp on a polyethyleneimine (pei)cellulose thin-layer chromatography (tlc) plat or by the decreasing rate of od 340 nm in an enzyme-coupled assay in which ntp hydrolysis was linked to nadh oxidation through activities of pyruvate kinase and lactate dehydrogenase as described previously (li et al., 2001b) . in principle, the reactions were carried out at 20°c in solution containing 50 mm tris [ph 7.5], 5 mm mgcl 2 , 5 mm dtt and indicated amounts of substrate and protein. for enzyme-coupled assays, the solution also contained 2 mm phosphoenol pyruvate, 0.2 mm nadh, 20 u of pyruvate kinase and 20 u of lactate dehydrogenase unless otherwise indicated. rna 5′-triphosphatase activity was analyzed by measuring the γ-phosphate released from 5′-[γ-32 p]rna on pei tlc plate using a phosphorimager (typhoon 9200). standard reaction was performed at 20°c in solution containing 50 mm tris [ph 7.5], 5 mm mgcl 2 , 5 mm dtt, 5 u of rnase inhibitor (takara) and specified amounts of protein and 5′-[γ-32 p]rna. reaction was stopped by adding edta to final 25 mm. tlc was developed using 0.15 m licl-0.15 m formic acid. inhibition experiments of rna 5′-triphosphatase were conducted in the presence of indicated amounts of atp, amppnp, or amp. the amount of radiolabeled products on tlc plate was quantified according to its pixel count against a standard curve of serial diluted 5′-[γ-32 p]rna versus their respective pixels. the reaction rate was estimated from a period of reaction during which no more than 15% of the initial rna substrate was hydrolyzed. proportional ratio between the product released and the enzyme applied under this criterion (fig. 4b) suggests that the rates estimated should be close to the initial rate. the mechaelis-menten constant, k m and v max were determined from lineweaver-burk plot using grafit software. inhibition of amppnp was assessed using lineweaver-burk plot in the presence of different fixed concentrations of amp-pnp. the inhibition constant, k i , was calculated according to the dependence of the slope, k m /v max , of the lineweaver-burk plot on the inhibitor concentration. catalytic constant, k cat , was calculated based on the equation v max = k cat × [e] 0 where [e] 0 is the molar concentration of the protein used in assays. plasmid pcbg and its derivatives were inoculated mechanically into plant leaves of 3-week-old c. quinoa according to the method described previously with minor modifications . in short, 2 μg dna in 10 μl aqueous solution was rubbed gently over the surface of the selected leaf that had been dusted with a thin layer of sterile carborundum. each plasmid dna was applied to at least 9 leaves, 3-4 leaves a plantlet and total 3 plantlets. fluorescent images of inoculated leaves were obtained 7 days post-inoculation with a fluorimager with an excitation filter of 488 nm and an emission filter using calibration files. spots with green fluorescence were further observed under a fluorescent microscope. local lesions that appeared later on leaves could be observed by naked eyes. putative rna capping activities encoded by brome mosaic virus: methylation and covalent binding of guanylate by replicase protein 1a reaction in alphavirus mrna capping: formation of a covalent complex of nonstructural protein nsp1 with 7-methyl-gmp enzymatic defects of the nsp2 proteins of semliki forest virus temperature-sensitive mutants expression, purification, and characterization of the rna 5′-triphosphatase activity of dengue virus type 2 nonstructural protein 3 characterization of the reovirus λ1 protein rna 5′-triphosphatase activity characterization of the nucleoside triphosphate phosphohydrolase and helicase activities of the reovirus λ1 protein structure and mechanism of the rna triphosphatase component of mammalian mrna capping enzyme rna helicase of semliki forest virus replicase protein nsp2 oligomerization and activity of the helicase domain of the tobacco mosaic virus 126-and 183-kilodalton replicase proteins adenylate levels, energy charge, and phosphorylation potential during dark-light and light-dark transition in chloroplasts, mitochondria, and cytosol of mesophyll protoplasts from avena sativa l yeast and viral rna 5′ triphosphatases comprise a new nucleoside triphosphatase family evolution of bamboo mosaic virus in a nonsystemic host results in mutations in the helicase-like domain that cause reduced rna accumulation sequences at the 3′ untranslated region of bamboo mosaic potexvirus rna interact with the viral rna-dependent rna polymerase mrna guanylation catalyzed by the s-adenosylmethionine-dependent guanylyltransferase of bamboo mosaic virus human coronavirus 229e nonstructural protein 13: characterization of duplex-unwinding, nucleoside triphosphatase, and rna 5′-triphosphatase activities multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase virus-encoded rna helicases the n-terminal half of the brome mosaic virus 1a protein has rna capping-associated activities: specificity for gtp and s-adenosylmethionine major domain swiveling revealed by the crystal structures of complexes of e. coli rep helicase bound to single-stranded dna and adp identification and characterization of the escherichia coli-expressed rnadependent rna polymerase of bamboo mosaic virus characterization of the adomet-dependent guanylyltransferase activity that is associated with the n terminus of bamboo mosaic virus replicase the helicase-like domain of plant potexvirus replicase participates in formation of rna 5′ cap structure by exhibiting rna 5′-triphosphatase activity a satellite rna associated with bamboo mosaic potexvirus nucleotide sequence of the genomic rna of bamboo mosaic potexvirus arg-16 and arg-21 in the n-terminal region of the triple-gene-block protein 1 of bamboo mosaic virus are essential for virus movement virus-specific mrna capping enzyme encoded by hepatitis e virus mapping the triphosphatase active site of baculovirus mrna capping enzyme lef4 and evidence for a two-metal mechanism mutagenesis of the dengue virus type 2 ns3 protein within and outside helicase motifs: effects on enzyme activity and virus replication virus-specific capping of tobacco mosaic virus rna: methylation of gtp prior to formation of covalent complex p126-m7gtp crystal structure of a dexx box dna helicase hepatitis c virus ns3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes an rna 5′-triphosphatase related to the protein tyrosine phosphatases rna-stimulated ntpase activity associated with the p80 protein of the pestivirus bovine viral diarrhea virus adenine nucleotide levels, the redox state of the nadp system, and assimilatory force in nonaqueously purified mesophyll chloroplasts from maize leaves under different light intensities identification of a novel function of the alphavirus capping apparatus: rna 5′-triphosphatase activity of nsp2 crystal structures of complexes of pcra dna helicase with a dna substrate indicate an inchworm mechanism brome mosaic virus 1a nucleoside triphosphatase/helicase domain plays crucial roles in recruiting rna replication templates rna-stimulated ntpase activity associated with yellow fever virus ns3 protein expressed in bacteria identification of the rna-binding sites of the 28 kda movement protein of bamboo mosaic potexvirus structure of the hepatitis c virus rna helicase domain this work was supported by grants, nsc 94-2752-b-005-012-pae, from the national science council, taiwan, republic of china. key: cord-257494-242k58ll authors: bastos, paulo; trindade, fábio; da costa, joão; ferreira, rita; vitorino, rui title: human antimicrobial peptides in bodily fluids: current knowledge and therapeutic perspectives in the postantibiotic era date: 2017-01-17 journal: med res rev doi: 10.1002/med.21435 sha: doc_id: 257494 cord_uid: 242k58ll antimicrobial peptides (amps) are an integral part of the innate immune defense mechanism of many organisms. due to the alarming increase of resistance to antimicrobial therapeutics, a growing interest in alternative antimicrobial agents has led to the exploitation of amps, both synthetic and isolated from natural sources. thus, many peptide‐based drugs have been the focus of increasing attention by many researchers not only in identifying novel amps, but in defining mechanisms of antimicrobial peptide activity as well. herein, we review the available strategies for the identification of amps in human body fluids and their mechanism(s) of action. in addition, an overview of the distribution of amps across different human body fluids is provided, as well as its relation with microorganisms and infectious conditions. human antimicrobial peptides (amps) represent approximately 10% of all curated amps catalogued to date. 1 human host defense peptides are an intrinsic part of the innate immune system and exhibit a broad activity spectrum against bacteria, fungi, viruses, and parasites while amps can be antibacterial (abps), antifungal, antiprotist, antiviral, anticancer, antiparasitic, insecticidal, spermicidal, chemotactic, antioxidant, protease inhibitors, or even exhibit wound healing properties (supporting information table s1), their scope of action overlaps considerably and some peptides show activity at several levels (fig. 2 ). [1] [2] [3] [4] [5] for instance, on the one hand, human plasma adrenomedullin-derived peptides are active against multiple bacteria species, human urinary and gingival fluid calcitonin gene-related peptide displays antimicrobial activity against several bacteria and fungi species, and human neutrophil peptides (hnps)/defensins isolated from most biological fluids display activity against several bacterial, fungal, and viral species (fig. 2) . on the other hand, dozens of human amps known to date display antimicrobial activity against the same human colonizers or pathogens, including escherichia coli, pseudomonas aeruginosa, and staphylococcus aureus (figs. 2 and 3) . also, it is intriguing to observe that even though some amps display broad activity spectra, others seem to be anywhere from species-to kingdom-specific (fig. 2) . while likely to be confounded global network depicting the distribution of human antimicrobial peptides across biological fluids and their microbial targets. rectangular nodes correspond to biofluids, circular nodes to target species, and each edge correspond to a given gene encoding one or more antimicrobial peptides. the thicker the edge, the stronger is the association of a biofluid to a pathogen, representing increased number of antimicrobial peptides defending against such pathogen in such biofluid. also, pathogens represented by bigger nodes (e.g., escherichia coli, candida albicans, staphylococcus aureus) represent those that are targeted by more antimicrobial peptides across different biofluids. by observational bias (whereby some species, e.g., e. coli, tend to be tested more frequently than others), this observation also reflects that the activity spectra of amps depend not only on their physicochemical properties (which group them according to unique families) but also on target properties and on the environment/biological fluid in which they are found. as the large majority of amps identified to date are abps (ß83%, figs. 1-3) , most concepts and examples provided in this review will be based on results from studies focusing on abps, closely followed by those derived from studies on antiviral and antifungal peptides. however, the same principles tend to apply across all amps. amps are very potent cationic molecules displaying minimum inhibitory concentrations (the minimum concentration that prevents bacterial growth) as low as 1-4 μg/ml, 6 which highlights their promise as broad-spectrum antimicrobial agents. furthermore, amps act more rapidly than conventional antibiotics and are not affected by the typical resistance mechanisms involving conventional drugs, making them very attractive for therapeutic purposes. in addition, amps are not mere microbicide agents, as their scope of action may encompass other functions, such as cellular development and immune system modulation. for instance, fetal keratinocytes express significantly more amps compared to neonatal and adult keratinocytes, despite the lower degree of exposition to the environment or to pathogenic agents. these a rough estimate of all amps catalogued to date (supporting information table s2 ) indicates 14.1% to be α-helices, followed by β-sheets (3.7%), and peptides presenting with both helices and β-sheets (3.7%). however, approximately 60% of all amps currently identified do not have a known 3d structure, while only 0.3% have a known structure that is neither a helix nor a β-sheet. excluding those that have unknown structures, 99 .2% of all amps are either α-helices and/or β-sheets, suggesting a highly conserved structure for a highly primordial biological function. secondary structures of human amps may include α-helices (e.g., ll-37), 34 β-sheets (e.g., defensin 1), 35 and extended peptides (e.g., indolicidin). 36 considering that β-sheets and α-helices in polypeptide chains typically contain three to ten amino acids per strand and 3.6 residues per turn, respectively, and that amino acid sequences can be virtually unlimited, one can easily envision that a given peptide can display a multitude of secondary structures and, thus, many diverse conformations (see 37 for protein secondary structures predictive approaches). among secondary structures, α-helices (in addition to being the most frequently encountered in nature) are the most regular and predictable, and, consequently, the most extensively studied. amps with linear α-helices are notably positively charged and amphipathic, but only adopt such secondary conformation upon binding to bacterial membranes. 38 therefore, their antimicrobial activity is directly dependent on the secondary structure. 39 in contrast, β-sheet amps possess well-defined conformations due to the presence of disulfide bridges formed between thiol groups in cysteines. in these peptides, such bridges are often required for antimicrobial activity. 40 contrariwise, bovine lactoferricin was shown to be effective against e. coli 0111 even when such bridges were blocked by pyridylethylation. 41 other human amps, such as human β-defensin 3 (hbd-3), present both α-helices and β-sheets. in the specific case of hbd-3, disrupting the three stabilizing disulfide bonds does not compromise its antimicrobial activity, but it does diminish its chemotactic properties. 42 furthermore, the correlation of the conformation with peptides' antimicrobial activity is not always as straightforward as one could imagine. for example, disruption of hbd-1's disulfide bonds results in peptides with free cysteines at the carboxy terminal that show increased antimicrobial activity against the pathogenic candida albicans and the gram-positive commensals bifidobacterium and lactobacillus. 43 moreover, linear extended peptides do not keep a fixed conformation in solution, and are enriched in one or more amino acids, particularly arginine, proline, and tryptophan. for instance, the amp histatin found in human saliva is enriched in histidine residues, 44 while prophenin is very rich in proline and phenylalanine residues. 45 when it comes to loop rich peptides, the presence of many prolines makes an amp much less likely to form amphipathic structures, adopting what is frequently referred to as polyproline helical type-ii (ppii) structures. these structures are specifically bound by sh3 domains, which are usually found in proteins that interact with other proteins and mediate assembly of specific protein complexes (upon proline-rich peptides binding). 46 human antibacterial peptides (abps), which compose the largest group of human amps, exhibit a weighted average net charge of +5.8 per peptide, which is considerably above the average net charge of all amps identified to date. also, despite being intuitive that the more positively charged an amp is, the more potency it will present, 47 human amps do not show greater potency or efficacy when compared to those of other species, suggesting that the environment and the presence of different amino acids (d and rare amino acids) also play a significant role. notwithstanding the large diversity of amps across human biologic fluids (figs. 2 and 3, table i ), it should be noted that amps are much more diverse across all species, than those identified in humans, which present rather conserved properties. in fact, some amps from other species are characterized by more intriguing sequences, structures, and physicochemical growth-regulated alpha protein properties. case in point, bacillus subtilis is known to produce several extremely potent lipopeptides (active at concentrations as low as 0.01-0.06 μm) with antimicrobial activity against several species. one of these, gageotetrin a is a very unique anionic amp that consists of only leucine and glutamic acid residues conjugated with a unique fatty acid, 3-hydroxy-11-methyltridecanoic acid. 48 another example is that of sonorensin from bacillus sonorensis, a broad-spectrum amp from the heterocycloanthracin subfamily, active against both gram-negative and gram-positive bacteria, including listeria monocytogenes and s. aureus. sonorensin displays an amino acid sequence with multiple copies of the same motif, making this peptide rather unique. 49 baceridin is a circular peptide from a plant-associated bacillus that is synthesized by ribosome-independent machinery and bears only six amino acids, 50% in the d configuration. 50 moreover, baceridin is a proteasome inhibitor, compromising cell cycle progression and inducing apoptosis in tumor cells by a p53-independent pathway. 50 copsin from coprinopsis cinerea is bactericidal against enterococcus faecium and l. monocytogenes by interacting with the peptidoglycan precursor lipid ii and interfering with the cell wall biosynthesis. 51 curiously, it is modified with a pyroglutamate, which confers a higher degree of thermal stability and resistance toward protease digestion. 51 even though some display fungicidal and virucidal properties, the chief activity of human amps is against gram-positive and gram-negative bacteria (figs. 1-3). furthermore, while amps represent an extremely diverse group of biological active molecules, most share common properties that contribute for their membranolytic or otherwise antimicrobial activity: positive charge, hydrophobicity, and amphiphilicity. therefore, the mechanism of action of most amps involves membrane disruption (fig. 4) , a process that is also required when their translocation is warranted and the respective targets are localized intracellularly. 39 the interaction of amps with bacterial membranes depends on the establishment of attractive electrostatic forces between these. bacterial species are known to carry negatively charged outer membranes due to phosphate groups of the lipopolysaccharides in gram-negative bacteria and to the lipoteichoic acids in gram-positive bacteria. 52 the fact that all gramnegative and gram-positive bacteria display these type of negatively charged lipids accounts for the lack of specificity of most abps, and promotes the attraction between amps and bacterial membranes while preventing their binding to most host cells membranes. moreover, the inherent hydrophobicity and amphiphilicity allow abps to form clusters at the membrane's surface once attached. 53, 54 at low concentrations (low peptide-to-lipid ratio), abps tend to adsorb onto the surface, adopting an orientation parallel to the membrane bilayer. however, clustering increases the peptide-to-lipid ratio, which subsequently promotes additional clustering. once a certain peptide-to-lipid ratio threshold is reached, abps adopt a perpendicular orientation relative to the bacterial membrane, which allows them to insert themselves into the membrane, 55 as will be further discussed (fig. 4) . peptides can adopt many different secondary structures and conformations, which depend on their amino acids sequence, as well as on the environment. consequently, different peptides and different environments favor the formation of unique structures, such as barrel staves, carpets, and toroidal pores. 40, 56, 57 when the peptides attach and are inserted into the membrane bilayer so that the hydrophobic regions align with the phospholipids' acyl groups and the hydrophilic regions create the central region of a pore, a barrel stave is formed. in addition, these structures act as a primer for the aggregation of new peptides, thus expanding the pore's diameter. 58 due to abps' cationic sites, membrane crossover would be very energetically unfavorable in their original parallel orientation. however, this orientation changes to a (1) cluster at the cell surface and cause membrane disruption by several different mechanisms (e.g., barrel staves, carpets, toroidal pores), (2) translocate into cells and impair intracellular organelle machineries, (3) impair protein-protein interactions, enzymatic cascades, and cytosolic signaling pathways, (4) interact with nucleic (not in the case of bacteria) acids, trap replication forks, and compromise nucleic acids as well as protein synthesis, (5) preclude several steps of viral replication, (6) inhibit genetic material trafficking, reverse transcriptase, and viral proteases, (7) block the interaction between virus and host cells (e.g., viral envelope glycoproteins gp120 and gp41 or coreceptor cxcr4), compromising virus binding and entry, and (8) cause membrane lysis on enveloped viruses. see text for detailed description. designed using servier medical art. perpendicular one upon abps' clustering, and the peptides are reoriented with their hydrophobic portions facing the membrane acyl groups and their cationic sites facing the interior of the pores. thus, clustering and pore formation provides a means for membrane disruption and the leakage of intracellular molecules from bacterial cells. 59 in contrast, if peptides remain parallel to the membrane surface with their hydrophobic residues facing the membrane while their hydrophilic residues face the surrounding milieu, a "carpet"-like structure is achieved. with such organization, membrane's rigidity weakens progressively as new peptides are added, culminating in its dissolution and breakup. mechanistically, this mode of action is less stringent because peptides do not need to adopt a specific structure. however, the necessary concentration of abps appears be much higher than that required for the formation of barrel staves. [60] [61] [62] for toroidal pores to form, peptides need to aggregate onto the membrane's surface and insert themselves in a perpendicular fashion. 63, 64 subsequently, abps are reoriented parallel to the membrane so that polar residues interact with the polar heads of the membrane lipids, thus causing membranes to bend. this induced bending of the bilayer causes the upper and lower leaflets to meet, and allows a mixture of peptides and lipid head groups to line with the interior of the pore, forming toroidal or wormhole structures. 60, 63, 65 abps may also inhibit bacterial growth and have a bactericidal effect by promoting the clustering of anionic lipids, such as phosphatidylglycerols (pgs) and cardiolipin. for instance, for ll-37, initially believed to act according to the "carpet" model, there is evidence that its fragments, namely kr-12, act as a magnet that competes for the negatively charged pgs, promoting their redistribution into "pg-rich domains." such phospholipidic reorganization may disturb cell signaling and compromise the activity of membrane-bound proteins, namely, the voltage-dependent potassium channels, which can seriously jeopardize bacteria survival. 34 likewise, n-acylated peptides derived from lactoferricin were shown to induce defects in e. coli cell division by rearranging the distribution of inner membrane phospholipids, essentially due to the clustering of cardiolipin and other anionic lipids. 66 the bias toward positive charges confers amps the ability to easily interact not only with biological membranes, but also with other negatively charged molecules (fig. 4) such as nucleic acids and those bearing phosphate groups. 39, 67 in addition, because this type of interaction is largely unspecific, amps can exhibit not only a broad-spectrum activity, but also multiple mechanisms of action (fig. 3) . 68, 69 for instance, lactoferricin is known to inhibit atp-dependent multidrug efflux pumps as well as dna, rna, and protein synthesis. 70 ten human amps derived from lactoferricin and lysozyme are thought (by sequence homology) to act by trapping the replication fork and preventing base recombination and dna repair. 71 furthermore, the presence of many prolines in some peptides (also known as ppii structures as aforementioned) hampers the organization of amphipathic structures. 72 these structures favor interactions between peptidic structures and between these and nucleic acids, thus playing a major role in signal transduction and complexes assembly. 73 however, ppii structures do not necessarily contain prolines, 74 and abps rich in arginine residues are also able to translocate across membranes and to interact with nucleic acids and proteins. hence, both proline and/or arginine-rich abps may be capable of inhibiting prosynthetic signaling pathways and enzymatic activity (fig. 4) , shifting the conformation of cellular structures and activating autolysins. however, this is a speculation that warrants further studying. in addition to targeting bacterial membranes, human defensins also bind to the peptidoglycan precursor lipid ii and inhibit cell wall biosynthetic enzymes in staphylococci species. 75 moreover, when stabilizing disulfide bridges between conserved cysteine residues in human amps with β-hairpin or β-sheet conformations are disrupted, the resulting linear peptides still maintain their antimicrobial properties despite losing membranolytic activity. 76, 77 the antifungal histatins act at multiple levels by mechanisms of action conserved across the histatin family of amps, with histatin 5 the most potent amp in this family. this amp binds the yeast transmembrane receptor heat-shock protein ssa1/2p, is internalized, and then targets primarily yeast mitochondria. 78, 79 by doing so, it leads to the formation of reactive oxygen species (ros), causes atp efflux, and inhibits oxidative phosphorylation. 80, 81 simultaneously, histatin 5 also interacts with the potassium transporter trk1p, causing release of k + ions and further atp efflux. 82 when the functional domain of histatin 5 was synthetically multiplied, its antifungal activities were significantly potentiated and it became significantly faster-acting compared to the normal histatin 5. 82 such an observation might prove itself very useful, because it indicates that modulating the number of functional domains per amp may enhance its antimicrobial activity without requiring the administration of increased quantities of amp. histatins may also exert their antimicrobial activities by inhibiting host and microorganismal proteolytic enzymes. histatin 5 is a strong inhibitor of human matrix metalloproteases 2/9 and metalloproteases from porphyromonas gingivalis. 83 thus, its inhibitory activity over proteolytic enzymes may attenuate tissue damage and microbial propagation during the onset of periodontal inflammatory disease. also, by inhibiting the cysteine protease clostripain from clostridium histolyticum, histatin 5 mitigates the virulence factors produced by this clostridium species and may therefore alleviate the complications associated with gas gangrene syndrome. 84 so far, the above-mentioned mechanisms of action have been discussed in the context of abps or antifungal peptides, and sometimes the same peptide is active against both bacteria and fungi. in turn, the action of antiviral peptides can diverge from these mechanisms and should thus be independently commented (fig. 4) , although some overlap does exist. extracellularly, most native antiviral peptides often present as α-helical structures, have primarily lytic activity on enveloped viruses, and can directly inactivate cell-free virions (fig. 4) . human lysozyme fragments are this group's prototype, capable of aggregating on the viral surface and causing disruption of membrane envelopes. 85, 86 similarly, cap37 peptide appears to exert its antiviral activity by destructing the virus envelope of type i herpes simplex virus (hsv) or even by disrupting the capsids of adenoviruses. 87 however, because membrane lipids of enveloped viruses derive from host cell membranes, special attention is required when considering the exploitation of this feature, as antiviral peptides may disrupt host cell membranes indiscriminately. alternatively, antiviral peptides may block the interaction between the virus and the host cell (fig. 4) . that is believed to be, at least in part, the mechanism of action of human defensins, θ -defensin, human α1-antitrypsin virip, cyclic lactoferricin, and serine protease inhibitor elafin. for instance, it has been demonstrated that all α-defensins [hnp-1, hnp-2, hnp-3, and hnp-4 and human α-defensin (hd)-5 and hd-6] and hbd-3 prevented hsv infection by blocking virus binding and entry. such antiviral activity was attributed to hnp-1-3, hd-5, and hbd-3's high affinity to hsv's glycoprotein b and hnp-4, hd-6, and hbd-3's avidity toward endogenous heparan sulfate. 88 similarly, protection against hiv is conferred by peptides with lectin properties that can bind viral envelope glycoproteins gp120 and gp41, crucial for viral attachment, fusion of envelopes with host membranes, and subsequent entry into host cells. 89 hnp-4 that binds and blocks the action of gp120, and θ -defensins (e.g., the pseudogene retrocyclin) together with the human α1-antitrypsin-derived 20-residue viral-inhibitory peptide both targeting gp41 represent examples of such peptides. [90] [91] [92] likewise, hsv-2 infection can also be blocked by retrocyclin, which binds tightly to glycoprotein b2. 93 the cyclic peptide lactoferricin seems to compete for the ligation to extracellular heparan sulfate or chondroitin sulfate, thus preventing the very first event in cytomegalovirus, hsv, and papillomavirus infection. [94] [95] [96] finally, elafin is thought to act indirectly in host cells by hampering viral attachment and probably by modulating host's antiviral and inflammatory responses toward hsv-2 infection. 97 synergistically with this mechanism, antiviral peptides can also bind and inhibit the activation of viral co-receptors, leading to further inhibition of glycoproteins-mediated viral attachment. not only that, hbd-2 and -3 inhibit viral infection by promoting the internalization of its co-receptor cxcr4. 98, 99 virucidal peptides may also preclude several steps of viral replication by interacting with intracellular targets (fig. 4) . this is the case of hnp-1 and hd-5, which may prevent hsv replication by binding directly to its dna. 88 also, hnp-1 arrests influenza virus replication by inhibiting protein kinase c phosphorylation, an essential step for nuclear trafficking events. 100 human cathelicidin (ll-37) derived peptides, in turn, inhibit reverse transcriptase and viral proteases, thus preventing integration of the viral genetic load into human dna. 101, 102 the few resistance mechanisms against amps demonstrated so far among microorganisms have been observed almost exclusively in bacteria. with very few exceptions, bacteria do not seem to develop complete resistance against cationic abps, though some strategies do exist among bacterial species for diminishing their potency and efficacy. the difficulty in acquiring resistance may stem mainly from the fact that abps target an essential cellular component for bacteria survival, which cannot be easily modulated by the cell without causing significant adverse effects. therefore, resistance mechanisms are not likely to be a limitation in the therapeutic utilization of abps. in light of this scarce resistance mechanisms de novo acquisition, what mechanisms do bacteria naturally develop/possess against abps? first, one should consider that the primary target of abps consists of bacterial cell membranes and bear in mind how abps depend on the electrostatic interactions between membrane negative charges and positive peptidic residues. accordingly, bacteria have learned how to modulate cell membrane charges and composition. gram-positive bacteria can partially modify the peptidoglycan's teichoic acid and gram-negative bacteria can partly change its lipopolysaccharide lipid a moiety, thus compromising this interaction. for instance, d-alanine-activating enzyme and d-alanine transfer protein are known to transport positively charged d-alanines to the surface anionic teichoic acids in gram-positive s. aureus, reducing the negative net charge of its outer membrane. 103 also in s. aureus, the five-component system graxsr-vrafg increases the expression of mprf and dltabcd genes upon exposure to cationic amps. when s. aureus strains are mutated so that the regulation of these genes is disturbed, these strains become more susceptible to daptomycin, polymyxin b, hnp, and platelet factor 4-derived peptide, and less infectious in vivo in a nonclinical endocarditis model. [104] [105] [106] the gene product mprf attaches lysine residues, which bear a positively charged ε-amino group, to the anionic phophatidylglycerols, making these less negatively charged and thus less susceptible to interactions with cationic amps. 105, 106 the gram-negative vibrio cholerae can increase its resistance to polymyxins more than 100-fold by modifying its lipopolysaccharides in a process mediated by almf. 107 the lpta gene product found in gonococcal species, such as the gram-negative bacteria neisseria gonorrhoeae, protects lipopolysaccharide lipid a moieties by attaching phosphoethanolamine, making these bacteria less susceptible to amps and increasing their capacity to modulate host's immune response by evading complement-mediated cellular death. 108, 109 moreover, in n. gonorrhoeae and neisseria meningitides, the presence of phosphoryl and phosphoethanolamine in lipid a moieties enhances the activation of the nf-κb pathway and the production of proinflammatory cytokines in human cells, which can amplify their virulence. 110 despite this modification-dependent virulence, the absence of such modifications in commensal neisseriae species allow these to colonize and coexist with the human host without inducing bactericidal host immune responses. 111 in addition, the adaptation of the composition and, hence, the electrostatic properties of the membrane in order to circumvent the host immune system is not an exclusive phenomenon of pathogenic bacteria. commensal bacteria of the intestinal tract can remove phosphate groups from their membrane's lipopolysaccharides, decreasing their negative charge and allowing greater survival and proliferation in the gut. 112 similarly to the above-mentioned paradoxical modification-dependent virulence, mutated commensal gut microbiota bacteroides that fail to remove phosphate groups from their lipopolysaccharides are killed during inflammation while normal strains keep their resilience. 112 therefore, there appears to be an inverse relationship between acquired resistance and bacterial virulence, so that induced/acquired modifications render cells less virulent while simultaneously allowing these to more easily colonize and coexist within the human host environment. second, bacteria are also able to adapt the fluidity of the membrane by increasing the hydrophobic interactions within their outer membrane, thus hindering pore formation. 113 third, bacteria can express proteases against abps or transporters and efflux pumps like atp-binding cassette (abc) transporters to circumvent such innate defense barriers. 114, 115 glu-c protease produced by s. aureus induces the loss of antibacterial activity of neuroendocrine peptides, and chromofungin, procatestatin, and human catestatin are enzymatically degraded when treated with bacterial supernatants. 116 in e. faecalis, bacitracin binds the abc transporter ef2050-2049, which interacts with the regulatory domain of the two-component system ef0926-27, increasing its expression and conferring resistance against bacitracin. 117 two-component systems regulate the expression of abc transporters and are constituted by a transport permease and a sensor kinase. however, at least in b. subtilis, the abc transporter itself is required for both sensing of and resistance to bacitracin, suggesting these transporters to also function as environmental sensors. 115 lastly, bacteria may also synthesize molecules capable of directly neutralizing abps, 118 which can provide a way for bacteria to bypass amps. however, such resistance mechanisms are very limited and resistance to amps does not take place to a large extent. r bastos et al. in humans, endogenous amps have been found in several fluids throughout the body, as depicted in figures 2 and 3 as well as in table i . as noted, multiple amps have been found in tears, saliva, milk, blood, urine, sweat (and others not-so-well-explored biofluids), reflecting not only a remarkable versatility but also a huge conservation that reinforces its essential role. the robust knowledge of amps on some fluids (e.g., tears, urine, saliva) over others (e.g., amniotic fluid, cerebrospinal fluid) most likely reflects their availability, but different exposure to pathogens from the environment (figs. 2 and 3) should also account for these discrepancies to a large extent. additionally, saliva and urine screening is of special interest, since both the oral cavity and the urinary tract represent the main doorways for microbial invasion and colonization, and are thus enriched with host defense peptides. likewise, milk screening is of high relevance due to the presence of particular proteins and amps that will boost neonate's immunity. despite underrepresented, amps from other fluids are equally relevant (figs. 2 and 3). for instance, amps collected from airway secretions, bronchoalveloar lavages, and nasopharyngeal aspirates can be valuable sources of information regarding the physiological response to pathogens of the respiratory tract and may, themselves, constitute raw models for new therapeutic drugs. also, other than circulating immune cells derived, blood has not been regarded as a rich source of amps, but this view can be questioned (figs. 2 and 3), most likely due to tissue-derived amps' passage to the blood circulation. however, it should be noted that human fluid reservoirs are not independent/isolated, and, consequently, peptides may be present throughout and cross between, possibly undergoing modifications between such reservoirs. amps may be present in fluids as a result of many different processes, including exocytosis, in situ proteolysis, tissue necrosis, and cellular lysis. therefore, in this section, we will characterize naturally occurring amps based on structural and functional similarities rather than their source of collection. defensins are cationic peptides rich in cysteine residues, presenting as β-sheet structures stabilized by disulfide bridges. 4 according to the alignment of these stabilizing disulfide bounds, mammalian defensins are classified into three subclasses, α-defensins, β-defensins, and θdefensins. disulfide bounds of α-defensins occur between cysteines 2-6, 1-4, and 3-5. these peptides consist of 29-35 amino acids and adopt a triple-stranded β-sheet conformation. human neutrophils express four distinct α-defensins, known as hnp-1 to hnp-4. despite having been initially isolated from peripheral blood leukocytes, hnp-1 to hnp-3 can also be found in bone marrow, spleen, and thymus. 119 the remaining two already identified human α-defensins, hd-5 and hd-6, are found only in paneth cells of the small intestine and in epithelial cells, thus being tissue specific. 120 expressed in the aforementioned cell types, amps can also be found in bodily fluids in direct contact with these cells. among the most recent discoveries, hd-6 has been found to be active against bifidobacterium adolescentis and other anaerobic gut commensals, but not (directly) bacteriostatic or bactericidal against most pathogenic bacteria, and may therefore play an important role in maintaining a balance among the microbiota of the gut. 121 similarly, human hd-5 is particularly potent against the gram-positive bacillus clostridium difficile, one of the most common pathogens responsible for nosocomial infections, whose highly virulent strains often attack small intestine mucosa. 122 in addition, α-defensins may show both cellular and regional specificity throughout the gastrointestinal tract, either secreted or active inside intracellular granules and constitutively expressed or amenable to induction. 15, 123, 124 it should also be emphasized that the activity spectrum of α-defensins is not limited to bacteria and fungi, exhibiting activity against several virus, including hiv, hsv, adenovirus, and human papillomavirus (hpv), and displaying several unique mechanisms of action (please refer to section 4). 4 it should be noted that the antimicrobial activity of α-defensins is influenced even by the difference in one amino acid, such as for hnp-1 to hnp-3. in fact, an additional acidic residue in hnp-3 lowers its effectiveness (most accurately, its potency) against s. aureus, p. aeruginosa, and e. coli. this is explained by the easily donated proton, which makes this defensin less positively charged (at least when considering interaction domains only), thus reinforcing the importance of a positive net charge for the antimicrobial activity of amps, 125, 126 as evidenced in section 4. in terms of disease, α-defensins are known to have a paramount role, particularly when the demands for defense mechanisms are increased. accordingly, a remarkable 15-to 25fold increase in the concentration of hnp-1 and hnp-2 (and hnp-3 to a smaller extent) in human tears as been reported after ocular surgery, an increase that allowed these peptides to reach the required concentrations for antimicrobial activity but that is followed by a decrease once healing has been completed and the demands for immune defenses have returned to baseline. 127 nonetheless, the goal of achieving this increased α-defensin concentration in tears is not restricted to the enhancement of the immune defenses, as it is also known to promote local cellular proliferation and tissue repair. 128 in parallel, salivary α-defensins are active against streptococcus mutans, but their secretion has been found deregulated in caries-positive human subjects. 129 in contrast, prolonged physical exercise has been shown to significantly increase the levels of salivary α-defensins, 130 showing that lifestyles may have a considerable impact on our amp-based defenses against microorganisms and it is possible to modulated the activity thereof. disulfide bounds of β-defensins occur between cysteines 1-5, 2-4, and 3-6. these defensins are longer than α-defensins, spanning from 36 to 50 amino acids, but also share a triple-stranded βsheet conformation. the first hbd was isolated from plasma samples and kidneys constitute the major source of hbd-1. 131 since then, peptides derived from human hbd-1 have been identified in tears, urine, vaginal lavage, sweat, and blood plasma, and these are effective primarily against e. coli. [131] [132] [133] [134] in turn, hbd-2 was isolated from psoriatic skin samples, 135 displaying bactericidal activity toward gram-negative e. coli, p. aeruginosa, and c. albicans, and bacteriostatic activity toward gram-positive s. aureus. despite very effective in vitro, both hbd-1 and hbd-2 present attenuated activities in vivo as a result of their sensitivity to physiological salinity. in contrast, hbd-3 is active against s. aureus and vancomycin-resistant e. faecium even at physiological salt concentrations. 136 the hbd-119 defensin has been found in seminal plasma, and it has been reported as potential efficacious against e. coli, s. aureus, and c. albicans. 137, 138 furthermore, hbd-114 has been identified in epididymal fluid and has been shown to regulate lipopolysaccharide-mediated inflammation toward e. coli, s. aureus, and c. albicans, protecting sperm from motility loss. 139 in disease conditions, increased concentrations of hbds in plasma and bronchoalveolar lavage fluid were reported in patients with diffuse panbronchiolitis. in particular, β-defensin 4a was suggested to play a role against p. aeruginosa. 140 also, while β-defensins are expressed throughout the gastrointestinal tract, these seem to have evolved together with changes in human gut bacteria, displaying characteristic expression patterns in response to certain microorganisms. their expression may be significantly altered in human diseases, most notably in inflammatory bowel diseases (for insightful reviews on this topic, please refer to 16, 141 and enhanced in inflammatory conditions of the ocular surface 134 ). with α-defensins, the role of the ph for amps' activity is considered. here, β-defensins serve to illustrate the role of salt concentration. in fact, tears contain a remarkable concentration of sodium chloride, which inhibits the activity of human amps (by ß40% in the case of hbd-2, an inhibition that increases up to 90% in the presence whole tear fluid, suggesting the presence of other inhibiting factors). 142 therefore, whenever there is an increase in the demand of amps activity, (e.g., upon infection or tissue damage), their concentrations must surge to reach the required protective levels. however, an excessive increase in the concentration of any amp may lead to tissue damage and local functions may be compromised. for this reason, a complex interplay of amps is required at surfaces like the ocular epithelia, which is achieved by a qualitative enrichment, in addition to the expected quantitative increase (for a depiction of the multitude of amps found in human tears, please refer to 143 ). similarly to α-defensins, the activity spectrum of β-defensins is not limited to bacteria and fungi, and they are active against several virus, namely, hiv, influenza a virus (iav), respiratory syncytial virus, and vaccinia virus, and display several unique mechanisms of action, depending on their target (see above section on mechanisms of action). 4 lastly, genomic studies have predicted additional β-defensins to be expressed in human cells, but their presence in human bodily fluids and their antimicrobial activity in vivo is yet to be demonstrated. 144 while αand β-defensins adopt triple-stranded β-sheet conformations, θ -defensins have shorter sequences and adopt a complete circular conformation, with stabilizing disulfide bounds between cysteines 1-6, 2-5, and 3-4. the peptide rhesus θ -defensin 1 is the prototype of this more recently discovered family of amps. like αand β-defensins, θ -defensins possess broadspectrum microbial activity against bacteria and fungi, while also being capable of protecting mononuclear cells from infection by hiv-1. 145, 146 moreover, these peptides display antiviral activity against hsv, hiv, iav, and severe acute respiratory syndrome (sars) coronavirus, acting by several mechanisms, depending on their target. 4 structurally, θ -defensin peptides are very interesting and very odd, representing the first cyclic peptide family discovered in animals, albeit not in humans. moreover, their circular structure appears to be required for antimicrobial activity. 145 these peptides were first isolated as antimicrobial octadecapeptides expressed in leukocytes of rhesus monkeys, and their formation results from the head-to-tail joining of two independent nine-amino acid peptides derived from truncated pro-α-defensins. 146 in contrast to the previously described classes of defensins, human θ -defensins are thought to be completely inactivated, despite the existence of mrnas encoding at least two θ -defensins expressed in the bone marrow. 145 while genes encoding θ -defensins in humans also encode premature stop codons (thus hampering θ -defensin expression), synthetic human θ -defensins (called retrocyclins) have been shown as promising antiviral agents. 147 in fact, retrocyclin-1 inhibits the entry of hiv, hsv, and iav into host cells. in other mammals, retrocyclin-1 also protects from infection by bacillus anthracis spores and the rhesus θ -defensin 1 protects mice from sars coronavirus infection. 147 cathelicidins are a very diverse group of cationic α-helical and amphipathic amps that exhibit a broad-spectrum activity against bacteria, fungi, and virus. 54 the term "cathelicidin" originally referred only to the entire precursor protein, though currently is commonly used for the whole family, including the resulting peptides with antimicrobial activity. in contrast to the mammalian trend in which cathelicidins and defensins are the main amps, there is only one human gene encoding multiple cathelicidin-related peptides, 144, 148 located on chromosome 3 and expressed in the airways, mouth, tongue, esophagus, epididymis, and small intestine. 149, 150 despite this tissue expression pattern, ll-37 is constitutively formed in spleen, liver, stomach, intestine, and bone marrow. ll-37 is highly positively charged (+6 charge at physiological ph 7.4), due to the high content of arginine and lysine amino acids, and adopts an α-helical structure in solutions with ionic composition similar to that of human plasma. 151 while defensins share common structural features, cathelicidin-related peptides are highly heterogeneous despite deriving from the same precursor. cathelicidins are characterized by a highly conserved n-terminal signal peptide (the so-called "cathelin domain") and a highly variable c-terminal antimicrobial domain that can be released after cleavage by proteinases. 148, 152 cathelicidin is cleaved into the antimicrobial peptide ll-37 by both kallikrein 5 and kallikrein 7 serine proteases, it is upregulated by vitamin d, and has been shown to significantly reduce the risk of death from infection dialysis patients. 153 ll-37 was shown to disrupt bacterial membranes through the formation of toroidal pores and carpet structures, 63 and to exhibit chemotactic properties, attracting leukocytes and activating secretion of chemokines. in addition, ll-37 is internalized by cells, acidified in endosomes, and activates the signaling pathway downstream to toll-like receptor 3 by interacting with double-stranded rna. 154 profiling of airway fluids collected from premature infants' tracheal aspirates during infection evidenced the production of ll-37 even at an early stage of development. 155 in these samples, ll-37 was found in significantly increased concentrations during pulmonary or systemic infections, suggesting this peptide to be as important to avoid infectious processes as to fight already established infections. 155 the concentration of ll-37 has been measured in seminal plasma samples from healthy donors and found to be up to 70-fold higher than in blood plasma. 149 furthermore, it was found to be attached to spermatozoa, eliciting a possible role in human fertilization or its precursor to be extensively cleaved by the high concentration of serine proteases present in seminal plasma. 149 ll-38, an alternative form of human cathelicidin peptides, contains one more alanine at the n-terminus than ll-37 and was initially found to be produced in female vaginal secretions due to the action of gastricsin on sperm precursor cathelicidin. 156 both ll-37 and ll-38 are active against bacteria such as e. coli, s. aureus, p. aeruginosa, and bacillus megaterium, and synergistically with peptides from seminal plasma play an essential defense role protecting the human reproductive system. 156, 157 interestingly, both citrullination and adp (adenosine diphosphate)-ribosylation of arginines can compromise the ability of ll-37 to prevent endotoxin-induced sepsis, perhaps because the attachment of these negatively charged molecules reduces the peptide's cationicity or by raising steric hindrance. 158, 159 histatins are a normal component of human saliva and part of the innate immune system, protecting against a broad array of infectious agents, especially against candida species, saccharomyces cerevisiae, cryptococcus neoformans, and neurospora crassa. [160] [161] [162] there are only two genes enconding histatins (both located on chromosome 4), and they are exclusively expressed in human salivary glands. 163 however, other active amps resulting from the cleavage of histatins 1 and 3 also exist. 164 histatins 1 and 3 are encoded by genes htn1 and htn3, respectively. 165 histatins 2, 4, and 5-12 are the products of posttranslational proteolytic cleavage of histatins 1 and 3, though histatin 5 can also result from posttranscriptional modification of histatin 3 mrna. 165, 166 peptides derived from histatin 1 bear distinct domains associated with specific functions, namely an n-terminal domain with antimicrobial activity and a c-terminal domain with wound-healing properties. 167, 168 histatin 5 has two important metal-binding motifs, consisting of an amino-terminal copper (ii)/nickel (ii)-binding motif and a zinc (ii)-binding motif, although they can also bind to cobalt (ii) ions. 169, 170 the ability to coordinate metal ions appears to be important for the stabilization of the secondary structure, particularly in the presence of negatively charged membranes. 170 nevertheless, histatins lack any defined secondary structure and are unordered when in aqueous solutions, assuming α-helical structures only in organic solutions. 171, 172 in addition, histatin 5 binding to copper (ii) and nickel (ii) ions induces the formation of ros, which contributes for its fungicidal activity. 80, 169 histatins can bind to a receptor on fungal cell membranes and induce cell death by disrupting the cell cycle and causing nonlytic loss of atp, as discussed above. 173 while histatins are effective, potent, and fast-acting amps against several antimicrobial species, these peptides are nontoxic to human cells and human microflora, which may be due to the fact that their translocation across the membrane is site and species specific. 78, 174 both parotid, submandibular, and sublingual glands secret histatins, and their concentration and secretion have been shown to decrease with age, even when accounted for total protein concentration. 175 moreover, oral candidal infections increase with age, suggesting a link between age-associated deficiency of human histatins and an increased risk of oral infections. 175 dermcidins are broad-spectrum anionic amps with no apparent homology to other known amps. peptides in this group comprise 110 amino acid residues, which are subsequently processed into smaller but still active ones. dermcidins are constitutively expressed in human eccrine sweat glands and secreted in sweat, exhibiting antimicrobial activity against common human pathogenic microorganisms such as s. aureus, staphylococcus epidermidis, e. coli, e. faecalis, and c. albicans. [176] [177] [178] this constitutive production contrasts with human defensins and cathelicidins, which are induced during inflammatory and stressful conditions. a small percentage of human breast cancer cells displays rna encoding dermcidin, and after oxidative stress induction different types of tumor cells leads to the production of diverse and biologically active proteolytically processed dermcidin peptides. 179, 180 therefore, the upregulation of dermicidins may confer human breast cancer cells a selective advantage compared to nonmalignant cells. in contrast, reduced expression of dermcidins in sweat from atopic dermatitis patients contributes to skin infections and altered skin colonization. 181 hence, the downregulation of dermicidins in atopic dermatitis patients may represents a suppression of an innate and fundamental defense mechanism, which, in turn, could confer an advantage to infectious microorganisms. unlike histatins, which are nontoxic to human cells and human microflora (see above), dermicidins can have serious deleterious consequences to the human host. dermcidin isoform 2 is known as a stress-induced oxidative protein capable of inhibiting the synthesis of nitric oxide in endothelial cells, insulin in pancreatic islet β-cells, and hepatic hormone synthesis. 182 also, hypertensive patients show significantly higher levels of plasma dermcidin, but salicylic acid has been shown to reduce dermcidin levels while simultaneously attenuating dermcidin-mediated insulin inhibition in patients with acute myocardial infarction. 183 therefore, the exploitation of dermcidin isoform 2 for antimicrobial therapeutic purposes is most likely not possible due to severe side effects. despite this, it may be a promising therapeutic target for hypertension and diabetes management. in animal models, dermcidin has been shown to induce platelet aggregation and coronary artery disease by activating platelet cyclooxygenase and inhibiting the constitutive form of nitric oxide synthase, respectively, with an efficiency 40 times higher than that of adp at activating cyclooxygenase. 182 notably, dermcidin was able to stimulate the development of coronary artery disease in one animal model, even at suboptimal concentrations of adp within 30 min. 183 together, these observations suggest that the ability of amps to fight infections depends on their induction by infectious microorganisms, the particular peptides generated, and the local environment/tissue in which they act. also, biological roles performed by human amps are more diverse than immediately expected, playing essential functions other than fighting infectious microorganism(s). in fact, dermcidin-mediated inhibition of insulin is unique in the sense that no other protein is known to arrest pancreatic insulin synthesis in such way, and similar observations might result in novel and unexpected applications of human amps. amps expressed by human liver (leap 1 and 2) were initially discovered in human blood, and subsequently found in urine. these peptides are known as hepcidins and are characterized by a high content in cysteine residues (approximately 32% of all residues, with eight disulfide bounds). 184 leap-1 or hepcidin-25 is essential for maintaining iron homeostasis, and, when mutated, leads to severe iron overload and juvenile hemochromatosis. 185 hepcidins are the main regulators of plasma iron concentration and its secretion is promoted during inflammatory responses, particularly by the action of interleukin 6, which is one of the main mediators of the acute phase response. 186 as iron bioavailability is a limiting factor for bacterial growth, hepcidins represent an organic mechanism for sequestering iron from invasive pathogens. however, when several iron sources are available, as it happens within the human host, eliminating a single reservoir may not be sufficient to attenuate microorganisms' virulence. moreover, bacteria are known to exploit almost every host iron-binding protein and reservoir and, consequently, human laeps/hepcidins are a rather limited source of antimicrobial activity. 187 for these reasons, the antimicrobial role of iron sequestration depends not only on hepcidins, but also on several other mediators, such as transferrin, lactoferrin, ceruloplasmin, haptoglobin, and hemopexin. mutations responsible for decreasing the levels of hepcidins or compromising the function of other regulators of iron homeostasis, such as the major histocompatibility complex class i like hereditary hemochromatosis protein (hfe), transferrin receptor 2, and ferroportin, lead to increased iron blood levels. when levels are high enough to produce hemochromatosis, individuals are at an increased risk of infectious complications, including bacteremia and meningitis caused by e. coli, 188 bacteremic cellulitis, and hemorrhagic bullous skin lesions caused by v. cholera, 189 multiple pyogenic abscesses in the liver parenchyma as a result of yersinia enterocolitica infection, 190 meningitis, endocarditis, and pericarditis resulting from infection with l. monocytogenes, 191 and fatal septicemia during infection with vibrio vulnificus. 192 in contrast, hemochromatosis can also be associated with decreased macrophage iron storages in the case of hfe-associated hemochromatosis. this depletion makes these cells more efficient in fighting salmonella enterica subsp. enterica, serovar typhimurium, and mycobacterium tuberculosis infections. 193, 194 however, these protective effects of macrophages iron storage depletion have only been demonstrated in animal models and appear to be mediated not by hepcidins, but rather by lipocalin-2, which reduces the availability of iron for salmonella, and transferrin and lactoferrin, both of which compromise iron acquisition by m. tuberculosis. 193, 194 neuropeptides and neuroendocrine peptides may exert mitogenic actions through which innate barriers are reinforced and may display neurotransmitter, immunoregulatory, and direct antimicrobial activity. vasostatin-1 was the first natural antifungal n-terminal chromogranin a derived fragment peptide, having been first isolated from chromaffin secretory granules from bovine adrenal medulla. 195 however, its sequence is highly conserved in humans (97% identity homology) and its microbial activity also overlaps, to some extent, with that of bovine origin. 196 irrespectively, human vasostatin-1 displays antimicrobial activities against gram-positive bacteria (micrococcus luteus, b. megaterium) and a large variety of filamentous fungi (n. crassa, aspergillus fumigatus, alternaria brassicola, nectria haematococca, fusarium culmorum, fusarium oxysporum, trichophyton mentagrophytes) and yeast cells (s. cerevisiae, c. albicans). 195 moreover, many autocrine and paracrine biological activities of chromogranin a, its precursor protein, have been attributed to peptides located along its sequence, which are co-released with catecholamines by chromaffin cells upon stimulation and can be found in human bodily fluids. 197, 198 therefore, the antimicrobial activity spectrum of vasostatin-1 may be extended by that of co-released peptides also formed by chromogranin a proteolytic cleavage, and some of the coproduced peptides are even more potent than vasostatin-1 itself. 195 in addition to the structural requirements for the antimicrobial activity of chromogranin a derived peptides, posttranslational modifications may also modulate their activity. for instance, s-pyridylethylation (an alkylating modification consisting of the addition of pyridylethyl moieties to the -sh functional groups of cysteine residues that disrupts disulfide bridges) renders it selectively active against b. megaterium but not m. luteus, while oxidation seems to render it inactive against bacteria but still active against fungi. in addition, chromogranin a derived peptides also undergo phosphorylation and glycosylation, which can modulate their physicochemical properties. 195 currently, vasostatin-1 is known to be stored in endocrine, neuroendocrine, and neuronal cells, and to be released from stimulated chromaffin and immune cells upon stress. moreover, the stimulation of polymorphonuclear immune cells induces the processing of chromogranin a and the secretion of vasostatin-1 and other chromogranin a derived peptides, 195 which may account for local inflammation. lastly, recombinant and synthetic human vasostatinderived fragments have been shown to inhibit vascular contraction induced by endothelin-1, parathyroid hormonal secretion, neuronal survival, expression of neurofilaments, and neuronal gaba uptake. 199, 200 adrenomedullin was first isolated from a human adrenal pheochromocytoma and its plasma concentration is raised under specific physiological conditions, namely, renal failure, 201 hypertension, 202 and sepsis. 203 this peptide has antibacterial activity against several grampositive and gram-negative bacteria, including propionibacterium acnes, s. aureus, m. luteus, p. gingivalis, actinomyces naeslundii, s. mutans, c. albicans, eikenella corrodens, actinobacillus actinomycetemcomitans, streptococcus pneumoniae, haemophilus influenzae, streptococcus pyogenes, bacteroides fragilis, e. coli, and helicobacter pylori. 204 human neuropeptide y displays antimicrobial activity against bacteria and fungi, but truncated fragments are tenfold more potent than the intact precursor neuropeptide y, perhaps because the net charge of the fragments is more positive than that of the intact neuropeptide. 205 encephalins and their derived peptides may either enhance or inhibit the immune response and the corresponding circulating immune cells/mediators in both humans and animal models. 206, 207 as well as their subsequent antimicrobial effects, may thus take place indirectly in immune system modulation. simultaneously, antibacterial assays have revealed that peptide b/enkelytin (a c-terminal fragment of proenkephalin a) specifically targets gram-positive bacteria, including m. luteus, b. megaterium, m. luteus, and s. aureus, while having no effect over gram-negative bacteria. 208, 209 classically, proenkephalin a has been ascribed to the secretory granules from adrenal medullary chromaffin cells and various brain regions, such as the striatum and the pituitary, according to some animal studies. 210, 211 despite this, it has also been demonstrated to be expressed in and secreted by polymorphonuclear neutrophils (accounting for its particular enrichment in wound fluids), 208 t and b lymphocytes, macrophages, and mast cells. 212, 213 however, it should be noted that the antimicrobial effects of encephalins and their derived peptides result mostly from animal studies and have not been adequately studied in human secretions, despite the high conservation of their sequences across species, which most likely contribute for the similar activity spectrum. even so, phosphorylation and glycosylation are characteristic of these peptides, 208, 214 and it is possible to envision that differences at the level of posttranslational modifications or as a result of gene divergence may result in distinct activity spectra. interestingly, peptide b/enkelytin is metabolized in vivo to opioid peptides, thus revealing an intricate relationship between innate immunity and pain modulation, and its plasma concentration is increased after coronary artery bypass grafting in humans. 215 because it is expressed in both immune and neuronal cells, it is possible that the same stimuli may act on both cells populations, thereby accounting for immune system modulation at the periphery and alterations in central nervous system signaling. however, the exact roles and mechanisms of this interplay are not clear. in addition, other amps found in epithelial secretions from the gut and the skin, for example, include peptides derived from alpha-melanocyte-stimulating hormone, which is active against s. aureus and c. albicans. 216 accordingly, evidences suggest that amps like these act by inhibiting mrna and protein synthesis rather than inducing direct membrane disruption, as is the case for most amps. 217 several cationic proteins display nonenzymatic antimicrobial activity, which is sustained by the resulting cleavage peptides after protein fragmentation. for example, lactoferrin is a glycoprotein enriched in milk and neutrophilic granules, which has the ability to sequester iron and thus prevent bacterial overgrowth. 218 when digested by pepsin, lactoferrin yields lactoferricin h (human), an amp able to neutralize bacterial toxins, regulate gene transcription, inhibit complement activation, viral infection, and tumor overgrowth, thus further extending the antimicrobial properties of its precursor. 76 human chemokines are secreted by macrophages and polymorphonuclear immune cells and, therefore, found in the blood circulation. 219 moreover, during allergic inflammatory responses to bacterial infections of the airways, the eosinophil-recruiting chemokines eotaxin-1/ccl11, eotaxin-2/ccl24, and eotaxin-3/ccl26 are generated by mast cell proteases, active against several airway pathogens, including s. pneumoniae, s. aureus, h. aeruginosa. 220 peptides resulting from the cleavage of pepsinogen a and c prosequences have also been shown to exert antimicrobial activity. although these were initially identified from the stomach of bullfrogs, synthesized human pepsinogen c prosequences with similar structural characteristics (amphipathic and helical structures) to these cleavage peptides have also exhibited antimicrobial activity. 221 calcitermin is an amp that results from the cleavage of a 15 amino acids long sequence from the c-terminal region of protein calgranulin c, isolated from human airway secretions. 222 by adopting an alpha-helical conformation in bacterial membranes, calcitermin is able to target gram-negative bacteria (e.g., e. coli and p. aeruginosa) and fungi (e.g., c. albicans). 222 furthermore, the precursor protein and fragment peptide may act synergistically to protect the human host against foreign microorganisms present in the airways. on the one hand, the cleavage peptide calcitermin inserts into membranes of microorganisms. on the other hand, the precursor protein calgranulin c has a proinflammatory activity. by acting as a dangerassociated molecular pattern molecule and binding to the receptor for advanced glycation end products in innate immune cells, it activates the map-kinase and nf-κb signaling pathways, leading to the production of proinflammatory cytokines and histamine, upregulation of cell adhesion molecules, recruitment of leukocytes, and degranulation of granulocytes. 223, 224 dermcidin-derived peptides such as dcds, ssls, and leks (the letters correspond to the first three amino acids of the corresponding precursor dermcidin protein) are also formed in eccrine sweat glands (but not in apocrine sweat glands) at these body sites with high probability for contact with pathogens (e.g., palms, face, arms) and exhibit antimicrobial activity against a large number of microorganisms, including s. aureus, e. faecalis, e. coli, s. epidermidis, pseudomonas putida, methicillin-resistant s. aureus, and rifampicin/isoniazid-resistant m. tuberculosis. [176] [177] [178] such dermcidin-derived amps are regulated by proteolytic processing at several levels (see section 2) and are differentially expressed during inflammatory conditions. 225 these peptides are resistant to proteolytic degradation in sweat up to at least 40 hr and, interestingly, most dermcidin-derived peptides are anionic in nature. if such distinct properties may lead to different antimicrobial activity spectrum is unclear, though not surprising, as skin represents a harsh and particularly unique environment. as of specific interest, it should be noted that the consensual average skin surface ph has been decreasing in the last few years and is now generally accepted to be below 5. while showering and cosmetic products decrease skin's surface ph, plain tap water can increase the skin ph up to 6 hr. 226 noticeably, negative charges seem to be important for dermcidin-derived peptides' antimicrobial activity, in contrast with what would be expected for amps. in fact, most dermcidin-derived peptides are naturally anionic, but low ph (high h + concentration) leads to an increase in their net charge (toward positivity). therefore, while an acidic skin actually keeps the resident bacterial flora attached to it, a more alkaline skin compromises such attachment, suggesting that the more negatively charged such skin peptides are (alkaline skin), the more efficient these become. 226 despite the average length of 32.76 residues and net charge of +3.21 for the close to 3000 amps already identified, human amps tend to be longer and more positively charged. amps with more than 100 amino acids have been identified across the six kingdoms of life. curiously, most (approximately half) appear to be of human origin, presenting as amino acid sequences of 127 residues on average and suggesting large amps to be a human feature. moreover, compared to all the remaining amps, large human amps tend to be biased toward leucine (8.2%) and lysine (10.5%) residues, displaying nonpolar aliphatic and positively charged side groups, respectively (table i) . therefore, large human amps are enriched with amino acids that promote distinguishing features of amps: high overall positive charge, hydrophobicity domains, and amphiphilicity. nevertheless, this bias toward lysine residues is neither unique to human amps nor more pronounced among these. for instance, the amphibian cathelicidin rc-1 is composed by 32% lysine residues, and its homologous snake crotalicidin contains 38% lysine residues, showing high potency against s. pyogenes, acinetobacter baumannii, e. faecalis, s. aureus, e. coli, klebsiella pneumoniae, and p. aeruginosa. 227 as evidenced in table i , large human amps have been found in a variety of bodily fluids, including tears, saliva, urine, airways secretions, breast milk, blood, sweat, and epididymal fluids. in addition, other large amps are also expected to exist despite not constitutively found in biologic fluids, due to their inducible nature. for instance, paneth cells in the small intestine release amps-rich granules upon stimulation by cholinergic or bacterial stimuli, 228 and granulocytes are known to contain amps in granules destined for extracellular secretion. 229, 230 therefore, as the synthesis and secretion of large amps may not be constitutive but amenable to induction by microbial macromolecules and inflammatory cytokine stimuli, several other large amps may yet to be catalogued. hence, exploration of novel large antimicrobial human peptides is an ongoing work that may yield novel therapeutic agents. peptidomics is the complete study or global analysis of the peptides present in a biological sample, at a given time, under a particular condition. 231, 232 the identification of naturally occurring amps in human biological fluids is a laborious, daunting, and sometimes unsuccessful task. such naturally occurring peptides can directly result from gene encoding, from passage to bodily fluids as a result of cellular damage and renewal, or from extracellular proteolytic processing of precursor proteins. the latter, considered to be the most prevalent, hampers the identification and interpretation of biologically active amps because precursor proteins may themselves display antimicrobial activity, but may also be cleaved into multiple functional and nonfunctional peptides. 233, 234 moreover, owing to the large dependency of amps on their sequence and 3d structure, these peptides should not be subject to mass spectrometry (ms) based approaches employing enzymatic or chemical digestion, neither to any in vitro step that may cause their fragmentation. instead, top-down proteomic strategies are preferred. classically, different peptides fractions have been separated and tested for their biological activity, often without ever addressing the peptides' biological/antimicrobial activity, their sequence, target, or hypothetical mechanism(s) of action. however, current strategies are more focused, precise, and informative, capable of addressing all these parameters, sometimes in a single study (see below). 129, 235, 236 human biological fluids consist of complex samples, containing lipids, salts, proteins, carbohydrates, and other molecules that make the study of endogenous peptides extremely difficult. consequently, enrichment and separation steps are almost always required. of these biomolecules, proteins and larger peptides as well as salts are the most complicated to separate from the target peptides. luckily, salts can sometimes be removed in the same procedures applied for protein removal. first and foremost, proteases/peptidases have to be inactivated, which can be done by denaturing procedures (e.g., microwaving, boiling) or by adding protease/peptidase inhibitors. 237, 238 however, both of these can modify the peptide structure and, thus, these steps can be avoided when samples are resistant enough to further protease/peptidase activity. this is the case for urine samples, for example, as, when collected, have long been subject to proteolytic processing in the bladder. proteins are frequently removed by selective precipitation, most commonly by organic solvent (e.g., acetone) or acidic (e.g., trichloroacetic acid) precipitation, which also removes salts (left in the supernatant). 236, 239 however, precipitation-based proteins removal is not complete and may lead to the formation of peptide aggregates, which are consequently lost in the precipitate. 240 since peptides can present a great variety of sizes, sequences, structures, hydrophobicity properties, net charges, and other characteristics, their isolation is complex. still, such complexity allows for isolation steps to take place at multiple dimensions, increasing peptides isolation efficiency and accuracy. for instance, peptides can first be isolated based on their size and charge, 241, 242 bias toward specific amino acids (e.g., cysteines, tryptophans, methionines), [243] [244] [245] or on the presence of post-translational modifications (ptms) (e.g., phosphopeptides, glycopeptides). 246, 247 still, because of the aforementioned limitations, the search for endogenous amps in human bodily fluids has mostly relied on the identification of candidate amps by chromatographic techniques (e.g., high-performance liquid chromatography, hplc) and sequencing (e.g., edman sequencing), followed by their de novo chemical synthesis and in vitro antimicrobial activity testing. for these reasons, the identification and in vivo testing of novel amps is often time consuming and requires multiple studies/phases and resources. in order to mitigate this problem, candidate human amps have come to be first predicted in multiple ways, including mining of the entire human genome for particular motifs known to confer antimicrobial activity, and screening of human peptidomes (e.g., salivary, urinary) in order to find homologous peptides to other known amps. accordingly, several libraries and databases are currently available for data comparison, and some search programs can even combine both previously obtained data and de novo information. 248 even so, the identification of amps benefits the most from combinations of chromatographic and immunological techniques, genomics and proteomics. while genomics and proteomics both allow the prediction of novel amps and have the potential for contributing with a larger number of putative amps, peptides with proven in vivo antimicrobial activity have to be more accurately identified by combinations of chromatographic and immunological techniques, chemical sequencing and antimicrobial assays. though immunoassays can detect the intended target in a variety of complex biological samples, these techniques do not provide an integrative view of the peptidome, can suffer from cross-reactivity or lack of specificity issues and require previous knowledge of the target structure. 249, 250 moreover, this issue becomes a particular problem when considering that shorter or longer peptides resulting from the same precursor protein may all contain the same epitopes and thus be recognized as the same peptide fragment despite exhibiting distinct biological activities. electrospray ionization (esi) 236 and matrix-assisted laser desorption ionization (maldi) 251 have been the most frequently and successfully applied ionization techniques in the field of bodily fluids peptidomics. in line with these, peptides are most frequently previously separated by lc-based 251 and capillary electrophoresis (ce) based approaches, allowing different peptide fractions to be independently separated. 252 from this point onwards, almost any mass analyzer can be used, although in practice quadrupole time-of-flight (q-tof) and ion traps (it) instruments are the most frequently utilized ones in peptidomics, especially in exploratory studies. [253] [254] [255] in turn, fourier transform-ion cyclotron resonance-ms is more suitable for targeted/confirmatory analysis when dealing with fewer peptides. 256 because these apparatuses allow tandem ms to be performed, peptides can be readily sequenced in addition to their identification, circumventing the above-mentioned limitations concerning immunological assays and largely simplifying peptidomics workflows. in summary, when aiming at the isolation of a particular peptide, with predicted or known charge and molecular weight, an ms-based procedure can theoretically be very straightforward. first, fluid samples require (sometimes multiple) centrifugation steps to remove cells, cellular debris, wastes, and possible contaminants. then, proteins and peptides in the supernatant are separated and fractionated by multidimensional chromatographic techniques. within these procedures, proteins and peptides can be initially fractionated by gel filtration (size dimension), followed by cation-exchange (charge dimension) fractionation. cation-exchange chromatography uses a negatively charged ion exchange resin with affinity for molecules with net positive charge. a salt gradient is used to separate the peptides of interest from other bound peptides, based on their predicted isoelectric point. finally, peptides can be subject to ms analysis. they are typically ionized by esi or maldi and subsequently analyzed in a q-tof, it, or hybrid ltq. as amps tend to be highly basic peptides due to a bias toward arginine and lysine residues, these have a propensity to be particularly enriched when applying cation exchange chromatography based techniques. 257 however, because biological fluids are complex matrices, these sometimes require multiple fractionation, isolation, and extraction steps. these are laborious optimization techniques for the detection and quantification of amps and, despite their inherent complexity, they are often based in not always accurate functional and structural predictions. a recent approach has proved itself capable for the isolation of peptides present in bodily fluids that can bind directly to certain bacterial membrane components, such as lipopolysaccharide, and induce an inflammatory response against these. 129, 235 after obtaining a bacterial homogenate and extracting membrane components with an organic solvent such as n-butanol, functionalized beads with n-hydroxysuccinimidyl-sepharose (an agarose used in affinity and protein chromatography) are conjugated with these membrane bacterial components and incubated with a human biological fluid (e.g., saliva, urine). then, peptides bound to the conjugated beads are eluted and analyzed in a ltq-orbitrap hybrid fourier transform apparatus. moreover, functionalized beads conjugated with bacterial membrane components can be tested in vivo for their capacity to induce the production of inflammatory mediators. thus, this exploratory approach could result in the identification of potential human amps with selective properties capable of binding bacterial membranes and inducing an inflammatory response. recently, dallas and colleagues have attained the most complete and prolific analysis of naturally occurring peptides with potential antimicrobial activity in human milk by nano-lc quadrupole-tof tandem mass spectrometry (ms/ms), while searching against libraries of human milk peptides and known amps. 236 in order to remove milk peptides produced by in vitro proteolysis, fresh milk derived peptides were compared to control samples of the same origin but immediately subject to boiling. as boiling denatures proteases, in vitro proteolysis does not take place and peptides present in these controls could then be confidently assigned in the original analysis. moreover, masses fragmented in the first round of ms/ms were excluded from subsequent rounds of ms. 236 the authors realized that more than 80% of the +300 unique naturally occurring peptides identified with 99% confidence have yet unknown functions, which serves to illustrate how much there is still to discover regarding the biological roles of human endogenous peptides in biological fluids. nevertheless, the antimicrobial activity of the milk peptides (41 predicted amps) was confirmed in vivo by radial diffusion assays against the gram-negative e. coli and the gram-positive s. aureus. 236 furthermore, this study reinforced the observation that the background against which mass spectra are searched seems to be a more critical factor than the mass analyzer or the ionization technique used, when it comes to both the number of peptides identified and the confidence by which these can be assigned. in the same study, authors have emphasized and demonstrated the importance of avoiding in vitro hydrolysis of amps in human bodily fluids, which can be attained by adding protease inhibitors after sample collection or by adequately accounting for this phenomenon, as the authors did by using an appropriate control. in contrast, not all bodily fluids seem to be susceptible to this degree of hydrolysis, at least not to the same extent. urine, for instance, is thought to be more stable and not to require treatment with protease inhibitors. 258 however, by the time urine is collected it has already remained stagnant for considerably longer periods of time, and, hence, probably already extensively exposed to the action of proteases/peptidases inherently present in urine. therefore, how intact urinary amps actually are should always be questionable and these should ideally be interpreted considering the background of hydrolytic enzymes present in the same urine sample. also questionable is the inhibition of proteolysis in seminal plasma samples. in fact, the liquefaction process of human semen performed by seminin and other proteolytic enzymes is physiological, 259 and, therefore, naturally occurring amps should be analyzed in samples that were allowed to liquefy without the addition of protease inhibitors. curiously, seminin, which is the primary agent responsible for liquefaction of the seminal coagulum, is partially destroyed by freezing at −20°c, 259 reinforcing the vitality of analyzing fresh samples. similarly, the degradation of peptide fragments derived from semenogelins in seminal plasma samples is time dependent and responsible for the decline in hiv-enhancing activity of sperm, once again underscoring the importance of timely performing analyses of peptides in bodily fluids. 260 as previously stated, amps activity depends largely on their 3d structures, and their correct characterization becomes of paramount importance. the best experimental techniques have been and currently are x-ray crystallography 261 and nuclear magnetic resonance, 262 both of which allow for the study of structure-function relationships, though very expensive. therefore, currently, these are commonly replaced or complemented by bioinformatics prediction tools whenever possible. using such tools, the sequence of amino acids of a peptide allows for the accurate and immediate prediction of the presence of secondary structures (e.g., αhelices, β-sheets). then, the overall 3d structure is devised by algorithms employing energy minimization principles and molecular dynamics, sometimes resulting in multiple and not mutually exclusive 3d conformations, which is in accordance with peptides dynamic structure in biological fluids. 263, 264 the most common identification software referenced in the literature for peptide/precursor protein identification are sequest and mascot. 265, 266 currently, however, there is a considerable need for algorithms and software capable of correctly identifying amps and predicting their structure-function relationship. amps are a promising replacement for conventional antibiotics owing to their effectiveness against multiresistant bacteria and over multiple bacterial species simultaneously, invoking a diverse set of mechanisms that cannot be easily shortcut by bacteria, fungi, and/or virus. an ideal amp would be (i) highly selective against its target while leaving human host cells unaffected, (ii) not prone to resistance mechanisms, (iii) relatively easy to produce at low costs, and (iv) stable during storage or upon administration. drug design of such amps focuses on peptidic and nonpeptidic mimetic drugs, modulating the hydrophobicity, positivity, and amphiphilicity of endogenous amps to increase their antimicrobial potency. however, exploiting amps for therapeutic purposes is actually more complex than initially envisioned. while linear amps present a simpler design and a more predictable structure-function relationship, circular peptides may allow the design of longer acting and more resistant amps. such peptides have been designed by inserting the intended amp into other circular peptide (cyclotides) and have been proven efficacious against hiv and inflammatory diseases by circumventing their instability and poor bioavailability. 22, 267 similarly, while humans produce amps with l-amino acids and their antimicrobial activity may depend on the action of endogenous enzymes, which can only act upon l-type amino acids, producing amps with d-amino acids may render these more resistant to hydrolysis. rather than administering exogenous amps, it is possible and perhaps more secure and controllable to pharmacologically stimulate the production of endogenous amps. for instance, 1,25-dihydroxycalcypherol (active vitamin d3) has been successfully used to induce the expression of defb4 (defensin, beta 4) and camp (cationic antimicrobial peptide) genes, the production of both ll-37 and human β-defensin 2 in cell cultures of keratinocytes from diabetic foot ulcers and to promote wound healing. 268 accordingly, the persistence of functional milk human κ-caseinoglycopeptides in plasma of human infants after human milk feeding serves the purpose of exogenously administering amps without the requirement for their de novo synthesis. 269 amps can help preventing biofilm formation and microorganisms' growth. that is the case of ll-37 and its fragments, which were already shown to have antibiofilm formation properties against several methicillin-resistant s. aureus strains and p. aeruginosa, common pathogens of hospital-acquired infections, as well as against the melioidoisis' etiological agent burkholderia pseudomallei and the uropathogenic e. coli. [270] [271] [272] [273] another amp with antibiofilm formation activity is hbd-3, found to prevent the development of methicillin-resistant s. epidermidis and s. aureus biofilms, largely responsible for orthopedic implants associated infections. 274 likewise, amps may also inhibit the formation of fungal biofilms. for instance, histatin 5 has been demonstrated to successfully inhibit the growth of c. albicans biofilm on denture acrylic. 275 these examples may broaden the applications of amps in the clinical and biomedical fields by allowing functionalization of catheters. alternatively, implants and other medical devices can also reduce bacterial colonization, biofilm formation, and infection rates, presenting long lasting functionality, broad-spectrum activity, and minimal cytotoxicity against human cells. 276, 277 human amps could, theoretically, also be used as biosensors immobilized onto microchips for identification purposes, allowing the identification of microorganisms. however, such applications have only been exploited with animal or synthetic amps. these biosensors on electrical impedance alterations resulting from the presence of bacterial or fungal surface elements, or on the specificity of peptide nucleic acid probes against their targets, presenting higher versatility, lower costs, and lower sensitivity limits compared to other conventional methodologies. [278] [279] [280] alternatively, amps could be used as drug delivery systems in the form of conjugates in order to accurately target drugs and other agents to tumor sites or intended organs, as typified by monodisperse "endosomolytic" nanoparticles for in vivo gene delivery using intravenous injection, or by functionalized nanoparticles with higher membrane penetration targeted against gliomas. 281, 282 such functionalization may allow for deeper tissue penetration, increased antimicrobial potency, sustained release, and novel routes of administration to be achieved. ever since the isolation of the first peptide from the frog skin in 1983, 283 several breakthroughs were made in the isolation, synthesis, and application of amps. still, many challenges are yet to overcome in the field of amps peptidomics, 284 reflecting mainly their huge diversity and how little is known about their behavior in vivo and their structure-function relationships. in terms of costs, producing amps can be several hundred times more expensive than the production of conventional antibiotics. 285 moreover, amps' design can be very complex and the quest for the perfect amp a never-ending search. in fact, considering the possibility of working with only 20 amino acids, a small peptide with ten amino acids could have up to 20 10 = 1.02 × 10 13 different sequences, a larger peptide with 20 amino acids could present with up to 20 20 = 1.05 × 10 26 different sequences, and one with 100 amino acids could adopt any one of 20 100 = 1.27 × 10 130 different sequences. such possibilities do not even account for the presence of posttranslational modifications. then, each one of these peptides could shape into several different secondary structures and fall into a wide spectrum of activity, depending on the milieu conditions. furthermore, in a way to overcome the challenge of peptide stability, one has to consider the possibility to synthesize and to administer amps containing d-amino acids, thus decreasing their susceptibility to hydrolysis by human enzymes. 267 to deal with such large amount of amino acid combinations one should mind using peptide libraries and computational models (such as by quantitative structure-activity relationship analysis) in order to narrow large collections to few surrogate amps (for a review please see blondelle and lohner's paper. 286 another challenge to overcome is the lower amp's activity in vivo when compared to that observed in vitro. this has largely hampered the development of amps as therapeutic agents, as this decrease in activity stems from differences in ph and salt concentrations, the presence of proteases and corresponding inhibitors, and other interacting molecules hindering antimicrobial activity. accordingly, several amps (e.g., pexiganan, 287 iseganan, 288 neuprex 289 have reached phase ii clinical trials only to fail after approval for marketing because they did not evidence superior activity over already marketed conventional antibiotics. in early 2016, clinicaltrials.gov (a registry and result database of publicly and privately supported clinical studies of human participants conducted around the world) listed few clinical trials involving amps. for instance, ll37 is currently being studied for its efficacy in melanoma patients due to its ability to stimulate the immune system, while c16g2 and chromogranin a derived peptides are under scrutiny for their therapeutic potential against dental diseases. simultaneously, therapeutic strategies are also being studied to augment amps' expression in amp-deficient patients. this deficiency is believed to be due to an increase in th2 lymphocytes derived cytokines, il-4, il-13, and il-10, and a decrease in tnf-α, il-6, il-1, and interferonγ . one example of such strategy is pimecrolimus, a calcineurin inhibitor that binds with high affinity to macrophilin-12, preventing the translocation of nuclear factor of activated t cells to the nucleus and the transcription and release of such inhibitory cytokines (for more up-to-date trials, readers can access clinicaltrials.gov). another possible caveat concerns the cytotoxicity to mammalian cells when in concentrations above naturally occurring values. when bacteria produce amps, they also develop means to avoid their targeting by such peptides, such as efflux pumps and the sequestering of enzymes. however, eukaryotic cells are equally prone to damage if the administration of exogenous amps is carried out in high enough concentrations. therefore, administering amps not constitutively produced or at concentrations above those normally found in the human host environment can be toxic and very detrimental to human cells. furthermore, rapid metabolism and low bioavailability are characteristic of amps because these peptides can suffer extensive proteolysis in vivo, thereby accounting for their inactivation and short half-life. 22, 28, 290 similarly, for peptides to be available at sites distant from their origin, these would have to cross many cellular membranes. however, due to amps' polarity, membrane hydrophobicity may prevent them from doing so. 291 moreover, in contrast to other highly polar mediators (e.g., ionic salts and a few metabolites), endogenous peptides tend not to have a transporter, further compromising their bioavailability in different compartments and the subsequent impossibility to be absorbed through the gastrointestinal tract. therefore, the number of possibilities rapidly escalades, and each amp-based therapy can be further optimized based on the modulation of chemical properties and engineering of delivery systems. even acknowledging all these factors, the human host is most certainly an unpredictable environment and the same peptide may behave differently, displaying unique individual pharmacokinetics and pharmacodynamics. on a final note, due to amps' interaction with and dependence on other immune mediators, variability will most likely result from immune status alterations as those frequently found in human diseases, including infectious ones. taken together, the above discussed results support amps as constituting a paramount group of defense molecules in human biological fluids, being present in most fluids, contributing for the sterility of some of these, but being most notably enriched in biological fluids bathing tissues with the largest load of microorganisms (fig. 2) , such as saliva and urine. also, while some amps seem the display activity spectra against only a handful of microorganisms, others display broad-spectrum effects. thus, the expression and activity patterns of human amps enriched in biofluids may have suffered selective pressures resulting from exposers to microorganisms specifically present in each fluid (fig. 2) . this work was financed by national funding from fct (fundação para a ciência e a tecnologia) through the project uid/bim/04501/2013, uid/ic/00051/2013. rv thanks fct for if/00286/2015 fellowship. the authors report no declarations of interest. with saliva proteomics research. since then, he extended the application of proteomics to the characterization of other biofluids' proteome and peptidome in order to identify the biological pathways modulated by diseases such as diabetes mellitus and bladder cancer. currently, he works to a large extent with bioinformatics tools for the identification of enzymes involved in the regulation of proteome remodeling. additional supporting information may be found in the online version of this article at the publisher's web site: apd3: the antimicrobial peptide database as a tool for research and education anticancer efficacy of magainin2 and analogue peptides structure-activity analysis of thanatin, a 21-residue inducible insect defense peptide with sequence homology to frog skin antimicrobial peptides cationic host defence peptides: potential as antiviral therapeutics the n-and c-terminal fragments of ubiquitin are important for the antimicrobial 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realistic outlook optimization and high-throughput screening of antimicrobial peptides pexiganan acetate a multinational, randomized phase iii trial of iseganan hcl oral solution for reducing the severity of oral mucositis in patients receiving radiotherapy for head-and-neck malignancy prolyl peptidases: a serine protease subfamily with high potential for drug discovery bioavailability and transport of peptides and peptide drugs into the brain key: cord-274506-fzcuu4ma authors: jo, seri; kim, hyojin; kim, suwon; shin, dong hae; kim, mi‐sun title: characteristics of flavonoids as potent mers‐cov 3c‐like protease inhibitors date: 2019-09-12 journal: chem biol drug des doi: 10.1111/cbdd.13604 sha: doc_id: 274506 cord_uid: fzcuu4ma middle east respiratory syndrome‐coronavirus (mers‐cov) is a zoonotic virus transmitted between animals and human beings. it causes mers with high mortality rate. however, no vaccine or specific treatment is currently available. since antiviral activity of some flavonoids is known, we applied a flavonoid library to probe inhibitory compounds against mers‐cov 3c‐like protease (3clpro). herbacetin, isobavachalcone, quercetin 3‐β‐d‐glucoside and helichrysetin were found to block the enzymatic activity of mers‐cov 3clpro. the binding of the four flavonoids was also confirmed independently using a tryptophan‐based fluorescence method. the systematic comparison of the binding affinity of flavonoids made it possible to infer their scaffolds and functional groups required to bind with mers‐cov 3clpro. an induced‐fit docking analysis revealed that s1 and s2 sites play a role in interaction with flavonoids. the experimental and computational study showed that flavonol and chalcone are favourite scaffolds to bind with the catalytic site of mers‐cov 3clpro. it was also deduced that some flavonoid derivatives with hydrophobic or carbohydrate attached to their core structures have a good inhibitory effect. therefore, we suggest that flavonoids with these characteristics can be used as templates to develop potent mers‐cov 3clpro inhibitors. coronaviruses (covs) are a positive sense, single-stranded rna virus coated with viral particles (fehr & perlman, 2015) . together with arteriviridae and roniviridae, covs belong to the coronaviridae family in the order nidovirales. these covs can infect a wide variety of hosts, including avian, swine and humans. human coronaviruses (hcovs) represent a major group of covs associated with various respiratory diseases from common cold to serious pneumonia and bronchitis (mesel-lemoine et al., 2012) . today, hcovs are recognized as one of the most rapidly evolving viruses originated from their characteristic high genomic nucleotide substitution rates and recombination. in human, their infection is known to cause approximately one-third of common cold. severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) covs are highly pathogenic and caused a fatal first major outbreak in 2003 and 2012 , respectively (de wit, doremalen, falzarano, & munster, 2016 . sars-and mers-covs genomes contain two open reading frames orf1a and orf1b translated to two respective viral polyproteins pp1a and pp1ab by host ribosomes. orf1a encodes two cysteine proteases, a papain-like protease (plpro) and a 3c-like protease (3clpro). while plpro cuts the first three cleavage sites of its polyprotein, 3clpro is responsible for cleavage of the remaining eleven locations resulting in release of a total of 16 non-structural proteins (nsp) in both sars-and mers-covs. the homodimeric form of 3clpro is active in the presence of substrates. the crystal structure of 3clpro showed that each monomer is composed of three structural domains: domains i and ii form a chymotrypsinlike architecture with catalytic cysteine and are connected to a third c-terminal domain via a long loop (neddle, lountos, & waugh, 2015) . in the proteolytic site, all 3clpros prefer glutamine at p1 position and leucine, basic residues, small hydrophobic residues at p2, p3 and p4 positions, respectively (chuck, chow, wan, & wong, 2011) . at p1′ and p2′ positions, small residues are required. since the autocleavage process is essential for viral propagation, 3clpro is a good drug target for anti-coronaviral infection. in this study, we used a proteolytic method to probe mers-cov 3clpro inhibitory compounds with a synthetic peptide labelled with the edans-dabcyl fret (fluorescence resonance energy transfer) pair (liu et al., 2005) . since emission wavelengths of edans are widely overlapped with absorbance wavelengths of dabcyl, the energy emitted from edans will be quenched by dabcyl in a close proximity (10-100 å). therefore, an increment of fluorescence can be a sign to judge whether a substrate is cleaved or not in this design. hence from the fluorescence intensity change, the proteolytic activity of protease could be detected. with a synthetic peptide with the fret pair, a flavonoid library was screened to search mers-cov 3clpro inhibitory compounds. recent studies showed that some flavonoids have antiviral activity in some viruses (frabasile et al., 2017; jucá et al.,2018; kiat et al., 2006; yang, lin, zhou, liu, & zhu, 2017; zakaryan, arabyan, oo, & zandi, 2017) . therefore, we assayed flavonoids and tried to induce their structural property crucial to bind with mers-cov 3clpro. the coding sequence of mers-cov nsp5, a 3c-like protease (ncbi ref. seq. nc_019843.3) was synthesized chemically by bioneer and cloned into a bacteriophage t7-based expression vector. the plasmid dna was transformed into e. coli bl21 (de3) for protein expression. e. coli bl21 (de3) cells were grown on luria-bertani (lb) agar plates containing 150 μg/ml ampicillin. several colonies were picked and grown in capped test-tubes with 10 ml lb broth containing 150 μg/ml ampicillin. a cell stock composed of 0.85 ml culture and 0.15 ml glycerol was prepared and frozen at 193 k for use in a large culture. the frozen cell stock was grown in 5 ml lb medium and diluted into 2,000 ml fresh lb medium. the culture was incubated at 310 k with shaking until an od 600 of 0.6-0.8 was reached. at this point, expression of mers-cov 3clpro was induced using isopropyl-β-d-1-thiogalactopyranoside (iptg) at a final concentration of 1 mm. the culture was further grown at 310 k for 3 hr in a shaking incubator. cells were harvested by centrifugation at 7,650 g (6,500 rev min −1 ) for 10 min in a high-speed refrigerated centrifuge at 277 k. the cultured cell paste was resuspended in 25 ml of a buffer consisting of 50 mm tris-hcl ph 8.0, 100 mm nacl, 10 mm imidazole, 1 mm phenylmethylsulfonyl fluoride (pmsf) and 10 μg/ml dnase i. the cell suspension was disrupted using an ultrasonic cell disruptor (digital sonifier 450; branson). cell debris was pelleted by centrifugation at 24,900 g (15,000 rev min −1 ) for 30 min in a highspeed refrigerated ultra-centrifuge at 277 k. the protein was purified by affinity chromatography using a 5 ml hi-trap q column (ge healthcare) followed by a 5 ml hi-trap blue column (ge healthcare). the custom-synthesized fluorogenic substrate, dabcyl-ktsavlqsgfrkme-edans (anygen), was used as a substrate for the proteolytic assay using mers-cov 3clpro (kuo, chi, hsu, & liang, 2004) . this substrate contains the nsp4/nsp5 cleavage sequence, gvlq↓sg (wua et al., 2015) , and works as a generic peptide substrate for many coronavirus including mers-cov 3clpro. the peptide was dissolved in distilled water and incubated with each protease. a spectramax i3x multi-mode microplate reader (molecular devices) was used to measure spectral-based fluorescence. the proteolytic activity was determined at 310 k by following the increase in fluorescence (λ excitation = 340 nm, λ emission = 490 nm, bandwidths = 9, 15 nm, respectively) of edans upon peptide hydrolysis as a function of time. assays were conducted in black, 96-well plates (nunc) in 350 μl assay buffers containing protease and substrate as follows: for the mers-cov 3clpro assay, 1.84 μl of 0.19 mm protease containing 20 mm tris ph 8.0 was incubated with 8.75 μl of 0.1 mm substrate at 310 k for 2 hr before measuring relative fluorescence unit (rfu). before the assay, the emission spectra of 40 flavonoids were surveyed after illuminating at 340 nm to avoid the overlapping with the emission spectrum of edans. every compound was suitable to be tested. the final concentration of the protease, peptide and chemical used at the assay was 1, 2.5 and 20 μm each. at first, mers-cov 3clpro and chemical were mixed and preincubated at room temperature for 1 hr. the reaction was initiated by the addition of the substrate, and each well was incubated at 310 k for 2 hr. after 2 hr, we measured the fluorescence of the mixture on the black 96-well plate using the end-point mode of spectramax i3x where the excitation wavelength was fixed to 340 nm and the emission wavelength was set to 490 nm using 9, 15 nm bandwidth, respectively. all reactions were carried out in triplicate. among the first forty flavonoids (table s1 ), four of them were picked up to further assay at a concentration range of 2 μm-320 μm. ic50 value which is the value causing 50% inhibition of catalytic activity of mers-cov 3clpro was calculated by non-linear regression analysis using graphpad prism 7.03 (graphpad software). the proteolytic assay using mers-cov 3clpro in the presence of triton x-100 has been performed to differentiate artificial inhibitory activity of chemicals through non-specific binding with proteases by forming aggregate or complexation. the concentration used in this study was 0.01%. to confirm the feasibility of the assay method independently, the fluorescence spectra from tryptophans of mers-cov 3clpro with candidate inhibitors were investigated (lin, lan, guan, sheng, & zhang, 2009 ). the fluorescence measurements were recorded with a spectramax i3x multi-mode microplate reader (molecular devices) at excitation and emission wavelengths of 295 nm and 300-500 nm, respectively. the optimal excitation and emission wavelengths were determined by softmax pro. five tryptophans of mers-cov 3clpro showed a strong fluorescence emission with a peak at 340 nm at the excitation wavelength of 295 nm. in contrast, the flavonoids were almost non-fluorescent under the same experiment condition. each 40 μm chemical was incubated with 1 μm mers-cov 3clpro for 1 hr, and the fluorescence intensity of mers-cov 3clpro was measured. all the docking and scoring calculations were performed using the schrödinger suite of software (maestro, version 11.5.011). the compounds were extracted from the pubchem database in sdf format and were combined in one file. the file was then imported into maestro and prepared for docking using ligprep. the atomic co-ordinates of the crystal structure of mers-cov 3clpro (5wkj) were retrieved from the protein data bank and prepared by removing all solvent and adding hydrogens and minimal minimization in the presence of bound ligand using protein preparation wizard. ionizer was used to generate ionized state of all compounds at target ph 7.0 ± 2.0. this prepared low-energy conformers of the ligand were taken as the input for docking analysis. grids for molecular docking were built using receptor grid generation. compounds were docked using ligand docking mode with postdocking minimization including 5 poses per ligand. the docked poses were then refined using an xp (extra precision) option with the threshold value for rejecting minimized pose of 0.5 kcal/mol. the energies were calculated using the opls3 force field. the induced-fit docking protocol (sherman, day, jacobson, friesner, & farid, 2006) was run from the graphical user interface accessible within maestro 11.5.011. receptor sampling and refinement were performed on residues within 5.0 å of each ligand for each of the 20 ligand-protein complexes. with prime (jacobson et al., 2004) , a side-chain sampling and prediction module, the side-chains, as well as the backbone of the target protein, were energy minimized. a total of 20 induced-fit receptor conformations were generated for each of the eight test ligands. re-docking the test ligands into their respective 20 structures within 30.0 kcal/mol of their lowest energy structure. finally, the ligand poses were scored using a combination of prime and glidescore scoring functions . the amount of cell harvested for purification of mers-cov 3clpro was 3.01 g per 2,000 ml of e. coli culture. the protein was purified by ion chromatography using a 5 ml hi-trap q column (ge healthcare). the column was equilibrated with a buffer consisting of 20 mm bis-tris ph 7.0, and the pooled fractions were loaded. the column was eluted using a linear nacl gradient to 1.0 m nacl, and the protein was eluted at 0.16 m nacl. the pooled fractions were loaded on a 5 ml hi-trap blue column (ge healthcare) equilibrated with a buffer consisting of 10 mm sodium phosphate ph 7.0. the target protein was detected in unbound fractions. sds-page showed one band around 33(33,737.77da) kda, corresponding to the molecular weight of mers-cov 3clpro. the protein was concentrated to 31.14 mg/ml for protease assays in a buffer consisting of 10 mm sodium phosphate ph 7.0. the purified mers-cov 3clpro was different from the previous method (kumar et al., 2016) where the native form was obtained after removing of a his 6 -tag wih factor-xa. in our experiment, the enzyme activity was 10-fold decreased when the his 6 -tag protein was used (data not shown). that is due to the location of the his 6 -tag connected to the n-terminus of mers-cov 3clpro. the published crystal structure showed that the active site pocket might be easily hindered by the flexible n-terminal his 6 -tag. a flavonoid library consisting of ten different scaffolds was also built (figure 1 ). it contains three isoflavones, one isoflavane, six flavones, nine flavonols, four flavanols, five flavanones, one flavanonol, one prenylflavonoid, eight chalcones and two unclassified flavonoids (table s1 ). we applied the library to assay mers-cov 3clpro. since flavonoids are known to aggregate through a complexation and thus non-specifically inhibit various proteases, the assay in the presence of triton x-100 was performed. before assay, we tested the influence of triton x-100 on the catalytic activity of mers-cov 3clpro. as shown in the figure s1 , only a slight increment of the catalytic activity was observed even up to 0.1% triton x-100. therefore, we performed the experiment at the concentration of 0.01% triton x-100 where no significant interference was detected. using forty flavonoids, an inhibitory effect of each compound at 20 μm was tested. among them, herbacetin (3,4′,5,7,8-pentahydroxyflavone), isobavachalcone (2′,4,4′-trihydroxy-3′-(3-methyl-2-butenyl)chalcone), quercetin 3-β-d-glucoside (3,3′,4′,5,7-pentahydroxyflavone 3-β-d-glucoside) and helichrysetin (4,2′,4′-trihydroxy-6′methoxychalcone) were found to have prominent inhibitory activity (figure 2 ). the four compounds showed the severely reduced fluorescent intensity and thus represented their mers-cov 3clpro inhibitory activity. the experimental data were plotted as log inhibitor concentration versus per cent fluorescence inhibition (figure 2) . ic50 values obtained from the dose-dependent inhibitory curves of herbacetin, isobavachalcone, quercetin 3-β-d-glucoside and helichrysetin are 40.59, 35.85, 37.03 and 67.04 μm, respectively. in order to confirm the inhibitory activity of the flavonoids independently, a general tryptophan-based assay method was employed. tryptophan was well-known to emit its own fluorescence. therefore, if tryptophan is positioned adequately in proteins, the change in fluorescence intensity can reflect the binding state of chemicals and be used to judge interaction between proteins and chemicals. mers-cov 3clpro contains five tryptophan residues and thus displays a high fluorescence peak at 340 nm at the tryptophan excitation wavelength of 295 nm. we monitored the change in the fluorescence intensities of mers-cov 3clpro depending on the presence or absence of four chemicals. since each compound in the flavonoid library was almost non-fluorescent under the experiment condition, a change in fluorescence intensity reflects interactions between mers-cov 3clpro and chemicals. only the four inhibitory compounds obviously reduced the fluorescence intensity of mers-cov 3clpro (figure 3) . the decreased emission intensity confirmed the complex formation between mers-cov 3clpro and inhibitory compounds. in order to deduce the binding modes of the inhibitory flavonoids, an in-depth theoretical investigation through an induced-fit docking study was carried out. the interactions between mers-cov 3clpro and the inhibitory flavonoids were analysed to predict their binding affinities. top ranked structures (according to the glide scores) from the induced-fit docking results for herbacetin (−10.246), isobavachalcone (−9.364), quercetin 3-β-d-glucoside (−9.751) and helichrysetin (−9.953) were selected and hypothesized to be biological complexes. in order to compare binding affinities to their closest homologues ( figure s2 figure s3 . it is very obvious that the s1 site and hydrophobic s2 site of mers-cov 3clpro play a key role in predicted complexes. flavonoids are natural compounds with multiple pharmacological characteristics such as antioxidant, anti-inflammatory, analgesic, anti-carcinogenic, antibacterial infection, antifungal and antiviral properties (frabasile et al., 2017) . naringenin has therapeutic effects on various neurological, cardiovascular, gastrointestinal, rheumatological, metabolic and malignant disorders (rani et al., 2016) . it also represents antiviral function (zakaryan et al., 2017) . fisetin is a commercially available nutraceutical with anti-inflammatory, antioxidant, anti-tumorigenic, anti-invasive, anti-angiogenic, anti-diabetic, neuroprotective and cardioprotective effects (pal, pearlman, & afaq, 2016) . interestingly, fisetin has an anti-noroviral activity by inducing cytokines (seo & choi, 2017) . although some of flavonoids show an antiviral effect, a molecular mechanism of their antiviral activity was rarely known. in this study, we assayed the inhibitory activity of various flavonoids against mers-cov 3clpro. for the trial, a flavonoid library composed of nine different scaffolds plus one undefined group was constructed. they are classified based on a c6-c3-c6 skeleton and differ in the overall hydroxylation patterns and in the saturation of the heteroatomic ring c together with the position of the attached aromatic ring b (at the positions c-2 or c-3 of ring c) (jucá et al., 2018) . the flavonoid library was tested, and thus, a systematic analysis was possible. among the ten groups, isoflavones, isoflavane, flavanols and flavanones did not show any inhibitory activity over mers-cov 3clpro. the other groups contains some flavonoids displaying moderate inhibitory activity. however, four flavonoids, herbacetin, isobavachalcone, quercetin 3-β-d-glucoside and helichrysetin exhibited prominent inhibitory activity. the immediate inference of the primary scaffold required for binding with mers-cov 3clpro was as follows: first, the orientation of the aromatic ring b at the position c-2 of ring c is essential as shown in isoflavones and isoflavane. second, the saturated heteroatomic ring c is not preferred as shown in flavanols and flavanones. third, chalcone and flavonol scaffolds show a promising binding property. the more detailed structural comparison also provided valuable structural information required for the binding affinity of each flavonoid. helichrysetin is a chalcone derivative (van puyvelde et al., 1989) . therefore, we compared its inhibitory activity with a chalcone, cardamonin (2′,4′-dihydroxy-6′-methoxychalcone) which is the structurally identical homologue except one hydroxyl group at the 4-position of the benzyl moiety of chalcone. the inhibitory activity of cardamonin at the concentration of 40 μm was lower than that of helichrysetin ( figure 4 ). it implies that the 4-hydroxyl group of helichrysetin is functionally important to bind with mers-cov 3clpro. the structural comparison of cardamonin with isobavachalcone also indicated the modification effect of acetophenone ring of chalcone: the hydrophobic modification at the 3′-position of the acetophenone ring moiety of isobavachalcone improves its binding affinity to mers-cov 3clpro. intriguingly, the docking study shows that the 4-hydroxyl group of helichrysetin forms a hydrogen bond with the hydroxyl group of tyr54 of mers-cov 3clpro. since tyr54 is located deep inside of the hydrophobic s2 site, helichrysetin is inserted into deeper than cardamonin ( figure s3a ). as a result, helichrysetin seems to have a better affinity and thus represents a better inhibitory activity. in the flavonoid library, there are quite similar homologues of herbacetin, isobavachalcone and quercetin 3-β-d-glucoside. they are kaempferol (3,4′,5,7-tetrahydroxyflavone), 2,2′,4′-trihydroxychalcone and quercitrin (3,3′,4′,5,7-pentahydroxyflavone-3-l-rhamnoside), respectively ( figure s2 ). their mers-cov 3clpro inhibitory activity is lower than their corresponding compounds at 40 μm. herbacetin is a kaempferol derivative where one more hydroxide is attached at 8-position of kaempferol. the docking study indicates kaempferol derivatives occupy the s1 and s2 sites of mers-cov 3clpro ( figure s3b ). the hydroxyl group at 7-position looks important to bind at the s1 binding site. a f i g u r e 4 comparison of inhibitory activity between homologue flavonoids. the two homologue flavonoids are compared side by side. each of two bars represents the effect of two homologue inhibitory compounds at 40 μm against mers-cov 3clpro compared to the control. each bar is expressed as the mean ± standard error of the mean (n = 3). rfu = relative fluorescence units bit better inhibitory activity of herbacetin indicates the hydroxide at 8-position is favourable for its binding affinity to mers-cov 3clpro. in the predicted complex, the hydroxyl group at 8-position of herbacetin induces a hydrogen bond with his41 at the s1 site. the improved activity of isobavachalcone compared with 2,2′,4′-trihydroxychalcone again points out the importance of its hydrophobic substituent. coincided with the experimental result, the prenyl moiety of isobavachalcone makes hydrophobic interactions with met25 and leu49 at the hydrophobic s2 site ( figure s3c) . quercetin 3-β-d-glucoside is a homologue of quercitrin where rhamnoside is substituted for glucoside. the better inhibitory activity of quercetin 3-β-d-glucoside means that hydroxymethyl is preferred to hydroxide in this position of the glucoside to interact with mers-cov 3clpro. the docking study represents that the hydroxymethyl group of the glucoside moiety makes a hydrogen bond with glu169 and thus contributes its tighter binding to the s1 site than the rhamnoside moiety of quercitrin ( figure s3d ). helichrysetin and isobavachalcone inhibiting the activity of mers-cov 3clpro are chalcone derivatives. chalcone has immunomodulation, antibacterial, antifungal, antiviral, anti-inflammatory, antioxidant, anticancer and anti-diabetic activities (yadav, prasad, sung, & aggarwal, 2011) . recently, the non-competitive inhibition of cardamonin against the dengue virus protease has been reported (kiat et al., 2006) . as discussed above, cardamonin is a strong homologue of helichrysetin. the analysis of the crystal structures of three viral proteases, mers-cov 3clpro, dengue virus ns2b/ns3 protease and norovirus 3clpro were performed, and their active sites were compared ( figure s4 ) (erbel et al., 2006; nakamura et al., 2005) . despite their sequential divergence, their overall architecture resembles each other. furthermore, the spatial positions of their catalytic residues were also highly conserved. the observation implies that chalcone and flavonol can be used as templates to develop a potential antiviral agent by targeting these viral proteases. in this study, we showed that the antiviral effects of some flavonoids may be directly from their inhibition of main viral proteases and thus nullify a process of virus peptides. therefore, one of antiviral mechanisms of flavonoids may be originated from their direct binding capability to viral proteases. consequently, flavonoid derivatives can be used as a template to design not only for broad-spectrum but also for virus-specific antiviral agents. a further study is going on to develop better inhibitory lead compounds derived from this study. we built a flavonoid library to systematically search mers-cov 3clpro inhibitory compounds with a fret method. we found four flavonoids effectively reducing the proteolytic activity of mers-cov 3clpro. the binding of the flavonoids was independently proven by a tryptophanbased fluorescence method. the assay in the presence of triton x-100 also eliminated the chance of the false-positive result from the aggregation of flavonoids. the analysis of the four compounds with their homologs using an induced-fit docking study provided an insight of flavonoid scaffolds required to bind with mers-cov 3clpro. the prominent activity of helichrysetin and isobavachalcone indicates that the flexibility of the chalcone scaffold is good to bind with mers-cov 3clpro. in contrast, the comparable activity of flavones with a rigid γ-pyrone ring indicates that their spatial rearrangement together with various functional groups may be an alternative strategy to interact with mers-cov 3clpro. therefore, this study suggests that an antiviral effect of some flavonoids is directly from their structural characteristics binding to viral proteases. profiling of substrate specificities of 3c-like proteases from group 1, 2a, 2b, and 3 coronaviruses sars and mers: recent insights into emerging coronaviruses structural basis for the activation of flaviviral ns3 proteases from dengue and west nile virus coronaviruses: an overview of their replication and pathogenesis the citrus flavanone naringenin impairs dengue virus replication in human cells extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes a hierarchical approach to allatom protein loop prediction flavonoids: biological activities and therapeutic potential inhibitory activity of cyclohexenyl chalcone derivatives and flavonoids of fingerroot, boesenbergia rotunda (l.), towards dengue-2 virus ns3 protease identification, synthesis and evaluation of sars-cov and mers-cov 3c-like protease inhibitors characterization of sars main protease and inhibitor assay using a fluorogenic substrate spectroscopic investigation of interaction between mangiferin and bovine serum albumin screening of drugs by fret analysis identifies inhibitors of sars-cov 3cl protease a human coronavirus responsible for the common cold massively kills dendritic cells but not monocytes a norovirus protease structure provides insights into active and substrate binding site integrity structures of the middle east respiratory syndrome coronavirus 3c-like protease reveal insights into substrate specificity fisetin and its role in chronic diseases pharmacological properties and therapeutic potential of naringenin: a citrus flavonoid of pharmaceutical promise inhibitory mechanism of five natural flavonoids against murine norovirus novel procedure for modeling ligand/receptor induced fit effects isolation of flavonoids and a chalcone from helichrysum odoratissimum and synthesis of helichrysetin prediction and biochemical analysis of putative cleavage sites of the 3c-like protease of middle east respiratory syndrome coronavirus the role of chalcones in suppression of nf-κb-mediated inflammation and cancer activity of compounds from taxillus sutchuenensis as inhibitors of hcv ns3 serine protease flavonoids: promising natural compounds against viral infections the authors declare no conflict of interest. the data that support the findings of this study are available from the corresponding author upon reasonable request. https://orcid.org/0000-0002-2205-1453mi-sun kim https://orcid.org/0000-0002-4092-4203 key: cord-277541-ieex2xwx authors: sun, jian; wang, hexiang; ng, tzi bun title: trypsin isoinhibitors with antiproliferative activity toward leukemia cells from phaseolus vulgaris cv “white cloud bean” date: 2010-06-14 journal: j biomed biotechnol doi: 10.1155/2010/219793 sha: doc_id: 277541 cord_uid: ieex2xwx a purification protocol that comprised ion exchange chromatography on deae-cellulose, affinity chromatography on affi-gel blue gel, ion exchange chromatography on sp-sepharose, and gel filtration by fplc on superdex 75 was complied to isolate two trypsin inhibitors from phaseolus vulgaris cv “white cloud bean”. both trypsin inhibitors exhibited a molecular mass of 16 kda and reduced the activity of trypsin with an ic(50) value of about 0.6 μm. dithiothreitol attenuated the trypsin inhibitory activity, signifying that an intact disulfide bond is indispensable to the activity. [methyl-(3)h] thymidine incorporation by leukemia l1210 cells was inhibited with an ic(50) value of 28.8 μm and 21.5 μm, respectively. they were lacking in activity toward lymphoma mbl2 cells and inhibitory effect on hiv-1 reverse transcriptase and fungal growth when tested up to 100 μm. protease inhibitors have been purified from an array of leguminous and nonleguminous species encompassing torresea cearensis [1] , erythrina caffra [2] , dolichos lablab [3] , crotalaria paulina [4] , medicago scutellata [5, 6] , canavalia gladiata [7] , pisum sativum [8] , dimorphandra mollis [9] , swartzia pickellii [10] , psophocarpus tetragonolobus [11] , delonix regina [12] , poecilanthe parviflora [13] , adenanthera pavonina [14] , cajanus cajan [15] , dolichos biflorus [16] , phaseolus acutifolius [17] , arachis hypogaea [18] , leucaena leucocephala [19] , bauhinia bauhinioides [20] , bauhinia variegata [21] , bauhinia ungulata [22] , vigna unguiculata [23] , lens culinaris [24] , glycine max [25] , peltophorum dubium [26] , pithecellobium dulce [27] , glycine soja [28] , and barley [29] . protease inhibitors impair the activity of insect midgut proteases and thus adversely affect protein digestion and health in insects. they represent one of the multitude of entomotoxic proteins [30, 31] . in addition to insecticidal activity [32] [33] [34] [35] , protease inhibitors demonstrate antiproliferative and antitumor activities [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] . the objective of the present study was to isolate and characterize proteins with protease inhibitory activity from white cloud beans. inhibitor. an aqueous extract of the beans (250 g) was produced by blending in distilled water (3 ml/g) followed by centrifugation (14000 g for 25 minutes at 4 • c). the resulting supernatant was applied to a 5 × 20 cm column of deae-cellulose (sigma) in 10 mm tris-hcl buffer (ph 7.4). after elution of unadsorbed proteins (fraction d1), the column was eluted successively with 0.2 m nacl and 1 m nacl in the tris-hcl buffer. fraction d2 eluted with 0.2 m nacl was dialyzed to remove nacl and then subjected to affinity chromatography on a 5 × 15 cm of affi-gel blue gel (bio-rad) in 10 mm tris hcl buffer (ph 7.4). the unadsorbed fraction (b1) was dialyzed against 10 mm nh 4 oac buffer (ph 5) and then applied to a 2.5 × 20 cm column of sp-sepharose (ge healthcare). after elution of unadsorbed proteins (fraction s1), the column was eluted with a 0-1 m nacl concentration gradient in the nh 4 oac buffer. the first and second adsorbed fractions (sp2 and sp3) were then further purified by gel filtration on a superdex 75 hr 10/30 column (ge healthcare) in 0.2 m nh 4 hco 3 buffer (ph 8.5) using an akta purifier (ge healthcare). the second absorbance peak represented purified trypsin inhibitor. terminal sequence analysis. the molecular mass of the isolated proteins was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) following the procedure of laemmli and favre [46] . gel filtration on an fplc-superdex 75 column, previously calibrated with molecular mass marker proteins (ge healthcare), was employed to determine the molecular mass of the protein. the n-terminal sequence of the protein was analyzed by using a hewlett-packard hp g1000a edman degradation unit and an hp 1000 hplc system [47] . activity. the test sample (20 μl) was added to 160 μl of a 1% casein solution in 0.1 m tris-hcl buffer (ph 7.4). trypsin (20 μl of a 0.5 mg/ml solution) was then added and the mixture was incubated at 37 • c for 15 minutes followed by addition of trichloroacetic acid (0.4 ml, 5%) that was added to bring the reaction to an end. after centrifugation the absorbance of the resulting supernatant, which indicates the amount of casein fragments produced by trypsin, was read at 280 nm. the % inhibition of trypsin activity is equal to the % decrease in absorbance of the supernatant [48] . activity. the isolated trypsin inhibitor (2.5 μm) was treated with dithiothreitol (dtt) at the final concentration 2.5, 10 and 40 mm for 5, 20, and 80 minutes at 37 • c. soybean trypsin inhibitor from sigma (2.5 μm) was similarly treated and used as a positive control. the reaction was terminated by adding iodoacetamide at twice the amount of thiol functions at each dtt concentration. residual trypsin inhibitor activity was measured at ph 7.4 as described above in assay for trypsin inhibitory activity. the highest iodoacetamide concentration used in the test was devoid of any effect on trypsin activity and the trypsin inhibitory activity of isolated trypsin inhibitor and soybean trypsin inhibitor [47] . the antiproliferative activity of the purified trypsin inhibitor was assayed as described below. the cell lines l1210 (human leukemia) and mbl2 (murine lymphoma) were obtained from american type culture collection. the cell line was maintained in dulbecco modified eagles' medium (dmem) supplemented with 10% fetal bovine serum (fbs), 100 mg/l streptomycin, and 100 iu/ml penicillin, at 37 • c in a humidified atmosphere of 5% co 2 . cells (1 × 10 4 ) in their exponential growth phase were seeded into each well of a 96-well culture plate (nunc, denmark) and incubated for 3 hours prior to addition of the trypsin inhibitor. incubation was performed for an additional 48 hours. radioactive precursor, 1 μci ([ 3 h-methyl]-thymidine, from ge healthcare), was then introduced to each well and the incubation continued for 6 hours. the cultures were then harvested using a cell harvester. the radioactivity incorporated was measured in a liquid scintillation counter [49] . the assay for hiv reverse transcriptase inhibitory activity was carried out in view of the report that trypsin inhibitors manifest this activity [50, 51] . it was conducted according to instructions supplied with the assay kit from boehringer mannheim (germany). the assay makes following use of the ability of reverse transcriptase to synthesize dna, commencing from the template/primer hybrid poly (a) oligo (dt) 15. the digoxigenin-and biotin-labeled nucleotides in an optimized ratio are incorporated into one of the same dna molecule, which is freshly synthesized by the reverse transcriptase (rt). the detection and quantification of synthesized dna as a parameter for rt activity are based on a sandwich elisa protocol. biotin-labeled dna binds to the surface of microtiter plate modules that have been precoated with strepatavidin. in the following step, an antibody to digoxigenin, conjugated to peroxidase, binds to the digoxigenin-labeled dna. in the last step, the peroxidase substrate is added. the peroxidase enzyme affects the cleavage of the substrate, yielding a colored reaction product. the absorbance of the sample at 405 nm which is directly correlated to the level of rt activity can be measured using a microtiter plate (elisa) reader. a fixed amount (4-6 ng) of recombinant hiv-1 reverse transcriptase was used. the inhibitory activity of the trypsin inhibitor was calculated as percent inhibition compared to a control without the trypsin inhibitor [49] . integrase. the plasmid that expressed his-tagged wild-type hiv-1 integrase, pt7-7-his (y | tx)-hiv-1-in, was a generous gift from professor s.a. chow (school of medicine, ucla). to express the enzyme, a 1-liter culture of e. coli bl21 (de3) cells containing the expressing plasmid was grown at 37 • c until it reached od600 0.7-0.8. cells were induced by addition of 0.8 mm iptg (isopropyl-βd-thiogalactopyranoside) and harvested, after 4 hours of incubation, by centrifugation at 6000 g for 10 minutes at 4 • c. cells were suspended at a concentration of 10 ml/g wet cell paste in 20 mm tris-hcl buffer (ph 8.0) containing 0.1 mm edta, 2 mm β-mercaptoethanol, 0.5 m nacl, and 5 mm imidazole. lysozyme was added to a concentration of 0.2 mg/ml. after incubation at 4 • c for 1 hour, the lysate was sonicated and centrifuged at 40 000 g at 4 • c for 20 minutes. the pellet was homogenized in 50 ml buffer a (20 mm tris-hcl, ph 8.0, 2 m nacl, 2 mm β-mercaptoethanol) which contained 5 mm imidazole. the suspension was rotated at 4 • c for 1 hour and cleared by centrifugation at 40 000 g at 4 • c for 20 minutes. the supernatant was applied to a 1 ml chelating sepharose (ge healthcare) column charged with 50 mm imidazole. the column was eluted with five column volumes of buffer a containing 5 mm imidazole, and the protein was eluted with three column volumes of buffer a containing 200 and 400 mm imidazole, respectively. protein containing fractions were pooled, and edta was added to a final concentration of 5 mm, followed by dialysis against buffer b (20 mm hepes, ph 7.5, 1 mm edta, 1 m nacl, 20% glycerol) containing 2 mm βmercaptoethanol and then against buffer b containing 1 mm 2.9. assay for antifungal activity. this assay was performed since some trypsin inhibitors demonstrate antifungal activity [8] . the assay for antifungal activity toward botrytis cinerea and fuserium oxysporum was executed in 100 mm × 15 mm petri plates containing 10 ml of potato dextrose agar. after the mycelial colony had grown to a sufficiently large size, sterile blank paper disks (0.625 cm in diameter) were laid at a distance of 0.5 cm away from the edge of the mycelial colony. an aliquot (15 μl) of the trypsin inhibitor was added to a disk. the plates were exposed at 23 • c for 72 hours until mycelial growth had enveloped the disks containing the control and had produced crescents of inhibition around disks containing samples with antifungal activity [49] . anion exchange chromatography of the bean extract on deae-cellulose resolved it into three fractions, an unadsorbed fraction d1 together with two adsorbed fractions d2 and d3. trypsin inhibitory activity was confined to fraction d2. after affinity chromatography of fraction d2 on affi-gel blue gel, the activity appeared in the unadsorbed fraction b1 (data not shown). ion exchange chromatography of fraction b1 on sp-sepharose resolved it into a small unadsorbed fraction sp1 and three large adsorbed fractions (sp2, sp3, and sp4) of about the same size ( figure 1 ). trypsin inhibitory activity was detected in fractions sp2 and sp3. final purification of sp2 on superdex 75 produced two fractions, sp2su1 and sp2su2 (figure 2(a) ). gel filtration of sp3 on superdex 75 yielded two fractions sp3su1 and sp3su2 (figure 2(b) ). fractions sp2su2 and sp3su2, both with a molecular mass of 16 kda as determined by gel filtration on superdex 75, were the only fractions with trypsin inhibitory activity. the remaining fractions sp2su1 and sp3su1 were inactive. the yields of the various chromatographic fractions are presented in table 1 . both sp2su2 and sp3su2 displayed a molecular mass of 16 kda in sds-page ( figure 3 ) and gel filtration ( figure 2 ). their n-terminal sequences are shown in table 2 . these two white cloud bean trypsin inhibitors inhibited trypsin with an ic 50 of about 0.6 μm (figure 4) . inhibitor. dtt treatment curtailed the trypsin inhibiting activity in a doseand time-dependent manner ( table 3 ). the two trypsin inhibitors did not inhibit hiv-1 reverse transcriptase when tested at various concentrations up to 100 μm (table 4) . they lacked antifungal activity when tested up to 24 μg/disk (100 μm, 15 μl) (data not shown). the ic 50 values of the inhibitory effects of the trypsin inhibitors on l1210 cells were, respectively, 21.5 μm and 28.8 μm (table 4 ). however, there was no activity toward mbl2 cells when tested up to 100 μm. they showed marked homology to partial sequences of other leguminous trypsin inhibitors. the present study disclosed the production of two trypsin inhibitors with closely related n-terminal sequences chromatographic behavior and bioactivities by the white cloud bean variety of phaseolus vulgaris. the trypsin inhibitors demonstrate the same molecular mass; both are adsorbed on deae-cellulose and unadsorbed on affi-gel blue gel results are presented as mean ± sd (n = 3). different alphabets (e.g., a, b, and c) indicate statistically significant difference (p < .05) when (i) data at same time point and different dtt concentrations or (ii) data at same dtt concentration but different time point were analyzed by analysis of variance followed by duncan's multiple range test. figure 4 : inhibitory effects by the two white cloud bean trypsin inhibitors on trypsin. ic 50 = circa 0.6 μm. ic 50 for soybean trypsin inhibitor = 1.5 μm. results are presented as mean ± sd (n = 3). results are presented as mean ± sd (n = 3). ic 50 = 0.25 μm. different letters (a, b, c, d) next to the data points indicate statistically significant difference (p < .05) when the data are analyzed by analysis of variance followed by duncan's multiple range test. but can be separated by ion exchange chromatography on sp-sepharose. they have approximately the same trypsin inhibitory potency, and neither of them inhibits hiv-1 reverse transcriptase inhibitory activity, hiv-1 integrase inhibitory activity, sars coronavirus proteinase inhibitory activity, or fungal growth. both inhibitors exhibit an antiproliferative activity toward l1210 cells albeit with a small difference in potency, while there is little activity toward mbl2 cells. the difference in yields of the two trypsin inhibitors from the white cloud beans is only slight. the two trypsin inhibitors exhibit n-terminal sequence homology with those of other leguminous trypsin inhibitors such as inhibitors mungbean (vigna mungo), cowpea (vigna unguiculata), and lima bean (phaseolus lunatus). whereas leguminous antifungal proteins [48, 49] , like nonleguminous antifungal proteins [53] , are unadsorbed on deae-cellulose and adsorbed on affi-gel blue gel, white cloud bean trypsin inhibitors are adsorbed on deae-cellulose and unadsorbed on affi-gel blue gel. thus the purification procedure adopted in the present investigation can be conveniently used to separate trypsin inhibitors from antifungal proteins. the antiproliferative activity of white cloud bean trypsin inhibitors is consistent with similar observations on field bean trypsin inhibitor [39, 40, 42, 43, 54] . it is noteworthy that white cloud bean trypsin inhibitors do not exert a similar action on lymphoma mbl2 cells. thus the action of white cloud bean trypsin inhibitors is tumour cellspecific. the ribosome inactivating protein trichosanthin exerts different antiproliferative potencies toward different tumor cells [55] . in contrast to broad bean trypsin inhibitor [48, 56] , those from white cloud bean do not inhibit hiv-1 reverse transcriptase. a variety of trypsin inhibitors exhibit antifungal activity [48, 56] . however, white cloud bean trypsin isoinhibitors lack such activity. this is reminiscent of the finding that lentil and vigna mungo inhibitors have little or no hiv-1 reverse transcriptase inhibitory and antifungal activities [57, 58] . in summary, the isolation of two trypsin inhibitors with very similar biochemical and biological characteristics from white cloud beans was achieved in the present investigation. the presence of multiple trypsin inhibitors has previously been reported in momordica cochinchinensis seeds [59] . white cloud bean trypsin inhibitors demonstrate antiproliferative activity against tumor cells but do no inhibit mycelial growth or hiv-1 reverse transcriptase. previously isolated p. vulgaris trypsin inhibitors have not been so tested [60] [61] [62] [63] [64] . they reportedly have a molecular mass of about 9 kda [63, 65] or 13 kda [61] , smaller than the value of 16 kda obtained for white cloud bean trypsin inhibitors purification and primary structure determination of a bowman-birk trypsin inhibitor from torresea cearensis seeds the spectroscopic analysis, inhibition and binding studies demonstrate the equivalence of erythrina caffra trypsin inhibitor and the recombinant substitution variant rec-sereti purification and characterization of a proteinase inhibitor from field bean, dolichos lablab perpureus l isolation and characterization of a new trypsin inhibitor from crotalaria paulina seeds a trypsin inhibitor from snail medic seeds active against pest proteases effects of the medicago scutellata trypsin inhibitor (msti) on cisplatininduced cytotoxicity in human breast and cervical cancer cells complete amino acid sequences of three proteinase inhibitors from white sword bean (canavalia gladiata) pa1b, an insecticidal protein extracted from pea seeds (pisum sativum): 1h-2-d nmr study and molecular modeling purification and characterization of a new trypsin inhibitor from dimorphandra mollis seeds characterization of a kunitz trypsin inhibitor with one disulfide bridge purified from swartzia pickellii structure of a kunitz-type chymotrypsin inhibitor from winged bean seeds at 2.95å resolution primary sequence determination of a kunitz inhibitor isolated from delonix regia seeds trypsin inhibitor from poecilanthe parviflora seeds: purification, characterization, and activity against pest proteases a kunitz-type inhibitor of coleopteran proteases, isolated from adenanthera pavonina l. seeds and its effect on callosobruchus maculatus characterization of a proteinase inhibitor from cajanus cajan (l.) formation of bowman-birk inhibitors during the germination of horsegram (dolichos biflorus) unusual structural characteristics and complete amino acid sequence of a protease inhibitor from phaseolus acutifolius seeds cdna clone of a putative peanut (arachis hypogaea l.) trypsin inhibitor has homology with peanut allergens ara h 3 and ara h 4 molecular mechanism of enzyme inhibition: prediction of the three-dimensional structure of the dimeric trypsin inhibitor from leucaena leucocephala by homology modelling characterization of a tissue kallikrein inhibitor isolated from bauhinia bauhinioides seeds: inhibition of the hydrolysis of kininogen related substrates the complete amino acid sequence of a trypsin inhibitor from bauhinia variegata var. candida seeds human plasma kallikrein and tissue kallikrein binding to a substrate based on the reactive site of a factor xa inhibitor isolated from bauhinia ungulata seeds crystallization and preliminary x-ray diffraction studies of a bowman-birk inhibitor from vigna unguiculata seeds complete amino acid sequence of the lentil trypsin-chymotrypsin inhibitor lci-1.7 and a discussion of atypical binding sites of bowman-birk inhibitors phage display selection can differentiate insecticidal activity of soybean cystatins a novel trypsin inhibitor from peltophorum dubium seeds, with lectin-like properties, triggers rat lymphoma cell apoptosis isolation and properties of a kunitz-type protein inhibitor obtained from pithecellobium dulce seeds purification and characterization of proteinase inhibitors from wild soja (glycine soja) seeds crystal and molecular structure of the serine proteinase inhibitor ci-2 from barley seeds molecular characterization of a new arcelin-5 gene cloning and functional expression of a mungbean defensin vrd1 in pichia pastoris protein proteinase inhibitor genes in combat against insects, pests, and pathogens: natural and engineered phytoprotection effect of dietary cowpea trypsin inhibitor (cpti) on the growth and development of the tomato moth lacanobia oleracea (lepidoptera: noctuidae) and on the success of the gregarious ectoparasitoid eulophus pennicornis (hymenoptera: eulophidae) putative protein digestion in a sapsucking homopteran plant pest nilaparvata lugens: delphacidae)-identification of trypsinlike and cathepsin b-like proteases identification of potent inhibitors of helicoverpa armigera gut proteinases from winged bean seeds effects of protease inhibitors on growth of hamster tumor cells in culture inhibition of n-nitrosomethylbenzylamine-induced esophageal neoplasms by the bowman-birk protease inhibitor effects of various preparations of dietary protease inhibitors on oral carcinogenesis in hamsters induced by dmba field bean protease inhibitor preparations, unlike methotrexate, can completely suppress yoshida sarcoma tumor in rats inhibition of benzopyreneinduced forestomach tumors by field bean protease inhibitor(s) protein proteinase inhibitors in legume seedsoverview erratum: the field bean protease inhibitor can effectively suppress 72-dimethylbenz[a]anthracene-induced skin tumorigenesis in mice the field bean protease inhibitor has the potential to suppress b16f10 melanoma cell lung metastasis in mice protease inhibitors and carcinoma of the esophagus a soybean kunitz trypsin inhibitor suppresses ovarian cancer cell invasion by blocking urokinase upregulation maturation of the head of bacteriophage t4. i. dna packaging events the papaya kunitz-type trypsin inhibitor is a highly stable β-sheet glycoprotein a bowman-birk-type trypsinchymotrypsin inhibitor from broad beans gymnin, a potent defensinlike antifungal peptide from the yunnan bean (gymnocladus chinensis baill) pleureryn, a novel protease from fresh fruiting bodies of the edible mushroom pleurotus eryngii isolation and characterization of velutin, a novel low-molecular-weight ribosome-inactivating protein from winter mushroom (flammulina velutipes) fruiting bodies concurrent purification of two defense proteins from french bean seeds: a defensin-like antifungal peptide and a hemagglutinin isolation of cucurmoschin, a novel antifungal peptide abundant in arginine, glutamate and glycine residues from black pumpkin seeds treatment with field bean protease inhibitor can effectively repress ethylnitrosourea (enu)-induced neoplasms of the nervous system in sprague-dawley rats ribosome inactivating proteins (rips) from momordica charantia for anti viral therapy a new peptidic protease inhibitor from vicia faba seeds exhibits antifungal, hiv-1 reverse transcriptase inhibiting and mitogenic activities isolation and characterization of a trypsin-chymotrypsin inhibitor from the seeds of green lentil (lens culinaris) trypsinchymotrypsin inhibitors from vigna mungo seeds multiple trypsin inhibitors from momordica cochinchinensis seeds, the chinese drug mubiezhi trypsin isoinhibitors from garden beans (phaseolus vulgaris) the isolation and characterization of a trypsin inhibitor from kintoki bean (phaseolus vulgaris) isolation and characterization of some proteinase inhibitors from phaseolus vulgaris var. nanus fogo na serra" inhibitor and interactive surface modeling for the enzyme-inhibitor complex complete sequence, subunit structure, and complexes with pancreatic α-amylase of an α-amylase inhibitor from phaseolus vulgaris white kidney beans purification and characterization of an extracellular prolyl endopeptidase from agaricus bisporus this work was financially supported by national grants of china (nyhyzx07-008 and 2007bad89b00). key: cord-292286-ygomb3oi authors: zakaryan, hovakim; arabyan, erik; oo, adrian; zandi, keivan title: flavonoids: promising natural compounds against viral infections date: 2017-05-25 journal: arch virol doi: 10.1007/s00705-017-3417-y sha: doc_id: 292286 cord_uid: ygomb3oi flavonoids are widely distributed as secondary metabolites produced by plants and play important roles in plant physiology, having a variety of potential biological benefits such as antioxidant, anti-inflammatory, anticancer, antibacterial, antifungal and antiviral activity. different flavonoids have been investigated for their potential antiviral activities and several of them exhibited significant antiviral properties in in vitro and even in vivo studies. this review summarizes the evidence for antiviral activity of different flavonoids, highlighting, where investigated, the cellular and molecular mechanisms of action on viruses. we also present future perspectives on therapeutic applications of flavonoids against viral infections. throughout human history, thousands of biologically active plants have been identified and used in medicine. virtually all cultures around the world continue to rely on medicinal plants for primary health care. according to the world health organization report, about 80% of the world's population depend on medicinal plants to satisfy their health requirements [30] . furthermore, there are currently hundreds of modern drugs based on active compounds isolated from plants. plants have the ability to produce a wide range of compounds including flavonoids, phytoalexins, lignans, and tannins, which are responsible for key functions in plant growth and development. flavonoids or polyphenolics comprise the largest group of secondary metabolites found in vegetables, fruits, seeds, nuts, spices, stems as well as in red wine and tea (table 1 ) [88] . these compounds are synthesized in response to various abiotic stress conditions such as ultraviolet radiation and play an important role as defense agents against plant pathogens and insects [9, 84] . the first evidence of a biological activity of flavonoids was reported by albert szent-gyorgyii in 1938, who showed that citrus peel flavonoids prevent capillary bleeding and fragility associated with scurvy [109] . since then, a broad spectrum of biological activities such as anti-inflammatory, antioxidant, antibacterial, antiviral, anticancer, and neuroprotective has been described for flavonoids [40, 53, 65, 95, 137] . research for antiviral agents isolated from plants started in 1950s, when the activity of 288 plants against influenza a virus was evaluated in embryonated eggs [14] . during the last 60 years, several plants and plant-derived compounds with antiviral properties were identified. in this article, we review the results of both in vitro and in vivo experiments demonstrating the antiviral activity of flavonoids, especially focusing on those classes of flavonoids that have been extensively investigated. there are now more than 6000 varieties of flavonoids that have been structurally identified [35] . all these compounds comprise a flavan nucleus and a fifteen-carbon skeleton consisting of two benzene rings (a-and b-rings, as shown in fig. 1 ) connected via a heterocyclic pyrene ring (c-ring, as shown in fig. 1 ). flavonoids are divided into several classes such as anthocyanidins, flavones, flavonols, flavanones, flavan, isoflavanoids, biflavanoids, etc (table 1 ) [24] . the various classes of flavonoids differ in the level of oxidation and pattern of substitution of the pyrene ring, whereas individual compounds within the classes differ in the pattern of substitution of benzene rings. while in flavonoids the b-ring links to the c-ring at the c2 position, the b-ring of isoflavonoids is substituted at position c3 (fig. 1 ). biflavonoids comprise of two identical or non-identical flavonoid units conjoined through an alkyl-or alkoxybased linker (fig. 1) . in plants, flavonoids generally occur as aglycones, glycosides and methylated derivatives. they are biosynthesized through the phenylpropanoid pathway, transforming phenylalanine into 4-coumaroyl-coa, which then enters the flavonoid biosynthesis pathway [32] . depending on the plant species, a group of enzymes, such as hydroxylases and reductases, modify the basic flavonoid skeleton, resulting in the different flavonoid classes. finally, transferases modify the flavonoid skeleton with sugar, methyl groups and acyl moieties. these modifications alter the solubility and reactivity of flavonoids [6] . a large body of evidence supports the role of light in the regulation of flavonoid biosynthesis [156] . flavones constitute a major class in the flavonoid family based on a 2-phenyl-1-benzopyran-4-one backbone. natural flavones include apigenin, baicalein, chrysin, luteolin, scutellarein, tangeritin, wogonin and 6-hydroxyflavone. the antiviral activity of flavones is known from the 1990s, when it was showed that the simultaneous application of apigenin with acyclovir resulted in an enhanced antiviral effect on herpes simplex virus types 1 and 2 (hsv-1 and hsv-2) in cell culture [92] . apigenin is most commonly isolated in abundance from the family asteraceae. the organic and aqueous extracts from asteraceae plants with apigenin as a major compound were found to be active against hsv-1, poliovirus type 2 and hepatitis c virus (hcv) [85, 127] . apigenin isolated from sweet basil (ocimum basilicum) showed a potent antiviral activity against adenoviruses (adv) and hepatitis b virus in vitro [17] . besides these dna viruses, apigenin was found to exert antiviral effect against african swine fever virus (asfv), by suppressing the viral protein synthesis and reducing the asfv yield by 3 log [46]. apigenin is also active against rna viruses. for picronaviruses, it has been shown that apigenin is able to inhibit viral protein synthesis through suppressing viral ires activity [82, 107] . furthermore, apigenin affects enterovirus-71 (ev71) translation by disrupting viral rna association with trans-acting factors regulating ev71 translation [153] . shibata et al. [115] showed that apigenin has antiviral effect on hcv through the reduction of mature microrna122, a liver-specific microrna which positively regulates hcv replication. among flavones, baicalein and luteolin have been also extensively investigated with respect to their antiviral activity. baicalein significantly reduced the levels of human cytomegalovirus (hcmv) early and late proteins, as well as viral dna synthesis, although it had no effect on viral polymerase activity [23, 31] . baicalein impaired avian influenza h5n1 virus replication in both human lung epithelial cells and monocyte-derived macrophages by interfering with neuraminidase activity [116] . other studies showed that oral administration of baicalein to balb/c mice infected with influenza h1n1 virus decreased the lung virus titer and increased the mean time to death [139] . similar effects were recorded on mice infected with sendai virus [28] . these inhibitory effects in vivo were mediated by serum baicalin, a metabolite of baicalein which has a glucose residue [26] . baicalin alone exerts its anti-influenza activity by modulating the function of ns1 protein, which down-regulates ifn induction [99] . further studies indicated that baicalin can directly induce ifn-c production in human cd4 ? t cells and cd8 ? t cells and act as a potent inducer of ifn-c during influenza virus infection [19] . recently, novel baicalein analogs with b-rings substituted with bromine atoms demonstrated extremely potent activity against influenza h1n1 tamiflu-resistant virus, indicating that baicalein and its analogs can be favorable alternatives in the management of tamiflu-resistant viruses [21] . in vitro replication of hiv-1 was suppressed by baicalin when infected cells were treated during the early stage of the virus replication cycle [66] . hiv-1 envelope protein was found to be the target site of baicalin's antiviral action via the interference of interactions between the virus structural protein and specific host immune cells [75] . baicalein and baicalin were also investigated against dengue virus (denv). they exerted a significant virucidal effect on extracellular viral particles and interfered with different steps of denv-2 replication [91, 148, 150] . in silico studies revealed that baicalein has strong binding affinity with denv ns3/ns2b protein (-7.5 kcal/mol), and baicalin may interact closely with the virus ns5 protein at a binding affinity of -8.6 kcal/mol [47] . for baicalin, computational studies also showed a high binding affinity (-9.8 kcal/mol) against chikungunya virus (chikv) nsp3 protein, suggesting that baicalin can potentially interfere with chikv infection [114] . it was found that luteolin has antiviral effect on hiv-1 reactivation by blocking both clade b-and c-tat-driven ltr transactivation [87] . luteolin also showed significant inhibition of epstein-barr virus (ebv) reactivation in cells [133] ; it suppressed the activities of the immediate-early genes zta and rta by deregulating transcription factor sp1 binding. xu et al. [142] tested 400 highly purified natural compounds for inhibition of ev71 and coxsackievirus a16 infections and found that luteolin exhibited the most potent inhibition through disruption of viral rna replication. besides these antiviral activities, luteolin or luteolin-rich fractions showed antiviral effects against severe acute respiratory syndrome coronavirus (sars-cov), rhesus rotavirus, chikv and japanese encephalitis virus (jev) [33, 67, 94, 146] . flavonols are characterized by a 3-hydroxy-2-phenylchromen-4-one backbone. among flavonols the antiviral effect of quercetin was the most extensively investigated. early in vivo studies showed that oral treatment with quercetin protected mice from lethal mengo virus [44, 125] . furthermore, an enhanced protection was observed when quercetin was administered in combination with murine type i interferon (ifn) [125] . quercetin also demonstrated a dose-dependent antiviral activity against poliovirus type 1, hsv-1, hsv-2, and respiratory syncytial virus (rsv) in cell cultures [60, 83] . epimedium koreanum nakai, which contains quercetin as the major active component, has been shown to induce secretion of type i ifn, reducing the replication of hsv, newcastle disease virus (ndv), vesicular stomatitis virus (vsv) in vitro, as well as influenza a subtypes (h1n1, h5n2, h7n3 and h9n2) in vivo [18] . hung et al. [51] have suggested possible mechanisms whereby quercetin may exert its anti-hsv activity. they revealed that quercetin inhibits the infection of hsv-1, hsv-2 and acyclovirresistant hsv-1 mainly by blocking viral binding and penetration to the host cell. they also reported that quercetin suppresses nf-jb activation, which is essential for hsv gene expression. recent investigations also pointed out the antiviral activity of quercetin against a wide spectrum of influenza virus strains. it interacts with influenza hemagglutinin protein, thereby inhibiting viralcell fusion [136] . in addition, in silico analysis revealed that quercetin may be a potential inhibitor of the neuraminidase of influenza a h1n1 and h7n9 viruses [79, 80] . molecular docking analysis also found that quercetin may interact with hcv ns3 helicase, ns5b polymerase and p7 proteins [34, 86] . these results correlate with experimental studies showing the anti-hcv activity of quercetin through inhibition of ns3 helicase and heat shock proteins [4, 81] . besides these viruses, the inhibitory activity of quercetin and its derivatives have been reported for other viruses, including advs, arthropod-borne mayaro virus, porcine reproductive and respiratory syndrome virus, canine distemper virus, jev, denv-2, porcine epidemic diarrhea virus, and equid herpesvirus 1 [11, 16, 27, 38, 41, 59, 118, 149] . quercetin also possesses anti-rhinoviral effects by inhibiting endocytosis, transcription of the viral genome and viral protein synthesis [37] . in mice infected with rhinovirus, quercetin treatment decreased viral replication and attenuated virusinduced airway cholinergic hyper-responsiveness [37] . kaempferol is another flavonol extracted from different medicinal herbs. kaempferol and its derivatives bearing acyl substituents have shown inhibitory activity against hcmv [89] . kaempferol derivatives isolated from ficus benjamina leaves were more effective against hsv-1 and hsv-2 than their aglycon form [145] . kaempferol derivatives with rhamnose residue turned out to be potent inhibitors of the 3a channel of coronavirus, which is involved in the mechanism of virus release [112] . one of the kaempferol derivative, kaempferol 3-o-a-lrhamnopyranoside, obtained from zanthoxylum piperitum was shown to significantly inhibit the replication of influenza a virus in vitro [45] . behbahani et al. found that kaempferol and kaempferol-7-o-glucoside have strong hiv-1 reverse transcriptase inhibitory activity [5] . these compounds exerted their effects, at a concentration of 100 lg/ml, on the early stage of hiv replication in target cells. recently, kaempferol-3,7-bisrhamnoside isolated from chinese medicinal taxillus sutchuenensis was shown to have potent in vitro activity on hcv ns3 protease function [144] . antiviral activity of kaempferol on the influenza viruses h1n1 and h9n2 were mentioned in a study conducted by a group of researchers in south korea. mechanistic and structural studies suggested that the compound acts on the virus neuraminidase protein and specific functional groups are responsible for kaempferol's efficacy [57] . a study comparing the antiviral activities of kaempferol and an isoflavone, daidzein, showed that kaempferol exerted more potent inhibitory activities on jev replication and protein expression, than daidzein. jev's frameshift site rna (fsrna) has been proposed as the target site for kaempferol's inhibitory activity against this flavivirus [152] . seo et al. conducted a study comparing the potency of different classes of flavonoids against two rna viruses, namely murine norovirus and feline calicivirus. their findings demonstrated that, among the flavonoids tested, kaempferol exhibited the most potent inhibitory activity against these two viruses [113] . there are number of other flavonols and derivatives acting as antivirals. for example, sulfated rutin, which is modified from glycoside rutin, demonstrated significant activity against different hiv-1 isolates [123] . this compound inhibited hiv-1 infection by blocking viral entry and virus-cell fusion, likely by interacting with hiv-1 envelope glycoproteins. rutin at 200 lm concentration was shown to inhibit ev71 infection by suppressing the activation of mek1-erk signal pathway, which is required for ev71 replication of [129] . rutin and fisetin also inhibited the replication of ev-a71 by affecting the enzymatic activity of the 3c protease [76] . fisetin treatment caused a dose-dependent decrease in the production of chikv nonstructural proteins and inhibition of viral infection [73] . moreover, zandi et al. showed that denv-2 rna copy number was significantly reduced following addition of fisetin to infected cells [149] . yu et al. found that myricetin may serve as chemical inhibitor of sarscoronavirus because it affects the atpase activity of the viral helicase [147] . flavans are characterized by a 2-phenyl-3,4-dihydro-2hchromene skeleton. these compounds include flavan-3-ols, flavan-4-ols and flavan-3,4-diols. among flavan-3-ols, the antiviral activity of catechin and its derivatives epicatechin, epicatechin gallate, epigallocatechin (egc), and epigallocatechin gallate (egcg), which are found in tea, has been largely investigated [122] . among different viruses studied as potential targets, influenza virus has received the most attention after an initial report by nakayama et al. showing that tea catechins, particularly egcg, are able to bind to the haemagglutinin of influenza virus, preventing its adsorption to madin-darby canine kidney cells [98] . furthermore, it has been suggested that egcg may be able to damage the physical properties of the viral envelope, resulting in the inhibition of hemifusion events between influenza virus and the cellular membrane [66] . recently, colpitts and schang reported that egcg competes with sialic acid for binding to influenza a virus, thereby blocking the primary low-affinity attachment to cells [22] . another tea catechin, egc, exerted the inhibitory effect on the acidification of endosomes and lysosomes, thereby reducing viral entry via clathrin-mediated endocytosis [52]. a structure-function relationship analysis of tea catechins revealed the important role of the 3-gallolyl group of the catechin skeleton for its antiviral activity [120] . the results also showed that modification of the 3-hydroxyl position significantly affected the antiviral activity. catechin derivatives containing carbon chains at 3-hydroxyl position demonstrated potent anti-influenza activity in vitro and in ovo [121] . several reports have demonstrated that tea catechins have an antiviral effect against hiv infection. among tea catechins, egcg is the most effective because it exerts its antiviral effect throughout several steps of the hiv-1 life cycle. it directly binds to cd4 molecules with consequent inhibition of gp120 binding, an envelope protein of hiv-1 [62, 134] . these studies identified trp69, arg59 and phe43 of cd4 as potential sites for interaction with the galloyl moiety of egcg. the same residues are involved in interaction with viral gp120 [135] . furthermore, early studies from nakane and ono showed that egcg and ecg were effective at inhibiting hiv-1 reverse transcriptase in vitro [96, 97] . tillekeratne et al. modified the molecular structure of egcg to determine the minimum structural characteristics necessary for hiv-1 reverse transcriptase inhibition [124] . in their study, the gallate ester moiety was found to be important for inhibition. besides these effects, egcg has the ability to reduce viral production in chronically infected monocytoid cells [143] . the inhibitory effect was increased by approximately 25%, when egcg was modified with lyposomes. tea catechins are also effective against herpesviruses. egcg has been shown to block ebv lytic cycle by inhibiting expression of viral genes including rta, zta and ea-d [13] . further studies indicated that one of the mechanisms by which egcg may inhibit ebv lytic cycle involves the suppression of mek/erk1/2 and pi3-k/akt signaling pathways, which are involved in the ebv lytic cycle cascade [78] . isaacs et al. found that egcg can inactivate hsv virions by binding to the envelope glycoproteins gb and gd, which are essential for hsv infectivity [54] . the egcg digallate dimers theasinensin a, p2, and theaflavin-3,3'-digallate inactivated hsv-1 and hsv-2 more effectively than did monomeric egcg [55]. these dimers are stable at vaginal ph, indicating their potential to be antiviral agents against hsv infections. the inhibitory effect of green tea extracts against hbv has been reported [140] . in hepg2.117 cells, egcg inhibited hbv replication through impairing hbv replicative intermediates of dna synthesis, thereby reducing the production of hbv covalently closed circular dna [48] . in contrast, huang et al. found that egcg decreased hbv entry into immortalized human primary hepatocytes by more than 80% but had no effect on hbv genome replication [50]. furthermore, egcg is able to enhance lysosomal acidification, which is an unfavorable condition for hbv replication [155] . besides these viruses, egcg has been found to exert antiviral activity against hcv by preventing the attachment of the virus to the cell surface and suppressing rna replication steps [8, 15] . a recent study also showed inhibitory activity of egcg against another flavivirus, zika virus (zikv): in this study, foci forming unit reduction assays were performed to evaluate the antiviral activity of egcg on zikv at different stages of virus replication. foci observed showed more than 90% inhibition when the cells were treated with egcg during virus entry [10] . similarly, egcg is able to block chikv attachment to target cells, but has no effect on other stages of infection [132] . naringenin, which belongs to the flavanones class, has been shown to reduce the replication of a neurovirulent strain of sindbis virus in vitro [102] . it also reduced sindbis virus-and semliki forest virus-induced cytopathic effect in virus yield experiments [105] . interestingly, naringin, the glycoside form of naringenin did not have anti-sindbis virus activity, indicating that the rutinose moiety of this flavanone blocks its antiviral effect. naringenin is also able to block the assembly of intracellular hcv particles and long-term treatment leads to 1.4 log reduction in hcv [39, 64] . the alphavirus chikv was effectively inhibited when infected vero cells were treated with naringenin at the post-entry stage. in the same study, hesperetin, another flavanone which is found richly in citrus fruits, was found to exert most potent anti-chikv effect during the virus intracellular replication, with an ic 50 of 8.5 lm [1] . molecular docking and molecular dynamics studies by oo et al. also revealed strong and stable interactions between hesperetin and chikv nonstructural protein 2 (nsp2) as well as non-structural protein 3 (nsp3), suggesting that these proteins may be the target of hesperetin's anti-chikv activity [101] . genistein is an isoflavonoid found in a number of plants including soybeans and fava beans. as a tyrosine kinase inhibitor, genistein reduced bovine herpesvirus type 1 and new world arenavirus pichinde replication, by preventing the phosphorylation of viral proteins [2, 126] . kinase inhibitor cocktails containing genistein displayed a broadspectrum antiviral activity against arenaviruses and filoviruses [68] . genistein was shown to inhibit hiv infection of resting cd4 t cells and macrophages through interference with hiv-mediated actin dynamics [42] . furthermore, it may act against hiv ion channel since it has the ability to block the viral vpu protein, which is believed to form a cation-permeable ion channel in infected cells [110] . genistein also exerted its antiviral effects on the replication of hsv-1, hsv-2, and avian leucosis virus subgroup j, by inhibiting virus transcription [3, 106] . the antiviral activity of other flavonoids is presented in table 2 . in spite of the wide range of biological health benefits which flavonoids possess, in addition to their high availability in humans' daily diets, there are challenges ahead for researchers before these natural compounds can be applied as therapeutic options in the clinical setting. bioavailability, defined by the us food and drug administration as ''the rate and extent to which the active ingredient or active moiety is absorbed from a drug product and becomes available at the site of action'', has been the main stumbling block to further advances in the potential use of flavonoids in the medical community. intake of metabolic derivatives of flavonoids from various food sources leads to relatively large differences in the final amount being successfully absorbed and utilized by humans [71] . factors such as molecular sizes, glycosylation, esterification, lipophilicity, interactions with the enteric microorganisms, pka, and other metabolic conjugations along the alimentary tract, affect the absorption and bioavailability of flavonoids in humans [49, 56, 69, 90, 93, 111, 133] . hence, efforts in enhancing the bioavailability of flavonoids upon intake by humans are vitally necessary in order to develop these natural compounds into potential antiviral drugs. the following are a few examples of efforts being carried out to tackle this issue which can be used as platforms for further successes in the future. in the past, researchers have looked into alternative methods to improve the compounds' solubility or to switch the site of absorption in the gut, with the aim of enhancing their bioavailability. a structural modification to hesperetin-7-glucoside, which resulted in a change in site of absorption from the large to the small intestine, has successfully yielded a higher plasma level of hesperetin in healthy subjects [100] . wang et al. [130] formulated a way to increase the oral bioavailability of flavonols extracted from sea buckthorn, by forming a phospholipid complex via solvent evaporation method. relative to the parent compounds, oral bioavailability of the tested flavonols was 172% -242% higher when the phospholipid complex was administered into rats [130] . flavonoids loaded in engineered nanoparticles have also been tested for their bioavailability following oral consumption. improved stability of catechin and egcg in chitosan nanoparticles have been shown to result in a higher rate of intestinal absorption [29] . poly (d, l-lactide) (pla) nanoparticles and polymeric micelles contributed to a more sustainable release of quercetin, which has poor bioavailability and undergoes substantial first-pass metabolism, as well as of the poorly absorbed apigenin [70, 124, 151] . self-microemulsifying drug delivery system (smdds) is another technology which has been used to overcome the problem of low bioavailability of hydrophobic molecules. upon entering the lumen of the intestine, an oil-in-water microemulsion containing the drug will be formed. the microemulsion increases the intestinal absorption of the drug or compound by avoiding the dissolution process [60, 108] . puerarin, an isoflavone isolated from the root of pueraria lobata, exhibited 2.6-fold higher bioavailability when prepared using smdds [154] . however, it is worth noting that while the bioavailability of flavonoids can be increased via different methodologies, it is vital that their biological efficacies are not affected, but maintained or enhanced. for instance, phosphorylated icariin has been found to inhibit duck hepatitis virus a more effectively than the parent compound [138] . isorhamnetin is a methylated flavonol derived from the structure of quercetin. dayem et al. investigated the antiviral potency of isorhamnetin against influenza a h1n1 virus and discovered that the methyl group on the b ring enhances its antiviral activity compared with the other tested flavonoids [25] . the efficacy of isorhamnetin against influenza virus was also shown when in vivo and in ovo models were tested [25] . improvement in bioavailability will definitely enhance the efficacy of different biological effects of all classes of flavonoids. hence, in addition to discovering the hidden potentials of flavonoids, scientists should also aim to identify ways to increase the amount of flavonoids available for the health benefits of human beings. natural compounds have been the center of attention among researchers working in various fields, including those related with antiviral drug development, due to their high availability and low side effects. the phytochemicals flavonoids, which are abundantly found in our daily diets of fruits and vegetables, have been actively studied as potential therapeutic options against viruses of different taxa in the past decade. numerous positive findings have been reported on the in vitro efficacy of flavonoids, but less promising results have been obtained for most compounds in in vivo studies. multiple factors contributed to this scenario, and in vivo studies must be prioritized by researchers. it is well-known that flavonoids possess enormous potential to be included in the daily prescriptions by physicians treating illnesses ranging from infectious and oncogenic to inflammatory and chronic degenerative diseases. however, it is time for researchers worldwide to take the initiative in making these compounds a success not only in the in vitro stage of research, but also in animal models, as well as in subsequent clinical studies. biochemistry and mechanistic studies on the flavonoids' inhibitory activities can improve our understanding of how these natural compounds work and, on the other hand, identify the stumbling block that is hindering further improvements in flavonoids antiviral research. (7) inhibition of chikungunya virus replication by hesperetin and naringenin effect of genistein on replication of bovine herpesvirus type 1 antiherpes evaluation of soybean isoflavonoids suppression of hepatitis c virus by the flavonoid quercetin is mediated by inhibition of ns3 protease activity in vitro anti-hiv-1 activities of kaempferol and kaempferol-7-o-glucoside isolated from securigera securidaca glycosyltransferases: managers of small molecules structural basis of inhibitor specificity of the human protooncogene proviral insertion site in moloney murine leukemia virus (pim-1) kinase epigallocatechin-3-gallate is a new inhibitor of hepatitis c virus entry flavonoids and melanins: a common strategy across two kingdoms the green tea molecule egcg inhibits zika virus entry in vitro inhibition of canine distemper virus by flavonoids and phenolic acids: implications of structural differences for antiviral design effect of naringenin, hesperetin and their glycosides forms on the replication of the 17d strain of yellow fever virus inhibition of epstein-barr virus lytic cycle by (-)-epigallocatechin gallate the action of plant extracts on a bacteriophage of pseudomonas pyocyanea and on influenza a virus epigallocatechin-3-gallate inhibits the replication cycle of hepatitis c virus in vitro antiviral activities of caesalpinia pulcherrima and its related flavonoids antiviral activities of extracts and selected pure constituents of ocimum basilicum epimedium koreanum nakai displays broad spectrum of antiviral activity in vitro and in vivo by inducing cellular antiviral state role of baicalin in anti-influenza virus a as a potent inducer of ifngamma inhibitory effects of flavonoids on moloney murine leukemia virus reverse transcriptase activity synthesis and anti-influenza activities of novel baicalein analogs a small molecule inhibits virion attachment to heparan sulfate-or sialic acid-containing glycans eight flavonoids and their potential as inhibitors of human cytomegalovirus replication the chemistry and biological effects of flavonoids and phenolic acids antiviral effect of methylated flavonol isorhamnetin against influenza antiviral activity of baicalin against influenza a (h1n1/ h3n2) virus in cell culture and in mice and its inhibition of neuraminidase quercetin and quercetin 3-o-glycosides from bauhinia longifolia (bong.) steud. show anti-mayaro virus activity effects of baicalein on sendai virus in vivo are linked to serum baicalin and its inhibition of hemagglutinin-neuraminidase chitosan nanoparticles enhance the intestinal absorption of the green tea catechins (?)-catechin and (-)-epigallocatechin gallate the growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety human cytomegalovirus-inhibitory flavonoids: studies on antiviral activity and mechanism of action flavonoids: biosynthesis, biological functions, and biotechnological applications antiviral activity ofl uteolin against japanese encephalitis virus docking studies of pakistani hcv ns3 helicase: a possible antiviral drug target structure and function of enzymes involved in the biosynthesis of phenylpropanoids inhibition of infectivity of potato virus x by flavonoids quercetin inhibits rhinovirus replication in vitro and in vivo inhibition of hsp70 reduces porcine reproductive and respiratory syndrome virus replication in vitro naringenin inhibits the assembly and long-term production of infectious hepatitis c virus particles through a ppar-mediated mechanism polyphenols as mitochondria-targeted anticancer drugs in vitro assessment of the antiviral potential of trans-cinnamic acid, quercetin and morin against equid herpesvirus 1 genistein interferes with sdf-1-and hiv-mediated actin dynamics and inhibits hiv infection of resting cd4 t cells anti-hepatitis b virus activity of wogonin in vitro and in vivo effect of quercetin on the course of mengo virus infection in immunodeficient and normal mice. a histologic study antiviral effect of flavonol glycosides isolated from the leaf of zanthoxylum piperitum on influenza virus antiviral activity of flavonoids 2547 anti-h1n1 virus, cytotoxic and nrf2 activation activities of chemical constituents from scutellaria baicalensis antiviral activity of baicalein and quercetin against the japanese encephalitis virus development of self-microemulsifying drug delivery systems (smedds) for oral bioavailability enhancement of simvastatin in beagle dogs antiviral effect of flavonoids on human viruses epigallocatechin gallate, the main component of tea polyphenol, binds to cd4 and interferes with gp120 binding development of chemical inhibitors of the sars coronavirus: viral helicase as a potential target divergent antiviral effects of bioflavonoids on the hepatitis c virus life cycle fisetin: a dietary antioxidant for health promotion inhibition of influenza virus internalization by (-)-epigallocatechin-3-gallate an evaluation of the inhibitory effects against rotavirus infection of edible plant extracts inhibition of lassa virus and ebola virus infection in host cells treated with the kinase inhibitors genistein and tyrphostin chemistry and biological activities of flavonoids: an overview development of biodegradable nanoparticles for delivery of quercetin updated knowledge about polyphenols: functions, bioavailability, metabolism, and health antiviral activity of selected flavonoids against chikungunya virus -o-arylmethylgalangin as a novel scaffold for anti-hcv agents flavonoid baicalin inhibits hiv-1 infection at the level of viral entry fisetin and rutin as 3c protease inhibitors of enterovirus a71 structure-activity relationship of flavonoids as influenza virus neuraminidase inhibitors and their in vitro anti-viral activities epigallocatechin-3-gallate inhibition of epstein-barr virus spontaneous lytic infection involves erk1/2 and pi3-k/akt signaling in ebv-positive cells computational screen and experimental validation of anti-influenza effects of quercetin and chlorogenic acid from traditional chinese medicine molecular docking of potential inhibitors for influenza h7n9 quercetin: bioflavonoids as part of interferon-free hepatitis c therapy? apigenin inhibits enterovirus 71 replication through suppressing viral ires activity and modulating cellular jnk pathway antiherpetic activities of flavonoids against herpes simplex virus type 1 (hsv-1) and type 2 (hsv-2) in vitro phenolic acids act as signaling molecules in plant-microbe symbioses identification and evaluation of antihepatitis c virus phytochemicals from eclipta alba computational docking study of p7 ion channel from hcv genotype 3 and genotype 4 and its interaction with natural compounds a flavonoid, luteolin, cripples hiv-1 by abrogation of tat function the effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer evaluation of the antiviral activity of kaempferol and its glycosides against human cytomegalovirus metabolomics view on gut microbiome modulation by polyphenol-rich foods baicalin, a metabolite of baicalein with antiviral activity against dengue virus combined effects of flavonoids and acyclovir against herpesviruses in cell cultures absorption, excretion and metabolite profiling of methyl-, glucuronyl-, glucosyland sulpho-conjugates of quercetin in human plasma and urine after ingestion of onions anti-chikungunya activity ofl uteolin and apigenin rich fraction from cynodon dactylon neuroprotective effects of chrysin: from chemistry to medicine differential inhibition of hiv-reverse transcriptase and various dna and rna polymerases by somecatechin derivatives differential inhibitory effects of some catechin derivatives on the activities of human immunodeficiency virus reverse transcriptase and cellular deoxyribonucleic and ribonucleic acid polymerases inhibition of the infectivity of influenza virus by tea polyphenols antiviral activity of baicalin against influenza virus h1n1-pdm09 is due to modulation of ns1-mediated cellular innate immune responses bioavailability is improved by enzymatic modification of the citrus flavonoid hesperidin in humans: a randomized, double-blind, crossover trial silico study on anti-chikungunya virus activity of hesperetin anti-sindbis activity of flavanones hesperetin and naringenin anti-hiv-1 activity of flavonoid myricetin on hiv-1 infection in a dual-chamber in vitro model plant derived compounds having activity against p388 and l1210 leukemia cells inhibitors of alphavirus entry and replication identified with a stable chikungunya replicon cell line and virus-based assays genistein inhibits the replication of avian leucosis virus subgroup j in df-1 cells apigenin restricts fmdv infection and inhibits viral ires driven translational activity anti-inflammatory effect of quercetin-loaded microemulsion in the airways allergic inflammatory model in mice drugs of natural origin genistein as antiviral drug against hiv ion channel absorption and metabolism of polyphenols in the gut and impact on health kaempferol derivatives as antiviral drugs against the 3a channel protein of coronavirus comparison of the antiviral activity of flavonoids antiviral activity of flavonoids against murine norovirus and feline calicivirus computational approach towards exploring potential anti-chikungunya activity of selectedflavonoids the flavonoid apigenin inhibits hepatitis c virus replication by decreasing mature microrna122 levels differential antiviral and anti-inflammatory mechanisms of the flavonoids biochanin a and baicaleinin h5n1 influenza a virus-infected cells antiviral activity of chrysin derivatives against coxsackievirus b3 in vitro and in vivo quercetin 7-rhamnoside reduces porcine epidemic diarrhea virus replication via independent pathway of viral induced reactive oxygen species silymarin efficacy against influenza a virus replication antiviral effect of catechins in green tea on influenza virus biological evaluation of anti-influenza viral activity of semi-synthetic catechin derivatives tea catechins as a potential alternative anti-infectious agent in vitro anti-hiv and -hsv activity and safety of sodium rutin sulfate as a microbicide candidate simplified catechin-gallate inhibitors of hiv-1 reverse transcriptase synergistic action of quercetin and murine alpha/beta interferon in the treatment of mengo virus infection in mice genistein treatment of cells inhibits arenavirus infection in vitro antiviral activity of plant extracts from asteraceae medicinal plants multiple effects of silymarin on the hepatitis c virus lifecycle saururus chinensis (lour.) baill blocks enterovirus 71 infection by hijacking mek1-erk signaling pathway a phospholipid complex to improve the oral bioavailability of flavonoids antienterovirus 71 effects of chrysin and its phosphate ester the green tea catechin, epigallocatechin gallate inhibits chikungunya virus infection colonic metabolites of berry polyphenols: the missing link to biological activity? epigallocatechin gallate, the main polyphenol in green tea, binds to the t-cell receptor, cd4: potential for hiv-1 therapy kinetic and structural analysis of mutant cd4 receptors that are defective in hiv gp120binding quercetin as an antiviral agent inhibits influenza a virus (iav) entry. viruses biological activities of polyphenols from grapes determine the structure of phosphorylated modification of icariin and its antiviral activity against duck hepatitis virus a inhibitory effects of baicalein on the influenza virus in vivo is determined by baicalin in the serum green tea extract and its major component epigallocatechin gallate inhibits hepatitis b virus in vitro tangeretin from citrus reticulate inhibits respiratory syncytial virus replication and associated inflammation in vivo identification of luteolin as enterovirus 71 and coxsackievirus a16 inhibitors through reporter viruses and cell viability-based screening inhibitory effects of (-)-epigallocatechin gallate on the life cycle of human immunodeficiency virus type 1 (hiv-1) activity of compounds from taxillus sutchuenensis as inhibitors of hcv ns3 serine protease potent antiviral flavone glycosides from ficus benjamina leaves small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells identification of myricetin and scutellarein as novel chemical inhibitors of the sars coronavirus helicase, nsp13 extract of scutellaria baicalensis inhibits dengue virus replication antiviral activity of four types of bioflavonoid against dengue virus type-2 novel antiviral activity of baicalein against dengue virus preparation and in vitro evaluation of apigenin-loaded polymeric micelles anti-japanese-encephalitis-viral effects of kaempferol and daidzin and their rna-binding characteristics apigenin inhibits enterovirus-71 infection by disrupting viral rna association with trans-acting factors characterization and evaluation of self-microemulsifying sustained-release pellet formulation of puerarin for oral delivery epigallocatechin-3-gallate opposes hbv-induced incomplete autophagy by enhancing lysosomal acidification, which is unfavorable for hbv replication light-controlled flavonoid biosynthesis in fruits acknowledgements the authors acknowledge all reviewers whose detailed comments improved the review and apologize to those authors whose contribution to flavonoids antiviral research may have been inadvertently missed. ethical standards this study is supported by the ra mes state committee of science, in the frames of the research projects 15rf-081 and 16yr-1f064. all authors declare that they have no conflict of interest. this article does not contain any studies with human participants or animals performed by any of the authors. key: cord-303468-95btvr1v authors: verran, joanna; jackson, sarah; scimone, antony; kelly, peter; redfern, james title: biofilm control strategies: engaging with the public date: 2020-07-30 journal: antibiotics (basel) doi: 10.3390/antibiotics9080465 sha: doc_id: 303468 cord_uid: 95btvr1v there are few peer-reviewed publications about public engagement with science that are written by microbiologists; those that exist tend to be a narrative of an event rather than a hypothesis-driven investigation. however, it is relatively easy for experienced scientists to use a scientific method in their approach to public engagement. this short communication describes three public engagement activities hosted by the authors, focused on biofilm control: hand hygiene, plaque control and an externally applied antimicrobial coating. in each case, audience engagement was assessed using quantitative and/or qualitative methods. a critical evaluation of the findings enabled the construction of a public engagement ‘tick list’ for future events that would enable a hypothesis-driven approach with more effective communication activities and more robust evaluation. it is increasingly being recognised by 'experts' that science literacy is of key importance for the public [1] . at a time where antimicrobial resistance (amr) continues to pose significant public health threats (or indeed, at a time of a global pandemic), an understanding of statistics, epidemiology and microbiology is even more desirable. as a subject, microbiology offers many topics with which we can engage non-experts, such as microbial diversity (including fungi, algae, protozoa and viruses as well as bacteria), beneficial microbes (for example, probiotics, fermented foods, the human microbiome), and messages that can influence behaviour in a positive manner (including vaccination, hand hygiene, antimicrobial stewardship) [2] [3] [4] . biofilms (an assemblage of microbial cells that are irreversibly associated with a surface-not removed by gentle rinsing-and enclosed in a matrix of primarily polysaccharide material [5] ) are of great importance to microbiologists, but also to many other professionals (such as engineers, biocide manufacturers, architects), and are found in a variety of environments (water distribution systems, industrial processing, hospitals). biofilm research is multi-disciplinary, extensive and significant, with many applications. there are several research centres which focus on biofilm, such as the us-based centre for biofilm engineering (http://www.biofilm.montana.edu/) and the uk-centred national biofilm innovation centre (https://www.biofilms.ac.uk/), and conferences about biofilm are regular and not uncommon. some individual researchers, research groups and research centres are keen to engage with external public audiences through outreach activities, although evidence of such activities (websites, articles, learning materials and other peer-reviewed outputs) is not easy to find. but why do we want the public to know about biofilms? and what does the 'public' need to know about biofilms? 'now wash your hands' was developed as part of a university faculty family fun day during national science and engineering week/healthcare science week in the uk. the aim was to raise awareness of effective handwashing, whilst also engaging the participants in a discussion about the skin microbiome/biofilm. this event guarantees an audience of predominantly families who are likely to have an existing interest in science. hand hygiene activities are well established as interactive learning activities with demonstrable public health impact (for example, as an intervention in reducing the spread of coronavirus [6] ). in this activity, demonstrators (academic staff and student volunteers) engaged audiences to demonstrate surface contamination and effective handwashing ( figure 1 ). thus, visitors at this activity (in a walkway area) had their hands 'contaminated' with a uv hand gel (www.hand-washing.com). this kit uses a fluorescent dye and ultraviolet light to illustrate the transmission of 'germs' from hands to other surfaces (and vice versa) and the importance of handwashing. in addition, the participants were invited to press their hands onto large agar plates for subsequent incubation to reveal the culturable microorganisms present on their skin. of course, they were unable to see the results of this work until after incubation, thus images of plates pre-inoculated with microorganisms present on hands and mobile phones [7] were available to view, and post-incubation images of their own plates were uploaded to flickr, a social media site that hosts images (http://tinyurl.com/howcleanareyourhands, figure 2 ). within a week from results going online, almost 100 downloads were recorded (the participants were provided with a card/web address), equivalent to the number of plates inoculated. from this, we deduced that visitors demonstrated interest and engagement with the activity. throughout the activity, conversations were ongoing. it was unfortunate that these interactions were not noted in some form: informal observations revealed points of interest from the participants such as their inability to clean hands effectively (especially the adults!) and amazement at the mobile phone contamination. the handprint technique has been used as an engagement tool for other events, such as an art installation called 'hands across the cultures' for registrants to a qualitative research conference and as part of the 'bioselfies' project (https://blogs.bl.uk/science/2020/02/introducing-bio-selfies-11-february-2020.html) initiated by the university of salford. flickr has been used for other events that require incubation of plates [8, 9] , and download numbers have on occasion exceeded the number of images posted, showing that the participants may have been sharing the findings with others. the fluorescent hand technique was used to illustrate person-to-person transmission by handshaking prior to a screening of the movie contagion (directed by soderbergh, 2011). one person 'contaminated' his/her hands, shook the hand of their neighbour, who shook her/his neighbour's hand and so on. thus, the passing-on of fluorescence was used to illustrate the transmission of infection through poor hand hygiene, reinforcing the message as to how the movie pandemic was initiated (hand contact). hand hygiene activities are common in microbiology engagement, the aim of the activity being primarily to inform, and hopefully to change, participants' behaviour so that effective handwashing hand hygiene activities are common in microbiology engagement, the aim of the activity being primarily to inform, and hopefully to change, participants' behaviour so that effective handwashing hand hygiene activities are common in microbiology engagement, the aim of the activity being primarily to inform, and hopefully to change, participants' behaviour so that effective handwashing techniques are employed. explanation regarding the presence or importance of the antibiotics 2020, 9, 465 4 of 12 skin microbiome/biofilm are likely rare (especially if the results are not available until a later date): the activity is inevitably more focused on the removal of temporary contaminants and on the importance of good handwashing. some discussion could take place regarding the hygiene-versus-cleanliness hypothesis [10, 11] . the flickr method used for posting images and monitoring downloads at least gives an indication of interest, but much more could be made of this activity. it would also be interesting to know if the 'good handwashing' messages are retained and employed in the future. however, longitudinal studies are rare in this type of public engagement, probably because of the significant advanced planning required in terms of gaining approval for personal data access (e.g., emails) and also because only short-term awareness raising tends to be the primary aim of the activity. the plaque biofilm is one of the best-known medical biofilms [12, 13] , and oral hygiene advertising frequently provides cartoons of plaque being removed to demonstrate the effectiveness of a paste, mouthwash or brush. it is known that good toothbrushing helps to remove plaque [14] and should be carried out regularly. different dentifrices claim varying activities, but virtually all formulations include fluoride (to 'strengthen the teeth') [15] , and many contain antimicrobial agents (to reduce the number of microorganisms, with claims around gum health) [16] . 'plaque attack!' was a laboratory-based activity designed for children and their parents, taking place during manchester science festival's family fun day at manchester metropolitan university. the aim of the event was to encourage good oral hygiene but also to captivate visitors with the components of the plaque biofilm as well as the laboratory and its equipment. being time-consuming and space-limited, the participants had to register for the event, were limited to 3 groups of 20 participants, be escorted to the laboratory, provided with appropriate clothing and instruction and supervised at all times. oral microbiology is a key research area in our laboratories, and the delivery team thought it would be valuable for visitors to encounter activity in a working (teaching) laboratory. the delivery team comprised phd students, technical staff and an academic. several activities were conducted as part of a 'round-robin' activity: sampling plaque (microscopy demonstration and take-home photo [zip mobile printer, polaroid]); disclosing plaque (using commercially available disclosing tablets), with photographs taken before and after cleaning teeth (in a wash area adjacent to the laboratory); looking at cultures of oral bacteria on agar plates; investigating biofilm structure/building a biofilm (using 'model magic' [crayola bedford uk], a white air-drying modelling clay) ( figure 3a ); and destroying a biofilm (using a water pistol to remove plaque (whose microorganisms were pre-constructed from fimo, a multi-coloured clay which can be hardened in the oven [www.staedtler. com]) hampered by plaque matrix (a translucent hair gel) [17] (figure 3b ). the participants were provided with a basic information sheet on plaque and oral hygiene, onto which they could attach their polaroid images. they were also given a bag containing complimentary toothbrush and toothpaste (courtesy of unilever [www.unilever.co.uk]). at the end of the activity, they were asked for free text feedback on what they thought of the event, and the information was coded into categories to allow for comparison [18, 19] (figure 4) . the participants were particularly engrossed in the microscopy demonstration, being able to see their own plaque at high magnification. they also clearly had fun 'destroying' the biofilm but were less interested in the more passive/less exciting activity (agar plates demonstration, building a biofilm). the free text provided by the participants (allowing more thorough insight compared to multiple-choice or leading questions such as 'give three things you have learned', or 'smiley face/sad face' evaluations [18, 20] ) gave valuable qualitative information that was used to inform subsequent activities. (a/top) participants at the 'plaque attack!' event were encouraged to create their own oral bacteria flora from modelling clay, which was assembled into the oral biofilm representation here shown. (b/bottom) participants were encouraged to 'destroy a biofilm' by removing bacteria (coloured plastic pieces) encased in biofilm extracellular matrix (hair gel) with a spray bottle filled with water. there was a total of 19 comments that were coded based on their focus-with each comment possibly being coded into more than one category. our research into titanium dioxide coatings included a range of laboratory-based studies that compared different titanium dioxide concentrations in paint formulations [21] . the work described . themes identified from 'plaque attack!' feedback. there was a total of 19 comments that were coded based on their focus-with each comment possibly being coded into more than one category. our research into titanium dioxide coatings included a range of laboratory-based studies that compared different titanium dioxide concentrations in paint formulations [21] . the work described in this paper was to see whether the effect of a photocatalyst in paint could be detected by the human eye. thus, as part of a phd project investigating the activity of photocatalytic surfaces, one of the external walls of the university was used to illustrate the effectiveness of titanium dioxide paints in terms of self-cleaning and reduction of the formation of biofilm on the wall material. photocatalytic material such as titanium dioxide can exhibit self-cleaning, anti-fouling and antimicrobial properties in the presence of light, which makes these materials excellent candidates for incorporation into urban buildings and infrastructure [22] [23] [24] . the self-cleaning properties stem from their superhydrophilic nature-as, for instance, that of a liquid (e.g., rain) rolling off the surface of a continuous body. this sheeting carries away dirt and debris, cleaning the surface in the process-as seen in the sydney opera house [25] . thus, biofilm formation on the surface is delayed or prevented. in our study, the wall, comprising concrete panels (smaller panels 190 cm × 76 cm, larger panels 406 cm × 76 cm) on a 1970s university building, was west-facing (location on chester street, manchester, uk m1 5gd). six of the panels were painted with a siloxane external paint formulation that contained or lacked the photoactive pigment (kindly provided by tronox, www.tronox.com). our aim was to inform the passing public about our research (an interpretation panel was affixed to the wall), and on occasion, we encouraged passers-by to participate in a longitudinal subjective assessment of the impact of titanium dioxide-containing paint on the perceived cleanliness of the panel. this engagement activity was done directly by interview and indirectly using photographs at specific times over a 44-month period. initially there was no apparent difference in the brightness of the painted panels (figure 5a ). members of the public attending a manchester science festival event (october 2014) were asked to rank the painted panels in order of cleanliness/whiteness, with 1 being most clean, and 6 being least clean (n = 18). the experiment was also conducted via a social media platform (facebook), with participants asked to assess whiteness using photographs (n = 48). the direct assessment was repeated after three years (n = 21). in all cases, the participants ranked two or three of the photocatalytic panels as the 'whitest'. in 2014, around 60% of the participants selected the three photocatalytic panels correctly. in 2017, this figure rose to 78%. after six years, the test-paint panels appeared whiter than the control panels ( figure 5b, may 2020) . the presence of the wall with its accompanying information panel at the side of the university science and engineering building provided a useful pointer to introduce visitors to some of the research ongoing in the faculty. the use of the public to assess the cleanliness of the wall proved unnecessary within a few months, when the impact of the test paint was apparent. the fact that almost all participants could discriminate between the panels after less than 12 months was also of interest. this approach might therefore be useful in the future for the assessment of test formulations. figure 5 . images of the wall at manchester metropolitan university used in the study of photocatalytic paint (panels labelled 1-6). panels 1, 3 and 6 were painted with photocatalytic paint, whilst panels 2, 4 and 5 were painted with paint that did not contain the photocatalytic agent. the image on the top (a) was taken in 2014, eight months following the application of the paint: whiteness/brightness difference between the two paint types is hard to distinguish. the lower image (b) was taken six years later (2020); panels painted with photocatalytic paint are visibly brighter compared to control paint panels. antibiotics 2020, 9, 465 9 of 12 much was learned from each event (as noted above), particularly through observation, in terms of what components participants like and engage with when discussing biofilm. in addition, quantitative evidence of engagement was derived from the 'now wash your hands' event; qualitative evidence of enjoyment and engagement was obtained from 'plaque attack', and the potential for acquisition of research data was indicated by the photocatalytic wall activity. these various outcomes informed how subsequent events for the public would take place, with more focus on design, delivery and evaluation. more recently, there has been increasing effort to ensure that these criteria for effective public engagement are met. microbiology has a particularly dynamic approach to public engagement, and many teams are now publishing the outcomes of their public engagement research in peer-reviewed journals, magazines or online. yet, in a review of public engagement activity around amr, a rich bedrock of activity was found only through personal contacts and communication rather than through a literature search [4] . it is even more important when talking to audiences about biofilms that intended messages are clear. thus, we describe in table 1 the planning of a hypothetical public engagement event designed to inform a large number of adults about biofilm and amr. our focus was on the combination of the two phenomena, which occurs, for example, when biofilms on medical devices present increased resistance to antibiotics [26] . in order to address this combined effect, it was first necessary to define the two phenomena separately. we particularly wished to avoid intrusive aspects of evaluation, relying instead on observation and other (subjective and objective) indicators from participants. we hope that this checklist may be useful for others who might wish to engage audiences with their biofilm/antibiotic research. the national biofilm information centre has recognised the importance of public engagement and is providing a hub for the dissemination of biofilm-focused outreach and engagement activities, which will enable, over time, ideas, expertise and outcomes to be shared and developed, in order to improve the effectiveness of engagement encounters for scientists and their audiences alike. we hope that our experiences in the area are of interest in this context. public engagement activities can be designed with clear aims that enable effective evaluation using both quantitative and qualitative methods. this is particularly important for complex phenomena such as biofilms and amr. the urgent need for microbiology literacy in society simfection: a digital resource for vaccination education practical microbiology in schools: a survey of uk teachers raising awareness of antimicrobial resistance among the general public in the uk: the role of public engagement activities. jac-antimicrob role of hand hygiene in healthcare-associated infection prevention the microbial contamination of mobile communication devices fitting the message to the location: engaging adults with antimicrobial resistance in a world war 2 air raid shelter spreading the message of antimicrobial resistance: a detailed account of a successful public engagement event rsph and ifh call for a clean-up of public understanding and attitudes to hygiene 99th dahlem conference on infection, inflammation and chronic inflammatory disorders: darwinian medicine and the 'hygiene' or 'old friends' hypothesis composition of in vitro denture plaque biofilms and susceptibility to antifungals dental biofilm: ecological interactions in health and disease power toothbrushes: a critical review comparison of the effect of fluoride and non-fluoride toothpaste on tooth wear in vitro and the influence of enamel fluoride concentration and hardness of enamel antimicrobial efficacy of different toothpastes and mouthrinses: an in vitro study blast a biofilm: a hands-on activity for school children and members of the public transforming a school learning exercise into a public engagement event: "the good, the bad and the algae refreshing the public appetite for 'good bacteria': menus made by microbes research methods in education photoinactivation of escherichia coli on acrylic paint formulations using fluorescent light. dyes pigment photoinduced reactivity of titanium dioxide photocatalytic construction and building materials: from fundamentals to applications effect of process parameters on the photocatalytic soot degradation on self-cleaning cementitious materials the authors declare no conflict of interest antibiotics 2020, 9, 465 antibiotics 2020, 9, key: cord-285856-0sw3wt1i authors: naesens, lieve; vanderlinden, evelien; rőth, erzsébet; jekő, józsef; andrei, graciela; snoeck, robert; pannecouque, christophe; illyés, eszter; batta, gyula; herczegh, pál; sztaricskai, ferenc title: anti-influenza virus activity and structure–activity relationship of aglycoristocetin derivatives with cyclobutenedione carrying hydrophobic chains date: 2009-02-05 journal: antiviral res doi: 10.1016/j.antiviral.2009.01.003 sha: doc_id: 285856 cord_uid: 0sw3wt1i previous studies have demonstrated that glycopeptide compounds carrying hydrophobic substituents can have favorable pharmacological (i.e. antibacterial and antiviral) properties. we here report on the in vitro anti-influenza virus activity of aglycoristocetin derivatives containing hydrophobic side chain-substituted cyclobutenedione. the lead compound 8e displayed an antivirally effective concentration of 0.4 μm, which was consistent amongst influenza a/h1n1, a/h3n2 and b viruses, and a selectivity index ≥50. structural analogues derived from aglycovancomycin were found to be inactive. the hydrophobic side chain was shown to be an important determinant of activity. the narrow structure–activity relationship and broad activity against several human influenza viruses suggest a highly conserved interaction site, which is presumably related to the influenza virus entry process. compound 8e proved to be inactive against several unrelated rna and dna viruses, except for varicella-zoster virus, against which a favorable activity was noted. previous studies have demonstrated that glycopeptide compounds carrying hydrophobic substituents can have favorable pharmacological (i.e. antibacterial and antiviral) properties. we here report on the in vitro anti-influenza virus activity of aglycoristocetin derivatives containing hydrophobic side chainsubstituted cyclobutenedione. the lead compound 8e displayed an antivirally effective concentration of 0.4 m, which was consistent amongst influenza a/h1n1, a/h3n2 and b viruses, and a selectivity index ≥50. structural analogues derived from aglycovancomycin were found to be inactive. the hydrophobic side chain was shown to be an important determinant of activity. the narrow structure-activity relationship and broad activity against several human influenza viruses suggest a highly conserved interaction site, which is presumably related to the influenza virus entry process. compound 8e proved to be inactive against several unrelated rna and dna viruses, except for varicella-zoster virus, against which a favorable activity was noted. © 2009 elsevier b.v. all rights reserved. currently available drugs for the treatment of influenza virus infections comprise the m2 ion channel blockers amantadine and rimantadine, and the neuraminidase inhibitors oseltamivir and zanamivir (de clercq, 2006; moscona, 2008) . stockpiling of oseltamivir and, to a lesser extent, zanamivir has been advocated in the context of pandemic preparedness (schünemann et al., 2007) , yet the recent isolation of oseltamivir-resistant seasonal influenza virus mutants, even from untreated patients, warrants for continued caution (lackenby et al., 2008; van der vries et al., 2008) . additional anti-influenza virus compounds should be urgently developed, having a novel antiviral target that is highly conserved amongst influenza virus (sub)types and, hence, less prone to genetic variation and resistance selection. one of the attractive therapeutic strategies would be a blockade of the viral entry into the host cell. the cellular entry process of influenza viruses has been unraveled since many years (reviewed in skehel and wiley, 2000) . a key role is being played by the viral envelope glycoprotein hemagglutinin (ha), which contains the receptor-binding site for initial attachment to the sialylated cellular receptors, and governs the receptor specificity of human versus avian influenza virus subtypes (chandrasekaran et al., 2008; nicholls et al., 2008) . in addition, after cellular uptake of the virus by endocytosis, the ha mediates the low ph-induced fusion of the viral envelope with the endosomal membrane, leading to release of the viral ribonucleoprotein in the cytosol. although the ha has been extensively studied from a biochemical and epidemiological perspective, specific antiviral drugs blocking the ha-receptor interaction remain to be clinically developed. several reports are available on the in vitro activity of small-molecule inhibitors of influenza virus fusion, which act by preventing the conformational change of the ha at low ph deshpande et al., 2001; plotch et al., 1999) . unfortunately, their development has been slow due to their inferior activity against some human influenza virus (sub)types, rapid selection for resistance and/or unsatisfactory outcome in animal models (yagi et al., 1999) . glycopeptide compounds represent a large series of natural, semisynthetic or fully synthetic compounds, which are widely recognized for their potent activity against gram-positive bacteria (nicolaou et al., 1999) . several studies have demonstrated that hydrophobic derivatives of glycopeptide antibiotics (e.g. vancomycin and eremomycin), and/or their aglycones exert antibacterial activity against glycopeptide-resistant enterococci. (cooper et al., 1996; printsevskaya et al., 2002; pace and yang, 2006) . remarkably, some of these lipophilic glycopeptide compounds were found to have inhibitory activity against coronaviruses and hiv, the latter being ascribed to inhibition of the hiv entry process (balzarini et al., 2003 (balzarini et al., , 2006 preobrazhenskaya and olsufyeva, 2006) . we here report on the chemical synthesis, anti-influenza virus activity and structure-activity relationship of novel glycopeptide compounds carrying a hydrophobic side chain on an aglycoristocetin backbone ( fig. 1) . high-yield synthesis of aglycovancomycin (1) and aglycoristocetin (2) (fig. 1 ) was performed as originally described by wanner et al. (2003) and consisted of deglycosidation of the parent antibiotics with hydrogen fluoride in anisole at neutral ph ( fig. 2) (sztaricskai et al., 2006) . the aglycones were converted into the squaric acid amide esters (4 and 5) by coupling with dimethyl squarate (3). this was followed by reaction with primary amines (6a-j) to yield the corresponding asymmetric squaric diamides (7a-g, 8a-j), using a regioselective procedure without the requirement for a protecting group strategy (tietze et al., 1991; sztaricskai et al., 2006) . the primary amines were: 6aminohexanol (6a); 6-aminohexanecarboxylic acid (6b); triglycine (6b1); dopamine (6c); n-(4-aminophenyl)piperidine (6d) and 4phenyl-benzylamine (6e). based on the observation that compound 8e displayed favorable anti-influenza virus activity (see below), subsequent modifications were performed to study the impact of an increasing steric bulk in the rigid aromatic side chain of 7f-g and 8f-h, which were prepared with 1-naphthylamine (6f), 4-aminoterphenyl (6g) or 2-aminoanthracene (6h). it has been shown that introduction of hydrophobic substituents into glycopeptide antibiotics enhances their activity against glycopeptide-resistant bacteria (cooper et al., 1996; printsevskaya et al., 2002; pace and yang, 2006) , but, unfortunately, these products have lower water-solubility. to improve solubility, the squaric acid amide ester 5 was reacted (sztaricskai et al., 2007b) with d-glucosamine (6i) or d-galactosamine (6j), resulting in the asymmetric squaric diamides 8i and 8j, respectively. in these products, the carbohydrate moiety is linked to the aglycone in an unusual manner, i.e. through a cyclobutandione moiety, and not directly through one of the hydroxyl groups, as in the parent glycopeptide antibiotics. the structure, reaction yield and physico-chemical data for the aglycones, their squaric acid amide esters and corresponding asymmetric diamides, are summarized in table 1 . the homogeneity of all compounds was checked by tlc and hplc, and the structures were confirmed by mass spectrometry. (2) were first converted into their squaric acid amide esters (4 and 5, respectively), followed by conversion to the asymmetric squaric diamides (7a-g and 8a-j, respectively). see table 1 for the structures of the r1 group, as present in the primary amines 6a-j and the final products 7a-g and 8a-j. (2) into their squaric acid amide esters (4 and 5, respectively), these were converted to the asymmetric squaric diamides (7a-g, derived from aglycovancomycin, and 8a-j, derived from aglycoristocetin). b hplc conditions: instrument: waters 600 with uv230nm detection; column: lichrospher rp-8 (4 mm × 250 mm; 10 m); injection volume: 20 l (corresponding to 2 g compound); solvents: table 2 , several asymmetric squaric diamides derived from aglycoristocetin exerted marked activity against influenza virus, the most potent compounds being the phenylbenzyl derivative 8e [average antiviral ec 50 : 0.4 m; selectivity index (si), defined as the ratio of mcc to ec 50 : 50]; the hexanol deriva-tive 8a (ec 50 : 1 m; si: 14) and the naphthyl derivative 8f (ec 50 : 1.4 m; si: 10). their activity was 2-to 5-fold higher than that of the squaric acid amide ester of aglycoristocetin 5 (ec 50 : 2.4 m; si: 42). an intermediate activity (ec 50 : 5 m) was observed for the triglycyl derivative 8b1 which, surprisingly, was comparably active as unsubstituted aglycoristocetin 2. the 3,4-dihydroxybenzyl derivative 8c and the d-galactosamine derivative 8j were active against two of the three influenza virus strains tested, and the derivatives containing carboxypentyl (8b) and d-glucosamine (8i) substituents had activity against only one virus strain. the compounds carrying 4-aminophenylpiperidine (8d), terphenyl (8g) and anthracene (8h) substituents were completely inactive. thus, the intrinsic antiinfluenza virus activity of aglycoristocetin is markedly increased by squaric acid amide coupling and addition of a hydrophobic side chain, with the phenylbenzyl group being the optimal substituent. the favorable effect of this side chain appears to depend on different factors, namely: neutral charge (the alcoholic aliphatic derivative 8a is clearly more active than the corresponding carboxypentyl compound 8b) and steric bulkiness (8e and 8f are active while the more bulky compounds 8g and 8h are not). the aglycoristocetin backbone structure was shown to be critical for inhibition of influenza virus, since no activity was observed for compound 7e, which represents the aglycovancomycin analogue of 8e. aglycoristocetin and aglycovancomycin both contain a central heptapeptide core and nonproteinogenic phenolic amino acids, i.e. ␤-hydroxytyrosine (c and e units), 4-hydroxyphenylglycine (b and d units) and 3,5-dihydroxyphenylglycine (a unit) (fig. 1) . the main structural differences between both glycopeptide aglycones are as follows (fig. 1) : (i) whereas aglycovancomycin has five aromatic rings (a-e) and two known amino acids (l-aspartic acid and n-methyl-d-leucine), aglycoristocetin has seven aromatic moitable 3 cytotoxicity of selected aglycoristocetin derivatives in human and animal cell lines. a . cytotoxic concentration ≥100 >100 100 >100 a human embryonic lung (hel) fibroblasts; human cervix epithelial (hela); african green monkey kidney (vero); and crandell feline kidney (crfk) cells; nd: not done. b the cytotoxic concentration was defined as the minimum cytotoxic concentration (mcc) or 50% cytotoxic concentration (cc50); cf. legend to table 2. eties (a-g); (ii) the additional f and g rings of aglycoristocetin are interconnected via a diphenylether linkage (constituting the ristomycinic acid moiety); (iii) aglycoristocetin lacks the chloro substituents on the c and e rings, present in aglycovancomycin; (iv) the c-terminal carboxyl function is free in aglycovancomycin, but contains a methyl ester in aglycoristocetin and (v) aglycoristocetin has a primary amine function, whereas aglycovancomycin contains a secondary amine group (crowley et al., 2004) . at present, we cannot speculate on which of these structural components explain the antiviral specificity of the aglycoristocetin derivatives. our basic test panel of influenza viruses contained one chimeric virus (a/x-31; h3n2 subtype), one a/h3n2 virus and one b virus strain (table 2) . when the lead compound 8e was further evaluated for activity against a/h1n1 (strain a/puerto rico/8/34), its antiviral ec 50 value was 0.12 ± 0.04 m, which is in the same range as that for the a/h3n2 and b strains. determination of its cytostatic activity in mdck cells, using a cell counting assay, revealed an ic 50 value of 67 ± 19 m. thus, 8e emerged as a potent inhibitor of influenza virus replication, with broad activity against different (sub)types and favorable selectivity. the glycopeptide compounds were evaluated for activity against other viruses besides influenza virus. none of the following rna viruses was found to be significantly inhibited by any of these glycopeptides, as evaluated by cpe or plaque reduction assay: feline coronavirus [examined in crandell-rees feline kidney cells]; vesicular stomatitis virus, coxsackie b4 virus and respiratory syncytial virus [performed in human epithelial hela cells]; parainfluenza-3 virus, reovirus-1, sindbis virus and punta toro virus [tested in african green monkey vero cells]. in human embryonic lung fibroblast cells infected with various dna viruses, the only glycopeptide displaying some activity was compound 8e, with antiviral ec 50 values of 10 m (herpes simplex virus type 1; wild-type or thymidine kinase-deficient); 6 m (herpes simplex virus type 2); >100 m (cytomegalovirus) and 12 m (vaccinia virus), and a selectivity index of 10-17. of note, 8e was found to be highly active against varicella-zoster virus (vzv; wild-type or thymidine kinasedeficient), with an antiviral ec 50 value of 0.55 m and a selectivity index of 180. the diverse antiviral assays performed in human or animal cell lines permitted to obtain a more detailed insight into the cytotoxicity of the aglycoristocetin derivatives selected from the influenza virus experiments (table 3 ). all compounds were either not or minimally cytotoxic at a concentration of 70-100 m. the cytotoxicity values for the lead compound 8e were in the same range as previously obtained in mdck cells. some glycopeptides in this study were previously evaluated for antibacterial activity (sztaricskai et al., 2006) , with 8e emerging as a highly active compound. our present anti-influenza virus data thus agree with the view that hydrophobic substitution has a positive impact on the pharmacological (i.e. antibacterial and antiviral) activities of glycopeptide compounds (printsevskaya et al., 2002; balzarini et al., 2003 balzarini et al., , 2006 . with regard to the antiviral mode of action, time-of-addition studies suggested that 8e blocks the viral entry process, since optimal anti-influenza virus activity was obtained when the compound was added to mdck cells 30 min prior to or simultaneously with virus infection. a detailed analysis is currently ongoing to determine the effect of 8e on the virus-receptor interaction (i.e. binding of the viral ha to the sialic acid terminus of cell surface glycans), endocytosis or membrane fusion (i.e. fusion of the viral envelope with the endosomal membrane). whatever the precise mode of action, the subtype-independent activity of 8e provides strong support that the interaction site of 8e is highly conserved amongst human influenza virus strains. this is consistent with our observation that influenza virus fully retained its sensitivity to 8e after eleven sequential virus passages in mdck cells in the presence of 8e (at concentrations up to 25 m). within human influenza virus ha sequences, only few residues are fully conserved, in particular in the receptor-binding site and fusion peptide (skehel and wiley, 2000) . the aglycoristocetin compounds described here and represented by the lead compound 8e are not the first glycopeptides reported to have activity against influenza virus. in 1993, naruse et al. reported on the isolation, characterization and anti-influenza virus activity of two kistamicin antibiotics (naruse et al., 1993) . similarly to our compounds, both kistamicins showed strong activity against influenza virus and low activity against herpes simplex virus type 1. the kistamicins were reported to be inactive in hiv syncytium assays (naruse et al., 1993) . the observation that the anti-influenza virus activity of the kistamicins was higher when a lipophilic substituent was present at the terminal amine function, is reminiscent of our findings with the aglycoristocetin derivatives. in conclusion, the broad and robust anti-influenza virus activity of the aglycoristocetin derivatives described in this study creates a new avenue for the development of anti-influenza virus agents with a novel mode of action. the relatively narrow structure-activity relationship points to a highly specific interaction with the antiviral target, which is probably related to interaction of the influenza virus hemagglutinin with its cellular receptor. further studies to unravel the structure-activity relationship and precise mode of action are underway in our laboratories. antiretroviral 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for the formation of drug biopolymer conjugates synthesis of squaric acid ester amides and diamides fatal oseltamivir-resistant influenza virus infection a new and improved method for deglycosidation of glycopeptide antibiotics exemplified with vancomycin, ristocetin, and ramoplanin development of anti-influenza virus drugs. i. improvement of oral absorption and in vivo anti-influenza activity of stachyflin and its derivatives this study was supported by grants from the fonds voor wetenschappelijk onderzoek vlaanderen (fwo no. 9.0188.07), the international consortium for anti-virals (icav) and the hungarian national scientific research foundation (no. otka t 46744). we thank leentje persoons, frieda de meyer and vicky broeckx for dedicated assistance, and dr. sándor kéki (department of applied chemistry, university of debrecen) for recording the mass spectra. we thank dr. t. cihlar (gilead sciences, usa) for the generous gift of oseltamivir carboxylate. key: cord-262950-zv6qerhg authors: reinert, kassidy s.; higbee, thomas s.; nix, lyndsay d. title: creating digital activity schedules to promote independence and engagement date: 2020-06-19 journal: behav anal pract doi: 10.1007/s40617-020-00437-8 sha: doc_id: 262950 cord_uid: zv6qerhg photographic activity schedules have been demonstrated to be effective in helping individuals with autism and other developmental disabilities learn how to complete both simple and complex sequences of activities without prompting from adults. although the majority of research studies demonstrating the effectiveness of activity schedules have used schedule books composed of static printed pictures attached to physical pages, recently researchers have begun to demonstrate the effectiveness of technology-based activity schedules. in the current article, we provide a task analysis for creating both simple and complex digital activity schedules using google slides, a freely available, web-based technology that operates on a variety of digital platforms. we also provide suggestions for how behavior analysts can train parents to use this technology with their children using telehealth procedures. ). these studies demonstrate that activity schedules promote independence, increase learner engagement with appropriate activities, and decrease reliance on prompts. therefore, these tools are ideally suited for situations, such as the current covid-19 crisis, where access to direct, professional-led learning may be reduced and clients may be provided with longer periods of "downtime" than under typical circumstances. activity schedules are an attractive tool for promoting independence not only because of their effectiveness but also because of the broad range of individuals with whom they can be used and the wide array of skills they can be used to promote. they require relatively few prerequisite skills and conditions in order to be effective (e.g., picture-object correspondence, acceptance of physical prompts, identified reinforcers) and can be used to teach sequences of behavior as simple as independent play sequences (macduff et al., 1993) and as complex as group social games such as hide-and-seek (akers et al., 2018) . importantly, researchers have demonstrated that parents can be taught, via technology, to implement activity schedule teaching procedures with high degrees of fidelity (gerencser, higbee, akers, & contreras, 2017) . to date, most activity schedules used in published studies have been composed of three-ring binders with pictures attached to individual pages or strips of pictures on a single page. although inexpensive, binder-or paper-based schedules editor's note this manuscript is being published on a highly expedited basis, as part of a series of emergency publications designed to help practitioners of applied behavior analysis take immediate action to adjust to and mitigate the covid-19 crisis. this article was submitted on april 3, 2020, and received final acceptance on april 6, 2020. the journal would like to especially thank dr. thomas szabo for his expeditious review of the manuscript. the views and strategies suggested by the articles in this series do not represent the positions of the association for behavior analysis international or springer nature. may have some disadvantages, such as being cumbersome to transport (carlile, reeve, reeve, & debar, 2013) and, perhaps more importantly, potentially socially stigmatizing, as same-aged peers would likely not be carrying around books during activities (giles & markham, 2017) . given technological advances that have made portable electronic devices, such as tablet computers, more readily available to teachers and families of individuals with disabilities, there may be advantages to using digital schedules that could be displayed on these devices. first, scanned or photographic images of activities could be easily moved around within activity schedules, reducing the cost and effort of printing and laminating images for inclusion in binder-based or single-page printed schedules. second, digital schedules could easily be shared across multiple devices. for example, copies of the schedule could be updated by a teacher or therapist and then e-mailed to a parent for use at home. finally, tablet computers are now ubiquitous in school, work, and home settings, thus reducing potential stigmatization for digital activity schedule users. multiple researchers have investigated the effectiveness of various forms of digital activity schedules delivered on electronic devices, including computers running microsoft powerpoint (rehfeldt, kinney, root, & stromer, 2004) , ipods to increase on-task behavior (carlile et al., 2013) , video clips to increase dramatic play (dauphin, kinney, & stromer, 2004) , ipads to increase time on task and decrease transition time during centers (gourwitz, 2014) , and ipads to promote completion of leisure activities (giles & markham, 2017) . one published study has also indicated that, for some participants, digital activity schedules may be preferred over binderbased schedules (giles & markham, 2017) . although further research on the use of digital activity schedules is certainly warranted, a sufficient body of research exists to demonstrate the potential benefits of this technology. given the portability of digital activity schedules and the potential for behavior analysts to be able to design digital activity schedules and transmit them electronically to parents or caregivers for use with their children at home, we believe that they are ideally suited to the current therapeutic environment caused by the covid-19 pandemic, which limits direct contact between behavior analysts and the clients and families with whom they work. thus, the purpose of this article is to provide a detailed task analysis that both behavior analysts and parents can use to create and modify digital activity schedules using google slides, a freely available, web-based program that works on a variety of digital devices and platforms. although detailed descriptions of how to create and build independence using activity schedules are available elsewhere (e.g., higbee & brodhead, 2016; higbee & sellers, 2017; mcclannahan & krantz, 1999) , a brief summary of how to select activities for inclusion in a digital activity schedule and standard prompting/teaching techniques is included here. we refer the reader to the cited papers for more detailed descriptions and instructions. specific activities to be included in a beginning activity schedule, including a digital one, should be moderately preferred, close-ended (i.e., have a clear beginning and end), relatively brief, and already independently performed by the learner, as the focus during initial activity schedule training is on how to follow the schedule rather than on the activities themselves (higbee & sellers, 2017) . beginning activity schedules are usually composed of at least two to three activity pages plus a terminal reinforcer page (typically a small amount of a preferred edible) at the end. additional activities can be added as the learner demonstrates mastery of the schedule to form progressively longer response chains. the sequence of activities should be changed frequently to ensure that the learner's behavior comes under the control of the pictures displayed as opposed to becoming a sequence of rote behaviors under proprioceptor control of sensations repeated in a performance chain. activity schedules can be used in multiple environments. however, beginning play/leisure activity schedules often are used at a table or desk that is large enough to both hold the schedule and provide enough room to complete the activities (the floor could also be used in the absence of a desk or table). activity materials are typically placed on a shelf near the desk/table. they could also be placed directly on the table if it is large enough to hold all materials and still have a clear work space to complete activities. alternatively, materials could be placed on the floor near the table. when learners are first beginning to use activity schedules, the idea is to have the materials close at hand to reduce travel time and the potential for distraction. once learners have mastered the basic components of activity schedule following, materials can be placed in their natural locations. learners are taught, through a procedure called "graduated guidance," described in the next section on prompting procedures, to follow the activity schedule. the following sequence of behaviors constitutes "following the schedule": 1. retrieve the schedule (obtain the tablet or other digital device) and place it on the table/floor where activities will be completed. open the schedule file/program (steps 1 and 2 may be completed by a parent or teacher for a digital schedule, depending on the learner's level of familiarity with the technology). 3. touch the picture of the activity (an observing response). 4. obtain the materials necessary for completing the activity (e.g., retrieve the pictured puzzle from a shelf). 5. complete the activity. 6. return the materials back to their original location. 7. "turn the page" on the digital schedule to display the next activity by touching or clicking on the arrow in the bottom-right corner of the screen. 8. repeat steps 3-7 until reaching the terminal reinforcer page and consuming the reinforcer. 9. return the digital device to its original location or close the program. in an effort to reduce dependence on adult-provided verbal prompts and instructions, learners are taught to follow activity schedules exclusively via physical prompts delivered from behind the learner, so as not to block the learner's view of the relevant visual stimuli that should control their behavior (mcclannahan & krantz, 1999) . the only verbal instruction given to the learner should be the initial instruction to begin completing the schedule (e.g., "it's time to do your activity schedule. here you go!"). after that, the person assisting the learner to follow the schedule should not provide any additional verbal instructions, verbal prompts, or conversation until the schedule is completed (unless one of the included activities is a social activity during which verbal interaction would be appropriate). the amount of physical prompting provided will depend on each individual learner's abilities but will often begin with hand-over-hand guidance that should be provided in a way that allows the learner to complete the schedule with as few errors as possible. this physical guidance should be faded as quickly as possible to avoid prompt dependence. a typical fading sequence would begin with hand-over-hand guidance that is progressively faded to physical prompts at the wrist, forearm, elbow, and shoulder, followed by gradually increased physical distance between the prompter and the learner. we have found that light physical prompts from behind at the shoulders are particularly effective for guiding learners back and forth between the schedule and the activity materials. fading can take place dynamically within sessions or can be scripted across sessions. one practical method for prompt fading that has been particularly useful when parents are the primary implementers is that used by gerencser et al. (2017) , where test sessions are periodically run using a 5-s prompt delay procedure to determine the steps a learner can complete independently. prompts that the learner needed to complete the task are recorded on a data sheet, which then serves as a teaching guide for sessions until the next test session is conducted (see the appendix, fig. 28 , for a sample data sheet). behavior analysts who have the capacity to view sessions live through videoconference technology could also provide parents with fading suggestions in real time. when learners make errors, the type of error will determine how the implementer/prompter should respond. if the learner begins to drift off task or begins to engage in stereotypy with schedule materials, the implementer/prompter should simply interrupt this behavior and physically redirect the learner to complete the activity correctly. however, if the learner obtains the incorrect materials after touching the picture representing those materials, he or she should be redirected back to the schedule, physically prompted to touch the picture of the materials again, and physically guided to obtain the correct materials. this helps ensure that learner behavior comes under the control of the schedule. once learners have mastered following basic activity schedules with brief, close-ended activities, more complex activities can be introduced. for example, implementers can add choice pages in which the learner can select from two or more available activities. on a close-ended activity schedule that a learner previously mastered, choice pages could be added to the schedule to replace one or more of the close-ended activities. learners can also be taught to set and respond to digital timers using visual cues in order to include open-ended activities in the digital schedule. red circles representing the number of minutes for the activity and a green circle representing the start button are placed below the picture of the open-ended activity. the minute button on the digital timer is also colored red and the start/stop button colored green. the learner is physically prompted to touch each circle below the picture and the corresponding colored button on the timer to set and start the timer. responding to these more complex activity schedule pages can often be taught via graduated guidance within activity schedule sessions. if a learner requires more practice, modeling and teaching sessions with timers and choice pages could occur outside of activity schedule sessions (higbee & sellers, 2017; mcclannahan & krantz, 1999) . learner performance can be measured by scoring steps from the schedule-following sequence that were performed independently by the learner. a sample data sheet is included in the appendix, fig. 28 . whereas collecting data during each teaching session would be ideal during the initial stages of learning, gerencser et al. (2017) suggest using test sessions that may provide sufficient data to make decisions about prompt levels. an example of the data sheet that is similar to the one used by gerencser et al. is provided in the appendix, fig. 29 . also, when parents are implementing activity schedules, behavior analysts could score performance viewed live through videoconferencing technology or via recorded sessions sent electronically by parents. the following task analysis uses the web-based program google slides to create digital activity schedules. google slides is a freeware program that works on a variety of digital devices and operating systems. more information about google slides can be found at https://www.google.com/ slides/about/. a flowchart of essential steps in the task analysis is provided in the appendix, fig. 30 , to guide the reader and assist with troubleshooting. 1. open a web browser and sign in to your google account. 2. navigate to https://docs.google.com/presentation. 3. create a new blank presentation by clicking the multicolored plus sign in the navigation bar near the top of the page. 4. click "untitled presentation" in the top-left corner and type "activity schedule template." activity schedule setup and cover (figure 1) 1. click "file" and navigate to "page setup" near the bottom of the list. 2. resize the page by clicking "widescreen 16:9." select "custom" and change the dimensions to 5.5 × 8.5 in. click "apply." 3. click "background" on the toolbar and select a desired color for the schedule background. select "add to theme" and then click "done." 4. select the subtitle text box and press the "delete" key. 5. click on the title text box. blue squares should appear on the text box, as shown in fig. 2 . resize the title text box by dragging down the bottom-center square to make a large rectangle centered on the page. a. google slides provides red guide lines (fig. 2 ) that aid with centering shapes, objects, and so on. these guide lines pop up when an item is centered horizontally and/ or vertically within a slide or with adjacent items. 6. select the title text box and change the background to white by clicking the fill color icon (fig. 3) . 7. select "click to add title" and type "student's name." student‛s name next next 8. while the title text box is selected, click on the align icon (fig. 4) . center the text horizontally by clicking the center icon and vertically by clicking the middle icon. 9. select the shape tool and select the right arrow icon, as displayed in fig. 5 . draw in the bottom-right corner of the page. 10. double-click on the arrow and type "next" in the center of the arrow. the activity schedule cover should look like fig. 1 . creating close-ended activity pages ( figure 6 ) and the terminal reinforcer page (figure 7) 1. create a new blank page by clicking on the last page in the left column and pressing the "enter" key. 2. delete all text boxes. 3. insert a large white square in the center of the page. 4. insert a picture of a close-ended activity using the insert image icon (see fig. 8 ) to import desired pictures of activity schedule toys. 5. center and resize the picture to fit in the white square area. 6. repeat steps 1-5 to create multiple close-ended activity pages and one terminal reinforcer page. the close-ended pages of the activity schedule should look like fig. 6 , and the terminal reinforcer page should look like fig. 7 . 7. after creating all pages in a schedule, move the terminal reinforcer page to the last page in the schedule. creating simple navigation between slides 1. navigate to the cover page. 2. select the shape tool ( fig. 9) and draw a large rectangle over the entire page. 3. right-click on the rectangle. navigate to "order," then "send backward." now only the next arrow should be visible on the page. 4. select the next arrow and click the insert link icon (fig. 10) in the toolbar. a white pop-up bar will appear on the screen. click on "slides in this presentation." select "next slide" and click "apply." this will now make it so clicking or pressing the next arrow during a presentation transitions to the next slide. a. please note that the pop-up drop-down menu may run below the bottom of the browser page and may not be fully visible. in order to view all link options, resize the object or move to a higher location on the page. perform the link operation and then return to the previous size and location. 5. select the rectangle and navigate to the insert link icon (fig. 10) in the toolbar. a white pop-up bar will appear on the screen. click on "slides in this presentation," select the current slide number, and click "apply." this will disable all clicks or presses anywhere on the page other than a click on the arrow. 6. click on the large rectangle and then on the color fill and border color icons (fig. 11 ). change the color fill and border color to transparent. 7. copy the large rectangle from the cover page and paste on additional close-ended and terminal reinforcer pages. 8. then copy the "next" arrow from the cover page and paste it on additional close-ended and terminal reinforcer pages. 9. completed close-ended slides can also be replicated using copy and paste. links will continue to work. to edit the copied slides, select the large transparent rectangle and move it out of the way in order to edit or replace pictures. after editing, return the large transparent rectangle to its original position. 10. go to the present icon (fig. 12) , located in the top-right corner of the toolbar, to view and navigate through the slides. a. navigate through the presentation by clicking on the "next" arrows. they should advance you to the next slide in the sequence. i. if an arrow does not advance correctly, check the links by clicking on the arrow. when clicked on, each arrow should display a white link bar below with the words "next slide." if the slide linked to the arrow is incorrect, click "remove link" and return to step 4 in the section "creating simple navigation between slides." ii. if there is no link, return to step 4 in "creating simple navigation between slides." b. after all arrow transitions are working correctly, restart the slideshow. check that no other transitions occur when clicking elsewhere on the screen (anywhere other than the arrows). i. if transitions do occur, return to step 5 in "creating simple navigation between slides." creating choice pages (figure 13) 1. create a new blank page by clicking on the previous page in the left column and pressing the "enter" key. creating navigation for choice pages 1. in the left navigation bar, navigate to the last choice page with a large picture in the lower rectangle (fig. 18 ) and press the "enter" key to create a blank page. title this new slide "blank." 2. navigate to the first choice slide with an empty lower rectangle (fig. 13 ). there should not be a "next" arrow on this page. 3. draw a large rectangle over the entire page using the shape tool (fig. 9 ). 4. right-click on the rectangle. navigate to "order" and then select "send to back." now only the four toy pictures should be visible on the page. 5. click on the large rectangle and navigate to the insert link icon (fig. 10) in the toolbar. a white pop-up bar will appear on the screen. click on "slides in this presentation" and select the current slide number. click "apply." 6. click on the large rectangle and then on the color fill and border color icons (fig. 11) . change the color fill and border color to transparent. 7. right-click on the rectangle and copy it. 8. navigate to the remaining choice pages and paste the transparent rectangle on all four pages. a. to ensure correct navigation, click on the transparent rectangle on each subsequent choice page. the rectangle on each slide should display a white link bar on the lower left corner with the current slide number. if the slide linked to the rectangle is incorrect, click "remove link." return to step 5 in the section "creating navigation for choice pages." 9. navigate to the first choice slide with an empty lower rectangle (fig. 13) . 10. click on one toy picture and click on the insert link icon (fig. 10) in the toolbar. a white pop-up bar will appear on the screen. click on "slides in this presentation" and select the corresponding slide number that has the identical toy picture enlarged in the lower half of the page. click "apply." 11. repeat step 10 for the remaining three toy pictures. a. to ensure correct navigation, click on each picture in the first choice slide. when clicked on, each picture should display a white link bar below with the corresponding slide number (see the flowchart in the appendix, fig. 30 ). if the slide linked to the small picture is incorrect, click "remove link." return to step 10 in the section "creating navigation for choice pages." 12. navigate to the "student's name" page (on the cover of the presentation). 13. right-click on the "next" arrow and copy it. 14. in the left navigation bar, select the second choice page (the first slide with a large toy picture in the lower rectangle; fig. 18 ) and paste the arrow. 15. select the "next" arrow and click the insert link icon (fig. 10) in the toolbar. a white pop-up bar will appear on the screen. remove the previous link by clicking on the "x." click on "slides in this presentation." select the "blank" slide and click "apply." this will now make it so clicking or pressing the "next" arrow during a presentation transitions to the next slide. 16. right-click on the "next" arrow and copy it. 17. paste a copy of the arrow on the remaining three choice pages. each of these pasted arrow's navigation should automatically be linked to transition to the blank slide. a. to ensure correct navigation, click on the "next" arrow on each subsequent choice page. when clicked on, each next arrow should display a white link bar below with the blank slide number. if the slide linked to the arrow is incorrect, click "remove link." return to step 15 in the section "creating navigation for choice pages." 18. go to the present icon (fig. 12) , located in the top-right corner of the toolbar, to view and navigate through the choice slides. a. if there are errors with transitions from the initial choice slide to subsequent choice pages, refer to step 11 in the section "creating navigation for choice pages." b. if there are errors with the transition arrows on the subsequent choice pages, refer to step 17 in the section "creating navigation for choice pages." c. after all arrow and picture transitions are working correctly, restart the slideshow. check that no other transitions occur when clicking elsewhere on the screen (anywhere other than the arrows and the first choice-page pictures). if transitions occur, return to step 8 in "creating navigation for choice pages." three-min timed activity pages (figure 19) a. click "file" and navigate to "page setup" near the bottom of the list. b. resize the page by clicking "widescreen 16:9." select "custom" and change the dimensions to 5.5 × 8.5 in. click "apply." c. navigate to the "activity schedule template" slideshow. 17. click the insert image icon (fig. 8) , resize, and place a picture of an open-ended/timed activity on the page above the circles (as shown in fig. 19 ). creating navigation for a 3-min timer page 1. draw a large rectangle over the entire page using the shape tool (fig. 9 ). 2. click on the large rectangle and navigate to the insert link icon (fig. 10) in the toolbar. a white pop-up bar will appear on the screen. click on "slides in this presentation" and select the current slide number. click "apply." 3. click on the large rectangle and then on the color fill and border color icons (fig. 11) . change the color fill and border color to transparent. 4. create smaller dots (circles) on the corresponding timer buttons (fig. 19) . a. select the shape tool ( fig. 9) and draw one small button-sized red circle. (pressing shift while drawing the circle will keep it perfectly round.) rightclick on the circle and then copy and paste it once. use the color fill icon (fig. 3) to change the second dot to green. i. move the red dot on top of the timer's minute button. ii. move the green dot on top of the timer's start button. 5. right-click the timer page in the left navigation bar and copy it. 6. paste four more timer pages into the left navigation bar (similar to fig. 17) . 7. create a new blank page by clicking on the previous page (the final timer page) in the left navigation bar and pressing the "enter" key. 8. navigate to the first timer slide. 9. right-click on the green button dot. navigate to "order," then "send to back." 10. select the red button dot. navigate to the insert link icon (fig. 10) in the toolbar. click on "slides in this presentation" and select "next slide." click "apply." 11. repeat steps 9-10 for the next two timer slides. 12. on the fourth timer slide, right-click on the red dot. navigate to "order," then "send to back." 13. select the green button dot. navigate to the insert link icon (fig. 10) in the toolbar. click on "slides in this presentation" and select "next slide." click "apply." 14. navigate back to the second timer slide. select the text box icon (fig. 14) , create a text box, and type "1:00." place the time text box over the timer screen. adjust the text box size, font size, and color fill as needed. 15. select the text box with "1:00." copy the text box and paste it into the remaining timer pages. edit the text boxes on each subsequent timer page to increase the time shown by 1 min (fig. 23) . edit the timer text boxes on the fourth and fifth timer pages to both say "3:00." 16. after copying and editing the text boxes, right-click the timer text box on each page and click "order," then "send to back." 17. select the fifth timer slide in the left navigation bar. 18. click on the "insert" menu ( fig. 24 ) and select "video." 19. a youtube search bar will appear. type in "3 minute timer." choose the timer and click the "select" button. 20. a "format options" bar will appear on the right when the video is inserted. select "autoplay when presenting," as seen in fig. 25 . 21. select the "next" arrow from the cover page of the presentation (with "student's name"). copy and paste the arrow in the bottom right of the video page (fig. 26) . 22. after the timer video page, create a blank slide to continue making the schedule. a. to finish the schedule, see the section "creating a new schedule/modifying the schedule template." 23. go to the present icon (fig. 12) , located in the top-right corner of the toolbar, to view and navigate through the timed activity slides. a. navigate through the presentation by clicking on the timer dots in sequence and the "next" arrow. each click should advance to the next slide in the sequence. i. if the slides do not advance correctly, check the links by clicking on the linked object. when clicked on, each linked object should display a white link bar below with the words "next slide." if the slide linked to the object is incorrect (or missing), click "remove link" and return to the step corresponding to the specific object: fig. 16 background button 1) red dot: step 10 in the section "creating navigation for a 3-min timer page" 2) green dot: step 13 in the section "creating navigation for a 3-min timer page" 3) next arrow: step 21 in the section "creating navigation for a 3-min timer page" b. after all transitions are working correctly in sequence, restart the slideshow. check that no other transitions occur when clicking elsewhere on the screen (anywhere other than the linked objects). i. if transitions occur, check the following: 1) ensure that the transparent rectangle is linked correctly. if it is not, return to step 2 in the section "creating navigation for a 3-min timer page." 2) if an incorrect object is creating a transition, exit the slideshow and navigate to the slide with that object. right-click on the object, select "order," and then "send to back." to create a new schedule, copy and paste the template slide sets into a new google slides presentation. the template sequences will maintain links and other properties. for example, to create one choice activity, copy all five slides of the choice page. this task analysis will assist in creating a template that clinicians can then use to create client-specific schedules. each schedule should end with a terminal reinforcer that is fig. 30 ). to replace pictures in any of the pages, select the large transparent rectangle and move it out of the way. then edit or replace pictures and place the large transparent rectangle back in place. to test and ensure all pages and features are working correctly, we suggest selecting the present icon (fig. 12) to navigate and interact with the slides as the client would to check for errors. to assist with following the activity schedule template sequence, we provide a flowchart example in the appendix, fig. 30 . there are various options for sharing activity schedule files with caregivers and clients. one way is to send the caregivers a link to the activity schedule file. to create a link, select the yellow "share" button ( fig. 27) in the topright corner. a pop-up menu will appear that provides sharing options, including view-only or editable access. select "get shareable link" and then e-mail this link to the family/client. other examples of how to share the activity schedule include creating a google drive folder with multiple schedule variations or sharing it with other instructional materials using google classroom. for all sharing options, learners will need to view the activity schedules in the present form or the slideshow view. to access the presentation on mobile devices or tablets, they will need to download the google slides app. researchers have demonstrated that photographic activity schedules can be an effective tool to promote independence and reduce the need for adult prompts across a variety of behaviors and for many different types of learners. whereas much of this body of research has investigated the use of binder-based or single-page paper activity schedules, in recent years, researchers have demonstrated that technology-based digital schedules can also be effective and, in some circumstances, preferred by learners (giles & markham, 2017) . digital activity schedules have multiple potential advantages that make them well suited to situations such as the current covid-19 crisis, during which face-to-face contact between behavior analysts and their clients and families may be reduced or eliminated. another possible advantage includes the relative ease with which they can be modified and the potential for behavior analysts to create and disseminate digital activity schedules electronically. the task analysis included in this article may be a useful tool as behavior analysts create and disseminate digital activity schedules. conflict of interest the authors have no known conflicts of interest to disclose. informed consent as this is a technical article, no human participants were involved in the project. thus, no informed consent was necessary. directions: collect data on the learner's performance. correct responses will be scored as a plus (+) and incorrect response or responses you provided physical supports will be scored as a minus (-). retrieves schedule step 27 steps 1-5 steps 1-10 step 1 steps 2-11 steps 12-18 steps 1-10 steps 1-10 an evaluation of group activity schedules to promote social play in children with autism using joint activity schedules to promote peer engagement in preschoolers with autism the use of linked activity schedules to teach children with autism to play hide-and-seek using activity schedules on the ipod touch to teach leisure skills to children with autism. education and treatment of children effects of a photo activity schedule book on independent task changes by students with intellectual disabilities in community job sites using videoenhanced activity schedules and matrix training to teach sociodramatic play to a child with autism evaluation of interactive computerized training to implement photographic activity schedules with children with autism spectrum disorder comparing book-and tablet-based picture activity schedules: acquisition and preference ipads for students with asd: comparing delivery modes for visual activity schedule (doctoral dissertation promoting independence, verbal behavior, and social skills in individuals with autism through activity schedules and script fading handbook of social skills and autism spectrum disorder: assessment, curricula, and interventions teaching children with autism to use photographic activity schedules: maintenance and generalization of complex response chains activity schedules for children with autism: teaching independent behavior creating activity schedules using microsoft® powerpoint® publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-295543-nj4a640t authors: castañeda-babarro, arkaitz; arbillaga-etxarri, ane; gutiérrez-santamaría, borja; coca, aitor title: physical activity change during covid-19 confinement date: 2020-09-21 journal: int j environ res public health doi: 10.3390/ijerph17186878 sha: doc_id: 295543 cord_uid: nj4a640t background: the lockdown and social distancing caused by covid-19 may influence common health behavior. the unprecedent worldwide confinement, in which spain has been one of the most affected—with severe rules governing confinement—may have changed physical activity (pa) and sedentary habits due to prolonged stays at home. purpose: the aim of this study is to evaluate how self-reported pa and sedentary time (st) have changed during confinement in the spanish population. methods: 3800 healthy adults (age 18–64 years) residing in spain answered the international physical activity questionnaire short (ipaq-s) twice between 23 march and 1 april (confinement). data analysis was carried out taking into consideration meeting general pa recommendations before confinement, age and gender. results: self-reported pa decreased significantly during confinement in our sample. vigorous physical activities (vpa) and walking time decreased by 16.8% (p < 0.001) and 58.2% (p < 0.001), respectively, whereas st increased by 23.8% (p < 0.001). the percent of people fulfilling the 75 min/week of vpa recommendation decreased by 10.7% (p < 0.001) while the percent of people who reached 150 min/week of moderate activity barely changed (1.4%). the group that performed the most vpa before confinement showed the greatest decrease (30.5%, p < 0.001). men reduced time in vpa more than women (21% vs 9%, respectively) who even increased time in moderate pa by 11% (p < 0.05) and reported less increase in st than men (35% vs 25.3%, respectively). conclusion: the spanish adult population, especially young people, students and very active men, decreased daily self-reported pa and increased st during covid-19 confinement. on 11 march 2020, the world health organization (who) [1] declared a global pandemic caused by severe acute respiratory syndrome coronavirus (sars-covid-2), which has become a public health emergency of international concern. during its first phase of expansion outside china, italy and spain were the most affected countries reporting most cases and deaths. thus, they were the first nations to declare a state of emergency in europe. in spain it was declared on 17 march and the government ordered a lockdown to restrict travel and cancel non-essential services in order to stop the spread of coronavirus disease (covid-19) [2] . social distancing and confinement are fundamental in tackling the spread of coronavirus. however, the ongoing lockdown across the country has no precedent and it is unknown how this may affect the general population's health and wellbeing. in these circumstances, the sudden and stressful situation in addition to prolonged stays at home may imply a radical change in lifestyle behavior such as physical activity (pa), eating habits, alcohol consumption, mental health, quality of sleep, etc. [3] [4] [5] [6] [7] . likewise, there is a general concern about the negative health implications of inactivity and sedentary behavior [8] . the general recommendation for considering an adult to be physically active is to attain at least 150 min of moderate or 75 min of vigorous intensity activity per week or an equivalent combination of both [9] , and sedentary behavior is defined as any waking behavior practiced while lying down, reclining, sitting or standing, involving an energy expenditure ≤ 1.5 metabolic equivalents [10] . while the disease spreads around the world, healthy people are being requested to stay at home for prolonged periods of time and, as a consequence, covid-19 has radically modified the determining factors (individual, interpersonal, environmental, regional or national policies and global) [11] of both types of behavior thus, due to isolation and limitations in engaging in regular and common activities, fulfilling pa recommendations and reducing sedentary behavior during lockdown may pose a significant challenge, especially during the first weeks when the population has limited chances to find alternatives to ensure they remain active even at home. hence, although individuals were encouraged to remain physically active in their homes [12], the unprecedented confinement may give rise to two situations. (1) the active population may decrease their activity and (2) the inactive population may not be likely to increase their daily pa. despite the fact that lockdown has affected several countries, data are scarce about how people have changed their pa and sedentary behavioral patterns because of the specific cases of isolation in each country. therefore, the aim of this study was to analyze self-reported pa and sedentary behavior before and during lockdown caused by covid-19 in a spanish healthy adult population. healthy adults (age 18-64 years, age category defined by who) living in spain were asked to participate in this cross-sectional study. all the subjects were informed about the objective of the study and their free participation in it, being able to leave it whenever they wanted. ethics approval was obtained from the deusto university human ethics advisory group, and informed consent was obtained from participants. sociodemographic data (age, height, weight, gender, whether working and/or studying) and self-reported pa data were collected by questionnaires sent between 23 march and 1 april, just 10 days after the state of emergency was declared in spain. two questionnaires were sent together to be answered consecutively at the same time. the first collected retrospective data about habits during a normal week before lockdown, and the second asked about the following one or two weeks. the questionnaire used was the ipaq short version validated in spanish [13] . the ipaq short version asks about three specific types of activity undertaken during the previous 7 days in the four domains (leisure time, work, household activities and transport); items are structured to provide separate scores for walking, moderately intense and vigorously intense activities. the ipaq version also contains a question about the time spent on sedentary activity. the invitation to participate in the study was issued via social media, e-mail and mobile phone from the physical activity and sports sciences department, university of deusto. moreover, a national sports store (forumsport s.a.) and a biomechanical laboratory (custom4us) also sent the questionnaire to their customers by e-mail. the primary outcome was the change in time and intensity of self-reported pa and the sedentary time prior to the confinement situation and after it. the secondary outcome was the change in the percentage number of participants who fulfilled general pa recommendations according to age, gender, working status and baseline pa levels. a total of 4160 healthy subjects answered the questionnaire. a total of 360 were excluded due to exclusion criteria: not resident in spain (n = 16), age < 18 or ≥65 (n = 110) and extreme scores in vigorous, moderate and walking activities (≥6 h per day, 7 days a week). the exact number of participants excluded was as follows: n = 81 were excluded because the sum of the total active time was >6 h per day (vigorous + moderate + walking). within the pre-covid data, n = 17, n = 31 and n = 62 were excluded because they exercised > 180 min per session at a vigorous level of intensity, >180 at moderate intensity and >21 h (3 h per day, 7 days of week) walking, respectively. finally, within the during-covid data, n = 17, n = 9 and n = 17 were excluded because they exercised >180 min per session at vigorous intensity, >180 at moderate intensity and >21 h (3 h per day, 7 days of week) walking, respectively. a univariate analysis with paired t-tests was used to compare the differences in primary outcomes before and during confinement. furthermore, a chi 2 frequency test was used to assess secondary outcomes, which refer to the change in the percentage number of participants who fulfilled the amount of self-reported pa in the subgroups. a subgroup analysis was performed on different age groups (18-24, 25-34, 35-44, 45-54, 55-64) , gender (m, f), working status (students, active workers, people that study and work, those that reported they did nothing), and self-reported pa, categorized into vigorous activity groups (0-75, 75-150, 150-225, more than 225 min/week) and moderate activity groups (0-150, 150-300, 300-450, >450 min/week). the pa subgroup categories are based on world health organization (who) recommendations. we divided the data into different groups following in accordance with who recommendations for activity of moderate (150 min per week) and vigorous (75 min per week) intensity. within each of the two categories we divided the data into 4 groups as follows: (1) under the amount recommended (less than 150 min of moderate intensity or less than 75 min of vigorous intensity), (2) following recommendations (150 to 300 min of moderate intensity or 75 to 150 min of vigorous activity), (3) twice the amount recommended and (4) three times the amount recommended. this categorization was made to extrapolate the results to the amount of self-reported pa fulfilled by participants in the study, with α level set at 0.05. statistical analysis was performed using the spss data analysis version 23 (spss, inc., chicago, il, usa). table 1 shows the demographic characteristics of 3800 participants who were mostly male, (54%) active workers (78%) and aged 42.7 ± 10.4 years (mean ± sd). (5) data presented as mean ± sd, n (%) using the who stratification, we divided the sample into five bmi groups with n = 77 (2.1%) being the underweight group, n = 2621 (70.0%) normal; n = 897 (24.0%) overweight; n = 144 (3.8%) obese and n = 4 (0.1%) extremely obese (<18.5 kg/m 2 ; 18.5-24.9; 25-29.9; 30-39, 9 and ≥40 groups of bmi score, respectively). during confinement, the amount of time spent on moderate and vigorous activities by all the population decreased by 2.6% (p = 0.102) and 16.8% (p < 0.001), respectively. in addition, walking time was reduced by 58.2% (p < 0.001) whereas sedentary time increased by 23.8% (p < 0.001) ( table 2) . men reported a higher decrease in vigorous activities than women (21% and 9%, respectively) and both reduced walking time to a similar extent (58.5% men, 59.6% women) ( table 2 ). however, sedentary time was reported to have a higher increase in men (35%, p < 0.001) than in women (25.3%, p < 0.001) and accordingly, men significantly reduced moderate activities by 8.2% (p < 0.001) while women increased these activities by 11% (p < 0.05). the student group showed the highest decrease in moderate (16.1% p < 0.05), vigorous (24.3%, p < 0.001) and walking activities (66.9% p < 0.001), whereas unemployed or non-students were proved to be the most sedentary during confinement (47.7%, p < 0.001) ( table 2 ). the adult population (age 55-65 years) decreased the amount of time they spent on vigorous activities the most (22.1%) whereas for moderate activities and walking time it was the youngest subjects (18-24 years) who also evidenced the greatest increase in sedentary time (47.7% p < 0.001). regarding meeting pa recommendations, the number of subjects who failed to complete 75 minutes' activity of vigorous intensity per week before confinement increased by 10.7% (p < 0.001) during lockdown. (table 3 ). in addition, the most active population (>225 min/week of vigorous activity) decreased their activity significantly by 7.7% (p < 0.001). nevertheless, meeting 150 min of moderate activity per week barely changed (1.4%, p value 0.117). in the subgroup analysis, the most active subjects showed the highest decrease in vigorous activity time (30.5%, p < 0.001) ( table 2 ). however, the less active population increased the time they spent on these activities by 34% and this trend is also evidenced in the case of moderate activities. furthermore, walking time declined similarly for all self-reported pa levels, while in terms of sedentary time, the most active group reported the highest increase (40.3%, p < 0.001). self-reported pa decreased significantly during confinement in all the population, in which vigorous and walking activities declined the most and moderate activities barely changed. there were more inactive people who failed to fulfil the 75 min/week of vigorous activities during lockdown and sedentary time also increased considerably. the impact on active and sedentary behavior was particularly high in men, young people, students and the physically very active population. to our knowledge, there are limited previous studies that have analyzed the effect of confinement caused by a pandemic on self-reported pa. thus, it is difficult to assess whether the population we studied has reduced the amount of self-reported pa to a smaller or larger extent. the only available data we found about self-reported pa during confinement comes from activity trackers, where it has been shown that in europe the country with the greatest step count decrease recorded was spain, with 38% less, followed by italy with 25%. this reduction is similar to the falling trend in walking time shown in our study. in this regard, other studies carried out in similar lockdown situations (confinement during seasons with adverse weather conditions-cold winter or heat waves) also reported pa decrease during confinement, whereas sedentary time increased because poor or extreme weather becomes an environmental barrier to going outdoors [14] [15] [16] [17] . sedentary time increased considerably, most likely due to the exchange between common daily active behavior (walking, cycling or transport to work, etc.) and the prolonged stay at home. young people and students spent more time seated during confinement and this may be due to the forced e-learning which encourages sedentary behavior related to excessive time on screen-based activities [18] . likewise, according to the socioecological model [11] , in a comparable framework where social or environmental barriers promote an inactive lifestyle (social isolation, loneliness and, when season changes), more sedentary behavior and less time being spent on light, moderate and vigorous pa have been reported [19] [20] [21] . hence, an involuntary prolonged stay at home may encourage sedentary behavior as well as during confinement caused by covid-19. the most active subjects showed the highest decrease in vigorous activity time and this may be explained by two main reasons: the forced sudden inaccessibility to community resources (e.g., sports facilities, urban trails, parks, green spaces, etc.) and the lack of time to react during the first weeks of confinement to gather fitness resources, in order to continue engaging in regular activities at home. regarding moderate activities, our data showed that it barely changed in all the population and this could be attributed to the fact that some people may have been maintaining the minimum recommended time doing alternative activities at home. in this regard, the less active population increased vigorous and moderate activities during confinement, which could be related to the promotion of such activities by health institutions, fitness centers, the internet and television by posting daily online workout routines. to our knowledge, there are no scientific articles supporting the benefits of promoting pa established during confinement. some specific examples of such activities are spanish national television (morning workouts), the free online classes offered by several institutions and fitness centers (using online platforms such as zoom or google meet) and the recommendations made by health associations. our results showed that there are people who found new ways of being more active during this lockdown, which could become best practices in the future, should a pandemic of similar characteristics hit again. active behavior changed according to gender. both genders reduced walking time to a similar extent, although men reported a higher decrease in vigorous activities that may be related to the greater pa prevalence reported in women over the years. [22] [23] [24] [25] . in terms of sedentary behavior, sitting time increased more highly in men who also significantly reduced moderate activities, while women increased these activities. this could be attributed to the gender gap by historically demonstrated female inequality in household and child care tasks, in which men have shown a low level of involvement in spain [26] [27] [28] . regarding meeting pa recommendations, the global age-standardized prevalence among the inactive population was 27.5% in 2016 [23] . according to the eurobarometer (eurobarometer, 2014), 33.6% of the spanish adult population did not attain minimum levels of pa, and 36% spent most of the day sitting down (national health surveys in spain). in our population, our inactive population before confinement is lower (25.3% corresponding to 75 min/week vigorous pa) than that of the eurobarometer. that may be explained by the fact that the population recruited for the study was particularly more active as shown in the data for people undertaking >225 min/week of vigorous activities, and this could be attributed to the fact that the questionnaire was shared by institutions linked to sport, exercise and biomechanics. therefore, it is to be expected that we recruited a population that is commonly more active than the general population. a limitation of the current study is that we selected the ipaq short version instead of the ipaq-long one because of concerns that the length of the questionnaire would result in significant participant burden. in addition, although the questionnaire collects data about the last seven days, in our study we requested information beyond a week, which may be justified due to the unprecedented situation and the lack of time available to manage more appropriate study design and methods. in order to obtain as many participants as possible, sharing the questionnaire was sent to customers involved in sports and biomechanical issues in addition to being shared on social networks. therefore, a sampling selection bias may have occurred in the population analyzed due to their natural active habits. another limitation could be the cross-sectional design of the study since directionality of the associations cannot be established. finally, the way in which the data were collected in this study can be in itself considered a limitation, since participants reported data in a subjective way when answering self-rated questionnaires. on the other hand, one of the strong points of the study is that the questionnaire was sent just when the population started confinement (during the first two weeks), and so it is likely participants had their common activities in mind before confinement in a fresh and realistic way. to our knowledge, this is the first study carried out on a confined large sample size, which may be useful in designing different strategies in order for the confined population to reduce sedentarism and increase pa. in conclusion, healthy adults reduced daily self-reported pa and increased sedentary time during covid-19 confinement in spain. the impact was particularly high in men, young people, students and the very active population. strategies should thus be employed to increase pa and decrease sedentary behavior. in this study, we tried to ascertain the influence of confinement on the lifestyle of the population. this study may serve to show the effect of an extraordinary measure, such as confinement, on this lifestyle, as well as a greater awareness of the effect of confinement. moreover, the results may help design and target interventions aimed at increasing physical activity and reducing unhealthy levels of sedentary time associated with pandemic mitigation efforts. who director media briefing on covid-19. available online alcohol use and misuse during the covid-19 pandemic: a potential public health crisis? lancet public health 2020, 5, e259 a prospective study of holiday weight gain who mental health. mental health and psychosocial considerations during the covid-19 outbreak dealing with sleep problems during home confinement due to the covid-19 outbreak: practical recommendations from a task force of the european cbt-i academy physical exercise as therapy to fight against themental and physical consequences of covid-19 quarantine: special focus in older people a tale of two pandemics: how will covid-19 and global trends in physical inactivity and sedentary behavior affect one another? on behalf of sbrn terminology consensus project participants. sedentary behavior research network (sbrn)-terminology consensus project process and outcome correlates of physical activity: why are some people physically active and others not? validation of the international physical activity questionnaire-short among blacks the effect of season and weather on physical activity: a systematic review. public health physical activity levels of community-dwelling older adults are influenced by winter weather variables influence of seasonal variations on physical activity in older people living in mountainous agricultural areas comparison of summer and winter objectively measured physical activity and sedentary behavior in older adults: age, gene/environment susceptibility reykjavik study covid-19): the need to maintain regular physical activity while taking precautions associations between social isolation, loneliness, and objective physical activity in older men and women social isolation, loneliness, and health behaviors at older ages: longitudinal cohort study loneliness predicts reduced physical activity: cross-sectional & longitudinal analyses the active living gender's gap challenge: 2013-2017 eurobarometers physical inactivity data show constant higher prevalence in women with no progress towards global reduction goals worldwide trends in insufficient physical activity from 2001 to 2016: a pooled analysis of 358 population-based surveys with 1.9 million participants worldwide variability in physical inactivity a 51-country survey shifting the physical inactivity curve worldwide by closing the gender gap the gender revolution: a framework for understanding changing family and demographic behavior parentalidad y división del trabajo doméstico en españa el impacto de cuidar en la salud y la calidad de vida de las mujeres the authors gratefully acknowledge those who participated in this study and forumsport and custom4us for helping to disseminate the questionnaire. the authors declare no conflict of interest. key: cord-303646-uxewsyli authors: janas, roman m.; socha, jerzy; warnawin, krzysztof; rujner, jolanta title: further studies on aminopeptidase-m in blood in children with cholestatic liver diseases and viral hepatitis date: 1999 journal: dig dis sci doi: 10.1023/a:1026626822298 sha: doc_id: 303646 cord_uid: uxewsyli aim of this study was to determine and furthercharacterize the serum aminopeptidase-m in children withliver diseases. based on our new assay, we have showntwo fractions of the enzyme. activity of the first fraction is expressed in undiluted serumat ph adjusted from 8.5 (ph of storaged serum) to 7.4.activity of the second fraction (cryptic activity)appears in the serum (ph 7.4) as a result of dilution and/or addition of aniline naphthalene sulfonicacid. in children with alagille syndrome, extrahepaticbiliary duct atresia, byler's disease, and acutehepatitis due to hepatitis b virus infection, activities of both fractions are highly elevated ascompared to healthy children or those with chronic viralhepatitis. moreover, serum aminopeptidase-m seems toreflect other aspects of the pathological process than those reflected by the alanine aminotransferaseand gamma-glutamyltranspeptidase. due to increasedactivity and broad substrate specificity, the enzymeseems to be also a cofactor of cholestasis andhepatitis. membrane -bound aminope ptidase -m (ec 3.4.11.2) functions as an ectopeptidase at the cell surface in many tissue s and the re is also a soluble form of this e nzyme in serum and othe r body¯uids (1± 3). serum aminope ptidase -m activity is marke dly increased in patie nts with chole stasis and was shown to be a use ful marke r of he patobiliary dise ase (4 ± 7). the enzyme is thought to originate from the live r (2) . howe ver, its multiple mole cular forms pre sent in se rum may arise from othe r organs and tissue s (6, 8) . in he matology, aminope ptidase -m (cluster of dif-fe rentiation 13) is a well-recognize d marker of mye loid cell le uke mia (9 ± 11) . inte restingly, it has bee n found that aminope ptidase -m on alime ntary or re spiratory tract e pithe lial cells is a receptor for human coronavirus 229e (12) . in a large population of appare ntly healthy humans, the e nzyme activity se ems to e xhibit some variations that are depe nde nt on age , se x, alcohol consumption, smoking, and drug intake (13) . among the substrate s of aminope ptidase -m, including those involve d in growth and differe ntiation re gulation, phagocytosis, and bacte ricidal and tumoricidal activity (3, 4, 9, 14 ± 16) , both methionine (met) and le ucine (le u) enkephalins are rapidly de grade d afte r the ir administration to humans or animals (17± 28) . in circulation, the e ndoge nous enkephalins se em to be prote cted from de gradation through binding to carrier prote ins (19) . howeve r, a fre e fraction of the e nke phalins would be susce ptible to the e nzyme. enkephalins appe ar to be implicate d in chole static live r dise ase and may be an important pathoge nic factor in such complications as pruritus or e nce phalopathy. inde e d, plasma e nke phalin concentrations are highly elevate d in adult patie nts with chole stasis of diffe ring aetiologie s (7, 29 ± 32) , and in rats with an e xpe rime ntal mode l of acute chole stasis (33, 34) . accumulating in blood, e nke phalins may cross the blood± brain barrie r and evoke , via the ir incre ase d availability in the central nervous syste m, a syndrome of chronic opiate -receptor stimulation (35, 36) . preliminary experiments have shown that naloxone or nalme fene , opiate -receptor antagonists, ame liorate d or reve rsed pruritus in patie nts with chronic chole static live r disease and, in some patie nts, pre cipitate d a syndrome that rese mble s the withdrawal reaction of opiate addiction (37, 38) . recently, we have shown that in childre n with chole stasis of diffe ring e tiologie s incre ase d se rum met-enkephalin occurs concom itantly with highly ele vate d se rum aminope ptidase -m activity and that both parame ters were appare ntly not affe cted by a short-te rm treatment with ursode oxycholic acid (39, 40) . alternations in the aminope ptidase -m activity as well as the ir re le vance to the situation in chole stasis and viral he patitis are not fully unde rstood yet, and furthe r syste matic studie s see m to be worthwhile . there fore , in this study we attempted to furthe r characterize and compare the serum aminope ptidase -m activity in he althy childre n, childre n with chole static live r dise ase , and childre n with chronic or acute he patitis due to hepatitis b virus (hbv) infe ction. patients. the protocol of this study was approved by the hospital ethics committe e. the following groups of patie nts were included in the study: group 1, e ight children, 8 ± 14 ye ars old (me an 10 years), with alagille syndrome (arteriohe patic dysplasia) , alanine am inotransfe rase ( alat): blood sam p ling. v enous blood was sampled without anticoagulants for the diagnostic examinations; it was possible to use part of e ach sample in this study. blood samples were centrifuged at 3000g for 15 min at 4°c, and the sera ® ltered through a 0.2-m m ® lte r (sartorius-me mbran® lte r, gottingen, ge rmany) to exclude all residual cell debris, and stored at 2 30°c. serum ph was me asured using a ph microelectrode (sigma chemical co., st louis, missouri). assay of am inop eptid ase-m activity. equal aliquots (500 m l) of the sera we re pooled in groups 1± 6, respective ly, and used for characterization of the aminopeptidase-m. the e nzyme activity was also dete rmined in the individual serum samples from patients from groups 1± 6. dilution of the serum was performed using 25 mmol/liter bistris± tris± ace tic acid buffe r with or without 0.15 mol/liter nacl, at ph range 4.5± 9.0. to maintain the serum ph at a desired leve l, the endogenous hco 3 2 was hydrolyzed with a negligible volume of 1 mol/liter hcl (® nal concentration 25± 30 mmol/ liter). resulting co 2 was remove d under low pressure, and ph was adjusted with a small volume of 1 mol/liter nao h, hcl, or bistris± tris± ace tic acid under the control of a ph microelectrode. the incubation mixture consisted of 100 m l of undiluted or diluted serum and 5 m l of [ 1 25 i] incubation was performed at 37°c for 0 min (control) to 15 min, and terminate d by acidi® cation with 100 mmol/l ace tic acid. de gradation of the substrates was quanti® e d by the aluminum silicate binding assay as recently described (41) with slight modi® cations. a homogenous suspension (50 mg/ml) of aluminum silicate powder (lloyd reage nt, pfaltz and bauer, wate rbury, connecticut) was prepared in 1% acetic acid. aliquots of 2 ml were adde d to the tubes at the e nd of each incubation period. afte r vortexing for 1 min, the tubes were centrifuged at 3000g for 10 min. for [ 12 5 i]leu-enkephalin, the supernatants were discarded by aspiration, and the silicate pellets counted for the g -radioactivity. for the [ 3 h]leu-e nkephalin, 1.5-ml aliquots of the supernatants were mixe d with 5 ml aliquots of the scintillation cocktail and counted for the b -radioactivity. intraand interassay coef® cients of variation we re 4.9% and 10.5% for iodinated and 8.9% and 14.5% for tritiated e nkephalins, respective ly. the e nzyme activity was e xpressed as picograms of degrade d substrate per minute per milliliter whole serum. intact [ 12 5 i]-or [ 3 h]leu-enkephalin was almost complete ly adsorbed by the aluminum silicate (93 6 3% , me an 6 sem, n . 30). reve rse-phase high-performance liquid chromatography showed that silicate pellet-bound radioactivity re pre se nte d the intac t labe le d pe ptide , whereas 85± 90% of the radioactivity appearing in the supernatants was due to [ 1 25 i]-or [ 3 h]tyrosine. unidenti® e d, two to three amino acid fragme nts constituted about 10 ± 15% of the radioactivity and did not react with the speci® c leu-e nkephalin antibody (data not shown). effect of inhibitors an d oth er subs tan ces on am inop eptidase-m. degradation of the labeled leu-enkephalin by serum aminopeptidase-m was quanti® e d by dete rmining the percentage of intact label in the presence of increasing concentrations of speci® e d substances. the exte nt of the label degradation in the control probes, no substance added (35± 40% degradation afte r a speci® e d incubation period) was take n as 100% relative activity. before the assay, the inhibitors to be teste d were preincubated with the serum at ; 20°c for 15 min. the inhibitory constants (k i ) and michaelis constant (k m ) towards unlabeled leu-e nkephalin were determined as previously described (39) . the following substances used in the present study we re purchased from sigma: 1,10-phe nanthroline, bestatin, puromycin, leu-e nkephalin, me t-e nkephalin, bovine serum albumin, 8-aniline naphthalene sulfonic (ans) acid, taurocholic acid, ursodeoxycholic acid, and cholic acid. other chemicals we re of analytical grade. statis tical an alysis. v alues are e xpressed as means 6 se m unless otherwise speci® ed. the mann-whitne y u test was used to dete rmine the signi® cance of differences betwe en means; p , 0.05 was considered signi® cant. during storage , serum ph rise s to 8.5. the e ffect of ph on aminope ptidase -m activity in undilute d se rum pools from groups 1± 6 is shown in figure 1 . the ph curve s were symme tric and dynamic with an optimum at about 6.5± 7.3 for both iodinate d and tritiate d le ue nkephalin. the same data were obtaine d in the e xpe rime nts with dilute d se ra. the increase in serum aminope ptidase -m activity as a result of dilution with 25 mmol/lite r buffe r, ph 7.4, is shown in figure 2a ,b. as shown in figure 2a , the e nzyme activity increased from 45 6 5 (undilute d se rum, ph 7.4) up to maxim al value s of 880 6 90 pg/min/ml (80-fold dilute d serum; value s are means 6 sem) . there were no statistically signi® cant diffe re nce s in the aminope ptidase -m activity be twe e n he althy childre n and those with chronic active he patitis due to hbv infe ction (p . 0.05) . as shown in figure 2b , the e nzyme activity in the undilute d se ra, ph 7.4, from childre n with alagille syndrome , extrahe patic biliary duct atre sia, byler' s dise ase , and acute he patitis due to hbv infe ction was: 411 6 55, 480 6 39, 198 6 25, and 300 6 69 pg/min/ml, re spe ctive ly. in the sample s dilute d 50-to 80-fold, the activity re ache d maximal value s of 6100 6 800, 6900 6 950, 2150 6 280, and 3880 6 410 pg/m in/ml, re spe ctive ly. negligible dilution, up to 1.5-fold, was without effe ct (figure 2a,b) . the same data were obtaine d in the e xpe rime nts whe re buffe re d saline , ph 7.4, se rved as a se rum dilue nt (data not shown) . the effect of the incre asing concentrations of ans acid on aminope ptidase -m activity is shown in figure 3 . in the undilute d and 5-and 30-fold dilute d sera, the e nzyme activity incre ase d about twice in the prese nce of 5, 1 and 0.1 mmol/lite r ans acid, respe ctively. for any dilution, an optim al conce ntration (c) of the ans acid may be estimate d from the following e mpirical formula: 5/a 5 c (in millimole s pe r liter), whe re a is the -fold dilution of the serum. highe r ans acid conce ntrations were inhibitory, but the e nzyme activity did not decrease below its basal le vel (no ans acid adde d). inhibition and substrate spec-i® city data of serum aminope ptidase -m in the se rum pools in groups 1± 6 are shown in table 1 . in both undilute d and dilute d se ra, puromycin, be statin, and amastatin inhibite d the e nzyme with k i value s of about 70 m mol/lite r, 1.8 m mol/lite r, and 70 nmol/lite r, re spective ly. in the prese nce of ans acid, the enzyme se nsitivity to the inhibitors was change d to a certain de gre e; howe ver, pote ncy and se le ctivity range s of the inhibitors re maine d unchange d. the k m value , de te rmined with leu-e nke phalin as substrate , was 359 6 62 m mol/lite r in both undilute d and dilute d se ra from groups 1± 6 ( table 1) . aminope ptidase -m activity in the individual sera from groups 1± 6 was evaluate d unde r diffe re nt assay conditions, and the re sults are shown in table 2 . in the undilute d sample s with ph 8.5, the e nzyme activity was statistically signi® cantly unde restimate d (p , 0.005) as compare d to the same sample s with ph adjuste d to the physiological le vel. maximal enzyme activity was observe d in the samples that were dilute d 80-fold. the se rum aminope ptidase -m activity was statistically signi® cantly highe r in childre n with cholestasis and acute he patitis as compare d to he althy childre n or those with chronic active hepatitis (p , 0.005) . we atte mpte d to de te rmine and characte rize the e xpre ssion and prope rtie s of se rum aminope ptidase -m activity in childre n with live r diseases and in he althy childre n. we have shown the pre sence of two fractions of the enzyme . activity of the ® rst fraction (basic activity) is e xpre ssed in the undilute d or ne gligibly dilute d se rum at ph 7.4. activity of the se cond fraction (cryptic activity) appe ars in the serum as a re sult of dilution and/or addition of ans acid. in childre n with chole stasis and in childre n with acute he patitis due to hbv infection, both fractions of the e nzyme are highly elevate d as compare d to he althy childre n or those with chronic active he patitis due to hbv infection. since enkephalins are among the well-characte rize d, natural aminope ptidase -m substrate s (2, 3, 17, 19 ± 28) , in our assay we use d radiolabe le d le ue nkephalin at a concentration close to the peptide leve l in circulation. [ 3 h]le u-e nkephalin is biologically indistinguishable from the native pe ptide whe reas substitution of the iodine see mingly in¯ue nced the substrate ± e nzyme inte raction, re sulting in a slightly de crease d rate of hydrolysis ( figure 1 ). the enzyme ph optimum was narrow, about 6.5± 7.3, and the activity dramatically decreased outside that range . after one free ze± thaw cycle , the ph of se rum increases to 8.5 due to ne ar comple te co 2 evaporation. to avoid a se rious unde re stimation of the enzyme activity (20 ± 24, 26, 39, 40) , se rum ph must be carefully adjuste d to 7.4 or, alte rnative ly, to the enzyme ph optim um. maintaining the serum ph may be achie ve d by dilution of se rum with an appropriate buffe r. howe ver, as we have shown, a dilution is anothe r factor affe cting the serum aminope ptidase -m activity. whe the r ph plays a role in in vivo re gulation of the e nzyme activity re mains to be determined. aminope ptidase -m activity dynamically increased as a re sult of dilution of the serum with simple buffer or buffe red saline . the plate au of the activity was found in the sample s dilute d 50-to 80-fold, where as a negligible dilution, up to 1.5-fold, was without e ffe ct. in he althy childre n and childre n with chronic active he patitis due to hbv infe ction, enzyme activity incre ase d from 70 (undilute d serum) to 900 pg/min/m l (80-fold dilute d serum). in childre n with byler' s disease, the re spective value s were 240 and 2000 pg/min/ml. the highe st incre ase s were found in childre n with alagille syndrome (from 360 to 6300 pg/min/m l), e xtrahe patic biliary duct atresia (from 430 to 6600 pg/min/ml) , and those with acute he patitis due to hbv infection (from 300 to 4000 pg/min/ml) . in many re ports (6, 7, 11, 13, 19, 23, 27, 33) , including ours (39, 40) , serum dilution was not take n into account as a factor that may affect aminope ptidase -m activity. an increase of e nzyme activity as a result of dilution sugge sts that a large portion of the enzyme molecule s is not hydrolytically active and is prese nt in the se rum as a cryptic fraction. this may be due to prese nce of the enzyme natural inhibitor( s) that dissociate as a result of dilution. if this assumption is true , the n an increase of the se rum aminope ptidase -m in chole stasis and acute he patitis is not due to a de® cie ncy of such an inhibitor since both fractions of the enzyme are highly e levate d. activation of the e nzyme may occur in vivo. re cently, martine z and coworke rs ( 20, 26) have shown that serum aminope ptidase -m activity in rats is capable of changing rapidly in re sponse to environmental e xpe rie nce . howeve r, the mechanism of e nzyme activation as a re sult of se rum dilution re mains to be de te rmine d. addition of ans acid, capable of disrupting weak bonds, incre ase d serum aminope ptidase -m activity in a dose -de pe nde nt manne r ( figure 3 ). howe ver, the mechanisms of incre asing enzyme activity by ans acid and by dilution se em to be diffe rent, and both factors appe ar to act inde pe nde ntly on the e nzyme structure , on re gulatory factors, or both. o ur inhibition and speci® city data have shown substantially the same characte ristics of the e nzyme both in the undilute d and dilute d sera de te rmined with or without the pre sence of ans acid. o ur data sugge st that a re liable aminope ptidase -m assay may be performed using a picomolar conce ntration of the e nke phalin labe le d with iodine or tritium, adde d at a ne gligible volum e to an aliquot of the undilute d se rum with ph adjuste d to the physiological leve l. maximal e nzyme activity may be de te rmine d using 50-to 80-fold-dilu te d se rum. hemolysis must be avoide d since e rythrocyte s contain an abundance of e nke phalin-de grading activity (40) . use of e dta plasma, phosphate , citrate , or barbital buffers marke dly decrease s the e nzyme activity. for the monitoring of aminope ptidase -m activity, a varie ty of substrate s (6 ± 9, 11, 13, 18 ± 26, 28, 33, 42) and technique s may be applie d (7± 11, 20 ± 22, 24, 27, 42) ; howe ver, we have shown he re that a se nsitive , fast, and inexpe nsive alum inum silic ate binding assay is an e xce lle nt method for detection of degradation of 3 h-or 125 ilabe le d enkephalins. chole stasis is associate d with an accumulation of e nkephalins and othe r opiate peptide s in blood that may contribute to pathoge nesis of pruritus or encephalopathy (7, 29 ± 39) . whe the r the opioid syste m is alte re d in acute viral he patitis remains to be de te rmined. although the origin of e nkephalins in cholestasis is unknown (35) , it see ms that an impairm ent of the he patic degradation of these pe ptide s doe s not occur (40) . the refore, an increase of serum aminope ptidase -m in chole stasis may be speculate d to be a home ostatic atte mpt to preve nt unlim ited accumulation of opiate pe ptide s in circulation. howe ver, due to increase d activity, the e nzyme see ms to cause substantial change s in the turnove r of othe r, biologically active peptide s in the blood (43) . there fore , we suggest that the e nzyme is not only a marker but also a cofactor of the disease. the source of se rum aminope ptidase -m was not prove n, but the e nzyme is thought to originate from the live r (2), where it is a he patocyte -membrane -bound metallope ptidase virtually abse nt in the he patocyte cytosol. the e nzyme may re¯ect othe r pathways or aspe cts of the pathological proce ss than those re¯ected by se rum alat or g gtp. inde ed, our patie nts with byle r's disease (se e mate rials and me thods) had a normal le vel of the se rum g gtp, moderate ly increased alat, and high le vels of aminope ptidase -m. in contrast, childre n with active chronic he patitis had e levate d leve ls of g gtp and alat but normal aminope ptidase -m activity. among our six patie nts with acute he patitis due to hbv infe ction (alat . 600 units/lite r), two patie nts had mode rately elevate d g gtp and highly incre ase d aminope ptidase -m activity whe reas four othe r patie nts e xhibite d high incre ase of both parame ters. aminope ptidase -m in the serum e xhibits a high amplitude of change s both in chole stasis and acute viral he patitis. howeve r, its physiological and pathophysiological role , as well as its re levance to the situation in chole static live r dise ase s and viral he patitis will re quire furthe r studie s. our data se e m to provide ne w, important insights for furthe r studie s on a role of this e nzyme. immunocytoche mical localisation of aminope ptidase-m in rat brain and periphe ry: re lationship of the e nzyme localisation and enkephalin metabolism peptidase inactivation of enkephalins: design of inhibitors and bioche mical, pharmacological and clinical applications peptidases involved in the metabolism of bioactive pe ptides e lektrophoretische variante n der alan in± am inope ptidase in se rum be i he patobilia è re n erkrankunge n a radioimmunoassay for the me asure ment of aminopeptidase (microsomal) in human se rum clinical signi® cance of a new isoform of se rum alanine aminope ptidase; re lationship with live r disease and alcohol consumption the he patobiliary disease marker se rum alanine aminopeptidase predominantly comprises an isoform of the hae matological mye loid differentiation antige n and leukae mia marker cd-13/gp150 be hal fj: multiple mole cular forms of human alanine aminopeptidase: immunological properties metalloproteaze activity of cd13/ aminope ptidase-n on the surface of human mye loid cells human myeloid plasma membrane glycoprote in cd-13 (gp150) is identical to aminopeptidase-n gp 150; aminopeptidase -n): pre dominant functional activity in blood ce lls is localised to plasma and is not cell-surface associate d holme s kv : human aminope ptidase-n is a receptor for human coronavirus 229e alanine aminope ptidase in se rum: biological variations and refere nce limits mcde rmott jr: human brain leucyl aminope ptidase: isolation, characte risation and speci® city against some ne urope ptides possible action of human placental aminope ptidase-n in feto-placental unit guste rson ba: ce ll-surface pe ptidases as modulators of growth and diffe rentiation breakdown of small enkephalin deriviates and adrenal pe ptide e by human plasma characte risation of three aminope ptidases puri® ed from mate rnal serum me chanism protecting plasma peptides from e nzyme hydrolysis: a comparative study enke phalin hydrolysis in plasma is highly correlate d with escape pe rformance in the rat ]e nke phalin in rat plasma se nsitive method for me asuring hydrolysis of enkephalins in plasma, using high-performance liquid chromatography with e lectrochemical de tection wards pe: n-terminal degradation of low mole cular we igh opioid peptides in human ce rebrospinal¯uid furthe r characte risation of the in vitro hydrolysis of [le u]-and [me t]enkephalin in rat plasma: hplc-e cd me asurements of substrate and me tabolite conce ntrations uptake and me tabolism of [ 3 h]-le u-enke phalin following either its intraperitoneal or subcutaneous administration to mice e xperience -dependent regulation of le u-enkephalin hydrolysis in rat plasma shun-cun c, berrye r p: a radioche mical assay for aminopeptidase n mentle in r: enkephalin me tabolism by microglia aminopeptidase-n (cd13) opioid pe ptides and primary biliary cirrhosis is ascite s caused by impaired he patic inactivation of blood borne endogenous opioid peptides? methionine enkephalin is incre ased in plasma in acute live r disease and is prese nt in bile and urine plasma leucine enkephalin is increase d in liver diseases endogenous opioids accumulate in plasma in rat model of acute cholestasis. gastroe nterology 103:630 ± brain and plasma leve ls of opioid peptides are altere d in rats with thioace tamide-induced fulminant hepatic failure: implications for the treatme nt of he patic ence phalopathy with opioid antagonists the pruritus of chole stasis: from bile acids to opiate antagonists the pruritus of chole stasis: potential pathogenic and therape utic implications of opioids a controlled trial of naloxone infusions for the pruritus of chronic cholestasis effects of naloxone infusions in patie nts with the pruritus of chole stasis methionine enkephalin concentration and e nke phalin-degrading activity are e levate d in blood in children with cholestasis me thionine e nke phalin and aminopeptidase-m activity in blood in children with cholestasis before and afte r treatme nt with ursodeoxycholic acid an aluminium silicate binding assay for quantitation of de gradation of chole cystokinin octapeptide and other short pe ptides substance p and bradykinin are natural inhibitors of cd13/aminopeptidase-n is the e nzyme a marke r or co-factor of the disease? we thank dr. h. falk, falk foundation e .v. freiburg, germany, for a trave l grant funded to r.m.j., as a result of which we were able to present part of the present data at the x international congress of liver diseases, o ctober 19 ± 21, 1995, basel, switzerland. key: cord-271068-rwx171oj authors: mayer, alejandro m.s.; rodríguez, abimael d.; berlinck, roberto g.s.; fusetani, nobuhiro title: marine pharmacology in 2007–8: marine compounds with antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiprotozoal, antituberculosis, and antiviral activities; affecting the immune and nervous system, and other miscellaneous mechanisms of action date: 2010-09-03 journal: comp biochem physiol c toxicol pharmacol doi: 10.1016/j.cbpc.2010.08.008 sha: doc_id: 271068 cord_uid: rwx171oj the peer-reviewed marine pharmacology literature in 2007–8 is covered in this review, which follows a similar format to the previous 1998–2006 reviews of this series. the preclinical pharmacology of structurally characterized marine compounds isolated from marine animals, algae, fungi and bacteria is discussed in a comprehensive manner. antibacterial, anticoagulant, antifungal, antimalarial, antiprotozoal, antituberculosis and antiviral activities were reported for 74 marine natural products. additionally, 59 marine compounds were reported to affect the cardiovascular, immune and nervous systems as well as to possess anti-inflammatory effects. finally, 65 marine metabolites were shown to bind to a variety of receptors and miscellaneous molecular targets, and thus upon further completion of mechanism of action studies, will contribute to several pharmacological classes. marine pharmacology research during 2007–8 remained a global enterprise, with researchers from 26 countries, and the united states, contributing to the preclinical pharmacology of 197 marine compounds which are part of the preclinical marine pharmaceuticals pipeline. sustained preclinical research with marine natural products demonstrating novel pharmacological activities, will probably result in the expansion of the current marine pharmaceutical clinical pipeline, which currently consists of 13 marine natural products, analogs or derivatives targeting a limited number of disease categories. the current article presents the preclinical pharmacology of marine natural products during 2007-8 maintaining the format used in the previous reviews (mayer and lehmann, 2000; mayer and hamann, 2002 , 2005 mayer et al., 2007 mayer et al., , 2009 . the preclinical pharmacology of antitumor and cytotoxic marine compounds has been reported in separate reviews (mayer, 1999; mayer and lehmann, 2001; mayer and gustafson, 2003 , 2006 . we have restricted the current as well as previous reviews to peerreviewed articles reporting the bioactivity or pharmacology of structurally characterized marine compounds. as we have done previously, we have continued to use a modification of schmitz's chemical classification (schmitz et al., 1993) to assign marine natural product structures to six major chemical classes, namely, polyketides, terpenes, peptides, alkaloids, shikimates, and sugars. novel antibacterial, anticoagulant, antifungal, antimalarial, antiprotozoal, antituberculosis and antiviral preclinical pharmacology of marine metabolites are presented in table 1 with the corresponding structures shown in fig. 1 . marine compounds that affect the immune and nervous systems, as well as those with anti-inflammatory effects are grouped in table 2 , with their corresponding structures presented in fig. 2 . finally, marine compounds that have been demonstrated to affect a variety of cellular and molecular targets are exhibited in table 3 , and their structures depicted in fig. 3 . several articles were published during 2007-8 reporting the preclinical pharmacology of extracts or structurally uncharacterized marine compounds, and although these have not been included in the present review, they probably may warrant further investigation: antimicrobial effects of the turkish red alga jania rubens (karabay-yavasoglu et al., 2007) ; antimicrobial activity in portuguese marine cyanobacteria synechocystis sp. and synechococcus sp. extracts towards gram-positive bacteria (martins et al., 2008) ; antibacterial activity against human and fish bacteria from the sub-arctic colonial ascidian synoicum pulmonaria from northern norway (tadesse et al., 2008) ; strong antibiotic-producing potential in actinomycetes from sediments in the trondheim fjord in norway (bredholt et al., 2008) ; antibacterial activity of deep-sea bacteria from sediments of the west pacific ocean ; potent antimicrobial activity in the green alga enteromorpha intestinalis collected in gujarat, india (nair et al., 2007) ; a nonhemolytic antimicrobial lipopeptide derived from the marine bacterium bacillus circulans (das et al., 2008) ; a t-antigen binding lectin with bioactivity against a broad spectrum of gram-positive and gram-negative bacteria from the sea cucumber holothuria scabra (gowda et al., 2008) ; anticoagulant activity of a 100-500 kda polysaccharide isolated from the fermented red seaweed lomentaria catenata which was greater than the clinical anticoagulant heparin (pushpamali et al., 2008) ; antimycobacterial activity in long-chain fatty acids isolated from the red alga polysiphonia virgata (saravanakumar et al., 2008) ; antileishmanial and anti-trichomonal activity in organic extracts of several mexican red and brown algae moo-puc et al., 2008) ; anti-hiv-1 activity of novel sulfated galactans isolated from the chinese red algae grateloupia longifolia and grateloupia filicina ; significant anti-herpes simplex virus activity in a 30 kda polysaccharide isolated from the red alga gracilaria corticata ; immunomodulatory effects of an enzymatic extract from the south korean marine brown alga ecklonia cava on murine splenocytes (ahn et al., 2008) ; immunostimulant properties of a sulfated polysaccharide isolated from the brazilian red alga champia feldmannii ; antioxidant and antimicrobial activity of the indian red and brown seaweed methanolic extracts with high phenolic contents (devi et al., 2008) ; and decreased expression of key regulatory genes involved in cholesterol and fatty acid biosynthetic pathways by the lipid extract of the cyanobacterium nostoc commune var. sphaeroides kützing (rasmussen et al., 2008) . 2. marine compounds with antibacterial, anticoagulant, antifungal, antimalarial, antiprotozoal, antituberculosis, and antiviral activities table 1 presents preclinical pharmacology reported during 2007-8 on the antibacterial, anticoagulant, antifungal, antimalarial, antiprotozoal, antituberculosis, and antiviral pharmacology of the marine natural products (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74) shown in fig. 1 . contributing to the global search for new antimicrobials to combat antibiotic-resistant strains of pathogenic bacteria, marine ecological niches have been described recently as "particularly promising" (fischbach and walsh, 2009) . during 2007-8, 38 studies reported novel antibacterial marine natural products isolated from marine bacteria, fungi, sponges, worms and fish, a larger effort than the ones reported in previous years (mayer et al., 2009) , and previous reviews of this series. only six papers provided detailed mechanism of action studies with marine antimicrobial compounds. kanoh et al. (2008b) reported the discovery of the novel bioactive spirodioxynaphthalene ascochytatin (1) isolated from cultures of a marine-derived fungus ascochyta sp. ngb4, which inhibited b. subtilis growth (mid= 0.3 μg/disk) by targeting the function of the bacterial growth regulatory system tcs (yycg/yycf). kitani et al. (2008) discovered that the 120 kda acidic glycoprotein l-amino acid oxidase termed ssap (2), isolated from the rockfish sebastes schlegelli (kitani et al., 2007) , acted selectively on gram-negative bacteria (minimum inhibitory concentration (mic) = 0.078-0.63 μg/ml). while h 2 o 2 was shown to mediate the antibacterial action of ssap, electron microscopy analysis revealed that ssap induced cell surface damage and morphological changes in several bacterial species. reported that the 21-residue peptide arenicin-1 (3) isolated from the marine polychaete arenicola marina exhibited significant antibacterial activity against p. aeruginosa and staphylococcus aureus (mic = 2 μg/ml). interestingly, arenicin-1 induced release of calcein from pe/pg liposomes, thus suggesting that the bacterial cell membrane is the main molecular target of the peptide. jang et al. (2007c) extended the pharmacology of the alkaloid isoaaptamine (4), isolated from the marine sponge aaaptos aaptos. isoaaptamine inhibited sortase a (ic 50 =3.7 μg/ml), an enzyme involved in s. aureus cell wall protein anchoring and virulence, thus potentially providing a novel lead compound "for further development" of potent antibacterials. lee et al. (2008) isolated seven sesterterpenes (5, 6, 7, 8, 9, 10, 11 ) from a tropical sponge dysidea sp. which demonstrated antibacterial activity against b. subtilis (mic = 1.56-12.5 μg/ml) by inhibiting isocitrate lyase activity, a key enzyme in the glyoxylate cycle which is present in most prokaryotes, lower eukaryotes and plants, but not in vertebrates. kanoh et al. (2008a) described a new sulfoalkylresorcinol (12) isolated (kossuga et al., 2008) (continued on next page) from the marine-derived fungus zygosporium sp. knc52, which showed antimicrobial activity against methicillin-resistant s. aureus. the mechanism of action appeared to involve inhibition (ic 50 =12.5 μg/ ml) of the in vitro polymerization of ftsz, a protein which is a structural homolog of eukaryotic tubulin, and that participates in bacterial cell division. as shown in table 1 , several new marine antibacterials were also reported in 2007-8 ( fig. 1) with mics less than 10 μg/ml against antibiotic-resistant bacterial strains, although no mechanism of action studies were reported: the alkaloids ambiguine isonitriles h and i (13 14) isolated from a marine cyanobacterium fischerella sp. (raveh and carmeli, 2007) ; the polyketides ariakemicins a and b (15, 16) isolated from a marine gliding bacterium rapidithrix sp. (oku et al., 2008) ; ayamycin (17) isolated from a bacterium nocardia sp. alaa 2000 (el-gendy et al., 2008a) ; the alkaloids batzelladines l and m (18, 19) isolated from the caribbean sponge monanchora unguifera (hua et al., 2007) ; a diphenyl ether (20) isolated from the indonesian sponge lamellodysidea herbacea (hanif et al., 2007) ; essramycin (21) obtained from the culture broth of a marine streptomyces sp. isolate merv8102 (el-gendy et al., 2008b) ; a meroditerpene (+)-isojaspic acid (22) isolated from the papua new guinean sponge cacospongia sp. (rubio et al., 2007) ; the bisindole pyrroles lynamicins a-d (23, 24, 25, 26) isolated from a novel marine actinomycete marinispora sp. (mcarthur et al., 2008) ; lipoxazolidinones a and b (27, 28) isolated from a marine actinomycete marinispora sp. (macherla et al., 2007) ; marinopyrrole a (29) isolated from an obligate marine streptomyces strain (hughes et al., 2008) ; a furanosesquiterpene (-)-microcionin-1 (30) isolated from a marine sponge fasciospongia sp. (gaspar et al., 2008) ; a macrolide phomolide b (31) isolated from a fungus phomopsis sp. ; a sargaquinoic acid derivative (32) isolated from the brown alga sargassum sagamianum (horie et al., 2008) , and tauramamide (33), a lipopeptide isolated from the bacterium brevibacillus laterosporus png276 (desjardine et al., 2007) . furthermore during this period, several novel marine metabolites with moderate antimicrobial activity (mic or ic 50 ranging from 10 to 50 μg/ml, or 10 to 50 μm, respectively), were also reported, but their weaker antibacterial activity precluded their inclusion in either table 1 or fig. 1 : acetylmajapolene a (mic = 20 μg/disk) (vairappan et al., 2008) , callophycoic acids a, b, g and h (mic = 16-63.9 μg/ml) (lane et al., 2007) ; corallidictyals a, b, c and d (mic less than 20 μg/ ml) ; (s)-(+)-curcuphenol analogs (ic 50 = 34-44 μm) (gul et al., 2007) ; cyclomarazines a and b (mic = 13-18 μg/ ml) (schultz et al., 2008) ; dehydroxychlorofusarielin b (mic = 62.5 μg/ml) (nguyen et al., 2007) , nodosol (mic = 16 μg/ ml) (kontiza et al., 2008) ; paeciloxanthone (mic = 40 μg/disk) , palmitoleic acid (ic 50 = 10-20 μm) (desbois et al., 2008) ; puupehenone-metabolites (mic = 8-16 μg/ml) ; tripalea clavaria c-secosteroids (mic = 25 μg/disk) (rodriguez brasco et al., 2007) , shishididemniols a and b (mic = 20 μg/disk) (kobayashi et al., 2007b) , and zafrin (mic = 50-125 μg/ml) (uzair et al., 2008) . noteworthy were reports of novel marine antimicrobial peptides: dicentracin, a new component of the moronecidin family isolated from head kidney leukocytes from the sea bass dicentrarchus labrax (salerno et al., 2007) ; hepcidins, three novel antimicrobial peptides isolated from the tilapia oreochromis mossambicus, with mics (50-100 μg/ml) against listeria monocytogenes, s. aureus, and enterococcus faecium (huang et al., 2007) ; scygonadin, a novel anionic antimicrobial peptide from the seminal plasma of the mud crab, scylla serrata , and tunichromes, small dehydrodopaminecontaining peptides found in hemocyte cells of the ascidian ascidia nigra that are capable of crosslinking proteins in vitro (cai et al., 2008) . four articles published during 2007-8, reported anticoagulant marine natural products isolated from algae and clams, a number very similar to that reported in our previous review (mayer et al., 2009) , and other reviews of this marine pharmacology series. jung et al. (2007b) characterized a novel 7.7 kda anticoagulant polypeptide termed tgap with a partial sequence (34) from the muscle protein of the south korean bivalve tegillarca granosa. the anticoagulant polypeptide, which demonstrated low in vitro cytotoxicity to venous endothelial cells, specifically inhibited the blood coagulation factor va, as well as the molecular interaction between factor iia and factor va in a ascochytatin (1 thrombin as well as potentiated antithrombin iii-mediated inhibition of coagulation factor xa. ten studies during 2007-8 reported on the antifungal activity of several novel marine natural products isolated from marine algae, bacteria, sponges and sea cucumbers, a decrease from our last review (mayer et al., 2009) , and previous reviews of this series. as shown in table 1 , only one report extended the molecular pharmacology of novel antifungal marine metabolites. symphyocladia latiuscula bromophenols (192, 193, 194, 195 furthermore, several marine natural products showed significant antifungal activity (i.e. mics that were either less than 10 μg/ml, 10 μm, or 10 μg/disk) (table 1 and fig. 1; 38 , 39, 40, 41, 42, 43, 44) , although no mechanism of action studies were reported in the published articles: callipeltins j and k (38, 39), mic = 1 μm (d' auria et al., 2007) , the triterpene glycoside holothurin b (40), mic = 1.56 μg/ ml (kumar et al., 2007) , the macrolide neopeltolide (41), mic = 0.62 μg/ml , the cyclopeptide pedein a (42), mic= 0.6-1.6 μg/ml (kunze et al., 2008) , and pseudoceratins a and b (43, 44), mic = 6.5-8.0 μg/disk (jang et al., 2007b) . hopefully, future studies on the molecular pharmacology of these marine compounds will elucidate their mechanisms of action. finally, several novel structurally characterized marine molecules isolated from sponges demonstrated mics or ic 50 s greater than 10 μg/ ml or 10 μm, and therefore, because of the reported weaker antifungal activity, they have been excluded from table 1 and fig. 1 : eurysterols a and b (mic = 15.6-62.5 μg/ml) (boonlarppradab and faulkner, 2007) ; nagelamides m and n (mic = 33.3 μg/ml) (kubota et al., 2008) , nortetillapyrone (mic = 31-62 μg/ml) (wattanadilok et al., 2007) , and tydemania expeditionis triterpenoids (ic 50 = 26-55 μm) (jiang et al., 2008) . these marine compounds may yet provide additional pharmacological leads in the ongoing global search for clinically useful antifungal agents. as shown in table 1 an increase from our previous review (mayer et al., 2009) , and previous reviews of this series. as shown in table 1 , nine marine molecules were shown during 2007-8 to possess significant antimalarial activity, although mechanism of action studies were reported for only two compounds. tasdemir et al. (2007) reported that (e)-oroidin (45) and (e)-oroidin tfa salt (46) isolated from the turkish marine sponge agelas oroides potently inhibited cultures of multidrug resistant k1 strain of plasmodium falciparum (ic 50 = 3.9 and 7.9 μg/ml, respectively) with concomitant inhibition of fabi (enoyl-acp reductase), a key enzyme of the type ii fatty acid synthase cascade (ic 50 = 0.3 and 5.0 μg/ml, respectively). further studies revealed that oroidin free base appeared to bind to "the enzyme-substrate complex or enzyme-cofactor complex" by an uncompetitive mechanism, providing further pharmacological characterization of the "first antimalarial marine natural product that targets p. falciparum fabi". additional antimalarial activity was reported for seven marine compounds. two reports were contributed during 2007 by the panama international cooperative biodiversity groups project: mcphail et al. (2007) isolated a novel linear lipopeptide dragomabin (47) from the cyanobacterium lyngbya majuscula with moderate antimalarial activity (ic 50 =6.0 μm), and significant differential toxicity between the malarial parasite and mammalian cells. linington et al. (2007) discovered that the new cyclic hexapeptides venturamides a and b (48, 49) isolated from a panamanian marine cyanobacterium oscillatoria sp., demonstrated significant in vitro antiplasmodial activity against the w2 chloroquinone-resistant strain of the parasite (ic 50 =5.6-8.2 μm), with mild cytotoxicity to mammalian cells, the first "example of the identification of cyanobacterial peptides with selective antimalarial activity". kasettrathat et al. (2008) proved that a new tetronic acid, nodulisporacid a (50), from a marine-derived fungus nodulisporium sp. crif1 isolated from a thai soft coral, was moderately antiplasmodial (ic 50 =1-10 μm) against chloroquine-resistant p. falciparum strain 94. na et al. (2008) identified a new marine streptomyces sp. h668 polyether (51) from hawaii that showed in vitro antimalarial activity (ic 50 =0.1-0.2 μg/ml) against both p. falciparum chloroquine-susceptible (d6) and -resistant (w2) clones, with minimal cytotoxicity towards mammalian cells. clark et al. (2008) found that a novel acylproline derivative, tumonoic acid i (52) from the papua new guinean marine cyanobacterium blennothrix cantharidosmum, showed moderate antimalarial activity against p. falciparum (ic 50 =2 μm). pontius et al. (2008a) isolated a heterocyclicsubstituted xanthone, chaetoxanthone b (53) from cultures of a marinederived fungus chaetomium sp. that showed selective activity towards p. falciparum k1 strain (ic 50 =0.5 μg/ml). four marine compounds were reported to possess antiprotozoal activity. kossuga et al. (2008) re-isolated the previously reported plakortide p (54) from the marine sponge plakortis angulospiculatus. the compound displayed selective effects against both leishmania chagasi (ic 50 = 0.5-1.9 μg/ml) and trypanosoma cruzi (ic 50 = 2.3 μg/ ml), with low concomitant hemolytic and cytotoxic activities towards human macrophages. reportedly, the mechanism of action towards intracellular l. chagasi did not appear to involve nitric oxide. simmons et al. (2008) found that two novel lipopeptides viridamides a and b (55, 56) were nearly "equipotent" in inhibiting both leishmania mexicana (ic 50 = 1.1 μm) and t. cruzi (ic 50 = 1.5 μm). besides the antimalarial activity, pontius et al. (2008a) discovered that chaetoxanthone b (53) had selective effects towards t. cruzi (ic 50 = 1.5 μg/ ml), with minimal cytotoxicity towards rat skeletal l6 myoblasts (ic 50 = 47 μg/ml). ten new marine compounds were reported in the global search for novel antituberculosis agents, a considerable increase from our previous reviews (mayer et al., 2009) , and previous reviews of this series. ospina et al. (2007) isolated a bioactive oxapolycyclic diterpene bipinnapterolide b (57) from the colombian gorgonian coral pseudopterogorgia bipinnata which weakly inhibited growth of m. tuberculosis h 37 rv (66% inhibition at 128 μg/ml). zhang et al. (2008) identified two new dimeric naphtha-γ-pyrones 8′-o-demethylnigerone (58) and 8′-o-demethylisonigerone (59) from the marinederived fungus aspergillus carbonarius which showed weak antimycobacterial activity against m. tuberculosis (h 37 rv, mic = 43 and 21.5 μm, respectively). interestingly, the presence of conjugated c=c-c=o bonds in the pyrane ring appeared to be crucial for antifungal activity. as a result of a continued investigation of the caribbean sea whip pseudopterogorgia elisabethae, wei et al. (2007a) reported that the novel tricarbocyclic norditerpenes caribenols a (60) and b (61) weakly inhibited m. tuberculosis (h 37 rv, mic = 128 and 63 μg/ml, respectively). furthermore, wei et al. (2007b) discovered two novel ring b abeo-sterols parguesterols a (62) and b (63) in the caribbean sponge svenzea zeai, which inhibited m. tuberculosis (h 37 rv, mic = 7.8 and 11.2 μg/ml, respectively). hopefully future information on the selectivity index of these two compounds will provide additional information to support the notion that they might "constitute important lead structures for the development of novel tuberculosis drugs due to their strong activity, specificity, and low toxicity". berrue et al. (2007) noted that several bioactive polyketides, 24-norisospiculoic acid a (64), dinorspiculoic acid a (65), and norspiculoic acid a (66) from the caribbean sponge plakortis zyggompha also inhibited m. tuberculosis (h 37 rv, mic 99 = 50 μg/ml). although all of these studies demonstrate that marine terpenes and polyketides constitute potentially novel antituberculosis leads, they unfortunately did not provide detailed mechanism of action pharmacology at the time of their publication. as shown in table 1 , three reports were published on the antiviral pharmacology of novel marine natural products against severe acute respiratory syndrome (sars) corona virus, herpes simplex, and dengue virus during 2007-8. de lira et al. (2007) discovered that the new esculetin-4-carboxylic acid ethyl ester (67) from the brazilian marine sponge axinella cf. corrugata inhibited the sars 3cl protease (ic 50 =46 μm). this is a potentially significant finding because the 3cl protease is a "high profile target" in sars drug development as it appears to be involved in the release of replicative viral proteins as well as the rna polymerase. talarico et al. (2007) reported that a d,l-galactan hybrid c2s-3 (68) isolated from the brazilian marine alga cryptonemia crenulata showed potent antiviral activity against three clinical strains of dengue virus serotype 2 (ic 50 =0.8-16 μg/ml), together with low cytotoxicity. further mechanistic work determined that c2s-3 appeared to be a "promising denv-2 entry inhibitor". mandal et al. (2008) described a sulfated xylomannan isolated from the indian red seaweed scinaia hatei which inhibited hsv-1 and hsv-2 (ic 50 =0.5-1.4 μg/ml), with low cytotoxicity, probably interfering with the hsv-1 multiplication cycle. interestingly the "very good antiviral activity against the wide spectrum of hsv strains tested" suggested this compound might be a good candidate for further preclinical research. five reports contributed preclinical pharmacology of marine compounds active against the human immunodeficiency virus type-1 (hiv-1), the causative agent of the acquired immunodeficiency disease syndrome (aids), an increase from our previous reviews (mayer et al., 2009) , and previous reviews of this series. artan et al. (2008) reported that the phlorglucinol derivative 6, 6′-bieckol (69) isolated from the brown alga e. cava inhibited hiv-induced syncytia formation (ic 50 = 1.72 μm), viral p24 antigen production (ic 50 = 1.26 μm), and the activity of the hiv reverse transcriptase (ic 50 = 1.07 μm), with "no cytotoxicity" at concentrations that inhibited hiv replication "almost completely". cirne-santos et al. (2008) extended the molecular pharmacology of the diterpene dolabelladienetriol (70) isolated from the marine brown alga dictyota pfaffii. the dolabellane diterpene blocked both synthesis and integration of the hiv-1 provirus by noncompetitively inhibiting the reverse transcriptase enzyme (ki = 7.2 μm). the investigators proposed dolabelladienetriol (70) as a "potential new agent for anti-hiv-1 therapy". plaza et al. (2007) described three new depsipeptides mirabamides a, c and d (71, 72, 73) isolated from the sponge siliquariaspongia mirabilis that potently inhibited both hiv-1 in neutralization (ic 50 = 0.14-0.19 μm) and hiv-1 envelope-mediated cell fusion (ic 50 = 0.14-3.9 μm), suggesting these compounds act at an early stage of hiv-1 cell infection, "presumably through interactions with hiv-1 envelope proteins". lu et al. (2007) added a "novel mechanistic profiling" of the previously reported sulfated polymannuroguluronate (spmg) (74), a polysaccharide with an average molecular weight of 8.0 kda isolated from the brown alga laminaria japonica, that has been reported to be in phase ii clinical trials in china as an anti-aids drug candidate. spmg appeared to eliminate the viral gene product known as transactivator of transcription protein (tat)induced signal transduction as well as angiogenesis in aidsassociated kaposi's sarcoma cells. furthermore, in 2008, hui et al. (2008) demonstrated that spmg appeared to show a neuroprotective effect because it decreased apoptosis caused by tat-stimulated calcium overload in pc12 neuronal cells, thus suggesting spmg might warrant further clinical studies in hiv-associated dementia. table 2 summarizes the preclinical pharmacological research completed during 2007-8 with the marine compounds (75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132) shown in fig. 2. the anti-inflammatory pharmacology of marine compounds reported during 2007-8 showed a considerable increase from our previous review (mayer et al., 2009) , and previous reviews of this series. several marine natural products were shown in preclinical pharmacological studies to target arachidonic metabolism in neutrophils and macrophages. pearce et al. (2007a) reported two new tricyclic alkaloids ascidiathiazone a (75) and b (76) isolated from a new zealand ascidian aplidium species that affected superoxide production by human neutrophils in vitro (ic 50 = 0.44-1.55 μm), as well as murine peritoneal neutrophis ex vivo, with concomitant "low or nonexistent" liver and renal toxicity, thus suggesting that these two compounds might become "potential anti-inflammatory pharmaceutical" leads. chao et al. (2008) identified the new cembranolides crassumolides a and c (77, 78) from the soft coral lobophytum crassum which inhibited expression of inos and cox-2 (apparent ic 50 less than 10 μm). similarly, from the same university, cheng et al. (2008) found that new cembranolides from the soft coral lobophytum duru, durumolides a-c (79, 80, 81) inhibited both the inos and cox-2 proteins in lps-activated raw 264.7 cells in vitro (apparent ic 50 less than 10 μm), suggesting that the α-methylene-γ-lactone moiety of these compounds was necessary for the observed activity. shen et al. (2007) showed that new briarane-type diterpenoids frajunolides b and c (82, 83), isolated from the taiwanese gorgonian junceella fragilis, significantly inhibited superoxide anion and elastase generation from human neutrophils in vitro (apparent ic 50 greater than 10 μg/ml). dang et al. (2008) demonstrated that the previously described fatty acids (84, 85) from the south korean marine red alga gracilaria verrucosa inhibited release by lps-activated murine macrophages of the inflammatory nitric oxide, tumor necrosis factor α and interleukin-6 (apparent ic 50 less than 20 μg/ml), thus revealing that the conjugated enone moiety played a critical role in the antiinflammatory activity observed in vitro. bittencourt et al. (2008) contributed novel preclinical pharmacology on a previously reported 90 amino acid polypeptide (86) isolated from the red alga hypnea cervicornis. the mucin-binding agglutinin, which was extensively characterized with several in vivo models of nociception and inflammation, probably exerted its anti-inflammatory activity (apparent ic 50 = 0.1-1 mg/kg) via interaction of the polypeptide with the lectin carbohydrate-binding site, although the exact mechanism of action remains undetermined. el sayed et al. (2008) demonstrated that manzamine a (87), (−)-8-hydroxymanzamine a (88), and hexahydro-8-hydroxymanzamine a (89) potently inhibited txb 2 generation (ic 50 = 0.25, less than 0.1, and 1.97 μm, respectively) in brain microglia. these findings provide further support to the notion that manzamine alkaloids appear to be "viable anti-inflammatory leads" for the preclinical pharmaceutical development of agents to modulate both activated brain microglia and eicosanoid generation. treschow et al. (2007) investigated four novel ω-3 polyunsaturated fatty acids (90, 91, 92, 93) from the new zealand green-lipped mussel perna canaliculus with an in vitro bioassay that used stimulated human neutrophil 5-lipoxygenase, demonstrating that the putative antiinflammatory potential of these compounds might be related to inhibition of leukotriene and prostaglandin metabolite production. sansom et al. (2007) reported a bis-prenylated quinone (94) from the new zealand brown alga perithalia capillaris which inhibited superoxide anion production in human neutrophils (ic 50 = 2.1 μm) in vitro, with low toxicity. an explanation as to why the authors decided not to investigate the quinone "as an anti-inflammatory lead" remains unclear, although it was strongly cytotoxic towards human leukemia cells (ic 50 = 0.34 μm). sugiura et al. (2007) characterized a novel antiallergic phlorotannin phlorofucofuroeckol b (pff-b) (95) from the edible brown alga eisenia arborea. pff-b, inhibited β-hexosaminidase release from rat basophilic leukemia cells (ic 50 = 7.8 μm) in vitro, thus showing higher potency than tranilast (rizaben®) (ic 50 = 46.6 μm), a pharmaceutical agent used for treatment of allergic disorders such as asthma, allergic rhinitis and atopic dermatitis in japan and south korea. kossuga et al. (2008) demonstrated that the polyketide plakortide p (54) isolated from the brazilian sponge p. angulospiculatus, potently inhibited thromboxane b 2 release (ic 50 = 0.93 μm) from activated rat brain microglia, thus extending the preclinical pharmacology of this compound, which besides being antiparasitic as discussed earlier in this review, also appears to be a potentially "novel antineuroinflammatory agent". pearce et al. (2007b) examined a halogenated furanone rubrolide o (96) isolated from a new zealand ascidian synoicum n. sp., which inhibited superoxide anion production in human neutrophils (ic 50 = 35 μm) in vitro with low toxicity. although rubrolides have been reported to be cytotoxic and antibacterial, the authors proposed that the anti-inflammatory activity for these tunicate metabolites though moderate was "unprecedented". khan et al. (2007) identified a ω-3 polyunsaturated fatty acid stearidonic acid (97) from the south korean brown seaweed undaria pinnatifida, which was shown to be very active against pmainduced mouse ear inflammation symptoms, edema, erythema and blood flows (ic 50 = 160, 314 and 235 μg/per ear, respectively). the in vivo data compared well with indomethacin which was used as a positive control (ic 50 = 90, 172, 179 μg/per ear, respectively), and thus supported claims that this "seaweed can be used as a remedy for inflammation-related symptoms". kobayashi et al. (2007a) discovered a novel dimeric oroidin derivative carteramine a (98) in the marine sponge stylissa carteri, and showed that it inhibited neutrophil chemotaxis (ic 50 = 5 μm). the authors suggested that because carteramine a has no structural resemblance to known compounds that inhibit neutrophil chemotaxis, their finding provides a "novel platform to develop a new class of anti-inflammatory agents". taori et al. (2007) identified three new analogues of dolastatin 13, lyngbyastatins 5-7 (99, 100, 101), isolated from marine cyanobacteria lyngbya spp. from south florida. although the three compounds selectively and potently inhibited porcine pancreatic elastase (ic 50 = 3-10 nm), suggesting a potential therapeutic use in pathophysiological conditions where elastase overactivity is involved, no mechanism of action studies were reported at the time. oh et al. (2008) purified a novel polyketide salinipyrone a (102) from the marine actinomycete salinispora pacifica, which moderately inhibited interleukin-5 (ic 50 = 10 μg/ml) in a mouse splenocyte model of allergic inflammation, with low cell cytotoxicity. the pharmacology of the marine compounds reporting activity on the immune system during 2007-8 showed a considerable increase from our previous review (mayer et al., 2009) , and previous reviews of this series. kawauchi et al. (2007) investigated the red pigment cycloprodigiosin hydrochloride (103) isolated from the marine bacterium pseudoalteromonas denitrificans. interestingly, the compound suppressed activator protein 1 (ap-1) (apparent ic 50 = 1 μm), and downstream gene expression of interleukin 8, a chemokine involved in the innate immune system. courtois et al. (2008) assessed the effect of the known compound floridoside (104), isolated from the french red alga mastocarpus stellatus on the complement system. floridoside was observed to potently activate the classical complement pathway (apparent ic 50 = 5.9-9.3 μg/ml) by recruiting immunoglobulin m (igm), suggesting that the compound might be an "important step in the development of a potent new anticomplementary agent" useful in therapy aimed at addressing complement depletion. greve et al. (2007) reported that the novel iso-iantheran a and 8-carboxy-iso-iantheran a (105, 106) purified from the australian marine sponge ianthella quadrangulata demonstrated agonist activity at p2y 11 receptors (ic 50 = 1.29 and 0.48 μm, respectively). this finding represents an interesting contribution to ionotropic purine receptor p2y 11 pharmacology, because these receptors have been shown to affect the maturation and differentiation of dendritic cells. huh et al. (2007) extended the pharmacology of prodigiosin (107) isolated from the marine bacterium hahella chejuencis collected in south korea. prodigiosin inhibited inos mrna expression and no production (apparent ic 50 = 0.1 μg/ml) by a mechanism that involved inhibition of nf-κb and p38 mapk and jnk phosphorylation. he et al. (2007) characterized the pharmacology of a water soluble polysaccharide (108) isolated from the mollusc arca subcrenata lischke, a popular chinese seafood, and named it aslp. aslp stimulated mouse spleen lymphocyte proliferation in a concentration-dependent manner (apparent ic 50 less than 100 μg/ml), and with the "branches of aslp" being required for the immunomodulatory bioactivity. aminin et al. (2008) described the immunostimulant activity of frondoside a (109), a triterpene glycoside isolated from the sea cucumber cucumaria frondosa. the glycoside was shown to stimulate lysosomal activity and phagocytosis in mouse macrophages, as well as reactive oxygen species formation. oda et al. (2007) provided novel information on the molecular mechanisms affected by smenospongidine, smenospongiarine and smenospongine (110, 111, 112) , sesquiterpene quinones previously isolated from a palauan marine sponge hippospongia sp. the observation that at 10 μg/ml, these compounds promoted interleukin-8 release, a member of the c-x-c chemokine superfamily which is involved in tumor progression and metastasis, suggested that "the functional group at c-18" might play a yet undetermined role in the observed results. yamada et al. (2007) isolated the novel macrosphelide m (113) and peribysin j (114) from periconia byssoides, a fungal strain discovered from the sea hare aplysia kurodai. significantly, both fungal metabolites inhibited the adhesion of human promyelocytic leukemia hl-60 cells to human umbilical vein endothelial cells (ic 50 = 33.2 and 11.8 μm, respectively) more potently than herbimycin a (ic 50 = 38 μm). the latter compound, a benzochinoid ansamycin antibiotic isolated from streptomyces sp. was used as a positive control in these studies. lu et al. (2008) contributed a novel cembranolide querciformolide c (115) from the soft coral sinularia querciformis, which significantly inhibited the activation of the inos and cox-2 enzymes (apparent ic 50 less than 10 μm) in lps-activated murine macrophages in vitro. ponomarenko et al. (2007) discovered that new diterpenoids (116, 117) isolated from a northern cook islands marine sponge spongia (heterofibria) sp. moderately activated murine splenocytes lysosomes (apparent ic 50 greater than 100 μg/ml). oh et al. (2007) characterized a novel cyclic peptide thalassospiramide b (118) isolated from a marine bacterium thalassospira sp., which showed immunosuppressive activity in an interleukin-5 (il-5) inhibition assay (ic 50 = 5 μm); an interesting finding because il-5 is expressed both in eosinophils and mast cells in asthmatic patients. pharmacological studies with marine compounds affecting the nervous system involved three areas of neuropharmacology: the stimulation of neurogenesis, the targeting of receptors, and other miscellaneous activities on the nervous system. marine natural products reported to be neuritogenic, might be used to treat damaged neuronal cells, and potentially neurodegenerative diseases. as shown in table 2 , compounds (119, 120, 121, 122, 123, 124, 125, 126) isolated from sea stars and a brown alga were observed to enhance the neuritogenic properties of nerve growth factor (ngf), a compound that has a critical role in differentiation, survival, and neuronal regeneration. kicha et al. (2007b) contributed two new steroid glycosides, linckosides l1 (119) and l2 (120) isolated from the vietnamese blue sea star linckia laevigata. all three glycosides (apparent ic 50 = 0.3 μm) showed synergistic effects on ngf-induced (2 ng/ml) neurite outgrowth of murine neuroblastoma c-1300 cells. han et al. (2007) contributed the novel steroid glycosides linckosides m-q (121, 122, 123, 124, 125) from the japanese sea star l. laevigata. linckoside p (124) induced neurite outgrowth in 55% of rat pheochromocytoma pc12 cells at 10 μm in the presence of nerve growth factor (ngf), while linckosides m-o appeared less active (21-32%), suggesting the importance of "the xylose on a side chain". ina et al. (2007) characterized pheophytin a (126) purified from the japanese brown alga sargassum fulvellum as a novel neurodifferentiation compound. pheophytin a at 3.9 μg/ml was observed to synergize with ngf (50 ng/ml) in promoting neurite outgrowth in rat pheochromocytoma pc12 cells by a mechanism that appeared to involve activation of mitogen-activated protein kinase signaling. as shown in table 2 , during 2007-8, three marine compounds (127, 128, 129) were reported to target receptors in the nervous system. peng et al. (2008) reported on the preclinical pharmacology of the α4/7conotoxin lp1.1 (127) originally isolated from the marine cone snail conus leopardus. the conotoxin induced seizure and paralysis in goldfish, and selectively yet reversibly inhibited acetylcholine-evoked currents in xenopus oocytes expressing rat α6α3β2 and α3β2 nicotinic receptors (apparent ic 50 less than 10 μm). aiello et al. (2007) characterized two novel bromopyrrole alkaloids damipipecolin (128) and damituricin (129) isolated from the mediterranean sponge axinella damicornis that were observed to modulate serotonin receptor activity in vitro. although damipipecolin inhibited ca 2+ entry in neurons (apparent ic 50 =1 μg/ml), the serotonin receptor subtype involved in the mechanism of action remains undetermined. finally, as shown in table 2 , during 2007-8, additional marine compounds (74 and 130, 131, 132) were reported to have miscellaneous effects on components of the nervous system. contributing to the ongoing search for acetylcholinesterase inhibitors useful in the treatment of alzheimer's disease, langjae et al. (2007) reported a new stigmastane-type steroidal alkaloid 4-acetoxy-plakinamine b (130), isolated from a thai marine sponge corticium sp. that significantly inhibited acetylcholinesterase (ic 50 = 3.75 μm). compound 130 was reported to be the "first marine-derived acetylcholinesterase-inhibiting steroidal alkaloid" with a mechanism of action involving an unusual mixed-competitive mode of inhibition. choi et al. (2007) extended the molecular pharmacology of two known meroditerpenes, sargaquinoic acid (131) and sargachromenol (132) isolated from the brown alga s. sagamianum. sargaquinoic acid potently inhibited butyrylcholinesterase (ic 50 = 26 nm), a novel target for the treatment of alzheimer's disease, with potency comparable to or greater than anticholinesterases in current clinical use. hui et al. (2008) further characterized the neuroprotective effects of the polysaccharide sulfated polymannuroguluronate (spmg) (74), noted earlier as currently being in phase ii clinical trials in china as an anti-aids drug candidate. spmg appeared to decrease the tat-stimulated calcium overload in pc12 neuronal cells as well as concomitant apoptosis-signaling cascades, thus demonstrating a potential for further clinical development as a therapeutic intervention in hivassociated dementia. table 3 lists 65 marine compounds with miscellaneous pharmacological mechanisms of action, and their respective structures (133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197) are presented in fig. 3 . because during 2007-8 additional pharmacological data were unavailable, it was not possible to assign these marine compounds to a particular drug class as was possible with the compounds included in tables 1 and 2. table 3 shows 14 marine natural products that the peer-reviewed literature reported with a pharmacological activity, an ic 50 , and a molecular mechanism of action affected by the respective compound: azumamide e (133), 1-deoxyrubralactone (134), fucoxanthin (135), okadaic acid (136), saproxanthin and myxol (137, 138), sarcomilasterol (139), spirastrellolides c, d, and e (140, 141, 142), spongia sesterterpenoid (143), stylissadines a and b (144, 145) and symbiodinolide (146). in contrast, although a pharmacological activity was described, and an ic 50 for inhibition of an enzyme or receptor determined, detailed molecular mechanism of action studies were unavailable at the time of publication for the following 51 marine compounds included in table 3 : asterias amurensis saponin (147), botrytis sp. α-pyrone derivative (148) (185), sargassum siliquastrum meroditerpenoids (186, 187, 188, 189, 190, 191) , symphyocladia latiuscula bromophenols (192, 193, 194, 195) , and tenacibactins c and d (196, 197) . several reviews covering both general and specific subject areas of marine pharmacology were published during 2007-8: (a) general marine pharmacology: marine natural products with an emphasis on source organisms and relevant biological activities (blunt et al., 2007 (blunt et al., , 2008 ; the value of natural products to future pharmaceutical discovery ; the structures and bioactivities of secondary metabolites from cyanobacteria (gademann and portmann, 2008) ; bioactive natural products from marine cyanobacteria for drug discovery (tan l.t., 2007) ; bioactive compounds from fungi in the south china sea (pan et al., 2008) ; biochemical and pharmacological functions of β-carboline alkaloids (cao et al., 2007) ; pharmacological properties of lamellarin alkaloids (kluza et al., 2008) ; biological activity of brown seaweed fucoidans (kusaykin et al., 2008; ; potential pharmacological uses of the marine green alga polysaccharide ulvan (lahaye and robic, 2007) ; pharmacological properties of marine bis-and tris-indole alkaloids (gupta et al., 2007) ; (b) antimicrobial marine pharmacology: a renaissance of genomics in antibacterial discovery from actinomycetes (baltz r.h., 2008) ; mining marine genomics for novel drug discovery from phytosymbionts of marine invertebrates (dunlap et al., 2007) ; (c) anticoagulant and cardiovascular pharmacology: structure, biology, evolution and medical importance of sulfated fucans and galactans as potential antithrombotic compounds (pomin and mourao, 2008) ; (d) antituberculosis, antimalarial and antifungal marine pharmacology: natural product growth inhibitors of mycobacterium tuberculosis (copp and pearce, 2007) ; new advances in novel marine fatty acids as antimalarials, antimycobacterial and antifungal agents (carballeira n.m., 2008) ; depsipeptides from microorganisms as a new class of antimalarials (fotie and morgan, 2008) ; the manzamine alkaloids for the control of malaria and tuberculosis (hamann m.t., 2007) ; (e) immuno-and anti-inflammatory marine pharmacology: chemistry and biology of anti-inflammatory marine natural products (abad et al., 2008) ; marine natural products as targeted modulators of the transcription factor nf-κb (folmer et al., 2008) ; glycolipids as immunostimulating agents ); (f) nervous system marine pharmacology: marine-derived drugs in neurology (martinez a., 2007) ; neuritogenic gangliosides from marine echinoderms (higuchi et al., 2007) ; (g) miscellaneous molecular targets: enzyme inhibitors from marine invertebrates (nakao and fusetani, 2007) ; natural products as aromatase inhibitors (balunas et al., 2008) ; biology of the aeruginosin family of serine protease inhibitors (ersmark et al., 2008) ; and marine natural products affecting membrane and intracellular processes, and ion channels (folmer et al., 2007) . six years after the approval of the marine compound ziconotide (prialt®) by the u.s. food and drug administration (williams et al., 2008) , the global marine preclinical pharmaceutical pipeline remains very active. the marine pharmaceutical clinical pipeline is available at http://marinepharmacology.midwestern.edu/clindev.htm and has recently been reviewed (mayer et al., 2010) . the current marine preclinical pipeline review contributes to the annual review series which was initiated in 1998 (mayer and lehmann, 2000; mayer and hamann, 2002 , 2005 mayer et al., 2007 mayer et al., , 2009 ) and reveals the breadth of preclinical pharmacological research during 2007-8, which resulted from the global effort of natural products chemists and pharmacologists from argentina, australia, brazil, canada, china, egypt, france, germany, india, israel, italy, japan, the netherlands, new zealand, panama, papua new guinea, portugal, russia, south korea, spain, switzerland, taiwan, thailand, turkey, united kingdom, vietnam, and the united states. thus, it appears that the marine preclinical pharmaceutical pipeline continues to contribute novel pharmacological lead compounds that will probably enable a future expansion of the marine clinical pharmaceutical pipeline, which currently consists of 13 compounds in phase i, ii and iii of clinical development (mayer et al., 2010) . natural marine anti-inflammatory products chaetominedione, a new tyrosine kinase inhibitor isolated from the algicolous marine fungus chaetomium sp isolation and structure determination of malevamide e, a dolastatin 14 analogue, from the marine cyanobacterium symploca laete-viridis immunomodulatory effects of an enzymatic extract from ecklonia cava on murine splenocytes damipipecolin and damituricin, novel bioactive bromopyrrole alkaloids from the mediterranean sponge axinella damicornis immunomodulatory properties of frondoside a, a major triterpene glycoside from the north atlantic commercially harvested sea cucumber cucumaria frondosa isolation and structures of erylosides from the caribbean sponge erylus formosus anti-hiv-1 activity of phloroglucinol derivative, 6, 6′-bieckol, from ecklonia cava biological effects of a sulfated-polysaccharide isolated from the marine red alga champia feldmannii the value of natural products to future pharmaceutical discovery renaissance in antibacterial discovery from actinomycetes natural products as aromatase inhibitors the marine sponge plakortis zyggompha: a source of original bioactive polyketides disturbance of voltage-induced cellular calcium entry by marine dimeric and tetrameric pyrrole-imidazole alkaloids antinociceptive and antiinflammatory effects of a mucin-binding agglutinin isolated from the red marine alga hypnea cervicornis marine natural products marine natural products eurysterols a and b, cytotoxic and antifungal steroidal sulfates from a marine sponge of the genus euryspongia actinomycetes from sediments in the trondheim fjord, norway: diversity and biological activity the crosslinking and antimicrobial properties of tunichrome β-carboline alkaloids: biochemical and pharmacological functions new advances in fatty acids as antimalarial, antimycobacterial and antifungal agents protein phosphatase inhibitors isolated from spongia irregularis collected in papua new guinea monoglycerides from the brown alga sargassum sagamianum: isolation, synthesis, and biological activity cytotoxic and anti-inflammatory cembranoids from the soft coral lobophytum crassum polysaccharides from gracilaria corticata: sulfation, chemical characterization and anti-hsv activities durumolides a-e, anti-inflammatory and antibacterial cembranolides from the soft coral lobophytum durum anticholinesterase activity of plastoquinones from sargassum sagamianum: lead compounds for alzheimer's disease therapy studies on puupehenone-metabolites of a dysidea sp.: structure and biological activity the dolabellane diterpene dolabelladienetriol is a typical noncompetitive inhibitor of hiv-1 reverse transcriptase enzyme natural products chemistry and taxonomy of the marine cyanobacterium blennothrix cantharidosmum natural product growth inhibitors of mycobacterium tuberculosis floridoside extracted from the red alga mastocarpus stellatus is a potent activator of the classical complement pathway 19-epi-okadaic acid, a novel protein phosphatase inhibitor with enhanced selectivity isolation and structural elucidation of callipeltins j-m: antifungal peptides from the marine sponge latrunculia sp anti-inflammatory constituents of the red alga gracilaria verrucosa and their synthetic analogues antimicrobial potential of a lipopeptide biosurfactant derived from a marine bacillus circulans a sars-coronovirus 3cl protease inhibitor isolated from the marine sponge axinella cf. corrugata: structure elucidation and synthesis isolation and structural characterization of two antibacterial free fatty acids from the marine diatom, phaeodactylum tricornutum tauramamide, a lipopeptide antibiotic produced in culture by brevibacillus laterosporus isolated from a marine habitat: structure elucidation and synthesis bioprotective properties of seaweeds: in vitro evaluation of antioxidant activity and antimicrobial activity against food borne bacteria in relation to polyphenolic content three new antimicrobial metabolites of phomopsis sp highly brominated mono-and bis-phenols from the marine red alga symphyocladia latiuscula with radical-scavenging activity biomedicinals from the phytosymbionts of marine invertebrates: a molecular approach semisynthetic studies on the manzamine alkaloids cyp3a4 inhibitors isolated from a marine derived fungus penicillium species essramycin: a first triazolopyrimidine antibiotic isolated from nature novel bioactive metabolites from a marine derived bacterium nocardia sp. alaa chemistry and biology of the aeruginosin family of serine protease inhibitors antibiotics for emerging pathogens biomedical research tools from the seabed marine natural products as targeted modulators of the transcription factor nf-κb depsipeptides from microorganisms: a new class of antimalarials antileishmanial properties of tropical marine algae extracts secondary metabolites from cyanobacteria: complex structures and powerful bioactivities isomeric furanosesquiterpenes from the portuguese marine sponge fasciospongia sp t-antigen binding lectin with antibacterial activity from marine invertebrate, sea cucumber (holothuria scabra): possible involvement in differential recognition of bacteria new iantherans from the marine sponge ianthella quadrangulata: novel agonists of the p2y(11) receptor bioactive metabolites from the caribbean sponge aka coralliphagum chemical transformation and biological studies of marine sesquiterpene (s)-(+)-curcuphenol and its analogs bis-and tris-indole alkaloids from marine organisms: new leads for drug discovery the manzamines as an example of the unique structural classes available for the discovery and optimization of infectious disease controls based on marine natural products linckosides m-q: neuritogenic steroid glycosides from the okinawan starfish linckia laevigata polybrominated diphenyl ethers from the indonesian sponge lamellodysidea herbacea isolation and structural characterization of a novel polysaccharide prepared from arca subcrenata lischke biologically active gangliosides from echinoderms antibacterial quinone metabolite from the brown alga, sargassum sagamianum batzelladine alkaloids from the caribbean sponge monanchora unguifera and the significant activities against hiv-1 and aids opportunistic infectious pathogens three different hepcidins from tilapia, oreochromis mossambicus: analysis of their expressions and biological functions the marinopyrroles, antibiotics of an unprecedented structure class from a marine streptomyces sp prodigiosin isolated from hahella chejuensis suppresses lipopolysaccharide-induced no production by inhibiting p38 mapk, jnk and nf-kappab activation in murine peritoneal macrophages sulfated polymannuroguluronate, a novel anti-acquired immune deficiency syndrome drug candidate, decreased vulnerability of pc12 cells to human immunodeficiency virus tat protein through attenuating calcium overload pheophytin a, a low molecular weight compound found in the marine brown alga sargassum fulvellum, promotes the differentiation of pc12 cells tenacibactins a-d, hydroxamate siderophores from a marine-derived bacterium, tenacibaculum sp. a4k-17 pseudoceratins a and b, antifungal bicyclic bromotyrosine-derived metabolites from the marine sponge pseudoceratina purpurea aaptamines as sortase a inhibitors from the tropical sponge aaptos aaptos structures and absolute configurations of sulfateconjugated triterpenoids including an antifungal chemical defense of the green macroalga tydemania expeditionis sulfated polysaccharide purified from ecklonia cava accelerates antithrombin iii-mediated plasma proteinase inhibition a novel anticoagulant protein with high affinity to blood coagulation factor va from tegillarca granosa meroditerpenoids from the brown alga sargassum siliquastrum new sulfoalkylresorcinol from marine-derived fungus, zygosporium sp. knc52 ascochytatin, a novel bioactive spirodioxynaphthalene metabolite produced by the marine-derived fungus, ascochyta sp. ngb4 antimicrobial activity of volatile components and various extracts of the red alga jania rubens cytotoxic and antiplasmodial substances from marine-derived fungi, nodulisporium sp. and cri247-01 suppression of ap-1 activity by cycloprodigiosin hydrochloride isolation of two anti-inflammatory and one pro-inflammatory polyunsaturated fatty acids from the brown seaweed undaria pinnatifida sulfated steroid glycosides from the vietnamese starfish linckia laevigata new neuritogenic teroid glycosides from the vietnamese starfish linckia laevigata symbiodinolide, a novel polyol macrolide that activates n-type ca 2+ channel, from the symbiotic marine dinoflagellate symbiodinium sp identification of an antibacterial protein as l-amino acid oxidase in the skin mucus of rockfish sebastes schlegeli antibacterial action of l-amino acid oxidase from the skin mucus of rockfish sebastes schlegelii lamellarin alkaloids: structure and pharmacological properties carteramine a, an inhibitor of neutrophil chemotaxis, from the marine sponge stylissa carteri complete structure elucidation of shishididemniols, complex lipids with tyramine-derived tether and two serinol units, from a marine tunicate of the family didemnidae new metabolites with antibacterial activity from the marine angiosperm cymodocea nodosa antiparasitic, antineuroinflammatory, and cytotoxic polyketides from the marine sponge plakortis angulospiculatus collected in brazil potential cancer chemopreventive in vitro activities of monomeric xanthone derivatives from the marine algicolous fungus monodictys putredinis nagelamides m and n, new bromopyrrole alkaloids from sponge agelas species antifungal activity in triterpene glycosides from the sea cucumber actinopyga lecanora pedein a and b: production, isolation, structure elucidation and biological properties of new antifungal cyclopeptides from chondromyces pediculatus (myxobacteria) structure, biological activity, and enzymatic transformation of fucoidans from the brown seaweeds pf1270a, b and c, novel histamine h3 receptor ligands produced by penicillium waksmanii pf1270 structure and functional properties of ulvan, a polysaccharide from green seaweeds callophycoic acids and callophycols from the fijian red alga callophycus serratus acetylcholinesterase-inhibiting steroidal alkaloid from the sponge corticium sp inhibition of the pathogenicity of magnaporthe grisea by bromophenols, isocitrate lyase inhibitors, from the red alga odonthalia corymbifera solution structures and biological functions of the antimicrobial peptide, arenicin-1, and its linear derivative inhibition of candida albicans isocitrate lyase activity by sesterterpene sulfates from the tropical sponge dysidea sp natural bromophenols from the marine red alga polysiphonia urceolata (rhodomelaceae): structural elucidation and dpph radical-scavenging activity cephalosporolides h and i, two novel lactones from a marine-derived fungus, penicillium sp fucoidan: structure and bioactivity bromophenols from the marine red alga polysiphonia urceolata with dpph radical scavenging activity cathepsin b inhibitory activities of three new phthalate derivatives isolated from seahorse venturamides a and b: antimalarial constituents of the panamanian marine cyanobacterium oscillatoria sp two new steroidal compounds from starfish asterias amurensis lutken antioxidant alkaloid from the south china sea marine sponge iotrochota sp sulfated polymannuroguluronate, a novel anti-aids drug candidate, inhibits hiv-1 tatinduced angiogenesis in kaposi's sarcoma cells anti-inflammatory cembranoids from the soft corals sinularia querciformis and sinularia granosa lipoxazolidinones a, b, and c: antibacterial 4-oxazolidinones from a marine actinomycete isolated from a guam marine sediment anti-herpetic activity of a sulfated xylomannan from scinaia hatei new bioactive hydrogenated linderazulene-derivatives from the gorgonian echinogorgia complexa heparinoid-active two sulfated polysaccharides isolated from marine green algae monostroma nitidum marine-derived drugs in neurology antimicrobial and cytotoxic assessment of marine cyanobacteria -synechocystis and synechococcus lyngbyastatin 4, a dolastatin 13 analogue with elastase and chymotrypsin inhibitory activity from the marine cyanobacterium lyngbya confervoides pompanopeptins a and b, new cyclic peptides from the marine cyanobacterium lyngbya confervoides molecular insights into azumamide e histone deacetylases inhibitory activity marine pharmacology in 1998 antitumor and cytotoxic compounds marine pharmacology in 2000: antitumor and cytotoxic compounds marine pharmacology in 2001-2: antitumour and cytotoxic compounds marine pharmacology in 2003-2004: anti-tumour and cytotoxic compounds marine pharmacology in 2005-2006: antitumour and cytotoxic compounds marine pharmacology in 1999: compounds with antibacterial, anticoagulant, antifungal, anti-inflammatory, anthelmintic, antiinflammatory, antiplatelet, antiprotozoal and antiviral activities;affecting the cardiovascular, endocrine, immune, and nervous systems; and other miscellaneous mechanisms of action marine pharmacology in 2000: marine compounds with antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiplatelet, antituberculosis, and antiviral activities; affecting the cardiovascular, immune, and nervous systems and other miscellaneous mechanisms of action marine pharmacology in 2001-2002: marine compounds with anthelmintic, antibacterial, anticoagulant, antidiabetic, antifungal, anti-inflammatory, antimalarial, antiplatelet, antiprotozoal, antituberculosis, and antiviral activities; affecting the cardiovascular, immune and nervous systems and other miscellaneous mechanisms of action marine pharmacology in 1998: marine compounds with antibacterial, anticoagulant, antifungal, anti-inflammatory, anthelmintic, antiplatelet, antiprotozoal, and antiviral activities; with actions on the cardiovascular, endocrine, immune, and nervous systems; and other miscellaneous mechanisms of action marine pharmacology in 1999: antitumor and cytotoxic compounds marine pharmacology in 2003-4: marine compounds with anthelmintic antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiplatelet, antiprotozoal, antituberculosis, and antiviral activities; affecting the cardiovascular, immune and nervous systems, and other miscellaneous mechanisms of action marine pharmacology in 2005-6: marine compounds with anthelmintic, antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiprotozoal, antituberculosis, and antiviral activities; affecting the cardiovascular, immune and nervous systems, and other miscellaneous mechanisms of action the odyssey of marine pharmaceuticals: a current pipeline perspective lynamicins a-e, chlorinated bisindole pyrrole antibiotics from a novel marine actinomycete antimalarial linear lipopeptides from a panamanian strain of the marine cyanobacterium lyngbya majuscula evaluation of selected tropical seaweeds for in vitro anti-trichomonal activity a new antimalarial polyether from a marine streptomyces sp. h668 1-deoxyrubralactone, a novel specific inhibitor of families x and y of eukaryotic dna polymerases from a fungal strain derived from sea algae marine algae: screening for a potent antibacterial agent enzyme inhibitors from marine invertebrates scalarane sesterterpenes from a marine sponge of the genus spongia and their fxr antagonistic activity dehydroxychlorofusarielin b, an antibacterial polyoxygenated decalin derivative from the marine-derived fungus aspergillus sp promotion of il-8 production in pma-stimulated hl-60 cells by sesquiterpene quinones from a marine sponge, hippospongia sp thalassospiramides a and b, immunosuppressive peptides from the marine bacterium thalassospira sp salinipyrones and pacificanones, mixed-precursor polyketides from the marine actinomycete salinispora pacifica ariakemicins a and b, novel polyketide-peptide antibiotics from a marine gliding bacterium of the genus rapidithrix bipinnapterolide b, a bioactive oxapolycyclic diterpene from the colombian gorgonian coral pseudopterogorgia bipinnata review of bioactive compounds from fungi in the south china sea anti-inflammatory thiazine alkaloids isolated from the new zealand ascidian aplidium sp.: inhibitors of the neutrophil respiratory burst in a model of gouty arthritis e/z-rubrolide o, an anti-inflammatory halogenated furanone from the new zealand ascidian synoicum n. sp alpha4/ 7-conotoxin lp1.1 is a novel antagonist of neuronal nicotinic acetylcholine receptors mirabamides a-d, depsipeptides from the sponge siliquariaspongia mirabilis that inhibit hiv-1 fusion structure, biology, evolution, and medical importance of sulfated fucans and galactans spongian diterpenoids from the sponge spongia (heterofibria) sp antiprotozoal activities of heterocyclic-substituted xanthones from the marine-derived fungus chaetomium sp monodictyochromes a and b, dimeric xanthone derivatives from the marine algicolous fungus monodictys putredinis isolation and purification of an anticoagulant from fermented red seaweed lomentaria catenata lipid extract of nostoc commune var. sphaeroides kutzing, a blue-green alga, inhibits the activation of sterol regulatory element binding proteins in hepg2 cells antimicrobial ambiguines from the cyanobacterium fischerella sp. collected in israel new c-secosteroids from the gorgonian tripalea clavaria extending the record of meroditerpenes from cacospongia marine sponges radical scavenging and singlet oxygen quenching activity of marine carotenoid fucoxanthin and its metabolites cdna sequence and tissue expression of an antimicrobial peptide, dicentracin; a new component of the moronecidin family isolated from head kidney leukocytes of sea bass, dicentrarchus labrax an antiproliferative bis-prenylated quinone from the new zealand brown alga perithalia capillaris antimycobacterial activity of the red alga polysiphonia virgata antitumor and cytotoxic compounds from marine organisms biosynthesis and structures of cyclomarins and cyclomarazines, prenylated cyclic peptides of marine actinobacterial origin four new briarane diterpenoids from the gorgonian coral junceella fragilis diapolycopenedioic acid xylosyl ester, a novel glyco-c 30 -carotenoic acid produced by a new marine bacterium rubritalea squalenifaciens viridamides a and b, lipodepsipeptides with antiprotozoal activity from the marine cyanobacterium oscillatoria nigro-viridis anti-allergic phlorotannins from the edible brown alga, eisenia arborea isolation and characterization of okadaic acid binding proteins from the marine sponge halichondria okadai screening for antibacterial and antifungal activities in marine benthic invertebrates from northern norway an algalderived dl-galactan hybrid is an efficient preventing agent for in vitro dengue virus infection bioactive natural products from marine cyanobacteria for drug discovery lyngbyastatins 5-7, potent elastase inhibitors from floridian marine cyanobacteria, lyngbya spp kempopeptins a and b, serine protease inhibitors with different selectivity profiles from a marine cyanobacterium, lyngbya sp marine natural products from the turkish sponge agelas oroides that inhibit the enoyl reductases from plasmodium falciparum, mycobacterium tuberculosis and escherichia coli novel anti-inflammatory omega-3 pufas from the new zealand greenlipped mussel, perna canaliculus the isolation, purification and biological activity of a novel antibacterial compound produced by pseudomonas stutzeri antibacterial activity of halogenated sesquiterpenes from malaysian laurencia spp a new 9, 11-secosterol from the vietnamese sea soft coral, sarcophyton mililatensis, increases the function of osteoblastic mc3t3-e1 cells a malespecific expression gene, encodes a novel anionic antimicrobial peptide, scygonadin, in scylla serrata structural features and anti-hiv-1 activity of novel polysaccharides from red algae grateloupia longifolia and grateloupia filicina antifungal activity evaluation of the constituents of haliclona baeri and haliclona cymaeformis, collected from the gulf of thailand caribenols a and b, sea whip derived norditerpenes with novel tricarbocyclic skeletons novel ring b abeo-sterols as growth inhibitors of mycobacterium tuberculosis isolated from a caribbean sea sponge, svenzea zeai paeciloxanthone, a new cytotoxic xanthone from the marine mangrove fungus paecilomyces sp spirastrellolides c to g: macrolides obtained from the marine sponge spirastrella coccinea saliniketals a and b, bicyclic polyketides from the marine actinomycete salinispora arenicola ziconotide: an update and review neopeltolide, a macrolide from a lithistid sponge of the family neopeltidae glycolipids as immunostimulating agents antibacterial and antilarval activity of deep-sea bacteria from sediments of the west pacific ocean cell-adhesion inhibitors produced by sea hare-derived periconia sp. iii. absolute stereostructures of peribysins j and macrosphelide m a sulfated fucan from the brown alga laminaria cichorioides has mainly heparin cofactor ii-dependent anticoagulant activity a new α-pyrone derivative, 6-[(e)-hept-1-enyl]-α-pyrone, with tyrosinase inhibitory activity from a marine isolate of the fungus botrytis circumdatin i, a new ultraviolet-a protecting benzodiazepine alkaloid from a marine isolate of the fungus exophiala isolation, structure elucidation, and antimycobacterial properties of dimeric naphtho-γ-pyrones from the marine-derived fungus aspergillus carbonarius this review was made possible with financial support from midwestern university to amsm; nih-sc1 award (grant 1sc1gm086271-01a1) of the university of puerto rico to adr; and fapesp grant 05/60175-2 (são paulo, brazil) to rgsb. the content of this review is solely the responsibility of the authors and does not necessarily represent the official views of the nih. assistance with extensive searches of the 2007-8 marine pharmacology literature in pubmed, marinlit, current contents® and chemical abstracts®, as well as article retrieval by library staff members, and students from the chicago college of pharmacy, midwestern university, are gratefully acknowledged. the authors are especially thankful to ms. mary hall for the careful review of the manuscript. key: cord-283301-adjjkqt2 authors: awolade, paul; cele, nosipho; kerru, nagaraju; gummidi, lalitha; oluwakemi, ebenezer; singh, parvesh title: therapeutic significance of β-glucuronidase activity and its inhibitors: a review date: 2020-02-01 journal: eur j med chem doi: 10.1016/j.ejmech.2019.111921 sha: doc_id: 283301 cord_uid: adjjkqt2 the emergence of disease and dearth of effective pharmacological agents on most therapeutic fronts, constitutes a major threat to global public health and man’s existence. consequently, this has created an exigency in the search for new drugs with improved clinical utility or means of potentiating available ones. to this end, accumulating empirical evidence supports molecular target therapy as a plausible egress and, β-glucuronidase (βglu) – a lysosomal acid hydrolase responsible for the catalytic deconjugation of β-d-glucuronides has emerged as a viable molecular target for several therapeutic applications. the enzyme’s activity level in body fluids is also deemed a potential biomarker for the diagnosis of some pathological conditions. moreover, due to its role in colon carcinogenesis and certain drug-induced dose-limiting toxicities, the development of potent inhibitors of βglu in human intestinal microbiota has aroused increased attention over the years. nevertheless, although our literature survey revealed both natural products and synthetic scaffolds as potential inhibitors of the enzyme, only few of these have found clinical utility, albeit with moderate to poor pharmacokinetic profile. hence, in this review we present a compendium of exploits in the present millennium directed towards the inhibition of βglu. the aim is to proffer a platform on which new scaffolds can be modelled for improved βglu inhibitory potency and the development of new therapeutic agents in consequential. the world today is embattled with an increasing paucity of effective therapeutic agents or regimen for many pathological conditions, as well as the menace of drug resistance and adverse effects of available drugs [1] . as a result, smooth and efficient clinical practice is rigidly stymied, while global public health, social security and man's life expectancy are seriously threatened and trickles to a disquieting edge [2] . likewise, the burdens of developing new therapeutic agents to ameliorate the status quo has become heavier on all stakeholders in drug research. in this regard, molecular target therapy is fast becoming a spearhead in the search for new drugs with improved therapeutic effects. amongst many targets explored, glycosyl hydrolases (ghs) are notable due to their role in many important biological processes. their principal function is to catalytically cleave the glycosidic bond of glycans thereby eliciting different physiological responses. therefore, inhibitors of this class of enzymes have enjoyed intense research and development owing to their potentials as antiviral, anticancer and antidiabetic agents as well as therapeutic agents for some genetic disorders [3e5] . ghs have been classified using different indices [6] . for example, based on substrate specificity, those cleaving o-or sglycosides are grouped into ec 3.2.1 class, while hydrolases of nglycosides belong to ec 3.2.2 class. advancements in genomic science have also enabled classification into gh families based on their amino acid sequence similarities [7] . this system further groups gh families into clans, given the improved conservation of protein fold than the sequence [8] . accordingly, the reviewed enzyme, b-glucuronidase (ec 3.2.1.31) is classified into gh family 1, 2, 30, 79, 154 and gh-a clan. b-glucuronidase (bglu) is mainly a lysosomal hydrolase widely distributed in mammalian tissues, body fluids and microbiota; but significantly retained in the endoplasmic reticulum [9] . the enzyme is also found in plants, fishes, insects and molluscs. specifically, human bglu belongs to gh family 2. it is a 332 kda ellipsoidal and homotetrameric glycoprotein with each 75e78 kda monomer containing 651 amino acid residues (fig. 1a) . the monomer precursor is synthesized initially on membrane-bound ribosomes and suffers c-terminal proteolytic processing of 18 amino acid propeptide en route or after their transport to the lysosomes [10e13]. x-ray crystallography of the protein structure reveals a dihedral symmetry for the tetramer with two identical monomers in the asymmetric unit arising from disulphide-linked dimers. each monomer contains three structural domains (fig. 1b) . the first domain has a barrel-like structure with a jelly roll motif; the second domain exhibits a geometry identical to immunoglobulin constant domains; while the third c-terminal domain forms a tim barrel motif (b/a) 8 [14] . the active sites of human bglu ( fig. 1c) viz. catalytic acid glu451 (proton donor), catalytic nucleophile glu540 (carbonium ion stabilizer), asp207 (plausible role as glu540) and tyr504 (unclear catalytic role), are all housed in the third domain and in each of the four catalytic centres of the tetramer [14, 15] . moreover, the enzyme has an optimal activity at acidic ph~4.5, corresponding to its lysosomal environment and thermally stable up to 70 c [10] ; although hyperthermophilic variants exists in other media [16] . bglu is encoded by the gus gene. a deficiency arising from mutations in this encoding gene is associated with atherosclerosis [17] and lysosomal storage disease e sly syndrome or mucopolysaccharidosis type vii [18] . on the other hand, bacterial bglu, which is expressed in human gut microbiota and most strains of escherichia coli shows 45% sequence similarity with human bglu. also, it has a bacterial loop containing 17-amino acid residues not found in human bglu, an optimal activity at neutral ph and active site catalytic residues as glu413 (catalytic acid) and glu504 (catalytic nucleophile) [19] . consistent with the activities of lysosomal ghs, bglu deconjugates b-d-glucuronides to their corresponding aglycone and b-dglucuronic acid via an s n 2 reaction and "configuration retaining" mechanism ( fig. 2) . the catalytic mechanism is conceived to proceed as follows; catalytic glutamic acid residue glu451 (or glu413 in bacterial ortholog) protonates exocyclic glycosidic oxygen of glucuronide (1) hence releasing the aglycone via a putative oxocarbenium ion-like transition state (2) . 'back-side' nucleophilic attack by glutamate ion glu540 (or glu504 in bacterial ortholog) e the catalytic nucleophile, stabilizes the transition state and results in glucuronyl ester intermediate (3) with an inverted configuration. finally, hydrolysis through an inverting attack of water molecule on the anomeric centre releases glu540 to form b-d-glucuronic acid (4) and a concurrent overall retention of substrate configuration [14,15,19e21 ]. due to the increased expression of bglu in necrotic areas and other body fluids of patients with different forms of cancer such as breast [22] , cervical [23] , colon [24] , lung [25] , renal carcinoma and leukaemia [26] , compared to healthy controls, the enzyme is proffered as a reliable biomarker for tumour diagnosis and clinical therapy assessment [27] . this overexpression is also a potential diagnostic tool for other disease states such as urinary tract infection [28] , hiv [29] , diabetes [30] , neuropathy [31] and rheumatoid arthritis [32] . in this vein, empirical data update on clinical applications of bglu for these and other disorders is provided on brenda database [33] . bglu activity is also harnessed in prodrug monotherapy. in normal body systems, drugs and other xenobiotics are detoxified via glucuronidation, an s n 2 conjugation reaction and important pathway in phase ii metabolism, catalysed by udpglucuronosyltransferases (ugts). the resulting usually less active glucuronide metabolite is readily excreted by renal clearance due to increased polarity or sometimes via biliary clearance [34] . however, elevated levels of bglu activity reverts this process through deglucuronidation, which hydrolyses the phase ii metabolites to their active forms (fig. 2) . hence, glycosidation of a drug to give its glucuronide enhances selective release of the active form at necrotic sites via bglu-mediated deglucuronidation thus improving the drug's therapeutic potential [35] . bglu's postulated ability to increase t regulator cells (treg) is also applied in low-dose immunotherapy (ldi) for managing allergic diseases [36, 37] , lyme disease [38] and other chronic conditions. while it's hydrolytic activity on glucuronide conjugates is harnessed in forensic analysis [39] and assessment of microbial water quality [40] . nonetheless, enterobacterial bglu deconjugation of drug and xenobiotic glucuronides in the gastrointestinal (gi) tract has been implicated in colonic genotoxicity [41] and certain drug-induceddose-limiting toxicities. for example, the gi toxicity of anticancer drug irinotecan (cpt-11) [42] , enteropathy of non-steroidal antiinflammatory drug (nsaid) diclofenac [43] , tissue inflammation and hepatoxicity. furthermore, bglu is deemed a potential molecular target for; (1) anticancer chemotherapy considering its role in tumour growth and metastasis [44, 45] . (2) neonatal jaundice treatment due to its high expression in breast milk and role in enterohepatic bilirubin circulation (hyperbilirubinemia) [46, 47] . (3) diabetes mellitus management consequent to the positive correlations between the disease state and enzyme activity level as well as associated periodontitis [48, 49] . (4) anti-inflammatory agents development owing to its pro-inflammatory role following significant release from degranulated mast cells and neutrophils [50, 51] . expectedly, inhibition of bglu markedly alleviated these pathological conditions and their adverse effects hence improving regimens' efficacy. based on the foregoing, we extrapolate that the development of potent, specific and non-cytotoxic inhibitors of bglu is imperative to improving the clinical efficacy of therapeutic agents and effective disease management while bearing in mind the physiological significance of both human and bacterial orthologs of the glycosyl hydrolase. however, the fate of these inhibitors rests on their inhibition constants (k i ), since ghs are generally characterized by high rate enhancement (k cat /k uncat > 10 17 -fold). also, accumulating evidence suggests the dependence of inhibitory potency on the ability to mimic the highly enzyme-stabilized transition state of an enzymatic reaction (k i z 10 à20 m) en route to catalytic product [21, 52, 53] . considering the proven and encouraging potentials of enzyme inhibition and molecular target therapy in drug development, and in continuation of our exploits and expositions thereon [54e58], herein we present a comprehensive review of research undertakings in the present millennium (2000e2019) directed towards the development of potent inhibitors of bglu that are either natural products or synthetic scaffolds. apropos, before discussing the different inhibitors, this article will first highlight the potentials of bglu activity as a diagnostic tool within the defined period. however, therapeutic application in prodrug monotherapy and enzyme replacement therapy (ert) will not be covered as these have been excellently treated in other reviews [59e61]. hitherto, our search of extant literature revealed that, although there exists a plethora of scholarly research on potential inhibitors of bglu activity, no review article is exclusively devoted to the subject matter. the aim of this review is therefore to bring to light those bioactive frameworks bestowed with promising bglu inhibitory potency. our principal goal is to intimate the reader on key structural features of reviewed molecules crucial to their inhibitory activities and toxicity profiles, while establishing the comprehensive relationships existing between reported molecules. the availability of safe, easy to use, consistent, less-invasive and cost-effective tool for early diagnosis of diseases or appraisal of therapeutic interventions is of uttermost importance in clinical medicine. since most disease states are accompanied by elevated levels of specific enzymes in the diseased milieu (tissues, plasma and other body fluids), quantification of enzymes' activity levels is seen as a reliable biomarker of either disease status, severity, effects, susceptibility or exposure [62e64]. moreover, the substrate specificity and selective quantification of enzymes in the presence of other biomolecules makes them a tool of choice thereto [65] . a review of bglu activity as a biomarker of some physiologically important conditions is hereby presented together with a concise summary in table 1 . periodontal disease is a group of inflammatory disorders triggered by host's immune response to the actions of virulent subgingival plaque bacteria biofilms which activates the release of polymorphonuclear leukocytes and macrophages into the gingival crevice. this leads to gingivitis e an inflammation of periodontal tissues and distortion of periodontal histology that is reversible with improved oral hygiene; or, subsequent tissue destruction, alveolar bone resorption and tooth loss if left unattended i.e. periodontitis [66, 67] . therefore, a reliable tool to ascertain disease status, severity, risk or efficacy of administered therapy is highly desirous to clinicians. however, conventional diagnosis involving the measurement of periodontal clinical parameters such as probing depth (pd), clinical attachment level (cal), gingival index (gg-i), bleeding on probing (bop) and alveolar bone loss (abl), suffers from intrinsic limitations. they only define the status of patient's periodontium at the time of examination and not periodontal disease susceptibility or risk [68] . thus, since periodontitis is characterized by an influx of inflammatory mediators and corresponding enzymes into the gingival sulcus, the quantification of neutrophil-derived bglu activity in gingival crevicular fluid (gcf) or gcf's outflow into the oral cavity and subsequent less invasive estimation of bglu activity in saliva is considered a reliable biomarker for periodontal disease diagnosis. to this effect, the relationship between salivary bglu activity and periodontal clinical parameters (pd, cal and gg-i) was investigated in subjects with different stages of periodontal disease [69] . the mean pd and gg-i, number of sites with pd ! 5 mm and total number of white blood cells, blood neutrophils and monocytes all showed highly significant correlations with enzyme's activity, while cal had a weaker correlation. using logistic regression modelling and the presence of at least 1 or 4 sites with pd ! 5 mm as disease criterion, bglu activity showed promising potentials as a tool for periodontal disease screening or assessment of therapeutic intervention. the study also observed smoking status to be insignificant on enzyme activity. however, in a similar study, only pd, cal and lymphocyte count exhibited positive correlation with salivary enzyme activity while no significant relationship was observed for gg-i [70] . recently, subjects with chronic generalized periodontitis were also found to have significant increase in bglu activity (8-fold) compared to normal ones. although, a reduction in enzyme activity persisted in smokers regardless of periodontal status [71] . table 1 reported potential applications of bglu activity as a biomarker. [112] 284** ache activity chronic exposure (w) acute exposure (n) elevated in 16.5% and 60% of subjects with chronic and acute exposure respectively [113] ns: not specified; w: weak correlation; n: no correlation; pi: russell periodontal index; *threshold to distinguish culture positive from culture negative balf; ** acute exposure (5), chronic exposure (230) . the efficacy of therapeutic intervention using amoxicillin and metronidazole to downregulate amplified neutrophil activity was studied in 14 patients with aggressive periodontitis [72] . treatment involved seven consecutive days of antibiotic administration with concurrent scaling, root planning and surgical therapy and a total of 36 months posttreatment evaluation period. subsequently, a markedly downregulated neutrophil activity with approximately 50% inhibition of bglu activity in gcf was observed. periodontal health was also restored and maintained during posttreatment evaluations. in another study, bglu activity was posited as a better biomarker compared to alkaline phosphatase for evaluating the response to non-surgical periodontal therapy in patients with different stages of periodontal disease [73] . taken together, these results articulate the potentials of bglu activity as an indication of pd, tissue inflammation or destruction as well as a biomarker of neutrophil influx, disease risk, susceptibility, status, or severity for timely diagnosis of the inflammatory disorder. however, administration of doxycycline hyclate in 16 subjects with aggressive periodontitis was inefficient on salivary bglu activity [74] . surprisingly, an increase in enzyme activity was found even after 2 months of treatment in 12 patients and a decrease in 4. although, the authors concluded bglu concentration only facilitated the detection of periodontal inflammation and not worthy as biomarker of susceptibility, their contrasting result is linkable to periodontal pretreatment of subjects prior to examination and short treatment time using doxycycline. empirical evidence affirms the role of inflammatory mediators and signalling pathways in the pathogenesis of insulin resistance and b-cell dysfunction in diabetes mellitus [75e77]. in parallel, these inflammatory mediators e.g. cytokines and mmps are also produced in periodontal tissues [78e82]; hence, leading to compromised glycaemic control after accessing systemic circulation. the susceptibility to periodontitis is therefore heightened in persons with diabetes or a history of the hormonal imbalance and vice versa. putatively, an effective therapy for one affords an improved management of the other [83e85]. accordingly, bglu activity was found to be significantly elevated in the saliva of patients with chronic periodontitis and diabetes compared to nondiabetic ones [86] . a significant correlation to bglu activity was observed for pd and cal and not gg-i in nondiabetic subjects with periodontitis; whereas, these periodontal parameters were similar in diabetics. the increased disease burden was also established when bglu activity was measured in the sera of patients with both diabetes and periodontitis [87] . compared to controls, enzyme activity was 9-fold higher in diabetic subjects with periodontitis and only 2-fold higher in diabetic subjects without periodontitis. this difference was attributed to damaged lysosomal membrane and consequent enzyme leakage into the cytosol. the quantification of bglu activity in neutrophil leukocytes exposed to bacteria stimuli has shown that diabetic patients with chronic periodontitis have strikingly higher enzyme activity compared to nondiabetics burdened with periodontitis and healthy subjects [88] . using discriminant analysis, the study established that bglu activity has a diagnostic potential with great accuracy in distinguishing healthy subjects from diseased ones. bglu activity stimulated by nonopsonized staphylococcus aureus showed strongest correlation with the intensity of periodontal parameters compared to opsonized zymosan and prodigiosan, while the highest enzyme activity was stimulated by opsonized prodigiosan. the strength of association between salivary bglu activity, periodontitis and type 2 diabetes mellitus has been examined in dentate patients with different stages of periodontal disease, diabetic patients and edentulous patients [89] . in all subjects, diabetic status contributed significantly to bglu activity, while periodontal status had greater influence on enzyme activity. higher enzyme activity was also found in nondiabetic dentate patients with periodontitis compared to edentulous controls. overall, compared to il-1b, bglu activity level was more reliable as biomarker of disease severity for periodontitis than it was for the presence of diabetes. in a predating study [90] , gcf bglu activity also correlated strongly with pd, cal and bop regardless of diabetic status. lower enzyme activity was seen in diabetic subjects compared to those with periodontitis. the results suggested a lower release of bglu in response to systemic inflammation (diabetes) due to reduced deficiency in neutrophil activity, in contrast to amplified activity in response to local inflammation (periodontitis). despite landmark developments in oncology, the high rates of morbidity and mortality and increased medical costs associated with all forms of cancer coupled with patients' psychological trauma on disease diagnosis has remained a major threat to global public health. the successful disease management and sustained wellbeing of affected individuals is however subject to early disease diagnosis and constant evaluation of administered therapy. this in turn relies on efficient tumour markers to ascertain disease risk or status i.e. early stage or metastatic cancer [91] . although a vast number of biomarkers have been identified for cancer diagnosis, only few have gained clinical approval due to inconsistencies and false positives in their utility [92] . a successful biomarker is that which will not only be specific and selective but will also predict treatment response, while differentiating lethargic and aggressive tumours. the aetiology of cancer is known to be closely associated with inflammatory pathways and oxidative stress, which jointly create microenvironments favouring neoplasia [93, 94] . hence, in the tumour milieu, an increase in extracellular activity of lysosomal exoglycosidases responsible for the catalytic degradation of glycoconjugates occur, due to malignancy-mediated cell-death and/or lysosomal damage. in this vein, available practical data supports the overexpression of bglu in extracellular fluids and tissues around tumour sites as a prime factor in cancer aetiology [24] ; thus, suggesting the enzyme's viability as cancer biomarker [95] . for example, bglu activity was 2-folds higher in the blood samples of 21 patients with colorectal adenocarcinoma compared to healthy controls [96] . based on cell maturity and clinical grading, enzyme activity was highest in subjects with low or moderately differentiated cells and in subjects with tumours infiltrating surrounding tissues and organs or visceral peritoneum respectively. estimation of serum bglu activity in this case proved to be 80% sensitive and 82% specific in distinguishing diseased and healthy subjects. likewise, enzyme activity was elevated by 6-fold in the peritoneal fluids of patients with ovarian or endometrial cancer compared to women with infertility used as controls [97] . the stronger correlation between cancer stage and bglu activity, b-galactosidase and a-mannosidase, reiterated the improved clinical viability of bglu activity as biomarker of gynaecologic tumour status and severity. however, bglu activity was equally elevated in the peritoneal fluid of patients with pelvic inflammation hence compromising its application for differential diagnosis of gynaecologic cancer and pelvic inflammatory disease. bacterial peritonitis is a life-threatening inflammation of the peritoneum e the tissues lining of the inner abdominal walls. as with other inflammatory conditions, cellular damage and polymorphonuclear leukocytes activity increases the level of lysosomal enzymes in the extracellular space via enzyme leakage through the cellular membrane. thus, quantification of these enzymes provides a potential diagnostic platform. in fact, bglu activity in the peritoneal fluid of patients with culture positive bacterial peritonitis was 9 and 33-fold greater compared to that of patients with acute mesenteric lymphadenitis and controls respectively [98] . peritoneal fluid-bglu activity measurement in this case holds greater clinical potential compared to b-galactosidase and a-mannosidase for early disease diagnosis and evaluating patient's response to treatment. similarly, considering the current global prevalence of antimicrobial resistance, timely diagnosis of bacteria-mediated meningeal inflammation (bacterial meningitis) is crucial to the outcome of therapeutic intervention. it has been shown that bglu activity is elevated in cell-free cerebrospinal fluid (csf) of bacterial meningitis patients early in the disease pathogenesis, even when traditional laboratory parameters such as number of csf cells, csf-blood glucose ratio and protein concentration indicated normal status [99] . csf-bglu as biomarker was superior to these traditional parameters for early and sensitive prediction of patient's response to antibiotic treatment. csf-bglu activity in neonates and infants has also been studied as biomarker for differential diagnosis of sterile csf pleocytosis due to urinary tract infection (uti) or meningitis [100] . median bglu activity in csf of patients showed significant difference without overlapping in each disease state i.e., bacterial meningitis (168), viral meningitis (26.5) and uti with sterile csf pleocytosis (44.1); median enzyme activity was lowest (19.1) in febrile subjects without csf pleocytosis used as controls. this proffers an unambiguous diagnosis of each condition with 100% sensitivity and specificity. in contrast, broad overlapping was found with classic csf laboratory parameters viz., csf cell number, neutrophil number, protein concentration and csf-blood glucose ratio. recently, a case-control study concluded that bglu activity in bronchoalveolar lavage fluid (balf) is clinically useful as biomarker of bacterial lung infection [101] . in balf samples from 92 children, enzyme activity was significantly higher in patients with positive balf bacterial culture (cþ) compared to those with culture negative balf (ce). 43 nmol 4-methylumbelliferone (4mu)/ml/h was identified as optimum activity value, which allowed differential sample screening (i.e. cþ from ce) with 84.8% sensitivity and 78.3% specificity. moreover, receiver operating characteristics (roc) curve analysis established the superiority and higher prognostic value of bglu activity for bacterial lung infection compared to % polymorphonuclear cell count, human leukocyte elastase, il-8 and tnfa. repeated exposure to organophosphorus compounds (op) in pesticides is responsible for the lethal poisonings seen in agricultural and veterinary workers; particularly, those in developing countries. op exerts their fatal effects by inhibiting acetylcholinesterase (ache) in nervous tissues, leading to muscarinic and nicotinic effects with central nervous disturbance [102] . on the other hand, bglu is retained in liver microsomal endoplasmic reticulum by forming non-covalent binding complexes with its accessory 64 kda glycoprotein, egasyn, a carboxylesterase isozyme [103, 104] . liver intake of op however cleaves microsomal bgluegasyn complex thus elevating the level of bglu in plasma to consequently making it an alternative biomarker for op pesticide poisoning diagnosis [105e107]. a cross-sectional study of pest control workers [108] and plastic greenhouse workers [109] , established that plasma bglu activity was more sensitive as biomarker of op poisoning compared to butyrylcholinesterase (buche) and acid phosphatase (acp). the enzyme's activity was higher in subjects with increased exposure than those with low exposure and controls. bglu activity correlated significantly with buche and acp activities. likewise, in a case-control study, serum bglu activity was significantly increased in patients with severe poisoning compared to mildly affected patients and controls [110] . the difference in enzyme activity between the latter groups was insignificant. nonetheless, mildly exposed subjects have been reported to show elevated bglu activity than severely exposed and control subjects, which suggests the enzyme's utility for diagnosing lowlevels of op exposure. for instance, a study [111] , observed the order of serum bglu activity based on poisoning severity as mild > severe > moderate poisoning, while the order after 12 and 24 h of admission was mild > severe z moderate poisoning; strong correlation also persisted between serum bglu and buche activities. similar results were also obtained in a recent cross-sectional study [112] . therein, moderately exposed subjects showed higher enzyme activity than the highly exposed, while non-significant statistical difference persisted between control and highly exposed groups. notably, bglu activity correlated well with diabetes propensity, lipid profile, liver function and buche but not ache. in another cross-sectional study, significant difference in plasma bglu activity only exists between controls and subjects with chronic exposure (1e45 years) to op [113] . activity level was similar in controls and patients with acute poisoning; although 3 out of the 5 examined acutely poisoned patients showed increased level of plasma bglu activity. however, a case report of an acute op self-poisoned patient reached contrasting conclusion [114] . the opposing result can be linked to sample size and limited data on patient's medical history. moreover, the reduced susceptibility of bglu-egasyn complex to op in humans is another primal factor [115] . in contrast to murine bglu-egasyn interaction, binding in humans is independent of the c-terminal 18 amino acids propeptide in bglu and esterase active site of egasyn. rather, it involves the 51 amino residues in bglu internal segment i.e. residues 228 to 279. the progress and momentous achievements in drug discovery cannot be isolated from the wealth of chemical entities i.e. natural products, gifted to man by nature. many successful molecular candidates of pharmaceutical drug discovery programmes are indebted to the presence of natural product-derived/inspired fragments in their scaffolds [116] . therefore, the chemical space of natural products (both plants and microbes) has continued to be a much-researched depository for therapeutically significant molecules. in this section, we review some application of natural products including isolated compounds, whole plant extracts and natural product-inspired molecules as potent inhibitors of bglu. the structures of selected inhibitors (ic 50 5 mm) are presented in table 2 at the end of the section. the clarification [117, 118] that d-saccharic acid-1,4-lactone (5, fig. 3 ) (saccharolactone or d-glucaric acid-1,4-lactone) is the active and non-toxic bglu inhibitor responsible for the strong inhibitory potency found with saccharate solutions has precipitated an increased interest in the compound. despite the poor stability at physiological ph, d-saccharic acid-1,4-lactone (d-sal) has been explored extensively for its therapeutic significance. although an ic 50 value of 3.6 mm was reported in the pioneering work [117] , a value ca. 40 mm is recurrent in literature. nevertheless, at a concentration of 1 mm, d-sal completely inhibited the hydrolytic action of human liver-derived bglu on quercetin glucuronides [119] , while over 90% inhibition of bglu in breast milk was recorded at 10 mm [120] . also, using urine samples of male sprague-dawley rats, d-sal was identified via metabolomics strategy as one of the therapeutic constituents in liuweidihuang pills, a famous traditional chinese prescription for cancer treatment and prevention [121] . whereas, in a 6-days cumulative study, intraperitoneal pre or cotreatment of female wistar rats with 3 mg/ml, 10 mg/ml or 10 mg/0.5 ml d-sal reduced the severity of cpt-11 induced smallintestine mucosal damage assessed by the number of apoptotic cells or mitotic figures, compared to cpt-11 treated controls [122] . damage reduction was independent of treatment schedule. the synthetic and natural precursors of d-sal i.e., 2,5-di-oacetyl-d-glucaro-1,4:6,3-dilactone (d-sdl, 6, fig. 3 ) and d-glucurono-g-lactone (d-gl, 7) respectively, have also potently inhibited bglu activity in male fischer rats thereby providing chemopreventive effects against azoxymethane-induced colon carcinogenesis [123] . diet supplementation with 0.5 or 2% d-sdl for 5 weeks significantly reduced aberrant crypt foci formation (i.e. preneoplastic lesions) by over 48.6 and 55.3% respectively, compared table 2 most active natural product-derived bglu inhibitors with ic 50 to azoxymethane-controls. d-gl did not afford any significant reduction in colonic tumour incidence during this initiation phase. in addition, 32 weeks treatment with d-sdl or high dose (2%) of d-gl, after 3-weeks subcutaneous injections of 15 mg/kg azoxymethane, provided over 70% inhibition of colon carcinogenesis during the post-initiation phase. it was suggested that d-sdl is a blocking agent which may inhibit pre-neoplasia during the initiation phase, while d-gl is only active during post-initiation of colon carcinogenesis. the increased hydrolytic stability of d-sdl to d-sal in vivo might also be responsible for these observed phenomena [124] . in another study, d-sal was more therapeutically efficient compared to its natural precursor (d-gl), for reducing epidermal hyperplasia (lethargic tumour promoter) and inflammation in 7,12dimethylbenz(a)anthracene (dmba)-induced complete skin carcinogenesis of sencar mice [125] . pre and cotreatment of murine models with d-sal for 4-weeks (twice weekly) by topical administration (0.5e4 mg) or dietary treatment (0.5 and 1%), both significantly reduced epidermal hyperplasia and inflammation by up to 57% of dmba-controls in a dose-dependent manner. d-sal also inhibited the initiation of carcinogenesis by reducing dmbainduced oxidative dna damage (c-8 hydroxylation of guanine) and mutations in codon 61 of ha-ras gene, by up to 78%. in contrast, d-gl only inhibited epidermal hyperplasia with topical treatment and inflammation by dietary treatment (5% in diet); albeit with inferior potency compared to d-sal. interestingly, another lactone-based bglu inhibitor (8, fig. 3 ), exhibited 8-fold superior potency compared to d-sal [126] . isolated from the ethyl acetate extracts of aspergillus terreus, an endophytic fungus initially isolated from marine alga laurencia ceylanica, butyrolactone (8) with ic 50 ¼ 6.2 mm possesses 16-fold stronger potency than its prenylated isomer (9) . bglu in breast milk is believed to be involved in neonatal jaundice and hyperbilirubinemia, by increasing serum bilirubin levels via enterohepatic bilirubin circulation in breastfed newborns, in contrast to those fed with infant formulas [47] . therefore, the suppression of bglu-mediated deglucuronidation has been proffered as a practicable regimen. l-aspartic acid (10, fig. 3 ) in casein hydrolysate formulas was identified as the active bglu inhibitor responsible for the lower levels of neonatal jaundice observed in newborns receiving such formulae [127] . at 100 mm, the natural amino acid showed~86% inhibition of bglu that is, 100-fold more potent than d-isomer. moreover, in a randomized and double-blind clinical trial involving 64-newborns, supplementing breastfeeding in the first week of life with 6 doses of l-aspartic acid (180 mg/5 ml of water/day) was more potent against bglu activity than higher concentrations of enzymatically hydrolysed casein (infant formula containing the inhibitor) or whey/casein (routine formula lacking the inhibitor) [128] . l-aspartic acid supplementation at minimal aliquot concentration significantly lowered transcutaneous bilirubin levels (25% lower than control), leading to higher faecal bilirubin excretion and reducing neonatal jaundice, with no adverse effects. flavonoids are evidently an indispensable class of natural products due to their ubiquity in the vegetal domain and their therapeutic significance. found in a variety of plant parts (leaves, flowers, stems, nuts, seeds etc.), they perform important functions especially plant's growth and protection against pathogenic invasion. this intrinsic property encourages their utility as a major constituent of different local medicinal formulations and diets, while their acceptable toxicity profile and physiologic tolerance further presents them as druggable subjects. flavonoids are typified by the c6ec3ec6 ring system that is, their basic structural skeleton consists of two benzene rings (a and b) linked by heterocyclic pyran ring c (fig. 4) . variations on the chromane core (ring a and c) and attachment position of ring b due to biosynthetic origins lead to classification into flavones, flavanols, flavanones, flavonols, isoflavones, neoflavonoids, anthocyanins and chalcones [129] . this ring system and the presence of hydroxyl units has availed flavonoids with different pharmacological properties such as antidiabetic, antioxidant, antimicrobial, antiplasmodial, antiproliferative and particularly enzyme inhibition [130, 131] . luteolin (11, fig. 4 ), a dietary 3 0 , 4', 5, 7-tetrahydroxyflavone has been reported as a viable chemopreventive and anticarcinogenic agent plausibly by inhibiting bacterial bglu-mediated enterohepatic circulation of colonic carcinogens [132] . the 30-weeks cumulative study using male wister rats showed that pre or cotreatment with luteolin by intragastric gavage per os (p.o.) at 0.1, 0.2 or 0.3 mg/kg body weight/day, significantly reduced bacterial bglu activity thereby suppressing 1,2-dimethylhydrazine (dmh)induced colon adenocarcinomas in a dose-dependent manner compared to dmh-controls. although 0.2 and 0.3 mg kg à1 day à1 produced similar result. luteolin supplementation also reduced tumour size from 2 cm to 0.25 and 0.50 cm during the initiation and post-initiation stages respectively, as well up to 90% reduction in tumour incidence. in addition, luteolin (11), its 7-o-glycoside (12), apigenin-7-o-glycoside (13) and catechin (14) were identified as key constituents in the leaf extracts of pistacia terebinthus responsible for the high e. coli bglu inhibitory potency [133] . at the highest test concentration (8.2 mg/ml), the leaf extracts exhibited 97.2% inhibition of bglu activity, corresponding to an ic 50 value of 2.11 mg/ml. in another exploit, 32 natural flavonoids were evaluated for their inhibitory strengths against e. coli bglu [134] . it was established that luteolin (11) , similar flavones e baicalein (15), scutellarein (16) and its glucuronidated analogue scutellarin (17) , as well as dietary and ubiquitously occurring flavonol e quercetin (18) , are superior inhibitors (ic 50 ¼ 5.76e29.64 mm) compared to reference inhibitor d-sal (ic 50 ¼ 36.07 mm); isoflavones and dihydroflavones displayed weaker inhibition. overall, luteolin (11) and scutellarein (16) emerged the most potent flavone-based e. coli bglu inhibitors with ic 50 ¼ 8.68 and 5.76 mm respectively. sar analysis (fig. 4 ) revealed the importance of 5,6,7-trihydroxy (pyrogallol) unit to bacterial bglu inhibition, as unsubstitution or replacement of a hydroxy unit with methoxy or glycosyl unit led to significant loss of inhibitory activity. o-methylation at positions-6, 7 and 4 0 and installing oh unit at position-3 was also detrimental to potency, whereas the presence of hydroxy unit at c-4' favoured potency. in addition, molecular docking studies of luteolin and scutellarein in the active site of e. coli bglu showed hydrogen bonding (h-b) interactions of phenolic oh units at c-5 and c-7 with catalytic acid glu413 and arg562 as well as hydrophobic contacts with ser360 and leu361 in the bacterial loop. furthermore, subjecting the methanolic extracts of edible flower and pedicel of aquatic rhizomatous herb e nymphaea pubescens (water lily) to bioactivity-guided fractionation established the superior (3-fold) bacterial bglu inhibitory potency of crude flower extracts compared to pedicel extracts and silymarin e a marketed natural bglu inhibitor [135] . kaempferol (19) was subsequently identified as one of the active metabolites with promising activity; ic 50 ¼ 36.47 mm and 76-fold superior to silymarin. interestingly, the lower activity of kaempferol (19) compared to flavonoids (11, 15e18) affirmed the sar results described earlier. amidst 21 constituents found in commonly used chinese herbal medicines, two prenylflavonoids, sanggenon c (20, fig. 5 ) and kuwanon g (21) emerged as the most potent broad-spectrum inhibitors (>70% inhibition; ic 50 ¼ 12.5 and 7.4 mm respectively) of whole human gut bacterial bglu (consisting of eight bacterial isolates), compared to reference compound amoxapine (7.3% inhibition) [136] . compound 20 exhibited improved potency against recombinant bglu from e. coli (ecogus) and s. pasteuri (spasgus), ic 50 ¼ 0.40 and 0.33 mm respectively, compared to compound 21 (ic 50 ¼ 1.6 and 0.98 mm respectively). however, compound 21 was a stronger inhibitor of bglu from three representative bacterial isolates (e. coli, e. fergusonni and s. pasteuri) compared to compound 20. additionally, molecular docking studies of 20 and 21 in the binding pockets of ecogus and spasgus revealed both flavonoids bind (via their hydroxy groups) in the allosteric site and not the active site of the recombinant enzymes. the higher number of h-b interactions formed by compound 21 supported the overall increased potency compared to compound 20. the realization that pro-inflammatory mediators such as bglu, released from degranulated mast cells or neutrophils, play significant roles in inflammatory disorders has sparked increased interest in the search for potent inhibitors of such processes as antiinflammatory drug candidates. in this regard, dihydroxychalcones 22 and 23 (fig. 6 ) isolated from the roots of hypericum geminiflorum holds therapeutic promise [137] . compounds 22 and 23 potently inhibited the release of bglu from degranulated rat neutrophils with ic 50 values of 5.80 and 6.60 mm respectively, better than the reference inhibitor trifluoperazine. however, only compound 23 showed moderate activity against the enzyme's release from degranulated rat peritoneal mast cells with ic 50 ¼ 70.0 mm. conversely, an isomer of luteolin e norartocarpetin 24 (fig. 6) and flavonoid-like mornigrol d 25 (fig. 6) , both isolated from the barks of morus nigra (black mulberry), were moderately potent bglu inhibitors [138] . at 100 mm, compounds 24 and 25 showed 67.7% and 65.9% inhibition respectively against the release of bglu from activated rat neutrophils. however, chiral flavonoid alkaloids isolated from the ethanolic extracts of scutellaria moniliorrhiza and subsequently separated using chiral hplc, e scumonilines 26e29 (fig. 6) , are better drug candidates compared to compounds 24 and 25, but similar to compounds 22 and 23 [139] . scumonilines 26e29 displayed~63% inhibition at 10 mm against bglu release from activated rat neutrophils with ic 50 values of 5.21, 5.85, 5.47 and 5.16 mm respectively; better than reference inhibitor ginkgolide b (ic 50 ¼ 6.63 mm). compounds 26e29 were also more potent lead molecules compared to both glucuronate esters 30e34 (fig. 6 ) isolated from similar scutellaria regeliana [140] and chiral egeninebased alkaloids 35e37 isolated from the rhizomes of corydalis decumbens [141] . compounds 30e34 showed 43.7e47.1% inhibition at 10 mm, whereas compounds 35e37 were more inferior inhibitors at the same concentration with 32.4e41.3% inhibition. evidently due to its phycocyanin-rich content and potent reduction of zymosan-induced damage to knee joint histological architecture, edible blue-green microalgae e spirulina is deemed a therapeutically viable anti-arthritic agent [142] . an analysis of the synovial fluid of female of1 mice knee joints after 4-days intraarticular injection of zymosan, followed by 8-days oral administration of spirulina water-suspension at 100 mg/kg and 400 mg/kg, showed 78.7% and 89.2% inhibition of bglu activity respectively. subsequently, arthritic parameters such as tibial articular cartilage destruction, erosion of bone structure, articular tissue inflammation, loss of general joint architecture and pannus formation, were all markedly reduced in spirulina fed mice compared to those receiving zymosan only. these anti-inflammatory and anti-arthritic effects were comparable to reference drug triamcinolone, a tuber extracts of arisaema tortuosum (whipcord cobra lily) has shown moderate anti-inflammatory effect via bglu inhibition in a dose-dependent manner [143] . conceivably, the presence of quercetin, rutin and lectin, identified through chromatographic profiling, facilitated maximum inhibition of 92.9% at 100 mg/ml, superior to reference inhibitor salicylic acid. employing a similar approach with ginger rhizomes [144] , only 6-gingerol (38, fig. 7) was identified with 85% inhibition of bglu at 1 mm, comparable to salicylic acid (82% inhibition). it is also noteworthy that constituents profiling of essential oils extracted from the leaves of seven local varieties of piper betle l. identified eugenol (39) with an ic 50 value of 616.68 mg/ml similar to 794.62 mg/ml of silymarin; although the essential oils were only moderate bglu inhibitors with ic 50 ! 5.26 mg/ml [145] . however, metabolomics profiling of the leafy shoots-methanolic extracts of three swertia species viz. s. chirayita, s. decussata and s. bimaculate, presented s. chirayita as the strongest inhibitor of bglu and xanthones as the most active metabolites responsible for the swertia species' potency [146] . the c-2-glycoside of norathyriol, mangiferin (40) emerged as the most active and therapeutically promising xanthone with ic 50 ¼ 38 mm and 50-fold more potent than silymarin. shikunshito-kamiho (sktk), a traditional oriental formulation and fenugreek seeds (fgs), an indian spice, are natural ethnotherapeutic agents with potentials identical to luteolin (11) i.e., attenuation of colon carcinogenesis via potent inhibition of bacterial bglu activity [147, 148] . 5-weeks oral administration of spf male icr mice's diet supplemented with 20 mg/kg or 60 mg/kg sktk water extracts, after 10 weeks subcutaneous injection of dmh (weekly), equipotently reduced bglu activity during and after treatment by 14.9% and 21.3% of controls (dmh alone) respectively. tumour incidence in colon, measured by the number of aberrant crypt foci was also significantly reduced from 21.6 (distributed in sigmoid and descending colons) in controls, to 6.9 and 6.4 (distributed in rectum) at 20 mg/kg and 60 mg/kg doses respectively. similarly, diet supplementation with fgs significantly reduced intestinal bglu activity, leading to a marked reduction in tumour incidence from 93.3% in male wister rats receiving dmh alone for 15 weeks to 16.6% in rats receiving dmh þ 2 g/kg fgs water extracts for 30-weeks cumulative period. flavonoid, saponin and fibre rich content of fgs were presumed to be responsible for its anticarcinogenic activity. the hepatoprotective activity of chinese medicinal formulation e reduohanxiao-tang, used in the treatment of stroke and liver diseases has also been attributed to its ability to inhibit bacterial bglu activity in vivo [149] . water extracts of this local formulation and two of its ingredients viz. rhizomes of pueraria thunbergiana and scutellaria baicalensis potently inhibited the activities of e. coli and rat liver-derived bglu with ic 50 values ranging from 0.35 to 1.42 mg/ml. subsequently, oral administration of these extracts at 100 mg/kg alleviated ccl 4 -induced liver injury measured by significant reduction in serum aspartate aminotransferase (ast), alanine aminotransferase (alt), and lactic acid dehydrogenase (ldh) levels, compared to controls. the superior hepatoprotective effect of pueraria thunbergiana was accredited to isoflavone daidzein (41, fig. 7) , an aglycone metabolite of main components puerarin and daidzin by intestinal bacteria. daidzein inhibited e. coli and rat liver bglu with ic 50 ¼ 0.41 and 0.50 mg/ml respectively whereas puerarin and daidzin were inactive. fascinatingly, structurally similar tectorigenin (42), an aglycone metabolite of 7-o-glycoside e tectoridin (43), isolated from the flowers of pueraria thunbergiana, exhibited better inhibitory potency (ic 50 ¼ 0.30 mg/ml) than its congeners [150] . 50 mg/kg intraperitoneal pre-treatment of male icr mice with tectorigenin or 100 mg/kg oral administration of tectoridin provided better hepatoprotection than dimethyl diphenyl bicarboxylate (ddb) and daidzein, by significantly lowering serum ast, alt and ldh levels relative to ccl 4 -treated controls. the prodrug behaviour of tectoridin, identical to puerarin and daidzin above, was also established by its absence in the serum after 250 mg/kg oral administration (only tectorigenin was detected) and its failure to provide desired hepatoprotection after 100 mg/kg intraperitoneal administration. the triterpenoid 18b-glycyrrhetinic acid (44, fig. 8 ) also known as enoxolone, is the aglycone metabolite by human intestinal bacteria responsible for the hepatoprotective activity exhibited by saponin e glycyrrhizinic acid 45 [151] . glycyrrhizinic acid (45) is isolated from the rhizomes of glycyrrhiza uralensis e the sweetening agent in sktk. it exists as a natural glucuronide conjugate of 18b-glycyrrhetinic acid (44) hence making it (45) a substrate for bglu-mediated hydrolysis for consequent release of active inhibitor 44. in vitro evaluation against e. coli and rat liver bglu revealed stronger potency of glycyrrhizinic acid (ic 50 ¼ 12.15 and 97.21 mm respectively) compared to 18b-glycyrrhetinic acid (ic 50 ¼ 509.90 fig. 7 . natural bglu inhibitors profiled from plant extracts and ethnomedicinal preparations. and 42.49 mm respectively). moreover, at 100 mg/kg doses, oral treatment with glycyrrhizinic acid or intraperitoneal treatment with 18b-glycyrrhetinic acid, protected the hepatocytes of male wistar rats against ccl 4 -induced liver injury evident by the significant reduction in ast, alt and ldh levels compared to ccl 4controls. intraperitoneal administration of glycyrrhizinic acid also did not provide hepatoprotection akin to tectoridin (43) . in addition, structurally similar emodinol (46) , isolated from the roots of perennial herb paeonia emodi showed stronger e. coli bglu inhibition in vitro with ic 50 ¼ 63 mm compared to 18b-glycyrrhetinic acid [152] . microbial biotransformation of drugs and/or their precursors to new molecules with modulated pharmacological profile is considered an invaluable tool in drug design and development [153] . the natural product class e steroids, are key substrates in this regard [154] . accordingly, naturally occurring androstane steroid hormone, 5-dehydroepiandrosterone (47, fig. 8 ) and its macrophomina phaseolina metabolites were examined for their bglu inhibitory potentials [155] . the inferior potency/inactivity of metabolites compared to parent 5-dehydroepiandrosterone (ic 50 ¼ 77.9 mm) suggests that having an alkene unit at position-5 and cyclopentanone unit in the molecular architecture is crucial to inhibitory potency. conversely, the synthetic anabolic steroid, dianabol and its metabolites by filamentous fungus cunninghamella elegans or macrophomina phaseolina were inactive against bglu; except metabolite (48) with ic 50 ¼ 60.7 mm [156] . the e-oxime derivative (49) of an inactive metabolite however showed decent inhibitory potency with ic 50 ¼ 49.0 mm, equipotent with d-sal and 2-fold superior than its z-isomer. nonetheless, the therapeutic safety of these dianabol derived bglu inhibitors is questionable considering the increased risk of hepatotoxicity associated with oral administration of 17a-alkylated anabolic steroids [157, 158] . the fascinating ability of prebiotics and probiotics to positively influence the composition and behaviour of human intestinal microbiota, has been continuously explored with considerable success to improve human health [159e161]. precisely, lactic acid bacteria (lab) probiotics can selectively utilize non-digestible oligosaccharide prebiotics as carbon source to produce active metabolites, which modulates or stimulates the activities of certain intestinal bacteria hence, eliciting important physiological response and conferring health benefit [162] . consequently, lab e lactobacillus acidophilus csg afforded stronger inhibition of intestinal bacteria (including e. coli) producing bglu thus providing hepatoprotection to male icr mice, compared to lactobacillus brevis hy7401 and bifidobacterium iongum hy8001 [163] . when anaerobically cocultured with e. coli (hgu-3), l. acidophilus csg also potently inhibited bglu productivity of e. coli compared to other labs. as a result, oral treatment of the murine models with 500 mg/kg of l. acidophilus csg alleviated ccl 4 -induced hepatotoxicity by lowering ast and alt levels to 66% and 57% respectively, of ccl 4 control group. whereas, for t-bhpinduced hepatotoxicity, ast and alt levels were lowered to 62% and 48% respectively, better than reference hepatoprotective agent ddb. similarly, lactobacillus plantarum cfr 2194 was the most effective strain of lactobacilli metabolizing fructooligosaccharide prebiotics to short chain fatty acids, thereby altering bglu productivity of e. coli [164] . lactic acid (50, fig. 9 ) and especially nbutyric acid (51) were identified as the major short chain fatty acid metabolites in the culture filtrate of lactobacillus plantarum cfr 2194 and fructooligosaccharides responsible for the observed decrease in bglu activity. the most abundant and relatively stable thiosulfinate, s-methyl methanethiosulfinate (52, fig. 9 ), found in chinese chive (allium tuberosum rottier), has exhibited strong inhibitory potency (ic 50 ¼ 3.60 mm) against e. coli bglu [165] . compared to other disulphides viz., dimethyl, allyl methyl, and diallyl disulphides, compound 52 is more useful for alleviating drug induced toxicities or providing hepatoprotection. in parallel, a naturally occurring acetal isolated from red macroalga neodilsea yendoana, isogloiosiphone b (53), possesses similar potential, although with weaker potency (ic 50 ¼ 57.0 mm) compared to compound 52 [166] . the adverse role of inflammatory pathways and corresponding mediators including lysosomal enzymes during the pathogenesis of myocardial infarction again implicates bglu in the cardiovascular disease. in a study which examined the protective effects of thymol (54, fig. 9 ) on inflammation in isoproterenol-induced myocardial infarction using male albino wistar rats [167] , the therapeutic benefit of inhibiting the release of bglu, other lysosomal enzymes and pro-inflammatory cytokines was evident in the restoration of near-normal myocardial histology and function, compared to diseased controls. rats pre and cotreated with thymol by intragastric gavage at 7.5 mg/kg body weight/day for 7 days and subcutaneous injection of 100 mg/kg isoproterenol for 2 days (day 6 and 7), showed significantly reduced levels of cardiac troponin-t (a cardiotoxicity biomarker) and high-sensitive c-reactive protein in their sera, compared to those treated with isoproterenol only. most importantly, the activities of lysosomal enzymes i.e. bglu, bgalactosidase, cathepsin b and d in serum and heart were also potently inhibited to near-normal. these anti-inflammatory effects of thymol afforded a well-protected myocardium with stable histological architecture. polyhydroxylated piperidine and pyrrolidine alkaloids commonly referred to as iminosugars (fig. 10a) , are a noble class of sugar mimics in which the pyrano or furano endocyclic oxygen has been replaced with a basic nitrogen atom [168] . this class also include isoiminosugars (i.e. 1-azacarbasugars or 1-n-iminosugars) having a methylene unit in place of endocyclic oxygen and the anomeric carbon replaced with nitrogen. although these ring alterations render iminosugars metabolically inert, their protonated forms mimic the pyranosyl or furanosyl unit in gh substrates, especially the putative oxocarbenium ion-like transition state (2, fig. 2 ) in gh catalysed hydrolysis [21, 169] . this in turn facilitates their recognizability by ghs and other carbohydrate-recognizing proteins for corresponding enzyme inhibition. since the isolation of nojirimycin (55, first iminosugar) and siastatin b (56, first isoiminosugar) from streptomyces cultures in 1966 and 1974 respectively [170, 171] , these sugar mimics have been a recurring subject of intensive research owing to their extensive biological activities and therapeutic applications elicited majorly through potent inhibition of ghs [172] . the strongest iminosugar-based inhibitors of bovine liver-derived bglu reported so far (fig. 10b) , with promising antitumor potentials are siastatin b derivatives 57, 58 and 59 with ic 50 ¼ 65, 62 and 68 nm respectively [173] . therefore, this section reviews iminosugars and their analogues as natural productinspired bglu inhibitors, without undue repetitions of other monographs [4, 21, 168, 169] covering them. uronic analogues of nojirimycin bearing glycaro-1,5-lactams or the bicyclic analogues with imidazole and tetrazole units (i.e. tetrahydrotetrazolopyridine-5-carboxylates and imidazopyridine-5carboxylates respectively), were prepared to examine the effect of sugar-acid configuration and presence of lactam, tetrazole or imidazole moieties on inhibitory potency against bovine liver bglu [174, 175] . evidenced by the most potent inhibitor 60 ( fig. 11 ) with k i ¼ 12 nm, gluco-configured units, are stronger bglu inhibitors compared to galacto-and manno-analogues, due to their better mimicry of glucuronic acid (4). the imidazole ring also conferred stronger inhibitory potency than tetrazole or lactam units whereas glycarolactams and tetrazoles shared similar potencies; except galacto-tetrazole (64) with~200-fold inferior potency to galactolactam (62) . further, although sugar configuration at c-4 was found to be ineffective on bglu inhibition for glycaro-1,5-lactams 61 and 62, it was very significant for tetrazole and imidazole derivatives. gluco-tetrazole (63) was 300-fold better than galacto-tetrazole (64), while gluco-imidazole (60) was 600-fold more potent than galacto-imidazole (65) and 1200 superior to manno-imidazopyridine (66) , which differs at c-2. moreover, sugar configuration of acid moiety at c-5 also had significant influence on potency. lconfigured units 67, 68 and 69 were 20-50-fold weaker than their d-isomers 63, 62 and 61 respectively. more importantly, the 2-fold increased potency of gluco-imidazole (60) compared to gluco-tetrazole (63) was attributed to stronger interaction of gluco-imidazole (60) with catalytic nucleophile glu540 compared to glucotetrazole (63) . interaction with catalytic acid glu451 was compromised due to protonation of the imidazole ring in the zwitterionic form. conversely, similar bicyclic molecules of nojirimycin with cyclic carbamate, urea or guanidine pharmacophores are weaker bglu inhibitors [176] . in all, only cyclic carbamates 70e73 (fig. 12) showed moderate inhibitory activity against bovine liver bglu. cyclic carbamates with carboxylic acid unit at c-5 of nojirimycin ring were also better inhibitors than their hydroxymethyl analogues. thus, compounds 71 and 73 were the most potent overall with ic 50 ¼ 218 and 259 mm respectively. it is noteworthy that bicyclic indolizidine iminosugar 74, with hydroxymethyl unit is also a weak inhibitor (<50% inhibition at 1 mm), although it exhibits desirable selectivity (7-fold) for e. coli bglu [177] . surprisingly, carbasugar 75 shared a similar fate with ic 50 ¼ 170 mm against e. coli bglu, even though it is not an iminosugar [178] . taken together, these results partly suggest that ability to mimic d-glucuronic acid favours bglu inhibitory activity. accordingly, glucuronic acid analogue of naturally occurring hemiaminal calystegine b 2 (76, fig. 13 ), uronic-noeurostegine (77) , was synthesized in 27-steps from levoglucosan [179] . compound conferred with improved selectivity for e. coli bglu. this is partly due to their improved lipophilic balance for bacterial cell penetration afforded by their c-alkyl unit and improved chemical stability of cec bond at the pseudoanomeric c-1 centre [180] . liminosugar c-glycoside, (à)-adenophorine (80, fig. 14a ), an enantiomer of natural iminosugar c-glycoside (þ)-adenophorine (81), was prepared together with it analogues via skeletal rearrangement of corresponding azepanes [181] . however, compound 80 and analogues were found to be weak but selective inhibitors of e. coli bglu. the c-propyl analogue, compound 82 with ic 50 ¼ 586 mm showed total selectivity for the bacterial ortholog. it was also superior to compound 80, its azepane precursor 83, and other iminosugars in the study which showed moderate selectivity with 3.1e18.1% inhibition at 1 mm. however, increasing the lipophilicity of azepane scaffold 84 via c-2 or n-alkylation, while tuning the configuration of alkyl and hydroxy substituents at c-2 and c-6 respectively (fig. 14b) , birthed e. coli bglu inhibitors with markedly increased potency and highly conserved selectivity [182] . sar analysis revealed that a combination of (6s)eoh unit and n-alkylation with hexyl, nonyl or dodecyl produced stronger inhibitors and their potency increased with increasing chain length. in contrast, (2r, 6r)-c-butyl and (2s, 6r)-cnonyl derivatives, compounds 85 and 86 respectively, were the most active c-alkylated analogues with ic 50 ¼ 261 and 27 mm respectively. compound 85 was stronger than its n-alkylated analogue, while compound 86 showed a compromised selectivity (ic 50 ¼ 97 mm against bovine liver bglu). overall, highly lipophilic compound 87, having (6s)en-dodecyl framework was the strongest inhibitor with ic 50 ¼ 3.30 mm, 3-fold superior potency to (6s)e n-nonyl analogue 88 (ic 50 ¼ 10.0 mm) and absolute selectivity for e. coli bglu. furthermore, substituent type and configuration at c-6 also influenced the inhibitory potencies of noeuromycin azepane analogues [183] . (6s)-configured compound 89 and 90 were weakly potent, compared to their inactive (6r)-configured analogues. compound 89 (ic 50 ¼ 139 mm) was 4-fold more potent than its 6hydroxymethylated analogue compound 90. incorporating the acetamido unit in siastatin b (56), while retaining the iminosugar c-glycoside scaffold via similar skeletal rearrangement of azepanes, afforded l-configured c-glycosides of 1-deoxynojirimycin (91) with weak inhibitory potencies [184] . sugar configuration at c-1 and c-2 (fig. 14d) , significantly influenced selectivity for ghs. hence, (2r, 3r)-configured molecules were selective inhibitors of e. coli bglu but inactive against bovine liver bglu, a-n-acetylgalactosaminidase and b-n-acetylgalactosaminidase. whereas, (2s, 3s)-configuration infused selectivity for b-n-acetylgalactosaminidase. consequently, (2r, 3r)-configured compound 92 with a rigid benzyl substituent emerged as the best e. coli bglu inhibitor in the series with ic 50 ¼ 90.7 mm compared to 37.2% inhibition at 1 mm of compound 93 with (2s, 3s)-configuration and butyl unit. the authors attributed these inferior activities to stereochemical mismatch between the l-configured units and dconfigured substrates of ghs. we also conceive that the presence of hydroxymethyl and not carboxylic unit (to mimic glucuronic acid) might also be responsible for the weak inhibitory potencies. this was indeed the case for 3,4,5-trihydroxypipecolic acids, uronic analogues of 1-deoxynojirimycin [185] . d-isomers were stronger than corresponding l-isomers, while the most active bglu inhibitors were those with better mimicry of glucuronic acid viz. dgluco and d-galacto configured units; compounds 94 and 95 respectively (fig. 15 ). compound 94 showed 3-fold selectivity for bovine liver bglu (ic 50 ¼ 70 mm), whereas compound 95 displayed an exclusive inhibition (ic 50 ¼ 86 mm). azoles are a prominent class of heterocycles with at least one nitrogen atom in their 5-membered aromatic ring. due to their structural rigidity and amazing physicochemical properties, which confers highly coveted and broad pharmacological activity spectrum and therapeutic potentials, they have remained a targeted scaffold of many synthetic protocols. members of this class include pyrazoles, imidazoles, thiazoles, triazoles, oxadiazoles, thiadiazoles and tetrazoles together with their benzo analogues i.e., indoles, benzimidazoles, benzothiazoles and benzotriazoles. these compounds also enjoy increased hydrogen bonding (h-b) capability furnished by ring n and/or o atoms for biomolecular targets binding. consequently, this section is an overview of reported bglu inhibitors bearing one or more azole nuclei. the critical focus is on the most potent inhibitor(s) in a given series, the pharmacological profile, structure-activity relationship (sar) and molecular docking analysis. bglu inhibitory potency (ic 50 ¼ 1.20e44.16 mm) on a set of imidazolylethylaryl carboxylates compared to reference inhibitor d-sal (ic 50 ¼ 48.38 mm) [186] . compound 96 (fig. 16 ) emerged as the most potent bglu inhibitor (ic 50 ¼ 1.20 mm) with 40-fold superior potency to d-sal. sar analysis supported by in silico studies articulated that compounds with electron-donating groups (edgs) displayed inferior inhibitory activities compared to those with electron-withdrawing groups (ewgs). moreover, molecular hybridization with indole nucleus resulted in a potent bglu inhibitor compound 97 with ic 50 ¼ 2.10 mm and 23-fold increased potency than d-sal. compound 96 and 97 displayed strong h-b interaction of indole nh unit and p-p interaction with active site residues of bglu. in vitro screening of imidazolopyridines ( fig. 17 ) for their bglu inhibitory potentials presented dihydroxy-substituted derivatives as promising inhibitors of enzyme activity [187] . (fig. 19) . however, replacing oh unit with more lipophilic meo or eto groups led to significant loss of activity. molecular docking analysis also disclosed the importance of iminic nitrogen and proximity of thiazole nitrogen to catalytic acid glu451 for strong inhibitory potency. compound 102 with adjacent oh units had a favourable fit into bglu catalytic pocket for stronger interactions with amino acid residues glu540, glu451 and tyr508. however, installing pyren-1-ylmethylenehydrazinyl moiety on thiazole skeleton improved bglu inhibitory potency [190] . comwith br, me and meo substituents were weaker inhibitors [191] . as a result, compound 108 with ic 50 ¼ 2.16 mm (fig. 21) , was the most potent inhibitor. it was 2 and 22-fold stronger than compound 109 and d-sal respectively. docking studies revealed that the free amino nh unit in compound 108 formed strong h-b interaction with oh unit of catalytic acid glu451. the dichloro substituents also substituted phenyl units at position-2 of benzimidazole core [192] . activity was dependent on the presence and position of cl and oh substituents hence 3,4-dichlorophenyl analogue 110 (fig. 22 ) was found with strongest inhibitory potency (ic 50 ¼ 6.33 mm). interestingly, introducing 5,7-dichloro unit on benzimidazole nucleus gave remarkable results as previously inactive molecules gained significant activity [193] . compound 111 emerged as the most potent inhibitor overall with ic 50 ¼ 4.48 mm and 11-fold stronger than d-sal. moreover, replacing an oh unit of the dihydroxy substituent with meo led to significant or total loss of activity. f, me, no 2 , naphthyl and anthracenyl units also gave inactive compounds. [194] . molecules containing ewgs no 2 , cl and f groups were superior in the series (fig. 23) . thus, the best activity was found with orthoakin to imidazole-indole hybrid compound 96 with ic 50 ¼ 2.10 mm (fig. 16) , molecular hybridization of benzimidazole with amide bond bioisostere, 1,3,4-oxdiazole, resulted in an isopotent hybrid 113 ( fig. 24 ) with ic 50 ¼ 2.14 mm [195] . again, ohsubstituted derivatives were stronger inhibitors compared to those containing f, cl and no 2 , me and meo substituents. docking studies revealed that the molecular hybrids adopted a linear configuration in the active site of bglu. the oxadiazole nucleus and central-phenyl ring of compound 113 formed strong h-b interaction with nh 2 unit of asn484 and phenolic oh of tyr508 respectively. compound 114 (ic 50 ¼ 3.14 mm) on the other hand interacted with catalytic acid glu451 and tyr508 via its central phenyl and phenol rings respectively. in another comparative study using 113 as lead but replacing benzimidazole with benzohydrazone unit (fig. 25) , the importance of benzimidazole nucleus to bglu inhibitory activity was established by the pronounced loss of activity [196] . nevertheless, oh-substituted derivatives and benzenetriol compound 115 emerged as the strongest inhibitor with ic 50 ¼ 7.14 mm. inhibitory potency was dependent on substituent's position in the order; para ˃ meta ˃ ortho for oh, no 2 and cl groups, while the presence of me or meo groups gave inactive compounds. molecular docking studies showed that inhibitory activity correlated strongly with the strength of h-b interaction hence the improved potency seen with oh-substituted derivatives. notably, the benzenetriol oh unit on compound 115 formed h-b interactions with glu540, asp207, tyr508, his385, and asn450, while the benzohydrazide unit interacted similarly with tyr508, glu540 and tyr504. in like manner, replacing the pharmacophores in compound 115 i.e. hydroxyl for methoxy and imine for more polar sulfonamide unit (fig. 26) , led to stronger bglu inhibitory potency for the resulting oxadiazole-benzenesulfonamides [197] . the most potent inhibitors were compounds 116 and 117 with ic 50 ¼ 2.40 and 6.34 mm respectively; compound 116 showed similar potency as compound 113. inhibitory activity was improved for parasubstituted derivatives, while ewgs produced stronger inhibitors than edgs. binding interactions in the active site of bglu especially h-b interaction of benzamide nh unit with glu451 established the improved activity of the sulfonamides compared to benzohydrazones. compound 116 formed h-b interaction via sulfonyl oxygens with asp207 and asn450, while the benzamide oxygen interacted in similar fashion with tyr508. additional ionic bonding between no 2 groups in compound 117 and glu451 also accounted for the improved potency. molecular hybridization (fig. 27) inhibitor d-sal respectively (fig. 28) . in silico studies of these benzothiazoles revealed that they adopted a linear conformation which allowed appropriate fit into the binding groove of bglu. ortho-oh unit on compound 120 formed strong h-b interactions with glu451 while the para-oh unit provided h-b interaction with asp207. the enzyme-inhibitor complex was stabilized in the active site through hydrophobic interactions of benzenetriol ring with indole nucleus of trp587, his385, asn484, tyr504, his509, arg600, and lys606. most importantly, all the active inhibitors were noncytotoxic in a cytotoxicity assay using mouse embryo fibroblasts (3t3-l1) and wistar rat hepatocytes (cc-1). benzothiazole and benzimidazole are bioisosteres due to their structural and pharmacological similarities; therefore, bioisosteric replacement of one for the other has been consistently explored in drug development. accordingly, bioisosteric replacement of the benzimidazole moiety in compound 113, to give new benzothiazole-oxadiazole hybrids resulted in equally potent bglu inhibitors [200] . the most potent compound 122 (fig. 29 ) with ic 50 ¼ 2.16 mm, was equipotent with compound 113. fascinatingly, compound 123 (ic 50 ¼ 4.38 mm) was also isopotent with similarly substituted compounds 121 and 111. further, docking studies of compounds 122 and 123 showed that h-b interactions of phenolic oh units with active site residues viz., glu451, tyr508, and asp207, favoured their strong inhibitory potency. moreover, compound 122 formed hydrophobic interactions with tyr504 and lys606 via its thiazole core and benzene ring respectively. the reduced potency of compound 123 was established in the strength of these interactions as well as the absence of additional van der waal contacts of benzothiazole ring in compound 122 with catalytic residues glu451 and glu540. in silico pharmacokinetic properties modelling of these inhibitors predicted good cell permeability and solubility. the compounds were also non-cytotoxic to 3t3-l1 and cc-1 cell lines. following a similar molecular development strategy leading to compound 122, new benzothiazole-hydrazone hybrids were assembled by deleting the 2-oxadiazolylphenol fragment in compounds 122 and 115 (fig. 30) [201] . compared to the parent compound series, the new hybrids had marked reduction in potency. the recurring pattern of reduced or abolished activity also persisted with me and meo substituents. nevertheless, compound 124 was 3.2.1.7. triazole. the triazole ring also affords potent bglu inhibitors. in vitro evaluations of 1,2,4-triazole-schiff bases [202] presented isatin-containing compound 125 (fig. 31) as the strongest inhibitor with ic 50 ¼ 2.50 mm and 19-fold superior activity than d-sal. cl, oh and no 2 substituents at ortho-positions also gave appreciable activities whereas me, meo, cumyl and other aryl derivatives were inactive. docking simulations of compound 125 in the active site of bglu revealed strong h-b interaction between isatin nh and catalytic residues glu540 and tyr504. the phenyl ring also provided p-anion and p-p interactions with glu451 and tyr504 respectively while van der waal contacts with lys606, tyr508, val410 and asn450 stabilized the inhibitor-bglu complex. however, amide bond bioisostere, 1,2,3-triazole, is a stronger bglu inhibitor compared to its 1,2,4-triazole isomer. molecular hybridization with carbazole [203] delivered eight compounds having ic 50 < 3 mm (fig. 32) . compound 126 (ic 50 ¼ 0.55 mm) was the most potent hybrid in the series and 83-fold stronger than d-sal. all the carbazole-1,2,3-triazole tethers were non-cytotoxic to 3t3 cells. 3.2.1.8. indole-based hybrids. a library of indole analogues containing the hydrazone unit in compound 115 have been examined for bglu inhibition [204] . evinced by the increased potency, the indole pharmacophore delivers stronger bglu inhibitors compared to benzothiazole and oxadiazole. for instance, the most potent inhibitor 127 with ic 50 ¼ 0.50 mm (fig. 33) , was 100-fold more potent than d-sal and 42-fold stronger than similarly substituted oxadiazole variant from compound 115 library. compounds 128 (ic 50 ¼ 2.40 mm) and 129 (ic 50 ¼ 2.50 mm) were also 3-fold and 43fold stronger respectively than their corresponding oxadiazole variants. moreover, substitution at ortho and para-positions gave stronger inhibitors compared to meta-position, while potency in monosubstituted analogues followed the order; f ˃ oh ˃ cl ˃ pyridyl ˃ no 2 ˃ meo. the excellent potency of compound 127 was afforded by strong h-b interaction of position-5-oh with glu451 and glu540 as well as position-4-oh with tyr508. indole and benzohydrazone nh units formed h-b interactions with asn502 whereas the ligandreceptor complex was stabilized by p-alkyl interaction of indole ring with trp528. expectedly, the reduced activity of compound 128 was seen in its weaker interactions in the active site of bglu. based on the pharmacological significance of thiadiazole pharmacophore and the promising inhibitory activity of indole hybrid compound 127, hybrids of indole-thiadiazole were assembled as new class of bglu inhibitors [205] . generally, thiadiazole nucleus infused stronger inhibitory potencies on the resulting hybrids compared to hydrazone unit. dihydroxy substituted compound 130 emerged as the strongest inhibitor with ic 50 ¼ 0.50 mm and equipotent as compound 127. ortho-substitution was more beneficial to potency than para and meta-substitutions, while inhibitors' strength followed the order; oh ˃ f ˃ me ˃ pyridyl ˃ cl ˃ no 2 (fig. 34) . however, bioisosteric replacement attempt with oxadiazole proved that thiadiazole unit is preferred for stronger inhibition [206] . the strongest potency remained with 2,3-dihrdoxy substituted derivative 131 (ic 50 ¼ 0.90 mm), although 2-cl derivative had an outlying potency. bazedoxifene (fig. 35) is a novel indole-based inhibitor of il-6/ gp130 for the management of triple negative breast cancer [207] . it is also a selective estrogen receptor modulator administered as co-drug with conjugated estrogens (duavee) in estrogen replacement therapy for the prevention of postmenopausal osteoporosis. albeit, the high ratio of circulating estrogen metabolites and parent estrogen aided by bglu deglucuronidation activity in the gut is considered a risk factor for postmenopausal estrogen receptor positive (erþ) breast cancer. accordingly, a study on the therapeutic utility of this combination drug in an ovariectomized mouse model showed significant reduction in faecal bglu activity without altering faecal microbiota diversity [208] . the study articulated the significance of bacterial bglu inhibition to improving the outcome of long-term administered estrogens for postmenopausal women or breast cancer patients. consistent with the excellent inhibitory potencies of indolebased compounds, a library of bis-indoles (fig. 36) have been reported as strong bglu inhibitors [209] . once more, the importance of (di)hydroxy substitution to potency was prominent. compound 132 emerged as the most active molecule with ic 50 ¼ 1.62 mm and 30-fold superior to d-sal. in silico studies suggested that h-b donor-acceptor potential of oh units on compound 132 significantly influenced ligand-receptor interaction. ortho-oh formed strong h-b with amino acid residues asp207 and tyr508, while para-oh interacted similarly with his385. arene-arene interaction of indole ring with tyr504 also aided the favourable binding to bglu. in another study [210] , both compound 132 and its 2,3dihydroxy compound 133 (ic 50 ¼ 1.2 mm) were the strongest inhibitors of bacterial bglu; albeit with moderate cytotoxicity against 3t3 mouse fibroblasts. the presence of n-phenyl substituted thiosemicarbazide unit however introduced marked increase in bglu inhibition of 1hbisindoles [211] . inhibitory potency for monosubstituted analogues followed the order f z cf 3 ˃ cl ˃ br ˃ me ˃ meo and ortho-substitution produced the best result (fig. 37) . accordingly, 2-f to glu540, asn450, lys606, trp528. tyr508 also formed h-b interaction with amide nh and arene-arene interaction with phenyl ring of tolyl unit. chalcones are both a class of naturally occurring flavonoid family and synthetically obtainable compounds with extensive therapeutic applications [213] . the presence of a highly reactive a,b-unsaturated ketone unit and delocalized electrons makes them coveted precursors for many synthetic manipulations and pharmacological explorations. the pro-inflammatory role of bglu in inflammatory disorders motivated the design of a series of (di)hydroxychalcones (fig. 39) , to inhibit the release of bglu from stimulated rat mast cells and neutrophils [214e216]. it was established that hydroxychalcones were stronger and selective inhibitors of neutrophil-derived bglu over mast cell-derived bglu. the best inhibitors were compounds 139 (ic 50 ¼ 0.6 mm; ˃ 160-fold selectivity) and 140 (ic 50 ¼ 1.3 mm; 7fold selectivity). sar analysis showcased the importance of a,bcompound 141 (fig. 40 ) has emerged as the most potent inhibitor with 75% inhibition at 10 mm and only 3-fold stronger than standard salicylic acid, amongst a series of chalcone-mannich adducts [217] . adopting a similar molecular design [218] , a library of chalcone-benzamides were found with slightly improved potency and sar analysis established the preference of ewgs over edgs for better enzyme inhibition. compound 142 containing similar 3-br substituent as compound 141, emerged as the most potent molecule (84.68% inhibition at 1 mm); 3-fold stronger potency than salicylic acid (24.77% at 1 mm). it was also non-cytotoxic in cck-8 assay in contrast to 2-fold increased cytotoxicity of equally potent 3-cf 3 analogue. notably, activity modulation by substituting trimethoxy unit for indole was unsuccessful [219] , leading to prominent reduction in percentage inhibitory activity of the chalcones (˃ 8-fold). structural development of a coumarin (chromen-2-one) compound 143 (fig. 41 ) recovered from virtual screening delivered a set of moderately potent inhibitors of bglu activity [220] . superior activity compared to d-sal (ic 50 ¼ 48.40 mm) was recorded compounds 144e147 with ic 50 values of 9.90, 11.70, 21.40 and 34.20 mm respectively. in another attempt [221] , despite different molecular tunings including polar oh and cl groups, the coumarin pharmacophore remained inactive against bacterial bglu. conversely, structurally isomeric flavenone (chromen-4-one) bearing a pyranose appendage, showed stronger inhibition of bacterial bglu [210] . compound 148 (fig. 42) , the most active inhibitor thereof with ic 50 ¼ 4.50 mm and 10-fold superior potency to d-sal, was totally non-cytotoxic against 3t3 mouse fibroblasts. in silico studies further disclosed the importance of pyranose ring in h-b interactions with active site residues glu503, asp161 and glu413. fascinatingly, introducing 1,3,4-oxadiazole pharmacophore to a chromen-4-one scaffold (fig. 43) , led to significant increase in potency [222] . the new hybrids exhibited consistent trend in their potency based on substituent type and position in the order; f > cl > me > no 2 > pyridyl > meo and ortho > para > meta respectively. thus on the other hand, azacoumarins (1h-quinolin-2-ones) having a basic nitrogen atom in place of endocyclic coumarin oxygen, are conferred with stronger and selective inhibitory potencies against bacterial bglu. plausibly, their admirable activity is provided by the ability to mimic or interfere the oxocarbenium ion-like transition state akin to iminosugars; thus, allowing the alleviation of druginduced toxicities and prevention of colon carcinomas. renowned examples in this class of bglu inhibitors are the strongly potent azacoumarins 151 and 152 (fig. 44) , identified from high-throughput screening [19, 223] . the compounds exhibited in vitro ic 50 values of 0.28 and 0.37 mm respectively, over 1000-fold selectivity for e. coli bglu and 100-fold increased potency than reference inhibitor glucaro-d-lactam 61. the inhibitors also maintained their potency in living bacterial cells with ec 50 ¼ 0.018 and 0.028 mm. other similarly active compounds (ic 50 (once daily), alleviated irinotecan (cpt-11) induced diarrhoea evident by a protected gi epithelium of balb/cj mice compared to bloody diarrhoea due to damaged tissues and glandular structure in those receiving cpt-11 alone [19] . pre-treating c57bl/6j mice with 10 mg of inhibitor 151 before intraperitoneal administration of ulcerogenic dose of nsaids e diclofenac (60 mg/kg), indomethacin (10 mg/kg) and ketoprofen (100 mg/kg), also successfully protected the mice models against nsaid-induced small intestine mucosal damage [224, 225] . moreover, compound 151 reduced all enteropathy parameters by inhibiting gi bacterial bglu-mediated hydrolysis of nsaid-acyl glucuronide in a concentration-dependent manner (ic 50 z 0.164 mm). interestingly, the compound did not alter drug's systemic exposure, hepatobiliary excretion of glucuronides and gi microbiota diversity. although it possesses a short half-life (˂1 h) and pan-cytochrome p450-mediated poor bioavailability (21%). however, an attempt to reduce the inhibitor's serum exposure for increased gi residence by replacing ethoxy unit with hydroxyl or morpholinyl groups led to inferior pharmacological profile compared to parent compound 151 [226] . further, compound 152 exerted 2-fold reduction in the severity of diclofenacinduced anastomotic leakage, without affecting drug's plasma concentration in wistar rats receiving 0.8 mg of the inhibitor and 3 mg/kg of diclofenac [227] . in contrast, rats receiving diclofenac only suffered severe leakage thus suggesting the inhibitor's potential clinical utility to improve anastomotic healing. emerging from the screening protocol which birthed compound 151, three piperazine-containing compounds 153, 154 and 155 ( fig. 45) showed promising inhibitory potentials against bacterial bglu [228] . though with slightly weaker inhibitory strengths (ic 50 the therapeutic significance of fused pyridinone-furans as antiinflammatory drug candidates via inhibition of neutrophil-derived bglu has been reported [234] . compounds bearing meo and cf 3 substituents on ring b (fig. 47) showed over 70% inhibition at 1 mm with no cytotoxic effects (<32% at 10 mm) in cck-8 assay as compared to reference salicylic acid (25% inhibition). replacing ring b with furan, thiophene or pyridine however gave poor inhibitors (<45% inhibition). compound 158 was the most potent with 75% inhibition. the inhibitory potencies of dihydropyrimidinone carboxylates ( fig. 48) , against bovine liver-derived bglu has reiterated that small polar substituents are necessary for strong enzyme inhibition [235] . appreciable potency (ic 50 ˂ 20 mm) was exclusive to cf 3 ˃ f ˃ oh ˃ thienyl ˃ cl substituted compounds in that order. whereas, meo, br, benzyloxy, furanyl, aliphatic alkyl or aryl units either reduced potency when co-substituted with polar f or oh groups, gave inferior activity with monosubstitution or rendered the molecule totally inactive. therefore, the most active compound 159 with ic 50 ¼ 9.38 mm, found favourable fit in the active site of bglu to form strong h-b interactions with key residues. nh of asp207 was both a h-b donor and acceptor to pyrimidinone carbonyl oxygen and free nh at position-1 respectively. carboxylate carbonyl oxygen interacted similarly with arg600 whereas position-3 nh interacted with catalytic residues glu451 and tyr508. however, pyrimidinetriones (barbituric acid) [236, 237] , exhibited inferior potencies compared to standard d-sal (ic 50 ¼ 45.75 mm), despite their similar binding interactions as compound 159 with active site residues and non-cytotoxicity to 3t3 cells. in a predating study, the structural homolog of pyrimidinone, quinazolinone [238] , exhibited a unique trend in inhibitory potency against e.coli bglu with significant tolerance for lipophilic alkoxy groups, although poor activity with methyl substituent persisted. the most active compound 160 (fig. 49) intriguingly, replacing alkoxy units with more polar oh, no 2 and cl substituents led to significant reduction in potency. inhibitory potency therefore followed the order; meo ˃ eto ˃ n(me) 2 ˃ no 2 ˃ oh ˃ cl and ortho ˃ para ˃ meta. importantly, the quinazolinone-based inhibitors were all non-cytotoxic to 3t3 cells. it is also noteworthy that a quinazolinone compound (table si) from ref. [223] , also showed strong inhibitory potency (ic 50 ¼ 1.90 mm) against bacterial bglu. the quinoline nucleus can be regarded as a household name in medicinal chemistry, due to its inexhaustible pharmacological application and occurrence in many clinically important natural products and synthetic molecules. the desirable pharmacokinetic profile and ease of accessibility with wide substrate tolerance for different synthetic protocols, also encourages its utility in countless synthetic explorations. research endeavours aimed at quinolinebased bglu inhibitors are therefore presented in this section. quinoline-3-carbohydrazides [239] , prepared analogously as hydrazone tethers above also holds promising inhibitory activities. similarly, oh unit at ortho-position yielded stronger inhibitors than f > cl > no 2 > pyridyl > thiophenyl > furanyl z me > meo, in that order. moreover, ortho-substitution consistently gave superior inhibitors compared to para-position, while meta-substitution conferred the weakest potency. 2 to 3-fold reduction in potency persisted when oh unit is substituted for meo unit (fig. 50) . hence, compound 162 (ic 50 ¼ 2.11 mm) and 163 (ic 50 ¼ 2.60 mm) emerged as the most potent inhibitors overall. docking studies revealed that oh unit at position-4 of quinoline ring fostered important h-b interaction with catalytic acid glu451, while carbonyl unit of hydrazone moiety formed h-b interaction with nh of tyr504. in another quest for new anti-inflammatory agents, quinolinefuran hybrids (fig. 51) were designed as inhibitors of bglu release from activated neutrophils [240] . sar analysis, established that 2-(furan-2-yl)quinolines (type a) were stronger inhibitors of inflammatory mediators than tricyclic furo[2,3-b]quinolines (type b). whereas, formyl unit favoured potency compared to acetyl, oxime, methyl oxime as well as both a,b-unsaturated butan-2-one and its saturated variant. therefore, non-cytotoxic compound 164 (ic 50 ¼ 5.0 mm) was the strongest inhibitor of bglu release with 3fold superiority to reference trifluoperazine. acetyl isomer compound 165, also had similar non-cytotoxic and strong inhibitory potency (ic 50 ¼ 7.5 mm). akin to azacoumarins (1h-quinolin-2-ones) 151 and 152, new quinoline-pyrazole conjugates were prepared via hit development protocol of compound 166 (fig. 52) identified from highthroughput screening, for the inhibition of intestinal bacterial bglu's hydrolytic activity on sn-38g fo alleviate cpt-11 druginduced toxicity [241, 242] . interestingly, all the resulting derivatives showed good selectivity for e. coli bglu except 2-cl, 3-oh, 3-me and 4-nh 2 substituted derivatives. the trend in inhibitory potency, based on substituent's position varied in the order; meta < ortho < para, while the presence of strongly hydrophilic oh, no 2 and nh 2 units at para-position was detrimental to potency. following oral administration to balb/cj mice for 5 consecutive days compared to compound 168 (40%). whereas, pharmacokinetic profiling of compound 168 revealed its lower plasma concentration and increased gi residence for improved intestinal bglu inhibition compared to azacoumarin compound 151. most importantly, oral co-administration of compounds 167 or 168 at 3 mg kg à1 day à1 for 10 consecutive days with 50 mg kg à1 day à1 intraperitoneal injection of cpt-11, protected balb/cj mice models against cpt-11 drug-induced intestinal mucosal injury, without altering drug's therapeutic efficacy. furthermore, mechanistic studies established that the presence of electronegative and electroneutral surface charges on e. coli active site pocket and the protonation of n-1 and n-5 atoms avails a ph-dependent and selective inhibition for the compounds (fig. 52) . consequently, compounds 167 and 168 are clinically viable agents for protecting against gi e. coli bglumediated mucosal damage and preventing the release of chemical carcinogens crucial to the development of precancerous lesions for colon carcinogenesis. the 200-fold increased inhibitory potency of benzohydrazideen-cyanoethyl tethers [243] , compared to parent compounds articulates the significance of a balanced molecular lipophilicity to bglu inhibition. o-alkylation with benzyl, benzoyl or tosyl groups slightly influenced potency for oh-substituted units; benzylation produced the best result (fig. 53) . hence, 169 (ic 50 ¼ 1.60 mm) and 170 (ic 50 ¼ 2.20 mm) were the most active conjugates in the series. similarly, phenoxyacetohydrazones [244] have displayed 2 to 5-fold improved potency compared to d-sal (ic 50 ¼ 48.40 mm). compounds 171 and 172 (fig. 54 ) emerged as the the desirable therapeutic significance of fused thienothiophenes has stimulated the synthetic exploration of a thienothiophene skeleton 173 (fig. 55) for bacterial bglu inhibition [245] . from the resulting thienothiophene library with interesting structural diversity, only compound 174 showed inhibitory activity against bacterial bglu with ic 50 value of 0.9 mm; 51-fold superior potency to d-sal. notably, the predicted pharmacokinetic properties using molinspiration and osiris calculations suggested that the inhibitor possesses good bioavailability and no toxicity risk as bacterial bglu inhibitor. in vitro study of phenyl disulphideebenzenesulfonamide tethers [246] , disclosed compounds 175 and 176 (fig. 56 ) as potent inhibitors of bglu (ic 50 ¼ 2.20 and 3.34 mm respectively), analogous to similarly substituted oxadiazole-benzenesulfonamide compounds 116 and 117 (fig. 28) . interestingly, bulkier phenyl disulfides 175 and 176 shared similar potency with lower molecular mass thiosulfinate 52, fig. 9 (ic 50 ¼ 3.60 mm). however, substitution at ortho-position gave stronger inhibitors than para-and meta-positions, in contrast to compounds 116 and 117 series. docking studies further revealed that the strong inhibitory potency of compound 175 is aided by the h-b interactions of sulfonamide nh and oxygen with tyr205 and asp207 respectively. the efficacy of urea unit for bglu inhibition was also examined using a library of no 2 substituted n-phenyl ureas [247] . therefrom, appropriate positioning of polar no 2 unit was important to inhibitory potency of examined molecules. compound 177 (fig. 57) , emerged as the only inhibitor with promising activity with ic 50 value of 3.38 mm. however, thiourea analogues bearing meta-cl substituent on n-phenyl ring [248] , were stronger inhibitors compared to those with unsubstituted n-phenyl ring or no 2 substituted ureas (fig. 58 ). akin to compound 141, thioureas containing piperazine unit also exhibited improved potency compared to their morpholine and piperidine analogues, while n-alkylation of piperazine with n-(o or p-methoxyphenyl) or n-(2-pyridyl), extant empirical data identified bacterial bglu as a chelating agent, since enzyme activity was totally lost in the presence of cu 2þ , hg 2þ and ag þ metal ions and restored in the presence of other chelating agents e versene and cysteine [249] . therefore, metal complexes with coordinatable metal centres exhibit significant bacterial bglu inhibitory activities. to this end, pd(ii) complexes containing aniline and triphenylphosphine ligands were synthesized [250] . para-chloroaniline derived metal complex 182 (fig. 59) , displayed the strongest activity against bacterial bglu with 98.5% inhibition and ic 50 ¼ 15.40 mm. it was also 2-and 5-fold superior to meta-chloroaniline derived and n-methyl substituted metal complexes respectively. similarly, n-heterocyclic carbene (nhc) complexes of au(i) [251] , as well as bis (nhc) complexes of au(i) and au(iii) [252] , containing hydroxy, chloride, acetoxy and acetonyl ligands were equally potent bacterial bglu inhibitors with ic 50 ranging from 0.14 to 2.60 mm. the strongest au(i) nhc complex 183 (ic 50 ¼ 0.14 mm) was 327-fold more potent than reference d-sal and 7-fold superior to strongest bis (nhc) complexes 184. nonetheless, undesirable cytotoxicity against 3t3 cells discomfits the therapeutic significance of these metal complexes. parallel to iminosugars 60e74, the inhibitory potencies of structurally similar glycopolymers (fig. 60 ) was dependent on the presence of carboxyl unit, type and configuration of sugar unit. hence, glycopolymers with d-glucaric pendants linked to the polymer frame at c-1 (185), were stronger inhibitors compared to glycopolymers linked at c-5 (186) or those bearing d-gluconic (187) [253], d-mannaric (188) [254] and shorter chain d,l-xylaric (189) and l-tartaric (190) [255] pendants. however, these glycopolymers only show >60% inhibition at millimolar concentrations. patents and patent applications disclosing potent inhibition of bglu particularly bacterial bglu and the therapeutic significance thereof, have been documented. we therefore review in this section, patents applications filed in the present millennium relevant to bglu inhibition. we have also included the only biomarker application of the enzyme filed within the period [256] . however, some of the patents and applications [257e263] emanated from published papers [19, 210, 228, 229, 241, 262] , reviewed in previous sections; therefore, only their summary is presented in table 3 . considering the limitations associated with traditional clinical assessments for periodontal diseases diagnosis and the improved reliability of bglu as a biomarker, a simple method requiring less expertise has been claimed to quantify elevated levels of the enzyme in the saliva of diseased patients relative to healthy ones, as a biomarker of disease risk and status [256] . the invention involves adding known b-d-glucuronide substrates (4-methylumbelliferone or phenolphthalein b-d-glucuronides) to a sample of patient's saliva in bglu activity assay and thereafter quantifying the amount of aglycone produced via fluorometry, colorimetry or spectrophotometry. the addition of a labelled polyclonal or monoclonal antibody specific for bglu to saliva sample and subsequent estimation of the amount of labelled antibody forming bglu-antibody complex was also claimed, as well as a test-kit for claimed methods. bacterial bglu-mediated hydrolysis of steroid glucuronides on the skin leads to body odours hence inhibitors of the process were developed and applied in deodorant and antiperspirant formulations [264] . the invention claimed that deodorant formulations with described compounds only inhibits bglu activity without altering the natural composition of skin microbiota; in contrast to bacteriostatic or bactericidal mode of action of prior arts. amongst the claimed inhibitors of different class, the strongest inhibition of e. coli bglu was found with galactaric acid (99% inhibition at 0.1% test concentration in water), as well as zinc glycinate and zinc gluconate; 99% and 97% inhibition respectively at 0.25% test concentrations. in the same vein, potent inhibitors of bacterial bglu activity on urine have found non-therapeutic application in hygiene and sanitary products for suppressing the generation of urine odour [265] . at 0.1 wt% in dipropylene glycol, all the claimed inhibitors viz. macrocyclic ketones, ketones, macrocyclic lactones, macrocyclic oxalactones, esters, aldehydes, alcohols, ethers and terpenes, showed over 60% inhibition of e. coli bglu. macrocyclic ketones were stronger inhibitors than others. consequently, the invention's most active inhibitor was compound 191 with 100%, 99.9% and 95.8% inhibition at 0.1, 0.01 and 0.001 wt% respectively. it was also effective after 48 h in suppressing the increase in urine odour when incorporated into different hygiene and sanitary products. phenoxy thiophene sulfonamides [266] were designed as adjuvants to eliminate the dose-limiting gi toxicity (diarrhoea) of cpt-11, through potent inhibition of bacterial bglu-mediated deconjugation of active metabolite's glucuronide (sn-38g) in the intestine; thus, improving the drug's therapeutic efficacy. the synthesis of 76 bglu inhibitors and 18 analogues of brite-355252 (compound 192) , the most active compound (ic 50 ¼ 20 nm), together with their therapeutic application was claimed. sar study of compound 192 analogues revealed that inhibitory potency was markedly dependent on n-piperazinyl pendant at meta-position of phenoxy ring. over 500-fold reduced inhibitory potency occurred with n-methylation (akin to compound 156), 15-fold reduction when appended at para-position and total loss of activity when removed. uninstalling the chloro unit on thiophene ring also slightly diminished potency by 5-fold. whereas, replacing naphthyl with substituted phenyl units afforded inhibitors with similar potencies i.e. ic 50 < 50 nm (table si) ; as a result, 193 was equipotent with 192. bglu is a physiologically important lysosomal glycosyl hydrolase with appreciable therapeutic potentials such as biomarker for disease diagnosis, endogenous bioactivator in prodrug monotherapy and enzyme replacement therapy. the enzyme's role in cell proliferation and inflammation also renders it a potential target for anticancer and anti-inflammatory drugs development respectively. moreover, since a significant number of commercially available drugs are metabolised by glucuronidation, hydrolytic activity (i.e. deglucuronidation) by bglu in human intestinal microbiota has been linked to colon carcinogenesis and genotoxicity as well as drug-induced dose-limiting toxicities of anticancer agents and nsaids, which thwarts their therapeutic potential and clinical utility. therefore, potent inhibition of bglu holds enormous clinical importance. generally, our literature survey found that a significant number of natural products-derived inhibitors display moderate inhibition of bglu, and increased potency is found with inhibitors containing a flavonoid skeleton. the physiological tolerance, acceptable toxicity and favourable pharmacodynamic profiles of these natural inhibitors, posits them as worthy scaffolds warranting witty molecular developments. in addition, iminosugars distinguish themselves as a unique class of natural product-inspired molecules with promising potentials regardless of their usually multiple synthetic steps. notable amongst these are the nojirimycin analogues and iminosugar c-glycosides, both conferred with highly selective inhibition of bacterial bglu. however, the inhibitory potency of these carbohydrate mimics relies on several factors such as sugar configuration, mimicry of glucuronic acid substrate and correct lipophilic balance. hence, to harness their remarkable potential, ingenious manipulations of the synthetic medicinal chemist interested in exploring their chemical space is expressly required. on the other hand, purely synthetic inhibitors show fascinating diversity in their inhibitory potencies and pattern, due to the plethora of pharmacophoric enrichments possible for a single molecular design. based on the foregoing, a comprehensive list of potent synthetic inhibitors (ic 50 5 mm) in this review is provided in table si of supporting information. evinced by their nanomolar ic 50 values, quinoline-pyrazoles (167 and 168), piperazinecontaining compounds (156 and 157; 192 and analogues) as well as the indole nucleus, are conferred with the strongest inhibition of bglu activity overall. the order of potency for hydrazone tethers was found as; bisindole ˃ indole > quinoline > thiazole ˃ benzothiazole z oxadiazole. whereas, no 2 , cl, particularly f and oh substituents at ortho-or para-positions usually produce stronger bglu inhibitors compared to their meta-substitutions. taken together, these suggests that the presence of polar groups with strong hydrogen bonding potential is crucial to inhibitory potency, while an indiscriminate increase in molecular lipophilicity is detrimental to strong bglu inhibition. consequently, we envisage that the model bglu inhibitor will be that which is appositely tuned in its lipophilicity and polarity for easy cell penetration, favourable fit into the binding/catalytic pocket of bglu for energetically stable binding and strong enzyme inhibition. we also perceive that rationale molecular hybridization of those active class of natural products and synthetic molecules will furnish stronger inhibitors of bglu with improved selectivity and acceptable toxicity profile. furthermore, we found that due to the mixture of electroneutral and electronegative spots on the surface of bacterial bglu binding pocket at physiological ph, in contrast to the electropositive spots for human bglu, the presence of a protonatable n-atom confers improved selectivity for bacterial bglu inhibition. this was particularly true for piperazine based inhibitors. therefore, we believe further probing of this phenomenon will allow structureactivity guided molecular tuning to afford stronger bglu inhibitors. to the best of our knowledge, mechanistic studies on the behaviour of these inhibitors is scanty in 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concentration il-1b: interleukin-1 beta il-8: interleukin-8 k cat : rate constant of catalysed reaction kda: kilodalton k i : inhibition constant k uncat : rate constant of uncatalyzed reaction lab glucuronide conjugate of sn-38 tnf-a: tumour necrosis factor-a udp: uridine-5 0 -diphosphate ugt: udp-glucuronosyltransferase the national research foundation (nrf) of south africa is appreciated for kic grant and other financial support. supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmech.2019.111921. key: cord-309052-3h0g7s9v authors: alam, fiaz; khan, gul nawaz; asad, muhammad hassham hassan bin title: psoralea corylifolia l: ethnobotanical, biological, and chemical aspects: a review date: 2017-12-15 journal: phytother res doi: 10.1002/ptr.6006 sha: doc_id: 309052 cord_uid: 3h0g7s9v psoralea corylifolia l. (leguminosae) is a well‐known traditional medicinal plant used from ancient times for treatment of various ailments. it is widely distributed and an important part of therapeutics in ayurveda and in chinese medicines. the aim of this review is to present comprehensive and most up to date report on its ethnobotanical, ethnopharmacological, clinical, phytochemical, and side effects. studies on the ethnobotanical, ethnopharmacological, clinical, phytochemical, and side effects of p. corylifolia were published until year 2017 and were searched using various scientific databases. the scientific literature searched revealed that these plant species has been extensively investigated in vivo and in vitro for various biological and phytochemical studies. it has cardiotonic, vasodilator, pigmentor, antitumor, antibacterial, cytotoxic, and anti‐helminthic properties and locally used for alopecia, inflammation, leukoderma, leprosy, psoriasis, and eczema. so far, about a hundred bioactive compounds have been isolated from seeds and fruits, and most important compounds identified belongs to coumarins, flavonoids, and meroterpenes groups. this review article summarized the most updated scientific literature on bioactive phytochemical and biological activities of p. corylifolia. this article will be a useful addition to providing information for future research, and more standard clinical trials are needed for the plant to be used as therapeutic agent. the family leguminosae contains about 500 genera and is among the largest families of flowering plants. the plants of leguminosae are widely distributed, and in terms of number of specie, it is one of the largest terrestrial family of plants after orchidaceae and asteraceae (stevens, 2015) . the genera that belong to this family are important medicinally and contain a variety of biologically important molecules; p. corylifolia is an important part of therapeutics in ayurveda and in chinese medicines. the plant is cardio active and showed antimicrobial and cytotoxic properties. it is used as a pigmentor. the plant showed cytotoxicity against tumors and worms (baquar, 1989; rizvi, saeed, & zubairy, 2007; sharma, 2004) . in chinese medicines, the psoralea plant is considered warm by nature, and therefore shows many healing actions on kidney and spleen meridians (daiquan, 2000 (daiquan, -2006 . the seeds of p. corylifolia are used in indigenous medicine systems for healing of various ailments. the seeds are diuretic, aphrodisiac, laxative, anti-helminthic, and are used in febrile conditions. in ayurveda, the seeds are used in the form of paste and as an ointment for external as well as internal use for treatment of different conditions such as alopecia, inflammation, leukoderma, leprosy, psoriasis, and eczema (huang, 1998; judd, campbell, kellogg, stevens, & donoghue, 1999; sjc, 2015) . in india, the powder of seeds is mixed with haratalabhasma (yellow arsenic) and converted to paste with the urine of cow. this paste is used to treat leukoderma lesions. in another formulation, the mixture of powdered seeds with buttermilk have been used externally for treating ringworm and scabies. the seed oil is taken orally with betel nut leaf for treatment of leprosy. another skin condition, dermatosis, has been treated with adjuvants therapy of bakuchi (p. corylifolia) with other local plants such as amalaki and khadira. similarly, the oils of bakuchi and karanja are mixed with vaseline to treat chronic skin diseases (khare, 2008) . the seeds of the plant are also considered useful in bilious disorder, snakebite, and in scorpion sting. the seeds are also used in the production of perfumes (deshaprabhu, 1966; maisch, 1889; sharma et al., 2000) . the root of p. corylifolia has shown its effectiveness in dental caries. the fruits are aphrodisiac and have laxative effect, and the leaves are antidiarrheal (baquar, 1989; sjc, 2015) . the plant is also valuable in treating alopecia areata (dua, kumar, pandey, & kumar, 2013b) . in combination with haritka and gokshura, this plant is used for urinary frequency, and with ashwagandha and bala, it is used for treatment of reproductive diseases and cough. in chronic diarrhea and for treating cold symptoms, this plant is used in combination with nutmeg and haritaki (anderson & voorhees, 1980) . the seeds of bakuchi in powder form mixed with the decoction of bibhitaka (terminalia bellirica bark) and kaakodumbara (ficushispida) was effective to treat vitiligo. the infection of ringworm is treated with a combination of tila (sesame seeds) and bakuchi (gupta, dhar, & atal, 1978) . a combination of bakuchi with haratalabhasma provides relief against leukoderma when applied externally (khatune et al., 2002) . in japan, the plant extracted with alcohol has been used as an additive in processed foods and pickles (nadkarni, 1976) . when the fixed oils are removed, what is left after the seed cake are used as feed or manure due to the presence of nitrogen (6.7%) and the minerals (7.8%). volatile oils obtained from fruits have an irritant effect on the skin and mucous membranes and stimulate the voluntary muscles in high concentrations (chaudhuri, 2015; gidwani et al., 2010; huang, 1998) . p. corylifolia in the form of extracts have been used in various herbal formulations when combined with other herbs and used in handling psoriasis and many other skin disorders . this plant is also reported to be used in cardiac problems, asthma, and urinary discharge (kr, 1975) . it also has anti-leishmaniasis activity (ali, akhtar, sultana, baboota, & ahuja, 2008) . it is used to control watery stool, urinary frequency, and reproductive imbalances (pole, 2006) . in china, it is specially used against vitiligo disease (anderson & voorhees, 1980; khare, 2004) . p. corylifolia is available in the form of various ayurvedic marketed formulations in india and across the world, the most famous brands are safuf bars®, zimad kibrit®, svitrakaravati®, khadirarista®, algushadi yoga®, sarvangasundarigutika®, bhallatakawaleha®, maheshwa-raghrita®, ayorajodilepa®, brihatsomarajitaila®, somarajighrita®, bawchichurna®, etc. (ali et al., 2008; pole, 2006; qiao et al., 2007) . the investigation showed that p. corylifolia possessed a wide range of phytochemicals including flavones, coumarins, monoterpenes, chalcones, lipids, resins, stigmasteroids, and flavonoids. the volatile oils are also reported from this plant (zhang, zhao, wang, lu, & chen, 2016) . it is revealed that seasonal variation also effects the phytochemistry of p. corylifolia. mostly, the bioactive compounds are found to be concentrated in the seeds. the phytochemistry of this plant is discussed as follows. the whole plant of p. corylifolia was extracted with organic solvents such as petroleum ether and chloroform. the subsequent isolation methods lead to the purification of bioactive compounds, for example, psoralen, isopsoralen, corylifolin, corylin, and psoralidin (gupta, gupta, & gupta, 2013) . peng and colleague obtained a new compound identified as neo-psoralen from the whole plant of p. corylifolia in 1996 and elucidated its structure on the grounds of chemical indications and spectroscopic analysis (chaudhuri, 2015; gupta et al., 2013; table 1 and figure 2 ). it was already discussed that most of the active constituents isolated so far from p. corylifolia are part of the seed. sen and colleague extracted seed oil from p. corylifolia and found that the oil is unsaponifiable with boiling points 180-190°c. during the experiment, another compound was also identified as methyl glucoside with melting points of 105-127°c (chopra & chopra, 1933) . chopra and chaterjee, in 1927 , identified essential oil, a fixed oil and resin of dark brown color with some traces of alkaloids in p. corylifolia (chopra et al., 2013) . dymock studied the sugar contents, extractive matter, albumin concentraton, and ash value, and also found some traces of manganese in seeds of p. corylifolia. in continuation of research for compounds from the seeds of p. corylifolia, three more important components were fractionated and identified as bakuchiol a monoterpene phenol and the two novels dimeric monoterpenoids, bisbakuchiols a and b (panda, 1999) . a very important pharmacological compound known as bakuchiol has also been biosynthesized in 1983, and it was concluded that it is a derivative of phenylpropane pathway (banerji & chintalwar, 1983) . similarly, bisbakuchiols a and b structures were evaluated, and it was noted that dimeric monoterpenoid skeleton contains two monoterpenes, which are connected through a dioxane bridge (wu et al., 2007) . the low polarity ether extract of p. corylifolia seeds was investigated, and it revealed the presence of various ketones and aldehydes containing compounds such as corylinal, c-formylated chalcone, and isoneobayachalcone. a new isoflavone compound known as psorlenal was also identified in the seeds (gupta et al., 1978) . the same compounds, psoralen and isopsoralen, were also isolated by applying other chromatographic techniques such as high-speed counter-current chromatography (liu, li, sun, & kong, 2004) . the seed's sample has been the main focus for the search of bioactive compounds, and such an isolation procedure involving spectroscopic methods and crystal x-ray diffraction resulted in isolation of five novel compounds. these compounds are named as psoracorylifols a-e and chalcone and bavachromanol (yin, fan, dong, & yue, 2006) . similarly, three new flavonoid compounds named as corylifols a, b, and c and bavachalcone were also fractionated from p. corylifolia seeds. another compound known as bakuchicin was also identified from the seeds (yin, fan, wang, dong, & yue, 2004) . the seeds are also reported to contain some other flavonoids such as bavachinin (bcn), bavachin, isobavachin, and isobavachalcone (ibc). the glycosides identified in seeds of p. corylifolia were psoralenoside and isopsoralenoside, which were of the benzofuran type. p. corylifolia also reported to contain some polar compounds, namely, neobavachalcone, 7-methyl bavachin, and bavachromene. these compounds were isolated and identified from the insoluble portion of the ethanolic extract. these compounds identified as (khatune et al., 2004) 2 aryl coumarin coumarin seeds anticancer (limper et al., 2013) 3 astragalin flavonoid seeds antioxidant 4 bakuchiol meroterpene seeds/fruit anti-acne antibacterial (katsura et al., 2001; newton et al., 2002) antifungal (newton et al., 2002) , (hosamani et al., 2012; lau et al., 2010; lau et al., 2014; prasad et al., 2004; savoia, 2012; srinivasan & sarada, 2012; yang et al., 2006) retinal regeneration (seo et al., 2013) anti-aging (seo et al., 2013) estrogen receptor agonist, postmenopausal symptoms (lim et al., 2011) anti-diabetic (behloul & wu, 2013) lymphangiogenesis inhibition (jeong et al., 2013) anticancer (chen et al., 2010; li et al., 2016) 5 bavachinin flavone seeds antibacterial (khatune et al., 2004) estrogen receptor agonist (lim et al., 2011) lymphangiogenesis inhibition (jeong et al., 2013) osteoporosis (liu et al., 2014) anti-alzheimer (chen et al., 2013) carboxylesterase inhibitors 6 bakuisoflavone flavone fruit antibacterial (siva et al., 2015) 7 bakuflavanone flavone fruit antibacterial (siva et al., 2015) 9 bavachin flavnonoid seeds/fruit osteoblast 10 bakuchicin coumarin seeds topoisomerase inhibitor (sun et al., 2003) 11 bavachalcone chalcone seeds anticancer (shan et al., 2014) cvs protective effect (dang et al., 2015) 12 bavachinone a flavonoid fruit antibacterial (won et al., 2015) 13 bavachinone b flavonoid fruit antibacterial (won et al., 2015) 14 bavacoumestan c flavonoid fruit antibacterial (won et al., 2015) 15 corylifolinin chalcone seeds antibacterial (khatune et al., 2004) carboxylesterase inhibitors (sun et al., 2016) 16 corylifols prenyl flavonoid seeds antibacterial (yin et al., 2004) 17 corylifol a flavonoid seeds/fruit carboxylesterase inhibitors 18 corylifol b flavonoid seeds carboxylesterase inhibitors 19 corylifol c flavonoid seeds protein kinase inhibition (limper et al., 2013) anticancer (limper et al., 2013) 20 corylifol d flavonoid seeds anticancer (stomach; yang et al., 1996; teschke et al., 2014) 21 corylifol e flavonoid seeds anticancer (stomach; yang et al., 1996; teschke et al., 2014) 22 coryfolin flavonoid whole plant antioxidant, anti-diabetic (behloul & wu, 2013) 23 corylin flavonoid whole plant osteoblast wang et al., 2001) anticancer (shan et al., 2014) carboxylesterase inhibitors (sun et al., 2016) 24 coryaurone a flavonoid fruit antibacterial (won et al., 2015) 25 dadzin isoflavnoid fruit antioxidant (shinde et al., 2010) 26 dadzein isoflavnoid fruit antioxidant (shinde et al., 2010) antidiabetic (behloul & wu, 2013) topoisomerase inhibitor (sun et al., 2003) 27 dihydroxy coumestan essential oil component seeds insecticidal, genotoxic (khatune et al., 2002; dua et al., 2013b) 28 genistein isoflavone fruit ani-diabetic, anti-obesity (behloul & wu, 2013) , antioxidant (shinde et al., 2010) 29 hydroxy bukuchiol meroterpene seeds lymphangiogenisis inhibition (jeong et al., 2013) 30 hydroxypsoralenol a flavonoid fruit antibacterial (won et al., 2015) 31 hydroxypsoralenol b flavonoid fruit antibacterial (won et al., 2015) 32 types of esters were also studied in p. corylifolia, and psoralester and psorachromene were identified as two new metabolites. the lymphangiogenesis inhibition (jeong et al., 2013) anti-alzheimer (chen et al., 2013) carboxylesterase inhibitors 33 isobavachin flavonoid seed/fruit osteoblast (li et al., 2014) 34 isopsoralen furanocoumarin whole plant antiprotozoal 35 neobavaisoflavone seeds antibacterial (khatune et al., 2004) 36 psoralen furanocoumarin whole plant/root leucoderma, psoriasis anticancer (hao et al., 2014) , antioxidant , anti-alzheimer (somani et al., 2015) , collagengenesis 37 psoralidin coumarin whole plant/seed estrogen receptor modulator (liu et al., 2014; lim et al., 2011) antioxidant (wang, yin, zhang, peng, & kang, 2013b) , antibacterial (khatune et al., 2004) anti-diabetic (behloul & wu, 2013) , antiprotozoal anticancer (hao et al., 2014; limper et al., 2013; yang et al., 1996) , anti-depressent (farahani et al., 2015) 38 psoracorylifol d flavonoid seed lymphangiogenesis inhibition (jeong et al., 2013) psoracoumestan coumestans seeds essential oil anti-cancer (limper et al., 2013) 39 xanthoangelol chalcone seeds anticancer (limper et al., 2013) figure 2 structures of important compounds isolated from psoralea corylifolia psoralester is a 10-membered lactone compound and the latter is an isomer of already known compound bayachromene (tewari & bhakuni, 2010) . khatune and colleague isolated new coumestan derivative 6-(-3-methylbut-2-enyl)-6 n-7-dihydroxycoumestan while working on the chloroform soluble portion of the seeds of p. corylifolia (khatune et al., 2002) . coumestans-3, 5′-dihydroxy-6′, 6′-dimethyldihydropyrano (2′, 3′: 8,9) coumestan, 3-hydroxy-5′-(1hydroxy-1-methylethyl) 4′, 5′-dihydrofuro (2′, 3′: 8,9) coumestan, and sophoracoumestan a have been fractionated from the seeds of p. corylifolia. lin and kuo isolated two new benzofuran derivatives; corylifonol and isocorylifonol along with astragalin, p-hydroxy benzoic acid from p. corylifolia seed extract (lin & kuo, 1992) . in 1989, zaka and colleague investigated the lipid and fatty acid composition of p. corylifolia. the lipids recognized were triacylglycerols, diacylglycerols, free fatty acids, monoacylglycerols, hydrocarbons, wax esters, and polar lipids. the purified crude lipids of p. corylifolia seeds were analyzed by thin layer and gas chromatographic techniques. among the components, the major polar lipid was c18: the sticky and oily pericarp constitute the fruit of p. corylifolia, and chemical investigation revealed some similar compounds as already isolated from seeds. a new isoflavone, corylinin, (7,4′-dihydroxy-3′in another experiment, hplc protocol was applied to identify some isoflavonoids such as daidzein, genistein, and biochanin a (sehrawat, sangwan, & yadav, 2014) . further work on the fruit extracted with hexane showed the presence of a phenolic monoterpene known as bakuchiol (cui, taniguchi, kuroda, & hatano, 2015) . dried p. corylifolia fruit powder extracted with methanol and analyzed on hplc reverse column showed the presence of isoflavonoids known as genistein, daidzein, and biochanin a (hsu, wu, chen, yang, & wang, 2001) . raun and colleagues have isolated seven compounds from the fruit of p. corylifolia and established the structure of compounds after the spectroscopic analysis. the components identified were corylinin (new compound), psoralen, neobavaisoflavone, sophoracoumestan a, uracil, and daidzin (ruan et al., 2007) . in 2015, two more new flavonoids, bakuisoflavone, and bakuflavanone were isolated from the fruit of p. corlylifolia with antimicrobial activities (lee, kim, baik, ryu, & lee, 2015) . in one study, six new flavonoid compounds and a meroterpenoid were isolated and identified by spectroscopic methods from p. corylifolia fruits and displayed medium activity against staph. mutans (won et al., 2015) . the dried fruits of the plant was investigated, and n-hexane the roots of p. corylifolia has been investigated for bioactive compounds. it was found that furanocoumarins psoralen and isopsoralen isolated from a petroleum ether extract were responsible for the anti-feedant activity against instar spodoptera litura larvae (sah et al., 2006 ; figure 2 ). the above discussions about phytochemical investigation of different parts of p. corylifolia clearly indicates that this plant is a very useful source of variety of bioactive constituents including flavonoids, terpenes, glycosides, alkaloids, coumarins, and others. p. corylifolia is a widely used herb and have many diverse ethnopharmacological and medicinal applications. the numerous chemical and pharmacological research that have been carried out have resulted in the isolation of the diverse bioactive compounds that are summarized below. p. corylifolia proved a promising agent in anti-acne formulations due to the presence of phenolic compounds bakuchiol. it proved to be safe and non-irritant and can be used for longer periods of the day because it showed no irritation and is non-sensitized . one of the bioactive isolated compound "soralen" found to have the ability to stimulate the development of melanin, and therefore it is employed for leucoderma treatment (kim, shim, ahn, & jung, 2013) . the plant is also used against the skin disease known as psoriasis (chen, yang, & zhang, 2013) . in one experiment, seeds of p. corylifolia was extracted with hexane and oil in water, cream was prepared with stearic acid as a base. in the next step, an open clinical trial was conducted on 30 patients suffering from eczema for a period of 30 days. after 2 weeks of cream application, symptoms score reduced. final day observation revealed that the symptom scores for eczema reduced from 6.367 ± 1.098 to 0.333 ± 0.279 for length of the lesion, from 1.333 ± 0.994 to 0.165 ± 0.087 for exudation rate, and from 2.567 ± 0.504 to 0.165 ± 0.132 for the rate of itching. this formulation was compared with the placebo preparation, in which the formulated cream contained all the ingredients except for the hexane extract of p. corylifolia. this study concluded that this plant could be effectively used for the treatment of eczema (gidwani et al., 2010) . p. corylifolia has been tested for antibacterial activity. wang et al. nine compounds exhibited significant antibacterial activity against sa and s. epidermidis (yin et al., 2004 that showed 11.5 mm zone of inhibition (acharya et al., 2015) . p. corylifolia in the form of the mouth rinsing solution has prevented the growth of s. mutans bacteria in a very low concentration. along with mouth rinsing ability, the ethanol extract of p. corylifolia was also reported to inhibit the human gingival fibroblast. in the same series of experiments, it was concluded that the extract is safe to use and has no toxic effect in normal doses . in 2015, two more new flavonoids, bakuisoflavone, and bakuflavanone were isolated from the fruit of p. corylifolia and were tested against the strains of mrsa (mrsa481 and mrsa 584) and it was found that two compounds showed some good antibacterial effects with mic values of (>32 (>9.4) and >32 (>9.4)) and (>32 (>9.5) and >32 (>9.5 μg/ml)), respectively . tissue culture studies showed that jasmonic acid treated plants of p. corylifolia enhanced psoralen content in leaves and roots of p. corylifolia. the methanol extract of root sample displayed effective antimicrobial activity against tested bacterial and fungal pathogens in range 25, 50, and 75 μg/ml. most effective activity 28 mm zone of inhibition was observed against p. aerugenosa at 50 μg/ml (siva et al., 2015) . p. corylifolia was also tested against the microbes sa, pseudomonas aeruginosa, and candida albicans, which are known to contaminate the cosmetics products. in the 96-well microplate assay, it was found to have antimicrobial activity . the a phenolic compound bakuchiol extracted from p. corylifolia (seeds) exhibited antifungal activity against many strains of pathogenic fungi, (hosamani, lakshman, & sandeepkumar, 2012; lau et al., 2010; lau et al., 2014; newton et al., 2002; prasad, anandi, balasubramanian, & pugalendi, 2004; savoia, 2012; srinivasan & sarada, 2012; yang et al., 2006) . in another study, activity was found against other fungi such as alternari brassicae, aspergillus niger, fusarium oxysporum, and rhizoctonia cerealis, in which mycelial growth was inhibited (satish, raghavendra, & raveesha, 2009; vonshak et al., 2003; yang et al., 2006) . in one study, p. corylifolia significantly reduced the incidents of the anti-worm property of the seeds of p. corylifolia is clinically proven on roundworms and flatworms (gidwani et al., 2010) . the seeds and the leaves of p. corylifolia was extracted with water and alcohol, and were tested on the spontaneous movements of setaria cervi whole worm and again on the isolated nerve muscle preparations. the survival of the microfilariae was tested in vitro. the dose required to inhibit the movements of whole worm and nerve muscle preparations for alcohol extracts of leaves were 160, 30, and for seeds were 50, 20 μg/ml (maurya, singh, & seth, 2014; mendam, kavitha, & naik, 2015; qamaruddin, parveen, khan, & singhal, 2002) . volatile oil extracted from the seeds of p. corylifolia displayed strong toxicity against both larvae and adult of the southern house mosquito, the bioactivity guided isolation from chloroform extract of seeds leads to the identification of pure compound, 6-(-3-methylbut-2enyl)-6 n-7-dihydroxycoumestan and found active against larvae and adult cx. quinquefasciatus. the same compound has also showed activity against the red flour beetle, tribolium casteneum hebrst. in this detailed study, both the male and female adults were exposed to the compound for 24-72 hr period and a range of ld 50 were calculated for dose starting from 400 to 1600 ppm and doses were used in five replications. it was concluded that the isolated coumestan compound can be a useful botanical insecticide (dua, kumar, pandey, & kumar, 2013a; khatune et al., 2002) . an external protozoan parasite ichthyophthirius multifiliis (also called "ich") has been reported to infest freshwater fish species. the p. corylifolia extracted with methanol showed excellent activity against i. multifiliis theronts in concentration of 1.25 mg/l or more when was exposed for a period of 4 hr. the p. corylifolia extract at 5.00 mg/l concentration has caused 100% mortality of protomonts and 88.9% of encysted tomonts. it was found that longer period (24 hr) and higher concentration (5.00 mg/l) caused the significant reduction of the survival rate and reproduction of tomont of i. multifiliis, which were exited from the fish after in-bath handling in situ (ling et al., 2013) . p. corylifolia has been found to be an alternative to malachite green to control i. multifiliis, an external protozoan parasite. the screening showed that p. corylifolia extract have the maximum activity against i. multifiliis theronts. when the experiments were conducted in vivo, at 1.25 mg/l or more concentrations of methanol extract of p. corylifolia, it caused 00% mortality of theronts during the 4 hr of exposure (ling et al., 2013) . this study showed the damaging effect of p. corylifolia against i. multifiliis trophont in situ. the same study leads to the evaluation of the activity of antiprotozoal compounds extracted form p. corylifolia against i. multifiliis. the two bioactivity guided isolated compounds, isopsoralen and psoralidin, were assessed. in comparison, study of psoralidin and isopsoralen, the inhibitory activity of psoralidin, was found more efficient. further, when experimented in vivo, the compound psoralidin at 2.5 mg/l, efficiently reduced the theronts numbers in infected fish over an exposure period of 5 hr. this study showed that psoralidin could be a very good candidate for the formulation as a commercial drug against i. multifiliis . the compounds from p. corylifolia were found to have a protective effect when tested against retinal damage caused by oxidative stress. to evaluate the effects of bakuchiol on the cell viability of dif(park et al., 2005) . a recent and very useful research on the same subject of hepatotoxicity shows that the isolated compound bakuchiol when administered at a dose of 52.5 and 262.5 mg/kg for 6 weeks in rats, many abnormalities were observed in the bakuchiol-treated groups including suppression of weight gain and food intake, change of some parameters in serum biochemistry, and increased weight of liver. the mrna expression of cyp7a1, hmg-coa reductase, pparα, and srebp-2 decreased in bakuchiol-treated group, the expression of bsep increased in bakuchiol-treated low dosage, and the expression of bsep decreased in bakuchiol-treated high dosage. it was concluded that bakuchiol could induce cholestatic hepatotoxicity, suggesting potential hepatotoxicity. the mechanism may be related to effects on liver lipid metabolism (li et al., 2017) . in one study, six compounds isolated from p. corylifolia were studied for their binding affinities for estrogen receptors, erα and erβ, using the yeast transactivation test. among the compounds, bakuchiol was in another study, psoralidin activity as er (α and β) agonist was evaluated in endometrial and human breast cell lines. psoralidin at 10 μm was able to get the highest reporter gene expression conforming to that of e2-treated cells and such an activation of the ere-reporter gene by psoralidin was totally stopped by the treatment of a pure er antagonist, indicating that the biological actions of psoralidin are intermediated by er. psoralidin was also able to stimulate the endogenous estrogen-responsive gene (ps2) in mcf-7, the human breast cancer cells. it was observed that activation of the classical er-signaling pathway by psoralidin is mediated via induction of er conformation by psoralidin and direct binding of the psoralidin-er complex of the eres present in the promoter region of estrogenresponsive genes. lastly, molecular docking of psoralidin to the ligand-binding pocket of the erα exposed that psoralidin is able to mimic the binding interactions of e 2 , and therefore, in the cellular environment it could act as an er agonist (liu et al., 2014) . psoralen isolated from p. corylifolia fruits were investigated as an inhibitor of ache enzyme in an attempt to explore its potential for the management of alzheimer's disease. the concentration of psoralen used was 25-400 μg/ml. it inhibited the ache in a dose-dependent way in animal models. adult male wistar rats, weighing 180-250 g, were used in the study. while a molecular docking study was also carried out, which showed that psoralen binds well within the binding site of the enzyme showing interactions such as π-π stacking and hydrogen bonding (somani et al., 2015) . although the activity measured in this study was moderate when compared with the standard compound used, despite that, the compound could serve as lead for synthetic analog preparation to improve the inhibitory activity. p. corylifoia also found to possess antidepressant activity. marzieh sarbandi farahani and colleague mentioned the mechanism of action of the plants with antidepressant action and the chemical components isolated from them. they mentioned that psoralidin isolated from seeds of p. corylifolia modify the hypothalamic-pituitary-adrenal axis (farahani, bahramsoltani, farzaei, abdollahi, & rahimi, 2015) . a similar study was conducted on psoralidin by yi and colleague on the icr strain of male mice. the dose was administered orally in forced swimming test and they observed the increased levels of 5hydroxytryptamine and 5-hydroxyindoleacetic acid in the brain and an altered dopamine level. the mechanism for antidepressant activity was proposed to be through involvement of monoamine neurotransmitter and the hypothalamic pituitary adrenal axis systems (yi et al., 2008) . some previous studies are also available, for example, one study was conducted on mice models and it was concluded that furocoumarins were actually responsible for antidepressant activity. in this study, the well-established antidepressants were used as standards for comparison with the seed extract of p. corylifolia. the dose range used was 7.5 to 100 mg/kg in comparison with amitriptyline (10 and 20 mg/kg) and fluoxetine (13 mg/kg). this study was well designed and results indicate the potential of the seed extract as competitive antidepressant when compared to the conventional therapeutic agents (chopra et al., 2013) . p. corylifolia also has a wide range of antioxidant activity. different compounds isolated from p. corylifolia were tested for their antioxiin the understanding of the reputation of this plant species in medicines now, attention has been given to produce callus culture. in one study, relationship between isoflavone and antioxidant activity of p. corylifolia cultures were experimented, and it was found that root-derived callus cultures produced more daidzein, whereas leafdervied callus produced more genistein, and this enhanced production was related to enhanced antioxidant activities (shinde, malpathak, & fulzele, 2010) . one of the isolated compound, psoralen showed the promising antioxidant activity (ic 50 value = 1.10 ± 0.60 μg/ml) against the superoxide anion production by human neutrophils in response to formyl-lmethionyl-l-leucyl-l-phenylalanine/cytochalasin b (fmlp/cb; chen et al., 2011). p. corylifolia extract possesses anti-diabetic activity by its action as the protective effects on pancreatic β cells (behloul & wu, 2013) . a more detailed biochemical study was conducted on the aqueous extract of seed of p. corylifolia that caused a significant recovery in the activities of hexokinase, glucose-6-phosphatase, and glucose-6-phosphate dehydrogenase and antioxidant enzymes such as peroxidase, catalase, and superoxide dismutase, along with the lipid peroxidation level in liver tissue and serum transaminase, and corrected the fasting blood glucose level in streptozotocin-induced diabetic rats at a dose of 20 mg/0.5 ml water/100gm body weight (ghosh, bera, chatterjee, ali, & debasis, 2009 ). p. corylifolia has been the part of many ayurvedic formulation that are used for the treatment of various central nervous system conditions such as for neurotropic activity and as central nervous system protective agent (goel & ojha, 2015) . based on such report, a study was conin another study, it was revealed that ibc, a flavonoid from p. corylifolia, has the ability to ameliorate the neuronal injury in brain diseases related to inflammation, and this was accomplished through inhibition of lipopolysaccharide induced intercellular adhesion molecule-1 expression and leukocyte adhesion to brain endothelial cell by blocking toll-like receptor 4 signaling . various studies on animals showed that genistein has the ability to decrease body weight by decreasing food intake. it also reduced the fat pad weight and enhanced the apoptosis of adipose tissues. for example, one such study was conducted on ovariectomised mice. this well-known trihydroxyflavone, genistein, has also been isolated from p. corylifolia, exhibited a potential anti-obesity and obesity related low grade inflammation activities through multiple mechanisms and cell signaling pathways. p. corylifolia extract possesses anti-obesity and antdiabetic activity by its action on adipocyte life cycle, obesity-related low-grade inflammation, and oxidative stress (behloul & wu, 2013) . the well-known chinese herb p. corylifolia l. (scurfpea fruit) has been employed for the treatment of bone fractures and also for joint diseases for thousands of years. p. corylifolia also improved the pathological bone condition, hyperosteoidosis, by increasing the serum inorganic phosphate level at a dose of 30 mg/kg. it was observed previously that the extract markedly decreased osteoid volume and there has been improvement in bone calcification (miura, nishida, & linuma, 1996) . the flavonoids of corylin and bavachin has the osteoblastic proliferation stimulating activity in umr106 cell line cultured in vitro (cho et al., 2001; wang, li, & jiang, 2001) . p. corylifolia extract when administered orally to ovx rats, it was noted that there was a decrease in urinary calcium excretion and serum osteocalcin at a dose of 25-50 mg/kg body weight. the experiments in this study showed that the extract also increased the bone mineral density and bone formation at 50 mg/kg bw (tsai et al., 2007) . such experiment, which were extended to 3 months, it can be concluded that p. corylifolia extract can be used at postmenopause state to prevent the osteoporosis. a further in depth study is still needed to make a therapeutic candidate. this study leads to the isolation of the compound responsible for the mentioned activity by weng and colleagues. the isolated compounds from p. corylifolia, known as bakuchiol and bavachin, showed to prevent the estrogen deficiency by inducing the upregulation in primary human osteoblast differentiation (weng et al., 2015) . an advanced herbal formula containing psoraleae fructus has previously showed promising bone protecting effect when tested in rats, and later on showed excellent results in women with osteoporosis. this herbal formula could efficiently have promoted the osteogenesis and suppress the adipogenesis in mesenchymal stem cells (siu et al., 2013 ). an hplc analysis was carried out to study the important constituents in scurfpea fruit. in the method employed, the compounds were identified by comparing their retention indexes with standard substances and it resulted in the identification of 11 compounds. the biological methods such as mtt and alp were employed to study the osteoblasts proliferation and differentiation activity. bavachin and isobavachin showed significant cell proliferation by stimulation, the compound bakuchiol displayed higher effect to boost osteoblasts differentiation. from the results, it was hypothesized that prenyl group as a side chain might be responsible for the activity mentioned, because this structural component was found as a common entity among the compounds tested for activity (li et al., 2014) . previous surveys revealed bakuchiol and bavachin, the two important components of p. corylifolia l., showed osteoblastic property. both the compounds displayed the prevention of bone loss caused by deficiency of estrogen in overiectomized animal models such as rats. one of in vitro study proposed that bavachin and bakuchiol caused the induction of primary human osteoblast differentiation by up regulating the wnt signaling pathway. this research proposes that boneprotective role makes these two compounds a promising and safe estrogen supplement for the estrogen replacement therapy (weng et al., 2015) . in one investigation, the compound psoralen with different concentrations (1, 10, and 100 μm) was tested on chondrocytes isolated from rats at 3-and 9-day intervals. it was found that at the low dose concentration psoralen was safe toward chondrocytes; however, at higher dose suppression of chondrocytes, proliferation was observed. the compound psoralen also increased the synthesis of type ii collagen at 100 μm, by 0.48-fold on day 3 and 0.56-fold on day 9. this was a detailed study, including mts assay, alcian blue colorimetry, western blotting, and qrt-pcr. the compound psoralen was also tested for cytotoxicity and exhibited low cytotoxicity toward chondrocytes at a dose range of 1-10 μm. a much higher dose of 100 μm suppression of chondrocytes proliferation was observed. the compound psoralen caused the inhibition of the type i collagen in gene expression and also in protein synthesis. it was concluded that psoralen could be an important biological compound for triggering the cartilaginous cellular functions of chondrocytes . the (jeong et al., 2013) . this was the first kind of study on p. corylifolia. the plant p. corylifolia extract neutralized the coagulation of caused by naja naja karachiensis snakebite when compared with the antidote used as a standard. the snake venom was experimented on human plasma (citrated) to evaluate its effect on activated partial thromboplastin time (aptt), prothrombin time (pt), and thrombin time (tt). snake venom (200 μg/ml) was found to delay pt (13 ± 0.57 to 23 ± 0.57 sec), aptt (35 ± 1.52 to 48 ± 2.0 sec), and tt (13 ± 0.57 to 33 ± 0.57 sec). pt and tt were prolonged, and it suggested the occurrence of thrombin-like or plasminogen activating enzymes (asad et al., 2013; asad et al., 2014) . a further in depth study of this activity is still underway in the author's laboratory. the extract of seeds of p. corylifolia have been reported to have stimulant activity against natural killer cells when tested in mice. this study report that the extract also modulates the antibody dependent cellular toxicity. during tumor development, the seed extract also inhibited the antibody complement mediated cytotoxicity. the study was conducted on balb/c male mice. the dose of 100 mg/kg was administered intraperitoneally. blood collected from punctured heart and serum was separated to study the antibody complement-mediated cytotoxicity. the natural killer cells were removed from spleen, and antibodydependent cellular cytotoxicity was assessed (latha, evans, panikkar, & jayavardhanan, 2000) . the isolated compounds from p. corylifolia including arylcoumarin and psoracoumestan showed strong anticancer potential by strongly hepatocarcinoma cells, it showed its inhibitory activity by inducing the mechanism of apoptosis (guo, liu, ye, & han, 2011; jiang & xiong, 2014; khan, iqbal, ahmed, & jamil, 2015; mohammadparast, rustaiee, rasouli, zardari, & agrawal, 2014; nehybova, smarda, & benes, 2014; rajan, tripathi, variyar, & pandey, 2014; tang et al., 2011; wong & rabie, 2011; yang et al., 2012) . similarly, two more compounds from the same specie identified as ibc and bcn attenuate aβ42-induced cell toxicity. the investigation was carried out on yeast two-hybrid system (chen et al., 2013) . during this study, eight compounds were tested, and among them ibc (3 μm) and bcn (30 μm) were proved to be active at non-toxic concentrations. the results were confirmed by a standard tht fluorescence method. the compound ibc also exhibited strong inhibitory effect on tht fluorescence, and bcn showed more efficient activity, which was comparable with the reference. moreover, the determination of ic 50 of ibc and bcn in the tht assay were about 25 and 45 μm, respectively. psoralidin in another study, caused the generation of reactive oxygen and it also thought to cause the inhibition of a549 cell proliferation. the method adopted was mtt assay. this study was designed to study the relationship of time and concentration used. the ic 50 values obtained after 24-, 48-and 72-hr treatment were 19.2, 15.4, and 11.8 μm, respectively. (hao, zhang, zhao, & chen, 2014) . another evidence of anticancer activity came from the compound bakuchiol that suppressed the testosterone induced cell proliferation and gene expression in lncap cells. this study was designed to explore the bakuchiol action in the androgen-dependent pca cell line (lncap). mtt assay and real-time pcr method were employed. the ic 50 of bakuchiol to androgen receptor was 8.87 × 10 4 , which was similar to the standard flutamide (10.00 × 10 4 ; miao et al., 2013) . these experiments showed that bakuchiol is a useful agent for drug development for androgen dependent pca. the same compound, bakuchiol, has also showed a strong anticancer action against human lung adenocarcinoma cell line a549 and showed much better results than its analogue resveratrol. ic 50 of bakuchiol at 72 hr was 9.58 ± 1.12 μmol/l, much lower than that of resveratrol (33.02 ± 2.35 μmol/l). bakuchiol triggered the process of apoptosis to a higher level, compared with resveratrol. it was also noted that oxygen species related apoptosis also contribute the cytotoxic properties of bakuchiol, and therefore, it also supports the use of bakuchiol against non-small-cell lung cancer. (chen et al., 2010) . bakuchiol also showed very selective clearance activity in hepatic stellate cells by a mechanism involving apoptosis. this study showed that p. corylifolia also has anticancer activity in liver cancer (chen et al., 2010; yang, paik, cho, cho, & kim, 2008) . in search of cytotoxic compounds from p. corylifolia, two isoflavnoids were isolated, named as corylifols d and e from the ethyl acetate extract. similarly, psoralidin was also found active against stomach carcinoma cell lines (teschke, wolff, frenzel, & schulze, 2014; yang et al., 1996) . bakuchiol is also an active ingredient of the dried ripe fruit of p. corylifolia that and corylin from p. corylifolia has been investigated against udp-glucuronosyltransferases and showed strong inhibition (shan et al., 2014) . it also inhibits lipopolysaccharide-induced endothelial-mesenchymal transition via down regulation of the nf-κb-snail signaling pathway (jung et al., 2015) . in one investigation against dna polymerase enzyme, p. corylifolia extract showed strong inhibitory activity. importance of isolated components from p. corylifolia is obvious from the fact that compounds such as psoralen is being produced from callus derived from various plant portions. in one experiment, it was found that in the in vitro conditions, cinnamic acid proved a very strong precursor of psoralen pathway that induced a maximum amount of psoralen (mohammadparast, rustaiee, rasouli, zardari, & agrawal, 2014 succeeding experiments with many kinase inhibitors propose that pf-mediated degradation of cyclin d1 and cdk4 is dependent on erk1/2 and/or gsk3β (park, sung, song, & jeong, 2016) . the crude extract of p. corylifolia with solvent ethanol was found a strong dna polymerase inhibitor of dna replication enzyme in an activity directed isolation assay that resulted in the purification of novel compound corylifolin, bakuchiol, neobavaisoflavone, and resveratrol. in a similar enzyme assay, some topoisomerase ii inhibitors were also isolated, namely, daidzein and bakuchicin (sun, woo, cassady, & snapka, 2003) . many studies have been designed so far to study the behavior of p. corylifolia extract and isolated compounds on important enzyme. additionally, the inhibition kinetics were calculated by dixon and lineweaver-burk plots for the inhibitory activities toward ces1. the inhibition kinetic parameters (k i ) were calculated to be 5.3, 9.4, 1.9, 0.7, and 0.5 μm for neobavaisoflavone, corylifolinin, coryfolin, corylin and bcn, respectively. it was concluded that this inhibition by the constituents might be responsible for the possible adverse effects of fp through the disrupting ces1-catalyzed metabolism of endogenous substances and xenobiotics (sun et al., 2016) . cytochrome p450 (cyp) is an assembly of heme-containing enzymes fixed essentially in the lipid bilayer of the endoplasmic reticulum, and helps metabolize several drugs and carcinogens. the plant p. corylifolia has been reported to be used in cardiac problems in traditional medicine (kr, 1975) . bavachalcone, a compound from p. corylifolia, has reported to have increased the luciferase activity of the manganese superoxide dismutase (mnsod) promoter and enhanced mnsod mrna and protein expressions. further, it was found that bavachalcone suppressed the mitochondrial superoxide production in endothelial cells. on the other hand, bavachalcone (in concentration range of 1, 2.5, and 5 μmol/l for 24 hr) stimulated liver kinase b1 and ampkα phosphorylation in a converse manner. mrna interfering by using short hairpin rna (shrna) of amp-activated protein kinase (ampk) inhibited bavachalcone-induced mnsod expression. moreover, ampk reduced by shrna-ampk reversed the inhibition of bavachalcone on mitochondrial superoxide production in endothelial cells. these results showed that bavachalcone could shield the endothelial function by enhancing the ampk activity and mnsod expression and lowering the mitochondrial oxidative stress, which is considered to be the key etiological reason in cardiovascular diseases (dang et al., 2015) . many herbal remedies, including essential oils, contain psoralen, and methoxy psoralen are reported to cause photosensitivity in the form of erythema and blisters (koh & ong, 1999) . p. corylifolia has been the part of many herbal remedy used to treat vitiligo and patients using such treatments in the form of creams are also reported to experienced erythema when exposed to sunlight (maurice & cream, 1989) . p. corylifolia is frequently indicated for vitiligo in india, and it contains psoralen, isopsoralen, and psoralidin. its extracts when tested on guinea pig skin have been stated to possess potent sensitizing action (pathak, daniels, & fitzpatrick, 1963) . another study reported the gonadal toxicity. although the ethanol extract of p. corylifolia seeds are proposed to use in processed food preservation, but it showed toxicity when tested on male and female rats. the period of the experiment was 90 days, and various concentrations were administered were 0%, 0.375%, 0.75%, 1.5% or 3.0%. histopathological investigation showed atrophy of seminiferous tubules, leydig cells, seminal vesicles, and prostate cells were observed in male rats administered with the 1.5% and 3.0%. with the same concentrations, the female rats showed a reduced number of corpora lutea in the ovaries and less frequent endometrial glands in the uterus. it was suggested that the extract caused the hypothalamus-pituitary-gonadal axis (takizawa et al., 2002) . in one investigation, p. corylifolia and its natural compounds (bavachin, corylifol a, neobavaisoflavone, ibc, and bcn) were evaluated for its potential toxicity, and results showed it had a potent inhibitory effect against human udp-glucuronosyltransferase 1a1 (ugt1a1), and it is considered as main stimulant for p. corylifolia related toxicity, including hepatic injury and raised bilirubin levels . in another study p. corylifolia extract and fractionated compounds such as psoralen and isopsoralen were incubated with the recombinant cyp3a4 enzyme or differentiated huh-7 and heparg cells. p. corylifolia extract, psoralen, and isopsoralen caused the inhibition of concentration cyp3a4 activity in a dose dependent manner with different potency in vitro. it was also noted that none of the sample tested showed any toxicity (liu & flynn, 2015) . several compounds isolated from p. corylifolia have many industrial applications and are available commercially. some of the examples are the following: the compound angelicin (cas 523-50-2) is available as antifungal compound (sardari, amin, sekhon, micetich, & daneshtalab, 2000) . the compound psoralen has been used as photochemical probe in studies of dna mutation and repair mechanisms (zarmouh, eyunni, & soliman, 2017) . methoxsalen (8-methoxypsoralen; cas 298-81-7) has been known as a potent suicide inhibitor of cytochrome p-450 (toyooka & ibuki, 2009 ). bakuchiol (cas 10309-37-2) is available as a ptp1b and dna polymerase inhibitor (choi et al., 2015) . bavachin (cas 19879-32-4 ) is available as a weak antioxidant that stimulates bone formation (jing et al., 2017) . corylifol c and, to a lesser extent, xanthoangelol are potent protein kinase inhibitors (inhibitory concentration 50% values for epidermal growth factor receptor; limper et al., 2013) . a well-known compound daidzin (cas 552-66-9) isolated from many plant species is a potent inhibitor of human mitochondrial aldehye dehydrogenase that demonstrates chemopreventitive activities (keung & vallee, 1993) . the compound genistein (cas 446-72-0) is available as a highly specific inhibitor of protein tyrosine kinase (zarmouh, messeha, elshami, & soliman, 2016) . these examples clearly show the importance of bioactive compounds isolated from p. corylifolia, an important traditional medicinal plant. the above-mentioned summary about p. corylifolia clearly showed that it is a very important plant from ethnobotanical, pharmacological, and chemical point of view. to date, more or less hundreds compounds have been separated from p. corylifolia, which was also mentioned by zhang et al., (2016) . we here presented the latest version of scientific literature on p. corylifolia, including the research work carried out in the recent years. p. corylifolia contains a wide variety of chemical constituents belonging to various groups, including flavonoids, coumarins, and meroterpenes, which are more dominant. p. corylifolia l. (fabacese) is a fortified source of biological active compounds, which gives the plant with great value for its use in pharmaceuticals, health, and body-care products. sehrawat and colleagues reported that p. corylifolia is a rare and endangered herbaceous medicinal plant. because most of the plant materials are collected from naturally occurring stands and are therefore being depleted rapidly and there is a possibility of extinction. therefore, it is suggested that there is a need for in vitro propagation of this species (sehrawat et al., 2014) . authors declare that there is no conflict of interest. anti microbial activity of different dosage forms of bakuchi (psoralea corylifolia linn.) taila, an ayurvedic formulation medicinal plants of bombay presidency efficacy of seed hydropriming with phytoextracts on plant growth promotion and antifungal activity in maize antipsoriatic microemulsion gel formulations for topical drug delivery of babchi oil (psoralea corylifolia) phytochemical studies and antimicrobial screening of non/less-polar fraction of psoralea corylifolia by using gc-ms psoralen photochemotherapy of cutaneous disorders compensatory effects of medicinal plants of pakistan upon prolongation of coagulation assays induced by naja naja karachiensis bite anti-venom potential of pakistani medicinal plants: inhibition of anticoagulation activity of naja naja karachiensis toxin biosynthesis of bakuchiol, a meroterpene from psoralea corylifolia medicinal and poisonous plants of pakistan genistein: a promising therapeutic agent for obesity and diabetes treatment systema naturae antibacterial activity of psoralea corylifolia l. seed and aerial parts with various extraction methods bakuchiol: a retinol-like functional compound, modulating multiple retinol and non-retinol targets new isoflavones and bioactive constituents from the fruits of psoralea corylifolia isobavachalcone and bavachinin from psoraleae fructus modulate aβ42 aggregation process through different mechanisms in vitro anti-tumor effects of bakuchiol, an analogue of resveratrol, on human lung adenocarcinoma a549 cell line bakuchiol: a hepatoprotective compound of psoralea corylifolia on tacrine-induced cytotoxicity in hep g2 cells prediction of drug-induced liver injury in hepg2 cells cultured with human liver microsomes psoralea corylifolia l. 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of psoralea corylifolia (somraji) and seed of trigonella foenum-graecum l.,(methi) in separate and composite manner in streptozotocin-induced diabetic male albino rat evaluation of a novel herbal formulation in the treatment of eczema with psoralea corylifolia ashtang ghrita: a noble ayurveda drug for central nervous system inhibitory effects of osthole, psoralen and aconitine on invasive activities of breast cancer mda-mb-231bo cell line and the mechanisms. zhong xi yi jie he xue bao= quality standards of indian medicinal plants corylinal: a new isoflavone from seeds of psoralea corylifolia phytochemical analysis of methanol extracts of psoralea corylifolia psoralidin induces autophagy through ros generation which inhibits the proliferation of human lung cancer a549 cells antimicrobial activity of leaf extract of psoralea corylifolia l the presence of three isoflavonoid compounds in psoralea corylifolia the pharmacology of chinese herbs neuroprotective effects of psoralea corylifolia linn seed extracts on mitochondrial dysfunction induced by 3-nitropropionic acid cytotoxicity of corylifoliae fructus. ii. cytotoxicity of bakuchiol and the analogues studies on lymphangiogenesis inhibitors from korean and japanese crude drugs induction of apoptosis in human hepatocarcinoma smmc-7721 cells in vitro by psoralen from psoralea corylifolia isobavachalcone attenuates mptp-induced parkinson's disease in mice by inhibition of microglial activation through nf-κb pathway medicinal plants. oxford and ibh publishing medicinal plants. tropical horticulture plant systematics: a phylogenetic approach aqueous extract of psoralea corylifolia l. inhibits lipopolysaccharideinduced endothelial-mesenchymal transition via downregulation of the nf-κb-snail signaling pathway handbook of ayurvedic medicinal plants: herbal reference library in vitro antimicrobial activities of bakuchiol against oral microorganisms. antimicrobial agents and chemotherapy daidzin: a potent, selective inhibitor of 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application of citrus hystrix as a folk remedy indian medicinal plants the wealth of india: raw materials: vol. viii. ph-re. the wealth of india: raw materials cytotoxicity of corylifoliae fructus. i. isolation of the effective compound and the cytotoxicity immunomodulatory and antitumour properties of psoralea corylifolia seeds two antifungal components isolated from fructus psoraleae and folium eucalypti globuli by bioassay-guided purification anti-dermatophytic activity of bakuchiol: in vitro mechanistic studies and in vivo tinea pedis-inhibiting activity in a guinea pig model isobavachalcone attenuates lipopolysaccharide-induced icam-1 expression in brain endothelial cells through blockade of toll-like receptor 4 signaling pathways phytoestrogen bakuchiol exhibits in vitro and in vivo anti-breast cancer effects by inducing s phase arrest and apoptosis osteoblasts proliferation and differentiation stimulating activities of the main components of fructus psoraleae corylifoliae fructus psoraleae contains natural compounds with potent inhibitory effects towards human carboxylesterase 2 bakuchiol contributes to the hepatotoxicity of psoralea corylifolia in rats estrogenic activities of psoralea corylifolia l. seed extracts and main constituents compounds isolated from psoralea corylifolia seeds inhibit protein kinase activity and induce apoptotic cell death in mammalian cells analysis of bakuchiol, psoralen and angelicin in crude drugs and commercial concentrated products of fructus psoraleae two new benzofuran derivatives, corylifonol and isocorylifonol from the seeds of psoralea corylifolia antiprotozoal screening of traditional medicinal plants: evaluation of crude extract of psoralea corylifolia against ichthyophthirius multifiliis in goldfish antimalarial activity of artemisia annua flavonoids from whole plants and cell cultures preparative isolation and purification of psoralen and isopsoralen from psoralea corylifolia by high-speed counter-current chromatography psoralidin, a coumestan analogue, as a novel potent estrogen receptor signaling molecule isolated from psoralea corylifolia cyp3a4 inhibition by psoralea corylifolia and its major components in human recombinant enzyme, differentiated human hepatoma huh-7 and heparg cells useful plants of the genus psoralea granzyme a cleaves a mitochondrial complex i protein to initiate caspaseindependent cell death the dangers of herbalism potential medicinal plants for lymphatic filariasis: a review natural sources used for treatment and prevention of filariasis bakuchiol inhibits the androgen induced-proliferation of prostate cancer cell line lncap through suppression of ar transcription activity effect of crude fractions of psoralea corylifolia seed extract on bone calcification effect of crude fractions of psoralea corylifolia seed extract on bone calcification in vitro enhancement of psoralen as an important anticancer compound in psoralea corylifolia through precursor feeding 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activity of extracts from psoralea corylifolia (fabaceae) correlated with the presence of a flavonoid compound potential antifilarial activity of the leaves and seeds extracts of psoralea corylifolia on cattle filarial parasite setaria cervi chemical fingerprint and quantitative analysis of fructus psoraleae by high-performance liquid chromatography mechanism of cytotoxicity by psoralea corylifolia extract in human breast carcinoma cells medicinal plants history, cultivation and uses studies on the chemical constituents of psoralea corylifolia l screening for inhibition activity of plant extracts on microorganism contaminating in cosmetics isolation and identification of furocoumarins from the seeds of psoralea corylifolia linn antifungal activity of diplotaenia damavandica antifungal potentiality of some plant extracts against fusarium sp plant-derived antimicrobial compounds: alternatives to antibiotics psoralea corylifolia l. an endangered medicinal plant with broad spectrum properties protective role of psoralea corylifolia l. seed extract against hepatic mitochondrial dysfunction induced by oxidative stress or aging comparison of the inhibitory potential of bavachalcone and corylin against udp-glucuronosyltransferases database on medicinal plants used in ayurveda agro-techniques of medicinal plants determination of isoflavone content and antioxidant activity in psoralea corylifolia l. callus cultures the effects of an antiosteoporosis herbal formula containing epimedii herba, ligustri lucidi fructus and psoraleae fructus on density and structure of rat long bones under tail-suspension, and its mechanisms of action optimization of elicitation condition with jasmonic acid, characterization and antimicrobial activity of psoralen from direct regenerated plants of psoralea corylifolia l systema naturae the netherlands in vitro acetylcholinesterase inhibition by psoralen using molecular docking and enzymatic studies in vitro and in vivo assessment of the effect of antiprotozoal compounds isolated from psoralea corylifolia against ichthyophthirius multifiliis in fish antifungal activity of phenyl derivative of pyranocoumarin from psoralea corylifolia l. seeds by inhibition of acetylation activity of trichothecene 3-o-acetyltransferase (tri101) pharmacognostic evaluation of the root of berberis asiatica fabaceae" angiosperm phylogeny website, 13 ed. missouri botanical garden and university of inhibition behavior of fructus psoraleae's ingredients towards human carboxylesterase 1 (hces1) dna polymerase and topoisomerase ii inhibitors from psoralea c orylifolia gonadal toxicity of an ethanol extract of psoralea corylifolia in a rat 90-day repeated dose study psoralen stimulates osteoblast differentiation through activation of bmp signaling review article: herbal hepatotoxicity-an update on traditional chinese medicine preparations new constituents from psoralea corylifolia histone deacetylase inhibitor sodium butyrate enhances the cell killing effect of psoralen plus uva by attenuating nucleotide excision repair psoralea corylifolia extract ameliorates experimental osteoporosis in ovariectomized rats screening south indian medicinal plants for antifungal activity against cutaneous pathogens osteoblastic proliferation stimulating activity of psoralea corylifolia extracts and two of its flavonoids chemical constituents from psoralea corylifolia and their antioxidant alphaglucosidase inhibitory and antimicrobial activities. zhongguo zhong yao za zhi= zhongguo zhongyao zazhi= chemical constituents from psoralea corylifolia and their antioxidant alphaglucosidase inhibitory and antimicrobial activities identification and characterization of naturally occurring inhibitors against udp-glucuronosyltransferase 1a1 in fructus psoraleae (bugu-zhi) indian medicinal plants. a compendium of 500 species positive skeletal effect of two ingredients of psoralea corylifolia l. on estrogen deficiency-induced osteoporosis and the possible mechanisms of action bioactive metabolites from the fruits of psoralea corylifolia effect of psoralen on bone formation bisbakuchiols a and b, novel dimeric meroterpenoids from psoralea corylifolia effects of gaultheria yunnanensis on adjuvant arthritis in rats. zhongguo zhong yao za zhi= zhongguo zhongyao zazhi= psoralen activates cartilaginous cellular functions of rat chondrocytes in vitro psc-afp, an antifungal protein with trypsin inhibitor activity from psoralea corylifolia seeds resveratrol induces the suppression of tumor-derived cd4+ cd25+ regulatory t cells the cytotoxicity of psoralidin from psoralea corylifolia the osteoprotective effect of psoralen in ovariectomy-induced osteoporotic rats via stimulating the osteoblastic differentiation from bone mesenchymal stem cells antidepressant-like effects of psoralidin isolated from the seeds of psoralea corylifolia in the forced swimming test in mice psoracorylifols a-e, five novel compounds with activity against helicobacter pylori from seeds of psoralea corylifolia antibacterial prenylflavone derivatives from psoralea corylifolia, and their structure-activity relationship study lipid class and fatty acid composition of psoralia corylifolia seed the benzopyrone biochanin-a as a reversible, competitive, and selective monoamine oxidase b inhibitor evaluation of the isoflavone genistein as reversible human monoamine oxidase-a and-b inhibitor. evidence-based complementary and alternative medicine the chemical constituents and bioactivities of psoralea corylifolia linn.: a review psoralea corylifolia l: ethnobotanical, biological, and chemical aspects: a review key: cord-330660-tx20im6w authors: mahmoud, huda k.; asghar, basim h.; harras, marwa f.; farghaly, thoraya a. title: nano-sized formazan analogues: synthesis, structure elucidation, antimicrobial activity and docking study for covid-19 date: 2020-10-07 journal: bioorg chem doi: 10.1016/j.bioorg.2020.104354 sha: doc_id: 330660 cord_uid: tx20im6w three series of nanosized-formazan analogues were synthesized from the reaction of dithiazone with various types of α-haloketones (ester and acetyl substituted hydrazonoyl chlorides and phenacyl bromides) in sodium ethoxide solution. the structure and the crystal size of the new synthesized derivatives were assured based on the spectral analyses, xrd and sem data. the antibacterial and antifungal activities were evaluated by agar diffusion technique. the results showed mild to moderate antibacterial activities and moderate to potent antifungal activities. significant antifungal activities were observed for four derivatives 3a, 3d, 5a and 5g on the pathogenic fungal strains; aspergillus flavus and candida albicans with inhibition zone ranging from 16 to 20 mm. molecular docking simulations of the synthesized compounds into leucyl-trna synthetase editing domain of candida albicans suggested that most formazan analogues can fit deeply forming stable complexes in the active site. furthermore, we utilized the docking approach to examine the potential of these compounds to inhibit sars-cov-2 3clpro. the results were very promising verifying these formazan analogues as a hopeful antiviral agents. according to world health organization (who) reports, the misuse of the antibiotics have led to development of multidrug resistance among various strains of microorganisms [1] . accordingly, new antimicrobial agents acting on novel targets have to be developed to overwhelm the increased incidence of microbial resistance to antibiotic remedy. from the best validated antimicrobial targets are the aminoacyl-trna synthetase enzymes which are key enzymes in the protein translation, producing the charged trnas required for proper assembly of peptide chains. from these enzymes, leurs have been considered as a drug target in fungi and bacteria, it is reported to be inhibited in the editing site by the potent antifungal 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (an2690) which is in clinical phases [2] [3] [4] . recently, the outbreak of covid-19, a new coronavirus pneumonia, caused by a new coronavirus (sars-cov-2) has emerged as a pandemic according to who in march 2020 [5] . to date, millions of infections and thousands of deaths have been recorded all over the world. in this scenario, there is a crucial need for developing antiviral agents interfering with the life cycle of the virus or with the virus replication or membrane fusion [6] . many researchers have considered the sars-cov-2 major protease (mpro), in addition called chymotrypsin-like protease (3clpr-2) as a potential target to anti-covid-19 drugs [7] [8] [9] . literature survey demonstrates that a lot of formazans have been described to possess broad spectrum of biological activities and pharmacological applications [10] such as antimicrobial [11] [12] [13] [14] , antiviral [15] [16] [17] , anticancer [18] , and anti-inflammatory [19] [20] . as for instance, the formazan derivative i was reported to display 100% inhibition of the ranikhet diseases virus [21] . additionally, the two formazans ii and iii synthesized by misra and dhar [22] showed 87% and 83% protections against the ranikhet disease virus, respectively. furthermore, many formazans iv were reported by lakshmi et al. [23] to have significant antibacterial and antifungal activities. also, uraz et al. [24] synthesized some formazan derivatives v, vi with different substituents and evaluated their antibacterial and antifungal activities against some selected microorganism species. the results revealed high activity against c. tropicalis, c. kefir, s. cerevisiae and c. neofarmans (figure 1) . recently, the synthesized drugs in the nanoscale have demonstrated a superior ability to penetrate the dna of various diseases, which have increased their biological activity and their ability to inhibit the diseases [25, 26] . inspired by the promising previously reported antimicrobial and antiviral activities of formazans and the urgent need of new effective antimicrobial agent and antiviral drugs to act mainly against covid-19 and in continuation of our research work in synthesis of bioactive compounds [27] [28] [29] [30] [31] [32] , we synthesized herein a new series of nano-sized formazan analogues to study their potential as antibacterial and antifungal agents in vitro. additionally, molecular docking simulations were done to propose their mode of binding in the editing domain of leurs and to show the possibility of these compounds to act against sars-cov-2 main protease enzyme. the first series of formazan analogues 3a-f were synthesized in sodium ethoxide solution at the xrd diffraction is a good tool to predict the size of the solid sample and the degree crystal regularity. five sample 3d, 3f, 5g, 5h and 7b of the synthesized formazan analogues were screened over 10˚<2θ <80˚ range to determined their crystallographic features (figures 6a-e). all investigated formazan analogues revealed sharp peaks which indicated the crystalline feature of them. the size of the crystals of the five samples was calculated according to the reported debye-scherrer equation [33] and the calculated size was tabulated in table 1 . the results referred to the formazan analogues moieties synthesized in the nanometer-scale. in addition, sem is another tool useful to give excellent insight about the crystallinity as well as surface topography for the tested solid samples. figure 7 contains two sem images for two formazan analogues 5h and 7b as examples for the prepared series. the two images indicated that the crystals of the two derivatives are found in the nanometer-scale. antimicrobial activities were carried out at the regional center for mycology and biotechnology (rcmb), al-azhar university, cairo, egypt. target compounds 3a-e, 5a-h, 7b were evaluated for their in vitro antibacterial, and antifungal activities, by inhibition zone method against two gram-positive bacteria: staphylococcus aureus (rcmb 010010) and bacillus subtilis (rcmb 015), two gram-negative bacteria: escherichia coli (rcmb 010052) and proteus vulgaris (rcmb 004) and two fungi: aspergillus flavus (rcmb 002002) and candida albicans (rcmb 005003) using gentamycin and ketoconazole as reference antibacterial and antifungal drugs, respectively (table 2&3 ). concerning the antibacterial activity against gram-positive bacteria, most compounds showed mild to moderate activities. the best activities were seen for the acetyl derivatives 5a, 5b, 5c, 5g and 5h with inhibition zones values ranging from 15 to 18 mm, compared to gentamycin with iz = 24 and 26 mm for s. aureus and b. subtilis, respectively. regarding gram-negative bacteria, they were resistant to 3a and 3b, while 3d having 4-br group revealed moderate activity against p. vulgaris (iz=17 mm) compared to gentamycin (iz=25 mm). on the other hand, compounds 5a, 5b, 5g and 5h displayed reasonable antibacterial activities against p. vulgaris with iz values 16, 18, 17 and 18, respectively. additionally, most compounds revealed mild antibacterial activities against the gram negative e. coli. most of the tested formazan analogues exhibited promising antifungal activities with the exception of 3e, 5f and 7b that were inactive against used fungal strains. focusing on the antifungal activities of compounds 3a-e, compound 3a elicited comparable antifungal activity (iz= 17 and 18 mm against a. flavus and c. albicans, respectively) to ketoconazole (iz= 16 and 20 mm against a. flavus and c. albicans, respectively). also, significant activities were observed for compounds 3b and 3c (iz=13-16 mm). in addition, compound 3d demonstrated superior antifungal activity against a. flavus (z= 20 mm) that was more potent than ketoconazole. furthermore, by studying the antifungal activities of 5a-h, moderate to potent activities were displayed. among these compounds, compound 5g with 3-cl group showed iz= 20 mm against a. flavus and 18 mm against c. albicans that were higher than those of ketoconazole. additionally, compound 5a showed comparble activity (iz= 16 and 19 mm) to ketoconazole. these antifungal activities results were highly appreciated since a. fumigatus is the second main cause of invasive aspergillosis and is the first leading cause of cutaneous aspergillosis [34] . in addition to the increased incidence of infections by c. albicans, the most common cause of candidiasis, and the increased resistance of c. albicans to antifungal drugs [35, 36] . leucyl-trna synthetase (leurs) belongs to the family aminoacyl trna synthetases (aarss), group of central enzymes that play a crucial role in protein synthesis, which is vital for survival of micro-organism and hence its inhibition presented a novel and attractive target for developing antimicrobials [37] . many recent reviews have discussed the importance of aarss in the discovery and development of antibacterial and antifungal agents [38] [39] [40] [41] . in this study, molecular docking study of the newly synthesized formazan analogues 3a-f, 5a-h and 7a,b have been performed onto the active site of candida albicans editing domain of cytosolic leucyl-trna synthetase to demonstrate their binding affinity and orientation. results of the docking simulation were displayed in table 4 showing the docking scores and the different formed interactions such as hydrogen bond, pi-h and non-polar pi-cation interactions. for majority of compounds, ala315, lys407, and lys483 were recognized as key amino acid residues responsible for hydrogen bonds generation and thr316, leu317, arg318, asp421and tyr487 were identified for minor interaction. whereas, for pi-h and pi-cation interactions, lys407, ser419, lys483 and tyr487 were the only observed residues. focusing on the binding mode of the most active analogues as antifungals, 3a, 3d, 5a and 5g (figure 8) , it was observed that they are well stabilized into the active site through strong it an attractive target for developing anti-coronavirus drug [42] . the protein structure of 3clpro-2 consists of 9 α-helices and 13 β-strands making together three distinctive domains: domain i (residues 8-101), domain ii (residues 102-184) and domain iii (residues 201-306) connected to domain ii by an extended loop (residues 185-200). 3clpro-2 contains a catalytic dyad that is composed of conserved residues his41 and cys145 and the key substrate-binding site is formed as a split between domain i and domain ii [43] . to estimate the binding affinity of derivatives 3a-f, 5a-h and 7a,b with covid-19 3clpro (pdb code: 6lu7), molecular docking study was done. as shown in table 5 and gln189 indicating the importance of these hydrophobic moieties. molecular modeling study revealed that these compounds could bind properly to c. albicans leucyl-trna synthetase editing domain. additionally, docking simulation into the active site of covid-19 3cl protease showed superior fitting into the active site with binding scores from -5.6064 to -8.0555 kcal/mol. the melting points of all new formazan analogues were recorded using a smp3 melting all the microbial strains that used in the current study have been supplied from al-azhar university in cairo, egypt from the culture collection of the regional center for mycology and biotechnology (rcmb). the method used for recording the antimicrobial activity according to the literature method [44] . molecular docking was analyzed through using moe-dock 2014 software [45] . chemical structures of 3a-f, 5a-h and 7a,b were drawn by the moe builder followed by minimization using the force field mmff94x in this program. hydrogen atoms were then added and unwanted water molecules were cancelled. docking was then performed using london dg for rescoring 1 and gbvi/wsa dg for rescoring 2. at the same time, refinement was done through forcefield. "ligand interactions" was utilized for the 2d visualization o the proteinligand interactions. the best pose was then selected depending on the binding energy and the interactions found in the active site. cm -1 . 1 h nmr (dmso-d 6 ) 1.60 (s, 3h anal. calcd. for c 22 h 19 cln 6 os (450.94) calcd: c, 58 -nitrophenyl)-2-oxopropanehydrazonic-n',2-diphenyldiazenecarbohydrazonic thioanhydride (5e) ir ύ: 3479 (br. 2nh), 1662 (co), 1596 (c=n) ir ύ: 3436 (br 2nh), 1653 (co), 1594 (c=n) -chlorophenyl)-2-oxopropanehydrazonic-n',2-diphenyldiazenecarbohydrazonic thioanhydride (5g) orange solid, yield (0.38g, 85%), mp 160-162°с (ethanol /dioxane), ir ύ: 3437 (br 2nh), 1659 (co), 1593 (c=n) m/z (%) 452 (m + +2, 0.16), 451 (m + +1, 0.11), 450 (m + , 0.38) aminoacyl-trna synthetases as drug targets in eukaryotic parasites an antifungal agent inhibits an aminoacyl-trna synthetase by trapping trna in the editing site crystal structures of the human and fungal cytosolic leucyl-trna synthetase editing domains: a structural basis for the rational design of antifungal benzoxaboroles molecular investigation of sars-cov-2 proteins and their interactions with antiviral drugs putative inhibitors of sars-cov-2 main protease from a library of marine natural products: a virtual screening and molecular modeling study molecular docking reveals the potential of aliskiren, dipyridamole, mopidamol, rosuvastatin, rolitetracycline and metamizole to inhibit covid-19 virus main protease tmprss2 and is blocked by a clinically proven protease inhibitor functionalized formazans: a review on recent progress in their pharmacological activities synthesis, evaluation and docking studies of novel formazan derivatives as an enoyl-acp reductase inhibitors synthesis and evaluation of antimicrobial activity of some new schiff bases and formazans synthesis, characeterization and in vitro antibacterial activity of novel 3-(4-methoxyphenyl)-1-isonicotinoyl-5-(substituted phenyl)-formazans synthesis and biological studies of some novel formazans synthesis of some new formazans as potential antiviral agents synthesis and antiviral activity of some new formazans synthesis of 1-(20aryl-40-oxo-3hquinazolyl)-3-aryl-5-phenyl-formazans as potential anti-viral agents synthesis of some new formazans and their biological activity novel formazans as potent anti-inflammatory and analgesic agents antiinflammatory activity of quinazolinoformazans synthesis of some newer formazans and tetrazolium salts as antiviral agents synthesis of some newer formazans and tetrazolium salts and their effect on ranikhet disease virus and the vaccinia virus synthesis and their possible biological activities of few formazans of 3-amino-2-sulphanyl-2 discovery of thiazole-based-chalcones and 4-hetarylthiazoles as potent anticancer agents: synthesis, docking study and anticancer activity new azoloazine derivatives as antimicrobial agents: synthesis under microwave irradiations, structure elucidation, and antimicrobial activity new series of thiazole derivatives: synthesis, structural elucidation, antimicrobial activity, molecular modeling and moe docking zno nanoparticles catalyst in synthesis of bioactive fused pyrimidines as anti-breast cancer agents targeting vegfr-2 antitumor activity of pyrrolizines and their cu(ii) complexes: design, synthesis and cytotoxic screening with potential apoptosis-inducing activity new and efficient approach for synthesis of novel bioactive [1,3,4]thiadiazoles incorporated with 1,3-thiazole moiety elements of x-ray diffraction aspergillus flavus: human pathogen, allergen and mycotoxin producer mechanisms of pathogenic candida species to evade the host complement attack pathogenicity and drug resistance in candida albicans and other yeast species hybrid phenylthiazole and 1,3,5-triazine target cytosolic leucyl-trna synthetase for antifungal action as revealed by molecular docking studies aminoacyl-trna synthetases and their inhibitors as a novel family of antibiotics aminoacyl-trna synthetases: essential and still promising targets for new anti-infective agents aminoacyl-trna synthetase inhibitors as potential antibiotics aminoacyl-trna synthetase inhibitors as potent antibacterials molecular investigation of sars-cov-2 proteins and their interactions with antiviral drugs anti-hiv drug repurposing against sars-cov-2 molecular operating environment (moe) 2014.09, chemical computing group inc., 1010 sherbrooke street west, suite 910 ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work ☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: _______________________________________________________________________________ ______ nano-sized formazan analogues: synthesis, structure elucidation, antimicrobial activity and docking study for harras and thoraya a. farghaly a,b * key: cord-286136-gm6w590s authors: aleksic sabo, verica; knezevic, petar title: antimicrobial activity of eucalyptus camaldulensis dehn. plant extracts and essential oils: a review date: 2019-03-05 journal: ind crops prod doi: 10.1016/j.indcrop.2019.02.051 sha: doc_id: 286136 cord_uid: gm6w590s eucalyptus has become one of the world’s most widely planted genera and e. camaldulensis (the river red gum) is a plantation species in many parts of the world. the plant traditional medical application indicates great antimicrobial properties, so e. camaldulensis essential oils and plant extracts have been widely examined. essential oil of e. camaldulensis is active against many gram positive (0.07–1.1%) and gram negative bacteria (0.01–3.2%). the antibacterial effect is confirmed for bark and leaf extracts (conc. from 0.08 μg/ml to 200 mg/ml), with significant variations depending on extraction procedure. eucalyptus camaldulensis essential oil and extracts are among the most active against bacteria when compared with those from other species of genus eucalyptus. the most fungal model organisms are sensitive to 0.125–1.0% of e. camaldulensis essential oil. the extracts are active against c. albicans (0.2–200 mg/ml leaf extracts and 0.5 mg/ml bark extracts), and against various dermatophytes. of particular importance is considerable the extracts’ antiviral activity against animal and human viruses (0.1–50 μg/ml). although the antiprotozoal activity of e. camaldulensis essential oil and extracts is in the order of magnitude of concentration several hundred mg/ml, it is considerable when taking into account current therapy cost, toxicity, and protozoal growing resistance. some studies show that essential oils’ and extracts’ antimicrobial activity can be further potentiated in combinations with antibiotics (beta-lactams, fluorochinolones, aminoglycosides, polymyxins), antivirals (acyclovir), and extracts of other plants (e.g. annona senegalensis; psidium guajava). the present data confirm the river red gum considerable antimicrobial properties, which should be further examined with particular attention to the mechanisms of antimicrobial activity. the continuing increase in the degree of bacterial resistance to conventional antibiotics is a problem of global significance. the crisis of antimicrobial resistance has been ascribed to the misuse of these agents and today resistant strains are common, such as methicillin-resistant staphylococcus aureus, vancomycin-resistant enterococci, drug resistant streptococcus pneumoniae and mycobacterium tuberculosis, carbapenemresistant and extended-spectrum beta-lactamase producing enterobacteria, multidrug-resistant pseudomonas aeruginosa and acientobacter baumannii etc. it was estimated that the medical cost per patient with an antibiotic-resistant infection is up to $29,069 and infections are usually life treating (ventola, 2018; aslam et al., 2018) . similarly, fungi have become resistant to poliens, azoles and echinocandins, and the emergence of drug-resistant strains has been reported in all fungal species (robbins et al., 2017) . beside the noticeable emergence of resistant protozoa and viruses, there is a problem with camaldulensis is highly variable (table 1 ). in the past, this character has been used to break up the group into different varieties or subspecies. the entire complex is currently under revision and new varieties or subspecies may be described or extant ones rationalised. until this work is completed, euclid (2006) decided to adopt a conservative view of e. camaldulensis. used for centuries as a traditional aboriginal herbal remedy, eucalyptus leaves and their essential oils have found various applications in everyday life due to their antiseptic, anti-inflammatory and antipyretic properties (jeane et al., 2003; kumar et al., 1988) . ancient aboriginal society in australia used e. camaldulensis plant in medicines to treat gastrointestinal symptoms (including colic, diarrhea, and dysentery), respiratory disease (colds, coughs, asthma, laryngalgia, laryngitis, pharyngitis, sore throat, trachalgia), arrest bleeding, open wounds, and cuts, as well as its decoctions for the relief of spasms, aches, and pains in muscles, but also pains in joints and even tooth (duke and wain, 1981) . as previously stated, e. camaldulensis plant is also known as red gum eucalyptus, murray red gum, and river red gum, because it produces red kino i.e. red gum, in significant amount. thus besides different plant parts aborigines used its secondary products as folk remedies. they made incisions in the tree trunks for obtaining the red kino and applied it directly to abrasions and cuts. except fresh kino, the dried, dehydrated kino was prepared and used in the same way as fresh, but after softening in water (williams, 2011; clarke, 2014) . another significant folk remedy is young leaves which were used for smoke bath, where burning leaves smoke surrounds patient. the smoking medicine was used for fevers, colds, flu and general sickness (williams, 2011; pennacchio et al., 2010; duke and wain, 1981) . being useful for treating various health conditions, e. camaldulensis and its folk remedies then were transferred and introduced to other parts of the world, such as africa. in sudan the red kino was used for sore throat and diarrhea, while the smoke of burnt leaves was inhaled in case of respiratory problems. in senegal for stomach-ache decoctions from leaves were prepared with sugar, while in zimbabwe for cough, flu, and fever a decoction of e. camaldulensis leaves were combined with citrus limon (l.) burm. f. fruits and psidium guajava l. leaves (doran and wongkaev, 2008; maroyi, 2013) . further more, to prevent tooth decay and periodontitis in nigeria teeth cleaning sticks were made from tree (bukar et al., 2004) , and in traditional medicine for healing wound infections, poultice of leaves containing eucalyptus oil have been used (adeniyi et al., 2006) . nowdays, e. camaldulensis has been the subject of numerous studies, to confirm plant's usefulness in traditional medicine for the treatment v. aleksic sabo and p. knezevic industrial crops & products 132 (2019) [413] [414] [415] [416] [417] [418] [419] [420] [421] [422] [423] [424] [425] [426] [427] [428] [429] of various ailments (coelho-de-souza et al., 2005; ghani, 2003; ito et al., 2000) . its essential oils are reported to be anesthetic, antiseptic and astringent (jeane et al., 2003; kumar et al., 1988) . in addition, a decoction of the leaves is reported to be a remedy for sore throat and other bacterial infections of the respiratory and urinary tracts (bruneton, 1999) . due to numerous contemporary data regarding the antimicrobial activity of e. camaldulensis, this subject will be discussed in special section/chapter. except antimicrobial effects, e. camaldulensis plant extracts (pex) and essential oils (eos) and its constituents possess numerous other beneficial effects. one such effect is certainly gastrointestinal effect. in animal models, extracts of the leaves of e. camaldulensis and e. torelliana r. muell are reported to decrease gastric acid production and thus appear useful for the treatment of gastric ulcers (adeniyi et al., 2006) . it has been proven that e. camaldulensis leaves methanol extracts possessed ulcer-healing promoting effect when investigated in acetic acid induced-ulcer in rat (rattus norvegicus domesticus) (table 2) . similarly, the poultice of the leaves is applied over wounds and ulcers (gill, 1992) . the antioxidant effect represents one more useful e. camaldulensis characteristic. the free radical scavenging activities of the essential oils is assessed by measuring their scavenging abilities for 2,2-diphenyl-1picrylhydrazyl (dpph) radicals. the scavenging activity for the eucalyptus camaldulensis was characterized as high (81.9%) (ghaffar et al., 2015) . the results of e. camaldulensis eo antioxidant effect evaluation indicated that the eo had high potent ferrous ions chelating and total antioxidant activities comparing to ascorbic acid and bht (el-baz et al., 2015) . also, the e. camaldulensis var. brevirostris leaves ethanol extract possessed antioxidant activity, where the prevailing antioxidants in the extract were gallic and ellagic acid (el-ghorab et al., 2003) . there are several more studies dealing with antioxidant activity of e. camaldulensis (barra et al., 2010; salem et al., 2015; siramon and ohtani, 2007; olawore and ololade, 2017) , but it is interesting to mention here that e. camaldulensis flower eo inhibited melanogenesis through its antioxidant properties and by down-regulating both mitogen-activated protein kinases (mapk) and protein kinase a (pka) signaling pathways. this study indicated that the essential oil has the potential to be developed into a skin care product (huang et al., 2015) . thus, except the medical application, e. camaldulensis extracts are also currently used in cosmetic formulations, and leaf extracts have been approved as food additives (takahashi et al., 2004) . also, essential oils and their constituents have been used as flavoring agents in the formulation of different pharmaceutical products, cosmetics, and food industry (cowan, 1999) . except this, e. camaldulensis pex and eos have the potential to be used as antibacterial and antifungal agents in cosmetic and pharmaceutical products. it is interesting to mention here the activity of e. camaldulensis eos (2-8 mg/ml) on dental biofilm formation in vivo where detected inhibition was 14.5-39.2% after four weeks, comparing to chlorhexidine (2 mg/ml), which inhibited maximum 13.9% of dental biofilm formation . extracts and essential oils of this aromatic plant can be used as food preservatives in order to reduce the dependency on synthetic chemicals in food preservation. in australia, it is also used as sources of wild honey, providing bees with good quality pollens and heavy yields of nectar (boland et al., 1984) . moreover, in industry, the wood of e. camaldulensis has been used for heavy construction, railway sleepers, flooring, framing, fencing, plywood, and veneer manufacture, wood turning, firewood, and charcoal production (boland et al., 1984) . eucalyptus camaldulensis wood burns well and make a good fuel, also its dense wood and coppicing ability make it an excellent species for fuelwood production used in several countries such as brazil (jacobs, 1981; eldridge et al., 1993) . furthermore, eucalyptus biomass residues from agro-forest and pulping industries represent a valuable source of highvalue compounds such as triterpenic compounds (domingues et al., 2011; ferreira et al., 2018) . due to the diversity of e. camaldulensis beneficial effects, all effects reported in the literature are summarized in table 2 . beside the beneficial effects, essential oils and plant extracts can exert potentially unfavorable effects as complex mixtures of different compounds. a risk assessment of their hazard is always necessary before commercialization, so the estimation of eos toxicity has been already conducted resulting in human oral and dermal dose limit recommendations. for most eos the recommended dose is in range 1-4%, but for cineole-rich eucalyptus sp. eos, including e. camaldulensis, the limit dose is 10%, indicating a generally low application risk (lis-balchin, 2006) . the safe daily oral dose in human adults is 300-600 mg, while semisolid preparations for topical use may contain 5-20% of eucalyptus oil (blumenthal and busse, 1998; tisserand and young, 2014) . similarly, e. camaldulensis bark methanolic extract ld50 value for swiss albino mice (mus musculus) is very high -1120 mg/kg, indicating its low host toxic effects (islam et al., 2014) . the low toxicity and natural origin of e. camaldulensis essential oil and extracts, in contrast to synthetic antimicrobials, favor their application as antimicrobial agents. eucalyptus camaldulensis leaves contain 0.1-0.4% essential oil, of which 77% is 1,8-cineole. there is considerable amount of cuminal, phellandrene, aromadendren (or aromadendral), valerylaldehyde, geraniol, cymene, and phellandral (council of scientific and industrial research, 1948 -1976council of scientific and industrial research (s.i.r, 1948council of scientific and industrial research, 1948 -1976 slee et al., 2006) . leaves contain 5-11% tannin. the kino (a class of wood exudates), contains 45% kinotannic acid as well as kino red, a ebrahimi et al. (2013) repellency against the adult females of culex pipiens dried fruits essential oil two different treatment levels (5 and 10 μl) in six exposure times (15, 75, 135, 195 , 255 and 315 s) erler et al. (2006) anti-diabetic effect albino rats oral 500 mg/kg of body weight dawoud (2015) alloxan-induced diabetic rats leaves ethanol extract 500 mg/kg of body weight in distilled water orally, e. camaldulensis leaves supplement incorporated into the diet 5 g/kg/day dawoud and shayoub (2017) v. aleksic sabo and p. knezevic industrial crops & products 132 (2019) 413-429 glucoside, catechol, and pyrocatechol. leaves and fruits test positive for flavonoids and sterols. the bark contains 2.5-16% tannin, the wood 2-14%, and the kino 46.2-76.7% (watt and breyer-brandwijk, 1962) . some of the reported phytoconstituents of the tree included essential oils, sterols, alkaloids, glycosides, flavonoids, tannins, and phenols. a considerable variation in the yield of leaf essential oil from e. camaldulensis has been reported (boland et al., 1984; shieh, 1996; moudachirou et al., 1999; farah et al., 2002) , depending on multiple biotope factors, and also genetic and/or epigenetic characteristics of the plant. the yields of e. camaldulensis leaves eo (0.90-0.98%) originating from pakistan and morocco were similar (ashraf et al., 2010; farah et al., 2002) , while moudachirou et al. (1999) reported a variable oil content of 0.6-1.4% from different locations of benin. the oil yield of e. camaldulensis from jerusalem was 0.5% (chalchat et al., 2000) and significantly higher oil yield was reported for e. camaldulensis from taiwan: 2.3-3.0% with respect to different seasons (shieh, 1996) . similar eos yield (0.77-2.53%) has been reported for e. globulus, as one of the economically important plants for essential oil production (joshi, 2012; selvakumar et al., 2012; harkat-madouri et al., 2015) . the reported essential oil yield for other eucalyptus species is slightly higher, ranging from 1.2% to 3% (w/w): the highest yield was obtained from e. cinerea f. (sebei et al., 2015) . all the e. camaldulensis essential oil single compounds belong to chemical class of hydrocarbons terpenes, further devided according to the number of isoprene units (c 5 h 8 ) to monoterepenes (c 10 h 16 ), sesquterepenes (c 15 h 24 ), and longer chains of isoprene units. oxygenated terpenes (oxygenated monoterepenes and oxygenated sesquterepenes) are called trepenoids. according to the literature, in e. camaldulensis eos dominates 1,8-cineole (eucalyptol), trans-pinocarveol and terpinen-4-ol from chemical class of oxygenated monoterepenes. the oils also contain considerable amount of monoterpene hydrocarbons (β-pinene, α-thujene, γ-terpinene, p-cymene), while sesquterepene hydrocarbons and oxygenated sesquterepenes are detected in significantly lower amounts (table 3) . when the composition of five eucalyptus essential oils (eucalyptus camaldulensis, eucalyptus astringens maiden, eucalyptus leucoxylon, eucalyptus lehmannii and eucalyptus rudis endl) are compared, a high percentage of monoterpenes, mainly oxygenated compounds, with lower quantities of sesquiterpenes were recorded throughout the four seasons (ben jemaa et al., 2012) . in e. camaldulensis oil, monoterpenes were prevalent (34.6-56.3% for all seasons) while sesquiterpene hydrocarbons (6.6-16.5%) and oxygenated sesquiterpenes (2.1-11.1%) were present in less extent. eucalyptus astringens oil had resembling chemical characteristic to e. camaldulensis oil, the abundant quantity of monoterpenes, which represented more than 50% of the total oil amounts for the all seasons (53.4-63.6 %). similarlly, e. leucoxylon essential oil, contained mainly oxygenated monoterpenes with a prevalence of 1,8 cineole (13.1-17.6%), such as in e. camaldulensis oil (15.5-20.6%). the essential oil of e. lehmannii also was made up largely of monoterpenes (hydrocarbons 27.6-44.9% and oxygenated 30.9-62.8%), with smaller amounts of sesquiterpene hydrocarbons and absence of oxygenated sesquiterpenes, unlike the oils of other eucalyptus species (ben jemaa et al., 2012) . the difference in the chemical composition of the e. camaldulensis eos may be due to many reasons, which can generally be classified in five main groups: (1) the change in plant genes through generations and hybridizations (naturally and induced) may result in production a variety of volatile oils compared with those of different habitat; (2) nutrients of different soils and their accumulation in the leaves may result in different plant metabolism and consequently production of different bio-products and also eos made of diverse compounds in variable amounts; (3) acclimation of species to the australian environment in which it is growing in the past, compared with the introduced and/or worldwide planted trees on plantations; (4) different ecotypes of the e. camaldulensis and (5) differences may be due to plant part used for essential oils extraction and its stage of development (maturity). knowing that these factors express significant effect on the percent compositions of some eo components for this species, e. camaldulensis can be grown in the corresponding areas and in specific conditions to enhance eo production. the variations in the chemical composition of eucalyptus eos with respect to seasons have also been reported (tsiri et al., 2003) , but the most commonly detected as major components in the eucalyptus essential oil are 1,8-cineole, β-pinene, γ-terpinene, and p-cymene. it is important to emphasize here that according to chemical composition the eucalyptus camaldulensis eo generally can be divided in two different types. type i is a cineole-rich essential oil containing 80-90% 1,8-cineole plus pinene, and type ii is a cineole-poor essential oil, containing significantly less cineol (williams, 2011) . plant genotype is important factor influencing the final chemical composition of eo (djilani and dicko, 2012) . due to genetic and epigenetic factors same plant species can produce a similar eo, but with different chemical composition and therapeutic activities. brophy and southwell (2002) examined essential oils of two variations, eucalyptus camaledulensis var. camaldulensis and eucalyptus camaledulensis var. obtuse, and the main compounds of eucalyptus camaledulensis var. camaldulensis were pcymene (22%), cryptone (14%), and spathulenol (17%), while eucalyptus camaledulensis var. obtusa had different main compounds: 1,8cinole (52%), α-pinene (15%), and aromadendrene (3%), suggesting that these subspecies belong to different types of eucalyptus essential oils. the content of 1,8-cineole was also variable but in the same range for essential oils reported from greece (25.3-44.2%) (tsiri et al., 2003) , pakistan (34.4-40.0%) (ashraf et al., 2010) , mozambique (37.1-40.0%) (pegula et al., 2000) , nigeria (32.8-70.4%) (oyedeji et al., 1999) , and taiwan (34.0-68.2%) (shieh, 1996) . similarly, the major component of eucalyptus camaldulensis oils originating form burundi, morocco, and benin was 1,8-cineole ranging from 31.0 to 72.5% with no presence of cryptone (dethier et al., 1994; zrira and benjilali, 1991; zrira et al., 1992; moudachirou et al., 1999) . cryptone has been shown to be present in low-cineole varieties of eos from australia (bignell et al., 1996) , uruguay (dellacassa et al., 1990) and south florida (pappas and sheppard-hanger, 2000) . for example, the major constituents identified in the essential oil from south florida included p-cymene (35.0%), cryptone (13.7%), terpinen-4-ol (5.7%), spathulenol (4.3%), and cuminaldehyde (3.7%), with a very low amount of 1,8-cineole (2.7%) (pappas and sheppard-hanger, 2000) . it was proven that essential oils of different plant parts have different chemical composition (table 3) . previous studies on the essential oil of e. camaldulensis flowers revealed the presence of 1,8-cineole, βpinene, and spathulenol as the most abundant constituents (giamakis et al., 2001) . the essential oil of the leaves was found to contain pcymene, γ-terpinene, α-pinene, 1,8-cineole, terpinen-4-ol, α-terpineol, carvacrol, and thymol as the major components (siramon and ohtani, 2007) . the major components of the fruits essential oil were aromadendrene, α-pinene, drimenol, and cubenol (el-ghorab et al., 2002) . plant developmental stage and maturity of plant parts used for essential oil extraction also affect essential oil chemical composition. giamakis et al. (2001) analyzed the immature flowers and calli grown in athens (greece), and they found that the main monoterpenes produced in e. camaldulensis calli, cultured in darkness and under light conditions, were 1,8-cineole, 62.70 and 69.26% as well as β-pinene, 27.09 and 25.31%, respectively. in lower amounts α-pinene (1.2 and 1.1%, respectively) and the terpenoids, camphene, myrcene, isocineole, myrtenol, bicyclogermacrene, spathulenol, and trans-pinocarveol were present (less than 1%). apart from isocineole, these constituents were also determined in the essential oil from immature flowers. this is remarkable because giamakis et al. (2001) showed that undifferentiated calli are capable of producing high amounts of monoterpenoid compounds (approx. one third of that produced by the explant). this makes immature flowers from e. camaldulensis an interesting candidate for the development of calli able to produce high percentages of these two important monoterpenes, 1,8-cineole and β-pinene (giamakis et al., 2001) . also, the influence of light conditions on essential oil chemical composition showed that light conditions did not considerably affect the production of monoterpenes and sesquiterpenes compounds by calli developed from immature flowers (giamakis et al., 2001) . furthermore, plant culture conditions can influence the essential oil chemical composition. soil salinity is a key ecological stress that severely influences plant productivity (williams, 2011) . ashraf et al. (2010) showed that salinity had a considerable effect on the percent compositions of some components of e. camaldulensis leaves essential oil. the mean values of 1,8-cineole content of eo from saline and nonsaline provenances of pakistan were 34.42 and 40.05%, respectively (ashraf et al., 2010) . therefore, they recommend stressing of this species by growing in the saline areas to enhance essential oil production for its various medicinal and pharmacological uses. extraction represents the primary step in obtaining the crude mixture of compounds from plants. quality and quantity of the extracts dependent of the target compound structures, natural sources, and type of processes (karacabey et al., 2013) , explaining the different phenolic composition in the extracts obtained with different procedures. the most commonly plant extract have been obtained by conventional solvent extraction methods (infusion, decoction, digestion, maceration, and percolation) (azwanida, 2015) using solvents such as water, ethanol, methanol, chloroform, dimethyl-sulfoxide etc. however, these techniques are demanding regarding the extraction process duration, organic solvent consumption, and lack of extraction automation. the potential and powerful alternative to conventional liquid solvent extraction methods are ultrasonic-assisted extraction (uae) and microwave-assisted extraction (mae), especially in the case of plant material (hao et al., 2002; eskilsson and bjrklund, 2000) . interest in mae has increased significantly over the past 5-10 years in particular medicinal plant research, as a result of its inherent advantages as special heating mechanism, moderate capital cost, and its good performance under atmospheric conditions (ballard et al., 2010; chan et al., 2011) . in addition, mae technique possesses many advantages compared with other methods for the extraction of compounds such as bioactive compounds (sanchez-aldana et al., 2013) saving in processing time and solvent, higher extraction rate, better products with lower cost, reduced energy consumption (up to 85-fold savings), and waste generation (yan et al., 2010; yemis and mazza, 2012) . this is confirmed for e. camaldulensis extraction of phenolic and flavonoid compounds, where the compounds were extracted with mae for 5 min which was equivalent with uae (60 min) and traditional extraction (24 h) methods (gharekhani et al., 2012) . many authors reported the chemical composition of e. camaldulensis extracts. the leaves of e. camaldulensis from the zoo-botanical garden in giza (egypt) yielded 4 major fractions (singab et al., 2011) . the major components of the first fraction (eluted with water) were identified as hhdp-glucopyranose, chlorogenic acid, and phloroglucinol derivatives. the second 30% methanol fraction was found to contain different galloyl-hhdp-glucopyranose positional isomers and pedunculagin as major components. the third 60% methanol fraction was predominantly composed of digalloyl-hhdp-glucopyranose (tellimagrandin i) α and β anomers, while the last 100% methanol fraction was composed of a mixture of ellagitannin dimers. the profiling of the obtained fractions by hplc-pda-esi/ms/ms indicated that ellagitannins were the most predominant components of all three methanol fractions (singab et al., 2011) . the secondary metabolites screening of e. camaldulensis leaf extracts from nigeria confirmed presence of tannin, saponins, and cardiac glycosides (ayepola and adeniyi, 2008) . analyzed n-hexane, chloroform, and methanol extracts of e. camaldulensis stem bark and leaf also grown in nigeria showed the presence of tannins and saponins in the stem bark and in the leaf of e. camaldulensis with absence of alkaloids in all extracts (adeniyi et al., 2009) . furthermore, the crude methanol leaf extracts also from nigeria contained in addition volatile oils and balsam (gum) (babayi et al., 2004) . similarly, the phytochemical screening of ethanol, methanol, and petroleum ether leaf extracts from nigeria contained in moderate to high amount secondary metabolites: alkaloids, saponins, tannins, flavonoids, steroid, carbohydrates, and cardiac glycosides, and not anthraquinones (chuku et al., 2016) . crude methanol leaf extracts of e. camaldulensis from iran had saponins, tannins, volatile oils, and balsam (gum), while the components such as anthraquinones, hydrolysable tannin, flavonoid, alkaloid, and glycosides were not detected (jouki and khazaei, 2010) ; crude methanolic leaves extract of e. camaldulensis from india contained anthraquinones, flavonoids, saponins, and terpenoids, while alkaloids, cardiac glycosides, and tannins were not detected (singh and thakur, 2016) . phytochemical screening of the crude stem barks methanol extract of e. camaldulensis from bangladesh indicated presence of saponins, flavonoids, tannins, and also volatile oils, while anthraquinones, hydrolysable tannins, alkaloids, and glycosides were not present (islam et al., 2014) . the polyphenolic composition (flavonoids and phenolic acids and aldehydes) also was studied in the soluble fractions of the methanolic extracts of eucalyptus camaldulensis originating from two spain provinces, huelva and pontevedra: gallic, protocatechuic, vanillic and ellagic acids, and protocatechic aldehyde were identified, along with eriodictyol, quercetin, naringenin, vanillin, naringin, quercitrin, luteolin, and kaempferol (cadahia et al., 1997) . eucalyptus camaldulensis extracts are generally rich in tannins which vary qualitatively and quantitatively according to the origin of the samples, and consequently protoanthocyanidin levels were influenced by the geographical origin (cadahia et al., 1997) . antimicrobial activity of e. camaldulensis eo and extracts are well documented against many microorganisms listed in the table 4 . for easier comparison, all data commented here for eos were re-calculated from microliter per milliliter or microgram/milligram per milliliter to percentage (v/v or w/v), using the equitations presented in fig. 2 . we considered here only minimal inhibitory and minimal bactericidal concentrations, while results obtained using disc or agar diffusion methods were not discussed. however, mic/mbc results vary between several micrograms to several milligrams. such high variation does not seem as real, even taking into account variation in oil composition, and are rather a consequence of erroneous equalization of one microliter and one microgram, or typographical errors. for instance, there are some nonsense mics, such as 2000 μl/ml, that is practically impossible to obtain (salem et al., 2015) . in some manuscripts even a species was not precisely indicated (harkenthal et al., 1999; karpanen et al., 2008; warnke et al., 2009; tadtong et al., 2016) and these results were not considered in the present review. eucalyptus camaldulensis plant extracts and eos were tested against wide range of bacteria. the most frequently included gram positive bacterium in screening is s. aureus. minimal inhibitory concentrations in most studies were in range 0.07-0.5% indicating moderately high activity against this bacterium. beside s. aureus, antibacterial effect was confirmed against b. subtilis (0.17-0.34%), m. luteus (0.2-0.4%), and s. pyogenes (0.4-1.1%) akin et al., 2010; knezevic et al., 2016; khubeiz et al., 2016; reda et al., 2017; ostad asiaei et al., 2018) . activity of eos was examined aginst l. monocytogenes in only one study and mic was not obtained with the highest used concentration of 1.0%, and the same was reported for enterococcus durans (akin et al., 2010) . activity against gram negative bacteria has been documented in a greater extent and mics for the most frequently used model organism e. coli was in range 0.15-3.2%. sensitivity of other enterobacteria is similar, with mics in range from 0.05 to 0.32% for k. pneumoniae (khubeiz et al., 2016; ostad asiaei et al., 2018) , 0.16-0.32% for salmonella enterica subsp. enterica serovar typhimurium (khubeiz et al., 2016) ; 0.35-0.4% for serovars typhi, paratyphi, and 0.6% for s. enteritis (ostad asiaei et al., 2018). other enterobacteria are similarily sensitive: shigella soneii was inhibited with 0.3% of eo (ostad asiaei et al., 2018) , while proteus vilgaris was sensitive in a broad range from 0.25% (ostad asiaei et al., 2018) to more than 1.28% (khubeiz et al., 2016) . the published data for p. aeruginosa are in the same range as for p. vulgaris, which is not surprising, as both species are generally highly resistant to antimicrobial agents. on the contrary, a. baumannii which is also known to be multi-drug resistant, showed sensitivity to eo in a range 0.05-0.1% . the lowest mic among gram negative bacteria was recorded for a gastrointestinal pathogen v. parachaemoliticus (0.01%) (khubeiz et al., 2016) . although the gram positive bacteria are consider more sensitive to eos in comparison to gram negative, it cannot be applied for essential oils of e. camaldulensis, since the most sensitive bacteria are gram negative -a. baumannii and v. parahaemolyticus. the minimal bactericidal concentrations for most bacteria are equal to, or rarely up to 4 times greater than mics. this low range in mic/mbc < 4 indicates that e. camaldulensis eos act as bactericidal agents (pankey and sabath, 2004) . antibacterial effects of e. camaldulensis extracts have been examined in a wider extent than essential oils. inhibitory concentrations varied depending on extraction method, plant properties, and model organism, being in broad range from 0.08 μg/ml to 200 mg/ml. crude aqueous leaf extracts show lower activity against various bacteria (range 25-50 mg/ml) (abubakar, 2010) , while bark aqueous extracts were more effective, with mic from 0.1 mg/ml for propionobacterium acnes to 4.0 mg/ml for p. aeruginosa (mabona et al., 2013) . the antimicrobial activity of methanol, ethanol or petroleum leaf extracts of e. camaldulensis showed significant variation; for instance, mics against b. subtilis varied from 0.04 up to 200 mg/ml or against s. aureus 1.25-25 mg/ml (chuku et al., 2016; ayepola and adeniyi, 2008) . for most examined gram negative bacteria mics were in range 10-200 mg/ml, while p. aeruginosa was even more susceptible (mics 10-100 mg/ml). similar or better antibacterial effect showed acetone leaf extract, being active against examined bacteria in range 15-50 mg/ml. the highest activity was obtained with dichloromethen extracts, with mics for staphylococcus spp. in range 0.25-1.0 mg/ml, and even lower for b. subtilis, p. acnes and b. agri (mics 0.10-0.79 mg/ml). interestingly, generally highly resistant m. tuberculosis was sensitive to methanol, n-hexane or chloroform extract with mics in range 0.004-0.064 mg/ml, while m. bovis was slightly less sensitive with mics 0.01-0.05 mg/ml (lawal et al., 2012; gemechu et al., 2013) . the progresses made on the investigation of essential oils mode of action, especially against bacterial cell targets, gave new perspectives in this combat. the knowledge about the essential oils and their target(s) on bacterial cell is crucial to understand which parts of bacterial cell are affected. the essential oils antibacterial action is linked to oil hydrophobicity that increases cell permeability and consequent leakage of cell constituents (dorman and deans, 2000; lambert et al., 2001; helander et al., 1998; turgis et al., 2009; ultee et al., 2002; faleiro, 2011) . it is important to perceive that a disturbed cell structures may affect stability of other cellular structures in a cascade type of action (carson et al., 2002) . essential oils have several target sites on bacterial cells; however it seems that all are directly or indirectly connected to the primary effect of essential oils on the bacterial envelopes. first of all, eos may cause cell wall and membrane disturbance (lambert et al., 2001; oussalah et al., 2006) , which further can lead to the significant loss of intracellular atp (oussalah et al., 2006; turgis et al., 2009) , induction the synthesis of heat shock proteins (burt et al., 2007) , ph disturbance (turgis et al., 2009; oussalah et al., 2006) , and intracytoplasmic changes (e.g. coagulation, periplasmic space enlargement) (becerril et al., 2007) . although the number of studies dealing with the eo mechanisms of action is increasing, there are still many questions to be answered before the precise mechanism is revealed. this is at the same time one of the main limitations for eos and extracts wide usage as antimicrobials. thus, the so far knowledge must be further improved enabling the combat bacterial pathogens and its resistance. contemporary research of eos and extracts are also focused on mechanism that allows bacteria to regulate some physiological activities, such as virulence, competition amongst populations, motility, sporulation, conjugation, antibiotic production, and biofilm formation (rodriguez-garcia et al., 2014) . this system of intercellular communication, quorum sensing (qs), is based on production of the signal molecules, called autoinducers (abraham et al., 2011) . this communication system is relative to cell density and some compounds interfere with the qs communication system and attenuating the bacterial pathogenicity, in the phenomenon known as anti-qs compounds (abraham et al., 2011) . there are reports of anti-qs activity of various plants species eos from genus eucalyptus, such as eucalyptus globulus l. (luís et al., 2016; cervantes-ceballos et al., 2015) , eucalyptus radiata d. (luís et al., 2016) , eucalyptus citriodora hook., eucalyptus smithii r. t. baker and eucalyptus staigeriana f. muell. ex bailey (luís et al., 2016) . unfortunately, there is still no data regarding e. camaldulensis anti-qs activity. in one study, antibacterial and in vivo biofilm preventive efficacies of e. camaldulensis oil were significantly higher than that of m. spicata oil and chlorhexidine, suggesting that e. camaldulensis eo is capable of fig. 2 . schematic diagram for re-calculation from microliter or milligram per milliliter to percentage (v/v or w/v). affecting biofilm formation . considering the anti-qs activity of above mentioned eucalyptus species and also detected antimicrobial and anti-biofilm effect of e. camaldulensis it can be assumed that it possess similar or even higher anti-qs activity. however, future detailed studies of anti-qs and anti-biofilm e. camaldulensis activity are needed in order to confirm this assumption. the e. camaldulensis leaves extracts and eos have a potential as antifungal agents. they are able to act as a moderate antifungal agent against household molds, wood rot fungi (siramon et al., 2013) , and phytopathogenic fungi (gakuubi et al., 2017; mehani et al., 2014) . eucalyptus camaldulensis eo has been studied for antifungal activity and was active in concentration 0.125-1.0% against most model organisms. the most sensitive seems to be f. sporotrichioides with mic 0.125% (mehani et al., 2014) , while r. oryzae is the most resistant, as 1% of eo was ineffective against this fungus (siramon et al., 2013) . the best known human and animal pathogenic yeast candida sp. showed considerable sensitivity to e. camaldulensis eo, with mic approx. 0.5% (siramon et al., 2013; nasir et al., 2015) . most eos from sardinia inhibited growth of a. niger and b. cinerea in concentration of 20 μl/plate, while lower concentrations of the oils, such as 5 μl/plate were ineffective (barra et al., 2010) . it is interesting to notice that liposomes containing e. camaldulensis eo were prepared (moghimipour et al., 2012) for antifungal oil activity. the particle size varied from 40.5 to 298 nm for the different formulations, with approx. 95% of the essential oil entrapped. inhibition of microsporum canis, m. gypseum, trichophyton rubrum, and t. verrucosum was achieved with 125 μl, and the liposomal gel formulation of the eo was proposed to improve antifungal activity. aqueous and organic extracts of e. camaldulensis have been reported to have antifungal activity (table 4 ). methanol leaves extracts showed inconsistent activity against c. albicans: in one study it was in range 50-200 mg/ml (chuku et al., 2016) , while in another it was 0.2 mg/ml (babayi et al., 2004) , indicating difference in active concentration of one thousand times. the bark methanole extract was active in concentration 0.5 mg/ml. methanol leaf and bark extracts showed considerable activity against dermatophytes: 0.8-1.6 mg/ml against microsporum spp, 0.125-1.6 mg/ml against trichophyton spp., and 0.2 mg/ml against epidemophyton flocossum (takahashi et al., 2004; falahati et al., 2005; mabona et al., 2013) . beside the antifungal activity of eos and extracts of e. camaldulensis, it is worth noticing that this species, along with e. blakelyi m., e. gomphocephala a. dc, e. rudis endl., and e. tereticornis sm., is a reservoir of an emerging pathogenic fungus -cryptococcus gattii. this fungus is usually related to tropic and subtropic regions, affecting the respiratory and nervous systems of the immunocompetent humans and domestic animals (sorrell et al., 1996; chakrabarti et al., 1997; bielska and may, 2016; roe et al., 2018) . similarily, c. neoformans var. grubii can be isolated from flowers and bark of e. camaldulensis (gugnani et al., 2005) . according to the literature, e. camaldulensis is natural reservoir of cryptococcus and is considered responsible for occurring cryptococcosis worldwide. in this context, it is interesting to observe that c. neoformans is moderately sensitive to e. citriodora hook eos, with mic90 0.5% (wt/v) (pattnaik et al., 1996; luqman et al., 2008) or e. globulus labill. with mic 0.13% (wt/v) (suliman et al., 2010) . unfortunately, data on cryptococcus sp. sensitivity to eos and plant extracts of reservoir species of eucalyptus, including e. camaldulensis, remains unknown. although there are no reports regarding e. camaldulensis mode of antifungal action, according to previous studies with other essential oils and fungi, the plasma membrane and the mitochondria are the probable antifungal targets of eos. according to recent studies, the antifungal activity of eos results from its ability to disrupt the permeability barrier of the plasma membrane and from the mitochondrial dysfunction-induced ros accumulation in a. flavus and c. albicans (tian et al., 2012; chen et al., 2013) . the exact mechanism of e. camaldulensis antifungal activity is unknown and should be elucidated in the future. infections caused by viruses are very common and sometimes life threatening, especially in immunocompromised patients and neonates (snoeck, 2000; khan et al., 2005) . despite the recent significant progress in antiviral drug development, viral infections are considered as one of the major causes of death worldwide (müller et al., 2007; meyers et al., 1982) . thus, novel natural antiviral agents need to be found urgently. many natural products possess antiviral activity and some of them are already in use for treatment of human viral infections with both rna and dna viruses (e.g. myricetin against coronavirus, linalool, urosolic acid, and apigenin against coxsackievirus, quercetin and narasin against denge virus, curcumin against hepatitis b and c virus) (lin et al., 2014; kitazato et al., 2007) . numerous secondary plant metabolites such as essential oils, flavonoids, saponins, tannins, alkaloids, lignans, terpenes, and phenolic acids express significant antiviral activity against different viruses (jassim and naji, 2003; chiang et al., 2003; sanchez palomino et al., 2002) . recently few studies confirmed the e. camaldulensis eos and plant extracts antiviral activity (table 4) . eucalyptus camaldulensis eos reduce coxackie b4 and rotavirus wa multiplication for 50%, herpes simplex virus 1 for 90%, but have no effect on adenovirus 7 multiplication (el-baz et al., 2015) . similarly, methanolic extracts showed 50% inhibition of hsv 1 and 2 in concentration 0.1-0.3 μg/ml, and against varicella zoster virus at concentration 1.0 μl/ml (abu-jafar and huleihel, 2017). dimethyl sulfoxide (dmso) extracts inhbit multiplication of animal new castle virus in concentration range 50-500 μg/ml (al-hadid, 2016) . antiviral activity has been observed for e. camaldulensis methanolic extracts against polio virus, coxsackie b, and echovirus 6 (adeniyi et al., 2015) . the data on antiviral activity of e. camaldulensis eo and extracts, although scarce, indicate their great potential, and necessity for further studies in this context. despite the fact that many plant extracts and essential oils were previously reported for their antiviral activities, the mechanism of action still remains poorly understood. there are many factors that influencing the eos mode of action, which should be taken in consideration when antiviral activity of plant antimicrobials is examined. one of such factors is the difference between enveloped and non-enveloped viruses, because the observed antiviral effect has usually been greater for enveloped viruses (yamada et al., 2009; siddiqui et al., 1996) . in majority of the studies dealing with antiviral mode of action, the focus has been on either the inhibition of viral adsorption to host cells or examination of the plant antimicrobials effectiveness against intracellular virus multiplication (gilling et al., 2014) . so, most commonly described modes of antiviral actions are virus inactivation and the impaired virus adsorption to host cells, which is often difficult to distinguish. like in other antimicrobial modes of action, antiviral mechanism is also dependent on eos or extracts compounds activity. this is one more factor that should be considered, because it affects the final antiviral mechanism of action. for some compounds the potential mechanism is reported, i.e. carvacrol acts directly upon the virus capsid and subsequently the nucleic acid (gilling et al., 2014) . when eos antiviral mechanism of action is considered, eos may cause the loss of the viral capsid integrity, ultimately leading to exposure of the viral genome. in addition, some eos subsequently act directly upon the viral nucleic acid (dna or rna). according to one study, e. camaldulensis eos may be promising antiviral agent against rna viruses with no effect against dna virus (el-baz et al., 2015) . also, with shorter periods of exposure to the antimicrobial, the virus is able to adsorb specifically to host cells; however, it may or may not be able to cause successful infection depending upon the integrity of the viral genome. on the other hand, after exposure to some eos virus capsid and genome remains intact. these antimicrobials appear to exert their antiviral effect by coating the capsid and thereby preventing specific adsorption of the virus to host cells (gilling et al., 2014) . although, there are still no reports regarding e. camaldulensis eos antiviral mode of action, there are some promising results about its antiviral effect (table 4) . these promising results represent foundation for further research and potential application of e. camalulensis eos as potent antiviral agent. among all deverse benefitial activities, e. camaldulensis expresess also an antiprotozoal effect as well. the reports regarding this effect are listed in table 4 . eucalyptus camaldulensis methanolic and aqueous extracts were active against leishmania major with ic50 values (50% inhibitory concentration) 586.2 ± 47.6 and 1108.6 ± 51.9 μg/ml, respectively (nosratabadi et al., 2015) . this was characterized as moderate leishmanicidal activity, but considering fact that present therapy consist antimony compounds which are expensive, toxic, and drug resistance is prevalent, e. camaldulensis plant extract derivatives represent safe, inexpensive, and promising alterantive solution. there are also several reports regarding antiprotozoal activity against trichomonas vaginalis, a causative agent of trichomoniasis which is the most prevalent nonviral sexual infection. the first report on e. camaldulensis aqueous extract anti-trichomonas activity showed that extract was active at concentration 500 mg/ml after 24 h (mahdi et al., 2006) . however, recent studies reported better effect of the e. camaladulensis extracts (hassani et al., 2013; youse et al., 2012) . five different e. camaldulensis leaves extracts including total extract, diethyl ether, chloroform, ethyl acetate, and water fractions were active in concentration range 12.5-50 mg/ml, with growth inhibiton achived after 24-72 h (hassani et al., 2013) . ethyl acetate fraction showed the highest percentage of growth inhibition with the lowest concentration (12.5 mg/ml) after 24 and 48 h (hassani et al., 2013) . even better antitrichomonas activity was detedted for wather and ethanolic extracts, where concentration 60-90 μg/ml killed trichomonas vaginalis for 72 h (youse et al., 2012) . the mainstay medication for trichomoniasis is metronidazole; however some resistant strains to this treatment have been detected making these results of e. camaldulensis anti-trichomonas activity very promising as antiprotozoal agent. except extracts, significant antiprotozoal activity was reported for e. camaldulensis eos. the essential oils were found to possess antitrypanosomal activity in vitro in a dose-dependent manner in a short time. the decrease of trypanosoma evansi number over time was achieved in doses of 400 mg/ml for 3 min, 200 mg/ml for 4 min, and 100 mg/ml for 15 min. against trypanosoma brucei brucei eos was more potent in the concentration of 400 mg/ml, decreasing the number of parasite for 3 min, 200 mg/ml for 4 min, and 100 mg/ml for 11 min (habila et al., 2010) . such prompt decrease in parasite number in the in vitro tests for both trypanosoma brucei brucei and t. evansisuggests that the eos kill the parasites efficiently, but by an unknown mechanism. there are suggestions of some potential mechanisms of antiprotozoal eos action. the activity of eos could be due to the hydrophobic nature of the cyclic hydrocarbons, which allow eos to interact with the protozoans causing conformational changes in the parasite membrane structure, resulting in the loss of membrane stability (calsamiglia et al., 2007) . the essential oils also can act by inhibiting some key enzymes in the parasite glycolytic pathway (smith-palmer et al., 2004) . furthermore, some eos components inhibit acetylcholinesterase activity and act on other vulnerable sites, such as cytochrome p450 (maciel et al., 2010) . this multicomponent nature of plant eos is an advantage for several target sites on protozoans, which is of great importance. it was shown that eos of e. camaldulensis are more potent against b. subtilis, s. aureus, e. coli, p. aeruginosa, s. lutea, and p. carotovorum in comparison to eos from e. gomphocephala dc., but not against a. tumefaciens. furthermore, the e. camaldulensis var. obtuse showed even better effect than e. camaldulensis (salem et al., 2015) . among seven examine essential oils of various eucalyptus species, eo of e. camaldulensis was among the best against b. subtilis, a. niger, and r. solani, but not against e. coli and s. aureus (ghaffar et al., 2015) . similar activity of e. camaldulensis and e. torelliana f. muell. was recorded for extracts against six strains of h. pylori (adeniyi et al., 2009) . it is worth to notice that among 132 extracts from 42 plants growing in southern africa, e. camaldulensis bark extract showed considerable activity against bacteria and fungi, with an exception against p. aeruginosa and m. canis (mabona et al., 2013) . finaly, in many studies regarding antimicrobial activity of various eucalyptus species, e. camaldulensis was not always included (ashour, 2008; safaei-ghomi and ahd, 2010; mulyaningsih et al., 2010; elaissi et al., 2012; sebei et al., 2015) . due to rapid emerging of microbial resistance to conventional drugs, the necessity for efficient solution(s) is rising. the main strategy represents finding new antimicrobial agents; however another strategy goes in the direction of reducing degree of bacterial resistance and/or bacterial re-sensitization to conventional antibiotics. this can be achieved using combined therapies. the most of the tested combinations are dual combinations, but the combinations of three, four or more agents also can be efficient (lesjak et al., 2016) . this is in line with antimicrobial efficiency and potential of eos and plant extracts which are complex mixtures of numerous compounds. combination strategy could be very promising regarding the diversity of agents that could be tested: (1) conventional non-antimicrobial agent (e.g. anti-inflammatory or anti-psychotic drug) + conventional antimicrobial agent (antibiotic); (2) conventional antimicrobial agent (antibiotic) + natural antimicrobial agent (essential oil, plant extract, bacteriophage, antimicrobial peptide ect.); (3) conventional antimicrobial agent (antibiotic) + single compound isolated from natural antimicrobial agent (with previously confirmed antimicrobial activity); (4) combination of two or more single compounds isolated from natural antimicrobial agents (with previously confirmed antimicrobial activity). being a plant with already detected and evaluated antimicrobial activity, it can be assumed that e. camaldulensis also have a potential in combined therapy. this assumption is confirmed in some studies in vitro and in vivo (table 5) . eucalyptus camaldulensis eos and extracts in vitro reduced resistance of mdr a. baumannii in combination with conventional antibiotics: β-lactams, ciprofloxacin, gentamicin, polymyxin b . similarily, the increase of β-lactamase produing mrsa and e. coli sensitivity to cephalexin, cefuroxime, amoxicillin, and ampicillin has been obtained through combination with e. camaldulensis eos (chaves et al., 2018) . synergistic activity has been recorded between e. camaldulensis plant extracts and gentamicin or ceftriaxone against mdr s. aureus and p. aeruginosa (reda et al., 2017; ibrahim et al., 2014) , as well as in combination with ampicillin against s. aureus (ibrahim et al., 2014) . furthermore, the combination of e. camaldulensis extract with another plant extract psidium guajava l. was also efficient against mdr bacteria (bala et al., 2014) . except antibacterial activity, other activities of e. camaldulensis extracts in combination were detected and characterized as efficient. antiviral activity was confirmed for the combination of e. camaldulensis 80% methanol leaves extract and acyclovir against herpes simplex virus -1 and -2 and varicella-zoster virus (abu-jafar and huleihel, 2017). similaily, the combination of annona senegalensis l. leaf methanol extract and e. camaldulensis extract efficiently cured in vivo albino mice infection with parasite trypanosoma brucei brucei (lafia strain) (kabiru et al., 2012) . all these data are very promising, but the methods used in different studies and results interpretation vary (table 5) . as a consequence, the data should be taken with precautions, as can not be compared and properly discussed. to avoid this problem, the testing combinations of different agents should be conducted using standardized methods, such as time-kill method (clsi, 1999; verma, 2007) , checkerboard method (verma, 2007; eucast, 2000) , chou-talalay method (chou, 2010) or boik method (boik, 2010) . all these methods possess some shortfalls, such as timeconsuming, labor-intensive, limitations regarding the number of the agents in combination, etc. unfortunately, there is no one gold standard for synergy testing and prior the further application of different phytochemicals, this issue should be overcome. summarizing the available data on antimicrobial properties of eucalyptus camaldulensis essential oil and extracts, it is obvious that this plant is a valuable source of phytotherapeutics. the essential oil, as well as leaf and bark extracts are particularly valuable as antibacterial, antiviral, and antifungal agents, and their antiprotozoal activity should not be neglected taking into account current therapy cost, toxicity, and protozoal growing resistance. some e. camaldulensis plant characteristics such as easy cultivation, wide distribution by plantation, and rapid growth additionally support further examination of antimicrobial activity, in order to enhance the commercial production of e. camaldulensis based pharmaceuticals. the future studies should be focused on determination of the mechanisms of antimicrobial activity, paticularly potential anti-biofilm and anti-qs effects, as well as the activity enhancement in combination with other available agents. effects of eucalyptus camaldulensis and other antimicrobial agents in combination. two-dimensional checkerboard method (verma, 2007) synergism (fici ≤ 0.5) knezevic et al. (2016) leaf essential oil from brazil the mic of the antibiotics were determined in the presence and absence of sub-inhibitory concentrations (125 μg ml −1 ) of the essential oil (coutinho et al., 2010) combining the essential oil with β-lactams reduced the resistance of tested strains agar disc diffusion method was employed as described by kirby and bauer (1966) , adopted by yushau et al. (2009) and bashir et al. (2011) synergism bala et al. 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astragalosides in radix astragali optimization of furfural and 5-hydroxymethylfurfural production from wheat straw by a microwave-assisted process effect of echinophora platyloba, stachys lavandulifolia, and eucalyptus camaldulensis plants on trichomonas vaginalis growth in vitro in-vitro sensitivity pattern of some urinary tract isolates to carica papaya extracts the essential oil of the leaves and the fruits of e. camaldulensis essential oils of twentyseven eucalyptus species grown in morocco key: cord-336165-if8uxqha authors: remoaldo, paula; serra, jaime; marujo, noémi; alves, juliana; gonçalves, alexandra; cabeça, sónia; duxbury, nancy title: profiling the participants in creative tourism activities: case studies from small and medium sized cities and rural areas from continental portugal date: 2020-09-12 journal: tour manag perspect doi: 10.1016/j.tmp.2020.100746 sha: doc_id: 336165 cord_uid: if8uxqha although cultural tourists increasingly seek to experience cultural events actively and to directly engage in creative activities, empirical knowledge about the creative tourist remains limited. this study aims to characterize the motivations and profile of creative tourists. the data was collected through a survey of participants in creative tourism activities in portugal developed by 40 pilot institutions of the creatour project during 2017 and 2018, with 814 usable questionnaires collected and validated. the questionnaire had 30 questions and marked the first time this kind of research was conducted in portugal. the questionnaire included questions on: the composition of their travel companions, their previous participation in a creative tourism experience, reasons for visiting the destination, their characterization of the creative tourism experience, an evaluation of their creative tourism experience, and their socio-demographic profile. using a cluster analysis to analyse the data, three clusters were found: novelty-seekers, knowledge and skills learners, and leisure creative-seekers. in the 1980s, alvin toffler (1980) pioneered the concept of "prosumer" to refer to a type of consumer involved in product design and in production itself. with the diffusion of the concept of prosumer -a fusion of the words "producer" and "consumer" -since that time, the consumer has become the producer of many of the products and experiences he consumes. the diffusion of this concept coincides with the emergence of a new generation of tourists, also called "qualified consumers" or "creative consumers" (carvalho, ferreira, & figueira, 2016; mihajlović & koncul, 2016; pappalepore, maitland, & smith, 2014; richards, 2010a; richards & wilson, 2006; zhang & yu, 2018) , postmodern travellers (carvalho et al., 2016; jelinčić & žuvela, 2012; o'dell, 2007; pappalepore et al., 2014; tan, luh, & kung, 2014) or "creative tourists" (raymond, 2003; smith, 2016; tan et al., 2014; zhang & yu, 2018) . regardless of the term applied, they all refer to tourists who actively create their experience in the destinations they choose. this new consumer of experiences has played an increasingly active role in the economy, leaving aside the passivity characteristic of the 1980s, to play an essential role in market communication (egger, gula, & walcher, 2016; kotler, kartajaya, setiawan, & vandercammen, 2012; tan et al., 2014; zhang & yu, 2018) . for alvin toffler (1980) , the concept of prosumer defines a type of consumer of the future, involved in the design and production of products to make them more personalized and individualized. in addition, prosumers are more informed, more educated and with an aboveaverage level of demand. in this sense, the creative tourist can be seen as a prototype of the prosumer (fundação de serralves, 2008; tan et al., 2014; egger et al., 2016; zhang & yu, 2018) . there is a great diversity of definitions of creative tourists, ranging from those that refer to participants in dance art experiences or handicraft workshops, to those that include people temporarily residing in artistic residences in search of creativity. according to richards (2011) , creativity can be used to implement creative tourism as a tourist activity, which involves the active involvement of tourists in the creative activities of the places they visit, or as a backdrop for tourism, in which tourists place themselves in a chosen creative environment. nevertheless, despite these efforts, defining the concept of creative tourism and describing the motivations and profiles of those who https://doi.org/10.1016/j.tmp.2020.100746 received 4 may 2020; received in revised form 1 september 2020; accepted 2 september 2020 engage in creative activities during their holidays are not easy tasks. the term "creative tourism" is relatively young and began to draw attention in the scientific milieu in the 1990s. pearce and butler (1993) were the first to mention creative tourism as a potential form of tourism. the definition most used by the experts in this area is the one elaborated by richards and raymond (2000) , who define creative tourism as offering tourists the opportunity to develop their creative potential through active participation in courses and learning experiences that are the characteristics of the destination in which they are carried out. over the last two decades, tourist demand has become increasingly exacting, segmented and constantly changing (fundação de serralves, 2008; tan et al., 2014; carvalho et al., 2016; smith, 2016; zhang & yu, 2018) . this process points to the emergence of a new tourist profile and, consequently, a new pattern of consumption that is directed toward the use of creativity as an alternative to mass cultural tourism. creative tourists are not satisfied to only observe cultural events and passively visit cultural spaces, but seek to experience them actively. they seek memorable events (pine & gilmore, 1999) and usefulness rather than novelty (tan, kun, & luh, 2013) . there are mindful visitors (moscardo, 1996) who want to become a part of the destination's everyday dynamics (ilincic, 2014) and ask for active participation and greater involvement with the local community (carvalho et al., 2016; mihajlović & koncul, 2016; ohridska-olson & ivanov, 2010; richards, 2003; richards & raymond, 2000; richards & wilson, 2007; smith, 2016; zhang & yu, 2018) . generally, creativity is associated with urban areas, especially large cities. this relationship between creativity and the city derives from the fact that creative industries have been greatly responsible for dynamizing the attractiveness of the cities, making them more attractive for companies, for new inhabitants and, consequently, for tourists (argent, tonts, jones, & holmes, 2013; boes, buhalis, & inversini, 2015; dekker & tabbers, 2012; richards, 2011; richards, 2014a) . this new type of tourist results from the depleted model of massified cultural tourism which does not gives him/her opportunities to have an active role. this depleted model can be clearly observed in southern europe countries that have been suffering from a high pressure of visitors in recent decades. in portugal (the territory analysed in the present paper) massified cultural tourism began to occur in recent years, especially in large cities like lisbon and porto. creative tourism appears as an opportunity to reinvent the current tourism model and offer a form of tourism that is more sustainable and close to the local community. the profile of cultural tourism is well defined and supported by a long spectrum of scientific research, but little is known about the creative tourist. the few international studies dedicated to the profile of the creative tourist continue to highlight the complexity of this segment, which involves tourists from multiple generations (children, adults and the elderly) looking for authenticity, exclusivity, improving skills and desiring contact with the local community. noting that these insights are derived from a generalized international level, the profile of the creative tourist has not been clearly characterized in portugal, especially the one that visits small and medium-sized cities and rural areas. in the research presented in this article, we specifically analyse the profile of this kind of visitor. we studied the tourists who participated in creative tourism experiences carried out by 40 institutions involved in the creatour project, located in the four nuts ii regions of continental portugal (norte, centro, alentejo and algarve). the creatour project "creative tourism destination development in small cities and rural areas" (https://creatour.pt/en/) was in process from november 2016 to june 2020. as a research-andapplication project, creatour developed an integrated approach to creative tourism development in small cities and rural areas across portugal. on the research side, the project aimed to examine and reflect on the creative tourism activities, including development dynamics and patterns, reception experiences, and community impacts, using methodologies and theoretical perspectives from the fields of tourism, cultural development, and local/regional development. on the practice side, it aimed to catalyse creative tourism offers in small cities and rural areas in portugal, inform and learn from their development, and link them with each other through the development of a national network (duxbury, 2020) . the project was funded by feder through the joint activities programme of compete 2020 and the regional operational programmes of lisbon and algarve and co-funded by the portuguese foundation for science and technology (fct/mec). taking a cultural development approach, creatour fostered a diversity of "bottom-up" ideas and experimentation in which 40 pilot projects were independently designed, implemented, and managed, but coupled with knowledge-sharing and capacity-building through networking. the creative tourism activities developed ranged widely, including: craft workshops involving textile, pottery, ceramics, leather, metal, and wood; fine arts workshops such as painting, sculpture, drawing, and illustration; photography, video, and digital arts workshops; performing arts workshops and community-engaged, participatory artistic residencies; storytelling sessions and workshops; gastronomy-focused workshops in which visitors learn food-related cultures of a place as well as culinary techniques to take home; imaginative "walks & visits" involving creation activities; ancestral traditions workshops and active participation activities; and activities related to raw materials production and work cycles for making, for example, salt, linen, wool, clay, marble, wicker, and so forth. this paper aims to characterize the profile of the tourists who participated in these activities and their motivations, attending to the following three questions: who are the participants in creative tourism activities? what are the main motivations to attend a creative tourism activity? can motivation be used to segment creative tourism participants (i.e., are creative tourists motivation-driven)? for the analysis of the data, descriptive and multivariate statistics have been developed through the program statistical package for the social sciences (s.p.s.s. version 22.0). this paper is organized as follows. after the introduction, section 2 presents a literature review of creative tourism and the creative tourist. the research methods are presented in section 3. the results and discussion are presented in section 4. finally, in section 5 the conclusions and some policy and managerial implications are presented. the development of creative tourism phenomena is linked to recent developments in cultural tourism. cultural tourism corresponds to the tourism in which cultural attractions are the main reason to visit or stay in a certain destination (csapó, 2012; mousavi, doratli, mousavi, & moradiahari, 2016; richards & wilson, 2006; williams, 2010) and that offers the visitor an opportunity to understand and appreciate the culture and essence of a place (kajzar, 2013 (kajzar, , 2014 richards, 2014b) . creative tourism emerged in response to a market that has specific needs. although it is the central theme of a growing number of investigations, the concept of creative tourism still remains quite vague in the scientific environment (richards, 2011 (richards, , 2014b tan et al., 2013; hung, lee, & huang, 2016; smith, 2016; creative tourism network, 2018; remoaldo et al., 2019; remoaldo, matos, gôja, alves, & duxbury, 2020; duxbury & richards, 2019a) . many definitions of creative tourism are linked to cultural tourism, and is generally understood as a form of cultural tourism that allows for a more authentic approach between the tourist and residents (briggs, 2005; gordin & matetskaya, 2012; jelinčić & žuvela, 2012; king, 2009; ohridska-olson & ivanov, 2010; richards, 2010b; richards, 2014a; richards & raymond, 2000; virginija, 2016) . creative tourism is oriented to immaterial resources such as learning, developing experiences and traditions (ribeiro et al., p. remoaldo, et al. tourism management perspectives 36 (2020) 100746 2020). this tourist is someone who wants to not only see the region but to experience it (virginija, 2016) . in this sense, creative tourism is a participatory form of cultural tourism more appropriate to contemporary social and economic structures. this type of tourism makes use of the intangible resources of the destination (e.g., lifestyles, narratives, creativity, media) and enables the tourist to participate actively in leisure, cultural and artistic activities that reflect the characteristics of the destination visited. it is a special form of tourism that creates the necessary conditions for travellers to exercise their and participate in creative workshops and activities (e.g., arts and heritage -brouder, 2012; rudan, 2012; hung et al., 2016) , which allows for a truer and more authentic experience in the destination. this new demand for deep immersion in the experiences consumed reveals a new profile of tourist. although there are attempts to characterize this new tourist (carvalho, ferreira, & figueira, 2011; florida, 2002; mota, remoaldo, & ribeiro, 2012; remoaldo, vareiro, & ribeiro, 2017; richards, 2010a; richards & wilson, 2007; silberberg, 1995; tan et al., 2014) , no consensus has yet been found. this new tourist wants a more authentic experience, immersed in the local cultural capital and close to the community (guerreiro & marques, 2017; remoaldo et al., 2020) . this trend is a response to the saturation of mass cultural tourism (richards, 2010a) . in this sense, creative tourism develops the social and cultural capital of tourists, as they become co-authors of their tourism experience, contributing to authentic, immersive and exclusive experiences (pine & gilmore, 1999; richards, 2014b; virginija, 2016) . the characteristics of the cultural tourist are very close to the ones of the traveller seeking creative experiences. cultural tourists, for the most part, exhibit different expectations and motivations from those of the "modern" tourists (smith, 2003) . one of the characteristics that distinguishes cultural tourists from the people who engage in creative activities comes from the intensity of the motivations (which may be, to a greater or lesser extent) to live new experiences, as well as the degree of involvement and interaction that the tourist establishes with the local community (smith, 2016) . creative tourists seek to enjoy participatory and authentic experiences and, mainly, generate their own experiences (prahalad & ramaswamy, 2004) , exercising co-creation (binkhorst, 2007; puczkó, 2013) . throughout the twentieth century, the attitude of tourists has undergone several changes and, on the whole, tourists have become more demanding. they have begun to look for experiences during their holidays as a way to develop their skills and take an active part in the culture of a place (richards, 2010b) . this search for learning is not an absolutely new and intrinsic feature of the creative tourist. completely to the contrary, this characteristic has been gaining expressiveness over the last two decades and is a central element of the experiences in creative tourism (anderson, 2009; peters, frehse, & buhalis, 2009; raymond, 2007; tan et al., 2013) . these tourists wish to contact and learn more about specific aspects of the culture of a particular community through active participation with the local community and the development of their creative skills in workshops and other activities (cortada, 2006; raymond, 2003; richards, 2003; richards & raymond, 2000; richards & wilson, 2007) . this kind of tourist assumes the role of co-creator, co-producer and consumer of the experiences and skills of the promoters of experiences (e.g., trainers, local community), that is, the tourist is involved in the local culture through making artefacts or products in the destination (e.g., handicrafts, gastronomy, art) (anderson, 2009; binkhorst, 2007; maisel, 2009; peters et al., 2009; prentice & andersen, 2007; ray & anderson, 2000; raymond, 2007; richards & wilson, 2006; tan et al., 2013) . in addition, the participant in creative tourism activities wants higher levels of social, emotional and educational interaction with the community and to feel like part of the destination (binkhorst & den dekker, 2009; richards, 2014b; smith, 2016; stolarick, denstedt, donald, & spencer, 2010) . investigations previously carried out on the profile of the creative tourist reveal that the existing segments are different and show that the creative tourist covers a wide range of travellers. what we do know is that the creative tourist tends to appreciate authenticity and cultivates the desire to get to know the local culture in a more "alternative" way that is closer to the local community. despite these attempts, the definition of the creative tourist is still very generalist and imprecise, many studies that claim to be about the creative tourist do not actually correspond to that segment of tourism, and further research about the creative tourist is still needed (duxbury & richards, 2019b) . in the next section, some international case studies are reviewed that present more detailed information on the profile of the creative tourist. creative tourism seekers tend to belong to the creative class (florida, 2002) , that is, a cosmopolitan class from the middle and upper layers of society which shares higher levels of education. internationally, a few research surveys present the characteristics and behaviour of these contemporary tourists (see table 1 ), but the body of research is still very limited and needs further theoretical development. source: authors' elaboration. p. remoaldo, et al. tourism management perspectives 36 (2020) 100746 in the early 2000s, raymond (2003) , based on a case study in new zealand (table 1) , presented a proposal to segment and profile creative tourism seekers based on general demographic profiles, proposing three groups: baby-boomers and retired, tourists under 30 (e.g., students and backpackers) and new zealanders who are interested in learning more about different aspects of the culture of their country. in 2009, a survey of participants from two creative tourism pilot events in the uk concluded that the participants were predominantly female, aged 45 or more years of age. the other large group was made up of younger women aged between 22 and 30 with busy full-time jobs and no children (campbell, 2010) . in 2014, tan et al. identified five distinct groups of creative tourists in taiwan -novelty-seekers, knowledge and skills learners, those who are aware of their travel partners' growth, those who are aware of green issues, and the relax and leisure type -by analysing 46 q-statements about creativity and creative experiences. in 2015 in bali, indonesia, another attempt to establish the profile of the creative tourist in creative tourism experiences of rural communities was presented by blapp (2015) . in total, 15 tourists' groups were interviewed based on an opportunistic sampling strategy. the interviews were limited to western tourists given the scope of the research to focus on creative tourism geared toward the western market (blapp, 2015) . in 2016, ali et al. examined the effect of creative tourists' experiences on their memories, satisfaction and behavioural intentions. a total of 296 surveys were conducted with guests at six selected resort hotels in malaysian states of terengganu and kedah who participated in creative activities, cooking classes, handicraft classes, storytelling sessions of local tales and 'batik painting'. the largest number of tourists was aged between 31 and 40 years (60%). females were predominant (54%), with three-quarters of them being malaysian (72%) and around one-quarter being foreigners (28%). in 2019, huang et al. conducted an empirical study in three popular creative tourism attractions in taiwan: meinong, singang and yingge. the following creative activities were developed: pottery, handicrafts and arts making. the majority of the 395 tourists surveyed was aged between 31 and 40 years and females were also predominant (59.2%). in portugal, a study on the influence of the creative industries on tourism in the city of porto (city break predominant tourism segment) presented the tourist profile in this city. in the sample collected of 385 tourists, males were predominant and their motivations were broader than participating only in creative activities. the tourists were from spain, aged between 19 and 25 years, single, with a high academic degree (bachelors or masters) and with high annual income (€15,000-€22,499) (barbosa, 2014) . concerning their motivations, for 65% of the respondents the main reason for their trip to porto was a vacation, 18% visited the city to visit friends or relatives and 5% visited to attend cultural events or attractions. regarding attending cultural or creative activities, 47% of respondents in this research responded positively (e.g., participating in serralves em festa, nos primavera sound, são joão, queima das fitas, axa street art, verão na casa da música) while 53% did not attend any cultural or creative activity (barbosa, 2014) . another study developed by melo, correia, cardoso, and marques (2019) analysed the perspectives of visitors and suppliers concerning the experiences of creative tourism in guimarães, portugal (a unesco world heritage destination) and identified that the majority of the respondents had not heard about creative tourism before (82% in total; n = 115). the authors reported that 50.7% were male and between 38 and 49 years old, and highlighted that 80% of the tourists come from the following countries: 35.7% from brazil, 18.8% from spain and 12.5% from france. most respondents had higher education degrees and an income higher than 501 euros/month. although these portuguese studies are not precisely about the profile of the creative tourist, they are important contributions to the study of the profile and motivations of visitors and help us to understand the visitors' profile for portuguese destinations. as we have previously noted, unravelling the profile and motivations of the creative tourist is not an easy task. the case studies reviewed showed that research on the traits of those engaged in creative tourism profile is still a little explored path. one of the possible explanations for this omission is the complexity of creative tourism as a wide variety of activities can be consumed within the creative tourism label. one can ask the following questions: do the characteristics of this profile depend on the type of activity consumed? for example, does the tourist who participates in the estival festival (held in the centro region of continental portugal) have the same characteristics (or similar characteristics) and motivations of a tourist who participates in a pottery workshop (held in the alentejo region of continental portugal)? our research was carried out in 2017 and 2018 with the participation of four research groups located in four regions of continental portugal: norte, centro, alentejo, and algarve. overall, 814 questionnaires were completed by participants in the creative tourism activities implemented by the 40 pilot institutions that were selected to join the creatour project ( fig. 1) . a wide array of organizations located in small and medium-sized cities and rural territories had submitted project proposals to develop creative tourism offers, and the 40 organizations selected to be co-researchers in creatour were monitored within the project until the end of 2019. the research presented here segmented the tourists based on their motivation to participate in creative tourism activities and analysed the resulting profiles. the questionnaire was designed based on a literature review of creative tourism, tourist motivations and cultural tourist profiles. a total of 10 motivation items were considered in this study ( table 2 ). the questionnaire consisted of 31 closed questions oriented to the participant's profile, motivations, perceptions and evaluation of activities, as well as the impacts on the local economy (e.g., accommodation, meals and local commerce). in order to analyse this data, multivariate statistics was used to simplify the data, describing the information through a small number of dimensions of analysis (reis, 2001) . hair, anderson, and tatham (1998) state that there are traditionally three types of segmentation techniques, being mostly of an exploratory nature. some of these techniques are in a general area of multivariate data analysis traditionally known as data reduction or reduction of dimensionality. using spss statistics 22.0, the first procedure (the exploratory factor analysis) started with motivation variables (table 2) . after this procedure, a hierarchical cluster analysis, based on the tourists' motivation using ward's method and the squared euclidean distance, was carried out to identify homogenous groups of respondents. a three-cluster solution was identified based on the dendogram and the agglomeration schedule. from the analysis of the output, chi-square tests (for qualitative variables) and t-test (quantitative variables) were carried out to characterize the clusters and to identify statistically significant differences concerning the following topics: reasons to select the creative experience; socio-professional situation; marital situation; net monthly income of the household; educational qualification; age; local shop behaviour and general evaluation of the experience. a cross-tabulation analysis was also carried out to test the association between sociodemographic and travel behaviour dimensions (i.e., first time at destination, information source and travel companions) with the cluster membership of respondents (tables 5 and 6 ). these analyses are presented in the next section. p. remoaldo, et al. tourism management perspectives 36 (2020) 100746 4. results as a first stage of data analysis, an exploratory factor analysis was conducted to identify the dimensions of participants' motivation at creative tourism activities. both the kaiser-meyer-olkin (kmo) measure of sample adequacy (.801) and bartlett's test of sphericity (1202.103) results revealed that the data was properly fitted for principal component analysis and responses were factor analysed with varimax rotation. for factor extraction, the criteria of eigenvalues equal or above 1.0 was adopted, and factor loadings of at least .50 were accepted for item inclusion (hair, black, babin, anderson, & tatham, 2010) . none of the items presented low factor loadings (< .50), however "it is culturally motivating", "because of the location" and "i know the promoter of the activity" presented .536, .589 and .522, respectively evidencing a value only slightly higher than .50. results obtained a three-factor solution that accounted for 55.5% of the total variance ( table 2 ). the following names were attributed to the items attributed to each factor: creative; partners and family togetherness; and local community interaction. all factors had a sufficient reliability relying on cronbach's alpha coefficients ranging from .705 and .585. although two of the factors present a .60 value, according to hair et al. (2010, p. 124 ) the generally agreed upon lower limit for cronbach's alpha is .70, although it may decrease to .60 in exploratory research. a cluster analysis was applied to identify a collection of individuals, based on the detailed information obtained, in relatively homogeneous groups. finally, application of hierarchical clusters allowed patterns to be detected in individuals through categorical and continuous variables. in particular, the hierarchical clustering technique is a process that allows for the organization of data into nested groups (dash, liu, scheuermann, & tan, 2003) . in this way, categorical variables were added (reasons to select a creative tourism activity; socio-professional situation; marital status; educational qualifications; shop behaviour at local shops; general evaluation of the experience) as well as quantitative variables (counts) organized by groups (age and net monthly income of the household) in order to ascertain how many natural groups could be observed. a total of three groups were identified, with a total of 221 (33.6%), 211 (32.1%) and 225 (34.2%) obtained for groups 1, 2 and 3 respectively (table 3) . to identify statistically significant differences between the three-cluster solution, a kruskal-walis test was p. remoaldo, et al. tourism management perspectives 36 (2020) 100746 applied and it shows that each group was different from each other. considering results from a one-sample t-test with a test value of 4 (table 4) , respondents evidenced a positive motivation towards creative tourism activities, except "to accompany someone" with a mean score of 3.19 (t-value = −13.589, p-value < 0.000) and "i know the promoter of the activity" with a mean score of 2.63 (tvalue = −20.156, p-value < 0.000). table 4 compares the motivations of each cluster and tables 5 and 6 show the profile of each group with respect to select demographic and travel behaviour characteristics. the clusters are described below according to their main characteristics. cluster 1 (novelty-seekers): this cluster includes 33.6% of the participants, with high motivations in the factor dimensions of creative (higher mean score-"it is original") and local community seekers (higher mean score -"it is culturally motivating"). in terms of sociodemographic characteristics, most are from the 18-35 years old group (37.4%) and the 36-53 years old group (34.2%) and, in terms of marital status, 61.8% are single and 29.7% are married, which means that they are mostly young individuals and middle-aged couples. they are well qualified, with bachelor degrees (39.5%) and postgraduate degrees (26.1%). considering the travel behaviour dimension, results reveal that individuals travelled with someone (73.2%): travelling with their child (27.6%), with an organized group (35.2%) or with friends (34.3%). within this cluster, 79.0% of the participants stated that the creative tourism activity was the primary reason for their visit. this group was named novelty-seekers because they tend to highly score on motivations which can be relevant to their engagement in actions of originality, fun and that stimulate creativity while, at the same time, they seek contact with other participants and with the local community. cluster 2 (knowledge and skills learners): this cluster includes 32.1% of the participants and it scored between the other two groups except in "to stimulate my creativity"; "it permitted interaction with other participants" and "i know the promoter of the activity". however, it is evident that with a higher score within the local community seekers factor (higher mean score -"it is culturally motivating") and in the creative factor (higher mean score -"it is original"), additionally and comparing with the group 1, this cluster presents a higher mean score within the socializers factor ("to accompany someone"). in terms of sociodemographic characteristics, most are aged between 18-35 years old (28.3%) and 36-53 years old (37.6%) and, in terms of marital status, 53.1% are single and 39.3% are married. they are well qualified, with bachelor degree (28.3%) and postgraduate degree (34.6%). considering the travel behaviour dimension, results reveal that individuals predominantly travelled with someone (90%): travelling with their spouse/partner (40.1%), with their children (40.0%) and with their family (42.6). within this cluster, 67.8% of participants indicated that this creative tourism activity was the primary reason of their visit to the locale. this group was named knowledge and skills learners because they tend to highly score motivations that imply their engagement in p. remoaldo, et al. tourism management perspectives 36 (2020) 100746 actions of originality, fun, and to participate in creativity-stimulating activities together with their travel companions, while at the same time they seem to seek contact with the local community. cluster 3 (leisure creative-seekers): this cluster includes 34.2% of participants and scored high in every motivation, with the two higher mean scores in the local community seekers dimension ("it permitted interaction with other participants" and "it is culturally motivating"). middle-aged participants prevail (39.5%), but the young group of participants (18-35 years of age) also comprise an important percentage (30.5%) and the majority of individuals are single (54.9%). in terms of education, they are also qualified with a university degree (38.1%), but a considerable part of this group has high school education (33.5%). considering their travel behaviour, 83.8% travel with someone, and among those who travel with companions, 33.9% travel with spouse/partner and 36.5% with their family. the majority of participants indicated that this creative tourism activity was the primary reason for their visit (72.9%). this group was named leisure creative-seekers because they tend to highly score all motivations that imply their engagement in creative tourism activities is based on a perception of it as an educational and creative experience that can positively affect their emotions and stimulate them to activate their creative dimension during their travel experience. cluster 1 (novelty-seekers) and cluster 2 (knowledge and skills learners) consider "culturally motivating" as the most important motivator to select a creative tourism activity, whereas cluster 3 (leisure creative-seekers) consider "interaction with other participants" as the most important motivation. it is important to underline that "to accompany someone" is the least important motivation for cluster 1; however, this is one of the most important motivators for cluster 3. in the same vein, "i know the promoter of the activity" is the most important motivator for cluster 3, whereas for clusters 1 and 2 it is the least important motivator. these points highlight the main differences between each cluster concerning their motivations to select creative tourism experiences. overall, the sample is constituted by a considerable number of females (63.8%). in terms of marital status, 53.6% are single and 36.7% are married, and 63% are highly qualified with university degree (table 5) . within a professionally active age (78.9% aged between 18-65 years old), 23.4% are "specialists in intellectual and academic source: authors' elaboration. p. remoaldo, et al. tourism management perspectives 36 (2020) 100746 activities", 13.8% are "technicians and associate professionals" and 9.4% are "managers/professionals". the household's net monthly income from employment is between 1001€ and 2500€ (43%). in terms of nationalities, 75.4% are from portugal and 24.6% are from foreign countries. in terms of travel companions, 25.2% travel with their partner, 24.3% travel with friends, 19.0% travel in an organized group, 16.1% travel as a family and 15.4% travel with their child(ren). for a considerable number of people this was the first time participating in a creative tourism experience (68.1%) and the creative tourism activity was the primary reason for visiting the destination (74.2%). the majority of participants learned about this experience through family and friends (33.2%), social networks (25.6%) and the website of the activity organizer (11.5%) (see table 6 ). this article aimed to characterize the profile of creative tourists and their motivations in portugal. the research sought to answer the following questions: who are the participants in creative tourism activities? what are the main motivations to attend a creative tourism activity? can motivation be used to segment creative tourism participants? the main focus of the study is to identify and articulate the profile of creative tourism participants based on their sociodemographic characteristics, travel behaviour and motivations. since the present study has an exploratory nature, in order to characterize the profile of these tourists, different groups of participants in creative tourism experiences were identified. although a few previous studies (in various countries) identified several features of creative tourists, none of them clearly identified groups of individuals based on sociodemographic characteristics, travel behaviour and motivations in a context of small and medium-sized cities and rural areas. also, they used a much smaller sample size than the one used in the current study. to validate the motivations-item scale and consequently to support the designation of the identified dimension factors and clusters, findings from a variety of studies were incorporated into the analysis, such as ryan and glendon (1998) about the application of the leisure motivation scale to tourism; tan et al.'s (2014) taxonomy of creative tourists; and the work on profiling creative tourists conducted by raymond (2003) , among others. the creative dimension factor was identified primarily based on the stated motivations about originality, fun and stimulation of the participant's creativity. this dimension partially corroborates with the characteristics of this form of tourism described by brouder (2012) , rudan (2012) and hung et al. (2016) . the partners and family togetherness dimension emerged as the factor linking participation in creative tourism activities with the need to share it with several types of travel companions, such as one's family. this dimension is grounded on ryan and glendon's (1998) research concerning the importance of the source: authors' elaboration. p. remoaldo, et al. tourism management perspectives 36 (2020) 100746 item "be with others" to clarify the holiday motivation scale. furthermore, tan et al.'s (2014) taxonomy of creative tourists exerts the importance of items about "family togetherness" or to "take the opportunity to participate in an activity to be together with my family or friends". the local community interaction factor is the most relevant motivation that contains items similar to the studies by tan et al. (2014) , raymond (2003) and ryan and glendon (1998) . motivation items such as interaction with other participants; culturally motivated; and meet and interact with the local community are highly rated, corroborating these previous studies. following the identification of factor dimensions, a segmentation procedure was adopted based on sociodemographic, travel behaviour and motivation-based criteria. in the cluster analysis, a data-driven segmentation approach (dolnicar & grün, 2008; mazanec, 2000) was conducted that relied on analysing the data collected to determine market segment profiles. the results reveal that participants of creative tourism activities consist of three distinct clusters. a first cluster, called novelty-seekers, comprises a high involvement in creative motivation factors, such as looking for fun, originality and creativity, but also with an appreciative degree of socialization with other participants and with the local community in order to learn about its culture. this cluster meets a few of the characteristics of the typologies of crompton (1979) in what concerns motives to travel (push factors), particularly in the dimension of travel as a facilitator of social interaction. novelty-seekers are also framed into crompton (1979) , mainly in the dimension of travel to find novelty: "synonyms included curiosity, adventure, new and different. novel meant new experience but it did not necessarily mean entirely new knowledge" (p. 419). this segment corroborates also with tan et al.'s (2014) typology of the novelty-seekers perspective, mainly due to the attraction of new activities and searching for new "creative" activities. the second cluster, named knowledge and skills learners, comprises a high mean value inside three factor dimensions/ items: originality and culturally motivating, to accompany someone, and to meet and interact with the local community. this group is also characterized as the most academically qualified and more likely to travel with their family. previous studies about the profile of the creative tourist found evidence of these types of characteristics for tourists who stated they are motivated to participate in a creative tourism activity to gain knowledge (tan et al., 2014) . in the same vein, studies by richards (2014a) and smith (2016) also emphasized educational interaction with the community, similar to this knowledge and skills learners segment of creative tourists. the third segment, leisure creative-seekers, comprises participants who evidence a high mean value in all of the motivation items. however, if we consider the top three items, two of them are concentrated in local community interactors (interaction with other participants and culturally motivating) and the third is found in knowledge and skills learners (to accompany someone). it is also important to underline that this cluster classified all the motivation items highly (4 or above, in a 1 to 5 likert scale), which means that this cluster meets certain characteristics of the relax and leisure type of creative tourists found in the study by tan et al. (2014) but also follows mckercher, ho, du cros, and chow's (2002) cultural tourist typology, namely, the type of purposeful cultural tourist. the results also demonstrated an upgrade in several characteristics concerning the type of cultural tourists articulated by mckercher et al. (2002) . for instance, the leisure creative-seekers cluster highly ranked interaction with other participants and is motivated to participate in creative tourism activities to accompany someone, because it is original, because they know the promoter of the activity, and because it is suitable for the whole family. following from this, creative tourism participants considered to be leisure creative-seekers demonstrate a need to socialize and share the co-creation process with others during their experience. these results align with the conclusions of several studies concerning the tourist's involvement in the local culture through their participation in activities related with artefacts or other local products (e.g., anderson, 2009; cabeça, gonçalves, marques, & tavares, 2020; raymond, 2007; tan et al., 2013, among others) . creative tourism participants place great importance on co-creation in creative tourism activities, which involves processes involving tourists and residents as full participants and not passive subjects (binkhorst, 2008) . experiencing and interacting are key, with co-creation perceived as "a prerequisite for the definition of what a creative experience means and what it presupposes" (cabeça et al., 2020, p. 12) . finally, a brief remark on the sociodemographic and travel behaviour characteristics of the three clusters. as presented in tables 2 and 3 , only variables that present as statistically significant should be considered as features for each cluster. in all the clusters, the age dimension shows that more than 60% of creative tourism participants are between 18 and 53 years of age, a result that is concordant with results of studies conducted by raymond (2003) , campbell (2010) and chang et al. (2014) . in the same vein, education and marital status generally show similar results in all clusters. for instance, a considerable part of the three segments of creative tourists in this study are single and well educated, similar to the results gathered by tan et al. (2014) . in terms of their travel behaviour, in all clusters the tourists indicated that their primary reason to travel to that location was to participate in the creative tourism experience and almost all travel was accompanied. taking all this into account, creative tourism can be a strategic development priority for tourism and culture in portugal, especially in small cities and rural areas, and can be part of a tourism innovation model focused on local resources. there is a growing demand for local tourism products with higher added value, and interaction between participants and local residents as well as co-creation processes are key when choosing a destination. having these characteristics associated with it, creative tourism meets the diverse needs and motivations of contemporary travellers and can constitute a diverse offer, combining with various types of existing tourism (e.g., cultural tourism, nature tourism, gastronomic tourism). in order to improve the desired relations among the participants in creative experiences and with the local community, it is important to empower practitioners from the tourism and cultural/ creative sectors to collaborate as tourism experience providers as well as other community members who can act as local community facilitators. beyond the socio-demographic characteristics of these creative tourism participants, this study also analyzes motivational characteristics, finding them to be similar to those identified in other studies internationally. this study's results reinforce the importance of the interaction component, which is one of the main characteristics of creative tourism, that is, the socialization and interaction of the visitors with the local community and also among the participants themselves. understanding creative tourists' motivations enables promoters to develop activities that are more attractive and appropriate to meet travellers' expectations, thus constituting a more sustainable offer. in addition, the study introduces a strong element of desire for involvement in activities with one's travelling companions, that is, the search for creative tourism activities that promote activities for a family and/or among friends. the study results indicate a shift in creative tourism towards a more interactional dynamic in which the bonds between participants and cocreation processes are highly valued. placing emphasis on the interactional dimensions of creative tourism can amplify its meaning and prompt further research attention to the ways in which it materializes. this study aimed to better know the profile of the creative tourist because it has been widely recognized that there is no clear definition of the creative tourist at an international or national level (duxbury & richards, 2019b) . indeed, this is the first major study at the national or international level to investigate and segment the creative tourism market for activities in small and medium-sized cities and rural areas. beyond the socio-demographic characteristics of these creative p. remoaldo, et al. tourism management perspectives 36 (2020) 100746 tourism participants, this study also analyzes their motivational characteristics, finding them to be similar to those identified in other international studies. this study's results reinforced the importance of the interaction component, which is one of the main characteristics of creative tourism, that is, the socialization and interaction of visitors with the local community and also among the creative tourism participants themselves. in addition, the study introduces a strong element of involvement in activities among travelling companions, that is, a search for creative tourism activities that promote activities for a family and/or among friends. as with any other research, this study has some limitations that should be highlighted. a primary dimension of these limitations is related to the data and sample design. the sample has different weight sizes among the portuguese regions, focusing on the selected rural areas and small and medium-sized towns that are home to the creative tourism initiatives that were the focus of this research. the questionnaires were applied locally by the participating pilot organizations and centrally analyzed by the research teams. consequently, the adopted sample method was by convenience. a second dimension is concerned with the motivations listed in the questionnaire relating to the creative tourism activities. this research has an exploratory nature, and future studies should further explore and possibly identify other type of motivations based on qualitative research (such as adopting these items and other items tested in previous studies to conduct semistructured interviews with creative tourists) in order to test and validate a scale of creative tourism motivations. looking forward, future methodological research following from this study includes the need to validate a scale of motivations for participants in creative tourism activities. within the creatour project, it would be valuable to conduct further analysis of the creative tourism participant profiles and motivations data in relation to the different types of creative tourism activities in which the tourists were engaged. further additional quantitative and qualitative research involving international travellers would extend this research and also help to inform practitioners on how they can best prepare for and attract tourists to creative tourism activities based in small cities and rural areas. as the current study represents a snapshot of an emerging array of creative tourism initiatives in small cities and rural areas throughout continental portugal, it would also be valuable to extend this research over time so that a longitudinal perspective can be obtained as the initiatives develop, mature and evolve. as well, the extension of this research in a comparative framework involving creative tourism initiatives in other countries would be important to develop a more comparable data framework internationally for creative tourism. in the context of the covid-19 pandemic, creative tourism is well positioned to significantly contribute to post-pandemic tourism. creative tourism is, by nature, designed for small groups (e.g., families and social bubbles). it aligns well with the growing focus on domestic tourism and longer stays in one place -exploring one's city, region, or country with new perspectives on its diversities; pursuing personal interests and curiosities; and developing new skills. the nature of activities within creative tourism is diverse, and its transversality is also a key strength, complementing and extending the offers of other types of tourism (gonçalves, borges, duxbury, carvalho, & costa, 2020) . in smaller and rural communities, creative tourism can assist in providing activities for people staying for longer periods, and can be interconnected with nature and outdoors and restorative activities. the pandemic has also alerted us to the high degree of precariousness for workers in the tourism sector, the impacts of tourism on local communities and the importance of communities in the scope of tourism. altogether, there is a need to rethink tourism's traditional models. going forward, tourism-resident communities have to take a more active role in establishing tourism agendas and planning for their re-booted local development trajectories. this highlights another future research line -generative relations between tourism and local communities. from now on, it is clear that tourism will necessarily have to take the health and well-being of communities into account when determining tourism approaches and agendas, and to (re)consider how tourism brings benefits to communities. from this vantage point, creative tourism is also well positioned. as a widely applicable approach to place-sensitive tourism development (bakas, duxbury, silva, & vinagre de castro, 2020) , creative tourism can encourage and enable the diversification and differentiation of tourism offers in smaller places. the authors contributed equally to this work. influence of experiences on memories satisfaction and behavioral intentions: a study of creative tourism yester-morrow: using a region's heritage and culture for its economic future multivariate data analysis a creativity-led rural renaissance? amenity-led migration, the creative turn and the uneven development of rural australia connecting to place through creative tourism creative tourism dynamics: connecting travellers, communities, cultures, and places a influência das indústrias criativas sobre o turismo na cidade do porto. portugal: escola superior do porto creativity in tourism experiences: the case of sitges turismo de cocreación: valor añadido en escenarios turísticos agenda for co-creation tourism experience research creative tourism in bali's rural communities-examination of the current offer and advice 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crowds to creative tourism: a search for creative tourism in small and medium sized cities challenging "factor-cluster segmentation catalyzing creative tourism in small cities and rural areas in portugal: the creatour approach towards a research agenda for creative tourism: developments, diversity, and dynamics towards a research agenda for creative tourism: a synthesis of suggested future research trajectories open tourism: open innovation, crowdsourcing and co-creation challenging the tourism industry the rise of the creative class: and how it's transforming work, leisure, community and everyday life policy recommendations on creative tourism development in small cities and rural areas. coimbra: creatour project creative tourism in saint petersburg: the state of the art visita guiada à fábrica de antiguidades: sociologia, turismo e autenticidade. anais brasileiros de estudos turísticos multivariate data analysis detecting common method bias in predicting creative tourists' behavioural intention with an illustration of theory of planned behaviour creative experiences, memorability and revisit intention in creative tourism benefits of creative tourism-the tourist perspective facing the challenge? creative tourism in croatia greece as cultural destination. 16th international colloquium on regional sciences cultural tourism and world heritage creative tourism and cultural development: some trends and observations. paper presented at the cultural tourism conference, bonavista institute for cultural tourism marketing 3.0: produits, clients, facteurshumains creative tourism: a global conversation market segmentation activities-based segmentation of the cultural tourism market creative tourism experiences in guimarães: a twofold analysis of visitors' and suppliers' perspectives changes in consumer behaviour -the challenges for providers of tourist services in the destination mindful visitors: heritage and tourism criatividade: a construção de novos cenários para o turismo em ponte de lima defining cultural tourism. international conference on civil, architecture and sustainable development (casd-2016) creative tourism magazine tourist experiences and academic junctures creative tourism business model and its application in bulgaria prosuming creative urban areas: evidence from east london tourism research-critiques and challenges the importance of lifestyle entrepreneurship: a conceptual study of the tourism industry welcome to the experience economy the future of competition: co-creating unique value with customers creative tourism supply: creating culturally empathetic destinations visitor experiences in cultural spaces the cultural creatives: how 50 million people are changing the world cultural renewal + tourism: case study-creative tourism creative tourism new zealand: the practical challenges of developing creative tourism lisbon: sílabo o legado de guimarães capital europeia da cultura de 2012. a leitura dos residentes e dos visitantes good and not-so-good practices in creative tourism networks and platforms: an international review management practices in creative tourism: narratives by managers from international institutions to a more sustainable form of tourism changes in the guimarães visitors' profile and the city attributes perceptions in the post hosting of the 2012 european capital of culture geo-crowdsourcing contributions for cultural mapping cultural tourism research methods. amsterdam: great britain by tj international creative tourism: a global conversation tourism development trajectories: from culture to creativity? tourism & management studies creativity and tourism: the state of the art creativity and tourism in the city development of experiences in creative tourism creative tourism developing creativity in tourist experiences: a solution to the serial reproduction of culture? tourism management tourism, creativity and development razvojne perspective kretivnoga turizma hrvatske application of leisure motivation scale to tourism estudo macroeconómico desenvolvimento de um cluster de indústrias criativas na região do cultural tourism and business opportunities for museums and heritage sites issues in cultural tourism studies issues in cultural tourism studies creativity, tourism and economic development in a rural context: the case of prince edward county a model of creative experience in creative tourism a taxonomy of creative tourists in creative tourism the third wave interaction between cultural/creative tourism and tourism/cultural heritage industries mass tourism, culture and the historic city: theoretical perspectives urban tourism and the politic of creative class: a study of the chefs in macao this research was conducted within the project creatour, "creative tourism destination development in small cities and rural areas" (project 16437), which is funded by the portuguese foundation for science and technology (fct/mec) through national funds and cofunded by feder through the joint activities programme of compete 2020 and the regional operational programmes of lisbon and algarve. key: cord-304375-l5gvpat3 authors: singh, kamaljit; kaur, hardeep; chibale, kelly; balzarini, jan; little, susan; bharatam, prasad v. title: 2-aminopyrimidine based 4-aminoquinoline anti-plasmodial agents. synthesis, biological activity, structure–activity relationship and mode of action studies date: 2012-03-13 journal: eur j med chem doi: 10.1016/j.ejmech.2012.03.007 sha: doc_id: 304375 cord_uid: l5gvpat3 2-aminopyrimidine based 4-aminoquinolines were synthesized using an efficacious protocol. some of the compounds showed in vitro anti-plasmodial activity against drug-sensitive cq(s) (3d7) and drug-resistant cq(r) (k1) strains of plasmodium falciparum in the nm range. in particular, 5-isopropyloxycarbonyl-6-methyl-4-(2-nitrophenyl)-2-[(7-chloroquinolin-4-ylamino)butylamino] pyrimidine depicted the lowest ic(50) (3.6 nm) value (56-fold less than cq) against cq(r) strain. structure–activity profile and binding with heme, μ-oxo-heme have been studied. binding assays with dna revealed better binding with target parasite type at rich puc18 dna. most compounds were somewhat cytotoxic, but especially cytostatic. molecular docking analysis with pf dhfr allowed identification of stabilizing interactions. faced with the challenges of drug resistance, poor health systems, lack of affordable, safe and convenient treatment options, efficient treatment of malaria, one of the most devastating parasitic diseases, represents an unmet medical need. malaria is a major public health concern in more than 90 countries inhabited by more than 2.4 billion people e 40% of the world's population and is responsible for almost 1 million deaths every year [1] . the majority of malaria victims in developing countries are pregnant women or children under the age of five, possessing little or no immunological protection. the disease is estimated to result in w250 million new annual infections worldwide. though the majority of the cases and approximately 90% of the malaria deaths are found in sub-saharan africa, the disease is now increasing in asia and latin america. malaria is caused by protozoan parasites of the genus plasmodium that infects and destroys red blood cells eventually leading to death, if untreated. the persistent threat of emergence of multidrug resistant plasmodium falciparum, universal chloroquine resistance [2, 3] , suspected resistance to artemisinins [4, 5] , and lack of effective, appropriate and affordable treatment options have given a new impetus to the research leading to broadening of the range of therapeutic targets. thus, creating a new armamentarium of drugs with promising antimalarial activity coupled with understanding of their mode of action may lead to the development of a new generation of treatments both for malaria control and eradication. the common feature of the drugs based on quinine 1 is the presence of a quinoline unit, usually a 7-chloroquinoline (chloroquine 2 and amodiaquine 3, chart 1), and are known to cause parasite death by blocking the polymerization of the toxic heme, into an insoluble and non-toxic pigment, hemozoin, resulting in cell lysis and parasite cell autodigestion [6e8] . the mode of action of 2,4-diaminopyrimidine based drugs, typified by pyrimethamine 4 [9] and the lead compound wr99210 5 [10] is through the inhibition of p. falciparum dihydrofolate reductase (pf dhfr) enzyme, required for the biosynthesis of tetrahydrofolate involved in the biotransformation of thymidylate synthase-catalysed deoxyuridylate to deoxythymidylate (dump / dtmp), through a methyl group transfer reaction during dna biosynthesis [11e16] . in addition, a number of polyamines inhibit ornithine decarboxylase activity in p. falciparum through binding with plasmodial dna [17, 18] . recently, investigations in hybrid antimalarial agents, combining 4-aminoquinoline with other pharmacophore having antimalarial activity have been found to be less prone to resistance to parasite and has thus offered an effective means to overcome the problem of drug resistance. we envisaged that linking 7-chloro-4-aminoquinoline unit, critical for antimalarial activity through a diversely functionalized lateral side chain with other antimalarial moiety such as aminopyrimidine, might furnish conjugate hybrids [19] capable of showing useful antimalarial activity. in this communication, we report synthesis of a set of compounds that possess basic, hydrophobic as well as hydrogen bonding substituents, required for targeting either or both heme as well as dna, thus providing new antimalarial agents active against chloroquine resistant strains of p. falciparum. we have evaluated their anti-plasmodial activity, cytotoxicity and cytostatic activity, binding studies with dna and heme (monomeric as well as m-oxo dimeric) using uvevisible, fluorescence spectrophotometry as well as nmr analysis. compounds 10aes were synthesized as outlined in scheme 1, via a common intermediate 8. 3,4-dihydropyrimidin-2(1h)-ones 6 were prepared through hcl-catalyzed biginelli condensation of appropriate aldehyde (r 3 cho), alkylacetoacetate (r 2 ch 2 coor 1 ) and urea [20] . dehydrogenation of 6 using pyridinium chlorochromate in dcm furnished pyrimidinones 7 [21] . refluxing 7 with pocl 3 yielded 8 which upon nucleophilic substitution reaction with appropriate 4-amino-7-chloroquinoline 9 gave 10aes in a synthetically useful manner [22] . structures of 6e10 were established on the basis of spectral ( 1 h nmr, 13 c nmr, ms, ft ir) as well as microanalytical analysis. the yields of the 2-aminopyrimidines 10 are reported in table 1 . antiplasmodial activity of pyrimidines linked to cq as in 10 has not been described in literature albeit the related dihydropyrimidin-2(1h)-ones (dhpms) have previously been reported [23] . using the synthetic protocol shown in scheme 1 allowed considerable diversification around the pyrimidine core for conducting sar analysis. the in vitro anti-plasmodial activities of 10aes were determined in primary and secondary screening against cq s and cq r strains of p. falciparum. the half maximal inhibitory concentration (ic 50 ) of 10aes are summarised in table 2 (fig. 1) . evidently, the compounds have anti-plasmodial activity in the nm range and against the cq r strain of p. falciparum, in some cases activity was found to be even superior to cq. systematic variation of the length as well as nature of the spacer connecting the pharmacophores discerned useful trends in the anti-plasmodial activity of these analogs. comparing 10aeg, bearing linear alkyl spacers, revealed an increase in the anti-plasmodial activity with increase in length of the spacer up to 4 methylene groups (10ae10c, table 2 ). further lengthening of the spacer chain length resulted in significant reduction in activity against both the cq r as well as the cq s strains. replacing the c-4 phenyl group in 10c by a methyl group to create 10m resulted in nearly 5 times increase in anti-plasmodial activity (ic 50 42.1 nm) against the cq s strain. in fact, compound 10m was found (table 2) to be the most active compound of the series, against the cq s strain. however, 10m exhibited a high resistance factor (w39) within the series but is 3.5 times less active than cq, against the cq r strain. replacement of the ethyl ester with isopropyl ester (10nes), in general showed a decrease in in vitro antiplasmodial activity against the cq s strain, although a reverse trend was observed for the cq r strain, especially in case of 10s, 10p and 10r, where replacing c-4 phenyl by p-nitrophenyl (10s) or onitrophenyl (10p and 10r) groups, in addition to incorporating an isopropyl ester, resulted in an increase of activity in that order, rendering 10r as the most potent (ic 50 3.6 nm, 56 times more potent than cq, table 2 ) of all these compounds. these results are in accordance with the trend observed in case of n, n-bis(7chloroquinolin-4-yl)alkane diamines, wherein the alkyl spacer consisting of four carbon atoms showed optimum potency [24] . the better anti-plasmodial activity of phenyl-substituted pyrimidine compounds against the cq r strain may be attributed to optimal fitting of these compounds in the active site of pfdhfr leading to a favorable conformation for p-p interaction with the heme functionality. moreover, the introduction of a nitro substituent on the phenyl ring at the c-4 position of the pyrimidine core results in a significant increase in anti-plasmodial activity as well as resistance against the cq r strain, although it has little effect on activities against the cq s strain (table 2) . also, the corresponding o-, m-or p-nitro derivatives showed considerable variation in antiplasmodial activity ( table 2 ). comparison of compounds (10n, 10q, 10s) having an identical spacer reveals that the p-no 2 substituted 10s (ic 50 175.8 nm) is more active than the o-/m-no 2 substituted compounds 10q and 10n, respectively, but are less active than the unsubstituted ethyl ester analog 10b. further, comparison with the butyl spacer analog suggests that o-no 2 phenyl derivative 10r is more potent than the m-no 2 counterpart 10o, as well as its ethyl ester analog 10c, against the cq r strain. compound 10r was found to be the most active compound in this series against the cq r strain with anti-plasmodial activity (ic 50 3.6 nm), 56 times more than cq (ic 50 201.8 nm) and comparable to artesunate (ic 50 2.8 nm) ( table 2) . comparing 10j (ic 50 28096.7 nm) possessing the branched chain spacers with identical carbon linear spacer analog 10b (ic 50 52.2 nm, table 2 ) led to significant decrease in activity. thus, compound 10j displayed a 540-fold decrease in activity compared to 10b, against the cq r strain. likewise, 10k possessing a branched c-6 spacer depicted a decrease (ic 50 860.4 nm, table 2 ) in activity against linear alkyl (c-7) spacer analog 10d (table 2 ), but higher than 10j. comparing 10j and 10k, with branched chain spacers, not only was the anti-plasmodial activity of the latter against both cq s and cq r strains increased, but it also showed an increased resistance factor ( table 2 ). the replacement of linear chain spacers with o-and p-linked aryl groups (10h and 10i, respectively), led to much higher ic 50 (table 2 ) values compared to former against cq r strain, while only moderate activity was observed in case of 10i against cq s strain. these findings could be attributed to the increased steric bulk (10h more than 10i) affecting the interaction of the iron center of heme with the compounds. this has been corroborated by performing the titration of monomeric heme with both 10h and 10i. 1:4 molar ratio of heme and 10i. hence, the flexibility, chain length and steric constraints of the spacer linking quinoline moiety and pyrimidine unit seem to play a role in anti-plasmodial activity of these derivatives. although the in vitro activity of 10aec, 10p and especially 10r (ic 50 3.6 nm) was superior to cq (ic 50 201.8 nm) against the cq r strain, these compounds suffer from high clogp values (table 2) , which are suggestive of the fact that these possess limited aqueous solubility, which in fact is not a serious limitation in view of recent advancements in formulation methods. antiviral activity of the compounds 10aec, 10len, 10p and 10r, which were active against the cq r strain was also evaluated against (i) parainfluenza-3 virus, reovirus-1, sindbis virus, coxsackie virus b4, punta toro virus in vero cell cultures, (ii) herpes simplex virus à1 (hsv-1; kos), herpes simplex virus-2 (hsv-2; g), vaccinia virus, vesicular stomatitis virus, herpes simplex virus-1 (tk à kos acv r ), cytomegalovirus, varicella-zoster virus in hel cell cultures, (iii) vesicular stomatitis virus, coxsackie virus b4, respiratory syncytial virus in hela cell cultures, (iv) influenza a virus (h1n1 and h3n2) and influenza b virus in mdck cell cultures and (v) feline corona virus (fipv) and feline herpes virus activity in crfk cell cultures (si tables s1es7). unfortunately, no significant antiviral activity was noted at subtoxic concentrations. most compounds were somewhat cytotoxic to the different cell lines (confluent nonproliferating cultures), but especially cytostatic against proliferating (vero/mdck/crfk) cell cultures. chloroquine has been shown to inhibit hiv through blockade of viral entry via inhibition of endosomal acidification [25, 26] . compounds 10aec, len, p, r were also tested against hiv-1 as well as hiv-2 (si table s8 ) in human t-lymphocyte (cem) cell cultures. however, none of the compounds were active at subtoxic concentrations. generally, the compounds exhibit a relatively high cytostatic activity but displayed a fairly safe selectivity index (except 10m and 10n) ( table 2 ) in the range of 10.04e638 against mdck cell cultures. the most active compound 10r with an ic 50 value of 3.6 nm against cq r strain exhibited a highest selectivity index (si ¼ 638), 10l with an ic 50 value of 160.8 nm against cq r strain exhibited a high selectivity index (361.9). however, 10m having an ic 50 value of 1659.8 nm was most toxic with a selectivity index of 0.48. the plausible mechanism of in vitro anti-plasmodial action against cq r strain has been investigated for 10c and 10r, found to be most potent of the series of the compounds reported herein (ic 50 26.1 nm, 10c and ic 50 3.6 nm, 10r). these compounds are also expected to bind to heme [fe(iii)ppix] (hydroxo or aqua complex of ferriprotoporphyrin ix) in solution and inhibit aggregation to bhematin, in much the same way as cq itself. the higher clogp values ( table 2 ) of these compounds being higher than cq necessitated the use of aqueous dmso as solvent. also in 40% aqueous dmso solution, heme is expected to be monomeric, while in purely aqueous solutions, it exists in aggregated form. further, it is known that cq binds to heme dimer (m-oxo heme) in vitro and can also inhibit the formation of b-hematin, the in vitro analog of hemozoin [27e30] . therefore the binding assay was also extended to heme dimer also. the interaction of 10c and 10r with monomeric heme was followed by spectrophotometric titration at ph 7.4 and 5.6 (the approximate ph of food vacuole), as described previously [31] . the spectral changes (fig. 3a , si figure s1 ), upon addition of increasing amounts (0e70 mm) of 10r to a solution of heme (2.4 mm, hepes buffer ph 7.4) showed substantial decrease of intensity of the fe(iii) ppix soret band at 402 nm with no shift in the absorption maximum. the longer wavelength q-bands (494 and 537 nm) of the metalloporphyrin also decrease in intensity (si figure s2 ) [32] . a sharp isosbestic point was observed at 416 nm (ph 7.4) and at 412 nm (ph 5.6). the association constants for the complexes formed between monomeric fe (iii) ppix and 10r at ph 7.5 and 5.6 were calculated from titration data and are presented in table 3 . the association constant of 10c ( fig. 2a ) is found to be greater (log k 6.018) than 10r (log k 5.078). decrease in the apparent ph from 7.5 to 5.6 caused a fairly modest decrease in log k values of both 10c and 10r, indicating a strong binding to heme even at acidic ph. the binding of 10c and 10r was also assessed from their 1 h nmr titration with heme. thus, while 1 h nmr spectrum of 10r depicted considerable broadening of signals under the conditions (40% dmso-d 6 in d 2 o) of recording and was inconclusive, the variation in the chemical shift in signals corresponding to quinoline unit of 10c were clear (fig. 4 ), upon addition of heme. a 1:1 stoichiometry of the most stable complexes of both 10r and 10c with monomeric heme at ph 7.5 and 5.6 was deduced from the job's plot (si figure s3 ) [33, 34] . the absorbance at 402 nm got to maximum when mole fraction of 10r approached 0.5. this is in good agreement with the association constant (log k) values corresponding to the most stable 1:1:heme: 10r species present in solution, obtained by fitting the titration data in hyp spec -a nonlinear least square fitting programme. the measured log k values for both 10r and 10c (table 3) are also in good agreement with the values reported in literature for cq [32, 35, 36] . thus, the formation of synthetic fe(iii)ppix-10r complex, as demonstrated above is suggestive of inhibition of formation of b-hematin and presumably results in anti-plasmodial activity of these compounds in a fashion similar to that of cq. titration of constant concentrations of heme in aqueous naoh solution at the physiological ph 5.8 of plasmodial food vacuole [35] allowed understanding of the interaction of 10r and 10c with m-oxo heme. with addition of increasing amount of 10r to the solution of m-oxo heme, the absorbance of soret band (362 nm) decreased appreciably with significant red shift (362e405 nm, fig. 5a ). the association constants and stoichiometry for both 10r and 10c were determined from titration data and are presented in table 3 .the calculated association constant of 10r (log k 6.31) is greater than 10c (log k 6.09) and corresponds to 1:1 stoichiometry of the 10r:moxo heme complex. the comparison of association constants of 10r and 10c for monomeric and m-oxo heme reveals that both compounds bind strongly with m-oxo heme than monomeric heme and inhibit hemozoin formation by blocking the growing face of heme resulting in the observed anti-plasmodial activity. further, all the compounds were also screened for in vitro inhibition of bhematin formation using b-hematin inhibitory assay in order to further confirm their mechanism of action based on interference with the heme detoxification process [37, 38] . as shown in table 4 , there is a general correlation between antiplasmodial activity and inhibition of b-hematin formation, but the same generalisation does not hold for compound 10h, as, in spite of having 100% b-hematin inhibition (table 4) , it is one of the least active (ic 50 27320.9 nm, cq r and ic 50 14828.3 nm, cq s ) compounds of the series. however, it must be noted that anti-plasmodial activity not only depends upon the b-hematin inhibition but also on other factors such as degree of accumulation of drug in food vacuole. further, 10r showed dose dependent inhibition of bhematin formation (si figure s4 ) with an ic 50 value lower than those reported for cq and quinine, thus demonstrating a better ability to interact with fe(iii)ppix. overall, these compounds showed strong b-hematin inhibition which seems to be their preferred mode of action. dna binding has been considered previously as one of the possible mechanisms for anti-plasmodial activity and has been studied using spectrophotometry [39] , spectrofluorimetry [40] , dna melting [41] , viscometry [42] etc. recently, some 4aminoquinoline derivatives have been shown to interact with dna [37] presumably through ionic interactions between phosphate groups of dna and protonated amine sites resulting in stabilization of the helical configuration of dna against thermal denaturation [41, 43] . additionally, interactions between aromatic nucleuses of the drug with nucleotide bases might also contribute. binding of the plasmid dna with cq resulted in the elevation of the thermal melting temperature (t m ) of dna in addition to other effects [44e47]. we therefore investigated the binding of the most active of the series, 10c and 10r toward gc rich calf thymus dna (ct dna) as well as at rich puc18 dna through stepwise addition of small increments of dna to a solution the compounds at constant concentration as well as physiological ph (fig. 6a and b) . a progressive decrease in the characteristic quinoline ring absorptions at 320 and 340 nm attributable to the intercalation of the quinoline into dna was indicated. the isosbestic point at 350 nm indicated that the spectra of limiting systems (i.e. the spectra of free and completely bound drug) intersect and permitted the selection of a single wavelength for study of complex formation. the binding constant (log k: 10c 4.67 and 10r 3.39) was calculated from benesiehildebrand equation [48] and suggested strong interaction with ct dna. comparison of binding constants reveals that 10c binds 20 times more strongly with ct dna than 10r. altering the dna base composition to see its effect on the drug binding, we studied the interaction of 10r with gc rich ct dna by fluorometric titration. the emission band at 380 nm of 10r (fig. 7) , decreases in intensity with increasing concentration and eventually gets completely quenched. the binding constant (log k) calculated from the fluorescence data is 4.67. similar titration with at rich puc18 dna (log k ¼ 5.27) revealed higher affinity of 10r toward at rich puc18 dna unlike cq which is known to interact strongly with gc rich dna [46] . the strong interaction of the pyrimidine analog with at rich puc18 dna further suggests that these compounds might target parasite dna which has unusually a high at content [47] in addition to b-hematin inhibition. the binding of ligand to nucleic acid of dna (especially at rich dna) may possibly induce conformational changes depending upon the strength and mode of its interaction with nucleic acids and thus result in increase in the thermal denaturation temperature, as observed for cq. groove binding or electrostatic binding of the phosphate backbone of dna gives rise to small changes in thermal denaturation temperature compared to the intercalation pathway due to stabilization of watson crick base paired duplex [43, 48, 49, 50] . in the direction of evaluating strength of interaction of 10c and 10r with dna, the thermal behavior (si figure s5 ) of ct dna in the presence of drug was evaluated. the thermal denaturation temperature of ct dna (60 c), in the presence of 10c and 10r recorded an increase in dt m of 3 c and 2.5 c, respectively (si table s9 ), suggesting primary groove binding and/or partial intercalative nature of the interaction. many of the anti-plasmodial agents are known to interact with pf dhfr for their anti-plasmodial activity. especially table 2 in vitro anti-plasmodial activity, cytotoxicity, resistance factor and selectivity index of compounds 10a-s. ic 50 pyrimethamine (chart 1) is known to produce anti-plasmodial effect by binding to pf dhfr. the best known lead compound for this inhibitory activity is wr99210 (5) . it is worth comparing the performance of the compounds designed in this work with that of 5, using molecular docking methods. the crystal structure (pdb id: 1j3i, a structure of wild-type pf dhfr-ts complexed with wr99210, nadph, dump) was considered for molecular docking analysis. after appropriately preparing the protein structure of pf dhfr, docking of 5 and 10r were carried out. fig. 8 shows the top scoring binding pose of 10r in the active site of pf dhfr. the docking score for 10r is à24.5, which is much better than that of the lead compound 5 (à20.1). the origin of this improvement in docking score can be traced to the increased number of stabilizing interactions between 10r and pf dhfr, in comparison to the interactions between 5 and pf dhfr. 10r shows hydrogen bonding interaction with tyr170 and asn108 residues; these two interactions are common to 10r as well as to 5. in addition, 10r shows hydrogen bonding interaction with ser111 and leu46 residues. these additional stabilizing interactions ensure that 10r adopts a slightly different (and improved) pose in comparison to that of 5. these improved interactions can be considered as deterministic factors for the improved anti-plasmodial activity of 10r. an efficacious transformation for converting readily available 3, 4-dihydropyrimidinones (dhpms) to 2-aminopyrimidine based 4aminoquinolines is presented. the compounds showed anti-plasmodial activity identical to or superior to cq. structureeactivity relationship has been drawn leading to the identification of at least one compound 10r as lead compound in this series. the tested compounds did not depict any antiviral activity and were cytotoxic. binding interaction of representative potent compounds with heme and m-oxo heme using uvevisible and nmr experiments furnished log k values identical with cq binding and pointed to 1:1 stoichiometry of the most stable complexes in solution. dna (both gc and at rich) binding affinity using absorption and spectrofluorometric data indicated stronger interaction of 10c (20 fold) than 10r with ct dna, however, 10r showed higher affinity toward at rich puc18 dna suggesting targeting of parasite at rich dna by 10r in addition to b-hematin inhibition as possible mode of action of these compounds. dna melting experiments suggested primary groove binding and/or partial intercalative nature of interaction of 10c and 10r with ct dna. thus, the strong electrostatic interaction of newly designed pyrimidine analogs with at rich dna and blockage of heme polymerisation by complexation of drug with heme contribute to the observed antimalarial activity in nano molar range. further, molecular docking analysis of 10r in the active site of pf dhfr indicated superior binding compared to wr99210. thus, drug 10r acts on multiple targets (heme, enzyme involved in biosynthesis of dna (dhfr), parasite dna) which accounts for its high anti-plasmodial activity. all liquid reagents were dried/purified following recommended drying agents and/or distilled over 4 å molecular sieves. thf was dried (na-benzophenone ketyl) under nitrogen. 1 h nmr (300 mhz) and 13 c (75 mhz) nmr spectra were recorded in cdcl 3 on a multinuclear jeol ft-al-300 spectrometer with chemical shifts being reported in parts per million (d) relative to internal tetramethylsilane (tms, d 0.0, 1 h nmr) or chloroform (cdcl 3 , d 77.0, 13 c nmr). mass spectra were recorded from indian institute of integrative medicine (csir), jammu, under electron impact at 70 ev on a bruker daltonics esquire 3000 spectrometer. elemental analysis was performed on flash ea 112 (thermo electron corporation) analyzer at department of chemistry, guru nanak dev university, amritsar and the results are quoted in %. ir recorded on ftir shimadzu 8400 fourier-transform spectrophotometer in the range 400e4000 cm à1 using chloroform as medium. melting points were determined in open capillaries and are uncorrected. for monitoring the progress of a reaction and for comparison purpose, thin layer chromatography (tlc) was performed on precoated aluminum sheets merck (60f 254 , 0.2 mm) using an appropriate solvent system. the chromatograms were visualized under uv light. for column chromatography silica gel (60e120 mesh) was employed and eluents were ethyl acetate/hexane or ethyl acetate/ methanol mixtures. to the stirred solution of 8 (2 mmol) and potassium carbonate (5 mmol) in dry thf (30 ml), a solution of appropriate 4aminoquinoline 9 (1.0 mmol) in dry thf (50 ml) was added. the reaction mixture was stirred for 48 h at room temperature. the reaction mixture was filtered and thf was removed under vacuum. the residue was purified by column chromatography using meoh/ etoac as eluent to obtain corresponding 10, which was recrystallized from dcm/hexane. using this procedure the following compounds were isolated. to the stirred solution of appropriate 4-aminoquinoline 9 in dry thf (50 ml) mixture of 8 (in a 1:2 molar ratio) and potassium carbonate in dry acetonitrile was added. the reaction mixture was refluxed for 48 h and then filtered. acetonitrile was removed under vacuum and the residue was purified by column chromatography using etoac/hexane as eluent to give 10h and 10i which were recrystallized from dcm/hexane. yellow solid. rf: 0.9 (70% hexane/etoac two clones of p. falciparum are used: (a) 3d7 clone of nf54 which is known to be sensitive to all anti-plasmodials, (b) k1 strain originating from thailand that is resistant to chloroquine and pyrimethamine, but sensitive to mefloquine. the cultures were naturally asynchronous (65e75% ring stage) and were maintained in continuous log phase growth in rpmi1640 medium supplemented with 5% washed human aþ erythrocytes, 25 mm hepes, 32 nm nahco 3 , and albumaxii (lipid-rich bovine serum albumin) (gibco, grand island, ny) (cm). all cultures and assays are conducted at 37 c under an atmosphere of 5% co 2 and 5% o 2 , with a balance of n 2 . stock drug solutions were prepared in 100% dmso (dimethylsulfoxide) at 20 mg/ml. the compound was further diluted to the appropriate concentration using complete medium rpmi1640 supplemented with 15 nm cold hypoxanthine and albumaxii. assays were performed in sterile 96-well microtitre plates; each plate contained 100 ml of parasite culture (0.5% parasitemia, 2.5% hematocrit). each compound was tested in triplicate and parasite growth compared to control and blank (uninfected erythtocytes) wells. after 24 h of incubation at 37 c, 3.7 bq of [ 3 h] hypoxanthine is added to each well [51] . cultures were incubated for a further 24 h before they are harvested onto glass-fiber filter mats. the radioactivity was counted using a wallac microbeta 1450 scintillation counter. the results were recorded as counts per minute (cpm) per well at each drug concentration, control and blank wells. percentage inhibition was calculated from comparison to blank and control wells, and ic 50 values calculated using graph pad prism 4.0. the preliminary screen uses the 3d7 strain. the compounds were tested at 6 concentrations (30, 10, 3, 1, 0.3, and 0.1 mg/ml). if the compound did not affect parasite growth at 10 mg/ ml it was classified as inactive, between 10 and 1 mg/ml, the compound was designated as partially active, and if <1 mg/ml, the compound was classified as active and was further evaluated by three-fold serial dilutions in a repeat test. for secondary screening both the 3d7 clone and the k1 line were used. the compound was diluted three-fold over at 12 different concentrations with an appropriate starting concentration based on the preliminary screen. the ic 50 is determined by a sigmoidal dose response analysis using graph pad prism 4.0. for each assay, the ic 50 and ic 90 values for each parasite line were determined against the known anti-plasmodials chloroquine and artesunate. the antiviral assays [except anti-human immunodeficiency virus (hiv) assays] were based on inhibition of virus-induced cytopathicity in hel [herpes simplex virus type 1 (hsv-1), hsv-2 (g), vaccinia virus, and vesicular stomatitis virus], vero (parainfluenza-3, reovirus-1, coxsackie b4, and punta toro virus), hela (vesicular stomatitis virus, coxsackie virus b4, and respiratory syncytial virus) and mdck (influenza a (h1n1; h3n2) and b virus) cell cultures. confluent cell cultures in microtiter 96-well plates were inoculated with 100 cell culture inhibitory dose-50 (ccid 50 ) of virus (1 ccid 50 being the virus dose to infect 50% of the cell cultures) in the presence of varying concentrations of the test compounds. viral cytopathicity was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. the anti-hiv activity and antiproliferative activity were evaluated against hiv-1 strain iiib and hiv-2 strain rod in human t-lymphocyte cem cell cultures. briefly, virus stocks were titrated in cem cells and expressed as the 50% cell culture infective dose (ccid 50 ). cem cells were suspended in culture medium at w3 â 10 5 cells/ml and infected with hiv at w100 ccid 50 . immediately after viral exposure, 100 ml of the cell suspension was placed in each well of a flat-bottomed microtiter plate containing various concentrations of the test compounds. after a 4-day incubation period at 37 c, the giant cell formation was microscopically determined. compounds were tested in parallel for cytostatic activity in uninfected cem cells. binding stoichiometries of drug with monomeric & m-oxo dimeric heme were monitored by uvevisible spectrophotometry using job's method of continuous variation [33, 34] . the concentration of drug & heme in solution was kept constant and changes in absorbance at 402 nm (monomeric)/362 nm (dimeric) were monitored as a function of the mole fraction. 96 well plate containing mixture of 50 ml of 0.5 mg\ml of hemin chloride dissolved in dmso, 100 ml of 0.5 m sodium acetate buffer (ph 4.4) and 50 ml of different concentrations of drug solution or 50 ml of solvent (control), was incubated at 37 c for 24 h. the plate was centrifuged at 4500 rpm for 3 min, and supernatant was discarded. the remaining pellet was re-suspended with 200 ml of dmso to eliminate unreacted hemin [38, 39] . the plate was centrifuged again and supernatant similarly discarded. the precipitate was dissolved in 150 ml of 0.1 n naoh & absorbance was read at 405 nm. the percentage of inhibition of ferriprotoporphyrin ix biomineralisation was calculated using formula: inhibitionð%þ ¼ 100xf½ðabs of controlþ à ðabs of drugþ= ðabs of controlþg. ic 50 values were determined by using graph pad prism 4.0. 6.4. dna binding studies with 10c and 10r stock solution of 10c and 10r (2 mm) were prepared in ar grade methanol. the dna binding experiments were carried out by making dilution of the stock with 1:1 buffered methanol. stock solution of dna was prepared by dissolving dna pellet in te buffer (10 mm trisehcl, 0.1 mm edta, ph 7.4). the dna concentration was estimated from its absorbance intensity at 260 nm with a known molar absorption coefficient value of 6600 dm 3 mol à1 cm à1 . the purity of dna was established from ratio of absorbance intensity at 260 nm and at 280 nm. the observed ratio of 1.8 ensured that dna was free from protein. 260 nm at various temperature in the presence and absence of drug in a 5:1 ratio of the dna and drug with a ramp rate of 0.5 c/min in a 40% dmso/te buffer (ph 7.4) with 0.5 mm nacl on a shimadzu 1601 pc spectrophotometer equipped with a peltier thermo regulator. the thermal melting temperature was calculated by plotting da/dt vs temperature using microsoft excel. the molecular docking analysis was carried out using flexx molecular docking module [52, 53] (flexx 1.13.5) available in the sybyl 7.1 software package from tripose [54].this docking approach adopts an incremental construction algorithm for identifying appropriate pose of the substrate in the active site of the enzyme. it generates about 30 possible poses of the substrate in the active site. most of the 30 poses obtained in this analysis adopted similar binding mode in the enzyme cavity and hence the top scoring pose was considered in this analysis. to perform the above analysis, the crystal structure 1j3i (wild-type plasmodium falciparum dihydrofolate reductase-thymidylate synthase (pfdhfr-ts) complexed with wr99210, nadph, and dump) was downloaded from the protein data bank [55] (pdb) and subjected to protein preparation and receptor description [56] . the structures of the substrates were prepared by first building the molecules in 3d and optimizing the structures using tripos force field. the ionic, tautomeric states of substrates were considered while performing molecular docking analysis. the pose shown in fig. 7 includes the neutral state of the substrate in the active site of the enzyme. yield: 65%. ir (kbr): n max 772, 1270, 1700, 2998, 3300 cm à1 1 h (300 mhz 26 ( s, 16h, ch 2 ), 1.57 (d, j ¼ 6.9 hz, 2h, ch 2 ), 1.71 (d, j ¼ 6.9 hz, 2h, ch 2 ), 2.48 (s, 3h for c 35 h 44 n 5 o 2 cl: c, 69 .96; n, 11.45. ms: m/z 602 (m þ ) yield: 58%. ir (kbr): n max 716, 1127, 1700, 2940, 3446 cm à1 1 h (300 mhz 5-ethoxycarbonyl-6-methyl-4-phenyl-2-[5-(7-chloroquinolin-4-ylamino)-2-methylpentylamino] pyrimidine (10k) viscous liquid. rf: 0.6 (8% meoh/etoac) white solid 44 (s, 6h, c6 & c4ech 3 ), 3.44 (q, j ¼ 6.0 hz, 2h, ch 2 ), 3.67 (q, j ¼ 6.3 hz, 2h, ch 2 ), 4.36 (q, j ¼ 7.2 hz, 2h h, 5.93; n, 16.63. ms: m/z 413.5 (m þ ) white solid 41 (s, 6h, c6 & c4ech 3 ), 3.40 (q, j ¼ 6.9 hz, 2h, ch 2 ), 3.57 (q, j ¼ 6.0 hz, 2h .15; n, 15.99. ms: m/z 428.1 (m þ ) -ylamino)propyl amino]pyrimidine (10n) yellow solid. rf: 0.6 (2% meoh/etoac) 13 c nmr (75 mhz ylamino)butylamino] pyrimidine (10o) yellow solid. rf: 0.6 (5% meoh/etoac) 55 (s, 3h ylamino)ethylamino] pyrimidine (10p) yellow solid 67 (s, 3h -ylamino)propyl amino] pyrimidine (10q) yellow solid. rf: 0.6 (10% meoh/etoac) 57 (s, 3h, c6ech 3 ), 3.19 (br, 2h, ch 2 ), 3.41 (q, j ¼ 5.4 hz, 2h ylamino)butylamino] pyrimidine (10r) yellow solid. rf: 0.6 (5% meoh/etoac) 57 (s, 3h, c6ech 3 ), 3.13 (br, 1h, nh), 3.30 (m, 2h n, 15.25. ms: m/z 548.1 (m þ ) -ylamino)propyl amino] pyrimidine (10s) yellow solid new medicines to improve control and contribute to the eradication of malaria chloroquine transport via the malaria parasite's chloroquine resistance transporter plasmodium chloroquine resistance and the search for a replacement antimalarial drug artimesinin resistance in plasmodium falciparum malaria artimesinin-resistant malaria in asia falcipain-2 inhibitors quinoline antimalarial: mechanisms of action and resistance discovery of dual function acridones as a new antimalarial chemotype a pre-emptive strike against malaria's stealthy hepatic forms modelling and informatics in the analysis of p. falciparum dhfr enzyme inhibitors effects of pyrimethamine, chlorguanidine and primaquine against exoerythrocytic forms of a strain of chloroquine-resistant plasmodium falciparum from thailand 8-quinolinamines and their pro-drug conjugates as potent bloodschizontocidal antimalarial agents synthesis of 4-pyrido-6-aryl-2-substituted amino pyrimidines as a new class of antimalarial agents new antimalarial drugs a bifunctional thymidylate synthetase-dihydrofolate reductase in protozoa mechanism based inhibitors of deoxythymidine monophosphate synthesis as antineoplastic agents plasmodium berghei: inhibitors of ornithine decarboxylase block exoerythrocytic schizogony interaction of pyrimethamine, cycloguanil, wr99210 and their analogues with plasmodium falciparum dihydrofolate reductase: structural basis of antifolate resistance recent developments in the design and synthesis of hybrid molecules based on aminoquinoline ring and their antiplasmodial evaluation aldureids of ethylic acetoacetate and ethylic oxaloacetate an efficacious protocol for the oxidation of 3, 4-dihydropyrimidin-2(1h)-ones using pyridinium chlorochromate as catalyst facile transformation of 3,4-dihydropyrimidin-2(1h)-ones to pyrimidines in vitro evaluation as inhibitor of myobacterium tuberculosis and modulators of cytostatic activity synthesis, antimalarial and cytotoxic evaluation of reversed chloroquines based on the 3, 4-dihydropyrimidi-2(1h)-one scaffold -chloroquinolin-4-yl) alkanediamines with potential against chloroquine-resistant malaria chloroquine and hydroxychloroquine as inhibitors of human immunodeficiency virus (hiv-1) activity effects of chloroquine on viral infections: an old drug against today's diseases? an assessment of drug-haematin binding as a mechanism for inhibition of haematin polymerisation by quinoline antimalarials interactions of quinoline antimalarials with hematin in solution quinoline antimalarials decrease the rate of betahematin formation ferriprotoporphyrin ix fulfills the criteria for identification as the chloroquine receptor of malaria parasites thermodynamic factor controlling the interaction of quinoline antimalarial drugs with ferriprotoporphyrin ix structure-function relationships in aminoquinolines: effect of amino and chloro groups on quinoline-hematin complex formation, inhibition of b-hematin formation, and antiplasmodial activity application of jobs method of continuous variation to stoichiometry of protein-ligand complexes determination of binding stoichiometry by the continuous variation method-the job plot chloroquine analogues: influence of side chain length and quinolyl nitrogen pk a on activity vs chloroquine resistant malaria a spectroscopic investigation of the binding interaction between 4, 5-dihydroxyxanthone and heme new bis (2-aminoimidazoline) and bisguanidine dna minor groove binders with potent in vivo antitrypanosomal and antiplasmodial activity a non-radiolabelled ferriprotoporphyrin ix biomineralisation inhibition test for the high throughput screening of antimalarial compounds the interaction of chloroquine with nucleic acids and nucleoproteins interaction of the antimalarial drug fluoroquine with dna, trna, and poly-(a): 19 f-nmr chemical-shift and relaxation, optical absorption, and fluorescence studies dna: reaction with chloroquine reaction between dna and quinacridine and other antimalarials spectroscopic studies on the thermodynamic and thermal denaturation of the ct-dna binding of methylene blue structure-function correlation of chloroquine and analogues as transgene expression enhancers in nonviral gene delivery the genome of plasmodium falciparum i: dna base composition sequence preference of chloroquine binding to dna and prevention of z-dna formation pharmacogenomic analyses of targeting the at-rich malaria parasite genome with at-specific alkylating drugs a spectrophotometric investigation of the interaction of iodine with aromatic hydrocarbons spectrophotometric studies of the interaction of chloroquine with deoxyribonucleic acid antimalarial versus cytotoxic properties of dual drugs derived from 4-aminoquinolines and mannich bases: interaction with dna quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique multiple automatic base selection: proteinligand docking based on incremental construction without manual intervention a fast flexible docking method using an incremental construction algorithm insights into antifolate resistance from malarial dhfr-ts structures a common feature-based 3d-pharmacophore model generation and virtual screening: identification of potential pfdhfr inhibitors key: cord-276166-b1e0bbrp authors: li, shi-fang; zhao, fu-rong; shao, jun-jun; xie, yin-li; chang, hui-yun; zhang, yong-guang title: interferon-omega: current status in clinical applications date: 2017-10-12 journal: int immunopharmacol doi: 10.1016/j.intimp.2017.08.028 sha: doc_id: 276166 cord_uid: b1e0bbrp since 1985, interferon (ifn)-ω, a type i ifn, has been identified in many animals, but not canines and mice. it has been demonstrated to have antiviral, anti-proliferation, and antitumor activities that are similar to those of ifn-α. to date, ifn-ω has been explored as a treatment option for some diseases or viral infections in humans and other animals. studies have revealed that human ifn-ω displays antitumor activities in some models of human cancer cells and that it can be used to diagnose some diseases. while recombinant feline ifn-ω has been licensed in several countries for treating canine parvovirus, feline leukemia virus, and feline immunodeficiency virus infections, it also exhibits a certain efficacy when used to treat other viral infections or diseases. this review examines the known biological activity of ifn-ω and its clinical applications. we expect that the information provided in this review will stimulate further studies of ifn-ω as a therapeutic agent. interferons (ifns) were first reported by isaacs and lindenmann as antiviral proteins that are generated by cells in response to viral infection [1, 2] . ifns are composed of three subgroups: type i, type ii, and type iii ifns. the type i ifns, including ifn-α, ifn-β, ifn-ε, ifn-ω, ifnκ, ifn-δ, ifn-τ, and ifn-ζ, exert biological activity through common receptors (interferon-α/β receptor 1 [ifnar1] and ifnar2) [3, 4] . type ii ifn, namely ifn-γ, is produced by t lymphocytes and natural killer cells in response to the recognition of infected cells [5] . type iii ifns consist of ifn-λ1, ifn-λ2, and ifn-λ3, which regulate the immune response via a distinct receptor complex that uses a signaling pathway that is similar to that of type i ifns [6, 7] . ifn-ω genes were first found in humans in 1985 [8, 9] . ifn-ω diverged from the ifn-α gene approximately 130 million years ago, and it is produced primarily in leukocytes [10] . human ifn-ω genes include four pseudogenes and one full gene that is expressed in leukocytes, and human ifn-ω shares 62% amino acid sequence homology and similar functions with ifn-α, and 33% amino acid similarity with ifn-β [11] . similar to other ifns, ifn-ω is also produced by cells in response to viral infection, and it has biological activities because it binds to the same receptors and activates a pathway that is similar to that activated by ifnar [12] , while the activation of phosphoinositide-3-kinase/ protein kinase b (p13k/akt) signaling is also vital for biological responses mediated by ifn-ω [13] . however, its antigenic structure is distantly related to ifn-α, -β, and -λ, as it does not cross-react with antibodies against them [14] . as a result, treatment with ifn-ω can be effective for patients who are resistant to ifn-α [15] . although ifn-ω has been studied extensively, our knowledge of ifn-ω is still limited compared with that of interferon ifn-α and -β. here, we provide a broad review of what is known about the biological activities and potential clinical application of ifn-ω. overall, we hope that the information provided in this review will stimulate further studies of ifnω as a therapeutic agent. until now, ifn-ω has been identified in humans [8] , felines [16] , pigs [17] , horses [18] , rabbits [19] , serotine bats [20] , cattle [21] , and sheep [22] , while it has not been found in canines and mice [21] ; however, the characteristics of ifn-ω differ in different species. the characteristics of ifn-ω from different species are summarized in table 1 . despite such differences, there are common characteristics that exist in these species. generally, ifn-ω genes are intronless, and nglycosylation sites (not present in felines) and transcription factor binding sites, such as irfs, isres, and nf-κb, are present in the promoter regions of these genes [9, 10, 20, 30] . in addition, all mammalian ifn-ω genes lack the proline codon that precedes the final conserved cysteine codon [24] . the ifn-ω protein contains several conserved residues, such as arginine at position 161, cysteine residues at positions 1 and 99 and 100, seven prolines (four of which are located at positions 4, 26, 39, and 117 of the mature protein), while positions 29 and 139 and 140 form two disulfide bonds. these residues are present at similar locations in type i ifns, and they are vital for their biological activity [33] . other conserved structural motifs, such as the regions between amino acid residues 29-41, 71-82, and 123-142, are highly conserved among ifn-α proteins, and they play a key role in the biological activity of ifn-ω [26, 28, 29] . in addition to having a structure that is similar to that of type i ifns, ifn-ω also displays the same physicochemical characteristics as type i ifns, such as high sensitivity to trypsin, insensitivity to temperature, and stability at ph 2 [28, 29] . some studies revealed that ifn-ω loses its antiviral activities after being treated with 0.25% trypsin. however, it retains high antiviral activity against vesicular stomatitis virus after exposure to ph 2 for 24 h at temperatures ranging from 42°c to 63°c [26, 28, 29] . based on analyses of phylogenetic trees obtained from alignments of the ifn-ω protein sequences from these mammal species, it was proposed that all these proteins evolved from a common ancestor [20, 28] . type i ifns, including ifn-ω and ifn-α, display a common mechanism of action. ifns interact with specific cell-surface receptors, and then the expression of ifn-stimulated genes (isgs) is induced, some of which encode antiviral effectors or molecules, such as signaling proteins, transcription factors, and apoptotic proteins, while chemokines further regulate ifn signaling and other host responses in a positive or negative manner [28] . ifn-ω is not an exception. it has been proposed that ifn-ω shares antiviral activities with ifn-α because it binds to the same type i ifn receptor complex. studies have revealed that ifn-ω induces the transcription of the mx1, isg15, ifit3, and isg56 genes [20, 28] . however, different from ifn-α, it shows certain degrees of cross-species activity; therefore, ifns could exhibit different physiological functions in the host. for example, bovine ifn-ω (boifn-ω) protects madin-darby bovine kidney cells, primary embryo bovine lung table 1 ifn-ω in different species and the characteristics of the ifn-ω gene and protein. location characteristics of the ifn-ω gene and protein reference human human chromosome 9 human ifn-ω has at least five members, but only one member of the human ifn-ω family is a functional gene that results in a functional and glycosylated protein. this protein has six additional amino acids located at its carboxyl-terminus (172 amino acid residues) and a single polypeptide chain comprising two disulfide bonds and an n-glycosylation-linked site at amino acid 80. in terms of the primary structures of the type i ifns, there is approximately 75% amino acid sequence identity between ifn-α and ifn-ω. [8, 9, 12, 15, 23] cat no data felines ifn-ω (feifn-ω) includes 13 subtypes that contain an amino-terminal secretory signal sequence from residues 1-23. the mature sequence of ifn-ω contains four highly conserved cysteines (positions 1, 29, 100, and 140) and seven prolines (four of those at positions 4, 26, 39, and 117 of the mature protein), while the feifn-ω protein has six additional amino acids at its carboxyl-terminus compared with feifn-α. among these subtypes, feifn-ω2 and feifn-ω4 contain a seven-amino-acid insertion at position 109, and the insert location is comparable to that of the feifn-α, while it has higher antiviral activity than the other subtypes. interestingly, different from that in other mammalian subtypes, these 13 subtypes do not have an n-glycosylation recognition site. [16, 24] pig chromosome 1 porcine ifn-ω (poifn-ω) contains seven or eight members with variable open reading frame lengths. the most extensive similarity of the poifn-ω sequences are to bovine leukocyte ifn-ω, and the lowest one is to equine ifn-ω. it contains an ifabd functional domain, multiple putative binding sites for type i ifn receptor subunits, and one putative nglycosylation site (asn/asp-x-ser/thr). the subtypes of the ifn-ω protein, which have five alpha-helices, bear aminoterminal 20-to 30-amino-acid signal peptides and four conserved cysteine residues, cys24, cys52, cys122, and cys162, which are also found in ifn-α1. in addition, all ifn-ω subtypes, except ifn-ω1 (whose carboxyl-terminus is 11 residues shorter than the other poifn-ω subtypes), have 14 or 16 extended residues, while the pairs ifn-ω2/6 and ifn-ω3/5 share identical sequences. [17, 25, 26] horse chromosome 23 horse ifn-ω contains eight members plus four pseudogenes, which is a greater than the number of ifn-α genes and more diverse than the other type i ifn genes. the ifn-ω gene contains a putative glycosylation sequence, asn-thr-thr, at positions 78-80. the ifn-ω protein has six alpha-helices and it contains ifabd (cd00095), interferon (pfam00143), and ifabd (smart00076) domains, and multiple putative binding sites for the type i ifn receptor subunit 1 and 2 and an nglycosylation site. notably, the length of the open reading frame of each ifn-ω subtype is invariant. [18, 27] rabbit no data the rabbit ifn-ω family comprises at least eight genes that display the highest degree of homology with human ifn-ω and the lowest with murine ifn-αii. there are two irf-1 binding sites at nucleotide positions 85 and 55, the hexamer repeat sequence 5′-gaaann-3′ in the 5′ noncoding sequence, and a repetition of the 5′-ttatttat-3′ motif, which is similar to many 3′ downstream regions of genes encoding inflammatory cytokines. in addition, the immediate promoter region of the rabbit ifn-ω genes has a high proportion of purine residues. an atg sequence, (5′-gaaatg-3′), was found only in the promoter region of rbifnw48 at nucleotide position 66. similarly, ifn-ω includes four cys residues at amino acid positions 1, 29, 99, and 139. [19] cattle chromosome 8q15 the ifn-ω family in cattle (boifn-ω) consists of 24 members that contain two pseudogenes and 22 functional genes. among these subtypes, the nucleotide similarity is 91.3%-97.2%, and the amino acid identity is 84.4%-95.4%. most of these subtypes encode 195 amino acids, expect boifn-ω7 and boifn-ω11, which lack nine amino acid residues at their carboxyl terminus. in addition, 23 cells, feline kidney cells, porcine kidney cells, rabbit kidney cells, and primary bovine testicular cells from vesicular stomatitis virus challenge. however, the antiviral activities of boifn-ω are low in madin-darby bovine kidney cells, but high in porcine kidney and bovine testicular cells. additionally, no protective effects were found on madin-darby bovine kidney cells and baby hamster kidney cells [28, 29] . these results suggest that cells have a tendency to be insensitive to ifnω from distantly related species. the antiviral activities of ifn-ω also vary with the subtypes and viral strain used in challenges. in a previous study, the expression of poifn-ω2/-ω6, poifn-ω3/-ω5, and poifn-ω8 were upregulated, while poifn-ω1 (which has 11 fewer residues at its carboxyl terminus than the other poifn-ω subtypes) was not detected in porcine kidney cells challenged with pseudorabies virus or poly (i):poly(c). interestingly, the level of poifn-ω1 also increased when peripheral blood mononuclear cells were challenged with pseudorabies virus, and poifn-ω2/-ω6 was upregulated the most in these studies [26] . similarly, recombinant serotine bats ifn-ω can inhibit european bat lyssavirus type 1, european bat lyssavirus type 2, and rabies virus replication in a dose-dependent manner, and different biological activities were elicited (in the order of european bat lyssavirus type 1 < rabies virus < european bat lyssavirus type 2) [20] . the antiviral activities of ifn-ω have also been compared to those of other ifns. when a549 cells were respectively treated with ifn-ω and type i and type iii ifns, the cells treated with ifn-ω at a concentration of 10 ng/ml obviously repressed the replication of influenza a virus in a dose-dependent manner, and it resulted in larger reductions in viral titers, compared with those obtained with ifn-α2 [34] . however, its activity was much lower than the activity of ifn-β1, and it was slightly lower than the activities of ifn-λ1 and ifn-λ2. depending on the dose, pretreating caco-2 cells with human ifn-ω significantly reduced the loads of the h1n1 influenza virus [35] . interestingly, ifn-β and ifn-ω showed similar inhibitory activity against both ca/04 and bj/501 influenza viruses at every concentration tested. in addition, both were significantly more potent than ifn-α as far as the inhibitory effects on both stains at a given concentration. in addition to its in vitro activities, ifn-ω has been demonstrated to have antiviral activity in vivo as well. for example, a robust induction of mx mrna and the oas-1a protein was found in animals treated by ifn-ω compared with controls [35] . furthermore, following daily intravenous treatment with human ifn-ω (huifn-ω), the viral load of h1n1 influenza virus decreased significantly in the lung tissues of guinea pigs, and ifn-ω exhibited an identical inhibitory effect against the ca/04 influenza virus compared with ifn-α [35] . although ifn-ω has demonstrated promising antiviral activity against some viral infections in vivo and vitro, several factors could limit its antiviral efficacy, such as poor pharmacokinetics and a short half-life [36] . pegylation, a covalent conjugation of nontoxic polyethylene glycol (peg), has been demonstrated to improve the plasma half-life of a therapeutic protein and decrease its proteolytic sensitivity [37] . recently, yu et al. [38] found that pegylation improved the poor pharmacokinetics of recombinant huifn-ω (rhuifn-ω), despite the fact that its antiviral activity was decreased to some extent. it is worth noting that the bioactivity of pegylated rhuifn-ω was determined by examining its pegylation sites, in which residues at the amino-terminus had higher activities compared with those of residues lys 134 and lys 152 , while the poor pharmacokinetics were determined by the conjugated peg mass. in a previous study, amino-terminally pegylated rhuifn-ω with 40 kda of linear peg, which exhibited 21.7% of the rhuifn-ω biological activity with a half-life of 139.6 h, allowed some viral diseases to be treated by fewer doses at longer dosing intervals [38] . the use of human igg1 fc fusion proteins has also been demonstrated to be a simple and effective method for prolonging serum half-life [39] . some studies showed that compared with rhuifn-ω expressed in yeast with a specific activity of 7 × 10 7 iu/mg, rhuifn-ω-fc had a lower activity of 1.6 × 10 7 iu/mg when it was expressed in chinese hamster ovary cells. furthermore, the terminal half-life of rhuifn-ω-fc was 35-fold higher than that of rhuifn-ω, and it exhibited better pharmacokinetics characteristics [40] . perhaps rhuifn-ω-fc will become a new alternative antiviral drug for the treatment of chronic viral infections. several studies also revealed that glycosylation has an essential effect on the activity of ifns [41] , and glycosylated ifn-ω was more potent than ifn-ω against bovine viral diarrhea virus, yellow fever virus, and west nile virus [41, 42] . a possible explanation for its potency is that glycosylated ifn-ω could induce the activation of the sterol regulatory element binding transcription factor, the activating enhancer binding protein 2-like yy1 site, the interferon consensus sequence binding protein, the erythroid kruppel-like factor gene, the homeotic gene forkhead of drosophila 8/hepatocyte nuclear factor 3/ mouse forkhead lung protein, hnf-1α, the interferon conserved sequence-binding protein, and the lymphocyte-enriched dna binding protein lyf, which are not induced by ifn-α [41] . glycosylated ifn-ω also exhibits good clinical applications. previous studies showed that glycosylated ifn-ω + ribavirin (rbv) had a synergistic effect in terms of its antiviral activity in hepatitis c virus (hcv)-infected patients [43] . similarly, a synergy of the antiviral effects of glycosylated ifnω + rbv against bovine viral diarrhea virus was also obtained. interestingly, an antagonism of the cytotoxic effects of rbv by glycosylated ifn-ω was observed [43] . thus, the combination of glycosylated ifn-ω and rbv appears to be favorable for the synergy of antiviral activities and the antagonism of the cytotoxic effects of rbv. increasing evidence suggests that ifn-ω may be associated with the nonspecific immune response based on increased survival time, the presence of acute-phase proteins (serum amyloid-a, α-1-glycoprotein, and the c-reactive protein), the phagocytic activities of whole blood cells and macrophages, natural killer cell activities, and reduced concurrent viral excretion [44] [45] [46] [47] [48] . in general, increasing the number of isgs, mx proteins, and zaps, which could enhance pathogen detection and innate immune signaling, indicates that ifn-ω elicits an immune response. when cells are treated with ifn-ω, mx-1, isg15, ifit3, and isg56 expression was upregulated in a dose-dependent or time-dependent manner [20, 29] . more recently, studies from leal et al. [49] showed that interleukin (il)-6 production was affected considerably in feline immunodeficiency virus (fiv)-infected cats treated with recombinant feline ifn-ω (rfeifn-ω) by subcutaneous or oral protocols. specifically, il-6 plasma levels decreased and proviral loads increased in fiv-cats treated subcutaneously with rfeifn-ω, while il-6 mrna expression decreased in the oral group. however, the therapy did not affect viremia and the expression of other cytokines (il-1, il-4, il-10, il-12p40, ifn-γ, and tumor necrosis factor-α) [49] . thus, ifn-ω appears to be involved in innate immunity, and it could further inhibit viral replication and exert its antiviral activity. ifns have been demonstrated to have antiproliferative effects as a result of inducing cell-cycle arrest and apoptosis, which are independent pathways [50] . similar to ifn-α, ifn-ω also inhibits cell proliferation in a dose-dependent manner [20, 29] . however, ifn-α exhibits a higher antiproliferative activity than ifn-ω at elevated concentrations [29] . ifn-ω also exhibits antitumor activity in vitro in a cell-specific manner. feline mammary carcinomas are among the most common feline tumors, and they represent an important cause of mortality. when used to treat feline and canine mammary carcinoma cells and derived putative tumor-initiating cells, rfeifn-ω exhibited a dose-dependent, species-specific, target cell-specific action, and an additive effect was observed between rfeifn-ω and conventional anticancer drugs such as mitoxantrone, doxorubicin, and vincristine [51] . thus, rfeifn-ω may be used as a therapeutic agent in feline and canine mammary carcinomas. there is evidence showing that gene transfer would allow one to take advantage of the beneficial effects of type i ifns without their undesirable side effects [52] . for instance, huifnβ gene lipofection produces an equal or even superior effect than that of high doses of exogenously applied huifnβ protein. recently, a study conducted by villaverde et al. [52] demonstrated that felines feifn-ω gene lipofection exhibited an equal or higher cytotoxic effect than rfeifn-ω. this kind of effect was due to the induction of reactive oxygen species generation, mitochondrial membrane potential disruption, and calcium uptake, indicating that ffeifn-ω lipofection is an alternative approach for treating both sensitive and resistant phenotypes with an equal or superior outcome and with fewer adverse effects than recombinant ffeifn-ω therapy [52] . similarly, injecting the gene encoding interferon ω1 via the tail vein into mice bearing a hep-a-22 liver tumor via liposomal gene delivery also significantly decreased tumor weights [53] . interestingly, this effect was greater (43% tumor inhibition) only when the plasmid contained human thymosin alpha 1 [53] . a dna ladder (a hallmark of cells undergoing apoptosis) was observed in the tumor cells treated by ifn-ω. interestingly, the antitumor mechanisms involve increasing the expression of tα1 and ifn-ω, and a plasmid-liposome complex of ifn-ω and ifn-α together exerted greater potency in inhibiting the growth of hep-a-22 tumor cells than either compound alone [53] . because of its antiviral, immunomodulatory, anti-proliferation, and antitumor activities, ifn-ω has been explored as a treatment option for some diseases and viral infections in humans and other animals. huifnω displays antitumor effects in several models of human cancer in vivo. in addition, it has also been used to diagnose some diseases [54, 55] . when produced in silkworm larvae using a baculovirus vector that includes the feifn-ω sequence, rfeifn-ω, as an immunomodulator, has been registered in many countries (e.g., japan, australia, new zealand, and mexico) to treat canine parvovirus, feline leukemia virus (felv), and fiv infections (http://emea.europa.eu) [45, 56] . despite the fact that it is not licensed to treat other viral infections, it exhibits efficacy when used to treat other viral infections such as bovine enterovirus, infectious bovine rhinotracheitis virus, bovine viral diarrhea virus, vesicular stomatitis virus, pseudorabies virus, european bat lyssavirus, influenza virus, feline calicivirus (fcv), and feline herpesvirus-1 (fhv-1) [20, 28, 34, 57] . the rfeifn-ω licensed protocol consists of three therapeutic cycles of five daily subcutaneous injections (1 mu/kg), beginning respectively on days 0, 14, and 60 [58] . however, its widespread use is limited because this protocol is relatively expensive and time-consuming. some alternative subcutaneous and topical protocols, such as oral and intralesional administration, have been suggested [59, 60] . interestingly, no adverse effects were found in cats treated with rfeifn-ω following mucosal administration [58, 61] , while subcutaneous administration was accompanied by some mild adverse effects (e.g., fever, lethargy, vomiting, and diarrhea) in some trials [46] . thus, rfeifn-ω could become an option for treatment in veterinary clinical practice. hcv is a major public health problem with approximately 170 million infections worldwide [62] . many hcv-infected individuals cannot eliminate the virus, and persistent infection with hcv causes chronic liver disease, including cirrhosis and hepatocellular carcinoma [63, 64] . until now, a combination of ifn-α and rbv has been the standard of care for chronic hcv infections [65, 66] . however, it has many deleterious side effects. furthermore, some hcv chronic carriers are not suitable for this therapy, and new antiviral approaches are needed. some studies showed that ifn-ω was equally effective at inhibiting hcv replication compared to ifn-α and ifn-β. interestingly, the combinations of ifn-ω and ifn-α or ifn-β exhibited primarily antagonistic interactions [67] . in addition, other studies showed that the activity of non-glycosylated ifn-ω was comparable to that of ifn-α, while glycosylated ifn-ω was more potent in repressing hcv rna replicons. thus, ifn-ω can be used as a prospective antiviral candidate for the treatment of hcv infections [41] . in addition to being a potential therapy for hcv, ifn-ω could also be used to diagnose some human diseases. aps-1 is a monogenic disease that is caused by mutations in the autoimmune regulator (aire) gene [68, 69] . currently, many autoantibodies, including those targeting aromatic acid decarboxylase, tyrosine hydroxylase, nacht leucine-rich repeat protein 5, tryptophan hydroxylase, and ifn-ω, have been found in aps-1 patients [55] . a study has shown an anti-ifn-ω antibody was highly prevalent in aps-1 patients with mutated aire alleles, even though the presence of anti-ifn-ω antibodies preceded aps-1 clinical symptoms and the development of other autoantibodies [70] . therefore, antibodies against ifn-ω have become an essential diagnostic tool for aps-1. numerous available assays, such as immunoassays, radioligand binding assays, and antiviral neutralization assays, were designed to detect anti-ifn-ω antibodies [70] [71] [72] . however, all of them have drawbacks, including a lack of sensitivity, the use of radioisotopes, the requirement for facilities to grow viral cultures, as well as being time-consuming. some novels diagnosed methods were also developed. zhang et al. [73] reported that an ipa based on 125 i-labeled ifn-ω showed higher sensitivity to aps-1 than other anti-type 1 ifn antibodies when 48 aps-1 patients were examined. similar to the study by zhang et al., 48 aps-1 patients were also investigated in a study by oftedal et al. [74] , and these patients were also positive for anti-ifn-ω antibodies, as determined by an ipa based on 35 s-labeled ifn-ω, which also exhibit a greater correlation to aps-1 than other anti-type 1 ifn antibodies. these results suggest that out of the spectrum of anti-type 1 ifn antibodies, testing for anti-ifn-ω antibodies is more suitable for patients suspected of having aps-1. recently, larosa et al. [75] developed a novel, highly sensitive, and specific assay to measure anti-ifn-ω antibodies. they found that 125 i-labeled ifn-ω and 35 s assays resulted in 5/6 and 1/6 discrepant samples, respectively, which agrees with aire gene results [75] . regarding the measurement of anti-ifn-ω antibodies in the studied patients, the 125 i assay seems to have a better performance, as it is disease-specific, more reliable, and more precise than the 35 s assay, making it possible to assess patients with aps-1 and patients suspected of having aps-1 prior to an aire gene analysis. fcv, a member of the caliciviridae family, is an important and common pathogen for cats, which is characterized by clinical signs ranging from mild and severe oral ulcerations or upper respiratory tract disease to a severe fatal systemic disease [76, 77] . unfortunately, there are still no direct-acting antiviral drugs for the treatment of fcv. previous studies demonstrated that rfeifn-ω had a positive therapeutic effect in fcv-infected cats in some experiments or trials [46, 57, 78] . combination antiviral therapy has become a common practice. recently, researchers revealed that a combination treatment of rfeifn-ω and mefloquine resulted in additive effects with a reduction in the half maximal inhibitory concentration [79] . another study conducted by lee et al. [80] showed that the relative expression of ifn-ω, mx, and zaps mrna in crandell-reese feline kidney cells was upregulated significantly in fcv-infected cats. interestingly, after the cats were pretreated with a korean red ginseng (krg) extract or ginsenosides, the expression of these genes was higher than that in the group treated with fcv alone [80] . it is desirable to find a combination of a krg extract or ginsenosides and ifn-ω that effectively treats fcv-infected cats. because of its similar structural characteristics and genomic structure, fcv could be a representative human norovirus surrogate [81] ; thus, these therapies could also be used to treat human norovirus infections. fhv-1, a double-stranded dna virus that replicates in the nucleus of host cells and produces intranuclear inclusion bodies, is one of the foremost causes of feline upper respiratory tract disease [82] . it has been demonstrated that rfeifn-ω has a dose-dependent inhibitory effect on the replication of fhv-1 in vitro [83] . several studies showed conflicting results about the biological activity of rfeifn-ω against these two viruses in cats. for instance, 160 cats suffering from fcvassociated feline upper respiratory tract disease were treated with an intravenous dose of 2.5 or 5 mu/kg rfeifn-ω three times per day every other day [82] . while improved clinical signs were detected within 7 days, no control group was used in this study. in another study, 20 cats with fhv-1-associated ocular keratitis were treated five times per day with a dose of 0.5 mu/ml rfeifn-ω, and although a significant improvement of ocular signs was observed after 3 weeks of treatment, no control group was included [83] . in 2007, using a placebo as a control group, the effect of rfeifn-ω in cats that were pretreated with rfeifn-ω before challenge with fhv-1 was investigated [84] . unexpectedly, no beneficial effects were observed in these cats. ballin et al. [85] reported a similar finding; they found that compared to a control group treated with a placebo, cats treated with rfeifn-ω did not exhibit improved clinical signs of acute viral feline upper respiratory tract disease despite having lower fcv copy numbers than cats receiving rfeifn-ω [85] . more studies are needed to verify that rfeifn-ω has a therapeutic effect in vivo. felv, an endogenous retrovirus that belongs to the genus gammaretrovirus of the family retroviridae causes a variety of degenerative and immunosuppressive disorders such as severe anorexia, cachexia, and progressive weakness [86, 87] . studies indicated that rfeifn-ω affects the felv cycle at the post-transcriptional level. however, it did not alter protein synthesis [88] . treatment with rfeifn-ω lowered reverse transcriptase activity, which is used to evaluate the amount of infectious viral particles, in a dose-dependent manner in felv-infected fl74 cells [88] . furthermore, according to the half maximal inhibitory concentration, rfeifn-ω was more potent than rhuifn-α in inhibiting reverse transcriptase activity. in addition, this study showed that ifn-ω decreased the viability of felv-infected fl74 cells and increased apoptosis and mortality in infected cells [88] . fiv is a feline lentivirus that was first described in 1987, and many studies have focused on it because its complex genomic and immunopathogenic features that are similar to those of human immunodeficiency virus [89, 90] . furthermore, fiv coinfection was found to occur in 7% of felv-infected cats [91] . previous studies showed that rfeifn-ω significantly decreased clinical scores and rates of mortality, and improved abnormal hematologic parameters (red blood cell count, packed cell volume, and white blood cell count) in virus-infected cats [45] . these studies demonstrated that rfeifn-ω had significant therapeutic effects on clinical signs. evidence also showed that an effective immune modulation in fiv-cats could be obtained by treating them orally with rfeifn-ω. in a previous study [45] , fiv-infected cats that were orally administered rfeifn-ω at a dose of 0.1 mu/cat gained weight, although no dramatic changes were obtained regarding viral loads and the cd4 + /cd8 + ratio. however, this study did not assess any clinical benefits [45] . recently, oral protocols were also used to administer rfeifn-ω in naturally fiv-infected cats, and initial clinical scores were obtained that were similar to those obtained using the licensed subcutaneous protocol [92] . interestingly, similar to the use of the licensed protocol, rfeifn-ω also induced a significant clinical improvement of cats that were treated orally, despite viral excretion, while there were no significant variations in acute-phase proteins [92] . these results suggest that oral administration of rfeifn-ω can be an effective alternative therapy for fiv-infected cats. fcgs is a multifactorial disease. some infectious viral diseases, such as fiv, fhv-1, and felv, can also trigger fcgs by inducing immune suppression and dysregulation [93, 94] . some studies showed that oral treatment with rfeifn-ω was a beneficial therapy for fcgs. a study conducted by hennet et al. [55] found that the oral protocol correlated with an obvious clinical improvement of fcgs lesions (caudal stomatitis and alveolar/buccal mucositis). furthermore, the only difference between rfeifn-ω and corticosteroids were that better pain relief was obtained with the rfeifn-ω treatment. recently, two clinical cases showed that the use of oral rfeifn-ω was also effective in type ii diabetic cats with fcgs [95] . in this study, a clinical improvement of oral lesions and a concurrent reduction of the required insulin dose had a positive relationship with rfeifn-ω therapy, which agrees with hennet's work showing that there was an overall relief of pain in refractory cases of fcgs [55, 95] . parvovirus, a small, non-enveloped, single-stranded dna virus, infects humans and carnivores in a host-specific manner. canine parvovirus type-2 (cpv), feline panleukopenia virus, and mink enteritis virus share approximately 98% homology in their nucleotide and amino acid sequences [96] . there are several therapies used to treat cpv infections; however, many of them have drawbacks [50] . reports suggest that rifn-ω has a significant therapeutic effect on cpv-infected dogs [97, 98] . for instance, it improved parvoviral clinical signs (such as pyrexia, vomiting, anorexia, and diarrhea), and reduced severe parvoviral enteritis and mortality in cpv infections. a study by kuwabara et al. [48] showed that continuous immunoenhancement after rfeifn-ω treatment and whole blood cells counts > 5 × 10 3 /μl in dogs might be essential for improving severe clinical symptoms caused by cpv infection. cpxv, belonging to the orthopoxvirus genus, is most frequently found in domestic cats with > 400 cases having been reported, and it is endemic in northern europe and western areas of the former soviet union [99, 100] . however, until now, no antiviral drugs have been licensed to cure cats of cpxv infection. a recent study conducted by mcinerney et al. [101] showed that treatment with rfeifn-ω had a certain protective effect in cats infected with cpxv, although two of the four cats in the study died. nevertheless, rfeifn-ω may be potentially useful for treating cpxv-associated pneumonia and dermatitis in cats. of note, additional research is needed before this can be confirmed. fibrosarcoma, a common disease in cats, accounts for over 40% of all skin tumors in this species [102] . currently, few successful treatments for fibrosarcoma have been reported in cats, although treatment of feline fibrosarcoma with rfeifn-ω has been demonstrated to be safe, well tolerated, and easily performed. a study by hampel et al. [103] revealed that rfeifn-ω increased the expression of major histocompatibility i molecules on feline fibrosarcoma cells, while eosinophilia, neutropenia, and weight loss were remarkably reduced by rfeifn-ω treatment. however, a placebo-controlled trial is needed. cad is a common, allergic skin disease. some therapeutic protocols have been reported, such as allergen-specific immunotherapy, the use of topical and oral glucocorticoids and calcineurin inhibitors, essential fatty acid supplementation, and bathing [104] . however, none of them were highly efficacious, and some severe side-effects were observed occasionally. fortunately, a study showed that treating dogs with cad with subcutaneously administered rfeifn-ω had a comparable efficacy to orally-administered cyclosporin [105] . in later studies [106] , oral ifn therapy showed higher efficacy than the subcutaneous treatment in terms of the canine atopic dermatitis extent and severity index and total scores, although the improvement of pruritus and quality of life scores were not significant; however, in this study, serum antibodies against rfeifn-ω were not detected in any of the dogs. fip is a common immune-mediated disease that is triggered by infection with a feline coronavirus [107] . previous studies showed that ifn-ω is a beneficial therapy in fip-infected cats. however, these studies used fewer than 12 cats per group and did not include a control group. furthermore, the effect of ifn-ω was not reliably confirmed in fip [108, 109] . for instance, in a study of cats suspected of having fip, 12 cats were treated with 106 u/kg of feline feifn-ω every other day until remission, followed by injections every week; four of the 12 cats lived longer than 2 years [108] . in a subsequent study conducted by ritz et al. [109] , a control group was used, but there were no significant differences between the survival times of cats treated with feifn-ω and a placebo, although the feifn-ω treatment resulted in a significantly lower lymphocyte count. interestingly, one cat survived > 3 months in the feifn-ω group. these results suggest that feifn-ω could also have a certain therapeutic effect in fip-infected cats. as an ifn, ifn-ω has been demonstrated to have similar levels of biological activities as ifn-α and ifn-β, which are limited by their toxicity and side effects; thus, ifn-ω may be a useful and alternative antiviral agent. although rfeifn-ω has only been registered to treat cpv, felv, and fiv infections, it shows comparable efficacy when used to treat other viral infections or diseases. however, its safety (e.g., toxicity and side effects) when used at a high dose has not been investigated extensively. in addition, the antiviral activity of ifn-ω in some fatal infectious diseases also needs to be 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with feline infectious peritonitis the authors declare no conflicts of interest. key: cord-291559-h6czy5bh authors: koirala, prashamsa; jung, hyun ah; choi, jae sue title: recent advances in pharmacological research on ecklonia species: a review date: 2017-08-24 journal: arch pharm res doi: 10.1007/s12272-017-0948-4 sha: doc_id: 291559 cord_uid: h6czy5bh the genus ecklonia (lessoniaceae, phaeophyceae), commonly called kelp (brown algae), is abundant on the coasts of japan and korea. during the past few decades, ecklonia species have received tremendous attention for their wide range of therapeutic properties and multiple health benefits, such as great nutritional value and being rich in vitamins, minerals, dietary fiber, proteins, and polysaccharides. several novel functional ingredients with diversified biological activities have been isolated and possess antimicrobial, antiviral, hepatoprotective, cardioprotective, anti-inflammatory, neuroprotective, anticarcinogenic, immunomodulatory, hypolipidemic, anti-diabetic, and antioxidant therapeutic properties. the present review discusses the phytochemical, pharmacological, therapeutic, nutritional, and health benefits of different species of genus ecklonia, as well as their use in the prevention of disease and maintenance of good health. seaweed is used as a vegetable and traditional medicine in east-asian countries such as japan, korea, and china. it is an important resource of the marine ecosystem, containing active metabolites with probable nutraceuticals that are rich sources of novel bioactive secondary metabolites and harbor diverse classes of health-promoting molecules, including essential dietary fiber, fatty acids, essential amino acids, and vitamins a, b, c, and e (kolb et al. 2004) . taxonomic classification divides seaweed into three major classes based more on their pigments and coloration than their genetics: rhodophyceae (red algae), phaeophyceae (brown algae), and chlorophyceae (green algae, also known as macroalgae) (rajasulochana et al. 2009 ). brown algae have been used for thousands of years, but only in modern times have they been recognized to contain bioactive substances such as polysaccharides, lipids, and polyphenols, with various pharmacological properties (kumar et al. 2008) . ecklonia is a genus of kelp (brown algae) belonging to the family lessoniaceae that has an abundance of eckoltype phlorotannins. there are nine species: ecklonia biruncinata, ecklonia brevipes, ecklonia cava (ec), ecklonia fastigiata, ecklonia kurome (ek), ecklonia maxima (em), ecklonia muratii, ecklonia radiate (er), and ecklonia stolonifera (es) (hornemann 1828; moon et al. 2008) . ec, an edible marine brown alga, is used as a food ingredient, animal feed, and fertilizer, as well as a raw material in the production of fucoidan and phlorotannin. ec is also used as an herbal remedy in the form of an extract called seanol, a polyphenolic extract, and ventol, a phlorotannin-rich natural agent with two major constituents, phlorotannins and sterols (kang et al. 2003a) . es (turuarame), ek, and er are edible species traditionally eaten in japan and korea and are rich in phlorotannins and fatty acids. ecklonia species are known to exhibit antioxidant (heo et al. 2005) , anti-inflammatory , antibacterial , anti-diabetic (jung et al. 2008) , anticancer (kong et al. 2009 ), anti-photoaging (joe et al. 2006) , anti-hiv (artan et al. 2008) , anti-hypertensive (jung et al. 2006) , hepatoprotective (jung et al. 2014a) , and anti-allergic activities (le et al. 2009 ). due to these numerous health benefits, they have been a focal point for researchers eager to elucidate their pharmacological potential. plenty of information regarding the pharmacological activities of terrestrial plants is available; however, such information is limited for marine species (shibata et al. 2008) . a handful of excellent studies are available regarding the pharmacological activities of ecklonia (wijesinghe and jeon 2012; thomas and kim 2011; li et al. 2011; . also, jiao et al. (2011) have reported the chemical structures and bioactivities of marine algae. fourteen years of research and nearly $35 million of clinical studies demonstrate the importance of ecklonia species. ecklonia-derived polyphenols are unlike those found in land-based plants and are quite possibly the most powerful antioxidants found in nature, being 10-100 times more powerful than other polyphenols. the oxygen radical absorbance capacity (orac) score of such a polyphenol is more than 8300. the water-soluble polyphenols found in land-based plants have a half-life of about 30 min, and their antioxidant powers decrease very quickly (seanol 2007) . various phlorotannins, polysaccharides, enzymatic extracts, and organic extracts from ecklonia exhibit multifaceted beneficial effects when used in pharmaceuticals, nutraceuticals, cosmeceuticals, and functional foods. thus, this genus has been a target of special attention, and consumer-driven demand has led to the development of marine-derived medicines. our review summarizes the literature on the biological characterization and pharmacological bioactivity of various ecklonia species, focusing on recent developments in the therapeutic application of extracts and isolates. a shift in the balance between oxidants and antioxidants in favor of oxidants is called oxidative stress (table 1) . it arises when the balance between the production of reactive oxygen species (ros) and antioxidant defenses changes. human cells have an inherited antioxidative defense system in the form of various enzymatic and non-enzymatic pathways for removing ros. elevated production of ros increases oxidative stress, leading to cellular dysfunction, and it can eventually contribute to many pathological conditions, including neurological disorders (agostinho et al. 2010) , diabetes (ceriello 2008) , cancer (perse 2013) , asthma , and dermal disease (trouba et al. 2002 ). an ethanolic extract of ec attenuated h 2 o 2 -induced comet tail formation and phospho-histone ch2ax expression. furthermore, it enhanced the level of the phosphorylated form of nuclear factor erythroid 2-related factor 2 (nrf2) and its nuclear translocation, which was associated with the induction of heme oxygenase-1 (ho-1) and nad'ph-quinone oxidoreductase-1 (nqo-1). thus, ec provided cytoprotective effects against oxidative stress in muscle cells via up-regulation of nrf2-ho-1 and nqo-1 expression through the activation of the mitogen-activated protein kinases (mapk) pathway, which could be due to its phenolic content (choi 2016) . in another study, es and ek showed high total phenolic content (tpc) and high antioxidant activity, including 1,1-diphenyl-2-picryl-hydrazyl (dpph) radical and hydroxyl (•oh) radical scavenging, ferrous reducing power, and superoxide (•o 2 -) radical scavenging activity. those results suggest that macroalgal beach-casts could be used as a new natural source for functional foods, cosmetics, medicines, and fertilizer instead of being processed into landfills or incinerated (kuda and ikemori 2009) . enzymatic extracts from er demonstrated more potent antioxidant ability in the ferric reducing ability of plasma (frap) and orac assays (tpc of 4.4 g) than conventional acidic extracts (tpc of 3.4 g), showing their potential for value-added nutritional products (charoensiddhi et al. 2015) . inflammation is a pathological condition that produces highly reactive species. nitric oxide (no), a small diffusible molecule responsible for vasodilatation, neurotransmission, and inflammation, is produced by organisms at a basal concentration. however, under stimulation by pathogens, no is generated in higher amounts by the inducible nitric oxide synthase (inos) in activated macrophages (moncada et al. 1991) . nonsteroidal anti-inflammatory drugs are commercially available medications for inflammatory pain, but their side effects have limited their use. anti-inflammatory drugs from natural resources have been sought due to the persistent deleterious side effects of commercial medications. ec dose-dependently suppressed inos and cyclooxygenase-2 (cox-2) protein expression and subsequently reduced no content in phorbol-12 myristate 13-acetate enzymatic extracts nf-jb lee et al. (2013c) (30 nmol/l) and a23187 (1 lmol/l) (pmaci)-stimulated human mast cell line-1 cells. no production decreased by 25.4 and 46.6% with 50 and 100 lg/ml ec treatments, respectively. furthermore, ec dose-dependently inhibited both the mrna and protein expression of tumor necrosis factor (tnf)-a, interleukin (il)-1b, and il-6 in pmacistimulated human mast cell line-1 cells without any cytotoxic effects. the inhibitory effects of ec on il-1b (75.0%) and tnf-a (60.6%) production were greater than those on il-6 (23.0%) at 100 lg/ml. in addition, ec exerted anti-inflammatory action via the inhibition of the extracellular signal-regulated kinase (erk)/mapk signaling pathway, suggesting a potent and efficacious antiinflammatory agent against mast cell-mediated inflammatory diseases (kim 2014d . the world health organization (who) defines diabetes as a chronic disease that occurs when the pancreas does not produce enough insulin or when the body cannot effectively use the insulin it produces, which leads to an increased concentration of glucose in the blood, i.e., hyperglycemia. type 1 diabetes (previously known as insulin-dependent) is characterized by a lack of insulin production. type 2 diabetes (formerly called non-insulindependent) is caused by the body's ineffective use of insulin, often from excess body weight and physical inactivity. the total number of people with diabetes is projected to rise from 171 million in 2000 to 366 million in 2030 (rathmann and giani 2004) . intensive insulin therapy carries a high risk of side effects, especially the occurrence of severe hypoglycemia; therefore, research into antihyperglycemic agents has focused on plants used in traditional medicine because they could provide better treatment than the currently used synthetic drugs (mccall 2012) . kimchi has been a popular side dish in korea since ancient times. baechu kimchi is a salt fermented cabbage widely consumed in traditional korean foods. a recent study revealed that kimchi with added ec extract showed high inhibition against a-glucosidase and a-amylase, with ic 50 values of 0.58 and 0.35 mg/ml, respectively. both of those inhibitory activities of kimchi with added ecklonia extracts (ke) were higher than those of kimchi extract alone. the hypoglycemic effect of ke was higher than that of kimchi extract on starch loading. ke suppressed the postprandial blood glucose level in both streptozotocin (stz)-induced diabetic and normal mice, which indicated a delay in the absorption of dietary carbohydrates consumed . in another report, baechu kimchi with added ec extract protected human umbilical vein endothelial cells (huvecs) from damage induced by high glucose by restoring cell viability and reducing lipid peroxidation and intracellular ros in a dose-dependent manner. furthermore, it reduced the overexpression of inos, cox-2, and nuclear factor-jb (nf-jb) proteins in huvecs, indicating its potential as a treatment against high glucose-induced oxidative stress . ek inhibited carbohydrate-hydrolyzing enzymes, decreased postprandial blood glucose levels, and improved glucose tolerance, decreasing both fasting blood glucose and insulin levels (xu et al. 2012) . ek effectively down-regulated blood glucose in both db/db mice and prediabetic c57bl/6j mice, indicating the presence of the active compounds in the gametophytes. ek regulated metabolism by manipulating the balance among cytokines, including interferon-gamma (ifn-c) or leptin, resulting in the downregulation of blood glucose (dwiranti et al. 2012 ). the liver is involved in the metabolism of internal and external toxic agents. it has an astounding role in the performance, maintenance, and regulation of homeostasis in the body and is engaged in almost all biochemical pathways of growth, fight against diseases, nutrient supply, energy provision, and reproduction. it plays a vital role in the detoxification of xenobiotics and drugs (agrawal et al. 2013) . hepatic disease is a term that indicates damage to liver cells, tissues, structures, or function. because no drug currently available completely or effectively stimulates hepatic function, offers complete protection to the organ, or aids in regenerating hepatic cells, naturally derived hepatoprotective agents are needed to negate the factors that contribute to liver damage. a polyphenol-rich fraction of ec prepared in gijang prevented diabetes by regulating various metabolic processes, such as lipogenesis, lipolysis, inflammation, and the antioxidant defense system in livers and adipose tissues affected by nonalcoholic fatty liver disease in high fat diet (hfd)-fed mice. magnetic resonance imaging/magnetic resonance spectroscopy (mri/mrs) analysis showed that liver fat and liver volume in hfd-triggered obese mice decreased following gijang extract treatment. furthermore, the treatment reduced the mrna expression levels of inflammatory cytokines and hepatic lipogenesis-related genes and increased the mrna expression level of cholesterol 7a-hydroxylase 1, the key enzyme in bile acid synthesis. thus, gijang extract ameliorated hepatic steatosis by suppressing inflammation and improving lipid metabolism . apart from this, ec increased the activity of alcohol dehydrogenase, aldehyde dehydrogenase, and cyclic adenosine monophosphate (camp) concentration, suggesting that it plays a role in the activities of alcohol-metabolizing enzymes and their regulating mechanisms in ethanol-treated hepatocytes. ec inhibited cytochrome p450 2e1 expression related to the production of ros (yamashita et al. 2015) . a study investigated the protective effect of es in alcoholic fatty liver and found that es treatment suppressed adipogenesis and increased the expression of fatty acid oxidation-related genes, e.g., peroxisome proliferator-activated receptor (ppar)-a and cpt-1, but decreased the expression of sterol regulatory element-binding protein (srebp)-1, a triglyceride (tg) synthesis-related gene, suggesting that es extract could be useful in preventing fatty acid oxidation and reducing lipogenesis in ethanol-induced fatty liver (bang et al. 2016) . neurodegenerative diseases are expected to surpass cancer as the second most common cause of death among the elderly by the 2040s (lilienfeld and perl 1993) . alzheimer's disease (ad) is characterized by loss of memory and other cognitive functions and accounts for most of the deaths in the elderly. an increase in acetylcholinesterase (ache) level around b-amyloid plaques and neurofibrillary tangles is a common feature of ad neuropathology (lam et al. 2016 ). parkinson's disease (pd) is a multidimensional progressive disease with many motor and non-motor features, including cognitive dysfunction. adverse intra-and extracellular effects of toxic a-synuclein are believed to be central to the pathogenesis of pd and other nervous system disorders with lewy body pathology (goldman and postuma 2014; ingelsson 2016) . many categories of natural and synthetic neuroprotective agents have been reported. considering the devastating side effects of synthetic neuroprotective agents, there is growing interest in nutraceuticals or other herbal alternatives (pangestuti and kim 2011) . a study by kang et al. (2013d) demonstrated that ec n-buoh extract regulated the expression and activity of csecretase and a-secretase, leading to a reduction in ab production by the stable cells and a reduction in the basal nuclear location of the psen1 responsible for chromosome mis-segregation in neurodegenerative disease. ec and ek together inhibited ache and bace1 by 84.41 ± 1.70 and 81.17 ± 2.43%, respectively, reducing neuronal cell death and improving dementia, highlighting their synergistic potential. ek likewise showed the highest result of the 2,2 0 -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (abts) assay (ic 50 = 0.07 ± 0.01 mg/ml), which the researchers attributed to its tpc (son et al. 2016) . kim et al. (2014e) further highlighted the importance of ec for its potential analgesic effects in postoperative pain and neuropathic pain. ec extracts (300 mg/ kg) significantly increased the mechanical withdrawal therapy value. the number of ultrasonic distress vocalizations in the treated group of rats was reduced at 6 and 24 h after plantar incision operation (62.8%). ec also increased the paw withdrawal latency in hot-and coldplate tests in the plantar incision rats. after 15 days of continuous treatment, ec (300 mg/kg) alleviated the spared nerve injury-induced hypersensitivity response, which could revolutionize the use of natural resources for therapeutic treatment. matrix metalloproteinases (mmps), known as matrixins, are a large family of similar proteolytic enzymes involved in tissue remodeling associated with various physiological and pathological processes, such as morphogenesis, angiogenesis, tissue repair, arthritis, chronic heart failure, chronic obstructive pulmonary disease, chronic inflammation, and cancer metastasis. as a result, mmps are considered viable drug targets in the therapy of those diseases (dormán et al. 2010) . according to gelatin zymography results, extracts harvested from ec and ecklonia bicyclis showed higher inhibitory effects on mmp-2 and -9 activity than those from the other marine plants at concentrations of 10, 50, and 100 lg/ml (bae et al. 2015) . the global epidemic of bacterial resistance to existing antibiotics such as b-lactams and quinolones necessitates the discovery of potent candidates from natural resources, both terrestrial and marine. the evolution of antibacterial biomolecules alongside bacteria over millions of years allow them to overcome strains such as methicillin-resistant staphylococcus aureus (mrsa) and fluoroquinoloneresistant pseudomonas (ramanan et al. 2016) . therefore, exploring and developing cheaper and more effective natural antimicrobial agents with better potential and fewer side effects than existing antibiotics, good bioavailability, and minimal toxicity is a public health priority (pérez et al. 2016) . a recent study showed that ec with ciprofloxacin evinced potent antibacterial activity against enterococcus faecalis, showing synergistic effects. etoac exhibited the strongest antibacterial activity, with a minimum inhibitory concentration (mic) value of 128 lg/ml against e. faecalis strains. furthermore, the combination of ciprofloxacin and the etoac fraction resulted in a p fic min of 0.188 and p fic max range of 0.508-563, suggesting that the ciprofloxacin-etoac combination resulted in an antibacterial synergy effect against e. faecalis (kim et al. 2015c) . ec led to the strongest growth effects on three lactic acid bacteria and fish pathogenic bacteria in a dose-dependent manner. secondary metabolites produced by ec significantly inhibited the growth of pathogen bacteria. in a further in vivo study, the co-treatment of ec and l. plantarum improved the growth and mortality of edwardsiella tardainfected zebrafish by regulating the expression of inflammatory molecules such as inos and cox-2. altogether, ec played an important role as a potential prebiotic and protected against the infection caused by e. tarda injection in zebrafish . ec (1.0%) improved the growth and body weight of olive flounder and decreased its mortality from e. tarda without changing its biochemical profile. the supplementation of 1.0% ethanolic extract of ec also enhanced the innate immune response of the fish, as evidenced by a high respiratory burst and increased serum lysozyme and myeloperoxidase activity. thus, ec acted as a prebiotic by improving the innate immune response in fish infected with pathogenic bacteria (lee et al. 2016c ). seanol, a seaweed extract rich in phlorotannins, stimulated mineralization with calcium phosphate, increased antibacterial activity, and increased compressive modulus. seanol and alkaline phosphatase (alp) interacted in a non-covalent manner. seanol exhibited antibacterial activity against mrsa with comparable cytotoxicity toward mg-63 osteoblast-like cells, suggesting its mineralizability and antibacterial activity (douglas et al. 2016) . venkatesan et al. (2014) reported the rapid biological synthesis of gold nanoparticles (au nps) using ec by reducing chloroauric acid. fourier transform infrared (ftir) spectroscopic analysis showed that au nps functionalized with biomolecules (a primary amine group, a hydroxyl group, and other stabilizing functional groups) showed good antimicrobial activity and biocompatibility with a human keratinocyte cell line. the results indicate that aunps might have promising applications in drug delivery, tissue engineering, and biosensor development. er extract prepared by using celluclast-assisted extraction induced significantly higher production of butyrate (9.2 lmol/ml) and promoted the growth of beneficial bacteria such as bifidobacterium and lactobacillus, improving gut health (charoensiddhi et al. 2016 ). ec and ek had the strongest inhibitory effects when tested using the agar disk diffusion method, with an mic of 0.31 mg/ ml and no cytotoxicity even at 200 lg/ml, indicating their potential as therapeutic agents for acne vulgaris ). obesity is a leading preventable cause of death worldwide, with increasing rates in both developed and developing countries. as defined by who, it is a medical condition in which excess body fat has accumulated to the extent that it can have negative effects on health, and it is often comorbid with diseases such as type 2 diabetes, hypertension, coronary disease, and cancer. in 1997, who announced that obesity had reached epidemic proportions worldwide (kumar and rao 2013; caballero 2007) . commercial drugs, such as orlistat; lorcaserin; and a combination of phentermine, topiramate, and bariatric surgery, such as roux-en-y bypass or gastric banding, are available. however, concerns about perioperative mortality, surgical complications, and the frequent need for reoperation mean that those procedures tend to be reserved for morbid obesity (rodgers et al. 2012 ). thus, remedies from natural sources are vital, especially those from marine sources. the anti-adipogenic activity of ec was determined by measuring lipid accumulation in adipocytes. the n-buoh fraction particularly reduced lipid accumulation and glucose consumption; the adipogenic transcription factors pparc and srebp-1c; and the adipogenic specific genes fatty acid binding protein (fabp)-4, fabp-1, fatty acid synthase (fas), lipoprotein lipase (lpl), hormone-sensitive lipase (hsl), and acyl-coa synthetase 1 (acs1) . ec polyphenol extract regulated fat metabolism, inflammation, and the antioxidant defense system in hfd-induced obese mice. ec polyphenol extract supplementation reduced body weight gain, adipose tissue mass, plasma lipid profiles, hepatic fat deposition, insulin resistance, and the plasma leptin/adiponectin ratio derived from hfd-induced obesity. furthermore, ec polyphenol extract supplementation selectively ameliorated the hepatic protein levels associated with lipogenesis, inflammation, and the antioxidant defense system, as well as activation of ampk and sirtuin (sirt1) to inhibit obesity (eo et al. 2015) . there has been a worldwide increase in allergic diseases, including atopic dermatitis, asthma, allergic rhinitis, and food allergies, possibly because environmental factors are interacting with genetic factors to sensitize individuals (tanaka 2014) . numerous studies have been done in the search for anti-allergens, especially from marine resources. ec extract exhibited excellent inhibitory activity against crude histidine decarboxylase (hdc), reducing overall histamine production by 46.29% and thereby enhancing the safety of mackerel muscle (kim et al. 2014g) . another report presented a 32% inhibition of hdc at a concentration of 1 mg/ml, reducing histamine poisoning by decreasing histamine production in mackerel (jung et al. 2013b) . ec and ek together inhibited the degranulation of a rat basophilic leukemia cell line (rbl-2h3), mitigating allergic symptoms and highlighting their synergistic potential (yoshioka et al. 2013) . phlorotannin-rich es inhibited enzymatic activity and degranulation in stimulated rbl-2h3 cells in a dose-dependent manner. the meoh: chloroform (1:2, v/v) (m/c) extract of es also inhibited enzyme activity and degranulation in stimulated rbl cells in a dose-dependent manner. the active compounds in the m/c extract might be phenolic compounds, such as phlorotannins, because the m/c extract became inactive when the phenolic compounds were removed (sugiura et al. 2012) . ionizing radiation produces deleterious effects, deterministic or stochastic, on living organisms, though it can have health benefits in the form of radiation therapy for the treatment of cancer or thyrotoxicosis. the benefits of ionizing radiation are compromised by the side effects that result from radiation-induced damage to normal tissue, and the synthetic agents used to combat those side effects, wr2721 (amifostine), ok-432, and ethiofos, have their own serious side effects, including decreased cellular function, nausea, hypotension, and death (baliga and rao 2010; park et al. 2008) . therefore, investigators have directed their attention toward plants and other natural products. enzymatic extracts of ec exhibited radioprotective properties, including the modulation of apoptosis via inhibition of the nf-jb signaling pathway ). ecklonia species and their constituents exhibit pronounced inhibitory effects against oxidative stress (table 2) . ec phlorotannins, including phloroglucinol (pg), eckol, dieckol, eckstolonol, and triphlorethol-a (tpa), scavenged intracellular ros, inhibited lipid peroxidation, and suppressed 2,2 0 -azobis(2-amidinopropane) dihydrochloride (aaph)-induced cell death in zebrafish embryos. these phlorotannins maintained the positive changes in morphological phenomena; pericardial edema, yolk sac edema, and growth retardation in zebrafish embryos exposed to aaph were not observed in the groups also exposed to phlorotannins, indicating that the phlorotannins possess prominent antioxidant activity against aaph-mediated toxicity (kang et al. 2013a) . tpa further exhibited a protective effect against oxidative stress-induced dna-base damage, especially 8-oxoguanine (8-oxog), in v79-4 cells. decreased level of 8-oxog induced by h 2 o 2 were confirmed by an increase in ogg1 mrna and ogg1 protein levels. tpa restored the expression of nuclear nrf2, small maf protein, and the nrf2-maf complex and also increased nrf2 binding to are sequences and the resulting ogg1 promoter activity. sequentially, it maintained the levels of the phosphorylated forms of akt kinase downstream of phosphatidylinositol 3-kinase (pi3k) and erk, which are regulators of ogg1, suggesting that ogg1 induction by tpa involves the pi3k/akt and erk pathways . eckol from ec also attenuated the high intracellular ca 2? levels stimulated by h 2 o 2 , decreased the augmented levels of mitochondrial ros, recovered h 2 o 2 -diminished atp level and succinate dehydrogenase activity, and induced manganese superoxide dismutase through phosphorylated ampk and forkhead box o3a (foxo3a), which showed a cytoprotective effect on chang liver cells ). fucoidan extracted from ec exhibited prominent effects on peroxyl radical scavenging activity and 2, 2 0 -azobisdihydrochloride-induced oxidative stress in vero cells and reduced ros generation, lipid peroxidation, and cell death in a zebrafish model, proving its antioxidant capacities in vitro and in vivo despite being neither a polyphenol nor a flavonoid. although, it did not contain a benzene ring or conjugated structure, it exhibited antioxidant potential (kim et al. 2014c ). in another study, 6,6bieckol, 7-phloroeckol, dieckol, and phlorofucofuroeckol (pff-a) isolated from ec significantly inhibited high glucose-induced ros and cell death in zebrafish. dieckol significantly reduced heart rate, ros, no, lipid peroxidation generation, and cell death and also reduced the overexpression of inos and cox-2, thereby preventing oxidative stress . es could be used as a natural antioxidant and cytoprotective agent. es inhibited ros even at a concentration of 25 lg/ml, yielding five compounds (pff-a, dieckol, eckstolonol, pg, and eckol) that inhibited total ros, proving its use as a potent scavenger (kang et al. 2004) . three active compounds were isolated from es, pff-a, dieckol, and dioxinodehydroeckol (dhe), among which pff-a and dieckol significantly suppressed intracellular ros in lipopolysaccharide (lps)induced raw264.7 cells, and dhe scavenged dpph radicals. pff-a also significantly inhibited the lps-induced production of no and prostaglandin e2 (pge2) through the down-regulation of inos and cox-2 protein expression ). dieckol and pff-a obtained from boiling water-and organic solvent extracts of ec and es showed almost 9-and 7-fold stronger antioxidant activity than the standard butylhydroxytoluene, and 6-and 4-fold greater activity than l-ascorbic acid in molar concentration, anti-inflammatory activity ecklonia cava dieckol decreased blood glucose level kang et al. (2013b) insulin resistance lee and jeon (2015) akt up-regulation kim et al. (2016b) phlorofucofuroeckol-a a-glucosidase and a-amylase fucodiphlorethol g uvb induced oxidative stress kim et al. (2014f) respectively (chowdhury et al. 2014) . three known phlorotannins, eckol, pff-a, and dieckol, along with one new compound, eckstolonol, were obtained from es, and the new compound was found to be a potent radical scavenger through its elimination of dpph radicals (kang et al. 2003b) . eckol suppressed the production of intracellular ros and increased glutathione peroxidase (gsh) level in hepg2 cells. it inhibited the production of ros in h 2 o 2treated hepg2 cells in a dose-dependent manner, and the total relative level of 40 lm eckol was estimated to be 11.2 ± 0.85% compared to the non-treated group, making it a much stronger ros scavenger than n-acetylcysteine (nac). the intracellular gsh content in hepg2 cells was dose-dependently enhanced by eckol treatment, indicating higher antioxidant activity than nac. thus, eckol mediated the expression of ho-1 in hepg2 cells, which was regulated by nrf2 activation via the jun n-terminal kinases (jnk) and pi3k/protein kinase b (akt) signaling pathways, suggesting that it is a natural antioxidant and cytoprotective agent (jun et al. 2014 (fig 1) . ec extract and its major compound (dieckol) significantly increased the survival rate and attenuated liver and kidney damage in mice with a whole-body inflammatory condition by down-regulating pro-inflammatory factors (inos, cox-2, tnf-a, il-6, and hmgb-1) via a nik/tak1/ ikk/ijb/nf-jb pathway. additionally, ec increased nrf2 and ho-1 expression, reducing inflammation (yang et al. 2016) . dieckol (5 and 10 lm) inhibited the production of a macrophage-derived chemokine, c-c motif chemokine 22, induced by interferon-c (10 ng/ml) in a dose-dependent manner and inhibited the nuclear translocation of signal transducers and activators of transcription 1 (stat1). these results showed that dieckol produced anti-inflammatory effects via the down-regulation of stat1 activation . in addition, 8,8 0 -bieckol isolated from ec suppressed key inflammatory mediators such as no and pge2 in raw264.7 macrophages. the inhibition of no occurred by suppressing lps-induced expression of inos at the mrna and protein levels in primary macrophages and raw264.7 cells. likewise, 8,8 0 -bieckol reduced the production and mrna expression of the inflammatory cytokine il-6, but not that of tnf-a, in raw264.7 cells. furthermore, 8,8 0 -bieckol significantly reduced mortality in lps-induced septic mice, which indicates that the anti-inflammatory properties of 8,8 0bieckol are associated with the suppression of no, pge2, and il-6 via negative regulation of the nf-jb pathway and ros production in lps-stimulated raw264.7 cells ). dieckol extracted from ec suppressed lpsinduced inos expression in mouse leukemic macrophage raw 264.7 cells, decreasing both lps-induced no production and inos promoter-driven transcriptional activity in a dose-dependent manner. furthermore, it prevented lps-mediated nf-jb activity. it also diminished lpsmediated p65 nuclear translocation or ijba phosphorylation dose-dependently and reduced lps-induced phosphorylation of mapks, especially p38mapk. collectively, these findings suggest that dieckol acts as a negative regulator of lps-mediated inos induction by suppressing nf-jb activity, implying a mechanistic role for dieckol in the regulation of the inflammatory response . the in vivo anti-inflammatory effect of fucoidan from ec was studied using tail-cutting-induced recent advances in pharmacological research on ecklonia species: a review 991 and lps-stimulated zebrafish models; it inhibited tail-cutting-induced and lps-stimulated ros and no generation and also showed a protective effect against the toxicity induced by lps exposure in zebrafish embryos ). pff-a isolated from es showed potential antiinflammatory properties in macrophages stimulated by lps. for this, 20 lm of pff-a significantly inhibited inos and cox-2 mrna levels induced by lps stimulation. similarly, levels of pro-inflammatory cytokines such as il-1b, il-6, and tnf-a were significantly reduced. pff-a further inhibited the promoter activities of inflammatory mediators and transcriptional factors. thus, pff-a regulated inos and cox-2 expression through the nf-jbdependent transcriptional control associated with the inhibition of multiple signaling proteins, suggesting that pg derivatives could be potential treatments for inflammatory diseases . the etoac fraction of es, along with its isolated compounds 2-phloroeckol, 6,6 0bieckol, pff-a, pff-b, and 974-b, inhibited the production of lps-induced no and pge2 and reduced the expression of inos and cox-2 in a dose-dependent manner (wei et al. 2016 ). most of the investigations on phlorotannins, particularly those derived from brown algae, indicate their promising anti-diabetic effects. for example, 2,7 00 -pg-6,6 0 -bieckol isolated from ec improved postprandial hyperglycemia through a-glucosidase and a-amylase activity in stz-induced diabetic mice. it showed higher inhibitory activity than the positive control, acarbose (a-glucosidase ic 50 of 130.04 lm; a-amylase ic 50 of 165.12 lm) (lee et al. 2017a ). in addition, 6,6 0 -bieckol purified from ec at concentrations of 10 or 50 lg/ml significantly inhibited high glucose-induced glucotoxicity and dose-dependently reduced the level of thiobarbituric acid reactive substances (tbars), generation of intracellular ros, and the level of no. furthermore, 6,6 0 -bieckol prevented the apoptosis of rat insulinoma cells under high-glucose conditions, attributed to increased expression of the anti-apoptotic protein bcl-2 and reduced expression of the pro-apoptotic protein bax, establishing ec as a potential nutraceutical candidate for protection against glucotoxicity (park et al. 2015a ). 6,6 0 -bieckol from ec at concentrations of 10 or 50 lg/ml markedly suppressed high-glucose-induced cytotoxicity and dose-dependently decreased the increased levels of tbars, ros, and no caused by high glucose. in addition, it down-regulated the overexpression of inos, cox-2, and nf-jb proteins in huvecs, indicating its therapeutic ability to treat diabetic endothelial dysfunction and related complications ). ec-derived dieckol noticeably decreased blood glucose level, serum insulin level, and body weight. furthermore, it reduced tbars and increased the activities of antioxidant enzymes, including superoxide dismutase (sod), catalase (cat), and gsh-px, in liver tissue. in addition, western blotting analysis revealed that dieckol increased the phosphorylation levels of ampk and akt observed in muscle tissues, suggesting that dieckol could be a therapeutic agent for type 2 diabetes ). dieckol-rich extract from ec led to a significant decrease in postprandial glucose level, insulin, and c-peptide level after 12 weeks without any adverse effects. in other words, ec supplementation significantly contributed to lowering postprandial hyperglycemia and reducing insulin resistance . in addition, dieckol improved blood glucose regulation, hepatic glucose metabolic regulation, and akt up-regulation in alloxan-induced hyperglycemic zebrafish ). pff-a isolated from ec showed prominent inhibitory effects against a-glucosidase and a-amylase activities, with ic 50 values of 19.52 and 6.34 lm, respectively, which were higher than those of acarbose. moreover, the area under the curve was significantly lower after pff-a administration (2296 vs. 2690 mmol min/l) in diabetic mice, indicating its potent anti-diabetic activity (you et al. 2015) . the a-glucosidase inhibitory property of em and its isolated compounds pg, dibenzo [1,4] dioxine-2,4,7,9-tetraol, and eckol exceeded that of the positive control, suggesting their potency as oral anti-diabetic drugs or functional food ingredients, with a promising role in the formulation of medicines and nutritional supplements (rengasamy et al. 2013) . es exhibited inhibitory activity on glucose-mediated protein damage, advanced glycation end-products (age), and rat lens aldose reductase (rlar), hinting at potential antidiabetic activity. in spite of negligible activity against age, es phlorotannins, including eckol, dieckol, and 7-phloroeckol, possessed inhibitory activity on glycation, which indicates that pff-a could be used to prevent diabetic complications (jung et al. 2008) . likewise, fucosterol from es inhibited rlar, human recombinant aldose reductase, protein tyrosine phosphatase 1b (ptp1b), and a-glucosidase activity (jung et al. 2013a ). pff-a, dieckol, and 7-phloroeckol isolated from es were potent and non-competitive ptp1b inhibitors, with ic 50 values ranging from 0.56 to 2.64 lm, and a-glucosidase inhibitors, with ic 50 values ranging from 1.37 to 6.13 lm. interestingly, pff-a and 7-phloroeckol were non-competitive, whereas dieckol exhibited competitive inhibition in a a-glucosidase assay. thus, isolated phlorotannins from both algae possessed marked ptp1b and a-glucosidase inhibitory activity that could contribute to the development of therapeutic agents to control postprandial blood glucose level and prevent diabetic complications (moon et al. 2011) . published results clearly indicate the anti-diabetic potential of brown seaweed and its derived components, which could be used as nutraceuticals or functional foods to treat diabetes. pyrogallol-phloroglucinol-6,6'-bieckol fucoidan dieckol from ec exerted cytotoxicity in lx-2, hsc-t6, and hepg2 cells, with reduced fibrosis features (large, spread out, and flattened polygonal shapes) in lx-2 cells compared with the untreated control. in addition, it attenuated the expression of a-sma and tgf-b1, increased the sub-g1 phase population, induced caspase-3 activation, and cleaved parp in hepatic stellate cells. thus, dieckol suppressed liver fibrosis via caspase activation, micro-rna-mediated jnk activation, and via nf-jb inhibition ). an in vitro study of dieckol showed the strongest protective effect and lowest cytotoxicity against ethanol-induced cell apoptosis in chang liver cells. western blot analysis revealed reduced cell apoptosis through the activation of b cell lymphoma-extra large (bcl-xl) and parp and down-regulation of bax and caspase-3, providing evidence for this potential protective agent against ethanol-induced liver diseases. in an in vivo study in a zebrafish model, the dieckol-treated group scavenged intracellular ros and prevented lipid peroxidation and ethanol-induced cell death in embryos (kang et al. 2013c cytochrome c from mitochondria to the cytosol in a dosedependent manner (lee et al. 2012a ). pg isolated from es decreased the formation of lipid peroxide in acetaminophen (800 mg/kg, i.p.)-induced rats. though the activities of cytochrome p-450, aminopyrine n-demethylase, and aniline hydroxylase were unchanged, pg restored enzyme activity in the livers of pretreated-rats, suggesting that acetaminophen-induced hepatic lipid peroxidation could be reduced by enhancing the activity of glutathione s-transferase (gst) (park 2000) . all of these data suggest that ecklonia species could be potent hepatoprotective agents. dieckol isolated from ec suppressed the phosphorylation of erk in lps-stimulated bv-2 microglia (1 lg/ml), attenuated akt phosphorylation, and increased the expression of gp91 phox , a catalytic component of the nicotinamide adenine dinucleotide phosphate (nadph) oxidase complex responsible for microglial ros generation. furthermore, dieckol offered neuroprotection, as confirmed in an enhanced green fluorescent protein-transfected b35 neuroblastoma cell line (cui et al. 2015) . dieckol isolated from ec showed potent activity on rotenone-induced oxidative stress in sh-sy5y cells, a human dopaminergic neuronal cell line. it reduced rotenone-induced cell death and retarded rotenone-induced a-synuclein aggregation in asynuclein-overexpressing sh-sy5y cells, which prevented a-synuclein aggregation, adding to its role in the prevention of pd (cha et al. 2016) . pg (1,3,5-trihydroxybenzene) from ec attenuated the increase in ros accumulation induced by oligomeric ab1-42 treatment in the ht-22 hippocampal cell line and ameliorated the reduction in dendritic spine density induced by ab1-42 treatment in rat primary hippocampal neuron cultures. also, pg attenuated cognitive dysfunction in the hippocampal region, indicating its anti-ad effects (yang et al. 2015b) . another report showed that pff-a from ec had particularly potent inhibitory activity (ic 50 = 0.95 lm) for butyrylcholinesterase (bche), more than 100-fold greater than for ache. other polyphenols (pff-a, eckol, 6,6 0 -bieckol, 8,8 0 -bieckol, and dieckol) inhibited glycogen synthase kinase 3b, which is related to the formation of hyperphosphorylated tau and generation of ab. additionally, pff-a inhibited amyloid precursor protein biosynthesis and showed very strong bace1 inhibitory activity, with a submicromolar ic 50 , making it an interesting potential drug candidate for ad ). an oligosaccharide sugar chain derived from ek inhibited the toxicity induced by the ab protein in both primary cortical cells and the sh-sy5y cell line, inhibiting apoptosis and fibril formation and indicating its potency for ad (hu et al. 2004) . em exhibited potent activity against ache, as did its isolated compounds pg, dibenzo [1, 4] dioxine-2,4,7,9-tetraol, and eckol, highlighting its potential as a functional food ingredient for the management of neurodegenerative disorders (kannan et al. 2013) . a phlorotannin preparation (pp) containing eckstolonol from es and ec exhibited a hypnotic effect by modulating the benzodiazepine site of the c-amino butyric acid receptor. pp ([250 mg/kg) decreased sleep latency and increased non-rapid eye movement sleep (nrems). likewise, eckstolonol significantly decreased sleep latency ([12.5 mg/ kg) and increased the amount of nrems (50 mg/kg). in addition, the hypnotic effects were completely abolished by pretreatment with flumazenil, suggesting that the phlorotannins could potentially be used as an herbal medicine for insomnia and offering a promising structure for the development of novel sedative-hypnotics . fucosterol and fucoxanthin from es showed noncompetitive and mixed-type inhibition against b-site amyloid precursor protein cleaving enzyme 1 (bace1). furthermore, molecular docking simulation results demonstrated the effective binding of isolated compounds by the bace1 enzyme, suggesting that both compounds could be used beneficially in the treatment of ad and providing potential guidelines for the design of new bace1 inhibitors . isolated eckol and dieckol attenuated tnf-a induced expression of mmp-1 and basal expression, though the expression of timp-1 was not affected. however, they did reduce both nf-jb and ap-1 reporter gene activity, which strongly indicates their mmp inhibitory potential. one study demonstrated the inhibitory effect of eckol and dieckol isolated from es on mmp-1 expression in human dermal fibroblasts, suggesting the possibility of developing an agent to prevent and treat skin aging (joe et al. 2006) . obviously, marine sources outweigh terrestrial sources in abundance in the development of safe mmp nutraceuticals. anticoagulation occurs by inhibiting the key serine proteases thrombin and factor xa, facilitated by accelerating the activity of the major physiological serine protease inhibitor serpin-antithrombin iii. heparin, a highly sulfated polysaccharide present in mammalian tissues, is commercially used as a blood anticoagulant. it has antihemostasis, fibrinolytic potentiation, and anti-lipemic activity in addition to coagulation activity. though it is a primary anticoagulant, difficulty in isolating it and its hemorrhagic side effects limit its use and drive researchers to search for novel anticoagulants from natural resources free from cytotoxicity (shanmugam and mody 2000) . studies of the anticoagulant bioactivity of brown seaweeds suggest that they have more than one mechanism of action, including the direct and indirect inhibition of thrombin through the activation of thrombin inhibitors (e.g., antithrombin and heparin cofactor). interestingly, the algal fucans were found to have anticoagulant activity through a direct inhibition of thrombin, whereas the invertebrate fucans showed activity through an indirect inhibition of the enzyme, which required antithrombin and heparin cofactor ii, and that has driven the rapid discovery of anticoagulants from marine resources (jiao et al. 2011) . fucoidan isolated from ek significantly inhibited the generation of thrombin and factor xa in the intrinsic pathway. furthermore, it inhibited the formation of prothrombin-activating complex (i.e., prothrombinase); the ic 50 of thrombin generation was one-tenth to one-seventh that of the activity of the thrombin in plasma, whereas the antithrombin activity of fucoidan was mediated by heparin cofactor ii in plasma. this further highlights the relationship between the molecular weight of sulfated polysaccharides and their anticoagulant activity such that the higher molecular weight fucans show greater anticoagulant activity than sulfates with a lower molecular weight (nishino et al. 1999 (nishino et al. , 1991a . sulfated polysaccharides, mainly 3-linked and 3, 4-disubstituted fucopyranosyl residues isolated from ek, exhibited potent anticoagulant activity (nishino et al. 1991b) . some potent and novel antiplasmin inhibitors such as pff-a and eckol isolated from ek inhibited the action of alpha 2-macroglobulin (ic 50 = 1.0 lg/ml) and alpha 2-plasmin inhibitor (ic 50 = 0.3 lg/ml), the main plasmin inhibitors in plasma (fukuyama et al. 1990 (fukuyama et al. , 1989 . thus, the development of antithrombotic algal polysaccharides would be advantageous because their use would avoid the potential for contamination with prions or viruses present in commercial heparins, which are obtained from pig and bovine intestines. moreover, with more specific activities or targets, algal sulfated polysaccharides could find applications complementary to heparin. dieckol from ec significantly blocked the cleavage of sars-cov 3cl(pro) in a cell-based assay without showing any toxic effects and exhibited a high association rate in the spr sensorgram, forming strong hydrogen bonds to the catalytic dyad (cys145 and his41) of the sars-cov 3cl(pro) ). both phlorotannins from ec and isolated epibiotic bacteria showed potent antibacterial activity in close affiliation with the genus bacillus (kanagasabhapathy et al. 2006) . another novel bacterial strain, designated ec29t, was isolated from ec, and it showed potent antibacterial activity (kim et al. 2015d) . another study suggested that the compounds eckol, dieckol, pff, and 7-phloroeckol exhibited potent antiviral activity, with ic 50 ranging from 10.8 ± 1.4 to 22.5 ± 2.2 lm against porcine epidemic diarrhea. these compounds completely blocked the binding of viral spike protein to sialic acids at concentrations less than 36.6 lm by hemagglutination inhibition. pff and dieckol inhibited viral replication with ic 50 values of 12.2 ± 2.8 and 14.6 ± 1.3 lm, respectively, in the post-treatment assay and exhibited stronger inhibition of viral rna and viral protein synthesis in later stages (18 and 24 h) than in early stages (6 and 12 h), suggesting their potential as natural therapeutic drugs against coronavirus infection ). dieckol and 8,8 0 -bieckol (hexamers) isolated from ek were tested against the food-borne pathogenic bacteria campylobacter jejuni with an mic of 50 mg/l and 0.03 lmol/ml, respectively, which were effective against mrsa as determined by a broth microdilution method. the bactericidal effects of the phlorotannins were more pronounced than those of the catechins . one study showed that n-buoh fractions with isolated phlorotannins (tpa, eckol, and dieckol) increased glycerol secretion and reduced the regulation of adipogenic transcription factors, pparc, ccaat/enhancer-binding protein (c/ebpa), and tnfa. those phlorotannins also reduced the differentiation-dependent factor 1/srebp-1c and downstream genes such as fabp-4, fatty acid transport protein-1, fas, leptin, and acs1, whereas they increased the mrna expression of hormone-sensitive lipase while suppressing perilipin expression to treat obesity . dieckol from ec down-regulated the expression of pparc, c/ebpa, srebp-1, and fabp-4, showing anti-adipogenic effects on adipocyte differentiation through the activation and modulation of the ampk signaling pathway to improve obesity . enzyme-treated ec and its isolated compounds (eckol, dieckol and pff-a) exhibited potent adipogenic activity in 3t3-l1 adipocytes. dieckol was found to be the major compound in the enzymatic extract, with a concentration of 16 mg/g. in addition, 50 lg/ml of ec extract inhibited glucose utilization and tg accumulation, as confirmed by oil red o staining. additionally, it decreased the expression of ccaat/(c/ebp)a, srebp-1c, adipocyte-fabp, fas, and adiponectin. thus, ec prevented adipogenesis by affecting the activation of the c/ebpa signaling pathway and the resulting adipogenesis-related gene expression (kim and nam 2017) . in a similar report, dieckol from ec inhibited lipid accumulation via activation of ampka signaling and cell-cycle arrest in 3t3-l1 cells and mouse and zebrafish models . seapolynol derived from ec inhibited triglyceride synthetic enzymes such as diacylglycerol acyltransferase 1, gpat3, kruppellike factor 4 (klf4), klf5, c/ebpb, c/ebpd, and protein c-ets-2, whereas it up-regulated klf2, an anti-early adipogenic factor, preventing metabolic disorders . fucosterol was isolated from the strong anti-adipogenic ch 2 cl 2 -soluble fraction from es, with significant inhibition (40.5%) of intracellular lipid accumulation at a non-toxic concentration. the anti-adipogenic activities of es, along with the isolated fucosterol, reduced lipid content in a concentration-dependent manner without showing any cytotoxicity. fucosterol treatment also yielded a decrease in the expression of the adipocyte markers pparc and c/ebpa in a concentration-dependent manner. taken together, these results suggest that fucosterol inhibits the expression of pparc and c/ebpa, resulting in a decrease in lipid accumulation in 3t3-l1 pre-adipocytes, thereby indicating the potential of es and its bioactive component fucosterol as anti-obesity agents (jung et al. 2014b ). similarly, fucosterol isolated from es down-regulated the insulin-triggered pi3k/akt and erk pathways, which subsequently decreased the expression of adipogenic transcription factors, including pparc, c/ebpa, and srebp-1. in addition, fucosterol enhanced sirt1 expression and decreased phospho-foxo1 expression, which resulted in the activation of foxo1 and revealed that fucosterol inhibited adipogenesis of 3t3-l1 preadipocytes at concentrations of 25 and 50 lm through the modulation of the foxo1 signaling pathway (lee et al. 2017b) . compound 974-b isolated from ek inhibited the differentiation of mouse embryonic fibroblasts and 3t3-l1 cells into adipose cells by acting as the peptidyl prolyl cis/trans isomerase inhibitor responsible for the uptake of tg and the differentiation of fibroblasts into adipose cells in response to insulin stimulation without inducing cytotoxicity. this finding suggests that 974-b could be a lead drug candidate for obesity-related disorders (mori et al. 2014) . altogether, these results suggest that several ecklonia species possess potent activity that might be exploited in adjunct therapy for obesity. dieckol from ec inhibited mast cell activation and mast cell-mediated type i allergic reactions caused by igespecific antigen, mainly through the marked downstream signaling of fceri. a high dose of dieckol suppressed hypersensitive reactions, offering another target molecule for the prevention or treatment of mast cell-dependent allergic diseases (ahn et al. 2015c) . six phlorotannins isolated from ek, pg, an unknown tetramer, eckol, pff-a, dieckol, and 8,8 0 bieckol, inhibited hyaluronidase at concentrations of 280, 650, [800, 140, 120, and 40 lm, respectively. also, 8, , the strongest hyaluronidase inhibitor, acted as a competitive inhibitor with an inhibition constant (ki) of 3.5 lm . park et al. (2011) described the multi-faceted protection mechanisms of pg against oxidative stress caused by ionizing radiation in mice. pg inhibited apoptosis and strengthened hematopoiesis. it increased the viability of splenocytes without cytotoxicity and significantly enhanced the proliferation of splenocytes by limiting the increment of sub-g(1) dna contents via the inhibition of ros production in 2 gy-irradiated splenocytes. in addition, pg significantly decreased dna damage and the number of apoptotic fragments in lymphocytes during oxidative stress and increased the counts of endogenous spleen colony forming units (cfus) compared with control mice exposed to ionizing radiation. a similar study confirmed that pg protected against small intestine damage caused by ionizing radiation, raising the apoptosis threshold of jejunal crypt cells. pg regenerated the intestinal crypts and down-regulated the expression level of proapoptotic molecules such as p53, bax, and bak in the small intestine. pg further augmented antiapoptotic molecules such as bcl-2 and bcl-x(s/l) (ha et al. 2013) . cancer is the uncontrolled growth of cells that can invade and spread to distant sites of the body. cancer can have severe health consequences and is a leading cause of death. lung, prostate, colorectal, stomach, and liver cancer are the most common types of cancer in men, whereas breast, colorectal, lung, uterine cervix, and stomach cancer are the most common types among women. more than 30% of cancer deaths could be prevented by modifying or avoiding key risk factors. cancer results from a mutation in the chromosomal dna of a normal cell, which can be triggered by both external factors (tobacco, alcohol, chemicals, infectious agents, and radiation) and internal factors (hormones, immune conditions, inherited mutations, and mutations occurring in metabolism) (croce 2008; ferlay et al. 2015) . in korea, cancer accounts for one in four deaths (27.6%), and more than 200,000 new cancer cases were diagnosed in 2012 . although antineoplastic drugs and chemotherapy are available, the deleterious effects of those medications have driven researchers to derive new drug candidates from natural products. dieckol isolated from ec prevented n-nitrosodiethylamine (ndea)-induced rat hepatocarcinogenesis. dieckol administered orally (40 mg/kg body weight) for 15 weeks with 0.01% ndea through the drinking water reversed the activities of hepatic marker enzymes such as aspartate transaminase, alanine transaminase, alp, gamma glutamyl transferase, lactate dehydrogenase, a-fetoprotein, and total bilirubin and increased the elevation of cytochrome p450. dieckol also decreased lipid peroxidative markers (tbars, lipid hydroperoxides, protein carbonyl content, and conjugated dienes) and decreased the antioxidant cascade viz enzymatic antioxidants (such as sod, cat, glutathione peroxidase, gst, glutathione reductase) and non-enzymatic antioxidants (such as reduced glutathione, vitamin c, and vitamin e). dieckol was more effective at 40 mg/kg than at 10 and 20 mg/kg body weight and protected the liver from cancer (sadeeshkumar et al. 2016) . likewise, dieckol showed anti-breast cancer activity by regulating the expression of metastasis-related genes ). ec and its major phlorotannin (dieckol) increased the tumor growth-inhibitory effect of cisplatin and reduced cisplatin-induced nephrotoxicity and weight loss in skov3-bearing mice. furthermore, they enhanced cisplatin-induced apoptosis by stimulating caspases in skov3 and a2780 ovarian cancer cells via down-regulation of akt and nf-jb signaling. thus, ec suppressed cisplatin-induced ros production and cell death in normal hek293 kidney cells, and its major compound dieckol evidently enhanced the suppression of tumor growth by cisplatin with less weight loss and kidney damage in a mouse model . dieckol from ec exhibited cytotoxic effects on a2780 and skov3 ovarian cancer cells, induced the apoptosis of skov3 cells, and suppressed tumor growth without any significant adverse effect in the skov3-bearing mouse model. furthermore, it activated caspase-8,-9, and -3; caused mitochondrial dysfunction; and suppressed the levels of anti-apoptotic proteins. thus, dieckol enhanced intracellular ros, and the antioxidant n-acetyl-l-cysteine (nac) noticeably reversed the caspase activation, cytochrome c release, bcl-2 downregulation, and apoptosis caused by dieckol through akt and p38 ). in another investigation, ahn et al. (2015b) showed the anticancer effects of crude polysaccharides isolated from ec enzymatic extracts, using amyloglucosidase (amg), viscozyme, protamex, and alcalase enzymes against a colon cancer cell line. among the tested extracts, crude polysaccharides of protamex showed the highest inhibitory effect against the growth of ct-26 cells and dose-dependently increased the formation of apoptotic bodies and the percentage of sub-g1 dna content. furthermore, crude polysaccharides of protamex activated caspase 9 and parp to regulate the expression of bax and bcl-2 and showed the highest inhibitory effect against the growth of ct-26 cells, attributed to their high fucose and sulfated group content, demonstrating anticancer effects on colon cancer cells via regulation of the bcl-2/bax signal pathway. additionally, dieckol isolated from es reduced the number of viable cells and increased the number of apoptotic cells, increased the expression levels of cleaved caspases-(3, 7, 8, and 9) and cleaved poly(adp-ribose) polymerase, increased the permeability of mitochondrial membranes, and increased the release of cytochrome c from mitochondria into the cytosol with an apoptosis-inducing factor. thus, dieckol induced apoptosis via the activation of both death receptors and mitochondrial-dependent pathways in human hepatocellular carcinoma (hep3b) cells (yoon et al. 2013 ). the skin is the human body's largest organ and is colonized by diverse microorganisms, most of which are harmless or even beneficial to their host. it acts as an anatomical barrier that is tough when intact and prevents the entry and colonization of many microbes (grice and segre 2011) . however, continuous exposure to ultraviolet (uv) light leads to various complications correlated with various pathological consequences, such as photo-damage of the skin, which is characterized by distinct alterations in the composition of the dermal extracellular matrix and results in wrinkles, laxity, coarseness, mottled pigmentation, and histological changes that include increased epidermal thickness and connective tissue alteration (rittie and fisher 2002) . therefore, varieties of anti-photoaging or photoprotective compounds from algae and other marine organisms have been isolated. dhe from ec exerted a preventive effect against uvbinduced apoptosis in human keratinocyte (hacat) cells, indicating its benefit as a repair agent for skin damage caused by uvb (ryu et al. 2015) . fucodiphlorethol g from ec reduced uvb radiation-induced loss of mitochondrial membrane potential, the generation of apoptotic cells, and active caspase-9 expression. thus, it prevented oxidative damage-mediated apoptosis induced by uvb irradiation (kim et al. 2014f) . dhe from es inhibited cellular melanin content and the expression of melanogenesis-related proteins, including microphthalmia-associated transcription factor and tyrosinase and tyrosinase-related proteins trp-1 and trp-2, stimulating the phosphorylation of akt in a dose-dependent manner without affecting the phosphorylation of erk (lee et al. 2012b ). angiotensin-i-converting enzyme (ace) plays an important physiological role in the regulation of blood pressure by converting angiotensin i to angiotensin ii, a potent vasoconstrictor. some commercial drugs, such as captopril, ramipril, lisinopril, and enalapril, have unfortunate side effects like cough, taste disturbances, skin rashes or angioneurotic edema, so novel compounds such as phlorotannins have been derived from marine organisms as potential ace inhibitors . jung et al. (2006) reported that, among the seven enzymatically hydroxylated brown algal species, ec was a potent ace inhibitor with an ic 50 value of 0.3 lg/ml. eckol, pff-a, and dieckol from es showed potent ace inhibiting activity with ic 50 values of 70.82 ± 0.25, 12.74 ± 0.15, and 34.25 ± 3.56 lm, respectively, showing the importance of es. the human immunodeficiency virus (hiv) infects cells of the immune system by destroying or impairing their function, ultimately causing immune deficiency and opening the door to opportunistic infections, thereby causing acquired immune deficiency syndrome (aids). approximately 36.7 million people were living with hiv at the end of 2015. as of mid-2016, 18.2 million people were receiving antiretroviral treatment worldwide. seven out of ten pregnant women living with hiv received anti-retroviral treatment (who 2016). more than 30 approved commercial drugs are available for the treatment of aids. these drugs keep the disease under control; however, they are often associated with the emergence of cross-resistant hiv strains and various side-effects (sánchez and holguín 2014; haas et al. 2004 ). so, the need for potential nutraceuticals, especially from marine organisms, to fight this vicious disease is vital. 8,4 000 -dieckol from ec at noncytotoxic concentrations repressed hiv-1 activated syncytia formation, lytic effects, and viral p24 antigen production. furthermore, it selectively inhibited the activity of hiv-1 reverse transcriptase enzyme with 91% inhibition at 50 lm. therefore, it showed high potential and could be considered as a drug candidate for the development of a new generation of therapeutic agents (karadeniz et al. 2014) . other pharmacological activities include immunomodulatory, aphrodisiac, anti-hair loss, hearing repair, urinary tract infection remedy, hair growth performance, cosmetic whitening, osteoarthritis, and bone-related conditions. one study suggested that sulfated polysaccharides from ec induced t and b cell responses via both the jnk and nf-kb pathways . ecklonia bicyclis together with the novel drug tradamixina improved male sexual function in elderly men, particularly libido, mild-moderate erectile dysfunction, ejaculation function, and sexual quality of life (sansalone et al. 2014 ). purified polyphenols from ec increased fibroblast survival in human dermal papilla cells, preventing hair loss . dieckol isolated from ec suppressed ea.hy926 cell proliferation induced by vascular endothelial growth factor, demonstrating its anti-proliferative and anti-migratory effects through a mapk molecular signaling cascade and suggesting its potential as an anti-angiogenic candidate . dhe from ec enhanced osteoblast differentiation, as evidenced by increased cell proliferation, alkaline phosphatase activity, and intracellular cell mineralization, along with enhanced levels of osteoblastogenesis markers at 20 lm in mc3t3-e1 pre-osteoblasts. furthermore, dhe up-regulated phosphorylated erk and c-jnk, which were also stimulated by the bmp signaling pathway . a polyphenol-rich extract from ec showed potent radical-scavenging activity and decreased the abr threshold shifts, suggesting its potential as a preventive agent against temporary threshold shift . water-soluble sulfated fucans isolated from ec induced the degradation of ij-b and the phosphorylation of mapk in raw264.7 cells, implying that they might stimulate raw264.7 cells through the activation of the nf-jb and mapk pathways (cao et al. 2014) . the etoac fraction of ec resulted in elongation of the hair shaft in cultured human hair follicles and activated transition of the hair cycle from the telogen to the anagen phase in the dorsal skin of c57bl/6 mice. furthermore, it induced an increase in igf-1 expression in human dermal papilla cells (bak et al. 2013) . ec and its phlorotannins tpa, eckol, and dieckol attenuated the pathophysiological consequences of osteoarthritis and enhanced osteoblast differentiation, as indicated by increased alkaline phosphatase activity and raised levels of osteoblastogenesis, preventing osteoporosis . undoubtedly, ecklonia species and their active metabolites are potential candidates for drug development, as shown by their plethora of activities against various diseases. our review provides current information on the biological and pharmacological potential of this genus, which could be further used for the development of nutraceuticals. screening and study of the interactions between these algae and their constituents and human systems threatened by disease need to continue. therefore, we recommend rapid screening and isolation to develop novel functional ingredients from ecklonia species. the future of pharmaceuticals based on natural products seems promising. neuroinflammation, oxidative stress and the pathogenesis of alzheimer's disease review: hepatoprotective activity of spirulina species the jnk/nfjb pathway is required to activate murine lymphocytes induced by a sulfated polysaccharide from ecklonia cava dieckol, isolated from the edible brown algae ecklonia cava, induces apoptosis of ovarian cancer cells and inhibits tumor xenograft growth a sulfated polysaccharide of ecklonia cava inhibits the growth of colon cancer cells by inducing apoptosis dieckol, a phlorotannin of ecklonia cava, suppresses ige-mediated mast cell activation and passive cutaneous anaphylactic reaction dioxinodehydroeckol enhances the differentiation of osteoblasts by regulating the expression of phospho-smad1/5/8 anti-hiv-1 activity of phloroglucinol derivative, 6,6 0 -bieckol, from ecklonia cava evaluation of effective mmp inhibitors from eight different brown algae in human fibrosarcoma ht1080 cells ecklonia cava promotes hair growth radioprotective potential of mint: a brief review protective effects of ecklonia stolonifera extract on ethanol-induced fatty liver in rats the global epidemic of obesity: an overview water soluble sulfated-fucans with immune-enhancing properties from ecklonia cava possible role of oxidative stress in the pathogenesis of hypertension dieckol, an edible seaweed polyphenol, retards rotenone-induced neurotoxicity and a-synuclein aggregation in human dopaminergic neuronal cells protective effect of a purified polyphenolic extract from ecklonia cava against noise-induced hearing loss: prevention of temporary threshold shift improved antioxidant activities of brown seaweed ecklonia radiata extracts prepared by microwave-assisted enzymatic extraction impact of extraction processes on prebiotic potential of the brown seaweed ecklonia radiata by in vitro human gut bacteria fermentation marine polyphenol phlorotannins promote non-rapid eye movement sleep in mice via the benzodiazepine site of the gabaa receptor the cytoprotective effects of ethanol extract of ecklonia cava against oxidative stress are associated with upregulation of nrf2-mediated ho-1 and nqo-1 expression through activation of the mapk pathway in vitro antibacterial and anti-inflammatory properties of seaweed extracts against acne inducing bacteria, propionibacterium acnes suppression of nf-jb by dieckol extracted from ecklonia cava negatively regulates lps induction of inducible nitric oxide synthase gene dieckol, a major phlorotannin in ecklonia cava, suppresses lipid accumulation in the adipocytes of high-fat diet-fed zebrafish and mice: inhibition of early adipogenesis via cell-cycle arrest and ampka activation multifunctional activity of polyphenolic compounds associated with a potential for alzheimer's disease therapy from ecklonia cava comparison of ecklonia cava, ecklonia stolonifera and eisenia bicyclis for phlorotannin extraction oncogenes and cancer dieckol attenuates microglia-mediated neuronal cell death via erk, akt and nadph oxidase-mediated pathways enrichment of enzymatically mineralized gellan gum hydrogels with phlorotannin-rich ecklonia cava extract seanol ò to endow antibacterial properties and promote mineralization effects of gametophytes of ecklonia kurome on the levels of glucose and triacylglycerol in db/db, prediabetic c57bl/6j and ifn-c ko mice brown alga ecklonia cava polyphenol extract ameliorates hepatic lipogenesis, oxidative stress, and inflammation by activation of ampk and sirt1 in high-fat diet-induced obese mice cancer incidence and mortality worldwide: sources, methods and major patterns in globocan 2012 structure of an anti-plasmin inhibitor, eckol, isolated from the brown alga ecklonia kurome okamura and inhibitory activities of its derivatives on plasma plasmin inhibitors anti-plasmin inhibitor. vi. structure of phlorofucofuroeckol a, a novel phlorotannin with both dibenzo-1, 4-dioxin and dibenzofuran elements, from ecklonia kurome okamura premotor and non-motor features of parkinson's disease the skin microbiome phloroglucinol protects small intestines of mice from ionizing radiation by regulating apoptosis-related molecules: a comparative immunohistochemical study pharmacogenetics of efavirenz and central nervous system side effects: an adult aids clinical trials group study antioxidative effect of proteolytic hydrolysates from ecklonia cava on radical scavenging using esr and h 2 o 2 -induced dna damage om fucus buccinalis lin acidic oligosaccharide sugar chain, a marine-derived acidic oligosaccharide, inhibits the cytotoxicity and aggregation of amyloid beta protein alpha-synuclein oligomers-neurotoxic molecules in parkinson's disease and other lewy body disorders seapolynol extracted from ecklonia cava inhibits adipocyte differentiation in vitro and decreases fat accumulation in vivo chemical structures and bioactivities of sulfated polysaccharides from marine algae the inhibitory effects of eckol and dieckol from ecklonia stolonifera on the expression of matrix metalloproteinase-1 in human dermal fibroblasts eckol enhances heme oxygenase-1 expression through activation of nrf2/jnk pathway in hepg2 cells angiotensin-converting enzyme i inhibitory activity of phlorotannins from ecklonia stolonifera inhibitory activities of extracts from several kinds of seaweeds and phlorotannins from the brown alga ecklonia stolonifera on glucose-mediated protein damage and rat lens aldose reductase kinetics and molecular docking studies of an antidiabetic complication inhibitor fucosterol from edible brown algae eisenia bicyclis and ecklonia stolonifera inhibitory effects of histamine production in mackerel muscle by medicinal herbs and seaweed extracts protective effect of the edible brown alga ecklonia stolonifera on doxorubicininduced hepatotoxicity in primary rat hepatocytes anti-adipogenic activity of the edible brown alga ecklonia stolonifera and its constituent fucosterol in 3t3-l1 adipocytes kinetics and molecular docking studies of fucosterol and fucoxanthin, bace1 inhibitors from brown algae undaria pinnatifida and ecklonia stolonifera antibacterial activities of marine epibiotic bacteria isolated from brown algae of japan antioxidant and anti-inflammatory activities of ventol, a phlorotannin-rich natural agent derived from ecklonia cava, and its effect on proteoglycan degradation in cartilage explant culture a new phlorotannin from the brown alga ecklonia stolonifera inhibitory phlorotannins from the edible brown alga ecklonia stolonifera on total reactive oxygen species (ros) generation protective effect of marine algae phlorotannins against aaph-induced oxidative stress in zebrafish embryo dieckol isolated from brown seaweed ecklonia cava attenuates type ii diabetes in db/db mouse model protective effect of dieckol isolated from ecklonia cava against ethanol caused damage in vitro and in zebrafish model phlorotannin-rich ecklonia cava reduces the production of betaamyloid by modulating alpha-and gamma-secretase expression and activity dieckol, a component of ecklonia cava, suppresses the production of mdc/ccl22 via down-regulating stat1 pathway in interferon-c stimulated hacat human keratinocytes acetylcholinesterase inhibitory activity of phlorotannins isolated from the brown alga, ecklonia maxima (osbeck) papenfuss anti-hiv-1 activity of phlorotannin derivative 8,4 000 -dieckol from korean brown alga ecklonia cava phlorotannins suppress adipogenesis in pre-adipocytes while enhancing osteoblastogenesis in pre-osteoblasts role of erk/mapk signaling pathway in antiinflammatory effects of ecklonia cava in activated human mast cell line-1 cells enzyme-treated ecklonia cava extract inhibits adipogenesis through the downregulation of c/ebpa in 3t3-l1 adipocytes hepatoprotective constituents of the edible brown alga ecklonia stolonifera on tacrine-induced cytotoxicity in hepg2 cells isolation and identification of phlorotannins from ecklonia stolonifera with antioxidant and anti-inflammatory properties phlorofucofuroeckol a inhibits the lps-stimulated inos and cox-2 expressions in macrophages via inhibition of nf-jb, akt, and p38 mapk evaluation of inhibitory effect of phlorotannins from ecklonia cava on triglyceride accumulation in adipocyte triphlorethol-a from ecklonia cava upregulates the oxidant sensitive 8-oxoguanine dna glycosylase 1 cytoprotective effect of eckol against oxidative stress-induced mitochondrial dysfunction: involvement of the foxo3a/ampk pathway protective effect of fucoidan against aaph-induced oxidative stress in zebrafish model the edible brown seaweed ecklonia cava reduces hypersensitivity in postoperative and neuropathic pain models in rats fucodiphlorethol g purified from ecklonia cava suppresses ultraviolet b radiationinduced oxidative stress and cellular damage inhibitory effects of brown algae extracts on histamine production in mackerel muscle via inhibition of growth and histidine decarboxylase activity of morganella morganii protective effect of marine brown algal polyphenols against oxidative stressed zebrafish with high glucose first evidence that ecklonia cavaderived dieckol attenuates mcf-7 human breast carcinoma cell migration synergistic antibacterial activity of ecklonia cava extract against anti-biotic resistant enterococcus faecalis winogradskyella eckloniae sp. nov., a marine bacterium isolated from the brown alga ecklonia cava 0 -bieckol suppresses inflammatory responses by downregulating nuclear factor-jb activation via akt, jnk, and p38 mapk in lps-stimulated microglial cells a marine algal polyphenol, dieckol, attenuates blood glucose levels by akt pathway in alloxan induced hyperglycemia zebrafish model dieckol, a phlorotannin isolated from a brown seaweed, ecklonia cava, inhibits adipogenesis through amp-activated protein kinase (ampk) activation in 3t3-l1 preadipocytes evaluation of marine algae wakame (undaria pinnatifida) and kombu (laminaria digitata japonica) as food supplements induction of apoptosis by phloroglucinol derivative from ecklonia cava in mcf-7 human breast cancer cells edible brown alga ecklonia cava derived phlorotannin-induced anti-adipogenic activity in vitro minerals, polysaccharides and antioxidant properties of aqueous solutions obtained from macroalgal beachcasts in the noto peninsula periodontal disease and obesity seaweeds as a source of nutritionally beneficial compounds-a review in vitro antiviral activity of phlorotannins isolated from ecklonia cava against porcine epidemic diarrhea coronavirus infection and hemagglutination anti-cholinesterases and memory improving effects of vietnamese xylia xylocarpa inhibitory effects of polyphenols isolated from marine alga ecklonia cava on histamine release efficacy and safety of a dieckol-rich extract (ag-dieckol) of brown algae, ecklonia cava, in prediabetic individuals: a double-blind, randomized, placebocontrolled clinical trial isolation and identification of phlorotannins from ecklonia stolonifera with antioxidant and hepatoprotective properties in tacrine-treated hepg2 cells dioxinodehydroeckol inhibits melanin synthesis through pi3k/ akt signalling pathway in alpha-melanocyte-stimulating hormone-treated b16f10 cells recent advances in pharmacological research on ecklonia species: a review 1003 anti-inflammatory effect of fucoidan extracted from ecklonia cava in zebrafish model alleviating effects of baechu kimchi added ecklonia cava on postprandial hyperglycemia in diabetic mice radio-protective effect of polysaccharides isolated from lactobacillus brevis-fermented ecklonia cava effect of baechu kimchi added ecklonia cava extracts on high glucose-induced oxidative stress in human umbilical vein endothelial cells microrna134 mediated upregulation of jnk and downregulation of nf-kb signaling are critically involved in dieckol induced antihepatic fibrosis a prebiotic role of ecklonia cava improves the mortality of edwardsiellatarda-infected zebrafish models via regulating the growth of lactic acid bacteria and pathogen bacteria a prebiotic effect of ecklonia cava on the growth and mortality of olive flounder infected with pathogenic bacteria a phlorotannin constituent of ecklonia cava alleviates postprandial hyperglycemia in diabetic mice fucosterol, isolated from ecklonia stolonifera, inhibits adipogenesis through modulation of foxo1 pathway in 3t3-l1 adipocytes phlorotannins as bioactive agents from brown algae dieckol as a novel antiproliferative and anti-angiogenic agent and computational antiangiogenic activity evaluation projected neurodegenerative disease mortality in the united states caffeic acid phenethyl ester alleviates asthma by regulating the airway microenvironment via the ros-responsive mapk/akt pathway insulin therapy and hypoglycemia selecting australian marine macroalgae based on the fatty acid composition and anti-inflammatory activity nitric oxide: physiology, pathophysiology, and pharmacology protective effect of phlorotannin components phloroglucinol and eckol on radiation-induced intestinal injury in mice protein tyrosine phosphatase 1b and a-glucosidase inhibitory phlorotannins from edible brown algae, ecklonia stolonifera and eisenia bicyclis a high-throughput screen for inhibitors of the prolyl isomerase, pin1, identifies a seaweed polyphenol that reduces adipose cell differentiation bactericidal activity of phlorotannins from the brown alga ecklonia kurome the influence of sulfate content and molecular weight of a fucan sulfate from the brown seaweed ecklonia kurome on its antithrombin activity an anticoagulant fucoidan from the brown seaweed ecklonia kurome inhibition of the generation of thrombin and factor xa by a fucoidan from the brown seaweed ecklonia kurome cancer statistics in korea: incidence, mortality, survival, and prevalence in 2013 neuroprotective effects of marine algae effects of phloroglucinol isolated from ecklonia stolonifera on the acetaminophen-metabolizing enzyme system in rat radioprotective properties of eckol against ionizing radiation in mice phloroglucinol (pg) purified from ecklonia cava attenuates radiation-induced apoptosis in blood lymphocytes and splenocytes dieckol, a sars-cov 3cl(pro) inhibitor, isolated from the edible brown algae ecklonia cava 0 -bieckol isolated from ecklonia cava protects oxidative stress through inhibiting expression of ros and proinflammatory enzymes in high-glucose-induced human umbilical vein endothelial cells 0 -bieckol protects insulinoma cells against high glucose-induced glucotoxicity by reducing oxidative stress and apoptosis polyphenol-rich fraction of ecklonia cava improves nonalcoholic fatty liver disease in high fat diet-fed mice antimicrobial action of compounds from marine seaweed oxidative stress in the pathogenesis of colorectal cancer: cause or consequence? antibacterial activity of the extracts of marine red and brown algae algaebacteria interactions: evolution, ecology and emerging applications global prevalence of diabetes: estimates for the year 2000 and projections for 2030 potential antiradical and alpha-glucosidase inhibitors from ecklonia maxima (osbeck) papenfuss uv-light-induced signal cascades and skin aging anti-obesity drugs: past, present and future dioxinodehydroeckol protects human keratinocyte cells from uvb-induced apoptosis modulated by related genes bax/bcl-2 and caspase pathway protective effects of dieckol on n-nitrosodiethylamine induced hepatocarcinogenesis in rats drug resistance in the hiv-1-infected paediatric population worldwide: a systematic review alga ecklonia bicyclis, tribulus terrestris, and glucosamine oligosaccharide improve erectile function, sexual quality of life, and ejaculation function in patients with moderate mild-moderate erectile dysfunction: a prospective, randomized, placebo-controlled, single-blinded study jp renew distributors 1906 lombard street heparinoid-active sulphated polysaccharides from marine algae as potential blood anticoagulant agents inhibitory activity of brown algal phlorotannins against hyaluronidase antioxidant activities of phlorotannins isolated from japanese laminariaceae enhancement of human hair growth using ecklonia cava polyphenols in vitro screening for anti-dementia activities of seaweed extracts inhibitory effect of extracts from the brown alga, ecklonia stolonifera, on enzymes responsible for allergic reactions and degranulation in rbl-2h3 cells flavonoids for allergic diseases: present evidence and future perspective potential pharmacological applications of polyphenolic derivatives from marine brown algae oxidative stress and its role in skin disease marine algae-mediated synthesis of gold nanoparticles using a novel ecklonia cava isolation and identification of anti-inflammatory compounds from ethyl acetate fraction of ecklonia stolonifera and their anti-inflammatory action prevent hiv, test and treat all-who support for country impact angiotensin-i-converting enzyme (ace) inhibitors from marine resources: prospects in the pharmaceutical industry phlorotannins from ecklonia cava (phaeophyceae): biological activities and potential health benefits exploiting biological activities of brown seaweed ecklonia cava for potential industrial applications: a review anti-diabetic effect of polyphenols from brown alga ecklonia kurome in genetically diabetic kk-ay mice ecklonia cava polyphenol has a protective effect against ethanolinduced liver injury in a cyclic amp-dependent manner 0 -bieckol, isolated from edible brown algae, exerts its anti-inflammatory effects through inhibition of nf-jb signaling and ros production in lps-stimulated macrophages brown algae phlorotannins enhance the tumoricidal effect of cisplatin and ameliorate cisplatin nephrotoxicity phloroglucinol attenuates the cognitive deficits of the 5xfad mouse model of alzheimer's disease protective effect of brown alga phlorotannins against hyper-inflammatory responses in lipopolysaccharide-induced sepsis models dieckol, isolated from ecklonia stolonifera, induces apoptosis in human hepatocellular carcinoma hep3b cells inhibitory effect of chlorophyll c2 from brown algae, sargassum horneri, on degranulation of rbl-2h3 cells isolation and structural determination of two novel phlorotannins from the brown alga ecklonia kurome okamura, and their radical scavenging activities phlorofucofuroeckol a isolated from ecklonia cava alleviates postprandial hyperglycemia in diabetic mice acknowledgements this research was supported by the basic science research program through the national research foundation of korea (nrf), funded by the ministry of education (2012r1a6a1028677). conflicts of interest the authors declare no conflicts of interest. key: cord-286303-wo6356vq authors: khanna, varun; li, lei; fung, johnson; ranganathan, shoba; petrovsky, nikolai title: prediction of novel mouse tlr9 agonists using a random forest approach date: 2019-12-20 journal: bmc mol cell biol doi: 10.1186/s12860-019-0241-0 sha: doc_id: 286303 cord_uid: wo6356vq background: toll-like receptor 9 is a key innate immune receptor involved in detecting infectious diseases and cancer. tlr9 activates the innate immune system following the recognition of single-stranded dna oligonucleotides (odn) containing unmethylated cytosine-guanine (cpg) motifs. due to the considerable number of rotatable bonds in odns, high-throughput in silico screening for potential tlr9 activity via traditional structure-based virtual screening approaches of cpg odns is challenging. in the current study, we present a machine learning based method for predicting novel mouse tlr9 (mtlr9) agonists based on features including count and position of motifs, the distance between the motifs and graphically derived features such as the radius of gyration and moment of inertia. we employed an in-house experimentally validated dataset of 396 single-stranded synthetic odns, to compare the results of five machine learning algorithms. since the dataset was highly imbalanced, we used an ensemble learning approach based on repeated random down-sampling. results: using in-house experimental tlr9 activity data we found that random forest algorithm outperformed other algorithms for our dataset for tlr9 activity prediction. therefore, we developed a cross-validated ensemble classifier of 20 random forest models. the average matthews correlation coefficient and balanced accuracy of our ensemble classifier in test samples was 0.61 and 80.0%, respectively, with the maximum balanced accuracy and matthews correlation coefficient of 87.0% and 0.75, respectively. we confirmed common sequence motifs including ‘cc’, ‘gg’,‘ag’, ‘cccg’ and ‘cggc’ were overrepresented in mtlr9 agonists. predictions on 6000 randomly generated odns were ranked and the top 100 odns were synthesized and experimentally tested for activity in a mtlr9 reporter cell assay, with 91 of the 100 selected odns showing high activity, confirming the accuracy of the model in predicting mtlr9 activity. conclusion: we combined repeated random down-sampling with random forest to overcome the class imbalance problem and achieved promising results. overall, we showed that the random forest algorithm outperformed other machine learning algorithms including support vector machines, shrinkage discriminant analysis, gradient boosting machine and neural networks. due to its predictive performance and simplicity, the random forest technique is a useful method for prediction of mtlr9 odn agonists. toll-like receptors (tlrs) represent an ancient evolutionary host immune defense system. there are 13 expressed tlr genes in mice (10 in humans), and each is devoted to recognizing a distinct set of pathogen associated molecular patterns (pamps) that are not found in healthy vertebrate cells, making them an important tool to help fight infections [1] . tlrs 1, 2, 4, 5 and 6 are extracellular and are situated in the plasma membrane where they bind bacterial cell wall components such as lipoteichoic acids, lipopolysaccharides, lipoproteins, and flagella. tlrs 3, 7, 8, 9 are located in endosomes where they recognize specific nucleic acid sequences expressed by various pathogens [2] . the extracellular signaling domain of tlr9 forms a horseshoe shaped dimer that forms a sandwich that clasps two cpg oligonucleotides (odn) resulting in the cytoplasmic domains coming into close proximity thereby triggering downstream signaling [2] . upon activation, tlr9 triggers an innate immune response characterized by the production of pro-inflammatory cytokines such as tnf-α, il-1, il-6, and il-12. some synthetic single-stranded odns that contain unmethylated cpg motifs mimic bacterial dna and can bind and activate tlr9 leading to cytokine secretion and enhancement of adaptive immune responses. synthetic tlr9-active odns have shown utility as vaccine adjuvants and anti-cancer immunotherapeutic agents. to identify a good tlr9 ligand, typically a large library of odns needs to be synthesized and screened on cell lines, which is a time consuming and expensive task. we hypothesized that modern in silico high-throughput screening (hts) methods may improve the ability to identify novel highly active tlr9 ligands. in silico screening, also known as virtual screening (vs), has been widely used to enrich datasets with compounds that have a higher probability of binding to the target of interest [3] [4] [5] , and has an advantage over traditional screening or physical hts due to its massively parallel processing ability; hence millions of compounds can be assessed economically in parallel. this is particularly important when the search space for potential odns tlr9 ligands is taken into consideration. a typical singlestranded odn tlr9 agonist is 24 nucleotides in length, which amounts to 4 24 total number of possible odns. vs methods are of two major classes based on the availability of structural information. if the 3d structure of a receptor is known, structure-based virtual screening (sbvs) [6] techniques such as homology modeling, molecular docking and molecular dynamics can be used. however, if the structural information of the receptor is lacking, then ligand-based virtual screening (lbvs) [7] techniques such as quantitative structure-activity relationship and machine learning are more appropriate. sbvs involves molecular complex optimization to find the most favorable 3d binding conformation of the ligand. consequently, sbvs is unsuitable for highthroughput screening of ligands like 24-mer odns, which have over 100 rotatable bonds. on the other hand, lbvs is computationally inexpensive, easy to use and might therefore be useful in the screening of tlr9 activating odns. in a recent review, murgueitio et al. [8] discussed the use of various computational approaches to investigate the structure and function of tlr receptors. to discover potential tlr ligands. zatsepin et al. [9] screened a library of 1.8 million commercially available compounds to discover tlr9 antagonists by using computational chemistry and cell-based assays. the authors reported 21 potential tlr9 antagonists with ic50 lower than 10 μm, with five of them having ic50 values below 1 μm. zhou et al. [10] constructed a 3d structure of human tlr9 ectodomains, complexed with cpg odns using homology modeling, then used molecular docking to study the interactions between tlr9 and the odns. they reported that leucine rich region (lrr)-11 was the main region in tlr9 responsible for odn binding. the authors further reported that five positively charged residues within lrr11 were specifically involved in the odn binding to tlr9. nagpal et al. [11] reported a support vector machine model to predict odns with tlr9 activity with the model achieving a maximum matthews correlation coefficient of 0.75 with an accuracy of 87%. tlr9 ligand prediction tools require availability of well-annotated odn datasets with experimentally determined tlr9 activity data. machine learning (ml) based techniques such as decision trees, random forest, support vector machines and neural networks can then be applied to such odn datasets. ml is an umbrella term for statistical models built to discover patterns in existing data to explain unseen data. ml models are very powerful tools that have been used in the past to predict and classify the pharmacokinetics or toxicological profiles of compounds [12] , predict biological activities or toxicity [13] and assist in screening and optimization of compounds [5] . to our knowledge, this is the first report on the use of random forest-based approaches to predict novel mtlr9 ligands based on an in-house experimentally validated odn dataset, with 91% prediction accuracy shown by experimental validation. the main goal of this study was to build a ml model that could distinguish odns that have high activity for mtlr9 from odns with low activity. we used 117 odns with known high mtlr9 activity, as positive examples while 274 odns with low activity were used as negative examples. we first analysed the dataset to understand the occurrence of sequence motifs in mtlr9 activating odns. we observed an uneven distribution of motifs with a few motifs such as 'gg' or 'cc' present in 57% of the odns in the high activity group compared to only 13% of the odns in the low activity group. figure 1 shows the percentage of odns in the top 20 motifs arrange in a clockwise manner, based on the absolute difference in the percentage of occurrence in high and low mtlr9 activity groups of odns. all motifs having an absolute difference above 10% are shown in additional file 1. we further analyzed the effect of motif occurrence on the mtlr9 activity score in the high and low activity groups of odns in the dataset. using the mann-whitney u test we compared the median mtlr9 activity score of odns with a motif to those without the motif for the two classes and calculated the p values. the significance threshold was set at 0.05. figure 2 shows the effect of top 20 motifs occurrence in high (fig. 2a) and low (fig. 2b ) mtlr9 active group of odns. the darker colored bars stand for a significant difference in the median mtrl9 activity score (p < 0.05) due to the presence of the motif in the odns. the dotted line is the median mtlr9 score of 0.53 and 0.18 for the high and low activity groups of odns, respectively. within the low activity group (additional file 2), we found that presence of motifs such as 'cc', 'gg', 'ggc', 'gcc', 'cccg' and 'cggc' significantly increases the median mtlr9 activity score, while the presence of motifs e.g. 'tgt', 'cgcgt' and 'tct' further lowers the activity of odns. in contrast, we found presence of 'cgtt' motif to significantly improve while 'ag' motif to significantly decrease the median mtlr9 activity score of the odns in the high activity group (additional file 3). since there was no single motif that could account for the mtlr9 activity score of the odns, we surmised that the combination of motifs and their interaction with the tlr9 receptor was responsible for determining overall mtlr9 activity. mean classification levels achieved by all algorithms in different k-fold cross validation schemes when applied to 20 bootstrap test samples obtained using the down-sampling technique are shown in fig. 3 . we found that overall rf model either outperformed or was on par with the other prediction algorithms in all four cross validation schemes. in five-fold cross validation the best rates were achieved by the rf and svm model with a maximum balanced accuracy of 95.65% and mcc of 0.91 (additional file 4). the mean balanced accuracy and mean mcc for rf model in five-fold cross validation was 77.8% and 0.57, respectively, with standard deviations of 0.08 and 0.15, respectively (table 1 ). in ten-fold cross validation, rf and gbm achieved the best results with the maximum balanced accuracy and mcc of 89.13% and 0.78, respectively (additional file 5). the mean balanced accuracy and mcc for the rf model in ten-fold cross validation was 78.9% and 0.60, respectively, with standard deviations of 0.06 and 0.11, respectively (table 1 ). in 15fold cross validation the best results were achieved by rf and svm with the maximum balanced accuracy and mcc of 86.9% and 0.74, respectively (additional file 6). the mean balanced accuracy and mcc for the rf model in 15-fold was 77.0% and 0.55, respectively with standard deviations of 0.06 and 0.11, respectively (table 1 ). in 20fold cross validation random forest achieved the best result with the maximum balanced accuracy and mcc of 87.0% and 0.75, respectively (additional file 7). the mean balanced accuracy and mcc of rf model was 79.7% and 0.61, respectively, with standard deviations of 0.05 and 0.09, respectively (table 1) . overall, the rf algorithm outperformed in all other ml methods, for different cross-validation values. we therefore selected rf with the 20-fold cross-validation scheme, having maximum mean balanced accuracy and mcc and minimum standard deviation on both measures, to perform the fig. 2 the effect of top 20 motifs in the high (a) and low (b) mtlr9 activity group of odns in the dataset. the darker bars represent a significant difference in the median mtlr9 activity score due to the presence of motif in the odns. the dotted line shows the median mtlr9 activity of 0.53 and 0.18 for the odns in the high and low activity groups, respectively, in the dataset mtlr9 activity predictions for the randomly generated odn dataset. external validation is the final step to evaluate the realistic performance of any prediction model. in this technique, the performance of the model is evaluated on a new dataset not used in training or testing the model. to rigorously evaluate the performance of our model, we randomly generated 6000 24-mer odn sequences using an in-house written python script and then screened and ranked these randomly generated odn for mtlr9 activity using our rf model. these odns were not present in our original dataset of 396 odns used for model building or training, and as they were virtual we had no prior knowledge of their likely mtlr9 activity at the time of model prediction. our rf model predicted 545 of these 6000 random odns to be of high activity and we selected the top 100 for chemical synthesis, and then experimental tested them for mtlr9 activity using the raw-blue reporter cell line that expresses mtlr. ninety-one (91%) of the predicted high activity odns had a mtlr9 activity value above 0.4, confirming the high accuracy of the model in predicting odn sequences with positive mtlr9 activity (fig. 4 ). this demonstrates that our mtlr9-specific rf prediction model is rigorous, with a strong performance on making predictions on a completely independent dataset. in this study we demonstrated the feasibility of using an rf model for in silico screening of synthetic odns to detect high activity mtlr9 agonists. multiple sequence features such as simple counts of nucleotides, the distance between motifs and graphically derived features like the moment of inertia were calculated before building the rf model. we observed higher occurrence of several motifs such as 'cggc', 'cccg', 'gcc', 'cgg', 'ggc', 'ccg', 'ccc', 'gg' and 'cc' in high activity as compared to low activity odns. this means that these cytosine and guanine rich motifs along with the key unmethylated cpg dinucleotide contribute to strong mouse tlr9 activation. interestingly, this is in contrast with the thymine rich motifs reported for tlr9 fig. 3 mean and standard deviation of balanced accuracy rates of the five classifiers on the twenty bootstrap test samples using k-fold crossvalidation scheme. mean balanced accuracy rate of rf model was greater than all five algorithms in all the folds stimulatory odns by nagpal et al. [11] . this may be due the fact that our odn training set was mouse specific whereas the dataset used by nagpal et al. [11] was not specific to any organism. on further analysis we found 15 and 4 motifs which significantly increased, or decreased, respectively, mtlr9 activity in the low activity group (additional file 2), whereas, we found only 3 and 4 motifs in the high activity odns which significantly (p value < 0.05) increased or decreased, respectively, mtlr9 activity (additional file 3). furthermore, we discovered motifs which significantly decreased mtlr9 activity in both low and high groups. for example, 'cgcgtg' and sub motifs like 'gcgtg' and 'cgcgt', decreased the activity of odns in both the high and low groups. however, we were unable to identify motifs that increased mtlr9 activity for both groups of odns. this suggests that a combination of motifs might be required to increase activity of odns in the high group whereas the activity of low odns can be improved even by inclusion of a single motif. cooccurrence of motifs and their effect on mouse tlr9 activity can be analyzed in the future to discover combinations of motifs responsible for the increase in the activity of odns in both groups. the performance of the rf model was compared to other methods, which were trained on the same data. the average classification accuracy achieved by all the methods when applied to 20 bootstrap test samples in four different cross-validation schemes is shown in fig. 3 . the results demonstrated that the rf model had the superior performance on the test datasets in most of the scenarios. the gbm and svm classifiers also had reasonable classification accuracy rates, however, rf outperformed them in 20-fold cross validation scheme. the selected rf model on average correctly classified 79.1% of the odns in the training set with high activity for mtlr9 and 80.2% of odns with low activity. the rf thereby achieved an overall balanced accuracy of 79.7%. finally, the rf model was used to virtually screen 6000 randomly generated odns from which it predicted 545 odns to have high activity for mtlr9. due to large number of predicted positive hits, the top 100 odns were selected for synthesis and testing for mtlr9 activity in vitro. ninety one out of the 100 synthesized odns were found to have mtlr9 activity above the cutoff of 0.4 for high activity odns confirming the prediction potential of the rf model. however, fig. 4 shows that the majority of predicted ligands had an activity value ranging from 0.5 to 0.7, which indicates that the model might need to be further fine-tuned to get even higher activity ligands, with a much larger dataset than the randomly generated 6000 oligonucleotides screened to find high activity ligands. in this study we found several sequence motifs that help explain the mtlr9 activity of cpg odns. motifs including 'cgtt', 'ggc', 'gcc' and 'cccg' significantly improved, whereas motifs such as 'ag', 'tct' and 'cgcgt' significantly decreased, the activity of mtlr9 odns. further, we developed and validated an rf model for predicting odns with mtlr9 activity. the results showed that the rf method was well suited for predicting high activity mtlr9 specific odns and outperformed various other learning algorithms such as svm, sda, nn and gbm. the model was used to screen a random library of 6000 odns and correctly identified 91 out of 100 odns that were subsequently confirmed to have mtlr9 activity. this shows the power of machine learning models for discovering novel tlr9 agonists. the lead mtlr9 active odn candidates from the above studies are now being tested as vaccine adjuvants and anti-cancer agents in relevant mouse models. the quality of the training dataset determines the quality of the resulting machine learning model. missing or insufficient data, mislabeling of the target variable, and irrelevant features may complicate the learning task and hinder the performance of the trained model. the sequences of odns with experimentally determined mtlr9 activity were obtained from in-house data we generated on synthesized odns that were characterized using a mouse tlr9 expressing reporter cell line (raw-blue cells, invivogen, usa). the dataset consisted of 396 odns with mtlr9 activity values ranging from 0.0 (no activity) to 1.14 (high activity). the odns were grouped into two classes (fig. 5) based on their respective activity value (i.e. 0.4 and above: high activity and below 0.4: low activity), resulting in a high activity group (count 117) and a low activity group (count 279). to ensure data quality, it is customary to check and remove any outliers, impute the missing data, check, and assign the variables the correct datatype. our dataset had neither missing values nor outliers and therefore, no further action was required in cleaning the dataset. however, to avoid overtraining the model with similar odns, the diversity of the dataset was increased by limiting the similarity within the group. this was achieved by clustering the odns within a group using the binary fingerprint features we developed during this study and applying a clustering cutoff of 0.85 to remove similar odns. this resulted in the removal of five odns from the low activity group with 274 remaining. all odns in the high group (count 117) were dissimilar enough not to breach the similarity cutoff and were retained. in our training dataset, the number of odns with low mtlr9 activity was approximately 2.5 times more than the number of odns with high mtlr9 activity. therefore, we used the down-sampling technique to balance the dataset, so that 50% of the samples were derived from the set of odns with high activity and 50% from the set of odns with low activity. subsequently, the down-sampled dataset was subdivided into training (80%) and testing (also known as validation) sets (20%), using a random sampling technique and the odns in the test set were excluded from model training. in order to choose the best classifier with k-fold cross validation, the performance of our models were measured using 20 down-sampled test sets. the overall methodology adopted in the study is shown in fig. 6 . in table 2 , we present the composition of the dataset used in this study. for each instance, the training dataset was composed of 188 odns (derived from 94 odns with high and low mtlr9 activity each). the test dataset used to evaluate the performance of a model was composed of 46 odns (23 each from the two groups of high and low mtlr9 activity). for the prediction set, we used an in-house python script to randomly generate 6000 24-mer odns, to capture the diversity of the 24-mer cpg-odn universe. every odn in the prediction set was classified using the selected model and crossvalidation scheme in a loop. for the final prediction, a consensus of the 20 predictions were taken for each odn in the prediction set. finally, the top 100 high activity predicted odns were selected for synthesis and experimental testing using the raw-blue reporter cell line assay. the training and test set odns along with experimental activity information are available in additional file 8. the measured mtlr9 activity value of all the synthesized 24-mer odns in the dataset. the odns were divided into two groups of high (shown in purple) and low (shown in green) activity using a cutoff score of 0.4, based on the optimal density (od) results from the raw-blue reporter cell assay it is possible to generate a large number of features for the odn sequence data that can be used to construct machine learning models. however, there are several problems in using all the possible features as (i) some of the features may be highly correlated (ii) some may not be relevant and may contribute to the noise in the model and (iii) using a large number of features may lead to overfitting. additionally, constructing models with many features is computationally demanding [14] . therefore, one of the most important aspects of creating a good ml model is the choice of appropriate features that can help explain the behavior of interest based on occam's razor principle (i.e. simple models are more likely to be closer to reality than complex models.) [15] . while there are a variety of features used in bioinformatics for sequence data, we used the binary fingerprint features and numerical features, including count and position of motifs, distance of the motifs with respect to the start position and graphically derived features such as the moment of inertia and radius of gyration, to train the model [16] . to generate fingerprint features, a fasta formatted file containing all high activity odn sequences was analysed using an in-house perl subroutine, to chop each sequence into motifs of increasing length from two to six nucleotides and record the start positions of the motifs. for example, with a small hypothetical odn 'tcg' of three nucleotides, two dinucleotides motifs tc1, cg2 and a trinucleotide tcg1 motif were generated. finally, a dictionary of the motifs with at least 10% difference in the occurrence rate in low and high group of odns (count 67) was prepared. subsequently, the dictionary was used to generate the binary fingerprint pattern for each sequence, where 1 showed the presence of a motif while 0 indicated its absence. different patterns of nucleotide usage in odns may lead to varied mtlr9 activity. therefore, all nucleotide characters (a, t, g, c) were counted in a sequence and the perl built-in dictionary data structure, hash, was used to store the count of each nucleotide. ambiguous nucleotide characters or gaps were ignored if present. calculating the distance between motifs with respect to their start positions the most commonly occurring motifs were used to calculate the distance between motif features along with their specific location. to map the position of a motif in the odns, the sequence of each odn was scanned for the presence of a motif and all the positions where each motif occurs were recorded. using eqs. (1)-(3), the distance between the second and first, third and first and the third and second occurrence of the motifs were calculated for all the motifs. where d_motif is the distance, p3, p2 and p1 are the position 3, position 2 and position 1 of the motif respectively, and 'n' is the number of nucleotides before the latter motif. in the case of the absence of a motif, 0 was substituted in the equation. it is important to keep 'n' in the equation to provide the specific location of the motifs within an odn, because the calculated distance between motifs could be same in several odns. for example, in a sequence s1 = tatgcgttcg-tacttgatctgac, the distance between cg motifs is 9-5 = 4. similarly, for another sequence s2 = tgctttcttgtcgtgcgggctgt, the distance between the cg motifs is 16-12 = 4, again. however, the descriptor d_cg2_1 value for s1 and s2 are 12 and 19, respectively, with the addition of n to the simple distance formula of d_motif. the graphical representation of dna sequences have been used for many applications including assessing phylogenetic relationships [17] , characterization of neuraminidase gene in h5n1 avian flu [18] and for describing similarity/dissimilarity of dna sequences [4] . in order to derive features, the 24-mer odn sequences were represented as a 2d-graph, as previously described [16] . briefly, each base in the sequence is represented as a material point on the graph which is treated as a rigid body and follows the rules of newtonian dynamics. numerical features such as the center of mass (μ x , μ y ), the principal moment of inertia(i 11, i 22 ) and radius of gyration (r g ) were calculated for each sequence as described in [16] . there are several feature selection methods used in machine learning to remove redundant or irrelevant features. these can be broadly divided into filter methods (e.g. correlation matrix, information gain, chi-square score, principal component analysis, regression coefficients, variable importance) and wrapper methods (e.g. forward/backward selection, randomized methods that combine pls with the genetic algorithm or monte carlo algorithm) [19] [20] [21] . filter methods are easy to implement because there is no learning involved and depend only on the application of a cut-off value to reject features due to the low importance in the model construction. in the wrapper methods, the performance of a learning algorithm is evaluated to select the optimum subset of features therefore, it is a very computationally expensive process [19] and is best suited for a limited number of features. furthermore, filter methods work well for text mining [19] , and are applicable for odn features, which are essentially nucleotide "words." due to the large number of fingerprint features available (67 in total), we first filtered out the constant and near-constant features (features with < 0.3 standard deviation) also known as zero and near zero variance features using the caret package in r. constant or near constant features take a unique value across samples and are uninformative. this resulted in the removal of 26 features. since these features are binary in nature, we also checked and removed any linear combinations of features if present. this resulted in the removal of 31 features. to understand the distribution in the high and low group of odns we created a cricos plot using the circlize package in r [22] . for all numerical features in addition to removing zero and near zero variance features we also calculated the correlation matrix and filtered out features that were highly correlated. the correlation coefficient was set at 0.85 and features with correlation above the cutoff value were removed. we then normalized the remaining features using centering and scaling techniques to make them unit independent. subsequently, we merged the fingerprint and numerical features to give us a merged set of 40 features, listed in table 3 . in the current study, five ml algorithms, i.e. random forest, gradient boosting machine, shrinkage discriminant analysis, support vector machine and neural network were compared, and the best performing model was chosen for the prediction of novel mtlr9 active odns. to have a non-biased assessment of the performance, kfold cross-validation was followed where one instance of the down-sampled training data was further divided into k partitions. the value of k varies from 5, 10, 15 to 20. for each partition, odns not included in the training were considered part of the testing dataset. finally, the testing data of the instance was used to evaluate the classification accuracy of the model, with the best model selected for prediction on an independent validation dataset. a graphic representation of the general procedure is given in fig. 6 . the random forest (rf) algorithm was introduced by breiman in 2001 [23] and is one of the most powerful ensemble machine learning technique that make predictions by averaging over several independent base learners in order to identify the class label for unknown instances. the base learners are usually the classification and regression trees (cart) constructed using a sample with replacement from the training data with the [23, 24] . briefly rf takes advantage of two powerful statistical techniques, bagging and random feature selection. in bagging each tree is trained on a bootstrap sample (sampling with replacement) and the predictions are made by the majority vote of the trees. furthermore, in rf instead of using all the features, rf randomly selects a set of features to split at each node when growing a tree. to assess the performance of the rf algorithm, rf performs a type of crossvalidation using the out-of-bag (oob) samples (samples which are not included in the training set). the concept of variable importance is inbuilt in the rf algorithm and the importance is measured by the gini impurity criterion index [25] . we used the caret package in r to evaluate the performance and developed an ensemble of 20 different rf models for final prediction. the mtry parameter was tuned using the tunegrid argument in the train function. the accuracy of the five ml algorithms was measured by presenting the prediction results in the form of a confusion matrix and the variety of performance measures were calculated based on the following statistical measures: tp, true positivesthe total number of correctly classified high activity odns. tn, true negativesthe total number of correctly classified low activity odns. fp, false positivesthe total number of low activity odns incorrectly classified as high activity odns. fn, false negativesthe total number of high activity odns incorrectly classified as low activity odns. using the measures above, a series of statistical metrics were computed including sensitivity (se), specificity (sp), balanced accuracy (ba), matthews correlation coefficient (mcc) and precision. the recall rate for the members of the positive class (high activity odns) is given by sensitivity, in eq. (4): similarly, the recall rate for the members of the negative class (low activity odns) is given by specificity, in eq. (5): the balanced accuracy of the model was calculated based on the eq. (6): we then calculated the mcc from eq. (7); the coefficient returns a value between + 1 and − 1. the higher the value of the coefficient, the better the classification result. finally, the precision was computed to measure the reproducibility of the results, in eq. (8): mouse raw-blue tlr9 reporter cell assay raw-blue™ cells are derived from the murine raw 264.7 macrophage cell line with chromosomal integration of a secreted embryonic alkaline phosphatase (seap) reporter construct inducible by nf-κb and ap-1 and were acquired from invivogen. the presence of agonists of mouse tlr9 activates downstream signaling pathways leading to the activation of nf-κb and ap-1, and the subsequent secretion by the raw cells of seap. levels of seap in the culture supernatant are measured chromatographically using the detection medium quanti-blue™. raw-blue cells were cultured in dmem supplemented with 10% (v/v) heat-inactivated fetal bovine serum, penicillin-streptomycin 10,000 u/ml (gibco), and normocin 100 μg/ml (invivogen). subsequently, raw-blue cells were seeded at a density of approximately 1 × 105 cells/well in a volume of 180 μl/well in a flat-bottom 96well culture plate (greiner-one). odns were diluted in saline and added to the culture plate containing raw-blue cells to the total volume of 200 μl. after culturing the cells for 3 h, the levels of seap were determined in the supernatant with quanti-blue™ solution (invivo-gen) by reading the absorbance at wavelength of 650 nm. toll-like receptors: activation, signalling and transcriptional modulation the structural biology of toll-like receptors in silico approach to screen compounds active against parasitic nematodes of major socio-economic importance graphical representation and similarity analysis of dna sequences based on trigonometric functions discovering highly selective and diverse ppar-delta agonists by ligand based machine learning and structural modeling computational methods in drug discovery use of machine learning approaches for novel drug discovery balancing inflammation: computational design of small-molecule toll-like receptor modulators computational discovery and experimental confirmation of tlr9 receptor antagonist leads toll-like receptor 9 interaction with cpg odn--an in silico analysis approach vaccineda: prediction, design and genome-wide screening of oligodeoxynucleotide-based vaccine adjuvants applying machine learning techniques for adme-tox prediction: a review deeptox: toxicity prediction using deep learning the curse of dimensionality in data mining and time series prediction the problem of overfitting 2d-dynamic representation of dna sequences coronavirus phylogeny based on triplets of nucleic acids bases graphical representation and numerical characterization of h5n1 avian flu neuraminidase gene sequence a review of feature selection methods with applications a review of feature selection and feature extraction methods applied on microarray data a review of variable selection methods in partial least squares regression circlize implements and enhances circular visualization in r random forests random forests: from early developments to recent advancements the origins of the gini index: extracts from variabilità e mutabilità (1912) by corrado gini publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations this article has been published as part of bmc molecular and cell biology, volume 20 supplement 2, 2019: 18th international conference on bioinformatics. the full contents of the supplement are available at https:// bmcmolcellbiol.biomedcentral.com/articles/supplements/volume-20supplement-2. supplementary information accompanies this paper at https://doi.org/10. 1186/s12860-019-0241-0.additional file 1. sequence motifs in mtlr9 active odns having an absolute difference in the occurrences above 10% in high and low activity groups of odns, arranged in a clockwise manner. the width of the ribbon shows the average percentage composition of the motifs each group.additional file 2. the effect of odn motif occurrences on the median mtlr9 activity in the low activity group. the median raw-blue activity for all the odns in the low activity group was 0.18. increase or decrease in the median activity values due to the presence of a motif are coloured green and red, respectively, with statistically significant values in bold. the significance threshold was set at p-value < 0.05. the motifs are arranged in alphabetical order.additional file 3. the effect of odn motif occurrences on the median mtlr9 activity in the high activity group. the median raw-blue activity for all the odns in the high activity group was 0.53. increase or decrease in the median activity values due to the presence of a motif are coloured in green and red, respectively, with statistically significant values in bold. the significance threshold was set at p value < 0.05. the motifs are arranged in alphabetical order.additional file 4. results of five-fold cross-validation.additional file 5. results of ten-fold cross-validation.additional file 6. results of fifteen-fold cross-validation. additional file 8. odns used as test and training sets for building the prediction model, along with activity information.authors' contributions vk and np designed the research; vk, ll and jf performed the research; vk, ll, jf. np, and sr analyzed data; vk, ll, np, and sr wrote the paper. all authors have read and approved the final manuscript. this work was supported by funding by the national institute of allergy and infectious diseases of the national institutes of health under contract hhs-n272201400053c. the content is solely the responsibility of the authors and does not necessarily represent the official views of the nih. all data reported in this study are available as tables and supplementary data. the cell line used in the assay is commercially available from invivogen inc. [26] .ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. key: cord-022955-vy0qgtll authors: nan title: proteases date: 2005-06-20 journal: febs j doi: 10.1111/j.1742-4658.2005.4739_4.x sha: doc_id: 22955 cord_uid: vy0qgtll nan the incretin hormones glp-1 and gip are released from the gut during meals, and serve as enhancers of glucose stimulated insu-lin release from the beta cells. furthermore, glp-1 also stimulates beta cell growth and insulin biosynthesis, inhibits glucagon secretion, reduces free fatty acids and delays gastric emptying. glp-1 has therefore been suggested as a potentially new treatment for type 2 diabetes. however, glp-1 is very rapidly degraded in the bloodstream by the enzyme dipeptidyl peptidase iv (dpp-iv; ec 3.4.14.5). a very promising approach to harvest the beneficial effect of glp-1 in the treatment of diabetes is to inhibit the dpp-iv enzyme, thereby enhancing the levels of endogenously intact circulating glp-1. the three dimensional structure of human dpp-iv in complex with various inhibitors creates a better understanding of the specificity and selectivity of this enzyme and allows for further exploration and design of new therapeutic inhibitors. the majority of the currently known dpp-iv inhibitors consist of an alpha amino acid pyrrolidine core, to which substituents have been added to optimize affinity, potency, enzyme selectivity, oral bioavailability, and duration of action. various compound series and their sar relative to alpha amino acids will be presented. memapsin 2 (b-secretase, bace1) is the membrane-anchored aspartic protease that initiates the cleavage of b-amyloid precursor protein (app) leading to the production of amyloid-b (ab), a major factor in the pathogenesis of alzheimer's disease (ad). since memapsin 2 is a major target for the development of inhibitor drugs for the treatment of ad, its structure and physiological functions are topics of intense research interest currently. here we discuss the structural features of memapsin 2 and how do they contribute to the activity and inhibition of the protease. structural and kinetic evidence support the presence of 11 subsites for substrate or inhibitor binding in the activesite cleft of memapsin 2. subsites p3 to p2' are most useful in the design of transition-state analogue inhibitors. recent data indicated that subsites p7, p6 and p5 have strong influence of hydrolytic rate or inhibition potency. these subsites are, however, too far from the transition-state isostere for the design of drug-like transition-state inhibitors but can be utilized for the design of non-transition-state inhibitors that compete for substrate binding. besides carrying out proteolytic activity, the ectodomain of memapsin 2 also interacts with app leading to the endocytosis of both proteins into the endosomes where app is hydrolyzed by memapsin 2 to produce ab. a phosphorylated motif in the cytosolic domain of memapsin 2 is responsible for the recognition of gga proteins as part of the recycling mechanism that transports memapsin 2 from endosomes to trans-golgi then back to cell surface. these interactions may also be considered for the design of small-molecular compounds that interfere with memapsin 2 trafficking and thus reduce the production of ab. identification of human carnosinase -a brainspecific metalloprotease m. teufel biochemistry, exploratory research, sanofi aventis, strasbourg, france. e-mail: michael.teufel@sanofi-synthelabo.com metalloproteases form a large and diverse family of proteases and are molecular targets that represent an opportunity for therapeutic intervention. in particular, the development of potent inhibitors has made progress for the family of matrix metalloproteases (mmp). the sequencing of the human genome revealed that a significant percentage of the drugable genome is represented by proteases, many of them still with unknown function. in this presentation, data will be presented on the deorphanization of two previously unknown genes by means of bioinformatics and classical biochemistry. this work led to the identification of human carnosinase, a dipeptidase specifically expressed in the human brain and an ubiquitously expressed close homologue, characterized to be a non-specific dipeptidase. stimulating serpins with synthetic tailor-made oligosaccharides: a new generation of antithrombotics m. petitou thrombosis & angiogenesis, sanofi-aventis, toulouse, france. e-mail: maurice.petitou@sanofi-aventis.com we will discuss our research on synthetic oligosaccharides able to selectively activate the inhibitory activity of antithrombin towards various serine proteinases. we first synthesized pentasaccharides closely related to the antithrombin binding domain of heparin [1] (the active site), as well as analogues displaying different pharmacokinetic profiles. selective inhibitors of coagulation factor xa were thus obtained that represent a new class of antithrombotic [2] drugs currently being evaluated worldwide. we then designed larger oligosaccharides [3] that inhibit both factor xa and thrombin in the presence of antithrombin. they are devoid of undesired nonspecific interactions with blood proteins, particularly with platelet factor 4. clinical trials are ongoing to prove the therapeutic benefits of this new type of coagulation inhibitors. slow tight binding inhibitors in drug discovery: in the case of dppiv and elastase inhibitors z. kapui, e. boronkay, i. bata, m. varga, e. mikus, k. urban-szabo, s. ba´tori and p. ara´nyi discovery research, chinoin member of sanofi-aventis group, budapest, hungary. e-mail: zoltan.kapui@sanofi-aventis.com enzyme are extremely potent causing significant inhibition at very low concentrations that may be comparable to the concentration of the target enzyme. when this inhibition is studied in vitro, complexities arise because the concentration of the inhibitor is so low that it is altered significantly as a result of combination with the enzyme. this situation is referred to as tight-binding inhibition. partly as a result of their low concentrations, tight-binding inhibitors often show slow-binding characteristics. unlike conventional inhibitors that act almost instantaneously (or at least within the ms time scale), slow-binding inhibitors may take several seconds, minutes or even hours for their effect to be fully exhibited. this association between slow-binding and tight-binding is relatively common and slow tight-binding inhibitors are extremely potent and specific. proteolytic enzymes are involved in a multitude of important physiological processes. their intrinsic properties and activities are in the focus of wide-ranging research and they have a valuable role in experimental and therapeutic purposes. serine proteases are attractive targets for the design of enzyme inhibitors since they are involved in the etiology of several diseases. within the class of serine proteases, human leukocyte elastase (hle) is one of the most destructive enzymes in the body. the enzyme dipeptidyl peptidase iv (dppiv) is a serine exopeptidase that cleaves xaa-pro dipeptides from the n-terminus of oligo-and polypeptides. inhibitors of dpp iv are of increasing interest to pharmaceutical industry alike, as they may become established as the next member of the oral antidiabetic class of therapeutic agents. objective of our work was to develop reversible, slow, tight-binding inhibitors against these serine proteases. ssr69071 is a potent inhibitor of hle, the inhibition constant (k i ) and the constant for inactivation process (k on ) being 0.0168 ± 0.0014 nm. this inhibitor is reversible, slow, tight-binding inhibitor with k on = 0.183 ± 0.013 10 6 /ms, and k off = 3.11 ± 0.37 10 )6 /s. ssr69071 inhibits the solubilization of elastin by hle with 13 nm of ic50 value. this inhibitor is one of the most effective inhibitor of a serine proteinase yet described. ssr162369 is a potent, competitive and slow tight binding type inhibitor of the human dipeptidyl peptidase-iv enzyme (k i = 2 nm, t½ = 8 h). on the basis of kinetic properties, ssr162369 forms stable enzyme-inhibitor complex. these slow tight-binding inhibitors have unique inhibitory properties, they are extremely active, and selective, form stable enzyme-inhibitor complex, therefore they have long-lasting effect. their oral activity and long lasting in vivo biological potency agreed very well with stable enzyme-inhibitor complex. the advantages in drug discovery of slow tight-binding inhibitors are discussed in this presentation. enzyme inhibition trend analysis -a new method for drug design m. shokhen, n. khazanov and a. albeck the julius spokojny bioorganic chemistry laboratory, chemistry, bar lan, ramat gan, israel. e-mail: albecka@mail.biu.ac.il many of the drugs that are currently in use or at different stages of development are enzyme inhibitors. therefore, enzyme mechanism-based inhibitors could be developed into highly selective drugs. our novel enzyme inhibition trend analysis method com-bines experimental enzyme kinetics data and high level quantum mechanical modeling of enzyme-inhibitor chemical interactions. the method utilizes the principal catalytic reaction scheme of the target enzyme and does not require its 3d structure (a ligand based approach). the method is valid for the prediction of the trend in binding affinity of inhibitors not only for the specific enzyme for which the qsar model was optimized, but also for the whole enzyme family. the methodology would contribute significantly to overcoming the problem of fast mutational resistance developed by pathogens in response to pharmaceutical treatment. it can be used as a computational tool for expert analysis of various hypotheses about structure-activity relationships formulated for the design of new inhibitors. angiotensin-converting enzyme (ace, ec 3.4.15.1) is a key enzyme for blood pressure control and water-electrolyte homeostasis. a large number of highly potent and specific ace inhibitors are used as oral drugs in the treatment of hypertension and congestive heart failure. somatic ace consists of two homologous domains (n-and c-) within single polypeptide chain, each one containing a catalytic site. the two catalytic sites within somatic ace molecule were long considered to function independently. however, recent investigations indicate the existence of negative cooperativity between ace active sites. we studied the properties of bovine ace active centers by use of separate ace n-domain (n-ace) obtained by limited proteolysis of parent somatic enzyme and testicular ace, which represents c-domain. these results were compared with the data obtained for full-length somatic ace from bovine lungs. the results obtained demonstrate strongly dependent mechanism of action of ace active centers in the reaction of the hydrolysis of tripeptide substrates. however, the hydrolysis of decapeptide angiotensin i proceeds independently on n-and c-domains. the mechanism of inhibition of ace activity is also dependent on the length of the inhibitor: (i) random binding of the ''short'' inhibitor molecule (such as captopril, lisinopril) to one of the active sites dramatically decreases binding of another inhibitor molecule to the second site; (ii) ''long'' nonapeptide teprotid binds to both active sites without any difficulties. since the main physiological ace substrates in the organism are ''long'' peptides angiotensin i and bradykinin, the development of new class of inhibitors with prolonged structure would be beneficial for abolishing of ace activity. synthetic peptide studies on severe acute respiratory syndrome coronavirus (sars-cov) extensive proteolytic processing of the replicase polyproteins, pp1a (486 kda) and pp1ab (790 kda), by the sars-cov 3clike protease (3cl pro ). besides, the structural spike protein of sars-cov contains two heptad repeat regions (hr1 and hr2) that form coiled-coil structures, which play an important role in mediating the membrane fusion process. in this study, we focused on both 3cl pro and the hr regions of sars. previous studies demonstrated that the coronavirus 3cl pro cleaves the replicase polyproteins at no <11 conserved cleavage sites, preferentially at the lq sequence. the reported crystal structure of sars-cov 3cl pro provides insights into the rational design of anti-sars drugs. in order to understand the molecular basis of the enzyme-substrate binding mechanism, we employ the synthetic peptide and mass spectrometry-based approaches to investigate the significance of selected amino acid residues that are flanking both sides of the sars-cov 3cl pro cleavage site. in addition, previous studies indicated that the relatively deep hydrophobic coiled coil grooves on the surface of sars-cov spike protein heptad repeat regions (hr1 and hr2) may be a good target site for the design of viral fusion inhibitors. we have designed and synthesized five truncated peptide analogs derived from hr1 and hr2 peptides based on both bioinformatics and structural analysis. the biological activities of these truncated analogs will be studied using circular dichroism spectroscopy, multidimensional chromatography, protein cross-linking and mass spectrometry-based approach. the above investigation will definitely broaden our knowledge on the sars research and will reveal the feasibility of rational design of synthetic peptide-based drug in combating with sars disease. ras-transfection-associated invasion: involvement of matrix metalloproteinase(s) confirmed using a chicken embryo model and real time pcr during metastasis tumorogenic cells leave the primary tumour and intravasate into the blood/lymphatic system, exiting at a secondary site to establish a secondary tumour. ras-transfection of a parental, non-invasive mcf-10a cell line, established from a patient suffering with benign fibrocystic disease, gave rise to an invasive derivative cell line (mcf-10a-neot) exhibiting the phenotype of a pre-malignant, invasive tumour. invasion and metastasis are protease-assisted processes, proteases either being secreted by the tumour, or by the stromal cells under the influence of the tumour. here we demonstrate the involvement of matrix metalloproteinase(s) in the invasion of the ras-transfected mcf-10a cell line. tumour cells were inoculated onto the damaged surface of the upper chorioamniotic membrane (cam) of a vasculated 9-day old chick embryo. the tumour cells were allowed to invade, and the number of invading cells quantified using real time pcr. inhibitors specific for various proteases were applied to the upper cam, to block invasion, and hence identify the proteinases involved. the number of tumour cells invading into the vascular system was established by sampling the lower cam and quantifying the numbers of alu sequences (present only in human cells) in the dna, isolated from the embryonic tissue, using real-time pcr. using this method, the key role of an mmp was demonstrated. spectrum ptk inhibitor, genistein (100 lm) abolished the release of neutrophil mmp-9, in the presence and absence of extracellular calcium, and reduced the release of timp-1. both pp2 (10 lm), a src family ptk inhibitor, and piceatannol (30 lg/ml), a syk family ptk inhibitor, reduced mmp-9 release substantially, indicating that multiple ptk families might be involved in mmp-9 release. inhibition of either syk or src ptks by piceatannol or pp2 did not appear to influence timp-1 release. low levels of wortmannin (100 nm, inhibition of pi3k) abolished the release of mmp-9 in the absence of calcium, and reduced mmp-9 release in the presence of calcium. investigations into the signaling pathways involved in timp-1 release are continuing. we conclude that mmp-9 release induced by extracellular calcium may be mediated through pi3k and multiple tyrosine kinases, including src and syk family ptks. timp-1 granule release may also be mediated by tyrosine kinases, although src and syk family ptks do not appear to be involved. thermodynamical and structural analysis of cruzain/cruzipain2 complexed with e-64 by molecular modeling and dynamics simulations peptidases represent one of the most relevant enzyme classes targeted by therapeutic intervention. to contribute to the assignment of a physiological role to genomic-derived peptidases and to make them more accessible for the drug discovery process, we have undertaken a program consisting of mrna expression profiling, full-length recombinant expression in insect cells, purification and determination of the catalytic activity for the human proteolytic enzymes. a milestone in the process was the construction of a non-redundant comprehensive database for all human peptidases comprising 443 unique annotated entries, by assembling and filtering public domain information and in-house generated data. in order to get an informative picture on their expression profiling, a transcriptome database for 375 human peptidases was created using the microarray (affymetrix tm ) and taqman ò (applied biosystems) technologies. in parallel, we have set up the procedure for pcr amplification and cloning of the peptidase genes in 96 mtp format and we have already created a repository of 231 full-length human cdnas encoding for peptidases. besides, the conditions for miniaturized insect cell cultures have been established. experimental trials have defined a validated, reliable and fully-automated robotic procedure for the purification of recombinantly expressed peptidases in 96 mtp format. in a pilot study using the high-throughput approach, 85% of the chosen reference hydrolases (14) were secreted into the insect cell medium. of them, 66% have been proven to be catalytically active using fluorescent homogeneous assays in 384well format compatible with the high-throughput screening criteria. the application of this procedure to genomic-predicted peptidases is discussed. comparison of putative glutamate racemases from bacillus species glutamate racemase catalyzes the interconversion between l-and d-glutamic acid and is the cell's source of d-glutamate, a key component in the synthesis of both the bacterial cell wall and the glutamyl capsule. bacillus subtilis has two glutamate racemases in its genome, race and yrpc, while b. cerus and b. anthracis have two race genes, race1 and race1. interestingly, race in b. subtilis is the isoform that is essential and has the greater catalytic efficiency, but both race1 and race2 have higher sequence homology to race, 70 and 79% respectively and share less homology with the yrpc isoform, both at 53%. we have cloned, overexpressed, purified, and are characterizing the kinetic and biophysical properties of the two putative glutamate racemases, race1 and race2 from b. cereus and b. anthracis, and will utilize kinetic and biophysical information to design inhibitors that may result in a novel antibiotic. although these two isoforms share a high sequence similarity, their properties are unique. kinetic data indicates a fivefold difference in catalytic efficiency of race2 compared to that of race1 in the l-to d-glutamate reaction. also, the absence or presence of substrate has an effect on the oligomerization state, details of which will be reported. finally, our collaborators have demonstrated through genetic knock out experiments that only one of the race isoforms is essential for the growth of b. anthracis. we have crystallized the race2 isozyme and x-ray data have been collected to 2.3 å . we are currently solving the structure via heavy-atom derivatives. acknowledgment: this research was funded by nih grant u19 ai056575. anti-inflammatory effects of methionine aminopeptidase 2 inhibition on human b lymphocytes e. janas 1 , r. priest 1 , s. ratcliffe 2 and r. malhotra 1 1 rheumatoid arthritis biology, glaxosmithkline, stevenage, uk, 2 high throughput chemistry, glaxosmithkline, stevenage, uk. e-mail: eva.x.janas@gsk.com processing of n-terminal methionine is an essential post-translational modification in both prokaryotes and eukaryotes regulating the subcellular localization, stability and degradation of proteins. the cleavage of the initiator methionine is catalysed by a highly conserved family of metalloproteases, methionine-aminopeptidase 1 and 2 (met-ap2). human met-ap2 is the molecular target of fumagillin, a natural product with antiangiogenic properties, which covalently binds to his 231 in the catalytic site of met-ap2. although fumagillin has been observed to inhibit proliferation and to cause cell cycle arrest in endothelial cells, the mechanism of inhibition is still poorly understood. recent studies describe high expression of met-ap2 in germinal centre b lymphocytes. here, we investigate the effect of the met-ap2 inhibitor fumagillin on b lymphocyte proliferation and cell cycle progression and compare these results to those observed in hu-vec. in addition our work sheds light on the mechanistic aspects of met-ap2 inhibition by fumagillin and its derivatives. effect of distal mutations on the molecular dynamics of the hiv-1 protease l10i and l90m are the most common distal mutations found in the protease gene of the drug resistant hiv-1 strains. these mutations do not confer resistance by themselves, however induce a large synergy effect when added to active site mutations. understanding the impact of the l90m and l10i mutations on the hiv-1 protease resistance profile is still a challenge. assuming that their contribution to the resistance profile could be mediated by conformational dynamics we have modeled l10i, l90m and l10i/l90m mutants of hiv-1 protease. these unbound mutated and wild type proteases were subjected to 10ns molecular dynamics simulations and compared using an essential dynamics (ed) analysis protocol. the first eigenvector of the native protease describes the flap openning motion. following eigenvectors describe ''the catalytic assisting motions'' (cam) of the protease that becomes dominant upon complex formation with a substrate (piana s et al. j mol biol 2002; 319(2): 567-583). mutation of luecine to methionine residue at position 90 perturbs the protein packing at the dimerization domain. such perturbations affect the dimerization domain motions which correlate with flap opening and the cam. as result the first eigenvector corresponds to the rotational of the one subunit relative to another along axis connecting residues 60 and 60'. in other words l90m mutation mistunes essential motions of the enzyme while retaining its flexibility. this could be the cause of the reduced structural stability of the l90m mutant. in contrast, l10i mutation causes only redistribution of the correlated motions amplitude. the catalytic assisting motion becomes the most influential that results in stabilization of the closed conformation. in turn, the flap opening motions are reduced in l10i mutant. essential dynamics of the double mutant l10i/l90m could be described in the following terms. a strong propagation of the cam induced by l10i mutation is coupled with the altered conformational space caused by l90m mutation. as result the double mutant prefers cam motions that are close to the native protease but also account for the perturbed packing within the dimerization domain. results presented may help understanding hiv-1 protease resistance pathways and in developing more efficient inhibitors of known drug resistant mutants. glutamate carboxypeptidase ii as a cancer marker and therapeutical target: two faces of an enzyme glutamate carboxypeptidase ii is a membrane-bound metallopeptidase expressed in a number of tissues such as jejunum, kidney, prostate and brain. the brain form of gcpii (also known as naaladase) is expressed in astrocytes and cleaves n-acetylaspartyl glutamate, an abundant neurotransmitter, to yield free glutamate. gcpii thus represents an important target for the treatment of neuronal damage caused by excess glutamate. animal model experiments suggest that specific inhibitors of gcpii could be useful for the treatment of several neuropathic conditions, such as brain stroke, chronic neuropathic pain or amyotrophic lateral sclerosis. in the same time, the enzyme is known as prostate-specific membrane antigen since it is upregulated in prostate cancer. it is used for the diagnosis and experimental therapy of prostate cancer using monoclonal antibodies and specific inhibitors. in order to analyze this important pharmaceutical target, we established an expression system based on drosophila schneider's cells. we have also cloned, expressed and characterized its human homolog gcpiii and homologous carboxypeptidases from pig and rat. using specific monoclonal antibodies, we have been able to study the expression of gcpii in various healthy and malignant tissues. we analyzed the substrate specificity of the enzyme using peptide libraries and identified two novel peptide substrates. availability of a recombinant protein enabled to introduce a simple fluorescent activity assay and test specific inhibitors. furthermore, we have biochemically characterized the recombinant protein in terms of pharmacologic properties, oligomeric status, ph dependence and activity modulation by metal ions. we have shown that the glycosylation is indispensable for gcpii carboxypeptidase activity and analyzed the role of each specific n-glycosylation site for the gcpii activity and folding. using site-directed mutagenesis, we are able to identify the domains sufficient and necessary for gcpii activity and also suggest structural explanation for the substrate specificity of the enzyme. dozens of chemicals feature inhibition of proteolytically important tyrosine residue of 20s proteasome by forming covalent bond to hydroxyl group that abolished its catalytic function. in contrary, the approach we utilize here is based on hydrogen and hydrophobic interactions reversibly inactivating all three sites of 20s complexes. we performed flexible docking studies of analogues of a natural product tmc-95a using 1jd2 crystal structure to describe the active site of protein and the position of the ligand. the search yielded several amide-like derivatives that have been screened for superimposition with tmc-95a. few of them revealed similar orientation of propylene groups to the active site of 20s. second screen was performed to reveal the chemicals with the strongest hydrogen-bonding of the ligand to the protein backbone of the receptor. this screen resulted in two chemicals that had strong h-contacts with tyr21, ser129 and, importantly, with proteolytically active tyr1 residue. to access the validity of the predicted chemicals we undertook in vitro studies measuring the hydrolyses of fluorogenic substrate by the sds activated 20s proteasome isolated from hela cells. we obtained more than 85% inhibition of 20s proteasome activity upon incubation the above chemicals (0.5 lg/ml) with proteasomes. we then demonstrated the effectiveness of the obtained chemicals to stabilize the level of oncosupressors, including p53 in benign (mcf10a) and highly metastatic (mda231) cell lines. treatment with these compounds greatly restored the level of p53 in cancer cells. finally, we performed proliferation assay and proved that adding of this artificially synthesized chemicals to mda231 cell line significantly reduced the level of proliferation, whereas mcf10a cells treated at similar conditions have not revealed any abnormal reduction of proliferation below control level. thus, we report of a strategy to predict highly suitable proteasome inhibitors that act via inhibition of protease activity and may lead to creation of a new class of drugs for cancer therapy. localization and trafficking of prostate specific membrane antigen (psma) and its variant form psḿ glutamate carboxypeptidase ii, also known as prostate specific membrane antigen (psma), is a transmembrane glycoprotein highly expressed in maligant prostate tissues. it was shown to represent very useful diagnostic marker and also potential therapeutic target for prostate cancer. two forms of the enzyme were identified in the prostate: full-length transmembrane form consisting of 750 amino acids and a truncated form (called psḿ ), believed to represent spliced variant of psma. the cdnas of both forms are identical except for 266-nucleotide region near 5´end of psma that is absent in psḿ . this deleted region codes for signal peptide as well as for intracellular and transmembrane domains. we are able to detect two protein forms in prostate cancer model cells (lncap cells) and we also show that both forms are glycosylated suggesting that this truncated form might originate from the processing of full length transmembrane psma. number of methods including differential centrifugation, pulse-chase experiments, immunochemistry and gfp-fusion protein analysis were used to analyze the origin, cell localization and trafficking of psma and psḿ in the mammalian cells. we have investigated the substrate specificity of the ns2b(h)-ns3pro protease by using internally quenched synthetic peptides representing both natural cleavage sequences and their recombinant chimeras. synthetic peptides incorporating the o-aminobenzoic acid/3-nitro-l-tyrosine fluorescence donor-quencher pair were used to analyze the minimum substrate length requirement, residue preferences and the contribution of prime side residues for enzymatic cleavage by the ns3 protease. a series of peptides derived from the ns3/ns4a cleavage site was designed for the substrate length mapping study. amino acid truncations in the non-prime and prime side region differently affected rates of substrate hydrolysis and binding as shown by their km and kcat values. the optimal substrate identified was a heptapeptide spanning p4-p3'. chimeric substrates with all possible combinations of non-prime and prime side sequences derived from 5 polyprotein cleavage sites (c, 2a/2b, 2b/3, 3/4a and 4b/5) were assayed for reactivity with the ns3 protease. kinetic parameters revealed a strong impact of the non-prime side residues on km, whereas variations in the prime side region had greater effect on kcat. the fluorogenic derivative of tetrabasic peptide rrrr/gtgn (c/ns5) demonstrated the highest affinity, whereas the peptide kkqr/sagm (2b/c) had the highest turnover number. the one with the greatest catalytic efficiency was identified as rrrr/sltl (c/4a). in addition, we have shown that a ser at p1' is the most preferred residue. the discovery of ns3 substrates with maximized reactivity will be useful for inhibitor development in sensitive high-throughput assays. inhibiting the mtor pathway with cci-779 results in decreased production of vascular endothelial growth factor in a head and neck squamous cell cancer cell line c.-a. o. nathan 1,2 , n. amirghahari 1,2 , x. rong 1,2 and y. sun 1,2 1 nathan, otolaryngology, otolaryngology/head and neck surgery, louisiana state university health sciences center, shreveport, la, usa, 2 nathan, cancer center, medicine, feist-weiller cancer center, shreveport, la, usa. e-mail: cnatha@lsuhsc.edu introduction: overexpression of the proto-oncogene eif4e in surgical margins of head and neck squamous cell cancer (hnscc) patients is an independent predictor of recurrence and is associated with increase in vascular endothelial growth factor (vegf) expression. activation of eif4e in margins through the mtor pathway has led us to determine that cci-779 an mtor inhibitor has both in vitro and in vivo growth inhibitory effects in hnscc cell lines. we wanted to determine if these effects were associated with decrease in vegf production. material and methods: a hnscc cell line fadu was treated with 1 and 10 ng/ml of cci-779 (previously established ic50 = 1 ng/ml). elisa was used to determine vegf protein levels in conditioned medium at 30', 1, 2, 4, 6, 24 and 48 h after treatment with the drug and compared to control cells treated with the diluent for each of the time points. results: a significant decrease in vegf production of 70% was noted at 24 h and maintained at 48 h in treated cells when compared to control cells at the same time points. the decrease in vegf levels (39-47%) was noted within 6 h of treatment with the drug. the percent decrease in vegf protein levels was the same for both doses of cci-779. conclusions: overexpression of eif4e in hnscc increases translation of mrnas with long 5'utrs, one of which is an important angiogenic factor vegf. inhibiting the mtor path-way with cci-779 can potentially decrease vegf production. this has future clinical implications for arresting tumor progression in hnscc patients with molecular positive margins identified by cells overexpressing eif4e, also known as minimal residual disease. proteases from cell culture of jacaratia mexicana m. c. oliver-salvador 1 , g. barrera plant proteases are important in food industry and food technology. the latex of jacaratia mexicana, caricaceae, fruits contains a high level of cysteine proteases. in this work was established a cell suspension culture of j. mexicana. callus culture was initiated from stem explants of j. mexicana on medium consisted of ¼-strength and full-strength ms mineral salts (murashige and skoog, 1965), full-strength ms organics and 6 g/l agar supplemented with cytokinins: 6-benzylaminopurine (bap) at 0.5 mg/l and 6-furfurylaminopurine (kinetin) at 0.25 mg/l and various concentrations (0.25, 0.5 and 1.0 mg/l) of auxins: 2,4-dichlorophenoxyacetic acid (2,4-d) 4-amino-3,5,6-trichloropiridin-2-carboxilic acid (picloram) indoleacetic acid (iaa) a-naphthaleneacetic acid (naa). all of the treatments induced callus except for the iaa, ana and without added phytohormones. the best auxin concentration for callus development was determined to be 0.5 mg/l. and the best condition medium for callus development and proteolytic activity of callus was determined to be 0.5 mg/l 2, 4-d + 0.5 mg/l bap. cysteine proteases were produced on callus culture of j. mexicana and liberated in the medium. also in the cell suspension culture these enzymes were secreted. our results support that is possible the synthesis of proteases in vitro culture of j. mexicana. since protease is a primary metabolite, further improvement in enzyme production is possible by increasing the growth rate and yield of cell culture of j. mexicana. and arginine, from peptides and proteins at neutral ph. it is known to play an important role in the control of peptide hormones, growth factor activity at the cell surface, and in the membrane-localized degradation of extracellular proteins. therefore, the present work was carried out to clone and express carboxypeptidase m in pichia pastoris, aiming at developing specific inhibitors and to evaluate the importance of the enzyme in different physiological and pathological processes. for this purpose, the enzyme's cdna was amplified from total placental rna by rt-pcr and cloned in the vector ppic9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in pichia pastoris. the results show that the cpm gene, after cloning and transfection, integrated in the yeast genome, which started to produce the active glycosylated protein. the recombinant protein was secreted into the medium and the enzymatic activity was measured with the fluorescent substrate dansyl-ala-arg. the enzyme was purified by a two-step protocol including gel filtration and ionexchange chromatography, resulting in a 1761-fold purified active protein in a concentration of 400 mg/l of fermentation medium. sds-page showed that recombinant cpm migrated as a single band with molecular weight similar to native placental enzyme (62 kda). these results demonstrate for the first time the establishment of a method using pichia pastoris to express human carboxypeptidase m. mutational analysis of active site of glutamate carboxypeptidase ii human glutamate carboxypeptidase ii (gcp ii) is a membrane metallopeptidase expressed predominantly in the nervous system, prostate and small intestine. in the brain, gcp ii catalyzes cleavage of the abundant neuropeptide n-acetyl-l-aspartyl-l-glutamate (naag) to n-acetylaspartate and glutamate. gcp ii is a type ii transmembrane glycoprotein with a short cytoplasmic nterminal region (amino acids 1-18), a transmembrane domain (amino acids 19-43) and a large extracellular domain (amino acids 44-750) where the active site of the enzyme is situated. gcp ii, as a cocatalytic zinc metallopeptidase, has two zn 2+ ions in the active site which are necessary for its enzymatic activity. recently, the crystal structure of gcp ii was determined in our laboratory and amino acids arg210, asn257, lys699 and tyr700 were proposed to bind c-terminal glutamate of naag (mesters et al., manuscript in preparation). in the presented study, we carried out site-directed mutagenesis to assess the influence of these amino acid residues on the activity of gcp ii. in addition, glutamic acid in the position 424 which is proposed to be involved in proton shift during the catalytical hydrolysis of peptide bond, was mutated to alanine. all the mutant proteins were expressed in insect cells, purified to near homogeneity and enzymatically characterized. it was shown that a mutation in any of these positions lead to significantly reduced naag-hydrolyzing activity. the substitution of glu424 almost completely abolished the enzymatic activity, thus suggesting glu424 is crucial for enzymatic activity of gcp ii. kinetic characterizations of mutant proteins and their substrate specificities will be presented in comparison with wild type gcp ii. comparative study of mammalian homologues of human glutamate carboxypeptidase ii glutamate carboxypeptidase ii (gcpii) is a membrane-bound metallopeptidase. in homo sapiens, gcpii was shown to be expressed in various tissues, mostly in the central nervous system, small intestine and prostate. in brain it hydrolyses n-acetylaspartylglutamate (naag), which is the most prevalent peptide neurotransmitter in the mammalian nervous system, to form glutamate and n-acetylaspartate. in small intestine gcpii plays an important role in folate absorption. in prostate its function is still unknown. it was shown that inhibition of gcpii is neuroprotective in many neurodegenerative states. according to current knowledge of this enzyme, its role may also be important in prostate (and possibly other) cancers, where its expression is dramatically changed in comparison with healthy tissue. gcpii is thus becoming an important therapeutic target and diagnostic molecule. in order to analyze structure-activity relationships in related glutamate carboxypeptidases, we set to study the mammalian homologues of human gcpii: gcpii of rattus norvegicus, sus scrofa and mus musculus, which have approximately 90% dna sequence similarity to human gcpii. information on the biochemical properties, expression pattern and structural similarity is crucial e.g. for testing of gcpii inhibitors in animal models. we have cloned and expressed recombinant gcpii of r. norvegicus and s. scrofa in insect cells with the aim to obtain pure recombinant protein sufficient for structural analysis. data on biochemical comparison of rat, pig and human gcpii forms will be presented and interpreted in the light of the gcpii structure. structural analysis of pla protein from y. pestis: docking and molecular dynamics of interactions with mammalian plasminogen systemz e. ruback and p. g. pascutti laborato´rio de modelagem e dinaˆmica molecular, departamento de biofisica, universidade federal do rio de janeiro, rio de janeiro, r.j. brazil. e-mail: eruback@biof.ufrj.br the plasminogen (plg) system is an important mechanism for the cell migration through the tissues in the mammalian organisms. some bacterial agents can activate this system by proteases and lead an uncontrolled degradation of extracellular matrix components (mec), and make an invasive character of these infections. the y. pestis protein pla is a plasmid coded outer membrane protein, with aspartic-protease activity and is closely related with the proteolytic activation of plg in the serine-protease form called plasmin. exactly how the pla activate plg in plasmin remains unclear. we performed in this work the predicted interaction between the plg and pla protein by rigid-body docking with hex and evaluate the complex stability by molecular dynamics (md) using the gromacs. to evaluate the docking accuracy we use the crystal structure of complex plg-streptokinase. the md results show more stability in the docked plg-streptokinase complex than in crystal complex observed by the rmsd and rmsf calculations after 2 ns in simulation box. the pla model was constructed with spdb-viewer using the pdb structure of ompt as template and quality of model was evaluated with prochek. the docked complex of plg-pla show same interaction site predicted in mutagenesis studies. after 8 ns md (72 083 atoms in box), we observed the relax of beta barrel structure of pla and the progressive approximation and stabilization between the cleavage site of plg into the extracellular loops of pla, followed of the increase of hydrogen bonds number. in this study we report the possible aminoacids that can be participant in the active site and the sub sites of interaction. the total understanding of these interactions can be a important tool for drug design against bacterial proteases. glutamate carboxypeptidase ii (gcpii), also known as naa-ladase i, folylpolyglutamate hydrolase (folh) or prostate specific membrane antigen (psma) is localized in number of tissues. in brain astrocytes, it regulates neurotransmission by cleaving neurotransmitter n-acetylaspatylglutamate (naag) into n-acetylaspartate and most common excitatory neurotransmitter glutamate. inhibition of gcpii activity protects against cell death after brain stroke. in animal models it has been also shown that specific inhibitors of gcpii could be useful for the treatment of chronic neuropathic pain, amyotrophic lateral sclerosis and other pathologic situations when excess glutamate is neurotoxic. gcpii is identical to prostate-specific membrane antigen (psma), a tumor marker in prostate cancer. gcpii is also found in the membrane brush border of the small intestine where it acts as a folate hydrolase. this reaction expedites intestinal uptake of folate through hydrolysis of folylpoly-gamma-glutamates to monoglutamyl folates. gcpii inhibitors might thus be useful in the imaging and treatment of tumors where folate is required for their growth. therefore it was of interest to investigate whether gcpii might be upregulated in brain tumors as well. in order to analyze this possibility, we took 57 samples from 49 patients with brain tumors treated in faculty hospital motol during 1999-2004 and determined expression and activity of gcpii by western blots and immunohistochemistry using monoclonal and polyclonal antibodies developed against extracellular epitopes of gcpii. moreover, we characterized the enzymatic activity of the enzyme in human samples and correlated the expression of gcpii with the type and grade of the tumor. search for optimal isosteres in beta-secretase peptidic inhibitors alzheimer's disease is a widespread, neurodegenerative, dementia-inducing disorder. it is ascribed to the presence of a lesion in several brain regions, the neuritic plaques, which are extraneuronal accumulations of b-amyloid protein (ab), a 42-aa insoluble peptide that mixed with axons and dendrites of neurons, interrupt the synaptic process and cause neuronal death. the peptide ab is a product derived by proteolitic cleavage from a larger transmembrane cell protein termed amyloid precursor protein, app. two enzymes are involved in this cleavage: b-secretase and a-secretase. the first one cuts app between met671 and asp672 of app to generate the n-terminus of ab in the rate limiting step of the process, while the second one cleaves at various places within a sequence between amino acids 712 and 717 to generate the respective c-terminus. using a combination of molecular modeling techniques, we have designed a set of novel b-secretase peptidic inhibitors with a variety of isosteres starting from the available crystallographic structure of this enzyme bound to the inhibitor om99-2. some of the resulting ligands are predicted to have higher affinity for this enzyme than the starting compound. these inhibitors have been synthesized, their b-secretase affinity tested and cell essays have been performed to determine their ability to preclude the formation of ab peptides in cell cultures. schizophrenia and bipolar affective disorder (bd) are two neuropsychiatric diseases with high social and economic costs. in spite of the prevalence of these diseases, no effective long-term treatments are currently available. the enzyme prolyl oligopeptidase (pop) shows increased activity in both illnesses. this serine protease hydrolyzes peptide hormones and neuropeptides at the carboxyl end of proline residues. because of the relevance of pop as a therapeutic target, many specific inhibitors of this protein have been developed in recent years. the inhibitors ono-1603, jtp-4819 and s-17092-1 are currently in clinical trial phase. s-17092-1 has been administered safely to humans and has been proposed as a potential treatment for cognitive disorders associated with cerebral aging. our aim is to develop new peptide human pop inhibitors. to obtain the human brain pop required for our studies, the cdna corresponding to the enzyme was cloned and subsequently expressed in e. coli. pop activity was monitored by 19f-nmr using a new synthesized pop substrate labeled with 19f. this substrate allowed us to perform the inhibition assay avoiding the interference problems of colorimetric and fluorimetric assays and was suitable for high throughput screening of new pop inhibitors. different strategies were used to find putative human pop inhibitors: in silico screening and solid phase synthesis of candidates and screening with chinese medicinal plants extracts. furthermore, nmr studies were performed with the purified human enzyme by labeling the protein isotopically with 15n and d2o and by selective labeling of the residues methionine and tryptophan with 13c. nmr spectra of the labeled protein were obtained at 800 mhz by applying trosy techniques. nmr will provide structural information to perform structure-based drug design of new pop inhibitors in the future as well as to study the interaction of the candidates with the active site of the enzyme. the crucial regulatory function of the membrane type 1-matrix metalloproteinase (mt1-mmp or mmp-14) in connective tissue metabolism, pericellular proteolysis of extracellular matrix (ecm) components, zymogen activation and angiogenesis was demonstrated with the severe phenotype of the mt1-mmp-deficient mice. this membrane-anchored enzyme is not only essential for normal development of hard tissues, but highly expressed in different human cancers where its level frequently correlates with malignant parameters. in most cases the high level of mrna or elevated level of protein can be predictive for disease development but these parameters only partly reflect the expression and forms of mt1-mmp in pathological conditions. biosynthesis, trafficking, intracellular activation, internalization, protein-protein interactions, and the level of physiological inhibitors (timps) strictly influence the activity of mt1-mmp in cells and tissues. in our experimental system, we followed mt1-mmp processing and shedding and characterized the cell-associated and released forms of the enzyme (jbc 2000; 275: 12080-12089; jbc 2002; 277: 26340-26350 and biochem j 2005; 386: 1-10). we found active and inactive truncated forms of mt1-mmp as a result of treatments or experimentally generated imbalance with timps. we have also developed approaches to identify mt1-mmp forms in tumor tissues. here we present and discuss different strategies to identify mmp-14 in diverse biological samples. because mt1-mmp endows tumor cells with the ability to invade and metastasize, these strategies can provide valuable information on the role and function of this key protease. contribution of calpain to cellular damage in human retinal pigment epithelium cultured with zinc chelator y. tamada 1 , t. nakajima 1 , t. r. shearer 2 and m. azuma 1,2 1 research laboratories, senju pharmaceutical co., ltd., kobe, hyogo japan, 2 departments of integrative biosciences, oregon health & science university, portland, or, usa. e-mail: yoshiyuki-tamada@senju.co.jp purpose: we previously showed involvement of calcium-dependent cysteine proteases (calpains, ec 3.4.22.17) in neural retina degeneration induced by hypoxia and ischemia-reperfusion. aged macular degeneration (amd) is one of the leading causes for loss of vision. amd showed degeneration of neural retina due to dysfunction and degeneration of the retinal pigment epithelium (rpe). rpe performs critical functions in neural retina, such as phagocytosis of shed rod outer segments. the purpose of the present study was to determine the contribution of calpain-induced proteolysis to damage in human rpe. zinc chelator tpen was used to induce cellular damage since zinc deficiency is a suspected risk factor for amd. methods: third-to fifth-passage cells from human rpe were cultured with tpen. leakage of ldh into the medium was measured as a marker of rpe cell damage. activity of calpains was assessed by casein zymography, and proteolysis of calpain substrates was detected by immunoblotting. to confirm calpain-induced proteolysis, calpain in homogenized rpe was also activated by addition of calcium. results: tpen caused ldh to leak into the medium from rpe cells, and calpain inhibitor sja6017 inhibited the leakage. casein zymography and immunoblotting for calpain and a-spectrin showed activation of calpain in rpe cultured with tpen. proteolysis by activated calpain was confirmed by addition of calcium to homogenized rpe. conclusion: these results suggested that activation of calpain contributed to rpe damage induced by tpen in vitro. acknowledgments: dr shearer has substantial financial interest (research contract and consulting fee) in senju pharmaceutical co., ltd., and dr azuma is an employee of senju pharmaceutical co., ltd., a company that may have commercial interest in the results of this research and technology. this potential conflict of interest has been reviewed and managed by the ohsu conflict of interest in research committee. in vivo and molecular risk factors of chloroquine or pyrimethamine-sulfadoxine treatment failure in children with acute uncomplicated falciparum malaria the risk factors associated with chloroquine (cq) or pyrimethamine-sulfadoxine (ps) treatment failure were evaluated in 691 children enrolled prospectively in six antimalarial drug trials between july 1996 and july 2004 in a hyperendemic area of southwestern nigeria. following treatment, 149 (39%) of 389 children given cq and 64 (22%) of 302 children given ps failed treatment by day 7 or 14. in a multiple regression model, four factors were found to be independent risk factors for cq treatment failure at enrolment: age <7 years [adjusted odds ratio (aor) = 0.46, 95% confidence interval (ci) 0.26-0.84, p = 0.01], asexual parasitaemia >100 000/ll (aor = 0.46, 95% ci 0.23-0.93, p = 0.03), presence of gametocytaemia (aor = 0.48, 95% ci 0.26-0.88, p = 0.02) and enrolment after 4 years of commencement of the study, that is, after 2000 (aor = 0.47, 95% ci 0.25-0.77, p = 0.003). following treatment with cq, two factors were independent risk factors for failure of treatment: delay in parasite clearance >3 days (aor = 0.26, 95% ci 0.1-0.7, p = 0.014) and presence of gametocytaemia on day 7 or 14 (aor = 2.5, 95% ci 1.1-6.0, p = 0.03). in those treated with ps, two factors were found to be independent risk factors for ps treatment failure at enrolment: age <1.5 years (aor = 2.9, 95% ci 1.3-6.4, p = 0.009) and presence of fever (aor = 0.3, 95% ci 0.14-0.78, p = 0.01). following treatment with ps, delay in parasite clearance >3 days (aor = 0.39, 95% ci 0.18-0.84, p = 0.016) was an independent risk factor for failure of treatment. the quintuple mutants made up of triple dhfr (asn-108, arg-59 and ile-51) mutant alleles and double dhps (gly-437 and glu-540) mutant alleles were found in isolates obtained from 33% of patients, was significantly associated with ps treatment failure (p = 0.001), while pfcrt and pfmdr-1 mutant genes did not significantly predict cq treatment failure in these patients. these findings may have implications for malaria control efforts in sub-saharan africa where control of the disease depends almost entirely on antimalarial monotherapy. development of high-throughput assay of lethal factor using native substrate m.-y. yoon department of chemistry, hanyang university, seoul, south korea. e-mail: myyoon@hanyang.ac.kr designing of inhibitors for anthrax lethal factor (lf) is currently of interest as an approach for the treatment of anthrax because lf plays major roles in cytotoxicity of target cells. lf is a zincdependent metalloprotease that specifically cleaves the mitogen-activated protein kinase kinase (mapkk) family. current assay system for the screening of lf inhibitor use the optimized synthetic peptide coupled with various kinds of fluorophores, which enables fast, sensitive, and robust assays suited to high-throughput screening. however, lines of evidence suggest that the regions beside the cleavage site are also involved in specificity and proteolytic activity of lf. in the present study, we tried to develop high-throughput assay for lf activity based on native substrate, mek1. the assay system relies on the ecl signal resulting from a specific antibody against the c-terminal region of native substrate. a glutathione-coated multiwell plate was used as a solid support to immobilize the native substrate by its n-terminal gst-moiety. immobilized substrate increases the specificity and sensitivity lf-catalyzed substrate hydrolysis compared to the solution phase assay. this assay system would be expected to discover a wide spectrum of anthrax inhibitor. while significant progress has been made over the past decade in elucidating the structure and enzymatic mechanism of the 20s proteasome, our understanding of its assembly pathway and the role of the propeptides in the maturation process is still substantially incomplete. similarly, the mechanisms involved in the translocation of substrates into the central nanocompartment are only dimly understood at present. we have used the rhodococcus proteasome to dissect the assembly pathway, combining mutagenesis and crystallographic studies. for the thermoplasma proteasome we have established a ''host-guest'' interaction system which allows us to follow the translocation of specific substrates into the interior of the proteasome by electron microscopy, mass spectroscopy and x-ray crystallography. transferring substrates to the 26s proteasome in the fission yeast schizosaccharomyces pombe c. gordon mrc human genetics unit, western general hospital, edinburgh, uk. e-mail: colin.gordon@hgu.mrc.ac.uk the ubiquitin pathway is found in all eukaryotes. in this pathway, target proteins are covalently modified by the addition of ubiquitin, a 76 amino acid protein, to specific lysine residues. the ability of multi-ubiquitin chains to function as a signal to target proteins for degradation by the 26s proteasome is well documented. a key question is how is the multi-ubiquitin chain is recognized as a signal? fission yeast rhp23/rad23 and pus1/ rpn10 represent two families of multi-ubiquitin chain binding proteins that can associate with the proteasome as well as some e3 ubiquitin ligases. they seem to provide a link to shuttle ubiquitinated substrates from the e3 ubiquitin ligases to the 26s proteasome. a detailed characterization of their proteasome binding will be presented along with their potential role in ubiquitin conjugate dynamics. finally data will be presented indicating that an additional substrate presentation pathway exists in fission yeast which is also conserved in higher eukaryotes. non-proteasomal rpn10 raises the threshold for association of a ubiquitin-binding protein with the proteasome the ubiquitin proteasome pathway is responsible for the removal of the vast majority of short-lived proteins in the cell. in order to be degraded, a protein substrate is tagged with polyubiquitin and delivered to the proteasome where it is proteolysed. a slew of shuttle proteins is thought to mediate the delivery of polyubiquitinated substrates, although the mechanism remains elusive. one such family of proteins is comprised of rad23, dsk2 and ddi1, which all bind polyubiquitinated substrates through a ubiquitinassociated domain (uba) as well as the proteasome through their ubiquitin-like domain (ubl). another potential shuttle structurally unrelated to the ubl-uba family is rpn10. rpn10 is found as an integral subunit of the proteasome as well as an in an unincorporated pool. we characterized the interactions of these proteins with individual proteasomal subunits, as well as between themselves. we find unique relationships between the putative shuttle proteins and the proteasome, pointing to functional dissimilarity among them. strikingly, unincorporated rpn10 interferes with binding of dsk2 to the proteasome. thus, we propose that rpn10 might play a negative role in proteolysis through its action on dsk2. proteins modified by multi-ubiquitin chains are usually targeted for degradation by the proteasome. in other cases, ubiquitylation mediates protein sorting or regulates other functions. a striking example for a non-proteolytic role of ubiquitin is the rad6 dna damage bypass at stalled replication forks. key elements of this pathway are two ubiquitin-conjugating enzymes, rad6 and the mms2/ubc13 heterodimer, which are recruited to chromatin by the ring-finger ubiquitin ligases, rad18 and rad5, respectively. moreover, also the sumo-conjugating enzyme ubc9 is affiliated with the pathway and we discovered that proliferating cell nuclear antigen (pcna), a dna-polymerase sliding clamp involved in dna synthesis and repair, is a substrate. pcna is (i) mono-ubiquitylated by rad6/rad18, (ii) modified by lysine (k) 63-linked multi-ubiquitylation, which additionally requires mms2/ubc13/ rad5, and (iii) sumoylated by ubc9. all three modifications affect the same lysine residue of pcna, indicating that they label pcna for alternative functions. indeed, we discovered that monoubiquitylation of pcna promotes an error-prone replication bypass, whereas k63-linked multi ubiquitylation mediates errorfree replication across the lesions. in contrast, sumoylation, which occurs even in the absence of dna damage, prevents recombination between homologs at the replication fork. these findings indicate that mono-ubiquitin, k63-linked multi-ubiquitin chains, and sumo are crucial for decision making at the replication fork. ubiquitin-mediated proteolysis is the primary mechanism in eukaryotes for degrading unwanted and misfolded proteins. through the cascade of e1, e2 and e3 enzymes, ubiquitin monomers are attached sequentially to the target proteins, which are then recognized and degraded by the 26s proteasome. the selection and specific timing of polyubiquitination of the target proteins are conferred by different e3 ubiquitin ligases. the anaphase-promoting complex (apc) is one of the most extensively studied e3 ubiquitin ligases that plays essential role in the cell cycle and specific developmental processes. the core apc is composed of 11-13 subunits. except for apc2 and apc11, relatively little is known about the role of the other apc subunits or the assembly of the complex. two wd40-repeat activator proteins, cdc20 and cdh1 determine stage-specific activation of the core apc as well as selection and binding of the apc substrates. in plants, the apc activators are present in multiple copies. arabidopsis contains 5 cdc20 genes, 3 cdh1-type activators known as ccs52a1, ccs52a2 and ccs52b. our work has been focused on the function of apc activators in the cell cycle and plant development, identification of novel apc substrates and on the assembly of the apc complexes. apc activities, based on the expression profiles of the cdc20 and ccs52 genes, will be presented at organism level. by detailed protein interaction studies in yeast two hybrid system and arabidopsis protoplasts or transgenic plants, we shall demonstrate how the core apc interacts with the activators and substrates, and propose a model for apc assembly. characterization of substrate delivery to the saccharomyces cerevisiae proteasome by quantitative shotgun proteomics the proteasome is the central protein degradation machinery in the eucaryotic cell. in conjunction with the ubiquitin system, it is responsible for constitutive bulk protein turnover as well as the controlled degradation of regulatory proteins. the system is very well characterized, but the mechanism by which poly-ubiquitinated substrates are delivered to the proteasome remains unclear. recently our lab has proposed a number of proteins to be proteasome-based receptors for poly-ubiquitinated substrates in s. cerevisiae (rpn10p, rad23p, dsk2p; verma et al., 2004). others (e.g. richly et al. 2005) have put forward a complex model for the delivery of substrates from the ubiquitinating machinery to the proteasome involving the aaa atpase cdc48p. by analyzing the composition of affinity purified proteasome complexes from s. cerevisiae cells lacking these factors and/or exposed to specific proteasome inhibition, we hope to further elucidate the substrate delivery pathway. ubiquitinated proteins recruited to the proteasome are identified utilizing capillary chromatography in-line to electrospray ion trap mass spectrometry (mudpit; link et al. 1999). using a reference strain grown in minimal medium solely providing heavy nitrogen ( 15 n) as an internal standard, we are able to record even gradual fluctuations in sample composition. differences in the recruitment of substrates to the proteasome in varying mutant backgrounds will shed light on the specificity of proteasome substrate receptors and the topology of the substrate delivery mechanism. oxidative protein damage by reactive oxygen species (ros) produces cross-linking, fragmentation and biochemical modification of the amino acids resulting in biological dysfunctions. quercetin, a widely distributed bioactive plant flavonoid, possesses anti-cancer, antioxidants and free radical scavenging activities, as well as it binds with dna causing dna fragmentation. a little is known about protein oxidative damage and its modifications by antioxidants. therefore, the aim of the present work was to investigate the molecular mechanisms of antioxidant and prooxidant activities of quercetin toward proteins. the antioxidant activities of quercetin, such as superoxide dismutase (sod)-and catalase (cat)-mimetic as well as hydroxyl radical (aeoh) scavenging activities were possessed. bovine serum albumin (bsa) was incubated with different concentrations of quercetin. quercetin has highly sod-and cat-like and hydroxyl radical (aeoh) scavenging activities. its activities are concentration dependent. quercetin fragmentized bsa into specific fragments which they detected by sds/polyacrylamide gel electrophoresis. oxidative protein damage was assessed as tryptophan oxidation, carbonyl, quenone and advanced oxidation protein products (aopp) generation. the increase of protein oxidation products was in concentration dependent manner. the carbonyl and quenone contents and aopp were highly significantly elevated in querce-tin-treated proteins when compared with the control sample. the tryptophan fluorescence was highly decreased in treated protein than in the control sample. the mechanisms of antioxidant and pro-oxidant activities of quercetin have been discussed. these results demonstrate that antioxidant quercetin may potentiate protein damage via oxygen free radical generation, particularly .oh radicals by quercetin. protein stability mediated by a hyaluronanbinding deubiquitinating enzyme is involved in cell viability protein degradation by the ubiquitin system plays a crucial role in numerous cellular signaling pathways. deubiquitination, a reversal of ubiquitination, has been recognized as an important regulatory step in the ubiquitin-dependent degradation pathway. we have identified three novel genes encoding a deubiquitinating enzyme, vdub1, vdub2, and vdub3 (villi deubiquitinating enzyme 1, 2, and 3) from human chorionic villi by rt-pcr. their cdnas are 1,593 bp in length and encode an open-reading frame of 530 amino acids with a molecular weight of approximately 58 kda. expression analysis showed that vdub transcripts are highly expressed in the heart, liver, and pancreas. in addition, they are expressed in various human cancerous cell lines. amino acid sequence analysis revealed that they contain the highly conserved cys, his, and asp domains, which are required for the formation of active site for the deubiquitinating enzymes. in vivo and in vitro deubiquitinating enzyme assays indicated that vdub1, vdub2, and vdub3 have deubiquitinating enzyme activity. here, we show that the overexpression of vdub proteins leads to irregular nuclear morphology and apoptosis, suggesting that these vdubs play an important role in regulating signal transduction involved in cell death. interestingly, the sequence analysis showed that vdub proteins contain the putative hyaluronan/mrna-binding motifs, and cetylpyridinium chloride-precipitation analysis confirmed the association between vdubs and intracellular hyaluronan and rna. chemical cleavage of peptide (amide) bonds usually requires harsh conditions. as a result of side reactions and the lack of specificity, chemical amide bond hydrolysis is not a preferred means of protein digestion. we have discovered selective cleavage of peptide bonds in proteins under milder circumstances than any previously reported chemical method. hydrolysis takes place in aqueous buffers in a ph range of 410, and occurs c-terminal to the proteogenic non-natural amino acid azido-homoalanine (azhal), effected by a staudinger reaction after addition of the mild and biocompatible reagent tris(carboxyethyl)phosphine (tcep). key feature in the suggested reaction mechanism is the unprecedented nucleophilic substitution of the resulting gammaiminophosphorane by the flanking c-terminal backbone amide oxygen atom. after hydrolysis, the new c-terminal peptide is present as a homoserine lactone residue and the n-terminal peptide as its free amine. this new reaction may find application as a very mild and selective bio-orthogonal degradation pathway in biochemistry and biomaterials science. overexpression of proteasome b5 subunit increases amount of assembled proteasome and confers ameliorated response to oxidative stress and higher survival rates the proteasome is the major cellular proteolytic machinery responsible for the degradation of both normal and damaged proteins. proteasomes play a fundamental role in retaining cellular homeostasis. alterations of proteasome function have been recorded in various biological phenomena including aging. we have recently shown that the decrease in proteasome activity in senescent human fibroblasts relates to the down-regulation of btype subunits. in this study we have followed our preliminary observation by developing and further characterizing a number of different human cell lines overexpressing the b subunit. stable overexpression of the b5 subunit in wi38/t and hl60 cells resulted in elevated levels of other b-type subunits and increased levels of all three proteasome activities. immunoprecipitation experiments have shown increased levels of assembled proteasomes in stable clones. analysis by gel filtration has revealed that the recorded higher level of proteasome assembly is directly linked to the efficient integration of ''free''/not integrated b-type subunits identified to accumulate in vector-transfected cells. in support we have also found low pomp levels in b5 transfectants thus revealing an increased rate/level of proteasome assembly in these cells as opposed to vector-transfected cells. functional studies have shown that b5 overexpressing cell lines confer enhanced survival following treatment with various oxidants. moreover we demonstrate that this increased rate of survival is due to higher degradation rates following oxidative stress. finally, as oxidation is considered to be a major factor that contributes to aging and senescence, we have overexpressed the b5 subunit into primary imr90 human fibroblasts and we have observed a delay of senescence by 45 population doublings. in summary, these data demonstrate the phenotypic effects following genetic up-regulation of the proteasome and provide insights towards a better understanding of proteasome regulation. expression levels of the components of the ubiquitin/proteasome pathway in pisum sativum seedlings under anoxia stress change in gene expression: proteins produced under aerobic conditions are no longer synthesized and are replaced by the socalled anaerobic peptides. among those proteins synthesized under o 2 deficiency some enzymes of the glycolytic and fermentative pathways were identified in plants. upon reintroduction of air, the anaerobic mrnas disappear rapidly and the increased levels of those enzymes must return to the basal levels. the ubiquitin/proteasome system is a major pathway of proteolysis in eukaryotic cells and may contribute to controlling the intracellular levels of a variety of short-lived regulatory proteins. in this proteolytic pathway, proteins are covalently conjugated to ubiquitin, which flags them for rapid hydrolysis by the 26s proteasome. long polyubiquitin chains must be formed to target a protein for destruction by the proteasome. in plants, the ubiquitin-mediated proteolytic pathway is implicated in a variety of cellular processes, including stress responses. in this study, 3-dayold pisum sativum seedlings were subjected to: (i) 15 h of anoxia stress; (ii) 2 h of aerobic conditions after 15 h of anoxia stress and (iii) 4 h of aerobic conditions after 15 h of anoxia stress. the levels of free and conjugated ubiquitin were detected by immunoblotting using anti-ubiquitin polyclonal antibodies. the changes in the mrna levels of some components of the ubiquitin/proteasome pathway in the seedlings were determined by relative semiquantitative rt-pcr. the results suggest an involvement of the ubiquitin-mediated proteolytic pathway in the anoxia stress response. b2-012p involvement of the anaphase promoting complex in plant development controlled degradation of short-live proteins via ubiquitindependent proteolysis by the 26s proteasome is a key mechanism in eukaryotes that regulates nearly all fundamental cellular processes including cell cycle. polyubiquitination of the protein substrate is sufficient to target it for degradation by a large atp-dependent multicatalytic protease, the 26s proteasome. the selection and specific timing of ubiquitination of the target proteins are conferred by different e3 ubiquitin ligase. the anaphase promoting complex (apc) is one of the e3 ubiquitin ligases, which by ordered destruction of various cell cycle proteins has fundamental roles in the regulation of mitotic and endoreproduplication cycles. the apc functions also outside the cell cycle. in post-mitotic cells, the cdh1 adaptor protein ensures stage specific activation and substrate selection of the apc. in plants, two classes of the cdh1-type activators have been identified, ccs52a and ccs52b that display differential regulation during the cell cycle and plant development as well as differences in their substrate-specificities. in arabidopsis, transient and complimentary expression profiles of the atccs52a1, atccs52a2 and atccs52b genes indicate apc functions during flower development. to identify apc targets, yeast two hybrid screens were performed in the laboratory. out of about 200 interacting proteins, several proteins were transcription factors including a key a regulator of flowers development. data on the interactions of the ccs52 proteins and transcription factors in arabidopsis protoplasts will be presented as well as a model for the apc regulated pathways. novel effects of ubiquitin system and chaperone proteins on the prion ''life cycle'' in yeast t. a. chernova 1 , k. d. allen 2 , e. p. tennant 2 , k. d. wilkinson 1 and y. o. chernoff 2 1 department of biochemistry, emory university, atlanta, ga, usa, 2 school of biology and institute for bioengineering and bioscience, georgia institute of technology, atlanta, ga, usa. e-mail: tcherno@emory.edu yeast prion [psi + ], the self-propagated aggregated isoform of the translation termination factor sup35, is used as a model system to study neural inclusion disorders. prion aggregates and other neural inclusions in mammals were previously reported to sequester ubiquitin (ub). proteasome inhibitors affected the turnover of mammalian prion proteins. however, a role of ub-dependent proteolysis in the prion ''life cycle'' has not been clearly defined. chaperone proteins, which are also implicated in ub-dependent proteolysis, have been shown to influence the formation and propagation of the prion aggregates. our results uncover the connection between alterations of ub system and chaperone proteins in their effects on the maintenance of yeast prion. we have demonstrated that deletions of genes encoding deubiquitinating enzymes, that are critical for ub regeneration at the proteasome (ubp6) or the vacuole (doa4), cause pleiotropic phenotypic effects that are primarily due to decreased levels of free ub in the yeast cells. these alterations, as well as deletion of the gene encoding ub-conjugating enzyme, ubc4, decreases [psi + ] curing by the overproduced disaggregase hsp104, suggesting that ub system influences hsp104-dependent clearance of prion aggregates. spontaneous [psi + ] formation was also increased in the ubc4 depleted cells. we previously demonstrated that excess of cytosolic chaperone ssa of hsp70 family increases de novo formation of [psi + ]. both in vivo and in vitro experiments uncover direct interactions between sup35 and hsp70 proteins. the amount of sup35-bound to hsp70-ssa was increased in ubc4 deletion strain. we propose a model to explain roles of hsp104, hsp70 and ub system in the prion life cycle. effects of parkinson''s disease mimetics on proteasome activity and protein turnover in human sh-sy5y neuroblastoma cells it has recently been suggested that impairment of the ubiquitin/ proteasomal system contributes to the degeneration of dopaminergic neurons (dn) and lewy body (lb) formation in parkinson's disease (pd). mitochondrial dysfunction is also a key factor in pd and agents such as mpp + and dopamine, which inhibit mitochondrial electron transport, produce selective degeneration of dn in animal models. in this study the effects of treating sh-sy5y cells with mpp + or dopamine over 72 h on proteasomal chymotrypsin-like activity (cla) was monitored. mpp + (0.1mm) caused a sustained depletion of glutathione levels followed by a reduction in proteasomal activity. a reduction in atp levels, caused by higher levels of mpp + (2mm), exacerbated this effect. exposure to low dopamine concentrations (0.1mm) led to large reductions in atp without affecting cla or glutathione levels; whilst higher concentrations (2mm) caused marked reductions in cla, glutathione and atp levels. these results suggest that, under oxidative stress, glutathione levels are important regulators of proteasomal activity in this cell line. our group has shown that mpp + can destabilize the neurofilament network in shsy-5y cells, partly due to changes in phosphorylation of neurofilament (nf) chains. as nfs are important components of lbs, and their mode of turnover is uncertain, we tested the effects of proteasome inhibitors on nf levels. treatment with these inhibitors led to nf accumulation, which was enhanced when glutathione levels were artificially depleted, suggesting that nfs can be degraded via the proteasomal pathway. the effects of proteasome impairment on protein accumulation will be discussed. mitochondria and the hypoxia-inducible factor 1 (hif-1): regulation of hif-1 is independent of a functional mitochondrial respiratory chain k. doege, w. jelkmann and e. metzen insitute of physiology, university of luebeck, luebeck, germany. e-mail: doege@physio.uni-luebeck.de the hypoxia-inducible factor hif-1 is the ''master-regulator'' in adaptation to low oxygen concentration and induces the hypoxic expression of several target genes, e.g. erythropoietin and vascular endothelial growth factor (vegf). in normoxia hif-1a is constantly produced but also degraded by oxygen-dependent prolyl-hydroxylation. mitochondria consume most of the oxygen delivered to cells and have been implicated in oxygen sensing. firstly, mitochondria have been proposed to stabilize hif-1a by production of reactive oxygen species (ros) in hypoxia. secondly, inhibition of the respiratory chain, e.g. by nitric oxide, has been proposed to cause redistribution of intracellular oxygen followed by reactivation of the prolyl hydroxylases and inhibition of hif signalling. we have used cells depleted of mitochondrial dna (q0) and gas permeable cell culture dishes to eliminate all oxygen diffusion gradients affecting the cells. we show that these dishes neutralize all effects of mitochondrial inhibition. additionally, cellular hypoxia as assessed by pimonidazole staining has been evaluated in human osteosarcoma cells treated with inhibitors of the respiratory chain under hypoxia. these results demonstrate an elevated po 2 under hypoxic conditions after treatment with mitochondrial inhibitors correlating with an intracellular oxygen concentration which reduces hif-1 activation. thus, neither the absence of ros nor the redistribution of intracellular oxygen supply leads to the destabilization of hif-1a in hypoxia. our experiments provide evidence that an increased intracellular po 2 evoked by the absence of mitochondrial oxygen consumption reactivates the prolylhydroxylases and is therefore responsible for the degradation of hif-1a under hypoxic conditions. enzyme activity is generally higher in rhizosphere than in bulk soil, as a result of a greater microbial activity sustained by toot exudates or due to the release of enzymes from roots. negative effects of heavy metals on soil microorganisms and enzyme activities have been long recognized. the aim of this study was to assess the stimulatory effects of different low molecular weight organic compounds commonly present in root exudates (mres) on microbial activity and protease activities and , and how high cd concentrations affect such stimulatory effects. soils (arenic udifluvent) were sampled from the agir long-term field trials, contaminated with cd nitrate at rates of 0 (control soil), 20 and 40 mg cd per kg of soil. the mre solutions contained glucose, citric acid, oxalic acid, glutamic acid or a mixture of the four compounds, added to give a rate of 300 mg of mre-c per kg of soil. the effects were measured at 4 mm (bulk soil) distance from the mrs. protease activity was determined by hydrolysis of n-benzoylargininamide (baa). the results showed that different mres had different stimulatory effects on microbial growth and on the protease activities, mostly localized in the rhizosphere soil layer. in the control soil, the dsdna content was significantly increased by the addition of all mre in both rhizosphere and bulk soil layers. the 20 and 40 mg cd per kg of soil negatively affected on protease activity. the glucose, citric acid, oxalic acid, glutamic acid, mres mix in both rhizosphere and bulk soil layers, did not stimulate protease. the, microbial growth and protease activities were drastically reduced by high cd concentrations. participation of different digestive proteinases of the yellow mealworm, tenebrio molitor, in initial stages of hydrolysis of the main dietary protein insects generally have a wide spectrum of digestive proteinases. the knowledge about the impact of different proteinases to initial stages of hydrolysis of dietary proteins is essential for insect control by means of proteinase inhibitors and bacillus thuringiensis toxins. the larvae of a stored grain pest yellow mealworm, tenebrio molitor, were reared on milled oat flakes. the main dietary protein for these larvae was 12s globulin, the main storage protein of oat seeds. to study the initial stages of 12s globulin hydrolysis in vitro the reaction was performed in the physiological conditions of anterior midgut (am) (ph 5.6) by purified enzyme preparations from am: two fractions of cysteine proteinases cys ii and cys iii, chymotrypsin-and trypsin-like proteinases. total hydrolysis of 12s globulin was observed with cys ii. slightly less effective was hydrolysis by chymotrypsin-like enzyme. cys iii cysteine and trypsin-like proteinases produced only partial hydrolysis of seed globulin. in all cases high molecular mass (mm) intermediate products were formed testifying that hydrolysis of 12s globulin was sequential. incubation with both cysteine proteinase fractions led to formation of 31 kda product, while serine proteinases proin contrast to ''classical'' bioregulator peptides, peptides could be generated in the course of catabolic degradation of functional proteins. for 15 years, we have been interested in such particular group of peptides derived from blood hemoglobin, hemorphins. hemorphins consist in a family of opioid receptor-binding peptides from 4 to 10 amino acids that are released by proteolytic processing from the (32-41) segment of human hemoglobin betachain. they are prevalent throughout the peripheral and central nervous system and have been isolated in vivo from tissues or fluids. many in vivo physiological effects have been related (coronaro-constrictory, anti-tumorous, immunoregulatory activities) and several of the hemorphins interact at various levels of the reninangiotensin system (ras) by inhibiting angiotensin-convertingenzyme (ace), aminopeptidase n (apn) and dipeptidyl peptidase iv (dppiv) activities. in addition, some hemorphins and in particular lvv-hemorphin-7 (lvvypwtqrf), binds with high affinity to the brain (ic 50 = 4.15nm) and renal at4 angiotensin receptor subtype and is possible the main endogenous ligand from this receptor. in an attempt to characterize in vivo precise mechanisms for their release, our attention is focused towards tumoral and central nervous system environments. the last one is particularly interesting as all cellular components implicated in the release of hemorphins are present simultaneously: the haemoglobin precursor and localized brain proteases which might come in contact with blood haemoglobin. in this purpose, the examination of potentiality for this tissue to generate ''neuro''-hemorphins would be of interest since sources of hemorphins in the brain have not yet been definitively established. later, we showed that sgt1 interacts with calcyclin (s100a6) and other calcium-binding proteins of the s100 family (nowotny et al. j biol chem 2003). moreover, in collaboration with dr chazin's group, we found that in vitro sgt1 binds to hsp90 (lee y-t et al. j biol chem 2004). in this work we studied the expression and subcellular localization of sgt1 in mammalian cells by means of western and northern blots. among different cell lines examined human embryonic kidney hek293 and human glioma t98g cells exhibit highest expression of sgt1 protein. moreover, we found that in mouse and rat cells there is one isoform of sgt1, while in human cells two isoforms of this protein were found. to study the subcellular localization of sgt1 we chose the cells containing moderate level of sgt1 such as human epidermal hep-2 cells. by applying immunocytochemistry we found that this protein is present not only in the cytoplasm but also in the nucleus. at present we check the effect of intracellular ca 2+ concentration on subcellular localization of sgt1 and on its co-localization with target proteins. acknowledgements: this work was supported by grants: kbn 3 p04a 043 22 and firca/nih r13 tw006005. combining reverse genetics, reverse chemogenomics and proteomics to assess the impact of protein n-terminal methionine excision in the cytosol of higher eukaryotes in living organisms whatever the cell compartment, proteins are always synthesized with methionine (met) as the first residue. however, this first met is specifically removed from most mature proteins. in the course of protein n terminal met excision (nme), the free n terminal met is removed by met aminopeptidase (map) cleavage. three enzymes (map1a, map2a and map2b) have been identified in the cytoplasm of arabidopsis thaliana. by combining reverse genetics and reverse chemogenomics in transgenic plant lines, we have devised specific and reversible switches for the investigation of the role of cytoplasmic nme in a. thaliana and of the respective contributions of the two types of cytoplasmic map throughout development. in the map1a ko context (map1a-1), modulating map2 activity by treatment with various concentrations of the specific drug fumagillin impaired plant development. hence, (i) cytoplasmic nme is essential in plants, (ii) plant map1a and map2s are functionally interchangeable as a complete block of either map type activity does not cause any visible molecular or phenotypic effect, (iii) a minimal level of cytoplasmic map is required for normal development and (iv) the plant a. thaliana appears an excellent system to study nme and the associated-role of anti-cancer agents like fumagillin. proteomics was used to assess the impact of nme blocking induced by fumagillin. we used a wild-type plant and the map1a-1 variant grown in the presence of 100 nm fumagillin. the map1a-1 variant showed a dwarf phenotype. we compared by 2d gel electrophoresis the patterns of each protein extracts. protein spots were identified by tandem mass spectrometry. the data show that fumagillin induces many dedicated pathways, with a prevalence of those related to oxidative stress. prolyl endopeptidases from the midgut of the yellow mealworm tenebrio molitor side of proline residues. these enzymes were found in mammals, several higher plants, fungi and bacteria. it is suggested that the enzymes participate in the in vivo regulation of the action of biologically active peptides. we for the first time report about two prolyl endopeptidases in the larval midgut of a stored product pest yellow mealworm tenebrio molitor where they can participate in the proteolysis of one of the main dietary proteins of t. molitor larvae -rich in proline prolamines. characteristics of two prolyl endopeptidases are significantly different. optimum for hydrolysis of the substrate z-ala-ala-pro-pna (n-carbobenzoxy-l-alanyl-l-alanyl-l-prolyl-p-nitroanilide) by prolyl endopeptidase 1 was at ph 8.5, and prolyl endopeptidase 2 -at ph 5.6. prolyl endopeptidase 1 displayed high phstability in the ph range 7.0-10.0 and the rate of hydrolysis increased in the presence of kcl and cacl 2 . prolyl endopeptidase 2 demonstrated low stability in the whole ph range, the rate of hydrolysis strongly decreased in the presence of above mentioned salts, but increased in the presence of high concentrations of edta. the influence of cell growth media on the stability and antitumour activity of methionine enkephalin studies with cultured tumour cell lines are widely used in vitro to evaluate peptide-induced cytotoxicity as well as molecular and biochemical interactions. the objectives of this study were to investigate the influence of the cell culture medium on peptide metabolic stability and in vitro antitumour activity. the degradation kinetics of the model peptide methionine enkephalin (met-e, tyr-gly-gly-phe-met), demonstrated recently to play an important role in the rate of proliferation of tumour cells in vitro and in vivo, were investigated in cell culture systems containing different amounts of foetal bovine serum (fbs). the influence of enzyme inhibitors (bestatin, captopril, thiorphan) on the met-e degradation was also investigated. the results obtained in the dulbecco's modified eagle medium containing 10% fbs indicated a rapid degradation of met-e (t 1/2 = 2.8 h). pre-incubation of the medium with a mixture of peptidase inhibitors reduced the hydrolysis of met-e, as shown by increased half-life to 10 h. the in vitro activity of met-e against poorly differentiated cells from lymph node metastasis of colon carcinoma (sw620) and human larynx carcinoma (hep-2) cells was determined. tumour cells were grown 3 weeks prior to the experiment in a medium supplemented with 10, 5 or 2% fbs. statistically significant to mild or no suppression of cell proliferation was observed in all cultures. in both cell lines, a significant suppression of cell growth by a combination of peptidase inhibitors and met-e, compared with cells exposed to the peptide alone and cells grown in the absence of met-e, was observed. this study indicated that caution must be exercised in interpreting the antiproliferative effects of peptide compounds in conventional drug-response assays. protein metabolism in whole body and skeletal muscle of laboratory rats treated by proteasome inhibitors proteasome inhibitors are new agents which may be used in treatment of cancer and other severe disorders. one of the possible side effects of their administration is disturbance in protein metabolism which may affect outcome of the illness. two separate studies were performed using wistar rats. in the first study, m. soleus (sol) or m. extensor digitorum longus (edl) were incubated in medium containing 30 mmol/l mg 132 or 30 mmol/l adaahx3l3vs or without inhibitor (control). protein synthesis was evaluated using l-[1-14c]leucine. proteolysis was determined according to the rate of the tyrosine release into the medium during incubation. in the second study, proteasome inhibitor mg 132 diluted in dimethyl sulfoxide (dms) was administered intraperitoneally in dose 10 mg/kg b.w. controls consisted of dms treated animals. changes in protein and amino acid metabolism were estimated in steady-state conditions using continuous infusion of l-[1-14c]leucine 2 h later. mann-whitney (in vivo study) and paired t-test (in vitro study) were used for statistical analysis. in in vitro study, both mg 132 and adaahx3l3vs significantly decreased protein synthesis and proteolysis. however, in in vivo study, a significant increase in whole-body protein synthesis and proteolysis were observed in mg 132 treated animals. acknowledgements: the study was supported by a grant of gacr no. 303/03/1512. bioinformatical evidence for a prokaryotic ubiquitin-like protein modification system h. scheel, s. tomiuk and k. hofmann bioinformatics group, memorec biotec gmbh, ko¨ln, germany. e-mail: kay.hofmann@memorec.com until recently, the ubiquitin system has been considered a purely eukaryotic invention. by now, the bacterial moad/moeb and this/thif systems are known to be prokaryotic versions of a rudimentary activation system for ubiquitin-like proteins. however, similarities to the ubiquitin system end after the activation step, as moad and this are not conjugated onto target proteins but rather have a role in the biosynthesis of molybdopterin and thiamin, respectively. the eukaryotic protein urm1 is the closest homolog of moad and this. unlike its bacterial cousins, urm1 is conjugated onto target proteins and thus can be considered the founding member of the diverse eukaryotic ubiquitin family. by using a bioinformatics approach that integrates methods of sequence analysis, phylogenetics, phylogenomics and gene-order analysis, we were able to show that many bacteria possess a third ubiquitin-like activation system that most likely is used for protein modifications. the novel system uses a moad/this relative, which is more closely related to urm1 than the typical moad and this proteins. these bacterial urm1 (burm1) proteins typically require the proteolytic removal of a c-terminal extension, which masks the gg motif important for activation. many burm1 operons contain a mpn+/jamm domain protein (belonging to a bona fide ubiquitin-specific protease family), which is most likely responsible for this cleavage. as a third component, an e1-like enzyme is also part of typical burm1 operons. the burm1-associated e1 enzymes look more like uba4 (the eukaryotic urm1-e1) than like the bacterial moeb/thif e1 enzymes. interestingly, the mpn+/jamm protease is also conserved in those bacteria whose burm1 end with gg, suggesting that burm1 removal is important not only for the activation step b2-025p non-hypoxic induction of hypoxia-inducible factors by insulin and 2-deoxy-d-glucose hypoxia-inducible factors (hifs) are key mediators of the cellular adaptation to hypoxia, but also respond to non-hypoxic stimuli like insulin. to clarify involvement of all known hif subtypes in conditions resembling diabetes, we determined distribution of mrnas and proteins in rats subjected to in vivo hypoglycemia and glucoprivation. wistar rats were infused with either saline, insulin, or 2-deoxy-d-glucose (2-dg) to provoke hypoglycemia or impaired glucose assimilation. using real-time qpcr, mrna levels of hif subunits 1a, 2a, 3a, 1b, and of the target gene glut-1 were determined in various organs. cellular distributions of hif-a proteins were examined by immunohistochemistry. treatments with insulin or 2-dg resulted in a widespread increase in hif-3a mrna after 6 h, whereas mrna expression of other hif subunits remained unaffected, except for hif-2a which increased in lung and heart after 2-dg. in cerebral cortex and kidney, enhanced staining of all hif-a proteins was observed after insulin or 2-dg treatments. lung, heart and kidney showed enhanced levels of glut-1 mrna. both hypoglycemia and glucoprivation provoke functional activation of the hif system, with transcriptional up-regulation of hif-3a representing a typical response. our data indicate an involvement of the hif system, and hif-3a in particular, in the pathophysiology of diabetes. fragments of human salivary statherin and pb peptide underlying a furin-like pro-protein convertase action in the pre-secretory salivary fragmentation pathway the recent analysis of some derivatives of human salivary peptides and proteins [13], such as acidic and basic proline-rich proteins (prp) and histatins, allowed recognizing in the presecretory salivary fragmentation pathway the action of a furinlike pro-protein convertase of the kexin-subtilisin family, often followed by a carboxy-peptidase action. on the same line, the present study was carried out to search in human saliva the fragments generated from statherin and pb peptide by the action of furin-like proteinases, utilizing a selected-ion monitoring strategy based on hplc-it ms. the fragments and post-translational derivatives detected with high frequency in multiple samples were the following: (i) statherin (5380 amu), des-phe-43 (5232 amu), des-thr-42-phe-43 (5131 amu), des-asp-1 (5265 amu), mono-phosphor. (5300 amu), statherin sv2 (missing 6-15 residues; 4149 amu), fragm. 10-43 (4128 amu), fragm. 11-43 (3971 amu), fragm. 14-43 (3645 amu). moreover, the fragm. 6-57 (5215 amu) of pb peptide (5793 amu) was identified. the quantity of these fragments in salivary samples was usually <10% of the parent peptide. the identified fragments confirmed the action of a proprotein convertase on furin-like consensus sequences, being the cleavage at arg-9 (ekflr), arg-10 (lrr) and arg-13 (rrigr) for statherin, and at arg-5 (rgpr) for pb peptide. detection of statherin missing n-and c-terminus residues indicated also a pre-secretory exopeptidase action, already observed in other salivary peptides. the function of these statherin and pb derivatives in the oral cavity must be elucidated. cloning and expression of a pepstatin insensitive acid protease from thermoplasma volcanium in e. coli acid proteases, commonly known as aspartic proteases, are recognized by their specific inhibition by pepstatin. acid proteases are found in microorganisms both as intracellular and extracellular enzymes. there is very limited number of thermostable, pepstatin insensitive acid proteases isolated from bacterial sources. the only example of purified and cloned acid protease from archaebacteria is thermopsin, produced by sulfolobus acidocaldarius. this thermophilic enzyme represents a new class of acid proteases. to extend our knowledge on the microbial acid proteases with thermostable properties, in this study we have undertaken the cloning and expression of a thermostable, pepstatin insensitive acid protease from themoacidophilic archaeon thermoplasma volcanium. a primer set was designed based on nucleotid sequence of the predicted thermopsin gene and pcr amplification produced a 3080 bp fragment, which covered complete thermopsin gene with some upstream and downstream sequences. the amplified thermopsin gene was cloned in e. coli, using pdrive vector. the alignment of the amino acid sequences of thermopsins from various archaea revealed the highest homology (44%) between the tp. volcanium thermopsin and putative tp. acidophilum enzyme, thermopsin 1. there was a low degree of similarity (28%) between the tp. volcanium thermopsin and thermopsin from sulfolobus acidocaldarius. expression of the recombinant thermopsin was attempted using qia expression kit, where the cloned gene was ligated to pqe expression vectors to be expressed under the control of t5 promoter. in this system the protein was tagged with 6xhis residue at n-terminal end so that it could be selectively isolated using ni-nta metal-affinity chromatography. include various vital proteins with discrete functions in the propagation of apoptosis. our aim is to generate a caspase cleavage site predictor specific for each member of the caspase family in order to make subtype-specific predictions of new caspase substrates. we have used a set of experimentally verified proteins to generate sequence logos and train a neural network in order to predict caspase cleavage sites. machine learning techniques, such as artificial neural networks, are often well suited to integrate the subtleties of sequence variations. this approach also enables integration of structural information in the pattern recognition procedure which could possibly increase the predictive performance of the neural network. the identification of new caspase substrates can lead to further elucidation of several cellular processes involving caspases, including apoptosis, cell cycle regulation, cellular differentiation, and pro-inflammatory responses. in addition, the generation of caspase inhibitors could be greatly aided by a caspase cleavage site predictor. regulation of protein synthesis and autophagic-lyososomal protein degradation in isolated pancreatic acini a. l. kovacs and e. papp cell physiology laboratory, department of general zoology, eotvos lorand university, budapest, hungary. e-mail: alkova@cerberus.elte.hu a series of biologically active compounds (wortmannin, ly294002, 3-methyladenine, rapamycin, okadaic acid, theophyllin, insulin, glucagon, cholecystokinin) influencing protein synthesis and autophagic-lysosomal protein degradation by interfering with important signalization pathways were investigated. our results show that in exocrine pancreas cells phosphatidyl inositolkinases (pi3k-s) are activators, while the target of rapamycin protein (tor) is an inhibitor of autophagy. camp is an inhibitor of lysosomal protein degradation that acts through members of the pi3k family. okadaic acid inhibits lyososomal protein degradation without inhibiting the formation of autophagic vacuoles. the inhibition of pi3k-s and tor diminishes protein synthesis, inhibitors of these kinases reduce the synthesis stimulatory effect of insulin. cholecystokinin showed a biphasic stimulatory effect while glucagon was ineffective on protein synthesis. on the base of these results a possible signalization pathway is suggested for autophagic segregation and lysosomal protein degradation in pancreatic acinar cells. purification and characterization of a bifunctional protease from vibrio vulnificus in this study, we purified and characterized an extracellular protease showing dual functions as prothrombin activator and fibrinolytic enzyme from vibrio vulnificus atcc 29307. the purified enzyme had broad substrate specificity towards various bloodclotting associated proteins such as prothrombin, plasminogen, fibrinogen and factor xa. the cleavage of these proteins could be stimulated by addition of 1 mm mn 2+ . the protease could acti-vate prothrombin to active thrombin. however, the thrombin activity generated from prothrombin activation by the protease seemed to be transient, with further cleavage resulting in a loss of activity. interestingly, the enzyme could enhance the activity of thrombin during the initial rate of fibrin formation when purified fibrinogen was used as substrate. it could also actively digest fibrin polymer as well as cross-linked fibrin. these results suggest that the secreted protease functions as a prothrombin activator and a fibrinolytic enzyme to interfere with blood clotting as part of the mechanism associated with its pathogenicity in human. tumor invasion and metastasis are the major causes of treatment failure and death in cancer patients. one requisite for neoplastic cell invasion during tumorigenic processes is the remodeling events that occur within the stroma or extracellular matrix (ecm). cysteine cathepsins, most likely along with matrix metalloproteases and serine proteases, degradate the ecm, thereby facilitating growth and invasion into surrounding tissue and vasculature. clinically, the activity levels and localization of cysteine cathepsins and their endogenous inhibitors have been shown to be of diagnostic and prognostic value. the aim of our study was therefore both the determination of prognostic and diagnostic impact of cathepsins b, l and h from human tissues extracts (normal and tumor tissue) and extracellular fluids (such as plasma and urine) and a1-proteinase inhibitor (pi) in pathogenesis of different types of human brain tumors, and extraction and purification of cysteine cathepsin endogenous inhibitors from normal and tumor brains and studying of their physicochemical properties. it was found that the increasing of cysteine cathepsins b, l, h activity levels in brain tumors tissues depend on histostructure, histogenesis and tumor malignancy grade. increasing of cathepsins l and h activity levels was found in plasma and urine in depending on histogenesis. at the same time decrease in pi activity level was registered. besides, kinetic characteristics of extracted normal brain endogenous inhibitors of cysteine cathepsins were determined. in extracted tumor brain endogenous inhibitors, there were differences in physicochemical properties in comparison with normal. the data obtained contribute to understanding the participation of cysteine cathepsins and their inhibitors in mechanisms of cancer genesis and both become useful for solving the problem of improving of tumor therapy and provide the possibility of using their activity as diagnostic and prognostic markers. protein hydrolysates of sea origin as components for microbiological culture media dry hydrolysate was prepared from protein-containing waste of icelandic scallop chlamys islandicus processing (spw) by means of a proteinase complex from king red crabs hepatopancreas. the enzyme consist of the proteolytic enzyme complex from crab hepatopancreas, in which serine proteases dominate (collagenase, elastase and trypsin-and chymotripsin-like proteinases). as proteinases from king red crab hepatopancreas have high enzymesubstrate affinity to icelandic scallop proteins, a high degree of proteolysis can be achieved. the composition and properties of the material were investigated on enzymatic protein hydrolysate from spw obtained under the most technologically suitable conditions: 50-55 o c, ph 7.5, 6 h, the ratio between the protein material and the enzyme preparation being 1000:6. for comparison we examined the composition of commercial pancreatic hydrolysate from poor-quality fish species, mainly boreogadus and micromestistus. it was found that hydrolysate from spw significantly overpowered the commercial analog in the mass percentage of the target product (free amino acid and oligopeptides). the resulting product contains not <80% free amino acids and oligopeptides. predominant are aspartic acid, leucine, isoleucine, arginine and lysine, which account for >5% of the free amino acids. the potential usage of the protein hydrolysate as a nutrient for microorganism cultivation is estimated. microbiological studies have demonstrated that the hydrolysate from spw can be used as a protein component in nutrient media. the tested microbial strains satisfactorily grew on the media. the z variant alpha-1 proteinase inhibitor (a1piz) misfolds in the endoplasmic reticulum (er) and is a substrate for er-associated protein degradation (erad). we report here that a1piz degradation is also dependent on vps30/atg6, a gene that encodes a component of two pi3-kinase complexes that regulate membrane traffic; complex i is required for autophagy, complex ii is required for the cpy-to-vacuole pathway. to elucidate why vps30p participates in a1piz degradation, we tested the hypothesis that erad was saturated at elevated levels of a1piz expression and that excess a1piz was targeted to one of these alternative quality control pathways. overexpression of a1piz led to vacuole-dependent degradation and both complexes were required for delivery of the excess a1piz to the vacuole. when the cpy-to-vacuole pathway was compromised a1piz was secreted and the distribution of soluble vs. aggregated forms of a1piz was comparable with that of wild type yeast. however, disruption of autophagy led to an increase in levels of aggregated a1piz; suggesting that when erad is saturated the excess a1piz is selectively targeted to the vacuole via the cpy-to-vacuole sorting pathway, while excess a1piz that forms aggregates in the er is targeted to the vacuole via autophagy. together, these results reveal multiple pathways for recognition and removal of aberrant proteins and provide direct evidence that aggregated a1piz is removed by autophagy. our findings may have application in the understanding of, and treatment for, individuals with liver disease caused by the accumulation of er aggregates of a1piz. acknowledgements: the study was supported by national science foundation grants mcb-011079 and mcb-0110331. yeast and lactobacillus association generates peptides from acid goat whey proteins fermentation s. didelot, s. bordenave-juchereau, e. rosenfeld, l. murillo, j. m. piot and f. sannier laboratory of biotechnology and bioorganic chemistry, university of la rochelle, la rochelle, france. e-mail: lmurillo@univ-lr.fr our goal was to produce peptides from fermentation of unsupplemented acid goat whey by dairy micro-organisms. we used a lactobacillus, lactobacillus paracasei, and a yeast, candida parapsilosis, both previously isolated from a cheese microflora. when co-cultivated aerobically, both micro-organisms grew on unsupplemented goat whey and led to a medium acidification from 6 to ph 3.5. reversed phase (rp)-hplc analysis revealed a total alpha-lactalbumin hydrolysis after 96 h of fermentation, a modification of the beta-lactoglobulin elution peak, and 2.5-fold increase in peptide level compared with the non-fermented whey. in the absence of c. parapsilosis, l. paracasei grew poorly on whey and only a weak medium acidification from 6 to 4.5 was observed after 192 h of fermentation. rp-hplc analysis revealed a weak modification of beta-lactoglobulin elution peak, a truncated form of alpha-lactalbumin and no peptide generation. c. parapsilosis was able to grow on unsupplemented goat whey without modifying ph of the medium, but only 25% of proteins were hydrolysed (alpha-lactalbumin) or denaturated (beta-lactoglobulin) and, again, no peptides were detected. these results suggest that (i) c. parapsilosis is required for l. paracasei growth and (ii) the co-culture of both micro-organisms is needed to generate peptides from alpha-lactabumin hydrolysis. during co-culture on whey, the use of penicillin g and cycloheximide as bacterial and yeast growth inhibitors respectively, revealed that l. paracasei growth was required for medium acidification to ph 3.5 and alpha-lactalbumin hydrolysis. however, we demonstrated that the protease(s) responsible of alpha-lactalbumin hydrolysis was (were) synthesized by c. parapsilosis during the first stage of fermentation and that medium acidification (obtained either by l. paracasei growth or chemically) was required for yeast protease(s) activity. dengue virus causes widespread human diseases such as dengue fever, dengue hemorrhagic fever and dengue shock syndrome. the viral genome is a positive rna strand that encodes for a single polypeptide precursor. processing of the polyprotein precursor into mature proteins is carried out by the host signal peptidase and by ns3 serine protease. the three dimensional structure of ns3 protease domain ns3pro has been elucidated [1] . recently a new construct of the recombinant form of the ns3pro, was engineered [2] . we have expressed in e. coli the his-tag-cf40.gly.ns3pro protein a new construct of the recombinant form of the ns3pro linked to a 40 -residue co-factor, corresponding to a part of ns2b, via a non-cleavable, flexible non-apeptide (gly 4 sergly 4 ), and have currently optimized the purification procedure. chemically optimized substrates, peptides and depsipeptides, were designed and tested to afford an efficient in vitro activity assay, using hplc and fret spectroscopy. the data suggest that the amino-terminal region of the 40-amino acid co-factor domain is involved in additional charged interactions with ns3 that are essential for activity as previously described. this form showed catalytic activity and spectroscopic studies were performed to identify the folding of the protein. moreover, experiments of limited proteolysis have been performed to identify the essential enzymatic domain of the protein and to stabilize the role of the cofactor in the activity and in folding stabilization of the enzyme. after 2 h of the limited proteolysis with endoproteinase asp-n the product was analyzed by sds-page and activity assay, showing a high reduction of the molecular mass and only a loss of the activity of the 20%. cd and 15 n-1 h-hsqc spectra of this protein fragment were performed and other functional and structural characterizations are in progress in our laboratory. it is intended to obtain the structure in solution of the essential active domain of the uniformly 13 c, 15 n-labeled cf40.gly.n-s3pro by high-field 3d nmr spectroscopy. the solution structure of the enzyme will be used to answer yet unresolved questions about the mechanism of action, the role of its cofactor ns2b, and the observed substrate specificity. introduction: fish consumption is associated to nutritional benefits due to the presence of proteins of high biological value, minerals, vitamins and polyunsaturated fatty acids. most studies concerning the benefits of fish consumption on cancer prevention have focused on fish fatty acids but little is known about the potential bioactivity of fish peptides. the present study was then designed to assess the antiproliferative activity of various fish protein hydrolysates, in order to further purify and characterize anticancer peptides. methods: twenty-one fish hydrolysates (from seven species) produced within the framework of the european valbiomar programm. fish hydrolysates composition (protein, fat and salt content) was determined by standard methods (kjehldhal, soxhlet extraction and volhard respectively). cytotoxic and antiproliferative activity were assayed in vitro on mcf-7/6 and mda-mb-231 human breast adenocarcinoma cell lines, following a cell viability colorimetric assay (promega, france). antiproliferative activity of fish hydrolysates was compared with that of reference anticancer molecules with various cellular targets, namely actino-mycine d, cytosine-beta-d-arabinofuranoside, cyclophosphamide, etoposide, kenpaullone and roscovitine. results: composition analysis revealed that most hydrolysates contained more than 70% protein. three blue whiting hydrolysates containing 96% protein, 0.5% lipid and 0.2% salt induced a strong breast cancer cells growth inhibition when tested at 1 g/l for 72 h in cell culture medium. blue whiting hydrolysates 3, 4 and 5, respectively, induced a growth inhibition of 24.5, 22.3 and 26.3% on mcf-7/6, and 13.5, 29.8 and 29.2 % on mda-mb-231. these in vitro antiproliferative activities are in the range of that observed when the two breast cancer cell lines are treated for 72h with kenpaullone, roscovitine or cytosine-beta-d-arabinofuranoside 10 )6 m. further studies are engaged to fractionate and characterize the antiproliferative peptides contained in blue whiting hydrolysates. during recent years, it has been established that intracellular proteolysis in eukaryotic cells is largely accomplished by a highly selective non-lysosomal pathway that requires atp and a large (2.5 mda) multisubunit complex known as the 26s proteasome. the proteasome-mediated pathway plays vital regulatory functions. it degrades many important proteins involved in cell cycle control, in signaling pathway, and in general metabolism, including transcription factors and key metabolic enzymes. another function of the proteasomal system is the removal of abnormal, misfolded and oxidized proteins generated under normal and, in particular, stress conditions. to date, proteasomes from other than animal or plant cells were studied only in yeast. recently, in our laboratory, the proteasome-mediated pathway was shown to be involved in the regulation of ligninolytic activities in the white rot fungi trametes versicolor and phlebia radiata upon nutrient starvation (staszczak, enzyme microb technol 2002; 30: 537-540). it was the first report on proteasomes in fungi representing basidiomycota. white rot fungi are able to degrade lignin by the action of secreted enzymes, the best characterized of which are laccases, lignin peroxidases, and manganese peroxidases. the subject of lignin biodegradation has commanded attention for a considerable period of time mainly because of its ecological significance and wide industrial applications of bioligninolytic systems. heavy metal ions are important environmental pollutants which affect biodegradation processes performed by white rot fungi. in the present study, we investigated whether the proteasomal degradation pathway might be involved in the regulation of laccase production by t. versicolor in response to cadmium exposure. studies of cacybp/sip function using small interfering rna cacybp/sip was discovered as a protein that bound calcyclin (s100a6) in a calcium-dependent manner (filipek and wojda 1996; filipek and kuznicki, 1998) and its distribution and some biochemical properties have been studied. for instance, it has been shown that cacybp/sip binds calcyclin via its c-terminal fragment (nowotny et al. 2000) and that, beside calcyclin, it interacts with other calcium binding proteins of the s100 family (filipek et al. 2002) . originally, we identified cacybp/sip in ehrlich ascites tumour (eat) cells but it is also present in other mammalian tissues and cells. in particular, high expression of cacybp/sip was found in neuronal cells of mouse and rat brain (jastrzebska et al. 2000) . at present the distribution and structural properties of cacybp/sip are quite well described but its function remains obscure. there is only one paper published concerning the possible involvement of cacybp/sip in b-catenin ubiquitination and degradation (matsuzawa and reed 2001). to elucidate the biological role of cacybp/sip we have designed and synthesized sirna (small interfering rna) against this protein. this sirna was then used to transfect neuroblastoma nb-2a and embryonic kidney hek293 cells, expressing high and low amount of endogenous cacybp/sip respectively. the level of cacybp/sip was monitored in cell extracts by western blot technique. we found that sirna against cacybp/sip, which we designed, inhibited the expression of this protein, as its level in transfected cells was lower in comparison with control cells. at present, we checked the effect of diminished expression of cacybp/sip on b-catenin degradation and other cellular processes. acknowledgements: this work was supported by grants: kbn heavy metals are powerful poisons for living cells. it has been shown that exposure to arsenicals, either in vitro or in vivo, in a variety of model systems, causes the induction of a number of the major stress protein families, such as the heat shock proteins (hsp) (toxicol appl pharmacol 2001; 177: 132). the reasons for heavy metal toxicity in vivo are not fully understood, but they are known to contribute to the accumulation of aberrant proteins (bba,1995,1268, 59). in animal cells, arsenite has been reported to cause sulfhydryl depletion, to generate reactive oxygen species and increase the level of high molecular mass ubiquitin-protein conjugates (toxicol appl pharmacol 2003; 186: 101). in cells submitted to stress conditions, several components of the ubiquitin/proteasome pathway are activated. in this major, eukaryotic proteolytic pathway, multiple ubiquitin molecules are enzymatically ligated to proteins destined for catabolism by an enzyme system composed of three types of enzymes, commonly referred to as e1, e2, and e3. the large ubiquitinprotein conjugates thus formed are subsequently degraded by a very large protease complex, the 26s proteasome, in an atpdependent process. the changes in free ubiquitin (ub) and ubiquitin-protein conjugates (ub-p) levels were followed by immunoblotting during the incubation of the higher plant lemna minor l.(duckweed) in the presence of arsenite (as), at concentrations known to confer thermotolerance to the plants. the observed increase in the amount of large molecular mass ubiquitin-protein conjugates is indicative of a role for the ubiquitin/ proteasome pathway in the response of lemna to as stress. this outcome is primarily attributed to an increased availability in protein substrates during as treatment for three main reasons: an increase in protein carbonyl (a major marker for protein oxidation) content detected by immunoblotting; moderate increments (as determined by semi-quantitative rt-pcr) in the mrna levels of the codifying sequences for the ubiquitin pathway components: ubiquitin, e1, e2 and the b subunit and the atpase subunit of the 26 s proteasome; an identical pattern of variation for the large ubiquitin-protein conjugates is observed in the simultaneous presence of as and cycloheximide, indicating that the observed increase in ubiquitin conjugates does not depend on de novo protein synthesis. ageing and autophagy y. stroikin and a. terman experimental pathology, linko¨ping university, linko¨ping, sweden. e-mail: yurst@inr.liu.se life of aerobic cells is associated with continuous oxidative damage resulting in the formation of altered, non-functional macromolecules and organelles. intracellular accumulation of oxidized proteins defective organelles and lipofuscin inclusions are typical manifestations of ageing that preferentially affects long-lived post-mitotic or growth-arrested cultured cells. autophagy, an important biological mechanism for renewal of damaged intracellular structures, has been found decreased in ageing. to learn more about the role of autophagy in ageing, we studied the effect of the inhibitor of autophagic sequestration 3-methyladenine (3-ma) on human diploid fibroblasts and astrocytes. inhibition of autophagy in growth-arrested (confluent) fibroblasts for 2 weeks resulted in the accumulation of altered lysosomes displaying lipofuscin-like autofluorescence, especially when 3-ma exposure was combined with hyperoxia. the findings suggest that autophagy is indispensable for normal turnover of lysosomes, and lysosomal components may be direct sources of lipofuscin. the accumulation of oxidatively damaged intracellular structures (so-called biological ''garbage'') was associated with decreased cell viability. two-week-inhibition of autophagy with 3-ma resulted in a significantly increased proportion of dying cells when compared with both untreated confluent cultures and dividing (subconfluent) cells exposed to 3-ma. similar results were obtained when autophagic degradation was suppressed by the protease inhibitor leupeptin. the results support the idea that biological ''garbage'' accumulation is essential for ageing and age-related death of post-mitotic cells, which can be prevented by cell division. recently two family members of the tumour suppressor gene p53 have been described, p63 and p73, which seem to be necessary for specific p53-induced stress-response pathways. furthermore, p63 and p73 appears to be crucial to determine the cellular sensitivity to anticancer drugs, particularly in tumours lacking functional p53. here, we show that p63 and p73 isoforms are also regulated by proteasomal degradation. we have identified several e3-ubiquitin ligases responsible for the regulation of the stability of p63 and p73. we found that the regulation of p63 and p73 is isoform-specific. furthermore, we demonstrate that ubiquitination of p73 influences the cellular localization of p73 and of the respective e3-ubiquitin ligases. finally, we show that the expression of the various e3-ubiquitin ligases can be differentially induced by p73-isoforms. in addition, the e3-ubiquitin ligases can influence the apoptotic function of p73. our findings demonstrate that p63 and p73 are sent to degradation or stabilized by e3-ubiquitin ligases in an isoform-specific manner and we suggest a negative feedbackloop between p63, p73 and their regulators, as they also influence the function of p63 and p73. increased level of metalloproteases was shown to accompany tumor angiogenesis and active invasion in adjacent tissue [1] . development of different types of tumors is often accompanied by increased protease activity in blood [2, 3] . in the present study we compared protease activity of plasma and eluate from surface of blood cells in healthy donors and patients with breast tumor. we have demonstrated recently that in blood of healthy donors almost all circulating nucleic acids (cirna) are bound at the surface of blood cells. in patients with fibroadenoma cirna were found at cell surface whereas in breast cancer, no cell-surfacebound cirna were detected in blood [4] . conjugates of hydrophobic and hydrophilic peptides of cd34 receptor with biotin were incubated with avidin-coated 96-well eia microplates. avidinpeptide complex was incubated with samples under investigation and serial dilutions of proteinase k solution, which was used for calibration of protease activity. undegraded peptides were visualized by incubation with goat anti-peptide antibodies followed by conjugate of anti-goat immunoglobulins with peroxidase. blood plasma and eluate from surface of blood cells of cancer patients demonstrated increased level of anti-hydrophilic protease activity compared with healthy donors. increase of protease activity against hydrophilic peptide in blood correlate with decrease of cell-surface-bound cirna, indicating that blood proteases can affect concentration and distribution of circulated na. identification of cleavage site and natural substrate specificity of prta, a serralysin-type metalloprotease from the entomopathogenic microorganism photorhabdus prta, a secreted basic metalloprotease of photorhabdus, belongs to the m12b (serralysin) family of proteases. the biological function of these enzymes is not known, but in some cases they are supposed to have a role in virulence. serralysins are generally assumed to have broad substrate side-chain specificity. attempts toward the generation of a sensitive and specific substrate of these enzymes had limited success, and no such substrate is available for prt-a. through mass spectrometric analysis of prta cleavage products of oxidized insulin a and b chain, we found that prta has a welldefined cleavage site preference. based on this, we developed a sensitive and highly specific oligopeptide substrate through optimization of the amino acid composition and length. the kinetic parameters of prta isolated from photorhabdus luminescens ssp. laumondii strain brecon were measured on the best substrate, dabcyl-glu-val-tyr-ala-val-glu-ser-edans, giving a km of 8.8 · 10 )5 , a kcat of 2.1 · 10 )2 /s and a kcat/km of 2.4 · 10 6 . its poor hydrolysis by various proteases proved its specificity, while it was very sensitivity in measuring prta activity in hemolymph samples from photorhabdus infected galleria mellonella larvae. the substrate preference of prt-a was determined by in vivo digestion of hemolymph proteins from manduca sexta. six minor protein components were selectively cleaved, which were provisionally disthe epithelial sodium channel (enac) is an integral component of the pathway for na + absorption in epithelial cells. enac activity is mainly regulated by mechanisms that control its expression at the cell surface, such as ubiquitination. the ubiquitin ligases nedd4 and nedd4-2 have both been shown to bind to enac and decrease its activity. conversely, the serum-and glucocorticoid regulated kinase (sgk), a downstream mediator of aldosterone, is able to increase enac activity. this effect is at least partly mediated by direct interaction between sgk and nedd4-2. sgk binds both nedd4 and nedd4-2 but it is only able to phosphorylate nedd4-2. phosphorylation of nedd4-2 reduces its ability to bind to enac, and hence increases enac activity. the impact of the interaction between nedd4 and sgk remains unclear. nedd4-like proteins interact with enac via their ww-domains. these domains bind py-motifs (ppxy) present in enac subunits. nedd4 and nedd4-2 both have four highly homologous ww-domains. previous studies have shown that interaction between nedd4 and enac is mainly mediated by ww-domain 3. sgk also has a py-motif, therefore we tested whether the ww domains of nedd4 and nedd4-2 mediate binding to sgk. we show that single or tandem ww domains of nedd4 and nedd4-2 mediate binding to sgk and that, despite their high homology, different ww domains of nedd4 and nedd4-2 are involved. our data also suggest that ww domains 2 and 3 of nedd4-2 mediate the interaction with sgk in a concerted manner, and that in vitro the phosphorylation of sgk at serine residue 422 increases its affinity for the ww domains of nedd4-2. the stimulatory effect of sgk on enac activity is partly mediated via nedd4-2 and will decrease if competition between nedd4 and nedd4-2 for binding to sgk occurs. we show that nedd4 and nedd4-2 are located in the same subcellular compartment and that they compete for binding to sgk in vitro. the concerted or successive action of proteolytic enzymes has been described in a number of important biological processes in which proteins are degraded or matured, such as digestion, turnover (lysosomal, proteosomal...), blood coagulation, developmental remodeling or apoptosis, among others. the complementary action of proteases belonging to different families to achieve a more efficient o a better modulated hydrolytic mechanism is well documented. specific molecular associations or shared scaffolds between the involved proteases and/or protein inhibitors and defined three-dimensional structures have also been reported. however, only in a few cases such structures involved metallo.carboxy-peptidases or their inhibitors [1] . we shall review this subject and describe, in such a context, a new model found in a marine invertebrate organism in which such a fact takes place. in particular, the characteristics of a novel bifunctional molecule displaying the functionalities and structures of serine-and metallo.carboxy-peptidases will be presented. its structure is fully different than the ones previously reported by us and collaborative groups for metallocarboxypeptidase inhibitors [2] [3] [4] . regulating the activity of herpes virus proteases c. s. craik departments of pharmaceutical chemistry, pharmacology, and biochemistry and biophysics, ucsf, san francisco, ca, usa. e-mail: craik@cgl.ucsf.edu herpesviral proteases exist in a monomer-dimer equilibrium in solution. dimerization is required for activity and a comformational change communicates the oligomerization state of the enzyme to the active site of each intact monomer. each monomer has an active site, which is spatially separate from the dimer interface. kaposi's sarcoma-associated herpesvirus (kshv), encodes a protease (kshv pr), which is necessary for the viral lytic cycle. like those of other herpesvirues proteases, the dimer interface of kshv pr is composed primarily of a helix near the c terminus, of the protein. the helix of one monomer interacts with residues in the symmetrically related helix of the other monomer across the dimer interface as well as with neighboring helices. small molecule inhibitors, site directed mutagenesis and 2d nmr spectroscopy were used to compare the monomeric and dimeric forms of kshv pr and to investigate the relationship of the active site and the dimer interface of the enzyme. active site inhibition was shown to strongly regulate the binding affinity of the monomer-dimer equilibrium of the protease, shifting the equilibrium completely to the dimeric form of the enzyme. a previously undetermined conformational change provided insight in to the regulation of protease activity by dimerization as well as an explanation for the weak dimerization of a family of enzymes with a disparately large dimer interface compared to their measured binding affinities. using this information as a guide, protein grafting of the interfacial helix onto a small stable protein, avian pancreatic polypeptide, generated a small macromolecular inhibitor that successfully disrupted the dimer interface and inhibited enzymatic activity. these results provide direct evidence that peptide bond hydrolysis is integrally linked to the quaternary structure of the enzyme, validate the protease as a therapeutic target and suggest the dimer interface may be an alternative site for antiviral design. abteilung strukturforschung, max-planck-institut fu¨r biochemie, martinsried, germany. e-mail: huber@biochem.mpg.de proteolytic enzymes catalyze a very simple chemical reaction, the hydrolytic cleavage of a peptide bond. nevertheless they constitute a most diverse and numerous lineages of proteins. the reason lies in their role as components of many regulatory physiological cascades in all organisms. to serve this purpose and to avoid unwanted destructive action proteolytic activity must be strictly controlled. control is based on different mechanisms which i will discuss and illustrate with examples of systems and structures determined in my laboratory. the family of serine protease inhibitors known as the serpins is represented in all branches of life and predominate in the higher organisms, including man. they have evolved an extraordinary mechanism to inhibit proteases which distinguishes them from the 20 other families of serine protease inhibitors, and renders them uniquely qualified to control of the proteolytic pathways essential to life. the mechanism is best described as a spring-loaded mousetrap, where nibbling of the peptide loop bait springs the trap and crushes the unsuspecting protease. as with a mousetrap, the active state of a serpin is metastable, and the energy released upon conversion to its more stable form is used to trap the protease. the complexity of the serpin mechanism provides many advantages over the simpler lock-and-key type mechanism, utilized by all other serine protease families. serpins provide stoichiometric, irreversible inhibition, and the dependence on serpin and protease conformational change is exploited for signaling and clearance. the potential for regulation is also an inherent part of such a complex mechanism, as illustrated by the heparin activation of serpins antithrombin and heparin cofactor ii. however, with complexity of mechanism also comes susceptibility to disease causing mutations: both through loss-of-function, as with thrombosis caused by antithrombin deficiency; and gain-of-function, as with dementia caused by neuroserpin polymerization. many crystallographic structures of serpins have been solved over the past 20 years, and we now have a frame-by-frame cinematic view of the intricate conformational rearrangements involved in protease inhibition, modulation of specificity, and molecular pathology of the remarkable shape-shifting serpins. structural lessons of serine proteases: function and mechanism of the serine protease-like hgf as a growth factor in met signaling hepatocyte growth factor (hgf), a plasminogen-related growth factor, is the ligand for met, a receptor tyrosine kinase implicated in development, tissue regeneration and invasive tumor growth. hgf acquires signaling activity only upon proteolytic cleavage of single-chain hgf into its a/b-heterodimer, similar to zymogen activation of structurally related serine proteases. although both chains are required for activation, only the achain binds met with high affinity. recently, we reported that the protease-like hgf b-chain binds to met with low affinity this suggests that additional allosterically linked regions may be involved in the signaling process. furthermore, antibodies directed toward the b-chain or the hgf a-chain result in inhibition of met phosphorylation in a549 cells. these antibodies also inhibit proliferation in bxpc3 cells and baf3 cells. implications for dimerization mechanisms of hgf-dependent met receptor activation and signaling are presented. in addition, mutagenesis of the hgf b active site region has been investigated with respect to imparting enzymatic activity. thus while hgf has the function of a growth factor, the structural and receptor binding aspects of hgf are more akin to those of serine proteases. trypsinogen 4 with a 28 amino acid leader peptide on its n-terminus is the predominant form of the enzyme in human brain gene prss3 on chromosome 9 of the human genome encodes, due to alternative splicing, both mesotrypsinogen and trypsinogen 4. mesotrypsinogen has long been known as a minor component of trypsinogens expressed in human pancreas, while the mrna for trypsinogen 4 has recently been identified in brain and other human tissues. analysis of the gene encoding trypsinogen 4 predicted two isoforms of the zymogen: isoform a may have a 72 amino acid, while isoform b a 28 amino acid n-terminal leader sequence. the translation initiation site for isoform a is an atg codon, while the initiation site predicted for isoform b is a ctg codon. we measured the amount of trypsinogen 4 mrna and the quantity of the protein as well in 17 selected areas of the human brain. trypsinogen 4 could be localized in glial and neuronal cells using immunohistochemical methods. we purified human trypsinogen 4 by affinity chromatography. our results show that splice isoform b is the predominant if not the exclusive form of the zymogen in human brain. the n-terminal residue of the isolated protein was identified by amino acid sequencing as a leucine. at the same time the longest mrna we were able to isolate was barely longer than the one corresponding to splice isoform b. although the most trivial explanation of our results is that isoform a is proteolytically processed to result in isoform b, it cannot be excluded that leucine rather then methionine is used as translation initiator amino acid. search for endogenous substrates for prolyl oligopeptidase in porcine brain prolyl oligopeptidase (po) is a serine protease present in most tissues, which preferentially cleaves the peptide bond at the carboxyl site of proline residues. the function of po is unknown, but it has been associated with several disorders of the central nervous system, such as depression and alzheimer disease. the purpose was to look for endogenous substrates for the recombinant porcine po in porcine brain. we adapted a method to extract the proteins from the brain with special attention to the smaller polypeptides since po is not known to cleave peptides larger than 30 amino acids. subsequently we looked for a method to separate the protein mixture in less complex fractions. 2d-gelelectrophoresis, commonly used in proteomics, is only suitable for proteins with a molecular weight between 10 and 200 kda and an iso-electric point between 4 and 10. two-dimensional chromatography offers a suitable alternative for small peptides. we chose ion exchange chromatography as a first and reversed phase high pressure liquid chromatography as a second step. the resulting fractions were divided into two parts. one part was incubated with the purified po, the other served as a control. by looking for shifts in the mass spectrum between the control sample and the incubated sample, we identified peptides cleaved by po. different methods, such as esi-qtof-ms and maldi-toftof-ms, were used to sequence cleaved peptides by msms. these experiments allowed us to deduce the sequence requirements for po cleavage. serine protease subtilisin immobilized on novel mesoporous materials serine proteinases are widely used in protein mapping and peptide or ester bond formation. fixation of enzyme on solid support has many advantages, such as high stability, possibility of recovering and low product contamination by enzyme. subtilisin carlsberg, a protease from bacillus licheniformis, was immobilized on mesoporous silica (sba-15) and several organosilica supports via physical adsorption. the bifunctional mesoporous organosilicas containing ch2-ch2 or ch=ch bridges in combination with organic tethers bearing amino or hydroxyl functionalities were synthesized using supramolecular templating in the presence of non-ionic triblock copolymers and exhibited high surface area and large pore diameters in the range of 50-70 å suitable for the incorporation of subtilisin. the kinetics of immobilization was examined for six different carriers. it was shown that enzyme retained hydrolytic activity after the immobilization. the dependence of subtilisin loading on the starting concentration of the enzyme during adsorption shows the maximum loading (455 mg protein/g support) at [e] = 20 mg/ml. the ph dependences of loading and activity of immobilized biocatalysts were bell-shaped. for the organosilica support containing amino and hydroxyl groups the ph-dependence was shifted to the alkaline ph by 2 in comparison with the support containing ch2-ch2 bridges. the adsorbed subtilisin desorbs easily in aqueous media, while no leaching of the enzyme was observed in acetonitrile and dmf/acetonitrile mixture (6/4). the immobilized biocatalyst shows high hydrolytic activity after incubation in non-aqueous acetonitrile for 1 week and after 48 h incubation in 60% dmf/acetonitrile mixture. these data indicate a possible application of the obtained biocatalysts in low water media. purification, structural and biological characterization of protease inhibitors from acacia plumose seeds protease inhibitors have been used in many current medicines. therefore, there is a considerable interest inside the pharmaceutical industry in discovering new composites and mechanisms of protease inhibition, since these investments have led, for example, to new anti-hiv therapeutical tests, coagulation diseases treatment and tests with anti-carcinogenic drugs. serine protease inhibitors are found in all plant tissues, mostly in the seeds of the leguminosae subfamilies: mimosoideae, caesalpinoideae and papilionoideae. acacia genus is one of most important member of mimosoideae, and the presence of protease inhibitors in this genus was described in only three species and none of them were structurally characterized. in this sense, we are studying three new protease inhibitors from a. plumose seeds. from saline extract of triturated mature seeds the inhibitors were purified and presented anti-coagulant activity, serine protease inhibitory activity and action on growth of fitopathogenic microorganisms, in vitro. the purification steps included size exclusion chromatography on the superdex-75 column, equilibrated and eluted with pbs, a ionic exchange chromatography on mono-s (hr 5/5) column, equilibrated with the buffer sodium acetate 50 mm (ph 5.0), and eluted with the same buffer in a gradient of 0-0.5 m of nacl. three fractions (eluted around 0.18, 0.22 and 0.33 m of nacl) that presented anticoagulant activity and serine protease inhibition were separated and denoted apia, apib and apic. their apparent mws were around 20 kda, by sds-page in the absence of reducing agents. in the presence of reducing agents they shown two bands: between 14-22, and 8-6 kda. the n-terminal analyze of higher mw chains were tyafl (apia); kellvdne (apib) and telhdd (apic). the circular dichroism spectra of these inhibitors were very similar, presenting a maximum around 230 nm and a minimum in 202 nm, compatible with presence of unordered and beta elements of secondary structure. their nterminal, cd spectra and two-polypeptide chains linked by covalent bound, are compatible with kunitz type inhibitors. probably these inhibitors are three different isoforms that present different inhibition specificity degree on the serine proteases family. the ki to different serinoproteases (trypsin, plasmatic kalikrein, elastase, quimotrypsin) and specificity to the phytopatogenic fungus are being investigated. although the proteases were initially described as enzymes involved in the non-specific degradation of dietary proteins, today it is known that they can also act as highly specific enzymes that perform selective cleavage of specific substrates. thus, alterations in the structure, regulation or function of this type of enzymes underlie serious human disorders including cancer. to date, more than 550 protease and protease homologs are annotated in man, mouse, and rat genomes (www.uniovi.es/degradome). the increasing complexity of the proteolytic systems has led to the introduction of global concepts as the term degradome to define the complete set of proteases that are produced in a specific moment by a cell, tissue or organism. as part of our studies focused on the characterization of the mammalian degradomes, we have identified and cloned unusual mosaic proteases containing in tandem serine protease domains. the first, called polyserase-1 is synthesized as a transmembrane protein that undergoes post-translational events to generate three independent serine protease domains. the second polyprotease is the polyserase-2, a secreted protein that remains as integral part of the initial protein product. to date, it is difficult to understand the putative functional advantages derived from the complex polyproteases and, albeit extremely unusual, it is not an unprecedented situation. thus, the amphibians ovochymase and oviductin are polyserine proteases that contain three in tandem serine proteases. in humans, angiotensin-coverting enzyme and carboxypeptidase d are polymetalloproteases that exhibit some similarities to the polyserases. all these polyproteases constitute examples that illustrate an additional strategy for increasing the complexity of the degradomes. evolution of a genetic locus, expressing several protease inhibitors with homology to whey acidic protein (wap) a. clauss and å . lundwall department of laboratory medicine, lund university, malmo¨, sweden. e-mail: adam.clauss@klkemi.mas.lu.se we have previously described a locus on human chromosome 20 that gives rise to 14 proteins containing wap four disulphide core (wfdc) domains. among them are the elastase inhibitors elafin and secretory leukocyte proteinase inhibitor (slpi). both slpi and elafin are also known to be important components of the innate immune defence by displaying anti-microbial properties. in order to gain a deeper understanding of the biological role of the locus, we have now extended our investigations of its organization and evolution into non-human mammals. homologous loci were identified on mouse chromosome 2, rat chromosome 3 and dog chromosome 24. transcript sequences were generated by race technology or retrieved from the est databases. as in humans, the murine and canine loci are divided into two sub-loci separated by approximately 200 kb. the majority of genes are conserved in all species, but the comparison also showed gain and loss of genes, e.g. two human pseudogenes were identified due to the discovery of functional rodent genes, and in the rat several duplications has yielded four slpi genes. a most interesting finding was that there is no murine elafin gene. the different wfdc domains showed a highly variable species conservation. this was particularly striking in proteins containing multiple domains, where the aminoterminal wfdc generally displayed low conservation, whereas the opposite was true for the carboxyterminal wfdc. the difference could be due to the potential targets of the inhibitors, which might be either highly variable exogenous microbial proteases or conserved endogenous proteases. signaling mechanism of thrombin-induced human gingival fibroblast contraction thrombin is activated during gingival tissue injury and inflammation. thrombin and other bacterial proteases also affect the functions of adjacent periodontal cells via stimulation of proteaseactivated receptors (pars). we noted that thrombin and par-1 agonist peptide (20 lm) induced the gingival fibroblasts (gf)-populated collagen gel contraction within 2-h of exposure. however, par-3 and par-4 agonist peptide (<20 lm) show little effect on collagen gel contraction. u73122 (phospholipase c inhibitor) and 2-apb (ip3 antagonist) were effective in inhibition of gf contraction. thrombin-induced gf contraction was inhibited by 5 mm egta (an extracellular calcium chelator) and verapamil (a l-type calcium channel blocker). in addition, w7 (10 and 25 lm, a calcium/calmodulin inhibitor), ml-7 (50 lm, myosin light chain kinase, mlck inhibitor), and ha1077 (100 lm, rho kinase inhibitor) completely inhibited the thrombin-induced collagen gel contraction. thrombin also induced the phosphorylation of erk1/erk2 in gf. however, u0126 only partially inhibited the thrombin-induced gf contraction. similarly, wortmannin (100 lm), ly294002 (20 lm) (two pi3k inhibitors) and genistein, also showed partial inhibition. moreover, nac was not able to suppress the gf-contraction, as supported by slightly decrease in reactive oxygen species production in gf by thrombin. these results indicate that thrombin is crucial in the periodontal inflammation and wound healing by promoting gf contraction. this event is mainly mediated via par-1 activation, plc activation, extracellular calcium influx via l-type calcium channel, and the calcium/calmodulin-mlck and rho kinase activation pathway. survival of the anticarcinogenic bowman-birk inhibitor from soybean at the terminal ileum of cannulated pigs plant protease inhibitors (pi) of the bowman-birk class, a major pi class in legume seeds, have emerged as highly promising cancer chemopreventive agents, being capable of preventing or suppressing carcinogenic processes in a wide variety of in vitro and in vivo animal model systems. in order to exert their chemopreventive properties in vivo, plant pi have to resist and survive, at least to some extent, degradation by acidic conditions and digestive enzymes during gut passage. in this study, we have evaluated the survival rate of the bowman-birk inhibitor (bbi) in the terminal ileum of cannulated pigs fed defatted soybean. two different quantitative approaches have been carried out. firstly, a competitive indirect elisa assay using an antisera capable to detect bbi free and/or in complex with digestive proteases; secondly, we have carried out spectrophotometric measurements of trypsin and chymotrypsin inhibitory activities in ileal samples, where the presence of bbi metabolites and/or single active loops can be detected. according to the elisa method, ileal apparent digestibility of bbi was 58 %, which resulted in a recovery of 0.61 mg out of 1.5 mg/kg feed ingested. significantly higher ileal digestibility values (95 %) were found when trypsin and chymotrypsin inhibitor activities were evaluated. the results suggest that the immunoassay may be overestimating the presence of functional pi by detection of inactive bbi, but also that the presence of complexed bbi with digestive proteases, even if protein extraction was carried out under acidic conditions, could make bbi undetectable in activity assays. studies are in progress to overcome these drawbacks. the resistance of bbi to the acidic conditions and digestive enzymes of the upper gastrointestinal tract make these proteins very interesting candidates for evaluation as chemopreventive agents, in modulating cell viability and tumor progression within the gastrointestinal tract. a single amino acid change in a chymotrypsin prevents plant proteinase inhibitor binding plants have evolved economical strategies to combat insects, which on one hand involves the production of multi-domain pis that can target multiple enzymes with different specificities and on the other, pis that belong to structurally distinct families. solanaceous plants, produce both type i and type ii families of pis, which specifically target serine peptidases. this study showed that type i pis are better inhibitors of a particular class of chymotrypsins within the gut of helicoverpa species that is otherwise unaffected by the type ii class of inhibitors. homology models were used to identify a single amino acid substitution in the helicoverpa chymotrypsin that was likely to confer resistance to the type ii inhibitor. our hypothesis was further supported by recombinant expression and mutagenesis of this single amino acid in the type ii inhibitor-resistant chymotrypsin. we therefore propose that both type i and ii inhibitors are required to protect plants against lepidopteran insects. mobility of the sulphate protamin/ low molecular weight heparin complexes in an electrical field glycosaminoglycans low molecular weight heparin (lmwh) activated plasma serine proteases inhibitors. serine proteases play an important role in thrombogenesis, the process that leads to blood clotting and such as heart attack, stroke and other cardiovascular disorders. lmwh has been used to temporarily render the blood incoagulable during prophylaxis or treatment of thrombosis and sometimes result in serious bleedings and for the heparin anticoagulant activity neutralization used sulphate protamin. it was investigated relationship between new lmwh-sk derivatives (were generated through the controlled cleavage of porcine intestinal mucosa heparin with a mixture of chitinolytic complex from streptomyces kurssanovii) anticoagulant activities and lmwh-sk complexes with sulphate protamin mobility in an electrical field. with this purpose used biospecific electrophoresis in 1% agarose with protamin sulphate. precipitation zones (zones of the equivalent) in the ''rocket'' form were generated. scanning image was saved as jpg format. the ''rocket'' squares estimated with the help of bandscan program. results: lmwh-sk with molecular mass (mm) 14.0; 5.8; 5.4; 4.7; 4.0; 3.4 kd demonstrated antithrombin activities (aiia) 85-264 iu/mg, activities against factor xa (axa) has made 100-278 iu/mg, axa/aiia ratio -(0.8-2.2). correlation coefficients between mm and precipitation zone heights or squares consist 0.56-0.73 (p < 0.05), between axa activities and precipitation zone heights or squares consist 0.37-0.54 (p < 0.05). conclusion: lmwh-sk was obtained with the chitinolytic comlex hydrolisis help has ratio axa/aiia-2,2, it is necessary for antithrombotic preparations. with the mm decrease axa activity increase and precipitation zone heights or squares of the lmwh-sk complexes with sulphate protamin decrease. the role of extracellular proteases in supplying filamentous fungi with nutrient compounds is well understood and experi-mentally documented. however there is no definite answer on the question on the need and role of these proteases in pathogenesis. the study of differences in the spectra of extracellular enzymes of saprotrophic and pathogenic fungi performed on fusarium species revealed that activity of secreted serine proteinases of pathogenic f. culmorum strain was much higher (up to 20-fold) than that of saprotrophic strain. the use of f. culmorum strains differing in pathogenicity (strongly and weakly pathogenic) demonstrated that activity of secreted serine proteases of strongly pathogenic strain was significantly higher (1.5-8-fold) than that of weakly pathogenic strain. this tendency was preserved in calculations of activity towards protein content and dry weight of mycelium indicating on purposeful synthesis and secretion of extracellular proteases by strains with high pathogenicity. at that these differences were much higher when the substrate for trypsin-like proteinases bz-arg-pna was used than in the case of substrate for subtilisin-like proteinases glp-ala-ala-leu-pna. according to the data obtained it is proposed that the value activity of trypsin-like proteinases secreted by the fungi correlated with the degree of their pathogenicity and plays, apparently, an important role in pathogenesis. acknowledgment: this work was supported by grants from the russian foundation for basic research. conformational adaptation of a canonical protease inhibitor upon its binding to the target protease increases specificity atomic resolution crystal structure of sgti in complex with crayfish trypsin provided further data on the molecular basis of the inhibition mechanism of pacifastin type inhibitors. in complex with crayfish trypsin, sgti exhibits more or less continuous contacts in an extended region (through sites p 12 -p 5' ) of the molecule. the comparison of this complex with a simulated bovine trypsin-sgti one shows that more than half of the interaction energy surplus is originated from the extended region of binding. some of these contacts result from a conformational change of sgti that was induced by its binding to the enzyme which is strongly supported by the critical comparison of the crystal structure of crayfish trypsin-sgti complex with the free form of sgti. alignment of the nmr structure ensemble with the x-ray structure of complexed sgti and a careful comparison of the backbone j, w angles were carried out. additionally, noe-derived restraints and corresponding distances in the complex are also compared. local conformation of both p 12 -p 4 and p 4 '-p 5 ' regions of the inhibitor shows significant changes upon binding suggesting that either or both of these regions may act as molecular recognition sites. this comprehensive analysis of the local backbone properties of sgti in the free and in the complex form made possible to identify conformational similarities and differences responsible for its efficient binding to the enzyme, and provides a good basis for further studying the structural aspects of protease inhibitor specificity. as most of serine proteases enteropeptidase light chain contains four disulfide bonds and one nonpaired cysteine at 122 (chymotrypsinogen-derived residue numbering) position which forms disulfide bond linking the pro-and catalytic domains. a mutant of human enteropeptidase light chain cys122ser was constructed by site-directed mutagenesis. the recombinant wild type and mutant proteins were produced in escherichia coli bl21(de3) with expression vector pet-32a. the active proteins were obtained after solubilization and renaturation of the fusion protein thioredoxin/human enteropeptidase light chain from inclusion bodies. after autocatalytic cleavage of thioredoxin the active enzyme was purified on agarose linked soybean trypsin inhibitor. the yield of refolded active enzyme increased from 1.87 to 7.84% in case of cys122ser mutant. the wild type and c122s mutant showed similar kinetic parameters for cleavage of small synthetic substrate gly-asp-asp-asp-asp-lys-naphthylamide, small ester thiobenzyl benzyloxi-carbonil-l-lysinate (z-lys-sbzl) and fusion protein cleavage. both enzymes were inhibited by trypsin-like serine proteases inhibitors but not inhibitors of chymotrypsin-like, cysteineor metallo-proteinases. recombinant human enteropeptidase light chain and its mutant c122s were active between ph 6 and 9 with a broad optimum at about ph 7.54 and demonstrated quite high stability to different denaturating agents. both enzymes demonstrated secondary specificity to chromogenic substrate z-ala-phe-arg-na with km = 0.067 mm, kcat = 23 s-1. proteinaceous low molecular serine protease inhibitors from wood rotting fungi k. j. grzywnowicz and j. zuchowski biochemistry department, maria curie-sklodowska university, lublin, poland. e-mail: grzyw@hermes.umcs.lublin.pl proteolytic enzymes have been firmly established as main regulatory components in a number of cellular and physiological processes. the most important factors influencing the proteolytic enzymes are natural, proteinaceous protease inhibitors, which form complexes with target proteases. they have been extensively investigated from the points of view on physiological functions, as tools for protease enzymology, models for protein-protein interactions and on potential medical applications. there is growing interest in new inhibitors of proteases from various sources. among known protease inhibitors from fungi are, yeasts inhibitors of proteinases a (asparagine protease) and b (serine protease), and low molecular inhibitors of serine proteinases from fruiting bodies of mushrooms -pleurotus ostreatus and lentinus edodes as well as some undefined proteinase inhibitory activities from water extracts of some species of basidiomycetes. searching for new, bioactive metabolites of basidiomycetous fungi we isolated and characterized recently some low molecular, proteinaceous, natural inhibitors of serine proteases, from mycelia of wood rotting fungi -trametes versicolor, abortiporus biennis and schizophyllum communae. isolation of inhibitors was achieved by ion exchange and size exclusion chromatography. preliminary characterization of their inhibitory activity (against some serine proteases), ph and temperature optima of action, and molecular mass, were classically analyzed. analysis of n-terminal amino acid sequences of these inhibitors suggests a new family of serine protease inhibitors from fungi. more detailed characterization of inhibitors (including molecular modeling) and preliminary experiments with laboratory animals and with lines of human cells are in progress. the role of serine proteases in the lectin pathway of complement activation p. ga´l 1 , g. ambrus 1 , v. harmat 2 , b. ve´gh 1 , g. na´ray-szabo´2, r. b. sim 3 and p. za´vodszky 1 1 institute of enzymology, hungarian academy of sciences, budapest, hungary, 2 protein modeling group, hungarian academy of sciences, budapest, hungary, 3 department of biochemistry, university of oxford, oxford, uk. e-mail: gal@enzim.hu the complement system is a cascade of serine proteases, and mediates essential functions during infection as a part of the innate immunity. activation of the complement system culminates in the destruction and clearance of invading microorganisms and damaged or altered host cells. our view about the complement system has changed considerably in the recent years, due to the discovery of a new activation pathway of complement: the lectin pathway. we have recombinantly expressed and characterized the mannose-binding lectin associated serine proteases: masp-1 and masp-2. these are related mosaic serine proteases with similar domain organization but with different enzymatic properties. we showed that masp-2 is capable of autoactivation and it can cleave c2 and c4 complement subcomponents. masp-2, therefore, can initiate the complement cascade without the contribution of any other protease. we demonstrated that the complement control protein (ccp) modules, which associate directly with the serine protease domain, stabilize the structure of the catalytic region masp-2 and contain exosites for the large protein substrates. these results are in agreement with the crystal structures of activated and zymogen forms of masp-2. masp-1 is the most abundant mbl-associated serine protease but it cannot activate the complement system. we demonstrated that masp-1 has a more relaxed substrate specificity compared to masp-2 and the activity of both proteases can be blocked by c1-inhibtor. we concluded that the two mbl-associated serine proteases participate in evolutionary and functionally different pathways. comparative kinetic study on s2' trypsin variants l. gombos, j. tó th, p. medveczky, a. ma´lna´si csizmadia and l. szila´gyi laboratory of enzymology, department of biochemistry, eo¨tvo¨s lora´nd university, budapest, . e-mail: gl@ludens.elte.hu by far the most serine proteases have a glycine in position 193, which is part of the s2' subsite (the second subsite on the enzyme surface c-terminal from the scissile bond of the substrate). in contrast, human trypsin 4, the trypsin isoform expressed in human brain, possesses an arginine in that position. the bulky side chain of this amino acid is responsible for the inhibitor resistance, the most striking feature of this isoform, as it interferes with the binding of polypeptide inhibitors to the enzyme surface. a chimpanzee typsin also has an arginine193, while rat trypsin v bears a tyrosine in that position. there is also a snake venom plasminogen activator, a trypsin type serine protease, that contains an s2' phenilalanine. we created glycine, arginine, tyrosine and phenilalanine s2' variants of human and rat trypsins by site directed mutagenesis in order to investigate the effect of these amino acids on the kinetic behaviour. on small chromogenic substrates and synthetic inhibitors, which do not interact with the s2' residue, there is no signifi-cant difference between the various mutants in catalytic efficiency and inhibitory constants, respectively. however, on oligopeptide substrates the catalytic efficiency decreases 20-50-fold in the nonglycine variants. this effect is even more dramatic with polypeptide partners: the catalytic efficiency drops 200-500 times while inhibitory constants increase by 3-5 orders of magnitude. we conclude that the catalytic mechanism is not fundamentally influenced by the substitution of residue 193, although this amino acid is part of the oxyanion hole. bulky residues in the s2' subsite hinder mainly the binding to interaction partners. structural studies on masp-2: towards the understanding of the mechanism of autoactivation mannose-binding lectin-associated serine protease 2 (masp-2), is the key enzyme of the lectin activation pathway of complement, a major element of innate immunity. a dimer of masp-2 complexed with mannose-binding lectin (mbl) is able to perform its biological functions: upon recognition of the pathogen by mbl masp-2 undergoes autoactivation, and then initializes the complement cascade by cleaving c2 and c4. masp-2 is a mosaic protein containing a chymotrypsin like serine protease domain (sp) and further domains with binding sites of mbl or substrates. our present study focuses on the structural background of the ability of the zymogen form of masp-2 to undergo autoactivation. we solved the structures of catalytic fragment of masp-2 both in its zymogen and activated forms. comparison of the two structures reveals characteristic conformational differences in the classical activation domain and in some other loops lining the substrate binding region. loop 1 shows a unique conformation with arg192 blocking the s1 pocket. we docked the activation loop of masp-2 in the active site of the active enzyme and built a model of the complex of the active and zymogen forms. the model reveals extended regions of molecular recognition. while this model represents the second step of autoactivation (active form cleaves zymogen), the first step (zymogen cleaves zymogen) requires the stabilization of the zymogen enzyme in active-like conformation. we built a model of a zymogen-zymogen complex. favorable and unfavorable contacts of the two zymogen molecules help us to identify possible molecular switches, as well as contact regions stabilizing an active-like conformation of the zymogen enzyme in the complex. the deg/htra proteases are atp-independent serine endopeptidases which are present in most organisms, including bacteria, humans and plants. previous work in our laboratory has shown that the deg2 protease of the model plant arabidopsis thaliana selectively degrades the photodamaged d1 protein in the reaction center of photosystem ii (psii) in vitro. therefore, deg2 is thought to catalyze the primary cleavage of photodamaged d1 protein, which is an important step of the repair mechanism that restores functional psii. our present studies aim to elucidate the regulation of the deg2 protease activity, especially with regard to its d1 degrading activity. we found deg2 associated to the stromal side of the thylakoid membranes and as a soluble protein in the chloroplast stroma. the amount and distribution of deg2 protein remained unchanged after exposure to different light intensities, which suggest either a substrate regulation or a posttranslational regulation of the d1 degrading activity of deg2. recent advances on deg2 regulation and complex formation will be presented. novel peptide inhibitors of human kallikrein 2 (hk2) human kallikrein 2 (hk2) is a serine protease produced by the secretory epithelial cells in the prostate. it activates several other proteases that may participate in the proteolytic cascade mediating metastasis of cancer. thus, modulation of hk2 activity is a potential way of preventing tumor growth and metastasis. furthermore, specific ligands for hk2 may be potentially useful for targeting and imaging of prostate cancer. we used enzymatically active recombinant hk2 captured by a monoclonal antibody exposing the active site of the enzyme to screen phage display peptide libraries. six different peptides binding to hk2 were identified using libraries expressing 10 or 11 amino acids long linear peptides. three of these peptides were specific and efficient inhibitors of the enzymatic activity of hk2. alanine substitution analysis revealed that motifs of 5-7 amino acid determined the inhibitory activity of the peptides. the peptides are also of potential utility for development of immunopeptidometric assays for hk2, which is promising marker for diagnosis of prostate cancer. furthermore, these peptides are potentially useful for treatment and targeting of prostate cancer. the mechanism of autoactivation of the zymogen masp-2 residues on the surface of pathogens. we managed to recombinantly express and purify two forms of zymogen masp-2. one form is the wild type zymogen enzyme, which can be activated, while the other one is a stable zymogen mutant form of masp-2. we could prepare the zymogen form of wild type masp-2 under certain conditions which enabled us to examine the kinetics of activation. we demonstrated that activation of masp-2 is a true autocatalytic activation without the involvement of any other protease. we characterized the enzymatic properties of zymogen masp-2 using the stable zymogen form. we demonstrated that zymogen masp-2 cannot cleave small synthetic substrates but it can cleave large protein substrate (c4). a molecular model for the interaction between zymogen and activated masp-2 during activation has also been built based on the available 3d structures of zymogen and activated masp-2. influence of streptokinase on the fibrinolytic system proteins the present study is dedicated to the investigation of the effect of protein by bacterial origin -streptokinase (sk) on the activity and interaction regulation mechanisms of fibrinolytic system proteins. the study was carried out with use of porcine haemostasis system which plasminogen isn't activated by sk. especially we were interested in study of the changing fibrinolytic system parameters such as tissue type plasminogen activator (t-pa), plasminogen activator inhibitor (pai-1), plasminogen, a2-antiplasmin activities. also the main parameters of coagulation system such as fibrinogen, soluble fibrin, fibrin degradation products levels and thrombin activity and quantity were studied. it was used affinity chromatography, electrophoresis, western-blotting, elisa, determination of proteins activity. it has been determined an increased consumption of plasminogen on 15% in 4 h after streptokinase injection. it was shown that activity and concentration of t-pa were significantly increased in 3.5 times in 1 h. on the next stages of investigation this parameters tend to norm. after sk injection pai-1 quantity was increased in two times (16.7 ng/ml compared to normal 8.9 ng/ml). the interesting fact was the activation of prothrombin by sk without activation of coagulation system in vivo. the injection of sk causes the significant increase of t-pa activity and quantity possibly due to direct or/and indirect effect on endothelial cells. we can conclude that sk causes pai-1 secretion due to effect on platelets as 90% of pai-1 storage is in a-granules of platelets. thus analysis of the data displayed besides of well-known sk function the influence of sk on the changing of fibrinolytic system potential possibly due to its effect on endothelial cells and platelets. paracrystalline inclusions in the mitochondrial matrix or intermembrane compartment occur in several biochemically unrelated disorders such as myopathies, paragangliomas and steatohepatitis, and in various cell types under normal conditions, as well. however, little is known about the composition of the inclusions, the mechanism of their formation and their relation to disease processes. in this study we have described the helix-shaped structures in the intracristal compartments of rat liver mitochondria that have undergone ca 2+ -induced permeability transition. the filaments are anchored in opposing parts of the mitochondrial membranes and appear to support the cristae mechanically. a protein, that apparently is a component of these helical filaments, has been identified as serine protease lactb. this protein shows close sequence similarity to the class c bacterial beta-lactamases and is the only member of this class in animals. since lactb has not been studied previously we cloned its cdna for expression in e. coli as c-terminal his-tagged fusion protein. lactb underwent proteolytic processing in both e. coli and in isolated mitochondria resulting in several protein fragments. this is likely to be due to autocleavage and may be an activation/maturation process. 2d blue native gel electrophoresis indicated that lactb was part of a >600 kda protein supercomplex. in summary, the presence of the serine protease motive in lactb and its supposed ability to form helical filaments suggest that lactb might function not only as a component of 'mitoskeleton' in maintaining and rearranging the mitochondrial ultrastructure under certain conditions, but also might take part in apoptotic processes. novel psychrophylic trypsin-type protease from serratia proteomaculans proteinase with trypsin specificity from psychrophylic microorganism serratia proteomaculans was partly purified. it was shown that the properties of this enzyme (temperature and ph-stability, efficiency of substrate hydrolysis) correspond with the psychrophylic character. inhibitor analysis and study of substrate specificity indicate that this enzyme is serine trypsin-type protease. at the same time this enzyme is zinc-dependent. proteases of such type were unknown till now. secondary specificity of the studied enzyme differs from the bovine trypsin specificity -this protease hydrolyses the short substrates more efficient. zinc, cadmium (ii) and copper (ii) ions in mmolar concentrations inhibit the enzyme activity. the unusual character of calcium ions influence on substrate hydrolysis and inhibition by the bovine pancreatic trypsin inhibitor (bpti) was registered for the studied enzyme. in vitro by a neutral to basic ph change [2, 3] . the kinetics of the activation process can be followed by stopped flow fluorescence (sff) experiments while the structural features of the transition can be explored by in silico molecular dynamics (md) and targeted molecular dynamics (tmd) [4] simulations. to challenge the activation process, mutants were constructed and studied by sff measurements. subsequently, on these mutants multiple md/tmd simulations were carried out. our results indicate the existence of parallel activation pathways. they demonstrate the absolute necessity of multiple simulations and of proper statistics. they reveal the pros and cons of the tmd method. a simple method for the purification of a novel serine endoprotease from wheat triticum aestivum (cv. giza 164) has been developed. it consists of ion-exchange and gel filtration. the molecular mass of the enzyme was 58 kda by sds/page under reducing conditions and 57 kda by gel filtration on a sepharose 6b column. the enzyme had isoelectric point and ph optimum at 4.2 and 4.5, respectively. the substrate specificity of the enzyme was studied by the use of synthesized and natural substrates, azocasein, azoalbumin, hemoglobin, casein, gelatin and egg albumin. the enzyme appears to prefer azocasein with km 2 mg azocasein/ml. the enzyme had a temperature optimum at 50°c with heat stability up to 40°c. while co 2+ and mg 2+ accelerated the enzyme activity by 54 and 56%, respectively, ca 2+ and ni 2+ had very little effect. the enzyme was strongly inhibited by phenylmethylsulphonyl fluoride (pmsf), but not by the other protease inhibitors, suggesting that the enzyme is a serine protease. from the results it can be concluded from the characterization that the t. aestivum serine protease may be suitable for food processing. in vitro effects of a potent, selective dipeptidyl peptidase ii (dppii) inhibitor in leukocytes and u937-cells. the compound was able to penetrate the cell membrane and proved efficacy without evidence for acute cellular toxicity. there was a dosedependent inhibition of intracellular dppii activity without affecting the dppiv activity (maximal efficacy at 100 nm). these properties enable to differentiate between dppii and dppiv in biological systems and allow further investigation of the physiological function of dppii. in a second step, we have been investigating the involvement of dppii in apoptosis in human leukocytes by using this compound. preliminar results based on annexin v-/pi-staining using up to 1 lm inhibitor in u937-cells and pbmc did not show signs of apoptosis while dppii activity was inhibited for 90%. effect of calcium ions on hydrolysis of peptide substrates of general formula a-(asp/glu) n -lys(arg)-b, catalyzed by enteropeptidase (ec 3.4.21.9), differs depending on substrate type. for specific enteropeptidase substrates (n = 4) calcium ion exhibits the promotion of hydrolysis by the natural two-chain enteropeptidase. hydrolysis of atypical enteropeptidase substrates (n = 1-2) is as a rule less efficient; in addition calcium ion shows in this case the inhibition influence. therefore the regulation of the nondesirable side-hydrolysis during full-length enteropeptidase-catalyzed chimeric proteins processing is possible by means of calcium ions. on the contrary the hydrolysis of substrates of all type (n = 1-4) by enteropeptidase light chain as well as the enzyme containing the truncated heavy chain (466-800 or 784-800 fragments) is inhibited by calcium ions. hydrolysis of the natural enteropeptidase substrate, trypsinogen, is at least two orders of magnitude more efficient than any artificial substrate hydrolysis. we propose that this effect is caused by participation in trypsinogen coordination with enzyme of the addition secondary substrate binding site and/or calcium-binding site; both sites located on the n-terminal half (118-465) of the enteropeptidase heavy chain. one more mechanism of the regulation of the enteropeptidase activity by calcium ion is the unusual calciumdependent autolysis of the enteropeptidase heavy chain leading to the drastic loss of its activity towards trypsinogen. autolysis of enteropeptidase heavy chain and well-known autolysis of trypsin were compared; the second one serves as the natural defense mechanism against the undesirable premature proenzymes activation in pancreas leading to pancreatitis. the corresponding enteropeptidase inactivation in low ca 2+ ion environment might be the component of the same protective mechanism. b3-034p human trypsin 4 selectively cleaves myelin basic protein: is this brain protease involved in the pathomechanism of multiple sclerosis? demyelination, the breakdown of the major membrane protein of the central nervous system, myelin is involved in many neurodegenerative diseases. proteases participating in this process are potential targets of therapy in neurodegenerative diseases. in the present in vitro study the proteolytic actions of calpain, human trypsin 1 and human trypsin 4 (the product of gene prss3) were compared on lipid-bound and free human myelin basic protein as substrates. digestions only with calpain and human trypsin 4 actions may be of some physiological or pathological relevance, since these two are expressed in human brain. the fragments formed were identified by using n-terminal amino acid sequencing and mass spectrometry. the analysis of the degradation products showed that human trypsin 4 of these three proteases cleaved myelin basic protein most specifically. it selectively cleaves the arg80-thr81 and arg98-thr99 peptide bonds in the lipid bound form of human myelin basic protein. based on this information we synthesized region 94-104 of myelin basic protein, peptide ivtprtpppsq that contains the specific trypsin 4 cleavage site arg98-thr99. in vitro studies on the hydrolysis of this synthetic peptide by trypsin 4 confirmed our results with intact myelin basic protein. what lends some biological interest to the above finding is that the major autoantibodies found in patients with multiple sclerosis recognize sequence 80-96 of the protein. our results suggest that human trypsin 4 may be one of the candidate proteases involved in the pathomechanism of multiple sclerosis. enteropeptidase is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of its activation peptide following the sequence asp-asp-asp-asp-lys. its light chain alone is sufficient for an effective cleavage of fusion proteins with trypsinogen activation peptide analog. human enzyme possesses 10-fold specificity coefficient compare to bovine one, and an explanation of this fact can contribute a lot to the attempts of improving or modulating enzymatic properties. highly pure and active recombinant human enteropeptidase light chain (l-hep) was obtained by renaturation from inclusion bodies expressed in escherichia coli cells and the active l-hep was purified on agarose-linked soybean trypsin inhibitor. enzymatic activity of purified l-hep was studied through the cleavage of the synthetic peptide substrates and several fusion proteins. l-hep associated with soybean trypsin inhibitor slowly and z-lys-sbzl cleavage was inhibited with ki* = 2.3 nm. comparison of l-hep and bovine enteropeptidase inhibition by bovine trypsin inhibitor aprotinin has shown almost an order difference in ki*. ph dependence of the enzyme activity was measured and ph optimum point was found to be 7.54. enteropeptidase light chain amino acid sequence and crystal structure were analyzed for the presence of target regions for mono-and bivalent ions. unlike trypsin with predicted and experimentally proved calcium-binding sites and sodium-activated thrombin, l-hep was predicted to be deprived of any of such sites and an influence of these ions on the cleavage of different substrates was found to be confined primarily to a substrate binding. as a continuation of our efforts to fully elucidate the antisnake venom properties of mucuna pruriens and to further understand the molecular changes that occurred in mouse plasma proteome as a result of in vivo challenge test with venom and mucuna pruriens proteins (mpe), two dimensional polyacrylamide gel electrophoresis was done. plasma was pooled and gels were run in triplicate to eliminate both biological and experimental variations. analysis using imagemaster 2d platinum software and other statistical analysis tools showed significant differences in protein expression between all the treatments and the control group. some proteins were down regulated, some up-regulated, some completely disappeared while new protein spots were identified. the protein expression of plasma of mouse immunized with mpe for 3 weeks before challenge with lethal dose of venom and that injected with venom alone was more complex. some venom proteins like ecarin are serine proteases that activate clotting factors like prothrombin, causing haemorrhage and disseminated intravascular coagulation, on the other hand, the protease inhibitors from mucuna pruriens must have acted to antagonize these effects by direct proteolysis (cleavage products/spots appearing in the protein map) or other immunological mechanisms. the results obtained represents the first proteomics approach in studying all the plasma proteins involved in this phenomenon. we have only concentrated on protein spots showing interesting variations with respect to control. it is also an important step in the identification of the affected proteins, the kind of modifications/molecular mechanisms involved which is likely the basis of the in vivo protection the plant extract showed against the venom. the use of enzymes at low temperatures has great potential in terms of lower energy costs, therapeutic applications and to lower microbial contamination in industrial processes. low temperature proteases (cryophilic -or psycrophilic -proteases) are of particular interest for detergents and as wound debriding agents. at present, we are studying cryophilic proteases from antarctic krill (euphausia superba), which normally lives in the sea at temperatures near 0°c. we have isolated several low temperature proteases by chromatography. enzyme activities and stability were characterized at low temperatures and as a function of ph to find optimum conditions for different applications. a particular enzyme, named kt1, showed particularly high specific activity at 20°c, several times that of commercial preparations of proteases such as subtilisins. this protein showed a high degree of similarity with digestive trypsins isolated from various arthropoda species. using mrna molecules obtained from abdominal sections of e. superba and subsequently subjected to a reverse-transcription reaction, we identified, isolated and sequenced a dna molecule that codes for an inactive zymogen of the enzyme. cloning of this dna sequence in escherichia coli strains allowed the recombinant expression of the zymogen, followed by purification and activation of the zymogen, which lead to an active cryophilic trypsin. we performed a homology modeling procedure that conducted us to obtain a molecular model of the mature enzyme. the 3d model thus obtained was refined using energy minimization, hydrogen network optimization and residue-residue contact optimization techniques, leading to a reliable model of the enzyme. we used this model to identify many interesting and novel features of the enzyme molecule that could be related with its cryophilic character, and to propose site-directed mutagenesis strategies that could be used to improve the enzyme performance at low temperatures, its ph-activity profile, specificity, inactivation resistance and recombinant expression. in addition, the 3d model allowed us to design and experimentally obtain mutants that are resistant to auto-degradation and more readily activated. molecular cloning and expression of lactba mitochondrial serine protease mitochondria are thought to have originated from a symbiotic relationship between a bacterium able to perform aerobic metabolism ant the ancestor of eukaryotic cells. lactb is the only mammalian protein showing sequence similarity to bacterial serine proteases and belongs to c class b-lactamases. mouse lactb is 551 amino acids long and compromises a predicted mitochondrial import sequence, a short putative transmembrane segment, a b-lactamase homology domain containing the serine protease motif, -sxxk-, and a c-terminal d-transpeptidase domain. the physiological role of mammalian lactb is unclear. therefore, the purpose of this research work was to clone the gene of lactb for expression of lactb in e. coli for further biochemical and cell biological study. the full length lactb gene was cloned into the entry plasmid pentr/sd/d-topo. expression clones were created performing a recombination reaction between the entry clone and four destination vectors. expression constructs resulting in n-or c-terminal gst fusion protein and in n-or c-terminal his6-tag fusion protein were transformed into bl21 (de3) competent cells which are designed for use with bacteriophage t7 promoter based expression systems. when lactb was expressed as an n-terminal gst fusion protein, full-length lactb protein was recovered by glutathione-agarose affinity chromatography. expression of lactb as a c-terminal gst fusion protein or with either an n-or c-terminal his6-tag resulted in proteolytic degradation of the protein and we were not able to detect full-length lactb. these results show that the n-terminal gst fragment protects lactb from proteolytic processing and that lactb can undergo autoproteolysis, which may be a part of a physiological maturation or activation process. design and synthesis of retro-binding peptides active site inhibitors of thrombin thrombin is an important pharmaceutical target for the treatment and prevention arterial and venous thrombosis. biological active peptides are recognized to have significant therapevtic potential but serious limitations especially for oral dosing. the peptide stereomers could differ when forming productive complexes with an enzyme. moreover, the replacement of l-amino acid residues forming the hydrolyzed p1-p'1 bond by their enantiomers is known to result in either an uncleavable or a very slowly hydrolyzed analogue. this phenomenon is often used for the synthesis of the peptide's inhibitors stable to the degradation by the enzymes of organism. as the peptides containing d-amino acids, nor are subject to an enzymatic hydrolysis, the purpose of researches was synthesis of a retro -d-analogues of thrombin's substrates constructed from d-amino acids. the di-and tripeptides of the general formula x-d-arg-d-phe-ome [where x = z, tos, ac h, and z-d-arg-d-ala-(d), l-phe-ome (otbu)] were synthesized by conventional methods of peptide synthesis in solution. special features of their interaction with thrombin are investigated. their inhibitory action on reaction of splitting of fibrinogen by thrombin and on reaction of a hydrolysis by thrombin baee showed, that their inactivating action depends on the substituent on n-end of dipeptides and configuration of phenylalanine in a molecule of tripeptides. the relationship between structure and inhibitory action of the synthesized peptides is discussed. the successful application of d-amino acids for designing of biologically active peptide's analogues as a potential medicinal agent, steady to enzymatic degradation is shown. substrate specificity of mannose lectin binding associated serine proteinase 3 n. s. quinsey and r. n. pike department of biochemistry and molecular biology, monash university, melbourne, victoria australia. e-mail: noelene.quinsey@med.monash.edu.au the innate complement system is involved with the neutralization of pathogenic microorganisms. it plays a comparative role to that of the classic immune complement cascade. in the innate complement system, the oligomers of mannose lectins are able to bind to microorganisms. these oligomers have been shown to have mannose lectin binding serine proteinase (masps) attached, which once activated lead to the activation of the c3 convertase complex, which finally leads to the formation of the membrane attack complex. there have been three active masps identified in the human innate immune system-masp-1, masp-2 and masp-3. there is high homology between these three serine proteases especially in the n-terserpins are protease inhibitors that present their reactive site loop (rsl) to target proteases, followed by drastic conformational changes that inactivate the protease. the sequence of the rsl of serpins determines the target specificity. the drosophila melanogaster gene spn4 encodes multiple serpin isoforms each containing an individual rsl, thus enabling the attack of different proteases. variant spn4a contains a consensus recognition/cleavage sequence of furin within its rsl and is equipped with a signal peptide and an endoplasmic reticulum (er) retrieval signal (hdel). this suggested that the protein resides in the secretory pathway, like furin, a proprotein convertase that activates many cellular proteins and pathogens. our experiments demonstrate that spn4a forms sds-stable complexes with human furin that is inhibited with a second order rate constant of 5.5 · 10 6 /m/s. the rsl of spn4a is cleaved c-terminally to arg-arg-lys-arg, in accord with the enzyme's cleavage site. furthermore, the serpin is retained in the er of transfected cos7 cells as shown by immunofluorescence staining. a hdel deletion mutant was detected mainly in the medium of trans-fected cos7 cells, demonstrating the necessity of the hdel signal for the observed cellular localization. further experiments show that furin 1 and 2 of drosophila melanogaster are physiological targets for spn4a, since secreted forms of both enzymes form stable complexes with the serpin. together, the results demonstrate that spn4a is a potent inhibitor of furin that may meet the target at its natural location. experiments with the other rsl variants show that the spn4 gene represents a multipurpose weapon that is directed against different families of proteases. formation of the covalent tetrahedral complex (tc) with substrate is the first step of the catalytic process in the active site of serine proteases. his57 (chymotrypsin numbering) plays a role of a general base catalyst, activating the ser195 nucleophile by abstraction of its proton. it was experimentally observed that the pka of his57 ne in tc formed by serine proteases with transition state analog inhibitors is about 5 units higher than the corresponding pka in the free enzyme. this work demonstrates that the environmental change of the his57 in tc, induced by the substrate binding in the enzyme active site, is the dominant factor in the pka increase of his57 ne, and triggers the enzymatic processing of the substrate. these results are based on quantum mechanical modeling of the active site of free chymotrypsin and tc complex of chymotrypsin with trifluoromethyl ketone inhibitor in dft b3lyp/6-31+g** level of theory. the polar environment of the enzyme active site is accounted for explicitly in the microscopic model. the combined environmental effects of the bulk water solvation and the rest of the protein is implicitly accounted for by our scrf(vs) continuous solvation approach. the role of local polar effects, such as the oxyanion and the asp102-his57 hydrogen bond, on the pka of his57 ne in tc is analyzed. genome-wide analysis of subtilase (subtilisinlike serine protease) genes in microbial genomes limited to regions surrounding the asp, his and ser catalytic residues. pattern-searching methods using hidden markov models, based on conserved sequences surrounding the catalytic residues, were used to search for subtilases encoded in >200 bacterial and archaeal genomes, representing 177 species. more than 350 subtilases were found to be encoded in 109 genomes. subtilases are more commonly found in grampositive bacteria than in archaea or gram-negative bacteria, and it is more common to have multiple subtilase-encoding genes than a single gene. the majority of the subtilases have a predicted signal peptide for translocation across the cell membrane, and a sub-group of these secreted subtilases are predicted to have a carboxy-terminal cell-envelope anchor, mainly of the lpxtg type for covalent anchoring to peptidoglycan. the genomic context of the subtilase-encoding genes was analyzed to gain insight in putative functions for these proteolytic enzymes. by also taking into account the predicted intracellular or extracellular location of the encoded subtilases, it was possible to predict a function for many subtilases in either nutrition/growth, spore germination, surface protein processing/activation, bacteriocin/toxin processing, or sigma factor activation/regulation. the poisoning by botropics species makes a similar physiologic, one of systemic effects is the blood coagulation for several mechanisms, as direct action on fibrinogen; factor x activation or platelet activation, by toxins of venoms. in the last years were identifies in botropics venoms, serine proteases. this toxins are responsible by coagulant activity with direct action on fibrinogen. serine proteases are utility for hemostatic system studies and for therapeutics use. looking for new molecules models is very important to show the mechanism of action and search structural characteristics responsible for its activities. the present work has the objective of purification and characterization of a coagulant factor (cf) from b. pirajai venom. the purification was made using a gel filtration, hydrophobic chromatography and an affinity chromatography. the molecular filtration was made in sephadex g-75 with ammonium bicarbonate buffer (ambic) 0.05 m ph 8.1, resulting four fractions (p1-p4), the coagulant fraction was named p1. the p1 fraction was submitted in phenyl sepharose chromatography using triz buffer 10 mm ph 8.6 in a decreasing gradient of nacl (4; 3; 2; 1; 0.5; 0 m), and to finish the chromatography it was used distilled water, resulting six subfractions (fp1-fp6), the coagulant subfraction was named fp1. the fp1 sub fraction was submitted in benzamidine sepharose chromatography and eluted in the solutions: distilled water, obtained the subfraction bfp1, sodium phosphate buffer 20 mm ph 7.8, obtained the subfraction bfp2 and glycine buffer 20 mm ph 3.2, obtained the sub fraction bfp3 that is the cf. the cf displayed one band in sds-page (11%) showing a pure protein, it has 58 kda, the minim coagulant dose is 1.75 lg and has action on fibrinogen beta chain. the genome of arabidopis thaliana encodes 16 putative proteases from the deg/htra family. this group of atp-independent serine-proteases was well examined in other organisms, especially e. coli and humans, but only limited data is available for members from this protease family in plants. deg1 and deg2 have been shown to act as proteases in the chloroplast, but no deg/htra proteases from other compartments have been examined so far. the putative protease deg15 is predicted to be localized in the peroxisome. we cloned the gene encoding deg15 (at1g28320) in an overexpression vector for heterologous expression in e. coli. the tagged protein was purified by affinity chromatography and used to raise polyclonal antibodies. with these antibodies we investigated the intracellular localization of deg15 and the protein level under various stress conditions in order to evaluate the in planta function of this protein. the effect of site-directed mutagenesis on cold adaptation of vpr; a subtilisin-like serine proteinase from a psychrophilic vibrio-species psychrophilic enzymes have very similar 3d structures as their homologous enzymes from mesophilic and thermophilic organisms. main characteristics of enzymes from psychrophiles are their high catalytic efficiency (kcat/km values) and thermolability. a subtilisin-like serine proteinase from a psychrophilic vibrio-species (vpr) shows these characteristics when compared to homologous enzymes from mesophilic and thermophilic organisms. the vpr gene was cloned, sequenced and expressed in e. coli and recently the crystal structure was determined at 1.84 å resolution [1] . structural comparisons have been carried out which have led to hypotheses about some of the structural factors which may contribute to cold adaptation of vpr. some of these hypotheses have been examined using site-directed mutagenesis. the specific residue exchanges were selected with the objective to incorporate stabilizing interactions into the cold adapted enzyme which were deemed to be present in related thermostable homologues. these include incorporation of pro into loops, a new potential salt-bridge, as well as substitutions aimed at improving packing in the hydrophobic core and decreasing apolar exposed surface. we have also introduced ser to ala substitutions at three different locations in the cold-adapted enzyme, but these were the most frequent amino acid exchanges observed in sequence comparisons of the enzyme to those of more thermostable homologues. here we report on the catalytic and stability characteristics of the selected mutants. engineering of gfp for the screening of serine protease inhibitors site specific proteolysis has been an attractive target for the development of antiviral therapies based on selective viral inhibitors. it has been previously demonstrated that reporter proteins like beta-galactosidase could be very useful for the high-throughput screening of hiv-1 protease inhibitors through the display of an accessible protease target site on the enzyme surface. in this work, by using structural analysis, we have engineered the gfp protein from jellyfish aequorea victoria to accommodate in its surface the hcv virus ns5a-5b protease cleavage site edvvccsmsytwtg, in a manner that proper proteolysis results in a fluorescent activity decrease. the three resulting gfp constructions, carrying the protease cleavage site in positions 23-24, 102-103 and 172-173, were soluble expressed in escherchia coli. moreover, the hcv ns4 cofactor residues 21-34 fused in frame via a short linker to the amino terminus of the hcv ns3 protease domain (residues 2-181) were also expressed in e. coli and under 1mm iptg induction, at least 60% of soluble protein was recovered and further purificated by an histidin tag. the analysis of gfp proteolysis in front of hcv recombinant protease were performed either with bacteria crude extracts and purificated proteins. the results presented here indicated that proper solvent exposure of target sites on gfp carrier protein may be a critical factor for protease cleavage and for the observation of fluorescence activity variance, being an aspect of absolute relevance for further design and implementation of newer analytical tests. various kinds of stressors cause the group of metabolic changes defined as the general stress response, initiated by some intracellular signals, such as production of abnormal or denaturated proteins, enhanced generation of reactive oxygen species and others. proteolytic enzymes quickly modify proteins and as a consequence can regulate cellular metabolism. although the stress defense mechanisms have been very often described in the recent literature, in very few works were estimated stress response abilities of white-rot basidiomycetes, which produce two kinds of very important ligninolytic enzymes -laccase and peroxidases. our previous results showed that the addition of menadione to abortiporus biennis idiophasic cultures caused the significant increase of the extracellular laccase activity in comparison to the control. the aim of this study was to determine activities of serine proteinases and natural serine proteinase inhibitors in idiophasic cultures of basidiomycete a. biennis grown under menadione-mediated oxidative stress conditions. we investigated the changes of intracellular serine proteinases activities in the presence and absence of atp, using hemoglobin and fluorogenic substrates. the level of natural serine proteinase inhibitors in mycelia was also measured. a fungal inhibitor of trypsin was partially purified and used to in vitro experiments. an interesting correlations between serine proteinases, serine proteinase inhibitors and laccase activities in prooxidant treated cultures were also observed. it can suggest that the proteolytic modifications under oxidative stress conditions can act as a regulation way of laccase activity. serine proteinases, inhibitory and laccase activities were additionally analyzed by native page. calpain is a ca 2+ -regulated cytosolic cysteine protease, functioning as a ''modulator protease'', i.e. regulating/modifying functions/activities of substrates by limited proteolysis to modulate cellular functions. human has 14 calpain genes and potential substrates extend to various cytosolic proteins such as kinases, transcription factors, cytoskeletal and er proteins. in skeletal muscles, expression of p94 (also called calpain 3) predominates, playing an indispensable role for muscle functions in cooperation with ubiquitously expressed conventional calpains. for, a defect of p94 proteolytic activity originated from gene mutations causes muscular dystrophy. p94 localizes in myofibrils binding to connectin/titin, a gigantic elastic muscle protein connecting the z-and m-lines of sarcomere, the repetitive unit of myofibril, with a single molecule. in mdm (muscular dystrophy with myositis) mice, connectin/titin with a small deletion caused by natural mutation of the connectin/titin gene is expressed, resulting in severe muscular dystrophy phenotypes such as body weight less than a half of that of wild type, severely affected limb muscles with impaired walking ability and only 2-3 months of life time. the deletion in the mdm allele of the connectin/titin gene overlaps one of the binding sites of p94 in the n2-line, another electron-microscopically visible line between the z-and m-lines of sarcomere. the mdm phenotypes clearly indicate that connectin/ titin or p94 or both are essential for proper muscle functions. to elucidate physiological roles of connectin/titin and p94, we analyzed mdm mice in relation to calpain system. as a result, mar-ps (muscle ankyrin repeat proteins) were shown to be up-regulated in mdm muscle. marps bind to the n2-and z-line regions of connectin/titin and function as transcriptional regulators translocating into the nuclei. carp (cardiac ankyrin repeat protein), one of marps, binding site in the n2-line region is proximate to the p94 binding site, thus suggesting interactions of both molecules. possible signal transduction systems to modulate muscle functions revealed by the analyses will be discussed based on the results. inhibition and activation of calpain by its disordered endogenous inhibitor, calpastatin p. tompa 1 , z. mucsi 2 , o. gyo¨rgy 2 , c. sza´sz 1 and p. friedrich 1 1 institute of enzymology, biological research center, budapest, hungary, 2 research group of peptide chemistry, university of eo¨tvo¨s lora´nd, budapest, hungary. e-mail: tompa@enzim.hu calpains are a family of intracellular calcium-activated cysteine proteinases, implicated in the regulation of key cellular processes, such as cell division and programmed cell death. their activity is under tight control by an intracellular protein inhibitor, calpastatin, an intrinsically unstructured protein that contains four equivalent inhibitory domains. each of these comprise three conserved subdomains, of which subdmomains a and c anchor the inhibitor in a calcium-dependent manner, whereas subdomain b binds at the active site and inhibits the enzyme. in this work it is shown that the consequence of this mode of binding is that isolated a and c peptides promote calcium binding to calpain and thus activate the enzyme. this activation is manifest in the sensitization to calcium ion: the calcium required for half-maximal activity is lowered from 4.3 to 2.4 lm for l-calpain and 250 to 140 lm for mcalpain. in the physiologically significant sub-micromolar and low micromolar calcium concentration range this sensitization leads to a more than tenfold activation, which is of potential physiological importance as isolated calpain requires high calcium concentrations never realized in vivo. here we suggest calpastatin is degraded in vivo in a way that generates the activator peptides. due to the structural disorder of calpastatin, this unprecedented mode of action raises intriguing questions with respect to the generality of this ambivalent behavior. to address this issue, we have collected extreme cases, when the same protein elicits opposing, inhibitory and activatory, responses within the same molecular setting: structural predictions show that these proteins are largely disordered. as a conclusion, the possible general implications of this finding are discussed. meprins are oligomeric, brush border membrane or secreted zinc proteases that have unique and complex structures. they are composed of multidomain, highly glycosylated evolutionarily-related a and b subunits that form disulfide-linked homo-or heterooligomeric dimers. the homooligomeric form of meprin a forms very high molecular mass multimers of 1 000 000-6 000 000 da, among the largest extracellular proteolytic complexes known. meprins cleave cytokines, growth factors, bioactive peptides and extracellular matrix proteins, important compounds in inflammatory intestinal disease and in cancer metastases. to investigate the role of meprins in intestinal immune responses, inflammation was induced in mice by oral administration of dextran sulfate sodium (dss). the results showed that wild-type mice (c57bl/6 · 129) had a more severe reaction to dss than meprin b null mice on the same genetic background, as determined by body weight loss, intestinal bleeding and mortality. this implies that the presence of meprin b increases host damage caused by dss and that meprin b plays an active role in intestinal pathophysiology. meprins are also expressed in colon cancer cells (e.g. sw480, sw620, and caco-2). expression of meprin a appears to increase with increasing metastatic potential. in addition, meprin a is highly expressed in the human liver hepatoblastoma cell line hepg2 and abundantly secreted into culture media. examination of human tumor samples showed that meprin a is expressed in primary colon tumors and in tumors that have metastasized to the liver. this indicates that meprin a expression in gastrointestinal tumor cells contributes to the progression of the disease. biochemical pathways mediating necrotic cell death and neurodegeneration in caenorhabditis elegans n. tavernarakis, p. syntichaki, c. samara and k. troulinaki institute of molecular biology and biotechnology, foundation for research and technology, heraklion, crete greece. e-mail: tavernarakis@imbb.forth.gr necrotic cell death plays a central role in devastating human pathologies such as stroke and neurodegenerative diseases. elucidation of the molecular events that transpire during necrotic cell death in simple animal models should provide insights into the basic biology of inappropriate neuronal death, and facilitate the characterization of mechanisms underlying degeneration in numerous human disorders. various cellular insults, including hyperactivation of ion channels, expression of human beta-amyloid protein implicated in alzheimer's disease, constitutive activation of certain g proteins, hypoxia and possibly the ageing process, can trigger a degenerative, necrotic cell death in the nematode caenorhabditis elegans. we are genetically and molecularly deciphering the c. elegans necrotic death program. we have isolated mutations in several distinct genetic loci that bock degenerative cell death initiated by various genetic and environmental insults. by characterizing such suppressors, we have discovered that neuronal degeneration inflicted by various genetic lesions in c. elegans, requires the activity of specific calcium-regulated calpain proteases and acidic ph-dependent aspartyl proteases. although, it is believed that these proteases become activated under conditions that inflict necrotic cell death, the factors that govern the erroneous activation of such-otherwise benign-enzymes are largely unknown. we identified novel factors that modulate cellular ph homeostasis, which are required for necrosis and showed that targeting these factors effectively protects from necrotic cell death in c. elegans. our findings demonstrate that two distinct classes of proteases are involved in necrotic cell death and suggest that perturbation of intracellular calcium levels may initiate neuronal degeneration by compromising ph homeostasis and deregulating proteolysis. search for regulatory proteins which are controlled by proteolysis in escherichia coli based on microarray analysis j. m. heuveling ag hengge, institute of microbiology, fu berlin, berlin, germany. e-mail: joheuvel@zedat.fu-berlin.de the impact of controlled proteolysis on regulatory events in prokaryotes is increasingly recognized over the last decade. as in eukaryotic cells, proteolysis is more than just a garbage disposal but has been found to be implicated in the regulation of many vital functions of the bacterial cells, like cell cycle, stress responses and development (hengge r and bukau b. mol microbiol 2003) . conditional degradation of regulators shows a high potential of integrating a great variety of signals as is well studied for the degradation of sigma s. this sigma subunit of the rna polymerase, which triggers the general stress response in escherichia coli is digested rapidly by the clpxp protease in association with the phosphorylated response regulator rssb under non-stress conditions (stuedemann a. embo 2004). several other regulatory proteins have been found to be subjected to proteolysis, as lexa, a regulator of the sos response. also lon protease is involved for example in the degradation of rcsa, a regulator of the capsule biosynthesis and of sula, a cell division inhibitor (as a review: hengge-aronis r, jenal u. curr opin microbiol 2003). in order to find other regulatory processes in which proteolysis plays a role we pursued a global approach using the microarray technique. in mutants lacking functional clpp or lon proteases or either one of the clp recognition factors clpa and clpx, we searched for genes, which are differentially transcribed compared to the wildtype. we found some interesting groups of genes belonging to common regulons governed by known regulators -candidates for clp or lon mediated proteolysis. after confirmation of these results through lacz fusion studies of representative genes of these regulons, these regulators are presently examined in in vivo degradation studies using immunodetection methods. a distinct group of serine peptidases cannot hydrolyze proteins, but can readily cleave peptides that are up to about 30 amino acid residues long. the representative member of the family, prolyl oligopeptidase is implicated in a variety of disorders of the central nervous system. the enzyme consists of a peptidase domain with an a/b-hydrolase fold and its catalytic triad is covered by the central tunnel of a seven-bladed b-propeller. this domain makes the enzyme an oligopeptidase by excluding large structured peptides from the active site. in most propeller domains the circular structure is ''velcroed'' together in a mixed blade, where both amino and carboxy terminus are involved to form a four stranded antiparallel b-sheet. non-velcroed or ''open topology'' propellers are rare, and prolyl oligopeptidase was the first protein structure exhibiting a domain of this nature. the apparently rigid crystal structure does not explain how the substrate can approach the catalytic groups. two possibilities of substrate access were investigated: either blades 1 and 7 of the propeller domain move apart or the peptidase and/or propeller domains move to create an entry site at the domain interface. engineering disulfide bridges to the expected oscillating structures prevented such movements, which destroyed the catalytic activity and precluded substrate binding. this indicated that concerted movements of the propeller and the peptidase domains are essential for the enzyme action. biochemical characterization of thermoplasma volcanium recombinant 20s proteasome and its regulatory subunit g. baydar and s. kocabiyik molecular genetics, biological sciences, middle east technical university, ankara, turkey. e-mail: gozde_baydar@hotmail.com proteasome associated energy dependent proteolysis is not only involved in rapid turnover of specific proteins that could be important during periods of stress, but also engaged in the turnover of the short-lived proteins that regulate a variety of cellular processes in both procaryotic and eucaryotic cell. the universal distribution of proteasome homologs in archaeal genome provide insight into the vital role of archaeal proteasomes. 20s catalytic core of archaeal proteasomes in combination with various aaa atpases and membrane associated lon proteases may play role in stress response or turnover of the regulatory proteins. however, little is known about the potential physiological roles of archaeal proteasomes. this study presents the data on biochemical and biophysical features of recombinant 20s proteasome of a thermoacidophilic archaeon thermoplasma volcanium (tpv). pcr was performed to amplify dna fragments containing tpv genes encoding the a -and b-subunits of the proteasome from tpv genomic dna. the amplified a-gene (tpva) and b-gene (tpvb) together with their upstream sequences were separately cloned and then combined in puc18 vector. the resulting recombinant puc-skba plasmid was used for heterologous production of in vivo assembled 20s proteasome in e. coli. the recombinant proteasome was purified by combination of ammonium sulfate precipitation, gel filtration chromatography (sepharyl s-300) and ion-exchange chromatography (q sepharose). molecular masses of purified protein subunits were estimated as 23.71 kda (b-subunit) and 21.13 kda (a-subunit). substantial post-glutamyl peptide hydrolyzing activity and chymotrysin-like activity were detected as associated with recombinant proteasome. maximum chymotrypsin-like activity was measured at 85°c and ph 8.5. crystallographic studies of the gtp-dependent transcriptional regulator cody from bacillus subtilis. cody is a gtp dependent transcriptional regulator of early stationary phase and sporulation genes in bacillus subtilis. it is activated by gtp, during rapid cell growth it represses several genes whose products allow adaptation to nutrient depletion. when the cells pass from rapid growth to stationary phase, the intracellular concentration of gtp drops thus releasing the repressed genes. cod y is a 259-residue polypeptide containing a helix-turn-helix motif for binding to dna. it also has motifs common with small gtpases, but cody has a much lower affinity for gtp. crystals of the full-length cody have been grown in the presence and absence of gtp from sodium citrate buffered solutions using lithium sulphate as a precipitant and diffraction data have been collected to 3.5 å resolution. attempts to solve the structure using anomalous data from the semet derivative crystals of cody have been hampered by the large number (70) of methionines in the asymmetric unit and difficulties in reproducibility of angiotensin-converting enzyme (ace) is a zinc metallopeptidase critical for the generation of the vasoconstrictor peptide angiotensin ii. a homologue of ace, ace-2, has recently been identified, which appears to play a counter-regulatory role to ace by inactivating angiotensin ii. like ace, ace-2 is a type i membrane protein with its active site contained within the extracellular domain. the expression of ace2 protein is normally low and restricted primarily to endothelial cells of the heart and kidney, kidney epithelium and testis. recent evidence from ourselves and others indicates that ace2 is significantly upregulated in a number of pathologies, such as myocardial infarction, renal disease and hepatitis c-induced cirrhosis. given that ace can be proteolytically released from the cell surface in culture, ace2 may likewise be shed into plasma or urine. detection of elevated levels of ace2 in plasma and urine may be a useful biomarker for the diagnosis of hepatic, renal and vascular disease. using a specific quenched fluorescent substrate, we have detected ace2 activity in human urine. in contrast, ace2 activity could not be detected in human plasma; interestingly, however, we noted that plasma markedly inhibited the activity of recombinant ace2, thus compromising the possibility of measuring plasma enzyme activity. we are in the process of purifying this inhibitor, which preliminary results suggest is small and hydrophilic. we are also currently optimizing methods for its removal from plasma samples, thus allowing detection of low levels of soluble ace2 activity in normal human plasma. the identification of a potential endogenous inhibitor of ace2, the first for this family of metallopeptidases, could have significant consequences for ace2 function in vivo and the regulation of angiotensin peptides. future studies will examine whether plasma or urinary levels of ace2 are elevated in cardiovascular, renal or liver disease. molecular determinants of proteolytic processing of non-structural polyprotein of semliki forest virus. institute of molecular and cell biology, university of tartu, tartu, estonia. e-mail: lulla@ut.ee semliki forest virus (sfv) is a positive-stranded rna virus. the replication of sfv is performed by the rna-dependent rna replicase complex (rc) and regulated by proteolytic processing. during the course of the infection template preference of rc changes from rna plus-strand to minus-strand. it has been known for several years that this preference switch is due to the proteolytic processing of sfv non-structural polyprotein p1234, mediated by viral cysteine protease located in the carboxy-terminal domain of the nsp2 protein. tight temporal regulation of this template specificity switch is crucial for the viral replication, but, nevertheless, its mechanism remains unsolved. therefore, the mapping of the essential molecular determinants of the site-specific cleavage consensuses may provide necessary information, concerning the cleavage regulation as well as regulation of the rna replication. the results of our studies indicate that as little as 5 amino acid residues from the c terminus of nsp3 protein determine the specificity of the proteolytic cleavage of the nsp3/nsp4 junction. at the same time sequences laying downstream of the cleavage point (in nsp4 region) have only minor effect on the cleavage efficiency. the exact region required for the cleavage of nsp2/nsp3 junction is yet not known but the sequences, required from c-terminal part of nsp2 protein, are likely short as well. in contrast, sequence lying within 80-240 n-terminal amino acid residues of nsp3 is vital for cleavage of the nsp2/nsp3 junction. this region may represent the cofactor of the nsp2 protease that activates processing at the nsp2/nsp3 cleavage site. thus, as the result of current research, a principally new function -regulation of the proteolytic processing and rna replication -was mapped to the conserved n-terminal region of the nsp3. this finding significantly improves our understanding about the role of nsp3, which was enigmatic till now, in the virus life cycle. activation occurs within the specific dibasic motif hsiirrsl, suggesting the involvement of the proprotein convertases (pcs) in these process. this family of endoproteases are responsible for the activation of a large variety of regulatory proteins by cleavage at multi-basic recognition sites exhibiting the general motif (k/r)-(x)n-(k/r)(n = 0, 2, 4 or 6). cotransfection of the furindeficient colon carcinoma cell line lovo with provegf-c and different pc members revealed that furin, pc5 and pc7 are vegf-c convertases. the processing of provegf-c is blocked by the inhibitory prosegments of furin, pc5 and pace4, as well as by furin-motif variants of alpha2-macroglobulin and alpha1antitrypsin. accordingly, mutation of the vegf-c pc-site (hsiirrsl to hsiisssl) inhibited provegf-c processing. following zebrafish caudal fin amputation, the injection of control vector or vector containing wild vegf-c did not affect fin regeneration. in contrast, injection of muted vegf-c (pro-vegf-c) inhibited fin regeneration. these data highlight the importance of vegf-c processing in zebrafish fin regeneration and suggest that zebrafish can be used as a simple and useful model for studying the role of protein maturation by the pcs in physiological processes. thimet oligopeptidase (top) hydrolyzes a variety of bioactive peptides and is implicated in the regulation of neurological and other physiological processes. top is composed of two ''clamshell'' domains, with the substrate-binding pocket and catalytic site lying between these domains. it is speculated that conformational changes in loops and coil regions connecting the domains lead to changes in substrate specificity. the loop region (residues 599-611) is close enough to the active site to interact with even the smallest substrate. it contains three glycine residues and is expected to be quite flexible. in an effort to trap intermediate conformations of the loop, we have replaced gly 599, 603, or 604 with ala and have compared the activities of the three resulting protein constructs towards two quenched fluorescent substrates. all three enzymes had lower activity than wild type towards a bradykinin analog, with g599a, the most active of the mutants, possessing 1/3 wild-type activity. however, utilizing a smaller substrate, g603a was the most active, surpassing even wild type (fivefold increase in activity). g604a had little activity towards either substrate. these results are consistent with data that revealed increases in activity towards the larger substrate, when the enzyme is partially denatured and presumably, more flexible and with increased accessibility of the binding loop to proteolytic enzymes, when partially denatured. acknowledgment: this work was supported by hhmi, nih-ns39892 (mjg). the proteasome: paradigm of a self-compartmentalizing protease self-processing of subunits of the proteasome crystal structures of the rhodococcus proteasome with and without its pro-peptides: implications for the role of the pro-peptide in proteasome assembly handbook of metalloproteins bovine chymotrypsinogen-a x-ray crystal-structure analysis and refinement of a new crystal form at 1.8 a resolution equilibrium and rate constants for the interconversion of two conformations of a-chymotrypsin. the existence of a catalytically inactive conformation at neutral ph refolding transition of alpha-chymotrypsin -ph and salt dependence e-mail: saleh38@hotmail.com reference 1. arnorsdottir j, kristjansson mm, ficner r. crystal structure of a subtilisin-like serine proteinase from a psychrotrophic vibrio species reveals structural aspects of cold adaptation (which is an excellent substrate for adam-17), we observed 35% increase in enzymatic activity in the media of 1 mm 5-ht-treated cells compared to untreated cells. however, we did not see any increase in the fluorescence when we used ''cate1'', an adam substrate, which does not recognize adam-17. to further support a role for adam-17/tace, we designed silencing rnas against the enzyme, which were introduced into the mesangial cells using lentiviral infection. successful silencing was confirmed by western blotting 4 days after infection. control and tace silenced human mesangial cells were stimulated with 2-10 lm of serotonin for 5 min, and erk activation was assessed by western blotting baron-ruppert 2 and e. heymann 1 1 department of physiological chemistry (fb8/gw) e-mail: rebecca.lew@med.monash.edu.au b4-015p functional properties of p94/calpain3 and connectin/titin in mdm mouse skeletal muscle y bunkyo-ku the lectin pathway of complement system is an important component of the innate immunity. it provides the first line of defence against infection, since it is activated on the surface of invading pathogens. the activation of the complement system results in the destruction and clearance of foreign microorganisms. mannose-binding lectin-associated serine protease-2 (masp-2) is the enzyme which is responsible for the initiation of the lectin pathway of complement activation. masp-2 is a multidomain serine protease, which is synthesized as an inactive zymogen and become activated upon mbl binds to carbohydrate plant proteinase inhibitors are widely spread in the different plant species being a significant component of a defense system. somewhere a significant diversity of the proteins related to the same structural family of the inhibitors in the same species may be observed. the family of potato kunitz-type proteinase inhibitors (pkpis) exemplifies a group of proteins with the diverse properties and may be divided into three major homology groups: a, b and c. a lot of genes encoding different pkpiproteins of each group were found in various potato cultivars (solanum tuberosum l.). inhibition activity of plant invertase, cysteine and serine proteinase was found in proteins subgroup c. a set of gene copies were isolated by pcr from potato cv. istrinskii genome. dna sequencing analysis of these resulted in identification of 24 different dna sequences with a high similarity to potato kunitz-type inhibitors of group c (pkpi-c). cluster analysis demonstrated that this clones represented multiple copies of six new genes denoted as pkpi-c1, -c2, -c3, -c4, -c5 and c6. it can be supposed that at least two alleles containing pkpi-c genes are harbored in tetraploid genome of potato. one of new genes, namely pkpi-c5, exhibited 99% identity with known invertase inhibitor cdna (1423) from cv. provita. another pkpi-c6 gene was similar (98% identical residues) with cdna (p340) from potato cv. bintje encoding for a putative trypsine inhibitor. four other new genes demonstrated as much as 89-92% identity with known pkpi-c proteins from other potato cultivars. the n-terminal sequence of the protein encoded by the pkpi-c2 gene was identical to the n-terminal sequence of specific subtilisin inhibitor pksi isolated from cv. istrinskii.b3-049p regional distribution of human trypsinogen 4 in human brain determined at mrna and protein level j. to´th 1 , l. gombos 1 , e. siklo´di 1 , p. ne´meth 2 , m. palkovits 3 , l. szila´gyi 1 and l. gra´f 1 1 laboratory of enzymology, department of biochemistry, eo¨tvo¨s lora´nd university, budapest, hungary, 2 institute of immunology and biotechnology, university of pe´cs, pe´cs, hungary, 3 laboratory of neuromorphology, department of anatomy, semmelweis university, budapest, hungary. e-mail: july@ludens.elte.huproteases play an important role in many physiological and pathological processes in the central nervous system such as development, neurite outgrowth, neuronal plasticity and degeneration and cell signaling. a gene coding for such an enzyme might be prss3 on chromosome 9 of the human genome. it encodes due to alternative splicing both mesotrypsinogen, which is expressed in pancreas, and trypsinogen 4 whose mrna has been identified in different human tissues (initially in brain, recently in different epithelial cell lines from prostate, colon and airway). analysis of the gene prss3 predicted two isoforms of the zymogen: isoform a may have a 72 amino acid, while isoform b a 28 amino acid n-terminal leader sequence. in order to gain information on the possible role of human trypsinogen 4 we have determined its amount at the mrna and the protein level as well in 17 selected brain areas using real-time quantitative pcr and elisa.the highest transcript levels could be detected in cerebellar cortex, while low amounts were found, e.g. in cerebellar white matter samples. the distribution of the mrna in different brain areas measured by real-time pcr is consistent with the protein levels detected with elisa. the usage of different monoclonal antibodies specific for the 28 amino acid leader sequence and the protease domain allowed the separate detection of the zymogen and the active enzyme. in e.g. the hypothalamus the zymogen is the dominant form, while a significant degree of activation was found in the cerebellar cortex. our data indicate that the extent of activation varies with different areas. as human trypsinogen 4 is ubiquitous in the brain we conclude that it might play a role in general neurological processes. serine proteases are enzyme involved in the maintenance of the cell homeostasis. thus, this type of enzymes must be extremely regulated and it has been highly reported that serine proteases are involved in the growth and expansion of different cancers. in this regard, the type ii transmembrane serine proteases (ttsps) constitute a subfamily of membrane anchored serine proteases that are ideally positioned to carry out different interactions with other cell surface or extracellular proteins. among them, tmprss2 and tmprss3 proteins have been reported to be overexpressed in most prostate and ovarian cancers respectively, matriptase/mt-sp1 is expressed in a wide variety of benign and malignant tumors and hepsin is overexpressed in ovarian and renal cancers. desc-1 is a ttsp member found differentially expressed in squamous cell carcinoma (differentially expressed in squamous cell carcinoma gene 1) and differentially from other ttsps, its expression is found to be reduced in tumor tissues respecting to the normal tissue at rna level in head and neck squamous cell carcinoma (hnscc), what suggests a possible tumor protective function for desc-1. in order to shed light about the role of desc-1 in these processes, we have carried out the molecular cloning of the human full-length cdna and expression of the recombinant protein to delineate the implication of this protease in hnscc.diffracting crystals. therefore we used limited proteolysis and mass-spectrometry analysis to identify the domain boundaries of the protein and were able to determine the sequence of two principal proteolytic fragments corresponding to the n-and c-terminal domains of cody. these individual domains which were successfully cloned in escherichia coli, overexpressed as histagged proteins, isolated and purified. both domains have been crystallized. the crystals of the n-terminal domain grow from bis-tris buffered solutions at ph 6.5 containing polyethylene glycol and calcium acetate. the crystals of the c-terminal domain were obtained using ammonium sulphate as a precipitant. the crystals of n-terminus domain diffract to at least 2.3 å and crystals of c-terminus domain -to 3.2 å using an in-house diffractometer with a mar research image-plate as a detector. progress towards the determination of cody structure will be presented. the gram-positive bacterium listeria monocytogenes is a facultative intracellular parasite. interactions of l. monocytogenes with the host cell are provided by a number of secreted and cell surface proteins. one of the most important virulence factors, actinpolymerizing protein acta, is surface attached via the hydrophobic c-tailed membrane anchor. despite, the membrane anchor acta was found in comparable amounts both on the cell surface and in the culture supernatant. the aim of the work was to investigate the mechanism of acta release and the role of this process in l. monocytogenes virulence. maldi-tof ms analysis of trypsin released acta suggested releasing due to proteolytic cleavage between histidine and threonine residues in the close vicinity of the membrane anchor predicted by the htmm analysis. the substitution of histidine with proline prevented acta release into the culture supernatant, although did not disturb its surface presentation. in silico analysis of eight other l. monocytogenes membrane-anchored surface proteins suggested the role for asparagine and threonine residues in specific proteolysis. the prediction was experimentally tested by substitution of the residues with alanine. the l. monocytogenes spontaneous mutant strain, unable to release membrane-anchored proteins into the culture supernatant, was isolated. the mutation was mapped outside the acta gene and presumably affected the corresponding peptidase. the mutation impaired the invasion of l. monocytogenes into the human epithelial-like hela cells that suggested the effect of the released proteins on signaling events that result in induced phagocytosis of the pathogen by normally non-phagocytic cells. angiotensin ii (ang ii) has been proposed to act as a regulatory peptide in the epidermal layer of human skin. while the expression of receptors and peptide precursors have been demonstrated in epidermis, the formation of ang ii and its inactivation have not been studied in detail. thus we have established a model system with cultured keratinocytes to examine the metabolism of ang i, ii and related peptides by intact epidermal cells. cultures were incubated with peptides in a minimal medium, which sustained cell viability for at least 24 h and the metabolism of peptides was monitored by chromatography (rp-hplc). with ang i as peptide substrate five major products were detected in keratinocyte culture media after 12 h incubation. a half-life of about 9 h was estimated for ang i and the slow degradation supports results of earlier studies revealing low activities of exopeptidases in a microsomal fraction from keratinocytes as compared to fibroblasts. the degradation of ang i was not affected by inhibitors of alanyl aminopeptidase, peptidyl dipeptidase a and neprilysin. since a peptide product formed from ang i in keratinocyte cultures resembled ang ii in hplc analysis, the activity of peptidyl dipeptidase a in these cells was assayed with hip-his-leu and the presence of the peptidase was confirmed by its sensitivity to captopril. further experiments showed that ang ii, iii and related peptides were degraded in keratinocyte cultures with rates similar to ang i and these reactions interfered severely with the formation of ang ii. immunohistochemical studies showed a strong positive staining for neprilysin and alanyl aminopeptidase in the dermal layer of human skin and at the epidermal-dermal junction confirming the results obtained with the cell cultures. soluble angiotensin converting enzyme-2 present in human plasma and urine p94/calpain 3 is the skeletal-muscle-specific calpain and is considered to be a modulator protease in various cellular processes. a defect in the p94 gene causes limb-girdle muscular dystrophy type 2a (lgmd2a), suggesting that p94 functions are indispensable for proper muscle functions. in sarcomeres, p94 localizes at z-, n2-and m line regions. although the binding partner for p94 at z-line has not been identified yet, n2-and m-line localization of p94 are considered dependent on its interaction with the n2a and m-line regions of connectin/titin, respectively. connectin is a gigantic sarcomeric protein playing an important role as a molecular template for sarcomeric organization, an elastic element generating passive tension, a platform for various protein ligands, etc. in this study, we focused on the molecular components associated with the n2a region of connectin/titin to extend our understanding on p94. intriguingly, a recessive mutation in the mouse connectin gene, mdm (muscular dystrophy with myositis), causes muscular dystrophy. there are two remarkable phenotypes consequential to mdm mutation. first, the mdm mutation abolishes p94 binding activity of connectin n2a fragment. second, in skeletal muscle from mice homozygous for mdm mutation, upregulation of cardiac ankyrin repeat protein (carp) is observed. carp also binds to n2a connectin at the n-terminal proximity of the region mutated by mdm. the effect of mdm mutation on p94 activity and the properties of n2a connectin as well as carp were analyzed using both animal model and cell culture systems. semliki forest virus (sfv) is well known model virus, which has been studied for decades. the main topic of this research was characterization and analysis of sfv replication machinery using approach based on use conditional-lethal mutants of viruses. the direct aim of the present study was to sequence and functionally characterize a panel of independent sfv temperature sensitive mutants. from all putative ts-mutations, identified in this study, two were mapped to nsp1 protein, four were mapped to nsp2 protein and one was founded in nsp4 region. number of assays were used to verify phenotypic effects of revealed mutations: titration of virus stocks at different temperatures, leak yield experiments, analysis of viral rna synthesis and viral polyprotein processing at different temperatures. nsp2 mutants had clear viral protease defect and accumulated non-cleaved polyproteins on different stages. besides all, biotechnological branch of our research is already developing. it includes improving of existing sfv based expression vector system by use of ts-mutations for the temperature regulation of foreign gene expression in mammalian cells. dipeptidyl peptidase iv activity and/or structure homologues (dash) in brain tumors pathogenesis of many diseases, including cancer, often involves improper proteolytic post-translational modification of biologically active peptides. association of dysregulated expression pattern of novel group of ''dipeptidyl peptidase (dpp)-iv activity and/or structure homologues'' (dash) with cancer development and progression has been suggested by several authors, including us [1] . dpp-iv enzymatic action as a common attribute of most of dash members modifies signaling potential of their substrates, biologically active peptides, not only quantitatively, but due to the changes in their receptor preferences also qualitatively. in this study, we have investigated expression (by real time rt-pcr and immunohistochemistry) and enzymatic activity (by biochemical assays and enzyme histochemistry) of plasma membrane localized dash members, in particular dpp-iv, fibroblast activation protein-alpha (fap) and attractin in human gliomas. it was revealed that varying quantities of dpp-iv, fap and attractin mrnas and proteins were coexpressed in the studied tumors. the majority of dpp-iv-like activity in the glioma tissue could be attributed to the canonical dpp-iv. this activity, assayed biochemically and expressed per mg of protein, was increased in high grade gliomas. inhibition studies suggested lack of enzymatically active attractin in the examined glioma tissues. the results of our pilot study demonstrate for the first time that both enzymatically active and inactive dash molecules are coexpressed in gliomas and suggest prevailing association of increased dpp-iv activity with high grade tumors. acknowledgment: this work was supported by iga nr/8105-3 and msmt 0021620808 vegf-c is involved in the neovascularization processes, steps essential for wound healing, cancer progression and many other physiological functions. zebrafish vegf-c processing and key: cord-021419-nypnib0h authors: olsufyeva, evgenia n.; yankovskaya, valentina s. title: main trends in the design of semi-synthetic antibiotics of a new generation date: 2020-03-17 journal: nan doi: 10.1070/rcr4892 sha: doc_id: 21419 cord_uid: nypnib0h this review summarizes main advances achieved by russian researchers in the synthesis and characterization of semi-synthetic antibiotics of a new generation in the period from 2004 to 2019. the following classes of compounds are considered as the basis for modification: polycyclic antibacterial glycopeptides of the vancomycin group, classical macrolides, antifungal polyene macrolides, the antitumour antibiotic olivomycin a, antitumour anthracyclines and broad-spectrum antibiotics, in particular, oligomycin a, heliomycin and some other. main trends in the design of modern anti-infective and antitumour agents over this period are considered in relation to original natural antibiotics, which have been independently discovered by russian researchers. it is shown that a new type of hybrid structures can, in principle, be synthesized based on glycopeptides, macrolides and other antibiotics, including heterodimers containing a new benzoxaborole pharmacophore. the review addresses the influence of the length of the spacer between two antibiotic molecules on the biological activity of hybrid structures. a combination of genetic engineering techniques and methods of organic synthesis is shown to be useful for the design of new potent antifungal antibiotics based on polyenes of the amphotericin b group. many new semi-synthetic analogues exhibit important biological properties, such as a broad spectrum of activity and low toxicity. emphasis is given to certain aspects related to investigation of a broad range of biological activity and mechanisms of action of new derivatives. the bibliography includes 101 references. the review addresses the influence of the length of the spacer between two antibiotic molecules on the biological activity of hybrid structures. a combination of genetic engineering techniques and methods of organic synthesis is shown to be useful for the design of new potent antifungal antibiotics based on polyenes of the amphotericin b group. many new semi-synthetic analogues exhibit important biological properties, such as a broad spectrum of activity and low toxicity. emphasis is given to certain aspects related to investigation of a broad range of biological activity and mechanisms of action of new derivatives. the bibliography includes 101 references. antibiotics are commonly used in the treatment and prevention of various infectious diseases. one of the major problems of modern chemotherapy is the disappointing efficacy when using available drugs against resistant bacterial strains. natural antibiotics, i.e., antibiotics produced by various microorganisms, have been and continue to be an important source of new highly active antimicrobial and antitumour agents. one of the most relevant approaches to the design of new drugs relies on targeted chemical transformations of natural antibiotics. 1 in the world science, considerable efforts are currently underway to combat the problem of resistance of microorganisms to available drugs. however, in comparison with other drugs, the development of new antibiotics is not carried out sufficiently. the design of anti-infective drugs and the creation of marketable products are still a challenge. 2 since the development of medicines for the treatment of chronic diseases is much more profitable and because of high requirements for safety, large cap pharmaceutical companies (big pharma) shut down their antibiotic research projects. currently, small-and medium-sized enterprises are developing a majority of new drugs through investments, venture capital, etc. because of high demands for new anti-infective drugs and extremely high cost of these works, cross-country collaborations are needed for research in this field. only a few new compounds were approved for therapeutic use in human medicine or have completed phase-iii clinical trials. 3 the problems are compounded by the fact that the increasing percentage of the population, particularly in developed countries and russia, suffer from infections that were not earlier dangerous, i.e., from opportunistic infections. this is due to a significant decrease in the immune status of the population caused by natural or man-made factors. the translation of semi-synthetic antitumour antibiotics (e.g., doxorubicin) into clinics resulted in the development of gold standard chemotherapeutic agents. many antibiotics have made a great contribution to understanding of mechanisms of development of resistance in bacterial and tumour cells. due to high innate or acquired drug resistance of cancer cells, chemotherapy of malignant tumours is often ineffective. the international research community has focused its attention on the search for new, more effective and less toxic antitumour agents. one of the most rational approaches to the targeted therapy is based on the search for inhibitors of important tumour cell targets among natural products, primarily antibiotics. 4 even the repurposing of known antitumour antibiotics is considered in order to address the problem of antibiotic resistance of antiinfective agents. 5 researchers of the gause institute of new antibiotics (gina) headed by academician of the ussr academy of medical sciences g.f.gause in the 1960 ± 1990s made considerable contribution to the discovery of a series of original antibacterial and antitumour agents (a total of 15 compounds), their characterization and introduction to medical practice. major achievements of the institute during this period are considered in the review. 6 antibiotics comprise an important class of natural products with unique structural diversity. chemical transformations of natural antibiotics imply a change of particular functional groups of the starting molecules with preservation of structural elements responsible for biological activity. the structure determines the possibility of chemical transformation of the antibiotic and reaction conditions. for example, many antibiotics contain nitrogenous and(or) nitrogen-free sugars, which are easily eliminated in acidic or alkaline media. many antibiotics are sensitive to oxidants, are poorly soluble in organic media or, on the contrary, in aqueous solutions, etc. on the other hand, in the case of similar structures of certain moieties [e.g., the presence of nh 2 , co 2 h, oh, c(o), etc. groups)], methods developed for one class of antibiotics can be applied to antibiotics of another class. the goal of synthetic chemists is to transform natural products with preservation of the sites responsible for biological activity. besides, targeted modification can be performed to gain better understanding of the mechanisms of action, in particular in order to investigate the interaction between the antibiotic and the target. in this review, the following classes of compounds are considered as scaffolds for the synthesis of new antibiotics: polycyclic glycopeptides of the vancomycin ± teicoplanin group, classical macrolides, macrolides of the amphotericin b ± oligomycin group, anthracyclines, aureolic acid derivatives, heliomycin, synthetic benzoxaboroles and some other antibiotics. such representatives as eremomycin, carminomycin, olivomycin a, oligomycin a and heliomycin 6 . olivomycin a 364 6.1. modification of olivomycin a at the aromatic ring of the aglycone 364 6.2. modification of olivomycin a at the side-chain 2 h -keto group of the aglycone 366 6.3. modification of the side chain at the c(2 h )7c (3 h the discovery of vancomycin (1) and teicoplanin (2) (fig. 1 ) has given impetus to research on polycyclic glycopeptide antibiotics. 7 natural antibiotics 1 and 2 are still used in medical practice and are considered as reserve antibiotics. they are commonly applied for the treatment of infections caused by gram-positive cocci, particularly, methicillinresistant staphylococcus aureus (mrsa) strains. glycopeptide antibiotics bind with high affinity to the terminal d-ala-d-ala group of the growing peptidoglycan chain on the outer bacterial cell wall, thereby inhibiting the enzymes transpeptidase and transglycosylase. the vancomycin resistance in enterococcus strains (vre) (for vana and vanb phenotypes) arises due to replacement of the d-ala-d-ala group by d-ala-d-lactate, which weakly interacts with the antibiotic. semi-synthetic glycopeptide analogues, such as oritavancin, telavancin and dalbavancin, have recently been used worldwide in medicine. these drugs only partially solve the problem of the treatment of infectious diseases caused by vancomycinresistant enterococci. 7, 8 the search for more effective glycopeptide analogues is an ongoing process. 7 ± 9 eremomycin 3 (see fig. 1 ), the natural antibiotic of this group, was discovered in the gause institute of new antibiotics. 17 this compound differs from vancomycin (1) by the absence of a chlorine atom and the presence of the additional amino sugar eremosamine in the side group of amino acid 6 (aa6), as well as by the structure of the amino sugar (4 h -epivancosamine or eremosamine) at the d-glucopyranose moiety attached to aa4. eremomycin (3) is 3 ± 5 times more active against gram-positive bacteria than antibiotic 1; however, drug 3 is also ineffective against vre and vancomycin-intermediate resistant staphylococcus aureus (visa). in recent years, series of new semi-synthetic derivatives of eremomycin, vancomycin and teicoplanin active against resistant vre and visa strains were prepared. 8 ± 10 figure 1 presents main possible directions of modification of the cand n-terminal groups of the peptide core (a and f ), 3 h -amino sugar (b), the amide group of asparagine (asn) (c), sugar elimination (d ) and edman degradation (e) for antibiotics 1 ± 3. the presence of (benzotriazol-1-yl)oxytripyrrolidinophosphonium hexafluorophosphate (pybop) as the peptide coupling reagent afforded a series of new carboxamide derivatives of eremomycin 4 ± 6 (scheme 1, fig. 1 a3) . 11 ± 14 after the purification, these compounds were isolated in *50% ± 80% yields. eremomycin pyrrolidide (4) has high in vitro antibacterial activity against sensitive and resistant gram-positive bacterial strains, including mrsa, visa and vre isolates. 13, 14 besides, compound 4 is much more effective in the treatment of induced sepsis in mice compared to vancomycin (1) and does not cause a pseudoallergic reaction typical of many antibiotics of this group. compound 4 was successful in preclinical evaluation (in collaboration with the limited liability company`medicine technology') and was recommended for further clinical trials. 13 eremomycin n-adamantan-2-ylamide (5) was synthesized in a similar way as amide 4 (see scheme 1). 15 in in vitro assays, compound 5 exhibits activity against mrsa, visa, vre and bacillus anthracis strains. this compound is also effective against ciprofloxacin-resistant strains of bacillus anthracis. model in vivo assays in mice infected with s. aureus or bacillus anthracis showed that compound 5 provides a higher survival rate of animals compared to ciprofloxacin and has pharmacologically relevant properties, exhibiting an excellent distribution in tissues. the synthesis of eremomycin carboxamides containing bulky substituents, such as 2-aminoadamantane (2-ad) (compound 5), in the presence of pybop at ph * 8.5 afforded the previously characterized unsubstituted eremomycin amide (6) as a by-product. 16 compound 6 is produced by the competitive amidation reaction of the antibiotic with ammonia, which is eliminated through transpeptidation of asparagine-containing peptides in an alkaline medium. an original method was developed for the selective introduction of different amino acids containing a hydrophobic substituent into glycopeptide antibiotics 1 or 2 via selective aminoacylation of the 3 h -amino group of the amino sugar moiety of the disaccharide branch. 17 for instance, the reaction of vancomycin 1 with n-fmoc-(n-n-octyl-o-4benzyl)-l-alanine n-hydroxysuccinimide (osu) ester gave figure 1 . structures of vancomycin (1), teicoplanin a2-2 (2) and eremomycin (3) and directions of their chemical modifications: amidation (a), acylation (b), alkaline hydrolysis of the c(o)nh2 group to co2h followed by amidation (c), sugar elimination (d ), edman degradation (e) and modification of the n-terminal amino group of the peptide core ( f ). fig. 1 b1) . in this reaction, the n-terminal group of the peptide core of the antibiotic remains intact. the n-fmoc protecting group can easily be removed by the treatment with a 5% secondary amine solution. compound 8 exhibits high activity against sensitive and resistant clinical strains of gram-positive bacteria, including vre. 18 in order to study in detail the interaction between the antibiotic and the target in the intact bacterial cell by solid-state nmr spectroscopy using the rotational-echo double resonance (redor) technique, 15 nh 2 -or f-labelled substituents were introduced into amino acid residues 3 (aa3) and(or) 7 (aa7) of the peptide chain of the antibiotic eremomycin (3). 19 the 15 n label was introduced in the vicinity of the binding pocket of the antibiotic. this was accomplished using carboxyeremomycin (9) , which was synthesized previously by the selective alkaline hydrolysis of compound 3 in a saturated aqueous solution of ba(oh) 2 . under these conditions, vancomycin (1) decomposes. carboxyeremomycin [ 15 n]-bisamide 10 was synthesized by the reaction of compound 9 with appropriate amines in the presence of pybop (scheme 3, fig. 1 a3,c3) . 19 eremomycin 4-fluorophenyl-n-piperazide (11) was synthesized by the conventional amidation method in the presence of pybop. the redor experiments were performed using intact staphylococcus aureus cells, which were grown in a culture medium containing bioprecursors with isotope-labelled atoms (e.g., 13 c-amino acid). the 15 n-or f-containing antibiotic that was added to the medium inhibits bacterial growth by forming a stable complex with 13 c-labelled peptidoglycan moieties (see fig. 2 , hydrogen bonds are indicated by dashed lines). 19, 20 the study of the complex with compound 10 provides an estimate of the distance from the distance between the c-terminal [ 15 n]-amide of eremomycin (10) and l-[ 13 c(3)]ala 1 of the peptidoglycan stem is 3.5 # a (see fig. 2 , a solid arrow). 20 consequently, higher activity of eremomycin amide 10 (compared to antibiotics 1 or 3) against resistant visa staphylococci can be attributed to the fact that this compound interacts with the peptidoglycan not only via a classical model (i.e., with the d-ala-d-ala target) but also with the l-[ 13 c(3)]ala group of its stem. besides, there is an additional binding site of derivative 11 to the target, which can also account for its high antibacterial activity against vre and visa. 21 previously, it was shown that the elimination of sugars (see fig. 1 d ) and the introduction of a hydrophobic residue into the aglycone can give rise to aglycone derivatives of antibiotics exhibiting activity against different types of enveloped viruses. 22 the modification of the eremomycin aglycone (12a), its de-d-meleu analogues (hexapeptide, 13a), which was produced by the cleavage of amino acid 1 (aa1) using the edman method (see fig. 1 e), and the teicoplanin aglycone (14) gave a series of new hydrophobic derivatives. (1-adamantylmethyl)amide of the eremomycin aglycone (12b) and its hexapeptide analogue (13b) are derived by the reaction of 1-adamantylmethylamine with 12a or 13a in the presence of diphenylphosphoryl azide (dppa) (scheme 4). diphenylphosphoryl azide rather than pybop is the reagent of choice for the amidation of aglycones, because the reactions in the presence of pybop often afford by-products containing the pybop moiety in the phenol group of the aglycone at aa4. 23 the acylation of compound 14 with di-tert-butyl dicarbonate (boc 2 o) followed by amidation under standard conditions in the presence of pybop gives the disubstituted derivative ð (2-adamantyl)amide of the n-boc-teicoplanin aglycone (15) . the acylation of 14 with 1-adamantylmethyloxy carbonate (adoc 2 o) affords the n-adoc-teicoplanin aglycone (16) (see fig. 1 f, scheme 4). compounds 12b, 13b and 14 ± 16 exhibit high in vitro activity against different corona-and flaviviruses, in partic-ular feline infectious peritonitis virus (fipv) and the coronavirus (sars-cov). 24 the most interesting data were obtained when studying antiviral activity of (1-adamantylmethyl)amide of the eremomycin aglycone (12b) and its de-(d-meleu) analogue (13b) against human immunodeficiency viruses (hiv): ic 50 = 1.6 and 5.5 mmol l 71 for hiv-1, 7.0 and 3.5 mmol l 71 for hiv-2, respectively. compounds of type 13b are promising selective anti-hiv agents because they cannot bind to bacterial targets. 23 apparently, they cannot induce resistance of bacteria during long-term application and can be used in the future for the prevention of hiv infections. the doubly modified teicoplanin derivative ð n-bocprotected 2-adamantylamide of the teicoplanin aglycone 15 ð exhibits high in vitro activity against a series of flaviviruses: hepatitis c virus (hcv), 25 yellow fever virus (yfv), japanese encephalitis virus (jev), tick-borne encephalitis virus tbev) and dengue virus (denv). 25, 26 compound 15 is unique in that it can inhibit replication of the closely related denv and hcv viruses by different mechanisms: in the former case, the inhibition occurs in the stage of virus entry in the host cell; in the latter case, after virus entry. protein kinases play an essential role in the virus entry in the cell and virus replication. hence, 15 analogues of the eremomycin and teicoplanin aglycones (including compounds 12a,b, 13a,b and 14 ± 16) were tested on a panel of 12 recombinant human protein kinases (pks) and two rat liver pks (ck1 and ck2). 27 these compounds were shown to inhibit pk activity by 50% at a concentration of <10 mmol l 71 and by 90% at a concentration of 10 mmol l 71 . teicoplanin aglycone derivatives 15 and 16 exhibit higher activity against many pks compared to eremomycin derivatives 12b and 13b, which also correlates with their higher activity against many types of enveloped viruses. the kinetic analysis of the inhibition of protein kinase ck2a demonstrated that teicoplanin n-adoc-aglycone 16 does not compete with atp and peptide substrates. 27 on the available data, it was suggested that one of the mechanisms of antiviral activity of glycopeptide derivatives can be based on the inhibition of serine/threonine protein kinases. the synthesis of hybrid analogues containing covalently bonded compounds of different classes (dual-acting antibiotics) with different spectra of antibacterial activity is a promising approach to the search for new antibacterial agents to combat antibiotic resistance of bacteria. 28, 29 boronic acids and benzoxaboroles are compounds capable of interacting with various biologically important components of the living cell, such as alcohols, amino alcohols, carbohydrates, rna and some peptides. a new class of synthetic antibiotics possessing antifungal, antimicrobial and antiparasitic activity was designed and synthesized based on benzoxaboroles and is currently developed by anacor pharmaceuticals (usa). 30 certain starting benzoxaboroles used in the synthesis also exhibit biological activity (see below). series of hybrid analogues 17 ± 20 linked to the borole or benzoxaborole moiety either directly or through a spacer were synthesized for the first time from glycopeptides 1 and 3. to introduce a substituent containing a boronic acid moiety into molecule 1 or 3, it is necessary to employ picolinic acid as a protecting group, which is easily removed in a weakly acidic medium (scheme 5). 31 the amidation of the carboxyl group of antibiotics 1 and 3 with 4-or 3-aminomethylphenylboronic acid picolinate esters in the presence of pybop gave new carboxamides of these antibiotics (17a ± 20a). the hydrolysis of the picolinic group under mild conditions in a weakly acidic aqueous medium affords derivatives 17b ± 20b containing the unprotected boronic acid moiety. borole-containing derivatives 17 ± 20 were found to be as effective as the starting antibiotics 1 and 3. eremomycin derivative 19b exhibits the highest activity against gram-positive bacteria and is more effective against resistant staphylococcus strains (visa) compared to compounds 1 and 3. a series of vancomycin conjugates containing different types of benzoxaborole substituents were synthesized: amido derivatives 21a,b, n-acyl derivatives (22) n-alkyl derivatives (23) (scheme 6). similar schemes were applied to synthesize benzoxaborole derivatives of eremomycin (7) (see scheme 1) and the teicoplanin aglycone (scheme 7). 32 carboxamides of eremomycin (7) (see scheme 1), vancomycin (21a) (see scheme 6) and the teicoplanin aglycone (24a) (see scheme 7) were synthesized by a standard procedure based on the treatment of compounds 1, 3 and 14, respectively, with 3-(aminomethyl)benzo[c] [1, 2] oxaborol-1(3h)-ol in the presence of pybop. the reaction of compound 1 or 14 with o-amino-n-alkylamines affords the corresponding amides 21b and 24b,c (n = 2, 3 and 5) containing a longer spacer. the reactions with osu-activated esters of the same in situ generated compounds were used to synthesize n-[3-(1-hydroxy-1,3-dihydrobenzo[c] [1, 2] oxaborol-7-yl)propanoyl] derivatives of vancomycin (22) and the teicoplanin aglycone (25a). the alkylation of vancomycin (1) with appropriate aldehyde in the presence of nabh 3 cn affords n,n hdi(1-hydroxy-1,3-dihydrobenzo[c] [1, 2] oxaborole-6-methyl)vancomycin (23) in quantitative yield (see scheme 6) . 32 the amidation of n-acyl-substituted teicoplanin aglycone 25a with appropriate amine (in a similar way as the synthesis of amides 21 and 24a) gave disubstituted teicoplanin aglycone derivative 25b. the five-membered oxaborole ring cleavage is not observed in various reactions of benzoxaboroles (see scheme 7) . the presence of peaks at m/z 1619. 49 [m7oh], 1474.39 and lower (1312. 34 and 1284.34) in the tandem mass spectrometry (esi-ms/ms-mrm) spectrum of compound 22 indicates that the antibiotic molecule contains a substituent in the n-terminal amino group of the peptide core rather than in the amino sugar n(3 h ) group of vancosamine (fig. 3) . 32 vancomycin derivatives 21a, 22 and 23 proved to be less effective against gram-positive bacteria than the starting compound 1, except for amide 21a, which exhibits activity comparable with that of vancomycin 1. 32 hybrid derivatives, in which benzoxaborole and the teicoplanin aglycone are linked by a spacer with a particular length, exhibited the highest antibacterial activity against clinical isolates of gram-positive bacteria. 3-amino-n-(1hydroxy-1,3-dihydro[c] [1, 2] oxaborol-6-yl)propylamide of the teicoplanin aglycone (24c, n = 2) possesses particularly high activity, in particular against vancomycin-resistant strains. 32 besides, this compound exhibits moderate activity against vancomycin-resistant enterococci (vre); the minimum inhibitory concentration (mic) is 4 ± 8 mg ml 71 . an increase or a decrease in the spacer length and the introduction of two benzoxaborole substituents into the n-and c-terminal groups of the peptide were found to decrease antibacterial activity. the broad-spectrum antibacterial drug kanamycin a (26a) is an important antibiotic of the aminoglycoside (aminocyclitol) class, which is still used in medicine for the treatment of many infectious diseases and also in agriculture. 7 aminoglycosides are active against gram-positive and gram-negative bacteria. the mechanism of their action is related to the interaction with the decoding site (a site) of the 16s subunit of ribosomal ribonucleic acid (rrna), which leads to disturbance of translation, i.e., protein biosynthesis. according to the literature data, a number of semi-synthetic derivatives of a new generation were synthesized based on aminoglycosides. 33 various heterodimeric aminoglycoside conjugates with other antibiotics were reported, and some of them are used in medicine. 28, 29 the synthesis of hybrid kanamycin a conjugates with glycopeptides 1 and 3 was described for the first time in our publication. 34 kanamycin a (26a) was conjugated with compounds 1 or 3 via an amino group of the antibiotic at the 1 position of 2-deoxy-d-streptamine. the acylation of this amino group is known to reduce the risk of the development of resistance, because it prevents deactivation of the antibiotic by enzymes. the amino group at the 3 position of 2-deoxy-dstreptamine and the 6 h -amino group of 6 h -deoxy-6 h -amino-d-glucopyranose of aminoglycoside 26a were protected by the benzyloxycarbonyl group (cbz). 3,6 h -bis-(cbz)-kanamycin a (26b) was synthesized by the reaction of the zinc complex of compound 26a with cbzcl in the presence of a base (et 3 n) using a modified method 35 (scheme 8). (mic *2 ± 4 mg ml 71 for compounds 27, 28a,b) and vre stains (mic = 8 mg ml 71 for compound 28a). 34 based on the results of these studies, methods were developed for the selective introduction of functional groups at the amino sugar amino group of vancomycin (1) or eremomycin (3), with the terminal methylamino group of the peptide core of the antibiotic remaining intact. 17 the amidation of the terminal carboxyl group of these antibiotics with various amines, including amines with bulky substituents, was studied in detail. 12, 13, 15 an unusual byproduct of amidation (previously unknown for these classes of antibiotics) was isolated, and the optimal conditions were found for the amidation providing the target products in high yields. 15, 16 the introduction of various groups into glycopeptides at certain positions of the molecule can give new derivatives active against bacteria that are resistant to the initial antibiotics ð glycopeptide-resistant enterococci. 5, 10 this resulted in the discovery of compounds exhibiting high activity against glycopeptide-resistant enterococci (mic = 2 ± 8 mg ml 71 ) and staphylococcus aureus with intermediate resistance to glycopeptide antibiotics (mic = 1 ± 2 mg ml 71 ). generally, eremomycin derivatives possess higher in vitro antibacterial activity than analogous vancomycin derivatives and show significant advantages over vancomycin in the treatment of animals in a mouse model of staphylococcal sepsis. 10, 13 conditions were found for the selective introduction of isotopic labels at both the terminal carboxyl group and the asparagine residue (aa3) of the peptide core of eremomycin, with carbohydrate moieties and other labile functional groups remaining intact. 19 the investigation of interactions of these compounds with native cells of gram-positive bacteria by the redor technique confirmed the mechanisms of action of this group of antibiotics proposed in our previous studies. 20, 21 modifications of glycopeptide aglycones at the carboxyl and(or) amino group were performed in a series of studies. 22 ± 25 this resulted in the discovery and characterization of a new class of polycyclic peptides exhibiting antiviral activity at micromolar concentrations against hiv-1 and hiv-2, as well as against the enveloped viruses hcv, denv and many other. 22 ± 26 the correlation between antiviral and pk inhibitory activities was established for a class of hydrophobic derivatives of glycopeptide aglycones. 27 the first heterodimeric conjugates of glycopeptide antibiotics with boroles, 31, 32 and with kanamycin a 34 were synthesized. the conjugates exhibit activity against resistant vre and(or) visa strains. the synthesis of chimeric (heterodimeric) macrolide-based antibiotics is a promising area of research to search for new antimicrobial agents. 28 these antibiotics are among the most effective broad-spectrum antibacterial agents. the semi-synthetic antibiotics clarithromycin (29) and azithromycin (30) are commonly used in medicine for the treatment of various infectious diseases caused by many gram-positive and gram-negative bacteria (fig. 4) . azithromycin (30) has the best pharmacological profile among macrolide antibiotics. the mechanism of action of macrolides is based on the inhibition of protein synthesis. the target of macrolides is the peptidyl transferase centre on the large 50s subunit of bacterial ribosome. however, clarithromycinand azithromycin-resistant clinical isolates of bacteria were isolated. 36 researchers at the gause institute of new antibiotics have developed methods for the conjugation of macrolide antibiotics with benzoxaboroles or polycyclic glycopeptide antibiotics and synthesized series of new chimeric antibiotics. conjugates based on clarithromycin (29), azithromycin (30) and various substituted benzoxaboroles were synthesized by the targeted modification of macrolides at the c(9), c(2 h ) and c(4 hh ) atoms and the c(11) ± c(12) bond (see fig. 4 a ± d ). according to the literature data, the introduction of arylalkyl groups at the 4 hh position of the cladinose moiety may help antibiotics overcome resistance caused by methylation of the macrolide-binding site of 23s rrna of the large ribosomal subunit. 37 a method was developed for the introduction of aminobenzoxaboroles at the c(4 hh ) atom of the cladinose moiety of the antibiotic through the carbamoyl group. 38 hybrid structures containing hydroxamic acid-derived benzoxaborole at the c(9) keto group of the aglycone were prepared, because modification of this group does not lead to the loss of antibiotic activity. scheme 10 shows the synthesis of conjugates based on clarithromycin 29 via the introduction of benzoxaborole groups (a and b) into the antibiotic molecule at the c(9) atom of the aglycone or at c(4 hh )7o-cladinose with acetyl protection of the c(2 h )7oh group of desosamine. 38 the former approach is based on the treatment of clarithromycin (29) with aminoacetic acid giving intermediate clarithromycin 9-syn(anti)-(o-carboxymethyl)oxime (31a). the reaction of 31a with aminobenzoxaboroles ha figure 4 . structures of the macrolide antibiotics clarithromycin (29) and azithromycin (30) . arrows indicate the directions of modification: at the 9 position of the aglycone (a), at the c(2 h )7oh group of desosamine (b), at the c(4 hh ) atom of cladinose (c), the formation of c(11),c(12)-cyclic carbonate and modification of the c(11) atom (d ). or hb gives the corresponding amides of clarithromycin (e/z)-9-carboxymethoxime 31b,c (see scheme 10). another approach was accomplished by a modified procedure 39 involving the following four steps: the protection of the c(2 h )7oh group of desosamine by the acetyl group giving 2 h -oac-clarithromycin (32), the transformation of the latter into activated 2 h -oac-clarithromycin 4 hh -o-1h-imidazole-1-carboxylate (33) by the treatment with carbonyldiimidazole (cdi), the amidation of 33 with amines ha or hb in the presence of the peptide coupling reagent 1,8-diazobicyclo [5.4 .0]undec-7-ene (dbu) giving 2 h -oac-substituted carbamoyl derivatives of clarithromycin 34a,b, and the deacetylation of 34a,b by heating in methanol to form target unprotected carbamoyl derivatives of clarithromycin 35a,b. compounds 31b,c and 35a,b exhibit the inhibitory effect against staphylococci and streptococci comparable with the activity of starting compound 29. in these assays, derivatives at the c(4 hh )-cladinose position were found to be more effective than the derivatives at the c(9) position of the aglycone of antibiotics 31b and 31c. compound 35b is the most effective against the strains staphylococcus epidermidis atcc 12228 and streptococcus pneumoniae atcc 49619. clarithromycin analogues 31c and 35b possess an opposite activity against gram-negative bacteria, such as sensitive strains of e. coli and resistant strains of e. coli (tolc and tolc puc erm42). thus, compound 31c is more active than 35b. in the former case, e. coli tolc and tolc puc erm42 are bacterial strains containing the outer membrane protein tolc, which is responsible for antibiotic efflux from the cell. in the latter cases, the strain contains, apart from tolc, the puc plasmid cloning vector and methylase erm42. 38 attempts were made to extend the method developed for the synthesis of c(4 hh )-substituted clarithromycin ± benzoxaborole conjugates to the synthesis of related azithromycin conjugates (scheme 11). 39 it appeared that the success of introducing the aminobenzoxaborole moiety into a macrolide antibiotic depends on the structure of antibiotics 29 and 30, as well as on the structure of aminobenzoxaborole. the acetylation of azithromycin (30) giving the 2 h -oac derivative (36) followed by the treatment of the latter with n,n h -carbonyldiimidazole in the presence of et 3 n produces the desired activated imidazole derivative ð azithromycin 2 h -oac-4 hh -o-1h-imidazole-1-carboxylate (37) (see scheme 11) . however, unlike the synthesis of clarithromycin analogue 34a, the amidation of compound 37 with 7-(hydroxy-1,3-dihydrobenzo[c] [1, 2] oxaborole)methylamine in the presence of dbu does not give the desired outcome. the introduction of the latter amine was accomplished using the trisubstituted derivative, the 11,12-cyclic carbonate azithromycin 2 h -oac-4 hh -o-1h-imidazole-1-carboxylate 38 as the substrate. compound the same conditions as those used for 37 was not successful (see scheme 11) . 40 meanwhile, this reaction with another amine containing an aminoethyl spacer gives the corresponding azithromycin carbamoyl derivative 39 (see scheme 11) . 40 however, the elimination of the 2 h -oac group finally results in the decomposition of the deacetyl derivative during its purification on silica gel. an alternative procedure for the introduction of benzoxaboroles into molecule 30 using carboxy derivatives and diaminoalkane spacers (scheme 12) proved to be more successful. 40 the treatment of imidazole derivative 38 with stronger bases, such as diaminoethane or 1,3-diaminopropane, in the presence of dbu resulted in the formation of aminoalkylcarbamoyl derivatives 40 (n = 2) and 41 (n = 3). the subsequent acylation of the latter with various benzoxaborole acids under standard conditions (dcc, hobt) gives a series of acylaminoalkylbenzoxaborole-containing carbamoyl derivatives of 11,12-cyclic carbonate, 2 h -oacazithromycin 42a ± 44a (see scheme 12) . the deacetylation of these compounds affords the corresponding cyclic carbo-nates 42b ± 44b containing the free c(2 h )7oh group of the desosamine moiety (r 1 = h) in quantitative yields. compound 46b and its 2 h -oac analogue 46a were synthesized from azithromycin 2 h -oac-4 hh -o-1h-imidazole-1-carboxylate 37 through intermediate 2 h -oac analogues 45 (n = 2, 3) (scheme 13). 40 therefore, the synthesis of compound 46a and its 2 h -oac analogue 46b showed that the introduction of the acylaminoalkylbenzoxaborole moiety can be accomplished using a scheme described above without protection of the c(11)7oh and c(12)7oh groups by cyclic carbonate. the evaluation of antibacterial activity of 4 hh -o-substituted derivatives 39, 42a ± 44a (n = 2), 42b ± 44b (n = 2, 3) and 46b (n = 2, 3) compared with that of compound 30 showed that the activity of compounds 39, 42 ± 44 and 46 against gram-negative bacteria (5 isolates) is lower than that against gram-positive bacteria (8 isolates). for example, the activity of compounds 39, 42a (n = 2), 43a (n = 3) and 42b (n = 3) against the gram-positive strains streptococcus pyogenes atcc 19615 and propionibacterium acnes atcc 6919 is comparable with that of the starting compound 30, while conjugates 42b (n = 3), 43a (n = 2), 43b (n = 2) and 44b (n = 2) are more effective than compound 30 against the strains streptococcus pneumoniae atcc 49619 or enterococcus faecium. the presence of the 2 h -oac group or 11,12-cyclic carbonate in analogous hybrid antibiotics was found to have almost no effect on antibacterial activity. for compounds 42a (n = 2, 3), 43a (n = 2, 3), 44a (n = 3), 42b ± 44b (n = 3) and 46b (n = 2, 3), the mechanism of antibacterial activity was studied using the prfpcer-trpl2a reporter construct, which responds to inhibitors of translocation of the ribosome along the matrix nucleic acid (mrna). 40 all compounds were found to inhibit the peptide chain growth at the exit from the ribosome tunnel like typical macrolide antibiotics. it is worth noting that one of the starting benzoxaborole acids, 3-(1-hydroxy-1,3-dihydrobenzo[c] [1, 2] oxaborol-7-yl)propanoic acid (see schemes 12 and 13, marked with an asterisk), also exhibited activity in this assay. 3.3. azithromycin ± benzoxaborole conjugates at the 11-oh group of the aglycone 11,12-cyclic carbonate can be used not only as the protecting group for two hydroxyl groups but also as the activated group to introduce benzoxaborole substituents at the 11 position of antibiotics. 41 the reaction of 11,12-cyclic carbonate azithromycin 2 h -oac derivative 47, which was generated from azithromycin (30) in two steps (treatment with ethylene carbonate followed by acylation of the 2 h -o group of the desosamine moiety), with diaminoalkane gave 2 h -oacetylazithromycin 11-aminoalkylcarbamates 48 (n = 3, 5) (scheme 14). the acylation of compound 48 with benzoxaborole acids under standard conditions (dcc, hobt) produced a series of azithromycin 2 h -oac derivatives 49a ± 51a. the deacetylation of these compounds afforded target benzoxaborole derivatives 49b ± 51b. new hybrid antibiotics 49b ± 51b exhibit broader-spectrum antibacterial activity against gram-positive and gram-negative bacteria compared to azithromycin (30) and tobramycin. compound 50b proved to be the most effective compound in this series, but its activity is lower than that of compound 30. the modified antibiotics do not overcome the antibiotic resistance in mrsa strains (strain atcc 33591). higher activity of these three compounds against the sensitive strain s. pneumonia atcc 6301 compared to tobramycin (mic is 40.06 ± 0.25 versus 4 mg ml 71 ) is a particularly valuable property. 41 , or , or , dcc, hobt; dcc is n,n h -dicyclohexyl carbodiimide, hobt is 1-hydroxybenzotriazole; in the former case, the reaction of azithromycin 11-aminoalkylcarbamates 48 (n = 3, 5) with antibiotics 1, 3 or 13 in the presence of pybop affords the corresponding derivatives of vancomycin (52, 53) , eremomycin (54) or the teicoplanin aglycone (55, 56) (scheme 15). 42 in the latter case, 11,12-cyclic carbonate azithromycin 4 hh -o-alkylaminocarbamoyl derivatives 40 and 41 are amidated with antibiotics 1, 3 or 13 in the presence of pybop (scheme 16). after the removal of the 2 h -o-acetyl protecting group from compounds 57a ± 61a and the column chromatographic separation on silanized silica gel followed by sephadex lh-20 chromatography, five new conjugates 57b ± 61b were isolated in 35% ± 45% yields based on the corresponding starting antibiotic. 42 antibacterial activity of derivatives 52 ± 55 modified at the c(11)7oh group of the aglycone was evaluated compared to the starting antibiotics vancomycin (1) and azithromycin (30) on a panel of gram-positive and gramnegative bacterial strains (8 and 3 strains, respectively). 42 none of the conjugates exhibited activity against gramnegative bacteria, which attests to the absence of the effect of the azithromycin moiety active against gram-negative bacteria. generally, compounds 52 ± 55 display similar or somewhat lower activity against gram-positive bacterial strains compared to compounds 1 and(or) 30, the activity against staphylococci being higher than that against the streptococci s. pneumoniae 49619 atcc and s. agalactis 52-2. azithromycin ± teicoplanin aglycone conjugate 56 containing a long spacer (n = 5) exhibits higher activity against all tested gram-positive bacterial strains (staphylococci and 42 derivatives at the 4 hh -cladinose position show a similar tendency. thus, they are inactive against gram-negative bacteria. the fact that the hybrid structures are ineffective against e. coli 25922 atcc and other gram-negative bacterial strains indicates that they cannot penetrate the outer phospholipid layer of the bacterial cell. in all the tested gram-positive bacterial strains, compounds 52 ± 55 exhibited activity comparable to or higher than that of azithromycin (30) and vancomycin (1) . their activity is provided by the presence of the glycopeptide moiety. unlike hybrid vancomycin analogue 58b, hybrid eremomycin analogue 59b displays significant activity against the vancomycin-resistant enterococci (vre) strains enterococcus faecium 569 and enterococcus faecalis 560 (mic = 3.2 and 6.5 mmol l 71 ), which can be attributed to the effect of the azithromycin moiety attached to the c-terminal group of the peptide core of antibiotic 3. 42, 43 the mechanism of action against gram-positive bacteria was confirmed by quantum chemical calculations of the energy of interaction dg 298 in relation to hybrid antibiotics 58b and 59b with the model d-ala-d-ala ligand typical of glycopeptides. 43 these studies resulted in the development of methods for the synthesis of macrolide-based hybrid antibiotics containing benzoxaborole as a new pharmacophore. 38, 40, 41 the behaviour of the macrolide antibiotic aglycone in chemical reactions was found to be affected by its structure, in particular it depends on the presence of an additional methylamino group in azithromycin (30) . 38, 40 the position of the benzoxaborole substituent was shown to influence the antibacterial activity of antibiotics 29 and 30. it was established that the c(11)-substituted analogues are less effective inhibitors of gram-positive and gram-negative bacteria compared to 4 hh -substituted analogues. 40, 41 the presence of 11,12-cyclic carbonate or the 2 h -o-acetyl group in the azithromycin molecule was shown to have no significant effect on the antibacterial activity of the conjugates, while an increase in the spacer length generally leads to an increase in activity of the final compounds. a method was developed for the synthesis of a series of chimeric antibiotics based on glycopeptides and azithromycin (30) . 42 the activity of almost all the synthesized compounds against the tested gram-positive bacterial strains, including vancomycin-resistant strains, is similar to or higher than that of the starting antibiotic. the range of antibacterial activity of the resulting hybrid derivatives and quantum chemical calculations suggest that the antibacterial activity is determined by the presence of the glycopep carbohydrate-containing polyene macrolides are commonly used in medicine for the treatment of both superficial and systemic mycoses due to their high activity and a broad spectrum of action. 44, 45 the mechanism of action of polyene macrolides is related to their ability to interact with sterol-containing cytoplasmic membranes and form pores (channels) in these membaranes, by which ions leave the cell causing its death. the efficacy of polyenes against fungal pathogens is due to their stronger binding to fungal membrane ergosterols compared to cholesterol present in human and animal cell membranes. nystatin (62a), partricin and pimaricin are administered locally, whereas amphotericin b (amb, 63a) is the only polyene that is applied for the treatment of systemic mycoses. unfortunately, polyenes are rather toxic agents because of their low selectivity for fungal versus mammalian cell. poor solubility of polyenes in water, their high hematotoxicity and nephrotoxicity and a number of other adverse effects have stimulated an extensive search for new, less toxic and more effective agents. previously, it was shown that toxicity of compound 63a and other polyenes can be reduced by chemical modification, which leads to a decrease in side effects. 44, 45 the toxic effect of compound 63a on blood cells (haemolysis) is particularly dangerous. in order to improve antifungal properties, cytotoxic and therapeutic characteristics and to study the mechanisms of action, series of new semi-synthetic derivatives based on amb (63a) and bioengineered analogues s44hp (64a), bsg005 (65a), bsg022 (66a), bsg019 (67), bsg003 (68a) and bsg018 (69) were synthesized (in collaboration with the company biosergen, norway) (scheme 17). 46 ± 49 the structural diversity of the above-mentioned polyenes 64a ± 66a, 67, 68a and 69 was provided by using methods of genetic engineering to alter genes encoding the nystatin-producing strain streptomyces noursei. 50 new analogues compare favourably with nystatin (62a), primarily due to the presence of a double bond (instead of a single one) at c(28) ± c(29) characteristic of 63a and other polyenes 64 ± 69. the heptaene group of the aglycone imparts rigidity to the antibiotic structure and improves antifungal activity. new monosubstituted polyene macrolides at the terminal 16-co 2 h group of the aglycone were synthesized based on compounds 63a and 64a; monosubstituted polyene macrolides at the mycosamine 3 h -amino group were prepared based on 63a, 64a and 65a. 47, 48 the synthesis of doubly modified analogues of antibiotics 63a and 64a was reported. 48 the related amido derivatives (64b ± 64h) substituted in a similar way at the c(16)-carboxamide group of s44hp were prepared. 46 ± 49 the yields of c(16)-amido derivatives were *50% ± 90% depending on the structure of the starting amine and the antibiotic. the antibiotic structure was found to have almost no effect on antifungal activity against the fungal strains candida albicans (atcc 14053), cryptococcus humicolus (atcc 9949), aspergillus niger (atcc 16404) and fusarium oxysporum (vkm f-140). the activity in each pair of the derivatives containing the same substituents is almost the same in magnitude. carboxamides 63b,d,g and 64b,e,i were found to be the most effective against the above-mentioned strains (mic 50 & 0.5 ± 2 mg ml 71 ). the major directions of chemical modification of s44hp (64a) and bsg005 (65a) at the mycosamine 3 h -amino group in alkylation and aminoacylation reactions, the aminocontaining reagents are protected by the 9-fluorenylmethoxycarbonyl (fmoc) group. conventional 3 h -n-alkyl derivatives of polyenes, such as the 3 h -n-(4-dimethylaminobenzyl)-substituted compounds s44hp (64l) and bsg005 (65c) and the n,n-di(aminopropyl)-substituted compounds s44hp (64m) and bsg005 (65d), were synthesized by reductive alkylation of compounds 64a and 65a with appropriate aldehydes in the presence of nacnbh 3 (see scheme 19, conditions b and c). 47 the yields of compounds 64m and 65d with respect to the starting antibiotics are *12% ± 20%. 3 h -n-acyl derivatives 64n and 65e were synthesized by the reaction of antibiotics 64a and 65a with n a ,n e -(fmoc) 2 -l-lysine in the presence of pybop in *71% ± 74% yields (see scheme 19, conditions d ). the removal of the fmoc group from intermediate derivatives 64o,p and 65f,g with a 5% piperidine solution in dmso 47 affords the corresponding 3 h -n-aminoacyl derivatives 64m,n and 65d,e in *11% ± 20% yields. the evaluation of the influence of substituents in the terminal group at the c(16) atom of the aglycone and the mycosamine amino group on the antifungal activity of polyenes showed that the replacement of the co 2 h group by me has no significant effect on antifungal activity against the tested strains. 48 the activity of s44hp (64a) and its analogue 64k is similar to that of bsg005 (65c) and its analogue 65b. thus, the effect of the same modifications on the activity of the initial antibiotics s44hp and bsg005 with very close values of antifungal activity can be multidirectional. the mic 50 values are changed in the following series: 64a = 65a, 64k = 65b, 64l > 65c, 64m > 65d, 64n < 65e. (77) . the removal of the fmoc protecting group from compound 72a gave 3 h -n-(l-lysyl)-s44hp n-(3-dimethylaminopropyl)amide (72b). 49 an alternative scheme involves the initial synthesis of fmoc-protected 3 h -n-aminoacyl derivatives of s44hp followed by their transformation into the corresponding c(16)-carboxamides (scheme 21). the reaction of compound 64a with n-fmoc-4-aminomethylbenzoic acid in the presence of pybop affords 3 h -n-(n-fmoc-4-aminomethylbenzoyl)-s44hp (78) . the amidation of the latter with appropriate amines in the presence of pybop gives 3 h -n-(n-fmoc-4-aminomethylbenzoyl)-s44hp dmae-amide (79a) and 3-hydroxypropylamide (80a). known compound 64p, which was prepared by the reaction of 64a with n a ,n e -(fmoc) 2 -l-lys in the presence of dcc and hobt, was used to synthesize n-(2-dimethylaminoethyl)amide (81a) and 3-hydroxypropylamide (82a) of n a ,n e -(fmoc) 2 -l-lysyl-s44hp. the removal of the fmoc group from compounds 79a ± 82a under mild conditions gave target products 79b ± 82b containing free amino groups (see scheme 21) . 49 the evaluation of antifungal activity of doubly modified s44hp derivatives 70 ± 82 against the above-mentioned four fungal strains compared to the corresponding monomodified s44hp c(16)-carboxamides 64b,c demonstrated that the additional modification of the mycosamine 3 h -amino group of carboxamide 64b has no significant effect on antifungal activity against these fungal and yeast strains (mic *0.5 ± 2 mg ml 71 ). meanwhile, the corresponding modifications of s44hp 3-hydroxypropylamide lead to a considerable decrease in antifungal activity. in the series of doubly modified derivatives, s44hp 2-n,n-dimethylethylamides (73 and 74, respectively), prepared via the amadori rearrangement with d-glucose or d-galactose, exhibit the highest activity, similar to that of the starting antibiotics 63a and 64a. 49 experiments in animals play a significant role in the selection of lead antifungal agents. since only rather toxic amb (63a) is used for the treatment of systemic fungal infections, compounds exhibiting the highest in vitro activity are currently tested for the haemolysis and(or) acute toxicity. new genetically engineered polyene macrolides 64a, 65a, 66a and 68a, the semi-synthetic derivatives dmae-s44hp (64b), 3 h -n-lys-bsg005 (65e) and doubly modified 3 h -n-(1deoxy-d-fructos-1-yl)-s44hp dmae (73) were evaluated for antifungal activity in the treatment of candida albicansinduced sepsis in mice and tested for toxicity. 47, 49 the largest margin between the therapeutic and toxic doses was observed for compounds 64b and 73. compounds, the effective dose was 2% and 6% of the maximum tolerated dose (mtd), respectively, whereas amb (63a) is effective only at a dose of 62% of mtd. the antifungal activity of c(16)-methyl-c(16)-decarboxypolyenes against four fungal strains changes in the following order: bsg005 [c(7), c(10)] (65a) > bsg019 [c(7)] (67) > bsg018 [c(7), c(9)] (69) . 48 this confirms the pattern of changes in the activity against c. albicans observed previously in the series of c(16)-carboxy-containing antibiotics with a similar arrangement of hydroxyl groups at c(7)7c(10): s44hp (64a) > bsg022 (66) [c(7)] > bsg003 (68a) [c(7), c(9)]. 50 the following amides were synthesized from polyenes 64a, 66a and 68a and 2-(n,n-dimethylamino)ethylamine according to the conventional amidation method in the presence of pybop: dmae-s44hp (64b), dmae-bsg022 (66b) and dmae-bsg003 (68b) (see scheme 17) . 47, 48 the activity of these compounds, like that of the starting antibiotics, changes in a similar series: dmae-s44hp (64b) > dmae-bsg022 (66b) > dmpe-bsg003 (68b). it is worth noting that low antifungal activity of compounds 66b and 68b was confirmed also by animal experiments related to the treatment of murine candida sepsis. these studies clearly demonstrated that the c(7)7c(10) group of the polyol moiety plays a critical role in antifungal activity, although this group has not previously been considered of importance in the model of antibiotic binding to the target. compounds containing a single hydroxyl group at the 7 position of c(7)7c(10) (66a and 67) exhibit low activity against the tested fungal strains. polyenes containing two hydroxyl groups at the 7 and 9 positions (68a and 69) are inactive. 48 antibiotics and semi-synthetic derivatives containing hydroxyl groups at the 8 and 9 positions (amb, 63a,b) and at the c(7) and c(10) atoms [s44hp (64a,b) and bsg005 (65a,b)] displayed the best results. the design of hybrid analogues of antibiotics containing pharmacophore moieties, which affect targets different from those used by the starting antibiotics, is a promising line of research. 28, 29 some benzoxaborole-containing compounds exhibit pronounced antifungal activity. 51 methods were developed for the synthesis of dual-action antibiotics based on amb (63a) and different types of benzoxaboroles. the following five types of conjugates were synthesized depending on the nature of the functional group in benzoxaborole, which can be used to attach the latter to the amb molecule: c (16) 3 h -n-sulfo derivative 87 and 3 h -n-mono-and 3 h -n,n-dialkyl derivatives 88a and 89 (scheme 22). 52 amide 83 was produced by the standard procedure that was applied to prepare the above-described amides; however, the yield of 83 was low (10%). apparently, the amidation interferes with the formation of the aminoborole complex with amb. as mentioned above, the yields of amb carboxamides in this reaction using other amines are higher than 50%. 3 h -n-derivatives 84a ± 86a were prepared from amb (63a) using in situ generated osu-activated esters. 3 h -n-sulfo derivative 87 was synthesized by the reaction of 63a with the appropriate sulfochloride in the presence of pyridine (py); 3 h -n-alkyl analogues 88a and 89 were prepared by the reaction of 63a with the appropriate aldehyde in the presence of nabh 3 cn. 52 the amidation of derivatives 84a and 87a with n,n-dimethylethylenediamine in the presence of pybop gave 84b and 87b, respectively (scheme 23). an attempt to synthesize disubstituted 3 h -n-alkyl-dmae analogue 88b according to a similar scheme from 3 h -n-alkylamino derivative 88a failed (see scheme 22) . 50 nevertheless, compound 88b was prepared using an alternative approach by the alkylation of c(16)-amide derivative 63b with 1-hydroxy-1,3-dihydrobenzo[c] [1, 2] oxaborole-6-carbaldehyde in the presence of nabh 3 cn (see scheme 23). 52 tandem mass spectrometry (esi-ms/ms-mrm) studies showed that different fragmentation patterns are possible depending on the modification of the starting polyene (scheme 24). 52 in many cases, the introduction of the benzoxaborole substituent leads to a decrease in cytotoxicity and haemolytic activity with retention of high antifungal activity. these facts were confirmed by membrane activity assays. 53 the results are given in table 1 . semi-synthetic amb derivatives 63b,d,e, 84a,b, 85a, 87b and 88a,b were shown to have a significant pore-forming ability in artificially formed sterol-containing membranes. 53 compounds with high antifungal activity and low haemolysis have higher selectivity for ergosterol-containing fungal membranes (c chol /c erg ) versus cholesterol-containing human cell membranes compared to compound 63a. for example, the high selectivity (5.4 ae 1.0) of compound 84b correlates with low haemolysis of human erythrocytes (6%). on the contrary, high haemolysis (47%) is determined by low or even reverse selectivity (c chol /c erg = 0.5 ae 0.2). for amb (63a) and its derivative 63b, the corresponding parameters have intermediate values (c chol /c erg = 2.3 ± 2.4, haemolysis 15% ± 23%). 53 amphotericin b (63a) was selectively modified at the mycosamine 3 h -amino group or the c(16) carboxyl group. the developed approaches were extended to the related polyenes s44hp (64a), bsg005 (65) and other genetically engineered polyenes, which differ from the starting amb by the substituent at the c(16) atom and the positions of hydroxyl groups at c(7) ± c(10) of the aglycone moiety. the structure ± activity relationship analysis of new semisynthetic derivatives of polyene macrocycles revealed several general features of antifungal activity. in particular, antibiotics and, consequently, their semi-synthetic analogues containing two hydroxyl groups in this region at the 8 and 9 positions (amb, 63a) or the 7 and 10 positions (s44hp and bsg005) exhibit high activity. a series of new semi-synthetic derivatives were shown to have pore-forming ability in artificially formed sterol-containing membranes. it is worth noting that they have selective activity against ergosterol-containing fungal membranes and lower haemolysis compared to amphotericin b. the aim of chemical modifications of the antibiotic oligomycin a (90a) (fig. 6 ) acting as the f 0 f 1 -atp synthase inhibitor is to prepare new analogues possessing selective antitumour or anti-infective activity and to elucidate the mechanisms of sensitivity of microorganisms to this agent. oligomycin a (90a) (hereinafter oligomycin) belongs to macrolides ð 26-membered a,b-unsubstituted macrolactones. it is a highly specific inhibitor of oxidative phosphorylation in the mitochondria of eukaryotes. oligomycin inhibits adenosine triphosphate (atp) synthesis and causes cell death. note. cerg is the minimum concentration of the compound that causes the pore formation in the dphpc/erg bilayer; cchol/cerg is the ratio of the minimum concentrations of the polyene in the dphpc/chol and dphpc/erg bilayers (dphpc is 1,2-diphytanoyl-sn-glycero-3-phosphocholine, chol is cholesterol, erg is ergosterol). the oligomycin molecule (90a) was found to contain a functional group, the chemical modification of which can produce the largest number of semi-synthetic analogues with various biological activities. the docking study of the interaction between the antibiotic and the target of the enzyme f 0 f 1 -atp synthase showed that the c(32)7c (34) side chain is not directly involved in the formation of this complex. 54 a series of 33-substituted oligomycin derivatives were synthesized using 33-deoxy-o-mesyl oligomycin (91) as the key compound, which was prepared by the selective treatment of 90a with methanesulfonyl chloride in a dmap ± py mixture (scheme 25). 55 the nucleophilic substitution of the group r via the s n 2 or s n 1 mechanism using various reagents gave the following 33-substituted derivatives: 33-(s)-oligomycin a (90b), 56 33-deoxy-33-(s)-thiocyanooligomycin (92), 57 33-deoxy-33-(r,s)-bromooligomycin (93) 58 and 33-deoxy-33-(s)-azidooligomycin (94) 55 (see scheme 25) . the racemization is observed only for 33bromo derivative 93. the 33-epimer of this antibiotic, 33-(s)-oligomycin a (90b), was synthesized by the solvolysis of 33-(r)-deoxy-omesyl oligomycin (91) with an aqueous mixture of tiourea and methyl cellosolve on heating. the reaction involves the walden inversion of configuration at the c(33) atom through a plausible mechanism presented in scheme 25. the biological activity assay of compound 90b revealed that the inversion of the hydroxyl group decreases activity against the actinobacteria streptomyces fradiae, while the antifungal activity remains at the same level. 33-(s)-oligomycin a (90b) exhibits somewhat higher activity against tumour cells compared to the starting analogue 90a. both antibiotics are able to overcome different drug resistance phenotypes and have low toxicity to non-malignant cells. 56 the treatment of oligomycin 91 with 98% formic acid afforded c(33)-o-formyloligomycin (95) 59 (see scheme 25) . formylated derivative 95 retains the ability to inhibit tumour cell growth, whereas the activity against most other test cultures, including non-malignant cells, decreases. due to selectivity against tumour cells, c(33)-o-formyloligomycin (95) holds promise for further investigation. 33-deoxy-o-mesyl oligomycin (91) can be quantitatively converted into 33-dehydrooligomycin (96) by the kornblum oxidation in a dmso ± et 3 n mixture at 105 8c (see scheme 25) . 54 attempts to oxidize the oh group at the c(33) atom of oligomycin (90a) to the keto group using different oxidizing agents failed. the cited study is interesting because this structure was previously presented as a new natural compound. however, the structure of this compound was not described in detail and its biological activity was not evaluated. derivative 96 exhibits twice lower activity against s. fradiae atcc-19609 compared to com pound 90a; its activity against candida spp. and other filamentous fungi is very similar to that of compound 90a. docking studies of the binding of derivative 96 to f 0 f 1 -atp synthase also showed that the affinity for the enzyme decreases compared to the starting antibiotic 90a. 54 a method for the synthesis of 1,4-disubstituted 1,2,3triazoles of oligomycin was developed based on 33-azido-33-deoxyoligomycin 94. the method involves the regioselective [3+2]-dipolar cycloaddition of the 33-azido group to monosubstituted alkynes (scheme 26). 55 the reaction of azide 94 with alkynes (phenylacetylene, propiolic acid and methyl propiolate) in a tert-butanol ± water mixture (1 : 1) can be performed both in the presence of a catalyst (cu i ) or without catalysts. this approach was applied to synthesize 33-deoxy-33-(4-phenyltriazol-1-yl)oligomycin (97), 33-deoxy-33-(4-methoxycarbonyltriazol-1-yl)oligomycin (98) and 33-deoxy-33-(4-carboxytriazol-1-yl)oligomycin (99) in 53%, 52% and 50% yields, respectively. compound 99 served as the starting compound for the synthesis of water-soluble 33-deoxy-33-[(4-dmae-carbonyl)triazol-1yl]oligomycin amide (100) exhibiting selective antitumour activity. 55 oligomycin 90a undergoes a retro-aldol rearrangement accompanied by dehydration in the presence of bases (scheme 27). 60 the structure of alkaline degradation product 101a was established by the detailed 1 h and 13 c nmr study, including heteronuclear correlation, combined with tandem mass spectrometry (esi-ms/ms-mrm). the structure of the carbon skeleton of the starting antibiotic 90a undergoes a significant transformation at c(7)7c(12) (the cleavage pathways a, b and c) giving open-ring compound 101a. the mechanism of alkaline degradation of oligomycin 90a presented in scheme 27 accounts for the formation of derivative 101a (through intermediates 90c,d) but not for the formation of 101b (through intermediates 90e,f). 61 as opposed to compound 90a, compound 101a does not exhibit activity against proteasomal f 0 f 1 -atp synthase at a concentration of 1 mmol l 71 because of the loss of conformational rigidity. the hydrogenation of oligomycin (90a) on a palladium catalyst occurs both at the 2,3-unsaturated bond of lactone and the diene system at c (16) the hydrogenation of compound 90a under other conditions leads to the sequential selective reduction of keto groups ð first at the c(7) atom and then at the c(11) atom. thus, the use of nabh(oac) 3 latter with nabh 4 in ethanol afforded (7s,11r)-tetrahydrooligomycin (104) (see scheme 28) . 59 the hydrogenation of double bonds of the macrolactone ring causes a decrease in the activity of compound 90a both against actinobacteria and fungal and mammalian cells. 62 this may be attributed to the loss of conformational rigidity and the fact that the geometry of the macrocycle favourable for the interaction with the target (f 0 f 1 -atp synthase) is changed because of destruction of the diene system of the starting antibiotic 90a. the retention of activity of the starting compound 102 against some strains of candida spp. supports the previous suggestion that there are additional targets in yeast cells of this genus, the binding to which is apparently independent of the geometry of the macrocycle. the reaction of compound 90a with m-chloroperoxybenzoic acid (m-cpba) in dichloromethane on decreasing the reaction temperature to 717 8c allows the selective epoxidation of oligomycin at one of c = c bonds to form unstable intermediate epoxide 105 (scheme 29). 59 in the presence of formic acid, the latter compound gives a stable disubstituted oligomycin derivative ð 16,17-dihydro-16s,17s-dihydroxy-16,33-diformyloligomycin (106). previously, 16-bromo derivative 107 of this antibiotic was synthesized using n-bromosuccinimide as the brominating agent (see scheme 29). 63 the addition of hydroxylamine and related compounds to 90a was studied. 64 the comprehensive study of the resulting compound by 1 h and 13 c nmr spectroscopy, including heteronuclear correlation, made it possible to establish the structure of nitrone 108a and exclude the possible existence of isomer 108b. it was found that an additional ring involving the c(3) ± c(7) atoms is formed, the activity of the antibiotic against f 0 f 1 -atp synthase being reduced. the reaction of antibiotic 90a with aminopyridinium and 4-dimethylaminopyridinium iodides in pyridine affords cyclic derivatives of pyrazolo [1,5-a] pyridine and 4-methylpyrazolo[1,5-a]pyridine 109a,b, respectively, annulated to the macrocycle (see scheme 30) . 64 their structures were established by 1 h and 13 c nmr spectroscopy, including heteronuclear correlation, and structure 109c was excluded. biological assays of new derivatives of oligomycin (90a) demonstrated that, in most cases, modifications reduce biological activity of the starting antibiotic against s. fradiae. 54, 65 ± 67 the growth inhibition assay of the strain s. fradiae atcc 19609 sensitive to very low concentrations of oligomycin (<0.001 nmol ml 71 or 0.0005 nmol per disc surface) and hypersensitive to most of the known antibiotics was used to study the mechanism of resistance of microorganisms. 66 mutant strains of this microorganism sensitive to analogues of compounds 92 (ref. 67 ) and 108a, as opposed to the starting antibiotic 90a and other derivatives, were produced under experimental conditions. the results of assays demonstrated that antibiotic 90a has several biotargets. these data confirm the conclusion that both the diene system of the macrocycle and its hydroxypropyl side chain play an important role in biological activity. 65 therefore, the chemical modification of the antibiotic oligomycin (90a), a highly active f 0 f 1 -atp synthase inhibitor, was performed for the first time. more than 20 new semi-synthetic oligomycin derivatives were prepared and the mechanism of their action was studied. analogues with selective antitumour or anti-infective activity were synthesized. the chemical modification of antibiotic 90a, which enables the efficient modulation of its biological activity due to a decrease in the binding to f 0 f 1 -atp synthase, was found. this provides the possibility to optimize pharmacological properties. olivomycin a (110) (hereinafter olivomycin) is a highly effective antibiotic with a unique mechanism of antitumour activity discovered in the gause institute of new antibiotics 6, 68, 69 (fig. 7) . its structure consists of the aglycone olivin and two carbohydrate chains attached to the aglycone at the 2 and 6 positions. the antibiotic is a dna duplex minor groove ligand, which binds to the guanine-cytosine (gc)-rich region. compared to other antibiotics of the aureolic acid group (mithramycin and chromomycin a), olivomycin 110 has better therapeutic efficacy. in recent years, antibiotics of this group have attracted increasing interest. 70, 71 antibiotics of this group were shown to be able to prevent the development of resistance of tumour cells to other antitumour agents, in particular via a mechanism involving the inhibition of transcription of the mdr1 gene, overexpression of which is responsible for multidrug resistance of tumour cells. mithramycin is used to treat paget's disease and testicular carcinoma. chromomycin is a drug of limited use in japan for the treatment of gastrointestinal cancer. olivomycin (110) was applied in the ussr for the treatment of ovarian cancer, reticulosarcoma and some other tumours. however, serious adverse effects limit the therapeutic potential of these drugs. various aspects of antitumour activity of antibiotics of the aureolic acid group were addressed in detail; however, chemical modifications of this class of antibiotics are poorly known. selective modifications of olivomycin (110) at the c(5) atom (a), the c(8)7oh (b) and c(2 h ) = o groups (c), the c(2 h )7c(3 h ) bond (d ) and the residues a4 and e4 (e) (see fig. 7 ) were developed in the gause institute of new antibiotics. the selective acylation of compound 110 at the phenolic hydroxyl group at the c(8) atom with a ac 2 o7py mixture affords 8-o-acetylolivomycin (111) (scheme 31). 72 an investigation of the reaction of olivomycin (110) with aryldiazonium salts showed that the azo coupling gives aryldiazenes monosubstituted at the 5 position of the aglycone accompanied by the elimination of the disaccharide branch at the c(6) atom: 5-(phenyldiazenyl)olivomycin (112), 5-(4-sulfamidophenyldiazenyl)olivomycin (113), 5-(4methoxyphenyldiazenyl)olivomycin (114), 5-(3,4-dichlorophenyldiazenyl)olivomycin (115) and 5-(4-methylphenyldiazenyl)olivomycin (116) (see scheme 31) . 72 to explain the outcome of this reaction, the frontier electron density in the highest occupied molecular orbital (homo) was calculated by the semiempirical quantum chemical am1 method (fukui indices f). alternative directions of the nucleophilic attack by the phenyldiazonium cation were chosen by considering possible anionic forms of olivomycin (110a ± 110c) (scheme 32 a). it was found that the 5 position in anion 110a is the most favourable for electrophilic attack giving compound 112 (the fukui index f homo is 0.617). meanwhile, the nucleophilicities of the c(7) and c(10) atoms are lower (0.356 and 0.185, respectively). the formation of anion 110a in an alkaline medium is confirmed by the above-mentioned selective acylation of compound 110 giving acetate 111 at the same hydroxyl group at the c(8) atom in high yield (see scheme 32) . 72 the calculated energy parameters of alkaline hydrolysis of the disaccharide branch are in agreement with the experimental data. thus, the hydrolysis proceeds very rapidly in the presence of the diazenyl substituent at the 5 position, while the storage of 110 in an alkaline medium (used for the azo coupling) on cooling to 0 ± 5 8c for 2 h in the absence of diazonium salt does not lead to hydrolysis. the azo coupling of aryldiazonium salts with the aglycone of olivomycin ð olivin (117), which was synthesized by quantitative acid hydrolysis of compound 110, was investigated. aryldiazenyl derivatives of olivin 117a ± c containing the same substituents as compounds 112, 114 and 115 (scheme 33) were synthesized. 70 the geometric configurations of the most probable tautomeric structures a ± d of derivative 117a were determined (scheme 34) and the total energies of each tautomeric form were calculated by the density functional theory at the b3lyp/6-31g(d) level of theory. the 1 h nmr spectroscopic data also indicate that compounds 117a ± c exist as equilibrium mixtures of isomeric forms a ± d. the reaction of olivomycin (110) with (o-carboxymethyl)hydroxylamine affords 2 h -(carboxymethoxime)olivomycin (cm) 118 (scheme 35). 74 the introduction of the carboxyl group into molecule 110 makes it possible to subject this compound to further modification. the reaction of compound 118 with appro75 the introduction of the carboxyl group into molecule 110, as in the case of long acid 118, provides a route to its further modification. the reaction of short osa 124 with appropriate amines in the presence of pybop or diphenylphosphoryl azide (dppa) affords osa n-methylamide it is worth noting that the method developed for the synthesis of intermediate osa (124) can be applied to prepare the related derivative of mithramycin bearing a similar side chain at the c(3) atom of the aglycone. hence, short' mithramycin acid becomes more available compared to the combinatorial biosynthesis and can be used for further chemical modification of mithramycin. 76 olivomycin a (110) has two carbohydrate chains bearing the acetyl group at the a4 position of the oliose moiety and the isobutyryl group at the e4 position of the olivomycose moiety. a series of analogues of compound 110, which differ in the set of functional groups, were synthesized in order to elucidate the influence of acyl substituents in carbohydrate chains on biological activity. 77 two analogues, de-e4-isobutyrylolivomycin a (133) and de-e4-isobutyryl-de-a4-acetylolivomycin a (or de-e4isobutyrylolivomycin c) (134), were synthesized by selective alkaline hydrolysis of the e4-isobutyryl group of olivomycin a (110) or c (135), respectively (scheme 37). it is worth noting that the hydrolysis of the e4-isobutyryl group in olivomycin (110) antibiotics 135 (de-e4-isobutyrylolivomycin a) and 136 (de-e4-isobutyryl-e4-acetyl-olivomycin a or olivomycin b) were isolated from the natural olivomycin complex produced by fermentation of the streptoverticillum cinnamoneum strain. after the purification by silica gel column chromatography and semipreparative hplc, compounds 135 and 136 were isolated in 40% and 45% yields, respectively. 70, 77 these compounds are identical to the previously characterized natural olivomycins c and b, respectively. 78 the modification of antibiotic 110 at the c(8) phenol group of the aromatic ring (compound 111) was found to have no effect on antiproliferative activity against cancer cell lines and does not alter its ability to inhibit topoisomerase i. 72 the transformation of 110 giving diazenyl derivatives accompanied by elimination of the disaccharide branch from the aglycone (compounds 112 ± 116) leads to a sharp decrease in antiproliferative activity. compared to the starting olivomycin 110, compounds 112, 115 and 116 acquire considerable selective activity against human immunodeficiency viruses hiv-1 and hiv-2 in assays in the human t-lymphocyte cell line (cem). 72 the evaluation of antiproliferative activity of olivomycin (110) and its analogues modified at the side-chain 2 h -keto group of the aglycone (cm amides 119 ± 121 and 123) against the leukaemia cell lines k562 and l1210 showed that these compounds are more effective than the starting acid 118 but are less active than compound 110. 74 for derivatives of antibiotic 110 with a shorter aglycone side chain (osa, 124) and its amides 125 ± 132, the evaluation of antiproliferative activity in the human chronic myeloid leukaemia cell line k562 and the human colon 75 the evaluation of antiproliferative activity of olivomycin analogues 133 ± 136 against hct116 cells showed that acyl groups in carbohydrate chains of antibiotic 110 play a significant role. the activity decreases with elimination of acyl groups from molecule 110; ic 50 is 0.02 (for 110), 0.064 (for 136), 0.28 (for 133) and >50 mmol l 71 (for compounds 117, 134 and 135). 77 these results correlate with the data on the ability of these compounds to inhibit dna-dependent topoisomerase i. 79 in the absence of antibiotics, the relaxation of supercoiled (sc) dna leads to the disappearance of inhibitory activity and the formation of a set of topoisomers. the inhibition of topoisomerase i is detected from the presence of residual amounts of sc-dna and a decrease in the amount of rapidly migrating topoisomers. a decrease in the inhibitory activity of the enzyme is consistent with a decrease in the antiproliferative activity in the series of compounds 110 > 136 > 133. the removal of the e4-isobutyryl group from the trisaccharide branch has a lower effect than the removal of the a4-acetyl group from the disaccharide branch. a similar pattern of activity is observed in another group of olivomycin derivatives. compound 118 displays low antiproliferative activity and is inactive against topoisomerase i. 2 h -(carboxymethoxime)olivomycin n-(2-adamantyl)amide (121) is active in both assays. 2 h -(carboxymethoxime)olivomycin n-(tert-butyl)amide (122) and 2 h -(carboxymethoxime)olivomycin n-(1,3-dihydroxy-2-methylpropan-2-yl)amide (123) are even more effective inhibitors of the enzyme, capable of inhibiting sc-dna relaxation even at concentrations of 0.1 mmol l 71 . 74 the data on antiproliferative activity of some analogues of antibiotic 110 do not correlate with the activity against topoisomerase i. for example, the analogue of olivomycin osa (124), which exhibits weak antiproliferative activity, displays inhibitory activity against topoisomerase i similar to that of olivomycin a (110), whereas dmae-osa (127) possessing high antiproliferative activity is a weaker inhibitor of this enzyme. 74, 79 recently, it was shown that highly effective analogue 127 can act as a dna duplex minor groove ligand in another assay. it can disrupt the key epigenetic dna methylation process with the dnmt3a enzyme on an equal basis with antibiotic 110. both compounds inhibit the formation of the dna ± enzyme7intermediate covalent bond, required for the methylation, at nearly equal micromolar concentrations. 80 olivomycin complexes with model oligonucleotide ± dna duplexes were studied by circular dichroism (cd) and fluorescence titration (ft). according to hartree ± fock 3-21g calculations of the 3d structure of the dimer [110] 2 mg 2+ , the presence of the dmae group in compound 127 leads to an increase in the binding constant (k a ) of the mg 2+ complex with the dna duplex by an order of magnitude compared to the acid osa (124) (k a = 1.35610 5 versus 2.1610 4 mol l 71 , as evaluated by ft). however, the presence of the bulky adamantyl substituent in compound 121 results in a decrease in the binding constant of the dimer [121] 2 mg 2+ with the dna duplex by an order of magnitude (k a = 1.32610 4 mol l 71 ). 81 the elimination of one acyl substituent also leads to a decrease in the antiproliferative activity compared to that of antibiotic 110 due probably to a decrease in its affinity for dna. 77 the molecular docking of complex of 110 with dna shows that the antibiotic can bind only to gc-rich regions in the minor groove of the dna duplex. 82 carbohydrate chains of olivomycin interact with the sugar ± phosphate backbone of dna, and the aglycone interacts with nucleic acid bases. the sites of 110 responsible for the interaction with dna (an additional hydrogen bond with the nh 2 group of the g base) and the complexation of the antibiotic with dna were identified. the structural fragment, which is not directly involved in the interaction with dna but models the affinity of the antibiotic to the target ð the minor groove of the dna duplex, was determined. based on these data, a schematic model of the interaction between olivomycin a and dna was proposed (scheme 38). 73 two semi-synthetic olivomycin derivatives (121 and 127) with the modified aglycone side chain were chosen based on the evaluation of antiproliferative activity in animal assays. compound 121 is more effective in the treatment of p388 murine leukaemia than the starting antibiotic 110 due to a decrease in toxicity and the absence of the cumulative effect. 83, 84 compound 127 (olivamide) is also more effective in this assay compared to the starting antibiotic 110. 85 detailed preclinical assays in transplanted syngeneic tumours confirmed the efficacy of the agent based on compound 127 for further clinical trials. 85 ± 88 these studies enabled the development of methods for selective chemical modification of the antibiotic olivomycin a, resulting in the synthesis of a series of semi-synthetic derivatives of this antibiotic; besides, structure ± activity relationship analysis was performed. 70, 73 many of these compounds exhibited high antiproliferative activity in different tumour cell lines. some aspects of the mechanism of action of olivomycin a and its natural and semi-synthetic analogues were considered. 74, 75, 77, 79 ± 82 it was concluded that high antitumour activity of these compounds is related to their high affinity to the dna duplex. based on the results of in vitro assays, compounds were chosen for the evaluation of antitumour activity in in vivo assays. after the preclinical evaluation of antitumour activity and toxicity in laboratory animals, semi-synthetic olivomycin analogue 127 ð olivamide ð was recommended for further clinical trials. 85 ± 89 anthracycline antibiotics are important chemotherapeutic agents commonly used in the treatment of malignant tumours. daunorubicin (137a), which was independently discovered under the name of rubomycin in the gause institute of new antibiotics (gina), 6 is applied mainly for the treatment of leukaemia in children and adults. doxorubicin (14-hydroxydaunorubicin, dox) (138a) is used in combination chemotherapy of breast cancer, smallcell lung cancer, sarcoma, tumours in children and haemoblastosis. an original method for the one-pot preparation of semi-synthetic doxorubicin from rubomycin was developed in the gina. 6 carminomycin (139a), which was also discovered in the gina, has lower cardiotoxicity than other anthracyclines and can inhibit the growth of doxorubicin-resistant tumours (138a). 90 the structures of anthracycline antibiotics 137a, 138a and 139a are shown in fig. 8 . the mechanism of cytotoxic activity of anthracycline antibiotics is related mainly to the inhibition of nucleic acid synthesis via intercalation between nitrogeneous base pairs, dna damage and inhibition of dna topoisomerases. an adverse effect of anthracyclines is the potentially irreversible and cumulative dose-related cardiotoxicity, apparently associated with the free-radical damage of myocardial cell membranes. the chemical modification of anthracycline antibiotics was extensively studied in the gina for a long period of time. in recent years, efforts were focused on the synthesis of new semi-synthetic analogues of drugs with improved anticancer properties. 91 daunorubicim (137a) and carminomycin (139a) were modified at the c(14) atom (b), the c(13) = o bond (c) and the daunosamine 3 h -amino group (d, e) (see fig. 8 ). 3 h -n-alkyl or 3 h -n-aminoacyl derivatives exhibit high antitumour activity, because they retain the amine function at the daunosamine moiety. 90 this function is required for the primary interaction of the antibiotic with the sugar ± phosphate backbone of dna. however, the drawback of reductive alkylation of anthracyclines 137a and 139a with aldehydes in the presence of nabh 3 cn is that it is accompanied by the reduction of the c(13) = o group as the side reaction giving n-alkyl-13-dihydro derivatives of daunorubicin (137b) and carminomycin (139b), respectively (scheme 39). these analogues are less active than the related compounds containing the c(13) = o group. 92 ± 94 13-dimethyl ketals of 14-bromo derivatives of daunorubicin (137c) and carminomycin (139c) were used to avoid this side reaction (see scheme 39) . the reductive alkylation of compounds 137c and 139c in the presence of nabh 3 cn can be accomplished using d,lglyceraldehyde and aldoses (the monosaccharide arabinose and the disaccharide melibiose) to form the following 14bromo-substituted 13-dimethyl ketals in quantitative yields: the antiproliferative activity of compounds 139e and 143 against leukaemia cells k562 is virtually as high as that of derivative 137e, but is lower than that of carminomycin (139a). the activity of 14-hydroxycarminomycin derivative 143 evaluated in the same assay is an order of magnitude lower than that of the starting anthracycline 139a, and the activity of doxorubicin 137e is two orders of magnitude lower than that of compound 138a. it should be emphasized that 14-hydroxycarminomycin derivatives 142 and 143 are equally active against the wildtype human breast adenocarcinoma cell lines mcf-7 and k562 and resistant pgp-expressing cell lines (mcf-7dox, k562i/s9). the antitumour activity of compound 139e evaluated in the murine leukaemia cell line p388 compares favourably with that of analogue 138a. thus, an increase in life span (ils) of mice bearing p388 leukaemia after a single admin-istration of 10 mg kg 71 of compound 139e is the same as that observed after administration of analogue 138a at a dose of 8 mg kg 71 (ils 108%). 92 it was shown that anthracycline antibiotics 137a, 138a and 139a, which are known to inhibit dna topoisomerase ii at micromolar concentrations, can inhibit dna topoisomerase i at the same concentrations. 75 new derivatives 137e, 139e, 142 and 143 have higher inhibitory activity against topoisomerase i compared to compounds 138a and 139a. the introduction of polyhydroxylated substituents at the 3 h -amino group of the daunosamine moiety of anthracycline antibiotics increases the inhibitory activity of anthracyclines against topoisomerase a method was developed for the preparation of new watersoluble depot forms of doxorubicin (138a) ð conjugates with high-molecular-mass polysaccharides. 95, 96 galactomannan davanat (144) was used as the polysaccharide. the latter was prepared by controlled partial hydrolysis of water-insoluble high-molecular-mass 1,4-b-d-galactomannan isolated from seeds of cyamopsis tetragonoloba or guar gum. the molecular mass of compound 144, determined by gel chromatography on a sephadex g-200 column calibrated with pullulans, is *92 kda. to conjugate compound 144 with 138a, the former is activated by the oxidation with periodate using a deficient amount of the oxidizing agent (0.07 ± 0.11 equiv. with respect to the total amount of sugar residues). this gives rise to schiff bases between the aldehyde groups of the oxidized polysaccharide (145a ± 145c) and the 3 h -amino group of the antibiotic (scheme 40). different structures of the final products are possible (146a ± 146c). the content of the starting antibiotic 138a in conjugate 146 is 5 mass % (determined from the content of the chromophore per unit mass of the dried powder by uv spectroscopy at 490 nm). in order to increase the doxorubicin content in the conjugate with davanat 145, the antibiotic molecule was attached to the polysaccharide through a spacer giving 3 h -n-l-lysyldoxorubicin (147) (scheme 41). 96 compound 147 contains two amino groups (while doxorubicin contains one amino group), which may facilitate the formation of the schiff base. besides, it is known that a series of n-acyl conjugates of daunorubicin or doxorubicin with amino acids possess high antitumour activity. 90 3 h -n-l-lysyldoxorubicin (147) was synthesized by the acylation of the amino group of doxorubicin (138a) with n a ,n e -(fmoc) 2 -l-lysine osu-ester followed by the deprotection of the fmoc-protected intermediate with a morpholine solution. 96 the schiff base in the resulting conjugate can have either a linear (148a) or cyclic structure (148b). scheme 41 presents the possible structures for one of the oxidized subunits of activated davanat 145. it should be noted that 3 h -n-l-lysyldoxorubicin can be attached to conjugate 145 through either the a-amino-or e-amino group of 3 h -n-l-lysyldoxorubicin (is not shown in scheme 41). the introduction of the lysine spacer between compounds 138a and 145 allows an increase in the antibiotic content in the conjugate to 10 mass % with retention of water solubility. the conjugates were purified by gel chromatography on a sephadex g-200 column and dialysis against deionized water using a membrane with molecular weight cut-off (mwco) m w > 15 kda. the molecular masses of conjugates 146 and 148 evaluated by gel chromatography on a sephadex g-200 column calibrated with pullulan standards are * 95 and * 98 kda, respectively. the antiproliferative activity of the doxorubicin conjugates was tested in three tumour cell lines: the murine melanoma cell line b16-f1, the breast cancer cell line mcf-7 and the colon cancer cell line ht-29 (htb-38). 96 the ic 50 values for conjugate 146 (taking into account the percentage of antibiotic 138a in the conjugate) are 0.025 ± 0.04, 0.15 ± 0.22 and 0.65 ± 1 mg ml 71 , respectively; for antibiotic 138a, 0.01 ± 0.02, 0.08 ± 0.12 and 0.2 ± 0.3 mg ml 71 . despite the fact that the cytotoxicity of conjugate 146 is *1 ± 2 orders of magnitude lower (data were not reported) than that of doxorubicin (138a), these results indicate that conjugate 146 is an active depot form of doxorubicin (138a). the antiproliferative activity of the 3 h -n-l-lysyldoxorubicin ± davanat conjugate (148) (ic 50 > 50 mg ml 71 ) against these tumour cell lines is much lower compared to cytotoxicity of conjugate 146. this can be due to the fact that the imine bonds in the 3 h -n-l-lysyldoxorubicin ± da-vanat conjugate (148) are more stable than those in conjugate 146. it should be noted that 3 h -n-l-lysyldoxorubicin is not released in in vitro assays. therefore, a biological model, which would provide release of 147, is apparently required to evaluate the therapeutic potential of conjugate 148. based on these studies, a method was developed for the introduction of polyhydroxylated substituents of different types and different length into anthracycline antibiotics, which was used to synthesize a series of new hydrophilic 3 h -n-alkyl derivatives of doxorubicin and 14-hydroxycarminomycin, including those containing mono-and disaccharide residues. 92, 93 this modification was found to enhance the inhibitory activity of anthracyclines against topoisomerase i with retention of antitumour activity of the antibiotics. the new 14-hydroxycarminomycin derivatives, unlike doxorubicin derivatives, were shown to suppress the tumour cell growth insensitive to doxorubicin. 93 a new water-soluble depot form of doxorubicin ð a conjugate with the highmolecular-mass polysaccharide galactomannan dava-nat ð was prepared. 94, 95 this depot form exhibits antiproliferative activity against the above-mentioned three tumour cell lines. 91 the antibiotic heliomycin (resistomycin, 3,5,7,10-tetrahydroxy-1,1,9-trimethyl-1h-benzo[cd ]pyrene-2,6-dione) (149) with a broad spectrum of biological activity (scheme 42) was discovered in the gause institute of new antibiotics. 6 first of all, this antibiotic is highly effective against grampositive and some gram-negative microorganisms, including drug-resistant strains. more recently, heliomycin was found to exhibit antifungal 97 and antiviral (anti-hiv) activity. 98 besides, compound 149 can block proliferation of some tumour cells in in vitro assays. 99 in the soviet union, heliomycin (149) was produced on a commercial scale and was used as a gel for topical treatment of skin infections and healing of burn wounds. the chemical modification of heliomycin is poorly known. hence, structure ± activity relationship studies require the development of method for the synthesis of new semi-synthetic derivatives and evaluation of their biological properties. the main goal is to prepare series of derivatives with improved water solubility in order to expand their practical application. a method was developed for the synthesis of aminomethyl derivatives 149 at the 4 position of compounds 150 (where x is an amine moiety or a nitrogen-containing heterocycle). typical synthetic procedures are presented in relation to compounds 150a ± c (see scheme 42) . 100, 101 the mannich aminomethylation of compound 149 can be performed using amines and formaldehyde in dmf (see scheme 42 b ). an alternative procedure for the synthesis of derivatives 150 is based on the use of pre-prepared stable iminium salts (see scheme 42, conditions a). in the case of aminomethylation of compounds 149 with polyfunctional amines, the amino group is protected with boc (see scheme 42 c) and then deprotected with acid (see scheme 42 d ). the reaction of compound 149 with n,n-dimethylamino(methylene)ammonium chloride afforded 4-[(n,n-dimethylamino)methyl]-3,5,7,10-tetrahydroxy-1,1,9-trimethyl-1-h-benzo[cd ]pyrene-2,6-dione hydrochloride (150a). 100 4-[(tert-butylamino)methyl]-3,5,7,10-tetrahydroxy-1,1,9-trimethyl-1h-benzo[cd ]pyrene-2,6-dione hydrochloride (150b) is produced by the treatment of compound 149 with tertbutylmethylamine in the presence of formaldehyde. the treatment of compound 149 with tert-butylpiperidin-4-ylcarbamate in the presence of a formaldehyde solution gives 4-(4-boc-aminopiperidinomethyl)-3,5,7,10-tetrahydroxy-1,1,9-trimethyl-1h-benzo[cd ]pyrene-2,6-dione, which is quantitatively converted to the corresponding amine dihydrochloride (150c) upon the treatment with hcl7meoh. the antiproliferative activity (ic 50 ) was evaluated by the colorimetric determination of cell metabolic activity (mtt assay) using a standard procedure in eight tumour cell lines, including both drug-sensitive and drug-resistant cell lines. 100 the resulting compounds inhibit tumour cell proliferation in a low submicromolar to micromolar concentration range, similar to that of doxorubicin (138a). however, most of these compounds significantly outperform doxorubicin in terms of the drug-resistance index. these compounds block the growth of the wild-type cell lines k562 and hct116 and the following multidrugresistant cell lines: the k562/4 subline isogenic to p-glycoprotein (pgp)-positive multidrug resistance and the hct116p53ko subline with p53 gene deletion. the development of multidrug resistance in these tumour cell lines is the crucial factor responsible for a decrease in activity of antitumour drugs. as opposed to compound 149, derivatives 150a ± c show a high level of induced apoptosis in the t24 bladder cancer cell line model. the introduction of the 4-aminomethyl moiety enhances the dna-binding affinity and the inhibitory activity against dna topoisomerase i. hence, compound 150c is the most promising candidate for preclinical trials. based on these studies, methods were developed for chemical modification of heliomycin (149). series of new analogues, water-soluble salts of amino-containing derivatives, were synthesized and they were shown to exhibit high antiproliferative activity against many tumour cell lines and inhibitory activity against various targets. 100 the value of these studies is that the majority of aminomethyl derivatives of heliomycin are active against both wild-type and drugresistant cancer cells at micromolar or submicromolar concentrations. the development of new-generation drugs is a challenging problem because of the increasing risk of the development of drug resistance in microorganisms. different approaches to the search for new compounds were developed in recent years. particular attention is given to semi-synthetic derivatives based on available natural antibiotics, since there are numerous examples of the successful application of this approach. some semi-synthetic derivatives based on macrocyclic glycopeptides have advantages over the gold standard chemotherapeutic agent ð vancomycin. 3, 7, 10 the distinguishing features of new representatives of this class are selectivity against multidrug-resistant pathogenic bacteria and higher bioavailability. the new semi-synthetic vancomycin analogue telavancin (vibativ) (manufactured by theravance and astellas pharma, us) was approved for use in the united states and europe. two semi-synthetic derivatives of glycopeptide antibiotics have completed phase 3 clinical trials and were approved by the united state food and drug administration (fda): chloroeremomycin (discovered by eli lilly, acquired by the medicine co in 2009) and dalbavancin (discovered by lepetit; acquired by pfizer in 2005). 8, 10 the drawbacks of vancomycin are the poor accumulation within tissues, because of which it is not used in the treatment of, for example, pneumonia, and a pronounced pseudoallergic reaction typical of glycopeptides. research in international cooperation showed the promise of chemical transformations of the antibiotic eremomycin belonging to this group of glycopeptide antibiotics. eremomycin is a highly active domestic antibiotic that suppresses the growth of gram-positive organisms; it is 3 ± 5 times more effective than vancomycin but is inactive against drug-resistant strains of staphylococci and enterococci. 6 original approaches and methods were developed under the supervision of professor m.n.preobrazhenskaya in the gause institute of new antibiotics. these methods can be applied to prepare derivatives of antibiotics of this group with desired properties. in 2006, the method for the synthesis of glycopeptide analogues was protected by an international patent. 18 promising analogues, various n h -derivatives and carboxamides of eremomycin, were synthesized. these derivatives compare favourably in efficacy against drug-sensitive and drug-resistant strains of staphylococci and enterococci with the related vancomycin derivatives and they are even more effective in a number of assays. 12 ± 15 some carboxamides show no allergenicity. 13 other derivatives (e.g., the adamantane derivative of eremomycin) are promising as a protection against biological risks because they were found to be active against the bacterium bacillus anthracis, including fluoroquinolone-resistant strains. 15 high activity of the resulting hydrophobic glycopeptide derivatives can be attributed to the dual mechanism of action on gram-positive bacteria. 20, 21 modifications of aglycones of glycopeptide antibiotics with hydrophobic substituents resulted in the discovery of a new class of polycyclic peptides active against a large group of enveloped viruses, such as hiv 23 or hepatitis c virus. 25 studies on the mechanisms of antiviral activity are currently ongoing. it was suggested that protein kinase is one of possible targets for this agluco analogue, because antiviral activity was shown to correlate with the inhibition of serine/ threonine protein kinases. 27 comprehensive research on the synthesis and characterization of heterodimeric conjugates based on different classes of antibiotics was initiated in the gause institute of new antibiotics under the supervision of professor m.n.preobrazhenskaya. the following hybrid antibiotics were synthesized: glycopeptide ± macrolide, glycopeptide ± aminoglycoside and hybrid compounds containing the benzoxaborole chromophore. the literature data provide evidence that this line of research holds promise. 28, 29 macrolides modified through the carbamoyl group at the 4 hh position can acquire activity against drug-resistant bacterial cell lines. generally, conjugates containing a long spacer exhibit higher activity than the related compounds with a shorter spacer. 40, 41 investigations of interactions between different benzoxaboroles and antibiotics made a significant contribution to the chemistry of not only antibiotics with complex structures but also the relatively poorly studied borole compounds. benzoxaboroles were found to be quite stable under different reaction conditions. in particular, the acylation, amidation and reductive alkylation with reactive agents and on heating are virtually not accompanied by oxaborole ring opening. 32 the introduction of the benzoxaborole substituent into the polyene macrolide amphotericin b (amb) also enhances biological activity. the resulting compounds possess valuable properties, such as lower cytotoxic and haemolytic activity compared to amb combined with high antifungal activity. 52 the efficiency of the approach to the design of antibiotics of a new generation was demonstrated in relation to antifungal polyene macrolides. it is based on a combination of genetic engineering techniques and methods of organic synthesis 48 ± 50 and is protected by an international patent. 46 the chemical modification of polyene antibiotics, which were obtained via the genetic engineering of nystatin a1 biosynthesis at the norwegian university, gave a series of agents exhibiting lower toxicity and higher activity compared to amphotericin b in animal assays. the dependence of antifungal activity on the structure of the polyol region [c(7) ± c(10)] of these antibiotics was revealed for the first time by preobrazhenskaya et al. 49 pioneering studies on the development of methods for selective chemical modification of the unique macrolactone oligomycin a, a specific f 0 f 1 -atp synthase inhibitor, and the original antitumour antibiotic olivomycin a of the aureolic acid group were performed. in both cases, examples of chemical modifications of related compounds for these antibiotics are absent in the literature. these results are of great scientific value. investigations of f 0 f 1 -atp synthase inhibitors are of considerable interest because this enzyme is involved in the development of resistance in microorganisms. chemical transformations of oligomycin a using different reagents were studied in detail. alkaline hydrolysis reactions were performed and conditions for the selective reduction of double bonds and keto groups, cyclization, etc. were found. the replacement of the c(33)-hydroxyl group by the activated mesyl group has proved to be particularly successful. 55 this modification has no significant effect on biological activity of the antibiotic but provides a possibility to optimize its pharmacological properties. 65, 67 only transformations via the biosynthesis were described for the aureolic acid group antibiotics mithramycin and chromomycin. 70, 76 these natural antibiotics, which have a limited use in the treatment of some malignant tumours, have attracted increasing attention of researchers in different fields. 71, 73 the synthesized compounds were tested for antiproliferative activity. based on the results of in vitro studies of a series of new olivomycin a analogues of different types, compounds were selected for the in vivo evaluation. after preclinical trials in laboratory animals (evaluatrion of antitumour activity and toxicity), one olivomycin a analogue was recommended for further clinical trials. 87 ± 89 a systemic approach applied to series of new semisynthetic analogues of olivomycin a allows a detailed study of the molecular mechanism of antitumour action of this antibiotic. 79 ± 81 in particular, it was shown that olivomycin and its analogues, acting as dna duplex minor groove ligands, can inhibit topoisomerase i and the dnmt3a enzyme responsible for the disruption of the key epigenetic dna methylation process. 79, 80 the structural fragments of olivomycin a critical for antitumour activity were identified. a model of the interaction of the antibiotic and some its analogues with the dna duplex was proposed. 82 new analogues of anthracycline antibiotics with substituents of different types and different length, including those containing mono-and disaccharide residues, were prepared. the evaluation of the effect of polyhydroxylated moieties of doxorubicin and 14-hydroxycarminomycin on antitumour activity showed that this modification does not lead to the loss of activity of anthracyclines 93 ± 95 and enhances inhibitory activity against topoisomerase i. 92 14-hydroxycarminomycin derivatives proved to be particularly valuable because these compounds, as opposed to related doxorubicin derivatives, inhibit the proliferation of both doxorubicin-sensitive and doxorubicin-resistant tumour cell lines. 96 a new water-soluble depot form of doxorubicin with the high-molecular-mass polysaccharide galactomannan davanat was constructed and it was shown that it exhibits antiproliferative activity against three tumour cell lines. 94, 95 series of new heliomycin analogues ð water-soluble salts of amino derivatives ð were synthesized and these compounds were shown to have high antiproliferative activity against many tumour cell lines and inhibitory activity against different targets. 100 the value of these studies is that the majority of aminomethyl derivatives of heliomycin are active against both wild-type and drugresistant cancer cells at micromolar or submicromolar concentrations. the method is protected by a patent. 101 targeted chemical modifications of antibiotics are not only performed in order to prepare new potential drugs of a new generation but are used as an efficient tool for investigation of the mechanisms of action on bacterial or tumour cells. it should be emphasized that many potent antibiotics are indispensable tools for molecular biology research of the living world. such comprehensive studies can lead to the design and synthesis of new-generation drugs, which would be more effective than the starting antibiotics and which are of theoretical interest as new compounds with high biological activity. figures 2, 3 , 5 and 6 were composed by the authors based on the cited publications (the references are given in the figure captions). new approaches to natural anticancer drugs. springer briefs in pharmaceutical science and drug development antibiotics: challenges, mechanisms, opportunities markovnikov congress on organic chemistry (book of abstracts) handbook of experimental pharmacology doctoral thesis in chemical sciences, m.v.lomonosov institute of fine chemical technology glycobiology and drug design. (acs symp. ser. 1102) carbohydrates and drug design. (asc symp. ser. 932) key: cord-339227-2i9q9c8u authors: djakpo, odilon; yao, weirong title: rhus chinensis and galla chinensis – folklore to modern evidence: review date: 2010-11-22 journal: phytother res doi: 10.1002/ptr.3215 sha: doc_id: 339227 cord_uid: 2i9q9c8u the species rhus chinensis mill. (anacardiaceae) is an important representative of the genus rhus, which contains over 250 individual species found in temperate and tropical regions worldwide. rhus chinensis has long been used by folk medicine practitioners in asia. leaves, roots, stem, bark, fruit and particularly the galls on rhus chinensis leaves, galla chinensis, are recognized to have preventative and therapeutic effects on different ailments (such as diarrhea, dysentery, rectal and intestinal cancer, diabetes mellitus, sepsis, oral diseases and inflammation). however, it is critical to separate evidence from anecdote. fortunately, recent scientific research has revealed that rhus chinensis compounds possess strong antiviral, antibacterial, anticancer, hepatoprotective, antidiarrheal and antioxidant activities. moreover, compounds isolated from the stem of rhus chinensis significantly suppressed hiv‐1 activity in vitro. compounds from this plant were also found to inhibit enamel demineralization in vitro and enhance remineralization of dental enamel with fluoride. this review highlights claims from traditional and tribal medicinal lore and makes a contemporary summary of phytochemical, biological and pharmacological findings on this plant material. it aims to show that the pharmaceutical potential of this plant deserves closer attention. copyright © 2010 john wiley & sons, ltd. rhus chinensis belongs to the genus rhus and the family anacardiaceae (miller et al., 2001) . commonly called sumac, rhus consists of approximately 250 individual species of fl owering plants, with six species found (four endemics) in china. like most sumacs, rhus chinensis is a dioecious shrub that can reach 8 m in height. it bears odd pinnately compound leaves and creamywhite fl owers. the fruits (drupes) are orange or red in color at maturity and contain one seed (barkley, 1937; miller et al., 2001; tianlu and barfod, 2008) . the species grows in areas with marginal agricultural capacity, and is widely distributed in temperate, subtropical, and tropical regions, including china, japan, malaysia, taiwan and india (rayne and mazza, 2007; ren et al., 2008) . the species rhus chinensis has two distinct varieties, rhus chinensis var. chinensis (syn. rhus (tianlu and barfod, 2008; grin; tropicos) . galla chinensis or galla rhois is the term used to describe the gall caused by the chinese aphid, schlech-tendalia chinensis (bell), on the leaves of rhus chinensis (lee et al., 1997) . this gall is widely used as a separate drug. other species in this genus also produce galls that are considered to have an inferior quality. a plethora of traditional medicine references claim curative power for rhus chinensis, despite its widespread use, many of these claims of effi cacy were not supported by scientifi c evidence, whether for traditional use validation or for drug development endeavors. fortunately, recent scientifi c research on rhus chinensis has revealed promising health benefi ts, including anticancer, antiviral, antimicrobial, antidiarrheal and antiinfl ammatory properties (yang et al., 2005; gu et al., 2007; ahn et al., 1998; chen et al., 2009; kim et al., 2005) . in recent years, the chinese herbal medicine galla chinensis has been discussed widely as a new alternative for carious disease (chu et al., 2007) . so far, no comprehensive review has been compiled from the literature encompassing the effi cacy of this plant. widespread claims of the medicinal effectiveness of various rhus chinensis tree preparations motivated us to bridge the information gap in this area. among rhus species, rhus chinensis and its gall, galla chinensis, have a long history of use by indigenous peoples for medicinal care and others. numerous curative properties are ascribed to different parts of this tree, namely root, bark, stem, leaf, fruit, fl owers, seed and gall ( table 1 ). the leaves and the root are used as depuratives, stimulating blood circulation. its decoction is used in the treatment of hemoptysis, infl ammations, laryngitis, snakebite, stomachache and traumatic fractures (duke and ayensu, 1985; kao, 1988; ouyang et al., 2008) . the ripe fruits of this plant have long been used in asia to treat dysentery and diarrhea, as well as other gastrointestinal disorders (kala, 2005; pradhan and badola, 2008; bose et al., 2008) . the fruit produces a sour juice when boiled with water. this juice, when diluted with water or/and mixed with raw eggs, treats diarrhea and dysentery (pradhan and badola, 2008) . it is used for the treatment of colic (chopra et al., 1986) and also as a food preservative (pradhan and badola, 2008) . the seed is used in the treatment of cough, dysentery, fever, jaundice, malaria and rheumatism (duke and ayensu, 1985; abbasi et al., 2009) . the gall of rhus chinensis has long been considered to possess natural medicinal properties with numerous benefi ts . galla chinensis is used internally for its astringent properties to treat disease such as diarrhea and hemorrhage (duke and ayensu, 1985) . it is a frequent ingredient in polyherbal prescriptions for diabetes mellitus (duke and ayensu, 1985) . it has hemostatic effects, often used to promote clotting following traumatic injuries and to treat burns (yeung, 1985) . it is also used to treat rectal and intestinal cancer, prolapse of the rectum, seminal enuresis and hemorrhoids (yeung, 1985; gao et al., 2000) . in addition to its antiphlogistic and antiseptic uses for treating diseases such as persistent cough, galla chinensis also has antiinfl ammatory properties (tian et al., 2009) . it is also used to counteract ulcers in the mouth and to treat fever and malaria (duke and ayensu, 1985; gao et al., 2000) . phytochemical studies on rhus species have been reported earlier and resulted in the characterization of several compound groups such as fl avonoids (taniguchi et al., 2000; lee et al., 2005; lin et al., 2008) , triterpenoids (kuo, 1991; parveen, 1991; lee et al., 2005) , phenolics (parveen and khan, 1988; lee et al., 2005; ouyang et al., 2008) , tannins (takechi et al., 1985) and aromatic alkanes (kuo et al., 1991; lee et al., 2005; ouyang et al., 2008) . the galls on rhus chinensis leaves are rich in gallotannin (50-70%), a type of hydrolysable tannin (kee and walter, 1999; xiao et al., 2000) . gallotannins from galla chinensis consist of a central glucose core, which is surrounded by several gallic acid units, and further gallic acid units can be attached through depside bonding of additional galloyl residues. structures containing 1 to 14 galloyl residues result from such processes, yielding tri-, tetra-, penta-, hepta-and nonagalloylglucose, and others (xiang et al., 2007; tian et al., 2009b) . pentagalloylglucose [1], 3-galloyl-gallic acid and 4-galloyl-gallic acid isomers isolated from galla chinensis are reported to be the primary bioactive gallotannins, possessing numerous medicinal activities and health benefi ts sakai et al., 1990; bhimani et al., 1993; feldman et al., 2001; . rhus chinensis is rich in well known phenolic compounds, gallic acid [2] and methyl gallate [3] (ahn et al., 1998 (ahn et al., , 2005 bae et al., 1998; choi et al., 2009 ). according to buziashvili et al. (1973) galla chinensis is composed of nearly 20% gallic acid and 7% methyl gallate. a new benzofuranone, 5-hydroxy-3-(propan-2-ylidene)-7-(3,7,11,15-tetramethylhexade-ca-2,6,10, 11-tetraenyl)-2(3h)-benzofuranone [4], together with 16 known bioactive compounds, including 5-hydroxy-7-(3,7,11,15-tetramethylhexadeca-2,6,10,11-tetraenyl)-2(3h)-benzofuranone [5], 3-oxo-6β-hydroxyolean-12en-28-oic acid [6], 3-oxo-6β-hydroxyolean-18-en-28-oic acid [7] moronic acid [8], betulonic acid [9], gallicin [10], dihydroxytoluene [11] and dimethylcaffi c acid [12] , have been isolated from the root stem of rhus chinensis (gu et al., 2007; wang et al., 2008) . phenol glycosides and lariciresinol-based ligan glycosides compounds have been shown to be present in the butanol extract of rhus chinensis root (ouyang et al., 2007 (ouyang et al., , 2008 . 6-pentadecylsalicylic acid, an antithrombotic compound [13] (kuo, 1991) and fi setin (3,7,3-,4-tetrahydroxyfl avone) [14] an antiinfl ammatory, have also been isolated from the stem of rhus chinensis. the leaves of this plant are rich in essential oils, with palmitic acid, phytol and n-heptacosane as the major components (zhu et al., 2007) . the high level of gallotannins along with phenolic compounds, gallic acid and methyl gallate, known antimicrobial agents make galla chinensis very useful in bacterial control (wu-yuan et al., 1988; ahn et al., 1998; kang et al., 2008; tian et al., 2009a tian et al., , 2009b . extracts from galla chinensis inhibited several bacteria such as bacillus subtilis, b. cereus, escherichia coli, enterobacter cloacae, helicobacter pylori, klebsiella oxytoca, lactobacillus casei, l. acidophilus, l. salivarius, salmonella derby, s. minesota, s. typhimurium, s. enteritidis, shigella dysenteriae, staphylococcus aureus, streptococcus mutans, s. sobrinus, ureaplasma urealyticum, with the minimal inhibitory concentration (mic) in the range 0.5-8 mg/ ml (wu-yuan et al., 1988 , bae et al., 1998 choi ii et al., 2002; kang et al., 2008; zhu et al., 2008; choi et al., 2009; tian et al., 2009a) . tian et al. (2009b) reported that different gallotannins from galla chinensis separated according to the number of galloylglucose had signifi cant antibacterial activities on bacillus cereus and salmonella typhimurium. structure activity relationship studies indicated that antibacterial activity was positively correlated with the numbers of galloyl groups and generally, gallotannins with higher molecular weights had strong antibacterial activities (tian et al., 2009a (tian et al., , 2009b . a methanol extract of galla chinensis was shown to have signifi cant growth-inhibitory activity towards harmful intestinal bacteria (ahn et al., 1994 (ahn et al., , 1998 . activity-directed fractionation of the methanol extract of galla chinensis has led to the isolation of gallic acid and its derivative methyl gallate as the major components involved in the observed antimicrobial activity. it was also reported that methyl gallate and gallic acid from galla chinensis had inhibitory effects on periodontopathic bacteria (mic = 1 mg/ml) and signifi cantly reduced the in vitro biofi lm formation of s. mutans (methyl gallate, 1 mg/ml gallic acid, 4 mg/ml, p < 0.05) (kang et al., 2008) . anti-hiv activity. in a recent study, different fractions of rhus chinensis showed potent anti-hiv-1 activity (wang et al., 2006) . subsequent anti-hiv guided fractionation of rhus chinensis led to the isolation of 17 compounds with potent anti-hiv-1 activity (gu et al., 2007; wang et al., 2008) . among those compounds, a new class of benzofuranone-type compounds 5-hydroxy-3-(propan-2-ylidene)-7-(3,7,11,15-tetramethylhexadeca-2,6,10,11-tetraenyl)-2(3h)-benzofuranone [4] and 5-hydroxy-7-(3,7,11,15-tetramethylhexadeca-2,6,10,11-tetraenyl)-2(3h)-benzofuranone [5] were found signifi cantly to suppress hiv-1 replication (gu et al., 2007) . compound [4] possessed signifi cant anti-hiv-1 activity with a therapeutic index (ti) of 42.40, whereas compound [5] showed moderate anti-hiv-1 activity with a ti of 3.28 (gu et al., 2007) . furthermore, the action mechanisms of the two benzofuranonetype compounds were investigated by wang et al. (2008) . these authors found that both compounds [4] and [5] inhibited hiv-1 replication in chronically infected h9 cells and may target late-stages of the hiv-1 life cycle. betulonic acid [9] an analogue of betulinic acid, a well known anti-hiv-1 agent (kashiwada et al., 1996; soler et al., 1996) exhibited moderate anti-hiv-1 activity with a ti value of 5.27-8.94 μm (gu et al., 2007; wang et al., 2008) . 3-oxo-6β-hydroxyolean-12-en-28-oic acid [6], 3-oxo-6β-hydroxyolean-18-en-28-oic acid [7] and moronic acid [8] are oleanolic acid-related triterpenes previously reported to have potential anti-hiv-1 activity (pengsuparp et al., 1994; soler et al., 1996; kashiwada et al., 1998) . these compounds showed weak anti-hiv activity with ti values of 4.14, 4.74 and 8.22, respectively (gu et al., 2007; wang et al., 2008) . mengoni et al. (2002) described the anti-hiv and the mechanism of action for oleanolic acid, both of which suggested that oleanolic acid inhibits hiv-1 protease activity in vitro. gallicins [10], gallic acid derivate-type compounds, have been reported to inhibit hiv-1 integrase . the work of wang et al. (2008) confi rmed the result in cell lines with a therapeutic index of 5.11. dihydroxytoluene [11] had the same extent of anti-hiv-1 activity with a ti of 5.34. wang and coworkers also showed that dimethylcaffi c acid [12] , caffeic acid phenylethyl ester derivate, has potent anti-hiv-1 activity with a ti value of 19.07. these values are relatively low compared with the control azt (ti > 471883) but the resistance and the adverse side effects to available conventional anti-hiv drugs beg the need of identifi cation and development of additional small-molecule inhibitors that can be used in combination with currently available antiviral agents. in vivo studies performed in mice have shown that the hot-water extract of rhus chinensis had prophylactic and therapeutic efficacy against herpes simplex virus (hsv) type 1 (hsv-1) (kurokawa et al., 1993 (kurokawa et al., , 1995a (kurokawa et al., , 1995b (kurokawa et al., , 1997 . this extract was also effective against acyclovir-resistant hsv-1 and hsv type 2 (hsv-2) infections in mice (kurokawa et al., 1995b) and improved the therapeutic effi cacy of acyclovir in mice infected with hsv-1 (kurokawa et al., 1995a) . subsequently, nakano et al. (1998) also investigated the effi cacy of rhus chinensis extract in vivo, using a guinea-pig primarily infected intravaginally with hsv-2. prophylactic oral administration of rhus chinensis at the dose corresponding to human use signifi cantly reduced the incidence and severity of spontaneous skin lesions compared with latently infected guinea-pigs administered water. when recurrent hsv-2 infection was induced by ultraviolet irradiation 3 months after primary infection, prophylaxis with rhus chinensis was also signifi cantly effective in reducing the severity of ultraviolet-induced skin lesions. two terpene compounds, moronic acid [8] and betulonic acid [9], were separated from rhus chinensis and their subsequent anti-hsv activities were assessed in vitro and in vivo . the effective concentrations of moronic acid and betulonic acid for 50% plaque reduction for hsv-1 were consecutively, 3.9 and 2.6 μg/ml. the therapeutic index of moronic acid (10.3-16.3) was larger than that of betulonic acid o. djakpo and w. yao (6.2). oral administration of moronic acid thrice a day to mice infected cutaneously with hsv-1, signifi cantly retarded the development of skin lesions and/or prolonged the mean survival of infected mice without toxicity compared with the control. moronic acid exerted stronger anti-hsv-1 activity in the brain of hsv-1-infected mice than in the skin, similar to the hotwater extract of rhus chinensis (kurokawa et al., 1995a . anti-hcv and anti-sars-cov activities. screening a library of traditional medicines, duan et al. (2004) found that the etoac extract fraction from galla chinensis was effi cient in inhibiting the ns3 protease activity of hepatitis carcinoma virus (hcv). 1,2,6-tri-o-galloyl-βd-glucose, 1,2,3,6-tetra-o-galloyl-β-d-glucose and pentagalloylglucose [1] were identifi ed as the active compounds. tri-, tetra-and pentagalloylglucose inhibited hcv ns3 protease with ic 50 of 1.89, 0.75 and 1.60 μm, respectively (duan et al., 2004) . likewise, tetra-o-galloyl-β-d-glucose isolated from galla chinensis exhibited prominent inhibition against severe acute respiratory syndrome coronavirus (sars-cov) with a 50% effective concentration of 4.5 μm (yi et al., 2004) . liu et al. (2003) found that crude aqueous extract of galla chinensis has the ability to inhibit enamel demineralization in vitro. in another study, chu et al. (2007) evaluated the effects of compounds from galla chinensis on the remineralization of initial enamel carious lesions using an in vitro ph cycling model. the group demonstrated the potential of three different fractions of galla chinensis to affect net rehardening of artifi cial carious lesions under dynamic ph-cyclic conditions. furthermore, zou et al. (2008) , using the same protocol, demonstrated the potential of galla chinensis extract to inhibit the demineralization of initial enamel carious lesions. the chemical compounds of galla chinensis showed effects and combined effects with fl uoride on enhancing remineralization of dental enamel (cheng et al., 2008) . at this point, the active compound of galla chinensis involved in remineralization or demineralization is still unknown. chu et al. (2007) isolated gallic acid [2] and methyl gallate [3], both of which showed poor activity compared with the crude extract. this result was confi rmed by cheng et al. (2008) testing the combined effects of galla chinensis extract or gallic acid with fl uoride on remineralization of artifi cial early enamel caries. they found that both the crude extract of galla chinensis and gallic acid had synergistic effects with fl uoride on remineralization, but with apparent differing mechanisms. thus, it seemed that gallic acid was not the only possible active constituent of galla chinensis to enhance remineralization. zou et al. (2008) similarly attempted to determine which of the constituent chemical fractions of galla chinensis conferred a potential anticaries benefi t by comparing the effects of four different fractions of galla chinensis on demineralization of a bovine enamel model. the crude extract was the most active one, prone to some losses of other active compounds during the separation process. cai et al. (2004) screened 112 chinese medicinal plants for antioxidant activity; the results showed that the aqueous extract of galla chinensis contained the highest antioxidant concentration of 17674 μmol teac/100 g. more recently, two similar studies have investigated the antioxidant activity of gallotannins in four different systems, namely 1,1-diphenyl-2-picrylhydrazyl (dpph) radical scavenging, ferric reducing antioxidant power, β-carotene linoleic acid system and hydroxyl radical scavenging assays. tian et al. (2009a) tested the antioxidant activity of gallotannins with different polarities and found that all of the consecutive extracts of galla chinensis possessed remarkable antioxidant activity. for example, dpph radical scavenging activity, ec 50 were in the sequence ethyl acetate (1.22 μg/ml) > ether (1.44 μg/ml) > ethanol (1.55 μg/ml) > water (2.11 μg/ml). generally, all fractions showed better capacity to scavenge free radicals than the controls, bht and trolox. the same results trend was observed with ferric reducing activity. antioxidant activity increased when the polarity of extracts decreased, suggesting that extracts with weaker polarities contained higher molecular weight tannins, and thus had stronger antioxidant effects. aware of this fi nding, tian et al. (2009b) isolated different gallotannins, containing 1-10 galloylglucoses (gg), from galla chinensis and investigated their antioxidant activities in the above systems. generally, gallotannins of high degrees of galloylation (5-10 ggs) had stronger antioxidant activities than those of low degrees of galloylation (1-4 ggs).the same conclusion was drawn in earlier work by yokozawa et al. (1998) . similarly, methyl gallate and gallic acid have been shown through in vivo and in vitro studies to have antioxidant and radical scavenging activity (chen and zhang, 2003; whang et al., 2005; madsen and bertelsen, 1995; peyrat-maillard et al., 2000) . it has been demonstrated that pentagalloylglucose possesses antioxidant activity and protects rat neuronal cells from oxidative damage feldman et al., 2001; oh et al., 2001; pan et al., 1999) . piao et al. (2009) showed that pentagalloylglucose exerts antiapoptotic activity through antioxidant properties. yang et al. (2005) were the fi rst to report the anticancer activity of rhus chinensis extract on carcinogenic cdc25 phosphatases. several molecules found in rhus chinensis such as pentagalloylglucose and gallic acid have been shown to have anticancer activity (bhimani et al., 1993; madsen and bertelsen, 1995; chung et al., 1998; hu et al., 2008; kuo et al., 2009) . pentagalloylglucose has been shown to exhibit in vivo anticancer effects against prostate cancer (hu et al., 2008; kuo et al., 2009) , lung cancer (huh et al., 2005) and sarcoma (miyamoto et al., 1987) , and in vitro inhibitory effects on the growth and/ or invasion of breast cancer, leukemia, melanoma and liver cancer . pentagalloylglucose can exert anticancer activity via the inhibition of angiogenesis (lee et al., 2004; huh et al., 2005) and invasion of melanoma cells in metastasis (ho et al., 2002) . in vitro studies showed that pentagalloylglucose signifi cantly inhibited the proliferation and tube formation of bfgfphytother. res. 24: 1739-1747 (2010) treated human umbilical vein endothelial cells (huvec) with an ic 50 of 8 μm (huh et al., 2005) . the result is similar to the in vitro antiangiogenic activity of pentagalloylglucose in vegf-treated huvecs (lee et al., 2004) . daily injection of 4 and 20 mg/kg of pentagalloylglucose signifi cantly inhibited the growth of the highly angiogenesis-dependent lewis lung cancer allograft by 57% and 91%, respectively (huh et al., 2005) . similarly, pentagalloylglucose inhibited the invasion of highly metastatic mouse melanoma b16f10 cells in vitro in a dose-and time-dependent manner, with ic 50 of 15 μm (ho et al., 2002) . some other investigations have also demonstrated that derivatives of galloylglucose inhibit not only cancer cell growth (pan et al., 1999; hu et al., 2008) but also the invasion of ht1080 human fi brosarcoma cells (ata et al., 1996) . several studies of natural hepatoprotective agents have revealed that the extract of galla chinensis showed promising hepatoprotective activity tian et al., 2005) . based on an activity-guided separation scheme an et al. (2005) purifi ed pentagalloylglucose [1] and an equilibrium mixture of 3-galloyl-gallic acid and 4-galloyl-gallic acid isomer from the methanol extract of galla chinensis and validated their hepatoprotective activity. pentagalloylglucose [1] and the mixture compounds were found to have marked protective effects on tacrine-induced cytotoxicity in human liver-derived hep g2 cells with ec 50 values of 70.39 ± 5.4 and 29.51 ± 0.7 μm, respectively, and also inhibited nitrofurantoininduced cytotoxicity in hep g2 cells at 150.9 ± 6.4 and 23.81 ± 0.5 μm respectively. furthermore, pentagalloylglucose treatment was able to reduce both hepatocyte necrosis induced by tertbutyl hydroperoxide (4 and 20 μm) and apoptosis induced by glycochenodeoxycholic acid (3.125 to 50 μm) in primary rat hepatocytes (park et al., 2008) . diabetes mellitus is a chronic metabolic disorder characterized by high blood glucose level due to agents such as α-glucosidase enzyme, which boosts the digestion of carbohydrate to monosaccharides in the process of intestinal absorption. therefore, shim et al. (2003) tested the inhibitory effect of an aqueous extract from the gall of rhus chinensis on α-glucosidase activity in in vitro and in vivo models. galla chinensis inhibited bacillus α-glucosidase activity with an ic 50 of 0.9 μg/ml. its inhibition on α-glucosidase was determined to be noncompetitive and reversible when the enzyme-substrate mixture was simultaneously treated with galla chinensis. galla chinensis signifi cantly suppressed the increase of blood glucose level in rats after oral administration of sucrose. these results suggest that galla chinensis might exert antidiabetic effects by suppressing carbohydrate absorption from the intestine and thereby reducing the postprandial increase in the blood glucose. likewise, tannic acid, a mixture of gallotannins containing pentagalloylglucose, was found to have a hypo-glycemic effect in patients with type 2 diabetes (gin et al., 1999) . aware of this result, li and coworkers hypothesized that pentagalloylglucose could have antidiabetic activity. using synthetic pentagalloylglucose in vitro and in an animal assay, it was demonstrated that pentagalloylglucose effectively reduced blood glucose and insulin levels in vitro and in animal models (li et al., 2005) . unlike most antidiabetic drugs, pentagalloylglucose may reduce blood glucose without increasing adiposity. the methanol extract of the dried ripe fruit of rhus chinensis was tested in experimental models of castor oil-induced diarrhea in swiss albino mice (tangpu and yadav, 2004; bose et al., 2008) . at graded doses, the extract showed remarkable antidiarrheal activity evidenced by an 80.70% reduction in the rate of defecation of control animals at a dose of 600 mg/kg body weight. the extract also reduced intestinal fl uid secretion induced by mgso 4 and gastrointestinal motility after charcoal meal administration in albino mice (tangpu and yadav, 2004) . in the same way, galla chinensis extracts were found to be effective against enterotoxigenic escherichia coliinduced diarrhea that produces a heat-labile enterotoxin (lt), which binds to the ganglioside g m1 on the surface of intestinal epithelial cells (holmgren and svennerholm, 1992) leading to a massive loss of fl uids and ions from cells (chen et al., 2009) . using the patent mouse gut assay in vivo study, chen et al. (2006) found that galla chinensis extract exhibited an anti-lt-induced diarrheal effect, with an ic 50 value of 4.7 ± 1.3 mg/ml. competitive gm1-elisa assay showed that galla chinensis suppressed (ic 50 = 0.17 ± 0.02 mg/ml) ltinduced fl uid accumulation by blocking the binding of ltb to g m1 . thin layer chromatography suggests that the most active fraction that inhibited the binding of ltb to gm1 was composed of mainly phenolics, especially gallic acid which signifi cantly blocked the binding of ltb to g m1 , with an ic 50 value of 10.9 ± 0.3 mm, and suppressed the lt-induced fl uid accumulation in a dose-dependent manner, with an ic 50 value of 25.4 ± 11.6 mm. the work of kim et al. (2005) showed that galla chinensis had antiinfl ammatory activity in in vivo and in vitro models. galla chinensis could control all of the infl ammatory mediators, such as histamine, heparin, lipidderived mediators and various cytokines in the model of immediate-type allergic reaction in a dose-dependent manner through different mechanisms. latter activityguided fractionation and purifi cation of the etoac fractions of the galla chinensis indicated that the main antiallergic component in galla chinensis was gallic acid. fisetin a fl avonoid found in the root of rhus chinensis (lin et al., 2008) was also found to down-regulate infl ammatory reactions in stimulated human mast cells . similarly, pentagalloylglucose has been shown through in vivo and in vitro studies to exercise a strong antiinfl ammatory effect (oh et al., 2004; lee et al., 2007 lee et al., , 2003 kang et al., 2005) . 6-pentadecylsalicylic acid has been isolated from airdried stems of rhus chinensis by bioassay-directed fractionation of the n-hexane extract of the stem (kuo, 1991) . this compound showed antithrombotic activity at 50 μg/ml using the amidolytic method (kuo, 1991) . it also prolonged clotting time in a dose-dependent manner in the clotting assay of thrombin-fi brinogen interaction. rhus chinensis species have long been recognized by folk medicine practitioners as having value and have been revealed to have great medicinal potential, much of which was completely unknown to western scientists. over the past few decades, the research efforts on rhus chinensis extracts indicate that the extracts have promising potential as antiviral, anticarie, antidiarrheal, anticancer, antidiabetic and hepatoprotective agents, among others. although the work reviewed here substantiated most of the traditional claims on its health effectiveness, more research is required for validation of the uses of this plant. the available information on the different bioactive contents in samples of various parts of this medicinal plant is very limited, both qualitatively and quantitatively. the gall on rhus chinensis leaves has received much scientifi c attention because of its high gallotannin content and subsequent health potential. however, other parts, such as fruits, leaves, and seeds, can also be investigated based on traditional uses and the fi ndings in other rhus species. , 5-hydroxy-3-(propan-2-ylidene)-7-(3,7,11,15-tetramethylhexade-ca-2,6,10,11-tetraenyl)-2(3h)-benzofuranone [4], 5-hydroxy-7-(3,7,11,15-tetramethylhexadeca-2,6,10,11-tetraenyl)-2(3h)-benzofuranone [5], 3-oxo-6β-hydroxyolean-12-en-28-oic acid [6], 3-oxo-6β-hydroxyolean-18-en-28-oic acid [ so far, galla chinensis is the only medicine proven to remineralize a hard tissue like enamel. this is a unique potential for this plant, but the active constituent is still unknown. the mechanistic activity of rhus chinensis material as prophylactic, therapeutic, anti-hsv, anti-hiv and antidiarrheal medicine needs to be further examined. on the other hand, efforts should also be made to survey other sumac species to determine if these properties are generalized across the rhus genus. the safety of rhus chinensis still needs to be rigorously established, since cases of toxicity from intake of gallotannins found in rhus chinensis have been reported in the literature. different gallotannins such as tri-, tetra-, hexa-, hepta-, octa-, nona-and decagalloylglucose can reduce blood pressure and blood urea nitrogen, as reported in animal studies in the literature (feldman et al., 1999; hofmann et al., 2006; nishizawa et al., 1983) . nonetheless, tannins diminish protein digestibility when present in high levels in diets with low protein content and also inhibit human salivary α-amylase, thereby causing potential negative effects on starch digestion and food taste. this needs to be taken in account when testing the effi cacy of rhus chinensis compounds in human beings by clinical trials for drug use validation. 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effects on urea-nitrogen concentration in rat serum penta-o-galloyl-β-dglucose inhibits phorbol myristate acetate-induced interleukin-8 [correction of intereukin-8] gene expression in human monocytic u937 cells through its inactivation of nuclear factor-kappab in vitro anti-proliferative effect of 1,2,3,4,6-penta-o-galloyl-β-d-glucose on human hepatocellular carcinoma cell line, sk-hep-1 cells sesquiterpenes with hepatoprotective activity from cnidium monnieri on tacrineinduced cytotoxicity in hep g2 cells four new lariciresinol-based lignan glycosides from the roots of rhus javanica var. roxburghiana new phenol glycosides from the roots of rhus javanica var. roxburghiana induction of apoptosis by penta-o-galloyl-beta-d-glucose through activation of caspase-3 in human leukemia hl-60 cells anti-infl ammatory activity of fi setin in human mast cells (hmc-1) ,6-penta-o-galloyl-β-d-glucose from galla rhois protects primary rat hepatocytes from necrosis and apoptosis phenolic constituents 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matrix-assisted laser desorption/ionization time of fl ight mass spectrum of chinese gallotannins chinese medicinal herb iconograph i chemistry of traditional chinese medicine. shanghai science and technology publishing house screening the active constituents of chinese medicinal herbs as potent inhibitors of cdc25 tyrosine phosphatase, an activator of the mitosisinducing p34cdc2 kinase handbook of chinese herbs and formulas. institute of chinese medicine small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells study on the inhibitory effect of tannins and fl avonoids against the 1,1-diphenyl-2-picrylhydrazyl radical anti-cancer, anti-diabetic and other pharmacologic and biological activities of penta-galloyl-glucose chemical variation in leaf essential oils of rhus chinensis from eight locations in southern and eastern china microdilution inhibition test of chinese herbs to assess their effect against clinical strains of ureaplasma urealyticum in vitro chinese materia medica effect of galla chinensis extract and chemical fractions on demineralization of bovine enamel in vitro the authors have declared that there is no confl ict of interest. key: cord-294897-29diwyoe authors: pina-pérez, m. c.; rivas, a.; martínez, a.; rodrigo, d. title: antimicrobial potential of macro and microalgae against pathogenic and spoilage microorganisms in food date: 2017-11-15 journal: food chemistry doi: 10.1016/j.foodchem.2017.05.033 sha: doc_id: 294897 cord_uid: 29diwyoe abstract algae are a valuable and never-failing source of bioactive compounds. the increasing efforts to use ingredients that are as natural as possible in the formulation of innovative products has given rise to the introduction of macro and microalgae in food industry. to date, scarce information has been published about algae ingredients as antimicrobials in food. the antimicrobial potential of algae is highly dependent on: (i) type, brown algae being the most effective against foodborne bacteria; (ii) the solvent used in the extraction of bioactive compounds, ethanolic and methanolic extracts being highly effective against gram-positive and gram-negative bacteria; and (iii) the concentration of the extract. the present paper reviews the main antimicrobial potential of algal species and their bioactive compounds in reference and real food matrices. the validation of the algae antimicrobial potential in real food matrices is still a research niche, being meat and bakery products the most studied substrates. and germany as pioneers in research and investment in this area (guedes, barbosa, amaro, pereira, & malcata, 2011) . furthermore, the study of natural bioactive compounds obtained from marine organisms is a relatively new field of research (since the '90s), with enormous scientific potential (cakmak, kaya, & asan-ozusaglam, 2014; mehadi et al., 2015) . given that oceans cover three quarters of the earth's surface, these marine organisms are of potential interest not only as ingredients for the production of food but also as practical and necessary metabolites with medical and technological properties, such as lipids, enzymes, biomass, polymers, toxins, pigments, and clean fuel, which can be produced, concentrated, and successfully isolated from these small but efficient ''marine bioreactors". these organisms are a viable and economical source for the production of these substances, and are in great demand in the nutraceutical, pharmaceutical, chemical, food, and cosmetic industries because of their moisturizing, antioxidant, and regenerative properties (batista, gouveia, bandarra, franco, & raymundo, 2013; devi, suganthy, kesika, & pandian, 2008; rani, singh, & maheshwari, 2013) . the possibility of an efficient and sustainable use of marine resources offers an important way of providing staple food, animal feed, pharmacological products, functional ingredients, and medical solutions for a global population that is rapidly increasing. the term ''algae" comprises a complex and heterogeneous group of photosynthetic organisms characterized by their photosynthetic nature and their simple reproductive structures. the algae group is divided into multicellular organisms, macroalgae or seaweed, and unicellular organisms, known as microalgae (measuring from 1 mm to several cm). macroalgae are often fastgrowing, reaching sizes of up to 60 m in length. a standard classification has been established, dividing seaweed organisms into three groups on the basis of their pigmentation: i) brown seaweed (phaeophyceae); ii) red seaweed (rhodophyceae) and iii) green seaweed (chlorophyceae). seaweeds are mainly used for the production of food and the extraction of hydrocolloids. on the other hand, microalgae are microscopic bodies that normally grow in suspension, some with features characteristic of bacteria. these algae grow in seawater, in the region where light penetrates (photic zone), basically up to a depth of 200 meters. diatoms (bacillariophyceae), green algae (chlorophyceae), and golden algae (chrysophyceae) are the most important microalgae in terms of abundance, but blue-green algae (cyanophyceae) are also classified as microalgae (e.g. spirulina) . more than ten million algal species are estimated to exist nowadays, most of them microalgae, representing a virtually unexplored plant material. so far, only about 50 species have been studied in detail from a physiological and biochemical point of view, in the discipline of ''phycology". the remarkable biodiversity of algae, ranging from species found in the coldest regions of antarctica to those that grow in the hottest deserts, opens up a new research niche, mainly in the baseline of the ''functionality of these marine vegetables". among the various research fields in which macro-and microalgae are appearing, gastronomy and food technology are two of the most important areas. new trends in cooking are emphasizing the use of algae as healthy, tasty, colorful ingredients to accompany the most innovative dishes. france, ireland, canada, and the united states are particularly active in introducing seaweed into local cuisine, and the movement is spreading to cookery books that include recipes with algae as the main ingredient (fao, 2012) . consumers have a good perception of algae as ''natural products", like lettuce, chard, or broccoli, with well-known, accepted health benefits (honkanen, 2009) . humans have certainly been consuming algae for years: pet foods, baby foods, dairy products, instant soups, meat coatings (cooked ham), and many others commodities are clear examples of the presence of seaweed in our daily diet. nowadays, most of these algae ingredients correspond to eu labeled additives no 1333/2008). exciting organoleptic properties (color, flavor, aroma, and taste) are being developed by introducing algae into the formulation of novel products with additional technological functions, including preservative functions (antibacterial, antifungal, antiviral, bacteriostatic) (el shoubaky & el rahman-salem, 2014; sanmukh et al., 2014; tüney, çadirci, ünal, & sukatar, 2006) , structural functions (emulsifying, gelling, and thickening properties attributed to algae) (ursu et al., 2014) , and nutritional properties (vitamins, proteins, polyunsaturated fatty acids) (bishop & zubeck, 2012; el-baky, el baz, & el-baroty, 2008) . spirulina maxima, chlorella vulgaris, haematococcus pluvialis, diacronema vlkianum, and isochrysis galbana are some of the most interesting algae with potential bioactive properties (batista et al., 2013) . all of them are able to accumulate high amounts of bioactive compounds with functional and technological properties. several products, such as pasta, bread, and snacks, are being developed with the incorporation of algae extracts in their formulation, and expectations about this practice are promising for the food industry. in view of the development of antibiotic resistance in bacteria and international trade pressure to achieve a high level of consumer protection, new alternatives to traditional preservatives should be developed and introduced by the food industry, even in products with limited shelf life. the new line of preservatives should also accomplish two further objectives, (i) to preserve the quality and organoleptic value of the product, and (ii) to satisfy consumer demand for natural, functional, ready-to-eat meals. in this connection, marine algae are emerging as a new generation of potential preservatives, macro-and microalgae extracts, or pure ingredients with health benefits and demonstrated antibacterial, antifungal, and antiviral activities (dai & mumper, 2010; devi et al., 2008) . although intensive study of the antimicrobial potential of algae has begun [2005] [2006] [2007] [2008] [2009] [2010] [2011] [2012] [2013] [2014] [2015] [2016] , most of the studies that have been published are about therapeutic and antibacterial/antiviral capabilities of algae compounds, their ability to inhibit or kill clinical bacteria (mehadi et al., 2015; rajeshkumar et al., 2014; rhimou, hassane, josé, & nathalie, 2010) , but not about the effect of these bioactive molecules against foodborne pathogens and spoilage microorganisms commonly found in food matrices. among the major bioactive constituents of algae with demonstrated antimicrobial potential, proteins, polysaccharides, polyunsaturated fatty acids (pufas), especially epa and dha, amino acids, and antioxidants (polyphenols, flavonoids, and carotenoids) are the most important ones (al-saif, abdel-raouf, el-wazanani, & ibrahim, 2014; senthilkumar & sudha, 2012) . however, the identification of compounds directly responsible for the antimicrobial potential of algae is still a relatively incipient field of research, mainly owing to the new kinds of compounds found in recent years . the recent year 2014 was designated as ''protein year", underlining the importance of finding alternative sources of these valuable molecules, proteins of animal and vegetable origin. in this field, algae take a high position among the raw materials proposed as alternative protein sources, together with soy, beans, grains, and, recently, insects . algae protein quality has been considered superior to that of other plant sources, such as wheat, rice, or beans, but poorer than that of animal protein sources, such as milk or meat (mendes, lopes da silva, & reis, 2007) . however, interest in marine proteins might be directly correlated not only with intact protein but also with the possibility of generating bioactive peptides. small peptides are generally considered to be the most ancient antimicrobial agents because of their ubiquity and simple molecular structure. in general, bioactive peptides comprise relatively small molecules (<10 kda, or 12-50 amino acids) that do not present any bioactivity prior to being released from the intact parent protein. however, in various processes such as digestion or in vivo hydrolysis, or as a result of application of technological treatments such as high pressure processing, these peptides demonstrate many physiological functions, including antioxidant, antihypertensive or ace inhibition, anticoagulant, and antimicrobial properties (ngo, wijesekara, vo, ta, & kim, 2011) . antimicrobial peptides are recognized as being divided into three groups: (i) linear a-helical peptides; (ii) cysteine-rich peptides; (iii) certain amino-acid-enriched peptides. critical factors affecting the antibacterial activity and modes of action of antimicrobial peptides are size, charge, conformation/secondary structure, hydrophobicity, and origin (animal/plant or marine) (aneiros & garateix, 2004) . according to the studies of al-saif et al. (2014) , the higher antimicrobial potential of several algae strains against escherichia coli (atcc 25322), pseudomonas aeruginosa (atcc 27853), staphylococcus aureus (atcc 29213), and enterococcus faecalis (atcc 29212) was directly related to the greater protein content detected in them. according to those studies, the most effective marine alga against the bacteria that were tested was g. dendroides, with a protein content of 13.4%, followed by u. reticulata (5.8%), cladophora socialis (2.3%), and c. occidentalis (1.7%). lectins are a class of carbohydrate-recognizing proteins that bind to cells, promoting hemagglutination and an antimicrobial effect. smith, desbois, and dyrynda (2010) reported the antimicrobial potential of lectins obtained from algae, with two red algal species, eucheuma serra and galaxaura marginata, being responsible for an inhibiting potential against vibrio vulnificus and v. pelagicus. the antimicrobial activity of lectins obtained from red algae against some clinical microorganisms was also observed by alves-vasconcelos et al. (2014) . although the way in which lectins act against bacteria is not well defined, paiva et al. (2010) reported that the antibacterial activity of these compounds against grampositive and gram-negative microorganisms is related to the interactions that occur between lectins and other bacterial cell wall components, including teichoic acids, peptidoglycans, and lipopolysaccharides (table 1) . macroalgae are rich sources of dietary fiber (25-75%), of which water-soluble fiber constitutes approximately 50-85% (wet basis). polysaccharides are some of the most important constituents of seaweed (kraan, 2012) . phaeophyta, or brown algae, are specifically rich in polysaccharides, including alginates, laminarins, fucans, and cellulose. chlorophyta, or green algae, are mainly composed of ulvan. the principal polysaccharides found in rhodophyta, or red algae, are agars and carrageenans. these polysaccharides obtained from algae have been related to positive effects on gut microbiota, acting as prebiotics, a prebiotic being a ''selectively fermented ingredient that allows specific changes, both in the composition and/or in the activity of the gastrointestinal microflora, that confers benefits upon host well-being and health". reduction of enteric infections in pigs and cattle is possible by means of administration of prebiotic compounds in the diet which promote the activity and proliferation of beneficial gastrointestinal microflora to the detriment of pathogenic bacteria (uyeno, shigemori, & shimosato, 2015) . the use of marine algal prebiotics as sources to control and reduce pathogenic bacteria and to improve animal and human health is another possibility that this line of research offers (promya & chitmanat, 2011) . the polysaccharide ulvan is easily extracted from ulva rigida, and could be hydrolyzed to produce bioactive oligosaccharides (mišurcová, škrovánková, samek, ambrožová, & machů , 2012) . also, oligosaccharides from brown algae have demonstrated antimicrobial potential in vivo inhibiting salmonella enteritidis colonization in broiler chickens (asthon acton, 2012) . another very interesting group of polysaccharides obtained from algae with a demonstrated antimicrobial potential is fucoidans (polysaccharides from phaeophyta). among the most important properties of these polysaccharides are the following: anticoagulant, antithrombotic, antiviral, antitumor, immunomodulatory, antioxidant, and anti-inflammatory (li, lu, wei, & zhao, 2008; marudhupandi & kumar, 2013) . according to the studies of de jesus raposo, bernardo de morais, and santos costa de morais (2015), sulphated polysaccharides from seaweeds (among them alginates, fucoidans and laminaran) have demonstrated to be effective against e. coli and staphylococcus aureus (chaetomorpha aerea, 50 mg/ml of extract). moreover, also carrageenans and the sulphated exopolysaccharide (seps) from the red microalga porphyridium cruentum are specifically effective inhibiting one of the most relevant foodborne pathogens, salmonella enteritidis (pierre et al., 2011) . recent studies have also demonstrated the potential of fucoidans for preventing helicobacter pylori infection, one of the most concerning emergent foodborne pathogens affecting 50-80% of the worldwide population. the effect of fucoidan from brown algae as a potential antimicrobial against h. pylori thereby reduces the risk of associated gastric cancers (marudhupandi, kumar, senthil, & devi 2014) . laminaria spp. extract containing either laminarin or fucoidan, or a combination of both, resulted in a reduction of fecal e. coli populations in piglets fed with 0.3 and 0.24 g/kg, respectively (o'doherty, mcdonnell, & figat, 2010) , consequently reducing the initial bacterial load in derived raw meat products (table 2) . these studies point out the potential of prebiotics from algae origin not only as potential antimicrobial ingredients in food, but also such as antimicrobial agents in vivo to be regularly added to diet and inhibiting the pathogenic bacterial proliferation in intestine (e.g. in both animal and human fed). one of the most valuable nutritional properties of algae is related to their high content of polyphenols, carotenoids, and flavonoids, referred to as antioxidants. antioxidants act to protect the human body against damage by reactive oxygen species (ros), which can lead to health disorders such as cancer, diabetes mellitus, neurodegenerative diseases, and inflammatory diseases with severe tissue injuries (rani et al., 2013) . among the different metabolites in algae, antioxidants are the most extensively studied (manivannan, karthikai, anantharaman, & balasubramanian, 2011; senthilkumar & sudha, 2012) . antioxidants are very powerful tools with which to fight oxidative stress and thus improve the health status of the general population (rani et al., 2013) . phenols constitute the largest group of secondary metabolites identified in algal species. in recent studies, a broad spectrum of in vitro antibacterial activity has been associated not only with plant phenols but also with phenols from algae, specifically against staphylococcus aureus and bacillus spp. other antimicrobial phenolic compounds isolated from the marine environment include anthraquinones, coumarins, and flavonoids . rutin, quercetin, and kaempferol flavonoids have been identified in all the algal species with antimicrobial potential, being present in different ratios in different species. according to the studies of al-saif et al. (2014) , the alga g. dendroides showed the highest concentration of these three flavonoids (rutin, 10.5 mg/kg; quercetin 7.5 mg/kg; kaempferol 15.2 mg/kg), and was also the most effective of the flavonoids studied in inhibiting bacterial growth (e. coli, p. aeruginosa, s. aureus, e. faecalis). the first antibacterial compound isolated from a microalga, chlorella spp., was a mixture of fatty acids. this compound was effective against both gram-positive and gram-negative bacteria (vello et al., 2014) . membrane-derived fas from macro-and microalga species have been associated with microbicidal activity as a mechanism of defense against viruses, protozoans, and bacteria (leflaive & ten-hage, 2009 ). the characteristic saturated and unsaturated fatty acids profile in algae, with a predominance of myristic, palmitic, oleic, and eicosapentaenoic acids (epa), is a specific feature associated with the antimicrobial potential of algal species (el shoubaky & el rahman-salem, 2014) . furthermore, palmitic acid has been assumed to be primarily responsible for the antibacterial activity of algae (al-saif et al., 2014; bazes et al., 2009 ). according to al-saif et al. (2014) .2]%, respectively, were associated with the highest antimicrobial potential, and the alga with the highest percentage of palmitic acid (75.5%), g. dendroides, was the most effective one against the bacteria studied. according to guedes et al. (2011) , the mechanism of action of fatty acids as antimicrobials may be due to cell leakage derived from membrane damage. some efforts are now being focused on identifying the compounds directly responsible for the antimicrobial capability of macro-and microalgae, taking into account that this is a relatively unexplored field of study (sanmukh et al., 2014) . in general, it could be said that the most antimicrobial compounds in algae are mainly polyphenols and polysaccharides that act by inhibiting microbial growth, or directly by destroying the living structures of microorganisms (bajpai, 2016) . novel extraction technologies (araujo et al., 2013; esquivel-hernández et al., 2016) and chemical extraction procedures (adam, abert-vian, peltier, & chemat, 2012; hammed et al., 2013) , are both applied to obtain selective extracts of algae rich in desired functional/antimicrobial compounds (dai & mumper, 2010) . since the late '70 s, various processes for obtaining extracts from seaweed have been developed, based on solid-liquid extraction, mainly by combining organic solvents, for example, [chloroform: methanol] mixtures, or acetone. additionally, some operations such as ultrafiltration processes are added to enhance selectivity. other methods use ethanol in a first step to precipitate protein, followed by a second stage extraction with hexane, butanol, or ethyl acetate. recent studies show that it is possible to use ternary mixtures of solvents (ethanol-hexane-water [77:17:6]) to form a homogeneous solution, with the advantage of significantly increasing the extraction yield and purity of the compounds extracted by a single stage, based on the high solubility of the compounds in the extractant mixture (parniakov et al., 2015) . the antimicrobial potential of algae extracts is dependent on the capability of the solvent to extract certain bioactive compounds, and also dependent on the sensitiveness of bacteria or fungi to these selective extracted compounds (al-saif et al., 2014; cakmak et al., 2014) . in general, these traditional extraction techniques, such as soxhlet, solid-liquid extraction (sle), or liquid-liquid extraction (lle), are time-consuming procedures that use high volumes of solvents and obtain low extraction yields. growing concern about the use of clean technologies to extract bioactive compounds from algae has led the international scientific community and r & d engineers to invest in technologies such as supercritical fluid extraction, extraction using high intensity pulsed electric fields (pefs), t ultrasonically assisted extraction (use) (parniakov et al., 2015) , microwave-assisted extraction (mae), and accelerated solvent extraction (ase), using pressure and temperature (esquivel-hernández et al. 2016 ) to preserve as much as possible of the quality and bioactivity of the extracted compounds . degradation of cell walls by an enzymatic pathway has also been used in microalgal treatment (e.g. in chlorella vulgaris), but it is still too expensive to be applied widely in industry (hammed et al., 2013) . the process of supercritical fluid extraction using co 2 is one of the most advanced techniques for obtaining bioactive compounds from algae. it is a highly efficient and fast way of obtaining extracts with high purity and rich in the desired functional compounds, but it remains relatively expensive as an industrial-scale method (mendiola et al., 2007) ultrasonically assisted extraction (use) is based on mechanical acoustic cavitation effects exerted on algae cell walls. among its main advantages are the effectiveness of the extraction process at room temperature, and therefore minimal loss of bioactive compounds. in microalgae, the use of ultrasound is already beginning to be widely used, with good results (adam et al., 2012; araujo et al., 2013) . the use of high intensity electrical pulses (pefs) is a method in which the application of high voltage pulses disrupts cellular material, facilitating the release of components such as proteins, chlorophylls, and carotenoids, among others. it has been successfully applied in bioactive extracts from spirulina and chlorella species. the method is based on the theory of electroporation, as the conductivity and permeability changes that occur in the cell membrane favor the formation of small pores that allow release of intracellular components to the environment (parniakov et al., 2015) . various dunaliella genus microalgal species have been treated by mae to favor extraction of carotenoids. microwave-assisted extraction promotes extraction by ohmic heating and homogeneous temperature distribution. the use of this technology in dunaliella tertiolecta and cylindrotheca closterium species has resulted in a process that combined rapid extraction, reproducibility, and a high yield (pasquet et al., 2011) , and other antimicrobial compounds (kadam, tiwari, & o'donnell, 2013) . until now, the antimicrobial potential of algae has generally been tested in vitro, using the agar diffusion method (cakmak et al., 2014; manivannan et al., 2011; qiao, 2010) . the broth dilution method (gupta, rajauria, & abu-ghannam, 2010) has also been used, providing a robust quantitative estimation of minimum inhibitory concentration (mic) values in a large number of samples. in the context of the present state of the art, in response to the food industry's demand for novel and alternative ingredients with high technological potential, the possibility of using seaweed and microalgae as natural preservatives to be added in the formulation of ''clean labeled foods" will soon become a reality. nowadays, the antimicrobial potential of algae against the main foodborne pathogens and spoilage microorganisms is one of the most interesting fields of research regarding the use of marine vegetables as sources of staple food and bioactives for human nutrition. the scientific advances and research focusing on this field that have been published to date are detailed below in order to provide the scientific community with a useful overview to help to guide future research trends/needs of research and the establishment of innovative projects. according to najdenski et al. (2013) , scenedesmus obliquus, chlorella sp., and nostoc sp. ethanolic extracts showed antibacterial potential against s. aureus. similarly, according to the studies of danyal, mubeen, and malik (2013) , ethanolic extract of pithophora oedogonium was effective in inhibiting the s. aureus growth. methanol and acetone extracts of scenedesmus spp. also exhibited antibacterial activity against s. aureus according to guedes et al. (2011) . furthermore, ishaq, matias-peralta, and basri (2016) found significant inhibitory activity of scenedesmus spp. acetone extract (0.35 mg/ml-3.48 mg/ml) against staphylococcus aureus (atcc 25923). methanolic extracts from nostoc spp., microcystis spp., scenedesmus spp., oscillatoria geminata, and chlorella vulgaris exerted a high antimicrobial potential against s. aureus and b. subtilis, with inhibition zone diameters between 16 and 18 mm (salem, hoballah, ghazi, & hanna, 2014) . these results are in agreement with those obtained by prakash, marimuthu, and jeeva (2011) regarding the antimicrobial potential of oscillatoria sancta and lyngbya birgei against this foodborne pathogen. manivannan et al. (2011) tested the brown seaweeds turbinaria conoides and padina gymnospora for their antimicrobial potential against s. aureus. s. aureus proved to be more resistant to t. conoides extracts (3-15 mm inhibition zone), but was more sensitive to p. gymnospora (10-16 mm inhibition zone). methanol and ethyl acetate extracts of t. conoides were the two most inhibitory ones against s. aureus. however, when growing s. aureus cells were exposed to p. gymnospora, diethyl ether and acetone were the extracts that inhibited cellular proliferation most effectively. methanolic and ethanolic extracts of dunaliella salina showed the lowest mbc against s. aureus (1.25 mg/ml) compared with other solvent extracts, such as hexane (mbc = 10 mg/ml) (cakmak et al., 2014) . the greatest antimicrobial potential of algae extracts against s. aureus was reported by tüney et al. (2006) , who observed an inhibitory diameter zone > 50 mm due to exposure of s. aureus to diethyl ether extract (0.5 g/ml) of fresh enteromorpha linza (0.5 g/ml), and a 38 mm inhibition zone in the case of fresh ulva rigida. this antimicrobial capabiity was not detected when the same fresh alga material was extracted with ethanol solvent. this led the researchers to the conclusion that some active compounds responsible for the antimicrobial potential against s. aureus are effectively extracted in diethyl ether, which produces larger halo zones than methanol, acetone, and ethanol, in which antimicrobial compounds are not completely dissolved and extracted. among the most effective antimicrobial compounds found in these algal species are terpenes, (e.g. usneoidone e, zosterdiol a, zosterdiol b, zosteronol, and zosteronediol) responsible for the antimicrobial and antiviral activity attributed to them (plaza del moral & rodríguez-meizoso, 2013). the antimicrobial potential of algae extracts is frequently compared with the potential of other currently used preservatives (at food industry level) or antibiotics (at clinical level). for example, devi et al. (2008) observed the effectiveness of haligra spp. seaweed extract (50 mg/ml) against s. aureus (mtcc 96), and found that the inhibitory potential of this alga extract was higher than the antimicrobial capability of sodium benzoate applied at a higher concentration (200 mg/ml). to date, most of the antimicrobial in vitro studies regarding the potential of algae against s. aureus have not been validated in food matrices. however, the promising results obtained against s. aureus were validated by the devi et al. (2008) research group by including haligra spp. extract at low concentrations in skimmed milk, demonstrating the effectiveness of 5 mg/ml as the minimum inhibitory concentration (mic) against s. aureus growth in dairy products. ascophyllum nodosum (brown phaeophyceae alga, in the fucaceae family) has been described by several authors as being effective in reducing the prevalence of e. coli o157:h7 in cattle before harvest (wang, xu, bach, & mcallister, 2009 ). moreover, according to the studies of wang et al. (2009) , antimicrobial activity from a. nodosum phlorotannins (pts) against various rumen microbes was observed, and it affected ruminal fermentation. in 2009 they published a new research work in which the bacteriostatic and bactericidal effects of pts from a. nodosum were evaluated against e. coli o157:h7, and were then compared with the antimicrobial potential of other tannins from terrestrial plant sources. an mic of 25 mg/ml was required to inhibit growth of e. coli o157:h7 strains (edl933 and e318 n) for 24 h at 37°c. pt bacteriostatic concentrations of 25 lg/ml or higher, and a bactericidal concentration ! 50 lg/ml, were required against the e. coli o157:h7 strains studied. according to ngo et al. (2011) , the potential biological activities of pts can protect the quality of food products against oxidative degradation by means of their antioxidant properties, and they can improve the safety of non-sterilized products as a control measure against microbial proliferation in the food chain. the antimicrobial potential exerted by pts from a. nodosum was explained by wang et al. (2009) . the technique of transmission electron microscopy was used to reveal the mechanism of action of pts against e. coli cells. the results of wang et al. (2009) indicated that tannins acted primarily on the bacterial cell wall. it seems that the patterns of structural disorganization by means of pt intervention were higher than the pattern observed as a result of the effect of terrestrial tannins e. coli o157:h7 cells. the antimicrobial potential of chaetomorpha linum, a green seaweed from the southeast coast of india, was tested against escherichia coli (mtcc no. 443), salmonella typhimurium (mtcc no. 98), and bacillus cereus (mtcc no. 430). methanolic extracts (90% w/v) of algae were tested by means of the agar diffusion method, assessing not only their antimicrobial capability but also their antioxidant potential, using the 1, 1-diphenyl-2-picrylhydrazyl radical (dpph) method. bacterial suspensions containing 1.5 â 10 8 cfu/ ml were inoculated in mueller-hinton agar plates. algal extracts were prepared at concentrations of 100, 300, and 500 mg/ml. the petri dishes were incubated for 16 h at 37°c and the inhibition zone was examined. a high correlation was found between the dpph antioxidant potential of methanolic c. linum extracts and the microbial inhibition capability exerted. the highest phenolic content of c. linum extract was 672.3 mg/gae/100 g/extract. e. coli was the most resistant bacterial strain to the effect of c. linum methanolic extract (senthilkumar & sudha, 2012) . the antimicrobial potential of t. conoides and p. gymnospora (kutz) vicker was tested by manivannan et al. (2011) against several microorganisms, including e. coli. the extraction procedure was carried out successively during a period of 10 h, using the following solvents: methanol, acetone, petroleum ether, ethanol, ethyl acetate, chloroform, and diethyl ether. the study conducted by manivannan et al. (2011) revealed that p. gymnospora extracts were significantly more effective against e. coli (8-17 mm inhibition zone diameter) than t. conoides extracts (2-8 mm inhibition zone diameter). when the bacterial growth was exposed to p. gymnospora extracts, the diethyl ether extract showed the greatest potential for inhibiting bacterial growth in agar plates, with a 17 mm inhibition zone diameter. the antimicrobial effectiveness of methanol, acetone, diethyl ether, and ethanol extracts from eleven seaweed species, fresh extract and dried extract, against growth of e. coli was assessed by tüney et al. (2006) . the results revealed that diethyl ether extracts of fresh c. mediterranea, e. linza, u. rigida, g. gracilis, and e. siliculosus (0.5 g/ml) showed effective results against gram-positive and gram-negative bacteria. the best inhibition results obtained with fresh and dried diethyl ether seaweed extracts were assayed against various pathogenic microorganisms. u. rigida and e. linza diethyl ether extracts showed the highest inhibition potential against e. coli (22 mm inhibition zone diameter), being c. mediterranea and g. gracilis also effective, with inhibition zone values between 16 and 20 mm. all of the most effective inhibition results were obtained with fresh materials. with regard to microalgae, the antimicrobial potential of d. salina against e. coli has also been tested. among the extracts studied, methanol and ethanol extracts of d. salina showed the highest effectiveness, achieving the lowest mbc against e. coli, 2.50 mg/ ml, compared with other solvent extracts such as hexane, which had an mbc of 10 mg/ml (cakmak et al., 2014) . according to manivannan et al. (2011) , salmonella typhimurium was slightly affected by t. conoides extracts, and was particularly sensitive to diethyl ether and petroleum ether extracts, with inhibition zone diameters in the range [8] [9] [10] [11] mm. these results are in agreement with those obtained by thirumaran and anantharaman (2006) regarding the antimicrobial potential of dictyota dichotoma against salmonella spp., concerning which they remarked that diethyl ether extracts showed the greatest inhibitory potential against salmonella paratyphi. on the other hand, p. gymnospora extracts showed higher antimicrobial potential against s. typhimurium, with inhibition zones of [7-24] mm. among the p. gymnospora extracts, chloroform and methanol were the most effective ones against this foodborne pathogen. t. conoides ethyl acetate extract showed the lowest inhibitory potential against salmonella spp. (%3 mm inhibition zone). the same inhibitory potential was detected for t. conoides acetone extract against salmonella spp. (%2 mm inhibition zone). the ethanolic extract of pithophora oedogonium was particularly effective against salmonella spp. (sanmukh et al., 2014) . similarly, ethanol extract of d. salina showed the lowest mbc against s. enteritidis (1.25 mg/ml), followed by methanolic extract with an mbc of 2.50 mg/ml. hexane extract was the least effective against s. enteritidis, with a required mbc of 10 mg/ml (cakmak et al., 2014) . high inhibition potential against s. typhimurium was exerted by chaetomorpha linum methanolic extract with concentrations in the range [100-500] lg/ml, producing inhibition zone diameters in the range [15-17] mm (senthilkumar & sudha, 2012) . ethanol extracts of p. oedogonium and botrydiopsis arhiz algal species were tested to assess their antimicrobial capability against two salmonella species isolated food samples, egg and meat, and named salmonella 1 and salmonella 4, respectively. the results revealed that p. oedogonium ethanolic extract was effective in inhibiting salmonella 1 growth, with an mic of 4 mg/ml (danyal et al., 2013) . according to the studies of el-baky et al. (2008) , several extracts of spirulina maxima were obtained from cells grown under different nitrogen levels [0.625-2.5 g/l nano 3 ), and their antimicrobial potential was assayed against various strains of bacteria. the s. maxima extracts were effective against b. cereus, with an antibacterial activity that was dose-dependent. mics in the range lg/ml were obtained, depending on the s. maxima extract applied. s. maxima extracts from cells grown at higher n levels were the most potent against all bacteria, with mic values of 30 lg/ml. the antibacterial activity of s. maxima was attributed by el-baky et al. (2008) to the presence of certain active components in all organic extracts, such as lipophilic and phenol compounds. according to the studies of cakmak et al. (2014) , different extracts (ethanol, hexane, dichloromethane, and methanol) of the microalga d. salina teodoresco (dunaliellaceae) were tested against various microorganisms, including bacillus cereus rskk 863. a minimum bactericidal concentration of 0.65 mg/ml of ethanol extract was required against b. cereus, corresponding to 2.50 mg/ml of d. salina fatty acids (palmitic (c16:0) > linolenic (c18:3 x3) > oleic (c18:1 x9) ). the studies of jang and lee (2015) revealed the high antimicrobial potential of two korean domestic algae, laurencia okamurae yamada and dictyopteris undulata holmes, against foodborne pathogens. b. cereus, s. aureus, and l. monocytogenes were particularly sensitive to laurencia okamurae yamada and dictyopteris undulata holmes extracts, and the antibacterial potential of both algae extracts was higher than that of streptomycin. dictyopteris undulata holmes was slightly more effective against b. cereus than laurencia okamurae yamada, with inhibition zones of 5.0 ± 0.2 mm and 4.0 ± 0.2 mm, respectively. the most effective inhibition potential against b. cereus has been reported by senthilkumar and sudha (2012) . c. linum methanolic 500 lg/ml extract resulted in a 27 mm inhibition zone against this foodborne pathogen. the total phenolic content (tpc) of the methanolic c. linum extract was determined by means of the folin-ciocalteu method, yielding a tpc value equal to 672.3 mg gae/100 g extract, related to significant antioxidant activity (as ascorbic acid equivalents). the higher scavenging activity of c. linum may be attributed to the structure of phenolic compounds, specifically to hydroxyl groups. accordingly, it could be said that the antimicrobial potential exerted by this methanolic c. linum extract is closely related to its antioxidant activity as measured by the dpph method, with the antioxidant potential of the extract being dose-dependent, with an ic 50 value of 9.8 lg/ml. the antimicrobial potential of ecklonia cava (laminariaceae family) was tested by nshimiyumukiza et al. (2015) against listeria monocytogenes. although the biological activities of e. cava (including antioxidant, antimicrobial, and immunomodulatory properties) have been previously reported, the study conducted by nshimiyumukiza et al. (2015) is the first to reveal the potential that this algae specie has against this relevant foodborne pathogen. several solvents were assayed in the process of extraction (methanolic extract, dichloromethane fraction, ethyl acetate fraction, butanol extract, and water fraction) of bioactive antimicrobial compounds. the methanolic extract of ecklonia cava exhibited the highest antibacterial activity against l. monocytogenes kctc 3710, with the ethyl acetate (etoac) soluble fraction having an mic value of 256 lg/ml and an mbc value of 512 lg/ml. methanolic and ethanolic extracts of the microalga d. salina teodoresco (dunaliellaceae) were particularly effective against listeria monocytogenes atcc 7644, with a mbc of 2.5 mg/ml compared with 5 mg/ml for algal fatty acids (fas) (cakmak et al., 2014) . the antimicrobial potential of this alga has been attributed to its very valuable fa content, with an x3/x6 ratio equal to 2.15, higher than the proportion found in some fish (cakmak et al., 2014) . this is in agreement with the studies of ohta et al. (1994) , which emphasize the antimicrobial potential of fa-rich oil extracted from the microalga chlorococcum hs-101 against another important methicillin-resistant gram-positive bacterium, s. aureus. the antimicrobial potential of myagropsis myagroides (sargassaceae family in phaeophyta) against l. monocytogenes kctc 3569 was assessed by lee, kim, lim, and ahn (2014). this macroalga revealed antimicrobial activity against gram-positive bacteria, and the ethanolic extract was effective with an mic of 0.013 mg/ ml against l. monocytogenes. a liquid-liquid technique was used to obtain five fractions, and the chloroform (ch 4 ) fraction exerted a strong antimicrobial activity against l. monocytogenes, with an mic of 0.031 mg/ml. the effects of these fractions were analyzed by means of transmission electron microscopy (tem), which detected serious damage to the cell envelope, leading to leakage of cytoplasmic material. a significant release of adenosine triphosphate (atp) (>10 -6 m atp) was also observed in the ch 4 -treated bacterial population, and this was defined as one of the main causes of bacterial death. with regard to the antifungal effects of macro-and microalgae, there is a particularly important study by indira, balakrishnan, srinivasan, bragadeeswaran, and balasubramanian (2013) that assesses the antimicrobial potential of a seaweed (halimeda tuna) against a wide range of foodborne pathogens, including nine fungi: aspergillus niger, aspergillus flavus, alternaria alternaria, candida albicans, epidermophyton floccosum, trichophyton mentagrophytes, trichophyton rubrum, penicillium spp., and rhizopus spp. according to that study, the minimum fungicidal concentration (mfc) of all the seaweed extracts studied (ethanolic, methanolic, and chloroform extracts) was 500 mg/ml, while aqueous extracts had an mfc value ranging between 250 and 500 mg/ml. it can be concluded that h. tuna extracts were very effective against a. niger, a. flavus, a. alternaria, c. albicans, and e. floccosum, and the methanol extract was the most effective one against fungi. salem et al. (2014) studied the antimicrobial potential of nostoc spp., microcystis spp., scenedesmus spp., oscillatoria geminata, and chlorella vulgaris algal strains against several food bacteria, including aspergillus niger. dried algal biomass was sonicated and extracted with 95% methanol and 95% acetone. extracts were prepared in dmso for evaluation of antimicrobial activity. algal extracts from nostoc spp., microcystis spp., scenedesmus spp., and oscillatoria geminata revealed antifungal activity, and the methanol extract of nostoc (50 mg/ml) was the most effective one against a. niger, with an inhibition zone diameter of 22 mm. acetone extract was also effective against a. niger (30 mg/ml), with a 21 mm inhibition zone diameter. an acetone extract of t. conoides also exerted a mild antimicrobial capability against a. niger, with an inhibition zone diameter close to 3 mm (manivannan et al., 2011) . extracts of trichodesmium erythraeum (hexane and ethyl acetate 5 mg/disc) were also effective against a. niger and a. flavus. in a comparison of the hexane and ethyl acetate extracts, the latter had a higher antifungal potential than the hexane algal extract. ethyl acetate extract of t. erythraeum inhibited growth of a. niger and a. flavus at 1000 mg/ml (thillairajasekar, duraipandiyan, perumal, & ignacimuthu, 2009) . both extracts are rich in myristic, oleic, linoleic, and linolenic acids. there is an established relationship between the richness in polyunsaturated fatty acids (lauric, palmitic, linoleic, linolenic, stearic, and myristic) of algal extracts and the antibacterial, antiviral and antifungal potential exerted by them (el shoubaky & el rahman-salem, 2014) . the antifungal potential of two brown seaweeds was also tested against a. niger by manivannan et al. (2011) . according to their results, padina gymnospora chloroform, ethanol, and ethyl acetate extracts were extremely effective in inhibiting a. niger growth (inhibition zone [15] [16] [17] mm). on the other hand, a smaller antifungal potential was attributed to t. conoides extracts (3-11 mm), the only noteworthy result being the inhibition observed after exposure of a. niger cells to diethyl ether t. conoides extract, with an inhibition zone diameter equal to 11 mm. the antifungal activity of seaweed extracts has also been related to the presence of phenolic compounds and their impact on spore germination. some algal extracts have also been shown to inhibit fungal enzyme activity owing to the presence of bioactive metabolites (salem, galal, & el-deen, 2011) . there is a serious lack of information about the antiviral potential of algae against foodborne viruses. mainly, noroviruses (novs) and other commonly viral contaminants in food (hepatitis a (hav), human rotavirus (hrv), hepatitis e virus (hev), nipah virus, highly pathogenic avian influenza (hpai) virus, sarscausing coronavirus) are of high concern for the scientific community (koopmans, 2012) . the main substances from algae that have been related to antiviral potential are sulfated polysaccharides, including fucoidan, sulfoglycolipids, carrageenans, sesquiterpene hydroquinones, etc. (elizondo-gonzalez et al., 2012) . suppression of dna replication and inhibition of host cell colonization by the virus are some of the effects of algal polysaccharides (e.g. fucan, laminaran, and naviculan) as natural antiviral compounds (ahmadi, moghadamtousi, abubakar, & zandi, 2015) . focused on foodborne viruses inhibition by marine algae it is remarkable the research work of wang, wang, and guan (2012) . the study of wang et al. (2012) reviewed the anti-viral potential of polysaccharides from brown seaweeds, mainly alginates and fucans, revealing a significant inhibiting activity against hepatitis b virus (hbv) dna polymerase, and consequently affecting its replication. algae polysaccharides have exerted also anti-viral potential against rotaviruses. these viral agents are the most important causing gastroenteritis, mainly in children, with fatal consequences. the effect of sulfated polysaccharides interfering with the adsorption process of enveloped viruses has been considered in the invention developed by anderson, schaller, mazer, and kirchner (1997) to demonstrate that carrageenan, and particularly k-carrageenan, is an effective inhibitor of rotavirus infection in animal cells. crude metanol extract (70% v/v) of spirulina platensis also showed antiviral activity with 56.7%, inhibition rate against rotavirus wa strain (hetta et al., 2014) . according to the studies of hetta et al. (2014) , free fatty acids (ffa) -induced endoplasmic reticulum (er) stress and suppressor of cytokine signaling protein (socs3) levels are the two main factors responsible for the signaling reduction and impairing the antiviral response. according to the studies of eom et al. (2015) phlorotannins from eisenia bicyclis showed a strong antiviral potential against norovirus (murine norovirus, mnv), with 50% effective concentration (ec 50 ) of 0.9 lm. the inhibition of oxidative phosphorylation, and the ability of phlorotannins to bind with proteins, such as enzymes and cell membranes, are the main causes explaining the antiviral activity of these compounds, that finally act causing cell lysis (shannon & abu-ghannam, 2016) . although the high anti-viral potential of algae compounds (ffa and polysaccharides, mainly) against clinical and foodborne viruses, scarce information has been reported up to date (shannon, 2016; wang et al., 2012) . more research is need to cover the self-evident gap in knowledge regarding the anti-viral applications of algae compounds from both approaches, pharmaceutical and food safety. a wide variety of algae have been introduced in food formulations for various purposes. meat, dairy, and bakery products are some of the best-known examples of innovative products that include these healthy vegetable ingredients. in europe, for a long time interest in algae has focused on the extraction of phycocol-loids with gelling, thickening, and stabilizing properties for use in many applications in the food industry (enzing, ploeg, barbosa, & sijtsma, 2014) . the main limitations affecting the application of algae in food products are related to sensory (residual flavor and aroma) and toxicological aspects (high iodine levels, or accumulation of arsenic, heavy metals, and contaminants) (bouga & combet, 2015) . these risks must be minimized in order to maximize the technological, functional, and nutritional advantages that are associated with macro-and microalgae materials and bioactive compounds. the introduction of microalgae in animal feed has been a reality since the '70 s, and is a good way to improve animal health and the subsequent quality of meat-derived products. in aquaculture feed, algae supplements are also being used, with good prospects (yaakob, ali, zainal, mohamad, & takriff, 2014) . in food for human consumption, seaweed has been introduced in breads, pizza bases, and cheese (e.g. ascophyllum nodosum) (hall, baxter, fryirs, & johnson, 2010) , and has also been added to pasta (e.g. sargassum marginatum)) (prabhasankar et al., 2009) and meat products (cofrades, lópez-lópez, ruiz-capillas, triki, & jiménez-colmenero, 2011) . the development of algae-based lipid powders and flours is also one of the hot topics that has been included in novel cuisine, and they are even being used instead of eggs, with really promising results for the vegan market. however, with regard to preservation there is scarce literature about validating the functionality of these ingredients as antimicrobials in real food matrices (gupta et al., 2010; sȃlȃgean, pop, catrinoi, & nagy, 2015) . although some researchers are making efforts to introduce raw algae and processed materials in the formulation of innovative meat-derived products (e.g. sausages and hamburgers), some aspects such as quality and organoleptic acceptance of the final formulation still need to be analyzed in depth. with regard to the validation of the antimicrobial potential of algae in meat products, a noteworthy study has recently been published by sȃlȃgean et al. (2015) . among the main advantages that the introduction of algae in meat-formulated products aims at are the following: (a) the introduction of a protein source of vegetable origin, (b) lower content of cholesterol, calories and fat in the finished products, and the associated (c) beneficial bioactive compounds present in algae-derived ingredients. according to the results obtained by sȃlȃgean et al. (2015) , in the formulation of half-smoked sausages (75% first-quality beef, [10-15]% animal fat, and brown algae [10-15]%) the addition of algae in the formulation led to a final product with higher quality (physicochemical and organoleptic), with improved functionality resulting from the antimicrobial potential of brown algae against s. aureus and e. coli (storage 7 days, 10-12°c). furthermore, a recent industrial patent has been obtained for the introduction of algae in meatderived products. according to cofrades et al. (2011) , a new method for the formulation of healthier meat products has been developed, incorporating less than 5 wt.% of himanhatalia elongata brown alga. brown algae are more suitable for meat formulations than red or green algae, not only because of their nutritional profile but also because of the color balance between animal tissue (cattle, pigs, sheep, goats, horses, poultry) and these pigmented algae. the result shows a reduction in sodium levels and an improvement in the lipid profile of the final product (cofrades et al., 2011) . the antimicrobial capability of macroalgae has been reported to reduce the need for the addition of salt, especially against gramnegative bacteria. a reduction of nearly 1 log 10 cycle was observed against coliforms during the shelf life of frozen processed meat products (brownlee, fairlough, hall, & paxman, 2012) . similarly, in bread products the antifungal potential of green seaweed added to bakery products suppressed mold growth for up to 9 days in preservative-free bread, equivalent to the control bread containing 5 g of sodium chloride (brownlee et al., 2012) . in the research line of novel meat products with longer shelf life, edible films and coatings (efc) are a promising preservation technology that provides a good barrier against spoilage and pathogenic microorganisms when natural antimicrobials are added. according to sánchez-ortega et al. (2014) , the addition of edible algae films and coatings on meat products had the following main advantages: reduction of lipid oxidation, increase in stability of red meat color, prevention of moisture loss, reduction of spoilage and pathogenic microorganism load, and partial inactivation of deteriorative proteolytic enzymes on the surface of the coated meat. the development of red algae films applied to bacon impacted positively on the microbiological quality of the product reducing e. coli o157:h7 by 0.45 log cfu/g and l. monocytogenes by 0.76 cfu/g with respect to the controls. the application of edible coatings from algae to cheese also reduced e. coli o157:h7 and l. monocytogenes populations by 1.21 and 0.85 log cfu/g, respectively, compared with the control after 15 days of storage (shin, song, seo, & song, 2012; sánchez-ortega et al., 2014) . other algae derivatives such as alginate and j-carrageenan have also been used in the formulation of edible films with antimicrobial properties against l. monocytogenes, e.g. in cold smoked salmon (neetoo, ye, & chen, 2010) . effective antimicrobial potential has been demonstrated against foodborne pathogens by both macro-and microalgae compounds. however, some contradictions can be found in the literature as a result of the different strains used in the determination of antimicrobial activity, different methods of extraction applied, and different ranges of alga material concentration used in the assays. the promising results of bacterial growth inhibition and inactivation by raw and purified algal compounds, and the lack of studies carried out in real food matrices open a research niche with high application at food industrial level. a valuable relationship could be established between the antioxidant potential of algae and their antimicrobial capability. consequently, the twofold use of algae extracts as antioxidants and antimicrobials in food products has good prospects in response to the horizon 2020 call for 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escherichia coli to seaweed (ascophyllum nodosum) phlorotannins and terrestrial tannins y an overview: biomolecules from microalgae for animal feed and aquaculture key: cord-336578-5qzpd890 authors: sethiya, jigar p.; sowards, melanie a.; jackson, mary; north, elton jeffrey title: mmpl3 inhibition: a new approach to treat nontuberculous mycobacterial infections date: 2020-08-27 journal: int j mol sci doi: 10.3390/ijms21176202 sha: doc_id: 336578 cord_uid: 5qzpd890 outside of mycobacterium tuberculosis and mycobacterium leprae, nontuberculous mycobacteria (ntm) are environmental mycobacteria (>190 species) and are classified as slowor rapid-growing mycobacteria. infections caused by ntm show an increased incidence in immunocompromised patients and patients with underlying structural lung disease. the true global prevalence of ntm infections remains unknown because many countries do not require mandatory reporting of the infection. this is coupled with a challenging diagnosis and identification of the species. current therapies for treatment of ntm infections require multidrug regimens for a minimum of 18 months and are associated with serious adverse reactions, infection relapse, and high reinfection rates, necessitating discovery of novel antimycobacterial agents. robust drug discovery processes have discovered inhibitors targeting mycobacterial membrane protein large 3 (mmpl3), a protein responsible for translocating mycolic acids from the inner membrane to periplasm in the biosynthesis of the mycobacterial cell membrane. this review focuses on promising new chemical scaffolds that inhibit mmpl3 function and represent interesting and promising putative drug candidates for the treatment of ntm infections. additionally, agents (fs-1, smart-420, c10) that promote reversion of drug resistance are also reviewed. nontuberculous mycobacteria (ntm) are mycobacteria, other than mycobacterium tuberculosis (m. tb) and mycobacterium leprae, the causative agent for tuberculosis (tb) and leprosy, respectively, that are environmental pathogens and are commonly found in soil, dust, biofilms, and natural and municipal water sources. ntm are also described in literature as atypical mycobacteria or mycobacteria other than tuberculosis (mott) [1] . to quinolones, beta-lactams, and aminoglycosides. inducible resistance occurs through induced expression of erythromycin resistance methylase (erm) genes, which result in the translation of a methylase that weakens the binding of macrolides with the bacterial ribosomes. acquired resistance by mutations in the 23s rrna (rrl), 16s rrna (rrs), and rpob genes of the ntm species causes high-level resistance to the macrolides and linezolid, aminoglycosides, and rifampicin, respectively. bedaquiline also shows acquired resistance against mac pulmonary disease through the regulator gene of the mmps5/mmpl5 efflux system, and this mutation results in cross-resistance to clofazimine [26] . resistance occurs through induced expression of erythromycin resistance methylase (erm) genes, which result in the translation of a methylase that weakens the binding of macrolides with the bacterial ribosomes. acquired resistance by mutations in the 23s rrna (rrl), 16s rrna (rrs), and rpob genes of the ntm species causes high-level resistance to the macrolides and linezolid, aminoglycosides, and rifampicin, respectively. bedaquiline also shows acquired resistance against mac pulmonary disease through the regulator gene of the mmps5/mmpl5 efflux system, and this mutation results in cross-resistance to clofazimine [26] . the increasing incidence, limited efficacy of current treatment options, and significant risk of drug-resistance in ntm infections suggest an urgent need for new antimycobacterial agents. while several drug candidates, such as inhaled nitric oxide gas, gallium nitrate, interferons, il-12, clofazimine, bedaquiline, rifabutin, amithiozone, linezolid, tedizolid, and tigecycline, are under clinical trials for ntm infections, most of them are repurposed antibiotics drugs used for tb or other bacteria. the drug discovery pipeline is primarily focused on efficacy against tb, with few trials assessing agents effective for treatment of ntm infections. recently developed compounds targeting the mmpl3 transporter, a new and promiscuous target recognizing many structurally unique chemotypes, have proven to be effective in both tb and ntm infections. in this review, we focus on potential new compounds that inhibit mmpl3 with efficacy against ntm species. the mycobacterial cell wall is different from the gram-positive and -negative bacterial cell wall [27] . it is a complex structure, made up of covalently linked mycolic acids (ma)-peptidoglycan (pg)arabinogalactan (ag) (also known as magp) [28] . the peptidoglycan is the innermost layer and mycolic acids are the outermost layer of the mycobacterial cell wall [28] . mycolic acids are a major component and are considered a hallmark of the mycobacterial cell wall [27, 29, 30] . mycolic acids comprise a highly impermeable lipid-rich layer that protects the mycobacterial cell against various threats including antibiotics and the host's immune system and also contributes to virulence [30, 31] . chemically, mycolic acids are α-alkylalted, β-hydroxylated long-chain fatty acids. the α-alkyl chain comprises saturated c22-c26 carbons, and the β-hydroxy long meromycoloyl chain comprises c42-c62 carbons [32] . depending on the functional groups attached, the mycolic acids found in m. tb can be differentiated into α-, methoxy-, keto-, and/or hydroxy-mycolic acids. there are in total c66-c90 carbons in a mycolic acid chain length [27, 30] . even though mycolic acids are found in all mycobacterial pathogens and provide similar cellular protection, structural integrity, and virulence, the increasing incidence, limited efficacy of current treatment options, and significant risk of drug-resistance in ntm infections suggest an urgent need for new antimycobacterial agents. while several drug candidates, such as inhaled nitric oxide gas, gallium nitrate, interferons, il-12, clofazimine, bedaquiline, rifabutin, amithiozone, linezolid, tedizolid, and tigecycline, are under clinical trials for ntm infections, most of them are repurposed antibiotics drugs used for tb or other bacteria. the drug discovery pipeline is primarily focused on efficacy against tb, with few trials assessing agents effective for treatment of ntm infections. recently developed compounds targeting the mmpl3 transporter, a new and promiscuous target recognizing many structurally unique chemotypes, have proven to be effective in both tb and ntm infections. in this review, we focus on potential new compounds that inhibit mmpl3 with efficacy against ntm species. the mycobacterial cell wall is different from the gram-positive and -negative bacterial cell wall [27] . it is a complex structure, made up of covalently linked mycolic acids (ma)-peptidoglycan (pg)-arabinogalactan (ag) (also known as magp) [28] . the peptidoglycan is the innermost layer and mycolic acids are the outermost layer of the mycobacterial cell wall [28] . mycolic acids are a major component and are considered a hallmark of the mycobacterial cell wall [27, 29, 30] . mycolic acids comprise a highly impermeable lipid-rich layer that protects the mycobacterial cell against various threats including antibiotics and the host's immune system and also contributes to virulence [30, 31] . chemically, mycolic acids are α-alkylalted, β-hydroxylated long-chain fatty acids. the α-alkyl chain comprises saturated c 22 -c 26 carbons, and the β-hydroxy long meromycoloyl chain comprises c 42 -c 62 carbons [32] . depending on the functional groups attached, the mycolic acids found in m. tb can be differentiated into α-, methoxy-, keto-, and/or hydroxy-mycolic acids. there are in total c 66 -c 90 carbons in a mycolic acid chain length [27, 30] . even though mycolic acids are found in all mycobacterial pathogens and provide similar cellular protection, structural integrity, and virulence, subtle differences in carbon chain length an composition are found. for example, at the time when this review was written, keto-and methoxy-ma have not been detected in m. phlei, [29] , yet are common in m. tb. these structural differences may account for the variations in drug susceptibility among mycobacterial pathogens. the biosynthetic pathway of mycolic acids involves many catalytic enzymes that synthesize and functionalize long fatty chains that are condensed together, transported out and attached to the outer membrane ( figure 2 ). the biosynthesis of mycolic acids initially involves two enzyme complexes: fatty-acid synthase-i (fas-i) and fatty-acid synthase-ii (fas-ii). fas-i initiates de novo fatty acid synthesis cycle from an acetyl group to produce c [16] [17] [18] and c 24-26 acyl coas [29] . each cycle of fas-i synthase undergoes the addition of two new carbon atoms, thereby increasing the chain length [30] . fas-ii further elongates the short chain c 12-16 fatty acids to c 18-30 acyl-acyl carrier proteins (acps) [29] . fas-i synthesizes hexacosanoyl-coa (c 26 ) that ultimately becomes the α-chain of mycolic acids [30] . β-ketoacyl-acp-synthase iii (mtfabh) initiates the fatty acid synthesis by combining malonyl-acp and acyl-coa through claisen condensation. the product formed is β-ketoacyl-acp, followed by a reduction to β-hydroxyacyl-acp by β-ketoacyl-acp reductase (maba). β-hydroxyacyl-acp dehydratases (hadab and hadbc) convert the β-hydroxyacyl-acp into an enoyl-acp. this enoyl-acp is reduced to an acyl-acp by nadh-dependent trans-2-enoyl-acp reductase (inha). this acyl-acp is again taken up into the fas-ii cycle and further elongated until a mero-mycolic acid chain is formed. it is thought to undergo five cycles for alpha-mycolic acids and eight cycles for methoxyand keto-mycolic acid. elongation of the fatty acid chain takes place by β-ketoacyl-acp-synthase (kasa or kasb). marrakchi et al. report inha, maba, hadb, and kasa as essential proteins [29] . in addition, kasb is essential for the full extension of the mycolic acids, the acid-fastness property of the mycobacteria, and production of ketomycolic acids [27, 33] . the addition of cyclopropanes to the meromycolic acid is catalyzed by the s-adenosyl-methionine (sam)-dependent methyltransferases. cmaa1, cmaa2, pcaa, and mmaa2 are the methyltransferases identified in m. tb [29] , however, the exact ntm enzymes have not been determined. the product from the fas-i is carboxylated by accd4 (acyl-coa carboxylase) [29] , as accd4 and accd6 are essential genes in m. tb [34] . before the final condensation with the short α-chain, the long-chain meromycolic acid (c 50-60 ) gets activated through the fadd32 enzyme. fadd32 (fatty acid adenylating enzyme) is a member of the fatty acyl-amp ligase (faal) class that activates and transfers the activated long chain acyl adenylate to the pantetheine moiety of the n-terminal acyl carrier protein (acp) domain of polyketide synthase 13 (pks13) [29, 35] . pks13 is a member of the type-i pks family and is responsible for performing the final condensation step in the production of mycolic acids. the five domains of the pks13 are peptidyl carrier protein (pcp)-like domain, ketoacyl synthase (ks), acyl transferase (at), acp domain, and thioesterase (te). 4 -phosphopantetheinyl transferase pptt is essential for the activation of the acp and pcp domains of pks13 [29] . after the final condensation step, a reduction mediated through cmra produces mature mycolic acids and their pks13-mediated transfer to trehalose to form trehalose monomycolate (tmm) [29] . after acetylation of the β-hydroxy group on tmm by tmat, tmm is transported out of the cytoplasm to the periplasmic space through the mmpl3 transporter [36, 37] . subsequently, this glycolipid serves as a donor of mycolic acyl chains that either form a covalent linkage with the arabinogalactan layer of magp or esterify another molecule of tmm to form the outer membrane glycolipid, trehalose dimycolate (tdm, also known as cord factor) [38, 39] . the enzyme responsible for the transesterification of mycolic acids from tmm to their cell envelope acceptors are the mycolyltransferases (ag85 complex), fbpa, fbpb, and fbpc (also known as ag85a, ag85b, and ag85c). ag85c helps in the formation of magp whereas ag85a and ag85b help in the formation of tdm [32] . although tdm is present mainly as the outer layer, phthiocerol dimycocerosate (pdim) and a number of other acyltrehaloses (sulfolipids, diacyl-, and poly-acyltrehaloses) also contribute to form an outer layer [39] . mmpl3 transporters aid in the translocation of tmm across the plasma membrane for cell envelope biosynthesis. mmpl3 belongs to the resistance-nodulation-cell division (rnd) protein superfamily of inner membrane transporters. its translocation activity is dependent on the proton motive force (pmf). on the basis of a spheroplast-based translocation assay that can determine the topology of tmm in the plasma membrane, xu and colleagues proposed that mmpl3 functions as a tmm flippase [40] . in the mmpl family, mmpl3 is an essential mmpl transporter indicating that its function is unique. co-crystallography and native mass spectrometry studies indicated that mmpl3 not only binds tmm but also phospholipids such as phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin suggesting that it might be involved in the translocation of more than one lipid to the periplamic space [39] . determination of the crystal structure of mmpl3 from m. smegmatis indicates that mmpl3 functions as a monomer [39, 41] . although this observation is contradictory to earlier gel filtration and single-particle electron microscopy studies indicating that mmpl3 and its ortholog in corynebacterium glutamicum (cmpl1) form trimers, the same way prototypical rnd transporters from gram-negative bacteria do [42] . we believe this discrepancy to be due to the fact that the recombinant forms of mmpl3 that were crystallized were devoid of their cytoplasmic c-terminal domain which our preliminary results indicate is required for the proper oligomerization of the transporter [43] . the mmpl3 transporter is essential for the biosynthesis of tdm and the mycolylation of the cell wall arabinogalactan that are required for the replication and viability of mycobacterial cells. genetic or chemical silencing of mmpl3 leads to rapid cell death in vitro and the same is observed in cellular and in vivo acute infection models [44, 45] . therefore, mmpl3 is considered as an attractive drug target [38, [46] [47] [48] . the mmpl3 ortholog from m. tb is 99%, 64%, and 56% identical to the mmpl3 orthologs of m. bovis, m. smegmatis, and m. abscessus, respectively [49] . inhibition of the mmpl3 transporter results in the accumulation of tmm concentrations intracellularly, and a reduction in the levels of magp and tdm [36, 37, 39, 41] . there have been two proposed mechanisms by which an inhibitor can act on mmpl3. the first is that the inhibitor blocks the translocation of the tmm by directly binding to the transporter. the second mmpl3 transporters aid in the translocation of tmm across the plasma membrane for cell envelope biosynthesis. mmpl3 belongs to the resistance-nodulation-cell division (rnd) protein superfamily of inner membrane transporters. its translocation activity is dependent on the proton motive force (pmf). on the basis of a spheroplast-based translocation assay that can determine the topology of tmm in the plasma membrane, xu and colleagues proposed that mmpl3 functions as a tmm flippase [40] . in the mmpl family, mmpl3 is an essential mmpl transporter indicating that its function is unique. co-crystallography and native mass spectrometry studies indicated that mmpl3 not only binds tmm but also phospholipids such as phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin suggesting that it might be involved in the translocation of more than one lipid to the periplamic space [39] . determination of the crystal structure of mmpl3 from m. smegmatis indicates that mmpl3 functions as a monomer [39, 41] . although this observation is contradictory to earlier gel filtration and single-particle electron microscopy studies indicating that mmpl3 and its ortholog in corynebacterium glutamicum (cmpl1) form trimers, the same way prototypical rnd transporters from gram-negative bacteria do [42] . we believe this discrepancy to be due to the fact that the recombinant forms of mmpl3 that were crystallized were devoid of their cytoplasmic c-terminal domain which our preliminary results indicate is required for the proper oligomerization of the transporter [43] . the mmpl3 transporter is essential for the biosynthesis of tdm and the mycolylation of the cell wall arabinogalactan that are required for the replication and viability of mycobacterial cells. genetic or chemical silencing of mmpl3 leads to rapid cell death in vitro and the same is observed in cellular and in vivo acute infection models [44, 45] . therefore, mmpl3 is considered as an attractive drug target [38, [46] [47] [48] . the mmpl3 ortholog from m. tb is 99%, 64%, and 56% identical to the mmpl3 orthologs of m. bovis, m. smegmatis, and m. abscessus, respectively [49] . inhibition of the mmpl3 transporter results in the accumulation of tmm concentrations intracellularly, and a reduction in the levels of magp and tdm [36, 37, 39, 41] . there have been two proposed mechanisms by which an inhibitor can act on mmpl3. the first is that the inhibitor blocks the translocation of the tmm by directly binding to the transporter. there are no fda-approved and marketed antibiotics that inhibit mmpl3. however, mmpl3 inhibitors do not share a common pharmacophore, suggesting that mmpl3 is a promiscuous drug target [53] . this article focuses on the new scaffolds of mmpl3 inhibitor found to be active against ntm species. there are no fda-approved and marketed antibiotics that inhibit mmpl3. however, mmpl3 inhibitors do not share a common pharmacophore, suggesting that mmpl3 is a promiscuous drug target [53] . this article focuses on the new scaffolds of mmpl3 inhibitor found to be active against ntm species. indole-2-carboxamide derivatives are one of the most extensively studied classes of the mmpl3 inhibitors. the compounds of this class show potent action against m. tb and ntm species. the nitd compounds (shown in figure 3 ) are previously reported indole-2-carboxamides under preclinical evaluation [54] . structurally, the compound consists of an indole ring at the left-hand side (lhs) and a cycloaliphatic group at the right-hand side (rhs) connected through an amide linkage. the compounds containing indole-2-carboxamide pharmacophore are additionally reported to be effective against drug-susceptible and drug-resistant tb [55] . in 2017, franz and colleagues reported series of indole-2-carboxamides with activity against ntm species including m. abscessus, m. bolletti, m. massiliense, m. avium, m. smegmatis, and m. cholenae [56] . the compounds were synthesized into two miniseries: unsubstituted indoles and 4,6-dimethyl substituted indoles. from the first series, adamantyl-and isopinocampheyl-containing unsubstituted indoles show activity against rgm with mic <1 µ g/ml, except m. smegmatis. from the second series, the compound with the highest potency contained a cyclooctyl ring and 4,6-dimethyl indole (1, table 1 ), having an activity ranging from 0.0039 to 0.6 µ g/ml against rgm. compounds with cycloheptyl, isopinocampheyl, and 4-methylcyclohexyl head groups also showed significant activity against mycobacteria. replacing the head group with an aliphatic long chain, phenyl ring, or surprisingly, adamantyl group rendered the compound ineffective. compound 1 is also active against sgm m. avium with mic = 0.05-1 µ g/ml and m. xenopi with mic = 0.25 µ g/ml. in vitro cytotoxicity studies show no toxicity against thp-1 cells and the selectivity index (si) of >1910 for m. abscessus, m. massiliense, and m. bolletii [56] . in vivo studies with 1 conducted in mice demonstrated good oral bioavailability and were efficacious in m. abscessus infected mice [57, 58] . compound 1 was also indole-2-carboxamide derivatives are one of the most extensively studied classes of the mmpl3 inhibitors. the compounds of this class show potent action against m. tb and ntm species. the nitd compounds (shown in figure 3 ) are previously reported indole-2-carboxamides under preclinical evaluation [54] . structurally, the compound consists of an indole ring at the left-hand side (lhs) and a cycloaliphatic group at the right-hand side (rhs) connected through an amide linkage. the compounds containing indole-2-carboxamide pharmacophore are additionally reported to be effective against drug-susceptible and drug-resistant tb [55] . in 2017, franz and colleagues reported series of indole-2-carboxamides with activity against ntm species including m. abscessus, m. bolletti, m. massiliense, m. avium, m. smegmatis, and m. cholenae [56] . the compounds were synthesized into two miniseries: unsubstituted indoles and 4,6-dimethyl substituted indoles. from the first series, adamantyl-and isopinocampheyl-containing unsubstituted indoles show activity against rgm with mic <1 µg/ml, except m. smegmatis. from the second series, the compound with the highest potency contained a cyclooctyl ring and 4,6-dimethyl indole (1, table 1 ), having an activity ranging from 0.0039 to 0.6 µg/ml against rgm. compounds with cycloheptyl, isopinocampheyl, and 4-methylcyclohexyl head groups also showed significant activity against mycobacteria. replacing the head group with an aliphatic long chain, phenyl ring, or surprisingly, adamantyl group rendered the compound ineffective. compound 1 is also active against sgm m. avium with mic = 0.05-1 µg/ml and m. xenopi with mic = 0.25 µg/ml. in vitro cytotoxicity studies show no toxicity against thp-1 cells and the selectivity index (si) of >1910 for m. abscessus, m. massiliense, and m. bolletii [56] . in vivo studies with 1 conducted in mice demonstrated good oral bioavailability and were efficacious in m. abscessus infected mice [57, 58] . compound 1 was also identified as a screening hit performed by low and colleagues, where 1 is reported as mmv687146 with mic 50 = 0.8 µg/ml [59] . the sar was further explored by replacing the 4,6-dimethyl groups with halogen functional groups. as aromatic methyl groups are vulnerable to cyp-mediated benzylic oxidation, replacing them with halogens may result in compounds with higher metabolic stability and similar lipophilicity when compared to previous series [60, 61] . from the series of compounds tested, 2, 3, and 4 showed activity similar to compound 1 against m. abscessus, with mic values of 0.125 µg/ml. compound 2 is a 4,6-dichloro substituted indole, whereas 3 is a 4,6-difluoro substituted indole. compound 4 retains similar activity as a monosubstituted 6-bromo-indole. a 4,6-dibromo-substituted indole was not studied. the bulkier cyclooctyl ring is optimal for the activity against m. abscessus [62] . variation in the amide linkage can result in loss of activity. linking functionalities including ketoamide, oxamide, 1,1-diamide-amine, and thiazole-2-amide were assessed against m. tb but did not improve activity [60] . compounds 2 and 4 were found to be equally effective against 30 clinical strains including smooth (s) and rough (r) variants isolated from the cf and non-cf patients. both compounds act by inhibiting tmm transport and lack activity against ag85 complex enzymes. compound 2 has the capacity to permeate human macrophages to elicit its action. however, the adme properties of 2 show low permeability, high plasma protein binding, and high intrinsic clearance. in addition, the mmpl3 a309p mutant shows high resistance to 2 [62] . [63] . this interesting scaffold is thought to act pleiotropically, having multiple other cell membrane-embedded targets, including mmpl3. li and colleagues found that 5 disrupts the pmf which may contribute to the inhibition of mmpl3. in addition to reducing extracellular levels of tdm, it also decreases the level of other cellular lipids. however, 5 does not have a docking pose with the mmpl3 protein, is not cross-resistant with direct mmpl3 inhibitors, and retains activity against the mmpl3 resistant mutants [64] . compound 5 is an optimized analog of sq109, 1-geranylindole or (5-fluoro-[(e)-1-(3,7dimethylocta-2,6-dien-1yl)]-3-(piperidin-1-ylmethyl)-1h-indole) with moderate activity and selectivity (mbc 90 = 12 µm and ic 50 vero = 22 µm). substitutions at the 1-and 3-positions on 5 are essential to maintain antimycobacterial action. various side chains have been assessed at the 1-position, such as n-butyl, n-octyl, 3-cyanopropyl, n-substituted aminocarbonylmethyl, benzyl, phenpropyl, phenoxypropyl, and cyclohexylethyl. optimal action is produced by the n-octyl side chain. moreover, an increase in chain length up to 12 carbons does not affect the activity. next, the substitution of the saturated cyclic ring at the third position is important. replacement of the azepane ring with an aliphatic chain with a tertiary nitrogen decreases the activity. substitution with cyclopropyl or cyclopentyl causes a small drop in the activity, whereas 4-methylpiperidine and n-methyl cycloheptylamine retain activity. while morpholine, piperazine, and thiazine groups decrease the activity, the 1,4-dioxa-8-azaspiro [4.5] decane (m. bovis mic 90 = 5 µm) shows higher activity than 5 (m. bovis mic 90 = 6 µm). replacing the fluoro group with chloro, bromo, or methoxy maintains the activity. however, the position of the groups correlates with the activity. azaspiroketals with 6-methoxy substitutions are potent, whereas azapenes with 4-fluoro substitutions are potent compounds [63] . despite being active against mycobacteria, 5 also has activity against the gram-positive bacteria s. aureus (mic 50 = 8 µm), but is not active against the gram-negative bacteria e. coli, suggesting 5 interacts with additional pharmacological targets. however, 5 has some toxicity against hepg2 and vero cell lines (ic 50 = 19.2 µm and 29.2 µm, respectively). in vitro studies determined moderate solubility (35.9 µm) and good metabolic stability with a half-life of 560 min tested on rat liver microsomes [63] . considering the promising activity against m. avium, further optimization of this scaffold is warranted to reduce potential for toxicity. additionally, the azaspiroketal derivatives were found to be more potent than 5 against m. tb and directly bind mmpl3, as supported by cross-resistance with other mmpl3 inhibitors. in vivo pharmacokinetic studies in murine models have been reported, proving promising adme parameters, and screens against ntm species should be pursued [64, 65] . in 2014, gobis et al. reported 6 as an antimycobacterial agent with activity against m. tb and m. bovis [66] . the mechanism of action for this compound was later identified as mmpl3 inhibition [67] . recent in vitro studies of benzimidazole derivatives show bacteriostatic action of 6 against m. abscessus clinical isolates including s and r variants from cf and non-cf patients. unlike the optimal 4,6-disubstitution patterned indole-2-carboxamides, the highly potent compounds from this series are 5,6-disubstituted benzimidazoles. in addition, this series lacks the amide side chain and the bulky cycloaliphatic group such as cyclooctyl, adamantyl which is thought to be a characteristic feature of mmpl3 inhibitors. despite this, 6 has similar activity to that of previously reported indoles 2, 3, and 4 (mic = 0.125 µg/ml). similar to indole-2-carboxamides, the nh hydrogen is required to form an h-bond with the mmpl3 transporter. replacement of the hydrogen with the aryl sulfonyl group results in inactivity. in contrast to indole-2-carboxamide derivatives, mono-substitution at the 6 position of the phenyl ring of benzimidazole significantly decreases the activity. increasing the length or degree of unsaturation of the chain also decreases the activity. a small mini-series with a phenyl group replacing the cyclohexyl group on the rhs was also assessed. all compounds within the series were inactive except one with a 3,5-dichlorophenyl having the mic = 0.25 µg/ml [68] . benzimidazole derivatives show acceptable cytotoxicity with si of 712 against thp-1 macrophages. compound 6 decreases the intracellular bacterial load in infected macrophages and in embryonic zebrafish models. however, the study reveals that many mmpl3 mutants are resistant to benzimidazole derivatives. in assessing the mmpl3 mutants, the potency drops >64-fold for the a309p mutant while a 4-to 8-fold drop is seen for the other mutants. however, greater resistance was seen against au1235 than for 6. of note, 2 (indole-2-carboxamide) is effective against all mmpl3 mutants, except the a309p mutant. compound 6 is cross-resistant with compound 13 (piperidine derivative) but not with sq109. it is assumed that 6 acts by direct mmpl3 inhibition, based on the available data. all reported mutants, except v299a, have mutations located far from the binding site of mmpl3. it is thought that mutations in mmpl3 can induce long-distance structural rearrangements that can result in reduced drug-binding affinity [50, 68] . william and colleagues identified mmpl3 inhibitors through high-throughput screening. of the hit compounds, two contain the benzimidazole pharmacophore. compound 7, 2-[(5-chloro-1h-benzimidazol-2-yl)sulfanyl]-n,n-di(propan-2-yl) acetamide is the only active benzimidazole and was among the most potent compounds identified in the screen against m. abscessus. compound 18 (4-thiophen-2-yloxane-4-carboxamide analog) was the most potent compound against m. abscessus among the hits (described below). compound 7 contains a sulfur group in the amide side chain and lacks the cycloaliphatic ring system, instead having two isopropyl substituents on the amine. it shows 81.8% growth inhibition for m. abscessus at the single concentration of 20 µm (mic 50 = 25µm). the compound is selective against the mycobacteria species and lacks activity against gram-positive and gram-negative bacteria (>200 µm). compound 7 has been shown to disrupt the membrane potential, thereby, suggesting its activity may be through disruption of pmf. the bone marrow murine macrophage cytotoxicity study reveals it as a safe compound (cc 50 > 100 µm). additionally, 7 has good solubility at 178 µm and good microsomal stability of 71% remaining 30 min after administration. benzimidazoles with a 5-methyl instead of a 5-chloro group lack activity against m. abscessus, suggesting the importance of having an electron-withdrawing group on the benzimidazole ring. the methyl group is also susceptible to the cyp-mediated benzylic hydroxylation and resulted in reduced microsomal stability, dropping it from 71% to 25% [48] . recently, dal molin et al. reported additional benzimidazole derivatives, 8 and 9, as putative antimycobacterial agents targeting the mmpl3 transporter. using gfp-based high-throughput screening, several hits were identified against m. tb and m. abscessus. compound 8 showed an mic 90 of 0.98 µm against m. tb. the potency for 8 drops to 62.5 µm against the drug-resistant mutants. compound 9 is cross-resistant to 8, and similarly, has a higher mic of >62.5 µm against 8 resistant mutants. both compounds have an mic 90 of 31.25 µm against m. abscessus. compound 8 was also able to significantly reduce mycobacterial load in an infected macrophage. the pharmacophore for 8 and 9 is an aminobenzimidazole, which differs from 6 and 7. compound 8 differs from 9 by the substitution on the benzimidazole ring. compound 8 is 5,6-dimethyl substituted, whereas 9 is 5-chloro,6-methyl substituted. compound 8 and 9 lack the amide side chain, like 6, and lack the cycloaliphatic group at the rhs, like 7. instead, they each contain a 2-isopropyl-1-aniline ring attached to the 2-benzimidazole. however, 8 shows high toxicity against thp-1 macrophages (15.625 µm) [69] . there is no evidence of how 8 and 9 inhibit mmpl3, direct or indirect. moreover, there are no reported in vivo pharmacokinetic studies for 8 and 9. optimization is required to increase potency against ntm species. graham et al. report 11 as a potent antimycobacterial agent showing activity against m. abscessus atcc 19977, m. avium 101, m. intracellulare 1956, and m. tb h37rv. the pharmacophore of this benzothiazole derivative is different than the two previously discussed. the nh of the benzimidazole is replaced by a sulfur atom, comprising the benzothiazole aromatic ring system. moreover, the amide linkage is reversed here such that the nh of the amide is attached to the benzothiazole ring (lhs) instead of the alicyclic ring (rhs) as seen in other mmpl3 inhibitor classes. compound 11 is an optimized version of the previous hit 10, which contained a dimethylated adamantyl ring (m. abscessus mic = 1 µg/ml, m. avium mic = 2 µg/ml, m. tb mic = 4 µg/ml). various cycloaliphatic groups (rhs) were studied by replacing the adamantyl group because the adamantyl ring is lipophilic and may lead to nonspecific binding. this is a common problem for not only mmpl3 inhibitors, but many antimycobacterial drug discovery efforts as the mycobacterial cell wall is lipid-rich with mycolic acids, leading to initial lipophilic hits that can effectively penetrate the cell wall. out of the screening process, 3, 3, 5-trimethylcyclohexyl group is found to be most effective against m. abscessus. various mono-, di-, and tri-substitution patterns using functional groups such as x, ch 3 , cf 3 , ocf 3 , och 3 , scf 3 , nch 3 , and oh were assessed on the benzothiazole ring (lhs). high activity against m. abscessus was seen in the 5,7 di-substituted and 6 mono-substituted patterns, with halogens such as fluorine and chlorine proving most effective. however, activity is low against other mycobacteria. it was suggested that the 1-methylated cycloaliphatic ring improves activity against rgm and sgm. the 1-methylcycloheptyl derivative shows >530-fold and >8-fold increase in activity against m. abscessus and m. avium compared to that of the unsubstituted cycloheptyl [70] . the ineffectiveness of the unsubstituted cycloheptyl against m. abscessus using cip 104536 susceptible strain is reported in another study [62] . moreover, it retained activity against sgm such as m. avium 101 (2 µg/ml), and m. intracellulare 1956 (2 µg/ml), m. chimaera 1502055 (1 µg/ml), and m. tb h37rv mc 2 6206 (≤0.12 µg/ml). active mmpl3 inhibitors, must be lipophilic enough to penetrate the cell envelope. benzothiazole derivatives exhibited bacteriostatic action against m. abscessus and have no activity against gram-positive and -negative bacteria up to 32 µg/ml. in vitro cyp enzyme inhibition studies show that 11 does not inhibit cyp2b6 and cyp3a4 more than 50% at 10 µm, cyp2b6 and cyp3a4 are the potential cyp enzymes for pulmonary delivery. also, no hemolytic activity was determined. unsurprisingly, this class of compounds suffers from low solubility (<3 µm) and high plasma protein binding (>95%). an in vivo study of 10 using granulocyte macrophage-colony-stimulated factor knockout (gm-csf ko) mice established that there is a reduction of 0.64 log 10 cfu in comparison to the vehicle group, but there is no significant reduction observed when compared to the untreated group [71] . the pharmacokinetics in a mouse model for one of its derivatives show 75% oral bioavailability and 1.5 l/h/kg plasma clearance after iv administration (10 mg/kg). in vivo screening, pharmacokinetic studies, and development of an inhaled formulation is in progress for this class [70] . however, mic assays using clinically isolated s and r variants need to be completed. in 2014, shah and colleagues report n-arylalkylbenzo[d]thiazole-2-carboxamides as anti-mycobacterial agents. these compounds contain the benzothiazole ring as an aromatic group on the lhs and a substituted aromatic phenyl ring on the rhs. unlike indole-2-carboxamides, they contain an additional carbon between the nh amide and the aromatic group on the rhs. most compounds from the series possess a mic range of 3.125-50 µg/ml against m. tb, except two compounds with mics of 0.78 µg/ml (12) and 1.56 µg/ml (not mentioned). compound 12 has low toxicity on the hek-293t (human embryonic 25 kidney cell line) with 32.16% inhibition at 50 µg/ml concentration. surprisingly, even though it has mmpl3 inhibitor characteristics, molecular modeling suggests that it is a hisg inhibitor. currently, there are no reported studies assessing efficacy of 12 against ntm [72] . considering the potency of other benzothiazoles, 10 and 11, 12 should be screened against ntm species and assessed for mmpl3 inhibition. ex vivo studies against thp-1 cell macrophages suggest that the compound enters murine and human macrophages, and has potential to decrease intracellular bacterial replication. in vivo zebrafish infection models reveal that 13 significantly decreases the bacterial load in the treated group as compared to the untreated group. however, mab_4508 mutants show high level resistance to the piperdinol derivatives, decreasing the potency of 13 by 4-to 64-fold. this mmpl3 inhibition decreases tdm formation and ag mycolylation in the periplasm [74] . recently, the piperidinol class was explored which led to the identification of 14. the sar suggests the importance of two aromatic rings, ring a and ring b, on both sides of the piperidinol. the two substituents, -cf 3 and -cl, on ring a are essential for activity. removing or changing the position of one or both substituents will significantly decrease the activity. similarly, the substitution of the small ortho group on ring b is optimal for the activity. replacing the -ch 3 to -cl on ring b yields 14 with the same potency (mic 99 = 0.125 µg/ml), suggesting preference for a lipophilic group. similarly, orthoiodine or bromine-containing molecules have similar potency to that of 14, but replacement with a fluorine substituent shows an eight-fold decrease in activity. this supports the role of steric hindrance at the receptor site, with larger radii atoms being preferred over smaller radii atoms, suggesting a twisted bioactive conformation. molecular modeling of the piperidinol derivative reveals that the nitrogen of 13 interacts with the carboxylic group of d618 and the hydroxyl of 13 interacts with the hydroxyl of y219 through hydrogen bonding. ring b is required to interact with the phenyl ring of f262 and f622 through π-π stacking interactions [75] . in vitro cytotoxicity studies in a vero cell line show that 14 is safe, achieving selectivity indices (si) of 400, whereas 13 has a si of 200 against m. abscessus. a preclinical pharmacokinetic study was performed on balb/c mice with drug administered via intraperitoneal (ip) route. the highest dosage used in the study was 250 mg/kg, which resulted in a 0% survival rate after 48 h. however, for the 100 mg/kg and 50 mg/kg groups, the survival rates increased to 67% and 100% after 96 h, respectively. more pharmacokinetic parameters were assessed, such as clearance rate, volume of distribution, and elimination half-life, none of which differ significantly from the values obtained by low et al. the clearance rate and peripheral volume of distribution are 6.9 ml/min and 2.0 l, and the elimination half-life is about 3.2 h. compound 13 has poor oral bioavailability, calculated at less than 1% [59] . currently, there are ongoing research efforts to improve the metabolic stability and polarity of this class of compound [75] . recently [48] . chemically, 17 is a n-[2-(4-chlorophenyl)ethyl]-4-thiophen-2-yloxane-4-carboxamide. this class of compounds is thought to directly inhibit mmpl3 [76] . the ethyl phenyl side chain on the nh of amide is optimal for the activity, removal of ethyl linker drastically decreases activity against m. tb, except for 4-isopropylphenyl and 4-trifluoromethyl. replacing the para-chloro with other groups led to loss of activity [76] . in vitro studies show that 17 binds mmpl3 directly without disrupting the membrane potential. there is no cross-resistance observed with sq109, suggesting different binding interactions with mmpl3. compound 17 shows a bacteriostatic effect in macrophages and is safe up to 200 µm [76] . compound 17 has good kinetic solubility of more than 300 µm. however, it exhibits poor microsomal stability, with only 45% remaining after 30 min [48] . it is also reported that the replacement of the thiophene group is warranted as it is susceptible to in vivo metabolism [76] . this interesting class of compounds should be further explored, as it shows potent activity against rgm along with desirable physicochemical properties. although benzofuran derivatives are ineffective against m. tb [58] , they possess activity against m. abscessus drug susceptible strain cip 104536 with a mic of 0.5-1 µg/ml [62] . structurally, it resembles the indole-2-carboxamide pharmacophore, except with an oxygen instead of a nitrogen in the aromatic ring system. therefore, this class lacks a potential h-bond donor interaction with mmpl3 which would have been formed by the nh of the indole. as of now, three benzofuran derivatives have been studied against m. abscessus. the two potent compounds (18 and 19) contains the cyclooctyl group as the cycloaliphatic ring on the rhs (mic = 0.5 µg/ml). compound 18 contains 4,6-dimethylbenzofuran whereas 19 contains 5-chlorobenzofuran. the cyclooctyl group is optimal for the activity, as replacement with an adamantyl ring decreases the activity by two-fold, mic = 1 µg/ml [62] . currently, there are no cytotoxicity studies or pharmacokinetic data available for this class of compounds. alsayed et al. screened a mini-series of quinolones and quinoline derivatives and determined them to be mmpl3 inhibitors. two quinolone derivatives showed putative antimycobacterial activity against m. tb with mics below 10 µg/ml, whereas quinoline derivatives were ineffective. all the quinolone compounds contain mono-or di-substitution on the lhs and the cyclooctyl or 1-adamantyl ring on the rhs. compound 20 is a 5-bromo-quinolone linked with 1-adamantyl ring through an amide side chain (m.tb mic = 4 µg/ml). potency decreases by two-fold when the 5-bromo is changed to 7-bromo (m.tb mic = 8 µg/ml), and 5,7-disubstitution drops the potency by four-fold as compared with 20. similarly, 6 or 8 mono-substitution or other di-substitutions result in loss of activity. the activity is higher for the quinolones than its tautomer, 4-hydroxyquinoline, suggesting the importance of the nh in the ring system which may form a h-bond with the receptor. in addition, the carboxamide side-chain at the 2-position is essential for activity, as changing from quinoline-2-carboxamide to quinoline-4-carboxamide results in loss of activity. although 20 is potent among all other quinolones against m. tb it lacks activity against sgm m. avium and rgm m. abscessus (mic > 159 µm). it exhibits low toxicity against vero cells (ic 50 = 39.87 µm) and has a si value of 4. however, 20 was found to have poor pharmacokinetic parameters, including solubility of 0.007 mg/ml, caco-2 permeability of 186 × 10 −6 cm/s, and 98% plasma protein binding [60] . further optimization is required to obtain activity against ntm species. in contrast to previous findings that the quinolines are ineffective, dan molin and colleagues identified the quinoline derivative, 21, which was found to be active against m. tb having a mic 90 of 7.8-15.6 µm. however, activity against m. abscessus is minimal (mic 90 > 31.25 µm). the scaffold varies from other mmpl3 inhibitors and 20. the removal of the methoxy group results in complete loss of activity against m. abscessus. unfortunately, 21 is associated with high cytotoxicity in thp-1 (7.8125 µm) [69] . currently, there is no available data in vivo pharmacokinetics or on how 21 binds mmpl3. further optimization of the compound to reduce cytotoxicity and improve activity against ntm species is needed. naphthalene derivatives were designed and synthesized as bioisosteric replacements of indole derivatives and further expanded the quinoline and quinolone study. five naphthalene-2-carboxamide derivatives were synthesized and screened in this study. out of the five compounds synthesized, two unsubstituted naphthalenes (22 and 23) were found to have activity against m. tb. they are structurally similar to each other, except for the cycloaliphatic ring on the rhs. compound 22 contains a cyclooctyl group whereas 23 contains an adamantyl group. adding substitutions to the naphthalene ring system is detrimental for antimycobacterial activity. compounds 22 and 23 are active against drug-susceptible and mdr m. tb (mic = 2 µg/ml and 6.5-13 µm, respectively), and have a two-fold increased potency against xdr tb strains (3.27-3.55 µm). however, they lack activity against ntm species such as m. avium and m. abscessus (mic > 209 µm) [60] . a cytotoxicity study showed no toxicity against vero cell lines (>227 µm and >419 µm). the molecular docking study performed on the compounds reveals that there is one less h-bond interaction with the mmpl3 receptor as compared to indole-2-carboxamides, suggesting reduced binding affinity. this supports the importance of the nh hydrogen within the indole ring. despite the fact that naphthalene and quinoline derivatives both lacks the nh hydrogen; activity is high with the naphthalene derivatives. because of high lipophilicity of the naphthene ring, these compounds are likely to have poor pharmacokinetic properties. the solubilities of 22 and 23 are 0.002 and 0.007 mg/ml. furthermore, 22 and 23 have caco-2 permeabilities of 190 and 207 × 10 −6 cm/s and plasma protein binding of 95% and 97% [60] . recently, gajdár et al. reported the 1-hydroxynaphthalene-2-carboxanilides as potent antimycobacterial agents. the study was performed using cyclic voltammetry and activity was determined based on electrochemical potential. compounds 24 and 25 were found to be effective against m. marinum and m. kansasii with mics of 28.8 µm (24) and 28.4 µm (25), and were determined to have similar activity against m. tb. using spinach chloroplast, the study showed that these compounds inhibit photosynthetic electron transfer (pet). it is thought that these derivatives have a multi-target effect including atp synthase and cytochrome bc1 with the disruption of the energy state of the cell [77] . acetamides can be formed with the deletion of 3-carbon atom from the indole-2-carboxamide derivatives. like indole-2-carboxamides, acetamides contain two rings, the aromatic ring on the lhs and the cycloaliphatic on the rhs, linked together with an amide moiety. shetty et al. were the first to report acetamides as a new class of mmpl3 inhibitors. using a single-point m. bovis bcg screening assay, they identified 16 hits with mics below 50 µm. compound 26 was one of these hits, with an mic 50 to explore the sar of 26, ten acetamide analogs were synthesized using the hit as the main pharmacophore. of the ten acetamides, only four compounds showed an increase in activity (mic 50 = 3-7 µm), whereas the other six compounds showed a drastic decrease in activity (mic 50 = 10->100 µm). the lipophilic groups on the aromatic ring are essential for activity. replacing the 4-trifluoromethyl with polar moieties such as cyano or ester groups decreased the activity, while activity is retained or increased with 3,5-dichloro or 3-or 4-trifluoromethoxy. changing the 3-or 4-trifluoromethoxy to 2-trifluoromethoxy causes inactivity (mic 50 > 100 µm). cycloaliphatic rings such as cyclohexane or cycloheptane (27) showed highest potency against m. tb (mic 50 = 3 µm), suggesting preference for bulkier cycloaliphatic groups. replacement of 1-cyanocyclopentane with an aliphatic group such as 1-cyano-1-methyl-isopropyl causes only a slight decrease in activity (mic 50 = 10 µm) and replacement with 1-cyano-1-methyl-cyclopropyl results in similar activity to that of the 26 (mic 50 = 7 µm) [49] . no further sar was performed to determine the importance of the 1-cyano group on the cycloaliphatic group. this scaffold is interesting for further sar development to determine if the activity is correlated to quaternary carbon or cycloaliphatic groups. cytotoxicity studies on 26 were performed on hepg2 (human liver carcinoma), thp-1 (human monocytic leukemia), and vero (african green monkey kidney epithelia) cell lines. results of these studies support low cytotoxicity and si of >25 µm. using rat liver microsomes, the half-life of the compound was found to be 28.6 min and the intrinsic clearance rate was 80.7 µl/min/mg of protein. as 26 lacks or possesses less activity as compared to the indole-2-carboxamides, further optimization and screening against ntm species is warranted [49] . previous screens show that the 1-substituted cyclooctyl ring has the highest potency against m. abscessus. we anticipate 27 to potentially display activity against m. abscessus. bm212 (28) is a commonly known as 1,5-diphenyl pyrrole derivative. compound 28 has shown activity against replicating and non-replicating m. tb including mdr-tb strains (mic = 8 µm). interestingly, the activity of 28 is greater against nontuberculous mycobacteria (mic m.avium = 1 µm) than m. tb (mic = 5 µm). compound 28 shows moderate cytotoxicity in vero cell lines (ic 50 = 9.6 µm) and has a si value of 1.92. a series of compounds (>300) was generated to improve the potency and physicochemical properties and reduce the cytotoxicity including herg inhibition. inhibition of the herg channel is responsible for the cardiotoxicity which was also associated with the spiro analog ( figure 3 ). despite good in vivo activity, the pyrrole derivatives suffer from poor solubility and metabolic profile, high plasma binding, and high herg channel inhibition. the proposed reason is the highly sp 2 region and easily protonated nitrogen in the structure. a hit-to-lead approach led to the discovery of 29 with improved in vitro and in vivo activity along with good physicochemical properties. the early sar suggests that the para-fluorophenyl at the n1 increases potency and reduces toxicity compared to 28. to maintain activity, lipophilic groups such as fluoro or electron donating groups such methoxyl or methylsulfanyl are required on the c5 position of the phenyl ring. the (thiomorpholin-4-yl) methyl ring has higher activity than the n-methylpyperazinyl group, later replaced with the morpholine group because of the metabolic instability. adding n-methylpiperazino-methyl or (thiomorpholin-4-yl) methyl group at the c4 position was found to be detrimental to activity. the para-isopropyl phenyl at c5 showed the highest microsomal stability in the mouse model. the sar was further studied by removing or replacing n1-and c5-phenyl with alkyl or cycloalkyl in order to decrease the sp 2 character. surprisingly, alkyl substituents at the n1 position retain antimycobacterial action. therefore, new sar indicates only one phenyl ring at c5 is essential for the activity whereas the n1 phenyl can be replaced with an alkyl or cycloalkyl substituent. the morpholine group is optimal for activity at the c3 position [78, 79] . compound 29 has an isopropyl group at the n1 position and para-isopropylphenyl group at the c5 position along with c2-methyl and c3-methyl morpholine. the compound has a mic value of 0.15 µm and intramacrophagic mic of 0.16 µm against m. tb. the cytotoxicity study on hepg2 gave to × 50 of 20 µm with a si of 133. the studied pharmacokinetic properties include membrane permeability of 7.2 × 10 −5 cm/s, 94.11% hsa binding, and clnd solubility of 199 µm. however, optimization is required to reduce the cytotoxic herg inhibition (16 µm) . moreover, in vivo study report poor absorption and low bioavailability (f = 1.2%). compound 29 has moderate systemic clearance value of 16.9 ml/min/kg and a half-life of 1.7 h. at a dose of 50 mg/kg, 29 shows 1.5 log cfu reduction after 9 days of infection [79] . many pyrrole derivatives from the series have submicromolar activity against m. tb. ultimately, only 28 has shown potent activity against m. avium. given the medical need, the screening of pyrrole derivatives against mac and other ntm species is needed. alkyl or cycloalkyl in order to decrease the sp 2 character. surprisingly, alkyl substituents at the n1 position retain antimycobacterial action. therefore, new sar indicates only one phenyl ring at c5 is essential for the activity whereas the n1 phenyl can be replaced with an alkyl or cycloalkyl substituent. the morpholine group is optimal for activity at the c3 position [78, 79] . compound 29 has an isopropyl group at the n1 position and para-isopropylphenyl group at the c5 position along with c2-methyl and c3-methyl morpholine. the compound has a mic value of 0.15 μm and intramacrophagic mic of 0.16 μm against m. tb. the cytotoxicity study on hepg2 gave to × 50 of 20 μm with a si of 133. the studied pharmacokinetic properties include membrane permeability of 7.2 × 10 −5 cm/s, 94.11% hsa binding, and clnd solubility of 199 μm. however, optimization is required to reduce the cytotoxic herg inhibition (16 μm) . moreover, in vivo study report poor absorption and low bioavailability (f = 1.2%). compound 29 has moderate systemic clearance value of 16.9 ml/min/kg and a half-life of 1.7 h. at a dose of 50 mg/kg, 29 shows 1.5 log cfu reduction after 9 days of infection [79] . many pyrrole derivatives from the series have submicromolar activity against m. tb. ultimately, only 28 has shown potent activity against m. avium. given the medical need, the screening of pyrrole derivatives against mac and other ntm species is needed. m. abscessus = 0.063 µg/ml alkyl or cycloalkyl in order to decrease the sp 2 character. surprisingly, alkyl substituents at the n1 position retain antimycobacterial action. therefore, new sar indicates only one phenyl ring at c5 is essential for the activity whereas the n1 phenyl can be replaced with an alkyl or cycloalkyl substituent. the morpholine group is optimal for activity at the c3 position [78, 79] . compound 29 has an isopropyl group at the n1 position and para-isopropylphenyl group at the c5 position along with c2-methyl and c3-methyl morpholine. the compound has a mic value of 0.15 μm and intramacrophagic mic of 0.16 μm against m. tb. the cytotoxicity study on hepg2 gave to × 50 of 20 μm with a si of 133. the studied pharmacokinetic properties include membrane permeability of 7.2 × 10 −5 cm/s, 94.11% hsa binding, and clnd solubility of 199 μm. however, optimization is required to reduce the cytotoxic herg inhibition (16 μm) . moreover, in vivo study report poor absorption and low bioavailability (f = 1.2%). compound 29 has moderate systemic clearance value of 16.9 ml/min/kg and a half-life of 1.7 h. at a dose of 50 mg/kg, 29 shows 1.5 log cfu reduction after 9 days of infection [79] . many pyrrole derivatives from the series have submicromolar activity against m. tb. ultimately, only 28 has shown potent activity against m. avium. given the medical need, the screening of pyrrole derivatives against mac and other ntm species is needed. alkyl or cycloalkyl in order to decrease the sp 2 character. surprisingly, alkyl substituents at the n1 position retain antimycobacterial action. therefore, new sar indicates only one phenyl ring at c5 is essential for the activity whereas the n1 phenyl can be replaced with an alkyl or cycloalkyl substituent. the morpholine group is optimal for activity at the c3 position [78, 79] . compound 29 has an isopropyl group at the n1 position and para-isopropylphenyl group at the c5 position along with c2-methyl and c3-methyl morpholine. the compound has a mic value of 0.15 μm and intramacrophagic mic of 0.16 μm against m. tb. the cytotoxicity study on hepg2 gave to × 50 of 20 μm with a si of 133. the studied pharmacokinetic properties include membrane permeability of 7.2 × 10 −5 cm/s, 94.11% hsa binding, and clnd solubility of 199 μm. however, optimization is required to reduce the cytotoxic herg inhibition (16 μm). moreover, in vivo study report poor absorption and low bioavailability (f = 1.2%). compound 29 has moderate systemic clearance value of 16.9 ml/min/kg and a half-life of 1.7 h. at a dose of 50 mg/kg, 29 shows 1.5 log cfu reduction after 9 days of infection [79] . many pyrrole derivatives from the series have submicromolar activity against m. tb. ultimately, only 28 has shown potent activity against m. avium. given the medical need, the screening of pyrrole derivatives against mac and other ntm species is needed. alkyl or cycloalkyl in order to decrease the sp 2 character. surprisingly, alkyl substituents at the n1 position retain antimycobacterial action. therefore, new sar indicates only one phenyl ring at c5 is essential for the activity whereas the n1 phenyl can be replaced with an alkyl or cycloalkyl substituent. the morpholine group is optimal for activity at the c3 position [78, 79] . compound 29 has an isopropyl group at the n1 position and para-isopropylphenyl group at the c5 position along with c2-methyl and c3-methyl morpholine. the compound has a mic value of 0.15 μm and intramacrophagic mic of 0.16 μm against m. tb. the cytotoxicity study on hepg2 gave to × 50 of 20 μm with a si of 133. the studied pharmacokinetic properties include membrane permeability of 7.2 × 10 −5 cm/s, 94.11% hsa binding, and clnd solubility of 199 μm. however, optimization is required to reduce the cytotoxic herg inhibition (16 μm). moreover, in vivo study report poor absorption and low bioavailability (f = 1.2%). compound 29 has moderate systemic clearance value of 16.9 ml/min/kg and a half-life of 1.7 h. at a dose of 50 mg/kg, 29 shows 1.5 log cfu reduction after 9 days of infection [79] . many pyrrole derivatives from the series have submicromolar activity against m. tb. ultimately, only 28 has shown potent activity against m. avium. given the medical need, the screening of pyrrole derivatives against mac and other ntm species is needed. alkyl or cycloalkyl in order to decrease the sp 2 character. surprisingly, alkyl substituents at the n1 position retain antimycobacterial action. therefore, new sar indicates only one phenyl ring at c5 is essential for the activity whereas the n1 phenyl can be replaced with an alkyl or cycloalkyl substituent. the morpholine group is optimal for activity at the c3 position [78, 79] . compound 29 has an isopropyl group at the n1 position and para-isopropylphenyl group at the c5 position along with c2-methyl and c3-methyl morpholine. the compound has a mic value of 0.15 μm and intramacrophagic mic of 0.16 μm against m. tb. the cytotoxicity study on hepg2 gave to × 50 of 20 μm with a si of 133. the studied pharmacokinetic properties include membrane permeability of 7.2 × 10 −5 cm/s, 94.11% hsa binding, and clnd solubility of 199 μm. however, optimization is required to reduce the cytotoxic herg inhibition (16 μm). moreover, in vivo study report poor absorption and low bioavailability (f = 1.2%). compound 29 has moderate systemic clearance value of 16.9 ml/min/kg and a half-life of 1.7 h. at a dose of 50 mg/kg, 29 shows 1.5 log cfu reduction after 9 days of infection [79] . many pyrrole derivatives from the series have submicromolar activity against m. tb. ultimately, only 28 has shown potent activity against m. avium. given the medical need, the screening of pyrrole derivatives against mac and other ntm species is needed. 10 m. abscessus = 1 µ g/ml [70] m. avium = 2 µ g/ml m. abscessus = 0.03 µ g/ml [70] m. peregrinum = 0.03 µ g/ml m. massiliense = 0.03 µ g/ml m. fortuitum = 0.03 µ g/ml m. chelonae = 0.03 µ g/ml m. avium = 2 µ g/ml m. intracellulare = 2 µ g/ml n.d. [ 10 m. abscessus = 1 µ g/ml [70] m. avium = 2 µ g/ml m. abscessus = 0.03 µ g/ml [70] m. peregrinum = 0.03 µ g/ml m. massiliense = 0.03 µ g/ml m. fortuitum = 0.03 µ g/ml m. chelonae = 0.03 µ g/ml m. avium = 2 µ g/ml m. intracellulare = 2 µ g/ml n.d. [ an inhaled liposomal amikacin inhalation suspension (arikacye ® ) has recently been approved by the fda for the treatment of mac infections of the lung in adults with limited or no alternative treatment options. this formulation is part of a combination regimen in patients who fail to achieve negative sputum cultures after a minimum of six consecutive months of first-line multidrug antibiotic therapy. the treatment is associated with many adverse effects, with n.d. [79, 80] an inhaled liposomal amikacin inhalation suspension (arikacye ® ) has recently been approved by the fda for the treatment of mac infections of the lung in adults with limited or no alternative treatment options. this formulation is part of a combination regimen in patients who fail to achieve negative sputum cultures after a minimum of six consecutive months of first-line multidrug antibiotic therapy. the treatment is associated with many adverse effects, with nephrotoxicity being the most serious [81] . in addition, greater than a two-fold drop in potency has been observed in resistant strains of mac [82, 83] . combining antibiotics should be done cautiously, as some combinations result in synergistic activity and suppression of antimycobacterial resistance, whereas other combinations exhibit deleterious drug-drug interaction. in vitro synergistic action is observed with amikacin or clarithromycin when administered with clofazimine, and here, it also prevents the re-growth of m. abscessus and m. avium [84] . similarly, in vitro studies report an increase in the action of clarithromycin against m. avium when combined with tigecycline. moreover, the combination therapy with tigecycline inhibits development of clarithromycin resistance [85] . synergistic action against m. abscessus is seen when either clarithromycin or tigecycline is co-administered with rifabutin [86] . rifabutin is also known to inhibit the inducible resistance of clarithromycin in m. abscessus [87] . conversely, in vitro negative drug-drug interaction is observed with the combination of bedaquiline and beta-lactams such as imipenem and cefoxitin [88] . in addition, there are few agents such as fs-1, smart-420, and c10 that promote reversion of drug resistance and restore the sensitivity of antibiotics. fs-1 is an anti-mdr/xdr tb drug, approved in 2015 in kazakhstan [89] . fs-1 consists of synthesized polysaccharide micellar units containing triiodide complexes coordinated by metal ions and integrated into a dextrin-polypeptide moiety resulting in a 30-300 kda complex [89] . the polysaccharides act as solubilizing agents and aid in the release of free iodine when mixed with water [90] . the released free iodine causes oxidative stress in the bacterial cell, a mechanism that drug-resistant mutants are more susceptible to [89, 91] . fs-1 induces reversion of drug resistance in mdr-and xdr-tb isolates by making them more susceptible to the oxidative stress induced by fs-1. fs-1 creates high oxidative stress inside the bacterial cell and resistance mutations are more sensitive to the oxidative stress. this led to the reduction in the drug resistance mutations. the reversion of drug resistance to aminoglycosides such as amikacin, kanamycin, and capreomycin has been observed in mdr-tb strains. adding fs-1 to the multi-drug regimen of tb prevents the accumulation of new drug resistance mutations and reduces the initial level of drug resistance in mdr-tb infections, and can reduce the duration of mdr-tb treatment from 24 months to 6-12 months [91] . the mic against the xrd-tb m. tb strain scaid 187.0 is decreased from 27.7 ± 2.4 µg/ml to 21.1 ± 2.0 µg/ml after the administration of fs-1 for 60 days. surprisingly, available data suggest that clinical xdr-tb isolates are more sensitive than the drug sensitive m. tb h37rv. in vitro studies of fs-1 against xdr-tb strain report >2-fold decrease in the mics for rifampicin, ethionamide, and pyrazinamide, followed by a small mic decrease for streptomycin and no change in mic for isoniazid. like in vitro, in vivo studies using hartley guinea pigs showed increases in susceptibility to antibiotics against m. tb isolates after treatment for 45 days. fs-1 induces dose-dependent drug resistance reversion with no direct interactions between fs-1 and the antibiotics. fs-1 also prevents recurrence of infection, which is commonly observed with antibiotics within 30 days of recovery time [89] . studies in human lymphocytes and tumor cell lines (hela and caco2) show no dna damaging action [90] . moreover, it has been observed in animal models that fs-1 reduces the hepatotoxicity caused by some antimycobacterial agents [89] . biotransformation of the antibiotics causes rapid inactivation of antibiotics via enzymes such as beta-lactamases. on the other hand, biotransformation is also required for pro-antibiotics (prodrug antibiotics) for activation. mutations in the bioactivating enzymes makes the drug ineffective. for example, mutations in katg, pnca, and etha is observed in isoniazid (inh), pyrazinamide (pza), and ethionamide (eth)-resistant clinical isolates, respectively [92] . blondiaux et al. report small molecules aborting resistance (smart-420), a spiroisoxazoline compound, which restores the sensitivity of all eth-resistant, mdr-and xdr-tb clinical isolates (figure 4 ). smart-420 provides an alternative bioactivation pathway to eth, but does not elicit any antibacterial action. although ineffective in the absence of eth, smart-420 boosts the activity of eth against eth-sensitive and -resistant m. tb. in vivo efficacy studies in a murine model confirm that eth alone is ineffective against eth-resistant strains, whereas the combination of eth with smart-420 significantly reduces the bacterial load (4.6 log) in mouse lungs in a dose-dependent manner. the study proposed that smart-420 triggers cryptic drug-bioactivation pathways in drug-resistant pathogens [92] . these examples provide insight that drug resistance is not irrevocable and discovery of small molecules could reverse it. further studies to investigate additional compounds that reverse drug resistance and evaluate use of such agents in combination with antibiotics against drug-resistant ntm strains are warranted. the current treatment for the ntm infection includes three antibiotics for at least for 18 months or 12 months after the first negative culture. the regimen includes a combination of approved tb drugs, macrolides, and aminoglycosides. however, treatment outcomes are often poor, the medications used are associated with serious adverse reactions, and there is a high chance of relapse or reinfection to the cured patient. in addition, ntm species are rapidly acquiring resistance to currently available antibiotic options, with resistance being observed even in recently approved drugs [69] . the liposomal amikacin inhalation suspension was recently approved for the treatment of m. avium infections. however, the resistance to the amikacin causes drop in the potency by more than two-fold [82] . therefore, new drugs with novel mechanisms are urgently needed. recently, many compounds have been developed that show activity against tb and ntm infections that target the mmpl3. as mmpl3 inhibitors act at the level of the plasma membrane, it is possible that some if not all of them can avoid biotransformation by cytoplasmic enzymes. mmpl3 is an attractive and promiscuous target for tb drugs that plays an important role in cell envelope biosynthesis. tmm, the mycolic acid donor, is synthesized within the bacterial cell and translocated into the periplasm via a mechanism involving the mmpl3 transporter. mmpl3 utilizes the proton motive force to facilitate this translocation. inhibition of mmpl3 transporter increases tmm levels inside the cell and decreases mycolic acids in the periplasm, thus exerting a bactericidal effect. as mycolic acid biosynthesis is required for the viability in all mycobacteria, mmpl3 inhibitors are effective against both m. tb and ntm species. all inhibitors whose interaction with mmpl3 has been characterized to date appear to inhibit mmpl3 directly although some of them display additional disrupting activity on the pmf that may further potentiate their activity against the transporter and confer upon them activity against non-replicating bacilli [50] . the classes of mmpl3 inhibitors explored against ntm species are indoles, benzimidazoles, benzothiazoles, quinolines and quinolones, benzofurans, thiophenes, naphthalenes, acetamides, and piperidines. as per table 1 , indole-2-carboxamides (1, 2, 3, 4) , benzimidazole (6), benzothiazole (12), c10 inhibits m. tb pellicle formation (mic = 6.25 µm) and blocks hypoxia-induced tolerance to oxidative stress, acid stress, and isoniazid (inh) (figure 4 ). resistance against inh, a prodrug activated by katg enzymes, typically emerges as a result of mutations in katg. c10 has been found to reverse resistance to inh in m. tb katg mutants. c10 does not have activity over other antibiotics such as ethambutol, rifampicin, or streptomycin. moreover, c10 acts synergistically with q203, an etc inhibitor targeting cytochrome bc 1 which is undergoing clinical trials [93] . these examples provide insight that drug resistance is not irrevocable and discovery of small molecules could reverse it. further studies to investigate additional compounds that reverse drug resistance and evaluate use of such agents in combination with antibiotics against drug-resistant ntm strains are warranted. the current treatment for the ntm infection includes three antibiotics for at least for 18 months or 12 months after the first negative culture. the regimen includes a combination of approved tb drugs, macrolides, and aminoglycosides. however, treatment outcomes are often poor, the medications used are associated with serious adverse reactions, and there is a high chance of relapse or reinfection to the cured patient. in addition, ntm species are rapidly acquiring resistance to currently available antibiotic options, with resistance being observed even in recently approved drugs [69] . the liposomal amikacin inhalation suspension was recently approved for the treatment of m. avium infections. however, the resistance to the amikacin causes drop in the potency by more than two-fold [82] . therefore, new drugs with novel mechanisms are urgently needed. recently, many compounds have been developed that show activity against tb and ntm infections that target the mmpl3. as mmpl3 inhibitors act at the level of the plasma membrane, it is possible that some if not all of them can avoid biotransformation by cytoplasmic enzymes. mmpl3 is an attractive and promiscuous target for tb drugs that plays an important role in cell envelope biosynthesis. tmm, the mycolic acid donor, is synthesized within the bacterial cell and translocated into the periplasm via a mechanism involving the mmpl3 transporter. mmpl3 utilizes the proton motive force to facilitate this translocation. inhibition of mmpl3 transporter increases tmm levels inside the cell and decreases mycolic acids in the periplasm, thus exerting a bactericidal effect. as mycolic acid biosynthesis is required for the viability in all mycobacteria, mmpl3 inhibitors are effective against both m. tb and ntm species. all inhibitors whose interaction with mmpl3 has been characterized to date appear to inhibit mmpl3 directly although some of them display additional disrupting activity on the pmf that may further potentiate their activity against the transporter and confer upon them activity against non-replicating bacilli [50] . the classes of mmpl3 inhibitors explored against ntm species are indoles, benzimidazoles, benzothiazoles, quinolines and quinolones, benzofurans, thiophenes, naphthalenes, acetamides, and piperidines. as per table 1 , indole-2-carboxamides (1, 2, 3, 4) , benzimidazole (6), benzothiazole (12) , piperidinol (13, 14) , benzofuran (18, 19) show sub micromolar activity against m. abscessus. compounds 1, 5, and 12 must be explored further to determine the activity against m. avium. therefore, in vivo testing should be conducted on these scaffolds to further screen against other ntm species. indole-2-carboxamides are extensively explored via in vitro and in vivo studies and show promising results. however, poor physiochemical and pharmacokinetic properties remain a barrier to drug development. the challenge that most mmpl3 inhibitors face is low solubility due to high lipophilicity. compounds 7 and 17 show a good solubility with moderate activity, but 7 has high metabolic stability, in contrast to 17. other emerging novel targets such as pks13 or fadd32, enzymes involved in the mycolic acid biosynthesis, have proven effective against m. tb. however, not many compounds have been screened against ntm species. classes of pks13 inhibitors include thiophenes, benzofurans, beta-lactams, and coumestans [94] . despite thiophene (tp2) derivatives having high potency against lab and clinical strains of m. tb with mic = 1 µm , they are inactive against sgm and rgm (mic > 128 µm) [95] . similarly, pks13 inhibitors identified as hits have mic <1 µm against m. tb but >31.25 µm against m. abscessus [69] , which may explain the lack of screening attempts against other ntm species. conversely, diarylcoumarins, a class of fadd32 inhibitors, have potent action against sgm (mic of 0.83 µm and 2.4 µm against m. marinum and m. intracellulare, respectively) [96] . recently, a new class of fadd32 inhibitor, the quinolines, were reported to maintain potent antimycobacterial activity while displaying improved pharmacokinetic properties [97] . however, screening of these compounds against the ntm species has not yet been reported. in addition, fs-1 and smart-420 could be potential agents to reverse the susceptibility of the drug-resistant ntm species. both compounds show reversion of drug resistance in the mdr-tb and xdr-tb when co-administered with tb antibiotics. fs-1 significantly decreases mics of rifampicin, ethionamide, and pyrazinamide. moreover, fs-1 also alleviates the hepatoxicity caused by antibiotics. smart-420 alters etha-dependent drug-bioactivation pathways restoring susceptibility in eth-resistant pathogens. there is an urgent need for developing a drug discovery pipeline against ntm infections. the in vitro assays must include sgm and rgm species along with m. tb as part of the screening. available data suggest that tb inactive compounds may retain activity against ntm species, encouraging a broader perspective on potential candidates for treating ntm infections. in testing ntm species, different morphotypes should be assessed because of the potential for varied susceptibility to antimicrobials [26, 98] . in conclusion, mmpl3 inhibitors have potent activity against sgm and rgm. studies also report synergistic action of mmpl3 inhibitors with tb antibiotics in vitro and in vivo [99, 100] . as treatment of ntm infections involves multidrug regimens, adding an mmpl3 inhibitor to the current therapeutic regimen may be beneficial. therefore, more research is required to explore mmpl3 inhibitors against ntm species and to develop a constant pipeline of drug candidates for treatment of ntm infections. nontuberculous mycobacteria (ntm) infections |hai| cdc. available online non-tuberculous mycobacteria and the rise of mycobacterium abscessus the new phylogeny of the 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membrane transporter of trehalose monomycolate involved in mycolic acid donation to the cell wall core of mycobacterium tuberculosis new approaches to target the mycolic acid biosynthesis pathway for the development of tuberculosis therapeutics mmpl3 is a lipid transporter that binds trehalose monomycolate and phosphatidylethanolamine mmpl3 is the flippase for mycolic acids in mycobacteria crystal structures of membrane transporter mmpl3, an anti-tb drug target structure-function profile of mmpl3, the essential mycolic acid transporter from mycobacterium tuberculosis the mmpl3 interactome reveals a complex crosstalk between cell envelope biosynthesis and cell elongation and division in mycobacteria essentiality of mmpl3 and impact of its silencing on mycobacterium tuberculosis gene expression therapeutic potential of the mycobacterium tuberculosis mycolic acid transporter, mmpl3 a piperidinol-containing molecule is active against mycobacterium tuberculosis by inhibiting the mycolic 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novel pyrazole-containing compounds active against mycobacterium tuberculosis chapter three-development of mmpl3 inhibitors for tuberculosis treatment medicinal chemistry approaches to tuberculosis and trypanosomiasis improved bm212 mmpl3 inhibitor analogue shows efficacy in acute murine model of tuberculosis infection amikacin liposome inhalation suspension for mycobacterium avium complex lung disease. sr. care pharm antimycobacterial susceptibility testing of nontuberculous mycobacteria identification and drug susceptibility testing for nontuberculous mycobacteria clofazimine prevents the regrowth of mycobacterium abscessus and mycobacterium avium type strains exposed to amikacin and clarithromycin tigecycline potentiates clarithromycin activity against mycobacterium avium in vitro rifabutin acts in synergy and is bactericidal with frontline mycobacterium abscessus antibiotics clarithromycin and tigecycline, suggesting a potent treatment combination rifabutin suppresses inducible clarithromycin resistance in mycobacterium abscessus by blocking induction of whib7 and erm41 bedaquiline eliminates bactericidal activity of β-lactams against mycobacterium abscessus genomic insight into mechanisms of reversion of antibiotic resistance in multidrug resistant mycobacterium tuberculosis induced by a nanomolecular iodine-containing complex fs-1 investigations of genotoxic activity of antimicrobial/antiviral agent fs-1 in human lymphocytes and tumor cell constraints of drug resistance in-prospects for pharmacological reversion of susceptibility to antibiotics reversion of antibiotic resistance in mycobacterium tuberculosis by spiroisoxazoline smart-420 chemical disarming of isoniazid resistance in mycobacterium tuberculosis identification of novel coumestan derivatives as polyketide synthase 13 inhibitors against mycobacterium tuberculosis. part ii antituberculosis thiophenes define a requirement for pks13 in mycolic acid biosynthesis diarylcoumarins inhibit mycolic acid biosynthesis and kill mycobacterium tuberculosis by targeting fadd32. proc. natl. acad. sci discovery of heterocyclic replacements for the coumarin core of anti-tubercular fadd32 inhibitors mycobacterium abscessus, an emerging and worrisome pathogen among cystic fibrosis patients synergistic interactions of mmpl3 inhibitors with antitubercular compounds in vitro indole-2-carboxamide-based mmpl3 inhibitors show exceptional antitubercular activity in an animal model of tuberculosis infection this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license n.d. [49] 28 m. avium = 2 µ m [79, 80] 29 n.d. [79, 80] an inhaled liposomal amikacin inhalation suspension (arikacye ® ) has recently been approved by the fda for the treatment of mac infections of the lung in adults with limited or no alternative treatment options. this formulation is part of a combination regimen in patients who fail to achieve negative sputum cultures after a minimum of six consecutive months of first-line multidrug antibiotic therapy. the treatment is associated with many adverse effects, with nephrotoxicity being the most serious [81] . in addition, greater than a two-fold drop in potency has been observed in resistant strains of mac [82, 83] . combining antibiotics should be done cautiously, as some combinations result in synergistic activity and suppression of antimycobacterial resistance, an inhaled liposomal amikacin inhalation suspension (arikacye ® ) has recently been approved by the fda for the treatment of mac infections of the lung in adults with limited or no alternative treatment options. this formulation is part of a combination regimen in patients who fail to achieve negative sputum cultures after a minimum of six consecutive months of first-line multidrug antibiotic therapy. the treatment is associated with many adverse effects, with nephrotoxicity being the most serious [81] . in addition, greater than a two-fold drop in potency has been observed in resistant strains of mac [82, 83] . combining antibiotics should be done cautiously, an inhaled liposomal amikacin inhalation suspension (arikacye ® ) has recently been approved by the fda for the treatment of mac infections of the lung in adults with limited or no alternative treatment options. this formulation is part of a combination regimen in patients who fail to achieve negative sputum cultures after a minimum of six consecutive months of first-line multidrug antibiotic therapy. the treatment is associated with many adverse effects, with nephrotoxicity being the most serious [81] . in addition, greater than a two-fold drop in potency has been observed in resistant strains of mac [82, 83] . combining antibiotics should be done cautiously, an inhaled liposomal amikacin inhalation suspension (arikacye ® ) has recently been approved by the fda for the treatment of mac infections of the lung in adults with limited or no alternative treatment options. this formulation is part of a combination regimen in patients who fail to achieve negative sputum cultures after a minimum of six consecutive months of first-line multidrug antibiotic therapy. the treatment is associated with many adverse effects, with nephrotoxicity being the most serious [81] . in addition, greater than a two-fold drop in potency has an inhaled liposomal amikacin inhalation suspension (arikacye ® ) has recently been approved by the fda for the treatment of mac infections of the lung in adults with limited or no alternative treatment options. this formulation is part of a combination regimen in patients who fail to achieve negative sputum cultures after a minimum of six consecutive months of first-line multidrug antibiotic therapy. the treatment is associated with many adverse effects, with nephrotoxicity being the most serious [81] . in addition, greater than a two-fold drop in potency has n.d.[49] an inhaled liposomal amikacin inhalation suspension (arikacye ® ) has recently been approved by the fda for the treatment of mac infections of the lung in adults with limited or no alternative treatment options. this formulation is part of a combination regimen in patients who fail to achieve negative sputum cultures after a minimum of six consecutive months of first-line multidrug antibiotic therapy. the treatment is associated with many adverse effects, with funding: this project was supported by the national institutes of health/national institute of allergy and infectious diseases research grant ai116525 and an award from the cystic fibrosis foundation. the authors declare no conflict of interest. key: cord-340879-gu91cact authors: li, miao; liu, qin; cui, yajuan; li, dong; wang, hexiang; ng, tzi bun title: isolation and characterization of a phaseolus vulgaris trypsin inhibitor with antiproliferative activity on leukemia and lymphoma cells date: 2017-01-23 journal: molecules doi: 10.3390/molecules22010187 sha: doc_id: 340879 cord_uid: gu91cact a 17.5-kda trypsin inhibitor was purified from phaseolus vulgaris cv. “gold bean” with an isolation protocol including ion exchange chromatography on deae-cellulose (diethylaminoethyl-cellulose), affinity chromatography on affi-gel blue gel, ion exchange chromatography on sp-sepharose (sulfopropyl-sepharose), and gel filtration by fplc (fast protein liquid chromatography) on superdex 75. it dose-dependently inhibited trypsin with an ic(50) value of 0.4 μm, and this activity was reduced in the presence of dithiothreitol in a doseand time-dependent manner, signifying the importance of the disulfide linkage to the activity. it inhibited [methyl-(3)h] thymidine incorporation by leukemia l1210 cells and lymphoma mbl2 cells with an ic(50) value of 2.3 μm and 2.5 μm, respectively. the inhibitor had no effect on fungal growth and the activities of various viral enzymes when tested up to 100 μm. protease inhibitors have been isolated from the seeds of different monocots and dicots, including maize [1] , wheat [2] , wampee [3] , bitter gourd [4] , momordica cochinchinensis [5] , and legumes [6] [7] [8] . some of them display a variety of biological activities including antifungal [9] , immunomodulatory [5] and antitumor/antiproliferative [8, 10] activities. thus, they have drawn the attention of many researchers. one class of protease inhibitors is trypsin inhibitors. trypsin inhibitors are divided into kunitz type [11] , bowman-birk type [12, 13] , and squash type [4] . the three types have a molecular mass of about 20 kda, 8 kda, and 3 kda, respectively. the soybean produces both kunitz and bowman-birk inhibitors [6] , while melons belonging to family cucurbitaceal produce squash-type inhibitors [4] . leguminous seeds are a rich source of proteins, including protease inhibitors, lectins [14] , antifungal proteins [15] , ribosome inactivating proteins [16] , and arcelins [17] . some of the proteins have interesting biological activities, such as insecticidal [14] , immunomodulatory [5] , antifungal [18] , and antitumor [8, 10] activities. the intent of the present study was to isolate a trypsin inhibitor from the gold bean and to test it for inhibitory action on tumor cells, viral enzymes, and fungal growth. upon ion exchange chromatography on deae-cellulose, the bean extract was separated into three fractions of approximately equal size, an unadsorbed fraction d1 and two adsorbed fractions d2 and d3. trypsin inhibitory activity was located in fraction d2. when fraction d2 was subjected to affinity chromatography on affi-gel blue gel, the activity was recovered in the larger unadsorbed fraction b1 (data not shown). upon ion exchange chromatography on sp-sepharose, fraction b1 was resolved into a small unadsorbed fraction s1 and two large adsorbed fractions (s2 and s3) of approximately equal size. activity resided in fraction s2. further purification of s2 on superdex 75 yielded two fractions, su1 and su2. only fraction su2 exhibited trypsin inhibitory activity. su2 was the purified trypsin inhibitor (gbti). the yields of the various chromatographic fractions are presented in table 1 . the n-terminal sequence of the trypsin inhibitor is shown in table 2 . gbti exhibited a molecular mass of 17.5 kda in both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and gel filtration ( figure 1 ). its trypsin inhibitor activity remained unchanged in the temperature range 20-80 • c, was reduced by 60% at 90 • c, and, was totally abolished after exposure to 100 • c. upon ion exchange chromatography on deae-cellulose, the bean extract was separated into three fractions of approximately equal size, an unadsorbed fraction d1 and two adsorbed fractions d2 and d3. trypsin inhibitory activity was located in fraction d2. when fraction d2 was subjected to affinity chromatography on affi-gel blue gel, the activity was recovered in the larger unadsorbed fraction b1 (data not shown). upon ion exchange chromatography on sp-sepharose, fraction b1 was resolved into a small unadsorbed fraction s1 and two large adsorbed fractions (s2 and s3) of approximately equal size. activity resided in fraction s2. further purification of s2 on superdex 75 yielded two fractions, su1 and su2. only fraction su2 exhibited trypsin inhibitory activity. su2 was the purified trypsin inhibitor (gbti). the yields of the various chromatographic fractions are presented in table 1 . the n-terminal sequence of the trypsin inhibitor is shown in table 2 . gbti exhibited a molecular mass of 17.5 kda in both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and gel filtration ( figure 1 ). its trypsin inhibitor activity remained unchanged in the temperature range 20-80 °c, was reduced by 60% at 90 °c, and, was totally abolished after exposure to 100 °c. trypsin inhibitor-enriched fractions are highlighted in boldface. results represent mean ± sd, n = 2. trypsin inhibitor-enriched fractions are highlighted in boldface. results represent mean ± sd, n = 2. gbti inhibited trypsin with an ic 50 of 0.4 µm ( figure 2 ). dithiothreitol (dtt) treatment lowered the trypsin inhibitory activity in a dose-and time-dependent manner ( table 3 ). the ic 50 values of the inhibitory effects of gbti on l1210 cells and mbl2 cells were 2.3 µm and 2.5 µm, respectively (table 4) . there was no inhibition on hiv-1 reverse transcriptase when it was tested at various concentrations up to 100 µm ( table 5 ). the inhibitor had no effect on the activities of hiv-1 integrase (table 5 ) and sars coronavirus proteinase at 100 µm. there was no inhibitory action on fungal growth at 100 µm (table 6) . table 3 . inhibition rate (%) of dithiothreitol (dtt) on the activity of gbti and soybean trypsin inhibitor after incubation at 37 • c for different durations. results are presented as mean ± sd (n = 3). different letters (e.g., a,b and c ) indicate statistically significant differences (p < 0.05) when (i) data at the same time point and different dtt concentrations or (ii) data at the same dtt concentration, but different time points, were analyzed by analysis of variance followed by duncan's multiple range test. inhibition rate (%) of purified or soybean trypsin inhibitor at x mm dtt = (trypsin inhibitory activity of purified or soybean trypsin inhibitor−trypsin inhibitory activity of purified or soybean trypsin inhibitor in presence of x mm dtt)/trypsin inhibitory activity of purified or soybean trypsin inhibitor × 100%. results are presented as mean ± sd (n = 3). different letters (e.g., a,b,c and d ) indicate statistically-significant differences (p < 0.05) when data were analyzed by analysis of variance followed by duncan's multiple range test. gold bean trypsin inhibitor (gbti) is unadsorbed on affi-gel blue gel, but adsorbed on deae-cellulose. this chromatographic behavior is distinctly different from that of other leguminous antifungal proteins which are unadsorbed on the ion exchanger and adsorbed on the affinity chromatography media [15, 19] . hence, the purification procedure described herein provides a convenient means to separate trypsin inhibitors from antifungal proteins which may be present in the same legume. the trypsin inhibitor from gold beans demonstrate some sequence resemblance to other leguminous trypsin inhibitors including those of cowpea, mung bean, and garden pea. it is noteworthy that it exerts a potent antiproliferative action on both l1210 cells and mbl2 cells. some of the legume trypsin inhibitors, for instance, field bean trypsin inhibitors, inhibit in vitro proliferation of cancer cells and metastasis in vivo [20, 21] . gold bean trypsin inhibitor is dissimilar from broad bean trypsin inhibitor [9] in that the former lacks hiv-1 reverse transcriptase inhibitory activity. the lack of hiv-1 reverse transcriptase inhibitory activity in gold bean trypsin inhibitor is similar to the findings on lily bulb trypsin inhibitor [22] . gold bean trypsin inhibitor is also devoid of any inhibitory effect on hiv-1 integrase and sars coronavirus proteinase. the absence of antifungal activity in gold bean trypsin inhibitor is also in agreement with the observation on some trypsin inhibitors, such as lily bulb trypsin inhibitor [22] . the results demonstrate that hiv-1 reverse transcriptase inhibitory and antifungal activities of trypsin inhibitors have structural requirements different from those of trypsin inhibitory activity. the importance of the disulfide linkage in gold bean trypsin inhibitor to its trypsin inhibitory activity is revealed by the ability of the reducing agent dithiothreitol to reduce the activity. this is reminiscent of the results on trypsin-chymotrypsin inhibitor from vigna mungo seeds [23] . gold bean trypsin inhibitor (gbti) is unadsorbed on affi-gel blue gel, but adsorbed on deae-cellulose. this chromatographic behavior is distinctly different from that of other leguminous antifungal proteins which are unadsorbed on the ion exchanger and adsorbed on the affinity chromatography media [15, 19] . hence, the purification procedure described herein provides a convenient means to separate trypsin inhibitors from antifungal proteins which may be present in the same legume. the trypsin inhibitor from gold beans demonstrate some sequence resemblance to other leguminous trypsin inhibitors including those of cowpea, mung bean, and garden pea. it is noteworthy that it exerts a potent antiproliferative action on both l1210 cells and mbl2 cells. some of the legume trypsin inhibitors, for instance, field bean trypsin inhibitors, inhibit in vitro proliferation of cancer cells and metastasis in vivo [20, 21] . gold bean trypsin inhibitor is dissimilar from broad bean trypsin inhibitor [9] in that the former lacks hiv-1 reverse transcriptase inhibitory activity. the lack of hiv-1 reverse transcriptase inhibitory activity in gold bean trypsin inhibitor is similar to the findings on lily bulb trypsin inhibitor [22] . gold bean trypsin inhibitor is also devoid of any inhibitory effect on hiv-1 integrase and sars coronavirus proteinase. the absence of antifungal activity in gold bean trypsin inhibitor is also in agreement with the observation on some trypsin inhibitors, such as lily bulb trypsin inhibitor [22] . the results demonstrate that hiv-1 reverse transcriptase inhibitory and antifungal activities of trypsin inhibitors have structural requirements different from those of trypsin inhibitory activity. the importance of the disulfide linkage in gold bean trypsin inhibitor to its trypsin inhibitory activity is revealed by the ability of the reducing agent dithiothreitol to reduce the activity. this is reminiscent of the results on trypsin-chymotrypsin inhibitor from vigna mungo seeds [23] . a lectin, an antifungal protein and a trypsin inhibitor can be isolated from the gold bean [24, 25] . all three proteins can be regarded as defense proteins that protect the plant from pathogenic and predatory organisms. previously, a trypsin inhibitors have been isolated from phaseolus vulgaris. however, they have not been examined for biological activities other than trypsin inhibitory activity [26] [27] [28] [29] [30] [31] [32] . in conclusion, a new trypsin inhibitor was purified from gold bean. it dose-dependently inhibited trypsin, and this activity was reduced in the presence of dithiothreitol in a dose-and time-dependent manner, signifying the importance of the disulfide linkage to the activity. it inhibited [methyl-3h] thymidine incorporation by leukemia l1210 cells and lymphoma mbl2 cells. a water extract of gold beans phaseolus vulgaris cv. "gold bean" from mainland china (260 g) was made by homogenizing them in distilled water (6 ml/g). the homogenate was then centrifuged 4) . the unadsorbed proteins (fraction b1) were dialyzed against 10 mm nh 4 ac buffer (ph 5) and applied on a 2.5 × 20 cm column of sp-sepharose (ge healthcare, uppsala, sweden). after elution of unadsorbed proteins (fraction s1), the column was eluted with a 0-1 m nacl concentration gradient in the nh 4 ac buffer. the first adsorbed fraction (s2) was then subjected to gel filtration on a superdex 75 hr 10/30 column (ge healthcare) in 0.2 m nh 4 hco 3 buffer (ph 8.5). the second absorbance peak (su2) represented purified trypsin inhibitor (gbti). the assay for trypsin inhibitory activity was carried out by addition of test sample (20 µl) to 160 µl of a 1% casein solution in 0.1 m tris-hcl buffer (ph 7.4). trypsin (20 µl of a 0.5 mg/ml solution) was then added and the mixture was incubated at 37 • c for 15 min before 0.4 ml 5% trichloroacetic acid was added to terminate the reaction. after centrifugation, the absorbance of the supernatant, which reflects the amount of casein fragments, was measured at 280 nm [22] . the purified trypsin inhibitor was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page, woodinville, wa, usa) for molecular mass determination in accordance with the procedure of laemmli and favre [33] . gel filtration on an fplc-superdex 75 column (ge healthcare, uppsala, sweden), which had been calibrated with molecular mass markers including phosphorylase b (94 kda), bovine serum albumin (67 kda), ovalbumin (43 kda), carbonic anhydrase (30 kda), soybean trypsin inhibitor (20 kda) and α-lactalbumin (14.4 kda) (ge healthcare), was conducted to determine the molecular mass of the protein. the n-terminal sequence of the trypsin inhibitor was determined by using a hewlett-packard hp g1000a edman degradation unit (hewlett packard company, palo alto, ca, usa) and an hp 1000 hplc system (hewlett packard company, palo alto, ca, usa). the purified trypsin inhibitor was incubated in a water bath at different temperatures from 20 • c to 100 • c for 15 min, then immediately chilled on ice. the reaction mixture was then subjected to the assay of trypsin inhibitory activity. the purified trypsin inhibitor (2.5 µm) was incubated with dithiothreitol (dtt) at the final concentration of 2.5, 10, and 40 mm for 5, 20, and 80 min at 37 • c, respectively. for comparison, soybean trypsin inhibitor purchased from sigma chemical co., (st. louis, mi, usa) (2.5 µm) was similarly treated. the reaction was terminated by adding iodoacetamide at twice the amount of thiol functions at each dtt concentration. the remaining trypsin inhibitor activity was measured at ph 7.4, as described above. the highest iodoacetamide concentration used in the test was devoid of any effect on the activity of trypsin and the trypsin inhibitory activity of purified trypsin inhibitor and soybean trypsin inhibitor [34] . the antiproliferative activity of the purified protein was determined as follows. the cell lines l1210 (leukemia) and mbl2 (lymphoma) were purchased from american type culture collection. the cell line was maintained in dulbecco modified eagles' medium (dmem) supplemented with 10% fetal bovine serum (fbs) and 100 mg/l streptomycin and 100 iu/ml penicillin at 37 • c in a humidified atmosphere of 5% co 2 . cells (1 × 10 4 ) in their exponential growth phase were seeded into each well of a 96-well culture plate (nunc, roskilde, denmark) and incubated for 3 h before addition of the trypsin inhibitor. incubation was carried out for another 48 h. radioactive precursor, 1 µci ([ 3 h-methyl]-thymidine, from ge healthcare), was then added to each well and incubated for 6 hrs. the cultures were then harvested by a cell harvester. the incorporated radioactivity was determined by liquid scintillation counting [15] . the assay for hiv reverse transcriptase inhibitory activity was carried out in view of the report that trypsin inhibitors manifest this activity [35, 36] . it was conducted according to instructions supplied with the assay kit from boehringer mannheim (basel, switzerland). the assay takes advantage of the ability of reverse transcriptase to synthesize dna, starting from the template/primer hybrid poly (a) oligo (dt) 15. the digoxigenin-and biotin-labeled nucleotides in an optimized ratio are incorporated into one of the same dna molecule, which is freshly synthesized by the reverse transcriptase (rt). the detection and quantification of synthesized dna as a parameter for rt activity follows a sandwich elisa protocol. biotin-labeled dna binds to the surface of microtiter plate modules that have been pre-coated with strepatavidin. in the next step, an antibody to digoxigenin, conjugated to peroxidase enzyme, binds to the digoxigenin-labeled dna. in the final step, the peroxidase substrate is added. the peroxidase enzyme catalyzes the cleavage of the substrate, producing a colored reaction product. the absorbance of the sample at 405 nm can be determined using a microtiter plate (elisa) reader and is directly correlated to the level of rt activity. a fixed amount (4-6 ng) of recombinant hiv-1 reverse transcriptase was used. the inhibitory activity of the protein was calculated as percent inhibition compared to a control without the protein [15] . the positive control used was kale antifungal peptide. the plasmid that expressed his-tagged wild-type hiv-1 integrase, pt7-7-his (y|tx)-hiv-1-in, was a generous gift from professor s.a. chow (school of medicine, ucla). to express the protein, a 1-liter culture of e. coli bl21 (de3) cells containing the expression plasmid was grown at 37 • c until od600 reached 0.7-0.8. cells were induced by addition of 0.8 mm iptg (isopropyl-β-d-thiogalactopyranoside) and harvested, after 4 h of incubation, by centrifugation at 6000× g for 10 min at 4 • c cells were suspended at a concentration of 0.1 g/ml wet cell paste in 20 mm tris-hcl buffer (ph 8.0) containing 0.1 mm edta (ethylenediamine tetraacetic acid), 2 mm β-mercaptoethanol, 0.5 m nacl and 5 mm imidazole. lysozyme was added to a concentration of 0.2 mg/ml. after incubation at 4 • c for 1 h, the lysate was sonicated and centrifuged at 40,000× g at 4 • c for 20 min. the pellet was homogenized in 50 ml buffer a (20 mm tris-hcl, ph 8.0, 2 m nacl, 2 mm β-mercaptoethanol) containing 5 mm imidazole. the suspension was rotated at 4 • c for 1 h, and cleared by centrifugation at 40,000× g at 4 • c for 20 min. the supernatant was loaded onto a 1-ml chelating sepharose column (ge healthcare) charged with 50 mm niso 4 and was equilibrated with buffer a containing 50 mm imidazole. the column was washed with five column volumes of buffer a containing 5 mm imidazole, and the protein was eluted with three column volumes of buffer a containing 200 and 400 mm imidazole, respectively. protein containing fractions were pooled, and edta was added to a final concentration of 5 mm. the protein was dialyzed against buffer b (20 mm hepes, ph 7.5, 1 mm edta, 1 m nacl, 20% glycerol) containing 2 mm β-mercaptoethanol, and then against buffer b containing 1 mm dithiothreitol. aliquots of the protein were stored at −70 • c [19] . a non-radioactive elisa-based hiv-1 integrase assay was performed according to the dna-coated plate method. in this study, 1 µg of smal-linearized pbluescript sk was coated onto each well in the presence of 2 m nacl as target dna. the donor dna was prepared by annealing vu5br (5'-biotin-gtgtggaaaatctctagcagt-3') and vu5 (5'-actgctagagattttccacac-3') in 10 mm tris-hc1, ph 8.0, 1 mm edta and 0.1 m nacl at 80 • c, followed by 30 min at room temperature. integrase reaction was performed in 20 mm hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (ph 7.5) containing 10 mm mncl 2 , 30 mm nacl, 10 mm dithiothreitol and 0.05% nonidet-p40 (sigma). after the integrase reaction, the biotinylated dna immobilized on the wells was detected by incubation with streptavidin-conjugated alkaline phosphatase (boehringer-mannheim, mannheim, germany), followed by colorimetric detection with 1 mg/ml p-nitrophenyl phosphate in 10% diethanolamine buffer (ph 9.8) containing 0.5 mm mgcl 2 . the absorbance due to the alkaline phosphatase reaction was measured at 415 nm. the ribosome inactivating protein luffin was used as a positive control [16] . the activity of sars coronavirus (cov) protease was indicated by a cleavage of designed substrate which was composed of two proteins linked by a cleavage site for sars cov protease. the reaction was performed in a mixture containing 5 µm sars cov protease, 5 µm sample, 20 µm substrate, and buffer [20 mm tris-hcl (ph 7.5), 20 mm nacl and 10 mm beta-mercaptoethanol] for 40 min at 37 • c. after 40 min, the reaction was stopped by heating at 100 • c for 2 min. then the reaction mixture was analysed by sds-page. if sars cov protease is inhibited by the test sample, there is only one band, which is the intact substrate, shown in sds-page [19] . this assay was conducted in view of the report on antifungal activity of some trypsin inhibitors [9] . the assay for antifungal activity toward mycosphaerella arachidicola and fusarium oxysporum was carried out in 100 mm × 15 mm petri dishes containing 10 ml of potato dextrose agar. after the mycelial colony had developed, sterile blank paper disks (0.625 cm in diameter) were placed at a distance of 0.5 cm away from the rim of the mycelial colony. an aliquot (15 µl) of the purified trypsin inhibitor was added to a disk. the plates were incubated at 23 • c for 72 h until mycelial growth had enveloped the disks containing the control and had formed crescents of inhibition around the disks containing samples with antifungal activity [15] . the positive control used was kale antifungal peptide. a 17.5-kda trypsin inhibitor was purified from phaseolus vulgaris cv. "gold bean". it was adsorbed on deae-cellulose, unadsorbed on affi-gel blue gel, and adsorbed on sp-sepharose. it dose-dependently inhibited trypsin with an ic 50 value of 0.4 µm. about 80% and all of the trypsin inhibitory activity were abolished after treatment with 2.5 mm dithiothrietol for 5 min and 20 min of incubation, respectively. [methyl-3h] thymidine incorporation by leukemia l1210 cells and lymphoma mbl2 cells was inhibited with an ic 50 value of about 2 µm. mycelial growth in fusarium oxysporum and mycosphaerella arachidicola were unaffected. the activities of hiv-1 reverse transcriptase, hiv-1 integrase and sars coronavirus proteinase were unaltered after exposure to the trypsin inhibitor. amino acid sequence and secondary structural analysis of the corn inhibitor of trypsin and activated hageman factor primary structure and reactive site of a novel wheat proteinase inhibitor of subtilisin and chymotrypsin a homodimeric sporamin-type trypsin inhibitor with antiproliferative, hiv reverse transcriptase-inhibitory and antifungal activities from wampee (clausena lansium) seeds bitter gourd proteinase inhibitors: potential growth inhibitors of helicoverpa armigera and spodoptera litura isolation of a trypsin inhibitor with deletion of n-terminal pentapeptide from the seeds of momordica cochinchinensis, the chinese drug mubiezhi protein proteinase inhibitors in legume seed-overview trypsin inhibitor from poecilanthe parviflora seeds: purification, characterization, and activity against pest proteases effects of the medicago scutellata trypsin inhibitor (msti) on cisplatin-induced cytotoxicity in human breast and cervical cancer cells a bowman-birk-type trypsin-chymotrypsin inhibitor from broad beans wolfenstein-todel, c. a novel trypsin inhibitor from peltophorum dubium seeds, with lectin-like properties, triggers rat lymphoma cell apoptosis characterization and pharmacological properties of a novel multifunctional kunitz inhibitor from erythrina velutina seeds formation of bowman-birk inhibitors during the germination of horsegram (dolichos biflorus) reductive unfolding and oxidative refolding of a bowman-birk inhibitor from horsegram seeds (dolichos biflorus): evidence for "hyperreactive" disulfide bonds and rate-limiting nature of disulfide isomerization in folding a comparison of the short and long term effects of insecticidal lectins on the activities of soluble and brush border enzymes of tomato moth larvae (lacanobia oleracea) a mitogenic defensin from white cloud beans (phaseolus vulgaris) purification and characterization of novel ribosome inactivating proteins, alpha-and beta-pisavins, from seeds of the garden pea pisum sativum molecular characterization of a new arcelin-5 gene distribution of proteinases and carbohydrates in the midgut of the larvae of the sweet potato weevil cyclas formicarius and response of proteinase to inhibitors from sweet potato concurrent purification of two defense proteins from french bean seeds: a defensin-like antifungal peptide and a hemagglutinin treatment with field bean protease inhibitor can effectively repress ethylnitrosourea (enu)-induced neoplasms of the nervous system in sprague-dawley rats the field bean protease inhibitor has the potential to suppress b16f10 melanoma cell lung metastasis in mice isolation and characterization of a novel trypsin inhibitor from fresh lily bulbs trypsin-chymotrypsin inhibitors from vigna mungo seeds an antifungal peptide from phaseolus vulgaris cv. brown kidney bean purification and characterization of a lectin from phaseolus vulgaris cv. (anasazi beans) characterization of trypsin inhibitors from tora-mame seeds, one of the japanese cultivars of phaseolus vulgaris trypsin isoinhibitors from garden beans (phaseolus vulgaris) isolation and characterization of some proteinase inhibitors from phaseolus vulgaris var. nanus natural plant enzyme inhibitors: isolation and properties of a trypsin/chymotrypsin inhibitor from kidney bean (phaseolus vulgaris) the isolation and characterization of a trypsin inhibitor from kintoki bean (phaseolus vulgaris) the isolation of two proteins, glycoprotein i and a trypsin inhibitor, from the seeds of kidney bean (phaseolus vulgaris) enzyme inhibitory activities of two leguminosae: phaseolus vulgaris l. pods and vicia faba l. hulls. trypsin inhibitory activity maturation of the head of bacteriophage t4. i. dna packaging events baeyens-volant, d. the papaya kunitz-type trypsin inhibitor is a highly stable beta-sheet glycoprotein a novel lectin from pseudostellaria heterophylla roots with sequence simularity to kunitz-type soybean trypsin inhibitor marmorin, a new ribosome inactivating protein with antiproliferative and hiv-1 reverse transcriptase inhibitory activities from the mushroom hypsizigus marmoreus the authors declare no conflict of interest.molecules 2017, 22, 187 key: cord-300872-blycbi4u authors: saadeh, haythem a.; sweidan, kamal a.; mubarak, mohammad s. title: recent advances in the synthesis and biological activity of 8-hydroxyquinolines date: 2020-09-21 journal: molecules doi: 10.3390/molecules25184321 sha: doc_id: 300872 cord_uid: blycbi4u compounds containing the 8-hydroxyquinoline (8-hq) 1 nucleus exhibit a wide range of biological activities, including antimicrobial, anticancer, and antifungal effects. the chemistry and biology of this group have attracted the attention of chemists, medicinal chemists, and professionals in health sciences. a number of prescribed drugs incorporate this group, and numerous 8-hqbased molecules can be used to develop potent lead compounds with good efficacy and low toxicity. this review focusses on the recent advances in the synthesis of 8-hq derivatives with different pharmacological properties, including anticancer, antiviral, and antibacterial activities. for this purpose, recent relevant references were searched in different known databases and search engines, such as medline (pubmed), google scholar, science direct, scopus, cochrane, scientific information database (sid), scifinder, and institute for scientific information (isi) web of knowledge. this review article provides a literature overview of the various synthetic strategies and biological activities of 8-hq derivatives and covers the recent related literature. taken together, compounds containing the 8-hq moiety have huge therapeutic value and can act as potential building blocks for various pharmacologically active scaffolds. in addition, several described compounds in this review could act leads for the development of drugs against numerous diseases including cancer. 8-hydroxyquinoline derivatives are an important group of compounds with rich and diverse biological activities. these compounds incorporate the 8-hydroxyquinoline (8-hq) moiety, which is a bicyclic compound that consists of a pyridine ring fused to phenol, in which the hydroxyl group is attached to position 8 [1] . in this respect, the pyridine ring maintains its properties as an electron-deficient entity with a basic nitrogen. in addition, and due to the presence of the phenolic group, 8-hq displays typical phenolic properties that make it susceptible to numerous chemical reactions and structural modifications, such as electrophilic aromatic substitution, diazonium coupling, and molecular rearrangements. the close proximity of the hydroxyl group to the heterocyclic nitrogen makes 8-hydroxyquinolines good monoprotic bidentate chelating agents, which form four-and six-covalent complexes with a wide range of metal ions, including cu 2+ , zn 2+ , bi 2+ , mn 2+ , mg 2+ , cd 2+ , ni 2+ , fe 3+ , and al 3+ [2] . figure 1 is a schematic representation of the different sites of reactions of 8-hq. all of the aforementioned properties make 8-hq a privileged structure with a variety of structural modifications, possessing a rich diversity of physical, chemical and biological properties. 8-hydroxyquinoline has attracted the attention of chemists, medicinal chemists, and people in health sciences due to its unique physical and chemical properties. the interest in this compound and its derivatives has increased considerably in the last two decades [3] . 8-hydroxyquinoline and many of its derivatives have a wide range of pharmacological applications, e.g., as iron-chelators for neuroprotection, as anticancer agents, as inhibitors of 2og-dependent enzymes, as chelators of metalloproteins, as anti-hiv agents, as antifungal agents, as antileishmanial agents, as antischistosomal agents, as mycobacterium tuberculosis inhibitors, and as botulinum neurotoxin inhibitors [4] . furthermore, these compounds are used as electron carriers in organic light-emitting diodes (oleds) and as fluorescent chemosensors for metal ions [5, 6] . on the basis of the preceding discussion, and owing to the importance of 8-hq from a chemistry point of view and to the wide range of biological activities and pharmacological applications of 8-hq and derivatives, this review focuses on current knowledge of the synthetic methods of novel derivatives, along with the derivatives' biological activities and medicinal applications. in addition, this review summarizes the most recent literature pertaining to the synthesis and bioactivity of 8-hq and its derivatives, covering the period ranging from 2017 to the present. in addition, structure-activity relationships (sars) of these derivatives are discussed to provide directions for further development of novel 8-hq-based bioactive agents. for this purpose, recent relevant references have been obtained from different databases, such as medical literature retrieval analysis and retrieval system online (medline) (pubmed), google scholar, science direct, scopus, cochrane, sid, and scifinder. our intention is that the content and organization of this review will be valuable to the field and will greatly help researchers. below are details about the recent documented synthesis methods of 8-hq and its derivatives, as well as their biological activities against different diseases and disorders. due to the coronavirus 2019 (covid-19) pandemic and its devastating economic, social, and health effects, we decided to start this study with the antiviral activities of some recent 8-hq derivatives. a few publications have dealt with the antiviral activities of 8-hq and its derivatives. de la guardia et al. described the synthesis of novel 8-hydroxyquinoline derivatives (scheme 1) and investigated their activity against dengue virus [7] . these researchers prepared a number of compounds from 8-hydroxyquinoline n-oxide 2, which upon treatment with copper-catalyzed grignard reagents (rmgx; r = i-pr and i-bu) gave 2-alkyl-8-hydroxyquinoline 3. subsequent chlorination using n-chlorosuccinimide (ncs) under acidic conditions afforded the corresponding 2-alkyl-5,7-dichloro-8-hydroxyquinoline 4 in a good yield. scheme 1. synthesis of 2-isopropy, 2-isobutyl-5,7-dichloro-8-hydroxyquinoline 4. 8-hydroxyquinoline has attracted the attention of chemists, medicinal chemists, and people in health sciences due to its unique physical and chemical properties. the interest in this compound and its derivatives has increased considerably in the last two decades [3] . 8-hydroxyquinoline and many of its derivatives have a wide range of pharmacological applications, e.g., as iron-chelators for neuroprotection, as anticancer agents, as inhibitors of 2og-dependent enzymes, as chelators of metalloproteins, as anti-hiv agents, as antifungal agents, as antileishmanial agents, as antischistosomal agents, as mycobacterium tuberculosis inhibitors, and as botulinum neurotoxin inhibitors [4] . furthermore, these compounds are used as electron carriers in organic light-emitting diodes (oleds) and as fluorescent chemosensors for metal ions [5, 6] . on the basis of the preceding discussion, and owing to the importance of 8-hq from a chemistry point of view and to the wide range of biological activities and pharmacological applications of 8-hq and derivatives, this review focuses on current knowledge of the synthetic methods of novel derivatives, along with the derivatives' biological activities and medicinal applications. in addition, this review summarizes the most recent literature pertaining to the synthesis and bioactivity of 8-hq and its derivatives, covering the period ranging from 2017 to the present. in addition, structure-activity relationships (sars) of these derivatives are discussed to provide directions for further development of novel 8-hq-based bioactive agents. for this purpose, recent relevant references have been obtained from different databases, such as medical literature retrieval analysis and retrieval system online (medline) (pubmed), google scholar, science direct, scopus, cochrane, sid, and scifinder. our intention is that the content and organization of this review will be valuable to the field and will greatly help researchers. below are details about the recent documented synthesis methods of 8-hq and its derivatives, as well as their biological activities against different diseases and disorders. in a similar fashion, kos and coworkers reported the microwave-assisted synthesis of thirty-two mono-, di-, and tri-substituted 8-hydroxyquinoline-2-carboxanilides, as shown in scheme 2 [8] . condensation of activated 8-hydroxyquinoline-2-carboxylic acid (5) with substituted aniline 6 in the presence of by phosphorus trichloride yielded the desired target compound 7 in good amounts (61-79%) . molecules 2020, 25, x for peer review 3 of 26 the antiviral activities of the two novel quinoline derivatives (r = i-pr and i-bu) 4 were evaluated in vitro against the dengue virus serotype 2 (denv2). both exhibited significant inhibitory activities against this virus. the results indicated that the iso-pr-substituted derivative exhibits a half-maximal inhibitory concentration (ic50) of 3.03 µm and a half-maximal cytotoxic concentration (cc50) of 16.06 µm, for an estimated selectivity index (si) of 5.30. on the other hand, the iso-bu derivative was also active, showing a higher si value of 39.5, with an ic50 of 0.49 µm and cc50 of 19.39 µm. the mechanism of action was also investigated and the results showed that these two derivatives are not virucidal, but appear to act at an early stage of the virus lifecycle, reducing the intracellular production of the envelope glycoprotein and the yield of infectious virions in treated and infected cells. in a similar fashion, kos and coworkers reported the microwave-assisted synthesis of thirty-two mono-, di-, and tri-substituted 8-hydroxyquinoline-2-carboxanilides, as shown in scheme 2 [8] . condensation of activated 8-hydroxyquinoline-2-carboxylic acid (5) with substituted aniline 6 in the presence of by phosphorus trichloride yielded the desired target compound 7 in good amounts (61-79%). these prepared compounds were subjected to bioactivity screening against the highly pathogenic h5n1 avian influenza viruses, with the results expressed as percentages of growth inhibition, and to cytotoxicity evaluation against the a549 cell line. the lipophilicity and electronic properties were the molecular parameters used to determine the structure-activity relationship. the lipophilicity of the studied compounds was determined as a log k value using reversed-phase highperformance liquid chromatography (rp-hplc). based on the presented results, most monosubstituted derivatives did not exert any antiviral activity or demonstrated only moderate activity. for example, 8-hydroxy-n-(3-nitrophenyl)quinoline-2-carboxamide showed optimum virus growth inhibition activity and cytotoxicity values of 85.0% and 4%, respectively. furthermore, the results show that the antiviral activity is influenced by increasing the electron-withdrawing properties of substituents on the anilide ring and is positively influenced by increasing the lipophilicity; in this manner, the derivative showing values of r = 3-no2 and log k = ca. 0.41 showed maximal activity with insignificant cytotoxicity. di-and tri-substituted derivatives (r = 3,4-cl, 3,4,5-cl, 3-cl-2-f, and 2,4-no2) showed higher inhibition of h5n1 growth and simultaneous low cytotoxicity. for example, 3-cl-2-f and 3,4,5-cl exhibited virus growth inhibition activity values of 91.2 and 9.7% and cytotoxicity values of 79.3 and 2.4%, respectively; the log k values for these derivatives were 1.44 and 1.26, respectively. the study indicated that the antiviral activity linearly increases with increasing lipophilicity and is positively influenced by increasing the electron-withdrawing properties of substituents on the anilide ring. with the frantic search for drugs to treat covid-19 patients and ultimately for a covid-19 vaccine, the 8-hq nucleus could play a role in this due to its promising antiviral activity; however, more research is required in this area. the spread of the antibiotic-resistant bacterial strains constitutes a serious threat to public health. some of these strains have even become resistant to many antibiotics and chemotherapeutic agents; these prepared compounds were subjected to bioactivity screening against the highly pathogenic h5n1 avian influenza viruses, with the results expressed as percentages of growth inhibition, and to cytotoxicity evaluation against the a549 cell line. the lipophilicity and electronic properties were the molecular parameters used to determine the structure-activity relationship. the lipophilicity of the studied compounds was determined as a log k value using reversed-phase high-performance liquid chromatography (rp-hplc). based on the presented results, most mono-substituted derivatives did not exert any antiviral activity or demonstrated only moderate activity. for example, 8-hydroxy-n-(3-nitrophenyl)quinoline-2-carboxamide showed optimum virus growth inhibition activity and cytotoxicity values of 85.0% and 4%, respectively. furthermore, the results show that the antiviral activity is influenced by increasing the electron-withdrawing properties of substituents on the anilide ring and is positively influenced by increasing the lipophilicity; in this manner, the derivative showing values of r = 3-no 2 and log k = ca. 0.41 showed maximal activity with insignificant cytotoxicity. di-and tri-substituted derivatives (r = 3,4-cl, 3,4,5-cl, 3-cl-2-f, and 2,4-no 2 ) showed higher inhibition of h5n1 growth and simultaneous low cytotoxicity. for example, 3-cl-2-f and 3,4,5-cl exhibited virus growth inhibition activity values of 91.2 and 9.7% and cytotoxicity values of 79.3 and 2.4%, respectively; the log k values for these derivatives were 1.44 and 1.26, respectively. the study indicated that the antiviral activity linearly increases with increasing lipophilicity and is positively influenced by increasing the electron-withdrawing properties of substituents on the anilide ring. with the frantic search for drugs to treat covid-19 patients and ultimately for a covid-19 vaccine, the 8-hq nucleus could play a role in this due to its promising antiviral activity; however, more research is required in this area. the spread of the antibiotic-resistant bacterial strains constitutes a serious threat to public health. some of these strains have even become resistant to many antibiotics and chemotherapeutic agents; hence, the term "multidrug resistance" has been introduced in the literature. thus, the development of existing drugs and the discovery of new leads are major strategies used to combat the threat of these multidrug-resistant strains. in this context, hu and coworkers prepared new derivatives of the known potent antibacterial agent 2,6-difluoro-3-hydroxybenzamide (dfmba) [9] . the substituted 8-hq 8 was alkylated with 1,3-dibromopropane in the presence of an aqueous sodium hydroxide solution and tetrabutylammonium iodide (tbai) in dichloromethane to afford a mono-halogenated intermediate 9. target compound 10 was obtained via alkylation of dfmba with the corresponding 8-hq mono bromide (scheme 3). molecules 2020, 25, x for peer review 4 of 26 hence, the term "multidrug resistance" has been introduced in the literature. thus, the development of existing drugs and the discovery of new leads are major strategies used to combat the threat of these multidrug-resistant strains. in this context, hu and coworkers prepared new derivatives of the known potent antibacterial agent 2,6-difluoro-3-hydroxybenzamide (dfmba) [9] . the substituted 8-hq 8 was alkylated with 1,3-dibromopropane in the presence of an aqueous sodium hydroxide solution and tetrabutylammonium iodide (tbai) in dichloromethane to afford a mono-halogenated intermediate 9. target compound 10 was obtained via alkylation of dfmba with the corresponding 8-hq mono bromide (scheme 3). scheme 3. synthesis of 2,6-difluoro-3-hydroxybenzamide-8-hydroxyquinoline derivatives. the antibacterial activity of these derivatives was evaluated against several gram-positive and gram-negative bacteria using the zone-of-inhibition test. inhibition zones were compared to the standard antibiotic pc190723 (11) , which is an effective bactericidal cell division inhibitor that targets cell division protein (ftsz) in several gram-positive bacteria. the results showed that the antibacterial activities were lower than that of pc190723. in addition, the highest inhibition zone ratios (inhibition zone of the test compound/inhibition zone of the standard) of 0.25 and 0.18 against s. aureus were observed for derivatives where r = 5-cl and 5,7-dicl, respectively. moreover, when comparing the activity of 8-hq derivatives with 8-hq naphthalene analogues, the former were more active; this is probably due to the ring nitrogen, which may increase the polarity and water solubility of the compounds-factors that are required for antibacterial activity. krishna reported the synthesis of a series of new 5-amino-7-bromoquinolin-8-yl sulfonates 15 in good yield using a multistep synthesis method [10] . bromination of 8-hydroxquinoline 1 with nbromosuccinimide (nbs) in chloroform afforded 7-bromoquinolin-8-ol, which upon treatment with nano2/hcl followed by reduction with na2s2o4 in 1:1 tetrahydrofuran (thf) and water gave 5amino-7-bromoquinolin-8-ol 14. the target sulfonate derivatives 15 were obtained from 5-amino-7bromoquinolin-8-ol by reaction with various sulfonyl chlorides in dry thf in the presence of triethylamine (tea) (scheme 4). the antibacterial activity of these derivatives was evaluated against several gram-positive and gram-negative bacteria using the zone-of-inhibition test. inhibition zones were compared to the standard antibiotic pc190723 (11) , which is an effective bactericidal cell division inhibitor that targets cell division protein (ftsz) in several gram-positive bacteria. the results showed that the antibacterial activities were lower than that of pc190723. in addition, the highest inhibition zone ratios (inhibition zone of the test compound/inhibition zone of the standard) of 0.25 and 0.18 against s. aureus were observed for derivatives where r = 5-cl and 5,7-dicl, respectively. moreover, when comparing the activity of 8-hq derivatives with 8-hq naphthalene analogues, the former were more active; this is probably due to the ring nitrogen, which may increase the polarity and water solubility of the compounds-factors that are required for antibacterial activity. krishna reported the synthesis of a series of new 5-amino-7-bromoquinolin-8-yl sulfonates 15 in good yield using a multistep synthesis method [10] . bromination of 8-hydroxquinoline 1 with n-bromosuccinimide (nbs) in chloroform afforded 7-bromoquinolin-8-ol, which upon treatment with nano 2 /hcl followed by reduction with na 2 s 2 o 4 in 1:1 tetrahydrofuran (thf) and water gave 5-amino-7-bromoquinolin-8-ol 14. the target sulfonate derivatives 15 were obtained from 5-amino-7-bromoquinolin-8-ol by reaction with various sulfonyl chlorides in dry thf in the presence of triethylamine (tea) (scheme 4). the newly synthesized sulfonate 15 derivatives were tested against several bacterial strains, such as staphylococcus aureus and bacillus megaterium (gram-positive bacteria), klebsiella pneumoniae and pseudomonas aeruginosa (gram-negative bacteria), and against two gram-negative, antibioticresistant e. coli bacterial strains, namely mutant e. coli (streptomycin-resistant) and donor e. coli (rifampin-resistant), using the agar well diffusion method; amoxiclav (ac) was used as the standard drug. among the synthesized compounds, derivatives with an aryl group showed potent activity. for example, the compound with ar = biphenyl exhibited potent activity against staphylococcus aureus, with an inhibition zone of 22 mm compared to the reference drug (24 mm). on the other hand, derivatives with ar = 4-fph and 2-oh-5-no2ph displayed potent activity against pseudomonas aeruginosa, with inhibition zones of 22 mm and 23 mm, respectively, compared to the standard drug (24 mm), whereas the derivative with r = 4-ch3ph was potent against klebsiella pneumonia, with an inhibition zone of 25 mm compared to the standard drug (27 mm these compounds were tested as antibacterial agents against six pathogenic strains, including e. cloacae, e. coli, k. pneumoniae, p. aeruginosa, s. aureus, and a. baumanii. minimal inhibitory concentrations of these compounds were calculated and compared with three standard antibiotics, namely penicillin g, norfloxacin, and erythromycin, using the disk diffusion technique. some of these prepared compounds exhibited remarkable antibacterial activity against gram-positive and gramnegative strains compared to the standard antibiotics used. one derivative (r = no2, r′ = h) was more potent than the standard drugs and showed activity against e. cloacae, k. pneumoniae, s. aureus, the newly synthesized sulfonate 15 derivatives were tested against several bacterial strains, such as staphylococcus aureus and bacillus megaterium (gram-positive bacteria), klebsiella pneumoniae and pseudomonas aeruginosa (gram-negative bacteria), and against two gram-negative, antibiotic-resistant e. coli bacterial strains, namely mutant e. coli (streptomycin-resistant) and donor e. coli (rifampin-resistant), using the agar well diffusion method; amoxiclav (ac) was used as the standard drug. among the synthesized compounds, derivatives with an aryl group showed potent activity. for example, the compound with ar = biphenyl exhibited potent activity against staphylococcus aureus, with an inhibition zone of 22 mm compared to the reference drug (24 mm). on the other hand, derivatives with ar = 4-fph and 2-oh-5-no 2 ph displayed potent activity against pseudomonas aeruginosa, with inhibition zones of 22 mm and 23 mm, respectively, compared to the standard drug (24 mm), whereas the derivative with r = 4-ch 3 ph was potent against klebsiella pneumonia, with an inhibition zone of 25 mm compared to the standard drug (27 mm) . a study published by rbaa the newly synthesized sulfonate 15 derivatives were tested against several bacterial strains, such as staphylococcus aureus and bacillus megaterium (gram-positive bacteria), klebsiella pneumoniae and pseudomonas aeruginosa (gram-negative bacteria), and against two gram-negative, antibioticresistant e. coli bacterial strains, namely mutant e. coli (streptomycin-resistant) and donor e. coli (rifampin-resistant), using the agar well diffusion method; amoxiclav (ac) was used as the standard drug. among the synthesized compounds, derivatives with an aryl group showed potent activity. for example, the compound with ar = biphenyl exhibited potent activity against staphylococcus aureus, with an inhibition zone of 22 mm compared to the reference drug (24 mm). on the other hand, derivatives with ar = 4-fph and 2-oh-5-no2ph displayed potent activity against pseudomonas aeruginosa, with inhibition zones of 22 mm and 23 mm, respectively, compared to the standard drug (24 mm), whereas the derivative with r = 4-ch3ph was potent against klebsiella pneumonia, with an inhibition zone of 25 mm compared to the standard drug (27 mm) . a study published by rbaa these compounds were tested as antibacterial agents against six pathogenic strains, including e. cloacae, e. coli, k. pneumoniae, p. aeruginosa, s. aureus, and a. baumanii. minimal inhibitory concentrations of these compounds were calculated and compared with three standard antibiotics, namely penicillin g, norfloxacin, and erythromycin, using the disk diffusion technique. some of these prepared compounds exhibited remarkable antibacterial activity against gram-positive and gramnegative strains compared to the standard antibiotics used. one derivative (r = no2, r′ = h) was more potent than the standard drugs and showed activity against e. cloacae, k. pneumoniae, s. aureus, these compounds were tested as antibacterial agents against six pathogenic strains, including e. cloacae, e. coli, k. pneumoniae, p. aeruginosa, s. aureus, and a. baumanii. minimal inhibitory concentrations of these compounds were calculated and compared with three standard antibiotics, namely penicillin g, norfloxacin, and erythromycin, using the disk diffusion technique. some of these prepared compounds exhibited remarkable antibacterial activity against gram-positive and gram-negative strains compared to the standard antibiotics used. one derivative (r = no 2 , r = h) was more potent than the standard drugs and showed activity against e. cloacae, k. pneumoniae, s. aureus, a. baumanii, e. coli, e. cloacae, and e. cloaca, with minimum inhibitory concentration (mic) values (mg/ml) of 1 × 10 −6 , 1 × 10 −5 , 1 × 10 −5 , 1 × 10 −4 , and 1 × 10 −4 , respectively, while the mic for the standard drug was 1 × 10 −4 . derivatives with r = r = h and r = h, r = cl displayed similar activities against s. aureus, with mic values of 1 × 10 −4 . bioinformatic petra, osiris, and molinspiration (pom) analyses indicated that all compounds exhibit good bioavailability and less toxic profiles when compared with penicillin g. interestingly, two research groups have reported on the hybridization of 8-hydroxyquinoline with ciprofloxacin in a single step and evaluated the antibacterial activity of the hybrid molecule. fu et al. reported the synthesis of 5-chloro-8-hydroxyquinoline-ciprofloxacin 19 via the mannich reaction (scheme 6). thus, 5-chloro-8-hydroxyquinoline 18 reacted with ciprofloxacin in the presence of paraformaldehyde in ethanol to afford the hybrid product at 75% yield [12] . interestingly, two research groups have reported on the hybridization of 8-hydroxyquinoline with ciprofloxacin in a single step and evaluated the antibacterial activity of the hybrid molecule. fu et al. reported the synthesis of 5-chloro-8-hydroxyquinoline-ciprofloxacin 19 via the mannich reaction (scheme 6). thus, 5-chloro-8-hydroxyquinoline 18 reacted with ciprofloxacin in the presence of paraformaldehyde in ethanol to afford the hybrid product at 75% yield [12] . the hybrid 19 was screened against both gram-positive and gram-negative (staphylococcus epidermidis, s. aureus, enterococci faecalis, and e. faecium) bacterial strains, and the minimum inhibitory concentrations (mics) were determined via the agar dilution method. the results indicated that the hybrid shows exciting promise against both gram-positive and gram-negative bacteria. it displayed significant effects against both susceptible and drug-resistant strains, with mic values of 4-16 µg/ml. these values were lower than those of standard drug (ciprofloxacin), which has mic values of 0.125-0.5 µg/ml. however, the authors speculated that the introduction of a quinolone skeleton might be an effective way to modify these kinds of compounds into a novel class of broad-spectrum antibacterial agents with a specific mode of action. on the other hand, vu and coworkers prepared a hybrid compound 21 at 96% yield through coupling of 8-hydroxyquinoline-2-carboxylic acid 20 and ciprofloxacin by using 2-(1h-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (tbtu) and n,n-diisopropylethylamine (diea) (scheme 7) [13] . the hybrid 19 was screened against both gram-positive and gram-negative (staphylococcus epidermidis, s. aureus, enterococci faecalis, and e. faecium) bacterial strains, and the minimum inhibitory concentrations (mics) were determined via the agar dilution method. the results indicated that the hybrid shows exciting promise against both gram-positive and gram-negative bacteria. it displayed significant effects against both susceptible and drug-resistant strains, with mic values of 4-16 µg/ml. these values were lower than those of standard drug (ciprofloxacin), which has mic values of 0.125-0.5 µg/ml. however, the authors speculated that the introduction of a quinolone skeleton might be an effective way to modify these kinds of compounds into a novel class of broad-spectrum antibacterial agents with a specific mode of action. on the other hand, vu and coworkers prepared a hybrid compound 21 at 96% yield through coupling of 8-hydroxyquinoline-2-carboxylic acid 20 and ciprofloxacin by using 2-(1h-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (tbtu) and n,n-diisopropylethylamine (diea) (scheme 7) [13] . interestingly, two research groups have reported on the hybridization of 8-hydroxyquinoline with ciprofloxacin in a single step and evaluated the antibacterial activity of the hybrid molecule. fu et al. reported the synthesis of 5-chloro-8-hydroxyquinoline-ciprofloxacin 19 via the mannich reaction (scheme 6). thus, 5-chloro-8-hydroxyquinoline 18 reacted with ciprofloxacin in the presence of paraformaldehyde in ethanol to afford the hybrid product at 75% yield [12] . the hybrid 19 was screened against both gram-positive and gram-negative (staphylococcus epidermidis, s. aureus, enterococci faecalis, and e. faecium) bacterial strains, and the minimum inhibitory concentrations (mics) were determined via the agar dilution method. the results indicated that the hybrid shows exciting promise against both gram-positive and gram-negative bacteria. it displayed significant effects against both susceptible and drug-resistant strains, with mic values of 4-16 µg/ml. these values were lower than those of standard drug (ciprofloxacin), which has mic values of 0.125-0.5 µg/ml. however, the authors speculated that the introduction of a quinolone skeleton might be an effective way to modify these kinds of compounds into a novel class of broad-spectrum antibacterial agents with a specific mode of action. on the other hand, vu and coworkers prepared a hybrid compound 21 at 96% yield through coupling of 8-hydroxyquinoline-2-carboxylic acid 20 and ciprofloxacin by using 2-(1h-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (tbtu) and n,n-diisopropylethylamine (diea) (scheme 7) [13] . the hybrid was screened for cell toxicity against hela cells, a tumor cell line, primary cell cultures of mouse fibroblasts, and against non-cancerous mammalian cell line. it showed no cell toxicity at the concentrations tested (0-200 µm). then, the antibacterial activity was tested against gram-negative and gram-positive bacteria, including pathogenic bacteria present in the hospital environment that are difficult to treat (enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, and enterobacter species). the results indicated that that hybrid 21 exhibits more potent activity than the standard drug against staphylococcus aureus, with an mic (mg/ml) value of 0.0625 (mic of the standard drug = 0.125). however, the hybrid was less active against the rest of the pathogens as compared to the standard drug. in general, the two aforementioned hybrids were systematically less active than the parent antibiotic, ciprofloxacin. from these published data, it is obvious that 8-hq could be an important motif for future antibacterial drugs, especially against antibiotic-resistant strains. however, more research is required, including experiments with animals using newly synthesized antibacterial agents. fungal resistance to antifungal agents has become a problem and a challenge to scientists in recent years. this resistance has serious implications for morbidity, mortality, and health care costs worldwide. hence, attention has been given to understanding the mechanism of antifungal resistance in order to develop new treatments and strategies to manage infections caused by resistant organisms. along this line, le pan and colleagues reported the synthesis of conjugates of 7-hydroxycoumarin (umbelliferone) with several heterocylic rings, including 4-and 8-hydroxyquinolines [14] . 7-hydroxycoumarin was mono-alkylated in moderate yields via an excess of br-(ch 2 ) n -br (n = 2,4) in refluxing acetone in the presence of k 2 co 3 -naoh (2:1). bromoalkylated coumarins were then reacted with 4-and 8-hydroxyquinoline in the presence of 1 equivalent of koh and catalytic amounts of ki and tetra-n-butylammonium bromide (tbab) as a phase transfer catalyst in refluxing acetonitrile to give the corresponding conjugates at 55-80% yields (scheme 8). molecules 2020, 25, x for peer review 7 of 26 indicated that that hybrid 21 exhibits more potent activity than the standard drug against staphylococcus aureus, with an mic (mg/ml) value of 0.0625 (mic of the standard drug = 0.125). however, the hybrid was less active against the rest of the pathogens as compared to the standard drug. in general, the two aforementioned hybrids were systematically less active than the parent antibiotic, ciprofloxacin. from these published data, it is obvious that 8-hq could be an important motif for future antibacterial drugs, especially against antibiotic-resistant strains. however, more research is required, including experiments with animals using newly synthesized antibacterial agents. fungal resistance to antifungal agents has become a problem and a challenge to scientists in recent years. this resistance has serious implications for morbidity, mortality, and health care costs worldwide. hence, attention has been given to understanding the mechanism of antifungal resistance in order to develop new treatments and strategies to manage infections caused by resistant organisms. along this line, le pan and colleagues reported the synthesis of conjugates of 7hydroxycoumarin (umbelliferone) with several heterocylic rings, including 4-and 8hydroxyquinolines [14] . 7-hydroxycoumarin was mono-alkylated in moderate yields via an excess of br-(ch2)n-br (n = 2,4) in refluxing acetone in the presence of k2co3-naoh (2:1). bromoalkylated coumarins were then reacted with 4-and 8-hydroxyquinoline in the presence of 1 equivalent of koh and catalytic amounts of ki and tetra-n-butylammonium bromide (tbab) as a phase transfer catalyst in refluxing acetonitrile to give the corresponding conjugates at 55-80% yields (scheme 8). the antifungal activity of these new conjugates against four phytopathogenic fungi (a. alternate, a. solani, b. cinerea, and f. oxysporum) was evaluated by measuring the mycelial inhibition of radial growth on potato dextrose agar (pda) media compared to the commercial fungicide carbendazim. the inhibition percentages of radial growth against all of these fungi for umbelliferone-8hydroxyquinoline (n = 2), umbelliferone-8-hydroxyquinoline (n = 4), and umbelliferone-4hydroxyquinoline (n = 4) were 49-63, 57-90, and 18-41%, respectively, compared to carbendazim (95-99%) at 200 µg/ml concentration. when these derivatives were tested at a series of lower concentrations to measure the half-maximal effective concentration (µg/ml), umbelliferone-8hydroxyquinoline (n = 4) was the most active compared to the reference drug at 10.6 and 2.3 µg/ml, respectively. the rest of the 8-hydroxyquinoline derivatives exhibited weak activity. the results also showed that umbelliferone derivatives with a spacer of 4 carbon atoms exhibit better antifungal the antifungal activity of these new conjugates against four phytopathogenic fungi (a. alternate, a. solani, b. cinerea, and f. oxysporum) was evaluated by measuring the mycelial inhibition of radial growth on potato dextrose agar (pda) media compared to the commercial fungicide carbendazim. the inhibition percentages of radial growth against all of these fungi for umbelliferone-8-hydroxyquinoline (n = 2), umbelliferone-8-hydroxyquinoline (n = 4), and umbelliferone-4-hydroxyquinoline (n = 4) were 49-63, 57-90, and 18-41%, respectively, compared to carbendazim (95-99%) at 200 µg/ml concentration. when these derivatives were tested at a series of lower concentrations to measure the half-maximal effective concentration (µg/ml), umbelliferone-8-hydroxyquinoline (n = 4) was the most active compared to the reference drug at 10.6 and 2.3 µg/ml, respectively. the rest of the 8-hydroxyquinoline derivatives exhibited weak activity. the results also showed that umbelliferone derivatives with a spacer of 4 carbon atoms exhibit better antifungal activity than those with a spacer of 2 carbon atoms. the chain length may contribute to the activity by influencing the flexibility of the molecules. in addition, the position of the oh group affects the antifungal activity; derivatives with oh at position 8 were more active than those with oh at position 4. the structure-activity relationship suggests that modification of the umbelliferone-8-hysroxyquinoline analogues could help to develop highly selective and low phytotoxic fungicides. the phytotoxicity of effective compounds was evaluated at 200 mg/l with l. sativa, with the results showing that umbelliferone-8-hysroxyquinoline (n = 4) had no phytotoxic effects on the seedling growth of lettuce. yurras and coworkers have reported on the multistep synthesis of novel 3,4,5-trisubstituted triazole derivatives bearing 8-hydroxyquinoline 31, evaluating their antimicrobial activity [15] . synthesis was achieved by first reacting 8-hydroxyquinoline 1 with ethyl 2-chloroacetate in refluxing acetone in the presence of a base. treatment of the formed ester 26 with hydrazine hydrate in ethanol followed by phenyl isocyanate afforded the corresponding thiourea 28. ring closure using koh in refluxing ethanol led to the formation of 3-mercapto-1,2,4-triazole derivative 29. reaction of 3-mercapto-1,2,4-triazole with 2-chloro-n-(substituted (benzo)/thiazole)acetamide 30 afforded the target compounds, as depicted in scheme 9. molecules 2020, 25, x for peer review 8 of 26 activity than those with a spacer of 2 carbon atoms. the chain length may contribute to the activity by influencing the flexibility of the molecules. in addition, the position of the oh group affects the antifungal activity; derivatives with oh at position 8 were more active than those with oh at position 4. the structure-activity relationship suggests that modification of the umbelliferone-8hysroxyquinoline analogues could help to develop highly selective and low phytotoxic fungicides. the phytotoxicity of effective compounds was evaluated at 200 mg/l with l. sativa, with the results showing that umbelliferone-8-hysroxyquinoline (n = 4) had no phytotoxic effects on the seedling growth of lettuce. yurras and coworkers have reported on the multistep synthesis of novel 3,4,5-trisubstituted triazole derivatives bearing 8-hydroxyquinoline 31, evaluating their antimicrobial activity [15] . synthesis was achieved by first reacting 8-hydroxyquinoline 1 with ethyl 2-chloroacetate in refluxing acetone in the presence of a base. treatment of the formed ester 26 with hydrazine hydrate in ethanol followed by phenyl isocyanate afforded the corresponding thiourea 28. ring closure using koh in refluxing ethanol led to the formation of 3-mercapto-1,2,4-triazole derivative 29. reaction of 3mercapto-1,2,4-triazole with 2-chloro-n-(substituted (benzo)/thiazole)acetamide 30 afforded the target compounds, as depicted in scheme 9. some sulfonates reported by krishna (scheme 4) were also tested in vitro against aspergillus niger and penicillium spinulosum fungal strains by the poison plate technique; fluconazole was used as a standard drug for antifungal activity. two compounds with ar = biphenyl and ar = 2-nitro-5-hydroxybenzene exhibited the most potent antifungal activity against aspergillus niger, with inhibition zones of 10 and 13 mm, respectively, compared to fluconazole (15 mm); and against penicillium spinulosum, with inhibition zones of 12 and 10 mm, respectively, compared to fluconazole (12 mm). cancer is a dreadful disease that has become a global burden, causing thousands of deaths per year, despite the technological and pharmaceutical improvements over the past several years. it has emerged as one of the leading causes of mortality worldwide [16] . cancer treatments include surgery, radiotherapy, and anticancer drugs (chemotherapy), in addition to other specialized techniques. much scientific effort is being made every day in the fight against cancer, but successful treatment of some cancer types is still a challenge that needs more work [17] . on the synthetic front in the fight against cancer, two new series of derivatives have been prepared where the bioactive quinolone motif is incorporated, as shown in scheme 10 [18] . 6-bromo-8-methoxyquinoline (1) was prepared according to a published procedure [19] . afterwards, compound 32 was subjected to a suzuki cross-coupling reaction with p-formphenylboronic (33) acid to afford synthon 34 in excellent yield. all target products (35) were synthesized via a simple and effective method using a one-pot mannich-type reaction that involves a reaction of amines, carbonyl compounds, and dialkylphosphonate. molecules 2020, 25, x for peer review 9 of 26 some sulfonates reported by krishna (scheme 4) were also tested in vitro against aspergillus niger and penicillium spinulosum fungal strains by the poison plate technique; fluconazole was used as a standard drug for antifungal activity. two compounds with ar = biphenyl and ar = 2-nitro-5hydroxybenzene exhibited the most potent antifungal activity against aspergillus niger, with inhibition zones of 10 and 13 mm, respectively, compared to fluconazole (15 mm); and against penicillium spinulosum, with inhibition zones of 12 and 10 mm, respectively, compared to fluconazole (12 mm). cancer is a dreadful disease that has become a global burden, causing thousands of deaths per year, despite the technological and pharmaceutical improvements over the past several years. it has emerged as one of the leading causes of mortality worldwide [16] . cancer treatments include surgery, radiotherapy, and anticancer drugs (chemotherapy), in addition to other specialized techniques. much scientific effort is being made every day in the fight against cancer, but successful treatment of some cancer types is still a challenge that needs more work [17] . on the synthetic front in the fight against cancer, two new series of derivatives have been prepared where the bioactive quinolone motif is incorporated, as shown in scheme 10 [18] . 6-bromo-8-methoxyquinoline (1) was prepared according to a published procedure [19] . afterwards, compound 32 was subjected to a suzuki crosscoupling reaction with p-formphenylboronic (33) acid to afford synthon 34 in excellent yield. all target products (35) were synthesized via a simple and effective method using a one-pot mannichtype reaction that involves a reaction of amines, carbonyl compounds, and dialkylphosphonate. the cytotoxicity of these prepared compounds against esophageal (eca109) and hepatocellular (huh7) cancer cell lines was evaluated using sunitinib as a positive control. the results showed that most of these compounds exhibit moderate to high activity, with ic50 values ranging from 2.26 to 7.46 µmol/l for the two most promising derivatives containing 2-methylphenyl and 4-methylphenyl groups for r 2 and iso-propyl for r 1 ; some of these compounds exhibited inhibition activities comparable to those of sunitinib, which showed ic50 values of 16.54 and 5.27 µmol/l towards eca109 and huh7, respectively. furthermore, the results indicated that ethyl and isopropyl substituents of phosphonate have no major effects on the cytotoxicity activity, while substituents on the phenyl ring showed significant influence on the bioactivity. the cytotoxicity of these prepared compounds against esophageal (eca109) and hepatocellular (huh7) cancer cell lines was evaluated using sunitinib as a positive control. the results showed that most of these compounds exhibit moderate to high activity, with ic 50 values ranging from 2.26 to 7.46 µmol/l for the two most promising derivatives containing 2-methylphenyl and 4-methylphenyl groups for r 2 and iso-propyl for r 1 ; some of these compounds exhibited inhibition activities comparable to those of sunitinib, which showed ic 50 values of 16.54 and 5.27 µmol/l towards eca109 and huh7, respectively. furthermore, the results indicated that ethyl and isopropyl substituents of phosphonate have no major effects on the cytotoxicity activity, while substituents on the phenyl ring showed significant influence on the bioactivity. faydy and coworkers described the synthesis of new derivatives of 8-hydroxyquinoline (36-38) ( figure 2 ) in a multistep approach [20] according to published procedures [21] [22] [23] . the antioxidant activity of prepared products, along with l-ascorbic acid, was evaluated by means of the free radical scavenging method using the 2,2-diphenyl-1-picryhydrazyl (dpph) assay. the results revealed that all products showed low antioxidant activity, with ic 50 values of 0.8-2.49 mg/ml, whereas the ic 50 value of 1-ascorbic acid was 0.1 mg/ml. furthermore, the results showed that as the number of hydroxyl groups increases, the inhibition activity increases too. molecules 2020, 25, x for peer review 10 of 26 faydy and coworkers described the synthesis of new derivatives of 8-hydroxyquinoline (36-38) ( figure 2 ) in a multistep approach [20] according to published procedures [21] [22] [23] . the antioxidant activity of prepared products, along with l-ascorbic acid, was evaluated by means of the free radical scavenging method using the 2,2-diphenyl-1-picryhydrazyl (dpph) assay. the results revealed that all products showed low antioxidant activity, with ic50 values of 0.8-2.49 mg/ml, whereas the ic50 value of 1-ascorbic acid was 0.1 mg/ml. furthermore, the results showed that as the number of hydroxyl groups increases, the inhibition activity increases too. [24] following a published procedure [25] . the structures of prepared compounds were confirmed with the aid of a panel of spectroscopic methods, including 1 h nmr, 13 the prepared compounds were then subjected to cell viability evaluation against hela, mcf-7, a-549, and mda-mb-231 (triple-negative breast cancer cell line) cell lines using the mtt assay [26] [27] [28] at a concentration of 20 mm. the results revealed that only 4 compounds designated with r = phenyl, 3,5-dimethylphenyl, 4-fluorophenyl, and 4-trifluoromethylphenyl exhibited significantly reduced cell viability percentages towards the tested cancer cell lines. the ic50 values for these compounds were between 26.30 and 63.75 mm against selected cancer cell lines, whereas the ic50 of docetaxel (positive control) was in the range of 3.37-4.46 mm. a new series of glycoconjugates composed of various sugar units (40, 41) (d-glucose or dgalactose) and 8-hydroxyquinolines (42, 43) was prepared in an effective and simple method [29] . the connection between these units was accomplished by o-glycosidic bond or via o-methylene 1,2,3-triazole linker, as shown in scheme 11. sugar derivatives of 44 and 45 were prepared according to published procedures [30] [31] [32] ; sugar was used to enhance the bioavailability and solubility of potential drugs. (figure 3 ) in moderate to excellent yields [24] following a published procedure [25] . the structures of prepared compounds were confirmed with the aid of a panel of spectroscopic methods, including 1 h nmr, 13 c nmr, ir, and hrms. molecules 2020, 25, x for peer review 10 of 26 faydy and coworkers described the synthesis of new derivatives of 8-hydroxyquinoline (36-38) ( figure 2 ) in a multistep approach [20] according to published procedures [21] [22] [23] . the antioxidant activity of prepared products, along with l-ascorbic acid, was evaluated by means of the free radical scavenging method using the 2,2-diphenyl-1-picryhydrazyl (dpph) assay. the results revealed that all products showed low antioxidant activity, with ic50 values of 0.8-2.49 mg/ml, whereas the ic50 value of 1-ascorbic acid was 0.1 mg/ml. furthermore, the results showed that as the number of hydroxyl groups increases, the inhibition activity increases too. (figure 3 ) in moderate to excellent yields [24] following a published procedure [25] . the structures of prepared compounds were confirmed with the aid of a panel of spectroscopic methods, including 1 h nmr, 13 (42, 43) was prepared in an effective and simple method [29] . the connection between these units was accomplished by o-glycosidic bond or via o-methylene 1,2,3-triazole linker, as shown in scheme 11. sugar derivatives of 44 and 45 were prepared according to published procedures [30] [31] [32] ; sugar was used to enhance the bioavailability and solubility of potential drugs. (42, 43) was prepared in an effective and simple method [29] . the connection between these units was accomplished by o-glycosidic bond or via o-methylene 1,2,3-triazole linker, as shown in scheme 11. sugar derivatives of 44 and 45 were prepared according to published procedures [30] [31] [32] ; sugar was used to enhance the bioavailability and solubility of potential drugs. all prepared derivatives were tested against different cancer cell lines, including hela, hct 116, and mcf-7, in addition to a normal human dermal neonatal fibroblast (nhdf-neo). compound 2, designated with r = r 1 = h and r 2 = oac, was the most active, with ic 50 values of 30.98, 22.7, and 4.12 mm against hela, hct 116, and mcf-7, respectively. other derivatives exhibited low bioactivity. this could be attributed to the use of the quinolone hydroxyl group to form a glycosidic linkage, which impedes the chelation of metal ions due to steric hindrance. in this respect, it should be stated that the presence of the 1,2,3-triazole moiety improves the activity of glycoconjugates, whereas the type of sugar fragment did not affect the activity significantly. finally, conjugates with a sugar moiety and free hydroxyl group exerted better inhibitory potential than acetylated analogs. all prepared derivatives were tested against different cancer cell lines, including hela, hct 116, and mcf-7, in addition to a normal human dermal neonatal fibroblast (nhdf-neo). compound 2, designated with r = r1 = h and r2 = oac, was the most active, with ic50 values of 30.98, 22.7, and 4.12 mm against hela, hct 116, and mcf-7, respectively. other derivatives exhibited low bioactivity. this could be attributed to the use of the quinolone hydroxyl group to form a glycosidic linkage, which impedes the chelation of metal ions due to steric hindrance. in this respect, it should be stated that the presence of the 1,2,3-triazole moiety improves the activity of glycoconjugates, whereas the type of sugar fragment did not affect the activity significantly. finally, conjugates with a sugar moiety and free hydroxyl group exerted better inhibitory potential than acetylated analogs. fouda published a paper dealing with the synthesis, characterization, and cytotoxicity of new derivatives of halogenated 2-amino-4-aryl-4-pyrano[3,2-h]quinolone-3-carbonitrile (48) derivatives (scheme 12) [33] . these derivatives were prepared through interactions of various 8hydroxyquinolines (46) with α-cyanocinnamonitriles (47) . these prepared compounds were screened for potential anticancer activity against mcf-7, hct 116, hepg-2, and a549 using the mmt assay. structure-activity relationship (sar) results revealed that 6-chloroanalogues were the most active, whereas the 9-methylanaloguess were the least potent. in addition, the lipophilicity of the products increased in the presence of halogen atom substituents at positions 4, 6, and 9. the ic50 values (mg/ml) of these compounds were in the ranges of 0.9-38. all prepared derivatives were tested against different cancer cell lines, including hela, hct 116, and mcf-7, in addition to a normal human dermal neonatal fibroblast (nhdf-neo). compound 2, designated with r = r1 = h and r2 = oac, was the most active, with ic50 values of 30.98, 22.7, and 4.12 mm against hela, hct 116, and mcf-7, respectively. other derivatives exhibited low bioactivity. this could be attributed to the use of the quinolone hydroxyl group to form a glycosidic linkage, which impedes the chelation of metal ions due to steric hindrance. in this respect, it should be stated that the presence of the 1,2,3-triazole moiety improves the activity of glycoconjugates, whereas the type of sugar fragment did not affect the activity significantly. finally, conjugates with a sugar moiety and free hydroxyl group exerted better inhibitory potential than acetylated analogs. fouda published a paper dealing with the synthesis, characterization, and cytotoxicity of new derivatives of halogenated 2-amino-4-aryl-4-pyrano[3,2-h]quinolone-3-carbonitrile (48) derivatives (scheme 12) [33] . these derivatives were prepared through interactions of various 8hydroxyquinolines (46) with α-cyanocinnamonitriles (47) . these prepared compounds were screened for potential anticancer activity against mcf-7, hct 116, hepg-2, and a549 using the mmt assay. structure-activity relationship (sar) results revealed that 6-chloroanalogues were the most active, whereas the 9-methylanaloguess were the least potent. in addition, the lipophilicity of the products increased in the presence of halogen atom substituents at positions 4, 6, and 9. the ic50 values (mg/ml) of these compounds were in the ranges of 0.9-38.2, 1.3-45.5, 0.7-44.5, and 1.23-36.7 against mcf-7, hct 116, hepg2, and a549, respectively; while the colchicine reference showed ic50 values of 6.1, 2.6, 4.6, and 3.78 mg/ml, respectively. in a similar fashion, chhabra et al. (2017) described an amberlite ira 402(oh)-mediated synthesis of novel benzothiazole-quinoline conjugates with excellent yields [34] . a synthetic procedure (scheme 13) involved a condensation reaction between the amino group (nh2) in 49 and the carbonyl group in salicylic aldehyde (50), followed by intramolecular cyclization under a these prepared compounds were screened for potential anticancer activity against mcf-7, hct 116, hepg-2, and a549 using the mmt assay. structure-activity relationship (sar) results revealed that 6-chloroanalogues were the most active, whereas the 9-methylanaloguess were the least potent. in addition, the lipophilicity of the products increased in the presence of halogen atom substituents at positions 4, 6, and 9. the ic 50 values (mg/ml) of these compounds were in the ranges of 0.9-38.2, 1.3-45.5, 0.7-44.5, and 1.23-36.7 against mcf-7, hct 116, hepg2, and a549, respectively; while the colchicine reference showed ic 50 values of 6.1, 2.6, 4.6, and 3.78 mg/ml, respectively. in a similar fashion, chhabra et al. (2017) described an amberlite ira 402(oh)-mediated synthesis of novel benzothiazole-quinoline conjugates with excellent yields [34] . a synthetic procedure (scheme 13) involved a condensation reaction between the amino group (nh 2 ) in 49 and the carbonyl group in salicylic aldehyde (50), followed by intramolecular cyclization under a microwave approach to form the benzothiazole (51) . the reaction between 51 and 5,7-dialkyl-8-hydroxyquinoline as a phenolate ion in the presence of a catalytic amount of amberlite ira and an excess amount of dibromoalkane under microwave conditions afforded the target product 52; dibromoalkanes were employed as linkers between the two fragments, 8-hydroxyquinoline and 2-benzothiazol-2-ylphenol. the prepared compounds were then subjected to a cytotoxicity study along with cisplatin (a positive control) against a panel of cancer cell lines, including hela, mcf-7, a549, and human ovarian carcinoma (a2780), using the mmt assay. most of the prepared compounds were more potent than cisplatin, with ic 50 values in the ranges of 5-19, 7-49, 10-30, and 10-38 mm against mcf-7, hela cells, a2780, and a549, respectively. in addition, the target products were 3-25-fold more selective in cancer cell lines than normal fibroblasts. molecules 2020, 25, x for peer review 12 of 26 microwave approach to form the benzothiazole (51) . the reaction between 51 and 5,7-dialkyl-8hydroxyquinoline as a phenolate ion in the presence of a catalytic amount of amberlite ira and an excess amount of dibromoalkane under microwave conditions afforded the target product 52; dibromoalkanes were employed as linkers between the two fragments, 8-hydroxyquinoline and 2benzothiazol-2-ylphenol. the prepared compounds were then subjected to a cytotoxicity study along with cisplatin (a positive control) against a panel of cancer cell lines, including hela, mcf-7, a549, and human ovarian carcinoma (a2780), using the mmt assay. most of the prepared compounds were more potent than cisplatin, with ic50 values in the ranges of 5-19, 7-49, 10-30, and 10-38 mm against mcf-7, hela cells, a2780, and a549, respectively. in addition, the target products were 3-25-fold more selective in cancer cell lines than normal fibroblasts. gayathri and coworkers prepared a novel compound (figure 4 ) bearing three quinolinone moieties in a simple procedure that involved a reaction of 3,6-bis(bromomethyl)-2-chloroquinoline and 8-hydroxyquinoline at a ratio of 1:2 in acetone (aprotic polar solvent) under reflux conditions. the target product was fully characterized by using various spectroscopic techniques and singlecrystal x-ray diffraction method. this compound was screened against mcf-7 and hela cancer cell in addition, shamsi and coworkers prepared 16 quinoline-based 1,3,4-oxadiazole-triazole derivatives (scheme 14) based on the hybrid strategy of nitrogen-containing heterocyclic scaffolds [36] . these compounds were considered to be high-impact motifs with a wide range of biological activities [37, 38] . the synthetic strategy was based on the treatment of 1 with carbonate to produce the phenolate anion, which reacts with ethyl chloroacetate under sn2 conditions to afford the corresponding intermediate. reaction of this intermediate with hydrazine produced 54, which undergoes intramolecular cyclization in the presence of carbon disulfide and the base (koh) to give compound 55 (1,3,4-oxadiazole) after acidification. the thiol group (ar-sh) in 55 is acidic and gives the ar-sanion upon treatment with a base. then, the corresponding anion reacts with propargyl bromide to afford compound 56. finally, reaction of various azide derivatives of 56 yielded the target derivative 57 through a (3 + 2) cycloaddition mechanism. scheme 13. synthesis of novel benzothiazole-quinoline derivatives. gayathri and coworkers prepared a novel compound (figure 4 ) bearing three quinolinone moieties in a simple procedure that involved a reaction of 3,6-bis(bromomethyl)-2-chloroquinoline and 8-hydroxyquinoline at a ratio of 1:2 in acetone (aprotic polar solvent) under reflux conditions. the target product was fully characterized by using various spectroscopic techniques and single-crystal x-ray diffraction method. this compound was screened against mcf-7 and hela cancer cell lines using mmt assay, giving ic 50 values of 21.02 and 27.73 mm, respectively [35] . molecules 2020, 25, x for peer review 12 of 26 microwave approach to form the benzothiazole (51) . the reaction between 51 and 5,7-dialkyl-8hydroxyquinoline as a phenolate ion in the presence of a catalytic amount of amberlite ira and an excess amount of dibromoalkane under microwave conditions afforded the target product 52; dibromoalkanes were employed as linkers between the two fragments, 8-hydroxyquinoline and 2benzothiazol-2-ylphenol. the prepared compounds were then subjected to a cytotoxicity study along with cisplatin (a positive control) against a panel of cancer cell lines, including hela, mcf-7, a549, and human ovarian carcinoma (a2780), using the mmt assay. most of the prepared compounds were more potent than cisplatin, with ic50 values in the ranges of 5-19, 7-49, 10-30, and 10-38 mm against mcf-7, hela cells, a2780, and a549, respectively. in addition, the target products were 3-25-fold more selective in cancer cell lines than normal fibroblasts. gayathri and coworkers prepared a novel compound (figure 4 ) bearing three quinolinone moieties in a simple procedure that involved a reaction of 3,6-bis(bromomethyl)-2-chloroquinoline and 8-hydroxyquinoline at a ratio of 1:2 in acetone (aprotic polar solvent) under reflux conditions. the target product was fully characterized by using various spectroscopic techniques and singlecrystal x-ray diffraction method. this compound was screened against mcf-7 and hela cancer cell lines using mmt assay, giving ic50 values of 21.02 and 27.73 mm, respectively in addition, shamsi and coworkers prepared 16 quinoline-based 1,3,4-oxadiazole-triazole derivatives (scheme 14) based on the hybrid strategy of nitrogen-containing heterocyclic scaffolds [36] . these compounds were considered to be high-impact motifs with a wide range of biological activities [37, 38] . the synthetic strategy was based on the treatment of 1 with carbonate to produce the phenolate anion, which reacts with ethyl chloroacetate under sn2 conditions to afford the corresponding intermediate. reaction of this intermediate with hydrazine produced 54, which undergoes intramolecular cyclization in the presence of carbon disulfide and the base (koh) to give compound 55 (1,3,4-oxadiazole) after acidification. the thiol group (ar-sh) in 55 is acidic and gives the ar-sanion upon treatment with a base. then, the corresponding anion reacts with propargyl bromide to afford compound 56. finally, reaction of various azide derivatives of 56 yielded the target derivative 57 through a (3 + 2) cycloaddition mechanism. in addition, shamsi and coworkers prepared 16 quinoline-based 1,3,4-oxadiazole-triazole derivatives (scheme 14) based on the hybrid strategy of nitrogen-containing heterocyclic scaffolds [36] . these compounds were considered to be high-impact motifs with a wide range of biological activities [37, 38] . the synthetic strategy was based on the treatment of 1 with carbonate to produce the phenolate anion, which reacts with ethyl chloroacetate under s n 2 conditions to afford the corresponding intermediate. reaction of this intermediate with hydrazine produced 54, which undergoes intramolecular cyclization in the presence of carbon disulfide and the base (koh) to give compound 55 (1,3,4-oxadiazole) after acidification. the thiol group (ar-sh) in 55 is acidic and gives the ar-s − anion upon treatment with a base. then, the corresponding anion reacts with propargyl bromide to afford compound 56. finally, reaction of various azide derivatives of 56 yielded the target derivative 57 through a (3 + 2) cycloaddition mechanism. the anticancer activities of all of these derivatives were examined against four different human cancel cell lines, namely human lung carcinoma (a-549), hepatocellular carcinoma (hepg2), human cervical carcinoma-hpv18 (hela), and human cervical carcinoma-hpv16 (siha), using the mmt colorimetric assay; normal cells were used as controls, whereas doxorubicin was used as the reference drug. the results indicated that the product with an o-chloro substitution on the phenyl ring was the most potent, with an ic 50 value of 5.6 mm against the a-549 cell line, which is higher than that of doxorubicin (ic 50 = 1.83 mm). interestingly, this compound was not toxic towards normal cells (up to 200 mm concentration). the anticancer activities of all of these derivatives were examined against four different human cancel cell lines, namely human lung carcinoma (a-549), hepatocellular carcinoma (hepg2), human cervical carcinoma-hpv18 (hela), and human cervical carcinoma-hpv16 (siha), using the mmt colorimetric assay; normal cells were used as controls, whereas doxorubicin was used as the reference drug. the results indicated that the product with an o-chloro substitution on the phenyl ring was the most potent, with an ic50 value of 5.6 mm against the a-549 cell line, which is higher than that of doxorubicin (ic50 = 1.83 mm). interestingly, this compound was not toxic towards normal cells (up to 200 mm concentration). a series of styrylquinolines with various substituents was prepared as shown in scheme 15 [39] . in the first step of the synthesis, the hydroxyl group in 58 was protected by conversion into the acetyl analogue 59. then, a condensation reaction between a methyl group at position 2 of protected 8hydroxyquinoline and appropriate aromatic aldehydes as carried out using microwave heating or conventional procedures [40] . finally, deprotection of the acyl group was achieved with carbonate anion-methanol or pyridine-water mixture afforded the target products 60. a series of styrylquinolines with various substituents was prepared as shown in scheme 15 [39] . in the first step of the synthesis, the hydroxyl group in 58 was protected by conversion into the acetyl analogue 59. then, a condensation reaction between a methyl group at position 2 of protected 8-hydroxyquinoline and appropriate aromatic aldehydes as carried out using microwave heating or conventional procedures [40] . finally, deprotection of the acyl group was achieved with carbonate anion-methanol or pyridine-water mixture afforded the target products 60. the anticancer activities of all of these derivatives were examined against four different human cancel cell lines, namely human lung carcinoma (a-549), hepatocellular carcinoma (hepg2), human cervical carcinoma-hpv18 (hela), and human cervical carcinoma-hpv16 (siha), using the mmt colorimetric assay; normal cells were used as controls, whereas doxorubicin was used as the reference drug. the results indicated that the product with an o-chloro substitution on the phenyl ring was the most potent, with an ic50 value of 5.6 mm against the a-549 cell line, which is higher than that of doxorubicin (ic50 = 1.83 mm). interestingly, this compound was not toxic towards normal cells (up to 200 mm concentration). a series of styrylquinolines with various substituents was prepared as shown in scheme 15 [39] . in the first step of the synthesis, the hydroxyl group in 58 was protected by conversion into the acetyl analogue 59. then, a condensation reaction between a methyl group at position 2 of protected 8hydroxyquinoline and appropriate aromatic aldehydes as carried out using microwave heating or conventional procedures [40] . finally, deprotection of the acyl group was achieved with carbonate anion-methanol or pyridine-water mixture afforded the target products 60. all prepared compounds were screened for anticancer activity against the wild-type hct 116 p53 +/+ and hct 116 p53 −/− cells and for cytotoxicity against normal cell fibroblasts. the results revealed that derivatives that have hydroxyl or acyloxy groups at position 8 of the quinolone moiety exhibit moderate activities (4.60-25.00 mm) and (2.61-25.00 mm) towards p53 +/+ and p53 −/− , respectively. analogues that were based on dichloroquinone and oxyacyl groups were the most active in this series towards p53 +/+ and p53 −/− (0. 28-13.85 [41] . their synthetic procedure involved a knoevenagel condensation between malonitrile (61) and the respective aryl aldehyde 62 to afford the corresponding arylidene-malonitrile intermediates 63, as shown in scheme 16. then, the phenolate anion attacks the β-carbon via c-7 to produce an acyclic michael adduct, which undergoes cyclization reaction (6-exo-dig) and tautomerization to afford the target product 64. all prepared derivatives in the study (along with compound ly290181 used as the standard) have been tested against various cancer cell lines, namely pancreatic carcinoma 518a, colon carcinoma cells ht-29, dld-1, hct 116, cervix carcinoma kb-v1 vbl , and mcf-7 tobo breast carcinoma cell lines, in addition to non-malignant fibroblasts. the results revealed that that these derivatives exhibit remarkable activities, with ic 50 values in nanomolar concentrations, meaning these results are even better than the activity of ly290181. two compounds designated with r = ch 3 , r 1 = r 3 = h, r 2 = r 4 = f and r = ch 3 , r 1 = r 3 = r 4 = h, r 2 = no 2 were the most active among all examined molecules. the first one had ic 50 values of 20.1 and 14 nm against mcf-7 and kb-v1 vbl , respectively. on the other hand, the second compound had an ic 50 value of 20 nm against kb-v1 vbl , which is even better than the reference. in this regard, it is worth mentioning that the mechanism of action of these compounds may be associated with tubulin polymerization interference and ros formation, in which the molecule-induced ros generation could be responsible for their cytotoxicity, since ros overproduction may induce endoplasmic reticulum stress. all prepared compounds were screened for anticancer activity against the wild-type hct 116 p53 +/+ and hct 116 p53 −/− cells and for cytotoxicity against normal cell fibroblasts. the results revealed that derivatives that have hydroxyl or acyloxy groups at position 8 of the quinolone moiety exhibit moderate activities (4.60-25.00 mm) and (2.61-25.00 mm) towards p53 +/+ and p53 −/− , respectively. analogues that were based on dichloroquinone and oxyacyl groups were the most active in this series towards p53 +/+ and p53 −/− (0. 28-13.85 [41] . their synthetic procedure involved a knoevenagel condensation between malonitrile (61) and the respective aryl aldehyde 62 to afford the corresponding arylidene-malonitrile intermediates 63, as shown in scheme 16. then, the phenolate anion attacks the β-carbon via c-7 to produce an acyclic michael adduct, which undergoes cyclization reaction (6-exo-dig) and tautomerization to afford the target product 64. all prepared derivatives in the study (along with compound ly290181 used as the standard) have been tested against various cancer cell lines, namely pancreatic carcinoma 518a, colon carcinoma cells ht-29, dld-1, hct 116, cervix carcinoma kb-v1 vbl , and mcf-7 tobo breast carcinoma cell lines, in addition to non-malignant fibroblasts. the results revealed that that these derivatives exhibit remarkable activities, with ic50 values in nanomolar concentrations, meaning these results are even better than the activity of ly290181. two compounds designated with r = ch3, r1 = r3 = h, r2 = r4 = f and r = ch3, r1 = r3 = r4 = h, r2 = no2 were the most active among all examined molecules. the first one had ic50 values of 20.1 and 14 nm against mcf-7 and kb-v1 vbl , respectively. on the other hand, the second compound had an ic50 value of 20 nm against kb-v1 vbl , which is even better than the reference. in this regard, it is worth mentioning that the mechanism of action of these compounds may be associated with tubulin polymerization interference and ros formation, in which the molecule-induced ros generation could be responsible for their cytotoxicity, since ros overproduction may induce endoplasmic reticulum stress. matrix metalloproteinases (mmps) play significant roles in cancer diseases, with mmp-2 and mmp-9 being important types among the various mmps. for instance, they could induce the release of cell membrane precursors of growth factors (e.g., epidermal growth factor receptor) ligands, which promote tumor proliferation [42, 43] . along this line, chen and colleagues described the synthesis of two series of 8-hydroxyquinolines, as shown in schemes 17 and 18 [44] . in scheme 17, the first step involved protecting the amino group in 65 with tert-butyloxycarbonyl (boc), then the free carboxylic acid group in 66 reacted with the amino groups in various substrates leading, to the formation of carboxamide 67. other steps involved deprotection to liberate the free primary amine 68 [45] , which matrix metalloproteinases (mmps) play significant roles in cancer diseases, with mmp-2 and mmp-9 being important types among the various mmps. for instance, they could induce the release of cell membrane precursors of growth factors (e.g., epidermal growth factor receptor) ligands, which promote tumor proliferation [42, 43] . along this line, chen and colleagues described the synthesis of two series of 8-hydroxyquinolines, as shown in schemes 17 and 18 [44] . in scheme 17, the first step involved protecting the amino group in 65 with tert-butyloxycarbonyl (boc), then the free carboxylic acid group in 66 reacted with the amino groups in various substrates leading, to the formation of carboxamide 67. other steps involved deprotection to liberate the free primary amine 68 [45] , which reacts with 5-chloro-8-hydroxyquinoline-7-carboaldehyde or 8-hydroxycarbaldehyde through reduction amination to yield target product 69. furthermore, in scheme 18, the phenolic hydroxyl group in 70 was protected and reaction of the primary amino group in 70 with substituted carboxylic acids afforded derivative 71. finally, deprotection of the hydroxyl group yielded the target derivative 72. molecules 2020, 25, x for peer review 15 of 26 reacts with 5-chloro-8-hydroxyquinoline-7-carboaldehyde or 8-hydroxycarbaldehyde through reduction amination to yield target product 69. furthermore, in scheme 18, the phenolic hydroxyl group in 70 was protected and reaction of the primary amino group in 70 with substituted carboxylic acids afforded derivative 71. finally, deprotection of the hydroxyl group yielded the target derivative 72. ho all of these derivatives were screened as potential mmp-2/9 inhibitors. the results revealed that compounds (of series 1) that have substituents at c-7 on the quinolone moiety showed ic50 values (mm) in the range of 0.81-10, while for those with substituents at c-5, the ic50 values ranged from 5.7 to 10. on the other hand, derivatives belonging to series 2 showed ic50 values in the range of 6.5-10 against mmp-2. as for mmp-9, using the same sequence, the ic50 values (mm) were in the ranges of 1.3-10, 5.1-10, and ˃10. some selected compounds (those with substituents at c-7) were further screened against a panel of cancer cell lines (hl60, k562, kg1, a549, pc-3, and mcf-7), along with human umbilical vein endothelial cells. the results indicated that most of the tested compounds exhibited good bioactivity, with ic50 values in the range of 0.69-22 mm. finally, the positive control, all of these derivatives were screened as potential mmp-2/9 inhibitors. the results revealed that compounds (of series 1) that have substituents at c-7 on the quinolone moiety showed ic50 values (mm) in the range of 0.81-10, while for those with substituents at c-5, the ic50 values ranged from 5.7 to 10. on the other hand, derivatives belonging to series 2 showed ic50 values in the range of 6.5-10 against mmp-2. as for mmp-9, using the same sequence, the ic50 values (mm) were in the ranges of 1.3-10, 5.1-10, and ˃10. some selected compounds (those with substituents at c-7) were further screened against a panel of cancer cell lines (hl60, k562, kg1, a549, pc-3, and mcf-7), along with human umbilical vein endothelial cells. the results indicated that most of the tested compounds exhibited good bioactivity, with ic50 values in the range of 0.69-22 mm. finally, the positive control, all of these derivatives were screened as potential mmp-2/9 inhibitors. the results revealed that compounds (of series 1) that have substituents at c-7 on the quinolone moiety showed ic 50 values (mm) in the range of 0.81-10, while for those with substituents at c-5, the ic 50 values ranged from 5.7 to 10. on the other hand, derivatives belonging to series 2 showed ic 50 values in the range of 6.5-10 against mmp-2. as for mmp-9, using the same sequence, the ic 50 values (mm) were in the ranges of 1.3-10, 5.1-10, and >10. some selected compounds (those with substituents at c-7) were further screened against a panel of cancer cell lines (hl60, k562, kg1, a549, pc-3, and mcf-7), along with human umbilical vein endothelial cells. the results indicated that most of the tested compounds exhibited good bioactivity, with ic 50 values in the range of 0.69-22 mm. finally, the positive control, the hydroxamate-based mmp inhibitor nngh [46] , showed ic 50 values of 29-187 mm for antiproliferation activities against various cancer cell lines, and against mmp-2 and mmp-9 showed values of 0.0091 and 0.0088 mm, respectively. ökten and coworkers described the synthesis of 5,7-dibromo-8-hydroxyquinoline [47] in excellent yield via reaction of 8-hydroxyquinoline with two equivalents of bromine in chloroform. the target molecule exhibited ic 50 values (mg/ml) of 5.8, 17.6, 18.7, 5.4, 16.5, and >1000 against a549, fl, hela, ht29, mcf7, and hep3b, respectively. from all of these studies, one can see the importance of the 8-hq moiety in potential anticancer drugs. in addition, some of the prepared compounds could be leads towards the development of potent and safe drugs. however, more work is required in this category, which could involve the use of animals and possibly human subjects to evaluate the efficacy and safety profiles of the prepared compounds. alzheimer's disease (ad), characterized by a loss of cognitive ability and severe behavioral irregularities, is a chronic neurodegenerative disorder. it is most common among the elderly, and can be described as an irreversible brain disorder that breaks down memory and reduces the ability of a patient to carry out simple mental and cognitive functions, such as comprehension, solving simple problems, and trivial calculations. this disease is becoming a universal health problem, and it can eventually lead to death [48] . statistics indicate the presence of approximately 2.5 to 4.0 million alzheimer's disease patients in the united states, and 17 and 25 million worldwide [49] . published research findings indicate that cholinergic dysfunction could be associated with selective and irreversible deficiency of the neurotransmitter acetylcholine, which is controlled by hydrolysis of acetylcholine via acetylcholinestrase (ache) and butyrylcholinestrase (bche). additionally, it was suggested that ache predominates in a healthy brain, whereas bche is considered to play a minor role in regulating the brain's ach levels [49] . the approved prescribed commercial drugs for the treatment of ad, which provide slight improvements in memory, include donepezil, rivastigmine, and others; their action is based on the inhibition of acetylcholinesterase. it is also worth mentioning that other factors contribute to ad, such as β-amyloid (a β) deposits and oxidative stress. in this respect, several studies have shown that levels of redox-active metal ions, including cu 2+ and zn 2+ , are observed in the brains of ad patients [50] . these metal ions can interact with β-amyloid peptides to form insoluble plaques [51, 52] . in the search for potent buche and ache inhibitors, hirbod et al. (2017) designed and prepared eight novel compounds incorporating coumarin and 8-hydroxyquinoline moieties (73), as shown in the [53] . these researchers used various dibromoalkanes (n = 3-5) as cross-linkers between 8-hydroxyquinoline and coumarin rings in the presence of an aprotic solvent (n,n-dimethylformamide). in addition, the activity levels of these prepared compounds were evaluated against buche and ache using ellman's method. the results demonstrated that some of the prepared compounds exhibited potent ache and buche inhibition activities, with half-maximal inhibitory concentration (ic 50 (74 and 75) , as shown in figure 5 [54] . the target compounds were prepared by chloromethylation of 8-hq to give 5-chloromethyl-8-hydroxyquinoline. then, the former compound was reacted with tert-butylpiperazine-1-carboxylate followed by trifluoroacetic acid to remove the protected boc group. finally, the resulting compound was reacted with the appropriate cinnamic or hydroxycinnamic acids, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (edc). in this context, β-amyloid (aβ) significantly contributed to the progression of alzheimer's disease (ad), where elevated levels of aβ have been detected in the brains of ad patients. moreover, aβ a aggregation can be clearly observed in the presence of cu 2+ , zn 2+ , and fe 2+ ions, since these ions can readily bind to aβ through some of its specific residues. in addition, aβ can aggregate by itself. th prepared target compounds were tested for their inhibition of aβ aggregation using the thioflavon t-binding assay [55, 56] . furthermore, chelating studies of cu 2+ , zn 2+ , fe 3+ , and fe 2+ were conducted using the former prepared derivatives by means of a uv-vis spectrophotometer. the results revealed that the compound designated with r 1 = h, r 2 , r 3 = och 3 showed the maximum percentage inhibition of aβ 1→42 aggregation of 65.82% with an ic 50 of 5.64 mm compared to resveratrol, which showed corresponding values of 51.74% and 12.43 mm. in addition, the previous compound was selected as a representative to examine its metal chelation behavior; it exhibited significant activity in this context, producing even better results than clioquinol. molecules 2020, 25 [57] . synthesis of these compounds involved protection of the phenolic group in 76 using boc group to allow the reaction of the primary aromatic amine in 77 with substituted benzoyl chlorides in the presence of triethylamine (tea) as the base and catalyst to form the carboxamide group 78. in the final step, deprotection of the boc group was accomplished with hydrogen chloride to afford the desired products as salt 79. regarding scheme 20, the phenoxide anion in 80 was formed in situ by reaction of the phenolic group with potassium carbonate (base), which then reacts with benzyl bromide. the methyl group in 81 was then subjected to pinnick oxidation using seo2 to afford the corresponding acid 82. reaction of the carboxylic acid group in 82 with thionyl chloride and then with 3-(cyclopentyloxy)-4-methoxyaniline or 3,4-dimethoxyaniline afforded 83 upon removal of bn with hydrogen gas in the presence of the palladium catalyst yielded the desired product 84. on the other hand, synthesis of compound 87 was achieved by converting 1 into tert-butyldimethylsilyl (tbs)-protected 2-aminoquinolin-8-ol (85). this process was carried out by oxidation of the starting material using m-chloroperbenzoic acid (mcpba) to form n-oxide, which was subsequently followed by refluxing with dimethylsulfate, treatment with nh4oh, and finally protection with tert-butyldimethylsilyl chloride (tbscl). the obtained derivative 86 was treated with the corresponding benzoyl chlorides, then went through the deprotection process using tetrabutylammoniumfluoride (tbaf) to afford the phenolic group in the target derivative 87 (scheme 21). compounds 79, 84, and 87 are considered hybrids of clioquinol-rolipram and roflumilast as multitarget-directed ligands for the treatment of ad in terms of inhibition of phosphodiesterase 4d (pde4d), the oxygen radical absorbance capacity (orac) value, and the experimental potential of the blood-brain barrier (bbb) permeability (pe) of the selected compounds using parallel artificial membrane permeation assay (pampa). in this respect, pde4d is involved in the process of longterm potentiation and memory consolidation. one of the derivatives of 79, for which x = h, r 1 = cf2h, [57] . synthesis of these compounds involved protection of the phenolic group in 76 using boc group to allow the reaction of the primary aromatic amine in 77 with substituted benzoyl chlorides in the presence of triethylamine (tea) as the base and catalyst to form the carboxamide group 78. in the final step, deprotection of the boc group was accomplished with hydrogen chloride to afford the desired products as salt 79. regarding scheme 20, the phenoxide anion in 80 was formed in situ by reaction of the phenolic group with potassium carbonate (base), which then reacts with benzyl bromide. the methyl group in 81 was then subjected to pinnick oxidation using seo 2 to afford the corresponding acid 82. reaction of the carboxylic acid group in 82 with thionyl chloride and then with 3-(cyclopentyloxy)-4-methoxyaniline or 3,4-dimethoxyaniline afforded 83 upon removal of bn with hydrogen gas in the presence of the palladium catalyst yielded the desired product 84. on the other hand, synthesis of compound 87 was achieved by converting 1 into tert-butyldimethylsilyl (tbs)-protected 2-aminoquinolin-8-ol (85). this process was carried out by oxidation of the starting material using m-chloroperbenzoic acid (mcpba) to form n-oxide, which was subsequently followed by refluxing with dimethylsulfate, treatment with nh 4 oh, and finally protection with tert-butyldimethylsilyl chloride (tbscl). the obtained derivative 86 was treated with the corresponding benzoyl chlorides, then went through the deprotection process using tetrabutylammoniumfluoride (tbaf) to afford the phenolic group in the target derivative 87 (scheme 21). during ad [60] . the pe values of selected compounds were evaluated using pampa. the results indicated that a derivative of 87 with r 1 = ch3 and r 2 = cyclopentylmethyl exhibited a maximum value of pe 16.41 (± 0.44) × 10 6 cm s -1 , whereas other tested compounds showed values higher than 4.7 × 10 6 cm s -1 , indicating that these compounds may cross the bbb. the pe values for rolipram, roflumilast, and clioquinol were evaluated as (18.87 ± 0.57), (9.22 ± 0.62), and (5.20 ± 0.33) x 10 6 cm s -1 , respectively. during ad [60] . the pe values of selected compounds were evaluated using pampa. the results indicated that a derivative of 87 with r 1 = ch3 and r 2 = cyclopentylmethyl exhibited a maximum value of pe 16.41 (± 0.44) × 10 6 cm s -1 , whereas other tested compounds showed values higher than 4.7 × 10 6 cm s -1 , indicating that these compounds may cross the bbb. the pe values for rolipram, roflumilast, and clioquinol were evaluated as (18.87 ± 0.57), (9.22 ± 0.62), and (5.20 ± 0.33) x 10 6 cm s -1 , respectively. figure 6 [61] . compounds 88 and 89 were prepared from 8-hq by reaction first with formaldehyde in the presence of hydrogen chloride, followed by reaction with triethylphosphite to afford diethyl ((8-hydroxyquinolin-5-yl)methyl)phosphonate [56] . in this reaction, the phenolic group was protected via reaction with chloro(methoxy)methane to yield diethyl ((8-(methoxymethoxy)quinolin-5-yl)methyl)-phosphonate. subsequent reaction of the last compound with various 2-nitrobenzaldehydes in the presence of sodium hydride yielded derivatives of 8-(methoxymethoxy)-5-(2-nitrostyryl)quinolone. reductive cyclization of 2-nitrostyrenes with carbon monoxide followed by removal of the protecting group (methoxymethane) by hydrochloride solution led to the formation of the final product. for compound 89, diethyl ((7-chloro-8compounds 79, 84, and 87 are considered hybrids of clioquinol-rolipram and roflumilast as multitarget-directed ligands for the treatment of ad in terms of inhibition of phosphodiesterase 4d (pde4d), the oxygen radical absorbance capacity (orac) value, and the experimental potential of the blood-brain barrier (bbb) permeability (pe) of the selected compounds using parallel artificial membrane permeation assay (pampa). in this respect, pde4d is involved in the process of long-term potentiation and memory consolidation. one of the derivatives of 79, for which x = h, r 1 = cf 2 h, r 2 = cyclopentylethyl, exhibited an ic 50 of 0.399 mm against pde4d compared to rolipram as the reference compound (ic 50 0.621 mm), whereas the derivative showing x = h, r 1 = cyclopentylmethyl of family 84 exhibited the highest value among all tested compounds in the orac assay (its value is 1.98 expressed as trolox equivalents). in this respect, the orac value of antioxidant activities increases the ability of a given compound as the antioxidant becomes better [58] . clioquinol, rolipram, and roflumilast showed values of 0.60, 0.070, and 0.067, respectively. oxidative stress has a major effect in the production of excess free radicals, which lead to cell death and cytosceletal damage in ad [59] . finally, the bbb has a major role in the generation of chronic brain inflammation during ad [60] . the pe values of selected compounds were evaluated using pampa. the results indicated that a derivative of 87 with r 1 = ch 3 and r 2 = cyclopentylmethyl exhibited a maximum value of pe 16.41 (±0.44) × 10 6 cm s −1 , whereas other tested compounds showed values higher than 4.7 × 10 6 cm s −1 , indicating that these compounds may cross the bbb. the pe values for rolipram, roflumilast, and clioquinol were evaluated as (18.87 ± 0.57), (9.22 ± 0.62), and (5.20 ± 0.33) × 10 6 cm s −1 , respectively. an earlier paper by wang et al. (2018) described the synthesis of new 8-hydroxyquinoline derivatives, as shown in figure 6 [61] . compounds 88 and 89 were prepared from 8-hq by reaction first with formaldehyde in the presence of hydrogen chloride, followed by reaction with triethylphosphite to afford diethyl ((8-hydroxyquinolin-5-yl)methyl)phosphonate [56] . in this reaction, the phenolic group was protected via reaction with chloro (methoxy)methane to yield diethyl ((8-(methoxymethoxy) quinolin-5-yl)methyl)-phosphonate. subsequent reaction of the last compound with various 2-nitrob enzaldehydes in the presence of sodium hydride yielded derivatives of 8-(methoxymethoxy)-5-(2-nitro styryl)quinolone. reductive cyclization of 2-nitrostyrenes with carbon monoxide followed by removal of the protecting group (methoxymethane) by hydrochloride solution led to the formation of the final product. for compound 89, diethyl ((7-chloro-8-hydroxyquinolin-5-yl)methyl)-phosphonate was prepared by reacting diethyl ((8-hydroxyquinolin-5-yl)methyl)phosphonate with sodium hypochlorite, followed by the same previous steps. on the other hand, compound 91 was prepared through a series of steps that involved reacting 2-methyl-8-hydroxyqunolines 90 with acetic anhydride, then with 2-nitrobenzaldehydes, followed by reductive cyclization of 2-nitrostyerenes in the presence of the palladium (ii) acetate catalyst under carbon monoxide gas, and finally with a base (carbonate or methoxide ions, depending on the nature of x). compounds 88, 89, and 91 were tested against the orac assay, bbb permeability assay, and inhibition activity towards amyloid beta (aβ) self-induced aggregation. the results revealed that compounds 88 (r = oh), 89 (x = h, r 1 = oh, r 2 = h), and 91 (x = h, r 1 = oh, r 2 = ch 3 ) exhibited the highest activities among all prepared compounds (6.6, 5.3, 5.9, and 5.4, respectively). the presence of the phenolic hydroxyl group at c-5 of the indole moiety enhances the activity. on the other hand, the presence of a chlorine atom in compound 89 lowers the activity compared to a hydrogen. the reference standards, including clioquinol, melatonin, and a mixture of clioquinol and melatonin, exhibit the values of 0.5, 2.4, and 2.9, respectively. this test was performed based on a fluorescein (orac-fl) method with a trolox as the internal standard. similarly, permeation of the bbb is considered an important parameter for potential central nervous system (cns) candidates; this assay was accomplished using the parma method. the results showed that most of the target products could effectively permeate the bbb through passive diffusion. two compounds of series 91, designated as x = h, r 1 = oh, r 2 = h and x = h, r 1 = oh, r 2 = och 3 , showed higher bbb permeability activity (pe values 14.8 and 12.1, respectively) compared to other derivatives that have a hydrogen atom instead of the phenolic hydroxyl group. in contrast, compounds 88 and 89, bearing the indole moiety at position 5 of the quinolone scaffold, gave pe values of 9.1 and 6.8, respectively, even in the presence of a phenolic hydroxyl group. aβ represents the major component of amyloid plaques found in the brains of alzheimer's patients [62] . inhibition of aβ self-induced aggregation for the prepared 8-hydroxyquinoline-indole derivatives was examined using thioflavin fluorescence. one of the derivatives of 91, for which x = h, r 1 = oh, r 2 = h, caused 51.2% percent inhibition, which was the highest percentage among the prepared compounds. on the other hand, compounds 88 and 89 caused 57.2% and 68.7% inhibition, respectively; whereas clioquinol, curcumin, and resveratrol showed 1.9%, 36.7%, and 42.1% inhibition, respectively. in a recent publication, prati and coworkers [63] described the synthesis of a new series of 8-hydroxyquinoline derivatives (94) for which the products possess structural features of two commercial drugs, namely donepezil and clioquinol. depicted in scheme 22 are the steps involved in the synthesis of these compounds. in a recent publication, prati and coworkers [63] this synthesis was a multicomponent mannich reaction that involved a mixture containing piperazine (92), paraformaldehyde, and 8-hq or its 5-chloroanalogue under a microwave-assisted procedure to afford 7-(piperazin-1-ylmethyl)-8-hydroxyquinolines (93). this synthon was then reacted with various benzyl chlorides in dmf. compound 94 and its derivatives were assayed for potential inhibitory activity towards human anti-hache and anti-hbche. the results indicated that at a concentration of 40 mm, all derivatives exhibited inhibition, with values ranging from 9.0 to 63.8% and 49.2 to 89.1% for 5-chloro-8-hydroxyquinoline and 8-hydroxyquinoline derivatives, respectively, against hbche. however, these compounds were inactive or showed very weak inhibition activity against hache, suggesting selectivity of the target products towards hbche. in addition, these results highlighted the effect of the chlorine atom at position 5 of the 8hydroxyquinoline moiety on the activity compared to the hydrogen atom. the chemical references this synthesis was a multicomponent mannich reaction that involved a mixture containing piperazine (92), paraformaldehyde, and 8-hq or its 5-chloroanalogue under a microwave-assisted procedure to afford 7-(piperazin-1-ylmethyl)-8-hydroxyquinolines (93). this synthon was then reacted with various benzyl chlorides in dmf. compound 94 and its derivatives were assayed for potential inhibitory activity towards human anti-hache and anti-hbche. the results indicated that at a concentration of 40 mm, all derivatives exhibited inhibition, with values ranging from 9.0 to 63.8% and 49.2 to 89.1% for 5-chloro-8-hydroxyquinoline and 8-hydroxyquinoline derivatives, respectively, against hbche. however, these compounds were inactive or showed very weak inhibition activity against hache, suggesting selectivity of the target products towards hbche. in addition, these results highlighted the effect of the chlorine atom at position 5 of the 8-hydroxyquinoline moiety on the activity compared to the hydrogen atom. the chemical references donepezil, tacrine, and galantamine exhibited inhibition towards hbche, with values of 84.3, >90, and 65.8%, respectively. on the other hand, the inhibition activity of compound 10 and its derivatives towards the aβ 42 antiaggregating property was evaluated. the obtained results demonstrated that the inhibition potencies of all derivatives ranged from 19.1 to 65.0% at the concentration of 50 mm; both series (x = h, cl) had close activity rates. in addition, the metal chelating ability of the selected compounds was examined using cu 2+ and zn 2+ in phosphate buffer solution (ph 7.4). spectroscopic data showed that there is a bathochromic shift of about 18 nm from the original band (243 nm) upon complex formation; as the metal ion concentration increases (1.56 to 50 mm), the intensity of the absorption band also increases. finally, all derivatives showed antioxidant activity, whereby some prepared compounds exhibited higher activity than trolox. raj and padhi reported that the condensation of 8-hydroxyquinoline-2-carbaldehyde (95) with aromatic diamines (96) afforded quinoline-based benzimidazole (97) followed by intracyclization reaction to produce derivatives of 100. with aliphatic diamines (98), compound 95 produced bis-imines (99) without undergoing intracyclization reaction, as shown in scheme 23 [64] . products of these reactions have been well-characterized using various techniques, including ft-ir, nmr, ms, and single-crystal x-ray diffraction method. in the first step of both reactions, one equivalent of 8-hydroxyquinoline-2-carbaldehyde underwent a condensation reaction with one equivalent of primary amine to form the mono-imine product (schiff bases). however, in the second step and with the presence of aromatic amine, an intramolecular ring cyclization occurred, where the resulting precursor reacts further with the second equivalent of 8-hydroxyquinoline-2-carbaldehyde, followed by migration of hydride to afford compound 100. in the case of an aliphatic amine, the second step represents a second condensation reaction with another molecule of 8-hydroxyquinoline-2-carbaldehyde to give the final product 99. in a recent publication, prati and coworkers [63] described the synthesis of a new series of 8hydroxyquinoline derivatives (94) for which the products possess structural features of two commercial drugs, namely donepezil and clioquinol. depicted in scheme 22 are the steps involved in the synthesis of these compounds. this synthesis was a multicomponent mannich reaction that involved a mixture containing piperazine (92), paraformaldehyde, and 8-hq or its 5-chloroanalogue under a microwave-assisted procedure to afford 7-(piperazin-1-ylmethyl)-8-hydroxyquinolines (93). this synthon was then reacted with various benzyl chlorides in dmf. compound 94 and its derivatives were assayed for potential inhibitory activity towards human anti-hache and anti-hbche. the results indicated that at a concentration of 40 mm, all derivatives exhibited inhibition, with values ranging from 9.0 to 63.8% and 49.2 to 89.1% for 5-chloro-8-hydroxyquinoline and 8-hydroxyquinoline derivatives, respectively, against hbche. however, these compounds were inactive or showed very weak inhibition activity against hache, suggesting selectivity of the target products towards hbche. in addition, these results highlighted the effect of the chlorine atom at position 5 of the 8hydroxyquinoline moiety on the activity compared to the hydrogen atom. the chemical references scheme 23. synthesis of new derivatives of 8-hydroxyquinolie-based benzimidazole. on the other hand, kong et al. (2017) prepared novel barbituroquinoline derivatives 106 and 107 in a one-pot procedure by combining three components in water, namely 1, 2-thiobarbituric acid (104), and aldehyde (isatin) 105 [67] , as shown in scheme 25. this reaction was conducted under mild experimental conditions and without a catalyst. nitriles or acetonitrile in aqueous potassium hydroxide solution yielded 1,2,4-triazole-based quinolone (103 on the other hand, kong et al. (2017) prepared novel barbituroquinoline derivatives 106 and 107 in a one-pot procedure by combining three components in water, namely 1, 2-thiobarbituric acid (104), and aldehyde (isatin) 105 [67] , as shown in scheme 25. this reaction was conducted under mild experimental conditions and without a catalyst. the 8-hydroxyquinoline moiety can act as a building block for various pharmacologically active scaffolds. in the present work, we have reviewed the recent literature pertaining to the synthesis and bioactivity of numerous 8-hq derivatives as anticancer, antiviral, antimicrobial, antibacterial, antifungal, and anticancer agents. the results obtained from this review highlight the importance of numerous derivatives of 8-hq as possible chemotherapeutic agents and as possible leads towards the development of new drugs to treat various diseases, including cancer. we hope that data presented in this review could help researchers in the fields of medicinal chemistry and pharmacology in designing new active compounds and in the modification of existing compounds in the search for new drug leads. the development of drugs, either natural or synthetic, is gaining popularity in the fight against diseases, such as cardiovascular disorders, cancer insurgence, and immune dysfunction. there are certain nuclei such as 8-hq that are important building blocks in the medicinal arena. therefore, new synthetic methods for bioactive 8-hq derivatives should be pursued. in this respect, more biological testing, including in vivo studies, should accompany these syntheses. studies should also involve different pharmacokinetic parameters related to the safety profiles of potent derivatives. in this review, we have shown different synthetic strategies for pharmaceutically important chemicals that incorporate the 8-hq moiety. these compounds exhibited a wide range of biological activities and could be used as therapeutic agents against different diseases, including cancer. some of these compounds could be envisioned as leads in the development of drugs. compound 101 then reacted with alkenes or alkynes containing electron-withdrawing substituents under the mechanism of 1,3-dipolarcycloaddition and in the presence of a base (k 2 co 3 ) to afford 102. on the other hand, the reaction of 101 with aromatic nitriles or acetonitrile in aqueous potassium hydroxide solution yielded 1,2,4-triazole-based quinolone (103) 8-hydroxyquinoline and its derivatives: synthesis and applications immiscible polymers in double spin-coated electroluminescent devices containing phenyl-substituted tris (8-hydroxyquinoline) aluminum derivatives soluble in a host polymer 8-hydroxyquinolines in medicinal chemistry: a structural perspective 8-hydroxyquinoline: a privileged structure with a broad-ranging pharmacological potential synthesis, characterization, and anti-corrosion properties of an 8-hydroxyquinoline derivative synthesis and characterization of 8-hydroxyquinoline complexes of tin (iv) and their application in organic light emitting diode antiviral activity of novel quinoline derivatives against dengue virus serotype 2 8-hydroxyquinoline-2-carboxanilides as antiviral agents against avian influenza virus synthesis and antibacterial activity of 3-benzylamide derivatives as ftsz inhibitors chemoselective synthesis of 5-amino-7-bromoquinolin-8-yl sulfonate derivatives and their antimicrobial evaluation synthesis, antibacterial properties and bioinformatics computational analyses of novel 8-hydroxyquinoline derivatives synthesis and biological evaluation of quinoline derivatives as a novel class of broad-spectrum antibacterial agents in vitro activities of a new fluoroquinolone derivative highly active against chlamydia trachomatis antifungal activity of umbelliferone derivatives: synthesis and structure-activity relationships synthesis of some novel 3, 4, 5-trisubstituted triazole derivatives bearing quinoline ring and evaluation of their antimicrobial activity a comprehensive review on the chemotherapeutic potential of piceatannol for cancer treatment, with mechanistic insights a novel potent nicotinamide phosphoribosyltransferase inhibitor synthesized via click chemistry synthesis and structure-activity relationships study of a-aminophosphonate derivatives containing a quinoline moiety an extremely stable and orthogonal dna base pair with a simplified three-carbon backbone synthesis and investigation of antibacterial and antioxidants properties of some new 5-subsituted-8-hydroxyquinoline derivatives synthesis, spectroscopic characterization, x-ray analysis, and dft-hf calculations of 5-ethoxymethyl-8-hydroxyquinoline synthesis and antimicrobial activities of sulfonohydrazide-substituted 8-hydroxyquinoline derivative and its oxinates bioconjugation via azide-staudinger ligation: an overview synthesis and biological evaluation of 8-hydroxyquinoline-hydrazones for anti-hiv-1 and anticancer potential design, synthesis and biological evaluation of 2-substituted quinolines as potential antileishmanial agents design, synthesis and biological evaluation of 1, 3, 6-trisubstituted b-carboline derivatives for cytotoxic and anti-leishmanial potential co-delivery of docetaxel and gemcitabine by anacardic acid modified self-assembled albumin nanoparticles for effective breast cancer management novel gemcitabine conjugated albumin nanoparticles: a potential strategy to enhance drug efficacy in pancreatic cancer treatment synthesis of 8-hydroxyquinoline glycoconjugates and preliminary assay of their b1,4-galt inhibitory and anti-cancer properties synthesis and characterization of a new cationic galactolipid with carbamate for gene delivery click'assembly of glycoclusters and discovery of a trehalose analogue that retards aβ40 aggregation and inhibits aβ40-induced neurotoxicity chemoselective ligation of maleimidosugars to peptides/protein for the preparation of neoglycopeptides/neoglycoprotein halogenated 2-amino-4h-pyrano [3,2-h] quinoline-3-carbonitriles as antitumor agents and structure-activity relationships of the 4-, 6-, and 9-positions amberlite ira 402 (oh) mediated green synthesis of novel benzothiazole-quinoline conjugates as cancer theranostics comparative theoretical and experimental study on novel tri-quinoline system and its anticancer studies synthesis, anticancer evaluation and dna-binding spectroscopic insights of quinoline-based 1, 3, 4-oxadiazole-1, 2, 3-triazole conjugates synthesis and anti-breast cancer activities of substituted quinolines design, synthesis and biological evaluation of new 4-(4-substituted-anilino) quinoline derivatives as anticancer agents the synthesis and anticancer activity of 2-styrylquinoline derivatives. a p53 independent mechanism of action an efficient microwave-assisted synthesis of structurally diverse styrylquinolines new pyranoquinoline derivatives as vascular-disrupting anticancer agents matrix metalloproteinase-7 degrades all insulin-like growth factor binding proteins and facilitates insulin-like growth factor bioavailability review on epidermal growth factor receptor (egfr) structure, signaling pathways, interactions, and recent updates of egfr inhibitors design, synthesis and preliminary bioactivity evaluations of 8-hydroxyquinoline derivatives as matrix metalloproteinase (mmp) inhibitors synthesis of tetracyclic pyrrolidine/isoxazolidine fused pyrano [3,2-h] quinolines via intramolecular 1,3-dipolar cycloaddition in ionic liquid discovery of cgs 27023a, a non-peptidic, potent, and orally active stromelysin inhibitor that blocks cartilage degradation in rabbits quinoline-based promising anticancer and antibacterial agents, and some metabolic enzyme inhibitors acetylcholinesterase inhibition by flavonoids from agrimonia pilosa coumarin derivatives as acetyl-and butyrylcholinestrase inhibitors: an in vitro, molecular docking, and molecular dynamics simulations study cholinesterase inhibitors and beyond pet imaging of copper trafficking in a mouse model of alzheimer disease nanoprobing of the effect of cu 2+ cations on misfolding, interaction and aggregation of amyloid b peptide coumarin derivatives bearing benzoheterocycle moiety: synthesis, cholinesterase inhibitory, and docking simulation study novel 8-hydroxyquinoline derivatives targeting b-amyloid aggregation, metal chelation and oxidative stress against alzheimer's disease design, synthesis, and evaluation of multitarget-directed resveratrol derivatives for the treatment of alzheimer's disease design, synthesis, and evaluation of orally available clioquinol-moracin m hybrids as multitarget-directed ligands for cognitive improvement in a rat model of neurodegeneration in alzheimer's disease synthesis and evaluation of clioquinol-rolipram/roflumilast hybrids as multitarget-directed ligands for the treatment of alzheimer's disease profiling donepezil template into multipotent hybrids with antioxidant properties antioxidant capacity in the lipophilic fraction of alzheimer's brain tissues inflammatory events at blood-brain barrier in neuroinflammatory and neurodegenerative disorders: implications for clinical disease design, synthesis, and evaluation of orally bioavailable quinoline-indole derivatives as innovative multitarget-directed ligands: promotion of cell proliferation in the adult murine hippocampus for the treatment of alzheimer's disease the amyloid beta peptide: a chemist's perspective. role in alzheimer's and fibrillization novel 8-hydroxyquinoline derivatives as multitarget compounds for the treatment of alzheimer's disease synthesis, characterization, and structure of quinoline-based benzimidazole derivatives synthesis of pyrazolo-and [1,2,4] triazolo-[1,5-a] quinolin-9-ols by cycloaddition to 8-hydroxyquinoline n-imide o-mesitylenesulfonylhydroxylamine and related compounds-powerful aminating reagents convenient one-pot synthesis of thiobarbituro-quinoline derivatives via catalyst-free multicomponent reactions in water this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license funding: this research received no external funding. the authors declare no conflict of interest. key: cord-338727-1kodz527 authors: ilinskaya, o. n.; mahmud, r. shah title: ribonucleases as antiviral agents date: 2014-10-11 journal: mol biol doi: 10.1134/s0026893314040050 sha: doc_id: 338727 cord_uid: 1kodz527 many ribonucleases (rnases) are able to inhibit the reproduction of viruses in infected cell cultures and laboratory animals, but the molecular mechanisms of their antiviral activity remain unclear. the review discusses the well-known rnases that possess established antiviral effects, including both intracellular rnases (rnase l, mcpip1 protein, and eosinophil-associated rnases) and exogenous rnases (rnase a, bs-rnase, onconase, binase, and synthetic rnases). attention is paid to two important, but not always obligatory, aspects of molecules of rnases that have antiviral properties, i.e., catalytic activity and ability to dimerize. the hypothetic scheme of virus elimination by exogenous rnases that reflects possible types of interaction of viruses and rnases with a cell is proposed. the evidence for rnases as classical components of immune defense and thus perspective agents for the development of new antiviral therapeutics is proposed. ribonucleases (rnases) that catalyze rna cleav age play a key role in the regulation of vital processes in any organism, ranging from virus to human. with out the reactions of rna degradation, the maturation of mrna and noncoding rna, the functioning of global systems of rna interference, and the epige netic regulation of gene expression, as well as pro cesses of cell growth and differentiation, apoptosis induction, and protection against viral infection, are not possible. there are approximately 20 exo and endonucleases of various nucleotide sequence and structure specificity in a cell [1] . along with intracel lular rnases, there are rnases that may be secreted into cultural or tissue fluid. biological effects of exog enous rnases are of particular interest. the control of blood vessel growth, toxicity against tumor cells, and antiviral activity are all properties of rnases that may potentially be utilized in medicine and define the cur rent hot points in studies focused on these enzymes. in the review, we consider the best known rnase that possesses antiviral effects. molecular structures of these rnases are presented in fig. 1 ; obviously, they are not characterized by a significant homology. therefore, the structural similarity of the catalytically active proteins is not a determining feature for their antiviral effects. in the 1960s, a group of scientists under the guid ance of prof. p. i. salganik at the institute of cytology and genetics (siberian branch, russian academy of sciences) demonstrated that increased activity of rna cleaving enzymes is observed in the blood and cerebrospinal fluid of tick borne encephalitis patients [2] . rnases were hypothesized to be directly involved in biological mechanisms of antivirus protection. the activation of intracellular rnases in the presence of antiviral preparations was later detected in plants [3] . in particular, transgenic tobacco plants with increased activity of intracellular zinnia elegans ribonuclease zrnaseii possessed high stability against the tobacco mosaic virus [4] . bacterial endoribonucleases specific to certain nucleotide sequences, e.g., the rnase toxn, provide for the phage stability of the population, inducing death of cells due to cleavage of phage and cel lular rna [5] . cleavage of trna by an anticodon nuclease prrc of escherichia coli is a mechanism of bacteria protection from infection with t4 phage [6] . the involvement of rnases in the protection of cells and the organism from viruses has been con firmed by ample evidence. a considerable body of data has been accumulated and allows one to consider rnases to be not only components of immune defense, but also the basis for the development of new antiviral preparations. the antiviral effect of rnases is best studied in the case of a signaling system involving rnase l, which mediates the effect of interferon induced by viral infection. the key enzyme of the system is 2',5' oli goadenylate synthetase polymerizing atp with for mation of rnase l activator, 2',5' oligoadenylate, of the general formula ppp2',5' a n , where n = 2-10 ade nylate residues. rnase l mechanism of action is well studied, i.e., the active form of rnase l forms a dimer with endoribonuleolytic activity against both viral and cellular rna [7, 8] . products of rna cleavage less than 200 nucleotide long are recognized by protein factors rig i and mda5. therefore, the formation of rna fragments enhanced by rnase l, followed by their interaction with rig i and mda5, activates transcription factor nf κb and triggers transcription of interferon β gene, which prevents virus replication and stimulates the growth of immune system cells [9] . however, the system may not completely protect cells from virus. in enteroviruses of group c, a phylogenet ically conserved rna structure resistant to cleavage by rnase l and inhibiting the activity of its endoribo nuclease domain has been found inside an open read ing frame [10] . a neurotropic theiler's picornavirus inducing chronic infection of central nervous system and demyelination of nervous tissue produces a spe cies specific helper protein l* that inhibits rnase l through interaction with its ankyrin domain [11] . these viruses possess mechanisms that provide for their resistance to rnase l, because they may block either its activity or ability for dimer formation. inter estingly, dimerization contributes to the antiviral activity of rnases l, along with the catalytic activity, which is required for viral rna cleavage a priori. zinc finger proteins are known to possess an anti viral effect; due to the cleavage of the polya end of the mrna, they induce and enhance rna turnover in cell [12, 13] . the introduction of domains with high catalytic nuclease activity into these proteins pro motes enhancement of antiviral properties. for exam ple, a hybrid construct based on a synthetic protein that belongs to this group and staphylococcus nuclease was obtained that prevents the replication of the dna containing human papilloma virus, [14] . monocyte chemoattractant protein induced pro tein 1 (mcpip1) also belongs to zinc finger proteins. the protein contains two conserved domains, i.e., the ccch sequence (zinc fingers) and nyn nuclease domain. mcpip1 is involved in the regulation of the inflammatory response in cell, and it is the nuclease domain that binds and destroys the viral rna [15] . this requires rnase activity and dimerization of the protein. mcpip1 cleaves viral rna and cellular mrna, as well as the mcirorna precursor, in a mg 2+ dependent reaction [16] . an increased level of the protein induced by inflammatory cytokines (such as tumor necrosis factor α (tnf α), interleukin 1β, lipopolysaccharides) inhibits the replication of den gue fever virus and japanese encephalitis virus. how ever, three other proteins of the mcpip group that contain both the ccch sequence and nuclease domain, but have no proline rich domain and thus are unable to form dimers, possess no antiviral activity [15] . these data emphasize the requirement for the dimerization of the mcpip proteins for manifestation of antiviral activity independently of the presence of the zinc finger domain in the molecule. the main rnases associated with eosinophils, the eosinophil cationic protein (ecp) and eosinophil derived neurotoxin (edn), possess antimicrobial, antihelmintic, and antiviral activity caused by their ability to catalytically cleave ssrna. these rnases are considered as potential agents against viral infec tions of the lungs [17] . at the level of amino acid sequences, they possess certain homology with bovine pancreatic ribonuclease and belong to a large super family of rnase a, the members of which have disul fide bonds in their structures. ecp, or rnase 3, is the most cationic (pi = 11) and the least catalytically active protein of the family, and edn (rnase 2, pi = 9) possesses approximately 100 fold higher activity [17] . only edn that possesses a unique loop of nine amino acid residues (l7) that differs different from a similar loop in ecp at its c terminus exhibits high activity against the respiratory syncytial virus associated with the contribution of this structure to interaction of rnase with viral capsid and penetration inside the vir ion [18] . rosenberg [19] , who studies eosinophil associated rnases, supposes that respiratory viruses containing ssrna are evolutionarily established mobile targets for edn, which directly utilizes its ribonucleolytic activity for the antiviral effect. fur thermore, in experiments in vitro, edn decreased the infecting ability of another ssrna containing virus, i.e., human immunodeficiency virus (hiv 1) [20, 21] . previously, onconase, an rnases from oocytes of the leopard frog rana pipiens, efficiently suppresses the replication of hiv 1 due to the selective degrada tion of viral rna, which exhibits no pronounced cytotoxic effect on infected human cells [22] . later, onconase was found to be capable of degrading cell trna, but not the rrna and mrna protected with proteins. this accelerates the trna degradation/syn thesis cycle and degradation products, which can probably serve as primers for viral replication, are also destroyed by onconase, leading to the inhibition of virus replication [23] . an rnase from the r. catesbei ana frog, homologous to onconase, efficiently blocks the replication of an rna containing virus of japa nese encephalitis and stimulates activation of caspases 3, 8, and 9, inducing apoptosis of infected bhk 21 cells [24] . modern studies demonstrated that both onconase and rnase a are inefficient with respect to the respiratory syncytial virus [28] . at the same time, onconase and ramphinase 2 (a recombinant protein similar to onconase) inhibited the replication of dna containing viruses, such as herpes simplex virus types 1 and 2, epstein-barr virus, kaposi sarcoma associated herpes virus, cytomegalovirus, and roseolovirus, without causing infected cell death; the two latter virus types were the most sensitive to these rnases [25] . therefore, the antiviral effect, even of closely related amphibian rnases, does not cover all viruses; certain rnases are active against certain viruses. this is due to the specific features of the struc ture of various viruses, the versatility of the organiza tion, and the properties of the cell they target, as well as the variability of molecular structures and the level of rnase catalytic activity. rnase a the earliest studies on the antiviral activity of rnases were performed using pancreatic rnase as an agent that quickly normalized the state and decreased the symptoms of meningitis and cerebrospinal pleocy tosis in patients with tick borne encephalitis [26] . the first preparation was registered as amorphous ribo nuclease (registration number 68/333/22, registration date april 30, 1968 ). today, cattle pancreatic ribonu clease (rnase a) is produced in russia by the sam son med joint company under the trade name of ribonuclease (registration number ls 000391, date of registration april 10, 2010) in the form of tablets and lyophilizate for preparing injections and local adminis tration solution. the preparation is advised for inflam matory disorders of the airways (tracheitis, bronchitis, pneumonia, bronchiectatic disease, sinusitis), parad ontosis, osteomyelitis, thrombophlebitis, abscesses, viral meningitis, and tick borne encephalitis [27] . therefore, the only approved rnase based antiviral preparation is the preparation obtained from the bovine pancreas. today, the improvement of the antiviral agents based on rnase a is ongoing. conjugates of rnase a with ligand free human serum albumin that, unlike the initial enzyme, exhibit activity against dsrna and possess high activity against influenza a and b viruses have been created [28] . rnase a is a component of a complex with gold nanoparticles and oligonucleotides complementary to rna sequences (nucleotides at positions 322-339) of hepatitis b virus. the complex decreased the content of viral rna in mice with hep atitis b by 99% [29] . despite the high probability of inhibiting the catalytic activity of rnase a by cell cytosol inhibitor [30, 31] , its functions as of one of the major representatives of the wide family of mamma lian rnases in the evolutionarily developed system of non specific immunity are doubtless [32] . bovin pancreatic rnase does not possess consider able activity against hiv 1, while bovine seminal rnase (bs rnase) inhibited the replication of the virus in h9 leukemia cells [33] . it was demonstrated that cleavage of dsrna by bs rnase is enhanced in the presence of interferon γ, which probably contrib utes to the mechanisms of antiviral immune defense [34] . let us note that bs rnase is a natural dimer, monomers of which are linked by two intramolecular disulfide bonds. this structure imparts the molecule stability against cytosolic rnase inhibitor and decreases its toxic effects compared to the monomer [33] . it should also be noted that many rnases tend to form oligomeric structures, including rnase a lyo philized from a solution in 40% acetic acid forms dimers, trimers, tetramers, and higher order multim ers [36] . dimers are formed via the exchange of the terminal domains between the monomers (either c or n terminal dimers). an important factor that pro motes oligomerization is the hydrophobic nature of the c terminal part of the molecule and hydrophilic nature of the n terminal part [37] . in all studied crys tal structures of a microbial rnase binase, there are features that indicate dimer formation, in which active center of one of the subunits is closed due to interac tions between the subunits [38] . α helices of the mol ecules of bs rnase, rnase a, human pancreatic rnase h, and binase contain hydrophobic segments capable of participating in both the interaction with the lipid bilayer and dimerization [39] . among the rnases discussed thus far, only the bs rnase is an established natural dimer. however, the data on the need for dimer formation by the monomers of rnase l and mcpip1 [7, 8, 15, 16] for them to acquire the ability to destroy the viral genetic material evidence that the contribution of supramolecular organization of rnases to their antiviral activity has not yet been sufficiently studied. the clinical application of mammalian rnases is not always efficient, since a specific inhibitor present in practically all tissues and cells and necessary for cell protection from its own rnases [31] blocks their cat alytic activity. the inhibitor does not deactivate bacte rial rnases, and broad possibilities for the design of simple bioengineered constructs based on microbial rnases make them especially attractive for the devel opment of new therapeutic agents. a series of experimental works of the end of the 20th century was devoted to the comparison of antivi ral activity of pancreatic and microbial rnases, in particular, rnase from actinomyces rimosus. both native and dextran modified microbial rnase caused greater and more prolonged effect on a dna contain ing aujeszky's disease virus than rnase a [40] . binase, or rnase of bacillus intermedius (modern title b. pumilus [41] ), well studied by now, exhibited high activity against the rna containing street rabies virus in infected guinea pigs, rabbits, and mice when injected to the site of infection [42, 43] . therapeutic effect of binase was registered both 2-3 h after infec tion and 1 day later (57-67% animal protection); the enzyme did not affect the formation of vaccine immu nity. a single intraperitoneal injection of binase to rab bits infected with foot and mouth disease virus type o and type a22 550 decreased animal mortality by threefold [44] . binase also possesses activity against influenza a/bethesda/10/63, a/odessa/2882/82, and b/leningrad/369/76 viruses comparable to that of a classic antiviral agent, rimantadine. binase was found to be active against influenza virus types a and b, while rimantadine is not active against influenza virus type b [45] . recently, we demonstrated that, at the non toxic concentrations to epithelial cells, binase exhibited antiviral activity against the pandemic influ enza a/hamburg/04/09 (h1n1) virus, the causal agent of the epidemic in 2009, upon both single and multicycle virus reproduction. the short term treat ment of virus infectious particles (15-30 min) with binase at increasing concentrations proportionally decreased the viability of the virus, which manifested by weakening its ability to infect lung adenocarcinoma cells a549 by almost an order of magnitude (fig. 2 ) [46] . importantly, binase does not induce expression of specific marker of immune response antigen cd69 and synthesis of interferon γ in population of cd8+ and cd4+ t lymphocytes, which evinces that the enzyme lacks the ability to induce polyclonal t cell response of the superantigen type [47] . screening for bacterial rnases that possess new antiviral properties continues. works on the isolation and characteristics of secreted rnases by the bacteria of the genus pseudomonas are ongoing [48] . the intra cellular ribonuclease of b. cereus was found to be effi cient against the tobacco mosaic virus [49] . culture fluid of b. pumilus isolated from the sea sponge petro mica citrine possesses antiviral activity with respect to bovine diarrhea virus [50] and the b. pumilus strain var. pashkov from midland soils exhibits a wide spec trum of antagonist activity, including the antienteroviral activity [51] . most likely, the antiviral properties of the bacillus cultural fluid are largely caused by the secreted rnases that correspond or are similar to binase. starting in the late 1990s of the 20th century, syn thetic rna hydrolyzing molecules generated from peptides and containing l lysine, histamine, or histi dine methyl ester residues have been developed [52] . chemical conjugates of lysine moieties with imidazole model the active center of rnase, contain the rna binding and rna hydrolyzing domains, and may find application for inactivation of rna in gene targeting therapy [52] . mimetics of rnases of another class have been created based on the conjugates of diazabi cyclo [2.2.2] octane with imidazole; the rate of rna hydrolysis by these mimetics increases proportionally to the number of positive charges in the molecule [53] . recently, synthetic rnases were found to act not only on rna containing viruses through hydrolysis of viral rna [54] , but also on dna containing viruses, in particular cowpox virus, through the destruction of the virus envelope [55] . of antiviral rnases modern antiviral agents should be used with due regard to the data on mechanisms of action and the targets whose damage leads to virus elimination. the most general effect is produced by preparations con taining interferon as a stimulator of the cell's natural defense system against the virus or synthetic analogues of nucleosides that block the synthesis of viral nucleic acids. the effect of other agents is selectively targeted against various stages of viral infection development and the life cycle of the virus, i.e., adsorption, penetra tion, synthesis of virus components, and the exit of daughter virions form cell. agents that act upon virus genome are of particular interest; they include anti sense oligonucleotides [56] , ribozymes [57] , and rnases discussed here. these agents suppress virus production, but they are probably able to destroy latent virus infection. here, we consider three possible mechanisms of the effect of binase on rna contain ing viruses, which may be exerted at any stage of cell infection. at the first stage, when binase meets the virus outside cell, its catalytic activity is not inhibited by the natural rnase and it may destroy viral rna (fig. 3, c) . mechanism of binase penetration inside the virion is not clear, however it has been proven that virus infecting ability decreases upon direct treatment of virus particles with binase [46] . at the next stage, binase may interact with the virus inside an endosome since endosomal type of internalization is characteris tic for exogenous rnases, e.g. α sarcine [58] , rnase a [59] , bs rnase [60] , and binase [61] (fig. 1) . finally, penetration of binase into cell nucleus is of particular importance: here the enzyme may directly destroy viral rna (fig. 3, a and b) . today, localization of exogenous rnases in cell nucleus has been convinc ingly demonstrated only for bs rnase [60] and binase [61] . it should be noted that binase penetrated not all cells: for example, the enzyme did not enter the alveolar epithelium mle 12 cells expressing viral t antigen on their surface, but rather caused their death [62] . since a number of facts that indicate the interaction of rnases with cell surface structures has been accumulated [63] , the contribution of these structures to the internalization of rnases by infected cells should be taken into account. therefore, for the development of highly efficient antiviral preparations based on rnases, knowledge of the exact stage at which the enzyme affects the virus is obligatory. furthermore, careful attention is focused on the possibility of the intervention of exogenous rnases in the process of rna interference involved in protec tion against viruses [64, 65] . the leading role is played by the dicer rnase producing small interfering rnas (sirnas) and using dsrnas (an intermediate product of virus replication) as a substrate [64] . furthermore, it has been found that sirnas specific to a conserved region of influenza virus rna introduced into a cell decreased the titer of the virus [65] . antiviral potential of sirnas is increased upon application in a complex with polycationic carrier [65, 67] . the application of sirnas in antiviral therapy is not limited to the treat ment of the influenza virus. positive results have been obtained in experiments on laboratory mice infected with a coronavirus causing severe respiratory syn drome and syncytial virus [64] . so far, there are few explicit data on the participation of exogenous rnases in formation and destruction of sirnas. it was dem onstrated that onconase changes the sirna expres sion profile in several pleura mesothelioma cell lines through the destruction of precursors of these mole cules and thus decreasing the amount of substrate for the dicer rnase [68] . therefore, the mechanisms of the antiviral activity of rnases include both the direct effect on the nucleocapsid and nucleic acid and indi rect effects, i.e., intervention into the rna interfer ence, immunomodulation, and induction of apoptosis in infected cells. figure 3 demonstrates hypothetical models of the elimination of viral infection by an exogenous rnase in function of the type of interac tion with cell: independent penetration (a), joint internalization (b), or outside cell (c). considerable economic losses from yearly epidem ics cause constant search for new antiviral agents that become useless with time due to high variability of viruses. the study of the molecular mechanism of the action of antiviral rnases is undoubtedly an urgent task, the solution of which may promote the develop ment of novel antiviral preparations capable of pro tecting the organism independently of changes in the virus genome. exoribonucleases and their multiple roles in rna metabolism inhibition of rna synthesis and reproduction of tick encephalitis virus under the influ ence of ribonuclease effect of 1,3;1,6 β d glucan on infec tion of detached tobacco leaves with tobacco mosaic virus effective expression of the gene encoding an extracellu lar ribonuclease of zinnia elegans in the sr1 nicotiana tabacum plants ribonu cleases in bacterial toxin-antitoxin systems structure function rela tions in the ntpase domain of the antiviral trna ribotoxin escherichia coli prrc small self rna generated by rnase l amplifies antiviral innate immunity mecha nistic insights into rnase l through use of an mdmx derived multi functional protein domain influenza a induced cellular signal transduction pathways a viral rna competi tively inhibits the antiviral endoribonuclease domain of rnase l evasion of antiviral innate immunity by theiler's virus l* protein through direct inhibition of rnase l multiple modes of rna recognition by zinc finger proteins inhibition of filov irus replication by the zinc finger antiviral protein gene and protein delivered zinc finger staphylococcal nuclease hybrid for inhibition of dna replication of human papillomavirus mcpip1 ribonuclease exhibits broad spectrum antiviral effects through viral rna binding and degradation mcpip1ribonuclease antagonizes dicer and terminates microrna biogenesis through precursor microrna degradation eosino phils, eosinophil ribonucleases, and their role in host defense against respiratory virus pathogens an insertion in loop l7 of human eosi nophil derived neurotoxin is crucial for its antiviral activity ribonucleases, nucleic acids and molecular biology ribonuclease is partly responsible for the hiv 1 inhib itory effect activated by hla alloantigen recognition ribonucleases in hiv type 1 inhibition: effect of recombinant rnases on infection of primary t cells and immune activation induced rnase gene and protein expression inhibition of hiv 1 production and selective degradation of viral rna by an amphibian ribonuclease entry into cells and selec tive degradation of trna by a cytotoxic member of the rnase a family rana catesbeiana ribonuclease inhibits japanese encephalitis virus (jev) replication and enhances apoptosis of jev infected bhk 21 cells us patent ribonuclease treatment of tick borne encephalitis lekarstvennye sredstva: 5000 naime novanii preparatov i ikh form (svoistva, primenenie, vzaimodeistvie, protivopokazaniya) [medicines: 5000 names of preparations and their forms (properties, appli cations conjugates of pancreatic ribonuclease and ligand free human serum albumin nanoparticle based artificial rna silencing machinery for antiviral therapy cytotoxic ribonu cleases: molecular weapons and their targets x ray structure of two crystalline forms of a strep tomycete ribonuclease with cytotoxic activity rnasea ribunucleases: ancient components of nonspecific defense system of the respi ratory tract rnase inhibition of human immunodeficiency virus infection of h9 cells interferon gamma activates the cleavage of double stranded rna by bovine seminal ribonuclease cytotoxicity of bovine sem inal ribonuclease: monomer versus dimer oligomerization of ribo nuclease a: two novel three dimensional domain swapped tetramers increase of rnase a n terminus polarity or c terminus apolarity changes the two domains' propensity to swap and form the two dimeric conformers of the protein structure and functional studies of the ribonuclease binase glu43ala/phe81ala mutant a hydrophobic segment of some cytotoxic ribonucleases antiviral activity of modified rnases barnase and binase: twins with distinct fates antiviral activity of bacillus intermedius rnase in experiments on cda mice infected with street rabies virus protective activity of bacillus intermedius rnase in guinea pigs and rabbits infected with street rabies virus comparative analysis of antiviral activity of pancreatic and microbial rnases anti flu effect of bacterial rnase in experiment, and pharmacokinetic rationale for the method of its application antiviral activ ity of binase against the pandemic influenza a (h1n1) virus binase induces apoptosis of transformed myeloid cells and does not induce t cell immune response extra cellular ribonu clease production from pseudomonas species purification and some properties of an extracellular ribonuclease with antivi ral activity against tobacco mosaic virus from bacillus cereus antiviral activity of bacillus sp. iso lated from the marine sponge petromica citrina against bovine viral diarrhea virus, a surrogate model of the hepatitis c virus experimental study of antiviral activity of spore forming bacteria bacillus pumilus pashkov artificial ribonu cleases: 1. synthesis and properties of conjugates con taining an rna binding fragment with lys residues and an rna hydrolyzing fragment with imidazole res idue chemical ribonucleases: 2. design and hydrolytic activity of the ribonuclease mimics on the basis of diazabicyclo[2.2.2]octane with a differing number of positive charges inactivation of a non enveloped rna virus by artificial ribonucleases: honey bees and acute bee paralysis virus as a new experimental model for in vivo antiviral activ ity assessment novel amphiphilic compounds effectively inactivate the vaccinia virus preparation of azacrown functionalized 2' o methyl oligoribonucleotides, potential artificial rnases targeting mrnas by engineered sequence specific rnase p ribozymes cyto toxic mechanism of the ribotoxin alpha sarcin. induc tion cell death via apoptosis secretory ribonucleases are internalized by a dynamin independent endocytic pathway essential stations in the intracellular pathway of cytotoxic bovine seminal ribonuclease internalization of bacillus intermedius ribo nuclease (binase) induces human alveolar adenocar cinoma cell death il'inskaia o.n. 2012. binase penetra tion into alveolar epithelial cells does not induce cell death cell targets of antitumor ribonucleases rna interference against viruses: strike and counter strike inhibition of influenza virus production in virus infected mice by rna interference sirna for influenza therapy an influenza virus inspired polymer system for the timed release of sirna onconase downregulates microrna expression through targeting microrna precursors the work was performed according to the russian government program of competitive growth of kazan federal university and was supported by the russian foundation for basic research (project no. 2 04 01226 a) and russian scientific foundation (project no. 14 14 00522). key: cord-339716-1khdh9nf authors: munasinghe, sithum; sperandei, sandro; freebairn, louise; conroy, elizabeth; jani, hir; marjanovic, sandra; page, andrew title: the impact of physical distancing policies during the covid-19 pandemic on health and well-being among australian adolescents date: 2020-10-21 journal: j adolesc health doi: 10.1016/j.jadohealth.2020.08.008 sha: doc_id: 339716 cord_uid: 1khdh9nf purpose: physical distancing policies in the state of new south wales (australia) were implemented on march 23, 2020, because of the covid-19 pandemic. this study investigated changes in physical activity, dietary behaviors, and well-being during the early period of this policy. methods: a cohort of young people aged 13–19 years from sydney (n = 582) were prospectively followed for 22 weeks (november 18, 2019, to april 19, 2020). daily, weekly, and monthly trajectories of diet, physical activity, sedentary behavior, well-being, and psychological distress were collected via smartphone, using a series of ecological momentary assessments and smartphone sensors. differences in health and well-being outcomes were compared preand post-implementation of physical distancing guidelines. results: after the implementation of physical distancing measures in nsw, there were significant decreases in physical activity (odds ratio [or] = .53, 95% confidence interval [ci] = .34–.83), increases in social media and internet use (or = 1.86, 95% ci = 1.15–3.00), and increased screen time based on participants' smartphone screen state. physical distancing measures were also associated with being alone in the previous hour (or = 2.09, 95% ci: 1.33–3.28), decreases in happiness (or = .38, 95% ci = .18–.82), and fast food consumption (or = .46, 95% ci = .29–.73). conclusions: physical distancing and social restrictions had a contemporaneous impact on health and well-being outcomes associated with chronic disease among young people. as the pandemic evolves, it will be important to consider how to mitigate against any longer term health impacts of physical distancing restrictions. aged children, a move to the online delivery of schooling. authorities requested that people remain in their homes wherever possible and limit their travel to obtaining essential goods and services. this public health strategy was absolutely necessary and appears to be yielding the desired result in terms of "flattening the curve" in the australian context [1] . there are potential impacts of physical distancing and social isolation, particularly among younger people, where social connection is a key part of psychosocial development. the necessary policy responses to covid-19 may impact the determinants of poor mental health outcomes, including suicidal behavior [2] . previous studies have shown psychological and physical health impacts of social isolation during quarantine [3] , and more generally, social isolation has been shown to be associated with poor mental and physical health outcomes [4] . in addition, adolescents are likely to have reduced physical activity, particularly incidental physical activity, and increased screen time as a consequence of the physical distancing measures. previous studies have shown the impacts of sedentary behavior on health outcomes in young people [5, 6] and interrelated factors of diet, overweight and obesity, and well-being [5, 7, 8] . the impact of the public health interventions in response to covid-19 to the daily routine of young people in australia on key health and well-being measures known to be associated with chronic disease has not previously been investigated. accordingly, this study investigates whether the physical distancing policies and school closures in the state of new south wales (australia) were associated with changes in physical activity, dietary behaviors, and well-being during the early period of this policy. participants were recruited as part of a broader prospective cohort study of adolescents investigating determinants of health and well-being over time. young people were recruited via social media (instagram and facebook) from the general population aged 13e19 years of a sydney population catchment. promotional and recruitment materials were developed and modified by members of a youth advisory group, and the social media strategy targeted those residing in western sydney; however, participants from areas outside of this catchment were not excluded if they enrolled in the study. the western sydney population catchment is a socioeconomically and ethnically diverse population of approximately one million people. participants were followed prospectively over a period of 22 weeks, from november 8, 2019, to april 19, 2020, after a social media campaign that ran from november 8, 2019, to january 8, 2020. institutional ethics approval for the study was obtained from the western sydney university human research ethics committee (hrec approval number: h13302). the total reach of the social media recruitment campaign was 164,640 adolescents in the western sydney area, of which 61% were female (n ¼ 100,640) and 39% were male (n ¼ 62,944). the total number of impressions (i.e., the number of times advertisements were displayed in news feeds) was 1,389,957, and this was higher among females (n ¼ 955,418, 69%) than males (n ¼ 425,222, 31%). the total number of click-throughs to the study webpage was 11,048, with a higher level of interest among females (n ¼ 8,295, 75%) than males (n ¼ 2,680, 25%). of 11, 048 individuals who clicked through to the study website, a total of 1,298 participants enrolled in the study and completed the baseline questionnaire, from which 582 participants were selected who provided one or more responses to follow-up ecological momentary assessment (ema). participants were predominantly female and aged 16e18 years (table 1) , reflecting the higher engagement in instagram and facebook among females than males more generally [9, 10] . the ethica data smartphone app (https://ethicadata.com/ product) was used to collect data from questionnaires, emas, and smartphone sensors. mobile sensor data were collected automatically through the ethica app only from those participants who provided consent and included geolocation information (via gps, wi-fi, and bluetooth), pedometer, motion-based activity recognition (mbar) data, and screen state (whether the screen of the smartphone is "on" or "off"). a baseline questionnaire and a 16-week schedule of follow-up emas were triggered when participants enrolled in the study, with questions sent directly to each participant's smartphone. there were nine emas relating to psychological distress, well-being, positive emotion, social networks, relationships, diet, physical activity, sleep, and academic behavior. each ema, except psychological distress and well-being, was administered weekly, but on different days. emas relating to psychological distress or well-being were administered monthly. thus, participants received daily emas but received a different ema on each day. emas were sent to participants at random times between 8 a.m. and 10 a.m. or between 3 p.m. and 8 p.m. to avoid notifications during school hours and periods when participants may have been sleeping. the 16week schedule of emas resulted in weekly or monthly measures for each domain spanning the 22-week follow-up period. the primary outcome variables for this study included measures of physical activity, sedentary behavior, dietary behavior, and psychological well-being. self-reported physical activity at baseline was based on responses to the pace þ adolescent physical activity measures [11] , and sedentary behavior was based on the adolescent sedentary activities questions [12] with the tv and computer items modified to also capture information on internet streaming, mobile phone, tablet, or gaming console use. self-reported physical activity and sedentary behavior relating to the previous 24-hour period were also collected each week for the 22week follow-up period via an ema. questions included: (i) "in the past 24 hours, were you physically active for a total of 60 minutes or more? 'physical activity' is any activity that increases your heart rate and makes you get out of breath some of the time"; (ii) "in the past 24 hours, did you spend any time watching tv?"; and (iii) "in the past 24 hours, did you spend any time on the internet, social media (like instagram, youtube, or facebook), or playing computer games?" for participants who answered "yes" to this question, a follow-up question was asked: "if yes, how long did you spend on the internet, social media, or playing computer games?" additional information on physical activity was collected passively via smartphone sensors, including pedometer, screen state (i.e., whether the phone was "on" or "off"), and mbar. the daily number of steps for each participant was collected via the pedometer. screen state was used as a proxy measure of sedentary behavior, with the assumption that during periods where the phone screen was active, participants were less likely to be engaging in physical activity. mbar is a composite indicator of activity provided by the ethica data app, which combines information from the phone sensors, including accelerometer, gyroscope, gravity, and magnetic field [13] . the mbar indicator is a categorical variable that divides each moment into an activity type: "on foot," "walking," "running," "on bicycle," "in vehicle," "unknown," "still" (the device is not moving), and "tilting" (the device angle relative to gravity has changed significantly). each categorization is also ascribed a confidence level score between 0 and 100. in the present study, each participant's mbar category was weighted by this score, such that categories with high confidence level scores were considered a more accurate assessment of the type of activity. self-reported dietary behavior at baseline was measured using questions validated for adolescents by the nsw centre for public health nutrition [14] to allow comparisons with dietary guidelines for children and adolescents in australia [15] . selfreported dietary behaviors relating to the previous 24-hour period were also collected each week for the 22-week followup period via an ema. questions included: (i) "in the past 24 hours, have you eaten any serves of fruit?" if participants responded "yes," a follow-up question was asked: "how many serves of fruit? (a serve ¼ 1 medium piece or 2 small pieces of fruit or 1 cup of diced pieces)"; (ii) "in the past 24 hours, have you eaten any serves of vegetables?" if participants responded "yes," a follow-up question was asked: "how many serves of vegetables? (a serve ¼ 1/2 cup cooked vegetables or 1 cup of salad vegetables)"; and (iii) "in the past 24 hours, have you had any meals or snacks such as burgers, pizza, chicken, or chips from places like mcdonalds, hungry jacks, pizza hut, kfc, red rooster or local takeaway food places?" if participants responded "yes," a follow-up question was asked: "how many meals?" psychological well-being self-reported psychological distress was based on the kessler psychological distress 6-item scale (k6) [16] . response options for each k6 item included "none of the time," "a little of the time," "some of the time," "most of the time," and "all of the time" and were scored in the range of 1e5 respectively. a score 19 was used as indicative of probable mental disorder as recommended [16] ; however, it is important to note that this standard cut point may overlook those with more moderate levels of psychological distress that may still be important [17] . the engagement, perseverance, optimism, connectedness, and happiness (epoch) measure of well-being was also included in the study to capture information on positive psychological characteristics [18] using a 5-point scale from "almost never" to "almost always." the k6 and epoch questionnaires (supplementary materials) were completed by participants at baseline with follow-ups sent to each participant every 4 weeks and short emas relating to selected epoch items sent weekly [19] . in addition, social relationships were measured based on the question: "in the past hour, who were you with?" participants could respond to one or more of the following options: "alone," "mother," "father," "sister(s)," "brother(s)," "other relatives," "classmates, peers," "strangers," "boyfriend or girlfriend," "friends," and "other, please specify." for participants who answered "friends," an additional question was asked: "how many friends?" finally, self-reported sleep duration in the previous 24 hours was also collected at baseline via a weekly ema over the 22-week follow-up period. a range of sociodemographic and other health factors were also collected at baseline. these factors included sex, age, language spoken at home, current year of school and educational achievement, employment status, income, and body mass index (based on self-reported height and weight; table 1 ). the change in measures of physical activity, dietary behavior, and well-being was compared pre-and post-implementation of the nsw guidelines for physical distancing to determine whether this policy resulted in significant changes in these key health behaviors. these guidelines officially came into effect on march 31, 2020 [20] ; however, physical distancing began in the earlier period of march with the closure of pubs, clubs, gyms, cinemas, places of worship on march 23, 2020 [21] and evidence of parents keeping children at home from school. accordingly, the period for when physical distancing began to be implemented was defined as march 23, 2020. analyses were restricted to those participants who completed at least one ema over the follow-up period (n ¼ 582; table 1 ). participants were predominantly female, with a median age of 17 years (interquartile range, 16e18). most participants spoke english at home (86%), were either in their senior year of schooling (23%) or finished school (43%), and almost 60% worked in a job (mainly part time). these participants contributed 4,805 responses to emas over the 22-week follow-up period, including 301 responses in the period after implementation of physical distancing guidelines ( table 2 ). the mean number of emas per week for this group was 9.6 (standard deviation ¼ 5.8), and the median number of emas per week was 10 (interquartile range ¼ 3e16). comparisons of participant characteristics between (1) those who completed baseline and follow-up, (2) those who completed emas pre-and post-implementation of the physical distancing policy, and (3) those who provided or did not provide sensor are provided in supplementary tables 1 and 2 descriptive plots of trajectories of physical activity were examined over the 22-week follow period, based on daily pedometer data, mbar, and weekly self-report emas. trajectories of self-reported fruit, vegetable, and fast food consumption were also examined based on weekly emas, as were trajectories of psychological well-being based on distress, well-being, and sleep duration. multivariate multilevel mixed effect logistic regression models were conducted to investigate associations between the implementation of nsw guidelines (specified as a binary pre-post variable on march 23, 2020) and subsequent changes in physical activity, dietary behavior, and well-being measures. there were significant decreases in physical activity in the period after the implementation of physical distancing measures in nsw. adolescents were significantly less likely to report 60 minutes of physical activity in the previous 24 figure 1a ). declines in physical activity were also evident based on the average number of steps per day and mbar (figure 2a,b) . there was also a significant increase in sedentary activity postimplementation of physical distancing, with higher social media and internet use (or ¼ 1.86, 95% ci ¼ 1.15e3.0; table 3, figure 2a ) and also evidence of increased screen time based on participants' smartphone screen state ( figure 2c ). the implementation of physical distancing measures was associated with lower levels of happiness (or ¼ .38, 95% ci ¼ .18e.82) and positive emotions (or ¼ .23, 95% ci ¼ .14e.39), respondents reporting being alone in the previous hour (or ¼ 2.09, 95% ci ¼ 1.33e3.28), and slightly higher increases in psychological distress (or ¼ 1.48, 95% ci ¼ .74e2.95; table 3 , figure 2b ). there were also declines in fast food consumption following implementation of physical distancing (or ¼ .46, 95% ci ¼ .29e .73) but no substantial changes in fruit and vegetable consumption, tv watching, or sleep duration (table 3 ; figure 1c ). this study investigated the impact of physical distancing guidelines implemented in new south wales, australia, on a range of health and well-being outcomes among a cohort of adolescents aged 13e19 years in sydney. the implementation of physical distancing interventions was associated with decreases in physical activity and well-being, and increases in being alone and social media and internet use in the 4 weeks after the policy was implemented. there was also a decrease in selfreported fast food consumption in the 4 weeks after the policy was implemented, but little change in fruit or vegetable consumption. these findings suggest that the substantial changes to the way in which communities are currently functioning, particularly for young people, has had a contemporaneous impact on health and well-being outcomes associated with chronic disease. an important finding in the present study were the decreases in happiness reported after the implementation of the physical distancing guidelines and a higher likelihood of being alone during this period. social isolation is an important risk factor for poorer psychological well-being among young people and, conversely, peer-, family-and school-connectedness play key roles as protective factors [22] . these protective connections may not have been as accessible to young people during the period of physical distancing resulting in lower levels of psychological well-being. it will be important to ensure that protective connections and other strategies to support the well-being of young people are maintained, to mitigate the potential psychological impact on young people. in australia, covid-19 cases remain low at the time of reporting; however, it is possible that physical distancing restrictions and online education may need to be reinstated if a second or third wave of infections eventuates. the shift to online delivery of education in nsw and the requirement to defer any nonessential travel is reflected in the increase in social media and internet use for the corresponding period in this study. there was also a decrease in physical activity likely related to the suspension of school and community sport and potentially mediated by a lack of access to green space in home environments. recent reviews have suggested both positive and negative impacts of social media, determined by the type of involvement (e.g., passive use, high investment, or support seeking) as well as the amount of time spent on screenbased activity [7, 23, 24] . in addition, some studies have found that screen-based sedentary behavior supplants time spent sleeping or engaged in physical activity [7] . the present study did not directly examine the association between screen-based sedentary behavior and physical activity, but while the pattern of findings is consistent with the idea of displacement, this may only be relevant when time is constrained (such as during school term or nonholiday periods). the finding that sleep hours did not decline contemporaneously with increased screen time perhaps suggests study participants had more time to engage in sedentary behavior without disrupting sleep duration. it remains to be seen whether sedentary behavior observed during the period of physical distancing will revert back to levels observed before physical distancing measures. this will be an important focus for future research, given the evidence that sedentary habits in adulthood are typically established during adolescence [12] . an interesting finding was the decrease in fast food consumption in the context of limited changes to fruit and vegetable consumption. this likely reflects a decrease in opportunistic purchases of fast food during the day and traveling either to school or to work. previous research has found increased consumption of this food type among adolescents and young people where there is a high density of fast food outlets located near schools and transport hubs [25, 26] . since the initial period of physical distancing, many fast food outlets have moved to take away and home delivery; however, the reduced consumption observed may indicate that fast food consumption was opportunistic and more associated with connecting socially with friends [26] . future studies could consider the impact of these changes on food delivery on the consumption of fast food among younger people of different ages and with differing discretionary income and access to private transport. the present study also found that consumption of fruit and vegetables did not increase, suggesting either that similar food types were substituted or there was a decrease in overall caloric intake. consumption of calorie-dense foods can be positively associated with feelings of stress, and given the reduction in fast food consumption occurred in the context of increased social isolation and psychological distress, this might explain the lack of nutritional substitution implied in this finding. despite reduced consumption of these food types via fast food outlets, there may have been an overreliance on processed supermarket food during this periodda limitation to this finding was that more specific questions relating to processed or junk food (i.e., not fast food purchases) were not explicitly measured. australia experienced panic buying of processed foods resulting in supermarkets placing limits on a number of food items because of shortages. however, this was not observed for fresh fruit and vegetables. alternatively, it may be that the development of new healthpromoting behaviors takes time to develop, and the observation period of the present study was not long enough for this to emerge. there are a number of methodological limitations to this study. first, although there was a positive response to the study through instagram and facebook, participants who were more likely to engage were overwhelmingly female and more likely to be older in age (16e18 years). the higher proportion of females may reflect greater engagement in social media among females than males, a phenomenon that has been noted in representative studies of social media use in australia [9, 10] . despite the imbalance by sex, the distribution of responses by key dietary behaviors, physical activity, and wellbeing outcomes was not substantially dissimilar to other representative prevalence studies of adolescents [26, 27] . the higher proportion of older-age adolescents likely reflects that for those aged 13e15 years, parental or guardian consent was required before enrollment in the study. this involved additional steps in making contact with parents or guardians via email and to arrange for links to download the ethica app, which likely discouraged some younger potential participants from enrolling in the study. an additional limitation was the low ema and follow-up survey completion rate. despite the use of an incentive (aud $30 giftpay voucher), only 45% of baseline participants (n ¼ 1,298) completed one or more subsequent ema, and <1% completed all 96 emas over the follow-up period. the weekly schedule of emas may have been too burdensome for participants, and future research may need to consider different schedules or incorporation of personalized feedback to keep young people engaged. there is also the risk of recall bias in this study, given the self-reported nature of the baseline and follow-up questionnaires. however, emas in (near) real time potentially reduce the likelihood or recall bias, in that questions relate to the immediate 24-hour period. patterns of ema responses relating to physical activity and screen time were also consistent with objective measures of physical activity based on available mobile phone sensor data, and the results were also generally comparable with previous adolescent health surveys for some of the measures [26, 27] . smartphone sensor data, collected passively from participants, were also used as proxy measures of physical activity and sedentary behavior. this was an innovative aspect of the study design and allowed comparison with ema responses and investigation of trajectories of spatiotemporal movement among participants. however, a large proportion of participants either did not turn on some smartphone sensors (e.g., geolocation) or there were intermittent trajectories of movement, where sensor data were not collected. this resulted in complete sensor information being available on only 515 participants over the follow-up period, only 40% of baseline participants (n ¼ 1,298) . the reasons for participants choosing not to engage in this aspect of the study are unclear but may relate to concerns about individual privacy, among both participants and caregivers (who were required to give parental consent for young people aged <16 years). there is also likely to be misclassification in smartphone sensor data, where periods between the initiation and cessation of a given state (e.g., "walking" in the mbar sensor) were likely overestimated, as the smartphone did not register the cessation of the state but only the initiation of the subsequent state (e.g., "still" in the mbar sensor). this means that the overall level of activity as measured by the smartphone sensor will be an overestimate; however, relative changes over time are likely to reflect actual declines or increases, and changes in emas appear to be consistent with changes in activities as measured by smartphone sensors. the present study suggests the potential immediate impacts on health behaviors that are intermediary to chronic disease outcomes. however, it is not clear whether there will be longterm psychosocial and health impacts associated with the physical distancing policies. these policies will be slowly wound back over coming weeks, and education and employment experiences will return to a degree of normality. the wider economic impacts associated with physical distancing and other policies relating to the closure of businesses and entertainment precincts on health outcomes are also not known. it is unclear whether the changes in health and well-being documented in the present study will be transient or whether there may be ongoing impacts. additional research needs to establish longer term trends in these outcomes to inform public health policy and intervention responses. this study provided a unique opportunity to measure health behaviors and psychological well-being among australian adolescents during the covid-19 pandemic. data collection occurred pre-and post-implementation of widespread physical distancing regulations in the community. these public health interventions have successfully "flattened the curve" on covid-19 to date; however, there have also been important changes among young people on a range of health and well-being outcomes. further research is needed to monitor the longer term trends in these outcomes. as the pandemic evolves, it will be important to consider how best to support psychological and physical wellbeing for young people to mitigate against potential longer term negative impacts. supplementary data related to this article can be found at https://doi.org/10.1016/j.jadohealth.2020.08.008. australian government department of health. coronavirus (covid-19) current situation and case numbers 2020 suicide risk and prevention during the covid-19 pandemic the psychological impact of quarantine and how to reduce it: rapid review of the evidence an overview of systematic reviews on the public health consequences of social isolation and loneliness child and adolescent obesity: part of a bigger picture long-term impact of overweight and obesity in childhood and adolescence on morbidity and premature mortality in adulthood: systematic review the health indicators associated with screen-based sedentary behavior among adolescent girls: a systematic review longitudinal impact of sleep on overweight and obesity in children and adolescents: a systematic review and bias-adjusted meta-analysis office of the esafety commissioner. state of play -youth, kids and digital dangers yellow social media report. melbourne: a sensis company a physical activity screening measure for use with adolescents in primary care the reliability of the adolescent sedentary activity questionnaire (asaq) available at: https://learn.ethicadata. com/documentation/data-sources/motion-sensors recommendations for short questions to assess food consumption in children for the nsw health surveys. sydney: nsw centre for public health nutrition dietary guidelines for children and adolescents in australia. canberra: commonwealth of australia the psychometric properties of the kessler psychological distress scale (k6) in a general population sample of adolescents validity study of the k6 scale as a measure of moderate mental distress based on mental health treatment need and utilization the epoch measure of adolescent well-being a comparison of affect ratings obtained with ecological momentary assessment and the day reconstruction method please cancel travel to regional nsw coronavirus: australia to close pubs, cafes and places of worship 2020 social isolation, psychological health, and protective factors in adolescence social networking sites and associations with depressive and anxiety symptoms in children and adolescentsea systematic review. child adol ment health association between social networks and subjective well-being in adolescents: a systematic review obesogenic neighbourhoods: the impact of neighbourhood restaurants and convenience stores on adolescents' food consumption behaviours overweight/obesity, physical activity and diet among australian secondary students-first national dataset 2009-10. cancer forum. the cancer council australia key: cord-327521-g5vefajw authors: spisni, enzo; petrocelli, giovannamaria; imbesi, veronica; spigarelli, renato; azzinnari, demetrio; donati sarti, marco; campieri, massimo; valerii, maria chiara title: antioxidant, anti-inflammatory, and microbial-modulating activities of essential oils: implications in colonic pathophysiology date: 2020-06-10 journal: int j mol sci doi: 10.3390/ijms21114152 sha: doc_id: 327521 cord_uid: g5vefajw essential oils (eos) are a complex mixture of hydrophobic and volatile compounds synthesized from aromatic plants, most of them commonly used in the human diet. in recent years, many studies have analyzed their antimicrobial, antioxidant, anti-inflammatory, immunomodulatory and anticancer properties in vitro and on experimentally induced animal models of colitis and colorectal cancer. however, there are still few clinical studies aimed to understand their role in the modulation of the intestinal pathophysiology. many eos and some of their molecules have demonstrated their efficacy in inhibiting bacterial, fungi and virus replication and in modulating the inflammatory and oxidative processes that take place in experimental colitis. in addition to this, their antitumor activity against colorectal cancer models makes them extremely interesting compounds for the modulation of the pathophysiology of the large bowel. the characterization of these eos is made difficult by their complexity and by the different compositions present in the same oil having different geographical origins. this review tries to shift the focus from the eos to their individual compounds, to expand their possible applications in modulating colon pathophysiology. essential oils (eos) are a complex mixture of volatile compounds that are produced by aromatic plants as secondary metabolites. they are present in all plant organs and their main role is to defend plants from aggressions by bacteria, fungi and viruses, but also by insects. there are huge amounts of eos obtained from different plants around the world, and most of them have been at least partially characterized for their antimicrobial activity against gram-positive and gram-negative bacteria, but also against other microorganisms such as fungi and virus. the composition of the eos was selected by nature during an evolutionary process lasting millions of years. their activity is the result of the competitive selection process acting on their antibacterial, antifungal and antiviral activities in a continuous evolutionary conflict between the survival of plants and the microbial aggressions. the antimicrobial strategies of eos have been to develop multi-target mechanisms of action which make it very difficult for microorganisms to become resistant to these compounds [1] . the enormous potentials for the possible use of eos as therapeutic agents against human pathogen the excessive amount of reactive oxygen species (ros) is at the basis of degenerative processes such as lipid peroxidation, protein glycation/oxidation and nitration, enzyme inactivation and dna damages that occur in many non-communicable diseases, including colitis and neurodegenerative diseases [3, 4] . some eo single molecules, such as geraniol, easily cross the blood-brain barrier and then reach all the organs in which they can exert their antioxidant activities [5] . ros exert multiple modulating effects on inflammation and play a key role in the regulation of immune responses [6] . eos and their molecules are capable of modulating different signaling pathways that are overactivated or downregulated during acute or chronic inflammation responses [7] . the recent discovery of the complexity of the human intestinal microbiota, composed of bacteria, fungi and viruses, and its intricate pathophysiological relationships with the immune system and the enteric nervous system, makes eos truly interesting for their antimicrobial activities, often selective for the different microbial components. from this point of view, eos could be considered potential powerful modulators of the intestinal microbiota. unfortunately, most eo molecules are quickly assimilated into the small intestine and do not reach the colon, where most of the intestinal microbiota is known to reside. the release of eos into the colon, therefore, becomes a fundamental point to allow these compounds to effectively modulate the pathophysiology of the colon. from this point of view, our research group is one of the first in the world to have used a single eo molecule in human clinical trials, to modulate the microbiota and manage the symptoms associated to a functional bowel disorder such as the irritable bowel syndrome (ibs) [8] . this review explores, in a broad and modern way, the knowledge that can lead to the effective use of eos and/or their single molecules in the modulation of the physiology and pathology of the large bowel. the major components of the most common essential oils are shown in table 2 , together with their chemical structure and major biological activity. several studies have been conducted to evaluate the effects of eos in colon inflammation [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] . in particular, several preclinical studies have been conducted on validated models of colitis induced in rats or mice by administration of dextran sulphate sodium (dss), trinitrobenzenesulfonic acid (tnbs) or acetic acid. while dss and acetic acid induces chemical damage to the epithelial cells, with consequent disruption of mucosal barrier and activation of immune cells in the lamina propria by the intestinal microbiota, tnbs acts by directly haptenizing colonic autologous/microbial proteins [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] . from this point of view, dss and acetic acid are models used in a translational way for ulcerative colitis studies, while tnbs is a model with some characteristics that bring it closer to crohn's disease. these models allow researchers to evaluate the effects of different eos on clinical, histological, serological and molecular markers of colitis. clinical features of the experimental colitis are mainly represented by weight loss, changes in stool consistency and rectal bleeding, usually scored to obtain a disease activity index (dai). histological analyses are performed to evaluate the extent of ulcerated areas, alterations of mucosal architecture and inflammatory cell infiltrations. the molecular markers that are more often evaluated comprise some circulating pro-inflammatory cytokines and the colonic expression of a plethora of enzymes and factors involved in inflammation. they include cyclooxygenase-2 (cox-2), nitric oxid synthase (inos), peroxisome proliferator-activated receptor-gamma (pparγ), nuclear factor kappa-light-chain-enhancer of activated b cells (nf-kβ) or myeloperoxydase (mpo) [9] . the anti-inflammatory effects of eos on colitis models are summarized in table 3 . with regard to the mechanisms of action of eos and their single molecules at the cellular and molecular levels, we underline that it is almost impossible to understand which molecule may exert anti-inflammatory effects when eos are used. even in studies carried out on single eo molecules, it is not easy to define a main molecular target, because these compounds act in a multitarget manner ( figure 2 ). for example, the anti-inflammatory effect of these molecules could be linked to direct interaction with pro-inflammatory proteins, such as nf-kβ, pparγ cox-2 and inos, but this direct interaction still remains to be demonstrated for the majority of these molecules. a specific interaction between borneol, camphene and eucalyptol, major components of thyme eo, and pro-inflammatory enzymes such as inos and cox-2 has been only indirectly demonstrated in vitro [18] . the multitarget effect of geraniol has been clearly demonstrated in vitro, since it is capable of regulating wnt/β-catenin, p38mapk, nfκb, pparγ and cox-2 signaling pathways. the transcription factor nf-kβ seems to be a common target for many different eo molecules, such as geraniol, eucalyptol, α-pinene, and camphor [19] , even if a definitive demonstration of the binding between these molecules and their supposed target is still lacking and should be provided by using molecular modeling studies. conceptually, however, these studies on colitis should be divided between those that delivered eo as it is, without carrier or controlled release and those in which the eo was delivered with retarded release systems, in order to really reach the colon. only by using these controlled release systems can the effects of these eos be analyzed at the colon level, where the experimentally-induced inflammation mainly occurs. basing on available studies, we can conclude that eos and their constituents could be very effective against the inflammatory component of experimental colitis. unfortunately, eo complexity makes it difficult to identify all the possible molecular targets responsible for these strong anti-inflammatory effects. the majority of studies on the antioxidant effects of eos, available in the scientific literature, are based on in vitro approaches. a limiting factor of these studies is that eos can themselves be oxidized within the intestinal lumen or into the stomach, thus losing some of their antioxidant properties even before reaching the small intestine. geraniol showed good antioxidant proprieties in vitro: palmrose and citronella eos, mainly composed of geraniol, have demonstrated to have an effective antioxidant activity in vitro on human lymphocyte cells. in this model, geraniol-containing eos, at a relatively low concentration (125 ppm), protected lymphocytes from dna methylation damages induced by methyl methanesulfonate [20] . these serum doses of geraniol are easy to achieve with a diet rich in aromatic plants or with food supplements [21] . recent studies have shown that geraniol administration reduced the intestinal inflammation induced by dss [7] , but these anti-inflammatory effects could be also linked to its antioxidant activity, since its administration resulted in a decreased inos activity and a decreased lipid peroxidation, in a rat model of colitis [22] . geraniol seems to exert its antioxidant activity also indirectly, by increasing the synthesis of liver antioxidant enzymes after oral administration at 120 mg/kg, in mice [7] . ginger eo administration at 200, and 400 mg/kg, significantly reduced the intestinal lipid peroxidation, increased the expression of intestinal antioxidant enzymes and serum glutathione level in a model of colitis induced by acetic acid in rats. in this model, ginger eo was able to induce free radicals neutralization and to protect the cell membrane lipids from oxidation, in a dose-dependent manner [14] . a recent study has evaluated antioxidant proprieties of carvacrol (5-isopropyl-2-methylphenol) which is a major monoterpenic component of origan (oreganum vulgarea), a plant widely used in mediterranean cuisine. carvacrol was used in a model of experimental induction of colorectal carcinoma, by using 1,2 dimethylhydrazine and dss, in rats. carvacrol was orally administrated before and after tumor induction at a dosage of 50 mg/kg. results of this and other studies showed that carvacrol was an excellent antioxidant agent and reduced colonocyte damages caused by ros [23, 24] . thymol is a natural phenol monoterpene isomer of carvacrol. thymol is one of the major constituents (20-65%) of thyme (thymus vulgaris) eo. an in vitro study on colon epithelial cells showed that thymol, at low doses (12.5 ppm), was a protective compound against oxidative dna damage [21] . twelve aromatic molecules from basil and thyme eos have been analyzed for their antioxidant activities, and in particular eugenol, 4-allylphenol, thymol and carvacrol (5 µg/ml) have shown greater antioxidant activities, measured in vitro on by using the aldehyde/carboxylic acid assay [23] . basil eo, orally-administered, has shown beneficial effects in a model of colitis induced by acetic acid in rats, at two different doses of 160 mg/kg and 320 mg/kg die. these effects were also linked to the reduction of mpo activity in the colon wall, an enzyme clearly involved in the oxidative damages induced by colitis [15] . despite this, further confirmations should be provided by human clinical trials. basing on available data we can confirm that eos and their constituents have interesting antioxidant properties that could justify their use as therapeutic agents against chronic intestinal oxidative damages ( figure 3 ). . antioxidant effect of essential olis (eos) in the gut. the chronic low-grade inflammation or dysbiosis that very soon occurs into the gut and in the gut wall increases the level of reactive oxygen species (ros). their increased levels are effectively counteracted by eos that are able to reduce the activity and expression of enzymes, such as myeloperoxydase (mpo), cyclooxygenase-2 (cox-2) or inducible nitric oxide synthase(inos), which are the ones most responsible for ros production and for the oxidative damages related to them. there are approximately 100 trillion cells in the human body, and more than 90% of them are microbes. they make up the human microbiota, consisting of bacteria, fungi and even viruses, mainly located in the intestine where they form the intestinal microbiome. microbiota can be considered a complex human organ which closely interacts with the gut-associated linfoid tissue (galt) and with the enteric nervous system. it is involved in many digestive functions, but it is also able to modulate the physiology of the immune system both locally and in the whole body. quantitative and qualitative microbiota alterations, known as dysbiosis, may be involved in the development or in the chronicization of several diseases [24] . the analysis of the bacterial component of the intestinal microbiota, thought their 16s rrna sequences, allowed to identify 4 major phyla, firmicutes (79%), bacteroidetes (17%), actinobacteria (3%) and proteobacteria (1%). at a lower taxonomic level, the most represented bacterial genera were found to be faecalibacterium, ruminococcus, eubacterium, dorea, bacteroides, alistipes and bifidobacterium [25] . the microbiota normally represents an ecologically stable environment, but pathogenic bacterial strands or xenobiotics can interfere in this equilibrium and give rise to dysbiosis and/or colitis. the use of broad-spectrum antibiotics to counteract infectious diseases is often associated with the onset of antibiotic resistance phenomena, other than cause a transient dysbiosis in the gastrointestinal tract. in the last few years, a great effort has been made to find new strategies to overcome the rising issue of antibiotic-resistance. in this scenario, eos may have a consistent role thanks to their capacity to control and modulate bacterial growth, acting both as bacteriostatic or bactericidal agents [26] . in fact, due to their lipophilic properties, eos can penetrate membranes, and damage bacterial cell structure, making their membranes more unstable and permeable. membrane disruption may also lead to bacterial death caused by the significant leak of ions and other essential cytosolic components. these eo effects are generally more pronounced on gram-positive bacteria with respect to gram-negative ones [27] . however, it has been demonstrated that eos can also affect the bacterial cell-wall and restore antibiotic susceptibility in drug-resistant gram-negative bacterial strains [28] . several studies have explored the antibacterial properties of eo single molecules. among them, the most studied was certainly geraniol, for its interesting antimicrobial potential. geraniol antibacterial activity seems to be linked to his property to destabilize bacterial cell wall and damage transmembrane efflux pumps, thus restoring drug-sensitivity in different bacterial antibiotic-resistant strains, such as enterobacter aerogenes, escherichia coli, pseudomonas aeruginosa and acinetobacter baumannii [29] . despite being absorbed very quickly and in an active manner by the small intestine mucosa, geraniol is reported to positively modulate the colitis-associated dysbiosis when administered by oral route by using a controlled delivery system based on microencapsulation [7] . in mice but also in humans, geraniol has demonstrated to act as an excellent modulator of intestinal microbiota, capable to boost populations of butyrate-producer bacteria such as collinsella and faecalibacterium, normally reduced in the dysbiotic human intestinal flora of ibs patients [8] . it is interesting to note how geraniol antibacterial activities were selective for pathogenic bacteria and do not involved commensal species [30] . for these reasons, geraniol can be considered as an efficient positive modulator of the intestinal microbiota. another interesting eo molecule with antibacterial activities is eugenol (2-methoxy-4-(prop-2en-1-yl)-phenol), the major compound present in clove oil, but also found in many other eos. eugenol has demonstrated antimicrobial activities based on a non-specific permeabilization of the bacterial membrane with depletion of cytosolic molecules such as adenosin triphosphate (atp), necessary for bacterial metabolism and survival [31] . this effect has been observed against e. coli, listeria monocytogenes and lactobacillus sakei using the relatively low concentration of 10 mm [32] . eugenol antibacterial effects against the pathogen l. monocytogenes have been analyzed in-depth and the principal mechanism of action identified was the alteration of the respiratory bacterial chain associated with dna damages [32] . in mice, orally administrated eugenol improved the secretion of the intestinal mucus, creating a thicker intestinal layer associated with positive changes of the mucosal microbiota ecology. in particular, it has been shown that eugenol inhibited the intestinal adherence of citrobacter rodentium, a mice pathogen that shares several biochemical features with clostridium difficile in humans [33] . another interesting antimicrobial effect of eugenol was observed in p. aeruginosa and e. coli, where this compound was able to inhibit their chemical communication system, also known as quorum sensing [34] . cinnamaldehyde (2e-3-phenylprop-2-enal) is a phenylpropanoid naturally present in the plant of the genus cinnamon. cinnamaldehyde is one of the most studied eo molecules and it has been already approved as antimicrobial food preservative [2] . antibacterial effects of cinnamaldehyde have been demonstrated by using many different bacterial models, but only a few studies evaluated its impact on the whole intestinal microbiota. in vitro, cinnamaldehyde was capable to inhibit the growth of potentially pathogenic bacteria such as staphylococcus aureus, enterobacter cloacae, a. baumannii and l. monocytogenes [35] and it was able to kill a pathogenic strand of e. coli at a very low concentration (0.05% v/v) [36] . one of the proposed antibacterial mechanisms of cinnamaldehyde inhibition of e. coli growth was the inactivation of its acetyl-coa carboxylase enzyme [37] . other studies showed that cinnamaldehyde antimicrobial activity has a broad spectrum of action, being effective also against enterococcus faecalis, enterococcus faecium, e. aerogenes salmonella enterica and clostridium perfringens [38] . finally, with a concentration of 20 µg/ml, cinnamaldehyde was also capable to improve the bactericidal efficacy of the antibiotic clindamycin on c. difficile, significantly decreasing its minimum inhibitory concentration (mic) from 4.0 to 0.25 µg/ml [39] . in vivo, only a few studies have been conducted on cinnamaldehyde, perhaps because of its strong aggressiveness towards the mucosal epithelia. nevertheless, in animal experimental colitis, the oral administration of cinnamon eo (approx. 70% in cinnamaldehyde) at 10 mg/kg or 15 mg/kg lead to an improvement of the ecological biodiversity of the intestinal microbiota. short-chain fatty acids (scfa)-producing bacteria family, such as bacteroidaceae, were increased while intestinal helicobacter and bacteroides were reduced [40] . in broiler and duck farming, the supplementation of food with a mixture of thymol and cinnamaldehyde improved animal growth performances and positively modulated their intestinal microbiota composition, boosting healthy bacteria and reducing anaerobic coliforms and lactose-negative enterobacteria [41, 42] . thymol is effective at extremely low concentrations (as low as 300 ppm) against the growth of many species of pathogenic bacteria that colonize the human intestine, such as c. difficile, c. perfringens, propionibacterium shermanii, propionibacterium freudenreichii and bacteroides thetaiotaomicron. the negative aspect of its antimicrobial activity is that thymol was not selective, and could also have a negative impact on commensal bacteria [30] . on the other hand, eos in which thymol is a major component have clearly shown to have a wide-spectrum bactericide effects on different pathogenic species such as l. monocytogenes, e. coli, s. enterica, s. aureus, clostridium botulinum, c. perfringens, shigella sonnei, sarcina lutea, mariniluteicoccus flavus, brochothrix thermosphacta, listeria innocua, l. monocytogenes, pseudomonas putida and shewanella putrefaciens [26] . moreover, thymol seems to be effective also against the bacterial biofilm formed by β-lactamase-producing enteric bacteria [43] . it is still debated if thymol could be or not degraded by the intestinal microbiome since it was found to be not effective against fecal fermentation reactions [44] . nevertheless, in weaning piglets, thymol associated with carvacrol was capable of positively modulating their microbiota by increasing populations of the lactobacillus genus and by reducing populations of enterococcus and escherichia genera [45] . in vitro, carvacrol was showed to inhibit bacterial adhesion, invasion and biofilm development in cultured intestinal cells [46] . in the farmed broiler, treatment with carvacrol-rich eos was tested to control the pathogenic bacteria spreading inside the farms. the results of these studies demonstrated that carvacrol reduced the microbial counts of e. coli and different salmonella species in the small intestine of farmed chicken [46] . moreover, carvacrol administration to broiler chickens was capable of eliminating the intestinal presence of campylobacter spp. after 21 days of daily oral administration at 120-300 mg/kg. this effect was probably linked to the enhanced growth of bacteria of the lactobacillus genus, that were found to be increased in chicken microbiota, after carvacrol administration. e. coli growth in the cecum of chickens was found to be significantly reduced by carvacrol. for these reasons, this molecule is today the most used in organic breeding to positively modulate gut microbiota and improve the growth performance of farmed chickens [47] . in intensive breeding practices carvacrol is often associated with thymol, since there is strong evidence that the combination of the two was more effective in decreasing intestinal pathogens and increasing the growth performance of chickens [48, 49] . limonene (1-methyl-4-(prop-1-en-2-yl)-cyclohex-1-ene) is a cyclic monoterpene present in a high amount in eo of citrus fruit peels and, in smaller concentrations, in many other aromatic plant eos. limonene has widely demonstrated antimicrobial and anti-inflammatory effects in vivo. in mice, daily oral administration of 160 mg (8000 mg/kg) of orange oil, rich in limonene, modulates the mice microbiota by enhancing the relative abundance of the lactobacillus genus and reducing the presence of scfa-producing bacteria [50] . its particular ability to reduce the scfa synthesis has been exploited in a mouse model of obesity to reduce weight gain. mice fed with a high-fat diet (hfd) were treated daily with microencapsulated sweet orange oil for 15 days (2 ml of suspension of microcapsules containing 18 g/l of sweet orange eo rich in limonene). the result of this study showed a reduced body weight in treated mice, associated with a modulation of the intestinal microbiota. specifically, the bifidobacterium population was enhanced with an overall reduction of the intestinal chronic inflammation induced by the hfd, in treated mice [51] . despite the low toxicity of limonene, which would not rise concerns, it should be noted that these effects on the microbiota were obtained only with high dosages of this compound. eucalyptol (1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane) is a cyclic ether and a monoterpenoid. it is the major compound in eucalyptus eo, but it can be also found in many other officinal plants eos. a recent review indicates that eucalyptus eo has extraordinary antimicrobial activities. eucalyptus eo has shown to be effective against a plethora of bacteria species and among them s. aureus, e. coli, bacillus subtilis, klebsiella pneumonia, salmonella enteritidis and p. aeruginosa [52] . nevertheless, eucalyptus eo also contains high amounts of other antimicrobial components besides eucalyptol, therefore not all of eucalyptus eo antibacterial activity can be ascribed to the presence of eucalyptol. however, literature data regarding eucalyptol antimicrobial activity are very limited, and new studies focused on this interesting compound are needed. menthol (5-methyl-2-(propan-2-yl)cyclohexan-1-ol) is a chiral alcohol and the main molecule present in cornmint and peppermint eos. it has been well known for its use in foods as a cooling and minty-smell aroma. many in vitro studies, reviewed by kamatou and coworkers [53] focused on its antibacterial activities. interestingly, in vitro, menthol was capable to drastically decrease c. difficile viability at 18.8 mg/ml, with a dose-dependent effect. its mechanism of action seems to be due to the significant leakage of cellular atp induced by menthol in this pathogenic bacterium [54] . fungi were reported to represent about 0.1% of all the microorganisms present in the gastrointestinal tracts. maybe also for this reason, despite the presence of fungi in the intestine has been known for many years, in depth studies of the human mycobiome were only recently performed [55] . humans are hosts to a wide number of fungi species that coexist with the other microorganisms into the gut in a complex ecological relationship of interdependence [56] . together with bacteria, fungi contribute to the modulation of the intestinal immune system [57] . many of them have a clear pathogenic potential even if, physiologically, they are commensals in our bodies. only in some specific conditions their overgrowth can lead to well-known mycosis. the best known fungal pathogen of humans is certainly candida albicans, which is a normal component of the gut mycobiota but may causes candidiasis in case of its intestinal and vaginal overgrowth [58] . an altered intestinal mycobiota has also been observed in other human pathological conditions, such as ibs [59] , inflammatory bowel disease (ibd) [60] and also autism-spectrum disorders and rett syndrome [61] . since human mycobiome is altered in some diseases, perhaps contributing to their pathogenesis, the therapeutic manipulation of the mycobiome could be a useful approach to treat and/or prevent these diseases [62] . eos antimycotic activities are characterized by a broad spectrum of actions [63] . c. albicans is responsible for the majority of fungal infections in humans and is certainly the most studied mycobiota pathogen. the overgrowth of c. albicans is usually controlled by the immune system of the host; however, in particular conditions such as in immunocompromised patients, this microorganism may cause severe infections [64] . for this reason, c. albicans is one of the main target for studies focusing the antifungal effect of eos and their single molecules. the antifungal activities of eo obtained from thymus vulgaris, citrus limonum, pelargonium graveolens, cinnamomum cassia, ocimum basilicum, and eugenia caryophyllus have been evaluated against clinical isolates of c. albicans and candida glabrata. all of these eos exhibited both fungistatic and fungicidal activity toward these two candida species, but cinnamon oil demonstrated the highest activity [64] . since the most represented active compounds of cinnamomum eo is cinnamaldehyde, many studies have been addressed to analyse in depth its activity against c. albicans. the mic 90 at which cinnamaldehyde has been shown to inhibit c. albicans growht ranged from 125 to 450 µg/ml [65] . limonene has shown to possess strong antifungal properties [66] and in particular an excellent anti-candida activity. a recent study analyzed the efficacy of this compound against the growth of c. albicans isolates, including fuconazole-resistant strains. in this study, the in vitro growth of 35 clinical isolates of c. albicans were completely inhibited at doses ranging between 5 mm and 20 mm of limonene. furthermore, limonene inhibited the adhesion, development and maturation of the c. albicans biofilm with 50% of inhibition occurred at doses between 1.8 and 7.4 mm. at the dose of 20 mm, limonene was also capable to degrade 70% of mature biofilm [67] . mentha eos obtained from m. piperita, m. spicata, m. longifolia, m. pulegium, m. cervina, and m. suaveolens, have demonstrated good antimycotic activities against different fungi genera, including candida [68] . menthol and (+)-carvone are the major components of peppermint eos and both exhibited strong antifungal activity in vitro. these activities were evident only at a relatively high doses: peppermint oil inhibited the in vitro growth of c. albicans at 20 mg/ml in agar dishes, whereas caraway oil showed inhibitory effects at lower doses, in the range 5-10 mg/ml [69] . a patented peppermint and caraway oils formulation, called menthacarin ® , was tested in an ibs animal model, showing to be effective in alleviating abdominal pain in rats via mycobiome modulation, suggesting a possible role of mycobiota dysbiosis in the etiopathogenesis of ibs [70] . thymus vulgaris eo has also shown to be effective against fungi pathobionts capable to infect humans. a study on dermatophyte, fungi that can cause superficial infections of the skin, and on aspergillus, fungus genera that can cause respiratory infections, reported mic values for thymus vulgaris eo ranging from 0.16 to 0.32 µl/ml. higher mic values, between 0.32 and 0.64 µl/ml, were reported for candida spp. the antifungal activity of this eo has been attributed to its two major components: thymol and carvacrol, that accounted respectively 26% and 21% of thymus vulgaris eo [70] . both these phenolic compounds seem to act by disrupting the fungal cell membranes [27] . an interesting study evaluated the antifungal activity of thymol in comparison with miconazole, a classical antifungal medication, against c. albicans growth and biofilms formation. the results of this study demonstrated that these two molecules were equally effective against c. albicans with no statistically difference between the two treatments, confirming an extraordinary antimycotic effect of thymol. however, relatively higher concentration was necessary for thymol, with a mic that corresponded to 350 µl/ml vs. 75.15 µl/ml for miconazole [71] . thymol antifungal activity was tested against other candida species, and it showed to be effective also against c. tropicalis. [72] . clove eo has been traditionally used in dentistry for its anesthetic and antimicrobial activities [73] . its anti-fungal action has been attributed to eugenol, the major clove oil molecule. a recent study indicated that clove eo, at concentrations that ranged between 0.03% and 0.25% (v/v), inhibited the biofilm formation in many candida species, grown on different substrates [74] . for what the mechanism of action concerns, eugenol was able to cause permanent injury to the cell membranes of c. albicans and morphological alterations to its cell wall [27, 75] . the activity of eugenol against c. albicans was multitarget and also targeted enzymatic pathways, such as the h + -atpase in mitochondria [76] . eugenol treatment also induced an overall oxidative damage to the fungal cell (lipid peroxidation), and these multiple damages may finally lead to cell death [77] . the antiviral activity of eos has been established for different viruses, and it can be directed against the viral particle or against their intracellular replication process [78] . several studies demonstrated that the herpes simplex viruses, type 1 and 2 (hsv-1 and hsv-2), influenza a virus and coronavirus may be sensitive to eos and to their single molecules [79] [80] [81] . it is interesting to observe that many hsv strains, resistant to synthetic antiviral drugs, are instead sensible to eos and to their components. that is probably because they have a multitarget mechanism of action compared to specific drugs that usually target single virus component or single metabolic pathways. for this reason, it is reasonable to expect that antiviral drugs and eos could act in synergy [78] . bovine viral diarrhea virus (bvdv) is considered a good animal model for the human hepatitis c virus. this virus has been successfully treated with basil eo and single monoterpenes that are major components of this eo: camphor, thymol and eucalyptol. to perform the tests, basil eo and monoterpenes were used at a concentration of 64 mg/ml. results obtained in this in vitro study showed that while the basil eo did not demonstrated to have significant antiviral activities, its single monoterpenes significantly decreased bvdv infectivity on bovine kidney cells, acting directly on the viral particle [82] . these results are interesting because they suggest that for specific antimicrobial applications, it is much more useful to use individual components of eos rather than eos as such. since the antimicrobial and antiviral activity of single eo molecules are multitarget activities, it is very important to design new study focused on single eo constituents and not on blends of different eos, that are very difficult to analyze and to replicate. antiviral properties of carvacrol have been demonstrated on nonenveloped murine norovirus (mnv), a good model for the human norovirus. carvacrol 0.25% and 0.5% (v/v) reduced virus propagation on a murine monocyte cell line, demonstrating its ability to inactivate mnv acting firstly on the viral capsid and afterwards directly on its rna. this underlines the efficacy of carvacrol as a natural surface disinfectant and as a food preservative to control human norovirus, which are the most frequent cause of food-borne viral diseases in humans, causing non-bacterial gastroenteritis. [83] . to date, no study has been performed to understand the impact of eos on the intestinal virome. the main physiological viral component of the gastrointestinal tract is represented by prophages or phages [84] . the bacteriophage component is mainly composed by temperate virus of the caudovirales order, but most of the detected viral sequences in human gut virome could not be attributed to known viruses [85] and to date it is estimated that the number of viruses in human stools is up to 10 9 per gram [86] . despite this, it is clear that eos may impact the intestinal virome composition by modulating all the microbiota components, it could be really difficult to understand the direct impact of eos on the intestinal viruses and the consequences of this modulation on the intestinal ecology. to date, there is some evidence that eos may be effective against different virus replication, but there are still not enough data to predict what could be the impact of eos on viral infectious diseases of the gastrointestinal tract. the presence of a very complex microenvironment makes it difficult to understand if eos can really act against the pathogen virus particles or if they reach mucosa cells only after the infection. since eos are potentially able to act both on bacteria and viruses, in pathological conditions it could be difficult to understand the real targets of these compounds. for example, it has been demonstrated that norovirus persistent infection can be sustained by gut bacteria dysbiosis, and the use of antibiotics in infected mice is able to counteract the viral replication [87, 88] . nowadays, colorectal cancer (crc) is one of the most common tumours worldwide. it represents the second cause of cancer death in europe, even if mortality is decreasing due to the new screening programs and improvement in therapies [89] . the aetiology of crc is not only related to genetic and environmental factors but also to gut microbiota and chronic colonic inflammation. genetic factors include mutations in genes regulating enterocyte cell growth, proliferation, differentiation or cell cycle control or polymorphisms of several proteins involved in dna repair and transcription [90] [91] [92] [93] . among the environmental factors, the most important seems to be the high consumption of red meat, smoking and drinking alcohol in huge amounts [94, 95] . the involvement of the gut microbiota in crc is related to the production of noxious metabolites by bacteria, such as secondary bile acids, polyamines or genotoxins [96] . these metabolites may cause to colonocytes oxidative stress, direct dna damage or induce inflammation [97] . chronic inflammation condition, such as those linked to ibd, are also recognised as a risk factors for crc development [98] . crc manifestation can be sporadic or have a familial predisposition, that's the case of familial adenomatous polyposis (fap) and other syndromes like peutz-jeghers, serrated polyposis and lynch [99, 100] . classical crc therapies include surgical treatments, radiotherapy or chemotherapy [101] , which are associated with important side effects and with the development of drug resistance [102, 103] . recently new pharmacological approaches have been successfully developed for crc, including biological drugs, aimed at the treatment of previously diagnosed cancers [104] . despite the presence of different therapeutic strategies for crc, we must not forget the potential of natural anticancer substances, especially in the prevention of a neoplasia which requires long times to transform from a benign dysplasia to a malignant adenocarcinoma. several eos and their single components have been tested in vitro and/or in vivo by using crc models and have been proved to be valid antitumoral molecules for this cancer. carvacrol was tested on different crc cell lines (htc-116 and lovo), where it was determined the reduction of proliferation and cell cycle arrest in g2/m phase, associated with the reduction of cellular invasion and migration proprieties [105] . geraniol has shown a strong cytotoxic effect on colo-205 cell line [106] but it was not able to induce apoptosis on caco-2 cell line, where it only showed a cytostatic effect, by arresting their cell cycle in s phase [107] . this demonstrate how the same compound may not have the same effect on different cellular crc models. geraniol also demonstrated its anticancer property against the regulation of polyamine metabolism, that is another target of cancer therapies [107, 108] . in different crc cell lines, geraniol has been shown to downregulate the ornithine decarboxylase (odc) and to upregulate s-adenosylmethionine decarboxylase (adometdc), two enzymes involved in polyamine catabolism and elimination [107] . thymol has shown cytotoxic effect against htc-116 cell line by inducing ros production and dna damages. it also induced cell-death by affecting cancer cell mitochondrial pathways [109] . on the other hand, thymol, geraniol, nerolidol, and methyleugenol, at low doses, have demonstrated to have genoprotective effects against oxidative and dna methylation damages in ht-29 cell line [21] . these data underline the importance of the effective dosage of these substances that reaches the colon, since all of them are subject to intestinal absorption. cinnamaldehyde have been tested on several crc cell lines and, like others eo components, it caused the inhibition of proliferation and the induction of apoptosis (by pi3k/akt inhibition and by increasing bax/bcl-2 ratio) associated with a reduction of invasion and migration capability of sw-480, hct-116 and lovo cells (by increasing e-cadherin levels and downregulating mmp-2 and mmp-9 enzymes) [110] . for these reasons, cinnamaldehyde has been used for the development of an aspirin-like drug for the prevention of crc [111] . cinnamaldehyde has also been associated with camptothecin, a hydrophobic anticancer drug, and then incorporated in polymeric micelles to obtain a controlled release system based on ph-gradients. these micelles have shown to induce apoptosis and generate intracellular ros with synergistic anticancer effects between cinnamaldehyde and camptothecin both on in vitro and in vivo models of sw-620 human colon tumour cell or bearing mice [112] . so, the anticancer activities of eos compounds can reduce drug resistance and sensitize cancer cells to traditional chemotherapeutic agents, by acting synergistically with them. geraniol has been reported to sensitize caco-2 cell line to 5-fluorouracil (5-fu), by altering cell membrane potentials and facilitating the uptake of 5-fu [113, 114] . the synergistic effect of geraniol and 5-fu has been investigated both in vitro and in vivo. geraniol potentiates 5-fu growth inhibition activity on sw-620 and caco-2 cell lines by downregulating the thymidylate synthase and thymidine kinase, two enzymes related to 5-fu cytotoxicity. these data have been confirmed in vivo on nude mice grafted with the human colorectal cells tc-118 [115] . in a similar manner thymoquinone, a compound of nigella sativa eo, demonstrated-in association with doxorubicin-an improvement of drug-antineoplastic activities on ht-29 cell line [116] . thymoquinone also sensitized colo-205 and htc-116 cancer cells to cisplatin and increased cancer cell death by suppressing nf-kβ [117] . finally, it has been shown that thymoquinone was capable to sensitize resistant lovo colon cancer cells to the drug irinotecan by affecting erk1/2 pathway and increasing the membrane permeability and the autophagy process [118, 119] . β-caryophyllene is a natural bicyclic sesquiterpene found in many eos, particularly in clove oil. it has been studied in association with paclitaxel and doxorubicin on dld-1 and caco-2 cell lines, respectively. it facilitated the passage of paclitaxel through the plasma membrane, increasing its anticancer activity [120] . moreover, β-caryophyllene treatment induced an intracellular accumulation of doxorubicin that increased its anticancer activity [121] . β-caryophyllene has also been involved in the regulation of glucose homeostasis in crc cells, regulating genes involved in glycolysis and cell growth and finally leading to cell growth suppression and apoptosis [122] . taken together, these results suggest that there is a real wide possibility of using eos or their individual compounds in crc prevention, but also in reducing the doses of classic chemotherapy drugs adopted to treat crc patients. this would consequently reduce the side effects of chemotherapy with a real benefit for patients during anticancer therapy but without decreasing its therapeutic efficacy. considering that the development of crc can be determined by the presence of colonic chronic inflammation for a long time, the efficacy of these natural compounds is certainly maximum in terms of crc prevention, in all subjects at high risk of developing this cancer, both for familiar history or for pre-disposing pathologies, such as ibd or fap. in this context and within long-term therapeutic preventive strategies, it has to be stressed eos multi-target properties, that makes the development of resistant tumour cells very difficult. eos, but especially their single molecules, are effective multitarget modulators of the intestinal physiology and pathology. their use to date has been limited due to several factors that include testing complex mixtures that did not allow identifying the individual active components, the difficulty of releasing these compounds in the affected intestinal tract and their toxicity and aggression on the mucous membranes which complicates their in vivo administration. most eos have very strong, sometimes unpleasant flavors, and in several cases they are also aggressive towards the oral, esophageal and gastric mucosa. administration in controlled release formulations therefore often becomes a necessity. by using normal pharmacological forms such as tablets, capsules or soft gels it is possible to obtain gastro-resistant pharmaceutical forms. once reached the intestine, the chemical compounds contained in the oes are assimilated more or less quickly, with different bioavailability, depending on their chemical structure and pharmacokinetics. geraniol rapidly crosses the enterocytes and, once reaching the bloodstream, has shown a half-life of 12.5 min with a pseudo first-order kinetics [5] . on the contrary, eugenol is rapidly absorbed, but has shown a half-life of 18.3 h in blood and could accumulate after repeated daily administrations [123] . cinnamaldehyde has shown a half-life of 6.7 h. however, the 60% of cinnamaldehyde is oxidized to cinnamic acid rapidly [124] . carvacrol is only partially absorbed in the intestine and has shown to reach low concentration in plasma with a peak after 2 h of oral administration [125] . d-limonene is only partially absorbed by enterocytes and has shown a half-life of 3.20 h in plasma [126, 127] . similar behavior has been found for β-caryophyllene, that has shown a half-life of 4.07 h in plasma [128] . thymol is absorbed quickly in the gut, and it is present in plasma only as thymol sulfate, that reach a considerable concentration after 20 min, with an half-life of 10.2 h [129] . the current scientific knowledges regarding their activities against oxidative stress and their ability to modulate the microbiota and the intestinal inflammation are more than enough to plan clinical trials on humans and provide evidences for their use as therapeutic agents. the strong multitarget antitumor activity of some molecules present in eo make them potentially very effective therapeutic strategies against crc, also in combination with chemotherapies already in use. however, the maximum potential of these compounds seems to be expressed in the prevention of crc, inflammation and intestinal dysbiosis. table 4 summarize the main effects of oes divided according to their main activity in the intestine and with the indication of the individual compounds mainly responsible for the action. increasing antineoplastic effect of doxorubicin, increasing cell death by suppressing nf-kβ in association with cisplatin, induction of autophagy in association with irinotecan [116] [117] [118] [119] n.a. β-caryophyllene antitumoral effect in association with chemotherapeutic agents, regulation of glucose homeostatis (in vitro) increase of anticancer activity of paclitaxel and doxorubicin regulation of genes involved in glycolysis and cell growth, induction of apoptosis [119] [120] [121] a limit to their use is the need of controlled release systems that target these molecules in the intestine area where their action is required. in fact, all these compounds are absorbed in the small intestine, some of them very quickly, such as geraniol, while others with less efficient transport mechanisms, such as limonene [5, 123] . nevertheless, possibilities for a controlled release of these molecules already exist, and it has already been proven that they are effective in humans to obtain a controlled release [8] . further in vivo and clinical studies are necessary to understand their bioavailability, pharmacokinetics and mechanism of actions. unfortunately, the low cost that these eos have on average, and their non-patentability, make them little or not at all interesting for pharmaceutical industries, making missing the sponsors for the clinical studies necessary to finally validate their therapeutic efficacy in many different intestinal pathologies of the large bowel, such as inflammation, colitis, dysbiosis and crc. metabolic engineering strategies for enhancing the production of bio-active compounds from medicinal plants antimicrobial, antioxidant, and immunomodulatory properties of essential oils: a systematic review oxidant signals and oxidative stress metal dyshomeostasis and their pathological role in prion and prion-like diseases: the basis for a nutritional 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inhibits proliferation and induces apoptosis in human colon cancer cells. anti-cancer drugs geraniol and geranyl acetate induce potent anticancer effects in colon cancer colo-205 cells by inducing apoptosis, dna damage and cell cycle arrest geraniol, a component of plant essential oils, inhibits growth and polyamine biosynthesis in human colon cancer cells polyamine metabolism and gene methylation in conjunction with one-carbon metabolism thymol elicits hct-116 colorectal carcinoma cell death through induction of oxidative stress cinnamaldehyde affects the biological behaviour of human colorectal cancer cells and induces apoptosis via inhibition of the pi3k/akt signalling pathway novel cinnamaldehyde-based aspirin derivatives for the treatment of colorectal cancer dual acid-responsive micelle-forming anticancer polymers as new anticancer therapeutics perturbation by geraniol of cell membrane permeability and signal transduction pathways in human colon cancer cells geraniol, a component of plant essential oils, sensitizes human colonic cancer cells to 5-fluorouracil treatment geraniol, a component of plant essential oils, modulates dna synthesis and potentiates 5-fluorouracil efficacy on human colon tumor xenografts combinatorial effects of thymoquinone on the anti-cancer activity of doxorubicin thymoquinone chemosensitizes colon cancer cells through inhibition of nf-κb thymoquinone induces caspase-independent, autophagic cell death in cpt-11-resistant lovo colon cancer via mitochondrial dysfunction and activation of jnk and p38 inhibition of nf-κb and metastasis in irinotecan (cpt-11)-resistant lovo colon cancer cells by thymoquinone via jnk and p38 potentiating effect of β-caryophyllene on anticancer activity of α-humulene, isocaryophyllene and paclitaxel the influence of sesquiterpenes from myrica rubra on the antiproliferative and pro-oxidative effects of doxorubicin and its accumulation in cancer cells effects of beta-caryophyllene on arginine 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experimental colon carcinogenesis carvacrol exhibits anti-oxidant and anti-inflammatory effects against 1, 2 dimethyl hydrazine plus dextran sodium sulfate induced inflammation associated carcinogenicity in the colon of fischer 344 rats this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors thank alberto sardo for illuminating us on the infinite potential of essential oils. the authors declare no conflict of interest. key: cord-344902-bittqpyo authors: scott, jennifer; abaraogu, ukachukwu o.; ellis, graham; giné-garriga, maria; skelton, dawn a. title: a systematic review of the physical activity levels of acutely ill older adults in hospital at home settings: an under-researched field date: 2020-10-15 journal: eur geriatr med doi: 10.1007/s41999-020-00414-y sha: doc_id: 344902 cord_uid: bittqpyo purpose: the purpose of this review was to identify, evaluate and synthesise existing evidence reporting the physical activity levels of acutely ill older patients in a ‘hospital at home’ setting and compare this to patients with similar characteristics treated in a traditional hospital inpatient setting. functional changes and any adverse outcomes due to physical activity (e.g. falls) in both settings where pa was reported or recorded were also evaluated as secondary outcomes. methods: a search strategy was devised for the medline, cinahl, amed, pedro, ot seeker and cochrane databases. search results were title, abstract and full-text reviewed by two independent researchers. data were extracted from included articles using a custom form and assessed for quality and risk of bias using the appraisal tool for cross-sectional studies. results: no studies set in the hospital at home environments were identified. 16 hospital inpatient studies met the criteria for inclusion. older patients managed in inpatient settings that would be eligible for hospital at home services spent 6.6% of their day active and undertook only 881.8 daily steps. functional change was reported in four studies with both improvement and decline during admission reported. conclusion: there is a lack of published research on the physical activity levels of acutely-ill older adults in hospital at home settings. this review has identified a baseline level of activity for older acutely ill patients that would be suitable for hospital at home treatment. this data could be used as a basis of comparison in future hospital at home studies, which should also include functional change outcomes to further explore the relationship between physical inactivity and functional decline. electronic supplementary material: the online version of this article (10.1007/s41999-020-00414-y) contains supplementary material, which is available to authorized users. hospital at home (hah) is a model of healthcare delivery which provides an alternative to hospitalisation by delivering acute-level hospital services in a residential setting [1] . the hah care model has increased in prevalence in recent years, with well-established programmes providing services in western europe, north america, brazil, australia, israel and south east asia [2] . home-hospitalisation has also been advocated during the recent covid-19 pandemic as a means of increasing bed capacity, facilitating quarantine and reducing disease transmission to vulnerable groups [3] . research interest has also been growing, with a more than sixfold increase in hah-related citations between 1999 and 2019 [4] . a recent systematic review found that hah may be a clinically effective alternative to inpatient care for some older, acutely-ill medical patients [5] . furthermore, it suggested hah treatment may pose less risk of physical functional decline to patients than the traditional ward-based inpatient environment [5] . functional decline is a known adverse effect of hospitalisation, affecting between 30 and 56% of older inpatients between admission to hospital and discharge [6] [7] [8] [9] , manifesting as a loss of muscle mass, strength, physical function and/or ability to perform basic activities of daily living such as dressing, eating and maintaining hygiene and continence [10] [11] [12] . physical inactivity while hospitalised, combined with older age, are predictors of functional decline [13] . hospitalised patients are highly inactive, with acute medical and surgical inpatients spending between 93 and 98.8% of their time sitting or lying [14] , and older patients spending as little as 76mins per day in an upright position [15] . recently published draft recommendations on physical activity for inpatients have emphasised the importance of incorporating opportunities for physical activity into the daily care of older adults to improve clinical outcomes, focusing on function, independence and activities of daily living [16] . however, there are many institutional barriers to physical activity in hospital including lack of staff support, tethering to medical devices, lack of assistive devices, and unfamiliar surroundings, as well as a fear of injury [17] . treatment in a less restrictive home environment may overcome such barriers, providing more opportunity for patients to continue to perform regular activities of daily living [5] , thereby lessening the risk of functional decline. this review sought to investigate the hypothesis that older, acutely ill patients treated in a hah setting may be more active than hospital inpatients with similar characteristics. the aim was to identify, evaluate and synthesise primary research studies reporting cumulative physical activity levels in these populations and, where reported, evaluate reports of functional decline or adverse effects resulting from physical activity during admission. as will be reported, no studies conducted in hah treatment settings were identified, and functional change outcomes were largely absent. the review protocol was developed in accordance with preferred reporting items for systematic review and meta-analysis protocols (prisma-p) [18] guidelines and registered with the international prospective register of systematic reviews (prospero, registration number crd42019138822) [19] . the review followed the guidelines set out in the cochrane handbook for systematic reviews of interventions [20] where applicable and complies with the prisma statement [21] for the conduct and reporting of systematic reviews. a comprehensive search strategy was developed in accordance with the cochrane recommendations for health care review [22] and reviewed by a specialist medical librarian. the search was initiated in july 2019 and updated 19 january 2020 to ensure currency. search terms and appropriate synonyms were chosen in alignment with the research objective and combined using boolean operators, subject headings, truncations and wildcards where appropriate. filters limited results to peer reviewed, english language, human studies with available abstracts published since 1980. all study designs were acceptable. the databases medline (ovid interface), central, cumulative index to nursing and allied health literature (cinahl), allied and complementary medicine database (amed), pedro and otseeker were chosen as the most relevant to the subject matter. the full search strategies with database-specific syntaxes for all sources are included in online resource 1. once key papers were identified, reference lists were handsearched and subject experts were approached to identify any further resources. 'grey' literature including conference abstracts, reports, unpublished data and dissertations were not included. multiple publications using the same participant dataset were excluded and the most comprehensive or recent publication used. setting studies set in either an hah or acute medical inpatient environment were included, studies did not have to compare both groups. hah was defined as 'a service that provides acute, hospital-level care by healthcare professionals in a home context for a condition that would otherwise require acute hospital inpatient care' [1] . an acute inpatient setting was defined as 'a hospital (private or public) providing 24-h care for people who are unwell and had an unplanned admission' [23] . as hah is designed to treat acute episodes of transient rather than chronic medical illness [5] , studies set in non-medical or non-acute environments such as palliative care, respite, rehabilitation, mental health, long-term care or residential nursing home facilities were excluded. studies concerned with post-discharge hah services (e.g. 'step-down' hah), were also excluded, as the focus of the research project is hah as an alternative to hospital admission for the preservation of physical function. participants studies involving patients aged 60 and over diagnosed with an acute-onset medical condition that would fall within the scope of a hah service were included. hah services predominantly manage non-surgical, non-critical conditions such as infection, acute exacerbations of cardiac and respiratory conditions, haematological and metabolic disturbances, and acute kidney injury [1] . certain conditions are not appropriate for management in a home setting such as those requiring surgery (e.g. acute coronary syndromes, orthopaedics), critical care or advanced diagnostics and interventions (e.g. stroke). to ensure that intervention and comparison populations were similar, studies containing these large numbers of patients with such conditions were excluded unless these participants could be discounted from the results. a margin of ≤ 10% of patients under 60 and ≤ 10% with excluded conditions was allowed. where numbers exceeded this margin, or other pertinent information was required, study authors were approached via email on up to 2 occasions to request abridged results. where a custom dataset was provided, this was used in analysis over the published dataset. intervention and comparator the intervention of interest was treatment in a hah setting compared to standard inpatient acute care. as this review aimed to establish if there are differences in the cumulative activity levels of patients in each setting, trials of other interventions to increase patient activity such as exercise programmes or physiotherapy sessions over and above usual care were not suitable for inclusion unless the physical activity levels of the control group were available, as the intervention group would not be representative of the general older acute population. outcome the primary outcome measure was the cumulative level of pa performed by patients receiving standard medical care in a hah and/or inpatient setting. it was decided a priori that acceptable measures would include objective methods, such as activity monitor data, or subjective methods, such as direct observation, self-reported instruments or questionnaires. changes in functional independence (e.g. activities of daily living, dependent walking) and physical performance (e.g. handgrip test, timed up and go) from admission to discharge, as well as any adverse effects reported as a consequence of physical activity (e.g. falls) were selected as secondary outcomes. the inclusion and exclusion criteria are summarised in table 1 . literature search results and bibliographic records were exported into refworks to facilitate deduplication and screening of titles and abstracts. articles meeting the inclusion criteria were then subjected to full-text appraisal. all records were reviewed by the lead researcher (js) and independently second-reviewed by another (ds, ua, mg or ge). the decision for inclusion or exclusion was recorded along with reasons for exclusion. where there was disagreement between reviewers on inclusion at any stage, a third reviewer was consulted. sixteen articles were selected for inclusion in the review. this process for identifying these is documented in the prisma flowchart [21] below ( fig. 1 ). the process of data extraction was performed using a custom template which was developed and piloted to extract: (1) data relevant to the research question, and (2) data required to perform a quality appraisal and risk of bias assessment using the appraisal tool for cross-sectional studies (axis) [24] (data extraction table: online resource 2, axis appraisal: online resource 3). the axis tool comprises 20 questions and considers study design and reporting quality in addition to the risk of bias when appraising research studies [25] . the data extracted were spot-checked for accuracy by the review team (ds, ua, mg or ge). where studies reported results for participants that were excluded from this review (e.g. surgical, non-geriatric) these were separated and excluded from the analysis. separate datasets were requested and received from karlsen [26] and valkenet [27] containing only participants that met the inclusion criteria. the physical activity outcomes of the studies were grouped according to their method of measuring physical activity levels and reporting format. in accordance with duvivier [28] , standing and slow walking have both been categorised as physical activity and grouped together into 'active time' for the purposes of analysis. time spent sitting or lying down, including sleep time, has been classified as 'non-active' time. this classification allowed 3 categories to emerge; (1) active time recorded over 24 h, (2) active time recorded over variable timeframes, and (3) physical activity as step count. the percentage of time spent actively was selected as a common scale to enable comparison of data across the studies. studies using step count as a measure of physical activity were reported separately. results reported in minutes were converted into a percentage of 24 h. median and interquartile ranges were converted into mean values using the formula devised by wan [29] to allow results to be summarised as pooled averages. summary independent t-tests were used to examine whether physical activity or step count differed significantly from the pooled averages when grouped by medical condition or studies at lower risk of bias. analyses were performed using spss v26, p < 0.05 was considered significant and 95% confidence intervals are reported. study characteristics no suitable hah studies were identified. all 16 included studies were conducted in single-site [21] acute inpatient hospital environments. the studies were published between 2006 and 2019, and the majority (n = 13) were cross-sectional observational designs aiming to establish the physical activity levels of patients as a primary outcome. this design is consistent with the nature of the research question, which does not aim to evaluate the efficacy of an intervention. of the remaining three studies, two were validation/agreement studies [27, 30] , and one was a randomised controlled trial (rct) [31] . participants most studies concerned general acute medical patients (n = 11, mean sample size 114, range . five studies were exclusively concerned with patients with specific conditions; two each reported physical activity levels of patients with acute exacerbations of chronic obstructive pulmonary disease (mean sample size 13.5, range 10-17) [30, 32] , and heart failure (mean sample size 36, range 27-45) [33, 35] and whilst one reported on patients with mixed medical conditions plus mild-moderate cognitive impairment (sample size 20) [34] . primary outcome all included studies assessed physical activity levels using objective accelerometer-based methods, except belala [34] who used behavioural mapping. valkenet [27] also performed behavioural mapping in addition to accelerometery (dynaport movemonitor). a variety of monitoring devices and algorithms were used, with the activpal (pal technologies, glasgow, uk) being the most commonly used device in studies concerned with posture (5 uses), and the stepwatch activity monitor (modus health, washington, us) used most frequently for step count (4 uses). the validity of the methods used was reported by most studies, except for the mediwalk pedometer (terumo, japan), used by ueda [31] . the range and validity of outcome measures used is available in online resource 4. the included studies were assessed for risk of bias using the axis tool [24] (online resource 3) which was deemed appropriate due to the high proportion of observational studies identified. there is an inherent risk of bias in descriptive, observational study designs, which rank low on evidence hierarchies, however, a well-designed and conducted crosssectional study can be of some evidential value [35] . the axis tool prompts consideration of selection, instrumentation and reporting bias as well as reporting and study design quality. it was also suitable for the evaluation of the methodology used to acquire and report physical activity levels in the rct included in this review [31] . a domain-based risk of bias assessment indicates a low risk of instrumentation and reporting bias, with adequate measurement and reporting of physical activity levels, however, there is a high risk of selection bias within the identified research (fig. 2) . the studies that performed better in the analysis [34, [36] [37] [38] gave greater consideration to reporting information on non-responders (patients that were eligible for inclusion but declined to participate). in terms of quality assessment, overall reporting quality was high, however, study design considerations were less well evidenced, with a broad lack of consideration of sample size, and frequently vague reporting of ethics or consent protocols. active time recorded over 24 h the level of inpatient physical activity reported as a percentage of 24 h could be established in seven studies (table 2) . when averages were pooled, the mean proportion of time spent active was found to be 6.6% ± 6.3 (range 3.8-8.3%). active time recorded over a variable timeframe three studies collected results over shorter, variable timeframes (7-12 h periods), during waking hours, and with different populations and measurement techniques (accelerometery and behavioural mapping), which precludes pooling of results, however, it can be seen that daytime-only levels are higher than the mean for 24 h results, ranging from 8.8 to 13.9% (median 10.7%) ( table 3) . physical activity as step count eight studies used pedometers or accelerometers to record 24 h step count as a measure of physical activity ( functional change between admission and discharge was reported in 4 studies, the results extracted are summarised in table 5 . as will be discussed, the reported outcomes from these studies were highly heterogenous in terms of tools used, data collection protocols and presentation of data, such that no summative conclusions on of the impact of differing physical activity levels on the incidence of functional decline could be drawn from the data. adverse effects occurring during the period of monitoring were poorly reported, with only four studies reporting this outcome; two advised there were no adverse effects [34, 38] and two reported one death (unrelated to physical activity) [31, 32] during the course of their research. sub-group analyses were performed comparing studies at lower risk of bias (according to axis appraisal) and concerning only one medical condition to the overall physical activity and step count results. both sub-group analyses found no significant difference in results comparing these devices to the overall results (table 6) , indicating the general results are an accurate representation of pa levels. the aim of this review was to identify, evaluate and synthesise the evidence on the physical activity levels of acutely ill older patients undergoing treatment in an hah vs inpatient setting. no hah studies of older adults could be identified, representing a significant gap in the literature surrounding this treatment model. despite the lack of hah research in this field, this review has provided useful data on the baseline physical activity levels that could be expected for patients suitable for treatment in a hah model of care: when monitored for 24 h/day, such patients spend on average 6.6% of the time active, and walk as few as 881.8 steps per day. these findings are consistent with other research on hospitalised older adults, despite the strict hah-specific inclusion/exclusion criteria applied. baldwin [14] reviewed 42 studies reporting the activity levels of acutely admitted medical and surgical adult patients, and found patients spent between 93% and 98.8% of their entire stay sitting or lying, and that the majority of studies reported a daily step count of < 1000. similarly, fazio [40] , in a systematic review of standing/ walking activity in medical inpatients, found that patients were active for 70 min per 24 h (4.9% of the time). the baseline pa values provided in this review may be suitable for use as an inpatient comparator value in future hah pa studies. the low levels of activity reflected in our findings can result in functional decline, however, in our results only four of the studies measuring physical activity also measured functional change. this represents a missed opportunity to further explore correlations between physical activity and functional decline that should be addressed in future pa studies in hospitalised and hah patients. where functional changes were reported there was high heterogeneity in results between studies. agmon [41] established that walking less than 900 steps when hospitalised was strongly associated with functional decline in older adults. both ueda [31] and villumsen [39] reported a mean step count below this threshold, and while both reported results using the barthel index, measurements were taken at different points in the studies and the results were presented differently: ueda [31] reported the change in mean score, while villumsen [39] reported the percentage of participants who improved. in all, six different metrics were used in the four studies reporting functional change, with high variability in measurement tools (see online resource 4), data collection protocols and reporting formats, precluding meaningful synthesis of the results. assessing physical function in acutely ill older inpatients who may present with a wide range of medical conditions and functional levels is undoubtedly challenging, and research is ongoing to identify the most feasible tools to use in this patient group [42] . a consensus-driven core outcome set for studies of functional performance in either older or hospitalised populations has yet to be developed and should be a research priority to allow evaluation and meta-analysis of the findings of studies in this field. placing the findings of this review in the wider context of physical activity research is challenging again due to substantial differences in the methods and outcome measures used. the techniques most frequently utilised in the studies in this review (24 h recording, positional accelerometery) rarely feature in population or community-based research. including night-time activity is likely to present a more accurate picture of all activity undertaken, especially in a hospital setting where circadian rhythms may be disrupted [14] , but will result in lower average activity levels than studies of day-time pa or sedentary behaviour only. this is evident in the results for the three studies that conducted monitoring over a shorter, daytime, timeframe (table 3 ) which found physical activity ranged from 8.8 to 13.9% of the monitoring period. as a result of these different outcome measures, recording periods and a lack of objectively established normative values for the 24-h physical activity of healthy free-living older adults, it is challenging to establish how much activity drops when hospitalised. however, as the continuous objective monitoring of research participants becomes easier and cheaper with developments in accelerometery and wearable digital technology, it may be the case that normative values for pa in free-living older adults can be established. this would allow more accurate evaluation of the extent to which normal pa is impeded by acute illness, in both hah and inpatient settings. a strength of this review is that it followed a systematic approach following cochrane guidelines where applicable [20] and was reported in accordance with prisma statement, which reduces the risk of bias. a possible limitation of this review is its high specificity arising from highly refined inclusion and exclusion criteria. this led to some potentially relevant articles being excluded. for instance, two promising rcts were identified during the literature search and selection process which found that adult hah patients may around 2.6 times more active than inpatients [43, 44] , however, these studies were excluded as it was not possible to isolate the results for participants aged over 60 years-only. a further limitation of this review is the high risk of bias present in the studies identified, which may limit the representativeness of the findings. physical and functional decline, caused in part due to inactivity during hospital admission, can have a considerable impact on an older patient's health and ability to remain independent on discharge. hah may offer a treatment environment that preserves and facilitates physical activity in older patients, however, it has been demonstrated in this review that there is a lack of research evidence to confirm this. this review has provided an indication of the baseline activity levels of inpatients suitable for a hospital at home service, however primary objective research is needed in this treatment setting. this review also identified that functional change is infrequently measured along with physical activity, representing a missed opportunity to assess the impact of immobility in hospital on function. where they are reported, functional measures are highly diverse and data collection protocols vary, impeding comparisons between studies. a consensus-driven core outcome set for the investigation of functional decline in hospitalised patients would greatly facilitate the comparison and synthesis of research in this field. sedentary behaviour, defined as 'any waking behaviour characterized by an energy expenditure ≤ 1.5 metabolic equivalents (mets), while in a sitting, reclining or lying posture' [45] , was included in the search strategy as a related field to physical activity. no studies reporting sedentary behaviour as the primary outcome met the inclusion criteria, therefore, this concept is not discussed further in this review. funding the writing of this review was funded through a joint nhs lanarkshire-glasgow caledonian university phd studentship. the funders have had no input in the writing of this review or it's conclusions. hospital at home, guiding principles for service development world hospital at home congress. societies and programs why we should expand hospital-at-home during the covid-19 pandemic citation report for "hospital at home". clarivate analytics admission avoidance hospital at home. cochrane database syst rev no one size fits all-the development of a theory-driven intervention to increase in-hospital mobility: the "walk-for" study the effectiveness of inpatient geriatric evaluation and management units: a systematic review and meta-analysis trajectories and predictors of functional decline of hospitalised older patients predictors on admission of functional decline among older patients hospitalised for acute care: a prospective observational study reducing, "iatrogenic disability" in the hospitalized frail elderly the hospital elder life program: a model of care to prevent cognitive and functional decline in older hospitalized patients rethinking hospital-associated deconditioning: proposed paradigm shift. phys therapy daytime physical activity and sleep in hospitalized older adults: association with demographic characteristics and disease severity accelerometery shows inpatients with acute medical or surgical conditions spend little time upright and are highly sedentary: systematic review daily and hourly frequency of the sit to stand movement in older adults: a comparison of day hospital, rehabilitation ward and community living groups recommendations for older adults' physical activity and sedentary behaviour during hospitalisation for an acute medical illness: an international delphi study attitudes and expectations regarding exercise in the hospital of hospitalized older adults: a qualitative study preferred reporting items for systematic review and meta-analysis protocols (prisma-p) 2015: elaboration and explanation cochrane handbook for systematic reviews of interventions preferred reporting items for systematic reviews and meta-analyses: the prisma statement crd's guidance for undertaking reviews in health care targets for older adults' physical activity and sedentary behaviour during hospitalisation: an international delphi study development of a critical appraisal tool to assess the quality of crosssectional studies (axis) methodological quality (risk of bias) assessment tools for primary and secondary medical studies: what are they and which is better improved functional performance in geriatric patients during hospital stay measuring physical activity levels in hospitalized patients: a comparison between behavioural mapping and data from an accelerometer minimal intensity physical activity (standing and walking) of longer duration improves insulin action and plasma lipids more than shorter periods of moderate to vigorous exercise (cycling) in sedentary subjects when energy expenditure is comparable estimating the sample mean and standard deviation from the sample size, median, range and/or interquartile range assessing sedentary behavior with the geneactiv: introducing the sedentary sphere impact of oral treatment on physical function in older patients hospitalized for heart failure: a randomized clinical trial gosselink r (2006) physical activity and hospitalization for exacerbation of copd a pilot study examining activity monitor use in older adults with heart failure during and after hospitalization a pilot observational study to analyze (in)activity and reasons for sedentary behavior of cognitively impaired geriatric acute inpatients levels of evidence in medicine walking in hospital is associated with a shorter length of stay in older medical inpatients mobility activity and its value as a prognostic indicator of survival in hospitalized older patients very low levels of physical activity in older patients during hospitalization at an acute geriatric ward: a prospective cohort study 2020) how much do hospitalized adults move? a systematic review and meta-analysis association between 900 steps a day and functional decline in older hospitalized patients feasibility and inter-rater reliability of physical performance measures in acutely admitted older medical patients hospital-level care at home for acutely ill adults: a pilot randomized controlled trial hospital-level care at home for acutely ill adults: a randomized controlled trial sedentary behavior research network (sbrn): terminology consensus project process and outcome stepping toward discharge: level of ambulation in hospitalized patients acknowledgements thanks to dr alexandra mavroiedi of strathclyde university for advice given during the drafting of the protocol preceding this review and to julie smith, specialist librarian within the school of health and life sciences, glasgow caledonian university for assistance with devising the search strategy. thanks also to professor jon goodwin for advice on data analysis strategy and to dr phillipa dall for assisting with the full text review stage. js was funded to write this review through a joint nhs lanarkshire-glasgow caledonian university phd studentship.author contributions js, ua, ge and ds conceived the review. all authors contributed to the development of the search strategy and participated in the screening, review and selection of the included papers. js drafted the review and all authors reviewed, provided feedback and approved the final manuscript. the authors declare that they have no competing interests.ethical approval not applicable. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. key: cord-346060-ns6v76rb authors: degli espinosa, francesca; metko, alma; raimondi, marta; impenna, michele; scognamiglio, elena title: a model of support for families of children with autism living in the covid-19 lockdown: lessons from italy date: 2020-06-02 journal: behav anal pract doi: 10.1007/s40617-020-00438-7 sha: doc_id: 346060 cord_uid: ns6v76rb italy has been the european country most affected by the covid-19 pandemic to date and has been in social lockdown for the longest period of time compared to other countries outside china. almost overnight, italian behavior analysts were faced with the challenge of setting up remotely whole-family systems aimed at maintaining adaptive skills and low levels of challenging behavior to be carried out solely by caregivers. given these extraordinary circumstances, the protocols available from the applied behavior-analytic, parent training, and autism literature did not appear to fully meet the needs of parents having to be with their children under extreme levels of stress in a confined space with limited reinforcers for 24 hr a day, 7 days a week. to meet this unprecedented challenge, we developed a dynamic and holistic protocol that extended to the full day and that recognized the need for sustainable intervention delivered solely by parents, who were often looking after more than one child. these practices are presented in this article, together with a discussion of lessons we have learned thus far, which may be useful for behavior analysts working in other regions in which the effects of the pandemic are not yet fully realized. although somewhat unorthodox, we include some parent comments at the end with the goal of sharing the parent perspective in real time as this pandemic unfolds across the world. before discussing responses to the pandemic, it may be helpful to say a few words about the italian health system as it concerns intervention for autism based on applied behavior analysis (aba). italy has a national health system in which autism is recognized as a condition that falls under the care of the state. education is free for all, and mainstream schooling is mandatory and therefore accessible by all children. there are no specialist schools. the delivery of therapeutic and educational services is regulated by registered professional bodies (i.e., psychologists, professional educators, speech and language therapists, neuro-rehabilitation technicians). aba-based intervention is not formally recognized by the italian health authorities, nor is it routinely offered as part of the state autism provision, which typically includes 1 hr per week of psychomotor therapy, 1 hr per week of logotherapy (i.e., speech and language therapy), and school attendance with varying levels of one-to-one educational support. the profession of behavior analysts is not officially regulated. despite the lack of formal governmental recognition of aba interventions for autism and a corresponding professional body, the country has witnessed a steady increase in the number of professionals credentialed by the behavior analyst certification board (bacb) in the past 10 years, verified course sequences, and state-funded health editor's note this manuscript is being published on a highly expedited basis, as part of a series of emergency publications designed to help practitioners of applied behavior analysis take immediate action to adjust to and mitigate the covid-19 crisis. this article was submitted on april 5, 2020, and received final acceptance on april 10, 2020. the journal would like to especially thank julie kornack and courtney tarbox for their expeditious reviews of the manuscript. it is important to note that this article reports the approach taken by a particular group of clinicians operating under completely unprecedented circumstances in one of the hardest hit regions of the world. there are many ways to use the science of applied behavior analysis to support families, and neither the authors nor the journal suggests this is the only approach or the best approach. however, this approach produced positive results for this group of families, and the editorial staff at the journal believes that the rest of the world of applied behavior analysis may benefit from learning from their experience. the views and strategies suggested by the articles in this series do not represent the positions of the association for behavior analysis international or springer nature. rehabilitation centers offering low-intensity (4 to 15 hr per week) aba intervention. as a result of parental demand for aba services, a considerable number of health professionals are enrolling in aba master's programs and consequently incorporating aba-based methods in their therapeutic practices for autism. schools have also begun to open their doors to bacb-credentialed professionals to support the individualized education plans of their students. nevertheless, ababased intervention remains largely privately funded by individual families and is, for the most part, carried out at home during the hours in which the child is not in school. at the time of this writing, the italian authorities have yet to provide statewide guidelines or funding for the continuation of intervention (aba and non-aba) via telehealth for children with autism during the lockdown period. in some regions (e.g., campania, lombardy, marche, emilia romagna), and with considerable variability, individual state-funded centers have begun to set up systems to provide intervention and parental support remotely. italy was one of the first european countries, together with germany, france, and spain, to register the first cases of covid-19 at the end of january 2020 and subsequently to impose movement restrictions on its citizens. for some weeks, it was the second country after china with the largest number of covid-19 cases. presently surpassed by the united states and spain, it currently registers the highest number of deaths related to covid-19 (world health organization, 2020) . on january 31, 2020, the italian government declared a state of national emergency and imposed the first social-distancing restrictions on february 24, with a decree closing all schools and many commercial activities in the northern regions (lombardy and veneto). these restrictions were gradually expanded and eventually extended to the rest of the country, with the period of complete national lockdown commencing on march 9 (ministry of health, 2020) . at the time of writing this paper, italy continues to be in complete lockdown, which includes home isolation, with outside movement restricted to one person per household and solely for the purpose of purchasing food or medicine. deliveries to households are limited to essential goods. outside physical activity is no longer permitted. however, if a child has a disability, she or he can be accompanied outside for brief walks, provided the parent carries a written certificate signed by a health professional attesting to the child's diagnosis and need to be outside. social-distancing measures during the initial lockdown period were less restrictive and were expected to last for a couple of weeks. although children had ceased going to school, and most home sessions had been interrupted, aba practitioners viewed this as a period similar to the summer holidays, where children spend long periods of time with grandparents and are essentially given free access to reinforcement, and parents are given a skeleton program of maintenance to prevent significant skill loss. under usual circumstances, we would expect to see some increase in challenging behavior and some skill loss during the summer months, but not so significant that it could not be addressed within the first few weeks of resuming the typical school and home intervention schedule. by the third week of the lockdown, it became clear that the isolation period would not only be extended to the rest of the country, but that measures would also become much more restrictive. thus, a different approach was required, especially because many parents (all family caregivers in the home are referred to as "parents" hereafter, for brevity) reported that their children were no longer satisfied with the usual reinforcers; were becoming increasingly uncooperative; were engaging in high levels of stereotypy and problem behavior, likely due to being denied access to regular but now unavailable reinforcers (e.g., swimming, going to the playground, taking extended car rides, going to the cinema or ice-cream parlor, participating in physical activity); were demanding high levels of undivided attention; and were becoming more difficult to direct to independent activities. parents also reported that they were struggling to reconcile the demands of distant working required by their employers with the needs of round-the-clock sole care of their children with autism, of siblings, and of their households in the absence of any outside help. although some families lived in the countryside and had access to a privately owned garden (i.e., private yard), many families lived in city apartments, where time spent outside was either prohibited or substantially limited due to the closure of parks and the shared courtyards. even those of us who are not psychologists saw clear early signs of mental health decline, as well as increasing marital conflict. although the latter problems were outside the scope of our practice as behavior analysts who are not also psychologists, referral to paid online psychotherapy or counseling, given the dire financial situation some families were experiencing, was not an option. nonetheless, we believed that the tools of the science of behavior could be extended to the larger family context to alter the repertoires of all its members and to increase contact with positive reinforcement for all. because restrictions in italy happened gradually, with families in the northern regions being in the first cohort, by the time families in the southern region were in lockdown, we had gathered sufficient data on the effects of the first 3 weeks of isolation with minimal structure and free reinforcement access. whereas for the northern families our work in the third week was focused on reducing the negative effects of 2 weeks of "free time," we were able to implement strategies proactively with the southern families to minimize the negative outcomes we observed for the families in the first lockdown cohort. prior to the lockdown period, none of our children displayed severe or unmanageable levels of challenging behavior, and they all had effective behavior management plans in place. after the first 2 weeks of lockdown with limited structure and free reinforcement access, in some of our first cohort families we observed the following during our online meetings: high levels of escape from simple instructions, the loss of independence and communication skills (appropriate mands), satiation (significant reduction in the time children spent with favorite items), an increase in problematic interactions between parents and all children, and unmanageable levels of mands for attention (both appropriate and inappropriate). parents reported being struggling to find new things to entertain their children with autism and siblings. sourcing novel toys or items to create new interests was not possible due to limited deliveries of nonessential items. in the following sections, we describe the protocol that was shaped through the frequent interactions with parents during our online observations and discussions. currently, as a group of professionals, we are serving approximately 30 families with this model. we were consulting with these families prior to lockdown and had been running home-based programs for at least 6 months. in the absence of published literature on interventions that require parents to engage with their children 24 hr per day, 7 days a week, in a confined space for a prolonged and undefined time period, we approached the problem inductively, altering what we did based on principles of learning, on what each individual situation required, and on what parents reported they felt they were able to do. the model developed was a systemic one, in which the client was no longer just the child in sessions working on educational targets but, rather, the whole family in its unique context. we began with an assessment of risk and an evaluation of each child's level of verbal functioning to establish the type of telehealth provision required (direct sessions or parent coaching; see ferguson, craig, & dounavi, 2019, for a review). the two main risks we aimed to mitigate were (a) parental burnout and (b) an increase in socially mediated challenging behavior of the child and siblings. we were less concerned with behavior maintained by automatic reinforcement, unless it was self-injurious. none of our children engaged in such behavior. the assessment was based on our history with the family, data from the child's prelockdown intervention, and direct contingency manipulation and observation via telehealth. for example, we asked the parent to leave his or her child with an activity while he or she talked to us, and we calculated how long the child was engaged without demanding parental attention. the main items included in the risk assessment (see table 1) were parents' prelockdown level of instructional control and social engagement with their children, the duration of the child's and siblings' ability to engage in solitary activities (either reinforcement or instructional, e.g., worksheets, chores, functional play), and parents' tolerance of nondangerous selfstimulation (e.g., flapping, noises, walking up and down, jumping on the sofa). in addition, we considered the child's age, the ability to engage in back-and-forth verbal interaction, the presence of siblings with a disability, and the number of supportive adults always present (i.e., single parent, both parents at home, other family members). for each individual family, we assessed the risk as high, medium, or low. the highest risk families were ones with a single parent or a parent with limited instructional control, who was on his or her own most of the day with two young children, one of whom had autism. in the case of one family, the parent was alone for most of the day with two adolescents with autism, one who was minimally verbal and the other who was verbally interactive. although this situation could have been considered high risk, in this specific case we considered it medium risk because the parent had excellent instructional control over both youngsters, who were both able to engage in solitary activities. as a general rule, the higher the risk, the higher the level of support we provided for the parent and the greater the daily structure. low-risk families included ones with both parents at home who were willing to engage with the child and sibling. in some cases, parents took turns to be with both children, so that one parent could be free. alternatively, each parent looked after one child, and they swapped children every few hours or every day. in these cases, it became crucial to ensure that both parents were involved in the care of the child and sibling, so that the burden of household management did not fall solely on the primary caregiver (parent 1). to achieve the involvement of both parents, provided they were both equally available, we worked separately with each one, setting up individual targets with each and developing a family schedule in which the time of each member was clearly specified. we identified three main learner profiles of the children, in terms of their need for support and perceived amenability to direct sessions over telehealth: 1. preschool-age children (n = 6): children who had not yet started elementary school (up to age 7); 2. minimally verbal children: children with limited adaptive, independent, and verbal skills (n = 16); and 3. verbally interactive children (n = 8). together with the risk assessment, this classification determined the daily family structure we arranged, as well as the type and frequency of support we provided as professionals. verbally interactive children were defined as being able to discriminate wh-questions (tact and intraverbal), follow multiple-step instructions, self-administer tokens, selfmanage interruption of reinforcement based on a timer, and not manifest challenging behavior in and out of sessions. verbally interactive children were those who we predicted would be able to sustain a direct session with their aba tutor (also commonly referred to as a technician or therapist) via telehealth. although none of the tutors had ever delivered interventions in this manner, extensive training was not needed because of their experience with the program targets and their familiarity with the child's home environment. for these families and tutors, contact with the aba consultant (commonly referred to as a supervisor) and/or lead tutor occurred once or twice per week to review targets, ensure that novel problem behavior was not emerging, and continue to provide support to the parents in managing the day. verbally interactive children received sessions in two formats. the first format involved the tutor remotely sharing the computer screen with the child, so what was once the table became the desktop computer. all visual stimuli were placed in individual electronic folders or powerpoint presentations, and the child responded to the materials presented via the tutor's desktop. tutors did not hold up cards to the screen, as it was too cumbersome, and some tutors also did not have the relevant materials at home. the second format was implemented with adolescents who were working on producing written responses; in that case, it was the child who remotely shared his or her screen with the tutor. the data collection system for these children remained unaltered from the prelockdown period, as the only change was the medium of delivery. most children received two 50-min sessions per day. for the two remaining profiles, preschool age and minimally verbal, we implemented a parent coaching system (see parsons, cordier, vaz, & lee, 2017 , for a review). the first telehealth session lasted up to 3 hr and was conducted by the team's aba consultant, with the participation of both parents and the team's lead tutor. subsequently, the consultant or lead tutor met with the family every day during the first and second week and every other day during the third intervention week and thereafter. the following protocol applies to families with preschool-age or minimally verbal children. the youngest child was 4 years old and had been receiving intervention for approximately six months. most of our families had been running an aba home-based program with the support of an aba consultant and tutor(s) for a minimum of 2 years and some for as long as 10 years. parents were not asked to take data, as this seemed unrealistic given how demanding their day was. professionals collected data during the online coaching sessions on the following: & parental report of challenging behavior; & parental report of their ability to maintain the agreed structure; & direct measurements of children's adherence to parental instructions; & direct measurements of challenging behavior during the coaching session; and & parents' procedural fidelity. prior to the lockdown period, children had access to a range of environments, each associated with its unique set of stimuli, signaling a specific reinforcement contingency. for example, children had learned that at school or during home sessions, brief periods of reinforcement were provided contingent on engaging in instructional activities and exchanging tokens. thus, children spent most of their learning time in school and in home sessions, where reinforcement access was regulated. for most of our children, the domestic context with parents signaled prolonged and often uninterrupted access to reinforcement in the evenings and on weekends. although not ideal, prior to the lockdown period, we did not view this as a significant problem because in most of their daily contexts (e.g., school and home sessions), children were able to engage in educational activities appropriately. during lockdown, however, the household became the only living context for all family members. this new set of circumstances created the need to institute an economic system that was sustainable, easy to implement for parents, and positively reinforcing for all members. the system also needed to promote the maintenance of adaptive skills and positive interactions, as well as allow for time off from interaction. during the first consultation session with each family, we worked with parents on structuring the entire day for all children (the child with autism and sibling), dividing it into blocks of activities to meet primary needs (breakfast, morning snack, morning outside time, lunch, afternoon nap for the younger children, afternoon snack, afternoon outside time, bath, and dinner). all times in between were considered dead times and, therefore, high-risk times that needed to be filled with contextually appropriate activities alternated with reinforcement intervals. one important aspect of structuring the day was to decrease the number of waking hours to reduce the fatigue and behavioral irritability that occurred toward the latter part of the day. we achieved two periods of roughly comparable duration between the mornings and afternoons by pushing lunchtime from 12:00 to 13:30-14:00 and bringing bedtime forward to no later than 21:00. as is fairly typical of southern european countries, our children often went to bed around 22:30 prior to the lockdown. we worked with parents to manipulate the stimuli associated with the end of the day (e.g., supper, bath, pajamas, story) to occur earlier than usual and in accordance with the recommendations for the optimal number of sleep hours for the child's chronological age (hirshkowitz et al., 2015) . table 2 shows an example of a daily schedule for a family in which parent 1 was alone most of the day with a 5year-old child with autism with limited independent and selfentertainment skills and a 3-year-old typically developing sibling. the day consisted of a rotation of contextually relevant activities or tasks for both the child and the sibling. we asked parents to identify one target per day in any of the activities, 1-week objectives, and 3-month objectives. we formulated the questions in this way: what would be helpful for you that your child learned? what do you want to teach your child today? what do you want to have taught your child in 1 week? when all this is over, what would you want your child to be able to do? in 1 month? in 3 months? parents chose from four types of activities broadly defined as follows: 1. independent activity: this encompassed any instructional activity the child could engage in without adult support (e.g., puzzles and shape sorters, worksheets, coloring, educational computer programs, domestic skills). visual activity schedules were utilized where useful (mcclannahan & krantz, 1999) . 2. household chores: these included chores the parent felt he or she could carry out with his or her child, giving the child things to do. we asked parents to go to each room and list all the chores that needed doing in each, however big or small. we asked parents to list every chore and not just the chores they thought their child could do or was already able to do. we wanted to identify objectives that were appropriate to the context and in which the parent was more likely to engage their child, as they needed to be done anyway. 3. tabletop discrete-trial teaching (dtt): we did not ask parents to run acquisition targets but only to maintain existing skills, with particular focus on clean responding without behavioral accessories (e.g., stereotypy). although it would be desirable if children maintained specific skill targets in specific programs, our purpose for having parents run dtt was primarily to help ensure that the children maintained some contact with the dtt contingency of rapid and accurate responding. 4. adult-led or shared activity (not dtt): these included activities that required the parent to engage one or both children, in which responses could be more loosely defined. examples of shared activities were completing simple crafts or making cookies. these were not necessarily reinforcing activities for the child with autism but often were reinforcing for the sibling. parents were coached to divide their attention between the two children, shape appropriate attention mands, and provide attention contingent on participation and engagement with the material. the targets for the child with autism were simply to remain in the activity and engage in some relevant responding. we did not include activities based on natural environment teaching (net), in which the parent actively had to manipulate the child's motivation and materials to evoke mands or to generalize language targets. this was because parents reported that they found this type of approach to be too effortful under these extreme circumstances. they reported they did not wish to be in a position to have to follow their child's motivation and to have to signal when that was no longer possible, risking the occurrence of challenging behavior. they also could not risk having to say no to something their child requested because of a lack of materials. although, in general, the daily structure centered on adult-led or shared activities alternated with periods of solitary reinforcement, all parents learned to interact with their children and siblings in a way that worked for them and maintained low rates of problem behavior. it is important to note that previous research exists that supports remote training of parents in net procedures (nefdt, koegel, singer, & gerber, 2010) , so we are not suggesting that this would not be a good approach for some families. two main reinforcement systems were implemented throughout children's waking hours: a token-based economy and an activity-based economy. in the token-based system, tokens were earned throughout the day and exchanged for preferred items. in the activity-based system, engagement in a less preferred activity (i.e., contextually appropriate activities) produced access to a more preferred activity. whether token based or activity based, the common element in both procedures was a system in which contingent relations between target behavior and preferred objects and activities were maintained, and reinforcers were unavailable outside those particular settings. we aimed to help parents establish a closed economy, meaning that engaging with parent-led reinforcement contingencies was the only way in which children could access those reinforcers, which is known to generate higher levels of responding (kodak, lerman, & call, 2007; reed, niileksela, & kaplan, 2013) . because we had already witnessed in our northern families (first cohort) the negative impact of free and prolonged access to reinforcers on (i.e., loss of children's skills and parental fatigue), we organized the household economic system so that reinforcement was accessible contingent on the production of contextually appropriate behavior for all children. this included the siblings, if they were under the age of 10 and not involved in remote schooling. the first step in closing the economy involved teaching parents to be able to limit access to all reinforcers for the child and siblings in every room of the household. parents classified reinforcers in terms of solitary and social for all children. solitary reinforcers were those that the child and sibling could consume on their own, and social reinforcers were those that required an adult and, where possible, were incorporated in the shared and adult-led activities. to help parents identify effective reinforcers, we accompanied them virtually through each room of the house and asked them to identify things their child liked or might like or items with which they had witnessed their child spend some time. every item was removed and placed in boxes, inside wardrobes or bags, or whatever container was available. in some cases, parents took pictures of the items to produce a reinforcer menu for the child (and sibling), whereas at other times, they took the child to the "shop," the place where all reinforcers had been stored. food treats were not available from the shop or the reinforcer menu; these were available at specific times during meals. the reasoning behind this was that food treats were items to be consumed rapidly, so the child was engaged for a very short period of time. our rationale in discontinuing noncontingent access to solitary toys and activities was that these items would hold their value and keep the children occupied longer. if the children were therefore able to be engaged with preferred solitary activities, the parent could safely have time off from the child and sibling. the secondary effect was to reduce escapemaintained problem behavior for both the child and the parent. by engaging with children in structured activities first, parents could access time to themselves and, as a result of contingent reinforcement delivery, also gain greater instructional control. we wished to create a mutually reinforcing situation for both the adults and the children, where parents could experience success in delivering instructions and interacting with their child, given that they would need to engage in this behavior daily in a confined space, over extended periods of time without a break. of course, discontinuing free access to reinforcers could also have the effect of increasing motivating operations that evoke problem behavior, so it was critical to support the children to be frequently successful in meeting the criteria for positive reinforcement, and thereby continuing to earn reinforcement on a frequent basis. many parents implemented the new reinforcement contingencies effectively, and the children learned very quickly that reinforcers were only available after the completion of activities or upon meeting the token schedule requirements. access to the reinforcer menu or shop was not available at any other time. children's mands for these items when they were not available were significantly reduced. because there were clear signals for reinforcement availability, children stopped asking for these items at other times, preventing parents from having to say no, thus reducing the risk of challenging behavior. children learned to mand for items (and receive them) only when the reinforcer menu was presented or when they were taken to the shop. they used whichever communication modality (e.g., vocal, sign, pointing, or selection-based modality) had been achieved prior to the lockdown period. given the level of stress the parents were experiencing after several weeks of lockdown, the likelihood was extremely high that they would reinforce problem behavior by providing the denied item to interrupt contact with the aversive stimulation produced by the child. we wished to reduce the risk by implementing the simplest possible system and not burdening parents with having to teach their child to tolerate denial. we simply removed that risk by giving frequent but contingent access to those items. the token system was implemented mainly for children who had limited verbal and nonverbal skills and who required frequent contact with reinforcement to engage in parent-led activities and instructions. a token system had been in place for all such children prior to the lockdown period, either at school or during home-based sessions. because we had always worked at home, all parents were familiar with the basic techniques; in fact, it was generally parents who manufactured the token boards, so the concept of a token economy system was not new to them. the fact that the system needed to be extended to the entire day was new and, in this sense, paralleled early applications of comprehensive token economy systems (ayllon & azrin, 1965 , 1968 phillips, 1968) . our token schedules consisted of three interrelated components: 1. the token-production schedule (the schedule by which responses earn tokens); 2. the exchange-production schedule (the schedule by which exchange periods are earned); and 3. the token-exchange schedule (the schedule by which the tokens were cashed in for preferred items or activities; see hackenberg, 2018 , for a review). the initial token-production schedule was set at fixed ratio (fr) 1, in which each target response (carrying out the parent's instruction within the activity proposed) produced a token to produce rapid acquisition. this was implemented throughout the day each time the parent gave an instruction. fairly quickly, parents naturally moved to a variable ratio schedule, in which a variable number of responses was required to produce a token, and they learned to adjust it based on the time of day or the difficulty of the task. the exchange-production schedule was fixed at fr 10, in which 10 tokens were needed to reach an exchange period. we did not make this variable, as it would have been too difficult for the parents to manage. the token-exchange schedule was fr 1-handing over the token boardwhereupon the parent presented the reinforcer menu or took the child to the shop to choose one item. during the exchange period, only one item or activity was allowed at a time. if the child wished to change the activity, then tokens had to be earned again for the change to occur. reinforcement duration varied according to parental needs and what was established by the daily schedule. in general, there were two types of reinforcement duration: brief (between 1 and 5 min) and long (up to 30 min). if parents needed their child to be occupied for additional time, they still had to interrupt after 30 min of consumption, place the item back in the shop, run a quick token board, and then open the shop or present the reinforcement menu again. the activity-based system was implemented when the parent had more than one child to look after, and it was extended to all siblings under the age of 10 if they were not involved in remote schooling. the system was based on creating half-hour blocks in which the parent was coached to engage the child and sibling in an adult-led or independent instructional activity for 30 min in order to produce 30 min of reinforcement time for everyone (the child, the sibling, and the parent). in the present article, we have described a model of supporting italian families during the past 6 weeks of lockdown. as professionals, we realized that this was one of the greatest challenges we would face in our careers. it soon became apparent, however, that the published literature and other tools upon which we typically rely were insufficient to deal with the magnitude and urgency of the present crisis. substantial previous research had been published on aspects of aba service delivery via telehealth, but little or no previous research had evaluated systems for transferring entire aba programs from in person to telehealth overnight, especially in the context of families living under total lockdown. in the absence of specific guidelines, we relied on an inductive process dictated by the tradition of our science, adapted published protocols, and derived procedures from principles. the system we were called to develop needed to be comprehensive and efficient. it needed to recognize the complexity of each individual family dynamic and be, at the same time, simple, realistic, and sustainable to maintain parental engagement. some may find the nearly complete elimination of noncontingent reinforcement and the application of a token economy across all waking hours to be somewhat extreme. however, the primary problem reported to us by parents before making this change was a lack of structure and loss of child motivation due to continuous free access to reinforcers. by programming reinforcement contingent on active engagement with the household schedule, we empowered the parents to increase their child's motivation and provide clear direction for everyone involved. if the parents had not been effective in providing sufficient antecedent support in the form of prompting and setting task difficulty at an achievable level, then such a system could have resulted in inadequate access to positive reinforcement. however, with support from their aba consultants, parents were successful in bringing order to their homes and helping their children to be calm, productive, engaged, and happy. despite the difficulties we are all experiencing, as both professionals and human beings, we have learned some valuable lessons that we hope will shape our ability to serve our families more effectively in the future. we have tremendous respect for the courage and dedication shown by the families we work with, who, at a time of adversity and uncertain future, have remained focused on the present. although parents realize that we are all learning as we go, we have seen a level of parental engagement that we had not been able to generate previously. such change, although borne out of crisis, may have enduring positive effects. our task moving forward will be to maintain these novel repertoires under more positive contingencies. we have not yet been able to analyze the data so far collected. we have included ongoing assessment of social validity by asking parents to comment on their experiences so far. we present three representative translated excerpts of parents' feedback: i thought it would be difficult to maintain the daily schedule of alternating instruction with reinforcement, but it has been very successful. i have stuck to it, and it has been all very natural and not too effortful. i am also very happy because i am able to spend time with my 2year-old, who is also making progress. i am able to play more with him and to focus on his speech. it's going well. (vittoria, parent of b, a 6-year-old girl with autism, and g, a 2-year-old boy) we received very simple and clear instructions-take away all reinforcers, engage him all day in simple domestic chores, give him routines, and give the reinforcers only after completing a token board. we saw an immediate change, zero problem behavior, and collaboration from c. if this situation had not happened, my husband and i would never have had such an enriched experience. seeing c so calm and compliant is the biggest reinforcer. (giada and davide, parents of c, a 9-year-old boy with autism) the management of g became very difficult. all his routines and perception of time had been disrupted. g, who was never interested in playing, became satiated with technology and was constantly searching for food, becoming very anxious during mealtimes. creating a closed reinforcer economy and dividing the day in clear sequential moments as to not get to the point of acute problem behavior and prevent boredom were essential. as parents, even in the absence of tutors, we are able to manage our child calmly and maintain learned skills. (veronica and giorgio, parents of g, a 7-year-old boy with autism) conflict of interest the authors declare that they have no conflicts of interest. ethical approval this article does not contain research conducted with human subjects. the measurement and reinforcement of behavior of psychotics the token economy: a motivational system for therapy and rehabilitation telehealth as a model for providing behaviour analytic interventions to individuals with autism spectrum disorder: a systematic review token reinforcement: translational research and application national sleep foundation's updated sleep duration recommendations: final report evaluating the influence of postsession reinforcement on choice of reinforcers activity schedules for children with autism: teaching independent behavior coronavirus, le misure adottate dal governo, provvedimenti attualmente vigenti, approvati dal governo in seguito all'emergenza sanitaria internazionale the use of a selfdirected learning program to provide introductory training in pivotal response treatment to parents of children with autism parent-mediated intervention training delivered remotely for children with autism spectrum disorder living outside of urban areas: systematic review achievement place: token reinforcement procedures in a home-style rehabilitation setting for "pre-delinquent" boys behavioral economics coronavirus disease (covid-19) pandemic publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-345695-5vi9wibk authors: hicks, lorin l.; schwab, nathan a.; homyack, jessica a.; jones, jay e.; maxell, bryce a.; burkholder, braden o. title: a statistical approach to white-nose syndrome surveillance monitoring using acoustic data date: 2020-10-22 journal: plos one doi: 10.1371/journal.pone.0241052 sha: doc_id: 345695 cord_uid: 5vi9wibk traditional pathogen surveillance methods for white-nose syndrome (wns), the most serious threat to hibernating north american bats, focus on fungal presence where large congregations of hibernating bats occur. however, in the western usa, wns-susceptible bat species rarely assemble in large numbers and known winter roosts are uncommon features. wns increases arousal frequency and activity of infected bats during hibernation. our objective was to explore the effectiveness of acoustic monitoring as a surveillance tool for wns. we propose a non-invasive approach to model pre-wns baseline activity rates for comparison with future acoustic data after wns is suspected to occur. we investigated relationships among bat activity, ambient temperatures, and season prior to presence of wns across forested sites of montana, usa where wns was not known to occur. we used acoustic monitors to collect bat activity and ambient temperature data year-round on 41 sites, 2011–2019. we detected a diverse bat community across managed (n = 4) and unmanaged (n = 37) forest sites and recorded over 5.37 million passes from bats, including 13 identified species. bats were active year-round, but positive associations between average of the nightly temperatures by month and bat activity were strongest in spring and fall. from these data, we developed site-specific prediction models for bat activity to account for seasonal and annual temperature variation prior to known occurrence of wns. these prediction models can be used to monitor changes in bat activity that may signal potential presence of wns, such as greater than expected activity in winter, or less than expected activity during summer. we propose this model-based method for future monitoring efforts that could be used to trigger targeted sampling of individual bats or hibernacula for wns, in areas where traditional disease surveillance approaches are logistically difficult to implement or because of human-wildlife transmission concerns from covid-19. introduction north american bat species face several contemporary conservation challenges throughout their range, including threats to habitat from loss and alteration of forested environments, and direct mortality from development of wind energy infrastructure [1, 2] . however, transmission of pseudogymnoascus destructans (pd), the pathogen that causes white-nose syndrome (wns), has emerged as the most serious threat to over a dozen species of north american bats that use caves and mines for hibernacula [3] . the pd fungus invades skin tissues (e.g., wing membrane) which disrupts homeostasis and causes dehydration that corresponds to an abnormally high frequency of arousals from torpor [4] . bats with wns increase arousal [5] and acoustic activity rates [6] above baseline levels during the normal hibernation period, which depletes critical energy reserves and can ultimately cause mortality. wns has caused precipitous declines in populations of hibernating bats and has contributed to the federal listing of the northern longeared bat (myotis septentrionalis) as threatened under the u.s. endangered species act [7] and endangered listings for three bat species in canada under the species at risk act [northern long-eared bat, little brown bat (myotis lucifugus), and tri-colored bat (perimyotis subflavus)] [8] . monitoring for wns is a critical management action and primarily has focused on detecting the pd pathogen either directly from bats captured in hibernacula or with environmental samples from occupied caves and mines [9] . since its emergence in eastern north america in 2006, wns has spread westward with some models predicting wns to arrive to montana by 2026 [10] . in june 2020, sampling conducted at bridges in 3 eastern montana counties confirmed the presence of pd, but not wns [11] . further, wns was detected in a little brown bat in washington state in 2016 and pd detection continues to increase among sites and across different species within washington [12] . thus, there is a second wns epicenter, which could affect known and previously unrecognized susceptible bat species and increase the spread of wns through western north america from both the west and the east [13] . the presence of wns in the pacific northwest opens another front in critical efforts to combat bat declines and shortens the timeline for implementing conservation strategies to protect vulnerable bat species. this effort is complicated by the fact that winter hibernation behavior for most western species is poorly understood [14] , yet this is when wns has the greatest negative impacts on bat populations [2, 15, 16] . further, little is known about winter roost sites and activity patterns of many bats in western north america. a recent review indicates that wns-susceptible species rarely congregate in large numbers in the western u.s. and that known winter roosts may be relatively uncommon or difficult to identify [17] . thus, typical surveillance approaches of estimating hibernating bat numbers and sampling for pd implemented in eastern north america where large hibernacula are more common does not translate well to all ecoregions [18] . therefore, alternate approaches to facilitate pathogen surveillance, monitor disease impacts, and conduct mitigation efforts for wns are urgently needed [13] . in addition to wns, bats can be influenced by habitat alteration from anthropogenic activities, such as forest harvesting [19] . forested areas provide essential habitat features that support bat diversity, including roost sites (e.g., trees) and foraging areas (e.g., riparian zones, wetlands) [20] [21] [22] . sustainable management of these forest resources may minimize the environmental stress on bats outside of the winter season (when wns most severely impacts bats). despite a robust network of public lands managed for preservation in western north america [23] , bat species richness is low in some protected areas, such as glacier national park [24] and lower bat activity is commonly reported in undisturbed habitats compared to disturbed [19] . managed forests support many species of bats not found or poorly represented in gov/reqapp/usermain.asp. these data were produced as part of a regional bat acoustic monitoring network and we had no special privileges to access these data and obtained them via request from the montana natural heritage program. funding: funding was provided by national council for air and stream improvement, inc., plum creek timber, weyerhaeuser, stimson lumber, f. h. stoltze land and timber. weyerhaeuser provided access and logistical support to collect empirical data and provided support in the form of salary for authors lorin hicks and jessica homyack. weyerhaeuser also contracted with tetratech for data collection and analysis, completed by author nathan schwab. the funder provided support in the form of salaries for authors [lh, jh], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. plum creek timber, stimson lumber, f. h. stoltze land and timber also provided funding and support in the form of salary for lorin hicks. weyerhaeuser also contracted with tetratech for data collection and analysis and nathan schwab received support in the form of salary. the specific roles of these authors are articulated in the 'author contributions' section. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. weyerhaeuser provided access and logistical support to collect empirical data and provided support in the form of salary for lorin hicks and jessica homyack. plum creek timber, stimson lumber, f. h. stoltze land and timber also provided funding and support in the form of salary for lorin hicks. weyerhaeuser also contracted with tetratech for data collection and analysis and nathan schwab received support in the form of salary. this does not alter our adherence to plos one policies on sharing data and materials. protected areas on public lands [1] and some forest management practices that reduce understory vegetation such as commercial thinning and prescribed burning can increase bat activity and occupancy [19, 25, 26] . nationally, public and private forestlands are managed under state forest practices rules, water quality best management practices, and forest sustainability programs (i.e., sustainable forestry initiative, forest stewardship council, american tree farm) to protect water quality and quantity, which includes riparian buffers that often provide habitat for bats. we quantified presence of bats across forested sites in montana, usa to document baseline levels of species diversity and activity rates prior to presence of wns. our objective was to evaluate long-term acoustic monitoring as a surveillance approach to monitor for potential wns presence in overwintering bat populations on both managed and unmanaged forest. specifically, we developed a statistical method to evaluate whether activity data occur outside the range of normal conditions, thus warranting more intensive sampling for the presence of pd on bats or in the environment. we predicted that bat populations in the western u.s. would have low levels of activity during winter months, high levels of activity during the summer, and that acoustic monitoring could be used as an effective approach for disease surveillance in remote areas of the intermountain west. our study area included forested areas across montana, usa (fig 1) . the forested areas in the western portion of the state were douglas fir (pseudotsuga menziesii)/snowberry (symphoricarpos albus) forest type, which generally characterized the region [27] and included locations of our acoustic detector stations. these forests had a mixed overstory, including douglas-fir, ponderosa pine (pinus ponderosa), grand fir (abies grandis), western larch (larix occidentalis), lodgepole pine (pinus contorta), and engelmann spruce (picea engelmannii), with understory vegetation comprised of snowberry, huckleberry (vaccinium spp.) and beargrass (xerophylum tenax). westside elevations ranged from 800 to 1150 meters. the climate in these ecoregions was continental-maritime with relatively low annual precipitation, with most precipitation occurring as snow [28] . the forested locations in eastern montana were characterized by limber pine (pinus flexilus) and ponderosa pine (pinus ponderosa) forest with understory of idaho fescue (festuca idahoensis) and bluebunch wheatgrass (agropyron spicatum). eastside sample site elevations ranged from 850-1520 meters. climate in the eastern ecoregions was continental with dramatic fluctuations in winter temperatures and longer, hotter, more arid summers than western ecoregions [29] . we selected 41 sampling sites that were near lentic or lotic systems and had �25% forest cover within a 1-kilometer buffer, estimated from the montana land cover data set in a geographic information system [30] (fig 1) . we did not randomly select sites for acoustic detectors but installed them in areas with potential for high bat activity that included proximity to standing live and dead trees, talus and rock cliffs, and waterbodies. we located four sites on intensively managed forest owned and managed by plum creek timber company (after 2016, weyerhaeuser) for production of sawtimber and other forest products. the remaining 37 sampling sites were selected from a regional bat acoustic monitoring network organized by the montana natural heritage program [31, 32] . these locations are primarily represented by federal and state landownership, but also include some tribal and private land. we we installed a single, full-spectrum acoustic recording unit (aru) at each sampling site. not all arus recorded data simultaneously or across the entire 2011-2019 study period, but we installed units for year-round recording of bat echolocation. each aru station consisted of a sm3bat or sm2bat+ acoustic detector (wildlife acoustics, maynard, massachusetts) and a microphone (u1 or smx-us) mounted on a pole at a height of approximately 3 meters. the arus operated on a 6-or 12-volt charging system (20-watt solar panel, 36-amp hour battery, and a charge controller) and housed in a weather-proof container on the ground below the microphone. we set the arus to record from sunset to sunrise to maximize battery life and balance with other logistical restraints of year-round sampling in remote locations. the units employed a sampling rate (samples per second) of 196,000, 256,000 or 384,000 khz. all arus used a minimum frequency filter of 8 khz, a trigger window of 2 seconds and a maximum file length of 5-8 seconds. most arus recorded in the wac0 compression format while four arus recorded directly to.wav format. the individual aru equipment and settings varied between the hardware platforms (sm3bat vs. sm2bat+), with detection distances of approximately 20-30 meters [33] , depending on the frequency of the source and other environmental variables (i.e., temperature and humidity) [34] . the equipment and settings were consistent for a given site throughout the study. the sensitivities of all microphones were within the manufacturer's specifications at the beginning of the deployment at each sampling site and replaced approximately annually. we examined equipment for functionality, checked microphone sensitivity, and exchanged data storage cards approximately once per month during summer and up to 6 months between visits during winter due to logistical constraints of access. each aru recorded a site-specific temperature approximately once every 1 or 5 minutes. at the end of our study, we parameterized acoustic files with sonobat 4.1.0 (sonobat, arcata, california usa) using a high-performance computing cluster at montana technological university. the parameters were then input into the sonobat montana [20160912] classifier to automatically assign a species classification to each bat pass. the montana classifier uses two regional species suites, each tuned to the species potentially present in that portion of the state. in cases where a bat pass was unable to be classified to species, a generic "bat" label was assigned. similar to the north american bat monitoring program [35] , we defined a bat pass as a sequence of echolocation pulses separated by more than 2 seconds of silence. we limited the maximum file duration to 5-8 seconds to reduce potential for recording more than one bat per sequence [36] . to quantify activity rates of bats, we analyzed the number of bat passes per detector-night (the number of operational detectors multiplied by the number of nights). for a night to be included in our estimates of detector-nights, the detector needed to register an internal temperature in the status file. we excluded data that did not meet this criterion from analysis. we manually verified a subset of diagnostic bat passes (approximately monthly for each species) with sonobat to ensure that species diversity of bats was accurate at all sampling stations [37] . we calculated the average number of passes per night over each month, by species, site and year using the ratio of total monthly qualified passes and the number of nights in the month. similarly, we calculated the average number of passes per night across all species, including passes without a species classification (total bat activity). a mean monthly temperature (˚c) was estimated from the mean of temperatures recorded at each site between sunset and sunrise across all nights in a month. we used a statistical model to describe variation in monthly-average nightly bat activity associated with several sources. to address our research questions, we aimed to characterize the extent to which activity varied among sampling sites, across years, by season, and in association with temperature. we fit a linear mixed-effects model with fixed effects for monthlyaverage mean temperature, season, and their interaction. we included random intercepts by site and year and random slopes for the temperature effect by site and year. thus, this model allowed for different overall levels of bat activity by site and year, and different temperature trends by site and year, while estimating population-average effects and quantifying the variation among sites and years. bat activity was log-transformed prior to analysis, and a constant of 0.01 was added prior to transformation to accommodate values of zero. we defined season by assigning each month to one of four calendar-based seasons (winter = dec, jan, feb; spring = mar, apr, may; summer = jun, jul, aug; fall = sep, oct, nov). we fit all models in r [38] using package lme4 [39] . we excluded sites with < ten months with at least one bat detection across all species from data used to fit the model. additionally, only data from 2012-2016 were used to fit the model, as limited data were available outside of this range. we note that both data restriction choices were performed to ensure sufficient data were available to estimate site-specific effects in the mixed effects model, but that no such restrictions were used in our raw data summaries. in addition to using the fitted model to describe variation in bat activity, we also used the model to generate prediction intervals [40] for new or future observations of bat activity in our region. we generated prediction intervals based on the model output using the 'predictinterval' function in package mertools [41] . we collected acoustic data used for our analysis between 28 sept 2011 and 4 august 2019, totaling 24,850 detector-nights at 41 sites. stations operated for an average of 606 (sd = 307) nights through the study. there were gaps in recordings because of equipment malfunction and wildlife encounters, but more than 5.37 million bat passes were recorded. after removing months with erroneous data due to equipment malfunction, 868 months of bat pass and temperature data were analyzed (per site mean: 21.2 months, sd = 10.7 months). we manually vetted acoustic files to confirm 13 species across the study, 11 of which were observed on managed forest sites (table 1) . three species [big brown bat (eptesicus fuscus), silver-haired bat (lasionycteris noctivagans), and western small-footed myotis (myotis ciliolabrum)] had confirmed activity in all calendar months, indicating that some bat species maintained some level of flight activity year-round. three presumed migratory species-spotted bat (euderma [2] . due to the high percentage of missing species information, our analyses include both estimates of total bat activity, i.e., total activity over all species, including bat passes not identified to species, and estimates for individual species where possible. across 24,850 detector-nights of sampling ambient temperatures, mean monthly temperatures ranged from -16.5˚c to 23.3˚c. mean monthly temperatures (± sd˚c) in montana varied across seasons [winter -1.7˚c (± 3.9˚c); spring 5.8˚c (± 4.3˚c); summer 16.2˚c (± 3.2˚c); and fall 6.7˚c (± 5.7˚c]). bat activity tended to be highest during summer months with most sites averaging >100 passes/night during july and august (fig 2) . additionally, most sites showed some activity during december and january, with one site averaging >10 passes/night. species-specific results show similar trends. we observed a strong positive association between average total bat activity and mean monthly temperature (fig 3) . the relationship was approximately loglinear for all 41 study sites, although the level of activity varied by site. results from our fitted model indicated a strong positive association between mean monthly temperature and total bat activity that varied by season (figs 4 and 5) . the estimated trends during the fall and spring indicated approximately 12.0-and 8.5-times greater numbers of bat passes per day, respectively, for every 5˚c increase in mean monthly temperature, on average, after adjusting for site and inter-annual differences. the association between bat activity and temperature was less pronounced during the winter and summer, where in both seasons we estimated approximately 2.8-fold greater activity, respectively, for 5˚c increases in temperature. overall levels of activity at a similar temperature also varied by season, although the difference was strongly dependent on temperature (fig 6) . we used the model random effects to estimate among-site and among-year variation in the mean response and temperature trends. we estimated considerable among-site variation in total bat activity after adjusting for temperature and seasonal effects, with site-specific means varying approximately 3.3-fold (1 sd) and among-year variation within a site of approximately 1.8-fold (fig 4) . estimated temperature trends among sites and among years were generally consistent, with variation in slopes of approximately 12% and 1% respectively. we fit this same model form to three individual species-eptesicus fuscus, myotis lucifugus, and m. californicus-to explore the possibility of seasonal differences in temperature associations among a species less vulnerable to wns [e. fuscus; [43] ], one highly vulnerable (m. lucifugus; [44] and one of unknown vulnerability (m. californicus). we note that since the majority of the bat passes were unidentified, the species-specific estimates may contain substantial unknown biases, as an unknown proportion of the unidentified bat passes consist of these species. our results suggest that winter activity for m. californicus is less strongly associated with mean monthly temperature than e. fuscus, while the reverse is suggested for the fall (fig 5) . we used the model fit to e. fuscus activity data (i.e., detections classified to the species e. fuscus) to illustrate a proposed monitoring approach with a statistical model for a single wnssusceptible species. the model was fit to data from all 41 sites to estimate among-site and among-year variation in activity, but we use the results to generate prediction intervals for four forest sites to depict how a landowner might implement this approach for wns surveillance monitoring. most observed activity data points fell within the 95% prediction interval, as expected (fig 7) . the prediction intervals were constructed to incorporate the estimated among-year variation in activity within a site. future observations of activity for these sites, along with model predictions, could be plotted to compare with prediction intervals. rapidly evolving technologies to passively observe and monitor wildlife concurrent with computational improvements in processing and analyzing large quantities of data are shifting management and research techniques from an animal-in-hand approach to indirect measurements of presence, abundance, diversity, and health [45, 46] . although acoustic monitoring has been implemented to investigate habitat relationships and community composition of bats [36] , uses of this technology are expanding into other objectives. here, we describe how yearround activity rates of bat species derived from acoustic monitoring can be used for surveillance monitoring for wns. we developed a simple and repeatable statistical modeling approach for wns surveillance monitoring with bat activity data collected from a broad geographic and temporal scale prior to known occurrence of wns in the intermountain west. this statistical approach can be applied elsewhere and may be particularly relevant for geographic locations where prominent hibernacula are inaccessible, rare, or unidentified [18] . further, our long-term and year-round collection of acoustic data for bats expands on limited knowledge of activity rates of species through time, seasons, across locations, and prior to wns. very little is known about the winter ecology of many species, specifically in western north america because of the limited sampling that has occurred during that season. technological advances in passive monitoring devices and identification software and an interest in wns has contributed to increased monitoring for bats in winter, when previously it was assumed they were rarely active [6, 47] . across our broad selection of 41 forested sites in montana, we documented a diverse community of bats (13 confirmed species), including at sites with a history of forest management. several species maintained some level of activity through winter, despite cold temperatures that averaged -1.0˚c in this season, which was below temperatures where at least some insect prey could sustain flight [48] . as expected, bat activity was positively related to warmer temperatures, although the strength of the relationship varied by season, with the strongest relationships with temperatures in spring and fall. in more temperate climates, other species of bats also displayed year-round activity that varied by season, sites, and species, including both migratory and non-migratory bats [2, 6] . the implications of winter activity of bats is not well-understood, particularly as it relates to body condition and energy stores or susceptibility to wns [6] . the ecology of bats infected with pd may lead individuals to change behaviors, such as higher activity rates during winter when bats typically use torpor to conserve energy [6] . aberrant behaviors by bats, including daytime activity out of roosts or hibernacula and during subfreezing temperatures increased after detection of wns in the southeastern u.s. [6] . however, the effects of winter bat activity on individual fitness have not been well-quantified. in north carolina, it was posited that year-round activity of typically migratory bats, including l. borealis, whose non-migratory behaviors could increase survival by reducing mortality from threats during migration, such as strikes from wind turbines [2] . alternatively, year-round activity by bats could be energetically costly if food resources do not meet metabolic demands and reduce reproductive output by individuals [2] . where underground caves or human structures are uncommon as hibernacula (e.g., the intermountain west), bats may congregate in smaller numbers in rock crevices during winter ( [49] ; unpublished data from this study). these wintering behaviors may provide protection against bat to bat transmission of pd. however, talus slopes can provide suitable temperature and humidity profiles for growth of pd, albeit below optimal levels [50] . the variability of relationships among prey availability, yearround activity, individual fitness, and susceptibility to wns remain a knowledge gap for north american bats [51, 52] . in addition to describing presence and seasonal activity rates of bats across forested sites in montana, we developed a process for using acoustic monitoring for disease surveillance that can be applied elsewhere. whereas approaches to incorporate uncertainty in estimates of population trends from bat count data in hibernacula are established, less information is available for managers to identify early-warnings from acoustic monitoring data [18] . here, we suggest the same basic framework that we used for the descriptive model of bat activity could be applied to an acoustic monitoring program aimed at identifying changes in bat activity consistent with wns. the approach could be used for single or multiple species and on individual sites or a collection of sites. additionally, the modeling approach is useful for detecting aberrant behaviors that could be associated with wns, including higher than normal activity rates during seasons where bats should be in torpor, or low activity rates due to population-level declines in bats. the general approach we consider is as follows: 1. collect baseline (pre-wns) data within a season or seasons of interest, and across several years and optionally across several sites. 2. fit a mixed-effects model to the data, incorporating variation with temperature, among years and among sites. optionally include other covariate information predictive of bat activity that can be easily gathered. 3. generate a population or site-level prediction interval that includes among-year variation. 4. monitor the collection of new data for excessive departures from the model prediction interval. in particular, look for bat activity in excess of the predicted range during winter months followed by bat activity below the predicted range during the following spring and summer [53] . we emphasize that our modeling approach was descriptive in nature and that we only recorded bat activity during nighttime. however, the approximately log-linear relationship with mean monthly temperature was strong in our dataset and may be useful in other investigations of bat activity. bat activity also showed approximately log-linear relationships with mean daily-max and daily-min temperatures. the non-random selection of sites included in our analysis limits the interpretation of the estimated model random effects or their application to other populations of sites. another limitation of species-specific results in this study relates to the large proportion of bat detections where no species label was assigned, where unknown biases could impact model estimates. we suggest that future site-specific surveillance models may be improved by including acoustic monitoring in daytime and by obtaining more high-quality species-specific call data with updated microphone technology [6] . we used prediction intervals to characterize expected ranges of among-year activity at the site level. one advantage of this approach in a monitoring program is that one set of intervals can be applied across multiple years without the need for annual refitting, provided suitable baseline data were used for model fitting. it is important to note that prediction intervals for mixed-effects models can be defined for any level of the random-effects structure [41] , with the appropriate level dependent on the end-use application. alternatives to prediction intervals could be employed in a monitoring program, including refitting a model each year to directly compare new data with a reference time period. currently, most conservation and management actions related to wns in north america are related to known hibernacula. for regions where large hibernacula are uncommon or rare, but where bats may still be susceptible to this threat, alternative approaches are necessary to ensure early detection of wns outbreaks and prevent further losses with targeted management actions. we propose a proactive and simple monitoring approach that could assist in managing risk of further loss of bat populations, which is especially relevant given current restrictions on field research due to concerns about transmission of covid-19 from humans to north american bats and among humans [54] . deploying passive arus to quantify bat activity, developing a predictive model of activity rates for one or more sites (including managed and unmanaged forest), and examining future data for deviations from these prediction intervals can all assist with a rapid response, including wns confirmation that could assist in recovery efforts. strong geographic and temporal patterns in conservation status of north american bats winter activity of coastal plain populations of bat species affected by white-nose syndrome and wind energy facilities white-nose syndrome: the devastating disease of hibernating bats in north america wing pathology of white-nose syndrome in bats suggests life-threatening disruption of physiology frequent arousal from hibernation linked to severity of infection and mortality in bats with white-nose syndrome winter behavior of bats and the progression of white-nose syndrome in the southeastern united states endangered and threatened wildlife and plants; 4(d) rule for the northern long-eared bat bats and white-nose syndrome 2020 determinants of pseudogymnoascus destructans within bat hibernacula: implications for surveillance and management of whitenose syndrome spread of white-nose syndrome on a network regulated by geography and climate fungus that causes white-nose syndrome found in eastern montana bat-killing disease white-nose syndrome confirmed east of the cascade range in washington first detection of bat white-nose syndrome in western north america. msphere activity following arousal in winter in north american vespertilionid bats western crevice and cavity roosting bats winter foraging of silver-haired and california myotis bats in western washington a review of bat hibernacula across the western united states: implications for white-nose syndrome surveillance and management improved analysis of long-term monitoring data demonstrates marked regional declines of bat populations in the eastern united states bently wigley t. site occupancy of foraging bats on landscapes of managed pine forest use of modified water sources by bats in a managed pine landscape bats in forests: what we know and what we need to learn bat activity in harvested and intact forest stands in the allegheny mountains us protected lands mismatch biodiversity priorities a macroecological perspective on strategic bat conservation in the u bat response to prescribed fire and overstory thinning in hardwood forest on the cumberland plateau effects of fire and its severity on occupancy of bats in mixed pineoak forests department of agriculture, forest service, intermountain forest and range experiment station ecoregions of the conterminous united states forest regions of montana land cover/land use theme montana natural heritage program montana bat and white-nose syndrome surveillance plan and protocols 2012 -2016 a directory of reports on long-term acoustic monitoring for bats at sites across the northern rocky mountains and great plains do you hear what i hear? implications of detector selection for acoustic monitoring of bats weather conditions determine attenuation and speed of sound: environmental limitations for monitoring and analyzing bat echolocation a plan for the north american bat monitoring program (nabat) on the importance of articulating assumptions when conducting acoustic studies of habitat use by bats montana bat and white-nose sydrome surveillance plan and protocols r: a language and environment for statistical computing. r foundation for statistical computing statistial intervals: a guide for practitioners mertools: tools for analyzing mixed effect regression models white-nose syndrome response team. bats affected by wns 2020 the resistance of a north american bat species (eptesicus fuscus) to white-nose syndrome (wns) an emerging disease causes regional population collapse of a common north american bat species computational sustainability: computing for a better world anda sustainable future how to learn to stop worrying and love edna monitoring winter acoustic activity of bats in montana effects of the environmental temperature on aedes aegypti and aedes albopictus mosquitoes: a review. insects winter bat activity in the canadian prairies unsuspected retreats: autumn transitional roosts and presumed winter hibernacula of little brown myotis in colorado frequent arousals from winter torpor in rafinesque's big-eared bat (corynorhinus rafinesquii) identifying research needs to inform white-nose syndrome management decisions. conservation science and practice going, going, gone: the impact of white-nose syndrome on the summer activity of the little brown bat (myotis lucifugus) fish & wildlife health committee and wildlife resource policy committee, bat working group, guidance for bat-related activities in response to covid-19. association of fish and wildlife agencies key: cord-342634-4ouhdjsr authors: semrad, katharina title: proteins with rna chaperone activity: a world of diverse proteins with a common task—impediment of rna misfolding date: 2010-12-26 journal: biochem res int doi: 10.1155/2011/532908 sha: doc_id: 342634 cord_uid: 4ouhdjsr proteins with rna chaperone activity are ubiquitous proteins that play important roles in cellular mechanisms. they prevent rna from misfolding by loosening misfolded structures without atp consumption. rna chaperone activity is studied in vitro and in vivo using oligonucleotideor ribozyme-based assays. due to their functional as well as structural diversity, a common chaperoning mechanism or universal motif has not yet been identified. a growing database of proteins with rna chaperone activity has been established based on evaluation of chaperone activity via the described assays. although the exact mechanism is not yet understood, it is more and more believed that disordered regions within proteins play an important role. this possible mechanism and which proteins were found to possess rna chaperone activity are discussed here. among all biological macromolecules, rnas represent one of the most functionally versatile players in the cell. rna molecules fulfill many different tasks such as coding and transfer of genetic information; they play regulatory functions in various cellular processes and catalyze chemical reactions (like cleavage and ligations). in addition to its functional versatility, rnas are also able to fold into countless different structures, many of which have similar stabilities as the native structure and therefore compete with the native fold. furthermore, rna molecules often undergo transition states during their folding pathways before they reach the native and active structure. these transient structures can represent traps along the folding pathway from which the molecules might have a hard time to escape and which then end up being long-lived intermediates. the reason for this structural versatility is the fact that rna consists of only four different bases which are easily capable of forming stable helices, that are not necessarily the native structure. the threshold for rna molecules to be able to perform their functions is usually the accomplishment of reaching its native and active structure. in the cellular environment, rna molecules do not appear as "naked" nucleic acids but always are found in conjunction with proteins. in some cases, the rna molecule helps the protein partner to fold correctly; in others, the protein stabilizes the rna structure. and last but not least proteins with rna chaperone activity aid during the folding process of rnas. proteins with rna chaperone activity open up misfolded rna structures and do not require atp [1] . furthermore, after the rna has been folded into its native structure, the protein becomes dispensable. although the term "rna chaperone" has been used routinely to describe various proteins that are capable to assist rna folding in vitro, the term rna chaperone is reserved to describe proteins whose rna folding activity has been verified on its natural target rna in vivo. therefore, most of the proteins in this paper will be referred to as proteins with rna chaperone activity if their rna folding activity was only determined in vitro and/ or on nonnatural rna targets. this paper will focus on the diversity of proteins with rna chaperone activity and which experimental assays are in use to determine whether a protein has rna chaperone activity. i will present examples for proteins with rna 2 biochemistry research international chaperone activity and discuss possible mechanisms of rna chaperoning. chaperone activity and rna misfolding 2.1. what are rna chaperones and proteins with rna chaperone activity? the list of proteins with possible rna chaperone activity is growing constantly. proteins with different activities that support rna folding are classified in this group. the definition of a protein with rna chaperone activity is that the protein prevents rna from misfolding by opening up misfolded structures. proteins with rna chaperone activity do not require atp, which distinguishes them from rna helicases, another group of proteins that facilitate rna folding (e.g., cyt-19) [2] . proteins with rna chaperone activity interact only transiently with rna molecules and are supposed to be dispensable once the rna has been folded correctly. this was shown for e. coli proteins s12 and stpa [3, 4] . a transient interaction and weak binding to rna might be difficult to define because many of the identified rna chaperones interact strongly with their target rnas and are found in rnp complexes like ribosomal proteins, hnrnps, la protein, and others. however, it has been demonstrated that a mutant stpa that shows stronger binding towards rna shows decreased rna chaperone activity suggesting that strong binding could also be detrimental to rna folding [5] . in that way, proteins with rna chaperone activity are also distinguished from "stabilizers" that are proteins that bind and stabilize an rna structure and are required to stay bound in order to keep the rna's native structure. cyt-18, the trna synthetase from neurospora crassa, is a "stabilizer" for the mitochondrial self-splicing group i intron: its presence is required to keep the native structure of the intron which otherwise unfolds readily. in the growing database of "proteins with rna chaperone activity", there exists an increasing number of proteins that simply possess rna annealing activity. a prominent and intensively studied member of this group is the bacterial host factor hfq that showed annealing activity on random substrates. hfq in addition is an rna chaperone as it was further demonstrated that hfq does possess unwinding activity upon its native substrates [6, 7] . in brief, the group of proteins with rna chaperone activity includes proteins that, first, open up misfolded structures without requirement of atp and that, second, are dispensable once the rna has been folded. 2.2. rna misfolding. rna molecules are prone to misfold in vitro and are usually prevented from misfolding in vivo. rna basically encounters two folding problems: a kinetic folding problem, where the rna molecule has to surmount kinetic barriers during the search for its native structure. secondly, rna molecules meet a thermodynamic folding problem as the final native structure often has to compete with alternative folds that have similar energetic stabilities [1] . rna folding is a hierarchical process, and first secondary structure elements have to form. secondary structural elements form between regions within the rna molecule that are in close proximity. they are a-form helices consisting of watson-crick base-pairs. secondary structures are very stable. the stability of a base-pair depends on the stability of both of its neighbouring base-pairs. already any rna of a reasonable length is able to form alternative base-pairs leading to alternative helices that become folding traps. tertiary structures are higher order structures that are built by assembling the secondary structure elements into a more complex collapsed fold. they can also involve formation of helices. this is the case in pseudoknots where either a loop region interacts with a distant single stranded region or with another distant loop. pseudoknots possess similar stability as secondary structures. but tertiary structural elements involve also other non-watson-crick interactions where for example, not only the watson-crick site of the nucleotide interacts with another nucleotide but also the hoogsteen edge or the sugar edge of the nucleotide is involved in hydrogen bonding [8] . an often reoccurring tertiary structure motif is the a-minor interaction where an adenine interacts with the minor groove of the a-form helix [9] . tertiary structures are often less stable and depend on the formation of secondary structures. finally, monovalent or divalent metal ions play an important role in tertiary structure formation. the first studies on rna structure and folding were done in the 1960s with yeast trna molecules. already then it was demonstrated that trnas are able to adopt two distinct conformations of which only one is the native structure which can be aminoacylated [10, 11] . the rna folding problem becomes even more prominent in the case of large rnas such as group i introns or in the context of large protein-ribonucleic acid complexes such as rnase p and the ribosome. it was demonstrated that the self-splicing group i intron of the thymidylate synthase gene of phage t4 misfolds in the absence of translation: when the ribosome does not prevent base-pairing between exon and intron sequences, the intron is not able to fold correctly and cannot perform the splicing reaction [12] . a similar observation was made with the group i intron of tetrahymena thermophila ribosomal rna: a subset of molecules misfolds and accumulates into an inactive population [13] . misfolding depends on exon sequences that form stable hairpins and intervene with 5splice-site formation. in vivo, however, some group i introns require the assistance of proteins to splice efficiently and prevent misfolding. for an example, the cyt-18 protein in neurospora crassa mitochondira is a trna synthetase which stabilizes the p4-p6 domain of group i introns and recruits cyt-19, an rna helicase, which then unwinds folding traps and promotes splicing [2, 14] . for large rnp complexes such as the ribosome, a growing body of evidence suggests that several additional factors such as helicases exist that assist during the folding process in vivo. as proteins with rna chaperone activity are very heterogeneous concerning their structure and their way to resolve the folding of rna molecules, there are various rna chaperone assays available to measure different activities. in principle, the assays can be divided into in vitro and in vivo assays that use either simple oligonucleotide annealing or displacement reactions or that measure catalytic activities of correctly folded ribozymes. measuring an activity that might be targeted more specifically to a certain subset of substrates in the natural environment of the putative rna chaperone makes it difficult to evaluate rna chaperone activity using a single assay. the substrates in the in vitro assay (e.g., oligonucleotides) might differ in sequence requirements or structure requirements from possible native substrates and might lead to negative results. strand unwinding assays might give positive results in the case of single-strand binding proteins. furthermore, in vivo assays to measure rna chaperone activity can be negatively influenced by possible toxicity of the putative rna chaperone when overexpressed or can lead to secondary effects in the cell that give false positive results. therefore, it is recommended to measure the rna chaperone activity at least in more than one chaperone assay to be certain that no non-specific activity is measured. figure 1) 3.1.1. oligonucleotide annealing. in this assay, two complementary oligonucleotides (oligos) present in concentrations above their dissociation constant are incubated together in the absence and in the presence of the protein to be evaluated for rna chaperone activity. an increase in the rate of duplex formation is observed when the tested protein has rna annealing activity. in principle two complementary short rna oligos are used in this assay, although it has been a habit to choose dna oligos instead. in order to measure rna chaperone activity, however, i think that rna oligonucleotides should be the preferred choice. detection methods of duplex formation include native gel electrophoresis where the two complementary rna strands can either be visualized by radioactive or fluorophor labelling and has been used in (besides many many other publications) [15] [16] [17] . rna annealing can alternatively be measured by observing the fluorescence resonance energy transfer (fret) upon the closing up of the two fluorescently labelled oligonucleotides (e.g., see [18, 19] ). activities. the ability of a protein to open up and unwind an already formed rna duplex is measured. to measure rna chaperone activity in contrast to helicase activity, this assay is performed in the absence of atp or an alternative energy source. analogous to the annealing assays, melting activity can be detected by using native gel electrophoresis or by measuring loss of fluorescence energy resonance transfer (fret) that occurs upon dissociation of the complementary fluorophor labelled rnas. in addition to under single turnover conditions, substrate to ribozyme annealing is measured. under multiple turnover conditions substrate dissociation is measured. (d) in the trans-splicing assay, the group i intron is split in two halves h1 (upstream exon, 5 -part of the intron) and h2 (3 -part of the intron and exon2), and splicing at low temperatures in the presence of chaperones is measured. (e) shows the cis-splicing assay where an enhancement of splicing at 37 • c is measured in the presence of chaperones. the construct (shosho) contains short exon 1 (27 nucleotides) and short exon 2 (2 nucleotides) sequences. the above-described detection methods, new approaches to detect folding or unfolding of single molecules emerge and include time-resolved nmr, which becomes a powerful tool to study folding of small rnas. cleavage. the hammerhead ribozyme cleavage reaction and folding of the ribozymesubstrate 3-way helical junction have been studied in a great detail. therefore, this assay represents a suitable tool to study rna chaperone activity upon folding of the hammerhead ribozyme-substrate construct. using the hammerhead cleavage assay, both annealing and strand displacement can be studied independently of each other [3, 20] . the advantage on the well-studied assay is that depending whether singleturnover conditions or multiple turnover conditions are employed, it is possible to distinguish between annealing and strand dissociation activities. using single-turnover conditions, where an excess of ribozyme and low concentrations of substrate are applied, substrate annealing is determined as substrate annealing becomes the rate limiting step. on the other hand, using multiple turnover conditions with an excess of substrate over ribozyme, the whole cleavage reaction is monitored consisting of annealing and product release. since product release represents the rate limiting step, product dissociation is measured. self-splicing of the thymidylate synthase group i intron (td intron) of bacteriophage t4 has been characterized, and the td intron has been used lengthily to monitor rna chaperone activity of various proteins. splicing of different group i intron constructs that do not fold readily into the splicing competent structure in vitro is tested with and without chaperones. cis-splicing assay. in this td intron construct both, 5 and 3 exons are shortened for the upstream exon down to 27 nucleotides and the downstream exon shortened to only 2 nucleotides. this short exon construct (td shosho) splices at 37 • c but rna chaperones increase folding and as a consequence the splicing rate of the short-exon construct is increased as well [5] . trans-splicing assay. here, the td intron is split into two halves in the center of loop l6 in the p4-p6 domain, where in the wild type group i intron an open reading frame for an endonuclease is present. the upstream in vitro transcribed construct contains 549 nucleotides of exon1 and 131 nucleotides of the 5 -part of the intron. the downstream construct consists of the remaining 147 nucleotides of the intron and 23 nucleotides of exon2. correct and efficient folding of the trans-intron-constructs is significantly impaired at 37 • c but works fine at elevated temperatures (55 • c), which is monitored through splicing [21] . chaperones with strong annealing and unwinding activities such as ribosomal protein l1 or l19 from e. coli are capable to catalyze trans-splicing at 37 • c or even at lower temperatures, for example, hnrnpi increases splicing at 25 • c [22] . in vivo rna chaperone activity assays (see figure 2) 3.2.1. in vivo folding trap assay in e. coli. splicing of the group i intron within the thymidylate synthase gene of phage t4 occurs efficiently in vivo. though, when splicing and translation are uncoupled by introducing stop codons in the upstream exon, splicing is significantly reduced. this is due to alternative base-pairing of exonic and intronic sequences which prevent the formation of the intron's native fold [12] . the mutant td precursor construct tdsh1 consists of an exonic stop codon and has an additional intronic point mutation (c865u) which further destabilizes the native intron structure. the tdsh1 construct is significantly impaired in splicing in vivo. overexpression of rna chaperones in the presence of the tdsh1 mutant is used to evaluate if the rna chaperone is able to rescue the misfolded intron and restore splicing [23] . transcription read-through of the chloramphenicol acetyl transferase gene (cat) is inhibited due to the preceding transcription terminator stem. the stable hairpin secondary structure of the terminator inhibits the polymerase to transcribe the cat gene and as a consequence no chaloramphenicol resistance is achieved. the cells are chaloramphenicol (cm) sensitive. proteins with rna chaperone activity are able to melt the terminator stem and as a consequence read-through occurs and the cells become chloramphenicol resistant. the transcription antitermination assay was used for assaying cold shock proteins or if1 from e. coli [24] [25] [26] . proteins that possess rna chaperone activity are very divers and span from viral to bacterial and human proteins that are involved in many different cellular processes. the list of rna chaperones is permanently growing. recently, a database for proteins with rna chaperone activity was established where every lab is able to contribute their data for a newly identified chaperone http://www.projects .mfpl.ac.at/rnachaperones/index.html [27] . the following selection of proteins with rna chaperone activity is not complete but points out the most important groups or single proteins. the first viral rna chaperone activities were reported in the early 1990s and showed that nucleocapsid protein 7 (ncp7) of hiv increases hammerhead ribozyme cleavage significantly [20, 28] . only a few years later, another virus-encoded protein with rna chaperone activity has been identified, the hdv delta antigen, which was also monitored in the hammerhead ribozyme assay and furthermore shown to be dispensable after folding has occurred [29] . flaviviridae core proteins were also monitored and shown to possess rna chaperone activity in a hammerhead cleavage assay and/or rna strand annealing activities [30, 31] . interestingly, many of the viral proteins show exceptional high degree of disorder. in the case of the flaviviridae core proteins, it was also reported that heat denaturation still retained strand annealing activity suggesting that the disordered domains of the proteins are involved in chaperoning. nucleocapsid proteins from two members of the coronaviridae family have been investigated and hammerhead ribozyme cleavage was shown to be enhanced in their presence [32, 33] . and again both nucleocapsid proteins show a high degree of disorder in in silico predictions. a growing body of evidence suggests that there exist many more viral proteins that possess rna chaperone activity. the list here is not complete but proteins that were shown to have only dna annealing activities were left out in this list. the majority of these small viral proteins show strong propensity for disorder which suggests that disorder might be a mechanistic requirement for chaperoning. interestingly, studies on nc proteins demonstrated that these proteins not only possess rna chaperone activity in vitro but also are required for strand annealing and strand displacement activities on their target rnas in vivo [33] . recently, a specific template switching assay designed to study strand displacement in a retroviral-derived system demonstrated that nucleocapsid protein from coronavirus shows rna chaperone activity and most likely is an rna chaperone in vivo [34] . stpa. the e. coli transcriptional regulator stpa, a 15 kd basic protein, was isolated as a repressor of a splicingdeficient group i intron in thymidylate synthase of phage t4 [4] . stpa was furthermore shown to possess strong rna chaperone activity in vivo in the folding trap assay [35] . the protein was tested in vitro in a strand-annealing and strand-displacement assay and exhibited strong activities in both tests [36] . more detailed studies on stpa revealed that the protein binds transiently to rna with a preference for unstructured regions and that binding to rna is diminished in the presence of high ionic strength [5] . in an elaborate study applying in vivo dms modifications to the rna, in vivo folding of the group i intron was evaluated in the absence and presence of stpa [37] : schroeder and coworkers demonstrated that stpa opens up tertiary interactions of the td group i intron. while the loosening effect is advantageous in wild type or misfolded introns, overexpression of stpa in the presence of introns that were already destabilized in their 3d structure was detrimental. structure prediction of stpa suggested that this protein exhibits more than 70% disorder and it was suggested that this unfolded regions of stpa might play a role in chaperoning [38] . ribosomal proteins are required within every cellular organism to build up the bacterial 70s or the eukaryal 80s ribosome. many ribosomal proteins further regulate transcription or translation of their own operons. in addition, ribosomal proteins are also involved in various very different cellular processes and fulfill extraribosomal functions [39, 40] . ribosomal proteins are highly conserved among various species and many ribosomal proteins have unusual long unstructured extensions that wind their way through the ribosome [41] . the first observation that ribosomal proteins are capable of chaperoning rna folding came from the belfort lab: screening for cellular factors that increase trans-splicing of the thymidylate synthase group i intron revealed that many ribosomal proteins possess chaperoning activity, with ribosomal protein s12 from the small ribosomal subunit having the strongest activity [3] . furthermore, s12 significantly increased hammerhead ribozyme cleavage [3] . a systematic study on large ribosomal subunit proteins from e. coli showed that 1/3 of the tested proteins possesses strong rna chaperone activity in vitro in the trans-splicing assay [21] . in addition, ribosomal protein l1 orthologs from eukarya, bacteria, and mesophilic archaea also exhibited strong transsplicing and cis-splicing activities in vitro [42] . although it makes sense that the rna chaperone activity of ribosomal proteins could play a role during ribosome assembly, a definite proof for the requirement of this activity in vivo has not yet been provided. recently, it was demonstrated that e. coli ribosomal proteins l15, l16, l18, and l19, that showed rna chaperone activity in vitro, further possess protein chaperone activity comparable to other protein chaperones such as hsp90 [43] . it was suggested that intrinsically unstructured domains of ribosomal proteins could play a role in chaperoning. the exact mechanism, however, still remains elusive (see section 5). cold shock proteins (csps) are conserved throughout bacteria and plants. they are expressed during cold-shock, when misfolding of rnas becomes a major problem for the organism and function as transcriptional antiterminator at low temperature. many experiments that have been performed to describe chaperone activity of cold-shock proteins utilize dna helices (and only sometimes in addition rna duplexes) and refer to the activity as nucleic acid melting activity. however, it has to be mentioned that there are no elaborate studies on whether there is a difference between rna duplex and dna duplex melting and whether dna melting activity automatically is the same as rna melting. e. coli contains nine members of the csp family and cspa, the major cold-shock protein and cspe were identified to interact non-specifically with rna molecules and to possess nucleic acid melting activities [44] [45] [46] . cold-shock proteins in higher plants are highly conserved. glycine-rich and zn-finger containing proteins from arabidopsis thaliana have been monitored for their nucleic acid melting activity, and it was shown that grp7 (glycinerich protein) and csdp1 (cold shock domain protein) possess rna chaperone activity [47] . a recent study also demonstrated that out of six glycine-rich proteins in rice (oryza sativa), which are likely to be involved in adaptation to cold-shock, three of them exhibit rna-(and dna-) melting activities suggesting that grps in plants fulfill a chaperoning role during low temperatures [48] . in e. coli translation, initiation factor 1 (if1) is a small 71 amino-acid long peptide, which contains 5 rigid β-barrels and belongs to the ob (oligomer-binding)-fold proteins such as the cold shock proteins. n-and c-termini of if1 are highly flexible. it was demonstrated that e. coli if1 is capable of complementing for a cspb and cspc double knock-out in bacillus subtilis suggesting that if1 and csps have at least partially overlapping activities [49] . e. coli if1 exhibits rna chaperone activity in various assays including rna annealing of complementary oligonucleotides, transsplicing, in vivo folding trap assay, and transcription antitermination in vivo and in vitro [25, 50] . heteronuclear ribonucleoproteins encompass a group of about 20 polypeptides that are predominantly nuclear in localization and are involved in rna processing. the first observation that hnrnps possess rna chaperone activity came in the early 1990s when fractionated hela nuclear extract was tested for annealing activity of an mrna and its antisense partner. three proteins, hnrnp a1, c1 and u, were identified and hnrnp a1 was further shown to enhance hammerhead ribozyme cleavage in vitro [16, 20] . later, a detailed study on possible functions of ro rnps, which are ro ribonucleoprotein complexes, composed of a small noncoding cytoplasmic rna, termed y rna and its protein partners was conducted: besides the permanently associated proteins ro60 and la, subpopulations of ro-rnps also contain hnrnp i and hnrnp k, both of which exhibited strong rna chaperone activity in vitro in the transsplicing and the cis-splicing assay [22] . hnrnp i is identical to poly-pyrimidine binding protein (ptb) isoform 4 and was identified as a splicing suppressor in mammalian cells [51] . it regulates cap-independent translation, localization of cytoplasmic rnas, and poly-a-site cleavage [52] . ptb belongs to the ires transacting factors (itafs), which are host factors (like la, hnrnp k, nucleolin, unr and many others) that interact with viral rnas and induce conformational changes that then lead to translation initiation [53] . it was further reported that calcivirus replication requires ptb but only at lower or at higher temperatures than the permissive 37 • c, suggesting a chaperoning role of ptb [54] . members of the group of itafs have been implicated in rna chaperoning like unr, a cold-shock domain containing protein [55] , human la protein, and hnrnp k [22] . hnrnp k is also a multifunctional protein that is a transcriptional factor for c-myc and c-src [56] [57] [58] ; it enhances splicing [59] and is a translational regulator [60] . la proteins primarily bind rna polymerase iii transcripts and protect them from nuclease attack [61] . they also interact with pre-trnas at their uuu-3 oh ends and facilitate their maturation. la contributes to assembly of rnp complexes by retaining rnas in the nucleus. la is also involved in translation regulation. and human la was demonstrated to possess rna chaperone activity in vitro in the cis-splicing assay and in vivo in the folding trap assay [22] . hfq. the bacterial protein hfq was first discovered in the end 1960s as a host factor for bacteriophage qβ replication [62] . the bacterial protein is a pleiotropic regulator for gene expression in bacteria. it interacts with many small rnas and their mrna targets and regulates posttranscriptional regulation of small noncoding rnas such as dsra, sodb, oxys, rpra, and spot42 [63] [64] [65] [66] [67] . hfq preferentially binds to a/u rich, unstructured regions. hfq encompasses an sm-domain, which is highly conserved among various species and usually is found in eukaryotic spliceosomal rnps. crystallographic studies of escherichia coli, pseudomonas aeruginosa, and staphylococcus aureus hfq proteins showed that hfq forms homohexameric ring structures with a central cationic pore that forms the rna binding site [68] [69] [70] . using strand annealing and strand melting assays to measure rna chaperone activity of hfq, only strand annealing activity was observed [27] . however, a detailed study using rnase footprinting on hfq's interaction with its target rnas sodb mrna and the small noncoding regulator ryhb rna demonstrated that hfq indeed did loosen secondary structures within sodb mrna that lead to binding of its regulatory rna rhyb [7] . a similar observation was made using fluorescence labelled rpos mrna and dsra small noncoding rna [6] . by means of fret, it was shown that hfq induces annealing of dsra to rpos mrna and prior to the annealing event hfq disrupts rpos secondary structure elements. consequently, hfq is entitled to be called rna chaperone. the prion protein is a misfolded isoform of the essential component of prion diseases such as creutzfeldt-jakob disease in humans-one of several neurodegenerative diseases. the function of the human prion protein is not clearly understood. it was demonstrated that the prion protein has rna (and dna) annealing activity [71] ; however, it was not yet shown if it possesses also rna unwinding activity and may therefore be classified as an "annealer". interestingly, the prion protein contains an intrinsically unstructured n-terminal domain [72] . the fragile x mental retardation protein (fmrp) is linked to the fragile x syndrome as the disease is due to transcriptional silencing of the gene. fmrp possesses rna binding activity and its interaction partners include a large number of mrnas, micrornas, sirnas, and small noncoding rnas as well as a multitude of different proteins [73] [74] [75] [76] . it was demonstrated using hammerhead ribozyme cleavage that fmrp possesses rna chaperone activity [77] . and finally in line with many other proteins with rna chaperone activity, it is interesting to mention that fmrp consists of a highly disordered c-terminus suggesting that the substrate versatility of fmrp might be accomplished through its structural disorder [78] . chaperones provide a critical cellular activity. proteins with rna chaperone activity are very divers in structure as well as in function: stpa, a transcriptional activator and repressor of a multitude of bacterial genes, is a small (15 kd) bacterial protein with intrinsically unstructured regions. stpa has strong rna chaperone activity. on the other hand the bacterial protein hfq is a large multidomain protein complex (60 kd) and folds into a compact ring-like structure. among ribosomal proteins, many were shown to possess rna chaperone activity (e.g., one third of large ribosomal subunit proteins from escherichia coli show rna chaperone activity in vitro). ribosomal proteins are usually small proteins many of which have long unstructured domains and are highly basic proteins. proteins with rna chaperone activity do not require an external energy source as rna helicases do. this raises the question of how rna chaperones accomplish the rna folding task and where the energy for this process comes from. proteins with rna chaperone activity in most cases encompass two major activities: the annealing activity and the unwinding activity (see also figure 3 ). many proteins with rna chaperone activity are highly basic proteins and therefore interact readily with negatively charged rna molecules. in that way, they might stabilize folded states by bringing together distant regions of the rna molecule and as a consequence increase rna double-strand formation. this mechanism could be comparable to the action of chemical chaperones such as osmolytes which are small organic compounds, that do not interfere with the cellular metabolism but speed up folding processes enabled through a crowding effect [79] . another indication that a crowding effect might play a role at least to some extent during rna annealing is the following: when rna chaperone activity is measured in vitro, there is always an excess of protein over rna present in the assay. for example, in the trans-splicing assay, 200 nmols of rnas (leading to a 20 nm end-concentration) are tested for folding in the presence of 1-2 μm protein. it was shown that e. coli ribosomal protein l1 displays maximal rna chaperone activity starting from 400 nm up to 2 μm protein concentration [42] . this means that at least a 20-fold excess of protein to rna has to be present to achieve maximal chaperoning activity of ribosomal protein l1 from e. coli. in this line, it also has to be mentioned that in the in vivo chaperone assay, which uses the folding trap of a misfolded group i intron in the thymidylate synthase gene of phage t4, it is always necessary that the measured protein is overexpressed and available in higher concentrations [23] . for example, the e. coli protein stpa, which is found constitutively expressed in the bacterial cell, only shows its rna chaperone activity in vivo when stpa is additionally over-expressed from an expression vector, thus showing that the cellular concentration of stpa is not sufficient to increase folding of the misfolded group i intron. certainly, this observation might be due to the engagement of stpa in other regulatory functions in the bacterial cell; however, it also points to the direction that more than one molecule 8 biochemistry research international of stpa is required to assist folding of the td group i intron. as a consequence the question rises if and how it is possible to distinguish between rna chaperone activity and a nonspecific single-strand rna binding activity of the protein that might both prevent misfolding. using the in vivo folding trap assay, however, not only proteins with possible rna chaperone activity like stpa had been tested but also a viral single strand binding protein from influenza virus (np) was tested and did not show any increase in splicing suggesting that single strand rna binding might not be sufficient for chaperoning. furthermore, a detailed study on stpa wild type and mutants demonstrated that only the fulllength stpa was able to show rna chaperone activity by simultaneously interacting with two rna molecules [5] . rna chaperone activity of stpa has been studied for more than a decade. it was shown that stpa has strong in vivo and in vitro rna chaperone activities. in a mechanistical in vivo study of stpa, schroeder and coworkers demonstrated that stpa loosens tertiary contacts within the thymidylate synthase group i intron [37] . in contrast, the neurospora crassa trna synthetase cyt-18 that also increases group i intron splicing of td stabilizes tertiary interactions. but how is the opening of tertiary structure elements accomplished without the hydrolysis of atp? this strand unwinding activity is more difficult to explain as the question remains of how a protein can actively open up hydrogen bonds when no apparent source of energy is required. in the protein world, it became more and more visible that the classical structure-function paradigm does not necessarily hold for many proteins and their activities. a growing body of evidence suggests that a multitude of proteins do not fold into compact domains but are fully or at least partially unstructured [80] . in eukaryotes, for example, conservative estimations point out that 5%-15% of all proteins are completely disordered and 50% of the cellular proteins have at least long unstructured domains. an interesting study by tompa and csermely demonstrated that among chaperones a significantly high percentage of proteins show long unstructured regions [38] . among rna chaperones, the percentage of at least partially disordered proteins is even higher (54%) than in the group of protein chaperones (36.7%). disordered proteins and protein segments allow a broad versatility for interaction partners and in this case for interaction with different rna molecules. but it can also explain the ability of proteins with rna chaperone activity to multitask as so far no rna chaperone has been identified whose only task is to aid in rna folding. interestingly, it was recently demonstrated that some ribosomal proteins that possess rna chaperone activity and contain disordered regions are also capable to chaperone protein folding suggesting once again that disordered regions provide high versatility for substrate interactions [43] . the idea of disordered rna chaperones is especially attractive because there are many advantages of proteins with disordered regions over compact proteins: (1) the main advantage of a disordered region is that it can easily interact with a range of many different partners and is not limited to a single binding pocket or recognition element on a partner molecule. (2) the bigger surface of the unstructured protein might provide a "loosening effect" for the incorrectly folded rna molecule. (3) the troublesome question of where the energy for the rna unwinding might come from could be explained by the gain of compactness upon interaction with the rna and a simultaneous loosening of the rna structure (see figure 3 (b)). as a consequence, the rna gains another chance to fold correctly. (4) the intrinsically unstructured protein might provide a folding platform for the rna as the chaperone holds the rna molecule in close proximity. in future the research focus on rna chaperones will lie on the understanding of the molecular mechanism and how intrinsically unstructured regions in proteins might play a role in function. interestingly, two very closely related ribosomal proteins l1 from archaea, that encompass 70% aminoacid identity, possess opposite activities: the mesophilic l1 protein displays strong rna chaperone activity whereas the thermophilic one inhibits ribozyme assays [42] . a detailed mutation study will likely shed light on the different activities and explain the rna chaperoning mechanism at least for a subgroup of rna chaperones. the big challenge, however, will be to identify in vivo targets of rna chaperones. rna chaperones are evaluated in assays for their broad specificity but in vivo they might be specialized to supervise folding of only a subset of rna molecules. the specificity might possibly be conferred by a different domain than the chaperoning activity. rna chaperones and the rna folding problem a dead-box protein functions as an atp-dependent rna chaperone in group i intron splicing escherichia coli proteins, including ribosomal protein s12, facilitate in vitro splicing of phage t4 introns by acting as rna chaperones escherichia coli protein stpa stimulates self-splicing by promoting rna assembly in vitro rna chaperone activity and rna-binding properties of the e. coli protein stpa spectroscopic observation of rna chaperone activities of hfq in post-transcriptional regulation by a small non-coding rna hfq, a new chaperoning role: binding to messenger rna determines access for small rna regulator geometric nomenclature and classification of rna base pairs rna tertiary interactions in the large ribosomal subunit: the a-minor motif two interconvertible forms of tryptophanyl srna in e. coli native and renatured transfer ribonucleic acid a ribosomal function is necessary for efficient splicing of the t4 phage thymidylate synthase intron in vivo alternative secondary structures in the 5 exon affect both forward and reverse self-splicing of the tetrahymena intervening sequence rna a tyrosyl-trna synthetase recognizes a conserved trna-like structural motif in the group i intron catalytic core domain structure and rna annealing activity of the escherichia coli regulatory protein stpa rna annealing activities in hela nuclei hiv-1 nucleocapsid protein as a nucleic acid chaperone: spectroscopic study of its helix-destabilizing properties, structural binding specificity, and annealing activity coupling rna annealing and strand displacement: a fret-based microplate reader assay for rna chaperone activity dissecting rna chaperone activity an rna chaperone activity of non-specific rna binding proteins in hammerhead ribozyme catalysis rna chaperone activity of large ribosomal subunit proteins from escherichia coli rna chaperone activity of protein components of human ro rnps assaying rna chaperone activity in vivo in bacteria using a ribozyme folding trap escherichia coli cspa-family rna chaperones are transcription antiterminators transcription antitermination by translation initiation factor if1 assay of transcription antitermination by proteins of the cspa family rna chaperones, rna annealers and rna helicases protein enhancement of hammerhead ribozyme catalysis identification and characterization of the rna chaperone activity of hepatitis delta antigen peptides the hepatitis c virus core protein is a potent nucleic acid chaperone that directs dimerization of the viral (+) strand rna in vitro rna chaperoning and intrinsic disorder in the core proteins of flaviviridae role of rna chaperones in virus replication coronavirus nucleocapsid protein is an rna chaperone coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription assaying rna chaperone activity in vivo using a novel rna folding trap assays for the rna chaperone activity of proteins rna chaperone stpa loosens interactions of the tertiary structure in the td group i intron in vivo the role of structural disorder in the function of rna and protein chaperones how common are extraribosomal functions of ribosomal proteins? extraribosomal functions of ribosomal proteins atomic structures at last: the ribosome in 2000 biochemistry research international ribosomal proteins: phylogenetic conservation and splicing inhibition janus chaperones: assistance of both rna-and protein-folding by ribosomal proteins cspa, the major cold-shock protein of escherichia coli, is an rna chaperone cold shock induces a major ribosomal-associated protein that unwinds double-stranded rna in escherichia coli nucleic acid melting by escherichia coli cspe cold shock domain proteins and glycine-rich rna-binding proteins from arabidopsis thaliana can promote the cold adaptation process in escherichia coli glycine-rich rna-binding proteins are functionally conserved in arabidopsis thaliana and oryza sativa during cold adaptation process complementation of cold shock proteins by translation initiation factor if1 in vivo rna chaperone activity of translation initiation factor if1 regulation of alternative 3 splice site selection by constitutive splicing factors minireview: polypyrimidine tract binding protein antagonizes exon definition cellular internal ribosome entry segments: structures, trans-acting factors and regulation of gene expression feline calicivirus replication: requirement for polypyrimidine tract-binding protien is temperaturedependent the apaf-1 internal ribosome entry segment attains the correct structural conformation for function via interactions with ptb and unr heterogeneous nuclear ribonucleoprotein k is a transcription factor identification of the src pyrimidine-binding protein (spy) as hnrnp k: implications in the regulation of src1a transcription specific binding of heterogeneous ribonucleoprotein particle protein k to the human c-myc promoter, in vitro heterogeneous nuclear ribonucleoprotein (hnrnp) k is a component of an intronic splicing enhancer complex that activates the splicing of the alternative exon 6a from chicken βtropomyosin pre-mrna mrna silencing in erythroid differentiation: hnrnp k and hnrnp e1 regulate 15-lipoxygenase translation from the 3 end the la protein factor fraction required for the synthesis of bacteriophage qβ-rna a small rna regulates the expression of genes involved in iron metabolism in escherichia coli regulatory roles for small rnas in bacteria hfq: a bacterial sm-like protein that mediates rna-rna interaction hfq is necessary for regulation by the untranslated rna dsra the sm-like hfq protein increases oxys rna interaction with target mrnas structure of pseudomonas aeruginosa hfq protein sm-like proteins in eubacteria: the crystal structure of the hfq protein from escherichia coli structures of the pleiotropic translational regulator hfq and an hfq-rna complex: a bacterial sm-like protein the prion protein has rna binding and chaperoning properties characteristic of nucleocapsid protein ncp7 of hiv-1 scavenger, transducer, rna chaperone? what ligands of the prion protein teach us about its function microarray identification of fmrp-associated brain mrnas and altered mrna translational profiles in fragile x syndrome a drosophila fragile x protein interacts with components of rnai and ribosomal proteins rna and micrornas in fragile x mental retardation rna cargoes associating with fmrp reveal deficits in cellular functioning in fmr1 null mice the fragile x mental retardation protein has nucleic acid chaperone properties disordered rna chaperone proteins: from functions to disease protons, osmolytes, and fitness of internal milieu for protein function intrinsically unstructured proteins: re-assessing the protein structure-function paradigm both referees of the paper are thanked for their valuable comments. doris chen is thanked for critically reading and chaperoning the paper. k. semrad is supported by a hertha firnberg fellowship (t261) from the austrian science foundation (fwf). key: cord-299007-5m6lk409 authors: paterson, r. russell m. title: cordyceps – a traditional chinese medicine and another fungal therapeutic biofactory? date: 2008-05-31 journal: phytochemistry doi: 10.1016/j.phytochem.2008.01.027 sha: doc_id: 299007 cord_uid: 5m6lk409 abstract traditional chinese medicines (tcm) are growing in popularity. however, are they effective? cordyceps is not studied as systematically for bioactivity as another tcm, ganoderma. cordyceps is fascinating per se, especially because of the pathogenic lifestyle on lepidopteron insects. the combination of the fungus and dead insect has been used as a tcm for centuries. however, the natural fungus has been harvested to the extent that it is an endangered species. the effectiveness has been attributed to the chinese philosophical concept of yin and yang and can this be compatible with scientific philosophy? a vast literature exists, some of which is scientific, although others are popular myth, and even hype. cordyceps sinensis is the most explored species followed by cordyceps militaris. however, taxonomic concepts were confused until a recent revision, with undefined material being used that cannot be verified. holomorphism is relevant and contamination might account for some of the activity. the role of the insect has been ignored. some of the analytical methodologies are poor. data on the “old” compound cordycepin are still being published: ergosterol and related compounds are reported despite being universal to fungi. there is too much work on crude extracts rather than pure compounds with water and methanol solvents being over-represented in this respect (although methanol is an effective solvent). excessive speculation exists as to the curative properties. however, there are some excellent pharmacological data and relating to apoptosis. for example, some preparations are active against cancers or diabetes which should be fully investigated. polysaccharides and secondary metabolites are of particular interest. the use of genuine anamorphic forms in bioreactors is encouraged. too much about cordyceps is unsubstantiated. this literature is written to sell so-called medicines to potentially vulnerable people with serious diseases. on the other hand, there is convincing scientific information that indicates significant pharmacological properties which are worth assessing (see table 1 ). the present large review undertakes this task and deals with papers that use the name cordyceps sometimes in its most general sense, especially when the revision of sung et al. (2007) is considered. there follows an extended introduction to the topic. the title relates to another in this journal concerning the fungal traditional chinese medicine (ftcm), ganoderma (paterson, 2006) . in that case, the fungus was indeed a biofactory in the sense that numerous compounds have been reported from the fungus. what is the situation with another ftcm, cordyceps? the immediate answer is that the state of the art is considerably less developed (see table 2 for a list of secondary metabolites). there is a general impression that this fungus is being used in a modern context, before the benefits, and even what is being used, have been determined scientifically. cordyceps is one of a growing number of ftcm being considered as cures for modtable 1 medically related purported effects of various cordyceps taxa (or preparations) as described by various authors ern human diseases. many commercial products are available in the market (e.g. didanosine from cordyceps militaris). these nutraceuticals are considered to relieve the ''stress for humans of living in technologically developed societies" by stimulating basic and secondary responses of the immune system (lakhanpal and rana, 2005) . the fungus represents a genus of perithecial ascomycetes (phylum ascomycota) classified in the clavicipitaceae, a monophyletic group included in the order hypocreales. the genus contains over 400 species and the anamorphs of most are unknown. paecilomyces is considered traditionally to host the anamorphs but this has been disputed. sung et al. (2007) should be consulted for an up-to-date revision (and see later). cordyceps are parasites of insects or fungi, often exhibiting a high degree of host specificity (fig. 1) . however, the cordyceps species associated with lepidopteran hosts do not represent a monophyletic group. there is even a high degree of genetic variation within cordyceps sinensis which creates difficulties in verifying samples. a taxonomic review of the fungus is now available (see later section) a similar review is required for ganoderma (e.g. see paterson, 2006 . larval infection via meiotic and/or mitotic spores/conidia and multiplication within the insect is from yeast-like budding. however, the fungus grows through the insect by hyphae. the accumulation of the biomass eventually kills the host (and/or a toxin(s) may be involved). it would be interesting to determine the biochemical parameters that cause these changes but this is not reported in the literature. the fungus ruptures the host body following over wintering and forms the sexual perithecial stroma that are connected to the dead larva below ground which grow upward to emerge above the soil surface (fig. 1) . the complete insect/fungus combination is used traditionally, but not exclusively, for medicinal purposes. the present reviewer has seen no reports of the insect per se being given as a treatment. c. sinensis (berk.) sacc., is one of the most famous traditional chinese medicines (tcm) and health foods. the fungus parasitises larvae of moths (lepidoptera), especially hepialus armoricanus (and thitarodes), and converts each larva into a sclerotium, from which the stroma and fruitbody grows. the complex (including the larva body) has been used as a health food and traditional medicine to ''invigorate the lung and nourish the kidney" in china for hundreds of years, and at least from the 17th century (dong and yao, 2007; kuo et al., 1994) . although what these preparations actually represented is impossible to determine given the difficulties in taxonomy of even modern times (burnett, 2003; korf, 2005 ; and see sung et al., 2007) . understandably, conservation and sustainable harvest are important issues. there is need for (a) research on biological screening, (b) a better understanding of the status in natural habitats, and (c) artificial cultivation of the fungus. cordyceps and products are available in ''western" countries as over-the-counter medicine/tonics which advertise them as chinese herbs with anti-aging, ''pro-sexual", anti-cancer and immune boosting effects, although with poor supporting scientific evidence. the believe is that c. sinensis (cs) has various beneficial effects on humans, including those of a psychological nature. the ftcm, is also called dong chong xia cao in chinese (=winter worm summer grass) (li et al., 2006a) . primarily it is prescribed as a tonic for body strengthening after serious disease. more recently other treatments have been claimed such as for (a) respiratory, renal, liver, nervous system and cardiovascular diseases, and (b) tumours, aging, hyposexuality and hyperlipidemia (kuo et al., 2006; chen et al., 2006; wang and shiao, 2000) . it has been officially classified as a drug in the chinese pharmacopoeia since 1964. furthermore, the outbreak of the severe acute respiratory syndrome (sars) in china in 2003 has increased use considerably. this would have been an excellent opportunity to have determined how effective it was. however, this does not appear to have been undertaken. the market demand for cs is growing sharply in many countries (dong and yao, 2007) . they would surely be hailed as medical breakthroughs if the efficacy of any of these treatments were confirmed. nevertheless, the identities of active components have not been determined (in all cases) (li et al., 2006b) . research has shown that at least some of the traditional uses ''may" relate to pharmacological activities (zhu et al., 1998a,b) (if not pharmacological activities then what?). herbs have been used throughout history to enhance physical performance, but scientific scrutiny with controlled clinical trials has only recently been used to study such effects (bucci, 2000) . the authors mention that cordyceps remain untested which is surprising given the interest in the fungus. the fungus is endemic to the alpine habitats of the tibetan plateau above 3000 m in south-western china, and there has been large-scale harvesting of the wild material from nepal and india more recently. it is agreed generally table 2 example of the range of species and some of the low molecular-weight secondary metabolites from cordyceps secondary metabolites c. sinensis cyclic peptides, h1-a c. militaris cyclic peptides, cordycepin, 10-membered macrolides, cepharosporolides c, e and f, pyridine-2,6-dicarboxylic acid and 2carboxymethyl-4-(30-hydroxybutyl) furan, dipicolinic acid c. pseudomilitaris bioxanthracenes c. brunnearubra cordyformamide c. sinclairii (2s,3s,4r)-(e)-2-amino-3,4-dihydroxy-2hydroxymethyl-14-oxoeicos-6-enoic acid c. cicadae ergosterol peroxide c. nipponica cordypyridones a and b c. ophioglossoides ophiocordin, glycoprotein containing nacetylgalactosamine c. heteropoda cicadapeptins i and ii, myriocin the number of compounds is small compared to that for ganoderma (paterson, 2006) . to have been over-harvested. furthermore, the price of natural products of cs is over us$ 12,000 kg à1 (2006 prices) for only ''average quality" (how this is determined is not clear) in the market and increasing (sharma, 2004) . so one can understand the pressures on supply. the socioeconomic implications of the ftcm are highly significant to the regions where it is harvested. the fungus has officially been classified as an endangered species by cites management authority of china and china customers and this scarcity is of considerable concern to all. consequently, living strains have been isolated from natural cs and cultivated in large quantity by bioreactor technology which is a promising method to meet the needs of human consumption and to reduce the pressure on natural resources of the species (dong and yao, 2007) . in vitro culture of the fungus has been employed increasingly and yang et al. (2005) state, ''it is generally accepted that its cultivated cs fungi possess the same functions as cs natural ''herbs" (sic)". some other issues that require addressing are that natural c. militaris is not readily available and is costly. thus, a growing number of socalled cordyceps products that derive from mycelial cultures of the asexual forms of these fungi have become commercially available (hamburger, 2007) . mycelia cultivation has resulted in establishing a number of cultures derived from the holomorphic cs. these are referred to by the anamorphic names paecilomyces hepiali and cephalosporium sinensis, although the anamorph of cs appears to be hirsutella sinensis (and see later). however, the situation is confused with some taxonomist using outdated names. to paraphrase buenz et al. (2004 buenz et al. ( , 2005 : while these strains undoubtedly support ecologically sustainable use of cs, the actual similarities between the wild fungus and the cultures are not clear. the consumption of complimentary medicine has increased dramatically, with over 42% of people in the united states of america reported as ''users". sales were us$ 3.3 billion in 1999 (buenz et al., 2005) . an important factor was the passage of the dietary supplement health and education act in 1994 in the usa which opened the market for tcm (cooper and chang, 2001) . one can appreciate how journals advocating these have increased concomitantly. why have they not been developed by big pharmaceutical companies and made available to the public in pure compound form? no doubt there could be many reasons why this has not happened (e.g. not enough profit, ''sticky" intellectual property rights issues (see paterson, 2008) , difficulty in mass production or synthesis, etc.)apart from the possibility that they simply may not be effective. buenz et al. mention that ''one of the most interesting supplements is the not yet well-characterized c. sinensis (berk.) sacc.". cs has attracted much research interest for anti-oxidant activity and there is considerable evidence of this from the fungus as a treatment of a wide range of diseases. however, unauthenticated material has been used in some cases. for example, a polysaccharide was isolated which can protect pc12 cells against hydrogen peroxide-induced neuronal cell toxicity, but the cordyceps mycelia used was from the wan fong pharmaceutical factory (zhejiang, china) and derived from ce. sinensis chen sp. nov. this is a nomenclaturally illegitimate fungal name, which raised doubts as to its relationship to cs. in fact, it was later proved to be a different species (dong and yao, 2007) and this is a specific example of a general problem in the field. an example of another problem is cordymax cs-4, a commercial mycelial fermentation product that lowered fasting plasma levels of glucose and insulin, improved oral glucose tolerance and increased the glucose-insulin index, which measures insulin sensitivity, in rats (zhao et al., 2002) . however, the following statement is given on the web site of the product, ''these (health-related) statements have not been evaluated by the food and drug administration. this product is not intended to diagnose, treat, cure or prevent any disease". extracts from artificially cultivated fruit-bodies of cs from the xinhui xinhan artificial cordyceps factory (guangdong, china) could scavenge ros by inhibiting malondialdehyde formation by the peroxynitrite generator sin-1. these results have been since referred to uncritically (e.g. buenz et al., 2004 buenz et al., , 2005 li et al., 2003) . however, the fungal material may have been unauthentic, because reports exist that cultivation of fruit-bodies of this fungus was not repeatable and that the manufacturer is actually selling c. militaris. furthermore, li et al. (2001) compared the anti-oxidant activities of natural cs and cultured cordyceps mycelia from different sources and were able to show the similar effects of the cultured mycelia to the natural products. however, the cultured material used was derived from a wide range of strains, and not from a valid cs. some of these were products from the chinese medicine factory of jiangxi and hebei boding pharmaceutical factory. in addition, the ftcm are boiled in water or soaked in alcohol to drink for medications/health foods. obviously, the various solvents and temperatures may have resulted in different compounds (dong and yao, 2007) . nevertheless, guo et al. (2007) state that the ongoing exploration of cs has shown that the species can produce ''many" bioactive compounds, and the medicinal benefits of cs have been demonstrated extensively. in addition, there is (unbelievably for scientific journals) the use of words such as ''yin" and ''yang" as a basis for undertaking scientific research on material activities. canney (2006) discusses kidney ''yang" and one needs to ask what this is from a scientific perspective. why include this word when it has no scientific currency? the fungus is also referred to as a herb and indeed the title of the piece asks, ''c. sinensis animal, vegetable or both?", whereas it is neither. various bioactive constituents from cordyceps species have been reported. these include cordycepin and other anti-bacterial and anti-tumour adenosine derivatives, ophicordin, an anti-fungal agent, and l-tryptophan. recent reports have indicated that cs contains polysaccharides exhibiting anti-oxidant activity and nucleosides that inhibit platelet aggregation (wu et al., 2005) . the bioactive compounds involved in the activities claimed include polysaccharides, modified nucleosides, and cyclosporin-like metabolites which are produced by this fungus and related species. the beneficial effects on (a) renal and hepatic function and (b) immunomodulation-related anti-tumour activities are most promising and deserve great attention. an increasing number of studies have used cultured mycelia in investigations. more mechanism-based, disease-oriented pharmacological studies are required to ensure clinical efficacy for particular diseases. however, pang et al. (2002) state revealingly that studies have demonstrated repeatedly that many natural products marketed as nutraceuticals or health food do not deliver the health benefit as claimed and are inconsistent from batch to batch. in the popular mind, cs first gained worldwide attention when it was revealed that several chinese runners who broke world records in 1993 had included this fungus in their diet as part of their training program. although scientist need to desist from quoting such reports as they are unsubstantiated and far-fetched. purported and unsubstantiated effects of the fungus include use as an aphrodisiac, analgesic, immune modulator, and free radical scavenger. a review of the literature uncovers the predictable collection of general papers concerning medical mushrooms some with a distinctly ''alternative" flavour. i have no concerns about being alternative but are they scientific? these overviews often are written in breathless, overblown and unscientific terms; others are well balanced. dasilva (2005) talks about ''novel mushroom-based healthcare products and therapeutics licensed for medical use can contribute to the good health status and feeling of the povertystricken strata of urban societies and populations. indeed, mushroom cultural practices and medicines are being widely accepted as the integral skeins in the fabric of the human society of tomorrow". a valid response to this situation of a wide range in the quality of papers would be to only review those papers that have a high impact factor (or any impact factor). temporarily putting the debate about the value of impact factors to one side, the reader may find it beneficial to concentrate only on those journals reviewed herein that do indeed have such ratings. of course, the disadvantage is that valuable information may be missed. it may be worth mentioning that, asian nations on the world stage have realised that biomedicine offers a unique chance to develop new industries and markets. universities in singapore, korea, hong kong and china are appearing concomitantly in world tables for the best and citations data suggest they are producing well-regarded papers (ince, 2007) . the following review concentrates on the biological activity of various preparations of cordyceps spp. (the name as used in the essentially non-taxonomic papers) because of the quantity of data and importance of medicinal claims, at the expense of taxonomic and quality control issues. the various pure compounds, extracts, whole fungus and other preparations as they relate to pharmacological activities are discussed next, as these divisions are considered to be most relevant to a biochemical/phytochemical perspectives of the topic. data from pure compounds are the most revealing in terms of determining effects of the fungus/insect. it is noted that these reports are scarce. some compounds from cordyceps (as defined here) are not particularly unusual. ng and wang (2005) review the chemical constituents and pharmacological properties. the chemical constituents include (a) cordycepin (3 0 -deoxyadenosine) and its derivatives, (b) ergosterol, (c) polysaccharides, (d) a glycoprotein and (e) peptides containing a-aminoisobutyric acid. the activities ascribed to the fungus are anti-tumour, antimetastatic, immunomodulatory, anti-oxidant, anti-inflammatory, insecticidal, anti-microbial, hypolipidaemic, hypoglycaemic, anti-aging, neuroprotective and renoprotective effects: so a vast a range of properties from a narrow spread of compounds. polysaccharides account for the anti-inflammatory, antioxidant, anti-tumour, anti-metastatic, immunomodulatory, hypoglycaemic, steroidogenic and hypolipidaemic effects. cordycepin contributes to the anti-tumour, insecticidal and anti-bacterial activity. ergosterol (a universal fungal compound) exhibits anti-tumour and immunomodulatory activity. finally, a dnase has been characterized. these are not particularly novel compounds and one wonders why there are so many reports of the effects of crude extracts rather than much more work on the effects of novel pure compounds. cordycepin (fig. 2) , 3-deoxyadenosine, is a derivative of the nucleoside adenosine differing from the latter by the absence of oxygen in the 3 0 position of its ribose entity. as such is may be quite common. initially, it was extracted from cordyceps; however, it is now produced synthetically. some enzymes do not discriminate between adenosine and so it can participate in certain reactions. for example, it can be incorporated into rna molecules causing premature termination of its synthesis. it is classified as an anticancer compound. cordycepin inhibited the growth of clostridium paraputrificum and clostridium perfringens, but had no effect on bifidobacterium spp. and lactobacillus spp. (ahn et al., 2000) . in addition, larvicidal activity against plutella xylostella after 2-4 days of treatment was observed . it is interesting that cordycepin, a compound originally isolated from c. militaris as much as 60 years ago (cunningham et al., 1950) , is known to exert cytotoxic effects through nucleic acid methylation (kredich, 1980) , with possible implications for the pcr of these fungi (see paterson, , 2008 . if it is a truly useful compound it is surprising that it is not a well-known pharmaceutical by now. the presence of cordycepin in cs has been difficult to confirm, although it has been confirmed by nmr (chen and chu, 1996) . however, other groups have not been able to detect this compound . it is clearly important to confirm the presence of the compound in cs in terms of determining the active components of the fungus and ultimately for chemotaxonomic purposes. cho et al. (2007) state that cordycepin is isolated from c. militaris and is (claimed to be) an ingredient in tcm which is prescribed for various diseases, such as cancer and chronic inflammation (again note how vague this verbatim statement is). in this study, the novel effect of cordycepin inhibiting collagen-induced platelet aggregation was reported. the data suggests that the inhibitory effect of cordycepin might be associated with the down-regulation of [ca 2+ ]i and the elevation of camp/cgmp production. this result has obvious significance for prevention of thrombus formation. finally, cordycepin inhibited the growth of b16 melanoma cells inoculated subcutaneously into right murine footpads (yoshikawa et al., 2004) . cordyheptapeptide a (fig. 3) , a novel cycloheptapeptide, was isolated from a strain of cordyceps together with four known bioxanthracenes. there were only two previous reports on the isolation of cyclic peptides from this genus and these were from c. militaris and cs. the metabolite exhibited anti-malarial activity against plasmodium falciparum and cytotoxicity to vero cell lines. also, the anti-malarial and cytotoxic activities of the bioxanthracenes were reported (rukachaisirikul et al., 2006) . in an extensive and impressive report, compounds designated as es-242s, were isolated from a verticillium strain and identified as bioxanthracenes (isaka et al., 2007a) . these compounds were known to exhibit potent activity as n-methyl-d-aspartate (nmda) receptor antagonists. in addition, five novel es-242 analogues were isolated with nine known compounds from a cordyceps strain. a closely related strain provided cordyheptapeptide a, cordyheptapeptide b, and known es-242s. the structures of the novel bioxanthracenes were 6 0 -o-desmethyl analogues of the compounds described. furthermore, cordyheptapeptide b has an n-methyl-l-phenylalanine residue in place of the n-methyl-l-tyrosine. the isolation, structure elucidation, and anti-malarial activity of es-242s and their analogues from the insect pathogenic fungus cordyceps pseudomilitaris (from a lepidoptera larva) were reported previously. cycloheptapeptide, cordyheptapeptide a, and some known es-242s were isolated from a cordyceps strain from an elaterid larva. in a continuing search for bioactive compounds from insect pathogenic fungi it was noticed that culture extracts of six cordyceps strains, collected in the same location (from coleoptera larvae, at doi innthanon national park, chiang mai province, thailand), showed similar 1h nmr spectra. this suggested the presence of bioxanthracenes (es-242s) and cordyheptapeptide a as major constituents. two of these strains were subjected to mass fermentation (15 l) and chemical investigation. as a result, five new es-242 analogues and nine known compounds and cordyheptapeptide a were isolated from an undefined strain. cordyheptapeptide b, was isolated, together with other known compounds. some of these were tested for activity against p. falciparum and cytotoxicity to kb cells (oral human epidermoid carcinoma), bc cells (human breast cancer), nci-h187 cells (human small cell lung cancer), and noncancerous vero cells (african green monkey kidney fibroblasts). cordyheptapeptide a exhibited antimalarial activity, while cordyheptapeptide b was inactive and both cyclic peptides showed moderate cytotoxicity. furthermore, cordyformamide is a plausible biogenetic precursor of xanthocillin y, and was isolated from a culture broth of cordyceps brunnearubra bcc 1395. cordyformamide was found to exhibit activity against p. falciparum, whereas it showed weak or no cytotoxicity (isaka et al., 2007b) . production of the nonribosomal peptides cicapeptins i and ii (fig. 4) were reported by krasnoff et al. (2005) which was the first report from fungi of consecutive hyp or pro residues in a nonribosomal linear peptide. the compounds exhibited anti-bacterial and anti-fungal activity. a novel immunosuppressant was isolated from the culture broth of isaria sinclairii, the anamorph of c. sinclairii, and characterized as (2s,3s,4r)-(e)-2-amino-3,4-dihydroxy-2-hydroxymethyl-14-oxoeicos-6-enoic acid, which was identical to anti-fungal substances, myriocin and thermozymocidin (fujita et al., 1990) . the suppressive activity was found to be equal to, or higher than cyclosporin a which is used clinically. the activities of the 10 derivatives were also examined, indicating the following relationships between structure and activity -the: (a) lactone formation between the carboxy group at c-1 and the hydroxy group at c-4, and the reduction of the carbonyl group at c-14 to the hydroxy group do not affect the suppressive activity; (b) hydrogenation of the double bond at c-6 resulted in activity; and (c) acetylation of the amino group and the thioketalization on the carbonyl group at c-14 ''drastically" reduced the suppressive activity. also, the compound suppressed the production of anti-bodies to sheep red blood cells and induction of cytotoxic lymphocyte t cells more strongly than cyclosporin a. obviously, this is an important lead compound and hence one of the more satisfactory papers. chen et al. (1999) isolated a pure compound (h1-a) from cs and investigated whether autoimmune disease progression in mice was affected by administration of the metabolite. the authors are vague as to what the compound is and state that, ''it is a kind of ergosterol and looks like testosterone". the authors also provide a chemical structure which confirms that it is a common sterol and a systematic name could have been provided. their results demonstrated that mice treated daily exhibited a progressive reduction in anti-ds-dna production. in clinical presentation, the treated group had a reduction in lymphadenopathy, a delayed progression of proteinuria, and an improvement in kidney function. histological analysis of kidney tissue indicated that h1-a inhibited mesangial proliferation that was evident in lupus nephritis. however, there was no change in immune complex deposition. h1-a ''may be" useful for treating systemic lupus erythematosus in human patients. however, more work is required. h1-a was claimed to be effective in the treatment of autoimmune disorders (yang et al., 2003) . results demonstrated inhibition of cell proliferation and promotion of apoptosis of activated human mesangial cells in vitro: the activities were not a result of cytotoxicity. in addition, h1-a inhibited tyrosine phosphorylation of human mesangial proteins. these findings suggest that h1-a modulated some (unspecific) subcellular signal-transduction pathways and changed the balance between proliferation and apop-tosis of mesangial cells in vitro and in vivo. the conclusions were that h1-a may be effective in the management of autoimmune disorders, and the modulation of the signal-transduction proteins may represent a target for future pharmacologic interventions. more correctly, they probably do represent a target, and such vague statements should be avoided. in an older report, hi-a alleviated immunoglobulin a nephropathy (berger's disease) with histological and clinical improvement . hi-a inhibited the proliferation of human mesangial cells and promoted apoptosis by suppressing tyrosine phosphorylation of bcl-2 and bcl-xl (yang et al., 2003) and reduced antids-dna production and lymphadenopathy, delayed progression of proteinuria, improved kidney function and inhibited mesangial proliferation . moving on to more interesting compounds, cordypyridones a and b were detected from the uncommon species, c. nipponica. these are atropisomers, and demonstrated potent anti-malarial activity in vitro (isaka et al., 2001b) . shin et al. (2001) state that, ''in an effort to evaluate the pharmacological effects, including the anti-aging effect" of the fruiting bodies of the cultivated paecilomyces japonica fungus, ''a new type" of cordyceps sp. was investigated. two pure compounds were isolated as active principles from low molecular-weight fractions, and a protein-bound polysaccharide that showed a marked increase in the liver enzyme activities, and a significant inhibition of lipid peroxidation was found. boros et al. (1994) reported that ophiocordin, an anti-fungal antibiotic from cordyceps ophioglossoides (kneifel et al., 1977) and balanol from verticillium balanoides are structurally identical. this may indicate a particularly close taxonomic relationship between the two taxa. the structure of ophiocordin was falsely assigned and balanol was the compound of interest. balanol was under development as an anti-cancer agent as it established to be a selective inhibitor of protein kinase c (see paterson, 2008) . it is more common for pure compounds to be tested in the fields of anti-bacterial, anti-fungal, anti-malarial and insecticidal activity which is to be recommended more generally. ophiocordin is an anti-fungal antibiotic isolated from submerged cultures of c. ophioglossoides. however, it is devoid of anti-bacterial activity (kneifel et al., 1977) . bioxanthracenes (see also previously) were isolated from c. pseudomilitaris (isaka et al., 2001a; jaturapat et al., 2001) and appear to be anti-malarial. ten-membered macrolides, cepharosporolides c, e and f, cordycepin, pyridine-2,6-dicarboxylic acid and 2-carboxymethyl-4-(30hydroxybutyl) furan were reported from c. militaris by rukachaisirikul et al. (2004) . however, only cordycepin was anti-malarial. krasnoff et al. (2005) reported cicadapeptins i and ii (nonribosomal peptides containing aminoisobutyric acid), which were anti-bacterial and antifungal, and myriocin (anti-fungal) from c. heteropoda isolated from an australian cicada. finally, a glycoprotein containing n-acetylgalactosamine was isolated from c. ophioglossoides but activity data are not available (kawaguchi et al., 1986 ). an inhibitor of the prophenoloxidase activation was isolated from a culture filtrate of c. militaris and identified as dipicolinic acid (dpa). the production of dpa in a range of clavicipitaceae fungi was examined. entomogenous fungi that produce dpa were integrated into one group by a phylogenetic analysis based on 18s rdna. interestingly, it was suggested that the group acquired an ability to produce dpa during its evolution from plant pathogenic fungi to entomogenous fungi (watanabe et al., 2006) . in a useful comparison of crude extracts and pure compound, the anti-diabetic effect of various fractions of c. militaris, ccca (crude cordycepin containing adenosine), cmess (ethanol soluble supernatant), and cordycepin were evaluated in diabetic mice (yun et al., 2003) . cmess showed a potent inhibitory activity of 34.7% in starchloaded mice: cmess reduced blood glucose level by 35.5%. however, ccca, and cordycepin showed no difference. after 7 days administrations of these drugs, cmess, and cordycepin dramatically reduced blood glucose level. ccca with a high concentration of cordycepin did not reduce blood glucose level. proliferation of t-lymphocyte was significantly decreased; while no production was increased more than two-fold in the cordycepin-administered group. the proliferation of macrophages and no production were significantly decreased in the cmess administered group. cmess and cordycepin may be (a) useful tools in the control of blood glucose level in diabetes and (b) promising new drugs as an anti-hyperglycemic agent without the defects of lowered immune responses and other side effects, the authors suggest. furthermore, cordycepin, 3 0 -amino-3 0 -deoxyadenosine, homocitrullyl aminoadenosine, adenine, cordycepic acid and d-mannitol have been reported from cordyceps spp. (cunningham et al., 1950; chatterjee et al., 1957; kredich and guarino, 1961; guarino and kredich, 1963; kaczka et al., 1964; liu et al., 1989) . ergosterol peroxide isolated from c. cicadae inhibited phytohaemagglutinin-induced t cell proliferation, and arrested the progression of activated t cells from g1 to s phase of the cell cycle. early gene transcripts, in particular those of cyclin e, interferon, and interleukins were suppressed (kuo et al., 2003) . the glycosylated form of ergosterol peroxide from cs was more potent than the aglycone in inhibiting proliferation of tumour cells (bok et al., 1999) . however, ergosterol peroxide is widespread in fungi and cordyceps does not offer any particular advantage in its preparation. it is worth noting that eight different cordyceps species (cs. c. militaris, c. cicadae, c. ophioglossoides, c. heteropoda, c. pseudomilitaris, c. nipponica, c. sinclairii) are listed in the above paragraph indicating the extent of the possible diversity involved in the biology and activity (tables 1 and 2) . although whether they are distinct species is open to question. water-soluble crude polysaccharides were obtained from the fruiting bodies of cultured c. militaris by hotwater extraction followed by ethanol precipitation. the polysaccharides were successively purified by chromatography giving three polysaccharide fractions. in the in vitro anti-oxidant assay, p70-1 was found to possess hydroxyl radical scavenging activity. the polysaccharide is a heteropolysaccharide and is occasionally branched. the fundamental information obtained from this work is beneficial to the interpretation in the relationship of polysaccharide structure and its biological functions. this provides the ''experimental evidence and scientific explanation for the folkloric uses of c. militaris as a substitute for cs" (yu et al., 2007a) . the effect of an exopolysaccharide fraction (epsf) from anamorphic strains of cs on the immunocyte activity of tumour-bearing mice was investigated. epsf significantly inhibited the h22 tumour growth, and elevated the activity of immunocytes. it enhanced the phagocytosis capacity of peritoneal macrophages and proliferation ability of spleen lymphocytes. epsf promoted (a) tnf-a expression of macrophages, (b) the cytotoxicity of spleen lymphocytes, and (c) tnf-a and ifn-c mrna expression of splenic lymphocytes . cs possesses anti-tumour, anti-oxidation and stimulation of the immune system activities . however, the identity of active component(s) has not been determined . towards this end, a polysaccharide was isolated from cultured cordyceps mycelia which had strong anti-oxidation activity, and which contained glucose, mannose and galactose. the pre-treatment of the isolated polysaccharide on cultured rat pheochromocytoma cells demonstrated strong protective effect against hydrogen peroxide (h 2 o 2 )-induced insult. treatment prior to h 2 o 2 exposure significantly elevated the survival of pc12 cells in culture. this was the first report that identified a polysaccharide from cordyceps, which protected against the free radical-induced neuronal cell toxicity. a water-soluble polysaccharide fraction, a poorly water-soluble polysaccharide, and a protein fraction stimulated steroidogenesis (huang et al., 2001b) . interestingly, galactomannans isolated from the insect portion of c. cicadae demonstrate potent hypoglycaemic activity in mice (kiho et al., 1990) . in an investigation into a polysaccharide from cs mycelium hypocholesterolaemic and hypotriglyceridaemic activity in mice was exhibited (kiho et al., 1996) . chen et al. (1997) studied a polysaccharide fraction from cs as to its effect on the proliferation and differentiation of human leukaemia cells using an in vitro culture system. the conditioned medium had an activity that significantly inhibited proliferation. differentiated cells also possessed phagocytosis functions and supported superoxide production. antibody neutralization studies further revealed that the tumouricidal and differentiating effects of the compounds were mainly derived from the elevated cytokine concentrations. finally, galactosaminoglycan from c. ophioglossoides reacted with sera from patients with certain collagen diseases and its use as an index of serological activity is thus of diagnostic value (ikeda et al., 1993) . an aqueous extracted polysaccharide from cultured c. militaris demonstrated general anti-inflammatory activity (yu et al., 2004a) as did ethanolic extracts of cultured fruiting bodies and mycelia of c. militaris applied topically in the croton oil-induced ear oedema test in mice. the fact that in vitro fruiting bodies were employed rather than in vivo is interesting as most papers report using fruiting bodies in vivo and/or in vitro biomass. however, the paper is flawed as the details of the cultivation of the fruiting bodies were not provided. antioxidant activity in the xanthine oxidase, haemolysis and lipid peroxidation assay systems was demonstrated by li et al. (2001a) from water extracts, and a polysaccharide fraction, of cultured cs mycelia. interestingly, the fruiting body and the caterpillar parts of cs are claimed to be similar in chemical composition and hence anti-oxidant activity, because the fungus had presumable replaced the insect constituents with fungal . it would be interesting to determine (a) how this occurs in terms of insect substrate utilisation and optimisation of yields of bioactive fungal components, (b) when the preparation is at the correct stage for use as a medicinal treatment, and (c) if these data could be extrapolated to in vitro culture. an ambiguous statement is made by shin et al. (2001) , ''cordyceps is negative for its many biological activities and a tonic for restoring vital functions in traditional chinese medicine". it proceeds to state that p. japonica is a new type of cordyceps species, which is incomprehensible. it also mentions that p. japonica is (or produces) mushrooms. paecilomyces is considered to be an anamorph of cordyceps and so this appears to be incorrect and, what appear to be mushrooms, may be synnemata (compacted conidiophores). however, it is unwise to speculate what the material actually is as descriptions are vague. a protein-bound polysaccharide that inhibited lipid peroxidation and increased the activity of anti-oxidant was described from the fungus. finally, phaeochromocytoma cells were protected against h 2 o 2 -induced injury by a 210-kda polysaccharide from cs mycelium . yamada et al. (1984) reported that a water-insoluble extracellular glucan isolated from the culture filtrate of c. ophioglossoides suppressed potently the growth of sarcoma 180 solid-type tumours. remarkably, a protein-bound polysaccharide fraction from c. ophioglossoides extended the life of mice bearing ehrlich carcinoma or a syngeneic tumour . also, ohmori et al. (1989a,b) isolated a galactosaminoglycan that inhibited the proliferation of sarcoma 180 cells and the growth of a syngeneic solid tumour in vivo: it exhibited cytotoxicity against cancer cells in vitro. chen et al. (1997) observed that medium from blood mononuclear cells stimulated with the polysaccharide fraction from cs inhibited the proliferation of human leukaemic cells, and induced approximately 50% to differentiate into mature monocytes/macrophages expressing non-specific esterase activity and certain surface antigens. the anti-proliferation and differentiating effects were demonstrated to be caused by an elevated production of cytokines, i.e. a tumour necrosis factor and an interferon. the exopolysaccharide fraction of cs inhibited metastasis of melanoma cells and down-regulated concomitantly the levels of bcl-2 protein into the lungs and the liver . the exopolysaccharide fraction of cultivated cordyceps stimulated peritoneal macrophages to take up neutral red and splenic lymphocytes to proliferate . crude and neutral polysaccharides of cs exerted hypoglycaemic activity in normal mice. however, the polysaccharide did not affect the circulating insulin level in normal mice (kiho et al., 1993) . the compound lowered the plasma glucose level in diabetic mice (kiho et al., 1996) . another unspecific polysaccharide from a hot-water extract of mycelia also lowered the plasma glucose level in normal, adrenaline-induced hyperglycaemic and diabetic mice (kiho et al., 1999) . some cordyceps-like strains have been isolated from the fruiting bodies of wild cs that have been reported to show the same properties as the natural product. however, care in interpretation is required as these could conceivably be contaminants (see later). an exopolysaccharide fraction was prepared from cultivated cs . the results showed that it enhanced significantly the neutral red uptake capacity of peritoneal macrophages and spleen lymphocyte proliferation in melanoma-bearing mice. the metastasis of b16 melanoma cells to lungs and livers was significantly inhibited. moreover, the levels of bcl-2 in the lungs and livers were decreased. the results suggest that the polysaccharide has an immunomodulatory function and anti-tumour activity. however, yang et al. (2005) state that although certain polysaccharides from cs are bioactive, the anti-tumour effect has not been confirmed. the authors investigated the effects of the exopolysaccharide fraction of cultivated cs fungus on c-myc, c-fos, and vascular endothelial growth factor (vegf) expression of tumour-bearing mice. the expression in the lungs and livers of treated mice were found to be significantly lower than those of untreated mice. the authors suggest that the fraction had inhibited tumour growth in the lungs and livers of mice, and that it is an adjuvant in cancer therapy. in addition, yu et al. (2004a,b) isolated four polysaccharides from c. militaris, cps-1 was shown to possess a significant anti-inflammatory activity and suppressed the humoral immunity in mice but had no significant effects on cellular immunity and non-specific immunity. in a previous study using anti-oxidant activity-guided fractionation csp-1 from cultured cordyceps, mycelium was isolated. the hypoglycemic effect of csp-1 on mice and rats was demonstrated. csp-1 increased circulating insulin level in diabetic animals, which suggests that the compound(s) may stimulate pancreatic release of insulin and/ or reduce insulin metabolism. chen et al. (2006) undertook further work on the biological activity of the isolate: the polysaccharide from fungus and its anti-oxidant activity on h22-tumour bearing mice was investigated. the h22 tumour growth was inhibited and sod activity of liver, brain and serum and gsh-px activity of liver and brain in tumour-bearing mice were enhanced. in general, beneficial effects were observed in the liver and brain of tumourbearing mice. finally, four exopolysaccharides with different molecular masses ranging from 50 to 2260 kda were reported from c. militaris by kim et al. (2003b,c) as part of yield optimisation studies. an extracellular polysaccharide extracted from the mycelia of cs with hot-water indicated that this d-glucan consisted of a backbone composed of (1 ? 3)-b-d-glucosyl residues and carried a single (1 ? 4)-b-linked d-glucosyl residue: sugar residues were linked with b-glycosidic bonds (wu et al., 2005) . a lectin from c. militaris exhibited hemagglutination activity in mouse and rat erythrocytes, but not in human abo erythrocytes (jung et al., 2007) . however, the n-terminal amino acid sequence differed greatly from those of other lectins. it exhibited mitogenic activity against mouse splenocytes. the following section concerns solvent extraction of the fungi. in effect, this is often how the preparations will be consumed as a tcm. the significance of tests on extracts is much reduced compared to those of pure compounds. there is a great deal of data. in general, this type of work needs to be deemphasised in favour of that of pure compounds. reports on the metal chelating and reducing power from cs are not available in the scientific literature. therefore, there is a demand to obtain an overall measure of the anti-oxidant activity of extracts using reliable fungal material because of increasing interest in the relationship between anti-oxidants and diseases. the anti-oxidant activities from natural and cultured mycelia of cs were investigated in vitro. optimal effects were demonstrated on the inhibition of linoleic peroxidation. the results suggested that the cultured and natural mycelia have direct and potent activities and that the cultured mycelia could be used for the anti-oxidant activity which would tend to reduce the pressures on the natural fungus, which is, after all, an endangered species (dong and yao, 2007) . the anti-oxidant efficiency of c. militantis extract (cme) and cs extract (cse) in protecting lipid, protein, and lowdensity lipoprotein (ldl) against oxidative damage was reported (hui et al., 2006) . however, this study provoked a strong response from hamburger (2007) which was subsequently rebutted by one of the original authors (duh, 2007) . this is something of an unexpected bonus to a reviewer of the literature such as myself as another opinion is obtained. the questions are, what biological material is being worked with, and can other scientists obtain it to repeat the experiment? the current author has encountered this before (paterson, 2005) where commercial interests are involved. the hamburger response can be applied in a general sense to some of the other work cited in the present review. cme and cse showed weakly inhibitory effect on liposome oxidation. the inhibitory effect of cme on protein oxidation was inferior to that of cse. cme and cse showed inhibition of ldl oxidation. the contents of the bioactive ingredients cordycepin and adenosine in cme were higher than those of cse; however, cordycepin and adenosine showed no significant anti-oxidant activity. in addition, a polysaccharide present in cme and cse displayed anti-oxidant activity. the authors concluded that the protective effects of cme and cse against oxidative damage of biomolecules are a result of their free radical scavenging abilities. however, the experimental data and some of the conclusions need a critical comment (hamburger, 2007) . the author criticised the report on the bases of poor taxonomy, biochemistry and extrapolation of data to imply possible cures of diseases. in particular, the authors' claims of a potential treatment for human disease on the basis of in vitro data are called into question. the rebuttal by duh simply confirms that appropriate information about the strains was not provided; the comments on the inadequate analytical procedures are largely accepted. and there is no doubt that the conclusions could have been rewritten to indicate that the results were preliminary. hamburger (personal communication, 2007) stated that the rebuttal was evasive, an assessment with which i agree. kuo et al. (2005) describe the effects of cs against group a streptococcus infection in mice. the preparation protected by decreasing bacterial growth ''and dissemination", thereby increasing mouse survival rate. il-12 and ifn-gamma expression and macrophage phagocytic activity also increased. kuo et al. (2007) claim to demonstrate that cs increased phagocytosis in human monocytic cells and abrogated inhibition of phagocytosis by causing cytokine production. these two reports are sound and in good journals. however, the fungus used was from a company called simpson biotech and very few or no details are supplied about how the material was collected, identified, maintained, and grown. of course, this is unsatisfactory. shahed et al. (2001) refer somewhat unusually to cs as a ''black blade" fungus. their results showed that cs improved renal function and reduced the expression of inflammatory and apoptotic genes in rats. the authors make the very conditional statement that, cs extract may play a potential therapeutic role in renal transplantation"; on the other hand it may not play an actual role. after all, precision in what is written in such important areas of medicinal research is crucial. the authenticity of the fungal material can be questioned: it was obtained from a company from the united states of america and there is little indication about the standards of collecting, purity, identities and maintenance of the materials. basically, what level of accreditation applies to such organizations? a c. militaris inhibited the growth of human umbilical vein endothelial cells (huvec) and ht 1080 cells. it down-regulated, in dose-and time-dependent manners, bfgf gene expression in huvec cells and mmp-9 gene expression in ht 1080 cells. the growth of melanoma cells in mice was suppressed. in addition, anti-angiogenic activity was manifested (yoo et al., 2004) . it is gratifying that adequate details of the fungal material are provided in this chinese journal, which acts as a model for others generally. chiou et al. (2000) observed a hypotensive effect of cs in anaesthetized rats and a vaso-relaxant effect in isolated aorta. the fungus counteracted arrhythmia in rats and increased the dosage of ouabain required to produce arrhythmia in guinea-pigs. in addition, the heart rate in anaesthetized rats and the contractility of isolated papillary muscle or atria in guinea-pigs were decreased (mei et al., 1989) . cultured fruiting bodies of cs prevented deposition of cholesterol in the aorta of atherosclerotic mice by inhibiting free radical-mediated ldl oxidation in an investigation into hypolipidaemic activity (yamaguchi et al., 2000a) . a hot-water extract of mycelia (a) lowered the total cholesterol concentration, (b) reduced the concentration of cholesterol carried by ldl and very-low-density lipoprotein, and (c) elevated the high density lipoprotein (hdl)-cholesterol concentration in the serum of mice fed a cholesterol enriched diet (koh et al., 2003a) . water extracts of cs: increased survival time of mice inoculated with carcinoma cells or syngeneic fibrosarcoma cells (yoshida et al., 1989) ; inhibited spontaneously liver metastasis of carcinoma cells and melanoma in syngeneic mice and results suggested that the activity was not attributable to cordycepin (nakamura et al., 1999a) ; prolonged the survival period of mice inoculated with b16 melanoma cells when coadministered with methotrexate (nakamura et al., 2003) and caused apoptosis of melanoma cells (nakamura et al., 1999b ). an orally administered cs was considered to be ''quite" safe based on body weight gain, and liver/kidney weights of mice (nakamura et al., 1999a,b) . the authors concluded the extract could inhibit aortic cholesterol deposition in atherosclerotic mice by scavenging free radicals in vivo. these extracts ''may have" beneficial effects on the process of atherogenesis and aging with few side effects (yamaguchi et al., 2000a) . this is good news but surely more evidence is required. towards these ends, tsai et al. (2001) also demonstrated the hydroxyl radical scavenging activity of cs. whereas reduced lipid peroxidation in rats was demonstrated by shen and chen (2001) . antioxidant activity in the xanthine oxidase, haemolysis and lipid peroxidation assay systems was reported from water extracts, and a polysaccharide fraction, of cultured cs mycelia (li et al., 2001a) . as mentioned previously, the fruiting body and the caterpillar parts of cs are claimed to be similar in chemical composition and hence anti-oxidant activity . it would be interesting to determine (a) how this occurs in terms of insect substrate utilisation by the fungus with a view to optimising yields of bioactive fungal components, (b) when the preparation is at the correct stage for use as a medicinal treatment, and (c) if these data could be extrapolated to in vitro culture. cho et al. (2003) and wang et al. (2005) also reported radical scavenging activity with wang et al. reporting activity against colorectal tumour cells. the influence of cs on the immunoactivity of macrophages was determined to probe the mechanism of its alleged tonic effect. the phagocytosis of macrophages were enhanced significantly (jia and lau, 1997) and ''maybe" the tonic effect of cs is accomplished by an enhancing effect on the immune system. some chemical fractions had insulin like and insulin release promoting activity and ''could be developed" as an anti-diabetic agent (young et al., 2001) . cordyceps possessed a strong anti-oxidation activity in all assays tested by li et al. (2001a) . the cultured cordyceps mycelia had equally strong anti-oxidation activity compared to in vivo cordyceps. further, the anti-oxidation activities were increased â10-30 in the partially purified polysaccharide fractions. in an intriguing report, liu et al. (2006) mentions that identification of an effective non-toxic biological radioactivity protector is a ''matter of some urgency". orally administered cs protected mice from bone marrow and intestinal injuries after total-body irradiation. the levels of free radical species within cells are suggested to be a likely mechanism for the purported effects. the biochemical mechanisms of anti-proliferative effects of c. militaris in human leukemia cells were investigated in a convincing study involving cancer treatment . it was found that they inhibited cell growth in a dose-dependent manner, which was associated with morphological change and apoptotic cell death such as formation of apoptotic bodies and dna fragmentation. furthermore, the treatment caused a dose-dependent inhibition of cyclooxygenase-2 (see paterson, 2008) and prostaglandin e2 accumulation. taken together, these results indicated that the anti-proliferative effects were associated with the induction of apoptotic cell death through regulation of several major growth regulatory gene products. the extracts ''may have therapeutic potential" in human leukemia treatment. in addition, corticosterone output by cultured rat adrenocortical cells was increased without increasing the intracellular camp level. the steroidogenic effect was abolished by the protein kinase c inhibitor calphostin c, indicating that its action may involve stimulation of protein kinase c (wang et al., 1998) . c. militaris reduced the fasting serum glucose level and enhances glucose utilisation in skeletal muscles in rats . the fungus demonstrated cytotoxic activities on the three kinds of human cancer cell lines, stomachic adenocarcinoma, colorectal adenocarcinoma, and hepatocellular carcinoma (lim et al., 2004) . cytotoxic activity-guided isolation and identification of active fractions afforded the well-known, cordycepin as an active component (see above). koh et al. (2003b) reported that cs mycelia prolonged swimming endurance capacity and produced an anti-fatigue action in mice. cs reduced the hepatic content of malondialdehyde and the serum concentrations of transaminases and alkaline phosphatase in rats with hepatic fibrosis. treatment with the extracellular biopolymers resulted in a reduction in hepatic hydroxyproline content and normalization of morphological characteristics of the liver, indicating an anti-fibrotic action (nan et al., 2001) . finally, crystals of the fungus stimulated proliferation of erythroid progenitor cells in mouse bone marrow . in a very interesting report, the insect-body part (of the tcm) inhibited proliferation of enhanced human mononuclear cells (hmnc). any differences between the fungus and insect components are well worth further investigation. the production of interleukin-2 and interferon was stimulated by the aqueous methanolic extracts and inhibited by the methanolic extracts (weng et al., 2002) . treatment of patients with condyloma acuminata brought about an increase in interleukin-2 and a decrease in interleukin-10, indicating a recovery in the balance of th1/th2 cytokines. recurrence was also diminished (gao et al., 2000) . interestingly, the ergosterol esters concentrations were much higher in the (dead) caterpillar than the fruiting bodies (yuan et al., 2007) although ergosterol was similar. although why these compounds are of particular interest is ''mystifying" as they are universal in fungi. in a surprising paper, the following may be an example of the mystical nature of some of the reports (see paterson, 2006) . it represents a bizarre rational for undertaking the work and it is surprising that it was published from a scientific perspective. the rational for the work is that cs is a popular chinese tonifying herb, and was/is revered for being, what is referred to as, 'yin-nourishing' and 'yanginvigorating' in chinese medicine (siu et al., 2004) . in order to establish the pharmacological basis for the 'yinnourishing' and 'yang-invigorating' action of cordyceps, the effects of wild and cultured cordyceps on concanavalin a stimulated splenocytes, an in vitro bioassay for 'yinnourishment', and myocardial atp generation capacity, an ex vivo bioassay for 'yang-invigoration', were investigated in mice. the results indicated that wild and cultured cordyceps enhanced the con a-stimulated splenocyte proliferation in vitro and myocardial mitochondrial atp generation ex vivo in mice, with no significant difference in potency of action between the two types of cordyceps. while the immunopotentiating effect was associated with the increase in interleukin-2 production, the stimulation of myocardial atp generation was paralleled by an enhancement in mitochondrial electron transport. when compared with typical 'yin' and 'yang' tonifying chinese herbs, cordyceps was found to possess both 'yin-nourishing' and 'yang-invigorating' activities, with a lower potency in both modes of action. it is impossible to take reports such as these seriously from a scientific standpoint and is given considerable space here to indicate a general problem which may have motivated some of the published reports reviewed herein. this is without attempting to detract from the philosophical aspects of the concepts of yin and yang in terms of two mutually correlated opposites in a general sense. a comprehensive definition of the corresponding philosophy of science is inappropriate: it is enough to state that it is vitally important for science that the information about the surrounding world and the objects of study be as accurate and as reliable as possible. to continue, cs fruiting bodies inhibited various tumour cell lines (kuo et al., 1994) : two fractions were particularly potent. growth inhibitors other than cordycepin and polysaccharides may have been involved; two fractions significantly inhibited (a) the blastogenesis response, (b) nk cell activity and (c) il-2 production (kuo et al., 1996) . neither fraction was cytotoxic, and immunosuppressive ingredients were found to be intracellular. fruiting bodies inhibited human mesangial cells (hmc) activation by il-1 plus il-6 and liver toxicity or mutagenicity were not observed. the fraction was purified to obtain purified compound h1-a (see above). the authors claim a novel treatment for human berger's disease ''in the future". a fraction (a) dose dependently suppressed bronchoalveolar lavage fluids (balf) cells proliferation, (b) reduced interleukin production in lps activated balf cell cultures and (c) affected interleukin mrnas in various significant manners (kuo et al., 2001) . the purported therapeutic activity may be related to modulation of cells functions in bronchial airways. furthermore, the molecular mechanism of cordyceps pruinosa pharmacological and biochemical actions of macrophages in inflammation has not been clearly elucidated . the authors suggest that an extract suppresses inflammation through suppression of nf-jb-dependent inflammatory gene expression, and hence may be beneficial for treatment of endotoxin shock or sepsis. shim et al. (2000) wrote that they attempted to develop ''a new type cordyceps". to obtain this, they investigated the effects of the fruiting bodies of the cultivated fungus of p. japonica grown on silkworm larvae on hyperglycemia in rats and mice and on immunological functions in mice. as mentioned previously, it is not at all clear whether paecilomyces (even) contains anamorphs of cordyceps. therefore, this report is a cause of some concern. immunostimulating activity and a significant anti-fatigue effect in mice were observed. kuo et al. (1994) reported unidentified substances which inhibited tumour cells, but which were not cordycepin or polysaccharides, in the methanolic extract of cs -this report requires further investigation. c. cicadae ascocarps enhanced hmnc proliferation (weng et al., 2002) . in contrast, the insect-body portion suppressed hmnc proliferation. this is a most interesting result in the current author's opinion because of the different effects of the two components. the action mechanisms of the fractions may involve the regulation of interleukin and interferon production in hmnc. overall, the results demonstrated that c. cicadae contained growth modulators for hmnc. unfortunately, the compounds responsible were not characterised. koh et al. (2002) demonstrated that a hot-water extract modulated interleukin-6 production by activation of macrophages and augmented the secretion of haematopoietic growth factors. whereas aqueous methanolic extracts of the ascocarp stimulated proliferation of phytohaemagglutinin-induced proliferation of hmnc. of course, such differences indicate that different compounds are involved or the same compounds are at different concentrations. c. ophioglossoides mycelia prevented cell death in neuronal cells and memory deficits in rats (jin et al., 2004) . two fractions from fruiting bodies inhibited (a) the blastogenesis response, (b) natural killer cell activity, (c) interleukin-2 production and (d) tumour necrosis factor production in phytohaemagglutinin-stimulated human mononuclear cells (kuo et al., 1996) . the levels of interferon, interleukin-1 and tumour necrosis factor produced by cultured rat kupffer cells were increased by the fungus (liu et al., 1996a) . proliferation of cells in balf was inhibited which also reduced tumour necrosis factor (kuo et al., 2001) . in an effort to evaluate the pharmacological effects, including the anti-aging, of the fruiting bodies of the cultivated p. japonica fungus, a new type of cordyceps sp. was investigated (shin et al., 2001) . this statement by the authors is incomprehensible (see above). types of fungi are the typical specimens often held in culture collections. it is not proposed to discuss the described effects herein. hot-water extracts of cordyceps scarabaecola stromata exhibited potent intestinal immune system-modulating activity, while the methanol-soluble fraction manifested intermediate activity (yu et al., 2003) . xu et al. (1992) detected inhibition in melanoma colony formation in murine lungs by cs. the fruiting bodies of ''p. japonica" reduced tumour weight and volume and lengthened the life span of mice inoculated with sarcoma 180 cells (shin et al., 2003) but what this fungus represents in unclear. water and ethanol extracts of cs possessed a potent anti-oxidant activity (yamaguchi et al., 2000b) . anti-lipid peroxidation activities also were detected and accumulation of cholesteryl ester in macrophages was inhibited via suppression of ldl oxidation. hot-water extracts were particularly effective. ''p. japonica" exhibited immunostimulating activity. its ethanolic extract stimulated phagocyteosis and macrophage acid phosphatase activity (shin et al., 2001 (shin et al., , 2003 . c. militaris demonstrated general anti-inflammatory activity (yu et al., 2004a) in mice. the fact that cultured fruiting bodies were employed is interesting rather than extraction from those from the wild and most papers seen for this review either use fruiting bodies from the wild and/ or in vitro biomass. however, it is disappointing that details of the cultivation of the fruiting bodies are not provided as this is required. nitric oxide production and inos gene expression in lps-stimulated raw 264.7 cells are suppressed by ethanolic preparations (won and park, 2005) . a unique insight into how the cultured fruiting bodies were produced in this report and should be referred to: this detail is required in future reports. cs (as supplied by the ''xinhui xinhan artificial cordyceps factory" (sic)) inhibited mda generation via hydroxyl radicals induced by the peroxynitrite generator sin-1 and macrophage accumulation of esterified cholesterol (yamaguchi et al., 2000a,b) . the authors concluded that the cultured cs has anti-oxidant and anti-lipid peroxidation properties and inhibits accumulation of cholesteryl ester in macrophages via suppression of ldl oxidation. the authors conclude correctly that this cultured chinese medicine appears to merit further investigation as an anti-atherosclerotic. in the unusual use of this solvent to create an extract from cs mycelia, apoptosis in human pre-myelocytic leukaemia hl60 cells was induced. in addition, cell proliferation was inhibited . obviously, ethyl acetate may extract different compounds from the fungus compared to the more common water or methanol extracts and the constituents need to be determined. an alcoholic extract of cs inhibited abdominal aortic thrombus formation in rabbits by preventing platelet aggregation (zhao, 1991) . cs was extracted in pbs and dialyzed (chiou et al., 2000) the resulting macromolecule fraction was assayed in anesthetized rats for hypotensive effects and in isolated aorta for vasorelaxant effects. a constituent(s) in cs relaxed vascular beds directly. the in vivo and in vitro effects and its extracted fractions on the secretion of testosterone in mice were studied (hsu et al., 2003a) . cs, watersoluble protein, and poorly water-soluble polysaccharide and protein significantly stimulated in vitro testosterone production in purified mouse leydig cells. the authors concluded that it is ''possible" that cs ''might" contribute to an alternative medicine for the treatment of some reproductive problems caused by insufficient testosterone levels in human males, which is a large leap in conclusions. increase antigen expression was found in hepatoma cells and ''will" provide more effective host immune surveillance against tumour cells (chiu, 1998 ) from a cordyceps extract. in an interesting study, the beneficial effects of the ''traditional chinese medicine cs", on mice with hypoferric anaemia were evaluated by nmr spectroscopy (manabe et al., 2000) . the extract increased hepatic energy metabolism in anaemic mice and was concluded to be due to increased hepatic blood flow. zhang et al. (2006) concluded that ''their" extract ''is" effective in resisting the oxidative damage on liver mitochondria of diabetic mice. qiao and jian (2007) attempted to identify the signaling pathways for the induction of hl-60 cell apoptosis by cs mycelium extract (csme). csme induced nuclear fragmentation and dna degradation, two hallmark events of apoptosis, in the hl-60 cells within 12-24 h of treatment. concomitantly, several major events in the mitochondrial signal pathway occurred, including (a) the loss of mtp, (b) cytochrome c release into the cytoplasm, (c) the decrease in bcl-2 protein level, (d) the translocation of bax protein from cytoplasm into mitochondria, and (e) the activation of caspase-2, -3, and -9. however, caspase-8, the initiator caspase in the death receptor pathway, was not activated. these results suggest that csme induces apoptosis in hl-60 cell through the mitochondrial pathway rather than the death receptor pathway. treatment of d-galactose-induced-aged-mice with an unspecific cs extract resulted in (a) an improved learning ability and memory, (b) an increase in superoxide dismutase activity in erythrocytes, liver and brain, (c) an increase in catalase and glutathione peroxidase activity in blood, (d) reductions in malondialdehyde levels in brain and liver and (e) a reduction in monoamine oxidase activity in the brain . extracts enhanced the antibody response, restored the phagocytic activity of macrophages in tumour-bearing mice, and lengthened the survival period of the mice (yamaguchi et al., 1990 ). an extract down-regulated apoptotic genes in the rat kidney following ischaemia/reperfusion (shahed et al., 2001) . manabe et al. (1996 manabe et al. ( , 2000 found that a mycelial extract increased hepatic energy metabolism, as demonstrated by liver atp:pi value, in diet-induced hypoferric anaemic mice by increasing hepatic blood flow. the following section considers the use of the fungus as whole mycelium and/or fruit-bodies, although the prepara-tions may have to be prepared in water or ethanol in practise. cs down-regulated inflammation-related genes in the rat kidney following ischaemia/reperfusion (shahed et al., 2001) . a similar treatment improved lung function in guinea-pigs and airway inflammation in rats, suggesting a possible asthma treatment . c. pruinosa inhibited (a) gene expression of an interleukin tumour necrosis factor, (b) inducible nitric oxide synthase (inos) and cyclooxygenase-2, and (c) nuclear transcription factor nf-jb activation in a lipopolysaccharide (lps)-stimulated mouse macrophage cell line: this indicated a role in the treatment of endotoxin shock or sepsis . anti-ds-dna production was inhibited and improved survival in mice indicated that cs may be beneficial to patients with systemic lupus erythematosus, an autoimmune disease with involvement of multiple organ systems . the fungus inhibited lymphadenectasis, reduced proteinuria and plasma anti-ds-dna antibody and improved renal function in mrl 1pr/1pr mice (fu and lin, 2001 ). an oral dose of 2-4 g daily for 3 years prevented the recurrence of lupus nephritis and protected renal function in lupus nephritis patients (lu, 2002) . carcinogenesis in the murine forestomach was suppressed by cordyceps (lin, 1984) . results from liu et al. (1996) indicated that the levels of il-1 and inf, produced by cultured rat kupffer cells were increased from rats fed on cs. studies have demonstrated that polysaccharides extracted from these natural products have anti-hyperglycemic effects (lo et al., 2001) . these authors investigated the effects of intragastrically administered cordyceps sp. for alleviating fasting hyperglycemia in diabetic rats. animals had significantly increased serum levels of triglyceride, cholesterol and blood urea nitrogen, and significantly decreased body weight, serum albumin levels and weights of the thymus, lungs and gastrocnemius muscle compared to animals in the control group. in addition, blood glucose was significantly increased compared to the control group; these results suggest that enterally administered cordyceps sp. has potential anti-hyperglycemic ability. these findings reveal that the fungus ''may be used as a nutraceutical to alleviate hyperglycemia in diabetes". oral administration of cordyceps alleviates fasting hyperglycemia . recent evidence has shown that an extract has immunoregulatory activity. the objective was to investigate whether cordyceps has biological activity in regulating the lymphocyte subsets in diabetes. after 2 weeks, body weight, thymus weight, thymocyte number, and the percentages of total t and t helper cells in the thymus were significantly lower in the treatment groups than in the controls. results demonstrate that stz-induced diabetic rats had significantly decreased numbers and subsets of t cells in the thymus. however, oral administration of cordyceps did not improve these changes. the results suggested that oral administration had no significant effect on lymphocyte subsets in stz-induced diabetic rats. hsu et al. (2003b) demonstrated in a convincing study, that cs mycelium regulates mouse cell testosterone production and may suppress stimulated testosterone production via p450 scc enzyme activity. hsu et al. (2003a) demonstrated that the fungus and fractions from it were capable of stimulating testosterone production. the steroidogenic activity was observed in vivo in male mice after 7 days of treatment (huang et al., 2004b) . chen et al. (2005) found that protein kinase a and protein kinase c pathways are acted upon to stimulate steroidogenesis in ma-10 mouse leydig tumour cells. inhibitors of protein kinase a, protein kinase c and phospholipase c and calmodulin antagonists (see paterson, 2008) reduce leydig cell steroidogenesis induced by the fungus. c. militaris inhibited (a) the growth and metastasis of lewis lung cancer cells, and (b) the growth of sarcoma s180 cells implanted in mice. in addition, the survival period of the mice was increased (liu et al., 1997) . liu et al. (1991) showed that p. sinensis inhibited lipid peroxidation but increases the amount of glutathione peroxidase and superoxide dismutase in mouse liver. again the name of the fungus raises similar questions as to those raised for p. japonica and consequently as to what material is being used and whether the experiments could be repeated. finally, cordyceps is included in a list of anti-aging tcm . cs stimulated mitochondrial electron transport and atp production (siu et al., 2004) . the effect of the fungus on hepatic fibrogenesis induced in rats was studied by liu and shen (2003) . it was found that it delayed cirrhotic development and improves liver function by inhibiting expression of transforming growth factor-and plateletderived growth factor and deposition of procollagen i and iii. zhou et al. (1990) presented evidence for the beneficial effects of cs on chronic hepatitis b. the fungus increased dna synthesis in primary cultured rat tubular epithelial cells (tian et al., 1991) . proximal tubular cells were protected from the toxic effects of gentamicin. the possible mechanisms include protection of sodium pump activity, reduction of lipid peroxidation and attenuation of lysosomal over-activity in tubular cells due to phagocytosis of gentamicin (zhen et al., 1992; li et al., 1996) . also, rat kidneys were protected from cyclosporin-induced nephrotoxicity and ameliorated glomerular and interstitial damage (zhao and li, 1993) . bao et al. (1994) reported that ''old" patients were protected from amikacin sulphate toxicity as demonstrated by decreases in urinary nephroaminoglycosidase and microglobulin. also, inhibition by c. militaris of ldl-induced proliferation of cultured human glomerular mesangial cells, which are involved in the development of glomerulosclerosis was observed (zhao-long et al., 2000) . treatment of mice with c. militaris lengthened the swimming time to exhaustion (jung et al., 2004 ). an unspecified cordyceps is one of the components of fuzheng huayu recipe, which is used to control the development of post-hepatic cirrhosis or to prevent complications from the disease (liu et al., 1996b) . the macrophage-stimulating activity of natural fungus and cultured mycelia has been described by zhang (1985) . zhang and xia (1990) demonstrated immunosuppressant effects in the heterotropic heart allograft model in rats and determined that prolonged survival periods were possible. also, zhu and yu (1990) found prolonged mouse skin allograft survival time. the number of t helper cells was increased, as were lyt-1/lyt-2 (t helper cells to t suppressor cells) in peripheral blood and spleen . a mitogenic action was demonstrated on splenic lymphocytes and interleukin-2 from spleen cells of rats with chronic renal failure was augmented (cheng, 1992) . in addition, natural killer cell activity was enhanced (xu et al., 1992) . treatment of patients with post-hepatic cirrhosis resulted in (a) enhancement of natural killer cell function, (b) increased number and improved ratio of cd4þ and cd8þ cells, and (c) reduction in iga and igg levels (zhu and liu, 1992) . treatment of chronic hepatitis b resulted in increased cd4/cd8 ratios, and reductions in hyaluronic acid and procollagen type iii. the data indicate the usefulness of the fungus in adjusting the level of t lymphocyte subsets and treating hepatic fibrosis (gong et al., 2000) . liu et al. (1992) demonstrated increased peripheral natural killer cell activity from healthy subjects and leukaemia patients. improved renal function and augmented cellular immune function in chronic renal failure has also been observed (guan et al., 1992) . the fruiting body portion, but not the carcass portion, of cordyceps reduced weight loss, polydipsia and hyperglycaemia in diabetic rats (lo et al., 2004) . some other somewhat obscure papers are available on the effect of whole fungi (i.e. peizhong, 1984; chen, 1985; zhang, 1985; shao, 1985; liu and xu, 1985; zhang, 1987; chen, 1987; du, 1986; sun, 1985; li et al., 1993) . feng et al. (1987) reported the vasodilating effect of cultured mycelia of cs in an investigation of the cardiovascular system of dogs. thirty three cases of chronic hepatitis b patients treated with cultured cs mycelia have shown that the drug (a) improves liver function, (b) promotes negative transfer hbsag, (c) markedly helps to raise plasma albumin, (d) helps patients resist high gamma globulin and (e) adjusts body immunocompetence (zhou et al., 1990) . it is suggested that the fungus may be a medicine for chronic hepatitis b patients in adjusting protein metabolism and correcting inversion of albumin and globulin. since inflammation has been reported to be associated with chronic diseases inhibitory dietary factors such as the fungus may be beneficial in alleviating disease (hong and lin, 2004) . 17b-estradiol may directly influence the quality of maturing oocytes and thus the outcome of assisted reproduction treatment. cs mycelium is ''believed" to enhance libido and fertility in both sexes. however, the mechanism of its effect in women has not been determined. this is surely a rather thin basis on which to undertake research. huang et al. (2004a) concluded that treatment of granulosa-lutein cells with cs results in increased 17b-estradiol production due, in part, to increased star and aromatase expression. if these data are confirmed, cs may help in the development of treatment regimens to improve the success rate of in vitro fertilization, they state. zhu and liu (1992) found that cultivated cordyceps mycelia inhibited humoral immune hyperfunction and increase the serum complement level in patients with post-hepatic cirrhosis, and improved liver function. whole body insulin sensitivity in rats was increased (balon et al., 2002) . the fungus increased the basal plasma insulin level (zhang et al., 2003) and inhibited hepatic fibrogenesis in rats with ccl 4 -induced liver fibrosis. previous studies by lo et al. (2006) demonstrated that the fruiting bodies of cs attenuated diabetes-induced weight loss, polydipsia, and hyperglycemia in rats. rats were orally administered, fruiting bodies, fermented mycelia, spent broth, or mycelia plus spent broth of the fungus. the results revealed that the mycelia and spent broth had anti-hyperglycemic activities similar to those of the fruiting bodies. the authors , bold claim that the fermented products could be developed as potential anti-diabetic agents or functional foods for persons with a high risk of diabetes mellitus needs further confirmation. yang et al. (2006) mention that mycelium can inhibit tumour growth and induce tumour cell apoptosis. however, the antitumour mechanisms are not fully understood. so, the molecular mechanism was determined. the authors conclude that cell apoptosis is induced by activating caspase-8-dependent and caspase-9-independent pathways and downregulating nf-jb protein expression. mycelia induced human granulosa-lutein cells to produce 17b-estradiol by upregulating expression of steroidogenic acute regulatory protein (star) and aromatase (huang et al., 2004a) . also, steroidogenesis in mouse leydig tumour cells (huang et al., 2001a) was stimulated without involvement of star (huang et al., 2000) . testosterone production was inhibited by human chorionic gonadotropin or dibutyryl cyclic amp. thus, its effect on the signal-transduction pathway for steroidogenesis may be after the production of cyclic amp. it is revealing to read the web sites of some of the commercial products: these are often written in unscientific terms. the best of them at least contain warnings that, ''these statements have not been evaluated by the (us) food and drug administration. this product is not intended to diagnose, treat, cure or prevent any disease". colson et al. (2005) undertook a revealing experiment to determine the effect on male cyclists' performance using a combination of cs and rhodiola rosea ''herbs" -a tradi-tional herbal medicine. importantly, and unusually, the study followed a double blind, randomized, placebo-treatment, pre-post test design. essentially no effect was observed. this is an example of why scientists need to circumspect about making unsubstantiated claims about the powers of these preparations even if spin does help project funding. ''in this regard, the maintenance of a balance of yin and yang -two opposing components involved in life activities as exemplified by the antagonistic action of the sympathetic and parasympathetic nervous systems-is essential in achieving a healthy condition" (leung et al., 2005) . statements such as these leaves the current reviewer bewildered. why these terms are used in a scientific publication does not appear to merit comment. previous studies have shown that long-term treatment with a ''yang-invigorating" chinese herbal formula (vi-28) could increase red cell cuznsuperoxide dismutase (sod) activity in male human subjects. yet the authors conclude, the beneficial effect of vi-28 treatment on mitochondrial functional ability and antioxidant capacity may have clinical implications in the prevention of age-related diseases. ka wai lee et al. (2006) investigated a commercial preparation of a cultivated strain of cs. they state that the immunomodulatory activities have been renowned for centuries. the report describes positively the immunomodulatory features: in vitro results demonstrated that the fungus induced the production of interleukin (il)-1, il-6, il-10 and tumour necrosis factor alpha, augmented surface expression of cd25 on lymphocytes, and elevated macrophage phagocytosis and monocyte production of h 2 o 2 . the authors state that, ''our results possibly provide the biochemical basis for future clinical trials". the current author emphasises that such over extrapolation of data needs to be discouraged. dai et al. (2001) found that cordymax cs-4 (a mycelial fermentation product of cs) ''improved the bioenergy status" in the mouse liver. these findings may explain why cordymax cs-4 is claimed to alleviate fatigue and improve physical endurance especially in aged subjects. reports of athletes' performance being improved may also be supported from these types of reports. cordymax cs-4 lowered fasting plasma levels of glucose and insulin, improved oral glucose tolerance and increased the glucose-insulin index, which measures insulin sensitivity, in rats (zhao et al., 2002) . an unspecified cordyceps is reported to be a component of fuzheng huayu recipe, which is used to control the development of post-hepatic cirrhosis or to prevent its complications (liu et al., 1996b) . as part of the eternal search to find an excelsior of youth, many are interested in anti-aging activity but it is difficult to define what his means scientifically. are the authors stating that the aging process is slowed for example? do people live longer? in any case, this was published in the journal, chinese journal of integrated traditional and western medicine which is an admirable sentiment but difficult to do in practice. a prep-aration of cs referred to as jinshuibao capsule, increased the depressed superoxide dismutase activity and reduced elevated malondialdehyde (mda) level caused by aging when tested in senile patients. in addition, they enhanced the repair of damaged non human animal dna (zhang et al., 1997) . quite extraordinarily beneficial effects on senile patients are claimed and, of course, the constituents of the capsule may be difficult to verify. zhou and lin (1995) used ''jinshuibao", to restore cellular immune function and ''improve the quality of life" of patients with advanced cancer without affecting humoral immune function. obviously, quality of life is a subjective notion and is difficult to define. remarkably, a dried powder preparation of mycelia called bailing capsule (a) prevented rejection of renal transplants, (b) protected renal and hepatic function, (c) stimulated haematopoietic function, (d) improved hypoproteinaemia and hyperlipidaemia and (e) reduced the incidence of infections (sun et al., 2004) . however, such claims must surely carry considerable elements of doubt. in another case of a combined treatment with as many as six other components in addition to cs, the treatment was more effective at preventing acute renal failure than chronic in rats. obviously, it is difficult or impossible to assess the efficacy of the individual components (ngai et al., 2005) . inhibition of apoptosis is a novel area of clinical investigation with great promise and so merits a separate section in this review and will be of particular interest to pharmacologists. in one of the more critical papers, buenz et al. (2005) mention that there is a wide range of uses of cs in the literature, and those claiming altered apoptotic homeostasis are of the most intriguing. however, they emphasize problems created by the (a) difficulty of identifying the species of cordyceps and (b) many conflicting reports of pharmacological function. in response, the authors (a) outline what is known about the ability of cs to alter apoptotic homeostasis, (b) attempt to reconcile differences in function, and (c) identify the challenges and how to progress cs research. many disorders (e.g. stroke, myocardial infarction and hiv) incorporate apoptosis in their aetiology and pathogenesis. the ability to inhibit apoptosis has emerged as an important potential therapy (e.g. cancer). there are reports of cs extracts inhibiting and inducing apoptosis (shahed et al., 2001) ; this is not contradictory but allows a foundation to determine the molecular mechanism of activity. indeed, cordyceps may contain compounds that inhibit apoptosis; however, conflicting evidence has been obtained. the fungus can scavenge reactive oxygen species by inhibiting malondialdehyde formation by sin-1, the peroxynitrite generator, which has been confirmed by xanthine oxidase, hemolysis, and lipid peroxidation assays. furthermore, an isolated extract of cs, h1-a (this is reported as a pure compound (see above), inhibited induced apoptosis, by permeabilizing the cell membrane and upregulating nitric oxide synthase (trubiani et al., 2003) . on the other hand, extracts of cs did not inhibit hydrogen peroxide-induced apoptosis (buenz et al., 2004) , in a reactive oxygen species model (fauconneau et al., 2002) . alternatively, cs down-regulated apoptotic genes and modulated apoptosis in a rat kidney ischemia reperfusion model. reported anti-apoptotic effects of cs include in (a) the mouse (anti-cytotoxic activity, anti-oxidant activity, cell proliferation inhibition), (b) cell culture (anti-proliferation activity, cell proliferation inhibition, hemolysis inhibitory activity, lipid peroxide formation inhibition, radical scavenging effect), (c) human cells (proliferation inhibition, natural killer cell inhibition, tumour necrosis factor inhibition) and (d) the rat (gene expression inhibition). whereas the reported apoptotic effects of the fungus are in: (a) the mouse (anti-tumour activity, metastasis inhibition) and (b) cell culture (proliferation stimulation, cytotoxic activity). in reality these opposite effects are not incompatible. there are three prominent factors that may contribute to the apparent discrepancies (the first and third are fairly obvious). first, certain fungi contain different biologically active compounds. second, extracts may contain a prodrug; a metabolism step may be required to generate the biologically active form. third, different constituents may be assayed as there are multiple methods of extraction utilized in the literature. furthermore, decreases in fas, fas ligand and tumour necrosis factor expression and decreased caspase-3 activity have been demonstrated (shahed et al., 2001) . cs inhibited tnf-a expression (kuo et al., 1996) . however, when apoptosis was initiated via a fas agonist antibody (ch-11) (alderson et al., 1994) , aqueous and alcohol extracts of cs did not rescue cells induced by fas receptor ligation (buenz et al., 2004) . furthermore, cell cycle arrest and/or inhibition of proliferation yield cells resistant to apoptosis. papers concerning proliferation of leukemic u937 cells and glomerular mesangial cells inhibition (zhao-long et al., 2000; lin et al., 1999) may be explained by conferring a apoptotic resistance to cells: the alteration may involve p53 (fridman and lowe, 2003) or nf-jb (karin et al., 2004) . overall, aqueous, and organic extracts of cs have the ability to inhibit apoptosis. cells pre-incubated with cs extracts were equally sensitive to hydrogen peroxide and fas-mediated apoptosis. thus, the putative antioxidant and anti-apoptotic properties of cs were insufficient to rescue cells from apoptosis in vitro (buenz et al., 2004) . furthermore, cancer chemotherapeutics have involved the ability to induce apoptosis. hence the polysaccharide fraction (h1-a) induced apoptosis by inhibiting (a) phosphorylation of bcl-2 and bcl-xl and (b) apoptosis induced by dimethyl sulfoxide (yang et al., 2003) . these data require to be confirmed by further work (buenz et al., 2005) . finally, direct cytotoxic activity may be a factor (nakamura et al., 1999a,b; kuo et al., 1994; sato, 1989; buenz et al., 2005) . a factor which is not often considered is whether the activities of extracts and pure compounds are from the cordyceps of interest or from other contaminating fungi. the issues of anamorph/teleomorph associations are relevant here. for example, epicoccins a-d were isolated from cultures of a cordyceps-colonizing isolate of epicoccum nigrum (zhang et al., 2007b) . gliocladinins a and b are of an isolate of gliocladium sp. that colonized cs (guo et al., 2007) . it is essential that such activity is differentiated from that obtained from the traditional medicine or cordyceps per se. in a series of papers, (a) paecilomyces militaris is shown to possess militarinones a, b, c, d and (b) farinosomes and a deoxymilitarinone substance were detected from paecilomyces farinosus (schmidt et al., 2002 (schmidt et al., , 2003 cheng et al., 2004 cheng et al., , 2006 . the authors do not make statements as to the holomophic connections to cordyceps. hamburger (2007) does make this link to c. militaris (teleomorph) from p. militaris (anamorph) apparently on the basis of the same pigment production. however, it is has been suggested that paecilomyces does not contain anamophic species of cordyceps . hence, comment on these reports is avoided by the current author because the relationships between paecilomyces and cordyceps remain ill-defined. another complicating facet is that the medicine may contain a proportion of the insect host and so what does this bring to the activity? this is an area of investigation which simply is not reported. indeed does the healthy insect have interesting activities, and/or at what stage does the activity occur in the infected insect? could the activity be greater at these stages? the accumulation of bioactive plant metabolites by insects is well documented (brown and trigo, 1994) and insects are used as medicines . these aspects are almost totally ignored in the case of the medicinal cordyceps. it appears as if entomologists have simply not been involved in the field. does the insect have to be dead before the system works or are more active components produced by the fungus as it is in the process of killing the insect? research into these factors could be fruitful. hence, it is worth reporting what is known about the differences between the insect and fungus parts: interestingly, galactomannans isolated from the insect portion of c. cicadae demonstrate potent hypoglycaemic activity in mice (kiho et al., 1990) . the fruiting body portion, but not the carcass portion, of cordyceps reduced weight loss, polydipsia and hyperglycaemia in diabetic rats (lo et al., 2004) . aqueous methanol extracts of c. cicadae ascocarps enhanced human mononuclear cells (hmnc) proliferation (weng et al., 2002) . in contrast, the methanol (100%) extracts of the c. cicadae insect-body portion suppressed hmnc proliferation. interestingly, the ergosterol esters concentrations were much higher in the (dead) caterpillar than the fruiting bodies (yuan et al., 2007) although ergosterol was similar. finally, the fruiting body and the caterpillar parts of cs are claimed to be similar in chemical composition and hence anti-oxidant activity because the fungus had presumable replaced the insect constituents with fungal . little scientific evidence exists to support the numerous herbs used to improve diabetes-related metabolic disorders (lo et al., 2004) . the dual organism medicine (i.e. fruiting body and carcass) has been proposed to have multiple medicinal activities. in one investigation, the effects of the fruiting body and carcass of the preparation on hyperglycemia were investigated. diabetic rats had significantly lower weight gain and higher blood glucose response in oral glucose tolerance test than the control rats; and these changes were significantly reduced by administrating the fruiting body of cordyceps. the results revealed that the fruiting body (not the carcass) of cordyceps attenuated the diabetes-induced weight loss, polydipsia and hyperglycemia, and these improvements suggest that fruiting body has a potential to be a functional food for diabetes. the water extracts from the fruiting body and ''worm" of natural cordyceps were analyzed for their content of nucleosides and polysaccharides; the results showed that the worm had a chemical composition similar to the fruiting body . in addition, both the fruiting body and worm of cordyceps showed similar potency in their anti-oxidation activities in the xanthine oxidase assay, the induction of hemolysis assay and the lipid peroxidation assay. these results suggest that the function of the worm is to provide a growth medium for the fruiting body, and that eventually, the worm is totally invaded by mycelia. aqueous methanol extracts of c. cicadae ascocarps enhanced human mononuclear cells hmnc proliferation (weng et al., 2002) . in contrast, the methanol (100%) extracts of the c. cicadae insect-body portion suppressed hmnc proliferation. this is a most interesting result in the current author's opinion. the following are references concerning the chemical constituents as determined by analytical profiles (guo, 1985; xiao, 1983; . yu et al. (2007b) developed a method involving ''biospecific" extraction and hplc for potential immunological components in cs. the two active compounds were identified as guanosine and adenosine. li et al. (2001b,c) found that cultured cs mycelia have a much higher content of nucleosides than natural cs. guo et al. (1998) described an hplc method for quantitative determination of adenosine and deoxyadenosine. li et al. (1999) determined adenosine in fermented products of cordyceps by reversed-phase hplc. ergosterol in cs can be determined by hplc li et al., 2004) , and, of course, ergosterol is present in all fungi. when there are many questions as to what are the active components, a section on optimisation may seem premature. however, kim and yun (2005) investigated the optimal culture conditions for the production of exopolysaccharides (eps) and cordycepin during submerged mycelial culture of c. militaris and cs. this was not done in the paper hamburger (2007) comments upon. fermentations were performed in flasks and in 5-l stirredtank bioreactors. the concentrations of mycelial biomass, eps and cordycepin achieved in submerged culture of c. militaris were higher than those of cs. as the authors claim, comparative studies between the two fungi are not available and the paper was the first report on the optimum medium composition for submerged culture of cs. cordyceps nutans pat. is another entomopathogenitic ascomycete belonging to the family clavicipitaceae which is parasitic on hemipteran insects (sasaki et al., 2005) . very few investigations have been made with this fungus. in this research, optimum temperature and ph for mycelial growth was determined. however, it appeared that growth remained low and commercial exploitation perhaps limited. however, it would be interesting to include this species in any future taxonomic treatments of the genus. optimisation of culture conditions for mycelial growth and production of polysaccharides and cordycepin were described by park et al. (2001b park et al. ( , 2002 , xu et al. (2002) , kim et al. (2003b,c) , xiao et al. (2004) , mao and zhong (2004) and hsieh et al. (2005) , although cordycepin is usually produced synthetically at present. two cases of lead poisoning were reported (wu et al., 1996) . these two patients took cordyceps herbal medicine for treatment of underlying diseases. loss of appetite and anemic signs of lead poisoning were manifested in one patient with a high blood lead level, while the other patient was asymptomatic. the lead content in the cordyceps powder was found to be as high as 20,000 ppm. after cessation of intake in the asymptomatic patient, and cessation of intake and treatment with chelating agents in the symptomatic patient, the blood lead levels returned to normal range. this report raises concerns about lead poisoning from unusual herbal medicine in general. this present review is not intended to be a taxonomic paper, but is essentially a review of the papers that employ the name cordyceps more or less loosely, to describe the fungus used in medicinally related experiments. however, in a most impressive paper, sung et al. (2007) state that cordyceps comprise over 400 species in their modern phylogenetic classification of cordyceps and the clavicipitaceous fungi. cordyceps fr. host range is broad, ranging from 10 orders of arthropods to the truffle-like genus elaphomyces, although most species are restricted to a single host species or closely related host species. the main objectives of the study was to reassess the (a) morphological traits used currently, (b) taxonomic utility of the anamorphic forms and (3) classification in relation to phylogenetic relationships. it may be worth mentioning that pcrs may be subjected to inhibition, and nucleic acids may be affected by the medium in which the fungus was grown (see paterson, , 2008 . unfortunately, sung et al. (2007) do not provide details of how the fungi were grown for analysis and so it is difficult to conjecture. obviously, as discussed in this review, the fungus produces numerous bioactive compounds which may be inhibitors or mutagens and the effect of metabolite production on the dna preparations need to be determined or misleading results may be obtained. however, to continue, it is worth mentioning that c. militaris maintains its name in the study while c. sinensis is now classified as ophiocordyceps sinensis. the taxon was historically classified in the clavicipitaceae, based on cylindrical asci, thickened ascus apices and filiform ascospores, which often disarticulate into part-spores. the fungus was characterized by having a pathological ecology on elaphomyces and arthropods, with infrageneric classifications emphasizing ascospore morphology, host affiliation and arrangement of perithecia. the production of welldeveloped often stipitate stromata was typical. sung et al. re-classified on the basis of phylogenetic relationships between 162 taxa by employing five to seven genetic loci. three clavicipitaceous clades were determined which rejected the monophyly of cordyceps and clavicipitaceae and most diagnostic characters used in cordyceps were not supported as being phylogenetically informative. however, the most consistent characters with the phylogeny were pigmentation, morphology and morphology of stromata. cordycipitaceae was validated based on c. militaris, the type of cordyceps, which included most cordyceps species that possess bright, fleshy stromata. the new family ophiocordycipitaceae was proposed. the majority of species here produce stromata that often possess aperithecial apices and are darkly pigmented, tough to pliant. elaphocordyceps was proposed for a subclade of the ophiocordycipitaceae, which includes species of cordyceps that parasitize (a) the fungal genus elaphomyces and (b) arthropods. the family clavicipitaceae included the core clade of grass symbionts, and the entomopathogenic genus hypoc-rella and relatives. the new genus metacordyceps is proposed for cordyceps species that are closely related to the grass symbionts in the clavicipitaceae s. s. metacordyceps includes teleomorphs linked to metarhizium and other closely related anamorphs. lists of accepted names for species in cordyceps, elaphocordyceps, metacordyceps and ophiocordyceps are provided. obviously, this work needs to be consulted in future work on the fungus for medicinal properties as does stadler et al. (2003) . too often authors have used highly conditional statements such as; the preparation ''may" have ''possible" activity against, for example, cancer, which are meaningless. it is claimed that modern studies are demonstrating anti-oxidant, vascular, immune, and anti-inflammatory effects (meletis and barker, 2005) and the mechanisms by which these mushrooms work are being elucidated. however, the journal in which the paper is published is dedicated to complimentary and alternative medicines. this implies it is not mainstream scientific and so scientists may not give it as much weight in comparison to, for example, phytochemistry or the journal of natural products. nevertheless, the former reports could be considered as providing leads for more scientific research. in general, problems include too many chemically uncharacterised crude extracts, inadequate taxonomic vigour, and over extrapolation of in vitro data to imply therapeutic value. this needs to be avoided as it falsely raises hopes for cures for serious diseases. furthermore, there are no biochemical data on how the insect are converted into fungal components, or if the insect host per se has pharmacological properties. poor (i.e. inadequate or obsolete) biochemical procedures and assays can also because problems (see hamburger, 2007) . the biological material being used often is not well characterised and there are few indications of specimens being available for other workers to repeat the described work for example (i.e. no voucher specimens are available), with some notable exceptions. a rather limited range of solvents have been employed to extract the components and, for example, chloroform/methanol is particularly efficacious for fungal compounds (e.g. paterson and bridge, 1994) . there is very little on the use of non-polar solvents. paterson et al. (2004) , paterson (2006) and paterson (2008) have advocated a method which may have utility in standardising bioactive fungi. in this scheme, a ''common, readily identifiable" morphological character is determined and then the fungus is analyzed for particular metabolites. in this case the steps may be: 1. produces stroma on lepidopteron insects. 2. produces detectable concentrations of, for example, cordycepin. the analysis for other compounds would be desirable and some potential marker compounds are provided in table 3 . however, the phylogenic approach of sung et al. (2007) will set the standards for decades to come and will profoundly influence future publications in all fields relating to cordyceps. table 3 summary of markers currently used for quality control of cordyceps and associated activities (after li et al., 2006a) compound type pharmacological activities comments nucleosides anti-tumour activities; ca 2+ antagonist; the release of various neurotransmitters presynaptically and anticonvulsant activity; stimulate axon growth in vitro and in the adult central nerve system nucleosides, especially adenosine, are usually used as markers, and the profiles can be applied for authentication of cordyceps polysaccharides anti-oxidation, immunopotentiation, anti-tumour, and hypoglycemic activity; anti-inflammatory activity and suppress the humoral immunity in mice represents the most biological properties of cordyceps, and less used as marker for quality control. thus, it should be developed ergosterol and its analogs cytotoxic activity, anti-viral activity, and anti-arrhythmia effect; suppress the activated human mesangial cells and alleviate immunoglobulin a nephropathy (berger's disease) ergosterol can be used as marker for quality control mannitol diuretic, anti-tussive and anti-free radical activities it sometimes is used as marker for quality control peptides anti-tumour and immunopotentiation activities a potential marker for quality control cordycepin: selective growth inhibitor derived from liquid culture of cordyceps militaris against clostridium spp regulation of apoptosis and t cell activation by fas-specific mab a fermentation product of cordyceps sinensis increases whole-body insulin sensitivity in rats amelioration of aminoglycoside nephrotoxicity by cordyceps sinensis in old patients antitumor sterols from the mycelia of cordyceps sinensis comparison of balanol from verticillium balanoides and ophiocordin from cordyceps ophioglossoides multi-level complexity in the use of plant allelochemicals by aposematic insects selected herbals human exercise performance the traditional chinese medicine cordyceps sinensis and its effects on apoptotic homeostasis cordyceps sinensis extracts do not prevent fas-receptor and hydrogen peroxide-induced t-cell apoptosis fungal populations and species cordyceps sinensis animal, vegetable or both? cordyceps sinesis (berkeley) saccardo: structure of cordycepic acid platelet hemopoiesis and ultrastructure observations in mice treated with natural cordyceps sinensis and its cultured mycelia the effect of natural cordyceps sinensis and its cultured mycelia on murine immuno-organs and function of the mononuclear macrophage system effects of cordyceps sinensis on murine t lymphocyte subsets the effects of chinese herbs on improving survival and inhibiting anti-ds dna antibody production in lupus mice morphological and genetic characterization of a cultivated cordyceps sinensis fungus and its polysaccharide component possessing antioxidant property in h22 tumor-bearing mice recent advances in studies on traditional chinese anti-aging material medica nmr and ir studies on the characterization of cordycepin and 2 0 -deoxyadenosine. zhongguo kang sheng su za shi (chin cordyceps sinensis mycelium activates pka and pkc signal pathways to stimulate steroidogenesis in ma-10 mouse leydig tumor cells effect of cordyceps sinensis on the proliferation and differentiation of human leukemic u937 cells genetic diversity and taxonomic implication of cordyceps sinensis as revealed by rapd markers determination of the anamorph of cordyceps sinensis inferred from the analysis of the ribosomal dna internal transcribed spacers and 5.8s rdna effect of cordyceps sinensis on cellular immunity in rats with chronic renal insufficiency farinosones a-c, neurotrophic alkaloidal metabolites from the entomogenous deuteromycete paecilomyces farinosus novel tetramic acids and pyridone alkaloids, militarinones b, c, and d, from the insect pathogenic fungus paecilomyces militaris protein constituent contributes to the hypotensive and vasorelaxant activities of cordyceps sinensis cordyceps sinensis increases the expression of major histocompatibility complex class ii antigens on human hepatoma cell line ha22t/vgh cells cordycepin (3 0 -deoxyadenosine) inhibits human platelet aggregation in a cyclic ampand cyclic gmp-dependent manner antioxidant and memory enhancing effects of purple sweet potato anthocyanin and cordyceps mushroom extract improvement of insulin resistance and insulin secretion by water extracts of cordyceps militaris, phellinus linteus, and paecilomyces tenuipes in 90% pancreatectomized rats cordyceps sinensis-and rhodiola roseabased supplementation in male cyclists and its effect on muscle tissue oxygen saturation asian herbals: opportunities for marketing traditional chinese medicines in the west cordycepin, a metabolic product isolated from cultures of cordyceps militaris (linn.) link cordymax cs-4 improves steady-state bioenergy status in mouse liver mushrooms in medicine and culture in vitro evaluation of antioxidant activities of aqueous extracts from natural and cultured mycelia of cordyceps sinensis antitumor activity of cordyceps sinensis and cultured cordyceps mycelia rebuttal on comparison of protective effects between cultured cordyceps militaris and natural cordyceps sinensis against oxidative damage induction of heat shock proteins (hsps) by sodium arsenite in cultured astrocytes and reduction of hydrogen peroxide-induced cell death vasodilating effect of cultured cordyceps sinensis (berk) sacc. mycelia in anesthetized dogs control of apoptosis by p53 effect of cordyceps sinensis on inhibiting systemic lupus erythematosus in mrl 1pr/1pr mice a novel immunosuppressant, isp-i, of isaria sinclairii effect of cordyceps sinensis on the th1/ th2 cytokines in patients with condyloma acuminatum effects of cordyceps sinensis on t lymphocyte subsets and hepatofibrosis in patients with chronic hepatitis b effect of cordyceps sinensis on tlymphocyte subsets in chronic renal failure isolation and identification of 3 0 -amino-3 0 -deoxyadenosine from cordyceps militaris determination of adenosine and 3 0 -deoxyadenosine in cordyceps militaris (l.) link. by hplc bioactive pterphenyl derivatives from a cordyceps-colonizing isolate of gliocladium sp preliminary study of cordyceps barnesii -comparison of the chemical constituents of cordyceps barnesii and cordyceps sinensis comment on comparison of protective effects between cultured cordyceps militaris and natural cordyceps sinensis against oxidative damage evaluation of the anti-inflammation screening model of macrophages cell line by the proinflammatory mediators secretions medium optimization for polysaccharide production of cordyceps sinensis in vivo and in vitro stimulatory effects of cordyceps sinensis on testosterone production in mouse leydig cells regulatory mechanism of cordyceps sinensis mycelium on mouse leydig cell steroidogenesis effects of extracted cordyceps sinensis on steroidogenesis in ma-10 mouse leydig tumor cells upregulation of steroidogenic enzymes and ovarian 17b-estradiol in human granulosa-lutein cells by cordyceps sinensis mycelium effects of cordyceps sinensis on testosterone production in normal mouse leydig cells cordyceps sinensis and its fractions stimulate ma-10 mouse leydig tumor cell steroidogenesis in vivo stimulatory effect of cordyceps sinensis mycelium and its fractions on reproductive functions in male mouse comparison of protective effects between cultured cordyceps militaris and natural cordyceps sinensis against oxidative damage co-n reaction-a new serological activity index-on wegener's granulomatosis world university rankings. the times higher education supplement a xanthocillin-like alkaloid from the insect pathogenic fungus cordyceps brunnearubra bcc 1395 bioxanthracenes from the insect pathogenic fungus cordyceps pseudomilitaris bcc 1620. ii. structure elucidation es-242 derivatives and cycloheptapeptides from cordycepssp. strains bcc 16173 and bcc 16176 structures of cordypyridones a-d, antimalarial n-hydroxy-and nmethoxy-2-pyridones from the insect pathogenic fungus cordyceps nipponica bioxanthracenes from the insect pathogenic fungus cordyceps pseudomilitaris bcc 1620. i. taxonomy, fermentation, isolation and antimalarial activity the immuno-enhancing effect of chinese herbal medicine cordyceps sinensis on macrophage j774 mycelial extract of cordyceps ophioglossoides prevents neuronal cell death and ameliorates beta-amyloid peptide-induced memory deficits in rats a mushroom lectin from ascomycete cordyceps militaris effect of medicinal plant extracts on forced swimming capacity in mice immunomodulatory activities of herbsnsenses tm cordyceps -in vitro and in vivo studies identification of cordycepin, a metabolite of cordyceps militaris, as 3 0 -deoxyadenosine the ikk nf-kappa b system: a treasure trove for drug development occurrence of gal beta (1-3) galnac-ser/thr in the linkage region of polygalactosamine containing fungal glycoprotein from cordyceps ophioglossoides polysaccharides in fungi. xxxii. hypoglycemic activity and chemical properties of a polysaccharide from the cultural mycelium of cordyceps sinensis polysaccharides in fungi. xxv. biological activities of two galactomannans from the insect-body portion of chan hua (fungus: cordyceps cicadae) structural features and hypoglycemic activity of a polysaccharide (cs-f10) from the cultured mycelium of cordyceps sinensis polysaccharides in fungi. xxxvi. hypoglycemic activity of polysaccharide (cs-f30) from the cultural mycelium of cordyceps sinensis and its effect on glucose metabolism in mouse liver a comparative study on the production of exopolysaccharides between two entomopathogenic fungi cordyceps militaris and cordyceps sinensis in submerged mycelial cultures larvicidal activity against plutella xylostella of cordycepin from the fruiting body of cordyceps militaris methanol extract of cordyceps pruinosa inhibits in vitro and in vivo inflammatory mediators by suppressing nf-jb activation optimization of submerged culture process for the production of mycelial biomass and exo-polysaccharides by cordyceps militaris c738 production and characterization of exopolysaccharides from an enthomopathogenic fungus cordyceps militaris ng3 ophiocordin, an antifungal antibiotic of cordyceps ophioglossoides hypocholesterolemic effect of hot-water extract from mycelia of cordyceps sinensis antifatigue and antistress effect of the hot-water fraction from mycelia of cordyceps sinensis activation of macrophages and the intestinal immune system by an orally administered decoction from cultured mycelia of cordyceps sinensis reinventing taxonomy: a curmudgeon's view of 250 years of fungal taxonomy, the crises in biodiversity, and the pitfalls of the phylogenetic age cicadapeptins i and ii: new aib-containing peptides from the entomopathogenic fungus cordyceps heteropoda homocitrullylaminoadenosine, a nucleoside isolated from cordyceps militaris inhibition of nucleic acid methylation by cordycepin. in vivo synthesis of s-3-deoxyadenosylmethionine by wil2 human lymphoblasts abrogation of streptococcal pyrogenic exotoxin b-mediated suppression of phagocytosis in u937 cells by cordyceps sinensis mycelium via production of cytokines cordyceps sinensis mycelium protects mice from group a streptococcal infection differentiation of cordyceps sinensis by a pcr-single-stranded conformation polymorphism-based method and characterization of the fermented products in taiwan growth inhibitors against tumor cells in cordyceps sinensis other than corydcepin and polysaccharides cordyceps sinensis as an immunomodulatory agent regulation of bronchoalveolar lavage fluids cell function by the immunomodulatory agents from cordyceps sinensis activation and proliferation signals in primary human t lymphocytes inhibited by ergosterol peroxide isolated from cordyceps cicadae medicinal and nutraceutical genetic resources of mushrooms a yanginvigorating chinese herbal formula enhances mitochondrial functional ability and antioxidant capacity in various tissues of male and female rats experimental study on effect of cordyceps sinensis in ameliorating aminoglycoside induced nephrotoxicity determination of nucleosides in natural cordyceps sinensis and cultured cordyceps mycelia by capillary electrophoresis anti-oxidation activity of different types of natural cordyceps sinensis and cultured cordyceps mycelia the contents and their change of nucleosides from natural cordyceps sinensis and cultured cordyceps mycelia simultaneous determination of ergosterol, nucleosides and their bases from natural and cultured cordyceps by pressurised liquid extraction and high-performance liquid chromatography the fruiting body and its caterpillar host of cordyceps sinensis show close resemblance in main constituents and anti-oxidation activity quality control of cordyceps sinensis, a valued traditional chinese medicine hypoglycemic activity of polysaccharide, with antioxidation, isolated from cultured cordyceps mycelia a polysaccharide isolated from cordyceps sinensis, a traditional chinese medicine, protects pc12 cells against hydrogen peroxide-induced injury rp-hplc determination of adenosine in fermented cordyceps effect of cordyceps sinensis on erythropoiesis in mouse bone marrow determination of ergosterol in cordyceps sinensis and cordyceps black-bone chicken capsules by hplc antitumor activity of cordyceps militaris on human cancer cell line inhibition of activated human mesangial cell proliferation by the natural product of cordyceps sinensis (h1-1): an implication for treatment of iga mesangial nephropathy inhibitory effect of cordyceps on carcinogenesis of the forestomach in mice oral administration of cordyceps does not affect lymphocyte subsets in stz-induced diabetic rats effects of fermented cordyceps powder on pulmonary function in sensitized guinea pigs and airway inflammation in sensitized rats effects of cordyceps sinensis (cs) on in vitro natural killer cells immuno-pharmacologic activity of cordyceps sinensis (berk) sacc anticarcinogenic effect and hormonal effect of cordyceps militaris link chemical constituents of cordyceps militaris (l.) link effect of fuzheng huayu recipe in treating posthepatitic cirrhosis influence of cordyceps sinensis (berk.) sacc. and rat serum containing same medicine on il-1, ifn and tnf produced by rat kupffer cells protection against radiation-induced bone marrow and intestinal injuries by cordyceps sinensis, a chinese herbal medicine inhibitive effect of cordyceps sinensis on experimental hepatic fibrosis and its possible mechanism anti-oxidation of paecilomyces sinensis (s. pnov.) intragastrically administered chinese herbal medicine cordyceps alleviates fasting hyperglycemia in diabetic rats anti-hyperglycemic activity of natural and fermented cordyceps sinensis in rats with diabetes induced by nicotinamide and streptozotocin the anti-hyperglycemic activity of the fruiting body of cordyceps in diabetic rats induced by nicotinamide and streptozotocin study on effect of cordyceps sinensis and artemisinin in preventing recurrence of lupus nephritis effects of the mycelial extract of cultured cordyceps sinensis on in vivo hepatic energy metabolism and blood flow in dietary hypoferric anaemic mice effects of the mycelial extract of cultured cordyceps sinensis on in vivo hepatic energy metabolism in the mouse hyperproduction of cordycepin by twostage dissolved oxygen control in submerged cultivation of medicinal mushroom cordyceps militaris in bioreactors antiarrhythmic effects of cordyceps sinensis (berk.) sacc medicinal mushrooms: a selective overview combined effects of cordyceps sinensis and methotrexate on hematogenic lung metastasis in mice inhibitory effect of cordyceps sinensis on spontaneous liver metastasis of lewis lung carcinoma and b16 melanoma cells in syngeneic mice activation of in vivo kupffer cell function by oral administration of cordyceps sinensis in rats antifibrotic effect of extracellular biopolymer from submerged mycelial cultures of cordyceps militaris on liver fibrosis induced by bile duct ligation and scission in rats pharmacological actions of cordyceps, a prized folk medicine the nephroprotective effects of the herbal medicine preparation, wh30+, on the chemicalinduced acute and chronic renal failure in rats isolation of galactosaminoglycan moiety (co-n) from protein-bound polysaccharide of cordyceps ophioglossoides and its effects against murine tumors the correlation between molecular weight and antitumor activity of galactosaminoglycan (co-n) from cordyceps ophioglossoides antitumor activity of protein-bound polysaccharide from cordyceps ophioglossoides in mice problems in the use of herbal and natural substances, with a specific example concerning the cardiovascular system growth inhibition of u937 leukemia cells by aqueous extract of cordyceps militaris through induction of apoptosis optimization of submerged culture conditions for the mycelial growth and exobiopolymer production by cordyceps militaris effect of agitation intensity on the exobiopolymer production and mycelial morphology in cordyceps militaris the coronamycin producer: a case of mistaken identity? ganoderma -a therapeutic fungal biofactory ganoderma disease of oil palm -a white rot perspective necessary for integrated control internal amplification controls have not been employed in diagnostic fungal pcr hence potential false negative results fungal enzyme inhibitors as pharmaceuticals, toxins, and scourge of pcr biochemical techniques for filamentous fungi solutions to penicillium taxonomy crucial to mycotoxin research and health inhibitory effect of cordyceps on carcinogenesis of the forestomach in mice cordyceps sinensis mycelium extract induces human premyelocytic leukemia cell apoptosis through mitochondrion pathway a cyclopeptide from the insect pathogenic fungus cordycepssp. bcc 1788 10-membered macrolides from the insect pathogenic fungus cordyceps militaris bcc 2816 optimum temperature and ph for mycelial growth of cordyceps nutans pat. (ascomycetes) studies on anti-tumor activity of crude drugs. i. the effects of aqueous extracts of some crude drugs in shortterm screening test militarinone a, a neurotrophic pyridone alkaloid from paecilomyces militaris (+)-n-deoxymilitarinone a, a neuritogenic pyridone alkaloid from the insect pathogenic fungus paecilomyces farinosus down-regulation of apoptotic and inflammatory genes by cordyceps sinensis extract in rat kidney following ischemia/reperfusion treatment of hyperlipidemia with cultivated cordyceps -a double-blind, randomized placebo control trial trade of cordyceps sinensis from high altitudes of the indian himalaya: conservation and biotechnological priorities effect of cordyceps militaris on the damage of rats induced by n-hexane profiles of nucleosides and nitrogen bases in chinese medicinal fungus cordyceps sinensis and related species pharmacological activities of paecilomyces japonica, a new type cordyceps sp anti-tumour and immuno-stimulating activities of the fruiting bodies of paecilomyces japonica, a new type of cordyceps spp antioxidant and immunostimulating activities of the fruiting bodies of paecilomyces japonica, a new type of cordyceps sp pharmacological basis of 'yin-nourishing' and 'yang-invigorating' actions of cordyceps, a chinese tonifying herb chemotaxonomy of pochonia and other conidial fungi with verticillium like anamorphs clinical study on application of bailing capsule after renal transplantation cordyceps sinensis and cultured mycelia phylogenetic classification of cordyceps and the clavicipitaceous fungi effects of cordyceps sinensis, rhubarb and serum renotropin on tubular epithelial cell growth dmso modifies structural and functional properties of rpmi-8402 cells by promoting programmed cell death rapid and specific detection of hydroxyl radical using an ultraweak chemiluminescence analyzer and a low-level chemiluminescence emitter: application to hydroxyl radicalscavenging ability of aqueous extracts of food constituents free radical scavenging and apoptotic effects of cordyceps sinensis fractionated by supercritical carbon dioxide effects of a watersoluble extract of cordyceps sinensis on steroidogenesis and capsular morphology of lipid droplets in cultured rat adrenocortical cells pharmacological functions of chinese medicinal fungus cordyceps sinensis and related species an experimental study on anti-aging action of cordyceps extract entomogenous fungi that produce 2,6-pyridine dicarboxylic acid (dipicolinic acid) immunomodulatory functions of extracts from the chinese medicinal fungus cordyceps cicadae anti-inflammatory and related pharmacological activities of cultured mycelia and fruiting bodies of cordyceps militaris lead poisoning caused by contaminated cordyceps, a chinese herbal medicine: two case reports structural analysis of a neutral (1 ? 3),(1 ? 4)-b-d-glucan from the mycelia of cordyceps sinensis medicinal herbal extracts -renal friend or foe? part two: herbal extracts with potential renal benefits optimization of submerged culture requirements for the production of mycelial growth and exopolysaccharide by cordyceps jiangxiensis jxpj 0109 studies on chemical constituents of cordyceps sinensis i application of statistically based experimental designs for the optimization of exopolysaccharide production by cordyceps militaris ng3 effects of cordyceps sinensis on natural killer activity and colony formation of b16 melanoma structure and antitumor activity of an alkalisoluble polysaccharide from cordyceps ophioglossoides augmentation of various immune reactivities of tumor-bearing hosts with an extract of cordyceps sinensis inhibitory effects of water extracts from fruiting bodies of cultured cordyceps sinensis on raised serum lipid peroxide levels and aortic cholesterol deposition in atherosclerotic mice antioxidant activity of the extracts from fruiting bodies of cultured cordyceps sinensis cordyceps sinensis mycelium induces ma-10 mouse leydig tumor cell apoptosis by activating the caspase-8 pathway and suppressing the nf-jb pathway effects of exopolysaccharide fraction (epsf) from a cultivated cordyceps sinensis fungus on c-myc, c-fos, and vegf expression in b16 melanoma-bearing mice efficacy of a pure compound h1-a extracted from cordyceps sinensis on autoimmune disease of mrl lpr/lpr mice h1-a extracted from cordyceps sinensis suppresses the proliferation of human mesangial cells and promotes apoptosis, probably by inhibiting the tyrosine phosphorylation of bcl-2 and bcl-xl effects of cordyceps militaris extract on angiogenesis and tumor growth antitumor activity of an extract of cordyceps sinensis (berk.) sacc. against murine tumor cell lines antitumour activity of cordycepin in mice hypoglycemic effect of cordyceps militaris pharmacological activities of stromata of cordyceps scarabaecola isolation and biological properties of polysaccharide cps-1 from cultured cordyceps militaris isolation, purification and identification of polysaccharides from cultured cordyceps militaris structural characterization and antioxidant activity of a polysaccharide from the fruiting bodies of cultured cordyceps militaris. carbohydr macrophage biospecific extraction and high performance liquid chromatography for hypothesis of immunological active components in cordyceps sinensis simultaneous determination of free ergosterol and ergosteryl esters in cordyceps sinensis by hplc anti-diabetic effects of ccca, cmess, and cordycepin from cordyceps militaris and the immune responses in streptozotocin-induced diabetic mice prosecutable function of cordyceps sinensis extracts for hepatic mitochondrial oxidative injuries in diabetic mice induction of hl-60 apoptosis by ethyl acetate extract of cordyceps sinensis fungal mycelium activation of murine peritoneal macrophages by natural cordyceps sinensis and its cultured mycelia lewis lung cancer of mice treated with cordyceps sinensis and its artificial cultured mycelia effects of the exopolysaccharide fraction (epsf) from a cultivated cordyceps sinensis on immunocytes of h22 tumor bearing mice immunomodulatory and antitumour effects of an exopolysaccharide fraction from cultivated cordyceps sinensis (chinese caterpillar fungus) on tumourbearing mice dynamical influence of cordyceps sinensis on the activity of hepatic insulinase of experimental liver cirrhosis influence of cordyceps sinensis on pancreatic islet beta cells in rats with experimental liver fibrogenesis epicoccins a-d, pipolythiodioxopiperazines from a cordyceps-colonizing isolate of epicoccum nigrum clinical and experimental studies on elimination of oxygen free radical of jinshuibao capsule in treating senile deficiency syndrome and its deoxyribonucleic acid damage repairing effects cordyceps sinensis -i as an immunosuppressant in heterotopic heart allograft model in rats cordymax cs-4 improves glucose metabolism and increases insulin sensitivity in normal rats inhibitory effect of cordyceps sinensis and cordyceps militaris on human glomerular mesangial cell proliferation induced by native ldl investigation on distribution and habitat of blaps rynchopetera fairmaire (coleoptera: tenebrionidae) in yunnan cordyceps sinensis in protection of the kidney from cyclosporine a nephrotoxicity inhibitory effects of alcoholic extract of cordyceps sinensis on abdominal aortic thrombus formation in rabbits mechanisms and therapeutic effect of cordyceps sinensis (cs) on aminoglycoside induced acute renal failure (arf) in rats effect of jinshuibao capsule on the immunological function of 36 patients with advanced cancer short-term curative effect of cultured cordyceps sinensis mycelia in chronic hepatitis b modulating effects of extractum semen persicae and cultivated cordyceps hyphae on immuno-dysfunction of inpatients with posthepatitic cirrhosis the scientific rediscovery of a precious ancient chinese herbal regimen: cordyceps sinensis part ii the scientific rediscovery of an ancient chinese herbal medicine: cordyceps sinensis part i immunosuppressive effect of cultured cordyceps sinensis on cellular immune response acknowledgements r.r.m. paterson is funded by grant sfrh/bpd/ 34879/2007 from fundac ßão para a ciência e a tecnologia, portugal. professor nelson lima, universidade do minho is gratefully appreciated for his unstinting support, as are other colleagues therein. l.e. gilbert, university of texas, austin was generous for allowing the use of his images of infected insects (fig. 1c-f ). marc stadler, university of bayreuth, germany is thanked for discussions. key: cord-352668-qjlqsb2k authors: cabello, francisco; sánchez, froilán; farré, josep m.; montejo, angel l. title: consensus on recommendations for safe sexual activity during the covid-19 coronavirus pandemic date: 2020-07-20 journal: j clin med doi: 10.3390/jcm9072297 sha: doc_id: 352668 cord_uid: qjlqsb2k sexual activity offers numerous advantages for physical and mental health but maintains inherent risks in a pandemic situation, such as the current one caused by sars-cov-2. a group of experts from the spanish association of sexuality and mental health (aesexsame) has reached a consensus on recommendations to maintain lower-risk sexual activity, depending on one’s clinical and partner situations, based on the current knowledge of sars-cov-2. different situations are included in the recommendations: a sexual partner passing quarantine without any symptoms, a sexual partner that has not passed quarantine, a sexual partner with some suspicious symptoms of covid-19, a positive sexual partner with covid-19, a pregnant sexual partner, a health professional partner in contact with covid-19 patients, and people without a sexual partner. the main recommendations include returning to engaging in safe sex after quarantine is over (28 days based on the duration one can carry sars-cov-2, or 33 days for those who are >60 years old) and all parties are asymptomatic. in all other cases (for those under quarantine, those with some clinical symptoms, health professionals in contact with covid-19 patients, and during pregnancy), abstaining from coital/oral/anal sex, substituting it with masturbatory or virtual sexual activity to provide maximum protection from the contagion, and increasing the benefits inherent to sexual activity are recommended. for persons without a partner, not initiating sexual activity with a sporadic partner is strongly recommended. sexuality is one of the aspects of personality in which the degree of intimacy and privacy is great. asking patients about their sex life often arouses misgivings and feelings of shame and/or guilt [1] . however, scientific evidence shows that successful sexuality benefits males and females physically and emotionally to, having a favourable impact on their quality of life. there is evidence that sexual activity has advantages for humans, including increasing our longevity [2] [3] [4] and improving our immune system, among others [5, 6] . additionally, successful sexual activity increases psychic wellbeing by improving mood, even in depressed and high-anxiety patients [7, 8] , falling asleep [9] ; stress [10] ; relaxation [11] ; physical form and providing a younger body image [12] thereby contributing to the prevention of post-traumatic stress and anxiety disorders [13] . sexual experience regularises the menstrual cycle [14] , relieves dysmenorrhea and reduces the risk of endometriosis [15] . sexual dysfunctions can cause some interpersonal conflicts by deteriorating either self-esteem or partner relationships [16] . additionally, it may constitute an early sign of some organic pathology such as cardiovascular [17] , neurological or endocrine diseases. there is also some evidence that sexual inactivity correlates with an increased frequency of cancer, need for major surgery, worsening mental health and the increase of cognitive decline and cardiovascular disease risk factors such as diabetes, hypertension and hypercholesterolemia [18] . the effects of sars-cov-2 on human sexual and reproductive function, including whether the virus passes the blood-testis and ovary barriers and whether there is any effect on sexual hormone production, are still unknown [19] . additionally, some studies are currently seeking to identify similar impacts across the different populations impacted by hiv [20] . in the current sars-cov-2 pandemic situation, sexual activity during quarantine could be a relevant aid in reducing the onset of post-traumatic stress and anxiety disorders that were experienced in other previous pandemic confinements. in canada, during the outbreak of severe acute respiratory syndrome (sars) in 2003, a high level of acute stress was observed among health workers [21] . ebola virus confinement in africa also increased suicides [22] and gender-based violence [23] . during the australian equine fever quarantine, a high level of anxiety was observed in 34% of those confined compared to 12% in those not confined [24] . in china, some of the health workers quarantined during the sars epidemic-maintained symptoms of post-traumatic stress disorder three years later [25] . sexual satisfaction is a good predictor of global life satisfaction in young people and older adults [26] . in a large survey on sexual health in spain [27] , people were interviewed about their motivation for sexual intercourse, and the vast majority pointed out that the main reason was either the search for emotional intimacy or to satisfy the need to love and be loved. additionally, sexuality, as a basic aspect of mental health, is a current topic of interest for clinicians and researchers [28] . there is some literature indicating the potential benefits of increased sexual activity during periods of forced isolation indicating that those who maintain frequent in-person, but not remote, social and sexual connections have better mental health outcomes [29] . given the psychologically negative repercussions of previous quarantines and the preventive benefits of healthy sexuality, it is reasonable to maintain one's safe sexual frequency. however, sexual intercourse requires close physical contact, and sars-cov-2 is very easily transmitted with this level of closeness [30] . physical contact entails high viral exposure. when sharing a home with a covid-19-positive person, the virus has been detected in 63.2% of room air samples and 66.7% of corridor air samples [31] . other known coronaviruses do not appear to be sexually transmitted, but sars-cov-2 has been found in bodily fluids such as the saliva, mucus, and faeces of infected people, albeit slightly less in urine (6.9%). some recent studies have reported the virus to be present in the testicular seminal duct [32, 33] compromising the safety of sexual intercourse by persistence for at least 2 weeks postinfection in urine, faeces and nasopharynx secretions. considering that 80% of those infected have mild symptoms or are asymptomatic, it is advisable to take some precautions at least during quarantine. the use of condoms and noncoital behaviour that does not involve direct contact with semen is highly recommended [34, 35] . the virus was very recently found in the vaginal discharge of an infected 65-year-old female even while she was receiving oral lopinavir/ritonavir plus remdesevir. after two previous negative results, the vaginal swab tested positive via a real time reverse transcriptase-polymerase chain reaction on days 7 and 20 from symptom onset [36] . this new finding raises the possibility that sexual intercourse could be an additional direct vector of infection, adding to the recent evidence of a likely faecal-oral transmission vector [37] , or indirectly by exposure of the rectal mucosa to saliva [38] . additionally, patients can persistently test positive on rectal swabs even after negative results from nasopharyngeal testing [39] . thus sexual, transmission may be possible despite apparent clinical recovery. using real-time reverse transcription polymerase chain reaction to routinely test for sars-cov-2 in faeces was recently recommended [37] . patients' sexual habits should be routinely investigated in order to avoid direct sexual practices if infected with covid-19. physicians should always address these questions in epidemiologic surveys on transmission routes in order to determine effective strategies to control infection. information about changes in sexual habits in the isolated population and in those infected by the virus is scarce so far. an increase in both, sexual desire and the frequency of sexual intercourse during the current covid-19 pandemic, compared to the previous 6-12-month period, has recently been shown, although the quality of sex decreased significantly [40] . however, another recent study showed that during the covid-19 outbreak, the frequency of sexual activity in china decreased significantly in men and women, accompanied by a lowering of risky sexual behaviours [41] . the main objective is to avoid contagion by covid-19 and, at the same time, maintain, as far as possible, active sexuality, given the multiple advantages that healthy sexuality brings according to scientific evidence. due to the ease of contagion and the lack of information about the possible transmission of sars-cov-2, a group of experts from the spanish association for sexuality and mental health, covering the fields of sexology, psychiatry, psychology and medicine reached a consensus. the multidisciplinary panel included four experts in the fields of family medicine, sexology, epidemiology, psychology and psychiatry. a bibliographic search was performed in the medline, scopus, psycinfo and web of science databases without time limits. after searching the information sources, two reviewers independently preselected potentially relevant references using the keywords; sexual * and coronavirus or covid-19 or pandemic (645 refs). after preselection the search was refined, duplicates were removed, and 83 refs were found. after reading the complete articles, those that would ultimately form part of the review were selected (38) . based on the current knowledge of the scientific literature, and considering the absence of either clinical guidelines or recommendations in this regard, the authors have developed a consensus on some specific recommendations to maintain safe sexual activity and to prevent the transmission of covid-19. the authors carried out three preliminary drafts until a full consensus of the final text was reached. in order to avoid the risk of contagion, the main recommendation is that tongue kissing and oral-sex relationships should be avoided. as indicated by recent recommendations from the new york department of health, "you are your safest sexual partner" [42] . thus, during the pandemic, and until the end of quarantine, it is a good time to devote oneself to autoerotic growth, which means, improving sexual health, and therefore mood, by training to optimise sexual response. under confinement with a sexual partner (not always a household partner), including the full diversity of partner types (homo, hetero, bisexual, nonheteronormative couples with polyamory or those who maintain living apart), there are several possibilities we can recommend to improve safe sexual activities by avoiding the risk of contagion to the greatest extent. to clarify the concept of "safe quarantine", our recommendation includes avoiding contact with high-risk populations during quarantine and avoiding restarting sex when in contact with a confirmed or highly suspected case. we contemplate two main scenarios: (a) partners living in the same household and (b) partner/s not living in the same household/starting a new relationship or polyamory. a. partner/s living in the same household. a sexual partner after passing complete asymptomatic quarantine. a sexual partner during quarantine. a sexual partner with suspicious symptoms of covid-19. a sexual partner that is positive for covid-19 5. a pregnant sexual partner. a sexual partner in contact with covid-19 patients. b. partner/s not living in the same household/starting a new relationship or polyamory. current information indicates that the average incubation period for covid-19 is 5.1 days (95% ci 4.5-5.8 days) and that 97.5% of cases developed symptoms in 11.5 days (95% ci: 8.2-15.6 days). the incubation period before the onset of fever is 5.7 days, and 98% of those infected have a fever in the first 12.5 days. only 1% develop symptoms after 14 days; there is a small but real possibility of symptomatology at 28 days after infection [43] . the recommendation is that after 28 days of confinement without symptoms and without external contact, sexual intercourse can be carried out according to the usual habits of the couple, but while increasing hygiene before and after sexual activity. this would be a good time to enhance sexual creativity by increasing communication about one's preferences and desirable experiences, possibly by sharing fantasies and testing new scenarios, engaging in erotic readings, or viewing erotic films. before starting sexual activity, proper handwashing becomes essential to avoid virus transmission through fomites [44] and by touching the t-zones (mouth, nose and eyes) of the sexual partner's face [45] . it is also advisable to recommend strategies to avoid self-contact with one's own t-zones, as it happens without realisation at an average of 23 times per hour [46] . in this scenario, we must take into account that the severity of covid-19 depends on several factors, including viral load. it is possible that one member of the couple could be contaminated but asymptomatic as seen in a sample of 157 cases in singapore where 6.4% of presymptomatic transmission was identified [47] . in this case, sexual intercourse could be a major risk of contagion. the recommendation, before 28 days, is that one can practice safe sex using penetrative positions from behind, avoiding kissing, oral and anal sex, and by always using condoms. if a partner shows any suspicious symptoms, such as fever (which may be intermittent), cough, diarrhoea, severe and unexplained tiredness, sore throat, anosmia, hypogeusia, or other symptoms associated with covid-19, he or she may be in the incubation period or suffering from a mild form of the disease. the recommendation would be to avoid direct sexual activity. as a substitute, performing self-stimulation (masturbation while simultaneously keeping the safety distance of approximately 2 m), narrating erotic fantasies, sharing visualisations of erotic scenes, or using erotic board games might be suggested. if a sexual partner has tested positive for sars-cov-2, having sex is a possible source of contagion, so it is advisable to isolate in separate rooms. however, it is advisable to continue to practice some kind of sexual activity while avoiding physical contact, as recommended previously. one can, for example, use telematic applications to maintain erotic activities. it should be considered that people with active covid-19 infections may not want to engage in any sexual activity (even remote/virtual connections,) depending on the severity of their symptoms and personal preferences. once symptoms disappear, there is a high probability of contagion, as the virus persists in nasal secretions for an average of 9.5 days and in stool from 11 to 16 days (20 days if treated with corticosteroids) [48] . the duration for carrying sars-cov-2 in covid-19 (between 16-28 days) can increase up to 33 days in those over 60 years old, as well as in very severe cases [49] . waiting for this period of time before having coital sex and maintaining the recommended precautions for confined stable sex partners without passing quarantine are recommended. it is currently unknown whether pregnant individuals are more likely to get sick with covid-19, and there is still little information on the severity of the disease [50, 51] . other coronaviruses and viral respiratory infections, such as the flu, may carry a higher risk of developing complications or serious illnesses in pregnant women. therefore, since this is a population at risk, precautions should be increased [52] . pregnant individuals do not appear to be at greater risk than the general population, except for the presence of other associated factors, such as preeclampsia, hypertension, diabetes, and uterine atony. these individuals should follow the same preventative measures as those indicated for the general population. fetal transmission is unlikely as no viruses have been reported in either the umbilical cord, amniotic fluid, breast milk, or neonatal pharynx smears. however, in addition to the usual records, fetal ultrasounds and cardio recordings are recommended. in the case of maternal infection, there is a risk of postpartum transmission, for which screening tests will be needed [53, 54] . however, a recent joint review, including 37 infected pregnant women and 38 newborns in iran/china/usa aged 23-40 years-old, a placental transmission during the pregnancy was observed, while keeping normal amniotic fluid, vaginal discharge and milk secretion. no teratogenic effects were shown. however, the risk of infection with breast milk, cough or other vectors was high for 5-7 days after birth, so it is advisable to avoid breastfeeding if the mother is infected [55] . the recommendation is to use the same identified sexual strategies for stable sexual partners confined without passing quarantine, thus avoiding kissing, oral sex, and anal penetration, and using "a tergo" and posterior positions. the health workers, caregivers, and professionals in contact with infected people in hospitals, residential centres, or similar areas could spend a great deal of time exposed to the virus, possibly without adequate protection. thus, there is a high risk of accumulating a large viral load. the recommendation would be to follow the same strategy used for contaminated people (i.e., virtual sex). under no circumstances should sex be performed in vivo with a new partner unless there is the certainty that the partner has been immunised against the virus. for those who do not have a partner and for very erotophilic people, nonheteronormative couples with polyamory or those who maintain living apart, the recommendation is abstinence from sexual intercourse until the incubation time passes without symptoms after starting the coexistence. additionally, it might be worth using sexting or virtual sex. some swingers clubs organise erotic encounters through platforms in order to have virtual group sex. the measures that are generally taken to contain sars-cov-2 have undoubtedly decreased the professional activities of sex workers, and fewer casual sexual encounters in this context may affect individual risks for hiv infection and other stis [56] . in order to minimise the risk of infection when sporadic sexual intercourse occurs, condoms, dental dams or similar protection methods should be applied. in summary, we recommend maintaining an adequate level of eroticism and sexual activity, both in confinement and throughout the pandemic. this, along with other factors such as physical activity, social distancing or smoking cessation, will allow the minimisation of some part of its negative effects. however, it is necessary to meet the standards established by international organisations for the prevention of covid-19 [57] , given that sexual relations, regardless of sexual orientation-heterosexual, homosexual or bisexual-can facilitate its spread. a summary of the recommendations can be seen in table 1 , considering the previous different scenarios contemplated. our recommendations are comparable to those made by institutions such as new york citi health [53] , but so far, no specific recommendations have been published on safe sexual practices to avoid the transmission of the virus by other sources of trusted information such as the centers for disease control and prevention (cdc) or the world health organization. there is certainty that the vast majority of the global population will survive this pandemic, but associated stressors, such as uncertainty about the end of the pandemic, fear of contagion, and economic and social impairment, facilitate a weakening of the immune system [58, 59] . as there is evidence that sexuality promotes the immune system and physical and mental health, it is advisable to maintain an acceptable degree of sexual intercourse. there is some evidence that healthy sexual activity improves the immune system and serves as a more preventive factor against sars-cov-2. on the other hand, healthy, safe and frequent sexual activity might be attenuating (even if there is no evidence yet) the negative psychological effects associated with the infection. unfortunately, at this moment, there are no vaccines available for sars-cov-2 and we do not yet know how durable immunity is after an infection. currently, the epidemic is still spreading, and there are no effective means to prevent the infection. as most vaccines are under design and preparation, there is currently no possibility of having "certainty" around immunisation status [60] . additionally, sars-cov-2 may cause neurologic and psychiatric effects such as delirium in some patients in the acute stage and depression, anxiety and post-traumatic stress disorder in the long term [61] . therefore, the protective benefits of sexual activity and the maintenance of a suitable couple relationship could be beneficial to avoid the psychiatric and psychological deterioration that is secondary to the pandemic. on the other hand, it is very relevant not to stigmatise healthcare workers and consider them contaminated people. however, if the risk is high due to being in permanent contact with covid-19 patients, precautions should be taken to avoid infections due to the existence of frequent asymptomatic cases. in this sense, pregnant women should not be stigmatised either, although additional precautions are necessary to avoid possible consequences after infection in the mother and the fetus. undoubtedly, the pandemic has implied some big changes in the sexual behaviour of special groups such as sex workers, where it is difficult to issue recommendations because it is their only livelihood in low-income countries. the recommendation of sexual abstinence in this group is aimed at promoting changes to sex work due to increased potential exposure to infection and various health concerns, however, they could be very difficult to apply. innovative strategies launched by the leaders of sex workers and health workers, implementing new health protection guidelines and clinical spaces, are especially instructive [62] . additionally, other vulnerable populations such as men who have sex with men (msm), require special attention. a recent survey in the usa on sexual habits in msm during the pandemic showed that half had fewer sex partners and most had no change in condom access or use with increased difficulties in accessing hiv testing, prevention and treatment services. in addition to our recommendations to reduce the risk of sexual contact, it is necessary to apply other strategies such as telehealth and mailed testing and prevention supplies to avoid increased hiv incidence among msm during the covid-19 pandemic [63] . the risk of unwanted pregnancies is a consequence of the covid-19 pandemic [64] , because sexual and reproductive health services have remained closed during the pandemic. fortunately, in some countries, remote resources have allowed them to continue their preventive function by prescribing contraceptive methods, but unfortunately, in many others it has not been possible. we consider both, sexual and reproductive healthcare to be a priority, guaranteeing access to contraception as well as adequate information on sexual and reproductive health. some sexual intercourse-related scenarios may not be covered by these recommendations, and additionally, there is a lack of full information on virus behaviour and its impact on the sex life of infected patients and asymptomatic transmitters. however, keeping sex life healthy and safe is one of the biggest challenges for health prescribers and for the general population in this pandemic. furthermore, the peculiarities of sexual minorities during the pandemic are not yet known so these recommendations may not be widespread. there is not enough information about how the treatments received by infected patients affect their sex life or whether there are modifications to desire, arousal or orgasm compared to the previous stage. although many uncertainties still remain, the study of the sex life of this population could clear up scarce information about how the virus affects both the sexual and reproductive life. however, the benefits of maintaining a healthy sex life, harnessing its qualities and preventing contagion should be a priority in all sexual activity scenarios. these recommendations do not cover all minority sexual behaviours due to the great heterogeneity of human sexuality. however, they include most diversity of sexual activity regardless of orientation: heterosexual, homosexual or bisexual. our proposals for confinement include maintaining a security period of 28 days, before the couple can restart their regular sexual practices, since 1% of persons affected by covid-19 develop symptoms after an incubation period of 14 days. therefore, although we know that this could be a difficult recommendation for many couples to accept, we consider it relevant to safeguard security as much as possible. another relevant limitation is related to some still unknown facts, such as the duration of the immunity in people who have overcome the disease. this would be crucial to avoid reinfections when patients are supposedly immunised but do not respect the preventive measures implemented. further research is needed to obtain more precise data on the pathophysiology, prevention and treatment of covid-19, as well as to better understand the duration and level of immunity that overcoming the disease confers. with more scientific evidence, these are transcendental factors when recommending actions, that could avoid contagion through sexual activity. progress in the understanding of these aspects, through well-designed observational, case-control and experimental studies, will allow us to modify and enrich our current recommendations. staying at home and complying with strict confinement standards is the most active way to fight sars-cov-2. strengthening the immune system along with other factors such as increased positive interpersonal relationships and rewarding occupational activities, will help us fight the negative effects of the pandemic. this must be done without forgetting to reserve time for erotic activity that fosters creativity and communication. finally, confinement protects us from the risk of contagion, but at the same time, it can be an opportunity for the cultivation of eroticism through communication, fantasies, and exploration of a new self and heteroerotic scenarios, taking into account the health precautions established by international organisations against covid-19. predictors of the longevity difference: a 25-year follow-up five-year mortality in a 70-year-old urban population in relation to psychiatric diagnosis, personality, sexuality and early parental death retrospective cohort mortality study of roman catholic priests effects of sexual arousal on lymphocyte subset circulation and cytokine production in man oxytocin may have a therapeutical potential against cardiovascular disease. possible pharmaceutical and behavioral approaches sexual function and depressive symptoms among male north american medical students intimacy and belonging: the association between sexual 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offices: a cincinnati area research and improvement group (caring) network study face touching: a frequent habit that has implications for hand hygiene presymptomatic transmission of sars-cov-2-singapore persistence and clearance of viral rna in 2019 novel coronavirus disease rehabilitation patients duration for carrying sars-cov-2 in covid-19 patients information about coronavirus disease european centre for disease prevention and control. what about pregnant women? q & a on covid-19 advisory: novel coronavirus 2019 (covid-19) recomendaciones para el manejo del recién nacido en relación con la infección por sars-cov-2 (covid-19) an analysis of 38 pregnant women with covid 19, their newborns infants, and maternal-fetal transmission of sars-cov2: maternal coronavirus infections and pregnancy outcoms clinical features and outcomes of pregnant women suspected of coronavirus disease 2019 the impacts of isolation measures against sars-cov-2 infection on sexual health world health organization. coronavirus disease (covid-19) pandemic dna methylation: conducting the orchestra from exposure to phenotype? human social genomics progress and prospects on vaccine development against sars-cov-2 psychiatric and neuropsychiatric presentations associated with severe coronavirus infections: a systematic review and meta-analysis with comparison to the covid-19 pandemic the effects of covid-19 on the health and socio-economic security of sex workers in characterizing the impact of covid-19 on men who have sex with men across the united states in april contraception and reproductive planning during the covid-19 pandemic this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-356062-7q5n4t97 authors: nan title: cumulative pharmacological activity index volumes 1-30 date: 2005-12-31 journal: studies in natural products chemistry doi: 10.1016/s1572-5995(05)80101-2 sha: doc_id: 356062 cord_uid: 7q5n4t97 publisher summary this chapter lists the important subjects on pharmacological activity that are discussed in the publication studies in natural products chemistry, volumes 1–30, such as abdominal constriction test, acanthoic acid, acetaminophen, parkinson's disease, photodynamic activity, prostaglandins, and oleanolic acid. the terms are mentioned along with the page numbers in which they are discussed in the publication. use of psycotria colorata in 30:205 19(4--+ 3)-abeo-8 c~, 13(s)-epoxylabda-4(18), 14-diene 29:102 activity in ebv bioassay system 29:102 19-nor-abieta-4( 18),8,11,13-tetraen-7one 29:100 activity in ebv assay system 29:100 abieta-8,11,13-trien-7-one 29:99 activity in ebv assay system 29:99 18-nor11, 7a, 15triol 29:99 activity in ebv assay system 29:99 11, 15, activity in ebv assay system 29:99 abieta-8,11,13-triene-7o~,l 5,18-triol 7acetate 29:99 activity in ebv assay system 29:99 abietic acid 29:99 activity in tpa assay system 29:99 activity in crg (+)-5-epi-acetomycin 10:447 antitumor activity of 10:447 2(x-acetoxy-14-hydroxy-15-iso-valeroyloxy-9-oxo-costunolide 29:89 activity in nfkb assay system 29:89 activity in tnf(x assay system 29:89 9cx-acetoxy-4 ~, 15-epoxymiller-1 ( 10)zenolide 29:89 activity in nfkb assay system 29:89 3~-acetoxy-813-isobutyryloxy-reynosin 29:87 activity in mam-2 assay system 29:87 activity in dif assay system 29:87 15-acetoxy-9 ~-hydroxy-8 [3-methacryloyloxy-14-oxo-acanthospermolide 29:89 activity in nfkb assay system 29:89 15-acetoxy-9cx-methacryloyloxy-8 ~hydroxy-14-oxo-acantho-spermolide 29:89 activity in nfkb assay system 29:89 3-acetoxycostunolide 29:89 activity in nfkb assay system 29:89 15-acetoxy-eremantholide b 29:89 activity in nfkb assay system 29:89 activity in tnf~ assay system 29:89 9~-acetoxy-miller-1 ( 10) 30:191, 192, 203 development of 30:192, 203 use of 30:191 analgesic effects 26:401, 403, 404, 406, 416, 418; 30:205, 208 5:512,747;10:78, 117;11:134;12:398;14:145;20:245, 712 ;21:21,108,109,190,207,213,214, 230,237,262,266,267,275,284,601, 615 ;23:41,126,164,235,258,355,468, 375,472,473,531,534,800;24:573, 856,857;26:226,1002;28:140,688; 30:328 11:130;12:192; 17:283 ;21:188,202,223,239,364,437; 23:649;24:953-954 21:186,192,197, 203,206,213,214,218,221,226, 227,233-235,223,238,240,389, 391,399,404,405,435,599-662; 22:626;23:3,4,271,341,344,348, 468,473,798,801354,368 ;26:227, 228,553,1098;28:4;66,473,475,686; 30:327,738,629 against candida albicans 30:327,629 from hypericum roeperanum 30:629 of (e)-8(17), 12-1abdadiene-15,16-dial 23:798 of (e)-8b, 17-epoxylabd-12-ene-15,16-deol 23:798 of (e)-8 [3,17-epoxylabol-12-ene-15,1619:511,748;21:132, 133,149,252,269,664,665 ;23:316, 533,534,687,704 ;24:742,743 ;30:226, 396,397 13:647;17:137,376;18:775;21:130, 134,153,152,312,591,594,690,616, 656,657;22:93,119,307,345,361; 23:158,297,512,602,832;25:43; 26:340,341,343,391,398,400,401, 403,404,406,415,418,482,556,558, 741,759,1035 ;27:660,858 ;28:200, 257,293 ;671,844;30:206,207,224, 225,292,320,689 21:674,744; 26:229,790,813,887,789,790,791, 800,805,807,810,811,821,828,830 -microbial activity 5:752;10:443; 15:327-339;18:776-778;21:153, 235,257,401,571,573,591,596,597, 599,606;22:626;23:94,153,180,233, 237,249,262,268,267,346,353,522; 24:64,69,206,330,331-334,402,403, 404,406,417,418,443,470,485,859, 1091,1092,1189;26:64,330,401,402, 1189;27:807;28:62,224,257,293; 28:698 22:308,321,345 ;23:395,534,535,742, 772,773,845 ;25:59;30:173,224,225, 289,320,321,524,561,743,745; 26:250,255,509,556,672,763,768, 887,1005,1006;28:4,16,17,63,179, 257,293 1:275,316-320-408; 17:17 ;21:137,405,420,435,436,638, 704,733;22:676;23:74,96,126,132, 139,395,445,522,696,721; 24:272-275 ;24:288,845,847,849; 25:43,430,431,438,450-453;26:56, 482,706;27:803;28:211,517,519,559, 560,566;30:3,28,29,55,56,58,59,64, 65,224,314,395,589,690,777,840 :461,563;10:608, 609,619,620; 17:146 ;21:132,148, 150,393,435,600,615,662,690,718; 22:307,361 ;23:233,268,233,346,395, 473,512,531,703;25:114;26:169,222, 224,226,344,452,741,759;27:482; 28:257,293 ;30:224,324-326,393,394, 395,398,401-405,407,410-412 132,148,162, 223,236,238,263,265,269,284,297, 366,388,390,410,419,421,431,571, 578,591,598,601,603,620,694 ;23:63, 93,95,108,111,129,126,107-152,153, 158,179,267,271,285,395-453, 455-485,491,497,642-643 ;739;24:25, 799,845 ;27:178,377,431,421,443, 693,778,801,924 ;24:353-376; 27:185-225,332,659;28:109,212,228, 229,340;29:743 ;30:4,26,172,175, 200,224,303,310,395,404,483,484, 485,495,496,497,499,562,588,603, 637,831,840,843, acaricidal enzymatic activity 24:995-998 tx-cembra-2,7,11-triene-4,6-diol 29:100 activity in ebv assay system 29:100 activity in pkc assay system 29:100 activity in skin-1 assay system 29:100 activity in odc assay system 29:100 ~-cembra-2,7,11-triene-4,6-diol 29:100 activity in ebv assay system 29:100 activity in skin-1 assay system 29:100 activity in odc assay system 29:100 of (-)-deoxypodophyllotoxin 30:589 of (-)-hemone 30:590 of (-)-nymphone 30:590 of (-)-yatein 30:590 of (+)-corytuberine 30:589 of (+)-epiaschantin 30:590 of (+)-epimagnolin 30:590 of (+)-epiyangambin 30:590 of (+)-hernovine 30:589 of (+)-laurotetanine 30:589 of (+)-magnoflorine 30:589 of (+)-malekulatine 30:590 of (+)-n-formyldehydroovigerine 30:589 of (+)-n-formylnornantenine 30:589 of (+)-n-formylovigerine 30:589 of (+)-n-hydroxyhemangerine 30:589 of (+)-n-methylhemangerine 30:590 of (+)-ovigerine 30:590 of (+)-ovihernangerine 30:590 of (+)-vateamine-2' [3-n134,136,150, 174,257,259,260,262,263,265,267, 269,270,271,273,274,284,311,364, 410,634,643,656,661,690,704,744; 23:96,97,187,218,238,249,250,253, 255,272,274,277,290,292,299,308, 317,701,274 ;24:294,352,740,867; 26:212-213,1185 as anti-fungal agents 24:406 2 [3,5-epoxy-5,10-dihydroxy-6c~ange lo y-lo xy-9 [3-is obutylo xygermacran-8ot, 12-olide 29:89 activity in inos assay system 29:89 activity in nfkb assay system 29:89 7 a, 8 c~-ep oxy-6 or-hydro xyabieta-9(11), 13-dien-12-one 29:99 activity in ebv assay system 29:99 9,10or-epoxy-9,10-seco-abieta4-o-phenyl-2-propionyl-neu5ac-cx2me 27:123 4-t-butyl-dimethyl-silyl ether 27:127 5-azido-5-deamino-neu5ac-c~2me 27:119 5-n-benzyloxycarbonyl-neu-cx2me 27:119 5-n-propionyl-neu-c~2me 27:119 7-deoxy-neu5 ac-c~2me 27:123,127 8-amino-8-deoxy-neu5 ac 27:121,122 8-azido-8-deoxy-neu5ac 27:121,122 8-tosyl-neu5acme ester 27:121,122 9-amino-9-deoxy-neu5 ac 27:121,122 9-amino-9-deoxy-neu5ac-c~2me 27:119 9-azido-9-deoxy-neu5 ac 27:121,22 9-azido-9-deoxy-neu5ac-c~2me 27:119 9-o-acetyl-neu5 ac-( ,590 activity of (-)-deoxypodophyllotoxin in 30:589 activity of (-)-hemone in 30:590 activity of (-)-nymphone in 30:590 activity of (-)-yatein in 30:590 activity of (+)-corytuberine in 30:589 activity of (+)-epiaschantin in 30:590 activity of (+)-epimagnolin in 30:590 activity of (+)-epiyangambin in 30:590 activity of (+)-hernovine in 30:589 activity of (+)-laurotetanine in 30:589 activity of (+)-magnoflorine in 30:589 activity of (+)-malekulatine in 30:590 activity of (+)-n-activity of hydroxyhernangerine in 30:589 activity of (+)-n-formylnornantenine in 30:589 activity of (+)-n-formyldehydroovigerine in 30:589 activity of (+)-n-formylovigerine in 30:589 activity of (+)-ovigerine in 30:590 activity of (+)-ovihernangerine in 30:590 activity of (+)-vateamine-2'13-noxide in 30:590 activity of 7-formyldehydrohernangerine in 30:589 activity of 7-formyldehydronornantenine in 30:589 activity of 7-formyldehydroovigerine in 30:589 activity of 7-hydroxy-6-methoxy12-hydroxy -6,7-seco-abieta-8,11,13trien-6,7-dial 29:99 activity in ebv assay system 29:99 7-hydroxy-6-methoxy-1-methyliso22:510,512,513,516,518,519, 521,522,525-529,531,534 achyranthes aspera l 9,459,503,753,754,311; 17:313,441 ;20:108-113,273,613; 24:215,228-232,503-509,739-798; 26:229,790,813,887;30:560,562,567, 570,590 chemo-differentiation therapy 30:498 of acute monocytic leukemia chemopreventive activity 30:591,597, 766 in mouse mammary organ culture model 30:591 in vitro 30:597 in vitro bioassays for 30:591 of chlorella vulgaris 30:766 ofisoquinoline alkaloids 30:597 of lignans 30:597 chemopreventive agent 26:219 genkwanin as chemopreventive efficacy 27:386 of limonene carcinogenesis 27:386 chemoprophylaxis 26:674 chemosensitizers 30:595 antimalarial drugs as 30:595 chemotaxonomic markers 26:1128 chemotherapeutic agent 30:5,330,742 atovaquone 30:330 for colon cancer 30:5 for multi-drug resistant cancers 30:5 for ovarian cancer 5 for taxol-resistant breast cancer chemotherapeutic intervention 30:397 targets for chemotherapy drug 27:436 antitumor activity of 27:436 chest pain 30:203 use of ginseng root in 30:203 childhood disintegrative disorder (cdd) gram-negative bacteria 30:693 maytenus species against gram-positive bacteria 23:68;30:629, 693 actamycin against 23:68 antimicrobial activity of 30:629 inhibitor maytenus species against grandiflorolic acid 29:100 activity in ebv bioassay system grandiflorolic acid angelate 29:100 activity in ebv bioassay system grandisin 26:230 trypanosomicidal activity of grandmal seizures granulocyte phagocytosis 26:47,55 ofjenisseensosides c 26:47,55 ofjenisseensosides d green tea 27:417,420 biological activity of 757 effect of general analogues on pgf2 na-induced contraction griseofulvin 24:934 as anti-fungal agents grob-type fragmentation 24:728,731 growth 30:61 of tumor cells 30:61 growth inhibition 17:142 growth inhibitor activity 7:407 guaiazulene 5:363,369 as an anti-inflammatory agent guidimacrin 27:831 antitumor mechanism 584 neurodegeneration 30:224 reperfusion injuries 30:224 respiratory disorders 30:224 role of free radicals in 30:224 human growth hormone human hek 293 cell line 30:781 biological response of human hl-60 leukemia cells 30:496 differentiation of 30:496 inhibition of 30:496 proliferation of human leukemia cells 18:269 human leukocyte elastase 16:727 inhibitor human leukocyte elastase (hle) 30:830,831,843 homologous sequences of 30:831 plasminogen activator (u-pa) 30:830 human lung carcinoma 23:290;30:5 discodermolide against human lung carcinoma) 30:691 phenolic triterpenes against 30:691 human medulloblastoma 23:290 human melanoma 23:255 human mesangial cell proliferation 30:308 in vitro human monoblastic leukemia cells 30:498 effect of hydroxyurea (hu) 30:498 growth inhibition of 30:498 human monoblastic leukemia u937 cells 30:498 growth inhibition of human neuroblastoma nbla-n-5 cells tamoxifen activity against 12:390 human neuroblastoma sh-sy-5y cells 12:390 human neutrophils 12:390 human neutropil protein kinase c 12:389 human onchocerciasis 12:9 ivermectin for human papilloma virus 30:394 pathogenesis of 30:394 cause of hepatocellular carcinoma human papilloma virus (hpv) 23:97 human papilloma virus type 11 30:406 microbicidal compound against human papilloma virus type 40 30:406 microbicidal compound against 30:406 human platelets 12:390 study of protein kinase c in 12:390 by staurosporine 12:390 human rotavirus (hrv) 30:406,412 cause of dehydrating gastroenteritis 30:406 member of 30:406 reoviridae type of human serotonin transporter gene (slc6a4) 30:376 regulation of neuronal activity human spermatozoa motility 21:675 human synovial pla2 25:697 inhibitor anti-glycemic effects 24:902-904 anti-hyperglycemic effects of -o-methyl-20-hydroxyecdysone activity in 29:28 pinnatasterone activity in 29:28 polypodine b 2-o-cinnamate activity in muscarinic achr antagonists 21:56 muscarinic antagonist 22:19 muscarinic receptor 21:95 muscarinic receptor antagonists 22:734 muscarinic-l,2-receptor 21:95 muscle pain 24:898 aconitine for relieving activation effects of 24:906 medicinal uses of 24:906 protein kinase c activation effects 24:906 muscular p-388 (murine lymphocytic leukemia) 30:588 activity of (-)-deoxypodophyllotoxin against 30:588 activity of (-)-yatein against p-388 lymphocytic leukemia cell 25:764 p388 mouse leukemia 12:390 staurosperine activity against p-388 murine leukemia cells 21:411 pachyman 5:288,317 antitumor activity of 5:317 pachyrrhizus erosus 24:779,822-826 anti-feeding activity of 24:825 pachyrrhizus palmatilobis 24:822 biological activity of 24:823 paclitaxel 240 (+)-acetoxypinoresinol dimethyl ether as 26:241 arctigenin as 26:241 arctigenin methyl ether as 26:241 chamigrenal as 26:244 cinnamophilin as 26:240 dehydroschisandrol a as 26:245 denudatin as 458 fptase inhibition by 24:458 paw oederma 30:207 inhibition of 30:207 2-pectenotoxin 19:580 cytoxic activity of 19:580 pectinasic pectolinaringenin 30:725,748 antispasmodic activity of 30:748 pedunculariside 29:84 activity in cox assay system 29:84 activity in epp assay system pelargonidin (anthocyanidin) 29:585 effects on lela 29:585 effects on penates sp. 28:713 as ~-glucosidase inhibitor 28:713 penicillium turbatum 11:194 antibiotic a 26771b by 11:194 penstemide 7:441 activity against p-388 lymphocytic leukemia penstemon rosseus 24:838 anti-fungal activity pentacyclic triterpenes 30:207 protein kinases inhibition by 564 in lipid autoxidation 3,4,6-p enta-o-gallo yl-~ -d-g luc o s e (galloyl ester) 29:589 effects on 229 biological activity of 28:229 8-prenyleriodictyol 28:229 biological activity of 28:229 prenylflavones 28:225 anti-human immunodeficiency virus (hiv) activity of 28:226 prenylfiavonoid 30:207 antinocicieptive effect of antitumor promoting activities of 29:720 as cytomegalovirus protease inhibitors 29:720 gastro-intestinal problems 29:720 use in hodgkins disease 29:720 use in infections 29:720 use in lupus 29:720 use in osteomyelitis 29:720 use in parkinson's disease 29:720 use in psoriasis 29:720 use in respiratory problems 29:720 use in skin ulcerations 29:720 use in syphilis 29:720 6-prenylnaringenin prevention by chitosan 27:436 of myelotoxicity 27:436 primary biliary cirrhosis 25:463 primary granulosa cells 25:269 apoptosis in 25:269 primary solid-tumor growth 30:64 effect of triterpenoids on 30:64 pristimarin 30:692 cytotoxic activity of prl1 & prl2 proproteins 29:592 effects on cysteine protease 851 probes 27:332 activity of 27:346 procurcumenol 29:90 activity in tnfct assay system procyanidin b 1 (dimeric flavan-3-ol) 29:580 effects on ace procyanidin b2 (dimeric flavan-3-ol) 29:580 effects on ace procyanidin b-5 3,3'-di-o-gallate 29:580 effects on ace procyanidin c2 (trimeric flavan-3-ol) 29:580 effects on ace 29:580 procyanidin polymer (flavan-3-ol polymer) 29:580 effects on ace prodrug monotherapy 21:157 natural anthracyclines for 21:157 prodrugs 21:157 of natural anthracyclines pro-inflammatory cytokines 25:461 pro-inflammatory substances 30:206 in chronic painful inflammatory diseases 30:206 proliferation 30:368,378 effect of serotonergic neurotransmission on 30:368 serotonin action on 30:378 prolysine 25:388 prophylactic effects pro-nociceptive prostaglandins 30:192 in inflammation pro-nociceptive transmitter 30:194 release inhibition of 30:194 prooxidant activities 30:745 in thiobarbituric acid assays 30:745 of chemiluminescence 30:745 of tert-butyl hydroperoxide 30:745 prooxidant property 30:524 of carotenoids 30:524 prophylactic drugs prostaglandin e2-dependent flavonoids cytoprotection effect pgh2) 25:595 prostaglandin inhibitors 30:192 clinical use of 30:192 use in rheumatism 30:192 use in osteoarthritis 30:192 use in headache 30:192 use in dental surgery anticancer clavulones 16:366 as antiviral compound 24:541-544 as pain mediator 30:192 in mast cells 30:192 nociceptive effect of prostate cancer prevention 30:525 role of lycopene protease inhibitor proteins from plants 29:596 effects on metallo protease inhibitors 24:487,488 567 cereal bifunctionals as 29:567 chymotrypsin as 29:568 destructive potential of 29:568 in alzheimer's disease 29:567 in angiogenesis 29:567 in cancer inflammatory disease 29:567 in protozoal infection 29:567 in viral infection 29:567 kunitz as 29:567 mustard family of 29:567 of metallocarboxypeptidase 29:567 ofserine protease 29:567 pepsin as 29:568 phytocystatins as 29:567 potato type 29:567 proteins as 29:567 serpin as 29:567 trypsin as 29:568 proteases 24:1005;29:567 enzymatic activity 24:1005 in apoptosis 29:567 in blood clotting 29:567 in cell division 29:567 in digestion 29:567 in extracellular matrix digestion 29:567 in inflammatory responses 29:567 protein kinase a (pka) 192 in regulation of ionotropic receptors protein kinase activity 28:677 ofxestoquinolide protein kinase c (pkc) activator 30:70 protein kinase c inhibition 24:573; 25:46,488 by xestocylamine 24:573 protein kinase inhibition 30:207 by pentacyclic triterpenes protein phosphatase inhibitors 27:874 protein phosphatases 25:707 inhibitor of 25:707 protein synthesis 30:407 inhibitor of 30:407 protein transduction domain (ptd) proteinaceous receptor 25:371 proteinase activity 21:150 protein-coupled receptor 27:821 g-proteine kinase c activity 21:638 proteins protein-tyrosine kinase activity 27:842 offlavonoid aglycones 27:842 of glycosides 27:842 ofkoelreuteria henryi protein-tyrosine kinase inhibitory activities proteolytic activity 30:841 proteolytic systemin inactivation protium kleinii 30:206 anti-inflammatory effect of 30:206 antinociceptive effect of 30:207 protocatechuic acid (3,4-dihydroxybenzoic acid) 29:589 effects on proton pump inhibitors (ppis) 25:612 proton-translocating nadh:q oxidoreductase 28:435 high-affinity inhibitors protozoocidal activity 30:329,741,742 against leishmania 30:329 against plasmodium 30:329 against tryponosoma 30:329 genus baccharis 30:741 in vitro 30:329 of neo-clerodane diterpenoids 30:742 ofquinone derivative 30:329 protozoocidal compounds 30:740 use of proxyelocytic leukaemia (hl-60) prunella vulgaris 30:401 antiviral activity of psammaplin a (bisprasin) 28:693 effects on bacillus subtilis psammaplysilla purpurea 28:693 antimicrobial activity of 28:693 as tyrosine kinase inhibitor pseudobersana mossambicensis 20:478 bioactive steroids from pseudoguaiane 11 a,13-dihydrohelenalin acetate 29:90 activity in cro assay system 627 against cytomegalo virus 30:627 against human immunodeficiency virus-1 30:627 against influenza virus 30:627 as virucidal agents 30:627 retroviral activity of 7:421 pseudolaric acid b 13:653 pseudomonas aeruginosa 30:739 antibacterial activity against 30:739 antifungal activity against pseudorabies virus 30:404 in vitro replication of 30:404 role ofheparin 30:404 psii inhibitors 26:368,371 psk in mitosis 25:377 psk-receptor 25:378 psk-a activity 25:378 psoriasis 30:484 use of lc~,25(oh)2d3 30:484 psychiatric disorders 30:369 anorexia 30:369 bipolar disorder 30:369 bulimia 30:369 effect of circadian activity on 30:369 obsessive compulsive disorder 30:369 panic disorder 30:369 schizophrenia 30:369 seasonal effective disorder 30:369 unipolar depression 30:369 psychodelic drugs 26:820 cocaine as 26:820 morphine as 26:820 semisynthetic lsd as 26:820 psychovegetative disorders 22:643,684 psycotria colorata 30:205 to relieve abdominal pain puberulin a 26:243,244 as paf-induced inhibitor pulegone 29:83 activity in am assay system 29:83 pulmericin 16:299 antifungal activity of 16:299 antileukemic activity of 16:299 cytotoxic activity 16:299 pulmonary vascular injury 30:70 role of serine protease in 30:70 role of thrombin pumiliotoxin alkaloids 27:238,239 alio-ptx 323 b alkaloid 27:249,250 biological activity of punta toro virus 30:411 treatment of 30:411 purifying blood 26:50 herniaria hirsuta in 26:50 purine receptors putative receptor proteins 25:398 putative satiety factor 24:910 effect of cholecystokinin 24:910 pyranocoumarin 27:429 biological activity pyranoid carbasugars 29:466 as cellular messengers 29:467 as insulin release mediators 29:467 biological activities of 29:466 effects on glycosidase enzyme 29:467 effects on glycosyltransferase enzyme 29:467 galactosyltrans ferase inhibition by pyridoacridine alkaloids 28:639 cytotoxic effects on kb cells 524 glycosidase inhibitors of 10:524 pyrrolomycin antibiotics 25:794 pytressin-induced coronary spasm antileukaemic activity of 4'-quercetagetin (6-hydroxy quercetin hexahydroxyflavone) (flavonol) 29:573 effects on 4'-pentahydroxyflavone) 29:578;30:208,749 analgesic effect of quercetin 3-o-c~-l-arabinopyranoside 28:64 anti-oxidant activity quercetin 3-o-c~-l-rhamnopyranoside 28:64,65 anti-oxidant activity of quercetin 3-o-[3-d-galactopyranoside 28:64 anti-oxidant activity o-galloyl)-glucoside 29:580 effects on ace quercetin-3-o-ot-l-rhamnopyranoside 28:64 anti-oxidant activity quercitrin (quercetin-3-rhamnoside) 29:581 effects on ace quillaia 26:55 of adjuvant activity 26:55 quinidine 25:347 quinine 25:344 290 antitumor activity of 10:117 inhibitor of dna quinoline drugs 25:348 quinoline drug family quinoline-based antimalarials 25:344 quinoline-ring antimalarial drugs 25:327 quinolines 25:327 quinolizidine derivatives 27:284,285 testing against mycobacterium tuberculosis 541-544 as antiviral quinone methides 30:694 antiplasmodial activity of 30:694 quinones rabidaea platyphylla 22:513 for epilepsy 22:513 rabies virus 30:409 inhibitor of 30:409 rabies virus infection 30:409 in chicken-embryo-related cells 30:409 rabies virus replication 30:409 in nerve fibers rachitic bone healing potencies 484 of vitamin d2 30:484 of vitamin d3 30:484 of vitamin d4 30:484 of vitamin d5 30:484 of vitamin d6 30:484 of vitamin d7 radical scavenging activity 23:362 of coumarin 23:335 radioligands 30:814 from brain membrane synaptosomes ranitidine 25:612,617 ranp-triggered signal transduction raphe nuclei 30:368 role in cognitive functions ras oncogenes 22:621 rat brain synaptosomes 30:807 p-affinity rat mammary carcinogenesis model 30:592 cancer chemopreventive activity rat serum vitamin d-binding protein (dbp) 30:494 binding assay for rebaudioside a 29:101 activity in tpa bioassay system tpa bioassay system 8-receptor 22:296 ~-receptor 22:296 8-receptor affinity 30:814 binding properties of 30:814 ~t-receptor affinity 30:814 binding properties of 30:814 of glycopeptide 30:814 8-receptor agonists k-receptor agonists 30:797,806 pharmacological activity of -receptor agonists 30:797 pharmacological activity of 30:797 8-receptor antagonists 30:797 pharmacological activity of k-receptor antagonists 30:797 pharmacological activity of 30:797 ~t-receptor antagonists 30:797 pharmacological activity of 30:797 receptor binding 27:377 receptor binding assay 30:810 in vitro 30:810 mouse vas deferens (mvd) activity in 30:810 of guinea-pig ileum (gpi) 30:810 receptor binding properties 30:815 of tyr-d-ala-phe-[[3-d-glc(oac)4]tyr-pro-ser-nh2 30:815 of tyr-d-ala-phe-asp-val-val-gly-nh2 (deltorphin c) 30:815 of oac)4]-gly-nh2: 30:815 of tyr-d-ala-phe-gly-tyr-pro-ser-nh2 (deltorphin c) 30:815 of tyr-d-ala-phe-gly-tyr-pro-thr([3-d-glc)-gly-nh2 30:815 of tyr-d-ala-phe-gly-tyr-pro-thr c )-val-vai-gly-nh2 receptor tyrosine kinase (rtk) 25:512 receptors 27:363;30:421 and cell-cell adhesion 30:421 and cell-cell communication 30:421 antigenic determinants of 30:421 for bacteria 30:421 ofchiral proteins 27:363 with enzymatic activity 27:824 without enzymatic recombinant prourokinase (pro-uk) 30:839 as fibrinolytic enzymes recombinant tissue-type plasminogen activator 30:839 as fibrinolytic enzymes 30:839 red wine 27:430 biological activity of 27:430 redox enzymes redox inhibitors 30:225 of 5-1ipoxygenase repandusic acid (hydrolysable tannin) 29:573 effects on hiv-1 protease 29:573 repellent properties 28:395 of amblyomma variegatum 28:395 reperfusion injuries 30:224 role of free radicals rescorcinolic lipids 30:165-167 effects on enzymatic activity 30:165-167 interaction with proteins reserpine 25:538 as immunosuppressive drug resistance-modifiers 30:595 antimalarial drugs as resistant plasmodium species 30:595 in chloroquine sensitivity resorcinolic lipids 30:119,158,160,162-164 as antifungal fluids 30:160 as growth reulators 30:162-163 as hair restoration-lotion 30:160 used in gingival infections respiratory ailments 24:847 respiratory depression 30:799 due to analgesic alkaloid 30:799 respiratory disorder 30:224 role of free radicals in 30:224 respiratory infection 30:408 respiratory syncytial virus 30:395 respiratory toxins 24:1002,1063 respiratory tract infections 26:50 herniaria hirsuta in 26:50 response decay mechanism 25:491 response modifiers resveratrol 27:430 biological activity of 27:430 resveratrol 28:582 effects in llc-bearing mice 28:582 effects on tumor 597 treatment of 30:597 use of hernandia moerenhoutiana in 30:597 use of hernandia nymphaeifolia 596 antimalarial activity of 30:595 antiplasmodial activity of 30:596 as platelet aggregation inhibitor 30:594 effect on antiplasmodial activity 30:596 retinal 29:111;30:520,521 activity in am bioassay system 29:111 in visual signal transduction 30:520 retinoic acid 29:111 activity in am, ebv, skin-1, odc bioassay systems 29:111 retinoic acid 30:493,498,521 in cell differentiation 30:521 in development 30:521 in growth regulation 30:521 retinoid retinol 29:111 activity in am retinol acetate 29:111 activity in am bioassay system retinol palmitate 29:111 activity in am bioassay system 29:111 retroviral enzymes 30:397 inhibitor of 408 causative agent of immunodeficiency syndrome 30:394 inhibitor of rett's disorder (rtt) 30:372,373,384 by mutations in mecp2 gene 30:373 genetic basis of 30:373 reutericyclin 28:127 as antibiotic reverse transcriptase 30:226,395,397 role in hiv replication cycle 30:226 role in viral replication rhazinilams 29:362 as microtubule poisons rheedia gardneriana 30:207 antinocicieptive effect of 30:207 rheumatism 26:50 herniaria hirsuta in 26:50 rheumatism 30:192,691 use of non-steroidal antiinflammatory drugs (nsaids) 30:192 use of prostaglandin inhibitors 30:192 rheumatoid arteriosclerosis 30:70 rheumatoid arthritis 21:152;22:310; 26:170,486;30:70,204,225 in treatment of cph82 26:170 in rats 30:204 reagent-induced 30:204 treatment of 30:225 use of kalopanax pictus rhoifolin (5, 4'-dihydroxy -7-rhamnosyl-glucosyl-flavone) 29:573 effects on hiv-1 protease 29:573 rhombenone 24:456 fptase inhibition by ricinoleic acid 30:193 analgesic action of 30:193 rickettsial pathogens 28:394 cowdria ruminantium as rifamycin-type macrolides 24:545,546 antiviral microbial-derived compound 24 rift valley fever infection 30:411 treatment of rimantidine 27:108 for treatment of influenza in infections 814 activity of deltorphin b in 30:807 of glycopeptide 30:814 rna virus 30:394 arenavirus type of 30:394 bunyavirus type of 30:394 coronavirus 30:394 influenza virus 30:394 measles virus 30:394 parainfluenza virus 30:394 picornavirus type of 30:394 rabies virus 30:394 reovirus type of 30:394 rotavirus type of rna-dependent rna polymerase activity 30:407 virion-associated rna-directed dna-polymerases 29:127 inhibition by sf-9 cell line 29:33 activity in mam-3assay system 29:100 antileukemic activity 8:18 sarcoptes mites 28:410 cause of human scabies 28:410 symptoms sarcoptes scabiei 28:409 cause of mange mites 28:409 cause of scabies sargasm horneri 30:400,405 anti-cmv activity of 30:405 polysaccharide (ps) from 30:400 sarkomycin 8:150 as antitumor agent 8:150 satratoxins 30:743 antiviral activity of 204 scavenging effects 23:443 of tannic acid 23:443 sch 207278 24:451 fptase inhibition by 24:451 schisandra 26:183 display platelet activating factor antagonist activity 26:183 effect on cardiovascular system 26:183 effect on heart rate 26:183 for treatment of hepatitis schisandrol a 26:245 as paf-induced inhibitor schistosomiasis (bilharzia) 7:405 schistosomicidal activity 1:545 schizonticidal drug 26:837 schizophrenia 101 activity in crg bioassay system 29:101 from scoparia dulcis scropolioside a 29:84 activity in tpa assay system 29:84 scrovalentinoside 29:84 activity in tpa assay system scutellaria baicalensis 30:55,56,69, 254,289 in allergric inflammatory disease 431 sdb 21:695 inhibitory effect of 21:695 sdc 21:695 inhibitory effect of 21:695 sdz-249-665 30:201 antinociceptive activity of 30:201 anti-hyperalgesic activity of 30:201 seasonal affective disorder 30:369 effect of circadian activity on 30:369 seaweeds 30:404 anti-cmv activity of activity in ebv assay system 29:100 1,2-secoemetine derivatives 6:485 amoebicidal activity of 6:485 secoiridoids 26:330 antimicrobial activity of 26:330 second messenger 25:516 secondary metabolites 28:3,432,617 acaricidal activity of 28:432 biological activity of 28:617 secondary metastatic tumor growth 30:64 effect of triterpenoids on 599 arnica as 22:559 artocarpus heterophyllus as basilicum polystachyon for 22:514 sedative activity 21:673 sedative in convulsions 22:518 crocus sativus l. for 22:518 sedative in epilepsy 22:535 withania somnifera l. as 22:535 g-selective agonist 30:807 dermorphin 30:807 8-selective antagonist tyr-tic-phe-nh2 selective antitumor activity 15:355 selective cytotoxicity 13:648 8-selective opioid agonist 30:817 deltorphin b 30:817 g-selective opioid peptide 30:802 8-selective opioid peptide of ici 19944 30:811 ofk opioids 30:811 8,g-selectivity 30:819 in vivo 30:819 of glyco analogues semilicoisoflavone b 28:229 biological activity of 28:229 semiochemicals semisynthetic lsd 26:820 as psychodelic drug 10-dihydro xy-7-methoxy-naphtho sendai virus infection sensory neurons 30:194 hyperpolarisation of 30:194 8er264 inhibitors serine 27:850 activitiy of serine 30:836 c~2-macroglobulin inhibition by serine protease inhibitor proteins 29:617 arrowhead pis api-a & api-b as cp-thionin as 29:617 effects on barley malt cysteine endoproteinases 29:617 effects on chymotrypsin 29:617 effects on subtilisin 29:618 effects on subtilisin eleusine double-headed try-~x-amylase inhibitor i-2 as 29:617 from brassica nigra 29:618 from brassica napus 29:618 from cassia fistula 29:617 from eleusine coracana 29:617 from hordeum vulgare 29:617 from phaseolus angularis from sagittaria sagittifolia 29:617 from sinapis arvensis 29:618 from vigna unguiculata 29:617 hordeum lipid transfer proteins as 840 blood clotting factors as 29:570 cathepsin g as 29:570 chymase as 29:570 chymotrypsin as 29:570 granzymes as 29:570 in angiogenesis 29:570 in blood clotting 29:570 in cytosolic proteolysis 29:570 in digestion 29:570 in inflammation 29:570 in proprotein processing 29:570 in tissue remodelling 29:570 kallikrein as 29:570 plasmin as 29:570 prolyl endopeptidases as 29:570 role in blood coagulation 30:70 role in platelet activation 30:70 role in pulmonary vascular injury serine proteases (subtilases) 25:388 cysteine proteinase inhibitor 25:370 poly phenol oxidase 25:370 serine proteinase inhibitor serine proteinase inhibitor i 25:370 as defense proteins serine-threonine-specific receptor protein kinases serotonergic innervations 30:377 autoradiographic imaging 30:377 in cerebral cortex 30:377 in neonatal rats by immunohistochemical techniques 30:377 physiological role of 377 serotonergic neurons 30:368,376 in raphe nuclei 30:376 role in 30:regulation of neurogenesis 627 as hyperforin inhibitor 30:627 as neurotransmitter 30:367 effect on synaptogenesis 30:378 in apoptosis 30:367 in cell proliferation 30:367 in presynaptic neuron 30:381 morphogenic properties of 30:367 neurochemical connection of 30:368 receptor activity of -ht) receptors 30:376,378, 382,383 in brain 30:383 in limbic brain regions 30:383 pharmacological aspects of serotonin (5-ht) re-uptake inhibitors 30:368 mechanism of serotonin (5-ht) synthesis 30:370,376, 377 in autistic children serotonin (5-ht) 1ar receptor antagonists 30:378 in posmatal treatment -ht)sa receptor 30:375 in brain development 30:375 in phenotypic behavioral irregularities 30:375 in purkinje cells 30:375 mrna expression of 30:375 serotonin action 30:378 on apoptosis 30:378 on maturation 30:378 on neuronal functioning 30:378 on proliferation serotonin agonists 25:531 anticomplementary activity of 25:46 role of cyclic amp-dependent protein kinase 25:46 delayed-type allergy suppressant activity of 25 serotonin synthesis inhibitor 30:378 parachlorophenylalanine as serum tc 27:420 biological activity of 27:420 serum transaminase 27:415,417,420 biological activity of 27:415 serum transaminase activity 25:469 of curcumia longa l serum triglyceride 27:404 biological activity sesquiterpene lactone 7:426 sesquiterpene quinones 5:429 antimicrobial activity of 5:429 cytotoxic activity of sesquiterpenoids drimane-type synthesis 29:127 avian myeloblastosis virus (amv) inhibition by 127 by jauch 29:127 sex pheromone sexual potency 28:4 ofbroussoflavonol g 28:4 sgot 30:292 activities of 30:292 sgpt 30:292 activities of 21:654 shikimic acid derivatives 29:478 antitumoral activity of 29:479 shinpterocarpin 28:229 biological activity of 28:229 sialic acids 27:103 biological function of 27:103 model for sialyltransferase activity 16:81 side effect 30:194,799 of analgesic alkaloid 30:799 of opioids sigmoidin a 28:229 biological activity sigmoidin b 28:229 biological activity of 28:229 signal reception 30:377 in autistic children 30:377 signal transduction mechanisms 25:518 signal transduction pathways signal transductors/activators of transcription (stats) 25:519 silandrin 26:255 as antihepatotoxic agent 26:255 silchristin 26:255 as antihepatotoxic agent 26:255 sildenafil 30:155 used for erectile dysfunction silymonin 26:255 as antihepatotoxic agent 26:255 simocyclinon 29:319 antibiotic activity of 29:319 cytostatic effects of 29:319 sindbis virus 17:135 sinomenine 25:472,476 hepatoprotective effect glycoside) 29:590 effects on pep 29:590 skin cancer 5:747 skin diseases 30:732 skin inflammation 30:323 anthrones used for 30:323 skk moth 1:704-706 juvenile hormone from slaframine 27:252 biological activity slaframine alkaloids 27:250,255 as muscarinic agonist 27:255 sleeping sickness smenoquinone 5:434,425 antimicrobial activity of smooth muscle relaxation 24:875-925 as biological action 24:875 of kampo medicines snake bite 30:691 treatment of 30:691 use of crossopetalum gaumeri 30:691 sobrerol 29:83 activity in mam-2 assay system 29:83 activity in gst assay system 29:83 activity in ras assay system 29:83 social phobia sodium salt of caffeic acid tetramer 24:742,743 anti-hiv activity of 24:742 soilborne fungi 21:181 bioactive metabolites from soilborne phytopathogens 21:182 biological control of 21:182 solandelactones 24:455 fptase inhibition by solanidane-induced teratogenicity 23:573 solid tumors neovasularization 25:593 somatostain 25:265 as a~ adenosine agonists somatostatin receptor 21:72;22:26; 25:530 type of g-protein-linked receptor 25:530 sonodione 30:566 from hernandia sonora 30:566 sores 30:616 tricyclic acylphloroglucinols sorivudine 24:474,486 as anti-viral agent 24 sorocein f 28:230 biological activity of 28:230 sos chromotest 22:623 soyabean saponins 25:222,223 hypocholesterdemic effects soyasaponins 27:398 anti-obesity action of 27:398 biological activity of 27:399 soybean trypsin inhibitor (sbti) spasmolytic activity 23:358 ;28:257,293; 30:264,561,748 of hernandia moerenhoutiana 30:561 ofhernandia voyronii 30:561 of imperatorin 23:350,358 of plant polyphenols 28:257,293 of thymus satureioides 30:264 on guinea pig ileum 30:748 on smooth muscles specionin 10:425 antifeedant activity of 10:425 spermicidal activity 26:53 of gypsophila paniculata sphingolipid receptor ca 2+ channels 25:535 sphingolipid receptors 25:534 sphinxolide 10:153;17:17 antitumor activity of 17:17 spider mite 1:702 hatching inhibitor spiroalkyl analogs 28:357 biological activity spirulina platensis 30:399,404,408 anti-influenza activity of 30:408 biological activity of 30:404 calcium spirulan from 30:399 polysaccharide (ps) from spleen/adipose tissue weight 30:56,57, 66 1203 effect of fucoidan on 30:56,57 effect of oleic acid on 30:65 spontaneous apoptosis 26:926 spontaneous metastasis squarroside a 26:31,50 immuno-modulatory effect of 26:31 from vaccaria segetalis squash family serine protease inhibitors 29:614 bd-ti-ii as 29:614 cmti-i as 29:614 cmti-iii as 29:614 cmti-iv as 29:615 cpgti-i as 29:615 cpti-ii as factor xiia, kallikrein, trypsin 29:615 effects on lysyl endopeptidase 29:614 effects on trypsin 29:614 effects on trypsin 29:615 effects on trypsin 29:616 effects on xa, xiia, kallikrein, plasmin, trypsin 29:614 elti-i as 29:615 from bryonia dioica 29:614 from citrullus vulgaris 29:614 from cucumis melo 29:614 from cucumis melo 29:614 from cucumis sativus 29:614 from cucurbita maxima 29:614 from cucurbita maxima 29:614 from cucurbita pepo 29:615 from cucurbita pepo 29:615 from ecballium elaterium 29:615 from echinocystis lobata 29:615 from lagenaria leucantha 29:615 from luffa acutangula 29:615 from luffa cylindrica cucurbitaceae) 29:616 from tricosanthes 29:616 hmti-i as 29:614 lati as 29:615 lati-ii as 29:615 lldti-i as 29:615 lldti-ii as 29:615 mcei-i as stachenone 29:101 activity in ebv bioassay system 29:101 standishinal 29:100 activity in ebv assay system 29:100 standishinal diacetate 29:100 activity in ebv assay system staphyllococcus aureus 30:693 minimal inhibitory concentration (mic) of staphyllococcus epidermidis 30:693 minimal inhibitory concentration (mic) of staphyllococcus saprophyticus 30:693 minimal inhibitory concentration (mic) of staphyllococcus warnieri 30:693 minimal inhibitory concentration (mic) of staphylococcus aureus 30:628,739 antibacterial activity against 30:739 antibacterial properties of 30:628 antifungal activity against starfish fertilisation inhibitor 28:712 callyspongin b as 28:712 stavudin 24:474,486,488 as anti-viral agent 24:474 steganes 29:360,399 cytotoxicity of 29:360,399 tubulin assembly inhibition by 29:360 antitubulin activity of 29:399,400 microtubules assembly inhibition by sterculia urens roxb. 30:405 antiviral activity of 30:405 steroid hormones 27:823 steroid saponins 7:426 teratogenic metabolites of 7:21-24 toxic cardiac active steroids sterol biosynthesis 25:303 inhibitor of 25:303 6a-sterol sulfate 28:702 cytotoxicity against sterol sulfates haplosamate a 28:703 as hiv-1 integrase inhibitor 28:703 sterols 25:43 antitumor activity of 25:43 antiinflammatory activity of 25:43 stevia stevia extract 27:307,310,314 for diabetics stevia leaves 27:315 immunological activity stevia plant 27:315 allergenic activity steviol 27:304,305,307,312 acute toxicity tests of 27:307 effect on fertility 27:312 mutagenicity tests with tpa bioassaysystem 29:101 activity in skin-1 bioassay system 29:101 acute toxicity tests of 27:307 allergenicity problems of 27:315 chronic toxicity studies of 27:306,307,308 effect on fertility 27:309,310 for phenylketonuria 27:315 mutagenicity tests for stigmasterol 30:208,505,570,719 analgesic effect of stilbene derivatives 27:405,409;28:579 biological activity of 27:405,409 in llc-bearing mice 28:579 tumor growth inhibition by 28:579 stimulatory 24:845 biological activity 24:845 stimulant agent 23:642 ofteucrium sp stimulus-response mechanisms 25:515 streptococci 29:535 pathogenicity of 29:535 serological groups streptokinase/streptodomase 25:271 streptolygidin 28:130 antibiotic activity of 28:130 as bacterial rna polymerase 28:inhibitor 130 as terminal dna transferase striga asiatica 30:162 infection with ofbioactive metabolites 21:251 of scopadulan-type diterpenoids structure activity relationship studies (sar) 30:195,494,800 of anandamide 30:196 of aspirin 30:195 ofopioid analogues 30:800 of vitamin d analogues 30:493 structure dereplication 29:658 bioactive natural product database in 29:670 of melanin inhibition strychnine 25:530 as immunosuppressive drug 25:530 strychnopentamine 26:1062 anticancer activity strychnos bisindole 26:1062 anticancer activity strychnos monoindole 26:1061 anticancer/protozoal subacute toxicity 27:307 from animals 27:307 of stevioside 588 effects on furin 29:588 sudhl-4-1ymphoma 21:138 sugar cane cystatins 29:593 effects on cysteine proteases 29:593 suicide inhibition 27:348 of 8'-hydroxylase 27:348 suicide inhibitors 9 sulfatobastadin 13 28:695 as endothelin a receptor inhibitor sulfide-containing pyrroloiminoquinones 28:685 as antileukaemic agents sulfur-containing cyclic peptides 28:678 biological activities of 28:678 cytotoxicity of sulphated polysaccharide (ps) 30:398, 400,405 anti-cmv activity of 30:405 anti-hiv activity of 30:398 antiviral activity against hiv 30:400 effect on viral replication 30:400 from marine sources 30:400 inhibitory effect of 30:398 mechanisms of action sulphated polysaccharide (ps) extracts 30:399 inhibitory activity of sulphonylurea drugs 25:534 sunflower cystatin phytocystatin 29:593 effects on cathepsin h ficin 29:593 effects on papain 29:593 sunflower multicystatin 29:593 effects on papain supercritical fluid extraction 21:576 superoxide dismutase superoxide dismutase activity 21:669 suppressing activity 30:308 on cells proliferation zizyphus saponin i as 27:41 zizyphus saponin ii sweroside 25:471,476 serum alt activity of 25 symbiotic marine microorganisms 23:185 bioactive metabolites 23:185 sympathetic ganglia 30:782 blockade of 30:782 sympathetic nerve activity 30:787 sympathomimetic amines 12:411 synapic potential mediation 28:318 by non-nmda receptors 28:318 synaptic actions 30:381 of serotonin synaptic dopamine transporters 25:539 synaptic glycine transporters 25:539 synaptic serotonin transporters 25:539 synaptogenesis 30:368,377 effect of serotonergic neurotransmission on 30:368 role in rodents 30:377 role of serotonin in 30:377 syndrome type-la 26:1130 adipose tissue distribution as 26:1130 cerebellar dysfunction as 26:1130 liver insufficiency as 26:1130 peripheral neuropathy as 26:1130 psychomotor retardation as synergistic activity 21:673 synergistic effect 21:608,611; 30:498,596 ofhervelines b 30:596 ofhervelines c 30:596 of retinoids 30:498 of vitamin d derivatives 30:498 synergistic interaction 21:698 of acyclovir(acv) 21:698 ofganciclovir (gcv) 21:698 with ganciclovir synergistic purgative actions 30:306 of rheinanthrone 30:306 of aloe-emodin-anthrone 30:306 synthetic pharmacological agents 30:224 antioxidant activity of 30:224 synthetic podolactones 28:484 allelopathic activity synthetic psychotropic agents 24:1093 syringaresinol 24:741 anti-platelet aggregation activity of systemic wound response protein 25:402 systemin activity 25:373 assay for ]-val-val-gly-nh2 30:815 receptor binding properties of receptor binding properties tyr-pro-ser-nh2 (deltorphin c) 30:815 receptor binding properties of -d-glc)-gly-nh2 30:815 receptor binding properties of oac)4]-gly-nh2 30:815 receptor binding properties of -d-glc)-tyr-pro-ser-nh2 30:815 receptor binding properties of -d-g lc )-v a 1-v al-gly-nh2 30:815 receptor binding properties of tyrosinase inhibitory activity 21:587 tyrosine kinase tyrosine-specific kinase 19:178 inhibitor of tyr-tic-nh2 30:809 activity in functional bioassay tyr-tic-phe-nh2 30:806 as 5 selective antagonist 30:806 u2 66 myeloma cell 25:273 ucn-01-ucn-02 12:386 antitumor activity of 12:395 protein kinase inhibitor ulapualides a 19:609 antifungal activity unsaturated carbapyranoses 29:475 as enzyme inhibitors 29:475 as herbicidal 29:475 biological properties of 29:475 unsaturated ketonucleosides 4:253 tumor inhibition by u-plasminogen activator (u-pa) 30:844 urdamycin a 11:134 antifungal activity of 11:134 antitumor activity ureterolithiasis-drug-therapy 27:382 urinary tract infections 26:50 herniaria hirsuta in 26:50 urine retention 30:194 as opioid's side effect 30:194 uroterpenol 29:83 activity in mam-2 assay system 29:83 activity in ras assay system ursane triterpene 29:585 effect on chy 29:585 ursolic acid 28:18,40;29:588 effects on hiv-1 protease 29:575 effects on lela 29:588 inhibition of hiv-1 protease dimerization by ursolic acid methyl ester (ursene triterpene) 29:575 effects on hiv-1 protease 29:575 usambarensine 26:1062 anticancer activity of 26:1060 uterine tumor 30:27 effect of pironetin derivatives in 30:27 uusambarine 26:1062 anticancer activity uvaol (triterpene) 29:588 effects on urs-12-ene-3,28-diol) (ursene triterpene) 29:575 effects on vaccaria segetalis 26:50 imunomodulatory effect of 50 vaccination 30:408 for respiratory infection as neuraminidase inhibitors 29:487 as c~-amylase inhibitor 29:486 c~-glucosidase inhibitors of 7:46;10:518 yeast a-glucosidase inhibitor of 10:518 valinuoctins 24:459 fptase inhibition by valiolamine 13:189,195 yeast c~-glucosidase inhibitor valiolamines 29:484 as glucosidase inhibitor 29:484 biological potential of 29:484 in clinical trials 29:484 in treatment of diabetes 29:484 valproic acid 22:507 vancomycin 25:791 vancoresmycin 28:150 activity against gram-positive bacteria vanilloid receptor agonist 30:202 olvanil as 30:202 capsaicin as 30:202 structure of vanilloid receptor antagonist 30:201, 202 capsazepine as 30 varicella-zoster virus 30:394 cause of varroajacobsoni 28:387 toxicity of varroa mites 28:383 vascular physiological action 30:55,69 of natural products vasodilating activity 21:379 vasodilator 23:358 scoparone as vasodilator effect 25:595 of acetylcholine vasodilatory activity 30:275 ofsatujera obovata vasoprotective agent 22:443 vasoprotective effects 28:302 of green tea vateamine-2' [3-n-oxide 30:567, 590,595 activity in human cancer cell line 30:589 activity in murine cancer cell line 30:589 as platelet aggregation inhibitor 30:594 cytotoxic effect of 30:589 veinotonic activities veraguensin 26:230 trypanosomicidal activity verapamil 25:534,673 as caz+-channel blocker 25:487 vermisporin 28:125 antimicrobial activity vernonia amygdalina 28:400 tick toxicity of 28:400 verrucarins 30:743 arenavirus junin 30:743 herpes simplex virus type ii (hsv-2) verrucosin 26:228 ofantiviral activity 26:228 vertebrate toxicity 21:98 vesicant activity vesicular glutamate transporters 25:538 vesicular monoamine transporter type 2 (vmat2) inhibitor vesicular stomatitis virus (vsv) 30:743,752 replication of 30:743 vesititol 28:224 anti-microbial activity of 28:224 vestitol 28:243 anti-helicobacter pylori activity of 28:243 veterinary medicine 28:383 for ectoparasites control 28:383 vetiver grass 28:399 for controlling ticks viagra (sildenafil) 25:541 vicolides 29:89 activity in ctn bioassay system 458 anticancer activity of 14:805 viral diseases 30:732 viral dna 30:226 integration of 12:332;21:98,193, 263,270,312,398,430,585,589,591, 594,643,659,670,673,675,690;23:59, 216,808;26:217;28:481 ;30 :629 against bone resorption 21:720, 721 against h+,k+-atpase 21:719 against staphyllococcus aureus 30:629 mechanism action of 30:225 of (-)-(8,8a-di-epi-swainsonine 12:325 of (-)-swainsonine 12:327 o f (+) cyasterone activity in 29:32 2-deoxy-20-hyroxyecdysone activity in 29:32 14-deoxymuristerone a activity in 29:32 2 [3,3 [3-dihydro xy-513-chole st-7en-6-one activity in 29:32 3 [3,14a-dihydroxy-5 [3-cholest-7en-6-one activity in 29:32 ecdysone activity in 29:32 313-hydro xy-5 [3-cho le st-6-one activity in 29:32 20-hydroxyecdysone activity in 29:32 inokosterone activity in 29:32 kc cell bioassay as 29:30 makisterone a activity in 29:32 muristerone a activity in 29:32 podecdysone a activity in 29:32 ponasterones activity in 29:32 213,313,513,14 a-tetrahydro x ycholest-7-en-6-one activity in 29:32 2[3,3 ~ ,25-tridihydroxy-513cholest-6-one activity in 29:32 viticosterone e activity in 29:32 insect control 21:611 insect juvenile hormone analogues 14:391-397 from thujone 14:391-397 insect pheromones 11: [415] [416] [417] via diisopropylethanediol esters 11:415-417 insect repellant 5:757 insecticidal activity 18:229,704;21:252, 254,262,266,267,269,270,271,278, 285,379,595,611;23:666;667,670, 677,678;24:25,799,845,866;26:231, 232,426,471,479,485;28:477; 30:153-154 activity of (-)-deoxypodo phyllotoxin in 30:589 activity of (-)-hernone in 30:590 activity of (-)-nymphone in 30:590 activity of (-)-yatein in 30:590 activity of (+)-corytuberine in 30:589 activity of (+)-epiaschantin in 30:590 activity of (+)-epimagnolin in 30:590 activity of (+)-epiyangambin in 30:590 activity of (+)-hernovine in 30:589 activity of (+)-laurotetanine in 30:589 activity of (+)-magnoflorine in 30:589 activity of (+)-malekulatine in 30:590 activity of (+)-n-hydroxyhemangerine in 30:589 activity of (+)-n-formylnornantenine in 30:589 activity of (+)-n-formyldehydroovigerine in 30:589 activity of (+)-n-formylovigerine in 30:589 activity of (+)-ovigerine in 30:590 activity of (+)-ovihernangerine in 30:590 activity of (+)-vateamine-2' [3-noxide in 30:590 activity of 7-formyldehydrohernangerine in 30:589 activity of 7-formyldehydronornantenine in 30:589 activity of 7-formyldehydroovigerine in 30:589 activity of 7-hydroxy-6-methoxy1:467,468;2:278,279,286, 289;6:108;7:103,110,121-123; 17:235,244 ;29:87 ;30:193,206 activity in ebv assay system 29:87 activity in skin 1 assay system 29:87 activity in crg assay system 29:87 activity in cro assay system 29:87 activity in aa assay system 29:87 activity in brk assay system 29:87 activity in cps assay system 29:87 activity in htm assay system 29:87 activity in paf assay system 26:3,4,10,13,15, 18,27,30,42,54,57;30:407 anti-inflammatory activity of 26:27 antiviral activity of 30:407 saponins 1:305; 402; 7:155, 156, 190, [426] [427] [428] [429] [430] [431] [432] 434, 435; 18:649 ajugasterone c activity in 29:28 amarasterone a activity in 29:28 amarasterone b activity in 29:28 cyasterone activity in 29:28 ponasterone a 2p-glucoside activity in 29:28 pterosterone activity in 29:28 rubrosterone activity in 29:28 sengosterone activity in 29:28 stachysterone c activity in 29:28 capitasterone activity in 29:28 sarcophytol a 29:100 activity in ebv assay system 29:100 activity in colon-2 assay system 29:100 activity in liver-2 assay system 29:100 activity in lung-1 assay system 29:100 activity in mam-3 assay system 29:100 activity in pancr assay system 29:100 activity in skin-2 assay system 29:100 sarcophytol b 8:18;29:100 activity in skin-2 assay system 29:100 use of platycodon grandiflorum key: cord-017504-rtg7fs82 authors: lim, t. k. title: punica granatum date: 2012-11-03 journal: edible medicinal and non-medicinal plants doi: 10.1007/978-94-007-5653-3_10 sha: doc_id: 17504 cord_uid: rtg7fs82 nan the pomegranate tree is native from the middle east to the himalayas in northern india. it has been cultivated and naturalised since ancient times throughout the mediterranean region of asia, caucasus, northern africa and europe. the fruit has manifold uses as it is today and was featured in egyptian mythology and art, in the old testament of the bible and in the babylonian talmud. from its native range, it was introduced to central and southern india and southeast asia. it was reported growing in indonesia in 1416. it was introduced into latin america and california by the spanish in 1796, it is now grown in california and arizona. it has been widely cultivated throughout india and drier parts of southeast asia and tropical africa. the most important growing regions are egypt, china, afghanistan, turkey, syria, pakistan, bangladesh, iran, iraq, india, myanmar and saudi arabia. there are some commercial orchards in israel on the coastal plain and in the jordan valley. pomegranate is primarily mild-temperate to subtropical and naturally adapted to regions with cool winters and hot summers, but can also be grown in warm tropical areas, such as in southern india, southeast asia and various islands in the caribbean. areas with mean annual temperature of 20-24°c is ideal. it suffers severe, irrecoverable injuries at temperatures below -11.0°c. the plant thrives in a semi-arid condition with mean annual rainfall of 500 to 1,000 mm and is extremely drought-tolerant. it does not fl ower and fruit well in very humid and wet climates. it is cultivated up to altitudes of 2,000 m as occur throughout the western range (baluchistan, n. & s. waziristan, nwfp, kurram, dir, chitral) in pakistan. the species is adaptable to a wide range of soil types including soils on which other fruit species will not grow. it thrives on calcareous soil, alkaline soil, gravelly soil and on deep, acidic loams. for commercial cultivation well-drained, heavy, light and medium soils are preferred although it can withstand seasonal waterlogging. irrigation is required to sustain high yields in drier areas. the fruit is relished fresh, out of hand by quartering the fruit and lifting out the rind to exposed the juice-laden arils around the seeds, both of which are eaten. the fruit is also consumed as juice which is the basis for lemonades or a beverage similar to wine. in the middle east, caucasus and india, pomegranate juice is a very popular beverage. for beverage purposes, the juice is usually sweetened. pomegranate juice is widely made into grenadine syrup for use in mixed drinks, cocktails and often processed into wine. in saudi arabia, the juice sacs may be frozen intact or the extracted juice may be concentrated and frozen, for future use. the juice can be processed into jellies by the addition of pectin and sugar. pomegranate is also used in food and as a spice condiment. fresh pomegranate arils are used in preparation of curd rice dadhojanam (telugu) in andhra pradesh in india. in northern india, the reduced juice is used for desserts and for marinating and tenderising meat due to its proteolytic enzymes. dried pomegranate arils are used in various cuisines such as trail mix, granola bars, or as toppings for ice-cream, yogurt and salads. dried whole arils are commonly sold in ethnic indian subcontinent markets. they impart a subtle, sweet-sour and tart fl avour popular in punjab and gujarat. dried pomegranate seeds, ' anardana ', has culinary importance as spice for vegetable and legume dishes in northern india and in pakistani cuisine. these dried seeds are used as an acidic condiment for chutney and curry preparation. the dried seeds can also be ground and used, which results in a deeper fl avoring in dishes and prevents the seeds from getting stuck in teeth. seeds of the wild pomegranate variety known as daru from the himalayas are renown as quality sources for this spice. in turkey, pomegranate seeds are also used in salads and sometimes as garnish for desserts such as güllaç . in greece and cyprus, pomegranate is used to make kolliva , a mixture of pomegranate seeds and sugar. pomegranate juice can also be processed into a concentrate, syrup and sauces for juice in food dishes and desserts. in iran, a traditional recipe fesenjan is made from a thick pomegranate sauce and ground walnuts used for duck and poultry or in a popular pomegranate soup ash-e nar . in azerbaijan, pomegranate sauce narsharab , made from pomegranate juice, is served with fi sh or tika kabab (grilled, roasted or stewed meat). in turkey, pomegranate sauce called nar ekşisi is used as a salad dressing, to marinate meat, or simply to drink straight. pomegranate syrup used in muhammara , a roasted red pepper, walnut, and garlic spread popular in syria and turkey. pomegranate is also popular in greek cuisine such as kollivozoum i, a creamy broth made from boiled wheat, pomegranates and raisins, legume salad with wheat and pomegranate, traditional middle eastern lamb kebabs with pomegranate glaze, pomegranate eggplant relish, and avocadopomegranate dip. pomegranate is also processed into a liqueur and fruit confectionery used as icecream toppings or mixed with yogurt and jam on toast. a deciduous, much-branched, small tree or shrub 1.5-6 m high with a smooth, dark grey bark (plate 1 ). branches are terete, opposite and branchlets usually ending in spines. leaves are opposite, glabrous, coriaceous, glossy green, entire, simple, oblong-lanceolate (plates 1 , 2 and 3 ) to obovate or elliptic, 19-35(−50) × 8-12 (−15) mm, subpetiolate, apex sub-actue to obtuse. flowers are large, showy, scarlet red or white, bisexual, to 4 cm across, solitary or clustered at the shoot apex (plates 2 and 3 ). calyx campanulate, reddish or purplish with six triangular, persistent lobes, petals 6, broadly obovate, wrinkled, alternating with the sepal lobes, stamens numerous, multiseriate, persistent, inserted on fl ower tube, ovary subglobose, inferior with three cells in two-series, style one thick, reddish, stigma simple slightly bilobed. fruit globose to subglobose, 6-8 cm in diameter, pale red to scarlet to purple or brownish,; the rind thick and coriaceous (plates 4 , 5 , 6 and 7 ). internally, the fruit is partitioned by thin leathery yellow septa into compartments fi lled with transparent sacs (arils) fi lled with tart, fl avourful, fl eshy, juicy, red, pink or whitish pulp (plates 7 and 8 ). in each sac, there is one white, pink or red, angular, soft or hard seed 10-13 mm long. food value of raw, pomegranate fruit (refuse 44% skin and membrane) per 100 g edible portion based on the california wonderful variety is as follows (usda 2012 ) : water 77.93 g, energy 83 kcal (346 kj), protein 1.67 g, total lipid (fat) 1.17 g, ash 0.53 g, carbohydrate 18.70 g; fi bre (total dietary) 4 g, total sugars 13.67 g, minerals -calcium 10 mg, iron 0.30 mg, magnesium 12 mg, phosphorus those of seeds, except potassium which was 49.2 ppm in the juice. the seed lipids had a refractive index of 1.518, melting point 13.0°c, iodine value 74.2, acid number 1.1, unsaponi fi able matter 0.7%, saponi fi cation value 188.9, ester value 187.8 and glycerol content 10.3%. the lipids contained 11 fatty acids, with caprylic (36.3%), the predominant acid, followed by stearic acid (22.5%); linoleic acid (10.3%) and oleic acid (5.1%). the saturated fatty acids of the seed lipids constituted 83.6% of the total fatty acids content. vitamin c content in 20 different turkish cultivars of pomegranate had a range of 312-1.050 mg/100 g, oil content a range of 2.41-3.73%, sterol content a range of 5.78-8.43%, anthocyanin content a range of 2,100-4,400 mg/l, potassium a range of 250-1,200 ppm, calcium a range of 35-326 ppm, magnesium a range of 176-427 ppm, iron a range of 21-46 ppm, sodium a range of 35-76 ppm, and phosphorus a range of 12-43 ppm (dumlu and gürkan 2007 ) . elfalleh et al. ( 2009 ) found total sugars of pomegranate juice comprised about 7 g/100 ml fructose and about 8 g/100 ml glucose, soluble proteins about 7 g/l, 9.46 mg/ 100 ml of phosphorus, and 271.94 mg/100 ml of potassium. the peel contained 9.43 and 210.86 mg/100 g of phosphorus and potassium respectively. the sodium contents were nearly 7 mg/100 ml in both peel and juice. nutrient composition of the juice for most components was comparable to the whole fruit. protein and fat values were higher in the whole fruit compared to the juice due to the seeds, which are 10% of the aril (juice sac) weight (thomas and gebhardt 2008 ) . pomegranate aril juice was reported to provide about 16% of an adult's daily vitamin c requirement per 100 ml serving, and to be a good source of vitamin b5 (pantothenic acid), potassium and antioxidant polyphenols. the tocopherol ( a -tocopherol, g -tocopherol, and d -tocopherol) contents were, respectively, 165.77, 107.38, and 27.29 mg/100 g from dry pomegranate seed (elfalleh et al. 2011a, b ) . a total of 18 compounds were found in pomegranate aroma pro fi les, including monoterpenes, aldehydes, alcohols, monoterpenoids and linear hydrocarbons . the most abundant compounds were trans -2-hexenal, 3-carene, a -terpinene and a -terpineol. the total concentration of volatiles ranged from 1.7 to 10.9 g/kg. overall consumer preference of pomegranate juices was associated with the presence of monoterpenes such as a -pinene, b -pinene, b -myrcene, limonene and g -terpinene. the presence of aldehydes such as hexanol, hexanal and cis -3-hexenol was correlated with poor overall consumer liking. high overall consumer liking was associated with intense and acceptable fresh pomegranate odour and fl avour (high scores of satisfaction degree), medium intensity of red colour and low sourness. pomegranate is a fruit rich in polyphenols that include fl avonoids, tannins and hydrolyzable tannins (gil et al. 2000 ; seeram et al. 2005a, b ) . pomegranate contain a complex mixture of gallotannins, ellagitannins, ellagic acid and anthocyanins (madrigal-carballo et al. 2009 ) . pomegranate juice was found to be rich in tannins, anthocyanins, ellagic acid derivatives, and hydrolyzable tannins (gil et al. 2000 ) . the predominant organic acid in pomegranate was citric acid followed by malic acid (pande and akoh 2009 ) . the peel fraction had the highest total hydrolyzable tannins content (4,792.3-6,894.8 mg/100 g of fw). a total of 35 dimers of fl avanol-anthocyanin adducts were detected, consisting of mono-and disubstituted hexoside derivatives of the adducts between the fl avan-3-ols (epi)gallocatechin, (epi) catechin and (epi)afzelechin and the anthocyanidins delphinidin, cyanidin and pelargonidin in pomegranate juice (sentandreu et al. 2010 ) . anthocyanidins found in pomegranate fruit included: delphinidin, cyanidin, and pelargonidin (noda et al. 2002 ) . pomegranate fruit was reported to contain ellagic acid, gallagic acid, punicalins and punicalagins (reddy et al. 2007 ) ; ellagic acid, caffeic acid, luteolin and punicic acid (lansky et al. 2005a, b ) ; pelargonidin-3-galactose, cyanidin-3-glucose, gallic acid, quercetin, and myricetin (naz et al. 2007 ) ; gallic acid, methyl gallate, ellagic acid, (+) catechin, isoquerecitrin, d-mannitol, ursolic acid, oleanolic acid, b -sitosterol and daucosterol (rena et al. 2009 ) . pomegranate fruit was found to have a low level of indolamines (8-12 m g/g serotonin, 4-9 m g/g tryptamine, and 13-29 ng/100 g melatonin) (badria 2002 ) . gözlekçi et al. ( 2011 ) found that in all the turkish pomegranate cultivars the highest levels of total phenolic content were obtained from the peel extracts. the total phenolic content ranged from 1,775.4 to 3,547.8 mg gallic acid equivalent (gae)/l among the cultivars. however, the total phenolic content of pomegranate juice and seed extract ranged from 784.4 to 1,551.5 mg gae/l and 117.0-177.4 mg gae/l, respectively. four phenolic compounds were identi fi ed and quanti fi ed in pomegranate peel and pulp: 2 hydroxybenzoic acids (gallic and ellagic acids) and 2 hydroxycinnamic acids (caffeic and p -coumaric acids) (elfalleh et al. 2011a ) . ellagitannins isolated from pomegranate pericarp included: inhibitors punicalin, punicalagin, granatin b, gallagyldilactone, casuarinin, pedunculagin, tellimagrandin i, gallic acid, granatin a, corilagin and ellagic acid (satomi et al. 1993 ) . pomegranate fruit peel had been reported to be a rich source of hydrolyzable tannins called ellagitannins (ets); pomegranate ets were found to show potent antioxidant, antiatherosclerotic and anticancer activities (seeram et al. 2005b ) . the major fruit peel ets were punicalagin (80-85% w/w) and ellagic acid (ea; 1.3% w/w) and unquanti fi ed amounts of punicalin and ellagitannin-glycosides (hexoside, rhamnoside and pentoside). pomegrante fruit peel is currently an underutilized food by-product with potential to develop phytoceuticals with potential health bene fi ts or to develop products for use in the cosmetic and food biopreservative industries (seeram et al. 2005b ) . prodelphinidins and gallocatechins including gallocatechin, gallocatechin-(4-8)-catechin, gallocatechin-(4-8)-gallocatechin and catechin-(4-8)-gallocatechin were identi fi ed from pomegranate peels (plumb et al. 2002 ) . luteolin, luteolin 7-o -glucoside, kaempferol, kaempferol-3-o -glucoside, kaempferol-3-orhamnoglycoside and quercetin were found in the fruit peel (van elswijk et al. 2004 ) . of the ellagi-tannins isolated from pomegranate pericarp, seven namely punicalin, punicalagin, granatin b, gallagyldilactone, casuarinin, pedunculagin and tellimagrandin i were found to be active carbonic anhydrase inhibitors and four namely gallic acid, granatin a, corilagin and ellagic acid to be weakly active inhibitors. the type of inhibition by three and seven punicalagin and gallagyldilactone was found to be noncompetitive. a new antifungal peptide designated as pomegranin with a molecular mass of 11 kda, was isolated from fresh pomegranate peels (guo et al. 2009 ) . epicatechin, epigallocatechin 3-gallate, fl avan-3-ol, catechin were found in the fruit peel and juice (de pascual-teresa et al. 2000 ) . pomegranate fruit and juice were found to contain the following lignans: isolariciresinol, medioresinol, matairesinol, pinoresinol, secoisolariciresinol and syringaresinol (bonzanini et al. 2009 ). total lignin contents in the seeds was determined as 36.1 m g/g, in wood knots 17.8 m g/g, in fruit pulp 11.2 m g/g and in the endocarp.3.3 m g/g. syringaresinol was most abundant in the seed (23.5 m g/g), pinoresinol in knots (8.9 m g/g), pulp (7.4 m g/g) endocarp (3.3 m g/g) and juice (2.1 m g/g). lignans were also found in two concentrated juices and three pomegranate beverages at levels of 0.4-4.4 m g/g. in addition to the peel, mesocarp, and twigs, lignans were detected in two juices obtained from entire pomegranate fruits, four commercial juices, and three encapsulated pomegranate extracts (fischer et al. 2012 ) . isolariciresinol was the predominant lignan with contents of 5.0, 10.5, and 45.8 mg/kg dry matter in processed pomegranate mesocarp, peel, and twigs, respectively. six anthocyanin pigments identi fi ed as delphinidin 3-glucoside, delphinidin 3,5-diglucoside, cyanidin 3-glucoside, cyanidin 3,5-diglucoside, pelargonidin 3-glucoside and pelargonidin 3,5-diglucoside were found to be responsible for the red colour of pomegranate juice (cv 'mollar') (gil et al. 1995 ) . the fruit skin contained only the cyanidin and pelargonidin derivatives. generally, there was an increase in juice pigmentation with fruit ripening. the concentration of pigments in juice obtained from mature pomegranates ranged between 50 and 100 m g of anthocyanin per gram fresh weight of arils. six anthocyanin pigments delphinidin 3-glucoside and 3,5-diglucoside, cyanidin 3-glucoside and 3,5-diglucoside and pelargonidin 3-glucoside and 3,5-diglucoside were found to be responsible for the red color of pomegranate juice (hernández et al. 1999 ) . generally, juice pigmentation increased as the fruit ripened. in the early fruit-ripening stages, delphinidin 3,5-diglucoside was the major pigment, followed by cyanidin 3,5-diglucoside, while in the later stages, the monoglucoside derivatives cyanidin 3-glucoside and delphinidin 3-glucoside increased considerably. the pelargonidin derivatives were always present in small amounts. rp-hplc analysis of pomegranate arils' anthocyanins revealed mono-and diglucosylated delphinidins and cyanidins as the major anthocyanins and pelargonidins as minor components (borochov-neori et al. 2011 ) . anthocyanin accumulation changed inversely to the season's temperatures. cyanidins were generally more abundant but delphinidin accumulation was enhanced in cooler season. monoglucosylated anthocyanins prevailed at cooler temperatures and subsided during seasonal warming with a concomitant rise in diglucoside proportion. the major anthocyanins detected in the 15 iranian pomegranate varieties were as follows: delphinidin 3-glucoside (2.19-16.29 mg/l), delphinidin 3,5-diglucoside (2.36-63.07 mg/l), pelargonidin 3-glucoside (0.26-1.36 mg/l), pelargonidin 3,5-diglucoside (0.01-8.11 mg/l), cyanidin 3-glucoside (5.78-30.38 mg/l), and cyanidin 3,5-diglucoside (4.39-166.32 mg/l) (alighourchi et al. 2008 ) . the major anthocyanins in the juice of 6 iranian pomegranate cultivars were delphinidin 3,5-diglucoside (372-5,301 mg/l), cyanidin 3,5-diglucoside (242-2,361 mg/l), delphinidin 3-glucoside (49-1,042 mg/l) and pelargonidin 3,5-diglucoside (7-90 mg/l) (mousavinejad et al. 2009 ) . the cultivar, saveh black leather had the highest level of ellagic acid (160 mg/l). pomegranate juices obtained from six iranian pomegranate cultivars were found to have 15.77-19.56 total soluble solids content (brix), ph values of 3.06-3.74, titrable acidity concentration from 0.51 to 1.35 g/100 g, total sugars content from 16. to 22.76 g/100 g (faroogh), total antho-cyanins 7.93-27.73 mg/100 g, ascorbic acid 8.68-15.07 mg/100, total phenolics content 526.40-797.49 mg tannic acid/100 g, the total tannins level 18.77-38.21 mg tannic acid/100 g, condensed tannins from 12.14 mg to 12.57 catechin/100 g, antioxidant activity from 46.51 to 52.71% (zarei et al. 2010 ) . phenolics, fl avonoids, anthocyanins, and tannins of pomegranate juices, obtained from nine tunisian ecotypes were quanti fi ed by el kar et al. ( 2011 ) . phenolics ranged from 1,570 to 3,299 mg gallic acid equivalents/l and fl avonoids from 135 to 156 mg quercetin equivalent/l of juice. highest anthocyanin content was 156 mg cyanidin-3-glucoside equivalent/l and highest tannin content was 2,550 mg catechin equivalent/l of juice. tartaric and quinic acids were con fi rmed in pomegranate juice at concentrations of 1-5 and ~ 1 mg/l, respectively (ehling and cole 2011 ) . twenty-one volatile compounds were found in fresh pomegranate juices from nine spanish cultivars, including aldehydes, monoterpenes, and alcohols . the most abundant compounds were hexanal, limonene, trans -2-hexenal, and cis -3-hexenol. the presence of monoterpenes ( a -terpineol) was correlated with overall consumer preference of pomegranate juice while high aldehydes ( trans -2-hexenal) concentrations were correlated with poor overall consumer liking. 5-hydroxymethyl furfural was determined to be at a signi fi cant level in traditional sour concentrate of pomegranate juice (orak 2009 ) . pomegranate was known to contain estrogens (estradiol, estrone, and estriol) (mori-okamoto et al. 2004 ) . polysaccharide (psp001) was isolated from pomegranate rind (joseph et al. 2012 ) . pomegranate seed oil was found to have 8% saturated fatty acids, 10% monounsaturated, 10% diunsaturated and approximately 70% conjugated acid, most probably punicic acid (el-shaarawy and nahpetian 1983 ) . pomegranate seed was found to have high contents of a -tocopherol (161.2-170.1 mg/100 g) and g -tocopherol (80.2-92.8 mg/100 g). the seeds of punica granatum also contained ursolic acid and b -sitosterol along with a long straightchain hydrocarbon -nonacosene (ahmed et al. 1995 ) . presence of estrogens and glycosides were also detected. estrone, an estrogen, was identi fi ed in pomegranate seeds (heftmann et al. 1966 ) . cold pressed pomegranate seed oil was found to contain punicic acid (65.3%), palmitic acid (4.8%), stearic acid (2.3%), oleic acid (6.3%), linoleic acid (6.6%) and three unidenti fi ed peaks from which two (14.2%) were probably isomers of punicic acid (schubert et al. 1999 ) . pomegranate seed had an average lipid content of 19.2% with punicic acid as the predominant fatty acid (pande and akoh 2009 ) . pomegranate seed oil was found to be rich in 1-o -trans , cis , trans -9,11,13-octadecatrienoyl glycerol and also to have small amounts of 1-o -isopentyl-3-o -octadec-2-enoyl glycerol and the known cis -9-octadecenoic, octadecanoic and eicosanoic acids (fatope et al. 2002 ) . pomegranate seed oil (pgo) was reported to be rich in 70% cis (c)9, trans (t)11,c13-18:3 as conjugated linolenic acids (cla) (kohno et al. 2004 ) . a triglyceride, di-o -punicyl-o -octadeca-8 z ,11 z ,13 e -trienylglycerol, was isolated and characterized from the seeds of punica granatum from india and iran (yusuph and mann 1997 ) . four compound were isolated from pomegranate seeds namely . pomegranate seed oil from 21 pomegranate cultivars was found to have mainly unsaturated fatty acids (about 88%) (el kar et al. 2011 ) . the predominant fatty acid was linolenic acid (44.51-86.14%), followed by linoleic acid (3.57-13.92%), oleic acid (3.03-12.88%), palmitic acid (3.13-11.82%), stearic acid (1.68-15.64%), gadoleic acid (0.50-4.91%), lignoceric acid (<2.53%), arachidic acid (<1.70%) and myristic acid (<0.85%). pomegranate seed linolenic acid isomers, punicic acid and α-eleostearic acid were found in pomegranate seeds (tran et al. 2010 ) . a high yield (3.1-4.2%) of unsaponi fi able matter containing tocopherol, aliphatic alcohol (including policosanol), squalene, phytosterols and triterpene was obtained from pomegranate seed oil (caligiani et al. 2010 ) . the levels of squalene (up to 800 mg/kg), policosanol (118-185 mg/kg), b -sitosterol (up to 8,069 mg/kg) and cycloartenol (5,916-7,766 mg/kg) were found while b -and d -tocopherol were the most abundant vitamin e forms. the seed oil of p. granatum may be an interesting alimentary source of substances of nutraceutical value involved in the modulation of cholesterol metabolism. linolenic acid isomers like punicic acid and αeleostearic acid were reported from pomegranate seeds (tran et al. 2010 ) . qualitatively, the pomegranate fatty acid composition of 21 pomegranate cultivars (15 tunisian and 6 chinese) seed oil was identical comprising mainly unsaturated about 88% (elfalleh et al. 2011b ). the predominant fatty acid was linolenic acid (44.51-86.14%), followed by linoleic acid (3.57-13.92%), oleic acid (3.03-12.88%), palmitic acid (3.13-11.82%), stearic acid (1.68-15.64%), gadoleic acid (0.50-4.91%), lignoceric acid ( < 2.53%), arachidic acid ( < 1.70%) and myristic acid ( < 0.85%). ) isolated the following bioactive compounds from pomegranate seeds: coniferyl methy lellagic acid; 3,3 ¢ ,4 ¢ -tri-o -methylellagic acid; phenethyl rutinoside; icariside d1 and daucosterol. a new class iii chitinase (pomegranate seed chitinase) with a molecular weight of approximately 30 kda was isolated and puri fi ed from pomegranate seeds (yang et al. 2011 ) . this chitinase was found to naturally bind calcium ions with high capacity and low af fi nity, suggesting it to be a calcium storage protein. this enzyme was found to be widely distributed in the stroma of amyloplasts of the embryonic cells, suggesting that amyloplasts in seeds could serve as an alternative plastid for calcium storage. two new b -sitosterol esters elucidated as stigmast-5en-3 b -ol-3 b -dodecanoate ( b -sitosterol laurate) and stigmast-5-en-3 b -ol-3 b -tetradecanoate ( b -sitosterol myristate) along with the known com pounds n-tricosane, n-heptacosanyl n-hexanoate olean-5,12-dien-3 b -ol-28-oic acid and olean-12-en-3 bol-28-oic acid were isolated from pomegranate fl owers (bagri et al. 2009 b ) . a new polyphenol compound named pomegranatate, together with, ellagic acid, 3,3 ¢ ,4 ¢ -tri-o -methylellagic acid, ethyl brevifolincarboxylate, urolic and maslinic acids, and daucosterol were isolated from the ethanolic extract of the fl owers of punica granatum . maslinic acid exhibited antioxidant activity as evaluated by measurement of ldl susceptibility to oxidation. a taraxastane-type triterpene, punicanolic acid; two galloyl glucoses, 1,2,6-tri-o -galloyl b -d-glucopyranoside, 1,2-di-o -galloyl-4,6-o -(s)-hexahydroxydiphenoyl b -d-glucopyranoside; fl avones, luteolin; triterpnenes oleanolic acid, maslinic acid; and b -sitosterol were isolated from pomegranate fl owers (xie et al. 2008 ) . an alkaloid 2-(2-propenyl)-d 1-piperideine was isolated from pomegranate leaves (roberts et al. 1967 ) . pomegranate leaves were found to contain tannins granatin a, granatin b, corilagin, strictinin, 1,2,4,6-tetra-o -galloyl-b -d-glucose and 1,2,3,4,6 -penta-o -galloyl-b -d-glucose and an ellagitannin, punicafolin elucidated as 1, 2, 4-tri-o -galloyl-3, 6-(r)-hexahydroxydiphenoyl-b -dglucose (tanaka et al. 1985 (tanaka et al. , 1990 . pomegranate leaves were found to be rich in polyphenols: brevifolin carboxylic acid, brevifolin, corilagin, ellagic acid, 3,4,8,9,10-pentahydroxydibenzo[ b,d ] pyran-6-one, granatin-b and punicafolin (nawwar et al. 1994b ) ; n-(2 ¢ ,5 ¢ -dihydroxyphenyl) pyridinium chloride, as well as the known fl avone glycosides, apigenin 4 (nawwar et al. 1994a ) ; ellagitannin, punicafolin, tannins, granatins a and b, corilagin, strictinin, 1,2,4,6-tetra-o -galloyl-b -d-glucose and 1,2,3,4,6-penta-o -galloyl-b -d-glucose (tanaka et al. 1985 ) ; gallotannins, 1,2,4-tri-o -galloylb -glucopyranose and 1,3,4-tri-o -galloyl-b -glucopyranose together with the hitherto unknown ellagitannins, 1,4-di-o -galloyl-3,6-( r )-hexahydroxydiphenyl-b -glucopyranose and brevifolin carboxylic acid 10-monopotassium sulphate (hussein et al. 1997 ) . a hydroquinone pyridinium alkaloid in the form a mixture of a con jugated and a cross-conjugated heterocyclic mesomeric betaine was isolated from the leaves of punica granatum (schmidt et al. 2005 ) . balwani et al. ( 2011 ) isolated a novel compound, 2-methyl-pyran-4-one-3-o -b -d-glucopyranoside from pomegranate leaves. these alkaloid isopelletierine, methylisopelletierine, pelletierine, pseudopelletierine were isolated from pomegranate bark (chilton and partridge 1950 ; wibaut et al. 1954 ) and roots (chilton and partridge 1950 ) ; isopelletierine, methylisopelletierine and y pelleterine from bark (wibaut and hollstein 1957 ) ; and n-acetyl-sedridine from bark and root (neuhöfer 1990 ) . the bark is rich in punicotannic acid (about 22%) and also contains gallic acid, mannite and four alkaloids isopelletierine, methylisopelletierine, pelletierine, pseudopelletierine (grieve 1971 ) . the following alkaloids were isolated from pomegranate bark and roots: pelletierine, methylisopelletierine, pseudopelletierine and from roots norpseudopelletierine, sedridine, 2-(2 ¢ -hydroxypropyl) d 1-piperidine; 2-(2 ¢ -propenyl) d 1-piperidine, hygrine and norhygrine (neuhöfer et al. 1993 ) . tannins and related compound were isolated from pomegranate bark and included punicalin and punicalagin elucidated as to 4, 6-(s, s)-gallagyl-d-glucose (1) and 2,3-(s)hexahydroxy-diphenoyl-4,6-(s, s)-gallagyl-dglucose (2), respectively and a hydrolyzable tannin, 2-o -galloyl-4,6-(s, s)-gallagyl-d-glucose (tanaka et al. 1986a ) ; ellagitannins, punicacorteins a, b, c and d, punigluconin, casuariin and casuarinin (tanaka et al. 1986b ) . punicacor teins a, b, c and d were established as novel c-glycosideic ellagitannins, the former two possessing a unique tetraphenyl (gallagyl) ester group, and the latter two containing a galloyl group in place of the gallagyl group, while punigluconin was elucidated as 2,3-di-o -galloyl-4,6-(s)-hexahydroxydiphenoyl gluconic acid. a fl avonoid diglycoside, quercetin-3,4 ¢ -dimethyl ether-7-o -a -l-arabinofuranosyl (1 → 6)-b -dglucopyranoside, quercetin, pelargonidine-3,5diglucoside and ellagic acid were isolated from pomegranate bark (chauhan and chauhan 2001 ) . the heartwood of punica grantum was found to contain ellagitannins: diellagic acid rhamnosyl toumy and rauwald 2003 ) ; 3 ¢ -o -methyl-3,4methylenedioxyellagic acid, as well as eight known ellagitannins and gallotannins (el-toumy et al. 2001 ) . a new dimeric gallic acid glycoside named humarain was isolated from stem bark of punica granatum (tantray et al. 2009 ) . punica granatum is a unique medicinal plant with a long and extensive ethnomedicinal uses since ancient times. various parts of the plant viz. seed, aril, fruit juice, peel, leaf, fl ower, bark, and roots have been reported to contain bioactive phytochemicals with interesting medicinal values and pharmacological activities. the phytochemistry and pharmacological properties of pomegranate plant parts suggest a wide range of clinical applications for the treatment and prevention of ailments such as cancer as well as other diseases where chronic in fl ammation is believed to play an essential etiologic role . the synergistic action of the pomegranate constituents appears to be superior to that of single constituents. in the past two decade, numerous invitro, in-vivo and preclinical studies on the antioxidant, anticarcinogenic, and anti-in fl ammatory properties of pomegranate constituents have been published, focusing on treatment and prevention of cancer, cardiovascular disease, diabetes, dental conditions, erectile dysfunction, bacterial infections and antibiotic resistance, and ultraviolet radiation-induced skin damage (jurenka 2008 ) . other potential applications include infant brain ischemia, male infertility, alzheimer's disease, arthritis, and obesity. aqueous and ethyl acetate extracts of pomegranate arils, juice and peels exhibited good antioxidant activity (ricci et al. 2006 ) . pomegranate juice, peel, and seed oil antioxidants were con fi rmed by ferric reducing antioxidant power (frap) and oxygen radical absorbance capacity (orac) methods (elfalleh et al. 2011 ) . the highest values were recorded in peels with 25.63 mmol trolox equivalent/100 g and 22.08 mmol te/100 g for frap and orac assay, respectively. the tocopherol ( a -tocopherol, g -tocopherol, and d -tocopherol) contents were, respectively, 165.77, 107.38, and 27.29 mg/100 g from dry pomegranate seed. four phenolic compounds were identi fi ed and quanti fi ed in pomegranate peel and pulp: 2 hydroxybenzoic acids (gallic and ellagic acids) and 2 hydroxycinnamic acids (caffeic and p -coumaric acids). results showed that the antioxidant potency of pomegranate extracts was correlated with their phenolic compound content. in particular, the highest correlation was reported in peels. high correlations were also found between peel hydroxybenzoic acids and frap orac antioxidant capacities. identi fi ed tocopherols appeared to contribute in major part to the antioxidant activity of pomegranate seed oil. gil et al. ( 2000 ) found that the antioxidant activity of commercial pomegranate juices (18−20 teac) was three times higher than those of red wine and green tea (6−8 teac). commercial juices extracted from whole pomegranates showed higher antioxidant activity than in experimental juices obtained from the arils only (12−14 teac). further, they found that commercial juices contained the pomegranate abundant tannin punicalagin (1,500−1,900 mg/l) while only traces were detected in the experimental juice obtained from arils showing that pomegranate industrial processing extracts some of the hydrolyzable tannins present in the fruit rind. also, anthocyanins, ellagic acid derivatives, and hydrolyzable tannins were found in the pomegranate juices. the results of studies by tzulker et al. ( 2007 ) showed that the antioxidant activity in pomegranate aril juice correlated signi fi cantly to the total polyphenol and anthocyanin contents. however, the homogenates prepared from the whole fruit exhibited an approximately 20-fold higher antioxidant activity than the level found in the aril juice. unlike the arils, the antioxidant level in the homogenates correlated signi fi cantly to the content of the four hydrolyzable tannins in which punicalagin was predominant, while no correlation was found to the level of anthocyanins. pomegranate juice was found to be a potent inhibitor of superoxide anion-mediated disappearance of nitric oxide . it was much more potent than concord grape juice, blueberry juice, red wine, ascorbic acid, and dl-α-tocopherol. as little as three μl of a six-fold dilution of pomegranate juice, in a reaction volume of 5,000 μ l, produced a marked antioxidant effect, whereas 300 μ l of undiluted blueberry juice or nearly 1,000 μ l of undiluted concord grape juice were required to produce similar effects. pomegranate juice and other antioxidant-containing products were found to augment the anti-proliferative action of nitric oxide (deta/no) on vascular smooth muscle cell (rat aorta) proliferation. and other antioxidant-containing products were found to augment the antiproliferative action of no on vascular smooth muscle cell (rat aorta) proliferation. pomegranate juice was much more effective than the other products tested and elicited no effects when tested alone in the absence of added no. pomegranate juice elicited no effects on enos protein expression or catalytic activity and did not enhance promoter activity in the enos gene. the observations indicated that pomegranate juice possessed potent antioxidant activity that resulted in marked protection of nitric oxide against oxidative destruction pande and akoh ( 2009 ) in their study found the highest antioxidant capacity to be in pomegranate leaves followed by peel, pulp, and seed. the tannin rich mixtures from pomegranate by-product exhibited ic 50 values against reactive oxygen species (ros) generation at 0.8-19 m g/ ml. the antioxidant capacity (orac) of pomegranate juice was 2,860 m mol te/100 g pomegranate juice which was comparable to blueberry and grape juice (thomas and gebhardt 2008 ) . oral administration of fl avonoid rich fractions from pomegranate fruits to rats at a dose of 10 mg/kg/day exhibited potential antiperoxidative activity (sudeesh and vijayalakshmi 2005 ) . malondialdehyde, hydroperoxides and conjugated dienes levels in the liver were signi fi cantly decreased antioxidative enzymes catalase, superoxide dismutase (sod), glutathione peroxidase and glutathione reductase were signi fi cantly elevated. glutathione content in the tissues were also increased. pomegranate fermented juice and cold pressed seed oil exhibited potent antioxidant activity almost equivalent to butylated hydroxyanisole (bha) and green tea ( thea sinensis ), but signi fi cantly higher than that of red wine ( vitis vitifera ) (schubert et al. 1999 ) . flavonoids extracted from cold pressed pomegranate seed oil exhibited 31-44% inhibition of sheep cyclooxygenase and 69-81% inhibition of soybean lipoxygenase. flavonoids extracted from pomegranate fermented juice displayed 21-30% inhibition of soybean lipoxygenase but showed no signi fi cant inhibition of sheep cyclooxygenase. total polyphenols in cold pressed pomegranate seed oil showed a concentration by weight of approximately 0.015%. fatty acid composition in cold pressed pomegranate seed oil showed punicic acid (65.3%) along with palmitic acid (4.8%), stearic acid (2.3%), oleic acid (6.3%), linoleic acid (6.6%) and three unidenti fi ed peaks from which two (14.2%) are probably isomers of punicic acid. acetone extract (70%) of pomegranate fruit displayed scavenging activity against hydroxyl (·oh) and superoxide (o2·-) radicals (noda et al. 2002 ) . its three major anthocyanindins, delphinidin, cyanidin, and pelargonidin, scavenged o2·in a dose-dependent fashion with id 50 values of 2.4, 22, and 456 m m, respectively but did not effectively scavenge nitric oxide. the anthocyanidins inhibited a fenton reagent ·oh generating system. further, the anthocyanidins inhibited hydrogen peroxide-induced lipid peroxidation in the rat brain homogenates with id 50 values 0.7, 3.5, and 85 m m, respectively for delphinidin, cyanidin, and pelargonidin (noda et al. 2002 ) . in another study, pomegranate elagitanninsellagic acid, gallagic acid, punicalins and punicalagins from pomegranate fruit showed ic 50 values of 1.1, 3.2, 2.3 and 1.4 m m, respectively, against reactive oxygen species (ros) generation and no toxicity up to 31.25 m g/ml against hl-60 cells (reddy et al. 2007 ) . the good antioxidant action of punicalagin a high molecular weight polyphenol isolated from pomegranate fruit pith and carpellary membrane was expressed not only through its scavenging reactions but also by its ability to form metal chelates (kulkarni et al. 2007 ) . binding of punicalagin with bovine serum albumin and metal ions such as iron and copper revealed different binding af fi nities, whereas its binding with dna was very weak and non-speci fi c. in-vitro cytotoxic studies against three cell lines, namely, vero (normal african green monkey kidney cell line), hep-2 (human larynx epithelial cancer cell line), and a-549 (human small cell lung carcinoma cell line) showed that punicalagin, was toxic only at higher concentration. studies found that pomegranate peel had the highest antioxidant activity among the peel, pulp and seed fractions of 28 kinds of fruits commonly consumed in china as determined by frap (ferric reducing antioxidant power) assay (guo et al. 2003 ) . in a subsequent study pomegranate peel extract was shown to have markedly higher antioxidant capacity than the pulp extract in scavenging or preventive capacity against superoxide anion, hydroxyl and peroxyl radicals as well as inhibiting cuso4-induced ldl oxidation. the contents of total phenolics, fl avonoids and proathocyanidins were also higher in peel extract than in pulp extract. the large amount of phenolics contained in peel extract may cause its strong antioxidant ability. the authors concluded that pomegranate peel extract appeared to have more potential as a health supplement rich in natural antioxidants than the pulp extract. separate studies showed pomegranate peel extracts to have both antioxidant and antimutagenic properties and may be exploited as biopreservatives in food applications and neutraceuticals (negi et al. 2003 ) . all the pomegranate peel extracts (ethyl acetate, acetone, methanol and water) exhibited marked antioxidant capacity, but the water extract was the lowest. the order of antioxidant capacity varied because of differential responses at four concentrations (25, 50, 75 and 100 m g/ml) in each solvent (negi et al. 2003 ) . studies in male rats showed that pomegranate fruit peel extract decreased lipid peroxidation in hepatic, cardiac, and renal tissues and serum glucose concentration (parmar and kar 2008 ) . pomegranate peels were found to contain potent antioxidant contents, as evidenced by free radical dpph scavenging value of 3.58 m g/ml and abts scavenging value of 7.364 mm trolox equivalent antioxidant capacity/100 g dry weight (elfalleh et al. 2009 ) . aqueous and alcoholic extracts of pomegranate rind showed good antioxidant effect with ic 50 ranging from 34.78 to 135.27/ml for aqueous and 40.03-105.93 m g/ml for alcoholic extracts (rajan et al. 2011 ) . phenolic compounds, tannins and fl avonoids were the major phytochemicals present in both the extracts. the aqueous and alcoholic extract yielded 122.33 and 176 mg/g gallic acid equivalent phenolic content, 135.33snf 81.33 mg/g quercetin equivalent fl avonoid and 81.66 and 114.23 mg/g tannic acid equivalent tannins respectively. plumb et al. ( 2002 ) found that the prodelphinidin dimers from pomegranate peels were potent antioxidants in the aqueous phase, being much more effective than the gallocatechin monomer in scavenging of the radical cation of 2,2-azinobis (3-ethyl-benzothiazoline-6-sulphonate, abts) relative to the water-soluble vitamin e analogue trolox c (expressed as trolox c equivalent antioxidant capacity, teac). in the lipid phase, only one of the dimers (gallocatechin-(4-8)-catechin) was signi fi cantly more effective than the monomer in the inhibition of lipid peroxidation of phosphatidylcholine vesicles. the water, methanol, acetone and ethyl acetate (etoac) extracts of pomegranate peel phenolics showed enhanced inhibitory effect on lard peroxidation as the phenolic concentrations increased . acetone extract exhibited the highest antiliperoxidant activity followed by water, methanol and etoac extracts. acetone extract at 0.1% (w/w) and water extract at 0.2% (w/w) exhibited an antiliperoxidant effect close to that of tea polyphenols (0.02%, w/w) and higher than that of bht (butylated hydroxytoluene) (0.02%, w/w). at 0.2% (w/w), acetone extract exerted a higher inhibitory activity on lard oxidation than that of tea polyphenols and bht. studies by guo et al. ( 2007 ) showed that showed that red pomegranate peel extract had the best effect on the scavenging ability of superoxide anion with lowest ic 50 value (4.01 m g/ml) among all pomegranate extracts (peel, juice, and seed of three varieties). the peel extract of white pomegranate had the best scavenging ability on hydrogen peroxide with the lowest ic 50 value (0.032 m g/ml) of the nine extracts. the seed extract of white pomegranate could scavenge hydroxide radical most effectively of the nine extracts (the ic 50 value 1.69 m g/ ml). the seed extract of white pomegranate (the ic 50 value was 3.67 m g/ml) was the most powerful on the dna damage-preventing effect of the extracts. the results of studies by xu et al. ( 2008 ) indicated that pomegranate peel extracts exerted protective effects on oxidative stress in mice loaded with restraint stress which may be attributed to its free radical scavenging activity and lipid peroxidation inhibitory effect. the extract decreased alanine aminotransferase and malondialdehyde levels and increased antioxidant capacity in the liver and glutathione levels in plasma as compared with restraint stress control mice. the methanol fraction of pomegranate peel showed highest antioxidant activity by all the four in vitro assays viz. dpph free radical scavenging, phosphomolybdenum, frap (fe(3+) reducing power) and cuprac (cupric ions (cu(2+)) reducing ability) comparable to ascorbic acid and butylated hydroxy toluene (bht) followed by activity in ethanol, acetone, and ethyl acetate fractions (zahin et al. 2010a ) . in cell free-systems, preparations from various parts of pomegranate displayed displayed good antioxidant capacity as assayed by 1,1-diphenyl-2-picrylhydrazyl (dpph), chemiluminescence luminol/xanthine/xanthine oxidase and lipoxygenase assays, with relative potency sequence of rind extract > pomegranate juice > aril juice (sestili et al. 2007 ) . however, only the rind extract was capable of preventing the deleterious effects -cytotoxicity, dna damage and depletion of non-protein sulphydrils (npsh) pool, caused by treatment of cells with hydroxide peroxide, tert-butylhydroperoxide or oxidized lipoproteins (ox-ldl) via a mechanism which was postulated to involve both direct scavenging of radical species and iron chelation. the results suggested that the aril juice the major and tasty part of pomegranate fruit, did not contain ellagic acid and punicalagin (i.e. the polyphenols highly represented in the rind which appeared to be responsible for the antioxidant capacity) in amounts suf fi cient to exert cytoprotection in oxidatively injured, living cells. based on these results, the authors advocated that development and evaluation of rinds-only based derivatives of pomegranate for antiatherogenic preventive purposes in humans should be encouraged. the antioxidant activity (percentage of inhibition of on peroxidation in linoleic acid system) of cpj (traditional sour concentrate of pomegranate juice) was determined to be higher (85.91%) than that of pj (pomegranate juice) (79.06%) (orak 2009 ). during the concentration process, the reducing sugars, glucose and fructose level of cpj showed an increase to 46.46, 23.89, and 22.53%, respectively. in cpj the amounts of sodium, iron, zinc, copper and lead were found lower than those of pj. in contrast, potassium and magnesium mineral contents increased during concentration. the total phenolics were also found to be 3,246 and 9,870 m g/ml in pj and cpj, respectively. the total anthocyanin content of pj was found to be 492.9 mg/l but it was not determined in cpj. 5-hydroxymethyl furfural was determined to be at a signi fi cant level in cpj as a result of the heat process. sezer et al. ( 2007 ) found that pomegranate and red wines decreased low-density lipoprotein (ldl) diene levels following a 30-min incubation period compared with controls. however, pure pomegranate wine demonstrated a greater antioxidant effect on diene level (110 m mol/mg of ldl protein) than pure red wine (124 m mol/mg of ldl protein). the phenol levels of pomegranate and red wines (4,850 mg/l gallic acid equivalents and 815 mg/l gallic acid equivalents, respectively) were in accordance with their total antioxidant activity (39.5 and 33.7%, respectively). four compound from pomegranate seeds namely coniferyl (4) displayed antioxidant activity, which was evaluated by measurement of low-density lipoprotein (ldl) susceptibility to oxidation and by in-vitro determination of malondialdehyde (mda) levels in the rat's brain . ethanolic extract of pomegranate fl owers was found to contain a large amount of polyphenols and to exhibit potent reducing ability, both indicative of potent antioxidant ability (kaur et al. 2006 ) . the extract showed 81.6% antioxidant activity in dpph model system. the fl ower extract was found to signi fi cantly scavenge superoxide (o 2− ) (by up to 53.3%), hydrogen peroxide (h2o2) (by up to 30%), hydroxyl radicals ( − oh) (by up to 37%) and nitric oxide (no) (by up to 74.5%). the extract also inhibited ( − oh) induced oxidation of lipids and proteins in vitro. these results indicated pomegranate fl ower extract to exert a signi fi cant antioxidant activity in-vitro. daily consumption of pomegranate juices was found to be potentially better than apple juice in improving antioxidant function in the elderly (guo et al. 2008 ) . as the plasma ascorbic acid, vitamin e, and reduced glutathione contents did not differ signi fi cantly between the apple and pomegranate groups in the study, the phenolics may be the functional components contained in pomegranate juice that accounted for the observations. recent in-vitro studies and preclinical animal studies have shown that pomegranate extracts selectively inhibit the growth of breast, prostate, colon and lung cancer cells (adhami et al. 2009 ) . an initial phase ii clinical trial of pomegranate juice in patients with prostate cancer reported signi fi cant prolongation of prostate speci fi c antigen doubling time. some of these researches are further elaborated herein. various parts of the pomegranate fruit e.g. seed oil, juice, fermented juice and peel extract, had been shown to exert suppressive effects on human breast cancer cells in-vitro and in this context, three estrogenic compounds, i.e. luteolin, quercetin and kaempferol, were detected in the fruit peel extract (van elswijk et al. 2004 ) . studies showed pomegranate fruit possessed chemopreventive and adjuvant therapeutic potential for human breast cancer (kim et al. 2002 ) . polyphenols from fermented pomegranate juice, pericarp, and oil inhibited aromatase activity by 60-80% indicating its ability to effect a blockade of endogenous active estrogen biosynthesis. fermented juice and pericarp polyphenols, and whole seed oil, inhibited 17-b -hydroxysteroid dehydrogenase type 1 from 34 to 79%, at concentrations ranging from 100 to 1,000 m g/ml in the sequence seed oil > > fermented juice polyphenols > pericarp polyphenols. lyophilized fresh pomegranate juice elicited a 55% inhibition of the estrogenic activity of 17-b -estradiol; whereas the lyophilized juice by itself displayed only minimal estrogenic action. inhibition of cell lines by fermented juice and pericarp polyphenols was according to estrogen-dependent (mcf-7) > > estrogen-independent (mb-mda-231) > normal human breast epithelial cells (mcf-10a). in both mcf-7 and mb-mda-231 cells, fermented pomegranate juice polyphenols consistently displayed about twice the anti-proliferative effect as fresh pomegranate juice polyphenols. pomegranate seed oil elicited 90% inhibition of proliferation of mcf-7 at 100 m g/ml medium, 75% inhibition of invasion of mcf-7 across a matrigel membrane at 10 m g/ml, and 54% apoptosis in mda-mb-435 estrogen receptor negative metastatic human breast cancer cells at 50 m g/ml. in a murine mammary gland organ culture, fermented juice polyphenols effected 47% inhibition of cancerous lesion formation induced by the carcinogen 7,12-dimethylbenz[a] anthracene (dmba). pomegranate seed oil and fermented pomegranate juice polyphenols were found to have anti-angiogenic activity (toi et al. 2003 ) . in-vitro studies showed that these pomegranate fractions strongly suppressed vascular endothelial growth factor in normal human breast epithelial cells (mcf-10a) and in estrogen sensitive (mcf-7) human breast cancer cells, but upregulated migration inhibitory factor in estrogen resistant (mda-mb-231) human breast cancer cells. an anti-proliferative effect on angiogenic cells was shown in human umbilical vein endothelial cell (huvec) and in myometrial and amniotic fl uid fi broblasts, and inhibition of huvec tubule formation was also demonstrated in an invitro model employing glass carrier beads. additionally, they showed a signi fi cant reduction in new blood vessel formation using the chicken chorioallantoic membrane (cam) model in-vivo. in another study, pretreatment of mouse mammary organ culture with pomegranate fermented juice polyphenols (w), a high-performance liquid chromatographic (hplc) peak separated from w (peak b), or pomegranate seed oil prior to exposure to the to the carcinogen 7,12-dimethylbenz[a] anthracene (dmba) resulted in a 42% reduction in the number of lesions for w compared with control, peak b and pomegranate seed oil each effected an 87% reduction (mehta and lansky 2004 ) . both pomegranate extracts and genistein inhibit the growth of mcf-7 breast cancer cells through induction of apoptosis, with combination treatment being more ef fi cacious than single treatments (jeune et al. 2005 ) . more recent studies demonstrated that pomegranate fruit extract dose-dependently inhibited nf-kb-dependent reporter gene expression associated with proliferation, invasion, and motility in aggressive breast cancer phenotypes while suppressing rhoc and rhoa protein expression ) . the bioactive components of the fruit extract comprised mainly ellagitannins and phenolic acids in the aqueous fruit extract and conjugated octadecatrienoic acids in the lipid fruit extract derived from seeds. the results suggested a role of pomegranate fruit extract in lowering the metastatic potential of aggressive breast cancer species. pomegranate extract inhibited the proliferation and viability of mmtv-wnt-1 mouse mammary cancer stem cells in-vitro in a timeand concentration-dependent manner (dai et al. 2010 ) . its constituents ellagic ursolic acid and luteolin also caused a time-and concentrationdependent reduction of cell proliferation and viability, suggesting that they contribute to the inhibitory effect of the extract, while caffeic acid had no effect. the methanolic pomegranate fruit peel extract was found to reduce cell proliferation and induce apoptosis on mcf-7 breast cancer cells (dikmen et al. 2011 ) . in addition, expression of the pro-apoptotic gene bax was increased, and that of the anti-apoptotic gene bcl-2 was decreased after pomegranate extract treatment. the extract exhibited high antioxidant activity and yielded total phenolic content of 331.28 mg of gallic acid equivalents/g of extract with ellagic acid as the most abundant constituent. among the ten pomegranate ellagitanninderived compounds (namely ellagic acid, gallagic acid, urolithins a and b and their acetylated, methylated, and sulfated analogues), urolithin b (ub) was shown to most effectively inhibit aromatase activity in a live breast cancer cell assay (adams et al. 2010 ) . ub signi fi cantly inhibited testosterone-induced mcf-7aro cell proliferation. the remaining test compounds also exhibited antiproliferative activity, but to a lesser degree than ub. the results suggested pomegranate et-derived compounds to have potential for the prevention of estrogen-responsive breast cancers. pomegranate seed linolenic acid isomers, punicic acid and α-eleostearic acid were found to be selective estrogen receptor modulators (serms) in-vitro (tran et al. 2010 ) . punicic acid inhibited (ic 50 ) estrogen receptor (er) α at 7.2 m m, estrogen receptor β at 8.8 m m. α-eleostearic acid (aea) inhibited erα/erβ at 6.5/7.8 m m. punicic acid agonized erα/erβ (ec 50 ) at 1.8/2 m m, antagonizing at 101/80 m m. α-eleostearic acid antagonized erα/erβ at 150/140 m m. both isomers induced erα and erβ mrna expression in mcf-7 breast cancer cells, but not in mda-mb-231 breast cancer cells. punicic acid, an omega-5 fatty acid in pomegranate seed oil, was found capable of inhibiting breast cancer proliferation (grossmann et al. 2010 ) . proliferation was inhibited 92 and 96% for mda-mb-231 and mda-erα7 cells, respectively. further punicic acid induced apoptosis in the mda-mb-231 and mda-erα7 cells by 86 and 91%, respectively compared to untreated control cells and disrupted cellular mitochondrial membrane potential. the results suggested the breast cancer inhibitor properties of punicic acid were dependent on lipid peroxidation and the protein kinase c signalling pathway. treatment of human lung carcinoma a549 cells with pomegranate fruit extract resulted in a decrease in the viability of a549 cells and dosedependent arrest of cells in g0-g1 phase of the cell cycle (khan et al. 2007a, b ) . treatment of cells with pomegranate fruit extract inhibited (i) phosphorylation of mapk proteins, (ii) pi3k, (iii) phosphorylation of akt at thr308, (iv) nf-kappab and ikkα, (v) degradation and phosphorylation of ikappabα, and (vi) ki-67 and pcna. oral administration of pomegranate fruit extract (0.1 and 0.2%, wt/vol) to athymic nude mice implanted with a549 cells resulted in a signi fi cant inhibition in tumour growth. treatment of mice with pomegranate juice prior to exposure to carcinogens benzo(a)pyrene (b(a) p) and n-nitroso-tris-chloroethylurea (ntcu), resulted in statistically signi fi cant lower lung tumour multiplicities than mice treated with carcinogens only (khan et al. 2007a ) . treatment of cells with pomegranate fruit extract caused inhibition of (a) activation of nuclear factor-kappab and ikappabα kinase, (b) degradation and phosphorylation of ikappabα, (c) phosphorylation of mitogen-activated protein kinases (extracellular signal-regulated kinase 1/2, c-jun nh(2)-terminal kinase 1/2, and p38), (d) phosphatidylinositol 3-kinase (p85 and p110), (e) phosphorylation of akt at thr(308), (f) activation of mammalian target of rapamycin signaling, (g) phosphorylation of c-met, and (h) markers of cell proliferation (ki-67 and proliferating cell nuclear antigen) and angiogenesis (inducible nitric oxide synthase, cd31, and vascular endothelial growth factor) in lungs of b(a)p-and ntcu-treated mice. overall, the results suggested that pomegranate fruit extract could be a useful chemopreventive/chemotherapeutic agent against human lung cancer. flavonoid-rich polyphenol fractions from the pomegranate fruit had been reported to exert antiproliferative, anti-invasive, anti-eicosanoid, and pro-apoptotic actions in breast and prostate cancer cells and anti-angiogenic activities in-vitro and in-vivo (kawaii and lansky 2004 ) . they found that various fruit extracts had proportional inhibitory effects on human hl-60 promyelocytic leukemia cell proliferation. fermented pomegranate juice and aqueous extract of pomegranate pericarps were found to be strong promoters of differentiation in all settings, while fresh juice extract showed only a relatively mild differentiation-promoting effect. li et al. ( 2011 ) found that pomegranate ellagitannins bound with gelatin to form self-assembled nanoparticles. ellagitannins encapsulated in nanoparticles were less effective in inducing the early stage of apoptosis on human promyelocytic leukemia cells hl-60. but they had similar effects in inducing late stage of apoptosis and necrosis. differentiation refers to the ability of cancer cells to revert to their normal counterparts, and its induction represents an important noncytotoxic therapy for leukemia, and also breast, prostate, and other solid malignancies (kawaii and lansky 2004 ) . pomegranate emulsion treatment (1 or 10 g/ kg) to rats, started 4 weeks prior to the dietary carcinogen diethylnitrosamine (dena) challenge and continued for 18 weeks thereafter, showed striking chemopreventive activity demonstrated by reduced incidence, number, multiplicity, size and volume of hepatic nodules, precursors of hepatocellular carcinoma (bishayee et al. 2011 ) . both doses of the emulsion signi fi cantly attenuated the number and area of g -glutamyl transpeptidase-positive hepatic foci compared with the dena control. the emulsion also attenuated dena-induced hepatic lipid peroxidation and protein oxidation and elevated protein and messenger rna expression of the hepatic nuclear factor e2-related factor 2 (nrf2). the methanolic extract of punica granatum fl owers was exhibited inhibitory effect on tumour necrosis factor-α (tnf-α, 1 ng/ml)induced cytotoxicity in l929 (murine fi broblast) cells (xie et al. 2008 ) . a new taraxastane-type triterpene, punicanolic acid (1), was isolated from the active fraction (ethyl acetate-soluble fraction) together with four triterpenes (2-5), two galloyl glucoses (6, 7), two fl avones (8, 9) , and b -sitosterol. among the constituents, 1, oleanolic acid (2), maslinic acid (4), 1,2,6-tri (7), and luteolin (8) signi fi cantly inhibited tnf-α-induced cytotoxicity in l929 cells at 30 m m. four pure chemicals, ellagic acid (e), caffeic acid (c), luteolin (l) and punicic acid (p), all important components of the aqueous compartments or oily compartment of pomegranate fruit exhibited anticancerous activities by inhibiting human pc-3 prostate cancer cell invasion of matrigel arti fi cial membranes (lansky et al. 2005a ) . all compounds signi fi cantly inhibited invasion when employed individually. when c, p, and l were equally combined at the same gross dosage (4 m g/ml) as when the compounds were tested individually, a supra-additive inhibition of invasion was observed. pomegranate cold-pressed seed oil, fermented juice polyphenols (w), and pericarp polyphenols (p) each acutely inhibited in-vitro proliferation of human prostate cancer, lncap, pc-3, and du 145 human cancer cell lines (albrecht et al. 2004 ) . these effects were mediated by changes in both cell cycle distribution and induction of apoptosis. for example, the androgen-independent cell line du 145 showed a signi fi cant increase from 11 to 22% in g(2)/m cells by treatment with pomegranate oil (35 m g/ ml) with a modest induction of apoptosis. in other cell lines/treatments, the apoptotic response predominated, for example, in pc-3 cells treated with pomegranate pericarp polyphenols, at least partially through a caspase 3-mediated pathway. all agents potently suppressed pc-3 invasion through matrigel, and furthermore pomegranate pericarp polyphenols and seed oil demonstrated potent inhibition of pc-3 xenograft growth in athymic mice. overall, the study demonstrated signi fi cant antitumour activity of pomegranatederived materials against human prostate cancer. in another study, combinations of the anatomically discrete pomegranate fractions: fermented pomegranate juice polyphenols (w), pomegranate pericarp (peel) polyphenols (p) or pomegranate seed oil (oil) exhibited synergistic prostate cancer suppression (lansky et al. 2005b ) . supraadditive, complementary and synergistic effects were proven in all models. proliferation effects were additionally evaluated with compusyn software median effect analysis and showed a concentration index ci < 1, con fi rming synergy. pomegranate fruit extract (pfe) exhibited antiproliferative and proapoptotic activities against human prostate cancer cells (malik et al. 2005 ; malik and mukhtar 2006 ) . pfe (10-100 m g/ ml; 48 h) treatment of highly aggressive human prostate cancer pc3 cells resulted in a dosedependent inhibition of cell growth/cell viability and induction of apoptosis. immunoblot analysis revealed that pfe treatment of pc3 cells resulted in (i) induction of bax and bak (proapoptotic); (ii) down-regulation of bcl-x(l) and bcl-2 (antiapoptotic); (iii) induction of waf1/p21 and kip1/p27; (iv) a decrease in cyclins d1, d2, and e; and (v) a decrease in cyclin-dependent kinase (cdk) 2, cdk4, and cdk6 expression. findings established the involvement of the cyclin kinase inhibitor-cyclin-cdk network during the antiproliferative effects of pfe. oral administration of pfe (0.1 and 0.2%, wt/vol) to athymic nude mice implanted with androgen-sensitive cwr22rnu1 cells resulted in a signi fi cant inhibition in tumour growth concomitant with a signi fi cant decrease in serum prostate-speci fi c antigen levels. the results suggested that pomegranate juice may have cancer-chemopreventive as well as cancerchemotherapeutic effects against prostate cancer in humans. in a phase ii, simon two-stage clinical trial for men with a rising prostate-speci fi c antigen (psa), daily consumption of pomegranate juice was found to have a positive effect following surgery or radiation for prostate cancer (pantuck et al. 2006 ) . there were no serious adverse events reported and the treatment was well tolerated. mean psa doubling time signi fi cantly increased with treatment from a mean of 15 months at baseline to 54 months posttreatment. in-vitro assays comparing pretreatment and posttreatment patient serum on the growth of human prostate cancer lncap showed a 12% decrease in cell proliferation and a 17% increase in apoptosis, a 23% increase in serum nitric oxide, and signi fi cant reductions in oxidative state and sensitivity to oxidation of serum lipids after versus before pomegranate juice consumption. in further studies, a standardized ellagitannins (ets)-enriched pomegranate extract (pe), signi fi cantly inhibited lapc-4 xenograft growth in severe combined immunode fi cient (scid) mice as compared to vehicle control seeram et al. 2007 ) . ellagic acid and several synthesized urolithins were shown to inhibit the growth of human prostate cancer cap cells in-vitro. the chemopreventive potential of pomegranate ets and localization of their bioactive metabolites in mouse prostate tissue suggested that pomegranate may play a role in cap treatment and chemoprevention. the results of studies demonstrated that an ellagitannin-rich pomegranate extract could inhibit tumour-associated angiogenesis as one of several potential mechanisms for slowing the growth of prostate cancer in chemopreventive applications (sartippour et al. 2008 ) . a pomegranate extract standardized to ellagitannin content (pomx) inhibited the proliferation of lncap and huvec cells signi fi cantly under both normoxic and hypoxic conditions. hif-1α (hypoxia-inducible factor-1α) and vegf (vascular endothelial growth factor) protein levels were also reduced by pomx under hypoxic conditions. pomx decreased prostate cancer xenograft size, tumour vessel density, vascular endothelial growth factor (vegf) peptide levels and hif-1α expression after 4 weeks of treatment in severe combined immunode fi cient (scid) mice. studies showed that pomegranate extract inhibited androgen-independent prostate cancer growth through a nuclear factor-kappabdependent mechanism . pomegranate extract (pe) inhibited nf-kappab and cell viability of prostate cancer cell lines in a dose-dependent fashion in vitro. maximal pe-induced apoptosis was dependent on pe-mediated nf-kappab blockade. in the lapc4 xenograft model, pe delayed the emergence of lapc4 androgen-independent xenografts in castrated mice through an inhibition of proliferation and induction of apoptosis. the scientist also showed that pomegranate polyphenols inhibited gene expression and androgen receptor (ar) most consistently in the human prostate cancer lncap-ar cell line (hong et al. 2008 ) . therefore, inhibition by pomegranate polyphenols of gene expression involved in androgen-synthesizing enzymes and the ar may be of particular importance in androgen-independent prostate cancer cells and the subset of human prostate cancers where ar is up-regulated. koyama et al. ( 2010 ) demonstrated that pomegranate extract derived from rind and arils (minus seeds) inhibited cell proliferation and induced apoptosis in human lapc4 prostate cancer cells by modulation of the igf-igfbp (insulin growth factor -insulin growth factor binding proteins) axis. pomegranate extract treatment also decreased igf-1 mrna expression in a dose-dependent manner indicating that its actions also involved tumour-speci fi c suppression of igf-1. pomegranate peel extracts increased the levels of oxygen radical absorbance capacity (orac) in plasma and the density of lecithin and the levels of zn in prostatitic rats (kuang et al. 2009 ) . it decreased the levels of malondialdehyde of prostate and the activity of acid phosphatase and the number of white blood cell and adjusted the levels of no in plasma compared with the prostatitis model group. the results indicated that pomegranate peel extracts could markedly improve the protective function of oxidation resistance. pomegranate ellagitannins/microbial metabolites were found to have cyp1b1 (a target in prostate cancer chemoprevention) inhibitory activity in prostate cancer cells (kasimsetty et al. 2009 ) . urolithin a, a microbial metabolite, was the most potent uncompetitive inhibitor of cyp1b1-mediated ethoxyresoru fi n-o -deethylase (erod) activity, exhibiting two-fold selectivity over cyp1a1, while urolithin b was a noncompetitive inhibitor with three-fold selectivity. the punicalins and punicalagins exhibited potent cyp1a1 inhibition with 5-10-fold selectivity over cyp1b1. cellular uptake experiments demonstrated a fi ve-fold increase in urolithin uptake by 22rv1 cells. western blots of the cyp1b1 protein indicated that the urolithins interfered with the expression of cyp1b1 protein. thus, urolithins were found to display a dual mode mechanism by decreasing cyp1b1 activity and expression. wang et al. ( 2011 ) showed that in addition to causing cell death of hormonerefractory prostate cancer cells, pomegranate juice also increased cell adhesion and decreased cell migration of the unkilled cells. pomegranate juice was found to upregulate genes involved in cell adhesion such as e-cadherin, intercellular adhesion molecule 1 (icam-1) and down-regulated genes involved in cell migration such as hyaluranan-mediated motility receptor (hmmr) and type i collagen. in addition, pomegranate juice signi fi cantly decreased the level of secreted pro-in fl ammatory cytokines/chemokines such as il-6, il-12p40, il-1 b and rantes, thereby having the potential to decrease in fl ammation and its impact. pomegrante juice also inhibited the ability of the chemokine sdf1 a to chemoattract these cancer cells. faria et al. ( 2007 ) found that pomegranate juice consumption decreased total hepatic cytochrome p450 (cyp) content as well as the expression of cyp1a2 and cyp3a in male mice. prevention of procarcinogen activation through cyp activity/expression inhibition may be involved in pomegranate juice's effect on tumour initiation, promotion, and progression pomegranate juice showed greatest antiproliferative activity against all cell lines namely human oral (kb, cal27), colon (ht-29, hct116, sw480, sw620) and prostate (rwpe-1, 22rv1) tumour cells by inhibiting proliferation from 30 to 100% (seeram et al. 2005a ) . at 100 m g/ml, pomegranate juice, ellagic acid, punicalagin and a standardized total pomegranate tannin (tpt) extract induced apoptosis in ht-29 colon cells. however, in the hct116 colon cells, ellagic acid, punicalagin and tpt but not pomegranate juice induced apoptosis. the trend in antioxidant activity was pomegranate juice > tpt > punicalagin > ellagic acid. their data indicated the superior bioactivity of pomegranate juice compared to its puri fi ed individual polyphenolic active ingredients illustrating the multifactorial effects and chemical synergy of the action of multiple compounds. in further studies, they (adams et al. 2006 ) found that pomegranate juice signi fi cantly suppressed tnf-α-induced cox-2 protein expression by 79%, total pomegranate tannin extract (tpt) 55%, and punicalagin 48% in ht-29 colcon cells. in addition, pomegranate juice reduced phosphorylation of the p65 subunit and binding to the nfkappab response element 6.4-fold, tpt suppressed nfkappab binding ten-fold, punicalagin 3.6fold, whereas ellagic acid was ineffective. pomegranate juice also abolished tnfαinduced akt activation, needed for nfkappab activity. pomegranate fruit rich in ellagitannins may have bene fi cial effects against colon cancer. in the stomach and gut, ellagitannins were reported to be hydrolyzed to release ellagic acid (ea) and were converted by gut microbiota to urolithin a (3,8-dihydroxy-6h-dibenzopyran-6one) type metabolites (sharma et al. 2010 ) . they reported that pomegranate ellagitannin extract, ellagic acid, and their colonic metabolite, urolithin a inhibited wnt signaling, which plays a pivotal role in human colon carcinogenesis, suggesting that et-rich foods may have potential against colon carcinogenesis and that urolithins were relevant bioactive constituents in the colon. studies by gonzález-sarrías et al. ( 2009 ) showed that elagic acid and its colonic metabolites, urolithin-a (3,8-dihydroxy-6h-dibenzo [b,d] pyran-6-one) and urolithin-b (3-hydroxy-6hdibenzo[b,d]pyran-6-one), modulated phase i and phase ii detoxifying enzymes in colon cancer caco-2 cells. ellagic acid and urolithins may exert some blocking chemopreventive effects in the colon but this effect may be critically affected by interfering factors, such as the food matrix nature. saruwatari et al. ( 2008 ) found that pomegranate juice potently inhibited the sulfoconjugation of 1-naphthol in caco-2 human colon carcinoma cells. the inhibition was both doseand culture time-dependent, with a 50% inhibitory concentration (ic 50 ) value of 2.7% (vol/vol). punicalagin, the most abundant antioxidant polyphenol in pomegranate juice, was also found to strongly inhibit sulfoconjugation in caco-2 cells with an ic 50 of 45 m m. additionally pomegranate juice and punicalagin both inhibited phenol sulfotransferase activity in caco-2 cells. the data also suggested that constituents of pomegranate juice, most probably punicalagin, impaired the enteric functions of sulfoconjugation and that this may have effects upon the bioavailability of drugs and other compounds and may be related to the anticarcinogenic properties of pomegranate juice. pomegranate seed oil (pgo) rich in 70% cis (c)9, trans (t)11,c13-18:3 as conjugated linolenic acids (cla) could suppress by azoxymethane -induced colon carcinogenesis, and the inhibition was associated in part with the increased content of cla in the colon and liver and/or increased expression of peroxisome proliferator-activated receptor (ppar) γ protein in the colon mucosa (kohno et al. 2004 ) . pomegranate extract was found to induce cell cycle arrest and alter cellular phenotype of human pancreatic cancer cells panc-1 and aspc-1 (nair et al. 2011 ) studies by weisburg et al. ( 2010 ) showed that pomegranate extract exerted greater antiproliferative effects towards cancer (such as hsc-2 carcinoma), than to normal, cells, isolated from the human oral cavity. the antiproliferative mechanism of pomegranate extract was, in part, by induction of oxidative stress. the mode of cell death was by apoptosis, as activation of caspase-3, and cleavage of parp. reduction of caspase-3 activation and of parp cleavage in cells co-treated with pomegranate extract and either cobalt or pyruvate, respectively, as compared to pomegranate extract alone, indicated that apoptosis was through the prooxidant nature of pomegranate extract. pomegranate seed oil (5%) signi fi cantly decreased mice skin tumour incidence, multiplicity, and 12-o -tetradecanoylphorbol 13-acetate (tpa)-induced ornithine decarboxylase activity, an important event in skin cancer promotion (hora et al. 2003 ) . the results suggested the potential of pomegranate seed oil as a safe and effective chemopreventive agent against skin cancer. afaq et al. ( 2005a, b ) demonstrated that topical application of pomegranate fruit extract (pfe) prior to 12-o -tetradecanoylphorbol-13-acetate (tpa) application on mouse skin afforded signi fi cant time-dependent inhibition, against tpa-mediated increase in skin edema and hyperplasia, epidermal ornithine decarboxylase (odc) activity and protein expression of odc and cyclooxygenase-2. also, application of pfe resulted in inhibition of tpa-induced phosphorylation of erk1/2, p38 and jnk1/2, as well as activation of nf-kappab and ikkα and phosphorylation and degradation of ikappabα. pretreatment of pfe on tpa-induced skin tumour promotion in 7,12-dimethylbenz(a)anthracene-initiated cd-1 mouse substantially reduced tumour incidence and lower tumour body burden when assessed as total number of tumours per group, percent of mice with tumours and number of tumours per animal as compared to animals that did not receive pfe. skin application of pfe prior to tpa application also resulted in a signi fi cant delay in latency period from 9 to 14 weeks and afforded protection when tumour data were considered in terms of tumour incidence and tumour multiplicity. studies by george et al. ( 2011 ) suggested that pomegranate fruit extract (pfe) and diallyl sul fi de (das) in combination afforded better suppressive activity of mouse skin tumours than either of these agents alone. pfe and das alone delayed onset and tumour incidence by ~ 55 and ~ 45%, respectively, while their combination at low doses synergistically decreased tumour incidence more potentially (~84%,). further, regression in tumour volume was seen with continuous combinatorial treatment. the inhibition was associated with decreased expression of phosphorylated erk1/2, jnk1 and activated nf-k b/p65, ikk a , i k b a phosphorylation and degradation in skin tissue/ tumour. polysaccharide (psp001) isolated from pomegranate rind was found to have antioxidant, antitumour and immunomodulatory properties (joseph et al. 2012 ) . psp001 exhibited a dosedependent enhancement in antioxidant activity using concentrations from 10 to 1,000 m g/ml when evaluated using various assays such as, ferric reducing antioxidant power assay, linoleic acid emulsion thiocyanate assay, and superoxide, hydroxyl and nitric oxide radical scavenging assays except for the dpph assay for which the highest activity was obtained at 200 m g/ml. psp001 exhibited anticancer activity with ic 50 values of 97.21 and 52.8 m g/-ml following 72 h incubation for mcf-7 (breast cancer), and k562(leukemia) cells, respectively. all the pomegranate peel extracts (ethyl acetate (etoac), acetone, methanol and water) decreased sodium azide mutagènicity in salmonella typhimurium strains (ta100 and ta1535), either weakly or strongly (negi et al. 2003 ) . at 2,500 m g/ plate all the extracts showed strong antimutagenicity. the antimutagenicity of the water extract was followed by acetone, etoac and methanol extracts. the methanol pomegranate peel fraction with promising antioxidant activity showed antimutagenic activity against sodium azide and methyl methane sulphonate with percent inhibition of mutagenicity ranging from 66.76 to 91.86% in a concentration-dependent manner using the ames salmonella/microsome assay (zahin et al. 2010a ) . similar trend of inhibition of mutagenicity (81.2-88.58%) against indirect mutagens (2-amino fl uorene and benzo(a)pyrene) was also recorded. phytochemical analysis by hplc, lc-ms of total phenolic content revealed high content of ellagitannins which might be responsible for promising antioxidant and antimutagenic activities of p. granatum peel extract. methanol extract of punica granatum fl owers (15 mg/plate) showed the highest antimutagenic activity in salmonella typhimurium ta 98 and ta 100, respectively (wongwattanasathien et al. 2010 ) . the protective effects of these fl ower extracts might be due to the presence of antimutagenic components that were supposed to be fl avonoids. studies demonstrated that tannin from the pericarp of punica granatum was an effective agent against genital herpes simplex virus (hsv-2) (zhang et al. 1995 ) . the tannin not only inhibited hsv-2 replication, but also showed stronger effects of killing virus and blocking its absorption to cells. punica granatum extract showed anti-human herpes simplex virus type 1 (hsv-1) activity, which was possibly contributed by the polyphenolic compounds in the herbal extract (li et al. 2004 ) . studies by neurath et al. ( 2005 ) indicated that hiv-1 entry inhibitors from pomegranate juice adsorbed onto corn starch and the resulting complex blocked virus binding to cd4 and cxcr4/ccr5 and inhibited infection by primary virus clades a to g and group o. their results suggested the possibility of producing an anti-hiv-1 microbicide from inexpensive, widely available sources. pomegranate juice containing polyphenols, β-sitosterol, sugars and ellagic acid) was reported to inactivate hiv and further shown to inactivate in fl uenza, herpes viruses and poxviruses (kotwal 2008 ) . a formulation consisting of fulvic acid, a complex mixture of compounds was previously reported to render vaccinia virus, hiv and sars virus non-infectious. recently, both fulvic acid and pomegranate juice were shown to inactivate genetically diverse strains of in fl uenza including h5n1, further con fi rming the broad spectrum nature of these agents. sundararajan et al. ( 2010 ) found that the acidity of pomegranate juice and concentrated liquid extract contributed to rapid anti-in fl uenza activity, but this was not a factor with pomegranate polyphenols powder (93%) extract. studies using pomegranate powder extract showed that 5 min treatment at room temperature with 800 m g/ml pomegranate polyphenols resulted in at least a 3log reduction in the titers of in fl uenza viruses pr8 (h1n1), x31 (h3n2), and a reassortant h5n1 virus derived from a human isolate. however, the antiviral activity was less against a coronavirus and reassortant h5n1 in fl uenza viruses derived from avian isolates. electron microscopic analysis indicated that viral inactivation by pomegranate polyphenols was primarily a consequence of virion structural damage. pomegranate polyphenol extract was shown to have anti-in fl uenza virus properties (haidari et al. 2009 ) . of four major polyphenols in pomegranate polyphenol extract (ppe) (ellagic acid, caffeic acid, luteolin, and punicalagin) punicalagin was the effective, anti-in fl uenza component. punicalagin blocked replication of the virus rna, inhibited agglutination of chicken rbc's by the virus and had virucidal effects. further, the combination of ppe and oseltamivir synergistically increased the anti-in fl uenza effect of oseltamivir. the data showed ppe inhibited the replication of human in fl uenza a/ hong kong (h3n2) virus in-vitro. exposure of foodborne virus surrogates feline calicivirus (fcv-f9), murine norovirus (mnv-1), and ms2 (ssrna) bacteriophage to pomegranate juice and pomegranate polyphenols resulted in titer reductions after one hour at room temperature, suggesting promise for use in hurdle technologies and/or for therapeutic or preventive use (su et al. 2010 ) . ethanolic extracts of garcinia mangostana , punica granatum and quercus infectoria were found to have good antimicrobial activity of nine thai medicinal plants with mics for methicillin-resistant staphylococcus aureus (mrsa) isolates of 0.05-0.4, 0.2-0.4 and 0.2-0.4 mg/ml, respectively, and for s. aureus atcc 25923 of 0.1, 0.2 and 0.1 mg/ml, respectively (voravuthikunchai and d kitpipit 2005 ) . mbcs for mrsa isolates were 0.1-0.4, 1.6-3.2 and 0.4-1.6 mg/ml, and for s. aureus atcc 25923 were 0.4, 3.2 and 1.6 mg/ml, respectively. punica granatum was found to have anti-quorum-sensing activity and may be useful in combating pathogenic bacteria and reduce the development of antibiotic resistance (koh and tham 2011 ; zahin et al. 2010b ) . in another study the ethanolic extract of p. granatum exhibited bacteriostatic and bactericidal activities against two enterohemorrhagic escherichia coli strains (voravuthikunchai and limsuwan 2006 (pai et al. 2010 ) . ethanol extract of p. granatum exhibited strong antibacterial activity against escherichia coli (sharma et al. 2009 ) . studies showed that punica granatum (pomegranate) methanolic extract (pgme) dramatically enhanced the activity of all antibiotics tested (braga et al. 2005a ) . synergic activity was detected between pgme and the fi ve antibiotics tested, chloramphenicol, gentamicin, ampicillin, tetracycline, and oxacillin, ranging from 38 to 73%. using pgme (0.1 × mic) in combination with ampicillin (0.5 × mic), cell viability was reduced by 99.9 and 72.5% in methicillin-sensitive staphylococcus aureus (mssa) and methicillin-resistant staphylococcus aureus (mrsa) populations, respectively. pgme increased the post-antibiotic effect (pae) of ampicillin from 3 to 7 h. pomegranate extract inhibited staphylococcus aureus growth and subsequent enterotoxin production (braga et al. 2005b ) . of several thai medicinal plants, the ethanol extract of p. granatum fruit rind displayed the most outstanding in-vitro antibacterial activity with mic of 0.39 and 12.5 mg/ml and mbc of 1.56 and 12.5 mg/ml against staphylococcus aureus and escherichia coli respectively (chansakaow et al. 2005 ) . the extract was found to contain both hydrolysable and condensed tannins. the methanol pomegranate pericarp extract exhibited maximum antibacterial activity against salmonella typhimurium , salmonella typhi and shigella dysenteriae serotype 1 (pradeep et al. 2008 ) . studies showed that the antibacterial activity of pomegranate rind can be enhanced by the addition of metal salts and vitamin c (mccarrell et al. 2008 ) . pomegranate rind extracts (pre) exhibited activity against the gram positive organisms at 24 h were inactive against gram negative bacteria. addition of cu 2+ salts to pre solutions extended the activities resulting in no detectable growth being observed for the pre/cu 2+ combination against escherichia coli , pseudomonas aeruginosa and proteus mirabilis . minimal antimicrobial activity was observed following incubation with fe 2+ , mn 2+ or zn 2+ salts alone or in combination with pre against any of the organisms in the test panel. the addition of vitamin c markedly enhanced the activities of both pre/fe 2+ and pre/cu 2+ combinations against staphylococcus aureus . pelargonidin-3-galactose, cyanidin-3-glucose, gallic acid, quercetin, and myricetin isolated from the methanolic extract of pomegranate fruit exhibited appreciable activity against species of corynebacteria , staphylococcus , streptococcus , shigella , salmonella , bacillus subtilis , vibrio cholera , and escherichia coli (naz et al. 2007 ) . however, all these compounds were more inhibitory against gram-positive species. gallic acid exerted the highest inhibitory activity against all the tested bacteria. various tannin-rich fractions from pomegranate byproduct and the ellagitannins, ellagic acid (1), gallagic acid (2), punicalins (3), and punicalagins (4) displayed antimicrobial activity when assayed against escherichia coli , pseudomonas aeruginosa , candida albicans , cryptococcus neoformans , methicillin-resistant staphylococcus aureus (mrsa), aspergillus fumigatus and mycobacterium intracellulare (reddy et al. 2007 ) . compounds 2 and 4 showed activity against p. aeruginosa , c. neoformans , and mrsa. a new antifungal peptide designated as pomegranin, isolated from fresh pomegranate peels, was found to inhibit mycelial growth of the fungi botrytis cinerea and fusarium oxysporum with an ic 50 of 2 and 6.1 m m, respectively (guo et al. 2009 ) . lyophilized pomegranate juice (lpj) exhibited antilisterial activity in-vitro and in ground beef (lucas and were 2009 ) . against fi ve listeria monocytogenes strains, lpj had a mic of 1.50-1.75% (wt/vol). the lpj (0, 30, 60, and 120 min of heating) signi fi cantly inhibited growth of all fi ve l. monocytogenes strains in refrigerated ground cooked beef by 1.80-4.61 log cfu/g at day 21. heating did not negatively impact lpj antilisterial activity. ethanol peel extract of pomegranate exhibited in-vitro and in-vivo antimicrobial activity against salmonella typhimurium (choi et al. 2011 ) . the minimal inhibitory concentrations of their extract were in the range of 62.5-1,000 m g/ml. in a s. typhimurium infection mouse model. the extract was found to have signi fi cant effects on mortality and the numbers of viable s. typhimurium recovered from faeces. although clinical signs and histological damage were rarely observed in the treated mice, the untreated controls showed signs of lethargy and histological damage in the liver and spleen. the results of this study indicated that the peel extract had the potential to provide an effective treatment for salmonellosis. studies on patients with denture stomatitis showed that gel extract of p. granatum may be used as a topical antifungal agent for the treatment of candidosis associated with denture stomatitis (vasconcelos et al. 2003 ) . in subsequent studies, punica granatum phytotherapeutic gel and miconazole (daktarin oral gel) exhibited antimicrobial effect against three standard streptococci strains ( streptococcus mutans , streptococcus sanguis and streptococcus mitis ), s. mutans clinically isolated and candida albicans either alone or in association (vasconcelos et al. 2006 ) . the minimum inhibitory concentrations of adherence of punica granatum gel against the test organisms were: 1:16 for s. mutans (atcc), s. mutans (ci) and s. sanguis ; 1:128 for s. mitis and 1:64 for c. albicans . the minimum inhibitory concentrations of adherence of miconazole against the same organisms were: 1:512, 1:64, 1:4, 1:128 and 1:16, respectively. in experiments with three and four associated microorganisms, the punica granatum gel had greater ef fi ciency in inhibiting microbial adherence than the miconazole. the results of this study suggest that this phytotherapeutic agent might be used in the control of adherence of different microorganisms in the oral cavity. studies showed that the hydroalcoholic extract from punica granatu m fruits was very effective against dental plaque microorganisms, decreasing the colony forming units per milliliter (cfu/ml) by 84% (menezes et al. 2006 ) . while similar values were observed with chlorhexidine, used as standard and positive control (79% inhibition). however, another study found that the gel containing 10% punica granatum extract was not ef fi cient in preventing supragingival dental plaque formation and gingivitis (salgado et al. 2006 ) . methanolic extract of pomegranate peel exhibited antibacterial activity against oral pathogens: staphylococcus aureus and s. epidermidis (abdollahzadeh et al. 2011 ) . only at concentration of 8 mg/ml and 12 mg/ml the extract was effective against lactobacillus acidophilus , streptococcus mutans and streptococcus salivarius . the extract did not inhibit actinomyces viscosus and candida albicans . pomegranate rind extract (pre) singularly showed limited ef fi cacy against methicillin-sensitive and -resistant staphylococcus aureus ( mssa, mrsa) respectively but in combination with cu(ii) ions (cupric sulphate), it exhibited moderate antimicrobial effects against clinical isolates of mssa, mrsa and panton-valentine leukocidin positive community acquired mssa (pvl positive ca-mssa) isolates. (gould et al. 2009 ) . sastravaha et al. ( 2005 ) showed that adjunctive local delivery of extracts from centella asiatica in combination with p. granatum signi fi cantly improved clinical signs of chronic periodontitis such probing pocket depth, attachment level, gingival index at 3 and 6 months and of bleeding index at 6 months in the test group as compared to control. no signi fi cant differences in plaque index were found between the two treatment modalities. the test group also showed statistically greater reduction of interleukin il-1β at both 3 and 6 months and lower il-6 concentration. a study of young adults showed that 4 weeks of thrice daily mouth rinsing with the extract improved salivary measures relevant to oral health including gingivitis (disilvestro et al. 2009 ) . salivary changes observed included a reduction in total protein (associated with plaque forming bacteria readings), activities of aspartate aminotransferase (an indicator of cell injury) and α-glucosidase activity (a sucrose degrading enzyme). the changes also included increased activities of the antioxidant enzyme ceruloplasmin and radical scavenging capacity. pomegranate mouth-rinse was found to have an antiplaque effect (bhadbhade et al. 2011 ) . aggregatibacter actinomycetemcomitans , porphyromonas gingivalis , and prevotella intermedia strains in-vitro. pomegranate mouth-rinse could be explored as a long-term antiplaque rinse with prophylactic bene fi ts. probiotication improved the antioxidant activity of sweet pomegranate aril juice from 74.4 to 91.82%, and sour pomegranate juice from 82.64 to 97.8% (fazeli et al. 2011 ) . based on the ferric reducing antioxidant power (frap) value, the reducing power of the probioticated pomegranate juices was also much stronger than the nonprobioticated juices. the frap values for sweet and sour probioticated pomegranate juices were 97.34 and 120.7 mmol/l, respectively, which were notably higher than 85.87 and 93.4 mmol/l for sweet and sour nonprobioticated juices. total counts of lactobacillus casei gg increased by about three log in sweet and two log in sour juices after 48 h incubation. both fermentated and nonfermentated juices exhibited a potent and widespectrum antibacterial effect, with the highest activity against pseudomonas aeruginosa with the sweet juice showing wider zones of growth inhibition. the results showed that probiotication of sweet and sour pomegranate juices could add to their bene fi cial antioxidant activities. pomegranate byproducts and punicalagins inhibited the growth of pathogenic clostridia and staphylococcus aureus (bialonska et al. 2009b ) . the growth of probiotic lactobacilli and bi fi dobacteria were generally not affected by ellagitannins. the effect of pomegranate ellagitannins on bi fi dobacteria was species-and tannin-dependent. the growth of bi fi dobacterium animalis ssp. lactis was slightly inhibited by punicalagins, punicalins, and ellagic acid. pomegranate ellagitannin-enriched polyphenol extract (pomx) supplementation signi fi cantly enhanced the growth of bi fi dobacterium breve and bi fi dobacterium infantis. bialonska et al. ( 2009a ) found that products of the intestinal microbial transformation of pomegranate ellagitannins may account for systemic antioxidant effects. while moving through the intestines, pomegranate ellagitannins namely ellagic acid and punicalagins are metabolized by gut bacteria into urolithins that readily enter systemic circulation. their study found that the antioxidant activity of urolithins was correlated with the number of hydroxy groups as well as the lipophilicity of the molecule. the most potent antioxidants were urolithins c and d with ic 50 values of 0.16 and 0.33 m m, respectively, when compared to ic 50 values of 1.1 and 1.4 m m of the parent ellagic acid and punicalagins, respectively. the dihydroxylated urolithin a showed weaker antioxidant activity, with an ic 50 value 13.6 m m, however, the potency was within the range of urolithin a plasma concentrations. numerous laboratory research, animal and human pilot studies had reported on the effectiveness of pomegranate fruit, pomegranate juice and pomegranate fruit polypehnols in reducing heart disease risk factors ldl oxidation, blood pressure, serum angiotensin converting enzyme (ace) activity, cholesterol esteri fi cation, macrophage oxidative status, and macrophage foam cell formation, all of which are steps in atherosclerosis and cardiovascular disease (aviram et al. 2000 (aviram et al. , 2002 (aviram et al. , 2004 aviram and dornfeld 2001 ; kaplan et al. 2001 ; esmaillzadeh et al. 2004 ; fuhrman et al. 2005 ; de nigris et al. 2005 ; rosenblat et al. 2006a, b ; fuhrman and aviram 2007 ; bagri et al. 2009 ) . in healthy humans, pomegranate juice consumption decreased ldl susceptibility to aggregation and retention and increased the activity of serum paraoxonase by 20% (aviram et al. 2000 ) . paraoxanase an hdlassociated esterase, could protect against lipid peroxidation. in apolipoprotein e-de fi cient e o mice, oxidation of ldl by peritoneal macrophages was reduced by up to 90% after pomegranate juice consumption and this effect was associated with reduced cellular lipid peroxidation and superoxide release. the uptake of oxidized ldl and native ldl by mouse peritoneal macrophages obtained after pomegranate juice administration was reduced by 20%. further, pomegranate juice supplementation of e o mice reduced the size of their atherosclerotic lesions by 44% and also the number of foam cells compared with control e o mice supplemented with water. the potent antiatherogenic effects in healthy humans and in atherosclerotic mice may be attributable to its antioxidative properties. anti-atherosclerotic properties was attributed to pomegranate potent anti-oxidative characteristics. after consumption of pomegranate juice, a 36% reduction in serum angiotensin converting enzyme (ace) activity and a 5% reduction in systolic blood pressure were noted in hypertensive patients . similar dose-dependent inhibitory effect (31%) of pomegranate juice on serum ace activity was observed also in-vitro. additional studies showed that pomegranate juice supplementation to atherosclerotic mice reduced macrophage lipid peroxidation, cellular cholesterol accumulation and development of atherosclerosis (kaplan et al. 2001 ) . pomegranate juice supplementation reduced each of the proatherogenic variables. it signi fi cantly induced serum paraoxonase activity and reduced mouse peritoneal macrophage (mpm) lipid peroxide content compared with placebo-treated mice and control mice. pomegranate juice administration to apolipoprotein e-de fi cient e o mice signi fi cantly reduced the oxidized (ox)-ldl mpm uptake by 31% and mpm cholesterol esteri fi cation and increased macrophage cholesterol ef fl ux by 39% compared with age-matched, placebo-treated mice. pomegranate juice consumption reduced macrophage ox-ldl uptake and cholesterol esteri fi cation to levels lower than those in 4-month-old, unsupplemented controls. pomegranate juice supplementation to e o mice with advanced atherosclerosis reduced the lesion size by 17% compared with place botreated mice. in a separate study, supple mentation of young (2-month-old) e o mice for 2 months with a tannin fraction isolated from pomegranate juice reduced their atherosclerotic lesion size, paralleled by reduced plasma lipid peroxidation and decreased ox-ldl mpm uptake. studies indicated that the proatherogenic effects induced by perturbed shear stress in cultured human coronary artery endothelial cells could be reversed by chronic administration of pomegranate juice (de nigris et al. 2005 (de nigris et al. , 2007 . pomegranate juice concentrate and pomegranate fruit extract rich in punicalagin reduced the activation of redox-sensitive genes elk-1, p-jun, p-creb, and increased enos expression (which was decreased by perturbed shear stress) in cultured endothelial cells and in atherosclerosis-prone areas of hypercholesterolemic mice. furthermore, oral administration of pomegranate juice to hypercholesterolemic mice at various stages of disease reduced signi fi cantly the progression of atherosclerosis and isoprostane levels and increased nitrates. de found that pomegranate juice reverted the potent downregulation of the expression of endothelial nitricoxide synthase (nosiii) induced by oxidized low-density lipoprotein (oxldl) in human coronary endothelial cells. their data suggested that pomegranate juice could exert bene fi cial effects on the evolution of clinical vascular complications, coronary heart disease, and atherogenesis in humans by enhancing the nitric-oxide synthase bioactivity. aviram et al. ( 2002 ) reported that pomegranate polyphenols protected low-density lipoprotein (ldl) against cell-mediated oxidation via two pathways, including either direct interaction of the polyphenols with the lipoprotein and/or an indirect effect through accumulation of polyphenols in arterial macrophages (aviram et al. 2002 ) . pomegranate polyphenols were shown to reduce the capacity of macrophages to oxidatively modify ldl, due to their interaction with ldl to inhibit its oxidation by scavenging reactive oxygen species and reactive nitrogen species and also due to accumulation of polyphenols in arterial macrophages; hence, the inhibition of macrophage lipid peroxidation and the formation of lipid peroxide-rich macrophages. additionally, pomegranate polyphenols increased serum paraoxonase activity, resulting in the hydrolysis of lipid peroxides in oxidized lipoproteins and in atherosclerotic lesions. these antioxidative and antiatherogenic effects of pomegranate polyphenols were demonstrated in-vitro, as well as in-vivo in humans and in atherosclerotic apolipoprotein e de fi cient mice. dietary supplementation of polyphenol-rich pomegranate juice to atherosclerotic mice signi fi cantly inhibited the development of atherosclerotic lesions and this may be attributed to the protection of ldl against oxidation. subsequent studies indicated that that pomegranate juice consumption by patients with carotid artery stenosis cas decreased carotid carotid intima-media thickness (imt) and systolic blood pressure and these effects could be related to the potent antioxidant characteristics of pomegranate juice polyphenols (aviram et al. 2004 ) . for all studied parameters, the maximal effects were observed after 1 year of pomegranate juice consumption. further consumption of pomegranate juice, for up to 3 years, had no additional bene fi cial effects on imt and serum paraoxonase 1 (pon 1) activity, whereas serum lipid peroxidation was further reduced by up to 16% after 3 years of pomegranate juice consumption. the antiatherogenic properties of pomegranate juice (pj) were attributed to its antioxidant potency and to its capacity to decrease macrophage oxidative stress, the hallmark of early atherogeneis (rosenberg et al. 2006 ). pomegranate juice polyphenols and sugar-containing polyphenolic anthocyanins were shown to confer pj its antioxidant capacity. their study showed that pj sugar consumption by diabetic mice for 10 days resulted in a small but signi fi cant decrement in their peritoneal macrophage total peroxide levels and an increment in cellular glutathione content, compared to mouse peritoneal macrophages harvested from control diabetic mice administrated with water. these antioxidant/antiatherogenic effects could be due to the presence of unique complex sugars and/or phenolic sugars in pj. they further showed the anti-oxidative characteristics of pj unique phenolics punicalagin and gallic acid could be related, at least in part, to their stimulatory effect on macrophage paraoxonase 2 (pon2) expression, a phenomenon which was shown to be associated with activation of the transcription factors papr γ and ap-1 (shiner et al. 2007 ) . similar results were obtained by pomegranate byproduct (which includes the whole pomegranate fruit left after juice preparation) (17 or 51.5 m g of gallic acid equiv/kg/day) administration to apolipoprotein e-de fi cient mice that resulted in attenuation of atherosclerosis development as a result of decreased macrophage oxidative stress and reduced cellular uptake of oxidized low-density lipoprotein (rosenblat et al. 2006 ) . in-vitro studies showed that preincubation of j774.a1 macrophages with pomegranate juice resulted in a signi fi cant reduction in ox-ldl degradation by 40% (fuhrman et al. 2005 ) . macrophage cholesterol biosynthesis was inhibited by 50% after cell incubation with pomegranate juice. this inhibition, however, was not mediated at the 3-hydroxy-3 methylglutaryl coenzyme a reductase level along the biosynthetic pathway. it was concluded that pomegranate juice-mediated suppression of ox-ldl degradation and of cholesterol biosynthesis in macrophages could lead to reduced cellular cholesterol accumulation and foam cell formation. studies in iran reported that consumption of concentrated pomegranate juice may modify heart disease risk factors in hyperlipidemic ( cholesterol ³ 5.2 mmol/l or triacylglycerol ³ 2.3 mmol/l) patients (esmaillzadeh et al. 2004 (esmaillzadeh et al. , 2006 . after consumption of concentrated pomegranate juice, signi fi cant reductions were seen in total cholesterol, low-density lipoprotein (ldl)-cholesterol, ldl-cholesterol/high-density lipoprotein (hdl)-cholesterol, and total cholesterol/hdl-cholesterol. but, there were no signi fi cant changes in serum triacylglycerol and hdl-cholesterol concentrations. anthropometric indices, physical activity, kind and doses of oral hypoglycemic agents, and the intakes of nutrients and fl avonoid-rich foods showed no change during the concentrated pomegranate juice consumption period. rosenblat et al. ( 2006 ) reported that pomegranate juice consumption by diabetic patients did not affect serum glucose, cholesterol and triglyceride levels, but it resulted in a signi fi cant reduction in serum lipid peroxides and tbars (thiobarbituric acid reactive substance) levels by 56 and 28%, whereas serum sh (sulfhydryl) groups and paraoxonase 1 (pon1) activity signi fi cantly increased by 12 and 24%, respectively. in the patients versus controls monocytes-derived macrophages (hmdm), they observed increased level of cellular peroxides (by 36%), and decreased glutathione content (by 64%). pomegranate juice consumption signi fi cantly reduced cellular peroxides (by 71%), and increased glutathione levels (by 141%) in the patients' hmdm. the patients' versus control hmdm took up oxidized ldl (ox-ldl) at enhanced rate (by 37%) and pomegranate juice consumption signi fi cantly decreased the extent of ox-ldl cellular uptake (by 39%). they thus concluded that pomegranate juice consumption by diabetic patients did not worsen the diabetic parameters, but rather resulted in anti-oxidative effects on serum and macrophages, which could contribute to attenuation of atherosclerosis development in these patients. pomegranate juice was found to have potent antiatherogenic activity . in-vitro studies demonstrated a pomegranate juice dose-dependent antioxidant capability against lipid peroxidation in plasma (by 33%), in ldl (by 43%), and in hdl (by 22%). pomegranate juice consumption by hypertensive patients reduced their systolic blood pressure (by 6%), along with inhibition (by 40%) of angiotensin converting enzyme (ace). pomegranate juice supplementation to atherosclerotic apolipoprotein e-de fi cient (e°) mice reduced their atherosclerotic lesion size by 44% and the number of foam cells in their lesion. consumption of pomegranate juice by ten patients with carotid artery stenosis (cas) for 1 year reduced the patients' carotid intima-media thickness (imt) by 32%. these effects were associated with exvivo reduced lipid peroxidation in plasma and in isolated lipoproteins in humans and mice. furthermore, pomegranate juice consumption by humans increased the activity of their serum paraoxonase (pon1), an hdl-associated esterase that protects against lipid peroxidation. macrophage atherogenicity was studied in mouse peritoneal macrophages (mpm) harvested from e° mice. following pomegranate juice consumption, uptake of oxidized ldl and cell-mediated oxidation of ldl by macrophages was reduced by 88 and by 20%, respectively, in association with reduced cellular lipid peroxidation, reduced superoxide anion release due to decreased nadph-oxidase activation, and elevated glutathione content. in-vitro studies demonstrated that pomegranate juice reduced macrophage ox-ldl degradation by 40%, and macrophage cholesterol biosynthesis by 50%. overall, the results of the above studies demonstrated that pomegranate juice consumption had very potent antiatherogenic properties, which could be associated mainly with pomegranate juice hydrolysable tannin antioxidative properties. in a recent study ) pomegranate juice (pj), fruit peels (pomxl, pomxp), arils (poma), seeds (pomo), and fl owers (pomf), extracts all were found to possess antioxidative properties in-vitro. after consumption of pomegranate juice, fruit peel, aril and fl ower extracts the atherosclerotic lesion area in atherosclerotic apolipoprotein e-de fi cient (e 0) mice was signi fi cantly decreased by 44, 38, 39, 6, or 70%, respectively, as compared to placebo-treated group, while pomegranate seed oil had no effect. pomegrante fl ower consumption reduced serum lipids, and glucose levels by 18-25%. consumption of the extracts except for the seed oil resulted in a signi fi cant decrement, by 53, 42, 35, 27, or 13%, respectively, in mpm (mouse peritoneal macrophage) total peroxides content, and increased cellular paraoxonase 2 (pon2) activity, as compared to placebo-treated mice. the uptake rates of oxidized-ldl by e (0)-mpm were signi fi cantly reduced by approximately 15% after consumption of juice and the two fruit peel extracts. similar results were obtained on using j774a.1 macrophage cell line. finally, pomegranate phenolics (punicalagin, punicalin, gallic acid, and ellagic acid), as well as pomegranate unique complexed sugars, could mimic the antiatherogenic effects of the pomegranate extracts. rock et al. ( 2008 ) reported that after 4 weeks of pomegranate juice consumption by male patients, basal serum oxidative stress was signi fi cantly decreased by 35%, whereas serum concentrations of thiol groups signi fi cantly increased by 25%. moreover, hdlassociated paraoxonase 1(pon1), arylesterase, paraoxonase, and lactonase activities increased signi fi cantly after pomegranate juice consumption by 34-45%, as compared to the baseline levels. pon1 protein binding to hdl was signi fi cantly increased by 30% following pomegranate juice consumption, and the enzyme became more stable. in male patients that consumed pomegranate polyphenol extract and in female patients that consumed pomegranate juice, a similar pattern was observed, although to a lesser extent. these bene fi cial effects of pomegranate consumption on serum pon1 stability and activity could lead to retardation of atherosclerosis development in diabetic patients. results of a randomized, double-blind, parallel trial involving men (45-74 years old) and women (55-74 years old) with moderate coronary heart disease risk suggested that in subjects at moderate coronary heart disease risk, pomegranate juice consumption had no signi fi cant effect on overall carotid intima-media thickness progression rate but may have slowed carotid intima-media thickness progression in subjects with increased oxidative stress and disturbances in the triglycerides-rich lipoprotein/high-density lipoprotein axis (davidson et al. 2009 ) . lei et al. ( 2007 ) reported that the pomegranate leaf extract could inhibit the development of obesity and hyperlipidemia in high-fat diet induced obese mice. mice treated with the extract presented a signi fi cant decrease in body weight, energy intake and various adipose pad weight percents and serum, serum total cholesterol (tc), triglyceride (tg), glucose levels and tc/highdensity lipoprotein cholesterol (hdl-c) ratio after 5 weeks treatment. further, the extract signi fi cantly attenuated the raising of the serum tg level and inhibited the intestinal fat absorption in mice given a fat emulsion orally. the effects were postulated to be partly mediated by inhibiting the pancreatic lipase activity and suppressing energy intake. yamasaki et al. ( 2006 ) found that mice fed dietary pomegranate seed oil (pso) high in levels of punicic acid showed signi fi cant increases in serum triacylglycerol and phospholipid levels but not in total cholesterol. punicic acid could be detected in serum, liver, and adipose tissues in mice fed the 0.12 or 1.2% pso diet. oral administration of streptozotocin-induced diabetic wistar rats with of 250 and 500 mg/kg of aqueous pomegranate fl ower extract for 21 days resulted in a signi fi cant fall in fasting blood glucose, total cholesterol, triglycerides, low-density lipoprotein cholesterol , very low density lipoprotein, lipid peroxidation level (bagri et al. 2009 ) . pomegrante extract elevated levels of high density lipoprotein cholesterol (hdl-c), reduced glutathione (gsh) and the antioxidative enzymes, glutathione peroxidase (gpx), glutathione reductase (gr), glutathione-s-transferase (gst), superoxide dismutase (sod) and catalase (cat). mcfarlin et al. ( 2009 ) found that weight gain in high fat diet mice was associated with an increase in biomarkers of cholesterol pro fi le, glucose sensitivity, adipose tissue accumulation and systemic low-grade in fl ammation. despite a similar level of energy intake, high-fate diet mice had a greater concentration of leptin and a lower concentration of adiponectin compared to high fat + pomegranate seed oil diet mice. pomegranate seed oil, a rich source of 9-cis , 11-trans conjugate linolenic acid, only altered body weight accumulation, fi nal body weight, leptin, adiponectin and insulin. pomegranate seed oil intake was associated with an improvement in insulin sensitivity, suggesting that risk of developing type two diabetes may have been reduced; however, cvd risk did not change. lan et al. ( 2009 ) demonstrated that ellagic acid in pomegranate leaf tannins could be transported into hepg2 cells and this correlated with total cholesterol alteration in the cells. vroegrijk et al. ( 2011 ) found that pomegranate seed oil, a rich source of punicic acid, ameliorated high-fat diet induced obesity and insulin resistance in mice, independent of changes in food intake or energy expenditure. compared to high fat diet mice, its intake resulted in a lower body weight and improved peripheral insulin sensitivity but did not affect liver insulin sensitivity. in a randomized, double-blind, placebo-controlled clinical trial of 20 obese adult volunteer, pomegranate juice administration for 1 month did not modify insulin secretion and sensitivity in the obese patients, however, the natural evolution to increased weight and adiposity was halted (gonzález-ortiz et al. 2011 ) . rosenblat and aviram ( 2011 ) found that the inhibitory effect of pomegranate juice on triglyceride biosynthesis could be attributed to a direct effect of pomegranate juice on the activity of diacylglycerol acyltransferase 1 (dgat1) the rate-limiting enzyme in triglyceride biosynthesis. pomegranate juice and its constituent punicalagin signi fi cantly and dose-dependently decreased the triglyceride content and triglyceride biosynthesis rate in j774a.1 macrophages or in c57bl/6 mouse peritoneal macrophages. both pomegranate juice and punicalagin increased (1.7-fold) mouse peritoneal macrophages paraoxonase 2 (pon2) mrna expression, and pon2 was previously shown to inhibit dgat1 activity. however, the addition of pj or punicalagin (50 m m) to microsomes from pon2-de fi cient mouse peritoneal macrophages still resulted in a signi fi cant reduction (50-58%) in dgat1 activity. in a randomised block design study of student volunteers, supplementation of pomegranate juice caused a fall in diastolic blood pressure and this could be related to ros scavenging activity rather than to angiotensin-converting enzyme inhibitors (wright and pipkin 2008 ) oral administration of pomegranate juice extract (100 and 300 mg/kg) to angiotensin-ii treated rats for 4 weeks signi fi cantly reduced the mean arterial blood pressure and vascular reactivity changes to various catecholamines (waghulde et al. 2010 ) . pomegranate juice administration signi fi cantly decreased the serum levels of ace (angiotensin converting enzyme) and the levels of thiobarbituric acid reactive substances (tbars); while enzyme activity of superoxide dismutase (sod), catalase (cat), glutathione reductase (gsh) in kidney tissue showed a signi fi cant elevation in pomegranate juice treated angiotensin-ii induced hypertensive rats. the results suggested that pomegranate juice extract could prevent the development of high blood pressure induced by angiotensin-ii probably by combating the oxidative stress and antagonizing the physiological actions of angiotensin-ii. chronic administration of pomegranate fruit juice (pj) extract (100 and 300 mg/kg; p.o. for 4 weeks) reduced the mean arterial blood pressure and vascular reactivity changes to various catecholamines and also reversed the biochemical changes induced by diabetes and angiotensin ii (ang ii) (mohan et al. 2010 b ) . acute subcutaneous administration of angiotensin ii causes a rise in blood pressure in streptozotocin-induced diabetic wistar rats. pj treatment also caused a signi fi cant decrease in levels of thiobarbituric acid reactive substances (tbars) in the kidney and pancreas while activities of enzymes superoxide dismutase (sod), catalase (cat), and glutathione reductase (gsh) showed signi fi cant elevation. pj treatment prevented the tubular degenerative changes induced by diabetes. the results suggested that the pj extract could prevent the development of high blood pressure induced by ang ii in diabetic rats probably by combating the oxidative stress induced by diabetes and ang ii and by inhibiting ace activity. pomegranate in particular its fl owers, seeds, and juice have been employed for the treatment of various diseases in traditional unani and ayurvedic systems of medicine in india but only the fl ower has been prescribed for the treatment of diabetic disorders katz et al. 2007 ) . the mechanisms for it hypoglycaemic effects are largely unknown, though recent research suggested pomegranate fl owers and juice may prevent diabetic sequelae via peroxisome proliferator-activated receptor (ppar) α/γ binding and nitric oxide production (katz et al. 2007 ; huang et al. 2005a, b ; li et al. 2008 ; xu et al. 2009 ) . pomegranate compounds associated with such effects include oleanolic, ursolic, and gallic acids (katz et al. 2007 ) . another study suggested that punica granatum fl ower (pgf) extract inhibited increased cardiac fatty acid uptake and oxidation in the diabetic condition (huang et al. 2005b ) . pgf extract and its component oleanolic acid enhanced peroxisome proliferator-activated receptor (ppar)-α luciferase reporter gene activity in human embryonic kidney 293 cells. this effect was completely suppressed by a selective ppar-α antagonist mk-886, consistent with the presence of ppar-α activator activity in the extract and this component. the fi ndings suggested that pgf extract improved abnormal cardiac lipid metabolism in zucker diabetic fatty rats by activating ppar-α and thereby lowering circulating lipid and inhibiting its cardiac uptake. excess triglyceride (tg) accumulation and increased fatty acid (fa) oxidation in the diabetic heart contribute to cardiac dysfunction. in subsequent in-vitro studies, the scientists (huang et al. 2005a ) demonstrated that 6-week oral administration of methanol extract from pgf (500 mg/kg, daily) inhibited glucose loading-induced increase of plasma glucose levels in zucker diabetic fatty rats (zdf), a genetic animal model for type two diabetes, whereas it did not inhibit the increase in zucker lean rats (zl). the treatment did not lower the plasma glucose levels in fasted zdf and zl rats. further, rt-pcr results demonstrated that the pgf extract treatment in zdf rats enhanced cardiac ppar-γ mrna expression and restored the down-regulated cardiac glucose transporter (glut)-4 (the insulin-dependent isoform of gluts) mrna. these results suggest that the anti-diabetic activity of pgf extract may result from improved sensitivity of the insulin receptor. from the in-vitro studies, it was demonstrated that the pgf extract enhanced ppar-γ mrna and protein expression and increased ppar-γ-dependent mrna expression and activity of lipoprotein lipase in human thp-1-differentiated macrophage cells. phytochemical investigation demonstrated that gallic acid in pgf extract was mostly responsible for this activity. further in-vitro studies showed that punica granatum fl ower extract and its components oleanolic acid, ursolic acid, and gallic acid inhibited lipopolysaccharide-induced nf-kappab activation in macrophages. the fi ndings indicated that punica granatum fl ower extract reduced cardiac fi brosis in zucker diabetic fatty rats, at least in part, by modulating cardiac et-1 and nf-kappab signalling. recent studies suggested that pomegranate fl ower (pgf) medicine ameliorated diabetes and obesity-associated fatty liver, at least in part, by activating hepatic expression of genes responsible for fatty acid oxidation ) . pgf-treated zdf rats showed reduced ratio of liver weight to tibia length, hepatic triglyceride contents and lipid droplets. these effects were accompanied by enhanced hepatic gene expression of peroxisome proliferator-activated receptor (ppar)-α, carnitine palmitoyltransferase-1 and acyl-coa oxidase (aco), and reduced stearoyl-coa desaturase-1. in contrast, pgf showed minimal effects on expression of genes responsible for synthesis, hydrolysis or uptake of fatty acid and triglycerides. pgf treatment also increased ppar-α and aco mrna levels in hepg2 cells. li et al. ( 2008 ) reviewed the dual ppar-α/-γ activator properties of pomegranate fl ower and its potential treatment of diabetes and its associated complications. ppars are nuclear transcription factors and are the major regulators of lipid and glucose metabolism. ppar-α is involved in the regulation of fatty acid (fa) uptake and oxidation, in fl ammation and vascular function, while ppar-γ participates in fa uptake and storage, glucose homeostasis and in fl am mation. synthetic ppar-α or ppar-γ agonists have been widely used in the treatment of dyslipidaemia, hyperglycaemia and their complications. however, they are associated with an incidence of adverse events. given the favourable metabolic effects of both ppar-α and ppar-γ activators, as well as their potential to modulate vascular disease, com bined ppar-α/-γ activation has recently emerged as a promising concept, leading to the development of mixed ppar-α/-γ activators. hontecillas et al. ( 2009 ) demonstrated that punicic acid (pua), a conjugated linolenic acid isomer found in pomegranate, caused a dosedependent increase ppar α and γ reporter activity in 3 t3-l1 pre-adipocyte cells and bound although weakly to the ligand-binding domain of human ppar γ. dietary pua decreased fasting plasma glucose concentrations, improved the glucose-normalizing ability, suppressed nf-kappab activation, tnf-α expression and upregulated ppar αand γ-responsive genes in skeletal muscle and adipose tissue. pua improved glucose homeostasis and suppress obesity-related in fl ammation studies in india showed that pomegranate seed extract (150, 300 and 600 mg/kg) administered orally to streptozotocin (stz)-induced diabetic rats caused a signi fi cant reduction of blood glucose levels by 47 and 52%, respectively, at the end of 12 h (das et al. 2001 ) . kim et al. ( 2011 ) found that administration of pomegranate extract to streptozotocin (stz)-induced diabetic mice for 4 weeks improved postprandial glucose regulation. further elevated na(+)-dependent glucose uptake by brush border membrane vesicles isolated from stz mice was normalized by pomegranate treatment. the results suggested that pomegranate extract could play a role in controlling the dietary glucose absorption at the intestinal tract by decreasing sodium-coupled glucose transporter sglt1 expression, and may contribute to blood glucose homeostasis in the diabetic condition. oral administration of pomegranate fl ower (pgf) extract markedly lowered plasma glucose levels in non-fasted zucker diabetic fatty rats (a genetic model of obesity and type two diabetes), whereas it had little effect in the fasted animals, suggesting it affected postprandial hyperglycemia in type two diabetes . the extract was found to markedly inhibit the increase of plasma glucose levels after sucrose loading, but not after glucose loading in mice, and it had no effect on glucose levels in normal mice. in-vitro, pgf extract demonstrated a potent inhibitory effect on α-glucosidase activity (ic 50 : 1.8 m g/ml). these fi ndings strongly suggested that pgf extract improved postprandial hyperglycemia in type two diabetes and obesity, at least in part, by inhibiting intestinal α-glucosidase activity. postprandial hyperglycemia plays an important role in the development of type two diabetes and has been proposed as an independent risk factor for cardiovascular diseases. in a recent paper, bagri et al. ( 2009a ) reported that oral administration of pomegranate aqueous extract at doses of 250 and 500 mg/kg for 21 days to stzinduced diabetic rats resulted in a signi fi cant reduction in fasting blood glucose, total cholesterol (tc), triglycerides (tg), low-density lipoprotein cholesterol (ldl-c), very low density lipoprotein (vldl), and tissue lipid peroxidation levels coupled with elevation of high density lipoprotein cholesterol (hdl-c), glutathione (gsh) content and antioxidant enzymes in comparison with diabetic control group. the results suggested that pg could be used, as a dietary supplement, in the treatment of chronic diseases characterized by atherogenous lipoprotein pro fi le, aggravated antioxidant status and impaired glucose metabolism and also in their prevention. in-vitro studies showed that pomegranate juice polyphenols increased recombinant paraoxonase-1 binding to high-density lipoprotein (hdl) beyond their antioxidative effect (fuhrman et al. 2010 ) . further recombinant paraoxonase-1 was found to be associated more ef fi ciently with hdls isolated from diabetic patients after pomegranate juice consumption versus hdls isolated before pomegranate juice consumption. studies by ahmed et al. ( 2005 ) showed that pomegranate fruit extract or compounds derived from it may inhibit cartilage degradation in osteoarthritis and may also be a useful nutritive supplement for maintaining joint integrity and function. the extract inhibited interleukin (il)-1β induced expression of matrix metalloproteinases by suppressing the activation of mitogen-activated protein kinases and nuclear factor-kappab in human chondrocytes in-vitro. pomegranate methanol extract was found to dose-dependently inhibit tumour necrosis factor α (tnf-α) production in lipopolysaccharide (lps) stimulated cells (jung et al. 2006 ) . the data suggested that the extract may suppress lps-stimulated tnf production through inhibition of nfkappab in bv2 microglia cells. shukla et al. ( 2008b ) reported that consumption of hydrolyzable tannins-rich pomegranate extract potently delayed the onset and reduced the incidence and severity of collagen-induced arthritis in mice. pomegranate extract -fed mice had reduced joint in fi ltration by the in fl ammatory cells, and the destruction of bone and cartilage were alleviated. levels of interleukin il-6 were signi fi cantly decreased in the joints of pomegranate-fed mice with collagen-induced arthritis. in mouse macrophages, pomegranate extract abolished multiple signal transduction pathways and downstream mediators implicated in the pathogenesis of rheumatoid arthritis. in another study, rabbit plasma samples collected after oral ingestion of polyphenol rich pomegranate fruit extract were found to inhibit the il-1β-induced pge2 and no production in chondrocytes (shukla et al. 2008a ) . these same plasma samples also inhibited both cox-1 and cox-2 enzyme activity ex-vivo but the effect was more pronounced on the enzyme activity of cox-2 enzyme. the studies suggested that pomegranate fruit extract-derived bioavailable compounds may exert an anti -in fl ammatory effect by inhibiting the in fl ammatory cytokineinduced production of pge2 and no in-vivo. pomegranate extract rich in polyphenols was found to inhibit the interleukin-1 b -induced activation of mkk-3, p38 a -mapk and transcription factor runx-2 in human osteoarthritis chondrocytes (rasheed et al. 2010 ) . this pharmacological actions of pomegranate extract suggest that the extract or its derived compounds may be developed as mkk and p38-mapk inhibitors for the treatment of osteoarthritis and other degenerative/ in fl ammatory diseases. in a pilot12 week openlabelled study, pomegranate consumption reduced the composite disease activity index (das28) and tender joint count in rheumatoid arthritis patients, and this effect could be related to the antioxidative property of pomegranates (balbir-gurman et al. 2011 ) . the results suggested dietary supplementation with pomegranates may be a useful complementary strategy to attenuate clinical symptoms in rheumatoid arthritis patients. supplementation of obese zucker rats with pomegranate fruit extract (pfe) or pomegranate juice (pj) signi fi cantly decreased the expression of vascular in fl ammation markers, thrombospondin (tsp), and cytokine tgfβ1, whereas seed oil supplementation had a signi fi cant effect only on tsp-1 expression (de nigris et al. 2007a ) . plasma nitrate and nitrite (no(x)) levels were signi fi cantly increased by pfe and pj. in addition, the effect of pfe in increasing endothelial no synthase (enos) expression was comparable to that of pj. the data suggested possible clinical applications of pfe in metabolic syndrome (clinical conditions such as obesity, hypertension, dislipidemia, and diabetes). in-vivo studies revealed that aqueous pomegranate peel extract inhibited neutrophil myeloperoxidase activity and attenuated lipopolysaccharide-induced lung in fl ammation in mice (bachoual et al. 2011 ) . inhibition of myeloperoxidase activity by pomegranate extract could explain its antiin fl ammatory action. balwani et al. ( 2011 ) demonstrated that 2-methyl-pyran-4-one-3-o -b -d-glucopyranoside (mpg) isolated from pomegranate leaves, inhibited tnf a -induced cell adhesion molecules expression by blocking nuclear transcription factor-k b (nf-k b) translocation and activation. the results suggested that mpg could be useful as a novel lead molecule for developing future antiin fl ammatory agents. oral pomegranate extract decreased reactive oxygen species concentration and acute in fl ammation in the tympanic membrane in rats after myringotomy (kahya et al. 2011 ) . the density of in fl ammatory cells was signi fi cantly less in rats treated with the extract and the lamina propria thickness and vessel density were also signi fi cantly reduced. pretreatment of wistar rats with a methanolic extract of pomegranate peel followed by carbon tetrachloride treatment retained catalase, peroxidase, and superoxide dismutase to values comparable with control values, whereas lipid peroxidation was reduced by 54% as compared to control (chidambara murthy et al. 2002 ) . histopathological studies of the liver supported the hepatoprotective effects exhibited by the extract by restoring the normal hepatic architecture. kaur et al. ( 2006 ) demonstrated that pre-treatment of mice with pomegranate fl ower extract at a dose regimen of 50-150 mg/ kg body weight for a week signi fi cantly and dose dependently protected against ferric nitrilotriacetate (fe-nta)-induced oxidative stress as well as hepatic injury. the extract conferred up to 60% protection against hepatic lipid peroxidation and preserved glutathione (gsh) levels and activities of antioxidant enzymes viz., catalase (cat), glutathione peroxidase (gpx) glutathione reductase (gr) and glutathione-s-transferase (gst) by up to 36, 28.5, 28.7, 40.2 and 42.5% respectively. a protection against fe-nta induced liver injury was apparent as inhibition in the modulation of liver markers viz., aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphatase (alp), bilirubin and albumin in serum. the histopathological changes produced by fe-nta, such as ballooning degeneration, fatty changes, necrosis were also alleviated by the extract. the fl ower extract was found to signi fi cantly scavenge superoxide radicals by up to 53.3%, hydrogen peroxide by up to 30%, hydroxyl radicals by up to 37% and nitric oxide by up to 74.5%. the extract also inhibited (.)oh induced oxidation of lipids and proteins in vitro. the potent antioxidant property of the fl ower extract was postulated to be responsible for its hepatoprotective effects. in another study, pomegranate fl ower infusion was found to exhibit hepatoprotective and antioxidant effect against trichloroacetic acid (tca)-exposed rats (celik et al. 2009 ) . the infusion signi fi cantly decreased levels of aspartate aminotransferase and alanine aminotransferase; increased signi fi cantly glutathione s-transferase activity in the liver, brain and spleen and maintained superoxide dismustase level in the liver. intake of pomegranate juice by mice for weeks was found to confer hepatic protection against protein and dna oxidation (faria et al. 2007 b ) . there was also a signi fi cant decrease in gsh (reduced glutathione) and gssg (oxidized glutathione), without change in the gsh/gssg ratio. all studied enzymatic activities (superoxide dismutase (sod), glutathione peroxidase (gpx), glutathione s-transferase (gst) and glutathione reductase (gr) and catalase) were found to be decreased by pomegranate juice treatment. also, glutathione s-transferase and glutathione synthetase transcription were also decreased in this group. chronic pomegranate peel extract administration to rats alleviated the bile duct ligation (bdl)-induced oxidative injury of the liver and improved the hepatic structure and function (toklu et al. 2007 ) . plasma antioxidant capacity and hepatic glutathione levels were signi fi cantly depressed by bdl but were increased back to control levels in the pomegranate extract-treated bdl group. increases in tissue malondialdehyde levels and myeloperoxidase activity due to bdl were reduced back to control levels by pomegranate extract treatment. similarly, increased hepatic collagen content in the bdl rats was reduced to the level of the control group with extract treatment. sumner et al. ( 2005 ) showed that daily consumption of pomegranate juice may improve stressinduced myocardial ischemia in patients who have coronary heart disease in a randomized, placebo-controlled, double-blind study. after 3 months, the extent of stress-induced ischemia decreased in the pomegranate group (sds − 0.8 ± 2.7) but increased in the control group (sds 1.2 ± 3.1). this bene fi t was observed without changes in cardiac medications, blood sugar, hemoglobin a1c, weight, or blood pressure in either group. mohan et al. ( 2010a ) demonstrated that pre-treatment of male wistar rats with pomegranate juice (100 and 300 mg/kg, p.o.) and its butanolic extract(100 mg/kg., p.o.) for a period of 21 days signi fi cantly inhibited the effects of isoproterenol-induced myocardial infarction such as heart rate, pressure rate index, ecg patterns, levels of lactate dehydrogenase, creatine kinase, superoxide dismutase and catalase in the serum and vascular reactivity changes. treatment with pj and b-pj (100 mg/kg., p.o.) alone did not alter any of the parameters as compared to vehicletreated wistar rats. hassanpour et al. ( 2011 ) found that pomegranate fruit extract displayed cardioprotective doxorubicin (dox)-induced cardiotoxicity in rats. rats administered the extract showed decreased qt and increase in heart rate compared to the dox group signi fi cant decrease in creatine kinase-mb isoenzyme, lactate dehydrogenase and no such signi fi cant decrease in aspartate aminotransferase were observed as compared to the dox group. there was signi fi cant increase in the level of reduced glutathione, whereas inhibition of lipid peroxidation and increase in superoxide dismutase concentration was not signi fi cant in the extract treated group compared to the dox group. histopathological study of the extract -treated group showed slight protection against myocardial toxicity induced by dox. p. granatum fruit peel extract elicited 100% precipitation of ovine haemoglobin in-vitro and when orally administered to ethanol-induced gastric-damaged rats produced a signi fi cant decrease in gastric lesions (gharzouli et al. 1999 ) . the acid content of the stomach was signi fi cantly increased by pomegranate (368%) suggesting that monomeric and polymeric polyphenols could strengthen the gastric mucosal barrier. administration of 70% methanolic pomegranate rind extract inhibited aspirin-and ethanol-induced gastric ulceration (ajaikumar et al. 2005 ) . treated animals showed increased antioxidant levels of superoxide dismutase (sod), catalase, glutathione (gsh) and glutathione peroxidase (gpx) and decreased level of tissue lipid peroxidation. no erosion of gastric mucosa, sub-mucosal edema and neutrophil in fi ltration was observed in treated animals. pomegranate tannins (500, 150, 50 mg/ kg) signi fi cantly inhibited ulcerative formation induced by both water immersion stress and pylorus ligation, and decreased the gastric mucosa damages induced by intragastric absolute ethanol, in dose-dependent manner in rats (lai et al. 2009 ) . its antiulcer effect was found to be due to increasing secretion of adherent mucus and free mucus from the stomach wall, which may inhibit generation of oxygen-derived free radicals, and decrease the consumption glutathione peroxidase (gsh-px) and superoxide dismutase (sod), decrease absolute alcohol-induced elevation of malondialdehyde and maintain content of no at normal level. punica granatum peel extract (ppe) supplementation of irradiated rats reduced oxidative damage in the ileal tissues and protected against ionizing radiation-induced enteritis and leukocyte apoptosis in rats, probably by a mechanism associated with the decreased production of reactive oxygen metabolites and enhancement of antioxidant mechanisms (toklu et al. 2009 ) . ppe treatment reversed all these biochemical indices induced by irradiations such as the decrease in glutathione and total antioxidant capacity associated with increases in malondialdehyde levels, myeloperoxidase activity, collagen content of the tissue with a concomitant increase 8-hydroxy-2 ¢deoxyguanosine (an index of oxidative dna damage) and increases in pro-in fl ammatory cytokines (tnf-α, il-1β and il-6) and lactate dehydrogenase. histopathological alterations and the increase in leukocyte apoptosis and cell death induced by irradiation was also reversed by ppe. oral administration of aqueous methanolic extract of pomegranate (490 and 980 mg/kg bw) signi fi cantly reduced the ulcer lesion index produced by alcohol, indomethacin, and aspirin, at both doses in rats (alam et al. 2010 ) . in pylorusligated rats the extract signi fi cantly reduced the ulcer lesions, gastric volume, and total acidity and prevented the ulceration by increasing the ph and mucus secretion. oral administration of pomegranate extract and its ellagic acid rich fraction (100 and 200 mg/ kg) signi fi cantly attenuated dextran sulfate sodium -induced colonic in fl ammation in mice along with attenuation of histamine, myeloperoxidase and oxidative stress (singh et al. 2009 ) . the antiulcerative effect was comparable to sulphasalazine (100 mg/kg, p.o.) and sodium cromoglycate (40 mg/kg i.p). the authors stated that the antiulcerative effects may be attributed to mast cell stabilizing, antiin fl ammatory and antioxidant actions. pomegrante peel extracts exhibited remarkable in-vitro anti-helicobacter pylori activity against the clinical isolates of h. pylori (mean of inhibition zone diameter ranging from 16 to 40 mm/50 m g disc). helicobacter pylori infection causes lifelong chronic gastritis, which can lead to peptic ulcer, mucosa-associated lymphoid tissue (malt) lymphoma and gastric cancer. pretreatment of rats with hydroalcoholic extract of pomegranate fl owers (125 and 250 mg/kg p.o. twice daily for 3 days) signi fi cantly attenuated hypertonic glycerol-induced myoglobinuric renal dysfunction in a dose-dependent manner . the mechanism of renoprotective effects of punica granatum was found to involve activation of ppar-g and nitric oxide-dependent signalling pathway. pomegranate fruit rind powder at the dose of 100 mg/kg orally as aqueous suspension was found to stimulate the cell-mediated and humoral components of the immune system in rabbits (gracious ross et al. 2001 ) . the pomegranate powder elicited an increase in antibody titer to typhoid-h antigen. it also enhanced the inhibition of leucocyte migration in leucocyte migration inhibition test and induration of skin in delayed hypersensitivity test with puri fi ed protein derivative (ppd) con fi rming its stimulatory effect on cell-mediated immune response. punicalagin isolated from pomegranate fruit was found to be a potent immune suppressant, based on its inhibitory action on the activation of the nuclear factor of activated t cells (nfat). punicalagin downregulated the mrna and soluble protein expression of interleukin-2 from anti-cd3/anti-cd28-stimulated murine splenic cd4+ t cells and suppressed mixed leukocytes reaction (mlr) without exhibiting cytotoxicity to the cells. in vivo, the punicalagin treatment inhibited phorbol 12-myristate 13-acetate (pma)induced chronic ear edema in mice and decreased cd3+ t cell in fi ltration of the in fl amed tissue. the results suggested that punicalagin could be a potential candidate for the therapeutics of various immune pathologies. yamasaki et al. ( 2006 ) found that dietary pomegranate seed oil (pso) high in levels of punicic acid (9c, 11 t, 13c-octadecatrienoic acid), may enhance b-cell function in mice. splenocytes isolated from mice fed 0.12 or 1.2% pso produced larger amounts of immunoglobulins g and m but not immunoglobulin a irrespective of stimulation with or without phorbol 12-myristate 13-acetate and the calcium ionophore a23187. dietary pso did not affect the percentages of b cells or cd4-positive or cd8-positive t cells in splenocytes. a polysaccharide (psp001) isolated from pomegranate rind was found to have immunomodulatory activity (joseph et al. 2012 ) . psp001 showed in-vitro growth stimulatory effect on isolated normal lymphocytes, and a proliferative index of 1.21 at a concentration of 1,000 m g/-ml was obtained, indicating immunomodulatory activity. wistar rats with excision wounds treated with 5% water-soluble gel formulated from the methanolic extract of dried pomegranate rind, showed good complete wound healing after 10 days (chidambara murthy et al. 2004 ) . in comparison in rats treated with 2.5% gel, healing was observed on day 12, and in the positive control animals receiving the blank gel took 16-18 days for complete healing. collagen content in terms of hydroxyproline level increased by two-fold in the group treated with 5.0% gel. the gel extract was found to contain gallic acid and catechin as major components. aslam et al. ( 2006 ) found that pomegranate seed oil, but not aqueous extracts of fermented juice, peel or seed cake, stimulated human keratinocyte proliferation in monolayer culture. contrariwise, pomegranate peel aqueous extract (and to a lesser extent, both the fermented juice and seed cake extracts) stimulated type i procollagen synthesis and inhibited matrix metalloproteinase-1 (mmp-1; interstitial collagenase) production by dermal fi broblasts, but had no growth-supporting effect on keratinocytes. the results suggested that pomegranate peel aqueous extract could promote regeneration of dermis and pomegranate seed oil could promote regeneration of epidermis. pomegranate peel methanol extract-based ointment signi fi cantly enhanced wound contraction and the period of epithelialization as assessed by the mechanical (contraction rate, tensile strength), the biochemical (increasing of collagen, dna and proteins synthesis) and the histopathological characteristics in guinea pigs (hayouni et al. 2011 ) . the extract displayed antioxidant activity as potent as natural and synthetic compounds (trolox, butylated hydroxyanisole, quercetin). in addition, the extract exhibited signi fi cant antibacterial and antifungal activity against pseudomonas aeruginosa , staphylococcus aureus , escherichia coli , klebsiella pneumoniae , salmonella anatum , salmonella typhimurium , streptococcus pneumoniae , and fungi candida albicans , candida glabrata , trichopyton rubrum and aspergillus niger. the results suggested that the pomegranate formulated ointment may be used as skin repair agent without hazard to human health. the ethanol extract of p. granatum fl owers showed signi fi cant wound healing activity when topically administered in rats (pirbalouti et al. 2010 ) . the extract signi fi cantly increased the rate of wound contraction and collagen turnover. in-vitro studies using normal human epidermal keratinocytes, showed that pre-treatment with pomegranate fruit extract rich in anthocyannins and hydrolyzable tannins protected against the adverse effects of uv-b radiation by inhibiting uv-b-induced modulations of nuclear factor kappa b (nf-kappab) and mitogen-activated protein kinases (mapk) pathways (afaq et al. 2005a, b ) . similarly, they reported pomegranate fruit extract to be an effective agent for ameliorating uva-mediated skin damages by modulating cellular pathways in-vitro (syed et al. 2006 ) . uva-mediated cellular damage occurs primarily through the release of reactive oxygen species and is responsible for immunosuppression, photodermatoses, photoaging and photocarcinogenesis. pretreatment of normal human epidermal keratinocytes with the extract (60-100 m g/ ml) for 24 h before exposure to uva resulted in a dose-dependent inhibition of uva-mediated phosphorylation of signal transducers and activators of transcription 3 (stat3), protein kinase b/ akt and mitogen activated protein kinases (mapks) viz. extracellular signal-regulated kinase (erk1/2). the extract pretreatment also inhibited uva exposure-mediated increases in ki-67 antigen and pcna (proliferating cell nuclear antigen) and increased the cell-cycle arrest induced by uva in the g1 phase and the expression of bax and bad (proapoptotic proteins), while suppressing bcl-x(l) antiapoptotic protein expression. studies by zaid et al. ( 2007 ) showed that pretreatment of human immortalized hacat keratinocytes with polyphenol-rich pomegranate fruit extract inhibited uvb-mediated decrease in cell viability, decrease in intracellular glutathione content and increase in lipid peroxidation. immunoblot analysis showed that pretreatment of hacat cells with pomegranate fruit extract inhibited uvb-induced (1) upregulation of mmp-1, -2, -7 and -9, (2) decrease in timp-1, (3) phosphorylation of mapks and (iv) phosphorylation of c-jun, whereas no effect was observed on uvb-induced c-fos protein levels. the results suggested that pomegranate fruit protected hacat cells against uvb-induced oxidative stress and markers of photoaging and could be a useful supplement in skin care products. pomegranate fruit extract (5-60 mg/l) was effective at protecting human skin fi broblasts from cell death following uv irradiation (pacheco-palencia et al. 2008 ) . this photoprotective effect was postulated to be related to a reduced activation of the pro-in fl ammatory transcription factor nf-kappab, suppression of proapoptotic caspase-3, and an increased g0/g1 phase, associated with dna repair. higher polyphenolic concentrations (500-10,000 mg/l) were required to achieve a signi fi cant reduction in uv-induced reactive oxygen species levels and increased intracellular antioxidant capacity. pretreatment of reconstituted human skin "epiderm" with pomegranate-derived products pomx juice, pomx extract and pomegranate oil inhibited uvb-induced cyclobutane pyrimidine dimers (cpd), 8-dihydro-2 ¢deoxyguanosine (8-ohdg), protein oxidation and proliferating cell nuclear antigen (pcna) protein expression (afaq et al. 2009 ) . further, pretreatment of epiderm with pomegranate-derived products resulted in inhibition of uvb-induced collagenase (mmp-1), gelatinase (mmp-2, mmp-9), stromelysin (mmp-3), marilysin (mmp-7), elastase (mmp-12), and tropoelastin. mmp-2 and mmp-9 activities were also inhibited. overall, the results suggested that all three pomegranate-derived products may be useful against uvb-induced damage to human skin. park et al. ( 2010 ) using cultured human skin fi broblasts, demonstrated that pomegranate fruit rind extract rich in polyphenols catechin, quercetin, kaempferol, and equol signi fi cantly protected against uvb-induced skin damage. the synthesis of collagen was increased and the expression of mmp-1 was decreased. oral feeding of pomegranate fruit extract to mice provided substantial protection from the adverse effects of uvb radiation via modulation in early biomarkers of photocarcinogenesis (afaq et al. 2010 ) . the extract inhibited uvb-induced: skin edema; hyperplasia; in fi ltration of leukocytes; lipid peroxidation; hydrogen peroxide generation; ornithine decarboxylase (odc) activity; and odc, cyclooxygenase-2 and proliferating cell nuclear antigen protein expression. the extract enhanced repair of uvbmediated formation of cyclobutane pyrimidine dimers (cpds) and 8-oxo-7,8-dihydro-2 ¢deoxyguanosine (8-oxodg). the extract inhibited uvb-mediated nuclear translocation of nf-k b; activation of ikk a ; and phosphorylation and degradation of i k b a . additionally, the extract further enhanced uvb-mediated increase in tumour suppressor p53 and cyclin kinase inhibitor p21. in further studies, khan et al. ( 2012 ) reported that oral feeding of pomegranate fruit extract to skh-1 hairless mice inhibited uvbinduced epidermal hyperplasia, in fi ltration of leukocytes, protein oxidation and lipid peroxidation. immunoblot analysis demonstrated that oral feeding of pomegranate fruit extract to mice inhibited uvb-induced (1) nuclear translocation and phosphorylation of nuclear factor kappa b/p65, (2) phosphorylation and degradation of i k b a , (3) activation of ikk a / i k k b and (4) phosphorylation of mitogen-activated protein kinase proteins and c-jun. pomegranate fruit extract consumption also inhibited uvb-induced protein expression of (1) cox-2 and inos, (2) pcna and cyclin d1 and (3) matrix metalloproteinases-2,-3 and -9 in mouse skin. overall, the data showed that pomegranate fruit extract consumption afforded protection to mouse skin against the adverse effects of uvb radiation by modulating uvbinduced signalling pathways. in a double-blind, placebo-controlled trial involving female subjects age 20-40s, kasai et al. ( 2006 ) found that oral administration of an ellagic acid-rich pomegranate extract had an inhibitory effect on a slight pigmentation (stains and freckles, brightness of face) in the human skin caused by uv irradiation. methanolic pomegranate extract showed 53.4% invitro mushroom tyrosinase inhibitory activity (adhikari et al. 2008 ) . a pomegranate rind extract was found to have skin whitening activity (yoshimura et al. 2005 ) . the extract exhibited inhibitory activity against mushroom tyrosinase invitro comparable to that of the skin whitening agent, arbutin. when taken orally the extract inhibited uv-induced skin pigmentation on the back of brownish guinea pig. the results suggested the skinwhitening effect of the extract was probably due to inhibition of the proliferation of melanocytes and melanin synthesis by tyrosinase in melanocytes. a pomegranate polysaccharide fraction inhibited the formation of advanced glycation end-products (ages) by 28% and also inhibited the formation of fructosamine in the bsa/glucose system (rout and banerjee 2007 ) . the fraction inhibit 1,1-diphenyl-2-picrylhydrazyl (dpph) and 2,2 ¢ -azinobis[3ethylbenzothiazoline-6-sulfonate] abts(+) radical activities by 69 and 88%, respectively with 4 m g/ ml concentration it also inhibited mushroom tyrosinase by 43% at 10 m g/ml concentration suggesting its ef fi cacy as a potential skin whitener. pomegrante juice was shown to have a protective effect against ethylene glycol-induced nephrolithiasis in rats (tugcu et al. 2008 ) . ethylene glycol caused hyperoxaluria characterised by severe crystalization in renal tubules and granulovacuolar epithelial cell degeneration, marked elevation in malondialdehyde and nitric oxide levels and decrease of reduced glutathione (gsh) in rats there was no crystal formation in the rats treated with ethylene glycol and pomegranate juice. administration of pomegranate juice at medium and high dosage to rats was found to have inhibitory effects on renal tubular cell injury and oxidative stress caused by oxalate crystal deposition by reducing ros, inos, p38-mapk, and nf-kb expression (ilbey et al. 2009 ) . rats treated with methanol pomegranate seed extract exhibited signi fi cant inhibitory activity against castor-oil induced diarrhoea and pge2 induced enteropooling (das et al. 1999 ) . the extract also displayed a signi fi cant reduction in gastrointestinal motility in charcoal meal test in rats. pomegranate juice and polyphenol-rich pomegranate fruit extract reduced platelet aggregation, calcium mobilization, thromboxane a(2) production, and hydrogen peroxide formation, induced by collagen and arachidonic acid (mattiello et al. 2009 ) . the polyphenol -rich fruit extract was more potent in reducing platelet activation. studies showed that pomegranate fruit components (mainly ellagic acid) modulated human thrombin amidolytic activity (cuccioloni et al. 2009 ) . pomegranate fruit ethanol extract was found to signi fi cantly increase the growth of osteoblastic mc3t3-e1 cells and caused a signi fi cant elevation of alkaline phosphatase (alp) activity and collagen content in the cells (kim and choi 2009 ) . treatment the extract decreased the tnfα-induced production of interleukin il-6 and nitric oxide in osteoblasts. promprom et al. ( 2010 ) found pomegranate seed extract to be a potent stimulator of phasic activity in rat uterus. pomegranate seed extract and b -sitosterol, the main constituent of the extract (16%), increased spontaneous contractions of the rat uterus in a concentration-dependent manner. their data suggested that the uterotonic effect was due to nonestrogenic effects of b -sitosterol acting to inhibit k channels and sarcoplasmic reticulum calcium atpase and thereby increasing contraction via calcium entry on l-type calcium channels and myosin light chain kinase. two b -secretase (bace1) inhibitors (anti-dementia agents) were isolated from pomegranate rind and identi fi ed as ellagic acid and punicalagin with ic 50 values of 3.9 × 10 −6 and 4.1 × 10 −7 m (kwak et al. 2005 ) . ellagic acid and punicalagin were less inhibitory to α-secretase (tace) and other serine proteases such as chymotrypsin, trypsin, and elastase, thus indicating that they were relatively speci fi c inhibitors of bace1. b -secretase is an aspartic-acid protease involved in the pathogenesis of alzheimer's disease studies showed that ethanolic extract of p. granatum seeds signi fi cantly exhibited the anxiolytic activity animal models of elevated plus maze test, barbiturate-induced sleeping time, tail suspension test, hot-plate and tail-fl ick tests (kumar et al. 2008 ) . the extract (250 and 500 mg/kg) signi fi cantly increased the sleeping latency and reduced the sleeping time. tail suspension test showed that the extract (250 and 500 mg/kg) was able to induce a signi fi cant decrease in the immobility time, similar to imipramine, a recognized antidepressant drug. tail-fl ick and hot-plate tests exhibited antinociceptive property of pomegranate extract, similar to morphine, a recognized antinociceptive agent. phytochemical screening and measurement of reducing power revealed the central nervous system (cns) activity of ethanol extract of pomegranate seeds may be due to its antioxidative pro fi le. supplementation of pomegranate fl owers led to improvements in learning and memory performances of streptozotocin-induced diabetic rats (cambay et al. 2011 ) . supplementation of pomegranate fl owers restored the elevated levels of lipid peroxidation and decreased level of glutathione towards their control values. daily pomegranate fl ower supplementation to diabetic rats reduced the increase in glial-fi brilar acidic protein (gfap) contents induced by diabetes in the hippocampus. the observations suggested that pomegranate fl ower supplementation decreased oxidative stress and amelioratedimpairment in learning and memory performances in diabetic rats and may be clinically useful in treating neuronal de fi cit in diabetic patients. loren et al. ( 2005 ) found that maternal dietary supplementation with pomegranate juice was neuroprotective in an animal model of neonatal hypoxic-ischemic brain injury. dietary supplementation with pomegranate juice resulted in markedly decreased brain tissue loss (>60%) in all three brain regions assessed, with the highest pomegranate juice dose having greatest signi fi cance pomegranate juice also diminished caspase-3 activation by 84% in the hippocampus and 64% in the cortex. in further studies, the scientists showed that pomegranate polyphenols and resveratrol reduced caspase-3 activation following neonatal hypoxic-ischemic injury (west et al. 2007 ) . in separate study, transgenic mice (app(sw)/tg2576) treated with pomegranate juice had signi fi cantly less (approximately 50%) accumulation of soluble aβ42 and amyloid deposition in the hippocampus as compared to control mice (hartman et al. 2006 ) . mice administered pomegranate juice learned water maze tasks more quickly and swam faster than controls. the results suggest that pomegranate juice may be useful in alzheimer's disease and warrant further studies. choi et al. ( 2011 b ) found that the ethanol pomegranate extract mitigated h 2 o 2induced oxidative stress in pc12 cells. additionally, the extract inhibited neuronal cell death caused by a b -induced oxidative stress and a b -induced learning and memory de fi ciency in mice. studies showed that pomegranate fruit extract exhibited embryo protective effect against adriamycin-induced oxidative stress in chick embryos (kishore et al. 2009 ) . pre-administration of pomegranate fruit extract signi fi cantly ameliorated to normal, embryo gross morphological deformities and signi fi cant changes in the levels of biochemical parameters in amniotic fl uid observed in the adriamycin-treated group. pomegranate juice consumption by healthy male rats provided an increase in epididymal sperm concentration, sperm motility, spermatogenic cell density and diameter of seminiferous tubules and germinal cell layer thickness and antioxidant activity, and it decreased abnormal sperm rate when compared to the control group (türk et al. 2008 ) . a signi fi cant decrease in malondialdehyde level and marked increases in glutathione, glutathione peroxidase and catalase activities, and vitamin c level were observed in rats treated with different doses of pomegranate juice. studies showed that ethanolic extract of pomegranate could be useful for the treatment of the deleterious effect of lead acetate administration on sperm production in rats (leiva et al. 2011 ) . the extract exhibited antioxidant activity similar to that of ascorbic acid and prevented lead acetate -induced spermatogenic disruption in rats. its antioxidant activity could explain its capacity to reverse the damage produced by lead acetate on spermatogenesis. in a randomized, placebo-controlled, doubleblind, crossover study involving male patients with mild to moderate erectile dysfunction, of the 42 subjects who demonstrated improvement in global assessment questionnaires (gaq) scores after beverage consumption, 25 reported improvement in erectile function after drinking pomegranate juice (forest et al. 2007 ) . subjects were more likely to have improved scores when pomegranate juice was consumed. although overall statistical signi fi cance was not achieved, this pilot study suggested the possibility that larger cohorts and longer treatment periods may achieve statistical signi fi cance. studies in rabbits with atherosclerosis-induced erectile dysfunction showed that pomegranate extract signi fi cantly improved intracavernosal blood fl ow, erectile activity, smooth muscle relaxation and fi brosis of the atherosclerotic group in comparison with the atherosclerotic group receiving placebo, but did not normalize them to the agematched control levels . pomegranate extract appeared more effective in diminishing oxidative products, preventing superoxide dismutase and aldose reductase gene upregulation, and protecting mitochondrial, endothelial and caveolae structural integrity of the atherosclerotic group. the study showed that dietary antioxidants could improve arteriogenic erectile dysfunction. pomegranate known to contain estrogens (estradiol, estrone, and estriol) exhibited estrogenic activities in mice (mori-okamoto et al. 2004 ) . administration of pomegranate extract (juice and seed extract) for 2 weeks to ovariectomized mice prevented the loss of uterus weight and shortened the immobility time compared with 5% glucose-dosed mice (control). further, ovariectomy-induced decrease of bone mineral density was normalized by administration of the pomegranate extract. the bone volume and the trabecular number were signi fi cantly increased and the trabecular separation was decreased in the pomegranate-dosed group compared with the control group. some histological bone formation/ resorption parameters were signi fi cantly increased by ovariectomy but were normalized by administration of the pomegranate extract. these changes suggested that the pomegranate extract inhibited ovariectomy-stimulated bone turnover. the authors concluded that pomegranate may be clinically effective on a depressive state and bone loss in menopausal syndrome in women. studies in human volunteers, found that in human liver microsomes, the mean 50% inhibitory concentrations (ic 50 ) for pomegranate juice (pj) and grapefruit juice (gfj) versus cyp3a (triazolam α-hydroxylation) were 0.61 and 0.55%, (v/v) respectively without preincubation of inhibitor with microsomes (farkas et al. 2007 ) . after preincubation, the ic 50 for pj increased to 0.97% whereas the ic 50 for gfj decreased to 0.41% suggesting mechanism-based inhibition by gfj but not pj. administration of pj also did not affect c(max), total area under the curve (auc), or clearance of oral midazolam. however, gfj increased midazolam c(max) and auc by a factor of 1.3 and 1.5, respectively, and reduced oral clearance to 72% of control values. the results suggested pj did not alter clearance of intravenous or oral midazolam, whereas gfj impaired clearance and elevated plasma levels of oral midazolam. jarvis et al. ( 2010 ) reported a potential interaction between pomegranate juice and warfarin as laboratory studies hade shown that pomegranate juice inhibited cytochrome p450 enzymes involved in warfarin metabolism. in a an open-label, randomized, single-center, two-period crossover study in healthy japanese volunteers, a single subtherapeutic doses of midazolam following 2 weeks consumption of pomegranate juice did not signi fi cantly alter the pharmacokinetic pro fi le of midazolam compared with that of the control (misaka et al. 2011 ) . results of a 5-week randomized, double-blind, placebo-controlled study involving 30 patients suggested that polyphenol-rich pomegranate juice (pj) supplementation added no bene fi t to the current standard therapy in patients with stable chronic obstructive pulmonary disease (cerdá et al. 2006 ) . the high teac (trolox equivalent antioxidant capacity) of pj could not be extrapolated in-vivo probably due to the metabolism of its polyphenols by colonic micro fl ora. the understanding of the different bioavailability of dietary polyphenols was thus critical before claiming any antioxidant-related health bene fi t. elbow fl exion strength was signi fi cantly higher during the 2-to 168-h period post-exercise with pomegranate juice compared with that of placebo (trombold et al. 2011 ) . elbow fl exor muscle soreness was also signi fi cantly reduced with pomegranate juice compared with that of placebo and at 48 and 72 h post-exercise. isometric strength and muscle soreness in the knee extensors were not signi fi cantly different with pomegranate juice compared with those using placebo. the results indicated a mild, acute ergogenic effect of pomegranate juice in the elbow fl exor muscles of resistance trained individuals after eccentric exercise. seven highly active ellagitannin inhibitors against carbonic anhydrase, punicalin (2), punicalagin (3), granatin b (5), gallagyldilactone (7), casuarinin (8), pedunculagin (9) and tellimagrandin i (10), and four weakly active ellagitannin inhibitors, gallic acid (1), granatin a (4), corilagin (6) and ellagic acid (11), were isolated from pomegranate pericarps (satomi et al. 1993 ) . the type of inhibition by compounds (3) and (7) using p -nitrophenyl acetate as a substrate, was noncompetitive. carbonic anhydrase inhibitors are used as antiglaucoma drugs, and many potent carbonic anhydrase inhibitors have also been shown to inhibit the growth of several tumour cell lines in-vitro and in-vivo providing interesting leads for developing novel antitumour therapies (supuran et al. 2004 ) using the hot plate method in mice, pomegranate fl ower extracts showed signi fi cant analgesic activity at a dose of 50 mg/kg body weight (chakraborthy 2008 ) . maximum analgesic activity was observed at 60 min after drug administration, which was equivalent to the standard drug used morphine sulphate. two milliliters of aqueous extract of pomegranate roots exhibited higher activity on cultures from entamoeba histolytica than from entamoeba invadens strains, producing growth inhibitions of about 100 and 40% respectively (segura et al. 1990 ) . alkaloid concentrations of 1 mg/ml had no amoebicide activity, however tannins at concentrations of 10 m g/ml for e. histolytica , and 100 m g/ml for e. invadens were suf fi cient to produce an growth inhibition about 100%. tannic acid was also tested on the cultures of e. histolytica producing a high inhibitory activity on growth, this effect was produced at 0.01 mg/ ml similar to that observed with the tannin mixture. the methanolic extract of pomegranate was reported to in-vitro inhibit the growth of the malarial parasite, plasmodium berghei (dell'agli et al. 2009 ) . in another study, gallagic acid and punicalagin from pomegranate by-product exhibited antiplasmodial activity against plasmodium falciparum d6 and w2 clones with ic 50 values of 10.9, 10.6, 7.5 and 8.8 m m, respectively (reddy et al. 2007 ) . pomegranate extract exhibited strong antimalarial activity against plasmodium falciparum (valdés et al. 2010 ) . p. grantum plant extract also exhibited in-vitro activity against the vaginal parasite, trichomonas vaginalis (el-sherbini et al. 2009 ) . hydroalcoholic pomegranate extract inhibited the growth of intracellular amastigotes of leishmania amazonensis with ic 50 value of 69.6 m g/ml (garcía et al. 2010 ) . additionally, a low toxicity on macrophage from balb/c mice was observed. wibaut and hollstein ( 1957 ) found that the anthelminthic activity of pomegranate bark extract was mainly due to isopelletierine, methylisopelletierine while y pelleterine was less active the molluscicidal activity of p. granatum bark and canna indica root against the snail, lymnaea acuminata was found to be both time and dose dependent (tripathi and singh 2000 ) . the toxicity of p. granatum bark was more pronounced than that of c. indica . the 24 h lc 50 of the c. indica was 6.54 mg/l whereas that of the bark of p. granatum was 4.39 mg/l. the ethanol extract of p. granatum (24 h lc 50 : 22.42 mg/l) was more effective than the ethanol extract of c. indica (24 h lc 50 : 55.65 mg/l) in killing the test animals. p. granatum and c. indica may be used as potent molluscicides since the concentrations used to kill the snails were not toxic to the fi sh colisa fasciatus , sharing the same habitat with the snail. in a subsequent study, tripathi et al. ( 2004 ) reported that sub-lethal 24 h exposure to active fraction of pomegranate bark separately or in combination with canna roots signi fi cantly inhibited the activity of acetylcholinesterase, acid/alkaline phosphatase, na(+)k(+)atpase and lactic dehydrogenase in the nervous tissue of lymnaea acuminata. lei et al. ( 2003 ) found that ellagic acid, the principal bioactive component of pomegranate leaf extract, had poor absorption and rapid elimination after oral administration pomegranate leaf extract, and part of it was absorbed from stomach. studies in rats showed that only 3-6% of the ingested punicalagin was detected as such or as metabolites in urine and faeces (cerdá et al. 2003b ) . only traces of punicalagin metabolites being detected in liver or kidney. the transformation of ellagic acid derivatives to 6h-dibenzo[b,d] pyran-6-one derivatives in the rat was con fi rmed. studies of cerdá et al. ( 2004 ) found that the potential systemic biological effects of pomegranate juice ingestion should be attributed to the colonic micro fl ora metabolites rather than to the polyphenols present in the juice. neither the potent antioxidant punicalagin nor ellagic acid present in pomegranate juice were detected in both plasma and urine on ingestion of pomegranate juice. three microbial ellagitannin-derived metabolites were detected: 3,8-dihydroxy-6h-dibenzo[b,d]pyran-6-one glucuronide, an unidenti fi ed aglycone (tentatively, trihydroxy-6h-dibenzo[b,d]pyran-6-one) and hydroxy-6-hdibenzo [b,d] pyran-6-one glucuronide in the plasma and urine. the metabolites did not show signi fi cant antioxidant activity compared to punicalagin from pomegranate juice. in separate studies, ellagic acid was detected in human plasma at a maximum concentration (31.9 ng/ml) after 1 h post-ingestion of pomegranate juice but was rapidly eliminated by 4 h (seeram et al. 2004 ) . six hours post-ingestion of pomegranate juice, ellagic acid (ea) was detected in plasma of all healthy human volunteers with a maximum concentration of 0.06 m mol/l, area under concentration time curve of 0.17 ( m mol × h) × l(-1), time of maximum concentration of 0.98 h, and elimination half-life of 0.71 h . ellagic acid metabolites, including dimethylellagic acid glucuronide (dmeag) and hydroxy-6h-benzopyran-6-one derivatives (urolithins), were also detected in plasma and urine in conjugated and free forms. dmeag was found in the urine obtained from 15 of 18 subjects on day 0, but was not detected on d −1 (day before) or +1 (day after), demonstrating its potential as a biomarker of intake. urolithin a-glucuronide was found in urine samples from 11 subjects on d 0 and in the urine from 16 subjects on d +1. urolithin b-glucuronide was found in the urine of three subjects on d 0 and in the urine of fi ve subjects on d+1. the scientists asserted that urolithins, formed by intestinal bacteria, may contribute to the biological effects of pomegranate juice as they may persist in plasma and tissues and account for some of the health bene fi ts noted after chronic juice consumption. studies by seeram et al. ( 2008 ) found that pomegranate juice, pomegranate polyphenol liquid extract and pomegranate polyphenol powder extract provide similar levels of plasma and urinary ellagitannin metabolites such as urolithin a, in human subjects. there was a delay in time of maximum concentration of pomegranate powder extract compared to pomegranate juice and pomegranate polyphenol liquid. mertens-talcott et al. ( 2006 ) found ellagic acid from pomegranate extract to be bioavailable at 1 h after consumption by healthy volunteers. its metabolites urolithin a, urolithin b, hydroxyl-urolithin a, urolithin a-glucuronide, and dimethyl ellagic acidglucuronide were found in the plasma. the antioxidant capacity measured with the oxygen radical absorbance capacity (orac) assay was increased with a maximum effect of 32% after 0.5 h, whereas the generation of reactive oxygen species (ros) was not affected. vidal et al. ( 2003 ) found that in chick embryo model doses of hydroalcoholic pomegranate fruit extract of less than 0.1 mg per embryo were not toxic. the ld 50 of the extract, determined in of-1 mice of both sexes after intraperitoneal administration, was 731 mg/kg. con fi dence limits were 565-945 mg/kg. at the doses of 0.4 and 1.2 mg/kg of extract, the repeated intranasal administration to wistar rats produced no toxic effects in terms of food intake, weight gain, behavioural or biochemical parameters, or results of histopathological studies. cerdá et al. ( 2003a ) found that repeated oral administration of high doses of the pomegranate ellagitannin punicalagin to rats for 37 days was not toxic. punicalagin and related metabolites were identi fi ed in plasma, liver, and kidney. five punicalagin-related metabolites were detected in liver and kidney, that is, two ellagic acid derivatives, gallagic acid, 3,8-dihydroxy-6h-dibenzo [b,d] pyran-6-one glucuronide, and 3,8,10-trihydroxy-6h-dibenzo [b,d] pyran-6-one. feedstuff intake, food utility index, and growth rate were lower in punicalagin treated rats during the fi rst 15 days without signi fi cant adverse effects, which could be due to the lower nutritional value of the punicalagin-enriched diet together with a decrease in its palatability (lower food intake). no signi fi cant differences were found in punicalagin treated rats in any blood parameter analyzed (including the antioxidant enzymes glutathione peroxidase and superoxide dismutase) with the exception of urea and triglycerides, which remained at low values throughout the study. clinical studies by heber et al. ( 2007 ) demonstrated the safety of a pomegranate ellagitannin-enriched polyphenol dietary supplement in overweight individuals with increased waist size and provided evidence of antioxidant activity in humans re fl ected by a signi fi cant reduction in thiobarbituric acid reactive substances (tbars) linked with cardiovascular disease risk. patel et al. ( 2008 ) found that the no observed-adverse-effect level (noael) for a standardized pomegranate fruit extract was determined as 600 mg/kg body weight/day, the highest dose tested in rats. compared to the control group, administration of the extract did not result in any toxicologically signi fi cant treatmentrelated changes in clinical observations, ophthalmic examinations, body weights, body weight gains, feed consumption, clinical pathology evaluations and organ weights. toxicological evaluation of pomegranate seed oil (pso) showed that the no observable adverse effect level (noael) was 50,000 ppm pso (=4.3 g pso/kg body weight/day) (meerts et al. 2009 ) . no mutagenicity of pso was observed in the absence and presence of metabolic activation up to precipitating concentrations of 5,000 m g/ plate (ames test) or 333 m g/ml (chromosome aberration test). the acute oral toxicity study revealed no signi fi cant fi ndings at 2,000 mg pso/ kg body weight. results from reversion and gene-conversion test in microorganisms, sister chromatid exchanges, micronuclei and sperm-shape abnormality assays in mice, clearly showed that the hydroalcoholic extract of pomegranate whole fruit was genotoxic when tested both in-vitro and in-vivo (sánchez-lamar et al. 2008 ) . the bark of the roots, the fl owers, the rind of pomegranate fruit and the seeds, are of fi cial in many pharmacopoeias. various parts of the pomegranate plant have been extensively used for thousands of years in traditional medicine in the middle east, ancient greece and asia (burkill 1966 ; grieve 1971 ; stuart 2012 ) ; and in india such as in the ayurveda and unani systems of medicine (nadkarni and nadkarni. 1982 ; sharma et al. 2002 ; kapoor 2000 ; pradeep et al. 2008 ) . the fruit rind and stem bark have been used as a traditional remedy for diarrhoea, dysentery and intestinal parasites. pomegranate pericarp has been commonly employed as a crude drug in indian traditional medicine for the treatment of diarrhoea as well as for use as an astringent, antihelminthic, asphrodisacs, laxative, diuretic, stomachic, cardiotonic and refrigerant. the seeds and juice are considered as bitter and astringent and employed as a tonic for hear and throat ailments. the astringent qualities of the fl ower sap, fruit rind and tree bark are considered useful remedies for nose bleeds and gum bleeds, toning skin, (after mixing with mustard oil) fi rming-up sagging breasts and treating haemorrhoids. a syrup prepared from the fruit is useful in all bilious complaints. the juice of the fresh fruit is much esteemed in dyspepsia and as a cooling, thirst-quenching beverage in fever and sickness. the fruit juice is also found bene fi cial in leprosy. pomegranate fruit juice has been used as eyedrops to treat cataracts. dried, pulverized fl ower buds are employed as a remedy for bronchitis. pomegranate has been reported as a remedy for diabetes in the unani system of medicine practiced in the middle east and india. the ancient greeks and egyptians used the fruit rind, fl owers and root bark as astringents and the last as vermicide for treating tapeworms. in malaysia, the root bark is used as vermifuge and powdered root bark is administered to children for stomach pains. the root is also used for diarrhoea and tits sap used for treating sore-eyes. leaves are used in jamu preparations with a raft of other herbal ingredients for many medicinal complaints. pounded leaves are used in a complex bolus for stomach ache and the fruit juice is recommended for coughs. in singapore, the root bark has been used in as a component in a compound infusion or decoction taken by women for 40 days after childbirth. other traditional uses of the fruit rind and root include as a treatment for snakebite (jain and puri 1984 ) , diabetes (singh 1986 ) , burns (siang 1983 ) , leprosy and assorted gynecological problems (singh et al. 1980 ) . in sri lanka, the fresh fruit has been used as a refrigerant to lower fever (arseculeratne et al. 1985 ) . in the philippines, a decoction of the tender leaves is used as a gargle for affections of the buccal cavity. the rind of the fruit is used internally in decoction as anthelmintic and taenifuge. in mexico, a decoction of the fl owers is gargled to relieve oral and throat in fl ammation. in korea, traditional uses of the fruit and rind include as an anthelminthic and for phlegm, cholethiasis, tineapedis and laryngitis. punica granatum is a drought tolerant tree suitable for arid and semi-arid zone afforestation. pomegranate has deep rooting system and is used for erosion control, planted along rivers to stabilize banks. an ideal suitable ornamental plant for gardens and amenity parks. pomegranate grows along well as intercrop with grapes in mediterranean countries. the tree is sometimes used for fencing and planted as boundary plants. pomegranate leaf litter decomposes slowly and is suitable for mulching. the leaves are foraged by domesticated stock. ink can be made by steeping the leaves in vinegar. both the fruit rind and the fl owers yield dyes for textiles. the light-coloured wood is hard and durable, mostly used in making farm implements, walking-sticks and in woodcrafts as it is only available in small dimension. tree branches are used as fi rewood. the bark is used in tanning and dyeing providing the yellow hue for moroccan leather. root bark yields a black ink rich in tannins. in japan, an insecticide is derived from the bark. studies revealed that pomegranate peel can prepared as an adsorbent in treating industrial ef fl uents containing phenols and safely disposed of by stabilizing into cement (bhatnagar and minocha 2009 ) . studies showed that that pomegranate peel waste can be used as adsorbent bene fi cially for nickel removal from aqueous solution (bhatnagar and minocha 2010 ) . pomegranate husk when converted into activated carbon exhibited its ability to remove hexavalent chromium from wastewater (nemr 2009 ) . studies showed that the nutritive value and the antioxidant capacity of pomegranate peel could be enhanced by ensiling into a favorable healthpromoting constituent of feedlot beef cattle diet (shabtay et al. 2008 ) . dietary supplementation with fresh peels promoted signi fi cant increases in feed intake and a-tocopherol concentration in the plasma, with positive tendency toward increased weight gain of bull calves. the pomegranate fruit is steeped in religious and cultural signi fi cance in judaism, christianity, islam, hinduism religions, persian, armenian, azerbaijani and chinese cultures (wikipedia 2012 ) . pomegrante germinates readily from seeds and are established from seedlings, rooted hardwood cuttings, from air layering and suckers. screening of nepalese crude drugs traditionally used to treat hyperpigmentation: in vitro tyrosinase inhibition pomegranate fruit extract modulates uv-bmediated phosphorylation of mitogen-activated protein kinases and activation of nuclear factor kappa b in normal human epidermal keratinocytes paragraph sign anthocyanin-and hydrolyzable tannin-rich pomegranate fruit extract modulates mapk and nf-kappab pathways and inhibits skin tumorigenesis in cd-1 mice protective effect of pomegranate-derived products on uvb-mediated damage in human reconstituted skin oral feeding of pomegranate fruit extract inhibits early biomarkers of uvb radiation-induced carcinogenesis in skh-1 hairless mouse epidermis short communication: studies on punica granatum -i isolation and identi fi cation of some constituents from the seeds of punica granatum punica granatum l. extract inhibits il-1beta-induced expression of matrix metalloproteinases by inhibiting the activation of map kinases and nf-kappab in human chondrocytes in vitro the inhibition of gastric mucosal injury by punica granatum l. (pomegranate) methanolic extract protective effects of punica granatum in experimentally-induced gastric ulcers pomegranate extracts potently suppress proliferation, xenograft growth, and invasion of human prostate cancer cells anthocyanins characterization of 15 iranian pomegranate ( punica granatum l.) varieties and their variation after cold storage and pasteurization antimicrobial activity of pomegranate ( punica granatum l.) fruit peels studies on medicinal plants of sri lanka. part 14 pomegranate as a cosmeceutical source: pomegranate fractions promote proliferation and procollagen synthesis and inhibit matrix metalloproteinase-1 production in human skin cells pomegranate juice consumption inhibits serum angiotensin converting enzyme activity and reduces systolic blood pressure pomegranate juice consumption reduces oxidative stress, atherogenic modi fi cations to ldl, and platelet aggregation: studies in humans and in atherosclerotic apolipoprotein e-de fi cient mice pomegranate juice fl avonoids inhibit low-density lipoprotein oxidation and cardiovascular diseases: studies in atherosclerotic mice and in humans pomegranate juice consumption for 3 years by patients with carotid artery stenosis reduces common carotid intima-media thickness, blood pressure and ldl oxidation pomegranate phenolics from the peels, arils, and fl owers are antiatherogenic: studies in vivo in atherosclerotic apolipoprotein e-de fi cient (e 0) mice and in vitro in cultured macrophages and lipoproteins an aqueous pomegranate peel extract inhibits neutrophil myeloperoxidase in vitro and attenuates lung in fl ammation in mice flora of java (spermatophytes only), 1. noordhoff melatonin, serotonin, and tryptamine in some egyptian food and medicinal plants antidiabetic effect of punica granatum fl owers: effect on hyperlipidemia, pancreatic cells lipid peroxidation and antioxidant enzymes in experimental diabetes new sterol esters from the fl owers of punica granatum linn consumption of pomegranate decreases serum oxidative stress and reduces disease activity in patients with active rheumatoid arthritis: a pilot study 2-methyl-pyran-4-one-3-o -b -d-glucopyranoside isolated from leaves of punica granatum inhibits the tnf a -induced cell adhesion molecules expression by blocking nuclear transcription factor-k b (nf-k b) the antiplaque ef fi cacy of pomegranate mouthrinse adsorptive removal of 2,4-dichlorophenol from water utilizing punica granatum peel waste and stabilization with cement biosorption optimization of nickel removal from water using punica granatum peel waste urolithins, intestinal microbial metabolites of pomegranate ellagitannins, exhibit potent antioxidant activity in a cell-based assay the effect of pomegranate ( punica granatum l.) byproducts and ellagitannins on the growth of human gut bacteria pomegranate-mediated chemoprevention of experimental hepatocarcinogenesis involves nrf2-regulated antioxidant mechanisms climate effects on anthocyanin accumulation and composition in the pomegranate ( punica granatum l.) fruit arils synergic interaction between pomegranate extract and antibiotics against staphylococcus aureus pomegranate extract inhibits staphylococcus aureus growth and subsequent enterotoxin production a dictionary of the economic products of the malay peninsula, revised reprint, 2 vols. ministry of agriculture and co-operatives characterization of a potential nutraceutical ingredient: pomegranate ( punica granatum l.) seed oil unsaponi fi able fraction volatile composition and sensory quality of spanish pomegranates ( punica granatum l.) pomegranate ( punica granatum l.) fl ower improves learning and memory performances impaired by diabetes mellitus in rats hepatoprotective role and antioxidant capacity of pomegranate ( punica granatum ) fl owers infusion against trichloroacetic acidexposed in rats repeated oral administration of high doses of the pomegranate ellagitannin punicalagin to rats for 37 days is not toxic evaluation of the bioavailability and metabolism in the rat of punicalagin, an antioxidant polyphenol from pomegranate juice the potent in vitro antioxidant ellagitannins from pomegranate juice are metabolised into bioavailable but poor antioxidant hydroxy-6h-dibenzopyran-6-one derivatives by the colonic micro fl ora of healthy humans pomegranate juice supplementation in chronic obstructive pulmonary disease: a 5-week randomized, doubleblind, placebo-controlled trial analgesic activity of various extracts of punica granatum (linn) fl owers antibacterial activity of thai medicinal plant extracts on the skin infectious microorganisms flavonoid diglycoside from punica granatum studies on antioxidant activity of pomegranate ( punica granatum ) peel extract using in vivo models study on wound healing activity of punica granatum peel the partition chromatography of alkaloids: part iii.-the alkaloids of punica granatum in vitro and in vivo antibacterial activity of punica granatum peel ethanol extract against salmonella punica granatum protects against oxidative stress in pc12 cells and oxidative stress-induced alzheimer's symptoms in mice the wealth of india: a dictionary of indian raw materials and industrial products pomegranate extract inhibits the proliferation and viability of mmtv-wnt-1 mouse mammary cancer stem cells in vitro studies on antidiarrhoeal activity of punica granatum seed extract in rats studies on the hypoglycaemic activity of punica granatum seed in streptozotocin induced diabetic rats effects of consumption of pomegranate juice on carotid intima-media thickness in men and women at moderate risk for coronary heart disease bene fi cial effects of pomegranate juice on oxidation-sensitive genes and endothelial nitric oxide synthase activity at sites of perturbed shear stress pomegranate juice reduces oxidized low-density lipoprotein downregulation of endothelial nitric oxide synthase in human coronary endothelial cells the in fl uence of pomegranate fruit extract in comparison to regular pomegranate juice and seed oil on nitric oxide and arterial function in obese zucker rats effects of a pomegranate fruit extract rich in punicalagin on oxidation-sensitive genes and enos activity at sites of perturbed shear stress and atherogenesis quantitative analysis of fl avan-3-ols in spanish foodstuffs and beverages antiplasmodial activity of punica granatum l. fruit rind the antioxidant potency of punica granatum l. fruit peel reduces cell proliferation and induces apoptosis on breast cancer pomegranate extract mouth rinsing effects on saliva measures relevant to gingivitis risk antimicrobial activity of six pomegranate ( punica granatum l.) varieties and their relation to some of their pomological and phytonutrient characteristics elemental and nutritional analysis of punica granatum from turkey analysis of organic acids in fruit juices by liquid chromatography-mass spectrometry: an enhanced tool for authenticity testing pomegranate ( punica granatum ) juices: chemical composition, micronutrient cations, and antioxidant capacity physico-chemical properties and dpph-abts scavenging activity of some local pomegranate ( punica granatum ) ecotypes antioxidant capacities of phenolic compounds and tocopherols from tunisian pomegranate ( punica granatum ) fruits fatty acids from tunisian and chinese pomegranate ( punica granatum l.) seeds chemical composition of juice and seeds of pomegranate fruit studies on pomegranate seed oil ef fi cacy of two plant extracts against vaginal trichomoniasis two ellagitannins from punica granatum heartwood two new ellagic acid rhamnosides from punica granatum heartwood ellagiand gallotannins from punica granatum heartwood concentrated pomegranate juice improves lipid pro fi les in diabetic patients with hyperlipidemia cholesterol-lowering effect of concentrated pomegranate juice consumption in type ii diabetic patients with hyperlipidemia pomegranate juice effects on cytochrome p450s expression: in vivo studies effect of pomegranate ( punica granatum ) juice intake on hepatic oxidative stress pomegranate juice does not impair clearance of oral or intravenous midazolam, a probe for cytochrome p450-3a activity: comparison with grapefruit juice monoacylglycerol from punica granatum seed oil effect of probiotication on antioxidant and antibacterial activities of pomegranate juices from sour and sweet cultivars determination of lignans in edible and nonedible parts of pomegranate ( punica granatum l.) and products derived there from, particularly focusing on the quantitation of isolariciresinol using hplc-dad-esi/ms(n) ef fi cacy and safety of pomegranate juice on improvement of erectile dysfunction in male patients with mild to moderate erectile dysfunction: a randomized, placebo-controlled, double-blind, crossover study foundation for revitalisation of local health traditions pomegranate and cardiovascular diseases: pomegranate juice polyphenolic antioxidants protect against oxidative stress and atherosclerosis development pomegranate juice inhibits oxidized ldl uptake and cholesterol biosynthesis in macrophages pomegranate juice polyphenols increase recombinant paraoxonase-1 binding to high-density lipoprotein: studies in vitro and in diabetic patients screening of medicinal plants against leishmania amazonensis synergistic growth inhibition of mouse skin tumors by pomegranate fruit extract and diallyl sul fi de: evidence for inhibition of activated mapks/nf-k b and reduced cell proliferation effects of aqueous extracts from quercus ilex l. root bark, punica granatum l. fruit peel and artemisia herba-alba asso leaves on ethanolinduced gastric damage in rats arte's f, toma's-barbera'n f (1995) changes in pomegranate juice pigmentation during ripening antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing effect of pomegranate juice on insulin secretion and sensitivity in patients with obesity dissimilar in vitro and in vivo effects of ellagic acid and its microbiota-derived metabolites, urolithins, on the cytochrome p450 1a1 anti-microbial activities of pomegranate rind extracts: enhancement by cupric sulphate against clinical isolates of s. aureus , mrsa and pvl positive ca-mssa total phenolic distribution of juice, peel, and seed extracts of four pomegranate cultivars immunomodulatory activity of punica granatum in rabbits -a preliminary study punicic acid is an omega-5 fatty acid capable of inhibiting breast cancer proliferation antioxidant activities of peel, pulp and seed fractions of common fruits as determined by frap assay evaluation of antioxidant activity and preventing dna damage effect of pomegranate extracts by chemiluminescence method pomegranate juice is potentially better than apple juice in improving antioxidant function in elderly subjects pomegranin, an antifungal peptide from pomegranate peels pomegranate ( punica granatum ) puri fi ed polyphenol extract inhibits in fl uenza virus and has a synergistic effect with oseltamivir in vitro antibacterial activity of some iranian medicinal plant extracts against helicobacter pylori pomegranate juice decreases amyloid load and improves behavior in a mouse model of alzheimer's disease cardioprotective effect of whole fruit extract of pomegranate on doxorubicin-induced toxicity in rat hydroalcoholic extract based-ointment from punica granatum l. peels with enhanced in vivo healing potential on dermal wounds safety and antioxidant activity of a pomegranate ellagitanninenriched polyphenol dietary supplement in overweight individuals with increased waist size identi fi cation of estrone in pomegranate seeds evolution of juice anthocyanins during ripening of new selected pomegranate ( punica granatum ) clones pomegranate polyphenols down-regulate expression of androgensynthesizing genes in human prostate cancer cells overexpressing the androgen receptor activation of ppar gamma and alpha by punicic acid ameliorates glucose tolerance and suppresses obesity-related in fl ammation chemopreventive effects of pomegranate seed oil on skin tumor development in cd1 mice anti-diabetic action of punica granatum fl ower extract: activation of ppargamma and identi fi cation of an active component pomegranate fl ower improves cardiac lipid metabolism in a diabetic rat model: role of lowering circulating lipids pomegranate fl ower extract diminishes cardiac fi brosis in zucker diabetic fatty rats: modulation of cardiac endothelin-1 and nuclear factor-kappab pathways tannins from the leaves of punica granatum pomegranate juice protects nitric oxide against oxidative destruction and enhances the biological actions of nitric oxide effects of pomegranate juice on hyperoxaluria-induced oxidative stress in the rat kidneys possible interaction between pomegranate juice and warfarin anticancer activities of pomegranate extracts and genistein in human breast cancer cells evaluation of antioxidant, antitumor and immunomodulatory properties of polysaccharide isolated from fruit rind of punica granatum suppressive effect of punica granatum on the production of tumor necrosis factor (tnf) in bv2 microglial cells therapeutic applications of pomegranate ( punica granatum l.): a review antioxidant effect of pomegranate extract in reducing acute in fl ammation due to myringotomy pomegranate juice supplementation to atherosclerotic mice reduces macrophage lipid peroxidation, cellular cholesterol accumulation and development of atherosclerosis crc handbook of ayurvedic medicinal plants effects of oral administration of ellagic acidrich pomegranate extract on ultraviolet-induced pigmentation in the human skin effects of pomegranate chemical constituents/intestinal microbial metabolites on cyp1b1 in 22rv1 prostate cancer cells punica granatum : heuristic treatment for diabetes mellitus punica granatum (pomegranate) fl ower extract possesses potent antioxidant activity and abrogates fe-nta induced hepatotoxicity in mice differentiation-promoting activity of pomegranate ( punica granatum ) fruit extracts in hl-60 human promyelocytic leukemia cells oral consumption of pomegranate fruit extract inhibits growth and progression of primary lung tumors in mice pomegranate fruit extract inhibits prosurvival pathways in human a549 lung carcinoma cells and tumor growth in athymic nude mice pomegranate fruit extract impairs invasion and motility in human breast cancer pomegranate fruit extract inhibits uvb-induced in fl ammation and proliferation by modulating nf-k b and mapk signaling pathways in mouse skin stimulation of osteoblastic differentiation and inhibition of interleukin-6 and nitric oxide in mc3t3-e1 cells by pomegranate ethanol extract chemopreventive and adjuvant therapeutic potential of pomegranate ( punica granatum ) for human breast cancer inhibitory effect of pomegranate on intestinal sodium dependent glucose uptake embryo protective effect of pomegranate ( punica granatum l.) fruit extract in adriamycin-induced oxidative stress screening of traditional chinese medicinal plants for quorum-sensing inhibitors activity pomegranate seed oil rich in conjugated linolenic acid suppresses chemically induced colon carcinogenesis in rats genetic diversity-independent neutralization of pandemic viruses (e.g. hiv), potentially pandemic (e.g. h5n1 strain of in fl uenza) and carcinogenic (e.g. hbv and hcv) viruses and possible agents of bioterrorism (variola) by enveloped virus neutralizing compounds (evncs) pomegranate extract induces apoptosis in human prostate cancer cells by modulation of the igf-igfbp axis effect of pomegranate peel extracts on experimental prostatitis rats in vitro studies on the binding, antioxidant, and cytotoxic actions of punicalagin central nervous system activity of acute administration of ethanol extract of punica granatum l. seeds in mice beta-secretase (bace1) inhibitors from pomegranate ( punica granatum ) husk effects of pomegranate tannins on experimental gastric damages transport behavior of ellagic acid of pomegranate leaf tannins and its correlation with total cholesterol alteration in hepg2 cells punica granatum (pomegranate) and its potential for prevention and treatment of in fl ammation and cancer pomegranate ( punica granatum ) pure chemicals show possible synergistic inhibition of human pc-3 prostate cancer cell invasion across matrigel possible synergistic prostate cancer suppression by anatomically discrete pomegranate fractions immune-suppressive activity of punicalagin via inhibition of nfat activation pharmacokinetic study of ellagic acid in rat after oral administration of pomegranate leaf extract evidence of anti-obesity effects of the pomegranate leaf extract in high-fat diet induced obese mice effect of punica granatum (pomegranate) on sperm production in male rats treated with lead acetate antiviral activities of medicinal herbs traditionally used in southern mainland china punica granatum fl ower extract, a potent alpha-glucosidase inhibitor, improves postprandial hyperglycemia in zucker diabetic fatty rats evaluation of antioxidant properties of pomegranate peel extract in comparison with pomegranate pulp extract pomegranate fl ower: a unique traditional antidiabetic medicine with dual ppar-alpha/-gamma activator properties fabrication of nanoparticles using partially puri fi ed pomegranate ellagitannins and gelatin and their apoptotic effects maternal dietary supplementation with pomegranate juice is neuroprotective in an animal model of neonatal hypoxic-ischemic brain injury anti-listeria monocytogenes activity of heat-treated lyophilized pomegranate juice in media and in ground top round beef pomegranate ( punica granatum ) supplements: authenticity, antioxidant and polyphenol composition prostate cancer prevention through pomegranate fruit pomegranate fruit juice for chemoprevention and chemotherapy of prostate cancer effects of pomegranate juice and extract polyphenols on platelet function antimicrobial activities of pomegranate rind extracts: enhancement by addition of metal salts and vitamin c pomegranate seed oil consumption during a period of high-fat feeding reduces weight gain and reduces type 2 diabetes risk in cd-1 mice toxicological evaluation of pomegranate seed oil breast cancer chemopreventive properties of pomegranate ( punica granatum ) fruit extracts in a mouse mammary organ culture volatile composition of pomegranates from 9 spanish cultivars using headspace solid phase microextraction punica granatum (pomegranate) extract is active against dental plaque absorption, metabolism, and antioxidant effects of pomegranate ( punica granatum l.) polyphenols after ingestion of a standardized extract in healthy human volunteers effect of 2 weeks' consumption of pomegranate juice on the pharmacokinetics of a single dose of midazolam: an open-label, randomized, single-center, 2-period crossover study in healthy japanese volunteers cardioprotective potential of punica granatum extract in isoproterenol-induced myocardial infarction in wistar rats effect of pomegranate juice on angiotensin ii-induced hypertension in diabetic wistar rats pomegranate extract improves a depressive state and bone properties in menopausal syndrome model ovariectomized mice fruits of warm climates. julia f identi fi cation and quanti fi cation of phenolic compounds and their effects on antioxidant activity in pomegranate juices of eight iranian cultivars indian materia medica with ayurvedic pomegranate extract induces cell cycle arrest and alters cellular phenotype of human pancreatic cancer cells medicinal plants in the republic of korea nmr spectral analysis of polyphenols from punica granatum antibacterial activity directed isolation of compounds from punica granatum antioxidant and antimutagenic activities of pomegranate peel extracts potential of pomegranate husk carbon for cr(vi) removal from wastewater: kinetic and isotherm studies the existence of pelletierine derivatives in punica granatum alkaloids in the bark of punica granatum l. (pomegranate) from yugoslavia punica granatum (pomegranate) juice provides an hiv-1 entry inhibitor and candidate topical microbicide antioxidant activities of pomegranate fruit extract and its anthocyanidins: delphinidin, cyanidin, and pelargonidin evaluation of antioxidant activity, colour and some nutritional characteristics of pomegranate ( punica granatum l.) juice and its sour concentrate processed by conventional evaporation agroforestree database: a tree reference and selection guide version 4 protective effects of standardized pomegranate ( punica granatum l.) polyphenolic extract in ultraviolet-irradiated human skin fi broblasts antifungal ef fi cacy of punica granatum , acacia nilotica , cuminum cyminum and foeniculum vulgare on candida albicans : an in vitro study antioxidant capacity and lipid characterization of six georgia-grown pomegranate cultivars phase ii study of pomegranate juice for men with rising prostate-speci fi c antigen following surgery or radiation for prostate cancer antimicrobial ellagitannin from pomegranate ( punica granatum ) fruits extract of punica granatum inhibits skin photoaging induced by uvb irradiation medicinal values of fruit peels from citrus sinensis , punica granatum , and musa paradisiaca with respect to alterations in tissue lipid peroxidation and serum concentration of glucose, insulin, and thyroid hormones safety assessment of pomegranate fruit extract: acute and subchronic toxicity studies the wound healing activity of fl ower extracts of punica granatum and achillea kellalensis in wistar rats antioxidant properties of gallocatechin and prodelphinidins from pomegranate peel searchable world wide web multilingual multiscript plant name database antibacterial activity of punica granatum l. against gastro intestinal tract infection causing organisms the effects of pomegranate seed extract and {beta}-sitosterol on rat uterine contractions antioxidant potentials of punica granatum fruit rind extracts pomegranate extract inhibits the interleukin-1 b -induced activation of mkk-3, p38 a -mapk and transcription factor runx-2 in human osteoarthritis chondrocytes antioxidant, antimalarial and antimicrobial activities of tannin-rich fractions, ellagitannins and phenolic acids from punica granatum l studies on the chemical constituents from xinjiang punica granatum pomegranate extract inhibits androgen-independent prostate cancer growth through a nuclear factor-kappab-dependent mechanism antioxidant activity of punica granatum fruits the occurrence of 2-(2-propenyl)-d 1-piperideine in the leaves of pomegranate consumption of wonderful variety pomegranate juice and extract by diabetic patients increases paraoxonase 1 association with high-density lipoprotein and stimulates its catalytic activities pomegranate juice protects macrophages from triglyceride accumulation: inhibitory effect on dgat1 activity and on triglyceride biosynthesis anti-oxidative effects of pomegranate juice (pj) consumption by diabetic patients on serum and on macrophages pomegranate byproduct administration to apolipoprotein e-de fi cient mice attenuates atherosclerosis development as a result of decreased macrophage oxidative stress and reduced cellular uptake of oxidized lowdensity lipoprotein free radical scavenging, antiglycation and tyrosinase inhibition properties of a polysaccharide fraction isolated from the rind from punica granatum pomegranate juice sugar fraction reduces macrophage oxidative state, whereas white grape juice sugar fraction increases it antiplaque and antigingivitis effects of a gel containing punica granatum linn extract: a double-blind clinical study in humans assessment of the genotoxic risk of punica granatum l. (punicaceae) whole fruit extracts ellagitannin-rich pomegranate extract inhibits angiogenesis in prostate cancer in vitro and in vivo pomegranate juice inhibits sulfoconjugation in caco-2 human colon carcinoma cells carbonic anhydrase inhibitors from the pericarps of punica granatum l investigation of a betainic alkaloid from punica granatum antioxidant and eicosanoid enzyme inhibition properties of pomegranate seed oil and fermented juice fl avonoids bioavailability of ellagic acid in human plasma after consumption of ellagitannins from pomegranate ( punica granatum l.) juice in vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenols as found in pomegranate juice rapid large scale puri fi cation of ellagitannins from pomegranate husk, a by-product of the commercial juice industry pomegranate juice ellagitannin metabolites are present in human plasma and some persist in urine for up to 48 hours pomegranate ellagitannin-derived metabolites inhibit prostate cancer growth and localize to the mouse prostate gland pomegranate juice and extracts provide similar levels of plasma and urinary ellagitannin metabolites in human subjects growth inhibition of entamoeba histolytica and e . invadens produced by pomegranate root ( punica granatum l.) lc-dad-esi/ms(n) determination of direct condensation fl avanol-anthocyanin adducts in pressure extracted pomegranate ( punica granatum l.) juice pomegranate wine has greater protection capacity than red wine on low-density lipoprotein oxidation nutritive and antioxidative potential of fresh and stored pomegranate industrial byproduct as a novel beef cattle feed central council for research in ayurveda & siddha (2002) database on medicinal plants used in ayurveda antibacterial activity of medicinal plants against pathogens causing complicated urinary tract infections effects of fruit ellagitannin extracts, ellagic acid, and their colonic metabolite, urolithin a, on wnt signaling macrophage paraoxonase 2 (pon2) expression is up-regulated by pomegranate juice phenolic anti-oxidants via ppar gamma and ap-1 pathway activation bioavailable constituents/metabolites of pomegranate ( punica granatum l.) preferentially inhibit cox2 activity ex vivo and il-1beta-induced pge2 production in human chondrocytes in vitro consumption of hydrolyzable tannins-rich pomegranate extract suppresses in fl ammation and joint damage in rheumatoid arthritis use of combined traditional chinese and western medicine in the management of burns traditional medicine in fiji: some herbal folk cures used by fiji indians medicinal plants from ujjain district madhya pradesh. part ii exploring the ameliorative potential of punica granatum in dextran sulfate sodium induced ulcerative colitis in mice pharmacological investigations of punica granatum in glycerol-induced acute renal failure in rats philippine alternative medicine. manual of some philippine medicinal plants in vitro effects of pomegranate juice and pomegranate polyphenols on foodborne viral surrogates flavonoids from punica granatum : potential antiperoxidative agents punica granaum l effects of pomegranate juice consumption on myocardial perfusion in patients with coronary heart disease in fl uenza virus variation in susceptibility to inactivation by pomegranate polyphenols is determined by envelope glycoproteins designing of novel carbonic anhydrase inhibitors and activators photochemopreventive effect of pomegranate fruit extract on uva-mediated activation of cellular pathways in normal human epidermal keratinocytes punicafolin, an ellagitannin from the leaves of punica granatum xl: revision of the structures of punicalin and punicalagin, and isolation and characterization of 2-o -galloylpunicalin from the bark of punica granatum l tannins and related compounds. xli: isolation and characterization of novel ellagitannins, punicacorteins a, b, c, and d, and punigluconin from the bark of punica granatum l tannins and related compounds. c. reaction of dehydrohexahydroxydiphenic acid esters with bases, and its application to the structure determination of pomegranate tannins, granatins a and b humarain: a new dimeric gallic acid glycoside from punica granatum l. bark nutritive value of pomegranate fruit and juice preliminary studies on the anti-angiogenic potential of pomegranate fractions in vitro and in vivo pomegranate peel extract prevents liver fi brosis in biliary-obstructed rats punica granatum peel extract protects against ionizing radiationinduced enteritis and leukocyte apoptosis in rats pomegranate ( punica granatum ) seed linolenic acid isomers: concentrationdependent modulation of estrogen receptor activity molluscicidal activity of punica granatum bark and canna indica root enzyme inhibition by the molluscicidal agent punica granatum linn. bark and canna indica linn. root the effect of pomegranate juice supplementation on strength and soreness after eccentric exercise protective effect of a potent antioxidant, pomegranate juice, in the kidney of rats with nephrolithiasis induced by ethylene glycol effects of pomegranate juice consumption on sperm quality, spermatogenic cell density, antioxidant activity and testosterone level in male rats antioxidant activity, polyphenol content, and related compounds in different fruit juices and homogenates prepared from 29 different pomegranate accessions usda) (2012) usda national nutrient database for standard reference, release 25. nutrient data laboratory home page in vitro antimalarial activity and cytotoxicity of some selected cuban medicinal plants rapid dereplication of estrogenic compounds in pomegranate ( punica granatum ) using on-line biochemical detection coupled to mass spectrometry use of punica granatum as an antifungal agent against candidosis associated with denture stomatitis minimum inhibitory concentration of adherence of punica granatum linn (pomegranate) gel against s. mutans , s. mitis and c. albicans studies on the toxicity of punica granatum l. (punicaceae) whole fruit extracts activity of medicinal plant extracts against hospital isolates of methicillin-resistant staphylococcus aureus medicinal plant extracts as anti-escherichia coli o157:h7 agents and their effects on bacterial cell aggregation pomegranate seed oil, a rich source of punicic acid, prevents diet-induced obesity and insulin resistance in mice punica granatum attenuates angiotensin-ii induced hypertension in wistar rats bioactive compounds from the seeds of punica granatum (pomegranate) constituents of the fl owers of punica granatum pomegranate: constituents, bioactivities and pharmacokinetics cellular and molecular mechanisms of pomegranate juice-induced anti-metastatic effect on prostate cancer cells pomegranate extract, a prooxidant with antiproliferative and proapoptotic activities preferentially towards carcinoma cells pomegranate polyphenols and resveratrol protect the neonatal brain against hypoxic-ischemic injury investigation of the alkaloids of punica granatum investigation of the alkaloids of punica granatum l. by partition chromatography antimutagenicity of some fl owers grown in thailand pomegranates ( punica granatum ), kiwifruit ( actinidia deliciosa ) and blood pressure: a pilot study medicinal fl owers. xxiii. new taraxastane-type triterpene, punicanolic acid, with tumor necrosis factor-alpha inhibitory activity from the fl owers of punica granatum effect of pomegranate peel extracts on oxidative stress in restrained mice pomegranate fl ower ameliorates fatty liver in an animal model of type 2 diabetes and obesity dietary effect of pomegranate seed oil on immune function and lipid metabolism in mice chitinase iii in pomegranate seeds ( punica granatum linn.): a high-capacity calcium-binding protein in amyloplasts inhibitory effect of an ellagic acid-rich pomegranate extract on tyrosinase activity and ultravioletinduced pigmentation a triglyceride from punica granatum broad spectrum antimutagenic activity of antioxidant active fraction of punica granatum l. peel extracts screening of certain medicinal plants from india for their anti-quorum sensing activity inhibition of uvb-mediated oxidative stress and markers of photoaging in immortalized hacat keratinocytes by pomegranate polyphenol extract pomx studies on physicochemical properties and bioactive compounds of six pomegranate cultivars antiviral activity of tannin from the pericarp of punica granatum l. against genital herpes virus in vitro antiliperoxidant activity of pomegranate peel extracts on lard dietary antioxidants improve arteriogenic erectile dysfunction key: cord-354044-3ugc7odq authors: salazar-gómez, anuar; ontiveros-rodríguez, julio c.; pablo-pérez, saudy s.; vargas-díaz, m. elena; garduño-siciliano, leticia title: the potential role of sesquiterpene lactones isolated from medicinal plants in the treatment of the metabolic syndrome – a review date: 2020-09-16 journal: s doi: 10.1016/j.sajb.2020.08.020 sha: doc_id: 354044 cord_uid: 3ugc7odq metabolic syndrome comprises a cluster of metabolic disorders related to the development of cardiovascular disease and type 2 diabetes mellitus. in latter years, plant secondary metabolites have become of special interest because of their potential role in preventing and managing metabolic syndrome. sesquiterpene lactones constitute a large and diverse group of biologically active compounds widely distributed in several medicinal plants used for the treatment of metabolic disorders. the structural diversity and the broad spectrum of biological activities of these compounds drew significant interests in the pharmacological applications. this review describes selected sesquiterpene lactones that have been experimentally validated for their biological activities related to risk factors of metabolic syndrome, together with their mechanisms of action. the potential beneficial effects of sesquiterpene lactones discussed in this review demonstrate that these substances represent remarkable compounds with a diversity of molecular structure and high biological activity, providing new insights into the possible role in metabolic syndrome management. metabolic syndrome (mets) refers to a cluster of risk factors related to the development of cardiovascular disease (cvd) and type 2 diabetes mellitus (t2dm) (alberti et al., 2009 ). this pathological condition represents a public health concern in most nations due to its association with mortality caused by the cardiovascular and metabolic complications (ju et al., 2017) . indeed, mets is recognized as a risk factor that influences progression and prognosis of coronavirus disease 2019, the infectious disease caused by the most recently discovered coronavirus sars-cov-2 (costa et al., 2020) . lifestyle modifications remain the primary component of metss risk factors reduction and pharmacological therapies are indicated in special situations to improving more than one factor (larsen et al., 2018) . in latter years, secondary plant metabolites have become of special interest in the scientific community because of their potential role in preventing and managing mets (cicero and colletti, 2016; francini-pesenti et al., 2019) . many compounds, such as sesquiterpene lactones (slns), have been isolated from medicinal plants and their hypoglycemic, anti-inflammatory, and antioxidative properties coupled with their capacity to modulate key cellular functions have been confirmed by in vitro and in vivo methods (chadwick et al., 2013; chaturvedi et al., 2016; alonso et al., 2018) . despite these effects are linked to the pathogenesis of mets, the role of these compounds to avoid its progression is not well documented compared to many other compounds such as polyphenols into which a great deal of research has been conducted. therefore, in order to provide relevant information regarding the potential benefits of slns in preventing abbreviations: ace, angiotensin i-converting enzyme; ampk, activated protein kinase; apoc3, apolipoprotein c3; at, adipose tissue; cat, catalase; cox-2, cyclooxygenase 2; cvd, cardiovascular disease; ffa, free fatty acids; fn, fibronectin; g6pase, glucose-6-phosphatase; gk, glucokinase; gpx, glutathione peroxidase; gsh, reduced glutathione; hdl-c, high-density lipoproteins-cholesterol; ifn-g, interferon gamma; il-1b, interleukin 1 beta; il-6, interleukin 6; inos, inducible nitric oxide synthase; ir, insulin resistance; jnk, c-jun n-terminal kinases; ldl-c, low-density lipoprotein-cholesterol; lps, lipopolysaccharide; mapk, mitogen-activated protein kinases; mcp-1, monocyte chemoattractant protein 1; mets, metabolic syndrome; nf-kb, nuclear factor kappa b; no, nitric oxide; ros, reactive oxygen species; slns, sesquiterpene lactones; sod, superoxide dismutase; stat1, signal transducer and activator of transcription 1; stz, streptozotocin; t2dm, type 2 diabetes mellitus; tbars, thiobarbituric acid reactive substances; tc, total cholesterol; tg, triglycerides; tgf-b1, transforming growth factor beta; tlrs, toll-like receptor; tnf-a, tumor necrosis factor alpha; vldl, very-low-density lipoprotein and managing mets, this review addresses plant-derived slns that are potentially responsible for the positive effect in improving risk factors associated with mets. an electronic literature search was conducted on slns with hypoglycemic and/or hypolipidemic effects and isolated from plants used in traditional medicine for the same purposes. the search was done in databases of google scholar, science direct, and scifinder until june 2020 using the keyword sesquiterpene lactones and following terms: metabolic syndrome, hypoglycemic, hypolipidemic, antidiabetic, antihypertensive, and medicinal plants. additional information was identified from references located in the retrieved articles. although many slns were screened in some of the studies, only those considered active by the authors are included in this review. the plants included in this review were selected based on their ethnomedicinal records. for each species, the currently accepted name in the online "the plant list" database has been checked and the reported name has been replaced with the current one. compound class, plant source, biological activity and important aspects related with mets are summarized in the table 1 . mets is defined as a cluster of risk factors such as raised blood pressure, atherogenic dyslipidemia, raised fasting glucose, and central obesity, related to the development of cvd and t2dm (alberti et al., 2009) . since the world health organization (who) reported the first formalized definition of the mets, the diagnostic criteria have been sequentially developed by several public health organizations (engin, 2017) . nowadays, the most recognized criterion for the clinical diagnosis of mets (alberti et al., 2009 ) is based on identifying at least three of the following risk factors: (1) elevated waist circumference (population and country-specific definitions determined by local organizations); (2) triglycerides (tg) 150 mg/ dl (<1.0 mmol/l) or on drug treatment for elevated triglycerides; (3) high-density lipoproteins-cholesterol (hdl-c) <40 mg/dl (<1.0 mmol/l) in men or <50 mg/dl (<1.3 mmol/l) in women or on drug treatment for reduced hdl-c; (4) blood pressure 130/ 85 mmhg or on antihypertensive medication treatment, and/or a history of hypertension; and (5) fasting glucose >100 mg/dl (>5.6 mmol/l) or on drug treatment for elevated glucose. the mechanisms underlying pathogenesis of mets have not been completely explored, but obesity-induced adipocyte dysfunction and inflammation is associated with the progression of insulin resistance and metabolic disorders (kl€ oting and bl€ uher, 2014) . body fat mass accumulation in obesity depends on different factors including the relationship of energy intake to energy expenditure and body energy storage (gomez-hernandez et al., 2016) . adipose tissue (at) plays an important role as an energy storage organ, as well as an endocrine organ produces adipokines such as leptin, adiponectin, monocyte chemoattractant protein 1 (mcp1), tumor necrosis factor alpha (tnfa) and interleukin 6 (il-6), which circulate and regulate systemic metabolism and inflammation. the cell type composition of at includes adipocytes, fibroblasts, macrophages, stromal cells, monocytes and preadipocytes (r afols, 2014) . on further at expansion, hypertrophy of adipocytes and increased secretion of macrophage chemoattractants occurs, including the secretion of mcp-1, which recruits additional macrophages. these actions in turn result in local inflammatory state, enhanced basal lipolysis, increasing the leakage of free fatty acids (ffa) and a dysregulated secretion of several proinflammatory adipokines (longo et al., 2019; xu et al., 2019) . subsequently, these adverse signals reach metabolic tissues (e.g. liver, pancreatic islets, and skeletal muscle) and modify inflammatory responses as well as glucose and lipid metabolism, thereby contributing to a global metabolic effect of insulin resistance. chronic low-grade inflammation in obesity results from the activation of various inflammatory mechanisms through nuclear factor kappa b (nf-kb) and c-jun n-terminal kinases (jnk) pathways. these pathways represent important modulators of cytokine gene expression downstream of toll-like receptors (tlrs) in insulin target cells (catrysse and van loo, 2017; rogero and calder, 2018) . nf-kb is an important transcription factor involved in different processes of the immune and inflammatory responses. it is composed of p50 and p65 subunits and is found in the cytoplasm in complex with inhibitory proteins of the ikb family. cytokines and lipopolysaccharide (lps) can stimulate cell surface receptors including toll-like receptor (tlr4) to initiate a signaling cascade that converge on the activation of the inhibitor of kb kinase (ikk) complex (baker et al., 2011) . the ikk complex phosphorylates ikba and induces its degradation, leading to the release and nuclear translocation of nf-kb to promote transcription of target genes such as tnf-a, interleukin-1beta (il-1b), il-6, il-8, cyclooxygenase 2 (cox-2) and inducible nitric oxide synthase (inos) (knab et al., 2014; ruan et al., 2011) . on the other hand, jnk are members of the mitogen-activated protein kinases (mapk) that mediate cellular responses to a variety of intra-and extracellular stresses (zeke et al., 2016) . in the context of obesity, jnk pathways can be activated by proinflammatory cytokines, ffa and reactive oxygen species (ros), and regulate several nuclear and extra-nuclear substrates, specially the transcription factor activator protein 1 (ap1) which controls the expression of proinflammatory genes and protein synthesis (e.g. tnf-a, il-1b, il-6 and il-8) (pal et al., 2016; feng et al., 2020) . deregulated activation of nf-kb and jnk pathways results in increased transcription of il-6 and tnf-a, which reduce the sensitivity of insulin target cells towards insulin. thus, chronically activated nf-kb and jnk pathways leads to the promotion of insulin resistance (yung and giacca, 2020) . additionally, increased flux of ffa to the liver in the insulin resistant state stimulates production tg and secretion in the form of verylow-density lipoprotein (vldl). the resulting hypertriglyceridemia leads to lower hdlàc levels and normal or slightly elevated lowdensity lipoprotein-cholesterol (ldl-c) levels (iqbal et al., 2018) . this is related to the typical dyslipidemic profile in mets. consistent with a potential role in the pathogenesis of mets, slns interfering with these processes described above could be useful to prevent the onset of insulin resistance and the risk of t2dm and cvd in obese individuals. slns represent a diverse group of terpenoids with more than 5000 different elucidated structures. they are particularly diversified in the compositae (asteraceae) family, but also occurring in apiaceae, illiciaceae, magnoliaceae, solanaceae, and euphorbiaceae families (padilla-gonzalez et al., 2016) . numerous species of these families are used in traditional medicine and slns have been described as their primary active constituents. these compounds possess several biological activities such as anti-inflammatory, antidiabetic, antimalarial, anti-proliferative, anti-parasitic and antimicrobial (merfort, 2011; chadwick et al., 2013; chaturvedi et al., 2011). slns are characterized by the presence of a g-lactone ring. the lactone ring can be fused to the remaining skeleton in either a cis or trans configuration, being most common the trans configuration (fischer et al., 1979; fischer, 1990; . based on their carbocyclic skeleton, slns are mainly classified in four major groups: germacranolides (10-membered ring), eudesmanolides (6à6 bicyclic compounds), guaianolides and pseudoguaianolides (5à7 blood glucose levels (#), hba1c (#), plasma insulin ("), tissue glycogen (") tc, tg and ldl-c (#); hdl-c (") tbars levels (#), gsh ("), sod, cat and gpx (") in brain, liver, heart, kidney, and pancreas eliza et al., 2009a eliza et al., , 2009b eliza et al., , 2010 5 costunolide germacrolide costus speciosus 6 alantolactone eudesmanolide inula helenium antiinflammatory-associated to glucose intolerance and ir (in vitro) antiinflamatory-obesity-induced ir (in vitro) attenuates lipid accumulation (in vitro) antiinflamatory-associated to diabetic nephropathy (in vivo) stat3 inhibitor. inhibition of the tlr4 gene expression. inhibition of tlr4-jnk signaling. il-6 and mcp-1 (#). apoc3 expression at both mrna and protein levels (#) tnf-a and il-6 (#) in diabetic kidney. serum creatinine and urea nitrogen levels (#). kim et al., 2017a kim et al., , 2017b yang et al., 2018; zhu et al., 2020 7 tirotundin 3,10epoxygermacranolide antidiabetic (in vitro) dual ppara/g agonists lin, 2012 8 tagitinin a 9 tagitinin g anti-hyperglycemic (in vitro) glucose uptake in 3t3-l1 adipocytes ("). blood glucose levels (#), serum insulin and pancreatic insulin levels (") g6pase activity (#) and gk activity (") tc, tg, ldl-c and vldl (#); hdl-c (") tbars levels (#), sod, cat and gpx (") in liver, kidneys, and pancreas tnf-a levels (#). normalization of blood pressure hong et al., 1999 hong et al., , 2005 reduction (#); increment ("); thiobarbituric acid reactive substances (tbars); reduced glutathione (gsh); superoxide dismutase (sod); catalase (cat); glutathione peroxidase (gpx); insulin resistance (ir); toll-like receptor 4 (tlr4); c-jun n-terminal kinases (jnk); interleukin 6 (il-6); monocyte chemoattractant protein 1 (mcp-1); apolipoprotein c3 (apoc3); amp-activated protein kinase (ampk); peroxisome proliferator-activated receptors a and g (ppara/g); nuclear factor kappa b (nf-kb); transforming growth factor beta (tgf-b1); fibronectin (fn); glucose-6-phosphatase (g6pase); glucokinase (gk); cyclooxygenase 2 (cox-2); interferon-gamma (ifn-g); tumor necrosis factor a (tnf-a); angiotensin i-converting enzyme (ace). a. salazar-g omez et al. / south african journal of botany 135 (2020) 1à12 bicyclic compounds) and many subtypes of slns according to their skeletal arrangement (adekenov, 2017; padilla-gonzalez et al., 2016) . in many cases, the g-lactone ring contains an exocyclic double bond conjugated to the carbonyl group (a-methylene-g-lactone) but in some cases the exocyclic methylene is reduced or the double bond can be endocyclic (martínez et al., 2012; padilla-gonzalez et al., 2016; . it has been well documented that biological activity of the most common types of slns is mainly attributed to formation of covalent union between the a,b-unsaturated group in the exo-methylene-g-lactone and nucleophilic biological targets (e.g. free cysteine sulfhydryl) resulting in alkylation through michael-type addition (schmidt, 2006) . this chemical reaction induces steric and chemical changes in enzymes, receptors or transcriptional factors leading to a series of cellular events that culminate in diverse biologic responses. the number of alkylating structural elements present on the molecule define differences between the activity of each sln. another structural influences on the biological effects are the molecular size, hydrophobicity, chemical environment and the presence of other functional groups (e.g., epoxy groups, hydroxyls or hydroxyls esterified with acetate, propionate, isobutyrate, angelate, epoxyangelate, and benzoate) (chaturvedi, 2011; ivanescu et al., 2015; padilla-gonzalez et al., 2016; . the structural diversity and the broad spectrum of biological activities drew significant interests in the pharmacological applications of slns (moujir et al., 2020) . many slns have proved to have a significant anti-inflammatory, hypoglycemic and hypolipidemic activity therefore making them attractive for mets therapy (chaturvedi, 2011; chaturvedi et al., 2016) . 6.1. enhydrin from smallanthus sonchifolius (poepp.) h.rob smallanthus sonchifolius (poepp.) h.rob., commonly known as yacon, is a perennial herbaceous plant native to the andean regions of south america (caetano et al., 2016) . yacon is consumed as a safety dietary supplement and because of its low glucose content and high fructooligosaccharide levels putative antidiabetic effects were suggested (delgado et al., 2013) . indeed, this suggestion is supported by the hypoglycemic (aybar et al., 2001; baroni et al., 2008) and hypolipidemic (miura et al., 2004; habib et al., 2011) reported activities. also, a potential use in metabolic disorders could be proposed based on the anti-inflammatory and antioxidant properties (feltenstein et al., 2004; sousa et al., 2015) . besides the above mentioned evidences, enhydrin (1), a melampolide (cis,trans-germacranolide) and the major sesquiterpene lactone component in leaves of s. sonchifolius, has proven to be effective reducing post-prandial glucose levels and a useful compound for blood glucose control by the induction of a late increase in plasma insulin in streptozotocin (stz)induced diabetic rats (genta et al., 2010) . more recently, in vivo and in vitro experiments showed that enhydrin (1) is effective in the management of post-prandial hyperglycemia through inhibition of a-glucosidase in the small intestine (serra-barcellona et al., 2017) . the inhibition of this enzyme has been linked to the presence of the a,b-unsaturatedg-lactone ring system (yin et al., 2014) , similar to the intact exo-methylene-g-lactone group in 1 (fig. 1) . thus, the antia-glucosidase activity of enhydrin could be derived from this interaction. in addition, the alkylation of nucleophilic sites of factors involved in early stages of the inflammatory cascade described above allows to relate the anti-inflammatory properties reported for 1 with the presence of the exo-methylene-g-lactone ring system (feltenstein et al., 2004; ma et al., 2007) . regarding toxicological studies, it has been reported that a single oral administration of enhydrin (1) at doses of 1.6, 4 and 8 mg/kg body weight (b.w.) did not caused deaths or acute toxic effects within 7 days in male and female rats (genta et al., 2010) . in acute study in rats, there were no deaths or signs of toxicity observed after single oral administration of 1 at any dose level up to the highest dose tested (0.32 g/kg b.w.) within 14 days. in subchronic studies, after oral administration for 90 days at daily doses of 0.4, 0.8 and 8.0 mg/ kg b.w., did not caused hematological, biochemical and histological alterations in rats (serra et al., 2012) . smallanthus macroscyphus (heliantheae, asteraceae), is a perennial herb commonly known as ''wild yacon'' native from the south american region comprising from southern bolivia to northwestern argentina. this species is closely related to s. sonchifolius and possibly with similar antidiabetic properties. polymatin a (2) is the main sesquiterpene lactone isolated from s. macroscyphus (de pedro et al., 2003) . this melampolide exerts an effective inhibition of post-prandial blood glucose peak and hypoglycemic activity in stz-diabetic rats probably by the stimulation of insulin release or due to an insulin-like effect (serra-barcellona et al., 2014) . due to the close relationship in the polymatin a (2) and enhydrin (1) structures, the melampolide 2 could be effective in the management of postprandial hyperglycemia through inhibition of a-glucosidase. however, no studies of these effects were found. in contrast to the c4àc5 epoxy group in enhydrin (1), polymatin a (2) presents a double bond (fig. 1 ). c4àc5 epoxy groups in melampolides such as enhydrin have been reported to hinder the ability of these compounds to inhibit nf-kb dna binding responsible to cytokines expression (schorr et al., 2007) commonly observed in inflammatory response. conversely, the effect is favored by the presence of a double bond between c-4àc-5 in other melampolides (schorr et al., 2007) . hence, hopeful results could be expected in future studies to explore anti-inflammatory activity in polymatin a. acute oral toxicity of polymatin a (2) in normal healthy rats at doses assayed (0.7, 1.4 and 2.8 g dried powder/kg b.w.) do not produced deaths or acute toxic effect (changes in behavior or posture, presence of convulsions or occurrence of secretions) within 14 days. the doses were well tolerated and did not produce adverse nutritional effect. no gastrointestinal symptom such as diarrhea or constipation were observed at the doses assayed. volume, ph and urine specific gravity were within normal ranges. no nitrites, protein or blood were detected in the urine samples of the animal groups treated (serra-barcellona et al., 2014) . in subchronic studies, polymatin a (2) was orally administered to wistar rats for 90 days at daily doses of 7, 14 and 28 mg/kg b.w. no toxicity signs or deaths were observed. there were no changes in the behavior, body or organ weights, hematological, biochemical or urine parameters of the rats. no histopathological lesions were observed in the examined organs. the results indicate that polymatin a (2) from s. macroscyphus leaves may be considered as nontoxic substance at a wide range of doses (serra-barcellona., 2016) . the common sunflower, helianthus annuus linn (asteraceae), is a well-known plant with edible seeds, flower petals and tender leaf petioles. this plant was proposed to offer a variety of medical benefits (lim, 2014; guo et al., 2017) . to provide a scientific explanation for its use in nigerian traditional medicine, onoja and anaga (2014) reported a hypoglycemic effect on the fasting blood glucose level in alloxan-induced diabetic rats after a single dose of the methanolic extract of h. annuus leaves. this study led to the recent isolation of the heliangolide 20-dehydroeucannabinolide (3) (fig. 1) . (onoja et al., 2020) . heliangolide slns represent an isomeric form of germacranolides with a cyclodecadiene skeleton and double bonds at c1=c10 and c4=c5, which stereochemical configurations are trans, cis respectively (s€ ulsen and martino, 2018). the heliangolide 3 reduced the fasting blood glucose level at dose of 0.2 mmol/kg in alloxaninduced diabetic rats (onoja et al., 2020) . the authors suggested that this sesquiterpene lactone might have reducing effects in glucose absorption based on the heliangolide derivative structure, which presents an intact exo-methylene-g-lactone group. as mentioned above, a,b-unsaturated g-lactone ring system has an important relation in the inhibition of a-glucosidase activity, thus the heliangolide 3 may be inhibiting this enzyme similar to enhydrin (1). in addition to blood glucose regulation, it has also been demonstrated the positive effects of hydromethanolic leaf extract of h. annuus l. on other components of mets, including reduced ldl-c and tg levels as well as hepatoprotective and antilipid peroxidation activities of alloxan-induced diabetic rats (onoja et al., 2018) . since 20-dehydroeucannabinolide (3) is one of the major components of the extract, potential activity of this molecule in managing dyslipidemia may be proposed. however, additional studies are needed to ascertain the hypolipidemic property of this compound. no in vivo toxicity studies on 20-dehydroeucannabinolide (3) have been reported to date. costus speciosus (j. koenig) sm is a plant used in traditional medicine in south asia to treat diabetic patients by eating one leaf of this species per day to regulate blood glucose levels (waisundara et al., 2015) . also, the hypoglycemic effect of c. speciosus root crude extracts have been reported (daisy et al., 2008) . eremanthin (4) (fig. 1) is a guaianolide sesquiterpene lactone present in c. speciosus and other plants such as pterodon pubescens (benth.) benth., eremanthus elaeagnus (mart. ex dc.) sch.bip. and laurus nobilis l. (eliza et al., 2009a; waisundara et al., 2015) . another sesquiterpene lactone reported to be present in c. speciosus is the germacrolide (a trans, trans-germacranolide) costunolide (5) (fig. 1) (eliza et al., 2009b) , which has been also isolated from magnolia spp, laurus nobilis and saussurea costus (falc.) lipsch (koo et al., 2001) . studies in stz-induced diabetic rats showed that oral administration of 4 and 5 decreased plasma glucose level, glycosylated hemoglobin (hba1c), total cholesterol (tc), tg, ldl-c and at the same time markedly increased plasma insulin, tissue glycogen and hdl-c (eliza et al., 2009a (eliza et al., , 2009b . the specific mechanism of eremanthin (4) bioactivity is not completely characterized, but the experimental model used in both works allowed the authors to suggest that this molecule might exert an insulin-like effect on peripheral tissues by either promoting glucose uptake metabolism, by inhibiting hepatic gluconeogenesis or by absorption of glucose into the muscle and adipose tissues through the stimulation of a regeneration process and revitalization of the remaining b cells (eliza et al., 2009a (eliza et al., , 2009b ). in addition, costunolide (5) has shown to suppress lps-induced nf-kb activation that leads to the suppression of inos and a consequent nonproduction of no. this activity was stronger than the observed for parthenolide, a germacranolide sesquiterpene lactone (koo et al., 2001) . thus, the anti-inflammatory activity exhibited by 5 serve as a promising and expanding strategy for treatment of metabolic disease-associated inflammation. on the other hand, the possible inhibition of inflammatory processes has not been studied in eremanthin (4). however, this compound has a rigid skeleton and an intact exo-methylene-g-lactone group, previously related to anti-inflammatory properties (simonsen et al., 2013) . additionally, the absence of free hydroxyl groups in 4 could help to improve anti-inflammatory properties since an increasing number of free hydroxyl groups are reported to diminish nf-kb inhibition activity (simonsen et al., 2013) . taking together these structural characteristics, we suggest that eremathin (4) is a candidate to be tested for inhibition of inflammatory processes related to metabolic diseases. acute oral toxicity of eremanthin (4) and costunolide (5) in normal healthy male wistar rats at doses assayed (10, 20, 40, 80 and 160 mg/kg b.w.) do not produced behavioral changes on the rats and mortality was not observed during acute toxicity test (10 days) (eliza et al., 2010) . finally, the antioxidant activity of eremanthin (4) and costunolide (5) has been demonstrated by significantly decreasing of the thiobarbituric acid reactive substances (tbars) and lipid peroxidation markers levels as well as by increasing reduced glutathione (gsh) levels and enzymatic antioxidants (eliza et al., 2010) . this activity could indicate a protective effect of both on oxidative stress in diabetes. inula (asteraceae) is one of the most popular herbs in traditional chinese medicine. this genus comprises more than one hundred species widely distributed throughout asia, africa, and europe, and many of these have been used to treat a wide range of diseases such as bronchitis, diabetes, obesity, hypertension, and inflammation (seca et al., 2014) . extracts of inula spp. such as inula britannica l., inula viscosa [the accepted name is dittrichia viscosa (l.) greuter.], inula racemosa hook.f., inula helenium l., etc., have been documented for their potential effects on some components of mets, especially hypoglycemic, hypolipidemic and antioxidant activities (kobayashi et al., 2002; zeggwagh et al., 2006; shan et al., 2006; ajani et al., 2009 ) as well as inhibition of a-glucosidase enzyme (orhan et al., 2017) . regarding bioactive secondary metabolites, slns represent the largest group of compounds occurring in inula spp. many of these compounds were isolated from plants mentioned above and demonstrated to exert diverse biological activities (seca et al., 2014; wang et al., 2014) . the anti-inflammatory mechanisms of some slns have been related to their influence on directly inhibit the mapk and block the activation of nf-kb, thus achieving the prevention of pro-inflammatory signaling (han et al., 2001; park et al., 2013) . alantolactone (6) (fig. 1) is the most known eudesmanolide present in several inula species, including inula helenium l. used for hypoglycemic and antiobesity purposes (seca et al., 2014; wang et al., 2014) . this compound has been extensively studied for its antitumor, antioxidant and anti-inflammatory effects (moujir et al., 2020) . regarding anti-inflammatory activity, the eudesmanolide 6 suppresses no, pge 2 and tnf-a production in lps-stimulated raw 264.7 cells by modulating the activity of the nf-kb and mapk signaling pathways (chun et al., 2012) , and it also suppresses tnf-a and interferon gamma (ifn-g)-induced production of chemokines by blocking the signal transducer and activator of transcription 1 (stat1) phosphorylation in hacat cells (lim et al., 2015) . as previously mentioned, it is recognized the fact that tlrs are responsible for the activation of inflammatory pathways in obesity state. in vitro trials confirmed that alantolactone (6) prevents the increase of il-6 levels and regulates macrophage infiltration by reduction of mcp-1 concentrations via inhibition of the tlr4-jnk pathway in both, lean (adipocytes) and obese (adipocyte-macrophage) states (kim et al., 2017a (kim et al., , 2017b . besides the effect on adipocytes, alantolactone (6) can also act in skeletal muscle and liver cells. in this context, the compound 6 inhibits il-6-induced insulin-stimulated glucose intolerance and insulin resistance in the skeletal muscle through blockade the activation of stat3 and the abnormal expression of tlr4 (kim et al., 2017a (kim et al., , 2017b . in l02 cells, alantolactone (6) also inhibits oxldl-induced lipid accumulation trough regulating the apolipoprotein c3 (apoc3) expression partly by decreasing the activation of stat3 . thus, may be relevant to explore further its role in managing metabolic complications on e.g. modulating hepatic il-6/stat3 signaling in high-fat diet-induced metabolic disorder in animal models, since the physiological metabolic response to il-6 depends on the specific source of il-6 in vivo (han et al., 2020) concerning to in vivo experiments, no hypoglycemic effect was observed on stz-induced diabetic mice after oral administration of alantolactone. however, inflammation and renal abnormalities were suppressed via inhibition of nf-kb gene expression and the high glucose-induced overexpression of pro-inflammatory cytokines and macrophage adhesion in renal nrk-52e cells were inhibited. (zhu et al., 2020) . these results led the authors to propose a beneficial use for the treatment of diabetic neuropathy. taking together all evidences mentioned for alantolactone (6) activity, it is probably that this compound could reduce some components of mets by the regulation of inflammatory processes. alantolactone (6) did not induced significant hepatotoxicity and nephrotoxicity after daily administration (100 mg/kg b.w.) for 5 weeks in mice . this compound also is a contact allergen, and in vitro is cytotoxic in cancer cells and induces apoptosis (tisserand and young, 2014) . 6.6. 3,10-epoxygermacranolides from tithonia diversifolia (hemsl.) a. gray tithonia diversifolia (hemsl) a. gray, commonly known as tree marigold or mexican sunflower (asteraceae: heliantheae), is a shrublike perennial or annual invasive plant, native from north and central america. traditionally, its leaves are widely used by indigenous people for treating a wide spectrum of diseases, including diabetes mellitus. several in vitro and in vivo studies have stated the antioxidant antidiabetic, hypolipidemic and antiobesity effects of t. diversifolia (ajao and moteetee, 2017; tagne et al., 2018) . germacranolides, eudesmanolides and guaianolides are some of the major components occurring in this plant (ajao and moteetee, 2017; tagne et al., 2018) and because of their previously described activity, they apparently are involved in the important spectrum of bioactivity reported for t. diversifolia. tirotundin (7) and tagitinin a (8) (fig. 1) isolated from the ethyl acetate soluble fraction of t. diversifolia are related to the activation of peroxisome proliferator-activated receptors (ppars) (lin, 2012) . ppars are members of nuclear hormone receptors superfamily involved in the metabolic regulation of lipid and lipoprotein levels, blood glucose, and abdominal adiposity. in mammals, three isoforms of ppars have been recognized namely ppar-a, ppar-b/d and ppar-g. activation of ppar-a by hypolipidemic fibrate class of drugs decreases tg levels and raises hdl-c in dyslipidemic individuals, whereas activation of ppar-g by antidiabetic thiazolidinedione agents causes insulin sensitization to enhance glucose metabolism (botta et al., 2018) . currently, dual ppara/g agonists, which stimulate both ppara and ppar-g isoforms to similar extents, are gaining popularity since it is believed that they are able to ameliorate the unwanted side effects of selective ppar-a and ppar-g agonists; and may also be used to treat dyslipidemia and t2dm simultaneously (balakumar et al., 2019) . tirotundin (7) and tagitinin a (8) have been suggested being dual ppara/g agonists after the evaluation of their agonistic activity against ppars employing a transient transfection reporter assay in hepg2 cells . on the other hand, the germacranolides tagitinin g (9), tagitinin i (10), 1b-hydroxydiversifolin-3-o-methyl ether (11) and 1b-hydroxytirotundin-3-o-methyl ether (12) (fig. 1) isolated from the aerial parts of t. diversifolia were found to significantly elevate glucose uptake in 3t3-l1 adipocytes without any toxicity (zhao et al., 2012) . the authors suggested that these compounds had pparg agonist activity on glucose uptake, similar to pioglitazone, a ppar-g agonist used as a control compound in the study. no in vivo acute, subchronic or chronic toxicity studies on 3,10-epoxygermacranolides described above have been reported to date. the increasing abundance of tithonia diversifolia in conservation and agricultural areas over the past ten years in south africa has been of concern. this plant is considered as a scrub resulting in the initiation of a biological control program against its propagation (simelane et al., 2011) . therefore, utilization for medicinal purposes of this invasive plant could be important before major eradication takes place. the genus magnolia, previously known as michellia, reported to exert various biological effects, including anti-cancer, anti-anxiety, antidepressant, antioxidant and anti-inflammatory (lee et al., 2011) . in later years, magnolia species and their components have been related to ameliorate characters of obesity and diabetes, such as hyperglycemia, hyperlipidemia, and other complications . since terpenoids are one of the principal substantial compounds in magnolia species, slns may be related with its medicinal properties (sun et al., 2015) . anticancer activity investigation lead to the isolation of the guaianolide micheliolide (13) (fig. 1) from m. compressa (maxim.) sarg. (ogura et al., 1978) . this compound has also been obtained in a semi-synthetic way from a bf 3 -mediated rearrangement of parthenolide (castañeda-acosta et al., 1993; zhai et al., 2012) , presenting enhanced stability and remarkable antiinflammatory effect by suppressing activation of the nf-kb through inhibition of ikba degradation as well as for the inhibition of p65 translocation to the nucleus (viennois et al., 2014) . the compound also influenced the mapk and pi3k/akt signaling pathways in lpsstimulated bv-2 cells (sun et al., 2017) . further, an in vitro assay has revealed that 13 attenuate the high glucose-stimulated activation of nf-kb, the degradation of ikba, and the expression of mcp-1 in rat mesangial cells (jia et al., 2013) . in addition, the dimethylamino michael adduct of 13 known as dimethylaminomicheliolide is a pro-drug of this sesquiterpene lactone approved for clinical trials for glioblastoma treatment . this compound protects the kidneys against proteinuria, renal failure, histopathological injury, and inflammation by suppressing activation of the nf-kb signaling pathway in db/db mice . these results showed that dimethylaminomicheliolide intervention mitigated diabetic kidney disease in db/db mice without directly affecting hyperglycemia. furthermore, it has been demonstrated that micheliolide (13) alleviates hepatic steatosis in obese db/db mice, and the molecular mechanisms driving the therapeutic effects of this compound might involve ppar-g upregulation to consequently inhibit nf-kb mediated inflammation and activate ampk/mtor-dependent autophagy (zhong et al., 2018) . thus, it will be interesting to future work evaluate if this effect in hepatic steatosis impacts glucose metabolism. no in vivo toxicity studies on micheliolide (13) have been reported to date. byrsonima crassifolia (malpighiaceae), commonly known as "nanche", is a tropical tree widely distributed in mexico, central and south america. this tree is commonly harvested, both for its edible fruit and for its health benefits (b ejar and malone, 1993; duarte, 2011) . in folk medicine, the fruit or bark infusion have been used as hypoglycemic remedy (andrade-cetto and heinrich, 2005) and according to ethnomedicinal reports, the antihyperglycemic activity of hexane and chloroform extracts from fruits and seeds of b. crassifolia in stzinduced diabetic rats was reported (perez-gutierrez et al., 2010) . the phytochemical analysis of antihyperglycemic extract from the seed of b. crassifolia revealed that byrsonines a (14) and b (15) (fig. 2) , two dimeric guaianolides, are responsible for the antioxidant, hypoglycemic and hypolipidemic activities (guti errez and ramirez, 2016) . in particular, it has been proposed that the antihyperglycemic activity of these compounds may involve both pancreatic and extra pancreatic mechanisms, based on the observed increase in serum insulin level and the reduction of hepatic glucose output via decreasing glucose-6phosphatase (g6pase) activity and increasing glucokinase activity (guti errez and ramirez, 2016) . although the mechanisms underlying the antihyperglycemic activity of 14 and 15 have not been completely explored, this effect could likely be ascribed to the activation ampactivated protein kinase (ampk). activation of the ampk pathway in the liver regulates glucose homeostasis by inhibiting hepatic glucose production and downregulating the expression of gluconeogenic genes, including g6pase gene expression. targeting ampk by natural products demonstrated considerable success in lowering blood glucose levels (joshi et al., 2019) . nevertheless, more experimental research is needed to substantiate this claim. on the other hand, the induction of antioxidant enzymes in hepatic, renal, and pancreas tissues after the administration of 14 and 15, indicates a positive effect of these molecules in improving glycemic control in stz-induced diabetic rats (guti errez and ramirez, 2016), since oxidative stress takes an important role in the pathogenesis and progression of diabetic tissue injury. in this sense, the reported improvement in insulin resistance by byrsonines a and b can be related to the observed increase in the hdl-c levels and the decrease in tc, tg, ldl-c, and vldl (guti errez and ramirez, 2016). additionally, tnf-a levels were also decreased after treatment of byrsonines a and b in stz-induced diabetic rats. the bioactivity described for 14 and 15 makes them one of the most active guaianolides on different components of mets reported at this time. as described above, another molecule of this family with activity on different components of mets is eremathin. however, byrsonines a and b (14, 15) seems to be more effective, which could be due to their dimeric structure that provides two possible michaels acceptors. dimeric slns have been found to be more potently cytotoxic than their monomers toward human cancer cells, indicating that the antitumor potential of slns are improved by the presence of additional a-methylene-g-lactone ring (singh et al., 2011; ren et al., 2016) . this phenomenon could be studied in 14 and 15 for mets treatment due to their extended activity reviewed here. moreover, pharmacokinetics studies are needed to understand either byrsonines suffer biotransformation reactions and act as monomeric molecules or if they can show biological activity in their original dimeric forms. no in vivo toxicity studies on byrsonines a (14) and b (15) have been reported to date. various species of the genus lactuca are globally important edible plants containing valuable nutrients such as polyphenols, sterols, vitamins, minerals, and dietary fiber. l. sativa l., lactuca indica l. and l. serriola l. (syn. l. scariola l.) have been used in the treatment of diabetes mellitus, hypertension, and cardiac diseases (eddouks et al., 2002; subramoniam, 2016) . in vivo experiments of some lactuca species shown the ability to reduce several metabolic risk factors, especially hyperglycemia, tg and tc (nicolle et al., 2004; salih, 2019) . slns are commonly found in lactuca species and are represented as subgroups such as lactucin-type guaianolides and the eudesmanolide-type. the latter is commonly found in some species of lactuca such as l. sativa l. var. anagustata, l. saligna l. and l. canadensis l. (han et al., 2010; kisiel and gromek, 1993; michalska et al., 2013) . the dimeric guaianolide lactucain c (16) (fig. 2) , isolated from l. indica linn. showed moderate lowering of plasma glucose in stz-diabetic rats (hou et al., 2003) . nevertheless, there are no evidences about the possible mechanism of action and no further research has been made about this compound. as mentioned before, dimeric slns could show an important activity in components of mets derived from their double a,b-unsaturated g-lactone ring. because of its hypoglycemic properties and chemical structure, 16 is also a candidate to further studies that analyze deeply its effect in blood glucose levels and some other metss components. no in vivo toxicity studies on luctucain c (16) have been reported to date. 6.10. 8-deoxylactucin from cichorium intybus l cichorium intybus, commonly known as chicory, was historically cultivated by the ancient egyptians as a vegetable crop, a coffee substitute, and a medicinal plant. nowadays, it is appreciated for its bitter taste and widely used in india as a traditional treatment for diabetes mellitus (al-snafi, 2016; pushparaj et al., 2007) . the antidiabetic, hypolipidemic, hepatoprotective, antioxidant and anti-inflammatory effects of c. intybus have been widely studied (chandra and sk, 2016) . this broad spectrum of biological activities has been attributed to its high content of phytochemical constituents, being slns the most abundant in roots and the responsible of the bitterness of leaves (al-snafi, 2016). 8-deoxylactucin (17) (fig. 2) is a guaianolide found as a major component of chicory root and it is also common in species of lactuca. it has been demonstrated that 17 possess anti-inflammatory activity by inhibiting the nuclear transcription factor nf-kb and as a selective inhibitor of cox-2 (cavin et al., 2005 ). an important structural characteristic of 17 is the presence of a-methylene-g-lactone group and a,b-unsaturated cyclopentenone. compounds that possess these structures, such as helenalin, can react with sulfhydryl group of cys38 in nf-kb and prevent dna binding (lyß et al., 1998; widen et al., 2017) . thus, 8-deoxylactucin (17) could interact with nf-kb by a similar mechanism. no in vivo toxicity studies on 8-deoxylactucin (17) have been reported to date. 6.11. artemisinin from artemisia spp artemisinin (18) (fig. 2) is a cadinanolide endoperoxide sesquiterpene lactone originally isolated from artemisa annua linn in 1972 (tu, 2016) . nowadays, 18 and their derivatives are part of the protocols for malaria treatment (bridgford et al., 2018) . experimental studies have also established their effectiveness as an anti-inflammatory, antioxidant, anti-tumor, and vascular protection agent jiang et al., 2016; efferth, 2017) . regarding to vascular protection, cao et al. (2020) demonstrated that oral administration of artemisinin (18) effectively alleviated inflammatory response (mcp-1, ifn-g, il-6 and tnf-a), elevated macrophage autophagy, suppressed foamy macrophage transformation, and thereby inhibiting atherosclerotic plaque formation in high-fat diet treated apoe à/à mice. in addition, 18 has been discovered to be a potential therapeutic agent for ameliorating type 1 diabetes because of its ability to promote the conversion of pancreatic glucagon-producing a cells to insulinsecreting b cells in rats . nevertheless, the widespread application of artemisinin as an anti-malarial drug could contributed to the selection of resistant strains of the etiologic agent plasmodium, thus its use to combating non-communicable human diseases would raise the risk for this selection and lead to an important drug resistance development. some authors have suggested that unless necessary, artemisinin (18) should be replaced by other therapeutic agents for the treatment of versatile types of human diseases (dong-sheng et al., 2017) . it is important to note that some species of artemisia have been traditionally used in treatment of diabetes such as a. dracunulus l., a. minor jacq. ex besser, a. pallens wall. ex d.c., a. sphaerocephala krasch and a. herba-alba asso, (mohamed et al., 2010; subramoniam, 2016) . several eudesmanolides and guaianolides have been isolated from a. herba-alba, all of them having in common the absence of the a-methylene in the g-lactone ring as in artemisinin (18) structure. indeed the ethanol, chloroform and water extracts of this plant have showed important hypoglycemic activity (mohamed et al., 2010) . animal experiments show considerable toxicity after the application of artemisinin family (neurotoxicity, embryotoxicity, genotoxicity, hemato-and immunotoxicity, cardiotoxicity, nephrotoxicity, and allergic reactions), but large clinical studies and meta-analyzes did not show serious human side effects, although proper monitoring of adverse effects in developing countries may not be a trivial task. there is a paucity of large-scale clinical trials adequate to detect rare but significant toxicity. the lesson learned from animal and human studies is that long-term availability rather than short-term peak concentrations of artemisinin cause toxicity (efferth and kaina, 2010) . thus, the observation of the toxicity of artemisinin derivatives in animals, but not in humans, is most likely due to different pharmacokinetic profiles after different routes of administrations. 6.12. scoporanolide and estafiatin from artemisia scoparia waldst. & kit artemisia scoparia waldst. & kit. (redstem wormwood) is widely distributed in southwest asia and central europe. this species has been reported to possess anti-obesity (richard et al., 2014) and hypolipidemic (boudreau et al., 2018) properties. a. scoparia ethanolic extract reduces non-alcoholic fatty liver disease by enhancing hepatic insulin and amp-ampk signaling in diet-induced obese mice . a. scoparia hot water extract reduces blood pressure in spontaneously hypertensive rats via the inhibition of plasma angiotensin i-converting enzyme (ace) activity (cho et al., 2015) . the phytochemical analysis of this extract revealed that seven slns contribute to the antihypertensive effect inhibition of ace activity, highlighting the activity of scoporanolide (19) and estafiatin (20) (fig. 2) (cho et al., 2016) showing significantly higher ace inhibitory activities than the other isolated slns. no in vivo toxicity studies on scoporanolide (19) and estafiatin (20) have been reported to date. the guaianolide cumambrin a (21) (fig. 2) , isolated from chrysanthemum boreale (makino) makino, has shown a pharmacological effect on the normalization of blood pressure in spontaneously hypertensive rats (hong et al., 1999) . ex vivo study demonstrate that this compound is a potent relaxant of aortic smooth muscle at a concentration of 5 £ 10 à5 m (hong et al., 2005) . no in vivo toxicity studies on cumambrin a (21) have been reported to date. the potential beneficial effects of slns on metss risk factors discussed in this review clearly demonstrate that these substances represent remarkable compounds with a diversity of molecular structure and biological activity. these beneficial effects include normalization of blood glucose levels, improvement of blood lipid profiles, anti-inflammatory activity, improvement of insulin sensitivity and normalization of blood pressure. the underlying molecular targets mediating these effects have not been completely understood. regardless of that, it can be noticed that at least six molecular targets or pathways could explain the effect of slns on components of mets: furthermore, the evidence from published literature demonstrates that some slns belonging to guaianolides like eremanthin (4) and germacranolides such as costunolide (5) and guaianolide dimers byrsonines a and b (14, 15), show multifunctional benefits due to their action on many metss risk factors. these observations suggest that structural specificity may be a key for understanding the mechanisms of action of slns. looking forward, the effects of slns on mets should be explained based on the molecular targets and should be related to biochemical mechanisms. mets has become a major public health problem worldwide and represents a common clinical condition in countries with a high incidence of obesity and western dietary patterns. natural products, such as slns, are attractive drug candidates and more attention must be paid to their potential use in the treatment and prevention of mets. the authors declare no conflict of interest. sesquiterpene lactones with unusual structure. their biogenesis and biological activity evaluation of antidiabetic effect of methanolic extract of inula racemosa root in rats tithonia diversifolia (hemsl) a. gray. 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relevance essential oil safety. a guide for health care professionals, second ed artemisinin-a gift from traditional chinese medicine to the world (nobel lecture) micheliolide, a new sesquiterpene lactone that inhibits intestinal inflammation and colitis-associated cancer costus speciosus and coccinia grandis: traditional medicinal remedies for diabetes inula sesquiterpenoids: structural diversity, cytotoxicity and anti-tumor activity artemisia scoparia extract attenuates nonalcoholic fatty liver disease in diet-induced obesity mice by enhancing hepatic insulin and ampk signaling independently of fgf21 pathway targeting nf-kb p65 with a helenalin inspired bis-electrophile etiology of metabolic syndrome and dietary intervention alantolactone suppresses apoc3 expression and alters lipid homeostasis in l02 liver cells role of c-jun n-terminal kinase (jnk) in obesity and type 2 diabetes study of hypoglycaemic and hypolipidemic effects of inula viscosa l. aqueous extract in normal and diabetic rats jnk signaling: regulation and functions based on complex protein-protein partnerships biomimetic semisynthesis of arglabin from parthenolide three new sesquiterpenes from tithonia diversifolia and their anti-hyperglycemic activity extracts of magnolia species-induced prevention of diabetic complications: a brief review micheliolide alleviates hepatic steatosis in db/db mice by inhibiting inflammation and promoting autophagy via ppar-g-mediated nf-kb and ampk/mtor signaling alantolactone mitigates renal injury induced by diabetes via inhibition of high glucose-mediated inflammatory response and macrophage infiltration this work was partially supported by the secretaría de investigaci on y posgrado, instituto polit ecnico nacional. asg was cona-cyt (267721) and ipn-beifi fellow. julio c. ontiveros thanks conacyt for funding conacyt project no. 1069 "c atedras cona-cyt". key: cord-351932-dn60t7qa authors: salehi, bahare; sener, bilge; kilic, mehtap; sharifi-rad, javad; naz, rabia; yousaf, zubaida; mudau, fhatuwani nixwell; fokou, patrick valere tsouh; ezzat, shahira m.; el bishbishy, mahitab h.; taheri, yasaman; lucariello, giuseppe; durazzo, alessandra; lucarini, massimo; suleria, hafiz ansar rasul; santini, antonello title: dioscorea plants: a genus rich in vital nutra-pharmaceuticals-a review date: 2019 journal: iran j pharm res doi: 10.22037/ijpr.2019.112501.13795 sha: doc_id: 351932 cord_uid: dn60t7qa dioscorea species, known as “yams,” belong to family dioscoreaceae. this genus consists of more than 600 species distributed from africa, asia, the caribbean’s south america, and the south pacific islands. their organoleptic properties make them the most widely used carbohydrate food and dietary supplements. the underground and/or aerial tubers represent valuable sources of proteins, fats, and vitamins for millions of people in west africa. this review gives a shot of secondary metabolites of dioscorea plants, including steroids, clerodane diterpenes, quinones, cyanidins, phenolics, diarylheptanoids, and nitrogen-containing compounds. this review collected the evidence on biological properties of description dioscorea, including in-vitro and in-vivo studies. dioscorea species contain promising bioactive molecules i.e. diosgenin that support their different biological properties, including antioxidant, hypoglycaemic, hypolipidemic, antiantimicrobial, inflammatory, antiproliferative, androgenic, estrogenic, and contraceptive drugs. indeed, besides its nutrient values, dioscorea is a potential source of bioactive substances of interest in the prevention/treatment of several diseases, and thus represents a great challenge in developing countries. however, ethnomedicinal potential should be validated and further researches on pharmacological properties and phytochemical composition should be carried out. particularly, doing some studies to convert the preclinical results to clinical efficacy should be guaranteed. dioscorea, food plant, traditional use, phytochemistry, pharmacological activities dioscorea belongs to the family dioscoreaceae, sub-family dioscoreoideae, consisting of 600 species. dioscorea species are native to the old-world including high temperatures tropics and subtropic regions of the world. the major part of species occur in west africa, southern asia, and tropical america: they are herbaceous climbers with rhizome or tubers dioscorea species and, being usedin the formulation of pharmaceutical products, have a high medicinal, industrial and commercial importance (1) . indeed, dioscorea species account for the most important dietary supplements ingredients used in cosmetics and pharmaceutical industries. it has also been commonly used by local people, mostly those who are engaged in the trade of medical plants worldwide. indeed, tubers of different dioscorea species are used as a cure for different diseases and ailments (cough, cold, stomach ache, leprosy, burns, fungal infections, dysentery, skin diseases, rheumatism, arthritis, etc.) in several formulations, and even for birth control (1) . it is well known the potential of this plant is related due to the different phytochemical compounds found in dioscorea. tubers and roots contain steroidal sapogenins, mostly diosgenin as well as volatile compounds (2, 3) . chemical substances like diosgenin found in dioscorea are commercially used in the pharmaceutical industry. the high demand for the medicinal use of this plant in different parts of the world suggests a strong need for conservation strategies. however, the conservation might not be easy or simple as it must involve plant protection and wellcontrolled access to its resources. there still a huge need for more research and information on the food, nutraceutical, and medicinal value worldwide of this plant species. therefore, this review tends to fill this gap by summarizing data on the current medicinal importance of dioscorea species. researches have been brought new knowledge about chemical compounds in tubers of dioscorea species and possible medicinal usage. the most common secondary metabolites are saponins, and more than 100 steroidal saponins (based on aglycon part as stigmastanol, furostanol, spirostanol, cholestanol, ergostanol, and pregnanol glycosides (figure 1 )) were isolated from various dioscorea species. besides these steroids, clerodane diterpenes, phenolics, cyanidins, quinones, diarylheptanoids, and nitrogen-containing compounds in the tubers of dioscorea species were quantified (4) . steroidal saponins mainly consist of furostane, a pentacyclic ring system with a sixth open ring; spirostane, a hexacyclic ring system; and pregnane, a tetracyclic ring system. the sugar part is mainly composed of glucose and rhamnose in various proportions and linkages. steroidal saponins are glycosides consisting of an aglycone (diosgenin) and several glycosyl moieties. the most common sugars encountered in saponins are pentoses (arabinose, xylose, etc.), hexoses (glucose, galactose, etc.) and 6-deoxyhexose (rhamnose, etc.). the saponins of this plant species are both water-soluble and water-insoluble steroid saponins. dioscorea species are well known for containing steroidal saponins, which were used as marker compounds for quality control of the botanical products. as reported by jesus et al., 2016 , diosgenin (3-β-hydroxy-5spirostene) is the primary furostanol saponin found in several plants, including dioscorea species, and is described as a promising bioactive compound with several medicinal properties, i.e. hypolipidemic, antioxidant, anti-inflammatory, hypoglycaemic, and antiproliferative activities (5) . diosgenin obtained by hydrolysis of yam saponin were the main raw material for the industrial production of steroid drug i.e. anti-inflammatory, androgenic, estrogenic, and contraceptive drugs, by underlying its potential in the prevention/treatment of several diseases. dioscorea villosa l. roots and rhizomes, also are known as "wild yam", is a rich source of diosgenin. today, dietary supplements containing wild yam extracts are popular among women for the alleviation of menopausal symptoms and are widely used as alternatives to hormone replacement therapy (5) . dioscorea alata l. dioscorea alata l. is one of the most popular varieties of yams. dioscorea alata (d. alata) were implicated in the promotion of the health of postmenopausal women. wild mexican yam was marketed for the treatment of irritability, hot flashes, insomnia, and depression. cheng et al., 2007 , reported the isolation and identification of two new compounds, hydro-q 9 chromene, and γ-tocopherol-9; together with four known compounds (rrr-α-tocopherol, coenzyme q 9 , cycloartane, and 1-feruloylglycerol): these compounds had phenolic hydroxyl group in common with the known phytoestrogens and have also antioxidant activity. moreover, their estrogenic activity was evaluated based on the ligand-dependent transcription activation assay (6) . dioscorea antaly jum. and h. perrier is originated from madagascar, diffused in the west and north-west regions. the study of rakatobe et al., 2010 reported two clerodane diterpenoids, antadiosbulbins a and b and two furanoid 19-norclerodane diterpenes, 8-epidiosbulbins e and g along with the known diterpenoid diosbulbin e as well as nine known phenolics including five phenanthrenes and four flavonoids in the ethyl acetate soluble part of the methanolic extract of the tubers (7) . dioscorea bulbifera l. dioscorea bulbifera l. (d. bulbifera l.), commonly known as air potato, is originated from africa and southern asia. the rhizome of d. bulbifera l. is called as "huang yao zi" in traditional chinese medicine and used for the treatment of thyroid diseases and cancers. in the northern districts of bangladesh, this herb is used for the treatment of cancers and leprosy. this plant has two types of storage organs, namely bulbils in the leaf axils of the stem and tubers. bulbils have been used as a folk remedy to treat diarrhea, conjunctivitis, and the extracts of the plant exhibit anti-tumor activity. the mannose-binding lectin from bulbils of d. bulbifera was studied for its clinical potential in hiv and cancer research (8) . phytochemical studies reported as main bioactive compounds of this plant: flavonoids, clerodane diterpenoids, and steroidal saponins and phenolic compounds. as instance, liu et al. 2011 reported the isolation and identification of two new compounds, bibenzyl type-2,5,2′,5′-tetrahydroxy-3′methoxybibenzyl and diarylheptanone containing a dihydrophenanthrene moiety named as diobulbinone a along with sixteen known compounds (9) . nine norclerodane diterpenoids were isolated from the tubers of d. bulbifera l. including two new compounds (diosbulbin n and diosbulbin p), and a naturally occurring compound (diosbulbin o) along with six known diosbulbins a-d, f, and g (10) . another study also showed the molecular changes of liver dysfunction and reveal overall metabolic and physiological mechanisms of the subchronic toxic effect of dioscorea bulbifera rhizome (11) . lam.-holl known as "yellow yam" is native to tropical west africa and its rhizomes are used as food and as a remedy for treating burns and fevers. a new furostanol glycoside namely 26-o-β -d-glucopyranosyl-3β,26-dihydroxy-20,22-seco-25(r)-furost-5en-20,22-dione-3-o-α-l-rhamnopyranosyl-(1→4)-α -l-rhamnopyranosyl-(1→4)-[ α -l -r h a m n o p y r a n o s y l -( 1 → 2 ) ] -β -dglucopyranoside was isolated from the methanolic extract of the rhizome of dioscorea cayenensis growing in cameroon, together with the known spirostanol saponins described as methyl protodioscin, asperoside and prosapogenin a of dioscin (12) . prosapogenin a of dioscin having a spirostan skeleton showed antifungal activity against candida species with mic values between 6.25 and 25 mg/ml (12) . two new fatty acidspirostan steroid glycoside esters, progenin iii palmitate, and progenin iii linoleate, were isolated from the methanol extract of the rhizomes of dioscorea cayenensis (13) . it is worth mentioning the recent studies on the nutrient and antinutrient composition of yellow yam (dioscorea cayenensis) and their products; as instance raw dioscorea cayenensis tubers contained 66.79 g moisture, 2.62 g crude protein, 0.27 g lipid, 0.17 g fiber, 0.63 g ash, 29.69 g carbohydrates, 262.30 mg potassium, 61.53 mg magnesium, 0.79 mg iron, 0.39 mg zinc, and yielded 108.26 kcal energy with insignificant vitamin content/100 g edible portion (14) . dioscorea esculenta (lour.) burk. dioscorea esculenta also called as a "lesser yam" of dioscorea species, is widely distributed southern asia and the pacific. the tubers of d. esculenta were used traditionally in the treatment of various diseases. moreover, fiteen steroidal saponins were isolated from its tuber powder, and from the leaf of dioscorea esculenta (d. esculenta); four of them belonged to furostanol-type saponin,eleven as spirostane-type saponins (15) . the rhizome of dioscorea japonica thunb., known as "san yak" in korea, is used as food and medicine. the plant was used in traditional chinese medicine to control/eliminate diarrhea, dilute sputum, improve anorexia, and moisturize skin (16) . phytochemical studies on dioscorea japonica (d. japonica) showed the presence of active hypoglycemic compounds (dioscorans a-f), sesquiterpene, and acetophenone along with the steroidal constituents, including spirostane, furostane, and cholestane types (16) . the rhizomes of dioscorea membrancea (d. membranacea) pierre have been used in thai traditional medicine. as instance, thongdeeying et al. 2016 isolated from the cytotoxic chloroform fraction of the rhizomes a new steroid (epi-panthogenin b), a known steroid (panthogenin b), two napthofuranoxepins dioscorealide a and dioscorealide b, phenanthraquinone (dioscoreanone) and three phenanthrene; the same authors reported that also, three steroids (β-sitosterol, stigmasterol, and β-dsitosterolglucoside) and two steroidal saponins [(diosgenin-3-o-α-l-rhamnopyranosyl (1→2)-β-d-glucopyranoside and diosgenin-3 -o -β -d -g l u c o p y r a n o s y l ( 1 -3 ) -β -dglucopyranoside)] were obtained from d. membranacea (17) . makino is bitter-sweet in taste and warm in nature according to the traditional chinese medicine. the rhizomes of d. nipponica were traditionally used in china as herbal medicine and food supplement for more than four thousand years; particularly, these plant species mainly act on the liver, kidney, and lungs, displaying anti-rheumatic, analgesic, blood circulation-stimulating, lung channeldredging, digestive, anti-diuretic, anti-tussive, panting-calming, and phlegm-dispelling activities. medicinally, it is commonly used for the treatment of rheumatoid arthritis, pain in the legs and lumbar area, kashin-beck disease, bruises, sprains, chronic bronchitis, cough and asthma and communication, and adherence to therapy is one of the main concerns (4, 18 and 19) . recently, it has been used as an important industrial raw material for the synthesis of steroidal drugs. concerning the phytochemical profile, twelve cyclic diarylheptanoids were isolated from the rhizomes of dioscorea nipponica (20) , among which two new cyclic diarylheptanoids, diosniponol a and b; moreover, as reported by the same authors, these compounds were evaluated for their effects on nitric oxide production without cell toxicity in lipopolysaccharide-activated bv-2 cells. therefore, diarylheptanoid derivatives from d. nipponica may be potential candidates for the treatment of various neurodegenerative diseases associated with neuroinflammation (20) . another study described the watersoluble non-saponin components [benzyl 1-o-β-d-glucopyranoside, leonuriside a, icariside d2, pyrocatechol-1-o-β-dglucopyranoside, (+) syringaresinol-4-oβ-d-glucopyranoside, cyclo-(leu-tyr), and adenosine], and phenolic acid (piscidic acid) from the rhizomes of d. nipponica (21) . on the other hand, 7-oxodioscin a new spirostan-type steroid along with known dioseptemloside g, (25r)-dracaenoside g, orbiculatoside b, dioscin, progenin iii, gracillin were determined. the presence of (3β,22α,25r)-26were also confirmed as furastan-type steroids (21) . it is worth mentioning the study of that explore the chemical basis of the rhizomes and aerial parts of dioscorea nipponica makino by a combination of cheminformatics and bioinformatics, particularly their potential therapeutic and toxicity targets were screened through the drugbank's or t3db's chemquery structure search: the compounds in the rhizomes possessed 391 potential therapeutic targets and 216 potential toxicity targets (22) . dioscorea opposita thunb. dioscorea opposita thunb. has been cultivated in china, japan, and korea as a food, and widely used as traditional medicine, for a very long time. there are about 80 yam cultivars that are originated in china, including the so-called "trueborn" chinese yam. the important ingredient of the yam is sugar, which provides energy and sweetness and adds to the general flavor of foods. also, the chinese yam assists in food digestion and is beneficial in cases of stomach disorders (23) . phytochemical investigations of dioscorea opposite (d. opposita) have revealed many chemical components such as purine derivatives, phenanthrenes, stilbenes, sapogenins, and saponins. phytochemical studies of the chloroform soluble fraction of d. opposita thunb. have resulted in the isolation of four new compounds, two dihydrostilbenes: 3,5-dihydroxy-4-methoxybibenzyl, 3,3′,5trihydroxy-2′-methoxybibenzyl; and dibenzoxepins 10,11-dihydro-dibenz[b,f]oxepin-2,4diol and 10,11-dihydro-4-methoxy-dibenz[b,f] oxepin-2-ol, together with an additional fifteen known compounds. all the nineteen isolated compounds were tested in the dpph, superoxide anion radical scavenging assays and cyclooxygenases (coxs) inhibition assay. among them, 3,3′,5-trihydroxy-2′methoxybibenzyl showed the most potent cox-2 inhibitory activity (23) . bioassayguided fractionation of d. opposita extracts led to the isolation and identification of 23 phenolic compounds. of them, 15 compounds reduced porcine pancreatic lipase activity at ic 50 values of less than 50 mm and from them 3,3′,5-trihydroxy-2′-methoxybibenzyl and (4e,6e)-1,7-bis(4-hydroxyphenyl)-4,6-heptadien-3-one showed the highest inhibition with an ic 50 value of 8.8 mm and dose-dependently in the concentration range 5-100 mm (24) . these findings provide that phenolic compounds with stilbenoids are considered to play important roles in the lipase inhibition of the d. opposita extract. especially, the stilbenoids possessing 3,5-dihydroxybibenzyl moiety showed higher inhibitory potencies than the others. the lipase inhibitory activities of stilbenoids depend on the presence of the hydroxyl group in the c-3 position. the structural difference also influences the inhibitory effect of diarylheptanoids on pancreatic lipase. dihydrostilbene phenanthrene and diarylheptanoid structures were deemed to be responsible for lipase inhibition activity of this species (24) . d. opposita thunb. is also rich in starch, water-soluble polysaccharides, and mucilage defined as a polysaccharide with unique viscosity characteristics are widely used in the pharmaceutical and food industries as a thickening agent and emulsion stabilizer. these agents are important in the food industry as they improve the sensory quality, flavor, texture, palatability, mouthfeel, and general appearance of the final products. therefore, the mucilage of this plant species is a potential candidate for food emulsifier resource of a natural emulsifier, especially under alkaline conditions obtained from industrial processing waste such as gum arabic, yellow mustard, and chia (salvia hispanica l.) (25) . dioscorea preussii pax d. preussii pax collected in bambui, cameroon, was reported to have in-vitro cytotoxic, antileishmanial and antifungal activities. three new steroidal saponins, named as diospreussinosides a, b, c, along with two known compounds were determined as (25r)-17α-hydroxyspirost-5r h a m n o p y r a n o s y l -( 1 → 4 )α -l -r h a m n o p y r a n o s y l -( 1 → 4 ) ] -β -dglucopyranoside were isolated from rhizomes of d. preussii (27) . dioscorea pyrifolia kunth d. pyrifolia kunth is called also as "akar kemeyan paya" in malaysia. these plants grow wild and are diffused particularly in peninsular malaysia. sharlina et al. 2017 reported that the starch extracted from d. pyrifolia was characterized by the high amylose content (44.47 ± 1.86%); the same authors marked how d. pyrifolia kunth represents a very important source of starch for food and non-food applications, such as crispy food, coating materials, hydrogels, films, bio-membranes, and adhesives (28) . cholestane glycosides, dioseptemlosides a and b, together with six spirostane glycosides, dioseptemlosides c-h, were isolated from the rhizomes of d. septemloba. among these, spirostane aglycones containing hydroxyl group at c-7 were reported in the family dioscoreaceae. spirostane-type glycosides showed strong activity on nitric oxide production (29) . on the other hand, three new diarylheptanoids, dioscorol a, dioscorosides e 1 , e 2 ; two new stilbenes, dioscorosides f 1 and f 2 were isolated from the rhizomes of dioscorea septemloba. besides, 1,7-bis(4-hydroxyphenyl)hepta-4e,6e-dien-3-one, 1,7-bis(4-hydroxyphenyl)-1,4,6-heptatrien-3-one, 3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)heptane, (3r,5r)-3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)heptane 3-o-β-d-glucopyranoside, (3r,5r)-3,5-dihydroxy-1,7-bis(4-hydroxywere also obtained as known compounds. they have also shown a significant increase in the glucose consumption in differentiated l6 myotubes and displayed triglyceride inhibitory effects in hepg2 cells . eight new compounds namely, dioscorosides g, h1, h2, dioscorol b, dioscorosides i, j, k1, and k2, together with twelve known ones were isolated from the rhizomes of dioscorea septemloba. moreover, the authors showed that some of these compounds were found to display significant inhibition of nitrite production evaluated in-vitro anti-inflammatory potential using lps-stimulated raw 264.7 murine macrophages (30) . currently, he et al. 2019 have identified and elucidated the structure of a new phenanthropyran, dioscorone b, and a new phenanthrene, together with seven known compounds, is from the 75% ethanol extract of dioscorea septemloba rhizomes. moreover, the authors reported that new isolated phenanthropyran, tested for antioxidant activity, showed excellent activities with ic 50 values of 0.07 ± 0.10 μm and 0.13 ± 0.09 μm, respectively (31). dioscorea tokoro makino d. tokoro is one of those wild yams, and its rhizome was generally used for daily food.; in particular in the northern part of japan, the rhizome was used as health-promoting food. d. tokoro was characterized by steroidal saponins, such as dioscin and gracillin, but no data were found about its health-promoting effect. the acetonitrile extract of d. tokoro rhizome fractionation led to protodioscin as an antiproliferative compound to hl-60 leukemic cells (32) . the most well-known species of this genus is dioscorea villosa, also called "wild yam." two furanostane type saponins, namely methyl parvifloside, and protodeltonin, were isolated from d. villosa as well as two spirostane types deltonin and glucosidodeltonin (zingiberensis i), whereas minor saponins were methylprotodioscin, disoscin, and prosapogenin (33) . the same authors reported that two fla-van-3-ol glycosides were also isolated from d. villosa and fifteen steroidal compounds, including two new metabolites, were isolated from the tubers of d. villosa; these compounds were characterized as cholestane type steroidal glycosides named as dioscoreavillosides a and b (33). dong et al., 2012 described how the rhizomes were also afforded 14 diarylheptanoids, five of which were new compounds containing a tetrahydropyran ring in the heptane portion of the molecule (34) . in another study, three furostanol saponins, parvifloside, methyl protodeltonin, and trigofoenoside a-1 were isolated from the n-butanol soluble extract of d. villosa by using hsccc; moreover the authors reported that subsquent normal-phase hsccc separation also led to the identification of four spirostanol saponins identified as zingiberensis saponin i, deltonin, dioscin, and prosapogenin a of dioscin (35) . on the other hand, two series of lipidated steroid saponins were isolated from d. villosa (36) . the series a was identified as a mixture of five lipidated steroid saponins: the series b was characterized as a mixture of the following five compounds: 5-en-clionasterthese findings revealed two classes as new biomarkers: the diarylheptanoids and the lipidated steroid saponins. the rhizome of dioscorea zingiberensis c.h. wright is known as ''huang jiang" and represents an important source of diosgenin. d. zingiberensis were used for the treatment of lung heat, pyretic stranguria, anthracia, coronary heart disease, and swelling; in particular, the rhizome of dioscorea zingiberensis is extensively used for the extraction of diosgenin sapogenin and its glycoside dioscin, used for the synthesis of sex hormones and corticosteroids. it is worth mentioning the current study of zhang et al., 2018 that give comprehensive overview of the traditional usage and phytochemistry of the dioscorea zingiberensis c.h. wright: -more than 70 compounds have been identified; -several of these have been tested in preclinical assays and clinical trials; -a wide spectrum of biological effects including cardiovascular, anti-thrombosis, hyperlipidemia, neuroprotection, anti-inflammatory, and anthelmintic effect has been verified (37) . wang et al. 2010 reported how spirostanol based aglycon containing steroidal saponins are the representative steroidal saponins, and their quantity is higher than that of furostanol steroidal saponins in d. zingiberensis (38) . apart from the steroidal saponins, other kinds of constituents were also identified from d. zingiberensis. until now, four steroids have been isolated from this plant including β-sitosterol, sterol, zingiberenin f, and (25r)-3β-hydroxyspirost-δ5,20 (21) in addition to the common steroidal saponins, other constituents classified as alkaloid, phenyl-glycoside, and benzoic acid derivatives have also been determined. 4-aminomethyl-phenol, 2-o-β-d-glucopyranosyl-4-hydroxybenzoic acid and another nonsteroidal saponin called 2-o-β-d-glucopyranosyl-4-methoxybenzoic acid, were isolated and identified from the n-buthanol extracts of fresh rhizomes. following the regular phytochemical research procedure, five compounds were obtained from d. zingiberensis (38) . they consisted of two new phenanthrene derivatives, namely 2,5,7-trimethoxy-1,4-dione-9,10-dihydrophenanthrene and 2,5,6-trihydroxy-3,4-dimethoxy-9,10-dihydrophenanthrene; a new anthracenedione, i.e., 2,5,7-trimethoxy-1,4-dione-anthracene; and two known 9,10-dihydrophenanthrenes, called 5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene and 2,5-dihydroxy-3,4,6-trimethoxy-9,10-dihydrophenanthrene. one new bibenzyl (3,5-dihydroxy-4,40-dimethoxybibenzyl.) and one new phenanthrene (2,5-dihydroxy-4,6-dimethoxy-9,10-dihydrophenanthrene.), together with two known bibenzyls (3,5-dihydroxy-4-methoxybibenzyl 3,5′,-dihydroxy-3′,4-dimethoxybibenzyl and four known diarylheptanoids ((3r,5r)-3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)heptane, (3r,5r)-3,5-dihydroxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)heptane, (3r,5r)-3,5-dihydroxy-1-(4-hydroxy-3-methoxyphenyl)-7-(4-hydroxyphenyl)-heptane and (3r,5r)-3,5-dihydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-heptane were isolated from the dichloromethane soluble fraction of the rhizomes of d. zingiberensis (39) . all these phenolic compounds were evaluated for their anti-pancreatitic activities on sodium taurocholate-induced pancreatic acinar necrosis as inhibition of necrotic cell death pathway activation. among them, (3r,5r)-3,5-dihydroxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl) heptane which bears a trihydroxy-diarylheptane skeleton was shown to protect against pancreatic acinar cell injury through mediating novel potential therapy for pancreatic necrosis (39) . the tubers of different dioscorea species present a wide variation of bioactive compounds, responsible for their pharmacological activities. generally, the evaluation of bioactive components and the assessments of their interactions could be viewed as the first step for the determination of potential of a plant (40) (41) (42) (43) (44) (45) (46) . also, their ethnopharmacological importance promotes further investigations of dioscorea metabolites as a source of potential therapeutic agents to ameliorate different diseases (table 1) . cytotoxic activity d. bulbifera ethanolic crude extract and different fractions (hexane, ethyl acetate, and water) at concentrations of 50, 100, and 200 μg/ml were found to possess a potent cytotoxic activity against human colorectal carcinoma (hct116), human colorectal adenocarcinoma (ht-29), human lung carcinoma (a549), human breast carcinoma (mcf-7), human tuber immune-modulating activity (54) . anti-inflammatory activity (55) anti-severe acute respiratory syndrome associated coronavirus (56) . gastroprotective activity (57) enhance murine splenocyte proliferation ex-vivo and improve regeneration of bone marrow cells (58) immunomodulatory activity (59) anti-inflammatory activity (55) . neuroprotective activity (82) antioxidant activity (83) mediated synthesis of gold and silver nanoparticles with catalytic activity (84) neuroprotective activity (82) antidiabetic activity (85) cell shrinkage, and dna fragmentation as shown by flow cytometry. the authors also revealed that dbeaf induces apoptosis effects on hct116 cells through externalization of phosphatidylserine and by promoting the loss of mitochondrial membrane potential (mmp), dysregulation of the bcl-2 family proteins and the downregulation of the expression of procaspase -8, -9, -10 and -3 and parp protein expression. furthermore, their results suggested that the inhibitory effect of dbeaf on erk1/2 and the activation of jnk were closely associated with the apoptotic cell death induction in human colorectal carcinoma (60). rhizome antioxidant activity (86) antitumor activity (81) anti-hypercholesterolemic effect (87) anti-platelet aggregation activity (87) antifungal activity (88) antihyperlipidemic and antioxidative activity (81) dioscorea pentaphylla l. antioxidant activity and antibacterial activity d. collettii var. hypoglauca and its active metabolite protoneodioscin, a furostanol saponin, was also evaluated for its cytotoxic activity against 60 cell lines including human leukemia, melanoma, and cancers of lung, colon, brain, ovary, renal, breast, and prostate. protoneodioscin exhibited significant cytotoxicity with 50% growth inhibition (gi 50 ) ranging from 0.5 to 9 mm against all cell lines from leukemia and most solid tumors (64) . in the following year, the same research team evaluated two other compounds, namely; methyl protoneogracillin and gracillin. the results revealed that methyl protoneogracillin exhibited significant cytotoxicity with (gi 50 ) ≤ 2 mm. leukemia, cns cancer, and prostate cancer were the most sensitive subpanels, while ovarian cancer was the least sensitive subpanels. on the other hand, gracillin lacked selectivity against human cancer diseases (29). a different approach was undertaken by ashri and co-workers (2018) (71), who evaluated the cytotoxicity of modified d. hispida starchbased hydrogels on small intestine cell line (fhs-74 int) to ensure the safety of their use. their results revealed that the prepared hydrogels did not show any cytotoxicity and could be employed for future pharmaceutical use. a mucopolysaccharide of yam (d. batatas decne) (ymb) was found to increase the cytotoxic activity of mouse splenocyte against leukemia cell at 10 µg/ml concentration. however, it did not affect the viability of splenocytes. the production of ifn-γ was significantly increased in the ymp treated splenocytes. at 50 µg/ml concentration, it increases the up taking capacity and lysosomal phosphatase activity of peritoneal macrophages. in addition, ymp (10-100 µg/ml) significantly increased the viability of peritoneal macrophages (54) . dioscorin, the glycoprotein isolated from d. alata was reported to activate toll-like receptor 4 (tlr4) and induce macrophage activation via typical tlr4-signaling pathways at 100 µg/ml concentration. it induces tlr4downstream cytokine expression in bone marrow cells isolated from tlr4-functional c3h/hen mice but not from tlr4-defective c3h/hej mice. dioscorin also stimulated multiple signaling molecules (nf-jb, erk, jnk, and p38) and induced the expression of cytokines (tnf-a, il-1b, and il-6) in murine raw 264.7 macrophages (47) . different dioscorea species were used traditionally for the treatment of memoryrelated disorders and dementia, i.e. alzheimer disease and other neurodegenerative diseases (82) . the chloroform extract from d. opposite rhizomes significantly reduced the glutamateinduced toxicity in a dose-dependent manner with a maximum neuroprotective effect at 10 µg/ml (82) . kim (16) . moreover, the compounds isolated from d. nipponica rhizomes exhibited anti-neuroinflammatory and neuroprotective activities, the most active of which was 3,7-dihydroxy-2,4,6trimethoxy-phenanthrene. the later was the most potent nerve growth factor (ngf) inducer, with 162.36% stimulation, and strongly reduced nitric oxide (no) levels with an ic 50 value of 19.56 μm in lps-activated bv2 microglial cells. also, it significantly increased neurite outgrowth in mouse neuro2a (n2a) cells and therefore possessed a significant neuroprotective activity (76) . antidiabetic activity different extraction procedures as ultrasonic-assisted extraction, cold water extraction, warm water extraction and hot water extraction significantly influenced the antidiabetic potential of the rhizomes of d. hemsleyi as determined via both alphaglucosidase and alpha-amylase inhibition assays, where the ic 50 values of alphaglucosidase inhibition assay varied from 27.41 to 274.36 µg/ml while those of alpha-amylase inhibition assay varied from 3.66 to 47.57 mg/ ml (70) . the antioxidant activity of the extracts of dioscorea species received much scientific attention (67, 72, 73 and 83) . different extracts of rhizomes of d. hemsleyi, d. hispida dennst, d. opposite thunb., d. nipponica makino, d. esculenta (lour). burkill, d. japonica thunb. var. pseudojaponica and d. pentaphylla l. were all studied for their antioxidant properties by different in-vitro assays namely; reducing power assay, ferric reducing antioxidant power assay (frap), dpph radical scavenging assay, hydroxyl radical scavenging assay, superoxide radical assay, self-oxidation of 1,2,3-phentriol assay and antioxidant activity by radical cation (abts) assay. generally, the studied extracts from different dioscorea species showed a significant antioxidant potential which may account for their involvement in the treatment of various disorders via radical scavenging mechanisms. lipopolysaccharide-stimulated raw 264.7 cells were employed to determine the antiinflammatory properties of the ethanolic extract of tubers of d. japonica thunb. var. pseudojaponica and individual steroid glycosides isolated from d. septemloba rhizomes (29, 73) . d. japonica extract at doses of 500 and 1000 µg/ml significantly inhibited lps-induced inos and cox-2 protein expressions, also, the nitrite relative concentration percentage ranged between 11.3% to 107.5% for the steroid glycosides isolated from d. septemloba rhizomes. similar to the total ethanol extract of d. membranacea presented a potent inhibitory activity, with an ic 50 value of 23.6 µg/ml. its most potent metabolite included diosgenin-3-o-alpha-lrhamnosyl (1-->2)-beta-d-glucopyranoside that showed a powerful inhibitory activity with ic 50 value as low as 3.5 µg/ml (74) . the study of the mechanism of anti-inflammatory activity of the ethanolic extract from the bark of d. batatas decne showed that it inhibited both no and pge2 production with an ic 50 of 87-71 µg/ml respectively. it was also suggested that the extract act suppressing dna-binding activity and reporter gene activity as well as translocation of the nf-jb p65 subunit. it also down-regulated ijb kinase (ikk), thus inhibiting lps-induced both phosphorylation and the degradation of ijba. in addition, it also inhibited the lps-induced activation of erk1/2 (55) . a current study of hwang et al. 2019 reported the anti-inflammatory effect of aerial bulblets of dioscorea japonica thunb extract through inhibition of nf-κb and mapk signaling pathway in raw 264.7. antiallergic activity d. membranacea ethanolic extract and compounds showed antiallergic activities by inhibiting beta-hexosaminidase release as a marker of degranulation in rbl-2h3 cells, with an ic 50 value of 37.5 µg/ml. from this extract, dioscorealide b showed the highest activity with an ic 50 value of 5.7 µg/ml (77) . du et al. 2017 reported how several compounds were isolated from the rhizomes of d. zingiberensis i.e. (3r,5r)-3,5-dihydroxy-1-(3,4-dihydroxyphenyl) -7-(4-hydroxyphenyl) heptane at a concentration of 0.5 mm showed anti-pancreatitis activities on sodium taurocholate -induced pancreatic acinar necrosis with an inhibitory effect of 19.0%. moreover, the same authors marked that this compound's protective effects were mainly dependent on atp inhibition and excessive ros production and thus saving the cells from mitochondrial dysfunction (39) . another study reported that the steroidal saponins obtained from the rhizomes of d. zingiberensis showed anti-arthritic activities on the lps stimulated 264.7 macrophage cells through induction of both p65 and iκbα protein expressions (98) . the methanolic extract of tubers of d. pentaphylla l. showed antibacterial activity against s. pyogenes, s. mutans, v. cholera, s. typhi, and s. flexneri. the best activity was: 19.00 mm and 16.00 mm inhibition zone diameter observed (at 50 μg/disc and 200 μg/ disc) against s. pyogenes using disc diffusion, agar well diffusion assays, respectively. the mic values were 100 μg/ml for the extract against v. cholera, s. typhi and s. flexneri while it was 50 μg/ml against s. pyogenes and s. mutans. similarly, rajendra et al. reported the antibacterial activities of 5% and 10% methanolic extract of d. deltoidea wall ex griseb rhizomes against s. aureus and e. coli, but this concentration does not show activity against s. typhi and p. aeruginosa. it could be concluded that, in general, dioscorea species do not exhibit a potent antibacterial activity (86) . the study of cho et al. 2013 studied dioscin, isolated from wild yam d. nipponica root bark, for its potential as an antifungal agent via the propidium iodide assay and calceinleakage measurement, in addition to, its ability to disrupt the plasma membrane potential, using 3,3′-dipropylthiadicarbocyanine iodide and bis-(1,3-dibarbituric acid)-trimethine oxanol. as reported by cho et al. 2013 the results showed that dioscin exerts a considerable antifungal activity, where, the dye-stained cells showed a significant increase in fluorescent intensity after treatment with dioscin, demonstrating that dioscin possess an effect on the membrane potential. the auhors concluded that dioscin could act by disrupting the fungal membrane structure resulting in cell death (99) . globally a large number of dioscorea plants have investigated in-vivo. data on dioscorea species potency on the animal model are summarized in table 1 . the anti-constipation activity dioscorea opposita thunb. yam tuber is rich in starch, which has always been ignored and discarded during the isolation of bioactive compounds. huang et al., 2016 studied the anti-constipation effect of native yam starch (ns), dual enzyme-treated starch (des), and cross-linked carboxymethyl starch (ccs) in constipation model induced by loperamide in km mice. the modified starches (des and ccs) could benefit the bowel function by increasing stool frequency and defecation moisture as they have the good water-binding capacity and swelling ability, the modified starches could increase the water-holding capacity of stools to remain soft. des and ccs groups had improved small intestinal propulsion through the production of more short chain fatty acids (scfas) and decrease ph by increasing acetic acid and butyric acid concentration in the feces. the study done on mice with high-fat diet showed that both native and modified starch from yam had anti-hyperlipidemic activity through a significant decrease of both cholesterol and triglycerides and the liver index. the reduction of superoxide dismutase (sod) and increase malondialdehyde (mda) was observed in hyperlipidemic mice while they were both improved in all starches-treated mice proving their antioxidant effect (table 1 ) (100). four types of resistant starch (rs) including native (rs2), retrograded (rs3), crosslinked with sodium trimetaphosphate/sodium tripolyphosphate (rs4) and complexed with palmitic acid (rs5) resistant starches from winged yam (d. alata l.) showed the gastroprotective activity of in ethanol-induced gastric ulcer in mice. the increase in gastric emptying rate may help in reducing the contact time of ethanol and gastric mucosa and helps to prevent the gastric tissue damage. therefore, an increase in gastric emptying induced by rs is a possible mechanism of anti-gastric ulcer. moreover, rs3 and rs5 groups generated more short chain fatty acids which are produced through fermentation of the starch by the colon microflora. the fatty acids have many physiological and biological functions, such as increasing the immunity of the stomach. etoh-ulcer can result from the imbalance between the generated reactive oxygen species (ros) and the natural antioxidant power. thus, the free radical scavenging is one of the mechanisms for inhibition of gastric ulcer (101). mao et al. 2018 reported the decrease of sod activity in ethanol-treated rats with an increase in mda levels in gastric mucosa indicating severe oxidation reaction in ethanol-induced gastric ulcer mice. rs3 (6.4 g/kg) and rs4 (6.4 g/kg) exhibited the best antioxidant effect. therefore, the inhibition of oxidative stress by the four types of rs is a possible mechanism that may assist in decreasing the gastric mucosal damage. generally, the high dose groups of rs3 and rs5 (6.4 g/kg) showed the best activity (50) . a current study also reported the protective effects of dioscorea batatas flesh and peel extracts against an ethanol-induced gastric ulcer in mice (57) . li et al. 2010 reported the significant antithrombotic and anticoagulation effect of the total steroidal saponin extract (tsse) from the rhizomes of d. zingiberensis c.h. wright on inferior vena cava ligation thrombosis rat model and pulmonary thrombosis mice model. oral administration of the extract significantly inhibited adenosine diphosphateinduced platelet aggregation (pag) in-vivo, which was more effective than xue-sai-tong, a commercial product of triterpenoid saponins from panax ginseng having antithrombosis activity in china. in addition, tsse inhibited the thrombosis in a dose-dependent manner (more than 50% inhibition rate) which was more powerful than the standard xue-saitong at 64.7, and 129.4 mg/kg doses of tss, and markedly reduced thrombus weight. tsse also exhibited in a dose-dependent manner prolongation of thromboplastin time (aptt) (which is an intrinsic clotting index), prothrombin time (pt) (that evaluates the extrinsic clotting pathway), and thrombin time (tt) (which is a test of fibrin formation, the addition of thrombin directly induces that). the more pronounced effect of tsse on pt and tt than aptt indicates that it inhibits the extrinsic pathway of coagulation and fibrin formation, and cut down the thrombotic risk (102) . the ethanolic extract of the tubers of d. japonica thunb. var. pseudojaponica (eedj) showed in-vivo anti-inflammatory activity by significantly inhibiting the development of carrageenan-induced paw edema in mice after the 4th and 5 th hour of the treatment at 1.0 g/kg. it also decreased the levels of tnf-α, which is a mediator of carrageenan-induced inflammatory response and induced the release of kinins and leukotrienes. carrageenaninduced paw edema causes oxidative stress with the release of peroxidation products, including malondialdehyde (mda) and nitric oxide. mda increased levels of oxygen free radicals, which attack polyunsaturated fatty acids in cell membranes and causing lipid peroxidation. eedj was able to reduce nitric oxide which was excessively released after administration of carrageenan and also could reduce the levels of mda through increasing the enzymatic antioxidant defense systems such as catalase (cat), superoxide dismutase (sod) and glutathione peroxidase (gpx) in the paw oedema which are the natural protectors against lipid peroxidation (73) . the anti-arthritic effect of the total saponin extract from the rhizomes of d. zingiberensis c.h. wright (tsrdz) in freund's complete adjuvant (fca) induced arthritis in rats (98) . in this study, a significant increase in arthritis scores and ankle perimeter were observed after 4 days from intraplantar administration of fca, which reaches its maximum on day 16. administration of tsrdz at 100 and 200 mg/kg produced substantial dose suppressive significant effect in the treated groups. tsrdz at 100 and 200 mg/kg also caused a marked reduction of fca-induced elevated indexes of the thymus and spleen. the high dose of tsrdz caused only mild synovial infiltration with few inflammatory cells and no obvious damage in cartilage bone erosion, which appeared in histopathological study. the efficient regulation of tsrdz on the inflammatory cytokines at 200 mg/ kg, was almost equivalent to that caused by the standard methotrexate. moreover, the mitigation of tsrdz on the prostaglandins (pge2), at 200 mg/kg, was almost equivalent to that of standard methotrexate. furthermore, administration of tsrdz at 200 mg/kg resulted in attenuating the production of mda and no, while elevating the sod activity compared with the control group (98) . the total 80% ethanol extract of the tubercles of d. trifida l.f. as well as its three subfractions, dichloromethane, butanol, and aqueous fractions showed anti-inflammatory activity in food allergy induced by ovalbumin in balb/c mice (94) . ova-sensitized balb/c mice received the ova solution to develop local signs of inflammation characterized by eosinophil infiltration, edema, an increase in a number of mast cells in the intestinal submucosa, mucus secretion, an increase in serum anti-ova igg1 and ige, and weight loss because of hyper-catabolism caused by the production of inflammatory cytokines. the crude extract of d. trifida tested at the doses of 100 and 300 mg/kg/day corresponding to 36.6 and 109.8 mg/kg/day of allantoin, and 3.0 and 9.0 mg of diosgenin correlated substances/kg/ day, respectively did not affect the body weight, but they inhibited ige production compared to untreated groups. the inhibition tended to be more significant in aqueous fraction and butanol fraction, which contains allantoin, than dichloromethane, which contains diosgenin. the ethanolic extract and its fractions exerted a reduction in several mastocytes and edema, which was also probably due to the presence of diosgenin and allantoin (94) . diosgenin was also reported to reduce the production of anti-ova ige and the inflammatory infiltrate in allergic balb/c mice intestines, resulting in a reduction in edema (103) . balb/c mice having food allergy showed an increase in mucus production by the caliciform cells in the small intestine (104) . an important allergic response that is induced by interleukins il-4 and il-3 is hypersecretion of mucus. mucus has a protective effect on the intestinal cells by limiting the antigen absorption. the presence of eosinophil peroxidase (epo) indirectly reflects the infiltration of eosinophils into intestinal mucosa, which was reduced by administration of the ethanolic extract of d. trifida. mollica et al. also showed that d. trifida extract and fractions reduced all of the inflammatory parameters associated with food allergies in ova-sensitised animals and this activity is correlated with the presence of allantoin and diosgenin correlated substances (94) . the animals treated with ethanolic extract showed a reduction in mucus production, and this result was also observed for the other fractions. previous studies showed that diosgenin and allantoin activities reduce mucus production in animals allergic to ova (103) . the crude extracts of d. membranaceae and d. birmania, which contain high amounts of sapogenins, inhibited the production of nitric oxide (no) most probably through inhibiting the nitric oxide synthetase (inos) and tnf-α, with consequent reduction of intestinal edema (74, 77) . a study by olayemi and ajaiyeoba also associated the antiedematogenic activity of crude extract from d. esculenta to the presence of sapogenins (105) . in-vivo studies have demonstrated that oral administration of allantoin attenuates the production of ige, il-4, and il-5, has an anti-inflammatory effect and promotes healing (68) . the methanol extract of the rhizomes of d. deltoidea wall.ex griseb. at 200 mg/ kg inhibited the rat hind paw carrageenaninduced edema. the maximum percentage of inhibition was achieved at 3 h after the intraperitoneal administration of the extract (106) . in a study made by yang et al., the chloroform soluble extract of the rhizomes of d. opposita (csdo) showed cognitive enhancing effects on spatial memory and learning function of mice against scopolamineinduced amnesic deficits via morris water maze and passive avoidance tests. in morris water maze test, both acute treatment by csdo (200 mg/kg body weight, p.o.) and prolongedtreatment (10 days' daily administration of 50 mg/kg body weight, p.o.) exhibited significant shorter escape latencies in daily first trial than the scopolamine-administered group during a 4-consecutive-day training period, which suggested that csdo improves the impaired reference memory (long-term memory) induced by scopolamine. especially, the acute treatment group showed a marked decrease in escape latencies in each daily trial compared with the prolonged treatment group. in this study, csdo also showed significant enhancing effects on spatial memory retention in the probe trial. similar results were obtained in the passive avoidance test. csdo treatment significantly increased step-through latencies of scopolamine-induced amnesic mice compared to control untreated group. the results of the two animal behavioral tests demonstrated that csdo improves spatial learning and memory function against scopolamine-induced amnesia (82) . the study of jeon et al., 2014 investigated the neuroprotective effect of herbal mixture from euphoria longana, houttuynia cordata, and dioscorea japonica, by testing the hypothesis that administration of herbs reverses memory deficits and promotes the protein expression of brain-derived neurotrophic factor (bdnf) in the mouse brain: these herbs demonstrated an inductive effect on bdnf, cyclic-amp response element-binding protein (creb) and phospho-creb (pcreb) (107) . the study of the antinociceptive effect of the methanol extract of the bulbs of dioscorea bulbifera l. var sativa (medb) was performed by nguelefack et al. (2010) the effects of medb persisted for 5 days after two administrations in cfa-induced hypernociception at 250 and 500 mg/ kg. medb significantly inhibited acute lipopolysaccharides (lps)-induced pain at 500 mg/kg but did not affect thermal hypernociception and capsaicin-induced spontaneous nociception. the antinociceptive effects of medb in prostaglandin-e2 (pge2) model was antagonized by either nitro-l-arginine methyl ester (l-name) or glibenclamide. this indicated that the plant exerted its antinociceptive effect through partial activation of the no-cgmp-atpsensitive potassium channels pathway (62) . the intraperitoneal administration of n-butanol extract of the d. nipponica rhizomes at 50 mg/kg regulates the blood cholesterol, triglyceride, hdl and ldl than the chloroform (50 mg/kg) and water extracts (50 mg/kg) (80) . wang et al. 2012 reported the potential activity of trillin, a steroidal saponin isolated from d. nipponica rhizomes, as antihyperlipidemic and anti-oxidative agent invivo. the intraperitoneal administration of trillin (0.5 mg/kg dissolved in 0.5% dmso) enhanced the blood circulation and could increase the bleeding time for more than 50% and coagulation in the rats fed on a high-fat diet in which the blood viscosity would be changed resulting in shorter blood coagulation time. the improvement effect of trillin in restoring the blood coagulation abnormality in high-fat diet fed rat was due to its ability to reduce the levels of blood cholesterol, triglyceride and two critical lipoproteins (hdl and ldl). the level of lipid oxidation was increased in high-fat diet rats, causing oxidative stress. trillin exerted an anti-oxidative effect through lowering lipid peroxidation, via the enhancement of superoxide dismutase (sod) level in blood. thus, the combination of trillin and lovastatin in treating hyperlipidemia could represent a more effective therapy (wang et al. 2012 ). the authors of this research marked how these findings greatly support the significant role of d. nipponica in protecting the cardiovascular system in-vivo against hyperlipidemia (80). tang (87); the authors reported how high serum levels of lactate dehydrogenase (ldh), aspartate aminotransferase (ast) and creatine kinase (ck) indicated cell membrane damage and were thus elevated in the iso model group. intra-gastric injection of total saponins of the three species could restore the activities of myocardial injury marker enzymes to the same extent. oxidative stress plays an essential role in the pathogenesis of mi injury. sod, catalase (cat), gpx, and total antioxidant capacity (t-aoc) levels were reduced significantly in the iso model group with the increase in mda. the activities of these enzymes were normalized by intragastric injection of the total saponins of the three species. the study revealed that the anti-mi mechanism of dioscorea saponins is related not only to the enzymatic antioxidant, such as gpx and cat but also to nonenzymatic antioxidants and its effect on myocardial histology (87) . the current studies reported the beneficial effects of purple yam (dioscorea alata l.) resistant starch on hyperlipidemia in high-fat-fed hamsters (108, 109) . the total saponins from rhizoma dioscoreae nipponicae could effectively reverse potassium oxonate-induced alterations in renal mouse urate transporter 1, glucose transporter 9, organic anion transporter 1, and organic anion transporter 3mrna and protein levels, resulting in enhancement of renal urate excretion in mice (79) . moreover, the authors concluded that the total saponins from rhizoma dioscoreae nipponicae had a uricosuric effect on the regulation of renal organic ion transporters in hyperuricemic animals. in a further paper, the same authors here was the potent uricosuric effect of tdn on hyperuricemic rats by decreasing the expressions of renal rurat1 while increasing the expressions of renal roat1 and roat3 (78) . a double-blind, placebo-controlled, crossover study was conducted to evaluate the effects of a wild yam cream in 23 healthy women suffering from troublesome symptoms of the menopause. every female was treated with the active cream and a placebo for 3 months randomly. after 3 months treatment with topical wild yam extract in women suffering from menopausal symptoms it was found that the cream is free from side-effects, and has little effect on menopausal symptoms (110) . dioscorea species make a significant contribution both as root crops and vegetables to the diets around the world. despite their importance as a food source, dioscorea plant parts are quite useful in the treatment of various ailments and disorders due to the presence of several bioactive compounds such as diosgenin. however, several reported ethnomedicinal potential need to be validated and detailed investigations on dioscorea pharmacological properties and phytochemical composition should be carried out to standardize the formulations used. indeed, some pharmacological properties such as hypolipidemic, hypoglycaemic, antioxidant, anti-inflammatory, antimicrobial, antiproliferative, androgenic, estrogenic, and contraceptive have been reported. however, most of the active constituents have not been characterized. thus, authentication of all the secondary metabolites (alkaloids, saponin, flavonoids, tannins, and phenols) from dioscorea should be performed thoughtfully to standardize and validate its quality and biological potentials. moreover, investigations are warranted to address the poor conversion of the preclinical results to clinical efficacity. therefore, an attempt should be made to understand their mechanism of action, pharmacokinetics/pharmacodynamics, and bioavailability and to conduct clinical trials. overall, these findings suggested that dioscorea should not be ignored and should rather be considered as a good alternative source of active molecules that can prevent or alleviate both functional and infectious disease burden, representing a current big challenge in developing countries. the knowledge gaps in this review such as insufficient data on a clinical trial to support preclinical results will help the further initiative to turn dioscorea into drug and stimulate activity to promote their production and utilization as not only valuable components of a well-balanced diet but also for disease prevention. dioscorea spp. (a wild edible tuber): a study on its ethnopharmacological potential and traditional use by the local people of similipal biosphere reserve volatile compounds in intermittent frying by gas chromatography and nuclear magnetic resonance volatile components of the rhizomes of dioscorea japonica dioscorea nipponica makino: a systematic review on its ethnobotany, phytochemical and pharmacological profiles diosgenin: recent highlights on pharmacology and analytical methodology isolation and identification of novel estrogenic compounds in yam tuber (dioscorea alata cv clerodane and 19-norclerodane diterpenoids 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from dioscorea pyrifolia tubers anti-inflammatory steroids from the rhizomes of dioscorea septemloba thunb bioactive constituents from the rhizomes of dioscorea septemloba thunb a new phenanthropyran and a new biphenanthrene from the rhizomes of dioscorea septemloba and their antioxidant activities protodioscin, isolated from the rhizome of dioscorea tokoro collected in northern japan is the major antiproliferative compound to hl-60 leukemic cells cholestane steroid glycosides from the rhizomes of dioscorea villosa (wild yam) diarylheptanoids from dioscorea villosa (wild yam) application of high-speed countercurrent chromatography-evaporative light scattering detection for the separation of seven steroidal saponins from dioscorea villosa lipidated steroid saponins from dioscorea villosa (wild yam) an overview on its traditional use, phytochemistry, pharmacology, clinical applications, quality control, and toxicity anthelmintic activity of steroidal saponins from dioscorea zingiberensis 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peel extracts against ethanol-induced gastric ulcer in mice dioscorea phytocompounds enhance murine splenocyte proliferation ex-vivo and improve regeneration of bone marrow cells in-vivo immunomodulatory effects of phytocompounds characterized by in-vivo transgenic human gm-csf promoter activity in skin tissues dioscorea bulbifera induced apoptosis through inhibition of erk 1/2 and activation of jnk signaling pathways in hct116 human colorectal carcinoma cells phytochemistry and therapeutic potential of medicinal plant: dioscorea bulbifera antinociceptive activities of the methanol extract of the bulbs of dioscorea bulbifera l. var sativa in mice is dependent of no-cgmp-atp-sensitive-k+ channel activation the cytotoxicity of protoneodioscin (nsc-698789), a furostanol saponin from the rhizomes of dioscorea collettii var. hypoglauca, against human cancer cells invitro the cytotoxicity of methyl protoneogracillin (nsc-698793) and gracillin (nsc-698787), two steroidal saponins from the 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dioscorea zingiberensis ch wright in freund's complete adjuvant induced arthritis in rats the antifungal activity and membranedisruptive action of dioscin extracted from dioscorea nipponica preparation, physicochemical characterization and biological activities of two modified starches from yam novel role of zn (ii)-curcumin in enhancing cell proliferation and adjusting proinflammatory cytokine-mediated oxidative damage of ethanolinduced acute gastric ulcers anti-thrombotic activity and chemical characterization of steroidal saponins from dioscorea zingiberensis ch wright diosgenin attenuates allergen-induced intestinal inflammation and ige production in a murine model of food allergy a model of chronic ige-mediated food allergy in ovalbumin-sensitized mice anti-inflammatory studies of yam (dioscorea esculenta) extract on wistar rats anti-inflammatory and antimicrobial property of dioscorea deltoidea l from nepal alteration in brain-derived neurotrophic factor (bdnf) after treatment of mice with herbal mixture containing euphoria longana, houttuynia cordata and dioscorea japonica the beneficial effects of purple yam (dioscorea alata l.) resistant starch on hyperlipidemia in high-fat-fed hamsters the beneficial effects of purple yam (dioscorea alata l.) resistant starch on hyperlipidemia in high-fatfed hamsters effects of wild yam extract on menopausal symptoms, lipids and sex hormones in healthy menopausal women key: cord-353815-w35spqqt authors: huan, yuchen; kong, qing; mou, haijin; yi, huaxi title: antimicrobial peptides: classification, design, application and research progress in multiple fields date: 2020-10-16 journal: front microbiol doi: 10.3389/fmicb.2020.582779 sha: doc_id: 353815 cord_uid: w35spqqt antimicrobial peptides (amps) are a class of small peptides that widely exist in nature and they are an important part of the innate immune system of different organisms. amps have a wide range of inhibitory effects against bacteria, fungi, parasites and viruses. the emergence of antibiotic-resistant microorganisms and the increasing of concerns about the use of antibiotics resulted in the development of amps, which have a good application prospect in medicine, food, animal husbandry, agriculture and aquaculture. this review introduces the progress of research on amps comprehensively and systematically, including their classification, mechanism of action, design methods, environmental factors affecting their activity, application status, prospects in various fields and problems to be solved. the research progress on antivirus peptides, especially anti-coronavirus (covid-19) peptides, has been introduced given the covid-19 pandemic worldwide in 2020. alexander fleming discovered lysozyme in 1922, and this discovery marked the birth of modern innate immunity. since then, antibiotics and antimicrobial peptides (amps) have been discovered. a total of 3,240 amps have been reported in the antimicrobial peptide database (apd3 1 ) updated on august 24, 2020. different types of amps have the following commonalities: their number of amino acid residues is between 10 and 60 (average: 33.26), and almost all amps are cationic (average net charge: 3.32). however, several anionic amps also exist, and they have several acidic amino acids like aspartic acid and glutamic acid (malkoski et al., 2001; schittek et al., 2001; lai et al., 2007) . the anti-microbial resistance of microorganisms is becoming increasingly serious with the abuse of antibiotics in medicine, agriculture and animal husbandry, especially in developing countries. research from kenya has detected substantial amounts of antibiotic residues in edible meat (ayukekbong et al., 2017) . the prevalence of vancomycin-resistant enterococcus (vre) and methicillin-resistant staphylococcus aureus (mrsa) is increasing in clinical medicine, so the countermeasures are urgently needed to address these bacterial infections. however, from the 1 http://aps.unmc.edu/ap/ perspective of pharmaceutical companies, the development of new antibiotic drugs results in low profit. thus, replacing antibiotics has become a consideration in the pharmaceutical, agricultural, animal husbandry, and food industries. research on amps is continuously developing and considerable amounts of data on amps have been stored in amp databases. however, the mechanism of amps remains incompletely understood, and further work needs to be performed to determine the relationship between different physicochemical properties to obtain low-cost and highly safe amps with remarkable antimicrobial effects and the specificity and a high capacity for synergies of amps should also be further developed (lazzaro et al., 2020) . the diversity of natural amps causes difficulty in their classification. amps are classified based on (1) source, (2) activity, (3) structural characteristics, and (4) amino acid-rich species (figure 1 ). the sources of amps can be divided into mammals (human host defense peptides account for a large proportion), amphibians, microorganisms, and insects according to statistical data in apd3. the amps found in oceans have also attracted widespread attention. mammalian antimicrobial peptides are found in human, sheep, cattle, and other vertebrates. cathelicidins and defensins are the main families of amps. defensins can be divided into α-, β-, and θ-defensins depending on the position of disulfide bonds (reddy et al., 2004) . human host defense peptides (hdps) can protect human from microbial infections but show different expressions in every stage of human growth. for example, cathelicidin ll-37, a famous amp derived from the human body, is usually detected in the skin of newborn infants, whereas human betadefensin 2 (hbd-2) is often expressed in the elderly instead of the young (gschwandtner et al., 2014) . hdps can be identified in many parts of the body such as skin, eyes, ears, mouth, respiratory tract, lung, intestine, and urethra. besides, amps in human breast milk also play an important role in breastfeeding because it can decrease the morbidity and mortality of breastfeeding infants (field, 2005) . what's interesting is that casein201 (peptide derived from β-casein 201-220 aa), identified in colostrum, shows different levels in preterm human colostrum and term human colostrums . dairy is an important source of amps, which are generated through milk enzymatic hydrolysis. several amps have been identified from α-lactalbumin, β-lactoglobulin, lactoferrin, and casein fractions, and the most famous peptide obtained is lactoferricin b (lfcinb) (sibel akalın, 2014) . furthermore, whether the amps derived from dairy products can be used for dairy preservation is also an interesting subject to develop. in addition to antimicrobial activity, hdps, such as cathelicidins and defensins, also affect immune regulation, apoptosis, and wound healing (wang, 2014) . antimicrobial peptides from amphibians play an important role in the protection of amphibians from the pathogens that have induced the global amphibian population decline (rollins-smith, 2009 ). frogs are the main source of amphibian amps and the most famous amp from frogs is magainin; the skin secretions of frogs from genera xenopus, silurana, hymenochirus, and pseudhymenochirus under the pipidae family are rich in amps (conlon and mechkarska, 2014) . furthermore, cancrin, which has an amino acid sequence of gsaqpykqlhkvvnwdpyg, has been reported as the first amp from the sea amphibian rana cancrivora (lu et al., 2008) . this marks a broader source of amps of amphibians. figure 1 | classification of antimicrobial peptides. (semreen et al., 2018) . myticusin-beta is an immune-related amp of mytilus coruscus and a promising alternative to antibiotics (oh et al., 2020) . moreover, ge33, known as pardaxin, is a marine amp and the ge33-based vaccine has shown the ability to enhance antitumor immunity in mice (huang et al., 2013) . the activity of amps can be divided into 18 categories according to the statistics of the adp3 database. these categories can be summarized as antibacterial, antiviral, antifungal, antiparasitic, anti-human immunodeficiency virus (hiv), and anti-tumor peptides (figure 2 ). antibacterial peptides account for a large part of amps and have a broad inhibitory effect on common pathogenic bacteria, such as vre, acinetobacter baumannii, and mrsa in clinical medicine and s. aureus, listeria monocytogenes, e. coli in food and salmonella, vibrio parahaemolyticus in aquatic products. many natural and synthetic amps like nisin, cecropins and defensins have shown good inhibition activity to gram-positive bacteria and gram-negative bacteria. in recent research, amps p5 (yirkirrffkklkkilkk-nh 2 ) and p9 (syerkinrhfktlkknlkkk-nh 2 ), which are designed based on aristicluthys nobilia interferon-i, inhibit mrsa and show a low cytotoxicity . antifungal peptides are a subclass of amps that address fungal infections with enhanced drug resistance. many afps have shown excellent anti-fungal activities against common pathogenic fungi, such as aspergillus and candida albicans in clinical medicine, yeast, filamentous fungi (e.g., aspergillus flavus), mold in food and agriculture. except for brevinin, ranatuerin, cecropins, many synthetic peptides also show good antifungal activity. for example, aurh1, derived from aurein 1.2, can effectively treat c. albicans infection, which has a lethal rate up to 40% (madanchi et al., 2020) . aflatoxin, which is a carcinogen produced by a. flavus, is harmful to the human body. many afps can inhibit the growth of a. flavus. for example, an afp with a sequence of fpshtgmsvppp can inhibit the growth of a. flavus md3. a total of 37 antifungal peptides isolated from lactobacillus plantarum te10 and their mixture can reduce a. flavus spore formation in fresh maize seeds (muhialdin et al., 2020) . moreover, two chemically synthesized radish amps show a good inhibitory effect against different yeast species, such as zygosaccharomyces bailii and zygosaccharomyces rouxii (shwaiki et al., 2020) . viruses cause serious harm to human life and huge economic losses to the animal husbandry. the covid-19, which is the recent outbreak, has caused great loss of lives and properties. furthermore, foot-and-mouth disease virus, avian influenza virus (aiv), and hiv are long-term threats to human life. so, it is extremely urgent to solve these problems, and antiviral peptides provide new ways. antiviral peptides show a strong killing effect on viruses mainly by (1) inhibiting virus attachment and virus cell membrane fusion, (2) destroying the virus envelope, or (3) inhibiting virus replication (jung et al., 2019 ) (shown in figure 3 ). a recent report has shown that amp epi-1 mediates the inactivation of virus particles and has good inhibitory activity against foot-and-mouth disease virus . moreover, infectious bronchitis virus (ibv) is the pathogen of infectious bronchitis and the inoculation of swine intestinal amp (siamp)-ibv mixed solution remarkably reduced the mortality of chicken embryos compared with the ibv infection group, showing the good inhibitory activity of siamp on ibv (sun et al., 2010) . anti-hiv peptides are a subclass of anti-viral peptides. the most important examples of these peptides include defensins (including αand β-defensins, which have different mechanisms), ll-37, gramicidin d, caerin 1, maximin 3, magainin 2, dermaseptin-s1, dermaseptin-s4, siamycin-i, siamycin-ii, and rp 71955 (madanchi et al., 2020) and antiviral peptide fuzeon tm (enfuvirtide) has been commercialized as an anti-hiv drug (ashkenazi et al., 2011) . due to the global spread of the covid-19 (figure 4a ), the antiviral peptides against the coronavirus will be discussed in more detail. coronaviruses (covs) belong to the family coronaviridae; they are enveloped viruses with a positive-sense single-stranded rna genome and have a helical symmetry (franks and galvin, 2014) . covs, including severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) (mustafa et al., 2018) , and the recent outbreak of covid-19 have caused serious threats to human life and property. covs can cause lifethreatening respiratory diseases and the viral particle is formed by spike glycoprotein (s), the envelope (e), the membrane (m), and the nucleocapsid (n) (vilas boas et al., 2019) . it should be noted that their infectivity requires viral spike (s) protein. fusion inhibitor peptides combine with the s protein to interfere with its folding and prevent infection. besides, the s2 domain of the sars-cov s protein contains heptad repeat hr1 and hr2 sequences. peptide hr2 (hr2: sltqinttlldltyemlslqqvvkalnesyidlkel) and its lipid-binding peptide is highly similar or even identical to the near-membrane portion of s protein ferredoxin, which interferes with refolding into post-fusion fusion-catalyzing domains (fds) (du et al., 2009; park and gallagher, 2017) . according to recent research, the lipopeptide ek1c4, derived from ek1 (sldqinvtfldleyemkkleeaikkleesyidlkel), is the most effective fusion inhibitor against covid-19 s proteinmediated membrane fusion . homology modeling and protein-peptide docking showed that temporin has potential therapeutic applications against mers-cov (marimuthu et al., 2019) . two amps from the non-structural protein nsp10 of sars-cov, k12, and k29, can inhibit sars-cov replication (ke et al., 2012) . furthermore, rhesus thetadefensin 1 (rtd-1) treated animals have a marked reduction in mortality in the presence of sars-cov while the peptide alone shows airway inflammation and the one possible mechanism of action for rtd-1 is immunomodulatory (wohlford-lenane et al., 2009) . in general, amps against coronavirus can be roughly classified as i) peptides derived from hr1, hr2 and rbd subunits of the spike protein, ii) peptides derived from other amps, iii) peptides derived from non-structural protein (mustafa et al., 2018) . furthermore, molecular docking analysis indicated that peptides were employed to disrupt the interaction between covid-19 and ace2 (angiotensin-converting enzyme 2) to inhibit covid-19 entrance in cells ( figure 4b ) (souza et al., 2020) . finally, it should be noted that this therapy lacks clinical trials and the main method of animal experiments is an intranasal administration. this reminds us that nasal drug delivery (ndd) is a potential therapy for amps as anti-coronavirus drugs. besides, the antiviral database avpdb 2 includes numerous antiviral peptides. parasitic protozoa can cause diseases in human and animals through a variety of routes, including animal-to-person or person-to-person contact, water, soil, and food (chalmers et al., 2020) . and with the increase in parasite drug resistance, the need for new treatments has increased. antiparasitic peptides show their killing effect on parasites which cause diseases such as malaria and leishmaniasis (mangoni et al., 2005; rhaiem and houimel, 2016) and amps like cathelicidin, temporins-shd show high inhibition activity against parasites (abbassi et al., 2013) . in recent research, epi-1, a marine synthetic amp, can remarkably inhibit trichomonas vaginalis by destroying its membrane (neshani et al., 2019) . the peptide jellein derived from bee royal jelly which has introduced above and 4-amino acid amp kdel (lysine, aspartic acid, glutamic acid, and leucine) has shown a significant effect on the leishmania parasite (cao et al., 2019; zahedifard et al., 2020) . however, it should be noted that their mechanisms are not the same. cyanobacterial peptides differ from higher-eukaryote amps because their antiparasitic action depends on specific protein targets. thus, these target parasites can be distinguished accurately even though they belong to the same family or genus (rivas and rojas, 2019) . the acps show anticancer mechanisms by (1) recruiting immune cells (such as dendritic cells) to kill tumor cells, (2) inducing the necrosis or apoptosis of cancer cells, (3) inhibiting angiogenesis to eliminate tumor nutrition and prevent metastasis, and (4) activating certain regulatory functional proteins to interfere with the gene transcription and translation of tumor cells (wu d. et al., 2014; ma et al., 2020) . tritrpticin and its analogs induce considerable toxicity toward jurkat cells in vitro, whereas indolicidin and puroindoline a can also act as acps (arias et al., 2020) . it should be noted that both net charge and hydrophobicity play important roles in optimizing the anticancer activity of acps and they can constrain and influence each other. thus, achieving a balance between net charge and hydrophobicity is important for better anticancer activity. besides the peptide mentioned above, anti-inflammatory, anti-diabetic peptides, spermicidal peptides etc. have been noticed, but they are not the same as antimicrobial peptides. simply put, anti-inflammatory peptides decrease the release of inflammatory mediators and inflammatory cytokines (nitric oxide, interleukin-6, and interleukin-1β) and some of them also inhibit inflammatory signals like nf-κb, mapk, and jak-stat pathways (meram and wu, 2017; gao et al., 2020) . anti-diabetic peptides play their function by modulating the g proteincoupled receptor kinase (grk 2/3) or activating glucagonlike peptide-1 (glp-1), glucagon receptors (marya et al., 2018; graham et al., 2020) . however, it is not accurate to classify these types of peptides as amps and bioactive peptides may be more convincing. proline is a typical non-polar amino acid. pramps behave differently from other amps, that is, they enter bacterial cytoplasm by the inner membrane transporter sbma instead of killing bacteria through membrane destruction (mattiuzzo et al., 2007) . once in the cytoplasm, pramps target ribosomes and block the binding of aminoacyl-trna to peptidyltransferase center or trap decoding release factors on the ribosome during the termination of translation to interfere with protein synthesis (seefeldt et al., 2015) . for instance, tur1a, which is an orthologous amp of bovine pramp bac7 discovered from tursiops truncatus, interferes with the transition from the initial phase to the extension phase of protein synthesis by binding to ribosomes. in addition, different pramps lack a high sequence similarity but have short motifs containing repeating proline and arginine (arg) residues (e.g., -ppxr-in bac5 and -prpxin bac7) (mardirossian et al., 2018 (mardirossian et al., , 2019 . although pramps mainly kill gram-positive bacteria, ppr-amp1, a proline-rich amp identified from crab (scylla paramamosain), exhibits antimicrobial activity against gram-positive and gram-negative bacteria (imjongjirak et al., 2017) . besides, pieces of research have shown that pramps have immunostimulation activity (li w. et al., 2016) . tryptophan (trp), as a non-polar amino acid, has a remarkable effect on the interface region of the lipid bilayer, whereas arg, as a basic amino acid, confers peptide charge and hydrogen bond interactions, which are essential properties to combine with the bacterial membrane's abundant anionic component. and it seems that trp residues play the role of natural aromatic activators of arg-rich amps by ion-pair-π interactions (walrant et al., 2020) , thereby promoting enhanced peptide-membrane interactions (chan et al., 2006) . in addition to indolicidin and triptrpticin which both are famous amps that rich in arg and trp residues. octa 2 (rrwwrwwr) is also a typical trp-and arg-rich amp that inhibits gram-negative e. coli and pseudomonas aeruginosa and gram-positive s. aureus. and short trp-and arg-rich amps designed based on bovine and murine lactoferricin have also shown strong inhibitory action against bacteria (strøm et al., 2002; bacalum et al., 2017) . histidine is a common basic amino acid, and histidine-rich amps show good membrane permeation activity. hv2 is a histidinerich amp designed based on rr(xh) 2 xdpgx(yh) 2 rr-nh 2 (where x represents i, w, v, and f). this peptide increases the permeability of bacterial cell membranes to cause cell membrane rupture and death. in addition, hv2 inhibits bacterial movement in a concentration-dependent manner and shows a strong anti-inflammatory effect by inhibiting the production of tumor necrosis factor α (tnf-α) ). an amp designed based on octa 2 has shown good therapeutic potential by replacing its arg residues with histidine (bacalum et al., 2017) . furthermore, l4h4, which is designed based on the linear cationic amphiphilic peptide magainin, also shows good antibacterial activity and cell penetration properties by inserting four histidine sequences in leucine and alanine (lointier et al., 2020) . the r group of glycine is generally classified as a non-polar amino acid in biology. glycine-rich amps, such as attacins and diptericins, widely exist in nature (lee et al., 2001; kwon et al., 2008) . these peptides contain 14% to 22% glycine residues, which have an important effect on the tertiary structure of the peptide chain. a glycine-rich amp derived from salmonid cathelicidins activates phagocyte-mediated microbicidal mechanisms, which differ from the mechanism of conventional amps (d'este et al., 2016) . furthermore, the glycine-rich central-symmetrical gg3 is an ideal commercial drug candidate against clinical gramnegative bacteria . antimicrobial peptides can be divided into four categories based on their structures including linear α-helical peptides, β-sheet peptides, linear extension structure, and both α-helix and β-sheet peptides ( figure 5 ) (lei et al., 2019) . moreover, progressively cyclic peptides and amps with more complex topologies (including lasso peptides and thioether bridged structures) are reported (koehbach and craik, 2019) . the membrane-targeting mechanisms of amps can be described through models, including the pole and carpet models and the pole model can be further divided into the toroidal pore and barrel-stave models (figure 6 ). the toroidal pore model is also known as the wormhole model. in this model, amps vertically embedded in the cell membrane accumulate and then bend to form a ring hole with a diameter of 1-2 nm (matsuzaki et al., 1995 (matsuzaki et al., , 1996 . the typical examples of this model are magainin 2, lacticin q, and arenicin. furthermore, cationic peptides, including tc19, tc84, and bp2, compromise the membrane barrier by creating fluid domains (omardien et al., 2018) . antimicrobial peptides aggregate with each other, penetrate the bilayer of the cell membrane in the form of multimers, and form channels that result in the cytoplasmic outflow. in severe cases, amps can induce cell membrane collapse and lead to cell death (lohner and prossnigg, 2009 ). for instance, alamethicin performs its pore-forming activity by using this model. besides, hairpin amp protegrin-1 can form stable octameric β-barrels and tetrameric arcs (half barrels) in implicit and explicit membranes by simulations (lipkin and lazaridis, 2015) . antimicrobial peptides are arranged parallel to the cell membrane. their hydrophilic end faces the solution, and their hydrophobic end faces the phospholipid bilayer. amps will cover the membrane surface that similar to a carpet and destroy the cell membrane in a 'detergent'-like manner (oren and shai, 1998) . however, this pore-forming mechanism requires a certain concentration threshold and the required concentration of amps is high. human cathelicidin ll-37 exhibits its activity through this mechanism, and amps with β-sheet structure also play a role in this model (shenkarev et al., 2011; corrêa et al., 2019) . polarized light-attenuated total reflection fourier transform infrared spectroscopy (atr-ftir) was used to study the effect of amp cecropin p1 on the bacterial cell membrane and found that it was an applied flat on the surface of the pathogen's cell membrane to destabilize and eventually destroy the cell membrane . membrane targeting mechanisms (the cell membrane composition differences of bacteria and fungi shown in figure 7) can be further refined to address the large differences in the lipid composition of the cell membranes of bacteria, fungi, and mammals. the main lipids in cell membranes include glycerophospholipids (gpls), lysolipids, sphingolipids, and sterols. phosphatidylethanolamine (pe), phosphatidylglycerol (pg), and cardiolipin (cl) are the most common anionic lipids in bacteria, whereas phosphatidylcholine (pc), phosphatidylinositol (pi), pe, and phosphatidic acid (pa) are the main gpls in fungal cell membranes (ejsing et al., 2009; singh and prasad, 2011; li et al., 2017) . furthermore, fungal cell membranes are more anionic than mammalian cell membranes and have higher pc content. meanwhile, ergosterol is the sterol found in the plasma membrane of lower eukaryotes, such as fungi, whereas that of animals contains cholesterol (faruck et al., 2016) . many amps take advantage of differences in membrane components to exert their effects. antimicrobial peptides are promising to be anti-biofilm agents but it should be noticed that they are different from the cell penetrating peptides (cpps) which typically comprise 5-30 amino acids and can translocate across the cell membrane. cpps could be categorized according to physicochemical properties into three classes: cationic, amphipathic, and hydrophobic, but anti-biofilm peptides have stricter requirements for these physicochemical properties. anti-biofilm peptides target the biofilms by different mechanisms including (1) degradation of signals within biofilms; (2) permeabilize within cytoplasmic membrane/eps; (3) modulating eps production etc. and then can address chronic multi-resistant bacterial infections (pletzer et al., 2016; ribeiro et al., 2016; guidotti et al., 2017; derakhshankhah and jafari, 2018; rajput and kumar, 2018) . for instance, saap-148, synthesized based on ll-37, showed activity to prevent biofilm formation by s. aureus and a. baumannii (crunkhorn, 2018) . the way of amps entering cells is direct penetration or endocytosis. after entering the cytoplasm, amps will identify and act on the target. depending on the target, amps can be divided into the following categories. antimicrobial peptides affect transcription, translation, and assembly into functional peptides through molecular chaperone folding by interfering with related enzymes and effector molecules. for example, bac7 1-35 targets ribosomes to inhibit protein translation (mardirossian et al., 2014) , whereas tur1a inhibits protein synthesis in e. coli and thermus thermophilus by inhibiting the transition from the initial phase to the extension phase. however, the differences between tur1a and bac7 also lead to various ways of binding to ribosomes and interacting with the ribosomal peptide exit tunnel (mardirossian et al., 2018) . but some amps' have different targets. for instance, genome-wide transcription shows that the amp dm3 can affect many important intracellular pathways of protein biosynthesis . chaperones are key proteins for correctly folding and assembling newly synthesized proteins and make them have stereoisomerism, which makes amps have cell selectivity and can prevent cytotoxicity. according to a previous review: both pyrhocoricin and drosocin can prevent dnak from refolding misfolded proteins by inducing a permanent closure of the dnak peptide-binding cavity (kragol et al., 2001; le et al., 2017; wrońska and boguś, 2020) . antimicrobial peptides can affect key enzymes or induce the degradation of nucleic acid molecules to inhibit nucleic acid biosynthesis. indolicidin, a c-terminal-amidated cationic trprich amp with 13 amino acids, specifically targets the abasic site of dna to crosslink single-or double-stranded dna and it can also inhibit dna topoisomerase i (subbalakshmi and sitaram, 1998) . tfp (tissue factor pathway inhibitor)1-1tc24, which is an amp from tongues, enters the cytoplasm of target cells after the rupture of cell membrane and then degrades dna and rna (he et al., 2017) . many amps can inhibit various metabolic activities by inhibiting protease activity. for example, histatin 5 has a strong inhibitory effect on the proteases secreted by the host and bacteria. amps enap-2 and indolicidin inhibit microbial serine proteases, elastase, and chymotrypsin (le et al., 2017) . cathelicidin-bf is a peptide isolated from the venom of bungarus fasciatus, it can effectively inhibit thrombin-induced platelet aggregation and further block protease-activated receptor 4 (shu et al., 2019) . antimicrobial peptides inhibit cell division by inhibiting dna replication and dna damage response (sos response), blocking the cell cycle or causing the failure of chromosome separation (lutkenhaus, 1990) . for instance, app (glaraltrllrqltrqltra), which is an amp with 20 amino acid residues, can efficiently kill c. albicans because of its cell-penetrating efficiency, strong dna-binding affinity, and ability to induce s-phase arrest in intracellular environment . mciz, which has 40 amino acid residues, is an effective inhibitor of bacterial cell division, z-ring formation, and localization (cruz et al., 2020) . moreover, it has been reported that several afps have damaging effects on the organelles of fungi. for example, histintin 5 can interact with mitochondria, causing the production of ros, and inducing cell death (helmerhorst et al., 2001) . in addition to intracellular targets, differences in cell wall composition, such as lipopolysaccharide (lps), lipid a and mannoproteins, are potential targets for amps. specifically, gram-positive and gram-negative bacteria are classified based on their bacterial cell wall structure. gram-positive bacteria have a layer of cross-linked peptidoglycan, whereas gram-negative bacteria have an additional outer membrane with an inner leaflet containing only phosphatidic acid and an outer leaflet made of lps. lps has numerous negatively charged phosphate groups, which combine with a salt bridge with a divalent cation (e.g., ca 2+ and mg 2+ ) to form an electrostatic network (nikaido, 2003) . this electrostatic zone is the main barrier against hydrophobic antibiotics and causes the low permeability of gram-negative bacteria. the main components of the fungal cell wall are mannoprotein, β-glucans and chitin (polymers of 1,4-β-n-acetylglucosamine) and the mutations in the relevant genes of the lps pathway and phospholipid trafficking provide resistance to the amps (cabib, 2009; spohn et al., 2019) . mannoproteins in fungal cell walls include a variety of proteins, including structural proteins, cell adhesion proteins (floccrin and lectin) and enzymes involved in cell wall synthesis and remodeling (hydrolytic enzymes and transglycosylase). these proteins differ from human cell membrane proteins and are potential targets of afps (rautenbach et al., 2016) . furthermore, teichoic acid and lipoteichoic acid in the cell wall are also potential targets of amps and these theories could support the design of amps with low cytotoxicity. antimicrobial peptides have good application prospects. however, amps have the following problems. (1) amps damage the cell membrane of eukaryotes and cause hemolytic side effects; (2) rising production costs and technical problems limit their manufacture; (3) their stability is limited at certain ph; (4) amps have reduced activity under the presence of iron and certain serum; (5) amps are easily hydrolyzed by proteases. therefore, the ideal amp should meet the following characteristics: (i) high antimicrobial activity; (ii) low toxicity to mammalian membranes; (iii) high protease and environment stability; (iv) low serum binding capacity and (v) ease of access and low cost production . therefore, designing amps to achieve the desired effect has attracted increasing attention. the rational design of antibacterial peptides should focus on the following five aspects: chain length, secondary structure, net charge, hydrophobicity, and amphiphilicity and these have been mentioned in many studies and this review will focus more on several specific methods of antimicrobial peptide design. site-directed mutation refers to the redesign of natural antimicrobial peptides by adding, deleting or replacing one, or several amino acid residues (torres et al., 2019) . the de novo design of peptides attaches importance to the design of amphiphilic amps (guha et al., 2019) . for example, gala is a well-known de novo-designed amp. amphipathic α-helical peptide gala is created by placing protonatable glutamic acid residues in most positions with the spacing of i to i + 4 (goormaghtigh et al., 1991) . the repeated sequence (xxyy)n, where x 1 and x 2 are hydrophobic amino acids, y 1 and y 2 are cationic amino acids, and n is the number of repeat units, is designed based on the hydrophobicity cycle that mimics natural α-helical amps and successfully designs broadspectrum α-helical amps. sequences (lkkl) 3 and (wkkw) 2.5 have the highest selectivity (khara et al., 2017) . moreover, l l k m w 2 model peptides are also de novo-designed peptides. amphipathic helical properties were conferred by using leucines and lysines, and two tryptophan residues were positioned at the amphipathic interface between the hydrophilic ending side and the hydrophobic starting side. among the model peptides, l 4 k 5 w 2 has good anti-mrsa activity (lee et al., 2011) . sequence templates can be obtained by comparing a large number of structurally homologous fragments of natural amps (such as hdps) and extracting conservative patterns based on the type of residue (such as charged, polar, hydrophobic, etc.) (zelezetsky and tossi, 2006) . based on the modification, the parameters, such as helix formation tendency, cationic, amphiphilicity and overall hydrophobicity, can be systematically changed. for instance, cecropin, magainin, protegrin, and lactoferrin have all been used as amp templates (fjell et al., 2012) . peptides can form nanostructures, such as micelles, vesicles, nanotubes, nanoparticle nanobelt, and nanofibre nanotube, and can increase or impart antibacterial activity to amps during the self-assembly of peptides. for example, kld-12 (kld) is a self-assembling peptide with 12 amino acid residues that can adopt nanostructures and are known for their tissue engineering properties. the addition of arg residues in kld shows no remarkable change in its β-sheet secondary structure and the self-assembly characteristics of the forming nanostructures (tripathi et al., 2015) . dimer structure can also be used to enhance the antimicrobial activity of amps and reduce toxicity, but membrane-destabilizing effects are reduced after dimer formation (malekkhaiat häffner and malmsten, 2018). various chemical modifications of amps, including residue phosphorylation, the addition of d-amino acids or unnatural amino acids (homoarginine), cyclization, halogenation, acetylation, and peptidomimetics, have been used to improve the stability of peptides against proteases. given that the enzyme is stereospecific, the incorporation of unnatural d-amino acids into the amp sequence can reverse the stereochemistry and prevent protease degradation (zhong et al., 2020) . the so-called peptidomimetics, whose main elements mimic the structure of peptides, are usually produced by modifications, such as chain extension or heteroatom incorporation of existing peptides (patch and barron, 2002) . ornine, which is an unnatural residue with a positive charge and has a high resistance to protease activity, is also used in non-chemical modification. replacing trp residues with family residues, such as β-dihydrophenylalanine, can stabilize secondary structures and improve antibacterial properties (maurya et al., 2013) . halogenation is highly related to the activity, specificity, and stability of amps. in the latest report, halogen is introduced into jelleine-i which is a short peptide isolated from the royal jelly of honeybees (apis mellifera) by replacing phenylalanine with a halogenated phenylalanine analog, increasing the antibacterial activity in vitro and anti-biofilm activity. in addition, the proteolytic stability of jelleine-1 is increased by 10-100 times by halogenation (jia et al., 2019) . the halogenated peptidomimetic α,α-disubstituted β-amino amides are also promising bacteriostatic drugs that have inhibitory effects on more than 30 multi-resistant clinical isolates of gram-positive and gram-negative bacteria (paulsen et al., 2019) . halogenation is also related to the specificity of amps. the o-fluorine substitution in phenylalanine residues maintains the activity of temporin l on e. coli but leads to the loss of activity on s. aureus and p. aeruginosa (setty et al., 2017) . three modes of cyclisation, including cyclisation via disulfide bonds, head-to-tail cyclisation and internal bonding between side chains, have been found in natural amps. the synthesis of disulfide bonds often complicates the development of synthetic peptides. the circularisation of the main chain of arenicin-1 molecule resulted in increased activity against drug-resistant clinical isolates but caused no substantial effect on cytotoxicity (orlov et al., 2019) . the hdps tachyplesins i, ii, and iii and their cyclic analogs cti, ctii, and ctiii, respectively, have similar structures and activities and can resist bacterial and cancer cells. the cyclisation of the backbone reduces the hemolytic activity and improves the stability of the peptides whilst maintaining effective anticancer and antibacterial activities (vernen et al., 2019) . capping refers to the addition of specific motifs or modifications, such as amidation at the c-terminus and acetylation at the n-terminus, rendering amps with more natural peptide characteristics. post-translational modifications play an important role in the function of amps and are the most commonly used in peptide design. the c-terminal rana box (consisting of a c-terminal cyclic heptapeptide with a conservative disulfide bond) and amide group are important c-terminal capping methods. for example, the c-terminal amide group of maximin h5 can enhance antibacterial efficacy without increasing lytic ability (dennison et al., 2015) . the n-terminal lipidated analog c 4 vg 16 krkp shows enhanced antibacterial activity against various gram-negative bacteria. the functions of n-terminal lipidation include (i) increasing lps neutralization, (ii) increasing stability to proteases and peptidases, and (iii) reducing cytotoxicity (datta et al., 2016) . furthermore, hydrophobic end labeling is a commonly used method to increase the activity of antimicrobial peptides. acyl lipid peptides have a linear or cyclic structure in which one or more hydrocarbon tails are connected to the n-terminus of a short oligopeptide (chu-kung et al., 2010) . lipopeptides have covalently attached hydrophobic moieties, such as sterols or fatty acids. aromatic amino acid terminal labeling is also the main hydrophobic terminal labeling method. tryptophan (w) and phenylalanine (f) are the commonly used aromatic amino acids. their large and polarisable residues have an affinity for the interface, and the w/f tag is also sensitive to the differences between ergosterol and cholesterol and can prevent self-assembly. this condition results in low aggregation numbers and high critical aggregation concentrations (schmidtchen et al., 2014) . peptide conjugation has been the goal of most research in recent years to produce active and stable amps with high selectivity. different side chains or amp fragments can be used aside from the repetition of the same amino acid motifs. for example, conjugating fatty acids with a length of 8-12 carbon atoms to the 4th or 7th side chain of the d-amino acids of ano-d 4,7 improves antibacterial selectivity and anti-biofilm activity. in addition, the new peptide exhibits high stability against trypsin, serum, salt, and different ph environments (zhong et al., 2020) . the conjugation of different amps can also be performed. for example, the hybrid peptide (pa2-gnu7) constructed by the addition of pa2 to gnu7 has a high activity and specificity to p. aeruginosa . smamps include a broad family of molecular entities based on the structure and function of amps. however, their backbones are not entirely based on α-amino acids, including β-amino acid oligomers, arylamide oligomers, and phenylene ethynylenes (michael henderson and lee, 2013) . for instance, smamp10, which is a potential drug for intravenous treatment, causes no drug resistance and has a strong inhibitory effect on mrsa and vancomycin-resistant enterococcus faecium (tew et al., 2006) . peptoids are peptide isomers, in which the side chain is bonded to the main chain nitrogen instead of α-carbon or poly-nsubstituted glycine in which the side chain is connected to amide nitrogen instead of the α-carbon on the main chain (andreev et al., 2018) . for example, the cationic peptide sa4 (iowagolfolfo-nh2) and its poly-n-substituted glycine homolog spo (ninonwnangnonlnfnonlnfno-nh 2 ) inhibit the planktonic and biofilm formation of a. baumannii strains, which are susceptible to multi-drug resistance (sharma et al., 2019) . motifs with specific functions have been reported increasingly. these motifs can be repeated units for combining into new antimicrobial peptides, or specific amino acid combination units appearing at the end (such as capping) of or even in the peptide chain. this motif includes two tripeptide structures, including gly-gly-his or val-ile-his, which are added at the end of the peptide chain. atcun-containing amps in the presence of hydrogen peroxide and ascorbic acid combine with cu 2+ to induce the valence of copper ions between +2 and +3 oxidation states and form an atcun-cu (ii) complex, generating ros by fenton-like reactions. extracellular polymeric substances (eps) are important for biofilms and can enhance the resistance of cells to antibacterial agents (flemming, 2016) . atcun-amps have been used to degrade environmental dna, which is one of the major components of eps. several related practical applications have been reported. for example, the biological activity against carbapenem-resistant enterobacteriaceae is increased by adding this motif to the n-terminus of an alpha-helical amp (such as cm15). besides, the cu-atcun derivative of ov-3 containing a c-terminal ggc sequence showed high levels of membrane permeation and lipid peroxidation. the concept of catalytic metal drugs has attracted widespread attention although the concept is still in its infancy because of the role of metal ions (alexander et al., 2017; agbale et al., 2019) . rana box: rana box is a heptapeptide motif (cglxglc) from the nigrocin family. rana box consists of two cysteine residues that are separated by four or five other residues on the side and can form a cyclic disulfide bond. rana box peptide has shown structural analogies with polymyxin (colistin), and the primary structure of the rana box motif is important in determining bacteriostatic activity (kozić et al., 2015) . the deletion of the 'rana box' motif will cause the amp antibacterial effect to disappear, but replacing the natural 'rana box' sequence of amps with amidated phenylalanine can expand its efficacy against antibiotic-resistant microorganisms, including mrsa and p. aeruginosa, and reduce cytotoxicity. this phenomenon also shows that the effect of the motif on amps needs to be determined based on the specific situation and is not completely beneficial (bao et al., 2018) . the lps binding motif (g-wkrkrf-g) can produce a broad spectrum of antibacterial activity when introduced into the c-terminus of temporin-1 ta and temporin-1 tb (close isoforms of temporin) (mohanram and bhattacharjya, 2016) . antifungal peptides have a conserved gxc(x 3−9 ) c γ-core motif (residues 5-14, gkcykkdnic; d-isomer) at its n-terminus, which is a cation part of the ring. this conserved motif interferes with the integrity of the plasma membrane of the cell (yount and yeaman, 2004) . conserved γ-core motifs are directly involved in protein-membrane interactions and strongly contribute to membrane binding (utesch et al., 2018) . if replace d-phe1-pro2 sequence in peptide chain with d-phe-2-abz turn motif (2-abz is an abbreviation of 2-aminobenzoic acid d-amino acid) in amp tyrc a, and nuclear magnetic resonance shows that this change retains the β-hairpin structure. unlike the traditional β-turn motif, the d-phe-2-abz motif can be used as a tool for β-hairpin libraries. the hydrophobic peptide can be formed into the nucleated β-hairpin formation by adding the d-phe-2-abz motif. moreover, the inclusion of this part in two designed cationic amphiphilic peptides can produce broadspectrum antibacterial activity and low hemolysis rate (cameron et al., 2017; cameron et al., 2018) . the ngr motif is composed of asn-gly-arg, and amps with this structure have strong cytotoxicity ( table 1 ). the data indicate that the new amps containing ngr may bind to cd13+ or αvβ3+ tumor cells by binding to cd13 or αvβ3, respectively, to exert anti-tumor activity, especially on cd13+ tumor cells . the central gxxxg motif can induce strong self-assembly and have been already used in the design of amps (brosig and langosch, 1998; krauson et al., 2012) . bovine lactoferrin b is an amp composed of 25 amino acid residues and has antibacterial, antifungal, and antiparasitic activities. the multivalent molecules lfcinb (20-25) 2 and lfcinb (20-25) 4 contain the lfcinb (20-25) motif (rrwqwr) and show inhibition activity against e. coli, p. aeruginosa, and s. aureus. chimeric peptide chimera 3 containing two motifs, namely, the rrwqwr of lfcinb (20-25) and the rllr of bfii computer design includes simple statistical modeling, structureactivity relationships study (abdel monaim et al., 2018) , neural networks (müller et al., 2018) , deep learning (veltri et al., 2018) , word embedding (hamid and friedberg, 2018) and machine learning. for example, a machine learning method by matlab is proposed based on the concept of scoring the contribution of each amino acid's antibacterial activity (wu x. et al., 2014) . the genetic algorithm was used to design the amphiphilic α-helical peptide guavalin 2, which has an uncommon amino acid composition (three tyrosine and three glutamine residues) and interestingly causes membrane hyperpolarization, which is a different mechanism from those of other amps (porto et al., 2018) . two research methods have been developed based on the research background of quantitative structure-activity relationships: prediction method based on amp therapeutic index and the identification of novel potential amps from the expressed sequence tag database based on the principles of the highly conserved signal peptide subclasses related to amps (juretić et al., 2011) . in this way, a variety of amp variants can be obtained. if combined with high-throughput screening, it can effectively obtain the desired amp. for instance, some new amps are designed by the combinatorial peptide library of melittin and show higher activity and lower cytotoxicity (krauson et al., 2015) . cations, such as na + and mg 2+ , may affect amp activity . however, the different valences of metal ions have varied effects on amps. for example, divalent cations show stronger antagonism to bacteria than monovalent cations with thanatin and s-thanatin, which are insect amps (wu et al., 2008) . in the presence of nacl, the signal response during the association phase remarkably decreased in single-cycle and multi-cycle kinetic experiments, resulting in a decreased association rate. this occurrence may be caused by the shielding effect of nacl between the cationic peptide and the zwitterionic membrane. another possible reason is that na + can bind to the phospholipid bilayer, where the ions interact with the phosphate and the carbonyl oxygen of lipid head groups (sabapathy et al., 2020) . the reduced activity of synthetic peptide [rllr] 5 under high salt concentration is possibly caused by the destruction of its α-helix structure. table 2 shows that several amps, including histatin, myxinidin, and hepcidin, contain atcun motifs (amino terminal copper and nickel with xxh sequence). iron is the most abundant metal ion in human saliva, but the combination with this metal ion results in the loss of the α-helix of histatin 5 and greatly reduces its antifungal activity (puri et al., 2015) . however, the coordination of copper (ii) and nickel (ii) ions can induce the formation of ros, which is essential for bactericidal activity (jeżowska-bojczuk and stokowa-sołtys, 2018). anionic amps have a large number of negatively charged aspartic and glutamic acid residues (lakshmaiah narayana and chen, 2015) . they require zinc as a functional cofactor and the zinc complex shows stronger antibacterial activity (jiang et al., 2014) . several of these amps use metal ions to form cationic salt bridges with the negatively charged components of the microbial membrane to penetrate the membrane. anionic amps may attach to ribosomes or inhibit ribonuclease activity when in the cytoplasm (jeżowska-bojczuk and stokowa-sołtys, 2018). metal ions also affect the self-assembly of peptides. these ions can recognize specific amino acids, such as lysine and glutamic acid, and may form salt bridges between peptide molecules to induce peptide self-assembly. for example, zn 2+ can stabilize the aggregation of peptides on the cell membrane, which results in the enhanced antibacterial effect of dcd-1l in the presence of zn 2+ (tian et al., 2015) . ph many amps are stable and retain their antimicrobial activity in a wide ph range. amps have enhanced activity at low ph because of their basic properties. this condition is related to the protonation of histidine at acidic ph, which promotes electrostatic interactions with anionic surfaces, including lps and the anions of phospholipids, and subsequently enhances antibacterial properties. the effect of ph on the antibacterial activity of amps varies. for example, thanatin's activity at neutral ph is slightly higher than that under acidic conditions. by contrast, the activity of xylan on e. coli, listeria, and c. albicans is remarkably higher at ph 5.5 than at ph 7.4 (holdbrook et al., 2018) . the inactivation of the histidinecontaining amp c18g-his under low ph conditions involves ph-dependent changes in the state of the aggregates in the solution, because the aggregates, which are sensitive to ph and lipid composition, may be affected by binding and conformation. peptides can also enhance bacterial membrane permeability at low ph (hitchner et al., 2019) . thrombin-derived c-terminal peptides (tcps) will also change the mode of cd14 (a protein that is abundant in human plasma) from anti-inflammatory mode to bacterial elimination mode from ph 7.4 to ph 5.5 (holdbrook et al., 2018) . a dimer (e.g., p-113) can be created to provide amps with resistance to a higher ph range. the sensitivity of this ph-sensitive amp can be used to achieve a certain targeting effect in practical applications. in addition, charge interaction is one of the most important factors in peptide self-assembly. ph affects the charge state of amino acid and substituent functional groups. therefore, adjusting the ph is the most common method for controlling peptide assembly and disassembly (tian et al., 2015) . proteases have a strong destructive effect on amps. for instance, ll-37, which has the strongest inhibitory effect on chlamydial infection, is inhibited by the protease chlamydial protease-like activity factor (cpaf) secreted by chlamydia (tang et al., 2015) . studies have been focused on the design of amp carriers to solve this problem (lewies et al., 2017; nordström et al., 2019) . the presence of chitosan-silica solid support of kr-12 peptide can protect it be hydrolyzed by α-trypsin, and the degree of protection is increased by 38% compared with the free kr-12 (diosa et al., 2020) . however, several enzymes, such as protease 65, esterase 66 and phosphatase 67, cut the blocking group of the peptide and trigger the self-assembly of the peptide, which positively affects amps (tian et al., 2015) . antimicrobial peptides can regulate pro-inflammatory reactions, recruit cells, stimulate the proliferation of cells, promote wound healing, modify gene expression and kill cancer cells to participate in the immune regulation of human skin, respiratory infections, and inflammatory diseases (de la fuente-núñez et al., 2017) . for example, α-defensins hnp-1, hnp-2, and hnp-3 showed effective antibacterial activity against adenovirus, human papilloma virus, herpes virus, influenza virus and cytomegalovirus. pulmonary diseases, such as idiopathic pulmonary fibrosis, alveolar proteinosis, and acute respiratory distress syndrome, show elevated levels of amps (guaní-guerra et al., 2010) . likewise, amps secreted by the paneth cells in the mammalian gut are important to shape the gut microbiota (bevins and salzman, 2011) . the application of amps in medicine, such as dental, surgical infection, wound healing and ophthalmology is developing now. but there are only three amps that have been approved by fda including gramicidin, daptomycin, and colistin. dental caries, endodontic infections, candidiasis, and periodontal disease are common diseases in the human oral cavity. dental caries is a prevalent oral disease and some acidogenic bacteria like streptococcus sp. are the main cariesassociated pathogens (izadi et al., 2020) . several amps have good application potential. for instance, peptide zxr-2 (fkiggfikklwrslla) has shown potent activities against pathogenic bacteria of dental caries, streptococcus mutans, streptococcus sobrinus, and porphyromonas gingivalis and peptide pac-113 (clinical trial identifier: nct00659971) that has been sold over the counter in taiwan for treating oral candidiasis (chen l. et al., 2017) . in surgical infection and wound healing: surgical infection occurs after surgery, burns, accidental injury, skin disease, and chronic wound infections have a serious hazard to human life (thapa et al., 2020) . several amps have shown the therapeutic potential of these diseases. for example, amp pxl150 shows pronounced efficacy as an anti-infective agent in burn wounds in mice and amp d2a21 has been in the third phase of clinical trials for treating burn wound infections (björn et al., 2015) . in ophthalmology: human eyes are prone to be infected by several organisms including bacteria and fungi in which s. aureus, streptococcus pneumoniae, p. aeruginosa, aspergillus spp., and c. albicans are the most relevant pathogens (silva et al., 2013) . although amps such as lactoferricin b, protegrin-1 exhibited antimicrobial activity against these pathogenic bacteria, their application in the field of ophthalmology is only at the theoretical stage. with the popularity of contact lenses and the increase in cases of related eye infections, antimicrobial peptides have shown good application prospects in ophthalmology (khan and lee, 2020) . additional methods need to be performed for the application of amps as drugs in medicine. the main strategies include (1) constructing precursors to reduce cytotoxicity and improve protease stability, (2) using amps in combination with existing antibacterial agents, (3) inducing the correct expression of amps with appropriate drugs and using engineering probiotics as vectors to express amps. for example, in the field of wound repair, different formulation strategies, such as loading amps in nanoparticles, hydrogels, creams, gels, ointments, or glutinous rice paper capsules, have been developed to effectively deliver amps to the wound (borro et al., 2020; thapa et al., 2020) . in recent research, the sponges developed from modified starch and hs-peg-sh are covalently immobilized with amp showed effective antibacterial activity (yang et al., 2019) . more technical means, including pheromone-labeled amps, local environment-triggered amps (enzyme precursor drug release system, ph-activated amps, etc.), have been developed to improve the targeting mechanism of amps. furthermore, nanotubes, quantum dots, graphene, and metal nanoparticles have been proposed to be a potential method to enhance drug delivery of amps (magana et al., 2020) . hybrid peptides have also been used to build targeting peptides. for example, pa2, which is a p. aeruginosa-targeting peptide, was combined with gnu7 (a broad-spectrum amp) to construct a hybrid peptide (pa2-gnu7) that targets oprf protein and has good bactericidal activity and specificity . furthermore, some antibiotics, for instance, daptomycin (a lipopeptide), lugdunin which is a 21-membered cyclic peptide consists of 6 amino acid residues plus a thiazolidine moiety and telavancin (a glycopeptide) have been widely used for the clinic (durand et al., 2019; lampejo, 2020) . although they are antibiotics, they have provided broader ideas for the design of amps. food preservatives have potential harm to the human body. therefore, natural preservatives are being advocated by more people. amps have a good inhibitory effect on common bacteria and fungi in food, and many amps are resistant to acids, alkalis, and high temperatures are easily hydrolyzed by proteases in the human body. thus, amps are a promising alternative to preservatives. nisin is a bacteriocin produced by l. lactis subspecies. lactic acid bacteria have been widely used as food preservatives. nisin is categorized as generally recognized as safe (gras) by the us food and drug administration (fda) and is used as a food preservative in other countries (khan and oh, 2016) . however, only nisin and polylysine are currently approved by the fda as food additives (santos et al., 2018) . pedocin pa-1, a bacteriocin consisting of 44 amino acids produced by a diplococcus, is also used as a food preservative and is sold on the market under the trade name alta 2431. pedocin pa-1 is used as a food additive to inhibit the growth of l. monocytogenes, which can cause meat deterioration (settanni and corsetti, 2008) . enterocin as-48 is an amp used to preserve cider, fruit and vegetable juices, and enterocin ccm4231 is used to preserve soy milk (rai et al., 2016; santos et al., 2018) . encapsulating bacteriocins into liposomes is a new method used to overcome the problems of amps in food applications (such as proteolytic degradation or interaction with food ingredients) (da silva malheiros et al., 2010) . moreover, active packaging by adding amps is a novel packaging method that has great potential in the food industry. for instance, ε-poly-l-lysine is used in conjunction with starch biofilms to show good inhibitory effects on aspergillus parasiticus (aflatoxin producer) and penicillium expansum and nisin have the potential to be dairy preservative because it is a highly surface-active molecule (luz et al., 2018) . the european union banned the use of animal growth promoters in animal feed in 2006. thus, a new antibacterial strategy is needed. many amps are the potential to be used in poultry, swine, and ruminants breeding and aquaculture because they can improve production performance (liu et al., 2008; bao et al., 2009) , immunity and promote intestinal health and some of them have a stronger inhibitory effect on bacterial inflammation if used with antibiotics cote et al., 2020) . for example, siamp has a good effect on the treatment of ibv in chicken (sun et al., 2010) . by adding swine gut intestinal antimicrobial peptides (sgamp), broilers showed higher average daily gain and feed efficiency under chronic heat stress conditions (hu et al., 2017) . frog caerin 1.1, european sea bass dicentracin and nk-lysine peptides (nklps) have good inhibitory effects on nodavirus, septicaemia haemorrhagic virus, infectious pancreatic necrosis virus and spring viremia carp virus, which are devastating to fish farming (león et al., 2020) . the amp in soybean meal fermented by b. subtilis e20 effectively inhibits v. parahaemolyticus and vibrio alginolyticus and enhances the resistance level of litopenaeus vannamei against v. parahaemolyticus when added to feeds (cheng et al., 2017) . for agriculture, the plant pathogenic infection of bacteria and fungi causes the loss of economy, for instance, aspergillus flavus infection of corn and peanuts, citrus green mold caused by penicillium digitatum, gray mold disease caused by botrytis cinerea on strawberries and geotrichum citriaurantii infection of citrus fruit all cause great harm to the growth and post-harvest of agricultural products (liu et al., 2007; liu et al., 2019) . several afps have shown prospect to control these problems. however, the practical application of antimicrobial peptides in the transportation and preservation of agricultural products is still lacking, because the use of antimicrobial peptides will greatly increase the cost in the transportation of fruits and vegetables (application examples of amps in these four fields are shown in table 3 ). antimicrobial peptides constitute a global research hotspot, but many key issues in design and application need to be solved urgently. several restrictive factors hinder the application of amps. the interaction of multidisciplinary subjects, such as biology, materials science, chemistry, bioinformatics, molecular informatics and pharmacy can further develop prospective amps. computer molecular dynamics simulation, cell membrane simulation, and more methods are being applied to study the mechanism of amps. how to further understand the correlation between amps and various targets instead of conducting one-sided experimental research might improve experimental designs to obtain stronger systemic and scientific demonstrations. on this basis, further animal experiments are required instead of simple cell-level experiments to test the effect of amps under complex physiological conditions. several complicated methods, such as the chemical method of peptidomimetics and non-natural amino acid modifications, have been applied in designing amps to solve the problem of protease hydrolysis. most methods use chemical substrates, but the cost of these methods cannot be ignored in practice. in addition, chemical synthesis and the use of engineered bacteria are currently the mainstream for such procedures. finding a better biological preparation method, reducing the cost and increasing the yield is important problems in practical application. furthermore, studying the amp expression of the organism itself and finding a better expression vector are necessary for mass production in the future as more amps in nature are discovered. further research is needed on the reported amps to solve the problem on structure-function relationship. as a branch of peptide drugs, amps need to progress with the advancement of medical science against the background of the current low success rate of 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an antimicrobial peptide, magainin 2, induced rapid flip-flop of phospholipids coupled with pore formation and peptide translocation role of the escherichia coli sbma in the antimicrobial activity of proline-rich peptides mechanism of action of novel synthetic dodecapeptides against candida albicans anti-inflammatory effects of egg yolk livetins (α, β, and γ-livetin) fraction and its enzymatic hydrolysates in lipopolysaccharideinduced raw 264.7 macrophages promising antimicrobial agents designed from natural peptide templates lollipop'-shaped helical structure of a hybrid antimicrobial peptide of temporin b-lipopolysaccharide binding motif and mapping cationic residues in antibacterial activity antifungal activity determination for the peptides generated by lactobacillus plantarum te10 against aspergillus flavus in maize seeds recurrent neural network model for constructive peptide design current treatment options and the role of peptides as potential therapeutic components for middle east respiratory syndrome (mers): a review epinecidin-1, a highly potent marine antimicrobial peptide with anticancer and immunomodulatory activities molecular basis of bacterial outer membrane permeability revisited. microbiol degradable dendritic nanogels as carriers for antimicrobial peptides myticusin-beta, antimicrobial peptide from the marine bivalve bactericidal activity of amphipathic cationic antimicrobial peptides involves altering the membrane fluidity when interacting with the phospholipid bilayer mode of action of linear amphipathic α-helical antimicrobial peptides ponericins, new antibacterial and insecticidal peptides from the venom of the ant pachycondyla goeldii redesigning arenicin-1, an antimicrobial peptide from the marine polychaeta arenicola marina, by strand rearrangement or branching, substitution of specific residues, and backbone linearization or cyclization expression systems for heterologous production of antimicrobial peptides lipidation increases antiviral activities of coronavirus fusion-inhibiting peptides mimicry of bioactive peptides via nonnatural, sequence-specific peptidomimetic oligomers antimicrobial activity of amphipathic α,α-disubstituted β-amino amide derivatives against esbl -carba producing multi-resistant bacteria; effect of halogenation, lipophilicity and cationic character use of click chemistry for obtaining an antimicrobial chimeric peptide containing the lfcinb and buforin ii minimal antimicrobial motifs anti-biofilm peptides as a new weapon in antimicrobial warfare in silico optimization of a guava antimicrobial peptide enables combinatorial exploration for peptide design iron binding modulates candidacidal properties of salivary histatin 5 antimicrobial peptides as natural bio-preservative to enhance the shelf-life of food anti-biofilm peptides: a new class of quorum quenchers and their prospective therapeutic applications antifungal peptides: to be or not to be membrane active antimicrobial peptides: premises and promises targeting leishmania major parasite with peptides derived from a combinatorial phage display library new frontiers for anti-biofilm drug development cyanobacterial peptides as a tour de force in the chemical space of antiparasitic agents the role of amphibian antimicrobial peptides in protection of amphibians from pathogens linked to global amphibian declines revisiting the interaction of melittin with phospholipid bilayers: the effects of concentration and ionic strength nisin and other antimicrobial peptides: production, mechanisms of action, and application in active food packaging dermcidin: a novel human antibiotic peptide secreted by sweat glands effect of hydrophobic modifications in antimicrobial peptides the proline-rich antimicrobial peptide onc112 inhibits translation by blocking and destabilizing the initiation complex primary structures of six antimicrobial peptides of rabbit peritoneal neutrophils recent updates of marine antimicrobial peptides application of bacteriocins in vegetable food biopreservation modulating the antimicrobial activity of temporin l through introduction of fluorinated phenylalanine nkl-24: a novel antimicrobial peptide derived from zebrafish nk-lysin that inhibits bacterial growth and enhances resistance against vibrio parahaemolyticus infection in yesso scallop cationic antimicrobial peptide and its poly-n-substituted glycine congener: antibacterial and antibiofilm potential against a. baumannii the unique antimicrobial peptide repertoire of stick insects molecular mechanism of action of β-hairpin antimicrobial peptide arenicin: oligomeric structure in dodecylphosphocholine micelles and pore formation in planar lipid bilayers antimicrobial peptide cathelicidin-bf inhibits platelet aggregation by blocking protease-activated receptor 4. intern anti-yeast activity and characterisation of synthetic radish peptides rs-afp1 and rs-afp2 against food spoilage yeast dairy-derived antimicrobial peptides: action mechanisms, pharmaceutical uses and production proposals the importance of antimicrobial peptides and their potential for therapeutic use in ophthalmology comparative lipidomics of azole sensitive and resistant clinical isolates of candida albicans reveals unexpected diversity in molecular lipid imprints a molecular docking study revealed that synthetic peptides induced conformational changes in the structure of sars-cov-2 spike glycoprotein, disrupting the interaction with human ace2 receptor integrated evolutionary analysis reveals antimicrobial peptides with limited resistance antimicrobial activity of short arginine-and tryptophan-rich peptides mechanism of antimicrobial action of indolicidin swine intestine antimicrobial peptides inhibit infectious bronchitis virus infectivity in chick embryos chlamydiasecreted protease cpaf degrades host antimicrobial peptides antimicrobial peptides from different plant sources: isolation, characterisation, and purification antimicrobial activity of an abiotic host defense peptide mimic topical antimicrobial peptide formulations for wound healing: current developments and future prospects role of peptide selfassembly in antimicrobial peptides peptide design principles for antimicrobial applications variants of self-assembling peptide, kld-12 that show both rapid fracture healing and antimicrobial properties a computational modeling approach predicts interaction of the antifungal protein afp from aspergillus giganteus with fungal membranes via its γ-core motif synergistic bactericide and antibiotic effects of dimeric, tetrameric, or palindromic peptides containing the rwqwr motif against gram-positive and gram-negative strains deep learning improves antimicrobial peptide recognition characterization of tachyplesin peptides and their cyclized analogues to improve antimicrobial and anticancer properties antiviral peptides as promising therapeutic drugs evolutionary plasticity of insect immunity ionpair-π interactions favor cell penetration of arginine/tryptophanrich cell-penetrating peptides human antimicrobial peptides and proteins high specific selectivity and membrane-active mechanism of the synthetic centrosymmetric α-helical peptides with gly-gly pairs antimicrobial peptides: promising alternatives in the post feeding antibiotic era control of green and blue mold and sour rot in citrus fruits by the cationic antimicrobial peptide paf56 rhesus theta-defensin prevents death in a mouse model of severe acute respiratory syndrome coronavirus pulmonary disease heat shock proteins (hsp 90, 70, 60, and 27) in galleria mellonella (lepidoptera) hemolymph are affected by infection with conidiobolus coronatus (entomophthorales) peptide-based cancer therapy: opportunity and challenge effects of cations and ph on antimicrobial activity of thanatin and s-thanatin against escherichia coli atcc25922 and b. subtilis atcc 21332 in vitro and in vivo activities of antimicrobial peptides developed using an amino acidbased activity prediction method inhibition of sars-cov-2 (previously 2019-ncov) infection by a highly potent pancoronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion fabricating antimicrobial peptide-immobilized starch sponges for hemorrhage control and antibacterial treatment multidimensional signatures in antimicrobial peptides secretory production of antimicrobial peptides in escherichia coli using the catalytic domain of a cellulase as fusion partner comparative study of different forms of jellein antimicrobial peptide on leishmania parasite identification and biochemical characterization of a new antibacterial and antifungal peptide derived from the insect sphodromantis viridis alpha-helical antimicrobial peptides-using a sequence template to guide structure-activity relationship studies antimicrobial activity and mechanism of the human milk-sourced peptide casein201 an antimicrobial peptide containing ngr motif has potent antitumor activity against cd13+ and cd13-tumor cells antimicrobial peptides conjugated with fatty acids on the side chain of d-amino acid promises antimicrobial potency against multidrug-resistant bacteria isolation and structure of corticostatin peptides from rabbit fetal and adult lung characterization of antimicrobial activity and mechanisms of low amphipathic peptides with different α-helical propensity we thank the national key r&d program of china (2019yfd0901705). key: cord-270892-ycc3csyh authors: rollinger, judith m.; schmidtke, michaela title: the human rhinovirus: human‐pathological impact, mechanisms of antirhinoviral agents, and strategies for their discovery date: 2010-12-13 journal: med res rev doi: 10.1002/med.20176 sha: doc_id: 270892 cord_uid: ycc3csyh as the major etiological agent of the common cold, human rhinoviruses (hrv) cause millions of lost working and school days annually. moreover, clinical studies proved an association between harmless upper respiratory tract infections and more severe diseases e.g. sinusitis, asthma, and chronic obstructive pulmonary disease. both the medicinal and socio‐economic impact of hrv infections and the lack of antiviral drugs substantiate the need for intensive antiviral research. a common structural feature of the approximately 100 hrv serotypes is the icosahedrally shaped capsid formed by 60 identical copies of viral capsid proteins vp1‐4. the capsid protects the single‐stranded, positive sense rna genome of about 7,400 bases in length. both structural as well as nonstructural proteins produced during the viral life cycle have been identified as potential targets for blocking viral replication at the step of attachment, entry, uncoating, rna and protein synthesis by synthetic or natural compounds. moreover, interferon and phytoceuticals were shown to protect host cells. most of the known inhibitors of hrv replication were discovered as a result of empirical or semi‐empirical screening in cell culture. structure–activity relationship studies are used for hit optimization and lead structure discovery. the increasing structural insight and molecular understanding of viral proteins on the one hand and the advent of innovative computer‐assisted technologies on the other hand have facilitated a rationalized access for the discovery of small chemical entities with antirhinoviral (anti‐hrv) activity. this review will (i) summarize existing structural knowledge about hrv, (ii) focus on mechanisms of anti‐hrv agents from synthetic and natural origin, and (iii) demonstrate strategies for efficient lead structure discovery. © 2009 wiley periodicals, inc. med res rev, 31, no. 1, 42–92, 2010 human rhinoviruses (hrv) are the major cause of upper respiratory tract symptoms, the socalled common colds in humans. their name reflects the primary site of infection. because hrv are nonenveloped, icosahedral viruses of small size with a diameter of about 30 nm ( pico 5 small in latin) that consist of an rna genome, they were assigned to the family picornaviridae. currently, this virus family of the order picornavirales comprises the eight genera enterovirus, hepatovirus, cardiovirus, kobuvirus, teschovirus, erbovirus, aphthovirus, and parechovirus with 22 species and a multitude of serotypes. 1 because of high similarity in genome sequence and genome organization (fig. 1) , the former genera rhinovirus and enterovirus have been combined recently, keeping the existing name enterovirus (www.picornastudygroup. com/taxa/species/species.htm). an overview on the current taxonomy of picornaviruses pathogenic for humans as well as on newly proposed species of hrv is given in table i . at present the genus enterovirus includes four approved human enterovirus (hev) species (hev-a, -b, -c, and -d) and two approved hrv species (hrv-a and -b) (www. picornastudygroup.com/taxa/species/-species.htm). since 2007, the global distribution of highly divergent hrv strains was reported. [2] [3] [4] [5] based on the results of sequence, genomic, and phylogenetic analyses, it was proposed that these strains represent a new hrv species, hrv-c. [2] [3] [4] 6 in 2009, a further proposal concerning a new potential hrv-d species was published after sequencing and analysis of all known hrv genomes. 7 the approved and newly proposed species of the genus enterovirus share z70% homology (average amino acid identity) in the precapsid protein p1 as well as in 2c and 3cd. 3, [7] [8] [9] [10] different antigenic properties provide the basis for a further division of species into serotypes (table i) . about 100 rhinovirus serotypes are currently known. according to the currently approved taxonomy, most of them (75 and hrv hanks) belong to hrv-a and 25 of them to hrv-b. 11, 12 the genome of all known hrv-a and -b serotypes as well as of several field isolates of hrv-a, -b, and -c has been sequenced completely. 3, 7, [13] [14] [15] [16] [17] [18] [19] [20] viruses classified as hrv-c could not be grown in cell culture until now. phylogenetic analyses have been performed with partial sequences, 12, 21 as well as with the whole genome. 3, 7 the most recent and comprehensive analysis of all known hrv genomes revealed that (i) hrv-a and hrv-c share a common ancestor, which is a sister group to hrv-b, (ii) hrv-c represents a third species, and (iii) a basal divergence within hrv-a of three distinct strains that led to the proposal of a fourth species hrv-d. hrv-a and -b most often induce a mild, usually self-limited upper respiratory illness in humans characterized by nasal stuffiness and discharge, sneezing, sore throat, and cough. the conventional term is common cold. the common cold is a heterogeneous group of diseases caused by numerous viruses that belong to several different families e.g. rhinoviruses, coronaviruses, enteroviruses, and adenoviruses. 22 but, hrv represent the most common etiological agent worldwide. a large number of distinct strains circulate each year. 23 moreover, in a family or even in a single specimen, multiple hrv serotypes were detected simultaneously. [24] [25] [26] by using rt-pcr and culture, it was shown that hrv induce 22-50% of upper respiratory tract infections in adults as well as children. [27] [28] [29] [30] [31] higher incidence has been described from september to november, [32] [33] [34] and from april to may. 33, 34 in some years and perhaps some geographical areas, spring was a more important time for rhinovirus transmission. 33, 34 although overall rates of respiratory illness are lower in summer, rhinoviruses are the most frequently isolated at this time of year. 34 the incidence is inversely proportional to age. 25, 35 by age 2 years, 91% of the children have antibodies against rhinoviruses. 36 in addition to common cold, hrv are also involved in acute otitis media in children. 36, 37 moreover, data supporting a causative association with more severe lower respiratory tract infections of infants, elderly persons, and immunocompromised patients have been accumulated. [38] [39] [40] [41] [42] [43] [44] [45] studies of childhood and adult asthma have shown that hrv infections can also trigger exacerbations in patients with asthma, [46] [47] [48] [49] chronic obstructive pulmonary disease, [50] [51] [52] [53] [54] [55] and cystic fibrosis. [56] [57] [58] the recently discovered novel rhinovirus genotype hrv-c was associated with community outbreaks of influenza-like, acute upper respiratory infections and severe low respiratory tract infections of infants e.g. febrile wheeze, bronchiolytis, and asthma exacerbations, which peaked in fall and winter. [2] [3] [4] [5] [6] 26, 59, 60 in addition, the presence of hrv-c in the middle ear in patients with acute otitis media was demonstrated. 37 hrv spread occurs by means of virus-contaminated respiratory secretions that contain a high virus concentration. [61] [62] [63] besides direct hand-to-hand transmission, small-and largeparticle aerosol transmission of rhinoviruses has been shown. 61, 64, 65 children are important ''vectors'' for hrv transmission to family members. 25 moreover, studies with natural hrv-infected adults provided evidence that daily activities of infected people can lead to contamination of environmental surfaces with hrv e.g. light switches, telephone dial buttons and handsets, and virus transfer to fingers of healthy individuals for infection. 63, 66 because viral contamination of the hands plays an important role in transmission of hrv from person-to-person, interruption of this step of virus transmission presents a potential target for intervention. this was experimentally proved by treatment of hands by iodine 67, 68 or salicylic and pyroglutamic acid. 69 observations from experimentally induced infections in normal adult volunteers helped to understand the pathogenesis of hrv infections. [70] [71] [72] [73] [74] [75] the 50% human infectious dose of rhinovirus is low and the infection rate between 70 and 80%. after the deposition of hrv on nasal or conjunctival mucosa, viruses are transported to the posterior nasopharynx by mucociliary action of epithelial cells. specific receptors on epithelial cells in the adenoid area are used for binding and entry. already 8-10 hr after intranasal inoculation, infectious virus can be detected. 76 virus shedding peaks on the second day after infection and decreases rapidly thereafter. 75 but, small amounts of viruses were discovered in nasal secretions for up to 3 weeks after infection. virus and/or viral rna were demonstrated in the upper 70 as well as lower respiratory tract. 45, 72, 77, 78 using in situ hybridization, arruda et al. (1995) detected viral rna in a low number of ciliated cells in nasal biopsies. in the nasopharynx, a small portion of virus-positive ciliated as well as nonciliated cells was positive for viral rna. in 2000 papadopoulos et al. provided evidence that hrv may also lytically infect human bronchial epithelial cells in cell culture as well as in experimentally infected volunteers and induce the production of interleukin-6, -8, and -16. in agreement with these results, hrv rna was detected in 24-45% of children and 10-18% of adults with pneumonia. [79] [80] [81] [82] taken together, the results of natural cold studies as well as of experimental infection in human volunteers clearly demonstrate that hrv are able to replicate in the upper as well as in the lower airways. hrv infection triggers vasodilation and increased vascular permeability in the nasal mucosa, leading to nasal obstruction and rhinorrhoea. the mechanism is still incompletely understood because no histopathological changes were observed in nasal biopsy specimens from infected persons. 75 this led to the suggestion that clinical symptoms are primarily caused by the inflammatory response of the host to the virus infection and not by the cytopathic effect (cpe) of hrv. results of immunological investigations suggest a modest correlation between the concentrations of il-6 and il-8 in nasal secretions and the severity of symptoms in upper and lower hrv-induced respiratory tract disease. 78, 83 on day 2-4 after virus challenge, il-6 and il-8 concentrations were significantly greater in nasal secretions from experimentally infected symptomatic subjects than in those from infected asymptomatic or sham-challenged subjects. il-8 has been proposed as a mediator of neutrophile infiltrations that are observed during symptomatic infections. in experimental rhinovirus infection the onset of symptoms e.g. nasal stuffiness and discharge, sneezing, and cough was observed 10-12 hr after intranasal inoculation of the virus. 76 in contrast to rhinovirus infections in adults, fever is found in 15% of children with upper respiratory tract infections. 84 other symptoms in children and adults may be hoarseness, headache, malaise, and lethargy. sometimes viral infection is accompanied by bacterial complication, leading for instance to acute otitis media in about 20% of infected children, sinusitis, and pneumonia. [79] [80] [81] 85, 86 experimental infection was also used to study the causation between rhinovirus infection and asthma as well as copd exacerbations. 78, [87] [88] [89] [90] [91] [92] [93] it was shown that hrv infection enhances airway reactivity and predisposes allergic patients to develop late asthmatic reactions. 88, 91 rhinoviral colds were associated with an increase in histamine responsiveness that was accompanied by a bronchial mucosal lymphocytic and eosinophilic infiltrate. 89 in a recent study, an increased hrv-induced clinical illness severity in asthmatic compared with normal subjects was demonstrated. 93 strong relationships were shown between virus load, lower airway virus-induced inflammation, and asthma exacerbation severity. the results of this study also indicated that augmented th2 or impaired th1 or il-10 immunity are likely important mechanisms. mallia et al. provided evidence that low dose experimental rhinovirus infection in patients with copd induces symptoms and lung function changes typical of an acute exacerbation of copd. 92 viral replication and increased pro-inflammatory cytokine response were associated with symptomatic colds, increases in lower respiratory tract symptoms and reductions in forced expiratory volume in 1s or peak expiratory flow rate. the epidemiological data and pathology of hrv infections explain their high medical and socio-economic impact. millions of children and adults are taken ill with common cold every year, need medical consultations, are unable to attend school and go to work. 94, 95 direct costs include hospitalization, medical fees, and symptomatic treatment. moreover, exacerbations are the major cause of asthma and copd morbidity, mortality, and health care costs associated with these diseases. 92, 93 to date, specific drugs that prevent or reduce rhinovirus infection are not available. common cold can be treated only symptomatically with analgesics, decongestants, antihistamines, or antitussives and antibiotics are often wrongly prescribed. 96, 97 because of the large number of circulating hrv serotypes, treatment with specific antiviral drugs is considered to be more striking than vaccination. therefore, the search for new highly active synthetic and/or natural anti-hrv compounds is absolutely essential and represents an important area of antiviral research. such an anti-hrv drug would have to be (i) with broad spectrum activity because of the high number of hrv serotypes, (ii) administered very early in infection to demonstrate a good antiviral effect because of the fast infection kinetics, (iii) very safe because of the broad application by millions of people, and (iv) directed against a highly conserved target with low risk of resistance development. due to the very high error rates and the lack of proofreading ability in rna polymerases of picornaviruses, 98 naturally drug-resistant variants may exist in virus populations or resistant viruses can emerge under treatment. as with hiv, another highly variable rna virus, the risk of resistance development and/or selection of resistant virus variants could be minimized by applying combination of drugs directed against different targets. because clinical symptoms are suggested to be primarily caused by the inflammatory response of the host to the virus infection mediated by specific cytokines, a further advantage of drug combinations could be an additional immune-suppressive activity. the knowledge of structural components, nonstructural proteins that are necessary for viral multiplication, and stages of the viral life cycle is an essential precondition for the development of measures to prevent and treat hrv infection. the structure of hrv particles is well known. infectious virions consist of an icosahedral protein shell (capsid) that surrounds and protects the genome, a single positive-stranded rna molecule of approximately 7,400 nucleotides. the organization of the enterovirus genome is shown in figure 1 . the viral genomic rna is infectious and encodes a single, long, open reading frame flanked by untranslated regions (utr) at the 5 0 and 3 0 end. a small viral protein (vpg) is covalently linked to the 5 0 end. the 3 0 end is polyadenylated like cellular messenger rnas. structural components within these utrs e.g. the cloverleaf and the internal ribosome entry site (ires) of the 5 0 utr play an important role in rna replication as well as protein synthesis. 98 the nucleotide sequence of some regions within these structures is highly conserved among enteroviruses. their blockade could significantly inhibit viral replication. the molecular structure of hrv-1a, hrv-2, hrv-3, hrv-14, and hrv-16 was determined by x-ray crystallography. [99] [100] [101] [102] [103] [104] [105] the results show that the viral capsid is composed of 60 protomers of each of the three outer structural proteins vp1, vp2, and vp3 and of vp4 in the interior. a star-shaped plateau at the fivefold axis of symmetry, surrounded by a deep depression (canyon) and another smaller depression at the threefold axis were detected. moreover, a hydrophobic pocket was found beneath the canyon floor. with exception of hrv-14 and hrv-3, this pocket is occupied by a fatty acid, the so-called pocket factor. these host cell molecules have been suggested to play an important role in the viral life cycle by providing transient stability to the capsid during its movement from one host cell to another. 102 the outer surface of virions contains neutralization antigenic as well as host cell binding sites. the latter allow the virus to attach to molecules of the host cell membrane (adsorption), the receptors, and to start their life cycle. [106] [107] [108] based on their receptor use, two groups of hrv can be distinguished. the majority of hrv serotypes, the major group uses intercellular adhesion molecule-1 (icam-1) as their receptor. 109 the 12 viruses belonging to the minor group attach to low density lipoprotein (ldl) receptor, very-ldl (vldl) receptor, and ldlr-related protein on the cells whereat multiple receptors are involved. [110] [111] [112] hrv of the major group apply the canyon as attachment site for binding to icam-1. 113 in contrast, ldl receptors of minor group viruses bind near the tip of the five-fold vertex. 114 hrv-87 has been shown to utilize a sialylated glycoprotein as a cellular receptor. 115 furthermore, a hrv-89 variant as well as wild-type hrv-54 can use heparan sulphate proteoglycans for cell attachment in addition to icam-1. 116, 117 the interaction of rhinoviruses with their receptors leads to virus concentration on the cell surface. it induces the release of the pocket factor and conformational changes in the capsid and mediates viral entry via endocytosis. [118] [119] [120] [121] whereas icam-1 binding directly causes uncoating, 122, 123 release of the rna genome from the capsid of ldl-bound minor group rhinoviruses is triggered by acidification of the endosomal, ph-dependent pathway. [124] [125] [126] this detailed knowledge of the capsid structure and function as well as of the virus-receptor interaction offers a good possibility to develop antiviral drugs that interfere with the first steps of the viral life cycle, adsorption as well as uncoating. after uncoating, rhinovirus proteins are synthesized by the translation of a single, open reading frame using cellular ribosomes. the resulting polyprotein of approximately 250 kd is cleaved by viral proteases 2a pro and 3c pro into 11 final products (4 structural and 7 nonstrutural proteins) immediately after translation. 127, 128 at first, both proteinases release themselves from the polyprotein by selfcleavage. the primary cleavage of the viral polyprotein between p1 and p2 is mediated by 2a pro . thereafter, 3cd pro is released from the p3 precursor by autocatalytic cleavage. next, 3c pro and its precursor 3cd pro process proteins of the p1 (capsid proteins), p2, and p3 (nonstructural proteins) region. interestingly, 2a pro cleaves also the eif4gi/ii component of the translation initiation factor eif4f necessary for host cell protein synthesis, [129] [130] [131] and 3c pro and/or 3cd pro the rna polymerase transcription factors tfiid, tfiic, sl-1, and ubf. 98 therefore, effective inhibition of 2a pro and 3c pro would not only inhibit virus replication but could also prevent the shutoff of cellular protein and rna synthesis. moreover, the active site of proteinases is highly conserved among enterovirus serotypes. 132 this high conservation in conjunction with their important role in virus multiplication predestines these enzymes as targets for antiviral therapy. the viral rna polymerase 3d (3d pol ) represents another very important nonstructural protein of hrv. it forms a complex with both cellular and viral proteins, the rna replication complex. 98 this enzyme synthesizes viral minus-strand rna and uses it as template strand for the synthesis of genomic viral rna. vpg (3b) is the primer for negative-as well positive-strand rna synthesis. negative-strand, but not positive-strand rna synthesis, is stimulated by 2a pro . further viral accessory proteins include 2b, 2c, 2bc, and 3ab. besides 3d pol these proteins can play an important role in inhibition of viral rna synthesis by antiviral compounds. in summary, the knowledge of the structure of the viral capsid, proteases, and polymerase and their important function in the viral life cycle predestine these proteins as potential anti-hrv targets. hrv grow in several human and some primate cells expressing the minor group ldl receptors and/or the major group receptor icam-1. human cells susceptible to hrv infections include embryonic kidney, amnion, diploid fibroblasts from embryonic lung, tonsil, liver, intestine, and skin, adult fibroblast lines from aorta and gingival and the kb, hep-2, and hela continuous cell lines. 133 but, the susceptibility of hela cells and human fibroblasts to virus infection may vary. 32 hrv multiplication also occurs in primary human airway fibroblasts 134 and differentiated bronchial epithelium. 77, 78, 135 the proportion of infectible epithelial cells was shown to be between 3 and 10%. 77, 135 but, enhanced levels of viral production were detected in poorly differentiated in comparison to differentiated epithelial cells. 136 the degree of viral infection correlated with il-6 and il-8 induction in these cells virus growth causes a typical cpe characterized by ballooning, refractiveness, granularity, and shrinkage of infected cells. the hrv-induced cpe, infectious virus titers, viral protein expression, and rna synthesis can be chosen as parameters to evaluate the anti-hrv activity of compounds in cell-culture based assays. there are several methods for antiviral screening against hrv. the plaque reduction assay has been traditionally performed and accepted as the ''gold standard'' in antiviral testing. [137] [138] [139] however, this test is laborious, time consuming, and the evaluation is subjective. therefore, it is not suited for the routine antiviral testing. it was more and more replaced by methods based on quantification of protection from virus-induced cpe after drug treatment. so, the cpe in sample-treated and untreated cells has been compared by light microscopy. 137, 140, 141 but this evaluation is also subjective. another more objective approach is the spectrophotometric quantification of cpe results in neutral red or crystal violet uptake assays, 12,141-144 and the tetrazolium dye reduction method. 132, 145 it allows an excellent and rapid antiviral screening of large numbers of compounds using small amounts of extracts, natural, or synthetic compounds. active samples can be scheduled for additional testing using other assays e.g. virus yield or plaque reduction assays, and for studies on the mechanism of action. the activity of potential antiviral drugs has to be approved in vivo. because of the high degree of species-specific variations in icam-1 preventing infection by major group hrv, practical animal models have been absent for a long time. chimpanzees were infected with several hrv serotypes but without developing clinical signs. 133 hrv do not induce infection in rabbits, guinea pigs, and weanling mice injected with hrv by different routes. one minor group hrv, serotype 2, was adapted to grow in mouse fibroblasts and used in a mouse model of rhinovirus infection in which growth could be demonstrated. 146 based on the fact that the ldl receptor family is highly conserved between human and mouse, newcomb et al. examined whether hrv-1b, another minor group virus, may infect mouse airways in 6-to 8-week-old female c57bl/6 mice. 147 the authors demonstrated that this hrv serotype replicates and induces airway inflammation in vivo. these results strongly correspond to those of bartlett et al. who established three novel mouse models of rhinovirus infection in balb7c mice. 148 in the first model, 6-week-old balb/c mice were infected with hrv-1b. in the second model, transgenic balb/c mice, expressing a mouse-human icam-1 chimera, were inoculated with the major group hrv-16. rhinovirus-induced exacerbation of allergic airway inflammation is mimicked in the third model. due to the lack of a small-animal model for hrv infection until 2008, the experimental human challenge model has to be used to approve effects of potential antiviral drugs under controlled conditions in preclinical studies. volunteers were experimentally inoculated with various serotypes e.g. hrv-4, hrv-9, hrv23, hrv-29, and hrv-39 to examine the efficacy of potential antiviral drugs under standardized conditions. [149] [150] [151] [152] [153] [154] examples and results of these studies with capsid-binders, protease, and rna synthesis inhibitors, as well as interferons are described in the following sections. antiviral agents that inhibit virus attachment, capsid uncoating, protein and rna synthesis of picornaviruses are the best studied, 95, [155] [156] [157] and will be in the focus of this section. inhibition of virus attachment and/or uncoating interrupts the viral life cycle at its beginning and prevents hrv infection. options to prevent these early steps of the viral life cycle include (i) virus neutralization by hrv-specific antibodies, (ii) receptor blockade by antibodies directed against the cellular receptors icam-1 or ldl, (iii) by soluble receptor molecules, or (iv) by compounds interacting with the viral capsid. because of the high number of serotypes circulating often in parallel, application of hrv-specific antibodies is thought to be no promising approach for prevention or therapy of rhinovirus infection. in contrast, antibodies directed against the cellular receptor or soluble receptor molecules of major or minor group hrv could inhibit 90 and 10% of hrv serotypes, respectively. therefore, the strategy to prevent virus-receptor interaction by receptor antibodies or soluble receptor molecules has been extensively evaluated in vitro as well as in vivo. the antiviral activity of icam-and ldl-specific antibodies was confirmed in cell culture. 158, 159 furthermore, the prophylactic effectiveness and safety of intranasally administered rhinovirus murine icam-1 antibody was assessed in two double-blind, placebo-controlled, randomized studies of volunteers experimentally inoculated with hrv-39. 160 in the result, no toxicity related to antibody application was recognized. the higher dosage of 1 mg/subject of rhinovirus murine receptor antibody did not reduce overall infection or illness rates, but was associated with a 1-2 day delay in the onset of virus shedding and cold symptoms. viral titers and nasal symptoms were significantly reduced on the second day after challenge. in summary, the monoclonal antibody to the cellular icam-1 was demonstrated to be not effective enough. a new strategy was the creation of multivalent fab fusion proteins against icam-1. a new molecule, named cfy196 demonstrated a better avidity and in vitro potency against hrv over conventional mabs. 161 cfy196 is under development as nasal spray with the name of coldsol. antagonism of virus-receptor interaction was considered as another promising way to prevent hrv attachment to host cells. soluble forms of fully or truncated icam-1, [162] [163] [164] and ldl or vldl-receptor concatemers [165] [166] [167] exhibited antiviral activity against major and minor group hrv, respectively, in cell culture. soluble forms of icam-1 compete with receptor binding sites on the virus capsid, hinder an early infection event such as entry or uncoating, or directly inactivate hrv due to the formation of empty capsids. 163, [168] [169] [170] a soluble ldl receptor fragment neutralized viral infectivity by aggregation. 171 concatemers of the third ligand binding module of the vldl-receptor did not lead to viral aggregation but blocked the receptor binding sites and possibly inhibited viral uncoating by cross-linking the viral capsid subunits via multi-module binding. 166 the antiviral activity of a truncated, soluble form of icam-1 was proved in hrv-16 infected chimpanzee. 172 in randomized, double-blind, placebo-controlled trials, the safety and efficacy of intranasal administration of tremacamra, a soluble icam-1 in experimental hrv-39-induced colds in humans, was shown. 173 no further development was reported for these agents. 95 a further option to prevent virus attachment was described for low-molecular-weight compounds, the so-called capsid-binding agents, which enter the small hydrophobic pocket within viral capsid protein 1 beneath the icam-binding canyon of hrv. 174, 175 zhang et al. showed that drug may integrate into mature viruses by diffusion as well as into progeny viruses during assembly. 176 when hrv-14 and hrv-16 were grown in the presence of pleconaril, a higher occupancy occurred than when the drug was introduced into the alreadyassembled viruses. in doing so, capsid-binders induce conformational changes of the canyon of hrv-3 and hrv-14, hinder virus-receptor interactions, and prevent attachment to host cells. 105, 175, [177] [178] [179] in addition, uncoating of both hrv serotypes was shown to be inhibited as a result of a potential loss of flexibility of the viral capsid after drug binding. in contrast to hrv-3 and hrv-14, capsid-binding compounds did not prevent attachment of hrv-1a. results from x-ray studies showed that drug binding into the hydrophobic pocket of hrv-1a replaces the pocket factor but induces only very small conformational changes. 180 therefore, kim et al. suggested that the observed conformational changes are too small to affect receptor binding. but, capsid-binding compounds prevented attachment of hrv-16 possessing a pocket factor like hrv-1a without distinct deformation of the pocket. 102, 176, 181 further results from comparative antiviral studies with different capsid-binding compounds and hrv, representative for the major and minor group, did not reveal a correlation between inhibition of adsorption and receptor grouping or antiviral grouping. 182 the reasons for the difference in the mode of action of capsid-binding compounds related to attachment inhibition are not fully understood until now. taken together, inhibition of rna uncoating was found for all investigated serotypes after drug binding independent of receptor grouping whereas prevention of virus attachment was found to be an additional mode of action for individual viruses and/or drugs. till now, various potent compounds belonging to diverse chemical classes have been described as uncoating inhibitors. just to give an impression of diversity, the structures of disoxaril and pleconaril, 12,183-185 pirodavir and the oxime ether, 14, 141, 142, 186, 187 the isoxazole derivate compound, 19, 143, 188 the imidazole derivative sch 38057, [189] [190] [191] the chalcone ro 09-0881, 192, 193 4 0 ,6 dichloroflavan and isoflavan, 137, 194 the pyridine derivative mdl 20, 957, 195, 196 and the phenoxybenzene mdl-860 197, 198 that exhibit a potent anti-hrv activity (table ii) are shown as examples in figure 2 . they inhibit most of hrv serotypes and a couple of them also affect enteroviruses, however, with varying susceptibility. based on variability of susceptibility to capsid-binders of different length, hrv serotypes were classified into two different groups, a and b. 140 several of the given examples of compounds were also clinically tested. studying the development of clinically effective capsid-binders, the long road to the discovery of a clinically effective anti-hrv drug becomes apparent. one well-described example represents the discovery and optimization of capsid-binders from sterling winthrop pharmaceutical group, the so-called win compounds. 174 first inhibitors originated from juvenile hormone mimetics that demonstrated some activity against hrv-1a. determination of the x-ray structure of hrv-14 helped to understand the compounds' binding sites at the virus capsid. 103 results from subsequent x-ray studies of hrv-win compound complexes revealed the location and nature of binding sites and provided information concerning interactions within these sites. 175, 179, 180 this knowledge was used for optimization and design of new compounds. 199 optimized win compounds, for example disoxaril and pleconaril ( fig. 2) , consist of a methylisoxazol ring, a substituted phenoxy group, and a five-membered heteroatom ring and inhibit a broad spectrum of rhinoviruses and enteroviruses (table ii) . 12, [183] [184] [185] in 1985, the first broad-spectrum win compound disoxaril (win 51711) was tested in clinical trials. 139 the development of crystallurea in human volunteers treated with high doses as well as its low bioavailability (15%) prohibited subsequent development. thereafter, results from sar and qsar analysis were used to further enhance the potency and spectrum of activity. in 1992, another compound, win 54954, was clinically tested. it was not effective in humans infected with hrv-23 and hrv-29. 153 moreover, it was rapidly metabolized and induced a reversible hepatitis. consequently, the further clinical development was stopped. the better understanding of pharmacokinetic properties of capsid binders and synthetic chemistry efforts led to the discovery of pleconaril, an orally bioavailable, welltolerated capsid-binder that inhibits most rhinovirus as well as various enterovirus serotypes. 12, [183] [184] [185] in 2000, schiff et al. published the efficacy of pleconaril in an experimentally induced coxsackievirus a infection in humans. 200 in phase ii placebo-controlled, natural cold trials, the drug produced a moderate reduction of 1-1.5 day in the medium time to elevation of illness compared with placebo. 201 these results were confirmed in two subsequent pivotal studies. 202 besides the moderate clinical efficacy, these studies revealed that 13% of baseline isolates were not susceptible to pleconaril and 11% developed reduced susceptibility (defined as 10-fold increase in baseline value). in a subsequent study the relationship of pleconaril susceptibility and clinical outcomes in the treatment of common cold caused by rhinoviruses was demonstrated. 203 based on drug interaction, marginal treatment effect, and possibility of transmission of resistant viruses, the fda did not approve the applied oral administration of pleconaril for the treatment of common cold. the molecular mechanism of drug interaction of orally given pleconaril was shown to be based on hepatic cytochrome p450 3a activation. 204 to reduce adverse effects, shering-plough under license of viropharma completed a phase ii clinical trial with an intranasal formulation of pleconaril for the potential treatment of common cold in high-risk populations in 2007. the results were not published until now. pyridazine analogues developed by janssen research foundation represent another example for the long road to discovery of a clinically effective capsid-binder. 186 in 1992, the broad-spectrum activity of pirodavir ( fig. 2 ; table ii ) against rhinoviruses was published. 187 in the same year the results of a randomized, double-blind, placebo-controlled trail to assess the therapeutic efficacy of intranasal pirodavir in natural common colds were described. 205 possibly as a result of poor water solubility and rapid hydrolysis of pirodavir, no clinical benefit was found. the problem of ester hydrolysis was resolved by the development of oxime ether analogues of pirodavir by biota. an example is shown in figure 2 . like pirodavir these new analogues are potent inhibitors of rhinoviruses. 142 an advantage over pirodavir is their improved bioavailability. bta-798, an antiviral analogue with long half-life and good oral bioavailability, was scheduled to a phase ii clinical trial in 2008. the results have not yet been published. in summary, despite extensive research leading to the discovery of potent anti-hrv capsid-binders, no agent has been approved for prevention and/or therapy of rhinovirus-induced diseases so far. nearly, the same conclusion has to be drawn for protease inhibitors. because of their pivotal role for viral polyprotein processing and the high conservation of critical amino acids, 132 2a pro as well as 3c pro represent potential anti-hrv targets. results from cell culture-based assays provided evidence that inhibition of hrv replication is in principle possible. for example, processing of the hrv-2 polyprotein was prevented by pyrrolidine dithiocarbamate treatment in virus-infected hela cells. 206 in contrast to other enteroviruses, [207] [208] [209] [210] pretreatment of cell monolayers with different nitric oxide donors leading to s-nitrosylation of 2a pro and 3c pro had neither an effect on virus replication nor on hrv-induced il-8 elaboration. 211 the proteolytic activity of 2a pro of hrv-14 was specifically inhibited by two elastase-specific inhibitors, 212 and an antiviral peptide representing a derivative of the caspase inhibitor zvad.fmk. 213, 214 homophthalimides, e.g. ly353349 ( fig. 3 ; table ii) , were described as inhibitors of 2a pro as well as 3c pro . 215 in contrast to protease 3c, no structure-activity relationship studies have been reported for hrv 2a protease. moreover, protease 2a accomplishes only one cleavage in hrv polyprotein, while protease 3c performs all other cleavages. after elucidation of the crystal structure of 3c pro , 216 computer modeling of structural features of protease inhibitors became possible. 217 furthermore, structure-based design was used to develop mechanism-based inhibitors of the 3c protease with potent antiviral activity against multiple hrv serotypes. 218, 219 highly active compounds incorporate various michael acceptor moieties, irreversibly bind to 3c pro , and exhibit anti-hrv-14 activity in hela cells. 220, 221 structure-activity studies were performed to optimize protease inhibitors. 220, [222] [223] [224] [225] [226] these efforts resulted in the identification of a highly active anti-hrv compound, ag7088 (rupintrivir; fig. 3 ; table ii ) that entered clinical trials. in cell culture, ag7088 inhibited a broad spectrum of laboratory hrv as well as clinical isolates. 132, 183, 227 in a single-cycle, time-of-addition assay it demonstrated antiviral activity when added up to 6 hr after infection. 227 inhibition of hrv replication strongly correlated with reduction in the level of il-6 and il-8 release into cell supernatant, leading to the suggestion that this agent may not only block virus replication but also diminish symptoms. 228 the pharmacokinetics and safety of rupintrivir were proved in two double-blind, randomized, placebo-controlled studies. 229 intranasal rupintrivir, administered as single doses of 4 and 8 mg or every 3 hr, six times per day, for 7 days, was safe and well tolerated. three double blind, placebo-controlled clinical trials were conducted to assess rupintrivir nasal spray (2% solution) for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers. 230 rupintrivir prophylaxis reduced the proportion of subjects with positive viral culture by 26% and viral titers but did not decrease the frequency of colds. drug treatment led to the reduction of the mean total daily symptom score by 33%. subjects receiving rupintrivir also demonstrated significantly lower viral titers and rna levels than placebo-treated subjects on days 2, 3, and 5 and on days 2 and 3, respectively. there was no influence on the proportion of subjects with positive viral culture and the frequency of colds. clinical development was terminated because rupintrivir did not act in a subsequent natural infection study in patients. 231 in parallel research efforts, an orally bioavailable inhibitor of table ii) , was discovered. 225, 231 like rupintrivir, this compound is an irreversible inhibitor incorporating a michael acceptor moiety that forms a covalent bond with the 3c protease active site cysteine. it demonstrated an antiviral activity against all hrv and related picornaviruses tested. 231 in a phase 1 clinical study, compound 1 was shown to be safe and well tolerated. according to a publication of patick, no further clinical development was planned for this compound. 95 the blocking of viral rna synthesis during replication represents another site for chemotherapeutic interdiction. it was shown that, rhinoviral rna can be targeted in a sequence-specific manner by deoxyribozymes, 232 morpholino oligomers, 233 and small interfering ribonucleic acids. 234 the efficacy of the latter two approaches was confirmed in cell culture. in addition, 2-furylmercury chloride ( fig. 4 ; table ii ), 235 table iii) , 236 and pyrrolidine dithiocarbamate (fig. 4 ) interfered with rhinoviral rna synthesis and inhibited hrv replication in cell culture-based assays. 206, 237 the nucleoside analog ribavirin ( fig. 4 ; table ii ) that inhibits a broad spectrum of rna as well as dna viruses acts also against hrv-2 in hela cells. 238, 239 the cellular inosine monophosphate dehydrogenase that controls de novo synthesis of purine nucleosides represents the principal target in the mode of action of ribavirin. 240 moreover, when ribavirin is incorporated into picornavirus rna, it pairs equally well with either uracil or cytosine inducing mutations that can be lethal to rna viruses. 241 further identified mechanisms of action for ribavirin include inhibition of genomic rna capping, enhancement of host t-cellmediated immunity against viral infections through helping to switch the host t-cell phenotype from type 2 to type 1. 242 another compound with potent anti-hrv activity in vitro is enviroxime ( fig. 4 ; table ii ), a benzimidazole derivative. 243, 244 it inhibits viral plus strand rna synthesis. 245 in particular the 3a protein, which is involved in the initiation of plus strand rna synthesis, was implicated as likely target of drug activity. 246, 247 however, results from another study suggest that enviroxime targets a complex of proteins and/or cellular factors and that the exact mechanism remains to be studied. 248 although there was a statistically significant reduction in clinical score in a prophylactic study with hrv-9-infected volunteers, 152 enviroxime failed in experimentally induced hrv-4 and hrv-39 infection, 149, 151 and in clinical studies, 248-250 because of poor bioavailability and side effects. in an attempt to overcome the marked hydrophobicity, water insolubility, and toxicity, wyde et al. incorporated enviroxime into liposomes and then tested the anti-hrv activity and toxicity of the liposome-incorporated enviroxime in cell culture. 251 the liposome preparation of enviroxime inhibited hrv-1a and hrv13 as effective as the parent compound and was 10-to z50-fold less toxic. in contrast to free enviroxime, the liposome preparation was readily and successfully delivered by small-particle aerosol to the upper and lower respiratory tract of mice. in another attempt to overcome the disadvantages of enviroxime, several benzimidazole as well as nonbenzimidazole analogs were synthesized and studied. [252] [253] [254] [255] even though some compounds were better bioavailable and could be administered orally, 254 none of these compounds was tested in clinical studies. besides virus-specific targets, cellular inhibitors like interferons may represent a therapeutical approach. among other activities, interferons exhibit antiviral activity. the advantages of interferon application include the broad spectrum of activity and low risk of resistance development. human leukocyte and fibroblast as well as recombinant human a interferons prevent the hrv-induced cpe in cell culture whereas a variation in sensitivity was observed. [256] [257] [258] intranasally applied recombinant interferon a and interferon b have been shown to be effective in humans when provided prophylactically both in experimental and natural rhinovirus colds. [259] [260] [261] [262] [263] [264] significant reductions in illness frequency, mean symptom score, nasal secretion weights, and frequency of virus isolation were observed. in contrast, recombinant interferon g did not prevent hrv infection or illness and may enhance the symptoms. 265 little to no therapeutic effect was found in patients with common cold after interferon treatment. 150, 266 moreover, blood-tinged mucus and nasal bleeding were described as side effects. 263, 267 combining interferons with dichloroflavan, enviroxime, chalcone ro-09-0410 produced synergistic increases in antiviral activity in vitro against hrv-2 and hrv-9. 268 an attempt to demonstrate synergy between the anti-hrv effect of recombinant human rhuifn a and enviroxime in hrv-9 and hrv-14-infected volunteers failed. 269 according to the authors, the main reason for this failure may be the rapid removal of enviroxime from the nose when given intranasally. a. impact of natural products nature provides an astonishing pool of secondary metabolites biosynthesized from living organisms such as plants, fungi, protozoan, insects, and other animal sources. in contrast to synthetic compounds, natural products are characterized by an overwhelming chemical diversity. previously, the chemical diversity space between these two groups was evaluated with respect to drug substances by feher and schmidt. 270 it is shown that combinatorial compounds densely populate a small area, whereas natural products cover a wider range quite similar to the chemical space occupied by drug substances. the authors accordingly suggest that combinatorial libraries that mimic the distribution properties of natural products might be more biologically relevant. one may assume that secondary metabolites evolved as reaction to their target receptors related to defence, protection, attraction, and signalling. these adaptation processes have enriched not only the metabolites' structural diversity but have also optimized drug-like metabolic traits likely to have favorable pharmacokinetic properties. 271, 272 it is this evolutionary concept that gives the pool of natural products the greatest source of scaffold diversity with molecules of biological relevance. newman and cragg analyzed the number of drugs approved between 1981 and 2006 and circumstantiated that especially the anti-infective area is strongly dependent on natural products and structures derived from natural scaffolds. 273 the anti-infectives including the antiviral vaccines are with 22.8% or 230 launched drugs by far the major category with only about 30% being synthetic in origin. from 1981 to 2006, 78 vaccines and antiviral drugs have been approved. excluding the high number of vaccines (25) and biologicals (12) , most of the 41 small antiviral molecules are based on nucleoside structures or on peptidomimetics; only 16.7% are classified as totally synthetic drugs. however, till now, neither a synthetic nor a naturally derived anti-hrv drug substance has been approved for the treatment or prevention of hrv infections. intensive research and development efforts in the field of natural products revealed several inhibitors of viral attachment and entry, and inhibitors of viral protease from natural sources. the efficacy of natural products is not only reflected by statistics of launched drugs but also by empirical knowledge gained over centuries by successful application of naturalbased ethnomedicinal products such as plants, culinary herbs, and spices. phytochemical and pharmacological work performed with ethnomedicinal anti-hrvs mainly from plants revealed a high number of active metabolites from different chemical classes, e.g. coumarins, flavonoids, alkaloids, quinones, terpenoids, polyphenols, and polysaccharides. natural products include complex extracts and their chemical entities, which are biosynthesized by nature. for an unambiguous presentation of anti-hrv natural products it is of prior importance to first distinguish between a single chemical entity from nature, i.e. an isolated, purified natural compound, on one hand, and a natural preparation comprising hundreds to thousands of constituents, mainly secondary metabolites, on the other hand. if natural preparations are derived from plants, they might also be labelled as botanicals, phytoceuticals, or phytotherapeutic agents. these multicomponent preparations might show a varying profile of their constituents depending on the used species, origin, collection time, plant parts, extraction procedures, preparation methods, and manufacturing processes, just to mention a few important elements. these parameters affect the final product in terms of the qualitative and quantitative composition of chemical constituents, which may have an impact in biological activity. accordingly, studies performed with phytochemically not specified extracts or nonstandardized preparations often suffer from irreproducible and incomparable results a wide variety of natural preparations showed to be acting therapeutically in hrv and other viral infections with often complementary and overlapping antiviral mechanisms of action. [274] [275] [276] most of these remedies are described in ethnopharmacological sources or handed down for generations. they usually consist of simply prepared natural items whose chemical composition is complex. many of the contained secondary metabolites, possibly active principles, have never been examined chemically or biochemically using modern medical knowledge. they are however components of plant medicines, which have stood the test of time and as such may offer clues of great interest to medicinal chemists. a clear advantage of the application of these products is their absent or relatively low toxicity due to a usually long-term empirical trial. although the knowledge of the immuno-pathogenesis of rv-induced diseases remains limited, the host defense function of the airway epithelium plays an important role in the innate-immune response to hrv-infection. 277 host cells respond by the production of mediators with antiviral activity such as type i interferons and nitric oxide, and produce cytokines and chemokines that influence the subsequent induced innate-and specific-immune response. these processes are beneficial in facilitating clearance of virus from the respiratory tract, but also cause immuno-pathology. following hrv-infection, disease severity is dependent on direct, harmful effects of the virus as well as tissue damage as a result of the host antiviral immune response. accordingly, a number of agents with phenomenological effects against common cold have shown to exert their activity more in the field of regenerating tissue damage than on a direct anti-hrv effect. several herbal remedies consisting of a multitude of secondary metabolites from different chemical classes may attribute in a beneficial way for the treatment of common cold by reducing symptom severity and duration due to their immune-modulating, anti-oxidative, and anti-inflammatory properties. beside these commonly observed bioactivities of natural products, multicomponent mixtures like botanicals often show overlapping symptomatic effects as well as synergistic and/or additive properties. thus, it is a challenging endeavor to track down an observed phenomenological effect of a complex mixture on a molecular level. the following section explores the significance and current knowledge of selected botanicals for the prevention and therapy of common cold. questions about (1) clinical evidence of efficacy, (2) the constituents or at least the chemical classes that are involved in the observed anti-hrv effect, and (3) the involved pharmacological targets were covered as far as possible. echinacea preparations include expressed juice from aerial parts as well as extracts of roots or aerial parts, or both, from one or more species of the genus echinacea (e. angustifolia, e. purpurea, and e. pallida). they are the most recognized botanicals for prevention and treatment of common cold and flu, and account for the second top-selling herbal products in the us-market. 278 accordingly, echinacea has come under much scientific scrutiny. the high number of studies dealing with the effectiveness of echinacea for preventing and treating the common cold from clinical trials was recently reviewed by woelkart et al. 279 the authors summarized the findings of the meta-analyses regarding the 16 randomized controlled trials evaluated in the cochrane database, 280 the 14 randomized clinical trials analyzed by shah et al., 281 and the experimental hrv-infection studies pooled by schoop et al. 282 to sum up, the clinical data on echinacea so far are not fully consistent, mainly based on problems inherent in assessing the efficacy of echinacea preparations, such as lack of comparability of available preparations, study design, and outcome. nevertheless, the meta-analyses showed some evidence that preparations based on the aerial parts of echinacea purpurea might be effective for the early treatment of colds in adults. 280 echinacea showed to decrease the odds of developing the common cold by 58% and the duration of a cold by 1.4 days. 281 similarly, the evaluation of three induced rhinovirus prevention studies revealed the odds of experiencing a clinical cold were 55% higher with placebo than with echinacea. 282 stepping into a molecular level, several constituents found in echinacea species could potentially affect the symptoms of common cold. chemically identified substances include polysaccharides and glycoproteins, caffeic acid derivatives (especially cichoric acid and echinacoside), and lipophilic polyacetylenes and alkamides. pharmacological studies have shown that cichoric acid, alkamides, glycoproteins, and polysaccharides possess immunomodulatory activity. additionally, alkamides have been reported to exert not only antiinflammatory effects but also cannabinomimetic properties, which are suggested as molecular mode of action of echinacea alkamides as immunomodulatory agents. 283 raduner et al. showed that some echinacea alkamides exert cannabinoid type 2 receptor-dependent and independent immunomodulatory effects on cytokine expression. 284 different echinacea constituents were evaluated for their anti-oxidative effects measuring the inhibition of in vitro cu(ii)-catalyzed oxidation of human low-density lipoprotein. thereby, the major caffeic acid derivatives, cichoric acid and echinacoside, showed the highest anti-oxidative effects, which was even higher when combined with a natural mixture of alkamides. 285 sharma et al. used cytokine antibody arrays to investigate the changes in the pro-inflammatory cytokines and chemokines released from human bronchial epithelial cells exposed to hrv 14. 286 application of two chemically characterized echinacea extracts showed a reversion of the stimulated release of numerous pro-inflammatory cytokine-related molecules, e.g. for the cytokine il-6, and the chemokines il-8 and eotaxin. in a similar study, an echinacea extract rich in polysaccharides and another rich in alkamides and caffeic acid derivatives were as well able to neutralize the effects of hrv-infected epithelial cells. 287 using gene expression analysis both studies revealed the anti-hrv benefit of echinacea preparations being involved in multiple immune response signaling pathways. taken together, the numerous pharmacological findings from literature, the potential of echinacea preparations, and their constituents to combat or prevent common cold can be deduced to immune modulating, anti-inflammatory, and anti-oxidative properties that may also act in some combination of these event, rather than acting directly on hrv. garlic cloves have been used traditionally to treat a number of infectious diseases. however, only few confirmatory studies have been published regarding the traditional antiviral uses. the clinical effectiveness of garlic on the prevention of common cold was investigated by josling in 2005, who published a double-blind, placebo controlled study assessing 146 patients more than a 12-week treatment period with an allicin-containing garlic supplement. 288 common cold infections and symptoms were recorded in a daily diary. patients in the treatment group had significantly fewer colds than patients in the placebo group (24 vs. 65, po0.001) who also had a longer duration of symptoms (5.01 vs. 1.52 days, po0.001). as soon as the garlic is chewed, cut, or pressed, its main ingredient, the sulphur containing alliin, is broken down by the enzyme alliinase to the thiosulfinate allicin. by steam distillation allicin is transformed to diallyl disulfide and diallyl trisulfide that are responsible for the distinctive smell of garlic. further, allicin transformation compounds, such as e-and z-ajoene, are not found in fresh garlic, but in lipophilic extracts. by investigation of different garlic extracts and isolates against a number of different human pathological viruses, weber et al. could show that allicin was the most active virucidal component from fresh garlic and fresh extracts. 289 results of the direct pre-hrv-2-infection incubation assay let suggest allicin to bind to the viral protein capsid, leading to a subsequent inhibition of viral adsorption and penetration. although the garlic thiosulfinates are endowed with significant cytotoxicity, the antiviral effects were obtained in nontoxic concentrations. 289 beside the direct anti-hrv effect of fresh garlic extract and allicin, a number of human immune functions were found to be enhanced in vitro by aqueous garlic extract, its polar, and thiosulfinate fractions. 290 in north america, panax quinquefolium, the ginseng species indigenous to both canada and the united states, has been a popular herbal remedy to combat stress, and to modulate both natural and acquired immune responses. american ginseng root extracts, rich in poly-furanosyl-pyranosyl-saccharides, have been found efficacious in the prevention of upper respiratory infections in immunocompetent healthy adults. 291, 292 in a randomized, double-blind, placebo controlled trial, 200 mg of a proprietary american ginseng root extract was given to 43 community-dwelling elderly adults (age 465 year) twice a day more than a ''cold and flu'' season period of 4 months. one month into the study, all participants received an influenza vaccination. during the first two months, no significant differences in duration and incidence were observed when compared to placebo. however, during the last two months significantly fewer subjects of the ginseng group reported acute respiratory syndromes than the placebo group (32 vs. 62%). additionally, the duration of respiratory symptoms was reduced by 55% in the ginseng group. 291 in a similarly arranged trial, 323 healthy adults (ages 18-65 years) with a history of at least two colds the previous year commenced a 4 month study at the beginning of a cold and flu season. 292 they received two 200 mg capsules daily of standardized american ginseng root extract or a placebo. outcomes measured were number of colds including symptom severity and total number of symptomatic days. a therapeutic effect was reported regarding symptom severity and fewer symptom days that were 31 and 34.5% lower in the ginseng group than in the placebo group. a phase ii randomized, controlled trial of 2 dosing schedules of american ginseng root extracts, rich in poly-furanosyl-pyranosyl-saccharides, evaluated the safety, tolerability, and efficacy in a pediatric population already suffering from an upper respiratory tract infection. the results showed no serious adverse events and a good tolerability of both ginseng doses; however, frequency and severity of symptoms were not significantly different among each of the three treatment groups, i.e. standard dose, low dose, and placebo. 293 the most prominent constituents of the genus panax are the triterpene saponins ginsenosides. they are known to have numerous pharmacological activities such as anti-cancer, anti-diabetes, antiviral, and anti-atherosclerosis effects. some compounds of this chemical class showed to be responsible for the immunostimulant activity of ginseng. 294 on the other hand, the efficacy of a polysaccharide-rich extract of american ginseng was compared with an extract rich in ginsenosides on systemic and gut-associated immune function. the authors of this study investigated the lymphocytes isolated from spleen, mesenteric lymph nodes and peyer's patches, and immune cell proportions and cytokine production from sprague-dawley rats. they could show that the polysaccharide-rich ginseng extract modifies the rats' systemic immune responses and affects the gut-associated immunity in a manner distinct from that of the ginsenoside-containing extract of american ginseng. 295 a direct antiviral activity of ginseng constituents could be attested for the polysaccharides on rotavirus infection in ma104 cells. the triterpene saponins, however, did not exhibit any rotavirus infection-inhibitory activity in this study. 296 a moderate in vitro virucidal effect (id 50 62 mm) of the ginseng saponin chikusetsusaponin iii against herpes simplex virus type i was detected by fukushima et al. this compound exhibited an intracellular inhibitory activity, but could only marginally affect the viral proteins postinfection. 297 the ancient chinese formula bu-zhong-yi-qi-tang (japanese name hochu-ekki-to) is a traditional herbal medicine in china and japan that is composed of ten species of medicinal plants, namely astragali radix, atractylodis lanceae rhizoma, ginseng radix, angelicae radix, bupleuri radix, zizyphi fructus, aurantii nobilis pericarpium, glycyrrhizae radix, cimicifugae rhizoma, and zingiberis rhizoma. this formula is reported to have various immunomodulatory, [298] [299] [300] and anti-inflammatory activities. 301 yamaya et al. recently investigated the effects of hochu-ekki-to in cultures of human airway epithelial cells infected with hrv-14. 302 the output of virus, associated levels of viral rna, and the production of icam-1, cytokines and acidic endosomes in cells were measured. in airway epithelial cells hochu-ekki-to was able to decrease virus output and susceptibility to hrv infection by decreasing icam-1 and by blocking the entry of viral rna into the cytoplasm from the endosomes. glycyrrhizin, a major component of one herbal ingredient of hochu-ekki-to, i.e. glycyrrhiza glabra, was able to reduce supernatant virus titers dose-dependently, with a maximum effect between 0.12 and 0.6 mm. however, no clinical trials with representative numbers of subjects are published so far. the human rhinovirus k 63 5. umckaloabo (pelargonium sidoides) p. sidoides and p. reniforme form the origin of the popular drug umckaloabo. this herbal remedy from south africa has found entrance in western medicine mainly as aqueous ethanolic root extract from p. sidoides for the treatment of infections of the respiratory tract. the efficacy of umckaloabo compared with placebo has been evaluated in 103 adults suffering from common cold by lizogub et al. 303 the applied herbal preparation was well tolerated by the patients. the study demonstrated only a weak efficacy of umckaloabo compared to placebo after 5 days. after 10 days, however, the p. sidoides extract significantly reduced the severity of symptoms and shortened the duration of the common cold compared with placebo. just recently, timmer et al. selected randomized controlled trials examining the efficacy of p. sidoides preparations for the treatment of various acute respiratory infections and analyzed their efficacy and safety. 304 the authors concluded that umckaloabo may be effective in alleviating symptoms of acute rhino-sinusitis and the common cold in adults. it may be effective in relieving symptoms in acute bronchitis in adults and children, and sinusitis in adults. reliable data on the treatment for other acute respiratory infections however were not obtained. identification of the metabolites from umckaloabo revealed a high number of different chemical classes, such as phenolic and cinnamic acids, tannins, flavonoids, and coumarins. antibacterial activities of umckaloabo against different pathogens have been reported. phenols, coumarins, and tannins have been identified to contribute with moderate antibacterial activities, however, cannot explain the effect of the whole extract (reviewed by kolodziej 305, 306 ). additionally, p. sidoides extracts have been reported to significantly activate the nonspecific immune system by induction of tnf and no-release, and ifn-like activities. 305 these effects are assumed to contribute to the controversially discussed potential of p. sidoides extract for the treatment of upper respiratory tract infections. only one study reports a direct antiviral effect, i.e. a clear dose-dependent anti-herpes simplex virus acitivity for the aqueous root extract of p. sidoides. 307 further pharmacological studies are needed to elucidate potential direct anti-hrv properties of umckaloaba and its constituents. carrageenan, a mixture of different polysaccharides, which is mainly extracted from red seaweeds, has been extensively used in food, cosmetic and pharmaceutical industry as a thickener and gelling agent. it has previously shown an antiviral efficacy against several viruses. 308, 309 in a recently published study, lambda-, kappa, and iota-carrageenan were investigated for their anti-hrv inhibiting potential. at a concentration of 200 mg/ml iotacarragenan, a sulphated polysaccharide, was able to fully inhibit virus-induced cell death in hrv-2 infected hela cells. 310 based on their studies, grassauer et al. concluded that iotacarrageenan is effective against different hrv-serotypes on primary human epithelial cells. it is hypothesized by the authors that iota-carrageenan might create a hostile environment for hrv and thereby block viral entry and replication. because of its safe application and proved in vitro efficacy, iota-carrageenan deserves consideration as a candidate for clinical trials for prevention and therapy of hrv-induced common cold. the level of knowledge on the impact of the six botanicals on hrv-infection discussed above is different and heterogeneous. the best studied herbal remedy associated with common cold, i.e. echinacea, showcases the innate problem connected with multi-component mixtures: starting from the late nineties till june 2009 some 100 original articles have been published to this topic and tried to elucidate questions concerning efficacy, molecular mechanism, and bioactive ingredients of echinacea. although some evidence is provided for the effects of extracts, chemical classes as well as well-defined constituents on specific targets and pathways, the findings cannot be deduced to a common denominator. further, results from clinical trials often suffer from lack of comparability, because of using different study designs, outcome measures, and overall the application of different preparations. 279 a proper quality analysis and characterization of the preparation under investigation is mandatory and should follow the recommendations and guidelines for reporting clinical trials for herbal medicine. 311 as underlined before (sections 5.1. and 5.2.), the chemical complexity of a natural preparation might be beneficial in terms of synergistic, additive, and overlapping effects caused by the multitude of evolutionary trimmed metabolites, which may attribute with modulating multi-target effects. on the other side, exactly this fact is hardly compatible with the proper assignment of an activity to a defined chemical entity according to western medical practise. in contrast to a single compound (synthetic or naturally based), the chemistry of a botanical is not only complex but also varying. the analytical profile and in term the pharmacological profile of the investigated samples can differ substantially. accordingly, the quantitative and qualitative comparison of different studies resulting from botanicals is by far more complex than those performed with pure single compounds, and may also explain why so little emphasis from pharmaceutical industry has been put into the further development of even promising natural preparations. in general, the search for potent, selective, nontoxic compounds that might be developed further to a drug substance is a multidisciplinary, time-and cost-consuming process. therefore, strategies for a target-oriented discovery of lead-structures either from nature or synthesis are in high demand. some of them will be discussed in the following section, providing examples from anti-hrv research. as already mentioned, nature provides an extremely rich pool of bioactive natural products. it is however a challenging endeavor to find exactly those compounds that show an activity on the focused target. in natural product research hints from folk medicine are a valuable starting point to dig for lead structures of certain interest. the majority of active principles from higher plants has been discovered as a result of ethnopharmacologically directed pharmacognostic research. 312, 313 oral or written indications for a beneficial application of a natural material first need a critical evaluation as to the selection of the correct material, its medicinal preparation, the kind of application, and overall the pharmacological profile. a rational criticism of often anecdotal efficacy from traditional medicine is a mandatory attitude to avoid overinterpretation of handed-down information. 314 in case of pretended anti-hrv remedies, the reported biological efficacy obviously suffers from being restricted to respiratory diseases, which might be caused by a panel of bacterial or viral infections. thus, an approved remedy may affect the microbes, or show an immune modulating or even antiinflammatory effect, or a combination of these. the holistic access is an innate character of ethnopharmacology and needs to be tracked down to the specifically involved target/s. in a recently published study focusing on the discovery of hrv-capsid binders from nature using a pharmacophore-based virtual screening of an ethnopharmacologically biased 3d-multiconformational database, some 30 secondary metabolites were predicted to act as capsid-binding inhibitors of hrv. 144 for an in depth phytochemical and pharmacological investigation, it was mandatory to focus on one promising natural material. thus, we consulted the ethnobotanical source materia medica, which was written by pedanius dioscorides in the 1 st century ad. the treatise consisting of five books comprises some 1,000 or so drugs derived from minerals, animals, and the majority of them from plants (800). it represents a great repository of botanical, medical, and pharmacological lore. scrutinizing the natural materials underlying the obtained virtual hits we came across asafetida, which is a gum resin gained from the roots of a variety of foul-smelling ferula species from the apiaceae family. based on the descriptions given in the materia medica there is strong evidence that the juice (i.e. resin) of the popular ancient silphion originating from media and syria corresponds to asafetida (book iii, cap. 80). 315 yielded by incision of the root and stalk and frequently mixed with sagapenon, i.e. the resin of f. persica willd (book iii, cap. 81), it is reported to be effective in the context of upper respiratory diseases, e.g. ''for chronic harshness of the throat,'' ''it clears the voice,'' ''shrinks the uvulas,'' ''suitable for a cough,'' ''for pleurisy,'' ''for chest pain;'' sagapenon is described as follows: ''it clears thick matters from the lungs,'' ''given to those who are chilled.'' 315 these descriptions finally helped to prioritize those virtual hits, which have been reported to be constituents of asafetida. 316 the pharmacological investigation of asafetida and its constituents farnesiferol b and c ( fig. 5 ; table iii ) revealed a distinct anti-hrv-2 effect in the low micromolar range using a cpe inhibitory assay. 143 the results of this study provided a rationale for the ancient usage of asafetida for upper respiratory tract infections. on the other hand, the traditionally manifested evidence for asafetida for the treatment of common cold symptoms substantially helped in the selection of this plant material in the search for anti-hrv capsid binders. 144 typically, an ethnopharmacologically based discovery of an active (anti-hrv) extract is followed by a bioassay guided fractionation, and the isolation of those constituents that are responsible for the extracts' bioactivity. the concept of a bioassay-guided approach is the fractionation accompanied by simultaneous detection of the activity during the separation steps, which results in a continuous enrichment, and finally in the isolation of the active ingredient/s. in this way a large number of anti-hrv agents from natural sources have already been discovered. semple et al. investigated the active principle of the asteraceae plant pterocaulon sphacelatum, which has been used in traditional medicine of aboriginal people of australia as a favored treatment for respiratory infections, especially colds. by means of an antiviral activity-guided fractionation measuring the poliovirus-induced cpe assay, the authors identified the flavonoid 3,7,3 0 -trimethoxy-5,6,4 0 -trihydroxyflavone, i.e. chrysosplenol c ( fig. 5 ; table iii ) as potent and specific inhibitor of the picornaviral replication. 6,7,8-trimethoxycoumarin ( fig. 5 ; table iii) was also isolated as a major constituent from the ethanolic extract of p. sphacelatum, but showed no activity against poliovirus. 317 interestingly, this coumarin exhibited a significant effect in the hrv-2-induced cpe assay in a recently performed virtual parallel screening study performed in our laboratory. 318 in contrast to the isolated coumarin, chrysosplenol c was already known as a member of the 4 0 -hydroxy-3-methoxyflavones, which represent potent and specific inhibitors of picornaviral, especially rhinoviral replication. [319] [320] [321] in 1993, vanden berghe, haemers, and vlietinck provided a profound survey of antiviral agents from higher plants, and demonstrated the impact of ethnobotanical knowledge in their search for antiviral compounds from african medicinal plants. the selection of investigated plant species was mainly based upon their use in the treatment of viral diseases by african traditional healers. 320 the antiviral activity of 100 different plant species belonging to 33 families was investigated; thereof 21 species exhibited prominent antiviral properties against one or more of the tested viruses. the most pronounced activity against picornaviruses was recorded within the genus euphorbia. all compounds detected as antiviral constituents from the respective extracts were identified as 3-methoxyflavone derivatives, especially 3-methylethers of quercetin and kaempferol ( fig. 5 ; table iii ). they showed no significant cytotoxicity and were highly active in tissue culture against all human picornaviruses. in tissue culture, cells infected with different picornaviruses (among them also hrv) no cpe was observed, when cells had been treated with 2 mg/ml of these 3-methoxyflavones. 320 the selection of natural materials based on ethnopharmacology is a profound rationale for lead structure discovery and highly superior to random selection. this however rarely applies to marine organisms, fungi, and microbes. although these organisms are esteemed as highly valuable source for bioactive metabolites, 322 hardly any records from folk medicine are given for them. a medium-sized activity screening using a robust cell-based or in vitro-assay with reasonable effort as to time and costs is a strategy to get a first insight into the antiviral activities of extracts, fractions, and compounds from synthesis or nature. for the discovery of novel naturally based hrv 3c-protease inhibitors, singh et al. used a small peptide containing q-g scissile bond as substrate for the in vitro screening of extracts. the authors isolated the novel benzoisochromanquinone (1)-thysanone ( fig. 5 ; table iii ) from an extract of the fungus thysanophora penicilloides with potent hrv 3c-protease inhibitory activity. 323 a continued screening revealed a pronounced hrv 3c-protease inhibitory activity for the extract of the chinese herb polygonum cuspidatum. by bioassay-guided fractionation 2-methoxystypandrone ( fig. 5 ; table iii ), a naphthoquinone, with an ic 50 value of 4.6 mm, was isolated from the plant material. the total syntheses of this natural compound and further analogues allowed for a structure-activity relationship, and particularly the comparison between activities of ortho-vs. para-quinones. to measure the selectivity of the compound series against cystein proteases other than hrv 3c-protease, the compounds were evaluated against papain. the simple 9,10-phenanthraquinone ( fig. 5 ; table iii ) was the most active compound of the series and showed an ic 50 value of 1.4 mm with a distinctly higher degree of selectivity than 2-methoxystypandrone. 324 a cpe reduction assay was applied for the identification of raoulic acid, a bicyclic c25 terpene acid ( fig. 5 ; table iii ) isolated from raoulia australis (asteraceae). raoulic acid exerted an antiviral activity against coxsackie virus b3, b4, enterovirus 71, and hrv-2 and -3 with ic 50 values in the submicromolar range. no activity was recorded against influenza a and b viruses. 325 in the course of the systematic screening of microbial and natural products for anti-hrv activity, ishitsuka et al. identified 4 0 ,5-dihydroxy-3,3 0 ,7-trimethoxyflavone ( fig. 5 ; table iii) from the leaves of the chinese medicinal plant agastache rugosa (lamiaceae) as natural compound with high activity against all picornaviruses except mengovirus. the authors synthesized the orally active 4 0 ,5-diacetyloxy-3,3 0 ,7-trimethoxyflavone and performed investigations in tissue cultures and in mice to determine the compound's mode of action. this was assumed to be the process of viral replication, thus located between viral uncoating and initiation of viral rna synthesis. 326 the activity of flavan ( fig. 5 ; table iii ) was discovered serendipitously during a screening program by using a plaque inhibition assay. 194 based on these results, bauer et al. synthesized 4 0 ,6-dichloroflavan (bw863c, fig. 2 ; table ii) , which revealed an activity against a number of hrv serotypes in the range between 0.007 and 10 mm. as soon as an active lead compound is identified it is of utmost importance to scrutinize the derivatives' activity to (i) obtain insight into the chemical requirements mandatory for the focused biological activity, (ii) improve the compound's pharmacological profile in terms of potency, selectivity, cytotoxicity, etc., and to (iii) improve its bioavailability. the nonphenolic aporphine alkaloid glaucine is a prominent constituent of the aerial parts of gaucium flavum (papaveraveae). in a recently published study, spasova et al. investigated the antiviral potential of glaucine ( fig. 5 ; table iii) , glaucine derivatives, and its semi-synthesized 3-aminoethylglaucine cinnamoyl-and hydroxycinnamoyl amides. beside the anti-oxidative potential of the newly synthesized compounds, they all exerted an antiviral activity against the replication of hrv-14. the best anti-hrv activity was observed for oxoglaucine ( fig. 5 ; table iii; ic 50 table iii; ic 50 15 mm, si 10.3 and ic 50 13 mm, si 11, respectively). 327 the early findings of the anti-hrv active naturally derived 3-methoxyflavone inspired chemists and pharmacologists in the synthesis and the pharmacological evaluation of derivatives thereof decorated with various substitution patterns. several reports published during the last two decades reviewed the findings of antiviral flavonoid research. 320, 328, 329 by investigating the antiviral activity of a wide variety of naturally occurring flavonoids, tsuchiya et al. found chrysosplenol b and c ( fig. 5 ; table iii ) contained in chrysosplenium plants, and axillarin ( fig. 5 ; table iii ) as potent anti-hrv agents. based on their findings, the flavone skeleton decorated with a methoxy group in position 3 and a 5-hydroxyl group revealed as mandatory for an anti-hrv activity. 319 a series of antipicornaviral 4 0 -hydroxy-3-methoxyflavone derivatives was synthesized by de meyer et al. in order to establish a structure-activity relationship. thereby, different substitution patterns of the a-ring system with methyl, hydroxy, methoxy, halo, nitro, and amino was performed. their activity against polio and hrv was compared with those of naturally occurring flavonoids. further, the importance of the 4 0 hydroxyl-group and of the 3-methoxyl-group was confirmed by investigation of different derivatives lacking these features. 321 the results showed that 4 0 -hydroxy-3-methoxyflavones with a monosubstituted a-ring are less active than the corresponding compounds having a polysubstituted a-ring. within the tested series of compounds, 4 0 ,7-dihydroxy-3-methoxy-5,6-dimethylflavone emerged not only as noncytotoxic but also as most potent substance in both antiviral test systems. the lowest concentrations for this compound that protect 50% of the cells from cpe of 12 hrv serotypes were in the range from 0.016 to 0.5 mg/ml. in contrast to quercetin, this flavone was also reported to have no mutagenic properties (measured up to 2.5 mg/plate) in a short-term microbial assay. 321 the mechanism studies performed with 3-methoxyflavones have shown an interference with an early stage in the viral rna synthesis; no induction of resistance was observed. 320 in contrast to this mode of action, the anti-hrv chalcones and flavans are reported to interact directly with specific sites on the viral capsid proteins, thereby preventing uncoating and the consequent liberation of viral rna. 193, 330, 331 due to its anti-hrv potency, low toxicity, and promising bioavailability, dichloroflavan was evaluated for its protective efficacy against experimental hrv-infection in two clinical, double-blind, placebo-controlled trials. however, the drug candidate failed either when administered orally or intranasally. 332, 333 unfortunately, till now no clinical trials evaluating the efficacy of 3-methoxyflavones in common cold have been performed. the common idea of all computational approaches is to extract knowledge from a more or less large set of data in order to make predictions of new events. 334 within the lead discovery process, computational approaches, such as virtual screening, docking, quantitative structure-activity relationship, have largely enhanced the impact of computational chemistry and nowadays chemoinformatics plays a predominant role in drug research. 335 the key goal of the use of such methods is to reduce the overall cost associated to the discovery and development of a new drug by identifying the most promising candidates to focus the experimental efforts on. a number of books and reviews on the impact of computational chemistry for lead structure determination highlight these efforts. [336] [337] [338] [339] in general, in silico methods can be divided into (i) ligand-based approaches, which rely on known active compounds. based on their physicochemical properties crucial for biological affinity, activities are predicted by extrapolation on not-yet tested substances, e.g. machine learning techniques and classical quantitative structure-activity relationship (qsar). ligand-based approaches are invaluable tools in cases where no structural information about the pharmacological targets is available. (ii) on the other hand, structurebased approaches use experimentally determined 3d structures of the targets, such as molecular docking or structure-based pharmacophore modeling for virtual screening. these methods allow for gaining insight into protein-ligand interactions at an experimentally determined (static) level (however not considering flexibility). a unique platform containing 3d coordinates of experimentally solved protein structures (by x-ray crystallography or nmr) is the protein data bank (pdb) currently comprising more than 50,000 structures of biomolecules and protein-ligand complexes. 340 additionally, there are several pdb-related web services and tools, which enable to use the pdb-portal in a rich diversity of information services for students and scientists. 341 particularly in the early stage of drug development, such as lead discovery and lead optimization, computational approaches allow for a target-oriented and rationalized proceeding, and thus may substantially help to maximize the success rate. 338 a recently published review on the impact of computer-assisted approaches in antiviral research thoroughly describes underlying in silico techniques, and highlights the benefits of computational approaches for the discovery of antiviral lead structures. 342 in anti-hrv research the capsid protein and the protease 3c revealed to be promising targets (as described before). inhibitors are assumed to have a major impact for the treatment of hrv-infections. additionally, these targets are structurally elucidated, and some potent ligands are known as well. these facts enable the performance of sensible computer-assisted approaches, both ligand-and structure-based. some studies using an in silico approach for the discovery of potential anti-hrv agents focusing on the mentioned targets will be reported in the following paragraphs. for compounds acting as potential hrv-capsid binders, some classical qsar and 3d qsar studies have been performed. while in classical qsar, the relationship between 2d calculated properties derived from chemical structures and measured biological activities are explored statistically, 3d qsar techniques are aimed at deriving a correlation and in turn activity prediction based on spatial arrangements of chemical properties and atoms. applying the 3d qsar technique comfa (comparative molecular field analysis) statistical models are derived, which are visualized in color-coded contours around the molecule. therein spots indicate where electrostatic properties and spatial arrangements are favorable for biological activity. 343 in the studies of diana et al., artico et al., and verma et al., qsar techniques helped on one hand to analyze and rationalize the structural features of active compounds essential for the interaction to the hrv canyon's binding pocket, and on the other hand to search for new classes of capsid binders, thus to narrow the synthetic challenges for specific anti-hrv agents, respectively. [344] [345] [346] [347] [348] in all these investigations hydrophobicity was found to be one of the most important determinants of substance activity. qsar combined with simplex representation of molecular structure was applied by kuz'min et al. based on the selectivity index (cc 50 /ic 50 ) and the hrv-2 inhibitory concentration of a set of [(biphenyloxy)propyl]isoxazole derivatives. 188 on the basis of qsar analysis and computational design, three new isoxazoles with high activity prediction were selected and synthesized. they all revealed a strong coincidence between experimental and predicted anti-hrv activity and selectivity index. terminal benzene substituents with negative electrostatic potential and a molecule length of approximately 5.5-5.6 å have been suggested as mandatory features within this chemical class for a hrv-2 inhibitory activity. hrv-serotypes show a high level of conservation at the protease 3c binding site; sequence alignment and secondary structure predictions suggested an overall architecture and mechanism of hrv-3c proteases that correlates with cellular cystein-and serine proteases, such as chymotrypsin and trypsin. 349, 350 the identity among 3c proteases from different families is however modest and provides space for the development of specific inhibitors for hrv-3c protease. 350 in a recently published review on selective inhibitors of picornavirus replication, de palma et al. summarized all currently known chemical structures acting as peptidic or nonpeptidic inhibitors of this viral target. 156 in 2000, reich et al., used the hrv 3c protease co-crystal structure information to rationalize the target-oriented synthesis of hrv 3c protease inhibitors from the class of substituted benzamides. activity data and subsequent crystallographic studies pointed out important requirements for the inhibition of the 3c protease. 351 similarly, maugeri et al. used a structure-based approach, and performed docking studies based on the 3c protease crystal structure with a virtual library consisting of benzamide derivatives. quantum-mechanic calculation proposed substituents with most promising biological activities. this workflow guided the design and synthesis of substances virtually assumed to act as substrate analogues. synthesis of some of these compounds and biological testing confirmed the underlying hypothesis. 219 quantitative molecular modeling studies were performed to better define and predict interactions between bicyclic 2-pyridone derivatives that showed to be irreversible inhibitors of the 3c protease. in these studies molecular mechanics simulations to evaluate chemical rate of covalent bond formation and free energy calculation combined with crystallographic studies were applied to explain the differences in activity of some irreversible peptidomimetic inhibitors. these data were used as a basis for further optimization of these compounds. 224, 352 in a recent study performed by kuo et al. some 6,800 compounds were subjected to a high troughput screening in the search for novel inhibitors for both 3c and 3cl proteases from picornavirus and coronavirus, respectively. 217 five nonpeptides were identified with ic 50 values r10 mm against severe acute respiratory syndrome-coronavirus 3cl-protease; one molecule was found to additionally inhibit the 3c proteases of coxsackievirus, enterovirus, and rhinovirus. this compound (id 43146) contains a dihydropyrazole ring decorated with two phenyl groups and a lengthy n-butyl-benzimidazolylamino-toluene. it was used as starting point for the selection of further four analogs showing ic 50 values in the range of 0.5-10 mm against the tested viral proteases. by means of docking-based computer modeling, the authors tried to rationalize the binding discrepancies responsible for individual and common protease inhibitors, thus to provide a rational base for the development of nonpeptide multiple-function inhibitors against coronaviruses and piciornaviruses. in anti-hrv research, first application scenarios have been conducted using pharmacophore models. according to the official iupac definition by wermuth et al., 353 a pharmacophore describes the 3d arrangement of steric and electronic features necessary to trigger or block a biological response. pharmacophores can be represented by three-dimensional chemical features, which include hydrogen bond donors and acceptors, aromatic rings, hydrophobic groups, as well as positive and negative ionisable moieties. additionally, the shape of ligands can be represented by shape features, which essentially describe the van der waals radii of the ligand atoms. the pharmacophore concept has proven to be successful, not only in rationalizing structure-activity relationships but also by its large impact in developing appropriate 3d-tools for efficient virtual screening. 336, 354 steindl et al. elaborated ligand-and structure-based pharmacophore models implementing the essential feature of the covalent binding to the cysteine 147 in the active site of the hrv-2 3c protease. thus, the in silico approach focused on defining a new pharmacophore feature representing a target structure for nucleophilic addition in the ligands, which is a crucial step for protease inactivation. the generated hypotheses retrieved known 3c protease inhibitors in the virtual screening cycle, and proposed potential (unconfirmed) ligands of the 3d protease binding site from available databases. 355 the viral capsid of several hrvs has been elucidated by crystallization and resolution of the 3d-structure. the hrv coat protein complexed with its highly active inhibitor win 61209 was used as starting point for the generation of structure-based pharmacophore models by . the models were used for virtual screening of a large commercially available 3d database. for final selection of virtual hits worth to be subjected to biological testing, docking studies and principal component analysis were performed. six candidates were tested for their ability to inhibit hrv serotype 39 by multiple-cycle cpe inhibition assay. although all of them showed a certain antiviral potential, one longitudinal piperazine derivative inhibited the virus at a concentration below 10 mm. some of the test candidates showed difficulties in the interpretation of experimental results due to their relatively high cytotoxicity and bad solubility. this circumstance asks for more cautious estimation of molecular properties for compound selection as stated by the authors. 356 in a subsequently performed study, the best validated pharmacophore model was used for the identification of naturally derived hrv coat protein inhibitors. 144 for virtual screening experiments the in-house generated 3d-database dios was used (as described before). based on the virtually predicted ligands and considering knowledge from traditional use, sesquiterpene umbelliferons from the gum resin of ferula sp., i.e. asafetida, were finally selected as most promising candidates. for biological evaluation, the antiviral activities of asafetida and its isolated constituents were assessed by an exploratory determination of the inhibition of the cpe induced by hrv serotypes 1a, 2, 14, and 16. the results revealed a dose-dependent and selective anti-hrv activity against serotype 2 for asafetida and its virtually predicted constituents, farnesiferols b and c ( fig. 5 ; table iii ; ic 50 : 2.6 and 2.5 mm, respectively). to scrutinize the selectivity of these two compounds against hrv-2 in comparison to the other tested serotypes, the amino acid sequences of hrv-2 and hrv-16 vp1 were aligned. since all amino acid residues involved in ligand binding showed 100% match in both serotypes, the experimentally determined selectivity profile could not be explained by different binding pockets. the serotype alignment does however not reflect potential protein flexibility during binding, which might differ between the hrv serotypes. additionally, off-target effects could be a reason for the observed selectivity. in a recently performed virtual parallel screening approach, we tried to identify potential targets of human pathological relevance for 16 constituents isolated and identified from the medicinal plant ruta graveolens. 318 using the screening platform pipline pilot 357 included in discovery studio, 358 low-energy conformers of the 16 identified molecules from r. graveolens were subjected to parallel screening. for this purpose, the inte:ligand pharmacophore model collection was used. 359 it currently comprises 2.208 models covering 280 unique pharmacological targets. based on the predicted ligand-target interactions, the authors focused on three biological targets, namely acetylcholinesterase, the hrv coat protein, and the cannabinoid receptor type 2. virtual hits and nonhits were assayed on their respective targets for a critical evaluation of the performed target-fishing approach. beside other predicted bioactivities, determination of their cpes on hrv-2 revealed the virtual hit arborinine ( fig. 5 ; table iii; ic 50 : 3.2 mm) and the nonpredicted hit 6,7,8-trimethoxycoumarin ( fig. 5 ; table iii ; ic 50 : 12.9 mm) as the most active anti-hrv constituents. it could be shown that the applied in silico strategy has the capacity of catalyzing drug discovery profoundly for all those diseases where molecular targets or molecular ligands are well defined to create reliable pharmacophore models. hrv, a prominent member of the picornaviridae family, infects humans more frequently than any other virus. infections with hrv mainly lead to upper respiratory diseases, such as the common cold, but may also cause more severe lower respiratory tract disorders. although the symptomology and severity of the common cold is relatively mild and the course of disease self-limiting, the socio-economic impact is tremendous in terms of recouping lost productivity due to sick leave. in the last decades, a number of anti-hrv agents from different origin-synthetics, natural compounds, biologicals, botanicals, and nutritionals-have been discovered. different concepts have been used as strategy for their discovery either starting from a phenomenological effect, e.g. empirical knowledge from folk medicine, or from a targetbased molecular level, e.g. icams, capsid binders, and hrv protease inhibitors. some of the outcomes revealed potent and promising activities that partly have been evaluated for the management of hrv-induced common colds in clinical trials mainly with sobering benefit. since the hrv infection is not life-threatening in most cases, a potential therapy has to be safe and effective with an almost unrecognizable level of side effects. these preconditions render the search for anti-hrv-agents into a high challenging endeavor and explains why no antiviral agent is approved for the prevention or treatment of hrv-infection until today, despite the significant efforts. moreover, the high variability of rhinoviruses suggests the need of more than one active principle covering different modes of action for an effective treatment. therefore, there is a high need for an ongoing search for new synthetic as well as natural compounds on a molecular level. the increasing knowledge about the hrv life cycle, gene, and protein sequence combined with the improved technologies in the field of experimental and computational methods have continuously enabled further insights into the spectacular world of hrv. only with this fundamental research, virtual screening approaches, such as qsar, pharmacophore-and docking-based screening cycles, or computational compound design, are applicable on a rational base. with the gained expertise from different disciplines and an adequate infrastructure for further research, it is encouraging to hope that discovery and clinical development efforts will continue in the search for agents that may treat or prevent the annoying common cold. picornavirales, a 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serotype 2 (hrv2) human rhinovirus 3 at 3.0 a resolution the canyon hypothesis viral cell recognition and entry crystallographic and cryo em analysis of virion-receptor interactions a cell adhesion molecule, icam-1, is the major surface receptor for rhinoviruses the minor receptor group of human rhinovirus (hrv) includes hrv23 and hrv25, but the presence of a lysine in the vp1 hi loop is not sufficient for receptor binding members of the low density lipoprotein receptor family mediate cell entry of a minor-group common cold virus multiple receptors involved in human rhinovirus attachment to live cells structural studies of two rhinovirus serotypes complexed with fragments of their cellular receptor the cellular receptor to human rhinovirus 2 binds around the 5-fold axis and not in the canyon: a structural view the major and minor group receptor families contain all but one human rhinovirus serotype human rhinovirus type 54 infection via heparan sulfate is less efficient and strictly dependent on low endosomal ph human rhinovirus type 89 variants use heparan sulfate proteoglycan for cell attachment internalization of human rhinovirus 14 into hela and icam-1-transfected bhk cells viral evolution toward change in receptor usage: adaptation of a major group human rhinovirus to grow in icam-1-negative cells rhinovirus-mediated endosomal release of transfection complexes major and minor receptor group human rhinoviruses penetrate from endosomes by different mechanisms cryoelectron microscopy analysis of the structural changes associated with human rhinovirus type 14 uncoating formation of rhinovirus-soluble icam-1 complexes and conformational changes in the virion conformational changes, plasma membrane penetration, and infection by human rhinovirus type 2: role of receptors and low ph uncoating of human rhinovirus serotype 2 from late endosomes x-ray structure of a minor group human rhinovirus bound to a fragment of its cellular receptor protein the expression and purification of human rhinovirus protease 3c polypeptide 2a of human rhinovirus type 2: identification as a protease and characterization by mutational analysis the eif4g-eif4e complex is the target for direct cleavage by the rhinovirus 2a proteinase purification of two picornaviral 2a proteinases: interaction with eif-4 gamma and influence on in vitro translation 2a proteinases of coxsackie-and rhinovirus cleave peptides derived from eif-4 gamma via a common recognition motif conservation of amino acids in human rhinovirus 3c protease correlates with broad-spectrum antiviral activity of rupintrivir, a novel human rhinovirus 3c protease inhibitor fields virology rhinovirus infects primary human airway fibroblasts and induces a neutrophil chemokine and a permeability factor basal cells of differentiated bronchial epithelium are more susceptible to rhinovirus infection resistance of differentiated human airway epithelium to infection by rhinovirus effect of isoflavans and isoflavenes on rhinovirus 1b and its replication in hela cells enantiomeric effects of homologues of disoxaril on the inhibitory activity against human rhinovirus-14 in vitro activity of win 51711, a new broad-spectrum antipicornavirus drug two groups of rhinoviruses revealed by a panel of antiviral compounds present sequence divergence and differential pathogenicity an orally bioavailable oxime ether capsid binder with potent activity against human rhinovirus in vitro activity of expanded-spectrum pyridazinyl oxime ethers related to pirodavir: novel capsid-binding inhibitors with potent antipicornavirus activity novel [(biphenyloxy)propyl]-isoxazole derivatives for inhibition of human rhinovirus 2 and coxsackievirus b3 replication structure-based virtual screening for the discovery of natural inhibitors for human rhinovirus coat protein in vitro resistance study of rupintrivir, a novel inhibitor of human rhinovirus 3c protease establishment of a mouse model for human rhinovirus infection human rhinovirus 1b exposure induces phosphatidylinositol 3-kinase-dependent airway inflammation in mice mouse models of rhinovirus-induced disease and exacerbation of allergic airway inflammation prophylactic activity of intranasal enviroxime against experimentally induced rhinovirus type 39 infection intranasal interferon-alpha 2 treatment of experimental rhinoviral colds topical enviroxime against rhinovirus infection therapeutic activity of enviroxime against rhinovirus infection in volunteers efficacy of oral win 54954 for prophylaxis of experimental rhinovirus infection ineffectiveness of echinacea for prevention of experimental rhinovirus colds current status of anti-picornavirus therapies selective inhibitors of picornavirus replication inhibitors of 3c cysteine proteinases from picornaviridae isolation of a monoclonal antibody that blocks attachment of the major group of human rhinoviruses an antibody fragment from a phage display library competes for ligand binding to the low density lipoprotein receptor family and inhibits rhinovirus infection modification of experimental rhinovirus colds by receptor blockade viral receptor blockage by multivalent recombinant antibody fusion proteins: inhibiting human rhinovirus (hrv) infection with cfy196 comparative antirhinoviral activities of soluble intercellular adhesion molecule-1 (sicam-1) and chimeric icam-1/immunoglobulin a molecule a soluble form of intercellular adhesion molecule-1 inhibits rhinovirus infection spectrum of activity of soluble intercellular adhesion molecule-1 against rhinovirus reference strains and field isolates soluble ldl minireceptors. minimal structure requirements for recognition of minor group human rhinovirus neutralization of a common cold virus by concatemers of the third ligand binding module of the vldl-receptor strongly depends on the number of modules rhinovirus-stabilizing activity of artificial vldl-receptor variants defines a new mechanism for virus neutralization by soluble receptors in vitro studies of the antirhinovirus activity of soluble intercellular adhesion molecule-1 mechanisms of receptor-mediated rhinovirus neutralization defined by two soluble forms of icam-1 efficient neutralization and disruption of rhinovirus by chimeric icam-1/immunoglobulin molecules recombinant soluble low density lipoprotein receptor fragment inhibits minor group rhinovirus infection in vitro prevention of rhinovirus infection in chimpanzees by soluble intercellular adhesion molecule-1 efficacy of tremacamra, a soluble intercellular adhesion molecule 1, for experimental rhinovirus infection: a randomized clinical trial inhibitors of picornavirus replication the structure of antiviral agents that inhibit uncoating when complexed with viral capsids structural and virological studies of the stages of virus replication that are affected by antirhinovirus compounds structural analysis of antiviral agents that interact with the capsid of human rhinoviruses conformational change in the floor of the human rhinovirus canyon blocks adsorption to hela cell receptors the site of attachment in human rhinovirus 14 for antiviral agents that inhibit uncoating a comparison of the anti-rhinoviral drug binding pocket in hrv14 and hrv1a analysis of three structurally related antiviral compounds in complex with human rhinovirus 16 antiviral capsid-binding compounds can inhibit the adsorption of minor receptor rhinoviruses in vitro activity of pleconaril and ag7088 against selected serotypes and clinical isolates of human rhinoviruses activity of pleconaril against enteroviruses susceptibility of coxsackievirus b3 laboratory strains and clinical isolates to the capsid function inhibitor pleconaril: antiviral studies with virus chimeras demonstrate the crucial role of amino acid 1092 in treatment in vitro activity of r 61837, a new antirhinovirus compound in vitro activity of pirodavir (r 77975), a substituted phenoxy-pyridazinamine with broad-spectrum antipicornaviral activity quantitative structure-activity relationship studies of [(biphenyloxy)-propyl]isoxazole derivatives. inhibitors of human rhinovirus 2 replication sch 48973: a potent, broad-spectrum, antienterovirus compound antipicornavirus activity of sch 47802 and analogs: in vitro and in vivo studies sch 38057: a picornavirus capsid-binding molecule with antiviral activity after the initial stage of viral uncoating direct and specific inactivation of rhinovirus by chalcone ro 09-0410 comparative studies on the antirhinovirus activity and the mode of action of the rhinovirus capsid-binding agents, chalcone amides 4 0 ,6-dichloroflavan (bw683c), a new anti-rhinovirus compound 3,4-dihydro-2-phenyl-2h-pyrano[2,3-b]pyridines with potent antirhinovirus activity antipicornavirus activity of substituted phenoxybenzenes and phenoxypyridines activity of 2-(3,4-dichlorophenoxy)-5-nitrobenzonitrile (mdl-860) against picornaviruses in vitro application of crystallography to the design of antiviral agents clinical activity of pleconaril in an experimentally induced coxsackievirus a21 respiratory infection oral pleconaril treatment of picornavirus-associated viral respiratory illness in adults: efficacy and tolerability in phase ii clinical trials efficacy and safety of oral pleconaril for treatment of colds due to picornaviruses in adults: results of 2 double-blind, randomized, placebo-controlled trials relationship of pleconaril susceptibility and clinical outcomes in treatment of common colds caused by rhinoviruses the effect of oral pleconaril on hepatic cytochrome p450 3a activity in healthy adults using intravenous midazolam as a probe safety and efficacy of intranasal pirodavir (r77975) in experimental rhinovirus infection inhibition of polyprotein processing and rna replication of human rhinovirus by pyrrolidine dithiocarbamate involves metal ions nitric oxide inhibits dystrophin proteolysis by coxsackieviral protease 2a through s-nitrosylation: a protective mechanism against enteroviral cardiomyopathy an antiviral mechanism of nitric oxide: inhibition of a viral protease nitric oxide inhibition of coxsackievirus replication in vitro nitric oxide donors inhibit the coxsackievirus b3 proteinases 2a and 3c in vitro, virus production in cells, and signs of myocarditis in virus-infected mice effect of nitric oxide on rhinovirus replication and virus-induced interleukin-8 elaboration inhibition of proteolytic activity of poliovirus and rhinovirus 2a proteinases by elastase-specific inhibitors an antiviral peptide inhibitor that is active against picornavirus 2a proteinases but not cellular caspases antiviral activity of caspase inhibitors: effect on picornaviral 2a proteinase dual inhibition of human rhinovirus 2a and 3c proteases by homophthalimides structure of human rhinovirus 3c protease reveals a trypsin-like polypeptide fold, rna-binding site, and means for cleaving precursor polyprotein individual and common inhibitors of coronavirus and picornavirus main proteases structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3c protease with potent antiviral activity against multiple rhinovirus serotypes new anti-viral drugs for the treatment of the common cold structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 1. michael acceptor structure-activity studies structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 2. peptide structure-activity studies structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 6. structure-activity studies of orally bioavailable, 2-pyridone-containing peptidomimetics structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 3. structure-activity studies of ketomethylene-containing peptidomimetics structurebased design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 8. pharmacological optimization of orally bioavailable 2-pyridone-containing peptidomimetics structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 4. incorporation of p1 lactam moieties as l-glutamine replacements in vitro antiviral activity of ag7088, a potent inhibitor of human rhinovirus 3c protease inhibition of human rhinovirus-induced cytokine production by ag7088, a human rhinovirus 3c protease inhibitor pharmacokinetics and safety of an antirhinoviral agent, ruprintrivir, in healthy volunteers phase ii, randomized, double-blind, placebo-controlled studies of ruprintrivir nasal spray 2-percent suspension for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers in vitro antiviral activity and single-dose pharmacokinetics in humans of a novel, orally bioavailable inhibitor of human rhinovirus 3c protease gaining target access for deoxyribozymes a morpholino oligomer targeting highly conserved internal ribosome entry site sequence is able to inhibit multiple species of picornavirus small interfering rna molecules as potential anti-human rhinovirus agents: in vitro potency, specificity, and mechanism mode of action of 2-furylmercury chloride, an antirhinovirus compound methoxyflavones as potent inhibitors of viral-induced block of cell synthesis antiviral effects of pyrrolidine dithiocarbamate on human rhinoviruses broad-spectrum antiviral and cytocidal activity of cyclopentenylcytosine, a carbocyclic nucleoside targeted at ctp synthetase in vitro antiviral activity of ribavirin against picornaviruses another ten stories in antiviral drug discovery (part c): ''old'' and ''new'' antivirals, strategies, and perspectives the broadspectrum antiviral ribonucleoside ribavirin is an rna virus mutagen ribavirin: recent insights into antiviral mechanisms of action inhibition of rhinovirus replication in in organ culture by a potential antiviral drug synthesis of syn and anti isomers of 6-[[(hydroxyimino)phenyl]methyl]-1-[(1-methylethyl)sulfonyl]-1h-benzimidaz ol-2-amine. inhibitors of rhinovirus multiplication comparative studies on the modes of action of the antirhinovirus agents ro 09-0410, ro 09-0179, rmi-15,731, 4 0 ,6-dichloroflavan, and enviroxime the antiviral compound enviroxime targets the 3a coding region of rhinovirus and poliovirus sequence determinants of 3a-mediated resistance to enviroxime in rhinoviruses and enteroviruses evidence that enviroxime targets multiple components of the rhinovirus 14 replication complex controlled trial of enviroxime against natural rhinovirus infections in a community the activity of enviroxime against rhinovirus infection in man activity against rhinoviruses, toxicity, and delivery in aerosol of enviroxime in liposomes 2-amino-3-substituted-6-[(e)-1-phenyl-2-(n-methylcarbamoyl)vinyl]imid azo[1,2-a]pyridines as a novel class of inhibitors of human rhinovirus: stereospecific synthesis and antiviral activity short synthesis and anti-rhinoviral activity of imidazo[1,2-a]pyridines: the effect of acyl groups at 3-position synthesis, antiviral activity, and biological properties of vinylacetylene analogs of enviroxime synthesis and antiviral activity of c2 analogs of enviroxime: an exploration of the role of critical functionality sensitivity of rhinoviruses to human leukocyte and fibroblast interferons cell and virus sensitivity studies with recombinant human alpha interferons comparative susceptibility of respiratory viruses to recombinant interferons-alpha 2b and -beta intranasal interferon-alpha 2 for prevention of natural rhinovirus colds intranasal interferon alpha 2 for prevention of rhinovirus infection and illness interferon-beta ser as prophylaxis against experimental rhinovirus infection in volunteers an effective dosage regimen for prophylaxis against rhinovirus infection by intranasal administration of huifn-alpha 2 efficacy and tolerance of intranasally applied recombinant leukocyte a interferon in normal volunteers intranasally applied recombinant leukocyte a interferon in normal volunteers. ii. determination of minimal effective and tolerable dose recombinant human interferon-gamma as prophylaxis against rhinovirus colds in volunteers ineffectiveness of postexposure prophylaxis of rhinovirus infection with low-dose intranasal alpha 2b interferon in families a randomized controlled trial of glucocorticoid prophylaxis against experimental rhinovirus infection synergism between anti-rhinovirus antivirals: various human interferons and a number of synthetic compounds failure to demonstrate synergy between interferon-alpha and a synthetic antiviral, enviroxime, in rhinovirus infections in volunteers property distributions: differences between drugs, natural products, and molecules from combinatorial chemistry a new rapid and effective chemistry space filter in recognizing a druglike database rational selection of structurally diverse natural product scaffolds with favorable adme properties for drug discovery natural products as sources of new drugs over the last 25 years ethnomedicinal antivirals: scope and opportunity plant substances as antiviral agents: an update plant substances as antiviral agents host defense function of the airway epithelium in health and disease: clinical background herb sales down 6 percent in mainstream market echinacea for preventing and treating the common cold echinacea for preventing and treating the common cold. the cochrane database of systematic reviews evaluation of echinacea for the prevention and treatment of the common cold: a meta-analysis echinacea in the prevention of induced rhinovirus colds: a meta-analysis the role of alkamides as an active principle of echinacea alkylamides from echinacea are a new class of cannabinomimetics: cannabinoid type 2 receptordependent and -independent immunomodulatory effects synergistic anti-oxidative effects of alkamides, caffeic acid derivatives, and polysaccharide fractions from echinacea purpurea on in vitro oxidation of human low-density lipoproteins echinacea extracts modulate the pattern of chemokine and cytokine secretion in rhinovirus-infected and uninfected epithelial cells modulation of immune response gene expression by echinacea extracts: results of a gene array analysis preventing the common cold with a garlic supplement: a double-blind, placebocontrolled survey in vitro virucidal effects of allium sativum (garlic) extract and compounds enhancement of in vitro human immune function by allium sativum l. (garlic) fractions efficacy of cold-fx in the prevention of respiratory symptoms in community-dwelling adults: a randomized, doubleblinded, placebo controlled trial efficacy of an extract of north american ginseng containing poly-furanosyl-pyranosyl-saccharides for preventing upper respiratory tract infections: a randomized controlled trial safety and tolerability of north american ginseng extract in the treatment of pediatric upper respiratory tract infection: a phase ii randomized, controlled trial of 2 dosing schedules inhibitory effect of ginsenoside rg3 and its derivative ginsenoside rg3-2h on no production and lymphocyte proliferation effect of cvt-e002 (cold-fx) versus a ginsenoside extract on systemic and gut-associated immune function inhibitory effect of ginseng polysaccharides on rotavirus infection antiviral activity of dammarane saponins against herpes simplex virus i effect of a traditional japanese herbal medicine, hochu-ekki-to (bu-zhong-yi-qi tang), on immunity in elderly persons effects of five kampohozais on the mitogenic activity of lipopolysaccharide, concanavalin a, phorbol myristate acetate and phytohemagglutinin in vivo immunological restoration and anti-tumor effect by japanese herbal medicine in aged mice antiinflammatory effects of bu-zhong-yi-qi-tang in patients with perennial allergic rhinitis hochu-ekki-to inhibits rhinovirus infection in human tracheal epithelial cells efficacy of a pelargonium sidoides preparation in patients with the common cold: a randomized, double blind, placebo-controlled clinical trial kern winfried v. pelargonium sidoides extract for acute respiratory tract infections traditionally used pelargonium species: chemistry and biological activity of umckaloabo extracts and their constituents aqueous ethanolic extract of the roots of pelargonium sidoides-new scientific evidence for an old anti-infective phytopharmaceutical efficacy of an aqueous pelargonium sidoides extract against herpesvirus protective effect of a natural carrageenan on genital herpes simplex virus infection in mice polysaccharides as antiviral agents: antiviral activity of carrageenan iota-carrageenan is a potent inhibitor of rhinovirus infection recommendations for reporting randomized controlled trials of herbal interventions: explanation and elaboration ethnobotany and the search for new drugs: ciba foundation symposium 185 ethnobotany and natural products: the search for new molecules, new treatments of old diseases or a better understanding of indigenous cultures? how scientific is the science in ethnopharmacology? historical perspectives and epistemological problems pedanius dioscorides of anazarbus-de materia medica sesquiterpene coumarins from ferula assafoetida l antiviral flavonoid from pterocaulon sphacelatum, an australian aboriginal medicine in silico target fishing for rationalized ligand discovery exemplified on constituents of ruta graveolens l antiviral activity of natural occurring flavonoids in vitro antiviral agents from higher plants and example of structure-activity relationship of 3-methoxyflavones 0 -hydroxy-3-methoxyflavones with potent antipicornavirus activity biodiversity: a continuing source of novel drug leads structure and stereochemistry of thysanone: a novel human rhinovirus 3c-protease inhibitor from thysanophora penicilloides discovery, total synthesis, hrv 3c-protease inhibitory activity, and structure-activity relationships of 2-methoxystypandrone and its analogues antiviral activity of raoulic acid from raoulia australis against picornaviruses antipicornavirus flavone ro 09-0179 cinnamoyl-and hydroxycinnamoyl amides of glaucine and their antioxidative and antiviral activities antiviral activity of flavones and flavans present status and prospects of flavonoids as antiviral agents inhibition of an early stage of rhinovirus replication by dichloroflavan (bw683c) effect of dichloroflavan (bw683c) on the stability and uncoating of rhinovirus type 1b failure of intranasally administered 4 0 , 6-dichloroflavan to protect against rhinovirus infection in man failure of oral 4 0 ,6-dichloroflavan to protect against rhinovirus infection in man neural networks as data mining tools in drug design chemoinformatics and drug discovery methods and principles in medicinal chemistry the impact of informatics and computational chemistry on synthesis and screening enhancing drug discovery through in silico screening: strategies to increase true positives retrieval rates neural networks for chemists the protein data bank the protein data bank (pdb), its related services and software tools as key components for in silico guided drug discovery development of antiviral agents using molecular modeling and virtual screening techniques comparative molecular field analysis (comfa). 1. effect of shape on binding of steroids to carrier proteins investigation on qsar and binding mode of a new class of human rhinovirus-14 inhibitors by comfa and docking experiments synthesis and structure-activity studies of some disubstituted phenylisoxazoles against human picornavirus comfa analysis of the interactions of antipicornavirus compounds in the binding pocket of human rhinovirus-14 dihydro-2-oxazolyl)phenoxy]alkyl]-3-methylisoxazoles: inhibitors of picornavirus uncoating understanding human rhinovirus infections in terms of qsar site-directed mutagenesis suggests close functional relationship between a human rhinovirus 3c cysteine protease and cellular trypsin-like serine proteases the picornaviral 3c proteinases: cysteine nucleophiles in serine proteinase folds substituted benzamide inhibitors of human rhinovirus 3c protease: structure-based design, synthesis, and biological evaluation structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 6. structure-activity studies of orally bioavailable, 2-pyridone-containing peptidomimetics glossary of terms used in medicinal chemistry (iupac recommendations 1997) methods and principles in medicinal chemistry human rhinovirus 3c protease: generation of pharmacophore models for peptidic and nonpeptidic inhibitors and their application in virtual screening docking versus pharmacophore model generation: a comparison of highthroughput virtual screening strategies for the search of human rhinovirus coat protein inhibitors ca: accelrys. 358. discovery studio rollinger extended her studies to the fields of phytochemistry, ethnopharmacology, molecular modelling and its application in natural product science completing the habilitation thesis entitled ''the search for bioactive natural products rollinger is a project leader and an associate in various national projects, as well as executive guest editor for current pharmaceutical design. she has been awarded the phoenix science award (2005) and received several additional awards until 1994 she was a postdoctoral research fellow at the institute of immunology, university marburg, where she studied picornavirusimmune cell interactions and the virus-induced cytokine induction. thereafter, she was a postdoctoral fellow at the institute of virology and antiviral therapy key: cord-324335-eoabmyg7 authors: nicoletti, marcello title: new solutions using natural products date: 2020-08-21 journal: insect-borne diseases in the 21st century doi: 10.1016/b978-0-12-818706-7.00007-3 sha: doc_id: 324335 cord_uid: eoabmyg7 most antibiotics are derived from natural products, like penicillin, as well as recent insecticides, like pyrethroids. secondary metabolites are produced by plants as ecological chemical mediators, and can therefore possess intrinsic physiological properties against other organisms. these benefits are far from being fully explored. in particular, attention is here focused on the multipurpose neem tree (azadirachta indica), reporting several experiments of applications in the field of seed oil and neem cake. the latter product seems to be promising because of the low cost, the possible production on a large scale, and the selection of effects in favor of beneficial organisms. neem cake is able to act on different sites, as required by integrated pest management. several utilizations of neem products are reported and their potentiality evidenced. some considerations in this chapter may appear distant from the title of the book, but only by applying the general natural rules can the reason of the single phenomenon be understood. other studies on resistance mechanisms of plasmodium are enabling new possible methods of control always based on natural products activity. and biotic targets, which are important to the individual homeostasis and survival. one form of evidence in accordance with this interpretation is the strong correlation between natural products' producers and biodiversity, therefore indicating a matter of adaptation. however, it is possible to argue that if differences can characterize each organism from another, the some is not true in case of a molecule. once determined the structure, a molecule is a molecule, even if generated by the metabolism or by a synthetic route, and therefore the living rules cannot be applied to the molecular world. this is true only in part. we must consider, in particular for natural products, the conformational forms, which are different ways of the same molecule to react. an organic molecule can change easily its 3d structure adapting to the environmental needs, like we open and close our hands. furthermore, once inside the metabolism, the molecule, even if inorganic, is integrated in the organic network and it is forced to collaborate to an integrated living system. the cell environment is far different from the test tube of the chemist. in practice, the molecule is simply part of an advanced complex integrated dynamic system, limiting its freedom. beside the consideration that physical forces work in the same way in the assembling matter, as we can know and distinguish a giraffe from a wizard, we can study natural compounds and distinguish them on basis of the structures and assign their place and role. about the role, we must remember that natural products are chemical mediators inside the environment, meaning that they must act on the receptor of the target organisms. therefore, the structures of natural products are derived from their activity, the same argument we use in the consideration of a pharmacological drug. here, the importance of natural products inspiring all the molecules tailored to affect living organisms. in particular, among the detected activities in plant species, the defensive against herbivorous is highly reported. plants cannot counteract by movements like animals and therefore an arsenal of chemical weapons is essential in their fight for survival. therefore, it is highly possible to find natural products toxic or repellent to phytophagous, like insects, as well as active constituents against pathogens. natural products with such properties should be extracted and found in the complex and confused reservoir of secondary metabolites, but the presence into the plant is not sufficient. it is necessary to realize the mechanism of action and the potentiality to be used as marketed products. therefore, the pathway should comprehend the discovery, the isolation, the sources, the bioactivities, and the possibility to be obtained in high quantity, the method of utilization, the environmental impact and the cost of production. the natural products have been already reported for antibiotic activity. in particular, essential oils and phenols are stated in many papers as being responsible of antibacterial and insecticide properties (ghosh et al., 2012; gibbons, 2008) . however, the activity seems to be too general. most essential oils are composed of the same main constituents, the difference being mainly quantitative for each compound or hidden deeply inside the plethora of secondary constituents. furthermore, excluding the phenols typical of essential oils, the quantity of known phenols is very large and complicated, with thousands of structures reported and different activities connected. therefore, special research must be performed, including the possibility to test new substances never reported. among secondary natural products with insecticide activity, a special place must be assigned to essential oils. in this regard, several papers report the utilization of essential oils as the active ingredient against pests. the antimicrobial activity of essential oils is also well-reported and evident in nature. several plants accumulate essential oils in the inner parts of roots and rhizomes, in order to avoid the devastating attack of micropathogens, wherein the plant often accumulates precious reserve substances. in other cases, the aerial parts are focused on defensive or cooperative actions. some birds defend their clutch by surrounding the nest with aromatic plants. the human utilization of essential oils of different kinds against insects has a long story. herodotus reported the use in ancient egypt of mosquito nets and towers impregnated with fish odor to avoid mosquito bites. an essential oil is a complex chemical mixture of substances volatile at ordinary temperature (figs. 7.1 and 7.2), and therefore the constituents must have low molecular weight. in other words, they are micromolecules, with average molecular weight of 120-160 uma and hydrocarbon prevalence. essential oils can be extracted from the raw materials by utilizing their volatile properties, such as in the steam distillation method. the antimicrobial activity of essential oils is usually a consequence of the content of phenols, but other properties must be considered. on the basis of the structures of the active constituents, there are two types of essential oils. the first one, mainly present in less advanced angiosperm dicotyledons, like magnolidae, contains mainly root and fruit drugs rich in simple phenolic phenylpropanoids, which are mainly utilized by the plant in protection of pathogens. in rosidae and sympetalae, the terpenoids progressively become predominant. the new volatile constituents, in addition to the protective and toxic effects, afford a positive attraction on pollinatory agents, evidencing the plant position and allowing a memory of the selected species. in this way, the scenario of the interaction between animals and plants changes from defensive to collaborative. the new plants to be selected and appreciated can enrich the offer to the collaborative animals fig. 7 .1 a typical current apparatus for the production of essential oils by steam distillation. with fruits of inebriant flavors, colored flowers and other nice experiences. therefore, utilized essential oils are mainly complex mixtures of volatile plant secondary metabolism and consist mainly of monoterpenes and sesquiterpenes, which means lipid secondary metabolites, and, to a lesser extent, of aromatic compounds. the choice of essential oil depends firstly on the taxonomy of the selected plant and the effects depend on the nature of the constituents of the essential oil. the conclusion, based also on direct experiments, is the presence of a general antibiotic and insecticide activity; however, another real need is a selective toxicity in favor of the useful organisms. therefore, some kind of activity is expected for an essential oil, but there is a necessity to maximize its effectiveness. they are exploited in several fields, such as perfumery, food, pharmaceutics, and cosmetics, but essential oils have also long-standing uses in the treatment of infectious diseases and parasitosis in humans and animals. essential oils, currently more than 300 of which are known, are highly variable in their complex composition. usually, at least a mixture of more than main 30 different constituents of low molecular weight is present. among the single species, the qualitative composition of the essential oil is respected, although a quantitative variability is common between population according to the environmental pressures. however, some terpenes can be easily found, like the hydrocarbons (myrcene, pinene, terpinene, limonene, cymene, αand β-phellandrene) and the oxygenated ones, like the alcohols (geraniol, linalool, menthol, terpineol, borneol) , the aldehydes (citral, citronellal), ketones (menthone, pulegone, carvone), bicyclic monoterpene ketones (thujone, verbenone, fenchone), acids (citronellic acid, cinnamic acid), oxides (1,8-cineole) and esters (linalyl acetate), but the aromatic phenols (carvacrol, thymol, safrole, eugenol) are also common. a few essential oils may also contain sulfur-containing constituents, methyl anthranilate, coumarins, and special sesquiterpenes such as zingiberene, curcumin, farnesol, sesquiphellandrene, turmerone, nerolidol, etc. often these components are at low concentrations (less than 1% each), but the opposite is also possible, with major compounds that can represent up to 70% of the total volume of oil, as much as 90% like eucalyptol in eucalyptus or limonene in citrus or pinenes in turpentine of pinus. therefore, the antiparasitic activity of an essential oil can vary according to differences in its chemical composition, but it is usually present. nowadays, there is an increasing interest in the utilization of essential oils against endoparasites and ectoparasites of animals and humans, in particular when they are resistant to conventional drugs. however, the use of essential oils is in general restricted for the high cost and considering that usually they are not adequately specified for the considered target (bagavan and rahuman, 2010; shaalan et al., 2005; tikar et al., 2018) . again, also in the case of essential oils, the insurgence of the insecticide resistance must be considered (brown, 1986) , although usually less common in these cases. therefore, in consideration of their general but also weaker effectiveness of essential oils in comparison with synthetic insecticides, their utilization requires the insecticidal properties of essential oils to be investigated in different approaches of selection of the studied plants and their uses. some examples of researches, in which i had occasion to participate, involving essential oils in insect-borne diseases are reported here. the leading idea was to utilize the essential oil properties in an innovative way, such as in mixture or selected types. in 2017 (benelli et al., 2017a,b) , the activities of five essential oils were investigated. the essential oils were obtained from different plants: pinus nigra var. italica (pinaceae), hyssopus officinalis subsp. aristatus (lamiaceae), satureja montana subsp. montana (lamiaceae), aloysia citriodora (verbenaceae), and pelargonium graveolens (geraniaceae) against culex quinquefasciatus (diptera: culicidae), which is a vector of lymphatic filariasis and of dangerous arboviral diseases, such as west nile and st. louis encephalitis. the research was original in its focus on the potential synergistic and antagonistic effects, testing them in binary mixtures on c. quinquefasciatus larvae. mixtures of essential oils are very easy to obtain, since the constituents are perfectly soluble in the final solution and the selected oils were cheap and easy to find on the market. in such a way, knowing the composition, it is possible to combine constituents, enhancing the range and the quality of activity. the pool of the investigated species was highly varied, but this was considered a positive factor. first, the chemical composition of each essential oil was investigated by gc-ms analysis, which is the best analytic method in such mixtures of volatile compounds. therefore, it was also necessary to test the activity of each essential oil and later to try the best combination on the basis of its effectiveness. the highest effectiveness was obtained by s. montana subsp. montana essential oil (lc 50 ¼25.6 μl l à1 ), followed by p. nigra var. italica (lc 50 ¼49.8 μl l à1 ), and a. citriodora (lc 50 ¼65.6 μl l à1 ). it was possible to obtain an enhancement of the larvicidal activity by preparing simple binary mixtures of essential oils (ratio 1:1), such as s. montana+a. citriodora, which showed higher larvicidal toxicity (lc 50 ¼18.3 μl l à1 ). on the other hand, testing s. montana + p. nigra (1:1), an antagonistic effect was detected, leading to an lc 50 (72.5 μl l à1 ) higher than the lc 50 values calculated for the two oils tested separately. therefore, these results indicate the extreme need for innovation and imagination in natural products research, against many papers repeating the same procedure that change only the plant used. another work (pavela et al., 2016a,b) was based on geographic distribution and the traditional use. six medicinal and aromatic plants-azadirachta indica (see later in this chapter), aframomum melegueta, aframomum daniellii, clausena anisata, dichrostachys cinerea, and echinops giganteus-have been traditionally used in cameroon to treat several disorders, including infections and parasitic diseases. the aim was to evaluate the activity of the essential oils of these plants against trypanosma brucei tc221 and determine their selectivity with balb/3t3 (mouse embryonic fibroblast cell line) cells as a reference. essential oils from a. indica, a. daniellii, and e. giganteus proved to be the most active ones, with half maximal inhibitory concentration (ic 50 ) values of 15.21, 7.65, and 10.50 μg/ml, respectively. these essential oils were characterized by different chemical compounds, including monoterpenes and sesquiterpene hydrocarbons and oxygenated sesquiterpenes. some of their main components were assayed as well on t. brucei tc221, and their effects were linked to those of essential oils. in this way, the research partially confirmed the ethnopharmacological indications, validating their traditional use and confirming the utility of popular information in the search for useful plants. the synergic action of binary mixtures of similar constituents of essential oils against larvae of the filariasis vector culex quinquefasciatus was also the inspiration behind research (benelli et al., 2017a,b) on four apiaceae species: trachyspermum ammi, smyrnium olusatrum, pimpinella anisum, and helosciadium nodiflorum. initially, all the essential oils proved to be highly toxic to the larvae, but short-term exposure to both binary mixtures strongly reduced emergence rates, fertility, and natality of the c. quinquefasciatus that survived after the treatment at the larval stage. in addition, larvicidal acute toxicity of essential oils main constituents, i.e., germacrone, isofuranodiene, and (e)-anethole, were carried out, with lc 50 being 18.6 mg l à1 , 33.7 mg l à1 , and 24.8 μl l à1 . the results demonstrated the promise of these essential oils and their constituents to develop cheap and effective mosquito larvicides. in another paper (pavela et al., 2016b) published in the same year, the vector target was the same but the selection of the plant totally different, as endemic to madagascar. the reason is that in some parts of the world, there are interesting examples of endemic flora whose species could contain different essential oils and therefore different activity. for this reason, pharmaceutical companies often explore remote parts of amazonia or isolated zones in search of new active compounds. there were examples of exploitation of rare african rauwolfia species to obtain their indole alkaloids. working with endemic species is important considering that in many cases, populations are in limited numbers and at risk of extinction, and we need to identify their molecular treasure before they disappear. this was also the motivation for my trips to several parts of the world, focusing in particular on deserts and islands, in search of special plants. madagascar's fauna and flora are diverse and unique. when the unique gondwana continent braked up in several pieces, india started to move to asia living africa. however, a consistent block remained near to africa, becoming a great island, now known as madagascar. this happened more than 100 million years ago. the isolation of madagascar gave rise to a particular case of biodiversity. this is the story of the beginning of madagascar, as far as we know. here, it is important to report that the island is characterized by at least seven very different habitats, each with different endemisms. the potentiality of cinnamosma madagascariensis, an endemic species widely present in the forests of madagascar, was reported to us thanks to the exceptional collaboration with professor philippe rasoanaivo, who had a deep knowledge of the flora of the island and their economic importance. this plant has important traditional uses ranging from management of dementia, epilepsy, and headache to malaria (rakotosaona et al., 2015) . few data have been reported about the chemical composition of its essential oils, and no studies have been published on its bioactivity against mosquitoes. once again, we first investigated the chemical composition of essential oils extracted from stem bark and leaves of the plant, and later their larvicidal potential against the filariasis vector culex quinquefasciatus. the reason was that when you have little information, you must consider that different parts of a plant can contain very different essential oils. in fact, gc-ms analysis revealed differences between the volatile profiles of leaves and bark oils. in the former, linalool (30.1%), limonene (12.0%), myrcene (8.9%), and α-pinene (8.4%) were the major constituents, while in the latter one, β-pinene (33.3%), α-pinene (19.3%), and limonene (12.0%) were the most representative compounds. acute toxicity experiments conducted on larvae of the filariasis vector c. quinquefasciatus led to an lc 50 of 61.6 and 80.1 μl l à1 for the bark and leaf essential oils, respectively. overall, cinnamosma madagascariensis bark and leaf essential oils against filariasis vectors proved to be promising, since they are effective at moderate doses. the insecticidal activity of the essential oil of another malagasy plant was also studied (benelli et al., 2020) . hazomalania voyronii is popularly known as hazomalana and its use to repel mosquitoes and resist against insect attacks has been handed down from generation to generation in madagascar. the property of the essential oils obtained from the stem wood, fresh and dry bark of h. voyronii were able to repel important mosquito vectors (aedes aegypti and culex quinquefasciatus). furthermore, the toxicity of the aforementioned essential oils was investigated by who on three insect species of agricultural and public health importance (cx. quinquefasciatus, musca domestica, and spodoptera littoralis), respectively, as well as the adequate topical application methods and compared with the commercial repellent n,n-diethyl-m-toluamide (deet). repellence assay revealed almost complete protection (>80%) from both mosquito species for 30 min when pure fresh bark essential oil was applied on the volunteers' arms, while deet 10% repelled more than 80% of the mosquitoes up to 120 min from application. the research validated the traditional use of the bark essential oil to repel insects, although an extended-release formulation based on h. voyronii essential oils is needed to increase the repellent effect over time. furthermore, it evidenced the wide spectrum of insecticidal plants potentially useful in the fabrication of green repellents and insecticides useful to control mosquito vectors and agricultural pests, avoiding the utilization of synthetic products. another interesting study (benelli et al., 2017b) was dedicated to helichrysum faradifani (asteraceae), which is a perennial endemic shrub growing in rocky and sandy places of madagascar. the ethnopharmacological about malagasy traditional medicine reports that this plant is used as a wound-healing agent, disinfectant, and for the treatment of syphilis, diarrhea, cough, and headache. the chemical composition of the essential oil distilled from the aerial parts of h. faradifani, and analyzed by gc-ms, evidenced that monoterpene hydrocarbons (51.6%) were the major fraction of the essential oil, with bicyclic α-fenchene (35.6%) being the predominant component. sesquiterpene hydrocarbons (34.0%) were the second major group characterizing the oil, with γ-curcumene (17.7%) being the most abundant component. its insecticidal activity was evaluated against second, third, and fourth instar larvae of the lymphatic filariasis vector culex quinquefasciatus by acute toxicity assays. the most sensitive were second instar (lc 50 ¼85.7 μl l à1 ) larvae. for the third and fourth instar larvae, the estimated lc 50 were 156.8 and 134.1 μl l à1 , respectively. finally, a different approach to volatile substances was performed, considering that smoke is often traditionally used against mosquitos (ansari and razdan, 1996) . therefore, the larvicidal, pupicidal, and smoke toxicity of senna occidentalis and ocimum basilicum leaf extracts against the malaria vector anopheles stephensi were evaluated (murugan et al., 2015) . in larvicidal and pupicidal experiments, s. occidentalis lc 50 ranged from 31.05 (i instar larvae) to 75.15 ppm (pupae), and o. basilicum lc 50 ranged from 29.69 (i instar larvae) to 69 ppm (pupae). smoke toxicity experiments conducted against adults showed that s. occidentalis and o. basilicum coils evoked mortality rates comparable to the pyrethrin-based positive control (38%, 52%, and 42%, respectively). furthermore, the antiplasmodial activity of these plant extracts in antiplasmodial assays was evaluated against chloroquine (cq)-resistant (cq-r) and cq-sensitive (cq-s) strains of plasmodium falciparum. the s. occidentalis 50% inhibitory concentrations (ic 50 ) were 48.80 μg ml à1 (cq-s) and 54.28 μg ml à1 (cq-r), while those for o. basilicum ic 50 were 68.14 μg ml à1 (cq-s) and 67.27 μg ml à1 (cq-r). the high potentiality of the reported data must be considered. these smokes, as the essential oils, can be quite easily obtained in good quantity and low cost and therefore locally produced and directly utilized. this is important for countries with limited economic resources. the distribution of individuals in accordance with the boltzmann curve is the result of the current chemical-physical environmental pressure, concentrating the organisms of the species in the most adapted form. however, sooner or later situations are destined to change, and some of the individuals confined in the wings of the curve are ready to profit of the change and enter in the center, as soon as the conditions will be favorable to them. another consequence of this typical statistical distribution is the careful preservation of individual types inside the population. in practice, on the genetic point of view, the best species or the favored community do not exist in absolute, and any declaration or pseudo-scientific argumentation about the primacy of a race, also human, must be considered as a guilty stretch. as confirmation, this is also in accordance with the distribution of the constituents of matter at a subatomic level. the final consideration is that the chemical composition of a plant is limited, being the expression of the genome of the species, but it may change at any time in response to internal and external stimuli. let us use these concepts to evaluate insecticides used in insect-borne diseases. the interest in the use of biocidal products of natural origin began in the 1930s and grew until the 1950s, when it was obscured by the arrival of synthetic insecticides on the scene. for a long time, the pesticides scenario was dominated by synthetic products, until several factors caused a decline in their utilization. however, in the last 20 years, interest in natural products has reappeared intensely, especially for the control of noxious insects at larval stage. this situation has matured, as is well known, following the indiscriminate (and not always necessary) use of excessive amounts of pesticides which, once released into the environment, are difficult to eliminate, as evidenced by the paradigmatic case of ddt (see chapter 1). at the same time, incidence of insect resistance has increased, resulting in partial product inactivity and/or increasingly massive dosage requirements. all this led to a need for the formulation of a new generation of pesticides, and to focus research and production efforts on natural products. in 1997, the world health assembly reported in resolution 50.13, section 2.4, the need to develop bio-insecticides. slowly but inexorably, the pesticides market registered the rise of biopesticides from natural products. the change in favor of natural products is the result of two concomitant facts: the evidence of the environmental damage due to massive utilization of synthetic products, and a new and growing sensibility in favor of respect for habitats, asking for more compatible solutions. in the current search for the production of a new generation of pesticides, useful for mankind's battle against superbugs and other threats, to face challenges to food supply and health, plant sources play a relevant role. the current prevalence of natural products evidences that a consistent number of biocides and antibiotics have been obtained from substances produced by living organisms, which are part of the great book of mother nature, whose lessons are still useful. that probably means that attention in chemistry is finally moving from the free synthetic approach to natural products, already selected during the long story of molecular evolution. in an article that appeared in acs' journal of natural products, charles l. cantrell and colleagues pointed out the impact of natural productssubstances produced by living plants, animals, and other organisms-on the production of pesticides. the article reports the percentages for registered insecticides obtained from 277 new active ingredients in the period 1997-2010 (cantrel et al., 2012) . the paper's aim was focused on the impact of natural product and natural product-based pesticides on the u.s. market, obtained on the basis of nai registrations of new active ingredient registrations with the u.s. environmental protection agency (epa). the ingredients are categorized into four categories: biological (b), natural product (np), synthetic (s), and synthetic natural derived (snd). in particular, nps are considered substances produced by living plants, animals, and other organisms. the report evidences that nps, snds, and bs all have origins in natural product research. nps accounted for 35.7%, ss for 30.7%, bs for 27.4%, and snds for 6.1%, arising from the combination of conventional pesticides and biopesticides. in the registered conventional pesticides, the category of biopesticides alone registered an evident majority of nps (with 54.8%), followed by bs (44.6%), snds (0.6%), and ss (0%). in contrast, on the conventional pesticides alone, the category s clearly dominated with 78%, followed by snd with 14.7%, np 6.4%, and b 0.9%. the review indicates that in the same period, more natural products were registered as nais for conventional pesticides and biopesticides than any other type of ingredient. the authors report that when biological ingredients and natural products recreated in laboratories are included, more than 69% of all nais registered in that time frame have natural origins. more than two out of every three new insecticides approved in the last years are directly derived from natural substances produced in plants or animals or have significant roots in them. it is noteworthy that these numbers are very similar to those obtained if we compare with a similar projection concerning registered medical drugs in a similar period, and published in the same scientific journal. it is also noteworthy that a similar trend can be observed in the case of the registration of medical drugs in the period 1981-2014, as previously reported in the same scientific journal by david j. newman and gordon m. cragg (newman and cragg, 2016): 36% of registered drugs directly or indirectly derived from np of secondary metabolism, 16% from b, 11% from snd, and only 31% from s. again, the details reveal differences in the sectors, with np and b dominating in anticancer and antibiotics, whereas the opposite concerns antiinflammatories, with s clearly dominating. these data, obtained on a total number of 1562 new approved drugs, are the results of several reviews that confirmed these percentages. in particular, the authors stress the role of microbes in the production of new drugs derived from natural products: "we wish to draw the attention of readers to the rapidly evolving recognition that a significant number of natural product drugs/leads are actually produced by microbes and/or microbial interactions with the "host from whence it was isolated," and therefore "it is considered that this area of natural product research should be expanded significantly." in other words, the future of pharmaceutical drugs could be related to natural products obtained by natural synthesis. once conceivable that the shift from synthetic pesticides to biopesticides seems to be incontrovertible, as fueled by the resistance phenomenon and the general tendency for "natural," the key argument is the choice of the raw material for the best bioinsecticide. as evident from the above reviews, bioinsecticides could be extracted from a living organism, like a plant, or produced by a living organism, like a bacterial strain by hemisynthesis, or obtained by synthesis in accordance with the structure of the active natural product. here, the debate is open between those affirming that "a molecule is a molecule" and those in favor of "original" natural products. anyway, in the case of an extract, the complexity of ingredients cannot be reproduced or performed by synthesis. the main characters of a natural "ideal" insecticide should be: biodegradability, environmental care, sustainability (obtained from renewable materials) and selectively (harmful to beneficial insects). it should also satisfy some conditions to be economic appealing and relevant, like be easy to produce, low cost, derived from raw materials that available and abundant in the country where the insecticide should be utilized. the last conditions are important to avoid accumulation by multinational agencies, as is happening for coffee and cacao. finally, but not in order of importance, the ideal natural insecticide must be able to compete in the market with the insecticides currently in use. the research for the ideal bioinsecticide is open, starting from the plant to be used. evident that this role should be assigned to neem. at that time, the tree was practically unknown in the occident, since its distribution and utilization were confined to the indian subcontinent. therefore, i wrote an article about neem to explain the importance of this plant, and some years later, i was contacted by an italian industry using neem oil because of the curiosity and interest raised by that article. the references about this plant and its use appear since time immemorial, as reported in the ayurveda and unani systems of traditional medicines, and even in the earliest sanskrit writings referring to the medical uses of fruits, seeds, oil, leaves, roots, and bark (gupta, 2001) . neem can be found all over the indian subcontinent since the time of the sanskrit-speaking aryans. so important was the species for the aryans, they even mentioned and included it in their sacred ayurveda, which is the sacred book of indian medicine. the species was later dispersed throughout the old tropics, including indonesia, either naturally or brought back by the ancient austronesian sailors after visiting and trading in india at least around 2000 years ago. through the centuries (kumar and navaratnam, 2000) , the medical importance of neem never waned in the indian subcontinent and it is now considered the "village pharmacy" for its importance in the ordinary life of indians, who use this plant to treat several illnesses (nix, 2007; girish and bhat, 2008) . many other news and references increased my interest. the marvelous tree, the problem-solving tree, the divine tree, india's tree of life, nature's drugstore, the pharmacy tree, the panacea for all diseasesthese are just some of the terms used to evidence the respect for this plant and its importance (ruskin, 1992; brahmachari, 2004; puri, 1999; national research council, 1992) . neem's relevance and its beneficial properties has been reported by the who/unep (1989), which considered neem as an effective source of environmentally powerful natural pesticide and one of the most promising trees of the 21st century for its great potential in pest management, environmental protection, and medicine koul and wahab, 2007) . furthermore, the u.s. national academy of sciences dedicated a report to neem, significantly titled "neem-a tree for solving global problems" (nas, 1992) . the importance of neem has increased exponentially in recent years. considering the enormous quantity of results and scientific data concerning the validation of medicinal and biological properties, the international scientific community included neem on the list of the top 10 plants to investigate and use for the sustainable development of the planet and the health of mankind (tewari, 1992; foster and moser, 2000) . however, in the occident, insecticidal activity is the most common application for neem oil and its derived products. the plant, besides neem, is also known as nimba, nimtree, margosa, and indian lilac. in botany, neem is azadirachta indica a. juss and belongs to the meliaceae family. meliaceae are angiosperm rosidae, closely related to simaroubaceae and rutaceae (schumutterer, 2002) . the family includes 51 genera and c.600 species. these are woody plants, like trees, shrubs, and shrublets, pantropically present, with a few temperate representatives in china and south africa. it is estimated that c.39 million years ago, two subfamilies diverged, cedreloideae with 14 genera and melioideae with 37 genera, including aglaia, dominating with c.120 species, and azadirachta with only two species. meliaceae are commonly known as the mahogany family, being known mainly for some important timber species, such as the true mahogany (swietenia mahagony). however, losses due to overexploitation and genetic erosion, as well as toxic effects on workers, limited the use of this true mahogany, nowadays widely replaced by spanish mahogany (s. macrophylla). azadirachta indica is an evergreen tree that grows up to a height of 15-20 m, but in favorable conditions it can to a height of about 20-35 m, with a trunk diameter up to 2.5 m (fig. 7.3) . the leaves of neem, composed of 9-19 leaflets, meaning 4-8 leaflets with a single terminal, are abundant, suspended by a strong and long petiole which lacks stipules, crowded near the ends of the branches. the leaflets are toothed, deeply serrated, their margins irregularly serrated, sharply pointed, and curved like a scythe. young leaves are pale, tender green, and tinted with rust, but during the favorable season the tree profits from the fresh, green color and shining surface of the leaves, giving a delicate and charming appearance. white small flowers are abundant, very fragrant, bisexual, or staminate in male exemplars. they are arranged in clusters at the axils of the leaves. they present five separated petals arranged in the form of a star. they appear in spring, and open in the afternoon giving out a delicate smell, which increases during the night. the fruit is a smooth, yellow-green, small, round drupe with a sweetflavored pulp. during the monsoon, when the flowers have fallen and the tree is in full foliage, the curved, toothed leaves, massed round the branches, have a distinctive appearance, which is easy to recognize. from march to may, the flowers, with five whitish petals, appear in great numbers on long, drooping stems. flowers are used to produce a bitter honey. the fleshy fruits are purplish-black, single-seeded drupes, which turn yellowish when ripe. elliptical in shape, they have a sweet-tasting juice loved by birds and bees. however, after the rains, the fruits change, giving off a strong unpleasant smell. in autumn, fruits fall down in great quantities if not harvested. the tree is believed to be native at least of north-east india and burma, or indonesia, but now widely distributed in the indian subcontinent, and it grows naturally throughout the dry regions of the country. it is usually planted along roads and avenues in towns and villages, because it grows fast and easily, and has an irregularly rounded crown with a canopy of leaves, making it a useful shade tree ( fig. 7.4) . the central regions of india are considered the patria of neem. if you go to coimbatore, in tamil nadu, you can find neem trees everywhere, in towns and in the countryside. it is mainly used for shade, lining streets or in most people's back yards. in india, it grows throughout the states of uttar pradesh, bihar, west bengal, orissa, delhi, maharashtra, gujarat, and andhra pradesh, but the original natural distribution is obscured by widespread cultivation. cultivation is easy, since neem usually grown from seed but can be propagated also from cuttings or root suckers, and it is a fast-growing species. potential utilizations of neem concern human, animal, and environmental health. the last one is a recent but very important acquisition. neem is cultivated for two main reasons: environmental care and the production of seed neem oil. the tree's tolerance and adaptation to hot and dry climates has made it one of the most commonly planted species in arid and semi-arid areas (tiwari et al., 2014) . the survival capacity of neem is mainly due to its highly expanded root system. neem trees are extremely useful to counteract desertification and furnish the only source of wood in arid and nutrient-deficient zones. the plant does not need particular care and grows rapidly up to 30 m tall. neem trees attain maturity in 6-7 years in areas where the sunlight is intense, weather is warm, and good well-drained soil is found. the tree is stable in windy zones and can live for 200 years or more. to survive in arid climates, neem depends on a wide strong root system with a deep tap root and extensive lateral roots, which are ideal for soil conservation. furthermore, its planetary presence can contribute to positive carbon sequestration to minimize climate changes, considering that adult neem trees can retain ae2.2 g of co 2 per m 2 and per hour, which means 40-50 tons of co 2 per hectare. therefore, neem trees can be considered to be air purifiers as well as air fresheners. for all these reasons, neem is widely cultivated in warm countries, and its areal distribution is expanding rapidly by massive cultivations in sub-tropical regions of america (caribbean cuba, central and southern america), asia (nepal, pakistan, bangladesh, sri lanka, myanmar, thailand, malaysia, indonesia, iran, china, turkey, indonesia), africa (kenya, cameroon), and australia. the cultivation of neem trees is in particular increasing in the drought-prone areas, like in south arabia (arafat valley) and in the uae. in 1978, northern nigeria, thanks to the governmental project arid zone afforestation (azap), saw 700,000 neem trees being planted. in europe, some cultivations are reported only in southern spain and portugal (sara and folorunso, 2002) . the presumed current global neem trees presence and production are reported in table 7 .1. the data were obtained by cross-referencing several sources and are only indicative, considering that a real census was never completed in many countries (in particular in china) and many plantations are in progress. india is still by far the homeland of neem, but the scenario has changed rapidly in the last years and will continue to do so in the future, as can be deduced from the data in table 7 .1. visiting oman, i noted the presence of planted neem trees in areas where acacia was the only other woody plant (usually the only plant) able to survive. the neem tree is resistant to drought and it grows in many different types of soil, but it thrives best in well-drained deep and sandy soils. normally, it flourishes in areas wherein the neem is a life-giving tree, especially for dry coastal, southern districts. its capacity to survive in arid zones improved cultivation in sub-arid to sub-humid conditions, with annual rainfalls between 400 and 1200 mm. it can tolerate high to very high temperatures, but it does not tolerate temperatures below 4°c, making cultivation in temperate climate very difficult. however, the future worldwide distribution of neem is not predictable. indians are convinced that it will be difficult to obtain similar ideal conditions to their country and others in the orient; however, the story of the cultivation of cinchona by dutch botanists in indonesia tells us that the results of the cultivation can be successful and the results even superior. in the complete linnaean binomial name of neem we found a. juss, which is the mark of the author of this species, designating the scientist who first published the name and a complete and scientifically reliable description of the species. a. juss refers to antoine laurent de jussieu (1748-1836), a great french botanist, who was the first to publish a complete and valid natural classification of flowering plants, named genera plantarum, published in 1789, the same year as the french revolution, surpassing the sexual system presented by linneus. antoine laurent jussieu was the member of a family of a plant enthusiasts; his uncle was the botanist bernard de jussieu, whose transferred knowledge and unpublished work were the starting point of the book of antoine laurent, and his son andrien-henri also became a botanist. the merit of his work was the use of multiple characters to define taxa. in this approach, he achieved a significant improvement over the "artificial" system of linneus, mainly based on the number of the reproductive characters, i.e., stamens and pistils. many people know the work and name of linnaeus, but the impact of jussieu's work was fundamental in taxonomy, founding the principles that served as the foundation of plant classification in a natural system. many presentday plant families are still attributed to him, as the species is to linnaeus. that's what who can find in ordinary sources of information. the consequent idea is that jussieu, though living in france at a historical revolutionary time, was totally dedicated to botany, but his work was also revolutionary, through a strange pathway. deeper information about his life is a source of important lessons. his uncle, bernard de jussieu, invited the young antoine to paris, where he was trained in medicine for 4 years. however, his uncle, via his position as a demonstrator at the jardin du roi (royal garden), had other plans for antoine. he guided his nephew's studies and prepared him for a lecturer's position at the garden, which was soon to become vacant. at just 22-years old, antoine was transferred to that position and his botanical training was limited because the subject then was viewed only as an accessory to his medical course. the inexperienced jussieu had to study botanical topics by night, since he had to teach by day. using the plants in the garden to teach plant morphology, which were arranged according to the current artificial system of joseph pitton de tournefort, jussieu started to realize the inadequacy of that system. this progressively changed his interest into a true passion for botany, and classification began until, as part of an application for a place at the academy of sciences, he produced a treatment of the ranunculaceae, starting a complete revision of the plant taxonomy system. continuing in his study, it became apparent to jussieu that the artificial system of tournefort was inadequate, and from 1774 he began arranging the plants in the royal garden in his own way and finally transferring the knowledge in the construction of his own system of plant classification. despite the initial success obtained by linnaeus' sexual system, it was clear to jussieu that the swedish naturalist had used a counting method, whereas it was necessary to grade the characters, considering some of them more important than others, depending on how variable they are within a species. this was a necessary lesson to consider the variability of living organisms correctly. however, he continued practicing medicine, chiefly devoting himself to the health of very poor people. in 1790, he was put in charge of the hospitals and charities of paris. in the final years of his life, jussieu, by then almost blind as well as deaf, dedicated the last part of his extraordinary life to meditation and prayer. the neem tree has few diseases and enemies (boa, 1995; schmutterer, 1998) . in general, it is considered a very resistant and healthy plant . however, it is possible that after its spread around the world, something is changing. we must move our focus from india to the new settlements, in africa. in northern nigeria, neem now is planted in towns and villages, as a highly evaluated source of shade and firewood, as well in the establishment of shelterbelts. in nigeria, like in other parts of africa, the small twigs of neem are used to clean and whiten teeth, in consideration of its antibacterial properties. in particular, reports of pests and damages comes from east africa, like gall mites (phyllocoptes sp.) on older plants, but the most potentially dangerous pest is aonidiella orientalis, known as oriental scale (ofek et al., 1998; lale, 1998; elder et al., 1998) . this insect was widespread in western kenya but is not currently harmful. likely to be native to asia, it has been introduced to many regions via shipments of plants and then began its slow spread. some ports check for this and other scales in plant shipments. in africa, neem has been widely planted in the sahel region. oriental scale first appeared in the sahel during the mid-1970s and caused widespread damage to neem trees planted there. infestations were first detected in nigeria in 1987 along its border with cameroon and by the mid-1990s, widespread damage had been reported to neem trees throughout northeastern nigeria. the oriental scale is a flattened, circular or oblong insect, about 1.6 mm in diameter. it varies in color from yellow to light reddish brown. it frequently forms large colonies and sucks the sap of small stems and branches, which is phloematic sap, rich in sugars and other organic substances. infestations often spread to the foliage, fruits, and even seeds. feeding damage causes the foliage to die, giving infested trees a burnt appearance. this is followed by progressive dieback of branches and eventual tree mortality. the heaviest infestations appear to be on large trees located either in marketplaces or around human settlements. the female attaches to the surface of a plant and causes the disease by the larvae, which roam the plant, feeding on sap by inserting their stylets, sucking sap, and weakening the plant progressively. the physical damage includes discoloration and deformation of leaves. flowers and fruits fail to develop. it is noteworthy that the effects of the pest attacks are very similar to those already reported for olive trees, including exsiccation of leaves and wood. the lesson is that, in this outbreak, we have an explosive negative mixture of alien species and man's activity on the habitat. the enormous spread of neem trees in recent years is an unusual phenomenon, whose consequences should be better monitored. before going into detail about the constituents, we must consider the typologies of the forms of the marketed products containing natural products. we are referring to insecticides, but the same considerations are simply applicable to nutraceuticals, phytomedicines, cosmetics, and even food. it is possible to find the plant drug, meaning a part of the plant utilized. the plant drug is usually utilized exsiccated, or as a derived product, like an extract, resin or oil, which can be obtained as such, or be enriched in one or more constituents, which are considered responsible for the activity. in this sequence, the original starting point, which makes the plant useful, has been betrayed in favor of increased efficacy. however, following this treatment, there are products registered as food supplements containing 90% of a pure substance and usually the origin is not at all natural (although this is not declared on the label). in such cases, the product is more similar to a medicinal drug than an extract and it should be considered and used in medicinal form. the distinction between such marketed products is not evident and not reported to the unaware consumer, who may well prefer the product due to its apparently "natural" origin. knowledge of the chemistry is therefore fundamental and the basis of any decision about the appropriate use of a plant. however, we cannot know exactly what is inside a plant. all our methods of investigation are limited and may be misleading, although papers and books are full of information about the compounds contained in plants or their derived products. this is the consequence of plants' extreme chemical complexity. in a single leaf of hemp, more than 400 constituents have been detected, considering the secondary metabolites alone, and hemp can contain high levels of thc or cannabinoids can be practically absent. in such cases, the morphology does not give any help and only a reliable chemical analysis can indicate what kind of hemp we are handling. since its beginning, phytochemistry has sought knowledge of all natural products. in about one hundred of years of activity, innumerable analyses were made and an enormous quantity of data collected in a very useful data base, but recent advancement in analytical devices and novel interpretation ask for a revision of the result of this job (kaushik et al., 2014; forin et al., 2011) . we must remember that the molecular world is not detectable by our senses and therefore we have secondhand and probably only partial information. sometimes, this information is considered sufficient to assign the compound/activity relationship, but only until other analyses confirm the presence of other molecular candidates. the seeds of neem contain at least 100 identified biologically active compounds (govindachari, 1992) . among them, major constituents are nortriterpenes, named limonoids, i.e., azadirachtin, nimbin, nimbidin, nimbolides, and many others (ragasa et al., 1997) . however, each year other new limonoids are discovered. more constituents mean more possible activities. preparations from the leaves or oils of the seeds are used as general antiseptics (mossini et al., 2009) . due to neem's antibacterial properties, it is effective in fighting most epidermal dysfunction, such as acne, psoriasis, and eczema. ancient ayurvedic practitioners believed high sugar levels in the body caused skin disease. neem's bitter quality was said to counteract this sweetness. during the last desert locust plague in africa, it was noticed that these insect, schistocerca gregaria (forskal), ate almost any vegetal around, leaving a bare landscape when they fly away, except for neem trees, probably because of the antifeedant effect of the very bitter leaves, due to the presence of limonoids. traditionally, indians bathed in neem leaves steeped in hot water. since there have been no reports of topical application of neem causing adverse side effects, this is a common procedure to cure skin ailments or allergic reactions. neem also may provide antiviral treatment for smallpox, chicken pox, and warts, especially when applied directly to the skin. its twigs are commonly used to clean and disinfect teeth. the brushing of teeth with neem to prevent gum diseases and for teeth whitening is very common, and not only in india. there are also various kinds of natural toothpaste on the market that contain neem extracts. it is also possible to prepare a homemade toothpaste to achieve shiny, cleaner teeth. neem powder is made by grinding dried neem leaves, which is traditionally used by mixing one teaspoon of neem powder with one teaspoon of baking soda and enough water to make a useful paste. these preparations can help to avoid the plaque and tartar that build up in gums, which are the root causes of bad breath. in addition to cosmetic uses, neem's antimicrobial activity to maintain dental health is also worth noting (chava et al., 2012) . a preparation can be obtained by boiling neem leaves in water until they reduce to a quarter of the original volume. finally, gargling with this concoction contributes to good breath and whiter teeth, as it kills the bacteria inside the mouth. the teas of neem leaves are utilized in indonesia and oman for their digestive properties (sujarwo et al., 2016) . neem's effectiveness is due in part to its ability to inhibit pathogens from multiplying and spreading (benelli et al., 2016) . neem produces painrelieving, antiinflammatory, and fever-reducing compounds that can aid in the healing of cuts, burns, sprains, earaches, and headaches, as well as fevers (chopra et al., 1952) . several studies of neem extracts in suppressing malaria have been conducted, all supporting its use in treatment. neem has broad applications to human and animal health, as well as organic farming (bhowmik et al., 2010) . it is reported as a powerful antiviral and antibacterial, with peculiarities that set it apart from other herbs in that class of broad antimicrobials (sandanasamy et al., 2013) . neem oil is also commonly added to a variety of creams and salves. it is effective against a broad spectrum of skin diseases including eczema, psoriasis, dry skin, wrinkles, rashes, and dandruff. these are just a few examples of the possible utilization of neem, and the potentiality of neem is considered by everybody, including the onu and other institutions, to be very high, but so far little has been done to develop appropriate products from it to help mankind. in particular, considering insect-borne diseases, in vivo activity of neem seed oil (nso) against malaria plasmodium has also been reported (dahiya et al., 2016; trapanelli et al., 2016) . today's exploding growth in human population is seriously depleting the world's natural reserves and economic resources. unless the runaway human population growth rate is slowed down, there will be little hope for raising everyone out of poverty in the developing world. besides educational constraints, the nonavailability of inexpensive methods of contraception, which do not cause trauma or impose on the esthetic, cultural, and religious sensitivities of people, limit the success of birth regulation programs. however, recent findings indicate that some neem derivatives may serve as affordable and widely available contraceptives. a recent controlled study in the indian army proved the efficacy of neem as a contraceptive. in 2020, the report of the washington-based international food policy research institute predicted a world even more unequal than the present, with food surpluses in the industrialized world and with chronic instability and food shortages in the global south, particularly in african countries. the main product of neem seed oil (nso) is the fixed oil obtained by expressing the seeds, still enclosed in the kernels. therefore, the fleshy pulp is removed or dried, to obtain the inner part. nso can be obtained by different extraction methods. most nso is produced in india by small-scale producers at ordinary temperatures using very simple machinery, which is utilized in other periods of the years for other oily extraction, like arachnids or soya seeds (figs. 7.5-7.9). however, modern apparatus is also used in india and many other countries are now producing and refining nsos. therefore, considering also the possible different geographical origin of the raw material, combined pre-and postharvesting factors can result in great differences in constituents present in marketed nsos, as already reported. therefore, despite the common definition for all the oils obtained from kernels of neem, it is necessary to consider that there is no single nso, but many nsos differing in shape, color, viscosity, chemical constitution, and activity. medicinal and cosmetic utilizations are relevant and continuously increasing. cold pressed neem oil is commercially known as margose oil and considered as pressed directly from seeds. there are hundreds of marketed products worldwide based on margose oil. neem, or margose, oil has a brown color, a bitter taste, and a garlic/sulfuric smell. the oil is usually obtained by simple pressing, but extraction by organic solvents, like hexane, is also used, though in such cases traces of the residue of solvent are always present in the final product. the type of insecticide is commonly registered as biocide, insect repellent, and antifeedant, intended for use on outdoor and greenhouse agricultural food and ornamental crops as a repellent and insect growth regulator. the products are considered to have no risk to human health because of their low toxicity via all routes of exposure. there is no reason to believe that any nontarget organisms, including honeybees and other beneficial insects, would be adversely affected, as tested by the environmental protection agency (epa). the environmental protection agency (epa) is probably the most important agency in charge of environmental care and health. it is an agency of the united states federal government whose mission is to protect human and environmental health. the epa is also in charge of the regulations of carbon emissions from power plants, automobiles, and other contributors to climate change. the epa became popular in europe because of a civil enforcement case against volkswagen and other car manufacturers, subject to reservations set forth in each of the partial settlements. in 2017, the u.s. department of justice resolved a criminal case against volkswagen ag with a plea agreement for the offenses of conspiracy, obstruction of justice, and entry of goods by false statement, and the u.s. customs and border protection resolved civil fraud claims with volkswagen arising from the illegal importation of affected vehicles. the epa was established in december 1970 by an executive order of president richard nixon, with headquarters in washington, d.c., in response to widespread public environmental concerns that gained momentum in the 1950s and 1960s. epa is a giant public agency with a budget of 9 billion us $, and it is born as reaction to the public movement in favor of the environment due to the carson's book (see chapter 1). it is considered reliable because independent. the epa is responsible for creating standards and laws to protect and preserve the natural environment and improve the health of humans by researching the effects of and mandating limits on the use of pollutants. the epa's aims include the regulation of the manufacturing, processing, distribution, and use of chemicals and other pollutants. in addition, the epa is charged with determining safe tolerance levels for chemicals and other pollutants in food, animal feed, and water. the best presentation of neem insecticide properties and the rationale of its utilization is the report of the epa about the registration of cold pressed neem oil, concerning a product named plasma neem. the report has a significant subtitle: "reasons why neem oil is an effective way to control insect hoppers" (epa (us environmental protection agency), 2012). the target of the product cold pressed neem oil is insect hoppers, because of their special liking for cash crops. the consequence is a crisis because of insect hoppers infesting these cash crops by chewing and sucking the leaves, as reported by many farmers. the reasons to use neem are focused on the effects of chemical fertilizers, which "provide a remedy but tend to kill beneficial insects as well." the report identifies three reasons why neem oil should be considered an effective way to control insect hoppers which feed on cash crops. reason 1: selectivity. "neem oil is usually sprayed on the leaves and stalks of a cash crop. so it is aimed at only the insect hoppers who spoil the cash crop by chewing the leaves or biting off bits of the stalk. the insect hopper which attacks the plant by consuming it will end up consuming the neem oil as well and hence die as an aftermath. beneficial insects, who replenish the soil in a natural fashion, do not consume the plant and hence do not get affected by neem oil." reason 2: eco-friendly efficacy. the content of azadirachtin in neem oil is considered very good at controlling and eliminating insect hoppers, avoiding the negative effects on the productivity of cash crops. furthermore, the neem oil exerts no damage to the soil or disruption of the chemical composition of a fertile soil. the product affects biological functioning of the insect hopper, and therefore the insect hopper forgets to feed or breed after devouring a cash crop with neem oil sprayed on it. the conclusion about this point is clear: "this leads to the complete elimination of the insect hopper and the infestation cycle without any other adverse chemical side effects." the action is mainly larvicide, but also deterrent and adult insecticide in most species. reason 3: cost-effectiveness. the utilization of neem oil for preserving cash crops from insect hoppers is considered very cost-effective. in comparison to chemical fertilizers, neem oil is expensive, but other positive effects must be evaluated. there is another consideration about the cost/benefit effect: eliminating negative insects and worms by neem oil, farmers need not spend money on buying supplements for the enrichment of soil. the heavy investment to preserve soil quality is totally avoided when a farmer uses neem oil. in conclusion: "overall, it can be said that neem oil as a source to control insect hoppers in cash crops is extremely beneficial for farmers." the final consideration of the epa is the advice not to use the content of the report as an endorsement of nso. in fact, it is important to remember that the report is based on scientific and experimental data. the epa is not working in favor of the industry or the market, but its judgments must be considered objective although its aim is environmental improvement. nso, obtained by the cold expression method, is the only natural productderived insecticide, whose registration was approved by epa. the epa's authority is so far internationally recognized. therefore, in the report there are three main key items of information: the evaluation of the selective insecticide activity; the preference for synthetic products in consideration of the eco-friendly properties; and the indication of azadirachtin as necessary for the activity. let us consider the report as a guide and evaluate each of these points. later, we will add other considerations. it is noteworthy that all these indications can be found already reported and present in the information about the ethnobotany and traditional medicine of neem. in the cultivation of rice, when the plantlets are underwater, the addition of extracts of neem to the usual fertilizer, not only decreases the mosquitoes number, but also has beneficial effects on the production. the utilization of neem to increase soil fertility and treat medicinally plants and livestock is recommended in the upavanavinos, an ancient sanskrit book on agriculture. the dried leaves and the oil are used as preservatives against insects and microorganisms for the postharvesting conservation of foods. conservation is guaranteed for more than a year. panels derived by the extraction of the oil are used as food additives for livestock, and the animals are washed with this diluted oil to prevent the attacks of parasites or harmful insects. in the last decade, the focus was on the potentiality of a new analytical technique, hptlc (high performance thin layer chromatography) ( fig. 7.10) . hptlc is the last evolution of planar chromatography and allows the evidence of most of the constituents of an extract in an identifying track, named fingerprint, wherein identification of constituents can be obtained by direct comparison in the same plate with the correct standards, utilizing the rf value and the reaction with adequate derivatization (nicoletti et al., 2012a,b) . improvements in separation and visualization of the spots are obtained by reduction of the size of the particles of the silica gel, constituting the fixed phase. the mobile phase can be selected on the ample repertory of solvents and their mixtures, as well as the several methods of derivatization and detection. the final effect, in comparison with ordinary planar chromatography (tlc), is like a myopic person wearing glasses. the advantages in comparison to the tlc are in the total control of the environmental conditions and the automatization of the procedures. each step of the analysis is performed entirely by a series of devices, and the operator is only asked to produce the program of actions by the software. plates can be visualized and derived in several ways, obtaining multiple information (figs. 7.11 and 7.12). they can be easily preserved and stored as digitalized images inside the computer, and immediately sent everywhere or compared with a data bank. however, the most important feature of hptlc is that the results of the analyses are very clear, thanks to careful preparation work that enabled optimal chromatographic conditions, as demonstrated by the quality of the images. in other words, our idea was to "see the molecules" (nicoletti, 2013) and obtain simple and clear evidence of the metabolic production. molecules are too small to feel their presence with our senses or directly by any sort of device, but it is possible to evidence their chemical properties in the plate, recording the rf value, the fluorescence, and the color after derivatization. an hptlc or nmr graphic needs an expert for correct interpretation, such as any specialized analysis, like a cardiogram . in hptlc, without any knowledge of chemistry, the presence or absence of a determined spot is evident (figs. 7.11 and 7.12). hptlc was selected to obtain a metabolomic approach, meaning the study of many constituents as possible, focusing on secondary metabolites (toniolo et al., 2013) . metabolomic is one of the -omic sciences generated by the dissection of the dogma of biology, based on the sequence dna➔rna➔proteins. it is necessary to propose some comments about the dogma of biology and why its crisis generated a series of other points of view. first of all, the use of the word "dogma" should be avoided in biology, since the matter is more complicated than a simple sequence, as actually happened. the central dogma of biology was first proposed in 1958 by francis crick, as a consequence of his discovery of the structure of dna. the dogma describes process by which the instructions contained in dna are converted into a functional product. another definition is: "the coded genetic information hard-wired into dna is transcribed into individual transportable cassettes, composed of messenger rna (mrna); each mrna cassette contains the program for synthesis of a particular protein (or small number of proteins)" (sources: definition from chapter 1: the dynamic cell, of molecular cell biology). the flow of genetic information within a cell follows the sequence: dna codes for rna via the process of transcription (occurring within the nucleus), rna codes for protein via the process of translation (occurring at the ribosomes), and proteins are responsible for the synthesis of the other metabolites (proteins are spread everywhere). cell data are organized within the database of dna and reversed in the metabolic flux, through rna. although clearly deficient, the central dogma of biology dominated genetics for decades, but through ongoing research, many exceptions were discovered. for example, most dna is silent, since it does not encode proteins. retroviruses, which are relevant for our arguments, present the possibility that rna transcribes into dna through the use of a special enzyme called reverse transcriptase, and other cases of deviance can be reported. however, the biggest revolution consists in the direction of the arrows. it is necessary that information could follow also the reverse pathway, allowing an appropriate response by the genome potentiality. therefore, at least the dogma must be rewritten with two-way arrows. in principle, a metabolomics study should be the determination of the pathway of cell production from the genome through transcription, but the term "metabolome" is now used to evidence the whole pool of metabolites, in particular for natural products, whereas transcriptomics is related to proteins. transcriptomics involves serious difficulty to obtain reliable results. a protein seems perfectly comfortable inside the cytoplasm, but outside, irreversible denaturation causes definitive degradation and consequent difficulty in understanding the protein's functionality. in contrast, small molecules are more stable in any environment and their molecular structures at least can be determined by phytochemical analysis (nicoletti and toniolo, 2012; toniolo et al., 2014) . however, in the metabolome we have hundreds of thousands of different constituents to be studied, and the classic approach to study the molecules one by one is impracticable, and other methods must be utilized. the lesson is that the role of any metabolite cannot be discarded a priori, and also a secondary influence in the evaluation of the property of an extract can be important to definite and obtain the final reaction. once again, the "magic bullet" paradigm is under discussion, but the total utilization of plant extracts must also be considered an unsatisfactory solution. the aim of our approach was to adapt the method to other subjects outside the pharmaceutical applications. therefore, our first studies focused on the determination of adulterants in nutraceuticals and other pharmaceutical products, like the "green viagras." later, we adapted the method and the devices to use the metabolome as a source of information about what is going on in a complex system in which living organisms are acting. therefore, we are able to study the effect of environmental factors, like ozone, on the quality of wine (valletta et al., 2015) . however, probably the most impressive application was the study of the environmental effects of the costa concordia disaster (toniolo et al., 2018) . on the night of january 13, 2012, the costa concordia, a giant yacht with approximately 1500 cabins, 3229 passengers, and 1023 crew, was wrecked off the rocks of the italian coast a few hundred meters from the port of giglio, a little island on the tyrrhenian coast in tuscany. like an injured helpless mastodon, the cruise ship inclined dangerously, until the inclination stopped with most of its starboard side under water. because of the inclination and the amount of people, the overnight evacuation of the costa concordia was a challenging process, and 32 people died. the cost of removing the ship was us$799 million. for scientists like us, interested in environmental damage, it was a unique occasion. for 1 year, 9 months, and 4 days, the enormous hull of the boat altered the underlying marine habitat, interrupting the normal flow of sunlight over a surface of more than 10,000 m 2 . the seagrass posidonia oceanica was chosen as the target organism of the impact evaluation, since, like in other parts of the mediterranean sea, it forms large underwater meadows. using hptlc analysis, it was possible to determine the health of each collected plant and make a map of the metabolic damage, which accorded with the shadowed area. however, albeit the negative conditions, the rhizomes turned out to be mostly still alive and able to reproduce the meadow again. therefore, the final task of our research was simply to wait until nature carried out its work. however, there is a further chapter of this story, written after our study. to remove the ship, a platform was transported from north europe. the problem was that the bottom of the platform was full of mytilus. when the platform was exposed to the hot mediterranean sea temperature, the mussels died, releasing their bodies, covering down the sea background and causing a further source of damage. a clear example of human stupidity and superficiality. anyway, devoted to our task, we are now repeating our analyses to understand what happened and what is still going on, relying on the quality and reliability of our indisputable results. the study of neem oil was based on the experiences obtained by improving the hptlc devices via the metabolomics approach. the central idea was to collect as much information as possible about the constituents of the neem products, without any preference for any kind of metabolite, considering any product and any extract like a unique molecular system. in the hptlc analyses on nsos, the objective was to achieve the total chemical characterization of the used oil, and then the derived products, by means of the production of a chromatographic reference profile of the metabolites' production. this objective is not easy to achieve due to the complexity and variability of neem oil. neem products are subjected to great variation in composition, due to preharvesting factors, like environmental situation, genomic differences, influences of the habitat, and others, including postharvesting situations, like harvesting and stocking, treatment of the raw material, separation of different parts, extraction methods, production of the final product, and others. in fact, analyzing different marketed neem oil from several productions and countries, we decided that it was not possible to refer to a single neem oil, but to neem oils in the plural, due the great differences in composition. therefore, we decided to obtain and adopt a reliable reference metabolomic hptlc profile for the neem extracts or products to be utilized in our biological experiments in vitro and in the field. in fact, one of the typical problems in activity tests is the differences in raw material giving rise necessarily to different results in activity and utilization. another important aspect of our metabolomic study was that the complexity of the neem profile was even greater than expected. this result is the consequence of the generalist approach. in other molecular chromatographic or spectroscopic analyses, like hplc, the result is sub judice on the detector's settlement. therefore, if the molecule does not possess the adapt chromophore, the molecule, even if it is the main component, is invisible to the detector. in hptlc, there are universal derivatization methods, like h 2 so 4 , to reveal the organic substances, but it is possible when necessary to adopt a particular agent. in this way, it was possible to exclude the presence of a relevant percentage of azadirachtins and the occurrence of other constituents relevant for the activity. this is another recurring lesson for those studying natural products. although a plant has been the object of several phytochemical studies, new constituents can be obtained. an example is the discovery of gossypol in cotton oil. insecticidal activity is reported in a hundred or so published papers, concerning a wide range of species of arthropods, as confirmations of many traditional uses (schumutterer, 1995; amirthalingam, 1998; jones et al., 1989; van der nat et al., 1991; biswas et al., 2002) . leaves are used in houses to repel and keep away insects. when half of a sample of soya leaves are sprayed with nso and offered as food to the japanese coleopteran (popillia japonica), the insects feed only on the nontreated parts of the leaves. in nicaragua, farmers spray their cultures with an aqueous extract obtained by leaving the seeds for 12 h in water. in general, nso-based products have proven to be very effective against a huge range of pests of medical and veterinary importance, mainly including mosquitoes. the insecticidal properties of neem and its many formulations are based on experimented antifeedant, fecundity suppression, ovicidal and larvicidal activities, including growth regulation and repellence against a great number (around 600) of different insects, also at very low dosages, whereas useful insects were shown to be unaffected isman, 1997; sharma et al., 1993; schumetter and singh, 1995; forin et al., 2011) . the deterrent activity was also important, which can be easily determined as reported in fig. 7 .13. other studies, like the molting and the growing of the selection under investigation, need special devices, as those reported in fig. 7 .14. in particular, a concentrated extract of neem seeds, named mitestop, developed by the university spin-off company alpha-biocare (d€ usseldorf, germany), proved to be very effective against a huge range of pests of medical and veterinary importance, including ixodes and rhipicephalus ticks, house dust mites, cockroaches (blatta, blattella, and gomphadorhina), raptor bugs (triatoma), cat fleas, bed bugs, biting and bloodsucking lice, poultry mites, and beetle larvae parasitizing the plumage of poultry. neem leaves can also be used to protect stored woolen and silk clothes from insects. concerning mosquitoes, emulsified formulations of a. indica oil showed excellent larvicidal potential against different mosquito genera, including aedes, anopheles, and culex, also under field conditions. insect growth regulatory activity of neem-borne molecules alter or block the metamorphoses of larvae (toniolo et al., 2014) . neem weakens the cuticle defense system of the young instars, causing easy penetration of pathogenic organisms, or interferes with the molting mechanism. concerning biological control, an increase of the control of aedes populations was observed after the combined application of predatory copepods and neem-based larvicidal products, since repeated application of nso does not affect populations of predatory copepods. however, relevant limitations are related to the relatively high cost of refined products and the low persistence on treated surfaces exposed to sunlight. in the soil, the half-life of azadiracthins, meaning the time necessary to degradate the compounds, is from 48 min to 4 days, depending on the environmental conditions, like moisture, high temperature, and sun. the breakdown is faster on plant leaves, due to the exposure and the surface. in the attempt to assign the active constituents of nso, we must consider that the chemistry of neem is very complicated in terms of numbers and types of constituents. despite the great quantity of dedicated research, chemical research is far from complete. hundreds of compounds have been isolated and identified from various parts of neem, with seeds being the most investigated for their commercial value. the seeds may contain approximately 45% of a brownish yellow oil, mainly constituted by several fatty acids, i.e., oleic acid cis-9-ottadecenoic (50-60%), palmitic esadecanoic acid (13%-15%), stearic acid ottadecanoic (14%-19%), linoleic acid cis, cis-9,12-ottadecadienoic (8%-16%), and arachidic acid (1%-3%), although several other compositions have been reported. after a certain time, fatty constituents tend to separate and appear as white amorphous material. the main characteristics of the oil are its unpleasant strong alliaceous odor and acrid taste, attributed to sulfurous constituents. the shape and consistence can be very different according to the extraction method and the source. in fact, the composition of neem oil is highly variable, depending on preharvesting factors, like the cultivar, the geographic and environmental origin of the raw material, collection seasons, and postharvesting, like the extraction method, preservation, and conservation. extraction can be executed with different apparatus, temperatures, pressures, and methods, affecting the yield as well as the content. as later reported, these aspects have been deeply considered and hptlc analyses can be utilized to ensure the chemical composition of the neem oil utilized in the biological experiments. among the c.300 compounds characterized from the neem seeds, more than one-third of them are nortriterpenoids, which are triterpenoids lacking some carbon atoms (kaushik et al., 2014) . nortriterpenoids are chemotaxomically well located in a few related families of rosidae angiosperm dicotyledons, i.e., rutaceae, simarubaceae, cucurbitaceae, and meliaceae, within the rutales order. generally, in the plants of the rutales order, the partial loss of the lateral chain is followed by a complicated rearranging of the remaining part, giving rise to different polycyclic molecular skeletons, full of oxygenated functional groups, partially acylated. syntheses of complex natural compounds are costly and therefore they are usually used only for special activities. it is necessary to consider this point, which is in favor of the use of the plants as source of these compounds, since the synthesis can reproduce the chirality of nortriterpenoids only with extreme difficulty and cost. nortriterpenes are a very interesting part of the plant's chemical ability, that we call biosynthesis, to produce active complex molecules. nortriterpenes present very complicated structures and high numbers of active parts. we must remember that in natural products, activity is based on the presence of functional groups, made by heteroatoms, which means mainly o and/or n. if nitrogen is present, you have alkaloids, otherwise the range is higher, comprising phenols, alcohols, ketones, and others. however, the introduction of an oxygen inside a derivative usually is obtained to increase the activity, but also introduces instability in the molecule. we must remember that a natural product started from co 2 and is likely to become co 2 again at the end of its life. this is the necessary turnover of atoms and energy in organic matter. the first process accumulates energy and it is based on reductive reactions (endothermic reactions), whereas the second one is based on oxidation and produces energy (exothermic reactions). in other words, life is based on subtraction of negative entropy from the habitat, and at the end of its life, the organism releases this energy to the system. during its life, the molecule is expected to carry out its role inside the biosystem, which is the reason for its synthesis inside the plant. to understand the role, the nature of the target is essential. in insect-borne diseases, the natural product should interfere in the life of herbivorous insects or dangerous pathogens. in the case of neem, the activity is mainly larvicidal, blocking the metamorphosis to the next pupal stage. the larvae are unable to develop and change their state. to obtain this result, a lot of chemical and finalized activity are necessary, in this case consisting of hormonal interference in the insect metamorphosis process. in other words, the molecule must be able to mimic the internal complex chemical apparatus that allows drastic changes in the forms of the insect, until it stops the process. let us consider in particular the class of nortriterpenes. we have already had occasion to meet monoterpenes in the essential oil constituents. owing to their biosynthetic origin derived from progressive accumulation of isoprene units, each made by five carbon atoms, terpenes are classified according to the increasing molecular weight in monoterpenes (c10), diterpenes (c20), triterpenes (c30), and tetraterpenes (c40). squalene, the unique precursor of all triterpenes, is a linear unsaturated hydrocarbon, but its derivatives, for stability reason, are all cyclized with hexagonal and pentagonal cycles in the final structure. among triterpenes, the most famous class is certainly the steroidal one. steroids are present in any organisms, where they carry out several fundamental roles. without steroids, starting from the cholesterol stabilization of cell membranes to the influence on metabolism, no cell, and therefore no organism, could survive. however, each organism synthetizes its own steroids. in fact, animals possess simpler steroids usually with few oxygenated functional groups, bacteria produces steroidal triterpenes mainly dedicated to the stability of the cell envelope, and plants biosynthetize quite complex structures, named phytosterols. generally, phytosterols possess more cycles respect to the ordinary structure of the steroid model and an increase of the number of functional groups (roy and saraf, 2006) . animal steroids are based on a simple, easy-to-remember sequence of four fused cycles: three hexagonal and the last pentagonal. the basic structure of a steroid is quite easy to write and remember by students, including the stereochemistry, whereas the structures of limonoids are very complex and not so easy to remember. the problem is that in the basic structure of a steroid the sequence of the cycles is linear, whereas in limonoids there are complicated re-arrangements, causing a circular total structure. the insect's molt and metamorphosis are triggered and directed by hormones, usually consisting of steroids, such as prohormones (pheromones or juvenile hormones) and ecdysones. the term "ecdysone" was introduced by the german biochemist peter karlson (1918 karlson ( -2001 in 1956 in "chemische untersuchungen € uber die metamorphosehormone der insekten" (karlson, 1956) . the etymology of this word is interesting, from the greek ekdusis "shedding," or more precisely ἐκ(ς) ("external" or "from inner to out") + δύω ("dress oneself") + -si(s) ("action") + -ona ("hormone"). however, ecdysones have typical steroidal structures, whereas limonoids are the result of a complex chemical rearrangement. thus, to interfere in insect life, special triterpenes are necessary. several plants, like those in rutales and sapindales and related families, are specialized in the synthesis of such molecules, probably made to defend the plant from phytophagous insects. to obtain this result, great chemical ability is necessary. first, part of the typical hydrocarburic lateral chain, typical of most steroids, is lost ("nor" in chemistry means exactly this passage obtained by cutting c-c bonds), and the remaining part is both oxygenated and compressed in a complicated polycyclic structure, which is quite stable in the cell environment, but easily degradable in contact with atmospheric oxygen and sunlight. in this way, the nortriterpenes can be produced in the plant and transferred to the insect with mortal effects through a subtle toxic effect. the idea is to interfere with the growth regulators, interrupting the balance of hormones, named juvenile, in particular interrupting the transition process from the larval instar stages to pupae and adults by juvenile hormone analogues. therefore, on learning this lesson from nature, we can find solutions and inspirations. on the basis of these considerations and the structural diversity ( fig. 7.15 ), nortriterpenes can be divided into two main groups: limonoids (c26), with partial loss of the lateral chain (manners, 2007) , and quassinoids (c20 and c19), with total loss of the lateral chain (vieira and braz-filho, 2006) . in ancient times, plants containing these kinds of compounds were mainly famous for the bitterness of their drugs, utilized in the production of tonics, digestifs, and medicines. limonoids are part of our ordinary experience with some fruits and are crucial for the dissemination process. when we eat a citrus fruit, such as a lemon or an orange, we taste the agreeable flavor of the juice, but the seeds are discarded because they contain the nortriterpene limonin, which is very bitter and unpleasant. by throwing away the seed, we contribute to the reproduction of the plant. several other properties of limonoids have also been reported, including antioxidant, antimicrobial, and antitumoral activities, the insecticidal of neem being so far the most important. limonoids are considered the most active ingredient of insecticide neem products. they are classified into nine basic structures, with three main skeleton types: (a) azadiracthins, highly polioxygenated and acylated, with a saturated first ring, a tetrahydrofurane ring between the two first rings, and a final dihydrofurane ring chained with the other part of the molecule; (b) nimbins, less oxygenated and acylated with a skeleton evidently similar to that of the steroids, the furane ring with only a link with the remaining part of the molecule; and (c) a third type similar to the azadirachtins one, but the polycyclic part containing the dihydrofurane ring is less complicated, giving rise to a more linear general skeleton. such variability is necessary to sustain the large range of targets. in fact, these differences are just as important for the biological activity as for the decomposition. however, in terms of market considerations, azadirachtins, in particular azadirachtin a, are considered the reference constituents to evaluate the quality, and therefore the activity, of neem oil. azadirachtin a is a highly oxygenated c-secolimonoid, whose content in the seeds is highly variable (0.1%-1%), mainly depending on the producing zone and the seasonal trend. this compound acts as a biocidal on insects after ingestion or contact, with several effects: (a) interference on the growing processes, inhibiting the molting or blocking the hormone ecdysone synthesis; (b) antifeedant, with reduction of feeding; (c) negative effects on adult fecundity and egg fertility; and (d) diminution of the defense capacity of the cuticle, easing the penetration of pathogens. in particular, the larvicidal effect consists in the formation of the "permanent larvae," i.e., larvae are able to complete the molt as a consequence of destruction of the cuticle or of hormonal perturbation of the metamorphoses. this study consists in the careful observation of the larvae transformations and in the daily count of the consequence of azadirachtins and related compounds on the molting of phytophagous with buccal apparatus, either biting-sucking and chewing, comprised in all systematic categories: orthoptera including grasshoppers, locusts and crickets, etheroptera, homoptera, aphides, cicadellidae, hymenopterous, thysanoptera, aleurodidae, dipera, beetles, and others, including acarus and nematodes. neem's oil formulations usually show a range of different azadirachtins amounts, ranging from 1000 to 4000 mg/kg, meaning that products can be obtained either by using directly poor neem oil or a dilution process of neem extracts containing different quantity of azadirachtins, up to 5%. in addition to neem oil, azadirachtins are also marketed, in particular azadirachtin a. the amount of production of this substance amounts to about 64 tons, with 80% coming from india, and china as the second producer. other data about the activities of nso and its products can be found in the references at the end of this chapter. our first experiments clearly demonstrated strong larvicidal activity of nso and neem cake on asian tiger mosquito (nicoletti et al., 2012a,b) . however, our hplc and hptlc analyses showed a low content in azadirachtins in the nso and in the methanol extract . the result was interesting, since it is well-known that insecticidal activity is strictly related to the chemical composition, but in contrast to most reports evidenced a relation between the activity and the presence of these limonoids. this consideration prompted research to identify a relationship between composition and activity in the case of nsos marketed by different producers. first, the hptlc analysis indicated great differences in the fingerprints of the analyzed oils, with special reference to limonoids (nicoletti, 2011; toniolo et al., 2014) . a second analytical step consisted of a fractionation of three selected neem oils in three fractions of increasing polarities (i.e., ethyl acetate fraction (ea), butanol fraction (bu), and water (we)). the initial neem oil and the obtained fractions were evaluated for larvicidal toxicity and field oviposition deterrence against the asian tiger mosquito, aedes albopictus. the experiments showed good toxicity of the entire neem oil and ea fractions against a. albopictus fourth instar larvae (with lc50 values ranging from 142.28 to 209.73 ppm), while little toxicity was exerted by bu and we fractions. the differences of activity were in accordance with the results of hptlc analyses, since the nsos more concentrated proved to be more active. these results were confirmed by deterrence of a. albopictus oviposition in the field (effective repellence values ranging from 98.55% to 70.10%), while no effectiveness of bu fractions was found. concerning ovideterrent activity, no difference due to the production site was found. these experimental data evidenced the possible use of neem constituents against culicidae in the field. the constituents must be found in the apolar fraction, but the hptlc analysis showed a complex composition, wherein limonoids were not prevalent. therefore, neem oil and ea fraction seem promising, since they are effective at lower doses, if compared to synthetic products currently marketed, and could be advantageous alternatives to develop newer and safer mosquito control tools, but other studies are necessary to obtain a better definition of the active constituents and tailor the neem products in accordance with the required utilization (benelli et al., 2015c; . therefore, when we started our work on neem products, we found several incongruities between the reported studies in the literature and our results . in case of incongruence of the experimental data, two main interpretations are possible. the anomalous data could be the consequence of some error in the experimental procedure or the previous reported data must be reconsidered on the light of the new ones. in fact, many scientific important discoveries have been as consequences of unexpected results. there is a strong tendency in pharmacology to assign the activity of a plant drug to one constituent, or eventually a few of the same chemical class. this is mainly a consequence of the pharmacological tests, which are tailored on the magic bullet axiom and the difficulties in determining precisely the composition of an extract. however, in an extract, and consequently in the plant, there are hundreds of compounds, with effects on bioavailability, solubility, and synergic and antagonist activity. in opposition to the magic bullet, there is the approach of the phytocomplex, invoked by many researchers in phytochemistry and pharmacognosy. an important part of research on neem was dedicated to increase its availability and properties, focusing in particular on stability and cost, toward the production of the ideal insecticide. the first aspect was assigned to the production of nanobioparticles containing neem extracts, which demonstrated clear larvicidal and deterrent activity on vectors, like ae. aldopictus, also in field conditions (chandramohan et al., 2016; murugan et al., 2016) . several factors must be considered in the case of a product based on natural substances. in theory, the plant could be available for everyone and therefore it cannot be patented. therefore, so far natural products are available for everyone and thus have been explored very little. natural products are the chemical part of the environmental interactions between living organisms and therefore they are natural candidates for the production of active drugs. the chemical production of a plant is strictly subjected to the environmental conditions that can highly influence this production. first, the exactly determined species must be used and determined in composition. once the raw material is obtained, the process of transformation can significantly influence the composition of the product. the technological transformation is essential to the quality and efficacy of the product. therefore, this second step is vital for the success of the product. the third step consists of the target being assigned to the product and the consequent marketing. in future, natural products will be even more important in the production of new drugs and foods and feeds, able to face the challenges of a continuously changing market. technology is key to this. the natural products market is expanding rapidly in previously unexplored areas, in particular as an alternative to products based on synthetic compounds. the prospects and possibilities in this situation are immense, but knowledge of nature and activity of natural products must be revised utilizing recent devices and research approaches. importance and role of natural products will increase if the multidrug resistance continues, asking for new bacterial and insect possibilities of control. the common composition of a botanical product is based on a single herb or on the combination of more species based on recipes and formulae mainly derived from the historical literature and empirical experiences. the long and accurate work of phytochemistry based on the sequence extraction-separation-identification, derived from the correspondence of one drug to one illness, generated a huge catalogue of identified natural substances that can be employed as useful standards to determine the composition of the botanical drug. the knowledge about composition must be as complete as possible; not a single constituent should be unused, and utility depends strictly on the utilization. natural products are derived mainly from plants as the result of coevolution between organisms and environment (tehri and singh, 2013) . for this reason, they have been used for centuries in popular and traditional medicines, as well as often being employed as spices and insecticides. unlike modern pharmacology and drug development, which are based on a single chemical entity, natural product preparations are multiingredient. a single herbal drug contains at least 100 compounds making a complex matrix, named a phytocomplex, in which the single active constituent is not considered solely responsible for the overall efficacy. the utilization of the phytocomplex is based on experimental basis, since many data afford the validity of this approach, although further confirmations could be obtained using modern pharmacological devices. in other words, the same botanical raw material can be used directly, or extracted in different ways or used as a source of selected substances, or modified according to the product and target. in 2010, a mixed team of experts from mit (massachusetts institute of technology) and the broad institute of harvard university, both in the usa, reported an interesting and innovative study for a scientific evaluation of the effectiveness of natural products. the argument is strictly inherent to the endless debate about the role of natural products and their efficacy, causing a fighting contrast, but useless and boring, between supporters of "natural" versus defenders of synthetic drugs. the key aim of the study was to understand what is going on between the two main levels of the metabolism (primary and secondary), on the basis of the consideration of the functional connection between genes and gene products, as well as between genes and targets. an innovative feature of the study is that the researchers decided to commit the argument to neutral judgment, submitting the elaboration of collected data to the computational work of artificial intelligence. the work was based on the comparison of cumulative connectivity distribution of small molecules, natural or synthetic, grouped according to connectivity associated with the target. assuming that proteins form biological networks and that metabolism and health depend on these networks, we should be able to assign a role to the molecules considered as possible medicines. the result showed that natural products target the proteins with a high number of protein-protein functional interactions (higher network connectivity), whereas the synthetic ones act on a limited protein network. the conclusions of the study, based on a computational approach, were evident: "we observe that approved drug targets that are not also natural product targets exhibit a connection distribution much closer to the case for human disease genes that natural product targets, which remain the most highly connected targets." this sentence indicates a positive and useful consideration about the role and activity of natural products. natural products tend to target more essential and general protein networks to an organism than other groups of small-molecule targets, like those more related to specific disease genes. therefore, the dichotomy between natural and synthetic active constituents must be considered mainly as a consequence of a cultural heritage, unable to assign a complementary or differently appropriate role to the two classes of molecules. the results of the study are coherent with the nature of natural products, whose production is the consequence of environmental interactions, including defense against predators or pathogens. this kind of defense cannot be specific, and therefore natural products act on more highly connected network of proteins, interrupting or limiting the activity of the essential proteins in environmental competitors or invaders. they may be tailored for a positive or negative influence in physiologic activities and basic metabolism of an ample range of organic targets. these arguments are in favor of the potential use of natural products as insecticides. in any case, there are several difficulties in assigning the activity to single constituents, causing several cases of wrong or misleading assignments of all the activity to single substances in the case of a plant extract, like in valerian (valeriana officinalis), whose extracts are largely marketed and utilized for their mild sedative effects. with the discovery of valepotriates, the effects were assigned to these constituents, but after the evidence that extracts with low content in instable valepotriates also exerted similar action, the essential oil was considered additionally responsible for the effect. another case consists of a current debate about hemp. besides cannabinoids, its essential oil and other constituents are now considered important for the multiple activities of hemp. in other words, there are hundreds of marketed products of hemp and many related claimed activities, and this can be related to the complex cannabidioma and/or the different compositions of the products, although they are all derived from the same raw material. it is very important to stress that important new features can appear, also in the case of species highly studied in their chemical composition, as shown in the scientific literature. recently, a new cannabinoid was isolated from cannabis sativa (citti, 2019) . as is wellknown, (à)-trans-δ 9 -tetrahydrocannabinol (δ 9 -thc) is the main compound of hemp and it is considered the main one responsible for intoxicant activity. however, the chemical constitution of this species is subject to high differences in accordance with its varieties and cultivars. cannabinoids possess a unique structure, derived by junction of a monoterpene and a polyketide unit. most of them have a side alkyl chain, whose length influences the biological activity of this cannabinoid. in fact, analogues of δ 9 -thc with a longer side chain were synthetized and they have shown cannabimimetic properties far higher than δ 9 -thc itself (seven c against five). in this study, a new phytocannabinoid with the same structure of δ 9 -thc, but with a seven-term alkyl side chain, was isolated and identified, and its stereochemical configuration confirmed by a stereoselective synthesis. this new phytocannabinoid has been called (-)-trans-δ 9 -tetrahydrocannabiphorol (δ 9 -thcp). the binding activity of δ 9 -thcp against human cb 1 receptor in vitro (k i ¼1.2 nm) proved to be similar to that of cp55940 (k i ¼0.9 nm), a potent full cb 1 agonist. in the cannabinoid tetrad pharmacological test, δ 9 -thcp induced hypomotility, analgesia, catalepsy, and decreased rectal temperature, indicating a thc-like cannabimimetic activity. as confirmation, the corresponding cannabidiol (cbd) homolog with a seven-term side alkyl chain (cbdp) was also isolated and unambiguously identified by matching with its synthetic counterpart. the presence of this new phytocannabinoid could account for the pharmacological properties of some cannabis varieties that are difficult to explain by the presence of the sole δ 9 -thc and indicate the importance of the interaction between constituents of the so-called cannabidiome. therefore, we were not totally surprised when we found good larvicidal activity against aedes albopictus also in nsos with low content in azadirachtins . this was quite a novelty on the basis of the literature, but it is necessary to consider the importance of the metabolomics approach and the possibility with hptlc to obtain several views of the same plates. each view means a revelation of different compounds on the basis of their chemical structure and present functional groups. using an appropriate revelation agent, it is possible to see compounds that are not visible with another derivatization. this approach is contrary to the tendency of current analytical chemistry to focus on a single class of compounds or even unique constituents, which obtain perfect and reliable but limited results. another incongruence consisted of the presence of insecticide activity also in neem products after years of production, when limonoids should be highly degradated. the first experimental evidence we obtained on the activity of neem oil was the inability of the larvae of ae. albopictus to complete the molt from larva to pupa. the larvae proved to be initially immature, their bodies imperfect, and finally before the third instar, most insects died and none was able to fly. the delicate mechanism of the development stage was jammed and the cruel destiny of the unfortunate insects assigned. each organism has its weakness. mosquitos, like any arthropod, possess a rigid exoskeleton, which offers efficient strong and secure protection, also against pesticides, which is one of the reasons for the success of these creatures. the exoskeleton of insects is primarily made of proteins (sclerotin) and chitin (a polysaccharide), which are interwoven and linked together to form strong but flexible bundles. interestingly, chitin is also the main constituent of the fungal cell wall. the ratio of the components of the exoskeleton varies from one body part to another on an insect. however, the exoskeleton is too rigid, and acts like a cover that encases the entire insect, and being a nonliving formation, the exoskeleton does not change size and grow with the insect. the exoskeleton is too ridged to be recycled or modified, and it must be substituted, but it must also protect the insect until the new exoskeleton is ready. during the growth period, insects must shed the exoskeleton in order to assume a new form. as a result, it is necessary for the insect to shed its old exoskeleton to make way for a new, larger one through a process called molting. this is a hormone-controlled phenomenon. during the molting stages, the hormones are released to start and finalize each step of the metamorphosis, until the mature insect finally emerges. however, the chemical constitution of the exoskeleton is variable in each insect species and this is the reason for the selective toxic effects, such as those reported in the case of neem. regarding the structures of insecticides acting as growth regulators, albeit in the case of ecdysones the relation with insect hormones is evident, in other cases the similarity is not so clear, as well as the real mechanism of action. the stages between the subsequent molts are generally called instars. these correspond to altered body proportions, colors, patterns, and changes in the number of body segments or head width. for most insect species, an instar is the developmental stage of the larval forms, but an instar can be any developmental stage including pupa or imago. the larval stage is in particular a delicate stage of the insect metamorphosis. however, we were totally aware that confirmation of the neem insecticide activity, albeit with a demonstrated chemical constitution, in a laboratory experiment was a weak starting point. the open questions were numerous: (a) how to obtain the same result in the field; (b) whether the larvicidal activity could be connected to other properties, in order to improve the use; (c) what the cost of neem oil would be, considering the large-scale spread of the insects; (d) how limited the stability of the active ingredients of neem would be; (e) what determination of chemical content of neem oil would be required, to be connected to the determination of the activity; and (f) what the ambit of utilization would be and the possible damage to the habitat. other advantages arising from the use of neem-based products are the rare induction of resistance, due to their multiple mode of action against pests, the low toxicity rates that have been detected against vertebrates, and finally the necessary environmental care. there is a little confusion about the plant species named azedarach, and very similar denominations. the name azedarach was given by the famous persian physician avicenna (980-1037) to indicate some poisonous trees; however, azadirakhti literally means "free book of india." in 1753, linnaeus reported about melia azadarachta in his species plantarum (1: 385 with habitat: india). in the same book (1: 384), we can find melia azedarach (habitat: syria) and melia azedarach var. sempervirens (habitat: zeylona). actually there are two distinct species, azadirachta indica a. juss, attributed to neem (or nimba, meaning "who gives good health," as reported in the sanskrit books) and melia azedaracht linneus, attributed to melia, a very similar tree. this is the typical taxonomic situation in botany and zoology. the differences between taxa are often very narrow and only specialists are able to find them. in any case, the problem of the significance of these differences is always a matter of debate. god bless taxonomists, because they are necessary to obtain order out chaos, but please do not spend your precious intellect on endless discussions with no final consistent result! in fact, the matter is complicated by synonymous, parental disputes, errors of any type, including wrong transliteration (i.e., gingko and ginko), disputable rules of the international codices, and more. neem and melia are very similar, but there are several tricks to distinguish between the two species. the first is commonly known also as indian lilac and the second one as persian lilac or simply melia. neem has usually white flowers whereas melia presents an explosion of blue flowers; the fruits of the former have an elongated shape, whereas the latter's are totally rounded. if the trees do not have flowers or fruits, and you are not a botanist, you may be in trouble, but you can remember that neem cannot live in temperate climate regions, whereas melia can be easily cultivated in such places. therefore, if you are in europe or the usa, you can be 90% sure on the matter. melia azedarach is known by several common names, such as melia, chinaberry tree, pride of india, bead-tree, cape lilac, syringa berrytree, persian lilac, and others. it is usually a large tree growing up to 30 m tall, with leaves 2-pinnate, rarely 3-, with primary pinnae in two to six pairs, usually three to seven leaflets per pinna, narrowly ovate or subovate, serrate, acuminate, irregularly toothed, or crenate. flowers are abundant and small, sweet-scented, in large axillary panicles. all parts of this tree are reported to have medicinal uses, but in particular, in terms of insecticide properties, seedlings are reported to present aphid attacks. a leaf used as a bookmark will deter insect pests. in italy, the tree is known as the tree of rosary, since in the past, before the advent of plastic, its hard and round kernels were used to make the grains of a rosary. our research on melia azedarach, as well as the references on this plant, evidences a significant difference in the chemical composition. limonoids are present, but different from azadirachtins and other constituents make a marked relevant dissimilarity in composition. the initial conclusion was that melia probably cannot compete with neem as an insecticide, but other utilizations can be explored. however, once again a limit in the references is an irresistible task for a researcher in search of innovations. in addition to the insecticidal properties, we were initially particularly interested in the antimicrobial activity. people often associate antimicrobial activity with infection and effects on their health, but microbes are everywhere and most damage affects cultivation of plants. agricultural methods of reproduction of plants with economic value were totally transformed by the introduction of micropropagation and stem cell culture. micropropagation allows the rapid cultivation of selected cultivars, saving time and resources. however, although the first steps of micropropagation were performed in aseptic conditions, the possibility of infection of calla, shoots, and seedlings is high. avoiding the infection must be done via an appropriate and sensitive approach, avoiding damage to the delicate meristems-a typical job for natural products. the antibacterial study (marino et al., 2014) aimed to investigate the antibacterial activity of unripe fruits of melia azedarach collected in different periods. the activity was tested on the shoots of a hybrid of prunus cerasifera x prunus spinosa and calla lily of zantedeschia aethiopica against several bacterial species. the data reported evidenced a positive antibacterial activity and the absence of any negative effect on the growth of shoots surviving at the second subculture on a standard medium. hptlc analysis showed the prevalence of polyphenols, such as chlorogenic and caffeic acids, which, on the basis of the literature, are consistent with the antimicrobial activity. this activity is important considering that many plant species of economic relevance are now obtained by micropropagation, and this cultivation in vitro is necessary to avoid any sort of contamination. further research is essentially the rational collection of most of the arguments previously considered, as evident in the title: "green-synthesised nanoparticles from melia azedarach seeds and the cyclopoid crustacean cyclops vernalis: an eco-friendly route to control the malaria vector anopheles stephensi?" (anbu et al., 2017) . in this research, once a single-step greensynthesis of silver nanoparticles (agnp) using the seed extract of m. azedarach was obtained, we tested its mosquitocidal activity. in laboratory assays on anopheles stephensi, ag np showed lc 50 ranging from 2.897 (i instar larvae) to 14.548 ppm (pupae). in the field, the application of ag np (10 âlc 50 ) led to complete elimination of larval populations after 72 h. finally, we decided to test the nanoparticles on nontarget aquatic predators. the application of ag np in the aquatic environment did not show negative adverse effects on predatory efficiency of the mosquito natural enemy cyclops vernalis. the reason for this additional research lies in the fact that numerous aquatic arthropods attack and devour preparasites. as we already know, the utilization of the insecticides, though with plant-derived active constituents, could be dangerous for the environmental equilibria. in particular, it could affect the natural biological control, based on the presence predators of the vector in the common habitat, remembering that all the insect stages, except the adult insect, need water. in such sites, there is fresh water everywhere, such as lakes, pools, and similar places, enabling life along the plant-covered banks of stagnant and slow-flowing bodies of water. in such places, mosquitos can proliferate as can any other predator, which in an aquatic environment is fundamental to limit the proliferation of the vector. in fact, after coupling, and the consequent blood feeding necessary to assume the proteins necessary for the eggs maturation, the female is looking for an appropriate place for the deposition of 100-500 eggs. a single anopheles, like other insect, is able to produce a quantity of eggs and larvae enough to invade all the neighboring habitats, as in the classic case of a locust invasion. this is not possible only thanks to the natural enemies. the microaquatic environments are the scenario of a continuous fight for survival, where often two or more species of arthropods are involved, as predator or as prey. in our study, we selected the genus cyclops, which is one of the most common of freshwater copepods, comprising more than 400 species. copepods are very little crustaceans, commonly called water fleas. they have a single large eye, which may be either red or black, and therefore they are named for the cyclops of greek mythology. cyclops prefers fresh water, and is less frequent in brackish water, where it feeds on small fragments of plant material, animals, or carrion. it swims with characteristic jerky movements and has the capacity to survive unsuitable conditions by forming a cloak of slime, with an average lifespan of about 3 months. several microscopic crustaceous, including copepod species, feed small and very small preys. in high-density, unstructured environments such as eutrophic lakes, predatory copepods commonly coexist with certain smallbodied prey, where encounters are frequent with ineptness on the part of the predator and counter-tactics by the prey. in particular, laboratory studies showed that copepods are effective predators on early-instar culex larvae, involving an important role in suppressing mosquito populations, because of their feeding behavior and abundance. they are very efficient in this role, since the presence of alternative abundant food, like bacteria and protozoa, does not deter their attacks on their preferred prey. copepods are capable of killing and eating at least four preparasites within 13 min and a predator density of 53 copepods/liter is expected to reduce the mosquito larvae by 50%, with the rate of predation inversely proportional to the water volume. neem cake: from by-product of an industrial process to multipurpose resource for a sustainable agriculture chain during our research activity, we were highly interested in industrial plantborne by-products, since they can offer new products to the market with lower cost and high usefulness. our attention was immediately attracted by neem cake, a cheap by-product of nso extraction, obtained as a residue after mechanical pressing of the neem seeds, considered of low economic value and utilized to enrich the soil of some mineral components, such as nitrogen. the laboratory test indicated neem cake activity against aedes albopictus and a number of culicidae species (nicoletti et al., 2010) . in the case of biocidal treatments, it is important to demonstrate that insecticide activity is associated with antimicrobial activities in consideration of the high possibility of infection and the severe consequences for health, in particular in the case of the animals, both pets and livestock. the importance of insecticidal and antimicrobial activities for animal treatment has been evidenced in the experiments with nso and neem cake further reported, including larvicide, deterrent, and repellent activities (benelli et al., 2015a,b; nicoletti et al., 2012a,b) . the complex range of different compounds in the neem seeds open the possibility to utilize the derived products to solve many current problems. the challenge now is to obtain marketed products tailored for different utilizations. the reported experiments evidenced these potentialities, which are only waiting a realization and a wider utilization. despite diseases, wars, and environmental disasters, the human population is growing. first of all, more people will need more food. this forecast shows in particular a massive increase in animal protein demand, needed to satisfy the growth in the human population, wherein billions of people require an increase of caloric input and better food. therefore, attention is focused on the sources of feed protein and their suitability, quality, and safety for future supply. in addition, the quantitative production aspect is causing a series of problems. there will need to be a considerable increase in feed manufacture, requiring a thriving, successful, and modern feed industry, including a key aspect concerning the protection and preservation of the food produced and marketed. this aspect is strictly related to safety issues, which will remain paramount in the minds of consumers following recent food crises. it is time to consider that the need of more food to feed due to increasing planet population perhaps cannot simply be solved by massive production, but reduction of food waste and conservation can increase food availability by 30%-40%. "feed the planet and energy for life" was the theme of world expo 2015 in milan, italy. among the activities occurring at the expo, research and proposals concerning the utilization of neem products were presented in a call for projects in favor of sustainable progress and production of future foods. the neem project was selected as the best one due to its possible applications in the production of food and feed. the expo event projected feeding as the main challenge for humankind and showed the extreme urgency of elements of innovation in technology and science connected to the production and conservation of food. it was demonstrated how serious feed problems still plague several areas of the world today, and the possibility of new solutions was mentioned. the neem project was focused on the agricultural utilizations of neem cake concerning its advantages as soil fertilizer and as a natural ectoparasiticide for the treatment of sheep and goats. neem products were proposed as being able to affect the biotic composition of the soil. neem cake must be preferred to neem oil for its cost and its form as a powder immediately available. several experiments evidenced the improvements of the utilization of neem cake in agrarian ecosystems: (1) availability of nutrients, in particular nitrogen and phosphorus, more consistent with the requirements of the crop; (2) development of the microbial beneficial biomass of the soil, which increases in quantity and activity, but with selective influence against nematodes and other negative components. in agricultural practices, plants in addition to nutrients should count on a greater variety of useful microorganisms and on acquisition of nutrients themselves, through the activation of complex symbiotic systems. if you want to understand the state of health of a tree, you must look down, not up; and (3) development of pest control system of insects and other arthropods of agricultural and livestock interest. neem cake, as an industrial by-product, is a heterogeneous material that maintains a high added value due in large part to its chemical composition, which confers its biological activity. neem cake is widely available on the global market, considering the increasing presence of neem trees in the world and production of nso. the exploitation of its characteristics in the food chain to improve consumer health, increase the productivity of agricultural products, and feed the planet is the logical consequence of the urgent need to develop new sustainable agricultural systems in a world where many highly polluting pesticides are no longer allowed to be used. however, more research, in particular in field conditions, is necessary to understand the real value of its microbiological, insecticide, fertilizer, and nematocide activity, involving collaborations between different experts in individual sectors-import companies, organic farms, and research institutions-in order to determine the manner and timing of land application of this valuable product of "waste," still underestimated. neem cake could lead to a revolutionary improvement in the fertilization of agricultural plants, adding to the characteristics of chemical fertilizers those of a soil improver. in agriculture, we could define neem cake as a prompt nutrient-release fertilizer, effective in allowing rapid absorption of nutrients and promoting development of the plant, with the capacity to increase the activity of the microbial biomass and organic matter, favoring the sequestration of carbon. the idea was to join the fertilizer, insecticide, and antimicrobial utilities of neem cake. exploitation of the use of neem cake as an insecticide came from this first test: some pots of impatiens (impatiens balsamina) were fertilized with 3% by volume of neem cake, and 500 mosquito larvae were reared starting from eggs. the eggs were hatched in control and treated in pot saucers, but none of the newborn larvae survived in the water saucers of pots treated with neem cake, while in the water saucers of pots unfertilized with cake, the 500 control larvae completed their development in less than a week, becoming adult mosquitoes. other major beneficiaries of the use of neem cake as insecticide are undoubtedly sheep farmers, who can use an organic product of natural origin and low cost that is simultaneously effective against the larvae of culicoides and other pests, while respecting the natural biotic communities. direct beneficiaries of neem cake, as a fertilizer, are farmers seeking pest and nematode control, in particular for nematodes. currently, some highly toxic products are still on the market by virtue of the absence of suitable alternatives. particular attention must be focused on the changes on soil micro-composition, as evidenced in several field experiments. in conclusion, we can report the following important advances in the use of neem cake as a functional fertilizer: (a) energy saving flows from the use of a waste of an industrial chain; (b) environmental sustainability, as documented by the analyses attesting the absence of heavy metals, aflatoxins and residues of pesticides; (c) neem cake is an excellent alternative to methyl bromide (bm) (banned as being responsible for the "thinning" of the ozone layer of the atmosphere); (d) neem cake is an excellent alternative to temephos and other organophosphates used to treat water infested with disease-carrying insects including mosquitoes, midges, and blackfly larvae; (e) neem cake is a great alternative to nematicides, like 1,3-dichloropropene; and (f) neem cake in the field trials carried out in sardinia had efficacy similar to azadirachtin biological products already established in organic farming, but were very expensive and not really effective. in addition, neem cake showed very low effect on "nontarget" insects that live in the same environments as culicoides larvae. ectoparasites are organisms that inhabit the skin of another organism, causing significant infestations and pathologies. many micropathogens can profit from the work of ectoparasites, either to colonize the skin injury and lesion, or be inserted in the host during the feeding. the vast majority of ectoparasites are arthropods, e.g., insects and arachnids. again the triangle host-vector-etiological agent is reproduced. many ectoparasites are vectors of pathogens, which are typically transmitted while feeding on or from other hosts. several ectoparasites (e.g., most lice) are host-specific, including livestock, pets, poultry, fish, and bees, but others parasitize a wide range of hosts, including humans. typical effects of infection on the host are irritability, dermatitis, secondary infection (other parasites profit of the skin necrosis), fecal hemorrhages, blockage of orifices, inoculation of toxins, and exsanguination. as a consequence, the host's general health can be seriously affected with low weight gains, particularly important in livestock. subdermally located parasitic larval stages of certain flies can be favored by the ectoparasited infection, causing a condition termed "myiasis." when insects (order hemiptera) are involved, the infection mechanism is similar to that previously described for any insect-borne disease. the vector contains several hematophagous ectoparasites, including approximately 150 species of kissing ("cone-nose") bugs (reduviidae, triatominae) and bed bugs and bat bugs (cimicidae). these parasites make physical contact with the host principally when ingesting a blood meal. these kissing bugs usually prefer domestic animals, from which relatively large blood volumes may be imbibed; in such a way they can cause a great deal of damage and transmit important diseases. ectoparasites play a very detrimental role in terms of decreasing the productivity of livestock, such as sheep and goats. nso was utilized in the field as an antibacterial in the case of ectoparasites' stings and bites resulting from goat wounds. common external sheep and goat parasites include ticks, lice, and mites. they cause restlessness and irritation. weight loss and reduction in milk production may occur as a result of nervousness and improper nutrition, because animals spend less time eating. bites can damage sensitive areas of skin (teats, vagina, eyes, etc.) . some parasites feed on blood, causing anemia, especially in young animals. the bite and the sting of ectoparasites allow bacteria to proliferate in wounds from abrasions or lesions from scratching, and cause levels of tissue reaction of different entities, super-infection, and cervical lymphadenopathy. ectoparasites cause many problems in livestock production. they seriously damage sheep and goat skins, resulting in the rejection or downgrading of the skins. this causes huge economic loss, as this skin damage renders it unsuitable for the leather industry due to the decrease in quality. lower production of meat is also a typical consequence. pseudomonas aeruginosa wound infections are characterized by a change in the color of the skin around the wound area and the formation of lesions. the bacterium products and pigment cause yellow discoloration of wool and consequently reduced quality and market value. nso treatment in the field on as a natural ectoparasiticide for sheep and goats proved to be successful in preventing and curing the attacks of endoparasites ( fig. 7.16) . the experiments were performed on selected livestock (fig. 7.17 ) by a specialized team of crea researchers (de matteis et al., 2015) . the effects on the parasites were evident (figs. 7.18 and 7.19) and even after the first treatment with nso, protection against ectoparasites was obtained. more important, the health of the treated livestock improved, as testified by the hematological profile of goats. in vivo and in vitro tests on blood cells from siriana, sanen, cashmere, and maltese goat (capra hircus) breeds showed no significant difference (p <.05) between nso treated and untreated goat hematological parameters at each sampling time considered. in addition, the nso effect on goat pbmc cultured in rpmi medium was evaluated at 1:2â102 to 1:20â106 dilutions at 14, 21, and 40 h of exposure. the in vitro test revealed that the response of goat pbmc viability is dependent on concentration, incubation time, and nso dose. in conclusion, the nso should be considered useful, safe, and innovative for development of topical solutions for the care of wounds. among the most relevant typology of neem products, we focused on selectivity. the antibacterial activity of nso was assayed against 48 isolates of escherichia coli, considering that this bacterium can produce beneficial and pathologic populations. the molecular biology characterization showed that 14 isolates resulted in diarrheagenic e. coli. nso showed biological activity against all isolates. however, there were significant differences between the antibacterial activities against pathogenic and nonpathogenic e. coli, as well as between nso and ciprofloxacin activities. on the basis of the results obtained, nso is able to counteract e. coli and also influence the virulence of e. coli-viable cells after treatment with nso. the preservation of marketed food is an important aspect of the smart utilization of the produced food (maruchecka et al., 2011) . furthermore, the consequent waste of unutilized food is a relevant problem in overcrowded towns (hlpe, 2014) . a large quantity of food is lost or wasted throughout the supply chain, from initial agricultural production down to final household consumption (hlpe, 2014; kader, 2005) . the loss or waste for high perishable food, such as fresh fruit and vegetables, fish and livestock products, has been estimated at as much as half of all food grown before and after it reaches the consumer. approximately one-third of all ffvs produced worldwide are lost during food supply chain production. shelf life plays a central role in food spoilage. the impact of the enormous quantity of packaging is evident in any planet environments. increase of the shelf life means reduction of cost and waste. everything pivots around the material utilized for packaging, and new solutions are emerging (otoni et al., 2016; singh and singh, 2005; cooksey, 2005; appendini and hotchkiss, 2002) , including passive packaging (brockgreitens and abbas, 2016; ozdemir and floros, 2004) , active packaging (coma, 2008) , intelligent packaging (lee et al., 2015; de kruijf et al., 2002) , and smart packaging (dobrucka and cierpiszewski, 2014) . although the results are not evident in our ordinary life, the galaxy of packaging is rapidly moving and increasing in research and proposals, based on new technologies and advanced techniques recently available, like nanotechnology and molecular biology. efforts are focused on solving the food preservative problems, to extend the shelf life of perishable foods, by reducing the need for additives and preservatives. "smart packaging" is based on the production of functional methods to obtain the following goals: be tailored depending on the product being packaged, including several types of food, beverages, pharmaceuticals, household products, etc.; reduce food waste, increasing the shelf life; and maintain, and eventually enhance, product attributes (e.g., look, taste, flavor, aroma). the key words are protect, preserve, and present. several methods and approaches, such as oxygen scavenging and antimicrobial technologies associated to the production of modified films, have been considered . they are different solutions to serve the basic and fundamental properties of packaging. so far, the dominant packaging is the basic one, using low-cost material and involving no interaction with the food inside. this is passive packaging, wherein the traditional packaging systems are included, as the use of covering material characterized by some inherent insulating, protective, or ease-ofhandling qualities. usually, the ordinary packaging of food is mainly a used to method to attract and select the consumer, beside a preservation. the consequence is the enormous amount of waste, and the consequent damage to the environment. this situation is increasing due to the increasing numbers of consumers in emerging countries, where these consequences are not adequately considered. packaging is considered active when it can interact in the same way and/or react to various stimuli, in order to keep the internal environment favorable for the maintenance of product quality. several environmental, biotic, and abiotic factors must be considered, in order to respond to the degradation process successfully. the activity involved could be the presence of an oxygen scavenger (this can absorb high-energy oxygen inside a package and therefore increase the shelf life of a product) or an anti-ros (a scavenger of radicals by oxygen or other origins), such as in the typical case of phenolic natural products. smart packaging relies on the use of chemicals, electrical, electronic, or mechanical technology, or any combination of these. technology is used to modify the packaging by adding constituents to change its features and properties (kerry et al., 2006; malhotra et al., 2015) . active and intelligent packaging is particularly dedicated to the preservation of fresh products, like vegetables, in accordance with increasing requirements for this kind of food (nicoletti and del serrone, 2017; nicoletti, 2014a,b) . intelligent packaging systems monitor the condition of packaged foods to give information regarding the quality of the packaged food during transport and storage (aguilera et al., 2003) . probably the most innovative aspect of intelligent packaging is that it can be supported by the utilization of systems of detection in meat and meat products, obtained through the use of sensor technologies indicators (thakur and ragavan, 2013) , including integrity, freshness, and time-temperature indicators (ttis) and radio frequency identification (rfid). therefore, active and smart packaging performs additional functions to the basic one by the introduction of innovations in the design of packaging, with the aim of increase the shelf life, but also to add conveniences for the user and usefulness for the consumer, to be introduced also in the supply chain. in this way, the product can respond not only to the need for a longer life, but also make the product more available, more useful, and more safe. since our invisible enemies are asked to play their role again, antibiotic activity is required. packaging is mainly used to separate food from environmental conditions, utilizing simple material made of paper or plastic. however, it cannot prevent internal attacks by microorganisms, but can only limit or delay the effects. therefore, additional treatments are required to limit their action, like the utilization of low temperatures, which involves additional costs and energy consumption and pollution. a new idea is to associate to the packaging some antimicrobial agent. before and during packaging, storage, and shelf life, food is subjected to a continuous attack by microorganisms. these microorganisms are working to benefit themselves by demolishing progressively the molecular structure of the food, as soon and as completely as possible. therefore, by preserving the food, we are working in a thermodynamically unfavorable situation. in term of shelf life, the food is in competition with its natural recycling, and, working to maintain as possible this limbo, we can utilize the food efficiently as it is possible. the resistance phenomenon interests also zoonotic food-and waterborne pathogens becoming more resistant to antibiotics (del serrone et al., 2006) . resistant strains of pathogens have been isolated from food, causing an increasing incidence of food-borne diseases. through the food, these microorganisms could be entering the human gastrointestinal tract on an almost daily basis. the antimicrobial activity of nso and related products have already been reported (palanappian and holley, 2010; baswa et al., 2001; sairam et al., 2002) . a possible utilization of antibacterial activity of neem cake against meat spoilage bacteria was tested using a broth model meat system . the tests were positive, since the growth inhibition zone (mm) varied significantly (p!.05). with respect to ciprofloxacin activity, the antibiotic value ranged as follows: 11.33 ae0.58 to 22.67ae0.58 mm and 23.41ae1.00 to 32.67 ae2.89 mm, respectively. the percentage of bacterial growth reduction (gr%) also varied significantly (p !.05) in function of considered nce concentrations (1:10-1:100,000), with the highest gr% for 10 μg nce (79.75 ae1.53 to 90.73 ae1.53). the numbers of viable bacterial cells never significantly (p .05) exceeded inocula concentrations used to contaminate the meat. all the results of the experiments showed that neem cake is able to counteract the main microorganisms responsible of meat spoilage, like strains of gram-positive and gram-negative, as well as facultative anaerobic bacteria. the antimicrobial activity of neem products was confirmed also for nso against spoilage bacteria, such as carnobacterium maltaromaticum, brochothrix thermosphacta, escherichia coli, pseudomonas fluorescens, lactobacillus curvatus, and l. sakei. after the second day after nso, only c. maltaromaticum-viable bacterial cells were detected. these data could be used to create new intelligent packaging. utilizing a nanotechnology already employed for other materials, neem cake may be incorporated into the cavities of nanoparticles, maintaining its antiparasite activity. once incorporated into the packaging material, the neem cake, also in minimum quantity, should be able to effect its preservative food action, acting against the demolishing microorganisms. the increase of the shelf life of meat should compensate for the additional cost of the packaging material, not considering the decrease of waste. it is possible that the first activity of luca was to find the energetic source for survival, and the second was to compete with the other lucas. the results are an endless transformation of forms and production of new molecules. the living organisms had a long time to organize their molecular weapons and the secondary metabolites are there, produced and organized to be considered and utilized in the right way. the neem tree is an example of nature's treasure. the advent of homo sapiens (lucy) changed in part the rules of the natural game, but natural products still remain a necessity for our life. recently, parenteral artesunate has replaced quinine and many other antimalarial products for the treatment of severe malaria. however, several reports have demonstrated the emergence of resistance to the efficacy of artemisinin-based combination therapy monotherapy, such as in western cambodia and other regions in south-east asia. to face the phenomenon, artemisinin-based combination therapies are now recommended by the who. the aim is to reduce the morbidity and mortality associated with malaria with artemisinin-based combination therapies, including chloroquine plus other drugs, like sulfadoxine-pyrimethamine. meanwhile, with increasing resistance to chloroquine, quinine is reconsidered, being so far the only substance for which plasmodium did not develop resistance. the consequences are that in uganda quinine was prescribed for up to 90% of children younger than 5 years with uncomplicated malaria, and from 2009, 31 african countries recommended quinine as a second-line treatment for uncomplicated malaria, 38 as a first-line treatment for severe malaria, and 32 for treatment of malaria in the first trimester of pregnancy. recent surveillance data from other sites are in accordance. however, quinine was substituted due to its limits and therefore in 2010, who (2010) guidelines recommended reinforcing quinine's activity by combining it with other antimalarial agents, like doxycycline, tetracycline, or clindamycin as a second-line treatment for uncomplicated malaria (to be used when the first-line drug fails or is not available) or quinine plus clindamycin for treatment of malaria in uncomplicated cases and in the first trimester of pregnancy. the development of effective cocktails is a current trend of medical treatment of several diseases, including forms of cancer. in addition, the combination of natural products and synthetic drugs is recommended. natural products can be utilized as resistance-modifiers or chemosensitizers, and may be able to restore chloroquine sensitivity in resistant strains of plasmodium. the idea of 8 years of research from different research groups was that the antimalarial treatment combined with natural products could be based on lower doses of chloroquine, in order to minimize the resistance insurgence and to avoid the collateral effects in the case of prolonged use, necessary in areas where the disease is endemic. this approach came from an ethnopharmacological investigation by professor rasoanaivo (rasoanaivo et al., 1992) . most people consider ethnopharmacology to be a collection of ancient utilizations of natural sources, and as knowledge that is going to disappear. on the contrary, in addition to traditional uses there are new ones emerging, even as consequences of the utilization of modern drugs. considering that oms reports that 80% of the planet population relies on traditional medicine, the utilization of medicinal plants is not limited to ancient times and past populations, but it changes according to needs and evolution of treatments. ethnobotanical knowledge is still passed from one generation to another in the majority of populations living in rural areas, and in urban areas, where malaria has been revealed to be resistant or incurable by modern scientific medicines, people have turned to traditional treatments. it is therefore of paramount importance to preserve and transmit this ethnobotanical heritage. therefore, this discipline must be regarded as a multidisciplinary science in movement, where botany, chemistry, and pharmacology play central roles for scientific evaluation and validation of popular uses. however, economic and social aspects must also be considered, in order to develop new drugs and treatments of both old and new diseases. most antimalarial drugs currently in use belong to the classes of aminoquinolines (chloroquine, amodiaquine, primaquine), quimolinomethanol derivatives (quinine, mefloquine, halofantrine), diaminopyrimidines (pyrimethamine), sulfonamides (sulfadoxine, sulfadiazine), biguanides (proguanil and derivatives), antibiotics (tetracyclines, doxycyclin, clindamycin), sesquiterpenes (artemisinin, dihydroartemisinin, arteether, artemether, artesunate) , and naphtoquinones (atovaquone). among them, only quinine and artemisinins are natural products, but also a relevant part of the current antimalarial arsenal. the potentiality of natural products is very high. a review by willcox and bodeker (2004) on traditional herbal medicines for malaria in three continents reported 1277 plant species from 160 families. however, the clinical trials are largely lacking, since only eight clinically controlled trials have been reported, involving p. falciparum and p. vivax. in the case of malaria, alkaloids are the first candidates responsible for the activity. there is a long tradition in popular medicine of plants containing these compounds to control fever. these plants also have a bitter taste, which is usually connected to the alkaloid presence, as already reported for the aforementioned quinine bark case. two important considerations attracted our attention, in view of the possibility to explore new strategies: the special endemic flora of madagascar and the occurrence of information about a popular treatment of malaria as yet unreported. madagascar is a land of endemism, consisting about 13,000 species of vascular plants, of which 80% are endemic, and eight families totally endemic. malaria is practically endemic in all madagascar and therefore the population harbors a very rich and unique knowledge on antimalarial plants. after a resurgence of malaria in the early 1980s, as a consequence of plasmodium falciparum resistance and due to the high costs of conventional drugs, local populations returned to the uses of herbal remedies. two hundred and thirty-nine plant species, of which about 30% are endemic to madagascar, have been reported as having antimalarial uses in malagasy traditional medicine. prof. rasoanaivo discovered the use by some populations in madagascar of decoctions of some local plants in association with low doses of chloroquine to complement chloroquine action against chronic malaria . the lower use, one or two tablets of chloroquine (100-200 mg), is probably adopted to avoid collateral effects due to prolonged use of chloroquine, but such a dose could be considered inadequate to favor chloroquine resistance. therefore, we have a mixture of recent learning and ancient knowledge, evidencing the reality of ethnopharmacology. however, popular uses of medicinal plants need scientific validation with advanced tools. therefore, research started from the knowledge that some populations in madagascar use decoctions of some local plants in association with low doses of chloroquine to complement chloroquine action against chronic malaria. in such a way, resistance insurgence and collateral effects are both lowered. on the basis of the ethnobotanical work conducted by rasoanaivo and his collaborators, 24 plants were selected and investigated for in vitro and in vivo antimalarial activity and a chloroquine-potentiating effect. in the case of validation of the activity, the determination of the active constituents followed. the results of these selections were that the alkaloids of loganiaceae, menispermiaceae, and rutaceae were the most promising compounds showing significant effects, some of them potentiating the action of chloroquine. from a phytochemical point of view, alkaloids are in pole position among natural products utilized in traditional medicine against malaria. mono-and bis-indole alkaloids have been isolated from several plants that are traditionally used to treat malaria on different continents. the most active compounds are those that originate from plants belonging to the genera strychnos (loganiaceae) and alstonia (apocynaceae). a review covering the indole alkaloids that have high antiplasmodial activities in vitro and in vivo, and favorable selectivity indices (si¼cc 50 /ic 50 ), was published by frederich et al. (2003) . in the case of malaria, alkaloids are the first candidates as being potentially responsible for the activity. there is a long tradition in popular medicine of plants containing these compounds to control fever. these plants also have a bitter taste, which it is usually connected to the alkaloid presence, as already reported for the aforementioned quinine bark case. two important considerations attracted our attention, in view of a possibility to explore new strategies: the special endemic flora of madagascar and the occurrence of information about a popular treatment of malaria so far never reported. madagascar is land of endemism, consisting about 13,000 species of vascular plants, whose 80% are endemic, and even 8 families totally endemic. malaria is practically endemic in all madagascar and therefore the population harbours a very rich and unique knowledge on antimalarial plants. after resurgence of malaria in the early 80's, as a consequence of plasmodium falciparum resistance and due to high costs of conventional drugs, local populations back to the use of herbal remedies (blanchard, 1901; maggi et al., 2017) . two hundreds and thirty-nine plant species, of which about 30% are endemic to madagascar, have been reported as having antimalarial uses in the malagasy traditional medicine. prof. rasoanaivo discovered the use by some populations in madagascar of decoctions of some local plants in association with low doses of chloroquine to complement cq action against chronic malaria . the lower use, one or two tablets of chloroquine (100-200 mg) , is probably adopted to avoid the collateral effects due to a prolonged utilization of chloroquine, but such dose could be presumed inadequate to favour chloroquine resistance. therefore, we have a mixture of recent acquirement and ancient knowledge, evidencing the actuality of ethnopharmacology. however, popular uses medicinal plants need a scientific validation with advanced tools. therefore, the researches started from the knowledge that some populations in madagascar use decoctions of some local plants in association with low doses of chloroquine to complement chloroquine action against chronic malaria. in such way, resistance insurgence is lowered, as well as collateral effects. on the basis of the ethnobotanical work conducted by rasoanaivo and his collaborators 24 plants were selected and therefore investigated for in vitro and in vivo antimalarial activity and a chloroquinepotentiating effect (maggi et al., 2017) . in case of validation of the activity, the determination of the active constituents followed. the results of this selection were that the alkaloids of loganiaceae, menispermiaceae and rutaceae were the most promising compounds showing significant effects, some of them potentiating the action of chloroquine. mono-and bis-indole alkaloids are traditionally used to treat malaria in different continents (ramanitrahasimbola et al., 2001 (ramanitrahasimbola et al., , 2006 . the most active compounds were mainly related to the genera strychnos (loganiaceae) and alstonia (apocynaceae). a review covering the indole alkaloids that have high antiplasmodial activities in vitro and in vivo, and favourable selectivity indices (si¼cc 50 /ic 50 ) was published by frederich et al. (2003) . strychnos is a pantropic genus, with about 200 species, present in three continents: 75 in africa, 73 in america, and 44 in asia and oceania (only s. potatorum is present in both asia and africa). asiatic species are mainly small trees, whereas in the new world lianes are generally dominant. the most famous strychnos species is the asiatic s. nux-vomica, because of strychnine contained in the seeds with 12 other related alkaloids. strychnine is also known and used for its bitter taste. south american species are characterized by different mono and bisindole alkaloids, important as constituents of some curare preparations of indios amazonia tribes (see introduction) . during the preparation of curare, the tribe curandero selects local plants and extracts the mixture by hot water. finally, the extract is filtered on leaves and concentrated to obtain a paste, which is preserved into a container, like a calebassa or a tube, maiden by a cane, or a pottery. active constituents in curare are bis-indole alkaloids from bark of strychnos ssp. and bis-tetrahydroisoquinoline alkaloids from menispermaceae. the genus strychnos is represented in madagascar by 14 species, of which five are endemic to the island. among them, s. diplotrocha leeuwenberg and s. myrtoides gilg & bussse are used as antimalarial in the northeastern part of the country (rasoanaivo et al., 2004) . the phytochemical analysis allowed the separation and the structural determination of several indole alkaloids, some already known and others never reported, including mixtures of epimers, which is very unusual in the same plant (rasoanaivo et al., 1991 (rasoanaivo et al., , 1996 (rasoanaivo et al., , 2001 . the in vitro and in vivo chloroquine-potentiating effect of the crude extract of dried and powdered stem barks of s. myrtoides exerted chloroquine-potentiating effects on p. falciparum fcm29, but it was devoid of intrinsic antimalarial activity. the extract was also devoid of cytotoxic effects on hela and l 929 fibroblast cells. the two compounds exhibit a closely related structure but different basicity. therefore, the latter parameter can be excluded from the factors affecting the chloroquine-potentiating effect. these results were confirmed by other experiments, demonstrating that the crude extract of s. myrtoides showed higher chloroquine-enhancing activity than its major bioactive constituents. these data support the use of the plant as a phytomedicine to treat malaria, but minor components of the extract may act synergistically. among the main isolated alkaloids, malagashanine was very interesting. malagashanine is an unusual indole alkaloid of the strychnos type. its pentacyclic structure contains seven consecutive stereogenic centers and, most important, a transfusion between the c and d rings, against all the other similar natural alkaloids. therefore, malagashanine is the parent compound of a new type of indole alkaloids (fig. 7 .20) (kong et al., 2016) , named n b c(21)-secocuran, isolated so far from the malagasy strychnos species, which are traditionally used as chloroquine adjuvants in the treatment of chronic malaria (rasoanaivo et al., 1996a (rasoanaivo et al., , 2001 . malagashanine showed only weak in vitro intrinsic antiplasmodial activity (ic 50 ¼146.5ae 0.2 μm), but did display marked in vitro chloroquine-potentiating action against the fcm29 chloroquine-resistant strain of plasmodium falciparum. another study allowed clarification of the mechanism of action of the major constituent, malagashanine, being able to prevent chloroquine efflux from the cell, and stimulates chloroquine uptake into drug-resistant p. falciparum strains. malagashanine appears able to act more on plasma membrane than inside the parasite, allowing the toxicity of chloroquine against plasmodium, even at sublethal doses. in the attempt to confirm the reversal of chloroquine resistance by the bark of s. myrtoides, a double-blind randomized controlled clinical trial of a standardized alkaloid extract titrated at 20% malagashanine took place in a government-run outpatient clinic in the town of ankazobe (northwest central highlands of madagascar), but the results of the treatment showed no significant efficacy, indicating a need for other confirmations. however, in conclusion, the approach, in accordance with recent tendencies on multidrug resistance control, based on mixtures of natural products and classic antimalarial drugs, with a relevant coincidence between the ethnobotanical reports and the scientific evidence, may offer interesting possible solutions for the treatment of malaria. many aspects about the mechanism of action of malagashanine as chloroquine adjuvant to reverse the resistance need further study. malagashanine could increase drug accumulation by interacting with a dysregulated ion exchanger, avoiding the decrease inside the food vacuole, or acts by a mechanism related to drug binding to hematin (perisco et al., 2017; rafatro et al., 2000) . in particular, in relation to the ph role in the blood red cell, it would be necessary to determine if malagashanine acts inside or outside the food vacuole, including the membrane periphery. the capacity of malagashanine to reverse cq resistance may be related to the well-known properties of verapamil ( fig. 7 .21) and related substances (martin et al., 1987; martiney fig. 7 .21 verapamil and other compounds studied for cq-resistant reversal by membrane calcium channels blocking. adovelande et al., 1998) . verapamil was the first calcium channel antagonist to be introduced into therapy in the early 1960s. it is a phenylalkylamine calcium channel blocker used in the treatment of high blood pressure, heart arrhythmias, and angina. in short-term incubations, verapamil was found to increase chloroquine accumulation in the lysosome of erythrocytes infected with both chloroquine-sensitive and -resistant organisms, but only to affect the chloroquine susceptibility of the latter. verapamil works independently of the overall ph gradient concentrating cq into a trophozoite's digestive vacuole. the activity is therefore related to the inhibition of membrane ion channels, interfering in the chloroquine transit within the parasite's cytoplasm. other substances like chlorpheniramine and others are reported as candidates for cq-resistant reversers. in any case, again the key role of natural products and ethnoparmacology information, such as for quinine (cinchona sp.) and artemisinin (artemisia annua), is fully confirmed. another attempt to explain the activity of malagasy plants alkaloids explored the role of glutathione. l-glutathione reduced (gsh) (fig. 7.22) is a simple tripeptide, consisting of glutamic acid, cysteine, and glycine. it is considered one of the most powerful endogenous antioxidants, capable of preventing damage to cellular components caused by reactive forms of oxygen, radicals, and heavy metals, although its role in stress management and efficient defense against pathogens are still under study (mangoyi et al., 2010) . besides its antioxidant defense and free radical scavenging, glutathione regenerates important antioxidants such as vitamins c and e. gsh exists in every cell of the human body, but it is also present in many other organisms, including fungi and bacteria. there is a linkage between gsh and malaria. some parasites are superprotected by gsh. they are endowed with powerful and host-independent mechanisms, which de novo synthesize or regenerate gsh and protect the parasites from oxidative damage and other outside attacks. gsh in particular protects the gametocytes against oxidative stress and inhibits the action of arginine, which produces no and expels it from the food vacuole. at the trophozoite stage of p. falciparum in human erythrocytes, gsh takes part in detoxifying processes of heme, produced by hemoglobin digestion, by polymerizing some 30% of heme to insoluble hemozoin. some authors suggest that the nonpolymerized heme, existing in the food vacuole, is subsequently degraded by gsh, increasing the role of this metabolite. chloroquine could interact with gsh, competitively inhibiting the degradation of heme by gsh or allowing toxic heme to accumulate in membranes and damaging parasites. this argument merits some explanation. the prooxidant damage and inflammation process created by excessive heme, hemozoin, and fragments from rupture of the digestive vacuoles in blood vessels and plasma can be mitigated by glutathione. in other words, the inside of the infected erythrocyte glutathione is beneficial to the parasite; outside of the erythrocyte it reduces the negative effects of the malarial infection. high oxidative stress could actually be detrimental for the survival of young parasites (gallo, 2009; patzewitz and m€ uller, 2010) . glutathione transferases (gsts) are versatile enzymes involved in the intracellular detoxification of numerous substances. gsts have been investigated in parasite protozoans, like those involved in malaria, with respect to their biochemistry and as targets in synthesis of new antiparasitic agents. p. falciparum possesses high quantity of these enzymes (pfgst) and their activity was found to be increased in chloroquine-resistant cells, and it has been shown to act as a ligand for parasitotoxic hemin. pfgst represents a promising target for antimalarial drug development. a pfgst isolated from p. falciparum has been associated with chloroquine resistance. plant extracts have been found to act at different vulnerable metabolic sites of pfgst, disturbing gsh-dependent detoxification processes, increasing cytotoxic peroxides levels and possibly increasing the concentrations of toxic hemin in the parasites. in the case of s. myrtoides alkaloids, malagashanine was found to prevent chloroquine efflux from and stimulated chloroquine influx into drug resistant p. falciparum, suggesting that its effects are more on the plasma membrane than inside the parasite. malagashanine (100 μm) reduced the activity of pfgst to 80%, but showed a time-dependent inactivation of pfgst, suggesting a role of malagashanine as a chemomodulator in cases of pfgst overexpression in chloroquine-resistant strains. the malaria cycle of a parasite is based on two cycles, one involving the host and the other affecting the vector. during the mosquito cycle, again there are metamorphoses and reproduction by the parasite. in consideration of the resistance phenomenon, new transmissionblocking agents, able to interrupt malaria transmission, are required. these blocking drug components can be effective in reducing gametocyte density in the human host (gametocytocidal activity) or disrupting parasite development in the vector (sporontocidal activity), resulting in a reduced number of infective vectors and, as a consequence, decreased incidence of malaria cases. in other words, control malaria's parasites through the cure of the vectors infested by the disease. in the sexual stages of plasmodium parasites, gametocytes are critical for the transmission of the parasite to its vectors. p. falciparum gametocytes are also important in the disease diffusion, since being exceptionally long-lived, they cause clinically cured patients to be reservoirs of infection. the cycle of propagation of the malaria parasites starts when the female anopheles feeds on blood from an infected vertebrate. immediately, the first metamorphosis starts. by the ingestion, the mature male and female gametocytes, namely micro-and macrogametocytes, enter the mosquito host. immediately after reaching the mosquito's midgut, the two types of gametocytes undergo dramatic metamorphoses. we must remember that such transformations are a response to environmental stimulation, like a decrease in temperature, an increase in ph, and an influence of xanthurenic acid. within 10-20 min, the rounded macrogametes leave the erythrocytes and diffuse inside the blood, together with the flagellates microgametes. now comes the last change. within the next 24 h, the motile male gametes can fecundate the macrogametes, and round zygotes develop that mature to elongated motile ookinets and move to the outer midgut surface, completing early sporogonic development. these changes can be obtained by severe transformation inside the intrinsic cell organization, involving the cytoskeleton directly. an equatorial position of chromosomes in the metaphase plate in the middle of the spindle is necessary for mitosis and symmetric cell divisions. a symmetric metaphase plate position is essential for symmetric cell divisions, explaining why it is conserved in all metazoans, plants, and many fungi. control of this parameter is essential, since differences in cell size have been linked to cell fate and generate a class of anticancer drugs. movements of chromosomes are in charge of microtubules, which are elements of the cytoskeleton. the cytoskeleton is a network of protein fibers forming the "infrastructure" of eukaryotic and prokaryotic cells. in eukaryotic cells, protein filaments and motor proteins form a complex mesh of protein filaments and motor proteins. the cytoskeleton aids the inside cell movement and transportation of subunits, like organelles and molecule groups, stabilizes and maintains cell shape, and gives support and order. the cytoskeleton is not a static structure but it is able to disassemble and reassemble its parts in order to enable internal and overall cell mobility. intracellular movements include in particular manipulation of chromosomes during mitosis and meiosis from the equatorial plaque to the polar positions, in the formation of daughter cells, and also it is implicated in the immune cell response to pathogens. the cytoskeleton is composed of at least three different types of fibers: microtubules, microfilaments, and intermediate filaments. these fibers are distinguished by their size, with microtubules being the thickest and microfilaments being the thinnest. the assemblement of the proteins, tubulines a and b, makes microtubules, in form of long cave filaments. these hollow rods function primarily to help support and shape the cell and as "routes" along which organelles can move. therefore, without the action of microtubules, the cell is unable to reproduce. the cell is blocked in a limb, with part of the mitosis already done and the final act in progress. the result is a polyploid cell, meaning a cell with double or more than the normal number of chromosomes. because chromosomes cannot move alone, they must be dragged by the cytoskeleton. the mechanisms of action of several important antitumoral drugs derived from natural products are characterized by promotion of the assembly or disassembly of microtubules, meaning stabilization or destabilization of the tubules against depolymerization, resulting in mitotic arrest. treated cells have defects in mitotic spindle assembly, chromosome segregation and movements, and consequently in cell division. the main problem of the utilization of these compounds in combination chemotherapy for sensitive tumor types concerns their selectivity against malignant cells. cancer is basically a disease of uncontrolled cell division, including too-active mitosis, multiplying the cancerous mass. in most cases, these changes in activity are due to mutations in the genes that encode cell cycle regulator proteins. however, although cancer cells are a selected target, in consideration of their high level of mitosis, other tissues can be involved in the action of positive regulators of cell division. molecular agents of plant origin are of primary importance in cancer treatment. those acting on the cytoskeleton can be classified into two main groups: antimicrotubule agents like colchicine and the vinca alkaloids, which induce depolymerization of microtubules, and taxol and taxotere, which induce tubulin polymerization and form extremely stable and nonfunctional microtubules (rowinsky et al., 1990) . neem products have been seriously explored in recent years, in several sectors, mainly in the fight against insect-borne diseases. however, it seems that so far the potentiality of neem has been only lightly touched on. neemazal is a marketed neem product consisting of a quantified alcoholic extract obtained from azadirachta indica seeds, with a reported limonoid concentration of 57.7%, consisting of azadirachtin a 34%, azadirachtins b-k 17.7%, salanins 4%, and nimbins 2% (dembo et al., 2015; habluetzel et al., 2007) . neemazal completely blocks transmission of the rodent malaria parasite p. berghei to anopheles stephensii in vivo, when administered to gametocytemic mice at a corresponding azadirachtin a dose of 50 mg/kg. other in vivo transmission blocking studies suggested that na may have stronger transmission blocking activity than azadirachtin a alone, evaluating the activity of nonazadirachtin a constituents of neemazal. in an ex vivo assay, which exploits a major target process of azadirachtin a against p. berghei, microgamete formation inhibition of plasmodium was used to estimate the pharmacodynamics of two varying doses of neemazal and azadirachtin a. a team led by prof. g. chianese (university of salerno, italy) explored the possibility of influencing plasmodium gametocytes by neem products, demonstrating the potential of blocking the reproduction stages of the parasite. neemazal is a marketed neem product consisting in a quantified alcoholic extract obtained from azadirachta indica seeds, with a reported limonoid concentration of 57.7%, consisting in azadirachtin a 34%, azadirachtins b-k 17.7%, salanins 4%, and nimbins 2%. neemazal completely blocks transmission of the rodent malaria parasite p. berghei to anopheles stephensii in vivo, when administered to gametocytemic mice at a corresponding azadirachtin a dose of 50 mg/kg. other in vivo transmission blocking studies suggested that na may have stronger transmission blocking activity than azadirachtin a alone, evaluating the activity of nonazadirachtin a constituents of neemazal. azadirachtins exert relevant effects on microtubules assembly and organization, interfering with the expression and/or function of adhesive proteins during the genesis of microgametocytes, through disruption of the organization of mitotic spindles and cytoskeleton formation and activity. these molecules can interfere with cytoplasmic microtubule organization and distribution, causing severe depletion of actin levels. in this action, neemazal proved to be more effective than azadirachtin a. in confirmation, another study showed that the product completely inhibits the growth of p. falciparum field isolates in an. coluzzii mosquitoes at a dose of 70 ppm in direct membrane feeding assays. microorganisms have not finished producing surprises and breaking the boundaries reported in books. meanwhile researchers are investigating malaria parasites more and more deeply in search of their weak points, but their study is complicated by the parasite's metamorphosis, which involves not only the shape but also fundamental aspects of the metabolism (becker and kirk, 2004) . asexual stages of the parasite contain a single mitochondrion, whereas gametocytes can have several mitochondria. the energy production is very important. plasmodium falciparum, as well as other similar apocomplexa protozoans, possesses an intriguing nonphotosynthetic plastic, discovered in the 1970s. the surprise was that apicoplastides possess their own nucleic acid. regarding their role, they were considered by kilejian (1991) as "a source of some substrate essential for energy production of mitochondrion." in view of their other characteristics, they could be considered a possible bridge between organisms or the ancestral point of divergence from green algae and protozoans. in conclusion, apicoplastides could be part of the endosymbiosis pathway, wherein degenerated chloroplasts were useful to increase a mitochondrial efficiency still in evolution. thus, endosymbiosis started with the inclusion of the two main bacterial forms, the hetero-and the autotrophic one, but later the ancestral (green or red) primordial alga degenerated the chloroplast in favor of a clear evolution toward the heterotrophic metabolism ( fig. 7.23) . usually the shift to the eukaryotic cell is considered a consequence of environmental factors, such as the increase of oxygen in the oxidative atmosphere; however, it is possible that in some cases interactions between organisms could also have played an important role. the study on apicoplastides allowed researchers to evidence similarities (keeling, 2008; kilejian, 1975; k€ ohler et al., 1997) between different arthropod-borne diseases, such as avian malaria, eimoriosis, and toxoplasmosis, confirming once more the occurrence of common survival strategies in different organisms. other differences concern the enzymes network and the membrane transport mechanisms. the new knowledge about parasitespecific organelles could be of fundamental importance to the development of future antimalarial drugs, increasing efficiency and decreasing side effects, like resistance. another important research front full of possibilities is focused on the membrane mechanism of cq's extrusion by permeability pathways induced by the parasite in the host red blood cells (saliba et al., 1998) . this is related to the cq of interfering with the detoxification of toxic heme monomers. the studies showed that 12-16 h after the invasion by the parasite, the socalled new permeability pathways act on the interchanges, i.e., the entry of nutrients, as well as mediating the efflux of metabolic wastes. several groups advanced the hypothesis of a number of channel types, activated by particular stress or stimuli (ginsburg and stein, 2004; kirk et al., 1994; duranton et al., 2004; staines et al., 2004; thomas and lew, 2004) . all these references testify to and confirm the presence of a wide range of studies in search of an answer to the challenge of resistance. the front is still too large and undetermined, but every year the knowledge of host cell reaction is increasing and there is a high probability that the problem will be solved in the coming years. during the development of the arguments contained in this book, it was necessary link the insect-borne diseases argument to several collateral items. the idea in particular was concentrated on a possible utilization of this particular topic as an epiphany, meaning an enlightening subject, which allows a revision of the problem from a new perspective. the interpretation of a new and key piece of information can allow the process of significant thought about a problem, until, in accordance with the original significance of the term in ancient greek, the ἐπιφάνεια (epiphanea) appears like a manifestation, with a striking appearance. this book started with considerations about gaps and books. let us now return to these two points. the lesson from carson's book about the fundamental role of beneficial insects in the survival of mankind, has arguably not been understood. throughout all warm terrestrial ecosystems, insects are a dominant component and they are part of the lives of any organism. the insect-plant relationship is a fundamental biotic interaction, and plants account for a large part of the planet's biomass, many times the biomass of all animals together (new, 2002; jankielson, 2018; dunn, 2005) . the animal biodiversity is dominated by that of insects. they are a beautiful example of variability, in terms of both number of species (more than 1 million) and abundance (more than half of all living organisms), although at most only about 7%-10% of insects are scientifically described. this diversity, consisting of large numbers of individuals and great intra-and interspecific variety, is a consequence of the enormous functional significance of insects in habitats. primitive insects appeared very early in the silurian period, when plants and animals finally emerged from the sea and colonized dry land, and over the last 400 million years the number of insect families has been rising. they were able to colonize any part of the territory, including the sky. today, the number of reported insect families is about 600 and they have survived various negative major impacts, including the mass extinction event at the end of the cretaceous period. a review analysis, published in the journal biological conservation by francisco sánchez-bayo, at the university of sydney, australia, and kris wyckhuys, at the china academy of agricultural sciences, beijing china, attests a current insect collapse. the decline's hypothesis is based on a study of 73 recent selected studies. the causes and significant factors include intensive agriculture, the heavy use of pesticides, urbanization, and climate change. the loss of insect population is calculated in an annual 2.5% rate over the last 20-25 years, and the future tendency is evaluated to 25% in the next 10 years and increasing continuously until only half left in 100 years. this scenario is already underway. in puerto rico, a recent study revealed a 98% fall in ground insects over 35 years. the catastrophic cascade effects on the planet's ecosystems include ants, aphids, shield bugs, and crickets, which are the food for many birds, reptiles, amphibians, and fish that eat insects. there are many indicators supporting the scenario (sánchez-bayo and wyckhuys, 2019; diamond, 1989) . in england, between 2000 and 2009, the number of widespread butterfly species fell by 58% on farmed land, suffering the biggest recorded insect falls overall-though that is probably a result of this area being more intensely studied than most places. a particular alert concerning bees being seriously affected has also been raised in europe and the usa; for example, only half of the bumblebee species found in oklahoma in the usa in 1949 were present in 2013 (alburaki et al., 2015 (alburaki et al., , 2018 aizen, 2009 ). the number of honeybee colonies in the usa was 6 million in 1947, but 3.5 million have since been lost. in 2013, according to eu data, there were around 630,000 beekeepers and 16 million hives in the eu, producing 234,000 tons of honey per year, but the same source tells us that many insect pollinator populations are now in clear decline. there is similar news from brazil, with half a million bees dead. on one side, this is considered the effect of the use of some pesticides, toxic to bees. on the other side, it is a classic example of rapid and intense environmental change to improve agricultural intensification and pasture, with the systematic elimination of all trees and shrubs that normally surround the fields, so there are plain, bare fields that are treated with synthetic fertilizers and pesticides. dr. sanchez-bayo said: "we are not alarmists, we are realists. we are experiencing the sixth mass extinction on earth. if we destroy the basis of the ecosystem, which are the insects, then we destroy all the other animals that rely on them for a food source." he added, "it will collapse altogether and that's why we think it's not dramatic, it's a reality." the situation comprehends micro-and macroepisodes, like the continuous devastation of equatorial tropical forests, in particular the amazonia territory. the sequence is clear and well-known, and it always works: first, the fire destroys the vegetation, in particular the woody plants; second, the soil is cleaned, otherwise the plants could replace the habitat rapidly; and third, the territory is declared totally compromised and ready for further utilizations. however, as observed by samways in biodiversity and conservation (1993, and later confirmed by this author in a series of further papers) in a paper titled "insects in biodiversity conservation: some perspectives and directives," the main concerns are the "lack of human appreciation of importance, coupled with the general disregard and dislike of insects, is an enormous perception impediment to their conservation. this impediment coupled with the taxonomic impediment must be overcome for realistic biodiversity conservation management. as it is not possible to know all the species relative to the rate at which they are becoming extinct, it is essential to conserve as many biotopes and landscapes as possible." there is a sentiment of urgency for measures "essential to preserve species dynamo areas as an insurance for future biodiversity," such that "preserved areas must also be linked by movement and gene-flow corridors as much as possible." the last point of view is crucial. preservation must be considered not only as an opportunity to maintain the presence of species in selected habitats against their disappearance, but it must be considered changes as opportunities to perform a positive future. in this regard, entomologists are asked to contribute in control of vectors affecting humans, crops, and livestock, but also to take an active part in the consideration due to the beneficial species. the central task is the possibility to predict accurately the environmental effects of any intervention. once the inherent risks connected with traditional control methods have been considered, the consequences of new introductions must be carefully predicted, including any synergist effect. the rate of insect species extinction is estimated as being eight times faster than those of mammals, birds, and reptiles (barnosky et al., 2011; dirzo et al., 2014) . another important current gap concerns scientific information. most ordinary people do not have access to data obtained by the scientific community, as well as opinions and models produced by experts and scientists. information, when available, is usually distorted and adapted to the dominant axioms by a plethora of generalist supposed experts. the proposed idea is that these kinds of people are able to know and comment on everything. the distortion, sometimes voluntarily pursued and often a consequence of general confusion, generates progressive modification of the starting points and even the concealment of important facts. the recent phenomenon of fake news is clearly generated from the same situation. although most research information is now easily accessible and can be obtained directly from the internet, its utilization remains restricted to dedicated people. in contrast, some scientific information is amplified far away from its real impact. how many times did you read about the discovery of a definitive cure to cancer? or about the already obtained solution to any physiological problem using staminal cells? in our era of globalized knowledge, news are obtained and fluxed indirectly, without few possibility of checking the origin and the reliability. it is necessary to consider that more than 46% of the human population, consisting of 3.5 billion people, are connected via the internet, and 2.5 billion utilize social networks regularly. these numbers are likely to increase 10% every year. all these people have access to information only through selected channels and although they are in a condition to verify it, science and general information are on different and distant levels. the main problem is that the information is reduced to a few soundbites, and there is no place for elaboration or proposals of other possible interpretations or points of view. this is not a recent case, produced by digitalization of communication. beside the sources, the problem of the quality of scientific information was fully evidenced more than 30 years ago, in the "public understanding of science." this is the title of a report requested in 1985 by the royal society and prepared by a group of experts, whose leader was the geneticist sir walter fred bodmer. the report evidenced the general lack of knowledge about scientific themes. on one side, most of the population, accounting for two-thirds of europeans, was confident about science and technologies, considering that scientists were able to solve human problems and make human life "easier, healthier and comfortable." on the other side, the sequence "more communication ¼more knowledge¼more social adherence to scientific arguments" appears largely inadequate. the dominant problem about scientific communication is that ordinary people need an alphabetization to understand and meet the complexity of the scientific items. the conversion of the original scientific information is usually distorted and changed, at best "adapted," but more often polluted by political, social, and cultural interests. the result is a reductive metamorphosis, in the best case, or complete revision to be adapted and useful to already-made opinions. among the various examples of this operation we find neverending debates, such as those concerning ogm, vaccines, or the consequences of climate changes, without considering abnormal and artificially created themes, such as the contraposition between vegans and meat-eaters. the manipulation is based on a presumed "democratic" interpretation of scientific data. no vote is necessary to assure the consistency of a scientific law based on adequate experimentation, but the aim is that reliability must be obtained by public consensus and even agreement. independence has always been a necessary character of science, but manipulation was never pursued. history tells us that any political or social manipulation of science led to disaster. in contrast, priorities, when based on correct scientific information, as well as consequent implications and decisions, must be subject to the most ample democracy. at the end of this little journey through macro-, micro-, and nanoworlds, it is undeniable how long the road still is to understand and discover the mysteries of insect-borne diseases. in the meantime, we await the next surprises. the covid-19 pandemy dramatically evidenced all the current limits of science and technology to face this kind of challenges. the virus was faster and clever. predictively and prevention were insufficient. despite the potentiality, the debacle and medicine was evident and the consequent economic and social damages were enormous. microorganisms will continue to play their role inside the habitats and next time their target could be the industrialized sources of our food. however, it is clear that is society will continue to ignore the alerts of researchers and scientists, the next pandemy will be the worst one. synergy between two calcium channel blockers, verapamil and fantofarone (sr33557), in reversing chloroquine resistance in plasmodium falciparum active and intelligent packaging: an introduction how much does agriculture depend on pollinators? lessons from long-term trends in crop production neonicotinoid-coated zea mays seeds indirectly affect honeybee performance and pathogen susceptibility in field trials honey bee survival and pathogen prevalence: from the perspective of landscape and exposure to pesticides green-synthesised nanoparticles from melia azedarach seeds and the cyclopoid crustacean cyclops vernalis: an eco-friendly route to control the malaria vector anopheles stephensi? operational feasibility of malaria control by burning neem oil in kerosene lamp in beel akbarpur village, district ghaziabad, india review of antimicrobial food packaging evaluation of larvicidal activity of medicinal plant extracts against three mosquito vectors has the earth's sixth mass extinction already arrived? antibacterial activity of karanj (pongamia pinnata) and neem (azadirachta indica) seed oil: a preliminary report of malaria, metabolism and membrane transport old ingredients for a new recipe? neem cake, a low-cost botanical by-product in the fight against mosquito-borne diseases larvicidal and ovideterrent properties of neem oil and fractions against the filariasis vector aedes albopictus (diptera: culicidae): a bioactivity survey across production sites shedding light on bioactivity of botanical by-products: neem cake compounds deter oviposition of the arbovirus vector aedes albopictus (diptera: culicidae) in the field neem (azadirachta indica): towards the ideal insecticide? synergized mixtures of apiaceae essential oils and related plantborne compounds: larvicidal effectiveness on the filariasis vector culex quinquefasciatus say chemical composition and insecticidal activity of the essential oil from helichrysum faradifani endemic to madagascar ethnopharmacology in the fight against plasmodium parasites and brain disorders: in memoriam of philippe rasoanaivo insecticidal and mosquito repellent efficacy of the essential oils from stem bark and wood of hazomalania voyronii herbal remedies of azadirachta indica and its medicinal application biological activities and medicinal properties of neem (azadirachta indica) le paludisme à madagascar a guide to the identification of diseases and pests of neem (azadirachta indica). rap publ neem-an omnipotent plant: a retrospection responsive food packaging: recent progress and technological prospects insecticide resistance in mosquitoes: a pragmatic review natural products as sources for new pesticides emergency and mosquitocidal potential of neem cake-synthesized silver nanoparticles: genotoxicity and impact on predation efficiency of mosquito natural enemies the efficacy of neem extract on four microorganisms responsible for causing dental caries viz streptococcus mutans, streptococcus salivarius, streptococcus mitis and streptococcus sanguis: an in vitro study biological activities and medicinal properties of neem (azadirachta indica) a novel phytocannabinoid isolated from cannabis sativa l. with an in vivo cannabimimetic activity higher than δ 9 -tetrahydrocannabinol: δ 9 bioactive packaging technologies for extended shelf life of meat-based products effectiveness of antimicrobial food packaging materials in vitro and ex vivo activity of an azadirachta indica a. juss seed kernel extract on early sporogonic development of plasmidium in comparison with azadirachtin a, its most abundant constituent active and intelligent packaging: applications and regulatory aspects assessment of neem oil effect on haematological profile and towards peripheral blood mononuclear cells of goat antimicrobial activity of a neem cake extract in a broth model meat system evaluation of a mono-component and a multicomponent herbal extracts as candidates for antimicrobial packaging of fresh retail meat assessment of microbiological quality of retail fresh pork meat in central italy neem (azadirachta indica a. juss) oil to tackle enteropathogenic escherichia coli impact of repeated neemazal ® treated blood meals on the fitness of anopheles stephensi mosquitoes the present, past and future of human-caused extinctions defaunation in the anthropocene active and intelligent packaging food-research and development-a review modern insect extinctions, the neglected majority organic osmolyte permeabilities of the malaria-induced anion conductances in human erythrocytes successful parasitoid control of aonidiella orientalis (newstead) (hemiptera: diaspididae) on carica papaya l biopesticide registration action document. office of pesticide programs. cold pressed neem oil. pc code 025006. margosa extract pt-18 secondary metabolism as a measurement of the efficacy of botanical extracts: the use of azadirachta indica (neem) as a model insecticide status report on global neem usage indole alkaloids from strychnos species and their antiplasmodial and cytotoxic activities inherited glutathione reductase deficiency and plasmodium falciparum malaria-a case study plant extracts as potential mosquito larvicides phytochemicals for bacterial resistance-strengths, weaknesses and opportunities the new permeability pathways induced by the malaria parasite in the membrane of the infected erythrocyte: comparison of results using different experimental techniques neem-a green treasure chemical and biological investigations on azadirachta indica (the neem tree) plants myths and traditions in india impact of the botanical insecticide neem azal ® on survival and reproduction of the biting louse damalinia limbata on angora goats food losses and waste in the context of sustainable food systems neem pesticides the importance of insect in agricultural ecosystems focus on phytochemical pesticides. the neem tree increasing food availability by reducing postharvest losses of fresh produce chemische untersuchungen € uber die metamorphosehormone der insekten neemdb: convenient database for neem secondary metabolites bridge over troublesome plastids past, current and potential utilization of active and intelligent packaging systems for meat and muscle-based products: a review circular mitochondrial dna from the avian malarial parasite plasmodium lophurae spherical bodies transport of diverse substrates into malariainfected erythrocytes via a pathway showing functional characteristics of a chloride channel a plastid of probable green algal origin in apicomplexan parasites malagashanine: a chloroquine potentiating indole alkaloid with unusual stereochemistry neem: today and in the new millennium neem (azadirachta indica): prehistory to contemporary medicinal uses to humankind neem in the conventional lake chad basin area and the threat of oriental yellow scale insect (aonidiella orientalis newstead) (homoptera: diaspididae) current topics in active and intelligent food packaging for preservation of fresh foods not ordinary antimalarial drugs: madagascar plant decoctions potentiating the chloroquine action against plasmodium parasites glutathione transferase from plasmodium falciparum-interaction with malagashanine and selected plant natural products antimicrobial food packaging: potential and pitfalls citrus limonoids: analysis, bioactivity, and biomedical prospects antilarval activity of neem cake extracts against aedes albopictus composizione biologica con proprietà fortemente biocide a basso contenuto di azadiractina e procedimento per la sua realizzazione antimicrobial activity of melia azadirachta fruit extracts for control of bacteria in inoculated in vitro shoots of 'mrs-2/5' plum hybrid and calla lily and extract influence on the shoot cultures reversal of chloroquine resistance in plasmodium falciparum by verapamil verapamil reversal of chloroquine resistance in the malaria parasite plasmodium falciparum is specific for resistant parasites and independent of the weak base effect product safety and security in the global supply chain: issues, challenges and research opportunities effect of neem leaf extract and on penicillum growth, sporulation, morphology and ochratoxin a production mosquitocidal and antiplasmodial activity of senna occidentalis (cassiae) and ocimum basilicum (lamiaceae) from maruthamalai hills against anopheles stephensi and plasmodium falciparum in vivo and in vitro effectiveness of azadirachta indica-synthesized silver nanocrystals against plasmodium berghei and plasmodium falciparum, and their potential against malaria mosquitoes report of an ad hoc panel of the board on science and technology for international development natural products as sources of new drugs from 1981 to hptlc fingerprint: a modern approach for the analytical determination of botanicals traceability in multi-ingredient botanicals by hptlc fingerprint approach advances in production of functional foods and nutraceuticals advanced in production of functional foods and nutraceuticals intelligent and smart packaging hptlc fingerprint analysis of plant staminal products analysis of multi-ingredient food supplements by fingerprint hptlc approach toxic effects of neem cake extracts on aedes albopictus (skuse) larvae neem cake: chemical composition and larvicidal activity on asian tiger mosquito the modern analytical determination of botanicals and similar novel natural products by the hptlc fingerprint approach current mosquito-borne disease emergencies in italy and climate changes. the neem opportunity neem-borne molecules as eco-friendly control tools against mosquito vectors of economic importance professor philippe rasoanaivo neem tree-"the village pharmacy the control of the oriental red scale, aonidiella orientalis newstead and the california red scale, a. aurantii (maskell) (homoptera: diaspididae) in mango orchards in hevel habsor (israel) trends in antimicrobial food packaging systems: emitting sachets and absorbent pads active food packaging technologies use of natural antimicrobials to increase antibiotic susceptibility of drug resistant bacteria glutathione biosynthesis and metabolism in plasmodium falciparum traditional herbal remedies and dietary spices from cameroon as novel sources of larvicides against filariasis mosquitoes? chemical composition of cinnamosma madagascariensis (cannelaceae) essential oil and its larvicidal potential against the filariasis vector culex quinquefasciatus say the interaction of heme with plakotin and a synthetic endoperoxide analogue: new insights into the heme-activated antimalarial mechamism neem: the divine tree azadirachta indica reversal activity of the naturally occurring chemosensitizer malagashanine in plasmodium malaria tetranortriterpenoids from azadirachta indica effect of the leaf essential oil from cinnamosma madagascariensis danguy on pentylenetetrazol-induced seizure in rats biological activities of the plant-derived bisindole voacamine with reference to malaria malagashanine potentiates chloroquine antimalarial activity in drug resistant plasmodium malaria by modifying both its efflux and influx malagashanine and malagashine, the alkaloids of strychnos mostuoides medicinal plants used to treat malaria in madagascar revised structure of malagashanine: a new nb,c(21)-secocuran alkaloid reversing agents in treatment of drug-resistance malaria the co-occurrence of c(3) epimer nb,c(21)-secocuran alkaloids in strychnos diplotricha and strychnos myrtoides screening extracts of madagascan plants in search of antiplasmodial compounds taxol: a novel investigational antimicrotubule agent limonoids: overview of significant bioactive triterpenes distributed in plants kingdom neem, a tree for solving global problems anti-microbial activity of a new vaginal contraceptive nim-76 from neem oil (azadirachta indica) transport and metabolism of the essential vitamin pantothenic acid in human erythrocytes infected with the malaria parasite plasmodium falciparum insects in biodiversity conservation: some perspectives and directives worldwide decline of the entomofauna: a review of its drivers fatty acid composition and antibacterial activity of neem (azadirachta indica) seed oil sustainable industrial utilization of neem tree (azadirachta indica) in nigeria some arthropod pests and a semi-parasitic plant attacking neem (azadirachta indica) in kenya the neem tree: azadirachta indica a. juss. and other meliaceous plants, sources of unique natural products for integrated pest management, medicine, industry and other purposes the neem tree: source of unique natural products for integrated pest management, medicine, industry and other purposes the neem tree (azadirachta indica a. juss) and other meliaceous plants: sources of unique natural products for integrated pest management a review of botanical phytochemicals with mosquitocidal potential mosquito repellent action of neem (azadirachta indica) oil quality of packaged foods plasmodium falciparum-induced channels ethnobotanical uses of neem (azadirachta indica a. juss.; meliaceae) leaves in bali (indonesia) and the indian subcontinent in relation with historical background and phytochemical properties the role of botanicals as green pesticides in integrate mosquito management-review monograph on neem (azadirachta indica a. juss.). international book distributors biosensors in food processing plasmodium falciparum and the permeation pathway of the host red blood cell susceptibility of immature stages of aedes aegypti, the vector of dengue and chikungunya to insecticides from india neem (azadirachta indica) and its potential for safeguarding animals and humans the hptlc approach to metabolomic determination of neem products composition determination by hptlc of chemical composition variability in raw material used in botanicals costa concordia disaster: environmental impact from phytochemical point of view trasmission blocking effects of neem (azadiracha indica) seed kernel limonoids on plasmodium berghei sporogonic development ecophysiological and phytochemical response to ozone of wine grape cultivars of vitis vinifera ethnopharmacognostical survey of azadirachta indica a juss quassinoids: structural diversity, biological activity and synthetic studies public health impact of pesticides used in agriculture: reportage of a world health organization and u.n. environmental programme malaria treatment guidelines. world health organization key: cord-277802-f8pyn3rx authors: roman, gheorghe title: mannich bases in medicinal chemistry and drug design date: 2015-01-07 journal: eur j med chem doi: 10.1016/j.ejmech.2014.10.076 sha: doc_id: 277802 cord_uid: f8pyn3rx the biological activity of mannich bases, a structurally heterogeneous class of chemical compounds that are generated from various substrates through the introduction of an aminomethyl function by means of the mannich reaction, is surveyed, with emphasis on the relationship between structure and biological activity. the review covers extensively the literature reports that have disclosed mannich bases as anticancer and cytotoxic agents, or compounds with potential antibacterial and antifungal activity in the last decade. the most relevant studies on the activity of mannich bases as antimycobacterial agents, antimalarials, or antiviral candidates have been included as well. the review contains also a thorough coverage of anticonvulsant, anti-inflammatory, analgesic and antioxidant activities of mannich bases. in addition, several minor biological activities of mannich bases, such as their ability to regulate blood pressure or inhibit platelet aggregation, their antiparasitic and anti-ulcer effects, as well as their use as agents for the treatment of mental disorders have been presented. the review gives in the end a brief overview of the potential of mannich bases as inhibitors of various enzymes or ligands for several receptors. the classical mannich reaction, a three-component condensation between structurally diverse substrates (xeh) containing at least one active hydrogen atom, an aldehyde component (generally r 1 -cho) and an amine reagent leads to a class of compounds generally known as mannich bases 1 (scheme 1). because mannich bases may be regarded as derivatives of the substrate obtained through substitution by an aminoalkyl moiety, mannich reactions are also known as aminoalkylation reactions. in the particular instance when formaldehyde is employed as aldehyde component, the substrate is converted into the corresponding mannich base through an aminomethylation process. although primary amines and even ammonia (in the form of an ammonium salt) may be employed as amine reagents in aminomethylations or aminoalkylations, secondary aliphatic amines (r 2 nh) are the most commonly encountered as amine reagents in the mannich reaction. as formaldehyde is used to a great extent as aldehyde component in the mannich reaction, the structural diversity of mannich bases stems primarily from the miscellaneous types of the substrates that can be subjected to aminomethylation, and secondarily from the variety of amine reagents that can be potentially employed in the mannich reaction. regardless of their structural diversity, the substrates should all have an activating functional group as a crucial structural feature that is required to render the substrate active in the mannich reaction. the carbonyl function in ketones, the phenolic hydroxyl in phenols, the terminal carbonecarbon triple bond in alkynes, the heteroatom in heterocycles, or electronwithdrawing groups that substitute the carbon atom a to the carboxylate group in esters of aliphatic carboxylic acids are common examples of pairs of activating groups and corresponding substrates, but the list is far from being exhaustive. a general classification of the most common types of mannich bases with respect of the substrates from which they derive and the nature of the atom substituted by the aminomethyl function is given in fig. 1 . under normal reaction conditions, substitution of a substrate with a single aminomethyl function results in mono-mannich bases, but two aminomethyl groups may also be grafted onto a substrate containing more than one active hydrogen atom, leading to double mannich bases such as 2e5 derived from dialkyl ketones, alkyl aryl ketones, 4-substituted phenols and pyrrole, respectively (fig. 2) . also, the aminomethylation of substrate xeh with amine reagents other than secondary amines (such as ammonia, having three reactive hydrogen atoms at nitrogen, or primary amines renh 2 , having two reactive hydrogen atoms at nitrogen) may lead to tris-mannich bases 6 and bis-mannich bases 7, respectively (fig. 2 ). in addition, the capability of some polyfunctional substrates to aminomethylate chemoselectively at a single potential reaction site under the appropriate reaction condition, or aminomethylate indiscriminately at multiple reaction sites, or even undergo aminomethylation simultaneously with ring closure, contributes considerably to the structural variety of the resulting mannich bases. two excellent, albeit rather old reviews provide more details on the synthesis and reactions of mannich bases to the interested reader [1, 2] . mannich bases have found numerous practical applications in the treatment of natural macromolecular materials such as leather, paper and textiles, the production of synthetic polymers, as additives used by the petroleum industry, as products used in water treatment, analytical reagents, cosmetics, dyes, etc. [3] . nonetheless, the most important application of the mannich reaction lies in the field of medicinal chemistry, and this claim is supported by the substantial number of papers published on this topic every year. first of all, mannich bases could present interesting biological activities, many of these having yet to be discovered through a diligent screening process. second, aminomethylation of drugs could be used to improve their delivery into the human body. aminomethylation may increase the hydrophilic properties of drugs through the introduction of a polar function in their structure, the long-known rolicycline being one of the most common examples [4] . the solubility in water of a drug could be further enhanced through the quaternization of the nitrogen atom in its aminomethyl derivative and conversion into an ammonium salt. alternatively, the lipophilic properties of a drug could be tailored through a mannich reaction if the appropriate amine reagent is employed [5] . in addition, the aminomethylated drugs could act as prodrugs, releasing the active substance under controlled hydrolytic conditions via deaminomethylation [6] or deamination [7] . in spite of the tremendous potential of mannich bases in medicinal chemistry, the wealth of information from studies concerning the structureeactivity relationship (sar) involving mannich bases or the use of aminomethylated drugs as prodrugs does not appear to have inspired many recent literature reviews of consequence, to the best of our knowledge. the present review fills this void by providing a comprehensive coverage of the most relevant developments in the medicinal chemistry of mannich bases generated exclusively through aminomethylation, the information being ordered according to the reported biological activity. due the large number of articles published on this topic, the coverage of this review is limited to the last decade. anticancer properties and cytotoxicity of ketonic mannich bases (with an emphasis on mannich bases of type 8 derived from acetophenones [8] ) and of structurally related a,b-unsaturated ketones [9] were reviewed 15 years ago. these two groups of compounds were shown to exert their cytotoxic action through the alkylation of cellular thiols such as glutathione or cysteine, and may be useful in sensitizing tumor cells to antineoplastic agents, and even reverse drug resistance [10] . it is therefore no surprise that compounds having both a ketonic mannich base moiety and an activated unsaturated carbonecarbon double bond in their structure (for example, mannich bases of chalcones such as 9) have been considered as candidates for the evaluation of the sequential cytotoxicity theory [11] . this theory hypothesizes that the successive release of two or more cytotoxic agents will result in increased toxicity to malignant tissue rather than to normal cells [12] . in addition, mannich bases 10 of enones, which are easily accessible from alkyl aryl ketones in one synthetic step, also demonstrated marked toxicity towards numerous cancer cell lines [13] . furthermore, as ortho-phenolic mannich bases undergo deamination easily to yield ortho-quinone methides, mannich bases of chalcones derived from either phenolic aldehydes or ketones, a class of compounds for which structure 11 is prototypical, have been examined also as cytotoxic agents [14] . in the last decade, the quest for more potent anticancer agents amongst these four general types of cytotoxic mannich bases 8e11 (fig. 3 ) has steadily continued. cytotoxicity of ketonic mannich bases of type 8 with various substitution patterns in the aromatic ring and of a few types of their derivatives has been studied in detail. gul et al. have shown that the structural modification of single mannich bases 8 (r 1 ¼ r 2 ¼ h) derived from acetophenone and secondary aliphatic amines into double mannich bases 12 (fig. 4) generally results in increased cytotoxicity against mouse renal carcinoma (renca) and transformed human t-lymphocyte (jurkat) cell lines [15] to the extent that double mannich bases were more cytotoxic than reference drugs 5-fluorouracil or melphalan. the cytotoxicity of these single and double mannich bases 8 and 12, respectively, was reversed when the compounds were used in a brine shrimp bioassay, presumably due to the fast deamination of the double mannich bases before they could reach their target [16] . also, ketonic mannich bases 8 with dimethylamino, 1-piperidinyl, 4-morpholinyl as amine moiety and featuring either an unsubstituted, variously monosubstituted phenyl rings, or a thiophene ring were evaluated with respect of their cytotoxicity towards jurkat cells [17, 18] or androgen-independent prostate cancer (pc-3) cells [19] , and the cytotoxicity for some of these compounds was 2.5-to 5.2-fold higher than that of the standard 5-fluorouracil. in addition, mannich bases of type 8 derived from 4-aryloxyacetophenones were shown to display moderate cytotoxic properties towards murine l1210 cells as well as human molt 4/c8 and cem t-lymphocytes, and a number of these compounds possessed remarkable potencies towards seven human colon cancer cell lines [20] . the use of primary aliphatic amines in the mannich reaction leads to reaction products with diverse structures (fig. 4) . aminomethylation of acetophenones using methylamine as amine reagent afforded both bis-mannich bases 13 (r ¼ ch 3 ) and piperidinols 14 (r ¼ ch 3 ) , and the evaluation of their cytotoxicity towards jurkat cells showed that bis-mannich bases 13 were generally more potent than the corresponding piperidinols 14 or mono-mannich bases 8 [21] . besides their ability to alkylate cellular glutathione, compounds 13 (r ¼ ch 3 ) may exert their cytotoxic action through the inhibition of dna topoisomerase i; as the corresponding piperidinols 14 were generally devoid of dna topoisomerase i inhibitory action, the authors tentatively attribute the activity of bis-mannich bases 13 to their linear structure and the possibility of formation of hydrogen bonds with dna nucleotides [22] . on the other hand, aminomethylation of acetophenones using isopropylamine [23] and n-butylamine [24] as amine reagent yielded only secondary mono-mannich bases 15 (r ¼ ch(ch 3 ) 2 , (ch 2 ) 3 ch 3 ), whose cytotoxicity was evaluated against huh-7 hepatoma cells, human jurkat and rat skeletal muscle derived myoblasts (l6) cells. compared to reference drug 5-fluorouracil, these compounds were 2.1-to 2.8-fold more cytotoxic towards huh-7 hepatoma cells, 2.6-to 4.2-fold more cytotoxic towards human jurkat cells, and 1.2-to 2.2-fold more cytotoxic towards l6 cells. aminomethylation of acetophenones using phenethylamine as amine reagent could led under carefully controlled reaction conditions either to mono-mannich bases 15 (r ¼ ch 2 ch 2 c 6 h 5 ) [25] or to the corresponding piperidinols 14 (r ¼ ch 2 ch 2 c 6 h 5 ) [26] ; the cytotoxicity of these compounds towards androgen-independent prostate cancer (pc-3) cells ranged from 8.2 to 32.1 mm, whereas the best compounds from each series had an average value for dna topoisomerase i interference of approximately 40%. anticancer activity of ketonic mannich bases has been compared with that of derivatives of the carbonyl function (fig. 4) . a series of azines 16 of ketonic mannich bases 8 were designed as bifunctional cytotoxic agents, but their activity towards jurkat cells [19] or pc-3 cells [17] was less potent or, in the best of cases, equipotent to the parent ketonic mannich bases. on the other hand, hydrazones 17 were consistently more cytotoxic than the corresponding ketonic mannich bases [18] . noteworthy is the contribution of gul et al. to the understanding of the mechanism of the cytotoxic action of these compounds. his group has provided evidence that connects the anticancer activity of ketonic mannich bases of various structures or piperidinols 14 with their ability to alkylate glutathione [21,27e29] , whereas a few of the same compounds had mixed effects on thioredoxin, glutaredoxin, or heat shock proteins hsc70 and grp75 [30] . only a limited number of examples of mannich bases of a,bunsaturated ketones of type 9 with cytotoxic action are available in recent publications. a small series of mannich bases 18 of 1arylidene-2-tetralones ( fig. 5) were evaluated as cytotoxic agents using human molt 4/c8 and cem t-lymphocytes, as well as murine p388 and l1210 leukemic cells [31] . compared to the parent a,bunsaturated ketones, mannich bases 18 were consistently more potent, with half maximal inhibitory concentration (ic 50 ) values in the 0.2e10 mm range. furthermore, mannich bases 18 derived from aromatic aldehydes substituted with chlorine, carboxyl, methoxy or cinnamoyloxy groups exhibited significant potencies towards human tumor cell lines, with an emphasis on their antileukemic effect. in most instances, the compounds prepared in this study demonstrated selective toxicity to different cells, which further enhances their potential utility. in addition to compounds 18, simpler mannich bases 19 derived from 2benzylidenecyclohexanones were synthesized, and their evaluation against the same cell lines proved once more that mannich bases were more cytotoxic than the corresponding 2arylidenecyclohexanones, some of them showing growthinhibiting properties (ic 50 of approximately 2 mm) more potent than reference drug melphalan [32] . because n-myristoyltransferase is expressed in larger quantities in tumors than it is in normal cells, this enzyme has been under consideration as a molecular target for cancer [33, 34] . however, the substantially high ic 50 value of 500 mm towards n-myristoyltransferase for a representative compound of the series of candidates 19 suggests that the inhibition of this enzyme does not play an important role in the mechanism of the cytotoxic activity of these compounds. several compounds 19 were also tested against murine cancer cells mac13 (sensitive to most cytotoxic agents) and mac16 (resistant to most cytotoxic agents), and they demonstrated high cytotoxicity against the latter, but also against normal murine cells c2c12 and 3t3 [35] . on the other hand, mannich bases 20 showed no activity against mac16, which points to the importance of the double bond conjugated to the carbonyl function. the exploration of possible mechanisms of cytotoxic action of these compounds revealed that compounds 19 may interfere with a number of essential cellular mechanisms by alkylation of thiols on enzyme or proteins, by disrupting mitochondrial electron transport, or by creating holes in the cell membrane, and thus promoting atp leakage [35] . finally, mannich bases 21 had high cytotoxic activity against two human breast cancer cell lines (mcf-7 and mcf-7/adr) cells and human leukemia hl-60 cells, showed glutathione binding ability, and exhibited inhibitory action on glutathione-s-transferase p, whereas their analogues obtained through the hydrogenation of the double carbonecarbon bond were slightly less active [36] . the nature of the dialkylamino group did not seem to affect the cytotoxic activity of these compounds, while the substitution of the aromatic rings with a methyl selectively increased the cytotoxic effect on breast cancer cells, but not on immortalized mammary epithelial (184b5) cells. the literature reporting the anticancer activity of mannich bases of type 10 is even scarcer. given the significant antineoplastic properties of compounds 10 [13] , a novel series having substituents r 1 in the phenyl ring that were carefully selected with a view to impart a variety of physicochemical properties has been designed and synthesized [37] . since a gradual release of mannich base 10 from niosomes had improved its bioactivity in vivo, amino alcohols 22 (fig. 5) , which may slowly undergo dehydration to yield the desired mannich base 10, were also examined as cytotoxic agents. finally, in order to explore the hypothesis that cytotoxicity would be retained even when a thiol is liberated, the synthesis of adducts 23 was carried out. compounds 10, 22 and 23 ( fig. 5 ) were evaluated against both human widr colon cancer cells and human crl-2522 foreskin fibroblasts. ic 50 values lesser than 10 mm were obtained when compounds 10 were evaluated towards human widr colon cancer cells, and the corresponding candidates 22 also had ic 50 values in the low micromolar range. on the other hand, conversion of mannich bases 10 into the corresponding adducts 23 led to a 37-fold reduction in potency. in addition, compounds 10 and 22 demonstrated a preferential cytotoxicity to cancer cells compared to normal fibroblasts [37] . further studies showed that compounds 10 and 22 are cytotoxic towards a large number of human tumor cell lines, two important features of many of these compounds being their lethal effects toward promyelocytic leukemic hl-60 cells and their selective toxicity for the aforementioned cancer cell line (selectivity index of 10 or more) [38] . because divergence in the mechanism of action is required for drug candidates that are developed to be tumor-specific and spare normal tissues, it is noteworthy that a representative mannich base 10 caused apoptosis and activated caspase-3, caspase-8, and caspase-9 in hl-60 cells, but not in hsc-2 cells. cytotoxic phenolic mannich bases of chalcone analogues (type 11 in fig. 3 ) are well represented in the recent literature. one of the strategies that are available for the synthesis of this type of mannich bases consists in the aminomethylation of chalcone analogues derived from at least either a phenolic aldehyde or a phenolic ketone. in line with this strategy, a series of five mono-mannich bases 24 (fig. 6 ) were obtained through a mannich reaction of chalcone analogues derived from 4-hydroxyacetophenone, employing piperidine as amine reagent [39] . despite the use of an excess of both paraformaldehyde and piperidine, no double mannich bases analogous to 11 were isolated. the evaluation of compounds 24 against androgene-independent prostate cancer (pc-3) cell line showed that although three of them were more potent than the parent chalcone analogues, the most cytotoxic mannich base 24 was 2.5-fold less potent than the reference drug 5fluorouracil. based on correlations between cytotoxicity and hammet constant on one hand and cytotoxicity and partition coefficient on the other hand, the authors hypothesized that an increase in the general hydrophobicity of the molecule would result in enhanced cytotoxicity. therefore, a novel series of mannich bases 24 was designed to incorporate a dibenzylaminomethyl residue as a replacement for the piperidinomethyl group [40] . again, every attempt to obtain double mannich bases by varying the reaction conditions failed, and only mono-mannich bases could be isolated. when evaluated against androgene-independent prostate cancer (pc-3) cell line, these compounds consistently displayed lower cytotoxicity than that of the parent chalcone analogues. unexpectedly, cytotoxicity in the series of mannich bases with a dibenzylaminomethyl residue was also much lower than that of the corresponding mannich bases in the series containing a piperidinomethyl motif, thus invalidating the hypothesis put forth by the authors in the previous study. furthermore, no adduct between ethanethiol and a representative mannich base 24 could be detected after 48 h, whereas the incubation of ethanethiol with the corresponding parent chalcone analogue under the same conditions resulted in formation of small amounts of adduct. the authors concluded that no active cyclohexadienone species are formed following a potential deamination of mannich base 24, and that the introduction of the dibenzylaminomethyl group further reduces the ability of the chalcone moiety to undergo thiol addition. with a view to explore the effect of variation of dialkylamino moiety on the cytotoxicity of phenolic mannich bases of chalcone analogues, 27 candidates were synthesized through aminomethylation of three chalcone analogues derived from 4hydroxyacetophenone and diverse secondary aliphatic amines [41] . equimolar ratio of reactants afforded mono-mannich bases of type 24, which were evaluated against hepatocellular carcinoma (hepg2), human lung carcinoma (sk-lu-1), and human breast cancer (mcf-7) cell lines. mannich bases with 4-phenylpiperazine residue exhibited reduced cytotoxicity towards all three lines of cancer cells, whereas the candidates with 4-methylpiperazine or 4ethylpiperazine residues were the most active in each series, but less cytotoxic than reference drug ellipticine. with respect to the substitution pattern in the b phenyl ring, mannich bases 25 ( fig. 6 ) derived from 4-chlorobenzaldehyde (r 1 ¼ h, r 2 ¼ cl) or 2methoxybenzaldehyde (r 1 ¼ och 3 , r 2 ¼ h) were consistently more active than those derived from 4-methoxybenzaldehyde (r 1 ¼ h, r 2 ¼ och 3 ). the screening identified five compounds whose ic 50 values against mcf-7 cell line were lower than 2 mg/ml, whereas the most cytotoxic compound 25 (r 1 ¼ h, r 2 ¼ cl, nr 2 ¼ 4ethylpiperazinyl) had ic 50 values lower than 2 mg/ml against all three cancer cell lines used in this study. a larger library of phenolic mannich bases of chalcone analogues featuring the dialkylaminomethyl moiety either in ring a or ring b of the chalcone system was synthesized through the clai-seneschmidt condensation of the appropriately substituted mannich bases of phenolic aldehydes or ketones with heterocyclic ketones or aldehydes, respectively [42] . the use of 4-alkoxy-2hydroxyacetophenones as substrates in the mannich reaction yielded a mixture of 5-aminomethylated derivative with the isomeric 3-aminomethylated derivative, the former being the major reaction product in all cases. the adept tailoring of the ratio between the substrate, formaldehyde and morpholine in the mannich reaction of 4-hydroxyacetophenone resulted in the selective preparation of either single or double mannich base from this substrate. on the other hand, 3-hydroxyacetophenone afforded a mixture of 4-morpholinylmethyl derivative with 2morpholinylmethyl derivative and 2,4-bis(morpholinylmethyl) derivative, even when an equimolar ratio between the reagents was used. isovanillin, which underwent aminomethylation only at position 2 with morpholine and piperidine as amine reagents, was employed as an example of a phenolic aldehyde substrate in the mannich reaction. with the help of these intermediates, several small series of mannich bases of heterocyclic chalcone analogues 26e33 (fig. 6 ) were synthesized and evaluated for cytotoxic activity against four human cancer cell lines, namely pc-3, mcf-7, nasopharyngeal carcinoma (kb), and resistant nasopharyngeal carcinoma (kb-vin). the rich diversity within this library comprised of structurally related entities allowed interesting insight on the cytotoxicityestructure relationship. first, the presence of two phenolic groups in ring a seems to enhance the cytotoxic activity, as proven by a candidate of type 26 (r 1 ¼ h, het ¼ 2-pyridinyl), which was the most potent in the entire library against all four types of cancer cell lines. then, the presence of a methoxy group in series 27 (r 1 ¼ ch 3 ) appears to be generally preferable to ethoxy and isopropoxy, whereas a comparison of the cytotoxicity of similar compounds of type 26 and type 27 usually favors the candidates in the latter series, which have the aminomethyl group para to the phenolic hydroxyl. an analysis of the cytotoxicity within series of candidates of type 28 demonstrated that six-membered heterocycles (particularly a 2-pyridinyl residue) are preferred to five membered heterocycles as ring b moieties, and this observation was validated by the inspection of anticancer activity of compounds in series 29. however, the presence of a second morpholinylmethyl group appears to be detrimental to the cytotoxic activity of candidates 29. shuffling of hydroxy and morpholinylmethyl groups in mannich bases 28 and 29 led to compounds in series 30e32, which are generally less cytotoxic than their counterparts derived from chalcone analogues having a 4-hydroxy substituent in ring a. selective high cytotoxicity against mcf-7 cell line was displayed by the compounds in series 33 featuring the morpholinylmethyl moiety in ring b of the chalcone system. overall, more than 80% of the mannich bases in this library of mannich bases of heterocyclic chalcone analogues 26e33 are cytotoxic (ic 50 < 4 mg/ml), while four members of the library are highly cytotoxic (ic 50 < 1 mg/ml) against all four cell lines, and other four against at least three cell lines, with mcf-7 and pc-3 being usually more sensitive than kb and kb-vin cell lines. later, novel mannich bases of type 24 (ar ¼ 3pyridinyl) were evaluated against several cancer cell lines, and the candidates showed cytotoxicity in the low micromolar range only towards promyelocytic leukemic cells (hl-60) and oral squamosa cell carcinomas (hsc-2, hsc-3 and hsc-4), whereas the ic 50 values against non-malignant gingival fibroblasts, pulp cells and periodontal ligament fibroblasts were higher [43] . the tumor selectivity of these mannich bases may be the result of their proven ability to cleave poly[adp-ribose]polymerase-1 in hsc-2 cells, but not in gingival fibroblasts cells. in addition, the cytotoxic activity of mannich bases of chalcone analogues with an aminomethyl moiety in b ring structurally similar to 33 against a panel of breast cancer (mcf7), melanoma (uacc62) and renal cancer (tk10) cell lines was described in a patent [44] . most compounds were active, and some were potent mostly towards the first two cancer cell lines. a series of mannich bases 34 of bichalcone analogues (fig. 6) , in which the two chalcone units are linked through a bis(aminomethyl) function generated by the use of a bifunctional amine reagent such as piperazine, has also been synthesized and evaluated against 25 cancer cell lines [45] . aminomethylation of acetovanillone with piperazine afforded a bis-mannich base, which subsequently led to compounds 34 through a claiseneschmidt condensation with various aldehydes. surprisingly, compound 34 (ar ¼ 2-pyridinyl) was selectively cytotoxic to human tongue squamous carcinoma (cal-27) and human pharyngeal squamous carcinoma (fadu) cell lines, whereas 3-pyridinyl and phenyl analogues were the most cytotoxic compounds towards all cell lines. substitution of the phenyl ring (ar ¼ c 6 h 5 ) with methoxy groups stripped mannich bases 34 of their cytotoxicity towards most cell lines, whereas the decrease in cytotoxic activity induced by the presence of chlorine as substituent was not so drastic. replacement of phenyl with 2-furanyl or 2-thiophenyl led to compounds that are selectively cytotoxic to one or more cell lines, but further substitution with methyl of these five-membered heterocycle renders them devoid of cytotoxicity against all lines. despite a few notable examples of selectivity, the results obtained for this collection of compounds are not very encouraging, and they suggest that the incorporation of a second chalcone unit does not enhance the cytotoxicity of mannich bases derived from chalcone analogues. cytotoxic activity of mannich bases of bichalcone analogues was further explored using candidates with a modified design. the synthetic strategy comprised the synthesis of mono-phenolic mannich bases starting from 4-hydroxyacetophenone or acetovanillone as substrates and employing 1-(4-(piperazin-1-yl)phenyl) ethanone as amine reagent, then the bichalcone unit was generated through a claiseneschmidt condensation of both acetyl functions with aromatic aldehydes [46] . only mannich bases from bichalcone analogues featuring a pyridinyl moiety (such as candidate 35, fig. 6 ) were active towards prostate cancer (du145), non-small cell lung cancer (a549), ileocecal (hct) and nasopharyngeal carcinoma (kb) cell lines with ic 50 values between 0.7 and 4 mm; all other compounds had ic 50 values greater than 20 mm. compared to compound 35, mannich base 36 having a single chalcone unit was less active (ic 50 between 10 and 13 mm). an exploration of the mechanism of action for these compounds suggested that compound 36 most likely acted via the fas/cd95 apoptosis signaling pathway. ferulic acid and its derivatives provide another example of a type of substrate containing an a,b-unsaturated system activated by an electron-withdrawing group, similar to that in phenolic chalcone analogues, from which phenolic mannich bases can be synthesized. the growing body of evidence suggesting that 3 0 -azido-2 0 -deoxythymidine (azt), a known antiviral, also possesses anticancer activity [47e49] has sparked a study aiming at cytotoxic evaluation of a series of conjugates of azt and ferulic acid derivatives [50] . thus, propargyl ester and n-propargylamide of ferulic acid chemoselectively underwent aminomethylation ortho to the phenolic hydroxyl with various secondary aliphatic amines, and the resulting phenolic mannich bases 37 (x ¼ o, nh) reacted through the terminal alkyne moiety with azt via a cu(i)-catalyzed click chemistry process to afford the corresponding 1,2,3-triazoles. evaluation of cytotoxicity for both mannich bases 37 (fig. 7 ) and the related 1,2,3-triazoles against human breast adenocarcinoma (mda-mb-231), lung adenocarcinoma (sk-lu-1) and colon adenocarcinoma (sw480) cell lines showed that only some of compounds 37 were cytotoxic. despite the presence in their structure of an identical scaffold comprising a phenolic mannich bases moiety and an activated carbonecarbon double bond motif that has been deemed responsible for the cytotoxic effect of mannich bases 37, all of the corresponding 1,2,3-triazoles were inactive. out of eight mannich bases of propargyl ester of ferulic acid reported in this study, three were inactive against all three lines, while the rest had weak to moderate cytotoxicity, and the most active candidate (ic 50 ¼ 20e44 mg/ml) was mannich base 37 (x ¼ o) having a pyrrolidinylmethyl moiety. with the exception of mannich base 37 (x ¼ nh) with a piperidinylmethyl moiety, all others candidates derived from n-propargylamide of ferulic acid were inactive. other aminomethylated phenols with cytotoxic activity have been reported besides phenolic mannich bases of chalcone analogues. unfortunately, the wide structural diversity of phenolic substrates from which these phenolic mannich bases originate and the lack of a systematic and comprehensive search for structureecytotoxic activity relationships within a particular type of phenolic substrate undermine any efforts to discover good lead compounds for further development as drugs. phenolic substrates subjected to the mannich reaction with a view to obtain novel cytotoxic agents include both simple and very complex structures. examples that illustrate structurally simple phenolic substrates are 1-naphthol and 8-hydroquinoline, whose mannich bases with piperidine and 4-arylsulfonylpiperazines exhibited growthinhibitory effects towards a panel of carcinoma cell lines, including hela (cervical epithelioid carcinoma cell), bt483 (mammary gland adenocarcinoma cell), skhep (hepatocellular carcinoma cell), and ce81t (esophageal carcinoma cell) [51] . although the cytotoxic effect of aminomethylated naphthols and 8hydroxyquinoline derivatives has been known for some time [52, 53] , a mechanistic study presented in the aforementioned report showed that these phenolic mannich bases induce apoptosis by activation of caspase-dependent pathways. furthermore, upon addition of copper ions, these compounds dramatically stimulate production of reactive oxygen species and activate various kinases, a group of enzymes that are known to be important in the oxidative stress-mediated cell death. candidate 38 (fig. 7) was the most potent in this small series, with a concentration for 50% cell growth inhibition of 0.71 mm; this value decreased to 0.06 mm in the presence of 50 mm copper. a more detailed study [54] of the structureecytotoxic activity relationship within this class of compounds showed that either replacement of sulfonyl function in 38 with a methylene group, or replacement of piperazine ring with an ethylenediamino moiety (as in compound 39, fig. 7 ) led to a significant increase in cytotoxic activity. the cell lines used in this study exhibited selective sensitivity towards different mannich bases, which appeared to be modulated by the nature of the arylsulfonyl group. as for the 8-hydroxyquinoline part of the molecule, substitution at position 5 of the quinoline motif (especially with a nitro group) had a beneficial effect, whereas its replacement with phenol, 3-hydroxypyridine or 1-naphthol led to a decrease of cytotoxic activity [54] . cytotoxicity of enantiomerically-enriched mannich bases 40 (fig. 7) derived from 2-naphthol, aromatic aldehydes and either 4-piperidinol (r ¼ h) or its acetylated counterpart (r ¼ coch 3 ) against murine leukemic l1210 and human lymphoblast molt 4/c8 and cem cell lines has been examined by another study [55] . all of the compounds were only moderately cytotoxic, with ic 50 values in the middle micromolar range, and were also 10e70-fold less potent than that reference drug melphalan. substitution of the aryl group in 40 with r 1 ¼ 4dialkylaminoethoxy resulted in a series of compounds whose cytotoxicity potential against estrogen-responsive human mcf-7 breast cancer cells was found to be comparable to that of tamoxifen. however, removal of the 4-piperidinol moiety from mannich bases 40 led to benzylnaphthols with enhanced cytotoxicity against mcf-7 cells, which is most likely due to their binding and antagonistic effects against human estrogen receptor alpha [55] . flavones represent another type of phenolic substrate from which cytotoxic mannich bases have been synthesized. because flavones such as chrysin bear structural resemblances to androgens, mannich bases 41 fig. 7) have been designed as inhibitors of human aromatase, an enzyme which converts androgens to estrogens, and therefore represents a key target in the treatment of hormone-dependent tumors, including breast cancer [56] . several single and double mannich bases of chrysin were found to inhibit human aromatase more effectively than reference drug aminoglutethimide [57] . in addition, mannich bases 41 (r ¼ oh, r 1 ¼ h, r 2 ¼ aminomethyl) of apigenin ( fig. 7 ) have been prepared from aliphatic primary and secondary amines via chemoselective aminomethylation at c-8 in the benzopyran ring system [58] . antiproliferative activity of these mannich bases against four human cancer cell lines, namely human cervical (hela), human liver (hepg2), human lung (a549), and human breast (mcf-7) cancer cells, was determined using the standard 3-(4,5-dimethylthiazol-2-diphenyl-tetrazolium) bromide (mtt) assay. pyrrolidine mannich base of apigenin was the most promising compound in this series, its inhibition of cell proliferation being greater than 90% against all four cell lines at a concentration of 1 mg/ml. many natural or synthetic carbazoles, either simple or condensed with other heterocycles (such as pyridocarbazoles, indolocarbazoles, pyranocarbazoles, pyrrolocarbazoles, etc), have been reported as anticancer agents. a recent study examines the cytotoxicity of a series of oxazinocarbazoles, which were the major products arising from the mannich reaction of n-substituted 2-or 4-hydroxycarbazoles with primary amines (fig. 8 ) [59] . under the appropriate reaction condition, 4-hydroxycarbazoles yielded 2,3,4,7-tetrahydro [1, 3] oxazino [5,6c]carbazoles 42, whereas 2hydroxy-9-methylcarbazole led to a mixture of regioisomeric 2,3,4,7-tetrahydro [1, 3] oxazino [6,5-b] carbazoles 43 and 2,3,4,7tetrahydro [1, 3] oxazino[5,6-a]carbazoles 44. use of allylamine, 3,3-dimethylallylamine or benzylamine as amine reagents afforded 44 as the major product, while isomer 43 was the major component of the mixture when 2-pyridinylmethylamine was employed. in addition, small to moderate yields of bis-mannich bases 45 or 46 were isolated from n-substituted 4-hydroxycarbazoles and 2hydroxy-9-methylcarbazole, respectively, but only when 2pyridinylmethylamine was employed as amine reagent. evaluation of the antiproliferative action of these compounds against cem (t cell leukemia), jurkat (acute t cell leukemia), raji (burkitt's lymphoma), mcf-7 (breast cancer cells) and caco-2 (colorectal cancer) using the wst-1 colorimetric assay showed, after the primary screening at 100 mm, that bis-mannich bases 45 and 46 were less active than the oxazinocarbazoles. in the series of compounds 42, the best antiproliferative effect was observed for candidates having either an allyl or a prenyl group at the carbazole nitrogen atom and an allyl group at the oxazine nitrogen atom. thus, jurkat and raji cell lines. the majority of candidates 43 and 44 exhibited significant antiproliferative action at 100 mm, and compound 43 (r ¼ 2-pyridinylmethyl) was the most active towards jurkat and raji cell lines (ic 50 ¼ 12 mm) [59] . aminomethylated derivatives of hydroxycarbazoles have been also mentioned in a different study [60] , which described the synthesis of a small series of phenolic mannich bases 47 (fig. 8 ) obtained from 5-substituted 2-hydroxy-5h-benzo[b]carbazole-6,11-diones along with their in vitro anticancer evaluation at national cancer institute (nci) using an in-house developed screening panel of approximately 60 cell lines derived from nine different types of cancer. only one mannich base 47 (r ¼ 4-h 3 coc 6 h 4 ) was more active than the parent benzocarbazoledione, and a compare analysis [61] revealed that its mechanism of action is novel and does not resemble the known mechanisms of action for standard anticancer drugs, but this candidate was not selected for further studies concerning its interaction with dna. in a related report, phenolic mannich bases 48 of 5-hydroxy-1h-naphtho [2,3-g] indoles were found to be inactive in a similar screening [62] . quinones are a class of compounds that have been widely investigated as anticancer agents [63] . besides anthracycline antibiotics, other natural hydroxyquinones such as plumbagin [64, 65] , juglone [66, 67] or lapachol [68] and their derivatives have been reported to exhibit significant cytotoxicity. the mannich bases of another natural phenolic quinone, namely lawsone, and their pt(ii) complexes 49 ( fig. 9) have been synthesized and shown to be highly cytotoxic towards six cancer cell lines: mda-mb-435 (melanoma), hl-60 (promyelocytic leukemia), hct-8 (colon), sf-295 (brain), ovcar-8 (ovary) and pc-3 (prostate) [69] . the ligands and the complexes that have long alkyl chains (r ¼ n-heptyl or ndecyl) were the most active (the complexes were actually more cytotoxic than cisplatin), whereas the neutral complexes (x ¼ cl) were generally more cytotoxic than the corresponding charged complexes (x ¼ h 2 o or nh 3 ). examination of the mechanism of action for some of these complexes has shown that aqua complexes 49 were more efficient inhibitors of ethidium bromide intercalation into dna than amino complexes, and candidate 49 (r ¼ (ch 2 ) 3 ch 3 , x ¼ h 2 o) was more efficient than cisplatin [70] . the same aqua complex also induced dna strand breaks, while the corresponding amino complex was ineffective. the ability of these complexes to inhibit topoisomerase i was also examined. most chlorido and amino complexes were as active as reference drug camptothecin in the dna relaxation assay, and did not cause major unwinding of dna, with the exception of complex 49 (r ¼ n-decyl, x ¼ cl). in addition, cellular platinum accumulation was shown to increase with the increase of the length of the alkyl chain of the amino moiety in the mannich base ligand. the chlorido pt(ii) complexes were oxidized to the corresponding chlorido pt(iv) complexes, but the cytotoxicity of these new complexes was comparable to the cytotoxicity of the parent pt(ii) complexes, presumably owing to the rapid reduction of pt(iv) complexes before entering the cancer cells [71] . furthermore, miscellaneous aminomethylated phenols from structurally diverse substrates have been reported as anticancer agents (fig. 10) . thus, a small series of seven phenolic mannich bases 50 were synthesized through aminomethylation of naturally occurring antibiotic lasalocid, with simultaneous elimination of the carboxyl group neighboring the phenolic hydroxyl [72] . antiproliferative effect of mannich bases 50 was evaluated against mcf-7 (human breast adenocarcinoma), a549 (human lung adenocarcinoma), ht-29 (human colon carcinoma) and p388 (murine leukemia) using either mtt or sulforhodamine b (srb) assay, and four candidates were more cytotoxic towards a549, ht-29 and mcf-7 cell lines that anticancer drug cisplatin. these candidates also presented higher selectivity towards cancer cells than towards balb/3t3 (normal murine embryonic fibroblast) or hlmec (human lung microvascular endothelial) cell lines. the lack of activity of the other three mannich bases was attributed to the aralkyl or long alkyl chains in the amine moiety of these compounds [72] . camptothecin, another naturally occurring phenolic substrate and lead compound for a plethora of cytotoxic substances, was converted into the oxazino derivatives 51 (fig. 10 ) by means of the mannich reaction using primary aliphatic and aromatic amines [73] . evaluation of these novel hexacyclic camptothecin derivatives towards nine human cancer cell lines (bxpc-3, nci-446, mcf-7, hepg-2, a549, a2780, bel7402, ht-29, and kb), using mtt assay and camptothecin and topotecan as reference compounds, showed that most of them exhibit cytotoxicity towards several cell lines that is superior or comparable to topotecan, while only a few of the candidates presented cytotoxicity comparable to camptothecin. because candidates 51 (r ¼ c 2 h 5 or n-c 3 h 7 ) were the most potent antiproliferative agents in this series, the presence of a small alkyl group at the nitrogen in the oxazine moiety seems to be preferable for a high cytotoxicity [73] . a series of oxazino derivatives 52 (r 1 ¼ ch 3 ) (fig. 10) were also prepared from g-tocotrienol and primary amines via the mannich reaction, whereas d-tocotrienol afforded under the same conditions a mixture of oxazino derivatives 52 (r 1 ¼ h) and 53, in which the former is the major component [74] . no reaction occurred when secondary amines were used instead, but two phenolic mannich bases 54 were obtained indirectly from the corresponding oxazino derivatives 52. out of 42 candidates in this library, thirty compounds had greater antiproliferative activity against the highly metastatic þ sa mouse mammary epithelial cancer cells than that of the parent tocotrienols (ic 50 ¼ 3 mm), and seven candidates had ic 50 values in the nanomolar range. mannich bases 54 were less active than the corresponding oxazino derivatives 52 against nci's standard panel of 60 cell lines, which suggests that the oxazine ring is an essential pharmacophore for the cytotoxic activity of these tocotrienol derivatives. generally, the oxazino derivatives 52 of dtocotrienol were more active than the corresponding isomers 53 derived from g-tocotrienol, and a long alkyl chain at the nitrogen atom (preferably with a terminal hydroxyl group) proved beneficial for the antiproliferative activity. the same conclusions were drawn after the evaluation of structureeantimigratory activity relationship using the highly metastatic mdamb-231 breast cancer cell line [74] . novel g-quadruplex ligand/alkylating hybrid structures 55 ( fig. 10) were obtained by tethering a naphthalene diimide core having g-quadruplex recognizing properties to phenolic mannich bases using flexible spacer [75] . the assessment of cytotoxic effects of these compounds 55 (n ¼ 1, 2, or 3) and their corresponding methiodides against human embryonic kidney 293t cell line by mtt assay suggests that the length of the spacer between the core and the phenolic mannich bases moiety modulates the cytotoxicity: candidates 55 having two-carbon atoms and one-carbon atom spacers were the most active (ic 50 4.5 and 10.5 mm, respectively), whereas the mannich base 55 with a three-carbon atoms spacer was less active. cytotoxicity of these compounds parallels their ability to alkylate dna, and the grafting of the alkylating mannich base moiety to the central core contributes to the enhancement of the g-quadruplex folding induction and stabilization. a similar g-quadruplex ligand/alkylating hybrid structure was shown to significantly slow the growth of melanoma cells by causing telomere dysfunction and down-regulation of telomerase expression [76] , which suggests that these hybrids could be possible candidates for the development of novel targeted anticancer therapies. in connection to this, the methiodide of double mannich base 56 (fig. 10 ) with a quinazoline core was also shown to cross-link linear dna at concentrations as low as 1 mm, and to inhibit dna transcription almost completely at 10 mm [77] . phenolic mannich bases 57 of norvisnagin ( fig. 10 ) were prepared through direct aminomethylation, and their ability to interact with dna was evaluated using both a qualitative binding assay and a colorimetric microassay based on the displacement of methyl green from dna [78] . three of the candidates 57 (r ¼ pyridinyl-2-amino, diethylamino, and methylamino) showed moderate dna binding affinity, and could be potentially cytotoxic. also, mannich bases 58 (r 1 ¼ h) of 1,3-dihydroxyxanthone ( fig. 10 ) displayed moderate to good cytotoxicity against lung cancer (nci-h460), tongue squamosa cell carcinoma (tca-8113), liver cancer (bel-7402), hepatocarcinoma (hepg2), gastric carcinoma (sgc-7901) and urinary bladder carcinoma (t24) in an mtt assay [79] . several reports of mannich bases of indoles as cytotoxic agents are also available in recent literature. cytotoxicity of a few indole mannich bases 59 derived from 4-substituted piperazines (fig. 11 ) was evaluated against liver (huh7), breast (mcf7) and colon (hct116) cancer cell lines using srb assay, and some of these compounds had ic 50 values in the lower micromolar range. compound 59 (r ¼ 3,4-dichlorobenzyl) was cytotoxic towards all three cancer cell lines, and fared better than reference drug 5-fluorouracil (5-fu) [80] . on the other hand, n-mannich bases 60 of 3methylindole did not inhibit the growth of cancer cells or had high ic 50 values. in spite of this fact, a subsequent study [81] was dedicated exclusively to the investigation of cytotoxicity of a novel series of n-mannich bases of type 60 against the same cancer cell lines. the broadening of the nature of substituent at position 4 of piperazine proved favorable, as several compounds reported in this later study presented cytotoxic activity comparable to reference drug 5-fu. a comparison between the morphological features of cancer cells for which apoptosis was induced either by a selected 3metylindole n-mannich base 60 or by paclitaxel suggests that compounds 60 (r 1 ¼ ch 3 ) and paclitaxel share the same mechanism of action. design of novel cytotoxic mannich bases in which indole itself was the substrate for aminomethylation was also revisited using an extended panel of 4-substituted piperazines as amine reagents in aminomethylation [82] . the novel candidates 59 proved to be cytotoxic towards the same cancer cell lines (huh7, mcf7, and hct116); however, no cytotoxicity was observed for the candidates with an electron-withdrawing group (such as 4nitrophenyl, benzoyl or acetyl) as substituent at position 4 of piperazine. other c-mannich bases of indole derivatives have been claimed as potent inhibitors of isoprenylcysteine carboxyl methyltransferase (imct), an enzyme that plays an important role in the posttranslational modification of proteins that are involved in the regulation of cell growth, and therefore represents a potential therapeutic target in oncogenesis. among the few small molecules inhibitors of imct that were discovered so far, the most promising appears to be cysmethynil, an indol-3-ylacetamide derivative which impairs growth factor signaling and induces cell cycle arrest and autophagy. because cysmethynil suffers from poor water solubility and strong binding to plasma proteins, rational modification of this hit compound has been expected to yield more potent imct inhibitors with improved bioavailability. replacement of the acetamide function in cysmethynil with various aminomethyl moieties led to compounds 61 (r ¼ dialkylamino) with an inhibitory effect on icmt that was generally 2e3-fold more potent than that of the parent inhibitor [83] . evaluation of the effects of these candidates on viability of mda-mb-231 human breast cancer cells using the colorimetric tetrazolium assay confirmed the results for icmt inhibition. thus, compounds 61 ( fig. 11 ) were found to be more cytotoxic (ic 50 3e13 mm) than cysmethynil (ic 50 22 mm). other modifications of the lead compound 61 (r ¼ diethylamino) either preserved the potency of the candidates (e.g., shuffling of the methyl group on the phenyl ring), or resulted in a potency decrease (e.g., replacement of n-octyl with prenyl). however, the replacement of m-tolyl moiety in 61 with more polar heteroaromatic rings led to submicromolar ic 50 values in the icmt inhibition assay, and to ic 50 values in the antiproliferative assay on breast mda-mb-231 and prostate pc3 cell lines that are 2e3-fold lower than that of fig. 11 . cytotoxic indole and azaindole mannich bases. the lead 61 (r ¼ diethylamino) [84] . compound 62 (fig. 11 ) was the most potent compound in this series, and presented a series of improvements of the drug-like profile over cysmethynil, such as good solubility in water, acceptable permeability through an artificial membrane, and limited tendency to form light scattering aggregates. using naphtho[2,3-f]indole-5,10-dione as scaffold, a series of 3aminomethylated derivatives was synthesized, and four candidates were evaluated as antiproliferative agents against the standard panel of 60 human cancer cell lines at nci [85] . compounds 63 (r ¼ primary or secondary aliphatic amine residue, r 1 ¼ oh) (fig. 11 ) were less potent than doxorubicin against any of the cell lines, but multidrug resistant breast cancer cells were found to be more sensitive to 63 than to doxorubicin. in addition, mannich bases 63 showed potency for cancer cell lines that are otherwise resistant to anticancer drugs, such as the p-glycoprotein-positive subline of k562 leukemia cells or the p53-null subline of hct116 colon carcinoma cell line. replacement of phenolic hydroxyl groups r 1 in 63 (r ¼ dimethylamino) by 2-aminoethyleneamino moieties led to mixed results, as the potency improved for some of the cell lines and declined for others, but the sensitivity of multidrug resistant breast cancer cells to these modified candidates was completely lost [86] . furthermore, candidate 63 (r ¼ quinuclidin-3ylamino, r 1 ¼ oh) was shown to inhibit topoisomerase i-mediated relaxation of dna, but the suppression of the topoisomerase i activity is presumably the leading although probably not the only factor contributing to cytotoxicity of mannich bases of naphtho [2,3-f]indole-5,10-diones [87] . preobrazhenskaya et al. have also shown that a series of single and double mannich bases 64 (r 1 ¼ h or dialkylaminomethyl) of 3,4-bis(indol-1-yl)maleimides (fig. 11) , structurally related to rebeccamycin or staurosporine, were highly cytotoxic towards к562 and hcт116 cell lines, but their cytotoxicity does not correlate well with their ability to either inhibit protein kinase c-a or constrain activation of multiple drug resistance [88] . mannich bases of an indole isostere, namely 5h-pyrrolo[3,2-d] pyrimidine, have been designed as inhibitors of phosphatidylinositol-3-kinase a (pi3ka), a lipid kinase that modulates activity of the pi3k downstream effectors akt and mtor. since the consequences of biological activation of akt include tumor progression, proliferation, survival, growth, invasion, angiogenesis, and metastasis, pi3ka represents an attractive target for development of anticancer drugs. although aminomethylated pyrrolopyrimidine 65 (fig. 11 ) was an efficient inhibitors of pi3ka (ic 50 ¼ 20 nm) and showed good selectivity for pi3ka over mtor (170-fold) , this compound exhibited low cytotoxicity towards pc3 cancer cells [89] . in addition, all the other analogues of mannich base 65 were found to be even weaker inhibitors of pi3ka than the lead compound. isatin is nowadays a well recognized and privileged scaffold in the design of cytotoxic and anticancer compounds [90] . several isatin-containing substrates, namely isatin and its 5-halogenated analogues, the corresponding imine derivatives obtained from sulfadiazine, sulfadoxine and trimethoprim, and a hydrazone derived from isoniazid, were aminomethylated using gatifloxacin as amine reagent [91] . the resulting mannich bases were tested against nci's standard panel of 60 cell lines using srb assay, and compound 66 (fig. 12 ) emerged as an efficient anticancer agent that was generally more potent than reference drug etoposide against most cell lines in the panel. another library of mannich bases derived from isatin, 4-halogenated isatins and their schiff bases with 2-amino-6-methylbenzothiazole was evaluated for cytotoxic effects on three breast cancer cell lines (mda-mb468, mdamb231 and mcf7) using srb assay [92] . the introduction of a halogen as substituent at position 4 of isatin mannich bases led to an increase in cytotoxicity, and modification of isatins into schiff bases followed by aminomethylation resulted in mannich bases further enhanced the cytotoxicity of these candidates. compounds 67 and 68 ( fig. 12) were the most potent in each series (ic 50 < 20 mm), and more potent than reference drug cisplatin, while their cytotoxicity against normal cells was low. as far as their mechanism of action is concerned, compound 67 induced cell cycle arrest in g2/m phase at concentrations similar to those observed for cell growth inhibition, whereas compound 68 did not [92] . furthermore, another collection of mannich bases of isatin imines generated either from 2aminobenzimidazole or 2-amino-4,5-dihydrothiazole was evaluated against mcf-7 human breast adenocarcinoma cell line using srb assay, but the cytotoxicity of these compounds was moderate (ic 50 > 20 mm) and inferior to that of doxorubicin [93] . generally, aminomethylated schiff bases of isatin derived from 2aminobenzimidazole were more cytotoxic than their counterparts derived from 2-amino-4,5-dihydrothiazole, compound 69 ( fig. 11) being the most potent in this collection (ic 50 ¼ 22.6 mm). cytotoxic mannich bases of isatins were also designed employing hybridization of isatin with a 4-aminoquinoline scaffold to generate the substrate subjected to aminomethylation [94] . the cytotoxicity of these compounds towards breast cancer cell lines mda-mb468 and mcf7 was moderate (ic 50 values between 15 and 65 mm), but the activity improved slightly in the series of the corresponding thiosemicarbazones (ic 50 in the range of 10e55 mm). mannich bases 70 and 71 (fig. 12) were the most potent candidates in every series (2e3-fold more cytotoxic than cisplatin), and they preferentially inhibited the growth of cancer cell over normal cells. studies using flow cytometry also suggest that these compounds induce cancer cell death by apoptosis. beside isatin derivatives, several other classes of nh-azoles have been aminomethylated with a view to synthesize cytotoxic mannich bases. 2,3-dihydro-1,3,4-oxadiazole-2-thiones appear to be the preferred substrate within this category, most likely owing to their straightforward preparation. starting from methyl salicylate, good yields of the corresponding oxadiazolethione mannich bases 72 ( fig. 13) were obtained in three steps [95] . most compounds in this collection arise from primary aromatic amines diversely substituted in the aromatic ring, whereas mannich bases 72 derived from secondary aliphatic amines are poorly represented. selected candidates from this series have been initially evaluated by nci against a panel consisting of nci-h460 (lung), mcf7 (breast), and sf-268 (glioblastoma) cancer cell lines using srb assay. seven of these thirteen mannich bases 72, most of them having either chlorine or carboxy group as substituent in the aromatic ring of the amine moiety, reduced the growth of nci-h460 cell line to 30% or less, and they were further selected for the standard 60-cell lines panel assay. compounds 72 (r ¼ h, r 1 ¼ 3-clc 6 h 4 or 4-clc 6 h 4 ) presented higher cytotoxicity than reference drugs 5-fu or cyclophosphamide against most cancer cell lines in this panel [95] . the ability of several oxadiazolethione mannich bases 73 featuring variously substituted aromatic ring at position 5 of the oxadiazolethione ring (r 1 ¼ h and r ¼ no 2 , oh, ch 3 , or r ¼ r 1 ¼ cl) to inhibit the growth of tumors in vivo has been also investigated [96] . tumor volume and tumor weight in mice injected with ehrlich ascites carcinoma cells were reduced by 52e74% at a dose of 50 mg candidates 73 (fig. 13 ) per kg body weight, whereas similar dose of reference drug 5-fu inhibited tumor formation by 93%. mannich bases 73, especially those having hydroxyl, methyl or chloro substituents on the phenyl ring at position 5, were the most potent. also, the counts of red blood cells and leukocytes, as well as hemoglobin levels, have been restored almost to the normal values in mice treated with mannich bases 73 [96] . aminomethylated oxadiazolethiones 74 (fig. 13 ) were generally more cytotoxic against colon carcinoma (ht29) and less cytotoxic against breast cancer (mcf7) cells using srb assay, but the most potent candidate 74 (nr 2 ¼ nhc 6 h 4 ch 3 -4) was 5-fold less cytotoxic than reference drug doxycycline [97] . cytotoxicity of a series of mannich bases 75 (fig. 13 ) of a norharmaneoxadiazolethione structural hybrids was evaluated against a panel comprising melanoma (uacc-62), breast (mcf7), ovarian resistant (nci/adr), renal (786-0), lung (nci-460), prostate (pco-3), ovarian (ovcar) and colon (ht-29) cell lines using srb assay [98] . several of candidates 75 (r ¼ h or n(ch 3 ) 2 , r 1 ¼ isopropylamino or benzylamino) exhibited a broad spectrum cytotoxic activity, and aminomethylation of the parent oxadiazolethiones significantly enhanced the cytotoxicity of each resulting mannich bases 75 compared to that of the corresponding substrate. furthermore, mannich bases 76 of a fluoroquinoloneeoxadiazolethione hybrid were prepared using either secondary aliphatic amines or substituted arylamines as amine reagents [99] . the in vitro evaluation of cytotoxicity against hep3b cancer cells using mtt assay showed that compounds 76 ( fig. 13 ) were more potent than the parent fluoroquinolone pefloxacin. also, mannich bases in this series derived from aliphatic amines were generally more cytotoxic than those derived from arylamines, and candidate 76 (r ¼ n(ch 3 ) 2 ) was even more potent than reference drug bisantrene. 2,3-dihydro-1,2,4-triazole-3-thiones could also act as substrates for the preparation of cytotoxic n-mannich bases. schiff bases obtained from 5-aryloxymethyl-4-amino-3-mercapto-1,2,4-triazoles and 3(5)-substituted pyrazole-4-carboxaldehydes were aminomethylated using either morpholine or diphenylamine to afford mannich bases 77 (fig. 13) , whose cytotoxicity against hepg2 cell line was evaluated using mtt assay [100] . out of five tested candidates, mannich bases 77 having a morpholine moiety were more potent than those with a diphenylamine moiety, but they were still 2e3-fold less cytotoxic than reference drug doxorubicin. eleven dimethylamine mannich bases 78, that were obtained through aminomethylation of schiff bases derived from a 4-amino-1,2,4triazole-3-thione having at position 5 a moiety originating from fluoroquinolone ofloxacin, were screened for cytotoxicity against murine leukemia cell line (l1210) and human leukocytoma cell line (hl60) [101] . mannich bases 78 (fig. 13) were generally more cytotoxic than the corresponding parent schiff bases, and candidates 78 with a hydroxyl group in the aromatic ring of the azomethine function (r ¼ oh) were the most cytotoxic compounds in the series (ic 50 values in the range of 0.14e0.83 mm). mannich bases of thiazolidinone derivatives have also been investigated as cytotoxic agents. c-aminomethylation of two 4thiazolidinones using secondary aliphatic amines afforded mannich bases 79 (x ¼ o, ch 2 ) (fig. 14) , which were evaluated against colon (hct116) and breast (t47d) cancer cell lines by srb assay, but their cytotoxicity was generally moderate to low (ic 50 values between 13 and 50 mm) [102] . in addition, n-aminomethylation of two thiazolidine-2,4-diones (r ¼ cl, och 3 ) with morpholine, piperidine and variously 1-substituted piperazines yielded mannich bases 80 (fig. 14) , which were investigated at nci against the standard 60-cell lines panel using srb assay, and proved to be virtually inactive (growth inhibition for the most sensitive cell line between 18 and 33%) [103] . several examples of p-mannich bases derived from organic esters of phosphorous acid that were disclosed as cytotoxic agents are available in the literature. thus, a-aminophosphonates 81 were synthesized from alkyl phosphites (r 3 ¼ ch 3 , c 2 h 5 , n-c 3 h 7 , i-c 3 h 7 , n-c 4 h 9 ), fluorine-substituted benzaldehydes and 2aminobenzothiazoles, and their cytotoxicity was evaluated against pc3 (prostate), a375 (melanoma), a431 (epidermoid carcinoma), and bcap-37 (breast) cancer cells using mtt assay [104] . most mannich bases 81 ( fig. 15 ) exhibited low to moderate growth inhibition of a375 and bcap-37 cells, but their cytotoxicity towards pc3 and a431 cells was generally greater. the nature of the fluorine substituent appears to influence cytotoxicity, as candidates 81 having fluorine directly attached to the aromatic ring are more potent than those with a trifluoromethyl substituent. an improvement of cytotoxicity with the increase of the length of the alkyl residue r 3 from the initial phosphite was also noted. p-mannich bases 82 (r 1 ¼ ch 3 , c 2 h 5 , i-c 3 h 7 ) (fig. 15 ) were prepared using thieno [3,2-c] pyridine-2-carboxaldehyde as aldehyde component in the mannich reaction of dialkyl phosphites with variously substituted arylamines [105] . the majority of candidates 82 showed good cytotoxicity against esophageal cancer cells (ec109), but they were generally more potent against hepatocellular liver carcinoma cells (hepg2) at a concentration of 50 mg/ml. a few aaminophosphonates 83 (fig. 15 ) were obtained through a mannichtype process from diphenyl phosphite, 3-acetylpyridine and variously substituted anilines, and proved to be cytotoxic to hepg liver carcinoma cell line (ic 50~1 5 mm) and mcf7 breast adenocarcinoma cell line (ic 50~2 0 mm) using mtt assay [106] . although these candidates were approximately 7-fold less cytotoxic than reference drug doxorubicin, their ld 50 values were greater than the corresponding ic 50 values, making them safe to use. aminomethylation of diethyl phosphite with either aromatic aldehydes and aromatic diamines or terephthaldehyde and various amines led to bis(aaminophosphonates) 84 or 85 (fig. 15 ), respectively, whose cytotoxicity against jurkat (t-cell lymphoma), raji (burkit's lymphoma) and mcf-7 (breast cancer) cell lines was determined using mtt assay [107] . in this structurally diverse series of compounds, a few were devoid of cytotoxicity while most of them were moderately cytotoxic. compound 85, derived from tryptamine as amine reagent in the mannich reaction, emerged as the most potent in this series, its cytotoxicity being comparable to that of doxorubicin. furan belongs to the category of electron-rich heterocycles known to undergo electrophilic substitutions such as the mannich reaction with great ease. for example, aminomethylation of synthetic lactone of natural furanditerpene 6a,7b-dihydroxyvouacapan-17b-oic acid as substrate and using various secondary aliphatic amines led to the furan mannich bases 86 (fig. 16 ) [108] . antiproliferative effect of these compounds was more potent than that of the parent lactone against a panel of nine cancer cell lines (melanoma (uacc-62), breast (mcf7), ovarian expressing the resistance phenotype for adryamycin (nci-adr/res), kidney (786-0), lung, non-small cells (nci-h460), prostate (pc3), ovarian (ovcar-03), colon (ht-29), and k562 erythromyeloblastoid leukemia), as determined by srb assay. mannich bases 86 were also equipotent to reference drug doxorubicin against at least one, if not many, of these cancer cell lines, often with ic 50 values as low as 1 mg/ml. in addition, 1,2,4-triazolo[1,5-a]pyrimidine-7-amines having at position 2 a side chain capped with a furan ring were aminomethylated with various secondary aliphatic amines to yield a large series of furan mannich bases 87 (fig. 16 ) [109, 110] . cytotoxicity of compounds 87 against liver cancer (bel-7402) and fibro sarcoma (ht-1080) cells was established using mtt assay, and the results suggest that both the substitution of the arylamine moiety and the nature of the aliphatic amino residue in the aminomethyl function have a significant influence on the potency of these candidates. in particular, the presence of a 4-trifluoromethyl group or a 4-fluoro-3-trifluoromethyl substitution pattern in the arylamine moiety led to high cytotoxicity against both cell lines at levels comparable to that of reference drug cisplatin. also, the presence of dimethylamino, 1-piperidinyl or 1-pyrrolidinyl moieties as aliphatic amino residues in the aminomethyl group of mannich bases 87 appears to result in significant antiproliferative effects, while 4-morpholinyl or 4-methylpiperazinyl moieties drastically decrease or even abolish cytotoxicity. titanium-based chemical entities enjoy the reputation of having a tremendous potential against solid tumors. recently, a number of studies presented the synthesis and the cytotoxicity for a series of titanocenes, some of them featuring various aminomethylated fivemembered heterocycles as substituent of either one or both cyclopentadiene moieties [111e114]. thus, titanocenes 88 and 89 ( fig. 17) containing mono-and bis-aminomethylated pyrroles, respectively, or titanocenes 90 and 91 (fig. 17 ) having a dimethylaminomethyl group at position 1 and 3 of indole, respectively, as well as titanocene 92 (fig. 17 ) presenting an aminomethylated imidazole ring, have been screened against pig kidney epithelial cells (llc-pk1) or human renal cancer cells (caki-1) and found to have ic 50 values quite similar to that of cisplatin (in the range of 5e10 mm). the presence of at least one aminomethyl function in their structure is claimed to be crucial for the high cytotoxicity of these titanocenes. the aminomethyl groups are able to coordinate the titanium center, and could therefore stabilize the mono-or dication formed through hydrolysis of either one or both chlorine atoms inside the cell. this results in enhanced interactions between titanocene and dna, leading to cell death at low concentrations. aminomethylation of terminal alkynes has also been employed for the generation of mannich bases with potential cytotoxic effect. 10-(prop-2-ynil)phenothiazines underwent aminomethylation with secondary aliphatic amines to give propargylamines 93 (r 1 ¼ h, cl, cf 3 ) (fig. 18 ), which were first evaluated for cytotoxic activity using two hematological tumor cell lines, namely hl60 (promyelocytic leukemia) and the ccrf/cem (lymphocytic leukemia), and were afterwards tested, in combination with doxorubicin, for ability to revert activity in the corresponding multidrug resistant variants, hl60r and cem/vbl300 [115] . although most compounds 93 were devoid of significant antiproliferative effect on the sensitive cell lines, a few of them were highly cytotoxic for the resistant cell lines, and appear to arrest cells in g1 phase of the cell cycle, unlike classic anticancer agents. furthermore, several mannich bases 93 were able to restore sensitivity to doxorubicin of the resistant cell lines, an effect that was concentration-dependent and reached maximum at 10 mm. propargylamines 93 seem to induce apoptosis by activating the caspase cascade, although neither the extrinsic nor the intrinsic pathways appear to be involved in apoptosis [115] . betulin derivatives bearing an ethynyl function have also served as starting materials for acetylenic mannich bases with cytotoxic potential. for example, propargylamines 94 ( fig. 18 ) have been obtained from alkynes prepared through addition of an organometallic derivative of acetylene to the carbonyl function in betulonic acid esters [116] , and alkynes synthesized from betulin through a sequence comprising the oxidation of the primary alcohol function to aldehyde, followed by addition of an organometallic derivative of acetylene to aldehyde carbonyl, afforded propargylamines 95 (fig. 18 ) [117] . cytotoxicity of these mannich bases was evaluated on a panel of nine human cancer cell lines using srb assay, and the results prove that some of these compounds show considerable toxicity (ic 50 values as low as 4 mm). introduction of the aminomethyl group significantly improved the cytotoxicity of propargylamines 94 and 95 compared to that of the parent alkynes, presumably by enhancing their solubility and bioavailability. highly hydrophobic and sterically hindered amino moieties, such as dicyclohexylamino or dibenzylamino, led to a decrease in cytotoxicity of the corresponding aminomethylated alkynes. mannich bases of this type were shown to act by triggering apoptosis, although a complementary process of autophagy could also be involved. in an attempt to circumvent the resistance mechanism developed by cancer cells after prolonged administration of doxorubicin and address the issues of poor solubility, short lifetime and high toxicity of prodrug doxoform [118] , a second-generation, watersoluble prodrug of doxorubicin was developed by conjugation of the active drug with salicylamide by means of a mannich reaction [119] . doxorubicinesalicylamide conjugate doxaliform 96 (fig. 19 ) has a half-life of approximately one hour, and was more cytotoxic than doxorubicin against mcf-7 sensitive (4-fold) and mcf-7/adr resistant (10-fold) breast cancer cells. furthermore, doxaliform is amenable to functionalization with a view to provide a site for attachment of a releasable targeting group that might direct the conjugate to a specific receptor that is overexpressed by cancer cells. because many breast cancer cells overexpress estrogen receptor a, this receptor was chosen for targeting by doxasaliform having tethered a hydroxytamoxifen moiety, as in prototype 97 ( fig. 19 ) [120] . cytotoxicity of these candidates as a function of the length of the tether showed that a triethylene glycol unit provides a lead compound whose growth inhibition of four selected breast cancer lines (mcf-7, mcf-7/adr, mda-mb-231 and mda-mb-435) was enhanced up to 140-fold relative to doxorubicin. later work confirmed that uptake of hydroxytamoxifen-targeted doxorubicinesalicylamide conjugate is mediated by both the antiestrogen binding site and estrogen receptor [121] . also, several doxorubicineformaldehyde conjugates tethered to the nonsteroidal antiandrogen cyanonilutamide were designed, synthesized and evaluated as androgen receptor-targeted ligands for specific delivery of the conjugate to prostate cancer cells [122] . such a construct was later used in studies intended to evidence binding to androgen receptor in live pc3 prostate cancer cells and the subsequent translocation of the construct bound to the receptor to the nucleus, but the results were not very promising [123] . other efforts [124] were directed towards the conjugates of doxasaliform with the cyclic peptide neme-vrgdf (known as cilengitide), which is a potent antagonist of a v b 3 integrin involved in many cell-matrix recognition and cell adhesion phenomena, and plays an important role in angiogenesis and tumor metastasis. although the complete construct maintained a high affinity for a v b 3 integrin, the ic 50 for growth inhibition of mda-mb-435 cells was 2-fold greater than that of doxasaliform; the poor results have been tentatively blamed on limitation of drug delivery caused by the specific reduced abundance of receptors in this type of cell. eventually, because doxasaliform is not as active as prodrugs doxoform or doxazolidine, this line of research was terminated. however, the topic was later revisited by other authors, who reported the synthesis of constructs derived from either doxorubicinesalicylamide, daunorubicinesalicylamide or their 2-acyloxymethyl derivative, and amino-terminated poly(ethylene glycol), and their use for in vitro and in vivo studies [125] . these constructs presented cytotoxicities comparable to those of the parent drugs, and the lifetime of one of these constructs was determined to be longer than that of doxorubicin. also, the construct was more efficient than doxorubicin at reducing the weight of s-180 xenografted tumors [125] . cytotoxic mannich bases derived from miscellaneous, structurally unrelated substrates are grouped in the last paragraph of this section. 2-aminomethylated 9-alkyl-1,2,3,4-tetrahydrocarbazole1-ones 98 (fig. 20) show moderate to potent cytotoxicity towards a549 (human lung adenocarcinoma), sgc (human gastric cancer), k562 (human myelogenous leukemia), hct116 (human colorectal carcinoma), and kb-vcr (human oral cancer) cells using mtt assay [126] . one of candidates 98 (r ¼ c 2 h 5 , r 1 ¼ ch 3 ) was more cytotoxic than reference drug taxol against a549 cell line (ic 50 ¼ 70 nm), and at least one of its mechanism of action appears to be the inhibition of tubulin polymerization. a series of 3aminomethyl imidazo[2,1-b]benzothiazoles 99 (fig. 20) were evaluated for antiproliferative activity against hepatocellular carcinoma (hepg2), human breast (mcf-7) and human cervical (hela) cancer cell lines using mtt assay [127] . mannich bases 99 inhibited the proliferation of cancer cells at concentrations lower than 10 mm, arrested the cell cycle at g2/m phase while downregulating cyclin b and upregulating chk2 protein, and appeared to induce apoptosis based on the elevated levels of caspase-3. 6-cinnamoyl-benzoxazol-2-ones (r 1 ¼ h, ch 3 o) were the substrates used for the synthesis of mannich bases 100 (fig. 20) , which had high to moderate cytotoxicity (ic 50 between 5 and 40 mm) towards human pre-b-cell leukemia cell line bv-173 and chronic myeloid leukemia k-562, and appear to exert their cytotoxic action at least in part through induction of apoptosis [128] . replacement of the methoxy substituent in compounds 100 with chlorine led to a marginal improvement of ic 50 values [129] . a series of mannich bases 101 (r ¼ h, ch 3 ; x ¼ ch 2 , o, n-r 1 ) (fig. 20 ) of fused pyridazinone derivatives were synthesized and tested in vitro using srb assay to determine their growth inhibitory properties at a single 10 mm dose against sixty different human tumor cell lines. most of them showed no activity of all, but two candidates moderately inhibited the growth of non-small cell lung cancer (ekvx and hop-92) and glioblastoma (snb-75) cell lines [130] . an impressive number of articles dealing with the antibacterial activity of mannich bases have been published in the last decade. from a structural point of view, mannich bases reported in these studies are derived from nearly all of the major types of substrates capable of undergoing aminomethylation. these mannich bases have been evaluated against both gram-positive and gramnegative bacteria belonging to various families. the evaluation of the antibacterial activity was performed using different screening methods, and not always the standardized versions, and this complicates the interpretation and the comparison of results obtained in different studies. owing to the particular relevance of tuberculosis, the reports concerning the screening of mannich bases against mycobacterium species are presented in a separate section. ketonic mannich bases with antibacterial potential are at the heart of several studies within the period of time covered by this review. aminomethylation of a,b-unsaturated cyclic ketones with variable ring sizes afforded a library of compounds 102 (n ¼ 1e4) (fig. 21 ) derived from secondary cyclic aliphatic amines and having on the aromatic ring either no substituent at all, or a methyl group, or a variable number of methoxy groups [131] . evaluation of the antibacterial activity of these mannich bases against a panel of both gram-positive and gram-negative bacteria using the serial dilution method showed that the candidates derived from seven-or eightmembered a,b-unsaturated cyclic ketones were the least active of all, and that the nature of the amine moiety, or the number and the position of the methoxy groups on the aromatic ring, did not influence the antibacterial activity. none of the compounds were active against pseudomonas aeruginosa, and only a few were moderately active against escherichia coli. mannich bases 102 affected gram-positive bacteria (minimum inhibitory concentration (mic) < 12.5 mg/ml in most cases), but the candidates were less potent than reference antibiotics. another set of mannich bases 103 ( fig. 21 ) obtained from benzo-fused cyclic ketones (indanone, 1tetralone, benzosuberone) were tested under the same conditions against an extended panel of gram-positive and gram-negative bacteria [132] . in this collection, both the nature of the amine moiety and the alkoxy substituent r 1 influence the antibacterial activity, to a greater extent for gram-positive bacteria and to a lesser extent for gram-negative bacteria. indanone derivatives were active against both classes of bacteria (mic between 1.56 and 25 mg/ml), whereas tetralone and benzosuberone derivatives were mostly active against gram-positive bacteria (presumably due to the higher outer membrane permeability of these bacteria). also, a statistical comparison between the antibacterial activity of mannich bases 102 and mannich bases 103 showed that the latter were more active than the former [131, 132] . based on oxazolidinone antibacterials linezolid and eperezolid, a small series of mannich bases 104 (r ¼ nhcoch 3 or nhcsch 3 ) (fig. 21) derived from cyclohexanone or benzo-fused cyclic ketones was synthesized and evaluated against three selected gram-positive microorganisms using the standard serial dilution method [133] . all of the compounds having an acetamido group had low antibacterial activity, but the replacement of this group with thioacetamido restored the activity up to the level exhibited by the parent oxazolidinone antibacterial. further modification (r ¼ nhcsnh 2 ) produced a candidate whose antibacterial activity was superior to that of linezolid. 4,6-dimethoxybenzofuran-3(2h)-one double mannich bases 105 (fig. 21 ) had moderate to high antibacterial activity, as determined using disc diffusion method at a concentration of 0.5% in dmso [134] . the majority of compounds 105 were active against e. coli, and those derived from pyrrolidine, diethylamine and di-nbutylamine as amine reagents showed significant antibacterial activity (zone of inhibition 17e22 mm) towards most bacteria, comparable to that of reference drug gentamycin (zone of inhibition 18e24 mm). on the other hand, compound 105 derived from n-ethylmethylamine was inactive (no inhibition at concentrations higher than 100 mg/ml). also, structurally diverse types of mannich bases derived from acetophenones or 2-acetylthiophene, such as mono-mannich bases of type 8 (r ¼ ch 3 ) (fig. 3) , bis-mannich bases of type 13 (r ¼ ch 3 ) (fig. 4) , and piperidinols of type 14 (r ¼ ch 3 ) (fig. 4) , as well as their azine derivatives 16 (fig. 4) , are devoid of antibacterial activity against a wide range of human-and plant-pathogenic bacteria [135] . methiodide 106 (fig. 21 ) of dimethylamine mannich base of acetophenone was the sole active compound in this series, and only against staphylococcus aureus, but the diameter of the inhibition zone measured 9 mm (compared to 25 cm in the case of reference drug ofloxacin), and its mic was 500 mg/ml. the antibacterial activity of ketonic mannich bases generated from arylamines and aromatic aldehydes was also investigated by several groups. starting from cyclohexanone, a set of eleven compounds 107 was obtained, and screening against four common bacteria using serial dilution method showed that many of these candidates have excellent antibacterial activity, comparable to that of ceftriaxone [136] . however, the lack of rational design and systematic use of the aldehyde and amine components used to generate mannich bases 107 ( fig. 21 ) preclude any logical conclusions regarding sar in this collection. use of four differently substituted acetophenones, 4-fluorobenzaldehyde and several para-substituted anilines led to mannich bases 108 (fig. 21) , which were screened against four common bacteria [137] . most of these compounds were inactive, but candidate 108 (r 1 ¼ 2-br, r 2 ¼ f) was more potent than reference drug ampicillin against all four microorganisms, whereas another candidate 108 (r 1 ¼ 2-br, r 2 ¼ h) was more potent than ampicillin against e. coli and s. aureus. arylamine mannich bases 109 and 110 ( fig. 21 ) were obtained from 3-acetylcoumarine and 2-acetylbenzofuran, respectively, and evaluated against e. coli, s. aureus and p. aeruginosa using disc diffusion method [138] . since most compounds in this series are derivatives of 4-aminobenzoic acid, it has been expected that at least a few of these compounds have good antibacterial activity. three candidates 109 (r ¼ h, cl or f) had antibacterial activity comparable to that of reference drug streptomycin, but the rest were almost inactive. the series of candidates 110 provided only one remarkably active mannich base (r ¼ n(ch 3 ) 2 ), but the rest of the compounds with a benzofuran moiety are generally more active than coumarine-derived candidates 109. 3-(arylamino)-1-ferrocenyl-1-propanones 111 ( fig. 21) showed broad spectrum activity against both gram-positive and gram-negative bacteria, the highest degree of growth inhibition being obtained for s. aureus [139] . mic values varied between 0.2 and 12.5 mg/ml, making these compounds approximately 100-fold less active than reference drug tetracycline. because compounds 112 ( fig. 21) with a similar structure but with a phenyl ring instead of ferrocene have been reported to possess moderate to high antibacterial activity [140] , it appears that the replacement of phenyl in 112 with ferrocene could be responsible for the substantial decrease in activity recorded in the case of compounds 111. aminomethylated phenols have also been reported to have antibacterial activity. mannich bases 113 ( fig. 22 ) obtained from 4t-butylcatechol and secondary aliphatic amines have been screened for antibacterial activity against seven types of bacteria, but their potency was rather low to moderate [141] ; their corresponding copper(ii) complexes fared better, as two of them had mics comparable to that of chloramphenicol (12.5 mg/ml). the mannich reaction of a derivative of another dihydroxylic phenol with either secondary aliphatic amines or primary arylamines afforded compounds 114 (fig. 22 ), whose zones of inhibition for three common bacteria, determined at two concentrations (100 and 200 mg/ml) using disc diffusion method, were smaller than those observed for reference drug ofloxacin at 20 mg/ml [142] . because chemical modification of natural antibiotic novobiocin by means of aminomethylation was hypothesized to increase permeability through the outer membrane of gram-negative bacteria, phenolic mannich bases 115 (fig. 22) were designed, synthesized and evaluated against s. aureus (one of novobiocin's usual targets), francisella tularensis and e. coli [143] . candidates 115 were significantly less potent than novobiocin against these three microorganisms, the antibacterial activity of the most potent of these compounds being almost 100 times weaker than that of novobiocin. thirteen novel phenolic mannich bases 116 (fig. 22 ) derived from lawsone, benzaldehydes and primary aliphatic amines, together with their corresponding copper(ii) complexes, were tested for antibacterial activity against seven types of bacteria [144] . with a few exceptions, mannich bases 116 were generally more potent than the corresponding complexes, presumably due to the greater solubility of the pro-ligands. two mannich bases 116 had antibacterial activity comparable or higher to that of chloramphenicol against bacillus subtilis, e. coli and s. aureus, whereas the other compounds inhibited bacterial growth at concentrations greater than 200 mmol/l. antibacterial activity of fused oxazines obtained through the mannich reaction of phenols and naphthols with primary amines has also been examined. 6-acetyl-1,3-benzoxazines 117 ( fig. 22) were screened at 50 mg/ml against three common microorganisms using disc diffusion method, and they showed good growth inhibitory activity towards s. aureus, but only moderate potency against e. coli and b. subtilis [145] . the antibacterial activity was evaluated for 1,3-benzoxazines 118 ( fig. 22 ) substituted with chloro and methyl groups in the aromatic ring, bis(1,3-benzoxazines) 119 ( fig. 22) , naphtho[1,2-e]-1,3-oxazines 120 (fig. 22) , as well as naphtho[2,1-e]-1,3-oxazines 121 (fig. 22 ) using serial dilution method [146] . regardless of the aliphatic or aromatic nature of the moiety at the nitrogen atom, most compounds, and especially naphthoxazines 120 and 121, had mic values greater than 50 mg/ ml, but two benzoxazines 118 derived from 4-chlorophenol and aromatic amines were more potent than ampicillin against e. coli, whereas compound 119 derived from 2,4-dichlorophenol was equipotent to gentamycin against s. aureus. in addition, benzoxazines 118 and naphthoxazines 120 and 121 having benzazole moieties at the nitrogen atom were tested against four common types of bacteria [147] . although the majority of the compounds were inactive, the antibacterial activity of few candidates was 2-folde15-fold greater than that of reference drug tetracycline; the enhancement of activity for these candidates has been associated with the presence of a benzimidazole system at the nitrogen atom and at least one halogen in the phenolic starting material. in contradiction to the previously mentioned results, a number of naphtho[2,1-e]-1,3-oxazines 121 obtained from variously substituted arylamines were found to be quite active as antibacterial agents towards e. coli and b. subtilis [148] . these compounds were generally more active against the latter pathogen, and the candidates derived from 4-fluoroaniline, 4-ethoxyaniline and 2,4,6tribromoaniline were even more potent than reference drug streptomycin. the mannich reaction of phenols fused with heterocycles has also been investigated for the preparation of antibacterials. mannich bases of 8-hydroxyquinoline have been quaternized at the heterocyclic nitrogen atom with bromoacetic acid esters of superior alcohols (r ¼ n-c 12 h 25 , n-c 16 h 33 , n-c 18 h 37 ) to give compounds 122 (x ¼ o, ch 2 ) (fig. 22 ) [149] . the antibacterial activity of candidates 122, determined for three concentrations (1, 2.5 and 5 mg/ml) using disc diffusion method, showed that the compounds were active towards both gram-positive and gram-negative bacteria, but the presence in their structure of long alkyl chains rather than the aminomethyl function is most likely responsible for their activity. phenolic mannich bases 123 (r 1 ¼ h, br) ( fig. 22) were evaluated against e. coli and bacillus cirroflagellosus at 1 mg/ml using disc diffusion method, but their antibacterial activity was generally weak, with the exception of candidates derived from morpholine as amine reagent [150] . aminomethylated derivatives 41 (r ¼ oh, fig. 7 ) also manifested antibacterial activity in various degrees; the most active candidates were the mannich base derived from cyclohexylamine, which was as potent as tetracycline against b. subtilis (mic 3.9 mg/ml), and the mannich base derived from morpholine, which had the same antibacterial activity as ampicillin against s. aureus (mic 2 mg/ml) [58] . phenolic mannich bases derived from 3-hydroxy-4h-pyran-4ones received special attention as potential antibacterials. allomaltol (5-hydroxy-2-methyl-4h-pyran-4-one) was converted into mannich bases 124 (fig. 23) , whose antibacterial activity against four different gram-positive bacteria was only moderate (mic > 32 mg/ml) [151] . on the other hand, the antibacterial activity of mannich bases 125 ( fig. 21 ) derived from chlorokojic acid was superior to that of analogous 124, and the candidates were shown to possess a broad spectrum, while being more active against standard strains (mics between 1 and 32 mg/ml) than towards clinical, drug-resistant isolates [152] . there is no striking difference between the antibacterial activity of aminomethylated derivatives 125 having 4-benzylpiperazines and those having 4-substituted piperidines as amine moiety. the growth inhibition of grampositive bacteria (with the exception of enterococcus faecalis) occurred at mic values that were lower than those required for the growth inhibition of gram-negative bacteria. mannich base 125 with 1-(4-chlorobenzhydryl)piperazine as amine moiety seems to be best compound in this series, but its activity was consistently lower than that of any reference antibacterial drug. subsequently, a novel series of mannich bases 125 was synthesized, this time using various 4-(substituted aryl)piperazines as amine reagent, but the compounds in this collection were weaker antibacterials than analogous 125 derived from 4-benzylpiperazines [153] . screening of another novel set of compounds 125 with either 4benzylpiperazines of 4-arylpiperazines did not identify any compounds with improved or remarkable antibacterial activity, as the best candidates in this study had mic values between 4 and 16 mg/ ml [154] . however, when piperazinyl-substituted fluoroquinolones were employed as amine reagents in the mannich reaction of 3hydroxy-4h-pyran-4-ones as substrates, the resulting hybrids 126 (r ¼ cl) (fig. 23 ) had a broad spectrum and were highly active against a panel comprising both gram-negative and gram-positive bacteria [155] . generally, aminomethylated derivatives of chlorokojic acid (126, r ¼ cl) were more potent antibacterials than aminomethylated derivatives of kojic acid (126, r ¼ oh). mannich base 126 (r ¼ cl) derived from fluoroquinolone ciprofloxacin was the most potent candidate in the series. replacement of the quinolone scaffold in candidates 126 derived from norfloxacin with a naphthyridone moiety (as in mannich bases 126 derived from enoxacin) resulted in decreased antibacterial activity. among the bacteria used in this study, e. coli and klebsiella pneumoniae were the most sensitive microorganisms to hybrids 126. docking of the most active compound into topoisomerase ii dna-gyrase active site (which is the traditional target of quinolones) showed that mannich base 126 derived from ciprofloxacin binds in a manner similar to ciprofloxacin to the enzyme's active site, and that the 3hydroxypyran-4-one moiety is anchored with additional hydrogen bonding within the binding pocket, thus increasing the stability of the inhibitoreenzyme complex. mannich bases of isatin derivatives have also been investigated as antibacterial agents. aminomethylation of isatin semicarbazone with (hetero)arylamines afforded mannich bases 127 (fig. 24 ), whose antibacterial activity was moderate towards e. coli and good towards s. aureus [156] . in addition, a series of mannich bases 128 (r ¼ secondary aliphatic amines) (fig. 24 ) derived from a hydrazone of isatin incorporating the biologically relevant moieties of 1,3,5triazine, sulfa drugs and azacarbazole had moderate to good antibacterial activity against the previously mentioned microorganisms [157] . hydrazidones obtained from isatin and hydrazides of variously substituted acetic acids have also been aminomethylated with a view to synthesize antibacterial mannich bases. for example, mannich bases 129 24 ) derived from substituted isatins and morpholine, piperidine or 1-methylpiperazine generally showed poor antibacterial activity against the two aforementioned types of bacteria, with the exception of compound 129 (r ¼ 4-methylpiperazinyl, r 2 ¼ 5-br), whose inhibition determined by disc diffusion method was approximately half of that for reference drug gentamycin [158] . another collection of mannich bases 129 (r 1 ¼ 4-or 4,5-substituted 2-ethylphenyloxy) generated from substituted isatins as substrates and morpholine or piperidine as amine reagents exhibited weak to moderate antibacterial activity towards e. coli, s. aureus and shigella flemeri (zone of inhibition 12e20 mm) compared to norfloxacin (zone of inhibition 26e28 mm) [159] . schiff bases generated from isatin and various (hetero)aromatic amines represent another type of isatin-derived substrate that was subjected to aminomethylation to obtain potential antibacterial agents. the use of isatin schiff bases in the mannich reaction with 2-((2,6-dichlorophenyl)amino]) phenylacetic acid as the amine reagent yielded compounds 130 (r 1 ¼ substituted phenyl) (fig. 24) , which proved to be 3e12-fold less potent than reference drug ciprofloxacin against an array of bacteria [160] . when 3-chloro-4-fluoroaniline was used to generate schiff bases from isatin and 5-chloroisatin, aminomethylation with secondary aliphatic amines gave mannich bases 131 (fig. 24) ; these compounds had moderate to good activity against e. coli, s. aureus, p. aeruginosa and salmonella typhi in disc diffusion assay, and showed also moderate to good b-lactamase inhibitory activity [161] . schiff base of 5chloroisatin and trimethoprim was aminomethylated to give compounds 131 (r 1 ¼ 4-amino-5-(3,4,5-trimethoxybenzyl)pyrimidin-2-yl), but only mannich bases 131 derived from ciprofloxacin, lomefloxacin or gatifloxacin as amine reagents in the mannich reaction were at least as potent, if not more potent, than the corresponding parent fluoroquinolones against a large panel of bacteria [162] . antibacterial activity of mannich bases 131 (r 1 ¼ 5benzyl-3-thioxo-2h-1,2,4-triazol-4-yl) derived from schiff bases of isatin or 5-halogen-substituted isatins and an aminotriazolethione was generally good against four common microorganisms, as the size of the inhibition zones for some of the candidates (especially those derived from halogenated isatins and having piperidine or morpholine residues in the aminomethyl group) was comparable to the size of the inhibition zone for reference drug ciprofloxacin [163] . recently, mannich bases 132 ( fig. 24 ) obtained using schiff bases derived from 5-fluoroisatin and 4-arylideneaminoanilines as substrates and ciprofloxacin as amine reagent were shown to be generally less potent antibacterials than reference drug ciprofloxacin, although some candidates had mic values comparable to those of ciprofloxacin against the investigated bacteria [164] . a large number of papers published in the last decade report the antibacterial activity of mannich bases of 2,3-dihydro-1,2,4triazole-3-thiones. from a structural point of view, mannich bases from triazolethiones that were synthesized through aminomethylation in these reports fall into two major categories: 5substituted 2-aminomethyl-2h-1,2,4-triazole-3-thiones 133 and 5-substituted 2-aminomethyl-4-arylideneamino-2h-1,2,4triazole-3-thiones 134. the relevant information from these studies (i.e., substituents at position 4 and 5 of the triazole ring, amine reagents used in aminomethylation, microorganisms employed in the antibacterial evaluation) is presented in a condensed manner in table 1 . in the series of compounds 133a, the nature of the amine moiety in the aminomethyl group appears to be critical for the antibacterial activity, as mannich bases from piperazines were consistently more potent than mannich bases from arylamines, and some of them were even more potent than reference drug ampicillin [165] . a comparison between antibacterial activities of 133a and structurally related 134b suggests that the replacement of substituent at position 4 of the triazole ring in 133a with hydroxybenzylideneamino in 134b further enhances the antibacterial activity. although it was shown that substitution with chlorine of the phenyl at position 5 of the triazole ring improves the antibacterial activity in the series of compounds 133b and 133c, and that aminomethylation also increases the bacterial growth of mannich bases 133c compared to the parent triazolethione, no candidates with substantial antibacterial activity could be singled out from these two collections [166e168]. however, use of ciprofloxacin as amine reagent in the synthesis of mannich bases 133c and 133d led to a substantial increased antibacterial activity of these compounds, and some of them were as potent as reference drugs ciprofloxacin and vancomycin against several types of bacteria and methicillin-resistant s. aureus (mrsa), respectively [169] . mannich bases 133e or 134f having a diphenylsulfone moiety at position 5 of the triazole ring had only weak antibacterial activity [170] . a small number of mannich bases 133f, especially those having a halogen as substituent of the phenyl at n-4 of the triazole ring, inhibited the growth of gram-positive bacteria [171] . mannich bases 133g, especially candidates with morpholine, 4benzylpiperazine, n-methylpiperidine and trifluoromethylphenylpiperazine in the aminomethyl group, had very good antibacterial activity (mic 1.56e12 mg/ml) [172] , whereas analogous 133h having 2-furyl instead of 2-thiophenyl were less potent (inhibition zones of 6e13 mm, compared to 10e35 mm for ampicillin) [173] . the three mannich 133i bases reported in a study gave interesting results, as the candidates derived from 1methylpiperazine (inhibition zones of 23e30 mm for all bacteria) and 2-(4-morpholinyl)ethylamine (inhibition zones of 14e22 mm for some of the microorganisms under evaluation) were more potent than reference drug ampicillin, whereas the bis-mannich base 133i derived from piperazine was inactive [174] . however, further elaboration of 133i into compound 133p abolished the antibacterial activity [174] . mannich bases 133j, which feature a 3pyridinyl moiety at position 5 of the triazole ring, were generally poorer antibacterials than reference drug ampicillin, although there were isolated examples amongst these candidates that show more potency than ampicillin against certain bacteria [175] . furthermore, the replacement of 4-pyridinyl with 2-quinolinyl as substituent at position 5 of the triazole ring, and modification of phenyl into benzyl as substituent at position 4 of the triazole ring led to inactive mannich bases 133k [176] . both types of mannich bases 133l and 134l, featuring a 1,2,4-triazole moiety at position 5 of the triazolethione scaffold, showed good antibacterial activity, but compounds 134l were generally more potent than 133l, and two of candidates 134l actually had mic values comparable to those of reference drug ciprofloxacin [177] . mannich bases 133m and 133n (continued on next page) [165] were devoid of effective antibacterial activity [178, 179] , as was structurally related candidate 133o (a singular and notable exception is this compound's activity against bacillus cereus) [179] . mannich bases 133q were also inactive as antibacterials, except for a few candidates that were more potent against p. aeruginosa than reference drug ampicillin [180] . every member of the series of [197] h primary arylamines diphenylamine piperazines s. aureus s. pyogenes [198] mannich bases 134a was active against the tested bacteria, but not as potent as reference drug nitrofurazone [181] . in addition, the nature of substituent at position 5 of the triazole ring in the series of compounds 134a had no noticeable influence on their antibacterial activity, but the data (based on a single example) suggest that presence of chlorine in the aromatic ring of substituent x of the pyrazole moiety could improve the activity. mannich bases 134c were mostly active against s. aureus and b. subtilis, and candidates derived from ethyl piperidine-4-carboxylate were generally more potent than those derived from piperazines [182] . it should be noted that the presence of halogen in the structure of 134c (r 2 ¼ 2,6-difluorophenyl or 2,6-dichlorophenyl) did not enhance the antibacterial activity of these mannich bases. in contrast, substitution with halogen in compounds 134d (e.g., r 2 ¼ 2,4dichlorophenyl) led to the most potent candidates in the series, and their antibacterial activity was comparable to that of reference [203] drug ciprofloxacin [183] . use of 2,4-dichloro-5-fluorophenyl moiety at position 5 of the triazole ring in mannich bases 134e also produced candidates with good to excellent antibacterial activity [184] . compounds 134e having secondary aliphatic amines in the aminomethyl group were generally more potent than those having arylamines, even if the anilines used in aminomethylation were substituted with additional halogen atoms [184] . although mannich base 134g had no antibacterial activity, a close analogue of 134g such as compound 135 (fig. 25 ) was 1.5e3-fold more potent than ampicillin against most bacteria in the panel, with the exception of s. aureus [185] . replacement of 4-methylpiperazin-1yl with 4-morpholinyl in the aminomethyl group of mannich base 134g, and use of substituted phenyl other than 4-fluorophenyl as r 2 afforded structurally related candidates 134h with moderate antibacterial activity [186] . antibacterial activity of mannich bases 134i was moderate to good, but even the most potent compounds in this series (all of them having a 4-methylpiperazin-1-yl moiety in the aminomethyl group) were at least 4-fold less active than reference drug ciprofloxacin [187] . mannich bases 134j also had moderate to good antibacterial activity (inhibition zones of 12e22 mm), but were less potent than tetracycline (inhibition zones of 25 mm) [188] . overall antibacterial activity was reasonable in the series of mannich bases 134k, while a few compounds had mic values against various bacteria close to those determined for reference drugs [189] . a few mannich bases 134m were more potent against p. aeruginosa than reference drugs levofloxacin and amikacin, while most candidates exhibited broad, moderate to good antibacterial activity [190] . evaluation of mannich bases of 2,3-dihydro-1,2,4-triazole-3ones as antibacterial agents received little attention in recent literature. a small series of six mannich bases 136 (fig. 25 ) derived from 4-(substituted (hetero)arylidendeamino)-5-methyl-1,2,4triazole-2-ones was synthesized and tested against seven types of bacteria, and the antibacterial activity of the candidates ranged from moderate to excellent [191] . all compounds were more potent than reference drug ampicillin against e. coli and p. aeruginosa, and candidate 136 (r 1 ¼ 2-hydroxyphenyl, nr 2 ¼ 2-(4-morpholinyl) ethylamino) was at least as active as ampicillin against all types of bacteria used in the study, with the exception of s. aureus. out of mannich bases of type 137 (fig. 25) , only those derived from morpholine or piperidine were excellent, broad spectrum antibacterials, whereas other analogues were either inactive, or weak antibacterials, or moderately active against the tested bacteria [192] . a single mannich base 138 (fig. 25 ) of 1,2,4-triazole-3-ones having a 2-quinolinyl moiety as substituent at position 5 of the triazole ring was synthesized and evaluated for antibacterial activity, and its effect on e. coli and p. aeruginosa was comparable to that of ampicillin [176] . three mannich bases having a 3-pyridinyl moiety at position 5 of 1,2,4-triazole-3-one scaffold, analogous to compounds 133j derived from 1,2,4-triazole-3-thione, had moderate to good activity against most bacteria in the panel, but were not superior to reference drug ampicillin [175] . antibacterial activity of mannich bases 139 of 2,3-dihydro-1,3,4oxadiazole-2-thiones has been investigated extensively. the structural features of the compounds reported in these studies have been summarized in table 2 , along with the microorganisms used for screening. mannich bases 139a had moderate to good antibacterial activity (mic values between 6.25 and 25 mg/ml), but none was as potent as reference drug ciprofloxacin, and no definite sar could be inferred from the biological results [193] . the growth inhibition of mannich bases 139b appears to be modulated by the nature of the amine in the aminomethyl residue; thus, candidates derived from 3-trifluoromethylaniline were the most potent in this series and had antibacterial activity comparable by that of reference drug ciprofloxacin, followed in order by mannich bases 139b derived from morpholine, 2-chloro-5-methylaniline and 4-chloro-2-trifluoroacetylanline [194] . in the case of mannich bases 139c, candidates derived from secondary aromatic amines were inactive, whereas the remaining compounds were less active against gram negative bacteria, and more active against gram positive bacteria (mic values 2e8-fold lesser than those of reference drug ampicillin) [195] . both mannich bases 139d reported in this study [185] had moderate to good antibacterial activity, and both were more potent than ampicillin against e. coli and e. faecalis, but the candidate derived from 1-methylpiperazine was generally more potent than mannich base derived from 2-(4-morpholinyl)ethylamine. in the case of thiazole-containing mannich bases, antibacterial activity of compounds 139e was moderate (mic values between 16 and 62.5 mg/ml) [187] , whereas compounds two of compounds 139f emerged as effective antibacterials, especially against gramnegative bacteria, with mic values 2e4-fold lesser than those for reference drug chloramphenicol [196] . several mannich bases 139g (nr 2 ¼ nhc 6 h 4 f-4, nhc 6 h 4 cl-4, nhc 6 h 4 cf 3 -2, nhc 6 h 3 f 2 -2,5) had antibacterial activity comparable to that of reference drug penicillin, and, for some bacteria, even comparable to reference drug streptomycin [197] . mannich base 139h (nr 2 ¼ 1-piperazinyl) was equipotent to reference drug ciprofloxacin against s. aureus, but the antibacterial activity of the rest of candidates in this series was weaker than that of ciprofloxacin against the tested bacteria [198] . the compounds reported in a study, including mannich bases 139i, are claimed to exhibit moderate antibacterial activity, but lack of access to the original information (which is only provided in the online edition as supplemental material, and is missing) makes data assessment impossible [199] . phenylpiperazine-containing mannich base 139j was generally more potent that candidate 139j derived from ethyl isonipecotate, but both had moderate antibacterial activity compared to reference drug ampicillin [176] . three out of eight synthesized mannich bases 139k were screened for antibacterial activity, but their growth inhibition was only weak to moderate (inhibition zones of 12e26 mm) compared to that of reference drug chloramphenicol (inhibition zones of 37e44 mm) [200] . none of the 21 synthesized mannich bases 139l presented any antibacterial activity at the maximum concentration tested (250 mg/ml) [98] . mannich base 139m was the only compound with antibacterial activity in the structural diverse library evaluated in the study, but its effects were moderate at best [189] . screening results for mannich bases 139n were also disappointing, as they only barely inhibited the growth of e. coli at a concentration of 10 mg/ml [174] . out of two synthesized mannich bases 139o, the candidate having a morpholine residue in the aminomethyl group displayed excellent antimicrobial activity against all the tested microorganisms, while the candidate having a methylpiperazine residue had good or moderate activities against most bacteria in the panel, with the exception of e. coli and k. pneumoniae [190] . the growth inhibition exerted by mannich bases 139p was moderate compared to reference drugs, and it was uninfluenced by the nature of the amine moiety in the aminomethyl group [177] . nalidixic acid-based oxadiazolethione mannich bases 139q exhibited very weak antibacterial activity (mic values > 750 mg/ml) compared to reference drug streptomycin (mic ¼ 5 mg/ml), and only towards b. subtilis, but some of the structurally related mannich bases 140 of 2,3-dihydro-1,3,4-thiadiazole-2-thione ( fig. 25 ) were quite active (mic values as high as 6.25 mg/ml) against the microorganisms used in the study [203] . finally, mannich bases 141 having a 3,4dichlorophenyl moiety at position 5 of 2,3-dihydro-1,3,4oxadiazole-2-one ring (fig. 25) were inactive against s. aureus, e. coli, p. aeruginosa and bacillus megaterium [204] . two studies presented the synthesis and antibacterial evaluation of c-mannich bases 142 of thiazolidinones (fig. 26) having a thiazolyl-2-imino substituent at position 2 [205, 206] . while candidates 142 having alkyl or aryl groups as r 1 substituent at c-5 of thiazolidinone ring showed no antibacterial activity against a panel of eight bacteria, some of their analogues unsubstituted at c-5 (r 1 ¼ h) had weak antibacterial activity against s. aureus (mic values of 20e40 mg/ml). two of mannich bases 143 (r 1 ¼ 2-hoc 6 h 4 ) of thiazolidinones featuring a 6,8-dibromo-4oxoquinazolin-3-yl moiety (fig. 26 ) were 2-fold less potent against s. typhimurium (when nr 2 ¼ diethylamino) and 4-fold less potent against b. cereus (when nr 2 ¼ 1-piperidinyl) than reference drug ciprofloxacin [207] . antibacterial activity of mannich bases 144 (fig. 26 ) was found to be more potent (mic values between 6.25 and 100 mg/ml) than that of parent thiazolidinones, and the sar suggests that the nature of substituent r 1 influences significantly the ability of these compounds to inhibit the growth of various bacteria [208] . aminomethylation of pyrimidine-2-thiones and pyrimidine-2ones, substrates that can be easily obtained in a considerable structural variety through a simple biginelli reaction, has also been investigated in relation with the antibacterial activity of the resulting mannich bases. thus, pyrimidine-2-thione mannich base 145 (fig. 26 ) was obtained using natural alkaloid cytosine as amine reagent in aminomethylation, and its evaluation as antibacterial agent showed that it is a potent growth inhibitor of s. aureus and b. subtilis, and it has weak activity against p. aeruginosa and e. coli [209] . double mannich bases 146 (fig. 26 ) of pyrimidine-2-thiones (x ¼ s) were good antibacterial agents (zones of inhibition of 96 to 88% of the zone of inhibition for reference drug streptomycin) against e. coli and b. subtilis [210] , and analogous double mannich bases 146 of pyrimidine-2-ones (x ¼ o) were even more potent, with the exception of the candidate having a 4-methoxyphenyl moiety at position 4 of the pyrimidine ring (r ¼ 4-och 3 , r 1 ¼ h) [211] . taking advantage of the lability of the proton located at the nitrogen atom in the amide function, several n-mannich bases 147 of 5-chloro-2-methoxybenzamide ( fig. 27 ) obtained from either sulfonamides or common secondary amines as amine reagents have been synthesized and evaluated for antibacterial activity against a panel of bacteria [212] . candidates 147 derived from sulfonamides were more potent than the parent compounds, whereas mannich base 147 derived from morpholine was an antibacterial agent with broad spectrum and good activity. n-mannich bases 148 of glutarimides (r 1 ¼ h, c-c 5 h 9 , c-c 6 h 11 , 4-clc 6 h 4 ) (fig. 27 ) have been screened for antibacterial activity, and claimed to be potent very potent, although no comparison to currently-in-use antibacterials was available [213, 214] . compounds 148 derived from sulfonamides showed improved growth inhibition activity of the tested microorganisms over the corresponding parent amines, whereas analogues generated from secondary aliphatic amines were generally equipotent to those obtained from sulfonamides [213] . also, n-mannich bases 149 of succinimide (r 1 ¼ h or c 6 h 5 , nr 2 ¼ nhc 6 h 5 , 2-pyridinyl) (fig. 27 ) and an n-mannich base from phthalimide were weak to moderate antibacterials against e. coli, s. typhi and b. subtilis [215] . well-known antibiotics have also been subjected to aminomethylation with a hope of increasing their antibacterial activity or improving their bioavailability. hybrids 150 of tetracycline and sulfonamides ( fig. 28 ) have been generated using the mannich reaction, and their screening against salmonella enteritidis and pasturella multocida showed that their antibacterial activity is higher than that of the parent sulfonamides; unfortunately, no comparison with the activity of tetracycline or a reference drug is provided in the study [216] . aminomethylation is one of the modifications that have also been explored in the case of vancomycin. after an n-decylaminoethyl moiety was introduced into the structure of vancomycin with a view to restore antibacterial activity against vancomycin-resistant enterococci while retaining potency against mrsa, grafting a hydrophilic group has been considered as a way to reduce the overall lipophilicity of the n-decylaminoethylmodified vancomycin and to restore the initial favorable distribution properties of vancomycin. several types of amine reagents (amino acids, amino sugars, phosphonic acids) have been considered for the synthesis of mannich bases 151 (fig. 28) , and some of these modifications led to candidates active against vancomycinresistant bacteria, and ultimately to the discovery of telavancin (td-6424) as a lead compound with improved absorption, distribution, metabolism and excretion (adme) properties over ndecylaminoethylvancomycin [217] . (fig. 28 ) was subsequently screened against a large number of predominantly gram-positive isolates with developed antibacterial resistance, and showed to be highly active against methicillin-resistant staphylococci (mic 90 0.5e1 mg/ml), streptococci (mic values 0.12 mg/ml), and vanb-type enterococci (mic values 2 mg/ml) [218] . with respect to its mechanism of action, telavancin inhibited late-stage peptidoglycan biosynthesis in a substrate-dependent fashion, bound to cell wall with high affinity, and perturbed bacterial cell membrane potential and permeability [219] . aminomethylation of vancomycin has also been used as the first step in the construction of glycopeptide/b-lactam heterodimers via linkage of aminomethylated vancomycin and various cephalosporin cores through an adipic acid moiety [220] . these heterodimers were all found to exhibit excellent potency against a range of important gram-positive bacteria, and a subset of these candidates also demonstrated rapid bactericidal activity against mrsa, whereas two of the most attractive compounds have been shown to exhibit in vivo efficacy 40 fold greater than that of vancomycin. finally, a patent claims a series of mannich bases of vancomycin as compounds suitable for oral delivery and having enhanced antibacterial potency [221] . antibacterial activity of mannich bases derived from miscellaneous substrates is covered in the last paragraph of this section. mannich bases 152 (r ¼ ch 3 , oc 2 h 5 ) (fig. 29 ) derived from either ethyl acetoacetate or diethyl malonate as substrates, variously substituted benzaldehydes as aldehyde components and benzyl carbamate as amine reagent had poor to moderate activity against five types of common bacteria [222] . hydrazidones derived from isoniazid and 2-alkoxybenzaldehydes have been aminomethylated at position ortho to the alkoxy group to give mannich bases 153 (fig. 29) . a few members of this collection having an ethoxy group demonstrated at concentration of 100 mg/ml antibacterial activity superior to that of reference drug amoxicillin at 50 mg/ml [223] , whereas candidates having a propoxy group were less potent [224] . mannich bases 154 derived from 7-methyl-2-(substituted aryl) imidazo[1,2-a]pyridines (fig. 29 ) and having two halogens as substituents in the phenyl ring at position 2 of the fused heterocyclic system were the most potent in this series against e. coli, p. aeruginosa, s. aureus and streptococcus pyogenes [225] . mannich bases 155 derived from an imidazo[2,1-b]-1,3,4-thiadiazole ring system (fig. 29) showed weak activity against gram-positive bacteria and moderate activity against vibrio cholera, but their growth inhibition activity of e. coli was comparable to reference drug norfloxacin [226] . evaluation of the antibacterial activity of mannich bases derived from 2-alkyl-3-hydroxy-pyridine-4(1h)-ones showed that only one candidate 156 (fig. 29 ) had moderate activity against s. enteritidis (16 mg/ml) and s. aureus (16 mg/ml) compared to reference drug ciprofloxacin (1 mg/ml and 0.5 mg/ml, respectively, for the mentioned microorganisms) [227] . mono-mannich bases 157 (r 1 ¼ ch 3 ) and double mannich bases 158 generated from sydnones ( fig. 29) were both found to inhibit moderately the growth of four standard microorganisms, regardless of the nature of the amine reagent used in aminomethylation, but no useful conclusions regarding sar in this series could be drawn for further development [228] . also, most mannich bases 157 (r 1 ¼ och 3 ) had weak to moderate activity against both gram-positive and gramnegative bacteria, with the exception of candidate 157 (r 1 ¼ och 3 , nr 2 ¼ 4-nitrobenzothiazole-2-ylamino), which was as potent as reference drugs ciprofloxacin and ampicillin against gram-positive bacteria [229] . in contrast, aminomethylation of another sydnone derivative using various primary and secondary aliphatic amines was reported to occur in the at n-1 in the pyrazoline ring to give mannich bases 159 instead (fig. 29) , which had good antibacterial activity compared to reference drug ampicillin against four common bacteria, and particularly against b. subtilis [230] . the copper(ii) complex of pyrazole c-mannich base 160 (fig. 29 ) inhibited the growth of b. subtilis at concentrations as low as 1.25 mm, most likely by promoting mutagenesis and inducing cell death [231] . screening of a few of pyrazolone n-mannich bases 161 (fig. 29 ) against various bacteria resulted in inhibition zones that were comparable to those of reference drug ciprofloxacin, but the mic values for these candidates were ultimately far greater from those of ciprofloxacin [232] . some of the acetylenic mannich bases 162 (r 1 ¼ c 6 h 5 , 4-clc 6 h 4 ; r 2 ¼ c 6 h 5 , 1-c 10 h 7 ) (fig. 29 ) had antibacterial activity comparable to reference drug ofloxacin against s. aureus, fewer showed good activity against e. coli, and only one candidate was equipotent to ofloxacin against p. aeruginosa [233] . various substrates (e.g., benzimidazole, 3methylpyrazole-5(4h)-one, succinimide, phthalimide or naphthalimide) were aminomethylated using norfloxacin as amine reagent, and some of the corresponding mannich bases were more potent than the parent fluoroquinolone, especially against e. coli and p. aeruginosa [234] . sulfonamides and primary or secondary aliphatic amines were employed as amine reagents in aminomethylation of isoniazid to give mannich bases 163 (fig. 29) , and some of the candidates had antibacterial activity that was superior to that of the parent sulfonamides, whereas mannich base 163 (nr 2 ¼ nhch 3 ) was the most potent compound in the series against s. aureus and b. anthracis [235] . use of hydantoins as substrates in the mannich reaction led to 3-aminomethylated derivatives 164 or to 1-aminomethylated derivatives 165 (fig. 29) , depending on the substituent on n-3 of imidazole ring, and some of these mannich bases had moderate to good antibacterial activity against four common bacteria [236] . aminomethylation and aminoalkylation of saccharin using various types of amine reagents led to mannich bases 166 ( fig. 29 ) with moderate to excellent antibacterial activity against s. aureus (zones of inhibition between 12 and 50 mm, compared to 31 mm for reference drug chloramphenicol) [237] . a note reports in a concise manner the synthesis and antibacterial activity of mannich bases 167 (fig. 29 ) derived from a phenothiazine and primary aromatic amines, and some of the compounds in this study were potent and had a broad spectrum against the tested bacteria [238] . mono-mannich base 168 of quinoxaline-2,3(1h,4h)-dione (fig. 29 ) was screened at concentration of 100 mg/disc, and was found to be more potent than reference drug nalidixic acid at concentration of 50 mg/disc against four common bacteria, with the exception of proteus vulgaris [239] . moderate to good antibacterial activity was determined for mannich bases 169 of 3-aryl-2-thioxo-2,3-dihydroquinazolin-4(1h)one (fig. 29 ), but none of the candidates was as potent as reference drug amikacin against the four common bacteria used in this study [240] . in addition to their anticancer activity evaluation, p-mannich bases 83 (fig. 15 ) were also screened for antibacterial activity, and the mic values were generally approximately 10 mg/ml, regardless of candidate's structure and type of bacteria, but no comparison with reference drugs was provided in the study [106] . a paper claiming the synthesis of s-mannich bases 170 of 4,6-diaryl-2mercaptopyrimidine and secondary aliphatic amines (fig. 29 ) also reports their antibacterial activity weak to moderate, as the most potent candidates are 3-to 7-fold less potent than reference drug ciprofloxacin against four types of bacteria [241] . tuberculosis is a chronic disease whose magnitude and gravity can be gleaned from a 2010 study by world health organization stating that in 2009 alone, 9.4 million new cases of tuberculosis were reported, and 1.7 million deaths were caused by tuberculosis, out of which 0.38 million people were infected with both human immunodeficiency virus and mycobacterium [242] . continuous rise of the number of reported cases of drug-resistant and latent tuberculosis is also alarming and calls for urgent discovery of novel classes of compounds with antimycobacterial activity capable of overcoming the resistance of mycobacterium strains to current drugs. mannich bases are among these novel classes of antimycobacterial compounds that have recently caught the attention of researchers in the field. several examples of papers dealing with antitubercular ketonic mannich bases are available. thus, dimethylamine mannich bases 171 of 2-benzylidenecyclohexanones ( fig. 30 ) were reported to have mic values between 0.39 mg/ml and more than 12.5 mg/ml against mycobacterium tuberculosis h 37 rv (for candidate 171 (n ¼ 0, r 1 ¼ r 2 ¼ h) and candidate 171 (n ¼ 1, r 1 ¼ f, r 2 ¼ h), respectively), which is approximately 20e25% of the efficiency of reference drug rifampin against this microorganism [243] . in addition, the ability of the same candidates to inhibit the growth of mycobacterium avium at concentration of 12.5 mg/ml ranged from 0 to 87%. however, no significant correlations between the structural patterns of mannich bases 171 (e.g., presence or absence of the cinnamoyl double bond, substitution pattern in the phenyl ring, electronic, hydrophobic or steric properties of the substituents in the aromatic ring) and their antimycobacterial activity could be established. mannich base 171 (n ¼ 0, r 1 ¼ och 3 , r 2 ¼ h) emerged from this series as a viable hit compound, having a mic value of 0.78 mg/ml and an ic 50 value for vero cells of 16.4 mg/ml. this compound presents therefore a greater toxicity towards mycobacterium over normal mammalian cells, which has been tentatively explained by its ability to affect respiration in isolated rat liver mitochondria, and had mic values against several drug-resistant strains of mycobacterium that were similar or identical to the values determined for the h 37 rv strain [244] . isocitrate lyase is an enzyme in the glyoxylate cycle that catalyzes the cleavage of isocitrate to succinate and glyoxylate, and, together with malate synthase, bypasses the two decarboxylation steps of the tricarboxylic acid cycle [245] . glyoxylate cycle is essential for the survival of pathogens, including m. tuberculosis, inside the host [246] , and isocitrate lyase has been identified only in bacteria, fungi, and plants, but no analogous enzyme has been documented in humans. therefore, isocitrate lyase has been pursued as a promising target for the development of novel antimycobacterial agents [247] . a high-throughput screening of a large library of ketonic mannich bases against this enzyme led to the identification of candidate 172 (fig. 30 ) with a potent inhibitory activity (ic 50 ¼ 53.5 mg/ml), but this compound's activity against m. tuberculosis has not been actually evaluated [248] . phenolic mannich bases 173 ( fig. 30 ) derived from the hydrazone generated from isoniazid and 2 0 -hydroxyacetophenone were screened against m. tuberculosis h 37 rv, and found to inhibit its growth with mic values ranging from 0.56 to 4.61 mm [249] . six compounds in this series were more potent than isoniazid (mic ¼ 2.04 mm), and the most potent candidate 173 (nr 2 ¼ 1piperazinyl) was shown to be equipotent to isoniazid in decreasing bacterial load in lungs of infected mice at a dose of 25 mg/kg. anti-mycobacterium activity of novobiocin mannich bases 115 (fig. 22) was evaluated, and two of these candidates (nr 2 ¼ n-acetoxyacetyl-n-methyl and n-acetaminoacetyl-nmethyl) showed an increased potency over novobiocin, although no comparison to established antimycobacterial drugs was provided [143] . phenolic mannich bases 174 derived from allomaltol and piperazines (fig. 30 ) had good activity against mycobacterium smegmatis, with mic values between 4 and 16 mg/ml [250] . although the growth inhibition of mycobacterium by the candidates in the library was moderate to good (mic values between 4 and 128 mg/ml), mannich bases derived from 1-phenylpiperazine and its analogues substituted with halogens in the aromatic ring were among the most potent compounds in the series. departure from substitution of piperazine in candidates 174 with bulky, aromatic rings directly linked at n-4 generally led to decreased potency against mycobacterium, although some substituents (such as tbutoxycarbonyl, ethoxycarbonyl, 2-hydroxyethyl, cyclohexyl, benzoyl, furfuryl or 2-(dimethylamino)ethyl) appear to be tolerated better than others (e.g., isopropyl, allyl, 2-methoxyethyl, 2ethoxyethyl, phenethyl or benzyl). an extension of this study, aimed at evaluating against m. tuberculosis h 37 rv the library of mannich bases of type 124 (fig. 23 ) obtained by replacement of piperazines in compounds 174 with piperidines, led to valuable insight on the sar for this particular type of compounds [251] . inspection of the sar in the subset of mannich bases 124 suggested that the position of the substituents and, to a lesser extent, the nature of these substituents plays an important role in their antimycobacterial activity, as the most potent candidates in this subset are generally those substituted at position 4 of piperidine ring [251] . replacement of allomaltol with chlorokojic acid as substrate afforded piperazine mannich bases 125 (r ¼ 4-substituted piperazin-1-yl) (fig. 23) , analogous to both 124 and 174 and having comparable antimycobacterial activity (mic values between 4 and many papers reporting the antimycobacterial activity of mannich bases of pyrrole derivatives have been published in the last decade. the intensive research in this field stems from the discovery by italian researchers at universit a "la sapienza" in rome of bm212 175 (fig. 31) as a potent agent, not only against several collection strains and clinical isolates of m. tuberculosis, but also against non-tuberculosis mycobacterial strains and drug-resistant mycobacteria, with mic values that are comparable to those of reference drugs isoniazid and streptomycin [252] . although the quest for a more potent antimycobacterial agent through systematic modification of lead compound bm212 was not successful at first, it provided experimental proofs for the importance of the presence and nature of substituents at positions 1 and 5 of pyrrole ring [253] . subsequently, novel pyrrole mannich bases 176 analogous to bm212 (fig. 31) , derived from 1-methylpiperazine but also from thiomorpholine as amine reagents, and having either chlorine [254] or chlorine and fluorine [255] as substituents of the phenyl rings in positions 1 and 5 of pyrrole, have been synthesized. two candidates 176 (r 1 ¼ f, r 2 ¼ h or r 1 ¼ h, r 2 ¼ f) showed an improved activity (mic values of 0.4 and 0.5 mg/ml) against m. tuberculosis over mannich base 175 (mic ¼ 0.7 mg/ml) [255] . investigation of structureeantimycobacterial activity relationship in analogues of 175 and 176 was further pursued with a view to optimize the structure of these candidates. thus, synthesis of novel mannich bases of pyrrole using iterative introduction of more lipophilic 1-naphthyl or 2-fluoro-or 2-chlorophenyl moieties at either one of positions 1 and 5 in pyrrole ring of these lead compounds [256] , or a recombination through shuffling of any of the substituents employed so far on the phenyl rings at these two positions of the pyrrole ring [257] was undertaken. these studies confirmed the superior antimycobacterial activity of mannich bases containing a thiomorpholinyl moiety compared to the activity of the corresponding analogues with a 4-methylpiperazinyl moiety, evidenced the importance of fluorine substitution (especially at position 2 of the phenyl ring) for antimycobacterial activity, and proved the decrease of activity with the introduction of 1-naphthyl moieties in the structure of lead compounds 175 and 176. unfortunately, no novel candidates, more potent than mannich bases 175 and 176, have been identified during these investigations. however, replacement of one halogen in the structure of lead compound 175 with more lipophilic substituents (methyl, ethyl n-propyl, i-propyl) afforded candidates with improved antimycobacterial activity (mic values between 0.125 and 0.5 mg/ml) over 175 and even over reference drug streptomycin [258, 259] . in order to further increase the lipophilicity, replacement of the methyl group at position 2 of the pyrrole ring with ethyl was also examined, but these novel mannich bases 177 (x ¼ s or nch 3 ) (fig. 31) were generally less active against several m. tuberculosis strains than their methylsubstituted analogues, although their cytotoxicity towards normal cells was lower than that of the methyl-substituted counterparts [260] . further exploration of the relationship between the nature of substituents on phenyl rings at positions 1 and 5 of pyrrole ring and the antimycobacterial activity of pyrrole mannich bases led to the identification of a novel lead compound 178 (fig. 31) with a very high activity toward both m. tuberculosis 103471 and h 37 rv strains (mic ¼ 0.125 mg/ml) [261] . antimycobacterial activity of mannich base 178 was comparable to that of reference drugs streptomycin or rifampin, while the candidate demonstrated low cytotoxicity towards normal cells (selectivity index ic 50 /mic > 1000) [261] . subsequently a series of morpholine derivatives were designed and synthesized with a view to lower the clearance rate in mouse microsomal fractions associated with the presence of thiomorpholine [262] . although the replacement of thiomorpholine with morpholine generally resulted in a decrease in potency, candidate 179 (fig. 31 ) was found to be equipotent to any of the lead compounds, showed improved microsomal clearance and lower cytotoxicity to normal cells, and was ultimately selected for in vivo pharmacokinetic and efficacy studies in an acute murine tuberculosis infection model. mannich base 179 had an efficacious dose that results in a 99% colony-forming unit reduction in the lung of 49 mg/kg, which is within the range of commonly employed tuberculosis drugs. a recent study showed that mutations in the mmpl3 gene in mycobacterium strains are responsible for resistance to bm212, and suggested that products of this gene are cellular targets for bm212, which makes mmpl3, a member of the mmpl (mycobacterial membrane protein, large) family, a new potential druggable target for the treatment of tuberculosis [263] . point mutations in mmpl3 gene that confer resistance to bm212 and other analogous pyrrole mannich bases have been later identified in several mycobacterium mutants [262] . a large number of antimycobacterial pyrrole mannich bases were also employed to obtain a final multiprobe 3-d qsar model, which was shown to offer good predictions for antimycobacterial activity of an external, unrelated test set of compounds [264] . accounts at various stages of the endeavors concerning the synthesis of pyrrole mannich bases as antimycobacterial agents, along with the related biological results obtained by the italian researchers, have also made the object of several author's reviews along the years [265e268]. a patent claiming the antimycobacterial activity of pyrrole mannich bases derived from 1-arylpiperazines is also worth mentioning [269] . on the other hand, attempts to make a more drastic departure from these already established structural features of antimycobacterial pyrrole mannich bases (such as the introduction of an oxazolidinone moiety reminding of antibacterial linezolid) led to a series of compounds 180 (x ¼ acetamido of 4-(hetero)aryl-1,2,3-triazol-1yl) (fig. 31 ) that were at best 15e65-fold less potent than mannich base 178 [270] . in addition, pyrrole mannich bases 181 (fig. 31 ) were shown to act as inhibitors (ic 50 values ranging from 1.5 to 15 mm) of mycobacterium protein tyrosine phosphatase b, an enzyme that is essential for the survival of bacteria in the host, but no in vitro evaluation using mycobacterium strains was performed [271] . mannich bases of isatin and its derivatives have been evaluated as antimycobacterial agents as well. several isatin mannich bases 182 (fig. 32) containing a semicarbazide moiety were equipotent (mic ¼ 0.625 mg/ml) to isoniazid against m. tuberculosis, and two of the candidates were more potent in vitro than either isoniazid or rifampicin against a multidrug-resistant mycobacterium strain (mic ¼ 6.25 mg/ml) [272] . schiff bases obtained from 5-substituted isatins (r 1 ¼ f, cl, f) and lamivudine were aminomethylated using fluoroquinolones (r 2 ¼ ethyl, cyclopropyl; r 3 ¼ h, ch 3 ) as amine reagents, and the resulting mannich bases 183 (fig. 32) showed 92e100% growth inhibition of m. tuberculosis h 37 rv at a concentration of 6.25 mg/ml; inhibition for one selected schiff base was 82%, and lamivudine alone did not inhibit the growth of this mycobacterium strain at the given concentration [273] . several morpholine mannich bases 184 of 4-substituted thiosemicarbazones of 5-trifluoromethoxyisatin (r ¼ c-c 6 h 11 , 4-fc 6 h 4 , 4-clc 6 h 4 , 4-brc 6 h 4 ) (fig. 32) were potent antimycobacterial agents (mic values between 3 and 5 mg/ml) [274] . replacement of the trifluoromethoxy group in 184 with nitro led to a slight decrease in potency, which became more drastic when fluorine replaced the trifluoromethoxy group, whereas the substitution of morpholine with piperidine generally decreased the antimycobacterial activity of the candidates [275] . using fluoroquinolone gatifloxacin as amine reagent, sixteen mannich bases derived from isatins, isatin semicarbazones, isatin thiosemicarbazones, isatin hydrazones with isoniazid, and isatin schiff bases with sulfadiazine were prepared and tested for antimycobacterial activity [276] . mic values recorded for 90% growth inhibition of mycobacterium by these compounds ranged from 0.78 mg/ml to 12.5 ng/ml, whereas mic values between 0.78 and 0.1 mg/ml were determined for multidrug-resistant strains. promising results were obtained when the most potent compound in this series was tested in a murine model at a dose of 50 mg/kg, and the analysis of data for inhibition of m. tuberculosis dna gyrase suggested that this enzyme could be the target of these isatinefluoroquinolones hybrids. good results were obtained from the evaluation as antimycobacterial agents of mannich bases obtained from 5-substituted isatins and their semicarbazones and thiosemicarbazones as substrates and ciprofloxacin as amine reagent (mic values between 1.2 and 10.3 nm) [277] , and mannich bases generated from 5-substituted isatins, the corresponding semicarbazones, thiosemicarbazones and oxime ethers using 8methoxyciprofloxacin as amine reagent had comparable potencies [278] . analogous mannich bases derived from moxifloxacin as amine reagent were also potent (mic value of 1 mg/ml for m. tuberculosis, and between 4 and 16 mg/ml for multidrugresistant mycobacterium strains), but all of these isatinemoxifloxacin conjugates were generally less potent antimycobacterials than parent fluoroquinolone [279] . evaluation as antimycobacterial agents of aminomethylated 2,3dihydro-1,3,4-oxadiazole-2-ones, 2,3-dihydro-1,3,4-oxadiazole-2thiones and 2,3-dihydro-1,2,4-triazole-3-thiones has led to interesting results. a direct comparison between mannich bases 185 (x ¼ o) of 2,3-dihydro-1,3,4-oxadiazole-2-one and mannich bases 185 (x ¼ s) of 2,3-dihydro-1,3,4-oxadiazole-2-thione (fig. 33) , having both a 4-pyridinyl substituent at position 5 of the azole ring and derived from cyclic secondary aliphatic amines, showed that the former were significantly more potent against an isoniazidsensitive m. tuberculosis h 37 rv strain than the latter, thus suggesting that the presence of oxygen is crucial for the antimycobacterial activity of this type of compounds [280] . in a continuation of the initial study, the search for better antimycobacterial agents was restricted to mannich bases of 2,3dihydro-1,3,4-oxadiazole-2-ones, while the variety of amine reagents employed in aminomethylation was expanded to include n-(substituted benzyl)methylamines, a structural modification that preserved the good activity of the candidates (mic values of 4e8 mg/ml) [281] . however, replacement of the 4-pyridinyl substituent at position 5 of mannich bases 185 (x ¼ o) with phenyl, 3pyridinyl of pyrazinyl resulted in a significantly decreased antimycobacterial activity, whereas replacement with 2-pyridinyl did not generally affect the activity of the candidates. bis-mannich bases 186 (fig. 33 ) derived from dapsone as amine reagent, benzaldehyde and furan-2-carboxaldehyde as aldehyde reagents and variously 5-substituted 1,3,4-oxadiazole-2-thiones as substrates had excellent antimycobacterial activities against both isoniazidsensitive and isoniazid-resistant strains of m. tuberculosis, as they were 5-fold more potent than isoniazid against the sensitive strain and 10-fold more potent than isoniazid against the resistant strain [282] . a study allowed another direct comparison, this time between mannich bases 139e of 2,3-dihydro-1,3,4-oxadiazole-2thiones ( table 2) and mannich bases 134i (table 1 ) of 2,3dihydro-1,2,4-triazole-3-thiones having the same 4isopropylthiazole-2-yl substituent at position 5 of the aforementioned azoles. some of candidates 134i were 2-to 4-fold more potent than the most potent mannich base 139e against m. tuberculosis h 37 rv strain, but they were still 16-fold less potent than isoniazid [187] . on the other hand, mannich bases 187 (r 1 ¼ h, oh; r 2 ¼ c 2 h 5 , ch 2 ch]ch 2 , c 6 h 5 , 4-brc 6 h 4 ; r 3 ¼ ch 2 c 6 h 5 , c 6 h 5 , substituted phenyl, 2-pyrimidinyl) derived from 1,2,4-triazole-3thiones (fig. 33 ) had low antimycobacterial activity (mic values between 25 and 100 mg/ml) [283] . the majority of mannich bases derived from either 2,3-dihydro-1,2,4-triazole-3-thione or 2,3dihydro-1,2,4-triazole-3-one having a 3-pyridinyl moiety at position 5 of the triazole ring were more potent antimycobacterials (mic between 16 and 32 mg/ml) than reference drug streptomycin against m. smegmatis [175] . in addition, 2,3-dihydro-1,2,4-triazole-3-one mannich base 137 (nr 2 ¼ 1-piperidinyl) (fig. 25 ) was equipotent to reference drug streptomycin (mic ¼ 4 mg/ml) against m. smegmatis [192] . mannich bases 188 of imidazo[2,1-b]-1,3,4-thiadiazole derivatives ( fig. 34 ) have been screened for antimycobacterial activity. evaluation of a library of mannich bases 188 (r 1 ¼ c-c 6 h 11 , 2-furyl, 2-thienyl; r 2 ¼ h, br; nr 2 ¼ 1-pyrrolidinyl, 1-piperidinyl, 4morpholinyl) against m. tuberculosis h 37 rv showed that all of the candidates had mic >6.25 mg/ml [284] , but a second collection of aminomethylated imidazo[2,1-b]-1,3,4-thiadiazole derivatives 188 (r 1 ¼ n-c 3 h 7 , c-c 6 h 11 , 2-thienyl; r 2 ¼ cl, ch 3 , och 3 ; nr 2 ¼ 1-pyrrolidinyl, 1-piperidinyl, 4-morpholinyl) consisted of mannich bases with good antimycobacterial activity (52e99% growth inhibition) at a concentration of 6.25 mg/ml [285] . amongst mannich bases 188 (r 1 ¼ 4-ch 3 oc 6 h 4 ch 2 ), pyrrolidine-containing derivatives were consistently the best agents against m. tuberculosis h 37 rv [286] . also, compounds 155 (nr 2 ¼ 1-pyrrolidinyl, 1-piperidinyl, 4morpholinyl) (fig. 29 ) had moderate antimycobacterial activity (mic ¼ 25 mg/ml) [226] . in addition to the types of substrates presented so far, miscellaneous other substrates have been subjected to the mannich reaction with a view to synthesizing compounds with antimycobacterial activity. aminomethylation of the amide function in tetracyclines using fluoroquinolones as amine reagents afforded mannich bases 189 (fig. 34) with excellent activity against m. tuberculosis (mic values between 0.2 and 1.56 mg/ml) [287] . pyrazinamide was also aminomethylated at the amide function using piperazines (including four fluoroquinolones) as amine reagents; the resulting mannich bases 190 (fig. 34) were at least as potent as reference drug pyrazinamide against m. tuberculosis, and showed a significantly improved antimycobacterial activity over pyrazinamide against multidrug-resistant mycobacterium strain [288] . anti-hiv drug efavirenz was aminomethylated using either common piperazines or piperazine-containing fluoroquinolones as amine reagents to afford hybrids 191 (fig. 34) , but only the mannich bases derived from fluoroquinolones had good activity against m. tuberculosis (no comparison with standard antimycobaterial drugs was available though) [289] . investigation of antimycobacterial activity of a small number of indole mannich bases 59 obtained from 1-arylpiperazines (fig. 11 ) uncovered a few candidates that inhibited the growth of m. tuberculosis h 37 rv almost completely at a concentration of 6.25 mg/ml [290] . 159 (fig. 29 ) had good antimycobacterial activity (mic < 5 mg/ml) [230] , while mannich base 168 (fig. 29 ) was moderately active against m. tuberculosis h 37 rv [239] . finally, pyrazolone mannich base 161 (nr 2 ¼ nhcoc 6 h 5 ) (fig. 29 ) was more potent than reference drugs ethambutol and ciprofloxacin, but inferior to isoniazid, against m. tuberculosis h 37 rv [232] . the incidence of fungal infections of any variety has been steadily increasing over the last decades, and it has become one of the main causes of morbidity and mortality, especially in patient with compromised immune systems [293] . development of resistance to currently-in-use antifungal drugs also represents a major concern, and the discovery of novel antifungal agents, preferably outside any of the six major classes that are presently available for treatment, is one of the highest priorities for pharmaceutical industry. the latest advances in the evaluation of mannich bases with various structures as potential antifungals are presented in this section. first reports on the antifungal activity of ketonic mannich bases are relatively recent. a few mono-mannich bases of type 8 (fig. 3) and double mannich bases 12 (fig. 4 ) of acetophenone (r 1 ¼ r 2 ¼ h; nr 2 ¼ dimethylamino, 1-piperidinyl, 4-morpholinyl) have been shown to have weak antifungal activity, but the corresponding methiodides were more potent than reference drug amphotericin b against yeasts saccharomyces cerevisiae and candida krusei, and against dermatophytes trichophyton mentagrophytes, trichophyton rubrum and microsporum canis [294] . thus, quaternization of the nitrogen atom in mannich bases of type 8 appears to be a chemical modification leading to efficient antifungals [135] . various substitution patterns in the phenyl ring in ketonic mannich bases 8 also did not improve significantly the antifungal activity of these compounds [295] . bis-mannich bases 13 (r ¼ c 2 h 5 ) derived from various acetophenones (fig. 4) were only active against aspergillus fumigatus, but the antifungal activity of the related piperidinols 14 (r ¼ c 2 h 5 ) (fig. 4) was broader and slightly more potent compared to that of corresponding mono-mannich bases 8 and bis-mannich bases 13, especially against dermatophytes t. rubrum and m. canis [295] . however, a later study showed that a minor modification such as replacement of ethyl with methyl as substituent at nitrogen increased the antifungal activity of bis-mannich bases 13 (r ¼ ch 3 ), while it decreased the potency of piperidinols 14 (r ¼ ch 3 ) against t. rubrum and m. canis [296] . use of phenethylamine as amine reagent in the mannich reaction with various acetophenones afforded secondary ketonic mono-mannich bases 15 (r ¼ ch 2 ch 2 c 6 h 5 ) (fig. 4) along with piperidinols 14 (r ¼ ch 2 ch 2 c 6 h 5 ), but these compounds had relevant activity (most mic values were either 12.5 or 25 mg/ml) only against m. canis, a dermatophyte whose growth was not inhibited by reference drug nystatin in the concentration range used in this study [297] . most members of a library of mannich bases 10 of enones had antifungal effects 2e16-fold more potent than amphotericin b against at least one (if not many) selected plant fungi [298] . ketonic mannich bases derived from arylamines as amine reagents have also been reported to have antifungal effects. thus, mannich bases 108 (r 1 ¼ 2-br, r 2 ¼ h or f) (fig. 21) were almost twofold more potent than reference drug ampicillin against candida albicans [137] , whereas candidates 109 (r ¼ f or cl) derived from 3-acetylcoumarin and 110 (r ¼ cl or n(ch 3 ) 2 ) derived from 2-acetylbenzofuran (fig. 21) had mic values comparable to those of reference drug fluconazole against aspergillus flavus, chrysosporium keratinophilum, and c. albicans [138] . evaluation of mannich bases 194 (fig. 35 ) derived from cyclic ketones (n ¼ 1e4) showed that these compounds had good anti-candida and anti-saccharomyces activity, but they were less potent against aspergillus strains, and none of the compounds were as potent as reference drug amphotericin b [299] . the anti-candida potency of candidates 194 generally diminished with the increasing size of the ring, while the substitution pattern of the aromatic ring or the nature of the amine in the aminomethyl moiety did not influence the antifungal activity considerably. in addition, the a,bunsaturated ketone system is a structural requirement for the antifungal activity of mannich bases 194, as the amino alcohols obtained through the reduction of the carbonyl group in 194 were mostly inactive towards all the fungi used in this study. inhibition of ergosterol synthesis does not appear to be the mechanism by which mannich bases 194 exert their anti-candida activity, although they are able to influence the development of pseudohyphae and induce noticeable changes in the protein composition in c. albicans [299] . inspection of sar disclosed in the previous study also proved valid for mannich bases 195 derived from cyclic ketones fused with an aromatic ring (fig. 35) , except that the mic values for compounds 195 were consistently better than those for mannich bases 194 [300] . transcript profiling of c. albicans cells treated with candidate 195 (n ¼ 0, nr 2 ¼ 4-morpholinyl) showed that the transcriptional response is typical for oxidative stress and similar to that of a c. albicans cap1 transcriptional activator, which suggests that the ability of mannich bases 195 to directly trigger oxidative stress may be at least in part the reason for their antifungal activity [300] . mannich bases 196 of thiocroman-4-ones (fig. 35) obtained either from secondary aliphatic amines or from primary arylamines had good activity against several types of fungi, and two candidates 196 (nr 1 r 2 ¼ n(ch 3 ) 2 , r 3 ¼ 6-cl, r 4 ¼ 5-f or 8-cl) were more potent than reference drug ketoconazole against the fungi investigated in the study [301] . antifungal activity of double mannich bases 105 (fig. 21) against several fungi was also good, although the potency of compounds 105 against c. albicans was only moderate [134] . several studies reporting the antifungal activity of phenolic mannich bases are available. the hydrazone obtained from 2,4dinitrophenylhydrazine and salicylaldehyde was aminomethylated using various secondary amines to give mannich bases 197 (fig. 36 ) [302] the most potent anti-candida candidates 197 (nr 2 ¼ diphenylamino, 4-morpholinyl, 1-piperazinyl) were 5e10fold weaker than reference drug clotrimazole, whereas the growth of aspergillus niger was inhibited by the most potent compounds at mic values in the range of 1.56e3.12 mg/ml [302] . most aminomethylated 4-t-butylcatechols 113 (fig. 22) , along with their copper(ii) complexes, have significant antifungal activity (radial inhibition of mycelial growth ! 70%) against several plant pathogenic fungi, which is comparable, or even higher in a several cases, to that of reference drugs nystatin and terbinafine [141] . even at a concentration 10-fold greater than that of reference drug voriconazole, only three of mannich bases 114 (nr 1 r 2 ¼ 4methoxyphenylamino, 1-piperidinyl, 4-methylpiperazinyl) (fig. 22 ) barely showed anti-candida activity that was comparable to that of voriconazole [142] . aminomethylation of mulundocandin, an antifungal lipopeptide belonging to echinocandin antifungals, was undertaken with a view to improve its solubility in water; the resulting mixture of mannich bases 198 (r 1 ¼ ch 2 nr 2 , r 2 ¼ h) and double mannich bases 198 (r 1 ¼ r 2 ¼ ch 2 nr 2 ) (fig. 36) was separated before the antifungal activity of the compounds was evaluated in vitro, but only one candidate showed good inhibition against c. albicans and a. fumigatus [303] . nonetheless, in vivo testing of the collection was promising, as many compounds showed anti-candida activity comparable to parent mulundocandin, while a significant improvement in anti-candida activity compared to mulundocandin was observed for mono-mannich base 198 (r 1 ¼ 4-phenylpiperazinylmethyl, r 2 ¼ h), which was equipotent to fluconazole. three members of a collection of 6acetyl-1,3-benzoxazines 117 (r 1 ¼ ch 3 , r ¼ 4-br; r 1 ¼ h, r ¼ 2-och 3 or 4-f) (fig. 22 ) had inhibition zones against a. niger comparable to that of reference drug fluconazole [145] . benzoxazines 118 and 119, as well as naphthoxazines 120 and 121 (fig. 22) were either inactive or had weak activity against candida spp., a. fumigatus and cryptococcus neoformans, but a few compounds in this library had moderate activity against sporothrix schenckii and t. mentagrophytes [146] . in contrast, naphtho[2,1-e]-1,3-oxazine 121 (r ¼ 4-c 2 h 5 oc 6 h 4 ) was more potent than reference drug griseofulvin against c. albicans, whereas naphtho[2,1-e]-1,3oxazine 121 (r ¼ 3-ch 3 oc 6 h 4 ) was equipotent to griseofulvin against a. niger [148] . examples of mannich bases from phenols fused with heterocyclic rings that were investigated for antifungal activity include compounds 123 (fig. 22) , which had only moderate to weak activity against a. niger and penicillium spp. compared to reference drug griseofulvin [150] , and compounds 122 (fig. 22) , which were efficient against aspergillus spp. and c. albicans at concentrations as high as 5 mg/ml [149] . mannich bases 125 of chlorokojic acid (fig. 23) had good anti-candida activity (mic values between 4 and 16 mg/ml) [152e154], but the antifungal activity against dermatophytes was slightly poorer [304] . in contrast, mannich bases 174 (fig. 30) derived from allomaltol and 4substituted piperazines had poor antifungal activity against c. albicans and candida parapsilosis compared to that of reference drug fluconazole, but some members of the library were equipotent to fluconazole against c. krusei [305] . antifungal activity of various mannich bases of isatin derivatives has been examined jointly with their antibacterial activity. mannich bases 127 (fig. 24 ) obtained from isatin semicarbazone as substrate and (hetero)arylamines as amine reagents had good anti-candida activity (75e87% of the activity of reference drug fluconazole) [156] , whereas mannich bases 128 (r ¼ secondary aliphatic amines) (fig. 24 ) had moderate antifungal activity against aspergillus spp., and consistently lower than that of reference drug fluconazole [157] . for the most active mannich bases 129 (r 1 ¼ 4-or 4,5-substituted 2-ethylphenyloxy, r ¼ 1-piperidinyl or 4morpholinyl), the antifungal potency against c. albicans and a. niger was 2/3 of the potency of reference drug griseofulvin [159] . a couple of mannich bases 130 (r 1 ¼ substituted phenyl) (fig. 24) were moderately active against a. niger, but the activity against c. albicans and penicillium notatum was generally poor [160] . mannich bases 131 (r 1 ¼ 5-benzyl-3-thioxo-2h-1,2,4-triazol-4-yl) (fig. 24) were moderately potent against c. albicans and a. niger compared to reference drug fluconazole [163] . finally, in their evaluation against a. niger, mannich base 132 (ar ¼ c 6 h 4 oh-4) (fig. 24 ) was equipotent to reference drug ketoconazole against a, fumigatus, whereas mannich base 132 (ar ¼ c 6 h 4 n(ch 3 ) 2 -4) was twofold more potent than ketoconazole, and three other analogues were equipotent to ketoconazole [164] . evaluation of mannich bases of 2,3-dihydro-1,2,4-triazole-3thiones (table 1) as antifungal agents has been reported in conjunction with their antibacterial activity. in the series of mannich bases 133a, with the exception of candidate 133a (r ¼ c 6 h 4 cl-4, nr 2 ¼ nhc 6 h 3 f 2 -2,6) whose anti-candida activity was 2/3 of that of reference drug clotrimazole, all other compounds were inactive, and so were mannich bases 133b investigated in the same study [165] . compared to reference drug fluconazole, most active mannich bases 133h were 3-fold less potent against c. albicans and s. cerevisiae [173] , mannich bases 133j were either inactive or showed low antifungal activity [175] , whereas mannich bases 133k were inactive against c. albicans and s. cerevisiae [176] . mannich bases 133i lacked any anti-candida activity [174] , as did mannich bases 133n and 133o [179] , and most mannich bases 133q [180] . the majority of mannich bases 134a were equipotent to reference drug fluconazole against c. albicans [181] , and a small number of candidates 134c were moderately active against the same fungus, whereas the rest were inactive [182] . while most mannich bases 134d were moderately active against aspergillus spp., c. albicans and penicillium marneffei, two candidates 134d (r 2 ¼ 4-clc 6 h 4 or 2,4-cl 2 c 6 h 3 ) derived from 1-methylpiperazine as amine reagent were equipotent to reference drug ciclopiroxolamine [183] . several mannich bases 134e were also equipotent to reference drug fluconazole against aspergillus spp., t. mentagrophytes and p. marneffei [184] . neither compound 134g, nor candidate 135 (fig. 25 ) exhibited any anti-candida activity [185] , whereas mannich bases 134i had weak to moderate activity against candida tropicalis, s. cerevisiae and a. niger [187] . the antifungal activity of mannich bases 134j against aspergillus spp. and penicillium spp. was moderate to good, and mannich base 134j (x ¼ y ¼ ch 3 , r 2 ¼ 4-no 2 c 6 h 4 ) was more potent than reference drug fluconazole against c. albicans [188] . a few mannich bases 134k also showed good antifungal activity against c. albicans and fusarium solani [189] . the best anti-candida activity in the series of mannich bases 134m was recorded for the candidate with r 2 ¼ c 6 h 4 och 3 -4 (85% of the inhibition zone of reference drug fluconazole) [190] . in addition, 1,2,4-triazolo[5,1-b]-1,3,5-thiadiazines 199 (fig. 36) have been synthesized through a double mannich reaction starting from 5-benzyl-2h-1,2,4-triazole-3(4h)-thione and primary aliphatic and aromatic amines, and these compounds had good antifungal activity against chrysosponium tropicum and t. rubrum, but were completely inactive against a. fumigatus, a. flavus and microsporum nanum [306] . mixed results were obtained for other fungi, as a few candidates 199 were more potent than reference drug clotrimazole against a. niger, but were moderately potent against c. albicans or fusarium oxysporum, whereas the rest of the compounds in the series were totally inactive against these three fungi. as far as the aminomethyl derivatives of 2,3-dihydro-1,2,4-triazole-3-ones are concerned, candidates 136 and 138 (fig. 25 ) had no activity against c. albicans and s. cerevisiae [176, 191] , whereas in the series of mannich bases 137 featuring an imidazole moiety (fig. 25) , candidate 137 (nr 2 ¼ 1-piperidinyl) was twofold more potent against s. cerevisiae and twofold less potent against c. albicans than reference drug fluconazole [192] . antifungal activity of aminomethylated 2,3-dihydro-1,3,4oxadiazole-2-thiones ( table 2 ) has been reported in the same studies dealing with these compounds' antibacterial activity. several mannich bases 139a were equipotent to reference drug ciclopiroxolamine against t. mentagrophytes, a. flavus and a. fumigatus (mic 6.25 mg/ml), but not against p. marneffei [193] . mannich bases 139b derived from either morpholine or 3trifluoromethylaniline showed high activity against p. marneffei, t. mentagrophytes, a. flavus and a. fumigatus compared to reference drug ciclopiroxolamine, whereas those derived from other primary aromatic amines had only weak antifungal activity [194] . only a few of mannich bases 139c derived from piperazines had moderate anti-candida activity, and the rest of the collection was inactive [195] . oxadiazolethione mannich bases 139d [185] and 139j [176] , having at position 5 of the oxadiazole ring either a pyridine or a quinoline moiety, had no noticeable anti-candida activity, and the activity of mannich bases 139e against s. cerevisiae, c. tropicalis and a. niger was moderate at best [187] . most mannich bases 139f had weak antifungal activity against c. albicans, a. niger and aspergillus clavatus, but one candidate 139f (nr 2 ¼ nhc 6 h 4 och 3 -4) was 2-fold more potent than reference drug ketoconazole against the first two fungi, and almost equipotent to ketoconazole against the last fungus [196] . several mannich bases 139g had similar mic values as reference drug amphotericin b against c. albicans, a. fumigatus, t. rubrum and t. mentagrophytes [197] . most imidazole-containing mannich bases 139h exhibited better antifungal activity against c. albicans, t. rubrum and t. mentagrophytes than reference drug fluconazole [198] . the compounds reported in a study, including mannich bases 139i, are claimed to exhibit significant antifungal activity against c. albicans, but lack of access to the original information (which is only provided in the online edition as supplemental material, and is missing) makes data assessment impossible [199] . three mannich bases 139k were moderately active against aspergillus spp. and curvularia lunata [200] , while mannich bases 139l [98] and 139o [202] had no anti-candida activity, and mannich base 139m showed no antifungal activity against c. albicans and s. cerevisiae [201] . in addition, several aminomethylated 2,3dihydro-1,3,4-oxadiazole-2-ones 141 were more potent than reference drug nystatin against fungal plant pathogens aspergillus spp. ( [204] . anti-candida activity of c-mannich bases 142 of thiazolidinone (fig. 26 ) was found to be 16-fold weaker than that of reference drug terbinafine [205] . while three mannich bases 143 were moderately active against c. albicans and a. flavus, one candidate 143 (r 1 ¼ c 6 h 5 , nr 2 ¼ n(c 2 h 5 ) 2 ) (fig. 26) showed against the latter fungus a growth inhibition potency (mic ¼ 1.56 mg/ml) which was half of that determined for reference drug fluconazole [207] . also, aminomethylated thiazolidinones 144 (fig. 26) had moderate activity against aspergillus spp. and c. albicans compared to reference drug ketoconazole [208] . pyrimidine derivatives that have been aminomethylated with a view to evaluate the antifungal activity of the resulting mannich bases include pyrimidin-2-ones, pyrimidin-2-thiones and (thio) barbituric acid. double n-mannich bases 146 (x ¼ o or s) (fig. 26) had 67e77% of the anti-candida activity of the clotrimazolecontaining commercial drug imidil [210, 211] . c-mannich bases 200 of (thio)barbituric acid (fig. 37) , obtained using furan-2carboxaldehyde or indole-3-carboxaldehyde as aldehyde components and 2-aminopyridine or 4-aminoantipyrine as amine reagents in the mannich reaction, showed good activity against aspergillus spp. compared to reference drug salicylic acid [307] . antifungal evaluation of mannich bases from miscellaneous substrates that do not belong to any of the previously mentioned classes is the topic of several studies. p-mannich bases 83 (fig. 15 ) showed good activity against c. albicans and s. cerevisiae (mic ¼ 10 mg/ml) compared to reference drug amphotericin b (mic ¼ 15 mg/ml) [106] . various mannich bases of type 149 (fig. 27) derived from imides presented anti-aspergillus activity that ranged from weak to good, but no correlations between structure and activity for the small number of compounds in this series could be established [215] . anti-candida activity of the most potent mannich bases 152 (fig. 29 ) was only half of the activity of reference drug fluconazole [222] . aminomethylated hydrazidones 153 (r 1 ¼ c 2 h 5 ) derived from isonicotinic acid hydrazide (fig. 29 ) had good anti-candida activity [223] , but their analogues 153 (r 1 ¼ n-c 3 h 7 ) were less active, and their activity against a. niger was even poorer [224] . several dimethylamine mannich bases 154 derived from 7-methyl-2-(substituted aryl)imidazo[1,2-a]pyridines (fig. 29 ) were 4-folde2-fold more active against aspergillus spp. and c. albicans than reference drug griseofulvin [225] . when evaluated against the same fungi, mannich bases 157 (r 1 ¼ ch 3 ) and 158 of sydnones (fig. 29) were consistently less active than both reference drugs griseofulvin and nystatin [228] , and the majority of mannich bases 157 (r 1 ¼ och 3 ) behaved similarly, with the exception of candidate 157 (r 1 ¼ och 3 ; nr 2 ¼ 4-nitrobenzothiazole-2-ylamino), which was more potent than reference drugs fluconazole and nystatin against c. albicans [229] . all of the mannich bases 156 derived from 2-alkyl-3-hydroxy-pyridine-4(1h)-ones (fig. 29) were inactive against aspergillus spp. and c. albicans [227] . a few aminomethylated pyrazolines 159 (fig. 29 ) had anti-candida activity (mics between 1.5 and 4 mg/ml) comparable or even better than that of reference drug clotrimazole (mic ¼ 2 mg/ml) [230] , while several acetylenic mannich bases 162 (fig. 29 ) were equipotent to reference drug ketoconazole against c. albicans [233] . mannich bases 164 and 165 derived from hydantoins ( fig. 29) were generally moderate growth inhibitors for c. albicans and a. niger [236] , and phenothiazine mannich bases 167 (fig. 29) were all active against a. niger (sizes of inhibition zone between 12 and 21 mm) [238] . antifungal activity of mannich base 168 (fig. 29 ) of quinoxaline-2,3(1h,4h)-dione (100 mg/disc) against c. albicans and a. niger was superior to that of reference drug clotrimazole (50 mg/disc) [239] , while antifungal activity of mannich bases 169 of 3-aryl-2-thioxo-2,3-dihydroquinazolin-4(1h)-one (fig. 29) equipotent to reference drug fluconazole against c. albicans (mic 6.25 mg/ml) [240] . as much as 20% of the world population live in areas with high risk of contracting malaria, and millions of people suffer from acute malaria each year, while approximately half million died from the disease in 2012 [308] . malaria has been largely eradicated in many parts of the world, but the total number of recorded cases is still on the rise. one of the causes for this grave situation is the proliferation of malaria parasites that are rapidly becoming resistant to antimalarial drugs, especially those that are most frequently used, such as chloroquine. the development of novel antimalarials has stagnated since the 1970s owing to lack of interest in developed countries for this topic, and limited market potential for these drugs. initiation of numerous programs supported by public funding led to a surge of interest in drugs for neglected diseases, and antimalarials are certainly amongst these drugs. use of the mannich reaction for the synthesis of antimalarials has a long and rich history, the quest for aminomethylated substrates for treatment of malaria starting with amodiaquine 201 (fig. 38) , the first mannich base that was used to treat malaria. many of these early studies focused on derivatives of 4aminoquinolines, and the sar within this class of antimalarials suggested that the chlorine atom at position 7 of the quinoline scaffold and the phenolic mannich bases moiety are structural features that are essential for the antimalarial activity of these compounds. nowadays, the use of amodiaquine has declined owing to its considerable toxicity as a result of its in vivo conversion into a reactive quinone-imine metabolite by cytochrome p450. it was therefore hypothesized that swapping the phenolic hydroxyl and the aminomethyl side chain in amodiaquine should prevent the formation of toxic metabolites, and help circumvent the adverse side effects associated with the use of amodiaquine. this hypothesis led to the design and synthesis of several amodiaquine analogues that retain the antimalarial activity, while lacking the potential to form a quinone-imine derivative [309] . out of these analogues, mannich base 202 (the isomer of amodiaquine henceforth named isoquine, fig. 38 ) expressed in vitro activity against both chloroquine sensitive hb3 strain and highly chloroquine-resistant k1 strain of plasmodium falciparum below 10 nm, and was 20 times more potent that chloroquine. isoquine (as its diphosphate salt) was subsequently tested in vitro against the murine plasmodium yoelii ns strain, and found to be 3-fold more potent than amodiaquine when administered orally. moreover, no toxic metabolites or any excessive accumulation of isoquine have been noticed in animal models, but unacceptably high first pass metabolism of isoquine to n-dealkylated metabolites in four animal species compromised isoquine's activity against parasites, and complicated the development process of isoquine into a marketable drug. a novel candidate, n-tert-butyl isoquine (gsk369796) 203 (fig. 38) , was identified using isoquine as lead, and shown to exhibit low nm activity against chloroquine-resistant and sensitive parasites, as well as in vivo oral activity equivalent to amodiaquine, but with a significantly improved antimalarial exposure profile [310] . in addition, n-tert-butyl isoquine had a better overall preclinical safety profile than chloroquine or amodiaquine, and can be synthesized in a scalable and cost-effective way. the study of disposition of candidates 202 and 203 showed that isoquine 202 undergoes in vivo oxidative n-dealkylation to desethyl-isoquine, a metabolite that had an improved blood clearance over isoquine; unfortunately, desethyl-isoquine also had a more potent inhibitory action of cyp1a2 than isoquine [311] . on the other hand, n-tertbutyl isoquine 203 had human plasma protein binding ability and inhibition of cyp2d6 similar to those of isoquine and desethylisoquine, but a reduced rate of n-dealkylation and a higher oral bioavailability compared to isoquine and desethyl-isoquine, and these properties made n-tert-butyl isoquine 203 a better candidate than isoquine 202 for further development. a study of the in vitro activity of isoquine 202 against p. falciparum clinical isolates collected in kenya in relation to amino acid changes in pfcrt and pfmdr1, the two genes associated with 4-aminoquinoline resistance, showed that isoquine possesses high activity against field isolates (ic 50 was 9 nm, compared with 56 nm for chloroquine, and 8 nm for amodiaquine), and that isoquine's activity could be correlated with polymorphisms in pfcrt, but not in pfmdr1 [312] . starting from n-tert-butyl isoquine 203 as a template, three benzoxazines 204 (r ¼ c 2 h 5 , n-c 3 h 7 , ch 2 c 6 h 5 ) (fig. 38 ) were designed and generated through a double mannich reaction, but despite the good anti-plasmodium activity against both chloroquine-sensitive (ic 50 between 21 and 38 nm) and chloroquine-resistant (ic 50 between 31 and 72 nm) strains, the rapid ring opening of benzoxazine at low ph makes them unsuitable for further development [313] . novel analogues 205 (nr 2 ¼ aliphatic primary and secondary amines, aromatic primary and secondary amines) of amodiaquine have been tested in vitro against chloroquine-sensitive strains of p. falciparum, and while all of the compounds were active, only mannich base 205 (nr 2 ¼ 4morpholinyl) (fig. 38 ) showed good activity (mic ¼ 63 ng/ml) compared to chloroquine (mic ¼ 250 ng/ml) [314] . antimalarial activity of analogues 206 of isoquine (fig. 38) has also been evaluated, but regardless of the nature of the amine in the aminomethyl moiety, their activity was considerably lower than that of reference drug chloroquine [315, 316] . use of primary amines derived from bicyclic scaffolds for the synthesis of novel isoquine analogues led to the discovery of 207 (fig. 38) , which was approximately twofold more potent that either chloroquine or isoquine against chloroquine-sensitive and resistant p. falciparum strains [317] . a series of isotebuquine analogues 208 (r ¼ cl or cf 3 , r 1 ¼ h), the corresponding double mannich bases 208 (r 1 ¼ ch 2 nhc(ch 3 ) 3 ), as well as n-oxides derived from a mono-mannich base and a double mannich base of type 208 (fig. 38) were also evaluated against chloroquine-sensitive and resistant p. falciparum strains [318] . while only a few of candidates 208 were more potent than chloroquine against the chloroquine-sensitive strain, the majority of these mannich bases were more potent than chloroquine against the chloroquine-resistant strain, and mannich bases having one aminomethyl function were generally more potent than double mannich bases. pyronaridine 209 (fig. 39) is another example of antimalarial drug that feature a phenolic mannich bases moiety in its structure, but the aminoquinoline scaffold that was typical for amodiaquine and its congeners has been replaced in pyronaridine by a naphthyridine ring system. pyronaridine was first synthesized in 1970 at the chinese institute of parasitic disease, and has been used solely in china as treatment for malaria with good results for over 30 years [319] . beside the difficultly accessible literature in chinese, there are several recent studies that provide information on its physicochemical properties [320] and adme properties in rats [321] , interaction with other antimalarials [322] , potential use in combination therapy with artesunate [323] , or its mechanism of action based on inhibition of b-hematin formation and subsequent glutathione-dependent hematin degradation process [324] . based on the observation that substitution of the quinoline scaffold in amodiaquine with a napthyridine ring system in pyronaridine preserved the antimalarial action, g€ orlitzer initiated an ambitious synthetic program aiming at exploring the effect on the antimalarial properties of replacement of quinoline in quinoline-based antimalarials with other fused heterocyclic systems containing one pyridine ring. thus, single (r 1 ¼ h) and double mannich bases (r 1 ¼ ch 2 nr 2 ) structurally related to amodiaquine and pyronaridine, respectively, and featuring pyrido[3,2-b]indol-4-yl (compounds 210, x ¼ nh) [325] , benzofuro [3,2[1, 6] naphthyridin-5-yl (compound 215) [332] moieties were synthesized and evaluated against chloroquine-sensitive and resistant p. falciparum strains (fig. 39) . as expected, all of the mannich bases presented in these studies exhibited good antimalarial activity, and double mannich bases were generally more potent than single mannich bases. however, in spite of the variety of fused heterocyclic systems that were investigated, no compound with antimalarial activity at least comparable to that of chloroquine emerged, with the exception of one pyronaridine analogue (210, x ¼ nh, r 1 ¼ ch 2 nr 2 , nr 2 ¼ 1-pyrrolidinyl). this candidate had ic 50 ¼ 50 nm against chloroquine-sensitive strain 3d7, ic 50 ¼ 38 nm against chloroquine-resistant strain dd2, and its evaluation against plasmodium winckei in a murine malaria model (intraperitoneal dosage of 100 mg/kg) showed that no intact parasite could be noticed after three days of treatment [325] . the phenolic mannich base moiety in the previously mentioned antimalarial agents can be also found in candidates that were designed through its incorporation into hybrids with dual mode of action. thus, the combination of a tetraoxane and a phenolic mannich base moiety afforded hybrids 216 called mannoxanes (fig. 40) , which are active at low nanomolar concentrations and surpass the ability of artesunate, tetraoxane rka 182 and a peroxide/amodiaquine combination to cure malaria in mice at 10 mg/kg [333] . hybridization of artemisin with a phenolic mannich base moiety led to candidates 217 (fig. 40) , which are up to 3 times more potent than artemisin against p. yoelii nigeriensis, could be easily converted into water soluble forms, can be administered orally, are presumed to have good bioavailability, and lack the capability to form neurotoxic metabolite dihydroartemisinin [334] . hybrids 218 (fig. 40 ) are structurally very similar to 217, and have been shown to be more active than sodium artesunate against chloroquine-sensitive nf54 and chloroquine-resistant k1 p. falciparum strains [335] . screening of a collection of compounds containing a 2-hydroxyethylamino motif in their structures for inhibition of parasite growth in red blood cells infected with chloroquine-sensitive nf54 p. falciparum strain identified a hit compound, whose further elaboration led to several hybrids featuring several types of phenolic mannich moieties as the second pharmacophore [336] . their evaluation against nf54 strain, k1 strain, and the rodent parasite plasmodium berghei showed that the introduction of the phenolic mannich bases moiety resulted in candidates that were very potent against the parasites in the 72 h assay. for example, compound 219 (fig. 40) the design of hybrids with dual mode of action has also been broadened to include pyrrole mannich bases. thus, hybrids 220 from 4-amino-7-chloroquinoline and pyrrole mannich bases (fig. 40) were evaluated against chloroquine-sensitive d10 and chloroquine-resistant w2 p. falciparum strains, but they were all less potent than isoquine against both strains [317] . however, candidate 220 (r 1 ¼ 4-clc 6 h 4 , nr 2 ¼ n(c 2 h 5 ) 2 ) was equipotent to chloroquine against d-10 strain, and four of the candidates were also more potent than chloroquine against w2 strain [317] . several candidates from a small library of mannich bases 221 (r 1 ¼ h or ch 3 ) of c-10 pyrrole analogues of artemisin (fig. 40 ) had antimalarial activity that was superior to both artemisin and chloroquine against chloroquine-sensitive 3d7 p. falciparum strain, and selected mannich bases from this library were more potent than the aforementioned reference drugs against chloroquine-resistant k1 p. falciparum strain [337] . all of the compounds tested displayed good activity in peter's 4 day suppressive test using 30 mg/kg over days 1e3 post-infection, and candidates 221 ( showed complete elimination of parasites. these three mannich bases were further investigated in vivo, and had effective doses between 4.3 and 5.3 mg/kg that eliminate 90% of p. berghei in mice, whereas candidate 221 (r 1 ¼ ch 3 , nr 2 ¼ 4methyl-1-piperazinyl) reached 100% clearance of parasitemia 24 h after the last treatment and increased mouse survival to 9 days, a biological profile that render it superior to clinically used sodium artesunate [337] . phenolic mannich bases of chalcone analogues (such as 222, fig. 41 ) have been claimed to possess antimalarial activity against chloroquine-sensitive d10 p. falciparum strain at concentrations ranging from 0.3 to 3 mg/ml [44] . taking into account the structural similarity between phenolic mannich bases 222 and ketonic mannich bases of a,b-unsaturated ketones, a possible mechanism action for compounds 222 could be the inhibition of thioredoxin fig. 41 . antimalarial phenolic mannich bases derived from miscellaneous substrates. reductase in p. falciparum [338] . phenolic mannich bases 223 of 2,4dihydroxybenzaldehyde, its thiosemicarbazone and semicarbazone (fig. 41) , as well as related quinoline-containing derivatives 224 (fig. 41) have been screened against chloroquine-resistant w2 p. falciparum strain [339] . only candidates 223 derived from 1-(7chloroquinolin-4-yl)piperazine had antimalarial activity, while the remaining mannich bases 223 were inactive. all of the candidates 224 were potent antimalarial agents, with mannich base 224 (nr 2 ¼ 4-(7-chloroquinolin-4-yl)piperazin-1-yl) featuring two quinoline motifs in its structure being the most potent (ic 50 ¼ 77 nm) [339] . imines of 4(1h)-pyridones 225 (r 1 and r 2 ¼ h or ch 3 ) (fig. 41) having a phenolic mannich base moiety were active against chloroquine-resistant w2 p. falciparum strain (ic 50 values between 9 and 33 mm) and atovaquone-resistant fcr3 p. falciparum strain (ic 50 values as low as 8 mm), but their potency was lower than that of reference drugs chloroquine or atovaquone [340] . in addition, aminoalkylation of lawsone using various aldehydes and n-alkyl-ferrocenylmethylamines afforded mannich bases 226 (r 1 ¼ alkyl) (fig. 41 ) structurally resembling antimalarial drug atovaquone, but evaluation of three candidates 226 (r 1 ¼ n-c 6 h 13 , n-c 7 h 15 , n-c 8 h 17 ) against p. falciparum showed that they were less potent than atovaquone itself [341] . because of their self-limiting nature, most viral diseases (with the exception of those caused by human immunodeficiency virus, hiv) do not require specific therapy, although treatments are used to bring the condition or its symptoms to an end more quickly. currently available drugs are designed to target four main groups of viruses, namely herpes viruses, hepatitis viruses, influenza viruses, and hiv. recent investigations in the antiviral activity of mannich bases deal mostly with aminomethylated phenols and aminomethylated isatins, although examples of mannich bases with antiviral properties derived from other types of substrates are available in literature. phenolic mannich base arbidol 227 (fig. 42) [342] is a broadspectrum antiviral agent commonly used in russia to treat acute respiratory viral infections, which acts by inhibiting virus-mediated fusion with target membrane and subsequent blockage of virus entry into target cells [343, 344] . using arbidol as lead compound, a group at shenyang pharmaceutical university led by gong initiated a program aiming at investigating structureeanti-hepatitis b relationship for a series of phenolic mannich bases derived from various heterocyclic ring systems. in their first report on this topic, the chinese researchers presented a series of analogues of arbidol 228 (x ¼ so, r 1 ¼ ch 3 , c-c 3 h 5 , r 2 ¼ br) (fig. 42) having various substituents r 2 in the aromatic sulfoxide moiety, and presenting amino residues different than the original dimethylamino group in arbidol (e.g., secondary aliphatic amines, nh-azoles belonging to imidazoles and 1,2,4-triazoles) in the aminomethyl function in position 4 [345] . sar investigation was based on the ability of these compounds to inhibit replication of hepatitis b virus (hbv) and the production of surface antigen of the hepatitis b virus (hbsag) and extracellular form of hepatitis b core antigen (hbeag) in hepg2.2.15 cells infected with hbv, and showed that half of these mannich bases exhibited inhibitory effects on hbv that were superior to reference drug lamivudine. replacement of the original methyl group at n-1 in 228 with cyclopropyl led to an increase in antiviral activity, but enhanced the cytotoxicity of these candidates as well, whereas the presence of fluorine or chlorine as substituents in the aromatic sulfoxide moiety resulted in an improvement of the anti-hbv activity. departure from the original phenylmercapto function in arbidol reduced the cytotoxicity of mannich bases 228 (x ¼ so), leaving in the same time the anti-hbv activity practically unchanged. also, the nature of the amino group in the aminomethyl function appears to have little effect on the anti-hbv activity of the mannich bases reported in this study [345] . the relationship between the presence of a linker (containing two or three atoms between the sulfur atom in the side chain and the phenyl moiety) and the anti-hbv activity of mannich bases 228 (x ¼ soch 2 ch 2 y, y ¼ ch 2 , o or null, r 1 ¼ ch 3 , r 2 ¼ br) was also explored, and candidates having a phenethyl residue proved to be inactive, whereas candidates with a 3-phenylpropyl residue presented remarkable anti-hbv activity [346] . a subsequent study [347] of the anti-hbv activity of arbidol analogues 228 (x ¼ so or so 2 , r 1 ¼ ch 3 , c-c 3 h 5 , r 2 ¼ h) unsubstituted at position 5 of the indole scaffold claimed that the nature of the amino function plays an important role, and that pyrrolidine-and imidazole-containing candidates exhibited better anti-hbv activity than candidates having different amino residues. for another set of mannich bases 228 (x ¼ so or so 2 , r 1 ¼ ch 3 , r 2 ¼ h), the authors suggested that the presence of 4-methylpiperazin-1-yl residue as amino moiety, rather than 1-imidazolyl, 2-methyl-1-imidazolyl or 1-piperidinyl, afforded more potent anti-hvb agents [348] . replacement of the sulfinyl group in the side chain with sulfonyl appeared to enhance the antiviral activity while reducing cellular toxicity of these mannich bases [347] , although the results in the later study [348] supported the opposite conclusion. substitution of the indole scaffold with quinoline in mannich bases 229 (x ¼ s or so) (fig. 42 ) resulted in potent inhibition of hbv dna replication (10-to 66-fold compared to reference drug lamivudine) [349] . analysis of sar for compounds 229 revealed that the presence of 4-fluorophenyl moiety linked to sulfur in the side chain at position 2 of the quinoline scaffolds, in conjunction with amine moieties such as 1pyrrolidinyl, 1-piperidinyl, 1-imidazolyl (but not 4fig. 42 . phenolic mannich bases inspired by arbidol and useful as anti-hepatitis b agents. methylpiperazinyl or morpholinyl) in the aminomethyl function are required for good anti-hbv activity. evaluation of mannich bases 230 (fig. 42 ) derived from a tetracyclic ring system comprising the quinoline scaffold fused with a benzothiopyrane ring system that has various substitution patterns in the aromatic ring identified a significant number of candidates with good inhibition of hbv replication [350, 351] . the search for anti-hbv agents with similar structures continued with the evaluation of a series of phenolic mannich bases 231 (r 1 ¼ ch 3 , c 2 h 5 , ch(ch 3 ) 2 , r 2 ¼ h or f, x ¼ s or so) of 5-hydroxybenzimidazoles substituted at position 2 with 4-alkylmercaptophenyl residues (fig. 42 ) [352] . these compounds exhibited inhibitory effect on the secretion of hbsag that was superior to reference drug lamivudine, but lacked the ability to inhibit the replication of hbv dna in hepg2.2.15 cells at concentrations that were inferior to those corresponding to 50% of their cytotoxicity. the presence of fluorine at position 6 of benzimidazole scaffold and the presence of sulfinyl function appears to be critical for the inhibition of hbsag secretion of mannich bases 231. evaluation of another series of mannich bases 232 (x ¼ s, so, so 2 , r 1 ¼ h, 4-f, 4-och 3 , 4-ch 3 ) of 5-hydroxybenzimidazoles (fig. 42) showed that the thioethers in this series did not inhibit the replication of hbv dna, but the candidates with 4-methylpiperazin-1-yl as amino moiety were good inhibitors of secretion of hbsag, and the inhibitory effect significantly decreased upon oxidation of sulfur in thioethers to sulfinyl and sulfonyl [353] . phenolic mannich bases 233 (r 1 ¼ h, 4-f, 4-cl, 4-br, 4-ch 3 , 3-och 3 ) derived from 6bromo-8-hydroxyimidazo[1,2-a]pyridine-3-carboxylates (fig. 42) , especially those having 4-morpholinyl or 4-methylpiperazin-1-yl moieties in the aminomethyl function, inhibited the replication of hbv dna, but had no inhibitory effect on secretion of hbsag or hbeag [354] . the nature of substituents in the arylmercapto residue modulates the anti-hbv activity, as the decrease of bulkiness of the substituents (from bromine to fluorine, for example) and the presence of lipophilic substituents (e.g., a methyl group) enhance the inhibition of replication of hbv dna, whereas the presence of a 3-methoxy group further increases the activity. phenolic mannich bases 125 derived from chlorokojic acid (fig. 23 ) have been evaluated against herpes simplex virus type-1 (hsv-1) and parainfluenza-3 virus (pi-3). amongst the candidates reported in one study, only two mannich bases had inhibitory concentration in the range of 0.1e0.8 mg/ml against hsv-1, whereas all of the compounds were active in various degrees against pi-3 [152] . all of the mannich bases 125 derived from 1arylpiperazines as amine reagents inhibited both hsv-1 and pi-3, and one candidate 125 (r ¼ 4-(4-methoxyphenyl)piperazin-1-yl) was as potent as reference drug acyclovir against hsv-1 [153] . in addition, candidate 125 (r ¼ 4-(3-chlorophenyl)piperazin-1-yl) presented remarkable activity (0.025e0.4 mg/ml) against pi-3. other aminomethyl derivatives of various phenolic substrates have been tested against different viruses. only one phenolic mannich base 57 (r ¼ nhch 3 ) of norvisnagin (fig. 10) was moderately active against hsv-1 [78] . evaluation of aminomethylated 7-hydroxycoumarin derivatives 234 (fig. 43) against flaviviridae and other viruses led to mixed results [355] . phenolic mannich bases 234 (r 1 ¼ h, r 2 ¼ h or ch 3 ) were generally inactive against bovine viral diarrhea virus (bvdv), yellow fever virus (yfv) or respiratory syncytial virus (rsv), and a few other candidates that were active against bvdv or yfw were also cytotoxic. o-alkylated phenolic mannich bases 234 (r 1 ¼ n-c 3 h 7 , r 2 ¼ h or ch 3 ), and especially candidates having a methyl group at position 4 of the coumarin ring system, were active against bvdv, but they were inactive against yfv or rsv. o-acylated phenolic mannich bases 234 (r 1 ¼ coc 6 h 5 , r 2 ¼ h or ch 3 ) were generally inactive against all three viruses, but one candidate 234 (r 1 ¼ coc 6 h 5 , r 2 ¼ h, nr 2 ¼ 1,2,3,4-tetrahydroisoquinolin-2-yl) had a remarkable activity against rsv, comparable to that of reference drug 6-azauridine, and had also very low toxicity. compounds 234 were not active against a panel of viruses comprising hiv-1, coxsackievirus b2, sb-1 strain of marek's disease virus, vesicular stomatitis virus and a reovirus. in addition, screening of an extensive library of compounds identified mannich bases 235 of 5-chloro-8-hydroxyquinoline (fig. 43) as reactivators of latent hiv-1, which could prove helpful in eradicating the latent reservoir of hiv-1 in resting memory cd4þ t cells, either alone or in combination with other treatments [356] . antiviral activity of mannich bases of isatin and its derivatives is the topic of several investigations. screening of twelve mannich bases 236 derived from semithiocarbazones of 5-nitroisatin (fig. 43 ) against a panel of other viruses afforded no compound with antiviral properties, with the exception of candidate 236 (x ¼ o, r 1 ¼ allyl), which had weak activity against yfv (strain 17d) at subtoxic concentrations. this candidate was more potent than reference drug ribavirin, but was also more cytotoxic [357] . based on results of molecular studies aiming at designing inhibitors of hiv reverse transcriptase, a series of mannich bases 237 (fig. 43 ) have been synthesized [358] . candidates 237 having secondary aliphatic amino moieties in the aminomethyl function showed 92 to 69% inhibition of the enzyme, whereas mannich bases 237 (nr 2 ¼ nhc 6 h 5 ) showed no inhibition. aminomethylation of schiff bases obtained from 5-substituted isatins (r 1 ¼ f, cl, f) and lamivudine using fluoroquinolones (r 2 ¼ ethyl, cyclopropyl; r 3 ¼ h, ch 3 ) as amine reagents gave mannich bases 183 (fig. 32) , which were less potent against hiv than the parent schiff bases, and the most potent candidates 183 (r 1 ¼ f) were also 10-fold less potent than lamivudine itself against hiv [273] . starting from schiff bases of trimethoprim, mannich bases 238 (fig. 43) were obtained using fluoroquinolones as amine reagents. their evaluation against hiv and hepatitis c virus (hcv) showed that mannich bases 238 (r 1 ¼ cl) inhibited replication of hiv in mt-4 cells at effective concentrations (ec 50 ) ranging from 9.4 to 56 mg/ml, and most compounds were active against hcv rna replication (80% inhibition at 50 mg/ml) [162] . mannich base 238 (r 1 ¼ ch 3 , nr 2 ¼ 4-(4chlorophenyl)piperazin-1-yl) and three mannich bases 238 (r 1 ¼ ch 3 ) derived from fluoroquinolones as amine reagents showed inhibition against replication of hiv in mt-4 cells at ec 50 ranging from 11.6 to 28.4 mg/ml [359] . in addition, all of the compounds 238 (r 1 ¼ ch 3 ) were active against hcv rna replication (65% inhibition at 50 mg/ml) [359] . furthermore, a study reports candidate 238 (r 1 ¼ h, nr 2 ¼ 4-(4-nitrophenyl)piperazin-1-yl) as inhibitor of japanese encephalitis virus and west nile virus in vitro, and a remarkable inhibitor of japanese encephalitis virus in a murine model [360] . investigations into the antiviral activity of mannich bases derived from substrates other than phenols or isatins are available in literature as well. the majority of mannich bases 80 (r ¼ cl, och 3 ) (fig. 14) , obtained through n-aminomethylation of thiazolidine-2,4-diones with morpholine, piperidine and variously 1substituted piperazines, showed no activity against severe acute respiratory syndrome (sars) coronavirus [103] . also, all of these mannich bases had antiviral activity against types a and b of influenza virus, but virus inhibition occurred almost at cytotoxic concentration. aminomethylation of the amide function in tetracyclines using fluoroquinolones as amine reagents afforded mannich bases 189 (fig. 34) , and four candidates derived from tetracycline and one derived from minocycline were found to inhibit hiv replication with ec 50 values below 20 mm, while their toxicity against mock infected cem cell line was greater than 140 mm [287] . in addition, all mannich bases 189 showed moderate inhibition of both 3 0 -processing and strand transfer steps of hiv-1 integrase. evaluation of efavirenz mannich bases 191 (fig. 34) led to the discovery of three candidates that were at least as potent as the parent compound against hiv [289] . a large number of indole mannich bases 239 (r ¼ aminomethyl, 3-amino-1-propyn-1-yl) (fig. 43 ) derived from a pentacyclic core, along several acetylenic mannich bases with the same core, have been claimed to be active against hcv and members of the flaviviridae family of viruses [361] . none of the mannich bases 240 (fig. 43) derived from indophenazine as substrate and sulfonamides, anthranilic acid or 2aminopyridine as amine reagents either showed any activity against hiv above their cytotoxic concentrations, but some presented weak activity against hsv, vsv, or vaccinia virus [362] . anticonvulsants are drugs used in the treatment of seizures in epilepsy, a common chronic neurological disorder that affects around 50 million people of all ages worldwide. the discovery of novel antiepileptic drugs relies either on rational design based on the use of well-known pharmacophores (e.g., imides), or on random screening of libraries of compounds. ketonic mannich bases 8 (r 1 ¼ 4-substituted aryloxy, 4substituted arylthio, r 2 ¼ h, nr 2 ¼ n(c 2 h 5 ) 2 ; r 1 ¼ 4fluorophenyloxy, r 2 ¼ h, nr 2 ¼ dimethylamino, 1-piperidinyl, 4morpholinyl, 1-pyrrolidinyl) (fig. 3 ) and the related piperidinols 14 (r 1 ¼ 4-substituted aryloxy, r ¼ c 2 h 5 ) (fig. 4) were examined for anticonvulsant activity in the maximal electroshock (mes) and subcutaneous pentylenetetrazole (scptz) screens [20] . none of the compounds provided protection in the scptz screen, but several candidates 8 and 14 demonstrated anticonvulsant activity in the mes screen below their neurotoxic levels (30 mg/kg). four ketonic mannich bases of type 8 derived from acetophenone or 4hydroxyacetophenone as substrates and common secondary aliphatic amines as amine reagents, as well as the corresponding azines 16 (fig. 4) , were assessed as anticonvulsants using the same two tests. the results showed that anticonvulsant activity of candidates 8 was superior to that of azines 16, and compounds derived from 4-hydroxyacetophenone were active at a dose of 300 mg/kg in the mes test, but no candidate showed anticonvulsant activity in the cptz test [363] . bis-mannich bases 13 (r 1 ¼ h, 4-cl, 4-ch 3 , 2thienyl, r ¼ c 2 h 5 ) and the corresponding piperidinols 14 were protective in the mes test at 30 mg/kg and/or above, while candidate 14 (r 1 ¼ 4-cl, r ¼ c 2 h 5 ) was protective in the scmet test at 300 mg/kg after 4 h [364] . the presence of a 4-chlorophenyl moiety appears to be important for the anticonvulsant activity of these compounds, and analogues having the same moiety were identified as good anticonvulsant agents in a previous study [365] . anticonvulsant activity of phenolic mannich bases of 3hydroxy-4-pyranones has been investigated extensively by aytemir's group. screening of derivatives of allomaltol 174 having a 4substituted piperazin-1-ylmethyl group (fig. 30 ) using mes and scptz tests showed that candidate 174 (r ¼ 3-cf 3 c 6 h 4 ) provided excellent protection against pentylenetetrazole-induced seizures, but was neurotoxic at high dose (300 mg/kg), while candidate 174 (r ¼ 4-clc 6 h 4 ) had high anticonvulsant activity in mes test at all doses after 30 min, without being neurotoxic [305] . evaluation of another series of mannich bases of allomaltol revealed that candidate 174 (r ¼ 2,3-(ch 3 ) 2 c 6 h 3 ) was active in scptz test at 300 mg/kg after 4 h, along with candidate 174 (r ¼ 3-clc 6 h 4 ), which was active in mes test at 300 mg/kg after 5 h [366] . although mannich bases of allomaltol obtained using morpholine or 4-(1-piperidinyl)piperidine as amine reagents had no anticonvulsant activity [366] , a subsequent study dealing with novel mannich bases generated from piperidines as amine reagents reported that candidates 124 (r 1 ¼ 3-ch 3 , r 2 ¼ 5-ch 3 ; r 1 ¼ 4-hoch 2 ch 2 , r 2 ¼ h; r 1 ¼ 4-c 6 h 5 ch 2 , r 2 ¼ h) (fig. 23) were protective in scptz test, while only candidate 124 (r 1 ¼ 4-hoch 2 ch 2 , r 2 ¼ h) provided protection in mes test at 300 mg/kg [367] . use of other piperidines as amine reagent in the mannich reaction with allomaltol as substrate led to candidates 124 (r 1 ¼ 4-(un)substituted phenyl, r 2 ¼ oh, cn, coch 3 ), and two of these mannich bases (r 1 ¼ 4-brc 6 h 4 , r 2 ¼ oh; r 1 ¼ 4-clc 6 h 4 , r 2 ¼ oh) were active in scptz test at 300 mg/kg after 4 h, while all of them were active in mes test either after 0.5 h or after 4 h [368] . in contrast, when allomaltol was replaced with kojic acid as substrate in the mannich reaction, but the same piperidines were used as mine reagents, the resulting candidates 241 (nr 2 ¼ 4,4-disubstituted piperidines) (fig. 44 ) were all active in scptz test either after 0.5 h or after 4 h, but only two of them (r 1 ¼ 4-brc 6 h 4 , r 2 ¼ oh; r 1 ¼ 4-clc 6 h 4 , r 2 ¼ oh) were active in the mes test at 300 mg/kg after 0.5 h, and one candidate 241 (r 1 ¼ c 6 h 5 , r 2 ¼ coch 3 ) was active at any dose after 4 h [368] . mannich bases 241 (nr 2 ¼ 4-substituted piperazines) of kojic acid as substrate and piperazines as amine reagents were generally better anticonvulsants than analogous mannich bases 174 of allomaltol, or than mannich bases 241 derived from kojic acid and 4,4disubstituted piperidines [369] . the authors tentatively explain the enhanced anticonvulsant activity of mannich bases of kojic acid compared to the activity of mannich bases of allomaltol through the possible formation of an extra hydrogen bond in the former compounds. however, mannich bases derived from chlorokojic acid were also good anticonvulsants, and they present, like mannich bases of allomaltol, only one hydrogen bond in their structure [154] . hydantoin represents the core structure of the old generation of antiepileptic drugs, such as phenytoin, and substitution with an aminomethyl moiety was shown to improve activity against mes seizures in mice [370] . evaluation of a library of n-mannich bases 242 (fig. 44 ) derived from 5-cyclopropyl-5-arylhydantoins having 4-substituted piperazines in the aminomethyl function showed that the majority of candidates were effective in the mes or/and scptz screens, and quantitative studies in rats after oral administration showed that three mannich bases 242 (r 1 ¼ h, r 2 ¼ c 6 h 5 , c 6 h 5 ch 2 , 3-ch 3 c 6 h 4 ch 2 ) were more potent than phenytoin in mes test [371] . results from another study [372] showed that candidates 242 were generally more active in mes test than in scptz screen, and chlorine-substituted mannich bases (r 1 ¼ cl) were generally less active than those unsubstituted in the aromatic ring (r 1 ¼ h), whereas candidates derived from 1,2,3,4-tetrahydroisoquinoline as amine reagent in the mannich reaction were less potent than those derived from morpholine or piperazines. compared to candidates 242 obtained from arylpiperazines as amine reagents, mannich bases having alkylene, alkenylene, carbonyl or ester linkers between the piperazine moiety and phenyl ring presented enhanced anticonvulsant protection to pentylenetetrazole-induced seizures, which was noticeable not only after 0.5 h, but after 4 h as well [372] . taking into account the established anticonvulsant properties of many spirohydantoins [373e375], mannich bases 243 (x ¼ nh) of spirohydantoins (fig. 44) have been synthesized and evaluated as anticonvulsants, and while some of them were effective in mes or/ and scptz screens, and were more potent than reference drug phenytoin, their high neurotoxicity precluded further testing [376] . besides hydantoin, succinimide presents the structural requirements for the core structure of good anticonvulsant agents (namely, a nitrogen-containing heteroatomic system with a least one carbonyl group), and the well-established drug ethosuximide is an example for this class of antiepileptic drugs. the group of obniska has been developing novel anticonvulsants for a long time, and mannich bases derived from variously substituted succinimides has been one of the classes of compounds that provided some of the most interesting results reported by these researchers. based on the significant number of candidates that have been synthesized, aminomethylated derivatives 244 of 3phenylsuccinimides (fig. 44) [382] , generally showed protection in mes screen, but some of them were also effective in scptz screen. moreover, a few candidates presented activity not only 0.5 h after administration, but also after 4 or 5 h, which is indicative of quick onset and long duration of anticonvulsant activity. mannich bases 244 of 3-arylsuccinimides derived from other secondary aliphatic amines, such as morpholine, 4benzylpiperidine, 4-cyclohexylpiperazine, were generally effective in both screens [381, 382] . despite the large number of compounds evaluated in these studies, no consistent sars could be established. compounds with good anticonvulsant properties emerged from almost all of these studies, but none of these anticonvulsant mannich bases was deemed sufficiently promising to advance to clinical studies. aminomethylated spirosuccinimides have also been investigated as anticonvulsants. candidates 243 (x ¼ ch 2 ) derived from 4-(3-trifluoromethylphenyl)piperazine and 4-(3chlorophenyl)piperazine as amine reagents in the mannich reaction were the most potent anticonvulsants in this series in mes test, and replacement of substituted aryl with 2-hydroxyethyl rendered the candidates active in both tests [376] . the activity of mannich bases 245 of simpler spirosuccinimides (fig. 44 ) appears to depend [377] and candidates 245 (n ¼ 1 or 2) derived from 4-(2methylphenyl)piperazine [383] were devoid of anticonvulsant activity. on the other hand, mannich bases 245 (n ¼ 1 or 2) obtained from 4-(3-trifluoromethylphenyl)piperazine as amine reagent were efficient in mes screen, but not in scptz screen [383] . evaluation as anticonvulsant agents of mannich bases 246 of succinimides 3,3disubstituted with identical ( (fig. 44) has also been the topic of several recent papers [384e387]. generally, mannich bases obtained from 3,3diphenylsuccinimide were more potent than those obtained from 3-alkyl-3-phenylsuccinimides, which, in turn, were more potent than mannich bases derived from 3,3-dialkylsuccinimides, which were actually inactive in most cases. candidates generated from 4arylpiperazines as amine reagents in the mannich reaction were efficient only in mes test, and the nature and position of the substituent in the aromatic ring attached to piperazine modulate the anticonvulsant activity of these compounds. however, mannich bases 246 derived from 4-(2-hydroxyethyl)piperazine or 4benzylpiperidine were efficient in both screens [384] . the effect of mannich bases 246 with potent anticonvulsant activity on na v 1.2 sodium channel currents was also investigated as a potential mechanism of action for these compounds, and the results showed that the anticonvulsant activity of these candidates correlates nicely with their effectiveness as sodium channel blockers [385, 386] . in addition, there was no direct correlation between anticonvulsant properties and 5-ht 1a , 5-ht 2a , 5-ht 1a and/or 5-ht 7 serotonin receptor affinity [383, 387] . besides hydantoins and succinimides, other substrates featuring the ureido motif in their structure have been aminomethylated with a view to obtain anticonvulsant agents. barbituric acid and its thio analogue have been subjected to the mannich reaction with two amine reagents having a quinazolinone moiety, and the resulting candidates 247 and 248 (x ¼ o or s, r ¼ 6-br, 6-i, 6,8-br 2 ) (fig. 45 ) provided good protection (40e80%, and 50e90%, respectively) in both mes and scptz screens at a dose of 50 mg/kg, while lacking neurotoxicity, or sedative and hypnotic effects [388] . mannich bases 169 of 3-aryl-2-thioxo-2,3-dihydroquinazolin-4(1h)-one (fig. 29 ) showed significant anticonvulsant activity in mes test, as some of these candidates afforded results comparable to those obtain for reference drug phenytoin [240] . several studies concerning the anticonvulsant activity of fused seven-membered ring systems containing two heteroatoms are available in literature. aminomethylated 2,3-dihydro-1,5benzoxazepines 249 (fig. 46) provided protection in the mes test in the range of 30e90% at a dose of 30 mg/kg; candidate 249 (r 1 ¼ 3-och 3 -4-oh, r 2 ¼ 2-och 3 ) was equipotent to reference drugs phenytoin and lamotrigine in mes screen, and was also equipotent to reference drug valproate in scptz screen [389] . another study allowed a direct comparison between the anticonvulsant activities of analogous aminomethylated 2,3-dihydro-1,5-benzoxazepines 250 (x ¼ o) and 2,3-dihydro-1,5benzothiazepines 250 (x ¼ s) (fig. 46) [390] . at a dose of 30 mg/ kg, the latter provided more protection (20e90%) in mes test than the former, with mannich base 250 (x ¼ s, r 1 ¼ 2-cl, r 2 ¼ och 3 ) being the most active compound in this series. ketonic mannich bases 251 (r ¼ h, cl, no 2 ; r 1 er 2 ¼ r 3 er 4 ¼ (ch 2 ) 4 or r 1 ¼ r 2 ¼ r 3 ¼ ch 3 , r 4 ¼ h) having a 2,3-dihydro-1,5benzodiazepine scaffold as the amino moiety (fig. 46) were evaluated using a model in which the seizures were induced chemically, and some of these compounds provided protection after 0.5 h, whereas only candidates 251 derived from acetophenone (r ¼ h) provided protection up to 2 h [391] . inflammation is part of the complex, nonspecific immune response of vascular tissue that occurs in reaction to any type of injury, such as pathogens, irritants, damaged cells, etc. the initial response of the body to harmful stimuli is initiated by cells already present in all tissues, and is achieved by the increased movement of plasma and leukocytes (especially granulocytes) from blood into the injured tissue, where they release a series of cell-derived mediators, triggering afterwards a cascade of biochemical events that propagate the inflammatory response. nonsteroidal antiinflammatory drugs (nsaids) are commonly used for treatment of inflammation, along with its symptoms (fever, pain, swelling), but they present significant side effects after long-term usage, such as gastrointestinal lesions, kidney injury, and cardiovascular risk. along with many other classes of compounds, mannich bases of various structures have been investigated as part of the ongoing search for novel anti-inflammatory drugs. ketonic mannich bases 252 (r 1 ¼ h, ch 3 , so 2 ch 3 ) derived from 2arylidenecyclohexanones and aromatic amines (fig. 47) have been evaluated using xylene-induced ear swelling test and carrageenan-induced paw edema, and candidate 252 (r ¼ h) was superior to reference drug ibuprofen in both tests, whereas several candidates 252 (r ¼ ch 3 ) were less efficient, but still more effective than ibuprofen, in the same tests [392] . administration of ketonic mannich base 253 (fig. 47 ) to rats with a chronic inflammatory process induced by insertion of cotton pellets led to increased phagocytic capacity of peripheric neutrophils, enhanced activity of the serum complement system, and higher catalase activity, while a slightly decrease in the activity of superoxide dismutase was observed [393] . a few double mannich bases 105 of 4,6dimethoxybenzofuran-3(2h)-one (fig. 21) inhibited the production of pro-inflammatory cytokines tnf-a and il-6 by 76e100% at a concentration of 10 mm, but they were more cytotoxic than reference drug dexamethasone, which had comparable effects with candidates 105 on the production of the same cytokines at only 1 mm [134] . investigation of the anti-inflammatory activity of mannich bases 254 (r ¼ h, f, cl, br, or 2 ; r 2 ¼ och 3 and oc 4 h 9 -n) having amino acids isoleucine and methionine as amine moieties (fig. 47 ) was performed using both carrageenan-induced paw edema model and pellet-induced granuloma model, and resulted in the discovery of two anti-inflammatory agents 254 (r ¼ och 3 and oc 4 h 9 -n; r 1 ¼ ch 2 ch 2 sch 3 ) derived from methionine [394] . at a dose of 25 mg/kg, these two mannich bases 254 had 81% and 71%, respectively, of the efficiency of the reference drug diclofenac (dose of 10 mg/kg) on the reduction of paw swelling. at the same dose, these two candidates 254 also showed 88% and 79%, respectively, of the activity of reference drug indomethacin (dose of 3 mg/kg) on chronic inflammation. a continuation of this study was undertaken with a view to broaden the nature of amino acids (cysteine, threonine, methionine, isoleucine, asparagine, and glutamine) as amine moieties in mannich bases 254, and also the nature of the alkoxy substituents in the aromatic ring, but these novel candidates had no anti-inflammatory activity at 5 mg/kg, with the exception of two candidates 254 (r ¼ oc 2 h 5 ; r 1 ¼ ch 2 sh, ch 2 conh 2 ), which displayed mild activity in the acute inflammation model [395] . only one example of mannich bases obtained from simple phenols as potential anti-inflammatory agents is available in recent literature. thus, evaluation of four phenolic mannich bases 255 (fig. 48) using formalin-induced acute inflammation model in mice revealed that one candidate had reliable activity upon parenteral administration (50 mg/kg), whereas reference drug diclofenac sodium exhibited only two-thirds of its activity, albeit at 8 mg/kg [396] . phenolic mannich bases 256 of resveratrol analogues containing a pyridinyl moiety and either one, two or three aminomethyl residues (fig. 48) were tested using xylene-induced ear edema in mice at 200 mg/kg, and two of them had 80% of the efficiency of reference drug ibuprofen, whereas the remaining candidates were less potent [397] . the anti-inflammatory activity of mannich bases of polyhydroxylic phenols was also investigated. at a dose of 10 mg/kg, two candidates 113 (nr 1 r 2 ¼ 1-piperidinyl and 4-morpholinyl) derived from 4,6-diacetylresorcinol (fig. 22 ) had a slightly lower activity than that of reference drug indomethacin in carrageenan-induced paw edema [398] . three candidates 257 (r 1 ¼ 3-cl, nr 2 ¼ 4-methylpiperazin-1-yl; r 1 ¼ 2-br, nr 2 ¼ 1piperidinyl or 1-pyrrolidinyl) (fig. 48) were more efficient at inhibiting the production of tnf-a at a concentration of 10 mm than reference drug dexamethasone at 1 mm [399] ; under the same experimental conditions, most mannich bases in the study were also more efficient at inhibiting the production of il-6 than dexamethasone. the same researchers also investigated the action on enzymes that are involved in inflammation (such as cyclooxygenases (cox), trypsin and b-glucuronidase) of phenolic mannich bases 258 (r 1 ¼ 2-f, 2-cl, 2-br, 3-f, 3-cl, 3-br) of chalcone analogues (fig. 48) having the same substitution pattern in ring a as candidates 257 [400] . with one exception, none of the candidates inhibited the activity of trypsin, whereas most mannich bases in this study inhibited the activity of b-glucuronidase. candidates 258 derived from chalcone analogues having a halogen at position 3 of the b ring were generally more potent than those derived from chalcone analogues having a halogen at position 2 of the b ring. in addition, a few mannich bases 258 were poor inhibitors of cox-1, but excellent inhibitors of cox-2, and the majority of the candidates were more efficient at inhibiting cox-2 than reference drug aspirin. in a different study [401] , two out of four phenolic mannich bases of other chalcone analogues were found to reduce rat paw edema induced by carrageenan, and although these compounds were found to be good inhibitors of trypsin this time, no satisfactory correlation with their antioxidant, free radical scavenging, or lipooxygenase inhibition could be established. because inducible nitric oxide synthase (inos) generates high levels of nitric oxide that modulates inflammations through multiple pathways, the inhibition of this enzyme by phenolic mannich bases of heterocyclic analogues of chalcones with various structures was also investigated. this type of phenolic mannich bases was found to strongly inhibit no production, with ic 50 values ranging between 10.5 and 0.018 mm [402] . benzoxazines 117 (fig. 22) , easily obtained from 4hydroxyacetophenones through a double mannich reaction, inhibited the swelling of rat paw in various degrees when administered orally in doses equimolar to 20 mg/kg of indomethacin [145] ; compared to indomethacin, one candidate 117 (r ¼ 4-f) was more efficient, while another candidate 117 (r ¼ 4-och 3 ) was equipotent. chalcone analogues 259 (r ¼ 4-och 3 or 4-ch 3 ) derived from these benzoxazines (fig. 48) had a more pronounced antiinflammatory activity compared to candidates 117, and all of them displayed 70e90% of the efficiency of reference drug indomethacin [403] . phenolic mannich bases of naturally-occurring benzopyranones have also been tested as anti-inflammatory agents. most mannich bases 260 derived from 7hydroxycoumarin (fig. 48) were superior to reference drug indomethacin at reducing rat paw edema induced by carrageenan, and two candidates 260 (nr 2 ¼ 4-morpholinyl, 1-piperazinyl) were 1.6 times more efficient than indomethacin at 10 mm without significant inhibition of cox-1 [404] . irisolidone is an isoflavone which was isolated from pueraria spp., and was found to exert its antiinflammatory action through suppression of inos gene expression and pro-inflammatory cytokines in activated microglia [405] . chemical modification of irisolidone by means of the mannich reaction using primary aliphatic and aromatic amines as amine reagents led to candidates 261 ( fig. 48) with enhanced ability to inhibit nitric oxide production compared to parent irisolidone [406] . anti-inflammatory activity of mannich bases of 2,3-dihydro-1,2,4-triazole-3-thiones has also been investigated. several candidates 133f (table 1) have been evaluated using carrageenaninduced rat paw edema model, and the activity of one of them (133f, r 2 ¼ 2-ch 3 c 6 h 4 , nr 2 ¼ 4-morpholinyl) was comparable to that of reference drug ibuprofen [171] . mannich bases 133i with secondary aliphatic amino moieties (dimethylamino, diethylamino, 1-pyrrolidinyl) in the aminomethyl function were as efficient as reference drug celecoxib in reducing rat paw edema both after 1 h and after 2 h, and they also had lower ic 50 values (around 1 nm) for the inhibition of cox-2 compared to celecoxib (1.9 nm) [407] . as far as 5-substituted 2-aminomethyl-4-arylideneamino-2h-2,3dihydro-1,2,4-triazole-3-thiones 134 (table 1) are concerned, the majority of mannich bases 134c had, upon administration of either 20 mg/kg or 40 mg/kg, a lower anti-inflammatory activity in carrageenan-induced rat paw edema model than reference drug indomethacin (5 mg/kg), although one candidate (134c, r 2 ¼ 2,6-cl 2 c 6 h 3 , nr 2 ¼ 4-phenylpiperazin-1-yl) inhibited edema formation at the higher dose almost as efficiently as indomethacin [182] . starting from ibuprofen, two separate studies reported the synthesis and anti-inflammatory activity of mannich bases 262 having different arylideneamino moieties at position 4 of the 2,3-dihydro-1,2,4-triazole-3-thione scaffold (fig. 49) . the most potent compounds in this series were those having either a 4-morpholinyl or a 4-methylpiperazin-1-yl residue in the aminomethyl function. when common 4-substituted benzaldehydes (r 2 ¼ 4-clc 6 h 4 , 4-ch 3 c 6 h 4 , 4-brc 6 h 4 , 4-no 2 c 6 h 4 ) were used to generate the azomethine moiety, several candidates 262 had anti-inflammatory activity comparable with that of reference drug diclofenac, and were generally more efficient than ibuprofen at every time interval up to 3 h [408] . also, the most potent compounds in this series were those having either a 4-morpholinyl or a 4-methylpiperazin-1-yl residue in the aminomethyl function. on the other hand, when 3aryl-4-formylsydnones were used to generate the azomethine moiety, the resulting mannich bases 262 were consistently more potent than the analogues in the previously mentioned series of compounds, but all of these candidates were less efficient than reference drug indomethacin in reducing rat paw edema [409] . several mannich bases 134h showed a peak in their antiinflammatory activity at 1 h post-injection (from 50% to 130% edema inhibition compared to indomethacin), whereas the antiinflammatory activity of other candidates 134h peaked at 4 h post-injection (from 84% to 100% edema inhibition compared to indomethacin) [186] . all of the mannich bases 263 (fig. 49 ) had only moderate anti-inflammatory activity, with the most potent compound in the series showing approximately 80% of the efficiency of reference drug indomethacin [410] . a few mannich bases 264 and other structurally related aminomethylated 2,3-dihydro-1,2,4-triazole-3-thiones (fig. 49 ) reduced significantly the inflammatory response (maximum inhibition between 30 and 59%) compared to reference drug diclofenac (inhibition between 42 and 63%), and some of them presented fast onset of their antiinflammatory action, while others had a long-lasting anti-inflammatory effect [411] . the anti-inflammatory activity of mannich bases of 2,3-dihydro-1,3,4-oxadiazole-2-thiones has been scarcely examined. several mannich bases 139a (table 2) , especially those derived from ibuprofen as starting material for the 1,3,4-oxadiazole-2-thione substrate and generated from 4-arylpiperazines as amine reagents in the aminomethylation step, were as efficient as reference drug diclofenac at reducing rat paw edema [193] . also, mannich base 265 (fig. 49 ) had anti-inflammatory activity comparable to that of reference drug indomethacin in carrageenan-induced rat paw edema test [412] . several papers published by g€ okhan's group have reported the anti-inflammatory activity of mannich bases of 2benzoxazolinones. one of their studies showed that candidates 266 (r 1 ¼ ch 3 , r 2 ¼ 2-or 4-clc 6 h 4 co) having an acyl moiety at position 6 of the benzoxazolidinone scaffold were more potent than their analogues 266 (r 1 ¼ 2-or 4-clc 6 h 4 co, r 2 ¼ h) with the same acyl group at position 5 (fig. 49 ); in addition, the antiinflammatory activity of two of these candidates was equipotent to that of reference drug indomethacin [413] . substitution with fluorine in the phenyl ring of the acyl moiety appears to be less favorable for the anti-inflammatory activity of candidates 266 (r 1 ¼ h, r 2 ¼ difluorobenzoyl) [414] . mannich bases 266 (r 1 ¼ ch 3 , r 2 ¼ h) having 4-arylpiperazine residues in the aminomethyl function were all less potent than reference drug indomethacin in carrageenan-induced rat paw edema test, but the results suggest that the nature of the substituent in the aryl group of piperazines plays an important role in the anti-inflammatory activity of these compounds [415] . a few mannich bases 266 (r 1 ¼ no 2 , r 2 ¼ h) had an anti-inflammatory effect comparable to that of reference drug indomethacin 3 h after administration, but their efficiency in reducing the swelling reached a plateau afterwards, whereas that of indomethacin continued to increase in time [416] . two studies have reported the anti-inflammatory activity of mannich bases of isatins. aminomethylation of derivatives of 5methylisatin thiosemicarbazone afforded mannich bases 267 (r 1 ¼ r 2 ¼ h or ch 3 , nr 2 ¼ secondary aliphatic amines) (fig. 50) , which showed moderate anti-inflammatory activity (18e44% inhibition of edema at a dose of 100 mg/kg) compared to reference drug diclofenac (65% inhibition of edema at a dose of 45 mg/kg) [417] . several mannich bases 268 (fig. 50) , which were obtained from a derivative of isatin hydrazone, had anti-inflammatory activity comparable to that of reference drug diclofenac, and the highest level of activity was observed after 2 h [418] . a correlation in this series between anti-inflammatory activity and the nature of the amino moiety in the aminomethyl function was noticed, as the activity decreased with the increasing lipophilicity of the amino group. a series of n-mannich bases 269 of benzimidazole derivatives (fig. 50) were evaluated as anti-inflammatory agents using formalin-induced paw edema method. compared with reference drug diclofenac (50 mg/kg), they all caused significant reduction of paw edema, albeit at different doses (200 mg/kg for mannich base 269 (r 1 ¼ h or ch 3 ) and 40 mg/kg for mannich bases 269 of 2styrylbenzimidazole) [419] . also, several mannich bases 269 (r 1 ¼ c 2 h 5 ), derived from both secondary aliphatic amines and primary arylamines, showed moderate anti-inflammatory activity 4 h after administration (33e57% of the activity of reference drug aspirin at the same dose of 100 mg/kg) [420] . mannich bases obtained from substrates other than those mentioned so far have been examined as anti-inflammatory agents as well. thus, n-mannich bases 270 (nr 2 ¼ 1-pyrrolidinyl, 1piperidinyl, 4-morpholinyl) of pyrimido[1,6-a]azepine derivatives (fig. 51 ) had moderate activity (46e58% reduction of edema compared to that recorded for reference drug diclofenac) [421] , whereas n-mannich bases 271 of a tricyclic system derived from pyrimido[1,6-a]azepine (fig. 51) were less potent (anti-inflammatory activity of approximately 40% of that of diclofenac) [422] . two aminomethylated pyridazinones bearing a thiophene ring, namely compounds 272 and 273 (fig. 51) , showed 65% and 95% reduction of rat paw edema, respectively, compared to reference drug indomethacin [423] . even when administered in a higher doses relative to that of reference drug, none of the mannich bases 274 (r ¼ h, ch 3 , och 3 , cl, x ¼ o or ch 2 ) derived from isoxazolines (fig. 51) was as efficient as indomethacin in reducing rat paw edema [424] . out of the two acetylenic mannich bases 275 (x ¼ o or ch 2 ) with a betulonic acid scaffold (fig. 51 ) that were investigated as antiinflammatory agents, only the piperidine derivative was almost as efficient as reference drug indomethacin in reducing rat paw edema; the morpholine analogue had only half of the activity of the piperidine mannich base [425] . as pain is a symptom of inflammation, the anti-inflammatory and analgesic activities of novel candidates are usually evaluated at the same time. therefore, it is not surprising that many of the studies that report the anti-inflammatory activity of mannich bases also offer information on their analgesic potential. several ketonic mannich bases 252 (r 1 ¼ h, ch 3 , so 2 ch 3 ) derived from 2-arylidenecyclohexanones and aromatic amines (fig. 47) were equipotent to reference drug ibuprofen in both acetic acid-induced writhing test and hot plate test [392] . one of the most potent candidate was compound 252 (r ¼ r 1 ¼ h) derived from ptoluidine, but a few mannich bases derived from 2-(4methylsulfonylbenzylidene)cyclohexanone were also efficient as pain relievers. phenolic mannich bases of 1-and 2-naphthols substituted in either rings of naphthalene system with various functions, which were synthesized using preformed aminomethylation reagents (e.g., imonium salts obtained from aromatic aldehydes and secondary aliphatic amines), were claimed to have analgesic activity [426] . the claim is difficult to assess, because only two candidates have been evaluated, and no comparison with established analgesics was provided. in addition, while candidate 276 (x ¼ ch 2 ) (fig. 52 ) was efficient (92% inhibition of the writhing reaction), the second candidate 276 (x ¼ o) offered only modest protection against pain (30% inhibition of the writhing reaction). mannich bases of 2,3-dihydro-1,2,4-triazole-3-thiones with analgesic activity have been reported in several studies. candidates 133f (r 2 ¼ 2-ch 3 c 6 h 4 , nr 2 ¼ 4-morpholinyl; r 2 ¼ 4-ch 3 oc 6 h 4 , nr 2 ¼ 4-methylpiperazinyl) ( table 1) , which had good antiinflammatory activity, were also tested for analgesic activity; their efficiency, determined using tail flick method in albino rats, was comparable to that of reference drug ibuprofen [171] . several mannich bases 262 (fig. 49) showed analgesic activity (tail flick latency between 6.8 and 7.1 s) that was comparable with that of reference drug pentazocine (tail flick latency of 7.45 s), while the rest of the compounds were moderately active [409] . mannich bases 263 (fig. 49) were less efficient in the tail flick test, as the reaction time for the most potent compound in the series was approximately 80% of the latency provided by reference drug pentazocine [410] . two candidates 139a (table 2) , both derived from ibuprofen as starting material for the 2,3-dihydro-1,3,4oxadiazole-2-thione substrate and generated using either ethyl piperidine-4-carboxylate or 4-(4-fluorophenyl)piperazine as amine reagents in the aminomethylation step, were more efficient analgesics than reference drug diclofenac in hot plate test [193] , while mannich base 265 (fig. 49 ) was equipotent to reference drug diclofenac in acetic acid-induced writhing test [412] . analgesic activity of mannich bases of 2-benzoxazolinones was also determined for the same candidates that were investigated for anti-inflammatory activity. in the library of mannich bases 266 derived from 2-benzoxazolinones (fig. 49 ) carrying a benzoyl moiety in the aromatic ring (r 1 or r 2 ¼ substituted benzoyl), no significant difference in analgesic activity between the 5benzoylated and the 6-benzoylated analogues could be observed [413] . although most candidates provided moderate to low protection in either tests used to determine their analgesic activity (namely acetic acid-induced writhing test and p-benzoquinoneinduced abdominal constriction test), two mannich bases derived from 6-(substituted benzoyl)-2-benzoxazolinones were found to be as efficient as reference drug aspirin. thus, analgesic activity of mannich base 266 (r 1 ¼ h, r 2 ¼ 2,6-f 2 c 6 h 3 co, nr 2 ¼ 4-(4acetylphenyl)piperazin-1-yl) was equipotent to that of aspirin [414] , while analgesic activity of mannich base 266 (r 1 ¼ ch 3 , r 2 ¼ 4-clc 6 h 3 co, nr 2 ¼ 4-(4-fluorophenyl)piperazin-1-yl) was slightly poorer than that of aspirin [413] . in the series of mannich bases 266 derived from 5-methyl-2-benzoxazolinone, several compounds showed better analgesic activity compared to reference drug aspirin, and the analgesic activity for all the compounds was consistently higher than their anti-inflammatory activity, suggesting that these mannich bases might exert their analgesic activity centrally [415] . two mannich bases 266 derived from 5-nitro-2benzoxazolinone were also found to possess analgesic activity comparable to that of aspirin [416] . mannich bases 267 of derivatives of 5-methylisatin thiosemicarbazone (fig. 50) were moderately efficient (16e53% protection) in preventing acetic acid-induced writhing in mice at a dose of 100 mg/kg, while reference drug diclofenac provided 74% protection in the same test at a dose of only 45 mg/kg [417] . mannich bases 268 of isatin hydrazone (fig. 50) carrying a quinazoline moiety and derived from acyclic secondary aliphatic amines (dimethylamine, diethylamine) were the most potent; their analgesic effect was superior or comparable to that of reference drug diclofenac 2 h after administration, but it decreased rapidly afterwards [418] . n-mannich bases 269 (fig. 50 ) obtained from benzimidazole using dimethylamine or diethylamine as amine reagents were found to be moderate analgesics at a dose of 200 mg/kg relative to paracetamol (100 mg/kg), while mannich base 269 derived from 2methylbenzimidazole as substrate and diphenylamine as amine reagent was a poor analgesic candidate [419] . owing to their toxicity, mannich bases derived from 2-styrylbenzimidazole were administered at a lower dose (20 and 40 mg/kg), and their analgesic activity ranged from moderate (nr 2 ¼ 4-morpholinyl) to very good (nr 2 ¼ diethylamino or 1-piperidinyl) when compared to paracetamol [419] . n-mannich bases 269 of 2-ethylbenzimidazole had analgesic activity comparable to that of reference drug pentazocine only when administered in doses that were 25 times greater than that of pentazocine [420] . other mannich bases 269 derived from 2-substituted benzimidazoles (r 1 ¼ ch 2 nhnhc 6 h 5 or 2-hoc 6 h 4 ) also showed moderate to good analgesic activity in acetic-acidinduced writhing test (62e84% of the analgesic activity of diclofenac at the same dose) [427] . evaluation of a first series of n-mannich bases 277 derived from 2h-4,6-dimethyl-3-oxo-2,3-dihydroisothiazolo[5,4-b]pyridine ( fig. 52 ) led to identification of weak to moderate analgesic agents [428] . amongst them, candidates derived from 4-aryl-or 4benzylpiperidine and those having a 4-(2-substituted phenyl) piperazine as the amine moiety were the most efficient analgesics in writhing and hot plate tests. a second series of mannich bases 277 was subsequently synthesized, and some of the candidates displayed significant activity in the writhing test, with analgesic activity 2 to 10 times more potent than that of aspirin and 1.5 to 10 times weaker than that of morphine, used as reference drugs in the study [429] . excess of reactive oxygen species produced in living organisms can cause oxidative stress and damage cells by initiating chain reactions that lead to lipid peroxidation, dna damage or protein oxidation. besides the complex system of antioxidant metabolites and enzymes that naturally prevent cell damage, exogenous antioxidants may sometimes be required to keep reactive oxygen species at an optimum level. use of the mannich reaction to generate novel chemical entities capable of acting as antioxidants is presented in this section. ketonic mannich bases 109 and 110 (fig. 21) , which were obtained from arylamines as amine reagents and 3-acetylcoumarine and 2-acetylbenzofuran as substrates, respectively, were tested for antioxidant activity by evaluating their ability to scavenge 1,1diphenyl-picrylhydrazyl (dpph) radical [138] . the most efficient candidates in each series, namely 109 (r ¼ h) and 110 (r ¼ n(ch 3 ) 2 ), showed moderate potency in scavenging dpph radical (approximately 65%) compared to standard butylated hydroxytoluene (90%). substitution of the aromatic ring with electron-withdrawing groups appears to reduce the antioxidant ability of these mannich bases. a large number of phenolic mannich bases have been reported in the literature as potential antioxidants. antioxidant activity of two phenolic mannich bases 278 derived from thymol (fig. 53) has been assessed by means of xanthine oxidase inhibition test for the cell-free system, and by inhibition of lipid peroxidation using ratliver homogenate [430] . both candidates, and especially mannich base 278 having morpholine as amine moiety, presented enhanced antioxidant activity in both tests compared to parent thymol. three phenolic mannich bases 114 (nr 1 r 2 ¼ 4-ch 3 oc 6 h 4 nh, 1piperidinyl and 4-morpholinyl) derived from 4,6diacetylresorcinol (fig. 22) , designed primarily as antiinflammatory agents, were also evaluated for their ability to inhibit lipid peroxidation; results showed that these candidates were more efficient than indomethacin at preventing lipid peroxidation, and that the antioxidant activity may be correlated with their ulcerogenic activity [398] . only three mannich bases 257 (fig. 48) , all of them having piperidine as the amine moiety (nr 2 ¼ 1-piperidinyl), showed moderate to high ability in scavenging dpph radical (49e74% of dpph scavenging ability of standard gallic acid), while the rest of the candidates were either poor scavengers of dpph radical, or failed to react with dpph radical at all [399] . although all of the phenolic mannich bases 197 showed efficiency as antioxidants in various degrees, candidates 197 (nr 2 ¼ diphenylamino, 4-morpholinyl, 1-piperazinyl) (fig. 36) were the most potent scavengers of hydrogen peroxide, their activity being comparable to that of standard ascorbic acid, but inferior to that of standard butylated hydroxyanisole [302] . selected tacrinee8-hydroxyquinoline hybrids 279 (fig. 53) , that were developed primarily as agents for the treatment of alzheimer's disease, exhibited also potent peroxyl radical absorbance capacities (2.6e4.7 trolox equivalents/mmol of mannich base), as determined by means of orac-fl method (oxygen radical absorbance capacity by fluorescence) [431] . phenolic mannich bases of natural flavanones have been designed and synthesized with the view to improve antioxidant efficiency, bioavailability and water solubility of parent flavanones. only three mannich bases 41 (r ¼ oh, r 1 ¼ h, r 2 ¼ 1-pyrrolidinyl, diethylamino or diisopropylamino) of apigenin (fig. 7) exhibited antioxidant activity greater than that of apigenin, while the rest of candidates 41 presented antioxidant activity comparable to that of apigenin [58] . antioxidant activity of these apigenin derivatives was concentration-dependent, and dpph radical scavenging ability of the most potent mannich bases 41 was almost the same as that of the standard ascorbic acid at a concentration of 1.25 mg/ml. scutellarein was also aminomethylated to give mannich bases 41 (r ¼ r 1 ¼ oh, r 2 ¼ aminomethyl), which had the ability to scavenge 50% of dpph radical in the sample at concentrations ranging from 24 to 33 mm, but no comparison with a well-established antioxidant was provided in the study [432] . mono-and bis-mannich bases 280 fig. 53 ) were synthesized and evaluated as inhibitors of photooxidation of a2e (a pigment of the lipofuscin of retinal pigment cells, thought to play a role in macular degeneration) [433] . these candidates showed sufficient antioxidant ability to inhibit noncellular and intracellular photooxidation of a2e, and were superior as antioxidants to quercetin itself. on the other hand, phenolic mannich bases obtained from other known antioxidants such as sesamol or 1-(2-hydroxy-4-methoxyphenyl)-3phenylprop-2-en-1-one were ineffective inhibitors of a2e photooxidation at 200 mm [433] . phenolic mannich bases of chalcone analogues such as candidates 258 (r 1 ¼ 2-f, 2-cl, 2-br, 3-f, 3-cl, 3-br) (fig. 48) were modest to poor scavengers of dpph radical (3e58% reduction of dpph absorption) compared to standard quercetin (86% reduction of dpph absorption) [400] . results for dpph scavenging ability of phenolic mannich bases derived from other chalcone analogues obtained in a different study are also in line with the antioxidant data recorded for mannich bases 258, as the greatest reduction of dpph absorption was only 13% at concentrations as high as 1 mm of mannich base [401] . however, one candidate in this series scavenged superoxide radical efficiently, while another candidate was a potent inhibitor of heme dependent lipid peroxidation [401] . several mannich bases 133g derived from 5-(2-thienyl)-2,3dihydro-1,2,4-triazol-3-thiones (table 1) were as efficient antioxidants as standard ascorbic acid (over 90% reduction of dpph radical), and showed enhanced antioxidant activity over the parent 2,3-dihydro-1,2,4-triazole-3-thiones [172] . a series of mannich bases 139 [1, 4] dioxin-6-yl, nr 2 ¼ substituted primary arylamines) ( table 2) were designed as antioxidant agents, and were evaluated using scavenging dpph radical assay, scavenging 2,2 0 -azino-bis(3-ethylbenzothiazoline-6sulfonic acid) radical cation (abts) assay, and ferric reducing antioxidant power assay [434] . more than half of these mannich bases showed greater dpph radical scavenging ability than butylated hydroxytoluene (ic 50 ¼ 44 mm), and seven of them exhibited greater antioxidant activity than trolox (ic 50 ¼ 30 mm) in the same assay. a few candidates 139 with high dpph scavenging activity, along with other mannich bases 139, were also very good scavengers of abts radical cation (their activity was greater than that of trolox). in ferric reducing antioxidant power assay, three mannich bases 139 showed better activity than trolox, and other seven were more potent than butylated hydroxytoluene. mannich bases 139 nr 2 ¼ 3,4,5-f 3 c 6 h 3 ) were the only compounds to exhibit high potency in all three assays, and they were also more efficient than trolox in inhibiting lipid peroxidation in mice liver microsomes homogenate [434] . n-mannich bases 149 of succinimide (r 1 ¼ h or phenyl, nr 2 ¼ nhc 6 h 5 , 2-pyridinyl) (fig. 27) and an n-mannich base of phthalimide showed moderate dpph scavenging activity compared to standards vitamin c and vitamin e, while their ability to inhibit peroxidation of linoleic acid was moderate to weak [215] . also, several n-mannich bases 166 of saccharin (fig. 29) were antioxidants as potent as standard ascorbic acid in the abts assay, and more potent antioxidants than saccharin itself [237] . n-mannich base 281 derived from a 3,5-disubstituted pyrazoline (fig. 53) had dpph scavenging and nitric oxide scavenging activities comparable to standard antioxidants ascorbic acid and rutin; the presence of the phenolic hydroxyl group could be responsible for the antioxidant activity of this compound [435] . finally, antioxidant activity of two c-mannich bases 282 of uracil (fig. 53) was determined by means of measuring the rate of oxidation of 2-propanol [436] ; addition of compounds 282 to the reaction mixture reduces the rate of oxidation to levels comparable to the rate of oxidation in the presence of butylated hydroxytoluene. a small number of studies deal with the investigation of mannich bases as antihypertensive agents. phenolic mannich bases appear to be particularly remarkable as blood pressure-lowering substances. several single and double mannich bases of various phenols and having thiomorpholine as amine moiety showed a gradual effect on systolic, diastolic and mean arterial pressure, starting at a dose 0.1 mg/kg; in the case of reference drugs captopril, losartan and omapatrilat, the same effect was noticeable at 0.001 mg/kg [437] . in a manner similar to the aforementioned reference drugs, these phenolic mannich bases also induced a gradual (but significant) reduction of heart rate in anesthetized mice. double mannich base 283 (fig. 54 ) emerged as a valuable candidate, with one of the lowest effective dosage in this collection, while exhibiting the highest antihypertensive activity in conscious spontaneous hypertensive rat model [437] . the same researchers examined the antihypertensive effect of double mannich base 284 with a 1,4-dihydropyridine moiety in its structure (fig. 54) , but the results showed that the activity of this candidate was inferior to that of double mannich base 283, presumably due to the change in bulkiness of the substituent para to the phenolic hydroxyl [438] . the presence of two aminomethyl functions in the structure of 284 may have been essential in preserving the antihypertensive activity of this candidate to a reasonable level, while replacement of thiomorpholine in 283 with morpholine in 284 could be also the cause for the poorer antihypertensive properties of candidate 284 relative to those of mannich base 283. in an attempt to synthesize aminomethylated 4-(naphthyloxy) butanoic acids (e.g., compound 285, were obtained when 4-(naphthalen-1-yloxy)butanoic acid was subjected to aminomethylation [439] . at a concentration of 5 mg/ kg, candidate 285 (nr 2 ¼ 1-piperidinyl) lowered blood pressure in anesthetized cats similarly to propranolol, while compound 286 (nr 2 ¼ 4-phenylpiperazin-1-yl) was superior to propanolol at the same dose (fall of blood pressure of 60 mm hg, duration of the effect of 120 min). it is also worth mentioning that the antihypertensive effect of a c-mannich base of 2-naphthol, namely 1fig. 54 . mannich bases as agents for blood pressure regulation. pyrrolidinylmethyl-2-naphthol 287 (fig. 54) , was reported in an earlier publication [440] . a large collection of mannich bases 288 (r 1 ¼ h, ch 3 , c 2 h 5 , ch 2 ch(ch 3 ) 2 , c 6 h 5 , och 3 , cl) (fig. 54) was evaluated for antihypertensive activity by the non-invasive tail cuff method, and several candidates were found to significantly reduce mean arterial blood pressure, albeit at higher doses than reference drug hydralazine [441] . the presence of alkyl groups as substituent r 1 seems to be favorable for the antihypertensive activity, whereas the nature of the amine moiety does not appear to influence the activity. several mannich bases 289 (r 1 ¼ ch 3 , c 2 h 5 , n-c 4 h 9 , ch 2 c 6 h 5 ; r 2 ¼ ch 3 , c 6 h 5 ) of imidazo[1,2-a]benzimidazoles (fig. 54 ) were found to reduce arterial blood pressure in anesthetized rats with 20% at doses that were lower than the dose at which reference drug dibazole produces the same effect (22 mg/kg) [442] . analogous aminomethylated imidazo[1,2-a]benzimidazoles 290 (r 1 ¼ ch 3 , n-c 4 h 9 , ch 2 c 6 h 5 ) (fig. 54 ) were more efficient antihypertensive agents than mannich bases 289, as they reduce arterial blood pressure with 20% at doses lower than those recorded for candidates 289. several octahydroquinazoline derivatives 291 (r ¼ h, 2-f, 4-f, 4-cl, 4-br, 4-ch 3 , 4-oh) (fig. 54 ) were obtained through a double mannich reaction starting from 3-(4-chlorophenylamino)-5,5dimethyl-2-cyclohexenone and arylamines, and were shown to produce insignificant changes in both arterial blood pressure and heart rate at a dose of 5 mg/kg [443] . however, derivatives 291 (r ¼ och 2 conhn]chc 6 h 4 r 1 , r 1 ¼ h, 4-ch 3 , 4-no 2 ) of a candidate in the initial series afforded significant decreases in both systolic and diastolic arterial blood pressure, with rapid onset of action (5 min) and minor decrease of heart rate in anesthetized male adult albino rats [443] . on the other hand, candidate 291 (r ¼ 4-cl) produced a time-dependent significant increase in both systolic and diastolic arterial blood pressure, without causing tachycardia for 30 min, which makes this compound useful for treatment of hypotension. a small number of studies report the activity of mannich bases against parasites other than plasmodium spp. among these parasites, which are the cause of parasitic infections especially in developing countries, different species from the trypanosomatidae family are pathogenic to humans and cause african trypanosomiasis (sleeping sickness, trypanosoma brucei), american trypanosomiasis (chagas' disease, trypanosoma cruzi) or leishmaniasis (leishmania spp.). in addition, one study investigated the activity of mannich bases against entamoeba histolytica, and one study reported the anti-schistosoma activity of mannich bases derived from praziquantel. all parasitic protozoa belonging to trypanosoma spp. have a unique thiol metabolism based on the flavoenzyme trypanothione reductase. this enzyme could be therefore considered a promising target for rational drug design against african sleeping sickness, chagas' disease, and different forms of leishmaniasis, owing to its absence of in the mammalian host, the structural differences to related host enzymes, and its essential role for parasite survival. because unsaturated ketonic mannich bases react readily with thiols, and because several such compounds were shown to be efficient mechanism-based inhibitors of p. falciparum thioredoxin reductase [338] , a study concerning the ability of these compounds to interact with both trypanothione reductase and free trypanothione was undertaken [444] . candidates 292 (nr 2 ¼ dimethylamino, 1-piperidinyl, 4-morpholinyl) (fig. 55 ) inactivated trypanothione reductase, but only in the presence of nadph, suggesting that reduction of this enzyme prior to its interaction with the mannich base is essential. the divinyl ketone arising from the deamination of candidates 292 is the actual inhibitor, and its activity against trypanothione reductase was higher than that of parent mannich bases; unfortunately, this divinyl ketone was also too reactive to be considered a drug candidate. mannich bases 292 displayed only modest activity against all strains of intracellular parasites, which may be explained by reaction with glutathione present in millimolar concentrations in the cytosol of the mammalian host cells. nonetheless, they showed a significant effect against extracellular t. b. rhodesiense, which might (at least partially) be due to their high reactivity toward trypanothione reductase and trypanothione. with trypanothione reductase validated as a drug target in trypanosomiasis, design and synthesis of novel unsaturated mannich bases based on melaminophenyl arsenical drug melarsoprol was subsequently pursued [445] . candidates 293 (r 1 ¼ cl, h, ch 3 ) (fig. 55) were efficient in vitro trypanothione reductase inhibitors, and while they did not display any significant activity in cell-based assays against t. cruzi, they were active towards t. brucei. the presence of the melamine residue para to the enone motif did not result in any improvement in the trypanocidal potency of 293 (r 1 ¼ cl) compared to hit compounds 292. however, the presence of the melamine residue meta to the enone motif significantly lowered the ic 50 against the human cell line used in the study, which is indicative of these compounds' high cytotoxicity. on the other hand, phenolic mannich base 294 (fig. 55) was basically inactive towards all parasites. mannich bases 293 were taken up effectively into cells despite the absence of p2 and hapt1 carriers, which suggests that the main route of entry for these compounds was not through aminopurine transporters. stemming from the observation that heterocyclic pyrazolopyridine ring system can be considered analogous to quinoline, and because aminoquinoline derivatives are efficient antimalarials, several derivatives featuring the pyrazolopyridine scaffold were designed and tested against leishmania amazonensis, in the evolutive form of promastigotes [446] . thus, both mannich bases 295 (r 1 ¼ c 6 h 5 , r 2 ¼ ch 3 , c 6 h 5 ) (fig. 55 ) are active antileishmanial agents with ic 50 values of 390 and 120 nm, whereas aminoquinoline derivative amodiaquine had ic 50 ¼ 890 nm. from the structureeactivity point of view, the results suggest that pyrazolopyridine ring system is a bioisostere of quinoline, and that the presence of the carbethoxy group in the structure of candidates 295 does not influence significantly the biological activity. amebiasis is a contagious disease of the human gastrointestinal tract that is caused by parasitic protozoa e. histolytica. all the candidates in a small library of piperazine mannich bases 139d of 5-(4pyridinyl)-2,3-dihydro-1,3,4-oxadiazole-2-thione ( table 2) were active against hm1:imss strain of e. histolytica, but only three of them were more potent than reference drug metronidazole (ic 50 ¼ 1.81 mm) [447] . the amine residues in the aminomethyl function of these active mannich bases were 4-ethylpiperazin-1-yl (ic 50 ¼ 327 nm), 4-(4-fluorophenyl)piperazin-1-yl (ic 50 ¼ 245 nm), and 4-(2-methoxyphenyl)piperazin-1-yl (ic 50 ¼ 1.06 mm). in addition, cytotoxicity of these antiamoebic candidates was low (in the concentration range of 2.5e250 mm). out of four newly synthesized mannich bases 296 of praziquantel (fig. 55) , two candidates (nr 2 ¼ n(c 3 h 7 -n) 2 and nhch 2 ch 2 oh) exhibited significant in vitro anti-schistosoma activity (100% worm killing at 40 mm and 30 mm, respectively), but they were not as efficient as praziquantel itself (100% worm killing at 10 mm) [448] . platelets are crucial for hemostasis, as they connect to one another through receptor bridges, form aggregates, and finally plug the tear in the interrupted endothelium. however, the aggregation of platelets leading to formation of clots can also be triggered by irregularities on the vessel wall, resulting in abnormal clot formation, which is the primary factor in the development of thrombotic disorders such as unstable angina, myocardial infarction, stroke and peripheral vascular diseases, especially when it occurs in the coronary artery. platelet aggregation is also initiated by endogenous substances, such as collagen, thrombin, prostaglandin endoperoxides, thromboxanes, arachidonic acid, adenosine diphosphate (adp), etc. inhibition of platelet aggregation represents a promising strategy for the treatment of thrombotic diseases, and several studies have reported the activity of mannich bases as inhibitors of platelet aggregation. several types of phenolic mannich bases represented by structures 24e32 (fig. 6) were also evaluated as inhibitors of platelet aggregation induced by adp or collagen at concentrations of 20 mm or 10 mg/ml, respectively [449] . numerous candidates having various structures showed good inhibitory effects and were more potent than reference drug clopidogrel, whereas nine compounds in this collection exhibited inhibitory activity in the range of 90e100% in both models. good platelet aggregation inhibitory activity was associated with the presence of a pyridyl moiety as ring b in the structure of chalcone analogues, and also by the presence in ring a of a hydroxy group meta to the carbonyl function. phenolic mannich bases 41 (r ¼ r 1 ¼ oh, r 2 ¼ aminomethyl) of scutellarein (fig. 7) were investigated as thrombin inhibitors, and their influence on several parameters such as prothrombin time, activated partial thromboplastin time, thrombin time and fibrinogen was determined [432] . all of these candidates showed greater thrombin inhibitory activity compared to parent scutellarein. mannich base derived from scutellarein and containing a morpholinyl residue in the aminomethyl function was the most potent of all, and was subsequently selected for molecular docking experiments with thrombin. these experiments revealed that the morpholinylmethyl group occupies deep pocket s3 of the thrombin binding site, whereas the scutellarein part of the inhibitor's molecule is anchored by three hydrogen bonds within the active site. the effect of mannich bases 289 (r 1 ¼ ch 3 , c 2 h 5 , n-c 4 h 9 , ch 2 c 6 h 5 ; r 2 ¼ ch 3 , c 6 h 5 ) and 290 (r 1 ¼ ch 3 , n-c 4 h 9 , ch 2 c 6 h 5 ; (fig. 54 ) on adp-induced aggregation of rabbit thrombocytes was studied in vitro [442] . several candidates 289 and most candidates 290 were found to have lower effective concentrations (ec 50 ) at which they decrease the degree of aggregation by half than reference drug acetylsalicylic acid. cellular effects of thrombin, including platelet aggregation, are mediated through the activation of thrombin receptor par-1 belonging to the family of four g-protein-coupled receptors called protease-activated receptors. par-1 has become an attractive drug discovery target, and peptide-mimetics or small organic molecules with par-1 antagonist properties have already been designed and synthesized. an indole mannich base motif has been incorporated in the structure of a series of novel peptide-mimetics 297 (r ¼ 4-ch 3 oc 6 h 4 ch 2 , 3,4-f 2 c 6 h 3 ch 2 , naphthalen-1-ylmethyl, naphthalen-2-ylmethyl) ( fig. 56 ) capable to bind to par-1 [450] . the ability of these candidates to inhibit par-1-induced platelet aggregation has been tested by measuring the degree of aggregation of human platelets, and also by establishing the degree of inhibition of contraction of aortic rings. compounds 297 (r ¼ 4-ch 3 oc 6 h 4 ch 2 , 3,4-f 2 c 6 h 3 ch 2 ) belonging to the series of 6-aminoindole derivatives were the most potent candidates, with values of inhibitory concentration that were about 5 times lower than reference compound rwj54003 for 75% inhibition of platelet aggregation. in addition, these results were confirmed by the enhanced inhibitory activity of these two candidates on the contraction of aorta rings when compared to the activity of rwj54003, an effect that became more evident with the increase of par-1 dose. candidates in the series of 5-aminoindole derivatives were found inactive or weak inhibitors in both assays. anti-ulcer agents are a class of drugs used to treat ulcers in the stomach and the upper part of the small intestine. acid peptic disease is a chronic pathology that affects millions of people worldwide, and it has been estimated that 10% of the world population will develop this condition in their lifetime [451] . an imbalance between aggressive factors (such as gastric acid, helicobacter pylori infection, excessive intake of anti-inflammatory drugs, immoderate consumption of alcohol, high concentrations of reactive oxygen species) on one hand, and protective factors (e.g., mucus, bicarbonate anion, prostaglandins, good blood flow, efficient cellular repair, endogenous and exogenous antioxidants) on the other hand is considered to be the cause of ulcers. treatment of ulcers and their symptoms relies on pain relievers, antiacids and cytoprotective agents to allow the healing of ulcers, and agents to prevent the recurrence of ulcers, such as proton pump inhibitors, muscarinic antagonist pirenzepine or h2 receptor antagonist cimetidine. the ability of mannich bases to act as anti-ulcer agents has been reported in a small number of studies. anti-ulcer activity of saminomethyl derivatives 170 generated from 4,6-diaryl-2mercaptopyrimidines (fig. 29 ) as substrates and secondary aliphatic amines as amine reagents in the mannich reaction was evaluated in vivo using aspirin-induced ulcer model in albino rats [241] . based on the ulcer score, the mean ulcer index and the degree of protection were calculated and compared to those of reference drug omeprazole. five candidates 170 (r 1 ¼ 4-clc 6 h 4 , r 1 ¼ 3-no 2 c 6 h 4 or 4-ch 3 oc 6 h 4 ) offered more than 50% protection, while other five mannich bases 170 (r 1 ¼ 4-clc 6 h 4 , r 1 ¼ 4-clc 6 h 4 , 3-no 2 c 6 h 4 or 4-(h 3 c) 2 nc 6 h 4 ) had only moderate anti-ulcer activity (approximately 30% protection) compared to omeprazole (99% protection). aminomethylation of 1,4-dihydropyrimidines with sulfanilamide as amine reagent afforded mannich bases 298 (fig. 57) , whose ability to reduce the volume of acid secretion was determined by pyloric ligation method [452] . three candidates 298 (r ¼ 4-ch 3 oc 6 h 4 , 3-ch 3 o-4-hoc 6 h 3 , 2-furyl) presented good antiulcer activity (ulcer indices in the range of 0.18e0.35 at a dose of 10 mg/kg), while reference drug omeprazole had an ulcer index of 0.08 at a dose of 1 mg/kg. a small number of furo [3,2-g] flavones of type 299 (r 1 ¼ c 6 h 5 , 4-clc 6 h 4 , 3-pyridinyl; r 2 ¼ h or ch 3 ) and 300 (r 1 ¼ c 6 h 5 , 3-pyridinyl) (fig. 57) , aminomethylated either at position 6 or 9, have been evaluated as gastroprotective agents using the ethanol-induced gastric ulcer model in rats [453] . the mean values of the protection index for these mannich bases were in the range of 20e44%, and no comparison with an anti-ulcer reference drug was provided in the study. the best three candidates 299 (r 1 ¼ 3-pyridinyl, r 2 ¼ ch 3 , nr 2 ¼ 4-methylpiperazin-1-yl), 300 (r 1 ¼ 3-pyridinyl, r 2 ¼ och 3 , nr 2 ¼ 4-methylpiperazin-1-yl) and 300 (r 1 ¼ c 6 h 5 , r 2 ¼ h, nr 2 ¼ 4-morpholinyl) have a methoxy group at position 4 as a common structural feature; the corresponding mannich bases having a hydroxyl group instead of methoxy were significantly less active. mental disorders comprise a broad range of problems, with different symptoms, generally characterized by some combination of abnormal thoughts, emotions, behavior and relationships with others. common neurological conditions labeled as mental disorders include depression and anxiety, while schizophrenia and bipolar disorder stand out as mental disorders that are severe and disabling. untreated mental, neurological and substance use disorders exact a high toll, accounting for 13% of the total global burden of disease, while unipolar depressive disorder is the third leading cause of disease burden, accounting for 4.3% of the global burden of disease. current predictions indicate that depression will be the leading cause of disease burden globally by 2030 [454] . a range of different types of treatment for mental disorders are available, and the most suitable treatment depends both on the disorder and on the individual. a major option for many mental disorders is psychotherapy, while another option is psychiatric medication. amongst several groups of drugs that are currently employed in psychiatric medication, antidepressants treat clinical depression as well as anxiety and a range of other disorders, anxiolytics (including sedatives) are used for anxiety disorders and related problems such as insomnia, mood stabilizers are used primarily in bipolar disorder, and antipsychotics are used for psychotic disorders, notably for positive symptoms in schizophrenia. selective serotonin reuptake inhibitors (ssris) play an important role in pharmacotherapeutic treatment of depression. in an attempt to modify the general ssri structural motif of g-phenoxypropylamine, two ketonic mannich bases 301 (r ¼ cl, br) (fig. 58 ) derived from 4-chloro-and 4-bromoacetophenone as substrates and 4-benzylpiperidine as amine reagent were synthesized, the carbonyl function in these amino ketones was subsequently reduced, and the resulting secondary hydroxyl was then converted into an ether group through reaction with 1-chloro-4trifluoromethylbenzene [455] . although the designed ssri analogues targeted in this study showed no antidepressant activity, the intermediate ketone mannich bases 301 were as effective as reference drugs fluoxetine, sertraline or imipramine at similar dosage in a validated experimental model of depression in mice such as the forced swimming test. an innovative computer-assisted approach based on the prediction of activity spectra for substances (pass) has been applied for the discovery of new anxiolytics [456] . an initial database comprising 5494 structures was generated by virtual combinatorial design of highly diverse chemical compounds, including different types of heterocycles such as thiazoles, pyrazoles, isatins, fused imidazoles, with the view to increase the probability of finding new chemical entities as anxiolytics. out of the eight hits obtained from this database, four candidates were mannich bases. ketonic mannich base 302, mannich base 303 derived from an imidazo[2,1-b] benzo[d]thiazole and two mannich bases 304 (r 1 ¼ f, r 2 ¼ ch 3 ; r 1 ¼ no 2 , r 2 ¼ c 6 h 5 ) derived from imidazo[1,2-a]pyridines (fig. 58 ) were synthesized and tested as potential anxiolytics using the conflict situation test. all of the candidates showed an anxiolytic effect that was comparable or greater than that of reference drug medazepam. mannich base 304 (r 1 ¼ no 2 , r 2 ¼ c 6 h 5 ) was the most potent anxiolytic in this study, being two times more potent than medazepam. a collection of compounds sharing a benzoxepin scaffold as common structural feature was evaluated for their sedativeehypnotic effect using phenobarbital-induced sleep test [457] . among these compounds, three ketonic mannich bases 305 (nr 2 ¼ 1-piperidinyl, 4-methylpiperazin-1-yl, 4-morpholinyl) (fig. 58 ) decreased the onset of phenobarbital-induced sleep and prolonged the duration of hypnosis when administered in a dose equimolar to that of reference drug phenobarbital. the hypnotic activity of candidates 305 (nr 2 ¼ 1-piperidinyl, 4-methylpiperazin-1-yl) was comparable to that of phenobarbital, while mannich base 305 (nr 2 ¼ dimethylamino) exhibited only moderate activity, and mannich base 305 (nr 2 ¼ 4-(2-chlorophenyl)piperazin-1-yl) was virtually inactive. therefore, the nature of the amino residue in these mannich bases seems to have a significant impact on their hypnotic activity. in addition to biological activities of mannich bases presented so far, isolated studies were found to report various other biological activities for mannich bases. these singular results are covered in this section, without any attempt to arrange them in a systematic order. two phenolic mannich bases derived from 2,4-and 2,6-di-tbutylphenol as substrates and dimethylamine as amine reagent were evaluated as hepatoprotective agents against experimental toxic hepatitis induced by tetrachloromethane. the degree of liver damage was evaluated in terms of alanine aminotransferase activity in blood serum and malonic dialdehyde content in liver homogenates. even at a dose of 10% of the corresponding ld 50 , the ability of these two candidates to diminish the hepatotoxic action of tetrachloromethane was superior to that of reference compound emoxypine [458] . in addition, in an assay using the same tetrachloromethane-induced hepatitis model, two acetylenic mannich bases 275 (x ¼ o or ch 2 ) with a betulonic acid scaffold (fig. 51 ) were shown to decrease alkaline phosphatase activity and lower alanine aminotransferase and aspartate aminotransferase activities in blood serum compared to control [425] . acetylenic mannich base 306 (fig. 59 ) was tested on guinea pig spontaneously beating atria with a view to evaluate its negative chronotropic activity, but the potency of this candidate was approximately four times lower than that of bradycardic agent zatebradine, and was therefore excluded from subsequent testing as blocker of hyperpolarization-activated current [459] . ondansetron analogues 307 (r 1 ¼ ch 3 ) having a piperazine moiety instead of imidazole (fig. 59) were synthesized and evaluated as anti-emetic agents using a retching model [460] . all the candidates were effective to some extent when administered at a dose of 8 mg/kg, but only mannich base 307 (r ¼ 2-pyrimidinyl) had anti-emetic activity comparable to that of reference drug ondansetron at low dosage (2 mg/kg). several 7-aminomethylated b-thujaplicin analogues 308 ( fig. 59) were tested against oxidative stress-induced death of ht22 cells following exposure to glutamate [461] . piperazine-containing mannich bases 308 were more potent than parent b-thujaplicin in protecting glutamate-challenged ht22 cells, with ec 50 values ranging from 0.08 to 1.7 mm. because the most potent candidates were those having a chroman moiety as substituent at n4 in the piperazine residue, the high in vitro neuroprotective activity of these mannich bases may be due to the potent antioxidant effect imparted by the chroman moiety. analogous morpholine mannich base 308 was also active in protecting ht22 cells from oxytosis, but it was less active than the piperazine-containing b-thujaplicin derivatives. the presence of the isopropyl group also seems to be important for the neuroprotective activity of these compounds, since an analogous mannich base derived from tropolone was not active. estrogen deficiency after menopause is one of the most common causes of osteoporosis. hormone replacement therapy is widely used to prevent bone loss, although it presents potential drawbacks such as increased risk of uterine bleeding and/or hyperplasia, increased risk of endometrial, breast or ovarian cancer, higher occurrence of myocardial infarction, cardiovascular disease, etc. conjugate 309 of 17b-estradiol and iminodiacetic acid (fig. 59 ) was designed as an estrogen-containing, bone-seeking agent that could prevent bone loss with lesser side effects, and was subsequently synthesized by means of the mannich reaction [462] . mannich base 309 showed significant affinity for bone, but lower affinity for ovary and uterus than 17b-estradiol, while it maintained 92% of the affinity of 17b-estradiol for osteoblast estrogen receptors. candidate 309 did not induce uterine hypertrophy, and lower levels of biochemical markers of bone turnover (e.g., osteocalcin, alkaline phosphatase, c-terminal telopeptide fragment of type i collagen cterminus) were found in the group of rats treated with 309 than in ovariectomized rats, which suggests decreased bone turnover. administration of candidate 309 improved bone mineral density and trabecular architecture after ovariectomy, but did not suppress body weight increase. these results suggest that compound 309 is effective in preventing ovariectomy-induced bone loss while exhibiting exhibited fewer adverse side effects than 17b-estradiol, making mannich base 309 a better choice for the prevention of postmenopausal osteoporosis. three bis-p-mannich bases 310 (r 1 ¼ r 2 ¼ ch 3 ; r 1 er 2 ¼ (ch 2 ) 3 ; r 1 er 2 ¼ (ch 2 ) 4 ) (fig. 59) , derived from diethyl phosphite as substrate and amino acids as amine reagents in the mannich reaction, have been used to investigate the induction of adipogenic or osteogenic differentiation of mesenchymal stem cells isolated from human adipose tissue (hmads cells) [463] . candidates 310 did not affect the adipocyte differentiation, but induced the inhibition of osteoblast formation without any detectable cytotoxic effect, whereas reference drug sodium alendronate elicited a cytotoxic effect even at the lowest concentration used in the study (10 à7 m). therapeutic efficacy and toxicity profiles of ketonic double mannich base 311 (fig. 59) , a putative immunosuppressant which preferentially inhibited jak3 as opposed to several other kinases, were examined [464] . candidate 311 blocked il-2-induced activation of jak3 and its downstream substrates stat5a/b, while it failed to inhibit several other enzymes, including growth factor receptor tyrosine kinases, src family members, and serine/threonine protein kinases. mannich base 311 alone prolonged kidney allograft survival and induced transplantation tolerance, and its combination with cyclosporin a presented therapeutic synergism. candidate 311 showed no nephrotoxicity, did not affect hematopoiesis or lipid metabolism, and was not metabolized by the cytochrome p450 3a4 isoform. therefore, because mannich base 311 prolongs allograft survival without several toxic effects associated with current immunosuppressive drugs, it may provide significant clinical benefits for transplant patients. an important number of studies report the activity of mannich bases as enzyme inhibitors. although the studies covered in this section usually validate the importance of the topic through a rational and evidence-supported connection between the enzyme under scrutiny and a certain medical condition, they only examine the action of mannich bases as enzyme inhibitors, and do not provide any experimental evidence that these mannich bases could be actually useful agents for the treatment of specific medical conditions. starting from tacrine, the first drug approved for the treatment of alzheimer's disease and a potent inhibitor of both acetylcholinesterase (ache) and butyrylcholinesterase (buche), multifunctional compounds 279 (fig. 53 ) that combine neuroprotective, antioxidant, metal-binding properties, and dual inhibition of ache and buche in a single small molecule have been designed and synthesized through the mannich reaction [431] . tacrinee8hydroxyquinoline hybrids 279 were potent inhibitors of both ache and buche of bovine origin with ic 50 ranging from submicromolar to nanomolar concentrations. candidates containing an unsubstituted 8-hydroxyquinoline moiety and a methylene tether of 7e10 carbon atoms had the most potent inhibitory activities. selected mannich bases 279 were evaluated as inhibitors of human cholinesterases, and they exhibited ic 50 values in the range of 0.5e5.5 nm against ache, and in the range of 6.5e55 nm against buche. mannich base 279 (r 1 ¼ r 2 ¼ h, n ¼ 7) was the most potent dual inhibitor of human ache and buche in this library [431] . other mannich base hybrids 312 (fig. 60) were designed as dual binding site inhibitors of ache by combining tacrine (a recognized inhibitor of catalytic binding site of ache) with the indanone moiety of donepezil (known to be responsible for the binding of this drug to the peripheral site of ache) [465] . their evaluation as inhibitors of ache (bovine erythrocyte) and buche (human serum) using ellman's method showed that the activity of two of these candidates was modest, while the third candidate 312 (z ¼ h, r ¼ och 3 , x ¼ (ch 2 ) 3 ) exhibited moderate activity against ache (25 nm) and good activity against buche (0.6 nm). apparently, the difference in activity between these candidates is due to the lengthening by one methylene group of the alkyl chain linking the tacrine and indanone moieties. 2-benzoxazolone mono-mannich bases 313 (fig. 60 ) derived from secondary aliphatic amines or primary arylamines, and bis-mannich bases 314 derived either from primary aliphatic amines (x ¼ nr, r ¼ c 2 h 5 , r 1 c 6 h 4 ch 2 ch 2 , r 1 ¼ h, 4-cl, 3,4-(ch 3 o) 2 ) or piperazine have been evaluated for ache inhibitory activity using ellman's method. the degree of inhibition was in the range of 70e82% at a concentration of 1 mm, but decreased to 8e34% at 0.1 mm, whereas the degree of ache inhibition by tacrine was virtually the same at both concentrations (greater than 99%) [466] . the most potent inhibitor in this series at both concentration was bis-mannich base 314 derived from piperazine. inhibitory activity against ache and buche of phenolic mannich bases 58 (r 1 ¼ h, ch 3 , allyl, prenyl; nr 2 ¼ n(ch 3 ) 2 , n(c 2 h 5 ) 2 , 1pyrrolidinyl, 1-piperidinyl, 4-morpholinyl) of xanthone derivatives ( fig. 10 ) was found to be moderate to good compared to that of reference drug galantamine [467] . candidates 58 derived from diethylamine as amine reagent in the mannich reaction were generally the most potent inhibitors of both enzymes. the nature of alkyl moiety r 1 modulates the selectivity of inhibitors 58 towards one of these enzymes; thus, the most potent inhibitors of ache feature a methyl group as r 1 , whereas the increasing bulkiness of this substituent appears to favor the inhibition of buche. mannich base 58 (r 1 ¼ prenyl, nr 2 ¼ n(c 2 h 5 ) 2 ) was the most potent dual inhibitor in this series, and molecular docking studies were performed with the view to garner information on the binding mode of this inhibitor with both enzymes. several phenolic mannich bases 315 of naturally occurring flavanone oroxylin a (fig. 61) were more potent inhibitors of intestinal a-glucosidase than parent oroxylin a, but the same candidates were less potent than oroxylin a against yeast a-glucosidase [468] . nonetheless, the most potent inhibitor of intestinal aglucosidase in this series was still 5.5 less potent than reference drug acarbose. because all of the candidates 315 which failed to inhibit both enzymes were derived from acyclic secondary amines as amine reagents in the mannich reaction, the alicyclic nature of secondary amines may responsible for the good inhibitory activity against a-glucosidases in this series. ketonic mannich bases 316 derived from nabumetone (fig. 61) possess modest inhibitory activity against intestinal a-glucosidase (approximately 20 mm), in addition to modest inhibitory activity against another therapeutic target for type 2 diabetes mellitus, obesity and related states of insulin resistance, namely proteintyrosine phosphatase 1b [469] . however, two mannich bases 316 (r ¼ h or 4-oh) were good agonists of peroxisome proliferatoractivated receptors, which are major regulators of lipid and glucose metabolism. a novel series of inhibitors was designed by retaining the nabumetone moiety, while sulfanilamide and 3amino-5-methylisoxazole served as amine reagents in the synthesis of ketonic mannich bases of type 317 and 318, respectively (fig. 61 ) [470] . candidates 317 were generally more potent inhibitors of a-glucosidase than analogous 318, suggesting that sulfonamide moiety is a more potent pharmacophore than aminoisoxazole. mannich base 317 (r ¼ 4-br) inhibited 80% of a-glucosidase activity at a dose of 10 mg/ml. replacement of the sulfonamide moiety with 4-aminobenzoic acid, with a view to mimic the acidic, hydrophilic part of molecules with known antidiabetic activity, led to a novel series of mannich bases 319 (r 1 ¼ h). a second series of candidates 319 (r 1 ¼ c 2 h 5 ) (fig. 61) was also evaluated as a-glucosidase inhibitors, but their activity was generally poorer than that of mannich bases 319 (r 1 ¼ h) derived from 4-aminobenzoic acid [471] . for example, the most potent a-glucosidase inhibitor (67% inhibition) in the series derived from 4-aminobenzoic acid was candidate 319 purine nucleoside phosphorylase (pnp) catalyzes the phosphorolytic cleavage of inosine, guanosine and their 2 0 -deoxy analogues to (deoxy)ribose 1-phosphate and hypoxanthine or guanine. genetical deficiency in pnp leads to complete t-cell deficiency early in life and eventual death of infants from virus infections, and pnp has been identified as a target for the treatment of t-cell lymphoma, rheumatoid arthritis, psoriasis, multiple sclerosis, and other t-cell mediated disorders. a transition state analogue is a compound that occupies the catalytic site of an enzyme and induces the slow conformational changes that would normally occur to place the catalytic site in the appropriate geometry for the search that would locate the transition state with the normal substrate of the enzyme. schramm et al. have developed a series of mannich bases of 9-deazahypoxanthine as transition state analogues for purine nucleoside phosphorylase inhibition, generally called immucilins. although aminomethylated purines and analogues have been present within the firstand second-generation of pnp inhibitors [472, 473] , they have been obtained through reductive amination, and not through direct mannich reaction. because a more expeditious synthesis of second-generation pnp inhibitors by means of the mannich reaction has been later developed [474] , the third generation of these inhibitors included several examples of mannich bases 320 (fig. 62 ) obtained through direct aminomethylation of substrate 9-deazahypoxanthine with amino alcohols such as ethanolamine, 3-amino-propan-1-ol, 4-amino-butan-1-ol, diethanolamine and 3-(2-hydroxyethylamino)propan-1-ol as amine reagents [475] . unfortunately, all these candidates obtained through direct aminomethylation proved to be modest or poor inhibitors of pnp (dissociation constants k i in the range of 780 to 120,000 pm) compared to other mannich bases previously investigated, such as 321 (dadme-immucilinh, k i ¼ 8.5 pm) or 322 fig. 61 . mannich bases as inhibitors of a-glucosidase. (dadme-immuciling, k i ¼ 7.0 pm) (fig. 62) . nonetheless, this study identified two acyclic, simplified alternatives of immucilin analogues, namely 323 (k i ¼ 9.0 pm) having an open-chain amino alcohol with two asymmetric carbon atoms, and 324 (k i ¼ 5.0 pm) derived from achiral dihydroxyaminoalcohol seramide (fig. 62) , as potent inhibitors of pnp. because the enantiomers of both immu-cilinh and dadme-immucilinh 321 have been shown to have pnp binding properties that differ considerably [476] , and since access to enantiomerically pure starting azasugar employed for the generation of 322 requires a suitable enzyme-based route, the discovery of the two simpler mannich bases 323 and 324 represents a significant step forward in the development of clinically useful pnp inhibitors. sulfur-containing transition state analogues have been developed as well, originally as specific inhibitor of pnp from p. falciparum, but it was later shown that sulfur-containing candidates analogous to dadme-immucilinh 320 or mannich base 323 also bind to human native pnp [477] . although human pnp tolerates substitution of 5 0 -hydroxyl in transition state analogues 321 and 324 with alkylthio and arylthio groups, the slow-onset nature of inhibition is lost, and inhibitor dissociation constants increase by two to three orders of magnitude. fluorine-containing analogue 325 (fig. 62) of dadme-immucilinh 321 has also been evaluated as human pnp inhibitor, and its enantiomers exhibit slow-onset binding constants of 32 pm for [(3s,4s)-325] and 1.82 nm for [(3r,4r)-325] [478] . in addition, direct mannich reaction has been used to generate in a similar fashion potent transition state inhibitors for other enzymes, such as methylthioadenosine phosphorylase methylthioadenosine nucleosidase [479, 480] or purine specific nucleoside hydrolase [481] . a large library of a-, band g-amino ketones was screened with a view to discover inhibitors of transglutaminase, but only b-amino ketones (ketonic mannich bases) strongly inhibited this enzyme [482] . mannich bases derived from aliphatic ketones as substrates, or those derived from alkyl aryl ketones as substrates and primary amines as amine reagents were weak inhibitors (ic 50 > 30 mm) of transglutaminase. potent transglutaminase inhibitory activity resided with ketonic mannich bases derived from alkyl aryl ketones as substrates and secondary aliphatic amines as amine reagents. both the nature of the aryl moiety in the substrate and the nature of the alkyl groups attached to the nitrogen atom influenced transglutaminase inhibitory activity of these candidates. generally, heteroaryl-substituted substrates (2-thienyl, 2-benzothienyl, 2furyl, 3-furyl, pyridinyls, pyrazinyl) fared better than substrates having aryl moieties (phenyl, naphthyls), and t-butyl, isopropyl, benzyl or 2-hydroxyethyl groups (but not phenyl) as alkyl substituent at nitrogen afforded potent transglutaminase inhibitors, such as candidate 326 (ic 50 ¼ 81 nm) (fig. 63) . because disulfides are well-known transglutaminase inhibitors, a novel series of b-amino ketones 327 (r ¼ h, n ¼ 0; r ¼ cooch 3 , n ¼ 0; r ¼ cooch 3 , n ¼ 1) (fig. 63 ) derived from cystamine, dimethyl cystine ester, and dimethyl homocystine ester as amine reagents in the mannich reaction were synthesized and found to be approximately 300 times more active than the starting disulfides [483] . mannich bases derived from 2-acetylthiophenes were once more the most potent inhibitors in this series, while the nature of the disulfide-containing amine moiety did not significantly influence the activity of candidates 327 having the same heterocyclic or carbocyclic moiety r 1 . mannich bases 328 derived from 3,4-dimethylphenol ( fig. 63 ) have been evaluated in vitro as inhibitors of two human carbonic anhydrase (hca, ec 4.2.1.1) isozymes i and ii using the hydratase and esterase assays, respectively [484] . only two candidates exhibit weak hca ii inhibitory effects on esterase activity, whereas other two mannich bases 328 could be used as carbonic anhydrase activators. phenolic mannich bases 280 derived from quercetin (fig. 53 ), 329 derived from baicalein and 330 derived from 1-(2-hydroxy-4methoxyphenyl)-3-phenylprop-2-en-1-one ( fig. 63 ) were screened for inhibition of cyclin-dependent kinases using a biochemical assay method based on fluorescence resonance energy transfer [485] . candidates 280 and 330 are essentially devoid of inhibitory activity of cyclin-dependent kinases; on the other hand, baicalein mannich bases 329 were 6e20 times more potent than the parent flavone, suggesting that the presence of a nitrogen atom at c-8 is crucial for the inhibition of cyclin-dependent kinases, while the presence of a second heteroatom in the amine moiety (e.g., oxygen in morpholine, sulfur in thiomorpholine, nitrogen in n-methylpiperazine) further increases the potency of these candidates. virtual screening of a commercial library containing drugs and drug-like molecules against a 3d model of heparanase led to the identification of several candidates with high scores for binding affinity; they were subsequently tested in nmr competitive inhibition experiments using suramine as a known heparanase inhibitor [486] . one of these candidates is amodiaquine 201 (fig. 38) , and a subset of fourteen representative compounds that retained the characteristic 4-phenylamino-chloroquinoline scaffold of amodiaquine, but presented different functional groups in the phenylamino moiety, was further selected for nmr competitive inhibition experiments. although no candidate with improved heparanase inhibitory activity over that of amodiaquine emerged from this subset, several structural requirements for the binding of an inhibitor to the active site of the enzyme were obtained. four mannich bases 331 of nitroxoline (fig. 63 ) were found to inhibit efficiently the activity of methionine aminopeptidase-1 from burkholderia pseudomallei with ic50 values ranging from low micromolar to 30 nm, and a few candidates in this series also showed in vitro growth inhibition of burkholderia thailandensis [487] . hexacyclic indole mannich bases 332 (r 1 ¼ h or ch 3 , nr 2 ¼ n(c 2 h 5 ) 2 , 1-pyrrolidinyl) and 333 (r 1 ¼ ch 3 , nr 2 ¼ 4morpholinyl) derived from annelated maleimidoindolodiazepines (fig. 63 ) were tested against a panel of 25 human protein kinases, but they were inactive against all of the tested enzymes at micromolar concentrations [488] . out of four synthesized acetylenic mannich bases 334 (nr 2 ¼ n(c 2 h 5 ) 2 , 1-pyrrolidinyl, n-allyl-n-methyl, n-(2dimethylaminoethyl)-n-methyl) derived from 7-(prop-2-ynyloxy) chromen-2-one (fig. 63) , three were inhibitors of squalene-hopene cyclase [489] . candidate 334 (nr 2 ¼ n-allyl-n-methyl) was half as potent as the hoffmanela roche's anticholesteremic drug ro 48-8071, which is an effective inhibitor of both squalene-and oxidosqualene cyclases that features the same secondary amino group in its structure. thiazolidinone c-mannich bases 335 (r 1 ¼ 4-ch 3 c 6 h 4 , 4-no 2 c 6 h 4 , 2-furyl, 3,4,5-(ch 3 ) 3 c 6 h 2 ; nr 2 ¼ n(c 2 h 5 ) 2 , 4morpholinyl, 4-methylpiperazinyl) (fig. 63) were evaluated as inhibitors of schistosoma mansoni cercarial elastase, but they were all inactive [490] . oxadiazolethione n-mannich bases 336 having a naphthalen-1ylmethyl residue at c-5 ( fig. 63 ) were all active against mushroom tyrosinase, and 6 to 10 times more potent than reference compound kojic acid [491] . the presence of a thione group able to chelate with copper, and the ability of the cyclic secondary amine to form extensive hydrophobic contacts within the binding site of tyrosinase appear to be responsible for the activity. another oxadiazolethione n-mannich base, namely 139m (table 2) , presented moderate inhibitory activity against urease within the series of compounds investigated, but no comparison with the activity of a well-established urease inhibitor was provided [201] . 19.1. ligands of 5-hydroxytryptamine receptors 5-hydroxytryptamine receptors (5-ht receptors), also known as serotonin receptors, belong to the group of g protein-coupled receptors (with the exception of 5-ht 3 , which is a ligand-gated ion channel). they are found in the central and peripheral nervous systems, and their function is to mediate both excitatory and inhibitory neurotransmission. 5-ht receptors are activated by their natural ligand, the neurotransmitter serotonin. mannich bases 245 (n ¼ 1 or 2) (fig. 44) were evaluated as ligands for the types of 5-ht receptors that could be involved in the mediation of the anticonvulsant effect of these compounds, but the candidates exhibited only moderate to low affinity for both 5-ht 1a (k i ¼ 81e370 nm) and 5-ht 2a receptors (k i ¼ 126e1370 nm) [383] . the nature of the spiranic cycloalkyl group did not influence serotonin receptor affinity significantly, although cyclohexylsubstituted candidates 245 were slightly more active than cyclopentyl-substituted analogues. the nature of the substituent in the aryl moiety at n-4 of piperazine ring modulates the selectivity towards one of these 5-ht receptors; thus, mannich bases 245 (r ¼ 2-och 3 ) showed the highest affinity for 5ht 1a receptors and the lowest affinity for 5-ht 2a receptors, while the highest 5-ht 2a receptor affinity was shown by mannich bases 245 (r ¼ 3-cf 3 ). in addition, computer-aided design of novel mannich bases of imidazoline-2,4-diones led to the identification of several compounds with potential dual affinity for 5ht 1a receptors and serotonin transporter, whose synthesis and evaluation finally led to candidates 337 (r ¼ h or f) (fig. 64) having the desired pharmacological profile [492] . a series of quinoxaline-2-carboxamides n-substituted with phenolic mannich bases moieties having either one or two aminomethyl functions have been synthesized and tested for 5-ht 3 receptor antagonistic activity in longitudinal muscle-myenteric plexus preparation from guinea pig ileum against 5-ht 3 agonist, 2-methyl-5-ht [493, 494] . candidates 338 (r 1 ¼ cl) (fig. 64 ) exhibited mild to moderate antagonist activity, and double mannich bases were generally less potent than their mono-mannich bases counterparts, but mannich base 338 (r 1 ¼ cl, r 2 ¼ h, nr 2 ¼ 4-methylpiperazin-1-yl) had 5-ht 3 receptor antagonistic activity comparable to that of reference compound ondansetron [493] . replacement of chlorine with methoxy led to less potent candidates 338 (r 1 ¼ och 3 ), whereas candidates with an ethoxy group at position 3 of quinoxaline ring were even less potent [494] . the presence of piperazines in the aminomethyl moiety of candidates 338 seemed to be more favorable for 5-ht 3 receptor antagonistic activity than the presence of other cyclic secondary amines. dopamine receptors are g protein-coupled receptors that are widely distributed in the brain, and whose primary endogenous ligand is the neurotransmitter dopamine. there are at least five subtypes of dopamine receptors, but d 1-2 receptor subtypes usually predominate, as they are 10e100 times more numerous than d [3] [4] [5] subtypes. discovery of l-745,870 339 [495] and fauc 113 340 [496] (fig. 65 ) as potent and selective d 4 receptor ligands has fueled the search for novel chemical entities having the ability to bind to dopamine receptors. based on the observation that these two ligands and many other dopamine receptor ligands feature a methylene bridge between the piperazine ring and the (hetero)aromatic part of the molecule, various analogues have been designed and synthesized. however, the majority of these analogues were obtained either through nucleophilic displacement by an appropriately substituted piperazine of a halogen atom from a halomethyl derivative of a selected (hetero)aromatic substrate, or through reductive amination of a formyl-substituted (hetero)aromatic substrate in the presence of a piperazine. literature search revealed a few examples when the mannich reaction was employed to synthesize the ligands to be evaluated for dopamine receptor binding ability. in addition to pyrrolo [2,3-b] pyridine and pyrazolo [1,5-a]pyridine ring systems used to in the generation of ligands 339 and 340, imidazo[1,2-a]pyridine is another example of an azaindole ring system that was aminomethylated to yield mannich bases 341 (fig. 65) [497] . receptor binding profiles of candidates 341 and several mono-mannich bases 342 derived from pyrrole ( fig. 65) were determined in vitro by measuring their ability to compete with [ 3 h]spiperone for the cloned human dopamine receptor subtypes d 2 long , d 2 short , d 3 and d 4.4 , while d 1 receptor affinities were measured employing an assay that used d 1 selective ligand [ 3 h]sch 23390 and porcine striatal membranes. all of the mannich bases 341 and 342 were selective ligands for the d 4 receptor subtype, and the candidates with an imidazo[1,2-a]pyridine scaffold were more potent than their counterparts having a pyrrole ring. the most potent mannich bases 341 (r ¼ 2-c 2 h 5 o, 2,3-cl 2 , 3-cf 3 ) showed an affinity for the dopamine d 4 receptor in the low nanomolar range, and their selectivity for this receptor subtype exceeded that of reference drug clozapine [497] . several small series of candidates comprising mannich bases 343, 344 and 345 (r 1 ¼ h or cl) having a pyrrolo[2,3-d]pyrimidine scaffold (fig. 65 ) were designed and evaluated as ligands for dopamine receptor subtypes d 1 , d 2 long , d 2 short , d 3 and d 4.4 , but they had no appreciable affinity for any of the dopamine receptors tested [498] . the strongest binding in these series was recorded for candidates 343 (r ¼ 2-ch 3 o) and 345 (r 1 ¼ h, r ¼ 2-ch 3 o) at dopamine d 4.4 receptor, with k i values of 2.4 and 1.9 nm, respectively. novel heteroaromatic scaffolds have been employed in the synthesis of mannich bases 346, 347 and 348 (fig. 65 ) as potential d 4 receptor ligands [499] . candidate 346 derived from pyrrolo[2,3c]pyridine ring system bound selectively at d 4.4 receptor (k i ¼ 6.4 nm). evaluation of mannich bases 347 generated from imidazo [1,2-c] pyrimidines and having various substituents at positions 2, 5 and 7 led to different results, depending on the nature and number of these substituents. thus, mannich base 347 (r 1 ¼ r 2 ¼ r 3 ¼ h) derived from the unsubstituted scaffold and all of the mannich bases 347 (r 1 ¼ ch 3 , r 2 ¼ r 3 ¼ h) obtained from 7methylimidazo[1,2-c]pyrimidine had no effect on dopamine d 4 receptor. the results for the binding assay for mannich bases derived from dimethyl-substituted imidazo [1,2-c] pyrimidines showed that candidates 347 (r 1 ¼ r 3 ¼ ch 3 , r 2 ¼ h) have binding affinities to dopamine d 4 receptor in the range of reference drug clozapine, while candidate 347 (r 1 ¼ r 2 ¼ ch 3 , r 3 ¼ h) is a weak ligand. further modifications, such as introduction of a carbethoxy group in the structure of candidates 347 (r 1 ¼ r 2 ¼ ch 3 , r 3 ¼ cooc 2 h 5 ), or two methoxy groups in mannich bases 347 (r 1 ¼ r 2 ¼ och 3 , r 3 ¼ h), led to compounds without affinity for dopamine d 4 receptor. in the series of mannich bases derived from 2,5,7-trimethylimidazo[1,2-c]pyrimidine, only candidate 347 (r 1 ¼ r 2 ¼ r 3 ¼ ch 3 , r ¼ 2-och 3 ) had good binding properties for the dopamine d 4 receptor, whereas another candidate 347 (r 1 ¼ r 2 ¼ r 3 ¼ ch 3 , r ¼ 4-och 3 ) exhibited a slight selectivity for dopamine d 3 receptor. use of pyrrolo [3,2-d] pyrimidine as scaffold led to some of the most potent ligands of dopamine d 4 receptor in this study (k i values between 2.4 and 3.8 nm). the nature of the amino group at position 2 of pyrrolo [3,2-d] pyrimidine scaffold modulates the activity; thus, mannich bases 348 (nr 2 ¼ 1pyrrolidinyl) had good affinities for dopamine d 4 receptor, but replacement of pyrrolidine with morpholine resulted in significant loss of dopamine d 4 receptor binding affinity. two novel indole mannich bases 59 (r ¼ 2-c 2 h 5 oc 6 h 4 , 2,3-cl 2 c 6 h 3 ) (fig. 11) were synthesized through direct aminomethylation and evaluated as ligands for several dopamine receptor subtypes, along with other heteroarylmethylpiperazines [500] . both candidates were potent and selective dopamine d 4 receptor ligands, and mannich base 59 (r ¼ 2-c 2 h 5 oc 6 h 4 ) was the most potent compound in this library (k i ¼ 0.03 nm). in addition, mannich base 59 (r ¼ 2,3-cl 2 c 6 h 3 ) was the most potent candidate having a 4-(2,3-dichlorophenyl)piperazine moiety, suggesting the importance of indole as (hetero)aromatic moiety in this type of dopamine d 4 receptor ligands. the effect on the affinity to d 2 , d 3 and d 4 dopamine receptor subtypes of replacement of 4-arylpiperazinyl in mannich bases with other monocyclic or bicyclic amine residues was also investigated [501] . mannich bases 349 (x ¼ ch or n) (fig. 66) derived from 3-(4-chlorophenyl)-8-azabicyclo[3.2.1]octan-3-ol as amine reagent in the mannich reaction had modest affinity and moderate selectivity for dopamine d 3 receptor subtype compared to d 2 and d 4 receptor subtypes. pyrrolidinol analogues 350 (x ¼ ch or n) (fig. 66 ) had poor binding affinities to all subtypes of dopamine receptors, while homopiperazine analogue 351 (fig. 66 ) had moderate binding affinity to dopamine d 4 receptor subtype (k i ¼ 18.6 nm). candidate 352 derived from 3,9-diazabicyclo[4.2.1] nonane ring system as amine reagent in the mannich reaction ( fig. 66 ) bound poorly to all types of dopamine receptor investigated in this study. although candidate 353 (fig. 66 ) derived from 2,5-diazabicyclo[2.2.1]heptane ring system as amine reagent in the mannich reaction had the highest d 3 receptor affinity of all the compounds tested in this paper (k i ¼ 11 nm), it also had poor selectivity towards d 3 (k i d2 ¼ 62 nm, k i d4 ¼ 69 nm). mannich base 354 derived from 2-(2-pyrimidinyl)piperazine (fig. 66 ) had modest binding affinity to dopamine d 4 receptor subtype (k i ¼ 56 nm), but good selectivity over d 2 and d 3 subtypes. thyroid receptors are nuclear receptors belonging to a superfamily whose members function as hormone activated transcription factors. the regulation of transcription for thyroid receptors is controlled by their natural ligand, the thyroid hormone. both unliganded and liganded thyroid receptors can bind to and regulate genes under the control of thyroid response elements, but the liganded thyroid receptor complex can also recruit a particular coactivator protein out of many available, with a view to steer the course of transcriptional regulation. high-throughput screening of a library of compounds identified a number of hits for inhibitors of the interaction between thyroid receptor and its coactivator src2 (steroid receptor coactivator 2), and two of these hit compounds were ketonic mannich bases 355 and 356 (fig. 67) [502] . these candidates represent a new class of thyroid receptor antagonists that have exceptional selectivity towards b isoform and are active in the presence of thyroid hormone, while being able to silence its signaling. mannich bases 355 and 356 are most likely covalently bound to thyroid receptor as a result of an alkylation process, thus inhibiting its hormone-induced gene transcription. subsequent preliminary sar showed that a hydrophobic moiety para to the ketone function in other ketonic mannich bases analogous to 355 and 356 is an important structural feature of potent inhibitors of the thyroid receptorecoactivator interaction [503] . a second series of candidates explored the effect that the nature of the amino group in ketonic mannich bases analogous to 355 and derived from 4-nhexylacetophenone has on the interaction between coregulatory protein src2 and both isoforms of thyroid receptors. all the synthesized candidates inhibited the interaction in the low micromolar range, with a roughly 2-fold selectivity towards isoform b. these ketonic mannich bases most likely function as prodrugs for the true active species, namely the a,b-unsaturated ketones resulted through deamination. therefore, a collection of enones having various substituents at the carbonecarbon double bond was also tested with a view to provide an insight on the mechanism of action of ketonic mannich bases as inhibitors of this interaction. the results showed that the inhibition of the interaction between thyroid receptor and coactivator protein src2 depends strongly on the substitution pattern at the carbonecarbon double bond in these a,b-unsaturated ketones. the most potent was the unsubstituted enone 357 (fig. 67) , which formally arises from 355 through deamination, whereas all other substituted enones were weaker inhibitors than 357. subsequent investigations confirmed that enone 357 is the actual inhibitor, and that compound 357 is generated within the binding site, where it remains bound until it reacts with one of the local cysteine residues [504] . in addition, an attempt to examine a complex between thyroid receptor b and mannich base 355 by x-ray crystallography led to the detection in the activation function-2 cleft of thyroid receptor b of enone 357 instead of mannich base 355. although enone 357 did not react rapidly with thyroid hormone, it positioned close to several cysteine residues. several other experiments showed that, once formed, enone 357 slowly alkylates nearby cys298 residue to occlude activation function-2 pocket of thyroid receptor b. significant improvement of pharmacological properties (especially potency and therapeutic index) of inhibitors of thyroid hormone receptor coactivator binding based on ketonic mannich bases was achieved with a series of novel candidates having in the aromatic ring electron withdrawing substituents associated with high efficacy, and a sulfonyl group to reduce cytotoxicity. also, piperazinone and 4-acetylpiperazine were used as preferred amine moieties with a view to minimize ion channel activity of these inhibitors [505] . the most potent compounds 358 and 359 (r ¼ 2,3-cl 2 or 2,5-cl 2 ) (fig. 67) showed outstanding thyroid hormone receptor coactivator interaction inhibitory potency, good therapeutic index, while no inhibition of kcl-stimulated depolarization of hek293-herg cells was observed for these mannich bases. androgen receptor is a ligand-regulated transcription factor in the nuclear receptor superfamily. the receptor, which represents an important molecular target for the treatment of prostate cancer, is activated through the binding of its two natural endogenous ligands, testosterone or 5a-dihydrotestosterone. subsequent activation of androgen-responsive genes can be blocked by androgen receptor antagonists through competitive inhibition. highthroughput screening of a structurally diverse chemical library comprising 16,000 synthetic and natural compounds led to the identification of 130 hit compounds that exhibited more than 85% competitive inhibition of 5a-dihydrotestosterone binding to androgen receptor [506] . mannich base 360 (r 1 ¼ r 2 ¼ ch 3 , r 3 ¼ c 6 h 5 ) (fig. 68 ) was selected for further development owing to its consistent androgen receptor antagonist activities in a cellbased assay using monkey kidney fibroblast cv-1 cells, and fortynine analogues were designed based on its b-amino ketone core structure. nine of these analogues showed higher binding affinity to androgen receptor than 5a-dihydrotestosterone, and evaluation of the enantiomers of candidate 360 (r 1 ¼ no 2 , r 2 ¼ cl, r 3 ¼ 2furyl) showed that the androgen receptor-modulating activity and binding resided with the dextrorotatory isomer. sar within this library of ketonic mannich bases revealed that the presence of chlorine as substituent r 2 , preferably para to the amino group, is important for androgen receptor antagonistic effects, while the presence of an electron-withdrawing group as substituent r 1 is crucial for high-affinity binding to the receptor. good androgen fig. 67 . mannich bases as inhibitors of the interaction between thyroid receptors and their coregulators. receptor binding activity was found among analogues having phenyl, 2-furyl and 2-thienyl as substituent r 3 , and mannich bases with heteroaryl moiety exhibited less cytotoxicity towards hela cells than mannich bases with r 3 ¼ (substituted)phenyl. candidate 360 (r 1 ¼ no 2 , r 2 ¼ cl, r 3 ¼ 2-furyl) inhibited the growth of sc3 cells (a mouse mammary cell line that responds well to androgens), while its effects on the proliferation of pc3 cells (a human prostate cancer cell line that lacks measurable androgen receptors) were very weak. mannich bases of type 360 were subsequently disclosed as selective non-steroidal antagonists of another member of the nuclear receptor superfamily, namely progesterone receptor, and sar within a collection of mannich bases 360 showed that coordinating effects of substituents r 1 and r 3 can modulate the potency and selectivity for progesterone receptor over androgen receptor [507] . mannich bases that modulate the activity of acetylcholine receptors have been the topic of two recent studies. two quaternary salts 361 (r ¼ ch 3 , allyl) derived from bis-aminomethylated pyrazinodiindole (fig. 68) were synthesized in order to probe their binding to the allosteric binding domain of muscarinic receptor m 2 [508] . these quaternary salts showed only slightly higher binding affinity to muscarinic m 2 receptor subtype than the corresponding parent double mannich bases, which in turn exhibited relatively poor allosteric potency. due to the structural similarity of candidates 361 and neuromuscular-blocking agents, their binding affinity to the muscle-type of the nicotinic acetylcholine receptors was also determined. thus, compound 361 (r ¼ ch 3 ) had a 34-fold higher affinity for the muscle type nicotinic acetylcholine receptor than for the allosteric site of m 2 receptors. double mannich bases 362 (nr 2 ¼ 1-piperidinyl, 2-methyl-1-piperidinyl, 1-azepanyl) derived from phthalamide (fig. 68) had a competitive antagonistic effect to acetylcholine on isolated guinea pig ileum that was poorer than that of reference compound atropine, and behaved as noncompetitive antagonists at higher concentration [509] . in addition, candidates 362 (nr 2 ¼ 1-piperidinyl, 2-methyl-1-piperidinyl, 1-azepanyl) antagonize the motor effect of oxotremorine in mice, while mannich base 362 (nr 2 ¼ 2,6-dimethyl-1-piperidinyl) was inactive in both tests. indole mannich base 59 (r ¼ phenethyl) (fig. 11 ) had moderate binding affinity to both sigma-1 (k i ¼ 14.2 nm) and sigma-2 (k i ¼ 55.8 nm) receptors, and can be considered as a potent, but non-selective ligand for sigma receptors [80] . ketonic mannich bases 363 (fig. 68) derived from cyclic ketones such as a-tetralone (x ¼ ch 2 ch 2 ), chromanone (x ¼ och 2 ), indanone (x ¼ ch 2 ) or benzosuberone (x ¼ (ch 2 ) 3 ) as substrates and either 4benzylpiperidine (z ¼ ch 2 ) or 4-benzylpiperazine (z ¼ n) as amine reagents in the mannich reaction were also evaluated for their affinity towards sigma-1 and sigma-2 receptors [510] . generally, 4-benzylpiperazine-containing mannich bases were higher-affinity sigma receptor ligands than the corresponding 4benzylpiperidine-containing analogues. compared to other alkylpiperidines or alkylpiperazines that were evaluated in this study, ketonic mannich bases 363 had only moderate affinity for sigma receptors. the presence of the carbonyl group in their structure appears to be detrimental, especially for the affinity to sigma-1 receptors, and the binding affinity seems to be influenced by the size of the saturated ring fused to the aromatic ring. thus, sixmembered candidates 363 are more potent than mannich bases 363 derived from indanone or benzosuberone. however, candidate 363 (x ¼ ch 2 ch 2 , z ¼ ch 2 ) was one of the most selective compounds in this study, with a selectivity towards sigma-2 receptors of approximately 0.1 (k i-s1 ¼ 24 nm, k i-s2 ¼ 3.4 nm). in contrast, mannich bases 363 derived from 4-benzylpiperazine were consistently better ligands for sigma-1 than for sigma-2 receptors. mannich bases of indoles substituted in the carbocyclic ring with a carboxamide group were evaluated as human histamine h 3 receptor antagonists [511] . preliminary analysis of the binding affinity of candidates 364 (r ¼ i-c 3 h 7 , c-c 4 h 7 , x ¼ ch 2 , o, nc 3 h 7 -i, nc 4 h 7 -c) (fig. 68) to human h 3 receptor with respect to different positional isomers within this series established a correlation between the 3,6-substitution pattern in 364 and good human h 3 receptor affinity, while the 3,4-substitution pattern was the least favorable for h 3 receptor binding affinity. the presence of piperidine and morpholine in the aminomethyl group led to compounds with excellent histamine h 3 receptor affinity, whereas the presence of 4-substituted piperazines in the aminomethyl group was not well tolerated. several mannich bases 364 of indoles with a 3,7substitution pattern also presented reasonable potency as human h 3 antagonists; in this case, the presence of a cyclobutyl residue in the piperazine ring in the carboxamide group seems to be more favorable for human histamine h 3 binding affinity than substitution with an isopropyl residue. in the search for new ligands able to discriminate among the three a 1 -adrenergic receptor subtypes, a series of mannich bases 365 (r ¼ ch 3 , n-c 3 h 7 , ch 2 c 6 h 5 , ch 2 c 6 h 11 -c, c 6 h 5 ) derived from 1,2,3,4-tetrahydro-4h-carbazol-4-ones (fig. 68) , that are structurally related to the potent a 1 -adrenergic receptor antagonist heat, were synthesized and evaluated [512] . as a general trend, all candidates showed lower affinities than heat for all three a 1 -adrenergic receptor subtypes, and replacement of tyramine with 4-(2substituted phenyl)piperazines did not enhance the affinity towards any subtype in particular. in addition, mannich bases 366 derived from 3-acetylindole and the same amines ( fig. 68 ) were designed as "open chain" analogues of candidates 365, and their evaluation showed a slight improvement in the affinity for all three a 1 -adrenergic receptor subtypes. starting from a hit obtained by screening a large library of compounds, and employing subsequent stepwise refinement of structural features, a series of mannich bases 367 (r ¼ (substituted) phenyl) of pyrazolones (fig. 68 ) was designed as ligands of cc chemokine receptor 3 (ccr3), a potential molecular target for treatment of allergic asthma [513] . the evaluation of binding to human ccr3 receptor of candidates 367 showed that most compounds display ic 50 values around 100 nm, and that only two of these mannich bases (r ¼ 4-fc 6 h 4 and 2-pyridinyl) bound more tightly to ccr3 than the initial hit compound (ic 50 ¼ 32 nm). because the lead compound 367 (r ¼ 4-fc 6 h 4 , ic 50 ¼ 20 nm) suffered from poor water solubility and metabolic stability, less lipophilic analogues were prepared by the introduction of ionizable groups in different parts of the molecule. replacement in the structure of 367 (r ¼ 4-fc 6 h 4 ) of the 4-chlorophenyl moiety with 4-fluorophenyl, 4-nitrophenyl, 4-sulfonamidophenyl, 4aminophenyl or pyridinyl moieties, as well as substitution of the 2,6-diflurophenyl moiety with pyridinyl or pyridinyl-n-oxide led to a second series of pyrazolone mannich bases 368 ( fig. 68) with improved solubility in water and enhanced metabolic stability. although structural modulations in the r 1 moiety generally decreased the binding affinity of candidates 368 to ccr3, modifications in the r 2 part afforded potent compounds (ic 50 ¼ 12 nm). two n-mannich bases 369 (r ¼ h or f) of a pyrazole derivative (fig. 68 ) were reported in a study that examined the interaction of a series of 4-heteroaryl-2-phenylquinolines with neurokinin nk-2 and nk-3 receptors, but they were inactive (k i > 10 mm) [514] . throughout the last decade, a large number of novel mannich bases have been synthesized and evaluated as potential treatments for a multitude of diseases and medical conditions, as prodrugs, or as molecules eliciting a response from biological targets. the vast amount of data concerning the structureeactivity relationship for mannich bases derived from structurally diverse substrates has added to previous knowledge, thus allowing better insight into the design of more effective drug candidates based on the aminomethylation reaction in the future. tremendous progress has been made in the field of anticancer agents and antimicrobials, and the last decade has witnessed the chemical modification of many biologically-active, naturally-occurring substrates or well established drugs by means of the mannich reaction with a view to improve their biological activity. as such, the mannich reaction has earned its rightful place as a powerful tool in medicinal chemistry, both for the synthesis of novel chemical entities endowed with various and interesting biological properties, and for the modification of physico-chemical properties of a candidate, that ultimately influence the candidate's biodisponibility, performance and pharmacological activity as a drug. advances in the chemistry of mannich bases further advances in the chemistry of mannich bases mannich bases: chemistry and uses effect of aminomethyl (n-mannich base) derivatization on the ability of s 6 -acetyloxymethyl-6-mercaptopurine prodrug to deliver 6-mercaptopurine through hairless mouse skin prodrugs e an efficient way to breach delivery and targeting barriers gilmer, b-aminoketones as prodrugs with phcontrolled activation anticancer and cytotoxic properties of mannich bases bioactivities of chalcones cytotoxic thiol alkylators sequential cytotoxicity: a theory evaluated using novel 2-[4-(3-aryl-2-propenoyloxy)phenylmethylene]cyclohexanones and related compounds evaluation of some mannich bases of cycloalkanones and related compounds for cytotoxic activity cytotoxic and anticancer activities of some 1-aryl-2-dimethylaminomethyl-2-propen-1-one hydrochlorides cytotoxic activities of mannich bases of chalcones and related compounds cytotoxic activities of mono and bis mannich bases derived from acetophenone against renca and jurkat cells cytotoxic activities of some mono and bis mannich bases derived from acetophenone in brine shrimp bioassay cytotoxicity of some azines of acetophenone-derived mannich bases against jurkat cells synthesis of some mannich bases with dimethylamine and their hydrazones and evaluation of their cytotoxicity against jurkat cells evaluation of the cytotoxicity of some mono-mannich bases and their corresponding azine derivatives against androgen-independent prostate cancer cells cytotoxic and anticonvulsant aryloxyaryl mannich bases and related compounds biological evaluation and structureactivity relationships of bis-(3-aryl-3-oxo-propyl)-methylamine hydrochlorides and 4-aryl-3-arylcarbonyl-1-methyl-4-piperidinol hydrochlorides as potential cytotoxic agents and their alkylating ability towards cellular glutathione in human leukemic t cells effect of some bis mannich bases and corresponding piperidinols on dna topoisomerase i the design and cytotoxic evaluation of some 1-aryl-3-isopropylamino-1-propanone hydrochlorides towards human huh-7 hepatoma cells cytotoxicity of 1-aryl-3-butylamino-1-propanone hydrochlorides against jurkat and l6 cells synthesis of 1-aryl-3-phenethylamino-1-propanone hydrochlorides as possible potent cytotoxic agents biological activity of 1-aryl-3-phenethylamino-1-propanone hydrochlorides and 3-aroyl-4-aryl-1-phenethyl-4-piperidinols on pc-3 cells and dna topoisomerase i enzyme effect of acetophenone derived mannich bases on cellular glutathione level in jurkat cells. a possible mechanism of action syntheses and stability studies of some mannich bases of acetophenones and evaluation of their cytotoxicity against jurkat cells effects of mannich bases on cellular glutathione and related enzymes of jurkat cells in culture conditions the effects of some mannich bases on heat shock proteins hsc70 and grp75, and thioredoxin and glutaredoxin levels in jurkat cells cytotoxic mannich bases of 1-arylidene-2-tetralones cytotoxic and topographical properties of 6-arylidene-2-dimethylaminomethylcyclohexanone hydrochlorides and related compounds potential role of nmyristoyltransferase in cancer inhibition of protein n-myristoylation: a therapeutic protocol in developing anticancer agents synthesis and cytotoxic and mechanistic studies of a-arylidenecyclohex(pent)anone or aarylcyclohexanone a'-mannich bases and their deoxo bisaryl cyclohex(pent) ene analogs synthesis and anticancer activity of 2-alkylaminomethyl-5-diaryl-methylenecyclopentanone hydrochlorides and related compounds dimmock, a-substituted 1-aryl-3-dimethylaminopropanone hydrochlorides: potent cytotoxins towards human widr colon cancer cells 1-aryl-2-dimethylaminomethyl-2-propen-1-one hydrochlorides and related adducts: a quest for selective cytotoxicity for malignant cells synthesis of 4 0 -hydroxy-3 0 -piperidinomethylchalcone derivatives and their cytotoxicity against pc-3 cell lines synthesis and cytotoxicity of novel 3-aryl-1-(3 0 -dibenzylaminomethyl-4 0 -hydroxyphenyl)-propenones and related compounds design, synthesis and in vitro cytotoxic activity evaluation of new mannich bases design, synthesis, and biological evaluation of mannich bases of heterocyclic chalcone analogs as cytotoxic agents 1-(3-aminomethyl-4-hydroxyphenyl)-3-pyridinyl-2-propen-1-ones: a novel group of tumour-selective cytotoxins preparation of aminoalkyl substituted chalcones for use in anticancer and antimalarial pharmaceutical compositions preparation of a series of novel bichalcones linked with a 1,4-dimethylenepiperazine moiety and examination of their cytotoxicity new bichalcone analogs as nf-kb inhibitors and as cytotoxic agents inducing fas/cd95-dependent apoptosis azidothymidine is effective against human multiple myeloma: a new use for an old drug hłado n, synthesis and anticancer activity of 5 0 -chloromethylphosphonates of 3 0 -azido-3 0 -deoxythymidine (azt) antileukemic activity and cellular metabolism of the aryl phosphate derivative of bromo-methoxy zidovudine (compound whi-07) synthesis and in vitro cytotoxic activity evaluation of novel mannich bases and modified azt derivatives possessing mannich base moieties via click chemistry synthesis and pharmacological exploitation of clioquinol-derived copper-binding apoptosis inducers triggering reactive oxygen species generation and mapk pathway activation cytotoxicity and antimicrobial activity of some naphthol derivatives synthesis and cytotoxicity evaluation of some 8-hydroxyquinoline derivatives synthesis and structure-activity relationship study of 8-hydroxyquinoline-derived mannich bases as anticancer agents design, synthesis and bioevaluation of novel candidate selective estrogen receptor modulators recent advancement in nonsteroidal aromatase inhibitors for treatment of estrogen-dependent breast cancer facile synthesis of chrysin-derivatives with promising activities as aromatase inhibitors nitrogen-containing apigenin analogs: preparation and biological activity synthesis and antiproliferative activity of oxazinocarbazole and n,n-bis(carbazolylmethyl)amine derivatives synthesis, antitumour activity and structureeactivity relationships of 5h-benzo[b]carbazoles display and analysis of patterns of differential activity of drugs against human tumor cell lines: development of mean graph and compare algorithm 10-anthradiquinone as precursor for antitumor compounds the natural anticancer agent plumbagin induces potent cytotoxicity in mcf-7 human breast cancer cells by inhibiting a pi-5 kinase for ros generation anti-tumor effects of novel 5-o-acyl plumbagins based on the inhibition of mammalian dna replicative polymerase activity inhibitory effect of novel 5-o-acyl juglones on mammalian dna polymerase activity, cancer cell growth and inflammatory response cytotoxic activity of naphthoquinones with special emphasis on juglone and its 5-o-methyl derivative antiproliferative activity of synthetic naphthoquinones related to lapachol. first synthesis of 5-hydroxylapachol novel platinum(ii) complexes of 3-(aminomethyl)naphthoquinone mannich bases: synthesis, crystal structure and cytotoxic activities exploring the dna binding/cleavage, cellular accumulation and topoisomerase inhibition of 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinone mannich bases and their platinum(ii) complexes novel 3-(aminomethyl)naphthoquinone mannich base-platinum(iv) complexes: synthesis, characterization, electrochemical and cytotoxic studies one-pot synthesis and cytotoxicity studies of new mannich base derivatives of polyether antibiotic e lasalocid acid novel hexacyclic camptothecin derivatives. part 1: synthesis and cytotoxicity of camptothecins with an a-ring fused 1,3-oxazine ring optimization of tocotrienols as antiproliferative and antimigratory leads quinone methides tethered to naphthalene diimides as selective g-quadruplex alkylating agents hybrid ligandalkylating agents targeting telomeric g-quadruplex structures synthesis and biological activities of quinazoline derivatives with ortho-phenol-quaternary ammonium salt groups dna binding, antiviral activities and cytotoxicity of new furochromone and benzofuran derivatives synthesis and biological evaluation of 1,3-dihydroxyxanthone mannich base derivatives as potential antitumor agents synthesis and in vitro evaluation of novel indole-based sigma receptors ligands synthesis and cytotoxic activity of novel 3-methyl-1-[(4-substituted-1-piperazinyl)methyl]-1h-indole derivatives design, synthesis, and biological evaluation of indole-based 1,4-disubstituted piperazines as cytotoxic agents amino derivatives of indole as potent inhibitors of isoprenylcysteine carboxyl methyltransferase functionalized indoleamines as potent, drug-like inhibitors of isoprenylcysteine carboxyl methyltransferase (icmt) 3-aminomethyl derivatives of 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione for circumvention of anticancer drug resistance synthesis and structureeactivity relationship studies of naphtho[2,3-f]indole-5,10-dione aminoalkyl derivatives: a new class of topoisomerase i inhibitors synthesis of 4-substituted 3-[3-(dialkylaminomethyl)indol-1-yl]maleimides and study of their ability to inhibit protein kinase c-a, prevent development of multiple drug resistance of tumor cells and cytotoxicity synthesis and sar of novel 4-morpholinopyrrolopyrimidine derivatives as potent phosphatidylinositol 3-kinase inhibitors cytotoxic and anticancer activities of isatin and its derivatives: a comprehensive review from synthesis and in-vitro cytotoxicity evaluation of gatifloxacin mannich bases hybrid pharmacophore design and synthesis of isatinebenzothiazole analogs for their anti-breast cancer activity synthesis of novel isatin-thiazoline and isatin-benzimidazole conjugates as anti-breast cancer agents design and synthesis of anti-breast cancer agents from 4-piperazinylquinoline: a hybrid pharmacophore approach hydroxyphenyl)-3-substituted-2,3-dihydro-1,3,4-oxadiazole-2-thione derivatives: promising anticancer agents synthesis of some novel 3,5-disubstituted 1,3,4-oxadiazole derivatives and anticancer activity on eac animal model synthesis, quantitative structure-activity relationship and biological evaluation of 1,3,4-oxadiazole derivatives possessing diphenylamine moiety as potential anticancer agents synthesis, antitumor and antimicrobial activity of novel 1-substituted phenyl-3-[3-alkylamino(methyl)-2-thioxo-1,3,4-oxadiazol-5-yl]-b-carboline derivatives synthesis and antitumor activity of fluoroquinolone c3-isostere derivatives: oxadiazole mannich base derivatives synthesis, characterization and in vitro cytotoxic properties of some new schiff and mannich bases in hep g2 cells design, synthesis and antitumor activities of fluoroquinolone c-3 heterocycles (iv): s-triazole schiffemannich bases derived from ofloxacin facile synthesis and quantitative structureeactivity relationship study of antitumor active 2-(4-oxo-thiazolidin-2-ylidene)-3-oxo-propionitriles synthesis and biological activity evaluation of 5-pyrazoline substituted 4-thiazolidinones synthesis, xray crystallographic analysis, and antitumor activity of n-(benzothiazole-2-yl)-1-(fluorophenyl)-o,o-dialkyl-a-aminophosphonates synthesis and biological activities of aaminophosphonates derivatives containing thieno[3,2-c]pyridine (in chinese) synthesis, antimicrobial and anticancer activities of a novel series of diphenyl 1-(pyridin-3-yl) ethylphosphonates design and one-pot synthesis of a-aminophosphonates and bis(aaminophosphonates) by iron(iii) chloride and cytotoxic activity synthesis, antiproliferative activity in cancer cells and theoretical studies of novel 6a,7b-dihydroxyvouacapan-17b-oic acid mannich base derivatives synthesis and anti-tumor activities of novel synthesis and cytotoxicity studies of novel dimethylamino-functionalised and n-heteroaryl-substituted titanocene anticancer drugs: synthesis and cytotoxicity studies synthesis and cytotoxicity studies of new dimethylamino-functionalised and indolylsubstituted titanocene anti-cancer drugs synthesis and cytotoxicity studies of new dimethylamino-functionalized and azolesubstituted titanocene anticancer drugs synthesis and preliminary cytotoxicity studies of achiral indolyl-substituted titanocenes multidrug resistance reverting activity and antitumor profile of new phenothiazine derivatives synthesis of antitumoractive betulinic acid-derived hydroxypropargylamines by copper-catalyzed mannich reactions cytotoxic betulin-derived hydroxypropargylamines trigger apoptosis doxoform and daunoform: anthracyclineformaldehyde conjugates toxic to resistant tumor cells doxsaliform: a novel n-mannich base prodrug of a doxorubicin formaldehyde conjugate design, synthesis, and biological evaluation of doxorubicin-formaldehyde conjugates targeted to breast cancer cells antiestrogen binding site and estrogen receptor mediate uptake and distribution of 4-hydroxytamoxifen-targeted doxorubicin-formaldehyde conjugate in breast cancer cells rational design and synthesis of androgen receptortargeted nonsteroidal anti-androgen ligands for the tumor-specific delivery of a doxorubicin-formaldehyde conjugate studies of targeting and intracellular trafficking of an anti-androgen doxorubicin-formaldehyde conjugate in pc-3 prostate cancer cells bearing androgen receptor-gfp chimera doxorubicin-formaldehyde conjugates targeting a v b 3 integrin poly (ethylene glycol) prodrug for anthracyclines via n-mannich base linker: design, synthesis and biological evaluation synthesis and invitro antitumor activities of some mannich bases of 9-alkyl-1,2,3,4-tetrahydrocarbazole-1-ones synthesis and biological evaluation of novel mannich bases of 2-arylimidazo [2,1-b]benzothiazoles as potential anti-cancer agents cytotoxic mannich bases of 6-(3-aryl-2-propenoyl)-2(3h)-benzoxazolones new synthetic chalcones: cytotoxic mannich bases of 6-(4-chlorocinnamoyl)-2(3h)-benzoxazolone synthesis and cytotoxicity of novel hexahydrothieno-cycloheptapyridazinone derivatives synthesis and antibacterial study of unsaturated mannich ketones synthesis and antibacterial activity of fused mannich ketones novel mannich ketones of oxazolidinones as antibacterial agents synthesis and biological evaluation of a novel series of 2,2-bisaminomethylated aurone analogues as anti-inflammatory and antimicrobial agents evaluation of antimicrobial activities of several mannich bases and their derivatives synthesis and biological evaluation of aminoketones highly efficient one-pot synthesis, antimicrobial and docking studies of newer b-amino carbonyl derivatives catalyzed by silica sulfuric acid synthesis of b-amino carbonyl derivatives of coumarin and benzofuran and evaluation of their biological activity vuki cevi c, antibacterial 3-(arylamino)-1-ferrocenylpropan-1-ones: synthesis, spectral, electrochemical and structural characterization synthesis and characterization of some mannich bases as potential antimicrobial agents synthesis, characterization and antimicrobial evaluation of copper(ii) complexes of 5-tert-butyl-pyrocatechin-derived mannich bases synthesis and microbiological evaluation of mannich bases derived from 4,6-diacetylresorcinol mannich reaction derivatives of novobiocin with modulated physiochemical properties and their antibacterial activities novel aminonaphthoquinone mannich bases derived from lawsone and their copper(ii) complexes: synthesis, characterization and antibacterial activity synthesis, antiinflammatory and antimicrobial activity of some new 1-(3-phenyl-3,4-dihydro-2h-1,3-benzoxazin-6-yl)ethanone derivatives an eco-friendly synthesis and antimicrobial activities of dihydro-2h-benzo-and naphtho-1,3-oxazine derivatives synthesis and antibacterial evaluation of benzazoles tethered dihydro[1,3]oxazines ]oxazine derivatives catalyzed by zirconyl chloride and evaluation of its biological activities biocidal activity of some mannich base cationic derivatives some electrophilic substitution reactions on 1-substituted-3-acetyl/carbethoxy-5-hydroxy-2-methylindole and the antimicrobial activity of these new indole derivatives mannich base derivatives of 3-hydroxy-6-methyl-4h-pyran-4-one with antimicrobial activity a study of cytotoxicity of novel chlorokojic acid derivatives with their antimicrobial and antiviral activities synthesis and biological activities of new mannich bases of chlorokojic acid derivatives design, synthesis and in vivo/ in vitro screening of novel chlorokojic acid derivatives mannich bases of 7-piperazinylquinolones and kojic acid derivatives: synthesis, in vitro antibacterial activity and in silico study synthesis and antimicrobial screening of novel mannich bases of isatin derivative synthesis and antimicrobial evaluation of some novel trisubstituted s-triazine derivatives based on isatinimino, sulphonamido and azacarbazole synthesis and antimicrobial screening of certain novel mannich bases bearing 1,8-naphthyridine moiety synthesis and antimicrobial activities of oxadiazoles, phthalazines and indolinones synthesis and antimicrobial activity of mannich bases of isatin and its derivatives with 2-[(2,6-dichlorophenyl) amino]phenylacetic acid synthesis, antimicrobial screening and beta lactamase inhibitory activity of 3-(3-chloro-4-fluorophenylimino)indolin-2-one and 5-chloroindolin-2-one derivatives, turk synthesis, antiviral and antibacterial activities of isatin mannich bases synthesis and bioevaluation of schiff and mannich bases of isatin derivatives with 4-amino-5-benzyl-2,4-dihydro-3h-1,2,4-triazole-3-thione synthesis, characterization and in vitro antimicrobial activity of some novel 5-substituted schiff and mannich base of isatin derivatives synthesis and antimicrobial activity of novel 5-(1-adamantyl)-2-aminomethyl-4-substituted-1,2,4-triazoline-3-thiones microbiologically active mannich bases derived from 1,2,4-triazoles. the effect of c-5 substituent on antibacterial activity synthesis and antimicrobial activity of thiosemicarbazides, s-triazoles and their mannich bases bearing 3-chlorophenyl moiety synthesis and antibacterial activity of some novel n2-hydroxymethyl and n2-aminomethyl derivatives of 4-aryl-5-(3-chlorophenyl)-2,4-dihydro-3h-1,2,4-triazole-3-thione synthesis and in vitro activity of 1,2,4-triazole-ciprofloxacin hybrids against drug-susceptible and drug-resistant bacteria synthesis, characterization and antibacterial activity of some triazole mannich bases carrying diphenylsulfone moieties synthesis of some new s-alkylated 1,2,4-triazoles, their mannich bases and their biological activities synthesis and biological activities of some novel aminomethyl derivatives of 4-substituted-5-(2-thienyl)-2,4-dihydro-3h-1,2,4-triazole-3-thiones design, synthesis and antimicrobial activities of some azole derivatives synthesis of some new 1,2,4-triazoles, their mannich and schiff bases and evaluation of their antimicrobial activities syntheses and biological activities of new hybrid molecules containing different heterocyclic moieties alpay karao glu, preparation and antimicrobial activity evaluation of some quinoline derivatives containing an azole nucleus convenient one pot synthesis and antibacterial evaluation of some new mannich bases carrying 1,2,4-triazolyl moiety synthesis and antimicrobial activities of some new biheterocyclic compounds containing 1,2,4-triazol-3-one and 1,3,4-thiadiazole moieties synthesis of some new 1,3,4-thiadiazol-2-ylmethyl-1,2,4-triazole derivatives and investigation of their antimicrobial activities synthesis and biological activities of methylenebis-4h-1,2,4-triazole derivatives regioselective reaction: synthesis, characterization and pharmacological studies of some new mannich bases derived from 1,2,4-triazoles el-emam, synthesis, antimicrobial and anti-inflammatory activities of novel 5-(1-adamantyl)-4-[(arylidene)amino]-3-mercapto-1,2,4-triazoles and related derivatives convenient one pot synthesis and antimicrobial evaluation of some new mannich bases carrying 4-methylthiobenzyl moiety synthesis and biological activity of schiff and mannich bases bearing 2,4-dichloro-5-fluorophenyl moiety synthesis of some new 1,2,4-triazoles starting from isonicotinic acid hydrazide and evaluation of their antimicrobial activities synthesis, antiinflammatory, analgesic, and antibacterial activities of some triazole, triazolothiadiazole, and triazolothiadiazine derivatives synthesis and pharmacological evaluation of clubbed isopropylthiazole derived triazolothiadiazoles, triazolothiadiazines and mannich bases as potential antimicrobial and antitubercular agents regioselective reaction: synthesis of novel mannich bases derived from 3-(4,6-disubstituted-2-thiomethylpyrimidyl)-4-amino-5-mercapto-1,2,4-triazoles and their antimicrobial properties new class of triazole derivatives and their antimicrobial activity synthesis, characterization, and biological activity of novel schiff and mannich bases of 4-amino-3-(n-phthalimidomethyl)-1,2,4-triazole-5-thione alpay karao glu, reduction, mannich reaction and antimicrobial activity evaluation of some new 1,2,4-triazol-3-one derivatives preparation and antimicrobial activity evaluation of some new bi-and triheterocyclic azoles synthesis and biological evaluation of some 1,3,4-oxadiazole derivatives synthesis, characterization and antimicrobial activity of some disubstituted 1,3,4-oxadiazoles carrying 2-(aryloxymethyl)phenyl moiety synthesis and antimicrobial activity of new 5-(2-thienyl)-1,2,4-triazoles and 5-(2-thienyl)-1,3,4-oxadiazoles and related derivatives synthesis, antimicrobial and cytotoxic activities of some novel thiazole clubbed 1,3,4-oxadiazoles synthesis and antimicrobial activity of linked heterocyclics containing pyrazole and oxadiazoles synthesis and antimicrobial studies of some mannich bases carrying imidazole moiety synthesis and antimicrobial activity of some new 5-(3-chloro-1-benzothiophen-2-yl)-1,3,4-oxadiazole-2-thiol and their derivatives synthesis, antimicrobial and antiinflammatory activities of 1,3,4-oxadiazoles linked to naphtho[2,1-b]furan antimicrobial and antiurease activities of newly synthesized morpholine derivatives containing an azole nucleus synthesis and antimicrobial activities of some new 1,2,4-triazole derivatives synthesis of novel nalidixic acid-based 1,3,4-thiadiazole and 1,3,4-oxadiazole derivatives as potent antibacterial agents synthesis, characterization and antimicrobial activity of novel 3,5-disubstituted-1,3,4-oxadiazole-2-ones synthesis of mannich bases of some 2,5-disubstituted 4-thiazolidinones and evaluation of their antimicrobial activities synthesis, characterization and evaluation of antimicrobial activity of mannich bases of some 2-[(4-carbethoxymethylthiazol-2-yl)imino]-4-thiazolidinones novel 6,8-dibromo-4(3h)quinazolinone derivatives of antibacterial and anti-fungal activities synthesis of 3-{4-[4-dimethylamino-6-(4-methyl-2-oxo-2h-chromen-7-yloxy)-[1,3,5]triazin-2-ylamino]-phenyl}-2-phenyl-5-(4-pyridin-2-yl-piperazin-1-ylmethyl)-thiazolidin-4-one and their biological evaluation synthesis and biological activity of 3,4-dihydropyrimidine-2-thione aminomethylene derivatives based on the alkaloid cytosine synthesis and in vitro study of biological activity of heterocyclic n-mannich bases of 3,4-dihydropyrimidine-2(1h)-thiones synthesis and in vitro study of biological activity of heterocyclic n-mannich bases synthesis and in vitro study of novel mannich bases as antibacterial agents synthetic, spectral, antimicrobial and qsar studies on novel mannich bases of glutarimides evaluation of antimicrobial activity and qsar study of a molecule library of the mannich bases of glutarimides syntheses, spectral, crystallographic, antimicrobial, and antioxidant studies of few mannich bases synthesis and biological study of medicinally important mannich bases derived from 4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,6,10,12,12a-pentahydroxynaphthacenecarboxamide hydrophobic vancomycin derivatives with improved adme properties: discovery of telavancin in vitro activity of telavancin against resistant gram-positive bacteria telavancin, a multifunctional lipoglycopeptide, disrupts both cell wall synthesis and cell membrane integrity in methicillinresistant staphylococcus aureus exploring the positional attachment of glycopeptide/b-lactam heterodimers vancomycin derivatives iodine catalyzed three-component synthesis of b-amino-b-keto-esters and their antimicrobial activity deep, synthesis, characterization and antimicrobial evaluation of novel derivatives of isoniazid synthesis and evaluation of some novel derivatives of 2-propoxybenzylideneisonicotinohydrazide for their potential antimicrobial activity synthesis and antimicrobial screening of novel series of imidazo[1,2-a]pyridine derivatives synthesis and evaluation of antibacterial and antitubercular activities of some novel imidazo synthesis, antimicrobial evaluation and qsar study of some 3-hydroxypyridine-4-one and 3-hydroxypyran-4-one derivatives synthesis of novel biologically active methylene derivatives of sydnones synthesis, characterization, and antimicrobial screening of some mannich base sydnone derivatives facile syntheses of mannich bases of 3-[p-(5-arylpyrazolin-3-yl)phenyl]sydnones, as anti-tubercular and anti-microbial agents, under ionic liquid/tetrabutylammonium bromide catalytic conditions copper(ii) complex with the tridentate ligand n,n-bis(2-ethyl-4-methylimidazol-5-ylmethyl)phenylethylamine (biaq). x-ray crystal structure and biological activity on bacillus subtilis of synthesis, sar study and evaluation of mannich and schiff bases of pyrazol-5(4h)-one moiety containing 3-(hydrazinyl)-2-phenylquinazolin-4(3h)-one design, synthesis, antibacterial activity and physicochemical parameters of novel n-4-piperazinyl derivatives of norfloxacin in vitro study of some medicinally important mannich bases derived from antitubercular agent microwave-assisted synthesis of some novel and potent antibacterial and antifungal compounds with biological screening synthesis and antimicrobial and antioxidant activities of simple saccharin derivatives with nbasic side chains synthesis and antimicrobial activity of 10-arylaminomethyl-3-methoxyphenothiazine-9-carboxylic acid in vitro antitubercular and antimicrobial activities of 1-substituted quinoxaline-2,3(1h,4h)-diones synthesis of some new thioxoquinazolinone derivatives and a study on their anticonvulsant and antimicrobial activities synthesis and anti bacterial and anti-ulcer evaluation of new s-mannich bases of 4,6-diaryl-3,4-dihydropyrimidin-2(1h)-thiones world health organization antimycobacterial arylidenecyclohexanones and related mannich bases methoxyphenylcarbonyloxy)benzylidene]-6-dimethylaminomethyl cyclohexanone hydrochloride: a mannich base which inhibits the growth of some drug-resistant strains of mycobacterium tuberculosis major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence isocitrate lyase: a potential target for anti-tubercular drugs identification of mannich base as a novel inhibitor of mycobacterium tuberculosis isocitrate by high-throughput screening synthesis and in vitro and in vivo antimycobacterial activity of isonicotinoyl hydrazones 4h-pyran-4-one derivatives: leading molecule for preparation of compounds with antimycobacterial potential molecular modeling and antimycobacterial studies of mannich bases: 5-hydroxy-2-methyl-4h-pyran-4-ones bactericidal activities of the pyrrole derivative bm212 against multidrug-resistant and intramacrophagic mycobacterium tuberculosis strains synthesis and microbiological activities of pyrrole analogs of bm 212, a potent antitubercular agent new pyrrole derivatives as antimycobacterial agents analogs of bm212 importance of the thiomorpholine introduction in new pyrrole derivatives as antimycobacterial agents analogues of bm 212 antimycobacterial compounds. new pyrrole derivatives of bm212 antimycobacterial compounds. optimization of the bm 212 structure, the lead compound for a new pyrrole derivative class antimycobacterial agents. novel diarylpyrrole derivatives of bm212 endowed with high activity toward mycobacterium tuberculosis and low cytotoxicity botta, 1,5-diphenylpyrrole derivatives as antimycobacterial agents. probing the influence on antimycobacterial activity of lipophilic substituents at the phenyl rings -diaryl-2-ethyl pyrrole derivatives as antimycobacterial agents: design, synthesis, and microbiological evaluation identification of a novel pyrrole derivative endowed with antimycobacterial activity and protection index comparable to that of the current antitubercular drugs streptomycin and rifampin improved bm212 mmpl3 inhibitor analogue shows efficacy in acute murine model of tuberculosis infection mmpl3 is the cellular target of the antitubercular pyrrole derivative bm212 pharmacophore assessment through 3-d qsar: evaluation of the predictive ability on new derivatives by the application on a series of antitubercular agents bm 212 and its derivatives as a new class of antimycobacterial active agents new pyrroles with potential antimycobacterial, antifungal and selective cox-2 inhibiting activities. synthetic methodologies new derivatives of bm212: a class of antimycobacterial compounds based on the pyrrole ring as a scaffold developing pyrrole-derived antimycobacterial agents: a rational lead optimization approach pyrrole derivatives as antimycobacterial compounds, us patent 7 antitubercular agents. part 7: a new class of diarylpyrroleeoxazolidinone conjugates as antimycobacterial agents organocatalytic multicomponent reaction for the acquisition of a selective inhibitor of mptpb, a virulence factor of tuberculosis novel 1-(4-substituted benzylidene)-4-(1-(substituted methyl)-2,3-dioxoindolin-5-yl)semicarbazide derivatives for use against mycobacterium tuberculosis h37rv (atcc 27294) and mdr-tb strain synthesis, anti-hiv and antitubercular activities of lamivudine prodrugs synthesis and antituberculosis activity of 5-methyl/trifluoromethoxy-1h-indole-2,3-dione 3-thiosemicarbazone derivatives synthesis and structureeantituberculosis activity relationship of 1h-indole-2,3-dione derivatives gatifloxacin derivatives: synthesis, antimycobacterial activities, and inhibition of mycobacterium tuberculosis dna gyrase synthesis and antimycobacterial evaluation of various 7-substituted ciprofloxacin derivatives synthesis and in vitro antimycobacterial activity of 8-och 3 ciprofloxacin methylene and ethylene isatin derivatives synthesis and in vitro antimycobacterial activity of moxifloxacin methylene and ethylene isatin derivatives antimycobacterial activity of new 3-substituted 5-(pyridin-4-yl)-3h-1,3,4-oxadiazol-2-one and 2-thione derivatives. preliminary molecular modeling investigations antimycobacterial activity of new 3,5-disubstituted 1,3,4-oxadiazol-2(3h)-one derivatives. molecular modeling investigations oxadiazole mannich bases: synthesis and antimycobacterial activity augustynowicz-kope c, synthesis and tuberculostatic activity of some 2-piperazinmethylene derivatives 1,2,4-triazole-3-thiones synthesis and evaluation of antitubercular activity of imidazo mannich bases and novel benzothiazole derivatives of imidazo[2,1-b]-1,3,4-thiadiazoles and their biological evaluation synthesis & evaluation of antitubercular activity of novel mannich bases of imidazo newer tetracycline derivatives: synthesis, anti-hiv, antimycobacterial activities and inhibition of hiv-1 integrase synthesis of pyrazinamide mannich bases and their antitubercular properties efavirenz mannich bases: synthesis, anti-hiv and antitubercular activities indole mannich bases and their antimycobacterial effect synthesis of novel isothiazolopyridines and their in vitro evaluation against mycobacterium and propionibacterium acnes studies on pyrazine derivatives. 39. synthesis, reactions, and tuberculostatic activity of 3 epidemiology of invasive mycoses in north america antifungal activity of some mono, bis and quaternary mannich bases derived from acetophenone antimicrobial evaluation of some mannich bases of acetophenones and representative quaternary derivatives antifungal evaluation of bis mannich base derived from acetophenones and their corresponding piperidinols and stability studies synthesis and antifungal activity of 1-aryl-3-phenethylamino-1-propanone hydrochlorides and 3-aroyl-4-aryl-1-phenethyl-4-piperidinols synthesis and antifungal evaluation of 1-aryl-2-[(dimethylamino)methyl]-2-propen-1-one hydrochlorides antifungal unsaturated cyclic mannich ketones and amino alcohols: study of mechanism of action antifungal activity of fused mannich ketones triggers an oxidative stress response and is cap1-dependent in candida albicans synthesis and antifungal activity of 3-substituted thiochromanones deep, synthesis, characterization and evaluation of mannich bases as potent antifungal and hydrogen peroxide scavenging agents mannich reaction: an approach for the synthesis of water soluble mulundocandin analogues evaluation of bioactivities of chlorokojic acid derivatives against dermatophytes couplet with cytotoxicity synthesis and evaluation of anticonvulsant and antimicrobial activities of 3-hydroxy-6-methyl-2-substituted 4h-pyran-4-one derivatives synthesis and studies of triazolothiadiazines. an approach toward new biologically active heterocyclic compounds, phosphorus ecofriendly synthesis of novel antifungal (thio)barbituric acid derivatives world health organization malaria isoquine and related amodiaquine analogues: a new generation of improved 4-aminoquinoline antimalarials candidate selection and preclinical evaluation of ntert-butyl isoquine (gsk369796), an affordable and effective 4-aminoquinoline antimalarial for the 21st century comparative preclinical drug metabolism and pharmacokinetic evaluation of novel 4-aminoquinoline anti-malarials antimalarial activity of isoquine against kenyan plasmodium falciparum clinical isolates and association with polymorphisms in pfcrt and pfmdr1 genes mimicking the intramolecular hydrogen bond: synthesis, biological evaluation, and molecular modeling of benzoxazines and quinazolines as potential antimalarial agents synthesis and in vitro and in vivo antimalarial activity of novel 4-anilinoquinoline mannich base derivatives synthesis and antimalarial activity evaluation of some isoquine analogues synthesis and antimalarial activity study of some new mannich bases of 7-chloro-4-aminoquinoline novel amodiaquine congeners as potent antimalarial agents synthesis and antimalarial activity of new isotebuquine analogues review of pyronaridine anti-malarial properties and product characteristics determination of the physicochemical properties of pyronaridine e a new antimalarial drug absorption, distribution, excretion, and pharmacokinetics of 14 c-pyronaridine tetraphosphate in male and female spra-gueedawley rats in vitro interactions between piperaquine, dihydroartemisinin, and other conventional and novel antimalarial drugs, antimicrob anti-malarial efficacy of pyronaridine and artesunate in combination in vitro and in vivo targeting of hematin by the antimalarial pyronaridine pyrido [3,2-b]indol-4-yl-amine e synthese und prüfung auf wirksamkeit gegen malaria benzofuro[3,2-b]pyridin-4-ylamines e synthese und prüfung auf wirksamkeit gegen malaria benzothieno[3,2-b]pyridin-4-yl-amine e synthese und prüfung auf wirksamkeit gegen malaria thieno[3,2-c]chinolin-4-ylamine e synthese und prüfung auf wirksamkeit gegen malaria thieno[3,4-c]chinolin-4-ylamine e synthese und prüfung auf wirksamkeit gegen malaria naphthyridin-5-yl-arylamine e phenol-mannich-basen vom amodiaquin-, cycloquin-und pyronaridin-typ antimalarial mannoxanes: hybrid antimalarial drugs with outstanding oral activity profiles and a potential dual mechanism of action aromatic amino analogues of artemisinin: synthesis and in vivo antimalarial activity artemisinin derivatives bearing mannich base group: synthesis and antimalarial activity novel in vivo active anti-malarials based on a hydroxy-ethyl-amine scaffold modular synthesis and in vitro and in vivo antimalarial assessment of c-10 pyrrole mannich base derivatives of artemisinin mechanism-based inactivation of thioredoxin reductase from plasmodium falciparum by mannich bases. implication for cytotoxicity synthesis and biological evaluation of phenolic mannich bases of benzaldehyde and (thio)semicarbazone derivatives against the cysteine protease falcipain-2 and a chloroquine resistant strain of plasmodium falciparum design, synthesis and structureeactivity relationships of (1h-pyridin-4-ylidene)amines as potential antimalarials synthesis and in vitro activities of ferrocenic aminohydroxynaphthoquinones against toxoplasma gondii and plasmodium falciparum pershina, indole derivative having antiviral arbidol: a broad-spectrum antiviral compound that blocks viral fusion mechanism of inhibition of enveloped virus membrane fusion by the antiviral drug arbidol synthesis and in vitro anti-hepatitis b virus activities of some ethyl 6-bromo-5-hydroxy-1h-indole-3-carboxylates synthesis and in vitro anti-hepatitis b virus activity of some ethyl 5-hydroxy-4-substituted aminomethyl-2-sulfinylmethyl-1h-indole-3-carboxylates synthesis and in vitro anti-hepatitis b virus activities of some ethyl 5-hydroxy-1h-indole-3-carboxylates synthesis and in vitro-anti-hepatitis b virus activities of several ethyl 5-hydroxy-1h-indole-3-carboxylates synthesis and anti-hepatitis b virus evaluation of novel ethyl 6-hydroxyquinoline-3-carboxylates in vitro synthesis and in-vitro antihepatitis-b virus activity of 6h synthesis and in vitro anti-hepatitis b virus activity of 6h-[1]benzothiopyrano[4,3-b]quinolin-9-ols synthesis and in vitro anti-hepatitis b virus activity of 1h-benzimidazol-5-ol derivatives synthesis and biological evaluation of 1h-benzimidazol-5-ols as potent hbv inhibitors synthesis and in-vitro anti-hepatitis b virus activity of ethyl 6-bromo-8-hydroxyimidazo[1,2-a] pyridine-3-carboxylates activity of mannich bases of 7-hydroxycoumarin against flaviviridae novel structurally related compounds reactivate latent hiv-1 in a bcl-2-transduced primary cd4þ t cell model without inducing global t cell activation synthesis and primary antiviral activity evaluation of 3-hydrazono-5-nitro-2-indolinone derivatives design of potential reverse transcriptase inhibitor containing isatin nucleus using molecular modeling studies newer aminopyrimidinimino isatin analogues as non-nucleoside hiv-1 reverse transcriptase inhibitors for hiv and other opportunistic infections of aids: design, synthesis and biological evaluation n-methylisatin-beta-thiosemicarbazone derivative (sch 16) is an inhibitor of japanese encephalitis virus infection in vitro and in vivo condensed pentacyclic derivatives for use in the treatment of flaviviridae infections synthesis, antiviral and cytotoxicity studies of novel n-substituted indophenazine derivatives, indian synthesis of some mono-mannich bases and corresponding azine derivatives and evaluation of their anticonvulsant activity evaluation of anticonvulsant activities of bis(3-aryl-3-oxo-propyl)ethylamine hydrochlorides and 4-aryl-3-arylcarbonyl-1-ethyl-4-piperidinol hydrochlorides synthesis and evaluation of anticonvulsant activities of some bis mannich bases and corresponding piperidinols synthesis of some new hydroxypyranone derivatives and evaluation of their anticonvulsant activities synthesis of some novel mannich bases derived from allomaltol and evaluation of their anticonvulsant activities anticonvulsant and neurotoxicity evaluation of some novel kojic acids and allomaltol derivatives synthesis and anticonvulsant activity of new kojic acid derivatives synthesis of aminomethyl-substituted cyclic imide derivatives for evaluation as anticonvulsants synthesis and anticonvulsant activity of new n-mannich bases derived from 5-cyclopropyl-5-phenylhydantoins synthesis and anticonvulsant activity of new n-mannich bases derived from 5-cyclopropyl-5-phenyl-and 5-cyclopropyl-5-(4-chlorophenyl)-imidazolidine-2,4-diones synthesis and anticonvulsant activity of new n-1 0 synthesis and potential anticonvulsant activity of new n-3-substituted 5,5-cyclopropanespirohydantoins synthesis and pharmacological evaluation of novel 1 0 karolak-wojciechowska, design, synthesis, and anticonvulsant activity of new n-mannich bases derived from spirosuccinimides and spirohydantoins synthesis and anticonvulsant properties of new n-[(4-arylpiperazin-1-yl)-methyl] derivatives of 3-aryl pyrrolidine-2,5-dione and 2-aza-spiro synthesis and anticonvulsant activity of new n-[(4-arylpiperazin-1-yl)-alkyl] derivatives of 3-phenylpyrrolidine-2,5-dione synthesis, physicochemical and anticonvulsant properties of new n-[(4-aryl-1-piperazinyl) alkyl]-1-phenyl-2,5-pyrrolidinedione and n-[(4-aryl-1-piperazinyl)alkyl]-3-(3-methylphenyl)-2,5-pyrrolidinedione derivatives synthesis and anticonvulsant properties of new mannich bases derived from 3-aryl-pyrrolidine-2,5-diones synthesis and anticonvulsant activity of new n-mannich bases derived from 3-(2-fluorophenyl)-and 3-(2-bromophenyl)-pyrrolidine-2,5-diones. part ii design, synthesis and anticonvulsant properties of new n-mannich bases derived from 3-phenylpyrrolidine-2,5-diones duszy nska, synthesis, anticonvulsant activity and 5-ht 1a , 5-ht 2a receptor affinity of new n-[(4-arylpiperazin-1-yl)-alkyl] derivatives of 2-azaspiro synthesis and anticonvulsant properties of new mannich bases derived from 3,3-disubstituted pyrrolidine-2,5-diones. part iv synthesis and anticonvulsant properties of new n-mannich bases derived from 3,3-diphenyl-and 3-ethyl-3-methyl-pyrrolidine-2,5-diones. part iii synthesis and biological properties of new n-mannich bases derived from 3-methyl-3-phenyl-and 3,3-dimethyl-succinimides. part v anticonvulsant activity and 5-ht 1a /5-ht 7 receptors affinity of piperazine derivatives of synthesis of some newer derivatives of substituted quinazolinonyl-2-oxo/thiobarbituric acid as potent anticonvulsant agents synthesis and pharmacological evaluation of newer substituted benzoxazepine derivatives as potent anticonvulsant agents synthesis and evaluation of some new substituted benzothiazepine and benzoxazepine derivatives as anticonvulsant agents synthesis and anticonvulsant activity of various mannich and schiff bases of 1,5-benzodiazepines novel 2-(e)-substituted benzylidene-6-(n-substituted aminomethyl)cyclohexanones and cyclohexanols as analgesic and anti-inflammatory agents biochemical effects of some new mannich base from ortho-hydroxyaryl alkyl ketones on rats with chronic inflammation synthesis and pharmacological activity of n-[b-(para-substituted benzoyl)ethyl]isoleucine and -methionine synthesis and anti-inflammatory, analgesic, and antipyretic activities of n-[b-(p-substituted benzoyl)ethyl]amino acids anti-inflammatory activity of isobornylphenol derivatives synthesis and antiinflammatory activity of resveratrol analogs new 4,6-diacetylresorcinol mannich bases: synthesis and biological evaluation synthesis and biological evaluation of nitrogencontaining benzophenone analogues as tnf-a and il-6 inhibitors with antioxidant activity synthesis and biological evaluation of nitrogen-containing chalcones as possible anti-inflammatory and antioxidant agents synthesis and antiinflammatory activity of chalcones and related mannich bases inhibitory effects of mannich bases of heterocyclic chalcones on no production by activated raw 264.7 macrophages and superoxide anion generation and elastase release by activated human neutrophils synthesis of some new 3,4-dihydro-2h-1,3-benzoxazines under microwave irradiation in solvent-free conditions and their biological activity synthesis and antiinflammatory activity of coumarin derivatives anti-inflammatory mechanisms of isoflavone metabolites in lipopolysaccharide-stimulated microglial cells synthesis and no production inhibition of novel mannich base derivatives of irisolidone synthesis and in-silico studies of some diaryltriazole derivatives as potential cyclooxygenase inhibitors regioselective reaction: synthesis and pharmacological study of mannich bases containing ibuprofen moiety regioselective reaction: synthesis, characterization and pharmacological activity of some new mannich and schiff bases containing sydnone synthesis, characterization and pharmacological activity of 4-{[1-substituted aminomethyl-4-arylideneamino-5-sulfanyl-4,5-dihydro-1h-1,2,4-triazol-3-yl] methyl}-2h-1,4-benzothiazin-3(4h)-ones synthesis and anti-inflammatory activity of new polyheterocyclic schiff bases and mannich bases docking, synthesis, and pharmacological investigation of novel substituted thiazole derivatives as non-carboxylic, anti-inflammatory, and analgesic agents synthesis, analgesic and antiinflammatory properties of certain 5-/6-acyl-3-(4-substituted-1-piperazinylmethyl)-2-benzoxazolinones derivatives synthesis and evaluation of analgesic, anti-inflammatory and antimicrobial activities of 6-acyl-3-piperazinomethyl-2-benzoxazolinones some new mannich bases of 5-methyl-2-benzoxazolinones with analgesic and antiinflammatory activities analgesic and antiinflammatory activities of some new mannich bases of 5-nitro-2-benzoxazolinones synthesis and pharmacological evaluation of 1-(1-((substituted)methyl)-5-methyl-2-oxoindolin-3-ylidene)-4-(substituted pyridin-2-yl)thiosemicarbazide synthesis, analgesic, antiinflammatory and ulcerogenic properties of some novel n 0 -((1-(substitutedamino)methyl)-2-oxoindolin-3-ylidene)-4-(2-(methyl/phenyl)-4-oxoquinazolin-3(4h)-yl)benzohydrazide derivatives synthesis, pharmacological screening, quantum chemical and in vitro permeability studies of n-mannich bases of benzimidazoles through bovine cornea synthesis and biological evaluation of mannich bases of benzimidazole derivatives synthesis, antiinflammatory and ulcerogenicity studies of some substituted pyrimido[1,6-a]azepine derivatives potential antiinflammatory activity and ulcerogenicity study of some novel pyrimido synthesis and anti-inflammatory activity of certain piperazinylthienylpyridazine derivatives design and synthesis of 3-[3-(substituted phenyl)-4-piperidin-1-ylmethyl/-4-morpholin-4-ylmethyl-4,5-dihydro-isoxazol-5-yl]-1h-indoles as potent anti-inflammatory agents efficient synthesis of the first betulonic acideacetylene hybrids and their hepatoprotective and anti-inflammatory activity substituted 1 and 2-naphthol mannich bases synthesis and analgesic activity of novel derivatives of 1,2-substituted benzimidazoles synthesis and in vivo pharmacology of new derivatives of isothiazolo[5,4-b] pyridine of mannich base type synthesis, analgesic activity and computational study of new isothiazolopyridines of mannich base type thymol analogues with antioxidant and l-type calcium current inhibitory activity alzheimer's disease, with neuroprotective, cholinergic, antioxidant, and copper-complexing properties mannich bases of scutellarein as thrombin-inhibitors: design, synthesis, biological activity and solubility synthesis of antioxidants for prevention of age-related macular degeneration synthesis and antioxidant activity of novel mannich base of 1,3,4-oxadiazole derivatives possessing 1,4-benzodioxan design, synthesis, and in vitro antioxidant activity of 1,3,5-trisubstituted-2-pyrazolines derivatives synthesis and antioxidant activity of aminomethylated 6-methyluracil derivatives synthesis and antihypertensive effects of new methylthiomorpholinphenol derivatives antihypertensive and antiarrhythmic properties of a para-hydroxy[bis(ortho-morpholinylmethyl)] phenyl-1,4-dhp compound: comparison with other compounds of the same kind and relationship with logp values 1-and 2-substituted naphthalenes: a new class of potential hypotensive agents studies on the cardiovascular action of tpyb: an antihypertensive agent with antiplatelet activity synthesis, characterization and antihypertensive activity of pyridazinone derivatives synthesis and pharmacological activity of 2-and 3-(aminomethyl)imidazo [1,2-a]benzimidazoles new octahydroquinazoline derivatives: synthesis and hypotensive activity irreversible inactivation of trypanothione reductase by unsaturated mannich bases: a divinyl ketone as key intermediate unsaturated mannich bases active against multidrugresistant trypanosoma brucei brucei strains antileishmanial pyrazolopyridine derivatives: synthesis and structureeactivity relationship analysis mannich base derivatives of 1,3,4-oxadiazole: synthesis and screening against entamoeba histolytica in vitro antischistosomal evaluation of some newly synthesized praziquantel derivatives structureeactivity relationships of chalcone analogs as potential inhibitors of adpand collagen-induced platelet aggregation synthesis and pharmacological evaluation of peptide-mimetic protease-activated receptor-1 antagonists containing novel heterocyclic scaffolds the association of helicobacter pylori infection and nonsteroidal anti-inflammatory drugs in peptic ulcer disease synthesis and antiulcer activity study of 1,4-dihydropyridines and their mannich bases with sulfanilamide synthesis of 6-and 9-alkylaminomethyl furoflavones as gastroprotective agents submitted to the 65 th world health assembly synthesis and antidepressant-like profile of novel 1-aryl-3-[(4-benzyl)piperidine-1-yl]propane derivatives design, synthesis, computational and biological evaluation of new anxiolytics benzoxepin derivatives: design, synthesis, and pharmacological evaluation with sedativeehypnotic effect synthesis and hepatoprotector activity of water-soluble derivatives of aminoalkylphenols design, synthesis and preliminary biological evaluation of zatebradine analogues as potential blockers of the hyperpolarization-activated current synthesis and antiemetic activity of 1,2,3,9-tetrahydro-9-methyl-3-(4-substituted-piperazin-1-ylmethyl)-4h-carbazol-4-one derivatives synthesis of tropolone derivatives and evaluation of their in vitro neuroprotective activity bone selective protective effect of a novel bone-seeking estrogen on trabecular bone in ovariectomized rats a one step/one pot synthesis of n,nbis(phosphonomethyl)amino acids and their effects on adipogenic and osteogenic differentiation of human mesenchymal stem cells the mannich base nc1153 promotes long-term allograft survival and spares the recipient from multiple toxicities donepeziletacrine hybrid related derivatives as new dual binding site inhibitors of ache synthesis and acetylcholinesterase (ache) inhibitory activity of some n-substituted-5-chloro-2(3h)-benzoxazolone derivatives synthesis and biological evaluation of 1,3-dihydroxyxanthone mannich base derivatives as anticholinesterase agents synthesis and biological evaluation of novel 8-aminomethylated oroxylin a analogues as a-glucosidase inhibitors synthesis and evaluation of 5-(6-methoxynaphthalen-2-yl)-1-aryl-1-(4-(trifluoromethyl) phenylamino)pentan-3-ones as potential antidiabetic agents synthesis and antidiabetic performance of b-amino ketone containing nabumetone moiety synthesis of novel b-amino ketones containing a p-aminobenzoic acid moiety and evaluation of their antidiabetic activities exploring structureeactivity relationships of transition state analogues of human purine nucleoside phosphorylase synthesis of second-generation transition state analogues of human purine nucleoside phosphorylase synthesis of a transition state analogue inhibitor of purine nucleoside phosphorylase via the mannich reaction third-generation immucillins: syntheses and bioactivities of acyclic immucillin inhibitors of human purine nucleoside phosphorylase syntheses and bio-activities of the l-enantiomers of two potent transition state analogue inhibitors of purine nucleoside phosphorylases immucillins in custom catalytic-site cavities a bfluoroamine inhibitor of purine nucleoside phosphorylase azetidine based transition state analogue inhibitors of n-ribosyl hydrolases and phosphorylases design and synthesis of potent "sulfur-free" transition state analogue inhibitors of 5 0 -methylthioadenosine nucleosidase and 5 0 -methylthioadenosine phosphorylase augustyns, narylmethyl substituted iminoribitol derivatives as inhibitors of a purine specific nucleoside hydrolase potent transglutaminase inhibitors, aryl b-aminoethyl ketones potent transglutaminase inhibitors, dithio b-aminoethyl ketones synthesis and characterization of phenolic mannich bases and effects of these compounds on human carbonic anhydrase isozymes i and ii nitrogen-containing flavonoid analogues as cdk1/cyclin b inhibitors: synthesis, sar analysis, and biological activity hit identification of novel heparanase inhibitors by structure and ligand-based approaches discovery of inhibitors of burkholderia pseudomallei methionine aminopeptidase with antibacterial activity search for inhibitors of bacterial and human protein kinases among derivatives of diazepines[1,4] annelated with maleimide and indole cycles umbelliferone aminoalkyl derivatives, a new class of squalene-hopene cyclase inhibitors synthesis of new 4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine derivatives with an incorporated thiazolidinone moiety and testing their possible serine protease and cercarial elastase inhibitory effects with a possible prospective to block penetration of schistosoma mansoni cercariae into the mice skin synthesis and biological activity of oxadiazole and triazolothiadiazole derivatives as tyrosinase inhibitors novel mannich bases, 5-arylimidazolidine-2,4-dione derivatives with dual 5-ht 1a receptor and serotonin transporter affinity pharmacophore based synthesis of 3-chloroquinoxaline-2-carboxamides as serotonin 3 (5-ht 3 ) receptor antagonist synthesis and biological evaluation of a novel structural type of serotonin 5-ht 3 receptor antagonists ]pyridine: an antagonist with high affinity and selectivity for the human dopamine d 4 receptor azaindole derivatives with high affinity for the dopamine d 4 receptor: synthesis, ligand binding studies and comparison of molecular electrostatic potential maps phenylpiperazinylmethylheterocycle derivatives: synthesis and dopamine receptor binding profiles synthesis of 5-[(4-phenylpiperazin-1-yl)methyl]pyrrolo [2,3-d]pyrimidine derivatives as potential dopamine d 4 receptor ligands design, synthesis and dopamine d4 receptor binding activities of new n-heteroaromatic 5/6-ring mannich bases discovery of highly potent and selective d4 ligands by interactive sar study synthesis and evaluation of ligands for d 2 -like receptors: the role of common pharmacophoric groups discovery of small molecule inhibitors of the interaction of the thyroid hormone receptor with transcriptional coregulators inhibitors of the interaction of a thyroid hormone receptor and coactivators: preliminary structureeactivity relationships structural insight into the mode of action of a direct inhibitor of coregulator binding to the thyroid hormone receptor improvement of pharmacological properties of irreversible thyroid receptor coactivator binding inhibitors discovery and biological characterization of a novel series of androgen receptor modulators aromatic b-amino-ketone derivatives as novel selective non-steroidal progesterone receptor antagonists synthesis and biological evaluation of n 1 ,n 2 -bis-[4-(t-amino)-2-butynyl]phthalamides as oxotremorine and acetylcholine antagonists synthesis and structureactivity relationships of 1-aralkyl-4-benzylpiperidine and 1-aralkyl-4-benzylpiperazine derivatives as potent s ligands indole-and benzothiophene-based histamine h 3 antagonists new 1,2,3,9-tetrahydro-4h-carbazol-4-one derivatives: analogues of heat as ligands for the a 1 -adrenergic receptor subtypes pyrazolone methylamino piperidine derivatives as novel ccr3 antagonists synthesis of new 4-heteroaryl-2-phenylquinolines and their pharmacological activity as nk-2/nk-3 receptor ligands the author declares that there is no conflict of interest. key: cord-020010-q58x6xb0 authors: nan title: 19th icar abstracts: date: 2006-03-13 journal: antiviral res doi: 10.1016/j.antiviral.2006.02.001 sha: doc_id: 20010 cord_uid: q58x6xb0 nan the society was organized in 1987 as a non-profit scientific organization for the purpose of advancing and disseminating knowledge in all areas of antiviral research. to achieve this objective, the society organizes an annual meeting. the society is now in its 20 th year of existence, and has about 600 members representing 30 countries. for membership application forms or further information, please contact dr. amy patick, secretary, isar; pfizer global r&d, department of virology, 10777 science center drive, san diego, ca 92121; phone +1 858 622 3117; fax +1 858 678 8182; e-mail amy.patick@pfizer.com. membership application forms will also be available at the conference registration desk, or from our website www.isar-icar.com. enzymes of the pol gene of hiv have been identified as important viral targets for the discovery anti-hiv therapeutic agents. while the viral targets, hiv reverse transcriptase and hiv protease, have been successfully investigated for the development of clinically useful therapeutic agents, research efforts on drug discovery on the third enzyme of the pol gene, hiv integrase, have not resulted in a single fda-approved drug. nevertheless, as integrase is essential for hiv replication, it remains an attractive target for the discovery of new anti-hiv agents. in this presentation, we report the discovery of a conceptually new beta-diketo acid, constructed on a nucleobase scaffold, that is a potent inhibitor of both the 3 -processing and strand transfer steps of recombinant hiv integrase. this inhibitor and the positive control compound, azt, were tested in a pbmc cellbased, microtiter anti-hiv assay against the clinical isolate, hiv-1teki (nsi phenotype) and hiv-1nl4-3 (si phenotype), and in a magi-x4 assay against hiv-1nl4-3 with hela-cd4-ltr-beta-gal cells. our integrase inhibitor was found to have highly potent in vitro anti-hiv activity and efficacy. the discovery of this remarkably active molecule, representative of a unique set of diketo acids bearing nucleobase scaffolds, has uncovered a new chapter in the chemistry and biology of integrase inhibitors and their potential therapeutic applications. berta bosch 1 , imma clotet-codina 1 , julia blanco 1 , eduard pauls 1 , gemma coma 1 , samandhy cedeño 1 , francesc mitjans 2 , anuska llano 1 , margarita bofill 1 , bonaventura clotet 1 , jaume piulats 2 , jose este 1 1 retrovirology laboratory, fundacio irsicaixa, badalona, spain; 2 laboratorio de bioinvestigación, merck farma y química, barcelona, spain macrophages are key cells for hiv infection and spreading inside the organism. macrophages cultured in vitro can be successfully infected after differentiation with cytokines such as macrophage colony stimulating factor (m-csf). in the monocyte to macrophage differentiation process with m-csf, av-integrins are upregulated concomitantly to the capacity of hiv to generate a productive virus infection. in the present study we show that an anti-av antibody, 17e6, inhibited hiv-1 infection of primary macrophages. the effect of 17e6 on r5 or x4 hiv-1 replication in acutely infected macrophages was dose-dependent, with a 50% effective concentration (ec50) of 17 ± 2 g/ml in the absence of cytotoxicity. similarly, a monoclonal antibody targeting the avb6 integrin (14d9.f8) also inhibited hiv-1 infection in this cell type. 17e6 reduced the detection of hiv-1 bal proviral dna in acutely infected macrophages but was completely ineffective against hiv-1 bal production in chronically infected macrophages, suggesting that 17e6 inhibited hiv infection at an early stage of the virus cycle. finally, a small molecular weight antagonist of the avb6 integrin reduced hiv replication at subtoxic concentrations. therefore, our results suggest that av-containing integrins could play a role in hiv replication in macrophages and indicate that small molecular weight compounds may be developed to interfere with hiv replication in macrophages through the interaction with av integrins. andrew vaillant 1 , hong lu 2 , shuwen liu 2 , carol lackman-smith 3 , roger ptak 3 , jean-marc juteau 1 , shibo jiang 2 1 replicor inc., laval, que., canada; 2 f. lindsay kimball research institute, new york blood center, new york, ny, usa; 3 southern research institute, frederick, md, usa the sequence independent antiviral activity of phosphorothioate oligonucleotides in inhibiting hiv-1 by blocking interactions between the v3 loop and cd4 has been previously described. this activity was attributed to their polyanionic activity. here we show that ps-ons (and their fully 2 -o-methylated derivatives) are also potent inhibitors of hiv-1-mediated membrane fusion and hiv-1 replication in a sequence-independent, sizedependent (optimal size ∼50-60 bases) and phosphorothioation dependent manner (independent of stabilization). ps-ons interact with the heptad repeat regions of gp41 and the hiv-1 fusion inhibitory activity of ps-ons is closely correlated with their ability to bind to these heptad repeats and block gp41 six-helix bundle formation, a critical step during the process of hiv-1 fusion with the target cell. the requirement for ps-on interaction was also found to be dependent on phosphorothioation, suggesting that the v3 loop/ps-on interaction may also have a hydrophobic component. the increased hydrophobicity of longer (≥40 base) ps-ons may contribute to their inhibitory activity against hiv-1 fusion and entry because these longer ps-ons have a greater hydrophobicity and are more potent in blocking the hydrophobic interactions involved in the gp41 sixhelix bundle formation than shorter ps-ons (<30 bases). this novel antiviral mechanism of action of long ps-ons has important implications for therapy against infection by hiv-1 and other enveloped viruses with type i fusion proteins. chris meier 1 , soenke jessel 1 , bastian reichardt 1 , olaf ludek 1 , jan balzarini 2 1 university of hamburg, institute of organic chemistry, hamburg, germany; 2 rega institute for medical research, katholieke universiteit leuven, leuven, belgium carbocyclic nucleoside analogues like abacavir showed very interesting antiviral properties. therefore, we are interested in a convenient stereoselective access to this class of compounds as potential antiviral agents. by using a new convergent synthetic strategy, starting from a chiral cyclopentenol, enantiomerically pure carbocyclic thymidine (carba-dt) was obtained as a key intermediate for further variations at the 3 -position. this pathway allows an entry to d-and l-configurated nucleoside analogues. however, using this approach a mixture of side products avoids the formation of the product in very high yields. however, we will present that the side products can be recovered by a stereoselective hydroboration leading to one intermediate only that can be use as well for the synthesis of carbocyclic nucleosides. the condensation of the carbocyclic moiety and different pyrimidine and purine nucleobase was achieved by a mitsunobu reaction. various analogues have been prepared via this strategy, e.g. d-and l-carba-bvdu, nucleoside analogues known to be antivirally active against hsv-1. additionally, carbocyclic ␣nucleosides and carbocyclic iso-nucleosides are accessible by this reaction sequence. all new nucleoside analogues were tested for their antiviral activity. particularly carba-dt was found to be a potent anti-hiv active derivative showing no toxicity. however, it can not be excluded that a non-activity of a compound is related to a missing phosphorylation to the monophosphate. in order to prove that, all nucleosides were converted into their cyclosal-phosphate trimesters and transferred into the nucleotides. detailed chemistry, enzymatic and antiviral activity data will be presented. in some cases the nucleotide releasing system showed improved antiviral activity as compared to the parent nucleoside. michela pollicita 1 , candace pert 2 , maria-teresa polianova 2 , alessandro ranazzi 1 , michael ruff 2 , carlo-federico perno 1 , stefano aquaro 1 1 university of rome tor vergata, italy; 2 georgetown university, washington, dc, usa monocytes/macrophages (m/m) are a strategic reservoir of hiv-1 commonly infected by ccr5-using (r5) strains of hiv-1. ccr5 is an attractive target for inhibition of ccr5 mediated hiv-1 entry. thus, ccr5 antagonists are expected to be a power-ful new class of receptor-based therapeutic agents against hiv-1 infection. d-ala-peptide t-amide (dapta) is an octapeptide derived from the gp120 v2 region of hiv-1, able to bind ccr5. dapta acts as selective viral entry inhibitor, displacing the binding of gp120 with ccr5. dapta anti-hiv-1 activity was evaluated in m/m infected with two different hiv-1 r5 strains, bal and 81a, in presence of several doses of the compound. dapta inhibited hiv-1 replication in m/m (>80% compared to control), measured by the p24 gag ag released in the cell culture supernatants, at concentration of 10-3 nm. pcr analysis of integrated hiv-1 proviral dna on cultured m/m proved that dapta is able to block hiv entry and so, to prevent hiv infection in m/m. moreover, the capability of different hiv-1 r5 strains produced and released by infected m/m in affecting neuronal homeostasis was assessed in a neuroblastoma cell line, sk-n-sh, expressing ccr5. in sk-n-sh were evaluated cell morphology, propidium iodide binding and fluorescenceactivated cell sorting (facs) analysis. dapta, at concentration of 10-3 and 10-4 nm, strongly inhibited apoptosis in sk-n-sh of 58 and 56%, respectively, compared to control. unexpectedly, tak-779 (a nonpeptidic ccr5 antagonist with potent anti-hiv-1 activity) inhibited apoptosis only of 30% compared to control. our results suggest that the development of new anti-hiv-1 compounds, such as dapta, could be important in synergistic combination with other antiretroviral treatments in prevent both central nervous system hiv-infection and the consequent neural damage. the mechanisms of dapta inhibition may include both suppression of hiv-1 r5 strains in the brain as direct inhibition of hiv-1 replication in m/m and gp120 related damage by ccr5 binding. pradimicin a (prm-a) is an antifungal non-peptidic benzonaphtacenequinone antibiotic that specifically inhibits human immunodeficiency virus (hiv) in cell culture. it markedly suppresses a variety of different hiv-1 clades in pbmcs, in primary macrophages and several hiv-2 and siv strains in laboratory cell lines (range of 50% effective concentrations: 0.69-11 g/ml; 50% cytostatic concentration: >50 g/ml). prm-a also inhibits syncytium formation between persistently hiv-1-infected hut-78 cells and uninfected sup t1 cells. prm-a behaves as an artificial lectin that selectively binds mannose-containing glycans. consequently, biacore experiments revealed that it binds to gp120 of hiv-1/mn in the presence of ca 2+ . prm-a is endowed with a high genetic barrier with regard to drug resistance development against hiv-1. a variety of multiple mutations at n-glycosylation sites in hiv-1 gp120 are required before the virus looses marked sensitivity to the drug. there is no clustered pattern of hiv-1 gp120 glycan deletions that occur under prm-a drug pressure. the resistance spectrum and mode of action is unique among any of the existing anti-hiv drugs and warrant further (pre)clinical investigations. acknowledgement: this research was supported by the flemish "fonds voor wetenschappelijk onderzoek," the centers of excellence of the k.u. leuven (no. ef/05/15), and the european commission (empro). jan muench 1 , ludger ständker 2 , knut adermann 2 , axel schulz 2 , michael schindler 2 , raghavan chinnadurai 2 , wolf-georg forssmann 2 , frank kirchhoff 2 1 department of virology university of ulm, albert-einstein allee 11, 89081 ulm, germany; 2 ipf pharmaceuticals gmbh, feodor-lynen-strasse 31, 30625 hannover, germany a variety of components in human blood might influence hiv-1 replication in infected individuals. peptide libraries derived from hemofiltrate (hf), an aqueous blood solution, contain essentially all circulating blood peptides with a molecular mass below 30 kda, including chemokines, defensins, and cytokines. to identify the most potent natural occurring factors inhibiting hiv-1 replication, we screened a hf-derived peptide library for antiviral activities. the most active fraction contained a 20-residue peptide corresponding to a c-terminal fragment of ␣1-antitrypsin (␣1-at), a highly abundant serine proteinase inhibitor. further analysis of the corresponding chemically synthesized peptide, termed virus inhibitory peptide (virip), demonstrated that it inhibits infection by all hiv-1 variants tested, independently of their subtype and coreceptor usage. notably, virip also blocked multi-resistant hiv-1 variants and primary isolates. virip specifically inhibited hiv-1 env function, and did not affect infection by virions containing hiv-2, siv, mlv, hcv, ebola or vsv env proteins. the antiviral activity proved to be highly specific for the 20-residue virip sequence since structurally closely related peptides were inactive. we found that virip inhibits hemolysis of erythrocytes induced by the hiv-1 gp41 fusion-peptide (fp). nmr spectroscopy confirmed that virip interacts directly with synthetic gp41 fp. our observations are evidence that a naturally occurring human substance inhibits hiv-1 infection by a new mode of action, i.e. binding of the highly conserved fp. furthermore, we performed a structure-activity-relation study with more than 350 virip analogs and found that specific amino acid changes enhanced the antiviral potency of virip by up to two orders of magnitude. experiments in cell culture and animal models further demonstrated that virip exerted no cytotoxic effects. thus, virip derivatives might become a new class of hiv-1 entry inhibitors. stefano aquaro 1 , valentina svicher 1 , roberta d'arrigo 2 , ubaldo visco-comandini 2 , andrea antinori 2 , mario santoro 1 , giovanni di perri 3 , sergio lo caputo 4 , pasquale narciso 2 , carlo-federico perno 1,2 1 university of rome tor vergata, italy; 2 inmi l. spallanzani, italy; 3 university of turin, italy; 4 sm annunziata hospital, florence, italy to investigate gp41-variability and correlation with viroimmunological parameters in 54 hiv-infected patients (pts) receiving t20 added as a single active drug to a failing regimen. two hundred and ten hiv-gp41 sequences and clinical follow-up from 54 t20-treated patients were analyzed from baseline up to 48 weeks (weeks) of treatment. the association of mutations with viremia (vl)/cd4 count (c/ul) was assessed by mann-whitney test. the addition of t20 to the failing antiretroviral regimen induced at 4 weeks a significant vl decrease from 5.1 log (stable in the last 12 weeks prior t20) to 4.3 log (p = .0002) and a significant cd4 increase from 48 c/ul (decreasing in the last 12 weeks prior t20) to 106 c/ul (p = .008). while vl rebounded to 4.7-5 log at 12-48 weeks, respectively, cd4 increased to 129 c/ul at 48 weeks. t20 resistance mutations, absent at bl, occurred shortly after treatment and usually alone. v38a was the most common sign of t20 failure (27.8% of pts). the viroimmunological outcome of t20-treated pts varied according to gp41-mutations. v38a/e (38.5% of pts) was associated with a cd4 increase from bl (48 c/ul) of 4.5-fold (142 c/ul) at 24 weeks and 6.0-fold (196 c/ul) at 36 weeks (p = .004 and .02 compared without v38a/e, respectively). no significant correlation with vl was observed (from 4.9 log at bl to 4.4-4.8 at 24-36 weeks). by contrast, q40h + l45m (11.1% of pts) was associated with cd4 loss from 71 c/ul at bl to 26 c/ul at 36 weeks (p = .02), without significant changes in vl (from 5 log at bl to 5 log at 36 weeks). mutation n126k (observed in 6 pts, but never found at bl) abrogates the 4th gp41-glycosylation site and correlated with 1.7-fold cd4 increase at 24 weeks. conformational changes induced by v38a/e in the highly conserved giv motif of gp41-hr1, are tightly related with a loss of hiv-induced damage of immune system. this facilitates cd4-recovery through mechanisms that can be virus-(loss of fusion efficiency) and immune-mediated (exposure of new epitopes) not applicable to protease/rt-inhibitors, and thus important for innovative therapeutic strategies. the spread of highly pathogenic h5n1 influenza viruses in humans in asia, with high mortality rates among infected individuals is a major public health concern. in the absence of a vaccine antigenically matching the pandemic virus, antiviral drugs can play an important role. in the present study we reported the antiviral activity of neuraminidase inhibitor oseltamivir against lethal h5n1 influenza virus infection in ferrets, an appropriate animal model that closely resembles clinical signs of human influenza. inoculation of young adult ferrets with a viral dose as low as 10 2 eid 50 of a/vietnam/1203/04 (h5n1) influenza virus caused high fever (39.8-41.8 • c), weight loss (25.4% of initial), anorexia, extreme lethargy and death of animals on days 7-10 post-virus inoculation (p.i.). oral administration of oseltamivir at a dose of 5 mg/kg/day for 5 days twice daily initiated 4 h p.i. inhibited the febrile response, reduced weight changes (14.6% of initial) and, most importantly, completely protected ferrets from lethal h5n1 infection. in the treatment groups, virus replication in the upper respiratory tract of ferrets was prevented, whereas untreated animals shed virus at titers of 2.8-6.5 log 10 eid 50 /ml on days 3, 5 and 7 p.i. systemic spread of the h5n1 virus was observed in untreated ferrets: virus was detected in multiple internal organs, including the brain. treatment with oseltamivir resulted in complete inhibition of virus replication in the lungs and small intestine on day 5 p.i. in the brains of treated animals virus was detected in one of the two animals tested with >100-fold reduction of titer. sequence analysis showed no amino acid substitutions at conserved residues in na or ha1 subunit in viruses isolated from ferret's internal organs after treatment. these results suggest that oseltamivir earlier treatment can prevent h5n1 mortality in ferrets, however, further studies investigating optimal doses and treatment durations required to achieve protection against infection with highly pathogenic influenza viruses are much needed. natalia ilyushina, erich hoffmann, rachelle salomon, robert webster, elena govorkova st. jude children's research hospital, memphis, tn 38105, usa in the present study we tested in the mouse model the hypothesis that combination chemotherapy with drugs targeting dif-ferent virus proteins may lead to more potent and beneficial effects. we applied plasmid-based reverse genetics technique to generate two recombinant a/vietnam/1203/04-like (h5n1) viruses. one virus possessed asparagine at position 31 of the m2 protein that was found in the naturally circulating virus (rgvn-1203) and confers resistance to amantadine. the other recombinant virus possessed serine at that position and was sensitive to amantadine (rgvn-1203sens) . balb/c mice were administered oseltamivir (1 or 10 mg/kg/day) and amantadine (1.5 or 15 mg/kg/day) twice daily for 5 days by oral gavage; the first doses were given 24 h before inoculation with 10 mld50 of h5n1 virus. combination treatment with 10 mg/kg/day oseltamivir and 15 mg/kg/day amantadine was given on the same schedule. single-drug oseltamivir produced a dose-dependent antiviral effect against both recombinant h5n1 viruses (p < 0.01). treatment with oseltamivir at dosage of 10 mg/kg/day significantly inhibited virus replication in the lungs, brain, spleen, and blood of mice at days 3, 6, and 9 after inoculation (p < 0.05), but resulted in low survival rate (20%). single-drug amantadine showed dose-dependent effect only against rgvn-1203sens strain. notably, risk of death for mice that received 15 mg/kg/day of amantadine or 10 mg/kg/day of oseltamivir was similar (p < 0.05). in contrast, prophylactic treatment of mice with combinations of oseltamivir and amantadine completely inhibited virus replication in the animals infected with rgvn-1203sens (p < 0.05) compared to singledrug usage and protected 90% of animals. importantly combination chemotherapy completely protected h5n1 virus spread to the brain of the mice: virus was not detected in brain of treated animals on days 3, 6, and 9 after inoculation and neurological symptoms were not observed. our results suggest that combination chemotherapy provides an advantage over single-agent treatment. this strategy could be an option for the control of influenza virus infection, and combinations with other novel drugs should be explored. françois jean 1 , reid asbury 2 , meera raj 1 , david lawrence 3 , martin petric 3 1 the university of british columbia, vancouver, bc, canada v6t 1z3; 2 ge healthcare bio-sciences, piscataway, nj 08855, usa; 3 british columbia center for disease control, vancouver, bc, canada v5z 4r4 in late 2002, severe acute respiratory syndrome (sars) became the first new severe and easily transmissible human disease to emerge in the 21st century. although it abated after six months, sars serves as a modern paradigm for human emerging infections with 772 deaths reported from 29 countries. the causative agent was found to be a new sars-associated coronavirus (sars-cov) . while the sequence of sars-cov genome was first reported by the bc genome sciences center, the full set of viral and cellular proteins that compose the sars-cov virion remains unknown. to approach this problem, we have utilized two-dimensional gel electrophoresis and liquid chromatography-tandem ms (lc-ms/ms) to identify the viral and cellular proteins in purified sars-cov virions obtained from human infected cells [huh7: human liver] and primate (veroe6: monkey kidney) infected cells. interestingly, analysis of the proteins from purified sars-cov preparations has revealed that the enveloped virions contain not only the predicted viral structural proteins (e.g. spike glycoprotein, nucleocapsid protein, and membrane glycoprotein) but also an important number of differentially incorporated host cellular proteins into or onto the newly formed viruses. we have unambiguously identified over 50 host cellular proteins in sars-cov virions by lc-ms/ms. these proteins include members of the annexin superfamily, cytoskeletal proteins, chaperones, vesicular transport proteins, uracyl-dna glycosylases, and aldehyde oxidoreductases. this study provides the first comprehensive and comparative analysis of the viral and cellular proteins that compose infectious particles of sars-cov obtained from human and primate infected cells. the functions of these newly identified hostspecific proteins are currently being investigated using rna interfering systems; their contributions to structure, viral productive replication, and pathogenicity will be discussed. acknowledgement: supported by an early career ubc operating grant (f. jean) and cihr (m. petric) . dale barnard 1 , craig day 1 , robert montgomery 1 , kevin bailey 1 , matt heiner 1 , larry lauridsen 1 , robert sidwell 1 , kurt berg 2 1 institute for antiviral research, utah state university, logan, ut, usa; 2 panum inst., immi, the ifn-lab, copenhagen, denmark severe acute respiratory syndrome (sars) is a life-threatening respiratory illness caused by sars-cov. there are no approved therapies for sars. some drugs inhibit sars-cov replication in vitro including human interferons and selected antiinflammatory agents (chihrin and loufty, 2005. 3, 251-262) . interferons are very promising because of their potent in vitro inhibition of sars-cov. although anti-inflammatory agents are not very active in vitro, it is thought that they might be efficacious in reducing any deleterious inflammatory response associated with virus infections such as sars infections in humans. for example, troxerutin, a flavenoid with anti-inflammatory properties, is in clinical trials for treating rhinovirus (rv) infections, ameliorating rv-induced inflammation (turner et al., 2004. apmis 112, 605-611) . therefore, troxerutin was tested for inhibition of sars-cov replication in the lungs of infected mice using a mix of four hydroxyethylrutosides that included troxuretin. in addition, mouse interferon-alpha, used as a model compound for human interferon-alpha, was evaluated for inhi-bition of virus lung titers. both mouse interferon-alpha administered i.p. daily beginning 12 h pre virus exposure at doses of 100,000 and 10,000 iu and the hydroxyethylrutoside mix (100 and 10 mg/kg) administered i.p using the same schedule reduced virus replication in the lungs of mice to below detectable limits. when a hydroxyethylrutoside mix was given to mice in the drinking water at 1.32 mg/ml (likely equivalent to an i.p. dose of 100 mg/kg, assuming that the mice drank freely), virus lung replication was also completely inhibited. all treatments appeared to be well tolerated, since all groups of mice gained weight. we also report on the efficacy of various combinations of two doses of these drugs administered i.p., using the same dosing regimen as described. these data support the supposition that interferon might be a useful therapy for treating human sars infections and that hydroxyethylrutosides should be investigated further as a potential therapy. acknowledgement: supported by contract no. n01-ai-15435 from the virology branch, niaid, nih. treatment options for human respiratory syncytial virus (rsv) are limited. an effective vaccine is not yet available. neutralizing polyclonal antibody (respigam tm , medimmune) and a humanized monoclonal antibody (synagis tm , medimmune), are licensed for prophylactic use. ribavirin is the only approved antiviral against rsv, but its efficacy is controversial and its use is limited to treatment of high-risk patients. there is a clear need for new anti-rsv therapeutics with improved efficacy and ease-of-use. many early efforts to identify anti-rsv compounds focused on blocking the process of fusion. we have developed a cell-based screening platform to identify antivirals that inhibit rsv transcription and replication. the assay does not require infection with wild-type virus. it is based on an rsv subgenomic replication system in baby hamster kidney (bhk-21) cells that express the essential viral replication proteins (n, p, l and m2-1). the readout is expression of the reporter gene lacz from a subgenomic rna. screening of the apath small molecule library yielded 596 compounds (hit rate = 0.75%) with ec50 values ≤10 m and with selectivity index (si) values ≥10. seventy-two compounds demonstrated antiviral activity against wild-type rsv (strain a2) in a cytopathic effects inhibition assay (ec50 < 10 m; si > 10). these anti-rsv compounds represent nine different chemical classes. two compounds, a 4-aminoquinoline (ec50 = 0.25 m) and a thienopyrimidine (ec50 = 0.82 m), were shown to have desirable pharmacokinetic profiles and were chosen for efficacy testing in the cotton-tail rat model of infection. sar studies to identify the pharmacophore of the compounds have been initiated. preliminary studies to characterize the mechanism-ofaction in virological assays will be discussed. acknowledgement: supported by nih r44 ai047552-02. we have previously reported bicyclic furano pyrimidine nucleoside analogues (bcnas) as exquisitely potent and selective inhibitors of varicella zoster virus (vzv) , with subnanomolar activity for p-alkylphenyl substituted analogues such as lead compound cf1743(cf-1743) (1) . these compounds have entered preclinical development with fermavir pharmaceuticals. we now report the first chromatography-free synthesis of these agents, their scale up to multi-gramme amounts, and their pre-clinical characterisation. in addition, we were keen to address potential solubility and bioavailability issues of these highly lipophilic agents by the synthesis of more polar analogues in two categories; side-chain ethers (2) as new analogues in their own right, and 5 -phosphates (3) as potential more soluble prodrugs. we report data on both of these new families at this meeting. finally, we note the application of our phosphoramidate pro-tide approach to this family, with a series of bcna protides (4) designed as intracellular phosphate delivery forms to bypass the essential vzv thymidine kinase-mediated first phosphorylation step. graciela andrei 1 , joos van den oord 2 , pierre fiten 1 , ghislain opdenakker 1 , erik de clercq 1 , robert snoeck 1 1 rega institute for medical research, katholieke universiteit leuven, leuven, belgium; 2 pathology department, u.z. leuven, leuven, belgium varicella (chickenpox) , the primary infection caused by vzv, is characterized by viremia and skin lesions. reactivation of the latent virus results in skin lesions characteristic of herpes zoster (shingles). as keratinocytes are one of the main target cells for productive infection in vivo for vzv, human epithelial cells represent a relevant model for the study of vzv pathogenesis and evaluation of antiviral compounds. organotypic epithelial raft cultures permit full differentiation of keratinocytes via culturing of the cells on collagen matrix at the air-liquid interface. we have previously shown that the susceptibility of cultures to infection with vzv depends on the stage of differentiation of the rafts. we have now quantified the activity of reference anti-vzv compounds by measuring viral dna load by realtime pcr. quantitative pcr for vzv dna was performed by using specific primers and a mgb-probe for the orf29 gene (single-stranded dna binding protein) by the taqman method. two series of raft cultures were infected with the wild-type oka strain after 4 days of differentiation and treated with serial dilutions of the test compounds. at 12 days post-differentiation one series of the cultures was processed for histology and the other one for viral dna quantification. acyclovir (acv), penciclovir (pcv) and brivudin (bvdu) at 4 and 0.4 g/ml, foscarnet (pfa) at 40 g/ml and cidofovir (cdv) at 4, 0.4 and 0.04 g/ml inhibited viral dna content by more than 95%. these results were in agreement with histological examination of the rafts, no cytopathic effect being observed at these concentrations. as expected, only cdv and pfa inhibited the replication of the thymidine-kinase deficient (tk-) 07-1 strain. a correlation between the degree of protection as determined by histological examination and viral quantification could also be demonstrated for cdv and pfa against the tk-07-1 strain. since no animal model is available for the in vivo evaluation of antiviral agents against vzv, the organotypic cultures may be considered as a valuable ex vivo model to evaluate the efficacy of new anti-vzv antivirals. jae-seon hwang 1 , oliver kregler 1 , john c. drach 2,3 , leroy b. townsend 3 , elke bogner 1 1 institut für klinische und molekulare virologie, erlangen, germany; 2 department of biologic and materials sciences, school of dentistry; 3 interdepartmental graduate program in medicinal chemistry, college of pharmacy, university of michigan, ann arbor, mi, usa dna packaging is the key step in viral maturation and involves binding and cleavage of viral dna containing specific dnapackaging motifs. this process is mediated by a group of specific enzymes called terminases. we have previously demonstrated that the hcmv terminase is composed of two subunits, the large one encoding pul56 and the small pul89, where each protein has a different function. while the large subunit mediates sequence specific dna binding and atp hydrolysis, pul89 is only required for duplex nicking. inhibitors targeting pul56 and/or pul89 are attractive alternatives as hcmv antivirals since mammalian cell dna replication does not involve cleavage of concatameric dna. we now have screened several members of the benzimidazole ribonucleoside class of replication inhibitors in order to determine if a compound has the capacity to block the atpase activity of the large terminase subunit pul56. analysis by bioluminometric atpase activity assays identified bdcrb and one more compound [2-bromo-4,5,6-trichloro-1-(2,3,5-tri-o-acetyl-␤-dribofuranosyl)benzimidazole (btcrb)] with inhibitory effects. although only btcrb and bdcrb were inhibitors of the atpase activity, two other compounds, dbdcrb and cl4rb, inhibited virus replication in a plaque-reduction assay, thus indicating that those have a different mode of action. in addition, by electron microscopy of thin sections we observed that in the presence of btcrb only b-capsids and dense bodies were formed. furthermore, spherical capsids accumulated in the perinuclear cisternae indicating a block in nuclear egress thereby providing additional evidence that closely-related benzimidazole d-ribonucleosides may have differences in their antiviral modes of action. human cytomegalovirus (hcmv) is the cause of significant morbidity and mortality in a variety of immunocompromised patients. currently available anti-hcmv drugs interfere with dna replication; however, these drugs are highly toxic, pre-cluding their long-term use in humans. interrupting hcmv viral entry is largely unexplored as an antiviral drug development strategy and is potentially an ideal and tractable goal. hcmv is believed to rely upon formation of ␣-helical coiled coils in the viral glycoproteins gb and gh to promote virus-host membrane fusion; peptides encompassing heptad repeat sequences in these two proteins inhibit viral infection. we have explored nonnatural oligomeric molecules ("foldamers") that are designed to mimic elements of the putative ␣-helical segment of gb. this effort has led to the discovery of oligomers of ␤-amino acids ("␤-peptides") that block hcmv infection. the ␤-peptide scaffold offers several advantages for the design of protein-protein interaction inhibitors, as ␤-peptides are amenable to modular synthesis, resist proteolytic degradation, and can display large and tailored molecular surfaces. the most potent ␤-peptide inhibitor blocks hcmv infection with a micromolar ic 50 in a cell-based assay. these compounds show specificity for hcmv relative to closely related viruses. mechanistic studies suggest that these inhibitors interfere with membrane fusion between hcmv particles and host cells. current efforts are focused on understanding in greater detail the origin of the observed biological activity, exploring other foldamer scaffolds as bases for inhibitor design, and developing specific fusion inhibitors for other herpesviruses. previous reports have indicated that herpes simplex virus (hsv) activates nuclear factor-kappab (nf-kb) during productive infections. nonsteroidal anti-inflammatory drugs (nsaids) have significant inhibitory effects on nf-kb. therefore, two nsaids, indomethacin and aspirin, were assayed for antiherpetic effects and utilized as tools to further study the role of nf-kb in hsv-1 infection. we report that indomethacin and aspirin inhibited hsv-1 replication at non-cytotoxic doses. in vero cells, 500 um indomethacin and 20 mm aspirin reduced hsv-1 titers 99.999 and 99.5%, respectively. electromobility shift assays revealed that hsv-1 activation of nf-kb is inhibited by the nsaids at doses that coincide with reduction of hsv-1 titers. to investigate a pathway for nf-kb inactivation, protein levels of ikb-alpha, a cytoplasmic nf-kb inhibitor, were examined. ikb-alpha protein was present in uninfected samples, but decreased over time in all hsv samples, regardless of chemical treatment, suggesting localization of nf-kb to the nucleus. immunohistochemistry studies verified that p65, a component of the dimeric nf-kb complex, translocated to the nucleus of hsv-1 infected cells in the presence or absence of the nsaids. finally, direct effects on viral gene activity were assayed by real-time rt-pcr analysis. indomethacin and aspirin reduced mrna for icp4, an essential hsv immediate-early gene, 2.9and 2.5-fold, respectively, resulting in significant decreases of icp4 protein. but transcriptional analysis revealed that synthesis of mrna for thymidine kinase, an hsv early gene, was unaffected by chemical treatment. however, mrna for glycoprotein c, an hsv late gene was undetectable in indomethacin and aspirin treated samples. cumulatively, these data indicate that: (i) indomethacin and aspirin block hsv-1 replication and (ii) the in vitro anti-herpetic effects of nsaids may reside in their ability to block nf-kb activity within the nucleus, impairing activation of essentials hsv genes. increasing species-specificity constraints preclude study of human cytomegalovirus (hcmv) in animals, necessitating the use of rodent cmvs to model human disease. however, the susceptibility of animal cmvs to clinically useful antivirals is unpredictable. for example, the guinea pig cmv (gpcmv), a uniquely valuable virus for modeling congenital cmv infection, is highly resistant to ganciclovir (gcv) at medically relevant doses. we used a molecular virological approach to test the hypothesis that gcv susceptibility could be conferred on gpcmv by insertion of the human ul97 phosphotransferase gene into the gpcmv genome. the gpcmv genome, cloned as a bacterial artificial chromosome in e. coli, was modified by site-specific recombination, using a shuttle plasmid targeting the gp97 locus, and carrying the ul97 gene from hcmv strain towne. the resultant chimeric virus was replication competent, and was found to contain the hcmv ul97 by southern-blot and sequence analyses. northern-blot revealed that a hcmv ul97-specific transcript was expressed with late gene kinetics. western-blot, using a hcmv ul97-specific polyclonal antibody, detected protein in virus-infected cells. the chimeric virus was gcv-susceptible, compared to wild-type gpcmv, with an ic 50 of 15 m. chimeric virus also exhibited increased sensitivity to maribavir (mbv), exhibiting a 3-log reduction (compared to wild-type virus) in the presence of 50 m mbv, and an ic 50 of 5 m. to study the in vivo pathogenesis of chimeric virus, cyclophosphamide-immunocompromised strain two guinea pigs were challenged intraperitoneally, resulting in evidence of disseminated infection, and mortality. ganciclovir treatment (25 mg/kg/day) resulted in reduced weight loss, and mortality, compared to placebo. these studies confirm the key role of ul97 in cmv antiviral therapy, and demonstrate that a 'humanized' gpcmv can be generated with altered antiviral susceptibilities. genital herpes infections are a global health problem and impact hiv/aids epidemic. strategies to prevent transmission include treatment of infected subjects to suppress shedding and prophylaxis with vaginally-applied microbicides. we examined the in vitro and vivo activity of rep 9, a fully degenerate 40 mer phosphorothioated oligonucleotide against hsv-2 infection of human cervical cells and in a vaginal murine model. rep 9 has broad-spectrum anti-herpetic activity with potent in vitro activity against hsv-1, hsv-2, hcmv, vzv, ebv, and hsv-6 (vaillant et al., submitted for publication). at a concentration of 100 m, rep 9 inhibited 6-logs of hsv-2 infection if present during the entire experiment. synchronized infectivity assays demonstrate that, unlike sulfonated polyanions in clinical trials, which primarily block hsv attachment, rep 9 acts at multiple steps and inhibits binding, entry and post-entry gene expression. in our in vivo studies, mice were treated once intravaginally with rep 9 or pbs control at various times prior to vaginal challenge with a lethal dose of hsv-2 strain 186 (10 log 4 pfu). rep 9 prophylaxis provided protection to mice from hsv-2 infection and disease. protection was significant when challenged 30 min after treatment (p < 0.01). additionally, treatment with an analog of rep 9, which cannot activate tlr-9 mediated immune stimulation, was at least as active as rep 9, suggesting that direct antiviral activity and not stimulation of innate immunity is the mechanism of action in vivo. utilizing this analog, protection was significant when challenged 30 min after treatment (p < 0.001) with a trend toward protection when administered 60 min prior to challenge (p = 0.07). in summary, treatment with the rep 9 analog which has superior resistance to low ph and nuclease degradation was more effective than rep 9, in some experiments protecting 100% of mice from viral infection and disease. the testing of this ph resistant rep 9 analog in a gel formulation is currently underway. acknowledgement: supported by contract no1-ai-15438 from the virology branch, niaid, nih. a phosphorodiamidate morpholino oligomer (pmo) designed to hybridize to a highly conserved region including the aug translation start site of hcv, called avi-4065, has been evaluated for efficacy, toxicity, and pharmacokinetic properties. avi-4065 inhibits translation initiated at the aug start site with ec50 of 308 nm (2.1 ug/ml) and shows positive cooperativity. this pmo retains most of the activity in the presence of point mutations in the hcv genome. huh-7 cells were incubated with normal human serum (nhs) or hcv infected human serum (is) and hcv replication observed by rt-pcr. avi-4065 produced robust inhibition of hcv in a dose and sequence-specific manner. studies conducted in vivo with avi-4065 in the hcv infected trimera mouse (xtl) show reduction in viral titer which is dose dependent with approximately 90% of mice with undetectable viral titer and the remaining mice show 1 log reduction in viral titer with 0.1 mg/mouse/day for 7 consecutive days. the fractional bioavailability of avi-4065 from a sq dose is approximately 1. the apparent elimination half life in rat, nonhuman primate and humans was 2.3, 4.5 and 11.4 h, respectively. the volume of distribution ranged from 0.6 to 1.0 l/kg and the cmax is linearly related to the dose in mg/m 2 . a phase i study in healthy volunteers in which 14 daily sq doses of 50 and 100 mg has been completed. no serious adverse events have been observed. treatment of infected patients is currently planned. inhibition of hcv polyprotein synthesis is anticipated to contain therapeutic benefis of both protease inhibitors and polymerase inhibition. hcv infection can progress to fibrosis, reduced liver function, hepatocellular carcinoma, and death. currently, the standard treatment for hcv infection involves treatment with pegylated interferon in combination with the nucleoside analogue ribavirin. this treatment regimen effects a cure in approximately 40-60% of the genotype-1 (gt-1) population; therefore a significant unmet clinical need exists in hcv therapy. virus-encoded polymerases have proven to be excellent molecular targets for chemotherapeutic intervention in numerous viral mediated diseases. in the case of hiv, hbv and herpes virus infections, deoxy-nucleoside analogues, which act as chain terminating agents, have been shown to have invaluable clinical utility. by analogy, appropriate ribonucleoside analogues might be expected to inhibit the essential rna polymerase (ns5b) encoded by hcv. here we describe the preparation of nucleoside analogues as inhibitors of the hcv polymerase. in our design of nucleoside analogs as potential anti-hcv agents, we chose to investigate the effect of 4 -substituted ribonucleoside derivatives. we reasoned that after incorporation of a ribonucleoside containing a 4 -substituent, a disruption in elongation of the growing rna could be effected through either steric hindrance or via a conformational change of the carbohydrate moiety. our investigations on several such analogues will be presented. of particular interest is 4 -azido-cytidine, which shows good activity in the genotype 1b sub-genomic replicon (ic50 = 1.28 m) with no measurable cytotoxic or cytostatic behavior. in addition, we have shown that the triphosphate of 4 -azido-cytidine is a potent and highly selective inhibitor of ns5b (ic50 = 0.32 m). joanna e. boerner, sue ma, choilai tiongyip, michael p. cooreman, teresa compton, kai lin novartis institutes for biomedical research, 100 technology square, cambridge, ma 02139, usa current drug discovery efforts for hepatitis c virus (hcv) focus on developing specific inhibitors of two viral enzymes, ns5b polymerase and ns3-4a protease. however, resistant viral mutants are likely to emerge during therapy, compromising the effectiveness of these inhibitors. an alternative and complementary strategy is to target host factors that are also essential for viral replication. cyclophilins, a family of peptidyl-prolyl isomerases and the cellular targets of cyclosporin a (csa), present such an opportunity. it was reported recently that cyclophilin b bound to hcv ns5b polymerase and stimulated its rnabinding activity, and that these functions were blocked in the presence of csa (watashi k. et al., molecular cell 2005) . nim811, a csa derivative, is a more suitable candidate for hcv therapy because it binds to cyclophilins with higher affinity than csa while lacking the immunosuppressive activity associated with csa. using the hcv replicon system we demonstrated that nim811 exhibited potent anti-hcv activities in vitro. moreover, the combination of nim811 with a specific non-nucleoside inhibitor of hcv polymerase led to synergistic antiviral effects with no significant increase of cytotoxicity. resistant clones against both inhibitors were obtained in vitro, however, it was much more difficult to generate resistance against nim811 than the polymerase inhibitor. also, there was no cross-resistance between the two inhibitors. finally, addition of nim811 to the hcv polymerase inhibitor drastically reduced the emergence of resistance compared to polymerase inhibitor alone. taken together, nim811, with a novel mechanism of action and a favorable pharmacokinetics and safety profile, represents a promising clinical candidate for treating hepatitis c and provides a rationale for specific combination therapy. the nucleoside analog r1479 was identified as a specific inhibitor of hcv replication in subgenomic hcv replicon cells. r1479-tp is a competitive inhibitor of cmp incorporation by hcv polymerase ns5b. in a transient replicon system r1479 inhibited hcv rna replication driven by genotype 1b polymerase with similar potency as compared to that driven by genotype 1a polymerase. r1479-tp inhibited native hcv replicase and recombinant ns5b from genotype 1a and 1b with similar potency. in contrast, r1479-tp did not inhibit human dna polymerases alpha, beta or gamma, including reverse transcriptase activities of dna polymerases beta and gamma, which were highly sensitive to inhibition by azt-tp and 3tc-tp. no significant inhibition was observed with human rna polymerases i, ii and iii derived from hela cells. in addition, the functionally related native influenza virus rna dependent rna polymerase (rdrp) activity in vitro was not inhibited by r1479-tp at concentrations up to 1 mm, suggesting high selectivity for the hcv rdrp. thus, r1479 was identified as a potent and highly selective inhibitor of hcv polymerase mediated rna synthesis. guangxiang luo, zhaohui cai, chen zhang, kyung-soo chang, jieyun jiang microbiology, immunology, and molecular genetics, university of kentucky college of medicine, lexington, ky, usa the study of hepatitis c virus (hcv) replication and the search for specific antiviral agents against hcv infection have been hampered by the lack of an efficient stable cell culture system of hcv infection and propagation. we have successfully constructed stable human hepatoma cell lines that contain a chromosomally integrated-genotype 2a hcv cdna and constitutively produce and secrete high titers of infectious virus into the culture media. transcriptional expression of the full-length hcv rna genome is under the control of a cellular pol ii polymerase promoter at the 5 end and a hepatitis delta virus ribozyme at the 3 end. the resulting hcv rna was expressed and replicated efficiently, as shown by the presence of high levels of hcv proteins as well as hcv rna in the stable huh7 cell lines. hcv secreted from the stable cell lines was infectious, as determined by antibody neutralization, blockage of putative hcv receptors, and inhibition of hcv replication by interferon. our findings demonstrate the establishment of a stable cell culture system of infectious hcv production and propagation, which allows the study of the entire hcv infectious cycle. the stable hcv-secreting cell lines are now being pursued to develop high throughput screens for effective hcv inhibitors. additionally, we established a novel and powerful hcv replication system in the mouse hepatocyte and mouse embryo fibroblasts (mef). hcv rna was found to replicate efficiently in both pkr +/+ and pkr −/− mef cells, demonstrating that hcv rna replication in mef cells is a powerful system to study host-virus interaction by using diverse gene-knockout animals. interestingly, hcv rna replicates more efficiently in the pkr −/− cell than in the pkr +/+ cell, suggesting a role of pkr in the control of hcv rna replication. however, ifn inhibited hcv rna replication in the pkr −/− cell with an efficacy similar to that in the pkr +/+ cell, suggesting a pkr-independent antiviral mechanism. clearly both pkr-dependent and pkrindependent antiviral mechanisms are important for the control of hcv replication and the mediation of the ifn-induced anti-hcv response. our studies set a stage for the development of transgenic mouse models of hcv replication and open up new avenues to study hcv and host interactions in mefs derived from diverse gene-knockout animals. andrea cuconati 1 , haitao guo 2 , gael westby 1 , anand mehta 2 , timothy block 1,2 1 institute for hepatitis and virus research; 2 drexel institute for biotechnology and virology research, doylestown, pa, usa the high levels of hepatitis b surface antigen (hbsag)-bearing non-infectious particles in the serum of infected individuals is thought to play a role in suppressing hepatitis b virus (hbv)specific immune response by titering out hbv-specific antibodies and lymphocytes. current hbv therapeutics do not directly reduce this viral antigenemia. our group has focused on the enhancement of the immune response through the inhibition of viral antigen secretion in the infected hepatocytes, with the therapeutic goal being the use of hbv vaccination for the treatment of acute and chronic infection. the high-throughput screening of a small molecule library of 80,288 drug-like compounds was undertaken to discover novel inhibitors of hbsag secretion. using the stably hbv-transfected, human hepatoma cell line hepg2.2.15, we developed an hts-compatible elisa protocol for the detection of hbsag secreted in the culture media. the screen resulted in 1758 initially positive hits, a hit rate of 2.2%. subsequent retesting for activity and toxicity by mtt assay has narrowed the number of confirmed, non-toxic hits to 77, currently categorized in twelve chemical series. we have previously reported on a trio of related pyrazolo-pyridines with ec50 measurements below 5.0 m and cc50 measurements >50.0 m. nascent structure-activity relationship (sar) suggests that a central moiety of the molecules is essential to activity, with an aromatic side group contributing to potency. among recently confirmed inhibitors, two currently under investigation include: (1) an isobutyl-acetamide with an ec50 of 87.0 nanomolar, and a cc50 of >50 m, and (2) a carbothiamide with an ec50 of 1.6 micromolar and a cc50 of >50 m. measurement of secreted hbv l and m antigens and cellular markers indicated that the pyrazolo-pyridines are not specific inhibitors of viral antigens, while the isobutyl-acetamide and the carbothiamide are indeed specific. measurement of intracellular viral dna indicated that none of these molecules are inhibitors of replication. we will be reporting on our studies of the potency, specificity, and potential mechanisms of action of these novel anti-hbv compounds. background: entecavir (etv) is a potent competitive inhibitor of hepatitis b virus (hbv) polymerase with activity versus all three enzymatic functions including priming, minus, and plus strand dna synthesis. virologic rebound due to etv resistance (etvr) has only been observed in lamivudine resistant (lvdr) hbv (m204v/i ± l180m), and requires at least one additional change in the reverse transcriptase domain (rt) at residues t184, s202, or m250. these substitutions surround the dntp binding site or primer grip of rt. the objectives of this work were to further characterize etvr and its mechanism(s) using cell culture, in vitro enzyme, and molecular modeling studies. methods: hbv cell culture assays used transfected hepg2 cells and quantitation of released, immunocaptured hbv nucleocapsids. gradient-purified intracellular nucleocapsids were used for in vitro rt assays. a 3d homology model based on the hiv-1 rt structure was used to model resistance changes in hbv. results: reduced etv susceptibility of etvr hbv was observed both in culture and enzymatically in vitro. kinetic studies showed various etvr substitutions in lvdr hbv selectively reduced etv-triphosphate (etv-tp) binding (k i ) to rt without markedly changing the affinity for dgtp (k m ) or inhibition by ddgtp. etvr rts also displayed reduced enzymatic activity (k cat ) relative to wildtype and etvr hbv appeared growth impaired. modeling studies suggested a novel etv-tp binding pocket in hbv rt that became constrained with etvr changes. m250 changes in the primer grip region of rt were unique in that resistance was primarily seen during synthesis of minus strand dna. etvr changes in the absence of lvdr substitutions had greatly reduced impacts on etv susceptibility, confirming models suggesting etvr is imparted through lvdr changes. summary: etv provides a high genetic barrier to resistance, requiring additional changes at residues t184, s202 or m250 along with pre-existing lvdr substitutions m204v/i ± l180m. kinetic parameters and molecular modeling indicated that etvr substitutions selectively affected etv-tp binding and reduced the replication capacity of hbv. a nonhuman primate (nhp) model of classical, lesional smallpox has been used to test the efficacy of intravenous (iv) cidofovir treatment. cynomolgus macaques were infected with a high dose (10 8 pfu iv) of variola to produce an artificial primary viremia, and then treated with cidofovir at 0, 24, or 48 h postinfection (pi). later treatment times were not evaluated. treatment at 24 or 48 h pi halted increases in peak blood viral genome titers measured by quantitative taqman-mgb real-time pcr, which were more than 10-fold less in cdv-treated animals compared to placebo. historically, the number of pox lesions provided the best correlation with human smallpox clinical severity, and cdv treatment in our model significantly reduced maximum pox lesion counts by >90%; the number and size of skin lesions, and in untreated animals contributed significantly to the total viral burden with lesions containing 10 9 -10 10 genomes/g. to better understand the role of viral burden and disease progression in major organ systems, a serial sample study was undertaken. in untreated animals at 24 h pi, viral replication in spleen exceeded 10 9 genomes/g while liver and bone marrow yielded 10 8 genomes/ml. in comparison, titers in other tissues ranged between 10 5 and 10 6 genomes/g and blood yielded 10 4 genomes/ml at 24 h, suggesting that the liver, spleen, and marrow may be initial sites of replication. levels of virus in the bone marrow reached a peak of approximately 10 10 genomes/g at day 5, then decreased to quantities consistent with those in blood. viral load in the blood increased with time, peaking around days 7-9 at 10 8 genomes/ml. virus was also detected in intestine, skeletal muscle, and late in infection, testes. the ability to successfully treat with cdv 24 h pi despite early extensive organ infection in the accelerated nhp variola model suggests that this treatment could be effective in reducing viremia and mortality after onset of symptoms in human smallpox, which demonstrates a more protracted disease course. work involving variola virus conducted in who-sanctioned cdc, atlanta bsl-4 laboratory. earl kern 1 , kathy keith 2 , robert jordan 2 , dennis hruby 2 , debra quenelle 2 1 department of pediatrics, university of alabama school of medicine, birmingham, al, usa; 2 siga technologies, inc., corvallis, or, usa although cidofovir (cdv) has been approved as an investigational new drug for emergency treatment of smallpox, its lack of oral activity and dose limiting toxicity dictates a need for continued development of better therapeutic agents for this potential bioterror disease. it has been reported previously that st-246, a low-molecular weight compound, inhibits replication of all the orthopoxviruses in vitro and protects mice infected with vaccinia or ectomelia virus. in the present study, we have utilized cowpox virus (cv) and vaccinia virus (vv) infections in vitro and in vivo to evaluate the efficacy of st-246 for treatment of orthopoxvirus infections. in plaque reduction assays in human foreskin fibroblast cells, both cv and vv were inhibited by about 0.1-0.5 um of st-246. for in vivo studies, st-246 was administered once daily by oral gavage to mice using 100 mg/kg for 5, 7, 10, or 12 days beginning 4 or 24 h after intranasal inoculation with vv or cv. st-246 was highly effective (p < 0.001) in preventing mortality due to vv or cv even when treatment was delayed up to 24 h post-infection. a dosing duration of 5 days was adequate for vv infected mice, but duration of 7 days or longer was required for efficacy in cv infected mice. when st-246 was given once daily for 14 days at 100, 30, or 10 mg/kg daily at 24, 48, or 72 h post-cv inoculation, mortality was significantly altered at all dosage levels and time points. to determine the effect of treatment on virus replication in target tissues, mice were inoculated with cv or vv and treated once daily with 50 mg/kg of st-246. on various days post-infection tissues were harvested and assayed for virus. in cv or vv-infected mice, st-246 treatment successfully reduced virus titers from 3 to 5 logs 10 in liver, spleen, and kidney. little effect was noted in lung tissue. these results indicate that st-246 has significant activity against vv and cv infections in vitro and in vivo and may be a potential chemotherapeutic agent for treatment of human orthopoxvirus infections. cidofovir (hpmpc) is a broad-spectrum anti-viral agent that is used (vistide ® ) to treat aids-related cmv retinitis. currently, cidofovir is of particular interest as a potential therapy for orthopox virus infections, including smallpox. an important limitation of cidofovir and analogous nucleotide drugs in a therapeutic role is their low oral bioavailability and poor transport into cells. in principle, bioavailability of a drug can be improved by structural modification targeting transporters expressed in human intestine. to be effective, the transported prodrug must be cleaved by endogenous enzymes to its parent compound. we will present synthetic studies of novel cidofovir and cyclic cidofovir (chpmpc) prodrugs incorporating amino acids or small peptides, comparing different drug-amino acid linkage strategies. the compounds were evaluated for transporter-mediated uptake and cellular and plasma hydrolysis. the results will be compared with similar studies carried out on a series of peptidomimetic conjugates of foscarnet, the trisodium salt of phosphonoformic acid (pfa), an anti-viral agent that also has very low oral bioavailability and poor cell penetration. the question addressed in this study is if wnv-reactive antibody can improve disease signs in a hamster model after the virus is demonstrated to be in the brain. the hypothesis is based on the high activity of a humanized monoclonal antibody, he16, in a mouse model when administered later in infection (oliphant et al., 2005. nat. med. 11, 522) . in this study, virus was demonstrated to be in the brains of hamsters at 5 days post-viral injection (dpi) by cell culture assay, quantitative rt-pcr, and immunohistochemical staining of wnv in neurons. eighty percent of hamsters treated i.p. 5 dpi with 100 mg/kg of humanized monoclonal antibody, he16, survived wnv disease, whereas, 37% of placebo-treated hamsters survived ( *** p < 0.001). if administered at 2 dpi, 100% survived. we tested the hypothesis that he16 is effective if delivered directly into the brain instead of by peripheral administration. the antibody was delivered into the brain 5 dpi using convectionenhanced delivery through a cannula implanted into the brain. the he16 was detected in the cns, but none was detected in the kidney. the survival of he16-treated hamsters was 88% as compared to 22% of placebo-treated animals ( *** p < 0.001). for additional proof, the majority of hamsters having wnv in their cerebrospinal fluid, a marker for cns infection, were protected with he16 administered i.p. at 5 dpi. this humanized monoclonal antibody, therefore, is a possible treatment for the post-exposure, wnv-infected humans that develop signs of neuroinvasive disease. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih, and grant 1-u54 ai06357-01 from the rocky mountain regional centers of excellence, nih. hemorrhagic fever viruses are of serious worldwide health concern as well as potential biological weapons. lassa fever virus in particular annually infects several hundred thousand individuals in west africa, and the export of this pathogen outside of this region, either intentionally or unintentionally, presents a serious risk to the developed countries of the world. the cdc and niaid have identified lassa fever virus as a category a priority pathogen, indicating the highest degree of threat to public health. no arenavirus-specific antiviral drugs are currently approved for use in humans. the purpose of siga's biodefense program is to develop safe and effective drugs for preventing and treating diseases caused by category a viruses. to that end, a large and diverse library of small molecule compounds was screened using a viral pseudotype assay to identify inhibitors that target the essential lassa surface glycoprotein (gp) and thus block viral entry into the host cell. twenty-six compounds were identified as quality hits, as defined by potency, selectivity, and chemical tractability. antiviral activity against authentic lassa fever virus was assessed in cell culture through a collaboration with colleagues at usamriid. a number of these potent antiviral compounds and their related analogs have exhibited informative chemical structure-biological activity relationships (sar). two potential lead compound series have emerged from these studies, each with 50% effective concentrations (ec50s) of less than 100 nm against lassa fever virus and with ec50s of less than 2 nm against lassa gp-pseudotyped virus. characterization of the in vivo properties of these compounds is underway. the in vitro antiviral potency and selectivity, animal pharmacokinetics, and the development process will be presented. these inhibitors represent an important step toward the development of a small molecule antiviral drug for lassa fever virus. sven enterlein 1 , pramila walpita 1 , allison groseth 2 , heinz feldmann 2 , ramon flick 1 1 university of texas medical branch, department of pathology, galveston, tx, usa; 2 national microbiology laboratory, public health agency of canada, winnipeg, man., canada nipah (niv) virus (family paramyxoviridae) is a recently emerged human and animal pathogen that can cause severe encephalitis with fatality rates of up to 70%. since no treatment or vaccination is available, and cross-species spread was observed, the virus has been classified as biosafety level 4 (bsl-4) agent. to avoid bsl-4 containment for the study of cis-acting signals as target for antiviral strategies, we used an optimized plasmid-driven t7 minigenome rescue system (without the need for recombinant vaccinia virus mva-t7) as well as an newly established rna polymerase i-based approach. minigenome rescue is based on transfection of the minigenome niv-cat and the plasmids encoding for the three nucleocapsid proteins n (nucleoprotein), l (polymerase), and p (phosphoprotein) and measured by enzymatic cat assays. we used the established plasmid-based minigenome rescue systems to screen for potential antiviral compounds. in a first step we tried to determine the optimal strategy for the delivery of small hairpin (sh) interfering rna molecules. for this we compared three shrna delivery systems against another bsl4 agent-reston ebolavirus (family filoviridae); (i) plasmid-mediated pol i and (ii) pol iii-driven shrnas, and (iii) exogenously (t7) produced shrna, for their ability to induce gene silencing. interestingly, beside the in vitro-generated or pol iii-driven shrnas, pol i transcripts showed very efficient inhibition of minigenome rescue. however, the most efficient delivery method was transfection of in vitro transcribed shrnas. we will present the results of this comparison and, based on the most efficient approach, also first results of shrnas targeted either to niv n, p, and l genes or to the leader/trailer noncoding regions to interfere with minigenome replication. conformative data with live virus experiments under bsl4 conditions will be included. filoviruses, which include ebola virus and marburg virus, are among the most notorious human pathogens because they cause sporadic outbreaks of severe hemorrhagic fever. unfortunately, very few therapeutic agents are available to treat infections with these viruses. antiviral screening methods that determine the effect of compounds on viral replication involve working with infectious virus, which is obviously not practical for these biosafety level 4 (bsl-4) agents. we developed an antiviral screening method based on a cell-based, infection-independent, ebola subgenomic replication system in which the expression of an easily measurable enzyme is dependent on the rna replication and transcription factors of ebola virus. using this system we screened a synthetic compound library for antiviral activity against ebola virus and have identified a number of inhibitors. we also used it to identify a peptide inhibitor directed against vp30. anti-ebola virus activity for many of the inhibitors was confirmed in a viral replication assay using a gfp-expressing zaire '76 strain of ebola virus. fifty-two small molecule inhibitors from at least six classes of compounds had ec50 values in the low micromolar range and good selectivity. several of these compounds have promising chemical, biological, and pharmacological profiles to pursue as potential anti-filovirus drugs. we are currently preparing to test these compounds in a mouse model of ebola virus. we have also begun a lead optimization program to improve antiviral potency and selectivity of aryl sulfonamide and 4-aminoquinoline compounds. acknowledgement: supported by nih r44 ai052917-02 and r01 ai066502-01. human papillomavirus (hpv) has been a difficult virus to target by traditional antiviral methods due to its small size, its small number of obvious therapeutic targets, and its resistance to propagation in vitro. nevertheless, antiviral compounds that reduce hpv dna load have the potential to prevent carcino-genic progression in infected patients. to that end, we developed an approach that dramatically reduces the hpv episomal dna load of keratinocytes in vitro by targeting viral dna sequences. pyrrole-imidazole polyamides, with some containing fluorescent probes to aid in cell localization studies, were designed to recognize the hpv31 ori. all fluorescent compounds rapidly localized to the nucleus of cultured keratinocytes following addition to the culture media. the compounds were then tested for their ability to alter keratinocyte hpv31 episomal dna content. two of the 19 compounds caused a dose-dependent reduction in hpv31 episomes as measured by taqman tm realtime pcr. while control and vehicle-treated cells maintained ∼1000 copies of hpv31 per cell, compounds 2-ta and 4-ta both reduced hpv31 dna levels to below 50 copies per cell after 48 h incubation with 10 m compound. an alternative taqman tm amplicon within the hpv31 e7 gene produced identical results. a multiplexed taqman tm real-time pcr reaction that followed the ratio of hpv31 dna to the human apoe gene also demonstrated dramatic loss of hpv31 dna copies, further confirming our initial observations. finally, cells were treated with polyamides for 48 h, polyamide-containing media was removed, and episome levels were followed for 8 days. at day 6, 4 days after removal of polyamide and 3 days after sub-culturing of the cells, viral episome levels remained approximately 60% lower than control samples. by day 8, 6 days after removal of polyamide, viral dna levels were beginning to recover but still remained significantly lower than control samples. together our results demonstrate that targeting the hpv origin of replication with dna-binding compounds dramatically reduces episomal dna levels. small interfering rnas (sirnas) are potent tools for gene down-regulation but are minimally stable in cells. to improve the efficacy of sirna, we replaced non-bridging oxygens in the phosphodiester linkages of natural rnas with bh3 groups. the resulting boranophosphates have unique properties, including enhanced nuclease resistance, altered hydrogen bonding of the phosphate, different interactions with metal ions, and increased thermal stability of rna:rna and rna:dna duplexes. anti-egfp sirnas containing boranophosphate modifications were prepared by in vitro transcription with t7 rna polymerase from ribonucleoside 5 -(alpha-p-borano)triphosphates, as well as normal and phosphorothioate sirnas. after confirming the presence of the borane modifications with maldi-ms, several properties of borano-modified sirnas were investigated: (1) the double stranded rna with borane modifications maintained the a-form conformation characteristics according to the circular dichroism (cd) spectra; (2) the borane groups in the sirnas increased the thermal stability, with an enhancement of t m by 0.5-0.8 • c per modification; and (3) sirnas with borano-modifications were shown to be at least 10-fold more resistant to rnase a digestion than normal ones. when these modified sirnas were used to down-regulate egfp expression in hela cell cultures, it was found that: (1) borano-modified sirnas were consistently more effective than sirnas containing the corresponding phosphorothioate modifications; (2) borano-sirnas were more effective than normal sirnas provided that the center of the antisense strand was not heavily modified; (3) borano-sirnas were more potent than normal or phosphorothioate sirnas at lower concentrations; and (4) finally, the silencing activity of boranophosphate singlestranded sirna (ss-rna) was comparable to that of unmodified ds-sirna. the borano ss-rna had excellent maximum silencing activity and was highly effective at low concentrations, and silencing activity was durable up to one week after transfection. results with anti-hpv sirnas will be discussed. boranophosphate modification is a potential new class of anti-viral therapeutic agents. this report describes the antiviral structure activity relationships that led to the discovery of phosphonomethoxy-2 -fluoro-2 ,3dideoxydidehydroadenosine (fd4ap, gs9148), a novel ntrti, with an excellent resistance profile toward hiv-1 variants containing major n(t)rti resistance mutations. methods: phosphonomethoxy analogs on purine and pyrimidine dideoxydidehydro (d4) and dideoxy (dd) ribose scaffolds were prepared. antiviral activity was measured against wildtype and n(t)rti-resistant recombinant viruses using cytopathic assay in mt-2 cells. mitochondrial toxicity was assessed in hepg2 cells by measuring mitochondrial dna content. results: the d4 scaffolds displayed superior antiviral activity compared to the dd scaffold and adenine was superior to other nucleobases. phosphonomethoxy-2 ,3dideoxydidehydroadenosine (d4ap) inhibited hiv-1 replication with a mean ec50 of 2.1 m and an 0.8-, 2.9-, and 2.9-fold change in potency against viruses containing m184v, k65r, and 6 thymidine analog mutations (tams), respectively. further exploration of d4ap was limited by its mitochondrial toxicity, which was then addressed in 2 ways: (i) preparation of l-d4ap or (ii) 2 fluorine substitution. l-d4ap exhibited an ec50 of 5.9 m but had substantially reduced potency (14-fold) toward m184v mutant viruses. fd4ap exhibited an ec50 of 12.3 m, with 0.8-, 1.2-, and 3.5-fold change in potency against viruses containing m184v, k65r, and 6 tams, respectively. no cytotoxic effects were measured up to 1 mm in mt-2 cells and no effects on mitochondrial dna were detected up to 300 m in hepg2 cells for both fd4ap and l-d4ap. conclusion: fd4ap is a novel phosphonate ntrti with antiretroviral activity toward wild-type and resistant mutant hiv-1 strains. compared to d4ap, the 2 -fluorine atom significantly improved the in vitro toxicity profile while retaining the favorable resistance profile. in subsequent studies, the monoamidate prodrug strategy was applied to fd4ap to achieve optimal in vivo pharmacokinetic properties. entry inhibitors, and ccr-5 antagonists in particular, have become one of the most actively pursued treatments for hiv within the pharmaceutical industry. recently, multiple groups have disclosed piperidine-based ccr-5 antagonists that -to the medicinal chemist's eye -might appear to share a common three-point pharmacophore comprised of a tertiary amine, a phenyl ring, and a carboxamide or sulfonamide group. in several of these cases, these pharmacophoric elements are tethered together by a flexible, aliphatic chain. we sought to improve the potency of and introduce structural novelty into this class of compounds by rigidifying this tether. herein, we describe stereoselective syntheses and sar of a series of ccr-5 antagonists wherein the tether has been replaced with four stereochemical isomers of a rigidified cyclopropyl scaffold. the regulation of hiv transcription is a complex, multistage process that requires the concerted action of viral and cellular proteins. we discovered the n-aminoimidazoles (naims) as a unique class of hiv inhibitors targeted at the viral transcription level. a prototype naim, nr-818, prevents the reactivation of dormant virus by inhibiting both the hiv-1 p24 and viral mrna production from latently hiv-1-infected cell lines upon stimulation with tnf-␣, pma, or tsa. extensive research revealed that nr-818 was unable to inhibit the nf-b activation pathway or chromatin remodeling at the viral promoter, both known to be crucial for viral transcriptional activation. focusing on the viral transcription process, chromatin immunoprecipitation (chip) experiments revealed that nr-818 was able to inhibit the ser5 phosphorylation of the c-terminal domain (ctd) of rna polymerase ii. this step is mediated by the cdk9 subunit of p-tefb, which is recruited to the viral promoter by the hiv-1 tat protein. since we did not find an inhibition at the level of cdk9 activity or tat-mediated transcription in tat-expressing cell lines transiently transfected with a ltr-gfp construct, we infer that nr-818 must interfere with the transcription process by a unique mode of action. evidence points towards a kinase, not belonging to the cdk family, to be the target of the naims, resulting in an antiviral action at the level of retroviral transcription. clara e. cases-gonzález 1 , sandra franco 2 , miguel a. martínez 2 , luis menéndez-arias 1 1 centro de biología molecular "severo ochoa", csic-uam, madrid, spain; 2 fundació irsicaixa, hosp. university germans trias i pujol, badalona, spain a ser-ser insertion at codons 69-70 together with substitutions t69s and t215y in the reverse-transcriptase (rt)-coding region of hiv-1 are known to confer resistance to zidovudine (azt) and stavudine (d4t). phenotypic resistance correlates with increased atp-dependent phosphorolytic activity on inhibitor-terminated primers. we have previously shown that an rt derived from a clinical isolate (ss rt) that contained the insertion and 10 additional mutations related to drug resistance (including t215y) showed >10-fold increased unblocking activity on azt-and d4t-terminated primers, when compared with an rt containing the insertion together with mutations t69s and t215y, in an otherwise wild-type bh10 sequence. these results suggested that other mutations associated with the complex t69sss/t215y in clinically relevant rts contributed to increase atp-mediated excision activity and conferred high-level resistance to azt and d4t in phenotypic assays. to identify residues increasing the excision activity, we obtained recombinant enzymes bearing ss rt residues 1-135 and wild-type bh10 rt residues 136-560 (l1 rt), or residues 1-135 of the bh10 rt and 136-560 of the ss rt (l3 rt), as well as an l1 rt variant with the substitution t215y (l2 rt) and an l3 rt derivative with t69sss (l4 rt). additional rts containing mutations m41l, a62v, or k70r together with the combination t69sss/t215y in the bh10 background were also obtained. atp-mediated excision activities on azt-and d4tterminated primers were determined and the effects of mutations were tested in phenotypic assays using recombinant hiv-1. the l2 rt containing mutations t69sss/t215y and additional changes in the n-terminal region showed the highest atp-dependent phosphorolytic activity on blocked primers, giving values similar to those reported for the ss rt. results were consistent with phenotypic data. in contrast, l1, l3, and l4 rts displayed low-level activity. further experiments revealed that three amino acid changes at the n-terminal region of the polymerase (m41l, a62v and k70r) were responsible for the increased excision activity shown by rts bearing mutations t69sss and t215y. from a series of phenyl-substituted thiazolobenzimidazoles, several compounds were identified as selective inhibitors of coxsackie b virus replication in vero cells. a structure-activity relationship was established, from which the 6-trifluoromethyl substituted analogs emerged as the most potent congeners. the compounds were active against all six coxsackie b strains tested. the in vitro antiviral activity of one of the most selective compounds, i.e. chi-033, was assessed by (i) mts-based cytopathic effect assays, (ii) virus yield reduction assays, (iii) real-time quantitative pcr (rt-qpcr) and (iv) by monitoring viral antigen expression. in all assays a clear concentration-response effect was obtained. the 50% effective concentration (ec50) was 0.30 ± 0.18 g/ml, while the cc50 (50% cytotoxic concentration) of chi-033 for vero cells was more than 100 g/ml, thus resulting in a selectivity index of >500. detailed single cycle time-of-drug-addition studies (in which viral replication was monitored by means of rt-qpcr) revealed that the compound interacts with viral replication at a time that coincides with the onset of intracellular viral rna synthesis. chi-033resistant virus is being generated by culturing the virus in the presence of increasing drug concentrations. drug-resistant virus will be genotyped, which should allow us to identify the (putatively viral) molecular target of this class of compounds. retroviruses 42 hiromichi tanaka 1 , kazuhiro haraguchi 1 , hiroki kumamoto 1 , takao nitanda 2 , masanori baba 2 , ginger e. dutschman 3 , yung-chi cheng 3 1 school of pharmaceutical sciences, showa university, tokyo, japan; 2 center for chronic viral diseases, kagoshima university, kagoshima, japan; 3 school of medicine, yale university, new haven, ct, usa our recent research program on the development of synthetic methods for 4 -carbon-substituted nucleosides has led to a new strategy, ring opening of 4 ,5 -epoxy-nucleosides with organoaluminum and organosilicon reagents. this enabled us to introduce alkyl, alkenyl, and alkynyl groups to the 4 -position. as a result of this study, 4 -ethynylstavudine (4 -ed4t) was found to be more anti-hiv active than the parent compound stavudine (d4t). this compound (4 -ed4t) has several additional appeals as a promising anti-hiv agent: much less toxic to various cells and also to mitochondrial dna synthesis, better substrate for human thymidine kinase than d4t, very much resistant to catabolism by thymidine phosphorylase, its activity enhances in the presence of a major mutation k103n known for nnrti-resistant hiv. in this conference, we present the synthesis and sar studies of 4 -ed4t analogues modified mainly in the sugar portion. negatively charged polymers (np) possess a broad immunoadjuvant and antiviral activity topically useful for vaccine, drug, and microbicide development. but their efficiency is limited over a reversibility of electrostatic kind of interference with virusspecific nano-objects. to overcome this limitation the purposemade intra-molecular modifications of np were studied among non-toxic maleic acid co-polymers (npsa), dextran and chitin derivatives (npps) within varied alicyclic modifiers application. the configurationally flexible alkyls (i), as non-alicyclic control, are ineffective synergist for np antiviral potency. monocycles (ii) are moderate active too. on the contrary the hardconformation frame-structured spheroids (iii-vi) exhibit ability (at optimal macromolecular parameters) to be super-effective synergists for strength and diapason of np antiviral action. unlike small molecular iii/iv-containing prototypes (amantadin, rimantadin, deitiforin, etc.), narrowly-effective inhibitors mainly of influenza a viruses, the np-coupled modifications become effective also against many other viruses, including the drugs resistant strains [antivir. res. 46 (1), 44]. in focus of the anti-hiv potency the ivs provide a 10-100-fold elevation of np activity. the more available and less toxic iii species are similarly active, but iii* (with spatial-optimally contactable double bond due to the exo-configuration) turns out the best synergist 20-500-fold amplifying the anti-hiv-1 selectivity up to is∼10000. augmentation of the frame cycles from iii-iv toward v-vi results in no essential enhancement of antiviral activity, but stimulates toxicity. the recently involved in the investigation vii, cholesterol-like systems, as tools for novel raft-targeted strategy, demonstrate capacity for at least 10-fold amplification of anti-hiv-1 potency our earlier studies showed that esterification of cidofovir (hpmpc) with alkoxyalkanols increased antiviral activity by more that two logs and promoted oral bioavailability. to evaluate this approach with purine based nucleoside phosphonates, we synthesized several alkoxyalkyl esters of acyclic purine phosphonates such as 2,6,-diamino-(9-[2-phosphonomethoxyethyl]purine (pme-dap) and 2-amino-6-cyclopropylamino-(9-[2phosphonomethoxyethyl]-purine (pme-cpr-dap) these purine phosphonates have been reported to be active against a wide range of viruses such as human immunodeficiency virus (hiv-1), other retroviruses, herpesviruses, poxviruses and hepatitis b virus. for this study several alkoxyalkyl analogs of acyclic 2,6diaminopurine nucleoside phosphonates were synthesized and evaluated against hiv-1. the alkoxyalkyl esters were more inhibitory than the unmodified compounds in p24 reduction assays in mt-2 cells infected with hiv-1. for example, hexadecyloxypropyl (hdp) and oleyloxyethyl (ole) esters of pme-cpr-dap were >3 logs more active than unmodified pme-cpr-dap. in spite of increased cytotoxicity in mt-2 cells, the selectivity indexes are more than 10-fold higher then for unmodified compound. in conclusion, esterification of pme-dap and pme-cpr-dap with hexadecyloxypropyl-or oleyloxyethyl-residues greatly increased their antiviral activity and selectivity against hiv-1 in vitro. victor kuz'min 1 , eugene muratov 1 , anatoly artemenko 1 , ludmila koroleva 2 , vladimir silnikov 2 , v. lozitsky 3 , a. fedchuk 3 1 a.v. bogatsky physical-chemical institute, odessa, ukraine; 2 institute of chemical biology and fundamental medicine, novosibirsk, russian federation; 3 ukrainian mechnikov research anti-plague institute, odessa, ukraine "chemical" ribonucleases hold promise as tools for studying the structures of rnas and rna-protein complexes, as reactive groups in conjugates intended for cleavage of particular rnas, as therapeuticals inactivating virus genome rnas or certain mrnas, and as a promising antiviral agents. drug design and development of new medicines directed against hiv are permanently actual tasks. the usage of modern quantitative structure-activity relationship (qsar) methods could allow us to solve these problems more effectively. the objective of the present work is qsar analysis of antiviral activity of various tetrapeptides-artifical ribonucleases and consequent molecular design of new antiviral agents. qsar approach based on simplex representation of molecular structure (sirms) has been used for the solution of the formulated problem. usage of sirms allows us to develop the molecular design of the new effective antiviral agents. thorough researches of relationship between antiviral activity (hiv-1, % of rna p-o bond cleavage) and a structure of artifical ribonucleases have been carried out. statistic characteristics for pls (partial least squares model) are quite satisfactory (r 2 = 0.836, q 2 = 0.788). on the base of these models the molecular fragments with positive or negative influence on the explored property have been determined. thus, for example, guanidine and triethylenediamine fragments promote antiviral action. it gives a possibility to realize based on elucidated rules molecular design of compounds with the high level of antiviral activity. the results of prognosis are verifying by the experimental investigations. thus, quite adequate simplex qsar model "anti-hiv activity-artifical ribonucleases structure" was obtained and used for drug design. the cyclotriazadisulfonamide (cada) compound specifically down-modulates the cd4 receptor expression on the surface of lymphocytes and monocytes/macrophages, the primary receptors utilized by hiv for infection of its target cells. cada thus inhibits the entry of hiv and hhv-7 (vermeire et al., 2002. virology 302, 342-353) . cada chemotherapy may not be susceptible to the production of drug resistant strains of viruses, as its mechanism of action is completely different from those of any other anti-hiv drugs currently in clinical use. the cd4 down-modulating and antiviral potencies of more than 25 cada analogs have been described (vermeire et al., 2003. mol. pharmacol. 63, 203-210) . structural modifications of cada were made to increase potency, reduce cytotoxicity, and improve physical properties. several head group analogs were synthesized with polar groups and good leaving groups ( fig. 1) . the anti-hiv and cd4 down modulation activities of these compounds are being studied. some of these head groups may regenerate the double bond of cada by elimination reactions, potentially producing water-soluble pro-drugs. isocada (sa05), an isomer of cada, was synthesized by cyclization of 1,5,7-triazabicyclo-[4.4.0]dec-5-ene (tbd) (fig. 1 ). this structural modification may reveal a relationship between the symmetry of the molecule and its biological activity. two new fluorine-containing analogs were also synthesized by modifying the toluenesulfonamide side arms (fig. 1) . the anti-hiv and cd4 down modulation activities of these new cada analogs are summarized. the center for drug discovery, university of georgia, athens, ga 30602, usa drug discovery targeted at the elusive viral enzyme, hiv integrase, has not resulted in a single fda-approved drug. in this presentation we describe our molecular modeling studies with conceptually novel inhibitors of hiv integrase that also possess potent in vitro anti-hiv activity. docking was performed on the catalytic core of integrase represented by chain c of pdb structure code 1bl3. building of molecules and primary modeling was done with sybyl 7.1 on a silicon graphics onyx3 (r14000) workstation. the program gold 3.0 (genetic optimization for ligand docking) was used extensively in evaluating the docking poses of these compounds with the active site of hiv integrase and to give information on key residues involved in the recognition and binding of these ligands. the gold function consists of three basic components: protein-ligand h-bonding energy, protein-ligand van der waals energy, and ligand internal energy. post-processing gold output was done with the program silver 1.1, a utility program supplied with gold for evaluating hydrogen-bonding interactions, metal coordination and van der waals factors. for comparison purposes, additional docking was performed using other docking protocols, notably the sybyl module flexx. data obtained from these and related studies including binding poses, binding affinities, functional and conformational considerations, and gold function scores will be presented and explained. the center for drug discovery, university of georgia, athens, ga 30602, usa hiv integrase is essential for hiv replication and is an attractive target for drug discovery against aids. however, research efforts on drug discovery pertaining to hiv integrase have not resulted in a single fda-approved drug for which mechanism of action is inhibition of hiv integrase. recently, we have been exploring a novel class of diketo acids that are constructed on nucleobase scaffolds and that have a specific arrangement of the functional and hydrophobic group on the scaffold. these compounds are inhibitors both key steps of hiv integrase. one lead compound from this group has also been found to have remarkable in vitro anti-hiv activity. however, the syntheses of the inhibitors are quite challenging. this presentation will describe the synthetic methodologies specifically developed in our laboratory for the preparation of some representative examples of these integrase inhibitors. purification approaches to produce highly purified compounds for biological studies will be explained. structural, functional and conformational data obtained from extensive spectroscopic studies will be discussed. representative anti-hiv integrase data and in vitro anti-hiv screening results will be presented. we have recently reported the synthesis and antiviral activities of a ring-expanded ("fat") nucleoside analogue, called nz-51, that inhibits both hcv and hiv in vitro with ec 50 values ranging in micromolar concentrations or less, with little or low toxicity to the host cells. in this part i of the presentation on this subject, we report our preliminary findings on the mechanism of anti-hiv activity of this compound, along with the synthesis and antiviral activity of a few additional analogues. in view of the fact that a number of hiv patients also suffer from hcv as a major coinfection, and that a number of them ultimately die of end-stage hcv-related complications including liver cirrhosis and hepatocellular carcinoma, a drug with dual inhibitory characteristics against both viruses is highly desirable and timely. marina burshtein 1 , alexander serbin 2 , alissa bukrinskaya 1 d.i. ivanovsky institute of virology, moscow, russia; 2 health research and development found, moscow, russia introduction: amantadine is a well-known effective antiinfluenza drug. it was modified to enhance its antiviral activity by chemically linkage with the water-soluble polyanionic matrix via different spacer groups. the other group of used compounds was norbornene derivatives, as norbornene is an adamantane analogue on anti-influenza activity. methods: the absence of cytotoxic effect was shown by mtt test for estimating cytotoxic dose (ctd50). the antiviral effect of the compounds was analyzed in lymphoblastoid mt-4 cells and in hela cd4+/b-galactosidase cells ("magi" cells). the effect of the compounds was registered by immunoblotting of cell lysates and by measuring of b-galactosidase activity. results: the strong inhibition of hiv-1 replication was observed when the compounds were added with the virus and was expressed even when the compounds added with the virus were removed 1 h after infection. the anti hiv-1 effect of the compounds was gradually decreased if they were added 1 and 2 h after infection, no inhibition was observed when the compounds were added 4 h after infection. the compounds did not impair the virion structure. adamantane and norbornene derivatives were shown also to inhibit azt resistant viral strains. conclusion: adamantane and norbornene were shown to be active hiv inhibitors with the high selectivity index. the compounds are promising candidates for further investigation including preclinical studies. less is known about the effect of their intracellular half-lives on the maintenance of antiviral activity. to investigate this question, we developed a novel in vitro antiviral persistence assay. measurement of the antiviral persistence of tenofovir (tfv) and abacavir (cbv) was coupled to measurement of the half-lives of their tfv-dp and cbv-tp anabolites. methods: mt-2 cells or stimulated primary cd4+ t-cells were incubated with graded concentrations of tfv or cbv for 14 h (h); then extracellular drug was removed by washing. cells were further incubated without drug for 0-24 h and then infected with hiv-1 (iiib or bal). p24 was quantified on day 2; inhibition of hiv-1 replication due to intracellular drug persistence (pc50) was determined relative to a standard ec50. decay of intracellular dp/tps in cd4+ t-cells was measured using lc/ms/ms. results: in mt-2 cells, the pc50 value for tfv 12 h after drug removal remained unchanged relative to the ec50 (<3-fold shift) whereas the pc50 for cbv shifted >65-fold, indicating less persistence of cbv. in cd4+ t-cells, the pc50 value for tfv also showed a minimal shift relative to the ec50 (2.4-fold) 24 h after drug removal. cbv showed a much larger relative shift (>243-fold). quantification by lc/ms/ms of intracellular tfv-dp and cbv-tp in cd4+ t-cells in vitro demonstrated that tfv-dp had the longest intracellular half-life of the two drugs (tfv-dp, 21 h versus cbv-tp, 5 h). conclusions: a novel antiviral persistence assay was developed to study the relationship between intracellular nrti halflives and antiviral activity. in both mt-2 cells and primary activated cd4+ t-cells, tfv had the longest persistence of antiviral activity. in cd4+ t-cells, tfv-dp also had the longest half-life of the two nrtis. cbv-tp had a much shorter half-life than tfv-dp and showed less antiviral persistence. although both drugs are approved for qd dosing, the half-life of intracellular tfv-dp maintains antiviral suppression in vitro over a timeframe most consistent with qd dosing. karen m. watson, tracy l. hartman, lu yang, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa isis 5320 is a phosphorothioate oligonucleotide with a molecular structure of t2g4t2. the g-quartet possessing molecule has been shown to be a potent inhibitor of hiv attachment and cell-cell fusion and acts by specifically interacting at the v3 loop of gp120. mapping studies with monoclonal antibodies targeting epitopes in and around the v3 loop have been used to define the binding site of isis 5320. in vitro, isis 5320 inhibits all laboratory and clinical strains of hiv-1 and hiv-2 tested, including representative subtype viruses, drug resistant viruses (including mdr viruses) and viruses that utilize the cxcr4 and ccr5 chemokine receptors. serial passage of virus in the presence of increasing concentrations of the oligonucleotide did not result in the selection of drug resistant virus strains and combination assays resulted in additive to synergistic interactions with other approved hiv inhibitors. the antiviral and toxicity profiles of isis 5320 resulted in the performance of human clinical trials for the therapeutic use of the oligonucleotide to treat hiv infection. the antiviral properties and mechanism of action of isis 5320 suggest that it may be an excellent anti-hiv topical microbicide. isis 5320 was found to be highly active in a cervical explant model of hiv infection with highly significant inhibition of ccr5-tropic strains of virus. activity was also observed in cell-free and cell-associated virus transmission assays, as well as in cd4-dependent and cd4-independent acute infection inhibition assays. in microbicidal specific combination assays, significant efficacy has been observed with isis 5320 used in combination with other microbicidal compounds. the results of these studies suggest that isis 5320 may represent a new and novel anti-hiv topical microbicide. karen m. watson, tracy l. hartman, lu yang, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa though a variety of compounds are being developed as anti-hiv topical microbicides, such as polyanionic molecules, surfactants, natural products, peptides, proteins, heterocycles, and virucidal agents, clinical efficacy studies that demonstrate the ability of these agents to impact virus transmission are still in progress. it has been estimated that a microbicide that is only 60% effective would have the capacity to prevent millions of new infections each year. thus, one of the challenges in hiv drug development is the discovery of compounds that will inhibit the sexual transmission of infectious organisms between sexual partners. the rapid mutability of hiv and the known presence of drug resistant viruses in wild type virus populations suggests that microbicide development will suffer from the same problems that exist for all hiv therapies, namely the selection of resistant virus strains that will bypass the microbicide barrier and infect target cells in the vaginal or rectal environment even in the presence of the microbicide. thus, it is likely that haart-like combination drug therapies will become the most effective means of inhibiting the sexual transmission of hiv. we have evaluated a wide variety of anti-hiv and anti-sti compounds in vitro alone and in combination with one another and have demonstrated that certain patterns of inhibition (additivity, synergy, antagonism) occur between the various classes of compounds. recently, we have compared the combination anti-hiv activity of microbicide compounds in fresh human pbmcs infected with clinical isolates of hiv to the combination activity of the same test agents in cem-ss-based cultures. in general, these two assay systems yield similar combination assay results. to provide a rationale for the combination use of the compounds in a microbicide setting, the same combination of compounds was evaluated in a microbicide-like virus transmission assay. these combination results suggest that higher levels of synergy between virus attachment and reverse transcriptase inhibitors might be expected in the microbicide environment compared to levels predicted for the systemic therapeutic environment. the results of the combination assays with various microbicides will be presented. during the onset of the hiv disease, hiv rna is continually produced in the face of treatment with haart in circulating reservoirs and rt inhibitors are almost ineffective in the postintegration events. among the classes of anti hiv-1 drugs, protease inhibitors (pi) are the unique to inhibit the hiv-1 production in chronically infected macrophages. in the progression of hiv infection, the role of the monocytes-derived macrophages (m/m) is further confirmed as they represent chronologically the first cytotype where the viral replication restarts as a consequence of failure or interruption of antiviral therapy. aim of the work was to evaluate the rebound of hiv-1 production when pi have been removed in hiv-1 chronically infected m/m and, moreover, to verify the effect of this removal on virus maturation, infectivity and ability to trigger apoptosis in uninfected peripheral blood lymphocytes (pbl). a rebound of p24 gag protein was measured starting from 12 h after drug removal yet virus infectivity remained 1 log lower than control up to 1 week. inhibition of hiv-1 replication was still 48% and 18% upon amprenavir 20 and 4 m, respectively. these data were confirmed by western blotting and electronic microscopy showing production and release of immature viral particles. moreover, pi (amprenavir and indinavir) treatment dramatically reduced apoptosis of pbl co-cultured with chronically infected m/m and kept cd4/cd8 ratio above the levels of untreated controls until the 5th day of co-culture. taken altogether, these findings suggest a wide clinical importance for amprenavir and indinavir for their relevant long-lasting antiviral effect in persistently-infected reservoirs of hiv even in case of drug interruption and/or when hiv infection can restart in districts where drugs find not sufficient concentration. moreover, these results strengthen the evidence for an unique positive utilize of pi against ongoing and productive hiv infection. weili jin, salvatore santino, michael wang gilead sciences, foster city, ca, usa background: effective inhibition of hiv reverse transcriptase (rt) currently represents a crucial objective of antiretroviral therapy. capravirine is a second-generation non-nucleoside rt inhibitor (nnrti) that is capable of blocking the replication of certain nnrti-resistant strains of hiv and was recently in clinical development. in this study, we report on the in vitro selection and characterization of viral resistance to capravirine. methods: viral resistance selection experiments were performed in mt-2 cells with the hiv iiib isolate and increasing concentrations of capravirine. viruses were analyzed genotypically by population sequencing and by single genome sequencing (sgs). recombinant viruses with nnrti mutations were generated from proviral dna clones. phenotypic analyses were performed in mt-2 cells. results: capravirine resistance selections were initiated at 1 nm (ec 50 of 1.5 nm for capravirine). following nine passages in the presence of increasing concentrations of capravirine, the l100i mutation emerged in rt and additional passaging led to v179d and f227c mutations at higher concentrations (100-300 nm). further increases in capravirine concentrations led to the emergence of a l100i + v179d + f227c triple mutant, which confers >1000-fold resistance to capravirine. sgs of mixed viral populations from different passages showed that l100i, v179d and f227c were present on the same genome, with l100i as the primary mutation, and f227c and v179d were acquired sequentially at later passages. through sgs analysis, a l100i + k166r + v179d + f227c quadruple mutation on the same genome was also observed at higher capravirine concentrations (>1000 nm). recombinant viruses carrying these mutations were produced to assess their susceptibilities to capravirine. conclusions: after extensive in vitro passaging of hivinfected cells in the presence of capravirine, neither k103n nor y181c mutations in rt were observed. instead, the l100i mutation was initially acquired, followed by mutations f227c and v179d. addition of the k166r mutation to the triple mutant genome, l100i + v179d + f227c, appears to further enhance hiv resistance to capravirine. oluwafemi olawuyi 1 , adeyemi falegan 2 1 medical microbiology, university college hospital, ibadan, nigeria; 2 dentistry, university college hospital, ibadan, nigeria issue: the percentage of aids/hiv is increasing every year in the third worlds, and this is reinforced by the factor that majority of youth in third worlds do not know his/her hiv status. description: a self developed validated and reliable questionnaire [r = 0.87] was used to collect the data and percentage was used to analyze the data. the population of the study was made up of youth [female and male] in higher institutions, working places, market places and community streets in nigeria, 50,000-sample size, selected through simple random sampling technique. the mean age is 25.5 years old. relative risk [rr] calculated is 3.1, i.e. rr >1, indicating that the factor is the risk factor, and the confidential interval [ci] for rr at 95% significant level is 2.61 < 3.1 < 3.90 from the formula, ci lower limit < rr < ci upper limit. lessons learned: seventy percent of the sample population did know his/her hiv status and had had sexual intercourse in the past before, out which 20% had the unprotected intercourse once or more, 25% had protected sex while 25% were not sure of using protection means. while, 20% have knowledge about own hiv status and had had sexual intercourse before. ten percent have no knowledge about own hiv status and had no sexual intercourse before. conclusion: aids/hiv still remains a killer disease in the third world. however, the lack of knowledge of individual's hiv status remains the only highest risk factor for the spread of the disease in the third worlds. yuichiro habu 2,3 , jacob barnor 1,6 , norio yamamoto 4 , kahoko hashimoto 1,2 , naoko miyano-kurosaki 1,2 , koichi ishikawa 5 , naoki yamamoto 5 , david ofori-adjei 6 , hiroshi takaku 1,2,7 1 department of life and environmental sciences, chiba institute of technology, chiba, japan; 2 high technology research center, chiba institute of technology, chiba, japan; 3 japan foundation of aids prevention; 4 department of molecular virology, bio-response, tokyo medical and dental university, tokyo, japan; 5 aids research center, national institute of infectious disease, tokyo, japan; 6 department of virology, noguchi memorial institute for medical research accra-ghana, accra, ghana; 7 bach tech corp. rna interference (rnai) is a potentially strong gene interference tool, which had been successfully used to silence many pathogenic viruses including hiv. however, many recent reports have shown that, in long-term assay cultures involving rna viruses such as hiv, escape mutants breakthrough the silencing effect. in the light of this conundrum, it had been proposed that, vector designed to target multiple genes in a synergistic manner, may address the problem. hence, we designed a chimeric rna expression vector which express vif shrna and decoy tar rna by combining vif shrna and decoy tar rna with linker to which dicer was able to recognize for cleavage, as a second generation rnai expression vector system. the synergistic effect of these molecules enhanced the inhibition of hiv-1 replication in a long-term transduced pbmcs, h9, and jurkat cell culture assays (9 weeks) and prevented virus breakthrough associated with sirna-mediated escape variants. notably, hiv-1 replication was similarly suppressed in the control cells expressing only vif shrna for about 3 weeks, but an increase in virus replication was observed afterwards. hiv viral rna extracted and sequenced at this point indicated escape mutants in the cells expressing the vif target in hiv. we confirmed substitution of bases in the vif shrna target sequence. on the other hand, the incidence of mutation was not observed in a sequence of viral rna from the culture expressing the vif shrna-decoy tar rna at the fourth week. interestingly, virus production was inhibited for a long-term by an effect of decoy tar rna, through the rna-protein interaction. combining shrna with decoy tar rna as second-generation anti-hiv shrna may provide practical basis for applying sirna-based gene therapy to the treatment of hiv/aids. introduction: this is a designed efficient gene therapy against aids/hiv. the novelty of this aids vaccine design/concept is seen in the fact that the 'pol' gene encoding for nonstructural proteins (polyproteins that generate three enzymes: reverse transcriptase, integrase and protease) is cloned in a suitable retroviral vector and adult stem cells are transfected by this and reinfused into the circulation to effectively counter hiv replication and antigenic variation. method: the mrna are isolated from adult stem cells and transcribed into cdna with reverse transcriptase. the cdna are then cloned in a suitable retroviral vector (vacinia) carrying 'pol' gene that confers resistance to a strong reverse transcriptase inhibitor drug. the adult stem cells are transfected by the recombinant mixture, and reinfused into the circulation of hiv infected person. result: the transfected stem cells are reinfused to provide renewable source of more and better empowered normal blood cell types that would disrupt and half hiv replication in the circulation. there would be efficient induction of both humeral and cellular mediated immunity with prolonged expression of antigens and protective immunologic memory generation against hiv antigenic variation. conclusions: this aids vaccine design would lead to both efficient prophylactic and therapeutic therapy against aids in that it would effectively take care of the problematic factor of hiv antigenic variation which has long been the main obstacle to potent aids vaccine development. because of the real risk of interspecies transmission and/or reassortment between avian, swine and human influenza a strains, drug susceptibility monitoring of circulating avian and porzine virus strains appears to be warranted for effective application of antiviral drugs like amantadine. this study was designed to gain insight into amantadine susceptibility of 6 avian and 12 porcine influenza a viruses isolated in germany between 1981 and 2002. virus strains were isolated in embryonated chicken eggs and passaged one time in mdck cells. plaque reduction assays were applied to examine virus susceptibility to amantadine. genotyping was used to confirm drug resistance. in the result of these antiviral studies, only 3 of the 12 porzine isolates but all 6 avian isolates were shown to be amantadine-susceptible. interestingly, the three amantadinesensitive porzine strains were isolated between 1981 and 1987. all porzine influenza a viruses isolated later on were drugresistant and contained the aa substitutions g16e, s31n, and r77q in the matrix protein 2 (m2). additionally, l27a was detected in two h1n1 strains. s31n and/or l27a are well known amino acid substitutions in m2 that confer amantadine resistance. the role of the pig as an intermediate host of avian and human influenza a viruses, the possible involvement of genetic reassortment, and the high incidence of naturally amantadineresistant porcine influenza a viruses suggest a real risk of emergence of amantadine resistant human viruses. therefore, further studies are ongoing now to evaluate the circulation of the resistant phenotype in pigs, birds and human. recently much attention has been devoted to searching for effective chemotherapeutic agents and vaccines for eradication of this notorious disease. at present only chemotherapy is available to combat avian flu, for instance, tamiflu, approved for the treatment by the us-fda. development of a simple, novel molecule with potential antiviral activity against is essential to treat avian flu viral infection. isatin (2,3-dioxoindole), is a versatile lead molecule for designing of potential antiviral agents and its derivatives were reported to possess broad spectrum antiviral activity. methisazone (nmethylisatin-3-thiosemicarbazone) was first clinically approved for treatment of pox viral infections, and its derivatives were documented to have anti-influenza activity. based upon this evidence, the present work was initiated to determine the antiviral activity of novel isatin derivatives against avian flu (h5n1) in mdck cells. antiviral activity was studied by virus yield assay (ec90), and cytotoxicity by neutral red uptake assay by uninfected mdck cells. all five compounds of a series inhibited the replication of avian flu (h5n1) virus replication in mdck cells and compounds spiii-5h and spiii-5cl were most active (ec90 5.5 g/ml, cc50 >100 g/ml and si > 18). details of these studies and results of treatment of influenza-infected mice are discussed. acknowledgement: supported in part by contract noi-ai-15433 and noi-ai-30048 from virology branch, niaid, nih]. arginine-rich peptide conjugated phophorodiamidate morpholino oligomers (arp-pmo) are nuclease resistant antisense compounds that hybridize to target rna in a sequence-specific manner resulting in disrupted rna function. eight arp-pmo were designed to base-pair with various regions of a/pr/8/34 (h1n1) rna and were then evaluated by hemagglutination and plaque assays for their ability to inhibit fluav production in vero cell culture. arp-pmo targeting the aug translation start site of the np or pb1 segment mrnas, or the 3 -terminus of their respective vrnas, were highly effective, reducing influenza virus titer by 1-3 orders of magnitude in a dose-dependent and sequence-specific manner over a period of 2 days. two of the p-pmo, targeting the pb1 translation start site region (pb1-aug) and the 3 terminus of np vrna (np v3 ), were evaluated by endpoint dilution (tcid50) or elisa assays against another h1n1 strain (a/wsn/33), as well as a/memphis/8/88 (h3n2) and a/thailand/1(kan-1)/04 (h5n1). the pb1-aug arp-pmo generated over 85% specific reduction of virus level, regardless of viral subtype or methodology, at concentrations in the range of 10-20 m. the np v3 p-pmo yielded similar results, with the exception of considerably lower efficacy against the h3n2 strain, with which it has two base mispairings. studies are planned to further evaluate of at least two arp-pmos in animal models for h1n1 and h5n1 fluva subtypes. macroheterocyclic compounds containing crown fragments and nitrogen atoms show large-scale biological activity. we synthesized series of aza-crown ethers and their derivatives. we also studied anti-influenza and antiherpetic action of some of them. anti-hsv action of studied compounds was tested using cyto-morphological method. hep-2 cells were infected with hsv-1 strain us in dose 5 ifu/cell. the cells were incubated in eagle's medium that contained compounds in a dose of 10 −4 m in experimental samples, or without them in control samples. then cells were fixed with 96% ethanol and stained with 0.01% acridine orange solution. the amount of infected cells with dna-containing virus inclusion bodies was counted by fluorescent microscopy. anti-hsv activity of compounds was calculated as the difference between of the percentage of infected cells in treated cell cultures to the percentage of infected cells in untreated cell cultures. anti-influenza activity was studied on the model of replication of a/hong kong/1/68 (h3n2) strain in tissue culture of chorio-allantoic membranes of chicken embryos. compounds were used in a dose of 10 −3 m during the study of their anti-influenza action. diaza-18crown-6 and two of its derivatives have showmen neither anti-hsv nor anti-influenza activity. diaza-18crown-6 derivatives that contain 2-oxyethyl-or ethoxycarbonyl-fragments decreased amount of cells infected by hsv-1 by 13 and 29%, respectively. both of these compounds inhibited replication of influenza virus on 1.7 log 10 tid 50 aza-15crown-5 did not show antiviral activity, but both its derivatives proved to be active inhibitors of hsv and influenza virus reproduction. aza-15crown-5 derivatives that contain 2-amino-3-phenyl-propanoyl-or 5-benzyloxy-3-oxapentyl-fragments decreased amount of cells infected by hsv-1 with virus-specific intranuclear inclusions by 41 and 47%, respectively. first compound inhibited replication of influenza virus on 1.5 log 10 tid 50 and the second one decreased virus amount on 2.0 log 10 tid 50 . the results of this study show that aza-crown ethers are the perspective class of compounds for search of new antiviral agents. acknowledgement: this work was partially supported by stcu (grant # 3147) . robert w. sidwell 1 , kevin w. bailey 1 , min-hui wong 1 , donald f. smee 1 , dale l. barnard 1 , shanta bantia 2 1 institute for antiviral research, utah state university, logan, ut, usa; 2 biocryst pharmaceuticals, inc., birmingham, al, usa the cyclopentane neuraminidase inhibitor, peramivir (bcx-1812, rwj-270201) has striking inhibitory effects on a spectrum of influenza viruses in vitro, and has also demonstrated significant effects against influenza a (h1n1, h3n2) and b virus infections when administered orally to mice and ferrets. unfortunately, clinical trials with the drug administered orally were not successful, probably due to low blood levels obtained after oral administration. significant plasma drug levels of peramivir persist up to 6 h after intramuscular (i.m.) injection; more importantly, however, is the observation that peramivir remains tightly bound to influenza virus n9 neuraminidase for over 24 h, suggesting single i.m. or intravenous (i.v.) therapy with the drug may be highly effective against an influenza infection. experiments now in press have indicated that single i.m. peramivir therapy administered up to 48 h after virus exposure was protective to mice infected with influenza a (h1n1) virus. in the present study, peramivir was administered i.m. or i.v. in a single injection 1 h pre-virus exposure in separate experiments to mice infected with an influenza a (h5n1) virus; efficacy was compared to similar dosages of oseltamivir and oseltamivir carboxylate run in parallel. dosages of 20 and 10 mg/kg of peramivir administered by either route significantly prevented deaths, lessened arterial oxygen (sao 2 ) decline, inhibited development of lung consolidation, and inhibited lung virus titers. the lung assays were performed at varying times after virus exposure. oseltamivir and oseltamivir carboxylate, which do not have the same neuraminidase binding abilities seen with peramivir, were less efficacious in these experiments. delaying the single i.v. therapy up to 72 h after virus exposure also significantly inhibited the virus infection. peramivir appeared to be well tolerated in toxicity control animals run concomitantly with these studies. these data indicate parenterally administered peramivir may hold promise as a therapy for clinical influenza a (h5n1) virus infections. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih. hiroshi saitoh 1 , naoko miyano-kurosaki 1,2 , hiroshi takaku 1,2 1 department of life and environmental sciences, faculty of engineering, chiba institute of technology, chiba, japan; 2 department of life and environmental sciences, faculty of engineering and high technology research center, chiba institute of technology, chiba, japan background: influenza virus causes widespread infection in the human respiratory tract, but existing vaccines and drug therapy are of limited value. recently, small interfering rnas (sirnas) are a powerful tool for sequence-specific, post-transcriptional gene silencing and have a potential therapeutic and prophylactic application against cancer, as well as infectious diseases. here we show that short interfering rnas (sirnas) specific for conserved regions of the viral genome can potently inhibit influenza virus production in cell lines. the influenza virus np gene is a potential target for rnai technology. on the other hand, the baculovirus (acmnpv) can infect a variety of mammalian cells, facilitating its use as a virus vector for gene delivery in viral entry into cells. in this study, we describe the inhibition of influenza virus production by baculovirus-mediated shrna expression vectors. methods: the psv2neo-u6 plasmid vectors and pvl1393based baculovirus vectors were used in this study. the influenza virus a and b np genes were made into the target and the shrna expression plasmid vectors were constructed under the control of the human u6 pol iii promoter. the shrna expression plasmids or shrna expression baculovirus vectors introduced into mdck cells, and 24 h later the cells were infected with either a/pr8 or b/ibaraki virus at a moi of 0.01. at 72 h postinfection, culture supernatants were harvested and assayed to determine the virus titer by plaque assay. conclusion: the findings reveal that newly synthesized np proteins are required for influenza virus replication and provide a basis for the development of shrnas expression plasmids as prophylaxis and therapy for influenza infection in humans. julia serkedjieva, ekaterina krumova, tsvetanka stefanova, nadja nikolova, maria angelova institute of microbiology, bulgarian academy of sciences, sofia, bulgaria a semi-standardized polyphenol-rich extract (pre), obtained from geranium sanguineum l., inhibited the reproduction of influenza viruses types a and b in vitro and in ovo and protected mice from mortality in the experimental influenza virus infection (serkedjieva and manolova, 1992) . the selective in vitro virus-inhibitory activity of pre was fairly modest and this was in contrast with the significant protection in vivo. thus, the therapeutic effect of pre needed explanation. it was presumed that it might be attributed to a combination of more than one biological activities known for natural polyphenols. we have demonstrated previously that pre manifested strong antioxidant and radical-scavenging activities in model systems (sokmen et al., 2004) . the current study was undertaken to investigate the effect of the plant extract on the levels of the antioxidant enzymes superoxide dismutase (sod), catalase (kt) and peroxidase (po) in mice lungs during influenza virus infection as well as the effect of pre on the production of reactive oxygen species (ros) and reactive nitrogen intermediates (rni) by alveolar macrophages in influenza virus infected mice. mice were challenged intranasally (i.n.) with 5-10 ld 50 of a/aichi/2/68 (h3n2) influenza virus. pre was administered by i.n. instillation 3 h before infection in the dose of 10 mg/kg. it was established that influenza infection induced an increase in sod, kt and po production and on days 6 and 9 after infection their levels reached 140-160% of placebo control. the application of pre brought enzymes values to control levels. influenza infection caused also a significant increase of h 2 o 2 , o 2 •− and no production by alveolar macrophages; the generation of ros and rni peaked on day 9. pre-treatment before viral challenge reduced this excessive production. in conclusion, the obtained results outlined the antioxidant and radical scavenging properties of the plant extract; pre beneficially modulated the oxidative stress response in influenza virus-induced pneumonia. this alternative mechanism of action might contribute to the overall protective effect in the lethal murine experimental influenza infection. the antiviral activity of s11, a natural herb extract, ji-sun kwon 1 , hyun-jeong lee 1 , chi-ung moon 2 , jong-hwan kwak 3 , youn-jeong lee 4 , chang-seon song 1 1 avian disease laboratory, college of veterinary medicine, konkuk university, seoul, korea; 2 hanyang university, seoul, korea; 3 sungkyunkwan university, seoul, korea; 4 national veterinary research and quarantine services, seoul, korea the antiviral activity of s11, one of the traditional korean medical herb extract, against influenza virus was investigated. the 50% effective concentration (ec50) using plaque reduction assay was 31.25 ug/ml and the mean 50% cytotoxic concentration (cc50) using wst-1 assay in the mdck cells was 334 ug/ml. oral gavage treatment of the s11 to balb/c mice infected with a/pr/8/34 (h1n1) influenza virus showed the therapeutic effects as delaying clinical signs, significant inhibition of death and reduction of lung virus titers. to identify the lead molecules, the s11 was subjected to further fractionation, purification, and isolation of active compounds. the antiviral activity of these natural herb compounds will be discussed. these results suggest that the s11 is a possible candidate for the development of new antiviral medicine for influenza therapy. hiroshi takaku 1,2,4 , takayuki abe 3 , hitoshi takahashi 1 , naoko miyano-kurosaki 1,2 1 department life environ. sci., chiba inst. tech., chiba, japan; 2 high tech. res. center, chiba inst. tech., chiba, japan; 3 res. inst. microbial dis., osaka university, osaka, japan; 4 bach tech corp background: the baculovirus autographa californica nuclear polyhedrosis virus (acnpv) has long been used as a biopesticide and as a tool for an efficient recombinant protein production in insect cells. in this study, we examined the immunization of a recombinant baculovirus expressing the influenza virus hemagglutinin (ha) against lethal influenza infection in mice. protection was observed in mice immunized intranasally with not only the recombinant baculovirus but also a wild-type baculovirus. baculovirus was also shown to induce secretion of inflammatory cytokines, such as tnf-␣ and il-6, in murine raw 264.7 macrophage cell line. results: a varied route of immunization with a recombinant baculovirus expressing the influenza virus hemagglutinin protein of a/pr/8/34 (h1n1) virus against lethal influenza infection was examined in mice. the recombinant baculovirus encoding the hemagglutinin gene under the control of chicken ␤ actin promoter was inoculated twice, 2 weeks apart, at a dose of 1.1 × 10 8 pfu per mouse by intramuscular, intradermal, intraperitoneal, and intranasal routes. mice intramuscularly and intraperitoneally immunized with the recombinant exhibited higher level of production of serum anti ha antibody than those immunized via the other routes, but protection was only achieved by the intranasal immunization. surprisingly, mice immunized with a wild-type baculovirus with intranasal route were also protected from the lethal influenza virus challenge. sufficient protection in mice was achieved by the intranasal immunizations with 10 8 pfu of either the recombinant or wild-type baculovirus, as evaluated by the reduction of virus titer, production of inflammatory cytokines, and pulmonary consolidations in the lung. these results indicate that infection with a baculovirus induces a strong innate immune response and protection of mice from lethal influenza virus infection. conclusion: baculovirus (cpg motifs) induces a strong innate immune response and protection of mice from lethal influenza virus a and b infection. andrew vaillant 1 , annie lebel 2 , nathalie goyette 2 , guy boivin 2 , jean-marc juteau 1 , phil wyde 3 1 replicor inc., laval, que., canada; 2 chuq-chul and laval university, st. foy, que., canada; 3 baylor college of medicine, university of texas, houston, tx, usa potent antiviral activity of phosphorothioate oligonucleotides (ps-ons) was observed against influenza viral infections. antiviral activity was sequence-independent, size dependent (optimally active ps-ons were ≥40 bases in length) and dependent on the presence of the phosphorothioate modification (hydrophobicity). binding studies showed that rep 9 (a 40 mer degenerate ps-on) interacts with both neuraminidase and hemagglutinin although the sialidase activity of neuraminidase was not affected, suggesting that the structural interactions of these proteins required for influenza activity are the target for this compound. the requirement for hydrophobicity further suggests that the alpha helical regions of hemagglutinin are one of the regions of interaction. the antiviral activity of rep 9 was conserved in many influenza a and b strains suggesting potential therapeutic activity against avian flu and other newly emerging influenza strains. rep 9 aerosol has excellent characteristics for lung deposition and aerosol treatment with rep 9 was well tolerated and highly effective against infections with influenza a both in prophylaxis and 24 h after infection. these results demonstrate the therapeutic potential of aerosolized ps-ons against influenza infection. acknowledgement: supported by nih contract no1-ai-15437. irina v. alymova 1 , y. sudhakara babu 2 , allen portner 1 1 virology division, department of infectious diseases, st. jude children's research hospital, memphis, tn 38105, usa; 2 biocryst pharmaceutical, inc., birmingham, al 35244, usa bcx 2798 is a novel selective inhibitor of human parainfluenza virus infections, which design was based on the threedimensional structure of the hemagglutinin-neuraminidase (hn) protein of newcastle disease virus. compound exhibited striking activity against parainfluenza viruses in vitro and in vivo, and was efficacious in prophylaxis of lethal synergism between parainfluenza virus and streptococcus pneumoniae in a mouse model. present study was conducted to determine if bcx 2798's resistant variants of the recombinant sendai virus whose hn gene was replaced with that of human parainfluenza virus type 1 (rsev(hpiv-1 hn) could be selected in tissue culture and animals. for this purpose virus was serially passaged in llc-mk 2 cells at moi 0.1 in the presence of increasing (from 100 to 3200 m) concentrations of compound; infected 129×1/svj mice were treated with 10 mg/kg/day of bcx 2798 twice for five days. treatment started 4 h before infection. individual clones of viruses were analyzed for the presence of mutations. one mutation, e527k, on the globular head region of the hn protein was selected in tissue culture after the fifth and eleventh passages of rsev (hpiv-1 hn) . several mutations in hn gene of rsev (hpiv-1 hn) were selected in an animal model after the second passage of virus from mice treated with bcx 2798. two mutations, n23s and p29q, were located in the cytoplasmic domain of hn protein; mutations n173s and t553a were found on the globular head region of the glycoprotein. only nonconserved amino-acid residues of hn protein were involved in substitutions. all isolated mutant viruses were stable after the five passages in llc-mk 2 cells without drug; did not develop other substitutions in the presence of drug and displayed no resistance to bcx 2798 both in vitro and in vivo. infectivity of all mutants was not altered to compare with the wild type of rsev (hpiv-1 hn) virus. taking together our results indicate that prophylaxis/treatment of human parainfluenza virus infections with bcx 2798 may not lead to appearance of clinically significant variant of viruses. kie-hoon jung 1 , michelle mendenhall 1 , lawrence m. blatt 2 , robert w. sidwell 1 , brian b. gowen 1 1 institute for antiviral research, utah state university, logan, ut, usa; 2 intermune, brisbane, ca, usa hantavirus pulmonary syndrome (hps) is an acute human respiratory disease with remarkably high case fatality rates (30-50%) for which the etiological agents are members of the bunyaviridae family, genus hantavirus. maporal (map) virus is a recently identified hantavirus isolated in western venezuela, which is most similar phylogenetically to hantaviruses known to cause hps in southern regions of south america. despite the lack of evidence that map can productively infect humans and cause hps, infection of hamsters closely resembles disease manifestations associated with human hps. hantaviruses, in general, are known to produce little to no cytopathic effect (cpe) in cultured cell lines. unexpectedly, we found that map produces remarkable cpe in several vero cell lines facilitating the evaluation of known antiviral agents, ribavirin and interferon alfacon-1. both drugs were highly effective at reducing cpe, as determined by visual examination and neutral red dye uptake, associated with map infection. since much of the observed cpe may be due to apoptosis of uninfected bystander cells, we also developed a quantitative (q)rt-pcr assay to detect copies of map genomic sequence to more directly assess the inhibition of viral replication. data obtained using the qrt-pcr-based assay were consistent with the visual cpe reduction and neutral red-uptake cytotoxicity findings. the development of in vitro antiviral testing methods for map are essential to the evolution of the in vivo hamster disease model of hps. the latter is of utmost importance considering the current need for effective antivirals for the treatment of hps and the lack of a suitable model that does not require biosafety level 4 containment facilities. acknowledgement: supported by contract no1-ai-30048 from the virology branch, national institute of allergy and infectious diseases, national institutes of health. nucleoside analogues are widely used in antiviral and anticancer chemotherapy. for this class of drugs, intracellular conversion of the nucleoside analogue into the corresponding 5mono-, 5 -di-, and 5 -triphosphate after target cell penetration is a prerequisite for biological activity. because of the structural differences from natural nucleosides, this conversion is often inefficient and, as a consequence, therapeutic efficacy is sometimes limited. the free phosphates, or nucleotides, have limited utility in therapy on account of their poor membrane permeability and chemical stability. one approach to improve the therapeutic potential of nucleoside analogues is the delivery of the corresponding nucleotide entities via neutral, lipophilic prodrugs, or protides. the nucleoside aryl phosphoramidate approach, developed by mcguigan and co-workers (2004) has been successfully applied to a number of different nucleosides (azt, d4t, dda, d4a). the general structure of aryl phosphoramidates encompasses two masking groups, an amino acid ester and an aryl moiety bonded to the phosphate group. in order to apply this protide technology to nucleosides with the potential for anti-hepatitis c virus (hcv) activity, we have undertaken studies designed to probe the effect of varying the natural and unnatural amino acid esters and the aryl groups used as masking groups in the target phosphoramidates. these compounds have been synthesised and evaluated using genotipe 1b sub-genomic hcv replicon. we have prepared a variety of arylphosphoramidate derivatives from a range of 4 -substituted nucleosides, including 4azido-cytidine, 4 -azido-uridine, and 2 ,3 -protected variants. with certain nucleoside phophoramidates, we have observed dramatic enhancement (>1000-fold) of replicon activity relative to the parent nucleoside. the synthesis, biological activity and sar of these compounds will be presented. reference mcguigan, d. cahard, balzarini, j., 2004. mini-review. med. chem. 4, 371-382 . we have identified a series of novel anthranilic acid derivatives that are potent, reversible inhibitors of hepatitis c virus (hcv) ns5b polymerase, an essential enzyme for viral replication. the micromolar ns5b polymerase inhibitors belong to the n-phenoxyacetylanthranilic acid chemotype. x-ray crystallography determined that the inhibitors bound to ns5b between the thumb and palm regions adjacent to the active site. guided by crystallography, subsequent modifications to the hydrogen bonding and lipophilic regions of the inhibitors resulted in greatly improved activity against ns5b. further sar studies revealed a second, more potent sub-series where the phenoxy group was replaced by an anilino group. analogs in both subseries showed antiviral activity in a cell-based replicon model of hcv. andrea brancale 1 , dimitrios vlachakis 1 , maria chiara barbera 1 , romano silvestri 2 , colin berry 3 , johan neyts 4 1 cardiff university, the welsh school of pharmacy, cardiff cf10 3 xf, uk; 2 universita' degli studi "la sapienza", dipartimento di studi farmaceutici, 00185 roma, italy; 3 cardiff university, cardiff school of biosciences, cardiff cf10 3us, uk; 4 rega institute for medical research, k.u. leuven, b 300 leuven, belgium hepatitis c is a viral infection that affects 170 million people worldwide, including 4 million in the united states and 8 million in europe. the virus establishes a chronic infection in 55-85% of cases and 20% of affected individuals develop cirrhosis. at the moment there is neither a vaccine nor an effective antiviral therapy available and efforts to identify a specific anti-hcv inhibitor have dramatically intensified in the last few years. many research groups have focused their interest on the enzymes involved in the viral replication and, among these enzyme, the viral helicase/ntpase has proven to be a suitable target for developing novel anti-hcv compounds. compound 1 is a potent inhibitor of the hcv helicase and, although its mode of action is still uncertain, it has been proposed that it acts as competitive inhibitor of rna binding. starting from this hypothesis, we have prepared a series of novel compounds based on the structure of 1 where the benzimidazole moiety has been replaced by different chemical groups, including the negatively charged carboxylate moiety, which should mimic the phosphate backbone of the nucleic acid. the synthesis, the enzyme inhibition and the biological evaluation in replicon of these novel compounds will be presented and analyzed. dale r. cameron migenix inc., 3650 wesbrook mall, vancouver, bc, canada v6s 2l2 hepatitis c virus (hcv), a leading cause of liver disease, continues to be an attractive target for new drug development. among the more favourable approaches to developing new hcv drugs is to target the rna-dependant rna (rdr) polymerase (ns5b), which has been shown to be an essential enzyme for replication. there are several published non-nucleoside inhibitors of this polymerase (some in clinical development) and several published allosteric binding pockets on the protein they target. to be successful, traditional lead identification can be timeintensive, costly and have large infrastructure requirements. increasingly, a push towards effective computational-based screening has led to the development of virtual screening tools. such tools allow investigation of large quantities of compounds in silico for particular properties without the need for compound synthesis or high throughput screening. moreover, these techniques require only a modest infrastructure investment and are very efficient. we employed the openeye set of screening tools (omega, rocs and eon) in concert with publicly available hcv inhibitor information, and commercial databases to identify novel leads. the inhibitor coordinates from a protein-inhibitor complex crystal structure were utilized as the target. available compound databases (asinex and chembridge) were utilized as the testset of compounds. filtered compound conformers were generated using omega and compared with the template using rocs with post-analysis by eon. visual analysis to maximize particular desirable binding features while minimizing protein-inhibitor steric clash allowed the list of potential hits to be further narrowed. multiple classes of compound were identified from the above procedure and after sourcing a subset of the actual compounds or close analogs, they were tested for enzymatic inhibition activity and further characterized. iteration of the process resulted in the identification of a lead compound class containing multiple active compounds, one with reasonable replicon activity. in conclusion, readily available structural and database information and virtual screening tools can be successfully utilized to identify novel inhibitors of hcv rdr polymerase which, in turn, can serve as novel leads for developing new therapies for treating hcv. synthesis, antiviral activity, and cytotoxicity of some novel quinazolin-4(3h)-one derivatives a series of novel 6-bromo/6,8-dibromo-4-(4-oxo-2-phenyl-4h-quinazolin-3y-l)-benzenesulphonamides were synthesized by condensation of 2-substituted benzo[1,3]oxazine-4-ones and sulphonamide. their chemical structures were assigned by means of spectral analysis (ft-ir, pmr, ms). synthesized compounds were screened for in vitro antiviral activity against human pathogenic viruses (hiv, hcv, hsv, vv). 6-bromo-4-(4-oxo-2-phenyl-4h-quinazolin-3y-l)benzenesulphonamide (sps-ii) and 4-(4-oxo-2-phenyl-4hquinazolin-3y-l)-benzenesulphonamide (sps-i) inhibits the replication of hiv-1 in acutely infected mt-4 cells at a concentration of approximately 10 g/ml, while not being toxic to the host cell at a concentration of 60 or >125 g/ml (selectivity index: 8 and >12), respectively. in huh 5-2 cells sps-i inhibited hcv rna synthesis at ec50 of 8 g/ml, while at cc50 for cell growth 32 g/ml. sps-ii inhibited the virus-induced cytopathicity in human embryonic lung (hel) cell infection with hsv-1, hsv-2 or vaccinia (vv) at a concentration of 50 g/ml, while not being toxic to the cells up to a concentration of 400 g/ml. further molecular modification in this series of compounds may help in optimising their antiviral activity. wengang yang, yongnian sun, avinash phadke, milind deshpande, mingjun huang achillion pharmaceuticals, new haven, ct 06511, usa hcv nonstructural protein ns5b is the catalytic subunit of the replication complexes, possessing a motif characteristic of rna-dependent rna polymerases. biochemical assays using recombinant ns5b have been used to investigate ns5b nonnucleoside inhibitors. however, the inhibitory effect of compounds often varies with the forms of recombinant ns5b and the concentrations of the template and/or primer used in the assays. in addition, it does not always correlate to that obtained with replicon-containing cells. these observations have cast concerns about the validity of these cell-free assays. in the report, we explored replication complexes, isolated as crude membrane fractions from replicon-containing cells, for their competency to synthesize viral rna in vitro as well as their responsiveness to ns5b inhibitors. after optimizing the experimental conditions, two species of nascent viral rna, one double-stranded and the other single-stranded, were readily detected. the addition of ns5b nucleotide inhibitor blocked synthesis of both species. the presence of nonnucleoside inhibitors, however, inhibited mostly single-stranded rna (ssrna) synthesis. in addition, the replication complexes isolated from the cells containing a replicon that carried a resistant mutation in ns5b to the nonnucleoside inhibitor were able to synthesize the same amount of ssrna in vitro regardless of the presence or absence of the inhibitor, demonstrating that the phenomenon is due to the specific inhibitory effect of the compound on ns5b. combining with kinetic studies that ssrna synthesis was inhibited only when the nonnucleoside inhibitor was present during the pulse period, we conclude that ssrna synthesis catalyzed by the replication complexes in vitro is likely derived from the de novo initiation. we have recently reported the synthesis and antiviral activities of a ring-expanded ("fat") nucleoside analogue, called nz-51, that inhibits both hcv and hiv in vitro with ec 50 values ranging in micromolar concentrations or less, and little or low toxicity to the host cells. in this part ii of the presentation on this subject, we report our preliminary results on mechanistic studies of anti-hcv activity of this compound, along with the synthesis and antiviral activity of a few additional analogues in the series. in light of the fact that hcv is a major co-infection in patients infected with hiv, and that a number of them ultimately die of end-stage hcv-related complications including liver cirrhosis and hepatocellular carcinoma, a drug with dual inhibitory characteristics against both viruses is highly desirable and timely. nigel bourne, ronald veselenak, richard pyles, minkyung yi, stanley lemon the university of texas medical branch, galveston, tx, usa more than 170 million people worldwide are estimated to be infected with hepatitis c virus (hcv). in the majority of these people a chronic infection is established which can result in serious long-term liver damage including progressive fibrosis, cirrhosis and hepatocellular carcinoma. in fact, hcv is believed to cause more than 100,000 cases of liver cancer annually worldwide and accounts for at least 40% of liver transplants in the us. current treatment options are limited and there is a high treatment failure rate. thus, there is a real need for new treatment options. amantadine has been evaluated as a treatment for chronic hcv infection in a number of clinical studies both as a monotherapy and in combination with other therapeutics. however, the results of these trials have been contradictory and at this time the clinical potential of amantadine as a therapy for chronic hcv infection remains unclear. recent studies have shown that the small hydrophobic hcv p7 protein forms an amantadine sensitive ion channel providing a possible basis for antiviral activity. we examined the ability of amantadine to reduce hcv replication in both subgenomic and full-length hcv replicons of genotypes 1a strain h77c and genotype 1b strain n. in these studies amantadine failed to reduce viral rna replication in any of the replicons tested. further, in infectious virus assays using hcv genotype 2a strain jhf-1 10 um amantadine failed to reduce viral rna levels under any of the conditions tested. however, in these infectious virus studies, when the viral inoculum was treated with amantadine prior to infection of cell monolayers, or when the amantadine was added to cells 1 h after virus adsorption there was a significant reduction in the number of infectious viral foci observed after 72 h incubation (p < 0.05 and <0.01, respectively). these results suggest that even in the absence of a direct impact on rna replication amantadine has antiviral activity. we are currently evaluating amantadine for activity in infectious hcv genotype 1a assays to further define its antiviral spectrum of activity. studies to more fully define its mechanism of action in the virus life cycle are also underway. dominique dugourd, raymond siu, jeremy fenn migenix inc., vancouver, bc, canada celgosivir is an alpha glucosidase inhibitor that is being developed for the treatment of hepatitis c virus (hcv) infections in humans. the purpose of this study was to evaluate the in vitro antiviral activity of celgosivir and its primary active metabolite, castanospermine, when combined with current approved therapies (ribavirin, interferon ␣-2b, or both) in a surrogate model of hcv (bovine viral diarrhea virus (bvdv)). compounds alone or in combination were tested against bvdv in infected madin-darby bovine kidney (mdbk) cells. synergies were analyzed using isobolograms and volume of synergy measurements (macsynergy ii tm software). the celgosivirinterferon ␣2b combination was significantly more synergistic than the celgosivir-ribavirin combination (∼3-fold), or the ribavirin-interferon ␣2b combination (∼2-fold). similarly, the castanospermine-interferon ␣2b double combination was more synergistic than the castanospermine-ribavirin combination (∼5-fold), or the ribavirin-interferon ␣2b combination (∼3.3-fold). the combinations of celgosivir-interferon ␣2b or castanospermine-interferon ␣2b led to significant decreases in the ec50s of celgosivir (up to >20-fold) and castanospermine (up to >50-fold). the effective ec50s of celgosivir or castanospermine were further reduced by the addition of ribavirin. the cytotoxicity of the double and triple combinations was additive or less than additive, indicating that combinations of celgosivir or castanospermine with ribavirin and/or interferon ␣2b were generally less toxic than expected. these results indicate that the combination of celgosivir with interferon ␣2b or with interferon ␣2b and ribavirin may be effective in the treatment of hcv. pegylated interferon ␣ plus ribavirin is the current standard of care for the treatment of chronic hepatitis c virus (hcv) infections. this regimen results in sustained virologic response in only about 50% of patients and is associated with significant treatment-associated toxicities. a number of approaches are being used to identify novel therapeutic combinations with better tolerability and/or efficacy. inhibitors of endoplasmic reticulum (er) ␣-glucosidase have been shown to inhibit viral replication and secretion and may have utility as part of new multi-drug treatment cocktails. the ␣-glucosidase inhibitor celgosivir is currently being evaluated in combination with pegylated interferon ␣ and ribavirin in humans. the purpose of this study was to evaluate the antiviral effects of combinations of celgosivir and castanospermine, the primary active metabolite of celgosivir, with other antiviral agents having diverse mechanisms of action. the effect of the combination of celgosivir or castanospermine with the nucleoside analogue nm-107, amantadine, and another iminosugar, n-butyl-deoxynojirimycin (nb-dnj) was determined in a cytopathic assay using the hcv surrogate virus bovine viral diarrhea virus in madin darby bovine kidney cells. synergies were analyzed using isobolograms and volume of synergy measurements (macsynergy ii tm software). volumes of synergy indicated that the castanospermine and nb-dnj combination was additive, while the celgosivir and nb-dnj combination was synergistic at high nb-dnj concentrations (>100 m). celgosivir and castanospermine were synergistic with both amantadine and nm-107, with volumes of synergy between 60 and 150 m%. isobologram analysis confirmed these synergistic interactions. these results indicate that celgosivir could be considered in combination regimens containing drugs that directly target viral replication like nm-107. mechanism(s) of synergy are under investigation. department of biotechnology, yonsei university, seoul 120-749, korea hepatitis c virus (hcv) is an enveloped virus with positivestranded rna genome of approximately 9.6 kilobases and a major cause of non-a and non-b hepatitis, leading to liver cirrhosis and hepatocellular carcinoma. combination of interferon-␣ (ifn-␣) and ribavirin is the current standard therapy for the treatment of hcv infection, but there is no specific antiviral therapy available. the hcv viral genome encodes a single polyprotein of approximately 3010 amino acids, which is proteolytically processed by a combination of host and viral proteases into at least 10 distinct structural and nonstructural proteins. the structural proteins include c, e1, e2, and p7 and the nonstructural (ns) proteins include ns2, ns3, ns4a, ns4b, ns5a, and ns5b. as new hcv specific therapies, small-molecule inhibitors against hcv enzymes including ns5b protein, the viral rna-dependent rna polymerase (rdrp), and ns3 protease are in clinical tests. however, rapid emerging of drugresistant mutants has been hampering their practical clinical applications. recently, we have shown that phosphorylation of hcv rna polymerase by protein kinase c-like 2 (prk2) regulates virus rna replication. hcv rna replication was inhibited when prk2 expression level was down-regulated by using a prk2-specific sirna. in this study, we investigated the anti-hcv effect of prk2 inhibitors in an hcv subgenomic replicon system. treatment of the replicon cells with prk2 inhibitors suppressing the endogenous prk2 activity inhibited the phosphorylation of hcv rna polymerase and resulted in suppression of hcv rna replication in a dose-dependent manner. furthermore, the prk2 inhibitor in combination with ifn-␣ more effectively inhibited hcv rna replication than ifn-␣ alone. because the prk2 inhibitor did not show cytotoxicity in the cell-based drug inhibition studies and cellular proteins rarely get mutated, prk2 can serve as a cellular target for therapeutic intervention of hcv replication. specific inactivation of prk2 activity will provide an opportunity to interfere with hcv rna replication. haitao guo 1 , tianlun zhou 2 , ju-tao guo 1 , andrea cuconati 3 , anand mehta 1 , timothy block 1 1 drexel university college of medicine, doylestown, pa, usa; 2 nucleonic inc., irvine, ca, usa; 3 hepatitis b foundation, doylestown, pa, usa more than 400 million people worldwide are chronically infected with hepatitis b virus (hbv). the major complication of chronic hepatitis b is the development of primary hepatocellular carcinoma (hcc), which causes an estimated 500,000 deaths annually. currently clinical treatments (␣-interferon and nucleoside analogs) of chronic hepatitis b rarely cure the virus infection. this is due, at least in part, to their failure to eliminate viral covalently closed circular (ccc) dna from the nuclei of infected hepatocytes. hbv cccdna is essential to the virus life cycle by serving as the template for the transcription of the pregenomic rna and of the subviral rna species. its elimination during chronic infection is considered critical to long-term therapy. however, cccdna has not previously been targeted in high throughput screens of small molecule libraries. to screen compound libraries for antiviral drugs targeting cccdna, we set out to develop a cell-based assay suitable for high throughput screening. since cccdna is time-consuming to assay, it was desirable to use a viral gene product that could serve as a reporter for intracellular cccdna level. we predicted that the secretion of hbv e antigen (hbeag) by hepad38 cells, a hepg2-derived tetracycline inducible hbv expression cell line, would be cccdna-dependent. this is because a large portion of pre-core mrna leader sequence in the 5 terminus of integrated viral genome was deleted, preventing hbeag expression from transgene, but could be restored from the 3 terminal redundancy of pre-genomic rna during viral dna replication and subsequent cccdna formation. our experimental results showed that following induction, hepad38 produced and accumulated cccdna, which became detectable between 7 and 8 days. hbeag synthesis and secretion into culture fluid were dependent upon and proportional to the level of cccdna detected. therefore, the secretion of hbeag by hepad38 cells could potentially serve as a convenient reporter for the high throughput screening of novel antiviral drugs targeting hbv cccdna. kathy aldern, james beadle, karl hostetler university of california, san diego and the veterans medical research foundation, san diego, ca, usa (s)-hpmpa is a broad spectrum antiviral active against orthopoxviruses, hbv, cmv, hsv, and other herpes group viruses. we have shown that hdp-(s)-hpmpa has greatly enhanced antiviral activity against these viruses. in addition, while hpmpa itself is nearly inactive against hiv, we showed that hdp-(s)-hpmpa exhibited an ec50 >3 logs less than unmodified hpmpa in mt-2 cells by p24 reduction assay. to evaluate the metabolic basis for the increased antiviral activity, we studied and compared the cellular uptake of radiolabeled cdv, (s)-hpmpa and their hdp-esters and conversion to hpmpa-diphosphate (hpmpapp) and cdv-diphosphate (cdvpp) in mrc-5 human lung fibroblasts using hplc partisil sax ion exchange chromatography. cellular uptake of hdp-cdv and hdp-(s)-hpmpa was similar. however, when cells were exposed to the respective drugs for 6, 24 and 48 h, hpmpapp appeared much earlier than cdvpp and reached levels several fold greater than observed with hdp-cdv. drug wash out experiments were carried out in mrc-5 cells exposed to radiolabeled hdp-cdv and hdp-(s)-hpmpa. after 24 h, the culture medium was removed and replaced with complete medium without drug and the levels of hpmpapp and cdvpp were measured by hplc every 2 days for 0-10 days. levels of the diphosphates declined slowly with a t 1/2 of 5-10 days. in conclusion, hdp-(s)-hpmpa is converted to its diphosphate more rapidly than hdp-cdv and reaches higher intracellular levels. this may explain, in part, its greater antiviral activity. the antiviral activity and oral bioavailability of cidofovir (cdv) is enhanced when the phosphonate is esterified with various straight chain alkoxyalkyl groups. the length of this chain is an important determinant of antiviral activity and selectivity. however, in some cases, rapid metabolism to an inactive short chain metabolite was observed. to enhance the metabolic stability of these esters, we synthesized cidofovir alkoxyalkyl esters bearing methyl groups on the penultimate carbon of the alkyl chain. enzymatic stability of 15-me-hdp-cdv (1) was tested in liver s9 fractions from various species. in mouse and human liver s9 fractions, compound 1 was completely stable for 90 min while 15-20% of the straight chain hdp-cdv was metabolized. the branched alkoxyalkyl esters were then evaluated in cells infected with vaccinia, cowpox and ectromelia viruses. the branched methyl analogs were substantially more active than cdv and equal to or slightly more active than the straight chain analogs. compound 1 retained full activity compared to hdp-cdv and compound 2 showed greater activity against orthopoxviruses compared to its unbranched analog. we believe that the structural modification of the alkyl chain slows the formation of inactive metabolites, possibly by interfering with oxidation and may result in better pharmacokinetics and more potent antiviral activity against orthopoxvirus infection in vivo. cidofovir ([1-(s)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine, hpmpc) is a broad spectrum antiviral agent clinically used for treatment of aids-related cmv retinitis. cidofovir has limited oral bioavailability (<5%), attributed to ionization of its phosphonic diacid moiety under physiological conditions. we have shown that masking of this group by conjugation of the cyclic form of the drug (chpmpc) via a ser side chain p-o ester linkage with x-ser dipeptides, where x = a hydrophobic amino acid, can result in prodrugs 1 that afford significantly improved biological availability of parent drug in an animal model. here we describe the total synthesis of novel cyclic cidofovir prodrugs 2ab of l-val and l-phe using an alternative conjugation strategy, viz. via an ethylene glycol link utilizing p-o and c-o ester bonds. the preparation of the hpmpc synthon from r-glycidol used our modification of the literature procedure (brodfuehrer et al., 1994) , involving reaction of tritylated (r)-glycidol directly with unprotected cytosine to achieve regiospecific opening of the epoxide ring, followed by reaction with benzoic acid anhydride to obtain the desired n-benzoyl intermediate needed to continue the synthesis. pybop was used as condensing agent in a convenient, one-pot conversion of hpmpc to chpmpc and subsequent esterfication of the latter by the ethylene glycol-modified amino acids. the prodrugs 2 were converted to drug by cellular (caco-2, hff) and tissue (liver and intestinal) homogenates, but did not show enhanced oral bioavailability when evaluated in a rat model, suggesting that such compounds may be useful for understanding the effectiveness of 1 in drug delivery. cidofovir (cdv) is a broad-spectrum anti-viral agent that is used to treat aids-related cytomegalovirus (cmv) retinitis and other cmv infections. cdv has good in vitro activity against orthopox viruses, including smallpox; however, its use is limited because of the drug's low oral bioavailability and poor transport into cells. in order to improve its oral bioavailability, our group has synthesized a series of dipeptide and amino acid prodrugs of the cyclic analog of cidofovir (ccdv). in the current project, we examined the cytotoxicity and antiviral activity of the prodrugs, showing that the compounds are not cytotoxic and have diverse activity against hcmv and orthopox viruses (vaccinia and cow pox) with 50% inhibitory concentrations ranging from 0.1 to 0.5 and 10 m and greater, respectively for the two virus types. in vitro and in situ perfusion studies established that the permeability of the prodrugs is enhanced more than 30-fold and that the transport is mediated, at least in part, by the intestinal dipeptide transporter. we also have found that the bioavailability of the prodrugs is dependent upon the prodrug structure and that we can achieve up to an eight-fold increase in bioavailability over the parent compound in vivo. drug stability experiments showed that in gastrointestinal and liver homogenates, the ccdv prodrugs are enzymatically hydrolyzed to the parent compound. it is clear from this work that the biologically benign dipeptide moiety, strategically linked to the drug to mask its anionic properties, significantly enhances intestinal transport of ccdv, creating the possibility of an orally bioavailable form of ccdv with low toxicity. acknowledgement: supported by funds from tsrl inc, the university of michigan, and nih grants r43ai056864 and u01ai061457. lawrence trost 1 , bernhard lampert 1 , lloyd frick 2 , merrick almond 1 , george painter 1 1 chimerix, inc., durham, nc, usa; 2 dmpk advisor, chimerix, inc., durham, nc, usa foscarnet, a pyrophosphate analog approved for the treatment of cmv retinitis and acyclovir-resistant herpes infections in immunocompromised patients, is active against highly drug resistant strains of hiv-1. however, the clinical utility of foscarnet is limited because it requires controlled intravenous infusion and is associated with high risks of renal impairment and seizure caused by alterations in plasma minerals and electrolytes. lipid conjugation has been shown to increase the in vitro activity, improve oral bioavailability, and reduce the toxicity of several antiviral drugs requiring intravenous administration because of poor bioavailability. in the case of foscarnet, conjugation to methylbatyl alcohol (cmx012) decreases the apparent ec 50 value against hiv-1 by up to 40-fold. cmx012 was esterified to produce cmx052 in order to increase solubility and to protect against decarboxylation of the foscarnet moiety during passage through the stomach. here we present the results of a preliminary toxicology and toxicokinetic study of the methylbatyl alcohol conjugate of foscarnet methyl ester (cmx052). rats were given oral doses of 10, 30 and 100 mg/kg cmx052 daily for 7 days. there were no clinical signs of toxicity. body weight and food consumption were comparable to controls and serum biochemistry, hematology, coagulation parameters and urinalysis were normal. there were no gross findings at necropsy, no effects on organ weights and no findings by histopathological examination of a wide range of tissues. importantly, there were no changes in serum biochemistry parameters or histopathological examination that were indicative of the renal impairment or serum electrolyte changes that are associated with foscarnet. oral dosing resulted in significant plasma exposure to cmx012 (c max > 4 g/ml), the biologically active deesterified form of cmx052. in conclusion, cmx052 is absorbed after oral administration, converted to cmx012, and has a good preliminary toxicity profile. these results support the development of cmx052 for the treatment of drug resistant hiv infection. zhiqian wu 1 , julie breitenbach 2 , ulrika erickson 1 , john hilfinger 3 , john drach 2 , gordon amidon 1 1 department of pharmaceutical sciences, college of pharmacy, the university of michigan, ann arbor, mi, usa; 2 school of dentistry, the university of michigan, ann arbor, mi, usa; 3 tsrl, inc., ann arbor, mi, usa vaccinia virus is a surrogate model system for study of pox virology and development of antiviral therapeutics. the potent anti vaccinia virus activity and various shortcomings of vidarabine make it a good candidate for improvement by utilizing prodrug strategy. vidarabine is a polar nucleoside drug with low membrane permeability and rapid degradation by adonesine deaminase. 5 -monoester prodrugs of vidarabine with various amino acids promoieties (l-valine, l-isoleucine, l-phenylalanine. laspartic acid, l-proline) are synthesized and evaluated for their stability, permeability and activity against vaccinia virus. prodrugs exhibit different hydrolysis rate in caco-2 cell homogenate (t1/2: 2-40 min). 5 -l-isoleucyl and 5 -l-valyl monoester prodrugs exhibit comparable bio-conversion rate and hpept1mediated uptake as well as caco-2 permeability with valacyclovir, a commercially marketed oral amino acid ester prodrug. both prodrugs have potent activity against vaccinia virus and are resistant to ada1. preliminary animal study shows 5 -lisoleucyl vidarabine results in >10-fold increase in circulating vidarabine level. the results suggest that it may be possible to use amino acids prodrug strategy to improve vidarabine as anti vaccinia virus agent. vidarabine [9-␤-d-arabinofuranosyl)adenine or ara-a) was originally investigated as an anti-tumor agent and was later found to be active against herpes simplex virus (hsv) types 1 and 2. it was the first fda-approved drug for treatment of systemic hsv infections. although replaced by acyclovir and analogs for most applications, vidarabine remains an alternative therapy for acyclovir-resistant hsv and varicella-zoster virus infections. despite its proven efficacy, vidarabine suffers some limitations including: (i) metabolism by adenosine deaminase (ada) to its inactive hypoxanthine homolog (ara-h); (ii) low lipophilicity and membrane permeability and (iii) poor aqueous solubility, thus limiting options for parenteral and peroral delivery. our recent interest in vidarabine was triggered by our discovery that it was ∼5-fold more active against vaccinia (vv) and cow pox (cpv) viruses than was cidofovir in plaque reduction assays. its activity was enhanced about 10-fold by combination with 1 m 2 -deoxycoformycin (pentostatin, a potent inhibitor of ada) thereby providing significant superiority to cidofovir. from these results and our earlier studies on 5 -substituted vidarabine analogs (lipper et al., 1978. mol. pharmacol. 14, 366-369), we determined that minimizing metabolism of vidarabine by synthesizing 5 -amino acid substituted prodrugs gave a significantly more potent anti-pox virus agent. we found that amino acid ester prodrugs of vidarabine are active against vv at non-cytotoxic concentrations. further, using cell homogenates, purified enzyme and intact cell systems, we showed that the prodrugs are resistant to inactivation by ada. the prodrugs also had enhanced transport potential, most likely targeting the intestinal dipeptide transporter. finally, oral delivery of the prodrug to the small intestine resulted in a 10-fold increase in vidarabine plasma levels when compared to unsubstituted vidarabine. these properties make the prodrugs of vidarabine good candidates as orally bioavailable anti-pox virus agents that are stable in the presence of ada. acknowledgement: supported by funds from tsrl inc. and the university of michigan. ulf goerbig 1 , anne baum 1 , jan balzarini 2 , chris meier 1 1 university of hamburg, institute of organic chemistry, hamburg, germany; 2 rega institute for medical research, katolieke universiteit leuven, leuven, belgium the cyclosal pronucleotide system has been designed for an intracellular delivery of therapeutically active nucleoside monophosphates. as part of recent work on the cyclosal approach, the interaction of cyclosal nucleotides with cholinesterases has been investigated. it is known that organo-phosphates may act as irreversible inhibitors of cholinesterases (suicide mechanism). in the case of cyclosal nucleotides, cholinesterase inhibition could lead to unwanted side effects in a possible therapeutic application. there are two types of cholinesterase found in humans, the highly specific, physiologically important acetylcholinesterase (ache) and the much more unspecific butyrylcholinesterase (bche) of unknown physiological importance. fortunately, no inhibition of ache was observed for a variety of different cyclosal nucleotides. in contrast, bche inhibition was found in some cases. the anti-hiv-active 3,5-bis-tertbutyl-6-fluoro-cyclosal-d4t monophosphate is the first cyclosal derivative combining three desired properties: successful intracellular delivery of nucleotides, sufficient hydrolytic stability and strongly reduced inhibitor activity towards bche. because of the promising properties of this compound, we combined this mask developed for d4t with the antiviral active nucleoside analogues like d4a, dda, azt and acyclovir. in this contribution we present the synthesis, hydrolysis stability, inhibition behaviour towards bche and anti-hiv data of these new compounds. henning jessen 1 , wolfgang fendrich 1 , tilmann schulz 1 , jan balzarini 2 , chris meier 1 1 university of hamburg, institute of organic chemistry, hamburg, germany, 2 rega institute for medicinal research, katholieke universiteit leuven, leuven, belgium cyclosal-pronucleotides are used for the delivery of antivirally active nucleotides into cells via a ph triggered selective hydrolysis. to distinguish between intra-and extra-cellular environment enzyme-cleavable side chains were introduced in the aromatic moiety of the pronucleotide to enrich the compound inside cells. this behavior will further be described as "lock-in"effect. many other different cyclosal pronucleotides have been designed, all showing different hydrolysis properties and antiviral data, both originating from the nature of the cyclosal-moiety as well as of the nucleoside. to examine these differences, analytical tools of high accuracy and sensitivity were needed, being structurally as close as possible to the lead compounds. these requirements are met by intrinsically fluorescent nucleosides coupled to different cyclosal masking groups. for analysis of the purine-type nucleosides iso-da with high intrinsic fluorescence properties was chosen and converted into iso-a, iso-dda and iso-d4a. for the pyrimidine-type series the fluorescent nucleoside m 5 k was synthesized as well as the dideoxy-compound dm 5 k. these nucleosides were transformed into different cyclosalpronucleotides and tested for their suitability for fluorescence analysis. in fact, an improvement of sensitivity by a factor of 5000 compared to uv-detection was found for some of the compounds (pmol detection). for all compounds fluorescence and absorbance spectra were recorded to determine the absorption and emission maxima. the new compounds lacked activity against hiv-1 and hiv-2 strains. however, the compounds showed low cytotoxicity, which is important for their usability as fluorescent probes in cells. due to the analytical sensitivity, a simple model uptake study could be carried out, employing an u-tube with two aqueous phases, which were separated by an unpolar organic solvent simulating a diffusion barrier. the properties of the aqueous phases were varied and an enzyme-driven enrichment of a "lock-in"-modified intrinsically fluorescent cyclosal-pronucleotide passing the diffusion barrier could be simulated. nicolas gisch 1 , jan balzarini 2 , chris meier 1 1 university of hamburg, institute of organic chemistry, hamburg, germany; 2 rega institute for medical research, katholieke universiteit leuven, leuven, belgium cyclosal-pronucleotides efficiently deliver therapeutically active nucleoside monophosphates in human cells. "lock-in"-cyclosal-pronucleotides -the so-called second generation of cyclosal-compounds -have been designed to trap the compound by intracellular cleavage of esterase-cleavable moiety. one disadvantage of the "lock-in"-compounds is their high chemical stability, which leads to a delayed drug delivery. therefore, conceptually different, enzymatically activated cyclosalpronucleotides have been developed. in this concept lipophilic donor substituents attached to the aromatic ring are converted into a polar acceptor substituent by intracellular enzymatic cleavage. as a consequence the liberated acceptor group leads to a strong decrease in hydrolysis stability and a rapid formation of a charged intermediate is the result. from the phosphodiester intermediate the nucleotide is released subsequently. the concept, synthesis, characterization and in vitro antiviral evaluation of the third generation of cyclosal-pronucleotides will be presented. tomas cihlar 1 , richard mackman 1 , adrian ray 1 , dean boojamra 1 , lijun zhang 1 , deborah grant 1 , hon hui 1 , jennifer vela 1 , neil parkin 2 , yolanda lie 2 , kirsten white 1 , michael miller 1 , gerry rhodes 1 , manoj desai 1 1 gilead sciences, foster city, ca, usa; 2 monogram biosciences, so. san francisco, ca, usa background: n(t)rtis are currently used as a backbone of antiretroviral combination therapy. however, their long-term benefit can be limited by adverse effects, resistance development, drug-drug interactions, and sub-optimal efficacy in treatment-experienced patients. therefore, we searched for novel nucleotide analogs with improved pharmacological profiles. methods: phosphonomethoxy-2 -fluoro-2 ,3 -dideoxydidehydroadenosine (gs9148) was selected from a broad range of nucleoside phosphonate analogs. phosphoramidate prodrug technology previously explored with tenofovir was applied to gs9148, resulting in the identification of gs9131 (ethylalaninyl phenyl ester of gs9148). results: gs9131 exhibits potent anti-hiv-1 activity in primary lymphocytes and t-cell lines (ec 50 < 150 nm). low cytotoxicity (cc 50 > 100 m) was observed in multiple cell types including renal cells. diphosphate metabolite of gs9148 was shown to act as an obligatory dna chain terminator and a competitive inhibitor of hiv-1 reverse transcriptase (rt) (k i = 0.8 m). unlike ddi, d4t, or d4fc, neither gs9148 nor its prodrugs inhibited mitochondrial dna replication in hepg2 cells. in a phenosense assay, gs9148 retained its full activity against hiv-1 variants with k65r, m184v or l74v mutations in rt. viruses with ≥4 thymidine analog mutations showed ≤2fold reduced susceptibility to gs9148, a shift that was smaller than that of any other tested nrti. following an oral dose of 3 mg/kg gs9131 in dogs, the bioavailability of prodrug exceeded 20%, resulting in high intracellular levels (9.0 ± 2.3 m) and prolonged retention (t 1/2 > 24 h) of gs9148 diphosphate in blood lymphocytes. conclusions: both gs9148 and its prodrug gs9131 exhibit favorable in vitro pharmacological profiles including less resistance due to rt mutations than approved nrtis. gs9131 possesses good in vivo pharmacokinetic properties and thus represents an attractive development candidate with potential for clinical efficacy in both treatment-naive and nrti-experienced patients. martin mcdermott, gabriel birkus, ruth wang, holly macarthur, xiaohong liu, nilima kutty, tomas cihlar, craig gibbs, swami swaminathan, arnold fridland, william lee gilead sciences, inc., foster city, ca, usa gs-7340 and gs-9131 are alkylalaninyl phenyl ester prodrugs of tenofovir (tfv; 9-[(2-phosphonomethoxy)propyl]adenine) and a novel nucleotide analog fd4ap (phosphonomethoxy-2 -fluoro-2 ,3 -dideoxydidehydroadenosine), respectively. both gs-7340 and gs-9131 exhibit potent in vitro anti-hiv-1 activity, favorable resistance profile, and low cytotoxicity. compared to tenofovir disoproxil (viread), both prodrugs are significantly more stable in plasma and deliver >10-fold greater levels of active diphosphate metabolites into pbmcs in vitro and in vivo. the initial step in the intracellular activation of gs-7340 and gs-9131 is the hydrolysis of the alanine carboxyester by an unknown hydrolytic enzyme. the isolation and identification of this enzyme from human pbmcs is reported here. results: a major enzyme capable of cleaving gs-7340 and gs-9131 was purified from human pbmcs and was separable from esterases able to cleave alpha napthyl acetate (ana). the increase in specific activity of prodrug hydrolase achieved was 3500-fold. sds-page analysis showed the presence of a prominent protein band at 29 kda, which was identified by ingel tryptic digestion and ms/ms sequencing of the resultant peptides as lysosomal carboxypeptidase a (cathepsin a, ec 3.4.16.5, cata). the biochemical properties of purified prodrug hydrolase matched those of cata. recombinant cata and the isolated prodrug hydrolase displayed nearly identical susceptibility to hydrolase inhibitors and substrate preference against a panel of tenofovir phosphoramidate prodrugs. incubation of both enzymes with [ 14 c]gs-7340 and [ 3 h]difluorophosphonate resulted in the labeling of an identical 29 kda protein (catalytic subunit). both labeled bands reacted with polyclonal antibodies specific for human cathepsin a. finally, following incubation with gs-7340, approximately 6-9-fold lower intracellular concentrations of tfv metabolites were detected in fibroblasts from patients expressing non-functional cat a (cat a-cells) compared to normal control fibroblasts (cat a+ cells). center for drug delivery and nanomedicine, university of nebraska medical center, omaha, ne, usa nucleoside 5 -triphosphate (ntp) is the biologically active form of many antiviral nucleoside analogs capable of efficiently blocking the production of viral nucleic acids in infected cells. we describe novel microparticulate formulations for encapsulation of ntp, drug delivery and antiviral therapy of respiratory infections. polymer networks (poloxagels) consisted of crosslinked poloxamers and cationic polymer molecules were designed, synthesized and characterized by loading with ntp and interaction with cells. poloxagel-ntp formulations could be obtained by simple mixing of the aqueous solution of ntp with the aqueous dispersion of poloxagel and subsequent lyophilization. drug loading was equal up to 30% by weight. in this form, phosphates groups of ntp are complexed with amino groups of polycationic backbone of poloxagels, and the formulations could be stored at room temperature for many months without degradation of ntp. the particle size of aqueous poloxagel-ntp dispersions was low, with a hydrodynamic diameter of 0.1-0.2 m. the rate of passive drug release in physiological solution was from 33 to 50% of loaded drug during the 24-h period. these formulations were effectively consumed by many types of cells. significant amounts of drug and poloxagels were detected in the cellular interior after only 1-2 h of incubation. in the presence of cellular membranes drug release from poloxagel-ntp formulations was dramatically increased. we attribute this effect to the triggered release of the bound ntp as a result of competitive interaction of polycationic backbone of poloxagels with phospholipids of cellular membranes. mucoadhesive properties of poloxamers may additionally enhance binding of poloxagels with airways/lung epithelium. 5 -triphosphate of 1-␤-d-ribofuranosyl-1h-1,2,4-triazole 3-carboxamide (ribavirin) was synthesized using phosphorylation with a tris(imidazolyl) phosphate in a convenient one-pot approach. formulations of different poloxagels with the ribavirin 5 -triphosphate were prepared and characterized as prospective antiviral formulations for prophylactic and therapeutic treatments of respiratory infections including influenza a virus. aerosolic route of application of these antiviral formulations and associated problems are discussed. edwin gong 1 , ebrima gibbs 2 , joel oger 2 1 department of pharmacology and therapeutics, the university of british columbia, vancouver, bc, canada; 2 neuroimmunology lab, ubc hospital, department of medicine, the university of british columbia, vancouver, bc, canada ifn-alpha and ifn-beta are currently employed in the treatment of many viral diseases, especially chronic hepatitis. ifn-beta is also employed for the treatment of multiple sclerosis (ms), a chronic and often debilitating disease of the central nervous system. however, as with other protein therapeutics, long-term ifn therapy can lead to the development of binding and neutralizing antibodies to ifns and thus lead to deceased clinical effect of ifns. in order to measure the bioavailability of ifns and the level of neutralizing antibody, we have developed a realtime rt-pcr (taqman) assay by quantitating the expression of mxa (an ifn-induced protein) mrna. the nucleotide sequences of mxa deposited in the genebank were aligned, and a pair of primers and the hybridization probe were designed based on the conserved regions. a house keeping gene, gapdh, was used as a calibrator for relative quantitation. the rna standards were generated by in vitro transcription from cloned mxa gene in a plasmid vector. the reaction parameters were optimized. the assay was validated using pbmcs of ms patients that were treated with ifn-beta. for evaluation of the ifn bioavailability, the total rna was extracted from pbmcs and quantitatively detected by one-step rt-pcr for both mxa and gapdh. the results calculated by the 2 (− ct) method showed that the difference (signal-to-noise ratio) between samples with neutralizing antibodies and samples from untreated ms patients or healthy donors were approximately 60-70-folds. this indicates that our assay is a reliable method for determination of ifn bioavailability. v. lozitsky 1 , i. kravchenko 2 , v. larionov 2 1 research anti-plague institute, odessa, ukraine; 2 national university, odessa, ukraine transdermal delivery (td) of drugs is a novel method for treatment of diseases. td is carried out by the help of transdermal therapeutic systems (tts), which are multilayer plasters that contain active ingredients. td have a number of advantages, such as: (1) prolongation of the drug's action; (2) drug's concentration is maintained in therapeutic range; (3) there is no trauma to patient's skin while using td; (4) removing tts from the skin immediately stops drug's entering to the organism; (5) first-pass effect in the liver is reduced; and (6) many highly active drugs are irritating the gastro-intestinal tract if administered orally, others have a short half-life time-these drugs do not have downsides mentioned above if used as tts. in our previous research we elaborated tts containing rimantadine (ttscr) and studied its efficacy during experimental influenza in mice. we had established that transdermal delivery of rimantadine is more effective than oral administration. the aim of this work was to increase the efficacy of ttscr. to solve this task we studied the influence of some permeability enhancers on anti-influenza efficacy of ttscr during experimental infection. applied tts had adhesion hydrogel matrix (polyvinyl alcohol and 1.2-propylenglycol). they consisted of a base and a plastificator, which improves the administration of active substances through the skin and does not induce irritation. ttscr (1 mg/mouse) were applied on shaven backs of experimental animals. tts for other groups additionally contained one of such permeability enhancers as: 10 mg/mouse of dmso or 10 mg/mouse of octanol or 1 mg/mouse of papain. tts were applied on shaven backs of mice and stayed there from 1 day before infection to 10th day after challenge. mice of all groups were infected intranasally with influenza virus a/pr/8/34 (h1n1), which is highly pathogenic for them. challenge was carried out using four animals for each virus dilution within the range of 10 −1 to 10 −7 . deaths of animals were recorded for 14 days. the results showed that proteolytic enzyme papain increased the anti-influenza efficacy of ttscr on 1 log 10 tid 50 . dmso and octanol did not demonstrate such activity. mikhail dobrikov 1 , serguei vinogradov 2 , barbara ramsay shaw 1 1 department of chemistry, duke university, durham, nc, usa; 2 center of drug delivery and nanomedicine, and college of pharmacy, university of nebraska, omaha, ne, usa nucleoside reverse transcriptase inhibitors (nrti) are widely used in the antiviral chemotherapy. most nrtis require stepwise phosphorylation to the respective nucleoside triphosphates, which inhibit the viral dna synthesis. however, the emergence of hiv-1 reverse transcriptase-dependent drug resistance limits the effectiveness of treatment by nrtis. the ␣-p-borano-nucleotide analogues show several unique physico-chemical and biological properties: (i) enzymatic studies indicate that the rp-isomer of ␣-p-borano-2 ,3 -ddndps is a better substrate for cellular ndp kinase than the parent ddndp; (ii) neither isomer of the ␣-p-borano-ddndps is a substrate for mammalian pyruvate kinase and shows very poor inhibitory properties to this enzyme; (iii) the rp-(␣-p-borano)-ddntp isomers are better inhibitors of drug-and multidrug-resistant viral reverse transcriptases and are poor substrates for dnadependent dna polymerises; and (iv) after incorporation into viral dna the borano-ddnmp residues are more resistant to atp-dependent removal from viral dna than parent ddntps. to by-pass inefficient phosphorylation of the nrtis, several prodrugs of ␣-p-borano-nucleotide analogues have been previously synthesized. a more efficient delivery system for ␣-p-boranonucleotide analogues based on nanosized cationic polymeric gel (nanogel) is proposed. selective inhibition of drug-and multi-drug resistant viral rts, poorer inhibition of intracellular kinases and dna polymerases by the ␣-p-borano-nucleotide analogues, and their specific delivery into infected cells in the complex with nanogel particles suggest a new approach to the design of more powerful antiviral drugs. acknowledgement: this work was supported by the nih grant r01 al52061 to b.r.s. we have developed a high-throughput, cell-based assay to address the critical need for antiviral drugs for the treatment of influenza. in consideration of the demand to screen high volumes of compounds, we targeted the development of a 384 microtiter plate format for the assay. in this assay, the inhibition of the influenza-induced cytopathic effect (cpe) in mdck cells was assessed using the celltiter-glo luminescent cell viability assay by promega. this reagent measures the amount of atp present in cells, which is directly proportional to the number of metabolically active cells. validation studies were executed to establish optimal cell density, viral concentration, dmso tolerance for compound dilution, incubation time for virus-induced cpe and effective control drug concentration. additional parameters, such as assay variability, reagent and read stability, edge effects, and ic50 stability were also investigated during validation. we are currently initiating use of the assay to screen chemical libraries and will report our findings from library screens in addition to the aforementioned validation. we believe the approach will also provide a mechanism for discovery of new antiviral leads for influenza as well as avian flu. fundacio irsicaixa, hospital universitari germans trias i pujol, 08916 badalona, spain we have developed bacteriophage lambda based genetic screen that can be used to isolate and characterize site-specific proteases. this genetic screen system is based on the bacteriophage lambda ci-cro regulatory circuit, in which the encoded repressor ci is specifically cleaved to initiate the lysogenic-to-lytic switch. we have adapted this simple, safe and rapid genetic screening system to predict the activities and phenotypes of human immunodeficiency virus type 1 (hiv-1) proteases in the course of viral infection and antiretroviral therapy. a specific target for the hiv-1 protease, p17-p24, was inserted into the lambda phage ci repressor. the target specificity of the ci-hiv repressor was evaluated by coexpression of this repressor with an hiv-1 protease construct. upon infection of escherichia coli cells expressing the two constructs encoding the ci-hiv-1 repressor and hiv-1 protease, lambda phage replicated up to 1000-fold more efficiently than in cells that did not express the hiv-1 protease. this assay responds appropriately to well-known hiv-1 proteases inhibitors and can be used to search for new proteases inhibitors. the high level of specificity of this system, in which modest differences in catalytic efficiency can be quantified, should be also useful for the characterization of different mutant viral proteases. we further demonstrated the broad applicability of this protease assay using other viral proteases and their cognate cleavage sites, including hepatitis c virus (hcv) ns3 protease and severe acute respiratory syndrome (sars) coronavirus (cov) (scov) 3c-like protease. compared with other protease assay methods, this assay has the following advantages: safe, highly sensitive, highly specific, easy quantification, and rapid generation of different protease cleavage substrates using molecular cloning and expression. this system may be useful for the development of a screening method to identify viral protease inhibitors and should be also useful to characterize cellular, viral, or other infectious agent proteases with different activities and specificities. karen m. watson, todd b. parsley, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa a virus transmission and rapid resistance selection assay has been developed in order to quickly evaluate the biological properties of anti-infective test compounds and rationally prioritize them for further development. the transmission assay specifically evaluates the ability of test agents to suppress and clear virus replication from cultures during the serial passage of virus in the presence of fixed concentrations of the test compounds alone or in combination. the growth and expansion of virus in the infected cultures has been shown to occur through the replication of originally infected cells in the absence of virus spread, through direct virus to cell infection, and through cell to cell transmission. in order to sterilize a culture a test compound must possess the ability to specifically and potently interfere with virus replication by each of these three methods and must be able to inhibit the replication of resistant viruses which pre-exist in the viral inoculum and which rapidly grow in the presence of the fixed concentrations of the test agent. twelve pyrimidinediones being evaluated for potential use as both anti-hiv therapeutic agents and topical microbicides were evaluated for their ability to inhibit virus transmission and to define their ability to rapidly select for resistant virus strains. these compounds were evaluated in parallel with known anti-hiv agents that inhibit virus entry (t20) and reverse transcription (sustiva, uc781 and azt). the results of the transmission assays suggest that significant biological differences exist between antiviral compounds and even between highly related congeners of the same class of pyrimidinediones, suggesting that the transmission and rapid resistant selection assay measures important antiviral properties of anti-hiv agents. biological studies that evaluate the mechanisms of virus growth in the presence of high concentrations of test compounds will be described. laure deflubé 1 , kerstin angner 1 , anna overby 1 , david stein 2 , patrick iversen 2 , ramon flick 1 1 utmb, department of pathology, galveston, tx, usa; 2 avi biopharma, inc., corvallis, or, usa in the family bunyaviridae, several members on the genus phlebovirus have been reported to cause disease in humans or livestock. among these, rift valley fever (rvf) virus is an important human/animal pathogen. its widespread geographic distribution and its ability to produce severe human disease makes this virus a worldwide public health concern. the phosphorodiamidate morpholino-oligomers (pmo) are a class of dna-like antisense agents typically synthesized to a length of about 20 subunits and contain purine or pyrimidine bases attached to a backbone composed of six member morpholine rings joined by phosphorodiamidate intersubunit linkages. they have been shown to be effective antiviral compounds for different virus families, e.g. coronaviridae and flaviviridae. pmo bind to rna preventing translation of the viral rnas. we used our recently developed plasmid-based minigenome rescue systems for uukuniemi (phlebovirus model virus) and rvf viruses to screen antiviral compounds based on the morpholino antisense oligonucleotide approach. for this the antiviral compounds were appraised on the basis of reporter gene activity (fig. 1b) . the inhibitory effects of the same compounds were also tested by measuring reduction in virus titer (fig. 1a) , by monitoring changes in viral antigen production using an indirect immunofluorescence procedure and facs analysis, and analysis of genome transcription/replication by rt-pcr. indeed several pmos could be identified with interfering effect at a low ic50 on bunyaviral minigenome rescue as well as virus proliferation. based on these results, we plan to confirm antiviral activity of the most promising compounds in suitable animal models. we have shown that the fractal approach to the problem of viruscell interaction gives the unique possibility to process the data through the sequence of the direct and inverse fourier transforms. the studies were carried on the herpes simplex virus us-1 interacting with the hep-2 sensitive cell culture. the object was imaged as system of bright peaks formed as a result of laser diffraction on the structural elements of the virus-cell system. the whole virus-cell interaction information is inserted into computer in a fastest parallel way. the laser intensity peaks, which form the speckle image of the system under consideration, could be transformed into the hierarchical system of the circles (or squares) according to the choice of the researcher, but conserving the same d value, which depends only on the true intermolecular interaction potential. this potential, being characteristic for every stage of virus-cell interaction, is responsible for the given structure of the dynamic virus-cell system and the unique, but the typical form of the fractal cluster corresponding both to the system itself and its image as well, was processed by computer techniques. the hierarchical fractal design of the virus-cell system, proposed here for the first time, gives the universality, needed for the quantitative description of any possible combination of the virus and corresponding sensitive cell. it should be noted, as well, that the fractal microscope use for viruscell dynamic system imaging have all the properties, required from all other experimental tools of monitoring, including the reliability, reproducibility and preciseness. this device could be used in drug design biological test stages with the scope of time and efforts economy during the compounds libraries screening. the fractal microscope combined with the qsar drug design technique makes the antiherpetic drug design more competitive as compared to the regular approaches. acknowledgement: the authors are indebted to the partial support of the stcu grant #3147. we have investigated experimentally the fractal properties of diffraction images obtained by laser irradiation of virus-cell system. it was shown that the diffraction process is mathematically equivalent to the direct fourier transform of the said system's components modeled with simple geometrical figures (e.g. circles). each viral family could be coded and described quantitatively with the average size of the free viral particle and the type of its symmetry. we propose here to use the inverse fourier transform of the virus-cell system in order to get the real enlarged image of the viruses attacking the sensitive cell as well as the cell's structural transformation caused by the sequent stages of virus reproduction process. the set of bright and dark spots, which forms the virus-cell system's diffraction image, could be coded into set of numbers (matrix form of correlation vector-function) using the quantification procedure. the correlation function was used as presented in polar coordinates because the system has the axial symmetry (laser beam taken as main physical axis). the full information included into the image peaks' diameters and color index is transformed using inverse fourier technique into set of intersecting bright and dark circles. the full in vitro dynamics of the structural changes of the virus-cell system are described by the changes of circles' diameters and the area of their intersection. it was shown, also, that the magnification of the fractal microscope could achieve 10,000× to 100,000×, depending on the laser power used. proposed fractal microscope could be applied as well in vivo experiments until the required magnification will not make us to use projection laser with the output exceeding 25 mw. we have shown that the fractal microscope based on the inverse fourier transform could be applied successfully in pharmaceutical antiviral drug design, laboratory and clinical trials of new antiviral preparations, especially effectively in hierarchic qsar research. acknowledgement: authors are grateful to the support under the stcu grant #3147. we have previously reported bicyclic furano pyrimidines as potent and selective inhibitors of varicella zoster virus (vzv) , with subnanomolar activity for palkylphenyl substituted analogues . these compounds however are highly lipophilic and poorly soluble in water. we then reported a series of p-alkyloxyphenyl compounds containing a phenolic ether aiming to enhance water solubility whilst retaining antiviral activity (mcguigan et al., 2002) . we will now report the synthesis, characterisation and antiviral evaluation of a novel series of p-alkyloxyphenyls where there is at least one methylene spacer between the phenyl and ether group to potentially boost the pharmacokinetic profile. the alkyl chain length was fixed to retain a high clogp value, a parameter that has previously been shown to correlate with high antiviral potency . the target structures were prepared by the pd-catalysed coupling of a series of para-substituted alkoxyphenyl acetylenes with 5-iodo-2 -deoxyuridine, to give intermediate 5-alkynyl nucleosides, which were subsequently cyclised in the presence of cui to give the desired bicyclic systems. the antiviral activity, cytotoxicity, and solubility of these compounds are to be reported. we have previously reported on some novel nucleoside analogues containing and unusual furano bicyclic pyrimidine base and long side chain , which were discovered to be both potent exquisitely and selective towards the varicella zoster virus. following this discovery, three main sites for modification were identified and explored: (1) the side chain; (2) the bicyclic base; and (3) the sugar moiety. modification to the side chain by insertion of a phenyl ring, led to the most potent anti-vzv nucleoside to date (ec50 1 nm) . the investigation into modifications at the three sites stated above has continued and we herein report further adjustments to these analogues. replacement of the furo oxygen with sulfur on the parent nucleosides bearing an alkyl side chain has been reported to retain antiviral activity. however, those bearing a phenyl alkyl side chain are here shown to give a slight reduction in anti-vzv activity. modifications to the phenyl ring of the side chain have included halogen substitutions, and the fluorine in particular has produced some intriguing results in that, while the ortho and meta substitutions show some anti-vzv activity, the para analogue is completely devoid of antiviral activity. we now report further studies which include the di and tri substituted phenyl analogues. finally, we have also investigated sugar modification that has included substitutions of the 3 hydroxyl group. previous modifications which have replacements of the hydroxyl groups, resulted in loss of activity against vzv . we now present some new 3 substituted analogues which have provided interesting biological results. we have previously reported bicyclic furano pyrimidines as potent and selective inhibitors of varicella zoster virus (vzv) with subnanomolar activity for palkylphenyl substituted analogues srinivasan et al., 2001) . the sar is now further explored via the substitution of phenyl derivatives with electron withdrawing and electron donating groups. we now report the synthesis, characterisation, and biological evaluation of a novel series of mono substituted phenyl derivatives in order to probe the structure activity relationships in this region. the target compounds were synthesised under sonogashira conditions where a series of substituted phenyl acetylenes were coupled with 5-iodo-2 -deoxyuridine, to give intermediate 5alkynyl nucleosides that were subsequently cyclised in the presence of cui to give the desired bicyclic systems. diseases caused by herpes simplex virus (hsv) are widely distributed. prophylaxis and treatment of these infections are important health care tasks that require also the search, design and development of new antiherpetic drugs overcome drug resistance and toxic side effects of existing drugs. drug selection simply based on results of empirical screening is not very effective. computer-based technologies may help to optimize the structure of antiviral compounds as well as to design and develop new drugs. the objective of the present work is the quantitative structureactivity relationship (qsar) analysis of antiviral activity of various n,n -(bis-5-nitropyrimidyl)dispirotripiperazines in connection with consequent drug design. the well-established simplex representation of molecular structure (sirms) qsar approach has been used to fulfill this objective. it allows the molecular design of new effective antiviral drugs. thorough investigation of the relationship between: (a) cytotoxic (hela cells and gmk cells, cc 50 , g/ml); (b) antiherpetic activity (hsv-1 strain kupka, ic 50 , g/ml); and (c) selectivity index (ratio of cc 50 to ic 50 ) and the structure of 48 n,n -bis-5-nitropyrimidyl derivatives of dispirotripiperazine have been conducted. statistic characteristics for pls (partial least squares) models are quite satisfactory (r 2 = 0.816-0.991, q 2 = 0.637-0.868). the results are confirmed by experimental data. based on the obtained models, molecular fragments that promote and interfere with antiviral activity were defined. additionally, these models provide the possibility to predict molecular fragments that will enhance antiherpetic activity and to design new well tolerated highly virus-specific drugs. in summary, the developed simplex approach is an effective instrument for prediction and design of novel effective antiherpetic agents. several representatives of a series of 5-arylethynyl-2deoxyuridines (1a) bearing bulky aryl groups were recently shown to possess unexpected activity towards hsv-1. unlike common anti-hsv drugs, these compounds retain activity towards kinase-deficient acyclovir-resistant strains. therefore, an unusual mechanism of antiviral action is assumed. in order to investigate the mechanism and to discover more potent analogues we synthesized several novel 2 -deoxy (1a) and 2 -arabino (1b) uridine derivatives possessing different 5-arylethynyl substituents. dinucleosides 2 containing two uridine moieties coupled to a single polycyclic aromatic hydrocarbon (e.g. pyrene) represent another type of structural variation of nucleosides 1a. these compounds as well as some of 1a and 1b possess bright fluorescence that can be used in biological evaluations. cytomegalovirus (cmv) is a wide spread opportunistic pathogen which belongs to the beta subfamily of the herpesviridae. primary infection is generally asymptomatic resulting in life long latency. however, morbidity and mortality rates post-transplantation are greatly increased following reactivation or recrudescence of cmv. ganciclovir (gcv) and cidofovir (cdv) have both been successful in suppressing cmv viral replication in immunocompromised patients. although sustained use of these drugs has resulted in the emergence of multi-drug resistant strains of virus. in this study we used plaque reduction assays to determine the antiviral efficacy of two ribonucleotide reductase inhibitors, didox (dx; 3,4dihydroxybenzohydroxamic acid) and trimidox (tx; 3,4,5trihydroxybenzamidoxime) in inhibiting both wild type and drug-resistant strains of murine cmv (smith strain). the results presented here demonstrate that both dx and tx inhibit viral plaque formation in a dose dependent manner in both wild type and the resistant strain. a 43-and 39-fold increase in drug dose was required for cdv and gcv respectfully to inhibit plaque formation by 50% in the resistant strain (cdv wt: 0.03 m, r: 1.28 m/gcv wt: 3.17 m, r: 122.85 m). this compared to only a moderate increase in drug dose required for dx and tx to achieve 50% inhibition in the resistant strain (dx wt: 20.71 m, r: 28.88 m/tx wt: 7.12 m, r: 11.59 m), corresponding to a 1.4-and 1.6-fold increase respectfully. further work is currently underway to determine the possible mechanism of antiviral actions and toxicity profiles of these novel virostatics. in patients with human immunodeficiency virus (hiv) infection, coinfection with herpesviruses continues to be a problem for patients receiving antiviral hiv therapy. since treatment can be affected by the large number of drugs required for multiple infections, it would be useful to have antivirals that are active against both hiv and the herpesviruses. we reported previously that alkoxyalkyl ester prodrugs of cidofovir (cdv) are several logs more active against herpesvirus replication than unmodified cdv. to determine if this strategy would be effective for other acyclic nucleoside phosphonates which are active against hiv infections, hexadecyloxypropyl (hdp) esters were synthesized from 1-(phosphonomethoxyethyl)-cytosine (pme-c), 1-(phosphonomethoxyethyl)-5-bromo-cytosine (pme-5brc), 1-(phosphonomethoxyethyl)-5-fluoro-cytosine (pme-5fc), 9-(phosphonomethoxyethyl)-2,4-diaminopurine (pme-dap) and 9-(phosphonomethoxyethyl)-2-amino-4cyclopropylaminopurine (pme-cprdap) and assayed for activity against herpesvirus replication. overall, the hdp esters were more active than the unmodified acyclic nucleoside phosphonates, indicating that this is a useful strategy for increasing the antiviral activity of acyclic nucleoside phosphonates. one of the most active compounds was hdp-pme-cprdap which had ec 50 values of 0.2, 0.3, and 0.03 m in hff cells infected with hsv-1, hsv-2 or hcmv, representing a 12-26-fold increase in efficacy over the parent pme-cprdap. another promising compound was hdp-pme-dap, which had ec 50 values of 0.7, 0.2, and 0.6 um in hff cells infected with hsv-1, hsv-2, and hcmv, representing a 16-43-fold increase over the parent pme-dap. the results presented here indicate that modified acyclic nucleotides with antiviral activity against hiv also inhibit the replication of some of the herpesviruses. further evaluation of their activity against other herpesviruses that are a problem in hiv-infected patients, such as human herpesviruses type 6 and 8, is warranted and may provide new therapeutic options for patients with coinfections. julie m. breitenbach 1 , katherine z. borysko 1 , jiri zemlicka 2 , john c. drach 1 1 biologic & materials sciences, school of dentistry, university of michigan, ann arbor, mi, usa; 2 karmanos cancer institute, wayne state university school of medicine, detroit, mi, usa we previously described first (qiu et al., 1998. j. med. chem.) and second generation (zhou et al., 2004. j. med. chem.) methylenecyclopropane purines that have potent and selective activity against hcmv. strains selected separately for resistance to first-generation analogs (synadenol, synguanol) were 10-20-fold resistant to several first-generation purine analogs. similar resistance was observed to the second-generation guanine analog cyclopropavir [ic 50 's in plaque assays = 0.35 and 21 m, respectively for wild-type (wt) and synguanol-resistant (1982r) virus]. likewise a ul97 deletion mutant (prichard et al., 1996. j. virol.) was resistant to both first and second-generation compounds (ic 50 's = 2.1 and 0.25 m in wt; 100 and 15 m in ul97 del , respectively for synguanol and cyclopropavir). ul97 from the hcmv strain selected for resistance to the synadenol was sequenced and two mutations were identified: m460i and c603y. because hcmv with either m460i or the related c607y mutation alone was sensitive to synadenol and synguanol (baldanti et al., 2002 . antiviral res.), we hypothesize that two mutations are required for resistance to first-and second-generation analogs. this hypothesis was tested by construction of three strains of hcmv from hcmv ad169 bac with one, the other, or both mutations in ul97. as expected, the two strains with the single mutations were 3-to 6-fold resistant to ganciclovir but had little resistance to the first generation compounds synadenol and synguanol (0.5-to 1-fold). both strains were somewhat more resistant to the second-generation compound cyclopropavir (6to 8-fold) but less so than observed in the 1982r virus with two mutations. study of the resistance of the constructed virus with two mutations is underway. we conclude that a functional ul97 is required for activity against hcmv and that is likely that two mutations in ul97 are required for significant resistance. acknowledgement: supported by grants p01-ai46390 and r44-ai054135 from nih and funds from the university of michigan. svitlana zagorodnya 1 , nadiya nesterova 1 , inna alexeeva 2 , larisa palchikovskaya 2 , galina baranova 1 , alexander kobko 2 , anna golovan 1 1 zabolotny institute of microbiology and virology of nas of ukraine, kiev, ukraine; 2 institute of molecular biology and genetics of nas of ukraine, kiev, ukraine search of new effective preparations capable to inhibit herpesviruses reproduction is stipulated by their certain resistance to different groups of chemical preparations. new triazine bearing tricyclic bases and their n-glycosidic derivatives structures are widely used as potential antiviral agents. the objective of the present investigation was to study the activity triazine bearing tricyclic bases nos. 1 and 2, as well as n-glycosidic derivatives no. 3 against epstein-barr virus-lymphotropic and oncogenic virus from herpesviridae family. as a model of ebv-infection in vitro we used the line of lymphoblastoid b-cells raji, which infected by ebv. an inhibition of reproduction of ebv in a cell culture by no 1, no 2, and no 3 was determined by reduction of a number of genome-equivalents of ebv dna on a cell, which were revealed by quantitative pcr with use of primers and reagents "amply-senc-100 r" (russia). the first stage of investigation of substances was the analysis of their cytotoxicity for cell line raji. they have been studied in concentrations of 1000, 500, 250, 125, 64, 32, 16, 4 , 1, 0.5 and 0.1 g/ml. the concentrations that inhibited the quantity of live cells on 50% (id50) were equal to substances no. 1-750 g/ml, no. 2-625 g/ml and no. 3-125. the minimal inhibiting concentration (mic) of nos. 1, 2, and 3 was equal to 1 g/ml, because the amount of genome-equivalents of dna ebv on a cell was reduced with 28.0 up to 14. hence, the index of selectivity (is) was equal to 750 and 625 for triazine bearing tricyclic bases nos. 1 and 2, for n-glycosidic derivatives-125. in addition these compounds were also tested in transcription and replication model systems in vitro. our results indicate that bases and their n-glycoside derivatives effect rna and dna synthesis in different manner. r. sgarbanti 1 , l. nencioni 1 , g. macrì 2 , c. nucci 2 , u. benatti 3 , m. magnani 4 , e. garaci 5 , a.t. palamara 1 1 department public health sciences, university rome "la sapienza," rome, italy; 2 department biopathology, physiopathological optics, university rome "tor vergata," rome, italy; 3 department exp. medicine biochemistry section, university genova, genoa, italy; 4 inst. biochemistry, university urbino, urbino, italy; 5 department exp. med. biochem. sciences, university rome "tor vergata," rome, italy several studies have demonstrated that different viruses induce an imbalance in the intracellular redox state through a depletion of glutathione (gsh), the main intracellular antioxidant. the imbalance in the intracellular redox state represents a key event in the development of viral infection. indeed, our previous data showed that treatment with gsh prevents a decrease in intracellular gsh and inhibits replication of different rna and dna viruses in vitro and in vivo. our recent data demonstrated that a butanoyl derivative of gsh (gsh-c4), with increased hydrophobic properties, inhibited in vitro parainfluenza-1 and hsv-1 replication more efficiently than gsh. for this reason we evaluated the effectiveness of topical gsh-c4 administration in hsv-1-induced keratitis in rabbits. for infection, the corneal epithelium, previously scratched, was inoculated with 2 × 10 5 pfu/ml of hsv-1. gsh-c4, dissolved in a saline solution (150 mm, 100 ul/eye), was administered as eyedrops four times daily for ten days. a saline solution was used for the control group. the clinical evaluation of conjunctival and corneal involvement, performed by using 0.5% fluorescein sodium eyedrops and a slit lamp fitted with a cobalt blue filter, demonstrated that gsh-c4 treatment was effective in reducing the severity and progression of keratitis and conjunctivitis. moreover, in gsh-c4 treated animals, conjunctival hsv-1 titre, assayed by tcid50 on day 4 post-infection, was significantly reduced as compared to that of control animals (mean = 1.4 × 10 3 units/ml versus 1.1 × 10 5 units/ml, n = 10 for group). accordingly, similar results were obtained by measuring virus titre from the corneas of gsh-c4-treated animals versus placebo animals (mean = 3.5 × 10 3 units/ml versus 8.5 × 10 5 units/ml, n = 5 per group). such results highlight the antiviral activity of gsh-c4 in vivo and suggest that topical gsh-c4 treatment could be considered as complementary therapy of hsv-1-induced keratitis. debra quenelle 1 , deborah collins 1 , latisha pettway 1 , caroll hartline 1 , james beadle 2 , w. wan 2 , karl hostetler 2 , earl kern 1 1 university of alabama school of medicine, birmingham, al, usa; 2 department of medicine, university of california, san diego and veterans medical research foundation, san diego, ca, usa cytomegalovirus (cmv) can cause a wide variety of clinical manifestations in immunocompromised hosts or transplant recipients. we have utilized severe combined immunodeficient (scid) mice implanted with human fetal tissue and subsequently infected with hcmv or balb/c mice infected with mcmv to evaluate new antiviral therapies against cmv infection. in the current studies we used these two models to determine the efficacy of (s)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine ((s)-hpmpa), hexadecyloxypropyl-(s)-hpmpa (hdp-(s)-hpmpa), or octadecyloxyethyl-(s)-hpmpa (ode-(s)-hpmpa). in the hcmv model, human fetal thymus and liver (thy/liv) tissues were implanted under the kidney capsule of mice and inoculated 16-20 weeks later with 4700 pfu of hcmv. tissue samples were obtained at various time points for quantitation of hcmv titers by plaque assay. in general, replication of the toledo strain of hcmv in the implant tissue increased through 21-28 days and then gradually decreased to undetectable levels by 8 weeks post-infection. to determine efficacy of these compounds, oral treatment with vehicle or 10 mg/kg of (s)-hpmpa, hdp-(s)-hpmpa or ode-(s)-hpmpa was initiated 24 h after infection and continued for 28 days. cidofovir (cdv) at 20 mg/kg was administered i.p. daily as a positive control. results indicated that (s)-hpmpa, hdp-(s)-hpmpa and ode-(s)-hpmpa were highly effective in significantly reducing replication when compared to the vehicle control. in mcmv infected mice, hdp-(s)-hpmpa was highly effective in preventing mortality when administered orally at 30 or 10 mg/kg beginning 24 h post-viral inoculation and 30 mg/kg when treatment was delayed until 48 h postviral inoculation. these data indicate that these compounds were highly efficacious in two animal models of cmv infection and should be evaluated for use in hcmv infections in humans. cytomegalovirus (cmv) is a ubiquitous ␤-herpesvirus that asymptomatically infects immunocompetent individuals but leads to serious illness in immunocompromised individuals, such as transplant recipients, neonates and aids patients. thus, the need for well-tolerated and potent antiviral compounds with activity against cmv is well recognized. in our current studies, we have evaluated the in vivo activity of rep 9, a fully degenerate 40 mer phosphorothioated oligonucleotide against murine cytomegalovirus infection (mcmv) in mice. rep 9 has potent in vitro activity against hsv-1, hsv-2, hcmv, vzv, ebv, and hsv-6 (vaillant et al., submitted for publication). in our initial studies, infected mice were treated with rep 9 and compared to saline-treated infected control mice. compound was administered intraperitoneally for 5 consecutive days at 10 mg/kg, starting at 2 days prior to infection. mice were infected with 5 × 10 5 pfu mcmv on day 0, at 3 h post-treatment. sera were collected at −22 h, at 36 hpi, and at 3 dpi for elisa analysis of ifn␥ production. spleens and livers were collected at 3 dpi for determination of virus titers. at 3 dpi, virus titers in the spleens and livers were significantly reduced by rep 9 treatment as compared to control mice. splenomegaly was observed in infected mice treated with rep 9 but not in saline treated, infected mice or in rep 9 treated, uninfected mice. ifn␥ levels in mice treated with rep 9 peaked at 36 hpi compared to 72 hpi for saline-treated control mice. these data suggests that immune stimulation might contribute to the antiviral activity of ps-ons, perhaps through ifn␥ levels. a second study comparing the in vivo activity of rep 9 with two oligonucleotide analogs that do not activate tlr-9 mediated immune stimulation suggests that direct antiviral activity of rep 9 and the analogs was the predominant therapeutic mechanism in vivo. moreover, one rep 9 analog exhibited even greater antiviral activity than rep 9 while causing no splenomegaly. additional experiments are underway to provide insights into the mechanism of action against mcmv infection. acknowledgement: supported by contract no1-ai-15438 from the virology branch, niaid, nih. kathy keith 1 , joseph maddry 2 , namita bansal 2 , kochurani jacob 2 , secrist john 2 , earl kern 1 1 department of pediatrics, university of alabama school of medicine, birmingham, al, usa; 2 southern research institute, birmingham, al, usa a series of novel antiviral agents was prepared based on lead compounds related to acyclic nucleoside phosphonates. these agents consist of a purine nucleus bearing a pendant phosphonic acid group. the design strategy was two-fold: (1) following the approach of the hostetler group, to mask or partially mask the anionic phosphonate as a lipophilic ester and/or as an amino acid phosphonamidate prodrug that could enhance cellular uptake and be cleaved intracellularly; and (2) to investigate new substituents at the purine 2-and 6-positions. for proof of concept, a phosphonomethoxyethyl adenine (pmea) scaffold was employed. over 100 analogs of pmea substituted at the purine 2-or at the adenine n-6 site have been synthesized and evaluated for activity against orthopoxvirus infections. many n-6 substituents other than the previously recognized n-cyclopropyl have shown antiviral activity, and these structure-activity relationships are being investigated. an exciting finding has been that introduction of several novel moieties at the purine 2-position particularly the hydrazino, hydroxylamino, or the cyclopropylamino groups resulted in several compounds with excellent in vitro activity. for example, octadecyloxyethyl (ode) 2-amino-n(6)-cyclopropyl pmea had ec 50 values of 0.03-0.07 m and ode 2-hydroxylamino-n(6)-cyclopropyl pmea had ec 50 values of 0.3-1.6 m against cowpox and vaccinia viruses, respectively, using a plaque reduction assay in hff cells. under these conditions the parent molecule, pmea, was completely inactive. these two compounds had cc 50 values of 30-70 m giving selective indices of 200-1000. these studies indicate that several modifications in the pmea scaffold can result in good antiviral activity against orthopoxvirus infections in vitro and the most active compounds are currently being scaled up for evaluation in animal models. isatin (2,3-dioxoindole), a versatile lead molecule for designing of potential bioactive agents, and its derivatives have been reported to possess inhibitory activity against a variety of pathogenic viruses. methisazone(n-methylisatin-3thiosemicarbazone) was one of the first synthetic antiviral agents used clinically for the treatment of orthopox virus infections. the presence of the thiosemicarbazone, however, can result in immunosuppresion and we have attempted to replace the thiosemicarbazone with a sulphonamide in order to modify the antiviral activity. the present work was performed to evaluate the antiviral activity and cytotoxicity of some novel 4-[(1,2dihydro-2-oxo-3h-indol-3-ylidene)amino]-n-(4,6-dimethyl-2pyrimidiny)-benzene sulphonamides against pox viruses such as vaccinia and cowpox virus in human fibroblast cells and the activity was compared with cidofovir(cdv). among the compounds tested,4-[(5-methyl-1,2-dihydro-2-oxo-3h-indol-3-ylidene)amino]-n-(4,6-dimethyl-2-pyrimidiny)-benzene sulphonamide(spiii-5me), was the most active compound with an ec50 value of 18 mol, compared with cdv, which had an ec50 of 20 mol against vaccinia virus. all the compounds were non-toxic (>300 lm)using a neutral red uptake assay. substitution of a halogen atom in 5th position of isatin was found to abolish the antiviral activity. this compound should be evaluated in orthopox infections in animal to determine its potential for development as an effective agents for treatment of these infections. acknowledgement: supported in part by contract no1-ai-30049 from virology branch, niaid, nih, usa. evgeny belanov 1 , svetlana kotovskaya 2 , nikolay bormotov 1 , sergey balakhnin 1 , olga serova 1 , nataliya perova 2 , zina baskakova 2 , galina dzhumbaeva 2 , valerii charushin 3 , oleg chupakhin 2 1 state research center of virology and biotechnology "vector", koltsovo, novosibirsk reg., russia; 2 ural state technical university, yekaterinburg, russia; 3 institute of organic syntheses, yekaterinburg, russia during this study, we synthesized a series of 1,2,4-benzotriazine ( fig. 1) derivatives in order to evaluate the structural features required for anti-orthopoxviruses activity. these derivatives were tested for cytotoxicity and activity against the vaccinia, cowpox, mousepox, monkeypox, and in some experiments with variola viruses in vero and mk-2 cells. the results from studies of structure-activity relationship revealed that only compounds containing phenyl group at c-3 and the alkoxy and fluoro substitutes in the benzene ring of benzotriazines showed anti-orthopoxviruses activity. the antiviral activity was reduced or lost after substitution with other substitutes. thus, we find a new class of heterocyclic compounds with antiviral activity. acknowledgment: this research was funded by istc project #1989. yali chen, guang yang, kady honeychurch, dennis hruby, robert jordan siga technologies, inc., 4575 sw research way-suite 230, corvallis, or 97333, usa we have recently discovered a highly specific and potent antiorthopoxvirus compound (st-246) via high throughput screening (yang et al., 2005. j. virol. 79, 13139-13149) . marker rescue of st-246 resistant variants localized compound resistance to the f13l gene that encodes a major orthopoxvirus envelope protein (p37), which is required for extracellular viral particle formation. p37 participates in wrapping of intracellular mature virus (imv) in membranes derived from the trans golgi or late endosomal compartment to produce intracellular enveloped virus (iev) that are transported to the cell surface to form extracellular virus particles. to gain insight into the mechanism of action of st-246, we examined the effects of st-246 on the production of the extracellular viral particles in bsc40 cells infected with recombinant vaccinia virus containing a gfp-tagged p37 protein. in the presence of st-246, iev particle formation was dramatically reduced, plaque formation was almost completely inhibited, and imv particles appeared to be retained in intracellular vesicles as revealed by electron microscopy. furthermore, st-246 prevented the intracellular localization of p37 to the late endosome compartment as measured by confocal microscopy. in contrast, st-246 did not affect localization of p37 expressed from a st-246 resistant virus variant. more intriguingly, the compound did not affect the intracellular localization of p37 in transfected cells. these results suggest that st-246 inhibits an unknown virusspecific activity that requires f13l. this work underscores the exquisite specificity of st-246 and supports continued development of st-246 as a potential anti-orthopoxvirus drug. guang yang, chris harver, dennis hruby, robert jordan siga technologies, inc. 4575 sw research way, suite 230 corvallis, or 97333, usa st-246 is a potent, orally bioavailable anti-orthopoxvirus compound that is active in vitro and in vivo. the frequency of naturally occurring st-246 resistant variants was measured by fluctuation analysis and found to be 3.58 × 10 −6 . marker rescue of drug resistant variants localized changes associated with reduced compound susceptibility to the vaccinia virus f13l gene. the spectrum of mutations that confer st-246 resistance was determined using an error-prone pcr procedure to increase the frequency of compound resistance by 100-fold relative to the frequency of naturally occurring resistance. using this procedure, random point mutations were introduced into the f13l coding sequence by error-prone pcr and the mutated f13l alleles were transferred into wild-type virus genome by marker rescue. sequence analysis of the input error-prone pcr products prior to marker rescue identified numerous nucleotide changes in the f13l coding sequence, some of which created nonsense mutations. virus recombinants were selected that formed plaques in the presence of drug selection. this powerful selection procedure enriched for viruses that produced functional, st-246 resistant, f13l proteins. sequence analysis of the compound resistant f13l alleles identified numerous silent mutations scattered throughout the f13l coding sequence and 27 point mutations leading to amino acid changes that clustered around amino acid positions 258-302 within the f13l gene. seven of these mutations resulted in single amino acid changes and could be correlated with reduced compound susceptibility. taken together, these results suggest that: (1) mutations in at least 7 positions within f13l can confer resistance to st-246 and (2) st-246 resistant mutations cluster to a 58 amino acid domain in a region of the protein of unknown function. several 5-substituted pyrimidine analogs were identified as having antiviral activity against cowpox virus (cv) and vaccinia virus (vv) in primary human foreskin fibroblast cells. molecules containing benzopyran, cyanovinyl, and pyrazolone moieties at this position exhibited significant antiviral activity against both these viruses. three compounds in this series had ec 50 values below 10 m in a plaque reduction assay against both cv and vv. the antiviral activity of these compounds was also determined against herpes simplex virus (hsv) in a plaque assay. two compounds with cyanovinyl derivatives at the 5 position had ec 50 values below 15 m against both hsv-1 and hsv-2, whereas other substituents at this position exhibited weaker activity against one or both of these viruses. analogs containing the benzopyran substituents were the most effective against varicella zoster virus (vzv) and yielded ec 50 values below 10 m in a plaque reduction assay. none of the compounds were active against human cytomegalovirus. interestingly, all of the compounds were much less effective in a thymidine kinase (tk) negative strain of cv suggesting that the activation by this enzyme was important in their mechanism of action. tk deficient strains of hsv were also comparatively resistant to some of the compounds. the tk dependence of these compounds in cv and hsv taken together with the lack of activity against cytomegalovirus replication suggests that activation by a viral tk is important in their mechanism of action. these results indicate that pyrimidine analogs with large substituents at the 5 position are substrates for the distinct tk homologs encoded by the herpesviruses and orthopoxviruses and suggest that they may be effective against infections with these viruses. synthesis and testing of additional analogs is warranted and should help identify the most potent analogs for in vivo testing. department of pediatrics, university of alabama school of medicine, birmingham, al 35233, usa n-methanocarbathymidine ((n)-mct) is a conformationally locked nucleoside analog that is active against some herpesviruses and orthopoxviruses in vitro. this compound inhibits the replication of herpes simplex virus (hsv) with ec 50 values below 1 g/ml, and vaccinia virus (vv) and cowpox virus (cv) with ec 50 values of 0.55 and 1.5 g/ml, respectively. assays using a thymidine kinase (tk) negative strain of cv yielded ec 50 values 14-fold greater than a tk positive isolate. similarly, a tk negative strain of hsv-1 was 90-fold less sensitive to the drug than wild-type strains. thus, the antiviral activity of this molecule is dependent on the type i tk in hsv and the type ii tk expressed by vv and cv viruses, suggesting that it is a substrate for these divergent forms of the enzyme. the drug is also a good inhibitor of viral dna synthesis in both viruses and is consistent with inhibition of the viral dna polymerase once it is activated by the viral tk homologs. it is also possible that the phosphorylated forms of the drug may inhibit other enzymes such as thymidylate synthetase and inhibit viral dna synthesis indirectly. the interesting tk dependence of this molecule explains the rather unusual spectrum of activity that includes orthopoxviruses, alphaherpesviruses, epstein-barr virus (ebv), and human herpesvirus 8 (hhv-8), since these viruses all express molecules with tk activity that could phosphorylate and thus activate the drug. conversely, n-mct is ineffective against the betaherpesviruses because they do not encode tk homologs. the compound is also highly effective in reducing the mortality of mice infected with cv, vv, and hsv when treatment is initiated 24 h after infection and at doses as low as 17 mg/kg. these results indicate that (n)-mct is active in vitro and in vivo and its mechanism of action suggests that the molecule may be an effective and selective therapeutic for orthopoxvirus and certain herpesvirus infections and that it warrants further development. we have previously reported the isolation and characterization of drug-resistant mutants obtained following repeated passages of the vaccinia virus (vv, lederle strain) in the presence of increasing concentrations of cidofovir (cdv). cdvr mutants encoded two mutations (a314t and a684v) not related to genetic polymorphism. we have now introduced these mutations in the pathogenic strain w western reserve and characterized the drug-susceptibility profile of the recombinant viruses and their pathogenicity in mice. both the a314t and the a684v recombinant viruses proved to be resistant to cdv and related compounds, such as cyclic cdv and -propoxy]pyrimidine}. the virus bearing both substitutions proved to be more resistant to cdv than the single mutants. interestingly, the a314t and the a684v mutants differed in their sensitivity to phosphonoacetic acid (paa); the a314t and the a684v mutants being, respectively, hypersensitive and resistant to paa. in contrast, the double mutant showed no change in sensitivity to paa as compared to the wild-type strain. unlike the a684v mutant that showed only a two to three-fold decrease in susceptibility towards the 3-hydroxy-2-phosphonomethoxypropyl (hpmp) purine derivatives, the a314t mutant showed cross-resistance to the hpmp purine derivatives. it should be noted that in the process of selection of cdv-resistant mutants in the presence of increasing concentrations of the compound, the a314t mutation appeared before the a684v substitution, and the latter mutation only occurred in conjunction with a314t. when tested for virulence in a lethal intranasal infection model in mice, all cdvr recombinant viruses proved to be attenuated, suggesting that cdvr mutations are associated with reduced pathogenicity. furthermore, we found that cdv at a dose of 50 mg/kg/day for 5 days was still able to protect mice (in terms of body weight loss) against an intranasal challenge with the a314t + a684v recombinant virus. evaluating the use of cpg dna as an antiviral therapy amanda phelps, linda eastaugh, amanda gates, david ulaeto, arthur krieg 1 defence science and technology laboratories (dstl), salisbury, wiltshire, uk; 6 coley pharmaceuticals ltd., ottawa, ont., canada at present there are no licensed antivirals against orthopoxvirus infections such as variola or vaccinia virus (vacv). although a stockpile of smallpox vaccine exists and has utility as a post-exposure treatment to infection, it is a live viral vaccine and as such cannot be administered to those with contraindications. bacterial dna contains unmethylated motifs that, together with their flanking regions, can stimulate an innate immune response. synthetic cpg dna mimics the immunostimulatory activity of bacterial dna and is recognised by intracellular toll-like receptor 9. there are four classes of cpg dna all of which have different properties, eliciting distinct initial immune responses. previous studies using an established lethal respiratory model of poxvirus infection demonstrated that a class b cpg dna (7909) could provide protection from lethality against vacv in balb/c mice when administered up to 7 days prior to challenge. in order to evaluate efficacy balb/c mice were challenged intra-nasally with vacv and treated with doses of 7909 ranging between 15 and 100 ug/mouse. treatment was administered intra-nasally under light anaesthesia either on the day of challenge, 1, 2, 3, or 4 days post-challenge. efficacy was determined by percentage body weight loss post-challenge. the optimum survival rate observed was 60% when treated with 30 ug 1 day post-challenge (70 mld50 challenge). a survival rate of 80% was observed when treated with 50 ug 2 days post-challenge (40mld50 challenge). the delay of treatment to either 3 or 4 days post-challenge was ineffective, indicating that the window of opportunity for delivery of 7909 is within 2 days. multiple doses of 7909 were used to attempt to extend this window of opportunity, delivering 7909 twice within a 3-day period. interestingly, this had a considerable detrimental effect, actually accelerating the onset of disease and ultimately death. further work is required to optimise the use of cpg dna as a potential antiviral therapy, and there is evidence to suggest that they may have immense utility as part of a co-administration therapy with other antiviral compounds, an area of work currently under investigation. © crown copyright dstl 2005. department of virology, hebrew university, hadassah medical school, jerusalem, israel the pathogenicity and immunogenicity of the lister (elstree) strain of vaccinia virus, used for vaccination against smallpox, was studied in the mouse model. the virus did not reach the brain when inoculated intranasally, but when injected intracranially at a dose of 5 × 10 5 plaque forming units (pfu), was lethal for 50% of the mice. lower doses of virus caused the mice to initially loose some weight but they completely recovered thereafter. a significant level of protection against a lethal dose of the wr strain was achieved in mice following immunization with the lister strain, while higher doses and repeated vaccination procedure, were required with modified vaccinia virus ankara (mva). we found that the lister vaccine strain applied in israel is comprised of heterogeneous virus population. we isolated and plaque-purified three virus variants differing in their plaque size in bs-c-1 cell cultures. they were named: l-large plaque, m-medium plaque and s-small plaque variants. these isolates could be neutralized by rabbit antibodies prepared against the western reserve strain of vaccinia virus and their one-step growth curves in bs-c-1 cells were quite similar. however, they differ in their pathogenicity to mice following intranasal inoculation of 10 7 pfu, or an intracranial injection of 5 × 10 4 pfu; the s variant being more virulent than the other two variants and resembles the pathogenicity of the lister strain. activity was also determined against a thymidine kinase (tk) deficient vaccinia virus in mouse and monkey cells. the potency of (n)-mct was similar to that seen with wild-type virus, suggesting that a cellular enzyme may be more important than viral tk to phosphorylate the compound. mice were intranasally infected with cowpox and vaccinia viruses followed 24 h later by intraperitoneal treatment with (n)-mct (2x/day for 7 days) or cidofovir (1x/day for 2 days). (n)-mct treatment at 100 and 30 mg/kg/day resulted in 90 and 20% survival from cowpox virus infection, respectively, compared to 0% survival (placebo). statistically significant reductions in lung virus titers on day 5 occurred in 10, 30, and 100 mg/kg/day treated mice. these doses did not spare mice from lethal vaccinia virus challenge, however, but the 30 and 100 mg/kg/day treatments significantly reduced day 5 virus titers and lung weights, and the 100 mg/kg/day treatment reduced lung consolidation. cidofovir (100 mg/kg/day) protected all animals from death in both models. the evaluation of (n)-mct may be limited to mice based upon its greatly reduced efficacy in the cells of higher animals. acknowledgement: supported in part by contract no-1-ai-15435 from the virology branch, niaid, nih. chelsea byrd 1 , elena sbrana 2 , shu-yuan xiao 2 , marina siirin 2 , robert tesh 2 , dennis hruby 1 , robert jordan 1 1 siga technologies, inc., corvallis, or, usa; 2 university of texas medical branch, galveston, tx, usa st-246 is a potent small molecule inhibitor of orthopoxvirus replication that has been shown to protect mice from lethal challenge with vaccinia and ectromelia viruses. here we report the results of preliminary trials that show efficacy of st-246 against severe monkeypox virus infection in the ground squirrel model. ground squirrels infected with less than 1 pfu of monkeypox virus develop a fulminant disease resembling human hemorrhagic smallpox: the most severe and lethal form of the disease. oral administration of st-246 at 100 mg/kg once per day for 14 days protected ground squirrels from a lethal challenge with 100 and 1000 pfu of monkeypox virus. compound treated animals showed no weight loss or evidence of disease, and blood chemistry values were similar to uninfected animals. in contrast, placebo-treated animals showed elevated liver enzyme (alt and ast) levels and all animals died by day 12 post-infection. when treatment with 48, 72, and 96 h, 100% protection was observed in the 24, 48, and 72 h groups, and 66% protection in the 96 h group. severe pathologic changes were observed in the organs of the animals receiving placebo, especially in the lungs, liver, and spleen. in contrast, the organs of the animals receiving st-246 at 0, 24, 48, and 72 h postinfection appeared grossly and microscopically normal. thus, st-246 appears to be a promising candidate for continued development as a therapeutic agent for severe orthopoxvirus infection. inge vliegen 1 , guang yang 2 , dennis hruby 2 , erik de clercq 1 , robert jordan 2 , johan neyts 1 1 rega institute for medical research, k.u. leuven, belgium; 2 siga technologies, inc. corvallis, or, usa st-246 is a potent inhibitor of the replication of various orthopoxviruses. resistance of cowpoxvirus to st-246 maps to a mutation in v061, which is homologous to vaccinia virus f13l (yang et al., 2005. j. virol.) . the latter encodes the envelope protein p37 required for production of extracellular virus. deleting f13l resulted in a virus ( f13l-vac) that is replicationcompetent in cell culture but that produces smaller plaques than the wild-type wr-vac. whereas intravenous (i.v.) inoculation of nmri mice with 2 × 10 5 pfu of wr-vac resulted in 59 ± 11 pox tail lesions per mouse, the same inoculum of f13l-vac caused no lesions (p < 0.001). athymic nude (nu/nu) or scid mice inoculated iv with 2 × 10 5 pfu f13l-vac did not develop tail lesions. the mean day of death in nu/nu mice inoculated with f13l-vac was 22 ± 1 days as compared to 9 ± 1 days for wr-vac-infected mice (p < 0.001); scid mice survived the infection. we next studied whether f13l-vac is able to protect mice against a subsequent infection with wr-vac. to mimic the human vaccination protocol, nmri mice were infected intracutaneously (i.c.) by means of scarification at the lumbosacral area with 5 × 10 5 pfu f13l-vac or placebo. none of the infected mice developed lesions at the inoculation site. one week later, animals were infected ic with 5 × 10 5 pfu of wr-vac. all placebo animals, but none of the f13l-vac animals developed poxvirus lesions. in a second set of experiments, mice were again inoculated ic with placebo or f13l-vac and were infected one week later with 2 × 10 4 pfu of wr-vac by the iv route. placebo animals developed an average of 14 ± 4 pox tail lesions; no lesions developed in the f13l-vac animals (p < 0.001). in a third set of experiments, nmri mice were inoculated iv with either 2 × 10 4 pfu of f13l-vac or placebo, and none of the mice developed lesions. one week later, animals were inoculated iv with 2 × 10 4 pfu wr-vac. the placebo group developed an average of 11 ± 8 lesions as compared to 1.2 ± 0.7 lesions in the f13l-vac mice (p < 0.05). f13l-vac may thus be considered as a severely attenuated virus that may have potential for use as a smallpox vaccine. ji yuan 1 , travis lim 1 , shuan coughlin 1 , dexin qiu 1 , zhen liu 1 , dave stein 2 , decheng yang 1 1 the james hogg icapture centre for cardiovascular and pulmonary research, university of british columbia, vancouver, bc, canada; 2 avi biopharma, inc., corvallis, or, usa background: coxsackievirus b3 (cvb3) is the most common cause of viral myocarditis, but existing drug therapy is of limited value. antisense oligonucleotides (asons) designed to pair with viral rna could inhibit viral replication. however, the effectiveness of traditional asons is limited due to poor cellular uptake and degradation by nucleases. phosphorodiamidate morpholino oligomers (pmos) contain backbone modifications, which make pmos more resistant to nucleases. in addition, an arginine rich peptide (p007) is conjugated to the 5 end of the oligomer to improve its delivery into cells. these features make p007-conjugated pmos (p-pmos) promising candidates for the inhibition of cvb3 infection. methods: total 8 p-pmos targeting distinct regions of viral genome and one scrambled sequence were designed and chemically synthesized. fitc labeled p-pmos were used to observe their distribution of by confocal microscopy. viral protein vp1, viral titre, and cell viability were measured by western-blot, plaque assay, and mts assay, respectively. results: p-pmos showed increased cellular uptake compared to non-conjugated pmos. among the 8 p-pmos, p-pmo-6, targeting the internal ribosomal entry site in the 5 utr, showed the most potent anti-cvb3 ability in a dose-dependent manner. both infected hela and cardiomyocytes hl-1 cells treated with p-pmo-6 showed drastically reduced vp1 production and 3.0 log decreases in viral titres as compared to the controls. cell viability assay revealed that 83 and 89% of treated hela and hl-1 cells were still alive as compared to 11 and 10% of control-treated cells and 47% antiviral activity still existed after 5 days treatment. in addition, cells treated post-infection showed similar inhibition of viral replication. furthermore, the specificity of the p-pmos was demonstrated by their inability to inhibit rsv infection in hela cells. we have showed that p-pmos can effectively inhibit viral replication in vitro, providing a new possibility for antiviral intervention. picornaviruses are responsible for various human viral diseases including common cold, encephalitis, meningitis, myocarditis, etc. up to now, there is no specific antiviral therapy to treat or prevent such viral disease. the usage of modern computer technologies may help to solve this problem more effectively. the objective of the present study was the quantitative structure-activity relationship (qsar) analysis of antiviral activity of a set of [(biphenyloxy)propyl]isoxazole derivatives that inhibit cvb3 replication in hela cells. based on results from qsar, the structure of new potential antiviral agents should be predicted by using consequent molecular design. the qsar approach applied is based on simplex representation of molecular structure (sirms). the relationship between: (a) antiviral activity against the pleconaril-sensitive clinical cvb3 isolate 97-927 (ic 50 , g/ml); (b) cytotoxicity in hela cells (cc 50 , g/ml); and (c) selectivity index (si = ratio of cc 50 to ic 50 ), and structure of 21 [(biphenyloxy)propyl]isoxazole derivatives has been studied systematically. quite adequate qsar models (r 2 = 0.831-0.931, q 2 = 0.674-0.896) have been obtained using pls (partial least squares) method for all parameters studied. the models are in close correlation with experimental data. structural fragments with positive or negative influence on cytotoxicity as well as antiviral activity have been determined on the base of these models. for example, qsar analysis of antiviral activity of [(biphenyloxy)propyl]isoxazole derivatives revealed that the presence of m-nitrophenyl or p-trifluorophenyl fragment has distinctly negative influence on antiviral action. compounds with strong antiviral activity have to contain an oxadiazole fragment. moreover, our data allow the virtual screening and molecular design of new well-tolerated compounds with strong anti-cvb3 activity. ivanka nikolova 1 , roumena petkova 2 , boris atanassov 3 , stoyan chakarov 2 , angel s. galabov 1 1 institute of microbiology, bulgarian academy of sciences, sofia, bulgaria; 2 scientific technological service, ltd., sofia, bulgaria; 3 institute of organic chemistry, bulgarian academy of sciences, sofia, bulgaria analysis of the rna sequence of the disoxaril-resistant mutants of the coxsackievirus b1 was carried out. the wild-type disoxaril-sensitive strain (connecticut 5) and two disoxarilresistant mutants (one of them produced in fl cells and the other one isolated from brains of newborn mice) infected with coxsackievirus b1 and treated with disoxaril and a disoxarildependent mutant strain obtained from the resistant strain by 9 passages in cell culture were included in the present study. a rt-pcr assay with primer sets selected from a region of the coxsackievirus b1 genome coding for the capsid protein vp1 was carried out. a parallel comparative analysis of the sequences of resulting fragments from the disoxaril mutants studied and the genbank sequence of origin of the vp1 gene of coxsackievirus b1 was performed with the blast alignment tool. distinct alterations in the vp1 locus of the disoxaril-resistant and the disoxaril-dependent mutants compared to the sequence of origin from the genbank (namely, a deletion of uug at ntt. 2749-2751 and an insertion of uuu at nt.2769) were observed. high-degree similarity (97%) between the resistant mutant produced in cell cultures and the dependent strain was observed, while the similarity to the wild strain was only 91%. the resistant mutant obtained in mice was found to be very similar to the strain, developed in cell cultures. a putative 3-d model of the spatial folding of the target protein in disoxaril mutants is proposed. ralitsa vassileva-pencheva, angel s. galabov in previous study of ours we presented a new approach to combined application of antivirals-consecutive administration of the partners. this schedule could be considered especially suitable for treatment of enteroviral infections, in which the development of resistance is very rapid due to the extremely high viral mutation rate. this approach aims to restrict the resistance development in experiments in vivo, using antivirals with proved high efficiency in experiments in cell cultures. the screening of various double, triple, and quadruple combinations that we carried out showed that two of the triple combinations, namely disoxaril (win compound)/oxoglaucin (a new antiviral drug, developed in our laboratory)/ptu-23 (a classic enteroviral inhibitor) and disoxaril/oxoglaucin/guanidinehydrochloride (a classic enteroviral inhibitor) manifest significant effect of protection in newborn mice with neurotropic coxsackievirus b1 infection. in the current study the role of the chronology of arrangement of the antivirals included in the combinations was investigated. in the experiments carried out with the triple combination disoxaril/oxoglaucin/guanidine-hydrochloride, it was found that the optimal treatment course should start with disoxaril. the treatment course is quite successful when disoxaril is followed by guanidine-hydrochloride. the effect of the triple combination starting with oxoglaucine, followed by guanidine-hydrochloride was moderate. the course starting with guanidine-hydrochloride proved to be ineffective. furthermore, we studied the virus sensitivity to the inhibitorspartners (ic50 values) and some other phenotypic characteristics of the brain isolates, e.g. the size of the plaques and the pathogenicity for mice. recently our contribution to the development of new antipicornavirus agents has led to the discovery of 5methylthio-3-aryl-isoxazole-4-carbonitrile derivatives whose in vitro anti-coxsackievirus b1 activity were dependent on the nature of the substituents on the para position of the phenyl ring. particularly, compounds 5-methylthio-3-[4-(3-phenyl-1-propoxy)phenyl]isoxazol-4-carbonitrile (on-7) and 5-methylthio-3-[4-(4-phenoxy-1-buthoxy)phenyl]isoxazol-4-carbonitrile (on-10) exhibited an interesting antiviral activity with high selectivity indexes. in the present study, we investigated on the mechanism of action of these compounds. studies on time of addition experiments suggested that these compounds exert a different interference with an early step of the viral replicative cycle. in fact, compound on-7 was effective when added within 1 h after the end of the adsorption period and no reduction was observed if it was added during the adsorption period. whereas a reduction of virus titer was observed for on-10 when was added during the adsorption period, while no reduction was observed if the compound was added after this period (time 0). the influence of the compounds on virus adsorption step, studied by the infective center assays, indicated that on-10 primarily interferes with coxsackie b1 cellular attachment. at a concentration 100 times the id 50 , inhibition of adsorption of coxsackievirus by on-10 was complete, while similar concentration of on-7 had no effect. our experiments on neutralization of viral infectivity and on thermal stabilization demonstrated that the compounds were able to directly inactivate coxsackievirus, and the infectious titer was restored to the original value after extraction of the compound with chloroform. however, the compounds did not protect the viral infectivity against heat inactivation at the different concentrations used. the blood-brain barrier (bbb) fulfills a vital protective function by limiting entry of potential pathogens, toxins, and inflammatory cells into the central nervous system (cns). disruption of the bbb is a common component of many cns diseases, including viral diseases such as that caused by west nile virus (wnv). transforming growth factor-␤1 (tgf-␤1) has previously been shown to improve the function of an in vitro model of the bbb. we evaluated the role of the bbb in wnv infection in mice by determining the ability of intraperitoneally (i.p.) administered sodium fluorescein to move from the circulating blood to the central nervous system. to demonstrate bbb permeability a mean and normal range of permeability values was determined in 30 non-infected c57/bl6 mice. in subsequent experiments, any animal expressing a permeability value greater than 2 sd above the mean was considered abnormally high. we determined that elevations in bbb permeability can be detected in mice 8 days after subcutaneous inoculation with wnv. wnv inoculated animals were treated with doses of 3000, 1000, or 300 ng/kg/day of tgf-␤1 or with drug vehicle once daily via the i.p. route on 7 and 8 days post-virus inoculation (dpi), and then assayed for bbb permeability on 8 dpi. sixty-two percent (8/13) of placebo-treated animals had abnormally high permeability values, while animals treated with 3000 and 1000 ng/kg/day of tgf-␤1 had 29% (2/7) and 57% (4/7) of animals with abnormally high permeability values, respectively. in contrast, none of the animals treated with 300 ng/kg of tgf-␤1 (0/8) expressed abnormally high permeability values, which was significantly lower (p < 0.01) than placebo-treated animals. these results suggest that tgf-␤1 may improve the function of the blood-brain barrier in wnv infected mice. acknowledgement: supported by grant 1-u54 ai06357-01 from the rocky mountain regional centers of excellence, nih. and contract no1-ai-15435 from the virology branch, niaid, nih. people infected with west nile virus (wnv) usually see their physicians after showing symptoms suggestive of neurological infection. wnv infects the central nervous system (cns) of rodents 3-5 days after s.c. viral challenge. yet, most published animal studies begin therapeutic treatments before or soon after viral challenge. the question addressed in this study is if neuroprotective agents can be efficacious when administered early before brain infection or later after the virus is demonstrated to be in the brain. the drugs evaluated in wnvinfected rodents were nmda and ampa receptor antagonists, modulators of nitric oxide synthase (nos) and nitric oxide production, and riluzole for reducing glutamate excitotoxicity. serial doses of diethyldithiocarbamate (ddtc) and n(g)monomethyl-l-arginine (l-nmma), an inducer or inhibitor of nos, respectively, administered i.p. daily for 10 days beginning 4 h before viral challenge slightly improved survival of mice, but the difference was not statistically significant. tolerated doses of two nmda-receptor antagonists, flupertine (7 mg/kg) and mk-801 (1 mg/kg), and one ampa-receptor antagonist, gyki 52466 (10 mg/kg), were administered twice daily (b.i.d.) on 4 though 9 days post-virus inoculation (dpi). gyki 52466 slightly improved mouse survival and weight gain, but the difference was not statistically significant. talampanel, an ampa-receptor antagonist and a derivative of gyki 52466, slightly improved hamster survival (p ≤ 0.05) when treatment began on 5 dpi, but repeated experiments using different doses and slightly different protocols gave mixed results. riluzole, the only drug shown to improved survival of amyotrophic lateral sclerosis (als), presumably by reducing glutamate excitotoxicity, was not effective against wnv disease when administered b.i.d. beginning 5 dpi. overall, neuroprotective agents did not consistently improve wnv disease, although slight improvements in animal survival might be relevant to improvement of neurological sequelae in wnv-patients. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih. hamster and mouse models for west nile virus (wnv) disease were used in this study to identify infected cells of the central nervous system (cns) early in the course of infection. this information may be relevant to therapeutic strategies since most wnv-infected people visit their physicians after showing symptoms suggestive of neurological infection. we subcutaneously infected adult mice and hamsters using 105.3 tissue culture infectious doses of wnv. tissues of infected and control animals from 3 to 11 days post-viral injection (dpi) were fixed by cardiac perfusion using 4% paraformaldehyde. we localized wnv, neuronal and astroglial markers in the paraffin embedded tissue sections by immunofluorescence. the images were captured using the confocal microscope (bio-rad, mrc 1020). we observed the presence of wnv antigen in cns tissues of mice and hamsters as early as 3 and 5 dpi, respectively. a strong wnv-specific immunofluorescence staining was observed in the cytoplasm of neurons from the spinal cord, cerebellum, cerebral cortex, and midbrain of these rodents. the wnv-specific staining co-localized with neuron-specific markers; however, astroglial markers were not co-localized with wnv antigen in brain sections. the lack of tropism by wnv for astrocytes was also confirmed in primary murine astrocyte cultures. interestingly, infected neurons in the midbrain of 7-day infected hamsters co-localized with calbindin, which is a calcium-binding protein and mostly expressed in the interneurons of the cns. therapies were evaluated in hamsters or mice at a time-point when wnv-stained neurons were identified in the cns. acknowledgement: supported by grant 1-u54 ai06357-01 from the rocky mountain regional centers of excellence, nih. laboratory of virology, department of biological chemistry, school of sciences, university of buenos aires, argentina dengue virus (denv) is an arthropod-borne flavivirus that has re-emerged in recent years as an increasingly important public health threat with nearly 50 million infections occurring each year. at present neither specific antiviral therapy nor vaccine exists for the treatment and prevention of denv infections. carrageenans are sulfated galactans that can be extracted from red seaweeds and comprise diverse structures with a wide range of biological properties useful in biomedicine. in a previous study we have demonstrated the antiviral activity of commercialand -carrageenans against denv type 2 and 3 in vero (monkey kidney cells) and hepg2 (human hepatoma cells), showing inhibitory concentration 50% (ic 50 ) values in the range 0.14-1.1 g/ml and selectivity indexes (cc 50 /ic 50 ) in the range 1000-10000. in the present work we studied the mode of action of these polysulfates against denv-2 in vero and hepg2 cells, first analyzing the influence of time of addition of compounds on anti-denv activity by an infectious centre assay. the highest inhibitory effect was observed when the compounds were added during adsorption or at 1 h p.i., being ineffective at later times. then, the effect of compounds on virus adsorption and internalization was studied separately by a virus yield inhibition assay. significant antiviral efficacy was attained if compounds were present either only during denv-2 adsorption or internalization. the possible effect of carrageenans on viral protein synthesis, the subsequent stage of the virus cycle occurring during the first hour of infection, was analyzed by pulse-labeling with ( 35 s)-methionine. no alterations in denv protein synthesis in carrageenan-treated cells were observed. when cells were transfected with purified denv-2 rna in the presence of -carrageenan no inhibition in fluorescent cell focus formation and virus production was detected. besides, no significant direct virucidal effect on denv-2 was shown by the compounds. these results indicate that both denv adsorption and internalization seem to be the main target for these compounds, lacking effect on the steps that occur once the viral genome is inside the cell during in vitro infection of human and monkey cells. multiple members of the flavivirus genus of the family flaviviridae cause lethal hemorrhagic fever or encephalitis. the public health significance of the hemorrhagic fever and encephalitis caused by such flaviviruses is enormous and global and there is a tremendous need for antivirals. imino sugar glucosidase inhibitors have been shown to have selective antiviral activity against viruses such as bovine viral diarrhea virus (bvdv) and west nile virus (wnv) that have common requirements for their glycoprotein processing during virus production. we are developing imino sugar deoxynojirycin (dnj) derivatives through chemical synthesis of compounds with various alkyl side chains and antiviral testing against bvdv and wnv as well as in wnv subgenomic replicon assays. briefly, using a single step growth (virus yield reduction) assay for bvdv and wnv, a series of dnj derivatives containing various conformational locking side chains were shown to have antiviral activity. pre-liminary structure-activity relationships (sar) were obtained for further modification of the alkyl side chain and improvement of these dnj derivatives. the activity and mechanisms of action of these compounds will be presented. several flaviviruses cause life-threatening diseases in man. currently, there is no therapy available for these infections. in recent years, several highly selective inhibitors of the replication of hepatitis c virus (hcv) were designed. most small molecule inhibitors of hcv that are in preclinical or clinical development are either protease or polymerase inhibitors. most of these compounds are highly selective for hcv and are unlikely to exhibit activity against flaviviruses. nucleoside polymerase inhibitors, however, may have the potential to inhibit the replication of flaviviruses as well. we evaluated in vitro whether the active component of the anti-hcv compound valopicitabine, i.e. 2 -c-methylcytidine inhibits the replication of flaviviruses in cell culture. the compound was found to exhibit specific antiviral activity against yellow fever virus 17d (ec 50 = 3.1 g/ml in cpe reduction assays and >98% reduction at 5 g/ml as assessed by qpcr) and dengue fever virus type 2 (ec 50 = 16.5 g/ml in cpe reduction assays). the compound also efficiently inhibited west nile virus replication (>98% at 5 g/ml as assessed by qpcr and >98% by plaque reduction neutralization test at 50 g/ml). in the absence of any drugs for the treatment of flavivirus infections, it may be envisaged that nucleoside polymerase inhibitors, when marketed for the treatment of hcv infections, could be used off-label for the treatment of lifethreatening flavivirus infections. even if such drug would not be able to completely inhibit flavivirus replication, a partial reduction of the viremia during the acute phase of the infection may be sufficient to prevent the development of a fulminate disease and thus protect against virus-induced mortality. yuri klimochkin 1 , andrew shiryaev 1 , igor moiseev 1 , v sabynin 2 , larisa rustamova 2 , alexandr petkevich 2 1 state technical university, samara, russia; 2 institute of epidemiology and microbiology, minsk, belaruss arenaviruses are one of the most dangerous tools of bioterrorism in the view of pathogenicity and epidemiological threat. for the purpose of searching new remedies for treatment are-naviruses infections the synthesis of new derivatives of cage compounds has been carried out. the prepared compounds are bridgehead derivatives of cage compounds bearing different functional groups such as hydroxy, acylamino, alkoxycarbonylamino, alkylthiocarbonylamino groups as well as iminoalkyl adamantane derivatives, some adamantylated heterocycles and compounds containing two adamantane moieties in a molecule. the antiviral activity of the cage compounds has been studied in respect to arenaviruses lassa (sierra-leone strain) and pichinde (an-3739 strain) on the vero cells culture. different level of antiviral activity was shown by 15 compounds. the most active compounds are monosubstituted adamantane derivatives having sufur and nitrogen-containing substituent in the bridgehead position. the data on the activity confirm the availability of searching inhibitors of arenaviruses reproduction in the cage compounds series. brian gowen 1 , donald smee 1 , min-hui wong 1 , anne pace 1 , kie-hoon jung 1 , kevin bailey 1 , lawrence blatt 2 , robert sidwell 1 1 institute for antiviral research, utah state university, logan, ut, usa; 2 intermune, brisbane, ca, usa several arenaviruses endemic to the south american (junin, machupo, and guanarito) and african (lassa) continents are known to cause frequently fatal hemorrhagic fever. with the exception of ribavirin, which has demonstrated efficacy in cases of lassa fever, there are no other effective therapeutics for the treatment of arenaviral hemorrhagic fever. the outcome of treatment is ultimately dependent upon early diagnosis and the tolerability of ribavirin by patients at the high doses required for effective antiviral activity. we have recently demonstrated that consensus interferon-alpha (ifn-alfacon 1) can protect hamsters from lethal pichinde virus (pcv) infection (gowen et al., 2005 . antimicrob. agents chemother.), which serves as a model for acute arenaviral disease in humans. here we demonstrate highly effective therapy through the combined use of ribavirin and ifn alfacon-1 for the treatment of pcv infection in hamsters. ribavirin was given orally, twice per day for 7 days, and ifn alfacon-1 was administered intraperitoneally, once per day for 10 days. treatments were initiated 1-5 days post-infection with various dose combinations, many which were less than optimal when the drugs were given independently. combining suboptimal doses of ribavirin (5-10 mg/kg/day) with ifn alfacon-1 (5-10 mg/kg/day), we were able to show increased protection from mortality, reduced viral burden and liver disease, and greatly extended survival times as compared to treatments where drugs were administered alone. our data indicate that synergistic activity resulted from combination therapy and that this activity may slow down the progression of disease and decrease fatality rates seen with severe arenaviral infections. further, combination therapy reduces the effective dosage of ribavirin, which would serve to limit its toxicity. acknowledgement: supported by contract no1-ai-15435 from the virology branch, national institute of allergy and infectious diseases, national institutes of health. slobodan paessler 1 , laure deflubé 1 , andrew vaillant 2 , jean-marc juteau 2 , ramon flick 1 1 department of pathology, university of texas medical branch, galveston, texas, usa; 2 replicor inc., laval, que., canada rift valley fever virus (rvfv; genus phlebovirus, family bunyaviridae) is an arbovirus transmitted by many species of mosquitoes. this virus is a major public health concern in sub-saharan africa and egypt, which spread to yemen and saudi arabia. in this area, rvfv is responsible for dramatic epidemics/epizootics underlining the need for efficient antiviral/prophylactic measures. rep 9 is a 40 mer phosphorothioate oligonucleotide, which has previously been shown to have broad-spectrum activity in several viruses (vaillant et al., submitted for publication) . we used a vaccine strain (mp12) as well as the wild-type rvfv (zh501), to test the ability of rep 9 to inhibit bunyavirus proliferation. in vitro data showed reduction of virus titer for both strains using rep 9 at nanomolar concentrations. moreover, the absence of the phosphorothioate modification in a stabilized rep 9 analog resulted in a loss of antiviral activity, suggesting that as in other viruses, the increased hydrophobicity of rep 9 is essential for its antiviral activity. based on the inhibitory activity observed in vitro, we started with in vivo efficacy studies by utilizing a validated mouse model used in our laboratory. more animal experiments are ongoing to confirm the in vitro results and to evaluate the antiviral effect of the rep 9. adriana garozzo 1 , rossella timpanaro 1 , aldo stivala 1 , gianna tempera 1 , christian c.c. cutrì 2 , angelo castro 1 1 department of microbiological sciences, university of catania, via androne 8, 95124 catania, italy; 2 department of pharmaceutical sciences, university of catania, viale a. doria 6, 95125 catania, italy our previous studies described the synthesis and the antiviral activity of 3,4,5-trisubstituted isothiazole derivatives that were found to be particularly effective against picornaviruses. compound 3-methylthio-5-phenyl-4-isothiazolecarbonitrile (is-2) exhibited an interesting anti-poliovirus activity with high selectivity index. in the present study, we investigated on the mechanism of action of this compound. studies on the time of is-2 addition to poliovirus type 1 infected cells suggested that the compound may inhibit some early processes of viral replication. in order to determine its mechanism of action, we evaluated the rate of attachment and internalization of purified [ 3 h]uridine-labeled poliovirus to hep-2 cells in the presence or absence of is-2. no effect on poliovirus adsorption and internalization to host cells was detected. we also investigated the influence of the compound on virus uncoating using labeled poliovirus and measuring the radioactivity of oligoribonucleotides formed from viral rna susceptible to ribonuclease. these experiments demonstrated that poliovirus uncoating is influenced by is-2 action. justin julander 1 , aaron olsen 1 , john morrey 1 , lawrence blatt 2 , kristiina shafer 1 , robert sidwell 1 1 institute for antiviral research, utah state university, logan, ut, usa; 2 intermune, brisbane, ca, usa alpha togaviruses are medically important arboviruses, with clinical cases occurring each year in north, south, and central america. the recent increase in the threat of the use of these viruses as bio-terrorism agents has led to increased efforts to develop therapeutic agents for treatment of these viruses. venezuelan (vee) and western equine encephalitis (wee) viruses have been listed as category b priority pathogens by the national institute of allergy and infectious disease (niaid). the goal of these studies was to characterize animal models for vee and wee for use in evaluation of antiviral therapies. c3h/hen mice were infected through the intranasal (i.n.) route with a vaccine strain of vee, tc-83. virus was detected in the brain 2 days post-viral injection (dpi). brain titers increased to a peak titer of 10 9.5 50% cell culture infectious doses per gram tissue (ccid 50 /g) on 4 dpi, maintained a titer of 10 9 ccid 50 /g through 7 dpi, and dropped slightly to 10 8.6 ccid 50 /g by 8 dpi. virus was also detected in spleen, liver, and kidney. treatment of vee-infected mice with interferon alpha b/d, a human consensus interferon, resulted in 100% survival, whereas all placebotreated animals died by 9 dpi. syrian golden hamsters were infected with 10 3 ccid 50 wee through intraperitoneal (i.p.) injection. morbidity, including hind limb paralysis, tremors, nasal bleeding, and hunching, and some mortality were seen as soon as 4 dpi. the majority of deaths occurred on 5 dpi. virus was detected in all organs assayed (brain, liver, and spleen) with peak titers occurring 4 dpi. interferon alfacon 1 (ifn alfacon), a human consensus interferon, active in hamsters, was effective in significantly reducing mortality (p < 0.001 as compared to placebo). there was a trend for reduction of brain titers in ifn alfacon-treated animals (mean titer 10 4.8 ccid 50 /g) as compared with placebo (mean titer 10 9.5 ccid 50 /g), although this difference was not statistically significant. these models will be useful in screening potential antiviral agents for efficacy against vee and wee. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih. justin julander 1 , kristiina shafer 1 , john morrey 1 , lawrence blatt 2 , robert sidwell 1 1 institute for antiviral research, utah state university, logan, ut, usa; 2 intermune, brisbane, ca, usa yellow fever virus (yfv) has caused significant morbidity and mortality for centuries. primates were the only animal models for visceral yfv. recently, hamsters were found to have morbidity and mortality when injected with a hamster-adapted jimenez strain of yfv (tesh et al., j. infect. dis. 183, 1431 -1436 . the objective of this study was to characterize this model of yfv viscerotropic disease for the study of effects of antiviral compounds and to test compounds with known efficacy for use as a positive control. animals were challenged with a 10 −4 dilution (a dilution previously shown to cause high mortality) of a liver homogenate made from livers taken 3 days post-viral injection (dpi) from hamsters challenged with the jimenez strain. there was 66% mortality in animals challenged with the virus up to 9 dpi. virus titers in the liver peaked 6 dpi as determined by qrt-pcr. a significant increase in serum levels of alt (6 dpi), alkaline phosphotase (6 dpi) and bilirubin (6 dpi), and a significant decrease in amylase (8 dpi), albumin (6 dpi), and glucose (6 dpi) were observed. hepatic icterus was observed in hamsters that exhibited disease signs at the time of necropsy. hamsters were treated with ribavirin or interferon (ifn) alfacon 1, a consensus interferon. ribavirin and ifn alfacon 1 both significantly (p < 0.001) reduced mortality as compared with placebo-treatment. there was also significant reduction in weight loss with ribavirin (p < 0.05) and ifn alfacon 1 (p < 0.01) treatment as compared with placebo. disease signs, such as lethargy and lying prostrate, were also reduced with treatment of ribavirin and ifn alfacon 1. viral liver titers from treated animals were not significantly different from titers in placebotreated animals. the hamster model of yfv disease will serve as a suitable model for the evaluation of antiviral compounds for efficacy against the virus. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih. polyomaviruses are small dna tumor viruses that depend on the host cellular dna polymerase for their replication. three polyomaviruses have been associated with tumor formation in humans: jc virus (jcv), bk virus (bkv) and simian vacuolating virus (sv40). in addition, some of them have been associated with viral diseases. jcv can cause progressive multifocal leukoencephalopathy in immunosuppressed patients, while bkv is considered to be the causative agent of polyomavirusassociated nephropathy, which leads to kidney transplant failure. sv40 has not been associated with a well-defined disease, but viral dna sequences and protein expression have been detected mostly in central nervous system (cns) tumors which strengthens the evidence for the association of this virus with human cancer. the activity of various acyclic nucleoside phosphonates (anps) such as cidofovir and adefovir against murine polyomavirus and primate sv40 in vitro has already been demonstrated (andrei et al., 1998. antimicrob. agents chemother. 41, 587-593) . here, the activity of a new class of anp's, namely 6-[2-(phosphonomethoxy)alkoxy]-2,4diaminopyrimidines, against polyomaviruses was assessed. confluent uc1-b cells were infected with either of the four murine polyomavirus strains mn/rde toronto, pta, 2pta2 or lid-1, while bsc-1 cells were infected with either the primate sv40 strain a2895, the sv40 pml-1 strain ek or the sv40 pml-2 strain dar. after removal of the residual virus, serial dilutions of the test compounds were added. the viral cytopathic effect was recorded microscopically after 4-6 days (murine polyoma virus) or 5-7 days (sv40). hpmpo-dapy (2,4-diamino-6-(r)-[3-hydroxy-2-(phosphonomethoxy)propoxy]pyrimidine) and pmeo-dapy (2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]pyrimidine) were less active/selective than cidofovir and adefovir against the three sv40 strains tested. hpmpo-dapy and pmeo-dapy proved to be equally active as cidofovir and adefovir against the murine polyomaviruses. naresh sunkara 1 , sylvester mosley 1 , brian bakke 1 , joshua sadler 1 , katherine seley(radtke) 1 , sunny zhou 2 1 university of maryland-baltimore county, baltimore, md, usa; 2 washington state university, pullman, wa, usa inhibition of biologically significant enzymes critical to nucleotide metabolism and viral replication is a well-established chemotherapeutic approach to the treatment of many diseases. transcriptional 5 -capping of viral mrna has been implicated as an "elongation checkpoint" critical to the replication cycle of many viruses. this capping process is accomplished by various methyltransferases, therefore disruption of methylation becomes an attractive target for therapy. this can be accomplished in several ways; in particular, by direct inhibition of methyltransferases (metase) and/or indirect inhibition of s-adenosyl-l-homocysteine hydrolase (sahase), both established cellular targets for antiviral, antiparasitic and anticancer agents. modified nucleosides, in particular carbocyclic nucleosides, have exhibited potent inhibitory activity against both sahase and metase. inspection of the recent literature has revealed a close correlation between sahase inhibition and potent biological activity against negative stranded (−)-rna viruses (i.e. arenaviridae, paramyxoviridae, rhabdoviridae), double stranded (ą)-rna viruses (reoviridae), poxviridae, as well as hiv-1, thus supporting the importance of sahase as a viable chemotherapeutic target. herein we report the design, synthesis, and preliminary biological activity of a new class of structurally novel carbocyclic nucleosides. phosphorylation of ␣-p-borano substituted nucleoside diphosphates charlotta wennefors, mikhail dobrikov, barbara ramsay shaw chemistry department, duke university, durham, nc 27708-0346, usa most nucleoside antiviral agents require stepwise phosphorylation to their respective triphosphates in order to be activated in the cell. ␣-p-borano substituted nucleoside triphosphates are of interest because they have proven to be good substrates for hiv-1 reverse transcriptase (rt) and may therefore be useful antiviral agents. studies in our laboratory have indicated that the ␣-p-borano substitution of 2 3 -dideoxycytidine triphosphate (ddctp) resulted in a 28-fold increase in efficiency of incorporation by mmlv rt compared to non-substituted ddctp. however, the potency of these ␣-p-borano substituted nucleoside analogs as anti-viral drugs highly depends on their ability to be activated to nucleoside triphosphate (ntp). the phosphorylation of nucleoside analog diphosphates to their respective triphosphates has remained largely unexplored. here, the roles of several phosphorylating enzymes are examined. in our laboratory, nucleoside diphosphate kinase, creatine kinase, and pyruvate kinase are being evaluated for their specificity towards nucleoside analog diphosphates. the effects of nucleobase, ribose, ␣-phosphate substitution and stereochemistry of the boranophosphate group are of interest. the binding affinities of the substrates for creatine kinase (ck) and pyruvate kinase (pk) were determined using a fluorescence-quenching assay, which allowed us to investigate the substrate affinity in the pre-steady state. rabbit muscle ck and pk were titrated with a wide range of ndps and ntps by monitoring a decrease in enzyme intrinsic fluorescence. the affinities of these substrates were determined to establish a structure-activity relationship for ck and pk and to evaluate the effect of a substrate ␣-p-borano modification. ck showed stereospecificity towards the sp isomer of adp␣b whereas pk showed stereospecificity towards the rp isomer of adp␣b. negative cooperativity was observed for all studied substrates. steady-state experiments are also being performed directly following the product formation using uv-visible spectroscopy and high performance liquid chromatography (hplc). these kinases were investigated because they may serve as a means for activation of antiviral ␣-p-borano substituted ndps. traditional antiviral targets encoded by the small human papillomavirus (hpv) genome are lacking. for this reason, we chose to target dna sequences within the hpv genome in an effort to identify compounds that would block viral dna replication in cells. we chose compounds known as polyamides, which are related to distamycin and other natural products, as our dna binding agents. unlike many literature studies where polyamides were designed to block formation of the transcription complex for a particular gene, we chose to target sequences within the origin of replication (ori). thus, pyrrole-imidazole polyamides, with some containing fluorescent probes to aid in cell localization studies, were designed to recognize the hpv31 ori. the principles used to design these compounds will be described. we used "traditional" hairpin polyamides and some more unusual structures related to very recent literature reports. from the focused library that we prepared, two highly active molecules were identified. the rest of the molecules had minimal or zero activity. no cellular toxicity was observed, either in this project or in a related program where polyamides were used to affect cox-2 transcription (and subsequent expression) in rheumatoid synovial fibroblasts. of particular interest is the difference between the active molecules and two closely related compounds that were inactive: the active species bind and recognize two more hpv dna base pairs than do the related but inactive structures. this presentation will provide detailed chemistry background and structural information to complement our cell work that is also being presented at the meeting. discovery of the chemokine receptor ccr5 as a co-receptor for hiv-1 infections revealed a novel approach to hiv-1 treatments and preventions. ccr5, a member from the family of 7tm g-protein coupled receptors, thus became an attractive target pursued in the pharmaceutical industry. with the recent successful developments of several small molecules in clinic, these ccr5 antagonists hold great promise to be the next generation of anti-hiv medicines. this poster will describe our efforts at the n-terminal piperidine ring of template a to improve pharmacological properties of derived molecules. according to current models, proteolytic processing of hiv-1 gag precursor occurs within the virions which detach from infected cells. meanwhile, the viral protease is activated much earlier, and gag p55 cleavage initiates in infected cells. we followed the fate of matrix protein cleaved in infected cells (cma) in comparison with ma cleaved in the virions (vma) and showed that both forms differ in their localization in the infected cells and in the virions, both forms are involved into virus pathogenesis and represent the targets for antiviral compounds. mt-4 cells were labeled with [3h]-leucine or myristic acid, and 2 h after labeling protease inhibitor was added to separate the cleavage of cma from vma. cma was found in the nuclear and membrane fractions of infected cells while cca resided in cytoplasm. 18-20 h after labeling cma was found in the virions localizing in the cores. vma was located under lipoprotein envelope of the virions. new membranotropic antiviral compounds based on adamantane-and norbornene-related derivatives not toxic for the host cells were added to mt-4 cells before infection or 1-2 h later and at concentration 2-6 ug/ml blocked reverse transcription, the transport of cma into the nuclei, and the production of infectious virus. the compounds inhibiting very early step of virus life cycle are optimal candidates for microbicides. to enhance their antiviral activity, we plan to associate polyanionic matrix with ma imitating peptides and cholesterol-like fragments. kurt vermeire 1 , thomas bell 2 , sreenivasa anugu 2 , noah duffy 2 , roger le grand 3 , erik de clercq 1 , dominique schols 1 1 rega institute for medical research, katholieke universiteit leuven, leuven, belgium; 2 department of chemistry, university of nevada, reno, usa; 3 service de neurovirologie, fontenay-aux-roses, france the cyclotriazadisulfonamide (cada) compounds were shown to be potent inhibitors of hiv replication in human t-cell lines, pha-stimulated pbmcs, and monocytes/macrophages (ec 50 : 0.3-3.2 m). the prototype compound, cada, had consistent activity against laboratory adapted and primary clinical isolates of hiv-1, irrespective of chemokine receptor preference (r5, x4, r5/x4). cada acted synergistically when evaluated in combination with various other hiv drugs, such as reverse transcriptase (rt), protease, and virus entry inhibitors. flow cytometric analysis revealed a significant decrease in the cell surface and intracellular expression of the cd4 receptor in human cells after cada-treatment. moreover, the anti-hiv activity of cada correlated with its ability to down-modulate the cd4 receptor in human t-cells. here, we report the consistent antiviral activity of cada against: (i) drug-resistant viruses (i.e. viruses resistant to rt inhibitors, protease inhibitors, and enfuvirtide); (ii) different hiv-1 subtypes (a, b, c, d, a/e, f, h, o); and (iii) various hiv-2 strains examined. in addition, cada potently inhibited sivmac251 infection of pbmcs isolated from macaques (ec 50 : 1.6 m). comparable results were obtained in human cells infected with sivmac251. flow cytometric analysis also demonstrated a significant and dose-dependent down-regulation of the cd4 receptor expression at the cell surface of simian pbmcs after treatment with cada. the combination of cada with cellulose acetate 1,2benzenedicarboxylate (cap), an enteric coating polymer for capsules and tablets, resulted in a synergistic antiviral activity. in summary, our data indicate that cada may qualify as a potential anti-hiv microbicide drug candidate for the prevention of the sexual transmission of hiv. the preparation of gel formulations of cada (as single drug and in combination with cap) for vaginal administration in non-human primates is currently under investigation. department of micro & immuno, suny upstate medical university, syracuse, ny, usa varicella zoster virus (vzv, human herpesvirus 3) infection causes chicken pox, latency is established in neurons, and reactivation leads to shingles. acyclovir and its derivatives are the treatment of choice for both manifestations of vzv. new therapeutics are needed because acyclovir-resistant strains exist, and treatment must begin within 48 h. we have studied the anti-vzv properties of roscovitine, a cyclin dependent kinase (cdk) inhibitor. here, we tested more compounds that block the cell cycle and determined that vzv is acutely sensitive to them. their effects on vzv replication were tested in human foreskin fibroblasts (hffs) because these primary cultures should have a normal cell cycle (unlike tumor cell lines). the cytotoxicity of the drugs was determined by neutral red dye uptake assays. hffs were inoculated with a low moi (0.01) of vzvinfected cells, which remains entirely cell-associated, and then treated with drugs or diluent for 48 h. vzv spread and replication were measured by infectious focus assay and quantitative real time pcr. all of the drugs tested (acyclovir [acv], phosphonoacetic acid [paa], aphidicolin, aloisine a, purvalanol a, roscovitine, r-roscovitine, s-roscovitine, indole-3-carbanol [i3c], l-mimosine, dichloro-␤-d-ribofurano-sylbenzimidazole [drb]) had some anti-vzv activity. the selective indices of aphidicolin (833), purvalanol a (570), and i3c (1000) were greater than the positive controls acv (250) and paa (60). aphidicolin inhibits mammalian dna polymerase and is in clinical use for cancer; purvalanol a, a 2,6,9-trisubstituted purine, primarily inhibits cdk1; and i3c is derived from cruciferous vegetables and inhibits cell proliferation. the concentrations of these compounds that inhibited vzv replication were much less than those needed to cause cell cycle arrest, suggesting that vzv depends on the enzyme activities targeted by these compounds and not on cell proliferation per se. these three drugs will be studied next in skin organ culture and in the scid-hu mouse model of vzv pathogenesis. the results presented here demonstrate that targeting cell functions can inhibit vzv replication and help us better understand virus-host interactions. the viruses could be identified as supra-biopolymeric nanoscale complexes, parasitic intervention in cells of which occurs on an inter-polymeric reactions level. so the antiviral safety can not be fully provided without adequate nano-responsible antivirals (nav). here we discuss a strategy and methodology for the multi-functional nav development by rational macromolecular sar-cooperation of: (1) polyelectrolyte-specific interferon induction and immunomodulation; (2) electrostatic-selective prevention of viruses absorption on plasma membranes; (3) membrane-targeted blocking of post-absorption steps (fusion); (4) macromolecular prevention of structure-specific interactions of viral and cellular receptors; as well as (5) polymericassociated disruption of the latest stage of viral replication (virions assembly and maturation). a cooperation of the (1) and (2) functions was explored by synthesis and sar-optimization of succinate and carbohydrate polymeric derivatives modified with controllable combinations of anionic (a1/a2) groups. the immune-mediated protectors against tick-born, rabies, and other viral infections in vivo, and hiv-1 absorption inhibitors in vitro, were developed. this pre-nav generation was used as a macromolecular platform to step-by-step targeted design and synthesis toward high effective multi-functional nav where virusresponsible nano-selectivity was achieved by rational intra-or inter-molecular cooperation of virus-specific membranotropic vectors (bi), raft-targeted anchors (ci), and peptide-kind mimickers of virus usable receptors of human cells (pi), particularly ccr5/cxcr4. as a result, the novel nano-sensitive systems possessed unique wide multi-synergistic antiviral potency on a high level selectivity up to si = 20,000 (against hiv-1 strains) were created and purposed for advancement of antiviral vaccines, drugs, and microbicides. marina kukhanova 1 , alexander ivanov 1,2 , georgii galegov 3 , valeria andronova 3 , maxim jasko 1 1 engelhardt institute of molecular biology, russian academy of sciences, moscow, russia; 2 centre for medical studies, university of oslo, moscow, russia; 3 ivanovsky institute of virology, russian academy of medical sciences, moscow, russia novel acyclic purine phosphonate derivatives bearing a double bond conjugated with the nucleic base, namely, (z)-and (e)-9-[3-(phosphonomethoxy)prop-1-en-1-yl]purines, were synthesized, and their efficacies against hiv-1 and hsv-1 were evaluated in cell cultures. the activity of (z)isomer was higher against hiv than that of the reference 9-[2-(phosphonomethoxy)ethyl]adenine (adefovir) and comparable in respect to the activity of adefovir against hsv. the (e)-isomer showed low antiviral activity against both viruses. the compounds were less toxic towards cell cultures if compared to adefovir. the diphosphates (z)-and (e)-9-[3-(phosphonomethoxy)prop-1-en-1-yl]purines were evaluated as substrates towards hiv-1 reverse transcriptase and hsv dna polymerase. (z)-isomer was shown to be a more efficient substrate for both enzymes than the (e)-isomer. human dna polymerase alpha could incorporate neither of the diphosphates into the 3 -end of the growing dna chain. available to this virus. jev genome is an approximately 11-kb single-stranded positive-sense rna that has a cap structure at its 5 terminus but lacks a poly(a) tail at its 3 -terminus. the coding region of the genome is flanked by 5 -and 3 -untranslated region (utr). the 3 -utrs on both plus-and minus-strand jev genome contain important cis-acting elements required for the replication of the viral rna genome. peptide nucleic acid (pna) is a synthetic oligonucleotide, in which the phosphodiester backbone of dna/rna is replaced with a polyamine-(2-aminoethyl) glycine skeleton. pna offers a potentially powerful approach for recognition of rna and silencing of gene expression. in this study, we investigated the antiviral effect of the pnas targeted to the 3 -utr region of jev genome. to evaluate the pnamediated inhibitory effect on rna synthesis in vitro, the rnadependent rna polymerase (rdrp) of jev, ns5 protein, which plays a major role in replication of the viral genomic rna, was expressed in escherichia coli and purified to near homogeneity by sequential column chromatographies. the recombinant jev ns5 protein exhibited a primer-dependent rdrp activity in vitro on a homopolymeric template, poly(a). in addition, it was able to accept both plus-and minus-strand 3 -utrs as templates for rna synthesis in the absence of an exogenous primer. it could utilize the 3 -end 83-nt of jev genome as a minimal template. in vitro rdrp assays using this functional recombinant jev rdrp in the presence of the pnas targeted to the jev 3 -utr 83-nt showed a dose-dependent rna synthesis inhibition. delivery of the inhibitory pnas to the jev-infected cells suppressed jev replication, as determined by western-blot analyses and plaque assays. our results showed a sequence specific inhibition of jev replication by antisense pnas, suggesting the possible application of pna as a novel anti-jev agent. julia serkedjieva 1 , reneta toshkova 2 , milena nikolova 3 , reneta tsvetkova 3 , stefka antonova 4 , ivana roeva 1 , munnever sokmen 5 , bektas tepe 5 , medine gulluce 6 , fikrettin sahin 6 , atalay sokmen 5 1 institute of microbiology; 2 institute of experimental pathology and parasitology; 3 institute of botany, bulgarian academy of sciences; 4 faculty of biology, department of microbiology, sofia university, sofia, bulgaria; 5 faculty of art and science, department of biology, cumhuriyet university, sivas, turkey; 6 faculty of art and science, department of biology, atatürk university, erzurum, turkey natural products can be an important source of new pharmaceuticals. research on antivirals of natural origin is mainly focused on plants, since, among other reasons, they can be selected on the basis of their ethnobotanical use. plant extracts and natural plant products exhibit also a variety of biological activities with pharmacophoric utility. the population of the balkan peninsula, like people from all continents, has long applied poultices and imbibed infusions of hundreds of indigenous plants. the present report summarizes the antiviral screening study of 134 plant products, obtained from 67 bulgarian and turkish medicinal plants. they were tested for inhibitory effect on the reproduction of selected influenza virus (flu) strains in mdck cells and herpes simplex virus (hsv) strains in mdbk cells. the reduction of virus-induced cpe and infectious virus yields were used as measures of viral inhibition. fifteen samples (11.2%) inhibited flu reproduction, and twelve samples (7.5%) were active against hsv. the anti-flu activity was confirmed in vivo for all tested samples. the most effective products were tested further for their antiproteolytic, antioxidant and immunogenic capacities and for potential antibacterial and antifungal effects. the following correlations among the variety of biological and pharmacological activities of the plant products were observed: the anti-flu effect was associated with anti-hsv effect and vice-versa in 55.5%; the antiviral effect was connected with antioxidant activity in 100%; the anti-flu effect was associated with immunogenic properties in 100%; there was found no correlation between the antiviral effect and the antiproteolytic capacity, the anti-viral properties and bacterial or fungal inhibition, the anti-viral activity and the polyphenol contents. our previous investigations have revealed antiviral activity of some proteolysis inhibitors such as e-aminocaproic acid (e-aca) and para-aminomethylbenzoic acid (pamba) in vitro, in vivo and in clinic. construction of qsar computer-assisted hierarchical system for the effective anti-herpetic (anti-hsv) and anti-influenza (anti-flu) agents' selection as well as the elaboration of new methods of their synthesis are permanently the object of keen interest of our team. the objective of this study was to investigate the efficacy 2,6-di-substituted pyridines and their analogs combined with the fragments of proteolysis inhibitors in the framework of the qsar approach. molecules of new compounds consisted of "nucleus" (py or ar) and two symmetrical fragments: e-aca-carbonyl or pambacarbonyl taken from the inhibitors' molecules. anti-flu activity in dose 10-3 m was studied in vitro on the model of a/hong kong/1/68 (h3n2) reproduction in tissue cultures of chorioallantoic membranes of 12 days old chick embryos. anti-hsv activity in doses 10-4 m was studied on models of reproduction of hsv-1 on cell culture hep-2. compounds with py-nucleus, contained pamba-carbonyl or e-aca-carbonyl fragments, demonstrated sufficient anti-hsv activity (39.4 and 61% of reduction of intra-nucleus virus-specific inclusions on infected cells account accordingly). 3,5-dihydrazine-carbonyl-2,6-dimethylpyridine showed high anti-hsv (52%) activity. the efficacy of the designed antiherpetic compounds obtained with the combined efforts of qsar computer-assisted design, properties prediction, synthesis, and biological testing as well as the correction introduced after the iteration circle passsage have proven to be the efficient modern way of drug design. acknowledgement: this research was supported in part by stcu grant # 3147 and all the authors are indebted to stcu foundation courtesy. lubomira nikolaeva-glomb 1 , angelina trifonova 1 , stephan filipov 2 , angel s. galabov 1* 1 institute of microbiology, bulgarian academy of sciences, sofia, bulgaria; 2 institute of organic chemistry, bulgarian academy of sciences, sofia, bulgaria a series of aporphinoid alkaloids isolated from glaucinum flavum l. or obtained synthetically, were tested in vitro for antiviral activity against viruses belonging to picorna-, orthomyxo-, paramyxo-and herpesviruses. one of them, oxoglaucine, manifested a well-pronounced inhibitory effect on poliovirus 1 replication in fl cells measured by the semi-quantitative agardiffusion plaque-inhibition test. in virucidal activity testing the compound did not show direct virucidal effect on the extracellular virus. oxoglaucine's 50% inhibitory concentration for poliovirus 1 (mahoney) was found to be 0.188 g/ml in the cpeinhibition test and 0.041 g/ml in the classical plaque-inhibition test. similar values were obtained for the vaccinal poliovirus type 1 strain, lsc-2ab. the antiviral effect of oxoglaucine on the replication of viruses belonging to another enterovirus species was tested, i.e. coxsackie and echoviruses (hev-b). cva-9, the six coxsackie b viruses and 6 echoviruses were tested for their sensitivity against the antiviral effect of oxoglaucine by the endpoint dilution method in the multi-cycle cpe inhibition set-up in fl cells. oxoglaucine revealed a marked inhibitory effect on all tested enteroviruses. the concentrations that reduced virus titer by 1 lg ranged from 0.01 to 1.0 g/ml. selectivity index was greater than 100 and even greater than 1000 for some of the viruses tested. time-of-addition study by the one-step virus growth cycle set-up showed strong virus inhibition during the early periods of virus replication. milka mileva 1 , angel s. galabov 2 1 department of medical physics and biophysics, medical university, sofia, bulgaria; 2 institute of microbiology, bulgarian academy of sciences, sofia, bulgaria in this study an investigation and comparison of the effects of plant flavonoid polyphenols quercetin and its sugar-containing homologue (rutinoside) rutin on the "oxidative stress" in liver, isolated from influenza virus a/aichi/2/68 (h3n2) (2.0 of ld50) inoculated mice, is carried out. it was found that experimental influenza virus infection is accompanied with graduated oxidative disturbances in the liver of mice, despite the absence of virus and inflammation in this tissue. it was found that experimental influenza virus infection is accompanied with a significant increase of lipid peroxidation products, a decrease of natural antioxidants (vitamin e, glutathione) and cyp, an inhibition of cytochrome c-reductase and liver monooxygenases (analgin-ndemethylase and amidopyrine-n-demethylase) as compared to control (non-infected) animals. the preliminary (5 days) supplementation of mice with rutin, quercetin or its combination, and their subsequent virus inoculation influence significantly all analyzed parameters as compared to controls. the protective effect of rutin against influenza virus-induced lipid peroxidation and activities of cyp and liver monooxygenases in liver was better expressed than the effect of quercetin may be due to containing of rutinoside part or difference of its metabolism. hyun-jeong lee 1 , ji-sun kwon 1 , chi-ung moon 2 , jong-hwan kwak 3 , youn-jeong lee 4 , chang-seon song 1 1 avian disease laboratory, college of veterinary medicine, konkuk university, seoul, korea; 2 hanyang university, seoul, korea; 3 sungkyunkwan university, seoul, korea; 4 national veterinary research and quarantine services, seoul, korea one of the traditional korean medical herb extract named s22 was investigated to determine the anti-influenza virus activity in vitro and in vivo. the s22 showed potent antiviral activities against a/pr/8/34 (h1n1) influenza virus with the 50% effective concentration (ec50) values of 62.5 g/ml and the 50% cytotoxic concentration (cc50) values of 673.86 g/ml in mdck cells. treatment with the s22 appeared capable of significantly ameliorating the influenza virus infection in mice by oral gavage treatment. the s22 treated mice showed significantly higher survival rate and lower pathogenic index as well as lung virus titers than untreated control mice. further, the s22 was extracted with chcl3, etoac and n-buoh for isolation of active compounds. the anti-influenza effects of these active compounds will be discussed. the antiviral effect of aqeous and ethanol extracts of ocimum gratissimum (og), terminalia catappa (tc), gynostemma pentaphyllum (gp), newbouldia laevis (nl), aspilia africana (aa) and phyllantus amarus (pa) leaves was examined by cultivation of virus and extracts in embryonated chicken eggs. extracts were inoculated immediately after virus (zero time) or 2 h after virus inoculation. virus replication was compared with those of controls by haemagglutination assay. at zero time, aqeous extracts of og, tc, pa, and gp inhibited virus growth by 50, 61, 72, and 94%, respectively whereas those of nl and aa did not. ethanol extracts of og, tc, pa and gp at same time inhibited by 25, 100, 72, and 100%, respectively whereas nl and aa did not. at two h after virus inoculation aqeous extracts of og, tc, pa and gp inhibited virus growth by 92, 6, 67, and 100%, respectively whereas nl and aa had no effect. ethanol extracts of tc, pa and gp inhibited the virus by 94, 78, and 94%, respectively whereas those of og, nl, and aa did not. the herbs were studied because they were being used by some herbalists in the treatment of human infectious diseases. the 20th international conference on antiviral research will be held in the palm springs, california area. the conference will begin on sunday, april 29, 2007 and will end on thursday afternoon, may 3, 2007. all scientific sessions will be held at the westin mission hills resort, rancho mirage, ca. the purpose of the international conference on antiviral research is to provide an interdisciplinary forum at which investigators involved in basic, applied, and clinical research worldwide can meet to review recent developments in all areas of antiviral research. specific topics to be covered in the program include synthesis and chemistry, biochemistry and mechanism of action, molecular biology and drug targeting, in vitro evaluation, animal models, pharmacokinetics, toxicology, and clinical trials. within these areas of interest, there will be invited overview speakers, oral presentations, and poster presentations. the famous el paseo shopping district of palm desert and downtown palm springs offer not only a large variety of galleries, boutiques and shops too numerous to mention, but there are restaurants for virtually every palate. whether your tastes run to burgers, sushi, pizza, escargot, steak, mexican or continental you will find it here with a california flourish in every price range. we hope you will take advantage of this opportunity to combine an important learning experience with a magnificent travel experience and join us in palm springs, california for the 20 th international conference on antiviral research. isar conference committee future conferences acknowledgement: the study was supported by rfbs 05-04-49500 and russian ministry of sciences (lot 11). key: cord-004948-ad3i9wgj authors: nan title: 7th international congress on amino acids and proteins : vienna, austria, august 6–10, 2001 date: 2001 journal: amino acids doi: 10.1007/s007260170030 sha: doc_id: 4948 cord_uid: ad3i9wgj nan the raised concentration of protein bound homocysteine in homocystinuric (hcu) patients displaces protein bound cysteine and increases the free/bound cysteine ratio in plasma. this ratio is independent of albumin concentration. results from 31 hcu patients were compared to 40 controls. free cystine concentrations in hcu were poorly discriminated from the control range but the total cysteine results were almost invariably lower than control data. this appears to result from an increased free/bound cysteine ratio in hcu [mean (range) for control 0.50 (0.22-0.71) and for hcu 0.76 (0.32-1.75); p ϭ 0.0005]. ex vivo protein binding experiments in albumin solution revealed the free/bound cysteine ratio to be linearly related to the amount of homocysteine bound (r ϭ 0.907, p ͻ 0.001). we conclude that measurement of total cysteine is essential for assessment of the true cysteine status in hcu. however, any cysteine deficit, or alteration to free/bound cysteine ratios, does not obviously effect glutathione synthesis as assessed by measurement of plasma total glutathione. (339 nmol/g; 21.6%); sprague-dawley rat (rattus norvegicus, rodentia) (n ϭ 6; 98-381 nmol/g; 9.8-13.7%); rabbit (152 nmol/ g; 11.3%); pig (sus scrofa f. domestica, artiodactyla) (221 nmol/ g; 13.4%); bovine (bos primigenius f. taurus, artiodactyla) (284 nmol/g; 23.6%); seal (phoca vitulina, carnivora) (136 nmol/g; 3.8%), and rob (halichoerus grypus, carnivora) (218 nmol/g; 5.4%). from the date it is concluded that d-aas are common in body fluids and certain tissues of vertebrates. in order to determine the quantity of cyst(e)ine and methionine, the oxidation of cyst(e)ine and methionine (ϫ) refers to not detected or not determinable; asx ϭ asp ϩ asn; glx ϭ glu ϩ gln; his, arg, trp, cys not determined; a) feed fortified with dl-met; b) nmol/g lyophilized serum. mentation and subsequent purification. the separation of the enantiomers of cysteic acid, methionine sulphone, aspartic acid and glutamic acid is displayed on the chromatogram. into cysteic acid and methionine sulphone with performic acid is often applied before hydrolysis of protein. the authors examined the applicability of this process in case of quantification of cyst(e)ine and methionine enantiomers. the rp-hplc analytical method was developed for the determination of the amount of cysteic acid and methionine sulphone enantiomers. the rate of conversion during oxidation from cyst(e)ine into cystic acid and from methionine into methionine sulphone was determined. the racemisation of l-cyst(e)ine and l-methionine was negligible during oxidation with performic acid, therefore this process can be applied before hydrolysis during quantification of cyst(e)ine and methionine enantiomers. after the performic acid oxidation and the 6 m hcl hydrolysis of the protein, opa/tatg (o-phthaldialdehyde/tetra-o-acetyl-1-thio--d-glucopiranoside) precolumn derivatisation method was used, and the enantiomers of sulphur containing amino acids were separated by rp-hplc (lichrosphere 100 rp-18e, 125 ϫ 4 mm, 5 µm column, merck-hitachi lachrom hplc). the resolution of the peak of cysteic acid and methionine sulphone enantiomers was better than 1,5. the method was used to determine the amount of l-and d-cyst(e)ine and land d-methionine containing preparations prepared by ferd/l rate of aspartic acid and the individual age of specimens. a method for age determination based on d-aspartic acid content and on the racemisation of l-aspartic acid of teeth was developed. d-glutamic acid, beside d-aspartic acid, was found to be eminently suitable for the estimation of individual age, as it showed a sufficiently high sensitivity. calibration curves based on these investigations were used for the age estimation of 65 adults (39 males and 26 females) of unknown individual age from the avar period series of kereki-homokbánya (hungary). the age distribution of the sample was the following: 39 individuals (60%) belonged to the adult age group, 22 persons (34%) to the mature and 4 (6%) to the senile one. the correlation between our results and those obtained using standard paleoanthropological methods was over 0.9. quantitative determination of free and bound 3-nitrotyrosine in rat plasma and tissues using isotope dilution liquid chromatography-electrospray tandem mass spectrometry t. delatour, p. a. guy, j. richoz, j. vuichoud, and nestlé research centre, nestec ltd, vers-chez-les-blanc, lausanne, switzerland since 3-nitrotyrosine was reported to be readily formed in proteins by reactions with nitrite or nitrogen dioxide, it has been postulated to be a possible marker for investigating peroxynitrite-mediated nitration of proteins. thus, several methods were developed to assess nitration of tyrosine in proteins and determine 3-nitrotyrosine in physiological fluids. methods based on hplc or gc/ms techniques were described to quantify 3-nitrotyrosine within tissues or biological fluids. unfortunately, it has been demonstrated that an artifactual nitration of tyrosine occurs with gc/ms assays leading to an overestimation of the response. in the present work, lc-esi-ms/ms methods for quantification of free 3-nitrotyrosine in rat plasma as well as bound 3-nitrotyrosine in tissue samples are reported. plasma samples were spiked with 2,5,6-d 3 -3-nitrotyrosine and the following steps were applied prior to injection into the lc-esi-ms/ms system used in selected reaction monitoring (srm) mode (m/z 283 ae 181 for the analyte and m/z 286 ae 184 for the internal standard): protein precipitation, solid phase extraction on aminopropyl cartridge and derivatization in nbutanol in hcl 3 n. 3-nitrotyrosine butyl ester has lead to a dramatic increase of the sensitivity (ca. 5-fold) by comparison with 3-nitrotyrosine. under such conditions, calibration curves exhibited excellent linearity (r 2 ͼ 0.99) within concentration range 0.3 to 28.5 nm (equivalent to 47.3-4,730 fmol on column) and recoveries above 85%. inter-and intra-assay precision was determined below 15% over the concentration range 1.4 to 28.5 nm. no artifactual nitration of tyrosine occurring during sample clean-up was observed. this was unambiguously established by plotting experimental ratio of analyte response/ internal standard response versus expected within the range 0.3-28.5 nm. this curve strongly correlated with a linear model (r 2 ͼ 0.99) and slope was 1.07 ϯ 0.06 (mean ϯ sd). basal level of 3-nitrotyrosine in rat plasma was measured to be within concentration range ͻ lod to 1.5 nm. 3-nitrotyrosine basal level in rat plasma, kidney and liver proteins was established by performing enzymatic hydrolysis in order to avoid artifactual nitration of tyrosine which may occur under strong acidic conditions (hcl 6 n at 120°c). resulting hydrolysates were analysed by lc-esi-ms/ms and 3nitrotyrosine was monitored in srm mode (m/z 227 ae 181 for the analyte and m/z 230 ae 184 for the internal standard). t. guszczynski 1 , r. b. kapust 2 , d. s. waugh 2 , and t. d. copeland 1 1 basic research laboratory, and 2 macromolecular crystallography laboratory, national cancer institute at frederick, maryland, u.s.a. the set-can fusion gene was first detected as associated with acute undifferentiated leukemia. set (also called phap ii) is a nuclear phosphoprotein with a long acidic tail. set has been shown to inhibit phosphatase pp2a and is a substrate of human granzyme a. in order to determine any zn(ii) binding properties of set, we utilized affinity capillary electrophoresis (ace) to detect shifts in mobility as zn(ii) ions bind to the protein. we have earlier employed ace to measure the binding constants of zn(ii) to the nucleocapsid protein of hiv-1. with a constant concentration of recombinant set as a receptor and varying concentrations of zn(ii) as ligand in the sample buffer, we observed changes in electrophoretic mobilities of set when complexes were formed with zn(ii). scatchard analysis of the mobility provided the stoichiometry and binding constant of zn(ii) to set. interdisciplinary research center, institute of nutritional science, department of food sciences, university of giessen, germany peptaibols are defined as fungal polypeptides containing a high proportion of aib (α-aminoisobutyric acid) and a cterminal bound amino acohol. the mold trichoderma aureoviride (strain imi 91968; commonwealth mycological institute, kew, uk) was cultured in complex medium consisting of casein peptone, 17 g; soy peptone, 3 g; yeast extract, 5 g; dglucose, 2.5 g; nacl, 5 g; dipotassium hydrogen phosphate, 2.5 g in 1 l demineralized water adjusted to ph 6.8. fermentation was conducted in nineteen 2-l shake flasks, each containing 400 ml medium, for 7 d at 27°c. mycelia were obtained by filtration and extracted with meoh and meoh/chloroform. extracts were evaporated to dryness and subjected to sephadex and silica gel chromatography (eluent chloroform/meoh/acoh/water 65 : 25 : 3 : 4) yielding 2.83 g and 0.9 g, respectively, crude peptaibol mixture named trichoaureocins. the peptide mixture was uniform on tlc but could be separated by analytical (fig. 1 ) and semipreparative hplc (nucleosil 100 c-18; 250 ϫ 8 mm id; 3 µm). six peptides could be isolated each of which was subjected to sequencing using on-line hplc (fluorocarbon stationary phase) esi-ms/ ms (lcq, thermoquest, finnigan mat) as described for peptaibols trichovirins and antiamoebins. sequences are presented in fig. 2 . the 20-residue peptaibols represent a natural peptide library and cause hemolysis of sheep erythrocytes and exert antibiotic activity against bacillus subtilis and staphylococcus aureus. national institute of chemistry, ljubljana, slovenia wine consists of several hundred components present at different concentrations. the dominant ones are water, ethanol, glycerol, sugars, organic acids, and various ions, while amino acids are present at much lower concentration. the composition of amino acids is of great importance in wine production. they act as a source of nitrogen for yeast during fermentation, they influence the aromatic composition of wine and their composition can be used to differentiate wines according to vine variety, geographical origin, and year of production. among already established analytical methods high-field nmr has been shown to be a promising method for the nondestructive analysis of low-molecular mass compounds in complex mixtures like wine due to its selectivity and capability of simultaneously detecting a great number of compounds. 1 h and 13 c one-dimensional nmr spectra of wine are very crowded and many signals are overlapped. due to a great difference in concentration levels the signal intensities of particular compounds may vary for the factor of 25. the tails of the dominant frequencies of water, ethanol and glycerol obscure weak signals of minor compounds like amino acids in the near surroundings. the use of 2d homo-and heteronuclear experiments and the suppression of strong signals are a prerequisite for a successful 1 h and 13 c signal assignment. a complete assignment of 1 h and 13 c nmr resonances of seventeen amino acids commonly present in wine and of γ-aminobutyric acid at ph 3 was accomplished using gradient-selected cosy, tocsy, gradientselected hsqc and hmqc experiments with incorporated wet pulse sequence for the supression of large signals. unambiguous assignment of 1 h and 13 c nmr resonances of amino acids is necessary for the selection of appropriate signals in fast and simple one-dimensional nmr that can serve as parameters in the chemometric classification of wines according to the provenance, vine variety, and year of production. institute of medical biochemistry, jagiellonian university collegium medicum, kraków, poland highly sensitive colorimetric method for determination of aldehydes in the reaction with n-methyl benzothiazolone hydrazone (mbth) turned out to be not very specific for such carbonyl compounds. namely, it has been found that tryptophan and to higher degree its n-derivatives (n-acetyl-trp, ala-trp, gly-trp) and also tripeptides (gly-trp-gly and leu-trp-leu) in the reaction with mbth and fe 3ϩ are converted to coloured products, with maximum wavelength at 595 nm. the properties of the products and the kinetics of the reaction under defined conditions are described in the spectrophotometric procedure. proteins containing tryptophan are also substrates in the reaction with mbth. comparison of molar extinction coefficients of mbth-fe 3ϩ -treated various proteins with those of simple n-derivatives of tryptophan shows, that not all molecules of tryptophan in proteins are accessible to the reagents, and in order to determine all tryptophan moieties partial unfolding of protein has to be performed. it should be emphasized that aldehydes cannot be detected and accurately determined in the presence of tryptophan derivatives and protein, and also aldehydes interfere with determination of tryptophan derivatives. natural product laboratory, department of chemistry, the university of burdwan, w. bengal, india detection of protein amino is of utmost importance for the evaluation of protein structure and also their presence in numerous natural products. several specific and non-specific reagents have been used for their detection using thin-layer chromatography, an important tool for such purpose. of the reagents in general use, ninhydrin is the most popular for its high sensibility, however, nihydrin produces same purple color with most of the amino acids (only proline and hydroxproline produce yellow color). an endeavour has been made to resolve this color problem with a reagent which is capable of developing various distinguishable colors with many of the protein amino acids and also shows its high sensitivity comparable to ninhydrin. a probable mechanism for such color formation has also been proposed. measuring enrichments below the sensitivity range of conventional gc-ms. the gc-c-irms technique combines the resolution capabilities of gc with the accuracy and precision of irms. at low abundance gc-c-irms analysis it is superior in terms of time, labor, and sample requirement as compared to the conventional off-line analysis. we discuss some latest advancements and applications of gc-c-irms amino acid analysis related to nutrition research. plasma amino acids in omnivorous human subjects show a characteristic 15 n-isotopic pattern with phenylalanine and threonine showing the lowest abundance, whereas e.g. alanine and leucine are higher by 25‰ δ 15 n. in rats fed diets containing intrinsically labeled 13 c casein or the corresponding amino acid mixture labeled with 13 c leucine and 15 n lysine whole-body protein homeostasis is better supported by casein-bound than free amino acids. there is no adaptation to a low lysine diet by an enhanced bioavailability of intestinal microbial lysine to extra-splanchnic tissues in minipigs. highly selective hplc determination of tyrosine, tryptophan and their related compounds based on precolumn derivatization followed by intramolecular fluorescence resonance energy transfer detection h. nohta 1 , m. yoshitake 1 , h. yoshida 1 , t. yoshitake 2 , and m. yamaguchi 1 1 faculty of pharmaceutical sciences, fukuoka university, nanakuma, johnan-ku, fukuoka, and 2 chemical evaluation and research institute, ishii machi, hita, oita, japan we have developed highly selective hplc method for the determination of tyrosine, tryptophan and their related compounds (l-dopa, catecholamines, 5-hydroxytryptamine, etc.). the compounds were precolumn-derivatized with a commercially available fluorogenic reagent for amines by usual manner. each derivative afforded intramolecular fluorescence resonance energy transfer (fret) from the tyrosyl or tryptophoryl moiety (donor) to the labeled fluorophore (acceptor); the acceptor fluorescence was observed with the excitation of the donor at 280 nm. the derivatives were separated on a reversedphase column and then effectively detected by monitoring their fret. through the screening study of 11 fluorogenic reagents, o-phthalaldehyde (with 2-mercaptoethanol) and dansyl chloride gave the best results for the purpose. the fret detection method was highly selective and sensitive by comparison with the previous methods detecting native fluorescence of the compounds or typical fluorescence of the acceptor. the presented study was devoted to determination of the energetic effect of interactions in aqueous solutions between urea and neutral amino acid derivatives. the principal reason for studying of interactions of peptides with urea is the hope that such investigations will give insight into the factors affecting protein denaturation in aqueous solutions. the enthalpies of solution of n-acetylglycinamide, n-acetyl-l-alaninamide and n-acetyl-l-leucinamide were measured in water and in aqueous solutions of urea of molality 0.25 to 3.0 mol·kg ϫ1 using the "isoperibol" type calorimeter at 298.15 k. from the obtained standard dissolution enthalpies ∆ sol h ϱ m the enthalpic pair interaction coefficients h xy for urea-nacetylamino acid amide pairs in water were calculated. these parameters derived from mcmillan-mayer theory are regarded as a measure of effect of interactions between solute molecules in solution. the h xy values for the systems investigated suggest that the interactions between urea and amide molecules dominate the effects of dehydration of nonelectrolyte and of peptides. the replacement of the hydrogen atom in the hydrocarbon chain with a methyl group causes a positive change in the value of the enthalpic pair interaction coefficient. the obtained results were compared with those of earlier studies of interactions between electrolytes, namely sodium chloride, potassium chloride and sodium iodide and the same n-acetylamino acid amides. the effect of the solute type on the magnitude of the interaction parameter was also analysed. the side chains of amino acids in solution react in various ways with the water molecules which surround them as well as with other components of solution depending on the fact whether they possess non-polar, polar or ionic groups. many research laboratories carry out studies intended to describe precisely the intermolecular interactions with the participation of amino acid side chains. such a description may allow one to describe better the spatial structures of protein and the mechanisms of folding its surface area. the present work reports the results of calorimetric measurements of the dilution enthalpies of l-α-amino acids in water. using modified mcmillan-mayer's theory, these results served to calculate the enthalpic homogeneous interaction coefficients which characterise interactions between the amino acid zwitterions with the competitive participation of water molecules. thus, these coefficients illustrate the differences in amino acid molecules interactions both with the homogeneous amino acid molecules and water molecules around them, and consequently they may play the part of a parameter which differentiates the hydrophobic/hydrophilic properties of amino acid side chains. the enthalpic interaction coefficients of the homogeneous pairs of l-α-amino acids were compared also with the hydrophobicity parameters obtained by fauchere et al., which describe the side substituents of natural amino acids as well as aminobutiric acid (aba). based on the above statement, one may conclude that the obtained enthalpic homogeneous pair interaction coefficients of l-α-amino acids in water make it possible to systematise amino acid side chains according to their affinity to water or their hydrophobic-hydrophilic properties. thus the enthalpic homogeneous pair interaction coefficients may play the role of parameter describing the lipophilicity (hydrophobicity) of amino acid side chains. compounds (iii) with amino acid ligands. in this work we present results of x-ray investigation of fourth amino acid complexes of rhenium (iii), which have different coordination of amino acids around binuclear complexforming center -re 6ϩ . substances (glyh) 4 . 2h 2 o -in inner, but gaba has cisposition according to re -re bond. influences of fatty radical length in the amino acid ligand on week interaction between binuclear anion [re 2 cl 8 ] 2ϫ and protonized amino acid are discussed. role of hydrogen bonds in formation of crystal unit cell of investigated substances is shown. these two factors are the reason of formation of staggered conformation of an anion [re 2 cl 8 ] 2ϫ in the substance (glyh) 4 [re 2 cl 8 ]cl 2 together with existence of quadruple re -re bond that is described first. in the substance [re 2 (gaba) 2 cl 5 (h 2 o)]cl . 2h 2 o axial position of re 2 6ϩ fragment are substituted by ligands of different kind: h 2 o and cl ϫ -that says about possibilities to coordinate a substrate of biological nature exactly to these position. a precise, sensitive and reliable rp-hplc/uv method was developed to enable determination of α, and k caseins in cow's milk. the optimised method using a chrompack p-300-rp column allowed separation of caseins in 30 min. this column differs from conventional alkyl-bonded silica rp matrices in that it is an underivatised polystyrene-divinylbenzene matrix, a material which proved excellent chemical and ph stability. gradient elution was carried out at a flow rate of 1 ml/min and a temperature of 46°c, using a mixture of two solvents. solvent a 0.1% trifluoroacetic acid in water and solvent b was 95% acetonitrile-5% water-0.1% trifluoroacetic acid. the effluent was monitored by a uv detector at 280 nm. the determinations were performed in the linear range of 0.038-0.38 mg/ml for k-casein, 0.19-1.9 mg/ml for α-casein and 0.15-1.5 mg/ml for -casein. the detection limits were 0.037, 0.03 and 0.0075 mg/ml, respectively. the validity of the method was verified. the recoveries ranged from 91 to 100% for cow's milk. the precision of the method was also evaluated, the % cv being less then 3.67%. the developed methodology was also applied with success to the separation of caseins in ewe and goat milks. different chromatographic profiles were obtained for the three kinds of milk. department of aquatic biosciences, the university of tokyo, bunkyo-ku, tokyo, japan several aquatic crustaceans and bivalve molluscs accumulate a large amount of free d-alanine (3-50 µmol/g wet wt.) in their muscle tissues. during seawater acclimation from freshwater to 75% seawater, red swamp crayfish procambarus clarkii largely accumulated d-and l-alanine by 6.8-and 4.5fold, respectively, together with l-glutamine, l-proline, and glycine. the percentage of d-alanine to total alanine increased from 38% in freshwater to 48% in 75% seawater. these data indicate that d-and l-alanine are the major compatible osmolytes responsible for the intracellular isosmotic regulation of this species as well as other crustaceans. under anoxia stress for 12 h in freshwater, 50 and 75% seawater, crayfish increased d-and l-alanine in muscle and hepatopancreas in addition to the increase of lactate. the increase was much higher in seawater than in freshwater. thus, d-and l-alanine may be anaerobic end products during prolonged anoxia of this species. alanine racemase [ec 5.1.1.1] has been proved to catalyze the interconversion of d-and l-alanine in crustaceans and bivalve molluscs. this enzyme was isolated to homogeneity from the muscle of black tiger prawn penaeus monodon. the purification was 127,600-fold with 16% yield. the molecular weight of the enzyme was estimated to be 45 kda on sds-page and 90 kda on gel filtration, suggesting the dimeric nature of this enzyme. the amino acid sequences of the peptide fragments obtained from the isolated enzyme showed low homology below 50% with those of microbial enzymes. syntheses and immunological effect of thymic humoral factor-γ2 analogues research laboratory, global shinwa pharmaceutical co. ltd., yoriki, matsuomura, iwate-gun, iwate-ken, japan nine analogues of thymic humoral factor (thf)-γ2, were prepared by the solid-phase method and their in vitro restoring effect on the impaired blastogenic response of phytohemagglutinin (pha)-stimulated t-lymphocytes of uremic patients with infectious diseases were examined. the results were as follows: [arg6]-thf-γ2 exhibited higher restoring activity than that of our synthetic thf-γ2. phylos has developed a powerful combinatorial biology platform for peptide and protein selections. phylos' proprietary profusion tm technology enables the selection of peptides and proteins with desired properties. the fundamental advance represented in this unique platform is the in vitro covalent linkage of a peptide or protein (phenotype) to the encoding messenger rna (genotype). this linkage permits the selection of a protein based on its characteristics and allows the recovery and amplification of that protein through pcr, an efficient means of bring the desired proteins to easily detectable levels. profusion tm technology has routinely selected peptide and protein binders with affinity constants in the nanomolar to picomolar range. the starting library size of randomized peptide or protein profusion tm constructs is typically 10 13 . linear and constrained loop peptide libraries, for ligand generation, enzyme: substrate interaction, peptidomimetic design, and epitope mapping have been successfully used. randomized constrained loops have also been incorporated in a betasandwich scaffold, resulting in the successful selection of binders against targets of therapeutic interest. antigenic properties of three biological active de novo proteins were investigated by peptide scanning approach, using noncleavable multipin technology. a de novo protein albebetin (pid caa47376) was engineered to attain a pre-designed 3d structure and later modified by grafting short peptide fragments from human α 2 interferon (aag59605), and insulin molecules (aag59606). such protein constructs carrying important biological activities may be used in future as potential protein pharmaceuticals. despite artificial proteins are investigated for more than 10 years, immunological properties of these substances are not known. in our experiments we applied an innovative approach of raising antibodies in yolks of egg-laying hens. three continuous antigenic determinants with different immunogenic potentials have been revealed in two proteins with partially overlapping sequences. it was shown that the octapeptide interferon fragment is the immunodominant site in albeferon and albeferon-insulin molecules. on the contrary, the hexapeptides, corresponding to the insulin fragment displayed low immunogenic activity. thus we recognise that the fragments attached to the de novo frame could essentially govern immunological properties of resulting construct. no preference of any type of secondary structure was observed in antigenic determinants. nevertheless, all of them are located at the boundaries of the secondary structure elements and on the predicted surface-located sites of albebetin molecule. peptide fragments from human α 2 -interferon and insulin corresponding to the functionally important sites of their molecules were grafted into de novo protein albebetin (pid caa47376) engineered to attain a pre-designed tertiary structure with a unique topology that has not been observed in natural proteins. by means of genetic engineering the dna fragments corresponding to these peptides were inserted into the albebetin gene to obtain two variants of albebetin with antiviral fragment of human α 2 -interferon and two variants of albebetin with insulin-like peptide. the chimerical genes were expressed in escherichia coli in a fusion expression system with thioredoxin based on the plasmid pet-32 (novagen). the fusion proteins were digested by highly specific protease factor xa and the target chimerical proteins were purified and tested for their structure and biological activity. according to the cd spectroscopy study the chimerical proteins maintained the pre-designed structural properties of albebetin. toxicological testing of the proteins in the mtt-test did not reveal their cytotoxicity. antiviral activity of de novo proteins with human α 2 -interferon fragments was studied in vitro using human fibroblasts cell line l-41 and simian cell line vero. treatment of these cell lines with the proteins revealed the dose-dependent stimulated antiviral activity on fibroblasts and direct dose-dependent antiviral activity on the vero cells. one of two de novo proteins including insulin-like fragment (pid aag59607) acquired ability to stimulate glucose uptake by l-929 cells although the efficacy of stimulation was lower than that for the synthetic peptide and insulin. these results demonstrated that albebetin can be used as a scaffold for constructing of the functionally active de novo proteins possessing the pre-designed tertiary fold of albebetin and various biological activities. the identification of genes encoding unique tumor associated antigens (taas) has facilitated the development of novel immunotherapeutic strategies in cancer patients. clinical investigations have focused on targeting these cancer antigens for the generation of anti-tumor t-cell responses. taa epitopes come from differentiation antigens, from embryonal reexpressed or overexpressed proteins, from mutated proteins and from viral proteins in viraly associated tumors. we have recently developed a novel screening system for identification of immunogenic and antigenic ctl peptide epitopes using d b-/-x 2m -/double knockout mice, transgenic for a single-chain hla-a2-2m molecule (hhd mice). specific ctl were derived by immunization of hhd mice with tumor peptide extracts loaded on antigen presenting cells and with hhd transfected human tumor cell lines ctl induced against peptides from various tumors recognized tumor peptides more effectively than peptides extracted from normal tissues and also reacted with a serie of peptides derived from overexpressed candidate proteins, identified by differential display methods (sage, microarrays) comparison of ctl derived from hhd mice to ctl induced from patient's pbmc showed overlapping recognition of many candidate peptides. using these hhd mouse derived ctl we identified novel peptide sequences from prostate, bladder, breast and colon carcinomas, antigens pap and steap, from breast carcinoma antigens muc1 and ba46-1. analysis of tumor differentially expressed genes by the sage method in colon, followed by screening for hla-a2 binding peptides resulted in 500 candidate peptides for immunogenicity screening. we have identified 22 antigenic peptides of which 7 peptides were found to be immunogenic in hhd mice. interestingly 3 of these peptides are derived from the same protein. differential expression studies, using "dna chips" were performed on prostate and bladder tumors versus normal tissues. ten new candidate genes from tcc were analysed for expression and potential immunogenic peptides. novel peptides from uroplakins and from mage-8 were identified. surface plasmon resonance biosensing in the study of viral antigenic sites mimicked by synthetic peptides p. gomes 1 , e. giralt 2 , and d. andreu 2 1 centro de investigação em química da universidade do porto, portugal 2 department de química orgànica, universitat de barcelona, spain antigen-antibody binding has been regarded as one of the most representative examples of specific molecular recognition in nature. the simplistic view of antigenic recognition in terms of a lock-and-key mechanism is superseded, since it is now evident that both antigens and antibodies are flexible and can undergo substantial mutual adaptation. this flexibility is the source of complexities such as degeneracy and non-additivity in antigenic recognition. we have used surface plasmon resonance to study the effects of combining multiple amino acid replacements within the sequence of the antigenic gh loop of foot and mouth disease virus. our aim was two-fold: to explore to what extent can antigenic degeneracy be extended in this particular case, and to search for potential non-additive effects in introducing multiple amino acid replacements. combined analysis of one such multiply substituted peptide by spr, solution nmr and x-ray diffraction shows that antigenic degeneracy can be expected as long as residues directly interacting with the paratope are conserved and the peptide bioactive folding is unaltered. structural properties of creatine kinase from amphioxus, branchiostoma belcheri gray f. inoue, s. obase, t. suzuki, and t. imai department of physiological chemistry, faculty of sciences, toho university, funabashi, japan to further our knowledge of creatine kinase (ck) in the fields of molecular evolution and comparative enzymology, we analyzed the ck gene of the protochordate amphioxus. amphioxus is thought to be the phylogenetic predecessor of vertebrates and thus possesses characteristics, such as enzymological properties, that are associated with ancestral vertebrates. the results clarified the sequence of 789 bases including the active site. the homology of the active site and the surrounding 48 bases for the amphioxus ck gene to that of the human and electric ray ck-m gene was 89.6% and 87.5%, respectively. the amino acid sequence of this region of amphioxus ck was also identical to that of human and electric ray ck-m. in addition, the estimated secondary structure of amphioxus ck was compared to that of human and electric ray. there were no marked differences in the relative ratio of the α helix, sheet and turn structures for the peptide structure of ck consisting of 263 amino acid residues. there was a high degree of homology in the sequence of 25 amino acid residues (met271ϳhis295) near the active site of ck between amphioxus and other organisms, suggesting that this region of ck is functionally essential for transphosphorylation. gelsolin is a ca 2ϩ -activated and phosphoinsitide-regulated cytoskeletal actin-binding-and-severing protein, its fragments 135-142: ksglkykk (g135-142) and 150-169 khvvpnev vvqrlfqvkgrr (g150-169), are responsible for the binding of this protein to actin and the cellular messenger phosphatidylinositol 4,5-bisphosphate (pip2). the binding of peptides g135-142 and g150-169 to a cluster of four pip2 molecules in a dimyristoyl-phosphatidylcholine lipid was in vestigated by means of molecular-dynamics (md) simulations of 1,600 ps. the binding of the pip2 molecules to the peptides g135-142, g150-169 showed both electrostatic and hydrophobic nature: lysine residues of the peptides formed salt bridges with the phosphate groups of the pip2 molecules, while hydrophobic interactions occurred between the nonpolar residues of the peptides and the fatty-acid tails of pip2. during the binding some of the pip2 molecules were dragged out of the lipid, thus disrupting the bilayer. after the binding dissociated a draggen-out pip2 molecule tend to incooporate back to the lipid. division of applied physiology, institute of veterinary physiology, university of zürich, switzerland chemical modification of the proteins: bovine serum albumin, α-lactalbumin, -lactoglobulin and chicken egg white lysozyme by 3-hydroxyphthalic anhydride (3hp) yielded compounds which exerted antiviral activity in vitro as compared with the native unmodified proteins. of the three enveloped viruses tested: human herpes simplex virus 1 (hsv-1), bovine parainfluenza virus 3 (pi-3) and porcine respiratory corona virus (prcv), the 3hp proteins were shown to be active against human herpes simplex virus 1 only indicating that a perturbation of the viral envelope is unlikely. pre-incubation of vero cells with 3hp-albumin, 3hp--lactoglobulin and 3hplysozyme resulted in protection against hsv-1 infection whereas pre-incubation with 3hp-α-lactalbumin had no antiviral effect. however, all 3hp modified proteins showed a more significant inhibition when present during or after the viral infection step. thus multiple mechanisms appear to be involved in the inhibition of hsv-1 infection. the blocking of cell receptors may contribute to the antiviral activity as shown by the preincubation data. however, a direct interaction between the modified proteins and the hsv-1 glycoproteins responsible for viral entry and spread, seems to play a more important role, as indicated by the smaller ec 50 values obtained during and after the infection. a dummy or a protagonist on the stage of inflammation? r&d department, zambon group, bresso, milan, italy amino acids are usually present in large excess in healthy and the excess is used as source of calories. however, metabolic alterations are observed in ill patients and preferential retention of sulphur amino acids (saa) occurs during the inflammatory response. the metabolism of cysteine is modified during the acute phase of sepsis in rats. sulphate production is lower, whereas the higher liver production of taurine seems to play a protective role; glutathione concentration is greater in liver, kidney and other organs and cysteine incorporation into proteins was higher in spleen, lung and plasma (acute phase proteins) while albumin level decreases. another important phenomenon is the impairment of methionine conversion to cysteine during stressed condition. premature infants or hiv patients synthesise cysteine from methionine at a much lower rate. thus, the metabolic flow through the trans-sulphuration pathway may be insufficient to meet the glutathione and cysteine requirement in critical conditions. the pro-inflammatory cytokines, interleukin-1, interleukin-6 and tnf-α are the main initiates that alter protein and amino acid metabolism. in this complex picture, saa supply may contribute to the immune system regulation. our previous investigations showed some biological activity of newly synthesized cluster rhenium compoundtetrachlorodi-µ-(γ-aminobutirato)dirhenium(iii) chloride -i such as antitumour activity, cell-stabilizing activity against osmotic hemolysis, changing of morphology of cells, and other. there exists some information about stabilizing effects of some metal-organic substances with antitumour properties on the isolated ishaemic-reperfused rat heart (leperre a. 1995) throughout decrease of malonaldehyde (mda) production. some new investigations showed the influence of metal-organic substances on apoptotic processes (winter b. 1998 , syrkin a. 1998 , that are considered now as the main mechanism of such tissue damages as ishaemia, myocardial infarct, etc. thus we tried to analyze such activity of i. two models of hemolytic anemia was used: a -on rabbits by introducing of pbac 2 -solutions; this model permits to investigate dynamics of anemia in one experimental animal; bon rats by introducing of phenylhydrazine chloride. i was administrated as in solution as in lyposomic (lyp) forms. all measurements and models were accomplished according to described procedures. administration of i led to: increase of hemoglobin and resistance of erythrocytes and to prolonging of life for hemolytic animals; significant decrease in quantities of mda and increase in quantities of reduced glutathion (gsh), glutathionreductase (gsr) and glutathionperoxidase (gsp) in myocardium, blood, brain, liver, splenic and entherocites of anemic animals. the most effective was i in lyposomic form. mechanism of antioxidant action of rhenium cluster compound is speculated and experiments with some well-known antioxidants to compare with i are working out. at present problem of finding remedies against the mostly dangerous human disease -aids is one of higher interest. the aim of this work was the investigation of inhibiting effect of high-pure l-lysine-α-oxidase (lo) e.c.1.4.3.2, extracted from trichoderma sp., on hiv-virus reproduction, comparatively to azidotymidin (azt), being now in use for treatment of aids-patients. for studying of inhibiting effect of lo, the mt-4 cells, sensitive to citopathical action of virus, were used. the experimental studying has shown, that the enzyme at concentration 7-70 ng/ml suppresses hiv reproduction and synthesis of virus' proteins, not exerting toxical effect on mt-4 cells. toxical dose of lo has been determined preliminary. a comparison with standard preparation -azidotymidin, which causes suppression of virus reproduction at concentration 3 mkm (1,2 mg/ml) not exerting toxical effect on mt-4 cells. the same effect is attained having used lo in doses 7-70 ng/ml. using lower concentrations of enzyme leads to partial increasing of virus' titre comparatively to control cultures. obtained data allow to conclude that lo from trichoderma sp. is more high specific agent than azidotymidin, because it needs 1000 times lower concentration for the same action. comparison of azt and lo action on synthesis of virus' antigens presenting in cultural media of mt-4 cells infected with virus, leads to conclusion, that lo has inhibitory action both on virus' reproduction and virus' protein synthesis. department of microbiology, dokkyo university school of medicine, mibu, tochigi, japan our previous studies showed that the cellular amino acid composition obtained by amino acid analysis of whole cells, differs in various organisms. these results suggest that the difference in the cellular amino acid composition reflects biological evolution. however, the basic pattern of cellular amino acid composition is relatively constant in all organisms, and the cellular amino acid compositions of the archaeobacteria are quite similar to those determined from codon usage data, based on the complete genomes. in the present study, the free amino acid compositions in archaeobacteria, eubacteria, protozoa, blue-green alga, green alga, slime mold, plants and mammalian cells were analyzed, to investigate whether changes in their free amino acid compositions reflected biological evolution. cell homogenates were treated with 80-90% ethanol to separate cellular proteins and free amino acids contained in the cells. rat hepatoma cells (r-y121b) were cultured in eagle's minimum essential medium (mem) containing 5% serum or in a modified mem lacking arginine, tyrosine and glutamine. no significant difference in the free amino acid composition was observed between the two cell groups cultured under two different conditions. the patterns of the free amino acid compositions differed completely from those of the cellular amino acid compositions, and from each other in various organisms. characteristic differences were observed between plant and mammalian cells, and between archaeobacteria and eubacteria. the patterns of the free amino acid composition in blue-green alga, green alga, protozoa and slime mold differed from each other and from those of eubacteria and archaea cells. it has been suggested that the free amino acid composition reflects apparent biological changes as the result of evolution. 2) catalyzes the hydrolysis of gamma-glutamyl compounds such as glutathione, and the transfer of their gamma-glutamyl moieties to amino acids and peptides. we previously developed enzymatic methods for the synthesis of various gammaglutamylamino acids using the transfer reaction of ggt from e. coli k-12 as a catalyst. it has been reported that gamma-lglutamyltaurine has a potent and long-lasting antiepileptic action, and its chemical synthesis has also been reported, but it required protecting and deblocking of reactive groups. thus, the purpose of this study was to develop an enzymatic method for the synthesis of gamma-l-glutamyltaurine using ggt. the optimum reaction condition was 200 mm l-glutamine, 200 mm taurine and 0.2 unit/ml ggt, ph 10, and 1-hr incubation of 37°c. forty-three mm gamma-glutamyltaurine was obtained and the yield was 21.%. gamma-glutamyltaurine was purified by dowex 1 ϫ 8 column and c18 column, and then identified with gamma-l-glutamyltaurine by nmr and polarimeter. in this study the yield of gamma-l-glutamyltaurine was comparatively low because synthesized gamma-lglutamyltaurine was promptly converted into the by-product, gamma-l-glutamyl-gamma-l-glutamyltaurine. the production of antimicrobial peptides is an important aspect of host defense in animals ranging from insects to mammals. they do not target specific molecular receptors on the microbial surface, but rather assume amphipathic structures that allow them to interact directly with microbial membranes, which they can rapidly permeabilize. they are thus perceived to be one promising solution to the growing problem of microbial resistance to conventional antibiotics. insects express a battery of potent antimicrobial proteins in response to injury and infec-tion. until now, approximately 170 immune peptides have been characterized from insects and other invertebrates. an antimicrobial gene (md-cecropin) belonging to cecropin family was cloned from the bacteria-charged adult house fly, musca domestica. expressed in the vector pgex-4t1. mrna was isolated and degenerated primers were designed according to the conserved sequences of cecropins. the full-length cdna encoding md-cecropin was cloned by rt-pcr and 5ј, 3ј-race and sequenced. the deduced amino acid sequence indicated that a prepeptide with 63 amino acid residues is first translated and then processed to a mature peptide with 40 amino acids. the dna encoding the mature peptide was subcloned into expression vector pgex-4t1, and expressed efficiently in e. coli bl21 as a fusion protein. the fusion protein was purified and specifically digested and the md-cecropin was further purified to homogeneity and the activity spectrum was investigated. escherichia coli with metabolic engineering methods l. yun, x. zhang, s. wang, q. xu, and l. ma biotechnology laboratory, institute of beijing radiation medicine, beijing, p.r. china a bioengineering escherichia coli strain was obtained by metabolic engineering method. three genes related to the biosynthesis of phenylalanine, arog, phea, and tyrb encoded key enzymes: 3-deoxy-d-arabino-heptulonate-7-phosphate synthetase (ds), a bifunctional protein-chorismate mutase (cm)/prephenate dehydratase (pd) and aminotransferase (at), respectively. in this work, the feedback inhibition of ds and cm/pd were relieved by site-directed mutagenesis on bases of homology comparison of related sequences of the key enzyme. the feedback inhibition resistant genes encoding ratelimiting enzymes in the main and terminal pathways were amplified by co-expressed in order of arog-phea-tyrb on the plasmid by their own operator plpr, pl, and pr. in the recombinant strain showed great resistant to the l-phenylalanine analogues, the specific activities of ds, cm, pd and at were increased by 3.10, 3.29, 4.91 and 8.16 folds, respectively. as the result, the amount of phenyalalnine biosynthesis of the bioengineered strain was increased greatly compared with that of the host strain. an enzymatic approach for the mapping of phosphoproteins resolved on two-dimensional polyacrylamide gels hiroshima proteome laboratory, regional science promoter program, kagamiyama higashihiroshima, japan an enzymatic approach for high-throughput mapping of phosphorylated proteins resolved on two-dimensional (2-d) polyacrylamide gels is presented. proteins of cultured rat skin fibroblasts were divided into two aliquots, one of which was dephosphorylated using recombinant 1 protein phosphatase. the two aliquots were then subjected to 2-d electrophoresis. the phosphoproteins could be mapped on the 2-d gel by com-paring the gels of the phosphatase-and non-treated samples, because the dephosphorylated proteins shifted to more basic positions on the gel. this technique revealed that approximately 5% of the detectable proteins were phosphorylated. fifteen phosphoproteins were identified by mass spectrometry, including proteasome component c8 and small glutamine-rich tetratricopeptide repeat-containing protein. furthermore, the extent of phosphorylation of two actin-modulating proteins, destrin and cofilin, was found to be significantly reduced when the cells were chemically or enzymatically detached from the culture dishes. the presented technique can be applied to all biological materials because it requires no protein-labeling step, and is therefore useful for high-throughput mapping of phosphoproteins in proteome research. with the completion of the human genome sequence maldi-tof-ms is increasingly becoming an established method for identification of proteins separated by 2d gel electrophoresis. mono-isotopic peptide mass fingerprinting (pmf) has been previously shown to be amenable to full automation encompassing the process of acquisition, data processing and databank searching under full software control. until now the throughput of maldi-tof-ms for proteomics has been limited to several hundred samples in a working day and this represents approximately 5-10% of the total proteins resolved by a large format 2d gel. to reduce the number of proteins to be identified the 2d gels are imaged and analysed to determine differences in expression levels within a set of gels. although much of the image processing is semiautomated the comparison is labour intensive as manual pattern matching has a role in the gel alignments (land marking). increased ms sample throughput allows the possibility of identifying every protein spot in a 2d gel within a day. this could eliminate the potentially erroneous step of human gel image alignment, whereby land marking could be achieved using the ms data. increased sample throughput requires greater capacity and robust unattended instrument operation. in this poster we describe an integrated robotic multiple plate loader that allows overnight unattended ms operation. other improvements include an increased laser repetition rate that allows the data capture rate to increase four fold. sample tracking, data archiving and data reporting are essential attributes of this new technology and these aspects are outlined in the presentation. the proteinchip tm biology system for ciphergen biosystems: a novel proteomics platform for rapid biomarker discovery, validation and identification ciphergen biosystems ltd., surrey technology centre, the surrey research park, guildford, surrey, u.k. the proteinchip system uses seldi (surface enhanced laser desorption/ionization) proteinchip technology to perform the separation, mass detection and analysis of proteins at the femtomole level directly from biological samples. surfaces are based on either chromatographic based chemistry (ion exchange, reverse phase, imac etc.) that bind large classes of proteins or biologically defined surfaces (antibodies, dna, receptors, etc.) that are used to investigate specific proteininteraction events. as with conventional elution chromatography each type of surface is designed to bind a different subset of proteins from a crude mixture. sample complexity is reduced on the surface by washing with standard biological buffers compatible with the chosen proteinchip array. unlike elution chromatography, proteins are detected directly from the stationary phase using laser based mass spectrometry greatly increasing throughput whilst reducing sample loss and improving reproducibility. multiple proteinchip surface and wash conditions are explored with a small sample set to resolve hundreds of proteins and establish assay conditions that reveal candidate biomarkers or diagnostic protein profiles. the resulting custom built assay is then used to monitor disease processes or drug toxicity profiles by screening large banks of samples such as tissue extracts or physiological fluids (serum, urine, csf, etc.). pharmaceutical research, genomics technologies, f. hoffmann-la roche ltd., basel, switzerland to the present, samples representing the total protein mixture have been usually analyzed by proteomics technologies mainly only the abundant, hydrophilic components have been visualized. these proteins could be solubilized with reagents compatible with isoelectric focusing, for example urea and chaps. such an analysis provides us with a limited image of the proteome, which is insufficient for the detection of the majority of the proteins. in a 2-d gel, where about 1 mg of protein amount has been resolved, 1,000-3,000 protein spots can be detected, using coomassie blue staining. the spots represent the products of only 200-300 different genes. other gene products, not visualized, are most likely expressed at too low levels for detection or they can not be identified because of limitations of the current technology, they are too small, too large, basic or hydrophobic. here we will discuss protein enrichment approaches prior to the analysis, which we have applied for the enrichment of bacterial and eukaryotic proteins. proteomic analysis of the rat liver mitochondrial proteins m. fountoulakis 1 , j.-f. juranville 1 , and l. suter 2 1 genomics technologies, and 2 drug safety, f. hoffmann-la roche ltd., pharmaceutical research, basel, switzerland subcellular fractionation increases the probability of detection of low-abundance proteins. we prepared mitochondrial, microsomal and cytosolic protein fractions from total liver of male rats. the proteins of the three fractions were analyzed by two-dimensional electrophoresis using broad and narrow ph range immobilized ph gradient strips. the proteins were identified by martix-assisted laser desorption ionization mass spectrometry. in the mitochondrial fraction, 190 different gene products were detected. approximately 70% of the identified mitochondrial proteins are enzymes with a broad spectrum of catalytic activities. most of the identified proteins had been detected before in other samples as well, analyzed in our laboratory. eight gene products were detected for the first time. these were represented by one spot each, whereas most of the frequently detected proteins were represented by multiple spots. in average, approximately 15 spots corresponded to one gene product. centre for molecular medicine, university college london, u.k. three kinds of experiments have been carried out successfully in our labs. (1) identification of post-translational modifications of the endothelin a and b receptors (etar and etbr) including both phosphorylation and acylation. we have developed new, very efficient methods for single step isolation of highly pure etar and etbr from cells. this has allowed us to obtain evidence that the post-translational modifications are very complex and result in multiple phenotypes showing different forms of modification for receptor. as with other systems, e.g. insulin-like growth factors, it is probable that these multiple phenotypes of the et receptors correspond to different forms of signalling dependent on cellular state, e.g. the cell cycle. it is, for example, already clear from the phosphorylation of the receptor that a series of different kinases must be involved. (2) following stimulation of fibroblasts with endothelin, phosphorylation/dephosphorylation signalling cascades involving several hundred proteins have been observed by use of high resolution 2d electrophoresis and detection of phosphorylated proteins labeled with 32 p by autoradiography or immunological methods. the large number of proteins involved are being identified by mass spectrometric methods such as mass fingerprinting or sequencing by mass spectropmetry. (3) differential gene expression has been followed by using 35 s met pulse chase labelling concurrently with endothelin stimulation. at least 50 proteins showed significant changes in expression of 2d gels and these proteins are also being identified. these experiments demonstrate that it is now possible to use proteomics methods to investigate the integration of response to an extracellular signal at the levels of the receptor itself, the subsequent signalling cascades and the ensuing gene expression. the proteomics technology permits concurrent monitoring of large numbers of protein phenotypes (the forms and amounts of individual proteins and is therefore able to provide a global overview of signalling processes which greatly augments more traditional investigations of individual proteins or pathways. furthermore, these new methods will allow quantitative determination of the changes in protein phenotypes, which is very important in view of the highly non-linear amplification properties of such signalling processes. an integrated approach to automated high throughput protein identification by 2d gel electrophoresis and mass spectrometry d. gostick 1 , s. cohen 2 , p. young 1 , b. karol 2 , j. langridge 1 , j. randell 3 , t. slyker 3 , and a. jacobson 3 1 micromass, manchester, u.k. 2 waters corporation, milford, massachusetts, and 3 bio-rad laboratories, hercules, california, u.s.a. establishing the function of gene products is the major challenge of the post genomic era. the rate-limiting step in this endeavour is the speed with which proteins can be isolated and identified. separation of proteins from cell lysates or sub-cellular domains by 2d gel electrophoresis is an established method of visualising these complex systems. recently mass spectrometry has proved to be a powerful method of further characterising these proteins. from the mass spectrum of the enzyme digest of a 2d gel spot, the resulting digest map is compared with the theoretical maps from the databases and the protein identified when these correlate. maldi-tof is of great benefit in these studies since it requires a minimal amount of sample, is relatively tolerant to salts and other contaminants arising from the gel and may be configured for automated sample analysis. high sample throughput with automated analyses including data processing and client-server database searching are already available. our system automatically acquires the data and processes the maldi mass spectrum into a monoisotopic peak list. this peak list is then automatically sent to a networked database for protein identification. when proteins are not identified from the maldi analysis or an ambiguous result is obtained, then further analysis of the sample by electrospray caplc-ms-ms is required. the development of a hybrid quadrupole orthogonal acceleration timeof-flight mass spectrometer (micromass, q-tof) has facilitated the generation of unambiguous amino acid sequences from the ms-ms analyses of tryptic peptides. these ms-ms spectra can be automatically searched against protein, nucleotide or est databases. thus enabling protein identification from gel spots, despite non-specific enzymatic cleavage, protein co-migration and post transitional modifications. for organisms who's genome sequences are poorly represented in the data bases de novo amino acid sequencing may be required. inferring de novo peptide sequences from ms-ms data is complex and is often the rate-determining step in this method. however, it is now possible to interpret the ms-ms spectrum automatically. in our approach the raw ms-ms spectrum is reduced to the plausible single-charge, monoisotopic mass spectrum. sequence interpretation is achieved by generating "trial sequences" consistent with the experimentally determined molecular weight. a probabilistic fragmentation model is used to transform the trial sequences to predicted spectra for comparison to the single-charge, monoisotopic spectrum and to calculate the likelihood that the trial sequence would account for the observed data. the possible number of trial sequences for any peptide is large, for example there are 20 10 possible sequences for a peptide containing any of the 20 naturally occurring amino acids and having 10 residues. to reduce the scale of the problem a terminated markov chain monte carlo algorithm is used to produce sequences. this bayesian method simulates an exhaustive search of all sequences having the correct mass. the huge increase in genomic sequence information available, combined with the increased sensitivity and selectivity provided by mass spectrometry, has allowed large-scale protein identification. however the analysis of the post translational modifications present on the identified proteins is a more challenging problem. currently the approach that offers the most expedient and specific solution, to determine modified peptides, is precursor ion scanning. this approach has primarily been performed on a triple quadrupole mass spectrometer where the rear quadrupole, (ms2) is set to transmit only the fragment ion of interest. the ms1 quadrupole is then scanned across the appropriate mass to charge range. in this paper we describe a method that allows specific post translationally modified peptides to be identified and sequenced during the course of an hplc experiment on the q-tof mass spectrometer. during the hplc run the instrument is switched alternately at one-second intervals between low and high collision energy with argon in the collision cell. the quadrupole, ms1 is not mass selective, operating in the rf only mode. the first data set at low energy (4ev) shows only the normal pseudo molecular ions. the second at higher energy shows their fragments. wherever a product ion of interest occurs in the high-energy data all its possible precursors are revealed by the corresponding 4ev data. since the two data sets contain the entire set of precursor and product ions that can be formed it is clearly possible to generate the equivalent of a constant neutral loss scan. this is invaluable in the case of phosphorylated peptides where the neutral loss of 98da (h 3 po 4 ) occurs via -elimination from the phosphoserine and phosphothreonine residues. this allows the q-tof mass spectrometer to switch from the ms mode to the ms/ms mode of operation when a potential pseudo molecular ion exhibits a neutral loss of 98 da between the high energy and low energy data sets. the product ion ms/ ms spectrum can then be acquired on the phosphorylated precursor ion. in the case of phosphotyrosine, neutral loss of the h 3 po 4 moiety is not observed, however a low mass immonium ion at m/z 216 can be detected. this characteristic ion (from the high energy data) is used to direct the mass spectrometer to fragment potential phosphopeptide precursor ions, which are selected from the low energy data. in this case several precursor ions may require ms/ms interrogation at one decision making time-point. with the first draft of the human genome completed largescale protein identification by mass spectrometry, even for samples originating from higher organisms has become relatively straightforward. this requires a high throughput facility to identify proteins that have usually been separated by 2d page. the approach providing the highest level of automated sample throughput, in terms of samples per hour, is currently maldi-tof-ms. this technique provides a peptide mass fingerprint of the protein digests and allows the rapid and accurate identification of the parent protein by comparison to a databank. however, under some circumstances, for example if the number of peptides detected is small or if the sequence coverage is poor, it is advantageous to be able to include even a short piece of sequence information to provide added specificity. in a conventional maldi-tof-ms instrument post source decay (psd) can be used to try and generate sequence information, however this approach is notoriously unreliable in producing good quality ms/ms data. one reason for this is that the peptide ions do not undergo fragmentation in a controlled environment such as a gas cell with selected collision gas and collision energy. an alternative approach is to use the predictable fragmentation obtained from a hybrid quadrupole ortho maldi source has been fitted to a hybrid quadrupole orthogonal acceleration time-of-flight (q-tof) mass spectrometer. in contrast to a conventional maldi-tof-ms instrument the resolution and mass measurement accuracy of the data is comparable between the ms and ms/ms modes. this allows superior data acquisition in the ms-ms mode compared to conventional maldi-tof-ms. a number of modifications have been made to optimise the system for high throughput proteomics. the maldi source has been configured with a high-density target plate, compatible with a 96 well microtiter plate. the acquisition software has been modified for automated data acquisition in both the ms mode and the ms to ms/ms switching mode. dedicated processing software has been developed to fully automate the post acquisition and databank searching. this software has been optimised to consider the unique nature of the data acquired from this configuration of instrument. in this paper we demonstrate the how an maldi-q-tof instrument can be used for high throughput proteomics. we also compare and contrast is functionality in comparison with alternative strategies for high throughput proteomics, namely conventional maldi-tof-ms and electrospray lc-ms/ms. pseudomonas putida is an ubiquitous, metabolically and physiologically extremely variable soil bacterium. it is kown to be a good colonizer of plant roots and a plant growth promoter. now, after the sequencing of the total genomic dna has been finished we have focused on the functional analysis of this strain. plant growth promotion is achieved in different ways. one is the inhibition of fungal and bacterial phytopathogens, which is known to be a multifactorial mechanism. an important factor of this mechanism is the production of siderophores (iron-transport-agents), small linear or cyclic peptides, which are synthesized in a ribosomal-independent manner by special synthetases. the siderophore production is induced by iron limitation. the regulation of this process was investigated by pulselabelling with [ 33 p] inorganic phosphate. 2d-protein patterns generated from cells grown with and without fesupplementation were compared. proteins which were phosphorylated under iron limitating conditions were analysed by maldi-tof peptide mass fingerprint. for the identification of the proteins we used an in-house peptide mass database which has been built based on the genomic sequence data. bio-rad laboratories, inc., hercules, california, u.s.a. worksbase software for proteomics is a platform independent information management system encompassing laboratory experimental workflow and bioinformatics for protein and biochemical research. the worksbase system is designed to allow direct internal integration between laboratory experimental data and background biological knowledge found in reference and in-house data, such as gene, protein and functional annotation databases. worksbase provides a crossdisciplinary research infrastructure for drawing together multiple lines of evidence for characterization of proteins, and integration of this data with domains such as gene expression, pharmacological screening, structure and related areas. while the focus is on the biology underpinning the experimental work, the system is also designed with the capability of providing a sample and workflow tracking system for use in the wet lab, effectively a proteome lims (laboratory information management system). as experimentation proceeds in the laboratory, worksbase software can be used for development of hypotheses on protein, biochemical pathway, and post-translational processing involvement in biological systems and disease processes. as such, identifications that are derived from lab work and user observation can be used to augment the reference data repository. however, unlike databases ands systems where the methods and reasoning for assignment of annotations are obscure, by maintaining the link between the source data and the biological roles derived from them, the accuracy and integrity of any information stored in the worksbase system can be directly ascertained. changes in the brain protein levels following administration of kainic acid k. krapfenbauer 1,3 , m. berger 2 , g. lubec 3 , and m. fountoulakis 1 1 f. hoffmann-la roche ltd., pharmaceutical research, genomics technologies, basel, switzerland 2 institute of cancer research, and 3 department of pediatrics, university of vienna, austria kainic acid (ka), a potent neurotoxin and excitatory amino acid, leads to derangements and modulation of brain proteins. no global brain protein expression pattern induced by ka-treatment has been reported yet. we studied the effect of systemic ka administration on the levels of brain proteins. rats were injected placebo or ka intraperitoneally and brain was taken after one week. the mitochondrial and cytosolic fractions of the brain proteins were analyzed by proteomics technologies. heat shock protein hsp 27 was exclusively detected in brains of animals treated with ka. the levels of neurofilaments and alpha-internexin were significantly decreased and a fragment of tubulin alpha-1 chain was manifold increased in ka-brains. the mitochondrial enzymes dihydrolipoamide dehydrogenase, atp synthase beta chain and isocitrate dehydrogenase were reduced and pyruvate kinase m1 was increased following ka treatment. the results indicate altered regulation of heat shock proteins, neuronal death, cytoskeletal disruption and mitochondrial derangement by systemic ka administration. this report confirms and extends previous studies on the effect of ka on the expression of brain proteins and suggests that our analytical system can serve as a model for neurotoxicological, neurobiological and neuropathological proteome studies. the rat brain mitochondrial proteins genomics technologies, f. hoffmann-la roche ltd., pharmaceutical research, basel, switzerland we constructed a two-dimensional database for rat brain mitochondrial proteins. rat is a useful model of human diseases of the central nervous system. in order to detect alterations in the levels of the low abundance brain proteins, the mitochondrial, microsomal and cytosolic fractions were prepared. the proteins of each fraction were analyzed by two-dimensional electrophoresis, followed by martix-assisted laser desorption ionization mass spectrometry. approximately 500 proteins were identified in the mitochondrial fraction, which were the products of 165 different genes. about 75% of the identified proteins were detected in the mitochondrial fraction only and the rest were detected in the cytosolic and about 2% were found in the microsomal fraction as well. 98 of the 165 proteins had not been detected before in our laboratory. the identified proteins were in the majority enzymes or enzyme subunits with a broad spectrum of catalytic activities and heat shock proteins. whilst lc-ms/ms has been utilised for the identification of proteins from complexes and cell lysates (qualitative proteomics), the quantitative study of gene expression using differential display has until recently been the preserve of a 2d gel based proteomic experiment. however, recently a great deal of interest has been generated on the use of isotope coded affinity tags (icat) for the quantitative study of gene expression at the proteome level. the technique is based upon chemically modifying the cysteine residues of proteins isolated from cells in two different states with light and heavy isotopically labeled reagents. the two cell states are then combined, digested with trypsin and the cysteine containing peptides preferentially selected by binding to an avidin column, prior to analysis by mass spectrometry. the eluent from this column is then analysed by capillary lc esi-ms/ms. interrogation of the eluting peptides by tandem mass spectrometry and databank searching results in the identification of the associated protein. we describe how icat data analysis has been automated within a software environment. the ms and msms data acquired using the qtof instrument are processed and analysed using a new algorithm which recognises related isotope clusters and quantifies their relative intensities. based on a user defined ratio threshold the software will automatically carry out an lc-ms/ms experiment and databank search in a client-server mode and provide a report of the identified proteins and their expression ratio in the two cell states. deterioration of the transcriptional, splicing and elongation machinery in brain of fetal down syndrome b. lubec 1 and m. fountoulakis 2 1 department of neonatology, university of vienna, austria 2 gene technologies, cns research, f. hoffmann la roche, basle, switzerland perturbation of brain development i.e. regulation of gene expression, differentiation, growth and migration in down syndrom (ds) has been reported to occur early in life pointing to impairment of the complex system of transcription and or translation and indeed, altered expression of transcription factors has been reported in adult ds brain. we therefore decided to compare the transcriptional and translational machinery in cortex of brains of controls and fetuses with down syndrome in the second trimenon of gestation. we determined a series of transcription/translation factors by 2 d-electrophoresis followed by maldi -identification and quantification with specific software. the protooncogene c-crk, crk-like protein, elongation factor 1-alpha 1, elongation factor 2, elongation factor tu and two out of four spots representing ptb-associated splicing factor psf were significantly downregulated in brain of fetal ds fetuses as compared to controls. the finding of reduced transcription and translation factors may indicate deranged protein synthesis. the underlying cause for individual reduced transcription, splicing and translation factors may be explained by chromosomal imbalance or by posttranslational modifications as e.g. phosphorylation, known to be aberrant in ds. reduced expression of transcription factors in fetal ds during early life may be responsible or reflecting impaired brain development and deficient wiring of the brain in ds. r. mazzoli 1 , m. g. giuffrida 2 , e. pessione 1 , g. dellavalle 2 , c. barello 2 , e. griva 1 , and c. giunta 1 1 dipartimento di biologia animale e dell'uomo, università di torino, and 2 csaapz-cnr. c/o bioindustry park canavese colleretto giacosa (to), italy a fast phenol degrading acinetobacter radioresistens strain was isolated in our laboratories and selected for bioremediation applications. this bacterium is also able to grow on benzoate and catechol as sole carbon-energy sources, metabolizing them via the ortho route. in previous researches we detected, by means of proteome analysis, some marker enzymes of the phenol and benzoate degradative pathways. in the present work we extend the identification of the proteins involved in the aromatic-ring opening (the different components of the phenol hydroxylase and benzoate dioxygenase, the catechol dioxygenase isozymes) together with other satellite proteins specifically induced by the aromatic growth substrate. of these last proteins some are probably related to the cellular uptake of benzoate and phenol while others are ascribed to the groel family of heat-shock chaperonines, involved in proteins processing and folding. aromatic substrates may thus act as stress-agents like heat or cold. proteomic studies on rat body fluids i. miller 1 , r. wait 2 , l. sironi 3 , i. eberini 3 , m. gemeiner 1 , e. tremoli 3 , and e. gianazza 3 1 veterinärmedizinische universität, wien, austria 2 imperial college school of medicine, hammersmith, london, u.k. 3 universita' degli studi, milano, italy previously, we have characterized rat serum proteins, both under "normal conditions" and during experimental inflammation, using two-dimensional electrophoretic separation, densitometric quantitation and identification by mass spectrometry and immunological procedures (http://linux.farma.unimi.it/ homeframed.html). we have now extended these studies to the protein composition of cerebrospinal fluid (csf) and urine, and have identified several proteins specific to these fluids, including major urinary protein, uromodulin, and prostaglandin d synthase. these baseline data provide a useful comparison to the biological fluids of stroke-prone spontaneously hypertensive rats, an inbred strain, which develops cerebrovascular abnormalities following high blood pressure. our studies have detected signs of an inflammatory condition several weeks prior to stroke. we have confirmed the sharp rise in proteinuria preceding stoke onset, and have identified the excreted proteins. following stroke we observe a massive increase in csf protein concentration as serum proteins, even those of large molecular size, cross an impaired blood-brain barrier. as a first step to discover useful disease markers from the urinary proteome, we have developed a unique and systematic approach for detection of low molecular weight urinary proteins by using high resolution two-dimensional (2d) electrophoresis and mass spectrometric methods. unlike previous studies on urinary proteins, and most importantly as observed in present study, our results show that a large number of low molecular weight protein spots can be visualized in the 2d electrophoresis pattern. it was observed that protein concentration and fractionation methods were critical for our ability to detect many proteins in the gel pattern. therefore, several approaches were carefully considered to concentrate and fractionate proteins in urine samples. initially, urine specimens from normal individuals were concentrated by using centrifugation and ultrafiltration methods. the concentrated samples of urine proteins were then fractionated by size exclusion and immunoaffinity chromatography. the size exclusion method was used to generate two fractions of proteins based on their native molecular weights. further, this method allowed us to enrich concentrations of less abundant proteins for each fraction. the immunoaffinity method was used to specifically remove well-known abundant urinary proteins (such as albumin) from the above mentioned two fractions. that the 2d pattern includes many native low molecular weight proteins was confirmed by analyzing both protein fractions from size exclusion chromatography. a detailed mass spectrometric analysis of the protein spots is carried out to identify the proteins observed in 2d pattern. since urine is an ultrafiltrate of plasma, many factors in urine are present in proportion to their rate of synthesis in the body. these factors include many low molecular weight proteins that remain undiscovered due to their low abundance. therefore, the present analysis of urinary proteins would serve as the most useful guide for the discovery of novel diagnostic markers in urinary proteins. i. pucci minafra 1,2 , s. fontana 2 , p. cancemi 1 , g. alaimo 1 , and s. minafra 1,3 1 centre of experimental oncobiology, 2 department of cell biology and development, and 3 institute of histology and embryology university of palermo, italy breast cancer is one of the leading causes of death for cancer among women. there are different types of breast cancers, grouped as invasive and non-invasive types. among the invasive types "infiltrating ductal carcinoma" (idc) accounts for about 80% of all breast cancers. in order to study some biological properties related to this type of cancer, we have developed and well characterized an "in vitro" system, consisting of an idc-derived cell line, 8701-bc (minafra et al., br. j. cancer, 60, 185-192, 1989 ) and some of its cloned cell lines, selected for their high and low invasive activity in matrigel. using this model we are producing proteomic maps to compare with that of non-tumoral breast epithelial cells and with breast tissue fragments, existing in our collection or available at the expasy proteomics server. protein identification is currently done by means of gel matching, edman-microsequencing and immuno-detection. to rationalize data we grouped proteins into functional categories: a) cytoskeletal proteins, b) metabolic enzymes, c) chaperonins and other functionally related proteins, d) peptides and enzymes with regulatory functions. a fifth group consists of peptides with unknown identity. among these sets of proteins we found that glycolitic enzymes and some chaperonins are overexpressed in cancer cells. in addition, new isoforms of potential interest as biomarkers for breast cancer, were identified by means of microsequencing. a. santucci 1 , l. trabalzini 1 , d. soldateschi 2 , e. ferro 1 , a. paffetti 1 , and p. martelli 1 1 dipartimento di biologia molecolare, sezione di chimica biologica, universita' degli studi di siena, and 2 diesse diagnostica senese srl, siena, italy human cytomegalovirus (hcmv) is an ubiquitous virus, belonging to the herpesviridae family, betaherpesvirinae subfamily, able to induce morbidity in immunocompromised patients and congenitally infected new-borns. hcmv has the largest genome among the herpes-viruses (240 kbp): ad169 strain genome was completely sequenced, containing about 200 open reading frames encoding polypeptides, most of which are not characterized. the viral genes are activated in a cascade fashion: 1) alpha, immediate-early genes, coding for regulatory proteins necessary for the activation of 2) beta, early genes, needed for dna replication, and, finally 3) gamma, late genes, coding for structural proteins of the mature virions. this latter category includes the virus surface antigenic proteins responsible for the main immune response during hcmv infections. although the sequencing of hcmv genome has been completed, very little is known about the actual nature of the viral proteins. the most appropriate approach to characterize hcmv phenotype is to study its protein expression as it is carried out within the host cell. for this purpose, we analyzed by two-dimensional electrophoresis (2d-page) the protein phenotipic repertoire of human fibroblasts and compared it with that of the same cell type following infection with hcmv strain ad169. the phenotypic 2d map of human fibroblasts dramatically changes following infection with hcmv. a relevant amount of newly appeared spots is attributable to hcmv proteins, mainly of the structural category, since we analyzed host cells at the 7-9th day of infection, when the late, gamma genes are supposed to be the only to be activated. on the other hand, a marked decrease of protein synthesis can be easily evidentiated in the infected fibroblasts respecting to uninfected cells. a temptative mapping of the main structural viral proteins (those against which patients sera are directed) was carried out by immunoblotting, microsequencing and mass spectrometry. comparative proteomics of cultured cells: identification of genetic defects and molecular mechanism of apoptosis regulation v. seyrantepe 1 , k. landry 1 , s. taurin 2 , s. n. orlov 2 , and a. v. pshezhetsky 1 1 sainte-justine hospital research centre, and 2 research centre, chum, university of montreal, montreal, pq, canada we employed a comparative proteomics of cultured cells to study mechanism of genetic disorders and for identification of key proteins involved in cell proliferation, differentiation, and death. in particular, this technology proved to be very useful to understand molecular basis of severe inherited diseases resulting from deficiency of lysosomal membrane transporters, and a role of programmed cell death (pcd) of vascular smooth muscle cells (vsmc) in cardiovascular disorders. to increase sensitivity of the identification of cellular proteins we have either have isolated cellular organelles such as lysosomal membranes or performed the differential extraction of soluble, membrane and cytoskeletal proteins. by comparison of pro-teomic cell maps from normal controls and individuals affected with lysosomal transport disorders we have selected and identified several candidate disease-causing proteins, which have to be further studied by mutation analysis and functional expression. for the second group of disorders we identified proteins, which de-novo synthesis could result in survival of vsmc including a two members of hsp70 family, a molecular chaperone grp78, and so-called mortalin (grp75) highly expressed in non proliferative tissues and associated with mortal cell phenotype. two-dimensional polyacrylamide gel electrophoresis (2d-page) is the established technology employed for the separation of proteins from a cell lysate, sub-cellular organelle or tissue sample prior to identification of the excised protein spots by mass spectrometry. in the order of several hundred to several thousand proteins, can be separated and visualised on a 2d gel by conventional staining or utilising fluorescent labelling techniques. the advantage of performing a two dimensional gel based separation is the ability to obtain quantitative information by comparing and contrasting two samples in a differential display experiment, for example, between a healthy and diseased state. the last stage however stipulates that the gels are reproducible which can be both difficult and time consuming to achieve. the relativity poor dynamic range that the gels exhibit also limits quantification. other restrictions include the under representation of certain classes of proteins, such as membrane proteins, large or small proteins and very acidic/basic proteins. for these reasons, amongst others, alternatives to 2d-page are being investigated. advances in both lc and mass spectrometry instrumentation have allowed the analysis of protein complexes, which have not been separated on a 2d gel. in this case protein identification is achieved via database searching of esi-ms/ms data. this provides qualitative information on the proteins that are present and has recently been coupled with isotope dilution experiments to provide relative quantiative information. these experiments normally involve separation of the complex digest mixture by microcapillary liquid chromatography connected to an instrument capable of data dependant switching between the ms and ms/ms modes. using this approach it has been demonstrated that hundreds of ms/ms spectra can be acquired in a fully automated fashion, resulting in the identification of significant numbers of proteins, including low copy number proteins, from a single lc-ms/ms experiment. if, however, a complex protein mixture is to be investigated then a fractionation step prior to separation of the peptides on the basis of their hydrophobicity would be advantageous. we have, therefore, adopted a 2d lc-ms/ms approach using a capillary lc system (caplc) operating at nanoliter per min flow rates coupled to a q-tof 2 mass spectrometer. by replacing the standard sample loop within this system with a strong cation exchange (scx) cartridge followed by a c18 trap cartridge it is possible to pre-fractionate the peptides before separation on a c18 column. after loading the sample, discreet fractions are sequentially eluted from the cation exchange cartridge using a salt step gradient; the eluted peptides are then retained on the trapping c18 cartridge whilst they are desalted. finally the peptides are eluted from the c18 pre-column, at 200 nl/min, onto a 75 um id ϫ 10 cm waters symmetry analytical column for separation and elution into the mass spectrometer. this analytical approach will be discussed with examples where this methodology has been used for the analysis of standard protein mixtures and also for the analysis of cell lysates and sub-cellular fractions. monoclonal igg are commonly observed in various b cell disorders, the most clinically relevant being multiple myeloma. in a series of 73 serum samples, immunofixation identified igg 1 , igg 2 , igg 3 , and igg 4 in 63, 4, 5, and 1 cases, respectively. their light-chains were k in 45 cases and λ in 28 cases. these monoclonal igg were further characterized by high resolution two-dimensional polyacrylamide gel electrophoresis (2-de) with various isoelectric focusing conditions as well as by 3-de (2-de of the proteins extracted from agarose after serum protein agarose electrophoresis). after 2-de or 3-de, the monoclonal γ-chains were not visualized in 29 out 73 cases, whatever the isoelectric focusing conditions that were tested. in 6 cases, γ-chains were only detected using alkaline ph 6-11 gradients. monoclonal γ-chains and light chains were highly heterogeneous in terms of pi and mr. however, a good correlation (p ͻ 0.05) was observed between the index of migration of the monoclonal igg in agarose gels and the pi of their γand of their light-chains (r ϭ 0.735, multiple linear regression). because of the extreme diversity of the different γ-chains as well as of the k-and γ-chains, it appears that a classification of monoclonal igg based only on their electrophoretic properties is not possible. alzheimer's disease (ad) is one of disorders caused by protein conformational changes and recent studies have shown that several chaperone proteins are involved in this process. as information of chaperone expression in ad brain is limited, we aimed to study the expressional pattern of chaperones in several brain regions as this may be essential to understand how folding defects can lead to disease. we studied the concomitant expressional patterns of molecular chaperones in seven brain regions of adults with ad using two-dimensional polyacrylamide gel electrophoresis (2-de) and matrixassociated laser desorption ionization mass spectroscopy (maldi-ms). we unambiguously identified and quantified nine different chaperone proteins. six chaperone proteins, heat shock protein 60 (hsp 60), hsp 70 ry, heat shock cognate (hsc) 71, alpha crystallin b chain, glucose regulated protein (grp) 75 and grp 94 showed aberrant expressional patterns depending on brain region. hsp 70.1, grp 78 and t-complex 1 (tcp-1) epsilon subunit did not show any significant expressional change. these findings are compatible with neuropathological and biochemical abnormalities in ad brain and this report presents the first approach to quantify nine different chaperones simultaneously at the protein level in individual ad brain regions providing evidence for the relevance of aberrant chaperone expression to ad neuropathology. the mainstream approach to protein separation, visualisation and identification has been to use two-dimensional gel electrophoresis coupled to mass spectrometry for the identification of the separated proteins. however this approach is limited with the level of protein that may be loaded onto the 2d gel and the nature of the proteins that may be incorporated onto the first dimension (ipg strip). an alternative approach for the qualitative analysis of complex protein mixtures is the use of tryptic digestion followed by electrospray lc-ms/ms. this approach is dependent on a high degree of chromatographic separation prior to the mass spectrometer, such that ideally individual peptides are eluted into the source. if this is the case then the dynamic range of protein identification can be increased and low copy number proteins can be identified. often, however there is a large degree of redundant sequence information acquired, as in theory one peptide ms/ms spectrum is sufficient to identify a protein from a sequence databank. if a protein identification is obtained from a databank search of an ms/ms spectrum, it is potentially valuable to exclude the rest of the theoretical tryptic peptides to "mine" deeper into the protein complex being studied. we have introduced a new protein databank search engine capable of matching a tryptic peptide from the swissprot/ trembl databank to an ms/ms spectrum in one second. using this search engine we are able to generate dynamic tryptic peptide exclude and include lists, based upon the theoretical tryptic peptides from the identified protein, which can be passed to the acquisition software of our q-tof mass spectrometer in real time. thus, we are able to automatically steer the q-tof, during acquisition, to select and switch to the ms/ms mode only on those peaks that meet the modified selection criteria. experiments can be designed in which peaks that belong to a protein already identified during acquisition can be avoided. this exclusion list is based upon m/z, charge state and a user definable mass tolerance. the mass measurement of the data from the q-tof mass spectrometer is typically better than 10 ppm and as a consequence of this a tight mass tolerance can be selected, thus making the exclude list extremely specific. alternatively, in the case of samples derived from 2d gel spots, the mass spectrometer may abandon the current sample, re-equilibrate the lc column and move on to the next sample. to illustrate this methodology we show examples, both on standard samples and complex protein mixtures where q-tof data acquisition has been directed based upon the results from a databank search. this data will be compared and contrasted to data acquired in the normal automated lc-ms/ms mode. the specific anti-cancer activity of green tea (؊)-epigallocatechin-3-gallate (egcg) department of molecular biology, hebrew university-hadassah medical school, jerusalem, israel the effect of the green tea polyphenol-(ϫ)epigallocatechin-3-gallate (egcg) was tested in cultures of normal and transformed nih-patmras fibroblasts. in this system transformation can be induced at will by the addition of dexamethasone, which induces the expression of h-ras by activating the mammary tumor virus long terminal repeat (mmtv-ltr) promoter. this facilitates a reliable comparison of the susceptibility of normal and transformed cells to egcg. it has been shown that egcg inhibited the growth of transformed but not of the normal fibroblasts. in an attempt to elucidate the mode of the preferential inhibitory activity of egcg, its effect on growth promoting factors has been examined. the level of ornithine decarboxylase (odc, ec 4.1.1.17), which is a signal for cellular proliferation, was reduced by egcg in the transformed but not in the normal cells. egcg also showed strong inhibition of tyrosine kinase and mitogen-activated protein kinase (mapk) activities, without affecting the kinases in the normal cells. similarly, egcg also preferentially decreased the levels of the oncogenes ras and jun in transformed cell. egcg preferentially induced apoptosis in the transformed fibroblasts. in vitro chemosensitivity tests demonstrated that egcg inhibited the proliferation of leukemic cells. these findings suggest that egcg has a therapeutic potential in the combat against cancer. objectives: to develop a safo, affordable immune supportive therapy for hivϩ patients. design: a randomised, double blind, placebo-controlled study, testing an internationally patented l-methionine combination (lmc), in approximately 400 hivϩ patients; not yet on anti-viral treatment (cd4 count 200 to 500). methods: parameters measured included: cd4 count, total lymphocyte court, viral load, several clinical, as well as mechanistic parameters. the difference in the change from the baseline (active -placebo) was determined for each parameter. the study is ongoing. results: within 3 months, significant trends are noted. the cd4 count of the patients on the active therapy, presented with a slower rate of decrease, compared to the placebo group, mean difference (md) in this change from baseline; 15.1/cmm and 95% confidence interval (c1), this was confirmed by the total lymphocyte court values. after 12 months the placebo group was placed on active, causing the difference to disappear. conclusions: although further trials are needed, these results already indicate t-methionine as an important role player in the immune system of patients with impaired immune function. c. chiarla 1 , i. giovannini 1 , j. h. siegel 2 , g. boldrini 1 , and m. castagneto 1 1 centro di studio per la fisiopatologia dello shock cnr, catholic university, rome, italy 2 department of surgery, umdnj, newark, new jersey, u.s.a. in critical illness and sepsis, changes in amino acid plasma levels (aapl) have been assessed extensively, while little is known about the relationship with changes in other plasma components, such as those involved in fluid-electrolyte and osmotic balance; their investigation is also limited, in large clinical samples, by inter-patient variability. we analyzed the relationships between plasma sodium (na ϩ pl, meq/l) and aapl (µm/ l) in eighty consecutive measurements performed in one single patient with post-traumatic sepsis and severe, prolonged illness. unique feature of plasma taurine (tau) was maintenance of a highly significant inverse correlation with na ϩ pl (r 2 ϭ 0.48, p ͻ 0.001). all other aapl were correlated directly, or unrelated, to na ϩ pl, the only exception being a weak inverse correlation between tryptophan and na ϩ pl. tau was correlated, strongly and directly, also to phosphoethanolamine (pea), glutamate (glu) and aspartate (asp): tau ϭ 707.1 ϩ 7.3(pea) ϫ 4.6(na ϩ pl) r 2 ϭ 0.86, p ͻ 0.001 tau ϭ 710.2 ϩ 11.4(asp) ϫ 4.7(na ϩ pl) r 2 ϭ 0.61, p ͻ 0.001 tau ϭ 677.4 ϩ 30.7(logglu) ϫ 4.7(na ϩ pl) r 2 ϭ 0.59, p ͻ 0.001 and unrelated, or weakly and inversely related, to other aapl (measurements of beta-alanine were not included). co-variation of na ϩ pl and these aapl (particularly tau and pea) was influenced by severity of illness, and more complex regressions were needed to quantify this effect. these results provide useful information on interdependency of tau, na ϩ pl and other aapl in critical illness. the central nervous system (cns) shows an exceptionally high degree of vulnerability to reactive oxygen species. considerable evidence suggests that free radical formation and oxidative stress might play an important role in the pathogenesis of parkinson's disease (pd). moreover, it has been reported that the levels of glutathione and vitamin e increase in the brain of patients with pd as a compensatory mechanism to deal with oxidative stress. since vitamin e is an effective free radical scavenger in the brain, its neuroprotective function is the issue of new therapeutic approaches in neurodegenerative diseases. to elucidate the possible role of vitamin e in the pathogenesis of pd, we assessed the plasma levels of vitamin e, measured by high-performance liquid chromatography, in 54 patients with pd. vitamin e concentrations were also assessed in 93 age and sex matched normal individuals. the mean plasma levels of vitamin e did not differ significantly between these two groups (22.5 ϯ 8.15 mmoli/l for pd patients and 21.0 ϯ 7.9 mmoli/l for controls). the results of our study suggest that plasma vitamin e concentrations do not play a major role in the pathogenesis of pd. vitamin e and cardiovascular disease: nutritional and intervention approaches f. galli 1,2 1 institute of biological chemistry, university of urbino, italy 2 department of cardiovascular research, st thomas' hospital, london, u.k. vitamin e is represented by a family of eight natural vitamers (4 tocopherols and 4 tocotrienols) of which αtocopherol (α-t) form has the highest biological activity. this vitamin accounts for most of the lipid-soluble, chain-breaking antioxidant activity in mammalian tissues and plasma. in addition, it shows nonantioxidant properties through which it modulates cell signaling and the expression of specific enzyme in cell models playing a role in atherogenesis (e.g. endothelial and inflammatory cells). the preventive effect of vitamin e on acdv is still a matter of debate. the largest epidemiological investigations and 4 out of 5 main intervention studies at yet available have suggested a correlation between levels of vitamin e and incidence of atherosclerotic cardiovascular disease (acvd) and related mortality. an overall conclusion rising from these studies is that the major effect (if any) of vitamin e is to be found with intakes higher than 100 iu (100 mg all-rac α-tocopheryl acetate) per day. however, other investigations have failed to demonstrate a beneficial effect of vitamin e against acvd, suggesting the need for more studies on its metabolism and function. recently a family of tocopherol binding and transport proteins has been identified. they play a key role in the selective uptake and delivery of tocopherols to lipoproteins and tissues. genetic abnormalities of these proteins have been demonstrated to be responsible for conditions of vitamin e deficiency in humans. their tissue distribution and regulation are now under investigation. the information available on vitamin e metabolism and its response to supplements or diet changes are at yet poorly characterized. the synthesis of stable isotopes and the characterization of major metabolites of main vitamers provide important advances in this research. in the last years, both plasma levels and urinary excretion of relevant metabolites of α-t have been characterized. little information is available on metabolites formed by other vitamers. the emerging role of γ-t and its main catabolite 2,7,8-trimethyl-2-(b-carboxyethyl)-6hydroxychroman (γ-cehc) in the defense against nitrogen oxide species formed during the activation of inflammatory cells is now well established and suggests the need for further studies on the bioavailability and transformation of this homologue of vitamin e in humans. at the same time, an oxidation byproduct of α-t found in human plasma, namely α-tocopherylquinone, has been proposed to be also de novo synthesized from phenylalanine with a role in the genesis of a defective polyunsaturated fatty acid metabolism observed in phenylketonuric patients. this suggests a possible, and at yet unexplored relationship between vitamin e and phenylalanine/fatty acid metabolism which might have also a role in atherosclerotic process. r. gaspari 1 , s. mensi 1 , g. mercurio 1 , c. callà 2 , l. colacicco 2 , e. sacco 2 , and s. lippa 2 1 department of anaesthesiology and intensive care medicine, and 2 department of biochemistry and clinical biochemistry, catholic university of rome, italy four patients (3 females, 1 male; aged from 21 to 45 years) affected by severe liver failure, were treated by a new blood purification method, namely molecular adsorbent recycling system (mars). mars removes albumin-bound toxins using a specific membrane with a dialysate solution containing albumin. in the patients the plasma levels of methionine (meth), branched chain and aromatic amino-acids and liposoluble antioxidants were measured. the fischer's index did not show any significant variation, whereas the plasma levels of meth were well correlated with the levels of liposoluble antioxidants (vitamin e and coq10). in fact, in the patients receiving just branched chain amino-acids, the plasma levels of both meth and antioxidants progressively decreased. on the contrary, if meth and branched chain amino-acids were administered, the plasma levels of coq10 and vitamin e showed a positive correlation with the plasma meth levels (p ͻ 0.02; r ϭ 0.63 and p ͻ 0.005; r ϭ 0.77, respectively). since vitamin e and coq10 are mutually dependent-molecules, the administration of meth, essential substance for coq10 synthesis, may be effective to maintain a good antioxidant status in patients with severe liver failure undergoing mars treatment. we obtained new synthetic peptide preparation epitalon to be widely applied as a pharmaceutical due to its properties important in medical care. epitalon was found to stimulate repair processes in retinal diseases via restoring the retinal functions, in particular its photoreceptors. this promising peptide drug is a linear tetrapeptide of formula h-ala-glu-asp-gly-oh (alanyl-glutamyl-aspartyl-glycine). the substance was obtained by classic peptide synthesis in a solution (scheme: (1 ϩ 2) ϩ 1) with n-oxysuccinimide activated esters. coohgroups of lateral radicals of glutamic and aspartic acids were defended as benzyl esters, benzyloxycarbonyl (ala) and tert.butyloxycarbonyl (glu) n-defending groups were employed, deblockade conducted by trifluoroacetic acid and catalytic hydrogenolysis. preparative hplc on a reverse phase was applied for purification. the product was fully characterised by the data of analytical hplc (substance content -99%), amino acid analysis, ir-and hmr-spectra. the ready drug form is ampoules containing 10 µg of the substance in 1 ml of isotonic solution. epitalon application in patients with pigmented retinal degeneration stopped the pathology development in 100% and increased visual functions in 80% of the cases. in 70% of the patients visual acuity raised by 0.1-0.3. electroretinography confirmed the retinal functional activity increase. an increasing number of proteins are implicated in apoptosis and several of them have been shown to be altered in alzheimer's disease (ad) brain. because of this apoptosis is thought to be the underlying mechanism of neuronal cell loss in ad. to further substantiate this hypothesis we investigated the expression of a recently identified apoptosis related proteins and other apoptosis regulators in frontal cortex and cerebellum of ad by western blot and elisa techniques. quantitative analysis revealed unaltered levels of bax and raidd (receptor interacting protein associated ich-1 (caspase-2)/ ced-3 (caenorhabditis elegans death protease-3)-homologous protein with death domain) in both regions. zip (zipper interacting protein) kinase, bim/bod (bcl-2 interacting mediator of cell death/bcl-2 related ovarian death gene) and p21 were significantly increased only in ad frontal cortex (p ͻ 0.05, in all cases). cerebellar bcl-2 levels were significantly increased in ad (p ͻ 0.01) while in ad frontal cortex, although the levels tended to increase did not reach significance level. the results indicate that apoptosis indeed account for the neuronal loss in ad. however, it does not seem to involve bax and raidd. a. magyar 1 , m. brózik 3 , r. tóbi 1 , t. szabó 1 , j. szakonyi 2 , b. rojkovich 3 , p. gergely 2 , and f. hudecz 1 1 research group of peptide chemistry hungarian academy of science, budapest, 2 central laboratory of immunology, semmelweis university, budapest, and 3 national institute of rheumatology, budapest, hungary rheumatoid arthritis (ra) is a systemic autoimmune disease of unknown etiology. it is the most common of the inflammatory joint diseases, affecting 1-2% of the world population. anti-filaggrin antibodies (afa) directed against the epidermal protein, filaggrin, belongs to the most specific markers of ra. epitopes, containing citrulline within the sequence of filaggrin, have been recently identified as major antigenic sites recognised by afa. the aim of our study was to identify these epitopes of filaggrin derived-peptides targeted by ra specific antibodies to provide further information about the nature of the initial autoantigenic substance. the most immunogenic six sequences of filaggrin and further, on the n-and c-terminal, shortened version of the original peptide ( 306 shqestrgrsrgrsgrsgs 324 ) were synthesized. we used conventional solid-phase peptide synthesis (fmoc strategy) carried out on "multipin ncp" noncleavable kit. in elisa experiments the presence of afa was deter-mined using serum samples of ra patients and healthy blood donors. in conclusion our results provide further evidence that not simply the presence of citrulline but also the nature of its surrounding amino acids have important role in the creation of autoantigenic epitope reactive with anti-filaggrin antibodies. the autoimmune nature of multiple sclerosis (ms) has introduced cytokine genes as logical candidates for the loci determining susceptibility to the disease and/or influencing disease progression. interleukin (il)-1alpha and 1beta are major proinflammatory cytokines that have been related with several chronic inflammatory diseases such as ms. the il 1-receptor antagonist (il-1ra) is a protein structurally related to il-1beta that effectively inhibits the proinflammatory effects of il-1. a polymorphism in the 5ј-flanking regulatory region at ϫ889 of the il-1alpha gene, which may cause an overexpression of il-1alpha and a variable number tandem repeats (vntr) polymorphism in the il-1ra gene have been also associated with several inflammatory diseases. two biallelic base change polymorphisms in the il-1beta gene have been reported to influence the protein production: one is located in the promoter region at position ϫ511 and the other is in exon 5 at position ϩ3953. to analyze the contribution of il-1alpha, il-1beta and il-1ra genes in the genetics predisposition to ms, we have examined four polymorphic genetic markers in 132 italian patients with clinically definite ms and 130 healthy controls. in summary, no significant differences in genotypes and allele frequencies were found between ms patients and healthy controls. fibronectin -the extracellular matrix protein is oxidatively modified with oxygen reactive species (ros) in inflammation site. activated neutrophiles release the hypochlorite acid (hocl) and chloramines as products of myeloperoxidase/ h 2 o 2 /cl ϫ system. these reactive chlorine species chlorinate in turn matrix proteins. the resulting changes of tertiary protein structure could be evaluated by monitoring the antigen/antibody complex formation. the formation of the complexes between native/chlorinated fibronectin and igg class antibodies were examined by means of elisa with luminol chemiluminescence detection. the degree of fibronectin modification was monitored with spectroscopic methods. since the oxidation leads to the fibronectin aggregation -the tryptophane contents in resulting aggregates were evaluated with stern-volmer approach (acrylamide quenching). moreover, the aldehydes influence on the ag/ab complex formation was examined -since aldehydes are known products of amino acids n-chloramines deamination. also the native and modified fibronectin adherence to the matrix proteins was monitored with use of hrp labeled antifibronectin antibodies. the preliminary results suggest that chlorination impairs the ab/ag complex recognition but also prove that igg bounded chlorinated fibronectin promotes igg clusters formation. it was found also that mm concentration of the serine derived glycoaldehyde decreases the fibronectin/igg recognition and the effect could be attributed to the igg aggregates formation. we demonstrate also that hrp-labeled iggs detect the collagen and fibrynogen adherent fibronectin in a dose dependent manner-details of the elisa method are discussed. in subjects with rheumatoid arthritis (ra) oxidized low density lipoproteins (ldl) are supposed to serve as mediators for joint damage, further exacerbating the inflammatory process. to better understand mechanisms of ldl oxidation in ra a specific marker of oxidative modification of apolipoprotein (apo) b-100 proline and arginine residues, 5hydroxy-2-aminovaleric acid (hava), had been measured in plasma and synovial fluid ldl subfractions (ldl 1 , svedberg units (s f ) 7-12 and ldl 2 , s f 0-7) by gc-ms. paired knee synovial fluid and plasma samples were collected from 10 subjects with ra. additionally, plasma samples were collected from 10 healthy controls. the ldl 1 hava content in plasma was not different between the groups (ra, 0.004 ϯ 0.001 vs controls, 0.004 ϯ 0.001 mol/mol apob-100, p ϭ 0.748). the ldl 2 hava content in plasma was significantly higher in ra (0.145 ϯ 0.051 vs 0.013 ϯ 0.002 mol/mol apob-100, p ϭ 0.000). furthermore, synovial fluid ldl 1 and ldl 2 in ra contained elevated hava levels when compared with plasma concentrations (ldl 1syn , 0.023 ϯ 0.012 mol/mol apob-100 (p ͻ 0.001) and ldl 2syn , 0.434 ϯ 0.129 mol/mol apob-100 (p ͻ 0.001)). results suggest that proline and arginine residues of apob-100 are highly reactive toward oxygen radicals in both plasma and synovial fluid in ra. furthermore, susceptibility of apob-100 to oxidative modification increases along the lipoprotein metabolic cascade. particularly small dense ldl 2 were prone to direct oxidation of apob-100. correlation between hava content in plasma and synovial fluid ldl 1 and ldl 2 in ra may allow the use of hava as a clinical marker of antioxidant barrier impairment in ra. vascular collagen accumulation contributes to development of hypoxic pulmonary hypertension (ph). we have shown that injections of a polymer of the proline analogue cis-4-hydroxy-l-proline (chyp) in liposomes attenuated acute ph in rats (ajrccm 1997; 155 : 1384) . we now treated rats with established ph with a new polymer containing an increased "payload" of chyp. chyp was conjugated to a low mw poly(ethylene glycol)-lysine carrier {poly (peg1000)-lys-chyp} to increase the % by wt of the analogue. rats were exposed to 10% o 2 for 7 da to induce ph. on da 0, 7 and 14 after 7 da of hypoxia, animals were injected iv with chyp polymer in liposomes (hc) or bioinactive trans-hyp polymer in liposomes (ht). air controls received thyp polymer in liposomes (at). at 0 and 21 da, we measured mean right ventricular pressure (rvp) and hydroxyproline (hyp) content in main pulmonary arteries. on da 0, rvp (mmhg) was 9 ϯ 1 and hyp (µg/vessel) was 88 ϯ 6 in at. rvp and hyp increased to 17 ϯ 1* and 139 ϯ 4*, respectively, in hypoxic animals (n ϭ 4; *p ͻ 0.05 vs. at). on da 21, rvps were at 10 ϯ 1, ht 24 ϯ 1*, hc 15 ϯ 1* †; hyps were at 91 ϯ 9, ht 176 ϯ 15*, hc 122 ϯ 1* † (n ϭ 4; *p ͻ 0.05 vs. at; †p ͻ 0.05 vs. ht). from da 0 to 21, rvp did not increase and hyp decreased in the hc group vs. ht. we conclude that weekly injections of polymeric chyp prevented progression of established hypoxic ph and reversed hyp accumulation. targeted delivery of antifibrotic polymers may prevent and reverse the progression of ph. (support: phs, barbara cornwall foundation). glucosinolates are amino acid-derived natural plant products found throughout the capparales order, which includes agriculturally important crops such as oilseed rape, brassica vegetables and the model plant arabidopsis. glucosinolates and their degradation products have a wide range of biological activities, e.g. in plant defense as deterrents against insect and fungi and as attractants to insects that are specialized feeders in brassicaceae. the conversion of amino acids to oximes is a key step in glucosinolate biosynthesis. we have recently shown that cytochromes p450 belonging to the cyp79 family catalyze the conversion of aliphatic, aromatic as well as indole amino acids to the corresponding oximes. cyp71e1 catalyzes the oxime-metabolizing step in the biosynthesis of the cyanogenic glucoside dhurrin. we have recently shown that the oxime-metabolizing enzyme in the glucosinolate biosynthetic pathway is a cytochrome p450 homologous to cyp71e1. the post-oxime enzymes in the glucosinolate pathway have high substrate-specificity for the functional groups, and low substrate-specificity for the side chain. therefore, we have been able to metabolically engineer new glucosinolate profiles into arabidopsis by altering the level of endogenous cyp79s and by introducing new cyp79s. the approach has great potential for design of "biotech crops" with improved pest resistance and increased nutritional value. hypercalcemia as a potential threat in the dietary treatment of maternal phenylketonuria f. eyskens 1 and s. beernaert 2 1 pediatrician, metabolic diseases and 2 dietitian, azm-koningin paola childrens hospital, metabolic lab pcma, antwerp, belgium over 90% of infants born to mothers with blood phenylalanine (phe) concentrations above 1200 µmol/l exhibit evi-dence of foetal dammage, low birth weight, microcephaly, dysmorphic facies, slow postnatal growth and development and long-term intellectual impairment. keeping maternal phe concentrations below 250 µmol/l before conception and throughout pregnancy reduces significantly the risk of abnormalities in the offspring of women with phenylketonuria (pku). we describe a woman, 31 years old, who showed phe blood levels of 150-200 µmol/l under a strict diet (total protein content of 1.13 g/kg body weight/day with 0.54 g/kg natural proteins and 0.59 g/kg proteins provided by the aminoacid mixture pku3 (milupa, germany); 1,655 cal/day) at the beginning of her first pregnancy. the first weeks she developed vomiting which gradually increased in severity. at 8 weeks of pregnancy, she had diarrhea, severe bouts of vomiting and manifested a deficient nutritional status with intake of 0.2 g/kg bw proteins and 1,178 cal/day. she was hospitalized to start refeeding using continue drip feeding administered by nasogastric tube. after 2 days on this regimen she developed vomiting, heart palpitations and mental confusion. her serum calcium level, that was normal at admission in the hospital, showed an elevation to 6.5-7 meq/l (ref. value 4.2-5.1 meq/l). the feeding was stopped immediately and under an intravenous infusion and gradually introducing a feeding composed of pku3, carbohydrates and mct fats the serum calcium and the blood phe levels dropped to normal values. and volatile components of caramel obtained by heating commercial maltose solution for different time intervals. one sample containing maltose only was used as control, the caramelization was conducted at 130c° for total time period 90 minutes and subjected to sensory analysis and isolation of volatile components. the odour and colour sensory tests were evaluated according to the international standard methods (iso). the results showed that addition of lysine as a catalyst gave rise to a significant (p ͻ 0.05) increase in intensity of the whole flavour in comparison with the control sample. the sweet and caramel notes, the most characteristic attributes of caramel, showed remarkable increase. on the other hand the increase in heating time in presence of lysine as a catalyst resulted in high significant increase in browning rate of caramel solution. the volatile components of each sample were isolated by using the new technique, solid phase microextraction (spme) and subjected to gc and gc-ms analysis. over 250 volatile components were separated, however only the most important component for caramel flavour were reported. maltol and 5hydroxymethyl-2-furfural (hmf) and 4. h-pyran-4-one,2,3dihydro-3,5-dihydroxy-6-methyl (dihydro dihydroxy maltol), the main characteristic caramelization products were present in high concentration in samples contaning lysine heated for 60 minutes. in addition one pyrazine was only identified in the samples contaning lysine. a comparative study between the present results and those of our previous study concerning addition of alanine as a catalyst was carried out. short-term exposure of human umbilical vein endothelial cells (huvecs) to hyperglycemia increases l-arginine transport (system y ϩ /cats) and nitric oxide (no) production (via enos). it has been reported that enos could also be activated by a ca 2ϩ -independent mechanism involving phosphorylation of ser 1177 by a phosphatidylinositol 3-kinase (pi3-kinase) dependent pathway. we investigated the involvement of pi3kinase on the stimulatory effect of acute hyperglycemia on enos and l-arginine transport in huvecs. l-arginine transport, no synthesis and phosphorylation of ser 1177 in enos were increased by d-glucose (25 mm, 2 min). similar results were obtained in huvecs exposed to insulin. incubation of cells with wortmannin (pi3-kinase inhibitor) prevented the effects of d-glucose and insulin. no changes in the intracellular ca 2ϩ and enos protein levels were detected. thus, acute hyperglycemia increases l-arginine transport and enos activity through a pi3-kinase dependent, ca 2ϩ independent mechanism in huvecs. [ the hypercalcemia in this patient was due to a very high content in calcium of the feeding administered (2-3 times the adh value) associated with a high vitamine d concentration (see table) and a clinical state of dehydratation. the further pregnancy was uncomplicated and a healthy girl was born who developed normal. • the aminoacid mixtures used in the treatment of pku contain a high level of calcium, phosphate, magnesium and iron. they also contain a high concentration of vitamine d. • nutritional monitoring of pregnant pku patients should include the calcium, phosphate, iron, zinc and vitamins status. • vitamins a and d suppletion is contraindicated in these patients based on the high concentrations of these vitamins in the aminoacid mixtures used in the dietary treatment. flavour and aroma chemistry department, national research centre, dokki, cairo, egypt caramelization of various carbohydrates leads to product with a high tinctorial strength provided by different additives catalyzing the process. the present study was conducted to evaluate the catalytic effect of lysine on the sensory attributes administered pku3 (80 g ϭ adh 1, 2 g/kg/bw) lipoic acid is a prosthetic group of h-protein of the glycine cleavage system and e2 components of the pyruvate, 2oxoglutarate and branched-chain 2-oxoacid dehydrogenase complexes. in mammals, attachment of lipoic acid to these proteins requires two enzymes. lipoate-activating enzyme (lae) catalyzes the activation of lipoate to lipoyl-nucleoside monophosphate. then, lipoyltransferase transfers the lipoyl moiety to the specific lysine residue of the proteins. we purified lae from bovine liver mitochondria. lae activated lipoate with gtp at a 1000-fold higher rate than with atp. the reaction absolutely required lipoate and mggtp, and the reaction product was lipoyl-gmp. lae activated both r-and senantiomers of lipoate to the respective lipoyl-gmp although preference for r-lipoate was observed. lipoyltransferase equally transferred both r-and s-lipoyl moiety from respective activated lipoate to apoh-protein. however, only h-protein carrying r-lipoate was active in the glycine cleavage reaction. cdna clones encoding a precursor lae with a mitochondrial presequence were isolated. amino acid sequence of lae was identical with that of xenobiotic-metabolizing/medium-chain fatty acid : coa ligase-iii, but an amino acid substitution due to snp was found. these results indicate that the medium-chain acyl-coa synthetase in mitochondria plays a novel function with gtp, the activation of lipoate. instituto di chimica biologica "g. fornaini", università di urbino, italy nitric oxide (no) can modulate red blood cells (rbc) glycolysis by translocation of the enzyme glyceraldehyde-3phosphate dehydrogenase (gapd) [e.c. 1.2.1.12] from the cytosolic domain of the membrane protein band 3 (cdb3) in the cytosol. in this study we have investigated which no-reactive thiols might be involved influencing gapd translocation, and which is the role of glutathione (gsh) in this context. two highly reactive cys residues (k 2 ϭ 73.7 m ϫ1 s ϫ1 and 101.5 m ϫ1 s ϫ1 , respectively) were identified by transnitrosylation with nitrosoglutathione (gsno) of cdb3 and gapd. the cys 149 in the catalytic site of gapd is exclusively involved in this gsno-induced nitrosylation. reassociation experiments carried out at equilibrium with preparations of rbc membranes and gapd revealed that different no-donors may form ϫsno on, and decrease the affinity between, gapd and cdb3. in intact rbc, both the no-donors 3-morpholino-sydnonimine (sin-1) and peroxynitrite (onoo ϫ ) significantly increased gapd activity in the cytosol and glycolysis measured as lactate production and energy charge levels. however, we obtained data suggesting that onoo ϫ is the main no-derivative able to cross the rbc membrane leading to gapd translocation and ϫsno formation. both in cell-free experiments and intact rbc, diamide (a thiol oxidant able to inhibit gapd activity) was observed to reverse the effect of sin-1 on gapd translocation. the results demonstrate that cdb3 and gapd contain reactive thiols that can be transnitrosylation mainly by means of gsno, these can ultimately influence gapd translocation/ activity and the glycolytic flux. abteilung für allgemein-viszeral-und gefässchirurgie, kliniken dr. erler gmbh, nürnberg, germany new surgical procedures like minimal-invasive-surgery brought many advantages for the surgical patient: less pain and shorter hospitalization. regarding nutrition, patients gets normal food on the ward still on the operation-day and need only saline-infusions overnight for fluid and electrolyte substitution but no hypocaloric parenteral nutrition. hypocaloric parenteral nutrition had been developped as a peripheral intravenous nutritional concept for patients with a normal body mass index over a period not longer than 4-5 days. multiple clinical studies showed that bowel movements increase earlier after an early postoperative enteral feeding which allows an earlier discharge of the patient. the result is a remarkable decrease of costs and an increase in patient benefit. still some years before surgeons preferred in visceral surgery parenteral nutrition over a period of 4-5 days under the opinion not to stress an anastomosis. this opinion changed in the last years under the aspect that about 1,000-1,500 ml of bile fluid, 1,000-1,500 ml pancreatic juice and 1,000-1,500 ml gastric juice per day are passing a small intestine anastomosis without any complications. concerning colon-anastomoses, the colon is preoperatively washed out, so it lasts until 5 days until defecation. multiple studies also showed a benefit for the patient regarding immunostimulation by early postoperative enteral feeding. conclusion: in our hospital with 244 surgical patients we recommend postoperatively either early normal enteral feeding or a high caloric parenteral nutrition if parenteral nutrition is needed for longer than 5 days. if artificial nutrition is necessary for more than 14 days we recommend enteral nutrition given by a tube or peg (percutaneous endoscopic gastrotomy). department of food technology, national research centre, dokki, cairo, egypt in the near east, "frekeh" has been known for many centuries as a stable food made from wheat. it is generally claimed that "frekeh" is better than wheat regarding its storage stability. the protein quality of parched immature durum wheat (frekeh) produced from 2 variety was evaluated. frekeh from four maturing levels during the dough stage of the seed development, were analyzed for approximate analysis. results showed that "frekeh" produced at the beginning of the dough stage was of better nutritional value than that produced at the following maturity levels, since the former was higher in protein, fat, minerals and crude fiber as well as in reducing sugar content. in addition, it was shown that these results confirm well with the sensory quality evaluation of the cooked product. further more, it was found that the cooking time was suitable to produce a "freqkeh" meal with high levels of acceptability. the observed decrease in protein content with increasing maturity level raised the question of how the protein quality of "frekeh" versus that of nature wheat grains varied. in this investigation, the amino acid of "frekeh" was determined. dietary treatment and carnitine supplementation has greatly improved long-term outcome of patients with ppa and (vitamin b12 unresponsive) mma. however, metabolic decompensation may be frequent and final outcome in most patients show various handicaps. to investigate the usefulness of measuring free carnitine and acylcarnitines in dried blood by tandem mass spectrometry, we investigated 9 patients with ppa and 5 with mma in a period of 8 months by weekly capillary blood punctures performed by the parents. age of the patients were from 0.5 until 18 years. clinical status at the time of blood drawing was evaluated by regular phone calls. free carnitine in all patients substituted by oral carnitine treatment (50-100 mg/kg/day bw) was normal. the parameter best reflecting clinical status was the c 3 /c 16 -acylcarnitine quotient. mean value in mma and ppa patients showed a range of 11.5-29.4 (normal 1.5 ϩ/ϫ 0.36, n ϭ 18), there was no difference between ppa and mma patients. individual mean values of the patients significantly increased when the patient was ranked higher in the clinical score system or during decompensation. since measurement of acylcarnitines in dried blood by tandem mass spectrometry is easy to perform, this method may be used for home monitoring of patients with mma and ppa. influence of acute treatment with 1,2,3,4tetrahydroisoquinoline on the levels of glutathione and reactive oxygen species, and on the enzymatic activity of γ-glutamyl transpeptidase in dopaminergic structures of rat brain e. lorenc-koci 1 , m. sokoĺowska 2 , m. zapaĺa 2 , and l. wĺodek 2 1 institute of pharmacology, polish academy of sciences, kraków, and 2 institute of medical biochemistry, collegium medicum, jagiellonian university, kraków, poland 1,2,3,4-tetrahydroisoquinoline (tiq) and its derivatives generated considerable interest as molecular species that may be implicated in the pathogenesis of parkinson's disease (pd). in pd, apart from the lack of dopamine in the striatum, a decreased concentration of glutathione (gsh) is found in the substantia nigra (sn). it is also known that gsh depletion potentiates the toxicity of mptp and 6-hydroxydopamine. however, there are no data available on the tiq influence on gsh metabolism. the aim of the present study was to exemine the effect of acute tiq administration on the levels of gsh and reactive oxygen species (ros), and on the enzymatic activity of γ-glutamyl transpeptidase (γ-gt) in dopaminergic structures of rat brain. the investigation was carried out 4 h after a single dose of tiq (100 mg/kg i.p.). at that time, a marked increase in the tissue gsh level and simultaneous significant inhibition of γ-gt were found in the structures studied. in tiq-treated rats, the production of ros was reduced in the sn, but it was markedly enhanced in the striatum. our results suggest that the increase in gsh level in dopaminergic structures stems from inhibition of γ-gt and refers to the extracellular pool of this peptide. apparently, the tiq-mediated alterations in the levels of gsh and ros may have some implications for the etiology of pd. tetrahydrobiopterin-responsive phenylalanine-hydroxylase deficiency with mutations distant from the tetrahydrobiopterin binding site z. lukacs 1 , r. steinfeld 1 , a. kohlschütter 1 , j. zschocke 2 , and k. ullrich 1 1 department of pediatrics, university of hamburg, and 2 university-children's hospital, heidelberg, germany phenylalanine hydroxylase (e.c. 1.14.16.1) catalyzes the hydroxylation of phenylalanine to tyrosine in the presence of oxygen and the cofactor (6r)-l-erythro-5,6,7,8tetrahydrobiopterin (bh 4 ). mutations in the phenylalanine hydroxylase gene may cause phenylketonuria or hyperphenylalaninemia. alternatively, disorders in bh 4metabolism also result in an increase in phenylalanine concentrations but simultaneously affect other bh 4 -dependent enzymes, consequently, causing a severe neurological disorder. recently, several patients with a phenylalanine-hydroxylase deficiency but with normal bh 4 -metabolism were reported who showed a significant decrease in blood phenylalanine concentrations upon treatment with bh 4 . indeed, two such patients in our hospital were also sensitive to daily oral doses of 5-10 mg bh 4 /kg. the subsequent molecular genetic analysis revealed that patient 1 was homozygous for the widespread mutation y414c and patient 2 was compound heterozygous for the mutations a104d and k320n. it is striking, that all mutations are located distant from the known bh 4 -binding site and thus, should not be associated with bh 4 -sensitivity. additionally, further patients who share the same genotype are not sensitive to bh 4 . therefore, it must be concluded that factors independent of the phenylalanine hydroxylase gene, like e.g. individual chaperone proteins, influence the three-dimensional structure of the enzyme and thereby, enhance enzymatic activity in the presence of elevated concentrations of bh 4 . retrotransposons are structurally similar to retroviral gag and pol which are required for their replication via reverse transcription, and seem to be an ancestral form of specialized retroviruses. reverse transcription of retrotransposons was assumed to occur in virus-like particles as well as in retroviruses. rna-packaging in this particles suggests a possibility of infection. presumably, the formation of functional virus-like particles requires the interaction of gypsy rna with a protein encoded by gypsy first open reading frame (orf1) or a product of its processing. the objective of this work was to study whether the protein by this frame can bind with nucleic acids similarly to retroviral gag-protein and how phosphorilation of that protein may influence to this interaction. then gypsy orf1 was cloned and expressed in escherichia coli, and its protein product was purified by ion-exchange chromatography on deae-cellulose and affinity chromatography on heparin-sepharose and tested electrophoretically. it was shown that recombinant protein bound with its own mrna and with dna. the affinity for ssdna bing higher than for dsdna. the binding constant was estimated with rna. the method utilizes the ability of nitrocellulose to bind proteins but not nucleic acids. binding of 50% gypsy rna was achieved with about 100 ng of the protein in 50 ml of the reaction mixture. the binding constant was 5&times; 108 m-1, which is consistent. the structure of the putative nucleic acid-binding domain suggests that the protein is more similar to the core proteins of spumaviruses of the family retroviridae that to those of other retroviruses. phosphorilation of gag-like protein encoded by first open reading frame of retrotrasposon gypsy (mdg4) affects to interaction with nucleic acid. tryptophan (trp) in humans is catabolized by several pathways leading to various metabolites of kynurenine and indolic compounds formation. a number of diseases are connected with abnormalities in its excretion, but relationship of cause and effect is usually unclear. we introduced a two-step procedure for the detection of defects in metabolism of trp: 1) tlc is employed when starting the investigation, 2) two hplc methods were proposed and used at the next step, when pathological findings are to be proved and the individual metabolites quantified. the first hplc procedure enables the assessment of tryptophan, indolylacry-loylglycine (iag) and other five indolic compounds. the second method is intended to the monitoring of kynurenine and seven of its catabolites. the same sep-pack pre-treated sample of plasma and urine is used for all methods. the reference values and the excretion pattern in some groups of patients (350 in total) were assessed. hepathopathy, gastrointestinal defects, myopathy and seizures with other neurological symptoms were the conditions connected with changes in the excretion of some metabolites of trp. significant decrease of iag excretion was found in burn patients early after the injury. urine analyses were performed at patient with hartnup disease and benign xanthurenic aciduria, inherited metabolic defects of trp. in other experiments, trp effect on the decarboxylation of other aromatic amino acids in the liver was investigated; only weak inhibition under physiological conditions was recognised. ( two hypothetical proteins of escherichia coli, ybbq and yhae, show high sequence similarity to d-threonine dehydrogenase from pseudomonas cruciviae ifo 12047. we cloned each gene encoding ybbq and yhae into e. coli jm109. both ybbq and yhae showed no d-threonine dehydrogenase activity and showed significant activities for d-serine in the presence of nad. ybbq and yhae were purified to homogeneity from the e. coli clones. ybbq consisted of two identical subunits with a molecular mass of 31 kda, whereas yhae was a tetramer (native molecular mass, 124 kda). ybbq showed the maximum activity at ph 11.0 for the oxidation of d-serine. whereas optimum ph of yhae was ph 10.5. they catalyzed oxidation of glycerate and 3-hydroxyisobutyrate. d-glycerate was the best substrate for both enzymes. both enzymes also catalyzed reduction of tartronate semialdehyde in the presence of nadh. at physiological ph, the rate of tartronate semialdehyde reduction was much higher than that of d-glycerate oxidation. the ybbq gene is in the operon of glyoxylate utilization and the yhae gene is in the operon for d-glucarate/galactarate utilization. these results suggest that both ybbq and yhae are dglycerate 3-dehydrogenases and function physiologically in conversion of tartronate semialdehyde into d-glycerate. a serine protease inhibitor model: synthesis and biology z. mucsi 1 , á. bódi 2 , l. gráf 2 , a. perczel 1 , a. patthy 3 , and g. orosz 4 1 department of organic chemistry, 2 biochemistry, eötvös university, budapest, 3 agricultural biotechnology centre, gödölló´, and 4 research group of peptide chemistry, hungarian academy of sciences, budapest, hungary sgci is structurally related to the pmpd-2 family of canonical serine protease inhibitors. in these peptides, there is a p1-p1ј position which is responsible for reversible binding to chymotrypsin. their structure is characterized by structural compactness: the molecule contains three -sheets and three disulfide bonds. in the sgci molecule the p1-p1ј corresponds to lys-leu bond, which is cleaved by chymotrypsin extremely slowly. the question arises why an excellent substrate behaves at the same time as inhibitor. it was assumed that the threedimensional structure of the molecule is responsible for the inhibitory activity. a model was designed to include all the known features of the inhibitor: the structurally necessary -sheet structure and the fragment containing the p1-p1ј environment. three model peptide were synthesized. two model peptides had no inhibiting effect and were cleaved by chymotrypsin. one of the cleavage points is the expected p1-p1ј position, while the other positions found to be chymotrypsin preferred positions after the first cleavage. the three-dimensional structures of the model peptides were mapped by nmr. on the basis of nmr structures obtained it has been shown that the cyclopeptide part is more flexible in the models than in sgci. the initial process in the reaction mechanism of a bisubstrate enzyme, rat mercaptopyruvate sulfurttransferase: inactivation study by using chloropyruvate n. nagahara 1 , t. nakagawa 2 , and m. minami 1 1 department of hygiene and public health, nippon medical school, sendagi bunkyo-ku, tokyo, japan 2 institute for organic chemistry, darmstadt university of technology, darmstadt, germany to investigate the reaction mechanism of a bisubstrate enzyme, rat mercaptopyruvate sulfurtransferase (ec 2.8.1.2, mst), inactivation kinetics with 3-chloropyruvate (chloropyruvate) was studied; each inactivation reaction was completed in a preincubation procedure. chloropyruvate is an analog of 3-mercaptopyruvate (mercaptopyruvate) and irreversibly inhibits mst. the inactivation depended on incubation time and the concentration of chloropyruvate and showed saturation kinetics. the plot for the logarithm of % activity remaining versus preincubation time showed pseudo-first-order. the kinact is 8.0 ϫ 10 ϫ2 min ϫ1 and k1 is 3.1 mm. these suggest that chloropyruvate serves as a mechanism-based inactivator. mercaptoethanol , so that chloropyruvate can approach cys247 via the donor substrate route and acceptor substrate one, and a ternary complex may be formed prior to the inactivation. these findings suggest that a donor substrate enters the catalytic cavity prior to an acceptor one in the initial process of the mst reaction: mst follows an ordered sequential mechanism. polyketides are natural products of bacteria, fungi, marine organisms and higher plants, many of which have clinical usage. actinorhodin (1) is an antibiotic produced by streptomyces coelicolor via an iterative type ii polyketide synthase (pks) system. this consists of a multi-enzyme complex with a single catalytic function for each enzyme. departments of 1 chemistry and 2 pediatric surgery, school of medicine, fujita health university, toyoake, japan it has been reported that, in rats with a single intoxication of α-naphthylisothiocyanate (anit), acute liver injury develops with enhanced lipid peroxidation and neutrophil infiltration in the liver tissue. melatonin functions as an antioxidant. melatonin is known to inhibit neutrophil infiltration into damaged liver tissues. therefore, we examined whether melatonin exerts a protective or preventive effect on anit-induced acute liver injury. male wistar rats received a single i.p. injection of anit (75 mg/kg) and oral administraton of melatonin (100 mg/kg) at 12 or 24 h after anit injection. animals administered with melatonin at 12 and 24 h after anit injection were sacrificed 24 and 48 h, respectively, after the injection. liver injury appeared 24 h after anit injection and developed at 48 h. melatonin administered at 12 h after anit injection prevented liver injury formation with attenuation of increases in hepatic lipid peroxide level and myeloperoxidase activity, an index of neutrophil infiltration. melatonin administered at 24 h after anit injection prevented liver injury development with attenuation of further increase in hepatic lipid peroxide level. thus, melatonin protects against and prevents anit-induced acute liver injury in rats possibly through its antioxidant action and/or its inhibitory action against neutrophil infiltration in the liver tissue. k. okamura-ikeda, s. katayama, k. fujiwara, and y. motokawa t-protein is a component of the glycine cleavage system and catalyzes the tetrahydrofolate-dependent reaction. mutation in human t-protein (ht) gene results in clinical nonketotic hyperglycinemia (nkh). eight point mutations have been identified so far in nkh patients with t-protein deficiency. to understand the structure and function of ht, the wild-type (wtht) and three mutant t-proteins (g47r, g269d and r320h) were expressed in escherichia coli with chaperons groel and groes which facilitated the recovery of the expressed proteins as a soluble form. levels of expression of these proteins were similar but the recovered soluble forms of mutants were about one-third of wtht. g47r showed comparable specific activity to wtht, whereas g269d and r320h mutants exhibited remarkable reduction in specific activity. since homoallelism for g269d mutation and heteroallelism for g47r and r320h mutation were identified in typical and atypical nkh, respectively, these results suggest that g269 and r320h mutations are highly deleterious in the aspects of not only protein folding and/or stability but also catalytsis. on the other hand, g47r mutation might affect mainly on the protein stability. detailed characterization of these mutants is now in progress. laboratory of animal nutrition and biochemistry, miyazaki university, miyazaki-shi, japan the minimal actinorhodin pks, shown in black below, consists of the ketosynthase (ks), chain length factor (clf) and acyl carrier protein (acp) and is the minimum set of enzymes required for polyketide production. we have investigated the stoichiometry of the ks-clf complex and the ks-clf:acp minimal system using three methods: 1. native gel electrophoresis. 2. cross-linking of proteins using dibromoacetone. 3. radical cross-linking of proteins. this new method has also been used with wild type s. coelicolor cell free extract with 10% ks-clf in order to elucidate which proteins are in close proximity to ks-clf during in vitro actinorhodin production. in ruminant animals, essential amino acids have never been completely established, because of the difficulty of its estimation due to the presence of microorganisms such as bacteria and protozoa in the first stomach called rumen. in our previous paper, histidine was shown to be the first limiting amino acid in the rumen contents when evaluated by chemical score. recently we have also reported that rumen microorganisms cannot synthesize histidine from histidinol. on the other hand, there have been some reports which showed that nitrogen balance of ruminants was not improved by supplementation of histidine to rumen microbial protein together with methionine, lysine and threonine which had been known to improve. based on these facts, we have a hypothesis that histidine may not be an essential amino acid for ruminants. in the present paper, we will report about the abilities of cattle liver and kidney to synthesize histidine from histidinol comparing with those of swine liver and kidney. the ability was demonstrated by examining the activities of histidinol dehydrogenase (crude enzyme) by means of direct measurement of an increase in histidine and decrease in histidinol. the amount of histidine produced from histidinol by the enzyme seemed sufficient for meeting the histidine requirement of cattle. the browning reaction is the sequence of events which begins with the reaction of amino group in amino acids, peptides or proteins with glycosidic hydroxyl group of sugars; the sequence terminates with the formation of brown nitrogenous compounds or melanoidines. this reaction gives rise to tremendous number of components such as volatile alcohols, ketones, aldehydes, esters, ethers and sulfur and nitrogen containing heterocycles in addition to nonvolatile amadori compounds and complex brown pigments of medium to high molecular weights. the present study was designed to choose a currently occurring system (aspartic acid -fructose) as a model system, since aspartic acid was found to be one of the most important amino acids in many kinds of food varieties. the reaction was done under controlled conditions of reactants ratios, temperature and time. the reaction mixtures were subjected to successive extractions with suitable solvents where the obtained corresponding flavour concentrates were thoroughly investigated. the results indicated different classes of compounds such as aldehydes, furans, alcohols and alkylated pyrazines varying in quantities depending on the reaction conditions. these products were also investigated concerning their toxicological effects. so, such products of nonenzymatic reactions showed different chemical and biological properties. purification and characterization p. piyarat 1 , s. nagata 2 , h. misono 2 , and k. packdibamrung 1 1 department of biochemistry, faculty of science, chulalongkorn university, bankok, thailand 2 department of bioresources science, faculty of agriculture, kochi university, nankoku, kochi, japan nad ϩ dependent alanine dehydrogenase was purified 100 fold to homogeneity from aeromonas hydrophila. molecular mass of 230,000 daltons was estimated for alanine dehydrogenase by sephadex g-200 chromatography. sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified en-zyme showed 1 polypeptide band with molecular mass of 40,000 daltons, indicating that the enzyme is hexamer. the enzyme is highly specific for alanine and nad ϩ . sulfhydryl group of the enzyme plays an important role in the catalysis. the enzyme retained its activity on heating at 55°c for 16 h. optimum ph for reductive amination and oxidative deamination were 8.0 and 10.5, respectively. the steady state kinetic studies including product inhibition on the enzyme reaction indicated that the oxidative deamination proceeds through a sequential ordered binary-ternary mechanism in which nad ϩ binds first to the enzyme followed by l-alanine and products are released in the order of pyruvate, ammonia and nadh, respectively. the k m values for nad ϩ , l-alanine, pyruvate, ammonia and nadh were 0.17, 20, 1.33, 77 and 0.25 mm, respectively. an elevation of apolipoprotein (apo) b-100 concentrations is a particular feature of several metabolic disorders, such as type 2 diabetes (t2d), impaired glucose tolerance (igt), and familial combined hyperlipidemia (fchl). to further understand the in vivo turnover of apolipoprotein b-100 of very low density lipoprotein subfractions (vldl 1 , svedberg units (s f ) 60-400 and vldl 2 , s f 20-60) kinetic studies were performed in subjects with t2d, igt, fchl, and healthy controls using a tracer of either l-[ring-13 c 6 ]-phenylalanine or l-[5,5,5-2 h 3 ]leucine. these studies showed direct hepatic vldl 1 apob-100 secretion to be increased in patients with t2d and igt when compared with controls. in contrast, patients with fchl showed a discrete increase in hepatic vldl 2 apob-100 secretion. in all patients vldl catabolism is not essentially impaired. vldl 1 apob-100 secretion is associated with plasma insulin and free fatty acid (ffa) concentrations, resp., whereas vldl 2 apob-100 secretion is correlated with plasma mevalonate and lathosterol levels. in conclusion, vldl overproduction is supposed to be completely responsible for higher triglyceride (tg) levels found in patients with t2d, igt, and fchl. vldl 1 overproduction seems to be regulated by tg and ffa substrate and appears to be an indicator of decreased insulin sensitivity. in contrast, vldl 2 overproduction is more likely to be regulated by the availability of cholesterol substrate. these data give further in vivo evidence that vldl 1 and vldl 2 secretion is regulated independently. arabidopsis resulted in enhanced production of cysteine and glutathione graduate school of pharmaceutical sciences, chiba university, chiba, japan serine acetyltransferase (satase) catalyzes the formation of o-acetyl-l-serine (oas) which is the key intermediate of cysteine biosynthesis. oas is not only a dominant limiting factor but recently suggested as a possible signal molecule for gene expression in cysteine biosynthesis. in has been shown that the activity of cytosolic satase from watermelon was feedback inhibited by l-cysteine. to enhance the ability of cysteine biosynthesis in plants and to reveal the role of oas in the regulation of sulfur assimilation, we made the point-mutated watermelon satase gene (satg277c) whose product was not inhibited by cysteine, and introduced satg227c into arabidopsis. the contents of oas, cysteine, and glutathione in transgenic arabidopsis were increased significantly as compared to the wild-type arabidopsis. we are currently dealing with the expression analysis of sulfur-related genes in transgenic arabidopsis accumulating oas due to the overexpression of satase. certain amino acids as source of specific branched chain fatty acids in fish sauce manufacture n. g. sanceda 1 , e. suzuki 2 , and t. kurata 1 1 institute of environmental science for human life, and 2 department of human biological studies, ochanomizu university, tokyo, japan the source of some branched volatile fatty acids (vfa) during the fermentation process in the manufacture of fish sauce was investigated. we previously reported that straight chain volatile acids seemed to have been derived from fish fats but unlikely for branched fatty acids which was believed to be derived from other sources. to clarify the source of branched volatile acids, specific amino acids, alanine, leucine, iso-leucine and valine were used in this study. these amino acids were first mixed with salt and added to fish. the fish mixtures were then aerobically and anaerobically incubated for one and a half months. results showed that addition of valine significantly increased the production of iso-butyric and iso-hexanoic acids and leucine increased that of iso-valeric in the aerobically fermented fish mixtures. a similar tendency was observed in the anaerobically fermented fish mixture except that an increase in the amount of iso-hexanoic acid was observed in the leucine added mixture, which was not observed in the aerobically fermented one. it seemed that specific branched volatile fatty acids were derived from certain amino acids. glutathione (gsh) is an important component of the cellular defense mechanisms that protect cells from oxidative injury. in the retina, the glial (müller) cells have been shown to synthesize and transport gsh, and thus are likely to be involved in regulating gsh levels. in the present study, we have characterized gsh transport system in a müller cell line using 35 s-gsh uptake. the results showed that gsh was taken up in a na ϩ -and concentration-dependent manner with a k m of 0.31 mm. moreover, cellular gsh had no effect on the rate of gsh uptake. in related studies, we found that oxidative stress induced the expression of γ-glutamylcysteine synthetase (gcs) subunits, and that gcs mrna levels were correlated with the degree of gsh depletion. because organic anion transporters (oatps) have been implicated in glutathione cotransport, we examined expression of oatp members using rt-pcr. we found that the müller cell line expressed transcripts for oatp1, oatp2 and oatp3. these studies indicate that the müller cell plays important role in gsh homeostasis in the retina. in the active site of human porphobilinogen synthase (ec 4.2.1.24, pbgs), two zinc ions are coordinated by cys 122 , cys 123 and cys 132 , and his 131 and cys 223 , respectively. the fomer zinc ion, closer to catalytic site lys 252 , plays an important role in catalysis. on the other hand, a role of the latter (distal) one has not been clarified. interestingly, in human hep3b cell, his 131 was replaced with arg (h131r). to elucidate the role of his 131 in catalysis, the kinetic properties of wild type and h131r mutant enzymes were studied. these cdnas were cloned by rt-pcr with total rna from human peripheral lymphocyte and hep3b cell, respectively. each cdna encoding pbgs with 3ј non-coding region was inserted into pet-22b(ϩ) vector and then the constract was transformed into e. coli strain bl21(de3). the cells were cultured in lb medium containing 50 mg/ml ampicillin and 10 µm zn ion for 3 h at 37°c. after addition of 1 mm isopropyl--d(ϫ)-thiogalactopyranoside, cells were further cultured for 24 h at 20°c. the highly purified pbgss were obtained by ultora centrifugion, fractionation with ammonium sulfate and column chromatographies with deaecellulose, hydroxylapatite and superdex 200, serially. we are now investigating molecular properties of these pbgss. agriculture and agri-food canada, lacombe research centre, lacombe, alberta, canada handling and management procedures such as capture and restraint can be significant stressors for recently domesticated animals such as elk (cervidae elaphus). the objective of the current study was to investigate the use of pre capture nutritional therapy in attenuating hpa response and improving animal welfare. fourty eight adult male elk stags ranging in age from 2-4 years and raised on pasture were used in the study with 23 as control and 25 as nutritionally treated. twenty four hours prior to capture the elk were offered either 1 kg of a cereal grain based dietary supplement or 1 kg of a cereal grain based nutritional therapy product containing specified amino acids (usa patent # 5505968). the amino acid content of the nutritional therapy product was minimally 0.5 g per 500 kg animal weight of ala, lys, phen, meth, thre, isoleu, val and tryp plus 15 g per 500 kg weight of leu and 40 g/500 kg weight of glut. the animals were subsequently captured and held in appropriate facilities designed to handle elk. saliva samples were collected on all animals immediately following capture and salivary cortisol was monitored by ria. animals offered the nutritional therapy product containing the amino acid mixture displayed lower cortisol levels (11.8 nmol/l) compared to the untreated controls (14.9 nmol/l; p ͻ 0.05). the data suggest that amino acid therapy can be used to attenuate hpa response to a stressor in captured elk. department of bioengeneering and technology, delhi, new delhi, india resistance to analogues of methionine by corynebacterium lilium results in the partial de-repression of methionine biosynthetic enzymes. the levels of enzymes involved in methionine biosynthesis also increased step-wise by successive endowing the resistant markers, resulting in the overproduction of methionine. moreover, the repressibilities of the enzymes were also reduced by the addition of methionine analogue resistance. analogue resistant mutants were developed by uv induced mutagenesis of corynebacterium lilium (wild type) strain. the single analogue (norleucine) resistant mutant c. lilium nl-87 produced 372 µg/ml methionine in shake flasks with methionine yield at 0.068 g methionine/g glucose and specific methionine production at 0.237 mg/g dcw, while double analogue (norleucine and triazole) resistant mutant c. lilium nt-33 produced 521 µg/ml methionine. a triple analogue (norleucine, triazole and ethionine) resistant mutant c. lilium nte-99 produced 1.848 g/l methionine. the methionine yield was 0.248 g methionine/g glucose and its specific productivity was 1.04 g methionine/g dcw. clinical biochemistry, laboratory 22, luxemburg, grand duchy of luxemburg blood plasma glucose level was compared on fast and 60 minutes after oral administration of 1200 mg of acetylcysteine. in the group of 49 healthy persons the plasma glucose level feel by 7.6% over the 60 minute period. in the 30 diabetics on the contrary, the plasma glucose level observed 60 minutes after administration of acetylcysteine was 11.8% higher than in blood plasma taken on fast. similar tests were carried out "in vitro" to interpret these different results. the control group consisted of 1 ml of distilled water ϩ0.2 ml 10% glucose ϩ0.2 ml god pad (boringer mannheim gmbh). in the acetylcysteine group the distilled water was replaced by 1 ml 0.01% solution of acetylcysteine. in the glucagon group the distilled water was replaced by 0.01% solution of glucagen hypokit novo nordisk. spectrometric determination was carried out after 60 minutes of incubation. a 27% diminution of glucose was observed in the acetylcysteine group in comparison with the control group. a 32.2% increase in glucose was observed in the glucagon group in relation to the controls. the results with healthy persons and the tests "in vitro" indicate that acetylcysteine lowers the level of glucose. but it elevates the level of glucose in the blood plasma of diabetics. it may be presumed that acetylcysteine modifies the insulinglucagon balance in favour of glucagon. the objective of this study was to fortify yogurt with three oilseed protein hydrolysates prepared from soybean (glycine max), sesame (sesanum indicum) and rice bran (oryza sativa) flours. hydrolysis was carried with two enzymes one of plant origin (papain) and the other of microbial origin (alcalase). a yogurt fortification experiment was then carried using the previous hydrolysates. the hydrolysates were added to yoghurt at 5, 10 and 15% levels of fortification and the fortified yoghurt was analyzed fresh, and after 7 and 15 days of consuming period. fortified yogurt was chemically examined for fermentation activity (ph values, acidity and proteolysis) as well as its organoleptic properties. results of this experiment indicate that the addition of soybean hydrolysates with papain (8.9 units/g) for 5 minutes (tb) and rice bran hydrolysates with alcalase (27.6 units/g) for 5 minutes (te) to yoghurt can ex-ceed 5-10%, while fortification with sesame hydrolysed with papain (8.9 units/g) for 30 minutes (td) and soybean hydrolysed with papain (8.9 units/g) for 30 minutes (tc) can not reach up to 15%. it is well known that dna is fragile to reactive oxygen intermediates (rois) damage. evidences that dna fragmentation and apoptosis occur in cardiomyopathies, in the failing heart and in cultured cells under hyperbaric oxygen (hbo) stress, demonstrated that oxygen free radicals also play a critical role in heart failure. as a consequence, myocardial cell survival depends on response to oxidative stress. experimental data obtained in vitro suggested that polyamines, by acting as rois scavengers, play a role in prevention of endonucleasemediated dna fragmentation and inhibition of alkylating agents-mediated damage, potentially exerting a protective role against rois damage. thus we studied polyamine metabolism and superoxide dismutase (sod) expression in an in vivo model of heart oxidative stress, such as rats subjected to hbo. four experimental groups were used: 1) controls; 2) rats subjected to hbo for 15 min once and immediately sacrificed; 3) rats treated as group 2 but for 3 consecutive days and immediately sacrificed; 4) rats treated as group 3 but sacrificed 24 h later (recovery). northern blot analyses showed that odc mrna accumulation increased immediately (paralleled by activity) in groups 2-4, while ssat mrna decreased remarkably, thus leading to higher polyamine concentration in rois-stressed hearts. contrariwise, sod mrna level decreased rapidly in groups 2-4. this suggests that hbo-induced compensatory mechanism in rat heart is based on specific and rapid boosting of polyamine concentration, caused by coordinate induction of biosynthesis and inhibition of catabolism, and not of enzymes known to metabolise rois such as sod. amino acids oxidation was greater in tumor-bearing rats muscle. leucine is an important ketogenic amino acid that proves energy to the skeletal muscle. leucine supplemented diet was used to analyze the effects produced by walker 256 growing in pregnant rats which were distributed into six groups. three groups received normal diet (18% protein): control (c), tumor-bearing (w), pair-fed rats (cp). three groups were fed with diet supplemented with 3% leucine (15% protein plus 3% leucine): pregnant fed with leucine (l), tumor-bearing with leucine (wl) and pair-fed with leucine (lp). after 21 days, the animals were submitted to intestinal perfusion to measure leucine, methionine and glucose absorption. leucine absorption increased in w and wl groups. glucose absorption reduced in tumor-bearing. in pregnancy with cancer, metabolic changes provided both reduced fetal and tumor development. tumor-bearing rats showed increase in methionine and leucine absorption, probably diverting this nutrients to tumor cells. glucose absorption reduced in w and wl. leucine supplemented diet group promoted high leucine absorption which could be used by neoplasic cells, and mainly by fetus and host. probably, the transamination of the branch long chain amino acid provided energy substract for the skeletal muscle, keeping the nitrogen offered to host carcass. ( undernutrition cause several changes as body weight loss, in biochemical parameters, even microscopic alteration in absorptive epithelium. this means the nutrients absorption process has been harmfully and consequently increase the damages caused by malnourished. knowing leucine is used as a ketonic and oxidative amino acid our main propose was to recovery the malnourished young rats with normal (rc) and leucine supplemented diet (rl, 3% of leucine) for 60 days. it was measured body, liver, and muscle weight, intestinal absorption of glucose, methionine and leucine, and body chemical composition. the body weight gain in rc and rl was higher than control group, suggesting that nutritional replacement for these groups could provided nutrients to support the body weight recovery, reaching as the same weight as the control. methionine and glucose absorption was reduced in malnourished group, but it was recovered (glucose, methionine and leucine) after nutritional replacement. leucine supplemented diet promoted a good recovery of carcass collagen nitrogen, keeping the carcass structural nitrogen. further studies are necessary to investigate this mechanism. [financial support: fapesp ( we diagnosed the very rare autosomal recessive disorder hyperprolinaemia type ii (deficiency of ∆ 1 -pyrroline-5carboxylate dehydrogenase, ec 1.5.1.12) in a girl aged 20 months presenting with seizures and encephalopathy. l-∆ 1pyrroline-5-carboxylic acid accumulates in this disorder and there is a 10-15-fold increase in plasma proline. surprisingly, she also had vitamin b 6 deficiency. this was an unrecognised association, which was not explained by her diet or medications. we hypothesised that pyridoxal phosphate (vitamin b 6 coenzyme) was de-activated by l-∆ 1 -pyrroline-5-carboxylic acid. with high resolution 1 h nuclear magnetic resonance spectroscopy and mass spectrometry, we have shown that these two compounds react at ph 7.4 and 37°c in vitro to form three novel adducts, which we characterised. they are products of a claisen condensation (or knoevenagel type of reaction) of the activated c4 carbon of the pyrroline ring with the aldehyde carbon of pyridoxal phosphate. if this previously unreported interaction occurs in vivo, pyrroline-5-carboxylic acid is a unique endogenous vitamin antagonist. preliminary observations show that pyrroline-5-carboxylic acid also condenses with other biologically important aldehydes and ketones. some of these reactions may contribute to the brain disturbances in hyperprolinaemia type ii. we have already identified adducts with acetoacetic acid in urine from our child, which is evidence that condensation can occur in vivo. the kidneys are characterized by a high activity of γglutamyl transpeptidase (γ-gt), as well as by a high cysteine level. the present paper was aimed to obtain information on how the activity of γ-gt and the levels of non-protein sulfhydryl compounds (npsh) changed with age in rat kidneys. simultaneously, protein-bound cysteine (pb-cys) and sulfane sulfur compounds were estimated. the kidneys were from following rats groups: young (3-month-old), middle-aged (19month-old) and old (31-month-old). the obtained results showed that the activity of γgt and npsh levels in the kidneys fell with age. at the same time, a significant increase in the level of protein-bound cysteine was observed. on the other hand, the content of sulfane sulfur compounds was elevated in the group of the oldest animals. these findings indicate that -due to disturbances in the γ-glutamyl cycle -the capacity for extracellular glutathione degradation and, in consequence, the availability of cysteine for intracellular gsh biosynthesis may be impaired. the increased pb-cys level indicates potentiation of the thiolation reaction, i.e. development of protein-mixed disulphides, cysteine, sulfane sulfur compounds, oxygen reactive species. national research centre, dokki, cairo, egypt in the past few years, many attempts have been made to prepare a synthetic insulin. the biological activity of insulin is known to be closely related to the c-terminal octapeptide fragment of its b-chain. this does not necessarily mean, however, that each of the amino acid residues of the octapeptide fragment is essential for its activity. it was found that b 23 gly and b 24 phe were present in all insulins so far obtained from various animal species indicating the significance of these two residues. it would therefore seem desirable to study the effect of each of these two amino acid residues or both on biological activity of the octapeptiede fragment of the b-chain. weitzel et al. found that the substitution of arginine b 22 with another amino acid resulted in a very large decrease in biological activity, which indicates that it participates in the action of insulin. also it was found that the aromatic amino acid residues (b 24 -b 25 ) participate in the action of insulin. a heptapeptide arg-phe-tyr-thr-pro-lys-ala-och 3 , corresponding to (b 22 -b 30 ) insulin des gly 23 -phe 24 , and an octapeptide arg-phe-phe-tyr-thr-pro-lys-ala-och 3 , des gly 23 were synthesized using the solid phase method. the c-termenal ends of both peptide were converted to methyl ester by transesterification cleavage from the resin. the side chain protecting groups were removed by hf. manual counter current distribution method was used for purification of the free peptides. the way to solve the evaluation of tyrosine containing peptide was studied. the free methyl ester peptides were administrated for insulin-like activity test by glucose metabolism in the rat fat cells technique in vitro. nitric oxide synthase inhibitors influence dynorphin immunoreactivity in the rat brain following hyperthermia p. alm 1 and h. s. sharma 2 1 department of pathology, university hospital, lund university, lund, and 2 laboratory of neuroanatomy, department of medical cell biology, biomedical centre, uppsala university, uppsala, sweden nitric oxide (no) is a free radical gas that influences neuronal communication in the central nervous system (cns). recent reports suggest that no can influence dynorphin neurotransmission in the normal brain as well as in several pathological states. previous reports from our laboratory show that the enzyme nitric oxide synthase (nos) responsible for no formation is upregulated in several brain regions following hyperthermia. the present investigation was carried out to find, whether hyperthermia can influence dynorphin immunoreactivity in the brain, and if so, whether inhibition of nitric oxide synthesis will alter its distribution in heat stressed rats. rats subjected to hyperthermia at 38°c for 4 h in a biological oxygen demand incubator (bod) resulted in marked redistribution of dynorphin immunoreactivity in several brain regions e.g., cerebral cortex, hippocampus, cerebellum and brain stem. pretreatment with two potent nos inhibitors, l-name (50 mg/kg, i.p.) and l-nmma (30 mg/kg, i.p.) 30 min before heat stress significantly altered the dynorphin immunoreactivity in the brain. these drugs alone however, did not influence the peptide expression in normal rats. the results suggest that (i) hyperthermia has the capacity to influence dynorphin immunoreactivity in the brain, and (ii) inhibition of nitric oxide synthase considerably influences the dynorphin immunoreaction in hyperthermia, not reported earlier. the functional changes induced by uncompetitive and competitive nmda antagonists, memantine, amantadine and mk-801, and cgp 40116, respectively, were studied in both saline-pretreated and mptp-pretreated c57 bl/6 mice. the nmda antagonists were administered acutely by themselves or in combinations of either: nmda antagonist plus subthreshold l-dopa dose or nmda antagonist plus suprathreshold l-dopa dose, to either the mptp-pretreated or the salinetreated mice. activity-enhancing or functional restorative effects of the nmda antagonists were variable with memantine and mk-801 distinguished from amantadine and cgp 40116. in the study of long-term effect of nmda antagonists mk-801 was administered postnatally and spontaneous motor behaviour and motor activity in response to several pharmacological interventions was assessed. marked alterations associated possible with apoptotic penchance are discussed. t. archer 1 and a. fredriksson 2 1 department of psychology, university of göteborg, and 2 department of psychiatry, university of uppsala, ulleråkers hospital, uppsala, sweden synergistic antiparkinsonian actions of different classes of putative therapeutic agents co-administered with a subthreshold dose of l-dopa (5 mg/kg) in drug-naive mptp-treated mice as well as the restorative actions of those compounds in suprathreshold l-dopa-tolerant mptp-treated mice subjected to "wearing-off" of l-dopa efficacy were assessed in a series of experiments. the classes of compounds studied included the noncompetitive nmda antagonists, memantine, amantadine and mk-801, the anticonvulsive and putative anticonvulsive agents, lamotrigine, fce 26743, phenytoin, the monoamine oxidase inhibitors, l-deprenyl, amiflamine, α-ethyltryptamine, clorgyline and phenelzine, and the α 2 -adrenoceptor agonists, clonidine and guanfacine. in this final case, the restorative effects of clonidine and guanfacine were antagonised by the α 2 -adrenoceptor antagonist, yohimbine, but not the α 1adrenoceptor antagonist, prazosin. within each class of potentially therapeutic agents a differential restorative efficacy was obtained, but the combination of different doses of apomorphine with clondine failed to restore motor activity. in vivo proton mr-spectroscopy of the human brain: assessment of n-acetylaspartate (naa) reduction as a marker for neurodegeneration w. block 1 , f. träber 1 , s. flacke 1 , f. jessen 2 , ch. pohl 3 , and h. h. schild 1 1 department of radiology, 2 department of psychiatry, and 3 department of neurology, university of bonn, germany proton magnetic resonance spectroscopy ( 1 h-mrs) is a well accepted non-invasive method to investigate changes in brain metabolite composition in different types of cerebral disease. we performed proton spectroscopy in patients with dementia of the alzheimer's type (ad) and in patients with motor neuron disease (mnd) with the aim to detect a specific metabolic pattern for each of these two neurodegenerative disorders. overall, more than 150 spectroscopic data sets of patients with mnd and more than 100 data sets of ad patients were acquired within the last 5 years. in the mnd group we found a significant reduction of naa/tcr metabolite ratios in the central region, which correlates with the disease severity and the clinical lateralisation of neurological symptoms and increases in the time course of the disease. in ad patients a similar reduction in relative naa contents was observed in the medial temporal lobe. the observed regional metabolic alterations correlate well with the characteristic neurological symptoms in ad (dementia) and mnd (muscular palsy) and seem to follow the disease process over time. since naa is exclusively expressed in neurons as shown by immunohistochemical studies, reduced naa levels suggest neuronal loss or dysfunction in the observed regions. center for molecular imaging research, massachusetts general hospital, boston, massachusetts, u.s.a. non-invasive measurement of hemodynamic parameters and imaging neovasculature architecture during angiogenesis is highly important in determining tumor prognosis and in assessing treatment efficacy. we suggested a technique to map the tumor vascular (vvf) and interstitial volume fraction (ivf) noninvasively in vivo. a poly-l-lysine based macromolecular probe (mpeg-pl-gddtpa) with extended circulation in the bloodstream designed to shield chelated paramagnetic lantanide with poly(ethylene glycol) chains. we hypothesized that a magnetic resonance signal after intravenous administration of a vascular paramagnetic probe can be maximized so the signal change after administration of a second comound (gddtpa) reflects the ivf but not the vvf. the method and its assumptions were verified in animal models of cancer. tumoral vvf and ivf values were consistent with histology data and literature values. imaging showed heterogeneity of both parameters at submillimeter pixel resolution. this technique was used for characterizing differential angiogenesis in human mammary adenocarcinoma lines as well as for imaging anti-angiogenic drug effects. anti-angiogenesis was induced using synthetic d-reverse peptides derived from thrombospondin-1. this study showed that peptide treatment results in slower brain tumor growth due to inhibition of de novo blood vessel formation and synergistic anti-proliferative effect on tumor cells. in conclusion, in vivo mr imaging can be used for non-invasive treatment assessment of novel antiangiogenic drugs. wallenberg neuroscience centre, lund university, lund, sweden we have recently found that 6-hydroxydopamine lesioned rats gradually develop dyskinetic-and dystonic-like movements upon repeated administration of a therapeutic dose of l-dopa. such movements simulate the time course of peak-dose dyskinesia in parkinson's disease. in this rat model, the severity of l-dopa-induced dyskinesia is strongly correlated with an upregulated expression of the prodynorphin gene in striatal neurons. using antisense technology and gel-shift assay analyses, we have addressed the role of transcription factors which may mediate this response. we have found that the camp response-element binding protein (creb) is essential in maintaining a basal expression of prodynorphin mrna in the intact striatum, but it is not required for l-dopa to induce the prodynorphin gene in dopamine-denervated striatal neurons. we have thus addressed the role of fos-and jun family tran-scription factors, and found very high levels of fosb-and jundlike proteins in the striata of dyskinetic animals. these proteins could bind to both ap1 and cre sites in the prodynorphin promoter. moreover, intrastriatal fosb knockdown could inhibit both the upregulation of prodynorphin gene expression and the development of dyskinesias under chronic l-dopa treatment. we propose that dimers of fosb-and jund-like proteins mediate abnormal changes in striatal gene expression which are linked to the development of l-dopa-induced dyskinesia. department of pharmacology, grünenthal gmbh r&d, aachen, germany glutamate plays important roles in both normal and pathophysiological nocieception. upon physiological conditions, glutamate release from primary afferents in the spinal cord activates largely ampa receptors. as those are ubiquitously involved in fast transmission in the cns, ampa antagonists have a broad side-effect profile. prolonged activation of nociceptors by tissue damage, inflammation or nerve injury evokes a long-lasting release of glutamate and neuropeptides, activating nmda receptors in the spinal cord. this mechanism appears to play a key role in pain chronification. the nmda receptor is, therefore, an important target for chronic pain treatment. both animal and human studies confirm the efficacy of nmda antagonists in chronic pain, however, clinically available compounds are weak or have unacceptable side-effects. glycine b antagonists and compounds selectively blocking nr2b-containing receptors appear to be safer, the reasons for this remain unclear. central side-effects could potentially be avoided by using nmda antagonists with restricted central access. peripheral nmda receptors (as well as some other subtypes of glurs) could be activated by glutamate released from the site of injury, thus contributing to peripheral hyperexcitability. some other subtypes of glurs can also contribute to peripheral sensitisation. of ionotropic glurs, kainate receptors appear important in inflammatory and neuropathic pain. they can be activated by high intensity stimulation of nociceptive afferents, and may act as autoreceptors controlling release of glutamate. group i metabotropic glurs are also present on primary afferents and on second order neurones in the spinal cord, and may play a similar role. antagonists of these subtypes of glurs are active in some models of chronic pain. specific upregulation of group ii metabotropic glurs in some pain-relevant structures could reflect a possible adaptive role of these inhibitory receptors under chronic pain conditions; their selective agonists also have a potential for the treatment of chronic pain. we have performed a series of studies of the distribution and function of mglur subtypes in the basal ganglia that suggest that members of this receptor family could serve as targets for novel therapeutic agents that would be effective in treatment of pd. for instance, two group ii mglurs (mglur4 and mglur7) are localized on presynaptic terminals of striatal neurons in the globus pallidus where they could reduce gaba release. furthermore, activation of group i mglurs results in a depolarization and increased cell firing of neurons in the subthalamic nucleus (stn) and projection neurons of the substantia nigra pars reticulata (snpr). interestingly, this effect is mediated by mglur1 in snpr projection neurons and mglur5 in stn neurons. finally, activation of group ii mglurs results in inhibition of glutamate release from stn terminals in the snpr. furthermore, selective agonists of group ii mglurs inhibit haloperidol-induced catalepsy in rats, suggesting an antiparkinsonian effect of these compounds. the rich distribution and diverse physiological roles of mglurs in basal ganglia raises the possibility that these receptors may provide targets for novel therapeutic agents that could be used for treatment of pd and related disorders. a. cupello 1 , m. parodi 2 , and m. balestrino 2 1 centro di neurofisiologia cerebrale, cnr, genova and 2 dipartimento di scienze neurologiche, university of genova, italy in vitro rat hippocampal slices are commonly used to study the effects of hypoxia in the central nervous system, because they allow to differentiate the effects of hypoxia in the brain from that of systemic (e.g., respiratory and cardiac) failure that may accompany hypoxia. we used electrophysiology to monitor and evaluate the damage caused by transient hypoxia to the nervous tissue. a few minutes after oxygen deprivation brain tissue suddenly depolarizes. this event, which is termed ìanoxic depolarizationî is accompanied by dramatic metabolic changes: transmembrane ionic gradients disappear (na ϩ enters, k ϩ exits the neurons), neurons swell, there is intra-and extra-cellular acidosis. this event is caused by functional inactivation of (na ϩ / k ϩ )atpase caused by decreased atp content, as it is suggested by the fact that it is mimicked by ouabain treatment. one of us has contributed to show that if this event is not promptly reversed by reoxygenation it causes irreversible damage, mainly by determining a massive entry of ca 2ϩ into neurons. pretreatment of tissue with creatine (1 mm or more) both increases neuronal energy store by increasing neuronal phosphocreatine and protects brain tissue from irreversible damage. in vivo increase in phosphocreatine has been shown using lower (0.5 mm) creatine concentration, injected directly into the lateral ventricle. a different type of hypoxia-induced damage is observed when hypoxia is of shorter duration. in this case upon reoxygenation one does not observe disappearance of evoked potentials but their increase. this phenomenon, originally described as ìpost-hypoxic hyperexcitabilityî has been later called ìanoxic long-term potentiation (ltp)î. we showed that this event can be prevented by inactivating the nuclear protein s-100. while this damage is milder than that induced by anoxic depolarization, it may explain stroke-induced epileptic fits. we are currently investigating what role, if any, pretreatment with creatine may have in preventing also this type of damage. cytoskeleton is subject to continuous modification to yield changes in cell shape and function of plasmamembrane proteins linked to the cytoskeleton. gelsolin (gsn) depolymerizes filamentous actin and thus causes dynamic uncoupling of membrane ion channels. we have studied alteration of neuronal ca 2ϩ influx by the absence of gsn and its pathophysiological consequences during cerebral ischemia. cytosolic ca 2ϩ concentrations were determined ratiometrically in synaptosomes preloaded with fura-2am. glutamate release from synaptosomes superfused with krebs' buffer was measured by hplc. transient focal cerebral ischemia was induced by 2 h occlusion of the middle cerebral artery (mca). in gsn deficient mouse brain cortical synaptosomes [ca 2ϩ ] i increase in response to k ϩ (30 mm) depolarization was 42% higher than in wild-type. ω-agatoxin iva 0.2 µm decreased ca 2ϩ -influx in neocortical wild-type synaptosomes by 53%, and abolished differences between gsnϩ/ϩ and ϫ/ϫ genotype. k ϩinduced release of glutamate in neocortical synaptosomes was 78% higher and lesion size after mca occlusion was 45% higher than in wild-type. it is concluded that presynaptic ca 2ϩ influx is increased in gsn deficient nerve terminals which, together with subsequently increased glutamate release, increases neuronal vulnerability. in vivo assessment of tissue alteration in cerebrovascular and neurodegenerative diseases s. flacke, w. block, f. träber, p. mürtz, h. urbach, and h. schild the combined used of perfusion imaging (pi) and molecular diffusion imaging (dwi) are opening a new window into the processes that occur during the first hours of ischemia. dwi detects early changes of proton diffusion associated with cytotoxic edema. pi has the potential to characterize the degree of regional hypoperfusion. mismatches between dwi and pi, i.e. hypoperfused areas with normal adc are considered potentially salvageable. we present results of 11 patients with an angiographically defined thrombembolus in the middle cerebral artery and a spontaneous stroke evolution. whereas the infarct core was clearly visible on both dwi and pi, tissue at risk of infarction could only be detected by an increased blood volume and transit time. however only in a subgroup of patients (n ϭ 3) these areas were incorporated into the final infarct. in these patients perfusion parameter of tissue at risk of infarction were more pronouncedly altered than in those where the tissue at risk was spared from infarction (ratios of tissue at risk vs normal (rcbv 2.17 ϯ 0.59, mtt 1.67 ϯ 0.22, ttp 1.32 ϯ 0.11, p ͻ .05). these human data show that a detailed analysis of diffusion/ perfusion mismatches allow the identification of tissue at risk of damage. glucose deprivation enhances the sensitivity of cerebellar granule cells to die by excitotoxicity. we have previously reported that neither 70 min of glucose deprivation, a treatment that depletes cell energy resources, nor exposure to 20 µm glutamate (glu) for 30 min, induce significant cell death in cerebellar granule cell cultures, 24 h after treatment. in contrast, the combined treatment with 20 µm glu and glucose deprivation induces both cell death and choline (cho) release. we investigated whether the neurotoxic effect of this treatment is related with inhibition of phosphatidylcholine (pc) synthesis. we found that exposure to 20 µm glu alone for 30 min, glucose deprivation for 70 min, and the combination of both treatments inhibited pc synthesis when measured at the end of treatment by 71%, 92% and 91%, respectively. furthermore, we found that exposure to either 20 µm glu, glucose deprivation or 20 µm glu ϩ glucose deprivation decreased incorporation of [ 3 h]cho into phosphocholine and increased the intracellular content of free [ 3 h]cho, indicating that all these treatments inhibit the synthesis of pc by inhibiting choline kinase activity. since only the combined treatment with 20 µm glu plus glucose deprivation evoked cho release and excitotoxic cell death, the present results indicate that other factors in addition to inhibition of pc synthesis are required to induce cho release and excitotoxic cell death in cerebellar granule cells. (supported by cicyt, saf98-0063.) the role of striatal metabotropic glutamate receptors in degeneration of dopamine neurons k. goĺembiowska, j. konieczny, k. ossowska, and institute of pharmacology, polish academy of sciences, kraków, poland the present study was undertaken to characterize the effect of blockade of the mglu 5 receptor subtype by 2-methyl-6-phenylethynylpyridine (mpep), as well as the effect of stimulation of the mglu 2/3 receptor by (ϫ)-2-oxa-4aminobicyclo[3.1.0]hexane-4,6-dicarboxylic acid (ly379268) on spontaneous and stimulated dopamine (da) release in rat striatum using an in vivo microdialysis. mpep (100-500 µm), perfused through a microdialysis probe affected neither the basal nor the veratridine (100 µm)-stimulated striatal da release. however, mpep given intraperitoneally (5 mg/kg) diminished either the basal or the veratridine-evoked da release. ly379268 (100-250 µm) administered locally also inhibited the veratridine-evoked da release in rat striatum. antagonists of mglur-i and agonists of mglur-ii have been shown to have neuroprotective properties in several models of neurotoxicity in animals. we have approached this issue using a selective mglu 5 antagonist in an animal model of neurotoxicity induced by methamphetamine. in our preliminary experiments, methamphetamine (5 ϫ 10 mg/kg sc every two hours) decreased the tissue content of striatal da and its metabolites dopac and hva.mpep (5 ϫ 5 mg/kg ip) given before every methamphetamine injection reversed its action. the effect exerted by the mglu 5 antagonist mpep seem to be mediated by sites located outside the striatum due to relieving da neurons of the facilitatory influence of glutamate. in turn, the attenuation of da release from nigrostriatal terminals by ly379269 may be a consequence of activation of striatal mglu 2/3 receptors. reversal of the methampetamine-induced da depletion suggests a potential for neuroprotective activity of mpep. o. golubnitschaja 1 , h. h. schild 1 , and j. flammer 2 1 department of radiology, university of bonn, germany 2 university eye clinic, basel, switzerland glaucoma remains a major eye illness with unknown etiology. although elevated intraocular pressure has been shown to be the major risk factor, there is a cohort of relatively young patients developing normal-tension glaucoma (ntg). assymptomatic ischemic events in brain have been shown to be often attributable to galucoma. perfusion of the retina and optic nerve head suffering from observed vasospastic dysfunction may be further reduced by changes in the intraocular pressure. ocular ischemia developed due to these blood flow deficits may play a major role in initiation of glaucoma. possibly secondary to ischemia the autoimmunogenic capacity is activated by ntg patients having an increased prevalence of systemic autoimmune diseases. therefore, the determination of potential molecular markers in blood lymphocytes could be useful for early diagnostics of ntg. our recent study using "gent hunting"-techniques showed indeed altered gene expression in lymphocytes of ntg patients. the demonstrated downregulation of xpgc gene expression which subsequently leads to the accumulation of damaged dna and an elevated p53 expression, together with the upregulation of a new abc-transporter seem to be specific for the pathogenesis of ntg. molecular imaging of ntg provides insights in mechanisms of disease initiation and allows the early diagnostics and preventive treatment. (supported by "bio-rad" and "amersham pharmacia aggregate cell cultures prepared from fetal rat telencephalon were used to study neuronal amino acid consumption during glucose restriction. to that end, both mixed (neuronglia) and neuron-enriched cultures were grown in chemically defined medium and tested at an advanced maturational stage. it was found that 6 h of exposure to reduced glucose (0.25 mm instead of 25 mm) caused significant increases in the consumption of several amino acids and the accumulation of ammonia. it also greatly changed the intracellular level of several amino acids in neurons, particularly of aspartate and glutamate. irreversible neuron-specific damage was observed one week after the insult. elevated glutamine media concentrations (4 mm instead of 0.25 mm) during glucose restriction further increased ammonia production and neuronal damage, although the overall rate of glutamine metabolism remained practically unchanged. taken together, our findings suggest that glucose deficiency caused (i) the dysfunction of crucial transamination pathways; (ii) a shift towards the oxidative deamination of glutamine and several other amino acids used by neurons as alternative energy substrates; and (iii) the accumulation of neurotoxic ammonia levels. institute for brain research, university of vienna, austria the racemic (d,l) mixture of the naturally occurring neutral aromatic amino acid 3,4-dihydroxy-l-phenylalanine (l-dopa) was first synthetized in 1911. in 1913, the natural levorotatory isomer was isolated from vicia faba beans and declared to be biologically inactive. however, in 1930 l-dopa was observed to lower the blood pressure in the rabbit, an effect opposite to the vasopressor effect of adrenaline. following the discovery, in 1938, of the enzyme l-dopa decarboxylase, ldopa's conversion in tissues (by decarboxylation) to dopamine (da), the first biologically active substance in the biosynthetic pathway of catecholamines, was demonstrated. subsequent pharmacological studies, done between 1942 and 1957, showed that the biological actions of l-dopa were, in principle, due to da formed from it in the body. in 1957, the central antireserpine effect of d,l-dopa was described in mice and confirmed in 1960 with l-dopa in humans. following the demonstration of da's occurrence in the brain in the years 1957/58, d,l-dopa was found (in rabbits) to restore brain da levels, reduced by reserpine. in 1960, the severe brain da deficit, confined to patients with parkinson's disease (pd) was reported and a year later l-dopa's superior anti-akinesia effect in patients with pd demonstrated. finally, in 1967 the high-dose oral l-dopa regimen was successfully introduced into clinical practise. in contrast to these supreme achievements, two related early studies remained, for different reasons, without consequence. despite some initial doubts about its mechanism of action, there is now convincing evidence for l-dopa therapy being a classic example of a central neurotransmitter replacement therapy, with the severe brain da deficit furnishing a rational basis for the amino acid's clinical use and high efficacy in patients suffering from pd. b. jakobsen 1 , a. tasker 2 , and j. zimmer 1 1 anatomy and neurobiology, sdu-odense university, odense, denmark 2 department of anatomy and physiology, university of prince edward island, canada the toxicity of domoic acid (dom) was studied in rat hippocampal slice cultures, prepared from 7-days old rats and grown on semipermeable membranes for 2-3 weeks before exposure. dom (0.1-100 µm) was added to the medium, alone or together with the glutamate receptor antagonists ns-102, nbqx or mk-801, for 48 hrs followed by 48 hrs in normal medium. neuronal degeneration was monitored and ec 50 values estimated by densitometric measurements of the cellular uptake of the propidium iodide (pi) at 24, 48, 72 and 96 hrs. the lowest ec 50 values, obtained at 72 hrs, were: ca1 (6 µm), dentate granule cells (dg) and ca3ab (10 µm),ca3c (12 µm). protective effects of 10 µm nbqx at 72 hrs were seen against 3 µm dom in dg, ca1 and ca3c and against 10 µm dom in ca1 and ca3c. 10 µm ns102 and mk801 only displayed protective effects together with nbqx. mk801 thus significantly increased the protective effect of 10 µm nbqx in ca1 against 10 µm dom in combination with 10 µm nbqx and 10 µm ns102. we can confirm that dom neurotoxicity primarily involves ampa/kainate receptors, but also nmda receptors at high concentrations (glutamate release). department of physiology/neuroscience, medical university of south carolina, charleston, south carolina, u.s.a. although dopamine has been most clearly tied to the development of addiction to drugs of abuse, recent studies indicate that once addiction has been established the expression of addictive behaviors, such as drug craving, is mediated more by long-term neuroadaptaions in glutamate transmission. data will be presented which supports and extends this hypothesis. repeated cocaine injections were given for one week and three weeks after the last drug injection a number of molecular, neurochemical and behavioral neuroadaptations were measured. it was found that in the nucleus accumbens there is a increase in the expression of genes encoding mglur2/3 and glur1, and a decrease in the expression of mglur5 and its accompanying scaffolding proteins homer1bc. this was accompanied by an increase in the capacity of mglur2/3 receptors to regulate presynaptic glutamate release and a blunting in the effects of stimulating mglur1/5 receptors. in addition, there is reduced activity in the cystine/glutamate exchanger 3 wks after repeated cocaine. as a result of these changes there is a decrease in the basal release of glutamate, and a relative increase in the releasibility of glutamate upon stimulation. by using the reinstatement model of drug seeking behavior, it was shown that glutamate transmission in the projection from the prelimbic cortex to the core of the nucleus accumbens was particularly affected by the cocaine-induced changes in gene expression. taken together, these findings support the use of glutamate autoreceptor agonists as possible therapeutic adjuvants in treating the cravings associated with addiction. dopamine (da), a catechol that autoxidizes to an oquinone, is implicated as an endogenous pro-toxin, however, the following studies suggest that da has dual neurodegenerative and neuroprotective roles. in rats treated as neonates with 6-hydroxydopamine (6-ohda; 134 :g icv), there was a 99% reduction in striatal tissue da content in adulthood, and a 3 to 5fold increase in spontaneous hydroxyl radical (ho*) formation (indirect salicylate trapping method: dihydroxybenzoic acid analysis). additionally, systemic l-dopa (60 mg/kg i.p.) suppressed ho* formation. however, when glutamate (50 mm) was added to an in vivo microdialysate, ho* formation was increased substantially more in the microdialysate from dainnervated striatum. these findings indicate that da innervation is inherently neuroprotective, but in the presence of a high level of an excitatory amino acid, da innervation predisposes to formation of reactive oxygen species. ongoing neuronal activity is likely to interact with and to determine the role of da as a neurotoxic or neuroprotective substance. (supported by ns 39272.) the glutamate hypothesis of schizophrenia along with the dopamine hypothesis was intensively discussed in the past. the last years however suggest more and more that neither a hypofunction of the glutamatergic system alone nor a hypofunction of the dopaminergic system alone is responsible for symptoms found in schizophrenia. the basal ganglia (bg) as the critical structures mediating symptoms of schizophrenia are innervated by dopaminergic fibers from the mesencephalon as well as by glutamatergic fibers from limbic structures; like prefrontal cortex, hippocampus, entorhinal cortex and amygdala. thus, limbic input is able to modulate information processing in each structure of the bg and by this way control dopaminergic functions through feedback mechanisms. dysfunction in limbic structrues may result in an imbalance of information processing via the bg and terminates in behavioral symptoms of schizophrenia. we showed in recent neurochemical studies in combination with behavioral analysis that a simple, generalized hypofunction of limbic glutamatergic input on bg nuclei is not the key mechanism inducing schizophrenic behavior. a dysfunction of a particular limbic structure or pathway seems to be responsible for an imbalanced information processing via the bg and imbalanced behavioral adaptation terminating in schizophrenic symptoms. [ there is a need to identify subtype-specific ligands for mglu receptors to elucidate the potential of these receptors for the treatment of nervous system disorders. to date, most mglur antagonists are amino acid-like compounds acting as competitive antagonists at the glutamate binding site located in the large extracellular n-terminal domain. we have investigated novel subtype-selective mglur5 antagonists which are structurally unrelated to competitive mglur ligands. using a series of chimeric receptors and point mutations we demonstrate that these antagonists interact with novel allosteric binding sites in the tm domain via a noncompetitive mechanism of action. recent studies in animal models implicate mglu 5 receptors as a potentially important therapeutic target particularly for the treatment of pain and anxiety. vascular endothelial growth factor (vegf) is a major mediator in angiogenesis and vascular permeability. in central nervous system (cns) vegf plays pivotal roles such e.g., inductor of endothelial cell proliferation, migration and inhibition of apoptosis, as well as mediator of blood brain barrier (bbb) breakdown and subsequently of brain edema formation. these ubiquitous epiphenomena are major complications in several cns pathologies, including head trauma and stroke. reduced tissue oxygen tension (hypoxia) and hypoglycaemia triggers vegf expression that occurs in ischemic regions around postraumatic or postinfarct necrosis. after brain injury, the expression of vegf is increased contributing to disruption of the bbb. vegf increases the permeability of bbb via the synthesis/release of nitric oxide and subsequent activation of soluble guanylate cyclase. the immunohistochemistry shows an increase of stained astrocytes around a cortical micronecrosis. vegf participates in the response of the cns to injury in a dose dependent way. immunostaining correlates with infarct volume and clinical disability. vegf-antagonists reduce ischemic brain edema and injury, involving vegf in pathogenesis and eventually in treatment of stroke and related disorders. this cytokine also exerts a neuroprotective effect mediated by its receptor flk-1. functions related to the inflammatory response, co-expression with proteins of the ecm and interaction with the two main receptors, flk-1 and flt-1, will be discussed. n-methyl-d-aspartate (nmda) receptors can mediate excitotoxic or neuroprotective responses. one of the molecular mechanisms responsible for nmda neuroprotection involves the release of brain-derived neurotrophic factor (bdnf) which in turn binds to and activates its cognate receptor trkb. bdnf levels in the neuronal culture medium increased 2-fold when cells were preincubated for three hours with nmda. at three hours, the increase in bdnf protein levels in the medium was accompanied by a concomitant increase in bdnf mrna. thus, nmda elicited two temporally distinct responses: an early release of bdnf protein followed by a later transcriptional activation of dbnf mrna and protein release. these results suggest that nmda activates the trkb receptor via a bdnf autocrine loop resulting in neuronal survival. in addition, extracellular regulated kinases (erk 1/2) were rapidly activated, which peaked within six hours of nmda treatment. erk 1/2 activation is completely blocked by mk-801 and partially blocked by k252a, suggesting the nmda and trkb receptors act in a coordinated fashion to activate erk 1/2. as an extension of this work, we discovered a single nucleotide polymorphism in the human nr1 gene that, when transfected into hek cells, alters the electrophysiological properties of the nmda receptor complex. possible consequences of this nmda receptor variant in signaling will be discussed. this overview summarizes our recent knowledge of the role that tyrosyl radical (tyro • ) can play in neurochemical systems of brain and thereby lead to neural disorders (pd, ad, als). these could involve the interactions of tyrosine and tyro • with reactive oxygen species (ros) and reactive nitrogen species (rns), via radical mechanisms and chain processes in the presence of o 2 and endogenous brain antioxidants. concentrations of tyro • , ros and rns can increase dramatically under conditions of generalized stress: oxidative, nitrative or reductive. this in turn can directly damage (by lipid peroxidation) or indirectly damage (by protein oxidation and/or nitration) cellular substructures which ultimately can lead to apoptotic neuronal cell death or autoschizis. enzymatically (classical peroxidase mechanisms) or non-enzymatically formed tyro • can react with no • and this reversible and intrinsic "combination" acts to "buffer' tyro • concentrations. the reaction of tyro • with superoxide (o 2 •ϫ ) is a scavenging reaction which proceeds rather by addition, not by electron transfer; and major resultant products are tyrosine hydroperoxides (tyrooh). however, the decay of tyro • can be also terminated by self-termination (dimerization) resulting in dityrosine (dt) formation. tyro • can catalyze ldl oxidation, although the precise mechanisms of this reaction in vivo remain unknown. nitration of tyrosine to 3-nitrotyrosine (3-nt) requires a one-electron oxidation as a primary step, with formation of tyro • , followed by addition of the nitrogen dioxide radical (no • 2 ). the promoting effect of carbon dioxide on peroxynitrite-mediated tyrosine nitration (via radical mechanisms) (tyro • /no • /o 2 •ϫ /no • 2 system) is due to the selective reactivity of the putative carbonate radical anion, as compared to that of the oxidizing hydroxyl radicals ( • oh). moreover, once formed, 3-nt may act to promote repetitive redox cycling; it may be reduced to the corresponding nitroanion radical, which is then oxidized by molecular o 2 to o 2 •ϫ and parent 3-nt. one-electron oxidation of 3-nt can result in catalytically active imminoxyl radical. dt formation can outcompete tyrosine nitration at lowsteady state concentrations of peroxynitrite. it is unquestionable that very high fluxes of no • and o 2 •ϫ are requisite intermediates of peroxynitrite, a tyrosine nitration agent formed via tyro • . evidence for the existence of generalized stress within neurons includes the presence of protein peroxides (tyrooh), dt, and 3-nt. the nitration/denitration processes can be pathologic, but these also may play: 1) a signal transduction role; 2) a role of "blocker" for radical-radical reactions (scavenging of no • , no • 2 and co 3 •ϫ by tyro • ); or 3) a role of delimiting factors for peroxynitrite formation. it is still unknown whether oxidation/nitration of tyrosine (as dopamine precursor or protein residue) via tyro • formation, is a footprint of generalized stress and neuronal disorders, an important part of o 2 •ϫ and no • metabolism, or just a part of integral processes for maintaining neuronal homeostasis. the complete answer of these questions should be the first priority task of our recent search, wherein the problem of increased free radical formation in the brain and/or the imbalance of ratios: ros/rns/tyro • may be all important in determining neural cell and tissue injuries under pathological conditions resulted from generalized stress. [acknowledgements. this work was supported in part by kbn ( more than 50% of patients with type 2 diabetes have coronary heart disease, related to silent ischemia, caused by an autonomic denervation of the heart in diabetic patients. oxidative damage to dna has been well documented in cardiac cells isolated from diabetic patients and rats with streptozotocin-induced diabetes mellitus (dm) . this dmmodel shows already seven days after onset of disease structural changes in vascular tissue typical for the development of atherosclerosis. this study evaluates possible molecular mechanisms for early events in the development of dm-induced cardiomyopathy. methods: using "expression array" we examined the activation of cardiac cell death in heart of dm-rats. ms-pcr was used to examine a differential dna methylation. results: an increased expression of genes encoding renin, angiotensinogen and p53 was detected in heart of dm-rats. substantial changes in the methylation status of the p53dependent p21 waf1/cip1 -gene and the cyclin d1-gene were detected in dm-rats. conclusions: the renin-angiotensin system is upregulated with diabetes, and this may contribute to the development of cardiomyopathy via oxidative damage and p53-dependent activation of cardiac cell death. this pathway includes de novo methylation of the p53-inducible p21 waf1/cip1 -gene encoding a protein which binds to and inhibits a broad range of cyclincyclin-dependent kinase complexes. (supported by "bio-rad" and "amersham pharmacia biotech") department of pharmaceutical biosciences, uppsala university, uppsala, sweden during the past decade studies have indicated that growth hormone (gh) may exert effects on the central nervous system (cns). for instance, gh replacement therapy was found to improve the psychological capabilities in adult gh deficient (ghd) patients. furthermore, beneficial effects of the hormone on certain functions, including memory, mental alertness, motivation and working capacity have been reported. likewise gh treatment of ghd children has been observed to produce significant improvement in many behavioural problems seen in these individuals. studies also indicated that gh therapy affects the cerebrospinal fluid (csf) levels of various hormones and neurotransmitters. further support that the cns is a target for gh emerges from observations indicating that the hormone may cross the blood-brain-barrier (bbb) and from studies confirming the presence of gh receptors in the brain. it was previously shown that specific binding sites for gh are present in discrete areas in the cns of both humans and rats. in peripheral tissues gh is shown to elicit its effects through a second mediator insulin-like growth factor 1 (igf-1). igf-1 is well recognized as a protective agent against neural injury in the cns. the neuroprotective effect of this peptide has a broad spectrum affecting many brain regions and acts through its antiapoptopic effect. the production of igf-1 is upregulated in areas of brain damage and the igf-1 system may be an important part of an endogenous neuroprotective system. in spinal cord injuries, however, the content of igf-1 is reduced. we recently observed a neuroprotective effect of topical application of igf-1 in animals subjected to spinal cord trauma. the observed effect may be mediated via a mechanism involving nitric oxide. in the same animal model we have very recently observed a neuroprotective effect of gh. recent reports suggest that the level of gh is drastically reduced in patients with spinal cord injury. in victims of spinal cord injury the secretion of gh and igf-1, as well, is known to be decreased. therefore, exogenous substitution of gh and igf-1 might be a promising approach in the future therapy of spinal cord injury victims. in fact, there is one report indicating that prolonged treatment with synthetic gh of spinal cord injured rats attenuates some of the neurological motor dysfunction seen in these animals 3 weeks following trauma. in our animal model we observed that topical application of rgh significantly reduced traumainduced disturbances in the fluid micro-environment. we also noted that gh was capable of attenuating the trauma-induced depression of spinal cord evoked potentials. the mechanism by which gh exerts it neuroprotective effects will be discussed. chronically administered levodopa in parkinson's disease (pd) treatment is ultimately associated with alterations in motor response. in 6-hydroxydopamine lesioned hemiparkinsonian rats, chronic twice-daily administration of levodopa progressively shortens duration of contralateral turning and augments the period of turning at or below 20% of peak turning rate. the pathogenesis of the response alterations involves in part sensitization of the corticostriatal glutamatergic synaptic activity. characteristic changes involving interactions between striatal kinase and phosphatase signaling now appear to contribute to sensitization of spiny-neuron glutamatergic receptors. glutamate-mediated striatal dysregulation, subsequently, modifies basal ganglia output system in ways that favor the appearance of parkinsonian motor response complications. at a molecular level, transcriptional activation of striatal creb contributes to the persistent expression of the levodopa-induced motor response alterations. conceivably, a safer and more effective therapy for all stages of pd can be provided by drugs that target intracellularly on striatal kinases or phosphotases, or by agents that interact extracellularly on non-dopaminergic striatal receptors such as ampa and nmda, adenosine a2, adrenergic a2, opiod, and serotonergic 2b. the primary cause of parkinson's disease is a loss of dopamine in the corpus striatum. it has been postulated that this effect leads to disinhibition of the striopallidal pathway and, secondarily, to a functional shift towards glutamatergic stimulation. the aim of the present study was to find out whether inhibition of glutamatergic transmission at a level of metabotropic glutamate receptors (mglurs) in the striatum may alleviate parkinsonian-like symptoms in rats. the non-competitive antagonist of receptor subtype 5 (mglur5), mpep (1.0-10 mg/kg ip), or the agonist of group ii mglurs, ly354740 (5-10 mg/kg ip), reduced the haloperidolinduced muscle rigidity and catalepsy. intrastriatal injections of the antagonist of mglur1, (rs) aida (7.5-15 µg/0.5 µl), but not of the agonist of group ii mglurs, 2r,4r-apdc (7.5-15 µg/0.5 µl), inhibited the muscle rigidity induced by haloperidol. in order to search for an influence of mglurs on the striopallidal pathway, the effect of mpep or of the agonist of group ii mglurs, dcg-iv, on the preproenkephalin mrna expression in the striatum was examined. the obtained results suggest that blockade of group i mglurs, or stimulation of group ii mglurs may be important to the amelioration of parkinsonian symptoms. striatal mglurs may contribute to at least some of these effects. several lines of evidence suggest an important role of glutamate in depression. the involvement of group i mglurs in depression has also been proposed. thus, we decided to evaluate whether group i mglurs antagonists have antidepressantlike effects. we also investigated if antidepressant treatment influences group i mglu receptors in the brain. the experiments were performed on male wistar rats (200-250 g) and male c57bl/6 mice (22-26 g). aida (group i mglurs antagonist) given i.v. in the dose of 50 µg, decreased the immobility time in the despair test in rats. mpep (noncompetitive, systemically active mglur5 antagonist) given i.p., was not effective in the despair test in rats. however, in doses of 1.0, 10 and 20 mg/kg, it significantly decreased the immobility time of mice in the tail suspension test. moreover, the deficit in passive-avoidance learning, which was observed in bulbectomized rats, was reversed by chronic, but not acute mpep (10 mg/kg) treatment. prolonged imipramine treatment resulted in significant increase of the level of expression of mglu5 receptors in the ca1 field of the hippocampus, while prolonged electroconvulsive shock treatment (ect) enhanced significantly the chemiluminescence of mglu5 receptors in the ca3 field. the results indicate that group i mglu receptors are modified by chronic antidepressant treatment and that group i metabotropic glutamate receptors antagonists may play a role in the therapy of depression. (this study was supported by kbn grant no. 4.po5a.091.17) institute of pharmacology, polish academy of sciences, krakow, poland chronic exposure to nicotine, alcohol, opioids, sedatives, and cannabis results in development of drug dependence that becomes evident upon a cessation of drug administration and expresses itself as a withdrawal syndrome (with its physiological and motivational manifestations). adaptations at the nmethyl-d-aspartate receptor (nmda-r) complex have been observed in different brain areas during chronic exposure to, and upon withdrawal from, opioids, ethanol, benzodiazepines and barbiturates. behavioral studies employ the assessment of the effects of nmda-r antagonists on: a) the development of dependence (nmda-r antagonists are co-administered with the drug), b) the maintenance of dependence (nmda-r antagonists are administered to animals with pre-established dependence, and -most relevant to the clinical situation -c) on the expression of drug dependence (assessment of the withdrawal severity in subjects with nmda-r antagonists administered just before the expected emergence of withdrawal). the development of dependence to opioids and benzodiazepines is significantly retarded by nmda-r antagonists. studies from this laboratory demonstrate similar inhibition by nmda-r antagonists of the maintenance of opioid dependence. both in rodents and humans, the expression of opioid antagonist-precipitated as well as spontaneous (natural) withdrawal is inhibited by nmda-r antagonists, and animal data demonstrate similar inhibition of the expression of dependence produced by ethanol, barbiturates and benzodiazepines. the involvement of the excitatory amino acid, glutamate and the inhibitiory amino acid, gamma-amino butyric acid (gaba) in the pathophysiology of spinal cord trauma is not known in details. this investigation is focused on the involve-ment of glutamate and gaba in a rat model of spinal cord injury using immunohistochemistry. spinal cord injury induced by an incision into the right dorsal horn of the t10-11 segments resulted in profound edema formation and cell damage in the adjacent t9 and t12 segments at 5 h. pretreatment with h-290/51 (50 mg/kg, p.o.), a potent antioxidant compound, effectively reduced the edema formation and cell injury following trauma. at this time period, untreated traumatised rats exhibited a marked increase in glutamate immunoreactivity and a distinct decrease in gaba immunostaining in the t9 and t12 segments compared to the control group. the changes in glutamate and gaba immunoreactivity in traumatised rats were considerably attenuated by pretreatment with h-290/51. these results suggest that (i) oxidative stress contributes to alterations in glutamate and gaba in spinal cord injury, (ii) glutamate and gaba are contributing to edema formation and cell damage and (iii) the antioxidant compound h-290/51 has a potential therapeutic value in the treatment of spinal cord injuries. dov pharmaceutical, inc., hackensack, new jersey, u.s.a. both preclinical (i.e., behavioral despair models) and clinical studies indicate that compounds reducing transmission at nmda receptors are antidepressant. conventional antidepressants may be viewed as "monoamine-based", increasing the synaptic availability of serotonin, norepinephrine, and/or dopamine. however, chronic administration of of conventional antidepressants alters both mrna levels encoding nmda receptor subunits and radioligand binding to this family of ligand-gated ion channels in circumscribed areas of the cns indicating that nmda receptors may be a downstream target of these monoamine-based agents. we have recently reported (li, et al., neuropharmacology, in press ) that a class of ampa receptor potentiators also exhibits antidepressant-like actions in preclinical models. in this presentation, i will describe how these two distinct, and (at a cellular level) seemingly diametric approaches employing glutamatergic mechanisms converge on intracellular targets that are also impacted by chronic treatment with biogenic amine-based agents. kainic acid is an essential pharmacological tool for many forms of neurobiological research. until several years ago, all commercially available kainic acid was derived from a single biological source (digenia simplex). commercial isolation of kainic acid in japan ceased in 1999, creating a void in the marketplace. recently several different companies have become providers of kainic acid, but each uses a different source of the compound (2 biological and 1 synthetic) and different isolation procedures. our objective was to use three common assay systems to evaluate the comparative pharmacological and neurotoxicological properties of these three sources of kainic acid. dose response curves, both alone and in the presence of receptor selective antagonists, were constructed for each kainate formulation using (a) cerebellar granule neurons in culture, (b) isolated hippocampal slice preparations, and (c) whole animal behavioural toxicity studies. preliminary results reveal many similarities, but also distinct differences between the three formulations, especially when challenged with antagonists for different eaa receptors. full results will be presented and discussed with respect to their implications for both extending the known kainite literature and for future studies employing kainic acid as a ligand in both mechanistic investigations and in animal models of neurodegenerative disease. our results from in vitro studies further elucidate the role of cell-cycle related proteins in neuronal apoptosis induced by excitotoxins. exposure of primary cerebellar neurons to toxic concentrations of glutamate was found to produce a significant, short lasting increase in the expression of p53 and cdc2. transcriptional activity of p53 was shown by increased p53 dna binding activity and by the concomitant induction of the cdk inhibitor p21, the cell cycle regulator gadd 45 and the apoptotic induced bax. cell-cycle proteins are also expressed concomitantly to dna damage in neurons undergoing excitotoxic degeneration. we found that excessive activation of glutamate receptor by nmda results in the formation of 8-oh-deoxyguanosine, which is a marker of oxidative dna damage. in addition, the expression of the dna repair factor msh2 increases in cultured cerebellar neurons or in ca3 pyramidal cells that have been challenged with excitotoxins. excitotoxicity may thus provide a further example of how re-expression of cell-cycle proteins might be tightly connected to dna damage and repair in neurons. rush-presbyterian-st. luke's medical center, chicago, u.s.a. patients with parkinson's disease by definition benefit from levodopa therapy. however, after 5 years of therapy 50% of patients experience motor response complications (mrc's): the benefit from each dose becomes shorter (wearing-off), more unpredictable (on-off) and associated with involuntary movements (dyskinesias). when dyskinesias first arise, they are associated with high levodopa levels and may be prevented or minimized by lowering levodopa intake. later on, the therapeutic window of levodopa narrows progressively and dyskinesias occur at doses equal to those needed to induce an antiparkinson effect. while the pathogenesis of motor complications remains incompletely understood, recent clinical studies implicate mechanisms downstream from the degenerating nigrostriatal dopamine system, possibly involving glutamatergic projections to the basal ganglia. in a rat model of pd, blockade of striatal glutamate receptors of the n-methyl-d-aspartate (nmda) subtype reverses levodopa-induced motor fluctuations. similarly, in 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (mptp) lesioned primates, several nmda-antagonists reduce levodopa-associated dyskinesias. in parkinsonian patients the nmda-antagonists dextromethorphan, dextrorphan and amantadine improve dyskinesias as well. these findings have lead to the suggestion that hyperfunction of nmda receptors on striatal efferent neurons, as a consequence of chronic non-physiologic dopaminergic stimulation, contributes to the pathogenesis of motor response complications. protein misfolding and aberrant polymerization are salient features of virtually all central neurodegenerative disorders, including alzheimer's disease (ad), parkinson's disease, polyglutamine diseases, tauopathies, and prion diseases. in many instances, a single amino acid change can predispose to disease by increasing the production and/or changing the biophysical properties of a specific protein. possible pathogenic similarities among the cerebral proteopathies suggest that therapeutic agents interfering with the proteopathic cascade might be effective against a wide spectrum of diseases. however, testing compounds preclinically will require diseaserelevant animal models. numerous transgenic mouse models of ad-like pathology have now been produced. our studies have found that tg2576 mice overexpressing human -amyloid precursor protein (huapp695k670n/m671l) produce copious deposits of diffuse and compact -amyloid as they age, and that females are more susceptible than are males (callahan et al., am. j. pathol. 158, 1173 -1177 , 2001 . recently, we also found that the overexpression of p25 protein, an activator of the kinase cdk5, results in tau hyperphosphorylation, axonopathy and severe motor deficits in transgenic mice, in the absence of neurofibrillary tangles. none of the existing transgenic models of -amyloidosis or tauopathy fully recapitulates the pathology of ad. in an attempt to more authentically model the human disease, we infused dilute ad-brain extracts into tg2576 mice at 3-months of age (i.e. 5-6 months prior to the usual onset ofamyloid deposition). we found that infusion of ad brain extracts results in: 1) earlier and more abundant deposition of -amyloid in app-transgenic mice (kane et al., j. neurosci. 20, 3606-3611); 2) evidence for the spread of pathology to other brain areas, possibly by neuronal transport mechanisms; and 3) tau hyperphosphorylation (but not neurofibrillary pathology) in axons passing through the injection site. the seeding ofamyloid by ad brain extracts suggests pathogenic similarities between -amyloidoses such as ad and other cerebral proteopathies, and could provide a new model for studying the proteopathic cascade and its neuronal consequences in neurodegenerative diseases. supported by warner-lambert/pfizer. purpose: the effects of essential amino acid deficiencies on function of cornea and lens were investigated. methods: dietary deficiencies of tryptophane and methionine were studied in young rats over 3 months. transparency of cornea and lens were evaluated using slitlamp microscope and scheimpflug camera. after sacrifice, lens fresh weight and crystallin patterns were determined to evaluate effects on lens growth and protein synthesis. results: methionine deficiency had no effect on the parameters investigated. tryptophane deficiency caused severe loss of body weight in both rat strains (brown-norway, bn; sprague-dawley, sd), sd rats also lost their hair. they developed corneal neovascularisations and cortical cataracts. bn rats developed faint neovascularisations and a discontinuity zone in the lens. diet intermission arrested pathological processes restarting when feeding diet again. this observation is supported by lens fresh weight data. dna staining evidenced that tryptophane deficiency arrested lens fiber maturation. conclusion: a difference has been found for 2 essential amino acids in their effects on transparency of cornea and lens. tryptophane deficiency stimulated corneal neovasculariseration, but arrested lens fiber cell maturation. the difference in reaction of cornea and lens to tryptophane deficiency between bn and sd rat eyes remains to be elucidated. dynorphin is a neuropeptide that is present in the dorsal horn of the spinal cord. the peptide is actively involved in pain processing pathways. however, its involvement in spinal cord injury is not well known. alteration in dynorphin immunoreactivity occurs following a focal trauma to the rat spinal cord. infusion of dynorphin into the intrathecal space of the cord results in ischemia, cell damage and abnormal motor function. antibodies to dynorphin when injected into the intrathecal space of the spinal cord following trauma improves motor recovery and reduces edema and cell changes. however, influence of dynorphin on trauma induced alteration in spinal cord bioelectrical activity is still not known. spinal cord evoked potentials (scep) are good indicator of spinal cord conduction that are altered following trauma. therefore, in present investigation, influence of dynorphin antibodies on trauma induced changes in scep was examined in our rat model. in addition, spinal cord edema formation and microvascular permeability disturbances were also investigated. our results show that intrathecal administration of dynorphin antiserum prior to injury has a beneficial effect on trauma induced electrical activity, microvascular permeability disturbances, and edema formation. these observations indicate that dynorphin is somehow involved in the altered bioelectrical activity of the spinal cord and participates in the pathophysiological processes leading to cell injury. fatty-acid binding proteins (fabps) are involved in the intracellular binding, targeting and transport of long-chain fatty acids (fas) to modulate cell growth and/or differentiation. fabp form a family of proteins displaying tissue-specific expression. the expression of brain type fabp (b-fabp) is spatially and temporally correlated with neuronal differentiation during brain development. heart type fabp (h-fabp) is widely distributed and present in skeletal muscles, kidney, lung, brain and endothelial cells. it is neuron-specific in postnatal brain and participates in neurite formation and synapse maturation. epidermal type fabp (e-fabp) is expressed at high levels during neurogenesis, neuronal migration, and terminal differentiation. although all three fabps could be involved in normal brain function in prenatal and postnatal life, a neurobiological role of fabps in neurodegenerative diseases has not been reported yet. these made us evaluate the protein levels of fabps in brains from patients with down syndrome (ds) and alzheimer's disease (ad) and fetal cerebral cortex with ds using two-dimensional (2-d) gel electrophoresis with subsequent matrix-assisted laser desorption ionization mass spectroscopy (maldi-ms) identification and specific software for quantification of proteins. in fetal brain, b-fabp and e-fabp levels were comparable between control and ds. in adult brain, b-fabp was significantly increased in occipital cortex of ds, and h-fabp was significantly decreased in ds (frontal, occipital, parietal cortices) and ad (frontal, temporal, occipital and parietal cortices). we conclude that aberrant expression of fabps, especially h-fabp in neurodegenerative diseases could be involved in impaired neurite outgrowth and synapse maturation. in our previous paper, it was shown that gaba-a receptor antagonist picrotoxin suppressed etoh (ethanol) selfadministration. recently, several authors indicated that systemic injection of dopamine or serotonine agonists reduced ethanol drinking in rats. therefore, in the present study we investigated the effects of thip (4,5,6,7-tetrahydroizokasazolo, 5,4-c pyridin-3-ol) gaba-a receptor agonist in naive and long-term ethanol-experienced rats on etoh selfadministration and on cardiovascular system. adult 13-17week-old male, normotensive wistar-kyoto (wky) and spontaneously hypertensive rats (shr) were used. naive rats were examined according to smith method. long-term ethanolexperienced rats were studied according to boyle method. thip was injected in naive rats at a dose of 8 and 16 mg/kg i.p. metabotropic glutamate receptors are coupled to phospholipase c stimulation and adenylyl cyclase inhibition through g-proteins. c6 glioma cells, that endogenously express the phospholipase c coupled metabotropic glutamate receptor type, were treated with different specific agonists of these receptors and the effect of these treatments on different components of metabotropic glutamate receptor pathway was studied by radioligand binding, phospholipase c activity and rt-pcr assays. agonists treatment caused a decrease in l-[ 3 h]glutamate binding to intact cells and membranes in a time dependent manner being maximum at 3-6 hours and recovered at 24-36 hours. this decrease was associated with a significant increase in the mrna level coding mglurs. no changes on g q/11 mrna level were detected in any case. however, a significant decrease in l-glutamate stimulated phospholipase c activity was detected after agonist treatments in both membranes and intact cells. this decrease was not associated to significant variations in mrna level coding phospholipase c 1 isoform. all these results suggest that agonist exposure causes a desensitisation of glial metabotropic glutamate receptor decreasing not only receptors number but its functionality. in this study the interaction between these two nuclei were investigated by means of microinjection and microdialysis techniques in sprague-dawley rats. steroetaxic surgery was performed by placing intracerebral parenchymal microinjection cannula into the right dmh and microdialysis probe into the left pvn. iliac artery was also cannulated to monitor the pulsatile blood pressure and heart rate by means of pressure transducer connected to a polygraph microinjection of 50 pmol nmda into the dmh was performed and microdialysis perfusates were collected simultaneously from the pvn in conscious rat model. γ-aminobutyric acid (gaba) and l-glutamic acid levels were analyzed by an isocratic hplc (high pressure liquid chromatography) method with the aid of a fluorescent detector. microinjection of 50 pmol nmda into dmh produced significant increases in mean arterial pressure and heart rate. nmda microinjection into the dmh produced significant increase in l-glutamic acid release in the pvn, but no significant change in gaba release was observed. these results suggest that stimulation of dmh by nmda results in subsequent stimulation of the pvn. [this study was sponsored by marmara university research foundation (project no: 1998/sag/38).] and in long-term ethanol-experienced rats only at a dose 16 mg/ kg i.p. control group (cg) received saline 3 ml/kg i.p. as can be seen in fig. 1 and table 1 the lower consumption of ethanol in shr in comparison to wky rats was observed. systemic injection of thip decreased dosedependently etoh intake in naive rats of both strains. this effect was more pronounced in shr (fig. 1) . similar phenomenon was observed after thip injection in long-term ethanolexperienced rats. there were no effect on systolic blood pressure and heart rate after thip treatment. born-bunge foundation, university of antwerp, and university of ghent, belgium increased neuronal excitability may underlie some of the neurological complications in uremic patients. in an effort to identify candidate neuroexcitatory compounds, 17 different uremic retention solutes, including several amino acids and amino acid derivatives, were applied to mouse spinal cord neurons in primary dissociated cell cultures. using the tight-seal whole-cell technique, a few of the candidate toxins were shown to evoke whole-cell currents in cells clamped at ϫ60 mv. in a first survey, each of the solutes was briefly applied in a concentration of 5 mm. significant inward whole-cell currents were evoked by guanidinosuccinate, spermine, and 3-indoxyl sulfate, whereas phenol evoked an outward current. further experiments indicated that guanidinosuccinate-evoked whole-cell currents were due to activation of nmda-type glutamate receptors in concentrations similar to those found in uremic patients. high (mm) concentrations of spermine activated voltage-gated calcium channels, whereas low (µm) concentrations were found to potentiate guanidinosuccinate-evoked currents through its action on the nmda receptor-associated polyamine binding site. whole-cell currents evoked by 3indoxyl sulfate or phenol seemed to be due to complex interaction with several different ion channels. we conclude that guanidinosuccinate-evoked nmda receptor activation, possi-bly potentiated by the neuroexcitatory effects of polyamines and other putative uremic neurotoxins, could be an important mechanism underlying the increased neuroexcitability in uremic brain. glutamine (gln) is one of the key metabolites in the cns (energy metabolite, precursor of neurotransmitter amino acids, end product of ammonia detoxication, osmolyte), and as such is a routine supplement of cns cell culture media. c6 glioma cells relatively easily adapt to culturing in a gln-deprived medium. the present study investigated the effects of gln deprivation on the characteristics of the different systems that mediate gln cell membrane transport in the cells. in contrast to a variety of cns and non-cns cells, the absence of gln did not derepress the methyl-amino-isobutyric acid (meaib)-sensitive ("system adependent") uptake. system asc became relatively more-, and system n less active than in cells grown in the presence of gln, but the ion -and substrate specificity of the uptake remained unaltered. system asc in c6 cells grown in a glnsupplemented medium shows two features distinct from most other cell types: a) strong ph sensitivity and b) partial tolerance of lithium substitution, pointing to domination of system asct2 -an asc variant strongly expressed in cultured astrocytes. cells grown in gln-deprived medium lost lithium tolerance, but not ph-dependence of the uptake, their properties thus resembling system glnt (sat1), a neuron-specific variant of system a. by contrast, transport of threonine, a standard asc system substrate, was not affected by gln deprivation and showed neither ph dependence nor lithium tolerance, which is typical of an asc in all the non-cns tissues. (supported by scsr grant no. 4 p05a 060 18.) the classical the hypothalamic-neurohypophysial system (hns) is comprised of neurons originating within the supraoptic nucleus (son) which project to the neurohypophysis to release the nonapeptides oxytocin (oxt) and vasopressin into the blood after appropriate stimulation. previous experiments have shown that a single social defeat experience triggers the release of oxt from somata and dendrites into the extracellular fluid of the son, but not from axon terminals in the neurohypophysis. to further investigate the regulatory mechanisms underlying this dissociated release, we exposed male wistar rats to a 30-min social defeat experience and monitored the release of the inhibitory amino acids gamma amino butyric acid (gaba) and taurine into the son using microdialysis. social defeat caused a significant increase of the intra-son pre-stress basal release). to reveal the physiological significance of the intrahypothalamically released gababicuculline, a specific gaba a -receptor antagonist -was administered into the son by retrodialysis. this treatment increased significantly the release of oxt both within the son (200%; p ͻ 0.05 vs. pre-stress basal release) and -as measured via chronically implanted jugular venous catheters -into blood under basal and stress conditions (up to 200%; p ͻ 0.01 vs. prestress basal release). however, bicuculline did not affect plasma vasopressin. these data demonstrate that gaba is released within the son during social defeat to act as an inhibitor of both, central and peripheral oxt secretion during emotional stress. the mechanism described here contributes to the regulatory capacity of the hns to ensure the appropriate involvement of oxt in the stress response of the animal (supported by dfg, en 366-21). down syndrome (ds) is the most common human chromosomal abnormality caused by an extra copy of chromosome 21 and characterized clinically by somatic anomalies, mental retardation and precocious dementia. the phenotype of ds is thought to result from overexpression of a gene or genes located on the triplicated chromosome or chromosome region. reports that challenge this notion, however, have been published. to add to this body of evidence, the expression ofamyloid precursor protein (app), ets-2 and collagen α1 (vi) chain precursor, encoded on chromosome 21, was investigated in fetal brain by western blot and two-dimensional electrophoresis (2-de). western blot detected app and ets-2 that migrated at ϳ75 and 50 kda, respectively. subsequent densitometric analysis of app and ets-2 immunoreactivity did not produce any significant change between controls and ds. since the metabolic fate of app determines the propensity of amyloid production, the expression of the secreted forms of app (sapp) had been examined. neither the expression of sappα nor sapp showed any detectable changes among the two groups. collagen α1 (vi) chain precursor, a protein resolved as a single spot on 2d gel was identified by matrix associated laser desorption ionization mass spectroscopy. quantitative analysis of this spot using the 2d image master software revealed a significant decrease in fetal ds (p ͻ 0.01) compared to controls. linear regression analysis did not show any correlation between protein levels and age. the current data suggest that overexpression per se can not fully explain the ds phenotype. apoptosis is the mechanism by which cells are programmed to die under a wide range of physiological and developmental stimuli. accumulating evidence indicates that enhanced apoptosis (programmed cell death) in down syndrome (ds) may play a role in mental retardation and precocious neurodegeneration of the alzheimer-type. in this regard, alteration of several apoptosis related proteins have been reported in adult ds brain. fetal ds neurons exhibited increased reactive oxygen species leading to early apoptosis, however, expression of apoptosis related proteins in fetal ds, has never been considered. to address this issue, we investigated the expression of proteins involved in apoptosis including fas (cd95, apo-1), caspase-3, bcl-2 and annexins in the cerebral cortex of control and ds fetal brain by western blot and two dimensional electrophoresis. here, we report that no detectable changes were obtained in fetal ds brain in the expression of fas, caspase-3, bcl-2 and annexins (i, ii, v, and vi) compared to controls. in parallel experiment, we also examined the expression of neuron specific enolase (nse), a neuronal marker found to be decreased in adult ds brain, to see if there is any neuronal loss and no difference was observed between the two groups. protein expression did not correlate with age. the unchanged levels of fas, bcl-2 and annexins together with unaltered caspase-3 expression, a predominant caspase that executes apoptosis in the developing nervous system, suggest that enhanced apoptosis may not be apparent in fetal ds brain as demonstrated for adult ds brain. introduction. among the various metabolites indicating neuronal damage, amino acids are regarded particularly important. detection of amino acids by microdialysis is currently introduced as a neuromonitoring tool in patient care. here, we present changes in the extracellular concentrations of various amino acids in stroke patients and in experimental stroke in cats. method. cat focal ischemia was produced by occlusion of the middle cerebral artery (mca) for 1 h followed by 2 h reperfusion. glutamate, aspartate, gaba, taurine, glycine, serine, glutamine, methionine, threonine, tyrosine, asparagine, valine, phenylanaine, isoleucine and leucine were sampled by microdialysis in the ischemic core and subsequently analyzed by hplc. human microdialysis was performed in patients with large mca infarction. the microdialysis probes were inserted into primarily non-infarcted tissue in the border zone of the ischemic territory. results. transmitter amino acids rose immediately after occlusion in the cat model. correspondingly, these substances increased sharply in the human brain, when the tissue around the probes became infarcted, as shown by positron emission tomography (pet) and ct scan. in contrast, structural amino acids did not show marked increases or even decreased during severe ischemia in both, experimental ischemia and stroke patients. these substances did increase, however, when the brain tissue was only slightly ischemic, i.e. after reperfusion of the cat brain, when brain swelling occurred, or in human brain, when tissue did not show any infarction in the ct scan but hypoperfusion in the pet image. conclusion. extracellular amino acids detected by microdialysis can serve as markers for secondary ischemia. severe ischemia is reflected by rapid increases of transmitter amino acids, due to various mechanisms including synaptic release and reversal of reuptake systems. oligemia seems to be reflected by slow increases of structural amino acids, possibly due to a reduction in cerebral protein synthesis. apoptosis has been implicated in the selective neuronal loss of down syndrome (ds). apoptosis activates a family of cysteine proteases with specificity for aspartic acid residues referred to as, caspases that play a key role in dismantling a cell committed to die. caspases activity is regulated by a variety of proteins that possess a domain resembling the prodomains of caspases. little is known, however, about the changes of caspases and their regulatory proteins in ds. here, we investigated levels of nine such different proteins by western blot technique in frontal cortex and cerebellum of control and ds subjects. the protein levels of dff45 (dna fragmentation factor 45), and flip (fadd like interleukin-1 -converting enzyme inhibitory proteins) were significantly decreased whereas that of rick (rip-like interacting clarp kinase) increased in both regions of ds. in contrast, cytochrome c, apaf-1 (apoptosis protease activating factor-1), procaspase-9 and arc (apoptosis repressor with caspase recruitment domain) were unchanged. procaspase-3 and -8 were significantly decreased in frontal cortex but no significant change was observed in cerebellum. regression analysis revealed no correlation between postmortem interval and levels of the investigated proteins. however, inconsistent correlation was found between age and levels of proteins as well as amongst the density of individual proteins. these findings demonstrate that dysregulation of apoptotic proteins does exist in ds brain and may underlie the neuropathology of ds. the study further suggests that apoptosis in ds may occur via the death receptor pathway independent of cytochrome c. hence, therapeutic strategies that target caspase activation may prove useful in combating neuronal loss in this disorder. in order to examine the differential roles of nitric oxide (no) induced by either endothelial no synthase (enos) or neuronal no synthase (nnos) after transient cerebral ischemia, we investigated the effects of the relatively selective cnos inhibitor, l-n 5 -(1-iminoethyl)ornithine (l-nio), the relatively selective nnos inhibitor, 7-nitroindazole (7-ni) and the no scavenger, 2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazole-1-oxyl 3-oxide (ptio) on hippocampal dysfunction caused by cerebral ischemia. we measured no concentration, mean arterial blood pressure (mabp), hippocampal blood flow, direct current potential, ca1 population spike (ps) and release of amino acids from rat hippocampus after transient forebrain ischemia, which was induced by 4vessel occlusion for 10 min. l-nio (20 mg/kg), 7-ni (25 mg/kg) and ptio (1 mg/kg) were administered intraperitoncally 20 min before ischemia. ptio, 7-ni and l-nio reduced ischemiainduced no production in the hippocampus during the early period of reperfusion. the rank order of inhibitory potency was ptio ͼ 7-ni ͼ l-nio. l-nio, but not 7-ni, reduced hippocampal blood flow during ischemia and increased mabp before, during and after ischemia, compared with the vehicle group. ptio increased mabp during and after ischemia. ptio and 7-ni, but not l-nio, reduced amplitude of anoxic depolarization induced by ischemia. 7-ni recovered in part ps amplitude 60 min after ischemia. 7-ni, but not l-nio, reduced ischemiainduced release of aspartate and glutamate, but not taurine. the present study provides further evidence for the idea that in the early stages of transient forebrain ischemia, enos-derived no has a neuroprotective effect in the hippocampus, while nnos-derived no has a neurotoxic effect. the estrogen affects brain protein synthesis in ovariectomized female rats k. hayase 1 , m. tanaka 1 , k. tujioka 1 , e. hirano 1 , and h. yokogoshi 2 1 department of home economics, aichi university of education, kariya, aichi, and 2 laboratory of nutritional biochemistry, school of food and nutritional sciences, the university of shizuoka, japan the purpose of this study was to determine whether 17-estradiol affected the rate of brain protein synthesis in ovariectomized female rats. experiments were conducted on three groups of 12 wk old female rats: group 1. ovariectomized to reduce the level of plasma estradiol; group 2. ovariectomized and treated with estradiol; and group 3. sham-operated control. the fractional rates of protein synthesis in brain of ovariectomized rats treated with estradiol were significantly greater than in ovariectomized rats without estradiol treatment. in brain, the rna activity [g protein synthesized/(g rnaᮀd)] significantly correlated with the fractional rate of protein synthesis. the rna concentration (mg rna/g protein) was not related to the fractional rate of protein synthesis in any organ. the results suggest that estrogen treatment of ovariectomized female rats is likely to increase the rate of protein synthesis in the brain, and that rna activity is at least partly related to the fractional rate of brain protein synthesis. we have synthesized a series of new peptides that have demonstrated potent antidepressant activity in animal models for depression and in phase iia and iib clinical trials. mif-1 (prolyl-leucyl-glycinamide) an endogenous brain peptide has been reported to have some clinical activity in patients with unipolar depression with few apparent side effects. we have undertaken a study to determine the effect of molecular structural changes on the antidepressant activity of this peptide. we evaluated our new derivatives in a stress-induced animal model for depression, i.e. porsolt test, we have found that 4-f-phe-4-oh-pro-arg-gly-trp-nh 2 (inn 00835) is superior in all the statistical parameters used. in comparative testing inn 00835 was more active than prozac (fluoxetine) and zoloft (sertraline) in our antidepressant model. a u.s. patent has been granted on these compounds. the clinical results of inn 00835 show that it is effective in over 80% of depressed subjects when blood levels exceeded therapeutic threshold with no significant side effects. inn 00835 has a rapid onset of action, 3-5 days (vs. 2-6 weeks for currently marketed products) with sustained effects for months following 5 to 10 doses over 1-2 weeks. h. iwama 1 , 2 , a. umino 2 , a. hashimoto 2 , k. takahashi 2 , n. yamamoto 2 , and t. nishikawa 1,2 1 section of psychiatry and behavioral science, tokyo medical and dental university graduate school, and 2 department of mental disorder research, national institute of neuroscience, ncnp, tokyo, japan using an in vivo dialysis technique, we have studied the extracellular contents of endogenous free d-serine in comparison with that of l-serine, glycine and l-glutamate in the brain of the freely moving rat. a high amount of d-serine was detected in the dialysate obtained from the medial prefrontal cortex and striatum, whereas the cerebellar dialysate contained only a trace concentration of the d-amino acid. intra-medial prefrontal cortex perfusion of a sodium channel activator, veratrine, augmented the extracellular release of glycine and l-glutamate but a slight decrease in that of d-serine in a tetrodotoxin-sensitive manner in the prefrontal area. moreover, selective destruction of neuronal cell bodies in the medial frontal region by means of local infusion of an excitotoxin quinolinate resulted in a marked reduction of extracellular and tissue levels of d-serine in the infused prefrontal region. these findings suggest that endogenous d-serine might be liberated into the extracellular space from non-neuronal cells or certain exceptional neuronal cells probably by a carrier-mediated process in the mammalian prefrontal cortex. also, the endogenous d-amino acid has been indicated to be accumulated or synthesized, at least in part, in the neuronal cells. nucleoside diphosphate kinase (ndpk) catalyzes a transfer of the terminal phosphate from nucleoside triphosphates ((d)ntps) to nucleoside diphosphates ((d)ndps) and has been suggested to be involved in the regulation of wide variety of cellular functions. in addition, ndpk isoforms (a and b) are encoded by nm23 genes (h1 and h2) , which are related with the metastatic potential of some tumors. although ndpk/ nm23 has been also implicated to modulate neuronal cell proliferation, differentiation and neurite outgrowth, a neurobiological role of ndpk/nm23 in neurodegenerative diseases has not been reported yet. here we evaluated the protein levels of ndpk-a/nm23-h1 in brains from patients with down syndrome (ds) and alzheimer's disease (ad) using twodimensional (2-d) gel electrophoresis with subsequent matrixassisted laser desorption ionization mass spectroscopy (maldi-ms) identification and specific software for quantification of proteins. ndpk-a/nm23-h1 was significantly decreased in brain regions (frontal, occipital, parietal cortices) of both ds and ad compared to controls. we conclude that the down-regulated ndpk-a/nm23-h1 upon neurodegeneration could play a pivotal role in a wide range of neurobiological functions such as neurite outgrowth and consequently these could result in functional disturbance of the nervous system in ds and ad. brain α-endosulfine is manifold decreased in brains from patients with alzheimer's disease: a tentative marker? and drug target? α-endosulfine has the sulfonylurea-like ability to block atp-sensitive potassium (k atp ) channels and is expressed in a wide range of tissues. although the blockade of k atp channels has been reported to be involved in the release of neurotransmitters, the neurobiological role of α-endosulfine has not been studied yet. we examined the levels of αendosulfine protein in frontal cortex and cerebellum from patients with alzheimer's disease (ad). α-endosulfine was extremely decreased in both regions of ad compared to controls. this could result in the continuous opening of k atp channels with subsequent decrease of neurotransmitters release and change of potassium fluxes. this study is of great significance for providing a neurobiological function of α-endosulfine in brain and furthermore, α-endosulfine could serve as a useful marker for the diagnosis of ad and a target for drug treatment. children's hospital heidelberg, and department of pharmacology and toxicology, university of marburg, germany d-2-hydroxyglutaric aciduria is an inherited neurometabolic disorder of unknown etiology characterized by progressive neurodegeneration of vulnerable brain regions during infancy and early childhood, resulting in psychomotor retardation, hypotonia, seizures, macrocephaly, enlarged lateral ventricles, delayed cerebral maturation as frequent neurological presentation in affected children. the disease is biochemically characterized by the accumulation of d-2hydroxyglutarate (d-2), which is structurally similar to lglutamate (ϭ 2-amino-glutarate). we therefore investigated in primary neuronal cultures from chick and rat, whether d-2 induces excitotoxic neuronal damage. here we report that d-2 decreased cell viability in a concentration-and time-dependent fashion by disturbance of intracellular calcium homeostasis as determined by fura-2 measurement. furthermore, fluorescence microscopy using dihydrorhodamine-123 revealed an increased generation of reactive oxygen species (ros) elicited by expo-sure to d-2. n-methyl-d-aspartate (nmda) receptor blockade specifically prevented excitotoxic neuronal damage as well as increased calcium influx and ros production, suggesting that d-2 is an agonist at nmda receptors. patch-clamp investigation confirmed that d-2 activated recombinant nmda receptors in hek293 cells. furthermore, activity measurement of single respiratory chain complexes revealed a specific inhibition of complex v activity by d-2. we conclude that excitoxic mechanisms contribute to the neuropathology of d-2hydroxyglutaric aciduria, highlighting new neuroprotective strategies for this neurometabolic diseases. these studies were designed to determine the effects of aging and an aging intervention on nmda subunit expression. in situ hybridization and receptor autoradiography were performed on naïve or behaviorally-tested c57bl/6 mice of different ages (3, 10-15, and 26-30 months old) and diet groups (ad lib-fed and diet restricted). there were age-related decreases in both e2 and z1 mrna density in naïve, ad lib-fed mice. correlations were found between changes in e2 subunit mrna and agonist binding and z1 mrna expression and antagonist binding. diet restriction significantly improved learning ability, slightly spared glutamate binding to nmda sites and improved z1 mrna expression in older mice. significant correlations were found between agonist binding and both learning ability and e2 and e1 mrna density. learning ability in the old mice also correlated with the ratios of mrna expression for e1 and e2 and/or z1 subunits. these results suggest that changes in nmda receptor binding and the relationship between subunit expression levels are important for maintaining memory functions in older animals. extracts of st john's wort (hypericum perforatum l.) are widely prescribed for the treatment of mild to moderate depression and the putative antidepressant constituent is probably hyperforin. in this study the effect of hyperforin was investigated on the release of neurotransmitter amino acids. coronal cortical slices (400 mm) were cut and perfused with gassed (95% o2, 5% co2) acsf at 37°c. two-minute samples of perfusate were collected and aspartate and glutamate were assayed by hplc. potassium-and veratridine-stimulated release was elicited by administering 2 pulses of kϩ (60 mm) or veratridine (20 mm) 30 minutes apart. in control experiments the second kϩ pulse elicited glutamate release which was 80% of the first pulse. hyperforin (5 mm) perfused for 30 minutes prior to, and during, the second kϩ pulse significantly increased glutamate release to 170% (p ͻ 0.001, n ϭ 6-8). release elicited by the second veratridine pulse was 70% of the first pulse for both glutamate and aspartate. hyperforin (5 mm) increased this release to the second pulse to 160% and 130% respectively (p ͻ 0.001, n ϭ 6-8). when perfused on its own for 30 minutes, hyperforin (5 mm) increased the basal release of glutamate (p ͻ 0.001, n ϭ 4-5). in conclusion, the increase in the release of neurotransmitter amino acids observed following hyperforin is possibly mediated through a facilitatory action on voltage-operated ca2ϩ or naϩ channels. glaxosmithkline group, glaxo wellcome s.p.a., medicines research centre, verona, italy n-methyl-d-aspartate (nmda) receptors are ligand gated ion channels widely distributed in mammalian brain, which play a crucial role in important physiological mechanisms, such as excitatory transmission, neuronal migration and memory formation. a peculiar feature of nmda receptors is the absolute requirement of l-glutamic acid and glycine for the opening of the channel. noteworthy, these two aminoacids reciprocally modulate binding at their respective recognition sites. aim of this work was to study nmda receptor glycine/glutamate interactions in rat and human brain. binding of the nmda antagonist [ 3 h]cgp39653 to rat cerebral cortical membranes was inhibited by glycine. the overall effect of glycine consisted in a decrease of [ 3 h]cgp39653 affinity, with a parallel increase of the receptor affinity for glutamate. the glycine antagonist gv150526a competitively reversed glycine inhibition, proving that the modulation was via the glycine binding site. [ 3 h]cgp39653 binding to rat brain sections revealed the existence of regionally distinct nmda receptor subtypes with difference glycine/glutamate interactions. in most regions of the human brain nmda receptors presented the same allosteric modulation of [ 3 h]cgp39653 binding, as revealed by large section autoradiography technique. nevertheless, detection of any regional variation was not possible, probably due to the high intersubject variability. the effect of long-term high k ϩ -treatment on neuronal survival, cellular maturation, nmda receptor (nr) splice variant expression, and receptor function was investigated in primary cultures of rat cortical neurones. long-term incubation (up to 15 days) with 25 mm k ϩ significantly increased neuronal survival and induced multiple morphological changes associated with promoted cellular maturation. cultures grown in medium containing 25 mm k ϩ also exhibited multiple changes in nr1 splice variant expression according to rt-pcr studies performed with primer pairs flanking the alternatively spliced regions, in order to estimate the ratios of the corresponding 3ј and 5ј splice variants. nr1-1 and nr1-3 (each containing exon 21) were decreased, whereas nr1-2 and nr1-4 (each lacking exon 21) were increased, accordingly. the predominant expression of nr1-b was further increased. after administration of ttx, each of the k ϩ -induced changes on mrna expression was virtually abolished. in voltage-clamp recordings (holding potential: ϫ70 mv), nmda induced inward currents in a concentration-dependent manner with a maximum effect of ϫ745 pa under control conditions. neurones treated with 25 mm k ϩ showed a significantly diminished response to nmda (max. response: ϫ368 pa). in conclusion, the present data indicate that a sustained increase in neuronal activity induces adaptive changes in nr1 splice variant expression and a decrease in receptor function. thus, alternative splicing associated with a diminished receptorcytoskeletal linkage may be important compensatory mechanism in preventing cellular damage due to long-term activation of excitatory nr. it seems conceivable that this mechanism contributes to the promoting effects of 25 mm k ϩ on neuronal survival and maturation. (supported by bmbf grant 01gg9818/0) there is accumulating reports that kainate-induced seizures elicit expression of various heat-shock proteins (hsps) in the brain, such as hsp27, hsp32, and hsp70. however, no investigation has been carried out on changes in level of apg-2, a member of hsp 110 family, after excitatory amino acid-induced seizures. by means of an immunoblot assay, we determined the levels of hsp70 and apg-2 in discrete brain structures of mice after a single intraperitoneal injection of kainate or nmda. apg-2 level was significantly decreased in the frontal cortex, hippocampus, and striatum 3 days after kainate administration, while hsp70 level was increased in these regions following the administration. decreased level of apg-2 returned to the control levels in the three regions 10 days after kainate administration. no significant changes were observed in levels of both hsp70 and apg-2 in hypothalamus, midbrain, medulla-pons, and cerebellum of kainate-treated mice. by contrast, nmda administration did not significantly affect both levels in any of the regions examined. these results suggest that, unlike the case of hsp70, apg-2 expression could be temporarily down regulated by signals peculiar to kainate, but not by those peculiar to nmda, in murine telencephalon. high concentrations of glucagon-like peptide-1 (7-36) amide (glp-1) and its specific receptor (glp-1r) have been found in the rat hypothalamus. in this study the actions of glp-1 and its related peptides, exendin-4 (glp-1r agonist), exendin (9-39) (glp-1r antagonist) and glp-1(9-36)amide (major glp-1 metabolite) on levels of amino acids (glu, asp, gln, gly, tyr, trp, gaba) in the hypothalamus were investigated. 125 i-glp-1 binding in rat hypothalamic membranes was competed by the peptides in the following order of potency; glp-1 ͼ exending-4 ͼ exendin (9-39) ͼ glp-1(9-36)amide. intracerebroventricular (icv) glp-1 (4 nmoles) produced a statistically significant reduction in levels of all measured amino acids compared with saline injected controls, whereas 4 nmoles of exendin (9-39) was ineffective. exendin-4 produced a statistically significant reduction in the levels of trp, glu, and tyr. glp-1(9-36)amide showed a statistically significant increase in the level all the amino acids tested in this study. prior administration of exendin (9-39) or glp-1(9-36)amide blocked the effects of glp-1 on the levels of the amino acids. these data are consistent with exendin-4 being a glp-1r agonist and exendin (9-39) being a specific glp-1r antagonist. glp-1(9-36)amide, a primary metabolite of glp-1, appears to act as an endogenous antagonist at the glp-1r. department of biophysics, instituto de fisiología celular, universidad nacional autónoma de méxico, méxico city, méxico cholecystokinin (cck), a family of neuropeptides, seems to be involved in anxiety. evidence from several laboratories indicates that the ansiogenic effects of cck are mediated by cck b receptors. however it has been reported that cck a receptors have been found in brain and cck a receptor antagonists have ansiolytic properties. the aim of this work was to study whether or not cck a receptors are also involved in the modulation of anxiety. male rats were cannulated in the lateral ventricle and cck (9 fmol) and/or cck antagonists (900 fmol) were injected 7 days after surgery. anxiety was evaluated in the elevated plus-maze test and locomotion in an open-field test. ansiogenic effects were observed when cck b receptor agonists (cck8ns; cck4) or a mixed cck b and cck a receptor agonist (cck8s) were injected. in contrast, cck33, a cck a receptor agonist or cck 1-21 and cck 26-29 were uneffective. furthermore, the ansiogenic effects of cck8s were prevented by the previous (15 min) administration of l365,260 (cck b receptor antagonist) but not by devazepide (cck a receptor antagonist). no effects on locomotion were observed in any condition. these results indicate that cck a receptors are not involved in anxiety, as measured by the elevated plus-maze test. congenital conditions (i.e. neural tube defects: ntg) have a multifactorial aetiology. deficiencies in the folate and transsulfuration pathways have, in recent years, been positively linked to ntd and other dysmorhogenic syndromes. efficient one-carbon metabolism is crucial for the synthesis of dna precursors, the remethylation of homocysteine and biomethylation of dna. more than 80% of the one-carbon units that flow through the metabolic system in mammals and birds are derived from l-serine and glycine, the natural substrates for shmt. the mitochondrial glycine cleavage enzyme system (gces) can potentially compete with shmt for tetrahydrofolate (thf) in the generation of the methylenetetrahydrofolate pool. valproate (depakene, epilim), an anti-epileptic agent, appears to be strongly associated with hyperhomocysteinemia, several other induced metabolic conditions, the inhibition of the gces and an increased incidence of ntd in epileptic women of child-bearing age. the exact mechanisms of valproate-induced ntd are not yet clear. we investigated the association of the teratogenic properties of valproate with the inhibition of shmt and/or the gces in developing embryos. chicken embryos were treated with sodium valproate (vpa) and pregnant female mice (c57bl) received intraperitoneal injections of vpa, during the critical period of embryonic neural tube development. control embryos were treated with sterilised saline solution. harvested embryos were subsequently investigated for congenital abnormalities and hepatic shmt and gces activities quantified with radiometric assays. the effect of vpa on hepatic dna synthesis was monitored ( 3 h-thymidine incorporation into embryonic dna) and the dna-methylation status determined (dna n 5 -methylcytosine levels). dose-responsive incidences of ntd were observed in vpa treated embryos. very few defects occurred in control embryos. shmt and gces appeared to be inhibited in liver extracts of vpa-treated embryos. hepatic dna synthesis was significantly compromised and 5-mc levels were altered in vpa-treated embryos. the inhibition of either shmt and/or gces activities appeared to be associated with valproateinduced ntd in the chicken and mouse embryo models. the primary mechanism of this effect can probably be ascribed to a restriction in the flow of one-carbon units through the metabolic system, decreased synthesis of dna precursors and alterations in the methylation status of dna. department of neuroscience, university of cagliari, italy ethanol is long known to cause dose-related biphasic effects and we recently found that ethanol bidirectionally affects also working memory. the euphoriant and excitatory effects produced at low doses are associated with the rewarding action of ethanol and are thought to be mediated by the activation of the mesolimbic dopamine (da) system. however, ethanol monophasically stimulates mesolimbic da release in the nucleus accumbens, even at doses that cause hypnosis and coma. in contrast, ethanol biphasically modulates mesocortical da release in the prefrontal cortex (pfc). the changes in da release induced by ethanol are time locked with corresponding changes in extracellular glutamate levels. these biphasic effects of ethanol on pfc da and glutamate are matched by biphasic changes in the performance in a spatial delayed alternation task -a working memory test that is sensitive to proper function of the pfc -suggesting a link between da and glutamate transmission in the cognitive effects of ethanol. focal application in the pfc of the competitive ampa/kainate receptor antagonist cnqx suppresses both da release and the improvement of working memory induced by low doses of ethanol. these results suggest that ethanol may increase da transmission in the pfc and enhance working memory functions by increasing the release of glutamate, thereby stimulating non-nmda glutamate receptors. the enhancing effect on working memory by low, excitatory doses of ethanol may be perceived as rewarding and could constitute an important neurobiological mechanism for excessive ethanol drinking. physiology department, faculty of medicine, al-quds university, jerusalem, palestine glutamate and asparate are considered as the main excitatory neurotransmitters in brain and spinal cord, in addition to their role in energy metabolism, synthesis of proteins and detoxification of ammonia. glutamate and aspartate are centrally involved in basic mechanisms generating epileptic seizures and in epileptogenesis. stimulated release of glutamate and aspartate was detected in vivo and in vitro following neuronal depolarization. photic stimuli has increased glutamate release from visual cortex, and afferent brachial stimulation has increased the endogeneous release of glutamate from contra-lateral sensorimotor cortex compared to ipsi-lateral side. similar results were achieved after local application of tityustoxin or veratridine to the sensorimotor cortex. implantation of cobalt powder over the right sensorimotor cortex of rats produced an epileptogenic lesions characterized by contra-lateral fore and hind limb jerks and an increase in the frequency of eeg spikes. the jerks started after 6 days with maximum myoclonic activity (15 jerks/min). the concentration of glutamate in the epileptogenic focus was decreased significantly by 29% (p ͻ 0.01) compared to the non-epileptogenic area on the left sensorimotor cortex, which was dissected but not treated with cobalt. part of the decrease in glutamate could be related to the enhancement of in-vivo release from the epileptogenic lesion to the extra-cellular fluid. kindling is the best model for studying the development of the epileptic focus (epileptogenesis), it could be achieved by repeated intra-cerebral micro-injection of glutamate (1.5 µ mol), aspartate (0.75 µ mol) or nmda (2-4 n mol), or repeated electrical stimulations of specific brain regions. in addition, glutamate antagonists particularly those specifically acting on the nmda receptor type e.g. 2-amino-5-phosphonovaleric acid (ap5) and 2-amino-7-phosphonopheptanoic acid (ap7) have been found to inhibit seizures in epileptic animals and inhibit the development of electrically kindled epilepsy. pre-synaptic glutamate receptor agonists like (1s, 3s)-acpd the agonist of group ii, and l-ap 4 the agonist of group iii receptors has reduced ca ϩϩ uptake and glutamate release, thus it has inhibited epileptogenesis by preventing the increase in both seizure score and after-discharge duration. injection into fully kindled animals has produced an anti-epileptic effect by reducing the mean seizure score and by increasing the mean generalized seizure thresholds. this results suggest the mechanism by which pre-synaptically active glutamate receptor agonists block the development of the chronically epileptic state induced by electrical kinding, and indicate that their anticonvulsive activity is due to inhibition of pre-synaptic glutamate and/or asparate release following blockade of pre-synaptic ca ϩϩ entry. testing the changes in glutamate release from hyperactive brain tissues, and the effect of different glutamate agonists and antagonists, supports the role of glutamate in initiating the process of epileptogenesis, and contributes in developing new anti-epileptic agents. (this project was supported by a grant from alexo) the functional roles of cl(ϫ) and divalent cations in the na(ϩ)/cl(ϫ)/gaba cotransport were examined in xenopus oocytes expressing the human gat-1 (hgat-1) gaba transporter cdna. our results showed that cl(ϫ) was not absolutely required for na(ϩ)/gaba transport via the hgat-1 (loo et al., j biol. chem. 275:37414-37422, 2000) . the cl(ϫ) interacted with the transporter to modulate the binding of external na(ϩ). although hgat-1 transported cl(ϫ) across the membrane with a stoichiometry of 2na(ϩ) : 1cl(ϫ) : 1gaba, the transported cl(ϫ) did not contribute to the net charge translocated across the membrane, suggesting a cl(ϫ)/cl(ϫ) exchange mechanism during the gaba transport cycle. the gaba transport via the hgat-1 is also modulated by divalent cations. the uptake of [3h]-gaba was inhibited significantly when both ca(2ϩ) and mg(2ϩ) were removed from the uptake buffer. several divalent cations tested were individually able to sustain the gaba uptake. in contrast to uptake, the gaba efflux was enhanced significantly upon removal of both ca(2ϩ) and mg(2ϩ) from the efflux buffer. the gaba transporter inhibitor skf89976a blocked the enhanced efflux, suggesting that the hgat-1 operated faster in the reverse mode in the absence of external divalent cations. these results suggest a regulatory role for the divalent cations in gaba transport. merck sharp & dohme, neuroscience research centre, terlings park, harlow, essex, u.k. the role of alpha5 containing gaba a receptors in hippocampal synaptic function has been investigated using pharmacological and electrophysiological techniques, as well as following disruption of the alpha5 subunit gene in knockout mice (ko). in the ca1 region of the hippocampus the induction of long-term potentiation (ltp) is powerfully regulated by gaba mediated synaptic currents (ipscs). agents that inhibit gaba-mediated transmission potentiate ltp induction, whereas allosteric agonists such as benzodiazepine-site agonists which slow the decay kinetics of ipscs suppress ltp induction. in alpha5 ko mice paired pulse facilitation of the amplitude of excitatory synaptic potentials is selectively enhanced in the ca1 region but not dentate gyrus. likewise, the frequency and rise time of spontaneous ipscs were similar in wt and ko slices. however their amplitude was significantly smaller in ko mice. furthermore, a significantly greater proportion of ipscs were best fitted to a mono exponential function in ko mice compared to wt animals. thus alpha5 containing gaba a receptors contribute to functional postsynaptic receptors on ca1 pyramidal cells in the hippocampus and modulate a postsynaptic component of synaptic facilitation. pharmacological research institute, volgograd medical academy, volgograd, russia the purpose of the study is to investigate effect of phenil (karphedon, mephebut, gammoxin) and circle (pyracetam) gaba derivatives on reproductive function of stressed male rats. the adult male rats were stressed by immobilization exposure (2 hours) twice in week during 6 weeks. four from five groups of stressed males were given substances (daily) at doses: karphedon -50 mg/kg, mephebut -10 mg/kg, gammoxin -40 mg/kg, pyracetam -200 mg/kg. the treated males were mated with intact females during 12 days. after the mate the treated males and more in 10 days all the mated females were sacrificed and investigated. analysis of our data indicates that the time of spermatozoa motion and epididymal sperm counts were decreased 73.2% (p յ 0.05) and 49.1% (p յ 0.05) respectively when compared with their intact controls. gaba derivatives have a softening effect on functional parameters of spermatozoa stressed males. karphedon and pyracetam increased the time of motion spermatozoa 86.3% (p յ 0.05), karphedon and mephebut drew near sperm counts to intact control level. the result of mate show that pregnancy rate was increased (p յ 0.05) by stress exposure and pregnancy rate of females mated with gaba stressed males was some more (p ն 0.05) than that of intact controls. the general embryonic morality was increased twice by stress and so the number of embryos was reduced 48.8%. the gaba derivatives exposure to stressed male rats reduced the embryonic mortality of their posterity and increased the number of embryos to intact control level. our findings demonstrate that gaba derivatives administration has a protective effect on reproductive function of stressed male. transmission on the brain. the realization that glutamatergic pathways are involved in such diverse processes in epilepsy, ischemic brain damage and parkinsons' disease, is of a great practical interest. there are at least three functional classes of ionotropic glutamate receptors: n-methyl-d-aspartate (nmda), α-amino-3-hydroxy-5 methyl-4-isoxazolepropionic acid (ampa) and kainate (ka). other central neurotransmitter systems are under nmda influence. some data point on neuroprotective action of nmda antagonist on nigrostriatal pathway. in the present study female wistar rats were exposed during pregnancy with daily injected mk-801 (dizocilpine) 0.5 mg/kg sc. control rats received tap water only. behaviour of 3 month old male offsprings was investigated by several psychopharmacological methods. oral activity, yawning, locomotor activity, stereotypy and catalepsy were recorded following respective central dopamine receptors agonists and antagonists administration (skf 38393, quinpirole, apomorphine, haloperidol). our results indicate that mk-801 applied during pregnancy modulate reactivity of the central dopamine receptors in adult offspring rats. [ the development of mammalian ingestive behavior is characterized by a transition from suckling to chewing, two distinct motor behaviors. we hypothesize that this transition is accompanied by changes in brainstem circuitry underlying these movements. since glutamatergic neurotransmission is critical for the proper functioning of brainstem circuitry responsible for mastication, we investigated the development of glutamate receptors in trigeminal motoneurons (mo5) and mesencephalic trigeminal neurons (me5); neurons comprising the circuitry responsible for jaw movements. we conducted a series of receptor immunohistochemistry experiments that characterized the expression of iontotropic and metabotropic glutamate receptors (mglurs) during early postnatal development. the functional roles of nmda, ampa and mglurs in neonatal mo5 were investigated using in vitro electrophysiological experiments. results demonstrated that the spatial and temporal expression of ampa, nmda and group i and ii mglurs are developmentally regulated within and between mo5 and me5 during early development. electrophysiological data demonstrate that mglurs function pre-and postsynaptically to modulate synaptic transmission between trigeminal premotoneurons and mo5. furthermore, nmda induced bursting is developmentally regulated and coincident with the transition from suckling to chewing behaviors. our studies suggest that the transition from suckling to chewing is accompanied by changes in the composition and function of glutamate receptors. fetal life in down syndrome starts with normal neuronal density but impaired dendritic spines and synaptosomal structure r. weitzdoerfer 1 , m. dierssen 2 , m. fountoulakis 3 , and g. lubec 1 1 department of pediatrics, university of vienna, austria information on fetal brain in down syndrome (ds) is limited and there are only few histological, mainly anecdotal reports and no systematic study on the wiring of the brain in early prenatal life exist. histological methods are also hampered by inherent problems of morphometry of neuronal structures. it was therefore the aim of the study to evaluate neuronal loss, synaptic structures and dendritic spines in the fetus with down syndrome as compared to controls by biochemical measurements. 2 dimensional electrophoresis with subsequent mass spectroscopical identification of spots and their quantification with specific software was selected. this technique identifies proteins unambiguously and concomitantly on the same gel. fetal cortex samples were taken at autopsy with low postmortem time, homogenized and neuron specific enolase (nse) determined as a marker for neuronal density, the synaptosomal associated proteins alpha snap [soluble n-ethylmaleimidesensitive fusion (nsf) attachment protein], beta snap, snap 25 and the channel associated protein of synapse 110 (chapsyn 110) as markers for synaptosomal structures and drebrin (drb) as marker for dendritic spines. nse, chapsyn 110 and beta snap were comparable in the control fetus panel and in down syndrome fetuses. drebrin was significantly and remarkably reduced and not even detectable in several down syndrome brain samples. quantification of snap 25 revealed significantly reduced values in ds cortex and alpha snap was only present in half of the ds individuals. we conclude that at the time point of about 19 weeks of gestation (early second trimester) no neuronal loss can be detected but drebrin, a marker for dendritic spines and synaptosomal associated proteins alpha snap and snap 25 were significantly reduced indicating impaired synaptogenesis. early dendritic deterioration maybe leading to the degeneration of the dendritic tree and arborization, which is a hallmark of down syndrome from infancy. pathfinding of growing axons to reach their target during brain development is a subtle process needed to build up contacts between neurons. abnormalities in brain development in down syndrome (ds) are described in a couple of morphological reports but the molecular mechanisms underlying abnormal wiring in fetal ds brain are not yet elucidated. we therefore performed a study using the proteomic approach to show differences in protein levels involved in the guidance of axons between control and ds brain in early prenatal life. proteins obtained from autopsy of human fetal abortus were applied on 2-dimensional gel, identified and quantified. we quantified 5 members of the semaphorin/collapsin family, the dihydropyrimidinase related proteins 1-4 and the collapsin response mediator protein-5 (crmp-5) in 8 ds and 7 control cortex samples. drp-1 and crmp-5 levels were comparable in the control and ds samples. evaluation of drp-2, drp-3 and drp-4 revealed significantly decreased levels of 2 of the 15 spots assigned to drp-2 and increased levels of one spot assigned to drp-3 and increased drp-4 in ds brain. we conclude that as early as from the 19 th week of gestation pathfinding cues of the outgrowing axons are impaired in ds. these findings may help to elucidate mechanisms leading to abnormalities in neural migration of ds brain. inflammatory processes play an important role in the degeneration of basal forebrain cholinergic cells alzheimer's disease. the proinflammagen lipopolysaccharide (lps) was infused chronically into the basal forebrain of young rats. we then determined whether the administration of two novel nonsteroidal anti-inflammatory drugs or a pancaspase synthesis inhibitor, zvad, could provide neuroprotection from the cytotoxic effects of the neuroinflammation. we also determined whether the administration of the non-competitive n-methyl-d-aspartate (nmda) receptor antagonist, memantine, could provide neuroprotection from the cytotoxic effects of the neuroinflammation. chronic lps infusions decreased choline acetyltransferase activity and increased the number of activated microglia within the basal forebrain. caspases 3, 8 and 9 activity was increased in ventral caudate/putamen. non-steroidal antiinflammatory drug therapy attenuated the toxicity of the inflammation upon cholinergic cells and reduced caspases 3, 8, and 9 activity in the caudate/putamen. zvad significantly decreased the levels of caspases 3, 8 and 9 but did not provide neuroprotection for cholinergic neurons. memantine significantly attenuated the cytotoxic effects of chronic inflammation upon cholinergic cells. these results suggest that prostaglandins contribute to the degeneration of forebrain cholinergic neurons in alzheimer's disease and that the cytotoxic effects of prostaglandins occur upstream to nmda receptor activation. intracranial administration of n-methyl-d-aspartate (nmda) receptor antagonists block learning of classical and avoidance conditioning in goldfish. studies with goldfish have shown that nmda receptors are mostly dense in the telencephalon and telencephalon ablation impairs avoidance learning. the present study investigated amnestic effects of microinjection of nmda receptor antagonist ap5 to the goldfish telencephalon in avoidance conditioning. in experiment 1, fish received no injection or microinjections of saline or various doses of ap5 to their telencephalon 20 minutes before three semiweekly training sessions. fish were tested without injec-tions in session 4. a one-way anova with multiple comparisons on the test scores showed that ap5 produced anterograde amnesia in a dose-dependent manner. in experiment 2, fish received several training sessions and a microinjection of various doses of ap5 20 minutes before testing. the test scores showed that ap5 did not decrease avoidance responses, suggesting that microinjection of ap5 did not impair performance processes. in experiment 3, fish received microinjections of ap5 or saline to their telencephalon immediately following three semiweekly training sessions and were tested without injections in session 4. a one-way anova on the test scores showed that ap5 did not produce retrograde amnesia. (supported by gvsu grant-in-aid.) tryptophan modulates striatal serotonergic activity relative to fatigue t. yamamoto 1 and e. a. newsholme 2 1 health science laboratory, tezukayama university, nara, japan 2 department of biochemistry, university of oxford, u.k. we have been reported that mechanism of fatigue in the brain relates to enhanced extracellular tryptophan and serotonergic function. brain concentration of tryptophan is not only dependent on the change of tryptophan which originates from the centarl nervous system, but also enhance tryptophan entering the brain from the blood-brain barrier and peripheral circulating tryptophan which is a trigger. supplementation of ltryptophan (2 um) into the incubation medium with the synaptosomal striatum causes tryptophan to the extrasynaptosomal release by high kϩ stimulation. injecting l-tryptophan (1 mm/ 30 min) into the left striatum by microdialysis method can induce early fatigue for running time of rats. on the other hand, tryptophan deficiency rats (body weight average 200 g) were made by tryptophan free feeding for 2 weeks, and the rat's running time increased (ͼ100 min difference). these results suggests that tryptophan is a potent active substance for fatigue in the brain. the active zone may be presynaptic terminal and the tryptophan itself may be releasing neuromodulators. (we appreciate that tryptophan free diet was provided by ajinomoto co., inc., japan.) our recent studies on the distribution of free d-serine, together with the d-serine action on the glycine site of the nmda type glutamate receptor, suggest that the d-serine can be an endogenous modulator of the nmda receptor. to explore the possible removal systems for brain d-serine signaling, we have evaluated the uptake of [3h]d-serine into the synaptosomal p2 fraction from the rat cerebral cortex. the cortical p2 fraction was able to accumulate [3h]d-serine in a temperatureand ph-dependent and saturable manner. the kinetic analysis indicates that cortical d-serine transport occurs by an apparent single-component system with km value of 283 µm and a vmax value of 207 pmol/mg protein/min. depletion of na ϩ and cl ϫ ions remarkably decreased d-serine uptake into the cortical p2 fraction. the pharmacological profile of the inhibition of dserine uptake by various amino acids was different from those of glycine uptake system and other amino acid transporters reported. d-serine uptake activity was preferentially observed in the brain tissues such as cerebral cortex and cerebellum to the peripheral tissues. the present data support the view that the endogenous d-serine is taken up mainly through a carriermediated transport system to regulate the extracellular concentration in the mammalian brain. a. bocheva et al. the mammalian brain contains all the urea cycle intermediates, whereas enzymes participating in the conversion of lornithine (l-orn) into l-citrulline (l-cit) are absent, resulting in an incomplete urea cycle. the discovery of nitric oxide (no) synthase that catalyses the formation of no and l-citrulline as a co-product from l-arginine (l-arg) in the brain has indicated an additional pathway for l-arg metabolism. l-canavanine (l-cav), is a potent antimetabolite and structural analog of larginine, produced by legumes such as the jack bean, canavalia ensiformis. l-canaline (l-can) is a potent inhibitor of ornithine aminotransferase. our previous results indicated that l-cav, l-cit, l-arg, and l-orn exerted an antinociceptive effect, whereas l-canaline induced hyperalgesia in rat. l-canavanine exert stronger antinociceptive effect than l-arginine, l-ornithine and l-citrulline. the aim of the present study was to investigate are d-arg, l-cav and naloxone reversed the analdesic effects of l-ornithine, l-citrulline and l-arginine. the experiments were carried out on male wistar rats. the changes in the mechanical nociceptive threshold of the rats were measured by the radall-selitto paw pressure test using and analgesimeter (ugo basile). the amino acids were applied intracerebroventricularly (i.c.v.) at a dose 20 µg/rat. the present results shown that d-arg, l-cav and naloxone reversed antinociception. the regulation of lysine metabolism in cereal crops r. a. azevedo 1 , p. j. lea 2 , s. a. gaziola 1 , a. p. pellegrino 1 , and s. m. g. molina 1 1 departamento de genética, escola superior de agricultura luiz de queiroz, universidade de são paulo, brazil 2 department of biological sciences, university of lancaster, u.k. a major nutritional drawback of cereal seeds is a deficiency in some amino acids, in particular lysine. biochemical, molecular and genetic studies have considerably increased our knowledge concerning the regulation of the aspartate pathway, by which lysine is synthesized. among the enzymes involved in lysine metabolism, aspartate kinase (ak) and dihydrodipicolinate synthase (dhdps) control the regulation of lysine biosynthesis, whereas lysine: 2-oxoglutarate reductase (lor) and saccharopine dehydrogenase (sdh), have been shown to play a key role in the breakdown of lysine. in general, lysine overproduction can be obtained by altering the sensitivity of dhdps to lysine, but accumulation of this amino acid in cereal seeds requires further manipulation of lor and/or sdh. this suggestion is strongly supported by five main points: (1) cereal mutant or transgenic plants do not exhibit any significant accumulation of lysine in seeds, but only in other tissues. (2) the enzymes of lysine degradation, lor and sdh, are endosperm specific in cereals only. (3) the opaque-2 mutant, which exhibits higher concentration of soluble lysine and protein lysine in the seed, contains several-fold lower lor and 2-fold lower sdh activity when compared to the wild-type maize. this reduction in activity in the opaque-2 mutant is due to a reduced protein lor-sdh concentration by reduction of the zlkrsdh gene transcript. furthermore, the opaque-2 maize gene has been shown to regulate ak and lor activity. (4) intermediates of lysine catabolism accumulated in the seeds of soybean and canola lysine overproducing plants, suggesting the presence of reduced lor and/or sdh activities. (5) among cereals and although still below the recommend values by fao, rice exhibits the higher concentration of lysine, but lor and sdh are present in much lower activities. also, in phaseolus vulgaris, lor and sdh activities were shown to be around 10fold lower then in maize endosperm. the regulation of the lor activity is complex and involves a calcium dependent phosphorylation/dephosphorylation mechanism. it remains to be seen whether this latter mechanism can be controlled, so as to allow the production of more crop plants that contain elevated concentrations of lysine in the seed. the genetic progress for nue can be accelerated with the use of secondary traits that possess high inheritance and correlation with productivity. several traits have been studied such as chlorophyll concentration, plant height, leaf senescence, anthesis-silking interval, kernel number, activities of enzymes of n assimilation and loci of quantitative traits for assisted selection. (we are grateful for financial support from fapesp, brazil and the british council.) (termed hyperaccumulators) that grow on metalliferous soils, are able to translocate cd from the roots and accumulate it in high concentrations in the shoots. cd may be detoxified in plants by combination with a family of sulphur rich peptides termed phytochelatins. cd has the capacity to inhibit a range of enzyme activities in plants, in particular those of the calvin cycle and chlorophyll biosynthesis. evidence that cd causes the production of reactive oxygen species (ros) has also been obtained. we have investigated the antioxidant responses of radish, soybean and sugarcane to cd treatment. seedlings were grown in increasing concentrations of cdcl 2 , ranging from 0.01-1 mm, for up to 96 h in a hydroponic system. analysis of cd uptake indicated that most of the cd accumulated in the roots, but some was also translocated and accumulated in the leaves. roots and leaves were analysed for catalase (cat), glutathione reductase (gr) and superoxide dismutase (sod) activities. gr activity increased considerably in the roots of all plant species tested after exposure to the metal, indicating a direct correlation with cd accumulation. cat activity also increased in roots but to a much lesser extent when compared to gr and also varied depending upon the plant species. the analysis of native page enzyme activity staining, revealed several sod isoenzymes in leaves of all plant species, however, only in radish was a clear increase in activity observed. the results suggest that in these plants, the activity of antioxidant enzymes responds to cd treatment. the main response may be via the activation of the ascorbate-glutathione cycle for the removal of hydrogen peroxide, or to ensure the availability of glutathione for the synthesis of cd-binding proteins. (we are grateful for financial support from fapesp, brazil and the british council.) all plant cells, tissues and organs provide the biosynthetic machinery and capacity to synthesise aliphatic polyamines. however, in physiological conditions only some organs and tissues synthesise polyamines, such as apical buds and sprouts, root apex, lateral buds of branches and secondary roots, as well as superficial layers of young stems and leaves, like epidermis, subepidermis and parenchyma cells. apical roots can also synthesise polyamines, but these activities in physiological conditions are lower than that of the shoots. this patterns recalls the one of auxins. polyamines are accumulated in high concentrations in storage organs, such as seeds, but not in tubers like helianthus tuberosus, potato or tuberised roots such as the carrot. also some fruit, e.g. oranges, contain high level of free polyamines, putrescine in particular. all other organs obtain polyamines through translocation via phloem tubes and xylem vessels. in plants, in addition to free polyamines, many polyamines are conjugated to hydroxycinnamic acids, the hydroxycinnamic amines, that only rarely represented outside the plant kingdom. this compounds are paticularly abundant in solanaceae family, where they can represent as much as 90% of the total polyamine pool, but they can be detected in different concentrations in many other families. the role of free and conjugated polyamines and their importance in food is discussed. drought, salinity or other environmental stressors promote the accumulation of free amino acids, amines and other organic n-metabolites with low molecular weight. in this contribution the influence of drought on the accumulation of amino acids, polyamines and trigonelline in growing barley plants and barley grains was examined. in comparison to non-stressed plants we obtained in stressed plants, exposed to drought before flowering, a higher concentration of proline (increase: 4-fold), n-trimethylglycine (2-fold), histidine (3-fold), tryptophane (1,7-fold), putrescine (1,4-fold), spermine (1,7-fold) and trigonelline (2-fold) in the dry matter of barley sprouts. in addition to this, drought caused an increase of the n-content in the plant biomass (35%) as a result of growth inhibition (34%). six weeks later the content of soluble n-metabolites and protein was analyzed in non-stressed and pre-stressed barley plants again. during this reproductive period of plant development all the test groups were cultivated under the same moisture conditions. the analysis of n-metabolites in the ripening grains showed, surprisingly an after-effect of the drought stress. for example, in grains of pre-stressed barley the concentrations of free proline, histidine, tryptophane and asxϩglx were threefold to fivefold higher than in grains of non-stressed barley. depending on the resistance of barley cultivars to drought the biochemical response was different: in plants with low resistance the increase of amino acids and amines was higher than in resistant cultivars. however, resistant cultivars have already high genuine concentrations of n-metabolities in non-stressed plants. by treatments with choline or 2-aminoethanol the stresspromoted accumulation of amino acids and trigonelline was diminished. consequently, different biochemical responses of cereals to drought result in changes of product quality and nitrogen use. our goal is to increase the lysine content in corn. we have used genetic engineering to increase lysine synthesis and to prevent metabolic breakdown of lysine. to increase synthesis we circumvented the normal feedback control of a key enzyme in the lysine biosynthetic pathway, dihydrodipicolinic acid synthase (dhdps). lysine-feedback-insensitive dhdps, encoded by the corynebacterium dapa gene, was expressed from seed-specific promoters in transformed corn seeds. expression of dhdps in the corn embryo, but not in the corn endosperm, resulted in a 20 to 30-fold increase in the accumulation of free lysine in the seeds and the total seed lysine content nearly doubled. lysine breakdown products have been observed in transgenic seeds that accumulate high levels of free lysine. we isolated a corn gene for the bifunctional enzyme lysine ketoglutarate reductase (lkr)/saccharopine dehydrogenase (sdh), which catalyzes the first two steps in lysine breakdown. knockout of lkr/sdh in corn by either mutation or genetic engineering results in a 10-fold increase in seed free lysine. combination of feedback-insensitive dhdps with knockout of lkr/sdh results in 2 to 3-fold higher levels of free lysine than dhdps alone. no adverse effects on seed or plant agronomic performance are associated with the high lysine trait. biotechnology center for agricultural and the environment and the plant science department, rutgers university, new brunswick, new jersey, u.s.a. 5ј-adenylylsulfate (aps) reductase catalyzes a key reaction in the plant sulfate assimilation pathway leasing to the synthesis of cysteine and the antioxidant glutathione. in arabidopsis thaliana aps reductase is encoded by a family of 3 genes. in vitro studies revealed that the enzyme product derived from one of the aps reductase genes (apr1) is activated by oxidation, probably through the formation of a disulfide bond. redox titrations show that the regulation site has a midpoint potential of ϫ327 mv at ph 8.5 and involves a 2-electron redox reaction. exposure of a variety of plants to ozone induces a rapid increase in aps reductase activity that correlates with the oxidation of the glutathione pool and is followed by an increase in free cysteine and total glutathione. during the response to ozone the level of immuno-detectable aps reductase enzyme does not increase. treatment of a. thaliana seedlings with oxidized glutathione or paraquat induces aps reductase activity even when transcription or translation is blocked with inhibitors. the results suggest that a post-translational mechanism controls aps reductase. a model is proposed whereby redox regulation of aps reductase provides a rapidly responding, self-regulating mechanism to control the glutathione synthesis necessary to combat oxidative stress. in aspergillus nidulans the structural genes coding for nitrate reductase (niad) and nitrite reductase (niia), share a common promotor region of 1,200 bp. we have previously characterized in vitro and in vivo the physiologically relevant cisacting elements for the two synergistically acting transcriptional activators, nira and area. we have further shown that area is constitutively bound to a central cluster of four gata sites and is directly involved in opening the chromatin structure over the promoter region and thus making additional cis-acting binding sites accesible. here we show that the asymmetric mode of nira-dna interaction determined in vitro is also found in vivo. binding of the nira transactivator is not constitutive as in other binuclear c 6 -zn ϩϩ -cluster proteins but depends on nitrate induction and additionally, on the presence of a wild type area allele. dissecting the role of area further, we found that it is required for intracellular nitrate accumulation and therefore could indirectly excert its effect on nira via inducer exclusion. but in a strain accumulating nitrate independently of area nira binding and chromatin rearrangement is not triggered by nitrate in the absence of area. v. nikiforova 1 , m. zeh 1 , o. kreft 1 , s. maimann 1 , h. hesse 2 , and r. höfgen 1 1 max-planck-institut für molekulare pflanzenphysiologie, potsdam, and 2 institut für biologie, angewandte genetik, freie universität berlin, germany higher plants, being a source of reduced sulfur for animal nutrition, assimilate inorganic sulfate into cysteine which is subsequently converted to methionine, another sulfur-containing amino acid. in order to investigate the possible regulatory points of the cysteine and methionine biosynthesis pathway a series of transgenic potato plants was engineered using clones encoding enzymes of the branched pathway from serine to cysteine as a pathway intermediate and from aspartate further on to methionine. increased cysteine levels were obtained in the leaves of serine acetyltransferase (sat) sense and cystathionine -lyase (cbl) antisense transformants. furthermore, glutathione levels were elevated in sat plants while downregulation of cbl was desastrous for plant growth, eventually. increased methionine levels were successfully obtained in potato by antisense inhibition of threonine synthase (ts). accumulation of free methionine was not only observed in source leaf tissues but as well in tubers. this enzyme competes with cystathionine gamma-synthase for the common substrate o-phosphohomoserine at the branchpoint between threonine and methionine synthesis, respectively. important control points of the biosynthesis of cysteine and methionine in potato, thus, turned out to be sat and ts, while further studies on overexpression of cystathionine gamma-synthase, cbl and ms did not reveal any substantial effect on potato methionine biosynthesis. dniepropetrovsk national university, board of biophysics and biochemistry, ukraine amino acids in root exudates of plants may be chelate agents as an alpha-amino acid can act like a bidentate ligand, forming a five-membered heterocyclic ring with suitable metal cations thus increasing mobility of metals. recently we have showed that application of growth regulators led to sharp increase of root exudative activity of some cultural (zea maize l.) and wild cereals (festuca rubra l., lollium perenne l.) during first days of germination. in this work we present results obtained in experiments with lollium perenne l., grown on sterile sand and on soils contaminated with great quantities of zn. detailed analysis of amino acid content of root exudates of several types of maize (hybrid, several lines, an opaque-2 mutant line) showed that the specie had more certain amino acids (cysteine, aspartic and glutamic acids and their amides, serine) in root exudates than cultural ones. these amino acids has more possibility for chelation due to existance of one more polar or ionogenic functional groop. seeds of lollium perenne l. were treated with growth regulater and planted on soils contaminated with salts of zink. it was shown that during 15 days of germination quantity of zn in primary leaves increased from 121,46 to 243,75% and decreased in soil: in upper layer from 95,74 to 85,07, midde layer from 95,83 to 82,04, lower layer from 85,72 to 72,74 mkg/kg correspondingly. thus, it was shown that stimulation of root exudative activity by pretreatment with a growyh regulator may be succesful in cleaning of soils and basicly this is a good method for phytoremediation. erenol exerted the strongest effect. exercise completely abolished the levels of cysteine in the atrial heart muscle. propranolol, isoproterenol, caffeine and pentylenetetrazol increased the ratio of cysteine to the total free amino acids in the atrial muscle, while physical stress and all cardioactive drugs tested increased this ratio in the ventricle muscle. disappearance of cysteine from the heart's atrial muscle after intensive exercise may be attributed to its utilization for atrial natriuretic factor and/or for endothelin synthesis, during stress. on the other hand it seems that hypoxia and isoproterenol are strong stimulants of no production, and consequently decrease the tissue levels of l-arginine, which is the major endogenous donor of no acting as the endothelin antagonist. measurement of serum levels of vitamin b12 is a screening test for detection of deficiency of this vitamin but low levels do not always indicate a deficiency of the vitamin. measurements of serum homocysteine and methylmalonic acid (mma) are used to confirm this deficiency because two enzymes involved in their metabolism have been shown to require vitamin b12, but these results can also be inaccurate. vitamin b12 deficient white cells exhibit ultrascopic nuclear appenages which have been shown to contain dna; this finding could possibly be used as another confirmatory test of vitamin b12 deficiency. twenty-seven patients (mean age -76.6 years) with low serum b12 were studied by electron microscopic determination of the percent of neutrophils exhibiting these appendages and routine clinical parameters. only one patient did not have nuclear appendages; the others had a range of 0.8%-17.6% of neutrophils examined. there was a significant correlation of homocysteine (r ϭ .46, p ͻ .05) and mma (r ϭ .53, p ͻ .01) with serum b12 levels but no correlation of appendage number (r ϭ .18) with serum b12. there was no correlation of appendage number with homocysteine (r ϭ .25) or mma (r ϭ .04). these results suggest that b12-deficient white cell nuclear appendages do not measure the same metabolic pathways as homocysteine and methylmalonic acid and may be useful in confirmation of vitamin b12 deficiency. further extensive clinical evalution would be necessary to explore this possibility. the hypothesis: "l-theanine has relaxing effects of central nervous system of human beings", was verified by electroencephalographical methods. methods: 14 male, healthy sport-students, free of drugs or stimulants, participated weekly in a cross-over study. after exhaustive bicycle-ergometer test as an individual, reliable, stress model, the subjects recovered by lying in a segregated shaded room. three testdrinks with different l-theanine content (d1 ϭ placebo, d2 ϭ 50 mg, d3 ϭ 200 mg) were given in a randomised, double-blind order. all test-conditions were standardized strictly. eeg-recordings (closed eyes) were carried out (m1 ϭ 3 min. after stress/before testdrink, m2 ϭ 30 min.-, m3 ϭ 45 min.-, m4 ϭ 60 min.-, m5 ϭ 120 min. after testdrink) with the cateem ® system. absolute and relative eeg-spectralpower were examined. results: significant reductions in all frequencies (exception theta-power) were found in early recovery, being not significant influenced by testdrinks. qualitative different behavior trends were found in frontal-, central-, occipital-regions with increased alpha1, theta (frontal) and decreasing beta1 relative-power earlier in recovery with d3. these findings were related to relaxing effects. after ingestion of l-theanine alpha2-, beta1-power at occipital regions decreased faster (m2) to placebo recovery levels (m3/m4). thus it may be concluded that l-theanine has no pharmaceutical effect on the down regulation system but supports the physiological mechanisms during recovery after physical stress in human brain. arginine and cysteine in muscle cytosol of rats' heart after exercise, hypoxia or challenge with six selected cardioactive drugs r. brus 1 , j. gabryś 2 , j. konecki 2 , and j. shani 3 1 department of pharmacology and 2 department of histology and embriology, medical university of silesia, zabrze, poland 3 department of pharmacology, the hebrew university school of pharmacy, jerusalem, israel levels of the amino acid l-arginine (a major endogenous donor of nitric oxide-no), cysteine (sulfur-containing amino acid, important for atriopeptins and endothelins synthesis), and of total free amino acids, were assayed by gas-liquid chromatography in cytosols of rats' atrial and ventricular muscle cardiomyocytes. the tissues were assayed after the rats had been exposed to either exercise (swimming), hypoxia or one of six cardioactive drugs such as propranolol, digoxin, pentylenetetrazol, reserpine, isoproterenol and caffeine. physical stress and the examined drugs significantly reduced the total amount of cytosolic free amino acids in both cardiac muscles. in the cytosol of the heart atrial muscle, reserpine, propranolol and pentylenetetrazol increased the relative content of l-arginine, while hypoxia and digoxin decreased it. in the cytosol of the ventricular heart muscle, hypoxia and all six drugs used, decreased the relative levels of l-arginine. hypoxia and isoprot-addition of somatostatin-14 or of some of its analogs was found to cause a selective inhibition, up to 50%, of the uptake of large neutral amino acids by isolated brain microvessels. although the luminal and abluminal sides of brain endothelial cells are both capable of taking up large neutral amino acids, only the uptake from the abluminal side was apparently inhibited by somatostatin. the involvement of a type-2 somatostatin receptor was suggested by assays with a series of receptorspecific somatostatin agonists, and was confirmed by the inhibition release caused by a specific type-2 receptor antagonist. a type-2 specific mrna was indeed shown to be present both in bovine brain microvessels ex vivo and in primary cultures of endothelial cells from rat brain microvessels. hemorphins represent a bioactive peptide class which contents between 4 and 10 amino acids and generated from the proteolysis of an hemoglobin "strategic zone". many activities have been related to hemorphins such as in vitro anti tumour effect, analgesia effects in vivo, and a potential role in the renin angiotensin system (ras). as far as their activity towards the ras is concerned, it was demonstrated that they could inhibit angiotensin converting enzyme (ace) and aminopeptidase n activity. so they could reduce angiotensin ii formation and angiotensin iv degradation. moreover some hemorphins, lvv-hemorphin-7 and vvhemorphin-7, could behave like angiotensin iv receptor binding competitor. further it could be interesting to study the angiotensin iv potentiality to interact with ace. inhibition studies showed that it was possible that angiotensin iv could behave like a competitive inhibitor of ace. so some hemorphins could interact at different ras steps to inhibit ace. additionally to their inhibition of angiotensin i conversion, they could inhibit angiotensin iv degradation and consequently cause ace feedback inhibition. inhibition studies have been checked with ras natural substrate (angiotensin i) and confirmed that angiotensin iv, vv-hemorphin-7 and mainly lvv-hemorphin-7 could be natural ace inhibitors. so the hemorphins regulatory role in the ras appears to be more and more probable. the role of administration of each of methionine and finastride on the testicular function of both normal and prostate precancerous old male rats was investigated. for normal animals, neither methionine nor finastride has exerted any significant change in the hormonal profile after teatment for 20 days. however methionine alone could exert a significant change in both testosterone and prostatic specific antigen {psa} levels after treatment for 40 days. on the other hand, both methionine and finasteride significantly increased the levels of testosterone and androstenedione, whileas markedly reduced the levels of dihydrotestosterone and prostatic specific antigen {psa} after treatment of prostate precancerous old male rats for a period of 20 days. noteworthy, continuation of treatment for aperiod of 20 days realized marked improvement of hormonal profile of the prostate precancerous old male rats. several observations in our department point to some role of glycine in fatigue and exercise. 1) in the framework of a study on the involvement of one-carbon matabolism in patients suffering from a polymorphic episodic psychosis, amino acid loading tests with serine, glycine and alanine were performed. a few hours after the administration of glycine, approximately 40% of the patients reported overwhelming feelings of fatigue and/or showed vegetative symptoms. 2) in patients suffering from chronic fatigue syndrome, we found increased plasma levels of glycine in 20% of the female patients. moreover, 60-70% of these patients omplained about a distorted sensory perception of objects. 3) young soccer players were observed during a period of 10 months, while in the course of this period eight blood samples were taken for amino acid analysis. based on the number and severity of injuries this population was divided into injury-prone and not injury-prone soccer players. it was found that in injury-prone soccer players plasma glycine levels during the whole observation period were significantly lower than in subjects who were not injury-prone. the consequences of the above mentioned observations will be discussed. institute of sportsmedicine, university of paderborn, germany 50 percent of amino acids in green tea leaves are represented by l-theanine (5-n-ethylglutamine). previous rat experiments demonstrated effects of l-theanine to act on metabolism of neurotransmitters. it was therefore suggested that is causes the relaxing effects of green tea. to examine its influence as a component of a drink on the sympathetic nervous system after maximal physical exercise skin resistance measurements through electrosympathicography (esg) were used. after individual maximal exercise on a bicycle-ergometer testdrinks with different amounts of l-theanine (0, 50 and 200 mg) were administered to 14 healthy volunteers in a randomised cross-over double-blind distribution on a weekly base. esg was monitored before and immediately after exercise as well as 13, 30, 45, 60, 75 and 135 minutes after end of exercise. all testconditions were standardized strictly. a characteristic esgcourse with subsequent qualities could be shown: 1. decreasing skin resistances after exercise could be established in each volunteer. 2. esg-activation levels before exercise could not even be reached again after a period of regeneration of 2 1 / 4 hours. 3. maximal electrodermal activity did not appear immediately after exercise, but after 13 minutes. however, l-theanine could not significantly influence peripheral sympathetic electrodermal activity during the regeneration after maximal physical exercise. a. mero 1 and h. pitkänen 1,2 1 neuromuscular research center, department of biology of physical activity, university of jyväskylä, and 2 rehabilitation center of kankaanpää, finland essential amino acid leucine has many important roles in the body. therefore the purpose of the present study was to investigate if leucine supplementation has effects on serum amino acid profile and performance following training period or following single training sessions. all experiments were carried out in a randomized double blind cross-over procedure during a training season. thirty six adult male track and field power athletes served as subjects. in experiment 1 ten of them were given leucine (50 mg/kg body weight per day) as tablets. the concentration of leucine decreased significantly (20%) in the placebo group (p; n ϭ 10) during 5 weeks but not when leucine was taken. also total amino acids (taas) decreased strongly (19%) during 5 weeks when dally protein intake was 1.26 g/kg body weight. in experiment 2 the subjects (n ϭ 16) carried out a single strength training seasion (sts) and consumed a drink containing leucine 100 mg/kg body weight. following sts leucine in serum increased by 11% (ns) when leucine was taken but decreased strongly (30%) in p, in experiment 3 the subjects (n ϭ 12) underwent at 7 days interval two maximal anaerobic running exercise (mare) tests on treadmill (n ϫ 20s with a recovery of 100s between the runs) until exhaustion. the subjects consumed drinks containing leucine (200 mg/kg body weight) or placebo 50 min before the test runs. following mare the concentration of leucine strongly increased by 28% whereas isoleucine (25%) and valine (15%) strongly decreased with the supplementation but no changes occurred in p. there were no improvements in physical performance either in mare or in explosive strength (experiment 2) with leucine supplementation. the date suggest that leucine supplementation during a training period and before single training sessions prevents decreases in serum concentration of leucine and may have also effects on some other single amino acids. this may be beneficial during intensive training although improvements in performance were not observed in this study. since there are only limited data regarding effects of training period or training sessions on serum amino acid profile, the purpose of this study was to investigate serum amino acid changes following training period and following three different training sessions. the subjects consisted of 31 track and field adult male power athletes. in experiment 1 eleven of them performed a 5-week training period including a training sessions per week, which included sprint work, speed endurance work, endurance work, weight training, and jumps. significant decreases in the fasting concentrations of total amino acids (taas) (19%), branched chain amino acids (bcaas) (21%), essential amino acids (eaas) (19%) and leucine (20%) were observed following training with the daily protein intake of 1.26 g/kg body weight. in experiment 2 eleven subjects performed a short run session (srs) of 3 ϫ 4 ϫ 60 m with recoveries of 120 s and 360 s, and a long run session (lrs) of 20 s runs with recoveries of 100 s until exhaustion. there were no significant changes in taas following the sessions but bcaas decreased by 8% in srs and by 7% in lrs. leucine decreased by 10% following srs but only by 5% (ns) following lrs. the peak blood lactate concentrations after srs and lrs were 13.8 ϯ 1.9 mmol/l and 16.4 ϯ 1.3 mmol/l, respectively. in experiment 3 sixteen subjects carried out a strength training session (sts), which consisted of jumps and heavy resistance exercises (speed and maximum strength) during 90 minutes. the taas decreased significantly by 14%, bcaas by 24% and leucine by 30% following sts, while the peak blood lactate concentration was 2.2 ϯ 0.4 mmol/l. these data indicate that remarkable decreases occur in the concentration of amino acids during a training period with the daily protein intake of 1.26 g/kg body weight. the decreases in serum amino acids are more pronounced following a strength training session than following lactic anaerobic running sessions. glutamine acts as a multipurpose regulator of amino acid and peptide transport across the blood-brain barrier departments of cellular biotechnology and haematology and of biochemical sciences, university "la sapienza", rome, italy isolated brain microvessels, the in vitro equivalent of the blood-brain barrier, have distinct na ϩ -independent uptake systems for the uptake of large hydrophobic amino acids, of enkephalins and of deltorphins, as shown by the absence of reciprocal inhibition. both d-and l-glutamine were capable, if added to the extracellular buffer, of exerting a competitive inhibition on the uptake of all these substrates. a trans-stimulatory effect was instead induced, in all cases, by l-glutamine preloading of the microvessels -the d-stereoisomer being instead ineffective, probably because of only l-glutamine could be taken up, in a concentrative manner, by some na ϩ -dependent concentrative system(s). all the na ϩ -independent systems present in brain microvessels seem therefore to share some structural feature responsible for their common susceptibility to interference by l-glutamine. this amino acid, whose synthesis can take place in the astrocytes, in the pericytes and also in the endothelial cells of the microvessels, plays a critical role in regulating the movements of several different substrates across the blood-brain barrier. department of applied bioorganic chemistry, division of life science, graduate school of agricultural science, tohoku university, aoba-ku, sendai, japan isolation and structure analysis of two amino acids from bovine ligamentum nuchae elastin hydrolysates revealed the presence of pyridine cross-links in elastin. the structures of these amino acids were determined to have 3,4,5-and 2,3,5-trisubstituted pyridine skeletons both with three carboxylic acids and a mass of 396 (c 18 h 28 n 4 o 6 ), identified as 4-(4-amino-4-carboxybutyl)-3,5-di-(3-amino-3-carboxypropyl)pyridine and 2-(4-amino-4-carboxybutyl)-3,5-di-(3-amino-3carboxypropyl)-pyridine. we have named these pyridine cross-links, desmopyridine (desp) and isodesmopyridine (idp), respectively. structure analysis of these pyridine crosslinks implied that the formation of these cross-links involved the condensation reaction between ammonia and allysine. the elastin incubated with ammonium chloride showed desp and idp levels increased as the allysine content decreased. desp and idp were measured by hplc with uv detection and were found in a variety of bovine tissues. the desp/desmosine and idp/isodesmosine ratios in aorta elastin were higher than in other tissues. desp and idp contents in human aorta elastin were found to be gradually increased with age. the concentration of idp was significantly elevated in aorta elastin of rat with chronic liver cirrhosis induced by carbon tetrachloride when compared with normal rats. the provision of glutamine to marathon runners has resulted in a decreased, self-reported incidence of illness. increasing evidence -in vitro; and in vivo suggests that neutrophils in humans may benefit from exogenous glutamine. the provision of glutamine in vivo should replete the marked decrease in the blood concentration observed after stress such as clinical trauma or prolonged, strenuous exercise. beneficial effects of glutamine supplementation include increased phagocytic activity and reactive oxygen intermediate production in vitro; decreased neutrophilia and il-8 production (a chemoattractant for neutrophils) in vivo and ex vivo. the aim of the present study was to establish whether glutamine supplementation in vitro and in vivo affects neutrophil function at rest and after exhaustive exercise. in addition, it was planned to establish the presence of glutaminase in human neutrophils, which has not yet been achieved, although glutaminase is present in rat neutrophils. methods: blood samples were taken from marathon runners receiving either glutamine or placebo, immediately after and one hour after a race. measurements included the plasma concentration of glutamine (enzymatic assay), il-8 production (elisa), and neutrophil activity. the latter was measured with two different techniques for measuring oxidative burst in whole blood, one of which was a novel chemiluminescence assay (knight scientific ltd, u.k.) with the fluorescent label, pholasin, and two different stimuli, f-met-leu-phe (fmlp) and phorbol-myristate-acetate (pma). in addition, isolated whole cells and subcellular neutrophil fractions were assayed for the presence of glutaminase. results: the plasma glutamine concentration was reduced overall by 26% 1 hr after the race (p ͻ 0.02). there was an apparent decrease (only close to significance, p ͻ 0.13) in il-8 production in the glutamine group compared with the placebo group. neutrophil function did not change between groups at any stage. the incidence of illness was 46% higher in the placebo group than the glutamine group in the week after the race. neutrophils from four out of six subjects gave an increased response (39.2%) to fmlp when incubated with glutamine compared with no glutamine, and four out of four gave an increased response to pma (31.1%). in the fmlp experiments there were two individuals who did not respond to the addition of glutamine. however, the response was not diminished whether or not glutamine was present. in separate studies, the effect of glutamine on lipopolysaccharide-induced il-8 production was also monitored. conclusions: the provision of glutamine after prolonged, exhaustive exercise appears to modify exercise-induced neutrophilia via a reduction in il-8 production and to reduce the incidence of illness in the following week. in vitro data suggest a role for glutamine in neutrophil metabolism. disappointingly, little or no evidence of the presence of glutaminase was found in human neutrophils. the three different methods used, freeze-thaw, homogenisation, nebulisation were apparently not sufficient to break open the granules. current studies are addressing this problem. r. j. ward 1 and l. m. castell 2 departments of biochemistry, 1 university catholique de louvain, belgium 2 oxford university, oxford, u.k. it is essential that the developing muscle has adequate amino acids for the synthesis of actin and myosin as well as those required for a multitude of enzymes involved in muscle metabolism. with carbohydrates and lipids, the body is able to store a reserve as glycogen and triglycerides respectively; however this is not the case with amino acids creatine supplementation in increasingly being used as a dietary supplement by athletes during high intensity, short term exercise to improve physical performance since it is converted in the muscle to phosphocreatine. transporters which permit creatine to cross the muscle membrane namely crt1 and crt2 (a na ϩ and clј dependent mechanism) have now been identified. creatine uptake is enhanced by the ingestion of carbohydrate at the same time as supplementary creatine. this may be due to increased circulating levels of insulin or insulin-like growth factor 1. more recently attention has been focussed upon the various transporters for amino acids across the muscle membrane. certain criteria are needed for the amino acids to enter the blood which include the presence of specific carriers for its transport across cells of the gastrointestinal tract, such as enterocytes, as well as minimal metabolism within these cells. a wide number of different transporters has been identified, which include neutral amino acids and cationic amino acids. despite the evidence which suggests that supplementation with some amino acids can influence metabolism, and therefore athletic performance, much more experimental work is still required in this area. m. weiss 1 , t. barthel 1 , r. schnittker 1 , k. e. geiss 3 , w. falke 3 , and l. r. juneja 2 1 university of paderborn, germany 2 taiyo kagaku co., yokkaichi, japan, 3 isme gmbh mörfelden, germany in animal studies l-theanine was shown to influence neurotransmitter systems. thus it may be helpful in managing stress regulation. so we observed the down regulation after physical stress in the brain (measured by eeg-mapping) and in peripheral hormonal systems (plasma levels of catecholamines, cortisole, prolactine, serotonine, measured by hplc). n ϭ 14 healthy students consumed drinks containing either 0, 50 or 200 mg l-theanine in randomized double-blind trials in the min 6-14 after a near maximal bicycle step test. measurements were done directly after exercise (m1) and 30 (m2), 45 (m3), 60 (m4), 120 (m5) min after the drink. l-theanine seemed to accelerate the normalization of eeg spectral power in high frequency waves (barthel in this congress). the physiological return of increased hormon levels to basal levels / the circadianic rhythm up to m2 (catecholamines) or m5 (cortisole, serotonine, prolactine) was not influenced by the drinks. but in the l-theanine trials correlations between eeg spectral power and some hormones were altered (slow wave power/some catecholamines except norepinephrine/delta disappeared and new correlations with prolactine appeared). thus we conclude that l-theanine acts at the switch from the brain to the peripheral stress regulation and thereby supports physiological relaxing after severe exercise. polyamines the development from callus to plantlets, both activities increased, reaching the maximum at this latter stage. also sadenosylmethionine decarboxylase activity displayed a similar trend. all the activities were present in supernatant and in particulate fraction. higher activity of enzymes assayed in the small embryos rather than in the embryo with higher shape, was consistent with following polyamine accumulation. department of biology, laboratory of cell biochemistry, university of rome "tor vergata", rome, italy intracellular transglutaminase (tg, ec 2.3.2.13), which catalyzes the formation of ε-(γ-glutamyl)lysine isopeptide cross-links between polypeptides, has been related to a variety of important biological processes and in the development of senile cataract. the majority of the dry weight of the eye lens is composed of protein called crystallins. in the mammalian lens, these proteins are divided into three major classes: α-, -and γcrystallins. native -crystallins are oligomers, which elute in two or more size classes during gel filtration, ranging from 200-50 kda. they contain 7 different types of subunits, named b1, b2, b3, a1, a2, a3, ba4, ranging from 31-32 kda. in the rabbit eye lens two -crystallin subunits ( b2 and b3), among the water soluble proteins, have been shown to act selectively as acyl donors substrates for lens tg. calpains are cytoplamic ca ϩϩ -dependent cystine proteinases. the cleavage of αand -crystallins, the main substrates of lens calpain ii, has been associated to the increase of lens turbidity, due to insolubilization of peptides. we observed that tg-induced post-translational modification of b2-and b3-crystallins with polyamines, enhances their cleavage by calpain ii. this finding suggests that the enhancement of calpain ii activity, after conjugation of polyamines into -crystallins, could represent an important regulatory mechanism which may contribute to the opacification process of the eye lens, conducting to cataract formation. transglutaminases represent a family of enzymes, widely diffused in nature, from bacteria to plants and higher animals. the present discussion will focus on 4 isoenzymes in mammals, which have been well characterized from the structural and functional point of view. they act on tissular proteins catalyzing crosslinkage through isopeptide bonds at peptidyl glutamine and lysine residues or incorporation of small molecular weight primary amines, usually polyamines, in an irreversible, calcium dependent reaction. in several instances the expression of transglutaminases is regulated at the transcriptional level. these enzymes help in maintaining structural integrity of tissues intervening in wound repair and in cellular homeostasis at the levels of cell activation, receptor signaling, cell proliferation, differentiation and death. these general roles involve bis(guanylhydrazones) are a class of compounds known to interfere with the metabolism of polyamines by virtue of their ability to inhibit s-adenosylmethionine decarboxylase (samdc), a key enzyme of polyamine biosynthesis. this property has made them useful tools to study the biological functions of these compounds. a curious feature of bis(guanylhydrazones) is their structural relationship with two molecules involved in polyamine biosynthesis, namely spermidine and s-adenosylmethionine. the methylglyoxal derivative of bis(guanylhydrazones), mgbg, has been actively studied both in animal and plant systems. in the present work the male pollen from actinidia deliciosa has been utilized to investigate the role of polyamines on the pollen tube growth. the effect of several bis(guanylhydrazones) was tested on pollen germination, length of pollen tube, levels of free and conjugated polyamines and samdc activity. all bis(guanylhydrazones) tested (glyoxal-bis-guanylhydrazone, gbg, methylglyoxal-mgbg, methylpropylglyoxal-mpgbg, ethylmethylglyoxal-emgbg) inhibit pollen germination and their effect is dose-dependent. a clear reduction of spermidine, both in free and conjugated form, was observed, as well as a pronounced decrease in samdc activity. these results suggest that the mechanism by which bis(guanylhydrazones) reduce the germination of kiwi pollen is related to their effect on spermidine biosynthesis. molecules structurally related to polyamines (1,8diaminooctane, 1,9-diaminononane, 1,10-diaminodecane) and other inhibitors of their metabolism (cyclohexylamine, cha) are also tested on kiwi pollen germination. n. bagni 2 , d. bertoldi 1,2 , e. candioli 1 , l. martinelli 1 , and a. tassoni 2 1 istituto agrario, san michele a/adige, and 2 dipartimento di biologia, università di bologna, italy in the frame of the study aiming to enlighten developmental programs during regeneration in grapes, polyamine content (free and conjugated to hydroxycinnamic acids) and biosynthetic enzyme activities were assayed during somatic embryogenesis. aliphatic polyamines are growth regulators affecting plant growth and development both in vivo and during in vitro cultures, being involved in several morphogenic processes related ti their action in cell division. the study was conducted on samples of callus, embryogenic callus, embryo at different stages and plantlets of vitis vinifera brachetto and chardonnay cultivars induced from anthers and ovaries. polyamine content (putrescine, spermidine and spermine) free plus conjugated to percloric acid soluble fraction, referred to unit, was higher in the cv. brachetto than in the cv. chardonnay, and reached the higher levels in the fullydeveloped embryo stage. besides, ornithine decarboxylase activity resulted higher than arginine decarboxylase and during multiple catalytic processes: the receptor signaling activity (demonstrated only for isoenzyme 2) is related to an intrinsic gtp-ase activity of type 2 transglutaminase; the processes leading to control of cell proliferation, differentiation and death are mainly related to the protein crosslinking activity, while the cell activation is tentatively considered dependent on the polyamidation of endogenous proteins at glutamine residues. the knowledge on this last aspect lies far back in comparison to the other roles of transglutaminases and requires further accurate investigation, which must further extend to the role of the enzyme in human pathology. the examination of polyamine metabolism at the present time suggests that vitamin b12 is implicated in polyamines metabolism. literature data speak that spermine and spermidine stimulate activity of cobalamin-dependant methionine synthase, the enzyme that catalyses the recycling of homocysteine to methionine; polyamines inhibit methionine adenosyltransferase. beside the wellknow significance of vitamin b12, in transmethylation reaction, the significance of 5,-deoxyadenozyl cobalamin, except the conversion of methylmalonyl-coa to succinyl coa, is not well elucidates. methionine as s-adenosylmethionine (sam) is essential amino acid for polyamine biosynthesis. sam has frequently usage in treatment of liver diseases. according the mentioned facts the aim of our experiments is to exanimate the significance of application of vitamin b12 alone and altogether with methionine to rats without and with experimentally induced cholestasis. our preliminary results speak about the disturbance of polyamine metabolism in hepatic tissue of rats with cholestasis. application of methionine alone increases the amount of polyamine in rat liver tissue, in-group without cholestasis and with bile duct obstruction. the animal treatment with cobalamin has higher amount of polyamines and lower activity of polyamine oxidase in liver tissues in both groups. the effects of vitamin b12 may be in direct relation with the formation of 5,-methylthio deoxyadenosine (mta), the by-product of spermidine and spermine biosynthesis. the explanation the exact roles of vitamin b12 in polyamine metabolism of liver tissue need the futher investigations. department of molecular genetics, the weizmann institute of science, rehovot, israel exposure of mouse myeloma cells that massively overproduces ornithine decarboxylase (odc) but not of parental cells to ornithine results in a massive increase in the intracellular concentration of putrescine, followed by rapid cell death. the treated odc overproducing cells display fragmented nuclei, chromatin condensation and an oligonucleosome-size dna "ladder"; consequently, their death can be described as apoptosis. the apoptotic process induced by the accumulated putrescine involves the release of cytochrome c from the mito-chondria, activation of caspases cascades demonstrated by the cleavage of caspase-2 and parp, a substrate of caspase-3. the general inhibitor of caspases, bd-fmk, effectively inhibited parp cleavage but failed to inhibit cell death. the intracellular ca 2ϩ chelator bapta/am and the antioxidant bha inhibit parp cleavage. however, only bapta/am inhibit the induction of cell death. it seems that bha subverted the death into caspase independent pathway. treatment with bapta/am did not interfere with the accumulation of putresine following ornithine treatment, suggesting that the accumulated putrescine induces the elevation in the concentration of intracellular ca 2ϩ which then activates the apoptotic process. the dominant anti-apoptotic effect of bapta/am over egta suggests that internal stores are the main source of the elevated ca 2ϩ , but that putrescine is also capable of inducing influx of extracellular ca 2ϩ . extensive small intestine resection results in the loss of absorptive surfaces, acceleration of intestinal transit and, as a consequence, in malnutrition, weight loss, diarrhoea and other complications of short bowel syndrome. the availability of human recombinant growth hormone rgh and its stimulatory effects on gut growth suggested its use in the treatment of short bowel syndrome. the trophic response of gi tract epithelium to hormones such as growth hormone is mediated by polyamines, which are vital in cell proliferation. this study was undertaken in rats to: 1/ evaluate the effects of rgh by monitoring polyamine and amine metabolism parameters in the adapting short bowel and 2/ determine whether erythrocyte (rbc) polyamine concentrations reliably reflect the proliferative activity of the remaining bowel. seventy per cent resection of the small intestine of wistar rats was performed under ether anesthesia leaving equidistant lengths of bowel from pylorus and ileocecal valve. recombinant human gh (0.2 iu, s.c., saizen, serono, switzerland) was administered once daily for 5 or 10 days, to randomly selected rats on the second postoperative day. animals were sacrificed 8, 13 and 21 days after the operation. enzyme activities were measured with radioassays or fluorimetry. polymines were determined as dansyl derivatives by hplc/fluorimetry. gh treated animals had significantly higher intestinal odc and sat and lowel dao activities; higher (non-significant) mucosal growth index and polyamine concentrations than in untreated counterparts on 8 th postoperative day. thereafter the two groups did not differ in the investigated parameters. rbc polyamine concentrations were higher in operated verses control rats; rgh treatment had no significant effect. however, rgh treatment significantly reduced hepatic mao a and b activities. our results suggest gh accelerated the adaptive growth of the bowel remnant. they justify use of erythrocyte polyamine concentration measurement as the marker of small bowel proliferative activity. however, side-effects of this treatment must be considered. tissue transglutaminase (ttg) activity has been evaluated in different neural tissues, such as brain, spinal cord and peripheral ganglia, and appears to be expressed in cerebellar granule cells (cgn) as well as in astrocytes. the role of ttg in neuronal functioning is likely to be quite complex. other than the role during development, significant changes of enzyme activity have been evaluated in different neurodegenerative conditions. it is well known that nmda receptor activation may be able to trigger excitotoxicity. the nmda-induced injury is mainly associated to ion influx and subsequent calcium overload. the effects of nmda application to both, cerebellar granule cells and glial cell cultures, have been assessed. in cgn, ttg activity increased rapidly after a brief stimulation with 100 µm nmda, whereas in glial cell cultures, high levels of enzyme activity was obtained after incubation of 24 h in presence of the same concentration of nmda. such results rule out the possibility that excitotoxicity can modify numerous proteins making them better substrates of ttg, and this could contribute to enhanced ttg-modifications of proteins in response to excitotoxicity. the pote protein can catalyze both uptake and excretion of putrescine. the km values of putrescine for uptake and excretion (putrescine-ornithine antiport) are 1.8 µm and 73 µm, respectively. amino acid residues, cys62, trp201, glu207, trp292 and tyr425 are strongly involved in both activities, and that glu77, tyr92, cys210, cys285, cys286 and glu433 are moderately involved in the activities. mutations of tyr78, trp90 and trp422 mainly affected uptake activity, indicating that these amino acids are involved in the high affinity uptake of putrescine by pote. mutations of lys301 and tyr308 mainly affected excretion activity, indicating that these amino acids are involved in the recognition of ornithine. the putrescine and ornithine recognition site on pote was found to be located at the cytoplasmic surface and the vestibule of the pore consisting of twelve transmembrane segments. the cadb protein has 30% sequence homology with pote protein. cadb can catalyze both uptake and excretion of cadaverine. the km values of cadaverine for uptake and excretion (cadaverine-lysine antiport) are 20 µm and 300 µm, respectively. it was found that two glutamate residues (glu76 and glu204) and four tyrosine residues (tyr73, tyr89, tyr90 and tyr310) are involved in the both activities. the difference of the substrate recognition site on pote and cadb is discussed. a. lentini, b. provenzano, and s. beninati department of biology, laboratory of cell biochemistry, university of rome "tor vergata", rome, italy tissue transglutaminase (ttg, e.c. 2.3.2.13) is a protein cross-linking enzyme which catalyzes an acyl transfer reaction where the carboxamide group of a peptide-bound glutamine is the acyl donor, and a lysine residue the acyl acceptor. polyamines may act as acyl acceptors, leading to the formation of mono-and bis-(γ-glutamyl)derivatives. we provided evidence that ttg activity is directly associated to differentiation markers, and inversely related to cell proliferation and invasion. we have shown the in vivo reduction of experimental melanoma metastasis by i.v. injection of a plasmid (psg5) carrying the ttg gene sequence to c57bl6/n mice. tumor cell metastatization requires specific interactions with subendothelial basement membrane (bm) and migration through the endothelial wall, allowing the colonization of the target tissue. therefore, the investigation on the possible mechanisms responsible for ttg effects is focused on the posttranslational modification of bm proteins. we detected that "matrigel", a tumor-derived complex of bm proteins, modified with polyamines after ttg catalysis, reduces both melanoma cell adhesion and invasion in an in vitro metastatic assay. similar results were obtained using polyamines conjugated to laminin, one of the major bm components, as unique substrate. our findings suggest that the increase of bm proteins conjugated to polyamines may be responsible for impairments of the invasive properties of melanoma cells. we demonstrated that interferon-α (ifnα) induces apoptosis in human epidermoid cancer cells. tissue transglutaminase (ttgase) is an enzyme involved in the regulation of apoptosis through the inactivation of some cell components. among these eukaryotic initiation factor-5a (eif5a) is peculiar because its activity is modulated by the formation of the amino acid hypusine. recently, we found that growth inhibition induced by ttgase is paralleled by reduced hypusine levels. here we report the effects of ifnα on the apoptosis, ttgase modulation and eif5a activity in human epidermoid lung h1355 cancer cells. we have found that 48 h exposure to 2,500 iu/ml ifnα induces 50% growth inhibition and 15% apoptosis in h1355 cells. moreover, ifnα induced a 4-fold increase of ttgase activity and expression that already occurred after 24 h of exposure to the cytokine. this effect was paralleled by a 1.6-fold enhance of ttgase mrnas. ifnα induced also a 30% increased eif5a expression while an about 50% decrease of hypusine levels was observed. increased ttgase activity was paralleled by a decrease of hypusine content and of eif5a activity. therefore, ifnαinduced apoptosis could occur through an increase of ttgase activity and the mechanism by which ttgase regulates biological functions can be the reduction of eif5a activity. adometdc deficient mice k. nishimura 1 , f. nakatsu 2,3 , k. kashiwagi 1 , h. ohno 3 , t. saito 2 , and k. igarashi 1 1 graduate school of pharmaceutical sciences, chiba university, chiba, 2 graduate school of medicine, chiba university, chiba, and 3 cancer research institute, kanazawa university, kanazawa, japan the amd1 gene encodes s-adenosylmethionine decarboxylase (adometdc) that is one of the key enzymes of polyamine biosynthesis. to examine the physiological role of polyamines, we performed the targeted disruption of the gene in mice to generate spermidine-and spermine-free mice. although the level of adometdc mrna decreased by 50% in amd1 ϩ/ϫ mice, adometdc activity reduced only by 30% and spermidine and spermine contents did not change significantly. they showed normal phenotype and life span. to obtain amd1 ϫ/ϫ mice, we intercrossed amd1 ϩ/ϫ mice and determined the genotype of the resulting offspring. however, we could not obtain any amd1 ϫ/ϫ mice from 20 heterozygous intercrosses over. amd1 ϫ/ϫ embryos died early in development, between e3.5 and e6.5 days post coitum. in culture of blastocysts at e3.5, the shapes of all cell lines were normal, but amd1 ϫ/ϫ cells appeared to arrest the cell proliferation at day 3 after the onset of cell culture. the arrest of amd1 ϫ/ϫ cell proliferation was rescued by addition of spermidine. these data indicated that the lethal phenotype of amd1 ϫ/ϫ mice was caused by growth retardation by polyamine depletion at early developmental stage. the formation of active species such as h 2 o 2 and aldehydes during the oxidative deamination of biogenic amines by amine oxidases (ao) suggests for these enzymes a key role in cellular processes. the ability of bovine serum amine oxidase (bsao) to oxidase free amino groups of lysozyme and ribonuclease a has been observed indicating a possible ao involvement in the post-translational protein modification. furthermore, bsao inhibition by h 2 o 2 formed during substrate oxidation under limited turnover conditions was demonstrated, which may be relevant to cellular physiopathology. we have also observed that some inhibitors of mitochondrial amine oxidases (mao) protected human melanoma cell line (m14) against apoptosis. the protection by catalase of mao-substrates induced membrane permeability transition was also obtained in isolated rat liver mitochondria, thus confirming a role of mao-derived h 2 o 2 in apoptosis. enrichment in ao activity by treatment with vegetal ao has been obtained in a erythroleukemia cell line (k562), substaining the possibility to modulate the intracellular ao activity. an antiarrhythmic and cardioprotective effect of bsao has been also observed on isolated rat heart in reperfusion; a protective effect during anaphylaptic crisis has been shown "in vivo", thus suggesting aos as a possible therapeutic agents. tetrakis(3-aminopropyl)ammonium, a unique polyamine produced by an extreme thermophile, stabilizes nucleic acids at high temperature t. oshima and y. terui department of molecular biology, tokyo university of pharmacy and life science, hachioji, tokyo, japan an extreme thermophile, thermus thermophilus, produces tetrakis(3-aminopropyl)ammonium; a novel polyamine containing a quaternary ammonium nitrogen. to clarify the roles of the unique polyamine in thermophily, the effects of tetrakis(3aminopropyl)ammonium on biochemical reactions related to nucleic acids have been investigated. the unique polyamine stabilized both double and single stranded dnas and rnas. tm of a double stranded dna was raised by 8°c by the addition of 0.25 mm of tetrakis(3-aminopropyl)ammonium. at around the boiling temperature of water, depurination of dna takes place. other long polyamines produced by the thermophile such as caldopentaamine also stabilized dnas and rnas. we found that tetrakis(3-aminopropyl)ammonium prevents depurination most effectively. tetrakis(3aminopropyl)ammonium activated the protein biosynthesis catalyzed by a cell-free extract of the thermophile at high temperature. the effects of this unique polyamine on dna and rna polymerases are also being investigated and the results will be presented. tissue transglutaminase (ttg) catalyses the cross-link formation between glutamine (q) residues and nh 2 -donor molecules present in the cells (polyamines, lysine-donor proteins). recently, it has been correlated to neurodegenerative disorders characterised by polyglutamine (q n ) expansion, like huntington's disease. studies carried out on cell extracts revealed that glyceraldehyde 3-phosphate dehydrogenase (gapdh) was found covalently linked to q n domains. however, to date no structural data are available to solve the issue of which residues of gapdh are substrates for ttg. by coupling classical protein chemistry procedures and mass spectrometric techniques we achieved this goal by using as ttg substrates the substance p, an 11-aa peptide bearing the simplest q n domain (q 2 ), and polyamines of different size and shape as q-and nh 2 -donor, respectively. in the present study we report that out of the 26 lysines present in gapdh only three are sites of ttgasedependent cross-link formation in vitro. moreover, to characterize the ttg catalysed cross-link between gapdh and polyq protein we used a synthetic q 17 -peptide as ttg substrate in the catalysed reaction with polyamines. we found that any q residue is a potential ttg substrate, no matter the specific position in the sequence or the steric hindrance of the specific amine under investigation. cjf inserm 95-09, institut contre les cancers de l'apareil digestif (ircad), strasbourg, france as soon as the key role of odc in polyamine metabolism was recognised, it became the major target for selective inhibi-tion. s. harik presented in 1973 the first potent odc inhibitor, α-hydrazino ornithine. although efforts continued until today, with the aim to improve odc inactivation, 2-(difluoromethyl)ornithine (dfmo) remained the most important compound among all polyamine-directed drugs. a known anti-leukaemic drug, methylglyoxal-bis(guanylhdrazone), was recognised early on by g. williams-ashman and his collaborators as an inhibitor of adometdc, the other highly regulated biosynthetic decarboxylase, and served as matrix for more recent developments. in the course of the years selective inhibitors for all enzymes involved in polyamine biosynthesis and degradation were synthesised. moreover, a series of polyamineuptake inhibitors were reported. however, only some of these numerous compounds reached a stage above evaluation as growth inhibitors of cancer cells. owing to the sophisticated homeostatic regulation of the polyamines in cells and organs by de novo synthesis, degradation, uptake and release, and due to the fact that exogenous polyamines (i.e. gut polyamines) can be utilised by the vertebrate organism, the efficacy of selective enzyme and uptake inhibitors remained modest in cancer therapy. the fact the dfmo became the most important drug for the therapy of west and central african sleeping sickness relies on differences of vertebrate and parasite biochemistry. a novel approach, initiated by carl porter, involved the design and synthesis of structural analogues of spermidine and spermine, which do not share the growth-promoting effects of the natural polyamines. a very large variety of homologues, mostly of spermine, with different alkyl-substituents on the primary amino groups, have been studied systematically with regard to their ability to alter enzyme and polyamine patterns, and to inhibit cell growth. in addition polymine-like chains with interposed heteroatoms (0, s, si etc.), and analogues with rigid aliphatic chains (due to inbuilt double and triple bonds, or of small rings) have been explored. the structural analogues either mimic regulatory functions of the natural polyamines, and thus lead to the depletion of endogenous pools of putrescine, spermidine and of spermine, or they prevent growth effects of the natural polyamines by displacing them from functionally important binding sites. the later type may be considered as polyamine antagonists. the actual drugs usually exhibit to some extent polyamine mimetic and antagonist properties. at present several polyamine analogues are in clinical trial. however, after more than 25 years of active research, a polyaminerelated anticancer drug is still not available. one may conclude from this fact that the polyamines are an inappropriate target for cancer treatment. however, it is more likely that polyamine metabolism is a difficult target, because the differences between normal and cancer cells are mainly of quantitative nature. moreover, numerous mechanisms have developed in the course of evolution, which enable the vertebrate organism to prevent lethal polyamine losses. nine novel chemically modified polyamine (pa) analogs were evaluated for their ability to inhibit the pa biosynthesis in rat hepatoma g-27 cell-free system as well as the growth of caov tumor cells. the final concentration of oxy-and aminoadenosine pa analogs or two uracils modified pa analogs were 0.1 mm in the reaction mixture. bis(uracilyl)-analogs and 8-(2-oxyethyl)ami-no-9--d-xylofuranosyladenine supressed pa and putrescine synthesis and in the same conditions were more effective than dl-α-difluoromethylornithine (dfmo) -strong specific inhibitor of ornithine decarboxylase (odc). the other adenosine modified compounds could act both as activators of odc and inhibitors both diamine and polyamine oxidase activities in regenerating liver test system. in contrast to those mentioned above two uracils modified agents as well as dfmo were able to inhibit odc and to increase the rate of oxidative deamination of pa in the same system. thus bis(uracilyl) pa analogs were the most active and may be useful for further investigation as substances having potential antitumor and antiproliferative properties. several studies concerning the periodontal status in adult and adolescent patients treated with fixed ni-ti archwires have been performed, but until now it is not yet available any information about the influence of patient age on gingival tissue responses to ni-ti alloy. recently, researches by us demonstrated that the prolonged use for over 12 months of ni-ti appliances may contribute to local pathological proliferative processes early detectable only through salivary polyamine concentration increase. although other data from our laboratory showed that salivary polyamine amounts are age and sex-independent, nothing is known about the influence of the age on salivary polyamine content m subjects wearing ni-ti appliances. eighty patients, under orthodontic treatment for 12 months, were divided into four groups: the pre-, the mid-, the late-and the post-pubertal. salivary polyamine concentrations were determined by hplc. only the late pubertal group revealed a significant increase in both the spermine and spermidine content, while the other groups showed no modification. the results suggest that gingival pathological responses to a long-term appliance's use may be related to the endocrine modifications that occur in the late-pubertal age. sexual hormones appear to be in synergy with ni-ti alloy in promoting proliferative activity of gingival cells. the effects of polyamines on the synthesis of various σ subunits of rna polymerase were studied to determine how polyamines influence the functional specificity of transcription using western blot analysis. synthesis of σ 28 was stimulated 4.0-fold and that of σ 38 was stimulated 2.3-fold by polyamines, whereas synthesis of other σ subunits was not influenced by polyamines. stimulation of σ 28 synthesis by polyamines occurred at the level of transcription. since our hypothesis is that polyamines regulate macromolecular synthesis mainly at the translational level, we searched for a target protein, related to the polyamine stimulation of σ 28 synthesis, whose translation is altered by polyamines. stimulation of σ 28 synthesis was due to an increase in the level of camp, which occurred through polyamine stimulation of the synthesis of adenylate cyclase at the level of translation. polyamines were found to increase the translation of adenylate cyclase mrna by facilitating the uug codon-dependent initiation. analysis of rna secondary structure suggests that exposure of the shine-dalgarno (sd) sequence of mrna is a prerequisite for polyamine stimulation of the uug codon-dependent initiation. antitumor quinones are approved for clinical use and others antitumor quinones are in different stages of clinical and preclinical development. the efficiency of the quinonic compounds in inhibiting cancer cells growth is believed to stem from their participation in key cellular redox mechanisms with consequent generation of highly reactive oxygen species (ros). the ros is turn modify and degrade nucleic acids and proteins within the cells. recently, quinonic drugs were attached to the neurodecapeptide lh-rh and evaluated as potential drugs in the treatment of different tumours. we have synthesized several series of n-quinonyl amino acids in which five ω-amino acids are attached to p-quinones with different values of redox potentials. the attachment was made via michael-like reductive addition of the amino acids to the quinonic ring or via substitution of a chlorinated atom. the n-ω-quinonyl amino acids were characterized as to their ability to form semiquinone anion radicals by epr and cyclic voltammetry technique. the preparative methods, the redox potentials as well as the physical and spectral data ( 1 h-nmr, ir, uv-vis and hrms) of these n-ω-quinonyl amino acids will be presented. the de novo design of biologically active peptides and proteins, mostly has involved consideration and design of backbone conformations (secondary structures) such as α-helix, -sheets, -turns, etc. (η/ψ space). however, for many bioactive peptides and proteins, especially those critical for information transduction such as neurotransmitters, hormones, antigens, neurocrines, etc. molecular recognition via side chain moieties is of paramount importance. thus far, the specific three dimensional orientations of side chain groups ( angles; chi space) in terms of biological activity has received only modest attention. in part this may be due to the energetics of chi space compared to ramachandran space. in order to overcome the current limitations of evaluating the importance of chi space in critical biological functions related to disease and behavior, we have designed amino acids with novel structures and unique constraints in chi space ( 1 , 2 , etc.), with special attention to their ability to mimic the chi space of native proteins and peptides. we have developed novel and simple asymmetric synthetic methods for such amino acids, often with ees greater than 98%. incorporation of these novel amino acids into bioactive polypeptide neurotransmitters has provided ligands with unique biological activities that effect unique behaviors including feeding, sexual, pain, and addictive behaviors. (supported by grants from the usphs and nida.) protein technology, wallenberg laboratory ii, lund university, lund, sweden we describe a method for comparative quantitation and de novo peptide sequencing of proteins separated either by standard chromatographic methods or by one and two-dimensional polyacrylamide gel electrophoresis. the approach is based on the use of an isotopically labelled reagent to quantitate (by mass spectrometry) the ratio of peptides from digests of a protein being expressed under different conditions. the method allows quantitation of the changes occurring in spots or bands that contain more than one protein, and has a greater dynamic range than most staining methods. since the reagent carries a fixed positive charge under acidic conditions and labels only the n-terminal of peptides, the interpretation of tandem mass spectra to obtain sequence information is greatly simplified. the sequences can easily be extracted for homology searches instead of using indirect mass spectral based searches and are independent of post-translational modifications. dehydroamino acids and their derivatives play important roles as constituents of various natural products and as synthetic intermediates for the preparation of optically pure amino acids. a large number of amino acid derivatives containing a pyrazol-4-yl, isoxazol-4-yl and other heterocyclic moieties has been prepared as potential agonists or antagonists for central glutamate receptors in connection with (r,s)-2-amino-3-(3hydroxy-5-methylisoxazol-4-yl)propanoic acid (ampa), a bioisostere of (s)-glutamic acid. -hetaryl-α, -didehydroalanines might be considered as conformationally constrained ampa analogs and might be potential candidates for the synthesis of novel types of ampa analogs, for example, via their hydrogenation. compounds containing 2h-pyran-2-one ring are also very useful synthons in selective synthesis. recently we have shown their use for the preparation of (e)-α, -didehydroα-amino acid derivatives containing a pyrazolyl moiety (vraničar l, polanc s, kočevar m (1999) tetrahedron 55: 271). as a continuation of our investigation in this field we report here a detailed study of the transformation of 2h-pyran-2-one derivatives 1 with hydroxylamine (2, x ϭ o) and various hydrazines (2, x ϭ nr 2 ) towards novel types of (e)-and (z)α, -didehydroamino acid derivatives 3. in most cases, the reactions were performed under basic conditions in a mixture of ethanol and pyridine. depending on the substrate and the reagent used the reaction could be controlled to give either (e)-or (z)-isomers; in some cases decarboxylation to the corresponding enamines 4 also occured during the reaction course. some attempts to hydrogenate compounds 3 towards α-amino acid derivaties 5 by homogeneous or heterogeneous catalysis were also performed. analogs of endomorphin 1 and 2 were prepared to investigate the effect of the positional and c-terminal amide replacements and modifications on the biological activity. modifications in position 2 and 4 were studied. in position 2 several hydroxy-and serine related amino acids were incorporated, whereas in position 4 the amide bond was replaced by hydroxymethyl and allyl group. protected peptide derivatives were synthesized on 2chlorotrityl resin and further transformed to the corresponding derivatives in solution phase. among the analogs tested, in in vitro tests the most effective compound found was d-ser 2 -endomorphin ϫ2. quite surprisingly, the partial agonist/antagonist properties of the derivatives in receptor binding and g-protein stimulation tests have been shown behave differently. the differences in efficacy and receptor binding properties of the compounds may explain the discrepancies between the in vitro and receptor binding tests. we have been assessing the possible applications of substituted 2h-pyran-2-ones 1 containing α, -didehydroamino acid unit in their structure as dienes in [4ϩ2]-cycloaddition reactions. as dienophiles we have been using different acetylene derivatives as well as n-phenylmaleimide and maleic anhydride. as it is evident from the structure of 2h-pyran-2-ones upon the cycloaddition of acetylene derivatives the first intermediate formed (2) still contains the carbon dioxide bridge. in many cases 2 easily expels co 2 and substituted benzene derivative 3 is produced. when the alkenes are used, the first part of cycloaddition is the same as when acteylene derivatives are used, but after the extrusion of co 2 from the adduct 4 there are two possible paths: so formed cyclohexa-1,3-diene (5) is either aromatized into benzene derivative (3) or it acts as another diene with favourably positioned double bonds and unusual double cycloadducts (6) are formed. since co 2 -containing adducts are thermally unstable it is advantageous to use high pressure techniques. with the acetylene derivatives we have not been able to isolate co 2 -containing adducts (2), while with alkenes we have isolated, depending on the structure pattern of the compound 1, all three types of products: aromatized 3, co 2containing 4 and double adducts 6. especially the type 4 is suitable for further transformations into other heterocycles containing amino acid moiety. research group of peptide chemistry, hungarian academy of sciences, budapest, hungary among the opioid receptors family, the cloning of µ, k and δ receptors was followed by another member, named lc 132 or orl 1 . searching for an endogenous ligand for this receptor resulted in successful identification of a peptide (fggft garksarklanq) called noc or ofq. in vitro and in vivo studies have demonstrated that noc mediates a variety of biological actions. results from structure-activity experiments suggest that the whole sequence of noc is not required for binding to the lc 132 receptor and for full biological activities. noc(1-13)-oh seem to be the minimum and essential sequence for good interaction with the receptor. this neuropeptide, similarly other peptides, are unresisting for enzymatic degradation and the releasing metabolites are very weakly active or inactive. some previous experiments refer to that the c-terminal amidation may protect the peptide from degradation. we purposed to synthesize carbamoyl analogues of noc(1-13)-nh 2 , hoping that these derivatives retain the ability to bind lc 132 receptor and are resistant against biological degradation: phe-nh-co--ala-noc(4-13)-nh 2 phe--ala-nh-co-phe-noc(5-13)-nh 2 phe-gly-nh-co--hphe-noc(5-13)-nh 2 the first step in the synthesis of the carbamoyl analogues was the preparation of the building block [r-co-nh-co-nh-hc(rј)-cooh] by the classical method and then it was incorporated into the peptide by solid phase peptide synthesis. [ nonproteinogenic amino acids and their derivatives are valuable compounds from their pharmacological and biochemical effects. they can be used also in synthesis of peptides, as biomarkers, as the ligands in catalitically active transition metal complexes and so on. it is possible to prepare such amino acids by asymmetric hydrogenation of their prochiral precursors. however high enantioselektivities was reached only in the case of chiral phosphine-rhodium catalysts. recently we showed that high diastereoselectivity in the hydrogenation of linear dehydrodipeptides may be achieved over achiral catalyst in the catalytic system substrate -salts of ca ore mg -pd/c due to formation of dehydrodipeptides complexes with ions ñà 2ϩ or mg 2ϩ and hence increasing of the conformational rigidity of substrates. this phenomenon may as well happen in other dehydrodipeptides, containing nonproteinogenic amino acids. among unnatural amino acids those bearing heterocyclic rings have attracted considerable attention due to the possibility of the heteroatoms participation in coordination with ions of metals. we have received some n-acyldehydrodipeptides, containing in the prochiral unit of dipeptides nonproteinogenic dehydroamino acids. all this n-acyldehydrodipeptides form in alcohol solution complexes with cax 2 and mgx 2 where one metal ion binds together several (up to 5) substrate molecules. this kind of complexation leads to the increase of conformational rigidity and to the diastereoface shielding of cϭc bond. moreover the combination of cations (ca 2ϩ or mg 2ϩ ) and anions (x) and the sequence of their mixing with a substrate determine the assembly inside complex particles and hence the sign and degree of asymmetric induction. indeed hydrogenation of these complexes formed in situ over achiral heterogeneous catalyst (pd/c) gives two diastereomers of corresponding n-acyldipeptides with the substantial increase of the reaction diastereoselectivity (up to 80%). in living cells, glutamine represents one of the main storage forms of nitrogen and is a major physiological source of ammonia for the biosynthesis of aminoacids, aminosugars, purine and pyrimidine nucleotides and coenzymes. glutamine-dependent amidotransferases perform nitrogen transfer from the amide group of glutamine to various electrophiles. when the latter is fructose-6p, the product of the reaction catalysed by glucosamine-6p synthase is d-glucosamine 6-phosphate, a structural building block of peptidoglycane (bacteria) and of chitin and mannoproteins (fungi). fluorinated analogues of glutamine are expected to interfere with this biological process due to the strong electron withdrawing effect of fluorine atom (without significant steric consequence), inducing modulation of binding and/or electronic properties. these compounds might therefore behave as reversible or irreversible active site-directed enzyme inhibitors. synthesis of optically active 1 from d-serine will be described and first results in the biological evaluation on glucosamine 6-phosphate synthase will be included. o. melnyk 1 , d. bonnet 1 , e. loing 2 , l. bourel 1 , and h. gras-masse 1 1-umr 8525, 2-sedac-therapeutics, biological institute of lille, france lipopeptides, owing to their ability to cross passively the cell membrane or biological barriers, are unique tools for the intracellular delivery of bioactive peptides. the structure of the lipophilic moiety is known to have a profound effect upon the interaction with the membrane and its alteration. the stepwise solid phase synthesis of lipopeptides is limited by the necessity to perform a complex rp-hplc purification following the cleavage and deprotection step. in addition, the harsh conditions used during the final acidolysis procedure does not allow the introduction of unsaturated or sensitive fatty acids. to speed up the access to large lipopeptides modified by various fatty acid moieties or cholesterol derivatives, we have designed novel synthetic methods which involve the chemoselective reaction of fully deprotected and purified hydrazinopeptides with fatty acid succinimidyl esters or glyoxylyl derivatives. application of these methodologies to the c-terminal 95-135 portion of interferon (ifn)-γ allowed the selection of the optimal lipopeptide ifn-γ agonist, as determined by its ability to induce the expression of surface mhc-ii molecules through interaction with the intracellular components of ifn-γ receptor. graduate school of science, osaka city university, osaka, japan glutamate receptors in mammalian cns are implicated in the construction of memory and early learning as well as in the pathogenesis of neuron damage to cause various neuronal diseases. in recent years, we have studied the conformational role of l-glutamate when it binds to the receptors through the synthesis of l-2-(carboxycyclopropyl)glycines (ccgs) and their related analogs. the works have demonstrated that not only the receptors require a specific conformation of l-glutamate, but also these analogs can be used as important tools for the neuropharmacological research. among them, dcg-iv, a 3јsubstituted analog of ccg-i, is used as a potent and selective agonist of mglur2. as an extension of these works, next program was focused on the synthesis of α-substituted glutamate analogs which would enable to develop potent and subtype-selective ligands for mglurs and transporters. α-alkoxymethylglutamate and ly354740 and its c5 epimer were chosen for the synthetic targets, since the former slightly restricts the glutamate conformation to an extended form and the latter rigidly fix to an extended or a folded form on its bicyclo[3,1,0]hexane skeleton. the key to the synthesis was a stereoselective construction of the quarternary carbon center, which was efficiently performed based on an asymmetric version of the strecker synthesis. details of the synthesis and their neuropharmacological activities will be described. using a genetically modified organism a broad variety of linear unsaturated amino acids are now accessible in enantiomerically pure form via this methodology, which can be used as starting materials for the synthesis of highly functionalized pipecolic acid derivatives. these compounds can be used to restrict conformations in polypeptides or can serve as scaffolds in synthesizing libraries for drug discovery. the synthetic approach involved both a pd-catalyzed amidopalladation reaction of alkoxy-allenes, in which the nh is added across one allene double bond and a ruthenium catalyzed ring closing metathesis step, to form a benzyloxypipecolic acid. further reaction of this n-sulfonyl-iminium-ion precursor with a nucleophile results in the formation of cis-substituted pipecolic acids. due to the unique electronic properties of fluorine, incorporation of α-fluoroalkylated amino acids is a new approach to design biologically active peptides with increased metabolic stability and defined secondary structure and provides a powerful nmr label for spectroscopic investigations. the application of proteases especially for cn-ligations is an attractive alternative to chemical methods, because the enzymatic formation of peptide bonds is highly regio-and stereospecific and, therefore, does not require large efforts to protect side chains of trifunctional amino acids. recently, the enzyme-catalyzed incorporation of α-fluoromethyl amino acids into the p 2 , p 3 and p 2 ј-position (nomenclature according to schechter and berger) of peptide fragments has been successfully performed. carboxypeptidase y was now shown to be suitable to catalyze the incorporation of α-trifluoromethyl alanine into the p 1 position of peptides. furthermore, the general applicability of the substrate mimetic concept in enzymatic peptide synthesis was expanded to the transfer of c-terminal α-fluoroalkyl substituted amino acids. generally, each trifluoromethyl-and difluoromethyl amino acid 4guanidinophenyl esters can be applied as acyl donor in trypsin and chymotrypsin catalyzed peptide bond formation independently of the acyl moiety and the natural enzyme specificity, respectively. via these two approaches, incorporation of αfluoroalkylated amino acids into the p 1 position of peptides using enzymatic methods was successfully applied for the first time. this investigation was performed in search of new 2јdeoxynucleoside analogues modified at 3ј-and 5ј-positions with amino acids and possessing antiviral activity. substrate mimetics strategy: an efficient approach to protease-catalyzed peptide ligation n. wehofsky 1 and f. bordusa 1,2 1 max-planck-society, research unit "enzymology of protein folding", halle, and 2 institute of biochemistry, university of leipzig, germany two main drawbacks seriously restrict the synthetic value of proteases as reagents in peptide fragment coupling: (1) native proteolytic activity and, thus, risk of undesired peptide cleavage; (ii) limited enzyme specificities restricting the amino acid residues between which a peptide bond can be formed. the latter can be overcome by the use of substrate mimetics. contrary to common acyl donors, substrate mimetics bear a binding site specific ester leaving group instead of having a specific amino acid moiety at the c-terminus of the acyl residue. this replacement mediates the acylation of the protease by nonspecific acyl residues. deacylation of the artificial acyl enzyme intermediate by the amino component added results in peptide bond formation regardless of the primary specificity of proteases enabling nonspecific coded and noncoded amino acid derivatives and even non-amino acid-derived acyl moieties to be coupled. the successful application of these artificial substrates for model peptide ligations catalyzed by the argspecific trypsin, the glu-specific staphylococcus aureus strain v8 protease (v8 protease), and α-chymotrypsin, which is specific for aromatic amino acid moieties, will be demonstrated. new development in the tritium labelling of peptides and proteins using solid state catalytic isotopic exchange with spillover-tritium yu. a. zolotarev 1 , a. k. dadayan 1 , b. v. vaskovsky 2 , and n. f. myasoedov 1 1 institute of molecular genetics, russian academy of sciences, and 2 shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia the reaction of high temperature solid state catalytic isotope exchange (hscie) of hydrogen in peptides and proteins with spillover-tritium was studied. the reaction ability of amino fragments in hscie was shown to depend both of their structure and on the availability and the mobility of the polypeptide chain. [ 3 h] peptide analysis using 3 h nmr spectroscopy was carried out, and the modified fragment [ 3 h]actc 4-10 (met-glu-his-phe-gly-pro), with molar activity of 80 ci/mmol and [ 3 h] zervamicin iib (ac-trp-ile-gln-iva-ile-trh-aib-leu-aib-leu-hyp-gln-aib-hip-aib-pro-phl, where aib ϭ 2amino-isobutyric acid) with molar activity of 70 ci/mmol was produced. the obtained preparations completely retained their biological activity. with the -galactosidase protein from termoanaerobacter ethanolicus as example, the interrelation between the protein's tertiary structure and the isotopic label distribution incorporated due to the hscie reaction was used. the labeled protein with the molecular mass of 83 kda was brought to fragmentation by glu-proteinase. peptide fragments were separated by hplc and were identified by maldi mass spectrometry. a correlation between the position of the amino acid fragment in the protein tertiary structure and its reaction ability in the hscie reaction was obtained. data on the retention of thegalactosidase enzymatic activity in condition of tritium label introduction are supplied. taurine chloramine modulates cytokine production by peripheral blood mononuclear cells m. chorą z . y 1 , e. kontny 1 , j. marcinkiewicz 2 , and w. maśliń ski 1 1 institute of rheumatology, warsaw, and 2 jagiellonian university, cracow, poland objective. proinflammatory cytokines are produced in a cascade fashion, where monocyte-derived tnfα and il-1 trigger production of il-6 and il-8 also in the other cell types. we reported recently that taurine chloramine (tau-cl) inhibits production of the latter cytokines in fibroblast-like synoviocytes. in present study the effect of taurine (tau) and tau-cl on tnfα, il-1 and il-6 production was examined. methods. peripheral blood mononuclear cells from healthy volunteers were stimulated with lps (24 h) in the presence of tau or tau-cl (100-500 µm). cytokine production was measured in culture supernatants (secreted) and cell lysates (intracellular) using elisa. results. in lps-stimulated cells both secreted and intracellular il-1 and il-6 were inhibited by tau-cl with ic 50 ϸ 300 µm and 425 µm, respectively. however, tau-cl exerted dual effect on tnfα production, raising it slightly (1.5 times) at low (100-200 µm) while reducing it (ic 50 ϸ 450 µm) at higher concentration. tau did not significantly affect cytokine production. tau-cl modulates proinflammatory cytokine cascade and eventually might down-regulate it when present at high (ͼ300 µm) concentration. department of biology, division of general physiology, university of oslo, blindern, norway every living cell must deal with osmotic and hydrostatic pressure changes between its environment and its interior and counteract volume changes. swelling activated channels is one group of effectors in the cell membrane that is important in preventing excessive volume increases by releasing inorganic ions and organic solutes that include taurine. such channels are associated with several physiological processes, but little is known about their activation mechanisms. we have used a rat thyroid cell line (frtl-5) to investigate the activation of a swelling sensitive [3h]taurine efflux pathway. hypo-osmolality and thyrotropin (tsh, 500 µm) increased transiently the rate coefficient for [3h]taurine efflux with a similar pattern of activation. the phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine (100 µm) increased the swelling activated efflux rate coefficient 6.4 times above the control level and the camp analogue dibutyryl-camp (500 µm) activated the pathway. these results indicate that both swelling and tsh activation of the taurine efflux pathway are mediated by camp. other aspects of the signal transduction pathway will be discussed. based on the inclination of n-chloroamines to disproportionate, the endogenous bactericidal agent n-chlorotaurine (nct), mainly at ph ͻ 7, is accompanied by n, ndichlorotaurine (ndct). since pure ndct could be synthesized as crystalline sodium salt, a first evaluation of its chemical and bactericidal properties was possible. ndct-na (melting point: 162-4°c, decomp.) is very well soluble in water and poorly in ethanol where it can be recrystallized from. on storage the initial ph 5 of the aqueous solution decreases which correlates with a decrease of oxidation capacity of 1.8% per day, probably originated by the elimination reaction r-ch 2 -ncl 2 ae r-chϭncl ϩ h ϩ ϩ cl ϫ as a first step. contrary to nct-na an immediate decomposition occurs when ndct-na comes into contact with undiluted dmso. in aqueous solution, however, ndct does not react with dmso. the bactericidal activity of 55 mm ndct at ph 7 and 5 against the gram-positive bacteria s. epidermidiand two strains of s. aureus was the same as with equimolar nct though ndct bears twice the oxidation capacity. against the gramnegative bacteria e. coli, p. aeruginosa, and p. mirabilis, however, a significantly higher activity of ndct was observed at both ph. the mechanism of taurine chloramine inhibition of fibroblastlike synoviocytes growth e. kontny 1 , m. kurowska 1 , j. kowalczewski 1 , i. janicka 1 , j. marcinkiewicz 2 , and w. maśliń ski 1 1 institute of rheumatology, warsaw, and 2 jagiellonian university, cracow, poland objective. in rheumatoid arthritis (ra) enhanced proliferation of fibroblast-like synoviocytes (fls) leads to hyperplasia of synovial membrane (sm). therapeutic approaches to inhibit an excessive growth of these cells are not satisfactory. thus, we investigated the effect of taurine (tau) or taurine chloramine (tau-cl) on ra fls growth. methods. fls isolated from sm of ra patients were stimulated for 72 hours with rhpdgf or rhtnf-α. tau or tau-cl were added at 100-250 µm concentrations. cell proliferation was determined by incorporation of 3 h-thymidine into dna. expression of proteins regulating cell-cycle progression or apoptosis, was estimated by western blotting. results. at 250 µm concentration tau-cl inhibited (by ϸ 70%) both pdgf-and tnf-α-triggered cell proliferation and similarly reduced expression of pcna (a cofactor for dna polimerase δ). however, tau-cl affected neither the expression of cell-cycle inhibitors (p21, p27) nor anti-apoptotic bcl-2 protein. tau has no effect on tested responses. conclusion. we report that tau-cl inhibits proliferation of ra fls by affecting expression of pcna, that is critical for cell cycle progression. e. kontny 1 , k. szczepań ska 1 , j. kowalczewski 1 , m. kurowska 1 , i. janicka 1 , j. marcinkiewicz 2 , and w. maśliń ski 1 1 institute of rheumatology, warsaw, and 2 jagiellonian university, cracow, poland objective. proinflammatory cytokines play critical role in the pathogenesis of rheumatoid arthritis (ra). we reported recently that taurine chloramine (tau-cl), but not taurine (tau), inhibits production of il-6 and il-8 by fibroblast-like synoviocytes (fls). in present study the mechanism of tau-cl inhibitory action was investigated. methods. fls isolated from synovial membrane of ra patients were stimulated with rhil-1 . tau or tau-cl were added at 250-500 µm concentration. after 0.5-2 h or 4 h the dna binding activity of nfkb and ap-1 (emsa) and the expression of il-6 and il-8 mrnas (rt-pcr) was examined, respectively. results. il-1 raised nfkb and ap-1 activity, followed by the elevation of cytokine mrnas expression. tau-cl, but not tau, reduced both the expression of cytokine mrnas (il-6 ͼ il-8) and the activity of transcription factors (nfkb ͼ ap-1). conclusion. tau-cl inhibits transcription of il-6 and il-8 genes due to its ability to diminish the activity of key transcriptional factors, that regulate these proinflammatory cytokine expression. institut für organische chemie, universität bremen, germany the synthesis of taurine and hypotaurine from cysteine can be followed up in astroglia-rich primary cultures obtained from brain of neonatal wistar rats. using 1 h and 13 c nuclear magnetic resonance spectroscopy cell extracts of glia cells incubated with 13 c labelled cystein show the label subsequently in hypotaurine, taurine, lactate and gluthathione. within 72 h, 35% of the total intracellular hypotaurine and 22.5% of taurine were newly synthesized from cysteine. both metabolites were also released to the medium. neurones are capable to take up both metabolites from glia media to recruit their organic osmolite. part of newly synthesized glutathione and lactate are also exported to the medium. by this means lactate may serve as an energy substrate for neurons. in-vivo mrs of lactate is obscured by line splitting and signal overlay. using various two dimensional pulse sequences as spin preparation sequences prior to localized single voxel, in-vivo mrs or spectroscopic imaging sequences will provide homonuclear non-coupled resonance signal of taurine. these singlet signals are detectable and quantified. diffusion weighted spectroscopy is used to characterize the mobility of taurine in living tissue. department of pharmacology and ophthalmology and visual sciences, texas tech university health sciences center, lubbock, texas, u.s.a. taurine depletion, whether by removing taurine from the diet or by using a taurine transport inhibitor, has demonstrated various pathologies in various animal models including man. the first reported pathology associated with dietary taurine depletion was in the retina of the cat. in this animal model, taurine deficiency resulted in disorganization of the tapetum (the light reflecting membrane), disruption of the outer segments, photoreceptor dysfunction, and cell loss. when allowed to proceed for a number of months the result was blindness. subsequent studies demonstrated that taurine deficiency also had a profound effect on cardiac physiology. echocardiograms of the left ventricle of the cat heart depleted in taurine showed a dilated cardiomyopathy reflected in an extended end-diastolic diameter and an extended end-systolic diameter. dietary taurine supplementation resulted in the above parameters returning to normal. the cat is a difficult animal model to use for a variety of reasons and thus the rat was chosen to further probe the consequences of taurine depletion. unfortunately, the tissue taurine levels in the rat do not respond to dietary taurine depletion, and thus other experimental means had to be designed. guanidinoethanesulfonic acid (ges), an analogue of taurine and a taurine transport inhibitor, has been utilized for the last 20 years to deplete rat tissues of their taurine content (j. e. shaffer and j. j. kocsis, methods of reducing tissue taurine levels, and r. j. huxtable, h. e. laird, and s. lippincott, rapid depletion of tissue taurine content by guanidinoethyl sulfonate. in: the effects of taurine on excitable tissues, spectrum publications, new york, 1981) . ges, when administered to rats in their diet in the drinking water as a 1-1.5% solution, usually produces a significant decrease in the taurine content of all tissues within one week of treatment. within 3-5 weeks the levels of taurine reach their nadir (20-50% of control) and continued feeding of ges does not further reduce the levels of taurine. unfortunately, ges replaces taurine and thus one must always consider the effects of ges on physiological events that occur within the tissues in question. again, as in the cat, taurine depletion manifested itself in retinal pathology: disruption of the photoreceptor structure, dissociation of the disc membranes, and abnormal electroretinograms (erg). other animals models such as the monkey have also demonstrated structural disorganization of the photoreceptors and abnormal ergs. finally, the ultimate test is whether taurine deficiency has an effect in man. in 1985, koppel and associates (geggel et al., n. eng. j. med. 312: 142-146, 1985) demonstrated that children on long term parenteral nutrition devoid of taurine had abnormal erg. supplementation of the parenteral nutrition with taurine restored the ergs to normal in the majority of the children. because of these definitive studies, all infant formulae in the united states, europe and japan now contain taurine. (supported in part by a grant from the taisho pharmaceutical co., ltd., tokyo, japan.) department of pharmacology and ophthalmology and visual sciences, texas tech university health sciences center, lubbock, texas, u.s.a. it has been demonstrated previously in our laboratory that taurine inhibits the phosphorylation of an ϳ44 kdalton protein present in the mitochondrial fraction of the rat heart (j. mol. cell. cardiol. 26: 1675 -1689 , 1994 . upon administering 1.5% guanidinoethanesulfonic acid (ges) in the drinking water of rats for 6 weeks, the taurine levels in cardiac tissue decline by 75%. however, the phosphorylation of a ϳ44 kdalton protein in the mitochondrial fraction of the heart tissue increased by 85% (j. mol. cell. cardiol. 26: 1675-1689, 1994) . reversal of these effects could be accomplished by feeding the rat 1.5% taurine in the drinking water for 6 weeks. the ϳ44 kdalton protein was isolated by 1-dimensional polyacrylamide gel electrophoresis (page) using traditional glycine buffers followed by re-electrophoresing the cut out portion of the gel, which corresponds to the ϳ44 kdalton protein, on a tricine-buffered gel resulting in sufficient pure protein for digestion and sequence analysis. it was determined that the ϳ44 kdalton was pyruvate dehydrogenase (amino acids 12: 139-144, 1997) which indicates a significant regulatory role for taurine in energy metabolism in cardiac tissue. these data are of significant interest in that taurine may be an additional effector of this enzyme or of the enzyme complex. studies are in progress to determine if taurine has a direct effect on either the kinase (inhibition) or the phosphatase (stimulation) associated with the pyruvate dehydrogenase complex. it has also been demonstrated and now reported that taurine depletion utilizing ges in vivo in rats affects the phosphorylation of myelin basic protein (mbp). in these experiments the animals were given ges (1%) for 6 weeks in their water and then killed; the hearts were removed and homogenized. the homogenate was then incubated with buffer containing mbp (50 ì) and radioactive atp for 6 minutes. animals were also treated with taurine (1%) in their drinking water for 4-5 weeks or treated with taurine following the ges treatment. page of the incubation mixture, autoradiography on the dried gel, and densitometry of the mbp band gave the following results: relative % activity ϯ sem (normalized to mg protein) control 100 ϯ 20 (n ϭ 6) ges-treated 147 ϯ 28 (n ϭ 6) p ͻ 0.05 (paired 5-test) control 100 ϯ 16 (n ϭ 5) taurine-treated 95 ϯ 17 (n ϭ 5) p ͼ 0.05 control 100 ϯ 32 (n ϭ 4) ges followed by taurine 95 ϯ 37 (n ϭ 4) p ͼ 0.05 these data confirm previous reports that it is easier to deplete animals of their cardiac taurine content than it is to raise the levels of taurine. these data on the effects of taurine depletion (increase in mapkinase activity) and taurine supplementation (no change in mapkinase activity) on mapkinase activity reflect these past observations. (supported in part by a grant from the taisho pharmaceutical co., ltd., tokyo, japan.) act additively, or in the case of mpo negated each other's effects. regarding our results there is significance to pharmacological regimens which enhance the supply of propofol or taurine in whole blood. these regimens influence considerably pmn intracellular amino acid concentrations and it is this pmn "labile free amino acid pool" which may be one of the determinants in cell nutrition positively or adversely affecting pmn immune functions. taurine supplementation to pmn seems to interfere independently from the effects of propofol on pmn free amino acids and on immune functions tested. institut für hygiene, universität innsbruck, austria n-chlorotaurine (nct) is a long-lived oxidant produced by activated human leukocytes during the oxidative burst. it has activity against a broad spectrum of pathogens including bacteria, fungi, viruses and helminths. as a special feature, the killing of microbes by nct can be increased significantly in the presence of ammonium and also of some amino acids (alanine, glycine). this is explained by transfer of the active chlorine ("transhalogenation") from nct to ammonium and amino acids to form the corresponding, stronger microbicidal n-chloro derivatives monochloramine and n-chloro amino acids, respectively. especially addition of ammonium to nct provokes rapid inactivation of fungi and even mycobacteria. because of its good tolerability, nct solution can be applied to human tissue to treat infections. in ammoniumcontaining body fluids like nasal mucus and urine, fungi and bacteria are killed within minutes. therefore, amino compounds of human secretions can be transformed to the above quoted endogenous and highly microbicidal chloramines by nct via transhalogenation -a unique property of an antimicrobial agent. successful treatment in cases of urinary tract and otorhinolaryngological infections and conjunctivitis in phase iia clinical trials provides strong support for this concept. the endogenous sulfonated amino acid taurine has numerous functions in the central nervous system, including positive modulation of gaba a receptor function. recently we found that mice lacking protein kinase c -epsilon (pkcε) are behaviorally and biochemically supersensitive to ethanol and other positive allosteric modulators of the gaba a receptor. in addition, these mice consume 50-75% less ethanol and wildtype controls in two separate self-administration paradigms. microdialysis studies in pkcε-deficient mice revealed elevated extracellular levels of taurine, which may account for the supersensitivity of gaba a receptors in these mice and resulting decreases in ethanol intake. in light of the fact that the taurine derivative acamprosate (calcium acetylhornotaurinate) is moderately effective in reducing craving and relapse in detoxified alcoholics, we examined the effect of taurine-related compounds on acute ethanol consumption in a two-bottle choice paradigm in rats. taurine (10-200 mg/kg ip) was only slightly effective in reducing ethanol intake but not preference, while the highest dose of taurine (200 mg/kg) also suppressed water intake. the taurine precursor hypotaurine (10-100 mg/kg ip) was also weakly effective in reducing ethanol intake but not preference or water intake. the most effective compound tested was homotaurine (10-100 mg/kg ip), which suppressed ethanol intake and preference by approximately 50% without altering water intake. these data indicate that endogenous taurine may regulate sensitivity to ethanol and subsequent ethanol self-administration, and that taurine-related compounds may be effective in reducing alcohol intake in humans. we are currently exploring whether taurine and related compounds are able to suppress ethanol-stimulated mesolimbic dopamine release, a primary neural substrate of ethanol reinforcement. (this work was supported by funds provided by the state of california for medical research on alcohol and substance abuse through the university of california at san francisco.) organic osmolytes, such as taurine, regulate a cell's osmotic balance without directly altering either the cell's ionic composition or the membrane potential. this property of the organic osmolyte often renders the cell resistant to damage during a pathological insult. indeed, ischemia is associated with a massive efflux of taurine from the cell, an event that minimizes the severity of the osmotic imbalance that develops from the accumulation of lactate, inorganic phosphate and sodium. however, taurine depletion also activates specific signaling pathways that provide further protection to the cell. among the signaling pathways activated by taurine depletion is a pi 3-kinase (phosphatidylinositol 3-kinase) linked pathway that catalyzes the phosphorylation and inactivation of the pro-apoptotic factor, bad. taurine depletion also activates protein kinase c, which in turn elevates the intracellular content of the antiapoptotic factor, bcl-2. increases in the extracellular osmolality by either addition of 20 mm taurine or 25 mm mannitol to the incubation medium activates similar pathways. however, pi 3kinase assumes a more important role in the mannitol treated cell than the taurine depleted cell. moreover, p38 map kinase is activated by mannitol treatment but not by taurine depletion. despite these differences, both taurine depletion and mannitol treatment protect the cell against hypoxia-induced apoptosis. the data suggest that osmotic stress protects the cell against apoptosis by increasing cellular levels of bcl-2 and promoting the inactivation of bad. this work was supported by a grant from the american heart association. a dummy or a protagonist on the stage of inflammation? r&d department, zambon group, bresso, milan, italy amino acids are usually present in large excess in healthy and the excess is used as source of calories. however, metabolic alterations are observed in ill patients and preferential retention of sulphur amino acids (saa) occurs during the inflammatory response. the metabolism of cysteine is modified during the acute phase of sepsis in rats. sulphate production is lower, whereas the higher liver production of taurine seems to play a protective role; glutathione concentration is greater in liver, kidney and other organs and cysteine incorporation into proteins was higher in spleen, lung and plasma (acute phase proteins) while albumin level decreases. another important phenomenon is the impairment of methionine conversion to cysteine during stressed condition. premature infants or hiv patients synthesise cysteine from methionine at a much lower rate. thus, the metabolic flow through the trans-sulphuration pathway may be insufficient to meet the glutathione and cysteine requirement in critical conditions. the pro-inflammatory cytokines, interleukin-1, interleukin-6 and tnf-α are the main initiates that alter protein and amino acid metabolism. in this complex picture, saa supply may contribute to the immune system regulation. department of applied biological chemistry, the university of tokyo, japan the intracellular level of taurine is maintained not only by the taurine transporter that transports extracellular taurine inside cells but also by endogenous synthesis from methionine and cysteine. we therefore investigated the regulation of both the taurine transporter and the cysteine dioxygenase, one of the main taurine biosynthetic enzymes, in hepg2 human liver cells. the intracellular taurine content of hepg2 cells was extremely increased by culturing in a hypertonic medium. the activity of taurine transport was increased by hypertonic conditions, which was due to the increased expression of the taurine transporter gene. the expression level of the cysteine dioxygenase gene was also increased, suggesting that the expression levels of both the taurine transporter gene and the cysteine dioxygenase gene were regulated in harmony by hypertonic conditions to accumulate taurine inside cells. on the other hand, the activity of taurine transport in hepg2 cells was down-regulated on culturing the cells in taurine-rich medium, the expression level of the taurine transporter gene being also markedly decreased. however, the expression level of the cysteine dioxygenase gene was not significantly altered under taurine-rich conditions, indicating that the gene expression of the taurine transporter and that of the cysteine dioxygenase was independently regulated by extracellular concentration of taurine. the amino acid, taurine, is found in very high concentration in the heart. although its most important putative function is osmoregulation, it also serves as a regulator of cell growth. isolated cardiomyocytes exposed to medium containing 1 nm angiotensin ii undergo hypertrophy, a process blocked by 20 mm taurine. the amino acid also inhibits angiotensin iiinduced activation of c-fos, upregulation of atrial natriuretic factor and induction of tgf-betal. central to virtually all of these actions of angiotensin ii is the translocation and activation of key protein kinase c (pkc) isoforms. therefore, we proposed that taurine inhibited the hypertrophic actions of angiotensin ii by interfering with the translocation of one or more of the pkc isoforms. indeed, taurine and angiotensin ii exhibited different effects on the translocation of several pkc isoforms. while taurine promoted the translocation of pkcalpha, pkcdelta and pkcepsilon from the particulate fraction to the cytosol, the levels of the three isoforms in the particulate fraction were elevated following treatment with angiotensin ii. by contrast, both taurine and angiotensin ii increased the pkczeta content of the particulate fraction and the pkcbeta2 content of the cytosol. when the isolated cardiomyocytes were incubated with medium containing both angiotensin ii and taurine, the effects on pkc distribution were largely additive. these data support the notion that taurine prevents the hypertrophic effects of angiotensin ii by interfering with the translocation of either pkcalpha, pkcdelta, pkcepsilon or a combination of more than one of the isoforms. (the study was supported by a grant from taisho pharmaceutical co.) main final metabolites of l-cysteine in mammals are sulfate and taurine, and they are excreted in the urine. our previous studies in rats have shown that the ratio of urinary sulfate and taurine in rats fed diet containing sufficient methionine and cysteine is 10: 2-3. in the present study, we determined urinary sulfate and taurine in urine samples of 58 healthy japanese women after 12h starvation following usual meal. free (inorganic) and total (free ϩ ester) sulfate were determined with ion chromatography, and taurine by reversed-phase hplc after dabsylation. average excretions (micromols per mg of creatinine) were: total sulfate, 12.53 ϯ 3.85; free sulfate, 11.57 ϯ 3.69; ester sulfate, 0.96 ϯ 0.94; taurine, 0.78 ϯ 0.53; urea, 187.71 ϯ 66.13. the ratio of total sulfate and taurine was 10 : 0.62. this suggests that sulfate formation in humans is more dominant than taurine formation as in rats and this tendency is more evident in humans than in rats, which is in accordance with low cysteinesulfinate decarboxylase activity in humans. sum of sulfate and taurine excretions was significantly correlated with that of urea: correlation coefficient, 0.675. this indicates that sulfur metabolism in humans is in the state of sulfur equilibrium similar to that of nitrogen and reflects protein metabolism. h. yokogoshi 1 and h. oda 2 1 laboratory of nutritional biochemistry, school of food and nutritional sciences, the university of shizuoka, and 2 department of applied biological sciences, nagoya university, nagoya, japan the effect of taurine on hypercholesterolemia induced by feeding a high-cholesterol (hc) diet to rats was examined. when various amounts of taurine (0.25-50 g/kg) were supplemented to hc for 2 wk, serum total cholesterol gradually and significantly decreased in a dose-dependent manner, compared with the control (cholesterol free) diet group. by contrast, serum hdl-cholesterol was elevated by taurine supplementation. in the hypercholesterolemic rats fed the hc diet, the excretion of fecal bile acids and hepatic cholesterol 7α-hydroxylase (cyp7a1) activity and its mrna level increased significantly, and the supplementation of taurine further enhances these indexes, indicating an increase in cholesterol degradation. agarose gel electrophoresis revealed that, in hypercholesterolemic rats fed the hc diet, the serum level of the heavier vldl increased significantly, but taurine repressed this increase and normalized this pattern. significant correlations were observed between the time-and dose-dependent increases of cyp7a1 gene expression and the decrease of blood cholesterol concentration in rats fed the hc diet supplemented with taurine. these results suggest that the hypocholesterolemic effects of taurine observed in the hypercholesterolemic rats fed the hc diet were mainly due to the enhancement of cholesterol degradation and the excretion of bile acid. in vitro studies have shown that ammonia, which is responsible for neurological symptoms associated with hyperamonemia, causes a massive release of taurine from cultured cns cells and brain slices. in this study, taurine (tau) release was measured in vivo in rat striatum following direct application to the microdialysis tube of 60 mm ammonium chloride which renders the final ammonia concentration in the extracellular space of ϳ5 mm. various in vivo stimuli evoke taurine efflux either by opening osmosensitive anion channels and/or by a mechanism secondary to glu accumulation and its interaction with nmda or ampa receptors. the following compounds were coadministered with ammonia to distinguish between these mechanisms: anion/cation transport inhibitors -dids and furosemide, a glu transport inhibitor-pdc, and nmda and ampa/ka receptor antagonists dizocilpine and dnqx. ammonia stimulated tau accumulation in the microdialysates to ϳ250% of basal value. dids and furosemide moderately inhibited the effect of ammonia (furosemide by ϳ30%), albeit dids added alone induced massive accumulation of tau with a delayed onset as compared to ammonia. ammonia-dependent tau accumulation was increased by ϳ50% in the presence of pdc and reduced in an equal degree (ϳ35%) by dizocilpine and dnqx. none of the agents affected tau accumulation in the absence of ammonia. the results show that ammonia in vivo evokes tau accumulation both via anion channels, possibly secondary to cell volume changes, and in consequence of stimulation of both nmda and ampa/ka receptors. (supp. by a scsr grant no 4p05a05519 and cimo, the acad. of finland) biosciences department, university of hertfordshire, hatfield herts, u.k. the discovery in 1987 that endothelium-derived relaxing factor is nitric oxide (no) was followed a year later with reports that the cationic amino acid l-arginine is the physiological precursor for nitric oxide. it has since been established that the terminal guanidinium nitrogen of l-arginine is metabolised via a series of oxidation reactions resulting in no production, with citrulline being formed as a co-product. of interest was the parallel observation that uptake of l-arginine was enhanced in inos expressing cells and that this was due to de novo synthesis of carrier proteins. the precise signaling pathway that regulates the enhanced expression of these carriers has been the subject of intense studies in recent years. current literature suggests that activation of upstream signaling molecules such as protein kinase c may be critical. in addition, downstream kinases thought to be points of convergence for various signals originating from cell surface receptors have also been implicated. two of these downstream targets include the 42 and 44 kda forms of mitogen-activated protein kinase (mapk) and the stressactivated 38 kda mapk. it is worth noting however that the involvement of these different transduction pathways in the regulation of the induction of l-arginine transporters is not universal, and likely to be different from system to system. as a result there has been conflicting data on the relevance of these signaling proteins in inducing l-arginine transport in different cell. these issues will be discussed and the individual signaling pathways assessed on a cell type and species basis. moreover, the role of downstream signaling molecules will be examined in more detail, looking in particular at the critical dependency on the p38 mapk. this kinase currently exists in four different isoforms which are p38α, , γ and δ. the involvement of individual isoforms of p38 in enhancing the expression of carrier proteins for l-arginine will be discussed. gw274150 is an acetamidine derivative of heterosubstituted lysine which has been shown to have a marked selectivity for the human inducible nitric oxide synthase isoform (young et al. 2000. bioorg. med. chem. lett., 10: 6, 597-600) . the systems associated with transport of this compound have been investigated using the macrophage cell line j774. prior to each study, j774 cells were seeded in 96-well culture plates and allowed to adhere for 24 h in dulbecco's modified eagle's medium (dmem). transport studies were carried out using hepes buffered krebs solution (50 µl; 37°c) containing l-[ 14 c]gw274150 (1 µciml ϫ1 ) in the presence of either 0.1 mm or 0.025-1 mm unlabelled substrate. in parallel studies transport (1 µciml ϫ1 , 0.1 mm) was monitored in the presence of 1 mm excess of various other amino acids known to be substrates for distinct transport systems. time course experiments revealed that transport of 0.1 mm of l-[ 14 c]gw274150 occurred in a time-dependent manner and was linear for up to 5 min. in addition, uptake was only marginally dependent on extracellular na ϩ . kinetic studies revealed that transport was saturable, and michaelis-menten analysis revealed single affinity entry with an apparent k t of 0.31 mm and v max of 5.15 pmol·µg protein ϫ1 min ϫ1 . at 1 mm, 2-methylaminoisobutyric acid (meaib), lalanine, l-valine and -2-amino-bicyclo-(2,2,1)-heptane-2carboxylic acid (bch) caused little or no inhibition of l-[ 14 c]gw474150 (0.1 mm) uptake. in contrast, transport of l-[ 14 c]gw274150 was inhibited markedly by l-arginine, llysine, l-leucine, l-methionine, 6-diazo-5-oxo-l-norleucine (don) and l-glutamine. with the exception of l-arginine and l-lysine, the inhibition caused by the other substrates was critically dependent on extracellular na ϩ and was completely reversed when extracellular na ϩ was replaced with choline. in parallel kinetic inhibition experiments, transport of 0.1 mm l-[ 14 c]gw274150 was inhibited in a concentration dependent manner by l-arginine (ki ϭ 0.04 mm), l-leucine (ki ϭ 0.06), don (ki ϭ 0.18 mm) and l-glutamine (ki ϭ 0.13 mm). taken together, these data suggest that gw274150 may be transported, at least in part, by system y ϩ . however, the marked inhibition caused by l-leucine, l-glutamine and l-methionine, substrates for the relatively high affinity cationic amino acid transporter system y ϩ l, would suggest that this system may also contribute to the uptake of gw274150; if so, the monophasic substrate kinetics imply that the two systems handle gw274150 with similar affinity. other systems such as b 0,ϩ could be ruled out on the grounds that this transporter is critically na ϩ -dependent while uptake of gw274150 is largely (ϳ80%) na ϩ -independent. similarly, b 0,ϩ , another broadspectrum aminio acid transporter that may be capable of transporting gw274150 does not interact with l-glutamine and thus unlikely to be involved in transport of gw274150, at least in j774 cells. although a large number of different amino acid transporters have been identified on a molecular basis, some of themfunctionally described in mammalian cells -are still missing. in search of mammalian est sequences, which contain the signature of the aaap (amino acid/auxin permease) family, we identified a murine full length cdna, which encodes a membrane protein with 10-11 putative transmembrane domains. the transporter mrna is expressed in various murine tissues, including lung, heart and kidney. for functional characterization we used the xenopus laevis oocyte expression system and employed flux studies and electrophysiological analysis. oocytes injected with the crna showed an increased uptake of 3 h-l-alanine and 3 h-l-proline. detailed electrophysiological analysis revealed an electrogenic transport mode, independent of sodium and chloride ions. lowering the extracellular ph increased significantly substrate induced currents in crna injected oocytes. out of the 20 proteinogenic amino acids the transporter recognizes only small amino acids, such as gly, ala, pro and ser. distinct structural analogues of these amino acids also interact with the transporters substrate binding site. in conclusion, we describe the molecular and functional characteristics of the first electrogenic proton driven amino acid transporter of mammals. pharmacology department, dr. willmar schwabe gmbh, karlsruhe, germany it is now well established that transport of amino acid neurotransmitters (like glutamate, aspartate, gaba and glycine etc.) from and to the neurones is essential for their proper functioning. like in the case of other neurotransmitters, specific pre-and post-synaptic as well as vesicular transporters are involved in such processes. extensive efforts to clarify the mechanisms and processes involved in the control and/or proper functioning of the amino acid transporters are now, therefore, being made in numerous laboratories. such efforts have not only led to the identification of a few specific ligands and/or modulators of neuronal amino acid transporters, but also have started unravelling the complex and diverse processes regulating their functions. aim of this communication is to point out potential usefulness of some neuroactive constituents isolated from therapeutically used medicinal herbs for clarifying the mechanisms involved in neuronal amino acid transport. our interest in such studies was initially triggered by the observations made with hyperforin, i.e. quantitatively the major neuroactive component of hypericum perforatum extracts widely used for the treatment of mild to moderate depressive disorders. this acyl phloglucinol derivative not only modulate synaptic transports of biogenic amines but also of glutamate, aspartate and gaba. since it does not interact with any of the till now described transporters for these neurotransmitters, efforts were made to clarify the mechanisms involved in their observed effects (both in vitro and as well as in vivo). the results of the in vitro studies available to date strongly suggest that its effects on neuronal amino acid transport processes is mediated via some novel extracellular mechanism controlling the h ϩ (and/or other ionic) concentrations of neurones. these observations not only demonstrate that hyperforin represent a structurally and mechanistically novel class of therapeutically useful agent but also suggest that it could be useful tool for clarifying the complex mechanisms involved in the control of neuronal amino acid transport. these observations stimulated us to screen other putative psychoactive herbal extracts and their active constituents on neuronal amino acid transport and on the consequences of disturbances caused by malfunction of specific transporters. observations made with several such agents indicate that either modulation of mechanisms and/or processes involved in neuronal amino acid transport or reversal of pathologies caused by anomaly of transporter functions could be involved in their modes of actions. these observations reinforce our conviction that studies directed towards clarifying the effects of herbal constituents on neuronal amino acid transport might not only be a feasible way for identifying novel types of therapeutically interesting molecules but also could expedite our knowledge on these complex processes. glutamate-regulated sodium dynamics in cortical astrocytes: implications for cellular bioenergetics j.-y. chatton, p. marquet, and p. j. magistretti the mode of na ϩ entry and the dynamics of intracellular na ϩ concentration (na ϩ i ) changes consecutive to the application of the neurotransmitter glutamate were investigated in mouse cortical astrocytes in primary culture by video fluorescence microscopy. an elevation of na ϩ i was evoked by glutamate, whose amplitude and initial rate were concentration-dependent. the glutamate-evoked na ϩ increase was primarily due to na ϩ -glutamate cotransport. the rate of na ϩ influx decreased during glutamate application, with kinetics that correlate well with the increase in na ϩ i and which depend on the extracellular concentration of glutamate. a tight coupling between na ϩ entry and na ϩ /k ϩ atpase activity was revealed by the massive na ϩ i increase evoked by glutamate when pump activity was inhibited by ouabain. during prolonged glutamate application, na ϩ i remains elevated at a new steady-state where na ϩ influx through the transporter matches na ϩ extrusion through the na ϩ /k ϩ atpase. a mathematical model of the dynamics of na ϩ i homeostasis will be presented which precisely defines the critical role of na ϩ influx kinetics on the establishment of the elevated steady-state and its consequences on the cellular bioenergetics. indeed, extracellular glutamate concentrations as low as 10 µm approximately doubled the energetic demands of the astrocytes. department of biochemistry and molecular biology, faculty of biology, university of barcelona, spain in the last 5 years a new family of amino acid transporters composed by two different subunits has been defined. two heavy subunits (rbat and 4f2hc) and seven light subunits are known. rbat and the light subunits b0,ϩ at and y ϩ lat1 are responsible for the inherited aminoacidurias type i cystinuria, non-type i cystinuria and lysinuric protein intolerance, respectively. the heavy subunits are highly glycosylated type ii proteins, while light subunits are very hydrophobic unglycosylated membrane proteins, displaying a polytopic (generally 12 transmembrane domains) predicted structure. the specificity of the amino acid transport activity depends on the light chain expressed. this, together with its topology, indicates that the transport function mainly relies on the light subunits. i will summarize some of our current studies directed to the understanding of structure-function relationships of these heteromeric carriers, specially concerning their oligomeric structure and initial attempts to reconstitute them. ongoing work on the isolation of new rbat-associated light subunits and new b0,ϩ at-associated heavy subunits, which could also play a role in cystinuria, will also be discussed. department of pharmacology, joh. gutenberg university, mainz, germany mammalian cationic amino acid transporters (cats) catalyze the transport of basic amino acids through the plasma membrane. the cat family comprises at least five related carrier proteins (cat-1, -2a, -2b, -3 and -4) with cat-2a and -2b being splice variants. in humans, only the "old" members of the family have been characterized (hcat-1, -2a and -2b). hcat-1 and -2b exhibit high affinity for cationic amino acids and are sensitive to trans-stimulation, consistent with the classical system y ϩ . in contrast, hcat-2a is a low affinity carrier relatively insensitive to trans-stimulation. interestingly, hcat-2a and hcat-2b differ only in a region of 42 amino acids. cat-3, so far only identified in rat and mouse, exhibits also system y ϩ activity. however, the substrate recognition and maximal transport activity seems to differ from other y ϩ transporters. cat-3 expression has been reported to be restricted to the brain in adult animals. a cdna encoding for human hcat-4 has recently been isolated, however, the transport activity of hcat-4 has not been characterized. when optimally aligned, the amino acid sequence of hcat-4 shows only about 40% identity with the other hcat isoforms. in contrast, the amino acid sequences of hcat-1, -2(a or b) and -3 are about 60% identical. to elucidate which amino acids are responsible for the difference in the transport properties of the hcat proteins, we constructed chimeric proteins between hcat-1 and hcat-2a and performed site directed mutagenesis. using this approach, we identified two amino acid residues that are responsible for the different transport properties of hcat-2a compared to the high affinity cat-isoforms. to characterize the human cat-3, we cloned a cdna encoding hcat-3. when expressed in xenopus laevis oocytes, hcat-3 had a similar transport activity and affinity for l-arginine as hcat-1 or -2b. hcat-3mediated l-arginine transport was trans-stimulated and independent of extracellular na ϩ ions. expression studies demonstrated that hcat-3 is not only expressed in different regions of the human brain, but also in peripheral tissues. to investigate if hcat-4 also functions as an amino acid transporter, we measured the transport of cationic, neutral and acidic amino acids in xenopus laevis oocytes expressing hcat-4, but could not detect an transport activity for any substrate tested. a bright fluorescence could be detected in the plasma membrane of oocytes expressing hcat-4 with the green fluorescent protein attached to the c-terminus. therefore, hcat-4 might either need a complementary protein to function as an amino acid transporter or serve as a transporter for a yet unidentified substrate. renal amino acid reabsorption in immature and adult rats as a sensitive marker of heavy metal-induced nephrotoxicity (pt, cr, tl) institut für pharmakologie und toxikologie, klinikum der friedrich-schiller-universität jena, germany the effects of cis-platinum (cp; 0.6 mg/100 g b. wt. i. p.), sodium dichromate (cr; 1 mg/100 g b. wt. s. c.) and tl 2 so 4 (tl, 2 mg/100 g b. wt. i. p.) on renal amino acid excretion and plasma amino acid composition were investigated in 10-(both sexes) and 55-day-old (female) anaesthetised wistar rats (han : wist). on the basis of diuresis experiments on conscious rats (determination of urinary volume and protein excretion) the mentioned doses and times (1 st day after cr in both age groups and in 10-day-old rats after cp and 3 rd day after cp in adult rats; 2 nd [55-day-old rats] and 5 th -6 th day [10-day-old rats] after tl) were found out to be optimal for the characterisation of amino acid transport after heavy metal poisoning. interestingly, in conscious 10-day-old rats cr nephrotoxicity is not detectable after 1 mg/100 g b. wt. whereas all of the other experimental groups showed nephrotoxic effects of cr, tl and cp in conscious rats. urine volumes were lower, but not significantly, in anaesthetised immature rats, independently of the administered nephrotoxin. glomerular filtration rate (gfr) is significantly lower in 10-day-old rats compared to adults. after cp, cr and tl gfr is significantly reduced only in adult rats and age differences disappeared nearly completely. in principle the renal fractional excretion (fe aa ) of amino acids was distinctly higher in immature rats as a sign of lower amino acid reabsorption capacity. nevertheless, the amino acid plasma concentrations were relatively high in immature control rats. however, both cr and cp did not distinctly influence molecular cloning and functional characterization of ata3, a novel subtype of the amino acid transport system a medical college of georgia, augusta, georgia, u.s.a. recent molecular cloning studies have revealed that the amino acid transport system a consists of more than one subtype. two different system a subtypes, called ata1 and ata2, have been cloned and functionally characterized. ata1 is expressed primarily in the brain and placenta whereas ata2 is expressed ubiquitously. heterologous expression studies have shown that these two subtypes cannot be distinguished functionally based on substrate affinity nor substrate specificity. we have now cloned a third subtype of system a, designated ata3. it is expressed primarily in the liver. apart from the liver, detectable level of expression is noted only in the skeletal muscle. interestingly, ata3 can be easily differentiated from the other two subtypes of system a based on functional characteristics. we first isolated rat ata3 cdna from a skeletal muscle cdna library using rat ata2 cdna as the probe. rat ata3 consists of 547 amino acids and exhibits a high degree of homology in amino acid sequence to rat ata1 (47% identity) and rat ata2 (57% identity). interestingly, this new transporter also has a comparable degree of homology to sn1 and sn2, the two known subtypes of the amino acid transport system n. however, when expressed heterologously in xenopus laevis oocytes, rat ata3 transports α-(methylamino)isobutyric acid (meaib), a specific model substrate for system a, confirming that this transporter is definitely a subtype of system a. system n does not transport this system a model substrate. with two-microelectrode voltage-clamp technique, we have shown that exposure of rat ata3-expressing oocytes to neutral, short-chain aliphatic amino acids induces inward currents. the amino acid-induced current is na ϩ -dependent and phdependent. analysis of the currents with alanine as the substrate has shown that k 0.5 for alanine (i.e., concentration of the amino acid yielding half-maximal current) is 4.2 ϯ 0.1 mm and that the na ϩ : alanine stoichiometry is 1 : 1. subsequently, we have cloned the human homolog of rat ata3 from a liver cell line (hepg2) cdna library. human ata3 also contains 547 amino acids and shows 88% identity in amino acid sequence with rat ata3. the sequence identity of human ata3 with human ata1 and human ata2 is 47% and 57%, respectively. the homology of human ata3 with human sn1 and sn2 is also similar (56% and 51% identity, respectively). the gene coding for human ata3 contains 16 exons and is located on chromosome 12p13. in the human, ata3 is expressed almost exclusively in the liver. when expressed in mammalian cells heterologously, human ata3 mediates the transport of neutral amino acids, including meaib, in a na ϩ -dependent manner. interestingly, while characterizing the function of this clone, we have uncovered a unique feature of this system a subtype. human ata3 is capable of mediating the transport of cationic amino acids. in fact, the affinity of human ata3 for cationic amino acids is higher than for neutral amino acids. however, the human ata3-mediated cationic amino acid transport is na ϩ -independent. in this respect, ata3 is similar to transport system y ϩ l that also transports neutral amino acids in a na ϩ -coupled manner and cationic amino acids in a na ϩindependent manner. in contrast, ata1 and ata2 have not been shown to interact with cationic amino acids. in addition to this difference in substrate specificity, ata3 also differs from ata1 and ata2 in substrate affinity. ata1 and ata2 interact with meaib with a k t of ϳ0.3 mm whereas the affinity of ata3 for this model substrate is comparatively at least 20-fold lower (k t , ϳ8 mm). but, ata3 interacts with arginine with a k t value of 0.3 mm. since liver does not express any of the previously known high affinity cationic amino acid transporters, amino acid plasma concentrations. but in both age groups the administration of cr and cp significantly decreased amino acid reabsorption capacity (increase in fe aa ) as a sign of nephrotoxicity, most pronounced in adult rats after cp. on the other hand, after tl, the fe of amino acids was distinctly higher only in adult rats as a sign of lower amino acid reabsorption capacity and, thus, as a sign of higher nephrotoxicity. in immature animals fe aa was increased only for few amino acids. however, in both age groups tl administration significantly decreased plasma amino acid concentrations, more pronounced in immature rats. the investigation of renal amino acid handling confirmed: (1) cr, cp and tl were more nephrotoxic in 55-day-old animals compared to immature rats as could be demonstrated previously using other parameters for nephrotoxicity testing. (2) the extent of toxic effects of heavy metals on the kidney is related to the maturity of renal functions involved in the enrichment of the respective metal in renal tissue and in its toxicity mechanism. (3) changes in the fractional excretion of amino acids (reduction in renal amino acid reabsorption capacity, e.g. increase in fe aa ) and in amino acid plasma concentrations (especially decreases as a consequence of enhanced renal loss of amino acids) are early indicators of nephrotoxicity. (4) therefore, the determination of renal amino acid handling is a highly sensitive marker for nephrotoxicity testing, both in immature and in adult rats. the mammalian h ϩ /peptide cotransporter pept2 was initially identified in the brush border membrane of renal proximal tubular cells as a high affinity type ptr2-family member. here we describe the synthesis and functional analysis of novel high affinity inhibitors for pept2 that will be useful in further studies on structure and functions. starting from lys[z(no 2 )]-pro a series of different lysine-containing dipeptide derivates were synthesized and studied for interaction with pept2 based on transport competition assays in pichia pastoris yeast cells and in epithelial skpt cells, both expressing pept2. the twoelectrode-voltage-clamp technique in x. iaevis oocytes expressing pept2 was used to determine whether the compounds are transported electrogenically or block the uptake of dipeptides. synthesis and functional analysis of lys-lys derivates containing z(no 2 ) side chain protections provided a set of inhibitors that reversibly inhibited the uptake of dipeptides by pept2 with k i values as low as 10 nm. this is the highest affinity of a ligand of pept2 ever reported. moreover, based on the structure-function relationship we can conclude that the spatial location of the ε-amino protecting group in a lys containing dipeptide and its intramolecular distance from the alpha catom are key factors for the transformation of a substrate into an inhibitor of pept2. ata3 is likely to provide the major route for the uptake of arginine in this tissue. institute of pharmacology and therapeutics, faculty of medicine, porto, portugal the present study examined the nature and regulation of the l-dopa transporter in two functionally different clonal subpopulations of opossum kidney (ok lc and ok hc ) cells. the inward transfer of l-dopa was largely promoted through an energy-dependent and sodium-insensitive transporter, though a minor component (ϳ15%) was found to require extraceilular sodium. l-dopa uptake was insensitive to meaib, but competitively inhibited by bhc (ok lc , ic 50 ϭ 336 µm; ok hc , ic 50 ϭ 439 µm). l-and d-neutral amino acids and basic amino acids markedly inhibited l-dopa accumulation. l-dopa, lleucine, l-arginine, bhc or l-arginine plus bhc stimulated [ 14 c]-l-dopa efflux. the accumulation of l-dopa was significantly higher at an acidic ph, and incubation of cells with l-dopa (100 µm) resulted in marked intracellular acidification. modulators of pka, pkg, pkc and ptk failed to affect the accumulation of l-dopa. only the ca 2ϩ / calmodulin inhibitors inhibited l-dopa uptake. it is likely that system b 0,ϩ might be responsible for the sodium-dependent uptake of l-dopa in ok cells, whereas sodium-independent uptake of l-dopa may include systems b 0,ϩ and lat2, the activation of which results in trans-stimulation of l-dopa outward transfer. the trans-stimulation of l-dopa inward transfer by an imposed h ϩ gradient suggest that ok cells are provided with an l-dopa-h ϩ cotransport system. amino acids are essential nutrients for cell growth and maintenance. the essential amino acids arginine and lysine, are mainly transported via the cationic amino acid transporter 1 protein (cat1). the regulation of translation of the cat1 mrna during amino acid starvation was studied. an adaptive response to amino acid starvation and stress is a global decrease of protein synthesis, by phosphorylation of the translation initiation factor eif2a. translation of the transporter mrna increases when eif2a is phosphorylated, allowing synthesis of the essential for survival arginine/lysine transporter protein. the mechanism of increased translation of this mrna involves the induction of activity of a uorf-containing internal ribosomal entry sequence (ires). translation of the uorf and phosphorylation of eif2a are required for increased activity. we propose that eif2a phosphorylation triggers translational attenuation within the uorf, converting a relatively inactive, to a high activity ires. this study demonstrates that like yeast, mammalian cells have developed a sophisticated response to stress conditions: when expression of most genes decreases, synthesis of stress response proteins increases to support cell survival. amino acid transport, cell volume and the regulation of cell death f. lang, s. fillon, i. setiawan, p. lang, v. tanneur, d. häussinger, and s. bröer department for physiology, university of tübingen, germany cell volume regulatory mechanisms participate in a wide variety of cellular functions including regulation of epithelial transport, excitability, hormone and transmitter release, metabolism, migration, cell proliferation and apoptotic cell death. besides ion transport, polyols, betaine and glycerophosphorylcholine, cells utilize amino acids including taurine to balance extracellular osmolarity and regulate their volume. cells counteract shrinkage by uptake and swelling by release of amino acids including taurine. moreover, cell swelling stimulates synthesis and cell shrinkage favours breakdown of proteins which are osmotically less active than the sum of the amino acids thus generated. conversely, amino acid transport does influence cell volume. concentrative uptake of amino acids leads to cell swelling, amino acid release to cell shrinkage. through alterations of cell volume the amino acids participate in the regulation of protein metabolism. thus, concentrative amino acid transport inhibits and release of amino acids favours proteolysis. these mechanisms participate in the regulation of cell death. cd95 induced apoptotic death of jurkat t lymphocytes is paralleled by the release of taurine. the taurine release occurs with a delay of some 60 min following cd95 receptor triggering but immediately preceedes apoptic cell shrinkage and dna fragmentation. the signaling leading to taurine release is in large part elusive but requires at some stage activation of caspases. moreover, taurine release and apoptotic dna fragmentation are strongly inhibited by lowering of temperature. preloading of the cells with taurine retards cd95 induced dna fragmentation pointing to an active role of taurine in the regulation of apoptosis. peptide transporters of the ptr-family are integral plasma membrane proteins, that mediate the electrogenic protoncoupled transport of di-and tripeptides and peptide-like drugs across cell membranes. the physiological role of pept1, one member of this family in mammals, is mainly the uptake of small peptides into intestinal and renal tubular epithelial cells. in caenorhabditis elegans a homologue to mammalian pept1 is encoded by the pep-2 gene, which is expressed in the intestinal cells and a subset of sensory neurons in the head of the animal. to study the physiological role of the pep-2 transporter in vivo, a c. elegans pep-2 mutant was constructed. the animals deficient in pep-2 show a remarkable phenotype with pronounced signs of malnutrition, characterised by a delayed development, less eggs in the uterus, a smaller brood size and a prolonged mean life-span compared to wild-type animals. we rescued the phenotype by the expression of the wt pep-2 gene in the mutant. the observed starved phenotype in pep-2 mutants might be best explained by the reduced intestinal absorption of peptide bound amino acids that are required for protein synthesis and energy metabolism and provides the first direct evidence for the predominant role of the intestinal peptide transporter in amino acid absorption. adenosine is a potent vasodilator in many vascular beds and modulated tone via elevation of intracellular camp and/or release of nitric oxide (no). we have previously reported that adenosine (ado) stimulates l-arginine transport and no production in human cultured umbilical vein endothelial cells (sobrevia et al., j. physiol. 499, 135-140, 1997) , and here further characterise the signalling cascades. rt-pcr demonstrated that fetal endothelial cell possess mrna levels for a 2a , a 2b and a 3 -adenosine receptor subtype, whereas negligible levels were detected for the a 1 -receptor. adenosine (10 µm, 2 min) induced increases in l-arginine transport and no production were ca 2ϩ and camp independent and stimulated transport was abolished in cells depolarised with 80 mm k ϩ . whole-cell patch clamp experiments revealed that adenosine activated inward k ϩ currents, resulting in a membrane hyperpolarization and enhanced influx of the cation substrate l-arginine. adenosine induced l-arginine transport and no production were also abolished by inhibitors of tyrosine kinases (genistein), mek1/2 (pd98059, u0126) but unaffected by inhibitors of pkc (calphosin c) and pi-3 kinase (ly29002). these data suggest that adenosine induces membrane hyperpolarization by activating inward k ϩ currents, increasing the driving force for cationic amino acid influx via system y ϩ . the discovery of nocardicine a by aoki et al. and aztreonam showed that monocyclic -lactams, collectively known as monobactams, can have antibiotic activity. this activity is poor but compensated by the unique effect they can induce on certain microbial cell membranes. our quest for new non-conventional surfactants for various biomedical applications led us to synthesize bioactive compounds with structural similarities to nocardicins. we present here the preparation and the study of original trimodular biosurfactants of type i: spermine and amine oxidase induce a cytotoxic effect on multidrug resistant chinese hamster ovary cells e. agostinelli 1 , s. lord-fontaine 2 , e. przybytkowski 2 , and d. a. averill-bates 2 1 department of biochemical sciences "a. rossi fanelli", university of rome "la sapienza" and cnr, centre of molecular biology, rome, italy 2 department de chimie/biochimie and toxen (centre de recherche en toxicologie de l'environnement), université du québec à montréal, canada the occurrence of resistance to cytotoxic agents in tumor cells is a major obstacle to successful anticancer chemotherapy. multidrug resistance (mdr) is associated with several phenotypic alterations. cells with the mdr phenotype display decreased drug accumulation due to overexpression of pglycoprotein (p-gp), encoded by the mdr-1 gene, which acts as an energy-dependent pump involved in extrusion of drugs. we studied a new strategy to eliminate mdr cells using an enzyme, bovine serum amine oxidase, capable of forming cytotoxic products, h 2 o 2 and aldehyde(s), from polyamines (spermine). the involvement of both toxic products, formed by the bsao/spermine enzymatic system, in causing cytotoxicity was investigated in multidrug resistant chinese hamster ovary cells, ch r c5, at 37 and 42°c. we observed that hyperthermia, depletion of intracellular glutathione (by l-buthionine sulfoximine) and inhibition of glutathione s-transferase (by ethacrynic acid), sensitized ch r c5 cells to the cytotoxic effect of spermine enzymatic oxidation products. mdr cells showed no resistance to h 2 o 2 and aldehyde(s) relative to their drug-sensitive counterparts, auxb1 cells, in experimental conditions of: higher temperature, higher spermine concentration and longer incubation time. the inhibition of cellular detoxification systems led to increased cytotoxic effects of spermine enzymatic oxidation products on both mdr and sensitive cell lines. these results might be of great interest and suggest that toxic oxidation products formed from spermine and amine oxidase could be used in anticancer therapy, mainly against multidrug resistant tumor cells. [acknowledgements: this work was supported by cnr "target project on biotechnology", ministero della sanità tar these compounds present a hydrophobic part introduced by an ester or amide linkage with an aminoacid, a junction modulus which corresponds to -lactam, and a hydrophilic part which contains a triazole, well-known in pharmaceutical industry for its inhibitor effect against -lactamase. the compounds are synthesised from 2-hydroxymethyl-2methyl propionic acid in five steps. selective activation of one of the primary hydroxyl groups was accomplished by the formation of alkoxy tris(dimethylamino)phosphonium (atdp) salts 3 from the corresponding diol. treatment of 3 with excess potassium carbonate in refluxing anhydrous acetone yields the monobactams 4. activation by atdp salts followed by treat-ment with sodium azide and reflux in toluene gives the azido compound. the reaction with acetylenic derivatives allows to obtain the surfactants. the compounds show classical surfactant behavior and the evaluation of their biological properties give evidence for their antibacterial and antiviral activity, which corresponds apparently to antiprotease activity. a prodrug approach to glutathione derivatives with in vitro antiparasitic activity department of chemistry and materials manchester, faculty of science and engineering, metropolitan university, manchester, u.k. the potential chemotherapeutic activity of peptides are lost in many cases in vitro, due to their inability to cross cell plasma membranes. the recent identification of a series of glutathione diesters with high antiparastic activities in vitro against t.b.brucei (african sleeping sickness) lead us to investigate the determinants associated with their activity. a qsar study on some twenty-five diester derivatives against t.b.brucei and t.b. rhodesiense lead us to conclude that the mechanism of action of these compounds is related to membrane penetration and hydrolysis, controlled by hydrophobicity and steric factors. a hplc and sensor study have confirmed the de-esterified diacid as the active agent of these prodrugs. dietary taurine prevents oxidative stress and morphological alterations in the retina of diabetic rat f. franconi 1 , m. a. s. di leo 2 , s. caputo 2 , n. gentiloni silveri 2 , and g. ghirlanda 2 1 department of pharmacology, university of sassari, and 2 department of internal and geriatric medicine, catholic university, rome, italy diabetes mellitus can cause various complications including retinopathy, which is the earliest and most common complications of diabetes mellitus, affecting 90% of diabetics and progressing to blindness in about 5%. considerable evidence implicates oxidative stress in the pathogenesis of diabetic retinopathy. in fact, hyperglycemia generates reactive oxygen species and free radical defense is reduced in diabetic patients. thus, the prevention of oxidative stress may have important implications for pharmacological attempts to prevent diabetic retinopathy. at this regard, it has been found that taurine, a semi essential amino acid with antioxidant activity, is decreased both in type 1 and type 2 diabetes mellitus. moreover, taurine seems to have a peculiar role of taurine in terms of cellular physiology and pathophysiology of the retina. among others, taurine is thought to produce important physiological effects through osmoregulation, calcium modulation and antioxidant effects. therefore, we examined the effect of dietary chronic (4 months) taurine (2% and 5%) supplementation in diabetic rats in comparison with vitamin e (200 and 500 ui). dietary taurine supplementation, for 4 months, does not influence conjugated dienes (cd), lipid peroxides (lp) and na/k atpase activity in the retina of non diabetic rats. using rats streptotozocin (stz) induced diabetes of 4-month duration, we found that cd, lp are significantly increased and they remained elevated for 4 months. while, the na/k atpase is significantly decreased during the whole experimental time (4 months). moreover, an inverse correlation has been found among the cd and lp and atpase activity. in the retina of stz rats, these biochemical alterations are accomplished with marked profound morphological changes. in stz rats, taurine enriched diets decrease the lipid peroxidation and preserve the atpase activity, being 5% taurine more effective than 2% diets. the morphological examination reveals that in rats feed with 5% taurine no proliferative changes are present. moreover, the beneficial effects of taurine are more marked than of those of vitamin e. these results and previous findings encourage new investigations to evaluate the efficacy of taurine as an adjunctive agent ch ch 3 (ch 2 ) n xco iran applicated be (500 mg/kg -10 days) and the third -control. enzyme activities were determined spectrophotometrically in brain homogenate. results: polyamine oxidase activity decreased significantly lower dose of be didn't induce any significant change in diamine oxidase activity gaba-transaminase activity increased significantly (p ͻ 0.005; p ͻ 0.001) and dose dependently upon be treatment we have been examined the effects of propofol, taurine and propofol combined with taurine on free intracellular amino acid (aa) profiles, superoxide anion formation (o 2 ϫ ), hydrogen peroxide production (h 2 o 2 ) and released myeloperoxidase activity (mpo) in polymorphonuclear leucocytes (pmn). propofol led to significant changes in pmn free taurine, glutamine, glutamate, aspartate, methionine, basic, neutral (naa) and branched chain amino acid concentrations. exogenous taurine reduced pmn naa while increasing intracellular taurine. taurine supplemented to propofol significantly reversed the changes in taurine, naa and alanine only. regarding pmn immune functions propofol significantly decreased o 2 ϫ , h 2 o 2 formation and mpo. taurine decreased o 2 ϫ and h 2 o 2 production, while increasing released mpo. when propofol and taurine were combined they appeared to by reacting tyrosine with 1-nitroso-2-naphthol in the presence of nitric acid 1-2-benzyo-8-(alanyl)-3-phenoxazone (blp) an analog of actinomycin d is produced. the structural similarity of blp to actinomycin d prompted the national cancer institute (nci) to investigate its antitumor activities. the nci investigations revealed that blp exhibits growth inhibitory effects on various cancer cells and as a result blp has received the u.s. patent from the u.s. patent office. the purposed of this investigation was to synthesize similar benzo phenoxazone derivative by reacting 1-nitroso-2-naphthol with 4-(α-hydroxy -methylaminopropyl)phenol in the presence of nitric acid. during the study, it was found out that 1,2-benzo-3phenoxazone derivative is not produced but a hydrogenated form of 1,2-benzo-3-phenoxazone which is probably 1,2-benzo-8-(α-hydroxy -methylaminopropyl)-3-hydroxyphenoxazine (bhmhp) which has been suhhested from mass spectra obtained by electron ionization, ei, chemical ionization, ci and electro-spray ionization, esi, methods. 1 bhmhp was screened against various cancer cell lines by nci and has shown promising effect against three (3) breast cancer cell lines: mda-mb-435, mda-n and hs-578 t. the 50% growth inhibitory (gi 50 ) concentrations for these three cell lines were 4.60 ϫ 10 ϫ6 , 4.62 ϫ 10 ϫ6 and 5.74 ϫ 10 ϫ6 molar respectively. a. bocheva 1 and t. pajpanova 2 1 institute of molecular biology, bulgarian academy of sciences, and 2 institute of physiology, bulgarian academy of sciences, sofia, bulgariathe histamine is an endogenous substance with neurotransmitter and neuromodulator functions in the organism. its antagonists are used in the therapy of allergic diseases and inflammatory reactions and as antiulcer drugs.the limited potentialities of the antihistamine therapy together with the increasing number of the people suffering from allergic diseases give rise to the design and synthesis of new histamine analogues as a perspective area in the chemistry of therapeutic drugs.additionally, compounds containing the guanidine, oxyamino and sufonamide moieties are known to elicit a variety of pharmacological responses and are present in several marketing drugs or drug candidates.on the other hand, similar compounds, being a part of bigger structures (for instance peptides), can imitate the molecules of already known at ii-receptor antagonists.having in mind these data we aimed to synthesize new analogs of histamine containing sulfo-and oxy-guanidino groups with common formula: a. bocheva 1 , s. pancheva 2 , and t. pajpanova 2 1 institute of physiology, bulgarian academy of sciences, and 2 institute of molecular biology, bulgarian academy of sciences, sofia, bulgariathe problem of the efficient therapy of pain is important not only from clinical but from social and economic point of view. the great achievements in medicine are connected with the research on the development of antinociceptive drugs.melanocyte-inhibiting factor (mif) is a tripeptide (pro-leu-gly-nh 2 ) that was discovered in hypotalamus.the mif-1 exerted a weak analgesic effect. the synthesis of non-protein amino acids and their incorporation into biologically active peptides might become a powerful method for the design and development of modified analogues of natural peptides. having in mind these data we synthezied a number of new mif-analogues, containing unnatural amino acids such as cav, slys, sleu, slle and snie and in vivo experiments were performed to study their action on the nociception. the changes in nociceptive effects were examined in male wistar rats by the tail-flick (tf) and hot-plate (hp), as well as, the randall-seitto paw-pressure tests. the peptides were applied intaperitoneal (i.p) injection at a does 1 mg/kg. the results show that the newly sinthesized analogues exert an antinociceptive effects in all tests used. naloxone at a dose 1 mg/kg (i.p) antagonized the antinociceptive effects of mif-analogues. the interaction between platelets and fibrinogen is known to be mediated by the intergrin gp iib/iiia. the arg-gly-asp (rgd) sequence located on fibrinogen and other proteins of blood and extracellular matrix is the minimum requirement for cell attachment and adhesion. it has been found that peptides containing the rgd sequence can effectively inhibit the binding of fibrinogen to gp iib/iiia. in addition aspirin has been shown to be beneficial in the treatment of stable and unstable angina, acute myocardial infraction. aspirin acetylates and inhibits the enzyme cyclooxygenase, the first enzyme involved in thromboxane a 2 (txa 2 ) synthesis, an activator of platelet aggregation and adhesion.we have already reported that the combination in the same molecule of dipeptide amides, containing amino acid(s) of rgd sequence, with salicylic-residue 2-ro-c 6 h 4 -coϳ, {where rϭh or ch 3 co} at their n-terminal amino group have shown inhibitory activity on human platelet aggregation. continuing this research project on salicyl-peptides we have synthesized a series of rgd analogs, incorporating salicylic acid derivatives, by conventional solution techniques and/or by solid phase. the synthesized rgd analogs were identified by ir, nmr and es-ms spectra and tested for inhibitory activity on human platelet aggregation in vitro, by adding common aggregation reagents (collagen, adp, thrombin) to citrated platelet rich plasma (prp). platelets were obtained from venous blood of healthy donors and the prp was isolated by centrifugation at 200 g for 5 min at 37°c. the aggregation was determined using a dual channel electronic aggregometer. malonyl dialdehyde (mda) production was measured using thiobarbituric acid reagent. in order to confirm these results, flow cytometry with monoclonal antibodies against gpib, gpiib/iiia, gpiiia and gmp140 was used. the ic 50 values of the synthesized and tested compounds, as well as their mda production and flow cytometry results will be discussed. amino acids have a long tradition as building blocks, chiral auxiliaries and/or ligands in advanced organic synthesis and catalysis. at dsm an enzymatic kinetic resolution process has been developed, based on an aminopeptidase catalyzed stereoselective hydrolysis of racemic amino acid amides to form a mixture of l-amino acid and unchanged d-amino acid amide.several small peptides currently are under investigation as possible anti-tumor agents. neuropeptides such as substance p (sp) and neuropeptide y (npy), have been studied for their ability to prevent tumor growth or the proliferation of several cancer cell lines. these neuropeptides have been investigated for their effect to prostate cancer, small cell lung cancer (sclc) and breast cancer. the synthetic sp analog [d-arg 1 , d-phe 5 , d-trp 7,9 , leu 11 ]sp (antagonist d) and the c-terminal analog [arg 6 , d-trp 7,9 , mephe 8 ]sp 6-11 (antagonist g) inhibit sclc cell proliferation in vitro and in vivo, while the analogs [glp 6 , glu(bu t ) 11 ]sp 6-11 and [glp 5 , glu(bu t ) 11 ]sp 5-11 showed significant inhibition in the proliferation of the cancer cell lines hela and t47d.in the present study the c-terminal analogs of sp [glp 6 , d-trp 7 , glu(bu t ) 11 ]sp [6] [7] [8] [9] [10] [11] (1), [glp 6 , d-trp 7,9 , glu(bu t ) 11 ]sp [6] [7] [8] [9] [10] [11] (2), [glp 6 , d-trp 7,9 , mephe 8 , glu(bu t ) 11 ]sp 6-11 (3), [glp 6 , d-trp 7 , mephe 8 , glu(bu t ) 11 ]sp 6-11 (4), [glp 6 , trp 7 , mephe 8 , glu(bu t ) 11 ]sp 6-11 (5), [glp 6 , mephe 7 , d-trp 8 , glu(bu t ) 11 ]sp 6-11 (6), [glp 6 , d-trp 7 , mephe 8 , glu(bu t ) 11 -oh]sp 6-11 (7), [glp 6 , d-trp 7 , cys(acm) 11 -oh]sp 6-11 (8), [glp 6 , d-trp 7 , mephe 8 , cys(acm) 11 -oh]sp [6] [7] [8] [9] [10] [11] (9), [glp 6 , d-trp 7,9 , mephe 8 , cys(acm) 11 -oh]sp 6-11 (10) have been synthesized and tested for their antineoplastic properties in several cancer cell lines. they were also examined for their cytotoxicity to normal cells.the analogs 1-6 are peptide amides whereas the analogs 7-10 are peptide acids. they were performed using the stepwise synthesis either in solution, using the method of mixed anhydrides with carbonic acids or in spps using the fmoc/bu t methodology. the fragment condensation method in solution, using phosphonium reagents, such as pybop, was also applied. the analogs were purified (hplc) and identified (ft-ir, es-ms, 1 h-nmr).the antineoplastic properties of the analogs were studied using sister chromatide exchange (sce) and proliferation rate index (pri). as it is known the sce method is an indicator of dna damages or its repair mechanism, while the method of pri is a sensitive marker of cytotoxicity. the experiments were carried out using cultured human lymphocytes from healthy donors and these results will be discussed.semiempirical quantum chemical investigation of some thymidine derivatives modified with amino acids and peptides at 3ј, 5ј-positions j. velkov 1 , i. stankova 1 , a. ivanova 2 , and a. tadjer 2 1 department of chemistry, south-west university "neophit rilski", blagoevgrad, and 2 department of chemistry, sofia university "st. kl. ohridsky", sofia, bulgaria optimized geometry and electron charge distribution for some thymidine derivatives (3ј,5ј-bis-o-n-α-benzyloxycarbonyl-alanyl-, 3ј,5ј-bis-o-n-α-benzyloxycarbonyl-valyl ,3ј,5ј-bis-o-n-α-benzyloxy-carbonyl-glycyl-glycyl-glycyl,3ј,5јbis-o-n-α-benzyloxycarbonyl-phenylalanyl,3ј,5ј-bis-o-n-αbenzyloxycarbonyl-glycyl) were calculated at the semiempirical (am1) level. the choice of method is limited by the molecular size. in addition, the differences between the ground state energy of the compounds and that of the hydrolysis reaction intermediates were compared to the experimentally found stability towards hydrolysis.with a few notable exceptions, attempts to crystallise integral membrane proteins have failed due to the difficulties in finding appropriate conditions for proteins that have both hydrophobic and hydrophilic domains. thus structural information is largely limited to predictions of secondary structure from the amino acid sequence and computer modelling, neither of which can as yet give high resolution detail. thus alternative approaches are required, and one that we have employed is to look at the substrate binding/transport characteristics of compounds and predict what features the binding site might have. the membrane transport protein that we are interested in is the proton-coupled di/tri-peptide transporter, which has a wide range of natural substrates and is known to transport therapeutically important non-peptides such as ᮀ-lactam antibiotics and angiotensin converting enzyme inhibitors.the initial question that interested us was what makes a di/ tri-peptide a substrate, but not an amino acid? while the obvious answer is the peptide bond, studies with 'space mimic' compounds (which have the space filling properties of a dipeptide but no peptide bond) gave the surprising result that the peptide bond was not essential for binding and translocation. although these space mimics had n and c termini, studies from our laboratory and others have shown that the presence of free amino or carboxyl groups are not a prerequisite for binding or translocation either. this leaves the question of what does distinguish a pept1 substrate from a non-substrate?computer modelling of a large number of pept1 substrates has allowed the development of a substrate template, whereby potential substrates can be scored according to their predicted binding affinity. from this it is clear that it is a sum of energies derived from a number of substrate-transporter interactions that determine binding affinity, including the n-and c-termini, the peptide bond components and the substrate side-chain groups. further studies aim to refine this model through the complimentary approaches of novel substrate design and sitedirected mutagenesis of the transporter protein.why are we interested in this? a large number of promising therapeutic compounds are found to have little or no bioavailability. compared with most membrane transporters pept1 has a wide range of potential substrates, and amongst its non-peptide substrates are a range of peptidomimetic therapeutic compounds. the recent finding that a peptide bond is not a prerequisite for transport opens up the possibility of designing prodrugs to be substrates for pept1, and this has found to be an effective strategy for example with the antiviral drug valacyclovir.(we thank the wellcome trust for their generous support.) nuklearmedizinische klinik und poliklinik der technischen universität münchen, germanyaim: the high amino acid metabolism of tumor cells allows tumor imaging with radiolabeled amino acids as 11 c-methionine (met) by positron-emission-tomography (pet). however in recent experimental and clinical studies met uptake was also found in inflammatory tissue thus leading to false positive results. the aim of the study was to compare [ 18 f]fluoroethyltyrosine (fet), a new amino acid analogue, with met to assess their suitability for differentiating between tumor cells and inflammatory cells in vivo and in vitro.methods: popliteal lymph nodes of balb/c and dba/2 mice were stimulated either by streptocotocin (stz), causing chronic lymphadenitis, or by concanavalin a (con a), causing in acute lymphadenitis. tumor infiltrated lymph nodes were induced by inoculating cells from a lacz transfected t-cell mouse lymphoma line into the footpads of syngenic dba/2 mice. the uptake of met and fet was determined quantitatively in tumor infiltrated and inflammatory lymph nodes as well as in the lymph nodes of untreated mice. in vivo imaging of tracer uptake in mouse lymph nodes was performed using a high resolution (2.4 mm) small animal pet (madpet). in vitro the uptake of the amino acids met and fet was investigated in different cells, such as sw707 human colon carcinoma cells and c6 rat glioma cells, stimulated human lymphocytes and macrophages. about 5 ϫ 10 5 cells of each cell line were incubated in a buffered medium containing either different concentrations of unlabeled amino acids or con a (stimulation of lymphocytes) or the transport inhibitors 2amino-norbornane-carboxylic acid (bch, l-system), α-(methylamino)-isobutyric acid (meaib, a-system) or l-serin (asc-system). 0.37 mbq of each amino acid tracer were added and incubated. uptake was stopped by using ice-cold pbs, cells were washed three times and uptake was analyzed.results: in tumor infiltrated lymph nodes uptake of both tracers was higher than in control lymph nodes. met showed an increased uptake in both lymphadenitis models, whereas fet did not accumulate significantly. met and fet uptake in tumor infiltrated lymph nodes was also seen in madpet images, however inflammatory lymph nodes could only be detected in met images.the amount of tumor uptake was different in the various cell types investigated. c6 cells showed the highest uptake of all cells investigated and a slightly lower uptake was found in sw707 cells. in con a stimulated lymphocytes, the uptake of fet was negligible, while met uptake was significantly higher than in both tumor cell lines. since bch reduced the uptake of fet and met to approximately 10%, fet seems to be also predominantly transported into tumor cells by the l-system. the results indicate, that fet appears to differentiate between tumor and inflammatory tissue, as a result of the low uptake of fet in inflammatory cells. nuklearmedizinische klinik und poliklinik der technischen universität münchen, germanyover the past few years numerous studies have documented the high diagnostic accuracy of positron emission tomography (pet) using the glucose analogue f-18-fluordeoxyglucose (fdg) for detection and staging of malignant tumors. a significant limitation of fdg-pet, however, is that increased uptake is not only observed in malignant tumors but also in activated inflammatory cells. due to the high glucose utilization of the normal brain and the lower protein synthesis in the normal gray matter the radiolabelled amino acid c-11-methionine (met) gives higher contrast between brain tumors and normal tissue than fdg-pet. rapid uptake of met has been documented for several malignant tumors like gliomas, lung cancer, bladder cancer and malignant lymphomas since amino acid transport and protein synthesis are generally increased in malignancies. the application of met-pet however has been limited by the short half life of the radioactive label c-11 (20 min) in contrast to f-18 (110 min). amino acid analogous labeled with f-18 like f-18-fluoro-α-methyltyrosine (fmt), f-18-fluoro-ethyltyrosine (fet), f-18-fluoro-phenylanaline, f-18-fluore-proline will allow a more widespread application of amino acid pet in oncology. an other amino acid analogue i-123-iodo-α-methyltyrosine (imt) is of clinical interest because the radionuclid i-123 allows it applicability for single-photoemission-computer-tomography (spect). the uptake of the amino acid analogues can only be regarded as a measure for the increased amino acid transport in the tumor cells because they are not incorporated into proteins. clinical data show that radiolabelled amino acids that are only transported into the cells are not inferior to those that enter protein synthesis. this tracers may also help to differentiate tumor lesions from inflammatory lesions when the expression of the transport systems for amino acids in tumor cells and inflammatory cells is different.lysinuric protein intolerance: understanding the pathophysiology of a multi-system disorder of dibasic amino acid transport m. p. sperandeo 1,2 , v. fiorito 2 , a. pietrosanto 2 , a. pepe 2 , g. andria 2 , and g. sebastio 2 1 telethon foundation, rome, and 2 department of pediatrics, federico ii university, naples, italy lysinuric protein intolerance (lpi; mim 222700) is an autosomal recessive disease, mainly found in finland and italy. clinical findings of lpi include: vomiting, diarrhea, failure to thrive, hepatosplenomegaly, osteoporosis, episodes of coma, and mental retardation. a life-threatening lung involvement (alveolar proteinosis) and renal insufficiency were also reported. metabolic derangement of lpi includes: reduced intestinal absorption of cationic amino acids (lysine, ornithine, arginine, caa), increased renal excretion of caa and dysfunction of the urea cycle leading to hyperammonemia and orotic aciduria. most of the clinical findings cannot be explained by a selective deficiency of amino acid transport, as indeed observed for cystinuria (mim 220100), a cognate disease of lpi. the molecular basis of lpi resides in an abnormal caa carrier functioning at the level of basolateral membrane of epithelial cells in the intestine and the kidney. caa transport is mediated by y ϩ l system, that is exerted by heterodimers consisting of the 4f2 heavy chain (4f2hc) and a light chain represented by either the solute carrier family 7a, member 6 (slc7a6) or 7 (slc7a7). after excluding the 4f2hc as the causative gene of lpi, we identified slc7a7 as the lpi gene and characterized mutations in twenty-five patients from 21 families (16 italian, 2 japanese, 1 moroccan, 1 greek, and 1 pakistani; 34 independent alleles) affected by lpi. thirty-two of the 34 independent alleles (94.1%) were characterized and fourteen mutations were identified. only five mutations (namely 1625insatca, w242x, 1425delctct, ivs3 ϩ 1gaea, s386r) were identified in more than one independent family. most mutations are located in the slc7a7 coding region, except for two splicing mutations. the pathogenesis of some clinical findings of lpi, namely alveolar proteinosis and renal involvement, remains mostly unknown. we are currently investigating the role of slc7a6 gene in lpi, which, in addition to slc7a7, is responsible of the y ϩ l activity. in fact, the regulation of the y ϩ l system, exerted by either 4f2hc/ slc7a76 or 4f2hc/slc7a7, is still unknown. hypothetically, the activation of 4f2hc/slc7a76 in all tissues might be the "simple" way to a lpi gene-therapy.[acknowledgements: m. p. s. is supported by telethon-italy (grant n.29cp) and is an assistant telethon scientist.] pre-eclampsia (pe) is a potentially life threatening complication of pregnancy and is one of the leading causes of maternal and fetal morbidity and mortality. pe is associated with endothelial cell dysfunction and inadequate placental perfusion. fetal plasma l-arginine levels are decreased in pe and there is controversy as to whether nitric oxide (no) production is altered. we have investigated whether the kinetics of l-arginine transport via system y ϩ and no production are altered in fetal umbilical vein endothelial cells (huvec) from pe pregnancies. kinetics of l-arginine transport were similar in huvec isolated from normal, preterm and pe pregnancies, however nethylmaleimide inhibited transport in normal but not pe huvec. basal and histamine-stimulated no production was similar in normal and preterm huvec, whereas pe increased basal (25 ϯ 5 vs 5.3 ϫ 3 pmol/10 8 cell/5 min) and histaminestimulated (70 ϯ 12 vs 20 ϯ 5 pmol/10 8 /5 min) no production. whole-cell patch clamp measurements revealed similar inward rectifying k ϩ currents in normal and pe huvec, with resting membrane potentials of ϫ65 ϯ 4 and ϫ80 ϯ 18 mv in normal and pe huvec, respectively. increased enos activity in pe endothelial cells may serve as a compensatory mechanism to counteract the hypertension observed in pe, however, elevated no production is apparently not associated with enhanced larginine transport. department of pharmacology, university of cambridge, u.k.over the past years, concerns have heightened over the escalating numbers of pathogenic microorganisms that are resistant to multiple antibiotics. this phenomenon poses major problems in the treatment of patients with hospital or community-acquired infections caused by bacteria, yeast, fungi and parasitic organisms. particularly intriguing are the so-called multidrug transporters, which have specificity of compounds with very different chemical structures and cellular targets. this lecture will focus on the molecular properties of the atpbinding cassette multidrug transporter lmra in the lactic acid bacterium lactococcus lactis. lmra is a close homolog of the human multidrug resistance p-glycoprotein, overexpression of which is one of the major causes of resistance of human cancers to chemotherapy. surprisingly, lmra can even substitute for pglycoprotein in human lung fibroblast cells. recent biochemical and pharmacological studies on lmra suggest that the protein may operate by a two-cylinder engine mechanism to transport amphiphilic drugs from the inner leaflet of the plasma membrane. this mechanism will be discussed in more detail. bone and bone marrow are important sites of metastasis formation in breast cancer; so, we studied the level of bone sialoprotein (bsn) and fibronectin (fn), two key connective tissue antigens, in patients with metastatic breast carcinoma. our data reveled that bsn have a statistically significant association with bone metastases in that disease. fn level was also significantly changed in metastatic breast carcinoma when compared to the non metastatic cases. kharkov national university, radiophysical department, chair of molecular and applied biophysics, kharkov, ukraine * present address: institute of cell and molecular biology, university of edinburgh, edinburgh, scotland, u.k.in the work the temperature dependencies of dielectric parameters of human serum albumin (hsa) and fibrinogen solutions (0.15 m nacl, ph 7.2) were obtained in the temperature interval 5-50 c degrees. the measurements of the dielectric parameters were carried out at the frequency of 9.2 hhz, i.e. in the range of free water molecules dispersion. in contrast to dependencies for poor solvent, temperature dependencies of dielectric parameters for protein solutions are of nonmonotonous character; they have a number of peculiarities in the temperature ranges of 8-10, 22-24 and 34-36 c degrees. this fact means that at these temperatures redistribution of free and bound water in protein-water system occurs due to structural changes in protein molecules. the dependencies of hydration of hsa and fibrinogen on temperature were obtained as well.in the work the mechanism of temperature changes of spatial organisation of protein molecules was proposed. perhaps, this mechanism is responsible for maintenance of thermal stability of the functionally active conformation of native proteins. as peculiarities on temperature dependencies of dielectric parameters of solutions of globular (hsa) and fibrillar (fibrinogen) proteins were in the same temperature regions, one may to assume that the mechanism of proteins thermal stabilisation in physiological temperatures interval has a general character. laboratory of cell pharmacology, university of leuven, medical school, campus gasthuisberg (o&n), leuven, belgium n-pomc was purified from conditioned medium of att20 cells using a sequence of concentration, fractionation by ion exchange, rp-hplc and gel-filtration. twenty isoforms of n-pomc, for both 11 and 13 kda, were identified by means of mass spectrometry and n-terminal sequencing. these isoforms are assumed to be pomc1-74 or pomc1-95 with heterogeneous glycosylation.the n-pomc isoforms were tested on prolactin (prl) gene expression and lactotroph mitosis in pituitary cell aggregate cultures. prl mrna content was quantified by means of real time rt-pcr. three 11 kda n-pomc fractions enhanced prl mrna levels by 33-36%, while all other isoforms were inactive. this effect was abolished by immunoneutralization with n-pomc monoclonal antibody. only one fraction stimulated lactotroph proliferation (38.2 ϯ 7.5%) as assessed by brdu incorporation in prl-immunoreactive cells. several (but not all) 13 kda n-pomc fractions stimulated prl mrna level and lactotroph mitosis. on the other hand, all 11 and 13 kda isoforms activated the mc-3 and mc-5 receptor in cell lines in which these receptors were transfected. thus, att20 cells produce various n-pomc isoforms, only a part of which display an effect on prl mrna expression. even fewer isoforms affect lactotroph proliferation. since all isoforms activate the mc-3 and mc-5 receptor, it is suggested that the effect of the few isoforms on lactotrophs is mediated by (a) different receptor(s). are widely prescribed for the treatment of mild to moderate depression and the putative antidepressant constituent is probably hyperforin. in this study the effect of hyperforin was investigated on the release of neurotransmitter amino acids.coronal cortical slices (400 µm) were cut and perfused with gassed (95% o 2 , 5% co 2 ) acsf at 37°c. two-minute samples of perfusate were collected and aspartate and glutamate were assayed by hplc. potassium-and veratridine-stimulated release was elicited by administering 2 pulses of k ϩ (60 mm) or veratridine (20 µm) 30 minutes apart.in control experiments the second k ϩ pulse elicited glutamate release which was 80% of the first pulse. hyperforin (5 µm) perfused for 30 minutes prior to, and during, the second k ϩ pulse significantly increased glutamate release to 170% (p ͻ 0.001, n ϭ 6-8). release elicited by the second veratridine pulse was 70% of the first pulse for both glutamate and aspartate. hyperforin (5 µm) increased this release to the second pulse to 160% and 130% respectively (p ͻ 0.001, n ϭ 6-8). when perfused on its own for 30 minutes, hyperforin (5 µm) increased the basal release of glutamate (p ͻ 0.001, n ϭ 4-5).in conclusion, the increase in the release of neurotransmitter amino acids observed following hyperforin is possibly mediated through a facilitatory action on voltage-operated ca 2ϩ or na ϩ channels.glucagon-like peptide-1 (7-36) amide (glp1) is the main product of the glucagons gene expression in intestinal l cells into the cirulation in response to the ingestion of food and is the most potent stimulator of glucose-induced insulin secretion. glp1 receptors have also been detected in discrete areas of rat brain and intracerebroventricular glp1 has been shown to inhibit feeding in fasted rats. in this study hplc techniques were employed to evaluate the effects of glp1 on serotonin (5-ht) and γ aminobutyric acid (gaba) metabolism in rat brain. glp1 (0.5 µm) produced a significant decrease in levels of 5-ht by 20% after 15 minutes of incubation with combined hypothalamus and brain sterr. synaptosomes. levels of 5hydroxyindolacetic acid (5-hiaa), the principal metabolite of 5-ht, and tryptophan the amino acid precursor of 5-ht, were also decreased significantly by 21% and 37% respectively. gaba and its amino acid precursor glutamic acid were both measured at the same conditions as above, but a precolumn derivatization hplc technique was used. the increase in levels of gaba (14%) and glu (6%) by glp1 was not significant.the results suggest that decreased synaptosomal levels of 5-ht and 5-hiaa caused by glp1 are due to diminished availability of typtophan by the peptide. in experimental model of iron overload we obtained the following results: the concentration of carbonyl groups tended to increase, while mda level significantly increased after feso 4 treatment (1.66 ϯ 0.25 vs control 1.51 ϯ 0.50 µmol/mg prot.) and (2.13 ϯ 0.5 vs control 1.3 ϯ 0.3 nmol/mg protein p ͻ 0.01) respectively. it was associated with significantly increased iron content (0.89 ϯ 0.23 µg/mg prot. vs control 0.49 ϯ 0.17 p ͻ 0.001). it is clear that oxidative stress occurs in experimental iron overload, if sufficiently high levels of iron within hepatocytes are achieved. in group treated with feso 4 and spermine, iron content was significantly decreased (0.36 ϯ 0.07 p ͻ 0.01 compared with fe treated only) and carbonyl group content tended to be lower in comparison to feso 4 treated only (1.58 ϯ 0.24), but mda level didn't change (2.31 ϯ 0.72). in addition, treatment with spermine alone resulted in increase of mda level (2.74 ϯ 0.7 vs control p ͻ 0.01), iron content didn't change (0.59 ϯ 0.29), but carbonyl groups were decreased (0.99 ϯ 0.28 vs control p ͻ 0.05). feso 4 treatment increased gsh level (126.38 ϯ 34.11 nmol/mg prot. vs control 88 ϯ 22.77; p ͻ 0.05) while in combination with spermine this increase was more profound (235.48 ϯ 42.7; p ͻ 0.001 vs control, p ͻ 0.001 vs feso 4 ). spermine alone produced similar increase of gsh level (127.4 ϯ 34.11, p ͻ 0.05 vs control; p ͼ 0.05 vs feso 4 ). the results emanating from the human genome programme have required a reappraisal of protein science and have led to the rapid upsurge in interest in the area of proteomics. this sudden re-emergence of protein science, in fact, was predictable and should not have been surprising.recent experience of protecting group design with respect to lysine and aspartic acid will be discussed together with aspects of chemical synthesis of small proteins of biological significance and in the context of chemical synthesis methodology making contributions to the general field of proteomics. using a cell line permanently expressing the mouse taurine transporter (mtaut) as a fusion protein, we investigated the underlying mechanism by which the immunosuppressive drug cyclosporin a (csa) inhibits taurine transport. csa inhibited the recombinantly expressed mtaut function both in dose and time dependent manner. the inhibitory effect of csa was reversible. thus, washing out the csa resulted in almost complete recovery of taurine uptake. to obtain further insight, we examined the surface abundance of the mtaut as a function of csa treatment using a surface-labeling assay. our results demonstrated that csa treatment altered the surface expression of the mtaut without significantly altering its total expression level, and the reduction in the cell surface expression paralleled the decrease in taurine uptake. upon removal of csa, the virtual recovery in taurine uptake was due to the concomitant increase in the number of taurine transporters on the cell surface. taken together, our results suggest that csa induced inhibition of taurine uptake was either due to the impaired targeting of the taurine transporters to the cell surface or due to the removal of the transporters from the cell surface. polyamines are neuromodulators in a number of physiological and pathological conditions in cns. since application of ethylene glycols causes hypoactivity and lethargy of experimental animals, depression of cns and various neurological symptoms, the aim of this study was to examine the effects of 2butoxyetanol on polyamine and gaba catabolism, taking in account an alternative pathway of gaba synthesis from putrescine. methods: male wister rats were allocated into three groups: first treated by be (100 mg/kg -10 days), second key: cord-023208-w99gc5nx authors: nan title: poster presentation abstracts date: 2006-09-01 journal: j pept sci doi: 10.1002/psc.797 sha: doc_id: 23208 cord_uid: w99gc5nx nan background and aims: homodimerization of myd88 adapter protein is essential for nf-kb activation in the inflammatory pathway triggered by il-1 and tlr [1] . we designed a peptidomimetic of the myd88 tir domain consensus peptide arg-asp-val-leu-pro-gly-thr [2] , named st2825. here, we report its synthesis and biological activity. we also report the synthesis and biological activity of its enantiomer, st3511, and its diastereoisomers, st3489 and st3558. methods: the structure of the myd88 tir domain consensus peptide is subdivided into three distinct portions, the most important of which is a b-turn. in the peptidomimetic design we changed the b-turn with a tricyclic spirolactam [3] , already known [4] . we synthesized this building block, its enantiomer and two of 8 possible diastereoisomers by "in solution" synthesis. based on semiempirical calculation of heat of formation [5] , we could predict the right stereochemistry of the 4 products selectively obtained in the last cyclization step. results: these four compounds were tested for their biological activity by reporter gene assay (rga). some coimmunoprecipitation experiments were also carried out and we report their results. conclusions: the results show the activity of st2825 and its isomers on our target, with limited specificity towards their stereostructure. introduction of a methylene bridge between the cα(i+1) and the n(i+2) atoms in an open peptide (i) to mimic simultaneously the cαh(i+1) and hn(i+2) protons (β-lactam scaffold assisted design -β-lsad) has proven to be a practical tool for the preparation of monotopic β-turn peptidomimetics (ii, r2 = r3 = h), according to the principle of separation of constraint and recognition elements1. in this work we report a short, general, and stereocontrolled synthesis of multitopic β-lactam scaffolds of type vi. α-alkyl serinates iii are converted into the corresponding enantiopure nnosyl-aziridines iv which undergo "in situ" ring-opening with amino acids v. subsequent base-promoted cyclization affords the n-protected α-alkyl-α-amino-β-lactams vii. incorporation of the novel scaffolds into linear and cyclic peptides and their conformational features are also presented, most of them showing stabilized β-and γ-turn conformations. poly(amino acids) are emerging as promising therapeutic carriers finding widespread application in the field of drug delivery. in this context, polyproline polymers have been used to solubilize poorly water-soluble proteins, in affinity chromatography for the purification of platelet profilin, and more recently, in the design of dendrimers. poly(amino acids) are most conveniently synthesized by polymerization of the corresponding amino acid n-carboxyanhydride (nca). in spite of the interest of polyproline, the preparation of proline n-carboxyanhydride (pro-nca) renders poor synthetic yields. in this work a new method for the preparation of pro-nca in high yields and purities is described. amino acid n-carboxyanhydrides are obtained by the method described by fuchs. but, in the case of proline, the n-carbamoyl chloride does not cyclise spontaneously as it takes place with other amino acids, and the use of a non-nucleophilic base is required for the cyclisation. a tertiary amine, such as triethylamine, is commonly used but it renders a low conversion of the n-carbamoyl chloride to the expected pro-nca, together with the presence of the pro-pro diketopiperazine byproduct. in the present work, polymer-supported bases have been used instead of triethylamine. higher yields of pro-nca, and very low percentages of diketopiperazine have been obtained. in addition, no tertiary amine contamination was observed. polymer-supported bases could also be recycled and pro-nca yields were reproducible. in conclusion, we have developed an efficient method for pro-nca preparation with polymer-supported bases. the introduction of novel nonproteinaceous heterocyclic amino acids into peptides results in new compounds with interesting structural, physicochemical and biological properties. the transformation of amino acid side chains after the peptide assembly is a convenient method of generating such modified peptides. taking into account the biological activity and complexing abilities of nitrogen-containing heterocycles, we investigated the formation of imidazole, benzimidazole and quinoxaline moieties using condensation with various aldehydes and α-dicarbonyl compounds after classical peptide synthesis on solid support. the imidazole synthesis utilizes the n-terminal or side chain amino group of amino acids, whereas a derivative of phenylalanine, β-(4-amino-3-nitrophenyl)alanine, was developed for benzimidazole and quinoxaline synthesis. the modified peptides were purified by preparative hplc and characterized by esi-ms, uv and nmr. in conclusion, we developed a straightforward method of synthesis of peptides with specific ion affinity and spectral characteristic. the broad range of commercially available aromatic aldehydes and dicarbonyl compounds makes possible the synthesis of combinatorial libraries of modified amino acids and peptides. part of this work was supported by a grant no. 3t09a 110 28 from the ministry of education and science. nmda receptors belong to the ionotropic group of glutamate receptors. the activity of the receptor can be altered by compounds acting at binding sites. the (r,s)-(tetrazol-5-yl)glycine (tg) has been shown to be a highly potent nmda (n-methyl-d-aspartic acid) receptor agonist with exitotoxic effects [1] . the aim of our studies was to investigate the chelating ability of tg towards copper(ii) ions. copper is widely distributed throughout the body with a distinct concentration in the brain. copper enters cells as complex and seeks out targets requiring it to function. for these reasons it was interesting to evaluate stability and structure of tg -copper(ii) complexes. the equilibrium and structural properties of complex species were characterized by ph-metric and spectroscopic (uv-vis and epr) methods. in the system, polymeric species are dominant at acidic ph range having { nh2, coo-} coordination with possible ntetr bridging elements. monomeric complexes were found at physiological ph. the two tg molecules are bound to copper ion via four nitrogen donors. the formation of two {nh2, ntetr} donor sets results in very strong metal-ligand interactions and the complex species are very stable over a wide ph region. we have also performed an investigation on similar tetrazole compounds in order to compare the chelating ability of the tetrazole moiety . the targets of our studies were 1,5-diamino-1h-1,2,3,4-tetrazole [2] and tetrazole aspartic acid. references continuing work in that field, we synthesized oxytocins containing tetrazole analogues of amino acids. the 5-tetrazolyl group is widely used in medicinal chemistry as an isostere of the carboxyl group. compounds containing tetrazole ring appear to be metabolically more stable than their carboxylic analogues and have comparable acidity. we synthesized derivatives of aspartic, glutamic, and alpha-aminoadipic acids containing 1h-tetrazole ring in side chains. these derivatives were then used for syntheses of oxytocin analogues substituted in position 4. apart from above we also obtained two analogues with tetrazole analogue of glycine in position 9. the first one contains 1h-tetrazole ring, the second one has tetrazole ring substituted with methyl group in position 1. oxytocin analogues possessing amino acids with tetrazole ring in side chains were synthesized on amide resin using fmoc methodology. in the case of analogues with c-terminal tetrazole ring, fragments 1-7 were synthesized on resin and then coupled with suitable dipeptides in solution. all obtained peptides show no pressor and rather low uteronic activity. however, for some analogues the uterotonic activity when measured in the presence of magnesium ions was several times higher. in humans, two classes of defensins, α-defensin and β-defensin, have been identified on the basis of tissue specificities and structural features including their modes of disulfide pairing. in general, particular combinations with disulfide bonding in cysteine-containing peptides are critical for expressing their intrinsic biological activities. in the case of human α-and β-defensins, however, disulfide isomers without the native pairing were demonstrated to exhibit similar antimicrobial activity to that of the native defensins. therefore, to assess the biological activities of defensins as well as defensin-based therapeutics, extreme care is required in the chemical synthesis of these peptides to avoid ambiguity in quality. in the present study, we synthesized human α-defensin-1, -2 and -4, and human β-defensin-1, -2, -3 and -4 by employing boc chemistry, and determined the optimal conditions for folding the respective reduced peptides preferentially into a native conformation. among the factors affecting the oxidative folding in the presence of reduced and oxidized glutathione, the buffer concentration and reaction temperature were essential. all the synthetic human α-and β-defensins were confirmed to have the respective native disulfide pairing by sequential analyses and mass measurements with cystine segments obtained by enzymatic digestion. all the human α-and β-defensins could be efficiently oxidized to the α-and β-defensin-type disulfide structure, respectively, under several conditions determined in the present study. these synthetic peptides of high homogeneity were used to accurately assess the antimicrobial activity. native chemical ligation is based on the reaction of a peptide bearing a c-terminal thioester group with an n-terminal cysteinyl peptide, leading to the formation of an amide bond at the aa-cys junction. the key starting materials for native chemical ligation are unprotected c-terminal thioester peptides. thioester peptides are often prepared using boc/benzyl solidphase peptide chemistry. however, the widespread use of the fmoc/tert-butyl chemistry for peptide synthesis, over the boc/benzyl method, has stimulated the development of methods allowing the preparation of thioester peptides that are compatible with the basic treatments used to remove the fmoc alpha-amino protecting group. we report here a novel method for thioester peptide synthesis that is based on the use of the sulfonamide safety-catch linker. once the peptidyl chain is assembled by fmoc/tert-butyl chemistry, the thioester function is generated on the solid-phase through an intramolecular n,s-acyl shift. the procedure seems to be insensitive to the bulkiness of the amino acid directly attached to the sulfonamide linker. the thioesters were successfully used for native chemical ligations in solution or on the solid support. we optimized the recognition sequence of the substrate and the reaction conditions with respect to the yield. the sortase-mediated ligation was successfully applied to the synthesis of cellpenetrating peptide-pna conjugates which showed enhanced activity in antisense experiments compared to pna alone. this ligation strategy was also employed for the coupling of a chemically synthesized construct of the extracellular loops of the crf-receptor with the corresponding n-terminal receptor domain, which was expressed in e. coli. this 23 kda protein behaves like an artificial receptor, binding specifically natural ligands. linear gramicidins represent the most investigated family of antibiotic peptides forming ionic channels. gramicidins produced by bacillus brevis are hydrophobic peptides composed of 15 amino-acids with d and l configuration strictly alternate. the presence of d-amino acids in the sequence of gramicidin a (hco-val-gly-ala-dleu-ala-dval-val-dval-trp-dleu-trp-dleu-trp-dleu-trp-nhch2ch2oh) should possible make the peptide highly resistant to proteolysis [1] . striking features like ethanolamine group in c-terminus, the n-terminal n-formylated valine and the high hydrophobicity of the peptide sequence, make the solid-phase synthesis of gramicidin a very tricky. therefore, we followed a new synthetic strategy for peptide chain elongation assisted by microwave energy. in fact, microwave energy has been demonstrated to produce highpurity compounds with more rapid reaction times, enhancing coupling rates and efficiency in difficult syntheses [2] . however, microwave-assisted solid phase peptide synthesis (mw-spps) has not been yet extensively investigated. in this context, we synthesized gramicidin a by mw-spps in high yield and purity, enhancing reaction rate compared to the traditional spps. thermal disruption of peptide aggregation, induced by microwaves, is possible favorable for obtaining this particularly difficult sequence. gramicidin a was incorporated in synthetic lipid bilayers, self-assembled on mercury electrodes, characterized by hydrophilic spacers interposed between the metal and the lipid bilayer. we tested the behaviour of gramicidin a in biomimetic membranes using electrochemical impedance spectroscopy (eis), ac voltammetry and other electrochemical techniques [3] . [ csf114(glc) is an n-glucosylated peptide to be produced in large scale by peptlab because it is the active molecule of the first specific diagnostic/prognostic test for monitoring disease activity and guiding therapeutic treatments of multiple sclerosis patients [1] . in order to develop a synthetic protocol by an automated instrumentation, increasing yield, purity of the crude, and reaction time, a microwave-assisted solid phase peptide synthesis was validated comparing the use of the new generation of triazine-based coupling reagents (tbcrs) with a series of commonly used ones. activation of carboxylic acids by tbcrs is particularly effective because of formation of triazine "superactive esters". the usefulness of tbcrs as coupling reagents has been recently confirmed in the synthesis of z-, boc-, and fmoc-protected dipeptides, sterically hindered amino acids, in the synthesis of esters, in manual and automated spps of difficult peptide sequences, and head-to-tail constrained cyclopeptide analogues [2] . moreover, we also demonstrated tbcrs efficient in a microwave-assisted solution synthesis of the n-glucosylated building block fmoc-asn(glcoac4)-oh using a manual monomode microwave instrument [3] . this building block was used to obtain csf114(glc) comparing the efficacy of a monomode microwave automatic instrument with the traditional solid-phase peptide synthesizers such as the manual and automatic in batch systems, as well as the continuous-flow one. it is known that enzymatic peptide synthesis is more advantageous than chemical synthesis in many aspects; it is highly stereoselective, racemization-free and requires minimal side-chain protection. the method is, however, limited to the use of amino acid derivatives which meet the enzymatic specificity as a coupling component. this problem may be solved using enzymes which have wide specificity of substrate. but in this case, secondary hydrolysis of the resulting peptide may arise from the inherent nature of the protease. in this matter, ficin and ficin-like enzymes were used as cysteine protease to analyze the diminishment of specificity for the substrate. the cysteine protease-catalyzed peptide coupling reaction has been studied by using synthetic fourteen boc-amino acid phenyl and naphthyl esters as acyl donor. the reaction conditions were optimized for organic solvent, ph, and concentration of acceptor. the coupling reaction was carried out by incubating an acyl donor (1 mm) with an acyl acceptor (ala p-nitroanilide, 35 mm) and enzyme (0.1u) in a mixture of gta buffer (50 mm, ph 9.0) and dmso (3:2) at 37ْc. the progress of the coupling reaction was monitored by rp-hplc. the products were obtained in satisfactory yields. non-enzymatic glycosylation, also called glycation, is a common modification in living organisms formed by the reaction of carbohydrates with free amino groups of peptides and proteins. it is a slow chemical reaction yielding amadori products undergoing further oxidation and degradation reactions finally leading to advanced glycation end-products (age). amadori products are early markers for ageing, diabetes mellitus and alzheimer's disease. despite the clinical importance of these amadori products, universal protocols to synthesize amadori modified peptides are still missing. here we describe a solid phase strategy for the glycation of specific amino groups on partially protected resin bound peptides using a global post synthetic approach. the peptides were synthesized by standard fmoc/tbu-chemistry using carbodiimide activation. the lysine position to be modified was incorporated with a methyltrityl protected ε-amino group, which can be selectively cleaved after completion of the peptide synthesis with 1% tfa in dichloromethane. the partly deprotected peptide was glycated in methanol using a ten-fold molar excess of 2,3-4,5-di-o-isopropylidene-aldehydo-β-d-arabino-hexos-2-ulo-2,6-pyranose and nabh3cn for 18 h at 70°c. after cleavage the overall yields were in the range of 50 -70 % for the tested octapeptides. all byproducts were well separated by rp-hplc allowing a simple purification strategy even for medium-sized peptides. thus the general strategy presented here allows routine synthesis of amadori peptides at reasonable yields and purities using standard protocols established in most laboratories synthesizing peptides. 2-chlorotrityl chloride resins are recommended for the synthesis of c-terminal proline peptide acids to overcome diketopiperazine formation during chain assembly. however, we have found these (and similar) resins to be unsuitable for the synthesis of peptides greater than 20 residues. for example, the chemokine guinea pig eotaxin, (73 residues c-terminal proline) assembles poorly if not at all on a 2-chlorotrityl resin. we sought to circumvent these problems in the chemical synthesis of peptides and proteins, through the development of a resin-swap procedure. whereby the initial c-terminal protected tripeptide is assembled on a 2-chlorotrityl resin, liberated from the solid-support, then reattached to a resin that is suited for long chain peptide / protein synthesis. using this approach, the synthesis of guinea pig eotaxin is reported. the tripeptide fmoc-thr(but)-lys(boc)-pro-oh was assemble on 2-chlorotrityl resin, cleaved with 20% tfe in dcm and attached to wang resin using standard protocols. peptide assembly gave the gp eotaxin in 53% overall yield (as determined by uv monitoring). fmoc-on cleavage, purification and tag removal followed by folding gave the native chemokine in good yield. choice of resin is one of the most critical factors in ensuring a successful peptide synthesis, we have shown the superiority of wang resin over chlorotrityl resin in the synthesis of medium and long peptides and developed a method for the synthesis of c-terminal proline containing peptides which overcomes the problem of diketopiperazine formation. the technique is being applied to the synthesis of other c-terminal proline peptides e.g. human eotaxin and ip10. dimerization of cell receptors, involved in antigen presentation, is an essential step in several cellular signal transduction processes, therefore substances that are able to modulate such a process are of potential therapeutic value. dimeric peptide ligands could represent useful tools to cause dimerization of such receptors. a similar strategy applies dimerization of ligands, interacting with dimeric proteins or proteins with multiple binding sites, to design molecules with enhanced affinity. dimeric analogs of the immunosuppressory hla class ii fragments were synthesized using suitably modified, standard fmoc solid-phase protocols and mbha-resin. the dimerization was achieved by crosslinking n-terminal amino groups of the peptides with the commercially available mixture of poly(ethyleneglycol)biscarboxylic acid (average mw 600, length range 30-45å), activated by esterification with pentafluorophenol. the same procedure was applied to synthesize a series of dimeric analogs of c-terminal fragments of plexin-b, consisting of two undecapeptides, linked by the polyethyleneglycol spacers. other biand polyvalent linkers were also investigated. our results demonstrated that the amino-terminal dimerizations of the tested hla-fragments resulted in enhanced immunosuppressive activities, whereas interaction of pdz dimer with the plexin fragments led to about 20-fold increase in affinity, as compared to their monomeric counterparts. [background and aims] elucidation of alzheimer's disease (ad)-related aß1-42 dynamic events is a difficult issue due to uncontrolled polymerization. [methods] based on the "o-acyl isopeptide method" (chem. commun. 2004, 124; j. am. chem. soc. 2006, 128, 696), we have developed a novel photo-triggered "click peptide" of aß1-42 (1), e.g., "26-n-nvoc-26-aiaß42 (2)", in which a 6-nitroveratryloxycarbonyl (nvoc) group was introduced at ser26 in 26-o-acyl isoaß1-42 (26-aiaß42, 3). [results] i) the click peptide 2 did not exhibit the self-assembling nature under physiological conditions due to one single modified ester; ii) photo-irradiation of the click peptide 2 and subsequent o-n intramolecular acyl migration afforded the intact aß1-42 (1) with a quick and one-way conversion (so-called "click"); and iii) no additional fibril inhibitory auxiliaries were released during conversion to aß1-42 (1). [conclusions] this method provides a novel system useful for investigating the dynamic biological functions of aß1-42, such as the self-assembly and aggregation processes in ad. several insulin analogues have recently been introduced clinically for improved treatment of diabetes. industrial productions of such insulins are based on microbial expression systems, which are highly efficient, but generally limited to the 20 proteogenic amino acids. also, some sequences form inclusion bodies or fail to express. the total chemical synthesis of insulin in research scale was a landmark achievement in peptide science. however, the most commonly used method relies on recombination of a-and b-chains under "random" folding and pairing of the three disulfide bridges. this folding/oxidation step is difficult and low yielding. a general approach using a removable auxiliary which can direct correct formation of disulfide bridges is highly desirable. in the pancreas as well as in microbial expression systems, insulins are prepared and folded as single chain precursors, with a c-peptide connecting the a and bchains. the c-peptide helps direct the orientation of a and b-chains in obtaining the correct disulfide pairing and overall peptide folding. upon folding, the c-peptide is removed enzymatically. we report here a new method for total chemical synthesis of insulin by use of fmoc-based step-wise solid-phase synthesis of single-chain precursors followed by cpeptide directed folding and cleavage of c-peptide, thereby allowing total chemical synthesis of novel insulins with unnatural substitutions. 2-chloro-4-methoxy-1,3,5-triazines 1a-c anchored on cellulose, silica or wang resin were prepared by the treatment of 2,4-dichloro-6-methoxy-1,3,5-triazine with appropriate solid support in the presence of a base. immobilized, environmentally friendly triazine coupling reagents 3a-c were obtained by treatment of 1a-c with n-methylmorpholinium p-toluenesulfonates 2 in the presence of hcl acceptor. the loading of the solid carriers were calculated from n, s contents, determined by microanalysis. all prepared immobilized n-triazynylammonium toluenosulfonates 3a-c have been found stable at room temperatures. activation of carboxylic components afforded triazine activate esters 4a-c connected to the support. treatment of 4a-c with appropriate amino components gave amides or peptides. the final products, chromatographically homogenous amides and peptides, were isolated by filtration or extraction from the solid support. mutter's pseudoproline dipeptides and sheppard's hmb derivatives are powerful tools for enhancing synthetic efficiency in fmoc spps. they work by exploiting the natural propensity of n-alkyl amino acids to disrupt the formation of the secondary structures during peptide assembly. their use results in better and more predictable acylation and deprotection kinetics, enhanced reaction rates, and improved yields of crude products. however, these approaches have certain limitations: pseudoproline dipeptides can only be used for sequences containing serine or threonine, and the coupling of the amino acid following the hmb residue can be extremely difficult. to alleviate some of these shortcomings, we have prepared fmoc-ala-(dmb)gly-oh and fmoc-gly-(dmb)gly-oh. these dmb-dipeptides can be incorporated into peptides in place of ala-gly and gly-gly, resulting in peptides containing structure breaking (dmb)gly residues. by introducing the (dmb)gly residue as part of a dipeptide unit, the need to acylate the highly hindered secondary amino group of (dmb)gly is avoided. on treatment with tfa the dmb group is cleaved regenerating gly. to test the efficacy of our new derivatives in expediting the synthesis of hydrophobic peptides, we undertook the preparation of the challenging neurotoxic prion peptide 106-126 <b>1</b>; this peptide reportedly can not be made using fmoc spps methods. the dipeptides marked in bold were systematically substituted with the appropriate dmb peptides. the effects of the substitution were evaluated using conductivity monitoring and lc-ms analysis of the crude peptides. h-lys-thr-asn-met-lys-his-met-<b>ala-gly</b>-ala-ala-ala-<b>ala-gly</b>-ala-val-val-<b>gly-gly</b>-leu-gly-oh <b>1</b> th120 efficient dipeptide production form unprotected l-amino acids with the novel enzyme l-amino acid α-ligase. k. tabata 2 , h. ikeda 1 , m. yagasaki 1 , s. hashimoto 1 background and aims: application of α-dipeptides has been limited due to the lack of cost-effective manufacturing methods. the known methods require the protection of amino acid(s) to fix the order of the amino acids ( fig. 1) . furthermore, they usually accompany the formation of longer peptides. to establish the costeffective manufacturing method, a novel activity which synthesizes α-dipeptides from two unprotected l-amino acids was screened. methods and results: a gene was found in the genome of bacillus subtilis by in silico screening based on a putative reaction mechanism. the purified protein coded on the gene, i) catalyses α-dipeptide formation from unmodified l-amino acids with a specific order in an atp-dependent manner, ii) never forms tri-or longer peptides, and iii) takes a wide variety of l-amino acids but no d-amino acids. the enzyme was tentatively named l-amino acid α-ligase (lal). the whole cell reaction of a recombinant e. coli strain expressing lal and polyphosphate kinase (ppk) with two l-amino acids and polyphosphate (polyp) enable the efficient production of many dipeptides with a certain order of the constituent amino acids through the coupling reaction of lal and ppk (fig. 2) . conclusion: a novel enzyme, lal, enables to synthesize dipeptides cost-effectively directly from unmodified l-amino acids. t. ye 1 marine organisms continue to provide rich sources of structurally unique and pharmaceutically active compounds. due to the difficulties in the isolation of significant quantities of these natural products, synthetic chemistry serves an important role in their structural assignment and biological evaluation. antifungal agents have received considerable attention recently since the spread of hiv has left many people open to fungal infections, and there is a rapidly growing number of drug resistant strains of fungus emerging. ll-15g256gamma is a cyclodepsipeptide isolated from the marine fungus hypoxylon oceanicum and structurally assigned in 1998 by schlingmann. the structure of ll-15g256gamma was determined by a combination of chemical degradation, chiral chromatography and spectroscopic analysis. ll-15g256gamma uniquely combines a beta-ketotryptophan and a polyketide portion within a macrolactone ring. ll-15g256gamma has exhibited potent activity against fungal strains and as such, is an attractive compound to develop as a future therapeutic agent. to date, there have been no reported studies towards the synthesis of ll-15g256gamma. we have completed the total synthesis of ll-15g256gamma by employing the macrolactamization followed by a c-h oxidation as the key step. aspartimide (aminosuccinimide, asu) formation is the first step in the degradation of asp/asn containing peptides and proteins. the reaction is especially prevalent at asx-gly sites and results in a variety of rearranged and racemized products. the bases used in fmoc-tert-based spps promote the formation of asu and related products. we recently found that the dmb backbone protection efficiently prevents secondary structure formation at gg sites and is orthogonal with respect to standard fmoc spps. here we explore the use of dmb, tmb and nbzl groups (z) for the synthesis of "difficult"/asu-prone peptides, in three different schemes: a) fmoc-asx-(z)gly-oh dipeptide building blocks; b) fmoc-(z)gly-oh monomer building blocks, and c) two steps "submonomeric" approach for synthesis of substituted n-benzyl glycines on the resin. we tested the new methods on two model peptides vkd/ngyi and ha21-20hiv-tat48-57 (h-g1lfgaiagfi engwegmidg20grkkrrqrrr30-oh) fusion peptide. the yield and purity of the products reach and even exceed the level in control experiments obtained with hmb protection and the peptides were found free of asu/piperidides. the acid removal of the dmb protection is ~30% faster than that of hmb. the submonomeric route (strategy c) is especially simple, efficient, cost effective and it allows the use of different amines for halogen-displacement. the backbone protecting groups used were in many respects superior to the commercial reagents and applicable for synthesis of both peptide acids and peptide amides. the use of nbzl-nh2 for halogen displacement represents a new method for preparation of backbone-caged peptides. alkyl bonded silica gels historically have been the standard in reversed phase (rp) purification of biomolecules such as synthetic peptides, small proteins, and oligonucleotides. silica gels provided the resolving power needed for challenging separations and the mechanical stability required to be operated industrially under high pressure conditions. the chief disadvantage of silica gels is poor chemical stability under alkaline conditions, which limits their capability to withstand rigorous clean and sanitization -in-place (cip/sip) protocols. as a result, polymeric media have gained recent market attention because of their excellent chemical stability, which enables full compatibility with modern cip/sip protocols. however, first generation polymeric gels lacked both the resolving power and the mechanical stability to be compatible with industrial high pressure dynamic axial compression (dac) hardware. rohm and haas' advanced biosciences division recently introduced a new, monospheric, 10 micron, high performance polymeric rp material. unlike existing softer polymeric gels, this product has higher mechanical stability which enables it to be used effectively with industrial dac / hplc hardware. in addition, this material provides high resolving power for the most challenging industrial separations, because of its unique and selective pore structure, as well as its small monospheric particle size. finally, because of its excellent chemical stability, the media is not limited in the range of ph that can be used. the combination of mechanical stability for high throughput, chemical stability for long lifetime in use, and high resolution for high yield, together translate to an effective cost-in-use solution for industrial polishing processes. we have developed new types of peptide nucleic acids with improved water solubility by introducing ether linkages and pyrrolidine rings in the main chain; pyrrolidine-based oxy-pnas (popnas). in this work, cellular uptake and endosomal release of the trans-l-popna oligomers, one of stereoisomes of the popna, were investigated. the cellular uptake was achieved by combining the popna oligomer with an n-terminal 23-mer peptide of an influenza virus hemagglutinin protein (ha2) that is labeled with a rhodamine fluorophore at the n-terminal and covalently linked with a hepta-arginine unit at the c-terminal (rho-ha2-r7). the fluorescence images of the cho cells after incubation with fam-po(13) [fam-o-cag tta ggg tta g-gly-nh2] in the absence and presence of rho-ha2-r7 were observed with confocal laser-scanning microscopy. incubation with fam-po(13) alone, no internalization of the oligomer was observed. in the presence of rho-ha2-r7, however, fam-po(13) was successfully internalized into cho cells and, more importantly, the fluorescence spread over the whole cell. the fluorescence image indicates that the popna oligomer in combination with the ha2-r7 peptide was transferred into cytoplasm within 1 h. since both the red (rho) and green (fam) fluorescence spread over the cytoplasm, the popna oligomers that were taken up into endosomes together with the rho-ha2-r7 were released into cytoplasm as the disruption of the endosomes by the ha2 peptide. in summary, the popna oligomers were readily taken up into cytoplasm of cho cells, when combined with a ha2-r7 peptide. most of functional rnas have post-transcriptional modifications, some of which are quite important for their structure and function. thus, for studying such rnas, it is necessary to use purified raw rnas obtained from living organisms. isolation of native rna is necessary also in the case of analyzing the sequence and modifications of mature rna, which may be different from simple transcript of its gene. therefore, rna isolation method is required. many previous reports demonstrated isolation of rnas, especially trnas. most common and traditional purification methods are based on successive column chromatographies. it seems difficult to apply such method to every trna because effective combination of columns varies among individual trnas. to overcome the difficulty, a sequence-specific selection method using a solid-phase dna has been devised. in this method, a trna can be purified from rna mixture by a single step. however, this method needs high temperature treatment, which might assist hydrolysis of rna strand and might impair heat labile modifications. pna-rna hybrid has been known to be much more stable than dna-rna hybrid. thus pna-based rna purification method seems to be possible for wider variety of rnas in lower temperature, in comparison with dna-based method. in this study, we attempted to purify a single rna, such as a trna and a noncoding rna, from rna mixture by using immobilized pna. r. pipkorn 1 , w. waldeck 2 , h. spring 3 , j. jenne 4 , k. braun 5 background and aims: safe drug delivery technologies are pivotal for genetic interventions, but viral vectors baer the risk of inflammatory reaction. questions concerning the efficacy of delivery of the genetic substances, the desired topical gene activation and targeting must be answered. therefore we attempted to develop a membrane non-perturbing delivery system for transport of inactive functional genes into cells and tissues. genes can be subsequently activated at the target site. our concept bases on the use of peptide-nucleic-acids (pnas) resistant against proteases and nucleases, oligonucleotide derivatives, in which the phosphate-backbone has been replaced with ethylen-amin connected alpha-amino-ethylglycine-units. methods: peptides conjugates were composed and synthesized according to the solid phase synthesis and protecting group chemistry strategies. pna sequences were conjugated covalently, non cleavable, with a capronic acid spacer to the nls, pkkkrkv. pnas have gained broad attention in antisense/antigene experiments and as diagnostic tools. in principal, they can be synthesised with several activating reagents known from peptide synthesis. namely, hatu or pybop are often used. synthesis with hatu is more laborious, because preactivation is needed in order to avoid guadinylation of the n-terminus of the growing pna-chain. we wanted to use pybop, because preactivation should not be needed in this case, which is especially useful in automated synthesis. surprisingly, in the pybop-mediated syntheses of 18mer pnas we obtained products showing molecular masses approx. 67 da above the expected ones. detailed analysis revealed, that the modification occurred at the only guanine residue in the sequence. in order to further characterise the side reaction, a short pna fragment was synthesised using hatu and pybop activation, respectively, and cleaved from the resin with and without the n-terminal fmoc-group. while synthesis with hatu gave the desired products, pybop partly activates the aromatic carboxy group of the guanine residue, which is substituted by piperidine during subsequent fmoc cleavage. the modified sequences could be further characterised by ms/ms-fragmentation. our results show that care must be taken when synthesising pnas with pybop activation. on the other hand, this reaction possibly opens an opportunity to synthesise guanine derivatives. the opioid receptor system in the central nervous system (cns) controls a number of physiological processes including pain, reward, gastrointestinal and cardiovascular functions. as a consequence, most pain modulating compounds currently available cause a variety of side-effects. the endogenous ligands for the opioid receptors are a series of peptides that includes endomorphin-1. endomorphin-1 has been shown to elicit potent anti-nociception through the highly selective activation of µ-opioid receptors. it is this receptor that mediates supraspinal analgesia and thus, selectivity for this receptor results in analgesia without affecting other processes. therefore, endomorphin-1 is considered a promising lead compound for the development of a new, safer pain medication. we have synthesized a large number of lipid-and carbohydrate-modified endomorphin-1 analogues and screened these compounds for their binding and activation of µ-and δ-opioid receptors in sh-sy5y cells as well as caco-2 cell monolayer permeability and plasma stability. compounds conjugated with either a lipoamino acid or sugar moiety on the c-terminus lost binding affinity by several orders of magnitude, whilst n-terminal conjugations resulted in minimal loss of binding affinity. a number of analogues showed pm binding affinity and high apparent permeability, and of these compounds, one has been selected for assessment in nociceptive and neuropathic pain models. in addition to these pre-clinical studies, internalization and tolerance formation of these compounds has also been measured in an effort to synthesise a non-tolerant opioid agonist. endomorphin-1 analogues with a high degree of amphiphilicity cause increased receptor internalization and subsequently less tolerance formation. a. marcinkowska 1 , l. borovičkova 2 , j. slaninowá 2 , z. grzonka 1 carbohydrate moieties of glycopeptides and glycoproteins play different decisive roles in various biological phenomena. conformation and solubility of proteins are influenced by the oligosaccharide chains, which can also inhibit the proteolytic degradation. as a result, the synthesis of glycopeptides is an attractive field that contributes to understanding of mutual interactions between both moieties and for their biological interest. the synthesis of glycopeptides requires a combination of synthetic methods from both carbohydrate and peptide chemistry. moreover, this synthesis needs stereoselective formation of the glycosyl bond between a carbohydrate and a peptide (amino acid) part, and also an appropriate protecting group methodology that allows selective deblocking of only one functional group in these polyfunctional molecules. in the present work we modified the oxytocin and vasopressin structure with glycoamino acids. transformations of fmoc-protected serine and threonine derivatives into appropriate o-glycosylated precursors suitable for solid phase peptide synthesis were worked out. the -and -o-glycosides were synthesized from fmocserine and fmoc-threonine allyl esters and appropriate glycosyl bromide using hanessian's modification of the koenigs -knorr reaction. these n--fmoc-protected glycosides were used in synthesis of glycopeptides. eight analogues of oxytocin modified in position 4 were obtained. we have also prepared two types of lysin-vasopressin analogues modified with glycoamino acid, in which the glucuronic acid was attached to the ω-amino group of lysine in position 8 through the amide bond. glycosylated analogues of oxytocin and vasopressin display an increased stability towards enzymatic degradation, and retain some hormonal activities. supported by grants: ds/8350-5-0131-6 (zg) and z40550506 (js) according to many authors the formation of amadori products is a key stage in the glycation process. glycated proteins may show allergenic properties and potentially initiate autoimmunological processes. they may also serve as the markers of diabetes. to our best knowledge, all procedures concerning the synthesis of peptide-derived amadori products reported in literature are based on "in solution" approach which makes them tedious and time consuming. a modified method of the solid phase synthesis of peptide-derived amadori products based on direct alkylation of the deprotected ε-amino groups with 2,3:4,5-di-oisopropylidene-β-d-arabino-hexos-2-ulo-2,6-pyranose in the presence of sodium cyanoborohydride was proposed. isopropylidene groups, protecting the sugar moiety in the obtained conjugate, were removed with trifluoroacetic acid containing 5% water. studies on optimization of the reaction performed on the model peptide attached to a wang resin, fmoc-lys-leu-leu-phe-(resin), showed that the best yield of the product is attained with a two-fold excess of 2,3:4,5-di-oisopropylidene-β-d-arabino-hexos-2-ulo-2,6-pyranose and a five-fold excess of sodium cyanoborohydride. the identity of the product was confirmed by high resolution ms. the several side products were isolated and their structures will be discussed. our results prove that the synthesis of glycated peptides in the solid phase is feasible. the lack of homogeneous glycoproteins in sufficient quantities is an ongoing challenge in glycobiology. in order to solve this problem researchers have turned to a variety of approaches ranging from mutant eukaryotic strains to the highly demanding total synthesis of glycoproteins. [1] using rnase b as a model nglycoprotein [2] we have searched a path to assemble this enzyme employing a combination of chemical and recombinant methods. native chemical ligation [3] allows the coupling of protein segments of unrestricted size in a chemoselective manner. we have developed solid phase methods to produce the required thioester building blocks 1-25-sr (a) and glycopeptide thioester 26-39-sr (b) containing an n-glycan at asn34 on a dual linker pega resin. [4] the remaining segment 40-124 (c) was expressed in e. coli as a fusion protein and released by intein mediated protein cleavage. [5] sequential coupling of the three rnase segments requires the use of a protective group at the n-terminus of segment b compatible with the oligosaccharide part. dysfunctional mutations of antitrypsin can result in a loss of elastase inhibitory activity or allow self-aggregation to occur and cause emphysema and cirrhosis, respectively. insights of the mechanism of disease provide strategy to cope with the aberrant protein aggregation and may bring potential therapeutic agents. in the present work, we describe our effort to identify effective anti-protein polymerization ligands by the employment of combinatorial technology. antitrypsin from human plasma was purified by glutathione sepharose and mono q-sepharose column chromatography. both ala-scanning and peptide shortening were carried out systematically to explore the structural requirements necessary for binding. combinatorial chemistry was then employed to conduct the library screening experiments. assessment of peptide binding was achieved through an unique gel electrophoresis assay. the structural requirements and the minimal peptide length required for binding were revealed by our systematic approach. this information was critical for the design of combinatorial library and the discovery of antitrypsin binding peptides with much improved affinity and specificity. there is currently no effective cure for z antitrypsin related cirrhosis and emphysema. the synthesis and screening of combinatorial libraries offer avenues to increase throughput and ultimately lead to the discovery of inhibitory peptides to the polymerization of pathogenic antitrypsin. with the rapidly increasing number of biopharmaceuticals in the industrial pipeline the need for efficient and expedient purification procedures is growing ever greater. affinity chromatography is one of the most promising technologies in this regard, as it offers very high selectivity and can often replace lengthy and expensive traditional chromatographic procedures. the use of combinatorial split-and-mix libraries is a powerful tool for discovering new affinity ligands but the technique has been limited by the laborious spectroscopic and chemical analysis needed to identify the binding ligand. we have previously introduced a novel bead encoding technology based on a 3-dimensional image recognition of patterns made by fluorescent particles randomly distributed inside larger beads. [1] the beads are read prior to each chemical transformation by an instrument featuring three fluorescence microscopes at a rate of 5,000 beads per hour. we here present the development of small peptidomimetic affinity ligands for the human growth hormone (hgh) by the use of this technology. the library was sought enriched prior to synthesis by in silico screening of a virtual combinatorial library using a large number of diverse building blocks. binding ligands were identified by incubation with fluorescence tagged hgh. [ the cinnamic acids and their derivatives have been found to possess a variety of biological effects, including antiviral, antimicrobial, antitumor and antioxidant activity. for example, several hydroxycinnamic acid conjugates with amino acids, isolated from plant sources showed enhanced antioxidant activity. the synthesis of cinnamic acid amides and their opioid activity was also cited in the literature. however the synthesis and pharmacological properties of sinapoyl-peptide amides continues to be virtually unexplored. on the other hand, the synthesis and opioid activity of analogs of tyr-mif-1 has been well documented by us. herein we present a synthesis of a series of sinapoyl -peptide amides where sinapic acid were attached consecutively to both c-and n-end of the tyr-mif-1 peptide chain: sa-pro-xaa-gly-nh2; sa-tyr-pro-xaa-gly-nh2; pro-leu-gly-nh(ch2)nnh-sa sa=sinapic acid; xaa=leu, unusual aminoacid; n=2,3 to obtain the sinapoyl-peptide-amides, both fmoc-and boc-based spps approach were used. analgesic activity was determined by the randall-sellitto paw-pressure test. the antioxidant effects were examined by dpph test as well. studies to establish the importance of introducing the sinapoyl moiety in the tyr-mif-1 molecule for the antioxidant and opioid activities are underway. several proteins are involved in the transcription of dna to mrna, among which the basic leucine zipper (bzip) proteins. these transcription factors bind specific dna sequences by dimerization and inserting short alpha-helices into the dna major groove. because the dimerization domain is only required to obtain the correct geometrical positioning of the alpha-helices, we will replace it by a dipodal steroid scaffold with defined stereochemistry. due to orthogonal protecting groups, a unique feature of this scaffold is the possibility to design not only homodimers, but also heterodimers. therefore this strategy allows for the construction of both major/major groove and major/minor groove binding peptides, either mimicking naturally occurring proteins or designing peptides with new binding properties. native chemical ligation and staudinger ligation are both suitable for the construction of these peptide dimers. moreover, a combination of solution-and solid-phase chemistry allows for the generation of combinatorial libraries. the increasing number of antibiotic-resistant bacteria is a global health problem. therefore the development of new highly efficient drugs is one of the major tasks of this century. as an example of peptides, which inhibit the growth of e. coli, we demonstrate an easy and rapid method for finding peptides with optimized antimicrobial properties. as a first step we built a modular construct. this construct consists of a constant cationic and a variable module. the cationic module was choosen to achieve cellpenetrating properties. the variable module was expected to act as the virtual active part of the peptide. to increase the proteolytic stability of the peptide we synthesized them in cyclic form. in the first step we used the combinatorial approach to screen approximately 64.000.000 peptide sequences in the variable region in order to find highly active peptides against e. coli. to optimize the identified sequence, we substituted all amino acids of the sequence with other amino acids and building blocks. additionally, in order to increase stability we modified the bridging. in this way we were able to uncover peptides with high antimicrobial activity as well as proteolytic stability and reasonable solubility. a series of melanocortin active core tetrapeptide hfrw nonpeptide imitations has been prepared using a combination of solution and solid phase synthesis. most of them included residue of 3-(1-imidazolyl) propylamine or histamine as substitutes of histidine. phenylalanine residue, which is included in melanocortins was replaced by residues of derivatives of 4, 4'-disubstituted isopropylidenedicyclohexane, 4, 4'-disubstituted bicyclohexane, 1,4-disubstituted cyclohexane, 1,5-disubstituted cyclooctane, and 1,2-, 1,3-or 1,4disubstituted benzenes. instead of arginine, residues of oligomethylene diamines, 2-butyl-2-ethyl-1,5-pentanediamine, 4,4'-methylene-bis(cyclohexylamine), and 4,4'-diaminodiphenylmethane were introduced. 2-naphtyloxyacetyl-, (4-1h-indol-3-yl)-butyryl-, 2-phenyl-ethanesulfonyl-and naphthalene-2-sulfonyl-groups served as replacement of tryptophan residue. tested on binding assay on melanocortin receptors, active core imitations exhibited a micromolar affinity to them. isopropylidenedicyclohexane and bicyclohexane derivatives showed about 10 fold higher affinity compared with corresponding derivatives of cyclohexane, cyclooctane or disubstituted benzene. obestatin is a novel endogenous ghrelin-associate peptide, which is involved in the regulation of food intake and weight gain. it was shown to be anorexigenic, able to decrease food intake, gastric emptying and jejunal motility. although obestatin and ghrelin originate from a common prepropeptide of 117 residues, they are reported to exert opposing physiological roles, by binding distinct receptors belonging to the subgroup of type a gpcrs [1] . obestatin was found to be the natural ligand of the orphan gpr39 receptor, a gpcr, expressed in jejunum, duodenum, stomach, pituitary, ileum, liver and hypothalamus. as many other peptides involved in the obesity process, it is a new and interesting drug target for the discovery of new anti-obesity molecules. in particular, the first step for the design of new molecules with potential improved anti-obesity activity, is the elucidation of the obestatin conformational features. here, we present the synthesis and the conformational analysis by nmr and cd spectroscopies of obestatin and its related 13-mer c-terminal sub-fragment, in aqueous solution and in membrane mimicking environment. the data outline the obestatin c-terminal portion as the region characterized by significant conformational features potentially opened to interesting future developments. [ a total of 50 isolates of rhizobium were collected from root nodules of medicago sativa and melilotus officialis plants in different regions of isfahan province .all of isolates on ty medium formed white ,slimy colonies with smooth margins and their inoculation on to roots of young alfalfa plants produced spindly nodules . the nodules developed with some of the isolates were big and pinkish ,although the rest of isolates produced small and white nodules .the speed of nodulation for all the isolates was almost similar and the related nodules were appeared within two weeks . the production of brown pigments on aged colonies of some isolates on ty or ty supplemented with l_tyrosine and copper sulfate revealed that these isolates of s. meliloti are melanin-producing rhizobia.based on the motility and sensitivity to antibiotics tests ,all of the isolates formed a reasonably homogenous group .however a few of them were able to produce an anti-microbial compound which was found to inhibit a number of isolates of s. meliloti .the compound did not suppress the growth of other bacteria . partial purification and spectrophotometery of the compound suggested that it likely belong to the antimicrobial polypeptides .considering on their physiological and biochemical properties ,none of the isolates were selected as a superior and competitive strain ,although based on nodulation efficiency , melanin and antimicrobial compounds production capability the isolate s. meliloti sm2 and sa23 were nominated to investigate in details. cyclotides are a fascinating family of plant-derived peptides characterized by their head-to-tail cyclized backbone and knotted arrangement of three disulfide bonds. this conserved structural architecture, termed the cyclic cystine knot, is responsible for their exceptional resistance to thermal, chemical and enzymatic degradation. cyclotides have a variety of biological activities but their insecticidal activities suggest that their primary function is in plant defense. in this study we determined the cyclotide content of the sweet violet viola odorata, a member of the violaceae family. we identified 30 cyclotides from the aerial parts and roots of this plant, 13 of which are novel sequences. the new sequences provide information about the natural diversity of cyclotides and the role of particular residues in defining structure and function. as many of the biological activities of cyclotides appear to be associated with membrane interactions, we used hemolytic activity as a marker of bioactivity for a selection of the new cyclotides. the new cyclotides were tested for their ability to resist proteolysis by a range of enzymes and, in common with other cyclotides, were completely resistant to trypsin, pepsin and thermolysin. the results show that while biological activity varies with the sequence the proteolytic stability of the framework does not, and appears to be an inherent feature of the cyclotide framework. the structure of one of the new cyclotides, cycloviolacin o14, was determined and shown to contain the cyclic cystine knot motif. this study confirms that cyclotides may be regarded as a natural combinatorial template that displays a variety of peptide epitopes most likely targeted to a range of plant pests and pathogens. furthermore, the inherent stability of the framework makes it an excellent scaffold for protein engineering applications. warfarin is the most widely prescribed anticoagulant drug for the prevention and treatment of arterial and venous thromboembolic disorders.because of large interpatient variability in the dose-anticoagulant effect relationship and a narrow therapeutic index careful dosage adjustment based on inr is essential. warfarin is available as a racemic mixture of two enantiomers,(s)-and (r)-warfarin. in contrast to (r)-warfarin, which is metabolized by multiple cytochrome p450s(cyps), including cyp1a2 and cyp3a4,(s)-warfarin, is predominantly metabolized to 7-hydroxywarfarin by polymorphic cyp2c9. since the potency of (s)-warfarin is much higher than that of (r)-warfarin, about 3-to 5-fold,any change in the activity of cyp2c9 gene is likely to have a significant influence on the anticoagulant response. previous in vitro findings revealed that certain variants in the cyp2c9 gene are associated with large interindividual differences in the pharmacokinetic and pharmacodynamic outcomes of warfarin therapy. three major alleles have been found to date in humans:arg144/ile359, and cys144/ile359 and arg144/leu359, and arg144/leu359, which have been designated cyp2c9*1 (wild-type), cyp2c9*2, and cyp2c9*3, respectively. we have investigated this polymorphism in iranians that has not been described previously. genomic dna was isolated from whole blood. for detection of cyp2c9*2, and cyp2c9*3 variants, a protocol based on pcr technique and endonuclease digestion with kpni, ava ii was used. in this research work, we have studied a group of 56 patients, in which warfarin therapy was initiated. recently new 21-residue antimicrobial peptides -arenicins were isolated from coelomocytes of marine polychaeta arenicola marina and their sequences were determined [1] . there are two isoforms of arenicins which differ only with single amino acid. these peptides have no structure similarity to any previously identified antimicrobial peptides. we have synthesized and estimated the antibacterial properties of arenicin-1: rwcvyayvrvrgvlvryrrcw. the linear peptide was prepared by solid phase method using boc-technology without any problem. however the cyclization caused the appreciable difficulties. the following methods of oxidation were used: oxygen of air, k3fe(cn)6 and hydrogen peroxide in aqueous or organic media. the best results were obtained by using hydrogen peroxide in methanol, but and in this case the yield of the aim peptide did not exceed 5%. synthetic arenicin had the same hplc profile and maldi-tof spectra as a natural molecule. the peptide showed an antimicrobial activity against gram-positive bacteria: peptidergic hormones and neurotransmitters are known to be produced by the specific cleavage of their precursor proteins that per se have no biological functions. the neutrophil-activating peptides we recently identified, however, are the peptides cleaved from mitochondrial proteins by proteolysis. therefore, we named them "functional cryptic peptides" because they are hidden in protein sequences. some of these peptides activate gi type of g proteins directly, and neutrophils are suggested to be stimulated by the direct (i.e., not via gpcrs) activation of g proteins. these peptides had features, in common, in their distributions of charged and hydrophobic amino acid residues, but homologies in their primary structures were not apparent. in the present study, we predicted functional cryptic peptides that activate g proteins, based on the distribution of charged and hydrophobic residues. receptors for these peptides were also investigated by the direct cross-linking experiments between peptides and their targeted proteins. the finding of functional cryptic peptides is expected to lead to the identification of novel signaling mechanisms where such peptides are involved in the regulation of bio-functions. the fragment 81-88 of the precursor of human interleukin-1alpha (pil-1α) (gk-vlkkrr) appeared to have more than 80% homology with corticotropin fragment 10-18 (gkpvgkkrr). we have previously synthesized the octapeptide gkvlkkrr (referred to as leucocorticotropin, lct) and found its high affinity binding to corticotropin receptors on various immunocompetent cells in human and mouse. in this study we investigated the interaction of lct with rat adrenal cortex membranes and the effects of lct on the level of 11-oxycorticosteroids (cs) in rat adrenal glands and plasma in vivo. lct was labeled with tritium by the high-temperature solid-state catalytic isotope exchange reaction to specific activity of 22 ci/mmol. receptor binding studies revealed that tritium-labeled lct bound with high affinity and specificity to corticotropin receptor on rat adrenal cortex membranes (kd = 2.2 nm). lct at concentrations of 0.1 -1000 nм was found to have no influence on the adenylate cyclase activity in adrenal cortex membranes, while intranasal injection of lct to rats at doses of 10 -50 microg/kg was found to inhibit the secretion of cs from the adrenals to the bloodstream. thus, lct is an antagonist of corticotropin receptor. comarin derivatives such as warfarin are prescribed widely for treatment and prevention of thrombosis. warfarin is the widespread oral anticoagulant drug employed, but its required dose is highly variable both inter-individually and inter-ethnically. so it is desirable to develop strategies to predict the warfarin dose response in patients before initiation of anticoagulation. the vitamin k-dependent γ-carboxilation system, consists of the vitamin k-dependent γ-carboxylase, which requires the reduced hydroquinone form of vitamin k1 as a cofactor and the warfarin sensitive enzyme vitamin k1 2,3-epoxide reductase (vkor), which produces the cofactor. warfarin exerts its anticoagulant effect by inhibiting the vitamin k epoxid reductase enzyme complex (vkor) that recycles vitamin k1 2, 3-epoxide to vitamin k1 hydroquinone. a component of the vkor termed vkorc1, has now been identified as a therapeutic target site of warfarin. point mutations were identified within the gene encoding vkorc1 in individuals who required large doses of wafarin to maintain therapeutic anticoagulation. however the relationship between the primary structure of vkorc1 and the mechanism of action of warfarin is poorly understood. in previous works we have shown that naturally occurring functional protein fragments affect cell proliferation [1] . their mechanisms of action involve receptors of "classical" regulatory peptides, or are non-receptoric [1] . among protein kinases involved are pka, camkii, mapk [2] . in organism bioactive functional protein fragments could participate in maintenance of tissue homeostasis. in present work, homeostatic potential of functional protein fragments was studied in compare with classical regulatory peptides. the panel of test substances was formed, including signal transduction modulators (pka, pkc, ca2+-channel activators), classical regulatory peptides (bradykinin, somatostatin, met-enkephalin, endothelin, neurotensin) and fragments of functional proteins (β-actin fragments from (75-90) and (69-77) segments; valorphin (β-globin (33-39)); neokyotorphin (α-globin (137-141); short acidic peptides from multiple precursors). their activity was tested in cultures derived from similar sources but differing in transformation degree (e.g., mouse embryonic vs. tumor fibroblasts) and/or culturing conditions. the factors most affecting cell sensitivity to the test substances were (in order of the importance decrease): (1) cell type; (2) transformed vs. normal phenotype; (3) cell density; (4) serum supply. activity of fragments of functional proteins, showing general correlation with other test substances, was more influenced by the culturing conditions (i.e., cell population status). thus, fragments of functional proteins could be regarded as partners of "classical" homeostasis regulators, playing role of finer tuners of tissue proliferative status. the study was supported by ras presidium programme "molecular and cell biology" histone-like proteins in bacteria are small basic proteins that contribute to the control of gene expression, recombination and dna replication. they are also an important factor in compressing the bacterial dna in the nucleoid. among the hlps, hu protein is attracted to dna containing structural aberrations such as four way junctions or single stranded lesions. this protein plays an important role in binding as a dimer and bending dna. it also contributes to the beginning of the dna replication. in this study we showed that a 10-kda protein, probably hu exists in halobacillus karajiensis which is a novel gram positive moderate halophile bacteria that was recently isolated from surface saline soil of the karaj region, iran. since hu is purified and characterized in e.coli we used this bacteria as the control in this study. the 10-kda protein extraction was carried out by using pca 5% which is normally used for extracting histones from eukaryotic cells. the results of running the protein extracts on sds-page demonstrated a band around 10-kda which was seen in protein extracts of this protocol. these results supported the hypothesis of the existence of a 10-kda protein in halobacillus karajiensis. sufficient oxygen and nutrient delivery is a necessity for tumors. when oxygen supply decreases, tumors initiate growth of new blood vessels. low grade astrocytomas, a class of malignant brain tumors, grow along the existing vessels in a process called co-option. hypoxia is induced in the progression from grade iii to grade iv astrocytomas (glioblastoma multiforme, gbm) which in turn triggers the formation of a new and distinct tumor vasculature. the new vessels formed by tumor-triggered angiogenesis differ by molecular composition from their normal vascular counterparts. we are utilizing phage displayed peptide libraries to identify peptides that specifically home to either co-opted or angiogenic brain tumor vessels. furthermore, we aim at characterizing differentially expressed endothelial markers (receptor molecules) to get a better understanding of the molecular changes in the vasculature. several rounds of in vivo biopanning was performed in mouse models of astrocytomas to isolate a phage pool that has up to a hundred-fold homing to low grade tumor lesions. out of the selected pool we discovered peptides capable of homing and accumulating to the tumor islets and co-opted vasculature. the homing potential of our newly identified peptides has shown to be highly specific for clusters formed by the tumor cells and co-opted "early" vessels within these palisades. these homing peptides represent promising candidates to selectively target co-opted vessels and tumor lesions in the brain and act as lead compounds in identification of surface molecules (receptors) differentially expressed by co-opted tumor vessels. the αvβ3 integrin receptors play an important role in human tumor metastasis and growth. the inhibition of these receptors by antibodies or by cyclic peptides containing the arg-gly-asp( rgd) sequence may be used as selectively treatment to suppress the disease. our research group has previously described that the formal introduction of a single carbon atom to bridge the cα (i) and n(i+1) contiguous residues of a linear or cyclic peptide leads to α-amino-β-lactam peptidomimetics containing predictably placed β-turn and γ-turn motifs, respectively. the combination of these results with the well-known capacity of rgd tripeptide for inhibition of the biological answer in integrin led us to the design of the following cyclic peptide. the adhesion and cell-growth "in vitro" assays using human umbilical vein endothelial cells (huvec), as well as "in vivo" assays with xenograph mice revealed that the rgd peptidomimetic was active to micromolar concentrations, slightly better than the reference compound in this field: cilengitide®. whole saliva is composed of secretions from parotid, submandibular and sublingual glands, and smaller ones from saliva of minor salivary glands (e.g. palatal and labial). saliva contains a variety of proteins and polypeptides. one of them is statherin, a multifunctional 43-amino acid residue, phosphominiprotein. this peptide is present in human parotid and submandibular saliva . the aim of our study was to investigate the stability of statherin in extracts of the major salivary glands. submandibular, sublingual and parotid gland tissues were obtained at autopsy 12 h after death. samples of the gland tissues were homogenized, centrifugated (30,000 g, 30 min., 4 c) and the supernatants were frozen and stored at -70 c prior to analysis. synthetic statherin was added to the supernatants before analysis (45 microgram/ml). the samples were analysed for the presence of the peptide by the matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (maldi-tof ms}technique and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds page). statherin has been found to be decomposed in extracts of parotid and submandibular glands and also in the extract of sublingual glands. the dramatic increase in research for new anthrax therapeutic approach was prompted by potential use of the causative agent of anthrax bacillus anthracis as a biological weapon. anthrax toxin consists of three proteins, the protective antigen (pa) and the two enzymes lethal factor (lf) and edema factor (ef) that are carried through the membrane of the target cell upon binding to specific site on the membrane receptor-bound pa. lethal factor and edema factor were found to cooperate to promote immune evasion of the bacterium. here we describe the production of peptide inhibitors of pa-lf binding, obtained by selecting pa-binding peptide by a competitive panning of a phage peptide library, using recombinant lf. we selected several 12 mer peptides, which were synthesized in tetra-branched multiple antigen peptide (map) form, inducing resistance to proteolytic degradation (1) and maintaining biological activity of phage peptides. lead tetra-branched peptides were systematically modified by progressive shortening and residue randomization, to obtain an increase of peptide affinity and inhibitory efficiency. affinity maturation of lead sequences enabled selection of a peptide which has an ic50 at least one log lower that any other lethal-toxin-inhibiting peptide described so far and is effective for in vivo neutralization of anthrax toxin activity (2) . the same peptide can also efficiently inhibit the binding of ef to pa and ef-induced camp increase in different cell lines. microtubules are dynamic polymers that have important roles in eukaryotic cellular processes such as signal transduction, cell polarity, vesicular transport and chromosomal movement. the dynamic behavior of microtubules has been studied both in vivo and in vitro. the effect of arsenic trioxide on microtubule polymerization has been studied under in vivo experimentation shown that it inhibits formation of mitotic bundles. we studied the mechanism of arsenic trioxide effect on polymerization of microtubule protein purified from sheep brain in vitro. microtubule polymerization has been conducted by adding 1mm gtp to purified tubuline in pem buffer at 37oc for 30 minutes and simultaneously followed by measuring turbidity (350 nm). the results shown that lag time of polymerization (nucleation step) is affected by increasing concentrations of arsenic trioxide from 0-5 micromolar. moreover the rate of elongation step was decreased exponentially by increasing arsenic trioxide concentration. electron micrographs also showed microtubules length decrement due to arsenic trioxide. the results have shown the inhibitory effect of arsenic trioxide on microtubule polymerization via its effect on nucleation step as well as elongation rate. background and aims: alzheimer's disease (ad) is the major cause of dementia among the elderly. the increase in life expectancy worldwide demands new therapies for ad urgently. self-association of the amyloid ß-protein (aß) into neurotoxic assemblies, a seminal event in the etiology of ad, is considered to follow interactions of the c-terminus of the 42-residue form of aß (aß42). we hypothesized that molecules with high affinity for the c-terminus of aß42 will disrupt aß42 oligomerization. a series of c-terminal fragments (ctfs) of aß42, aß(x-42) with x = 28-39, has been prepared to study their potential to inhibit aß42 oligomerization and neurotoxicity. methods: attenuation of aß42 assembly by ctfs was studied by quantitative analysis of oligomer size distributions using a photo-cross-linking assay followed by sds-page. biological activity of ctfs themselves and as inhibitors of aß42-induced neurotoxicity was assessed by mtt reduction assay using differentiated pc-12 cells. the structure of the ctfs was studied by circular dichroism (cd) spectroscopy and ion mobility spectrometry-mass spectrometry (ims-ms) coupled with molecular dynamics (md) simulations. results: ctfs were found to inhibit aß42 oligomerization in a length dependent manner with minimal or no toxicity of the ctfs themselves. certain ctfs were found to inhibit aß42-induced neurotoxicity. cd spectra indicate that increasing peptide length results in growing ß-sheet content. structures based on experimentally determined cross-sections support the existence of a previously proposed turn around residues gly37-gly38. the data suggest that aß42 ctfs can serve as lead compounds for development of peptidomimetic drugs for treatment and prevention of ad. background and aims: human kallikrein hk2 is a prostate specific serine protease, which expression level is elevated in aggressive human prostate cancer suggesting a possible role in a tumour growth and spreading. since hk2 protease is highly prostate specific, inhibition of its activity is a possible method to prevent tumour growth without interfering the function of the other proteases. we have identified hk2 specific linear peptide inhibitor by using phage display techniques. in order to design peptide for in vivo studies we tested the protease stability of the linear and the cyclic forms of the peptide. methods: the prerequisite of the binding was studied by using conventional ala-replacement method and the most optimal sequence was selected for further studies. the stability of the original linear form, acetylated form, peptide with cystein bridge and head-to-tail cyclic peptide was tested with modified trypsin (sequencing grade) and with human plasma. results: both linear versions and peptide with cystein bridge were unstable and were degraded during the first 30 minutes in both stability tests. head-to-tail form of the peptide was stable in both tests during the first 180 minutes. conclusions: since our peptide contains arginine there was a possibility that our peptide is sensitive to trypsin and other serum proteases. indeed both linear and one cyclic from degraded in our tests. only head-to-tail peptide was stable during the first 3 hours suggesting protease resistant folding. background and aims: a large number of anticancer agents has been developed in recent years. however, these agents have very little or no specificity which leads to systemic toxicity. among them paclitaxel is considered to be one of the most important drugs in cancer chemotherapy; however, this agent has also lack of selectivity to the tumor tissue. therefore, development of tumor-targeting prodrug is highly promising. methods: to activate cytotoxic agent specifically at the tumor tissue, we developed a new prodrug strategy based on o-n intramolecular acyl migration, which is a well-known reaction in peptide chemistry, and photodynamic therapy. results: we synthesized a prodrug which has a coumarin derivative conjugated to the amino acid moiety of isotaxel (o-acyl isoform of paclitaxel). the prodrug was selectively activated by visible light irradiation (430 nm) leading to cleavage of coumarin. finally, paclitaxel was released by subsequent o-n intramolecular acyl migration. conclusion: we synthesized and evaluated a novel type of paclitaxel prodrug. this prodrug showed promising kinetic data. therefore, we believe that photoactivation can be promising novel strategy for design of tumor-targeting prodrugs. the search of new immunosupressants, exhibiting the mechanism of action characteristic for cyclosporine a (csa) and fk-506 is an important challenge for medicinal chemistry. cyclolinopeptide a (cla) natural cyclic nonapeptide [cyclo(leu-ile-ile-leu-val-pro-pro-phe-phe)] possesses a strong immunosuppressive activity comparable with that of csa in low doses. the possibility of practical application of cla as a therapeutic agent is limited due to its high hydrophobicity. it has been suggested that the tetrapeptide sequence pro(6)-pro(7)-phe(8)-phe(9) is responsible for the interaction of the cla molecule with the proper cellular receptor. in order to evaluate the role of this tetrapeptide unit for biological activity of native peptide, we decided to modified this fragment. in this communication we present linear and cyclic cla analogues in which phenylalanine residues in position 8 and/or 9 have been replaced with amphiphilic; alphahydroxmethylphenylalnine 1 or homophenylalanine 2. the synthetic strategy and biological activity will be evaluated. resistance to currently used small molecule antibiotics develops at an alarming rate. while resistance to β-lactams in clinical isolates is primarily due to hydrolysis of the ring by β-lactamases, when bacteria develop resistance to fluoroquinolones or aminoglycosides, the sequences of the target biopolymers are altered. earlier we developed a family of antibacterial peptide derivatives that kill bacteria by inhibiting protein folding and are active in animal models of infection. in the current study we examined the synergy between antibiotics acting by different modes of action. inhibition of properly folded active resistance enzymes was completely efficacious to recover the activity of amoxicillin, a β-lactam antibiotic against strains that were originally resistant to this molecule. some activity of ciprofloxacin was also recovered by reducing the load of the induced self-defense dnak protein, but the synergy between the antibacterial peptide and the fluoroquinolone did not yield full bacterial killing. the mode of action of the synergy is indeed inhibition of protein folding because no such effect could be observed with kanamycin where resistance involves changes in the target protein sequence. as opposed to current β-lactamase inhibitors and combination therapies that work against only a limited number of strains, inhibition of all protein folding in bacteria is a universally applicable treatment option. elimination of resistance to β-lactams by proline-rich peptide derivatives may represent a viable avenue to give second life to these antibiotics for which large stockpiles are available for pharmaceutical companies in both patented and generic forms. the integrin αiibβ3 is the major integrin-adhesion receptor on platelets. in unstimulated platelets αiibβ3 is present in a resting conformation state. upon platelet activation by agonists, αiibβ3 receives intracellular signals (inside-out signaling) that allow its rapid conversion to a high-affinity state capable of binding soluble ligands, resulting in platelet aggregation. the intracellular signals include proteins that bind to the cytoplasmic tails of the two subunits α and β of the integrin, or integrin-associated membrane proteins. in vivo charge swapping mutation studies suggested that αiib and β3 tails have a direct site of interaction between αiib (r995) and β3 (d723). peptides derived from the cytoplasmic tail sequences can specifically induce or block αiibβ3 activation in platelets. the aim of this study is to develop peptide analogues based on the cytoplasmic tail sequences of both αiib and β3 subunits that could inhibit platelet thrombus formation by specifically disrupting the inside-out signaling pathway. peptide analogues of the αiib and β3 subunits spanning the sequences αiib-989-1008, αiib -997-1003, αiib -997-1008, αiib-1000-1008 and β3-743-750, β3-743-756, β3-749-756 were synthesized in their free state, palmitoylated and/or tagged with the tat fragment 48-60 and carboxyfluorescein-labeled, in order to investigate their membrane permeability, as well as their inhibition of the platelet aggregation. inflammatory pain begins when noxious stimuli (thermal, chemical or mechanical) excite sensorial neurons called nociceptors. the activation of nociceptors leads to the opening of some ionic channels and depolarization of the cell membrane. one of these channels is trpv1, which is directly implied in thermal hyperalgesia associated to inflammation. in previous work it has been found that peptoid h-arg-15-15c ( fig. 1) inhibits the activation of trpv1 by blocking the pore entrance. however, this compound showed toxicity in vivo. the aim of our work is the design and synthesis of new compounds, based on the structure of h-arg-15-15c, with better therapeutic properties. we synthesized some new non-competitive antagonists of trpv1 that exhibit notable anti-inflammatory and analgesic activity in vivo. th. skarlas, e. panou-pomonis, d. krikorian, m. sakarellos-daitsiotis, c. sakarellos erf is a transcriptional repressor with tumor suppressor activity regulated by the ras/erk signaling pathway. it has been shown that erf interacts with, and is phosphorylated by erks in vitro and in vivo. this phosphorylation determines its subcellular localization and biological function. erf exhibits a high degree of specificity and sensitivity for erks. the major objective of our study is to provide proof of principal for a specific anti-cancer approach targeting the ras-pathway, which is commonly activated in human tumors, via the stimulus of the downstream effector erf. this will be attained by modeling specific peptide inhibitors that block the erf phosphorylation and inactivation by the ras/erk signaling pathway. we present the design and synthesis of peptide inhibitors incorporating the fsf and fkf motifs, known to play a critical role in the erf/erk interaction, in their free forms or conjugated to a carrier. ubiquitinium is a well known mechanism in protein degredation of eukaryotic cells ,in which many obsolte and corrupted three dimentional structure protein ,become marked by covalent attachment of ubuquitin through a multi-step enzymatic pathway.ubiquitin is a small ,8.5 kda peptide of 76 amino acid residues that targets such substrtes for proteolysis in proteasome .recnt studies showed that an extra cellular ubiquitination process also taking place in the epididymes of humans and other animals marks protein on the surface of the defective sperm .it appears that structurally and functionally defective sperm become surface ubiquitinated by epididymal epithelial cells. a certain portion of ubiquitin -labeled sperm is phagocytosed and the remaining is ejaculated .hence ubiquitin on the sperm surface could be a good marker of semen quality control in men. the aim of present study is to purify ubiquitin from packed blood cells , to produce and purify antiubiquitin antibodies,to design an immunofluorescence assy for detection of defective sperm, to compare the percentage of ubiquitinated sperm in oligoasthenotertozoospermia and normozoospermia and finally to determine correlations between sperm parameters and sperm ubiquitination. p. vakalopoulou, ch. anastasopoulos, g. stavropoulos, s. yiannis c-terminal analogues of substance p (sp) have been studied for their ability to prevent tumor growth or the proliferation of several cancer cell lines. the incorporation of damino acids into the sequence of sp and n-methylation of peptide bonds have shown to protect sp from the action of plasma and tissue peptidases. aiming to design and prepare more potential antagonist of cancer cells proliferation and taking into account that all the metabolites of the c-terminal hexapeptide analogue [arg6, d-trp7,9, mephe8]sp6-11 (antagonist g) possess the n-me group and d-trp residue, we proceeded to the synthesis of peptoid-peptide hybrids. they are oligomeric peptido-mimetics containing the residue [-ν(bzl)-ch2-co-]=(nphe). the incorporation of n-substituted glycine in peptide chains has been proved to improve their stability against proteases and give biologically active peptides. thus, the tetrapeptoid-peptide hybrids h-arg1-d-trp2-nphe3-d-trp4-oh and h-d-trp1-nphe2-d-trp3-leu4-oh, corresponding to metabolites of antagonist g and also the hexapeptoid-peptide hybrids glp1-d-trp2-νphe3-d-trp4-leu5-glu(obzl)6-νη2 and glp1-d-trp2-νphe3-d-trp4-leu5-glu(obzl)6-oh have been synthesized. the latter have incorporated the amino acid residues glp at the n-terminal and glu(obzl) at the c-terminal of the analogue, which have shown to give to the analogues increased resistance and biological activity. all the products were purified (hplc), identified (esi-ms) and set about for study their biological properties and activity against cancer cells proliferation. a chemokine receptor cxcr4 has multiple critical functions in normal and pathologic physiology. cxcr4 is a gpcr that transduces signals of its endogenous ligand, cxcl12 (stromal cell-derived factor-1, sdf-1). the cxcl12-cxcr4 axis plays an important role in the migration of progenitors during embryologic development of the cardiovascular, hemopoietic, central nervous systems and so on. this axis has recently been proven to be involved in several problematic diseases, including hiv infection, cancer cell metastasis, leukemia cell progression, rheumatoid arthritis (ra) and pulmonary fibrosis. thus, cxcr4 is a great therapeutic target to overcome the above diseases. fourteen-mer peptides, t140 and its analogs, were previously found to be specific cxcr4 antagonists that were identified as hiv-entry inhibitors, anti-cancer-metastatic agents, anti-chronic lymphocytic/acute lymphoblastic leukemia agents and anti-ra agents. cyclic pentapeptides, such as fc131 [cyclo(d-tyr-arg-arg-l-3-(2-naphthyl)alanine-gly)], were previously found as cxcr4 antagonist leads based on pharmacophores of t140. in this symposium, we would like to report the development of low molecular weight cxcr4 antagonists involving fc131 analogs and other compounds with different scaffolds including leaner-type structures. erythropoietin (epo) controls the proliferation and differentiation of red blood cells. it activates epor by inducing dimerization and reorientation of two receptor chains. peptides mimicking the action of epo, epo mimetic peptides (emp), have been discovered by phage display, interacting with the receptor on the active site and competing with the hormone [1] . another peptide, epor derived peptide (erp), was reported to activate the receptor through an alternative site distant from the hormone binding site, and to have synergic action with epo [2] . we report the design of new synthetic epo-r agonists by dimerization of active peptides. pegbased polyamide linkers of precise length were used to link the molecules, using oxime chemistry [3] . these peptides include emps that have been homo-dimerized through their n-or c-terminus. a hetero-dimer of one emp and one erp peptides was also created. biological characterization of the molecules is currently under investigation. [ the envelope spike (s) glycoprotein of the severe acute respiratory syndrome associated coronavirus (sars-cov) mediates the entry of the virus into target cells. recent studies point out to a cell entry mechanism of this virus similar to other enveloped viruses, such as hiv-1. as it happens with other viruses peptidic fusion inhibitors, sars-cov s protein hr2 derived peptides are potential therapeutic drugs against the virus. it is believed that hr2 peptides block the six-helix bundle formation, a key structure in the viral fusion, by interacting with the hr1 region. it is a matter of discussion if the hiv-1 gp41 hr2 derived peptide t20 (enfuvirtide) could be a possible sars-cov inhibitor given the similarities between the two viruses. we used fluorescence spectroscopy techniques to test the possibility of interaction between both t20 (hiv-1 gp41 hr2 derived peptide) and t-1249 with s protein hr1 and hr2 derived peptides. our biophysical data show a significant interaction between a sars-cov hr1 derived peptide and t20. however the interaction is only moderate (kb = (1.1±0.3) × 105 m-1). this finding shows that the reasoning behind the hypothesis that t20, already approved for clinical application in aids treatment, could inhibit the fusion of sars-cov with target cells is correct but the effect may not be strong enough for application. [1] were used to investigate the structure, dynamics and thermodynamics of the known complex between erythropoietin mimetic peptides (emp) and erythropoietin receptor (epor). with gromacs 3.2 bioinformatics software, we have obtained from the known emps about the key functional amino acids required for effective epo mimetic action. then we systematically altered the amino acids in those peptides, and simulated the complex to observe the differences between the altered peptides with the original ones. based on these results, we designed new emps of potential significance. in order to fast identify the mimetic action of these new peptides, we synthesized these peptides and labeled the epor binding peptide (ebp) with quantum dots [2] , to study the binding of these new emps to epor. our results illustrate a principle for fast identifying receptor-specific sites importance for receptor internalization, and for enhancing sensitivity to hormones and other agonists. blood vessel formation largely contributes to the pathogenesis of numerous diseases, including ischemia and cancer [1] [2] . in this regard therapeutic strategies aim to stimulate vascular growth in ischemic tissues and suppress their formation in pathologies like in tumour and diabetic retinopathy. placental growth factor (plgf), an homolog of vascular endothelial growth factor (vegf), (42% amino acid sequence identity), stimulates angiogenesis and collateral growth in ischemic heart and limb. whereas vegf exerts it biological function through the binding to both vegf receptor-1 (vegfr-1or flt1) and vegfr-2 (or kdr) plgf binds specifically to flt1. the complex plgf/flt1 constitutes a potential candidate for therapeutic modulation of angiogenesis and inflammation [3] . the binding between plgf and flt-1 has multipunctual features [4] and potential antagonist must have a sufficient molecular surface to spatially distant contact points. we have used an elisa-like screening assay to select antagonists of plgf/flt-1 complex from a large random library of tetrameric unnatural peptides (complexity: 3^30=27.000 molecules) identifying two active molecules with an about 10 m ic50. the relative stability of identified peptides were assessed in human serum and their inhibitory properties were tested in a capillary-like tube formation assay performed with human umbilical vein endothelial cells (huvec the αvβ3 integrin is a cell adhesion receptor involved in angiogenesis and tumor cell invasion. the tripeptide motif rgd is the αvβ3 minimal recognition sequence and many rgd-containing peptides have been investigated as radiopharmaceuticals for targeting angiogenesis and tumor metastatic phenotype. since rgd sequence binds also to other integrins, the aim of the present study was to develop and characterize a selective αvβ3 ligand suitable for imaging. a novel peptide containing the rgd loop covalently linked to an echistatin domain (crgdechi) was designed, synthesized and then tested for selective binding to αvβ3 integrin. a panel of peptides were used for comparison. adhesion assays showed that the novel peptide was able to inhibit adhesion of αvβ3 overexpressing cells but not αiibβ3 and αvβ5 overexpressing clones. in conclusion the novel peptide showed a high affinity and specificity for αvβ3 integrin. the design of new molecules, based on the lead compound presented here, is currently ongoing with the aim at developing novel anticancer drugs and/or new class of diagnostic noninvasive tracers as suitable tools for αvβ3 -targeted therapy and imaging. background and aims: short peptides like leu-pro-phe-phe-asp (lpffd) and leu-pro-tyr-phe-asp-amide (lpyfda) can influence the structure and aggregation of ß-amyloid peptides. soto's pentapeptide lpffd has been published as a ß-sheet breaker (bsb). it is necessary to gain more information about the nature of the interaction of aß and the pentapeptides mentioned above for the understanding of their action and for the possible development of future therapeutic agents. methods: in this study radioligand binding assay, diffusion ordered nmr spectroscopy, dynamic light scattering, circular dichroism and ft-infrared spectroscopy was used. results: it was shown by radioligand binding assay and diffusion ordered nmr spectroscopy that both pentapeptides bind to aggregated aß. dynamic light scattering, circular dichroism and ft-infrared spectroscopy revealed, that after the treatment of the aß with the pentapeptides aß fibrils are still present. conclusion: both peptide can bind to aß and can cause small conformational changes of aß, however, they cannot prevent completely the formation of aß fibrils in 50-100 micromolar concentration using 1:1 molar ratio of aß and the bsb peptide. peptide arrays are convenient tools for the analysis of antibodies, protein binding domains and to address other biological questions. here we present a new method to produce identical copies of arrays on microscope slides. the peptides are synthesized on modified cellulose-discs, using a variation of the spot-method introduced by ronald frank more than 10 years ago [1] . the new array format overcomes several limitations of the spot-method, e.g. the low throughput with only one copy of the library and the large sample volumes that are needed for membrane incubations. for the presented arrays modified cellulose discs with covalently bound peptides are dissolved after synthesis. the resulting solutions can be spotted onto glass slides by conventional spotting techniques. three dimensional layers of cellulosepeptide molecules are formed on the surface of the supports used for spotting. a virtually unlimited number of identical arrays can be printed and assays are performed with a sample volume of 100 µl or less. as application example we show mapping experiments of the streptavidin recognition site with a peptide library containing histidine-proline motives. because of the much higher peptide loading compared to conventional arrays, the formed 3-dimensional structure might be superior for protein-interaction studies with even low binding constants. [ the interaction between the cap binding protein, eukaryotic initiation factor (eif) 4e, and the scaffolding protein eif4g is critical for the formation of the heterotrimeric eif4f translation initiation (ti) complex (eif4e/eif4g/eif4a). elevated levels of eif4e and eif4g found in several human solid tumor cancers and the induction of malignant transformation in animal models by overexpression of eif4e and the reversal of this phenotype by treatment with anti-sense rna, suggest the importance of the eif4e/eif4g interaction in the excessive translation of oncogenic proteins. eif4g binds to eif4e through a conserved eif4e-binding motif yx4l (non-specified (x) and hydrophobic ( ) amino acids) that interacts with an hydrophobic hot spot on eif4e. we report here the identification of a putative eif4e anionic exosite that is distinct from the hot spot and contributes to the binding of eif4g-derived ligands. our strategy focuses on in situ eif4e-templated click reaction-mediated assembly of hybrids comprised from an anchoring minimal eif4g-derived peptide fragment, which binds to the hot spot, and a series of complementing positively charged fragments targeting the anionic exosite. we synthesized a training set of [1, 2, 3] triazole-containing hybrid peptides that are potent inhibitors of eif4e/eif4g interaction. moreover, we achieved in situ eif4e-templated assembly of these hybrids from the corresponding fragments via click reaction in the absence of cu(i) catalysis. as such, we demonstrate a proof-of-concept for a new paradigm in the development of inhibitors of protein-protein interaction merging click reaction with fragment-based and in situ target-templated approaches. goodpasture disease is an autoimmune pathology caused by the accumulation of reactive autoantibodies against the alpha-3 of collagen iv. goodpasture antigenbinding protein (gpbp) is a ser/thr protein kinase that phosphorylates the alpha-3 chain and might be important in human autoimmune pathogenesis [1] . we are carrying out in our laboratories the biophysical and functional characterization of gpbp protein. in the presence of some proteins and at specific experimental conditions, gpbp participates in structurally ordered intra-and inter-protein aggregation processes. structure prediction programs identify four different domains for gpbp: an n-terminal domain showing pleckstrin homology (ph domain); a central domain with high tendency to form coiled-coils; a domain with ww features; and a c-terminal start domain ('star-related lipid-transfer'). using the tango algorithm [2] , we have identified several aminoacid sequences in the gpbp start domain of vertebrates with high tendency to participate in protein aggregation. in this work we present the synthesis and structural characterization of a collection of peptides derived from the sequences described above. we recently developed a combinatorial library screening protocol to identify hpq-containing cyclopeptides that bind streptavidin more tightly than its linear analogues. the relative affinities in ic50 of these structurally constrained ligands and its linear counterparts were measured by a captured enzyme-linked immunosorbent assay. however, their intrinsic binding kinetics remained to be elucidated. in this work, surface plasmon resonance (spr) was employed to directly determine the kinetics and thermodynamics of the ligands binding to a streptavidin chip. solid-phase peptide synthesis was carried out using standard fmoc chemistry. spr experiments were carried out using biacore3000 optical biosensor. streptavidin was immobilized onto a cm5 sensor chip using the standard amine coupling procedure. the equilibrium dissociation constants and kinetic on/off rates of n-to-side chain and n-to-c cyclopeptides were deduced by scatchard analysis and computational simulation, respectively. it was found that both cyclopeptides exhibited similar binding kinetics and bound streptavidin far more avidly than its linear form (1000-fold). in addition, the reversed (qph) linear and cyclic peptidyl ligands were hardly recognized by streptavidin. not only the binding specificity was distinguished qualitatively, but also the entropic advantage acquired by the pre-organized conformation over its linear analogues was demonstrated quantitatively by spr in this study. the mutation of tumour suppressor genes in the progression of cancer is well characterized. for example, p53 is found to be mutated in approximately 50% of cancers and the loss of this proteins activity has been shown to lead to the deregulation of cell growth and apoptosis. the potential of peptide aptamers to inhibit protein/protein interactions in a highly specific manner makes them very attractive as research reagents or as target validation tools in anti-cancer drug discovery. more interestingly, these molecules have the potentially to inhibit the activity of proteins which are key regulators of cancer cell growth and therefore could act as synthetic tumour suppressor proteins. we used peptides based on known protein/protein interactions, as well as peptides isolated using display technologies, for the design of protein aptamers that were used to analyze pathways critical in controlling cancer cell growth. a range of scaffolds were used to present these peptides in an effort to optimize the peptides activities. data relating to the activity of these peptide aptamers in vitro as well as in cellular systems will be discussed. the cyclic undecapeptide urotensin ii (u-ii) is the endogenous agonist for the u-ii receptor (ut), a gq coupled gpcr. current views suggest that binding by agonist, but not antagonist, leads to induction of stabilization of an active receptor conformation. we have previously probed the interactions of urotensin ii with rat ut (rut) using a series of photolabile u-ii analogues containing p-benzoyl-l-phenylalanine (bpa). it was found that the c-jun n-terminal kinases (jnks) are important mitogen-activated protein kinases. these ser/thr protein kinases are activated by various growth factors, cytokines, and cellular stresses. jnks have been shown to play a key role in phosphorylation of proteins in signal transduction of different diseases including cancer, neurodegenerative, cardiovascular, and inflammatory diseases. therefore, these enzymes are considered as important therapeutic target proteins. the interactions of jnk with peptides are of special interest for development of novel specific atp-noncompetitive inhibitors. interactions of this kinase and its mutants with various substrates were demonstrated in vivo using yeast ras-recruitment system. bioinformatical tools have been developed to predict optimized binding peptides as well as to correlate sequence position and amino acid with binding effiency to extract binding determinants. biomolecular interaction analysis have been performed for selected peptide sequences using surface plasmon resonance (spr) technology. real time measurements of the binding of peptides to the different isoforms jnk2 and jnk3 resulted in the determination of affinities as well as kinetic constants for association and dissociation. experimental results and their bioinformatic analysis are discussed with respect to critical features of potential atpnoncompetitive inhibitors. the b-domain of is one of the five nearly homologous domains of staphylococcal protein a. this domain contains three alpha-helices which are assembled in an anti parallel three-helix bundle. the b-domain binds the fc region of mammalian immunoglobulins through the n-terminal fragment that contains two alpha-helices. the c-terminal helix does not interact with fc but it is necessary for the correct folding and immunoglobulin recognition of the b-domain. to search for new peptide analogues of the c-terminal helix that bind the n-terminal fragment, a ¨one-bead one-compound¨ library of 300 peptides was designed based on the sequence of the c-terminal helix. active peptides were obtained after incubation of the library with the n-terminal fragment and rabbit immunoglobulin g labelled with fluorescein. new peptides were found and their sequences identified by maldi tof-tof mass spectrometry. the synthesis of the two most active peptides was carried out and the binding with the n-terminal fragment was confirmed by cd spectroscopy. the nterminal fragment peptide showed an increase in helicity when the c-terminal wild type peptide or some analogues were present in solution. the complete domains with the c-terminal fragment mutations were synthesized and structurally characterized by cd and nmr spectroscopy. the wild type and the new mutants adopt predominantly an alpha-helical structure. the interaction between rabbit immunoglobulin g and the wild type b-domain and the new analogues was investigated using surface plasmon resonance. although compared with the wild type, the mutants exhibited different kinetics, they were able to bind the immunoglobulin with high affinity. a. jaśkiewicz, e. bulak, h. miecznikowska, k. rolka sfti-1, a strong trypsin inhibitor, was isolated in 1999 from seeds of sunflower. it is homodetic 14-amino-acid-residue peptide containing a disulfide bridge. because of its small size and strong trypsin inhibitory activity, this inhibitor became an interesting model for studying enzyme -inhibitor interactions. sfti-1 possesses one reactive site located at the lys5-thr6 peptide bond and therefore is able to interact with the enzyme in a 1:1 stoichiometry. in this report we describe chemical synthesis and kinetic studies of a series of sfti-1 analogues containing double sequences of the wild inhibitor. their structures contain combinations of disulfide bridges and/or head-to-tail cyclization. each of these analogues contains two trypsin-specific reactive sites. we expect that kinetic studies should answer the question whether such dimeric analogues are able to interact simultaneously with more then one trypsin molecule and how this fact affects their inhibitory potency. in addition, we alsopresenttwo analogues in which we substituted the disulfide bridge with a carbonyl one. since carbonyl bridge has not been previously introduced into molecules proteinase inhibitors, we decided to check its impact on the activity and proteolytic stability of such modified analogues. ubiquitination, the covalent attachment of one or multiple polymerized ubiquitins is a post-translational modification of proteins, which has manifold functions. it mainly determines the protein for degradation, but also activation, deactivation or substrate alteration. due to its ubiquitinous distribution in all eucarionts no high-affinity antibodies could be originated. highaffinity ligand peptides are of interest to study ubiqutination. based on bioinformatical considerations and investigations of ubiquitin-interacting proteins short peptide sequences were selected. by using a peptide array specific ubiquitin binding was monitored and quantified with label free detection based on reflectometric interference spectroscopy (rifs). the results from rifs were confirmed by detection of binding in solution with fluorescence correlation spectroscopy (fcs) using carboxyfluorescein and s0387-labelled peptide amides. binding constants were determined by isothermal calorimetry (itc) and rifs. finally 1h,15n-nmr chemical shift analyses of the peptides with the highest affinity were carried out, which allowed the localisation of the interaction site of ubiquitin with the peptide the results from all four methods correlated very good. they showed fast equilibria within 30 s and binding constants down to the low micromolar range. nmr results revealed hints for discrimination possibilities between lys48 and lys63 polymerized ubiquitins. ( 101f is a potent neutralizing mab that binds the human respiratory syncytial virus (hrsv) f protein and is a promising candidate for clinical development. the majority of neutralizing antibodies to hrsv f protein map to two regions of the protein designated site ii and site iv,v,vi. to further characterize the 101f epitope, we employed a trypsin digestion of a hrsv f protein-101f mab complex, followed by mass spectrometry analysis of the resulting recovered mab bound peptide. one peptide at m/z 3330 was captured by the 101f mab. sequence assignment was based upon mass and matched with the database from a virtual digest. this peptide was assigned as residues 420-445 [tkctasnknrgiiktfsngcdyvsnk] of the hrsv f protein which spans antigenic site iv,v,vi. to further delineate the epitope, the binding of 101f mab to a series of peptides corresponding to antigenic site iv,v,vi in the hrsv f protein was determined. based on the peptide elisa data, the 101f-binding region could be reduced to 422-436 sequence [ctasnknrgiiktfs] . as demonstrated by the substitution analysis, r429 and k433 significantly contribute to epitope binding, but another positively charged residue, k427 makes a minor contribution to the binding. both, the peptide elisa and proteolytic digestion of the mab-antigen complex approaches identified the same region of hrsv f protein as being critical for the binding of 101f. furthermore, these data confirm the results obtained using complementing genetic approaches using a panel of mutations in recombinantly expressed f protein and selection of antibody escape virus mutants (data not presented). the recently identified uracil-dna specific nuclease (ude) is the first representative of a new family of nucleases. the protein sequence has no detectable homology to other proteins except a group of sequences present in genomes of other pupating insects (vertessy et al, submitted). to analyze the physiological function of this protein, peptide conjugates were prepared to serve as synthetic antigens for the generation of antibodies against isoforms of dutpase, an enzyme inherently involved in preventing the synthesis of uracil-dna [1, 2] . we used poly[lys(seri-dl-alam)] (sak) as a synthetic branched polypeptide [3] or bovine serum albumin (bsa) as a natural macromolecular carrier. peptides were prepared by solid phase method utilizing syro2000 (mulltisyntech gmbh, germany) peptide synthesizer, using fmoc chemistry and dipci/hobt-mediated coupling on rink-amid mbha resin. a c-terminal cys(acm) was added to the native sequences for incorporation of sh group into the peptide. in case of sak choroacetylated polypeptide was conjugated with sh-peptide to form thioether linkage. the maleimidobenzoyil-n-hydroxyszukcinimid (mbs) derivative of bsa was used to introduce the peptide into the macromolecule. antibodies have been developed as diagnostic tools and therapeutics for many different diseases. however, the isolation and preparation of intact specific antibodies is often very tedious or even unfeasible. recent studies have shown that single paratope peptides might be well capable to mimic corresponding antigen ligands [1, 2] , suggesting that paratope peptides from a native antibody might have many advantages, e.g. for molecular vaccine design and targeting. we have developed a new method for identification of paratope-containing peptides by proteolytic affinity-proteome analysis in combination with high resolution fticr-mass spectrometry (fticr-ms) [3] . in the present study we used hen eggwhite lysozyme (hel) and a polyclonal rabbit anti-lysozyme antibody (helpab) as a model system. the direct determination of paratope peptides was obtained by selective binding of a dtt-cleavage mixture of the anti-lysozyme antibody to immobilised hel, followed by proteolytic digestion of the antibody-antigen complex (paratope excision, parexprot). two specific paratope peptides were identified by maldi-fticr-ms, and the corresponding peptide sequences were identified by database search within a 1-2 ppm threshold. additionally, the identified paratope peptides were synthesised and characterised by affinity mass spectrometry, which ascertained their full binding specificity to lysozyme. the propeptide blocks the active site of inactive zymogen of cathepsin d and is cleaved off during maturation. we have designed a set of peptidic fragments derived from the propeptide structure and evaluated their inhibitory potency against mature cathepsin d using kinetic activity assay. the mapping localized two segments in the propeptide involved in the inhibitory interaction with the enzyme core: n-terminus of propeptide plays a major role and the active site anchor plays a minor role according to their respective ki values. in addition, a fragment derived from the mature n-terminus of cathepsin d displayed inhibition, which supports its proposed regulatory role. the mechanism of interaction of both propeptide segments was characterized by the mode of inhibition and by spatial modeling of propeptide in cathepsin d zymogen. using fluorescence polarization measurements, kd in nanomolar range was determined for the n-terminal propeptide segment. the inhibitory potency of the active site anchor segment was modulated by ala38val mutation that was reported to be associated with cathepsin d pathology. . by comparing the resulting low-energy conformations using different sets of atoms, specific conformational features common only to the high/medium affinity compounds were identified. they included the spatial arrangement of the three most important pharmacophoric side chains tyr2, arg4, and nal5 as well as the orientation of the xaa3-arg4 amide bond, which together represent a "minimalistic" 3d pharmacophore model for binding of the cyclopentapeptide antagonists to cxcr4. this model rationalizes the data for the cyclopentapeptides as well as for the peptidomimetic cxcr4 antagonist krh-1636. automated docking of the pharmacophore model to the 3d structure of the tm region of cxcr4 revealed that the pharmacophoric groups of the cyclopentapeptide ligands were involved in favorable interactions with their counterparts in cxcr4. for instance, the hydroxyl group of tyr2 formed a hydrogen bond with lys38, the guanidino group of arg4 formed a salt bridge with glu288, and the backbone carbonyl of xaa3-arg4 formed a hydrogen bond with lys282. this finding gives additional support for the suggested 3d pharmacophore model, and also provides opportunities for rational design of cxcr4 mutants to map potential contacts with peptide ligands. with the successful completion of the human genome project, the next challenge is to assimilate enormous amount of genetic information generated and to assign functions to a large number of proteins encoded. although the dna chip technology to detect the abundance of mrnas has been established, it is known that the abundance of mrnas and proteins does not correlate. thus, protein detection methods for reproducible and quantitative investigation of protein networks are strongly required. we attempted to establish a novel protein detection system based on a fluorescent measurement that does not require labeling of target molecules and preparation of secondary antibodies. we focused on a steric hindrance caused by the interaction between a target protein and a specific capture agent. when a target protein interacts with a specific capture agent immobilized on solid surface, we assumed that a steric hindrance in the vicinity of a capture agent increases. in order to detect the differences in the steric hindrance, we utilized a fluorescent system with the staudinger reaction. this reaction is a chemical ligation between a phosphine and an azide group. these two functionalities are unreactive with protein surfaces under biological conditions. we incorporated an azide group into an immobilized capture agent and investigated the efficiency in the staudinger reaction between the azide and an external triphenyl phosphine derivative. it was found that a target protein bound to the capture agent immobilized onto the solid support interferes with the efficiency in the staudinger reaction. the major histocompatibility complex (mhc) has a crucial role to initiate the immune response via the binding of the peptide fragments (epitopes) of foreign antigens and their presentation to the t-cell receptors (tcr). the co-receptor molecule cd4 enhances the binding between tcr and mhc ii. small molecules that mimic surfaces of mhc-ii may lead to blockage of the autoimmune response and the development of drugs for immunotherapy. hla-dqa1*0501/dqb1*0201 (dq2) and hla-dqa1*0501/dqb1*0301 (dq7) are highly correlated to autoimmune diseases as sjogren syndrome (ss) and systemic lupus erythematosus (sle). the non polymorphic β regions of the modelled hla-dq7, which are exposed to the solvent and may disrupt the interaction of dq7 with cd4+ t lymphocytes were determinated using the getarea program. it was found that the regions 133-140 (arg-asn-asp-gln-glu-glu-thr-thr) and 59-66 (glu-tyr-trp-asn-ser-gln-lys-glu) display the highest solvent accessibility. peptide analogs of these regions were synthesized, by the fmoc/otbu solid phase strategy, purified by rp-hplc and characterized by mass spectrometry esi-ms. the dimeric analogs of the peptides, designed to mimic the superdimeric nature of the immunosuppressory fragments of hla class ii molecules were also synthesized and investigated. conformational studies were performed with cd spectroscopy and biological experiments are in progress. background and aims: aggregates of β-amyloid peptide (aβ) play central role in the etiopathology of alzheimer's disease (ad). short peptides like c. soto's pentapeptide lpffd and lpyfd-amide synthesized in our laboratory are neuroprotective agents against aβ assemblies both in vitro and in vivo. however, the mechanism of their neuroprotective effect has not yet been fully understood. methods: transmission electron microscopy (tem), cd, ft-ir, diffusion ordered nmr spectroscopy, dynamic light scattering, and radioligand binding assays were used. results: all the methods applied showed that the pentapeptides mentioned above do not break the fibrillar structure of aβ, that is these molecules are not real β-sheet breakers (bsb). the pentapeptides bind to aβ fibrils and cause small structural changes by intercalating into the aβ assemblies. fibrils of aβ survive one week treatment with the pentapeptides using them in 2 to 5-time molar excess. conclusion: all the results in our laboratory show that the short peptides have long-term interaction on aβ-assemblies. in the first step they bind tightly to the aβ surface and prevent further interaction of aβ fibrils with the neuronal membranes. after this step the short peptides can be built into the structure of aβ-assemblies with intercalation causing a less ordered β-conformation. proteolytic enzymes (neprilisin, ide) could cleave and hydrolyze aβ peptides after this structural change, therefore the short peptides are good drug candidates for the treatment of ad. cellular processes in normal and pathogenic cell states are regulated by external stimuli via complex networks of catalytic and non-catalytic protein-protein interactions. we have developed methodology for the synthetic variation of peptides and peptidomimetics using polymer reagents including linker reagents enabling polymer-supported cacylations. [1] in combination with the virtual screening of protein subsites, we have demonstrated the application of the novel synthetic methods to inhibitor optimization for various proteases including plasmepsin ii, hiv protease, and sars coronavirus main protease. [2, 3] moreover, multivalent peptide polymers have been developed for the intracellular targeting of proteins. [4] this methodology was now extended to the inhibition of peptide-protein interactions by small molecules. for this purpose, we have composed a library of 20,000 small molecules by algorithmic searching of a database of bioactive molecules with virtually designed substructures (fragments). high throughput assays were developed on the basis of fluorescence and fluorescence polarization detection. despite the scepticism regarding the inhibition of protein-protein interactions with small molecules, efficient hit molecules have been developed for several intracellular targets and were subjected to synthetic variation and cellular follow-up assays. the essential event in platelet adhesion to the blood vessel wall after injury or in thrombosis is the binding to sub-endothelial collagen of plasma von willebrand factor (vwf), a protein which interacts transiently with platelet glycoprotein (gp) ibalpha , slowing circulating platelets to facilitate their firm adhesion through other collagen receptors, e.g. integrin alpha2beta1 and gpvi. to locate thevwf-binding site in collagen iii; we synthesized 57 overlapping triple-helical peptides which comprise the whole native sequence of collagen iii . peptide #23 (gpogpsgprgqogvmgfogpkgnd (o is hydroxyproline)) alone bound vwf, with an affinity comparable to that of native collagen iii. immobilized peptide #23 supported platelet adhesion under static and flow conditions, processes blocked by an antibody which prevents the vwf a3 domain from binding full-length collagen. truncated and alaninesubstituted triple-helical peptides derived from #23 either strongly interacted with both vwf and platelets, or lacked both vwf and platelet binding. thus, we identified the sequence rgqogvmgf as the minimal vwf-binding sequence in collagen iii. the present work completes our understanding of the collagen-vwf interaction, providing information on crucial sequences in collagen that perfectly complements our existing knowledge of the collagen-binding site in vwf and may assist in targeting the collagen-vwf interaction for therapeutic purposes solid phase assay systems such as enzyme-linked immunosorbent assay (elisa), surface plasmon resonance (spr), and overlay gels are used to study processes of protein-protein and protein-peptide interactions. the common principle of all these methods is that they monitor the binding between soluble and surfaceimmobilized molecules. following the use of bovine serum albumin (bsa)-peptide conjugates or isolated synthetic peptides and the above-mentioned solid phase assay systems, we were able to demonstrate that positively charged peptides, which would be expected to repulse each other, can interact with each other. both the elisa and spr methods showed that the binding process reached saturation with kd values ranging between 1 and 14 nm. no interaction was observed between bsa conjugates bearing positively charged peptides and conjugates bearing negatively charged peptides or with pure bsa molecules, strengthening the view that interaction occurs only between positively charged peptides. however, interactions between the same peptides were not observed in solution when was monitored by nuclear magnetic resonance (nmr) or by native gel electrophoresis. thus, it appears that for positively charged molecules to interact one of the binding partners must be immobilized to a surface, a process that may lead to the exposure of otherwise masked groups or atoms. the relevance of our findings for the use of solid phase assay systems to study interactions between biomolecules will be discussed. the hematopoietic progenitor kinase 1 (hpk1), a mammalian hematopoiesis-specific ste20 kinase, contains a cluster of four proline-rich sequences called p1, p2, p3 and p4 located after the kinase domain. these pro-rich regions play an important role in the interactions of this kinase with different adapter proteins. previous studies showed that p1, which contains the canonical pxxpxr motif, and p2 and p4 with the canonical pxxpxk motif interact with the c-terminal sh3 domain of hematopoietic lineage cell-specific protein 1 (hs1) even if with different affinity. hs1 protein shares a high amino acid sequence and structural similarity to cortactin although their functions differ considerably. here we report the results of our investigation on the interaction between the c-terminal sh3 domain of cortactin and the four proline-rich motifs of hpk1. these interactions were analyzed by non-immobilized ligand interaction assay by circular dichroism (nilia-cd). upon peptide addition, the binding was monitored by the cd changes of the trp side-chains of the conjugate gst-sh3cort. the dissociation constants kd were determined analyzing the cd data at 294 nm using a nonlinear regression method. the results demonstrate that gst-sh3cort displays an affinity binding higher than that found with the corresponding hs1 domain and that the four hpk1 pro-rich regions are not equivalent. p2 appears to bind with the highest affinity (kd=0.5 µm), followed by p1 (kd=10 µm) and p4 (kd=33 µm), whilst p3 does not interact at all. the generation of a fibrin clot is mediated by the regulated activation of a series of serine proteases and their cofactors. factor viii in its activated form, fviiia, acts as a cofactor to the serine protease fixa, in the conversion of the zymogen fx to the active enzyme fxa. both fviii and fix are essential for normal coagulation, deficiencies of either are associated with the bleeding diatheses hemophilia a and b, respectively. the role of fviiia is to bind factor ixa, generating the phospholipid-dependent intrinsic factor xase complex. at least two interactive sites have been identified for the enzyme-cofactor interaction. the ser558-gln565 region within the a2 subunit has been shown to be crucial for viiia-ixa interaction. in an attempt to study this interaction, we synthesized a series of peptides of 558-565 loop of the a2 subunit. the syntheses of these peptides were carried out by using spps and fmoc/but methodology. the synthesized compounds were purified by rp-hplc and lyophilized to give fluffy solid, identified by ft-ir, nmr and es-ms spectra. these compounds were tested for inhibitory activity on human platelet aggregation in vitro, by adding common aggregation reagents to citrated platelet rich plasma (prp). the aggregation was determined using a dual channel electronic aggregometer by recording the light transmission. and 120 ci/mmol, respectively. both tritiated aβ peptides were used in cat brain( in vivo) experiments and it was found that the peptide aggregates enter the neurons within 30 min (electronmicroscopic autoradiography). this transport is most probably an endocytotic pathway. aβ aggregates could interact also with cytoplasmic proteins such as 3-phosphoglyceraldehyde dehydrogenase etc. we suppose that aβ assemblies can interact both with membrane receptors (nmda, ampa, ach) and with cytoplasmic proteins triggering neuronal dysfunction and death. background and aims: ww domains are the shortest known protein domains and contain a stable three-stranded b-sheet, which presents the binding site for prolinerich ligands. this interaction is mediated by hydrophobic interactions between aromatic and hydrophobic residues of the domain, and the polyproline core of the ligand. as part of our ongoing efforts aimed at synthetically mimicking conformationally defined protein binding sites (1), we have designed and synthesized linear and cyclic peptides covering the binding site of the ww domain of human yes-associated protein (hyap-ww), whose structure in complex with a proline-rich ligand had been solved by nmr spectroscopy (2) . methods: peptides were synthesized by spps, purified by hplc, and characterized by 2d-nmr spectroscopy, as well as by molecular dynamics calculations. affinities of the peptides to a hyap-ww ligand were determined in direct and competitive binding assays. results and conclusions: a cyclic peptide covering the sequence stretch of hyap-ww that contains its primary contact residues for proline-rich ligands, was found to compete with the domain for binding to a hyap-ww ligand. long-ranging noes identified in the nmr spectra of this peptide indicate a conformation, in which sequentially distant residues are brought into spatial proximity, likely through formation of a beta-sheet. these result demonstrate the feasibility of functional, as well as structural, mimicry of conformationally defined protein binding sites through synthetic peptides. the rockefeller university, new york, ny, usa integrins constitute a family of transmembrane cell surface receptors. they are involved in cell-cell and cell-extracellular matrix interactions. thus, they participate in many physiological and pathophysiological processes and are of crucial importance for the living organism. integrins possess two non-covalently bound subunits, &alpha; and &beta;, that jointly participate in ligand binding. these dimeric proteins show very high specificity in recognition of natural ligands. for example, α4β1 integrin recognizes vcam-1 (vascular cell adhesion molecule 1) and fibronectin through binding amino acid motifs tqidspln and ldv, respectively. on the other hand, fibronectin is a classical ligand for α5β1 integrin with the recognition motif rgd. as shown, identification of the integrin ligands occurs through small recognition amino acid sequences (mostly tripeptides). thus, small cyclic peptides possessing a recognition motif in the appropriate three-dimensional conformation are able to interfere with the integrin-ligand interactions and act as inhibitors. the aim of this investigation is the characterisation of small cyclic peptides containing the rgd motif and the determination of the selectivity and specificity of these inhibitors. two new pentapeptides with 3-amino-cyclopropane-1,2-dicarboxylic acid monomethyl ester ((+/-)acc) were synthesized and tested. peptides were characterized in biological assays with living cells (k562 and wm115) and in surface plasmon resonance binding studies. experiments have shown that cyclic peptide cyclo-(arg-gly-asp-(+)acc-val) is a very potent inhibitor (ic50-value in nm range) of interaction between vitronectin and αvβ3 or αvβ5 integrins. when preparing biotin-labelled peptides as ligands for avidin-based assays, it is chemically most expedient to locate the biotin label on the n-terminal group of the peptide. this is done without any regard to how this may affect peptide-target interactions, biotin-avidin binding, and the solubility properties of the resultant peptide. in many instances, the products are poorly soluble, have little biological activity, and poor affinity for avidin. problems can also arise during the synthesis of such nterminally biotinylated peptides due to the poor solubility and reactivity of many of the reagents used for biotin introduction. to overcome these limitations, we have developed an extremely simple method for synthesising peptides c-terminally with biotin. peptides can now be easily prepared by standard solid phase techniques either n-or c-terminally labelled, and screened to determine the optimum presentation for the biotin. in cases studies using protein-protein interaction and kinase assays, peptides c-terminally labelled with biotin gave better sensitivity. y. yang 1 , j. eble 2 , n. sewald 1 many bacterial pathogens bind and enter eukaryotic cells to establish infection. invasin is an outer membrane protein required for efficient uptake of yersinia into m cells. invasin mediates its entry into eukaryotic cells by binding to members of β1 integrin family that lack insertion domains (i domains), such as α3β1,α4β1,α5β1,α6β1, and αvβ1. this type of peptide-protein interaction is an ideal subject for the rational design of inhibitors. the integrin binding motif consists of one loop region with a conservative asp911 residue and two synergistic regions. the aim of this project is to synthesize cyclic peptides based on the invasin binding epitope sdms. this sequence has to be positioned in a β-turn with asp in i+1 position for optimal activity of the peptide. also the arg883 and asp811 residue, which are about 27.29å and 31.54å respectively away from the asp911 residue of the sdms loop in invasin, should be investigated. peptides that mimic these recognition sites have been synthesized and tested as ligands for the integrin peptide-dna cross-linking is a very powerful tool for studying peptide-dna complexes. it transforms non-covalent complexes into covalent complexes, which renders characterization of the adduct by classical techniques (mass spectroscopy, nmr,…) much easier. the aim of our research is to develop a new method for peptide-dna cross-linking involving the incorporation of a furan moiety. the strategy is inspired by the naturally occurring process of oxidative furan ring opening by cytochrome p450. the resulting cis-butene-1,4-dial has been shown to react with amino-and sulfhydryl groups of macromolecules such as proteins and dna. in our research, dna binding peptides are modified with a furan moiety and then chemically oxidized into a reactive enal. this enal can react with dna to form a covalent peptide-dna complex. previous attempts to selectively oxidize furan modified minor groove binding peptides consisting of n-methylpyrrole building blocks failed. we are now applying the same strategy on major groove binding peptides consisting of natural amino acids. currently the oxidation conditions are being optimized so that the furan moiety undergoes selective oxidation. these optimized conditions will be applied to known dna binding peptides, in order to obtain a peptide-dna cross-link. we coupled octanoyl or palmitoyl group to the n-terminus of an analogue of sv40 nuclear localization signal (nls) peptide, sv126-133(ser128) to investigate the effect of fatty acid chain length on the conformation of the lipopeptide-antisense oligodeoxynucleotide (odn) complexes and to establish the optimal peptide/odn molar ratio (rm) for the effective delivery of odn into the cells. the odns used in this study were targeted towards either the green fluorescent protein (gfp) mrna and the junction sequence between ews and fli1 genes. the conformational change of odn at different rm values was followed by circular dichroism (cd), attenuated total reflection-fourier transform infrared (atr-ftir) spectroscopy and atomic force microscopy (afm). the sv40 peptide-mediated odn transfer into nih/3t3 cells was studied by epifluorescence microscopy. the interaction between the hiv-1 regulatory protein rev and rev responsive element (rre) of hiv-1 mrna has emerged in the last decade as an important target in antiviral therapy. the rev-rre interaction is essential for the replication of hiv. the rev protein binds to the rre site located in the env coding region of the full length viral mrna and facilities the export of the rna from the nucleus, while protecting it from the cell's splicing machinery. in the published nmr structure of the rre/rev-derived peptide complex, an -helical segment of rev binding domain recognises a specific region of rre. an approach is described to design a new class of -hairpin peptidomimetic ligands for hiv-1 rev protein, which inhibit its binding to the rre rna. a model -hairpin peptide served as a scaffold to pre-organise side chains into a geometry similar to that seen in a helical peptide. a library of peptidomimetics was prepared by grafting sequences related to the rna recognition element in rev onto a hairpin-inducing d-pro-l-pro template. the electrophoretic mobility shift assay (emsa) revealed that all of the designed peptidomimetics bind to rre and the best examples show affinities (kd) in a nanomolar range. these new ligands show a novel approach to designing rev peptidomimetics, represent interesting leads for the development of more potent hiv rre/rev inhibitors and permit more detailed studies of the mechanism of binding to rna. a. napiorkowska, a. sawula, m. olkowicz, p. mucha, p. rekowski tat (trans-activator of transcription) is the protein which controls the early phase of hiv-1 replication cycle. it is a potent viral trans-activator containing from 86 to 101 amino acid residues which binds to tar rna. the fundamental role of tat is promoting effective elongation of viral mrna (vmrna). binding of tat to tar is mediated by a 9-amino-acid, highly basic arg49-lys-lys-arg52-arg-gln-arg-arg-arg57 sequence of the arm (arginine rich motif); the key role in these interactions is played by arg52. the goal of our research was to investigate the interaction of 27-nucleotide tar rna with synthesized tat peptide analogues using capillary electrophoresis (ce), a powerful analytical technique of biochemical studies. changes in electrophoretic mobility of the tar peak are employed for monitoring tar-tat complex formation. ce experiments were performed using lpa-coated capillary and a physical gel containing buffer. native arm fragment tat(49-57)nh2, its analogues ac-tat(49-57)nh2 and ac-[lys52]tat(49-57)nh2 and analogues substituted in position 52 with alanine-, homoalanine and lysine-derived amino acids containing nucleobases (adenine, guanine, cytosine, uracil, thymine) and nucleosides (adenosine, guanosine, uridine and cytidine) in the side chain were studied. specific interactions and complex formation were observed for both the native arm peptide fragment and selected tat analogues. the research is aimed at improving our understanding of the molecular mechanism of peptide-nucleic acid interaction, as well as evaluating the usefulness of selected nucleobase-containing amino acids as point probes for investigating peptide-rna interactions. interactions between proteins and dna are important to all living organisms. the goal is to investigate the molecular recognition between dna and the transcription factor phob of e. coli on the single molecule level and to identify amino acids required for dna binding. phob is composed of a transactivation domain (amino acids 1-127) and a dna binding domain (amino acids 123-229) that binds to specific dna sequences (pho boxes) containing a tgtca sequence. [1] chemical synthesis of peptide epitopes present in the dna binding domain of phob and isolation of the whole dna binding domain of phob was performed. the protein was purified using intein mediated protein purification. an additional cysteine residue was ligated to the protein using intein mediated ligation reactions and will be used for immobilization and labeling. in single molecule force spectroscopy (afm) experiments it has been shown that both a peptide with a native phob-sequence and the recombinant protein bind to dna. competition experiments were performed to prove specific dna binding. [2] mutated peptides and proteins where strategic amino acids were replaced by alanine have also been examined to reveal the contributions of single residues to molecular recognition. the binding contribution of the proteins is determined by surface plasmon resonance, electrophoretic mobility shift assays and fluorescence correlation spectroscopy. we investigated the biophysical characteristics and the pore formation dynamics of naturally occurring and synthetic peptides forming membrane-spanning channels by using isolated rod outer segments (os) of reptilia and amphibia recorded in the whole-cell configuration. once blocked the two os endogenous conductances (the cgmp channels by light and the retinal exchanger by removing one of the transported ion species from both sides of the membrane, i.e. k+, na+ or ca2+), the os membrane resistance (rm) could be >5 gώ. therefore, any exogenous current can be studied down to the single channel level. macroscopic currents of amplitude of ~300 pa were recorded in symmetric k+ or na+ (>100 mm) and ca2+ (1 mm) from the commercially available alamethicin mixture, the synthetic alamethicin f50/5 (a major component of the natural mixture), and selected analogues applied at 1 µm concentration at -20 mv. once applied and removed the peptide, the current activates and deactivates with a time constant of about 160 ms. the synthetic analogues [glu(ome)7,18,19] and [glu(ome)18,19] produce a current of about 100 pa at 1 µm concentration, and they show a strong activation by hyperpolarization as alamethicin f50/5 itself. clear single channel events were observed when the concentration of all of the alamethicin peptides is reduced to <250 nm.<br> these results indicate that the three gln residues at positions 7, 18 and 19 of alamethicin f50/5 are not a key factor for pore formation and its conduction properties. in general, the pore assembly and disassembly are very fast and cooperative events. the translocation mechanism of penetratin (rqikiwfqnrrmkwkk) is not clear, but the involvement of cell membrane was supposed. recent studies with phospholipid model membranes have shown that penetratin interacts only with negatively charged liposomes. we aimed to analyse the effect of penetratin on liposomes composed of different phospholipids (dppc/dppg 2:8-8:2) by fluorescence spectroscopy. in the first set of experiments, liposomes labelled with fluorescent markers (dph, ans and tma-dph) were incubated with penetratin and the fluorescence polarisation was determined as a function of the temperature. in the range of 15-200 mol/mol phospholipid/penetratin ratio, no change in the transition temperature was observed indicating that penetratin has no influence on the membrane structure. next, we have analysed the interactions between phospholipids and penetratin through changes in the intrinsic fluorescence of the peptide due to the presence of two w residues in its sequence. comparing the emission spectra corresponding to penetratin in aqueous media or in presence of vesicles one can clearly appreciate a blue shift. this indicates that that tryptophan residues are mainly exposed to a hydrophobic environment. analysis of the main band shows low values of polarization suggesting a free motion of the peptide chain. on the contrary polarization measured for penetratin mixed with liposomes results in higher values. this indicates that hydrophobic residues, like trp, are inserted into the bilayer and their motion is restricted. these data suggest the presence of interation sensed strongly by trp properties. cyclopeptide antibiotic gramicidin s (gs) has antimicrobial activity against gram-positive and gram-negative bacteria and some fungi. but non-specific action of gs and its high lytic potential limits therapeutic application of gs. we attempted to elucidate in which way gs molecule could be modified to lose its haemolytic side effects. gs molecule interacts directly with membrane phospholipids due to electrostatic and hydrophobic interaction. naturally, changes in the state of a lipid bilayer cause changes in the gs molecule binding to a bilayer. we studied the effect of gs on human blood platelets and the effect of platelet membrane state on the gs-induced disaggregation of cells with the help of turbidimetric and microscopy techniques. we modified the membrane state by temperature, osmotic stress, ionizing irradiation, lipid oxidation. depending on concentration gs causes platelet shape change and activation. when added to preliminary aggregated (in response to physiological agonists -thrombin, epinephrine, adp) platelets, gs causes crumble of cells aggregates. the rate and extent of platelet disaggregation under the effect of gs non monotonously depends on temperature (range of 5-40°c) and irradiation dose (up to 200 gy). parameters of the gs interaction with membranes are determined by the mobility of membrane lipids. factors modifying the lipid bilayer change the degree and the speed of the gs interaction with platelet membranes. results obtained permit to use gs for testing the state of membrane lipids and on the other hand allow to suppose ways of gs molecule modifications to achieve its tolerance to blood cells. g. bai 1 , p. gomes 1 , r. seixas 1 , m. hicks 2 , m. prieto 3 , m. bastos 1 eukaryotic antibiotic peptides (eaps) have been widely studied for the past years as an alternative to conventional antibiotics due to emergence of multi-resistant microbial strains, and significant efforts targeting increasingly potent and specific antimicrobial peptides are being made. one interesting approach in peptide antibiotics is based on hybrid sequences derived from natural eaps, with ca ( resistance to conventional antibiotics has stimulated a search for alternative therapeutics for microbial infections, a possible source that has gained much interest in recent years are antimicrobial peptides. antimicrobial peptides target the cell membrane directly, which is a key feature as evolution has shown bacteria have had difficulty in altering their membrane composition and organization to mount a suitable defence against these peptides. a common theory is that peptides that bind strongly exhibit high biological activity, but our real-time quantitative binding studies via surface plasmon resonance (spr) have shown that this correlation does not always hold. as more information on the molecular details of membrane disruption is required, we have used atomic force microscopy (afm) to visualise peptide insertion and changes in membrane morphology by a range of antimicrobial peptides in situ. interaction studies were performed with a series of phospholipid mixtures that mimic either mammalian cells (high in phosphatidylcholine and cholesterol) or microbial cells (high in phosphatidylethanolamine and phosphatidylglycerol). the present study may assist in the design of new specific antimicrobial peptides with high antimicrobial activity and low host toxicity. proportions of popc and popg as models. very high molar ratio partition constants ((18.9+-1.3)x10^3 and (43.5+-8.7)x10^3) were obtained for the bacterial models (popg:popc 4:1 and 2:1, respectively), these being about one order of magnitude greater than the partition constants obtained for the less anionic mammalian model systems ((3.7+-0.4)x10^3 for the 100% popc system). at low lipid:peptide ratios there were significant deviations from the usual hyperbolic-like partition behavior of peptide vesicle titration curves, especially in the case of the most anionic systems. membrane saturation was shown to be related to such observations and mathematical models were derived to further characterize the peptide-lipid interaction under these conditions. the calculated peptide-to-lipid saturation proportions, together with the determined partition constants, suggest that the minimal inhibitory concentrations of omiganan pentahydrochloride could represent the conditions required for bacterial membrane saturation to occur. the hemolytic pore-forming toxin sticholysin ii (stii) produced by the sea anemone stichodactyla heliantus belongs to the actinoporin protein family. the n-terminal domain of these proteins is required for interaction with membranes. to investigate the role of stii´s n-terminal domain in membrane binding and in the molecular mechanism of hemolysis, peptides corresponding to residues 1 to 35, or shorter fragments from this region, were synthesized. in some peptides leu was replaced by trp. all peptides exhibited hemolytic activity, albeit to a lesser extent than the whole protein. moreover, peptides lacking the 1-14 hydrophobic stretch were less active. the longer peptides were also able to permeabilize phospholipid vesicles. conformational studies were performed in aqueous solution and in membranemimicking environments. cd spectra showed that, while the shorter, more hydrophilic peptides, displayed a random conformation, the longer peptides underwent aggregation with increasing concentration, ph, and ionic strength. in the presence of trifluoroethanol and upon binding to detergent micelles and phospholipid bilayers, the peptides showed a propensity to acquire -helical conformation, as expected for the sequence comprising residues 14 to 26. fluorescence spectra demonstrated that the first residues of stii´s n-terminus penetrate more deeply into the bilayer, whereas residues 14-26 are located more superficially. this is in agreement with the predicted amphipathic nature of the helix formed by these residues and corroborates the existing hypotheses for the role of the n-terminal domain in the process of membrane insertion and pore formation. among a great number of antibacterial peptides a group of trp-rich peptides is of special interest. taking into consideration, that in most of proteins tryptophan is not frequently occurring amino acid, the biological meaning of a high content of tryptophan in structure of these antimicrobial peptides is particularly interesting. in the present study we carried out the investigation of antimicrobial and hemolytic activities of selected trp-rich peptides and their action on microbial membrane: ilpwkwpwwpwrr-nh2 indolicidin (i) pitwpwkwwkgg-nh2 3b3 (ii) plswffprtwgkr-nh2 gsp-1a (iii) fpvtwrwwkwwkg-nh2 puroindoline (iv) vrrfpwwwpflrr-nh2 tritrypticin (v). sunflower trypsin inhibitor sfti-1 is the smallest and the most potent known peptidic trypsin inhibitor from the bowman-birk class of proteins [1] . this head-to-tailcyclized 14-amino-acid peptide contains one disulfide bridge and a lysine residue (lys5) present in the p1 position, which is responsible for inhibitor specificity.as was reported by us and other groups, sfti-1 analogues with one cycle only retain trypsin inhibitory activity. very recently we have shown [2] that introduction of nsubstituted glycine residues mimicking lys and phe (denoted as nlys and nphe) in the p1 position of monocyclic sfti-1 with disulfide bridge yielded potent trypsin and chymotrypsin inhibitors, respectively. in this novel class of proteinase inhibitors contains completely proteolytic resistant p1-p1' reactive site. in the present communication we report chemical synthesis and determination of trypsin and chymotrypsin inhibitory activity of a series of ten sfti-1 analogues modified in the p1 position by these peptoid monomers (nlys and nphe). each of the synthesized peptomeric (peptide-peptoid hybrid polymer) sfti-1 analogues contains one of the following cycles: head-to-tail, disulfide bridge formed by cys, by pen and by cys/pen residues. the impact of the different cycles introduced into sfti-1 structure on proteinase inhibitory activity will be discussed. s-protein contains a proteolytic processing site and two interacting heptad repeat regions denoted as hr-n and hr-c. following processing of s-protein mediated by host cellular protease/s, the c-terminal s2-fragment fuse with host cell membranes via its hr-n and hr-c domains that form coiled coil 6-helix bundle (trimeric of dimers)-crucial for its receptor-mediated viral fusion. our objective in this work is to study the proteolytic site using model peptides and also to examine the interaction of hr-n and hr-c domains using fluorescence microscopy and other techniques. thus we synthesized an intramolecularly quenched fluorogenic peptide containing the proposed cleavage site [abz-eqdrntr761 evfatyx, abz=2-amino benzoic acid and tyx=3-nitro tyrosine] and showed by kinetic measurements that this cleavage is mediated most efficiently by furin, followed pc5 and pc7. other potential substrates were also tested and compared. above cleavage can be blocked by specific-pc-inhibitors in a dose-dependent manner. in addition using fluorescent-labeled peptides derived from hr-n and hr-c domains, circular dichroism spectra and surface assisted laser desorption mass spectral interest has grown to develop specific and potent inhibitors of this enzyme. our objectives in this study are to generate soluble recombinant human (h)ski-1 enzyme, design potent inhibitors and study its 3dmodel structure. we have successfully expressed hski-1 enzyme lacking its transmembrane domain in hek-293 cells and purified the enzyme via chromatography. in addition we developed ski-1 inhibitors by using pseudo-and multi-branch peptide approaches. in first approach we inserted dipeptide isosteres amino oxy acetic acid (aoaa) or 8-amino 3, 6 dioxa octanoic acid (adoa) at scissile p1-p1' position ((r175 l) of hski-1. a typical example is 167gryssrrl(adoa)aip179. other dipeptide isosters were also incorporated at the cleavage site of either ski-1 prodomain or lassa virus glycoprotein. in second approach we prepared 2 and 4-branch peptides containing hski-1128-137 segment. these peptides inhibit ski-1 in competitive manners with varying degrees ranging from low m to high nm ic50. circular dichroism spectra indicated strong interactions of inhibitors with ski-1 consistent with observed inhibition profile. a 3d-model structure of catalytic domain of ski-1 indicated a broad catalytic pocket cysteine proteases are of great importance in biochemical processes and these enzymes are used in biotechnology, food industry and agriculture. in this connection synthesis of high selectivity and high specificity substrates for cysteine proteases is of importance. enzymatic synthesis of peptides is a good tool for obtaining different biologically active peptides. immobilized serine proteases, subtilisin carlsberg and α-chymotrypsin immobilized on poly(vinyl alcohol) cryogel (pvacryogel), proved to be a convenient biocatalyst for such kind of syntheses. the high specific chromogenic substrate for cysteine proteases assay glp-phe-ala-pna was obtained with high product yields (up to 88% in 24 h) using subtilisin and chymotrypsin immobilized on pva-cryogel. the reaction was carried out according to the following scheme: glp -the residue of pyroglutamic acid, pna -p-nitroanilide. the influence of initial concentrations of components, the reaction mixture composition, the biocatalyst content and time on product yield was studied. it was shown that the optimal conditions are: dimethylformamide-acetonitrile mixture 20:80 (v/v), initial concentrations -85 mm, and enzyme-to-substrate ratio -1:3900. this approach was used in order to synthesize analogous substrates, containing different fluorogenic and chromogenic groups as well as other amino acids in p1position. the obtained substrates were tested for the papain assay. peptidyl-a-ketoaldehydes 3 represent attractive lead compounds and intermediates in the development of potent protease inhibitors due to their structural similarity with peptide aldehydes, previously known to be excellent inhibitors of serine-and cysteine protease. recently, we demonstrated the application of polymer cyano methylene-and carboxylato methylene phosphoranes in the assembly of a-hydroxy-b-amino esters (norstatines), a,b-diketoesters, and a,b-unsaturated ketones. [1, 2, 3] we now present a further development of our reagent linker 2 approach employing peptidyl-a-ketoaldehydes 3 and diamino propanoles 4. carboxylato methylen phosphoranes 1 derived from bromo acetic esters which are readly acylated without racemization, play the key role in our synthetic concept. herein we show the oxidative cleavage to peptidyl-a-ketoaldehydes 3 using dimethyldioxirane (dmd) in acetone as oxidant, after saponification and decarboxylation on the solid support. diamino propanoles 4 were furnished via the reductive amination of resin-bound peptides. over the past few years nuclear magnetic resonance has emerged as a powerful means for lead molecular identification and optimization.on the other hand, the 19f nmr has been used succesfully in several structural studies, protein folding studies and for the identification of active compounds, using a very similar methodology that the one used in the present work. the methodology required the labeling of the substrate with a cf3 moiety. the enzymatic reaction is performed with the cf3 substrate and quenched, using an enzyme inhibitor. 19f nmr is then used to monitor the evolution of both substrate and product. only two peaks are observed, the starting substrate and the cleaved substrate. this nmr method has some advantages: fluorine nmr is very sensitive, 0,83 times that of the proton. there are no spectral interference from protonated solvents, buffers or detergents typically present in the enzymatic reactions.the 19f isotropic chemical shift is extremely sensitive to small structural perturbations resulting in different chemical shift for the signals of the substrate and product. isotopic labeling of the protein is not required. as a model, caspase-8, which play a critical role in the initiation of apoptosis process5 and hiv-1 protease were chosen. two different kind of libraries were screened: one based on natural products from plant and animal extracts used in tradicional chinese medicine and a second one corresponding of a synthetic library with two sublibraries of 160 and 144 compounds.with this methodology it has been possible to identify some compounds with very promising inhibitory properties. background and aims: human kallikrein 2 (hk2), a prostate specific serine protease, regulates the activity of several factors that may participate in proteolytic cascades promoting tumor growth and metastasis. thus, inhibition of its enzymatic activity is a potential way of preventing growth and metastasis of prostate cancer. moreover, specific ligands for hk2 have potential use for targeting and in vivo imaging of prostate cancer and for development of novel assays. methods: to find peptide ligands we panned several phage display peptide libraries against active recombinant hk2 captured by a monoclonal antibody exposing the active site of the enzyme. alanine scanning and amino acid deletion analyses were performed to elucidate the motifs required for hk2 inhibition. results: from libraries expressing 10 and 11 amino acid long linear peptides we isolated six different hk2-binding peptides. three of these peptides are specific inhibitors of the enzymatic activity of hk2. amino acid substitution and deletion studies indicated that motifs of 6 amino acids are necessary for the inhibitory activity. conclusions: we have developed specific hk2 inhibitors by phage display technology. these novel hk2 specific peptides are potentially useful for treatment and targeting of prostate cancer. peptidylarginine deiminase iv (padiv) catalyzes the citrullination of arg residues in various peptides and proteins, such as histone, resulting in the production of citrullinated proteins in granulocytes [1, 2] . the citrullination mechanism of histone subunits and its functional effects in cells are not well known yet in detail. recently, it has been reported that the protein deimination/citrullination by pad iv plays a role in rheumatoid arthritis [3] . this implicates that the citrullination of histone may be related to rheumatoid arthritis. in order to further study the citrullination mechanism of histone, we explored the citrullination sites of histone h2a and h3 by pad iv using a series of synthetic peptides. recently, hagiwara et. al. reported that pad iv only citrullinates the arg3 of histone h2a as well as the arg3 in histone h4 [4, 5] . in order to investigate the citrullination mechanism, the n-terminal peptides of histone h2a and h3 were chemically synthesized and examined the citullination by pad iv. the n-terminal acetylation effect of the n-terminal synthetic peptide was also estimated on the citrullination by padiv. the velocity of each arg residues in the n-terminal peptides were estimated in vitro. the results indicated that padiv recognizes the specific arg residues in the synthetic peptide, and that the n-terminal acetylation of the histone peptides dramatically affects on the substrate recognition of padiv. in addition, the cd spectra of the n-terminal peptides were measured to elucidate the structural specificity for the recognition of pad iv. background and aims. prolyl oligopeptidase (pop) is a serine peptidase that cleaves oligopeptides after prolyl residues. it has been associated with cognitive disorders. pop inhibitors have been shown to enhance cognition in monkeys (1) and to improve performance in verbal memory tests in humans (2) . in the present study, the p2 l-prolyl residue of pop inhibitors was replaced by two l-proline mimetics, the 5-t-butyl-l-prolyl group and the (r)-cyclopent-2-enecarbonyl group. the effect of the mimetics on in vitro potency, lipophilicity and binding kinetics were studied. methods. the l-proline mimetics were synthesized according to the published procedures (3, 4) with minor modifications. the ic50 and ki values and the binding kinetics were determined for porcine pop. the log p values were determined with the shake-flask method. results. the replacement of the p2 l-prolyl residue by the l-proline mimetics gave compounds which were equipotent with their parent structures. both l-proline mimetics increased lipophilicity but the effect of the 5-t-butyl-l-prolyl group was more pronounced. while the 5-t-butyl-l-prolyl group increased the dissociation half-life of the enzymeinhibitor complex, the (r)-cyclopent-2-enecarbonyl group decreased it. conclusions. both l-proline mimetics perfectly mimicked l-proline at the p2 position of pop inhibitors. these mimetics can be used to modify the lipophilicity and the binding kinetics of pop inhibitors. the proteasome is an essential multicatalytic protease of the ubiquitin proteasome pathway. as a prime executor of regulated proteolysis, the proteasome controls almost all aspects of cell metabolism from signal transduction to cell cycle and differentiation. pharmacological intervention into proteasome activity leads to cell apoptosis. this observation was applied to successfully treat multiple myeloma, since the cancer cells exhibit substantially higher sensitivity to competitive inhibition of proteasome than normal cells. however, the complete shutting down of the proteasome catalyzed proteolysis leads to serious side effects resulting from the disruption of proteolytic homeostasis even in noncancerous cells. here, we show an alternative approach to control the proteasome activity using peptide based noncompetitive regulators. the cathelicidins derived peptides rich in proline and arginine (pr) residues have been found to affect activity of all the proteasome complexes both in vivo and in vitro, likely by binding to the face of the enzyme. mechanism and structural constrains of the pr peptides dictating their influence on the proteasome remain elusive. our results indicate that there are three sequence related properties of the pr peptides controlling their effectiveness as proteasome regulators: length of the peptide, distribution of a set of positive charges at the peptide n-terminus, and positioning of proline residues. far uv cd spectroscopy demonstrates that these properties also correlate with the structure of pr peptides. in particular, it seems that structural propensity of the pr peptides to form beta-turns are required to bind to proteasome as regulatory competent molecules. our work is focused on the search of selective, low-molecular cathepsin b peptide inhibitors acylated with the (e)-3-(benzylsulphonyl)acroyl group (bsa). the double bond, embedded in the bsa moiety is activated by two electron-withdrawing groups and may be a good target for the michael-type addition of the catalytically active -sh group. three series of peptide derivatives possessing general structures: bsa-phe-asn(r)-oh, bsa-ile-x(oh)-n(ch3)2 and bsa-x-pro-oh were synthesized in solution and characterized by enzyme kinetic studies against papain, cathepsins b and k. it should be noted that all the investigated compounds were competitive and reversible inhibitors of the enzymes examined. using 2d 1h nmr (tocsy, cosy, roesy) and 13c nmr spectroscopy along with theoretical calculations (analyse program) we determined the conformational properties of two most potent and selective cathepsin b inhibitors. this work was supported by grant ds/8350-5-0131-6. background and aims: we have developed peptides inhibiting human kallikrein-2 (hk2) activity. as hk2 is overexpressed in prostate cancer tissue, these peptides are potentially useful for treatment and diagnosis of prostate cancer. two of the potential physiological substrates for hk2 are proform of prostate specific antigen (propsa) and insulin-like growth factor-binding protein-3 (igfbp-3). both of these might participate in the regulation of prostate cancer growth: igfbp-3 by inhibiting igf-dependent tumor growth and psa by degrading extracellular matrix. we aimed to study whether our hk2-inhibiting peptides inhibit also hk2 activity towards natural protein substrates, i.e. activation of propsa and degradation of igfbp-3. methods: the effect of the peptides on the activation of propsa by hk2 was studied by preincubating the peptides with hk2, followed by addition of psa and specific psa substrate. igfbp-3 degradation was studied by two specific immunoassays, one detecting only native igfbp-3, while the other one also detected proteolytically cleaved forms of the protein. results: hk2-inhibiting peptides were found to inhibit propsa activation and igfbp-3 degradation by hk2 in a dose dependent fashion. conclusions: we have developed new peptides inhibiting hk2 activity towards natural substrates, like propsa and igfbp-3. the peptides might be useful for treatment of prostate cancer and other diseases associated with increased hk2 activity. from the seeds of garden four-o'clock and spinach we isolated two serine proteinase inhibitors (mjti i -mirabilis jalapa trypsin inhibitor and soti i -spinacia oleracea trypsin inhibitor), which are probably representatives of a new family of inhibitors. the purification procedures of these inhibitors included affinity chromatography on immobilized methylchymotrypsin in a presence of 5 m nacl, ion exchange chromatography and/or preparative electrophoresis and finally rp-hplc on c18 column. their primary structures (fig. 1 ) differ from those of known trypsin inhibitors, but showed significant similarity to one another, as well as to the antimicrobial peptides isolated from the seeds of mirabilis jalapa (mj-amp1, mj-amp2), mesembryanthemum crystallinum (amp1) and phytolacca americana (amp-2 and pafs-s) and from hemolymph of acrocinus longimanus (alo-1, 2 and 3). the equilibrium association constants (ka) of mjti i and soti i with bovine -trypsin were found to be about 107-109 m-1. mjti i and soti i have been synthesized using solid-phase method. the synthesized inhibitors and inhibitors isolated from plants have similar properties. the disulfide bridge pattern in both inhibitors was established after digestion with thermolysine, followed by the maldi-tof: cys1-cys-4, cys2-cys5 and cys3-cys6. s. cosgrove, l. rogers, c. hewage, j.p. malthouse aspartyl proteases are required for the multiplication of the aids virus and for producing the amyloid protein which causes alzheimer's disease. hiv protease inhibitors have been highly effective in treating aids patients and it is hoped that potent inhibitors of the beta secretases will also prove effective in treating alzheimer's disease. therefore inhibitors of the aspartyl proteases have great therapeutic potential. we have shown that the peptide glyoxals are potent inhibitors of the thiol protease papain and of the serine proteases subtilisin and chymotrypsin. using 13c-nmr we have been able to show that glyoxal inhibitors react reversibly with an active site nucleophile in these enzymes to form a tetrahedral adduct which is tightly bound by the enzyme. in the present work we synthesise 13c-enriched peptide glyoxals, we assess their inhibitor potency, and use 13c-nmr to examine how the inhibitors interact with the aspartyl protease pepsin. z-ala-ala-[2-13c]phe-glyoxal was synthesised from [1-13c]phenylalanine which was converted to its methyl ester. this was then coupled with z-ala-ala to give z-ala-ala-[2-13c]phe-ome which was hydrolysed to the free acid. this was converted to the diazoketone and transformed into z-ala-ala-[2-13c]phe-glyoxal using dimethyldioxirane. nmr spectra at 11.75 t were recorded with a bruker avance drx 500 standard-bore spectrometer. we show that peptide glyoxal inhibitors can be potent inhibitors of pepsin and that pepsin only binds one of the four glyoxal forms (one non-hydrated, one fully hydrated and two partially hydrated forms). alzheimer`s disease (ad) is the most common cause of dementia in older people. a major factor in the pathogenesis of ad is the cerebral deposition of amyloid fibrils, consisting of amyloid β peptides (aβ), as senile plaque. the 40-to 42 amino acid long aβ is generated by the proteolysis of β-amyloid precursor protein (app) by β-and γ-secretases. since bace1, a unique member of the pepsin family of aspartyl proteases initiates the pathogenic processing of app by cleaving at the n-terminus it is a molecular target for therapeutic intervention in ad proteolytic activity was found to occur, to a variable degree, in digestive organs of all studied organisms over the entire ph range. the common feature was the existence of two activity peaks, in the acid (ph 2.5 -3.5) and alkaline (ph 7.5 -8.5) zones, as well as a similar protease set containing e and d cathepsins, a trypsin-like enzyme, elastase, and collagenolytic proteases. proteolytic activity in the hepatopancreas of crab and sea star was found to be an order higher than in other study objects. high protease activity in crab hepatopancreas is an evolutionary mechanism compensating for a poor differentiation of digestive system, low substrate specificity of enzymes, and cold environment. trypsin activity in digestive organs of invertebrates suggests that a trypsin-like enzyme is a genetically old one, an evolutionary origin of all serine proteases. a difference of kind between vertebrates and invertebrates is that the latter have cathepsine activity (absent in vertebrates) and no pepsin activity. it is of interest to develop enzyme inhibitors containing a light activated switch that can be used to control binding and inactivation of an enzyme. several inhibitors containing the azobenzene photoswitch group have previously been developed and have shown changes in activity of around two times on photoswitching. this study aimed to improve this switching by more extensive derivatisation of azobenzene to closer resemble the peptide substrates of proteases. a series of peptidomimetics containing the azobenzene photoswitch group were synthesized and assayed against the protease alpha-chymotrypsin. these compounds contained azobenzene, linked to a known chymotrypsin inhibitory group (either a trifluoromethylketone or boronate ester), and otherwise designed to be peptide-like. in some cases both ends of the azobenzene moiety were derivatized in order to increase the impact of photoswitching on the shape of the compound and thus its enzyme binding strength. assays showed that most compounds were reversible inhibitors of chymotrypsin, with low micromolar inhibition constants (ki or ic50). up to four times increase in enzyme inhibition on light activated switching of the azobenzene group conformation was obtained. a number of peptidyl derivatives structurally based upon the inhibitory sites of cystatins has been synthesized. these compounds are prone to proteolytic degradation, are rapidly excreted and poorly bioavailable. the majority of this problems might be overcome by use of peptidomimetics with structures resembling those of previously synthesized peptidyl derivatives. among the peptidomimetics are azapeptides, in which alpha-ch group of amino-acid residue is replaced by a nitrogen atom. the azapeptides have recently been demonstrated as potent and selective inhibitors of cathepsins b and k. it was shown that azapeptide inhibitors bind along the active site cleft of cathepsin b in a bent conformation. this bent structure is likely to result from the mobility of the bonds in the vicinity of the inserted azaamino acid residue as well as from the interaction with enzyme. in our present work we have studied the peptide of a sequence: z-arg-leu-arg-gly-ile-val-ome, which is characterized by one major and three minor conformations. the replacement of alpha-ch group in the gly residue of peptide chain of z-arg-leu-arg-gly-ile-val-ome by the nitrogen atom likely results in rigid conformation. our aim was a comparison of structure of the parent peptide z-arg-leu-arg-gly-ile-val-ome and a selective cathepsin b inhibitor z-arg-leu-arg-agly-ile-val-ome by using 1h-nmr. severe acute respiratory syndrome corona virus associated main protease (sars cov mpro protease), alternatively known as chymotrypsin-like protease (3clpro), is a mediator of virus infection cyclus and from there a therapeutic target. a peptide aldehyde library targeting the sars corona virus main protease (sars-cov mpro, alternatively known as 3clpro) was designed on the basis of three different reported binding modes and supported by virtual screening. a set of 25 peptide aldehydes were prepared by a newly developed methodology and investigated in an inhibition assay against sars-cov mpro. [1] protected amino acid aldehydes furnished by the racemization-free oxidation of amino alcohols with dess-martin periodinane were immobilized on threonyl resins as oxazolidines. following boc-protection of the ring nitrogen yielding the n-protected oxazolidine linker, peptide synthesis was performed efficiently on this resin releasing deprotected products under mild hydrolysis conditions. the library was tested in a new fluorimetric enzyme assay for sars cov mpro. via immobilization of the fluorophor, 2-(7-amino-4-methyl-3-coumarinyl)-acetic acid, the substrate actsavlq-amca was prepared, surprisingly displaying a higher affinity than the native substrate. several potent inhibitors were found with ic50 values in the low micromolar range. interestingly, the most potent inhibitors seem to bind sars-cov mpro in a non-canonical binding mode. currently, the initial screen is extended towards the discovery of small molecule inhibitors of sars corona virus main protease. literature: a method of bromelain cleavage of surface glycoprotein hemagglutinin (ha) from the influenza a virions was initially employed for ha ectodomains crystallographic study [1] . the remaining spikeless subviral particles were used by us earlier for ha2 c-terminal fragment extraction and mass spectrometric (ms) investigation [2] . now sds-page analysis of the subviral particle preparations revealed several additional bands in a range of 9-23 kda together with major viral proteins comparing to intact virions (figure, m1matrix protein, f1-f5-m1 protein fragments, np-nucleoprotein). maldi-tof ms analysis of the in-gel trypsin hydrolyzates has shown that the additional bands are fragments of м1 protein. this was confirmed by n-terminal sequencing of the protein fragments electroblotted from the bands. concentration of sh-reducing reagent in bromelain digestion reaction influenced on the m1 fragment bands intensity. we conclude that due to membrane destabilization during ha spikes removing, m1 protein localized under viral membrane inside intact virions becomes accessible to limited proteolysis by bromelain. [ dipeptidyl peptidases (dpp's) sequentially release dipeptides from polypeptides. among those enzymes, dppiv, fapα, dpp8, dpp9 and dppii cause the release of n-terminal dipeptides containing proline or alanine at the penultimate position. they are all members of clan sc, a group of serine proteases that contains proline-specific peptidases. dipeptidyl-peptidase iv (dppiv) is the best studied member of this group of enzymes and has become a validated target for the treatment of type 2 diabetes over the last years. the development of inhibitors for the related enzymes (i.a. dppii) has only started recently. this poster presents selected products synthesised to further elaborate the structure-activity relationship for dpp ii inhibitors with a 2,4-diaminobutyrylpiperidine basic structure. this class of compounds was described earlier by our group as the hitherto most potent and selective inhibitors of dpp ii. starting from n4-p-chlorobenzyl-substituted uamc00039, our lead compound, two types of modifications were proposed: • the synthesis of n4-(di)alkyl-and arylalkyl analogues; • the synthesis of 3-methyl analogues. in our previous study, we reported potent and small-sized bace1 inhibitors containing phenylnorstatine [(2r,3s)-3-amino-2-hydroxy-4-phenylbutyric acid; pns] at p1 position as a transition-state mimic. in developing more active compounds, we focused our attempts on the p1 position, where we replaced the pns by its thioderivative. herein, we present the synthesis of a novel phenylthionorstatine [(2r,3r)-3-amino-2-hydroxy-4-(phenylthio)butyric acid; ptns] as a p1 moiety with hydroxymethylcarbonyl (hmc) isostere, and then an application to the bace1 inhibitors design. we have synthesized ptns starting from readily available n-benzyloxycarbonyl-serine and after multistep reaction (including weinreb amide formation, thiophenyl group introduction, through cyanohydrin derivative the transformation into the 2-hydroxy ester and then acid). purification was done by column chromatography and rp-hplc. peptides were synthesized by the fmoc based solid phase method and characterized by maldi-tof ms. the peptide inhibitors were adopted to enzyme assay using a recombinant human bace1 and a fluorescence-quenching substrate. bace1 inhibitory activity was determined based on the decrease% of the cleaved substrate by the enzyme.we have synthesized ptns and then the (2r,3r)-enantiomer was applied to spps (solid phase peptide synthesis). we synthesized octa-and pentapeptide-type inhibitors of bace1 containing pns or ptns at the p1 position. these compounds were enzymatically tested and showed high bace1 inhibitory activity. a novel derivative of pns, ptns, was synthesized, and evaluated in comparison to corresponding pns. the inhibitors with ptns exhibited a slightly higher inhibitory activity against bace1 comparing to those with pns. this study suggests possibilities of the application of ptns to design other aspartyl proteases inhibitors. the αν β3 integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis, mainly by interacting with matrix proteins through recognition of an arg-gly-asp (rgd) motif. inhibition of the αν β3 integrins with a cyclic rgd peptide impairs angiogenesis, growth and metastasis of solid tumours in vivo. the aim of this study was to investigate the effects of replacement of amino acids by aza-β3-amino acid analogs in cyclic rgdpeptides as αν β3 -integrin antagonist on angiogenesis, microcirculation, growth and metastasis formation of a solid tumour in vivo. the selectivity profile of these antiadhesive cyclopeptide is rationalized by a special presentation of the pharmacophoric groups. thr rgd motif resides in position i to i+2 of a regular γ-turn. we synthetized linear and cyclic aza-β3 rgd-peptide with the purpose to examine the effect on the conformation and the activity. are aza-β3 amino acids γ-turn mimetics? the preferred conformations were determined by nmr. prostaglandins are involved in a large number of biological activities mediated by their g-protein coupled receptors (gpcrs). the prostaglandins pgf2 alpha receptors are found specifically in uterine muscle, where they initiate parturition and labor. the pgf2 alpha receptor plays a key role in preterm labor, for which medical and social costs are estimated at $ 9 billion per year in the usa (the highest per patient cost of any disorder). peptide mimics have been developed in our laboratory (1, 2) , that serve as allosteric antagonists of the pgf2 alpha receptor. the importance of the turn geometry of the central residue in these peptide mimics has been investigated using enantiomeric indolizidin-2-one beta-turn mimics which can respectively induce type ii and ii' geometry. our presentation will discuss the synthesis and biology of these novel allosteric modulators of prostaglandin pgf2 alpha receptor activity. it was shown that luteinising hormone -releasing hormone (lhrh) receptors are overexpressed in the most of adenocarcinoma cells in contrast to their low content in normal tissues. these data create the basis for lhrh analogues application in therapy of breast, ovary, prostate, lung, intestine, liver and kidney cancers. both agonists and antagonists utility for the targeting of cytotoxic moiety to the tumor cells is well documented. however, the number of lhrh analogues possessed their own cytotoxic activity is still very limited. we nicotianamine (na) that was first isolated from the leaves of nicotiana tabacum l [1] , is known as a key biosynthetic precursor of phytosiderophores. various studies have proved that nicotianamine plays a significant role in plants as an iron, nickel, zinc ... transporter [2] . the aim of our study was to synthesize unnatural analogues of na via peptide intermediates, to investigate the mechanisms of metal transport and accumulation within the plant. we found that the strategy developed for na synthesis could not be applied when the azetidine ring was changed for pyrrolidine ring and we investigated a new route to synthesize such analogue. these synthetic pathways will be discussed. the primary physiological roles of arginine vasopressin (avp), [cycle1-6 (h-cys1-tyr2-phe3-gln4-asn5-cys6-pro7-arg8-gly9-nh2)], involve vasoconstriction of vascular smooth muscles, via v1a receptor, and antidiuretic action in kidney (blood osmolality regulation) via v2 receptor. binding of avp to the v1a receptor subtype also stimulates glycogenolysis in the liver and promotes platelet aggregation. in addition, activation of the v1b (also known as v3) receptor causes adrenocorticotropic hormone release from the anterior pituitary. v1b receptors are also present in the brain where avp functions as a neurotransmitter. in the recent years by the salivary glands of several bloodsucking animals like, teaks, leeches, vampire bats and so forth are isolated plenty of proteins and peptides with different molecular weight and well established anticoagulant activity. many of the strongest anticoagulants isolated by bloodsucking animals are found in the extract of salivary glands of different kinds of leeches. such leech is the haementeria officinalis, from which is isolated the most active inhibitor of factor xa -ats. in order to study the role of some amino acids in the process of interaction among peptides mimetics and the active site of serine proteinases, some fragment analogues of ats's active site by replacement of some amino acids with the other with similar structure or with unnatural amino acids were synthesized. in the present work the synthesis and the anticoagulant activity according to the aptt and ic50 of the newly synthesized peptides and structure-activity relationship will be discussed. rational design of peptides is a challenge which would benefit from a better knowledge of their rules of sequence-structure-function relationships. peptide structures can be approached by spectroscopy and nmr techniques but data from these approaches too frequently diverge. structures can also be calculated in silico from primary sequence information using three algorithms: pepstr, robetta and peplook. the most recent algorithm, peplook introduces indexes for evaluating structural polymorphism and stability. the method uses a de novo search of energy minima by an iterative boltzmann-stochastic procedure and using a combination of 64 phi/psi couples derived from the structural alphabet for protein structures proposed by etchebest et al. for peptides with converging experimental data, calculated structures from peplook and, to a lesser extent from pepstr are close to nmr models. the peplook index for polymorphism is low and the index for stability points out possible binding sites. for peptides with divergent experimental data, calculated and nmr structures can be similar or, can be different. these differences are apparently due to polymorphism and to different conditions of structure assays and calculations. the peplook index for polymorphism maps the fragments encoding disorder and the mean force potential score indicates which residues will be most available for interactions with partners. this should provide new means for the rational design of peptides. several diseases like cancer metastasis, rheumatoid arthritis and chronic lymphocytic b-cell leukemia are linked to the interaction of the cxcr4 chemokine receptor to its natural ligand, the 68 amino acid protein stromal cell-derived factor-1α (sdf-1α). [1] one strategy for the treatment of these diseases could be to block the interaction between cxcr4 and sdf-1α with small cxcr4 antagonists. furthermore, radiolabeling of suitable compounds with appropriate radioisotopes could provide agents for imaging of cxcr4 expression in vivo via pet. previous studies by fujii et al. on cxcr4 antagonists led to a high affinity cyclic pentapeptide with the sequence cyclo[gly-d-tyr-arg-arg-nal]. [2] to further improve this structure, different approaches have been chosen with respect to metabolic stability, bioavailability, conformational rigidity and chemical versatility for radiolabeling. first, an n-methyl scan of the backbone amides was performed to influence conformational freedom and to increase metabolic stability and bioavailability. n-methylation of arginine residues yielded peptides with moderate affinity (ic<sub>50</sub>-values: 23nm (n-me)arg<sup>3</sup> and 31nm (n-me)arg<sup>4</sup>, resp.) whereas n-methylation of other amino acids significantly decreased the affinity (ic<sub>50</sub>>100nm). by substitution of arg<sup>3</sup> by ornithine, the affinity was mostly retained. [3] the amino group of orn can be alkylated or acylated via radiolabeled groups containing short lived isotopes. moreover, the bioavailability should be improved as the high basicity of the two guanidino groups could be reduced. first ornithine-acylated derivatives showed ic<sub>50</sub> values between 11-35nm enabling for the first time <sup>18</sup>f-radiolabeling of small cxcr4 antagonists for pet imaging in vivo. binding of ligands to integrins plays a major role in cell adhesion, migration, and signal transduction of cells. these interactions are important not only for normal cell functions, but also in pathogenic processes. the v 3 integrin for example is involved in tumor cell adhesion and osteoporosis. the association of ligands is specific and requires minimal recognition sequences. therefore, suppression of integrin activity using competitive inhibitors bears great pharmacological potential. the tri-peptide sequence rgd is a prominent recognition sequence of integrin ligands. two new cyclic pentapeptides were synthesized containing the tripeptide sequence rgd as well as 3-amino-cyclopropane-1,2-dicarboxylic acid monomethyl ester (acc) and valine varying only with respect to the stereochemistry of acc. both the (+) (all r) and (-) (all s) isomers of acc were incorporated. acc is a cyclic -amino acid as well as a cyclopropyl analogue of aspartic acid. biological tests with cell lines expressing mainly v 3 and v 5 integrin show a higher inhibitory activity of cyclo-(-arg-gly-asp-(+)acc-val-). in order to derive a structure-activity relationship of these two isomers, solution structures in dmso-d6 were investigated by nmr spectroscopy. subsequently, structural information was obtained by applying distance restraints derived from the nmr spectra in distance geometry/simulated annealing and molecular dynamics calculations. due to the rigidity of the cyclopropyl unit in acc, the structure of the cyclopeptide is significantly influenced by the integrated propane ring, thus explaining the different biological properties. integrins are an important family of cell adhesion molecules. currently, 24 members are known. among other functions, integrin α 4 β 1 is involved in inflammatory processes, leukocyte migration and tumor angiogenesis. the structure of its natural ligand vcam-1, including the binding loop sequence tqidspln, has been determined by x-ray crystallography. therefore, it is possible to apply the concept of spatial screening: using small cyclic peptides with structure inducing building blocks, the binding motif is presented in different well-defined structural arrangements. for this study, a series of cyclic penta-and hexapeptides based on the tqidspln sequence has been synthesized. β-homoamino acids, i.e. β 3 -amino acids with proteinogenic side-chains, have been incorporated as structure inducers for spatial screening. although β 3 -amino acids are supposed to prefer the central position of ψγ-turns, less data exist than for e.g. d-amino acids. apart from the structural characterization of potential high affinity ligands for integrin α 4 β 1 , a major goal of this work is to provide a better understanding of the influence of β 3 -amino acids on the structure of cyclic peptides. the structures of the peptide library have been investigated by nmr spectroscopy, followed by dg/sa and md calculations. the results substantiate the γ-turn inducing capability of β-homoamino acids, but also prove the formation of different turn structures in certain cases. a comparison to the x-ray structure of vcam-1 shows that the structure of the binding sequence has been successfully approximated by some of the peptides. biological activity tests should lead to meaningful structure-affinity relationships. neuropeptide y (npy) is a 36-amino acid peptide amide and binds to the so-called y receptors. its most dominant element is the c-terminal alpha-helix spanning amino acid residues 12-36. residues 1-10 form a polyproline helix with highly conserved proline residues at positions 2, 5 and 8, followed by a loop structure. the importance of the polyproline helix strongly varies between different receptor subtypes. it obviously plays no role in ligand binding at the y2 receptor subtype, whereas the n-terminal segment is of importance for ligand binding at y1 and y5. in order to further study the importance of the polyproline helix we introduced a conformationally constrained pyridone dipeptide mimetic at different single positions by solid phase peptide synthesis using fmoc/tbu strategy. the resulting peptides have been investigated in cell lines that selectively express y1 and y5 receptor, respectively. different methods including radioactive competitive binding assay, cd and nmr have been applied to investigate conformation and interaction of receptor and ligand. loss of affinity at the y1 receptor is independent of the position and about 10-, 20-and 30-fold, respectively, when introduced once, twice and thrice. introduction of the building block in position 8/9 leads to the most reduced affinity at the y5 receptor subtype but, surprisingly, affinity can partially be regained by introduction of the dipeptide at two additional positions. the position of the dipeptide is of greater importance at y5. these novel peptides clearly indicate the importance of proline residues and the structure of the n-terminus for ligand binding. interactions of src homology 2 (sh2) domains with phosphotyrosine (py) containing ligands is critical for regulating cellular processes. the cytosolic protein tyrosine phosphatase shp-1 contains two sh2 domains. an intramolecular interaction of the n-terminal sh2 domain with the catalytic (ptp) domain renders the enzyme inactive in the native state. binding of a py-ligand to shp-1 n-sh2 leads to a conformational shift and the dissociation of the sh2-ptp complex [1] . in previous studies we investigated the topographical and conformational preferences of the n-sh2 domain of shp-1 using conformationally restricted linear and cyclic peptides derived from the natural interaction partner ros py2267 [2] . we identified peptides that showed an increased binding affinity for the n-sh2 domain and partially inhibited ros-mediated shp-1 activity. on the basis of these results we hypothesized that an imperfect fit of the py+1 and py+3 side chains might be responsible for the inhibitory effect. in order to confirm this hypothesis we synthesized a new series of peptides and evaluated their biological activity. to better understand the role of each individual sh2 domain in the activation process we also determined the binding affinity against the c-sh2 domain and the activation profile of different shp-1 mutants. pull-down assays of the interactions of the py-ligands with full length shp-1 confirmed the results obtained for the binding to the individual sh2 domains. proteins are targets for photo-destruction due to absorption of incident light by endogenous chromophores. mass spectroscopic data presented evidence that structural modification observed upon irradiation of goat alpha-lactalbumin at 290 nm results from tryptophan (trp) mediated cleavage of disulfide bonds [1] . the aim of the recent studies is to define structural elements that direct the destructive influence of near-uv light on the disulfide bridges of proteins. most of the proteins of the immunoglobulin superfamily contain a so called triad, consisting of two s atoms, forming a disulfide bridge, and a single trp in their close vicinity [2] . we have indications that this arrangement gives rise to a photolytic degradation similar to that described in our earlier studies for goat alpha-lactalbumin [3] . we therefore investigated the influence of uv light on the single chain variable fragment (scfv) of a monoclonal antibody (82d6a3) [4] which contains two triads. the results showed that after irradiation of the wild type scfv (i) new bands (degradation products) appeared in electrophoresis experiments and (ii) the affinity for its antigen, von willebrand factor decreased. by site-directed mutagenesis, we modified the critical trp-residues to perform a parallel study on these mutants. background and aims: it is known that thrombomodulin has important function which prevents thrombus. we found kmylcvckn (m, n >= 2) peptides derived from thrombomodulin had strong anti-thrombus activity in our recent studies. these peptides formed two structures, parallel and anti-parallel, as dimers, we examined the relation between structure and activity. methods: two peptides of kkkylc(acm)vckkk and kkkkylcvc(acm)kkkk were synthesized by fmoc chemistry. dimer peptides were made by removing acm with iodine, after dissolving in 0.1 m tris hcl buffer (ph 8.0) and oxidizing the mixture of these synthesized peptides spontaneously. then three peptides shown in figure were separated using rp-hplc. the peptide concentration in normal human pooled plasma was 10 micro moles / l when measuring aptt (activated partial thromboplastin time). results: the anti-parallel formed peptide, peptide b, was prolonged aptt approximately 2.7 times, although two parallel formed peptide, peptide a and c, were not significantly different from the aptt of normal plasma. conclusions: these peptides have structure-activity relationship, we observed that the anti-parallel formed peptide had strong anti-thrombus activity. insect kinins share a highly conserved c-terminal pentapeptide sequence phe-xaa-xbb-trp-gly-nh2, where xaa can be tyr, his, ser or asn and xbb can be ala but is generally ser or pro. they are potent diuretic peptides that stimulate the secretion of primary urine by malpighian tubules, organs involved in the regulation of salt and water balance (1). the insect kinins preferentially form a cis-pro, type vi β-turn. insect kinin analogs containing tetrazole (1) and 4-aminopyroglutamate (2), both cis-peptide bond, type vi β-turn motifs, demonstrate significant activity in the in a cricket diuretic assay. in this study, we compare the diuretic activity of insect kinin analogs incorporating the four stereochemical variants of the 4-aminoglutamate (apy) motif. three of the insect kinin analogs incorporating the stereochemical variants, ( the need for new effective and to mammalian cells non-toxic antifungal agents increases in parallel with the expanding number of immunocompromised patients at risk for invasive fungal infections. in our laboratory we have produced a serie of low-molecular peptide derivatives of the general structure: x-arg-leu-nh-ch(ipr)-ch2-nh-y (where x and y were acyl groups with aromatic carbocyclic system). we have found and earlier reported that some of these display high antimicrobial activities against several clinically important gram-positive pathogenic bacteria. in this study we have by solution methods synthesized a group of low-molecular compounds and investigated their antifungal activity. the study included both candida and aspergillus species. we have found that some of the compounds were highly fungicidal. we also made a conformational study in which the residues were separately replaced by selected hydrophobic amino acids and their equivalents. the conformational study showed that the desirable stable intramolecular structure could only be formed in the presence of some vital components. this work was supported by grant ds/8350-5-0131-6. increased resistance of bacterial pathogens to currently employed antibiotics has resulted in efforts to develop antimicrobial compounds with new mechanisms of action. previously, we have synthesized some high potent antimicrobial compounds based upon the n-terminal binding fragment of human cystatin c. some derivatives of the general structure: x-arg-leu-nh-ch(ipr)-ch2-nh-y (1) (where x and y were acyl groups with aromatic carbocyclic system) have displayed the broad antibacterial spectrum and high activity against several clinically important gram-positive pathogens, including multi-resistant staphylococci. herein, the synthesis and structure -antibacterial properties relationship for two series of analogues of 1 are presented. the x and y groups in 1 were replaced by selected substituents with various geometry and distance between aromatic moieties and carbonyl. we have established the general structural features which the discussed class of peptide derivatives should possess in order to displaying the particular antimicrobial activity. this work was supported by grant ds/8350-5-0131-6. we have synthesized beta-endorphine-like decapeptide immunorphin sltclvkgfy which corresponds to the 364-373 sequence of the heavy chain of human igg. immunorphin was found to be a selective agonist of non-opioid (naloxone-insensitive) beta-endorphin receptor. the purpose of this study was to prepare [3h]immunorphin and characterize by its using the non-opioid beta-endorphin receptor on mouse peritoneal macrophages and membranes isolated from various rat organs. by use of tritium-labeled immunorphin ([3h]sltclvkgfy) with specific activity of 24 ci/mmol, non-opioid beta-endorphin receptors were revealed and characterized on mouse peritoneal macrophages and rat myocardium, spleen, adrenal, and brain membranes. since dehydroamino acids are quite reactive and various thiol nucleophiles are known to add to their double bonds [1, 2] , we hoped that these compounds might act as alkylating inhibitors of cathepsin c (dipeptidyl-peptidase i). its main function is protein degradation in lysosymes, but it is also found to participate in the activation of neuraminidase and proenzymes of serine proteinases (leukocyte elastase, cathepsin g, granzyme a) [3, 4] . it is well known, that phosphonodipeptides structural analogues of synthetic substrates of cathepsin c are the model substances in designing the new inhibitors of this enzyme. for that reason we have undertook the synthesis, theoretical and structural investigations of phosphonic analogues of dehydropeptides. gly-∆zphe-abupo(ome)2 gly-∆zphe-alapo(oet)2 gly-∆zphe-leupo(ome)2 gly-∆zphe-valpo(oet)2 gly-∆zphe-glypo(ome)2 gly-∆zphe-nbupo(oet)2 the structure and conformational preferences in this group of peptides had been investigated by mean of nmr techniques. in order to find the interactions between compounds-enzyme (cathepsin c) and interpret the results of biological test, the molecular modelling methods had been used. the interaction of v 3 integrin receptor with its ligands is selectively implicated in various processes, like angiogenesis, bone-formation, tumor genesis and tissue-genetic migration of embryonic cells. several cyclic rgd pentapeptides are known as selective ligands for v 3 integrin receptor. the aim of this study was to prepare a new conjugate, composed of the cyclo[rgdfc] derivative and a branched chain polycationic polypeptide, poly[lys(dl-alam)] (ak). the cyclopeptide was prepared on 2-cl-trityl chloride resin by fmoc/tbu strategy. the "head-to-tail" cyclisation was achieved in a diluted solution of dmf in the presence of bop and hobt coupling reagents and diea base. coupling of the cyclopeptide to ak polymer was carried out by thioether linkage. adhesion properties of soluble cyclic rgd peptides and their plate-immobilized forms were studied. free cyclopeptides evoke aggregation of cultured primary neural and cloned neural stem cells, while their plate-immobilized forms fail to support cell adhesion. on the contrary, in case the newly synthesized ak-c(rgdfc) conjugate such induction of cell aggregation was not observed. whereas immobilizing this derivative to either glass, or plastic was found to support cell-attachment in case of various cell types. in addition, all cell lines investigated -including also the primary neural cells -attached to ak-c(rgdfc) coated surface and survived, grew or differentiated even in the absence of serum. our data suggest that cyclic rgd -polypeptide conjugates represent a new tool to investigate selective cell adhesion and may provide a novel scaffold-material for directed cell-seeding. in the ph-induced channel closure in combination with the pip2 interactions. however, their detailed regulations are still remaining unclear. therefore, in the present study, we investigate these crucial residues with electrophysiological recordings and rationally designed mutagenesis based upon our structural analysis of kir1.1 tetramer. lys-80 is located fairly close to the intracellular channel gate and protrudes its long side chain positive charge into the pore. this may interfere with the potassium flow by providing repulsion charge while ph is lowered, which pushes the channel towards its closed state. mutation to met-80 therefore reduces such ph-sensitivity. on the other hand, arg-188 is supposed to be responsible for the maintenance of channel opening in the presence of pip2. loss of positive charge at this site may lead to the enhanced ph-sensitivity due to an abolished or reduced pip2 interaction. more interestingly, the double mutant for both sites reveals a compensation scenario. in combination with the discussion for the role of previously known r-k-r triad, our data provide very clear structural explanation for the exact functional roles of these basic residues in the regulation of ph-sensitive channel gating. mouse obese cart peptides are neurotransmitters involved in feeding, stress and endocrine regulation. leptin, a long-term adiposity signal, upregulates expression of cart in the hypothalamus. recent findings of co-localization of cart and cholecystokinin (cck)-a receptor (responsible for satiety effect of cck) in brain and gastrointestinal tract suggest a neurochemical link between cart peptides and cck. in normal fasted mice, cart(61-102) peptide decreased food intake after intracerebroventricular (icv) administration in a dose-dependent manner. anorectic effect of cart peptide was enhanced by peripherally administered cck-8, while cck-a receptor antagonist, devazepide blocked the effect of cart peptide on food intake. we used two mouse obesity models in this study: monosodium glutamate (msg) and diet-induced obese (dio) c57bl mice. both dio and msg mice had substantially increased fat to body mass ratio compared to their controls and were hyperleptinemic. msg mice were hypophagic and neither cart peptide nor cck-8 and devazepide had any effect on food intake of these mice. dio mice fed high-fat diet showed slightly decreased sensitivity to central administration of cart peptide, effect of cck-8 on food intake was preserved. in conclusion, cart peptide and cck-8 showed a synergistic effect on feeding in control mice that pointed to their probably integrated action in the central nervous system. analogously, devazepide suppressed cart anorectic effect. in msg obese mice, effects of both cart peptide and cck-8 on food intake were diminished due to disrupted signaling in hypothalamus. in dio mice, additive effects of cart and cck-8 were partly preserved inspite of hyperleptinemia and increased adiposity. b. chini 1 , s. stoev 2 , l.l. cheng 2 , m. manning 2 , were subsequently shown not be selective for the rat v1b receptor [2] . peptides a-d served as excellent leads to the design of selective agonists for the rat vp v1b receptor [3] . replacement of the arg8 residue in a-d by lys, orn, dap and dab, led to the first potent and selective agonists for the rat v1b receptor [3] . we now report that three of these; d the aim of the de novo peptide synthesis and the incorporation of cofactors is the construction of artificial protein models. these model systems can be used for understanding the structure-function relationship of native proteins and might open a way for possible applications. protegrin-1 (pg-1) is an 18-amino acid peptide with an amidated c-terminus, which forms an antiparallel beta-sheet, constrained by two disulfide bridges. the native sequence of pg-1 is highly cationic, containing six positively charged arginine residues. it was found that the structural features such as amphiphilicity, charge and shape are important for the cytolitic activity of pg-1. in this study we investigate the sar (structure activity relationship) of two pg-1 analogues: rglcycrgrfcvcvg-nh2 (bm-1) and rglcyrprfvcvg-nh2 (bm-2). our antimicrobial activity studies of these peptides show that the bm-1 peptide is active against microbe species as well as the native pg-1, whereas the bm-2 is completely inactive. the bm-1 analoque is shorter than native pg-1 and contains only three arginine residues, therefore is much cheaper in the chemical synthesis, what could be an advantage of this antimicrobial peptide. the conformational studies of both analogues were performed by using 2d 1h-nmr technique (in dmso-d6) and molecular dynamics studies. the 3d solution structure of both analogues was established using interproton distances and torsion angles. for simulated annealing calculations the xplor program was used. our conformational studies show that the bm-1 forms a regular beta-hairpin structure, which is very similar to that of the native pg-1 peptide, whereas the bm-2 analogue is very flexible, what could be a reason of the antimicrobial inactivity. copper amine oxidases (ec 1.4.3.6) catalyze the oxidative deamination of primary amines to the corresponding aldehydes, ammonia and hydrogen peroxidase. these enzymes are ubiquitous, occurring in micro-organisms, plant and animals. activity of this enzyme increases under various stress conditions including thermal and water stresses. although lsao is not a thermostable enzyme, it is in maximum stability and activity above physiological temperatures. in this study we have investigated the kinetics of thermal denaturation of lentil seedling amine oxidase (lsao) by measuring its denaturation constant (kden) at various temperatures from 37 to 67 degrees centigrade in 100 mm phosphate buffer, ph 7.0. the results of thermal inactivation curves as well as measuring of a280 at various temperatures were used to calculate kden. moreover, activation energy (ea) for denaturation reaction was obtained from corresponding arrhenius plot. our results showed that unfolding process started to occur at 56 degree centigrade and ea of denaturation was changed at 65 degree centigrade proving a dominant conformational change of the enzyme at this temperature. the results of the kinetic study are coincident with previously reported equilibrium studies denoting the optimum and melting temperature of the enzyme are 56 and 65 degree centigrade, respectively. development and advancing of enzymatic processes used for production and modification of natural polysaccharides are now major biochemistry challenges. the paper investigates enzymatic systems in invertebrates, in particular, an enzymatic complex obtained from the hepatopancreas of red king crab paralithodes camtschaticus, and clarifies its effect on the mechanism of chitin and chitosan hydrolysis. chitinolytic activity was estimated with spectrophotometer using 4-(dimemylamine)-behzaldehyde method by the concentration of n-acetyl-d-glucosamine which is educed under chitinolysis. total glycolytic activity was defined by the sum of n-acetyl-d-glucosamine and d(+)-glucosamine in the reaction with potassium hexaferricyanide (iii). content of d(+)glucosamine in the hydrolysates of chitin and chitosan was estimated by highly effective reverse-phase liquid chromatography (helc) of aminosaccharides with ortho-phthalaldehyde. the paper studies the process of chitin and chitosan glycolysis and the effects of different factors (ph, temperature and time of incubation, enzyme/substrate ratio) on the total glycolytic activity of the enzymatic complex from crab hepatopancreas, which is compared with a previously studied proteolytic and exochitinase activities. a mechanism of enzymatic hydrolysis of chitin and chitosan is suggested. study results allowed the following conclusions concerning glycolytic and deacetylase activity of ep: 1) ep induces the formation of a monomer (n-acetyl-d-glucosamine) and oligomers (chitin and chitosan) with low deacetylation. thus, ep is characterised by a marked endochitinase (endochitosanase) activity; 2) n-acetylglucosamine deacetylase and, apparently, exochitosanase activity was not revealed; 3) it was found that chitinase and protease activities of ep are associated with different enzymes. [background] in opioids, the n-terminal amino acid 2',6'-dimethyl-l-tyrosine (dmt) enhances bioactivity by orders of magnitude. c-terminal modification of the dmt by a methyl group, h-dmt-nh-ch3, exhibited µ-opioid receptor affinity (kiµ = 7.5 nm) equivalent to that of morphine; however, antinociception was only 0.64-0.85% [1] . dmt plays an important role in the message domain to anchor opioid ligands into the active site of opioid receptors, specifically to trigger biological activity by the µ-opioid receptor. [methods] dimerization of dmt through diaminoalkanes [2] or 3,6-bis-(aminoalkyl)-2(1h)-pyrazinone produced potent opioidmimetics with high affinity for µ-opioid receptors (kiµ = 0.02-0.115 nm), agonism (gpi, ic50 = 1.3-1.9 nm), and antinociception in mice after systemic and oral administration, which verified passage through the epithelial membranes of the gastrointestinal tract and blood-brain barrier [3] . (1-aminocycloalkane-1-carboxylic acid) . in this case the ring consists of five atoms. knowing that acylation of the nterminus of several known b2 blockers with a variety of bulky groups has consistently improved their antagonistic potency in the rat blood pressure assay, the apc substituted analogues were also synthesized in n-acylated form (with 1-adamantaneacetic acid (aaa)). the activity of eight new analogues was assayed in isolated rat uterus using a modified holton method in munsick solution and in rat blood pressure tests. the results clearly demonstrated the importance of the position in the peptide chain into which the sterically restricted apc residue was inserted. apc at positions 7 led to preservation or reduction of antagonistic qualities, respectively. acc at position 8 enhanced antagonistic qualities in blood pressure test and led to preservation of activity in antiuterotonic test.. in most cases acylation of the n-terminus led to enhancement of antagonistic potencies. our findings offer new possibilities for designing new potent and selective b2 blockers. background: during the course of developing opioidmimetic analgesics, data revealed that the n-terminal residue 2',6'-dimethyl-l-tyrosine (dmt) plays an important role in anchoring opioid ligands in the active site of opioid receptors. as a single residue c-terminally extended with an aminomethyl group exhibited µ-opioid receptor affinity (kiµ = 7.5 nm) similar to morphine; however, antinociception was only 0.64-0.85% [1] . in order to develop potent µ-opioid agonists, the dimerization of tyr or dmt through diaminoalkanes [2] or 3,6-bis-(aminoalkyl)-2(1h)-pyrazinones [3] resulted in production of unique opioidmimetics with high receptor affinities and potent biological activities [3] . methods: the synthesis of opioids and opioidmimetics and the determination of their receptor binding characteristics were performed as described previously [1] [2] [3] . results and conclusion: newly synthesized 3-(tyr-nh-butyl)-6-(tyr-nh-propyl)-2(1h) pyrazinone and 3-(tyr-nh-propyl)-6-(tyr-nh-butyl)-2(1h) pyrazinones (i and ii) exhibited fairly high binding affinity towards µ-opioid receptor (kiµ = 7.6 and 27.4 nm, respectively). replacement of tyr with dmt in i and ii gave opioidmimetics iii and iv (kiµ = 0.021 and 0.051 nm, respectively); they exhibited 361-and 537-fold higher binding affinity than the tyr derivatives. while iii is a dual µ-/δ-opioid agonist, iv is only a µ-opioid agonist. these findings pave the way to design additional µ-opioid receptor agonists and antagonists for therapeutic application. divalent cations have been known for a long time to influence significantly binding to receptors and biological activity of the peptide oxytocin (ot). there is very low binding of 3h-ot to the receptors in the absence of these ions. it has been speculated where the divalent cations work. recently an article appeared showing formation of a complex divalent cation-ot and stressing the importance of n-terminal amino group for binding and activity [1] . however deamino analogues of ot are also very active and their binding is also influenced by divalent cations. we have studied ot, deaminooxytocin (dot) and an ot antagonist (antag) by means of electrospray ms and we have observed that all these compounds form molecular adducts with zn2+, mg2+, mn2+ and ca2+. in binding experiments using 125-i antag, the quantity of tracer bound to membranes of hek cells having stable expressed human ot receptor strongly depends on the character and concentration of divalent ions. displacement curves using unlabelled antag do not change in the absence or presence of 10 mm of tested divalent ions. on the other hand, displacement curves using unlabelled ot and dot are shifted to the left in the presence of mg2+ and mn2+, and to much lesser extent by zn2+ and ca2+. all this points to the idea that the divalent ions do work on the site of membrane receptors. biologically active peptides exhibit multiple conformations in solution. thus, the synthesis of conformationally restricted analogues is a valuable approach for determining structure -activity relationships. restrictions can be imposed e.g. through the formation of cyclic structures within the peptide framework by disulfide bridges, or by substitution of chosen amino acid residues that limit conformational freedom, thus forcing the peptide backbone and/or side chains to adopt specific orientations. in recent years, conformationally constrained analogues of bioactive peptides seem to be a feasible approach to providing useful informations concerning threedimensional structure of such compounds which, in turn, could rationalize our knowledge about structure-biological activity relationships and thus help to design peptides with desired pharmacological properties. steric restrictions can be introduced by the formation of cyclic structures within the peptide backbone or by incorporation of amino acids with limited conformational freedom, which in turn results in specific orientations of the peptide backbone and its side chains. another approach to reduce the flexibility of the analogue is substitution of chosen amino acids with various types of pseudopeptides prepared trough short-range cyclizations. the present work is a part of our studies aimed at clarifying the influence of sterical constraints in the n-terminal part of arginine vasopressin (avp) and its analogues on the pharmacological activity of the resulting peptides. we describe the synthesis of four new analogues of avp substituted at positions 2 and 3 or 3 and 4 with two diastereomers of 4-amino-pyroglutamic acid and four peptides in which we combined the above modification with the placement of 3mercaptopropionic acid (mpa) at position 1. all the peptides were tested for their in vitro uterotonic, pressor and antidiuretic activities in the rat. different strategies to modulate shp-1 activity protein tyrosine phosphatase shp-1 consists of two sh2 domains n-terminal to the catalytic (ptp) domain and a short c-terminal tail. the binding of a py-ligand to the n-sh2 domain is required for an efficient activation of shp-1 phosphatase activity. the specificity of the shp-1 sh2 domains is determined by the py-residue (position 0) and residues at positions -2, +1 and +3. combinatorial peptide library methods revealed different classes of consensus sequences for both sh2 domains [1, 2] . in addition, the importance of residues c-terminal to py+3 (+4 to +6), in particular for binding to the n-sh2 domain, has been demonstrated [3] . together with investigations of the determinants for optimal sh2 domain binding and stimulation/inhibition of shp-1 activity [4] , these informations were useful for the generation of different strategies for effectors of shp-1 activity. peptides cyclized between different positions of the general consensus py-2 to py+3 were synthesized and evaluated with respect to n-sh2 domain binding and stimulation of phosphatase activity. structure-activity studies have revealed that the specificity of an integrin towards its rgd-containing ligands can be evaluated through the distances between the cβ atoms and/or the distance between the charged centers of arginine and aspartic acid as well as, the pseudo-dihedral angle (pdo), composed by the r-cζ, r-cα, d-cα and d-cγ atoms, which defines the relative orientation of the arg and asp side chains. in a previous study [1] , the antiaggregatory activity of rgd peptide analogues, i.e. their ability to act as fibrinogen receptor αiibβ3 antagonists, was correlated with the above structural criteria. our results suggested that the fulfillment of the criterion -45ο < pdo < +45ο is a prerequisite for an analogue to exhibit activity. in the present study, we examine the above criteria to rgd-containing 15peptides, derived from the active sites of the ecm proteins fibrinogen, fibronectin and vitronectin, as well as, from the cryptic rgd site of von willebrand factor. the correlation of the structural data with the biological activity of compounds, are in good agreement with the previously mentioned -45ο < pdo < +45ο criterion. furthermore, our results show that the differences in activity of compounds, which display similar distances between the charged centers of arg and asp, can be better evaluated by the pdo structural criterion. acknwolegments : this work was supported by grants from eu and the hellenic ministry of education ( heraklitos). references : the gpiib/iiia receptor, which is a member of the integrin family, is the most abundant receptor in the surface of platelets and can interact with a variety of adhesive proteins including fibrinogen, fibronectin and von willebrand factor. fibrinogen binding on gpiib/iiia is an event essential for platelet aggregation and thrombus formation. mapping of the fibrinogen binding domains on gpiib subunit suggested the sequence 313-332 as a putative binding site [1] . this region was restricted to sequence gpiib 313-320 (ymesradr) using synthetic octapeptides overlapping by six residues [2] . the ymesradr octapeptide inhibits adp stimulated human platelets aggregation and binds to immobilized fibrinogen. in this study we present the conformational analysis of three synthetic analogues yaesradr (a2) ymesaadr (a5), and ymesraar (a7), using nmr spectroscopy and distance geometry calculations. common structural characteristic of peptides a2 and a7 is the interaction between the side chains of arg5 and glu3, however in a2 the guanidino group of arg5 seems to form salt bridges with both glu3 and asp7. peptide a5 is stabilized only by a week interaction between arg8 and glu3 side chains. the interactions between the residue side chains provoke different overall shape of the three molecules. the most populated structural family of a2 exhibits a π backbone shape, a5 a turn around -s4a5-, while a7 an almost extended shape. background and aims: endomorphin-2 (em-2: h-tyr-pro-phe-phe-nh2), endogenous opioid peptide isolated from bovine and human brain, has high affinity and selectivity for the mu receptor and produces potent and prolonged analgesia in mice [1] . in this presentation, the incorporation of ethylene-bridged phe-phe unit (eb[phe-phe]) or piperidine carboxylic aid (pic) in position 2 was carried out to obtain more potent agonist or antagonist with stability against dipeptidyl peptidase iv (dpp iv). methods: the synthesis of eb[phe-phe] unit was achieved according to the procedure of lammek b. et al. [2] protected peptides were synthesized by a solution method using boc-chemistry. the final products were identified by maldi-tof mass spectrometry and elemental analyses. the receptor binding affinity of peptides was assessed by radio-ligand receptor binding assay using mu and delta opioid receptors from rat brain membranes or cos-7 cell membranes expressing each opioid receptors. muc2 glycoprotein, produced by the epithelium of the colon, built up mainly of repeat units of 1ptttpitttttvtptptptgtqt23, can be underglycosylated in colon carcinoma. we have been studying the epitope structure of the muc2 repeat unit with the mucin peptide specific mab 996 monoclonal antibody. this antibody recognizes the 18ptgtq22 sequence as minimal, and 16ptptgtq22 as optimal epitope. our interest lies in the modification of this epitope with maintained or enhanced specificity, and we aim to clarify the effect of different epitope modifications on mab 996 antibody binding: a) amino acid changes in the flanking region, b) glycosylation in the epitope core and in the flank. for this we have prepared a) libraries of ax(1)ptgtqaa and atptgtqx(2)a peptides, and x(1)ptgtqx(2) heptapeptides based on the antibody binding properties of the libraries; and b) glycopeptides pt(galnac)ptgtq, ptpt(galnac)gtq and ptptgt(galnac)q. the peptides were prepared by solid phase synthesis; after purification, esi-ms and amino acid analysis characterisation their antibody binding properties were studied by competitive elisa. our results show that a) although all amino acids in positions x(1) and x(2) resulted in antibody binding; in position x(1) hydrophobic, in x(2) aromatic residues provided stronger binding than that of the native peptide; b) glycosylation on thr(17) did not influence the binding of mab 996, but on thr(19) the presence of n-acetyl-galactosamine, interestingly, slightly increased the antibody recognition. these findings could be useful in designing synthetic peptide vaccines for tumour therapy. histidines play essential role in binding of biological metal ions, either in small or macromolecular chelating molecules, e.g. in metalloenzymes. therefore the low molecular weight polyhistidine type ligands are of potential importance as model substances. continuing our investigations on a novel branched oligopeptide type ligand -(his)4(lys)2lys-nh2 -prepared by solid phase peptide synthesis, we investigated the metal ion binding properties with zinc(ii) and copper(ii). the eight primary metal-binding sites are the four imidazole and four ammine groups on the ligand. phpotentiometric titrations revealed, that up to ph 8 all these donor atoms loose their protons on increasing ph. the competition between the protons and the metal ions results the decrease of pka values to about 1-3 in the case of copper(ii) and to about 4-6 in case of zinc(ii) ion. this reflects the higher stability of the complexes formed with copper(ii) in spite of the weak axial coordination that seems to occur in zinc(ii) complexes. combined potentiometric, spectrophotometric, cd and nmr spectroscopic methods were utilized to investigate the speciation and the structure of the complexes formed in aqueous solution. the prepared cu(ii) complexes cleaved dna, but it is not known whether in oxidative or in hydrolytic manner. because of this ambiguity further studies with zn(ii) complexes will be undertaken. this work has received support through sapstclg97697 nato collaborative linkage grant and from the hungarian science foundation (otka t43232). lgr8. further studies have shown that in both male and female gonads, insl3 and lgr8 represent a paracrine system important for meiosis induction in the ovary and male germ cell survival in the testis. thus insl3 may have clinical applications in fertility management. we undertook to determine the key structural elements responsible for its unique actions. methods: alanine-scanned analogues of human insl3 and mimetics of the b-chain alone were prepared by solid phase peptide synthesis. each was subjected to cd spectroscopy for secondary structure analysis and assayed for in vitro lgr8 binding and activation activity. the tetrapeptide h-dmt-d-arg-phe-cbp was found to be a selective µ agonist [ic50 (gpi) = 78.8 ± nm] with 40-fold lower potency than the corresponding, highly potent tiq4-tetrapeptide, but with still 3-fold higher potency than leu-enkephalin. in conclusion, we developed selective, cbp-containing δ antagonists and µ agonists with significant potency. recently, we described the syntheses and biological activities of several opioid peptide analogues that contained the n-terminal sequence 1-4, common to dermorphin and deltorphin. some of them showed very high agonist potency both in the gpi assay and in the mvd assay [1, 2] . in this work, we designed new analogues in which the sequences were elongated at the c-terminal to obtained the full sequences of dermorphin (a) and deltorphin (b). the syntheses of compounds and their biological activity profiles will be discussed. background and aims: endomorphin-2 (em-2: tyr-pro-phe-phe-nh2) is very potent endogenous opioid peptide, which exhibits high affinity and selectivity for the mu-opioid receptor [1] . previously, we had reported that [ac3c2]-em-2 containing 1-aminocyclopropane-1-carboxylic acid (ac3c) exhibited higher affinities than em-2 for the mu-opioid receptor [2] . in order to clarify that the substitution of 1-aminocycloalkane-1-carboxylic acids (acnc: n indicates the number of carbon atoms in a ring) for pro in position 2 of em-2 is efficient to obtain higher affinity for the mu-receptor, we synthesized . therefore, the replacement of pro to ac3c and ac4c will be efficient to make these analogs adopt bioactive conformation and exhibit high affinity for mu-receptor. in the past few years, many attempts have been made to prepare a synthetic insulin. the biological activity of insulin is known to be closely related to the c-terminal octapeptide fragment of its b-chain.it was found that b23 gly and b24 phe were present in all insulins so far obtained from various animal species indicating the significance of these two residues.it would therefore seem desirable to study the effect of each of these two amino acid residues or both on biological activity of the octapeptide fragment of the b-chain. a heptapeptide arg-phe-tyr-thr-pro-lys-ala-och3, corresponding to (b22-b30) insulin des gly23-phe24, and an octapeptide arg-phe-phe-tyr-thr-pro-lys-ala-och3, des gly23 were synthesized using the solid phase method. the c-terminal ends of both peptide were converted to methyl ester by transesterification cleavage from the resin. the side chain protecting groups were removed by hf. manual counter current distribution method was used for purification of the free peptides. the way to solve the evaluation of tyrosine containing peptide was studied. the free methyl ester peptides were administered for insulin-like activity test by glucose metabolism in the rat fat cells technique in vitro. aim of this study is to develop peptides as useful tools for degradation of synthetic dyes, which are often pollutants. we focused our interest in peroxidases, a class of enzymes reported to efficiently degrade azo and anthraquinonic dyes. in particular, the fungus versatile peroxidase (vp) of pleurotus eryngii can perform this degradation. therefore, our goal is the synthesis of a peptide based on this peroxidase able to emulate its biological function. the linear and cyclic peptide sequences were derived by the theoretical model of vp [pdb: 1a20], which determined the amino acids fundamental for the desired function of the active sites. in particular, the residues instrumental for the coordination of the heme, the mn binding site, and the long range electron transfer pathway [1] , were pin-pointed. moreover, we calculated the radius of the heme cavity. the next step was the synthesis of these peptides in order to verify the coordination of the heme and optimize their sequences. the syntheses were carried out by solid-phase following the fmoc/tbu strategy. because of purification difficulties of the fully-protected peptide, we undertook an alternative synthetic pathway, based on a solid phase head-to-tail cyclisation strategy, following the fmoc/tbu/allyl three-dimensional protection scheme [2] . next steps will be to test the coordination properties of the synthetic peptides, with respect to the heme, and further computational studies based on the new model of pleurotus the calcium plays an important role in biochemical pathways. it binds to enzymes and proteins in a different process. aspartic (asp, d) and glutamic (glu, e) acid side chains are the main ligands of calcium, but the contribution of the backbone carbonyl groups in the binding is also important. generally the binding places in the proteins are an unstructured loop between two helixes (310-or alpha-helix). the common sequence is the so-called ef-hand motif, which contains 12 amino acids [1] . it is already known that some proteins also bind calcium with a non-ef-hand loop. for example alpha-lactalbumins have a ten amino acid long sequence for binding [2] . it is an asp rich sequence where 5 asps are closer to each other than in ef-hand motif (-k79fldddltdd88-) but only 3 asps side chains take part in calcium coordination. we constructed a series of cyclopeptides to mimic the loop structure of alpha-lactalbumin [3] . in this study we focus on determining the importance of conservative amino acids within the ca2+ binding loop of this protein, using microcalorimetry (itc). the itc measurements were performed in different organic solvents and at different temperature. the synthesis of fatty acids in adipose tissue. in this article, we present the solution structure of gip in water and tfe/water determined by nmr spectroscopy. the calculated structures are characterised by the presence of an -helical motif between residues ser11-gln29 and phe6-gln29 respectively. the helical conformation of gip is further supported by cd spectroscopic studies. six gip(1-42)ala1-7 analogues were synthesised by replacing individual n-terminal residues with alanine. alanine scan studies of these n-terminal residues showed that the gip(1-42)ala6 was the only analogue to show insulin secreting activity similar to that of the native gip. however, when compared with glucose its insulinotropic ability was reduced. for the first time, these nmr and modelling results contribute to the understanding of the structural requirements for the biological activity of gip. a knowledge of the solution structure of gip and of the role of its individual residues will be essential in the understanding of how they interact with the gip receptor. efrapeptins are pentadecapeptides produced as a mixture of six closely related analogues (efrapeptin c-g) by the fungus tolypocladium niveum and other members of this species. they consist predominantly of the nonproteinogenic amino acids -aminoisobutyric acid (aib), isovaline (iva), -alanine ( ala) and pipecolic acid (pip), have an acetylated n-terminus and bear an unusual cationic c-terminal headgroup derived from leucinol and 1,5-diazabicyclo[4.3.0]non-5-ene. efrapeptin c is a competitive inhibitor of the f1-atpase and active against the malaria pathogen plasmodium falciparum. an anti-proliferative effect was also reported. conformational analysis of efrapeptin c in trifluoroethanol and dimethylsulfoxide was conducted to obtain structure-affinity relationships. the absence of amide-and -protons resulted in an imperfect assignment and unsatisfying conformational study. specific deuteration of methyl groups in aib did not simplify the assignment. cd and ft/ir spectra hint to helical or beta-turn secondary structures as main structure elements. residual dipolar couplings (rdc) were measured in a stretched cross-linked poly(dimethylsiloxane) gel in dichloromethane. the impact of the rdc on the conformational analysis led to an improved high resolution structure from simulated annealing protocols and consolidated the formation of a helical structure of efrapeptin c in nonpolar solution which is comparable with the binding pocket of the f1-atpase. finally, the dynamics of the resulting structures was studied using the gromos96 force field in explicit solvent. serotonin selective reuptake inhibitors (ssris) are currently among the most frequently prescribed therapeutic agents of depression. their therapeutic use includes also obsessive-compulsive disorder, panic disorder, bulimia. the serotonin transporter (sert) is the target of serotonin selective reuptake inhibitors (ssris). altough the inhibition is the proximal event in antidepressant action, the clinical benefit of antidepressant medications requires weeks of continuous dosing, indicating that their mechanism of action involves events downstream from acute transporter blockade. long-term effects of ssri treatment may be due to changes in intrinsic properties of sert structure, function, or regulation. thus, understanding the mechanism of action of sert remains a primary goal in the search for developing novel treatments for diseases associated with serotonergic dysfunction. in the present study experimentally determined ligand selectivity of the buspirone analogues toward the serotonin transporter was theoretically investigated on the molecular level. the model of serotonin transporter based on the crystal structure of bacterial homologue from aquifex aeolicus (leutaa) was constructed using the traditional homology modelling approach. a series of docking experiments with ssri's were conducted, using interactive molecular graphics techniques combined with energy calculations and analysis of the transporter-ligand complexes. structural information about the serotonin transporter and its molecular interactions with ssri's is important for understanding the mechanism of action of these drugs and for development of drugs with improved potency and selectivity. the protein kinase c (prkc) is a member of a super-family of the eukaryotic receptor protein kinases. it forms dimers and is anchored in the membrane, with a cytoplasmic kinase domain and an external domain, presumably acting as a sensor. prkc enables formation of biofilms of bacillus subtilis which show a high degree of spatial organization. they colonize various surfaces and produce complex antibiotic resistant communities. prkc acts as a ser/thr kinase with features of the receptor kinase family of eukaryotic hanks kinases. our current study involved theoretical modeling of the protein kinaze prkc complexes with the modified atp. the ligands were selected from a set of molecular probes developed by k. shah and coworkers [1] . each modified atp molecule was docked to the active site of the kinase molecule using autodock genetic algorithm procedure. the optimized structures of the complexes were submitted to the molecular dynamics simulations in the amber force field. we obtained four optimized structures of prkcc complexes in water. the results suggest the great similarity of our complexes with human cyclin-dependent kinase 2 [1] complexes. background and aims. indolicidin is a 13-residue antimicrobial peptide, which was isolated from bovine neutrophils. this molecule possesses a wide spectrum of antibacterial, antifungal and antiviral activity, furthermore it has also haemolytic effect. data derived from structural investigations led to considerably diverse conclusions regarding the secondary structure of this peptide, therefore the aim of this study was to examine the effect of cis-trans isomerization on the conformational properties of this antimicrobial peptide. methods. the conformational analysis of indolicidin containing cis or trans xxx-pro peptide bonds was performed by simulated annealing calculations with the use of amber force field. results. for the conformers of indolicidin with cis or trans xxx-pro peptide bonds, the evolving secondary structural elements were examined and poly-proline ii helix and type vi beta-turn were identified. in the case of this peptide, various intramolecular interactions may play an important role in stabilizing the structure of conformers. therefore the presence of the h-bonds between backbone atoms, the aromatic-aromatic interactions between the side-chains of trp amino acids and the proline-aromatic interactions between the side-chains of trp and the pyrrolidine rings of pro amino acids was investigated. conclusions. the conformational comparison of the peptides possessing cis or trans xxx-pro peptide bonds resulted in different secondary structural elements for both isomers, which are the poly-proline ii helix and type vi beta-turn for the trans and cis isomers of indolicidin, respectively. the occurrences of various intramolecular interactions are in agreement with the observed secondary structures. we have shown the monte carlo conformational search using macromodel is useful for conformational study of oligopeptides prepared from alpha, alphadisubstituted alpha-amino acids. moreover, we have studied conformational analysis of oligopeptides containing chiral alpha, alpha-disubstituted alphaamino acids to predict the helical screw sense of helical structures. here we report computational study on conformation of oligopeptides containing cyclic alpha, alpha-disubstituted alpha-amino acids with side-chain chiral centers. background and aims. the homopolymeric amino acids (hpaas) are polypeptides consisting of the same amino acids. some of them play a relevant role in the formation of several neurodegenerative diseases. most probably the poly-(ala) and poly-(gln) are the best representatives of these peptides because of their important biological effects. our aim was to perform conformational analysis and structural investigation of these two hpaas. methods. to explore the conformational spaces of the peptides, simulated annealing (sa) and random search (rs) calculations were carried out using amber force field. two different forms of the hpaas were modelled: either with charged n-terminal amino group and c-terminal carboxyl group, or with the n-and c-termini blocked by acetyl and n-methyl amide groups, respectively. results. for the conformers obtained by sa and rs calculations, the occurrences of various secondary structural elements like different types of beta-turns, gammaand inverse gamma-turns, alpha-helix, 310-helix, poly-proline ii helix and beta-strand were investigated. in the cases of various helices and beta-strand, segments with different lengths characterized by these secondary structures were determined along the entire sequence of peptides. for the conformers of the hpaas, the intramolecular h-bonds formed between the backbone atoms as well as between the backbone and side-chain atoms were identified. the vasopressin and oxytocin receptors (v1ar, v2r and otr) are membrane-embedded proteins belonging to the large family a g protein-coupled receptors (gpcrs). they are involved in crucial physiological functions as the regulation of water metabolism, control of blood pressure and stimulation of labor and lactation, mediated via v2r, v1ar and otr, respectively. as such, they are involved in a number of pathological conditions and are important drug targets. understanding their inhibition and activation mechanisms may improve design of ligands capable of selective stimulation or blockade of the respective receptors presenting the therapeutic targets. to investigate the otr, v1ar and v2r interactions with agonists and antagonists thirty computer models of receptor-ligand complexes have been modeled via docking and molecular dynamics (md) and analyzed in details. the receptor models were built on rd crystal structure template or using the coordinates of mii-gtα(338-350), for non-active and activated models, respectively. the ligands (arginine vasopressin, oxytocin, desmopressin, atosiban ([mpa1,d-tyr(et)2,thr4,orn8]ot) and barusiban (mpa1,d-trp2,ile3,allo-ile4,asn5,abu6,mol7) were docked into the receptors. the complexes have been embedded into the hydrated popc bilayer and submitted to 1ns unconstrained md in the amber force field. the relaxed systems have been obtained and analyzed in details. the receptor residues responsible for agonists/antagonists binding have been identified and mechanism of binding involving the highly conserved residues has been proposed. a three-dimensional models of the neurohypophyseal hormone receptors were constructed using a multiple sequence alignment and either the crystal structure of bovine rhodopsin or the complex of activated rhodopsin with gta c-terminal peptide of transducin rd*-gt(338-350) prototype to obtain nonactive or activated receptor models, respectively. analogs were docked to v1ar, v2r and otr, both non-active and activated models. the low-energy receptor-ligand complexes, with properly docked analogs were submitted to the constrained simulated annealing (csa), in vacuo. the relaxed receptoranalog models were obtained. the residues responsible for analogs binding to v1ar, v2r and otr have been identified and presumable biological activity of these compounds was determined. n-methyldehydroamino acids belong to non-standard amino acids found in nature. n-methyl-(z)-dehydrophenylalanine was found in tentoxin, a selective weed killer, having been produced by several phytopathogenic fungi of the alternaria genus. n-methyl-(z/e)-dehydrobutyrine and n-methyldehydroalanine are components of nodularins and microcystins, families of hepatoxins produced by species of freshwater cyanobacteria, primarily nodularia spumingena and microcystis aeruginosa. the simplest n-methyl dehydropeptides, ac-delta(me)xaa-nhme (where xaa = ala, (z/e)-abu, (z/e)-phe, and val) and, for comparison, the saturated ac-l-(me)ala-nhme analogue were investigated using computational methods. cis-trans b3lyp/6-31+g**//hf/3-21g ramachandran potential energy surfaces were created. the conformers found were optimised at the b3lyp/6-31+g** level. the effect of the electrostatic solute/solvent (water) interaction on the solute energies was investigated within the scrf method using the polarisable continuum model (pcm) on the geometries of solutes in vacuo. it was found that for all the studied dehydropeptide molecules the lowest conformer (phi, psi = ~ -109°, 10°) has the cis n-methyl amide bond. this feature seems to be independent of the dehydroamino acid moieties, the c-beta substituent and the z/e configuration. the pi-electron conjugation as well as the n-h···n hydrogen bond play the dominant role in the stability of this conformer (see figure) . the preliminary nmr investigations into the conformational preferences of the studied molecules in solution confirm the theoretical results obtained. the strong tendency of the n-methyl amide bond to adopt the cis configuration seems to be the reason why n-methyldehydroamino acids are found in small natural cyclic peptides, where they ensure the conformational flexibility necessary for biological action. the purpose of this study was to determine the potentials of mean force (pmf) of the interactions between models of nonpolar amino acid side chains in water. the potentials of mean force (pmf's) dependent on orientation were determined for systems forming hydrophobic and diagonal complexes composed of side-chain models of alanine, valine, leucine, proline and iso-leucine, respectively, in water. for each hydrophobic pair in water a series of umbrellasampling molecular dynamics simulations with the amber force field and explicit solvent (tip3p water model) were carried out and the pmfs were calculated by using the weighted histogram analysis method (wham). in all cases a characteristic shape of pmf plots for hydrophobic association were found, which was manifested as the presence of contact minima and solvent separated minima. depths of contact minima for all systems studied were about 1 kcal/mol. in this work we compared the ability of two theoretical methods of ph-dependent conformational calculations to reproduce experimental potentiometric-titration curves of two models of peptides: ac-k5-nhme in 95% methanol (meoh)/5% water (h2o) mixture and ac-xx(a)7oo-nh2 (xao) (where x is diaminobutyric acid, a is alanine, and o is ornithine) in water, methanol (meoh) and dimethylsulfoxide (dmso), respectively. in theory, in all three solvents, the first pka of xao is strongly downshifted compared to the value for the reference compounds, the water and methanol curves have one, and the dmso curve has two jumps characteristic of remarkable differences in the dissociation constants of acidic groups. the predicted titration curves of ac-k5-nhme are in good agreement with the experimental ones; better agreement is achieved with the md-based method. the titration curves of xao in methanol and dmso, calculated using the md-based approach, trace the shape of the experimental curves, reproducing the ph jump, while those calculated with the edmc-based approach, and the titration curve in water calculated using the md-based approach, have smooth shapes characteristic of the titration of weak multifunctional acids with small differences between the dissociation constants. quantitative agreement between theoretically predicted and experimental titration curves is not achieved in all three solvents. the poorer agreement obtained for water than for the nonaqueous solvents suggests a significant role of specific solvation in water, which cannot be accounted for by the mean-field solvation models m337 a. papakyriakou 1 , g.f. vlachopoulos 2 , g.a. spyroulias 2 , e. manessi-zoupa 3 , p. cordopatis 2 angiotensin-i converting enzyme (ace) belongs to the m2 family of the ma clan of zinc metallopeptidases and can act either as a dipeptidyl carboxypeptidase, which catalyses the proteolytic cleavage of dipeptides from the carboxy terminus of a wide variety of peptides, or as an endopeptidase, which hydrolyses peptides bearing amidated c-termini. among the former category of ace peptide substrates, the most distinguished are those involved in blood pressure regulation, such as angiotensin i (angi) and bradykinin (bk). in the latter category falls the gonadotropin-releasing hormone (gnrh) in an attempt to analyze molecular interactions at atomic level we simulated the ace-substrate complexes, using the recently determined 3d crystal structure of ace testis isoform and a knowledge-based docking method in order to insert the peptide substrate (angi, bk and gnrh) of ace into its catalytic cleft. in order to introduce the effect of protein mobility and gain information about enzyme-substrate recognition and interaction we have sampled the conformational space of these complexes via molecular dynamics simulations with explicit solvent representation. we have also performed molecular dynamics calculations with tace-inhibitor complexes, such as lisinopril, as well as with tace mutated at specific sites, such as the ligands of the two buried chloride ions that have been shown to affect substrate activity. our results provide new insights into the role of specific domains of tace and their implication in the enzyme activity, which is not readily apparent from the available crystal structures. two main mechanisms for the propagation of action potential in myocytes are: 1) the free flow of local circuit current through gap junctions and 2) the effect of electrical field. here we study effect of each mechanism and their importance during action potential propagation. method: we simulated the cardiac myocyte by the orcad software, then used the model of sinoatrial node to stimulate the myocytes model and studied the propagation of action potential with and without gap junction. result: our results show that, although gap junction solely is not able to mimic physiological condition, but it is necessary for normal cardiac functioning. on the other hand, electric field is not sufficient for successful propagation of action potential and the existence of gap junction is necessary. anthrax is a disease of animals and humans, caused by the bacterium bacillus anthracis. anthrax toxin (at) consists of three proteins, one of which is the anthrax lethal factor (alf). alf is a gluzincin zn-dependent highly specific metalloprotease (~90.000 kda), which belongs to the m34 family of the ma clan of zinc metalloproteases. alf cleaves most isoforms of mitogen-activated protein kinase (mapk)-kinases (meks) close to their amino termini, leading to the inhibition of one or more signaling pathways. no data are available on the enzyme-substrate interaction at the molecular level. therefore, we performed classical molecular dynamics simulations on the alf-mkk/mek complexes in order to probe protein-substrate interactions. the simulations pinpointed specific hydrophobic as well as electrostatic alf-peptide substrate interactions and these data were exploited in the building of virtual combinatorial libraries of di-and tri-peptides using the twenty native aminoacids. by applying docking simulations to anthrax zn-metalloprotease around 1.000 peptide substrates were virtually screened according to their binding affinity. data suggest that complexes of alf with peptides substrates bearing arg, trp, lys and phe aminoacids, exhibit the highest binding affinity providing evidence for electrostatic interactions between negatively charged residues of alf's active site and positively charged side-chains of di/tri-peptides. new libraries of substrates were built incorporating non-protein residues, organic moieties and chelating groups. alf-substrate complexes with the best score (in terms of binding energy) are further analysed. in the present studies we designed and synthesised seven new bradykinin (bk) analogues and evaluated them in the in vivo rat uterotonic assay using a modified holton method in munsick solution on a strip of rat uterus and in blood pressure test. we used [arg0, hyp3, thi5, 8, d-phe7]bk, the b2 antagonist of vavrek and stewart as a model, when designing our analogues. in all cases, the n-terminus of our peptides is acylated with bulky substituent. we previously reported that acylation of the n-terminus of several known b2 antagonists with various kinds of bulky acyl groups has consistently improved their antagonistic potency in rat blood pressure assay. on the other hand, our earlier results seem to suggest that effects of acylation on the contractility of isolated rat uterus depend substantially on the chemical character and size of the acyl group, as we observed that this modification may either change the range of antagonism or even transform it into agonism. the peptides were synthesized by the solid-phase method using the fmoc-strategy the modifications proposed either preserved or increased the antagonistic potency in the rat blood pressure test. on the other hand, the seven substituents, differently influencend the interaction with the rat uterine receptors and except one led to decrease of antiuterotonic activity. in both cases acylation of the n-terminus led to enhancement of antagonistic potencies. our results may be of value in the design of new b2 agonists and antagonists. the formations of amyloid fibrils have been reported as for various amyloidosis. several structural models of fibrils are proposed for respective proteins so far. however, their common basic structures and universal features to induce amyloid fibril formations are not known in detail. previously, we examined intermolecular interactions among the several amino acid residues in barnase, which is known to form amyloid-like fibril. based on the experimental results using a series of mutant barnase, we discovered that the interactions between hydrophobic side-chains are the most essential driving force to form the fibrils and that both intermolecular and inter-sheet interactions in the fibril maintain highly ordered molecular packing. in the present paper, we describe a novel prediction method for core regions of various fibril-forming proteins and show the verification of the above possible structural principle. at first, we calculated the interaction's score between side-chains in the antiparallel orientation of beta-strands. next, the peptides with predicted sequences of fibril cores, a couple of high-scored regions with a designed turn moiety to induce a hairpin-like form, were chemically synthesized by spps. as a result, the formation of amyloid fibrils was confirmed for most of high-scored sequences. in addition, we also applied this method to prion protein, we could predict 4 possible beta-strands with hetero-paired orientation. some synthetic peptides involving these strands were proved to have fibril-forming ability. thus, we have developed the novel method to predict the core regions that induce amyloid fibrils. a principal factor analysis (pfa) is a very efficient way of identifying patterns in the data sets even if the patterns are hard to find (e.g. in the high dimensional data sets). this is the reason why the pfa method can be powerful tool for analyzing molecular dynamics (md) trajectories. it is possible to reduce dramatically the trajectory size without loosing significant structural information by applying the pfa procedure. we used this tool for interpretation of results from the molecular dynamics simulations of the model of the transcription factor nf-kb. nf-kb is a protein involved in the numerous biological processes such as regulation of immune response, inflammation, various autoimmune diseases and is used by many viruses, including human immunodeficiency virus (hiv), to activate transcription of their own genes. only the trajectory of the backbone atoms of the nf-kb were subjected to the further analysis. peptides contain many basic sites such as side chains of basic amino acid residues, oxygen and nitrogen atoms of amide groups, and terminal amino groups. these parts can interact with protons. this interaction can change conformational behaviour of peptides and, consequently, their biological functions. the interaction becomes even stronger in the gas phase. in that case, the stability of the peptide chain is influenced, which may have impact on peptide fragmentation during mass spectroscopy analysis of peptide structures. in this study, we will present the interaction of proton with carbonyl oxygens in the model of alanine tripeptide. quantum chemical calculations employing density functional theory using hybrid b3lyp functional and 6-31++g** basis set were used to describe this interaction and also to find possible pathways of proton transfer among interaction sites. two different mechanisms of proton transfer were found. the first mechanism is represented by an isomerization of the proton around the double bond of the carbonyl group. the second mechanism is based on the large conformational flexibility of the tripeptide model where all carbonyl oxygens cooperate. the later mechanism exhibits nearly half energy barrier of the rate-determining step compared to the first one. we focus our attention on situation, in which methyl groups attached to alpha atoms in tripepetide model influence the conformational behavior. results will be presented for all four possible stereochemical configurations. a. papakyriakou 1 , p. galanakis 2 , p. gazonis 2 , g.a. spyroulias 2 p53 protein is one of the most effective defensive weapons of human body against carcinogenesis, due to its tumor suppression properties. it has been noticed, in many types of cancer, that the functions of p53 are being downgraded or even suppressed and this fact is ought to the presence of mutated forms of p53 or to the complete absence of the protein. the suppression of p53 levels is being indirectly regulated by the protein itself, which activates the expression of a gene, the oncogene mdm2 (murine double minute 2), which expresses the mdm2 protein, known as human-mdm2 or just hdm2. hdmx protein is a homologue protein to hdm2 and is being implicated, through various biological processes, in the suppression of p53. however, recent experimental evidence suggests that hdm2 and hdmx proteins are not the only ubiquitin ligases that negatively regulate p53 through ubiquitin pathway. two recently discovered e3 ligases, cop1 and pirh2, are also proposed to promote p53 for degradation. all these proteins function as e3 ligases bearing a ring finger domain. these domains are characterized by their high content in cysteines and the binding of two zn(ii) ions while they catalyze the latter stage of protein signaling for proteolysis by the 26s proteasome, through the ubiquitin pathway. the structure variation and the stability of these ring fingers is studied through molecular dynamics simulations of 2-5 ns and structure variations are analyzed in a structure-function correlation basis. semax is a synthetic analogue of adrenocorticotropic hormone acth 4-10. it is a nootropic agent containing seven amino acids met-glu -his-phe-pro-gly-pro without hormonal (adrenocorticotrophic) activity. semax is neuroprotective via a mechanism involving the regulation nitric oxide (no) and lipid peroxidation. semax proved to be highly effective in abating the rise in no and restoring neurologic functioning [1] . it was found to improve intellect and memory in healthy human. it is effective in rehabilitation of people with memory and motor disorders, parkinson's and hantington's diseases, after cerebral stroke and head trauma [2] . to study conformation dynamics in connection with in vivo activity of semax the molecular dynamics method of standard protocol was applied [3] . semax and about twenty its analogs were studied. using cluster analysis method semax was found to be more labile among various synthesized analogous (met-gln -his-phe-pro-gly-pro; gly-glu-his-phe-pro-gly-pro; lys-glu-his-phe-pro-gly-pro; glu-his-phe-pro-gly-pro; his-phe-pro-gly-pro). because of collective degree of freedom it has one more stable configuration that is unreachable in analogs. singularities of semax and analogous were studied using 2-d, 3-d poincare maps, auto and crosscorrelation functions of special type in terms of topological structure of energy hypersurface. this work was supported by rfbr (pr. 04-04-49645), russian ministry of education and science, moscow government and crdf. rhodopsin (rd) is the only representative of g-protein coupled receptors (gpcrs) whose structure has been described with high resolution. thus, it has become the structural prototype for other gpcr. these receptors are involved in transduction of various signals into the cell and actions of many hormones and neurotransmitters. about 50% of all drugs act through gpcr. growing evidence that rd and related gpcrs form functional dimers/oligomers, followed by direct proof (using atomic force microscopy -afm) that in the retina rd associates into a paracristalline network of rows of dimers, need models of rd-transducin (g t -heterotrimeric protein) complex that would envision an optimal rd dimer/oligomer amenable to satisfy all well documented interactions with gt. current model includes tetramer built of two activated (metaii) and two inactive rd molecules, ligands stabilising metaii: gtα(ile338-phe350) and gtγ(asp60-cys71)farnezy, lipid bilayer built of 36 pc (phosphatidylocholin head groups) , 6 ps (phosphatidyloserine) and 30 pe (phosphatidyloetanolamine) (all three types of phospholipids contain the polyunsaturated docosahexaenoyl chain -dha) and water. experimental data concerning shape of oligomer, conformational changes in metaii, proper interactions and distances among residues have been looked upon. the poster shows results of the molecular dynamic carried in amber force field for ~6000ps in the periodic box. conformational changes which took place during simulation caused proper adaptation one another monomers in tetramer and ligands to activated receptors. the human cystatin c (hcc) is a one of known domain swapping proteins. during this process, one of the hcc β-hairpins (β2-l1-β3) changes its conformation forming long β-strand. this conformational transition destabilizes the monomer structure and leads to domain-swapped dimer. the causative force for changing the βhairpin conformation is assumed to be the alleviation of distortions of the l1-loop val57 amino acid residue's backbone. following the above assumption and our previous conformational studies of the hcc β-hairpin peptide we investigated the influence of the point mutations, v57d, v57p and v57n of the val57 residue, on the β-hairpin peptide structure. the conformational studies by means of cd spectroscopy and molecular dynamics studies were performed. the study revealed that the hcc peptide with the wild-type sequence has the strongest tendency from all studied peptides to form a β-hairpin structure. on the basis of these results we conclude that the presence of distortions in the val residue of l1-loop is unlikely to cause the 3d domain swapping of the human cystatin c. acknowledgments: this work was financially supported by the ministry of scientific research and information technology of poland under grant 1t09a10430. temporin a (ta) (flpligrvlsgil-nh2) and temporin l (tl) (fvqwfskflgril-nh2 ) are small, basic, hydrophobic, linear antimicrobial peptides amide found in the skin of the european red frog, rana temporaria. these peptides have variable antibiotic activities against a broad spectrum of microorganisms, including clinically important methicillin-sensitive and resistant staphylococcus aureus as well as vancomycin-resistant enterococcus faecium strains. to gain further insight into the mechanism of action of these small antimicrobial peptides, we have investigated their conformational behaviour in different environmental conditions. more specifically, we deeply investigated by solution nmr spectroscopy in water and water/dmso (8:2) solutions as isotropic solutions and 200 mm aqueous solution of dpc (dodecylphosphocholine) was used as membrane mimetic environment. understanding the basis of the interactions of temporins with membranes could be crucial for the design and synthesis of potent antimicrobial agents. cripto is the founding member of a family of soluble and cell bound growth factors known as egf-cfc [1] distinguished by the presence of an n-terminal signal peptide, two distinct cysteine-rich domains (crd) and a c-terminal hydrophobic region involved in cell surface attachment by a post-translational gpi modification. the characteristic crds, known as egf-like and cfc domains (from the first members cripto, frl1 and cryptic), both span about 40 residues with 3 disulfide bridges [2] each, which, presumably, beside a possible functional modularity, confer them also a structural independence. in this work we have focused our attention on the cfc domain of mouse cripto. the domain has been produced by ssps, along with variants bearing mutation on h104 and w107, that have been described as crucial for alk4 receptor recognition. the two variants have been purified and refolded, achieving the correct disulfide bridges, and then comparatively analyzed by cd spectroscopy under different ph conditions; thus obtaining experimental insights on the structural arrangements of this new class of protein domains. furthermore, the binding properties of wild type and mutants cfc domains to alk4 receptor have been determined by using an elisa-based assay. our results demonstrated that the cfc domain alone can directly bind alk4 in the absence of additional ligands and, furthermore, confirmed a role of h104/w107 in cripto/alk4 interaction. there is considerable interest in the pharmacology of the two cholecystokinin (cck) receptors ccka-r (or cck-1) and cckb-r (or cck-2) that mediate the biological action of the cck hormone. they are membrane receptors belonging to the superfamily of g-protein coupled receptors (gcpr) and are predominantly located in the gastrointestinal tract and in the central nervous system, respectively. a library of 14 cyclic peptide analogues derived from the octapeptide c-terminus sequence of the human cholecystokinin hormone [cck(26-33), or cck8] has been designed, synthesised and characterised. the 14 peptide analogues have been rationally designed to specifically interact with the cck type b receptor (cckb-r) on the basis of the structure [2] of the bimolecular complex between cck8 and the third extracellular loop of cckb-r [namely, cckb-r(352-379)]. the new ligands showed binding affinities generally lower than that of parent cck8. anyway, structure activity relationship data underline that preservation of the trp30-met31 motif is essential, and that the phe33 side chain and a carboxylic group close to the c-terminal end must both be present. the nmr conformational study in dpc micelles of the compound endowed with maximal binding affinity (cyclo-b11, ic50=11 m) shows that this compound presents the turn-like conformation, centred at the trp30-met31 segment, as planned by rational design, and that such conformation is stabilised both by the cyclic constrain and interaction with the micelle. cripto is the founding member of a family of extracellular growth factors called egf-cfc found in mouse, human, chicken, xenopous and zebrafish [1] . these proteins are characterized by the presence of an n-terminal signal peptide, a c-terminal hydrophobic region and two highly conserved cysteine-rich domains, the egf-like (epidermal growth factor) and the cfc (cripto/frl1/cryptic). cripto is strictly required in the early embryonic development and contributes to deregulated growth of cancer cells in adults, since it is highly over-expressed in many solid carcinomas. it has been proposed that each single domain of cripto could bind different protein partners, playing different functional roles [2] . on this grounds, investigation of the single domains 3d-structures can have also strong functional implications. we present here an extensive conformational analysis of the mouse cfc domain (96-134 sequence) and of the w107a mutant based on nmr data. sequences have been synthesized by spps and refolded reconstituting the correct disulfide bridges [3] . the molecular models have been built by computational methods using the nmr data collected under both acidic (ph 3) and nearly physiological (ph 6) conditions. both domains show a globally extended folding with three strands linked by the three disulfide bridges and two connecting loops, in which h104 and w107, key residues in receptor binding, are exposed to the solvent urantide, a selective antagonist. thus, we carried out a study aiming at the characterization of conformational arrangement and affinity properties of ut extracellular segments.we measured by surface plasmon resonance (spr) technology the binding affinities of the three ligands, u-ii, urp and urantide towards the three extracellular loops of ut. furthermore, the secondary structures of the synthetic receptor fragments in presence of dodecylphosphocholine micelles and interaction with ut ligands were analysed using nmr spectroscopy. spr data showed that the ec loop ii was able to recognize the ligands u-ii, urp and urantide with similar affinities while none of these two ligands were able to interact with the extracellular loop i. furthermore, the absence of binding of urantide, a peptide antagonist, suggested strongly that loop iii would be involved in the signal transduction process and implies that u-ii and urp, but not urantide, would bind to ut according to a common pattern. moreover, the results indicate that potent ut antagonists could be designed by producing highaffinity ligand targeting the extracellular loop ii. also, the spr and nmr studies revealed that the synthetic structural ut domains contained some of the conformational and chemical features essential for the binding of hu-ii, urp and urantide to hut. synthetic cysteine-rich replicates of naturally occurring peptides such as hormones, neurotransmitters, enzyme inhibitors, defensins and toxins often can be oxidatively folded in high yields to their native structure. the presence of identical cysteine patterns in the sequence were found to lead to identical disulfide connectivities and homologous spatial structures despite significant variability in the non-cysteine positions. therefore, it is generally accepted to attribute the disulfide connectivities based on the homology of their cysteine pattern. minicollagen-1 from the nematocysts of hydra is a trimeric protein containing n-and c-terminal cysteine-rich domains involved in the assembly of an intermolecular disulfide network. examination of three-dimensional structures of peptides corresponding to these folded domains by nmr spectroscopy revealed a remarkable exception from the general admitted rule [1] . despite an identical cysteine pattern, they form different disulfide bridges and exhibit distinctly different folds. additionally, comparative analysis of the oxidative folding revealed for the c-terminal domain a fast and highly cooperative formation of a single disulfide isomer, the n-terminal domain proceeding mainly via an intermediate that results from the fast quasi-stochastic disulfide formation according to the proximity rule. to our knowledge, this is the first case where two short peptides with identical cysteine pattern fold uniquely and with high yields into defined, but differing, structures. therefore, these cysteine-rich domains may well represent ideal targets for structure calculations to learn more about the elementary information encoded in such primordial molecules. the conformational change of the cellular prion protein, prpc, to its virulent "scrapie" form, prpsc, is believed to be responsible for prion infectivity. and several studies suggest that the prion disulfide bond is important for the stability, structure, and propagation of prion oligomers. to test this hypothesis, we selected two conserved peptides flanking the disulfide bond in the sheep prion protein, and measured the secondary structure of these peptides with circular dichroism, hydrogen/deuterium exchange, and molecular dynamics simulations. our preliminary data suggests that the two peptides do not adopt stable secondary structure, native or otherwise. thus, the folding intermediate of a prion protein seems unlikely to comprise local structure around the disulfide bond. the conformationally labile cα-tetrasubstituted α-amino acid residue bip possesses non isolable (r) and (s) atropoisomers. we have previously reported that in the linear dipeptides boc-bip-α-xaa*-ome with α-xaa* = ala, val, leu, phe, (αme)val and (αme)leu residues at the c-terminal position of bip, the onset of an equilibrium between diastereomeric conformers with unequal populations could be observed by cd and 1h nmr. the phenomenon of induced circular dichroism (icd) represents the basis for the "bip method", an easy and fast configurational assignment for chiral α-amino acids. in search for an extension of the bip method, we investigated the boc-bip-β-xaa*-ome dipeptide series with β-xaa* = β3-hala, β3-hval, β3-hleu, β3-hpro, β3-hphe, or the cyclic β2,3-amino acids (1s,2s)/(1r,2r)-achc and (1s,2s)/(1r,2r)-acpc. low-temperature (233 k) 1h nmr spectra in cd3od revealed the presence of two conformers. significant d.r. (diastereomeric ratio) values were observed for all combinations of bip with both β3-and cyclic β2,3-amino acids. cd analysis in meoh solution of the boc-bip-β-xaa*-ome dipeptides allowed us to conclude that the cd resulting from the induced axial chirality in the biphenyl core of the bip residue gives clear information on the β-xaa* configuration for both β3-and cyclic β2,3-amino acids (except the aromatic β3-hphe), with a p torsion of the biphenyl axial bond of bip being preferentially induced by (l)-β3-xaa* as well as cyclic (1s,2s)-β2,3-xaa* c-terminal residues. we have recently reported that the induced circular dichroism (icd) of the biphenyl core of boc-bip-xaa*-ome dipeptides based on the conformationally labile cαtetrasubstituted α-amino acid residue bip could allow an easy and fast configurational assignment for both α-and β-xaa* amino acid residues. in search for other biphenyl/xaa* architectures in which a transfer of central to axial chirality could result in a potentially useful icd, we considered n-substituted 6,7-dihydro-5hdibenz[c,e]azepine (daz) derivatives from α-and β-amino acids as interesting candidates. in the present communication, we report the syntheses, and the 1h nmr and cd analyses of a series of (daz)xaa*-ome amino esters derived from α-, β3-, and cyclic β2,3-xaa* residues, namely dβ-peptide molecules possess interesting conformational characteristics and biological properties. they may represent a new class of rigid foldamers potentially useful as templates or spacers. 3d-structures of β-peptides have been experimentally investigated using x-ray diffraction and various spectroscopic techniques, but they have never been doubly spin labelled and studied by epr. a terminally protected β-hexapeptide, based on trans-(3r,4s)-β-toac and trans-(1s,2s)-achc, synthesized using classical solution methods, was found by ft-ir absorption and cd techniques to adopt the 3-14-helical conformation. a set of four, terminally blocked, hexapeptide sequences, each characterized by four strongly helicogenic aib residues and all combinations of the two isomeric ile/allo-ile residues at positions 2 and 5 was synthesized by solution methods and fully characterized. a detailed solution (by ft-ir absorption, nmr, and cd) and solid (crystalline)-state (by cd and x-ray diffraction) conformational investigation allowed us to validate our assumption that all four peptides are folded in well developed 3-10-helical structures. however, the most relevant conformational conclusion extracted from this 3d-analysis is that the handedness of the 3-10-helical structures formed does not seem to be sensitive to the configurational change at the β-carbon atom of the constituent ile versus the diastereomeric allo-ile residues (in other words, the dominant control on this important structural parameter appears to be exerted by the chirality of the amino acid α-carbon atom). these results complement published findings on the diverging relative stabilities of the intermolecularly h-bonded β-sheet structures generated by ile versus allo-ile homo-oligopeptides. taken together, these data represent an experimental proof for the intuitive view that potentially different conformational properties are magnified in a strongly self-aggregated homo-peptide system (as compared to weakly self-aggregated, helical, host-guest peptides such as those investigated in this work). in a first approach to β-sandwich proteins the hydrophobic core between two symmetrical sheets each with four antiparallel β-strands was computationally designed by packing of amino acid side chain conformations (rotamers) in an initially given backbone structure. the proteins were synthesized by coupling four peptides with β-hairpin structure to a cyclic decapeptide template (tasp). an aggregation observed by equilibrium ultracentrifugation with the first designed proteins was decreased to a dimer by increasing the surface charge in two further variants of this protein from -1 to +3 and +5. replacement of l-pro by d-pro in the loops and the template proved to stabilize the β-structure. these results led us to an improved design of an asymmetric core with algorithms for selection of proteins with a minimal number of atom clashes and cavities in the core, and a maximum number of hydrogen bonds after energy minimization. this protein termed beta-mop (modular organized protein) was synthesized in amounts to allow a characterization by cd, ftir, tryptophan fluorescence during reversible unfolding, and by high resolution nmr. nmr measurements of diffusion indicate a dimeric structure. the β-structure is stable up to 80 °c (353 k) as determined by 1d 1h nmr showing sharp resonance lines. the 2d 1h,1h dqf-cosy spectrum at 750 mhz shows a typical βsheet distribution extending well into the characteristic regions >8.5 ppm (for amide protons) and >5.0 ppm (for hα signals). all data indicate a well folded protein with β-structure. a.s. galanis 1 , z. spyranti 1 , n. tsami 1 , g.a. spyroulias 1 , e. manessi-zoupa 2 , g. pairas 1 , i.p. gerothanassis 3 , p. cordopatis 1 angiotensin-i converting enzyme (ace) belongs to the m2 family of the ma clan of zinc metallopeptidases and can act either as a dipeptidyl carboxypeptidase, or as an endopeptidase. among the ace peptide substrates, the most distinguished are angiotensin i (angi) and bradykinin (bk) due to their role in blood pressure regulation. despite the fact that biological data strongly suggest that the two active sites exhibit different selectivity and activity towards physiological and exogeneous substrates none experimental evidence for the interaction of angi and bk with ace catalytic sites, is available so far. a dual approach for studying the structure and physicochemical determinants of ace-angi/bk interaction has been performed. the first involves the application of molecular dynamics simulations (presented elsewhere in this book) and the second is making use of the solid-phase synthesized 36-46 aa ace catalytic site maquettes (csm) bearing the native sequence and the application of the nmr spectroscopy, and presented herein. therefore, high-resolution multinuclear nmr spectroscopy was applied to analyze the conformational features of ace substrates angi and bk in dmso or aqueous mixtures. then titration experiments were conducted and ace csms were titrated by angi/bk peptides, while monitored by nmr. 2d 1h-1h tocsy and noesy experiments were used in order to map the interaction site of both substrates and csm through chemical shift perturbation and comparison of noe signal differentiation. competitive binding studies were also carried out through titration studies of csm-angi/bk and known ace inhibitors. a. carotenuto 1 , p. grieco 1 , l. auriemma 1 , e. novellino 1 , v.j. hruby 2 the melanocortine receptors are involved in many physiological functions, including pigmentation, sexual function, feeding behavior, and energy homeostasis, making them potential targets to treat obesity, sexual dysfunction, etc. understanding the conformational basis of the receptor-ligand interactions is crucial for the design of potent and selective ligands for these receptors. the conformational preferences of the cyclic melanocortin agonists and antagonists mtii, shu9119, [pro6]mtii, and pg911 (table 1) when two chromophores are chirally oriented and close enough to one another in space, their excited states couple and become non-degenerate. this phenomenon, termed exciton coupling, produces a typical bisignate cd curve. the intensity of the cd couplet is dependent on the molar extinction coefficient and the distance between the interacting chromophoric moieties, while the sign is governed by the angle between the effective electron transition moments. in particular, exciton coupling over a long distance can be observed only with strongly absorbing chromophores, e. g. porphyrin derivatives, characterized by their extremely intense and sharp soret band near 415 nm. in this work we examined by the exciton coupled cd method the combined distance and angular dependencies, generated by the seven conformationally restricted β-turn and 3-10-helical spacer peptides -l-ala-[l-(αme)val]n-(n = 1-7) on a system formed by two intramolecularly interacting 5-carbamido-5,10,15,20-tetraphenylporphyrin chromophores. these porphyrin derivatives are confirmed to be excellent reporter groups. we find that not only the centerto-center separation (from 19 to 34 å) between the two chromophores, but the orientation (roughly parallel or perpendicular) between the directions of their effective transition moments as well, are responsible for the onset or even for the modulation of the intensity of the exciton coupling phenomenon. in particular, the porphyrin…porphyrin interaction is still clearly detectable over the long distance of ca. 30 å when the two chromophores are about perpendicularly oriented. a. hetényi 1 , g.k. tóth 2 , c. somlai 2 , t.a. martinek 1 , f. fülöp 1 β-peptides are probably the most thoroughly investigated peptidomimetic oligomers. to extend the field of β-peptides towards the construction of possible new secondary structures, the replacement of the cα and cβ atoms of the β-amino acid with heteroatoms could be an attractive modification, for example cβ-atom of β-peptides by an nr moiety, leading to hydrazine peptides. in the literature, there are only a few studies [1] [2] [3] about hydrazine peptides, and hydrazine peptides with cyclic side-chain have not been studied yet. in order to determine the secondary structure preference of 1-amino-pyrrolidine-2s-carboxylic acid homo-oligomers (figure 1 ), their potential energy hypersurface were probed at the ab initio b3lyp/6-311g** level. the calculations predicted the 8-strand to be the most stable structure. the hydrazino-peptides in question were synthetized on solid support, and their structures were characterized by nmr and cd methods. the results were found to be in good accordance with the 8-strand structure. cathepsin c [ec 3.4.14.1](1), which belongs to family of cysteine proteases, catalyzes hydrolysis of n-terminal peptide, preferential glyphe. this enzyme may play a part in chronic airway diseases (2) . also increaser level of enzyme was found in case of cancer, rheumatism and muscle's distrophy (3) (4) (5) . for this reason we have undertook investigations of peptides containing two dehydroamino acid residues, which could act as alkylating inhibitors of this enzyme. to define structure and conformation of investigated peptides we were used different methods of nmr spectroscopy, including standard 1d experiments, protonproton correlations, proton-carbon correlations, and 2d noe experiments. to complete structural research computational chemistry methods had been used. in order to predict the biological activity of investigated peptides, the simulation of docking process of these peptides to enzyme active site had been made and after that correlated with results of enzymatic test.. the obtained results suggest, that investigated peptides containing two ∆phe residues (z and e isomers respectively) in solution have bent conformation, which is stabilized by intermolecular hydrogen bonds. these results are confirmed by the results of theoretical calculations. also simulation of docking process have showed two possible peptide's orientation in active site of cathepsin c and allowed the rational interpretation of biological test's results. turns are important elements of secondary structure in peptides and proteins. different types of turns are distinguished according to the number of residues involved. the most abundant is the β-turn, which involves four consecutive amino acids with the co at position i hydrogen-bonded to the i+3 nh. the γ-turn is centred at a single residue and is generally stabilized by a hydrogen bond between the i co and the i+2 nh. model dipeptides rco-l-pro-xaa-nhr' are the smallest systems able to adopt the β-turn conformation, which is favoured by the presence of proline at i+1. a peptide of this series, incorporating a cyclopropane amino acid (xaa), has been shown to accommodate two consecutive γ-turns in the solid state [1] , instead of the expected β-turn conformation. the double γ-turn encountered is unique among crystalline short linear peptides. in fact, the γ-turn is observed almost exclusively in low-polarity solvents, and only a few oligopeptides of cyclic structure exhibit a γ-turn in the crystal. this is the first time that the strong tendency of pro-xaa dipeptides to adopt a β-turn in the solid state has been switched to the γ-turn. theoretical calculations [2] also show the high preference of this cyclopropane amino acid for the γ-turn conformation. [ oxidative stress plays an important part in the development of cardiovascular disease (cvd). haptoglobin is a hemoglobin-binding protein that has a major role in providing protection against heme-driven oxidative stress. there are two common alleles for haptoglobin (1 and 2), and the three phenotypes, haptoglobin 1-1, haptoglobin 2-1, and haptoglobin 2-2, differ in their ability to function as antioxidants. we determined whether there was a relation between the haptoglobin phenotype and the development of coronary artery diseases. haptoglobin (hp) phenotypes were determined in iranian patients with coronary artery diseases. we performed haptoglobin (hp) genotyping by polymerase chain reaction (pcr) using allele-specific primer-pairs. in multivariate analyses controlling for conventional cvd risk factors, haptoglobin phenotype was a highly statistically significant, independent predictor of cvd. the odds ratio of having cvd in patients with the haptoglobin 2-2 phenotype was 5.0 times greater than in patients with the haptoglobin 1-1 phenotype. an intermediate risk of cvd was associated with the haptoglobin 2-1 phenotype. these results suggest that haptoglobin phenotype is an important risk factor in determining susceptibility to cardiovascular disease which may be mediated by the decreased antioxidant and antiinflammatory actions of the haptoglobin 2 allelic protein product. the special feature of proteins involved in alzheimer's or prion diseases is their ability to adopt at least two different stable conformations. the conformational transition that shifts the equilibrium from the functional to the pathological isoform can happen sporadically. it can also be triggered by mutations in the primary structure, changes of different environmental conditions, or the action of chaperones. elucidation of the molecular interactions that occur during the transformation from α-helix to β-sheet and the consecutive formation of amyloids on a molecular level is still a challenge. therefore, the development of small peptide models that can serve as tools for such studies is of paramount importance. we succeeded in generating model peptides that, without changes in their primary structure, predictably react on changes of diverse environmental parameters by adopting different defined secondary structures. these de novo designed peptides strictly follow the characteristic heptad repeat of the α-helical coiled coil structural motif. furthermore, domains that favour β-sheet formation and aggregation can be generated.1 alternatively, those peptides can be equipped with functionalities that allow either the binding of metal ions or the interaction with membranes. as proof of our concept we showed that the resulting secondary structure of such peptides will strongly depend on environmental parameters. thus, this system allows to systematically study the interplay between peptide / protein primary structure and environmental factors for peptide and protein folding on a molecular level. the pathogenesis of alzheimer's disease (ad) is strongly linked to neurotoxic assemblies of the amyloid β protein (aβ). aβ is a soluble component of human plasma which by an unknown mechanism becomes aggregated and neurotoxic. some genetic mutations within the aβ sequence cause very early onset of ad-like diseases, probably by facilitation of aβ assembly into neurotoxic species. recently, it was found that not amyloid fibrils, but smaller aβ assemblies initiate a pathogenic cascade resulting in ad. therefore, preventing the folding of nascent aβ monomers would have therapeutic benefit. to uncover details of structural changes accompanying the aggregation process, especially its initial stage, we have decided to study the aβ(11-28) fragment and its mutation-related variants. our recent studies on this aβ fragment using the cd method and the aggregation test have proved it a good model for structural studies. the obtained results confirmed that the aggregation process follows the scheme with an α-helical intermediate and pointed out differences in the behaviour of aβ variants. to further confirm the scheme of the structural changes accompanying aggregation we have applied ftir spectroscopy and analysed aggregation-induced changes of the amide i band which is directly related to peptide backbone conformations. the ftir spectra analysis indicate that water addition provoked conformational changes are strongly dependent on the aβ(11-28) variant and in some cases the formation of α-helical intermediate seems to be preceded by 310 helix formation. to verify this hypothesis the temperature dependent atr ftir spectra will be analysed. supported by ug bw grant. amino acid octarepeats present in the prion protein bind to cu2+ and are considered as a potential periplasmic copper ion transporters. this octarepeat is located in the unstructured region of the prion protein, which is supposedly not intricately involved in prion aggregation. our group is involved in exploring the function of octarepeats with a special emphasis on their possible role in amyloid fibril formation and aggregation.1 in this context, we have prepared truncated peptide constructs derived from the prion protein octarepeat phgggwgq and have reported their fibrillation activity. we will present aggregative behavior of a truncated bis-pentapeptide, containing gggwg segment, when tethered with a flexible linker diaminobutane. fibrillar architectures were observed by this bis conjugate after incubation in water which was probed by different microscopic and steady state fluorescence techniques. further investigations with ki revealed a homogeneous environment of the two tryptophan moieties in the conjugate. in the absence of other side-chains, it is likely that fibril formation involves hydrophobic interaction between tryptophan indole moieties and main chain backbone interactions. interestingly, a facilitator role for aromatic-glycine motifs for amyloid aggregation has been proposed based on bioinformatics search of the swiss-prot and trembl databases. collagens are known to fold into a highly ordered rode-shaped triple helix with stretches of lower and higher suprastructural stability and even disruptions to modulate recognition by other proteins that interact with the extracellular matrix [1] . to increase understanding of folding and stability of the collagen triple helix, we have adressed the design of photocontrolled collagenous peptides. our aim was to crosslink two side chains of the repetitive (xaa-yaa-gly) sequence motifs of collagen model compounds via an azobenzene chromophore in analogy to our previous studies on photomodulation of the conformational preferences of cyclic peptides and more recently of hairpin-peptide model systems [2] . molecular modeling experiments suggested appropriate sequence positions that could result in triple-helical peptides with conformational stabilities that can be modulated by cis/trans isomerization of the azobenzene moieties. as light switchable crosslinker azobenzene-4,4'-n-(4-iodo-2-butynenyl)carboxyamide was synthesized for reaction with two (4s)-mercaptoproline residues placed in suitable xaa and yaa positions, respectively. by this approach a fully folded triple helix was obtained upon thermal relaxation, and unfolding was induced by irradiation at 350 nm. the favorable optical properties of the azobenzene derivative together with the regular suprastructure resulted in a valuable model system that allows for ultrafast time-resolved studies of collagen folding and unfolding. amyloid formation is connected with alzheimer's disease, parkinson's disease, finnish familial amyloidosis. after protein misfolding short peptide sequences act as "hot spots" providing the driving force for protein aggregation in amyloid fibrils. we have identified one of these sequence stretches in the abl-sh3 domain of drosophila (dlsfmkge) whereas the human homologous region (dlsfkkge) is predicted to be less amyloidogenic. the possible reason for the difference of amyloid formation propensities of the two peptides was investigated by molecular dynamics (md) of β-sheet structures. the antiparallel alanine β-sheets consisting of two and ten strands were constructed, minimized, and mutated to the sequences dlsfmkge and dlsfkkge. all four systems: 1) dlsfmkge -two strands, 2) dlsfkkge -two strands, 3) dlsfmkge -ten strands, 4) dlsfkkge -ten strands, were surrounded by 10 å layer of water molecules over the solute and subjected to md, amber 8.0 force field, ntp protocol. the md runs were started at the temperature of 10 k and the temperature was elevated stepwise by 10 degrees till 300 k. the results show considerably higher hydrogen bond percentage for dlsfmkge than that one for dlsfkkge during the course of the simulation, thus suggesting that dlsfmkge is a potential fibril-maker, but dlsfkkge is not. two strand β-sheet systems were stable until 170 k. the ten strand β-sheets are more stable. angiotensin-i converting enzyme (ace) has a critical role in cardiovascular function, which consists of cleaving the carboxy terminal his-leu dipeptide from angiotensin-i producing a potent vasopressor octapeptide, angiotensin-ii. there are two isoforms of ace. the somatic isoform is present in all human cells except the testis cells, where the testicular isoform is produced. the major difference between these two types is that, the somatic form has two active sites, at the n-and c-end respectively while the testicular has only one, which is almost identical to the somatic c-terminal active site. here we report the structural study of a 108aa peptide (previously expressed in bacteria), which corresponds to an extended domain of the human somatic n-terminal active site of ace (ala361-gly468) by circular dichroism experiments, and the overexpression in bacteria, purification and structural study, using circular dichroism techniques, of a 108aa peptide which corresponds to an extended domain of the human somatic c-terminal active site of ace (ala958-gly1065). following the subclonning into an appropriate expression vector and the expression, the peptide was isolated from the inclusion bodies using chromatography techniques. the recombinant protein fragment had a molecular weight, measured by esi-ms, of 12102 kda which was in consistence with the theoretical calculation based on the dna sequence. the recombinant peptides acquired their theoretically calculated secondary structure only when 1,1,1-trifluoroethanol is present at a concentration of ~70%. in order to elucidate their structures, solutions of these peptides, labeled with 15n and/or 13c, will be studied by nmr spectroscopy. aggregation of peptides is believed to trigger various degenerative diseases but it also plays an important role for the preparation of peptide fibres and peptide-based biomaterials. it is therefore extremely important to understand the mechanisms involved in peptide aggregation and be able to control them. studies were performed on a library of amphiphilic peptides, designed around the sequence of a model antimicrobial peptide rich in leucine and lysine. the library also included peptide hybrids in which natural amino acids were replaced by non-proteinogenic omega-amino acids, such as 6-aminohexanoic acid and 9-aminononanoic acid. the aim was to estimate the aggregation and its correlation with the biological activity by using a fluorescence technique commonly employed to calculate the cmc (critical micelle concentration) of surfactants. peptides and peptide hybrids were synthesized on solid support using the fmoc polyamide protocol. they were purified by semi-preparative rp-hplc and characterized by esi-ms and analytical rp-hplc. the aggregation behaviour of the synthesized molecules was investigated in water by steady-state fluorescence measurements using pyrene as fluorescent probe. peptides were dissolved in water/pyrene or water/pyrene/0. fluorescence spectroscopy has become an extremely valuable technique for conformational studies of biopolymers, the development of peptide-based chemosensors, and biochemical research in general. in this connection, synthetic amino acids as fluorescent probes to be incorporated into a peptide chain may exhibit significant advantages over the related protein (trp and tyr) residues in terms of potentially different and ameliorated properties. we recently designed and prepared a new fluorescent amino acid, antaib, based on a planar anthracene core and belonging to the class of achiral, ciα↔ciα cyclized, cα-tetrasubstituted α-amino acids (strong β-turn and helix inducers in peptides). peptides based on antaib combined with (l)-ala residues were synthesized and subjected to a conformational analysis. more specifically, the protected derivatives boc-antaib-oet (oh) and fmoc-antaib-otbu (oh) were prepared in seven steps from 1,2, 4 conformational transitions in peptides and proteins emerge to play the major role in the genesis and evolution of prion related diseases and alzheimer's disease (ad).1 in this context, conditions influencing this transition and the following aggregation process are of paramount interest. peptides and proteins that are involved in aggregation processes contain potential metal binding sites. the concentration of metal ions in the brain tissue is naturally high and zn in the mm range has been found in ad amyloid plaques. thus, it is widely accepted that metal complexation is one of the key incidents that lead to conformational transitions and aggregation. we present here a coiled coil based model peptide system with an intrinsic amyloid forming tendency which can be used to study the impact of different metal ions on secondary structure and aggregation. metal complexing histidine residues were incorporated to create potential binding sites which, depending on their position and the nature of the metal ion, dictate folding and aggregation. the time dependent conformational transition was monitored by cd-spectroscopy. aggregates were characterized by cryo tem. high resolution fticr-ms experiments revealed information on the stoichiometry of the peptide-metal complexes. in the absence of metal ions the presented peptides formed amyloids in a time range of weeks. depending on the his positions and milieu conditions, the nature of the metal ion determines folding and aggregate morphology. furthermore, metal binding was shown to inhibit the amyloid formation. a challenge to our understanding of protein folding is the design of a protein from first principle, i.e. starting from geometric restraints and applying properties of amino acids expected to be essential for folding to a defined structure. we developed a program to calculate the backbone coordinates of antiparallel strands to match the surface of an elliptical cylinder. various parameters like the number and shearing of the strands and the ellipticity of the structure can be varied. the relative orientation of the β-strands and the geometrical features of the hydrogen-bonds were derived from statistical analyzes of natural β-sheet structures. iterative cycles of core-packing with amino acid rotamers, molecular dynamics simulation and energy minimization with the charmm forcefield are used to include backbone movements and to minimize the risk of trapping an energetically unfavorable structure. the quality of residue packing is assessed with the help of criteria which proved to be successful in our design of β-sandwiches. at the protein surface, a network of salt-bridges with an excess of positive charges has been designed to increase the stability and the solubility of the protein. the final sequence is synthesized by standard solid phase fmoc-chemistry. insights gained from the analysis of the synthesized structure with ftir and cd spectrometry should help us to refine the parameters for subsequent designs. with this strategy, we hope to contribute to a better understanding of protein folding. the immunoglobulin binding protein g (1igd) from streptococcus species consists of 61 amino acids residues, which form two antiparalell-packed beta-hairpins and an alpha-helix in the middle of the sequence packed to the beta-sheet. the second hairpin was found to be stable in isolation. this fragment is therefore likely to be the first folding initiation site of the protein which could provide an adequate nucleation center on which the rest of the polypeptide chain would find a favorable environment to fold. thus, among the two beta-hairpins, the 48-59 fragment of 1igd corresponding to the c-terminal beta-hairpin was synthesized. in our studies, we investigated different environmental and temperature conditions for formation of the 48-59 beta-hairpin structure. its structure was examined by means of cd spectroscopy in water, buffer solutions (ph = 3 to 9) and in aqueous solutions of trifluoroethanol. additionally, its structure was investigated in the solid state by ftir spectroscopy. the cd studies revealed that the 48-59 fragment of 1igd in water forms mainly a statistical-coil structure, whereas the ftir technique shows formation of a regular beta-sheet structure. nmr spectroscopy and calorimetric measurements were carried out at various temperatures. our studies show that the 48-59 fragment at low temperature exists in an equilibrium between two conformations -a regular beta-hairpin and a statistical-coil. although increasing temperature resulted in shifting the equilibrium in the direction of the statistical-coil structure, the overall beta-hairpin shape of the 48-59 fragment was maintained. prion diseases are characterized by the conversion of the physiological cellular form of the prion protein (prpc) into an insoluble, protease-resistant abnormal scrapie form (prpsc) with an highly beta-sheet content [1] . the analyses of intrinsic structural propensities of the prp c-terminal domain showed an high conformational flexibility for the αhelix 2 fragment which indicates that this region may be particularly important in the prpc→prpsc transition [2] . therefore conformation-based approaches focalized on helix 2 region appear to be the most promising for the study of prion protein misfolding. recent studies on tetracycline properties showed that this molecule binds and disrupts prp peptide aggregates and inactivates the pathogenic forms of prp [3, 4] . a fluorometric titration of the fluoresceinated peptide corresponding to prion protein helix-2 with tetracycline has been carried out to determine the value of the apparent dissociation constant of this interaction (estimated to be 189 ± 7 nm). remarkably, the fluoresceinated peptide exhibits in water a canonical α-helical cd spectrum, that is maintained even in presence of tetracycline. accordingly, docking calculations and molecular dynamics simulations suggest that tetracycline interacts preferentially with the c-terminal end (residues 183-195) of helix-2 with a significant involvement of the treonine rich region. in the last decades, a series of discoveries have shed light on the role played by the carbohydrate moiety in glycoproteins. it has been shown that covalently linked sugar moieties influence peptide/protein properties such as hydrophobicity, conformation, biostability and bioactivity. the design of carbohydrate-peptide analogs with increased, retained or modified biological activity requires an understanding of their conformational preferences both in solution and in the receptor-bound state. in our recent work we have created two classes of well-structured linear and cyclic carbohydrate-modified analogs of opioid peptides, leu-enkephalin and leuenkephalin amide. the first class represents a group of compounds in which the linear peptide is alkylated at the n-terminal position by 1-deoxy-d-fructose unit, while its cyclic analog possesses an ester bond between the c-6 hydroxy group of the sugar moiety and the c-terminal carboxy group of peptide, 1-deoxy-dfructofuranose acting as a bridge between the leu-enkephalin terminal parts. the rigid 5-membered imidazolidinone ring is characteristic for the second class of compounds. in these adducts an imidazolidinone moiety connects the acyclic sugar residue with the linear peptide chain. in the corresponding bicyclic imidazolidinone analogs 19-membered ring is formed through an ester bond between the primary hydroxyl group of the d-gluco-pentitolyl residue and the cterminus of peptide. this work reports the comparative cd and ftir spectroscopic properties of the prepared glycopeptides in comparison with data on the non-modified flexible parent peptides performed in different solvents in order to expose the structural and conformational differences caused by a keto-sugar, rigid 5membered imidazolidinone ring and/or cyclization. the α-helical coiled coil structural motif consists of two to five α-helices which are wrapped around each other with a slight superhelical twist. the simplicity and regularity of this motif have made it an attractive system to study the role of complementary interactions for protein folding. here we present a systematic study showing that intermolecular electrostatic interactions between positions e and g of the helices are in competition with the intramolecular interactions between positions e/b and g/c. those competitive interactions affect folding and stability of the motif which were monitored by temperature dependent cd-spectroscopy. incorporation of oppositely charged amino acids in positions e/b and g/c reduced considerably electrostatic repulsion between equally charged amino acids in positions e and g. in addition coiled coil stability can be increased by the alkyl part of the amino acid side chains in positions e and g. studies with natural and unnatural amino acids showed that the longer this alkyl part the better is the hydrophobic core protected from solvent. therefore the repulsion of equally charged amino acids in positions e and g can be overruled by involving them either into attractive intrahelical electrostatic interactions or into hydrophobic core formation. human cystatin c (hcc) is a 120 amino acid residues protein that reversibly inhibits papain-like cysteine proteases. this inhibitor belongs to the amyloidogenic proteins shown to oligomerize through 3d domain swapping mechanism. the crystal structure of hcc reveals the way the protein refolds to produce symmetric dimer while retaining the secondary structure of the monomer. the monomeric form of hcc consists of a core with a five-stranded antiparallel β-sheet wrapped around a central α-helix. the hcc dimerization is preceded by an opening movement of l1 loop from β2-l1-β3 hairpin and separation of the β1-helix-β2 fragment from the remaining part of the molecule. the amino acid sequence of β1-helix region suggests additional possible partial unfolding in the n-terminal part of helix. in order to investigate the structural stability of β1-helix region the peptide corresponding to the helix and its n-terminal truncated analogs was synthesized along with the peptide analogs of helix containing point mutations that could stabilize helical structure of the n-terminus. the peptides were synthesized by the solid-phase method using fmoc/tbu tactics. purified products were identified by maldi-tof. the secondary structure content was calculated from the cd spectra using selcon3. the random coil was the predominant structure of the peptide corresponding to the α-helical fragment of hcc and its n-truncated analogs. however, an increase of αhelix content was observed in some of the peptides corresponding to the helix containing point mutations. we expect that these mutations could stabilize the hcc monomer and suppress dimerization. the tumor suppressor protein p53 is a trarnscription factor that triggers cell-cycle arrest and apoptosis in response to genotoxic stress signals. the tetramereric structure of the p53, which is essential for its activity as a transcription factor, is formed as a dimer of dimers. while the primary dimer is constructed from inter molecular formation of a two-stranded anti-parallel b-sheet and a two anti-parallel a-helix bundle, the secondary dimer is stabilized through interactions between residues on the surface of the primary dimer. from various substitution experiments on p53, it has been shown that hydrophobicity of phe341 is critical for the tetramer formation of p53. also we have substituted three phenyl groups of p53 with cyclohexylalanine (cha) and showed that phe341cha is dramatically stabilized against temperature, chemical denaturant, and organic solvent by cd measurements. here, to clarify the mechanism of the stabilization of phe341cha, we analyzed its three dimensional structure using x-ray crystallography. we obtained two kinds of crystals, one is a hexagonal bipyramid crystal in the space group of p<i>6<sub>4</sub>22</i> with <i>a</i>=50.12 å, <i>b</i>=50.12 å, <i>c</i>=48.18 å diffracted to about 2.0 å, and another is tabular crystal in the space group c<i>2</i> with <i>a</i>=77.25 å, <i>b</i>=50.04å, <i>c</i>=55.10 å diffracted to about 2.1 å. in these crystals, the peptides formed tetramers which are very similar to those observed in the wild-type. the structure of the pocket where the side chain of cha341 is incorporated was defined to elucidate the hydrophobic interaction to determine the stability. helices shown in proteins, as a secondary structure, almost always form right-handed screw sense. this right-handedness is believed to result from the chiral center at the αposition of proteinogenic l-α-amino acids. among proteinogenic amino acids, l-isoleucine and l-threonine possess an additional chiral center at the side-chain β-carbon besides the α-carbon. however, no attention has be paid how the side-chain chiral centers affect the secondary structures of their peptides. recently, we have reported that sidechain chiral centers of chiral cyclic α,α-disubstituted amino acid (s,s)-ac(5)c(dom) affected the helical secondary structure of its peptides, and the helical-screw direction could be controlled without a chiral center at the α-carbon atom. herein, we synthesized a chiral bicyclic α,α-disubstituted amino acid (r,r)-ab(5,6)c and its homopeptides, and studied the relationship between the chiral centers and the helical-screw handedness of peptides. contrary to the left-handed helices of (s,s)-ac (5) four threefinger-toxins (tfs) have been purified from the pooled venom of golden krait (bungarus fasciatus, i.e. bf) from thailand and studied previously. these peptide toxins contain 60-65 residues and 4 or 5 pairs of disulfide bonds, and are rich in β-structure. we herein analyzed the tf-isoforms in bf venoms from kolkata (eastern india), hunan province (eastern china) and indonesia to study the geographic variations and structure-function relationships of the venom polypeptide family. a total of five or six tfs of low lethality were purified from each of the geographic venom samples, the n-terminal sequences and accurate masses of the peptides were determined. the cdnas encoding some of these tfs were also cloned and sequenced. full peptide sequences were deduced and match with those of the tfs purified from crude venom. intra-species variations of the venom tfs were found to be surprisingly high since sequence-identities between the majorities of orthologous toxins in different geographic samples are only 75-80 %. most of the bf proteins were not neurotoxic by electrophysiological assays using chick biventer-cervicis and mouse diaphragm neuromuscular tissues. the toxins appear to be associated with weak toxins or non-conventional snake venom tfs as analyzed by a phylogenetic tree. the reason behind their lack of neurotoxicity would be discussed. v. moussis, e. panou-pomonis, c. sakarellos, v. tsikaris peptides involved in neurodegenerative diseases can adopt at least two different stable secondary structures. amyloid-forming proteins can experience a conformational transition from the native, mostly α-helical structure, into a ß-sheet rich isoform. the latter conformation is probably present in intermediates for the formation of amyloids. the conformational change can be triggered by protein concentration or environmental changes. therefore, our aim was to generate a de novo designed peptide that contains structural elements for both, stable α-helical as well as ß-sheet formation. this model peptide can be used to elucidate the conformational changes dependent on concentration and ph.1 the design is based on the well studied α-helical coiled coil folding motif. the conformation and structure of the resulting aggregates were characterized by cd-spectroscopy and cryo transmission electron microscopy. as a result, three distinct secondary structures can be induced at will by adjustment of ph or peptide concentration. low concentrations at ph 4.0 yield globular particles of the unfolded peptide whereas, at the same ph but higher concentration, defined ß-sheet ribbons are formed. in contrast, at high concentrations and ph 7.4, the peptide prefers highly ordered α-helical fibers. in conclusion, we successfully generated a model peptide that, without changes in its primary structure, predictably reacts on environmental changes by adopting different defined secondary structures. thus, this system allows to systematically study now the consequences of the interplay between peptide primary structure and environmental factors for conformation on a molecular level. cells respond simultaneously to a multitude of different signals. inside a cell signals from activated receptors are integrated by networks of enzymatic reactions and molecular interactions, leading to a spectrum of cellular responses. in order to understand the relationship between a specific cellular stimulus and a cellular response, methods are required to detect in parallel the pattern of molecular interactions. a large number of molecular interactions is mediated by protein domains, binding to linear peptide motifs. lysates of activated and resting cells were incubated on peptide microarrays carrying peptides corresponding to such binding motifs of signalling domains. binding of proteins to a spot of the array was probed by immunofluorescence. jurkat t cells were stimulated either with the phosphatase inhibitor pervanadate or with antibodies directed against cell surface receptors. upon activation of t cells, numerous changes in the pattern of molecular interactions were detected for a total of 10 proteins and 30 peptides. these changes were caused either (i) by masking or unmasking of a binding domain which resulted in a reduced or increased binding of a protein to the microarray or (ii) by recruitment of a protein into a complex that in turn bound to the microarray. the changes were dependent on the nature of the stimulus. the human melanocortin receptor 1 (hmc1r) was constructed to contain a flag epitope and a hexahistidine tag at the amino-terminus as well as at the carboxyl terminus to facilitate purification. stably transfected hmc1r in human embryonic kidney (hek293) cell lines that expressed the receptor resulted in a kd value of 0.1 and 0.2 nm respectively in each case when the super potent agonist mtii was competed with [125i]ndp-α-msh. treatment of the tagged receptors in the hek293 cells with agonist resulted in down-regulation which indicates that these tagged receptors retain their biological functions. the hmc1r was solubilized from cell membranes with n-dodecyl-β-d-maltoside and purified at a nickel chelating resin and a newly constructed affinity column. the purified hmc1r was a glycoprotein that migrated on sds/page with a molecular mass of 58 kda. the results from matrix-assisted laser desorption ionization-time of flight (maldi-tof) mass spectrometry was used to identify and characterize peptides derived from the hmc1r following in-gel digestion with chymotrypsin. the phosphorylation sites were identified on the purified human melanocortin receptor 1 with agonists (peptide vs small molecule) treatment. the discovery of antibiotics in the 1930s has been one of the most revolutionary events in the history of medicine. however, during last decades, the increase of antibiotic resistance has significantly hampered the application of antibiotics. therefore, further scientific effort to find new antibiotics with novel mechanisms of action is of high importance. insects are the largest and the most diverse group of living animals on earth. they have potentially been confronting high variety of microorganisms. as a result, they have evolved powerful defense system, thus representing vast source of novel potential therapeutics. we chose larvae of fleshfly neobellieria bullata for identification and characterization of new promising molecules, peptides or proteins, which participate in immunity response against microbial infections. the hemolymph of the third-instar larvae of neobellieria bullata was used for isolation. the larvae were injected with bacterial suspension of escherichia coli or staphylococcus aureus to induce antimicrobial response. the hemolymph was separated into crude fractions, which were subdivided by rp-hplc. isolated fractions were characterized by uv-vis spectroscopy, amino-acid analysis, mass spectroscopy, 1-d and 2-d sds electrophoresis, capillary zone electrophoresis, ion-exchange hplc, tryptic digests and n-terminal sequencing. we found out significant antimicrobial activities against escherichia coli, staphylococus aureus or pseudomonas aeruginosa in several fractions. using real-time pcr, we followed and compared levels of mrna of different proteins and peptides in induced and non-induced larvae. despite the fact that many technological advances are currently involved in proteome analysis, like two-dimensional gel electrophoresis and mass spectrometry, there is still a great need for the development of novel engineered chemical probes for proteomics and interactomics. here, we describe our approach concerning the study of proteome and interactome of proteins involved in cell-matrix interactions. it relies on the use of a small synthetic inhibitor chemically modified to allow for its immobilisation to magnetic beads or affinity chromatography materials. proteins will be detected together with their native interaction partners because of nondenaturing conditions. this general procedure is applied for the enrichment of metalloproteinases, especially matrix metalloproteinases, which are potential target in tumour therapy. hydroxamic acids are known to be potent inhibitors of metalloproteinases. marimastat is a reversible inhibitor with a good potency and shows activity towards a wide range of metalloproteinases. the synthesis of new marimastat derivatives will be reported. the parent compound is modified with a linker to allow immobilisation on a solid surface. binding studies were performed using surface plasmon resonance. this approach is not only appropriate for the generation of metalloproteinase proteome subsets by affinity column or using magnetic beads, but also to enrich and isolate interaction partners of the target proteins. the present report summarizes the latest data devoted to theoretical and experimental investigation of the high temperature solid state catalytic isotope exchange reaction (hscie) that takes place in peptides and proteins by the action of deuterium and tritium [1] . the available ms-procedures, designed to estimate the amount of protein, are aimed at derivatization at different stages of sample preparation, and as the best result, it is only possible to achieve quality comparison of the objects involved. the hscie reaction allows the production of evenly deuterium labelled proteins and peptides, and their application makes it possible to create a qualitative mass spectrometry method for protein analysis. introduction of definite amounts of these deuterium-labeled proteins into biological objects, prior to isolation, separation and trypsinolysis, will generate quantitative information concerning the composition of the proteins under study. tritium labelled proteins produced at a temperature of 100-120oc carry the isotopic label in all the peptide fragments and completely retain their enzymatic activity. the proteins' reactivity is dependent on their three-dimensional structure. the hscie reaction has been shown to be used both in the production of tritium labelled proteins and in the investigation of spatial interactions in protein complexes. in addition, evenly deuterium and tritium labelled peptides can be used in studies of the kinetics and transformation paths of peptides in the organism's tissues. immunization of mice with type ii collagen (cii) from rat leads to development of collagen-induced arthritis (cia). susceptibility to cia is associated with the major histacompatibility class ii protein h-2aq that binds the glycopeptide epitope cii260-267 (1) and presents it to helper t cells. [1] to explore the interactions in the system and to stabilize 1 towards in vivo degradation, amide bond isosteres have been introduced in its backbone.glycopeptide 1 was virtually docked into the binding site of a comparative model of h-2aq. based on the hydrogen bonding network between the peptide backbone and h-2aq, the amide bond between ala261-gly262 was chosen for isosteric replacement. to vary the geometric and hydrogen bonding properties, mimetics of the dipeptide were synthesized with the amide bond replaced by ψ[ch2nh], ψ[coch2] and ψ[(e)-ch=ch], respectively. these were introduced in 1 using solid-phase synthesis to give glycopeptidomimetics that were biologically tested for their ability to bind to the h-2aq protein and for recognition by t cells. shown to be a potent neuroprotective factor in various pathophysiological models. despite its therapeutic potential in diverse neurodegenerative diseases, its short in vivo half-life limits its utility as a useful clinical agent. moreover, the development of a peptidomimetic that reproduces the pharmacological activity of pacap is unlikely since the pharmacophores are spreaded throughout the entire peptide chain. therefore, the development of pacap analogues with lower susceptibility to proteolysis represents a first step toward clinical applications. in the present study, derivatives of both pacap27 and pacap38 with particular chemical modifications were developed targeting specific peptidase sites of action. results indicate that the incorporation of an acetyl or a hexanoyl group at the n-terminus and modifications at the ser2 residue contributed to increase stability against dipeptidyl peptidase iv, the major enzyme involved in pacap degradation. moreover, after determination of pacap metabolites in human plasma, the amide bond between residues 21 and 22 was substituted by a ch2nh surrogate and this derivative showed increased plasma stability. all modified peptides were tested for their ability to induce pc12 differentiation. the effects of pacap analogs on pc12 cells are mediated through the pac1 receptor which is the major receptor involved in the neuroprotective effects of pacap. this study exposes interesting data concerning pacap metabolism in isolated human plasma and demonstrates the possibility of increasing the metabolic stability of pacap without significantly reducing its biological activity. species of the fungal genus trichoderma are commercially used as bioprotective agents against fungal plant diseases. more than 400 strains were collected from their natural habitats and evaluated for biocontrol properties. seven of the most active isolates exhibiting strong biological activity towards eutypa dieback and esca diesease of grapevine were classified as trichoderma brevicompactum, or shown to be closely related to that species. these strains were screened for production of peptaibiotics. the formation and synergistic action of hydrolytic enzymes and peptaibiotics were to play an important role in mycoparasitism. after background and aims: conotoxins are short, disulfide-rich neurotoxins that target various ion channels and receptors. these peptides have desirable pharmacological properties to become therapeutics for neurological disorders; several conotoxins have already reached clinical development stage. our long-term goal is to improve bioavailability, metabolic stability and pharmacokinetics of conotoxins using a variety of chemical modifications. methods: we designed and chemically synthesized conotoxin analogs containing two distinct types of backbone modifications: (1) peptide-peptoid chimeras (conopeptoids) of alpha-conotoxin imi and (2) peptide chimeras of mu-conotoxin kiiia containing non-peptidic "backbone spacers". results: conopeptoid-imi, containing ala9 replaced by n-methyl glycine potently blocked activity of nicotinic acetylcholine receptors. in mu-conotoxin kiiia, aminohexanoic acid or amino-3-oxapemtanoic acid were inserted to be a part of the peptide backbone. the two oxidized analogs containing "backbone prosthesis" differed in their hydrophibicity profile, but they both potently inhibited neuronal sodium channels. conclusion: our results suggest that backbone engineering may become an effective method of producing conotoxin analogs with modified bioavailability. to increase the stability and the therapeutic efficacy of peptide sequences from myelin oligodendrocyte protein (mog) that act as multiple sclerosis (ms) antigens, we grafted them onto a framework of a particularly stable class of peptides, the cyclotides. they are a recently discovered family of cyclic plant peptides with superb intrinsic stability. the limitations of linear peptides as drugs due to their instability and poor bio-availability can be overcome by using the cyclotide scaffold as a framework for novel drug design. peptide epitopes from mog protein were incorporated onto the framework of the model cyclotide kalata b1 by means of boc-spps approach. after successful backbone cyclisation and oxidation of the cysteine residues, the peptides were purified to high purity with rp-hplc. nmr chemical shift analysis was used to assess whether the grafted analogues have a stable scaffold, similar to that of kalata b1. a structure of a representative peptide was determined and it shows remarkable resemblance to the native scaffold of kalata b1. the activity of the bioengineered peptides has been tested in vivo. a group of mice injected with one of the peptides have shown a depression in the clinical score and have not fallen ill. this is an exciting result that shows the first active bioengineered cyclotide in an animal model of disease. the structural information from nmr studies will be used in conjunction with the results from the activity studies in a feedback loop to design second-generation lead molecules. a.d. de araujo, p.f. alewood conotoxins are small, disulfide-rich peptide neurotoxins produced in the venom of marine cone snails that enable these molluscs to capture their prey. these compounds exhibit a high degree of selectivity and potency for different ion channels and their respective subtypes, making them useful tools in the investigation of the nervous system. due to the key role of ion channels in many physiological processes, conotoxins are also excellent drug lead candidates in drug discovery, with some examples currently undergoing clinical trials and one recently approved drug (prialt®). like other peptide drugs, the use of conotoxins in drug development is limited by the low oral bioavailability of these compounds due to pre-systemic enzymatic degradation and poor penetration through the intestinal mucosa. peptide analogs that mimic the native structure and incorporate rationally designed non-natural modifications in the disulfide framework or peptide backbone may exhibit increased resistance to proteolytic degradation. in biological environments, disulfide linkages are susceptible to attack by enzymes and reducing agents (such as glutathione). therefore we carried out the synthesis of redox-stable conotoxin analogs in which intrinsic disulfide bonds were replaced by thioether linkages. in this work, wehave explored two solid-phase synthetic routes in the preparation of thioether conotoxin mimetics: the first route based on peptide assembly using a tetra-orthogonally protected lanthionine building block, and the second route based on intramolecular on-bead cyclisation between cysteine and betabromoalanine residues. thyrotropin releasing hormone (trh, l-pglu-l-his-l-pro-nh2), a tripeptide synthesized in the hypothalamus, operates in the anterior pituitary to control levels of tsh (thyroid-stimulating hormone) and prolactin. the thyrotropin-releasing hormone (trh) receptor (trh-r) belongs to the rhodopsin/β-adrenergic receptor subfamily of seven transmembrane (tm)-spanning, g protein-coupled receptors (gpcrs). the two g-protein-coupled receptors for trh, trh receptor type 1 (trh-r1) and trh receptor type 2 (trh-r2), have been cloned from mammals and are distributed differently in the brain and peripheral tissues. the trh receptor subtype-1 appears to mediate the hormonal and visceral effects, whereas trh receptor subtype-2 has been implicated in its central stimulatory actions. identification of critical features of the trh, separation of its multiple activities through design of selective analogues and affinity labels have been elusive and unfulfilled goals for more then 30 years. this presentation will highlight our studies on effect of the biological activity of trh with the introduction of alkyl groups of varying sizes at the n-1 and c-2 position of the centrally placed histidine residue of trh peptide. the requisite n--boc-dialkyl-l-histidines as building scaffolds have been synthesized in six steps from l-histidine methyl ester dihydrochloride and used for the synthesis of various trh analogues. ghrelin, the natural ligand for the growth hormone secretagogue receptor (ghsr-1a), has received a great deal of attention due to its ability to stimulate growth hormone secretion and to control food intake. during these last years, ghrelin analogues or mimetics gained interest for their implication in food intake regulation. in the course of our studies concerning ghrelin analogs, we developed new ligands of the ghsr-1a based on heterocyclic structures. interestingly, one heterocyclic family presented high affinity for the ghrelin receptor. a structure activity study was performed within this family and led to potent ghsr-1a agonists and antagonists. the binding affinities were determined by displacement of radiolabelled ghrelin and the agonist or antagonist character was measured by intracellular calcium mobilisation. the first in vivo results revealed that in vitro activities and in vivo responses were not correlated. particularly, binding to ghsr-1a and in vivo gh release and food intake control were not fully correlated. these results strongly suggest the presence of receptor subtypes that modulate ghrelin actions. some examples will be reported. further investigations are going on in the laboratory. background and aims: alzheimer's disease (ad) related beta-amyloid (aβ) peptides possess high propensity towards aggregation. nowadays one of the major directions of the drug-design against ad is the synthesis of putative amyloid aggregation inhibitor molecules (aai) which are able to hinder the formation of these toxic amyloid aggregates. in the present work we report the design, synthesis and investigation of putative aais derived form the peptide sequence aiigl identical to aβ(30-34). methods: aβ(1-42) peptide and putative aggregation inhibitors were synthesized manually using fmoc-chemistry and dcc/hobt activation. studies on both the aβ aggregation and the effect of the aais were performed with several instrumental techniques. the total amount of the aggregates was determined by thioflavine-t binding assay, while their size distribution was characterized with dynamic light scattering (dls). aggregated aβ forms were visualized with transmission electron microscopy (tem). the binding affinity of the aais to aβ fibrils was studied in saturation transfer nmr experiments, while in vitro viability assays were performed on cultured human sh-sy5y neuroblastoma cells to monitor the neuroprotective effect of the amyloid aggregation inhibitors. results and conclusions: 32 derivatives of aβ(30-34) were synthesized and tested concerning their neuroprotective effect against aβ-mediated toxicity. the most promising candidates were examined with physico-chemical methods to characterize their aggregation altering ability. the pentapeptide riigl-amide proved to be the most powerful neuroprotective compound, and it was also able to alter considerably the aggregation kinetics of aβ(1-42) . this molecule might serve as a lead compound in the drug-design against ad in the future. m. mattiuzzo, a. bandiera, m. benincasa, i. samborska, m. scocchi, r. gennaro antimicrobial peptides (amps) are ancient molecules of the innate immune defence system. most of them kill bacteria by lysing their membranes. the proline-rich group of amps represents an exception, as they act via a permeabilization-independent mechanism that is likely based on recognition of molecular targets/transporters. however, specific internal targets have not yet been identified for most of these amps. bac7 is a pro-rich amp isolated from bovine neutrophils that is capable to translocate across membranes to target hypothetical intracellular proteins. in this study, we used a molecular approach to identify putative targets for bac7 by selection of peptide-resistant mutants followed by identification of the genes responsible of this resistance. to this aim, an e. coli strain susceptible to the fully active fragment bac7(1-35) was subjected to chemical mutagenesis and a number of peptide-resistant mutants was obtained. a pool of genomic dna from these mutants was used to construct a plasmid library that was used to transform a susceptible strain. this approach allowed the identification of 14 different clones that provided a high level of resistance to bac7. sequence analysis revealed the presence of genes originating from five different regions of the e. coli chromosome. among them, one clone contained ptrb, a gene coding for a serine peptidase broadly distributed among gram -bacteria, which could be involved in resistance by degrading the peptide. other resistanceconferring clones, which encode for membrane proteins that may be involved in peptide translocation across the memmbrane, are currently under investigation. lycotoxins are peptides from the venom of the wolf spider that were predicted to have an amphipatic alpha-helical structure and confirmed to possess significant antimicrobial and pore-forming activities. [1] we became interested in these peptides as potential leishmanicidal agents against leishmania donovani promastigotes and leishmania pifanoi amastigotes. thus, lycotoxin i (lyi) and lycotoxin ii (lyii), [1] and shortened analogues lyi1-19, lyi1-15, lyii1-21 and lyii1-17, were synthesized as c-terminal carboxamides. short-and long-term parasite proliferation measurements showed all peptides except lyi1-5 to be active against both promastigotes and amastigotes at the micromolar range, and suggest peptide effects on parasites to be irreversible. lyii, that showed the lower activity, became more leishmanicidal upon residue trimming, whereas the most active lyi displayed the opposite behaviour. a set of complementary techniques showed that lycotoxin peptides are membrane-disruptive to promastigotes. electron microscopy showed that two populations of promastigotes, one with intact parasites and the other extensively damaged, are formed upon addition of the peptides at their ec50. all peptides were non-hemolytic for sheep erythrocytes below 20 micromolar. tissue transglutaminase (tg2) is an enzyme that plays a key role in the pathogenesis of the celiac disease. tg2 is the main autoantigen recognized by the antiendomysium antibodies and, furthermore, catalyzes the deamidation of strategic glutamine to glutamic acid within the sequence of immunodominant gliadin epitopes. recently, another unexpected role for surface tg2, in the innate immune response in the celiac disease, has been suggested. it follows that tg2 inhibitors might represent a potential attractive pharmacological alternative to the gluten-free diet that, nowadays, is the only possible therapy for celiac patients. starting from the sequence of the heptapeptide pqpqlpy, known to be a high affinity substrate of human tg2, we have synthesized new analogs replacing pro3 with different constrained amino acids (d-pro, pip, chg, ind, deg, inp, hyp, thz)) with the aim to develop specific inhibitors of tg2. actually, proline residues present in the gluten epitopes are important in determining the immunogenicity of the epitopes and the specificity of tg2. herein, we describe the preliminary conformational studies of the synthesized analogs by cd and nmr spectroscopy and molecular modeling methods. the structural features of the peptides have analyzed in different environment. given the role of the domain pqpqlpy in the gliadin proteins, structural analysis on its analogs are of considerable interest. the results of our studies might be useful to clarify the role of the proline residues in the interaction of the gluten epitopes with tg2 and, consequently, to gain new insight in the molecular mechanism of celiac disease. carnosine and related histidine-containing dipeptides are known to react with high efficiency with the products of lipid peroxidation, namely 4-hydroxy-trans-2,3nonenal (hne) and other alpha, beta-unsaturated aldehydes, preventing their reaction with nucleophilic residues in proteins and nucleic acids. histidyl hydrazide alone or conjugated with aminoacids, long chain fatty acids, cholesterol and alpha-tocopherol synthesized in our laboratories demonstrated higher aldehydesequestering efficiency than carnosine, and were also efficient in protecting sh-sy5y neuroblastoma cells and rat hippocampal neurons from hne-mediated death. the cytoprotective efficacy of these compounds suggests their potential as therapeutic agents for disorders that involve excessive membrane lipid peroxidation. lantibiotics are antibiotic natural products that are synthesised ribosomally and undergo extensive post-translational modification, resulting in multiple thioether bridges and dehydro amino acids in the mature peptide. one such lantibiotic is mersacidin, which is produced by bacillus sp. and exhibits extremely promising in vitro and in vivo efficacy versus a number of clinically relevant pathogens, most notably methicillin-resistant staphylococcus aureus (mrsa). mersacidin is believed to function by binding to the bacterial cell wall precursor lipid ii, thereby preventing its incorporation into the growing peptidoglycan network. in an attempt to better understand this mode of action and develop more active analogues, we have embarked upon its chemical derivatisation. some of these modifications resulted in an altered antibacterial spectrum, permitting some insight into tentative structure-activity relationships. the characterisation of these structurally complex compounds via a combination of multidimensional nmr and tandem mass spectrometry will also be presented. conjugation of peptides with different moieties, such as peg, lipids, carrier proteins and toll-like receptor ligands is an established approach to improve their pharmacokinetic profile for drug use and/or to enhance their immunogenicity as subunit vaccines. the development of suitable conjugation strategies for peptides of any complexity is therefore fundamental for their effective use in human therapeutic applications. here we describe our strategy to engineer a trimeric coiled coil to obtain a covalently linked, structurally stable construct endowed of extra functionality for further derivatization. we previously showed that covalent stabilization of the designed trimeric coiled coil izn17, by interchain disulfide bonds, yielded an extremely potent and broad inhibitor of viral infection, (ccizn17)3, [1] . this potent inhibitory activity makes (ccizn17)3 not only attractive as an antiviral compound, but also as an immunogen to elicit a neutralizing antibody response [2] . we have now developed an alternative synthetic strategy to obtain the covalently-linked izn17 trimer, which allows the presence in the molecule of a free thiol for subsequent chemoselective reactions. first, we showed that stable interchain thioether bonds can be effectively substituted for the disulfides. second, we devised an orthogonal cysteine protection scheme which allows formation of the thioether bonds, while leaving an extra free cysteine on demand. this thiol group can be used for conjugation of the trimeric coiled coil to adjuvant/carrier proteins or, as reported more specifically here, to a peg40kda moiety. human igg is a most abundant type of immunoglobulin in serum and most of antibody drugs applied for therapy of cancer and autoimmune diseases also belong to this group of human immunoglobulin. specifically binding peptide to human igg is very useful for detection and purification as an affinity ligand of human igg. although some previous reports described such peptides, we tried here to isolate high-specific and high affinity ligands by employing random peptide-displayed t7 phage library. our t7 random peptide phage library possesses a total diversity of 10 powered 10th consisting of different sequence length constrained by disulfide bond. by biopanning against human igg, we isolated several igg specific clones from our library. the peptides displayed on these phages shared some common sequences in the limited region surrounded by cys residues, which suggests they are essential for binding. these clones bound only to fc region of igg and did neither other types of human immunoglobulin nor igg of other animals. a synthetic peptide derived from a phage clone showed a sub-nanomolar of kd value in binding to human igg fc on spr analysis. these results indicate that the peptide motifs obtained here are a strong candidate of human igg-specific affinity ligand for detection and purification of igg. therefore, we are now going on constructing detection and purification system using modified and improved peptide motifs. synthesis of glp-1 analogues as potential agents for blood glucose control p. kanda, r.p. sharma, c.p. hodgkinson in this study, we synthesised a panel of glp-1 analogues stabilised against dpp-iv degradation through either selective amino acid substitutions for ala8, or introduction of amide bond surrogates into the peptide backbone between ala8 and glu9. each was made by standard fmoc or boc chemistry, purified by hplc, and characterized by electrospray mass spectroscopy. all derivatives except one bearing a hydrazine modification were stable to dpp-iv proteolysis for up to 48 hours at ph 7.5, 37o. each was tested for its ability to augment insulin release from a glucose-sensitive, murine insulinoma-derived tc-6 cell line in culture. it was found that each compound acted as a glp-1 agonist to varying degrees, with some exhibiting higher activity than native glp-1 toward promoting insulin release. the most active analogues have been chosen as candidates for stabilisation against renal clearance in efforts to develop new glp-1 analogues with therapeutic potential. the high propensity of the glucose regulatory hormone human islet amyloid polypeptide (iapp) to misfold and aggregate into cytotoxic beta-sheets and fibrils is strongly associated with beta-cell degeneration in type ii diabetes (t2d) and precludes its pharmacological use for the treatment of diabetes. iapp analogs that combine solubility, lack of cytotoxicity, and bioactivity with the ability to block iapp aggregation and cytotoxicity could thus be of high biomedical interest. here we apply a minimalistic conformational restriction strategy to redesign the extremely insoluble and amyloidogenic 37-residue iapp sequence into a soluble, nonamyloidogenic, noncytotoxic, and bioactive iapp mimic (yan et al., pnas (2006) ). the designed mimic has nearly the same sequence as iapp but is highly soluble, nonamyloidogenic, noncytotoxic and a full iapp receptor agonist. in addition, the mimic binds with high affinity iapp and blocks and reverses with nanomolar activity its cytotoxic self-assembly which makes it to the most potent known iapp cytotoxic self-assembly inhibitor. due to its bifunctional nature, the mimic might find therapeutic application for the treatment of diabetes both as an inhibitor of amyloid cytotoxicity and as a soluble iapp receptor agonist. our findings offer a proof-of-principle of a chemical design approach for generating a novel class of highly potent inhibitors of polypeptide cytotoxic selfassembly which are nonamyloidogenic mimics of the native amyloidogenic sequence as well. such reengineered biomolecules -the design of novel mimics is in progress-are of high biomedical significance for understanding the mechanism of protein aggregation diseases and for the development of prospective therapeutic treatments. peptides that are capable of targeting abnormal changes of living tissue can be very useful in early detection or diagnosis of, e.g., cancer. conjugating a functional agent, an effector unit, to such a peptide may provide the agent with improved pharmacodynamic properties.the specificity of a thiol group for reactive groups offer a unique way to attach effector units to cysteine-free linear or cyclic peptides. tumor targeting peptides were synthesized by fmoc-type solid phase methods. peptides cyclic by cystine were modified by lactam bridges. fluorescein, metal (e.g. lanthanide) chelates and cytotoxins were coupled to tumor targeting peptides via e.g. a peg-type spacer, or the conjugates were immobilized on plates for adhesion assays. the method was uncomplicated and gave stable conjugates with good solubility. the approach is useful in making stable peptide-effector conjugates and sets of them for e.g. detection assays such as delfia method and has prospective use in development of diagnosis and therapy. antibacterial proline-rich peptides -synthesis and antibacterial activity d. knappe, r. hoffmann the antibacterial proline-rich peptide family, originally isolated from insects, shows remarkable activity against diverse bacterial and fungal pathogens. while more and more bacterial pathogens become resistant to common drugs, this part of insect innate immunity provides a new promising approach to develop future peptide-drugs. proline-rich peptides possess a significant sequence homology and share a common mechanism of action. in addition oglycosylated threonine residues of drosocin and formaecin appear to be necessary for full antimicrobial activity, although the significance of the carbohydrate moiety in interaction with intracellular targets is still unknown. we synthesized analogues of different antibacterial peptides on solid phase by the fmoc-tbustrategy. the combination or insertion of sequence regions from different native antibacterial peptide sequences offers several advantageous effects, including further reduction of toxicity and broadening of the antimicrobial activity. furthermore, mimicking the o-glycosylation site and changing the carbohydrate moieties, may yield new synthetic approaches to increase both the activity and the selectivity of these oligopeptides. new immunotherapeutic approaches have been developed for treatment of neurodegenerative diseases of the alzheimer's dementia (ad) type. the identification of a ß-amyloid-plaque specific epitope, aß(4-10) (frhdsgy) [1] , recognised by therapeutically active antibodies from transgenic ad mice, provides the basis for development of new ad vaccines and for molecular ad diagnostics. in order to produce immunogenic conjugates, the aß4-10 epitope was attached via thioether linkage to synthetic carriers of well-defined structures, such as tetratuftsin derivative (ac-[tkpk(clac)g]4-nh2) and its elongated version by a helper t-cell epitope (ac-fflltriltipqsld-[tkpk(clac)g]4-nh2); sequential oligopeptide carrier (ac-[lys(clac)-aib-gly]4-oh) and multiple antigenic peptide (clac-lys(clac)-lys(clac-lys(clac))-arg-arg-ßala-nh2). the epitope conjugates containing a cysteine residue either at the c-or n-terminus, and the chloroacetylated carriers were prepared by spps according to boc/bzl strategy. conjugation reactions were performed in solution under slightly alkaline conditions, and monitored by hplc and high resolution-ms. structures and molecular homogeneities of all epitope peptides, carriers and conjugates were ascertained by hplc, maldi and esi-fticr-ms. conformational preferences of the synthesized compounds in water and in tfe were examined by cd spectroscopy. comparative binding studies of the conjugates with a mouse anti-amyloid protein beta-(1-17) monoclonal antibody were performed by indirect elisa. experimental data showed that the chemical nature of the carrier, the epitope topology and the presence of a pentaglycine spacer between the epitope peptide and the carrier, had significant effects on the antibody recognition and on the secondary structures of the conjugates. the new cla analogue 1, containing ethylene bridge between phe nitrogen atoms, was found to exhibit unexpected stimulatory effect in the model of the in vitro humoral immune response in mice. to disclose the structure-activity relationship the nmr solution conformational analysis was carried out. the solid-state and solution conformational analysis of native cla indicated the existence of this cyclic system as a complex mixture of conformations [1] . the nmr spectra recorded at 600 mhz in chloroform at 214 k showed the different conformational behaviour of both cyclopeptides: cla exists as one isomer [1] , peptide 1 is in an equilibrium among at least of three conformers. the picornaviruses are small nonenveloped rna viruses with a single positive strand rna. the virus replication cycle starts after the penetration of the virus in the cytoplasm of the host cell. there are several stages of the virus life cycle used for attack. one of the most useful strategies for attacking of the virus includes inhibition of important for the virus lifecycle enzymes. the key enzymes in the replication of the picornaviruses are 3c and 2a proteases. changes in the active center of these enzymes make them incapable to produce polyprotein in vitro, therefore the inhibition by low molecular weight molecules could stop the viral replication in vivo. 3c protease plays a major roll in the enzymatic proteolysis of the initial viral polyprotein. the target compounds were based on structural modifications in the known as crucial for the 3cp inhibition activity dipeptides phe-gln by incorporation of additional amino acid and pyrrole moiety. the synthesis was cared out as multiple-peptide synthesis in parallel using stepwise spps, fmoc-strategy. the obtained compounds were tested for antiviral activity by agar-diffusion plaque inhibition test against coxsackievirus b1 replication in fl cell and on this base some structure-activity interpretations were made. histone deacetylases (hdac) play important roles in various aspects of regulation such as proliferation, differentiation, and aging by counteracting with histone acetyl transferases. the hdacs categorized in class-i and ii have a metalloprotease-related mechanism in its catalytic activity. these enzymes could be inhibited by small molecules bearing various zinc ligands such as hydroxamic acid and mercaptan. based on the structure of chlamydocin, which has a cyclic tetrapeptide framework, cyclo(-l-aoe-aib-l-phe-d-pro-), where aoe is (2s,9s)-2-amino-8-oxo-9,10-epoxydecanoyl, we have developed potent hdac inhibitors as shown in the figure. in the present study, we examined the effects of the chlamydocin hydroxamic acid (1) and ss-hybrid (2) on the diabetes model mice, kk-ay. the peptide (1) exhibited satisfactory activity to reduce both the blood glucose and blood insulin levels comparable to or even superior to those by pioglitazone, a pparγ agonist. the ss-hybrid (2) , which is expected to be reduced inside of cells to generate the corresponding thiol-containing cyclic tetrapeptide, also showed a significant effect but less than (1). the effect was dose-dependent from 3 mg/kg to 30 mg/kg. the effects of hdac inhibitors were also confirmed by the observation of in vivo histone hyperacetylation induced in the lymphocyte cells. synthetic peptides have a number of advantages over current vaccines. however, exploitation of synthetic peptides as vaccines has been limited by the small size; copy number, inefficient delivery, poor peptide immunogenicity and mhc restriction. we have developed chemical methodologies that overcome these limitations by synthesising and polymerising vinyl-peptides. protective b-cell and t-cell peptide epitopes from the oral pathogen porphyromonas gingivalis were identified for three different mhc restricted mouse strains using pepscan techniques. fmoc chemistry for spps was used to synthesize these peptides and a vinyl moiety was incorporated at the n-terminus using acryloyl chloride. after rp-hplc purification free radical polymerisation using ammonium persulphate and temed was used to polymerise the vinyl-peptides in the presence of either acrylamide or other vinyl-monomers to produce single peptide or multi-peptide polymers. size exclusion chromatography indicated that the peptide polymers were >2 million da. the peptide polymers were used to immunise each mouse strain (balb/c, cba and c57bl6) and the t-cell response induced was evaluated using proliferation and cytokine (elispot) assays. the peptide polymers were found to be highly immunogenic, the single peptide polymers were found to only induce a response in their respective mouse strain, however, the multi-peptide polymer containing all of the t-cell epitopes was found to induce a response in all three mouse strains. in conclusion, our data shows that the polymerisation method overcomes all of the limitations in developing a peptide vaccine and most importantly that of mhc restriction. cytotoxic substances are auspicious weapons in tumour therapy. this compounds inhibit cell division and proliferation, hence, they affect all cells that are able to divide. however, all these compounds act intracellularly, i.e. at first they have to enter the tumour cell efficiently. this is a serious obstacle when using highly effective cytostatica and the cause of severe adverse effects by using higher doses. our aim is to overcome this problem by using synthetic hybride molecules composed of the cytostatic agent, in our case derivatives of arglabin, covalently linked to shuttle peptides. in order to identify the most effective possibility we tested two different strategies. by using the peptide hormone npy, whose specific y receptors are often overexpressed by tumour tissues, we intended to address the chemotherapeutic selectively to y receptor expressing tumour cells via receptor mediated endocytosis. on the other hand, the cytostatic agent was covalently bound to a cell penetrating peptide derived from human calcitonin (hct). recently, a c-terminal fragment of human calcitonin was found to internalize into excised nasal epithelium while the receptor activating n-terminal part is lacking. for the class of hct derived carrier peptides previous studies suggested a receptor independent "lipid raft" mediated endocytotic mechanism of uptake. here we present comparative data investigating both strategies, the highly selective receptor mediated delivery and the highly efficient receptor independent delivery. we investigated the peptide uptake by various cell lines and examined the release of the cytostatic agents inside the cells and its toxic effects. background and aims: synthetic peptides provide a straight-forward access to rationally designed inhibitors of molecular interactions based on structural information of proteins. poor membrane permeability may be overcome via cell-penetrating peptides, but low stability remains a major drawback. an increase of stability and bioactivity of peptides by coupling to polymers is intended. methods: for the development of peptide-functionalized cell-penetrating polymer conjugates, peptides were coupled chemoselectivly to hpma (n-(2hydroxypropyl)methacrylamide, average mw 28.5 kda), as an inert backbone polymer by native chemical ligation. hpma is water soluble and its capacity in drug delivery has been demonstrated. peptide-functionalized polymers and free peptides were incubated with proteases or cell lysates and proteolytic break-down was determined by fluorescence correlation spectroscopy (fcs) deriving information on the number and size of fluorescent particles based on temporal fluctuations of a fluorescence signal caused by diffusion of particles through a femtoliter-size confocal detection volume. the diffusion time depends on molecular size. therefore this technique is suited for the detection of proteolytic fragments. results: efficient chemoselective conjugation of unprotected fluorescein-labelled peptides was accomplished by means of native chemical ligation. our fcs investigations revealed that conjugation of peptides to hpma increased their biostability. this data also indicate that this effect is peptide-dependent. a proapoptotic peptide coupled to hpma and introduced into mammalian cells by electroporation retained its bioactivity. cofunctionalization of hpma with this peptide and nonaargine yielded an efficient cellular import. macrolides, a rather large group of natural or semi-synthetic antibiotics, are widely known translation inhibitors whose structure is based on 14-16-member lactones with carbohydrate substitutes attached. macrolides bind to ribosomal tunnel (rt) in a way that their lactone ring is located orthogonally to the long axis of the rt, covering most of its cleft. carbohydrate residues of the macrolides are spread along the walls of the rt. hence, the mechanism of protein synthesis inhibition by macrolides relies on the mechanical obstruction they provide to the passage of nascent polypeptide chain through the rt. the goal of this study was to design and obtain peptide derivatives of macrolides interesting both as antibacterial agents and potential probes for investigation of nascent peptide chain topography in the rt. tylosin ( in order to reach target sites inside the cns, neurotherapeutic candidates must overcome the blood-brain barrier (bbb). while several transport mechanisms occur at the bbb, this work has focused on the passive diffusion mechanism. the prediction of a peptide's ability to cross the bbb is not a simple task; hence there exists the need for the rational study of the relevant factors that affect the movement across this physiological barrier. here we present two new approaches based on in vitro non cellular assays for this type of studies. firstly, the evaluation of compound mixtures on parallel artificial membrane permeability assays (pampa). this approach increases the throughput of the study and structure-activity relationships can be easily establish. secondly, the transport and biological activity evaluation in a single assay. this second approach has been applied to the search of inhibitors for cns proteases involved in different neurological diseases (such as prolyl oligopeptidase for schizophrenia) able to cross the bbb. these two new approaches allow assaying compound's permeability in the early stages of a drug development project, and then designing novel analogues with improved bbb transport properties or using blood-brain-barrier shuttles for their delivery. two newly synthesized compounds are derivatives of l-valin and are positional isomers of nicotinic (m-6) and isonicotinic acid (p-6). these functional groups, as well as established good lipid solubility suggest that the main target for their biological action probably will be central nervous system. the presence of aminoacid l -valin, supposes their low toxicity, confirmed by our earlier experiments. the aim of the present work is to study their pharmacological activity as putative drugs. methods: male albino mice (18-20g, 10 in groups) were used. next neuropharmacological parameters were studied: analgesic effect (acetic acid test), neuromuscular coordination (rota-rod test), orientation ("hole board" test) and learning and memory (passive avoidance-step down test). results: significant analgesic effect of both compounds was established (more pronounced by dose 250 mg/kg i.p.). slight depressing effect on the orientation reactions in animals was registered, but the neuromuscular coordination and locomotor activity of treated animals were not changed. good dose-dependent effect on learning and memory was established and м-6 had stronger effect than р-6. the compounds modified the effects of some model substances with central nervous activity. hexobarbital sleeping time was significantly prolonged by p-6, but was antagonized by m-6. pentileneterazole threshold was increased significantly and suggests some anticonvulsant activity of both compounds. conclusion: as positional isomers m-6 and p-6 demonstrated some variations in their pharmacological activity probably due to the differences in their kinetics, metabolism and excretion. registered significant neuropharmacological activity, accompanied by low toxicity motivates the new synthesis and future experimental studies. the glyproline family of regulatory peptides includes pro-gly-pro, gly-pro, pro-gly and also the simplest peptides with hyp substituted for pro. a distinctive feature of these peptides is that they exhibit a broad spectrum of biological activities: the antiulcer activity, the inhibition of blood clotting and thrombosis, the reduction of degranulation activity of mast cells, and the normalization of stressogenic behavioral disorders. alzheimer's (ad) and parkinson's (pd) disease are progressive neurodegenerative disorders which are characterized by amyloid plaques. the main components of the plaques are β-amyloid peptides (aβ1-40 and aβ1-42) and α-synuclein. we have previously shown that small peptides structurally related to the sequence of aβ(1-42) protect against the neurotoxicity of aβ peptides. recent studies by other groups have shown that β-synuclein can counteract the aggregation of α-synuclein in the neurodegenerative process of pd, hereby might protect the central nervous system from the neurotoxic effects of alpha-synuclein. it was found that a tetrapeptide (kegv) protected against the neurotoxicity of aβ peptides in vivo. based on the previous findings, the following sequences of β-synuclein have been synthesized: kegv-nh2 and kegv-oh. after comparing the sequences of α-and β-synuclein, we found common sequences which are keqv, regv, kqgv, keqa. we have synthesized these tetrapeptides in amide forms at their c-termini. the peptides have been synthesized on mbha resin using a manual solid phase peptide synthesis equipment and boc-chemistry. the neuroprotective effects of peptides have been investigated in vitro in mtt test in differentiated neuroblastoma cell culture (sh-sy5y) and in electrophysiological test on rats using multibarrel electrodes in vivo. the neuroprotective peptides might stop neuronal death and can influence ad and pd progression. the present study was carried out to determine antinociceptive effect in vivo of plant peptide hormone phytosulfokine-alpha (h-tyr(4-so3h)-ile-tyr(4-so3h)-thr-gln-oh ) (i) and its selected analogues, such as h-phe(4-no2)-ile-tyr(4-so3h)-thr-gln-oh (ii), h-d-phe(4-so3h)-ile-tyr(4-so3h)-thr-gln-oh(iii), h-tyr-ile-tyr(4-so3h)-thr-gln-oh(iv), h-tyr(4-so3h)-ile-tyr-thr-gln-oh(v) and h-tyr-ile-tyr-thr-gln-oh (vi) in rats. peptides were injected into the lateral brain ventricle at the dose of 100 nmol. in the preliminary investigation we found the psk-as well analogues ii and iii induced a significant antinociceptive effect determined in the test of hot plate. the probable mechanism of this effect was discussed. a.b. bozhilova-pastirova 1 , b.a. landzhov 1 , p.v. yotovski 1 , e.b. dzambazova 2 , a.i. bocheva 2 the tyr-mif-1 family of peptides (tyr-mif-1`s) includes mif-1, tyr-mif-1, tyr-w-mif-1 and tyr-k-mif-1, which have been isolated from bovine hypothalamus and human brain cortex. all these peptides interact with opioid receptors and in addition bind to non-opiate sites specific for each of the peptides. data in the literature suggest that tyr-mif-1`s have antiopioid and opioid -like effects. we used wistar rats to study distribution and density of the tyrozine hydroxylase (th) imunoreactive fibres and nadph-d reactive neurons in the rat ventral and dorsal striatum. our results showed that neuropeptides mif-1 and tyr-mif-1 may affect them. opioid peptides have been recognized as modulators of reactive oxygen species (ros) in mouse macrophages and human neutrophils. data in the literature suggest that peptides of the tyr-mif-1 family -mif-1 and tyr-mif-1 have antiopioid and opioid -like effects. these neuropeptides are isolated from bovine hypothalamus and human brain cortex. so far no data about direct scavenger properties of tyr-mifs peptides were available. in this study we tested the hypothesis that they may scavenge ros in vitro. the antioxidant activity of these two substances was studied in the concentration range of 10-6 -10-4 mol/l. we investigated the luminol-dependent chemiluminescence to test their ability to scavenge the biologically relevant oxygen-derived species: hydroxyl radical, superoxide radical, hypochlorous acid in vitro. we found that tyr-mif-1 was a powerful scavenger in all tested systems. the effects were higher for hypochlorous anion and weaker for superoxide radical. mif-1 had no scavenge activity against the hydroxyl and superoxide radicals and showed a moderate scavenger effect on hypochlorous anion. we have compared different strategies to increase the immunogenicity of an antigenic hiv peptide as a vaccine candidate. our selected b-cell epitope comprises 15 amino acids (317-331) of the v3 region of hiv-1, jy1 isolate (subtype d), and is in tandem with a t-helper epitope corresponding to the 830-844 region of tetanus toxoid. several presentations, including oligomerization, map dendrimer, conjugation to dextran beads or to other macromolecular carriers, have been synthesized and evaluated. murine sera from the different presentations of the v3 epitope have been compared with regard to antibody titers and cross-reactivity with heterologous hiv subtypes. the map dendrimer version of the peptide, conjugated to recombinant hepatitis b surface antigen protein, was a better immunogen than the dendrimer alone, and showed higher immunogenicity than other multimeric presentations, or than the peptide alone conjugated to dextran beads. the map dendrimer version, either alone or conjugated to hbsag, enhanced cross-reactivity towards heterologous v3 sequences relative to monomeric peptide. group a streptococcus (gas) responsible for critical diseases (eg. acute rheumatic fever and rheumatic heart disease) are classified over 100 serotypes according to their surface virulence m proteins. development of vaccine to prevent infection with gas is hampered by the widespread diversity of circulating gas strains and m protein serotypes, and multivalent vaccine strategy would contribute to prevention against various gas infections and provide better protective immunity. we have studied the efficacy of incorporating four different epitopes derived from gas m protein into a single synthetic lipid core peptide (lcp) construct, in inducing broadly protective immune responses against gas following parenteral delivery to mice. peptide vaccine was synthesized on mbha resin by manual spps in situ neutralization/hbtu activation in boc-chemistry. immunisation with the mono-or multi-epitopic lcp vaccine led to high titers of antigen-specific systemic igg responses, and the production of broad protective immune responses as demonstrated by the ability of immune sera to opsonise multiple gas strains. systemic challenge of mice after primary vaccination showed that mice were significantly protected against gas infection in comparison with control mice demonstrating that vaccination stimulated long-lasting protective immunity. these data support to the usefulness of lcp system in the development of synthetic multiepitope vaccine to prevent gas infection. glycoprotein d represents a major immunogenic component of the virion envelope of herpes simplex virus and able to induce high titres of neutralizing antibodies. one of its optimal epitopes is the 9-22 region (9lkmadpnrfrgkdl22). several cyclic peptides possessing thioether bond and different ring size have been already prepared and some of them were conjugated with tetratuftsin derivative (ac-[tkpkg]4-nh2) by thioether bond formation using selectively removable cys protecting groups. antibody recognition results suggested that the size of the cycle has considerable influence on antibody recognition, however, the replacement of met in position 11 by nle is permitted. conjugation of cyclic peptide might increase the antibody recognition, but the binding depends on the structure and/or conjugation site of the cyclic peptide. conjugate with the best binding capacity (7.2 pmol/100ul) as well as the conjugate containing the linear (9-22c) epitope (0.7 pmol/100ul) were selected for immunization. in order to increase the production of antibodies a new group of conjugates was prepared. in these constructs promiscouos t cell epitope peptide derived from tetanus toxoid (ysyfpsv) was attached to both amino groups of lysine residue coupled to the n-terminus of the carrier (ac-ysyfpsv-k(ac-ysyfpsv)-[tkpk(clac)g]4-nh2). the cys containing linear and cyclic epitope peptides were conjugated to the carrier in solution (0.1m tris buffer, ph 8). this work was supported by grants of the spanish-hungarian intergovernmental program and cost chemistry action. background. primary hyperparathyroidism (pht) is characterized with increased parathyroid hormone (pth) secretion and in 70% of pht patients with hypertension. it was previously shown that pro-analogue of pth with a reversed sequence (which include strong alkali sequence -arg-lys-lys-) induced significant hypertensive response at dose 10-10m/kg b.w. one of the hypothesis attributed hypertension in pht patients to the presence of fragments of degraded pth possessing -arg-lys-lys-sequence. aim. to compare influence on mean arterial blood pressure (map) of analogue of 25-34 pth fragment (amide) and 25-34 fragment of pth, with -arg-lys-lys-sequence and also responsible for binding to pth receptor. methods. chosen peptides were synthesized manually by a solid phase peptide synthesis method. the purity of the products was tested by reversed-phase high performance liquid chromatography. the synthesized peptides showed the right molecular mass. the influence on map of synthesized peptides was tested in wistar rats. sequential increasing boluses of each peptide: 10-10, 10-9 and 10-8m/kg.b.w. in the same animal were given i.v. blood pressure was measured continuously in carotid artery. results. injection of synthesized analogue of 25-34 fragment of pth does not show influence on map vs. control group. synthesized 25-34 fragment of pth increased map in 92min. of experiment for 12mmhg ± 3mmhg vs. time of administration of first dose and for 17mmhg vs. control group. conclusion. it seems to be possible, that in case of alternate degradation of pth, accumulation of 25-34 fragment of pth may partially play role in the mechanism inducing hypertension in pht patients. biologically active domains of a high affinity receptor for ig е (fcεri) were determined, the fragments 111-114 and 111-117 of the receptor. the program of research of biological properties of synthesized tetrapeptide rnwd and heptapeptide rnwdvyk included the study of their binding with ige, which was contained in standard solutions and in patients' blood serum. the binding of peptides with ige was explored by the ifa method using ige antibodies labeled with horse-radish peroxidase (hrpo). peptides in the concentration of 100 mkg/ml were used for sorption on immunological plotting boards. higher correlation between the ige concentration and the optical density of the solution after introducing monoclonal antibodies labeled with hrpo and substrate chromogenic mixture (r=0.99) was found in the diagnostic system with sorption rnwdvyk-peptide than in the diagnostic system with sorption rnwd-peptide (r=0.94). similar investigations were conducted with the diagnostic systems with sorption rnwd and rnwdvyk peptides, but rwnd peptides conjugated with hrpo were used as antibodies against immunoglobulin e, instead of hrpo-labeled monoclonal antibodies. almost equal correlation was found between the concentration of ige in standard serum and serum of allergy patients with the known concentration of ige and the optical density of the solutions after introducing the rnwd peptide, conjugated with hrpo. after introducing allergy patients' blood serum in the holes on the plotting board, the heptapeptide bound 23.79% more ige than the tetrapeptide. our experiments demonstrated a high ige binding activity of synthetic rnwd-and rnwdvyk-peptides. in this study, the information spectrum method (ism) of the ha1 subunit of the h5 hemagglutinin protein of the influenza virus, h5n1, of different reference isolates was performed in order to identify possible antigenic determinants resistant to virus mutations. results of this analysis demonstrated that the primary structures of ha1 subunit of h5 hemagglutinins encode a common information corresponding to one characteristic frequency in their iss, which is probably important for the biological function of these proteins, including their possible recognition by the immune proteins targeting this molecule. besides, comparison of the iss of ha1 proteins of h1 "spanish flu" and h5n1 isolates demonstrated an informational similarity between them. based on these results, a segment of the n-terminus of ha1 h5n1 was identified to play a crucial role in the inhibitory and immunological properties of all possible h5n1 variants. the identified core segment, being highly conserved in all h5 strains, was selected as an antigenic determinant and coupled to the sequential oligopeptide carrier (socn), (lys-aib-gly)n, to the lys-nεh2 groups, in order to develop a diagnostic immunoassay and formulate a vaccine candidate for the highly pathogenic h5n1 influenza virus. background and aims: thrombin plays a key role in various disorders such as arterial thrombosis, atherosclerosis, restenosis, inflammation and myocardial infarction. insights into the way in which thrombin interacts with its many substrates and cofactors have been clarified by crystal structure and site-directed mutagenesis analyses, but until recently there has been little consideration of how its non-proteolytic functions are performed. we investigated cardiovascular effects of seven modified proline-and rgd-containing peptides designed from three surface-exposed sites of prothrombin, corresponding to residues 218-223, 332-347, and 445-454. methods: cardioprotective effects of synthetic peptides were tested on the two rat models of heart failure produced by coronary artery occlusion (10-or 45-min) and reperfusion (30-or 240-min). arterial blood pressures from left carotid artery, heart rate and ecg ii standard lead were measured throughout experiment. at the end of second experiment hearts were morphologically investigated by light microscopy and electron microscopy methods. results: on animal model with short-term ischemia investigated peptides did not protected from myocardium ischemia during occlusion, however, tp-l13, bk-mc and tp-h7 protected rat hearts from ventricular fibrillation, contributed more significant functional recovery during reperfusion and raising survival rate. on the model with prolong ischemia, acceptable cardioprotective effect revealed tp-h7 and bk-mc. these peptides significantly diminished necrotic zone of left ventricle, protected hearts from ischemia-reperfusion induced functional and morphological changes. conclusions: investigated proline-containing peptides revealed activity on cardiovascular system -decreasing of blood pressure, cardioprotective properties and improved recovery from ischemia. r. mansi, d. tesauro, c. pedone, e. benedetti, g. morelli the widespread use of compounds containing the gamma-emitting radionuclide 99mtc in nuclear medicine for the scintigraphic imaging, as well as the recent introduction of the beta-emitting radionuclides 188re and 186re in radiotherapy, have led to a rapid development of their chemistries, in order to produce novel radiopharmaceuticals. we have developed new peptide based radiopharmaceuticals based on a scaffold in which the radioactive metal ion is complexed by two different peptides that are able to bind two target receptors (see figure) . the 3+1 mixed-ligand approach has been used for the preparation of neutral oxotechnetium(v) and oxorhenium(v) peptide complexes. the complex preparation requires the simultaneous action of a dianionic tridentate ligand and a monoanionic monodentate thiolato on a suitable metal precursor. the dianionic tridentate ligand is based on the snn donor set able to stabilize the metal complex. the chelating agent (hsc(ch3)2conhch2ch(co-r)nh2) was coupled step by step to a bioactive peptide synthesized on solid phase. the second ligand, based on monodentate thiolato moiety, was coupled on n-terminus of the second peptide. labelling procedures and biological tests on tumour cells overexpressing receptors are described for 99mtc(o) complexed by cck8 and octreotide peptide derivatives. background: endostatin inhibits the proliferation of endothelial cells and induces their apoptosis. the measurement of serum endostatin can predict tumor vascularity. tumor angiogenesis is a strong prognostic factor in patients with hepatocellular carcinoma(hcc). significantly high levels of endostatin were noted in patients with trabecular-type tumors , and with hepatitis infection. methods : 20 patients with hcc, 16 patients with git malignancies, 8 patients with liver metastasis and 8 without metastasis, and 10 normal persons . all subjects were tested for alfa-feto protein (afp) , ca19.9 , carcinoemberyonic antigen (cea), and endostatin by elisa results : endostatin in normal control persons was 47.5 ± 14.22 ng/ml with a significant elevation (p< 0.001) between the hcc group and all the other tested groups .afp was 1.9 ± 0.98 ng/ml in normal persons with a significant elevation between hcc and all the other tested groups ( p< 0.01). ca19.9 was 8.14 ± 1.89 u/ml in normal persons with a significance elevation ( p< 0.01) relative to hcc., and a significance of ( p< 0.001 ) relative to git cancers with metastases. cea was tested to be 1.12 ± 0.71 µg/l in normal persons , and had a significance of ( p< 0.001) relative to git metastases. endostatin was elevated in 2 of 8 patients with git cancers not proved to have metastasis. conclusion : endostatin can be used to denote metaplasia and can also detect possibilities of metastasis or liver cell affection even before the frank development of metaplasia affibody® molecules are a novel class of affinity proteins which are generated by combinatorial engineering of the 58 aa three-helix bundle scaffold, originating from the b domain of staphylococcal protein a. we have used fmoc/tbu chemistry for total chemical synthesis of the affibody zher2:342, binding with picomolar affinity to the cell surface receptor her2. the synthetic protein was investigated for molecular imaging of her2-overexpressing tumours. in vivo detection of her2 in malignant tumours provides important diagnostic information which may influence patient management. to enable gamma camera imaging of the tumours, a panel of potential 99mtc-chelating sequences was designed and introduced into the affibody. the well-studied tc-chelating sequence mercaptoacetyltriglycyl (mag3) was compared to serine-containing sequences with increased hydrophilicity, such as mercaptoacetyltriserinyl (mas3). the total synthetic yield was 14-16 % and the her2-binding affinity of the affibody conjugates were all in the range 200-400 pm. binding specificity of tc-labelled affibody molecules was determined on her2-expressing skov-3 ovarian carcinoma cells. all variants showed receptor-specific binding. the tumour-targeting properties were studied in skov-3 tumour-bearing nude mice. all conjugates demonstrated high tumour uptake, quick blood clearance and low uptake in most other organs. the biodistribution results further showed that the more hydrophilic, serine-containing chelators resulted in a reduced hepatobiliary excretion, which significantly decrease the background in the abdomen area and provide for more sensitive detection. gamma camera images of mice with grafted tumours showed clear visualization of her2-expressing tumours using the 99mtclabelled mas3-affibody conjugate, suggesting a potential future application of this agent for diagnostic imaging. antimicrobial peptides are molecules with a unique mechanism of action. they are widespread in nature and play the role of an effective weapon of innate immune system against bacteria, fungi and viruses. the purpose of this study was to investigate the in vitro activity of natural antimicrobial peptides: citropin, piscidin, protegrin, temporin, uperin and the analogues of antimicrobial peptides: iseganan, pexiganan and omiganan. the peptides were synthesized using the solid-phase method and purified by high-performance liquid chromatography. the peptides were subjected to microbiological tests [mic (minimal inhibitory concentration) and mbc (minimal bactericidal concentration)] on reference strains of bacteria, according to the procedures outlined by the national committee for clinical laboratory standards (nccls). for comparison, conventional antibiotics (vancomycin, rifampycin, piperacillin, chloramphenicol) were included in this research. both the natural antimicrobial peptides and the analogues inhibited the growth of bacteria, but at higher concentrations than did conventional antibiotics. nevertheless, both natural origin of antimicrobial peptides and their low toxicity constitute a considerable advantage and this is an argument for considering the antimicrobial peptides as good candidates for medicines. the linear hexapeptide cypate-grdspk (compound 1; the cypate moiety is a near-infrared fluorescent label) whose rgd sequence was rearranged to grd showed high uptake in the αvβ3 integrin-positive tumor tissues in vitro and in vivo. despite low affinity of 1 to the integrin in the binding assays, the uptake was inhibited by equimolar amounts of the cyclic peptide c(rgdfv), which possesses high affinity to αvβ3 integrin. these observations led to hypothesis that cell internalization of compound 1 may be mediated mostly by only one of the integrin subunits, as the β3 one. indeed, blocking of αv integrin by the specific antibody did not inhibit the internalization of 1 in tumor cells, which was in the contrast with successful blocking the cell internalization by the anti-β3 integrin antibody. similar results were obtained in immunocytochemical assays employing the anti-αv and anti-β3 integrin antibodies. also, studies utilizing the β3-knockout and wild-type mouse cell lines demonstrated that deletion of the β3 subunit markedly decreased internalization of compound 1 in the β3knockout cells. the preferential interaction of compound 1 with the β3 subunit of integrins relative to the αv subunit was supported also by molecular modeling studies. summarizing, the bulk of our experimental and modeling data emphasizes interaction with the β3 integrin as the primary mechanism of the uptake of cypate-grdspk by tumors. since this compound showed the superior biodistribution profile in vivo, our results may provide a strategy to image and monitor the functional status of the β3 integrin in cells and live animals. background and aim: a growing tumor is accompanied by tumor intoxication development. intoxication independs on tumor size and intensity of its break-up. tumor intoxication is one of variant of endogenous intoxication. concentration of tyrosine-contained peptides (tcp) in blood plasma have been proposed as biochemical marker of endogenous intoxication at different organs cancers. our aim was to determine the tcp concentration in blood plasma patients with ovary tumor and its association with the severity of tumor. materials and methods: 178 patients with ovary tumor, mean age is 53 years, were studied. the control group consisted of 20 healthy women without tumor. patients were divided into 2 groups: people with non-malignant and people with malignant ovary tumor. tcp content in blood plasma was estimated by our technique. results: tcp concentration in the control group were 0,32±0,13 mmol. the tested marker was present in increased concentration in blood plasma of the patients with ovary tumor. the mean concentration tcp in patients with non-malignant tumor was 0,53±0,16 mmol. the content of this marker in blood plasma of patients from second group was increased 1,32±0,20 mmol compared with healthy control group. after treatment a significant decrease in tcp content was observed. conclusions: the result indicate that content tcp in blood plasma depends of the type of tumor. it could be suggested that determination of tcp concentration in blood plasma could be useful for improve the diagnostic of ovary tumor and monitoring of its progression. c. strongylis 1 , ch. papadopoulos 1 , k. naka 2 , l. michalis 2 , k. soteriadou 3 , v. tsikaris 1 troponin is a structural protein complex, which is responsible for the regulation of skeletal and cardiac muscle contraction. it consists of three components: troponin i (24kda), troponin c (18kda), and troponin t (37kda), each of which carries out different functions in the striated muscles. cardiac troponins are released into the bloodstream of patients after the onset of a cardiovascular damage. even minimal elevations over the normal values, of serum troponin t and i are being used to diagnose acute myocardial infarction and also to rule out the patients' condition. the development and commercialization of highly specific biological assays for the detection of cardiac troponins is based on the production of specific antibodies against the whole complex or individual subunits. however, the specificity and sensitivity of these assays vary due to problems mainly originated from the fact that cardiac troponins have a high homology with the skeletal isoforms. the aim of this work is to select and synthesize appropriate regions of the cardiac isoforms of troponin i, c and t, suitable for the production of more sensitive and specific cardiac troponin detecting reagents. in order to construct the immunogenic complexes, the selected sequences were conjugated to the tetrameric sequential oligopeptide carrier (soc4), either by the classic solid phase step-by-step methodology or by chemoselective ligation reactions. using the carrier conjugated troponin sequences, anti cardiac troponin complex specific antibodies in high titers were produced. the increasing problems with the reproductive systems of man and animals are recently linked to the presence of polluting chemicals with endocrine activity, the so called endocrine disrupting chemicals or edc's . the family of edc's is a heterogeneous one and consists of natural and synthetic hormones (like estradiol, ethynylestradiol and diethylstilbesterol), phyto-estrogens (like genistein and coumestrol) and industrial chemicals (like bisfenol-a, ftalates and various pesticides). because of the complexity of the environmental matrices and the low physiologically active concentrations of the edc's there is still a need for an efficient routine analysis protocol. we want to develop a solid-phase bound receptor that possesses a high selectivity for edc's and thus can be used in a simple solid-phase extraction protocol. this receptor must have the right functional groups that bind the edc's with a strong affinity and must be able to create a cavity in which the edc's can fit. by looking at nature's own estrogenic receptor for humans we have found the different amino acids responsible for the specific interactions . in order to create the cavity which mimics the behaviour of the hormone-binding domain of the human estrogenic receptor we have made a tripodal scaffold. this tripodal scaffold has three orthogonal protected amino groups that will allow the generation of three independent peptide chains. milk proteins are a source of opioid peptides. these peptides are liberated from milk proteins during enzymatic hydrolysis. some of these peptides are characterized with agonistic (β-casomorphins) and some with antagonistic (casoxins) properties. the aim of the investigations was to determine the presence of opioid peptides with antagonistic properties in milk products. the experimental material included cheeses, yogurts and kefirs. peptides were extracted with a methanol-chloroform mixture (2:1 v/v). the peptide extracts were purified by spe method on c18 or stratax columns and characterized by sds-page electrophoresis. the agonist opioid peptides (casoxins) were identified by hplc using standard agonist peptides. the opioid activity was measured by examining the effects of peptide extracts on the motor activity of isolated rabbit intestine. the results of sds-page electrophoresis indicated the presence of 5 to 9 fractions in peptide extract derived from cheeses and yogurts and 17 to 20 ones from kefirs. the presence of casoxin a (0.22-0.68 µg/mg of extract) was proved in all examined the milk products. lactoferroxin a (0.31-1.88 µg/mg of extract) was identified only in kefirs and yogurts. those products were also found to contain trace amounts of casoxin c. all peptide extracts showed the antagonistic activity in the relation to motor activity of isolated rabbit intestine. the highest antagonistic activity was reported of peptide extract from kefirs (3.62-17 .20%) and gouda cheese (15.68-16.36%), as compared to morphine. the physiological and nutritional function of these antagonist peptides requires elucidation. a. péter 1 , r. berkecz 1 , f. fülöp 2 the past decade has seen a growing interest in β-amino acids, which are important intermediates for the synthesis of compounds of pharmaceutical interest and can be used as building blocks for peptidomimetics. oligomers of β-amino acids (β-peptides) fold into compact helices in solution. recently, a novel class of β-peptide analogues adopting predictable and reproducible folding patterns (foldamers) was evaluated as a potential source of new drugs and catalysts. studies on synthetic β-amino acids can be facilitated by versatile and robust methods for determining the enantiomeric purity of starting materials and products. highperformance liquid chromatography (hplc) is one of the most useful techniques for the recognition and/or separation of stereoisomers including enantiomers. the aim of the present work was to evaluate hplc methods for the separation of enantiomers of eighteen 3-amino-3-aryl-substituted propanoic acids (β-amino acids). direct separations were carried out on different macrocyclic glycopeptide based stationary phases, such as ristocetin a containing chirobiotic r, teicoplanin containing chirobiotic t, teicoplanin aglycon containing chirobiotic tag, vancomycin containing chirobiotic v columns and on a chiral crown ether based column. the effects of different parameters on selectivity, such as the nature of the organic modifier, the mobile phase composition, the flow rate and the structure of the analytes are examined and discussed. the separation of the stereoisomers was optimized by variation of the chromatographic parameters. the efficiency of the different methods and the role of molecular structure in the enantioseparation were noted. the elution sequence of the enantiomers was determined in most cases. a rational approach to evaluating peptide purity a. swietlow 1 , r. lax 2 1 amgen, inc., pharmaceutics, thousand oaks, ca 2 polypeptide laboratories, inc., torrance, ca, usa recent years have seen an enormous increase in interest in peptide therapeutics. new peptide leads are often chosen by screening procedures using microgram to milligram quantities of peptides, frequently provided by specialized manufacturers utilizing automatic synthesizers to maximize output. the purity of the resulting compounds is often not very high. the use of spps synthetic procedures predetermines that most impurities are closely related and difficult to resolve by reversephase purification. these factors, combined with the use of generic analytical methods not specifically optimized for the peptide in question (e.g. the ubiquitous 0.1% tfa/water/acetonitrile system), lead to erroneous results that frequently severely overestimate the purity of the peptide. the use of poorly characterized materials in pharmaceutical development leads to significant risks of obtaining false negative or false positive results that may cause potential leads to be overlooked or misinterpretation of their pharmacological profiles. we describe a rapid, systematic and reliable hplc procedure for evaluation of peptide purity. utilizing the increased separation efficiency by increasing the column temperature and adjusting the gradient in two steps in reverse-phase buffers containing tfa, naclo4, or ion-exchange buffers containing kcl, we demonstrate that methods -suitable for preclinical research -can be developed rapidly. the proposed approach will be illustrated with examples of peptides ranging between 9 and 28 amino acids and a model peptide vypnga. it will be demonstrated that peptides showing an hplc purity close to 100% are often 10 -25% less pure. to approach a high-throughput cell assay format using peptides, we attempt to design and construct a peptide microarray for examination of cell activities of peptides including apoptotic cell death. peptides were immobilized onto solid surfaces via a novel multi-functional linker. the linker enable us to examine various types of peptide cell assays in an array format. we also designed and synthesized peptidyl capture agents on the basis of the cell-active sequences suitable for the peptide microarray. the utility of targeted nanoparticles as fluorescent probes for tissue imaging has recently been subject to widespread interest. one exciting prospect is the further development of nanoparticles conjugated to both targeting peptides and cytotoxic cargoes. these nanodevices could preferentially bind to specific cells and/or tissues to provide effective tools for drug delivery. hence, such multifunctional nanoparticles could provide both diagnostic and therapeutic functions by acting as fluorescent probes that offer targeted delivery of therapeutic agents. we have coated the surface of quantum dots (qdots) with cell-penetrating peptides (cpp) to target and label u251m cells for fluorescence imaging. qdots were initially coupled to polyethylene glycol linkers via carboxyl functionalities on their surface. a heterogeneous mixture of poly-arginine peptides of varying lengths (arg(6)-arg(10)) were covalently coupled via amide bonds to the polyethylene glycol linkers, conferring a cell penetrating capacity to the modified qdots. fluorescence imaging of u251m cells, after incubation with the conjugated qdots at concentrations of 20nm, gave clear signals indicating cell binding and internalisation of the modified qdots across the plasma membrane. we aim to further expand this work by employing racemic mixtures of cpp and cytotoxic agents to engineer conjugates that will facilitate both imaging and the therapeutic delivery of cytotoxic moieties. the ability of cell penetrating peptides (cpp) to deliver biologically active cargoes into different cell types has been successfully applied in several experimental systems. despite the progress and growing number of described cpps, reports about the internalization mechanisms and the intracellular routes of cpps still remain controversial. we have characterized the membrane interaction and cellular localization of proteins delivered into hela cells by cell penetrating peptide transportan (tp) and its shorter analogue tp10 on ultrastructural level. our previous results obtained by transmission electron microscopy showed that complexes of transportans with gold-labeled streptavidin translocated into cells inducing large invagination of plasmamembrane, suggesting the uptake by macropinocytosis. the complexes of transportan with gold-labeled neutravidin, in contrary, were taken into cells mostly via caveosomes and clathrin-coated vesicles. the cell-transduced transportanprotein complexes localized mainly in the vesicular structures of different size and morphology. most of the complexes-containing vesicles in the perinuclear area contained also lamp2 protein, marker of late endosomes and lysosomes. still, the transportan-protein complexes were not confined in the membrane-surrounded vesicles, but spread in the cytosol suggesting the escape of transportan-protein complexes from endosomes. our findings show the involvement of different endocytic pathways in the transportan-mediated uptake process of proteins. the concentration of a cpp and the properties of cargo protein seem to determine the pathway for the cellular uptake of a particular construct. rgd peptides (r = arginine; g = glycine; d = aspartic acid) have been found to promote cell adhesion upon interaction with alphav-beta3 receptors, which are strongly overexpressed during neoangiogenesis by solid tumor associated cells compared to healthy cells. in this study we designed new targeting motifs aimed to deliver various antitumoral drugs specifically to cells involved in tumor vascularization. we inserted this short rgd sequence in tetracyclopeptides closed with various means. we expect these new cyclotetrapeptides to be more specific for the targeted receptor. moreover, these new type of cyclic peptides were multimerized on different scaffold to further improve the receptor avidity. our purpose is first to scrutinize and to quantify the efficient cellular uptake of these molecules and second, to address the specific cell targeting of a fluorescent cargo by differents tools such as fluorescence activated cells sorting (facs) analysis or fluorescence microscopy. these new targeting units were evaluated on two different cell lines: human umbilical vein endothelial cells (huvec) with an over expression of the alphav-beta3 integrin receptor and a549 cells expressing a much lower level of this receptor. preliminary results about the selectivity and the efficacy of these new targeting units will be presented. we have recently developed new approaches for obtaining highly immunogenic peptide conjugates: synthetic polyelectrolytes (pe) were used for the conjugation with peptide molecules in which pe carry out the carrier and adjuvant roles simultaneously. in this study, 4 epitopes of antigenic parts of surface antigen of hepatitis b virus (2-16, 22-35, 95-109 and 115-129 of the s gene.) had been synthesized.the synthesis of peptides was performed by explorer pls ® automated microwave synthesis workstation (cem). peptide conjugates of synthetic anionic polyelectrolytes (copolymers of acrylic asid and n-vinylpyrrolidone) were synthesized by carbodiimide condensation following the modification procedures described early. composition and structure of bioconjugates were characterized by hplc (shimadzu), nanospr-3, zetasizer nano zs, steady state fluorescence spectrometer qm-4 and viscotek tda 302 size exclusion chromatography. it was obtained that a single immunization of mice with pe-peptide conjugates without classical adjuvant increased the primary and secondary peptide-specific immune response to hbsag. moreover, these conjugates possess own selectivity for recognizing the antibody in blood sera of hepatitis virus injected people. tissue engineering requires delivery of transplanted cells to organ sites needing repair/regeneration. we have demonstrated that several active laminin peptide-conjugated chitosan membranes enhanced the biological activity and promoted cell adhesion in a cell-type specific manner. the most active laminin peptide (ag73: rkrlqvqlsirt)-conjugated chitosan membrane could deliver keratinocytes to a wound bed. when human keratinocytes were seeded onto the ag73-chitosan membranes under serum-free condition, more than 70% of the cells attached within 2 hrs. the membranes carrying keratinocytes were stable enough for handling with forceps and were inverted onto the muscle fascia exposed on the trunk of nude mice. keratinocyte sheets were observed after 3 days and colonies appeared after 7 days on the fascia of host mice. these cells were multilayered on day-3 and expressed various keratinocyte markers, including cytokeratin-1, involculin, and laminin ?2-chain. these results suggest that the ag73-conjugated chitosan membrane is useful as a therapeutic formulation and is applicable as a cell delivery system, such as delivering keratinocytes to the wound bed. the peptide-chitosan approach may be a powerful cell transplantation tool for various tissues and organs. the fluorescent semi-conductive (cdse/zns) nanocristals possess very attractive optical properties that could be used for tracking individually biological receptors in vivo. our aim is to design functionalized water-soluble semi-conductive nanocristals (or quantum dots) that interact selectively with lipidic or biological membranes. to valid our approach, the interaction between the decorated qd and giant vesicles were observed by optical fluorescent and dark field microscopies. in view to solubilize and selectively bind fluorescent nanocristals to a lipid membrane, heterobifunctionalized peptidic ligands (liipe) that presented an adhesion domain for the nanocristal surface, an hydrophilic spacer and a terminal recognition function, were synthetized. the colloïdal stability of the water-soluble nanocristals (nc-lipe) was checked by dynamic light scattering, optical and electron microscopies the interaction of grafted nanocristals (nc-lipe) with positive or neutral giant vesicles was observed by optical fluorescence and dark field microscopies. as shown in figure, negatively charged nanocristals (nc-lipe) selectively adsorbed onto the surface of positively charged giant vesicles without altering the morphology of the vesicle. the nanocristals appeared as fluorescent patches growing on the surface of the vesicle until completely recovering. therefore these ligands (lipe) permitted to chemically functionalize the nanocristals by keeping their colloïdal stability and their fluorescence in water. furthermore it was possible to selectively label vesicle membrane. creatine analogues for treatment of obesity: us patent 5 bioactive peptides containing pairs of basic amino acids are rapidly metabolized as a result of cleavage by trypsin-like enzymes. to increase the metabolic stability of opioid peptides containing arg-arg and arg-lys pairs, the arg residues were replaced by homoarginine (har) kenes international leprince 1 , f. cavelier 2 , p. gandolfo 1 , m. diallo 1 , h. castel 1 , l. desrues 1 m304 new bradykinin analogues modified with 1-aminocyclopentane-1-carboxylic acid effect on rat blood pressure and rat uterus o. labudda-dawidowska 1 , d. sobolewski 1 , m śleszyńska 1 , i derdowska 1 synthesis and heparin binding sites identification f. baleux 1 , f. arenzana-seisdedos chemokines are small proteins involved in numerous biological processes (inflammation, immunity, morphogenesis, tissue repair, and tumour development) the general goal of our project is to elucidate the role that hs plays in vivo in the physiologic and pathologic activities of the chemokine cxcl12/stromal derived factor-1, and to characterise the molecular and structural determinants accounting for the interaction. three sdf isoforms, alpha (68 aa), beta (72 aa) and gamma (98 aa) have been identified. we previously identified the major heparin binding site on sdf alpha and demonstrated the importance of hs/sdf interaction in hiv entry cell inhibition (1,2). sdf gamma amino acids sequence corresponds to the sdf alpha sequence extended by a c-ter 30 amino acids sequence containing putative heparin binding sites. in order to determine sdf gamma heparin binding sites, wild type and mutants proteins were synthesised by stepwise solid-phase peptide synthesis using fmoc chemistry prusiner proc. natl. acad. sci here we describe xenome's drug development process for the chi family, conopeptide -mria[1] of the predatory marine snail conus marmoreus, leading to a suitable drug candidate (xen2174). xen2174 is highly selective for the norepinephrine transporter (net) compared to other transporters, such as dopamine and serotonin, and inhibits net via an allosteric mechanism. xen2174 is currently in a phase i/iia clinical trial for the treatment of severe pain. an intensive synthetic analogue and screening program around -mria, incorporating early stage animal data xen2174 isomers were synthesized via selective disulfide bond formation to identify the active connectivity. data from alanine-scans, single amino acid mutations and probing of backbone interactions combined with the full 3d nmr structure, led to the development of a pharmacophore for xen2174. this model is refined from further studies where structure-activity relationships were developed utilising binding and functional assay data for a range of peptides anti-cancer drug design anti-cancer drug design published structural data for hdac-like protein, a bacterial enzyme sharing high homology to the hdacs in its active site, confirmed that this protein contains a zinc in the active site. for the discovery of specific hdac inhibitors, a number of hydroxamic acis and related compounds have been designed based on the ligating function to the zinc atom. the mechanism also involves an appropriate nucleophile in the active site. chlamydocin is a cyclic tetrapeptide on the other hand, we have been focusing the cyclic tetrapeptide to develop the potent and specific inhibitors of hdacs. in the present report, we employed the chlamydocin scaffold and successfully introduced the series of thioether as the functional group to the cyclic tetrapeptide. it is well argued that the strong inhibition of hdac requires the best combination of zinc ligand, capping group, and appropriate spacer between them jerusalem 3 department of biological regulation 4 department of organic chemistry 5 department of biological chemistry, weizmann institute of science, rehovot, israel estrogen has a key role in the regulation of skeletal growth and maintenance of bone mass. the use of estrogen and selective estrogen receptor (er) modulators in treatment of osteoporosis is limited due to substantial risks for breast cancer. recently, we developed peptides having estrogen-like activity peptide emp-1 had no effect on bone growth in normal mice, and did not influence the ovx-induced bone-loss. we then developed a new µct methodology to evaluate uncalcified and calcified growth-plate parameters. in the ovx mice, peptide emp-1 reduced volume and thickness of the uncalcified growth-plate, a possible cause for the inhibition of bone longitudinal growth. based on a reported enhancement of er-in female mice during protein biosynthesis, after the release of the nascent polypeptide chain, ribosome recycling factor (rrf) disassembles the post-translational complex. rrf has been shown to be essential for bacterial growth. thus, we are attempting to design suitable compounds to inhibit the rrf function as candidates for new-type antibiotics. we have determined the structure of rrf with 185 amino acid residues by nmr and x-ray analyses and shown that rrf has two domains domain i with three stranded helical bundles and domain ii with &beta;/&alpha;/&beta furthermore, we recently determined the structures of the 70s ribosome-rrf complex by cryo-electron microscopy and the 50s ribosome-rrf domain i complex by x-ray analysis using the results of these experiments, we elucidated the interaction profiles between rrf and ribosome and found that the cationic center consisting of three arginine residues on the surface of the helical bundle, which we have shown to be essential for the activity of rrf, is bound to helix 69-71 of 23s ribosomal rna. we synthesized the rna and peptide fragments around this interacting site and characterized them by physico-chemical analyses. the results of cd and biacore experiments to investigate the details of the interactions between them showed that a 27 mer of rna fragment is bound to rrf <i>biochemistry</i> <b>42</b> causes an increase of pain by inhibiting the opioid response [1]. recent research has shown further that melancortin receptors, mainly subtype mc4r, produce an increase in response to pain stimuli [2]. based on this previous work, we are developing chimeric ligands which will be of benefit to therapeutic pain treatment with enhanced opioid efficacy by acting as agonists at opioid receptors and antagonists at both cck and melanocortin receptors cck (i) and melanocortin (ii) pharmacophores were overlapped by trp, and different profiles of opioid pharmacophores (iii) were linked to the n-terminal of the melanocortin pharmacophore (figure). the designed ligands showed moderate to high biological activity at all three receptors depending on their respective structures. design considerations and structure-activity relationships will be discussed in detail along with in-vivo assay results china synthetic exendin-4 is a 39-amino acid peptide that exhibits potent anti-diabetic and dose-dependent glucose-regulatory activity. exendin-4 is susceptible to degradation in plasma, so its activity is limited. our aim is to find sites in exendin-4 that are susceptible to cleavage and provide information for designing new exendin-4 analogues. in this study the stability of exendin-4 in human plasma was evaluated in vitro. exendin-4 was incubated in plasma at 37 ℃, extracted with sep-pak octadecyl columns and subsequently analyzed using high performance liquid chromatography (hplc). the results showed that exendin-4 was slowly broken down in plasma with a half-life of 9.57 h. the degradation products were identified by quadrupole time of flight mass spectrometry (q-tof-ms) with electrospray ionization pharmacology of exenatide(synthetic exendin-4): a potential therapeutic for improved glycemic control of type 2 diabetes one of the proposed solutions for the pharmacotherapy of obesity, a major health problem in the western world, is to regulate the biochemical pathways that control the metabolic balance in the body. the melanocortin pathway regulates energy balance by binding of the catabolic endogenic neuropeptide αmsh to its mc4 receptor and thus causes a decrease in food intake. we have synthesized a backbone cyclic peptide library, based on the minimal αmsh sequence phe6-d-phe-arg-trp9 [1], that activates the mc4 receptor. all the members of the library shared the same sequence analysis using colorimetric liposomes confirmed that bbc-1 penetrate the intestinal cells by the transcellular mechanism. moreover, bbc-1 have high metabolic stability to intestinal enzymes (100% recovery after 5 hr). ec50 analysis showed that bbc-1 selectively binds and activate the mc4 and mc5 receptors (ec50 3.97±0.63 and 7.27±0.40 respectively). oral administration of bbc-1 in mice showed reduces food intake melanocortin tetrapeptides modified at the n-terminus, his, phe,arg and trp positions 2 department of experimental and health sciences graduate school of pharmaceutical sciences 2 graduate school of agriculture 3 graduate school of medicine consisting of 54 amino acids, is a product of the metastasis suppressor gene kiss-1. this c-terminally amidated peptide was identified as the endogenous ligand of an orphan g-protein coupled receptor metastin-gpr54 signaling may regulate gonadotropin secretion and negatively regulate cancer metastasis. it is interesting that activation of gpr54 signaling negatively regulates the function of sdf-1-cxcr4 axis in cho and hela cell transfectants we conducted the structure-activity relationship (sar) study on kisspeptin-10 using the neuropeptide-derived rw-amide scaffold to identify five-residue peptide amides as novel gpr54 agonists equipotent to kisspeptin pro-), where aoe is (2s,9s)-2-amino-8-oxo-9,10-epoxydecanoyl. in continuation of our study to design and synthesize analogues bearing a zinc ligand to develop potent inhibitors of histone deacetylase (hdac) inhibitors, we shifted the aromatic ring of phenylalanine at the aminoisobutric acid (aib) position and also at the imino acid position. the aim is to explore the interaction of cyclic scaffold with the rim of hdac paralogs. we replaced the epoxyketone moiety of aoe with sulfhydryl group, which is protected as disulfide hybrid, as zinc ligand. benzene ring was introduced to aib structure to design amino-1-indane carboxylic acid and amino-2-indane carboxylic acid. aromatic ring-containing imino acids, such as d-tic were also employed to replace d-pro. the cyclic tetrapeptides were profiled by the inhibition of hdac1, hdac4, and hdac6 in adult goats, however, the infection remains unapparent and the virus may cause abortion, vulvovaginitis or balanoposthitis. the use of a vaccine could provide a powerful tool for the control of cphv-1 infection. synthetic peptide-based vaccines have advantages of being selective, chemically defined and safe. in order to further localize immunogenic epitopes, glycoproteins b, c and d of cphv-1 were analyzed with several prediction programs. peptide conjugates incorporating t and b cell determinants in multiple copies in branched architecture are better immunogens for the preparation of goat vaccines, we synthesized peptide conjugates bearing t cell epitope on the n-terminal of the core. b cell epitopes were conjugated via a thioether bond on the ne-amino group of four choroacetylated lysine residues of the core. elisas confirm that the b cell epitopes and the conjugates t celltetratuftsin induce epitope-specific and antibody responses two recorded electrodes implanted bilaterally. eeg recorded after the mirror focus was arises. trh applicated intranasal in ultra low doses (10-9 m and 10-12 m) or intravenously in high doses (25mg, 50mg, 100 mg). for eeg registration and analysis the computer system conan was used and new modification of fractal analyze of quantized eeg. results: the synchronization of epileptic activity between primary and mirror focuses observed on the third day after operation. intranasal application of trh induced reduction of spontaneous focal epileptic activity as in primary cobalt damage focus as in the mirror focus more than 1h. the inhibition of mirror focus was more expressed. quite the contrary intravenous trh administration provocated the epileptic discharges in both local focus. the intense synchronized generalized activity was record during 30-40 min deraos 1 , t.v. tselios 1 , i. mylonas 2 , g.n. deraos 1 it is known that cyclic peptides are more stable in enzymatic degradation and conformationally restricted compared to linear. the cyclization was achieved using o-benzotriazol-1-yl-n,n,n',n'-tetramethyluronium tetrafluoroborate (tbtu) and 1-hydroxy-7-azabenzotriazole, 2,4,6 collidine allowing fast reaction and high yield final product. the purification was achieved using reversed phase high performance liquid chromatography (rp-hplc) and the peptide purity was assessed by analytical hplc and mass spectrometry (esi-ms). the synthesized cyclic plp analogue was found to exhibit lower agonist eae activity compared to linear plp139-151 epitope in sjl/j mice. this implies that the conformation of cyclic analogue does not trigger autoimmune reaction in the central nervous system and therefore encephalomyelitis the netherlands 3 department of experimental and health sciences on the other hand, in both central and peripheral nervous system, cck acts as neurotransmitter. recently, cck is focused at modulation effects on feeding especially. in this study, we tried to establish a sensitive and specific enzyme immunoassay (eia) for detecting cck and to investigate the effect of some dopamine receptor antagonists using this eia, we measured plasma cck-like immunoreactive substance (is) levels in five healthy human subjects after single oral administration of some prokinetics. the minimum amount of cck detectable by our eia system was 2.0 pg/ml, and the ic50 of the calibration curve was 75 pg/ml. we revealed that domperidone and itopride caused significant decreases in plasma cck-is levels but metoclopramide and sulpiride did not. we established a sensitive and specific eia for cck. furthermore pro-pro-phe-phe-), isolated from linseed oil [1], possesses a strong immunosuppressive activity comparable at low doses with that of cyclosporin a [2]. it has been postulated that the tetrapeptide sequence pro-pro-phe-phe is important for biological activity of cla on the basis of this information we have synthesized a series of cla analogues in which the alpha-proline residue was replaced by beta2-iso-proline and beta3-homo-proline residues, respectively (fig.1). the synthesis of titled beta-amino acids has been achieved according to the literature procedure italy synthetic peptides are largely used as antigens in solid-phase immunoenzymatic assays (elisa) for recognition of antibodies (abs) in biomedical research and, most importantly, in the set up of diagnostic methods. it is well known that the method of peptide immobilization on the solid support is very important for a correct ab recognition atherosclerosis in patients infected with helicobacter pylori (hp) we synthesized appropriate oligopeptides immobilized on cellulose via n-or c-termini, using standard -alanine linkers as well as a new linker, developed for this particular studies glc)ghsvflapygwmvk) we found the strongest recognition when the peptide was linked to the cellulose support via the c-terminus. however, in the case of ureb f8 hp urease smallest epitope (sikedvqf), and epitope ub-33 (321-339 hp fragment: chhldksikedvqfadsri) the strongest reactions with sera of atherosclerosis suffering patients were obtained for n-terminally anchored peptides the synthetic dipeptide gamma-d-glutamyl-l-tryptophan (scv-07) has been shown to stimulate t-lymphocyte differentiation and specific immune responses, and enhance il-2 and inf-gamma production. due to this preferential activation of th1 cytokine production, scv-07 may show utility in treatment of infectious diseases cba mice at a dose of 2,500 µg/kg. the same animals were used for all three methods of administration with a dosing interval of 2 weeks. blood samples were taken from the right retro-orbital sinus. for determination of the scv-07 concentration in blood samples, an "eia-scv-07" competitive solid-phase immuno-enzyme assay was developed (loq 20 ng/ml) with a mean residence time of 10 minutes. 5 minutes postdose, indicating very rapid absorption. mean concentrations then declined and were measurable through 1.5 and 3 hours postdose (mrt 20 and 50 minutes, for i/p and p/o, respectively). the estimated bioavailability of scv-07 after i/p and p/o administration was almost equivalent gastrin-17 (g17) is a peptide which promotes gastric acid secretion, cell proliferation, and occasionally gastrointestinal cancer in the gastric antrum. g17 also promotes the growth of cancerous colonic epithelial cells, but the cck2/gastrin receptor, which mediates its activity, is largely not expressed on such cells. instead, our previous studies have shown that some other receptor mediates stimulation of proliferation of dld-1 and ht29 human colonic carcinoma cells by ncarboxymethyl gastrin (g17gly) at namomolar concentrations and inhibition at micromolar concentrations, indication separate binding sites. we have shown previously that g17(1-12)-oh stimulates cell proliferation of ht-29 cells -6)-nh2, in order to determine their selectivity for and activation of the putative proliferation-stimulatory receptor. the results revealed that g17(1-12)-oh is not selective for a single receptor, but binds both sites as do g17 and g17gly. g17(1-6)-nh2 promotes dose-dependent and non-biphasic proliferation of dld-1 cells and binds a single receptor with low affinity. m484 comparative study of ige-binding activity of synthetic peptides rnwd and rnwdvyk v th486 involvement of l-name in the antinociceptive effects of newly synthesized analogues of tyr-mif-1 peptide in rats tyr-mif-1 is able to interact with opioid receptors with a higher potency at m sites as well as to its specific non-opiate receptors in the brain. nitric oxide and tyr-mif-1`s are potent modulators of opioid activities. involvement of no in nociceptive effects is well documented in various physiological and pathological processes in the cns. l-name when administrated i.c.v. or systemically exhibit antinociceptive activity in rats as evaluated by the pp test. in the present study, we investigated the involvement of l-name in the antinociceptive action of newly synthesized analogues of tyr-mif-1 peptide: nα-(me)tyr-mif-1, d-tyr-(me)-mif-1, tyr(cl2)-mif-1 and tyr(br2)-mif-1. the experiments were carried out on male wistar rats (180-200 g). the changes in the mechanical nociceptive greece paclitaxel is one of the most important anticancer drugs used mainly in treatment of breast, lung, and ovarian cancer and is being investigated for use as a single agent for treatment of lung cancer, advanced head and neck cancers, and adenocarcinomas of the upper gastrointestinal tract. however, the development of resistance to paclitaxel, the side effects and low solubility of this drug remain major obstacles for its optimal use in the clinical practice. in this work, we present the synthesis of various analogues in which paclitaxel is covalently bound to peptides or as multiple copies to synthetic carriers. these peptide-paclitaxel derivatives possess greater solubility in water, could be suitable in producing anti-paclitaxel antibodies and inhibit the proliferation of human breast, prostate and cervical cancer cell lines. although, no major differences were found concerning the extent of the antiproliferative effect between various paclitaxel derivatives and paclitaxel, the analogue with four molecules of paclitaxel covalently bound to synthetic carrier [ac-(lys-aib-cys)4-nh2] when used at low concentrations inhibited cell proliferation more potently than paclitaxel th489 involvement of the histaminergic system in the nociceptin-induced pain-related behaviors in the mouse spinal cord the purpose of the present study was to determine whether histamine-containing neurons in the spinal cord are involved in nociceptin-induced behaviors in mice. the i.t. injection procedure was adapted from the method of hylden and wilcox. immediately following the i.t. injection, the time spent for nociceptive behaviors including scratching, biting and licking were measured. the i.t. administration of nociceptin resulted in nociceptive behavioral responses, which were eliminated by the i.t. co-administration of opioid receptor like-1 (orl-1) receptor antagonists. the nociceptive behaviors were significantly attenuated by the i.t. co-administration of the h1 receptor antagonists, but not the h2 receptor antagonists. i.t. co-administration of the h3 receptor antagonist significantly increased the behavioral responses, whereas the behavioral responses were completely attenuated by the i.t. co-administration of the h3 receptor agonist. an antiserum against histamine injected i.t. reduced the nociceptin-induced behavioral responses. the same result was observed by i.p. pretreatment with histidine decarboxylase inhibitor. in conclusion, i.t.-administered nociceptin elicits the orl-1 receptor-mediated nociceptive behavioral responses. the activation of the orl-1 receptor by nociceptin may induce the release of histamine in previous studies, we demonstrated that highly constraint cyclic (s,s)-cxaac-containing peptides inhibit platelet aggregation and fg binding [1,2]. cyclization reduces the allowed conformations, of both the backbone and the side chains, and possibly induces a favourable for the biological activity orientation of the charged side chains. conformational studies revealed that orientation of the charged side chains toward the same side of the molecule increase the anti-aggregatory activity of the inhibitor. in this work we present the synthesis and the inhibitory activity of new cyclic compounds. for the design of the studied compounds we combined the available information from the -cdc-containing inhibitors and the gpiib 313-320 (ymesradr) sequence which has been shown to inhibit the adp induced human platelet activation however, its pharmacological effects and physiological functions are still unclear. the present study was designed to characterize the nociceptive behaviours induced by intrathecal (i.t.) administration of hk-1 in mice. the i.t. administration was made in conscious mice according to the method described by hylden and wilcox. immediately after the i.t. administration, the accumulated time for nociceptive behaviours was measured for 10 min. the i.t. administration of hk-1 dose-dependently produced characteristic nociceptive behaviours consisting of scratching, biting and licking, which peaked at 0-5 min and almost disappeared by 15 min after injection. the subcutaneous pretreatment with morphine dose-dependently attenuated the hk-1-induced nociceptive behaviours. the nociceptive behaviours elicited by low-dose of hk-1 were significantly inhibited by i.t. co-administration with nk1 receptor antagonist, however, the nociceptive behaviours elicited by high-dose of hk-1 were not affected by i.t. coadministration with nk1 receptor antagonist. on the other hand, nmda receptor antagonists significantly suppressed both high-and low-doses of hk-1-induced nociceptive behaviours in a dose-dependent manner. these results suggest that the nociceptive behaviours induced by low-dose of hk-1 may be mediated through both nk1 and nmda receptors, whereas high-dose of hk-1 may induce the nociceptive behaviours through nmda receptor the bacterial lp are strong modulators of the innate immune system. until recently, it was generally assumed that triacylated lp, like the synthetic pam3cys-sk4, are recognized by tlr2/tlr1 heteromers, whereas diacylated lp, like fsl-1, induce signalling through tlr2/tlr6 heteromers. contrary to this model, we could show that depending on the peptide moiety, diacylated lp also signal in a tlr6-independent and tlr1-dependent manner. the aim of this study was to analyse more closely the structural basis of this heteromer usage. the synthesis of lp was carried out by fully automated solid phase peptide synthesis and fmoc/tbu chemistry on tcp or rink amide resin. information on the structural factors determining the tlr2/tlr1 versus tlr2/tlr6 heteromer usage was obtained by testing of ligands with cells obtained from tlr2 , tlr6 , and tlr1-deficient mice. when stimulating b-lymphocytes of wild-type mice we found that ester-bound long-chain fatty acids are necessary to induce considerable responses. for triacylated lp with long chain length ester-bound acyl residues (like pam3c-ssnask4) the response in tlr1-deficient cells was only slightly decreased, whereas for lp with short length ester-bound fatty acids (like pamoct2c-ssnask4) the response was completely abolished. in summary, a tri-acylation pattern is necessary but not sufficient to render an lp tlr1-dependent and a di naples 2 department of organic chemistry "ugo schiff sera from patients suffering from autoimmune disorders often contain multiple types of autoantibodies, some of which can be exclusive of a disease and thus used as biomarkers for diagnosis. identification of these autoantibodies, as disease biomarkers, should be achieved using native antigens in simple biological assays. however, post-translational modifications, such as glycosylation, may play a fundamental role for specific autoantibody recognition. in line with these observations, we previously described synthetic glycopeptides able to detect high autoantibody titers in sera of patients affected by multiple sclerosis, an inflammatory, demyelinating disease of the central nervous system. we also demonstrated that glycopeptides able to reveal high antibody titers in multiple sclerosis sera are characterized by a type i we describe here the result of a conformationally driven rational design exercise, which led to the preparation of new, optimized glycopeptides endowed with enhanced antigenic properties. most importantly, the same approach, based on structure alignment, was used to shed light on the native antigen(s), target of pathogenetic autoantibodies involved in demyelination processes vitro and in vivo evaluation for cholecystokinin-b receptor imaging istituto nazionale per lo studio e la cura dei tumori ome (f0)<br> (aib, α-aminoisobutyric acid) to investigate its binding properties to tb(iii) ions. according to our published spectroscopic results f0 populate a set of ordered conformations involving 310/α-helical segments and compact structures generated by the formation of a turn around the flexible gly5-gly6 central motif. cd experiments showed that the binding of tb(iii) to f0 gives rise to a structural transition of the peptide chain from a helical to a folded conformation. peptide binding is also responsible for the dramatic increase in the tb(iii) fluorescence intensity, suggesting that the tb(iii)/f0 complex may represent an interesting system for imaging applications or bioanalytical sensing the 16 kda protein of mycobacterium tuberculosis provokes specific immune response, therefore related epitope peptides and peptide-conjugates can be considered as potential diagnostics. in our previous study we have determined the functional human t-cell epitope within the 91-110 region. based on this we synthesised two groups of peptides: a) nand c-terminal alanine and beta-alanine elongated variants of the 91-104 epitope and b) 91-104 peptides with alanine substitution at different position according to the hla dr and tcr binding sites. peptides were prepared by solid phase synthesis using boc/bzl or fmoc/tbu strategy. the homogeneity and the primary structure of peptides were checked by analytical rp-hplc, amino acid analysis and esi-ms. the t-cell stimulatory activity of the compounds was investigated using in vitro assays (proliferation and ifn-gamma production) on the 91-110 epitope specific human t-cell clones and pbmc (peripherial blood mononuclear cells) from patients and healthy (ppd+, ppd-) subjects. the effective peptides were conjugated to branched polypeptides with polylysine backbone (sak, eak), tetratuftsin derivative (h-[thr-lys-pro-lys-gly]4-nh2) and lysine dendrimer (h-lys-lys(h-lys)-arg-arg-beta-ala-nh2) (map) carrier via thioether bond formation. the subtitution degree of the conjugates was determined by amino acid analysis. pbmc and human t-cell clones were stimulated with the free peptide alone or with peptide-conjugates containing an equimolar amount of peptide or with a mixture of free peptide and carrier italy we demonstrated, for the first time, that an aberrant post-translational modification (ptm, n-glucosylation) is possibly triggering autoantibody response in multiple sclerosis. this was possible because of a "reverse approach", which led to csf114(glc), a structure based designed glycopeptide, as the first multiple sclerosis antigenic probe accurately measuring high affinity autoantibodies (biomarkers of disease activity) in sera of a statistically significant patients' population universal peptide scaffold" to be modified with a series of glycosyl amino acids (different in sugars and linkages), in the aim of developing personalized diagnostic/prognostic tests. the csf114 beta-turn structure, exposing at the best the aberrant ptm specific for antibody-mediated forms of other autoimmune diseases, will lead to a family of antigenic probes to be used in diagnostic this information is encoded by the distribution of the electron-ion potential (eiip) of amino acids along the sequence and is represented by the frequency components in is. proteins with the same biological functions or interacting proteins (e.g. antibody/antigen) share the information corresponding to the common frequency components in their iss. investigation of the hiv-1 envelope glycoprotein gp120, as a model system for hypervariable proteins, revealed that this information is strongly conserved and is not significantly affected by natural mutations. the c-terminus of the second conserved region (c2) of gp120, encompassing ntm peptide is important for infectivity and neutralization of hiv-1, while human natural anti-vasoactive intestinal peptide (vip) antibodies reactive with gp120 play an important role in control of hiv disease progression. ntm/vip multiple copies were coupled to an artificial sequential oligopeptide carrier for developing an immunoassay (elisa) as a reproducible, reliable and sensitive tool for the detection of anti-ntm/vip derived antibodies these peptides have been utilized in an immunopeptidometric assay for specific measurement of active, noncomplexed psa. however, this assay has not been sensitive enough for the measurement of active psa in clinical samples. therefore, we aimed to develop an improved assay utilizing the same principle as previously, but using a more sensitive detection method based on proximity ligation assay. methods: in the assay, psa is first captured on a solid phase by a psa antibody czech republic rapidly increasing knowledge of new gonadotropin-releasing hormones (gnrh)of different species of the animal kingdom induces the need to prepare new synthetic derivatives and fragments of these peptides with higher potency and metabolic stability and suitable for the formulation of new immunogens. the species related differences in the sequence of the native mammalian gnrh pglu-his-trp-ser-tyr-gly-leu-arg-pro-glynh2 concern predominantly the positions 5, 7 and 8, particularly tyr in position 5 is replaced for his or leu, leu in position 7 by val or trp, and arg in position 8 is substituted by lys, ser, asn or gln. several gnrh derivatives with with the above substitution and gnrh fragments were prepared by solid phase peptide synthesis and purified by rp-hplc. purity of the synthetic peptides was checked by capillary zone electrophoresis (cze); peptides were analysed as cations in acidic backround electrolytes (ph 2.25 -2.quantitative analyses for determination of their effective electrophoretic mobilities and the estimation of their effective charges.supported by grant of ministry of agriculture of cr-nazv qf 3028 by rants of ga cr nos we use peptaibiomics for the structural determination of peptaibiotics from fungi grown on single agar plates thus avoiding time-consuming isolation and purification procedures. the method comprises fast and effective solid-phase extraction followed by on-line rp-hplc coupled to tandem esi-ms. here we present a survey of the peptaibiome of hypocrea species. in extracts of hypocrea semiorbis, h. vinosa, h. dichromospora, h. gelatinosa, h. nigricans, h. muroiana and h. lactea a multitude of short and long-chain peptides containing aib could be characterized. the formation of new and known peptaibiotics could be established by comparison with sequences stored in data bases japan process scale rp hplc purification of peptides and proteins is increasingly important in bio-pharmaceutical production. besides selectivity, other crucial factors are loadability, recovery loadability is believed to depend on the surface area of the packing material. consequently, smaller pores providing larger surface area should lead to increased loadability. this principle is misleading in the case of large molecules, because they cannot penetrate smaller pores. therefore the chromatographically accessible surface area has to be taken into account. recovery problems like irreversible adsorption or aggregation are frequently caused by hydrophobic surface properties of ods phases. the less hydrophobic c8 is a substituent to avoid considerably these problems. however c8 is less durable than ods under extreme acidic conditions. our new proprietary c8 modification technology combined with a perfect end-capping minimizes the presence of residual silanol groups and protects the silica surface sp-200-c8-bio demonstrates high mechanical stability by no obvious alteration of back pressure and particle size after 10 repeated packing cycles in dac columns. by overcoming the common weaknesses of the conventional c8 rp silica phases, daisogel sp-200-c8-bio opens new avenues for process scale separation of peptides and proteins. m514 determination of peptide: protein molecular ratio in conjugates by seldi-ms method synthetic peptides are widely used as antigens in various research and practical areas of biology and medicine. peptides with molecular masses < 5000 kda should be conjugated with carrier proteins in order to ensure their immunogenicity and protect from proteolysis. in these cases the comparison of peptide immunogenicities and immunotest system development should be performed having in mind exact peptide-to-protein ratios. 23 conjugates of peptide fragments of hepatitis c virus envelope protein e2 with ovalbumin, bovine serum albumin, and myoglobin were prepared using glutaraldehyde (ga), m-maleimidobenzoyl n-hydroxysuccinimide ester, dimethyl suberimidate (dms), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide as conjugating reagents. the rough evidence of the peptide-protein conjugate formation was obtained by page. the exact peptide:protein molar ratio was estimated in all 23 conjugates by seldi-ms. almost all conjugates had oligomeric structures due to the formation of intermolecular linkages between proteins. the peptide : protein molar ratio in conjugates varied from 1:1 to 13,6:1. conjugates obtained with the ga were more diversified in the number of peptide molecules linked to carrier proteins (peptide:protein ratios ranged from 3:1 to 13:1) than other conjugation reagents mazur-marzec 1 poland posttranslational modifications (ptms) like phosphorylation, acetylation, or methylation have been shown to play a significant role in directing the function of various proteins [1]. in eukaryotes, most of proteins have been shown to be posttranslationally regulated by a variety of different modifications. many effects of ptms include a change of enzymatic activity capillary electrophoresis (ce) has been used to study electrophoretic behavior of ptm-peptides gal-nh2, gal(1-15)-nh2 by capillary electrophoresis. using a phosphate buffer most of ptm-peptides were poorly separated at acidic or neutral ph. the best results were obtained using trifluoroethanol containing separation buffers. optimization of ce separation of maps of peptides containing ptms should allow to detect ptm-proteins and characterize their role in the living cell. comparison of modification events occurring in diseased and healthy cell may iran the purpose of this study was to use the application of multiplex reverse transcription polymerase chain reaction(rt-pcr) assay for detecting the two most common leukemia translocations t(1;19) and t(9;22) in childhood acute lymphoblastic leukemia in iranian children. 32 cases of leukemia patients were screened with the rt-pcr assay. this assay will identify the all type bcr-abl transcripts encoded by the t(9;22) and all described variants of the e2a-pbx1 transcript encoded by the t(1;19). rna was isolated from leukocyte cells of patient's samples. through the construction and optimization of specific primers for each translocation,we have been able to set up multiplex rt-pcr reactions.then pcr products was electrophoresed on agaros gel and were compared with size markers and expected fragtments key words: acute lymphoblastic leukemia -multiplex rt-pcr tu521 study on the syntheses and lc/esi-ms analyses of the glutathione conjugates of bile acids t. wakamiya 1 , m. sogabe 1 a carboxylic acid-containing drug, is metabolized to a glutathione (gsh) conjugate in vivo, and the conjugate is excreted in human urine [1]. although bile acids, compounds with carboxylic acid in molecules, are also expected to form gsh conjugates in liver, no evidence is so far obtained to confirm such metabolism, since there are no suitable standard samples for the research. in the present paper we report the syntheses of the gsh conjugates of main bile acids in human, i.e., cholic acid (ca), chenodeoxycholic acid (cdca), deoxycholic acid (dca), ursodeoxycholic acid (udca) and lithocholic acid (lca) as shown below, and the detailed analyses of these synthetic conjugates by means of linear ion trap lc/esi-ms. furthermore, the evidence for conversion of cholyl adenylate [2] and ca-coa thioester into ca-gsh conjugate will be presented these peptides do no exhibit such strong side effects as csa, but their practical application is hindered because of their poor solubility in water. the 49-57 fragment of tat protein and its analogs, including oligoarginine sequences, are known for their unusual ability to cross cell membranes, skin, and blood-brain barriers. moreover, these peptides are able to transport other substances into cells. this strategy was successfully applied in cases of csa, taxol, and other drugs to improve their bioavailability. now we have synthesized a series of analogues of cyclolinopeptide a, clx, and the immunosuppressive fragment of ubiquitin, covalently bound to the cell-penetrating fragment of tat and its analogues. the ability to cross the biological membranes and the immunosuppressive activities of these conjugates were tested. the conformation of the peptides was determined by circular dichroism methods: we used fluorescein-labelled cationic cell-penetrating peptides and analyzed the uptake efficiency (flow cytometry) as well as the intracellular distribution (confocal laser scanning microscopy). the bioactivity of a proapoptotic cargo-peptide, delivered into the cells either via electroporation or via cpps was quantified using a caspase-3 activity assay and cellular assays. to address the integrity of cpps during their trafficking, a fluorescent double-labelled antp peptide was designed and used as an intracellular fret-sensor. results: endocytosis-mediated uptake of the cpp-cargo conjugate led to a significant reduction of cargo bioactivity compared to its direct transfer via transient membrane permeation. this finding was related to the sequestration of peptides within endocytic vesicles but also, in the case of the tnf response, to the induction of receptor internalization during cell entry. moreover, during endolysosomal passage peptides undergo significant proteolytical degradation. conclusions: the endocytosis-dependent uptake mechanism of cholesterol pullulan (cp), in which maltose moieties are partially modified by cholesterol, is unique in forming self-assembled nanoparticles (20-30 nm) in water. combination of these characteristics is considered to be promising for development of effective non-viral vectors without toxicity. a conjugate of hiv-tat and cp was synthesized and its gene expression efficiency was evaluated. fully protected hiv-tat-(48-57)-cys(snm)-gly-nh-r was obtained by conversion of the corresponding cys(acm) peptide which was synthesized by the solid-phase method [snm: (n-methyl-n-phenylcarbamoyl)-sulfenyl] [1]. the sulfhydryl function was introduced to the hydroxyl groups of cp by acylation with trt-3-mercaptopropionic acid followed by acid treatment. resulting 3-mercaptopropionyl-cp was coupled with cys(snm) peptide to form disulfide bridge and the protecting groups of the peptide were removed to give the cp-tat conjugate. cp-tat and pcmv-luc complex was transfected into cos7 cells and luciferase activity was analyzed after 24 h. cp-tat elicited remarkable cytoplasmic luciferase activity and low toxicity this finding provides a possibility to use gnrh-iii as a targeting moiety for intracellular drug delivery. therefore we have prepared methotrexate and doxorubicine conjugates of gnrh-iii. the drug molecules were attached to the lys side chain in position 8 of gnrh-iii by thioether bond formation through the gflg spacer elongated with cys either at the c-or n-terminus. since we found earlier that the dimer derivative of gnrh-iii was more effective, new dimer derivatives with a combination of antitumour agents were also prepared. branched gnrh-iii derivative (pyrhwshdwk(clac-glfgc(acm))pg-nh2]) was synthesised by spps. the drug molecules were attached to this compound by thioether bond and finally disulfide bridge was formed between two peptide chains. the cytotoxicity of new derivatives was characterised by mtt test th531 oligopeptide antifungals are exceptionally active against multidrug-resistant yeast previous studies have demonstrated that longer sirnas that are processed by dicer can result in more potent knockdown than the corresponding standard 21-mer sirnas. dicer-substrate 25-27-mer sirnas were conjugated with different structural classes of peptides and their cell uptake properties evaluated. peptides were conjugated to the 5' end of the sirna sense or antisense strand via a thioether bond under denaturing conditions to prevent aggregation and precipitation. the ability of conjugates to translocate fluorescently-labeled sirna across the plasma membrane was evaluated by flow cytometry. the results indicate that some peptides can mediate higher efficiency uptake of sirna into cells compared with lipofectamine or cholesterol-conjugated sirna. the peptide-sirna formulation with 27-mer sirna conjugates showed higher knockdown of tnf-alpha mrna and protein levels in activated human monocytes in vitro compared to the conjugated 21-mer sirna species. the products resulting from in vitro digestion of peptide-conjugated rna duplexes with recombinant human dicer were identified using esi-ms and consisted predominantly of the desired 21-mer sirna several peptides containing the sequence arg-gly-asp (rgd) were studied and developed for their nanomolar affinity to the membrane receptor alfa v beta3 and alfa v beta 5 integrins, which are over-expressed by endothelial cells during proliferation and by tumor cells. to improve the pharmacological profile of some camptothecin derivatives (cpts), five conjugates were designed, where the cytotoxic drugs were covalently attached to the rgd peptide analogues for preferential uptake into tumor cells. the peptides to be used have been selected among a series of new pentacyclic peptides bearing at 5-position a trifunctional pseudoamino acid with a carboxy-terminal side-chain. peptide analogues showing the highest affinity to alfa v beta 3 and alfa v beta 5 integrins were coupled with cpts at different positions. the conjugates have been optimized for binding to the receptors, proteolytic stability and an overall improvement in tumor selectivity. the nature of the linkage between rgds and cpts has a major impact on stability and biological activity of the conjugates. the conjugates with amide bond, but not those with ester bond, are sufficiently stable and show in vitro antitumor activity against a498 and a2780 cell lines combination of amaranth protein with other plant proteins (cereals) enables to formulate the composite protein (near to milk or beef protein, but exclusively on vegetal basis). it is shown on graphs. the aim of the projects' proposals is a development and realization of the technology for fractionation of amaranths defatted flour product is a top protein obtained by removing starches and next polysaccharides decomposed on soluble monosaccharide by specific enzymes. there are shown the chromatograph measuring. we can see complete disintegration of starch and the unchanged proteins. the separate solution monosaccharide is usable for others fermentative processes or as a nutrient solution for yeasts there are description methods for isolation amaranth protein -extraction processes, enzymatic removal starch. the product is a isolate protein rich in essential amino acids. the waste monosaccharide solution was used to production yeasts biomass rich in proteins vitamins amaranth protein isolate have high nutritional value and can be used as food ingredients, for functional, probiotic formulation to begin with, we made epitope mapping with the highly sensitive spot array method (2) in order to study antigenic regions of parvovirus b19 vp1 and vp2 capsid proteins. epitope mapping identified highly reactive, immunodominant early epitope on parvovirus surface that centered to kyvtgin residues of vp1. in the subsequent phases we developed the kyvtgin epitope type specific (ets) igg serodiagnostics. a correlation between enhancing igg avidity to b19 capsid and a transient reactivity with the point-of-care kyvtgin peptide was clear. together the two assays enhanced the value of early diagnosis of b19 infections (3) acute phase-specific heptapeptide epitope for parvovirus b19 diagnosis synthetic peptide arrays on membrane supports-principles and applications human parvovirus b19 infection during pregnancy--value of modern molecular and serological diagnostics 1 laboratory of peptide & protein chemistry & biology 2 department of organic chemistry "ugo schiff" and cnr-iccom 3 department of chemistry 4 department of pharmaceutical sciences glc) able to recognize specific autoantibodies in multiple sclerosis (ms) patients' sera has been developed by the laboratory of peptide & protein chemistry and biology [1and bio-plex suspension array system, biorad. the biosensor technology and bio-plex suspension array system will offer advantages such as rapid analysis, and high sensitivity for a high throughput screening. immobilisation will be based on different strategies that are anchoring the synthetic antigen on different solid supports such as polystyrene well plates (elisa) optimisation of the different techniques was performed with anti-csf114(glc) autoantibodies isolated using affinity chromatography from ms patients' sera. the analytical parameters such as specificity, sensitivity, and matrix effect were evaluated. the different technologies have been used for a high throughput screening of ms sera which control specific cell adhesion.2 here we discuss the route for preparation of amphiphilic block copolymers composed of hydrophobic polylactide and hydrophilic polyethylene oxide (peo) blocks, carrying various cell-adhesion oligopeptide sequences at the end of peo block. fully protected peptide fragments were prepared by solid-phase peptide synthesis by using fmoc strategy and chlortrityl resin. the side-chain protected peptides were cleaved from resin by 25% hfip solution in dcm. the copolymers peptide-polytehyleneoxide were prepared by coupling of the activated peptide fragments with α-amino-ω-hydroxy-peo in dmf using pypop as an activation reagent. subsequently, the polylactide block was grafted to the ω-hydroxy end group of the peptide-peo copolymer via a controlled rop polymerisation of lactide 2r)-2-aminocyclopentanecarboxylic acid (acpc) and beta-methylphenylalanine (beta-mephe) were designed and synthesized to obtain more potent and selective mu-opioid receptor ligands with higher stability against proteolytic enzymes. we have prepared the peptides by spps methods using racemic amino acids. the diastereomeric peptides were separated by hplc. the configuration of the unnatural amino acids in the peptides was determined by chiral tlc using enantiomeric standards. radioligand binding assays and in vitro gpi and mvd assays indicated that several analogues showed high, subnanomol affinity and high selectivity for mu-opioid receptors having agonist or antagonist properties. the incorporation of alicyclic amino acids into the endomorphins resulted in enzyme resistant peptides. the most promishing analogues (dmt-pro-phe-phe-nh2 and tyr-(1s,2r)acpc-phe-phe-nh2) were labeled with tritium using precursor peptides containing dehydroproline or dehydro-(1s,2r)acpc amino acids and tritium gas and pd/baso4 catalyst. the novel peptides and their radiolabelled analogues with high specific radioactivity (1.4-2.8 tbq/mmmol) have become useful pharmacological and biochemical tools for the opioid research iran background and aims: injectable drug delivery based on polymer solution platforms has gained in resent years, particulary for protein-based therapies. the influence of polymer molecular weight (rg 502h, rg504h) on the morphology, erosion of matrices and also on their in-vitro drug release behavior over a period of 28 days was assessed for leuprolide acetate in this study. methods: each formulation was composed of 33% (w/w) polymer and 3% (w/w) leuprolide acetate dissolved in nmp. release studies were performed in a home-made diffusion cell at 37°c. the polymer erosion was studied using two different methods as follows. (a): l-lactic acid detection (b): ph change study. the morphology of the matrices was then analyzed by scanning electron microscope as is shown, the lower molecular weight polymer formulation shows higher porosity and pore diameter due to a rapid phase inversion phase i) can be divided into three more phases with different release rates. results showed that burst effect for rg 502h, 32%, was significantly (p<0.05) higher than rg 504h (13%) italy fabrication of photocurrent-generating systems based on bioinspired organic-inorganic hybrid materials is currently of great interest. more specifically, the photoelectronic properties of nanometric films formed by peptide self-assembled monolayers have been actively investigated. in this work interdigitated gold microelectrodes were modified by covalently linking a hexapeptide ester functionalized by a lipoic acid (lipo) at the n-terminus. the peptide chain [lipo-(aib)4-trp-aib-otbu] comprises five α-aminoisobutyric acid (aib) residues and one trp, a fluorescent amino acid with strong absorptions in the uv region. due to the very high percentage of conformationally constrained aib residues in the chain, the peptide adopts a rigid 3-10-helical structure. cyclic voltammetry measurements indicate that the peptide forms a homogeneously and densely packed monolayer on the gold surface, while current/voltage curves exhibit interesting rectifying properties of the peptide sam. photocurrent generation experiments, performed on the peptide-layered microelectrode, show peculiar modifications of the spectrum. at 240 nm a notably higher photocurrent/voltage response was observed for the peptide-modified electrode, suggesting that a photoinduced electron transfer process from trp to gold does take place with high efficiency this may lead to randomly orientated enzymes and subsequently limited activity. the aim of this work is to selectively activate enzymes at their c-terminal position in order to allow specific immobilisation. we chose akr1a1, an enzyme of the aldo/keto reductase superfamily, for the synthesis of an artificially monolabeled redoxprotein. akr1a1 is a monomeric enzyme and catalyzes the nadph dependent reduction of aliphatic/aromatic ketones and aldehydes. to produce monofunctionalized enzymes we applied the strategy of expressed protein ligation (epl). accordingly, we used the impact®-system and cloned the aldo/keto-reductase as fusion protein with an additional intein/chitin binding domain. through intein mediated splicing we could produce c-terminal thioester of the akr1a1. in the next step, the thioester was coupled to a biotin containing peptide by native chemical ligation. this specifically modified enzyme was immobilised on avidin coated surfaces. the attachment on the surface was tested by tryptic digestion, followed by maldi-tof-ms analysis since safe organic solvent waste disposal is an important environmental problem, we aimed to perform peptide synthesis in water. we have reported solid phase peptide synthesis in water using water-soluble n-protected amino acids, such as 2-[phenyl(methyl)sulfonio]ethoxycarbonyl and 2-(4-sulfophenylsulfonyl)-ethoxycarbonyl amino acids. following to study on water-soluble n-protected amino acids, we developed a new technology based on nanochemistry for solid phase peptide synthesis in water. the new technology is based on coupling reaction of suspended nanoparticle reactants in water. fmoc-amino acids are used widely in peptide synthesis, but most of them show poor water-solubility. we prepared well-dispersible fmoc-amino acid nanoparticles in water by pulverization using a planetary ball mill in the presence of poly(ethylene glycol) (peg). the size of fmoc-amino acid particles was 300-500 nm. to evaluate the utility of this technique, leu-enkephalin was prepared using the nanoparticulate fmoc-amino acids on a peg-grafted rink amide resin in water supramolecular structures formed from n-lipidated oligopeptides immobilized in the regular pattern on the cellulose surface are able to bind ligand molecule, thus acting like artificial receptors. due to the conformational flexibility of lipidated oligopeptide chains, the supramolecular structure is highly flexible, forming the cavities with the shape and prosperities adjusted most effectively to requirements of the guest molecule. structural requirements for a peptide providing the most efficient fit the guest molecules are not known, therefore an array of the artificial receptors have been synthesized and used in the studies. thus, even in the case, when the single receptor in an array does not necessarily have selectivity for a particular analyte, the combined fingerprint response can be extracted as a diagnostic pattern visually, or using chemometric tools in order to improve the sensitivity of the competitive binding and to study the mechanism of molecular recognition, experiments involving fluoresceine and fluoresceine marked acp fragment were performed. we found that λmax and intensivity of fluorescence depends on the structure of the peptide motif and lipidic fragment of receptor this mts was linked with dhhp-6 by disulfide bond, and the new molecular was named mts~dhhp-6. the peroxidase activity of mts~dhhp-6 (2.1x103 u•µmol-1) was tested and similar to that of mp-11 (4.2x103 u•µmol-1). mts~dhhp-6 coated with quantum dots (qds) [3] were observed to accumulate into neonatal rat cardiomyocytes (nrcms) of wistar rats and co-localized with mitotracker red in mt. these results suggest that mts~dhhp-6 is an excellent apx mimics and may have potential proceedings of the twenty-eighth european peptide symposium, kenes international, israel, 2005, 551. references: 1 elastin-based polypeptide, poly(val-pro-gly-val-gly), undergoes self-assembly called coacervation, in which microcoacervate droplets with approximately 1000 nm diameters are formed [1]. nanoparticles cross-linked by cobalt-60 γ-irradiation of these microcoacervate droplets are useful as drug release devices. to investigate the size optimization of nanoparticles, the stability of nanoparticles in the treatment of enzyme, and the drug release profiles from nanoparticles, the three copolymers; poly[10(val-pro-gly-val-gly), (val-ala-pro-gly-val-gly)], poly[4(val-pro-gly-val-gly) application of polyelectrolytes and theoretical models the synthetic heptapeptide rnwdvyk is a fragment of a high affinity receptor (fcεri) for immunoglobulin e (fragment 111-117). it is the active domain for binding with ige. the program of studies of biological properties of the heptapeptide included the investigation of its binding to ige contained in standard solutions and in patients' blood serum. the binding of rnwdvyk with ige was investigated by the ifa method using the ige antibodies labelled with horse-radish peroxidase (hrpo). we determined the optimum sorption concentration of the peptide in this experimental immunoenzyme system to be 100 mkg/ml. the ability of synthetic rnwdvyk peptides to bind with ige was studied as a function of ige concentration in standard serum (0.47 to 60 ng/ml ige). a high correlation was found between the ige concentration and the optical density of the solution after introducing monoclonal antibodies labeled with hrpo and the substrate chromogenic mixture (r=0.99). similar investigations were conducted using the allergy patients' blood serum. the serum with a known concentration of ige was added to immunological plotting boards with sorbed synthetic rnwdvyk peptides. a high correlation was also found between the concentration of ige in the patients' blood serum and the optical density of the solution after introducing monoclonal antibodies labelled with hrpo and substrate chromogenic mixture (r=0.94). our experiments showed the high ige binding activity of synthetic rnwdvyk peptides. we demonstrated the possibility of construction of diagnostic systems for the quantitative determination of ige and ige-antibodies. the fusion protein nucleocapsid-dutpase is present in virions of mason-pfizer monkey betaretrovirus and in virus-infected cells where it potentially contributes to rna/dna folding and reverse transcription (barabas, et al., 2003; bergman et al., 1994; berkowitz, et al., 1995) . in addition to trimeric dutpase core, the protein possesses flexible n-and c-termini consisting of the nucleocapsid segment, and a peptide motif conserved in dutpases. to analyze the function of the flexible cterminal peptide segment, reconstitution experiments were designed with truncated enzyme lacking the c-terminal 14mer oligopeptide and the synthetic oligopeptide prepared on rink-amid resin by solid-phase peptide synthesis, using fmoc strategy. the truncated enzyme proved to be practically inactive. addition of the synthetic 14mer (pyrgqgsfgssdiy) at 100fold molar excess resulted in partial complamentation of the catalytic activity (to 10% of original). a mixture of the truncated enzyme and the 14mer oligopeptide (this latter at 100fold excess) was put to crystallization trials. we conclude that the c-terminal 14mer is essential for catalytic activity. antifolate drugs are inhibitors directed to interfere with folate metabolic pathway. methotrexate (mtx) and pemetrexed (alimta®) are known folic acid analogues used in cancer treatment. different peptide conjugates of mtx have been prepared for intracellular delivery. (1) in octaarginine conjugates one of the carboxylic groups of mtx was attached to the n-terminal of the peptides. (2) however, as results showed, that both carboxylic groups are required to the biological effect of mtx. therefore we decided to synthesize peptide conjugates of folic acid analogues in which the carboxylic groups are untouched. octaarginine, penetratin and a cyclic peptide cgnkrtrgc, which can deliver a cargo molecule in the lymphoid system, were used as delivery peptides. we introduced squaric acid or aminoxy acetic acid as linker moiety between the peptides and cargo molecules. the conjugation was monitored by rp-hplc, the crude products were purified by rp-hplc and were identified by mass spectrometry. the biological activity of the conjugates was evaluated in vitro on sensitive and resistant human leukemia (hl-60) cell lines. besides its endocrine activity, trh (the tripeptide pglu-his-pro-nh<sub>2</sub>) has also been long recognized as a modulatory neuropeptide with broad range of physiological and pharmacological activities in the central nervous system (cns). although numerous centrally active and metabolically stable analogues and peptidomimetics have been synthesized using trh as a template, selectivity of their cns action has remained an issue to be addressed. we aimed at discovering novel analogues with enhanced cns-selectivity by incorporating pyridinium building blocks. the design also allowed for enhancing transport across the blood-brain-barrier and increasing residence time in the cns through prodrug strategy. solid-phase chemistry was used to prepare the analogues and novel methods previously not used to incorporate pyridinium moieties into resin-bound peptides such as the zincke reaction were also introduced. comprehensive evaluation included measurement of affinity to trh-receptor, acetylcholine-releasing, analeptic and antidepressant-like activity in animal models, as well as prediction of membrane affinity, determination of in vitro metabolic stability, and pharmacokinetics and brain uptake/retention studies that employed in vivo microdialysis sampling. a strong connection between acetylcholine-releasing potency and analeptic effect in animals was obtained for close analogues of trh, while pyridinium compounds designed from the structurally related pglu-glu-pro-nh<sub>2</sub> maintained the antidepressant-like effect of the parent peptide, while showing significant decrease in analeptic action.in conclusion, an increase in the selectivity of cns-activity profile was obtained by the incorporation of pyridinium moieties. we have also demonstrated the benefits of the prodrug approach on the pharmacokinetics, brain uptake and retention of the analogues upon systemic administration. the use of small radiolabelled compounds such as peptides is a very attractive tool for the diagnosis of several different pathologies, specially cancer, through the use of nuclear medicine techniques.1 among the various membrane receptors, the two cholecystokinin receptors ccka-r and cckb-r are very promising biological targets for radiolabelled compounds due to their overexpression in many tumours2. in order to develop radiolabelled peptide derivatives able to target these receptors, the binding mode of the c-terminal cholecystokinin octapeptide (cck8), toward the two cholecystokinin receptors ccka-r and cckb-r has been, recently, structurally characterized. 3 the structural data suggest that modifications on the n-terminal end of cck8 obtained by introducing chelating agents and their metal complexes should not affect the interaction of the derivatized cck8 peptides with both ccka-r and cckb-r. here we report the labelling procedures and the in vitro and in vivo characterization of new 99mtc cck8 derivatives. a stable 99mtc-nitrido complex is obtained by using the coordinative set formed by: 1) the n-terminal amino group and the sh cystein of the cck8 derivative cys-gly-cck8 peptide; and 2) a pnp aminodiphosphine used as coligand. several phosphines are used in order to define the best labelling procedures and to optimize the in vivo biodistribution properties of the 99mtc labelled peptides.references various combinations of pore size and chemistry of silica-based materials were investigated for high performance liquid chromatography (hplc) of peptide separation. incorrect pore size and ligands have been suggested to cause peak broadening, poor resolution and poor recovery. our study suggests that an appropriate combination of pore sizes and ligands is necessary to obtain the most efficient usage of reversed-phase hplc columns according to the molecular weights of peptides and proteins. we will also show the possibilities of an improved method development for the separation of complex peptide mixture by ph or additives.the development of new biopolymer materials as drug delivery systems is of enormous interest on biomedicine. dendrimers are polymers with particular properties; they are highly branched polymers with well-defined chemical composition, and show compact globular shape, monodisperse size and controllable surface functionalities. peptide dendrimers incorporates amino acids in their structures and have additional features such as biocompatibility and biodegradability.in previous studies we described the solid-phase synthesis of a new class of polyproline-based dendrimers. these biopolymers have the capacity to cross the mammalian cell membrane and moderate toxicity. these promising results open up the possibility to explore these dendrimers as delivery systems.to design more versatile polyproline dendrimers, we have developed a methodology that involves the combination of solid-phase and solution strategies. diverse multivalent peg-and proline-based cores were synthesized to attain dendrimers with distinct topologies. dendrimers were synthesized by iterative building block addition [(glypro5)2imdoh], around an inner core, using a peptide solution convergent approach. a variety of coupling methodologies and protecting groups for the n-terminal function were explored. the novel high throughput protein detection system using designed peptide arrays has been successfully indicated on their capabilities as the "protein-chip" [1] [2] [3] [4] . our concept has many advantages especially for high-quality industrial production and practical applications compared to arrays with antibodies or recombinant proteins. the deposited peptide solution can be dried without covalent immobilization, although, when the resulted arrays are exposed in protein-solution they showed planned conformation [5] . based on these basic results, several hundreds of α-helical, β-loop and β-sheet peptides, which involved a cysteinyl residue for covalent immobilization and tamra as a fluorescent label, were successfully synthesized, characterized and used as capture molecules. a novel material for chips made from amorphous carbon suitable for our concept has been developed, which has significant advantages over conventional glass or polymer plates, such as no selffluorescence, mechanically more stable, easy manufacturing in the aspects of precised and high throughput processing. thus, chip-plate with nano-l wells could also be easily manufactured. peptides were deposited on these chips surface covalently as well as non-covalently in 350 picol/spot (diameter: ca 200 µm). the resulted chips were used for protein detection. a part of this work was funded by the okinawa-bio-project and nedo-grant. key: cord-023225-5quigar4 authors: nan title: posters date: 2012-08-21 journal: j pept sci doi: 10.1002/psc.2449 sha: doc_id: 23225 cord_uid: 5quigar4 no abstract is available for this article. laboratory of molecular biology and immunology, department of pharmacy, university of patras, patras, greece antimicrobial peptides (amps) are an important component of innate immune system of most living organisms. they have recently gained much attention as new anti-infective drugs with new modes of actions and few or no side effects. their antimicrobial spectrum covers gram-positive and -negative bacteria as well as fungi and certain viruses 1 . fish have proven to be a rich source of antimicrobial peptides. three chrysophsin peptides (chrysophsin-1, -2, -3) have been identified in the gills of the red sea bream, chrysophrys major, which are all bactericidal to pathogenic bacteria at low concentrations 2 . they are cationic α-helical peptides, rich in histidine residues and all end in an unusual rrrh motif. however, in addition to its high antimicrobial potency, chrysophsins have considerable hemolytic activity. the development of new analogues which would preserve high antimicrobial potency, but would lack the undesired hemolytic activity, could be a useful tool with possible commercial and clinical applications. in the present study, we synthesized a series of analogues of chrysophsin-1 with different ratios of lys and leu residues, utilizing the fmoc/but solid phase methodology 3 . the synthesized analogues were purified and isolated by rp-hplc. the antimicrobial properties of the above peptide analogues are currently testing in 3 gram positive (s. aureus, s. epidermidis, e. faecium) and 2 gram negative (e. coli, p. aeruginosa) bacteria. the goal is to identify the minimum bacteriostatic and bactericidal concentrations of the analogues, under conditions that simulate the best possible that of the human organism. hemolytic or cytotoxic activity of the peptides will also be determined. 1 the rise of antibiotic resistance demands the development of new antimicrobial agents. these should exhibit a novel mechanism of action so as to overcome the resistance and be invulnerable to 'not yet acquired resistance mechanisms'. such criteria are difficult to meet. however, cationic host defence peptides (hdps) have emerged as promising candidates. hdps target and disrupt bacterial membranes. in order to evade such a threat a bacterium would need to make substantial changes to its membrane composition disfavouring the development of resistance (1) . however, exact role and mechanism of hpds in the regulation and monitoring of microbial invasions remain to be established. herein we will present new potential mechanisms of antimicrobial regulation by helical hdps using de novo (2) and native systems (3) . biophysical and microbiology aspects of the experimental designs will be discussed. the low number of the newly discovered antibiotics, emergence of multiple-drug resistance, and the alarming death rate due to the infection disease led to development the alternative means to combat the infections. the researchers accumulate information about antimicrobial drugs that could be result of the innate immunity mechanisms. armed only with the innate immunity, the insect has developed into the most widespread class in living kingdom. they produce several antimicrobial peptides with complementary and rapid mode of action. so far there are hundreds of antimicrobial peptides isolated from insect and lot of them are waiting to be discovered. the fleshly neobellieria bullata was chosen for isolation of these active compounds. its larvae in the third instar were squeezed to collect the haemolymph, which was gradually centrifuged and precipitated by acidified methanol. supernatant was subsequently separated by chromatographic methods (spe column, rp-hplc) to obtain fractions of short peptides. identification and characterization of these fractions were performed by tricine electrophoresis, mass spectrometry maldi-tof analysis and n-terminal sequencing. several fractions showed antimicrobial activity against institute of chemical kinetics and combustion, 630090 novosibirsk, russian federation in this work, we extracted 3d-structural information on newly synthesized, medium-length, double spin-labeled peptaibiotics using peldor spectroscopy. we investigated the magnetic dipole-dipole interactions between spin labels and the orientation selectivity effects. in particular, the medium-length peptaibiotics tylopeptin b 1,2 and heptaibin 3 , double spin-labeled with the nitroxyl probe toac (4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-4-carboxylic acid), were studied by means of x-band peldor spectroscopy. this study was conducted on tylopeptin labeled at positions 3 and 13 (t313) and heptaibin labeled at positions 2 and 14 (h214) in frozen glassy methanol solutions at 77 κ. peldor data analysis was carried out using the theory developed for short interspin distances. the distance distribution functions between spin labels for τ313 (maximum at 1.78 nm, halfwidth of 0.08 nm) and η214 (maximum at 2.30 nm, half-width of 0.05 nm) were determined. the intramolecular distances observed between the labels allowed us to assign an essentially α-helical conformation to τ313 and a largely prevailing 310-helical structure to η214 under the aforementioned experimental conditions. are amidated at the c-terminus, as a result of a posttranslational enzymatic reaction. temporins are particularly active against gram-positive bacteria and are not toxic to eukaryotic cells. in this study we designed a series of analogues of tb with the aim to improve the peptide antimicrobial activity against both gram negative and gram positive strains and then to structurally elucidate the mechanism of interaction of active peptides with lps. the peptides have been synthesized substituting one or two amino acids with an alanine and lengthening the sequence with positively charged amino acids. among the 16 designed peptides, one of the analogues, tb_kkg6a, showed highly increased activity against gram negative bacteria and also a slightly increased activity against gram positive bacteria with a total lack of hemolytic activity. to develop ll-37-derived short amps with prokaryotic selectivity and lipolysaccharide (lps)neutralizing activity, a series of amino acid-substituted analogs based on ig-19 (residues 13-31 of ll-37) were synthesized. analog a4 showed the highest prokaryotic selectivity, but much lower lps-neutralizing activity compared to ll-37. the analogs, a5, a6, a7 and a8 with higher hydrophobicity displayed lps-neutralizing activity comparable to that of ll-37, but much lesser prokaryotic selectivity. these results indicated that the proper hydrophobicity of the peptides is crucial to exert the amalgamated property of lps-neutralizing activity and prokaryotic selectivity. to increase lps-neutralizing activity of the analog a4, we synthesized trp-substituted analogs (a4-w1 and a4-w2), in which phe 5 or phe 15 of a4 is replaced by trp. despite their same prokaryotic selectivity, a4-w2 displayed much higher lps-neutralizing activity compared to a4-w1. this result suggested that the effective site for trp-substitution when designing novel amps with higher lps-neutralizing activity, without a remarkable reduction in prokaryotic selectivity, is the amphipathic interface between the end of the hydrophilic side and the start of the hydrophobic side rather than the central position of the hydrophobic side in their α-helical wheel projection. furthermore, d-enantiomeric peptides (a4-w1-e and a4-w2-e) of a4-w1 and a4-w2 possessed not only more improved prokaryotic selectivity and retained lpsneutralizing activity compared to a4-w2 but also protease stability. taken together, a4-w1-e and a4-w2-e can serve as promising templates for the development of therapeutic agents for the treatment of endotoxic shock and bacterial infection. department of zoology, faculty of science, charles university, prague, czech republic antimicrobial peptides (amps) are among the most promising lead compounds for developing medicines in the fight against resistant pathogenic bacteria. we have already shown that the venom of wild bee is a rich source of pharmacologically interesting antimicrobial peptides [1] [2] [3] [4] . from the venom of solitary bee macropis fulvipes, we isolated and characterized the novel antimicrobial peptide named macropin (mac-1). by edman degradation and mass spectrometry, its primary sequence was established as gfgmalkllkkvl-nh2. mac-1 possesses potent antimicrobial activity against both gram-positive andnegative bacteria and moderate hemolytic activity against human red blood cells. cd spectra confirmed that mac-1 can form an amphipathic α-helical secondary structure in the presence of membrane-mimicking substances as sodium dodecyl sulfate or organic solvents like trifluoroethanol. we prepared a series of mac-1 analogs to study the effect of incorporating d-amino acid residues into the sequence in various positions on antimicrobial and hemolytic activity, α-helicity and serum stability. the substitution of l-amino acid residues at n-terminal part of sequence by d-amino acid residues led to the improving hemolytic activity with maintaining or increasing antimicrobial activity. these modifications increased peptide stability in human serum. effect of the incorporation of d-amino acid residues into the mac-1 sequence on its α-helical structure will be discussed. the neutralization of endotoxins (lipopolysaccharide, lps) by suitable compounds has been shown to be a key step in the treatment of infectious diseases, in particular in the case of gram-negative bacteria. the active endotoxic center of lps is lipid a, its lipophilic part. an effective antimicrobial peptide against gram-negative bacteria is magainin 2, which was originally found in the skin of an african frog. here, we studied the interaction of hexa-acyl bisphosphoryl lipid a 1 prepared from erwinia carotovora lps with magainin 2 with some minor substitutions in the amino acid pattern. by using fourier-transform infrared spectroscopy, the gel to liquid crystalline phase transition of the acyl chains of lipid a, the conformation of their phosphate groups due to peptide binding, and the profile of the secondary structure of the peptides was investigated. the zeta potential of lipid a aggregates in the presence of the peptides was determined by measuring the electrophoretic mobility. small-angle x-ray scattering was performed for the elucidation of the aggregate structures in the absence and presence of the peptides, and isothermal titration calorimetry was applied for evaluating the thermodynamics of binding between peptides and lipid a. the data show that asp-or glusubstituted peptides improved the binding activity to lipid a correlated with characteristic changes in the physical parameters, which were stronger expressed for the aspsubstituted peptide. the new hydrogen bond connection between glu and asp by carboxylic acids apparently leads to a more pronounced -structure of the peptide. the conformation change of the peptide enhanced the activity of incorporation into the lipid a aggregates, along with changes in biochemical and biophysical parameters. royal college of surgeons, dublin, ireland cationic antimicrobial peptides (caps) have been reported to exhibit anticancer activity 1 . one such peptide, p18, has been shown to inhibit the growth of several cancer cell lines, with inhibiting concentration (ic50) in the range of 10 to 20 μm 2 . however the concentration at which p18 and other caps act is too high to be clinically relevant. the enhancement of their activity can be achieved through the modification of their amino acid composition or the addition of other molecules. conjugation of naturally produced hydroxylated fatty acids to p18 showed a 10-fold improvement in its anticancer activity on a variety of human-derived cell lines. in addition to the enhancement of activity we wished to understand the mechanism of action of the peptide and conjugates. we investigated the uptake of conjugated and unconjugated peptides into hela (cervical) and miapaca (pancreatic) human cancer cells and the localisation of the peptide in the cell once taken up. we investigated the effect of altering the carbon number of the hydroxylated fatty acids ranging from hydroxyhexanoic acid (r6) to hydroxydodecanoic acid (r12) conjugated to p18 peptide and tested on hela and miapaca cell lines. circular dichroism studies were performed to investigate the effect on α-helical content due to amino acid composition alteration and hydroxyalkanoic acid conjugation. the effect of the position of the hydroxyl moiety on enhancement of activity was also investigated. in the current study p18 and its derivatives also lacked haemolytic activity with concentrations up to 66 fold higher than ic50 values needed to observe any haemolysis. when current antibiotics become less efficient, there is a promise that some antibiotics can be replaced by other nature's substances, e.g. peptides. halictines are novel antimicrobial peptides isolated from the venom of the eusocial bee halictus sexcinctus. we obtained four analogues of the native peptide hal 1 from iocb av cr. they already characterized structural properties of these peptides and their antimicrobial activity against selected bacteria 1 . the analogues were prepared by point mutations of native peptide, which could increase antimicrobial activity and decrease undesirable hemolytic activity. our aim was to characterized membrane permeation activity of halictines through the use of a basic model of biological cells -large unilamellar phospholipid vesicles luvs. we prepared two basic types of leakage assays based on luvs with free dyes entrapped inside and one assay with laurdan content. we used classical steady state fluorescence spectroscopy 2 and advanced fluorescence methods 3 for study of dyes escape from luvs and we also used laurdan generalized polarization technique gp 4 for better understanding peptide insertion into membrane. in this way we received complementary information and we can conclude that the most active peptides are the native hal 1 and analogue hal 1/6. however hal 1/6 requires presence of negatively charged phospholipids in membrane which may explain its higher selectivity against bacteria. furthermore, fcs results have shown that the leakage happens via pore formation. results from gp revealed that peptide insertion in the membrane do not lead directly to formation of pores. against a wide range of microorganisms, mainly by perturbing the permeability of bacterial membranes through the formation of pores. however, amps effects on membrane properties probably extend beyond poreformation. we performed a systematic spectroscopic analysis of the effects on membrane structure and dynamics of two very different amps: the cationic pmap-23, which creates pores according to the "carpet" model 1 , and alamethicin, which forms "barrel-stave" channels 2 . by using fluorescence anisotropy measurements on liposomes comprising probes localized at different depths in the bilayer, we measured peptide effects on membrane fluidity and order. laurdan spectral shifts provided indications about water penetration in the bilayer. in the case of pmap-23, it was possible to focus specifically on the lipids surrounding the peptide by following the membrane-probe fluorescence due to fret from the peptide trp residues. finally, peptide-induced perturbation of lateral mobility and domain formation were determined by several methods. all experiments were compared with liposome-leakage measurements: while for pmap-23 all membrane-perturbing effects are correlated with the vesicle leakage process, alamethicin does not significantly influence membrane dynamics at the concentrations in which it forms pores. surprisingly, in all cases the most significant peptide-induced effect is a reduction in membrane fluidity. we have reinvestigated 20-residue peptaibols named metanicins from an ascomycetous fungus originally described as metarhizium anisopliae strain cbs 597.80 (cbs = centraalbureau voor schimmelcultures, utrecht, the netherlands). however, due to unusually shaped conidia and based on rna-sequencing of its internal transcribed spacer (its) region, the identification of cbs 597.80 as metarhizium has been withdrawn and this particular strain is currently under taxonomic reinvestigation 2 . sequencing of four isolated peptides by fab-ms, esi-ms and edman degradation of partial hydrolysates revealed structural relationship to 20-residue peptaibol antibiotics paracelsins from trichoderma reesei (=hypocrea jecorina). sequences determined are: ac-u-a-u-a-u-a(u)-q-u-v-u-g-l-u-p-v-u-u(j)-q-q-fol (exchange positions in parenthesis; ac, acetyl; u, aib, α-aminoisobutyric acid; j, d-isovaline; fol, l-upmc univ paris 06 laboratoire des biomolécules; cnrs umr 7203; ens lbm; address: laboratoire des biomolécules, ens dpt de chimie, 24, rue lhomond f-75005, paris, france current data suggest that the cellular uptake of cellpenetrating peptides (cpps) occur by two processes: direct translocation across the plasma membrane and endocytosis 1 . the large diversity of cpp sequences described in the literature (derived either from fragments of proteins, structurally constrained synthetic peptides, peptide libraries 2 or dendrimers) has hampered the identification of general rules for their efficacy of internalisation. we have used a reductionist approach, restricting the cpp functional groups (amide and guanidinium) and tailoring cpp amphiphilic properties. two families of cpps have been designed: 1) primary amphiphilic cpps corresponding to tetra-arginines functionalised with fatty acid chains of different lengths and 2) secondary amphiphilic cpps containing arginine and alanine or tryptophan residues 3 . these cpps were linked by a disulfide bridge to a peptide inhibitor of protein kinase c (pkci). the efficiencies of internalisation of the conjugates were quantified by a method based on maldi-tof mass spectrometry previously developed in our group 4 . the mechanism of internalisation was studied by comparing the amounts of cell-surface bound and internalized pkci cargo on cho-k1 cells and glycosaminoglycan-deficient cho cells at 4 o c and 37 o c. conjugates were found to enter by both direct translocation and glycosaminoglycandependent endocytosis. in addition, the primary amphipathic cpps were found to be more efficient than the secondary amphipathic ones. furthermore, structural or mechanistic novelty does not guarantee immunity from resistance, with strains resistant to linezolid identified prior to fda approval. therefore, modifying existing antibiotics to overcome resistance mechanisms presents an opportunity to rationally develop effective new drugs more rapidly than screening for new structures. vancomycin is a glycopeptide commonly used as a front line treatment for infections caused by methicillinresistant staphylococcus aureus (mrsa). the emergence of vancomycin-resistant enterococci (vre), vancomycinintermediate s. aureus (visa) and vancomycin-resistant s. aureus (vrsa) has prompted the development of semisynthetic glycopeptides 3 . we have generated a variety of glycopeptide derivatives that show superior antibacterial activity against mrsa and vre compared to vancomycin and second generation lipoglycopeptides. this was undertaken by employing a combination of solid phase and solution phase chemistry to attach a membraneassociative element that selectively binds to bacterial membranes in preference to eukaryotic membranes, thus increasing the local concentration at the lipid ii d-ala-d-ala peptidoglycan cell wall precursor target site. three novel antimicrobial peptides, named panurgines (png), were isolated from the venom of wild bee panurgus calcaratus. one of them is dodecapeptide with sequence lnwgailkhiik-nh2 (png-1). the next two peptides are almost identical. these are cyclic peptides containing 25 amino acid residues and two intramolecular disulfide bridges ldvkkiicvackixpnpackkicpk-oh (x=k png-k and x=r png-r). all peptides exhibited antimicrobial activity against gram-positive bacteria and gram-negative bacteria, antifungal activity and low haemolytic activity against human erythrocytes. we prepared 11 analogues of α-helical amphipathic png-1 with the aim to improve its biological properties and a linear analogue of png-r to elucidate the importance of disulfide bridges for its activity. in the second part of the study, we followed the effect of panurgines on the degree of membrane disruption by observing the leakage of fluorescence dye (calcein) entrapped in artificial phospholipids vesicles [1] . specifically, we investigated membrane interactions of pngs with the vesicles made from negatively charged 1:2 dopc/dppg and 1:2 dopc/dopg vesicles as a general model of bacteria membrane and 15:80:5 dopc/dopg/cl as a possible model for a membrane of bacillus subtilis. the membrane interaction of pngs was also investigated on uncharged dopc vesicles as potential model membrane for erythrocytes. pngs exhibited weak dye-leakage activity for neutral vesicles, while they effectively induced dye leakage in the presence of negatively charged vesicles. these results indicate that pngs have stronger potency to disrupt bacteria-mimicking anionic membranes than those which mimic eukaryotic cell membrane. department of biochemistry and toxicology, university "lucian blaga", 550012 sibiu, romania a common tool to bias the conformation of linear peptides is the insertion of side-chain modified amino acids or sidechain/main-chain conformationally restricted building blocks. an alternative approach is a simple backbone modification. in this connection, backbone amide replacements with (almost) isosteric surrogates were extensively used. these modifications may impart resistance to enzymatic degradation and better bioavailability to the peptides, but also influence the secondary structure. a thioamide (ψ[cs-nh]) is perhaps the closest structural mimic of an amide. however, it possesses different and attractive features: (i) its nh group forms stronger hydrogen bonds, being more acidic than that of the amide. (ii) its c-n bond undergoes cis/trans isomerization by irradiation at 260 nm (π→π* transition). (iii) it may act as a "minimalist" fluorescence quencher. for all these reasons, we started a programme aimed at exploring how the endothioamide bond affects peptide folding and bioactivity. in this communication, we describe the synthesis and conformational results of the three analogs of the membrane-active peptaibiotic trichogin ga iv listed below: n-octanoyl-aib-gly-ψ[cs-nh]-leu-aib-gly-gly-leu-aib-gly-ile-leu-ome (2/3) n-octanoyl-aib-gly-leu-aib-gly-ψ[cs-nh]-gly-leu-aib-gly-ile-leu-ome (5/6) n-octanoyl-aib-gly-leu-aib-gly-gly-leu-aib-gly-ψ[cs-nh]-ile-leu-ome (9/10) the syntheses of the three peptides were accomplished in solution according to a fragment condensation approach. appropriate thioamide-containing tri-or tetrapeptides were prepared by treating the corresponding all-amide precursors with the lawesson reagent. ft-ir absorption, 2d-nmr and cd conformational investigations on the three analogs were conducted in comparison with the naturally occurring peptaibiotic. all three analogs maintain the capability to interact with the dope/dopg model phospholipid membranes and exhibit a comparable bioactivity against s. aureus. peptide-peptide interaction of lactococcin g class iib two peptide bacteriocin h. etayash, w.soliman and k. kaur* faculty of pharmacy and pharmaceutical sciences, university of alberta, edmonton, alberta, t6g 2e1 lactococcin g, a class iib two-peptide bacteriocin, consists of two complementary peptides lcng-α and lcng-β that act as one functional unit with optimal antimicrobial activity achieved by the presence of both peptides in approximately equal amounts. in this study we have investigated the mechanism of pairing of the two complementary peptides as well as explored any specific interaction that could take place between the peptides. molecular dynamics (md) simulation was employed to study the interactions at the atomistic level. four different md simulations with the peptides in a lipid bilayer system were conducted. md results from these simulations confirmed and pointed out that (i) the two putative gxxxg motif, g7xxxg11 in lcng-α and g18xxxg22 in lcng-β, were attracted and came closer to each other, showing the role of these motifs in attracting the two peptides to each other. closer views, however, showed no clear interactions between these two motifs. most likely, nonspecific interactions play a role in bringing the two peptides together; (ii) variations and loss in the secondary structure in both the peptide fragments were confirmed among the four simulations. on the contrary, stability of helical regions was identified between residues w3-g9 and d26-q29 in lcng-α and v9-e14 in lcng-β; and (iii) role of tryptophan at the n-terminal regions in positioning and setting the peptide orientations were confirmed which matched the previous reported results. faculty of pharmaceutical sciences, unesp -univ. estadual paulista, araraquara, sa͂ o paulo, brazil antibiotic resistant bacterial strains represent a global health problem. antimicrobial peptides (amps) are promising novel antibiotics because they have displayed little or no resistance effects. it is well known that the charge, amphipathicity, hydrophobicity and helicity of the peptide are fundamental for the biological activity. in addition, covalent dimerization appears as a new parameter to be studied. in this way, several bioactive sequences were dimerized obtaining pharmacotechnics advantages like enhanced antimicrobial activity, solubility and proteases resistant. however, the effect of this modification is unclear since dimeric versions of some amps are toxic 1 . to evaluate the effects of dimerization on the structure and biological activity of the amp aurein 1.2, the monomeric version (au) and the c-and n-terminal dimers, (au)2k and e(au)2, respectively, were synthesized. circular dichroism results indicated that dimeric versions showed more defined structures in aqueous solution. e(au)2 showed "coiled coil" structure while (au)2k an αhelix structure. in contrast, au displayed typical spectra for disordered structures. in tfe and lpc, all the peptides acquired a high amount of α-helix structure. the antimicrobial activity against bacteria and yeast decreased with dimerization. however, dimeric peptides promoted the aggregation of c. albicans. hemolytic and vesicle permeabilization assays showed that au has a concentration dependence activity, an effect that can be assigned to a "carpet" like mechanism peptide, whereas this effect was less pronounced for dimeric versions, suggesting that dimerization may change the mechanism of action. in conclusion, our studies showed that the effects of amp dimerization are complex and still unclear. 1 , the first antimicrobial peptide generated in vivo and isolated from the gut contents of the cattle tick boophilus microplus 2 . we have shown that these peptides are equally lethal to candida albicans mdm8 and practically not active on human erythrocytes 1 . to examine the properties and mode of action of hb40-61a, we synthesized it and its fluorescently labeled analogue (fam-hb40-61a) by the solid-phase method at 60 o c, purified them by rp-hplc and characterized their purified forms by lc-esims. at low salt concentration, both peptides were found to inhibit the growth of candida albicans atcc 90028, candida parapsilosis atcc 22019 and candida krusei atcc 6258, but hb40-61a was two-fold more active (mics of 12.5; 12.5 and 50.0 μm, respectively). at those concentrations, both peptides also kill the fungi. assays with human erythrocytes showed that, likewise hb40-61a, fam-hb40-61a present activity lower than 25% at 100 μm. apparently, hb40-61a targets the membrane cell because confocal microscopy analysis revealed that, at the half of mic value, fam-hb40-61 accumulates on the fungal cell membrane. in contrast, fluorescence activated cell sorting (facs) analysis revealed that, at the mic, more than 90% of the fam-hb40-61a penetrates into the cell. membrane permeability assay using hb40-61a, c. albicans atcc 90028 and the kit live/dead funga light confirmed progressive membrane damage associated with an increase in peptide concentrations. the use dibac4 (5) and facs analysis showed that hb40-61a alters the plasma membrane potential, leading to cell death. supported by fapesp, cnpq and capes. lasso peptides form a growing class of 16 to 21 residue ribosomally-synthesized and post-translationally modified peptides produced by bacteria. they share a rigid and compact interlocked structure consisting of a macrolactam ring at the n-terminus and a c-terminal tail that is looped back and threaded through the ring, forming a typical [1] rotaxane 1, 2 . the macrolactam is formed by condensation of an asp8 or glu9 side-chain with the free amino group of a gly1 or cys1. the lasso fold is stabilized either by steric hindrance assumed by bulky amino acid side-chains and/or by disulfide bonds between cysteines from the tail and the ring. given this structure, lasso peptides display a high stability against proteolytic and chemical degradation. they are biologically active on various enzymatic targets, which confer them in some cases an interesting antimicrobial activity. given its characteristics, the lasso scaffold thus represents a promising tool for biotechnological application in the development of bioactive peptides. until now, nine peptides had been structurally characterized as lasso peptides. based on a genomics-based approach, we identified a novel lasso peptide from streptomyces sviceus that we termed sviceucin. it was produced in high yield by heterologous expression in s. coelicolor and submitted to structural analysis by mass spectrometry and nmr. sviceucin is 20residue long and stabilized by two disulphide bonds. their connectivities were identified mainly from the typical noes between the beta-protons of the cysteines. the lasso structure of sviceucin was obtained by nmr-based molecular modelling. sviceucin was shown to exhibit antibacterial activity directed against gram positive bacteria, while gram-negative bacteria and fungi showed resistant. the penicillium chrysogerum antifungal protein (paf) is a cysteine-rich, cationic protein that inhibits the growth of a variety of filamentous fungi without toxic effect on mammalian cells 1 . although paf is used to be produced in p. chrysogerum or a similar microorganism, preparation of analogues of the protein for structural and functional investigations requires an efficient chemical method. the unsuccessful continuous synthesis of the 55-mer small protein prompted us to use native chemical ligation 2 . the syntheses of the fragments were performed by solid-phase method applying tboc chemistry. using the acid-labile tboc protecting group, the thioester end of the n-terminal fragment remains intact during the course of the synthesis. the first attempt was the synthesis of peptides with pmethylbenzyl groups on the side chains of all of the six cysteine residues. under no circumstances oxidative folding provided the natural disulphide bridge pattern. the failed attempts led us to orthogonal protection of the sulphydryl groups. different sets of protecting groups were tried and evaluated. our experiments showed that basic treatment triggered rearrangement of the previously formed disulphide pattern. thus, base-labile protecting groups (such as 9-fluorenylmethyl, fm) have to be avoided in the synthesis of paf. the alarming increase and spread of antibiotic resistance among bacterial pathogens has stimulated the development of new antibacterial agents with innovative mode of action. antimicrobial peptides with broad spectrum activity are widely distributed in nature and play an important role in innate immunity in several species, including humans. tigerinins are a unique family of 11-to 12-residue antimicrobial peptides found in skin secretion of the indian frog rana tigerina 1, 2 . characterized by a disulfide-bridged loop composed of nine amino acids, tigerinins do not show primary structural homology to any known antimicrobial peptides from amphibians. tigerinins could provide novel lead compounds for the design of effective antimicrobial peptides with a new mode of action. the peptide murdp1 has been identified after the screening of phage display libraries against pseudomonas aeruginosa cell wall biosynthesis murd amide ligase enzyme 3 . murdp1 is a low micromolar range inhibitor of murd enzyme and showed good antimicrobial activity. composed of nine amino acids, it is also characterized by a nine residues disulfide-bridged loop containing two prolines. this great similarity with tigerinins, led us to investigate if murd enzyme could be a potential target for these peptides. in silico analyses using modelling, molecular dynamics and docking with p. aeruginosa murd showed that murdp1 and tigerinin-1 and -2 make similar interactions in the binding site. these results suggest that murd may be an intracellular target for tigerinin-1 and tigerinin-2. synthesis, murd enzymatic inhibition assay, antibacterial activity evaluation and structure-activity relationships of murdp1 and tigerinins analogs will be presented. h. etayash, l. norman, t. thundat*, k. kaur* faculty of pharmacy and pharmaceutical sciences, department of chemical and materials engineering, university of alberta, edmonton, alberta t6g 2e1, canada listeria monocytogenes is a gram positive bacterium that accounts for about 28% of the deaths resulting from food borne illnesses in north america. moreover, l. monocytogenes is considered one of the most difficult bacteria to detect in contaminated food products. while standard microbiological and biochemical assays currently used are accurate and sensitive, they are time consuming and often require specialized instruments operated by a trained user making on-site testing difficult. to this end, we propose the development of an antimicrobial peptide (amp) or peptide fragment sensor for the on-site detection of l. monocytogenes. leucocin a, which is a naturally occurring amp consisting of a 37 amino acid sequence, is known to exhibit specific activity against l. monocytogenes at pico to nanomolar concentrations. for this reason, we have synthesized a shorter peptide fragment of leucocin a consisting of 24 amino acids using solid phase peptide synthesis. the peptide was purified by reversed phase hplc and maldi-tof mass spectrometry indicates the desired biological entity was achieved. by including an n-terminal cysteine group, the tailored amp was readily immobilized at a gold interface. the resulting thickness and molecular orientation, determined by ellipsometry and grazing angle infrared spectroscopy, respectively, indicate that the helical peptides were adsorbed on the interface with a preferred orientation parallel to the surface. the bacterial specificity of the anchored leucocin a fragment was tested against three gram positive bacteria and results reveal that the adsorbed amp exhibits a limit of detection of approximately one bacterium/μl which is a clinically useful detection range. faculty of science, university of south bohemia, české budějovice, czech republic during the last few years we have identified three novel defensins from arthropods. two of them, lucifensin 1 and lucifensin ii were purified from various tissues of lucilia sericata and l. cuprina larvae, respectively. larvae of these flies are routinely used in the hospitals around the world for the treatment of non-healing infected wounds in the procedure known as maggot therapy. these 40 amino acid residues and three disulfide bridges peptides differ from each other only in one amino acid residue in position 11 (val-ile). linear precursor of lucifensin was prepared by fmoc-spps chemistry which was then subjected to the oxidative folding yielding a peptide with a pattern of disulfide bridges identical to that of native lucifensin and other insect defensins. this was examined by the identification of the fragments resulting from the thermolysin digestion of lucifensin by means of mass spectrometry. however, this cyclization reaction proceeded via an intermediate having incorrect pairing of disulfide bridges. from the hemolymph of blood sucking tick dermacentor marginatus (d.m.) we purified defensin containing 38 amino acids and three disulfide bridges. its sequence determined by edman degradation and mass spectrometry was identical to that previously determined by molecular biology methods 2 . sequence of d.m.defensin shows no homology to insect defensins. by spps prepared linear precursor of d.m.-defensin was subjected to oxidative folding under the open air. the linear peptide was straightforwardly folded into cyclic one which was identical to the native peptide. in antimicrobial assay using a set of different bacteria all three studied defensins show activity preferentially against gram-positive bacteria including staphylococcus aureus but are inactive against gram-negative ones. the importance of disulfide bridges on tertiary structure of defensins and their antimicrobial activity will be presented. recently, the chemical structure and conformation of pseudodesmin a has been determined through x-ray diffraction and nmr spectroscopic analysis 1 . in this way pseudodesmin a was identified as a new member of the viscosin group of antimicrobial peptides (amps). in addition, it was demonstrated that individual molecules self-assembly in apolar environment into a supramolecular pore-like structure, providing structural support for its biological activity 2, 3 . to further explore the structure-function relationship, a viable synthesis strategy for pseudodesmin a analogues was developed, based on side-chain attachment of the first amino acid to the solid support, followed by stepwise fmoc solid-phase synthesis of the linear peptide precursor and on-resin head-to-tail cyclization. nmr study confirmed the molecular structure and thus the development of an efficient and successful synthesis of this type of amp's. these results and the synthesis route will be presented. trichogin ga iv, isolated from the fungus trichoderma longibrachiatum 1 , is the prototype of lipopeptaibols, a subclass of short-length peptaibiotics exhibiting membranemodifying properties. its primary structure is as follows: n-oct-aib 1 -gly-leu-aib-gly-gly-leu-aib-gly-ile 10 -lol, where n-oct is n-octanoyl, aib is α-aminoisobutyric acid, and lol is the 1,2-amino alcohol leucinol. this peptaibol is predominantly folded in a mixed 310-/αhelical conformation with a clear, albeit modest, amphiphilic character 2 . in this work, we synthesized by solution and solid-phase methodologies a set of trichogin ga iv analogs in which the four gly residues, lying on the poorly hydrophilic face of the helical structure, are substituted by one (or more) strongly hydrophilic lys residues. moreover, we synthesized another set of analogs where one (or more) aib residues are replaced by leu. the conformational preferences of these analogs were assessed by x-ray diffraction, cd, and 2d-nmr techniques 3 . we tested the role played by the substitutions on the peptide bioactivity, e.g. protease resistance, cytotoxicity, and hemolysis. cytotoxicity was tested using three in vitro cell-based assays: (i) human red-blood cells lysis; (ii) cell mortality in total human blood leukocytes and in separate subpopulations; (iii) cell mortality in three tumor-derived stable cell lines (hela, a541, and a431). our data show that some of our trichogin analogs are active against tumor cells, leaving the leukocytes unaffected. a convenient post-screening ring opening approach for the decoding of one-bead one-compound cyclic peptide libraries a. girard, e. biron* faculty of pharmacy, université laval and chuq research center, quebec, canada combinatorial chemistry has been widely used as an effective method for the generation and screening of synthetic peptide libraries. amongst the different combinatorial methodologies to discover new bioactive peptide-based compounds, we were particularly interested in the one-bead one-compound (oboc) approach 1 . this powerful approach fully exploit the great molecular diversity accessible with peptides and has been used to identify a great number of ligands and modulators for a wide variety of biological targets. however, its use with cyclic peptides is limited by difficulties in sequencing hit compounds by edman degradation or tandem mass spectroscopy due to the lack of free n-terminal amine and complicated fragmentation patterns, respectively. this problem has been overcome by pei and coworkers by using a bead segregation strategy in which the outer layer exposes the cyclic peptides and the inner layer the linear counterpart for sequencing 2 . more recently, lim et al. reported an elegant method to prepare and sequence oboc cyclic peptoid libraries without encoding by using a ring opening approach with triazine-based cyclic derivatives 3 . unfortunately this method is incompatible with amino acids bearing some functionalized side chains. based on this strategy, we have developed an efficient method to prepare oboc cyclic peptide libraries that does not require encoding by using a simultaneous ring opening/cleavage approach. the procedure is compatible with commonly used amino acids and allows rapid and efficient sequencing of selected hits after on-bead screening. the synthesis of an oboc cyclic peptide library, ring opening methodology and sequencing by mass spectrometry will be presented. cyclotides are a very abundant class of plant peptides that display immense sequence variability around a conserved cystine knot motif and a head-to-tail cyclized backbone conferring them with remarkable stability 1 . their intrinsic bioactivities combined with tools of peptide engineering make cyclotides an interesting template for the design of novel agrochemicals and pharmaceuticals 2 . however, laborious isolation and purification prior de novo sequencing limits their discovery and hence their use as scaffolds for peptide-based drug development 3 . here we extend the knowledge about their sequence diversity by analyzing the cyclotide content of a violet species native to western asia and the caucasus region 4 . using an experimental approach, which we named 'sequence fragment assembly' by maldi-tof/tof-based peptidomics, we were able to characterize novel cyclotides from viola ignobilis. amino acid sequencing of various enzymatic digests of cyclotides allowed the accurate assembly and alignment of smaller fragments to elucidate their primary structure, even when analyzing mixtures containing multiple peptides. using in-source decay and high energy collision induced dissociation of digested cyclotides allowed to distinguish isobaric residues ile and leu. overall this work underlines the immense structural diversity and plasticity of the unique cyclotide framework. the presented approach for the sequence analysis of peptide mixtures facilitates and accelerates the discovery of novel plant cyclotides. glycation is a nonenzymatic reaction occurring between reducing sugars and reactive amino groups of biomolecules. the reaction leads to a formation of a heterogeneous mixture of compounds which are classified as early, intermediate or advanced glycation end products (age). these compounds, especially advanced glycation end products, are involved in many pathological processes, mainly diabetic complications, and could be markers of certain diseases. detection of early products of glycation (amadori products) is a relatively easy task and can be performed by various methods including e.g. ms/ms techniques, isotopic labeling and affinity chromatography on immobilized boronic acid 1,2 . however, the diverse structures of ages make detection of these compounds more challenging. the aim of the study was testing a new method of ages identification based on isotopic 13 c labeling. a model protein (hen egg lysozyme) was modified with an equimolar mixture of [ 12 c 6 ]glc and [ 13 c 6 ]glc. then the glycated protein was subjected to reduction of the disulfide bridges followed by enzymatic hydrolysis. the obtained digest was analyzed by lc-ms methods. the glycation products were identified on the basis of characteristic isotopic patterns resulting from the use of isotopically labeled glucose. this method allowed for identification of 41 early maillard reaction products and 8 different structures of the glycation end products. isotopic labeling technique combined with lc-ms is a new and very sensitive method for identification of the advanced glycation end products even if their structures are unknown. this method could be also used as an alternative method of detection of amadori products. in the course of a project aimed to assess the significance of antibiotics for the producing organism(s) in the natural habitat, we screened a specimen of the fungicolous fungus hypocrea phellinicola growing on its natural host phellinus ferruginosus 1 . using a peptaibiomics approach 2,3 , we detected 19-and 20-residue peptide sequences by (u)hplc/hr-esi-qqtof-ms. structures of peptaibiotics found were independently confirmed by analyzing the peptaibiome of an agar plate culture of h. phellinicola cbs 119283 (ex-type) grown under laboratory conditions. notably, h. phellinicola could be identified as a potent producer of 18-, 19-, (culture) and 20-residue (specimen) peptaibiotics of the suzukacillin-type4. minor components of the 19-residue peptaibols, herein named suzukacillins c, are assumed to carry a c-terminal residue tentatively assigned as tyrosinol (tyrol). in addition, the previously isolated suzukacillin b 4 was sequenced and shown to be a microheterogeneous mixture of 11-residue peptaibols. in order to further investigate the significance of antibiotics for the producing organism(s) in the natural habitat, we screened specimens of the fungicolous fungus hypocrea pulvinata growing on its natural hosts piptoporus betulinus and fomitopsis pinicola 1 . using a peptaibiomics approach2, we detected 17-, 18-, 19-(major sequences), and 20-residue peptide sequences in the five specimens analyzed by (u)hplc/hr-esi-qqtof-ms. structures of peptaibiotics found were independently confirmed by analyzing the peptaibiome of pure agar cultures obtained by single-ascospore isolation from the specimens 1 . major, 19-residue peptaibols were assigned as deletion sequences of the trichosporins b3 lacking the ala/aib residue in position 3 . our results corroborate that: i) peptaibiotics are, indeed, biosynthesized in the natural habitat, thus, ii) their membrane-perturbing formation of ion channels may support the parasitic life style of a fungicolous fungus. based on methodology that we have developed in our lab 2 , we identified specific and selective substrates for these serine proteases. we used a 10,000 membered pnaencoded peptide library to screen 10,000 possible peptide substrates in a single experiment. the library was incubated with the protease of interest and then hybridized on a custom designed dna microarray. microarray scanning and data analysis allowed the measurement of the changes in fam/tamra ratios resulting from the protease activity and the determination of the protease specificity. to verify the predicted activity and specificity, fret peptides were synthesized, incubated with the enzymes and the hydrolysis reaction was followed by monitoring fluorescence emission. specificity constants kcat/km were calculated and the cleavage sites of the peptides were identified. dubs were, moreover, found to be associated with several diseases and as such are emerging as potential therapeutic targets 2 . several directions have been pursued in the search for lead anti-dub compounds. however, none of these strategies have delivered inhibitors reaching advanced clinical stages due to several challenges in the discovery process, such as the absence of a highly sensitive and practically available high-throughput screening assay 3 . in this study, we report on the design and preparation of a fret-based assay for dubs based on the application of our recent chemical method for the synthesis of ub bioconjugates 4 . in the assay, the ubiquitinated peptide was specifically labeled with a pair of fret labels and used to screen a library comprising 1000 compounds against uch-l3. such analysis identified a novel and potent inhibitor able to inhibit this dub in time-dependent manner with kinact = 0.065 μm and ki = 0.8 μm. our assay, which was also found suitable for the uch-l1 enzyme, should assist in the ongoing efforts targeting the various components of the ubiquitin system and studying the role of dubs in health and disease. 3 . more recent work based on rna interference experiments on a mouse model suggested that isoform-specific inhibitors against nmt1 might be effective anti-cancer agents as a knockdown of nmt1 inhibits the tumour growth, whereas knockdown of nmt2 has no effect 4 . if residual nmt2 activity can compensate for loss of nmt function in healthy cells, potential toxicity may also be minimised. we developed a method to identify peptide or protein substrates of nmt1 and/or nmt2. peptides/ proteins are exposed to nmt1 and/or nmt2 and an alkyne-tagged analogue of myristoyl coa. subsequent azide-alkyne "click" cycloaddition allows visualisation of the myristoylated substrates in fluorescence or chemiluminescence, using a fluorescent or a biotin moiety on the capture reagent. this labelling technology was applied to peptide libraries prepared on microarrays to investigate nmt1/2 isozyme substrate specificity using recombinant nmt1 and nmt2. peptides made of the first 8 or 15 amino acids at the nterminus of known myristoylated proteins were functionalised with a biotin moiety at the c-terminus and immobilised on an avidin-functionalised glass plate before being screened for activity. selective peptide substrates will be developed as isozyme-specific inhibitors and applied in cancer cell lines. using chemical proteomics and the labelling technology, a selective nmt1 or nmt2 inhibitor could also be used to identify protein substrates of one isozyme. for this purpose computer programs are created which can generate fragments of one compared structure and to reveal homology by their scanning along the amino acid sequence of another. our analysis was performed by comparing the primary structures of all possible protein fragments with the amino acid sequences of all presently known natural regulatory oligopeptides. the oligopeptides were extracted from the erop-moscow database1 which at the time of analysis contained data on the structures and functions of more than 10,000 natural oligopeptide regulators. the structure-function analysis was performed using a specialized software package. the input data were the complete amino acid sequences of the proteins used as a source of fragments with a specified length. then the initial sequence was fragmented in a stepwise manner. for example, in the case of dipeptide fragments, this procedure produced fragments with the following numbers of amino acids from the n-terminus -1-2, 2-3, and so on until the fragment that started at the second residue from the c-terminus. the cases when the amino acid sequence of a fragment coincided with part of the primary structure of a natural oligopeptide were recorded in the total protein chemical synthesis requires a case by case design and optimization which is governed by factors such as the solubility of the individual peptide segments, their primary sequence and in particular the presence of "difficult" amino acid residues at ligation junctions such as proline or the location of cysteines. usually, a subset of chemical tools are selected among a vast array of methodologies to match the specificities of the target protein. in this context, methods enabling the assembly of three peptide segments in the n-to-c and c-to-n direction play a central role and must be considered as complementary as they can be selected for building subdomains of the target protein. to date, most of the proteins were assembled in the c-to-n direction. only few methods are available for the n-to-c sequential assembly of proteins, whose design is highly challenging. we have recently reported that sea ligation, that is the reaction of a bis(2-sulfanylethyl)amido group (called sea) with a cysteinyl peptide, allows the formation of a native peptide bond in water and at neutral ph 1 . in this communication we will show that native chemical ligation and the unique chemical properties of sea group 2,3 can be combined in order to design a highly efficient one-pot three segments protein assembly procedure, working in the n-to-c direction 4 amylin is one of the most amyloidogenic peptides, its fibrils are responsible for causing type ii diabetes. amyloid formation mechanism is investigated both to find amyloid inhibitors as potential medical drugs, and to use amyloids as potential self-assembling biomaterials [1] . amyloid formation of amylin 10-29, its reverse and designed analogue beta-sheets and beta-sheet stacks was studied by molecular dynamics (md), amber 9.0, f99 force field. md revealed that for amylin 10-29 and its reverse analogue both the parallel and antiparallel beta-sheet and beta-sheet stack structures are stable suggesting that this could explain the high tendency of amylin to form amyloid fibrils. parallel amylin 10-29 beta-sheet stacks are kept together by two hydrophobic cores, while for the antiparallel system the dominating is the backbone hydrogen bonding between neighbor strands. also the bent form of the amylin 10-29 beta-sheet is stable. this is in concordance with transmission electron microscopy (tem) experiments stating that all three peptides, amylin 10-29, its reverse and designed analogues, exhibited significant fibrillar polymorphism [2] university of gdansk, poland molecular dynamics (md) of two peptides dlsfmkge (mk) and dlsfkkge (kk) not related to any known disease was run to investigate the mechanism of the amyloid formation. the parallel and antiparallel [1] betasheets of mk and kk peptides were simulated by molecular dynamics (md), amber 9.0, f99 force field, ntp protocol. it was found that antiparallel beta-sheets both of mk-and kk-peptides show much higher stability than the corresponding parallel beta-sheets. this md result was supported by atr-ftir spectroscopy [2] . the betasheet stacks built from six ten stranded antiparallel beta-sheets of mk-and kk-peptides: 10x6xmk and 10x6xkk, were subjected to md. it was found that the mk-system, 10x6xmk, is strongly kept together due to hydrophobic core built from two metionines, two phenylalanines and two leucines, but the kk-system, 10x6xkk, which differs only by one mutation m5k dissolves already at 20 ns of md run, because the separate beta-sheets don't hold togather in the betasheet stack due to lost hydrophobic core. the hydrophobic core of the mk-system consists of hydrophobic units centered on the two phenylalaninetwo metionine hydrophobic interactions, and two leucines from the both sides stabilize the unit. this mechanism could be used in amyloid based biomaterials. urokinase plasminogen activator (upa) is a serine protease involved in the metastasis of several tumor types. upa is therefore an interesting target in cancer therapy. upain-2 is a new analogue of a highly specific peptidic inhibitor (upain-1) of upa. the peptide contains twelve amino acids and is cyclized through the cysteines at its termini (s 1 -s 12cyclo-ac-cswrglenhaac-nh2). upain-2 inhibits upa with a ki of approximately 40 μm. 1 one method to improve binding affinity is multivalent exposure of the inhibitor, where the local concentration at the binding site is increased. fusion of upain-1 to the trimeric tetranectin showed improved binding affinity compared to the single peptide. 2 here, we report efforts towards novel chemically linked upain-2 peptides to allow multivalent display. the ki value of an upain-2 dimer, linked by a short peg chain through the n-termini, was almost halved compared to that of the single peptide (23 μm). this motivated us to explore the role of the site (n-or cterminal) and the size of the linking segment on the binding affinity. additionally, the influence of the number of upain-2 peptides in the molecule (two vs. four) was investigated by synthesizing a carboprotein that displayed four upain-2 peptides. we present two novel nmr spectroscopic approaches to study reversible self-assemblies in solution. both methods were applied on the self-assembling pseudodesmin a, a pseudomonas produced cyclic lipodepsipeptide that has the capacity to form pores in cellular membranes. 1, 2 the first method is based on the dependence of the 13 c α relaxation rate constants on the anisotropy of the assembly. 3 when the monomer conformation is known and the multiple ch bonds in the monomer sufficiently sample all orientations, the rotational diffusion coefficients can be assessed, revealing assembly shape information. in addition, the orientation of the monomer within the assembly is obtained. the second method is based on fitting translational diffusion coefficient data as a function of concentration in a model-free way, i.e. without assuming an oligomer shape beforehand. here, it is assumed that the diffusion coefficient's dependence on the oligomer size behaves as a power law, which dramatically simplifies the expression for the average diffusion coefficient (measured by pfg-nmr) as a function of concentration. the fitted value of the exponent of the power law fully embeds all shape information of the assembly, and may be related to the socalled fractal dimension of the oligomer. moreover, this approach reveals mechanistic information concerning the assembly formation. both methods thus allow structural information of the assembly to be obtained, even when there is little or no prior knowledge available on the mechanism of the selfassembly. nucleotides and α-amino acids are crucial building blocks for living organisms. these chiral molecules are the biosynthetically precursors of two of the most important classes of biopolymers, dna and proteins, respectively. the 3d-structures of biomolecules are currently studied using a variety of techniques, while helical handedness is routinely detected by means of light pulses of opposite circular polarization. the difference in the uv absorption of these two circularly polarized pulses is called electronic circular dichroism (ecd). in nature, biomolecules explore a wide range of conformations with intrinsically strong ecd signals in the 200-300 nm region, but these signals are essentially absent in the visible. nanomaterials such as metallic nanoparticles (depending on their sizes) display absorptions in the visible region but are achiral. as a result, when biomolecules are co-assembled with nanomaterials their chirality is transferred to create a plasmon-induced ecd signal in the visible region. in this work, we present our results which underscore the occurrence of moderately strong ecd bands in the range 300-550 nm resulting from a series of appropriately thiolfunctionalized peptide oligomers (based on alternating l-ala and aib residues) covalently anchored to 2-2.5 nm sized gold nanoparticles. we related the (positive or negative) signs of the ecd plasmonic signal with the oligopeptide length, that in turn is strictly associated with their secondary structure. this latter property was simultaneously monitored via ecd in the 200-250 nm range. we believe that in our systems a peptide-tometallic surface chirality transfer would take place. light can be controlled with high temporal and spatial precision. if a specific molecule is made light-sensitive, then a precise spatiotemporal control of some of its properties can be achieved. azobenzene is the most widely used photochromic group due to its propensity to pass reversibly from the cis to the trans state under irradiation with light of the appropriate wavelength. the cisand transazobenzene isomers exhibit different spatial arrangement of the aromatic moieties that give rise to significantly distinct physical and chemical properties. the design of novel azobenzene-based molecules with precisely placed photochromic groups able to induce photomodulation of macroscopic properties is currently attracting much interest. in this work, we explored the behaviour of the conjugate formed by linking each of the four hydroxyl groups of pentaerythritol to the carboxylic function of bis[p-(phenylazo)benzyl]glycine. this c α -tetrasubstituted α-amino acid bears two side-chain azobenzene groups. the resulting system exhibits tetragonal symmetry, with a total of eight azobenzene moieties, and can be viewed as a central core surrounded by a shell of azobenzene groups at the periphery. up to eleven (out of the possible fifteen) discrete states produced by sequential trans-to-cis isomerization of the individual azobenzene units have been observed depending on the time of exposure to uv-light. this process is fully reversible (cis-to-trans) under vis-light irradiation for several cycles. in addition, this compound has been shown to exhibit photomodulated physical properties, such as polarity and hydrodynamic volume. moreover, it shows a high propensity to self-assemble in aqueous solution, giving rise to supramolecular vesicles. light-scattering and electron microscopy experiments confirmed that a conformational reorganization of the vesicles can be triggered under exposure to uv or vis light. the total chemical synthesis of native or modified proteins is gaining increase importance in the study of protein function, but also in the development of protein therapeutics. it is usually achieved by assembling in water unprotected peptide blocks using so-called native peptide ligation methods. recently, our group has developed a novel native peptide ligation method based on a peptide featuring a bis(2sulfanylethyl)amido (sea) 1 group on its c-terminus in reaction with a cysteinyl peptide in water at ph 7. we will discuss in this communication the scope and limitations of sea native peptide ligation. for this, model sea peptides featuring all the possible proteinogenic amino acids were synthesized. their rate of sea native peptide ligation with a model cys peptide were determined in the absence of presence of guanidinium hydrochloride or other additives frequently used in ncl. we will present also experiments intended to clarify the mechanism of sea ligation such as the effect of ph on the rate of ligation, or the ability of the transient thioester sea form produced by in situ n,s-acyl shift to participate in thiol-thioester exchange 2,3 . overall, the data show that sea ligation is an interesting method for native peptide ligation at various x-cys junctions, and thus an interesting alternative to ncl. plga copolymers were used as the support for inducing controlled biomarkers releasing system. visualization of the penetration in the hippocampus of mice with confocal microscope was carried out by testing both peptide-free and peptide-bearing nanoparticles, previously labeled with the phthalocyanine fluorescent probe. the encapsulation degree of the larger (12-24) segment was less effective than the others thus stressing the importance of the peptide length to this internalization process. the results showed that all peptide-containing nanoparticles were able to cross the blood-brain-barrier thus indicating improved bioavailability and uptake for peptide delivery into the brain. in regard to the radiolabeling approach, the 99m tc radioisotope was used to label the peptide sequences at his residues, as previously described 3 . stable metal-peptide complexes were obtained in 10 -5 -10 -6 m peptide concentration range. noteworthy, higher metal labeling yield was achieved with peptide segments bearing his residues at peptide c-terminal position, thus pointing to a positiondependent effect for the 99m tc coupling reaction. in conclusion, the findings indicate potentials for the proposed encapsulation and radiolabeling strategies applicable for in vitro and in vivo diagnostic assays with these peptides for the study of amyloid plaques. we have used bifunctional short peptides (ac-cg n c-nh 2 , n=2, 4, 6) to selectively link gold nanorods in an end-to-end manner. additionally, we have manipulated the gap distance between the rods by changing the length of the peptide linker. the presence of the peptide in the gaps was shown by incorporating a propargylglycine residue in the sequence, which was detected with surface-enhanced raman spectroscopy (sers). the acetylene moiety will allow further chemical modification of the linker in the gaps, opening a wealth of interesting molecular systems to be placed and studies inside self-assembled nanogaps. 1 in this work, the fragmentation pathways of alitame, neotame and andvantame in comparison to those of aspartame and aspartame-d3, were studied by negative ion electrospray ionization (esi) high resolution mass spectrometry (thermo orbitrap mass analyzer). accurate mass spectra of the dipeptides allowed proposing specific fragment ions. neotame and advantame, which are the n-(3,3dimethylbutyl) and n-[3-(3-hydroxy-4-methoxyphenyl)propyl] derivatives of aspartame, presented similar fragmentation to that of aspartame. for neotame and advantame, the "diketopiperazine'' pathway seemed to be the major one, while a pathway resulting to the formation of a pyrrolidine-2,5-dione derivative, through the involvement of the side chain carboxyl group of aspartate, was also observed. for alitame, the "pyrrolidine-2,5-dione" pathway was recorded. similarities in the fragmentation using either orbitrap or triple-quadrupole mass spectrometry have been observed. elucidation of the fragmentation is very useful for the trace-level determination of the artificial dipeptide sweeteners in complex matrices. generation of silver nanoparticles in the presence of oligoproline derivatives p. feinäugle, h. wennemers* eth zürich, switzerland in the last years, the generation of silver nanoparticles (agnps) attracts, due to its unusual physical and chemical properties, more and more attention. agnps offer great opportunities for applications in molecular electronics, catalysis, imaging and for antimicrobial coatings. 1 the characteristics depend on their shape and size. 1 many efforts have been made to optimise the generation process by, for example, varying the reducing agents, which usually are used for the synthesis or using manifold additives which should guide the nucleation and also stabilize the resulting particles. nevertheless, the generation of agnps in defined sizes and shapes still remains a challenge. we address this goal by utilizing functionalized oligoprolines that form a conformationally well-defined and rigid helical secondary structure (ppii) 2 as additives. recently, we showed that by decorating this template with aldehydes which allow for in situ reduction of the silver, they act as scaffolds in the generation process and allow the formation of defined nanoparticles. 3 we will report the results of the generation of agnps with various oligoprolines as additives, which differ in the attached functional groups as well as in the length of the peptides. laser desorption/ionization mass spectrometry (ldi-ms) using specific inert surfaces to promote ion formation has been widely investigated the last decade [1] . in addition to porous silicon through the original dios technique, different materials were tested as potent ldi-promoting agents. we explored a variety of inert silicon-based uvabsorbing materials that were presenting different physico-chemical properties for the analysis of peptides [2] [3] [4] . both material architecture (amorphous powders, structured particles, structured surfaces) and material hydrophilic/hydrophobic character tuned by specific chemical derivatization (oxidation, silanization) were probed as crucial parameters for achieving efficient and robust detection of an home-made array of model peptides covering a wide structural and mass diversity. through this set of experiments, we were able to compare the performances of all investigated silicon-based supports, especially taking into account peptide detection sensitivity (down to femtomolar concentrations) and reproducibility/repeatability (intra-spot/inter-spot signal variations) as well as the method robustness using conventional maldi-tof/tof instrument. having illustrated the capability to achieve both peptide detection and sequencing on these ionizing surfaces in the same run, high-throughput identification of protein tryptic digests by a rapid ms profiling and subsequent ms/ms analyses was achieved. comparison of the ms and ms/ms data with those obtained with sample conditioned in organic matrix [5, 6] showed a great behavior for low mass responses demonstrating the capability of ldi on nanostructured silicon supports to be a complementary method to maldi in proteomic workflow. the dipeptides aspartame, alitame, neotame and advantame are low caloric artificial sweeteners. advantame 1 , which is the n-[3-(3-hydroxy-4-methoxyphenyl)propyl] derivative of aspartame, is the most recent among them. an application for its approval has been applied in usa, australia and new zealand. such sweeteners are used in food products and beverages and they can help in managing body weight and disorders like obesity and diabetes. 2 in this work, the simultaneous determination of aspartame, alitame, neotame and advantame by negative and positive electrospray ionization (esi), under hydrophilic interaction chromatography (hilic), is presented. advantame, neotame and intermediates were synthesized in our laboratories for the present application. the key-step for the synthesis of advantame and neotame was the reductive amination of h-asp(obu t )-phe-ome with 3-(3-hydroxy-4methoxyphenyl)propanal and 3,3-dimethylbutanal, respectively. the chromatographic behavior of the artificial sweetener dipeptides was studied on two hilic columns: kinetex hilic (a fused core silica column) and zic-hilic column (a sulfoalkylbetaine column). the separation of dipeptides was achieved on kinetex hilic using 5 mm ammonium formate buffer ph 3.5 / methanol / acetonitrile (15/10/75), with a flow rate of 100 μl/min at 50 o c column oven temperature. at this ph, silica is neutral and the dipeptides are in positively charged form. the retention mechanism of all analytes seems to be partition to the water layer as well as hydrogen bonding. 3 département de pharmacologie, université de sherbrooke, sherbrooke, qc, canada plasma and in vivo stability are essential requirements for the successful development of potential drug candidates or diagnostic imaging probes. rapid degradation of compounds in plasma may result in insufficient concentration to produce the desired pharmacological activity or to be used as a diagnostic agent. there are several strategies to improve plasma half life of peptides including pegylation, modification of nand cterminal fragments of peptide, replacement of labile amino acids, and cyclization 1 . we have previously reported on probes which specifically detect matrix metalloproteinase-2 (mmp-2) activity with magnetic resonance and optical imaging 2,3 . mmps are zincdependent endopeptidases degrading the extracellular matrix (ecm) and involved in cancer progression. the main goal of this work was to find more stable probes without sacrificing enzyme specificity. we have selected specific mmp-2 substrates 4 and their stability was evaluated in three different conditions: in plasma, in plasma with a mmp inhibitor and in a mmp-2 solution. the samples were analyzed by hplc to detect the degradation pattern of our compounds and by lc-ms to determine the molecular mass of peptide fragments. based on these studies, the most stable peptide was selected and incorporated in a solubility switchable probe with radiolabelled (68)ga-dota. its in vivo stability was estimated up to 30 minutes, making it a suitable candidate for further investigations. cancer of thyroid gland is the most common malignancy of the endocrine system. the treatment improvement could be achieved by early diagnosis. the aim of the study was to identify cancer specific markers using the libraries of artificial receptors immobilized on the cellulose. an array of supramolecular structures formed from n-lipidated peptides attached to cellulose via aminophenylamino-1,3,5-triazine was prone to formation of monolayer of "holes" and "pockets" in dynamic equilibrium. this selforganized structures were found capable of binding small guest molecules very efficiently recognizing the shape, size, and polarity of ligands, thus resembling arti cial receptors 1 . recognition and binding properties of guest molecules by artificial receptors depends mainly on the character of peptidic pockets and structure of the fatty acid. proper construction of the binding pocket allows selective binding components of mixtures of compounds from a living organism 2 . the preliminary data indicates that it is possible to construct an array of artificial receptors with diversified structures of peptidic pockets which are able to distinguish between components of homogenates from tumor and normal tissue. century. most of opioid alkaloids and their derivatives have μ-opioid affinity, while endogenous enkephalins are rather δ-than μ-selective. morphine is still the drug of choice for treating severe pain caused by cancer or surgical operation, but its side effects are the reason for the searching and development of new, selective mor agonists. the aim of our study is to choose within recently published crystallographic structures templates for homology modeling of the human μ-opioid receptor. we generated several models using different templates and all of them were evaluated by docking procedure (gold 5.1) ligands used in this investigation were synthesized and evaluated for their biological activity in our previous studies. they are enekphalin analogues with substitutions in second position. the best model of the human mu-opioid receptor was chosen according to data obtained from docking and in vitro biological activity of analogues and endogenous enkephalins. acknowledgments: this work was supported by nfsr of bulgaria project dvu 01/197 and cost action cm0801 project do 02-135/31.07.2009. pneumoniae, h. pylori, proteus sp. are considered as important factor contributory to development of rheumatoid arthritis (ra). the aim of this study was to investigate the level and specificity of antibodies binding to the synthetic peptides corresponding to the bacterial ureases "flap" region sequences in the rheumatoid arthritis patient's sera. for these investigations, peptides with amino acid sequences derived from "flap" regions of different ureases were synthesized using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate 1 (dmt/nmm/bf4) as coupling reagent. peptides were immobilized on a cellulose membrane. the level of antibody binding as well as specificity of them was analyzed by quantitative dot blot method using sera 40 sera from rheumatoid arthritis patients (rap) and sera from 38 volunteer blood donors (vbd). the results of studies suggest that "flap" region may be involved in arising antibodies participating in autoimmunological processes but not to fight infection. this effect indicates that the peptides analyzed by us could be useful for investigation of ra pathogenesis. this suggestion was confirmed by the antibodies absorption experiment which indicates that specificity of antibodies present in rap serum is slightly lower in comparison with vbd serum. it has been found that antibodies present in rap serum recognize not only a specific peptide but also peptides containing fragments with different amino acid sequences. it means that immune system of rap is unstable and may produce a wide spectrum of antibodies recognizing not only a specific epitope but also a set of similar structures. autoantigen-specific t-cells also play a crucial role in the initiation and perpetuation of dsg3/dsg1-specific t-cell responses. t-cells recognize epitopes from dsg3 protein and produce different cytokines, e.g. interferon-γ (ifnγ). functional t-cell epitopes of dsg3 protein have outstanding importance in immunopathological research, development and the design of novel diagnostic tools. our previous studies have shown that certain t-cell epitope peptides are able to stimulate the peripheral blood monomorphonuclear cells (pbmc) of pv patients more effectively than those of healthy donors. our aim was to select a set of t-cell epitope peptides as potential synthetic antigens which are reliably able to distinguish between donors based on the in vitro t-cell stimulating activity. we have prepared synthetic dsg3 oligopeptides by fmoc/tbu solid phase methodology. after cleaving from the resin with tfa the peptides were purified by rp-hplc, and they were characterized by rp-hplc, mass spectrometry and amino acid analysis. pbmc of pv patients and healthy donors were isolated; and the cultures were stimulated by dsg3 peptides in a concentration of 0.025 mm for 20 hours, and the rate of ifnγ production was determined from the supernatants in sandwich elisa. synthetic dsg3 oligopeptides induced different in vitro ifnγ production rate on pbmc obtained from pv patients and healthy controls determined by elisa. our approach identified a synthetic antigen set as a promising biomarker for pemphigus vulgaris. [1] . in particular, cap2b (pelyafprvamide) has been shown to elicit antidiuretic activity in the green stink bug acrosternum hilare [2] , an important pest of cotton and soybean in the southern united states. analogs of cap2b containing either an (e)-alkene, cispro or a transpro isosteric component [3] were synthesized and evaluated in an in vitro stink bug diuretic assay, which involved measurement of fluid secretions of malpighian tubules isolated from a. hilare [2] . at a concentration of 1 μm, the conformationally constrained transpro analog demonstrated significant antidiuretic activity, whereas the cispro analog failed to elicit any activity. the results provide strong evidence for adoption of a trans orientation for the pro in cap2b neuropeptides during interaction with the receptor associated with the antidiuretic process in the stink bug. the work further identifies a scaffold with which to design biostable mimetic cap2b analogs as potential leads in the development of environmentally favorable pest management agents capable of disrupting cap2bregulated diuretic systems. the enkephalins are pentapeptides (tyr-gly-gly-phe-met/leu) with a proven antinociceptive action. it is believed that the interaction between them and the lipids composing the membranes is important for converting the peptides into a "bioactive" conformation 1,2 . using langmuir's monolayer technique the interaction of a synthetic methionine-enkephalin (met-enk) and its amidated derivative (met-enk-nh2) with mixed lipid monolayers composed of palmitoleoylphosphatidylcholine (popc), sphingomyelin and cholesterol was studied. the surface pressure-area (π-a) isotherms with regard to πmin, π max and the hysteresis curve shape of the pure lipid monolayers and after the addition of the respective enkephalins were detected. in addition, by using brewster angle microscopy (bam), the surface morphology of the mixed lipids-enkephalins monolayers were determined. our results suggest that there is a strong penetration effect of the enkephalins studied into the mixed monolayers. moreover, our results demonstrate the potential of lipid monolayers formed in langmuir's through in combination with bam to be successfully used as an elegant and simple membrane models to study lipid-peptide interactions at the air/water interface. acknowledgments: this work was supported by bulgarian ministry of education, youth and science, projects n do02-107/08, drg 02/5 and my-fs-13/07. dept pharmacolgy, temple univ, philadelpha, pa 19140, usa bioinformatic algorithms has predicted the existence of several potential hormone-like peptides transcribed from the ecrg4 gene 1 . previous publications indicated a highly level of gene expression of ecrg4 products has been found in the pancreas 1 , choroids plexus, epithelial cells, leukocytes, and macrophages 2 . however, the presence in the hypothalamus and the major form of derived peptides in each tissue haves not been clearly identified. knowingledge of the precise peptide generatesd within a given tissue is essential to understanding its functions. we have generated the peptide specific antibodies to against ecrg4-derived pprepro-augurin(71-147) and developed a specific ria kit for the quantification of the such peptide in question. a method for the purification of endogenous ecrg4-derived peptides from bovine hypothalamus also has been established. using ria to monitor the immunoreactive fractions and maldi-tof to identify the endogenous peptides, we foundhave detected the presence of ecrg4 derived molecular of bovine preproaugurin(71-147) from the homogenates of bovine hypothalamus. immunohistochemistrycal staining by antibody aalso confirmed the presence of thee peptide in some of the hypothalamic cells. of hypothalamus. the amount of prepro-augurin(71-147) in the hypothalamus although not soas high as pancreas, but is one third of the augurin level of the pituitary. conclusions: the native peptide derived from augurin preproteinecrg4 has been discovered.identified. we have confirmed the property of purified peptide,s prepro-augurin(71-147), along with the synthetic peptide standards. the present study provides the necessary procedures such as the elution from (1) c18 column, (2) p6 sizing column, and (3) a further purification conditions for hplc in order to enhance the immunoreactivity from tissue fractions and yield enough amount for identification. this dsip-related peptide (kn-dsip or knd) differs from dsip by only 2 amino acid residues in positions 2 and 5. we do not consider the homology between dsip and knd as accidental, bearing in mind functional significance of histone demethylases of the jmjc-group. methylation-demethylation of histones is known as an important mechanism of posttranslational modification playing a prominent role in epigenetic regulation of chromatin structure and gene transcription. dsip is also known as an effective "normalizer" and protector from homeostatic disorders induced by stress related disturbances. we suggest that histone demethylase of the jmjc-group containing dsip-related region can be considered as a possible protein precursor of endogenous peptides with dsip-like activity. in order to test our hypothesis we synthesized knd and studied its biological effects. in a preliminary assay cited below [1] knd showed similar and probably more pronounced effects than dsip as an agent that stimulates endurance and stress-resistance of animals in the forced swimming test. also knd provided a more active detoxifying action after administration of a semi-lethal dose of the cytostatic agent. in the present work we assessed neuroprotective and antioxidative potency of both peptides in vivo and confirmed the higher efficiency of knd. this study is supported by the moscow government. is a tridecapeptide (pglu 1 -leu 2 -tyr 3 -glu 4 -asn 5 -lys 6 -pro 7 -arg 8 -arg 9 -pro 10 -tyr 11 -ile 12 -leu 13 ) highly expressed in the central nervous system. this peptide elicits an analgesic response following peripheral or central administration. importantly, nt exerts a more potent analgesia than morphine at an equimolar dose, without having the associated side effects of opioid drugs. 1 structure-activity studies have identified the c-terminal fragment nt(8-13) as the biologically active minimal sequence. however, nor the full or truncated peptides cross the blood-brain barrier (bbb), thus hampering its clinical development. the substitution of pro 10 by an unnatural amino acid silaproline 2 (sip) increased bioavailability and plasma stability. structural properties conferred by the pro 10 were also retained as determined by nmr and ir. 3 aiming at delineating the mode of action of cl, three new cl derivatives bearing suitable labeling moieties, i.e the fluorescent molecule fitc, the streptavidin-counterpart biotinyl-group and the 99m tc-radiometal chelating unit dimethylgly-ser-cys, were designed, synthesized, purified, and characterized to be applied in in vitro and in vivo evaluation studies. the structure of the cl derivatives in aqueous solutions was studied with nmr, in parallel and in comparison with the parent molecule cl, in order to examine whether the presence of the labeling moieties has induced changes to the structure of the biologically active part of cl. cell survival assays with cl and the cl derivative bearing the fitc moiety were conducted in the pc12 cell line in order to explore their rescue effect. in parallel, the cl derivative bearing the dimethylgly-ser-cys moiety was successfully radiolabeled with 99m tc and its stability was assessed over time in its synthesis reaction mixture and in plasma. this 99m tc-radiolabeled derivative was subsequently administered to swiss albino mice in order to determine the biodistribution of cl in the living organism and its route of excretion, a study that has not been carried out so far for any peptide of the humanin family. furthermore, the potential interaction of cl with β-amyloid peptide, the hallmark of ad pathogenesis, was explored with circular dischroism. the results of this multifaceted approach to the biological action of cl will be presented. institute of biochemistry and biotechnology, martin-luter university, halle-wittenberg, germany kinins, such as the nonapeptide bradykinin, are important mediators of various physiological and pathophysiological responses including inflammatory disease, asthma, rhinitis, cell division, pain, vascular permeability, allergic reactions, pathogenesis of septic and endotoxic shock. there are two types of receptors for kinins, known as b1 and b 2 . b 2 receptors are constitutively expressed in wide variety of cells and required entire bk sequence for recognition, while b1 receptors have normally very limited expression and respond to [desarg 9 ]bk. b 1 receptors gene is turned on following either tissue damage or inflammation. accumulated evidence indicates that most of the clinically relevant effects of bk are functions of b2 receptors this being the reason why research on their antagonists is a topic of great interest. in our previous study we described the synthesis and some pharmacological properties of four new analogues of bradykinin (bk), designed by substitution of position 7 or 8 of the known [d-arg 0 ,hyp 3 ,thi 5,8 ,d-phe 7 ]bk antagonist with l-pipecolic acid (l-pip) (both analogues were also prepared in n-acylated form with 1-adamantaneacetic acid (aaa)). our results showed that presence of l-pip in position 7 slightly increased antagonistic potency in the blood pressure test, but it turned the analogue into an agonist in the rat uterus test. replacement of thi by l-pip in position 8 also enhanced antagonism in the rat pressure test but preserved the antagonism in the rat uterus test. in the present study we continue our previous investigations to find structural requirements which in the case of bk analogues result in high b2 antagonistic activity. several new bradykinin analogues modified in their cterminus with d-pipecolic acid were synthesized using spps method. the biological properties of the analogues were assessed by their ability to inhibit vasodepressor response of exogenous bk in conscious rats and by their ability to inhibit the contractions of isolated rat uterus evoked by bk. acknowledgements this work was supported by the university of gdansk (ds/8453-4-0169-2). peptides with beta-turn structure in peptide/mhc complexes a. stavrakoudis department of economics, university of ioannina, greece major histocombatibility complex (mhc) molecules interact with small peptides and form complexes. in most of the cases, peptide's structure in these complexes is found in extended conformation. however, notable exceptions exist where the peptide forms a beta-turn structure. this happens mainly in the central part of the peptide in class i complexes [1] , or at the c-terminal of class ii complexes [2] . several peptide/mhc complexes were, derived with xray studies, were extensively subjected to molecular dynamics simulations [3] in order to investigate the stability of this turn-like structural feature and to explore the factors that possibly contribute to this stability. it was found that both intra-peptide and peptide/mhc interactions might be responsible for peptide's conformation. the peptides were found to undergo several structural transitions indicating conformational plasticity and not a completely rigid structure inside the mhc groove. the results might be of special importance in designing defective peptide vaccines and beta-turn pharmaceuticals. the heptapeptide met-enkephalin-arg6-phe7 (merf) with the sequence of yggfmrf is a potent endogenous opioid located at the c-terminus of proenkephalin-a (penk), the common polypeptide precursor of met-and leuenkephalin. our systematic bioinformatic survey revealed considerable sequence polymorphism at the heptapeptide region of different penk prepropeptides among 56 vertebrate animals. four orthologous heptapeptides with single or double amino acid replacements were identi ed among 15 animals, such as yggfmgy (zebra sh), yggfmry (newt), yggfmkf (hedgehog tenrek) and yggfmri (mudpuppy). each novel hepta-peptide, together with the mammalian consensus merf and metenkephalin, were chemically synthesized and subjected to functionality studies, using radioligand binding competition and g-protein activation assays in rat brain membranes [1] . equilibrium binding af nities changed from good to modest as measured by receptor type selective [ 3 h]opioid radioligands. the relative af nities of the heptapeptides reveal slight mu-receptor (mop) preference over the delta-receptors (dop). [ 35 s]gtpγs assay, which measures the agonist-mediated g-protein activation, has demonstrated that all the novel heptapeptides were also potent in stimulating the regulatory g-proteins. all peptides were effective in promoting the agonist induced internalization of the green uorescence protein-tagged human mu-opioid receptor (hmop-egfp) stably expressed in hek293 cells. thus, the c-terminally processed penk heptapeptide orthologs exhibited satisfactory bioactivities, moreover they represent further members of the so-called "natural combinatorial neuropeptide library" emerged by evolution. corticotropin releasing factor (crf) exerts most of its physiological and pathophysiological actions by interacting with its type 1 receptor (crf1) and activating different intracellular signalling pathways. the crf1 is a plasmamembrane protein, which belongs to the family b of g-protein coupled receptors (gpcrs) and like the other gpcrs consists of an amino-terminal extracellular region, a carboxyl-terminal intracellular tail and seven, mostly hydrophobic, membrane-spanning segments (tm1-tm7), connected by alternating intracellular (il) and extracellular loops (el). binding of crf and its related peptides, such as sauvagine, to the extracellular regions of crf1 is associated with receptor activation and subsequent activation of different g-proteins and regulation of diverse signalling pathways. using a mutagenesis approach in combination with a radioligand binding study we found that trp259 and phe260 in the second extracellular loop of crf1 interacted with the amino-terminal portion of crf and sauvagine. interestingly only the interaction of sauvagine with trp259 and phe260 is important for crf1-mediated stimulation of camp accumulation. in marked contrast the interaction between crf and the residues trp259 and phe260 was unimportant for the activation of adenylate cyclase. thus it is possible for trp259 and phe260 of crf1 to regulate distinct signalling pathways, or different sets of them, after their interaction with different peptides. we are now performing experiments to fully elucidate the signalling pathways that are regulated by the interaction of crf and sauvagine with trp259 and phe260. these studies will advance the development of crf1-selective selective signalling-specific peptides that would be extremely useful for the elucidation of the role of crf1 in many physiological and pathophysiological situations, and possibly for the treatment of several crf1-related diseases. thymus humoral factor gamma-2 (thf-γ2), an octapeptide, purified from crude thf, retains essentially all the biological properties of thf [1] [2] . it regulates clonal expansion, differentiation and maturation of t-cell precursors, stimulates the production of lymphokine, maitains the normalization of impaired ratios between helper(cd4+) and suppressor / cytotoxic (cd8+) subsets and augments il-2 production in spleen cells. thf-γ2 has a calculated molecular weight of 918 and has the following amino acid sequence: leu-glu-asp-gly-pro-lys-phe-leu. its poor stability towards protein enzyme limits its extensive application. with the inte ntion to promote its bioavailability, bioactivity and develop ideal immunoregulatory drug candidat, four series of derivatives of thf were designed and synthesized: 1. n-and cterminal acylation. 2.restitution the flexible segment gly-pro by unnatural amino acids 6-aminohexanoic acid (aca) in order to shorten the synthetic steps and simultaneity improve the bioavailability and biostability of peptide; 3. reserve protected group of some amino acid residus as spot mutation. 4. mannich-based cyclization was carried out on resin [3] , phe was replaced by tyr serving as the active hydrogen component, a proline was introduced at the n terminal as the amine component and formaldehyde was used as the only component in solution. the bioactivity of synthesized products were detected. the leukocytopenia model in mice was induced by cyclophosphamide intraperitioneal injection. white blood cell count, thymus index and spleen index were detected to evaluate the immune function of compounds in mice. the results show that those compounds play a significant role in improving immune function in mice. the activity of compound lhl21 and lhl22 are also better than authentic compound tp-5 and tα1. marine organisms have been recognized as a promising source for the development of new pharmaceuticals. in the course of screening for antitumor substances from marine organisms, we found cyclic peptides containing many nonribosomal amino acids such as hydroxyasparagine, hydroxyleucine, or other supporting a hydrophobic side chain that were shown to be a key element for their biological activity. the laxaphycine b, a cyclic lipopeptide isolated from marine cyanobacteria anabaena torulosa harvested in french polynesia 1 constitutes an example of this peptide class. this compound has attracted our attention because of its micromolar cytotoxic activities on different cancer cell lines as well as its antiangiogenic properties which seems to be due to an interaction with the vegf receptor-1 2-3 . the synthesis of the non-natural amino acids 4-5 and of laxaphycine b analogues will be presented along with their preliminary biological activities. immune response suppressors are used in the medical praxis to prevent graft rejection after organ transplantation and in the therapy of some autoimmune diseases including dermatology. cyclolinopeptide a (cla) c(pro 1 -pro 2 -phe 3 -phe 4 -leu 5 -ile 6 -ile 7 -leu 8 -val 9 -), a cyclic, hydrophobic nonapeptide isolated from linseed, possesses strong immunosuppressive and antimalarial activity. 1 it has been suggested that both the pro-pro cis-amide bond 2 and an 'edge-to-face' interaction between the two aromatic rings of adjacent phe residues 3 in tetrapeptide unit are important for biological activity. this edge-to-face interaction can be influenced when phenyl rings are replaced by naphtyl substituent. in this communicate new analogues of cla modified by 2naphtylalanine (2-nal) in positions 3 or 4 or both 3 and 4 (1-3 linear analogues, 4-6 cyclic analogues) will be presented. the synthetic strategy and biological activity as well as conformational analysis will be evaluated. the onset of type ii diabetes mellitus (t2dm) coincides with the deposition of fibrillar material in the islet of langerhans in the pancreas that is a clinical hallmark of more than 95% of patients suffering this disease. 1 the main component of the pancreatic amyloid deposits is a 37-residues polypeptide hormone called islet amyloid polypeptide (iapp) or amylin. 2 in this work we have examined, by means of cd spectroscopy and tht-fluorescence, the conformational polymorphism of both full-length 1-37 hiapp, and the related fragment hiapp17-29, and compared the results with the respective rat counterparts. moreover, the cytotoxic activity was determined toward different pancreatic β-cells lines in the attempt to correlate iapp's fibrillogenic properties with the ability to mediate cell death. together the results suggest that β-sheet conformational transition, that generally preludes to fibril formation, is not a prerequisite for eliciting toxicity toward β-cells cultures. interestingly, confocal microscopy indicated that both hiapp1-37 and hiapp17-29 can enter the cell and might exert their toxic action at intracellular level. acknowledgments: this work was supported by miur, firb-merit project rbne08hwlz. due to its physiological functions, 26s proteasome is considered the target molecule in overcoming several diseases [1] . its core particle 20s has three types of active sites: chymotrypsin-, trypsin-and caspase-like. many natural and synthetic compounds were tested for their ability to inhibit proteasome. a recent report describing the inhibition of 20s by the serine proteases inhibitor -bovine pancreatic trypsin inhibitor -was considered by us with great attention [2] . our scientific interest is focused on peptide inhibitors and their interaction with serine proteases. sunflower trypsin inhibitor (sfti-1) is the smallest and the most potent peptide inhibitor in the bowman-birk family. owing to its size and the rigid structure (disulfide bridge and "head to tail" cyclisation) sfti-1 is willingly chosen as the lead structure in the search for new inhibitors [3] . its sequence is shown below (lys 5the p1 residue responsible for specificity): & 1 gly-arg-cys(& 2 )-thr-lys 5 -ser-ile 7 -pro 8 -pro-ile-cys(& 2 )-phe-pro-asp& 1 since native sfti-1 is not able to inhibit 20s [2] , we have designed its monocyclic analogues (with disulfide bridge only) with lys or arg in position 5 (p 1) and at least one basic amino acid (lys or arg) in positions 7 (p 2') and/or 8 (p3'). all analogues inhibit chymotrypsin -(ic50 at the range of 1÷3 μm) and caspase-like (ic 50 at the range of 0.7÷7μm) activities in vitro, whereas their activity towards trypsin-like specificity is much weaker. in several rat tissues, our view on ras has changed. metabolism of the ang-(1-12) may represent alternative pathway of ang ii formation, importantly, independent on renin and ace activity 1,2 . ahmad 2 et al. have described metabolism of ang-(1-12) by human atrial tissues and showed that ang ii is formed mainly by chymase. this renin-inependent ang ii production could explain the "resistance" regarding use of ace inhibitors in patients with hypertension or diabetic nephropathy. noteworthy, the role of ang-(1-12) in circulation is still unclear and there are no information about possible pharmacological modulation of its metabolism. in our study, we compared the ex vivo metabolism of angiotensinogen (fragment 1-14) in hypertensive (shr) and normotensive (wky) rats in organ bath of aorta and heart using lc-ms method 3 . surprisingly, we identified ang-(1-12) formed via reninindependent pathway to be a main product of angiotensinogen metabolism in rat aortic tissue and heart. in this setting, ang-(1-12) appeared to be not only prevalent metabolite of angiotensinogen, but also served as a substrate for generation of ang i and ang ii. as compared to wky rats, formation of ang ii, from ang-(1-14), was much higher in shr aortas but not in the heart. the functional consequences of these findings require further investigation. this study was supported by the grant n n401 293939 polish ministry of science and higher education. the lysosomal cysteine protease cathepsin c (cat c), also known as dipeptidyl peptidase i (dppi), activates a number of granule-associated serine proteases with proinflammatory and immune functions by removal of their inhibitory n-terminal dipeptides. activity of this protease is associated with several pathologies in human body [1] . in this work the characterization of cat c specificity using combinatorial chemistry methods will be described. the main goal of this work was to determine of substrate specificity of the prime region of this enzyme. the chemical synthesis and deconvolution of two libraries will be described. the hemostatic mechanism has the crucial role to prevent loss of blood from injured blood-vessels. this loss is prevented by the integrity of the vessel walls, by platelets aggregation or by blood coagulation, which in normal conditions is limited onto the local trauma of the vessel wall. in generally, the blood coagulation mechanism is important for maintaining vascular integrity and thus for the precaution of an organism from bleeding, which may also occur by blood coagulation caused by thrombin production. the diversion rate of this production leads to an expansion of thrombin to the general blood circulation. thus, when thrombin generation is not controlled by the mechanisms of inhibition, a widespread undesirable intravascular thrombosis is occurred. the whole process of platelets adhesion requires the presence of clotting factor viii (fviii), a necessary for the blood coagulation cascade glycoprotein, which takes part in the intrinsic pathway and acts as a coenzyme for the activation of factor ix, a serine protease depended on the thrombin production. the target of the present research is the synthesis of biologically active cyclic, head to tail, peptides, analogs of the sequence 558-565 of a2 subunit of fviii, which are potentially capable to block fviiia-fixa complex, reducing the thrombin production and thus the blood coagulation. the synthesized peptides are investigated for their inhibitory activity and tested for clotting deficiency by measuring the chronic delay in the activated partial thromboplastin time (aptt) and the reduction of the % value of the fviiia, which they generate in samples containing recombinant fviiia, in vitro. the blood coagulation is part of an important host defence mechanism, which under pathological conditions results in inappropriate intravascular coagulation when thrombin is produced. clotting sequence is the result of a cascade of two biochemical pathways, intrinsic pathway, so called because all components are present in blood, and extrinsic pathway, in which tissue factor is required in addition to circulating components. the activated form of factor viii (fviiia) is a key component of the fluid phase of the blood coagulation and plays an important role formatting a trimolecular complex with factor ixa, ca 2+ and negatively charged phospholipids of the cells membrane. this complex is called tenase and participates in activation of prothrombin, which acts on fibrinogen to generate fibrin monomer, polymerized rapidly to form fibrin clot. the fviii is comprised of a heavy (a1-a2-b) and a light (a3-c1-c2) peptide chain, both cleaved by proteases at three sites, resulting in alteration of its covalent structure and conformation. its deficiency is known as haemophilia a. our research effort is focused on the synthesis, identification and biological evaluation of peptide analogs, expected to inhibit selectively the increasing of thrombin production. their sequence is based on the regions in which the fviii interacts with fix, specifically on the sequence 558-565 of the a2 subunit. the synthesized peptides are examined for their activity and tested for clotting deficiency by measuring the chronic delay in the activated partial thromboplastin time (aptt) and the reduction of the % value of the fviiia, which they generate in samples containing recombinant fviiia, in vitro. inhibitor with the following structure: ac-llllrvkr-nh2, which has potent effects on the proliferation of prostate cancer cells. the potency and stability of this compound was subsequently enhanced by substitution of arg residue in position p1 with its conformationally restricted mimetic -4-amidinobenzylamine (amba). nevertheless, the specificity toward pace4 was significantly reduced by this modification. thus, in order to improve its selectivity without sacrificing inhibitory potency we decided to use positional scanning approach. in this study we present synthesis of two series of peptide libraries, which were designed by substitution of leu in the p5, p6 position of our control peptide (ac-llllrvkr-amba) with each of nineteen amino acid residues in order to verifying its influence on activity and selectivity of the resulting analogues. all peptides were synthesized by a combination of solid phase peptide synthesis and solution synthesis and tested for their inhibitory potency against furin and pace4. the p2-p8 fragments were synthesized by fmoc/tbu spps strategy on hydrazinobenzoyl or acid labile 2chlorotrityl chloride resin. then coupling of the 4-amidinobenzylamine · 2 hcl was performed. the best modifications were combined to give as several multipoint substituted inhibitors. we believed that our work, will provide new important information about structure-activity relationship of these class of analogs in order to obtain potent and highly specific pace4 inhibitor. institute for research in biomedicine, parc cientific de barcelona, barcelona -spain a bacterial toxin-antitoxin (ta) system is composed of two genes organized in an operon encoding a toxin and an antitoxin that regulate the growth and death bacterial cell under various stress conditions. the operon parde encode a ta system formed by pare toxin and its antitoxin pard. pare is a 12 kda protein that inhibits dna gyrase activity and thereby blocks dna replication. however the pare-gyrase interactions and the gyrase activity inhibition mechanism have not been explored. as an approach for understanding of this mechanism and to elucidate the pare region responsible for protein-protein interactions we have designed and synthesized a series of linear analogues of pare and investigated the ability of peptides to inhibit dna topoisomerases activity. so, based on structural data inferred from pare three-dimensional model 1 , 12 peptides were synthesized by solid-phase method. four peptides (parelc3, parelc8, parelc10 and parelc12), showed complete inhibition of dna gyrase supercoiling activity, by gel electrophoresis assay 2 , with an ic100 of 20 to 50 μmol.l -1 . in addition, intrinsic fluorescence and fluorescence anisotropy assays showed that inhibition process must occur by interaction with the gyra subunit. differently of wild type pare, the peptide analogues were able to inhibit the dna relaxation of topoisomerase iv with lower ic100 values. interesting was that only parelc12 displayed inhibition of the relaxation activity of human topoisomerase ii. our results suggest a new class of molecules with simultaneous inhibitory activity in dna gyrase and topoisomerase iv. furthermore, we have obtained the first example of a synthetic peptide from a bacterial toxin with inhibitory activity on human topoisomerase ii. institute of experimental endocrinology, slovak academy of sciences, bratislava, slovakia the renin-angiotensin system (ras) has long been recognized as an important regulator of systemic blood pressure and electrolyte homeostasis. our understanding of ras has experienced remarkable change over the past two decades. besides, angiotensin ii, the new biologically active peptides [e.g. ang-(1-7), ang-(1-12), ang iv, ang-(2-10)] and pathways [e.g. angiotensin converting enzyme 2 -ace2] have been described 1 ; some of them, like ang-(1-7) may oppose many actions of ang ii. importantly, despite all components of classical ras are found in adipose tissue 2 , the data about fat formation of various angiotensins remain scarce. in our study, we compared the ex vivo metabolism of angiotensinogen, ang-(1-12) and ang i in hypertensive (shr) and normotensive (wky) rats in organ bath of retroperitoneal and periaortic fat tissue using lc-esi-ms method. additionally, qpcr measurements of mrna expression of main enzymes involved in ang i metabolism were performed. both in the periaortic and epidydymal fat, the formation of ang-(1-7) was higher than production of ang ii. fat tissue formation of two main ang i conversion products, ang ii and ang-(1-7), differed significantly between shr and wky rats. compared to wky rats, the formation of ang-(1-7) in periaortic fat tissue was decreased in shr. in opposite, in epidydymal fat tissue formation of ang-(1-7) and ang ii was higher in shr. interestingly, there were no differences in aorta formation of ang ii and ang-(1-7) between shr and wky rats. our results suggest that in hypertension visceral fat production of angiotensin peptides is increased, while generation of "beneficial" ang-(1-7) in periaortic fat is decreased. however, the functional importance of such finding require further investigation. department of chemistry and biochemistry, university of washington, usa phospholipases a2 (pla2) are a superfamily of enzymes involved in various inflammatory diseases. 1 in particular, human secreted giia spla2 is an attractive target for the development of novel medicines. 1 we have shown that 2oxoamides based on γ-or δ-amino acids are potent inhibitors of cytosolic giva pla2. very recently, we have demonstrated that a long chain 2-oxoamide based on (s)leucine displays inhibition of human and mouse giia spla2s (ic50 300 nm and 180 nm, respectively). 2 a combined experimental/computational study was undertaken to further understand the role of the α-amino acid of 2-oxoamides for the inhibitor-enzyme binding. the crystal structure of giia spla2s reveals a highly conserved ca 2+ -binding loop and a catalytic dyad consisting of his47/asp91. 2-oxoamides based on hydrophobic αamino acids showed better binding score prediction compared to polar α-amino acid derivatives. a number of new 2-oxoamides based on α-amino acids were synthesised and tested for their inhibitory activity against giia, gv and gx pla2. the 2-oxoamide based on (s)-valine displayed potent inhibition of giia spla2 (ic 50 218 nm) in accordance with the predicted docking score. docking results reveal that (s)-valine-based inhibitor forms key interactions with the active site of the enzyme. the carboxylic group participates in a hydrogen bonding with gly31 and lys62, and 2-carbonyl group with gly29. furthermore, both carbonyl groups are in the proximity with ca 2+ . the side chain of (s)-valine adopts a suitable orientation to interact with tyr51 and lys62. the long aliphatic 2-oxoacyl chain is accommodated in the hydrophobic region of the active site and creates proximal contacts with leu2, ile9, his6 and phe5. the search for novel classes of pharmaceutical molecules with enhanced therapeutic power has been the subject of numerous research groups all over the world. moreover, systems of immobilization and controlled release which are adapted to these new classes of molecules, has proven to be an area of extreme importance to provide the same therapeutic efficacy. using solid-phase chemistry a series of ccdb toxin analogous peptides were synthesized and were synthesized and tested against the capacity of inhibition of bacterial enzymes dna gyrase and topoisomerase iv (topo iv). subsequently those peptides were detained in drug delivery systems (dds) to be tested against the inhibition of growth of different bacterial species. in this data we could observed that the analogue ccdbsg2 could inhibit only dna gyrase and not the topoisomerase iv. in the other hand the analogue ccdbsg1 presents a hard inhibition potential against topo iv specially because of their structural difference. is possible conclude that topoisomerase iv presents the tertiary structure very similar to dna gyrase, but those mechanisms of action must be clearly distinct 1 . in the in vitro studies, as expected, results revealed that the drug delivery systems are the key to the power efficiency of peptide analogues against the bacterial growth inhibition which cannot be observed when the peptides are free in solution. some of the different lipid compositions of the dds are demonstrating to be more efficient in the membrane cell transverse and this data previously assumes that it is possible to apply different types of dds to promote the peptide molecules transport across the cellular membranes according to several specific therapies. with this studies we have obtained more knowledge about the interaction system of enzyme-toxin and hopes which helps in future studies to development a new antimicrobial molecules class. it is urgent to develop less toxic and more efficient treatments for leishmaniases and trypanosomiases. we propose to target an ancestral form of the proteasome, the hslvu protease, which is present in the parasite's single mitochondrion, essential for the growth of these organisms and has no analogue in the human host. originally discovered in eubacteria, this complex is constituted by two central hexameric hslv protease rings sandwiched between two hexameric hslu atp-ase rings. as hslv shares a similar enzymatic mechanism with the host proteasome, we propose to inhibit the assembly of the complex in order to be selective. according to studies on bacterial hslvu, 1,2 the c-terminal segment of hslu is essential in hslv activation and in complex assembly, therefore representing a privileged target. we produced recombinant hslv, which is inactive alone, and showed that a synthetic c-terminal hslu peptide was able to induce the digestion by hslv of a fluorogenic substrate that we developed. with this enzymatic test in hands, we started the characterization of the interaction of the c-terminal portion of hslu with hslv. we will present the results obtained with various series of analogues of the original c-terminal hslu peptide, including truncated forms, ala scan, constrained analogues and multivalent constructions. helped by molecular modelling studies, the aim is to establish structural requirements, which could lead to high affinity and stable ligands able to inhibit the interaction between the hslu and hslv rings, obligatory for the degradation of proteins by the hslvu complex. finally, we checked that hslv was inhibited by classical active-site directed proteasome inhibitors like bortezomib. f. babos a,d , e. szarka b , gy. nagy c , z. majer d , g. saŕmay b , a. magyar a , f. hudecz a,d citrullinated filaggrin peptide (ccp) were detected in ra sera and anti-ccp positivity is widely used for diagnostic purposes. identification of new epitopes of filaggrin 1 would be useful in the diagnosis of anti-ccp3 seronegative patients. in order to achieve optimal immune recognition of biotinylated epitope peptides it is important to analyse the effect of the labelling moiety on antibody binding. for these studies 5-as well as 19-mer peptides with nor c-terminal biotin were synthesised manually by spps, using fmoc/ t bu strategy. biotinylation was performed by using biotin, biotinyl-6-aminohexanoic acid or 4,7,10-trioxa-1,13-tridecanediamino succinic acid linker 2 modified biotin. labelled peptides were used in an indirect elisa, on neutravidin pre-coated plates and the binding was detected by anti igg-hrp. to examine the role of the presence/position of biotin in the secondary structure of the peptides, electronic circular dichroism (cd) method was used. we found that the ccp3+ serum samples specifically recognized the c-terminally biotinylated 5-mer filaggrin peptides, while showed no binding with the n-terminally biotinylated compounds. in case of the 19-mer epitope peptides there was no difference between the recognition of nand c-terminal biotinylated analogues. data presented suggest that the position of the biotin in case of the short filaggrin epitope peptides markedly influence the serum antibody binding. upon activation process, they are released from the granules and then involved in immunoresponse of the organism. when out of the cell those enzymes remain in free form or become associate with the cell membrane. the physiological role of this proteases is manifestated in several processes such cytokine and chemokine processing, platelet activation, and degradation of extracellular matrix's proteins [1] . in this work results of the specificity of two members of nsps pr3 and hne evaluated using the combinatorial chemistry methods will be presented . both enzymes share primary specificity and to obtain the selective substrate that will be recognized only by one enzyme, the prime sites should be investigated. the general formula of the designed library is as follows: where in positions x1', x2' and x3', the set of 19 proteinogenic amino acids (except cys) was introduced. abz is 2-amino benzoic acid served as donor of fluorescence and 3-nitro-l-tyrosine as acceptor. eukaryotic proteasome is a highly organized protease complex comprising a catalytic 20s core particle (cp) and two 19s regulatory particles (rp), which together form the 26s structure. the main function of this large intracellular protease is to degraded ubiquitine labeled proteins. the catalytic particle of the proteasome displays three distinct enzymatic activities: trypsin-like, chymotrypsin-like and glutamyl-like. the increase activity of the proteasome is associated with several disease including cancer [1] . the main aim of this work is to synthesized the cell permeable fret displaying peptides that will selective cleaved by single proteasome activity. additionally each peptide when independently cleaved by the proteasome subunit, should emit the fluorescence energy in a different spectral region. our intention was designing substrates which would allow to monitor simultaneously (in a single experiment) and independently of three proteasome activities in this report, we will describe the chemical synthesis of several peptides modified at on cand n-termini by synthetic fluorescent amino acids the general formula of these peptides is as follows: where x is a non proteinogenic amino acid that serve as a donor of fluorescence, y amino acid that is a acceptor of fluorescence. the obtained fluorescent peptides were examined for their ability to cross the cell membrane. also kinetic parameters (k cat, km, kcat/km) with proteasome will be presented. approximately 100 dubs are encoded in the human genome and are involved in a variety of regulatory processes, such as cell-cycle progression, tissue development, and differentiation. recently, several groups have introduced various methods for linking ubiquitin to different substrates via nonhydrolyzable isopeptide bonds, which resist the action of dubs. using these methods, one could explore the function and the mechanism of dubs and apply them in activity based profiling. here we present a new and convenient strategy for preparing nonhydrolyzable ubiquitinated peptides and proteins by nmethylating the isopeptide bond. using this method we prepared several nonhydrolyzable ubiquitinated peptides with different lengths derived from ubiquitinated h2b and examined their affinity to different dubs. f.i. nollmann, c. dauth, d. reimer, h.b. bode* goethe universität, frankfurt, germany bacteria of the genus xenorhabdus and photorhabdus are gram negative gamma proteobacteria that live in symbiosis with nematodes of the genus steinernema. undergoing their partly entomopathogenic life cycle these bacteria not only produce antibiotics 1,2 and insecticides 3 but also several different small molecular compounds and peptides. 4 for the most part the biological benefits of these secondary metabolites have not fully been understood yet. 5 with the help of inverse feeding experiments, hr-ms and nmr as well as molecular engineering we were able to characterize and/or isolate some of these peptides. since they are mostly produced in trace amounts, we synthesized them in order to make them accessible to continuative testing. given that not only linear but also highly methylated or cyclic peptides are produced, the synthesis was quite challenging. nevertheless, we were able to establish in our laboratory a general synthesis route for cyclic peptides 6 and depsipeptides 7 , as well as highly methylated hydrophobic linear sequences. testing several of these peptides has revealed activity against insect cells and against the causative organisms of neglected tropical diseases. cyclotides are a large class of plant peptides defined by a head-to-tail cyclized backbone and three conserved disulfide bonds in a knotted arrangement. these unique structural features confer them with remarkable stability and due to a range of bioactivities they are extensively investigated as templates in drug discovery 1 . based on the use of oldenlandia affinis in traditional african medicine for its uterotonic principle we investigated crude plant extracts and semi-pure cyclotide fractions for the ability to induce uterine contractions using a collagen-gel contractility model2. pharmacological analysis of the effects led to the identification of the oxytocin receptor, a representative of the g-protein coupled receptor (gpcr) family, as a molecular target for cyclotides. mass spectrometry-based sequence analysis of 'active' fractions revealed cyclotides with high similarity to the human oxytocin (h-ot) peptide that exhibited weak binding to the human oxytocin receptor. we further analyzed synthetic cyclotide-derived small ot-like peptides and grafted the h-ot sequence into the stable cyclotide frame. these peptides showed increased binding and activation as compared to native cyclotides. these findings may open new avenues for the discovery of gpcr ligands from natural peptide sources. gpcrs are promising drug targets and ~5 0% of currently used drugs act via binding to these receptors. natural combinatorial peptide libraries are likely to play an important role in identifying novel gpcr ligands 3 . particularly plant cyclotides cover a large chemical space based on their high sequence diversity. together with their range of bioactivities and unique stable structure suggests that cyclotides are of current and future interest for drug discovery and development. acknowledgements: this work is funded by the austrian science fund fwf (p22889). drosha and dicer are two key endonucleases for biogenesis of micrornas (mirnas) that regulate target mrna. drosha converts pri-mirna to ~7 0 nucleotide (nt) pre-mirna in nucleus and dicer converts pre-mirna to linear ~2 2 nt single-stranded mirnas in cytosol. even though dicer is potentially important to control availability of mature trans-acting rnas in cytosol, the enzyme itself does not seem to be the suitable target controlling mirna processing due to the lack of its substrate specificity. nature, however, might be intelligent enough to differentiate a variety of pre-mirna, so that a certain specific pre-mirna is converted to mature mirna in case it needs. therefore, other component(s) in the enzyme complex could be involved in recognition of auxiliary proteins from out sources to give extra specificity. we have synthesized trp-containing amphiphilic peptides against several pre-mirna. peptide 2b showed a picomolar binding affinity and a large specificity against pre-let7a-1. in vitro mirna processing, dicer activity was also selectively enhanced in the presence of this peptide. on treatment with this peptide on hct116 colon cancer and p19ec cell lines, let7a-1 mirna was more processed than reference mirnas. the toxicity of furan is known to rely on its selective oxidation in the liver by cyt p540 enzymes transforming it into the very reactive butenedial, which quickly reacts with proximate nucleophiles. 1 this principle was used in our laboratory to develop a high yielding dna interstrand crosslinking methodology. 2 in view of the demonstrated site-selectivity, the method further holds promise for sitespecific crosslinking dna to its binding proteins, which is highly relevant in the study of transient protein-dna interactions. furthermore irreversible dna binding can be achieved through such a covalent linkage, which is potentially useful for new generation therapeutics. 3 the reactive furan moiety can in principle be incorporated either in the dna or in the protein. in the former case, a furan modified nucleotide was built into an oligonucleotide positioning the furan moiety at the periphery of the dna, to avoid interstrand crosslinking. for the latter approach, we initially chose to synthetically access a furan modified dna binding protein mimic. next to a previously described non-covalent gcn4 mimicking dimer, 4 we have also investigated a new type of steroid-based dipodal dna binders. 5 synthesis of the latter constructs has proven challenging in view of the immobilization of two peptide chains with helix forming tendency at close distance on the template. results, showing the power of microwave assistance will be discussed. in an alternative approach, a full length protein was modified with furan by amber suppression based on the structural similarity between a furan modified amino acid and pyrrolysine. 6 pharmaceutical institute, university of bonn, an der immenburg 4, 53121 bonn, germany human matriptase-2 is a 80 kda protein with trypsin like specificity. this protein exhibits a domain organization similar to family of membrane-bound serine proteinases known as type ii transmembrane serine proteinases. among many ascribed function in human body, this enzyme is a potent negative regulator of hepcidin, the peptide involved in iron homeostasis [1] . matriptase-2 has a similar fold as other tmsp members, however their detailed specificity still remain unclear. the aim of this study was to determine the substrate specificity of this physiological important enzyme using combinatorial chemistry approach. in order to characterize the matriptase-2 specificity, the tetrapeptide library with c-terminal amide of aminocoumarin (acc-nh2) that serve as a fluorophore, was synthesized. its general formula is given below: x4-x3-x2-x1-acc-nh2, where in position x4-x2 the set of proteinogenic amino acid residues are present, whereas in position x1 lys or arg was introduced. deconvolution of such library was performed using iterative approach in solution. the results obtained indicate that matriptase-2 display diverse p4-p2 specificity as compare to matriptase-1. the most efficient hydrolyzed amino acid residue in position p4 appear to be ile, that is followed by arg in p3 and ser in p 2 . the arg in position p1 is 30% faster hydrolyzed then lys. for selected substrates, the kinetic parameters (kcat, k m ) were determined. amyotrophic lateral sclerosis (als) is a chronic progressive disease. it is characterized by degeneration of upper or lower motor neurons, but its pathogenesis is still unknown and no effective treatment currently exists. it is known that antibodies to gangliosides have been found in some als patients, and these antibodies are also well known to be present in the patients affected by a variety of autoimmune diseases including multiple sclerosis. up to now anti-gangliosides antibodies are detected in clinical immunology laboratories using isolated non consistent antigen mixtures. therefore, we are interested in developing reliable and univocally characterized synthetic antigens for efficient antibody detection. csf114(glc) is a family of structure-based designed glycopeptides that we previously developed as multiple sclerosis (ms) synthetic probes. these n-glucosylated peptides are able to detect specific autoantibodies in the sera of an antibody-mediated form of ms. 1 autoantibody recognition was favored because of the exposition of the sugar amino acid on the tip of type 1' β turn structures. aim of this study is the introduction, in the type 1' β turn peptide structure, of the sugar moiety specific for anti-gangliosides antibody recognition by synthesizing specific building blocks. these building blocks are amino acids carrying glycans mimicking the biological activity of complex oligosaccharides. we selected sialic acids (in particular the n-acetylneuraminic acid -neu5ac) 2 because they are involved in a significant number of biological events. neuraminic acid and its derivates are widely distributed in animal tissues and in bacteria, especially in glycoproteins and gangliosides. therefore, we synthesized fmoc-l-asn(neu5ac)-oh and fmoc-l-ser(neu5ac)-oh. these building blocks will be introduced in the type 1' β turn structure for the detection of anti-gangliosides antibodies in als. as a distinct pattern of ms could involve an antibodymediated demyelination, identification of autoantibodies as specific biomarkers is a relevant target. even if interesting data focused on the diagnostic and prognostic role of the detection of antibodies to myelin oligodendrocyte glycoprotein (mog) in adults' serum, its value remains dubious due to many other contrasting results. our research group identified csf114(glc), an nglucosylated peptide, able to detect disease-specific autoantibodies in the sera of a statistically significant number of ms patients. 1, 2 since this synthetic antigen may be considered as a mimic of aberrant post-translational modification (i.e. n-glucosylation) of myelin protein(s) triggering autoimmunity in ms, our goal is to obtain the extracellular domain of mog properly glucosylated thanks to a simplified native chemical ligation approach. 3 for this purpose, the n-glucosylation will be introduced in a synthetic peptide fragment following the building-block approach by spps. the other protein fragment bearing an n-terminal cysteine will be expressed in e. coli after introduction of a selective point mutation into mog. finally, our aim is to test the semi-synthetic protein by sp-elisa to study the ability to detect autoantibodies in ms patients' sera and to find a potential cross-reactivity with csf114(glc). this peptide is an endogenous ligand of the opioid receptorlike 1 (orl1), previously referred to as "orphan" receptor, structurally and functionally related to the classical opioid receptors. also the hexapeptide ac-ryyrwk-nh2 is shown to be a selective ligand for the nop receptor with marked analgesic effect. with a view to developing ligands for the nop receptor with more potent analgesic activity, new series of the ac-rfmwmk-nh2 and ac-ryyrwk-nh 2 , modified at position 4 and 5 respectively with newly synthesized β 2tryptophan analogues were synthesized 1 . the aim of the present study was to examine the effects of naloxone (nal) and jtc-801 (nop receptor antagonist) in the analgesic activity of newly synthesized hexapeptide analogues. all peptides (10 μg/kg), nal (1 mg/kg) and jtc-801 (0,5 mg/kg) were injected intraperitoneally (i.p.) in male wistar rats. antinociceptive effects were evaluated by two nociceptive tests -paw-pressure (pp) and hot-plate (hp) and statistically accessed by anova. the results will be discussed compared to the referent compound in both tests used and mechano-and thermo-receptors are involved. [1] . socs1 and socs3 have many similarities as well as some intriguing differences. both can block signalling by direct inhibition of jak enzymatic activity yet apparently require different anchoring points within the receptor complex. while the primary socs1 interaction is with a critical py residue within the jak catalytic loop [2] it interacts also with py residues in the ifnαr1 and ifn r1 subunits in a jak1-independent manner; the socs3-sh2 domain also interact with y1007 in jak2, albeit with slightly lower affinity, but subsequent studies demonstrated a high affinity interaction with py residues located within receptor subunits [3] . mutagenesis studies identified small regions at the n-termini of the socs1 and socs3-sh2 domains, and at the c-terminus of the socs3-sh2 domain, which were critical for phosphotyrosine binding. in order to gain insights in molecular discriminants for the interaction of both socs1 and socs3 toward jak2 and tyk2 we designed and synthesized peptides encompassing regions involved in proteins recognition. we set up a spr assay to evaluate the affinities of complexes formation. then through an alascanning approach we have designed new peptide sequences containing un-natural amino acids that are able to better recognize wild sequences and whole proteins. cellular experiments on stat1 activation signaling suggest their potential application as modulators of disorders involving socss overexpression. targeting proapoptotic death receptors (drs) to trigger apoptosis in cancer cells is a promising anticancer therapeutic approach. trail (tnf-related apoptosis inducing ligand) is a transmembrane homotrimeric protein belonging to the tnf family that triggers selective tumour cell apoptosis upon binding to its cognate receptors dr4 and dr5. several strategies are being developed to exploit the unique cancer selectivity of the trail-dr pathway in therapy, including the use of recombinant trail targeting dr4 or dr5. [1] recently, a disulfide-bridged macrocyclic 16-mer peptide (derived from phage display) that binds selectively to dr5 has been identified. [2] oligomeric versions of this macrocyclic peptide display increased binding avidity to the receptor and exhibit the capacity to activate the trail apoptotic pathway both in vitro and in vivo. [3] however, disulfide bonds are susceptible to reduction and scrambling in vivo potentially resulting in the loss of the desired biological activity. among alternative linkages with increased redox stabilities, lanthionine thioethers, in which one of the sulfur atoms of the disulfide bond is removed have previously been introduced into biologically active peptides with some success. [4] disulfide bridges can undergo a -elimination in alkaline conditions, followed by a michael addition to give a thioether bridge. optimization of this reaction led to the desulfurized analogue of the dr5-binding peptide. the native dr5-binding peptide and its desulfurized analog have been compared for their structural (nmr conformational analysis) and biological properties (affinity to dr5 and signaling pathways). the apelin/apj complex has been detected in many tissues and is emerging as a promising target for a number of pathophysiological conditions. in the central nervous system, apelin/apj was detected in brain regions involved in spinal and supraspinal control of pain, such as the amygdala, hypothalamus, dorsal raphe nucleus and spinal cord. we propose the hypothesis that apelinergic agonists represent a potential new approach to pain modulation and that the synthesis of stable analogues would lead to compounds with antinociceptive properties. there is currently little information on the structure/activity relationship (sar) of the apelin hormone. in an effort to better delineate sar, we synthesized analogs of apelin-13 modified at selected positions with unnatural amino acids, with a particular emphasis on the c-terminal portion. analogs were then tested in binding and functional assays by evaluating gi/o mediated reduction in camp levels and by assessing β-arrestin2 recruitment to the receptor. the plasma stability of new analogs was also assessed. several were found to possess increased binding and higher stability compared to the parent peptide. there is compelling evidence that the neuropeptide 26rfa and its cognate receptor gpr103, are involved in the control of food intake and bone mineralization. 1 among the gpcrs whose structures have been solved, gpr103 exhibits the highest sequence homology with the beta2adrenergic receptor. the aim of this work was to experimentally characterize predicted ligand-receptor interactions by site-directed mutagenesis of gpr103 and design of point-substituted 26rfa analogs. starting from the x-ray structure of the beta2-adrenergic receptor, a 3d molecular model of gpr103 has been built. the bioactive c-terminal octapeptide 26rfa(19-26), kggfsfrf-nh 2 , 2 was subsequently docked in this gpr103 model and the ligandreceptor complex was submitted to energy minimization. in the most stable complex, the phe-arg-phe-nh2 part was oriented inside the receptor cavity whereas the n-terminal lysine remained outside. a strong intermolecular interaction was predicted between the arg 25 residue of 26rfa and the gln 125 residue located in the third transmembrane helix of gpr103. in order to study this interaction, we have investigated the ability of 26rfa and arg-modified 26rfa analogs to activate the wild-type (wt) and the q125amutant receptors transiently expressed in cho cells. the platelet receptor αiibβ3 plays a critical role in the process of platelet aggregation and thrombus formation. upon platelet activation its conformation changes leading to an increased affinity for fibrinogen. the αiibβ3 activation is regulated by "outside-in" and "inside-out" signaling. among the protein-protein interactions, which contribute to «inside-out» signaling, the most important is that of talin with the β3 cytoplasmic tail. it has been recently suggested that talin-mediated αiibβ3 activation relies on the cooperative interaction of the membrane proximal (mp) and the membrane distal (md) β 3 regions with talin f3 domain and that the -n 744 ply 747 -motif of β3, which can be phosphorylated at y 747 , plays a critical role in this process. 1 to evaluate the interaction of talin with the β3 tail of integrin we designed and synthesized three peptides corresponding to the md and mp parts of β3 in their carboxyfluoresceinlabeled form (md: cf-r 736 akwdtannplyke 749 -nh 2 , cf-n 743 nplykea 750 -nh 2 and mp: cf-k 716 llitihdrke 726 -nh2). emission and anisotropy fluorescence spectroscopy was used to quantitatively assess the affinity of these peptides for talin. furthermore, to challenge the role of the y 747 phosphorylation in talin-α iib β 3 interaction we also studied the binding of talin to the modified analogues of md, cf-r 736 akwdtannpl(ptyr)ke 749 -nh 2 and cf-n 743 npl(ptyr)kea 750 -nh 2 . our experiments revealed that the md and mp parts of β3 bind tightly to talin and that y 747 phosphorylation has an inhibitory effect on this binding. functionalized oligoprolines as multivalent scaffolds in tumor targeting p. wilhelm, h. wennemers* eth zurich, zurich, switzerland oligoprolines are known to be structurally well-defined molecular scaffolds. in aqueous media, even short chain lengths of six proline residues adopt a polyproline ii helix (ppii). this secondary structure is a highly symmetric helix where every third residue is on top of each other in a distance of about 9.5 å. [1] the incorporation of azidoproline (azp) allows facile and versatile functionalization either via copper-catalysed azide-alkyne cycloaddition (cuaac) or an acylation that followed a staudinger reduction. [2] based on the structural integrity of the oligoproline scaffold, targeting vectors can be conjugated via coppercatalysed azide-alkyne cycloaddition in defined distances. recent studies on radiolabeled oligoproline-bombesin conjugates, to target the gastrin-releasing peptide receptor (grp-r), showed in vitro and in vivo superior internalization in prostate cancer cells compared to the established monovalent ligands. [3] a facile route to synthesize alkynylated ligands has been developed successfully. we are currently expanding this concept to the integrinligand c(rgdyk) as well as to [tyr 3 ]-octreotide, which binds to somatostatin-receptors. the monovalent analogue of the latter, dota-[tyr 3 ]-octreotide (dotatoc), is well established for diagnosis [4] and therapy [5] of somatostatinpositive tumors such as neuroendocrine tumors. the cu i -catalyzed azide-alkyne addition 1 (cuaaa), the useful variant of "click chemistry," has emerged as a powerful technique for specific addition. that chemistry is also commonly used for conjugation, and cyclization of peptides. it is known that cyclization can increase the metabolic stability of peptides, as well as enhance potency or selectivity. another useful application of the cuaaa, which we are reporting, is the n-terminal crosslink of two synergic peptides to gain their potency. cuaaa reaction is performed on solid phase (merrifield resin) where one of the peptide components with azido group on the linker (6azido-hexanoic acid) is "clicked" with second peptide component in solution, made by fmoc strategy in partially protected form containing at n-terminal side alkyne group (fmoc-l-propargylglicine). cuaaa coupling is performed in dmf/t-buoh/h2o with presence of cui and sodium ascorbate when reacting mixture was degassed. linked peptides are cleaved finally from resin and purified. as an application example we picked two endothelin active peptide analogues: bq123 derivative 2 (a highly potent and selective eta antagonist) and irl-1620 derivative 3 angiogenesis is a key step in the transition of tumors from a dormant state to a malignant state. the vascular endothelial growth factor (vegf) is a major contributor to tumor angiogenesis. its pro-angiogenic activity is mainly mediated through binding to two tyrosine kinase receptors located predominantly on the surface of endothelial cells: vegfr-1 and vegfr-2. vegf binding to these receptors triggers the activation of different signal transduction pathways responsible for the proliferation, survival and migration of endothelial cells 1 . vegf/vegfr system constitutes a target to stop tumour growth. an attractive approach is the development of peptides, or small-molecules, with a high affinity for the extracellular domain of the receptors to prevent vegf binding. based on the x-ray structure of vegf and the d2 domain of vegfr-1 2 , cyclic peptides had been developed in our group 3 . such peptides, mimicking simultaneously the 3-4 loop and helix ·1 of vegf, can bind to d2 domain of vegfr-1 and inhibit receptors phosphorylation and thus map kinase pathway 4 . we describe here our strategies to optimize peptidic antagonists of vegfr-1. chemical modifications are made in order to better mimic peptide conformations and to increase their receptor binding affinities. we introduce a hydrophobic functional group at the c-terminal of the original cyclic peptide 4 , some of such modified peptides reveal improved vegfr-1 binding affinity. otherwise, as the helix ·1 presents most of the important residues in vegfr1 binding according to alanine-scan in the literature5, we try to stabilize the helical conformation by insertion of aib residues or by peptide cyclisation. the peptides affinities are evaluated by an elisa test developed previously 3 . institute of chemistry and biochemistry, freie universität berlin, thielallee 63, d-14 195 berlin, germany new polypeptide was isolated from the azemiops feae viper venom by combination of gel filtration and reversephase hplc and called azemiopsin. its amino-acid sequence (dnwwpkpphqgprpprprpkp) was determined by means of edman degradation and mass spectrometry. it consists of 21 residues and does not contain cysteine residues. according to circular dichroism measurements, this peptide adopts a β-structure. peptide synthesis was used to verify the accuracy of the determined sequence and to prepare sufficient peptide amount for biological activity studies. azemiopsin efficiently competed with α-bungarotoxin for binding to torpedo nicotinic acetylcholine receptor (nachr) (ic50 0.18±0.03 m) and with lower efficiency to human α7 nachr (ic50 22±2 μm). ala scanning showed that amino-acid residues at positions 3-6, 8-11 and 13-14 are essential for binding to torpedo nachr. in biological activity azemiopsin resembles waglerin, a specific blocker of muscle-type nachr from tropidechis wagleri venom. however the sequences of these peptides are markedly different, and azemiopsin is the first natural toxin to block nachrs that does not possess disulfide bridges. laboratory of peptide science, nagahama institute of bio-science and technology, nagahama, shiga 526-0829, japan while neutrophils infiltrate into damaged sites immediately after tissue injury, endogenous factors which induce their acute transmigration and activation have not been thoroughly elucidated. for the candidates, we recently identified two novel neutrophil-activating cryptides, mitocryptide-1 (mct-1) and -2 (mct-2), which were hidden in mitochondrial cytochrome c oxidase and cytochrome b, respectively [1] [2] [3] . in addition, the presence of many neutrophil-activating peptides other than mct-1 and -2 was observed during their purification. these findings suggest that neutrophils are regulated by many unidentified peptides. here, we purified a novel neutrophil-activating octadecapeptide whose primary structure was identical to mitochondrial cytochrome c (68-85) from porcine hearts. we named this functional peptide as mitocryptide-cyc (mct-cyc). the structure-activity relationships of cytochrome c on β-hexosaminidase release from neutrophilic differentiated hl-60 cells demonstrated that cytochrome c (70-85) was the most potent cryptide among cytochrome c-derived peptides. since cytochrome c is known to be involved in the apoptotic process, our present results suggest that cryptides produced from cytochrome c play an important role in scavenging toxic debris from apoptotic cells by neutrophils. anthracis spores are very resistant and can remain dormant in soil for decades. therefore, an effective detection system for b. anthracis is urgently needed. recently, it was found that one of the components of the b. anthracis exosporuim is a collagen like protein whose carbohydrate portion is composed of the tetrasaccharide with the highly specific monosaccharide upstream terminal, named anthrose. 1 since anthrose was not found on other bacterial spores, including those closely related to b. anthracis, this monosaccharide is an attractive target for the development of new b. anthracis detection and identification methods. peptide cyclization represents particularly interesting approach for the design of artificial receptors for anthrose, because cyclic peptides provide the possibility of having a spherical lipophilic binding site of appropriate size and shape for a particular carbohydrate substrate. 2 the presence of hydrogen donor/acceptor groups within a three-dimensional structure permits carbohydrate substrates to be encapsulated, thereby allowing their binding in water. in order to determine whether the cyclic peptide receptor can selectively detect the anthrose, we have successfully prepared cyclic peptide combinatorial library (total 6859 peptides) by the process of divide, couple and recombine ("tea-bag" technology) using standard fmoc solid-phase peptide synthesis. 3 prepared combinatorial library is screened for anthrose binding in fluorescence-based assay, and individual cyclic peptides with enhanced affinity toward anthrose are identified by the positional scanning deconvolution process. 3 cyclization of linear sequences is a well-known approach used to restrict the flexibility of peptides. cyclization often increases selectivity of peptides towards one specific receptor type, increases metabolic stability and generally increases lipophilicity, which often improves the bloodbrain barrier permeability of peptides. in our previous study [1] we have reported on the synthesis of a cyclic endomorphin-2 (em-2) analog, tyr-c(d-lys-phe-phe-asp)-nh2, which elicited analgesia after peripheral administration. encouraged by the fact that this analog was able to cross the blood-brain barrier we designed and aliskiren is the first orally active, direct renin inhibitor to be approved for the treatment of hypertension. its structure and conformational analysis were explored using molecular dynamics (md) simulations. for the first time, md calculations have also been performed for aliskiren at the receptor site, in order to reveal its molecular basis of action. it is suggested that aliskiren binds in an extended conformation and is involved in several stabilizing hydrogen bonding interactions with binding cavity (asp32/255, gly34) and other binding-cavity (arg74, ser76, tyr14) residues. of paramount importance is the finding of a loop consisting of residues around ser76 that determines the entrapping of aliskiren into the active site of renin. the details of this mechanism will be the subject of a subsequent study. additionally molecular mechanics poisson-boltzmann surface area (mm-pbsa) free energy calculations for the aliskiren-renin complex provided insight into the binding mode of aliskiren by identifying van der waals and nonpolar contribution to solvation as the main components of favorable binding interactions. adamantyltripeptides and phospholipids in liposomal bilayers 1 . now, we were primarily interested to study incorporation profile of mannosylated adamantyltripeptides. we have demonstrated that the adamantyl moiety, due to its liphophilic properties, penetrates into the lipid core of the bilayer while the hydrophilic part with the mannosyl moiety is exposed on the liposome surface. after concanavalin a (con a), a lectin, which specifically binds α-d-mannosyl residues, was added to the liposome preparation, increase in liposome size and appearance of aggregates has been observed. the enlargement of liposomes was ascribed to the specific binding of the con a to the mannose present on the surface of the prepared vesicles. the afm analysis revealed that the adamantyltripeptide molecules grouped into small domains that raise above the bilayer surface. the molecule size and molecular geometry, as well as the hydrophilic and hydrophobic surfaces in the structure of mannosylated adamantyltripeptides, are responsible for arrangement of molecules in the lipid bilayer. this approach might be a useful model for investigation of specific protein interactions with membrane receptors. also, the adamantyl moiety may be considered as a potential membrane anchor for different carbohydrate or other molecules of interest, which could be bound on it and thus exposed on liposome surfaces and as such used in targeted drug delivery. the assay is carried out in a 96 well format p122 and images are captured throughout the course of the assay, thus we can not only determine a ligand's propensity to induce internalization, but also its efficacy and internalization rate. addition of test compound, followed by the standard agonist at a later interval, enables differentiation between agonist or antagonist activities. in the positional scanning format [1] , while the possibility of agonists and antagonists working against each other within a mixture exists, the effects are minimized in screening the whole library as there are as many arrangements of the sub-libraries as there are defined positions. therefore while an agonist and antagonist might be present in a particular mixture in one sub-library they will be in different mixtures in all other sub-libraries. we have used this assay format to simultaneously screen for novel agonists and antagonists against the orexin 2 receptor. assay development and library screening will be presented. [1] . since the pro residue in position 2 of em2 is very important in the proper conformational alignment of the two aromatic residues tyr 1 and phe 3 in em2 molecule at the receptor site, it is possible that structural modification around the pro 2 residue yields compounds with unique biological properties and improved metabolic stability. in the present study, we synthesized seven em2 analogues containing isopro or constrained residues with oxopyrrolidine or oxopiperadine ring, instead of pro residue in position 2. all peptide analogues were synthesized solid phase method. incorporation of oxopyrrolidine and oxopiperadine rings were carried out on a solid support by the methods of gellerman, et al. [2] and mohamed, et al. [3] , respectively. opioid receptor binding activity for μ and δ-receptors using the development of resistance to mainstay cancer therapies has become a major limitation for the treatment of many cancers. there is an urgent need to develop new antineoplasic agents with innovative anticancer approaches. to overcome resistance to cancer therapies, our attention has turned to proteins that regulate multiple signalling pathways essential for tumour survival. among the few known nodal proteins upregulated in cancer cells and involved in many hallmarks of cancer, we are interested in survivin. an essential regulator of cell proliferation and apoptosis, survivin is sharply overexpressed in cancer cells and plays a major role in resistance. 1 being a small protein, its bioactivity is relies mainly on protein-protein interactions (ppi) with different partners. a critical point for its multiple functions in cancer is its association with hsp90, which is required for its stability. a nonapeptide from survivin called shepherdin has been shown to modulate the interaction of survivin with hsp90 by binding to hsp90 and to induce death of tumour cells. 2 unfortunately, shepherdin is not cell permeable, has low proteolytic stability and shows poor bioavailability, limiting its use as anticancer therapeutic agent. to improve pharmacological properties of shepherdin, cyclic and peptidomimetic analogs of shepherdin have been synthesized followed by structure-activity relationship studies. in hsp90 binding studies, some cyclic hexa-and heptapeptidic analogs showed increased affinity compared to shepherdin. the synthesis of cyclic and peptidomimetic analogs and the results from the binding assays and the conformational analyses will be presented. the hexapeptides with formula ac-ryyr/kw/ir/k-nh2 have been identified as shortest peptide sequence with high nop receptor affinity, selectivity and marked analgesic effect. it was found that the following peptides act as partial or full agonists or antagonists of nop receptor in different in vivo and in vitro systems. these hexapeptides were used as chemical templates in sar studies 1,2 . the aim of the present study was the synthesis and the biological screening of new analogs of ac-rfmwmk-nh2 and ac-ryyrwk-nh 2 , modified at position 4 and 5 respectively with newly synthesized β 2 -tryptophan analogues 3 . these non natural amino acids were prepared using reaction of asymmetric friedel-crafts alkylation of various indoles with a chiral nitroacrylate to provide optically active β-tryptophan derivatives. the four newly synthesized ligands for the nociceptin/orphanin fq (n/ofq) receptor (nop) have been prepared by solidphase peptide synthesis-fmoc-strategy. these compounds will be tested for agonistic activity in vitro on electrically stimulated smooth-muscle preparations isolated from vas deferens of wistar rats. bacterial infections are a common problem associated with dermal wounds. these infections can prolong or impair wound healing. hydrogel materials that display inherent activity against bacteria can be used to directly treat accessible wounds to prevent or kill existing infection. in this work, we describe the design and utilization of injectable gels prepared from self-assembling β-hairpin peptides having a high content of arginine. these gels were found to be extremely effective at killing both gram-positive and gramnegative bacteria, including multi-drug resistant p. aeruginosa. importantly, no added antibacterial agents are necessary since the nanostructure of the gel, itself, is the active agent. using self-assembling peptides for material construction allows facile structure-activity relationships to be determined since changes in peptide sequence at the monomer level are directly transposed to the bulk material's antibacterial properties. structure-activity relationships studies show that arginine content largely influences the hydrogel's antibacterial activity, and influences their bulk rheological properties. these studies culminated in an optimized gel, composed of the peptide pep6r. pep6r gels prepared at 1.5 wt % or higher concentration, demonstrate high potency against bacteria, but are cytocompatible towards mammalian mesenchymal stem cells. the general mechanism by which pep6r exerts its action was explored and it is suggested that involves membrane disruption that occurs when cells come in contact with the gel's surface. atomic force microscopy (afm) was used to study the effect of the gel on the cell envelope morphology of e. coli. rheological studies indicate that the gel is moderately stiff and displays shear-thin recovery behavior, allowing its delivery via simple syringe. 1 they are intimately involved in the molecular process leading to the delicate nano-patterned silica shells of diatoms. deciphering the mechanisms of silica-biogenesis in diatoms will inspire the development of novel routes for the biomimetic synthesis of silicon-based materials under mild conditions and expand the scope of biotechnological applications, e.g. for immobilization of enzymes in silica matrices. we synthesized silaffin peptides derived from c. fusiformis that carry posttranslational modifications such as phosphorylation or polyamines linked to lysine side chains. a distinct alteration of silica precipitation activity depending on the particular modifications of the silaffins emerged. 2 these modified silaffin peptides were covalently linked to recombinant proteins by expressed protein ligation leading to stable protein-silaffin conjugates. 3 using egfp as model protein, we could show that egfp-silaffin conjugates can induce biomineralization of silica and ensure an efficient and homogeneous immobilization of egfp into silica particles, superior to simple co-biomineralization approaches. moreover, a significant stabilization of immobilized egfp against denaturing agents was observed. we established a method for controlled immobilization of biomolecules based on covalent attachment of silaffin peptides with well-defined silica precipitation properties. currently this method is applied to the immobilization of biotechnological relevant enzymes in order to test their activity and the stabilization effect. herein, we present the covalent functionalization of multiwalled cnts (mwcnts) with organocatalysts based on proline or proline derivatives carrying either a dipeptide or a sulfonamide moiety. two different approaches were followed, namely, covalent grafting of the organocatalysts either at the tips or at the sidewalls of the cnts. for the former approach, mwcnts were oxidized in order to introduce carboxylic units at their tips and make them easily dispersed in aqueous solutions. then, oxidized mwcnts readily reacted with proline-based derivatives carrying a free amino unit yielding the corresponding hybrid materials. for the latter approach, the functionalization methodology based on in-situ generated aryl diazonium salts was followed. in this context, mwcnts were modified with aryl units carrying free amino terminal groups, which were subsequently conjugated with proline-based derivatives carrying a free carboxylic unit. all newly formed hybrid materials were fully characterized with complementary spectroscopic (atr-ir, raman), thermal (tga) and microscopy (tem) techniques. the catalytic evaluation of the activity of the cnt-based organocatalysts in aldol reactions is in progress. financial support from gsrt/εσπα 2007-2013 συνεργασια through 09συν-42-691-νανοκαταλυση project is acknowledged. novel organogels based on self assembly of rationally designed pseudopeptides c. pappas, n. sayyad, a.g. tzakos, i. plakatouras section of organic chemistry and biochemistry, department of chemistry, university of ioannina, ioannina, gr-45110, greece self-assembly is becoming a rather intriguing way to build an array of nano-and micro-structured materials. low molecular weight organogelators can self-assemble into various architectural types in organic solvents through weak intermolecular interactions. such organogelators have potential applications in the generation of novel materials for nanobiotechnology1. herein, we report the synthesis of rationally designed pseudopeptides and the conditions to form organogels. the obtained gels are responsive to temperature, and the sol-gel process is thermoreversible. the architecture of the constructed organogels was characterized via tem and spectroscopic techniques. diffusion ordered nmr spectroscopy (dosy) was further utilized to determine differences in the molecular shape of the different pseudopeptides. applications of the resulted compounds in nanotechnology will be reported. since 2000, organocatalysis has met such a great rate of expansion that is nowadays considered the third major branch of modern asymmetric catalysis along with the transition metal catalysis and biocatalysis. after the seminal work of list, lerner and barbas on the enantioselective aldol reaction between acetone and 4-nitrobenzaldehyde catalyzed by proline, it became clear that amino acids and peptides could serve as an abundant pool full of potential to develop novel organocatalytic motives. following our recent report that the combination of a prolinamide with a thiourea group having as a spacer a chiral diphenylethylenediamine leads to an efficient organocatalyst for the aldol reaction, 1 we recently considered the possibility to couple the prolinamide unit with an urea moiety. one of our main interests was the substitution of the diphenylethylenediamine spacer by a gem diamine derived from an α-amino acid. the gem diamine is easily synthesized via a curtius rearrengement of the corresponding acyl azide. after synthesis and evaluation of a number of potential catalysts, the prolinamide derivative bearing a gem diamine derived from (s)-phenylalanine and an aryl urea moiety proved to provide the best results in the reaction between cyclic ketones and aldehydes. utilizing 10 mol% of our organocatalyst, the aldol products were obtained in high to quantitative yields (up to 98%), high to excellent diastereoselectivities(up to >98:2) and high to excellent enantioselectivities (up to 99% ee). peptide self-assembled monolayers are of current interest to study physicochemical properties of modified metal (e.g. au) surfaces. rigid peptide scaffolds could enhance the interaction between gold surfaces and labels by reducing and precisely monitoring the distance between the supported monolayers and gold. the c α -tetrasubstituted αamino acid 4-amino-1,2-dithiolane-4-carboxylic acid (adt) 1 , which contains a cyclic disulfide system, is interesting in this respect because it may allow the parallel binding of the peptide helical chain to the metal surface. adt occurs in nature 2 and has been utilized in medicinal chemistry 3 and in a model compound of [fefe] hydrogenase. 4 we synthesised a series of constrained helical peptides, based on the ala-ala or the ala-aib sequence, containing one or two adt residues. these peptides were functionalised with spectroscopic or opto-electronic labels. among the large number of reactions involving the formation of carbon-carbon bond, the addition of ketones to nitroolefins is a powerful tool for the synthesis of γ-nitrocarbonyl compounds, useful intermediates for pharmaceutical industry. our recently reported primary amine-thioureas based on tert-butyl esters of natural amino acids exhibit excellent performance for the michael reaction of ketones with nitroolefins providing the products quantitatively and almost stereospecifically (>99% ee). 1, 2 using this methodology, enantiopure baclofen and phenibut (analogs of gaba) have been synthesized. 2 polymersupported organocatalysts constitute a great challenge for the michael reaction. in the current study, we report the immobilization of amine-thiourea catalysts containing (1s,2s)or (1r,2r)-diphenylethylenediamine and tert-butyl aspartate, on various polymer supports, either directly or through spacer units. the solid-supported catalysts evaluated in the reaction between acetone and βnitrostyrene and highlighted the importance of the choice of the polymer as well as the presence of the spacer or not. the direct attachment of the primary amine-thioureaaspartate to a crosslinked polystyrene-divinyl benzene resin containing a uniform distribution of aminomethyl groups provides a supported catalyst that affords the product of the reaction between acetone and β-nitrostyrene quantitatively and in high enantioselectivity (91% ee a. theodorou, g.n. papadopoulos, c.g. kokotos* laboratory of organic chemistry, department of chemistry, university of athens, athens, greece after the pioneering report that proline can catalyze efficiently the intermolecular aldol reaction between acetone and a variety of aromatic aldehydes, 1 it became evident that amino acids and peptides 2 can afford a plethora of different structural scaffolds for novel catalysts. along the first decade of its life, organocatalysis has grown to such an extend that now it is considered the third major branch of asymmetric catalysis. recently, researchers have paid special attention to other amino acids rather than proline. some primary amino acids have already been applied to a number of transformations with success. 3 usually improved catalytic properties are observed when derivatives of primary amino acids are utilised. we have undertaken a study on the application of simple and cheap primary amino acids and amino acid derivatives, either commercially available or easily obtained, as organocatalysts for the asymmetric α-amination of aldehydes. in the present work, we report that the use of simple derivatives of primary amino acids like phenylalanine and aspartic acid can efficiently catalyze this transformation leading to products in high to quantitative yields and enantioselectivities up to 94% ee. the majority of the organocatalysts developed up to now for asymmetric organic transformations employ more than one functionalities in the catalytic mechanism that act through either covalent or non-covalent interactions. for example, proline employs the pyrrolidine nitrogen and the carboxylic acid group, while chiral thioureas combine the thiourea functionality with a tertiary or a primary amino group. we have recently shown that an amide of proline with a diamine carrying a thiourea group is a very good catalyst for the enantioselective aldol reaction. 1 trying to improve the activity, we have found that a tripeptide-like thiourea having as building blocks (s)-proline, (1s,2s)diphenylethylenediamine and (s)-di-tert-butyl aspartate provides the products of the reaction between ketones and aromatic aldehydes in high to quantitative yields and high stereoselectivities (up to 99:1 dr and 99% ee). a number of structural modifications of the catalyst were undertaken in order to understand the role of the hydrogen bond donors of the catalyst, i.e. the prolinamide hydrogen and the two hydrogen atoms of the thiourea group. we have come to the conclusion that the importance of the hydrogen bond donors of the catalyst follows the order: thiourea hydrogen originated from aspartate › amide hydrogen › thiourea hydrogen originated from diphenylethylenediamine. g eldrug s.a., patras 26504, greece a convenient and facile synthesis and in vitro biological evaluation of n-substituted 5-butylimidazole derivatives as potent angiotensin ii (ang ii) receptor type 1 (at1) antagonists have been reported in the present study. a series of imidazole based compounds bearing the biphenyl moiety at the n-1 position, a halogen atom at the c-4 and polar substituents such as hydroxymethyl at the c-2 position were synthesized. 1,2 these compounds were evaluated for binding to human at1 receptor and for ang ii antagonism in vitro on isolated rat uterus. in particular, 5butyl-1-[[2΄-(2h-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole derivatives complexed with the at1 receptor and showed high binding affinity. these analogues were also found to be active in the rat uterotonic test. importantly, their binding affinities and potencies were comparable to those of losartan. these results indicate that the hydroxymethyl at the c-2 position of the imidazole ring is favorable for high affinity binding and antihypertensive activity and in line with the activities of the losartan counterparts. experimental findings are in good agreement with docking studies, which were undertaken in order to investigate ligand/at1 receptor interactions. z-leu-glu-his-asp-aluc, suc-leu-leu-val-tyr-aluc) are good substrates for bioluminescence assays, for example in the detection of caspase activity during apoptosis 1 . these substrates generally offer significant advantages, such as increased sensitivity, ease of use, and high throughput screening capacity. luciferase-based assays are typically 10-to 100-fold more sensitive than the comparable fluorescent assays (rhodamine 110, 7-amino-4-methylcoumarin (amc) and 7-amino-4trifluoromethylcoumarin (afc)). the synthesis of different type peptide-amino-luciferin conjugates and their precursors have been published 2 and some of them are commercially available. however, because of their high price the in vivo application of these conjugates is limited. to solve this problem we successfully worked out a new, easier and more convenient and economical method for the preparing these derivatives starting from 2-chloro-benzothiazole. moreover this products have excellent purity (>99%) and adequate yield (82-93%). major health problems arising from bacterial resistance towards existing antibiotics make discovery of antibacterial drugs with new mechanisms of action pertinent. although proof of concept for a novel antimicrobial approach using peptide nucleic acid (pna) antisense targeting of essential bacterial genes was obtained a decade ago, this technology is still limited by the lack of carriers that facilitate effective bacterial delivery and confer optimal pharmacokinetic properties to the prospective drugs. [1, 2] in the past two decades, parallel efforts of exploiting naturally occurring antimicrobial peptides (amps) as drugs have been made. the cationic amp subclass appears to be directly involved in the innate immune response towards microbial infections. [3] so far only few cell-penetrating peptides, with activity on mammalian cells, and other membrane-active peptides, have been investigated as potential vehicles for bacterial delivery. for instance, cationic amps with an internal target appear not to have been investigated for bacterial delivery of antibiotics. the aim of this project is to develop highly potent genetic antibiotics by exploiting naturally occurring antimicrobial peptides as potential delivery vehicles for antisense peptide nucleic acid oligomers. the amps are chosen from amps reported to act via intracellular targets, and thus must possess an inherent ability to permeate bacterial cell membranes without direct killing of the bacteria. faculty of chemistry university of gdansk, gdansk, poland azt (3'-azido-2'3'-dideoksythymidine), a modified nucleoside used in antiretroviral therapy and peptide plant hormone -systemin were used as substrates of 1,3-dipolar cycloaddition (click chemistry). systemin is 18-aa peptide defense hormone released in response to plant (tomato, tobacco) damage or pathogen attack. we examinated whether systemin's fast movement through plant tissues could be used for cargo (azt) transport. the huisgen cycloaddition also known as 1,3-dipolar cycloaddition is a chemical reaction belonging to the larger class of cycloadditions. reaction between organic azide and alkyne appended substrates allows the synthesis of the desired conjugate in high purity and yields irrespective of the sequences and functional groups on either of the two substrates [1, 2] . conjugate of azt-systemin has been synthesized by click chemistry, using systemin modified at n-terminus with propiolic group and azt. the conjugation was catalyzed by cu(i). the reaction was fast, efficient and regioselective. its progress was easily monitored by capillary electrophoresis (ce). ce was also applied for characterization of systemin and azt-systemin stability and movement throughout tomato leaf and stem. despite the fact that systemin moves rapidly through tomato tissues, our calorimetric (itc) studies showed that the peptide does not interact with liposomes-cell membrane model. universitätsklinik für nuklearmedizin, inselspital, bern, switzerland regulatory peptides (e.g., somatostatin, bombesin) have been shown to be suitable vectors for the specific delivery of radioactivity to tumors for diagnostic and therapeutic applications in nuclear oncology. 1 a potential drawback of such vectors is their inherent instability in vivo. thus, new strategies are needed for the stabilisation of radiopeptides in order to improve their bioavailability and, consequently, increase their accumulation in the targeted tissue. it has been suggested that 1,2,3-triazoles, readily obtained by cuaac, are suitable amide bond surrogates which are resistant to proteases. 2 in the present study, we report the synthesis and pharmacological evaluation of radiolabelled, triazole-containing analogues of the gastrin releasing peptide receptor (grpr) targeting peptide bombesin (bbn). to study the effect of backbone modifications in the minimal grp-binding sequence, we synthesized a series of analogues of [nle 14 ]bbn (7) (8) (9) (10) (11) (12) (13) (14) , in which each amide bond is individually replaced by a 1,4-disubstituted 1,2,3triazole. after radiolabelling of the peptidomimetics, their binding affinity and internalization kinetics were determined using pc-3 cells. metabolic stability was evaluated in blood serum. a number of the novel tumor-targeting peptide analogues presented exhibit both a retained high affinity (nm) towards the grpr and an improved serum stability. first preclinical data on the in vivo evaluation of the most promising candidate will be presented. to the best of our knowledge, this is the first report of the systematic replacement of amide bonds with 1,2,3triazoles within the binding sequence of linear, high affinity peptides. the methodology can be applied to a variety of peptide vectors and thus, holds great potential for the development of novel, stabilized peptide-based radiopharmaceuticals. dna is the molecular target for many of the drugs that are used in cancer therapeutics, and is viewed as a nonspecific target of cytotoxic agents. although this is true for chemotherapeutics, other agents that were discovered more recently have shown enhanced specificity. 1 the development of new site-specific dna binders, which are associated with the recognition of the dna major groove, are based on the design of transcription factor mimics that bind the dna as a dimer 2 , and prevent specific genes from being transcribed. these could ultimately result in interesting biomedical applications as designed genome interfering agents or diagnostics. 3 in order to approach this biological constructs, we choose the bzip leucine zipper transcription factor as a model to mimic. as the entire structure cannot be synthesized without expensive, complicated and time-consuming biotechnological methods, the substitution of the dimerization domain by a less complex scaffold is the first step in the design. thus, we consider a steroid based scaffold as a candidate. the specific choice of the steroid scaffold as substituent is inspired by its known ability to enhance proteolytic stability of attached peptides, by its conformational properties ensuring correct positioning of the two appended chains and by its potential to increase bioavailability. this transcription factor binds specific dna sequences by dimerization and inserting short α-helices into the dna major groove. in order to attach the peptides to the scaffold, different strategies were studied. firstly, applying the well-known click chemistry, functionalizing the scaffold with an alkyne moiety, the peptide with an azide and viceversa. secondly, via the unknown resin to resin transfer reaction (rrtr), which has not been applied on peptide chemistry so far. this unprecedent methodology consists on the reaction of a peptide, which is attached on a safety-catch resin, with a second resin bearing a nucleophilic amino terminus resulting in amide bond formation. during the process, the peptide on solid support undergoes cleavage. an hexapeptide was synthesized on a preloaded safetycatch resin. deoxycholic acid derived scaffold with orthogonally protected amines was attached to tentagel resin that acts as acceptor resin. rrtr experiments were performed at both c12 and c3 positions of the deoxycholic acid derivative. in addition, this convergent strategy can be applied to other different peptide conjugated systems. we recently described a new kind of cyclized peptide in which the cyclization is performed between the side-chains of two diaminoacyl residues via a diversely substituted guanidine bridge. 1 we showed that the degree of bridge substitution could impact on the orientation of the bridge inside the cycle and therefore the peptide conformation. we prepared two series (22 and 15 atoms cycle size) of cyclic enkephalin analogues to assess the potential effect of this kind of bridge on the biological activity. the compounds were synthesized on the solid support via the formation of a thiourea bridge and with the variable substituent being introduced at the last step before cleavage. it is noteworthy that the synthesis afforded at least two stable and separable conformers for each analogue of the shortest cycle series. generally, one major and one minor species were recovered. but in the case of di-substituted compounds with a cyclic moiety (pyrrolidine or piperidine substituents), three significant species were obtained. analogues were submitted to various biological assays (binding to μ and δ opioid receptors and functional assays). we observed a significant variation in affinity and selectivity for the receptors as a function of the degree of bridge substitution. a structural analysis by 2d nmr has been undertaken and correlated the variation in activity with a variation in conformation. the origin of the multiple conformers observed for the analogue with a pyrrolidine susbtituent was also investigated. this kind of cyclization could represent a useful tool to easily modulate the conformation and biological activity of a unique peptide sequence. the t-cell response is triggered by the formation of the trimolecular complex between the major histocompatibility complex (mhc), the immunodominant myelin protein epitopes and the t cell receptor (tcr). herein, we report the design and synthesis of non-peptide analogues with the ability to mimic the immunodominant epitope 83-99 of mbp 1,2 . the mimetics were designed to block the formation of the trimolecular complex and therefore the t-cell activation 3, 4 . more specifically, indole analogues were synthesized with substitution at positions 2 and 4 or 6. these molecules contain a carboxyl or an ethyl ester group in position 2 and a benzylamino or phenylamino group in position 4 or 6. the synthesis of the indole ring was achieved by fischer reaction followed by catalytic hydrogenation, reductive amination or arylation and ester hydrolysis. the synthesized molecules were purified using liquid chromatography, and they were identified by mass spectrometry and 1h-nmr. laboratory of peptide science, nagahama institute of bio-science and technology, nagahama, japan amyloid β peptide (aβ), the main component of senile plaques in the brain of alzheimer's disease (ad) patients, is formed by proteolysis of amyloid precursor protein (app). as β-secretase (bace1: β-site app cleaving enzyme 1) triggers aβ formation by cleavage at the aβ domain nterminus, it is a molecular target for ad therapeutic intervention. 1 previously, we reported potent pentapeptidic 2 and non-peptidic 3 bace1 inhibitors containing a substrate transition-state mimic. although these inhibitors exhibited potent inhibitory activities, their molecular-sizes appeared a little too big (mw>600) for developing practical drugs. in this study, we designed a series of small molecular peptides, with bace1 inhibitory activity, lacking the p1-p1' region on the basis of the conformational structure bound in bace1. 4 design and synthesis of new 3'-peptidyl-trna analogues, in particular "hydrolysable" analogues, which represent covalent conjugates of peptide-nucleic acid (pna) with "stop-peptides," were carried out. such compounds are of interest as tools to study the ribosome functioning and as inhibitors of protein biosynthesis. (2 aminoethyl)glycine pna models 3'-end trna sequence cca in designed structures. computer simulations showed the formation of watson-crick pairing of the pna cytosine residues with 23s rrna nucleotides g2251 and g2252 involved in interactions with peptidyl-trna during its specific binding in p site of the ribosomal peptidyl transferase center (ptc). short "stop-peptides" were planned for conjugation with pna. these peptides form stable complexes with the ribosomal tunnel (rt) that leads to ribosome stalling and translational arrest. structures of "hydrolysable" 3'peptidyl-trna analogues that could form peptide bond with amino acid residue of aminoacyl-trna in a-site of ptc included 2'-deoxyriboadenosine instead of the pna adenine containing residue. such conjugates would permit to identify the chemical nature of specific sites localized in rt and responsible for interactions with amino acid residues of the nascent polypeptide chain. pna and "stop-peptide" as well as pna-"stop-peptide" conjugates were prepared by solid phase synthesis on sasrin polymer using fmoc/bhoc(boc) strategy. synthesis of "hydrolysable" conjugates included modification of the 3'hydroxyl of 5'-protected 2'-deoxyadenosine by n-blocked "stop-peptide", deprotection of the 5'-hydroxyl, its conjugation with n-protected pna and removal of protecting groups from the resulted conjugate. the binding of the new 3'-peptidyl-trna analogues with ribosome will be tested by chemical probing and in the cell free translation system. this study was supported by the russian foundation for basic researches (grant 10-04-01187-a). a close structural similarity of endomorphin-2 and another atypical opioid peptide, morphiceptin, which both have a phe residue in the third position, encouraged us to study antinociceptive activity of these two peptides and their analogues. in order to improve the affinity and chemical stability of these opioid peptides, we have designed, synthesized, and analyzed 9 novel analogues. the first modification included endomorphin-2 and morphiceptin analogues, where halogenated phenylalanines in position 3 or 4 were incorporated as surrogates of the native phenylalanine. another important modifying element is non-protein amino acid canavanine (cav) and its analogue (sarg). it is well documented that cav and sarg exhibit strong analgesic activity. two new morphiceptin analogues were synthesized by introducing cav and sarg in position 3. we further characterized their antinociceptive activities by the paw pressure (pp) test. the experiments were carried out on male wistar rats (180-200 g), treated with i.p. doses of 1 mg/kg. e eldrug s.a., patras 26504, greece the renin angiotensin system (ras) has been a prime target for the therapy of cardiovascular diseases. angiotensin ii type 1 (at1) receptor mediates vast majority of biologically detrimental actions. non-peptide at1 receptor blockers are presently the most specific means to block the ras enzymatic cascade. the dupont group was the first to develop losartan (dup 753), an orally effective angiotensin ii receptor blocker, which is metabolized in vivo to the more potent antagonist exp 3174. herein, we report on the preparation of e-urocanic1 acid based analogs, focusing our attention on the introduction and structural modifications of the substituents on the imidazole ring as well as the modifications on the acrylic side chain. in particular, we have designed and synthesized a series of urocanic acid analogs bearing the biphenylmethyl tetrazole moiety at the n-1 of the imidazole ring.2 additionally, the rigid acrylic chain was lengthened by esterification resulting in the ethyl ester and on the other hand the latter was readily converted to the corresponding acrylic alcohol or aldehyde which may proved to be effective structural elements for enhancing biological activity. finally, a lipophilic alkyl chain such as the n-butyl group was introduced at the 2-position of the ring which may possibly enhance the antihypertensive activity. docking studies and biological evaluation of the synthesized analogs are being undertaken. university of athens, department of chemistry, laboratory of organic chemistry, 15701, panepistimiopolis zografou, athens, greece the backbone modification of bioactive peptides with replacement of a scissile peptide bond in enzymatic hydrolysis is a well-established strategy for developing protease inhibitors. 1 in particular, for zinc metalloproteases, which contain a zinc atom in their active site, several successful modifications have been reported over the past years. 2 phosphinic pseudopeptides are among the best candidates when addressing the challenge to potent and selectively inhibit zinc proteases. a thorough search in the literature3 revealed the absence of any reference regarding thiophosphinic pseudopeptides. we thought that this class of compounds would add a valuable tool in the field of zincbinding groups. in the present study, we describe in detail the first synthesis of a new class of phosphorous compounds, thiophosphinyl dipeptide isosters (tdis). we prepared several fully protected thiophosphinate pseudodipeptides of the general formula pg-phe-ψ[p(s)(ox)ch2]-gly-pg' starting from the corresponding phosphinate pseudodipeptide using lawesson's reagent. selective deprotection of these compounds was also studied and the results are disclosed. these compounds can be used as building blocks for the synthesis of longer thiophosphinic pseudopeptides after suitable deprotection and elongation as well as transition transition state-mimicking inhibitors for several zinc metalloproteases. in the last decade, trypsin inhibitor sfti-1 isolated from sunflower seeds [1] has become one of the most studied peptidic inhibitors of serine proteases. owing to its small size and a strong trypsin inhibitory activity (ka = 1.1×10 10 m -1 ), sfti-1 is considered to be a very attractive template for designing proteinase inhibitors with the potential use as pharmacological agents [2] . it could also serve as an affinity probe for the isolation of trypsin like (sfti-1) or chymotrypsin like ([phe5]sfti-1) proteinases. following this idea, we decided to synthesize a set of cell-permeable monocyclic sfti-1 analogues with a fluorophore moiety attached at their n-termini. the presence of the fluorophore in the molecule enabled us to show that the analogues can cross the cell membrane. the cell penetration assay was performed using multiple cell lines (hela cells and human fibroblasts cell line (46br.1n) was obtained from european collection of cell cultures (ecacc)). for all the obtained peptidomimetics, we determined the association constants with cognate proteinases. selected peptides were also used as a probes for the detection of inhibitor -proteinase complex, which was achieved by the means of gel filtration chromatography equipped with fluorescence detector and acrylamide native gel electrophoresis. the functional reconstruction of folded protein surfaces with peptide-based mimics is an enormous scientific challenge. the majority of proteins show activity through a small area of their folded surface: "the binding site". however, linear peptides are too flexible and seldomly adopt the correct 3d-structure of the binding site spontaneously. therefore, they show limited or no activity at all 1 . crucial for activity is to control the secondary (αhelix, β-sheet and/or β-turn) and tertiary structure (relative orientation of subdomain structures). we present the development of a new type of watersoluble scaffolds that have the potential to control both secondary and tertiary structure of discontinuous (i.e. double-loop) protein mimics. the new scaffolds contain a first pair of reactive functionalities to constrain the linear peptide conformation via a 'clips' reaction2, stabilizing the secondary structure. next to this, a second functionality allows for ligation of two dissimilar constrained peptides to form a discontinuous binding site mimic via oxime-ligation or click-reaction. these ligations offer the ability to position different peptide loops in 3d, thus mimicking the tertiary structure of the native protein. most unique to our approach is the fact that all chemical conversions are performed in aqueous media, using side-chain unprotected peptides 3 . growth hormone-releasing peptide 6 (ghrp-6) is a synthetic hexapeptide (his-d-trp-ala-trp-d-phe-lys-nh2), which interacts with two kinds of receptors: growth hormone secretagogue receptor 1a (ghs-r1a) and cluster of differentiation 36 (cd36). the latter is a membrane glycoprotein member of the class b scavenger family, and decreases the internalization of oxidized lipids into macrophages, as well as causes inhibitory effects on angiogenesis associated with binding to thrombospondin. to increase activity and selectivity for the cd36 receptor, different analogues of ghrp-6 were synthesized. in particular, substitution of trp 4 in ghrp-6 by aza-amino acids has given selective analogs, due likely to induction of a β-turn secondary structure. for aza-peptide synthesis, a submonomer solid-phase approach has proven effective to introduce side chains onto the semicarbazide residue. studying influences of benzylidene, benzhydrylidene and fluorenylidene residues during the alkylation of the semicarbazide, superior conversion was observed with fluorenone derivative, and mild alkylation conditions employing et4noh as base have improved yields and minimized racemisation. our presentation will focus on the improved submonomer synthesis method for optimization of selective and potent cd-36 ligands with antiatherosclorotic and anti-angiogenic effects. for instance, the integrin αvβ3, vitronectin receptor, is expressed in a number of cell types and has been shown to mediate adhesion of osteoclasts to bone matrix, vascular smooth muscle cell migration, and angiogenesis. integrin αvβ3 also play a significant role in tumor growth, invasion and metastasis, and is a receptor for the extracellular matrix proteins with the exposed arginine-glycine-aspartic (rgd) tripeptide sequence. rgd has been shown to be potent antagonist of the integrin αvβ3, and has excellent anti-angiogenic properties including its suppression of tumor growth in animal models. in this context, drug design based on the rgd structure may provide new treatments for diseases such as thrombosis, osteoporosis, and cancer. we designed and synthesized series of short rgdmimetics containing the sequence xaa-gd, where xaa is arg-mimetic. as promising candidates we have chosen canavanine (cav) and canaline (can) instead of the basic residue arg. in order to improve antitumor activity of the parent molecule, c-terminal modifications were also applied. their cellular uptake was determined on human breast (mcf7) cancer cell lines. furthermore, the in vitro cytostatic effect was evaluated by mtt assay on human liver hepatocellular carcinoma (hepg2) and human breast (mcf7) cancer cell lines after 24, 48 and 72 hours of treatment. in the case with the human tumor cell lines (hepg2, mcf7) and c-modified analogues, statistically reliable results were achieved for the most of concentrations used. acknowledgements: this work was supported by bulgarian ministry of education and science, project my-fs-13/07. microwave assisted solid phase synthesis of urea and urea/amide based foldamers k. pulka, c. douat-casassus, g. guichard* european institute of chemistry and biology, university of bordeaux 1 -cnrs umr 5248, pessac, france foldamers 1 are fully arti cial molecules that structurally and functionally mimic variety of biopolymers. among them, aliphatic n,n'-linked oligoureas with proteinaceous side chains can adopt extremely robust helical folds stabilized by intramolecular three-centred h-bonds. 2 owing to their resistance to enzymatic degradation, diversity of side chains and structural predictability urea-based foldamers represent unique scaffolds to elaborate functional mimetics of α-polypeptides. of note, heterogenous oligo(urea/γamides) backbones obtained by substituting nh groups by ch2 display very similar folding propensities. in our laboratory we are investigating the solid phase synthesis of urea and urea/γ-amide oligomers. urea bonds are incorporated into the growing chain by reaction of active succinimidyl carbamates. previously we have applied two different strategies involving fmoc-or bocchemistry, but both methodologies suffer some limitations. 3 therefore a new strategy (compatible with the use of tfa sensitive linkers and side chain protecting groups) featuring azide as a masked amine group has been developed. the synthesis of 15 new azido protected succinimidyl carbamate building blocks is reported. they were obtained in 6 steps from α-amino acids (12-45% overall yield). the staudinger reduction with pme3 was successfully applied to restore the amine group after urea formation on solid support. in addition, microwave irradiation has been found to dramatically accelerate the synthesis. overall, this azide-strategy combined with microwave irradiation was found to be very effective for the solid phase synthesis of oligoureas and related hybrids, surpassing previously developed approach utilizing fmoc chemistry. these antibiotics should have a mechanism different from currently used antibiotics to circumvent existing resistance mechanisms 1 . previous results have shown that "genetic" antibiotics operating by gene silencing in bacteria via rna interference may be successful new candidates. efficient silencing requires efficient crossing of cell membrane. this step can be alleviated using cell penetrating peptides (cpp) as carrier of drug candidates, such as peptide nucleic acids (pnas) which inherently have poor internalization properties 2 . the aim of this study is to elucidate mechanisms of uptake in bacteria using pna-cpp conjugates, which previously have shown promising antibacterial effects 2 . the fate of the pna and cpp parts of the conjugates, once inside the cell, is investigated regarding localization and possible degradation within the cell. furthermore, a method for toxicity testing of pna-cpps is being developed using histamine release in rbl-2h3 cells as a quantitative measure of allergenicity of pna-cpps. the prospect of this information is to define boundaries within which cpps can be found, thereby rationally designing novel efficient antibacterial biomolecular drug delivery systems. oxytocin and its fragments have the potential to influence behavioral and cognitive functions, including their disturbances in some brain disorders. therefore, there is an interest to synthesize new peptide-steroids chimeras for potential therapeutic use. oxytocin analogue was synthesized in solution by coupling azido-phenylalanyl residue or p-azidopegylated handle to the n-terminal end of oxytocin molecule. its c-terminal fragment pro-leu-gly-nh2 (mif-1) was elongated at proline residue by the same type of azido handles as well. both peptides were marked for fluorescent detection of their possible binding on brain slices. peptide chimeras with the suitable steroids were prepared via azide click to the triple bond on the modified steroid counterpart like (17α)-17-hydroxypregn-4-en-20-yn-3-one, 24-norchol-5-en-22-yn-3β-ol. steroidyl-peptides were then used in the trials using rat-brain slices. the sites of the peptide-steroids chimeras bound to the brain tissue were identified with the aid of fluorescent microscopy. the suitable chimeras will be tested for their penetration through blood brain barrier for the pharmacological effects. indicating that the orientation of the n-butyl group is of primary importance. docking studies revealed that the highly active analog affords an additional hydrophobic binding feature compared to losartan which fits to an extra hydrophobic cavity. these results may contribute to the discovery of new biologically active molecules by a convenient and cost effective synthetic strategy. the context of pain research, the co-administration of opioid agonists and nk1 antagonists previously led to an enhanced antinociceptive potency, 1 and recently largent-milnes and co-workers have shown that a hybrid opioid-nk1 octapeptide was able to attenuate tolerance development, related to sustained opioid treatment. 2 our group has prepared a compact opioid agonist-nk1 antagonist peptidomimetic chimera dmt-d-arg-aba-gly-nme-3',5'-bn(cf3)2 1 that served as a lead structure. 3 we report a solid phase method for the synthesis of the 4amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (aba) structure, which is used as a central unit in the investigated dual ligands. this method allowed the rapid assembly of new bifunctional ligands containing the aba structure. variations of the d-arg 2 , gly 4 and n-benzyl substituents were made. the introduction of d-cit 2 , a gly 4 → β-ala4 substitution and the removal of the trifluoromethyl substituents in 1 caused considerable shifts in receptor binding. the obtained structure-activity relationships will be presented. hence, a promising approach for the treatment of dmd is the use of drugs to force ptc readthrough. (+)-negamycin 1 is a dipeptidic antibiotic containing a hydrazide structure. although (+)-1 was not clinically developed due to some toxicity, it was recently reported that (+)-1 restore dystrophin expression in the muscles of mdx mice, an animal model of dmd. 1 therefore, (+)-1 is a promising therapeutic candidate for diseases caused by nonsense mutations. based on our own efficient total synthetic method of (+)-1, 2 structure-activity relationship (sar) study was perfromed to discover derivatives with a potent readthrough-promoting activity. we found a derivative, (5r)-5-hydroxy-6-aminohexanoyl-glycine exhibited not antimicrobial activity but a similar readthrough activity to (+)-1, suggesting that the ptc readthrough mechanism can be distinguished from the antimicrobial mechanism. 3 moreover, we synthesized 5-epi-negamycin and found that this analog exhibited a similar activity to (+)-1 in in vitro readthrough assay. this result hence prompted us to synthesize a 5-dehydro-derivative, e.g., 5-dehydro-3-epinegamycin 2, which is a natural product with little antimicrobial activity. surprisingly, we found that 2 showed a higher in vitro readthrough-promoting activity than (+)-1. this result suggests that mother nature independently evolved readthrough-promoting products like suppressor trna, in distinction from aminoglycosides, which show both antimicrobial and readthrough-promoting activities. agricultural university of athens, athens, greece high interest has been paid to synthetic structural motifs that promote specific conformations because of their importance for the development of new therapeutic peptidomimetics. 1 in addition, such motifs may show catalytic activity for asymmetric organic transformations. during the last two decades, various synthetic structural motifs that promote reverse turns have been studied. following our interest on chiral prolinamide-thioureas that present interesting organocatalytic activity, 2 we have undertaken a combined experimental/computational study to understand the structural features that may stabilize a reverse turn in short-length peptidomimetics containing a thiourea functionality. compounds with the sequence r-pro-diphenylethylenediamine-thiourea-asp(obut)-obut (r: boc or fmoc, or boc-ala), were synthesized and studied by nmr spectroscopy (tocsy, 1h-13 c hsqc, noesy, roesy spectra) for the sequential assignment and the exploration of the dipolar connectivities. sampling of the conformational space was driven by the noe intensities while molecular dynamics simulations were further applied to the consistent with the experimental data conformers in order to monitor the stability of the formed hydrogen bonding interactions in the course of time. energy refined produced conformers were subsequently modified by applying all combinations of d-and l-amino acids at each site in a stepwise manner. the modelled structures were studied in silico aiming to explore the combinations of heterochiral residues which would promote a folded structure and would favour the potential of β and γ turn motif. the most promising combinations were chosen for synthesis and subsequent nmr characterization. 3 in this research project we will deal with chemical strategies to produce suitable surface modifications in order to induce multidirectional cellular migration along gold surfaces. to achieve this objective we want to use and characterize self-assembled monolayers (sams) of thiolated dna chains (dna-sh) adsorbed on gold surfaces through the hybridization with complementary modified single-stranded pnas. pna is a structural dna mimic obtained by polymerization of n-(2aminoethyl) glycine monomers that replace the ribose-phosphate backbone characteristic of natural nucleic acids. it is an achiral, uncharged, and relatively rigid biopolymer of high biological and chemical stability, 4 and it can bind complementary dna strands with higher affinity than the corresponding dna sequences.for all these reasons we have chosen pna as a key molecule to promote and assist the movement of cells. by producing a chemical gradient of dna-sh along a gold surface in the presence of a chemotactic molecule it will be possible to obtain and control a directed cellular migration. the norwegian structural biology centre and the centre for theoretical and computational chemistry, department of chemistry, university of tromso, 9037 troms , norway renin is a highly selective aspartic protease which catalyzes the hydrolysis angiotensinogen, a protein secreted from the liver, to the decapeptide angiotensin-i. 1 angiotensin-i is further processed by the relatively nonspecific angiotensin converting enzyme (ace) to give the octapeptide angiotensin-ii, a potent vasoconstrictor and the dominant peptide produced by the reninangiotensin system. renin catalyses the rate determining step in the formation of angiotensin-ii, and has for several decades been an established therapeutic target for drug development in relation to hypertension. 1 in the search for renin inhibitors, substituted piperidine derivatives have been identified as promising, 2-4 and piperidines have proven to be efficient scaffolds for the development of novel non-peptide aspartic protease inhibitors, particularly towards renin. [5] [6] [7] we herein describe a series of 4-triazolyl substituted piperidine derivatives that have been synthesized from n-boc protected trans-4-ethynyl-3-hydroxy piperidine and tested as novel renin inhibitors. piperidine derivatives containing a 1-substituted 1,2,3-triazol-5-yl substituent were found to be most active and molecular docking experiments provides a rank order that is in very good agreement with experimental data. the cxcr4/sdf-1 axis is involved in many biological processes such as hematopoiesis, immune cell migration, as well as in cancer metastasis. cxcr4 also mediates the infection of t-cells with x4-tropic hiv functioning as a coreceptor for the viral envelope protein gp120. cxcr4, as a pharmaceutical target, is of utmost importance but the lack of synthetic agonists has seriously slowed down drug development. it has been recently described by our research group 1 , that grafting the sdf-1 n-terminus onto a side-chain of the inverse agonist t140 2 . generated high affinity synthetic agonists as well as partial agonists for the chemokine receptor cxcr4. to remain stable towards proteases and act as useful pharmaceutical tools, the pk-adme properties need to be improved with a gradual transition to peptidomimetic structures. medicinal chemistry witnessed major advances with the discovery of small synthetic molecules that mimic the natural peptidic substrates. these small molecules do not undergo proteolytic degradation, an advantage they hold over natural counterparts. in order to improve stability against proteases, part of the sdf-1 chain was replaced with variable lengths of polyethylene glycol and unnatural amino acids at differents positions. here, we have produced a series of compounds, most of which showing nanomolar affinities for cxcr4 and some are displaying partial agonistic properties. tlrs are the innate immunity receptors that recognize the epitopes found on surfaces of various cells and therefore they initiate and sustain the atherogenic inflammatory response [1, 2] . we assume that the use of small stat1 mrna−binding pna−inhibitors to manipulate the activity and expression of stat1 could prove an attractive therapeutic strategy in treatment of atherosclerosis. to that end we synthesized a specific stat1 mrna−binding pna inhibitor as well as a non-specific pna to compare their inhibition of gene expression. in our work we developed effective method of synthesis of pna−peptides conjugates by means of "click chemistry". determination of optimal conditions for conjugation (connection of pna with the peptide) will allow for the design of compounds useful in gene therapy. the specificity of pna hybridization to complementary dna fragment was verified by capillary electrophoresis (ce). as an artificially synthesized somatostatin analogue, tyr 3octreotate (toca) can specifically bind to somatostatin receptor (sstr), which are usually over-expressed on many tumor cells. carbohydration of n-terminus of toca has resulted in improved pharmacokinetics and tumor targeting (1) . 18 f is an ideal nuclide for positron emission tomography (pet) imaging; there may be significant uses of 18 f labeled glucitol-toca and its analogues as tumor probes for the diagnosis of sstr-positive tumors. in order to explore a novel pet probe for diagnosis of sstrpositive tumors, we designed a synthetic route to synthesize n-gluc-lys(nota)-toca, which uses 1,4,7-triazacyclononane-1,4,7-triacetic acid (nota) as the chelating reagent. n-gluc-lys([al 18 f]nota)-toca is radiosynthesized quickly and efficiently using the chelation reaction of al 18 f complex and n-gluc-lys(nota)-toca. the aim of this study is to develop an efficient method for the synthesis of monomers of triazolic nucleic acid (tna), a new class of artificial nucleic acids. but-2-yne-1,4-diol and nucleobases derivatives will be substrates of the monomers synthesis. tna oligomers could be used as specific inhibitor of tar rna hiv-1, the regulatory rna structure crucial for hiv replication. "click chemistry" based on 1,3-dipolar cycloaddition will be used to conjugate an alkyne and azide derivatives of monomers subunits. a ru (ii) complex will be used as a catalyst of internal alkyne (but-2-yn-based) cycloaddition. the reaction gives exclusively of 1,4,5-trisubstituted derivative of triazole ring 1 . the monomers will be characterized using rp hplc, capillary electrophoresis (ce) and 1 h and 13 c nmr. the resulting monomers containing fmoc-protected amino group and a free carboxyl group will be used for the classical spps method to synthesize tna oligomers. tna sequences will be designed against tar's bulge and an external loop. 1 through the recognition that the repertoire of polypeptide conformations can be greatly expanded by the creation of structures incorporating β-amino acids. 2 moreover, the numerous advantages of hybrid (mixed α-and β-) backbone peptidomimetics with respect to homogeneous ones were quite recently outlined. 3 we describe here various β-amino acid-based β-hphe-β-hphe dipeptide derivatives, also conformationally constrained, and their application to the synthesis and biological evaluation of hybrid analogues of the opioid endogenous peptide endomorphin-2 (em-2). the opioid system mediates a wide variety of pharmacological and physiological processes, including pain perception and modulation. the amidated tetrapeptide em-2 has been shown to be μ-opioid receptor (mor) agonist exhibiting a very high μ-receptor affinity and selectivity, and it is an important model in the search towards new analgesics. 4 structural investigation of em-2 reveals the high conformational freedom of the phe side chains and also the inherent flexibility of the peptide backbone, indicating many probable bioactive conformations, ranging from βturns to extended conformations. with the aim of better clarify the relevant role of the proper spatial orientation of the aromatic rings and in particular of the benzyl side chains at position 3 and 4, 1 h nmr studies, molecular modelling, and molecular docking to a homology mor model of our hybrid analogues are currently under way. the lantibiotics represent a class of antimicrobial peptides, in which the unusual amino acids dehydroalanine and dehydrobutyrine and the intramolecular thioether bridges (lanthionines) are important structural features for bioactivity.the lipid ii -nisin complex is responsible for pore-formation since the c-terminal part of nisin is inserted into the bacterial cell membrane which ultimately results in cell leakage and collapse of vital ion gradients. in order to increase the metabolic stability of nisin, the oxidationsensitive thioether bridges can be replaced by metabolically stable dicarba moieties, as successfully demonstrated by the synthesis of nisin ab(c) analogs containing alkane/alkene bridges [1] . to obtain more insight into the importance of the cross-bridged de-ring structure (i→i+3, i+2→i+5 connectivity) on nisin's bioactivity, we synthesized a series of all four diastereomers of the crossed alkene-bridged de-ring mimic, using ring-closing metathesis. all four diastereoisomers were obtained by hplc and structurally characterized by nmr spectroscopy. an orthogonal protection scheme was used, to enable the independent n-or c-terminal modification of the bicyclic hexapeptides with azide/alkyne functionalities. via cu(i)-catalyzed cycloaddition chemistries, alkyne-functionalized natural abc-fragments of nisin, which were obtained by tryptic digestion of full length nisin followed by hplc purification, have been conjugated to synthetic de-ring mimics to obtain novel nisin derivatives and their affinity toward lipid ii and pore-forming capacity have been studied. herein, we report on the details of the synthesis and characterization of the geometric isomers of the synthetic de-ring mimics, and their use as synthons in cu(i)-catalyzed click chemistry to obtain newly designed nisin hybrids as potential novel peptide antibiotics. università di ferrara, dipartimento di biochimica e biologia molecolare, ferrara, italy mirnas play an important role in regulation of gene expression, being involved in numerous processes such as cell proliferation, cell differentiation, apoptosis and also in the progress of diseases as cancer and cardiovascular disorders. mirnas associated to diseases recently become targets for the development of new drugs based on antisense oligonucleotides or analogues complementary to the chosen mirna, in order inhibit the binding of the mirna to its mrna target. therapeutic silencing of mirna has been also observed in several animal disease model. 1 in this work we propose a new approach to interfere in the mirna function, based on peptide nucleic acid (pna) oligomers designed to be complementary to selected regions of the mirna precursor (pre-mirna). as the pre-mirna bases belonging to the stem are not perfectly complementary, we hypothesized that the mismatched duplex of the pre-mirna could be opened by pnas inhibiting of its maturation into mirna. two pna sequences, targeting respectively the "sense region" and the " 5' end region" of the pre-mir210 were designed. pnas were conjugated to different carrier peptides, hiv-tat, r8, k4 and two nuclear localization signal (nls and binls), in order to increase their cellular uptake. to verify the ability of the designed pnas to give strand invasion on the pre-mirna, we conjugated also pnas to the thiazole orange, a probe which lights-up upon hybridization 2 the development of privileged molecular scaffolds efficiently mimicking reverse turn motifs has attracted remarkable interest when structural constraints are exploited to increase both binding and selectivity of model peptides. one of the successful approaches to restrict peptide conformation is the disubstitution in the α position of an α-amino acid, leading to a conformational constraint and a stereochemically stable quaternary carbon center. in particular, spirocyclic scaffolds are able to provide, upon the attachment of appropriate functional groups, useful high-affinity ligands, relevant to the field of drug discovery. 1 at present, we are interested to spirocyclic tryptophan (trp) analogues, in order to develop new reverse turn nucleating moieties able to be inserted into pharmacologically relevant peptidomimetic compounds. among peptides sharing a tryptophan-containing β-turn motif of which the trp residue is critical for binding, we looked at the hormone peptide somatostatin, 2 acting in various organ systems as a neuromodulator and a neurotransmitter, as well as a potent inhibitor of various secretory processes and cell proliferation. 3 somatostatin and its analogue octreotide (sandostatin® drug, clinically used for the treatment of endocrine tumors and acromegaly) are thought to interact with the sst1-5 receptors mainly by inserting a β-turn substructure, carrying a lysine (lys) and a trp side chain into a pocket of the g protein-coupled somatostatin receptor. we report here the preparation and structural characterization of a new 1,2,3,4-tetrahydro-β-carboline (thbc)-based spirocyclic lactam as type-ii β-turn model compound and the application of its core structure to the synthesis of a somatostatin mimetic, whose biological evaluation is under way. the analogues of sfti-1 modified in the p 1 position by, βand γ-amino acids and n-substituted β-alanines r. lukajtis, a m. filipowicz, a a. legowska, a d. debowski, a a. lesner, a k. rolka a a faculty of chemistry, university of gdansk, 80-952 gdansk, poland serine proteinases play very important roles in many physiological processes in humans, such as: food digestion, fertilization of the ovum, blood clotting and dissolution of blood clots, immune response. however, their uncontrolled activity can evoke serious pathological conditions. therefore, serine proteinase inhibitors are considered to be a promising class of therapeutic agents. trypsin inhibitor sfti-1, on which we focused our attention in the last decade, is an attractive template for the design of such compounds. its primary structure is shown below: & 1 gly-arg-cys(& 2 )-thr-lys 5 -ser 6 -ile-pro-pro-ile-cys(& 2 )-phe-pro-asp& 1 the inherent feature of natural peptides and proteins is their low stability towards proteases, which seriously reduces their bioavailability. there is a growing need for the development of artificial biopolymers with diverse side chains, capable of mimicing peptide function. β-and γpeptides are an interesting class of peptidomimetics with significant chemical and biological properties. the present communication describes the chemical synthesis and inhibitory activity of a series of trypsin inhibitor sfti-1 monocyclic analogues (with disulfide bridge only) modified in p1 position by βand γamino acids and n-substituted β-alanine (β-peptoid units). the following mimetics of proteinogenic lys or phe were used: β 3 hlys, β 3 hphe, γ 4 hhlys, γ 4 hhphe, βhnlys, βhnphe. all compounds were synthesized manually on solid support. β-peptoid monomers were introduced into the peptide structure by two steps method [1] . newly obtained sfti-1 analogues modified in p1 position by β-derivatives of lys and phe were able to inhibit bovine β-trypsin and bovine αchymotrypsin, respectively, whereas the remaining ones (except for [βhnphe5]sfti-1) appeared to be inactive. the notion that early soluble aß intermediates are endowed with cytotoxic effects suggests that a major effort should be directed toward the inhibition of amyloid aggregation at very early stages. inhibiting aß self-oligomerization could, therefore, provide a useful approach to treating and controlling the pathogenic pathways underlying alzheimer's disease (ad). likely, agents that target the basic molecular recognition process preceding the formation of early intermediates are the most valuable candidates. we have conjugated a trehalose moiety to the known ß-sheet breakers pentapeptides lpffd. 1 trehalose has received a special interest because it has been found to be effective in the treatment of neurodegenerative diseases associated with peptide or protein aggregation. 2 the glycosidic moiety was covalently linked to different regions of the peptides' primary sequence, including the n-terminus or c-terminus or the aminoacid side chain. this new class of peptides showed an increased resistance to proteases. 1 in this work, the inherent ability of these peptides to recognize and bind the monomeric form of recently reported a d-amino acid-containing hiv protease inhibitor with a sulfonyl group showed an activity enhancement against drug resistant viruses. x-ray crystallographic study of the derivative revealed existence of four bridging water molecules. we suggest that the additional indirect interactions through water molecules induced the inhibitor's flexibility in binding conformation, keeping the affinity with the mutated proteases. oxalyamide, so-called oxamide, has two carbonyl oxygen atoms as hydrogen bonding acceptor similar to sulfonyl group, which is promising to interact with water molecules. to increase the numbers of bridging water molecules, we built-in two oxamide structures to both terminals of pseudo-symmetric compounds with hydroxymethylcarbonyl-hydrazide isostere. the derivatives were tested for inhibitory activity using wildtype hiv protease and a highly mutated protease with lopinavir resistance. we found that the loss of potency against the mutated protease was relatively small in the oxamide derivatives. the molecular dynamic simulations suggested the ability of bridging water formation of the two oxamide groups. optimization of the pna-synthesis using different bases for fmoc-deprotection s. rawer 1 , k. braun 2 , r. pipkorn 2 1 life technologies, darmstadt, germany 2 dkfz, heidelberg,germany pna (peptide nucleic acids) are considered as highly sensitive and specific tools for antisense strategies especially conjugated with cell penetrating peptides. individual designed shuttle systems can be applied in cancer diagnostics and possible therapy (1) . it is, however, undisputed that proper pnas' syntheses prove to be a challenge for coupling and fmoc-deprotection. due to the structure-formation the success of the synthesis depends strongly from parameters, like activator's quality and deproctection kinetics correlating to the length of the pna polymer spps product. using the example of the spps pna synthesis' results of the coding sequence of c-myc human exon ii, different bases, acting as fmoc-deprotection reagents, are compared and analyzed aiming at optimizing the pna synthesis strategy (2) peptidoglycans are central structural components of the cell wall of bacteria. several plant receptors are known to recognize peptidoglycan fragments. it is believed that these receptors form part of the defense mechanism against bacterial infections in several plant species. peptidoglycans consist of long chains of alternating β(1-4)linked glcnac and murnac moieties that are crosslinked by short, non-ribosomal peptides. these peptides consist of several d-amino acids and the symmetrical (r,s)diaminopimelic acid (meso-dap). in particular, the latter complicates the synthesis of peptidoglycan fragments due to the requirement for individually addressing the two pairs of functional groups. some chemical syntheses of peptidoglycan fragments have been reported [1] [2] [3] [4] , hhich involved multi-step formation of an orthogonally protected dap moiety, and elaborate oligosaccharide synthesis. here we present a new and simple approach to peptidoglycan synthesis which is based on the use of commercially available building blocks for the dap and oligosaccharide components. this allows easy access to a range of peptidoglycan fragments for structure-activity studies. the introduction of solid-phase peptide synthesis (spps) and the subsequent refinement of resins, linkers, coupling reagents and amino acid protecting groups allowed access to a wide range of peptides. therapeutic peptides, in particular, have benefitted from the maturation of spps, as complex peptides can be synthesized more efficiently in comparison to conventional solution phase synthesis. however, peptides containing multiple disulfide bonds often still remain difficult to make due to a lack of orthogonal cysteine protecting groups that can be used in routine spps. the cysteine protecting group s-tertbutyl mercapto (s-tbu) is commercial and orthogonal to other cysteine protecting groups. removal of the protecting group is facilitated by reducing agents (e.g. thiols or phosphines) and is stable to tfa and piperidine, hence compatible with fmoc/o-tbu peptide synthesis. however, the protecting group cannot be used in routine spps due to long deprotection times (4-24h) . in certain cases it has been shown to be impossible to remove due to proximity of bulky protecting groups and sensitivity to certain sequences. additionally, reports of desulfurization of s-tbu protected cysteine to dehydroalanine, by the use of prolonged exposure to reducing agents, show the limitations of this protecting group. the concept of cysteine protecting groups labile to reducing agents is promising due to orthogonality to other cysteine protecting groups and the limitations of s-tbu initiated an investigation into novel reductive cysteine protecting groups. herein, we introduce s-tmp as a novel cysteine protecting group that is very labile to reducing agents. the increased lability, in comparison to s-tbu, allows utilization of reducing agent labile protecting groups in routine peptide synthesis of disulfide containing peptides. as modern automated spps protocols allow the assembly of larger and increasingly complex peptides, a precise control of the coupling reactions is a crucial prerequisite in peptide synthesis. monitoring the progress of synthesis allows the detection of undesirable products caused by side reactions, incomplete couplings or deprotections. 1 although different methods have been developed for monitoring spps, we observed that the use of colorimetric test or continuous-flow uv absorbance of the reaction column effluent was not informative enough to identify difficult steps in the synthesis. in this study we demonstrate the usefulness of the combination of a mw-assisted mini-cleavage protocol and the uplc-esi-ms analysis for monitoring the quality of the reaction steps. as a proof of concept, based on this strategy, we monitored the synthesis of pthrp(1-34)nh 2 (synthesised by fmoc/tbu rt-spps, liberty™, cem), characterised by a cluster of arginine residues in the 19-21 region. 2 by the use of mw irradiation during the mini-cleavage protocol, we optimized time for mini-cleavages particularly in case of multi-arginine containing peptides, protected by pbf group. the results obtained by uplc-esi-ms showed that the complete removal of the pbf groups from the arginine sidechain residues required 1h at rt. on the other hand, the mw-assisted mini-cleavage monitoring let us to obtain final results just in 15 min, confirming that the use of microwave irradiation in mini-cleavages is an efficient strategy to monitor also difficult peptide couplings, such as multiarginine peptides. identification of some deletion sequences was helpful to recognise critical couplings in order to adopt more efficient coupling strategies and therefore to optimise the final yield and purity of the crude peptide. development of green sustainable chemistry is currently regarded as a challenge in science and technology to reduce the use of organic solvents and utilize less toxic solvents instead. water and aqueous-based solvent systems represent an increasingly significant choice for replacing traditional solvents in synthetic chemistry. until recently, peptide synthesis in aqueous solution has remained largely unexplored. this is because the most common building blocks are sparingly soluble in water and are considered inappropriate for in-water peptide synthesis. we have developed a method for solid-phase peptide synthesis in water, which utilizes water-insoluble fmoc-amino acids that are converted to water-dispersible nanoparticles. in this way, the solubility problem is overcome. our technology, which uses suspended nanoparticle reactants for the coupling reaction, offers many advantages in terms of reaction efficiency over inwater synthesis using water-soluble or non-disperse reactants. however, there are two main problems with this nanoparticle approach; (i) slow reaction rates compared to general peptide synthesis in ordinary organic solvents (ii) poor yields for the synthesis of long peptides because the protected peptide chains on the resin have a tendency to aggregate in water. mw assisted spps is particularly attractive because of the widespread availability of the new technology, including automated peptide synthesizers. a trial of mw assisted inwater solid-phase synthesis using non-disperse boc-amino acids has been reported by albericio previously. currently, fmoc-amino acids are routinely used as building blocks for solid-phase peptide synthesis. with this in mind, we have developed a mw irradiation procedure aimed at reducing reaction time and increasing reaction yield for in-water solidphase synthesis using water-dispersible fmoc-amino acid nanoparticles. and we demonstrated in-water solid-phase synthesis of difficult sequence peptide with mw irradiation. m. lebl, z. flegelova spyder institute praha, czech republic cotton was shown as a convenient solid phase support earlier 1-3 , but did not find wide acceptance by the peptide community. we decided to try its application as (i) a support of choice for the synthesis driven by combination of capillary forces and gravity, (ii) support for synthesis utilizing in situ neutralization boc based protocol, (iii) support for combinatorial synthesis based on easy labeling and physical separability of cotton substrate, and (iv) support for multisupport synthesis. -we have built a simple synthesizer in which the cotton carrier (functionalized thread) is placed inside the capillary tubing and the appropriate reagents are introduced by connecting the inlet with appropriate reagents. the speed of "pumping" the reagents is driven by the difference between the elevation of the inlet and outlet of the capillary tubing. -we have shown that boc solid phase synthesis utilizing in situ neutralization is compatible with cotton substrate and provides high quality products. combining with the fact that cotton by itself 4 acts as the self-association breaking agent, makes cotton a suitable carrier for synthesis of "difficult" sequences. -labeling of individual solid support particles can be easily based on the length of the cotton thread pieces, number and positions of knots, or their attachment to a secondary carrier. in addition, it is possible to synthesize peptides differing by the partial structure (alternative linkers, terminal modifications, etc.) in a mixture of classical resin with labeled cotton carriers, which are easily separable at the end of the synthesis. . use of microwave irradiation provides peptides in a fraction of time compared to conventional methods, and the peptides are also often generated in higher yield and purity. while microwave technology is particularly suited for the synthesis of "difficult" to synthesize peptides, this tool can routinely be used for the synthesis of a wide variety of peptides without the need for extensive method optimization. the focus of this study is to demonstrate how a peptide can be synthesized on a small scale (for example 25 μmol) up through larger scales (>1 mmol) with ease. as a biologically relevant model peptide the last 13 residues of the human platelet factor 4 protein (hpf4 57-10) was selected due to its significant antimicrobial activity. 1 however, the problem of developing a robust fmoc thioester method is that the deprotection of the fmoc group with base at each cycle is not compatible with an active ester at the c-terminus. many ingenious approaches have been developed to generate the required thioester peptide. 2, 3, 4 the most popular has been to use an nacylsulfonamide as a base and acid stable (safety-catch) linker for peptide synthesis. alkylation of the sulfonamide after peptide assembly makes the linker labile to cleavage with nucleophiles. 5 whilst popular, it has been plagued by notoriously low yields which originate from the incomplete acylation of the resin-bound sulfonamide with the c-terminal residue, incomplete alkylation of the sulfonamide and the incomplete thiolysis of the resin-bound protected peptide. in this poster we describe the development of a novel dual linker strategy 6 , involving anchoring of the sulfonamide linker to a standard acid-labile resin. this variation overcomes many of the current limitations of the sulfamylbutyryl linker approach and provides a simpler and scalable method for peptide ligation via fmoc spps. m. ziovas, d. tataraki, p. manousou, n. parveri, f. satoglou, d. gatos and k. barlos department of chemistry, university of patras, 26500 patras, greece solid phase peptide synthesis is traditionally performed by the attachment of the c-terminal amino acid through its α-carboxyl function on a suitable solid support and elongating peptide chain towards the amino terminal of the peptide by adding sequentially the amino acid residues in the gradually growing peptide chain. several thousands of publications and patents describe this methodology and its application for the production of peptide pharmaceuticals. in contrary to the attachment of the c-terminal carboxyl function, attachment of amino acids and peptides through an amino acid side-chain on suitable resins and their application in spps is described in a small number of publications and patents. most of these publications describe the attachment of the amino acids through a side-chain carboxyl group of asp and glu. in the present work peptides were synthesized very efficiently in high yield and purity by anchoring of side-chain hydroxyl, amino or thiol groups of amino acids, amino acid amides, amino alcohols or small peptides on resins of the trityl or benzhydryl type. several peptides of pharmaceutical interest, such as exenatide, octreotide, pramlintide, calcitonin, bivalirudin, insulin b-chain and others were produced as examples of this technology, either by the step-by-step procedure or by fragment condensation in solution and on solid phase. step given that some of these diseases are caused by a mutation and/or malfunction of an essential protein, a better understanding of the structure and function of such proteins will allow us to prevent, slow down or even cure these diseases. to increase our knowledge, the synthesis of the target protein, a fragment involved in its activity or interacting peptides that modulate the protein activity is often required. in some cases the preparation of a protein analogue that improves its efficacy is envisage. however, conventional solid-phase peptide synthesis methods have some limitations when attempting to achieve these complex sequences of considerable length. using novel technologies, such as a microwave-assisted solid phase synthesis, commonly found in many peptide synthesis labs, here we performed the step-wise solidphase synthesis of a protein holding more than 100 residues (d-vegf). this synthetic achievement indicates the suitability of this approach for synthesis of long proteins or their analogues. the detailed synthesis of the enatiomeric version of vegf and the selenomethionine substituted analogues of huprp (106-140),1 proteins involved in angiogenesis and prion protein amyloidoses respectively, are described as study cases, where the use of microwaves allow us to obtain them in a fast and efficient manner. therefore, the development of novel peptide analogues with enhanced in vivo stability could potentially provide therapeutic alternatives. the pharmacological evaluation of a bioactive peptide [des-gly 10 ,tyr 5 (ome),d-leu 6 ,aze-nhet 9 ]gnrh, analogue 1, is presented herein. in vitro (kidney mouse membranes) and in vivo (clinically relevant pharmacokinetic mouse model) bioassays were coupled to liquid chromatographytandem mass spectrometry. analogue 1, an agonist of the gnrh receptor with a binding affinity in the nanomolar range, caused testosterone release in mice that was acutely dose-dependent, an effect blocked by cetrorelix. repeated dosing studies in mice demonstrated that analogue 1 was well tolerated and had potency similar to that of leuprolide, based on plasma and testis testosterone reduction and histopathological findings. analogue 1 also shared with leuprolide similar significant antiproliferative activity on androgen-dependent prostate cancer (lncap) cells. on the basis of pharmacokinetic advantages, we expect that analogue 1 or analogues based on this new design will be therapeutically advantageous for the treatment of cancer and endocrine disorders. cortexin is a polypeptide drug isolated from cattle and porcine brain cortex. cortexin is effective in monotherapy and in combination with traditional methods of treatment. cortexin produces tissue-specific, regulatory, and reparative effects on the brain cortex and contains active low-molecular-weight neuropeptides (<10 kda) penetrating through the blood-brain barrier. cortagen is a tetrapeptide h-ala-glu-asp-pro-oh (geropharm) produces nootropic and neuroprotective effects. oleylcortagen is a lipophylic analog of cortagen c 17 h 32 o-ala-glu-asp-pro-oh was created for increased proteolytic stability and increased penetrating through the blood-brain barrier. the main aim of our investigation is the analysis of psychopharmacological profile of 3 peptide preparations in comparison with piracetam. have been shown oleylcortagen (1 mg/kg) and piracetam (200 mg/kg) possess activating effect on motor and research components of behavior in «open field» test. two of peptides (oleylcortagen, cortexin) decreased period of immobilisation and demonstrated antidepressant effects on rat behavior in the porsolt's test, on other hand cortagen demonstrated depressant action. therefore, the significant psychoactivating properties are typical for oleyl-cortagen, cortexin. the mechanism of the action of these peptides can be explained from the viewpoint of the regulatory cascade. they produce a direct information impact on cell structures of the brain, and then promote release of the regulatory peptides, which in turn, induce the release of the next group of peptides. neurology, queen square, london wc1n 3bg, uk one of the hypotheses of alzheimer's disease neuropathology involves beta-amyloid (βa) binding with proteins on neuronal cell surface which leads to cell lysis and amyloid plaque formation. according to the latest data α7-type of the nicotinic acetylcholine receptor (achr) and the prion protein can be the target for betaamyloid toxicity [1, 2] . aggregated βa causes many pathological changes in cultures of mixed neurons and astrocytes such as sporadic cytoplasmic intracellular ca 2+ -signal, activation of reactive oxygen species (ros) production and cell death. in the present work we demonstrated the ability of affinity purified antibodies to synthetic fragment 173-193 of α7-subunit of the achr (achrabs) or to peptide 95-123 of the prion protein (prpabs) to protect cells from βa induced cell death. we also showed that both antibodies did not block βa induced ca 2+ -signal in astrocytes. however, preincubation of cortical co-culture of neurons and astrocytes with achrabs or prpabs significantly reduced the rate of caspase 3 activation and the rate of βainduced ros production via modulating nadph-oxidase. more detailed research of involvement of α7-type achr revealed that α-bungarotoxin was also very effective in the inhibition of caspase 3 activation and superoxide production. the observed positive effect of antibodies to α7-type achr or to the prion protein gives an additional explanation regarding the involvement of these proteins in ad pathology and provides new approach into an anti-ad vaccine design. capturing and macrophage-aided clearance of amyloid beta by surface modified proteineous particles m. richman, s. rahimipour department of chemistry, bar-ilan university, ramat-gan 52900, israel imbalanced homeostasis and oligomerization of amyloidβ (aβ) peptide in the brain are hallmarks of alzheimer's disease (ad). microglia and macrophages play a critical role in ad progression by clearing aβ from the brain or inducing inflammation. recent evidence suggests that the phagocytic pathway of aβ may be defective in ad microglia/macrophages that contributes to the build-up concentration of aβ in the brain. 1,2 therefore, efforts have been directed toward developing treatments that trigger these cells to clear aβ through alternative mechanisms. we have recently demonstrated that protein microspheres modified at their surface with multiple copies of an aβrecognition motif can strongly bind aβ, inhibit its aggregation and directly reduce its toxicity by sequestering it from the medium. 3 here, we describe how the aβ-bound microspheres can stimulate microglial cells and be phagocytosed through a mechanism that is distinct from that of aβ. the phagocytosis was mostly effective with microspheres having diameter size of about 0.7-1 mm and introduction of polyethylene glycol to the surface of the microspheres changed the kinetics of the phagocytosis. moreover, while aggregated aβ induced a significant inflammatory response that was manifested by the release of tnf-α from the microglial cells, the aβ-bound microspheres dramatically reduced the amount of the released cytokine. our data suggest that surface-modified microspheres could be utilized to detoxify other pathogenic or misfolded proteins that their accumulation may lead to genesis of other diseases. vasoactive intestinal peptide (vip) and its derivatives have been thought to be promising drug candidates for airway inflammatory diseases. however, the therapeutic potential of vips is highly limited because of rapid metabolic degradation and systemic side effects following systemic administration. previously, to overcome these drawbacks, our group developed a novel vip derivative, [arg 15, 20, 21 , leu 17 ]-vip-grr (ik312532), with improved metabolic stability (1), and respirable powder (rp) formulation of ik312532 (ik312532-rp) for pulmonary administration (2) . these attempts successfully led to enhanced pharmacological effects of ik312532 in the airway system and reduced systemic exposure; however, further chemical modification of ik312532 with a focus on metabolic stability might provide better clinical outcome. the present study aimed to design a pegylated vip derivative with improved metabolic stability and to develop its respirable powder (rp) formulation for inhalation therapy. ik312532 was chemically conjugated with peg (5 kda, p5k), the physicochemical and biochemical properties of which were characterized by cd spectral analysis, binding assay, and metabolic stability. the rp formulation of pegylated ik312532 (ik312532/p5k) was prepared with a jet mill, and in vitro inhalation performance and in vivo pharmacological effects in antigen-sensitized rats were also evaluated. the cd spectral analysis demonstrated that peg conjugation had no impact on the solution structure of ik312532. although receptor-binding activity of the ik312532/p5k (ic50: 82 nm) was estimated at ca. 30-fold less than that of ik312532 (ic50: 2.8 nm), metabolic stability for the ik312532/p5k was highly improved. according to the laser diffraction and cascade impactor analyses, ik312532/p5k-rp had fine in vitro inhalation performance. insufflation of ik312532/p5k-rp (150 μg of ik312532/p5k) in antigen-sensitized rats resulted in marked attenuation of inflammatory events, as evidenced by significant decrease of inflammatory biomarkers and granulocyte recruitment in pulmonary tissue at 24 h after antigen challenge. from these findings, pegylation of vip derivative, as well as its strategic application to the rp formulation, might be a viable approach to improve its therapeutic potential for treatment of airway inflammatory diseases. the previous studies have shown that trkb (tropomyocin receptor kinase) acts as an oncogenic agent and its binding to bdnf (brain derived neurotrophic factor) activates signaling angiogenesis of tumor proliferation [1] . for finding the most stable and potentially effective peptides against the trkb, we applied the following protocol. at the first step of this protocol we designed a peptide library by using sequence tolerance method in rosetta 3.3 package, then peptide energy optimization performed by backrub protocol for finding the most stable peptides. the five best peptides in energy optimization selected based on backrup scores by using r package [2] . 3d-structure prediction of the selected peptides was performed by using molecular dynamic in hyperchem 7 software. docking of peptides with trkb receptor was carried out in haddock software. we used cyclotraxin, a selective trkb inhibitor as positive control for this protocol. cyclotraxin and the peptides were compared by anova or t-test. the peptides are going to be tested against the trkb in an in vitro model. dirucotide (mbp 82-98 ) is a synthetic peptide analog of , that consists of 17 amino acids and tested in a phase trial were failed to reach his tolerance level on previous phase ii in rpms patients. one of the major disadvantages of peptide therapy is the activation of proteolytic enzymes, leading to peptide degradation. to address this problem cyclic peptide analogues have been synthesized. thus we synthesise a linear and cyclic analogues of dirucotide. the two analogues were synthesized by changing the amino acid residue at position 91 from to the synthesis of the linear peptide, as well as of the cyclic one, was carried out by the fmoc/tbu methodology, utilizing the 2-chlorotrityl chloride resin (cltr-cl). the purification was achieved using semi-preparative rp-hplc and the identification was assessed by analytical rp-hplc and by mass spectrometry (esi-ms). the linear and cyclic peptide analogues will be used in human t-cell cultures to test their immunogenicity in patients versus healthy controls. 1 in the first approach a reporter moiety was introduced to diagnose and treat cxcr4 related diseases. therefore, an anchor point to attach additional molecules to the ligand was elucidated. using sar studies to optimize the linker from the ligand to the detectable moiety the excellent receptor affinity could be retained. in a final step the reporter moiety was introduced to give a ligand for diagnosis via pet imaging and for possible endoradiotherapeutic applications. 2 originating from the dimeric motif present in many active cxcr4 ligands several dimers were prepared using a monomeric ligand identified in the prior study. comparison of monomers and dimers yielded a possible subsite binding mode explaining why the dimers exhibit enhanced affinity using a model derived from the origins of the monomer. 3 several peptidomimetic modifications were introduced to the ligand to reduce the peptidic structure. in a conformationally guided approach introduction of a peptoid motif could enforce a single active conformer that was enhanced through subsequent modifications. this yielded a compound 100 times better than the original cxcr4 antagonist which is the most affine cxcr4 ligand reported so far. our previous studies have demonstrated that pace4 represents a potential therapeutic target for the treatment of prostate cancer 1 . moreover, we have developed potent and selective pace4 inhibitor, (20-fold specificity over furin), known as multi-leu or ml peptide, which has a significant inhibitory effect on the proliferation of prostate cancer cell lines. peptide-based drug candidates can be limited by their poor metabolic stability and low bioavailability. thus, we performed structure-activity relationship studies to improve the pharmacokinetic properties of our ml inhibitor. we have designed and synthesized new ml peptide analogs having various chemical modifications. first, based on our previous results, we combined the most effective modifications of position p8 (d-amino acids) and p1 the arginine mimetic 4amidinobenzylamine (amba) to improve overall properties of our leading compound. second, the n-terminus of the resulting analogs was modified with a fatty acid, in order to enhance their cell permeability properties. third, we modified the inhibitors with a peg moiety to increase their stability and bioavailability. we tested the inhibitory activity, stability in plasma, cellular uptake, and cytotoxicity of each inhibitor. the results of this study demonstrate that the presence of the n-terminal extension (c8 or peg8) does not affect activity of our inhibitors. on the other hand, we show that introduction of the peg moiety does not increase cytotoxicity of ml analogs. it is interesting to note that the pegylated analog ac-peg8-d-leu-lllrvkr-amba has better cell-permeability activity than its counterpart without peg unit. this combination of pharmacological properties makes our new ml analogs promising candidates for the development of potential anti-prostate cancer agents. [1] peripheral coadministration of rf9 with opioid analgesics led to confirm the involvement of npff receptors as a part of the antiopioid system. indeed, rf9 was able to reverse the opioid induced hyperalgesia in rat (randall selitto test). then, a complete structure-activity relationships analysis was performed with rf9, assessing the involvement of each moiety for affinity and selectivity towards both npff receptors. a first exploration of the n-terminus part of rf9 (>80 synthesized derivatives) led to replace the hydrophobic adamantane moiety by a hindered aromatic group, providing a subnanomolar npff1 ligand with more than two log-units selectivity against npff2. then, the removal of the cterminal amide function led to reduce the dipeptide arg-phe-nh2 into an arginine derivative. in spite of an initial loss of affinity, optimization of the phenethyl moiety at the cterminus part of arginine led to non-selective nanomolar ligands for both npff1 & 2 receptors. next, we applied efficient methodologies in order to synthesize non-natural analogs of arginine, leading to various compounds exhibiting selectivity for either npff1 or npff2 receptors. in particular, compound rf313 was identified as a selective npff1 antagonist (npff1, ki = 64 nm; npff2: ki > 13 μm). lacking of analgesia properties, oral administration of rf313 (1 mg/kg per os) to rats was able to fully reverse fentanylinduced hyperalgesia. rf313 is the first orally available npff1 antagonist capable of reversing opioid induced hyperalgesia at low dose. moreover, this result allows identifying for the first time npff1 receptor as a key-partner of the anti-opioid system. administration of multiple antiplatelet agents has become the mainstay in the treatment of acute coronary syndromes in everyday clinical practice. we have previously reported significant antiplatelet effects of novel synthetic peptides' single administration on experimental carotid artery thrombosis in rabbits 1 . in the present study we sought to investigate the peptides' effects when administered in marginally effective doses (significantly lower than those utilized in the past), in animals that had previously received low doses of aspirin. the peptides when co-administered with aspirin preserved the carotid artery's blood flow, in contrast to the total artery occlusion observed in animals receiving aspirin and placebo. blood flow at 90 min after electrical stimulation was reduced to 56.7±7.9% and 33.2±0.3% in the ymesradr and psrcdcr-nh2 groups respectively (p<0.001 vs aspirin and control). thrombus weight was significantly reduced in animals receiving ymesradr and psrcdcr-nh2 versus aspirin and control (3.9±0.3 mg and 3.1±0.4 mg, vs 7.8±2.2 mg and 5.7±0.8 mg respectively, p<0.05). platelet aggregation was significantly inhibited in the ymesradr and psrcdcr-nh2 groups by 36.0±14.1% and 45.0±12.0% for adp (p<0.05 vs aspirin and control), and 35.5±6.3% and 54.2±5.6% for aa (p<0.05 vs aspirin and control), respectively. blood loss did not significantly differ among the various groups. administration of novel synthetic peptides, even at marginally effective doses, in animals previously treated with low doses of aspirin results in enhanced antiplatelet effects in an experimental model of arterial thrombosis. the study of peptide metabolism is particularly important when examining anticancer peptides; it can provide pivotal clues for the evaluation and improvement of stability of a peptide drug leading to enhanced pharmacokinetics and efficacy. gnrh analogues are used for the treatment of prostate cancer. as with other peptides a drawback is their short half-life due to their metabolic degradation. in order to examine the stability of these analogues we have developed several in vitro peptide stability and metabolism assays using specific tissues, isolated membranes, cancer cells that are analyzed subsequently using lc-ms/ms based approaches. such in vitro studies are followed up with pharmacokinetic studies in mice in order to establish the correlation between in vitro and in vivo approaches. the kidney is the main metabolic organ for peptide metabolism and for that reason we employed a peptide stability and metabolism assay previously described by our group using isolated mouse kidney membranes for the evaluation and comparison of different gnrh analogues. we tested gnrh analogues in other tissues such as mouse plasma, which is the "distributing" tissue for these drugs and mouse brain homogenate, a tissue known for its abundance in peptidases and relevance for centrally mediated effects. stability studies were also performed in cancer cells. in all cases lc-ms/ms based assays were developed for measurement of peptide drug and the resulting metabolites. for triptorelin, and a series of gnrh analogues, degradation and metabolite formation was studied by our mouse kidney membranes assay. studies of coadministering the peptide of interest with inhibitors that are presumed to block the metabolism in mice are ongoing. the vulnerability of gnrh analogues was verified after incubation with plasma and brain homogenate and by metabolite identification we obtained clues about the key cleavage sites. the described in vitro/in vivo protocols provide valuable information that could lead to the synthesis of more stable anticancer peptides with improved anticancer efficacy. in this work, we explored the use of a high-throughput synthesis and screening approach with peptide arrays to identify and structurally optimize shortened hiv-1 fusion inhibitors. the peptide array technology involves miniaturized synthesis of immobilized peptides, followed by affinity testing with a five-helix bundle, as a mimetic of the fusogenic gp41 protein. this exercise resulted in the identification of a class of truncated peptides which demonstrates a surprisingly high antiviral potency, despite the absence of the pocket-and the lipid-binding domain. the propensity of these peptides to adopt the bioactive α-helical conformation in solution as determined by circular dichroism, could be the key factor for this unexpected potency. these peptides are promising leads for the treatment and prevention of hiv. the pathological role of platelets in cardiovascular disease (cvd) is well established. platelets secrete adp to recruit additional platelets to a thrombotic site. we have previously identified novel cell-permeable peptide modulators of platelet function by using a bioinformatic screen based on patterns of evolutionary conservation in transmembrane proteins 1 . in this study we further explored peptides derived from cadherin cell adhesion molecules. we explored a range of 14 overlapping peptides derived from different cadherins varying from 6-15 amino acids long. peptides are synthesized and analyzed in a high-throughput platelet adp secretion assay. peptides (0.05-50μm) were assessed in the presence and absence of thrombin receptor activating peptide (trap;4μm). we identified cadh-5 and 6 proteins, but not cadh 1 or 2 in human platelets using western blotting and mass spectrometric analysis. peptides derived from paralogous juxta-membrane, cytoplasmic regions of these cadherins are potent modulators of platelet secretion. by systematically deleting amino acids from c or n-terminus of active peptides, we established that the minimal functional sequence for biological activity was a short six-residue motif, which corresponds to the known catenin-binding region of cadherin tails (kepllp) 2 . peptides alone have no biological activity. however, they potentiate the response induced by the platelet agonist trap at low doses. thus we have identified a cadherin-derived peptide that can modulate platelet secretion events. this highlights a previously unknown role of cadherins in the regulation of platelet function. agents that interfere with cadherin signaling in platelets might have a therapeutic role in cvd. ageing of the brain leads to impairments in cognitive and motor skills, and is the main risk factor for several common neurological disorders such as alzheimer's disease (ad) and parkinson's disease (pd). altered protein handling (proteolysis, repair system, chaperones) forms a basis for a large number of protein conformational disorders. extra-and intracellular, as well as intranuclear accumulation of abnormal proteins, in the form of protein inclusions and aggregates, and dysfunction of the quality control mechanisms are common in all these disorders. alterations in protein homeostasis occur with age, causing molecular changes such as protein misfolding and aggregation. many biologically active proteins lack stable secondary and tertiary structure, these are called intrinsically disordered proteins (idps), some of them (e.g. β-amyloid, α-synuclein) are coupled to neurodegenerative disorders. idps exist as assemblies of rapidly fluctuating structures undergoing coupled folding and aggregation process. protein aggregation is characterized by polymorphism, where a mixture of soluble oligomers, amyloid fibrils and amorphous aggregates is the final product. soluble oligomers are inevitable formed during the self-association process and might initiate the neurodegenerative cascades of ad, pd and similar diseases. the emerging consensus that protein misfolding (leading to idps) is the cause of several neurodegenerative disorders now offers the opportunity to develop a general therapy. soluble oligomers with id regions are potential drug targets. recently short peptide fragments and small peptidomimetic molecules have been found also in our laboratory; these molecules bind to the id regions of β-amyloid and are putative drug candidates. precise control of bleeding is ensured by anticoagulant mechanisms, which under normal conditions prevail over coagulants mechanisms. disrupting the balance between procoagulant and anticoagulant systems due to congenital or acquired defects leading to thrombotic disorders. anticoagulants are substances that inhibit blood clot formation. their action consists in inhibiting the synthesis of prothrombin, the substances forming thrombus as well as some coagulation factors. many peptides and proteins with different molecular weight, such as antistasin (ats), ghilantens, hirudin, etc. showing high anticoagulant activity are isolated from salivary glands of ticks and leeches. they inhibit the action of serine proteinases from blood coagulation cascade. this creates an opportunity for targeted synthesis of low molecular weight analogues of some of these proteins, which can be used in the prevention and treatment of thrombotic disorders. ats is competitive, slow-binding inhibitor of factor xa. it is well known that blood coagulation could be blocked at different stages of the coagulation cascade through inhibition of various enzymes. therefore, it is interesting to determine the place of action of newly synthesized antithrombotic agents in the blood coagulation cascade. this can be done by determining the inhibitory constants of newly synthesized peptides on different enzymes of this cascade. herein we report on our kinetic investigation of newly synthesized peptide amides, analogues of isoform 2 of ats 1 . ki, km, vmax and type of inhibition on the factor xa, thrombin and plasmin were determinate. some interesting differences between type of inhibition of ats, free acids and amide analogues of ats were revealed. to evaluate the relative anti-platelet aggregation activities of each peptide, the lebetins were chemically synthesized and fully characterized. here we described the synthesis, the solution structure of lebetin g1-g38 from the venom of vepera lebetina by 1 h bidimensional nmr and the relation structure-activity. this peptide has been demonstrated to be associated with a potent anti-platelet aggregating activity. the g1-g38 three dimensional structure consists in a compact β-bulged hairpain core from which emerges one loop and the cterminus and the n-terminus. we report on an approach whereby ligands are designed to bind and stabilize the 13-26 region of aβ in an α-helical conformation. these ligands reduce aβ toxicity to cells in culture and to hippocampal slice preparations. in addition, when these inhibitors are administered to drosophila melanogaster expressing human aβ (1-42) in the central nervous system, a prolonged lifespan, increased locomotor activity, and reduced neurodegeneration is observed 1 . stabilization of the central aβ α-helix appears to counteract polymerization into toxic assemblies and indicates that this approach holds promise for the development of orally available compounds against alzheimer's disease. encouraged by the above results we are currently developing a second generation of designed ligands. this involves synthesis of different new peptoids and unnatural amino acids. additional support for the concept comes from recent molecular dynamics simulations that also uncover details of the mechanism of unfolding of the aβ central helix 2 as well as retardation of the folding in presence of ligands designed to interact with the native helical conformation 3 . synthesis and methodology for new ligands, which includes synthesis of novel amino acids, will also be presented. triostin a is a well-known natural product with antibiotic, antiviral, and antitumor activities. it inhibits rna synthesis by bifunctional intercalation into dna base pairs through its two quinoxaline units showing cpg selectivity. triostin a must adopt an altered conformation upon dna bisintercalation that is substantially different than its preferred native x-ray or solution conformation. this fact suggests that the destabilizing conformational change in the cyclic octadepsipeptide counteracts much of the gains derived from a second intercalation. nonetheless, the wide range of pharmacological activities exhibited by this compound prompted interests in identifying novel and additionally potent lead compounds with improved pharmacokinetic properties for clinical applications. herein, a library of twelve simplified triostin a analogues has been synthesized by solid-phase peptide synthesis. the introduction of the key quinoxaline units was carried out in solution. the analogues' conformation corresponds to the staple form that bisintercalator cyclic (depsi)peptides adopt when binding to dna and, in addition, some of the synthesized compounds showed improved solubility. our library was evaluated for its antiproliferative activity against four human cancer cell lines and one analogue showed greater cytotoxicity than triostin a and even comparable activity to doxorubicin, a very commonly used drug in cancer chemotherapy nowadays. surprisingly, little is known about its mechanism of degradation in solution or the degradation products. a recent study identified monomeric polysulfides and dimeric degradation products, postulated to derive from β-elimination followed by deamidation and dimerization. 1 we recently reported that degradation of oxytocin and its analogues in aqueous solution at ph 7.4 produced monomeric polysulfides with up to 6 sulfur atoms as well as dimeric products. 2 unexpectedly, incubation of ot or of various analogues modified in position 1 resulted in identical dimeric degradants. we concluded that β-elimination via breakage of the c-s bond of cys1 must be a key step of the process, and that the resulting δala 1 residue would have to undergo further modifications to yield the same dimeric products independently of the substitution of the n-terminal nitrogen. here we further clarify the degradation mechanism and propose a structure for the dimers. we postulate that hydrolysis of the δala 1 residue yields an n-pyruvoyl linear peptide, which loses one sulfur atom and subsequently forms dimers, which we found are linked by one disulfide bridge and one non-reducible bond. the putative linear n-pyruvoyl oxytocin intermediate was synthesized and found to degrade to the same dimers as the ones in the incubations of ot. a [u-13 c]cys 1 ot analogue gave degradation products with 13 c-nmr spectra consistent with a non-stereospecific aldol-type condensation. detailed experimental procedures, structures of the degradants and the postulated mechanism of ot degradation in near neutral solutions will be presented. inserm u765 paris, france αiibβ3 is the main platelet integrin and is responsible for platelet aggregation. a lipid-modified peptide corresponding to αiib intracellular sequence 1000-1008 (palmitoyl-k-l 1000 eeddeege 1008 , pal-k-1000-1008), is platelet permeable and inhibits human platelet aggregation induced by thrombin 1 . ymesradr, a peptide corresponding to the extra-cellular sequence 313-320 of αiib, is a platelet activation and aggregation inhibitor 2 . the aim of the present study was to investigate the cooperativeness of the intracellular and extra-cellular peptides on platelet aggregation and their effect on the phosphorylation of fak and erk. pal-k-1000-1008 together with the extracellular ymesradr peptide, at concentrations lower than their ic50 values, showed cooperative inhibition of platelet aggregation. the peptide combination inhibited also fibrinogen and pac-1 binding to activated platelets. fak phosphorylation is a postaggregation event related to outside-in activation of the receptor. the combination of peptides inhibited fak phosphorylation. erk phosphorylation is independent from platelet aggregation, and is enhanced by rgd-peptide inhibitors. the combined peptides inhibited erk2 phosphorylation. ovarian cancer (oc) is considered a rare disease and represents the fifth most common cause of death from cancer in women. the standard first-line treatment consists of a combination of paclitaxel and carboplatin (ddp) or carboplatin alone. in the case of progressive disease or drug resistance to platinum-based agents, either alone or in combination, investigational compounds should be used (1) . the mechanisms behind acquired resistance to ddp and its derivatives are not clear yet, although it is evident that the process is multifactorial, including enhanced dna repair processes. some peptides designed from the interface subunit of the human thymidylate synthase (ts) have been identified recently (2), as effective anticancer agents against sensitive and resistant oc cells. one of them was also able to recover the cellular sensitivity towards cisplatin in resistant oc cells in the μm range. to improve its potency and selectivity structural studies have been performed in combination with cellular assays aimed at understanding the mechanism of action. a label-free quantitative proteomic approach has been undertaken to study the effects of the peptide on the proteins involved in the modulated metabolic pathways, in particular those involved in the folate metabolism. structure-activity relationships (sar) have been performed to improve the lead peptide pharmacodynamics. all the compounds have been assayed and a protein profile set was studied to mark and validate their behavior as inhibitor of oc cell growth. hepatitis c is a liver disease provoked by a virus known as hcv. the disease is insidious. hcv causes anorexia, nausea, vomiting, fever, fatigue and jaundice. in about 40% of sufferers the disease is short, but others become chronic. in the chronic form in about 20% of cases the final result is cirrhosis of the liver and in the remaining 20% it leads to liver cancer. hcv is a very serious problem today. about 3% of people infected with hcv worldwide, i.e. about 4 million are residents of europe. 170 million people carry the disease as a chronic illness with the potential to develop into cancer in their liver. all these people represent a "reservoir" for storage and distribution of hcv. ribavirin, the nucleoside analog 1-β-d-ribofuranosyl-1,2,4triazole-3-carboxamide, known by the trade name virazole (also known as rebetron in combination with interferon-α), exhibits antiviral activity against a variety of rna viruses (paramyxoviruses, flaviviruses, etc.) as well as some dna viruses. in humans ribavirin is currently used in combination with interferon-α to treat hcv infections. this lack of strict specificity and a broad spectrum of activity are due to its multifunctional mechanism of action against viruses. these characteristics have made ribavirin a drug of substantial research interest. unfortunately, ribavirin shows a significant toxicity, causing bleeding in accumulation [1] . herein, we report a total synthesis of modified in position 5' of ribose residue, ribavirin in order to be further linked to cell penetrating peptides (cpps). in addition the synthesis of some cpps as well as bonding between two parts of final hybrid molecules will be reported. in our case the design of new hybrid molecules is done in order to: (a) vectorize ribavirin into liver cells; (b) transport ribavirin molecule trough cell membrane and (c) decrease toxicity of ribavirin. to obtain oligomeric aβ peptide, our laboratory uses a precursor depsipeptide of aβ. this precursor, termed as "iso-aβ" has an ester bond between the side chain of ser 26 and gly 25 . at physiological ph this ester bond becomes an amide bond via an o→n acyl shift. binding partners by which the oligomeric aβ mediates its toxic effect has not been yet investigated in the proteome or subproteome level. we used protein array technology to study the interaction of oligomeric aβ with 8163 recombinant human proteins, immobilized on a protein chip. aβ binding proteins were identified with the aid of a monoclonal aβ antibody. altogether 324 proteins were found to interact with our aβ-oligomers. these proteins were grouped according to their function. one of the major groups contained 24 proteins participating in translation. these proteins were found in ribosomes. to prove our proteomic results ribosomes from rat hippocampus were isolated. elisa experiment revealed that aβ binds to ribosomes in a dose-dependent manner. using the sequence of the 324 aβ-binding proteins a homology search was performed to find oligopeptides, that possibly bind to aβ. based on these sequences a peptide chip containing hexapeptides was prepared. aβ interacting peptides were identified with a monoclonal antibody. several peptides were synthesized and tested on mtt assay. two out of four compounds inhibited the toxicity of aβ on rat hippocampal slices. summarizing our results aβ binding proteins and peptides were identified. knowledge about aβ binding proteins can help to understand the pathogenesis of ad, such us the possible involvement of ribosomes. oligopeptides can be lead compounds of future drug development. huge proteolytic complex named proteasome catalyzes protein degradation in every eukaryotic cell. it consists of 31 subunits forming four stacked rings and one or two regulatory caps. two inner rings of the proteolytic part contain three catalytic β-subunits that possess different substrate specificity. higher vertebrates can express γinterferon-inducible immuno-β-subunits. proteasome plays an essential role in continual turnover of intracellular proteins and in antigen processing. autoimmune diseases such as multiple sclerosis and its murine model eae are believed to rise from breakdown of tolerance of the immune system. it assumed that immunoproteasome could play an important role in autoimmune diseases. several classes of chemicals proved to be inhibitors of proteasome and the most active are boronate peptide derivatives. these inhibitors totally inactivate proteasome and result in full stop of intracellular protein turnover and cell death via apoptosis. another class of inhibitors, epoxy ketones, was shown to be more selective for immunoproteasome and could be used not for full stop of proteasome function, but for fine tuning of altered proteasome functioning. we examined properties of several inhibitors of four different classes, namely peptide boronate bortezomib, peptide aldehyde mg132, lactam lactacystin, and peptide epoxyketones epoxomicin, mg132ek, uk101 and pr-957. for inhibition experiments we used proteasome isolated from eukaryotic cell lines cho, nso and hek, treated and non-treated with γ-interferon, as a model cells contatinig constitutive and immunoproteasome. the upregulation of proteasome immunosubunits was revealed in cho and nso cells treated with γ-interferon. the ic50 values for all studied inhibitors were obtained, and ki in some cases were calculated. the epoxyketones were shown to selectively inhibit in submicromolar concentrations the proteasome sample which contain high amount of immunosubunits. in order to find an effective antimalarial, this study refers to some angiotensin ii (aii) analogues which were considered the important physicochemical characteristics described by silva et al. 1 to verify the biological activity against plasmodium gallinaceum and to understand the hydrophobic cluster influence, explained by tzakos et al. 2 these analogues were synthesized and characterized as described by silva 1 , as well as the biological assays and comprises, to verify: the hydrophobic cluster activity -a) drvyhipf; b) drvypr; c) ryhipf and d) fphiyvrd; the importance of these residues in aii molecule -e) rypf; the importance of aromatic residues -f) yhpf and the action of these hydrophobic residues, when interacting with the parasite membrane -g) vipf. it was observed that in a (94% of bioactivity), the phenol group of tyr is close to imidazole group of his that could promote a hydrogen bond formation. besides that, could occur van der waals interactions between ile and phe residues due its proximity and non-polar characteristic. these interactions could not be effective in native aii (88%) 3 , because ile residue promote a steric influence on the organization of his and tyr residues 4 that not exist in b (57%). in c (74%) and e (72%) analogues, the influence of the arg residue could promote a cation-π interaction with tyr residue 5 and the cluster may have suffered slight destabilization and its antiplasmodial activity was compromised subtly. in d (12%), the electrostatic change, obtained with the total inversion can have disordered its interaction with parasite membrane, since it is not related to membrane receptors, because d-aii presented 91% of biological activity. moreover, hydrophobic and aromatic residues importance was confirmed through the results obtained 93% and 89% of activity, with g and f, respectively. we conclude that hydrophobic cluster modifications and interactions of amino acid side-chain influences in the biological activity. closed joint-stock company "vertex", st-petersburg, russia creatine (cr), a small molecule synthesized in the kidney, liver and pancreas plays important role in atp synthesis, replenishing its store even in the absence of oxygen. cr is able to protect brain cells against ischemic damage; however it has poor ability to penetrate the blood-brain barrier without specific carrier protein. thus, synthesis of stable hydrophobic derivatives capable of crossing the bbb by alternative pathway is of great importance for the treatment of different neurological diseases including stroke, traumatic brain injury and hereditary crt deficiency. here we describe the synthesis and biological activity of new hybrid compounds -creatinyl amino acids. originally the title compounds were synthesized by guanidinylation of sarcosyl peptides. however, for large scale synthesis better results can be obtained using direct cr conjugation with amino acid or peptide derivatives by isobutyl chloroformate method. addition of equivalent amount of ptoluenesulfonic acid as lipophilic counterion ensures efficient cr dissolution in dmf along with its simultaneous protection towards intramolecular cyclization. it excludes the application of expensive guanidinylating reagents and permits to simplify the synthetic procedure. purification of final product and its conversion into appropriate salt form can be achieved by iec followed by crystallization from organic solvents. synthesized creatinyl amino acids and peptides exhibited significant biological activity in different assays including platelet aggregation test, ischemic stroke and nano2-induced hypoxia model. one of the most effective compounds -creatinyl-glycine ethyl ester increases life span of experimental animals more than two times in hypoxia model and has neuroprotective action in brain stroke model when applied both before and after ischemia. these data evidenced that creatinyl amino acids can represent promising candidates for the development of new drugs useful in stroke treatment. the efficient recognition and destruction of tumor cells via specific cellular markers is a major goal in cancer therapy. various growth factor receptors such as egfr, hgfr, vgfr and their downstream signaling networks have been proven to be effective molecular targets, as they are frequently involved in cancer proliferation and metastasis. downregulation of these receptors and/or blocking their signaling pathways have clear anti-tumoral effects. 1 drugs based on monoclonal antibodies (mab) targeting such cell surface receptors have attracted a lot of attention as a new generation of therapeutics. however, their production is costly and identifying new, variable routes to modified molecules with similar properties is currently a major focus. 2, 3 here we present an approach to chemically synthesize a molecule that combines the mode of action of antibodies with the advantages of smaller, chemically accessible molecules. these "synthetic antibody" (sab) molecules contain a chemoattractant that activates the innate immune response and resembles the fc domain of a typical antibody. specificity is imparted by two binder peptides that assume the function of the variable antibody domains and bind to a cell surface target. the fc and fab domains of the sab molecules are connected via polyethylene glycol linkers. sab molecules are prepared by solid phase synthesis, a flexible technique that allows fast production, full control of their properties and targeting two different cell surface receptors (bispecific tumor targeting). they are currently tested in vitro and in vivo for their effect on the innate immune system, general toxicity and selective binding to cancer cells. the key enzyme in the processing of polyproteins translated by viral rna genome of sars-cov is a 33kda protease called 3c-like protease (3cl protease). sars 3cl protease is a cysteine protease containing a cys-his catalytic dyad, and cleaves precursor poly proteins at as many as 11 conserved site involved a conserved gln at the p1 position and a small amino acid (ser, ala, or gly) at the p'1 position. due to its functional importance in the viral life cycle, sars 3cl protease is considered to be an attractive target for drug design against sars. recently, we found tetrapeptide aldehyde, ac-thr-val-cha-his-h, showed high inhibitory activity with ic50 value of 98 nm toward 3cl-r188i mutant protease 1,2 . to compare the inhibitory activity of small compounds with those containing active functional groups, we synthesized serine-derivatives within the essential functional groups and evaluated its inhibitory activity. the synthetic scheme was started from fmoc-ser(tbu)-oh, following modification of c-terminal carboxyl group with p2, n-terminal amine with p4 and side chain alcohol with p1 functionalities. 5 steps overall reaction led to obtain 44 novel serine derivatives for the small molecular inhibitors of sars 3cl protease. the assay with 3cl r188i mutant protease was examined to evaluate the inhibitory activity of the synthetic serine derivatives. then, molecular docking study of complex of 3cl protease with the ligand was carried out. docking simulation experiment with r188i (pdb id: 3aw0) and the inhibitor, which has the best activity in the serine derivatives, indicated that p1 fitting s1' pocket. at the result of assay, p1, p2 and p4 positions of the inhibitor should be modified by benzoyl group, cyclohexyl group and cinnamoyl group, respectively. their bioactivities are underpinned by their distinctive structure with exceptional stability, thus making cyclotides exciting, not only for agricultural and pharmaceutical purposes, but also as a template in drug design. in all of the reported activities, cell membranes seem to be the primary target for cyclotide activity. to unravel the importance of lipid membranes on the reported activities of cyclotides, a set of cyclotides belonging to möbius and bracelet subfamilies were compared in their mode of action. the lipid selectivity and membrane affinity were compared with their efficiency against different target cells (e.g. red blood cells, bacteria, hiv particles). we have found that the bioactivity of cyclotides is dependent on the lipid composition of the target cell membrane and independent of a protein chiral receptor. in particular, all the native cyclotides tested target the cell membrane through specific binding to phospholipids containing phosphatidylethanolamine (pe)headgroups, but the membrane binding affinity is further modulated thorough non-specific peptide-lipid hydrophobic interactions, which are dependent on the specific cyclotide. in addition, the bioefficiency of cyclotides broadly correlate with their ability to target and disrupt the cell membrane. overall, we have shown that even with a common specificity for membranes containing pe-phospholipids, a fine selection was found across the family. in particular, each cyclotide inserts and disturbs the membrane in a distinct way, which explains the diversity of this family but also their distinct activities. the observation that all the tested cyclotides have a preference for a specific lipid makes this family truly intriguing and brings insights to optimize the use of the cyclotide template in drug design. malaria is a disease that affects around 500 million people causing 0.5-1 million of deaths annually. based on our previous studies, angiotensin ii (aii) presented antiplasmodial activity against plasmodium gallinaceum, but due to pressure activity, it cannot be used as an antimalarial drug. in an attempt to increase antiplasmodial activity and reduce hypertensive activity, we synthesized by solid phase method, cyclic analogues of aii with i-(i+2) and i-(i+3) lactam bridge scaffold 1 using asp and lys residues. the bridge was more effective when inserted next to n-terminal extremity 1 , probably this insertion, on another portion of the peptide, provides a change in the conformation of the molecule and its hydrophobic cluster formed by tyr, ile and his 2 , which may have influence in the peptide-membrane interaction. thus, we have focused in the n-terminal extremity, testing new analogues, using glu/asp/orn/lys residues as bridgeheads components in i-(i+4) lactam bridge scaffolds, which showed that antiplasmodial activity is increased using glu residue and that larger lactam rings are better to biologically active. therefore, new restrict peptides by i-(i+2) and i-(i+3) lactam bridge were designed, using glu residue as bridgehead element, but the same effect was not verified, getting a maximum of 65% of bioactivity. on the other hand, we promoted an increase in the hydrophobic character of the molecule, replacing the asp residue of aii sequence by fmoc-glu and asp(ofm), in order to improve the interaction of these compounds in the sporozoite membrane. the replacement by fmoc-glu provided a decrease of activity, while that asp(ofm) kept the aii activity, because there are changes of charge in the peptide, which may have modified the conformation in physiological medium. this kind of approach may offer the basis for development of new drugs and chemotherapy against malaria. animal venoms are complex chemical cocktails, comprising a wide range of biologically active reticulated peptides that target with high selectivity and efficacy a variety of membrane receptors such as ion channels or g-protein coupled receptors. venoms can therefore be seen as large natural libraries of biologically active molecules that are continuously selected and highly refined by the evolution process. the vision associated with the venomics project is to investigate in depth the enormous structural and pharmacological diversity of venom peptides through the development, integration and implementation of a novel research paradigm combining cutting-edge "omics" technologies in a high-throughput workflow. this new paradigm enclosed in venomics aims at replicating in vitro the diversity of venoms to generate original peptide banks to be used in drug discovery programs. herein, we show the different strategies we adopted for efficient solid phase synthesis and folding with an easy purification of peptides rich in cysteine and containing posttranslational modifications (ptm). angiogenesis depends on the adhesive interactions of vascular cells. the adhesion receptor integrin av b3 was identified as a marker of angiogenic vascular tissue. the αν β3 integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis, mainly by interacting with matrix proteins through recognition of an arg-gly-asp (rgd) motif. inhibition of the αν β3 integrins with a cyclic rgd peptide impairs angiogenesis, growth and metastasis of solid tumours in vivo. the aim of this study was to investigate the effects of replacement of a-amino acids by aza-β 3 -amino acid analogs in cyclic rgd-peptides as αν β3 -integrin antagonist on angiogenesis, microcirculation, growth and metastasis formation of a solid tumour in vivo. the selectivity profile of these antiadhesive cyclopeptide is rationalized by a special presentation of the pharmacophoric groups. we synthesized cyclic rgd peptidomimetics that include aza-β 3 -amino acid residues. modifications were added to the rgd skeleton in order to optimize the peptide activity. then, we investigated the pharmacokinetics activity of these pseudopeptides in hek (human embryonic kidney 293) and endothelails cells huvec (human umbilical vein endothelial cells) cell by analyzing cell viability and protein involved in the angiogneisis processes. since tenascin c is a factor expressed highly in the tumorassociated matrix, targeting it would be a desirable first step for targeting the tumor-specific microenvironment in fact, a high level of tenascin c expression has been reported in most solid tumors, including lung cancer, colon cancer and glioblastoma. therefore, the targeted binding of tenascin c in tumor stroma would inhibit tumor metastasis by modulating cancer cell growth and migration. we isolated a peptide that bound to tenascin c by phage display peptide library selection, and the selected peptide specifically recognized tenascin c protein in xenograft mouse tissue. we also observed exclusive staining of tenascin c by the selected peptide in tumor patient tissues. moreover, the peptide reduced tenascin c-induced cell rounding and migration. we propose that the tenascin c targeting peptide may be useful as a specific anti-cancer diagnostic and therapeutic tool for most human solid tumors. radiolabeled pansomatostatins are expected to enhance hsst1-5 tumor-uptake and to broaden clinical indications as compared to currently established sst 2-prefering radioligands. previous experience has revealed [111in-dota 0 ,dtrp 8 ]ss-14 ([ 111 in]at2s) as a true pansomatostatin analog, exhibiting however poor in vivo stability. in order to enhance metabolic stability, we introduced a second disulfide bridge to the at2s motif by formation of extra 6/12-amino acid (aa) or 8/12-aa ring generating at5s and at6s, respectively. the orthogonally protected sequences were assembled on the solid support, deprotected and cleaved from the resin with tfa. the first cys 6 -cys 11 (at5s) or cys 5 -cys 12 (at6s) cyclization was performed in dmso, while the second was completed with iodine oxidation after in situ deprotection of cys 3 (acm) and cys 14 (acm). during hsst 1-5+-autoradiography, at5s showed unexpected total loss of sst 1-5 affinity, whereas at6s showed high affinity (ic 50 in nm) to all hsst1-5 (hsst1= 11.5±3.3; hsst2= 6.3±0.6; hsst3= 9.7±3.6; hsst4= 5.4±0.8; and hsst5= 25.7±7.0). consistent with this finding, only at6s stimulated sst2 internalization during immunofluorescence-based internalization assays, showing agonistic properties for sst2. furthermore, [111in]at6s internalized rapidly and specifically in sst2+ ar4-2j and hek293-hsst3+-cells. hplc analysis of 5 min ex-vivo mouse blood samples revealed that >98% [111in]at6s remained intact. after injection in scid mice bearing ar4-2j and hek293-hsst3+ tumors [111in]at6s specifically localized in the rsst2a+ (1.9±0.2%id/g vs. 0.8±0.05%id/g + 100 nmol tate at 4 h postinjection (pi)) and in hsst3+ implants (3.7±0.4%id/g vs. 0.3±0.05%id/g + 80 nmol ke108 at 4 h pi). this study has shown that introduction of an extra disulphide bridge in at2s confers high metabolic stability. however, in a 6/12member ring combination it leads to total loss of affinity. the reasons for this effect are currently investigated by nmr conformational studies. transporter compounds are useful tools to solubilise and increase the delivery of therapeutic molecules in the human body. one system to improve the cellular uptake of such therapeutic molecules are cell-penetrating peptides (cpps). these short peptide chains are either polycationic (containing several arg and lys) or show a more amphiphatic structure. 1 it is known that the multivalency effect -the presentation of several copies of a cpp motif on a single molecule -can increase the cellular uptake. 2 peptide dendrimers represent a group of tree-like, multivalent macromolecules, which are synthesized for different chemical and biological applications in our group. 3 we now combine linear cpps with peptide dendrimers to get a well defined branched molecule made up of only natural amino acids. in our systematic study of peptide dendrimers decorated with different cpps we found that the potency of the single cpp as a transporter for small molecules can be increased and that these peptides show usually low cytotoxicity. additionally we designed new dendritic cell penetrating peptides with similar activities like linear cpps. all compounds are covalently linked to fluorescein for visualization with flow cytometry and confocal analysis. the results show that the peptides can transport efficiently a hydrophobic cargo into the cells. chemical stability of esters of acyclovir with amino acid and cholic acids k. chuchkov, r. chayov, i. g. stankova* south-west university "neofit rilski", blagoevgrad, bulgaria amino acid esters of antiviral drugs are a very good solution for improving oral bioavailability of the actual medicine. one of the most effective and tolerant prodrugs is valine ester of acyclovir -valaciclovir. taken orally exhibits three to four times higher bioavailability of acyclovir. the chemical stability of amino acids (4-fphenylalanine) (r,s) and bile acids (deoxycholic acid and chenodeoxycholic acid) esters of acyclovir was studied in experimental conditions simulating some relevant biological medias (ph 1.0 and 7.4, 37°c).the chemical stability experiments revealed that the examined amino acid ester of acyclovir were relatively unstable in acidic ph, but bile acid ester is stable in the same ph. the examined amino acid and bile acid esters of acyclovir in neutral ph are relatively stable. in ph 7,4 all of tested compounds are more stable than valacyclovir (t1/2 = 13 h) -the first effective prodrug of acyclovir. in acidic ph acyclovirdeoxycholat and acyclovirchenodeoxycholat are more stable than valacyclovir. acyclovirchenodeoxycholat is the most promising anti-ebv prodrug candidate with high activity and satisfying chemical stability. cell-penetrating peptides (cpp) have become efficient tools for the cellular internalization of bioactive molecules due to their ability to cross the plasma membrane of diverse cells and cell lines. [1] we recently reported that the cpp sc18, which consists of the residues 106-121 of the c-terminal region of the cationic antimicrobial peptide cathelicidin (cap18), is an effective carrier peptide for small organic molecules like fluorophors and toxic peptide sequences into various cell lines [2] . however, in general linear peptides are more susceptible to proteolytic degradation than their cyclic analogs [3] . therefore, we investigated the cyclization of cpp derived from sc18 by means of cui-mediated azidealkyne cycloaddition (cuaac) [4] . furthermore, we examined their conformation and proteolytic stability as well as their internalization efficiency and toxicity against various cell lines, in comparison to their linear equivalent and to other cpp. looking for the proper prodrug: a peptidomimetic approach to identify and inactivate bacterial mono-adp-ribosyltransferase toxins m. beich-frandsen, r. jørgensen division of microbiology and diagnostics, statens serum institute, copenhagen, denmark mono-adp-ribosylation is an endogenous posttranslational modification in eukaryotic cells, simultaneously utilized as virulence strategy by deadly secreted bacterial toxins. many bacterial toxins have been found to act as mono-adp-ribosylating enzymes, targeting anything from g-proteins to the actin skeleton. the diphtheria toxin from c. diphtheriae and exotoxin a from p. aeruginosa, both target the diphthamide-group of a unique modified histidine in eef2, inhibiting protein synthesis by ribosome mimicry 1,2 . we aim to inactivate these nad+-utilizing toxin enzymes by nad-conjugated peptidomimetics, in a target-specific prodrug-approach. the adp-ribosylation reaction follows a random third-order s n 1 mechanism. in the proposed model for the transition state of the reaction, the cleavage of the n c1-nn1 bond of nad + releases strain and generates a oxacarbenium ion intermediate with a positively charged nicotinamide (n)ribose, subject to a nucleophilic attack from the substrate 3,4,5 . adp-ribosylating toxins are commonly characterized by a artt-motif involved in substrate recognition 2 . studies suggests conformational rearrangement of the residues surrounding the substrate binding site to be required for optimal geometry 5 of the initial glycosidic nc1-nn1 bond cleavage within nad + . subtype specific nad-conjugated peptides, designed based on previous structural analysis of the adpribosylation reaction, act as substrate for the enzymatic adp-ribosyl-transfer, and hereby attach covalently to and inactivate the nad + -utilizing toxin. relying on previous structural studies, and established ligand-binding and kinetic data, an initial peptide library, designed by bioinformatics and evaluated for specificity of common targets in-silico identifies initial leads. lead-scaffolds are implemented in rational peptide-design, based on high-resolution structural-and biophysical studies of multiple peptide-enzyme complexes, to identify possible prodrug-strategies for enzyme inactivation. nanoparticles play a crucial role in medicine for their potential application as in vivo carriers of active principles [1] . liposome display unique pharmacokinetic properties slowly releasing drugs loaded in the inner aqueous cavity. in the last years we have developed supramolecular aggregates labeled by bioactive peptides able to recognize overexpressed receptors on tumour cells membrane delivering doxorubicin chemiotherapeutic drug [2] . neurotensin(nt), a 13 amino acid peptide, has dual functions of neurotransmitter or neuromodulator. the cterminus short fragment 8-13 preserve the activity but the half life of wild type form in vivo is very short. nt receptor type 1 (nts1) is overexpressed in severe malignancies such as small cell lung cancer and colon, pancreatic, and prostate carcinomas. we have designed new amphiphilic molecules containing in the hydrophobic moiety two aliphatic chains and in the hydrophilic moiety a the bioactive portion able to aggregates with phospholipid molecules achieving liposome. we have synthesized neurotensin wild type sequence, the truncated form and the tetra-branched neurotensin(nt1-13) or a truncated form(nt8-13) tetrabranched peptides(nt4) adopting an opportune synthetic strategy on solid phase. all liposome were formulated adding the neurotensin amphiphilic monomer in ratio 5:95 with dopc in order to evaluate the capability to recognize selectively receptors overexpress on cell membrane surface. the liposomes size was determined by dynamic light scattering measurements, values for the hydrodynamic radius(rh). the selective internalization and cytotoxicity of fully doxorubicin loaded liposomes as compared to pure dopc liposomes, was tested in ht29 human colon adenocarcinoma and te671 human rhabdomyosarcoma cells. recently, small interfering rna (sirna), one kind of rna interference (rnai) technology represent the most common and, to date, the most effective method to inhibit target gene expression in human cells. it is also a common recognition that non-toxic delivery of sirna is an urgent problem for the therapeutic application of sirna. for the efficient gene silencing in vivo, prolonged circulation of sirna with take efficient and non-toxic cellular uptake and resistance against enzymatic degradation are indispensably required. 1 ) telomerase activity has been regarded as a critical step in cellular immortalization and carcinogenesis and because of this, regulation of telomerase represents an attractive target for anti-tumor specific therapeutics. in this paper, we present the efficient and non-toxic cellular uptake of sirna using novel amphiphilic peptides and the application to silencing of htert in human cancer cell lines. in the present study, we investigated the intracellular delivery of sirna using some amphiphilic peptides and the silencing effect of sirna targeting htert mrna in 3 human cancer cell lines, jurkat, hela and k562. the complex of sirna and a specific amphiphilic peptide or its hybrid with an intracellular transport signal peptide could be effectively taken up into cells. the complex also showed a high silencing effect against htert mrna. moreover, the combination of sirna-nes conjugates and the amphiphilic peptides improved silencing effects up to 95.2 %. the amphiphilic peptides and their hybrids showed almost no cyto-toxicity and protected sirna against intracellular nuclease digestion in 10% fbs (half life time was over 48h). tumor targeting with the decapeptide gonadotropinreleasing hormone (gnrh) or its analogues is based on the discovery that gnrh receptors are overexpressed in many tumor cells, compared with their expression in normal tissues. using these peptides as carriers/targeting moieties in a conjugate with therapeutic agents can increase the selectivity and the stability of the conjugates, or eliminate the toxic side effects of the drug. gnrh-iii (<ehwshdwkpg-nh 2) is especially favoured as a targeting moiety because of its antiproliferative effect and weak endocrine activity in mammals. the aim of our work was to synthesize novel short-chain fatty acid (scfa) acylated daunorubicin-gnrh-iii bioconjugates with enhanced enzymatic stability, cellular uptake, in vitro and in vivo antitumor activity. ser in position 4 of gnrh-iii was replaced by lys and its ε-amino group was acylated with various fatty acids. gnrh-iii( 4 lys(x), 8 in conclusion, glu 6-10 /dglu 6-10 substitution in gi-derived radiopeptides enhanced stability and reduced renal values by ~2 .5 fold. on the other hand, removal of the (glu)5sequence in mg11 suppressed renal uptake but undermined stability. both modifications, however, compromised tumor targeting, demonstrating that more efficacious structural changes in the gi motif are warranted to improve the in vivo profile of resulting radioligands. synthesis and characterization of a 99mtc-labeled rhodamine-conjugated angiotensin peptide as a potential cardiac function imaging agent s.m. okarvi king faisal specialist hospital & research centre, cyclotron and radiopharmaceuticals department, riyadh 11211, saudi arabia angiotensin ii (ang ii) is an 8-amino acid peptide that has been known to play a vital role in cardiovascular system. the actions of ang ii have been implicated in many cardiovascular conditions, such as coronary heart disease and heart failure. in an effort to develop a cardiac imaging agent with enhanced targeting capacity, we attached ang ii to rhodamine (rh), a lipophilic cation that specifically accumulates in the myocardium. the rh conjugated ang ii was evaluated for its potential as a heart imaging agent. rh-lys-gly-cys-asp-arg-val-tyr-ile-his-pro-phe-conh2 was prepared by solid-phase peptide synthesis according to fmoc/hbtu methodology. nhs-rh was attached to the ang ii peptide via the free amino group of lys residue by manual synthesis. rh-ang ii conjugate was labeled with tc-99m by the stannous-tartrate exchange method. in vitro stability was determined in human plasma and in excess of cysteine. in vivo biodistribution and biokinetics were performed in balb/c mice and sprague dawley rats. the rh-ang ii conjugate radiolabeled efficiently with tc-99m (>75% labeling efficiency) as determined by hplc analysis. tc-99m-rh-ang ii exhibited good chemical stability against cysteine transchelation and sufficient metabolic stability in human plasma. in mice, the bioconjugate displayed efficient clearance from the blood and excreted mainly through the renal route with some excretion by the hepatobiliary pathway. the uptake in the heart was 1.8±0.5% id/g as early as 30 min post-injection; whereas, the uptake in the lungs, liver, stomach and kidneys varied between 1-10% id/g. in rats, the bioconjugate displayed relatively better pharmacokinetic characteristics, with low uptake in the major organs (<4% id/g). the uptake in the heart (1.7±0.4% id/g) was found to be higher than the uptake in the blood and muscle, resulting in good heart-to-blood and heart-to-muscle uptake ratios. this initial study towards the development of an effective cardiac imaging agent advocates that the use of hybrid conjugates appears to hold a great promise as a new and attractive approach for rapid and efficient imaging of heart. in humans two isoforms of gnrh are exist, gnrh-i (<ehwsyglrpg-nh 2, where <e is pyroglutamic acid) and gnrh-ii (<ehwshgwypg-nh2). in contrast to gnrh-i, exact function of gnrh-ii is not well identified. however, it was indicated that gnrh-ii derivatives have more potent antiproliferative activity on human endometrial and ovarian cancer cells in vitro than gnrh-i analogs. some kinds of tumor cells (e.g. breast, prostate, colon) produce gnrh-i and gnrh-ii, and express their receptors. however, the presence of functionally active gnrh-ii receptors on cancer cells is still not clear 1-2 . on the other hand, gnrh-ii analogs, both agonists and antagonists, induce apoptosis in many different cell types. in contrast to gnrh-i agonists, gnrh-ii agonist [d-lys 6 ]gnrh-ii does not activate nfκb, therefore does not protect the cancer cells from apoptosis [3] [4] [5] . in the regulation of apoptosis, not only the pro-and antiapoptotic proteins play a role 6 , but other molecules also attend (e.g. ck2, casein kinase ii enzyme) 7 . our aim was to synthesize new proapoptotic peptide or enzyme inhibitor containing [d-lys 6 ]gnrh-ii based derivatives and investigate their in vitro cytotoxic, cytostatic and apoptotic effects. the sythesis of [d-lys 6 ]gnrh-ii, which is one of the most potent gnrh-ii agonist, was carried out on solid phase using fmoc/tbu strategy. different proapoptotic peptides or enzyme inhibitors were attached directly to the gnrh-ii derivative. in vitro cytostatic and cytotoxic effects of gnrh-ii conjugates were investigated by mtt-assay using different human breast (mcf-7 and t47-d) and colon carcinoma (ht-29) cell cultures. early apoptotic effect of gnrh-ii conjugates was studied by flow cytometry on mcf-7, t-47d and ht-29 cell lines. according to our results, these conjugates might be potential candidates for cancer treatment, because the combination of a targeting moiety (gnrh derivative) with proapoptotic peptides or enzyme inhibitors and drug molecules might have synergistic activity resulting in highly potent multifunctional therapeutic agents. department of pharmaceutical and pharmacological sciences, university of padua, padua, italy melanoma is the most deadly skin cancer with an increasing incidence. its therapy continues to be a challenge since, regardless of the treatment used, longterm survival is quite uncommon. melanoma cells are characterized by high levels of glutathione s-transferase p1-1 (gstp1-1) isozyme. this enzyme is also highly expressed in solid tumours and drugresistant cells, where a role for this protein in the regulation of cell proliferation has been described. 1 in the past, lyttle et al. synthesised glutathione (gsh)linked anticancer prodrugs activated by physiological concentration of gsts. 2 structure and mechanistic studies showed that in the gst-gsh complex, the gst tyr residue placed near the s atom of the cys in gsh, is in the phenoxide form to facilitate the deprotonation of the sulfhydryl group in gsh, making it able to react with electrophilic species. 3 by taking advantage of this active-site geometry, we synthesised two tyrosinase activated melanoma prodrugs (4-methoxyphenol and n-acetyl-4-s-cysteaminylphenol) linked to gsh. in these compounds an ethoxycarbonyl group was placed between the gsh sulphur, oxidate to sulfone, and the tyrosinase-activated prodrugs. in this way the tyr phenoxide (tyr7 in gstp and tyr6 in gstm isozyme) would be able to abstract one of the acidic methylene protons linked to the sulfone moiety. such a deprotonation should trigger a beta-elimination, resulting in the release of melanoma prodrugs. the stability of the obtained putative prodrugs has been evaluated in different ph buffers and in rat liver and kidney cytosolic fractions. synthetic peptides represent first choice biomolecules, with respect to proteins and mab, for the monitoring of population variability and receptor functionality associated to a tumor pathology due to their favorable pharmacokinetic profile. it has been demonstrated that the surface molecule integrin alfa-v beta-3 could be an ideal target for the interaction with melanoma directed radiolabeled peptides. this integrin is indeed overexpressed both in tumor cells and in tumor vessels endothelium, while it is not expressed in the majority of normal tissues. cyclic peptides with the sequence arg-gly-asp (rgd), are known to bind the integrin alpha-v beta-3 with high affinity and selectivity and could conveniently target a radiotherapeutic to melanoma cells. 1 we previously demonstrated that the cyclic rgdfk peptide interact with high affinity to a375 human melanoma cells. 2 the aim of this work was to conjugate the 99mtc to the peptide by using the [ 99m tc(n)(pnp)] 2+ system (pnp=aminodiphosphines) to obtain a radiolabeled peptide useful in melanoma imaging. 3, 4 spectrum. a nh(i)→βch(i-1) cross peak more intense than the nh(i)→βch(i) cross peak is observed when the peptide adopts the 2.05-helixl conformation. by contrast, an opposite trend of intensities of the same noe cross peaks indicates the occurrence of a 310-helical conformation. by using this method, we were also able to determine that polar solvents, such as mecn or meoh, may induce a 310helical structure in deg homo-peptides which otherwise adopt the 2.05-helix conformation in cdcl3. this finding allows us to rapidly switch these peptide series from "short" (310-helix) to "long" (2.05-helix) by changing the solvent only, thus making them extremely attractive tools. school of biological sciences, university of auckland, auckland 1142, new zealand naturally occurring anti-freeze proteins (afps) enable organisms like polar fish to survive the freezing temperatures of their natural habitat. 1 as well as being cryoprotective, afps exhibit properties of ice recrystallization inhibition (ri). 2 the ability of afps to influence the size, morphology and aggregation of ice crystals can be used in food technology, where the growth of ice crystals in frozen foods is of primary concern. 3 chemical synthesis of afps enables access to pure material for structural and functional studies. our new research programme in the 'food and health' area, prompted us to develop tailor made anti-freeze peptides for potential applications in the frozen food industry. 'multiple salt-bridge analogues' of the naturally occurring winter flounder afp were synthesized and evaluated for their antifreeze activity in comparison to the natural sequence. details on the molecular modelling, structural characterization and anti-freeze activity analysis on the synthetic afps will be presented. several disorders are now believed to be conformational diseases caused by misfolding in the structure of specific proteins leading to the gradual aggregation and deposition of these abnormal proteins. attempts to develop effective therapies for these proteopathies (such as alzheimer's, parkinson's and huntington's disease) have been directed toward reducing the production of the proteins, inhibiting their aggregation, or promoting their removal. conformation-dependent antibodies that specifically target misfolded proteins are proving to be useful tools to study the pathogenesis and as possible treatments of these diseases. we have previously shown that active immunization with tetrapalmitoylated aβ1-15 peptide embedded in liposomes (aci-24) was capable of eliciting a conformation-specific immune response in mice. 1 the antigenic peptide was shown to mimic the aggregated β-sheet conformation of the pathological a β42 peptide on the surface of the liposomes by cd, ssnmr and tht assay. we have also reported the modulation of the conformation of aβ 1-15 derived sequences by means of different lipidation patterns, lipid chain lengths and liposome surface charge. here we present the characterization of this panel of liposomal peptide constructs by atr-ir and correlate the results with previous studies by cd. in summary, atr-ir secondary structure analysis of a battery of amyloid-derived lipopeptides allowed to study the key parameters responsible for the adopted conformation on the lipid bilayer. the presented strategy to control the conformation of liposomal peptide immunogens could be applied to the rational design of vaccines against a range of protein misfolding diseases, such as alzheimer's disease. cyclic lipodepsipeptides (clps) are secondary metabolites produced by various bacteria, most prominently bacillus and pseudomonas. these often display potential antimicrobial properties, frequently ascribed to the ability to disrupt cellular membranes or form ion-pores. clps are a structurally diverse group of molecules always consisting of an oligopeptide chain cyclized by a lacton bond and a fatty acid moiety. they are divided into several groups based on sequence similarity. one such group is the viscosin group, which contains clps produced by pseudomonas that all feature nine amino acids and a conserved sequence pattern of hydrophobic and hydrophilic residues. 1 recently, we have extensively described the self-assembly properties of one viscosin group member, pseudodesmin a. 2, 3 we have shown that in non-polar organic solvents this clp forms anisotropic supramolecular structures of unrestricted size, reminiscent of the ability to form ionpores in the non-polar cellular membrane environment. here, we report on how the most prominent structural variations within the viscosin group affect the conformation and self-assembly properties. since the well-defined monomer conformation of pseudodesmin a is key to this self-assembly, the most striking variation within the viscosin group is the variability of the leu5 stereochemistry. for the first time, a conformational study will be reported for clps featuring an l-leu5 residue, such as viscosin and viscosinamide. the results of this study contribute to a deeper understanding of the structure-function relationship of clps in general. effective design of peptide mimicking protein helical segments is a great challenge. an intriguing approach to improve the helical content in a peptide sequence is achieved through the "peptide stapling" strategy using a hydrocarbon cross-linker (staple) 1 . in this study we have investigated the effect of the introduction and location of staples on secondary structure of sequences derived from a reported peptide, mimicking cullin3 binding region to kctd11 2,3 , to gain an insight into the designing of new molecular entities able to modulate the kctd11 oncosuppressor activity. indeed, kctd11 plays a crucial role in the ubiquitination of hdac1 by acting, in complex with cullin3, as an e3 ubiquitin ligase, thereby affecting hedgehog activity and medulloblastoma growth. in the designed peptides the stapling has been achieved by ring-closing olefin metathesis between two s-2-(4'pentenyl) alanine residues incorporated at i and i+4 positions and into different sites within the peptide chains. the stapling position has been chosen on the basis of cullin3-kctd11 complex model. circular dichroism studies have highlighted that the stapled peptides exhibit remarkable and different changes in secondary structure contents in comparison with the linear precursors and the unmodified sequence. a nmr conformational analysis will allow the determination of the conformational preferences of the stapled peptides at atomic resolution with also relevant implications for the kctd-cullin3 recognition. based on our findings from protease-mediated ligation of cleavage-sensitive peptides and proteins using ionic liquids (ils) as additives 1 and from chemical acylation reactions of peptides in ils, 2 we hypothesized direct and unique interactions between biomolecules and ils as the molecular reason for il-mediated structural effects. to verify this, we performed systematic structural investigations of suc-ala-xaa-pro-phe-pna and ala-xaa-pro-phe model peptides dissolved in aqueous imidazolium-based ils using high resolution magic angle spinning (hr-mas) nmr spectroscopy. highly resolved nmr spectra are obtained that allow for complete 1 h resonance assignments of the peptides as solutes and the ils as solvents. the changes in the proton chemical shifts δδ( 1 η) of a peptide on dissolution in ils arise from direct interactions between both, including il-induced structural/conformational changes of the peptide molecules. 3, 4 the strength, position, and type of these interactions are influenced by the nature of the amino acid residues in the peptide sequence as well as the nature and type of the il anions. downfield shifts indicate strong interactions between the ils and the peptide backbone, whereas upfield shifts point to hydrophobic interactions and stacking effects with the il imidazolium ring. the pattern of δδ( 1 η) along the peptide chain reveals the sites and the type of interactions. the extent of δδ( 1 η) implies a significant effect on the structure of the peptides. different δδ( 1 η) were obtained for cis-and trans-configured peptide isomers and point to a conformer-specific il/peptide interaction. calcitonin is a 32-amino acid polypeptide hormone, which takes part in calcium metabolism in bones. it belongs to a protein family whose members may form amyloid fibrils. these fibrils (or pre-fibrillar aggregates) are related to serious diseases known as amyloidoses, whereas amyloid-like fibrils may have natural functional roles in many organisms. the amyloid form of human calcitonin is related to medullary thyroid carcinoma. using our online consensus prediction tool for 'amyloidogenic propensity' of protein molecules "amylpred", a novel hexapeptide of human calcitonin was predicted as a novel, possible amyloidogenic peptide. we investigated experimentally its ability to form amyloid fibrils, utilizing tem, x-ray fibre diffraction, atr ft-ir spectroscopy and polarized light microscopy. analysis of the results shows that this peptide self-assembles into amyloid-like fibrils and that amyloid fibrillogenesis is mediated via nuclei of liquid crystalline nature, known as spherulites. we thank the university of athens for the financial support. school of pharmacy, university of norwich nr4 7tj ; 3 inserm u982, université de rouen, 76821 mont saint aignan 26rfa, a rfamide neuropeptide, is the ligand of gpr103 and the system 26rfa/gpr103 is involved in feeding behaviour [1] . a structural study by nmr showed that 26rfa, in a membrane mimetic medium, possesses a helix p4-r17 -linker k18-g21 -turn f22-r25 motif. sar studies on truncated fragments of 26rfa indicate that, in addition to the turn motif, additional residues are required to maintain a correct biological activity (26rfa ec50=10,4nm; 26rfa10-26 ec50=37,5nm; 26rfa13-26 ec50=95,3nm; 26rfa16-26 ec50=237nm) [2] . our aim is to synthesize new gpr103 ligands using an iterative process combining rational conception, peptide and modified amino acid syntheses, biological and structural analyses. structural and biological results will allow us to find new modifications and conceive more potent analogues. using nmr, we demonstrated that 26rfa11-26 encompasses a nascent helix and a turn while 26rfa13-26 possesses only the c-terminal turn structuration. the destabilization of the helical structure on fragments with a shorter n-terminal region could explain the decrease of the biological activity of 26rfa fragments. we decided to focus on the importance of a stabilized helix on the biological activity. we chose the hydrogen bond surrogate method which stabilizes a helix by replacing the main hydrogen bond with a covalent link without modifying the side-chains [3] . syntheses, nmr characterization and molecular modelling of analogues will be discussed. acknowledgments: this work is supported by erdf funding through the is:ce-chem project and interreg iv a france-(channel)-programme. firstly, it induces a constraint by restricting the ı o-dihedral angle to values around -60°. secondly, the xaa-pro peptide bond is subject to cis-trans isomerization characterized by an increased cis population and an activation energy that is low when compared to the other amino acids. besides, the five-membered ring of the pro residue can adopt two main distinct conformations (up-puckered or downpuckered) that are almost equally abundant in peptides and proteins. a variety of mimics and analogs have been designed in order to control the conformation of the peptide backbone and/or to alter the cis/trans ratios and the rotational barriers for cis-trans isomerization. in this presentation, nmr studies 2 and theoretical calculations 3 have been performed on model peptides ac-ser(ψpro)-nhme, (s,s)-ac-ser(ψh, cf3 pro)-nhme, and (r,s)-ac-ser (ψ cf3 ,hpro)-nhme. 4 their thermodynamic and kinetic features have been analyzed in chloroform, dmso, and water, allowing a precise description of their conformational properties. we found that trifluoromethyl c δ -substitutions of oxazolidine-based pseudoprolines can strongly influence the cis-trans rotational barriers with only moderate effects on the cis/trans population ratio. in chcl3, the configuration of the cf3-c δ entirely controls the ψ-dihedral angle, allowing the stabilization of γ-turn like or ppi/ppii-like backbone conformations. moreover, in water and dmso, this c δ -configuration can be used to efficiently constrain the ring puckering without affecting the cis/trans population ratio. theoretical calculations have ascertained the electronic and geometric properties induced by the trifluoromethyl substituent and provided a rational understanding of the nmr observations. in contrast to formation of the native state, aggregation of misfolded protein molecules into so-called amyloid fibrils is a strongly irreversible, kinetically-controlled process which may lead to structural variants of fibrils composed of chemically-identical protein chains. this poorly understood aspect of polymorphism of amyloid fibrils has important implications in vivo in the course of amyloidassociated neurological disorders -e.g. in the form of prion strains in creutzfeldt-jakob disease. in this paper, we examine cross-seeding behavior of a model amyloidogenic polypeptide -bovine insulin (bov-ins) and recombinant lys b31 -arg b32 insulin (kr-ins). fibrils of bov-ins and kr-ins exhibit characteristic "fingerprint" features in the amide i' infrared region. these spectral traits are transferred to daughter generations of fibrils upon cross-seeding of native bov-ins with kr-ins fibrils and vice versa. this study highlights an interesting aspect of a non-anfinsenian behavior of aggregating insulin: the memory effect. our results indicate that fine infrared features in the amide i region of fibrils from closely related proteins may not reflect different amino acid composition but rather the fact that a particular amino acid sequence favors an amyloidogenic pathway that determines a certain and spectrally-distinct stacking mode of β-strands. an encounter with an alien seed on the amyloidgenic pathway would hijack the yetnative monomers and force them to follow the assembly pattern (and resulting ir characteristics) of the preformed fibril. phosphorylation and oglcnacylation are dynamic intracellular protein post-translational modifications that often are alternatively observed on the same protein serine and threonine residues. phosphorylation and oglcnacylation commonly occur on residues in natively disordered regions of proteins, and often have opposing functional effects. in the microtubule-associated protein tau, hyperphosphorylation is associated with protein misfolding and aggregation as the neurofibrillary tangles of alzheimer's disease, whereas oglcnacylation is stabilizing. a series of peptides derived from the prolinerich domain (residues 174-239) of tau was synthesized, with the free ser/thr hydroxyls, with phosphorylated ser/thr, with oglcnacylated ser/thr, and with the dietylphosphorylated ser/thr. phosphorylation and oglcnacylation were found by cd and nmr to have opposing structural effects, with phosphorylation favoring polyproline helix (ppii) formation, with oglcnacylation opposing ppii, and with the free hydroxyls intermediate. phosphorylation of serine and threonine residues in prolinerich sequences was found to induce ppii in other proline-rich sequences, with significant conformational restriction at phosphoserine and phosphothreonine residues observed by nmr. notably, serine and threonine residues are common in proline-rich domains of proteins, and ppii is a common conformation observed for phosphorylated ligands in protein-protein interactions. the ser/thr diethylphosphate was found to be a practical mimic of oglcnacylation, as an easily synthesized neutral amino acid with similar sterics to oglcnac and strongly opposing ppii. dept. of biology, university of padova, padova 35121, italy we recently established that designed hairpin peptides interfere with the amyloidogenesis of both human pancreatic amylin and α-synuclein ( α-syn), two 'unstructured' polypeptides associated with human diseases. 1 in the case of α-syn, the most potent 'amyloid inhibitors' act by diverting α-syn to non-amyloid aggregates. the binding sites for these agents and nonhairpin peptides of similar sequence have been determined by 2d-nmr-monitored titration studies using universally 15 n-labeled α-syn. in the case of the hairpins that produce non-amyloid aggregates from α-syn, initial binding occurs in the non-amyloidogenic c-terminal segment of α-syn and the hairpin peptide is incorporated in the resulting aggregates. nmr studies also serve to define binding interactions and conformational changes that may be essential early steps in aggregation and pre-amyloid state processes. mt-ii is a long acting, non-selective super-agonist of melanocortin gpcr receptors, 1 characterized by lactam bridge between residues i and i+5 stabilizing a type-ii β turn structure. 2 the recently introduced cui-catalyzed azide-alkyne 1,3-dipolar cycloaddition, presents a promising opportunity to develop a new paradigm for an orthogonal bio-organic and intramolecular side chain-toside chain cyclization. we introduced this [1,2,3]triazolyl-mediated cyclization in a pthrp model peptide, and demonstrated that i-to-i+4 side chain-to-side chain cyclization offers a powerful approach for generating stable helix mimetic structures. 3, 4, 5 in the current study we explored the potential of the i-to-i+5 side chain-to-side chain 1,4-disubstituted [1, 2, 3] triazolyl-bridge to stabilize -turn conformations. we designed a series of [1,2,3]-triazolyl containing cyclo-heptapeptides, derived from the model peptide lactam mt-ii, differing in the size of the bridge, the location and orientation of the [1,2,3]-triazolyl moiety. conformational and biological studies, performed on this series demonstrate that [1, 2, 3] triazolyl-mediated side chain-to-side chain cyclization results in the conformational stabilization of type-i β turn generating heterodetic cyclopeptides with enhanced in vitro potency and improved receptor subtype selectivity. this study confirms the versatile nature of the [1, 2, 3] triazolyl-mediated side chainto-side chain cyclization. moreover the chemical accessibility, functional compatibility, and metabolic stability suggest that the [1, 2, 3] international associated laboratory samuel de champlain, 5 regional platform for cell imaging of haute-normandie (primacen), institute for medical research and innovation (irib), university of rouen, 76821 mont-saint-aignan, france scorpion toxins are polypeptidic molecules capable of selectivity binding to ion channels with the highest affinities and represent natural peptide blockers that are good tools to characterize new or particular types of ion channels. astroglial cells appear to express all ion channels species even voltage-gated ion channels previously suspected to be present only in excitable cells; but these channels are poorly studied and their function are not still completely elucidated.thus, the purpose of the present study was to investigate for the first time, the global effect and the potential protective effect of one scorpion toxin acting on kv channels, the pi4,on h2o2-induced oxidative stress and cell death in rat astrocytes. pi4 is a 38-residue toxin crosslinked by 4 disulfide bridges, isolated from the venom of pandinus imperator1 and is one of the most potent and selective inhibitor of shaker b and kv1.2 channels2. in our study, pi4 was obtained by solid phase peptide chemical synthesis(spps) and named spi4. the identity and homogeneity of spi4 were also verified by hplc, mass spectrometry analysis(maldi-tof) and an enzyme-based cleavage to determine the assignment of spi4 half-cystine pairings.the synthetic spi4 was found to be indistinguishable from the natural toxin. incubation of cultured astrocytes with graded concentrations of h2o2 for 1h provoked a dose-dependent reduction of the number of living cells as evaluated by flow cytometric analysis and lactate dehydrogenase assay. interestingly, pre-treatment of astrocytes with very low concentrations of spi4(10-8 to10-12 m),dose-dependently prevented cell death induced by h2o2, and the effect of spi4 was accompanied by a marked attenuation of ros accumulation. also, spi4 stimulated sod and catalase antioxidant activities in a concentrationdependent manner,and blocked h2o2-evoked inhibition of sod and catalase activities. in addition, at low concentration (10-8 to10-14 m) the toxin exhibits no toxic effect on astrocytes.taken together these data indicate that the spi4 acts as a protective agent on astrocytes from oxidative assault and then might have therapeutic value in the potential treatment of a number of neurodegenerative diseases. [4] . in order to select small active peptides, the library was kept at the minimum size and was built using only 12 selected d-amino acids to avoid structural redundancies. to further increase the chemical diversity [5] a set of different carboxylic acids were coupled to the n-terminus. the iterative screening led to the identification of an n-terminally modified tetrapeptide with a very high radical-scavenging activity. by analyzing several peptide variants, we found that antioxidant properties were strongly sequence-and structure dependent, because scrambled or proline-scan variantswhose secondary structures are deeply altered -are much less effective. data on peptide structure-activity relationship will be presented. an investigation of the in vitro effects produced in cellular models of oxidative stress are also in progress. structural analysis of peptide-analogues of zp proteins with amyloidogenic properties: insights into mammalian zona pellucida formation. n.n. louros, v.a. iconomidou, s.j. hamodrakas* department of cell biology and biophysics, faculty of biology, university of athens, athens 15701, greece a filamentous structure surrounding mammalian oocytes, called zona pellucida, is responsible for species-specific sperm binding. this extracellular porous structure is composed of 3-4 glycoproteins, termed zp1-4, which assemble into filamentous polymers that are cross-linked by disulfide bonds. all zp proteins present a unit with high sequence homology at their c-terminal end, designated as the zp domain. this structural module is found in a family of extracellular proteins with various functions and from a wide variety of organisms (zp domain proteins). it is a binary structure of approximately 260 amino acids, divided into two domains, the n-terminal region named zp-n and the c-terminal region known as the zp-c domain. the zp-c block is associated with protein function, whereas the zp-n domain is responsible for protein polymerization. structural analysis revealed a possible polymerization model based on backbone interactions between specific parts of the zp-n units. peptide-analogues of the aforementioned segments were synthesized, since the ''amylpred'' application, an 'amyloidogenic determinant' prediction algorithm developed in our lab, predicted them as being potentially 'aggregation prone'. our experimental data clearly indicate that these peptides do actually fold and self-assemble into amyloid-like fibrils. therefore, all the derived data suggest that mammalian zona pellucida is a novel functional amyloid, similar to its biological equivalent, silkmoth chorion that has been established to be a natural, protective amyloid. we thank the university of athens for the financial support. biostrucutres and bioimmaging institute of national research council, naples, italy the definition of the molecular basis of human diseases is one of the most important goals of structural biology. nucleophosmin (nmp1) is an ubiquitously expressed protein is a nucleolar chaperon and has a key role in several biological processes from ribosome biogenesis, to cell-cycle regulation and is a frequently target of oncogenic mutations in acute myeloid leukaemia (aml) [1] . these mutations occur predominantly at the c-terminal domain of npm1 compromising its stability and causing the aberrant translocation of npm1 to the cytosol. this domain is structurally characterized by the presence of three alpha helices, and the folding of cter-npm1 proceeds via an extended nucleus and the denatured state retains significant malleable structure at the interface between the second and third helices [2] . here, we have further investigated the structural determinants in the folding process of the c-term npm1 domain and on the basis of available structural and composition data, several peptides covering the three helices were designed and synthesized. their conformational properties were investigated by cd and nmr spectroscopy: these studies demonstrated that once removed from the protein architecture helix1 and helix2 substantially miss fully ordered conformations while helix3 retain a high helical content also in mutated variants. our observations may help structure-based drug-design inserm u829 university of evry-val d'essonne, bâtiment maupertuis, 91025 evry, france sl21 (stop-like protein with mass of 21 kda) is a neuronal protein with a calmodulin-binding and microtubulestabilizing activity. microtubule binding is inhibited by calmodulin 1,2 . the lack of the stops gives rise to defects in synaptic plasticity, as observed in schizophrenia 3 . to study the sl21 protein's structure and its interaction with calmodulin, we have used a 44 residue peptide derived from sl21, containing the microtubule binding motif mn. the structure of the peptide has been analyzed using circular dichroism (cd) spectropolarimetry. the cd spectra showed that the peptide's secondary structure consists in a predominantly β-sheets, while the tertiary structure consists in rather folded 3d-structure. the temperature dependence of cd indicated that the secondary structure of the peptide remained very stable between 5 °c and 95 °c, while the tertiary structure changed at about 35 °c. the interactions between the sl21 peptide and calmodulin have been studied using the isothermal titration calorimetry (itc). the results showed that calmodulin binds the peptide with the affinity of 104 m-1, in a ca 2+ -independent manner, the reaction being driven by entropic contributions. our results might contribute to understanding the microtubule stabilizing activity of the sl21 protein. peptides 4, 5 . based on the structure in polar environment a transition from 310-helical to an α-helical conformation at the water/lipid interface in bilayer membranes is hypothesized 3 the efficient method development for the analysis of proteins, peptides, and synthetic oligonucleotides with a novel hybrid reversed phase column f. becker *1 , n. shoji *2 , a. matsui *2 , c. yokoyama *2 , t. sato *2 , t. takai *2 , and n. kuriyama *2 *1 ymc europe gmbh, *2 ymc co., ltd. the recent development of bio-pharmaceutical industry has been remarkable and an effective analytical method with higher sensitivity, superior selectivity, and increased speed has been required in the characterization of peptides, proteins and oligonucleotides by highperformance liquid chromatography (hplc). the method development of reversed phase hplc requires optimization of several conditions, such as bonded-phase, column efficiency, solvent type, ph and temperature. especially ph and buffer type is a most important parameter to control retention of biomolecules which have multiple ionic functional groups. furthermore, temperature often becomes a key tool to achieve better peak shapes and resolution for larger molecular-weight compounds. although silica based reversed phase columns have been widely used for separation of biomolecules, they have low stability under alkaline conditions and a limited usable ph range. to improve the chemical stability at an expanded ph and temperature range , we have developed a new type of organic/inorganic hybrid reversed phase column, ymc-triart c18 and ymc-triart c8. the novel technologies of manufacturing particles and surface modification provide outstanding chemical stability and excellent peak shape for many types of compounds under a variety of mobile phase conditions. in this poster, we will show characteristics of this new hybrid column, and some example cases of efficient method development in separation of the biomolecules such as peptides, proteins and synthetic oligonucleotides. the fully-extended peptide conformation (2.05-helix) is the longest possible for a single α-amino acid as it is characterized by an axial translation per residue of about 3.85 å. therefore, it is extremely attractive for its application as a molecular bridge. however, it is known that the stability of this type of helical structure (relative to the competing, much shorter 3 10-helix) appears to be dramatically governed by the choice of the c-terminal functionality (only esters seem to be appropriate). in this work, we examined the series pyrac-(deg)n-o-(pno2)bzl [where pyrac is the fluorophore 1-pyrenylacetyl, deg is cα,α-diethylglycine, n=1-5, and o-(pno2)bzl is para-nitrobenzoxy] with the deg homo-peptides as fully-extended bridges in a static fluorescence study. to perform a very stringent comparison with our ft-ir absorption and nmr results, the solvent used was chcl3. the trend observed in the quenching efficiencies for the shortest members of the series, the (deg)1-3 peptides, steadily decreases from 0.97 (n=1) to 0.89 (n=2), and 0.64 (n=3). this finding is compatible with the conclusion that these three peptides are all essentially fully-extended in chcl3 solution. however, the quenching efficiencies for the longest deg homo-peptides. 0.68 for n=4 and 0.65 for n=5, are not further reduced. the inevitable conclusion is that the tetra-and pentapeptides populate at least two different conformations (most probably, the 2.0 5-helix and the 310helix), in which the probe dyads are located at different distances. notably, these results fit well with those extracted from our ft-ir absorption and nmr studies in the same solvent of low polarity. we conclude that a combination of terminally aromatic groups, as in the deg homo-peptide series investigated in this work, is not eligible for the fabrication of a stable fully-extended conformation. the surfactant protein c (sp-c) is a 35-residue highly hydrophobic peptide with two covalently linked fatty acyl chains that adopts a mainly helix structure while associated with the membrane. however, it misfolds into a β-rich structure under certain environmental conditions, which suggests that sp-c is in a metastable state 1 . the slow α→β transitions are likely to be caused by the membranedissociation, deacylation1 and exposure to the neutral ph 2 of the alveolar subphase, leading to the formation of amyloid aggregates associated with pulmonary alveolar proteinosis. we have been studying the ph-dependent conformational behaviour of both the acylated and deacylated isoforms of sp-c in a membrane mimetics chloroform/methanol/water mixture using a constant-ph molecular dynamics method [3] [4] [5] . our simulations identify the increase of ph as a promoter of peptide-peptide interactions, which may contribute to the α→β transitions observed at neutral and alkaline ph conditions in the experimental results 2 . we also observe that the loss of structure is more prone to begin at the c-terminal region and that the β motifs start to form between the n and cterminal disordered regions of the sp-c. in addition, the presence of the acyl groups results in a slightly higher structural stabilization of the sp-c helix and in a decrease of the n-terminal plasticity at acidic and neutral ph. since a comprehensive conformational analysis and the elucidation of the mode of association between sp-c and the membrane are essential to understand the interactions involved in structure stabilization and in some specific functions of the peptide, we are now studying both sp-c isoforms in a membrane environment. these studies will not only be the most relevant for the in vivo situation but will also answer to some questions left open by the previous work. were rather small. vp plays a very important role in controlling resorption of water by the distal tubules of the kidney and in regulating the osmotic pressure of blood in mammals. vt, while exhibiting both oxytocin and vasopressin activities, has not changed over the last 300 million years until appearance of the mammals. currently, it occurs naturally in the non-mammal vertebrates [1] . here we report on studies of arginine-vasopressin (cyfqncprg-nh2, avp) and arginine-vasotocin ([ile3]avp, avt) in a mixed dodecylphosphocholine (dpc) and sodium dodecylsulfate (sds) micelles. dpc micelles are considered good models of eukaryotic cell membrane [2] , while the sds micelle improves peptide/micelle solubility [3] . for these studies we use 2d nuclear magnetic resonance (nmr) supported with molecular modelling methods. conformation-activity considerations relationships and the role of the peptide-micelle interactions on the structure of the analyzed peptides will be described and discussed. vibrational and chiroptical spectroscopic investigation on diamide peptide models k. knapp, a m. hollósi, a e. vass, a zs. majer a a eötvös lor nd university, institute of chemistry, laboratory for chiroptical structure analysis, budapest, hungary understanding of peptide folding has great importance in many fields of chemistry. since amino acid residues are the simplest building blocks of peptides, the investigation of their conformational properties could help us in understanding the structures of peptides or in designing peptides of specific 3d structure. describing the conformational building blocks of even the simplest peptide models (e.g., amino acid diamides) can serve as a good starting point to decipher higher-level structural properties of their polymers. among the simplest compounds which include the essential structural elements of polypeptide backbone are the n-methylamides of n'acetylamino acids. these compounds have been the subject of extensive spectroscopic analyses (ftir, vcd, ecd, nmr) [1, 2] . the conformational landscape of these model compounds were analyzed by solution phase vcd and ecd spectroscopy and density functional theory (dft) computations. this work reports systematic vcd and ecd spectroscopic studies in different solvents on synthesized diamide models (ac-α/βhomo-xxx-nhch3, xxx = ala, pro, phe). vcd and ecd data, combined with quantum chemical calculations performed at the b3lyp/6-31++g** and 6-311++g** level of theory was particularly useful for identifying the preferred dipartimento delle scienze biologiche, università di napoli "federico ii", napoli, italy c dipartimento di scienze ambientali, seconda università di napoli, caserta, italy β-hairpin is an important secondary structural element used by many proteins for biomolecular recognition, and thus is an attractive tool for mimetic design 1 . aβ -hairpin motif consists of two antiparallel strands linked by a turn region. in this work we aimed to stabilize a β-hairpin conformation by an interstrand covalent bridge, made through a click chemistry reaction, which yields 1,4-disubstituted 1,2,3, triazole, starting from alkyne and azide reactive groups. in particular, we explored the conformational stability of a series of β-hairpin peptides containing a 1,4-disubstituted 1,2,3 triazole bridge with variable length. we employed the well structurally characterized trpzip2 peptide as a convenient b-hairpin model system. 2 as alkyne amino acids were introduced propargylglycine (pra), homopropargylglycine (hpg) or bishomopropargylglycine (bpg), while the following azide amino acids were inserted: lβ-azidoalanine (dap (n3)), l-γ-azidohomoalanine (dab (n3)), and l-δ-azidoornitine (orn (n3)). all peptides were synthesized and purified. successively, the linear unprotected peptides were cyclized in solution by the cu(i)-catalyzed azide alkyne cycloaddition (cuaac) reaction and purified prior to the spectroscopic characterization. linear and cyclic peptides were analyzed by a combination of cd and nmr techniques. 3 have been investigated. 1 their behaviour has been compared with that of proline in homochiral and heterochiral dipeptide sequences including l-or dphenylalanine. nmr and ir studies as well as x-ray diffraction analysis provide evidence that the additional β-phenyl substituent does not disrupt the tendency of proline to occupy the i+1 position of a β-turn. moreover, the β-turn conformation is stabilised, with reference to l-pro, when l-cis(βph)pro or l-trans(βph)pro are combined with d-phe, that is, in the heterochiral sequences. the pyrrolidine conformation is significantly affected by the presence of the β-phenyl group. the puckering modes that alleviate most the steric hindrance introduced by the bulky phenyl substituent are preferred. the l-cis(βph)pro residue shows a marked propensity for the cγ-endo/cβexo half-chair arrangement whereas the trans derivative exhibits a higher flexibility, with different pyrrolidine shapes being accessible. interactions between the aromatic ring of (βph)pro and that of the contiguous l-or d-phe residue are observed in all the dipeptides crystallised. in solution, they seem to occur only for the heterochiral sequences. this intramolecular aromatic-aromatic interaction may be responsible for the higher β-turn ratio observed for such sequences and also seems to affect the conformational preferences of the pyrrolidine ring. in fmoc chemistry the trt and acm groups are widely accepted as a protecting group for cys. upon incorporation of these cys derivatives onto the growing peptide chain, however, considerable base-catalyzed racemization of cys is known to always occur at the activating and coupling steps with phosphonium or uronium reagents such as pybop or hbtu, respectively. in addition to this, racemization of the c-terminal cys esterified to resins also occurs during the repetitive n α -fmoc deprotection using piperidine. in order to find an s-protected cys derivative applicable for fmoc chemistry that can efficiently suppress racemization of cys both when its incorporation is conducted by phosphonium or uronium reagents and when it is linked to a resin via ester linkage, we introduced the 4methoxybenzyloxymethyl (mbom) group into the thiol function on cys. the mbom group was found to substantially supress racemization of cys (0.4%) during its incorporation mediated by phosphonium or uronium reagents in comparison with the trt and acm groups (8.0% and 4.8%, respectively). even after a 6-h treatment of the peptide resin, in which its c-terminal cys was linked to a trt-type resin, with 20% piperidine in dmf, the mbom group caused a significant reduction in the level of racemization with the c-terminal cys (6.4%) while trt and acm groups led to a considerable extent (30.2% and 27.9%, respectively). next, we demonstrated the usefulness of the mbom group on cys by applying it to ncl chemistry involving the coupling of a peptide thioester and a cys-peptide. in the synthesis of the latter peptide, however, a diastereoisomer with racemization of the nterminal cys tends to be difficult to separate from the intact peptide as its chain length increases. thus, this measure using fmoc-cys(mbom) can facilitate the avoidance of ambiguities in the quality of the synthesized peptides. labeled peptides are important tools in chemical biology to obtain information on their biological function in, for example, cellular communication processes as well as to study the molecular basis of diseases. the functionalization of bioactive peptides with biophysical reporter groups like strongly absorbing chromophores, is most often performed via post-synthetic chemoselective biocojugation reactions, involving amino, thiol, or azide specific reactions. an alternative approach for the installation intense chromophoric organic molecules, is the coupling of nonchromophoric precursor molecules to obtain chromophoric properties in the final product. recently, we have shown that the [2+2] cycloaddition-cycloreversion reaction between peptide-functionalized electron-rich alkynes and electrondeficient cyanovinyl derivatives results in the formation of a new class of π-conjugated peptidic donor-acceptor chromophores. 1, 2 the chromophoric properties of the final product can be tuned by varying the p-electron conjugation pattern. herein, we describe the synthesis of the versatile building blocks, n-a-(4-ethynylphenyl)-n-a-(methyl)-glycine and n-a-(4-(1,2,2-tricyanovinyl)benzoyl)-glycine, their subsequent application in spps to functionalize peptides with d-p-a chromophore precursor molecules, and their uvvis absorption characteristics. as a typical example, alkyne-functionalized aβ peptides, val-ala-ile and lys-leu-val-phe-phe-ala-glu, have been reacted with a series of cyanoolefins to obtain far-red intense imaging chromophores for amyloid-rich peptide aggregates. a novel method for regioselective disulfide formation in mini-proteins by kinetic oxidation control t. lindner, u. haberkorn, w. mier department of nuclear medicine, university hospital heidelberg, im neuenheimer feld 400, heidelberg, germany rigid frameworks are essential for the development of peptide-based alternatives to immunoglobulins. [1] disulfideconstriction covalently stabilizes tertiary structures, which allows further miniaturization of protein-derived drugs by chemical synthesis. while the amino acid assembly steps in solid phase peptide synthesis are highly efficient, regioselective formation of multiple disulfide bonds is still a challenging and time demanding task. even after applying laboriously optimized folding conditions and extensive purification, a successful synthesis of the correctly folded isomer is not guaranteed. [2] to circumvent these problems, a procedure that takes advantage of the different reactivity of free vs. acmprotected thiols during iodine oxidation was developed. this protocol to sequentially form two disulfide bridges in a one-pot reaction was applied to different peptides, such as (a) min23, a mini-protein scaffold for phage-display assisted drug design; [2] (b) α-conotoxin imi, a neurotoxic peptide isolated from the venom of marine cone snails; [3] and (c) endothelin-1, a peptide involved in blood pressure regulation. [4] the presented technique tolerates precarious residues within the to-be-folded-peptide, such as fluorescent dyes or metal chelators as used for bioactivityassays. compared to the individually optimized literature procedures, this novel approach offers significant improvements: overall yields >30% are obtained in first attempts and stepwise formation of the disulfide bridges is performed within a few hours instead of days. in recent thirty years, c-terminal modified peptides have been proved to have greater potential as apis (active pharmaceutical ingredients) due to their increased chemical and enzymatic stability and improved pharmacodynamic properties 2-4 . a prominent example, octreotide 5-6 , an octapeptidoalcohol, has witnessed as a potent anti-cancer agent targeted for gastro-entero carcinomas. in view of synthetic methodology, peptidoalcohol can not be directly prepared by standard spps protocol becouse of the c-terminal structure released from resin are not alcohol but always peptidoacid or peptidoamide. to overcome this problem, a novel protocol of shortened n-1 coupling cycles on merrifield resin and then the ammonolysis of peptedyl resin by an aminoalcohol as the c-terminal residue getting peptido-alcohol as targetting product has been devoloped in our lab. because of the cleavage treatment of peptidyl merrifield resin is not under acidic condition, such as hf or tfmsa, but ammonolysis, some side-chain producting groups(spg) related to boc chemistry like bzl, clz, tos…; must be avoided in sequence assembly. therefore a hybrid orthogonal protection (hop) of boc/fmoc protocol was adopted for the sake of producing naked peptidyl (without any spg) resin before ammonolysis. fifteen peptidoalcohols with different terminal alcohols were conveniently prepared, most of them released form resin with very good yields. due to its cyclic structure, proline is the coded amino acid with a more restricted conformational flexibility. the incorporation of additional groups into the pyrrolidine ring is a useful means to produce new amino acids that combine the conformational properties of proline with sidechain functionality. this is the case of β-phenylproline, (βph)pro, that can be regarded as a proline-phenylalanine hybrid in which the orientation of the aromatic substituent is dictated by the conformation of the five-membered ring and the cis or trans configuration of the phenyl group relative to the carbonyl moiety. accordingly, cis(βph)pro and trans(βph)pro combine the conformational properties of proline with an aromatic side-chain functionality that is rigidly oriented with respect to the peptide backbone, and this may be useful in the design of biologically active peptides and other applications relying on specificallyoriented side-chain moieties. we have developed synthetic procedures for the preparation of the cis(βph)pro and trans(βph)pro stereoisomers in enantiomerically pure form. the methodology is based on the preparation of racemic precursors of each amino acid and their subsequent hplc resolution on chiral columns. multigram quantities of the target amino acids have been isolated in optically pure form and suitably protected for use in peptide synthesis. the importance of peptide cyclization for studying peptide conformation, creating new structures, or for developing peptide therapeutics is well established. in particular, sidechain lactam bridges linking two amino acid residues that are several residues apart in the linear sequence or headto-tail backbone peptide cyclization enable rigidification of the structure and improvement of in vivo stability. native chemical ligation (ncl) is now an established method for producing backbone-cyclized peptides or proteins. the application of ncl to the synthesis of sidechain cyclized peptides is less frequent. head-to-side-chain cyclization by ligating a c-terminal thioester with a cys residue located on a lysine side-chain was used by few authors. the alternative tail-to-side-chain cyclization mode is rare, probably due to the difficulty of installing a thioester group on amino acid side-chains such as aspartic or glutamic acids the reaction of a bis(2-sulfanylethyl)amido (sea on ) group with an n-terminal cysteine residue in water and at neutral ph results in the formation of a native peptide bond. [1] oxidation of sea on results in a cyclic disulfide called sea off having a 1,2,5-dithiazepan-5-carbonyl structure. [2] sea off is a self-protected form of sea on . we show here that bis(2-sulfanylethyl)amido side-chain acid(dab), ornithine and lysine were selected as building block; 1a and n,n'-cbz-1-amidinopyrazole (1b) were selected as guanidinylating reagents for specific situation. for synthesis of n-terminus local cyclo-guanidine peptide, designated peptides were assembled on acid labile solid support such as rink amide resin by fmoc strategy. then either fmoc-dab(boc)-oh, fmoc-orn(boc)-oh or fmoc-lys(boc)-oh was incorporated respectively at n-terminus. fmoc was removed followed by guanidinylating by 1b and then peptide was cleaved by acid. by neutralizing with nmm in acetonitrile solution, side chain amino group and a-guanidine would form 6, 7 or 8 membered local cycloguanidine. the remaining cbz could be removed by hydrogenation. for synthesis of backbone side chain cyclic peptide, bis-fmoc-daa was introduced in the peptide previously on resin followed by removal of fmoc. selective guanidinylate side chain aminogroup by 1a followed by peptide assembling with an insertion of orthogonal protected daa at 1-4 aa apart from first daa. for synthesis of a-nh2sidechain cyclic peptide, first daa should be introduced with orthogonal protected form. 1b was used to guanidinylate a-nh2. after cleavage and neutralization of those two kinds of intermediates, guanidine-bridged marco-cyclic peptide was formed. the resin is also a multipurpose tool for the synthesis of carboxylic acids, esters and thioesters. when the synthesis is completed, the fully protected peptide hydrazide resin is oxidized with either n-bromosuccinimide (nbs) or copper(ii) acetate in pyridine. the resulting acyl diazene resin is then cleaved by peptide displacement at the c-terminus with amine. the fully deprotected peptide amide is finally obtained by treatment with trifluoroacetic acid (tfa). in our approach, we used a 4-fmoc-hydrazinobenzoyl am novagel resin to synthesize a peptide-substituted amide in the c-terminus. first, the oxidative cleavage was carried out with nbs in pyridine and a nucleophile [a protected 4 (aminomethyl) benzimidamide (amba)]. however, the yield of the reaction was very poor. in the next step, we applied copper(ii) acetate in the presence of pyridine and amba. following optimization, the efficiency of the process was significantly improved. herein we discuss the conditions needed to obtain a reasonably high efficiency of the oxidative cleavage in the synthesis of our c-terminal modified peptides using the aryl hydrazine resin linker. blood vessels on tumor tissues, similarly to integrin receptors. this observation suggests cd13 as a selective target for targeted delivery of drugs and nanoparticles to tumor neovasculature using ngr peptides as homing motif. in our work, new cyclic-ngr peptides containing a thioether linkage were prepared. the influence of their structure on the speed of succinimide ring formation and deamidation was evaluated and compared with the previously published data on cyclic-ngr derivatives containing amide bond or disulfide bridge in the cycle (c[kngre]-nh2 and c[cngrc]-nh 2 ). to avoid the deamidation under the conditions used for cyclization, the synthetic routes were optimized. the influence of the ph, ionic strength and temperature of the solution on their chemical stability was investigated. the structure of the cyclic peptides was investigated by circular dichroismand nmr-spectroscopy. receptor binding ability and the influence of the cyclic peptides on the cell adhesion and motility were also evaluated. this work was supported by grants from the hungarian national science fund (otka nk 77485 and k 81596) and the national innovation office (bio_surf, om-00146/2008). clickable peptides and their attachment to oligonucleotides m. wenska, m. alvira, r. strömberg department of biosciences and nutrition, karolinska institutet, novum, se-141 83 huddinge methodology for the ready conversion of peptides into "clickable" azido-peptides with the possibility of selecting either n-terminus or c-terminus connection is presented. 1 synthesis of peptide-oligonucleotide conjugates (poc's) include conjugates of oligonucleotides with peptides known to be membrane penetrating and nuclear localization signals. a general procedure, based on a new activated alkyne linker, for the preparation of poc's has been developed. 2 with this linker, conjugation is effective at room temperature in mm concentration and submicromolar amounts. this is made possible since the use of a readily attachable activated triple bond linker speeds up the cu(i) catalyzed 1,3-dipolar cycloaddition ("click" reaction). the main scheme for conjugate preparation involves sequential conjugation to oligonucleotides on solid support of i) an h-phosphonate based aminolinker ii) the triple bond donor p-(npropynoylamino)toluic acid (pata) and iii) azido-functionalized peptides. the method gives excellent conversion of oligonucleotide to the poc on solid support, and only involves a single purification step after complete assembly. the procedure which makes use of a low concentration of copper ions leads to a product with very little copper left (similar or less than in drinking water). the synthesis is flexible and can be carried out in non-specialist laboratories without the need for specific automated synthesizers since it has been designed to utilize commercially available oligonucleotide and peptide derivatives on solid support or in solution. comparison of alternative deprotection reagents to piperidine for the synthesis of a poly-alanine peptide on the tribute® peptide synthesizer m.a. onaiyekan,* j.p. cain, c.a. chantell, m. menakuru protein technologies, inc. tucson, az, usa in peptide synthesis, piperidine is a common agent for fmoc removal. however, piperidine is a controlled substance which requires special handling and cannot be used in some countries. therefore, it would be useful to identify alternative deprotection reagents to piperidine for fmoc removal. it is well known that poly-alanine sequences have a high propensity to aggregate after the fifth residue. in this application, (a)10k-oh was synthesized using the tribute®'s intellisynth uvmonitoring and feedback system to compare the efficiency of fmoc removal by piperidine vs. three alternative bases (pyrrolidine, cyclohexylamine, and tertbutylamine) in the last 5 cycles of the synthesis. it was found that pyrrolidine produced a higher purity product with fewer deprotection repeats and shorter deprotection times per cycle than piperidine, proving it to be a highly efficient, viable alternative to piperidine for fmoc removal. the endogenous tripeptide gpe also nammed "glypromate" is made up by the three n-terminal residues (glycine-proline-glutamate) of the insulin-like growth factor 1 (igf1). this tripeptide is a partial glutamate antagonist and showed good results in different neuroprotective in vitro and in vivo experiments. 1,2 gpe also binds to glial cells regulating neurotransmitter levels in the brain. 3, 4 however, gpe suffers from poor lipophilicity and a short half-life in vivo. that's why there is a need for more lipophilic and protease resistant analogues of gpe. in this poster we present the synthesis of trifluoromethylated analogues of gpe based on the 2 or 5-cf3-pseudoproline residues. introduction of fluorine atoms on bioactive compounds is known to deeply modify their physico and biochemical properties increasing lipophilicity and resistance to protease. 5 thus, developing a trifluoromethylated analogues, we intend to increase the bioavailability of gpe, keeping the benefit of its neuroprotective properties. our research team is strongly involved in the synthesis of trifluoromethylated alpha-amino acids. recently we published the synthesis of 2-trifluoromethyl-1,3oxazolidines derived from fluoral and (l)-serine and we demonstrated that these five membered ring 5-cf3pseudoprolines are hydrolytically stable and can be considered as proline analogues. 6 that's the reasons why we are interested to replace the proline residue of gpe by those trifluoromethylated compounds. the development of original coupling conditions and the detailed synthesis of two pseudoprolines analogues of gpe will be presented in this poster. modifications. in combination with automated spps, unprecedented access to large peptides and small proteins for biological research has been achieved. we demonstrate the application of this methodology to the synthesis of a variety of peptides on the prelude® peptide synthesizer. exploring the space of fluorine-labeled α-amino acids for solid state 19 f-nmr structure analysis of peptides: rational design, synthesis and applications p. solid state 19 f-nmr is a powerful method to study membrane-active peptides, as it can reveal their conformation, orientation and dynamics when embedded in biomembranes. 1 for this purpose the native peptide has to be selectively labeled with a suitable 19 f-containing amino acid at several different positions. the resulting battery of singly 19 f-labeled analogues is then analyzed by solid state 19 f-nmr. the main limitation to this approach currently lies in the poor arsenal of available 19 f-labels. we have therefore rationally designed and synthesized several specific amino acids bearing a cf3-reporter group, which fulfil all strict criteria to a "proper" 19 f-label. 2, 3 to allow a geometry-based structure calculation, the cf3group has to be rigidly attached to the peptide backbone. we thus rigidified the side chain using either a [1.1.1]bicyclopentane moiety, a cyclobutane ring, or the intrinsic proline framework. this way, suitable cf3-labeled analogues were created as substitutes for bulky hydrophobic amino acids (leu/ile/val/met), for aromatic residues (phe), for polar side chains (ser/thr), and for proline (pro). by now we have applied the developed 19 f-labels for a comprehensive structure analysis of more than ten different membrane-active peptides (gramicidin s, pgla, mag 2, kigaki, sap, temporin a, bp100, etc). recently, several new activators have been introduced into the market, and they were evaluated along with some older activators for their ability to synthesize a range of peptides with shorter and longer reaction times on the symphony® peptide synthesizer. it was found that hdmc, pyclock, comu, hctu, and hatu worked well at shorter reaction times (2x1 min), but pyoxim and tffh only worked well at longer reaction times. the performance of pybop at shorter reaction times was poor only for more difficult sequences. these results are important for selecting an appropriate activator for fast spps applications. the plant cyclotides form the largest family of cyclic peptides 1 . they contain a signature motif referred to as the cyclic cystine knot, which is derived from the cyclic backbone and three inter-knotted disulfide bonds. intriguingly, cyclotides can be boiled, treated with chemicals or enzymes without disrupting their overall fold. thus, they are sometimes labeled as ultra-stable proteins. in addition, cyclotides are tolerant to mutations, and as a scaffold they can successfully accommodate foreign bioactive epitopes of variable sequences 2 . cyclotides share many of these properties with another disulfide containing cyclic plant peptide, the sunflower trypsin inhibitor 1 (sfti-1) 3 . emerging evidence indicates that cyclotides and sfti-1 are valuable not only as peptide stabilizing scaffolds; in combination with their cell penetrating properties, these disulfide rich cyclic peptides have significance as intracellular drug carriers. although both peptides are genetically encoded, studies to ascertain the exact mechanisms of their biosynthesis are currently on going. thus, the synthesis of cyclotides and sfti-1 are currently restricted to chemical means. we have recently adapted a fmoc-spps method for cyclic peptide synthesis, via n-acylurea intermediates with the assistance of microwave irradiation. this method is a safe and convenient alternative to boc-spps and has the ability to be automated conveniently. using this method, parent scaffolds as well as several cyclotide and sfti-1 analogues with potential antimicrobial and matrix metalloprotease activities were synthesized. with the rising interest in the cyclization concept as a tool to impart stability on unstable peptides, the cyclic peptide synthesis method adapted herein is anticipated to have numerous applications. fixed configuration. the nonnatural oligomers have an extended conformational space and are supposed to adopt non-canonical secondary structures 2 . in addition, the backbone modification makes these molecules more stable towards proteolytic degradation. the majority of proteins in nature are post-translationally modified, and the most abundant modification is the protein glycolysation, which introduces wide structural variety to proteins. glycoproteins have an important role in the biological recognition process, such as immunodifferentiation, cell adhesion, cell differenciation and regulation cell growth 3 . new aza-β 3 -amino acids bearing either an azide instead of amine on lys and orn chain or an alkyne group will be described and used in solid phase synthesis to finally performed a click chemistry to cyclize pseudopeptides or to introduced a glycosylated function 4 . true for a series of peptides that display strong corticotropin releasing factor (crf) antagonistic activity. seminal studies by rivier et al. have shown that the incorporation of a lactam bridge in the crf-sequence resulted in an enormous increase in activity and potency, due to stabilization of the bioactive a-helical conformation of the peptide; and the newly designed peptide was called astressin. 1 based on the astressin sequence, we started a truncation and deletion study to arrive at astressin analogs with a reduced size but still remain active as crf antagonists. this study resulted in the smallest active crf antagonist, astressin(30-41). 2 this sequence was further optimized by the introduction of novel covalent constraints, other than the well-known lactam bridge. as a first approach, the alkene/alkane bridge, which can be introduced via ring-closing metathesis via alkenesubstituted amino acid side chains, and as a second approach, the triazole bridge ('click' macrocyclization), via either a cu(i)-or a ru(ii)-catalyzed cycloaddition reaction between azide-and alkyne-derivatized amino acid residues were explored. herein, we will present the details of the synthesis of the alkene-, azide-, and alkyne-functionalized amino acids, their use in spps, and the optimized approaches for macrocyclization. furthermore, the peptides have been characterized by hplc, nmr, lcms, and studied by circular dichroism spectroscopy to obtain insight into the helical propensity of the peptides in relation to the cyclic constraint. the synthesis of a nitronyl nitroxide, c α -tetrasubstituted αamino acid (a class of sterically restricted amino acids that promote the formation of peptide β-turns and helical structures) was achieved by derivatisation of racemic 2amino-5-cyano-indan-2 carboxylic acid [aic(cn)]. racemic boc-aic(nn)-oh was prepared by bis(alkylation) of ethyl isocyanoacetate under phase transfer conditions with 3,4-(bis)bromomethyl benzonitrile as alkylating agent, followed by acidic hydrolysis, n α -boc protection, and saponification of the ester function. resolution was achieved through formation of the diastereomeric amides of (s)-phenylglycinol with chromatographic separation and mild acidic hydrolysis. reduction of the nitrile group to an aldehyde was carried out with raney nickel in the presence of sodium hypophosphite. condensation with 2,3-diamino-2,3-dimethylbutane gave the corresponding tetramethylimidazolidine, which was oxidised with 3chloroperbenzoic acid to the desired nitronyl nitroxide. the uv-vis absorption and epr spectra of the amino acid were recorded and its magnetic properties were examined. in order to develop the synthesis of this peptide using the fmoc solid-phase peptide synthetic methodology, orthogonally protected β-hydroxyaspartic acid was needed. more precisely we wish to dispose of (2r,3r)-n -fmoc-3-tbdm-silyloxy-aspartic acid α -allyl ester instead of the recently reported dmab ester 2-3 indeed, in preliminary assay using this protective group we experienced difficulties during the final cyclisation step 4 . the synthesis was developed starting from inexpensive l(+) dimethyltartrate and extended to the others stereoisomers of the β-hydroxyaspartic acid. structure. for that, we chose to replace proline by silaproline to afford polysilaproline. this study shows the comparison of two polyamino acids: polyproline and polysilaproline polymers. homopolypeptides were synthesized by polymerization of corresponding amino acid n-carboxyanhydride 3 . multicomponent reactions (mcrs) represent a chemical process involving at least three reactants for the formation of several covalent bonds in one operation 1 . by definition mcrs are chemo-and regioselective, convergent stepefficient procedures and take place with high atom economy. the copper(i)-catalyzed 1,3-dipolar cycloaddition of organic azides and terminal alkynes (cuaac) reported by meldal 2 and sharpless3 has been involved in various fields of chemistry and biochemistry research. however only few reports describe the implementation of cuaac and mcrs. 4, 5 recently our research focused on a novel threecomponent reaction based on a cu(ii)-triggered aminolysis of peptide hydrazide resin and an azide-alkyne cycloaddition sequence. 6 copper(ii)-induced oxidative aminolysis of hydrazides generates cu(i), catalyst of the azide-alkyne cycloaddition. this feature was exploited to design a solid phase detaching three-component reaction. the mcr process requires a peptide hydrazide resin, an amino azide linker and an alkyne, resulting in the formation of peptide modified at the c-terminus through an amino 1,2,3-triazole linker. this method can potentially be applied to the synthesis of a large variety of peptide derivatives starting from fmoc-spps assembled peptidyl resins. furthermore, it is not practical to compare hplc spectra from different resin samples (e.g., before and after reaction) directly. a comparison by analyzing the same (mg) amount of resin would involve tedious sample preparation that is extremely error-prone and would be impractical because factors resulting from the increase or decrease of the molecular weight of the resin-bound compounds may have a significant influence on the results. the use of internal reference compounds allows rapid assessment of reactions performed on solid supports. the internal reference compound is bound to the resin together with the substrate and cleaved with the products after completion of the reaction. commercially available compounds can be used for this purpose, or likewise, the reference compound can be generated from the substrate by partial capping of a functionality. the peak integration of the reference compound in the hplc-uv spectra can be correlated directly to those of the rest of the compounds present in the reaction mixture and therefore a quantitative interpretation of the spectra with respect to conversion and yield is possible. here we demonstrate the proof of principle as well as the accuracy of this method. modifier proteins such as ubiquitin are conjugated to protein substrates in cells and thereby mediate various biological processes. 1 of high interest, is the ubiquitin fold modifer 1 (ufm1, 83 residues) which has structural similarity to ubiquitin but has no sequence similarity. unlike ubiquitin, ufm1 has not been extensively studied and little is known about its biological role. to understand ufm1's biological functions, access to pure, homogeneous natural and modified ufm1 protein is essential. chemoselective ligation techniques are suitable for providing such proteins. recently, a variation of the α-ketoacid-hydroxylamine (kaha) ligation 2 was developed, which utilizes the chemoselective reaction between a c-terminal peptide αketoacid and a n-terminal 5-oxaproline. 3 this modified form of the kaha ligation furnishes a native peptide bond and a homoserine residue. this ligation is useful for the synthesis of proteins from two unprotected protein segments in aqueous buffers. for the synthesis of larger proteins, a sequential ligation strategy is necessary. using ufm1 as the model system we have developed a sequential ligation procedure using kaha ligation with 5-oxaproline. applying the new sequential ligation strategy we have prepared ufm1 by total chemical synthesis. we have also prepared a cterminal thioester surrogate of ufm1 protein, which is suitable for conjugation to proteins of interest. the syntheses required the development of a bifunctional peptide segment bearing an α-ketoacid and an orthogonally protected 5-oxaproline. the preparation of the protein segments, their intermediates, the deprotection, and sequential kaha ligations towards the syntheses of ufm1 protein and c-terminal modified thioester ufm1 protein will be discussed. affinity and biological activity, we have designed and synthesized new analogues by multiple n-methylation of hut-ii(4-11) backbone amide bonds. all the peptides were performed by a novel synthetic approach, in which the introduction of n-methyl groups occur during regular solidphase peptide synthesis. on these new ligands we evaluated the binding affinity and biological activity at the ut receptor and performed preliminary nmr conformational studies. since that time a number of different machines have been used to automate peptide synthesis. modern machines are following two general setups; the so called "single approach" and the "parallel approach". in the single approach, the machine is developed to synthesize one or few peptides simultaneously. the user is able to optimize the synthesis conditions on each single peptide and each single coupling step. the maximum product quality regarding purity and yield is the major task of this approach. in the parallel approach, the machine is developed to synthesize a huge number of different peptides in the same single setup and time-frame. the user always has to find a synthesis protocol appropriate for the needs of each peptide to reach the maximum quality, knowing that there will be always a number of failed peptides. as a result you will find both types of peptide synthesizers in laboratories all over the world: the single machine, for the complicated peptides, and the parallel machine, allowing generation of multiple peptides with standardized protocols for each. the tetras is the first instrument combining the advantages of both machine types and allows the user to synthesize up to 106 different peptides in parallel. each peptide can have its own individual synthesis protocols, separate of all others. the user can combine different synthesis scales, peptide lengths, and activator reagents in one run. finished peptides can be removed and new peptides can be started while the tetras is still running. the tetras allows the user to establish an uninterrupted production shop using one instrument only. siemion, i. z.; peptide res the peptides: analysis, synthesis and biology monitoring peptide folding in membrane-active peptides: a time-resolved spectroscopic study e. gatto a cordopatis p. 31st european peptide symposium sar studies of triazolyl-containing cyclopeptides: a defined -turn structure increases potency and selectivity to melanocortin receptor subtypes c. testa, a,b proc. natl. acad. sci proc. natl. acad. sci usa multicomponent reactions microglobulin: a "difficult" protein s. abel, m. beyermann 1 the protein ß2-microglobulin constitutes the noncovalently bound light chain of the major histocompatibility complex class i (mhc) and plays an essential role in the dialysisrelated amyloidosis. [1, 2] to examine the amyloid fibrils of the ß2-microglobulin (ß2-m) via infrared spectroscopy we intended to synthesize 13-c-labeled ß2-m. [3] due to the two cysteine residues in positions 25 and 80 we used the native chemical ligation (ncl) strategy for assembling the 99-mer protein. this necessitates the synthesis of three segments which was accomplished on solid phase using the fmoc/t-bu chemistry. the preparation of the segments had to be optimized with respect to aggregation, aspartimide and piperidide formation, trifluoracetylation, and s-tert-butylsulfonium formation. additionally, ncl steps had to be optimized, because of "internal" thioester formation, dimerization and the formation of side-products of the activated n-terminal segment peptaderm inc., krakowskie przedmie cie str. 13, warsaw, poland immunosuppressors, such as cyclosporine a (csa) and tacrolimus®, are routinely used in prevention of graft rejection after organ transplantation and in therapy of some autoimmune diseases, including skin inflammation. a naturally occurring in linseed oil cyclolinopeptide a (cla, c(-pro-pro-phe-phe-leu-ile-ile-leu-val) 1 possesses a strong immuno-suppressive activity, comparable at low doses with that of csa 2 , but is much less toxic. we synthesized new cla analogs, containing instead of one proline residue its six-membered mimics, pipecolic acid (pip): c(-pip-pro-phe-phe-leu-ile-ile-leu-val) (1) and c(-pro-pip-phe-phe-leu-ile-ile-leu-val) (2). the incorporation of pipecolic acid residue led to different conformational behavior of the nonapeptide cycle. nmr experiments in cdcl 3 solution showed that cla analogue 1 with the pipecolic acid residue in position 1 was much more flexible than cyclopeptide 2. the new peptides were devoid of toxicity up to 100μg/ml with regard to human peripheral blood mononuclear cells (pbmc), did not inhibit tumor necrosis factor alpha production in blood cell culture, but exhibited dosedependent, anti-proliferative actions for phytohemagglutinin a-activated pbmc. since peptide 1 was more potent it was tested for growth inhibition of l-1210 lymphatic leukemia. the peptide was found to strongly inhibit the cell growth even at low concentration (63% inhibition at 5μg/ml). hiv-1 has emerged as the largest and the most devastating public pandemic in our days, affecting approximately 70 million people worldwide 1 . development of an effective, safe and preventive hiv vaccine remains an urgently needed priority. epitopes for hiv-specific antibodies in elite controllers, a subgroup of long term non progressors, encompassing segments of mper of gp41 and for the v3 loop of gp120 were identified using the phage display technology 2 . immunization experiments with epitopes conjugated to an artificial sequential oligopeptide carrier (soc4), formed by four repeats of the tripeptide lys-aib-gly in tandem, or to the palmitoyl group are currently in progress. all syntheses were performed on a rink amide resin following the fmoc technology. conjugation of epitopes to the soc4 carrier was realized via a chemoselective ligation approach, which generates an oxime bond between the h2n-o-groups of the modified lysine residues and the aldehyde group of each epitope 3 institute of immunology and experimental therapy, polish academy of sciences, 53-114 wrocław, poland 4 peptaderm inc., krakowskie przedmieście 13, 00-071 warszawa, poland nonproteinogenic amino acids have been a tool to modify the structures of natural peptides since a long time 1 . bioactive peptides involved in a physiological and biochemical processes cannot be applied in the therapy because of their instability in physiological conditions. that's why the synthesis of their stable active analogues is a challenge for medicinal chemistry nowadays. 4-trans-hydroxyproline (hyp) is an important building block of natural collagen. it is responsible for the stabilization of collagen super helix, forcing the trans amide bonds configuration with preceding amino acids 2 . at the same time the impact of trans-4-hydroxyproline on the conformation other than the collagen peptide chains of biologically important compounds is little known. it is known that immunosuppressive activity of cla is comparable with cyclosporine a and is associated with the presence of the tetrapeptide fragment pro-pro-phe-phe containing pro-pro cis amide bond. 3 now we present synthesis, conformation and biological activity of new analogues of cyclolinopeptide a (cla), containing 4-transhydroxyproline instead of proline residues in position 6 or 7. we expected that the introduction of the hydroxyl group in the pyrrolidine ring might influence the biological activity and conformation of the native peptide due to its hydrophilic character and hydrogen bonding ability. the linear precursors of modified cla analogues were prepared manually by standard solid-phase procedure "step by step" on wang resin using fmoc/tbu strategy and tbtu as coupling reagent. the cyclizations of linear peptides have been made under high dilution conditions by means of edc/hobt coupling reagents. the biological activity of newly synthesized compounds as well as the conformational study will be evaluated. dip. di scienze ambientali, seconda università di napoli, caserta, italy nmr spectroscopy is a powerful method to perform structural studies on peptides. to completely fulfill the potential of nmr, peptides labeled with stable isotopes ( 15 n, 13 c, 2 h) are essential. 1 peptides are easily prepared on solid-phase but chemical synthesis becomes prohibitively expensive when applied to the incorporation of isotopes. an alternative cost-effective strategy is the recombinant expression of peptides in e. coli as fusion constructs with carrier proteins. 2 the main problem of this approach is the need of chemical reagents or proteases to cleave the target peptide from its fusion partner after purification. proteases may determine the heritage of extrasequence amino acids at the peptide n-or c-terminus, while chemical reagents require harsh reaction condition that may modify target peptides. an interesting solution is represented by the use of inteins as fusion partner. inteins are protein elements that can catalyze their self-excision from a flanking sequences in mild conditions, by adding nucleophilic agents such as thiols or simply by shift of ph and temperature, bypassing the use of proteases or chemical reagents. 3 we used the self-cleaving mxegyra mini-intein as fusion partner for the preparation by recombinant means of two isotope labeled peptides, hplw and qk. 4, 5 the two peptides target vascular endothelial growth factor receptor (vegfr) and have been described to modulate vegf-dependent angiogenesis. our expression and purification scheme allows to obtain homogeneously isotope labeled peptides. the availability of isotope labeled hplw and qk opens the way to nmr studies aimed to characterize the folding dynamics of the two peptides and their structures in complex with vegfr. an nmr method to discriminate between the fullyextended and different helical conformations in a spacer peptide c. peggion*, m. crisma, f. formaggio, c. toniolo icb, padova unit, cnr, department of chemistry, university of padova, 35131 padova, italy the ideal fully-extended, α-peptide conformation, also known as 2.05-helix, is characterized by φ = ψ = ω = 180°t orsion angles. the repeating motif of this foldamer is a pentagonal (pseudo)cyclic structure (called c5), stabilized by an intraresidue h-bond. the n-h and c=o groups in the 2.05-helix are not involved in intermolecular h-bonds. multiple c5 conformations were observed in homopeptides made up of c α,α -dialkylated glycines with both side chains longer than a methyl. this is the case for c α,αdiethylglycine (deg), the residue studied in this work. it is known that deg homo-peptides can adopt the 2.05-helix 1 or the 310-helix depending on environmental factors and nand/or c-terminal moieties. 2, 3 in this communication, we introduce an nmr method to discriminate between the 2.05-helix and the 310-helix based on the observation of cross-peak intensities in the noesy human serum amyloid a (saa) is a highly conserved apolipoprotein produced by the liver under inflammatory conditions accompanying e.g. atherosclerosis, cancer and amyloidosis [1] . it is also known that saa1α isoform has the amyloidogenic properties [2] . till now it is little known about structure of human saa, as it hampers structural studies due to its facile aggregation. the analysis of protein sequence and cd data together with theoretical studies revealed a typical globular structure of the protein [3] . the c-terminal sequence of saa contains three proline residues, which probably are responsible for the unordered structure. recent in vitro studies involving saa and human cystatin c (hcc) revealed direct interactions between the (86-104) fragment of saa and the (93-120) sequence of hcc. the results of elisa test for the (86-104) saa fragment have shown that it binds to hcc very well. the nmr studies for the wild (86-104) sequence found an unordered structure in phosphate buffer. based on these data we decided to check how the point mutations pro→ala in (86-104) saa fragment could influence the peptide's structures. we synthesized four peptides with pro→ala point mutations and we performed cd experiments at different conditions. the results show that two of them contain disordered structure and two α-helical structures. in this project we analyze the solution structures of these peptides at the atomic resolution using 2d nmr supported with molecular dynamics. design and conformational analysis of stapled peptides mimicking cullin3 binding region to kctd11. i. de paola, a l. pirone, a e. pedone, a s. di gaetano, a l. vitagliano, a r. fattorusso, b g. malgieri, b l. zaccaro*, a acknowledgement: this study was supported by eu within the european regional development fund (poig. 01.01.02-00-007/08-04). model of angiotensin ii bound to the at1 receptor in the lipid bilayer environment m.t. matsoukas, t. tselios* department of chemistry, university of patras, gr-26504, patras, greece the renin-angiotensin system () plays a major role in blood pressure regulation. a sequence of enzyme reactions leads to the release of angiotensin ii which interacts principally with the type-1 angiotensin ii receptor (at1), a 359-residue, which belongs to the g protein-coupled receptor family. in the present study, the human at1 3d model was constructed using modeler for the sequence alignment and loop refinement tools. on this basis, the crystal structure of bovine rhodopsin, (pdb code 1u19), was used as a 3d template. the gromacs software and amber99sb forcefield were utilized for molecular dynamics calculations [1] in order to evaluate the binding mode of angiotensin ii. the role of the critical amino acids of the binding site v108, n111, l112, a163, k199, s252, h256, n294 and y292 is being studied. moreover, newest information on the role of the 2 nd extracellular loop by unal et. al. [2] have been implemented on the model, therefore we propose the contribution mechanism of the residues f170-q187 for binding of angiotensin ii to the at1 receptor for activation and signaling. a. stavrakoudis department of economics, university of ioannina, greece one key step in the immune response against infected or tumor cells is the recognition of the t-cell receptor (tcr) by class i major histocompatibility complexes. it has been found [1, 2] that such peptide/mhc complexes can interact with antibodies as well. this happens mainly in the central part of the peptide in class i complexes [1] , or at the cterminal of class ii complexes [2] . in some cases, the same peptide/mhc complex has been found to interact with both tcr and antibodies [3] . in these study a series of supermolecular complexes have been studied with stateof-art molecular dynamics simulations [4] the dipeptide kyotorphin (tyr-arg, kyo) plays a role in pain modulation in the mammalian central nervous system (cns), and is one of the most investigated neuropeptides. the tyr-arg motif exists widely throughout the brain not only as kyotorphin, but also as the n-terminal part of several endogenous analgesic peptides 1,2 . also, this peptide is very rapidly degraded by aminopeptidases 3 . one of the successful strategies in the design of neuropeptides with enhanced stability and improved delivery to the cns is that with the use of non-protein amino acids, like canavanive (cav), a structural analogue and antimetabolite of arginine (arg trichogin ga iv (noct-aib-gly-leu-aib-gly-gly-leu-aib-gly-ile-lol, in which noct is n-octanoyl and lol is leucinol) is an antimicrobial lipopeptaibol, a unique group of membraneactive compounds of fungal origin, characterized by a high content of the nonproteinogenic ca,a-disubstituted glycine aib (a-aminoisobutyric acid). owing to the gem-dimethyl substitution on the c a atom, aib exhibits a strong propensity to induce β-turns and 310/α-helical conformations in peptides. we have previously reported on a fluorescent analog of trichogin ga iv, the primary structure (and acronym) of which are: fmoc-aib-gly-leu-aib-gly-gly-leu-toac-gly-ile-leu-ome (f0t8) where fmoc is fluorenyl-9-methyloxycarbonyl, toac is 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid, and ome is methoxy. the double substitution of an energy donor (fmoc) at the n-terminus and an acceptor (toac) in the trichogin sequence enabled us to make use of time-resolved optical spectroscopies, spanning from the nanosecond to the microsecond time regime, to investigate the conformational propensity and the dynamical features of f0t8. experimental and computational results indicated that the 3d-structural and dynamical properties of f0t8 are characterized by a transition from an elongated helix to a more compact conformation mimicking a helix-turn-helix motif. to further investigate the role of the flexible gly5-gly6 central motif we synthesized a new trichogin analog having the gly6 residue substituted by aib: fmoc-aib-gly-leu-aib-gly-aib-leu-toac-gly-ile-leu-ome (f0a6t8) experimental and computational results indicated that also the f0a6t8 peptide populate two conformations, the dynamics of which were studied at different temperatures using time-resolved spectroscopic measurements. this replacement was demonstrated to stiffen the peptide backbone by reducing the flexibility around the crucial -gly5-gly6-dipeptide unit. the antigen α4β1, a member of the integrin family, is involved in the migration of lymphocytes through endothelium to the site of inflammation. 1 thus, α4β1 antagonists may be useful tools for the treatment of various inflammation disorders such as asthma and inflammatory arthritis. in addition, recent studies indicate that α4β1 integrin promotes angiogenesis by allowing the invasion of myeloid cells into tumors, while α4β1 antagonists prevent monocyte-induced angiogenesis, macrophage colonization of tumors and tumor angiogenesis. 2 aiming to the discovery of novel α4β1 antagonists, a series of new peptide analogues cyclized through cysteine disulphide bonds were synthesized and tested in vivo against angiogenesis in chicken embryo chorioallantoic membrane (cam model) 3 . sar results indicated that: yr-c(cdpc)-conh2 promoted angiogenesis at the higher studied concentration and showed slight inhibition at the lower one, sal-r-c(cdpc)-oh, sal=salicylic acid, showed important inhibition of angiogenesis at dose-dependent manner, yr-c(cdpc)-oh and sal-yr-c(cdpc)-oh both showed no activity on angiogenesis. nmr spectroscopy was applied for the sequential assignment as well as for the elucidation of specific conformational features. experimental noe data were further imposed as distance constraints to a thorough conformational search by applying molecular dynamics simulations. energy refined produced conformers were used as template for the generation of the pharmacophore model associated with the antagonistic activity. such studies are intended to drive a rationalized design and development of this class of inhibitors. hynes r. o. cell, 1992, 69, 11-25. scaffold discovery by phylomers: a novel cd40l specific scaffold derived from glycyl trna synthetase s.r. stone [1] , k. hoffmann [1, 2] , n. milech [1, 2] , p. t. cunningham [1] , m. kerfoot [1] , s. winslow [1] , y-f, tan [1] , m. anastasas [1] , c. hall [1] , m. scobie [1] , p.watt [2] , and r. hopkins [1, 2] [1] drug discovery technology unit, telethon institute for child health research, 100 roberts road, subicao, 6008, western australia [2] phylogica pty ltd, 100 roberts road, subiaco, 6008, western australia biopanning of phylomer 1 phage display libraries against human cd40l yielded a cluster of highly specific overlapping peptide fragments, from three bacterial genomes, corresponding to the highly conserved catalytic domain from the tetrameric gα2β2 class of glycyl trna synthetases. structural analysis of the overlapping peptide fragments described a scaffold consisting of a central βsheet, comprising 4 anti-parallel β-strands, flanked by nand c-terminal α-helices. further structural analysis revealed that these key structural features, which also encompass the crucial atp-binding motifs of the catalytic domain, are conformationally conserved across both tetrameric gα2β2 and dimeric gα2 glycyl trna synthetases, yet importantly, there is only limited sequence conservation across these classes. given the identical function of the described domain and it's structural conservation, we postulated that members of the dimeric gα2 class would display similar cd40l specific binding as the tetrameric gα2β2 class, despite the sequence dissimilarity. to test this hypothesis, structurally equivalent peptide fragments of representative bacterial, archaeal and eukaryotic genomes comprising the dimeric gα2 class were tested for cd40l binding in a process we termed ortholog scanning. the results showed that both archaeal (p. horikoshii) and eukaryotic (h. sapiens) structurally equivalent peptides bound to cd40l with reasonable specificity and inhibited the cd40:cd40l interaction with comparable ic50's to the primary gα2β2 class sequences. similar results were also observed for the representative bacterial gα2 class peptides. that the sequentially diverse orthologous peptides display cd40l specific binding has important implications to the affinity enhancement strategies to develop the scaffold as a therapeutic agent, and in improving its "drug-like" properties. we have initiated 1 an investigation related to the effect of radical species upon structures of some peptide segments. in the proposed experimental protocol, aqueous peptide solution was submitted to gamma ray irradiation in controlled 1-15 kgy doses. the generation of peptide analogues, possibly induced by reactive oxygen species were examined by electrospray triplequadrupole tandem mass spectrometry (collision induced dissociation approach) and amino acid analysis of crude and/or purified by-products. noteworthy, the gamma irradiation process induced, regardless of the peptide sequence, a non-linear and progressive degradation of all peptides assayed. furthermore, these peptides could be classified in some different classes according to their halflife dose. for instance, the vasoactives angiotensin ii (aii), ang (1-7), bradykinin (bk) and some related peptides were more stable than the melanocyte-stimulating hormone α-msh, substance p or the bk 's (305-325) b2 receptor fragment (lvyvivgkrfrkksrevyqai). usually, the most prominent derivatives generated from this experimental protocol revealed that they are likely induced by oxidation process, yielding a variation of +16 da in their molecular weight. the main source of peptide modifications seems to lie either on the phe (hydroxyl group insertion at o-, m-or p-positions of its aromatic side chain) or met oxydation. in the former case, only phe 8 and not phe 5 is oxidized in the bk structure whereas substance p generates an analogue bearing metsulfoxide without modifying its phe 7,8 residues. thus, collectively, these findings clearly stress the complexity of factors involved in peptide structural modifications induced by gamma ray-type strong electromagnetic irradiation experiment. an additional target of this approach lies indeed, in the production of unusual peptides for further structure-function investigations. university of bern, bern, switzerland linear peptides are typically poor drug candidates due to their low bioavailability and rapid proteolysis. these limitations can be overcome by rigidifying their structure through head-to-tail or side chain-involving cyclizations. cyclic constraints may also increase biological activity by stabilizing secondary structures and by reducing the entropic penalty of binding to a protein target. the use of multiple branching amino acids in a peptide sequence, like diamino acids (as used in peptide dendrimers 1 ) or amino diacids, allows to design peptides resembling polycyclic alkanes, a type of topology only rarely found in nature (e.g. amatoxins and lantibiotics). bicyclic homodetic peptides such as "norbornapeptides" (bicyclo[2.2.1]heptapeptides) were prepared using an orthogonal protection scheme: the first cyclization is performed on resin after selective deprotection of a glutamic acid residue, whereas the second ring closure is achieved by amide bond formation at high dilutions. these peptides are structurally well-defined and cover an almost pristine area of peptide topological space. 2 their conformational rigidity was investigated by means of 2d-nmr and x-ray crystallography and may offer a platform to design drugs tackling protein-protein interactions. the interaction of peptide ligands with protein receptors face peculiar challenge in recognizing binding surfaces due to availability of a multitude of conformations. therefore it is essential to constrain the peptide conformations for the recognition of receptors and thus finding the bioactive conformation. the cell surface receptor protein family integrins recognize "rgd" sequence which is present in different proteins. to determine the bioactive conformation required to bind with receptor αiibβ3, the peptide sequence "riprgdmp" from kistrin was inserted into cdr 1 loop region of rei protein (rei-rgd34). it helps out in finding the possible bioactive conformation of peptide by restricting the sampling space. the activity of rei-rgd34 was studied and found that as the temperature increased rei-rgd34 showed a higher affinity towards the receptor αiibβ3. the proposed mechanisms for the increased activity of rei-rgd34 at higher temperature were justified in either of two ways. the modified complex forces the restricted peptide to adopt a bioactive conformation or it unfolds the peptide in a way that opens its binding surface with high affinity for receptor. in this study we model the conformational preferences of "rgd" sequence in octapeptide "riprgdmp" at two different temperatures (250 o c and 420 o c) using multiple md simulations. we found that at higher temperature "rgd" sequence from "riprgdmp" adopt turn conformation, while a bend conformation was observed at low temperature. the analysis of various pharmacophoric parameters hint that the turn conformation of "rgd" sequences adopted at higher temperature could be the potential bio-active conformation, and helps out in designing of antagonists for cell surface receptor αiibβ3. the 14-residue peptaibol antibiotic trichovirin i-4a (tv) of the linear, covalent structure ac-aib-asn-leu-aib-pro-ala-val-aib-pro-aib-leu-aib-pro-leuol (ac, acetyl; aib, α-aminoisobutyric acid; leuol, l-leucinol) has been synthesized1 and very thin (~25 μm) hair-like crystals were obtained from a methanolacetonitrile-water mixture. diffraction data were collected at 100 k at the diamond light source england, using the microfocus beamline 2 i24 and a x-ray beam focused to a size of 10 μm full-width-half-maximum. two independent molecules (a) and (b) were located in the crystal's asymmetric unit 3 . both chains assume 4 complete turns of a curved 310 right-handed helical conformation stabilized by intramolecular hydrogen bonds. up to now tv represents the longest right-handed 310-helix of a natural peptaibol sequence complementing those of synthetic, protected homooligo-aibinsulin is a protein hormone that plays a key role in regulation of blood glucose levels and, thus, has widespread impact on lipid and protein metabolism.insulin is known to act through binding to the insulin receptor (ir); however, the structure of the insulin-ir complex is not known. the crystal and nmr structures of insulin represent only inactive storage forms. it is widely acknowledged that insulin must undergo structural changes in the c-terminus of the b-chain upon binding to the ir. in addition, the n-terminus of the bchain may adopt two different conformations in hexamer, known as t-and r-states. the r-state of the n-terminus of the b-chain creates a long b1-b19 central a-helix. the t-state of n-terminus is in an extended conformation. however, the biological relevance of the t/r forms remains elusive 1 . in this study, we have focused on the synthesis of new insulin analogues modified at the nterminus of the b-chain and subsequently correlated their biological activities with their 3d-structures. the invariant residue glyb8 seems to be critical for the t/r transition. glycine can adopt wide range of dihedral angles (φ/ψ) and it occupies significantly diverse dihedral angles in t-and r-states. a-aminoisobutyric acid (aib) is an amino acid with a high helical propensity, which often folds into right-or left-handed α-helix. we have introduced aib at position b3, b5 and b8 with the aim to induce the r-state of the hormone. in contrast, as d-pro and nmeala are not able to adopt the φ/ψ angles of the right-handed α-helix we have introduced these amino acids at position b8 to obtain the t-state of insulin. peptide dendrimers are tree-like molecules formed by alternating functional amino acids with branching diamino acids such as lysine. 1 unfortunately these molecules have not yielded to structural characterization and little is known about their molecular-level structure. computational methods seem to be an adequate tool to address these issues.herein we present a comprehensive structural characterization of peptide dendrimers using molecular simulation methods. 2 multiple long molecular dynamics (md) simulations were used to extensively sample the conformational preferences of several third-generation peptide dendrimers, including some known to bind aquacobalamine. we used several conformational analysis procedures (clustering, energy landscapes and multivariate analysis) to analyze conformational changes that can be correlated with particular structural trends.the results point to a high conformational flexibility of these molecules, with no clear "folded state", although two markedly distinct behaviours were identified. some dendrimers favour mainly loose conformations, while others prefer more compact configurations. through a series of computational mutations we investigated the influence of the presence and placement of charged residues in dendrimer topology, finding that electrostatic interactions among charged residues are a major determinant in structure acquisition by peptide dendrimers. these conclusions bring new insight into the conformational behaviour of these systems and may provide better routes for their functional design. acid-mediated prevention of aspartimide formation in solid phase peptide synthesis t. michels, a r. dölling, b u. haberkorn a , w. mier*, a a department of nuclear medicine, university hospital heidelberg, 69120 heidelberg, germany; b biosyntan gmbh, robert-rössle-straße 10, 13125 berlin, germany aspartimide formation is one of the major obstacles that impede the solid phase synthesis of large peptides and proteins. the main reason for aspartimide formation is the piperidine-catalyzed fmoc cleavage of peptides containing aspartic acid. several side chain protecting groups have been developed 1 but the complete prevention of aspartimide formation can only be achieved using n-(2hydroxy-4-methoxybenzyl) (hmb) as backbone protecting group. 2 however, hmb-protected building blocks are difficult to synthesize and only the dipeptide containing glycine (fmoc-asp(tbu)-(hmb)gly) is commercially available. until now, no cost effective strategy to suppress this side reaction has been developed. formally, aspartimide is the result of an attack of an amidate species at the carbonyl carbon of the otbu protected side chain carboxylate of aspartic acid, which might be prevented by protonation of the amidates with piperidinium ions. in this work the suppression of aspartimide formation by adding small amounts of organic acids to the deprotection agent piperidine was studied. this procedure was shown to efficiently prevent the formation of aspartimide side products in several peptides, i.e. pres9-33-y, a 26-mer peptide derived from the hbv surface antigen and a peptide parathyroid hormone (pth) fragment. the testing of a series of 18 different acids covering a broad range of pka values showed that this effect is virtually independent of the acid strength. since aspartic acid is found in most oligopeptides, the authors recommend to generally add 5% (v/v) formic acid to piperidine based fmoc cleavage mixtures. decomposition of the resin linkers during tfa cleavage of peptides in fmoc-strategy leads to alkylation of sensitive amino acids 1 . this side product formation is a crucial drawback, especially during the synthesis of biologically important cys-containing peptides on wang support. through a battery of approaches (1h-nmr, uv and lc/esi-ms) we detected an unexpected alkylation of the sulfhydryl group of cysteine side-chain residues by the phydroxyl benzyl group from the wang resin linker. herein, we present the feasibility for s-alkylation of cys-containing peptides from wang linker decomposition. this sidereaction occurs during the final tfa cleavage of the peptide from the solid support, while the position of the cysteine residue within the peptide sequence as well as the resin's substitution influence the extent of cys-alkylation. the stephan angeloff institute of microbiology, bulgarian academy of sciences, sofia, bulgaria influenza viruses cause epidemics and pandemics all over the world. therefore, the development of virus resistance to drugs, leads to search for novel derivatives and approaches to chemotherapy for human influenza infection. antioxidant therapy is known to be one potential approach. the application of combination therapy of antioxidants with antiviral drugs could reduce the complications and lethal effects, caused by an influenza virus 1 . in our study, amino group of neuraminidase inhibitor -oseltamivir, which belong to second generation anti-flu drugs, was covalent conjugated with known antioxidantscysteine, histidine. tryptophan and etc. the study of the role of the modified by antioxidants oseltamivir on proliferation of influenza virus is in progress. recently we reported a short synthesis of 5 or 6-membered cyclic guanidine via intramolecular reaction of alkyl diamine with n,n'-cbz-methylisothiourea(1a). here we report a further application of synthesis two types of cyclo-peptide guanidine-bridged cyclopeptides utilizing this mechanism -n-terminus local cyclo-guanidine peptide and backbone guanidine-bridged marco-cyclic peptide. three n,n'protected diaminoacids (daa) including 2,4-diaminobutyric p334. antifreeze glycoproteins (afgps) are found in the deep sea teleost fish in arctic and antarctic oceans. these biomolecules are able to inhibit the growth of ice crystals and depress the freezing temperature of the blood serum in fish enough to keep them from freezing in their sub-zero environments while the melting temperature remains unchanged 1 . despite afgps have been consider as a potent cryopreservation, obstacles to develop afgps as medicinal and industrial application are mainly due to the lack of access to pure form from natural sources and the problem of understanding how afgps inhibit ice crystal growth. as a result, a considerable progress toward the design and synthesis of afgp analogues has been made several groups 2 . in the course of the studies on the structure-activity relationships of afgps, we are interested in peptoids as mimics of α-peptides and synthesized monoglycosylated peptoid analogues by substituting the glyco-thr residue as afgps mimics. in this presentation, we will show our studies on how the insertion of peptoid residue into afgp backbone affects the afgp activity by measuring both thermal hysteresis (th) and ice recrystalliztion inhibition (iri). [1] , both for diagnosis and endoradiotherapy. for this application the peptides can be attached with chelating agents that bind radioactive metals such as 67 ga, 68 ga or 111 in for imaging or therapeutic radiometals such as 90 y and 177 lu. the chelating agent most frequently applied is the macrocyclic ligand 1,4,7,10-tetraazacyclododecane-n,n',n'',n'''-tetraacetate (dota), it is commonly introduced as the tris(tbu ester). the cleavage of the tbu protecting groups on dota is known to be sluggish [2] . several attempts have been made to synthesize dota with protecting groups that can be removed under mild conditions. however, these derivatives have not yet found widespread application. our new approach was to prepare a protecting group for dota-based prochelators that is convergently cleaved under the cleavage conditions of the amino acid protecting groups of the peptide. o-phenylisopropyl (opp) esters are more sensitive towards acid than tbu esters. deprotection occurs with 2% trifluoroacetic acid in dichloromethane [3] . therefore, a synthesis of the prochelator dota-tris(opp ester) was developed. the copper-catalyzed azide-alkyne cyclization (cuaac), the most commonly recognized variant of "click chemistry," has emerged as a powerful technique for ligation, conjugation, and cyclization reactions of peptides. it is known that cyclization can increase the metabolic stability of peptides, as well as enhancing potency or selectivity by stabilizing an active conformation. one application of the cuaac that has generated interest is the use of this reaction to replace a disulfide bridge with the product triazole, which among other complementary properties may prevent in vivo redox chemistry. in this poster, we synthesize a new analogue of the cyclic cancertargeting peptide cngrc where we replace the disulfide bond with a triazole linkage using click chemistry and a fully automated, on-resin method using the single-shot delivery feature on the prelude® peptide synthesizer. unnatural amino acids including d-amino acids are manufactured mainly by the enzymatic process. however, one enzyme can produce only one amino acid due to its high specificity and it takes a long time and a lot of expenses to develop the appropriate enzyme itself. arca (alanine racemase chiral analogue) is an organic catalyst1 which can overcome these drawbacks and can produce almost all kinds of amino acids efficiently. the amine functionality of l-threonine is freely reacted with the aldehyde group of arca to form the corresponding imine, which is easily epimerized in the presence of organic base due to the acidity of the alpha proton of imine. the difference in the stability between the imines of the optical epimers rendered them to be shifted to d-allo-threonine derivative dominantly. once the epimerization reaction reached equilibrium, the reaction mixture was hydrolyzed under acidic condition to give d-allo-threonine and arca, which could be recycled repeatedly without significant loss in yield or purity to produce more d-allo-threonine from lthreonine in excellent yields. optimization of the reaction conditions with various bases and solvents is discussed and mass production of optically active d-allo-threonine including optical purification is described. the manufacuring process for the preparation of arca will be shared as well. our group is interested in the development of efficient synthetic routes for the preparation of enantiopure atrifluoromethylated amino acids (a-tfm-aas) starting from chiral cf3-oxazolidines or imines 1 and their incorporation into a peptide chain. 2 these non-natural amino acids are very attractive compounds for the design of biologically active molecules, particularly peptides, due to the unique physical, chemical and biological properties impart by the cf3 group. 3 as conformationally constrained cyclic amino acids have recently gained considerable interest, we are particularly focused on the preparation of pyrrolidine-type a-tfm aas. 1, 4 incorporation of proline derivatives is known to restrict the amino acyl-proline cis/trans isomerization, to limit the protein folding and consequently to modulate the biological activity of peptides. based on these observations, mutter's group introduced pseudoproline building blocks (ψpro) into a peptide sequence as reversible protecting groups for ser, thr and cys. 5 the ψpro residues proved to be versatile tools for overcoming the aggregation caused by hydrophobic interactions encountered during solid-phase peptide synthesis (spps). they also turned out to be inducers of βturns containing predominantly cis-amide bond and useful tools in peptide cyclization. here, we report the results obtained for the preparation of various hydrolytically stable trifluoromethylated pseudoprolines (cf3-ψpro) as well as the methodological studies developed to optimize the synthesis of various c-and n-terminal cf3-ψpro containing dipeptides. rennes, france protein strructure and function rely on a still not fully understood interplay of energetic and entropic constraints defined by the permutation of the twenty genetically encoded amino acids. many attempts have been undertaken to design peptide-peptide interaction pairs and synthetic receptors de novo by using special building blocks. 1 a rational approach starting from hydrazine to create new building blocks based on a tailored metalchelating amino acid analogues was envisaged. to create chemical recognition units, which bind oligohistidine tags with high affinity and stability, several supramolecular entities containing one to three nitrilotriacetic acid analogue (ynta) moieties were synthesized. these new building blocks additionally contained an amino group or an acido group, which can be flexibly introduced into peptide in n or c-termini or into the peptidic chain by solid phase chemistry in fmoc/t-bu strategy. these multivalent chelators were characterized and the corresponding metalchelating peptides could act as metal sensors and synthetic receptors for histidine-tagged proteins. the potential of peptides as drug candidates is often limited by their pharmacokinetic properties. structural modification of the peptide backbone via n-methylation is a powerful medicinal chemistry tool that confers oral bioavailability to these molecules. n-methylation exerts a strong effect on the backbone conformation and, as a result, many n-methylated peptides show enhanced biological activity and higher receptor selectivity. another approach to increase the solubility of peptides is by conjugation of peg to a derivatizable functionality. by combining these two approaches we have developed n-oegylation. this novel form of peptide modification consists of the attachment of oligoethylene glycol (oeg) chains to the amide bonds. many bioactive peptides comprise one or more n-me amino acids which are essential for their activity. thus, we consider that replacement of a backbone n-me group by an oeg chain may imply a minimal structural perturbation and may lead to n-oegylated peptides with preserved biological activity. furthermore, our strategy is a promising way to improve the bioavailability of cyclic peptides that do not have any site where a peg could be attached. as a proof of principle, several n-oeg analogs of two bioactive cyclic peptides were synthesized in spps. first, we performed a full n-oeg scan of the sansalvamide a peptide. next, several analogs of cilengitide were prepared by replacing the n-me of valine by oeg chains of different length. depending on its size, the oeg residue was incorporated by using an n-oeg derivative as building block or, alternatively, using an n-substituted amino acid bearing an attachment site where a peg was conjugated post-synthetically.the biological activity of all the n-oegylated peptides was evaluated. some of the sansalvamide a peptide analogs exhibited cytotoxicity within the same range as the original peptide, which suggests that backbone amide groups may be useful oegylation sites in bioactive cyclic peptides. the modification of peptides is an important step in pharmacology to vary the affinity and the stability of peptidic drugs. whereas a wide range of strategies exists for the functionalization of the n-terminus and the side chains, facile variation of the c-terminus remains an important challenge. we consider peptidyl-phosphoranes as a promising platform to enable orthogonal and mild introduction of a great variety of chemical functionalities at the c-terminus. a convenient method for the synthesis of soluble peptidyl-phosphoranes has been presented by our group recently. 1 in this, 2-bromo-acetyl bromide was coupled to a wang-resin followed by alkylation of triaryl-or trialkyl phosphine moiety. deprotonation to the phosphorus ylide and subsequent acylation with an fmocamino acid created the basis for assembly of the peptide by spps. final acidic cleavage produced a decarboxylated and unprotected, soluble peptidyl-phosphorane. from this point, a variety of orthogonal modification reactions at the peptides c-terminus is possible, e.g. click reaction with azides allow for the incorporation of triazoles as peptide bond mimetics. the wittig reaction opens up another interesting portal for c-terminal modification, as vinyl ketones are formed by reaction with aldehydes. the described chemistry was applied to modify caspase-3 inhibitors. in order to address the s1 site of caspase-3, the commonly known devd inhibitor was varied at the c-terminus by introduction of different residues. the devd motif was synthesized as peptidyl-phosphorane and modified in wittig reactions. the resulting c-terminal vinyl ketones were obtained by the reaction of aliphatic and aromatic aldehydes. a small library was generated, and 12 novel compounds were tested for their potential to inhibit caspase-3. semmelweis university, department of biophysics and radiation biology, budapest, hungary considering the impact of uv irradiation on the structure and function of proteins 1 , it is a matter of utmost importance to resolve the conditions of photolysis more deeply. we think that a protein, as a complex unit, gives multiple responses to all impacts therefore the analysis of these responses is a rather complex problem. the main goal of our research is the deeper understanding the tryptophan-mediated photolysis of disulphide bridges in bio-active proteins upon near-uv irradiation using cyclic peptide models, as small protein units, to define the caused functional damage. cation -pi interaction is increasingly recognised as an important noncovalent binding interaction which plays a role in establishing the final structures of proteins. within a protein, cation -pi interactions can occur between the cationic side-chains of either lys or arg and the aromatic side-chains of phe, tyr or trp 2 . our earlier results with gla indicate that new covalent bonds are also formed between cys and lys during illumination, which is also a reason why the lys residue is planned to be included in the sequence of the models. our aim is to study whether the cation -pi interaction can have an influence on the ss-bridge splitting in small cyclic pentapeptide models. here we report about the conformational analysis, synthesis and spectroscopic investigation of lys-and arg-containing model peptides. the azide functionality is very popular mainly due to azidealkyne click chemistry 1 used in many peptide ligation strategies. azido-peptides are usually prepared by incorporation of azide containing residues or azide functionalization of aldehyde resins affording c-terminal azido-peptides. 2 alternatively, the n-terminus can be converted into an azide via a cu(ii)-catalyzed diazotransfer reaction using triflyl azide. 3 recently, a number of safer, shelf-stable and easily prepared diazotransfer reagents has been developed, of which imidazole-1-sulfonyl azide has been used to introduce azide moieties in proteins under copper-free conditions. typically, it has not been reported to be used on the solid phase. 4 we provide a very easy, fast and efficient method for conversion of amines into azides on a solid phase support which in our opinion has major benefits over earlier reported methods making using of less stable reagents that require a metal ion catalyst. we demonstrate how the diazotransfer reaction can be performed on a solid phase support using the imidazole-1sulfonyl azide reagent without the need for a metal ion catalyst. using a model peptide we studied the effect of stoichiometry, added base and solvent. in addition we examined the effect of the nature of the n-terminal residue on the efficiency the diazotransfer reaction. finally, we found that the optimal conditions to perform the reaction also depend on the nature of the solid phase support the reaction is performed on. the novo nordisk foundation center for protein research, university of copenhagen, copenhagen, denmark site-selective strategies for post-translational modification of peptides and proteins are essential tools for many areas of research in the life sciences, yet remain a chemical challenge due to the multiplicity of functional groups present. there are powerful chemoselective reactions, however, they aim at introducing only one functionality at each reaction site. here we present a one-pot, threecomponent dual-functionalization of peptides or proteins based on a 1,3-dipolar cycloaddition between a functionalized malemide, an n-hydroxylamine and a peptide or protein with an n-terminal serine residue at the n-terminus, which is selectively oxidized to a 2-oxoaldehyde. most common moieties for labeling, e.g. fluorophors and peg-chains, are commercially available as maleimides. nitrones were easily obtained by condensation of peptide-aldehydes and primary nhydroxylamines under aqueous conditions. the chemoselective 1,3-dipolar cycloaddition reaction between the peptide-nitrone, and a functionalized maleimide proceeded in aqueous solution at room temperature or with gentle heating, which provided the stable isoxazolidine product. we envision that this 'one site -two functions' method can be used widely to introduce two separate moieties. the method was used to introduce two separate ligands in a range of other peptides. for example, new multimodal molecular imaging techniques depend on facile chemical methods for site-selective dual-functionalization. we used our new methodology to synthesize a cyclic rgd-peptide for combined pet and optical molecular imaging. finally, the small protein ipb3 was successfully n-terminally modified, including with a peg-chain, using this new, general method. multiple sclerosis (ms) is the most known chronic, inflammatory, demyelinating disease of the central nervous system (cns), characterized by a progressive neurodegeneration, caused by an autoimmune response to self-antigens in genetically susceptible individuals. it is nowadays known that post-translational modifications may affect the immunogenicity of self-protein antigens, triggering an autoimmune response and creating neoantigens; in particular aberrant glycosylations affect various parts of the immune response and have profound effects on immune tolerance. in previous studies we demonstrated the value of the glycopeptide csf114(glc) which, by virtue of the particular type i' β-turn structure, optimally exposes the minimal epitope asn(glc) to autoantibody recognizing in elisa in multiple sclerosis patients' sera 1 . elisa assays allowed to conclude that the ability in detecting autoantibodies in multiple sclerosis sera was stricktly linked to saccharidic moieties and to conformation around minimal epitope of the antigenic glycopeptide. herein, taking advantage of such considerations, we focused our attention on the synthesis of a little library of lysine branched multiple antigen peptides (maps), containing the minimal epitope asn(glc), in an attempt to increase the antigenicity of linear peptide sequences 2 . with this aim, we performed the spps of glucosylated maps via the building block approach, studying the role of different long spacers on the dendrimeric core, and the role of different peptide sequences around the sugar moiety, in order to optimize the synthetic process and to evaluate the influence on the affinity and specificity in sp-elisa. environmentally induced co-or post-translational modifications of autoantigens are hypothesized to break immune tolerance leading to self reactivity in pbc. 1 it has been previously reported that the use of synthetic post-translationally modified peptides, introducing fmoc-l-lys(nε-(±)-α-lipoic acid)-oh, as peptidomimetics of natural neoantigens allowed to detect autoantibodies in the sera of patients affected by pbc, and they might be useful diagnostic tools that can be used in earlier stage patients and possibly to monitor disease activity. 2 only the r-(+)-enantiomer of α-lipoic acid exists in nature and is an essential cofactor of four mitochondrial enzyme complexes. 3 but it remains unclear if the tridimensional structure of the lipoic acid is of any importance in the interactions antibody-peptide during the indirect elisa tests. therefore, it is necessary to synthesize each peptide separately with one absolute configuration of the lipoic acid. herein, we describe the synthesis of the two diastereoisomers fmoc-l-lys(nε -(r)-α -lipoic acid)-oh and fmoc-l-lys(nε -(s)-α-lipoic acid)-oh that have to be used in fmoc/tbu spps as building blocks for the synthesis of post-translationally modified peptides. 2 recently it has been reported the introduction of a new generation of cd diagnostics based on a unique antigen approach, consisting on human ttg cross-linked with gliadin peptides coated on the elisa plates 3 . on the basis of experimental data obtained by mass spectrometry and indicating which are the fragments of these two proteins that are supposed to be involved in the antibody recognition, we were able to select the most representative ttg and gliadin fragments 4, 5 to design and synthesize by fmoc-spps nine cross-linked eoepitopes. aim of our study was the characterization of autoantigenic epitopes by testing, in celiac patients' sera, the reactivity of these nine synthetic peptides. these neoepitopes were tested in elisa to evaluate the iga and igg response against ttg-gliadin adducts in celiac patients' sera in order to develop a new elisa test based on peptides as an even more powerful diagnostic tool in terms of specificity and sensitivity. 1 more than 80 analogs have already been described, wherein the hydroxy acid and the amino acid constituents were replaced by d-amino acids and/or n-methyl amino acids with preserved or altered side chains. for certain types of cancer cells, several of these analogs were found to be more active than the natural product itself. 2 however, it does appear that many of these compounds have limited solubility in water. 2b here we report the synthesis of 5 novel analogs of the sansalvamide a peptide bearing an n-oligoethyleneglycyl (oeg) chain attached to the different backbone positions. attachment of this chain is aimed to enhance the hydrophilicity of the original peptide. our synthetic strategy to modify the backbone with the n-oeg group relies on the use of n-oeg amino acids, which were synthesized in solution and then used as building blocks in spps. as expected, couplings to the n-oeg residues were found to require special conditions. methods for the coupling to nmethyl amino acids were applied and this enabled to obtain the different linear pentapeptides, which were cyclized in solution. both the synthetic strategies of these demanding peptides as well as the preliminar evaluation of their biological activity will be deeply discussed. glycoconjugates such as glycoproteins and glycolipids have important roles in cell functions, for example, intercellular recognition, cell proliferation control, and information transmission. in order to study the structurefunction relationship, synthesis of these glycoconjugates is essential. glycoproteins and glycopeptides are classified into two categories: n-and o-glycosylated derivatives. the n-acetyl-α-d-galactopyranosylated ser or thr derivatives [ser/thr(α-d-galnac)] are important intermediates for o-glycopeptide synthesis. however, the synthesis of ser/thr(α-d-galnac) derivatives by chemical glycosylation is difficult because of the decreased nucleophilicity of hydroxy function in the glycosyl acceptor due to an unfavorable hydrogen-bonding pattern between the oh and α-nh groups 1 . several approaches to overcome this problem have been reported 1, 2 . in addition, the o-glycosidic bond is cleaved easily in acidic conditions. in this study, we assumed that the formation of a cyclic structure containing an α-nh group would increase the reactivity of oh function. thus, we focused on the n, n'isopropylidene derivatives of ser/thr containing dipeptides 3 . we found the reaction of mannopyranosyl trichloroacetimidate and the n, n'-isopropylidene dipeptide in the presence of tmsotf in dichloromethane produced the desired glycosylated dipeptide in good yield. however the selective intermolecular disulfide bond formation is a very difficult and complicated synthetic problem. in this work we report on synthetic approaches for the formation of conjugates with intermolecular thioether or disulfide bonds. for the disulfide bond formation, we use two activation approaches: i) activation of the four cys residues of the carrier testing two activating reagents, in both solid and liquid phase respectively and ii) activation of the cys containing bioactive molecule. as bioactive molecule we selected the r 997 ppleed 1003 sequence derived from the intracellular part of the αiibplatelet integrin receptor. this region is critically involved in platelet aggregation and is a target of intervention for developing antithrombotic agents 2 . the ac-[lys-aib-cys(ch2co-αiib997-1003)]4-nh2 and ac-[lys-aib-cys(cys-αiib997-1003)]4-nh2 conjugates were synthesized and examined for their ability to inhibit platelet aggregation. the biological assays indicated that the synthesized conjugates penetrate the platelet membrane and inhibit human platelet aggregation, in contrast to the corresponding free peptide analogues. the molecules were reported to exhibit broad-spectrum cytotoxicity against the tumor cell lines. although these peptides contained the novel β-methoxytyrosine, lipton et al. reported the synthesis and cytotoxicity of desmethoxycallipeltin b, in which substitution of d-tyrosine for β-methoxytyrosine did not substantially affect the cytotoxicity of callipeltin b 3, 4 . however, a structure-activity relationship study of the molecules has not been shown to date in detail. in the course of our recent research regarding the synthetic study of cyclic depsipeptides, we conducted studies on the synthesis of callipeltins supposed to be efficient structures for ccr5 inhibitors as anti-hiv drugs or anti-cancer agents. in the present study, we report the synthesis of cyclic depsipeptides of callipeltin b analogues consisting of l-, d-amino acids and/or n-methyl amino acids, for a structure-activity relationship study of linear-and cyclic depsi-peptides against hela cells5. in the assay of synthetic peptides, all of the synthetic callipeltin b analogues exhibited no cytotoxicity. we supposed that dimethylpyroglutamic acid of callipeltin b was essential structure to show the cytotoxicity against hela cells. monash university, melbourne, australia protein-protein interactions represent a significant portion (15-40%) of all interactions within the cell; 1 as such these interactions are ideal targets for drug discovery. while difficult to target using small molecules, these interactions can be disrupted using a small section of the protein's binding partner. these short peptides must retain the defined secondary structure associated with the protein binding interface in order to inhibit their protein targets. as the secondary structure adopted by the parent protein is not always exhibited by its derived peptides, constraints are introduced as necessary to help define the structure of the peptide. inducing secondary structure reduces the energy required for organisation, decreasing the energy of binding and has the potential to increase stability with respect to degradation by proteases. 2 solid phase peptide synthesis was used to make several small peptides corresponding to the structured sections of the binding partners for three protein-protein interactions. these peptides were designed to target heart disease, prostate cancer, and liver cancer respectively. secondary structure was introduced using lactam bridge constraints. for the αhelical peptides, a side chain constraint approach was used to nucleate helix formation. as hydrogen bonding between the c=o of the i th residue and the nh of the (i+4) th residue stabilises native α-helices, constraints were introduced linking the side chains such that the residues were held in close proximity. for β-pleated peptides, an antiparallel β-sheet arrangement was achieved by introducing turn regions into the peptide in such a way that the β-strands were aligned. constraints were again introduced using lactam bridges between lys and arg or glu side chains. these peptides were characterised by nmr and cd spectroscopy to verify the correct secondary structure had been induced. göttingen, germany different properties can be combined in a single molecule by using a scaffold arranging functional groups in a predefined topology. the tasp (template-assembled synthetic proteins) concept describes templates to reinforce and direct the folding of designed molecules into a predetermined topology. [1] due to their resistance to proteolytic degradation and their rigid basic structure, cyclic β -tripeptides are suitable carrier molecules for bioactive compounds; they are further known to form tubelike structures by stacking of the peptide rings leading to higher organization of functionalized peptides. [2] with this scaffold different inhibitory systems were synthesized that feature cell penetrating and fluorescent properties. the signal transducer and activator of transcription 3 (stat3) protein, which has been described as an oncogenic protein, was selected as the first target. [3] a peptide sequence which targets the sh2 domain of stat3 was used in two different approaches. it was either directly attached to the cyclic β-tripeptide via a huisgen [2+3]cycloaddition or the peptide was incorporated into the inhibitor loop of the cystine knot microprotein omcoti-ii, which was also attached to the cyclic-β -tripeptide. [4] further, sodium channels are addressed usingconotoxines. first, an alkyne functionalized conotoxin siiia was synthesized applying different folding methods. the alkyne linker will be used to attach a fluorophore or to functionalize a cyclic-β-tripeptide. using single molecule imaging the spatial distribution, local concentration and organization of the ion channels in neurons will be imaged. further, the cyclic-β -tripeptide templating effect will be used functionalizing with μ-conotoxines. those proteins are folding helper proteins. together with chaperones, they form receptor complexes. they catalyze the isomerization of prolyl bonds in various folding states of target proteins. indeed, their role has been implicated in refolding of denatured proteins, de novo protein synthesis and the biologically active conformation of proteins 1 . among them, the fkbp subclass comprises the small ppi calstabin 1 and 2. it is of interest to try to understand the way those proteins act, in order to help the overexpression of various types of membrane proteins, aiming at the renaturation, purification and crystallization attempts of receptors. we chose to work on calstabin 2 because this short (107 aa) protein has been described as a sub-family comprising 4 isoforms (from ~3 0 to ~1 00 aminoacids), some of them not being fully described to date. the relative shortness of those proteins together with the fact that the two higher molecular weight ones are catalytically active as prolyl isomerases, facilitate the characterization of the synthetic proteins.in order to obtain the full length calstabin 2, a native chemical ligation (ncl) approach was chosen 2 . an optimized stepwise elongation allowed the obtention of the c-terminal segment up to the cys 22. moreover, several methods were compared for the synthesis of peptide 1-21 opportunely functionalized at its c-terminus for the ncl.the ligation at thr site between peptide 1-21 featuring a bis(2-sulfanylethyl)amino 3 the chemical diagnostics of paintings is a relevant topic in the field of chemical sciences applied to the conservation and safeguard of cultural heritage. chromatography is a highly sensitive and suitable technique for accurate methods of analysis of the limited amount of sample material typically available from works of art. paint media deriving from proteins traditionally include egg, milk, animal and fish collagen glue. egg yolk (egg tempera), egg albumin (glair) and casein (a blend of related phosphoproteins commonly found in milk) are traditionally used as pigments binders. we propose the uplc-based amino acid analysis as diagnostics technique on non pre-treated or submitted to extraction processes model samples, showing that good results can be achieved with very scarce sample manipulation and great advantage. we applied the amino acids analysis carried out by the accq•tag™ ultra performance liquid chromatography to the standard and model samples. in particular, after protein hydrolysis (24h, 114 °c, 6m hcl) of the samples, the amino acid derivatization by 6-aminoquinolyl-n-hydroxysuccinimidyl carbamate allowed a reproducible amino acids analysis characteristic of the protein type. the results obtained confirmed the reliability of the data achieved and demonstrated that the accq•tag™ ultra uplc method could be a powerful technique to be applied to the relevant field of protein binders diagnostics for paintings conservation. moreover a multivariate analysis that offers a wide variety of tools and methods mainly concerned with mathematical models for the representation of multidimensional data has been proposed and the high model efficiency has been established for sample containing mixture of proteins. reactions performed on solid supports, such as resin, are commonly monitored by hplc-uv after cleaving the products from the support. however, uv-absorption coefficients may differ between compounds, and therefore the relation of the area percentage values of the peaks may not directly reflect the molar concentrations of the corresponding compounds. it is for this reason that, for example, in solid-phase peptide synthesis it is difficult to calculate the yield of the coupling of a fmoc-amino acid or the removal of the fmoc-group because of its high absorbance. recently, we reported the identification of minimal phosphopeptides that specifically interact with the pbd of human plk1, but not those of the closely related plk2 and plk3 1 . comparative analyses of the crystal structures of the plk1 pbd in complex with the minimal phosphopeptides revealed that the c-terminal spt dipeptide functions as a high-affinity to the interaction. in an attempt to obtain the adequate cellular permeability and stability in vivo, we have accomplished the peptide-peptoid hybrid or peptomers cyclization using various methods like formation of amide, thioether and triazole and screened the plk1 inhibition activity on the first cyclic peptomers liibrary using pbd-binding assay. based on our first screening results, we also carried out the detailed investigation to further increase the activity and also to understand the significance of peptoid mimics as plk1 inhibitors. the mode of interaction between the cyclic peptomers and pbd might provide a template for designing therapeutic agents that target plk1. a synthetic 83 amino acid long peptide corresponding to the minimal metacaspase catalytic domain induces cell death in leishmania major c. servis, h. zalila*, i. gonzalez, l. lozano, n. fasel department of biochemistry, university of lausanne, epalinges, switzerland despite a lot of controversy during the last decade, there is increasing experimental evidence that cell death (cd) is genetically programmed in lower eukaryotes.in the cd proteolytic cascade of plants and protozoa, caspases are likely replaced by metacaspases that are cysteine peptidases recognizing arginines or lysines in p1position. metacaspases have been found to control cell death in plants. the human protozoan parasite leishmania major expresses a single metacaspase (lmjmca) harboring a central domain with the catalytic dyad histidine and cysteine as found in caspases. metacaspase could therefore be one of the executioners of the death pathway in leishmania.in this work we showed that, in stress conditions, lmjmca precursor forms were extensively processed into soluble forms containing the catalytic domain and this domain was sufficient to enhance sensitivity of parasites to hydrogen peroxide by impairing the mitochondrion function. we tested different lengths of the lmjmca catalytic domain and found that the overexpression of the polypeptide corresponding to amino acids 136-218 was sufficient to sensitize l. major mitochondria to oxidative stress.we synthetized an 83aa long peptide corresponding to the minimal metacaspase catalytic domain (aa136-218) and showed that it has specific metacaspase activity in vitro.we are currently investigating its activity on possible target proteins, which have been identified in a yeast two-hybrid screen. identifying proteins involved in the metacaspase signaling pathway will shed light on the understanding of cd in leishmania and open new perspectives in drug target investigation to fight leishmaniasis and other major infectious diseases. s. alasibi, g. ashkenasy department of chemistry, ben-gurion university of the negev, beer-sheva, israel various factors can affect the conformations and folding states of protein molecules and as a consequence their activity. these factors include amino acid mutations, interactions with other macromolecules, binding to regulatory molecules, and also external changes such as ph jump or shining light. in order to control the folding states and to modulate the functions of peptides and proteins by light, photocleavable groups are usually incorporated into specific residues to mask critical interactions. for example, introducing caging groups into coiled-coil proteins recognition interface affects complex formation and template-assisted ligation reactions, in which the coiled-coils serve as templates to catalyze the condensation reactions between two short peptide fragments 1 . our research group has been studying peptides replication networks, which were made of coiledcoil peptides and analyzed the response of such networks to light as external trigger 1 . it was shown that even replicating networks made up of a small number of molecules can possess complex behavior, considering the wealth of catalytic pathways and transformations. hence, boolean logic operations can provide valuable means to analyze and interpret their behavior 2 . herein, we describe the use of chemical inputs and uv light to manipulate peptides folding and functionality within new synthetic networks. these networks perform complex behavior and, as a result, selective product formation is used to implement boolean operations that have not been achieved before. institute of bioorganic chemistry of ras, moscow, russia earlier, we have shown that n-acylated amino acid nitriles and amides react with ethylene derivatives forming the 3amino-and 3-hydroxypyridines and pyrroles [1] . a possible reaction mechanism is the geterodienic condensation of 5aminooxazole derivatives to dienophiles. the higher yields were observed when used the dicarboxylic acids as dienophiles and 5-amino or 5alkoxyoxazole as geterodienes. while the same reactions with the fullerene derivatives, as dienophiles, gave low yields. the nitrile groups of specified pyridines possess ability to react with amino groups of peptides and proteins even at room temperature. in view of high activity of nitrile groups such pyridines can form tetrapyridotetrazoporphyrins and self-condensation products giving appropriate dendrimers (possible due to mobile hydrogen in the 6th position). high molecular weight dendrimers were identified by massspectrometry, gel electrophoresis and dynamic light-scattering. catalytic oxidizing properties of tetrazaporphyrin derivatives and phtalocyanin were used in synthesis of cyclic peptides and for the s-s bonds formation. the transformation of peptides into heterocycles via an intramolecular reaction of nitrile groups was used to determine the sequence of some peptides, which favored the resistance of transformed compounds to hydrolysis and to the electron impact at mass spectrometry. diazotization of peptides and their derivatives facilitates identification of amino acid sequence by mass spectrometry due to the peculiarities of their fragmentation. in addition, an amino acid analysis of the diazotized peptide makes it easy to determine the n-terminal amino acid. we present here a multi-disciplinary approach combining x-ray crystallography, computational analyses, and immunological tests to identify epitopes of the oligopeptide-binding protein a (oppa bp) from the gramnegative pathogen burkholderia pseudomallei, the etiological agent of melioidosis. 3 computational analysis on oppa crystal structure was used to design potential consensus epitopes, that once synthesized as free peptides (comp 1-3) were found to be immunoreactive against sera from melioidosis patients. notably, one of the predicted peptides allowed to distinguish between seropositive, seronegative and recovered groups, underlining its potential for diagnostic purposes. parallel experimental epitope mapping, based on proteolysis and mass spectrometry, allowed us to identify linear peptide epitopes (exp 4-6) localized in similar protein regions as comp1-3. moreover, the match between theoretical and experimental mapping of epitopes was improved by expanding our computational approach, i.e. including an energy based decomposition procedure to divide oppa bp into separate fragments. overall, our results illustrate the successful development of a novel integrated structurebased approach for the discovery, design and preparation of epitopes. nonetheless, given antigen crystal structures, our method is expected to be broadly applicable in the design and generation of new epitope candidates, as being confirmed by on going experiments on different antigens. the application of peptide thioacids as reactive intermediates and building blocks has received considerable attention recently. the chemical ligation reaction between thioacids and azides has been reported for the synthesis of small to larger peptides as well as for the modification of proteins. 1 fmoc based methods for the preparation of peptide thioacids have to our knowledge not been extensively researched and a facile approach to their synthesis is desirable.we have recently shown that t-butyl thioesters are robuster than previously reported, and can be used for the fmoc based solid-phase preparation of peptide thioesters being also easily cleavable with thiolates. 2 peptides attached via a 4-mercapto 4-methylpentanol (mmp) resin can be cleaved using 3-mercaptopropionitrile to obtain protected thioacid peptides with a ß-elminable cyanoethyl group.the thioacid peptides could then be obtained in situ after treatment with dbu (6.5 % in dmf) and further reacted with sulfonyl azides in the presence of 2,6-lutidine in a one pot reaction. by treating the cyanoethyl peptide thioesters with (nh4)2s in a sodium phosphate buffer (ph = 9), various model penta-peptide thioacids could be obtained cleanly at room temperature in up to 75 % overall yield based on initial coupling. these peptides were then further ligated with electron deficient sulfonyl azide functionalized peptides.larger peptide thioacids could also be obtained using this protocol. a 16mer derivative of penetratin-1, a cell-penetrating peptide from the third helix of the homeodomain of the antennapedia protein, was prepared as a peptide thioacid in a 51 % yield (based on coupling of the first amino acid). in this report, a sensitive, selective and rapid uplc-ms method was developed for the determination of the [lys-gly]5-mog35-55 peptide in order to control the conjugation of mannan with the [lys-gly]5-mog35-55 peptide. the separation was performed on an acquity uplc system with a beh c18 column packed with 1.7 μm particles. the total run time was 3 min. calibration curve based on peak area ratio was linear at the concentration range of 20 -90 μg/ml, with a detection limit of 5 μg/ml. the method showed satisfactory reproducibility and confirmed the entire conjugation between oxidized mannan and peptide sequence. the development of simple, low-cost and fast methods for protein purification is of increasing importance both for academic and industrial applications. a very promising approach is inverse transition cycling (itc) that exploits the temperature dependent aggregation properties of elps. 1 elastin-like polypeptides (elp) are artificial polypeptides composed of pentameric repeats (val-pro-gly-xaa-gly) derived from mammalian elastin. 2 elps are characterized by a specific transition temperature (t t ) that depends on the amino acid composition of the pentarepeat; they are water-soluble below and aggregate reversibly above this narrow temperature range (t t ). 3 these properties are transferred to target proteins by n-or cterminal fusion with elps. 4 during itc these fusion proteins precipitate, while other components remain in solution. repeated cycles of heating and cooling allow simple recovery of the target protein.we synthesized various elps consisting of 5 to 10 pentameric repeats and including different guest residues. the transition temperature of all synthetic elps was determined using photometric assays and measuring turbidity. 3 in order to test if elp properties can be efficiently transferred, we fused elp to a small recombinant protein (ras-binding domain, rbd) by expressed protein ligation. 5 this approach will allow the incorporation of elps with unnatural amino acids and other chemical modifications into target proteins. currently we are focusing on biotechnologically relevant enzymes that constitute a major cost factor in industrial processes. the authors thank süd-chemie/clariant for their financial support. department of pharmacy, university of patras, rio, greece peptides penetrating the cell membrane, known as cell penetrating peptides (cpps), as well as their mimics, used as delivery agents to cells have been reported 1,2 . cpps can be natural sequences or artificial constructs designed to capture the features of natural formations. cpps are particularly important in the delivery of peptides, proteins, nucleic acids, small molecule drugs or imaging agents. incorporation of a heterocyclic motif into a peptide or peptide-like backbone introduces conformational constraints and/or latent reactivity related to the heterocycle's structural profile. heterocycle-based cpp mimics are, thus, promising candidates for therapeutics 3 protected synthetic non-ionic peptides, which are for example synthetic intermediates for the production of api's, are often very hydrophobic and not soluble in most common solvents. they are thus difficult to purify by preparative rp-hplc, classically used for industrial production. it is then challenging to develop alternative purification chromatographic processes using suitable solvents and providing good yields, high purity and sufficient productivity. the technique of support free liquid-liquid chromatography 1 , including both its hydrostatic (centrifugal partition chromatography or cpc) and its hydrodynamic (counter-current chromatography or ccc) declensions, are mainly involved in phytochemical studies 2 but has also been applied to peptide purification 3 . the previously developed biphasic solvent systems are not adapted to the purification of highly hydrophobic protected peptides. to overcome this problem, two new scales of biphasic solvents systems and a ternary biphasic solvent system were developed to overcome solubility problems often encountered with those peptides. the new systems composed of heptane/thf/ch3n/dmso/water, heptane/me-thf/nmp/water, and cmpe/dmf/water were efficiently used for the cpc purification of a 39mer protected exenatide and a 8mer protected peptide intermediate of bivalirudin synthesis. the developed scales show a wide range of polarity and should be useful for general use in cpc for the separation of hydrophobic synthetic free or protected peptides. the progressive aggregation of β-amyloid peptide (β-ap) into insoluble amyloid fibrils ultimately leading to formation of toxic amyloid plaques is widely considered to be the central pathogenic cause of alzheimer's disease. in the last decade accumulating evidence suggests that soluble oligomeric non-fibrillar forms of β-ap are neurotoxic as well. consequently, inhibiting the aggregation of β-ap is one of the therapeutic strategies against alzheimer's disease and a number of small molecules have been identified as inhibitors of β-ap aggregation and neurotoxicity. among these, curcumin, the phenolic yellow pigment and active ingredient of the turmeric herb, is receiving special attention because of its rich pharmacology that includes in vitro and in vivo inhibitory action against alzheimer's disease insults. in the current work the interaction of β-ap(1-40) with curcumin is investigated with fluorescence, cd, and nmr spectroscopies in water and water-methanol mixtures and at various β-ap(1-40):curcumin ratios. in nmr studies in 100% methanol curcumin behaves like a macromolecular species with a change in the sign of its noe signal providing direct indication of its association with β-ap(1-40). in 50% methanol the presence of β-ap(1-40) results in great broadening of the 1 h peaks of curcumin, indicative of a complete change in its solution state. additionally, the fluorescence of curcumin in 50% methanol shows a blue shift with enhanced intensity, observations consistent with a hydrophobic modification of curcumin environment upon interaction with β-ap. finally, in water the induced circular dichroism spectrum of curcumin in the near uv region provides clear evidence for the loss of symmetry of curcumin molecule due to changes in its microenvironment generated by interaction with β-ap(1-40). our experimental findings support the direct interaction of β-ap(1-40) with curcumin and establish its importance as a potential aggregation inhibitor of β-ap. [1] . based on its sequence, we synthesized h-tyr-d-trp-nh-1-ada (1-adamantane) (yo-14) and h-tyr-d-trp-nh-2-ada (2-adamantane) (yo-13) and reported they had potent antiproliferative activity on cancer cells (a-430 and sw480), which were comparable to tt-232 and cycloheximide. a structure-activity relationship analysis revealed that lipophylicity of yo-14 and -13 could be responsible for their antiproliferative activity. now, we described the substitution of tyr of yo-14 and -13 by tyr(bzl), phe, 1-nal(1-naphthylalanine), 2-nal (2-naphthyalanine) and the anticancer and dna polymerase inhibitory activities in order to explore the effect of hydrophobic substituent. among the compounds, yo-113 and -114 had the highest lipophilicity judging from their retention time and lipophilicity index (yo-113: 45.5 min, 59.9; yo-114: 44.6 min, 55.9). yo-113 and -114 exhibited strong dna polymerase inhibitory activity as well as antifroliferative activity on hct116 cells at 100 m. these activities were greater than those of yo-14 and -13. antiproliferative activity of the compounds containing 1-ada such as yo-14, -89, -109, -111 and -113, was comparable to that of the compounds containing 2-ada such as yo-13, -90, -110, -112 and -114. these findings suggest that the lipophilicity well correlates with dna polymerase inhibitory activity and antiproliferative activity on hct116 cells. further structureactivity relationship study is progressing in our group. multiple sclerosis (ms) is a chronic autoimmune disease of the central nervous system (cns) 1,2 . our aim was to immunologically control the attack of the myelin sheath in ms patients without the total suppression of the immune system. anthraquinones (mitoxantrone, ametantrone) are widely used in cancer therapy as immunosuppressants.mitoxantrone is also used to treat several forms of advancing ms, including secondary progressive ms, progressive relapsing ms, and advanced relapsingremitting ms 3 . more specifically, mitoxantrone is an inhibitor of the type ii topoisomerase, which disrupts dna synthesis and dna repair in both healthy cells and cancer cells. herein, we report the synthesis of an anthraquinone type compound conjugated to the immunodominant 35-55 myelin oligodendrocyte glycoprotein (mog35-55) for the selective immunosuppression of the encephalitogenic t cells in ms patients. the anthraquinone was synthesized by a friedel-crafts acylation of hydroquinone from phthalic anhydride, followed by reduction of the resulted quinizarine to its leuco form, addition of the appropriate diamine and air oxidation 4 . the synthesized molecules were purified using liquid chromatography, and they were identified by mass spectrometry and 1 h-nmr. the synthesis of the mog35-55 was performed under microwave irradiation 4 and its conjugation with the anthraquinone was performed in solution. the final analogue was purified by rp-hplc and identified by esi-ms. benzopyrans, diketopiperazines and 1,5benzodiazepin-2,4-diones are well-known and widely investigated scaffolds, e.g. the latter showing anxiolytic and antiarrhythmic effects. now, we propose a new potential "privileged structure" containing a triazole moiety mimicking the cis-amide bond within the 1,5-benzodiazepin-2,4-dione motif 2 .molecules based on this [1,2,3]-triazolo [1,5-d] benzo-1,4diazepin-2-one scaffold are synthesized and decorated via a modular approach on wang resin using α-amino acids, 2-ethynylaniline building blocks and n-alkylating agents resulting in five points of diversity. the methodology involves the attachment of α-amino acids onto a solid support, subsequent removal of the fmoc group followed by an optimized diazotransfer reaction of the resulting amine yielding a resin-bound azide. conversion of the latter into a 1,5-disubstituted 1,2,3-triazole moiety is achieved quantitatively by addition of a range of 2-ethynylaniline building blocks using a ru(ii)-catalyst. the desired scaffold can be obtained in high crude purities (>95%) in solution via an acid catalyzed one-step cyclisation-release strategy. solution-phase n-alkylation finally affords the fully diversified scaffold. interestingly, n-alkylation induces atropisomeric effects which can be studied via 1 h nmr spectroscopy.taking into account future screening results of the synthesized libraries, a well-thought decoration of this scaffold leading to discovery of new lead molecules is within reach. peptide symposium in the wonderful small seaside town of porto carras. maurice manning and lajos balaśpiri were nominated as captains of the two teams, the rest of the world and europe. similar matches were organized at subsequent european peptide symposia. now, in greece, the two captains would like to hand over their roles to younger scientists [professors gabor mezö(hungary) and laśzlóötvös (usa)] to continue this tradition at the coming european and possibly american peptide symposia. it seems best to play in the free time (in the evenings after the excursions). necessary conditions: good weather; a nice large soccer field; a soccer ball, preferably new; and jerseys and shorts (different colours), organized as always by the organizer commettee. the captain of the winning team will receive a trophy at the end of the 32 nd european peptide symposium. the teams will remember two earlier excellent referees: professor lajos kisfaludy in porto carras [1986] and the soccer professor + ferenc puskaś (hungary) in budapest (1998). in the poster session, the results from the past 25 years will be presented in about 15-20 pictures. these pictures may possibly be bought free, 0% can be saved at the poster session. all participants are welcome at the new party in athens. conclusion will be presented by the players and fans in athens. the human lactoferrin-derived peptide, hlf1-11, was proven to be highly active against antibiotic-resistant bacteria 1 . however, the clinical use of this antimicrobial peptide (amps) is hampered by the peptide low stability due to fast degradation or to peptide aggregation, as the use of higher peptide concentrations results on higher toxicity levels. amp immobilization onto a biomaterial surface could be the pathway to overcome these difficulties 2 . the aim of this work is the development of an antimicrobial surface by covalent immobilization of hlf1-11 onto the surface of chitosan thin films. chitosan ultrathin films were prepared through the spincoating of a 0.4% chitosan solution in gold substrates. hlf1-11 immobilization was performed through an ss bound between hlf1-11 terminal cysteine and an n-acetyl cysteine previously coupled at chitosan films. surfaces were characterized using ellipsometry (thickness), infrared reflection absorption spectroscopy (irras) and x-ray photoelectron spectroscopy (xps). bacterial adhesion studies were performed using methicillin-resistant s. aureus (atcc33591). chitosan films were incubated with this bacterial suspension at 37ºc for 6h and 24h. the viability of the attached bacteria was evaluated using live/dead® bacterial viability kit (baclight tm ) and fluorescence microscopy. hlf1-11 peptide was successfully covalently immobilized onto chitosan thin films. both soluble and attached peptide presented a higher antimicrobial activity than the control chitosan. identified as a potent vasoconstrictor that binds with high affinity to ut receptor. 1 the cysteine-linked cyclic region, hut-ii(4-11), is responsible for the biological activity and has been widely used to elucidate the structure-activity relationship of hut-ii. 2 with the aim to investigate the role of hydrogen bond and the effects of a peptide backbone constraint on binding key: cord-352844-wggg3ynb authors: annunziata, francesca; pinna, cecilia; dallavalle, sabrina; tamborini, lucia; pinto, andrea title: an overview of coumarin as a versatile and readily accessible scaffold with broad-ranging biological activities date: 2020-06-29 journal: int j mol sci doi: 10.3390/ijms21134618 sha: doc_id: 352844 cord_uid: wggg3ynb privileged structures have been widely used as an effective template for the research and discovery of high value chemicals. coumarin is a simple scaffold widespread in nature and it can be found in a considerable number of plants as well as in some fungi and bacteria. in the last years, these natural compounds have been gaining an increasing attention from the scientific community for their wide range of biological activities, mainly due to their ability to interact with diverse enzymes and receptors in living organisms. in addition, coumarin nucleus has proved to be easily synthetized and decorated, giving the possibility of designing new coumarin-based compounds and investigating their potential in the treatment of various diseases. the versatility of coumarin scaffold finds applications not only in medicinal chemistry but also in the agrochemical field as well as in the cosmetic and fragrances industry. this review is intended to be a critical overview on coumarins, comprehensive of natural sources, metabolites, biological evaluations and synthetic approaches. coumarins are a wide family of secondary metabolites found in various species of plants (more than 1300 coumarins have been identified from natural sources, especially green plants) but also fungi and microorganisms [1, 2] . the main pathway of coumarin biosynthesis occurs by shikimic acid route, via cinnamic acid, through phenylalanine metabolism [3] . the history of these natural products began 200 years ago-the name of the class derived from the plant coumarouna odorata (dipteryx odorata) from which the simplest member of this family, coumarin itself ( figure 1) , was isolated by vogel in 1820 [3, 4] . chemically speaking, coumarins are organic heterocycles and their nucleus is represented by benzo-α-pyrone (2h-1-benzopiran-2-one), whose systematic nomenclature was established by international union of pure and applied chemistry (iupac) [5] . natural coumarins are subdivided in different classes based on their chemical diversity and complexity-simple coumarins, isocoumarins, furanocoumarins and pyranocoumarins (both angular and linear), biscoumarins and other coumarins such as phenylcoumarins (table 1 ) [6] . coumarins have several attractive features, such as low molecular weight, simple structure, high in a recent work, eight coumarin metabolites, which had not been identified previously, were detected by means of uplc/quadrupole-tof tandem mass spectrometry in human urine [22] . among them, positional isomers of 7-hydroxycoumarin glucuronide and 7-hydroxycoumarin sulphate were found. it was proposed that such isomers should bear the substituent in position 5, 6 or 8. metabolites coming from a double hydroxylation and subsequent conjugation with glucuronic acid or sulphate group (and their isomers) were detected as well. another metabolite was the one obtained by a double hydroxylation of the coumarin ring, followed by methylation and glucuronidation at the two newly generated hydroxyl groups. finally, the n-acetylcysteine conjugated metabolite was identified. in this work, o-hydroxyphenylacetic acid was also detected in the samples, whereas free coumarin and o-coumaric acid were not found, indicating that coumarin was completely metabolized before excretion, after oral administration. this meant that o-coumaric thunberginols (antidiabetic) [19] phenylcoumarins int. j. mol. sci. 2020, 21, x for peer review 3 of 83 thunberginols (antidiabetic) [19] phenylcoumarins isodispar b (anti-inflammatory) [20] this privileged scaffold serves as an important platform for the design of compound libraries in the search for new drug candidates. here, we critically review the actual state of the art on natural and synthetic coumarins, focusing on the biological activity, structure-activity relationships (sars), pharmacokinetics (pks) and their potential applications in the pharma and agri-food sectors. furthermore, an overall overview of the most common synthetic routes applied to obtain simple coumarins are provided, together with a further discussion on alternative, innovative and green synthetic methodologies. coumarin is metabolized by cytochrome p450-linked mono-oxygenase enzyme (cyp2a6) system in liver microsomes, which leads to hydroxylation; subsequently, the hydroxylated metabolite follows phase ii conjugation reactions. although coumarin could potentially be hydroxylated at all six possible positions (i.e., carbon atoms 3, 4, 5, 6, 7 and 8) (figure 1 ), 7hydroxycoumarin and 3-hydroxycoumarin are the main metabolites. the former one faces phase ii conjugation reaction resulting in the glucuronide derivative, whereas 3-hydroxycoumarin can be further metabolized by ring splitting to form two products, o-hydroxyphenyllactic acid and ohydroxyphenylacetic acid ( figure 2 ) [4, 21] . since the expression of cyp2a6 varies between individuals, due to genetic and environmental factors, an inter-individual variation in the metabolism of coumarin drugs is possible [4] . in a recent work, eight coumarin metabolites, which had not been identified previously, were detected by means of uplc/quadrupole-tof tandem mass spectrometry in human urine [22] . among them, positional isomers of 7-hydroxycoumarin glucuronide and 7-hydroxycoumarin sulphate were found. it was proposed that such isomers should bear the substituent in position 5, 6 or 8. metabolites coming from a double hydroxylation and subsequent conjugation with glucuronic acid or sulphate group (and their isomers) were detected as well. another metabolite was the one obtained by a double hydroxylation of the coumarin ring, followed by methylation and glucuronidation at the two newly generated hydroxyl groups. finally, the n-acetylcysteine conjugated metabolite was identified. in this work, o-hydroxyphenylacetic acid was also detected in the samples, whereas free coumarin and o-coumaric acid were not found, indicating that coumarin was completely metabolized before excretion, after oral administration. this meant that o-coumaric isodispar b (anti-inflammatory) [20] this privileged scaffold serves as an important platform for the design of compound libraries in the search for new drug candidates. here, we critically review the actual state of the art on natural and synthetic coumarins, focusing on the biological activity, structure-activity relationships (sars), pharmacokinetics (pks) and their potential applications in the pharma and agri-food sectors. furthermore, an overall overview of the most common synthetic routes applied to obtain simple coumarins are provided, together with a further discussion on alternative, innovative and green synthetic methodologies. coumarin is metabolized by cytochrome p450-linked mono-oxygenase enzyme (cyp2a6) system in liver microsomes, which leads to hydroxylation; subsequently, the hydroxylated metabolite follows phase ii conjugation reactions. although coumarin could potentially be hydroxylated at all six possible positions (i.e., carbon atoms 3, 4, 5, 6, 7 and 8) (figure 1 ), 7-hydroxycoumarin and 3-hydroxycoumarin are the main metabolites. the former one faces phase ii conjugation reaction resulting in the glucuronide derivative, whereas 3-hydroxycoumarin can be further metabolized by ring splitting to form two products, o-hydroxyphenyllactic acid and o-hydroxyphenylacetic acid ( figure 2 ) [4, 21] . since the expression of cyp2a6 varies between individuals, due to genetic and environmental factors, an inter-individual variation in the metabolism of coumarin drugs is possible [4] . int thunberginols (antidiabetic) [19] phenylcoumarins isodispar b (anti-inflammatory) [20] this privileged scaffold serves as an important platform for the design of compound libraries in the search for new drug candidates. here, we critically review the actual state of the art on natural and synthetic coumarins, focusing on the biological activity, structure-activity relationships (sars), pharmacokinetics (pks) and their potential applications in the pharma and agri-food sectors. furthermore, an overall overview of the most common synthetic routes applied to obtain simple coumarins are provided, together with a further discussion on alternative, innovative and green synthetic methodologies. coumarin is metabolized by cytochrome p450-linked mono-oxygenase enzyme (cyp2a6) system in liver microsomes, which leads to hydroxylation; subsequently, the hydroxylated metabolite follows phase ii conjugation reactions. although coumarin could potentially be hydroxylated at all six possible positions (i.e., carbon atoms 3, 4, 5, 6, 7 and 8) (figure 1 ), 7hydroxycoumarin and 3-hydroxycoumarin are the main metabolites. the former one faces phase ii conjugation reaction resulting in the glucuronide derivative, whereas 3-hydroxycoumarin can be further metabolized by ring splitting to form two products, o-hydroxyphenyllactic acid and ohydroxyphenylacetic acid ( figure 2 ) [4, 21] . since the expression of cyp2a6 varies between individuals, due to genetic and environmental factors, an inter-individual variation in the metabolism of coumarin drugs is possible [4] . in a recent work, eight coumarin metabolites, which had not been identified previously, were detected by means of uplc/quadrupole-tof tandem mass spectrometry in human urine [22] . among them, positional isomers of 7-hydroxycoumarin glucuronide and 7-hydroxycoumarin sulphate were found. it was proposed that such isomers should bear the substituent in position 5, 6 or 8. metabolites coming from a double hydroxylation and subsequent conjugation with glucuronic acid or sulphate group (and their isomers) were detected as well. another metabolite was the one obtained by a double hydroxylation of the coumarin ring, followed by methylation and glucuronidation at the two newly generated hydroxyl groups. finally, the n-acetylcysteine conjugated metabolite was identified. in this work, o-hydroxyphenylacetic acid was also detected in the samples, whereas free coumarin and o-coumaric acid were not found, indicating that coumarin was completely metabolized before excretion, after oral administration. this meant that o-coumaric in a recent work, eight coumarin metabolites, which had not been identified previously, were detected by means of uplc/quadrupole-tof tandem mass spectrometry in human urine [22] . among them, positional isomers of 7-hydroxycoumarin glucuronide and 7-hydroxycoumarin sulphate were found. it was proposed that such isomers should bear the substituent in position 5, 6 or 8. metabolites coming from a double hydroxylation and subsequent conjugation with glucuronic acid or sulphate group (and their isomers) were detected as well. another metabolite was the one obtained by a double hydroxylation of the coumarin ring, followed by methylation and glucuronidation at the two newly generated hydroxyl groups. finally, the n-acetylcysteine conjugated metabolite was identified. in this work, o-hydroxyphenylacetic acid was also detected in the samples, whereas free coumarin and o-coumaric acid were not found, indicating that coumarin was completely metabolized before excretion, after oral administration. this meant that o-coumaric acid found in human plasma [23] underwent a biotransformation process before being eliminated, probably leading to o-hydroxyphenylacetic acid. in a healthy human body, normal metabolic processes produce free radicals and other highly reactive species such as ions, molecules with unpaired electrons, reactive oxygen, carbon, nitrogen or sulfur species (ros, rcs, rns or rss). when these species are overproduced, oxidative processes might cause cellular damage, affecting cellular components and causing ionic imbalance or mitochondrial disfunction [24] . the role of oxidative stress in different pathologies is well know: inflammation, cardiovascular diseases, cancer, diabetes and even neurodegenerative disorders often count oxidative damage among their pathological features [25] [26] [27] . therefore, exogenous antioxidants might be useful in order to maintain the right concentration of radicals, reducing the amounts of free radicals and avoiding oxidative stress [28] . the antioxidant potential of natural and synthetic coumarins has been deeply investigated in the last years and it became clear that poly-hydroxy or phenolic coumarins are efficient antioxidants in biologicals systems [29] . here below the most recent updates in this field are reported. in 2019, couttolenc and collaborators studied the radical scavenging activity of three hydroxy-4-methylcoumarins (1-3, figure 3 ) by means of experimental and theoretical methods [30] . int acid found in human plasma [23] underwent a biotransformation process before being eliminated, probably leading to o-hydroxyphenylacetic acid. in a healthy human body, normal metabolic processes produce free radicals and other highly reactive species such as ions, molecules with unpaired electrons, reactive oxygen, carbon, nitrogen or sulfur species (ros, rcs, rns or rss). when these species are overproduced, oxidative processes might cause cellular damage, affecting cellular components and causing ionic imbalance or mitochondrial disfunction [24] . the role of oxidative stress in different pathologies is well know: inflammation, cardiovascular diseases, cancer, diabetes and even neurodegenerative disorders often count oxidative damage among their pathological features [25] [26] [27] . therefore, exogenous antioxidants might be useful in order to maintain the right concentration of radicals, reducing the amounts of free radicals and avoiding oxidative stress [28] . the antioxidant potential of natural and synthetic coumarins has been deeply investigated in the last years and it became clear that poly-hydroxy or phenolic coumarins are efficient antioxidants in biologicals systems [29] . here below the most recent updates in this field are reported. in 2019, couttolenc and collaborators studied the radical scavenging activity of three hydroxy-4-methylcoumarins (1-3, figure 3 ) by means of experimental and theoretical methods [30] . firstly, the scavenging activity of the compounds was evaluated on abts (2,2′-azino-bis (3ethylbenzothiazoline-6-sulfonic acid) diammonium salt), dpph (2,2-diphenyl-1-picrylhydrazyl) and galvinoxyl radicals as trolox (a vitamin e analogue) equivalent antioxidant capability (teac). the results showed that, whereas 1 did not exhibit radical scavenging activity, 2 resulted more active than trolox against the abts•+ radical (ec50 30.83 μm) and 3 displayed better antioxidant activity than trolox against abts•+, dpph and galvinoxyl radicals (ec50 values of 39.98, 150.99 and 13.19 μm, respectively). it is likely that such differences in antioxidant activity may rely on the differences in the relative positions of hydroxy groups [31] . then, compound 3, which showed the best scavenging activity, was evaluated for its primary antioxidant capacity. in this step, three reaction mechanisms were considered: single electron transfer (set), hydrogen transfer (ht) and radical adduct formation (raf), involving •ooh, •ooch3 and •ch(oh)ch3 as radicals. these experiments were carried out in lipidic and aqueous media, in order to mimic membrane and intra-cellular environment [32] , [33] . the results indicated that different mechanisms are involved depending by the medium and that positions 4, 7 and 8 of compound 3 are probably involved in ht mechanism, whereas positions 3, 4, 5 and 7 could be involved in raf mechanism. finally, the secondary antioxidant activity of compound 3 in aqueous media at physiological ph was evaluated. in the haber-weiss reaction (i.e., the reduction of copper in aqueous media and subsequent copper-catalyzed hydroxy radical formation), 3 was able to inhibit copper (ii) reduction avoiding oxidative stress. it was found that hydroxy groups in position 7 and 8 are fundamental for the primary and secondary antioxidant activity in both lipid and aqueous media. recently, wang and co-workers investigated the antioxidant activity and the mechanism of the antiradical action of six coumarin-fused coumarins (4-9, figure 4 ) previously synthesized by xi and liu [34, 35] . firstly, the scavenging activity of the compounds was evaluated on abts (2,2 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), dpph (2,2-diphenyl-1-picrylhydrazyl) and galvinoxyl radicals as trolox (a vitamin e analogue) equivalent antioxidant capability (teac). the results showed that, whereas 1 did not exhibit radical scavenging activity, 2 resulted more active than trolox against the abts•+ radical (ec 50 30 .83 µm) and 3 displayed better antioxidant activity than trolox against abts•+, dpph and galvinoxyl radicals (ec 50 values of 39.98, 150.99 and 13.19 µm, respectively). it is likely that such differences in antioxidant activity may rely on the differences in the relative positions of hydroxy groups [31] . then, compound 3, which showed the best scavenging activity, was evaluated for its primary antioxidant capacity. in this step, three reaction mechanisms were considered: single electron transfer (set), hydrogen transfer (ht) and radical adduct formation (raf), involving •ooh, •ooch 3 and •ch(oh)ch 3 as radicals. these experiments were carried out in lipidic and aqueous media, in order to mimic membrane and intra-cellular environment [32] , [33] . the results indicated that different mechanisms are involved depending by the medium and that positions 4, 7 and 8 of compound 3 are probably involved in ht mechanism, whereas positions 3, 4, 5 and 7 could be involved in raf mechanism. finally, the secondary antioxidant activity of compound 3 in aqueous media at physiological ph was evaluated. in the haber-weiss reaction (i.e., the reduction of copper in aqueous media and subsequent copper-catalyzed hydroxy radical formation), 3 was able to inhibit copper (ii) reduction avoiding oxidative stress. it was found that hydroxy groups in position 7 and 8 are fundamental for the primary and secondary antioxidant activity in both lipid and aqueous media. recently, wang and co-workers investigated the antioxidant activity and the mechanism of the density functional theory (dft) calculations were performed, followed by the examination of the primary mechanisms, including ht, electron transfer-proton transfer (set-pt) and sequential proton loss transfer (splet). the most stable conformation of all the compounds was a non-planar structure, due to the steric repulsion of the groups in positions 5 and 5′. such conformation was retained by the correspondent anions and cation radicals (aro − , aroh +• ). ht process resulted the most significant in gas or non-polar phase, where compound 9 showed the highest activity. the ht path was possible for compounds 7, 8 and 9, having two or three oh groups, whereas 4 resulted inactive due to the absence of oh groups; compound 5, with only the 6-oh group, was less active than other compounds and 6 could merely trap dpph radical with a small rate constant. a second hat process was possible only for compound 9 and this finding could explain the higher activity of this molecule. in polar media, splet mechanism was favored-in this case the studied compounds were more efficient than trolox in the deprotonation step and, among them, compound 7 resulted to be the most promising, being more prone than the other compounds to deprotonate in a polar environment. the antioxidant potential of coumarin nucleus can be exploited in the production of new hybrid compound with enhanced antioxidant activity. a recent example of this strategy is the synthesis of a new series of chitosan derivatives (10a-d, figure 5 ) containing the coumarin nucleus, achieved by li and collaborators [36] . the antioxidant potential of compounds 10a-d was investigated by evaluating the inhibition of lipid peroxidation, metal ion chelation and free-radical scavenging activity. since both chitosan and coumarins have antioxidant properties themselves, the synthesized compounds were expected to show a stronger activity. lipid peroxidation inhibition was determined by quantifying thiobarbituric acid-reactive substance (tbars), using linoleic acid as a reference compound [37] . the results displayed the ability of the synthesized molecules to inhibit tabrs in a concentration-dependent manner; compound 10d emerged as the most active (ic50 = 0.11 mg/ml), showing a more efficient scavenging activity than chitosan alone (ic50 = 0.38 mg/ml). then, the radical-scavenging activity was the antioxidant potential of compounds 10a-d was investigated by evaluating the inhibition of lipid peroxidation, metal ion chelation and free-radical scavenging activity. since both chitosan and coumarins have antioxidant properties themselves, the synthesized compounds were expected to show a stronger activity. lipid peroxidation inhibition was determined by quantifying thiobarbituric evaluated. for this purpose, free radicals •oh, dpph and o2• − were used. compounds 10a-d showed a stronger •oh scavenging activity (ic50 = 0.09-0.12 mg/ml) compared to that of chitosan. these results suggested that the coumarin moiety strongly enhances chitosan antioxidant properties. since the chelating ability of antioxidants prevents oxidative stress by avoiding •oh production, iron-chelating properties of the new compounds were also evaluated through measurements of inhibition of ferrozine-fe 2+ complex formation. again, the obtained results (ic50 = 0.02-0.04 mg/ml) were better than those of chitosan alone. finally, cytotoxicity of the synthesized compounds was tested: no toxic effects were detected on 3t3-l1 and hhl-5 cells. interestingly, 3t3-l1 cells showed an increase in their viability, probably because of the antioxidant activity of the tested compounds. a similar approach was followed by popova and co-workers, who designed and synthesized a series of 4-methylcoumarins with tert-butyl, isobornyl and isocamphyl substituents (11, 12, 13-17, figure 6 ) [38] . the synthesized compounds were evaluated in terms of antioxidant, membrane-protective (mpa) and radical-scavenging (rsa) activities in vitro. all the tested compounds, at a concentration of 100 μm, exhibited good inhibitory activity against lipid peroxidation products (lpo) formation (ic50 = 3.33 -7.12 nm), whereas 7-hydroxy-4-methylcoumarin, used as reference compound, showed no activity. the scavenging activity, evaluated using dpph, strictly depended on the structure: only isobornyl derivatives showed moderate activity in the dpph assay (compound 15 showed rsa% = 57.48 ± 0.60 at 100 μm; rsa% = 87.95 ± 0.22 at 500 μm). moreover, the protective activity towards cell membrane was assessed, measuring the inhibitory activity against h2o2-induced hemolysis of red blood cells (rbcs). in all the experiments, the most promising compound was 15, having two isobornyl moieties. from these studies, it appears clear that coumarin derivatives show a great potential as antioxidant, membrane-protective and radical-scavenging compounds and that their activity depends mainly on the number and the position of the hydroxy groups. the term "cancer" defines a wide range of diseases caused by the accumulation of mutations and characterized by a multi-step process, involving many different factors which may not directly cause cancer themselves but can increase the chances of genetic mutations [39, 40] . recently, maleki et al. have synthesized eighteen o-prenylated coumarin derivatives and tested them on hela cervical cancer and hdf normal cells by mtt assay [41] . the most promising compounds are reported in figure 7 . the synthesized compounds were evaluated in terms of antioxidant, membrane-protective (mpa) and radical-scavenging (rsa) activities in vitro. all the tested compounds, at a concentration of 100 µm, exhibited good inhibitory activity against lipid peroxidation products (lpo) formation (ic 50 = 3.33-7.12 nm), whereas 7-hydroxy-4-methylcoumarin, used as reference compound, showed no activity. the scavenging activity, evaluated using dpph, strictly depended on the structure: only isobornyl derivatives showed moderate activity in the dpph assay (compound 15 showed rsa% = 57.48 ± 0.60 at 100 µm; rsa% = 87.95 ± 0.22 at 500 µm). moreover, the protective activity towards cell membrane was assessed, measuring the inhibitory activity against h 2 o 2 -induced hemolysis of red blood cells (rbcs). in all the experiments, the most promising compound was 15, having two isobornyl moieties. from these studies, it appears clear that coumarin derivatives show a great potential as antioxidant, membrane-protective and radical-scavenging compounds and that their activity depends mainly on the number and the position of the hydroxy groups. the term "cancer" defines a wide range of diseases caused by the accumulation of mutations and characterized by a multi-step process, involving many different factors which may not directly cause cancer themselves but can increase the chances of genetic mutations [39, 40] . recently, maleki et al. have synthesized eighteen o-prenylated coumarin derivatives and tested them on hela cervical cancer and hdf normal cells by mtt assay [41] . the most promising compounds are reported in figure 7 . the results represent a good starting point for the design of novel derivatives, because most of the examined compounds exhibited selective toxicity on hela cells (ic50 values between 136.4 ± 1.90 μm and 172.2 ± 1.80 μm after 24 h), whereas no negative effects on hdf normal cell's growth was detected. sar studies proved that the substitution on c6 position of coumarin nucleus provided the best anticancer activity, followed by substitution on c8. in addition, it was found that the cytotoxic properties of o-prenylated coumarins depends on the length of the prenyl chain, which increases the lipophilicity of the molecule, thus facilitating its penetration into the cells. the cytotoxic activity of prenylated-coumarin derivatives had been evaluated also by other groups. recent studies have shown a functional role of lipoxygenases (loxs) in carcinogenesis, precisely in prostatic cancer and the capability of 5-farnesyloxycoumarin (18, figure 8 ) to inhibit 15-lox-1 [42] [43] [44] starting from these observations, orafaie and collaborators investigated the inhibitory activity of compound 18 on 15-lox-1 [45] . the cytotoxic effects of 18 were evaluated by means of mtt assay on two carcinoma cell lines (pc-3 and du145) and a normal cell line (hff3), using 4-mmpb (4methyl-2-(4-methylpiperazinyl) pyrimido [4,5-b] benzothiazine) as reference compound. when pc3 and du145 human pca cells were treated with different concentrations of both 4-mmpb and 18 for 24, 48 and 72 h, a dose-dependent and time-dependent decrease in the survival of the cells was exhibited. pc3 cells resulted to be more sensitive than du145 cells to both inhibitors (ic50 in μg/ml for compound 18 on pc3 cells: 24 h, 40 19.52 ± 4.92) . moreover, compound 18 had no significant anti-proliferative activity on normal cells. concerning the mechanism of action, it was found that 5-farnesyloxycoumarin acts as a cytotoxic agent causing chromatin condensation and dna damage and induces the arrest of the cell cycle in g0/g1 phase. a similar study was carried out on 8-farnesyloxycoumarin (19, figure 8 ) by hosseinymehr and collaborators [46] . again, the coumarin derivative showed inhibitory activity on 15-lox-1 in pc3 and du145 cell lines, thus inducing apoptosis of the cancer cell, with the same mechanism of the results represent a good starting point for the design of novel derivatives, because most of the examined compounds exhibited selective toxicity on hela cells (ic 50 values between 136.4 ± 1.90 µm and 172.2 ± 1.80 µm after 24 h), whereas no negative effects on hdf normal cell's growth was detected. sar studies proved that the substitution on c6 position of coumarin nucleus provided the best anticancer activity, followed by substitution on c8. in addition, it was found that the cytotoxic properties of o-prenylated coumarins depends on the length of the prenyl chain, which increases the lipophilicity of the molecule, thus facilitating its penetration into the cells. the cytotoxic activity of prenylated-coumarin derivatives had been evaluated also by other groups. recent studies have shown a functional role of lipoxygenases (loxs) in carcinogenesis, precisely in prostatic cancer and the capability of 5-farnesyloxycoumarin (18, figure 8 ) to inhibit 15-lox-1 [42] [43] [44] . the results represent a good starting point for the design of novel derivatives, because most of the examined compounds exhibited selective toxicity on hela cells (ic50 values between 136.4 ± 1.90 μm and 172.2 ± 1.80 μm after 24 h), whereas no negative effects on hdf normal cell's growth was detected. sar studies proved that the substitution on c6 position of coumarin nucleus provided the best anticancer activity, followed by substitution on c8. in addition, it was found that the cytotoxic properties of o-prenylated coumarins depends on the length of the prenyl chain, which increases the lipophilicity of the molecule, thus facilitating its penetration into the cells. the cytotoxic activity of prenylated-coumarin derivatives had been evaluated also by other groups. recent studies have shown a functional role of lipoxygenases (loxs) in carcinogenesis, precisely in prostatic cancer and the capability of 5-farnesyloxycoumarin (18, figure 8 ) to inhibit 15-lox-1 [42] [43] [44] starting from these observations, orafaie and collaborators investigated the inhibitory activity of compound 18 on 15-lox-1 [45] . the cytotoxic effects of 18 were evaluated by means of mtt assay on two carcinoma cell lines (pc-3 and du145) and a normal cell line (hff3), using 4-mmpb (4methyl-2-(4-methylpiperazinyl) pyrimido [4,5-b] benzothiazine) as reference compound. when pc3 and du145 human pca cells were treated with different concentrations of both 4-mmpb and 18 for 24, 48 and 72 h, a dose-dependent and time-dependent decrease in the survival of the cells was exhibited. pc3 cells resulted to be more sensitive than du145 cells to both inhibitors (ic50 in μg/ml for compound 18 on pc3 cells: 24 h, 40 19.52 ± 4.92) . moreover, compound 18 had no significant anti-proliferative activity on normal cells. concerning the mechanism of action, it was found that 5-farnesyloxycoumarin acts as a cytotoxic agent causing chromatin condensation and dna damage and induces the arrest of the cell cycle in g0/g1 phase. a similar study was carried out on 8-farnesyloxycoumarin (19, figure 8 ) by hosseinymehr and collaborators [46] . again, the coumarin derivative showed inhibitory activity on 15-lox-1 in pc3 and du145 cell lines, thus inducing apoptosis of the cancer cell, with the same mechanism of starting from these observations, orafaie and collaborators investigated the inhibitory activity of compound 18 on 15-lox-1 [45] . the cytotoxic effects of 18 were evaluated by means of mtt assay on two carcinoma cell lines (pc-3 and du145) and a normal cell line (hff3), using 4-mmpb (4-methyl-2-(4-methylpiperazinyl) pyrimido [4,5-b] concerning the mechanism of action, it was found that 5-farnesyloxycoumarin acts as a cytotoxic agent causing chromatin condensation and dna damage and induces the arrest of the cell cycle in g 0 /g 1 phase. a similar study was carried out on 8-farnesyloxycoumarin (19, figure 8 ) by hosseinymehr and collaborators [46] . again, the coumarin derivative showed inhibitory activity on 15-lox-1 in pc3 and du145 cell lines, thus inducing apoptosis of the cancer cell, with the same mechanism of compound 18. pc3 cells resulted to be more sensitive to 19 inhibition than du145 cells; ic 50 halawa et al. synthesized and characterized a new series of 4-arylamino-3-nitrocoumarin analogues from 4-hydroxycoumarin and tested them on the human cervix carcinoma cell line kb-3-1 [47] . these compounds were found to target the dna-topo i (human topoisomerase i) complex, thus blocking cell replication and leading to cell death. among this series, thiazolidinylidene derivative 20 ( figure 9 ) containing a malononitrile fragment exhibited the best cytotoxic activity with an ic 50 value of 21 µm. the cytotoxicity of 20 was explained also by docking studies which highlighted that it forms important h-bonds with arg364, asp533, gln633 and 5 -thio-2 deoxyguanosine phosphonic acid of the dna backbone. [47] . these compounds were found to target the dna-topo i (human topoisomerase i) complex, thus blocking cell replication and leading to cell death. among this series, thiazolidinylidene derivative 20 ( figure 9 ) containing a malononitrile fragment exhibited the best cytotoxic activity with an ic50 value of 21 μm. the cytotoxicity of 20 was explained also by docking studies which highlighted that it forms important h-bonds with arg364, asp533, gln633 and 5′-thio-2′ deoxyguanosine phosphonic acid of the dna backbone. herrera et al. synthesized a series of 3-and 7-styrylcoumarins, some of which showed antiproliferative activity on sw480 human colon adenocarcinoma cells [48] . among them, 7-(4-hydroxy-3,5-dimethoxystyryl)-2h-chromen-2-one (21, figure 10 ) showed the highest activity (ic50 = 1.01 μm) as it was capable of inducing apoptosis in sw480 cells, probably by modulating the tumor-suppressor protein p53. the new compound was also tested in vivo, demonstrating to be able to inhibit the early progression of colon adenocarcinoma [49] . as coumarin nucleus can be widely decorated, it can be used in the synthesis of hybrid compounds, targeting different proteins involved not only in tumor growth but also in metastatic and angiogenetic processes. in this context, diao and collaborators synthesized a series of diethylene glycol tethered isatin-1,2,3-triazole-coumarin derivatives (22a-l, figure 11 ) [50] . several isatin-based or 1,2,3-triazole or coumarin-based compounds (semaxanib, carboxyamidotriazole and stx64) are involved in clinical trials or have already been used the treatment of various cancers (as colon-rectal, prostatic, endometrial and breast cancer) [51, 52] , whereas isatin-1,2,3-triazole-coumarin derivatives showed activity against different cancer types. in addition, sar studies demonstrated that the linker between isatin and 1,2,3-triazole influences the activity [52, 53] and that hydrogen bonds are fundamental for the biological activity [54] . these evidences guided diao and collaborators in the choice of diethylene glycol as a linker [47] . ten compounds were tested on hepg2 (liver carcinoma), hela (cervical cancer), a549 (lung adenocarcinoma), du145 (prostatic cancer), skov3 (ovarian carcinoma), mcf-7 (breast cancer) and drug-resistant mcf-7/dox (doxorubicin-resistant mcf-7) herrera et al. synthesized a series of 3-and 7-styrylcoumarins, some of which showed anti-proliferative activity on sw480 human colon adenocarcinoma cells [48] . among them, 7-(4-hydroxy-3,5-dimethoxystyryl)-2h-chromen-2-one (21, figure 10 ) showed the highest activity (ic 50 = 1.01 µm) as it was capable of inducing apoptosis in sw480 cells, probably by modulating the tumor-suppressor protein p53. the new compound was also tested in vivo, demonstrating to be able to inhibit the early progression of colon adenocarcinoma [49] . [47] . these compounds were found to target the dna-topo i (human topoisomerase i) complex, thus blocking cell replication and leading to cell death. among this series, thiazolidinylidene derivative 20 ( figure 9 ) containing a malononitrile fragment exhibited the best cytotoxic activity with an ic50 value of 21 μm. the cytotoxicity of 20 was explained also by docking studies which highlighted that it forms important h-bonds with arg364, asp533, gln633 and 5′-thio-2′ deoxyguanosine phosphonic acid of the dna backbone. herrera et al. synthesized a series of 3-and 7-styrylcoumarins, some of which showed antiproliferative activity on sw480 human colon adenocarcinoma cells [48] . among them, 7-(4-hydroxy-3,5-dimethoxystyryl)-2h-chromen-2-one (21, figure 10 ) showed the highest activity (ic50 = 1.01 μm) as it was capable of inducing apoptosis in sw480 cells, probably by modulating the tumor-suppressor protein p53. the new compound was also tested in vivo, demonstrating to be able to inhibit the early progression of colon adenocarcinoma [49] . as coumarin nucleus can be widely decorated, it can be used in the synthesis of hybrid compounds, targeting different proteins involved not only in tumor growth but also in metastatic and angiogenetic processes. in this context, diao and collaborators synthesized a series of diethylene glycol tethered isatin-1,2,3-triazole-coumarin derivatives (22a-l, figure 11 ) [50] . several isatin-based or 1,2,3-triazole or coumarin-based compounds (semaxanib, carboxyamidotriazole and stx64) are involved in clinical trials or have already been used the treatment of various cancers (as colon-rectal, prostatic, endometrial and breast cancer) [51, 52] , whereas isatin-1,2,3-triazole-coumarin derivatives showed activity against different cancer types. in addition, sar studies demonstrated that the linker between isatin and 1,2,3-triazole influences the activity [52, 53] and that hydrogen bonds are fundamental for the biological activity [54] . these evidences guided diao and collaborators in the choice of diethylene glycol as a linker [47] . ten compounds were tested on hepg2 (liver carcinoma), hela (cervical cancer), a549 (lung adenocarcinoma), du145 (prostatic cancer), skov3 (ovarian carcinoma), mcf-7 (breast cancer) and drug-resistant mcf-7/dox (doxorubicin-resistant mcf-7) as coumarin nucleus can be widely decorated, it can be used in the synthesis of hybrid compounds, targeting different proteins involved not only in tumor growth but also in metastatic and angiogenetic processes. in this context, diao and collaborators synthesized a series of diethylene glycol tethered isatin-1,2,3-triazole-coumarin derivatives (22a-l, figure 11 ) [50] . several isatin-based or 1,2,3-triazole or coumarin-based compounds (semaxanib, carboxyamidotriazole and stx64) are involved in clinical trials or have already been used the treatment of various cancers (as colon-rectal, prostatic, endometrial and breast cancer) [51, 52] , whereas isatin-1,2,3-triazole-coumarin derivatives showed activity against different cancer types. in addition, sar studies demonstrated that the linker between isatin and 1,2,3-triazole influences the activity [52, 53] and that hydrogen bonds are fundamental for the biological activity [54] . these evidences guided diao and collaborators in the choice of diethylene glycol as a linker [47] . ten compounds were tested on hepg2 (liver carcinoma), hela (cervical cancer), a549 (lung adenocarcinoma), du145 (prostatic cancer), skov3 (ovarian carcinoma), mcf-7 (breast cancer) and drug-resistant mcf-7/dox (doxorubicin-resistant mcf-7) human cancer cell lines. all the synthesized compounds showed weak to moderate in vitro activity against all the cell lines, therefore further sar studies are necessary for the obtainment of new, more active compounds. human cancer cell lines. all the synthesized compounds showed weak to moderate in vitro activity against all the cell lines, therefore further sar studies are necessary for the obtainment of new, more active compounds. figure 11 . general structure of diethylene glycol tethered isatin-1,2,3-triazole-coumarin derivatives 22a-l. cai et al. developed a series of fluorescent coumarin-benzo[b]thiophene 1, 1-dioxide conjugates [55] . these compounds act on stat3, which is involved in the regulation of mitochondrial apoptotic pathway [56] . in particular, they hypothesized that the inhibition of phosphorylation of tyr705 and ser727 might prevent stat3 activation. four stat3 over-activated human cancer cell lines (i.e., human breast carcinoma mda-mb-231 and mcf-7 cells, human colonic carcinoma hct-116 cells and human hepatocellular carcinoma hepg2 cells) were selected to assess the biological activity of the newly-synthesized compounds. compound 23 ( figure 12 ) showed high potency in inducing cancer cell apoptosis and ros generation, inhibiting stat3 phosphorylation on tyr705, affecting mitochondrial membrane potential and preventing stat3 dna-binding activity. in addition, 23 inhibited implanted 4t1 breast cancer growth in vivo. the antiproliferative effects of compound 23 on normal cells were investigated by mtt assays comparing it to the stat3 inhibitor stattic, which showed growth inhibition activity in both tumor and normal cells, whereas compound 23 exhibited selective inhibitory activity against cancer cells (ic50 14.62 μm for mcf-10a cells and ic50 35.60 μm for l02 cells), indicating a higher selectivity than stattic. carbonic anhydrases (cas) are ubiquitous metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and protons, which is an essential physiological reaction [57] . thus, this enzyme is involved in a wide range of physiological and pathological processes (ph regulation, co2 homeostasis, respiration, bone resorption and tumorigenesis) [58] and its deregulation by means of carbonic anhydrases inhibitors (cais) may be useful in the treatment of many disorders [59] [60] [61] . the ideal ca inhibitor would selectively act against those isoforms (hca ix, xii, for instance) related to a certain disease [62, 63] . in this context, coumarins emerged as potential figure 11 . general structure of diethylene glycol tethered isatin-1,2,3-triazole-coumarin derivatives 22a-l. cai et al. developed a series of fluorescent coumarin-benzo[b]thiophene 1, 1-dioxide conjugates [55] . these compounds act on stat3, which is involved in the regulation of mitochondrial apoptotic pathway [56] . in particular, they hypothesized that the inhibition of phosphorylation of tyr705 and ser727 might prevent stat3 activation. four stat3 over-activated human cancer cell lines (i.e., human breast carcinoma mda-mb-231 and mcf-7 cells, human colonic carcinoma hct-116 cells and human hepatocellular carcinoma hepg2 cells) were selected to assess the biological activity of the newly-synthesized compounds. compound 23 ( figure 12 ) showed high potency in inducing cancer cell apoptosis and ros generation, inhibiting stat3 phosphorylation on tyr705, affecting mitochondrial membrane potential and preventing stat3 dna-binding activity. in addition, 23 inhibited implanted 4t1 breast cancer growth in vivo. the antiproliferative effects of compound 23 on normal cells were investigated by mtt assays comparing it to the stat3 inhibitor stattic, which showed growth inhibition activity in both tumor and normal cells, whereas compound 23 exhibited selective inhibitory activity against cancer cells (ic 50 human cancer cell lines. all the synthesized compounds showed weak to moderate in vitro activity against all the cell lines, therefore further sar studies are necessary for the obtainment of new, more active compounds. figure 11 . general structure of diethylene glycol tethered isatin-1,2,3-triazole-coumarin derivatives 22a-l. cai et al. developed a series of fluorescent coumarin-benzo[b]thiophene 1, 1-dioxide conjugates [55] . these compounds act on stat3, which is involved in the regulation of mitochondrial apoptotic pathway [56] . in particular, they hypothesized that the inhibition of phosphorylation of tyr705 and ser727 might prevent stat3 activation. four stat3 over-activated human cancer cell lines (i.e., human breast carcinoma mda-mb-231 and mcf-7 cells, human colonic carcinoma hct-116 cells and human hepatocellular carcinoma hepg2 cells) were selected to assess the biological activity of the newly-synthesized compounds. compound 23 ( figure 12 ) showed high potency in inducing cancer cell apoptosis and ros generation, inhibiting stat3 phosphorylation on tyr705, affecting mitochondrial membrane potential and preventing stat3 dna-binding activity. in addition, 23 inhibited implanted 4t1 breast cancer growth in vivo. the antiproliferative effects of compound 23 on normal cells were investigated by mtt assays comparing it to the stat3 inhibitor stattic, which showed growth inhibition activity in both tumor and normal cells, whereas compound 23 exhibited selective inhibitory activity against cancer cells (ic50 14.62 μm for mcf-10a cells and ic50 35.60 μm for l02 cells), indicating a higher selectivity than stattic. carbonic anhydrases (cas) are ubiquitous metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and protons, which is an essential physiological reaction [57] . thus, this enzyme is involved in a wide range of physiological and pathological processes (ph regulation, co2 homeostasis, respiration, bone resorption and tumorigenesis) [58] and its deregulation by means of carbonic anhydrases inhibitors (cais) may be useful in the treatment of many disorders [59] [60] [61] . the ideal ca inhibitor would selectively act against those isoforms (hca ix, xii, for instance) related to a certain disease [62, 63] . in this context, coumarins emerged as potential carbonic anhydrases (cas) are ubiquitous metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and protons, which is an essential physiological reaction [57] . thus, this enzyme is involved in a wide range of physiological and pathological processes (ph regulation, co 2 homeostasis, respiration, bone resorption and tumorigenesis) [58] and its deregulation by means of carbonic anhydrases inhibitors (cais) may be useful in the treatment of many disorders [59] [60] [61] . the ideal ca inhibitor would selectively act against those isoforms (hca ix, xii, for instance) related to a certain disease [62, 63] . in this context, coumarins emerged as potential atypical hca ligands that, unlike classical hcais, do not need to chelate the prosthetic zinc ion but, after binding the catalytic site, are hydrolyzed to the corresponding 2-hydroxy cinnamic acid derivatives, the actual inhibitors [64, 65] . some studies highlighted that coumarins are capable of binding at the entrance of hca catalytic site, blocking the enzyme activity. furthermore, 7-hydroxycoumarins derivatives showed good selectivity toward ix and xii isoforms over hca i and ii and, in some cases, they exhibited cytotoxic activity on cancer cells [66] [67] [68] [69] . many efforts have been made in this direction and the most recent results are here reported. fois and collaborators have recently investigated the selective inhibitory activity towards ca ix and ca xii of extracts of magydaris pastiacea seeds, from which they isolated ten linear furocoumarins, four simple coumarins and a new angular dihydrofurocoumarin [70] all the mentioned compounds were tested against four isoforms of hca: both extracts showed selective activity towards ca ix and xii whereas no effect was seen on isoforms i and ii at a concentration of 100 ng/ml. the most promising compound was umbelliprenin (24, figure 13 ), highly active (k i = 5.8 nm) against ca xii and highly selective over isoforms i and ii. atypical hca ligands that, unlike classical hcais, do not need to chelate the prosthetic zinc ion but, after binding the catalytic site, are hydrolyzed to the corresponding 2-hydroxy cinnamic acid derivatives, the actual inhibitors [64, 65] . some studies highlighted that coumarins are capable of binding at the entrance of hca catalytic site, blocking the enzyme activity. furthermore, 7hydroxycoumarins derivatives showed good selectivity toward ix and xii isoforms over hca i and ii and, in some cases, they exhibited cytotoxic activity on cancer cells [66] [67] [68] [69] . many efforts have been made in this direction and the most recent results are here reported. fois and collaborators have recently investigated the selective inhibitory activity towards ca ix and ca xii of extracts of magydaris pastiacea seeds, from which they isolated ten linear furocoumarins, four simple coumarins and a new angular dihydrofurocoumarin [70] all the mentioned compounds were tested against four isoforms of hca: both extracts showed selective activity towards ca ix and xii whereas no effect was seen on isoforms i and ii at a concentration of 100 ng/ml. the most promising compound was umbelliprenin (24, figure 13 ), highly active (ki = 5.8 nm) against ca xii and highly selective over isoforms i and ii. umbelliprenin (24) was then tested on hela cancer cells in order to evaluate its cytotoxic activity and resulted to possess a moderate cytotoxicity (ic50 =75 μm) proving to be able to inhibit tumor growth, angiogenesis and metastasis formation in mice (after i.p. administration). it is noteworthy that ca ix and xii are overexpressed in tumor cells under hypoxic conditions, whereas the mentioned tests were carried out under normoxic conditions, which could explain the moderate cytotoxic activity of the isolated compound. in 2019, starting from 4-methylumbelliferone (25, figure 14 ), buran and co-workers synthesized a series of 8-substituted coumarin-based compounds bearing alkylpiperazine and arylpiperazine chains (26-37, figure 14 ), and evaluated their inhibitory activity against hca i, ii, ix and xii [71] . all the tested compounds were able to inhibit hca isoforms ix and xii, without showing any inhibitory activity towards the cytosolic isoforms i and ii up to a 10 μm concentration. the test pointed out that these compounds had higher affinity for hca xii over ix and, except for compound 36 (ki (hca xii) = 26.4 nm), they all had ki values comparable to those of the reference drug acetazolamide (ki (hca xii) = 5.7 nm). it is remarkable that the substitution of c8 position of 4-methylumbelliferone (25) umbelliprenin (24) was then tested on hela cancer cells in order to evaluate its cytotoxic activity and resulted to possess a moderate cytotoxicity (ic 50 = 75 µm) proving to be able to inhibit tumor growth, angiogenesis and metastasis formation in mice (after i.p. administration). it is noteworthy that ca ix and xii are overexpressed in tumor cells under hypoxic conditions, whereas the mentioned tests were carried out under normoxic conditions, which could explain the moderate cytotoxic activity of the isolated compound. in 2019, starting from 4-methylumbelliferone (25, figure 14 ), buran and co-workers synthesized a series of 8-substituted coumarin-based compounds bearing alkylpiperazine and arylpiperazine chains (26-37, figure 14 ), and evaluated their inhibitory activity against hca i, ii, ix and xii [71] . all the tested compounds were able to inhibit hca isoforms ix and xii, without showing any inhibitory activity towards the cytosolic isoforms i and ii up to a 10 µm concentration. the test pointed out that these compounds had higher affinity for hca xii over ix and, except for compound 36 (k i (hca xii) = 26.4 nm), they all had k i values comparable to those of the reference drug acetazolamide (k i (hca xii) = 5.7 nm). atypical hca ligands that, unlike classical hcais, do not need to chelate the prosthetic zinc ion but, after binding the catalytic site, are hydrolyzed to the corresponding 2-hydroxy cinnamic acid derivatives, the actual inhibitors [64, 65] . some studies highlighted that coumarins are capable of binding at the entrance of hca catalytic site, blocking the enzyme activity. furthermore, 7hydroxycoumarins derivatives showed good selectivity toward ix and xii isoforms over hca i and ii and, in some cases, they exhibited cytotoxic activity on cancer cells [66] [67] [68] [69] . many efforts have been made in this direction and the most recent results are here reported. fois and collaborators have recently investigated the selective inhibitory activity towards ca ix and ca xii of extracts of magydaris pastiacea seeds, from which they isolated ten linear furocoumarins, four simple coumarins and a new angular dihydrofurocoumarin [70] all the mentioned compounds were tested against four isoforms of hca: both extracts showed selective activity towards ca ix and xii whereas no effect was seen on isoforms i and ii at a concentration of 100 ng/ml. the most promising compound was umbelliprenin (24, figure 13 ), highly active (ki = 5.8 nm) against ca xii and highly selective over isoforms i and ii. umbelliprenin (24) was then tested on hela cancer cells in order to evaluate its cytotoxic activity and resulted to possess a moderate cytotoxicity (ic50 =75 μm) proving to be able to inhibit tumor growth, angiogenesis and metastasis formation in mice (after i.p. administration). it is noteworthy that ca ix and xii are overexpressed in tumor cells under hypoxic conditions, whereas the mentioned tests were carried out under normoxic conditions, which could explain the moderate cytotoxic activity of the isolated compound. in 2019, starting from 4-methylumbelliferone (25, figure 14 ), buran and co-workers synthesized a series of 8-substituted coumarin-based compounds bearing alkylpiperazine and arylpiperazine chains (26-37, figure 14 ), and evaluated their inhibitory activity against hca i, ii, ix and xii [71] . all the tested compounds were able to inhibit hca isoforms ix and xii, without showing any inhibitory activity towards the cytosolic isoforms i and ii up to a 10 μm concentration. the test pointed out that these compounds had higher affinity for hca xii over ix and, except for compound 36 (ki (hca xii) = 26.4 nm), they all had ki values comparable to those of the reference drug acetazolamide (ki (hca xii) = 5.7 nm). it is remarkable that the substitution of c8 position of 4-methylumbelliferone (25) it is remarkable that the substitution of c8 position of 4-methylumbelliferone (25) did not have any influence on inhibition of hca xii, suggesting that no significant interaction may be achieved between the side chains of compounds 26-37 and the catalytic site of isoform xii. on the other hand, alkylpiperazine derivatives showed better activity on hca ix when compared with the other synthesized compounds, being compound 30 the one with the highest activity among them (ic 50 = 27.1 nm). similar results were obtained by many other groups that have recently synthesized coumarin-based compounds and evaluated them as hca ix and xii inhibitors. sulphocumarins, bis-coumarins and coumarins 1,3,4-oxadiazole derivatives are some examples [72] [73] [74] . the battle against infections and multidrug resistant bacteria is almost certainly one of the most challenging issue that the scientific community and the whole mankind will face in the near future. multidrug resistant (mdr) bacteria are defined as non-susceptible strains to one or more antimicrobials on three or more antimicrobial classes, whereas strains that are non-susceptible to all antimicrobials are classified as extremely drug-resistant strains [75, 76] . the plant kingdom provides a virtually endless source of novel chemicals and scaffolds, such as polyphenols and coumarins [77] . in 2005, the antibacterial potency of nearly 50 coumarin derivatives, natural and synthetic, was evaluated and then correlated by a sar study. bacterial susceptibility to coumarins was evaluated by determining the minimal inhibitory concentration (mic) and minimum bactericidal concentration (mbc), considering active compounds exhibiting mic values ranging from 62.5 to 2000 µg/ml. among the active compounds, osthenole (a natural coumarin having also the anti-inflammatory activity) showed the most potent activity with a mic of 62.5 µg/ml against s. aureus and b. cereus [78] . in 2015, nagamallu and co-workers exploited the vilsmeier-haack reaction to obtain a series of novel pyrazole-containing coumarins and then evaluated their antioxidant and antibacterial activities [79] . among the series, two compounds (38 and 39, figure 15 ) showed a good antibacterial and antifungal activity, with mic values comparable with the ones of ciprofloxacin (positive control against bacteria species) and fluconazole (positive control against fungi strains). the battle against infections and multidrug resistant bacteria is almost certainly one of the most challenging issue that the scientific community and the whole mankind will face in the near future. multidrug resistant (mdr) bacteria are defined as non-susceptible strains to one or more antimicrobials on three or more antimicrobial classes, whereas strains that are non-susceptible to all antimicrobials are classified as extremely drug-resistant strains [75, 76] . the plant kingdom provides a virtually endless source of novel chemicals and scaffolds, such as polyphenols and coumarins [77] . in 2005, the antibacterial potency of nearly 50 coumarin derivatives, natural and synthetic, was evaluated and then correlated by a sar study. bacterial susceptibility to coumarins was evaluated by determining the minimal inhibitory concentration (mic) and minimum bactericidal concentration (mbc), considering active compounds exhibiting mic values ranging from 62.5 to 2000 μg/ml. among the active compounds, osthenole (a natural coumarin having also the anti-inflammatory activity) showed the most potent activity with a mic of 62.5 μg/ml against s. aureus and b. cereus [78] . in 2015, nagamallu and co-workers exploited the vilsmeier-haack reaction to obtain a series of novel pyrazole-containing coumarins and then evaluated their antioxidant and antibacterial activities [79] . among the series, two compounds (38 and 39, figure 15 ) showed a good antibacterial and antifungal activity, with mic values comparable with the ones of ciprofloxacin (positive control against bacteria species) and fluconazole (positive control against fungi strains). following the idea that the introduction of an additional coumarin nucleus on a parent coumarin molecule could improve the pharmacological activity (i.e., dicumarol as anticoagulant), in 2017, chougala and colleagues designed and synthesized a series of bis-coumarin derivatives [80] . figure 16 shows the common scaffolds of the bis-coumarins synthesized using l-proline as catalyst in a multi-component reaction approach. following the idea that the introduction of an additional coumarin nucleus on a parent coumarin molecule could improve the pharmacological activity (i.e., dicumarol as anticoagulant), in 2017, chougala and colleagues designed and synthesized a series of bis-coumarin derivatives [80] . figure 16 shows the common scaffolds of the bis-coumarins synthesized using l-proline as catalyst in a multi-component reaction approach. the antibacterial potency of the compounds was evaluated against gram-positive and gramnegative strains, comparing the mic with ciprofloxacin and most of the newly synthesized compounds showed modest to good inhibiting activity against the tested microorganisms. compounds 40a-e ( figure 16 ) were highly active and more potent than ciprofloxacin against s. aureus and e. faecalis, whereas 41c and 41d were active only against gram-positive e. faecalis. actually, compounds 41a-e were the most promising against e. coli. once established the broad spectrum of action of the coumarin nucleus, various researchers focused their attention on the activity against multidrug resistant strains, in particular on the eskape pathogens, the coterie that escape the lethal action of antibiotics: enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumanii, pseudomonas aeruginosa and enterobacter species [81] . in 2017, naik et al. synthesized and evaluated against s. aureus and other bacterial strains a series of 3,4-dihydropyrimidinone-coumarin analogues, measuring the mic values and comparing them to ciprofloxacin [82] . the structures and the mic values for compounds 42-49, the most promising derivatives, are reported in figure 17 : the substitution at c6 position seemed to be excellent for the activity and able to modulate the potency, decreasing the efficiency in the order of -ch3 > 7,8-benzo > -cl > -och3. furthermore, it was revealed from docking studies that that one hydrogen atom and two oxygen atoms of 3,4-dihydropyrimidinone substituted coumarins form interactions with the active site residues of s. aureus gyrase, indicating that the presence of hydrogen bond acceptor and donor groups may enhance antimicrobial activity. in 2018, chavan and hosamani proposed a facile method for the microwave assisted synthesis of a series of pyrazole-containing coumarins and tested their antibacterial and anti-inflammatory activities [83] . the researchers evaluated the in vitro antibacterial activity of the newly synthesized the antibacterial potency of the compounds was evaluated against gram-positive and gram-negative strains, comparing the mic with ciprofloxacin and most of the newly synthesized compounds showed modest to good inhibiting activity against the tested microorganisms. compounds 40a-e ( figure 16 ) were highly active and more potent than ciprofloxacin against s. aureus and e. faecalis, whereas 41c and 41d were active only against gram-positive e. faecalis. actually, compounds 41a-e were the most promising against e. coli. once established the broad spectrum of action of the coumarin nucleus, various researchers focused their attention on the activity against multidrug resistant strains, in particular on the eskape pathogens, the coterie that escape the lethal action of antibiotics: enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumanii, pseudomonas aeruginosa and enterobacter species [81] . in 2017, naik et al. synthesized and evaluated against s. aureus and other bacterial strains a series of 3,4-dihydropyrimidinone-coumarin analogues, measuring the mic values and comparing them to ciprofloxacin [82] . the structures and the mic values for compounds 42-49, the most promising derivatives, are reported in figure 17 : the substitution at c6 position seemed to be excellent for the activity and able to modulate the potency, decreasing the efficiency in the order of -ch 3 > 7,8-benzo > -cl > -och 3 . furthermore, it was revealed from docking studies that that one hydrogen atom and two oxygen atoms of 3,4-dihydropyrimidinone substituted coumarins form interactions with the active site residues of s. aureus gyrase, indicating that the presence of hydrogen bond acceptor and donor groups may enhance antimicrobial activity. the antibacterial potency of the compounds was evaluated against gram-positive and gramnegative strains, comparing the mic with ciprofloxacin and most of the newly synthesized compounds showed modest to good inhibiting activity against the tested microorganisms. compounds 40a-e ( figure 16 ) were highly active and more potent than ciprofloxacin against s. aureus and e. faecalis, whereas 41c and 41d were active only against gram-positive e. faecalis. actually, compounds 41a-e were the most promising against e. coli. once established the broad spectrum of action of the coumarin nucleus, various researchers focused their attention on the activity against multidrug resistant strains, in particular on the eskape pathogens, the coterie that escape the lethal action of antibiotics: enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumanii, pseudomonas aeruginosa and enterobacter species [81] . in 2017, naik et al. synthesized and evaluated against s. aureus and other bacterial strains a series of 3,4-dihydropyrimidinone-coumarin analogues, measuring the mic values and comparing them to ciprofloxacin [82] . the structures and the mic values for compounds 42-49, the most promising derivatives, are reported in figure 17 : the substitution at c6 position seemed to be excellent for the activity and able to modulate the potency, decreasing the efficiency in the order of -ch3 > 7,8-benzo > -cl > -och3. furthermore, it was revealed from docking studies that that one hydrogen atom and two oxygen atoms of 3,4-dihydropyrimidinone substituted coumarins form interactions with the active site residues of s. aureus gyrase, indicating that the presence of hydrogen bond acceptor and donor groups may enhance antimicrobial activity. in 2018, chavan and hosamani proposed a facile method for the microwave assisted synthesis of a series of pyrazole-containing coumarins and tested their antibacterial and anti-inflammatory activities [83] . the researchers evaluated the in vitro antibacterial activity of the newly synthesized in 2018, chavan and hosamani proposed a facile method for the microwave assisted synthesis of a series of pyrazole-containing coumarins and tested their antibacterial and anti-inflammatory activities [83] . the researchers evaluated the in vitro antibacterial activity of the newly synthesized compounds through agar-well diffusion method against two gram-positive (bacillus subtilis (atcc no. staphylococcus aureus (atcc-29213)) and two gram-negative (escherichia coli (atcc-25922) and pseudomonas aeruginosa (atcc no.25619)) bacterial strains [84] . the mic values were compared to those of ciprofloxacin and all the compounds showed significant antibacterial activity. in particular, compounds 50 and 51 ( figure 18 ), showed an excellent activity against s. aureus (mic 0.78 µg/ml and mic 1.562 µg/ml, respectively). docking studies were in good agreement with the biological results. compounds through agar-well diffusion method against two gram-positive (bacillus subtilis (atcc no. 23857) and staphylococcus aureus (atcc-29213)) and two gram-negative (escherichia coli (atcc-25922) and pseudomonas aeruginosa (atcc no.25619)) bacterial strains [84] . the mic values were compared to those of ciprofloxacin and all the compounds showed significant antibacterial activity. in particular, compounds 50 and 51 ( figure 18 ), showed an excellent activity against s. aureus (mic 0.78 μg/ml and mic 1.562 μg/ml, respectively). docking studies were in good agreement with the biological results. in 2017, madeiro and co-workers focalized their interest towards the antibiotic activity of coumarins isolated from rutacea species ( figure 19 ) [85] . bergapten, xantoxin, isopimpinellin and imperatorin did not exhibit any antibacterial activity, even at the highest concentration, against s. aureus strains resistant to tetracycline, erythromycin and norfloxacin. however, their role as modulator of other antibiotics seemed quite promising, because isopimpinellin and imperatorin reduced the mic of erythromycin, tetracycline and norfloxacin. nevertheless, more detailed research is necessary in order to enable the use of these natural products as adjuvants to antimicrobial agents. in 2018, widelsky and co-workers isolated some similar linear furanocoumarins from the fruits of peucedanum luxurians tamamsch, with more encouraging results. plants of the peucedanum genus have been used for centuries as antibacterial agents and, for some of them, the activity was confirmed by biological and pharmacological studies on plant extracts and on a few isolated compounds [86] . all the six isolated compounds showed a broad growth-inhibitor activity against several bacteria strains but three of them ( figure 20 ) resulted particularly interesting. 6′,7′-dihydroxybergamottin was the most active against all the bacteria strains tested, probably because of the aliphatic chain in c5 position; similar activity was noticed for peucedanin and officinalin isobutyrate. in 2017, madeiro and co-workers focalized their interest towards the antibiotic activity of coumarins isolated from rutacea species ( figure 19 ) [85] . bergapten, xantoxin, isopimpinellin and imperatorin did not exhibit any antibacterial activity, even at the highest concentration, against s. aureus strains resistant to tetracycline, erythromycin and norfloxacin. however, their role as modulator of other antibiotics seemed quite promising, because isopimpinellin and imperatorin reduced the mic of erythromycin, tetracycline and norfloxacin. nevertheless, more detailed research is necessary in order to enable the use of these natural products as adjuvants to antimicrobial agents. compounds through agar-well diffusion method against two gram-positive (bacillus subtilis (atcc no. 23857) and staphylococcus aureus (atcc-29213)) and two gram-negative (escherichia coli (atcc-25922) and pseudomonas aeruginosa (atcc no.25619)) bacterial strains [84] . the mic values were compared to those of ciprofloxacin and all the compounds showed significant antibacterial activity. in particular, compounds 50 and 51 ( figure 18 ), showed an excellent activity against s. aureus (mic 0.78 μg/ml and mic 1.562 μg/ml, respectively). docking studies were in good agreement with the biological results. in 2017, madeiro and co-workers focalized their interest towards the antibiotic activity of coumarins isolated from rutacea species ( figure 19 ) [85] . bergapten, xantoxin, isopimpinellin and imperatorin did not exhibit any antibacterial activity, even at the highest concentration, against s. aureus strains resistant to tetracycline, erythromycin and norfloxacin. however, their role as modulator of other antibiotics seemed quite promising, because isopimpinellin and imperatorin reduced the mic of erythromycin, tetracycline and norfloxacin. nevertheless, more detailed research is necessary in order to enable the use of these natural products as adjuvants to antimicrobial agents. in 2018, widelsky and co-workers isolated some similar linear furanocoumarins from the fruits of peucedanum luxurians tamamsch, with more encouraging results. plants of the peucedanum genus have been used for centuries as antibacterial agents and, for some of them, the activity was confirmed by biological and pharmacological studies on plant extracts and on a few isolated compounds [86] . all the six isolated compounds showed a broad growth-inhibitor activity against several bacteria strains but three of them ( figure 20 ) resulted particularly interesting. 6′,7′-dihydroxybergamottin was the most active against all the bacteria strains tested, probably because of the aliphatic chain in c5 position; similar activity was noticed for peucedanin and officinalin isobutyrate. in 2018, widelsky and co-workers isolated some similar linear furanocoumarins from the fruits of peucedanum luxurians tamamsch, with more encouraging results. plants of the peucedanum genus have been used for centuries as antibacterial agents and, for some of them, the activity was confirmed by biological and pharmacological studies on plant extracts and on a few isolated compounds [86] . all the six isolated compounds showed a broad growth-inhibitor activity against several bacteria strains but three of them ( figure 20 ) resulted particularly interesting. 6 ,7 -dihydroxybergamottin was the most active against all the bacteria strains tested, probably because of the aliphatic chain in c5 position; similar activity was noticed for peucedanin and officinalin isobutyrate. liu and colleagues made a huge effort to synthesize and identify coumarin-pyrazole carboxamide derivatives as potential topoisomerase-ii inhibitors: 70 novel compounds were obtained and evaluated [87] . three of them (52-54, figure 21 ) were endowed with promising antimicrobial activities. in particular, compound 52 showed a considerable inhibitory activity compared with ciprofloxacin against escherichia coli and compound 53 exhibited excellent antibacterial activity against salmonella typhi. the selected compounds exhibited also potent inhibition against topo ii and topo iv with ic50 values in the range 9.4-25 mg/l. fungal diseases are a well-known plague for animal and plant worlds but a more hidden menace for human health. more than 90% of all reported fungal-related deaths results from species that belong to one of four genera: cryptococcus, candida, aspergillus and pneumocystis [88] [89] [90] . coumarin derivatives are endowed with antifungal activity, potentially useful in both pharma and agri-food sectors. in this liu and colleagues made a huge effort to synthesize and identify coumarin-pyrazole carboxamide derivatives as potential topoisomerase-ii inhibitors: 70 novel compounds were obtained and evaluated [87] . three of them (52-54, figure 21 ) were endowed with promising antimicrobial activities. in particular, compound 52 showed a considerable inhibitory activity compared with ciprofloxacin against escherichia coli and compound 53 exhibited excellent antibacterial activity against salmonella typhi. the selected compounds exhibited also potent inhibition against topo ii and topo iv with ic 50 values in the range 9.4-25 mg/l. liu and colleagues made a huge effort to synthesize and identify coumarin-pyrazole carboxamide derivatives as potential topoisomerase-ii inhibitors: 70 novel compounds were obtained and evaluated [87] . three of them (52-54, figure 21 ) were endowed with promising antimicrobial activities. in particular, compound 52 showed a considerable inhibitory activity compared with ciprofloxacin against escherichia coli and compound 53 exhibited excellent antibacterial activity against salmonella typhi. the selected compounds exhibited also potent inhibition against topo ii and topo iv with ic50 values in the range 9.4-25 mg/l. fungal diseases are a well-known plague for animal and plant worlds but a more hidden menace for human health. more than 90% of all reported fungal-related deaths results from species that belong to one of four genera: cryptococcus, candida, aspergillus and pneumocystis [88] [89] [90] . coumarin derivatives are endowed with antifungal activity, potentially useful in both pharma and agri-food sectors. in this fungal diseases are a well-known plague for animal and plant worlds but a more hidden menace for human health. more than 90% of all reported fungal-related deaths results from species that belong to one of four genera: cryptococcus, candida, aspergillus and pneumocystis [88] [89] [90] . coumarin derivatives are endowed with antifungal activity, potentially useful in both pharma and agri-food sectors. in this paragraph, we will focus on the recent progresses in the development of novel antifungal drugs for human use whereas agri-food applications can be found in section 3.2, food systems. diseases by candida species, a family of fungi that normally live on the host epithelial species but can initiate a fatal infection in particular cases like immunodeficiency, are the fourth most common cause of nosocomial blood-stream infections. despite several species of candida can cause disease, candida albicans prevails in term of incidence [88, 91, 92] . therefore, in 2016, shaik and colleagues designed a novel series of coumarin derivatives conjugated with 1,2,3-triazole moieties, on the basis of a previous work by shi and zhou and of the common use of azoles as antifungal drugs [93, 94] . the antifungal potency of the novel compounds was tested against candida albicans and other four fungal pathogens (i.e., fusarium oxysporum, aspergillus flavus, aspergillus niger and cryptococcus neoformans); mic values were evaluated and compared to mic of the reference compounds miconazole and fluconazole. compounds 55-57 and 59 ( figure 22 ) were found to be equipotent to miconazole against c. albicans and compound 58 was found to be two-fold more active compared with miconazole and equipotent to fluconazole. paragraph, we will focus on the recent progresses in the development of novel antifungal drugs for human use whereas agri-food applications can be found in paragraph 3.2, food systems. diseases by candida species, a family of fungi that normally live on the host epithelial species but can initiate a fatal infection in particular cases like immunodeficiency, are the fourth most common cause of nosocomial blood-stream infections. despite several species of candida can cause disease, candida albicans prevails in term of incidence [88, 91, 92] . therefore, in 2016, shaik and colleagues designed a novel series of coumarin derivatives conjugated with 1,2,3-triazole moieties, on the basis of a previous work by shi and zhou and of the common use of azoles as antifungal drugs [93, 94] . the antifungal potency of the novel compounds was tested against candida albicans and other four fungal pathogens (i.e., fusarium oxysporum, aspergillus flavus, aspergillus niger and cryptococcus neoformans); mic values were evaluated and compared to mic of the reference compounds miconazole and fluconazole. compounds 55-57 and 59 ( figure 22 ) were found to be equipotent to miconazole against c. albicans and compound 58 was found to be two-fold more active compared with miconazole and equipotent to fluconazole. furthermore, molecular docking studies showed that these compounds have a high affinity toward the active site of enzyme p450 cytochrome lanosterol 14α-demethylase and analysis of adme parameters confirmed their drug-like properties [94] . in 2017, fifteen novel coumarin derivatives were synthesized by tiwari et al., under solvent free conditions and exploiting the ionic liquid [et3nh][hso4] as a catalyst [95] . the compounds were tested both for their antifungal and antibacterial activities and, among the series, compounds 60 and 61 resulted to be the most potent as fungicides ( figure 23 ). the mic values observed for compound 60 and 61 were comparable to the standard drug miconazole. furthermore, molecular docking studies showed that these compounds have a high affinity toward the active site of enzyme p450 cytochrome lanosterol 14α-demethylase and analysis of adme parameters confirmed their drug-like properties [94] . in 2017, fifteen novel coumarin derivatives were synthesized by tiwari et al., under solvent free conditions and exploiting the ionic liquid [et 3 nh][hso 4 ] as a catalyst [95] . the compounds were tested both for their antifungal and antibacterial activities and, among the series, compounds 60 and 61 resulted to be the most potent as fungicides ( figure 23 ). the mic values observed for compound 60 and 61 were comparable to the standard drug miconazole. paragraph, we will focus on the recent progresses in the development of novel antifungal drugs for human use whereas agri-food applications can be found in paragraph 3.2, food systems. diseases by candida species, a family of fungi that normally live on the host epithelial species but can initiate a fatal infection in particular cases like immunodeficiency, are the fourth most common cause of nosocomial blood-stream infections. despite several species of candida can cause disease, candida albicans prevails in term of incidence [88, 91, 92] . therefore, in 2016, shaik and colleagues designed a novel series of coumarin derivatives conjugated with 1,2,3-triazole moieties, on the basis of a previous work by shi and zhou and of the common use of azoles as antifungal drugs [93, 94] . the antifungal potency of the novel compounds was tested against candida albicans and other four fungal pathogens (i.e., fusarium oxysporum, aspergillus flavus, aspergillus niger and cryptococcus neoformans); mic values were evaluated and compared to mic of the reference compounds miconazole and fluconazole. compounds 55-57 and 59 ( figure 22 ) were found to be equipotent to miconazole against c. albicans and compound 58 was found to be two-fold more active compared with miconazole and equipotent to fluconazole. furthermore, molecular docking studies showed that these compounds have a high affinity toward the active site of enzyme p450 cytochrome lanosterol 14α-demethylase and analysis of adme parameters confirmed their drug-like properties [94] . in 2017, fifteen novel coumarin derivatives were synthesized by tiwari et al., under solvent free conditions and exploiting the ionic liquid [et3nh][hso4] as a catalyst [95] . the compounds were tested both for their antifungal and antibacterial activities and, among the series, compounds 60 and 61 resulted to be the most potent as fungicides ( figure 23 ). the mic values observed for compound 60 and 61 were comparable to the standard drug miconazole. further studies demonstrated that compound 60 acts by inhibiting ergosterol biosynthesis in c. albicans. molecular docking studies revealed, as for previous study on compounds 55-59, a highly spontaneous binding ability of the tested compounds to the active site of lanosterol 14α-demethylase, which suggests that the tested compounds inhibit the synthesis of this enzyme. moreover, in silico admet properties were evaluated and demonstrated that all the compounds exhibited a good percent absorption (% abs) ranging from 84.9% to 100%. moreover, these compounds had proven to be safe after in vitro toxicity, in vivo acute oral toxicity and behavioral studies. coumarin-based antifungal azoles had been further investigated by elias and co-workers, who, in 2019, developed a series of 11 coumarins conjugated with 1,2,4-triazole and imidazole motifs [96] . the analysis of the biological effects of these novel chemical entities highlighted that imidazole-based derivatives ( figure 24 ) were more efficient against several candida strains compared to 1,2,4-triazole derivatives. moreover, the mode of action of the two classes of compounds were different. whereas the antifungal activity of the triazole-based azoles was dependent on expression of cyp51, the target of the azole antifungals, imidazole-based compounds displayed antifungal activity against a mutant lacking cyp51, indicating that imidazole-based azole antifungals have additional modes of action. this peculiarity could be favorable in the struggle against drugs-resistant fungal strains. further studies demonstrated that compound 60 acts by inhibiting ergosterol biosynthesis in c. albicans. molecular docking studies revealed, as for previous study on compounds 55-59, a highly spontaneous binding ability of the tested compounds to the active site of lanosterol 14α-demethylase, which suggests that the tested compounds inhibit the synthesis of this enzyme. moreover, in silico admet properties were evaluated and demonstrated that all the compounds exhibited a good percent absorption (% abs) ranging from 84.9% to 100%. moreover, these compounds had proven to be safe after in vitro toxicity, in vivo acute oral toxicity and behavioral studies. coumarin-based antifungal azoles had been further investigated by elias and co-workers, who, in 2019, developed a series of 11 coumarins conjugated with 1,2,4-triazole and imidazole motifs [96] . the analysis of the biological effects of these novel chemical entities highlighted that imidazole-based derivatives ( figure 24 ) were more efficient against several candida strains compared to 1,2,4-triazole derivatives. moreover, the mode of action of the two classes of compounds were different. whereas the antifungal activity of the triazole-based azoles was dependent on expression of cyp51, the target of the azole antifungals, imidazole-based compounds displayed antifungal activity against a mutant lacking cyp51, indicating that imidazole-based azole antifungals have additional modes of action. this peculiarity could be favorable in the struggle against drugs-resistant fungal strains. furthermore, it should not be forgotten the contribution of natural coumarins to the fight against candida infections. ferulago trachycarpa boiss. is one of the species of ferulago w. koch., common in anatolian region, exploited in traditional medicine but also in culinary field. previous studies showed that coumarins are the main compounds found in ferulago but only in 2018, dikpinar and colleagues conducted the first antimicrobial activity guided isolation of the molecular constituent of this particular species of ferulago [97] . four coumarin compounds ( figure 25 ) were isolated and purified and then three of them were tested against bacterial and fungal strains; in particular, marmesin senesioate, suberosin and crenulatin showed antifungal activity with 625 mg/l mic against candida albicans. the antifungal potency of coumarin itself against candida albicans had been previously evaluated, in particular its antibiofilm activity [98, 99] . recently, xu and co-workers focused their attention on the possible way to prevent the adhesion and formation of biofilm by candida albicans on implanted medical devices. the research group observed that coumarin not only suppresses biofilm formation but also affects the structure of the mature biofilm; moreover the adhesion, the cell surface hydrophobicity (csh) and the filamentous growth of c. albicans significantly decreased after coumarin treatment, indicating that coumarin inhibits biofilm formation by suppressing adhesion [100] . further studies demonstrated that compound 60 acts by inhibiting ergosterol biosynthesis in c. albicans. molecular docking studies revealed, as for previous study on compounds 55-59, a highly spontaneous binding ability of the tested compounds to the active site of lanosterol 14α-demethylase, which suggests that the tested compounds inhibit the synthesis of this enzyme. moreover, in silico admet properties were evaluated and demonstrated that all the compounds exhibited a good percent absorption (% abs) ranging from 84.9% to 100%. moreover, these compounds had proven to be safe after in vitro toxicity, in vivo acute oral toxicity and behavioral studies. coumarin-based antifungal azoles had been further investigated by elias and co-workers, who, in 2019, developed a series of 11 coumarins conjugated with 1,2,4-triazole and imidazole motifs [96] . the analysis of the biological effects of these novel chemical entities highlighted that imidazole-based derivatives ( figure 24 ) were more efficient against several candida strains compared to 1,2,4-triazole derivatives. moreover, the mode of action of the two classes of compounds were different. whereas the antifungal activity of the triazole-based azoles was dependent on expression of cyp51, the target of the azole antifungals, imidazole-based compounds displayed antifungal activity against a mutant lacking cyp51, indicating that imidazole-based azole antifungals have additional modes of action. this peculiarity could be favorable in the struggle against drugs-resistant fungal strains. furthermore, it should not be forgotten the contribution of natural coumarins to the fight against candida infections. ferulago trachycarpa boiss. is one of the species of ferulago w. koch., common in anatolian region, exploited in traditional medicine but also in culinary field. previous studies showed that coumarins are the main compounds found in ferulago but only in 2018, dikpinar and colleagues conducted the first antimicrobial activity guided isolation of the molecular constituent of this particular species of ferulago [97] . four coumarin compounds ( figure 25 ) were isolated and purified and then three of them were tested against bacterial and fungal strains; in particular, marmesin senesioate, suberosin and crenulatin showed antifungal activity with 625 mg/l mic against candida albicans. the antifungal potency of coumarin itself against candida albicans had been previously evaluated, in particular its antibiofilm activity [98, 99] . recently, xu and co-workers focused their attention on the possible way to prevent the adhesion and formation of biofilm by candida albicans on implanted medical devices. the research group observed that coumarin not only suppresses biofilm formation but also affects the structure of the mature biofilm; moreover the adhesion, the cell surface hydrophobicity (csh) and the filamentous growth of c. albicans significantly decreased after coumarin treatment, indicating that coumarin inhibits biofilm formation by suppressing adhesion [100] . the antifungal potency of coumarin itself against candida albicans had been previously evaluated, in particular its antibiofilm activity [98, 99] . recently, xu and co-workers focused their attention on the possible way to prevent the adhesion and formation of biofilm by candida albicans on implanted medical devices. the research group observed that coumarin not only suppresses biofilm formation but also affects the structure of the mature biofilm; moreover the adhesion, the cell surface hydrophobicity (csh) and the filamentous growth of c. albicans significantly decreased after coumarin treatment, indicating that coumarin inhibits biofilm formation by suppressing adhesion [100] . the antifungal potency of coumarin-based triazoles against other fungal strains in addition to candida species had been evaluated by dharavath and colleagues in 2020. coumarin-based 1,4-disubstituted 1,2,3-triazole derivatives were synthesized using a highly efficient, eco-friendly protocol via a copper(i)-catalyzed click reaction between various substituted arylazides and terminal alkynes. the in vitro antifungal activity was tested against aspergillus niger, aspergillus flavus and fusarium oxysporum by using the disc diffusion method and the results were compared with those of clotrimazole, the reference drug. compounds 63-68 ( figure 26 ), characterized by the presence of a para-substituted phenyl moiety, directly linked to the triazole ring, showed comparable activity in respect to the reference compound clotrimazol [101, 102] . the antifungal potency of coumarin-based triazoles against other fungal strains in addition to candida species had been evaluated by dharavath and colleagues in 2020. coumarin-based 1,4disubstituted 1,2,3-triazole derivatives were synthesized using a highly efficient, eco-friendly protocol via a copper(i)-catalyzed click reaction between various substituted arylazides and terminal alkynes. the in vitro antifungal activity was tested against aspergillus niger, aspergillus flavus and fusarium oxysporum by using the disc diffusion method and the results were compared with those of clotrimazole, the reference drug. compounds 63-68 ( figure 26 ), characterized by the presence of a para-substituted phenyl moiety, directly linked to the triazole ring, showed comparable activity in respect to the reference compound clotrimazol [101, 102] . 2020 has been a crucial year for the timeless war between human and viruses: world health organization (who) declared the outbreak of sars-covid-19 a public health emergency of international concern on 30 january 2020 and on 11 march who characterized covid-19 as a pandemic [103] . whole developed countries have been quarantined and generations that never faced medical crisis are now struggling with the consequences of the viral diffusion. human history is afflicted by the cyclic succession of pandemic events and the research of new antivirals is still ongoing, due to the ability of viruses to mutate and the continuous appearance of new viruses on the medical scenario [104] . the natural world is a priceless source of valuable medical compounds and also in the fight against viral diseases there are several natural molecules which exhibit antiviral activity [105] [106] [107] [108] [109] . coumarins, likewise other polyphenolic compounds, exert a remarkable antiviral activity [110, 111] . the antiviral activity of coumarins explicates through different mechanisms which affect the life cycle of viruses and their biological activities could be changed depending upon the combination of various substituents and conjugates [104, 112] . coumarins appears to be active against several viruses, like hiv, influenza, hepatitis, dengue and chikunguya [104] . liu and co-workers after a phytochemical study on the stem of clausena lenis isolated three new and nine known prenylated coumarins [113] . all the prenylated coumarins were evaluated both for their anti-inflammatory and anti-hiv reverse transcriptase (rt) activities. in this last case, the inhibition assay for the cytopathic activities of hiv-1 (ec50) as well as cytotoxic activity assay against c8166 cell line (cc50) according to mtt methods were applied [114, 115] . the three new isolated compounds (69-71, figure 27 ) showed the best inhibitory activity with an ec50 of 0.29, 0.68 and 0.17 μm, respectively. furthermore, no cytotoxicity was observed against c8166 cell line (cc50 > 200.00 μm). 2.6. antiviral activity 2020 has been a crucial year for the timeless war between human and viruses: world health organization (who) declared the outbreak of sars-covid-19 a public health emergency of international concern on 30 january 2020 and on 11 march who characterized covid-19 as a pandemic [103] . whole developed countries have been quarantined and generations that never faced medical crisis are now struggling with the consequences of the viral diffusion. human history is afflicted by the cyclic succession of pandemic events and the research of new antivirals is still ongoing, due to the ability of viruses to mutate and the continuous appearance of new viruses on the medical scenario [104] . the natural world is a priceless source of valuable medical compounds and also in the fight against viral diseases there are several natural molecules which exhibit antiviral activity [105] [106] [107] [108] [109] . coumarins, likewise other polyphenolic compounds, exert a remarkable antiviral activity [110, 111] . the antiviral activity of coumarins explicates through different mechanisms which affect the life cycle of viruses and their biological activities could be changed depending upon the combination of various substituents and conjugates [104, 112] . coumarins appears to be active against several viruses, like hiv, influenza, hepatitis, dengue and chikunguya [104] . liu and co-workers after a phytochemical study on the stem of clausena lenis isolated three new and nine known prenylated coumarins [113] . all the prenylated coumarins were evaluated both for their anti-inflammatory and anti-hiv reverse transcriptase (rt) activities. in this last case, the inhibition assay for the cytopathic activities of hiv-1 (ec 50 ) as well as cytotoxic activity assay against c8166 cell line (cc 50 ) according to mtt methods were applied [114, 115] . the three new isolated compounds (69-71, figure 27 ) showed the best inhibitory activity with an ec 50 prenylated coumarins came to the fore in 2019 thanks to a work by liu et al., who revealed for the first time the presence of these type of derivatives in the fruits of manilkara zapota, an ever-green tropical tree. also in this case, three new derivatives were identified (72-74, figure 28 ) together with seven known compounds and the team evaluated their anti-inflammatory and anti-hiv activities, exploiting the methods described above [116] . prenylated coumarins came to the fore in 2019 thanks to a work by liu et al., who revealed for the first time the presence of these type of derivatives in the fruits of manilkara zapota, an ever-green tropical tree. also in this case, three new derivatives were identified (72-74, figure 28 ) together with seven known compounds and the team evaluated their anti-inflammatory and anti-hiv activities, exploiting the methods described above [116] . prenylated coumarins came to the fore in 2019 thanks to a work by liu et al., who revealed for the first time the presence of these type of derivatives in the fruits of manilkara zapota, an ever-green tropical tree. also in this case, three new derivatives were identified (72-74, figure 28 ) together with seven known compounds and the team evaluated their anti-inflammatory and anti-hiv activities, exploiting the methods described above [116] . the new prenylated coumarins showed the highest anti-hiv rt effect among the prenylated coumarins derived from the fruit of manilkara zapota; in particular, compound 72 displayed the most powerful effect with an ec50 value of 0.12 μm. comparative studies between compounds 72-74 and the other coumarin derivatives isolated from the fruits highlighted the importance of the isopentenyl group as a substituent at c6 and 2-methylbut-3-en-2-yl group as a substituent at c3 for the anti-hiv rt effect. in the same year, jesumoroti and co-workers approached the problem from another point of view, both regarding the target and the origin of the coumarin derivatives [117] . first of all, a different viral target was chosen, hiv-1 in, which is essential for a stable infection. moreover, there are no homologous enzymes in the host cell [118] . secondarily, the coumarin derivatives were obtained decorating the coumarin nucleus with an hydrazide group, in order to achieve a synergistic effect against-hiv-1-in and reducing the toxicity correlated to the salicylhydrazide, which was however essential for the activity [119] [120] [121] . the synthesized compounds were evaluated for their in vitro hiv-1 in inhibitory activity using chicoric acid (ca) as a reference compound, according to the method described by mccoll et al. [122] . compounds 75-78 ( figure 29 ) appeared to be the most active among the whole series showing an inhibition of extracellular in (evaluated in vitro according to the method described by mccoll et al. [122] ) in a range from 95% to 86 % and ic50 between 13 nm (compound 76) and 31 nm (ca ic50=10 nm). furthermore, the cytotoxicity of all the obtained compounds was tested and derivatives 75-78 showed low or negligible cytotoxicity. the new prenylated coumarins showed the highest anti-hiv rt effect among the prenylated coumarins derived from the fruit of manilkara zapota; in particular, compound 72 displayed the most powerful effect with an ec 50 value of 0.12 µm. comparative studies between compounds 72-74 and the other coumarin derivatives isolated from the fruits highlighted the importance of the isopentenyl group as a substituent at c6 and 2-methylbut-3-en-2-yl group as a substituent at c3 for the anti-hiv rt effect. in the same year, jesumoroti and co-workers approached the problem from another point of view, both regarding the target and the origin of the coumarin derivatives [117] . first of all, a different viral target was chosen, hiv-1 in, which is essential for a stable infection. moreover, there are no homologous enzymes in the host cell [118] . secondarily, the coumarin derivatives were obtained decorating the coumarin nucleus with an hydrazide group, in order to achieve a synergistic effect against-hiv-1-in and reducing the toxicity correlated to the salicylhydrazide, which was however essential for the activity [119] [120] [121] . the synthesized compounds were evaluated for their in vitro hiv-1 in inhibitory activity using chicoric acid (ca) as a reference compound, according to the method described by mccoll et al. [122] . compounds 75-78 ( figure 29 ) appeared to be the most active among the whole series showing an inhibition of extracellular in (evaluated in vitro according to the method described by mccoll et al. [122] ) in a range from 95% to 86 % and ic 50 between 13 nm (compound 76) and 31 nm (ca ic 50 = 10 nm). furthermore, the cytotoxicity of all the obtained compounds was tested and derivatives 75-78 showed low or negligible cytotoxicity. prenylated coumarins came to the fore in 2019 thanks to a work by liu et al., who revealed for the first time the presence of these type of derivatives in the fruits of manilkara zapota, an ever-green tropical tree. also in this case, three new derivatives were identified (72-74, figure 28 ) together with seven known compounds and the team evaluated their anti-inflammatory and anti-hiv activities, exploiting the methods described above [116] . the new prenylated coumarins showed the highest anti-hiv rt effect among the prenylated coumarins derived from the fruit of manilkara zapota; in particular, compound 72 displayed the most powerful effect with an ec50 value of 0.12 μm. comparative studies between compounds 72-74 and the other coumarin derivatives isolated from the fruits highlighted the importance of the isopentenyl group as a substituent at c6 and 2-methylbut-3-en-2-yl group as a substituent at c3 for the anti-hiv rt effect. in the same year, jesumoroti and co-workers approached the problem from another point of view, both regarding the target and the origin of the coumarin derivatives [117] . first of all, a different viral target was chosen, hiv-1 in, which is essential for a stable infection. moreover, there are no homologous enzymes in the host cell [118] . secondarily, the coumarin derivatives were obtained decorating the coumarin nucleus with an hydrazide group, in order to achieve a synergistic effect against-hiv-1-in and reducing the toxicity correlated to the salicylhydrazide, which was however essential for the activity [119] [120] [121] . the synthesized compounds were evaluated for their in vitro hiv-1 in inhibitory activity using chicoric acid (ca) as a reference compound, according to the method described by mccoll et al. [122] . compounds 75-78 ( figure 29 ) appeared to be the most active among the whole series showing an inhibition of extracellular in (evaluated in vitro according to the method described by mccoll et al. [122] ) in a range from 95% to 86 % and ic50 between 13 nm (compound 76) and 31 nm (ca ic50=10 nm). furthermore, the cytotoxicity of all the obtained compounds was tested and derivatives 75-78 showed low or negligible cytotoxicity. seasonal influenza claims around 0.29-0.65 million victims annually, even if several drugs are commercially available [123] . the flu is a contagious respiratory disease caused by influenza viruses, manifesting major epidemics with no predictable periodicity or pattern and all different from one to another [124] . for these reasons, a constant effort in the development of new drugs for the treatment of this disease is of greatest importance. in 2019 osman and co-workers combined two bioactive moieties into a single molecule in order to obtain new bioactive compounds with antibacterial and antiviral effects [125] . in particular, based on a previous study of the same research team, in which coumarin scaffolds and thiazole moiety were combined leading to compounds endowed with both antibacterial and antiviral activities, the potentiality of this combination was further explored [126] , [127] . four new molecules of the series showed a remarkable antiviral activity against h1n1 virus. compounds 79-82 ( figure 30 ) seemed to be promising agents, having ic 50 values of 4.84, 19.72, 6.12 and 9.13 µg/ml, respectively, against the h1n1 virus. commercially available [123] . the flu is a contagious respiratory disease caused by influenza viruses, manifesting major epidemics with no predictable periodicity or pattern and all different from one to another [124] . for these reasons, a constant effort in the development of new drugs for the treatment of this disease is of greatest importance. in 2019 osman and co-workers combined two bioactive moieties into a single molecule in order to obtain new bioactive compounds with antibacterial and antiviral effects [125] . in particular, based on a previous study of the same research team, in which coumarin scaffolds and thiazole moiety were combined leading to compounds endowed with both antibacterial and antiviral activities, the potentiality of this combination was further explored [126] , [127] . four new molecules of the series showed a remarkable antiviral activity against h1n1 virus. compounds 79-82 ( figure 30 ) seemed to be promising agents, having ic50 values of 4.84, 19.72, 6.12 and 9.13 μg/ml, respectively, against the h1n1 virus. bizzarri et al. exploited the regioselective oxidation of coumarin derivatives with 2iodoxybenzoic acid (ibx) in order to obtain catechol and pyrogallol moieties [129] . after cytotoxicity studies that confirmed the safety of the series of derivatives, the antiviral activity against influenza viruses a/pr8/h1n1 was evaluated and compounds 84-89 ( figure 32 ) resulted able to inhibit the viral replication. interestingly, pyrogallol derivatives 88 (ic50 = 69.9 μg/ml) and 89 (ic50 = 47.9 μg/ml) turned out to be more active than catechol derivatives 84 (ic50 = 106.5 μg/ml) and 85 (ic50 = 91.5 μg/ml). moreover, pyrogallol and catechol derivatives were more active than the monohydroxycoumarins 6-hydroxycoumarin and 7-hydroxycoumarin (ic50 110 μg/ml for both the parent compounds). commercially available [123] . the flu is a contagious respiratory disease caused by influenza viruses, manifesting major epidemics with no predictable periodicity or pattern and all different from one to another [124] . for these reasons, a constant effort in the development of new drugs for the treatment of this disease is of greatest importance. in 2019 osman and co-workers combined two bioactive moieties into a single molecule in order to obtain new bioactive compounds with antibacterial and antiviral effects [125] . in particular, based on a previous study of the same research team, in which coumarin scaffolds and thiazole moiety were combined leading to compounds endowed with both antibacterial and antiviral activities, the potentiality of this combination was further explored [126] , [127] . four new molecules of the series showed a remarkable antiviral activity against h1n1 virus. compounds 79-82 ( figure 30 ) seemed to be promising agents, having ic50 values of 4.84, 19.72, 6.12 and 9.13 μg/ml, respectively, against the h1n1 virus. bizzarri et al. exploited the regioselective oxidation of coumarin derivatives with 2iodoxybenzoic acid (ibx) in order to obtain catechol and pyrogallol moieties [129] . after cytotoxicity studies that confirmed the safety of the series of derivatives, the antiviral activity against influenza viruses a/pr8/h1n1 was evaluated and compounds 84-89 ( figure 32 ) resulted able to inhibit the viral replication. interestingly, pyrogallol derivatives 88 (ic50 = 69.9 μg/ml) and 89 (ic50 = 47.9 μg/ml) turned out to be more active than catechol derivatives 84 (ic50 = 106.5 μg/ml) and 85 (ic50 = 91.5 μg/ml). moreover, pyrogallol and catechol derivatives were more active than the monohydroxycoumarins 6-hydroxycoumarin and 7-hydroxycoumarin (ic50 110 μg/ml for both the parent compounds). bizzarri et al. exploited the regioselective oxidation of coumarin derivatives with 2-iodoxybenzoic acid (ibx) in order to obtain catechol and pyrogallol moieties [129] . after cytotoxicity studies that confirmed the safety of the series of derivatives, the antiviral activity against influenza viruses a/pr8/h1n1 was evaluated and compounds 84-89 ( figure 32 ) resulted able to inhibit the viral replication. interestingly, pyrogallol derivatives 88 (ic 50 = 69.9 µg/ml) and 89 (ic 50 = 47.9 µg/ml) turned out to be more active than catechol derivatives 84 (ic 50 = 106.5 µg/ml) and 85 (ic 50 = 91.5 µg/ml). moreover, pyrogallol and catechol derivatives were more active than the monohydroxycoumarins 6-hydroxycoumarin and 7-hydroxycoumarin (ic 50 110 µg/ml for both the parent compounds). one of the possible advantages of oxidized coumarins could be their mode of action against viruses. indeed, due to their antioxidant activity, coumarins derivatives could affect intracellular redox-sensitive pathways useful for viral replication, independently from the variability of the strains [129] . as already mentioned, coumarins have been studied also for their potential application as antihepatitis agents. tsay and co-workers studied the activity against hepatitis c virus (hcv) of some unnatural imidazole-coumarin conjugates [130] . above all, three compounds (90-92, figure 33 ) showed a noteworthy antiviral activity against hcv with ec50 values ranging from 5.1 to 8.4 μm and selective indices (si = cc50/ec50, which is a measure for the therapeutic window of the compound in an assay system) higher than 20. huang and co-workers focused their attention on the investigation of the potentiality expressed by esculetin (or 6,7-dihydroxycoumarin) against hepatitis b virus (hbv) [131] . the results suggested that esculetin efficiently inhibits hbv replication both in vitro and in vivo, which provides an opportunity for further development of the compound as antiviral agent. inflammation is a general physiological response that aims, firstly, to the circumscription of the harmful factors, which could be both endogenous (e.g., cancer, ischemia, autoimmune diseases) and exogenous (e.g., viral or bacterial infections, trauma), secondarily, to the removal of the causes of the damage and finally to the reparation of the tissues and restoration of the functions. nevertheless, the inflammation and the consequent restorative process could become harmful for the organism itself when there is a persistent stimulation and the phases of inflammation and reconstruction are contemporary activated, causing tissue injuries and fibrosis [132] . during an inflammatory process, many inflammatory effectors and mediators are produced and involved, often with a common effect on vascular system and on the recruitment of leukocytes [133] . frequently, inflammatory mediators are the target of anti-inflammatory drugs [134] [135] [136] [137] . one of the possible advantages of oxidized coumarins could be their mode of action against viruses. indeed, due to their antioxidant activity, coumarins derivatives could affect intracellular redox-sensitive pathways useful for viral replication, independently from the variability of the strains [129] . as already mentioned, coumarins have been studied also for their potential application as anti-hepatitis agents. tsay and co-workers studied the activity against hepatitis c virus (hcv) of some unnatural imidazole-coumarin conjugates [130] . above all, three compounds (90-92, figure 33 ) showed a noteworthy antiviral activity against hcv with ec 50 values ranging from 5.1 to 8.4 µm and selective indices (si = cc 50 /ec 50 , which is a measure for the therapeutic window of the compound in an assay system) higher than 20. one of the possible advantages of oxidized coumarins could be their mode of action against viruses. indeed, due to their antioxidant activity, coumarins derivatives could affect intracellular redox-sensitive pathways useful for viral replication, independently from the variability of the strains [129] . as already mentioned, coumarins have been studied also for their potential application as antihepatitis agents. tsay and co-workers studied the activity against hepatitis c virus (hcv) of some unnatural imidazole-coumarin conjugates [130] . above all, three compounds (90-92, figure 33 ) showed a noteworthy antiviral activity against hcv with ec50 values ranging from 5.1 to 8.4 μm and selective indices (si = cc50/ec50, which is a measure for the therapeutic window of the compound in an assay system) higher than 20. huang and co-workers focused their attention on the investigation of the potentiality expressed by esculetin (or 6,7-dihydroxycoumarin) against hepatitis b virus (hbv) [131] . the results suggested that esculetin efficiently inhibits hbv replication both in vitro and in vivo, which provides an opportunity for further development of the compound as antiviral agent. inflammation is a general physiological response that aims, firstly, to the circumscription of the harmful factors, which could be both endogenous (e.g., cancer, ischemia, autoimmune diseases) and exogenous (e.g., viral or bacterial infections, trauma), secondarily, to the removal of the causes of the damage and finally to the reparation of the tissues and restoration of the functions. nevertheless, the inflammation and the consequent restorative process could become harmful for the organism itself when there is a persistent stimulation and the phases of inflammation and reconstruction are contemporary activated, causing tissue injuries and fibrosis [132] . during an inflammatory process, many inflammatory effectors and mediators are produced and involved, often with a common effect on vascular system and on the recruitment of leukocytes [133] . frequently, inflammatory mediators are the target of anti-inflammatory drugs [134] [135] [136] [137] . huang and co-workers focused their attention on the investigation of the potentiality expressed by esculetin (or 6,7-dihydroxycoumarin) against hepatitis b virus (hbv) [131] . the results suggested that esculetin efficiently inhibits hbv replication both in vitro and in vivo, which provides an opportunity for further development of the compound as antiviral agent. inflammation is a general physiological response that aims, firstly, to the circumscription of the harmful factors, which could be both endogenous (e.g., cancer, ischemia, autoimmune diseases) and exogenous (e.g., viral or bacterial infections, trauma), secondarily, to the removal of the causes of the damage and finally to the reparation of the tissues and restoration of the functions. nevertheless, the inflammation and the consequent restorative process could become harmful for the organism itself when there is a persistent stimulation and the phases of inflammation and reconstruction are contemporary activated, causing tissue injuries and fibrosis [132] . during an inflammatory process, many inflammatory effectors and mediators are produced and involved, often with a common effect on vascular system and on the recruitment of leukocytes [133] . frequently, inflammatory mediators are the target of anti-inflammatory drugs [134] [135] [136] [137] . among the numerous biological activities shown by coumarin derivatives, the anti-inflammatory effect could not certainly miss. dawood et al., in 2015 , developed a new series of coumarin derivatives hybridizing two pharmacophoric moieties-the coumarin scaffold itself and thiazoline or thiazolidinone groups, both showing cyclooxygenase 2 (cox-2) inhibitor effect [127] , [138] [139] [140] [141] [142] [143] . the compounds were evaluated in vivo for their systemic effect, in vitro for their ability to inhibit human cox-1 and cox-2 and also to evaluate the ulcerogenic potential compared to the reference drug, indomethacin, always following standard methods reported in literature. most of the new compounds showed significant in vivo anti-inflammatory activity with a superior gastro-intestinal safety profiles (0-7% ulceration) as compared to indomethacin. the ic 50 among the numerous biological activities shown by coumarin derivatives, the antiinflammatory effect could not certainly miss. dawood et al., in 2015 , developed a new series of coumarin derivatives hybridizing two pharmacophoric moieties-the coumarin scaffold itself and thiazoline or thiazolidinone groups, both showing cyclooxygenase 2 (cox-2) inhibitor effect [127] , [138] [139] [140] [141] [142] [143] . the compounds were evaluated in vivo for their systemic effect, in vitro for their ability to inhibit human cox-1 and cox-2 and also to evaluate the ulcerogenic potential compared to the reference drug, indomethacin, always following standard methods reported in literature. most of the new compounds showed significant in vivo anti-inflammatory activity with a superior gastrointestinal safety profiles (0-7% ulceration) as compared to indomethacin. the ic50 values of all the bioactive compounds ranged between 0.31 and 0.78 mm, showing an in vitro high affinity and selectivity toward the cox-2 isoenzyme, compared to the reference celecoxib. ethyl thiosemicarbazone 93, thiazoline derivatives 94-98 and the thiazolidinone compounds 99-100 exhibited the highest in vivo and in vitro anti-inflammatory activities with good cox-2 selectivity ( figure 34 ) [143] . nevertheless, cyclooxygenases are not the only enzymes involved in the inflammatory process. actually, 5-lipoxygenase (5-lox) catalyzes two different steps in the arachidonic acid metabolism that brings to the production of leukotriene a4, which is successively metabolized into leukotriene b4 [144] . molecular inhibitors of leukotrienes competitively bind the active site of 5-lox and are divided in three category: redox-active compounds (i.e., coumarins), iron-ligand inhibitors with weak redoxactive properties and non-redox-type inhibitors [145] . in 2016, srivastava and colleagues evaluated the anti-inflammatory and analgesic effect of a series of synthesized 7-substituted coumarins and, consequently, the most active compounds were assessed in vitro for 5-lox inhibition [146] . compounds 101 and 102 ( figure 35 ) resulted the most promising derivatives, also in the ulcerogenic risk evaluation when compared to acetylsalicylic acid. in vitro kinetic study of compound 102 (ic50 = 2.09 nm) showed mixed or non-competitive type of inhibition of 5-lox. the presence of a substituent on c6 position of benzothiazole ring was found very important for increasing the activity. nevertheless, cyclooxygenases are not the only enzymes involved in the inflammatory process. actually, 5-lipoxygenase (5-lox) catalyzes two different steps in the arachidonic acid metabolism that brings to the production of leukotriene a 4 , which is successively metabolized into leukotriene b 4 [144] . molecular inhibitors of leukotrienes competitively bind the active site of 5-lox and are divided in three category: redox-active compounds (i.e., coumarins), iron-ligand inhibitors with weak redox-active properties and non-redox-type inhibitors [145] . in 2016, srivastava and colleagues evaluated the anti-inflammatory and analgesic effect of a series of synthesized 7-substituted coumarins and, consequently, the most active compounds were assessed in vitro for 5-lox inhibition [146] . compounds 101 and 102 ( figure 35 ) resulted the most promising derivatives, also in the ulcerogenic risk evaluation when compared to acetylsalicylic acid. in vitro kinetic study of compound 102 (ic 50 = 2.09 nm) showed mixed or non-competitive type of inhibition of 5-lox. the presence of a substituent on c6 position of benzothiazole ring was found very important for increasing the activity. in 2018, liu et al. identified ten new coumarin derivatives (3 monomers and 7 dimers) isolated in a phytochemical study on murraya exotica, a dwarf tree that is native of the tropical and subtropical areas of asia and traditionally used in the treatment of inflammatory-related diseases [147] . previous studies had shown that the main active components in m. exotica were coumarins; also bis-coumarins were isolated from the plant. a further investigation on the 95% aqueous etoh extract of the roots allowed the obtainment of new coumarins, together with other bioactive molecules. the compounds were tested for their inhibitory effect on no production and compound 103 ( figure 36 ) stood out among the coumarins in the inhibition against lps-induced no production in bv-2 microglial cells with ic50 value of 8.6 μm. the nuclear factor-kb (nf-kb) family of transcription factors is composed by a set of related, evolutionarily conserved dna-binding proteins. in response to multiple stimuli such as inflammatory cytokines, bacterial lipopolysaccharide (lps), viral infection or stress, a series of cascade reactions bring towards the entry on nf-kb into the nucleus and to the activation of several genes participating in immune and inflammatory responses, cell adhesion, growth control and regulation of apoptosis [148, 149] . in 2019, fan and co-workers evaluated in vivo and in vitro the antiinflammatory activity of osthole ( figure 37 ) [150] , a natural prenylated coumarin from cnidium monnieri (l.) cuss. but also found in other medicinal plants, which has already shown different biological and pharmacological properties such as neuroprotective, osteogenic, immunomodulatory, anticancer, hepatoprotective, cardiovascular protective and antimicrobial activities [13] . the team established that osthole inhibited the production of no, pge2, tnf-a and il-6 in lps-induced macrophages and suppressed the activity of inos and cox-2, probably by blocking the activation of nf-kb and p38/mapk signaling pathways. in 2018, liu et al. identified ten new coumarin derivatives (3 monomers and 7 dimers) isolated in a phytochemical study on murraya exotica, a dwarf tree that is native of the tropical and subtropical areas of asia and traditionally used in the treatment of inflammatory-related diseases [147] . previous studies had shown that the main active components in m. exotica were coumarins; also bis-coumarins were isolated from the plant. a further investigation on the 95% aqueous etoh extract of the roots allowed the obtainment of new coumarins, together with other bioactive molecules. the compounds were tested for their inhibitory effect on no production and compound 103 ( figure 36 ) stood out among the coumarins in the inhibition against lps-induced no production in bv-2 microglial cells with ic 50 value of 8.6 µm. in 2018, liu et al. identified ten new coumarin derivatives (3 monomers and 7 dimers) isolated in a phytochemical study on murraya exotica, a dwarf tree that is native of the tropical and subtropical areas of asia and traditionally used in the treatment of inflammatory-related diseases [147] . previous studies had shown that the main active components in m. exotica were coumarins; also bis-coumarins were isolated from the plant. a further investigation on the 95% aqueous etoh extract of the roots allowed the obtainment of new coumarins, together with other bioactive molecules. the compounds were tested for their inhibitory effect on no production and compound 103 ( figure 36 ) stood out among the coumarins in the inhibition against lps-induced no production in bv-2 microglial cells with ic50 value of 8.6 μm. the nuclear factor-kb (nf-kb) family of transcription factors is composed by a set of related, evolutionarily conserved dna-binding proteins. in response to multiple stimuli such as inflammatory cytokines, bacterial lipopolysaccharide (lps), viral infection or stress, a series of cascade reactions bring towards the entry on nf-kb into the nucleus and to the activation of several genes participating in immune and inflammatory responses, cell adhesion, growth control and regulation of apoptosis [148, 149] . in 2019, fan and co-workers evaluated in vivo and in vitro the antiinflammatory activity of osthole ( figure 37 ) [150] , a natural prenylated coumarin from cnidium monnieri (l.) cuss. but also found in other medicinal plants, which has already shown different biological and pharmacological properties such as neuroprotective, osteogenic, immunomodulatory, anticancer, hepatoprotective, cardiovascular protective and antimicrobial activities [13] . the team established that osthole inhibited the production of no, pge2, tnf-a and il-6 in lps-induced macrophages and suppressed the activity of inos and cox-2, probably by blocking the activation of nf-kb and p38/mapk signaling pathways. the nuclear factor-kb (nf-kb) family of transcription factors is composed by a set of related, evolutionarily conserved dna-binding proteins. in response to multiple stimuli such as inflammatory cytokines, bacterial lipopolysaccharide (lps), viral infection or stress, a series of cascade reactions bring towards the entry on nf-kb into the nucleus and to the activation of several genes participating in immune and inflammatory responses, cell adhesion, growth control and regulation of apoptosis [148, 149] . in 2019, fan and co-workers evaluated in vivo and in vitro the anti-inflammatory activity of osthole ( figure 37 ) [150] , a natural prenylated coumarin from cnidium monnieri (l.) cuss. but also found in other medicinal plants, which has already shown different biological and pharmacological properties such as neuroprotective, osteogenic, immunomodulatory, anticancer, hepatoprotective, cardiovascular protective and antimicrobial activities [13] . the team established that osthole inhibited the production of no, pge2, tnf-a and il-6 in lps-induced macrophages and suppressed the activity of inos and cox-2, probably by blocking the activation of nf-kb and p38/mapk signaling pathways. in the same year, mu and colleagues synthesized a series of eleven 7-substituted coumarins and evaluated their anti-inflammatory activities and their ability to exploit the nf-kb pathway [151] . all the screened compounds showed a relevant anti-inflammatory activity. in the series, compound 104 ( figure 38 ) was identified as a hit and further studies of molecular docking were conducted confirming that 104 could bind to the active site (nls polypeptide) of nf-κb p65. its binding affinity was further confirmed by surface plasmon resonance (spr) analysis. moreover, western blot assay showed that 104 remarkably blocked the nf-κb signaling pathway in vitro. alzheimer's disease (ad) is the most common form of dementia (60-70% of cases of dementia are cause by ad) and consists in a neurodegenerative disorder characterized by a slow, progressive and irreversible loss of cognitive function and memory [152] [153] [154] [155] [156] [157] . the current therapeutic approach, mainly based on the use of acetylcholinesterase (ache) inhibitors (rivastigmine, donepezil, galantamine) or n-methy-d-aspartic acid (nmda) receptor inhibitors (memantine), is merely symptomatic and does not counteract degeneration progression. new innovative approaches, such as multi-targeted strategies, are urgently required in order to cure cognition and motor dysfunctions, neurodegeneration and depression. among their biological activities, coumarins showed the ability to inhibit some biological targets involved in ad. with this in mind, some recent works aimed at investigating coumarins potential in the treatment of ad are discussed below. in 2019, karakaya and collaborators investigated the antioxidant and ache/buche inhibitory activity of aerial parts, fruits, flowers and root extracts from ferulago cassia boiss [158] . root's and fruit's dichloromethane extracts showed the highest antioxidant potential in tba assay. the evaluation of anticholinesterase activity was carried out by means of the ellman's method [159] : dichloromethane extracts showed significant inhibition against buche (96.56% ± 2.98 and 82.33% ± 2.69, respectively) at 20 μg/ml and appreciable inhibition against ache (53.24 ± 1.22 and 31.38 ± 5.41%, respectively) at 20 μg/ml. four coumarins were isolated from ferulago cassia-peucedanol, suberosin, grandivitinol and umbelliferone ( figure 39 ). therefore, f. cassia can be considered a starting point for the development of new compounds with antioxidant and anticholinesterase activity. in the same year, mu and colleagues synthesized a series of eleven 7-substituted coumarins and evaluated their anti-inflammatory activities and their ability to exploit the nf-kb pathway [151] . all the screened compounds showed a relevant anti-inflammatory activity. in the series, compound 104 ( figure 38 ) was identified as a hit and further studies of molecular docking were conducted confirming that 104 could bind to the active site (nls polypeptide) of nf-κb p65. its binding affinity was further confirmed by surface plasmon resonance (spr) analysis. moreover, western blot assay showed that 104 remarkably blocked the nf-κb signaling pathway in vitro. in the same year, mu and colleagues synthesized a series of eleven 7-substituted coumarins and evaluated their anti-inflammatory activities and their ability to exploit the nf-kb pathway [151] . all the screened compounds showed a relevant anti-inflammatory activity. in the series, compound 104 ( figure 38 ) was identified as a hit and further studies of molecular docking were conducted confirming that 104 could bind to the active site (nls polypeptide) of nf-κb p65. its binding affinity was further confirmed by surface plasmon resonance (spr) analysis. moreover, western blot assay showed that 104 remarkably blocked the nf-κb signaling pathway in vitro. alzheimer's disease (ad) is the most common form of dementia (60-70% of cases of dementia are cause by ad) and consists in a neurodegenerative disorder characterized by a slow, progressive and irreversible loss of cognitive function and memory [152] [153] [154] [155] [156] [157] . the current therapeutic approach, mainly based on the use of acetylcholinesterase (ache) inhibitors (rivastigmine, donepezil, galantamine) or n-methy-d-aspartic acid (nmda) receptor inhibitors (memantine), is merely symptomatic and does not counteract degeneration progression. new innovative approaches, such as multi-targeted strategies, are urgently required in order to cure cognition and motor dysfunctions, neurodegeneration and depression. among their biological activities, coumarins showed the ability to inhibit some biological targets involved in ad. with this in mind, some recent works aimed at investigating coumarins potential in the treatment of ad are discussed below. in 2019, karakaya and collaborators investigated the antioxidant and ache/buche inhibitory activity of aerial parts, fruits, flowers and root extracts from ferulago cassia boiss [158] . root's and fruit's dichloromethane extracts showed the highest antioxidant potential in tba assay. the evaluation of anticholinesterase activity was carried out by means of the ellman's method [159] : dichloromethane extracts showed significant inhibition against buche (96.56% ± 2.98 and 82.33% ± 2.69, respectively) at 20 μg/ml and appreciable inhibition against ache (53.24 ± 1.22 and 31.38 ± 5.41%, respectively) at 20 μg/ml. four coumarins were isolated from ferulago cassia-peucedanol, suberosin, grandivitinol and umbelliferone ( figure 39 ). therefore, f. cassia can be considered a starting point for the development of new compounds with antioxidant and anticholinesterase activity. alzheimer's disease (ad) is the most common form of dementia (60-70% of cases of dementia are cause by ad) and consists in a neurodegenerative disorder characterized by a slow, progressive and irreversible loss of cognitive function and memory [152] [153] [154] [155] [156] [157] . the current therapeutic approach, mainly based on the use of acetylcholinesterase (ache) inhibitors (rivastigmine, donepezil, galantamine) or n-methy-d-aspartic acid (nmda) receptor inhibitors (memantine), is merely symptomatic and does not counteract degeneration progression. new innovative approaches, such as multi-targeted strategies, are urgently required in order to cure cognition and motor dysfunctions, neurodegeneration and depression. among their biological activities, coumarins showed the ability to inhibit some biological targets involved in ad. with this in mind, some recent works aimed at investigating coumarins potential in the treatment of ad are discussed below. in 2019, karakaya and collaborators investigated the antioxidant and ache/buche inhibitory activity of aerial parts, fruits, flowers and root extracts from ferulago cassia boiss [158] . root's and fruit's dichloromethane extracts showed the highest antioxidant potential in tba assay. the evaluation of anticholinesterase activity was carried out by means of the ellman's method [159] : dichloromethane extracts showed significant inhibition against buche (96.56% ± 2.98 and 82.33% ± 2.69, respectively) at 20 µg/ml and appreciable inhibition against ache (53.24 ± 1.22 and 31.38 ± 5.41%, respectively) at 20 µg/ml. four coumarins were isolated from ferulago cassia-peucedanol, suberosin, grandivitinol and umbelliferone ( figure 39 ). therefore, f. cassia can be considered a starting point for the development of new compounds with antioxidant and anticholinesterase activity. thanks to their simple structural architecture and chemical stability, coumarins can be easily synthesized and modified in order to produce more active and selective compounds. najafi and coworkers synthesized a series of tacrine-coumarin derivatives linked to a 1,2,3-triazole moiety and evaluated their activity in terms of ache and butyrylcholinesterase (buche) inhibition, keeping donepezil and tacrine as reference drugs [160] . in addition, their beta-secretase 1 (bace1) inhibitory activity and neuroprotective potential were evaluated. since tacrine is a well-known inhibitor of the catalytic site of ache, whereas coumarins showed affinity for the peripheral anionic site (pas) [161] , this new compounds may be potential dual-and therefore more powerful -inhibitors of ches. the in vitro ache and buche inhibitory activity was evaluated using the ellman's method [159] ; among all the tested molecules, compound 105 resulted the best in ache inhibition (ic5 0= 0.027 ± 0.009 μm; tacrine ic50 = 0.048 ± 0.011 μm, donepezil ic50 = 0.039 ± 0.097 μm) and compound 106 was the most promising buche inhibitor (ic50 = 0.006 ± 0.002 μm; tacrine ic50 = 0.010 ± 0.004 μm, donepezil ic50 = 8.416 ± 0.628 μm) ( figure 40 ). thanks to their simple structural architecture and chemical stability, coumarins can be easily synthesized and modified in order to produce more active and selective compounds. najafi and co-workers synthesized a series of tacrine-coumarin derivatives linked to a 1,2,3-triazole moiety and evaluated their activity in terms of ache and butyrylcholinesterase (buche) inhibition, keeping donepezil and tacrine as reference drugs [160] . in addition, their beta-secretase 1 (bace1) inhibitory activity and neuroprotective potential were evaluated. since tacrine is a well-known inhibitor of the catalytic site of ache, whereas coumarins showed affinity for the peripheral anionic site (pas) [161] , this new compounds may be potential dual-and therefore more powerful -inhibitors of ches. the in vitro ache and buche inhibitory activity was evaluated using the ellman's method [159] ; among all the tested molecules, compound 105 resulted the best in ache inhibition (ic 50 thanks to their simple structural architecture and chemical stability, coumarins can be easily synthesized and modified in order to produce more active and selective compounds. najafi and coworkers synthesized a series of tacrine-coumarin derivatives linked to a 1,2,3-triazole moiety and evaluated their activity in terms of ache and butyrylcholinesterase (buche) inhibition, keeping donepezil and tacrine as reference drugs [160] . in addition, their beta-secretase 1 (bace1) inhibitory activity and neuroprotective potential were evaluated. since tacrine is a well-known inhibitor of the catalytic site of ache, whereas coumarins showed affinity for the peripheral anionic site (pas) [161] , this new compounds may be potential dual-and therefore more powerful -inhibitors of ches. the in vitro ache and buche inhibitory activity was evaluated using the ellman's method [159] ; among all the tested molecules, compound 105 resulted the best in ache inhibition (ic5 0= 0.027 ± 0.009 μm; tacrine ic50 = 0.048 ± 0.011 μm, donepezil ic50 = 0.039 ± 0.097 μm) and compound 106 was the most promising buche inhibitor (ic50 = 0.006 ± 0.002 μm; tacrine ic50 = 0.010 ± 0.004 μm, donepezil ic50 = 8.416 ± 0.628 μm) ( figure 40 ). concerning the anti-buche activity, structure-activity relationship studies showed that cl and me substituents, as well as the methylene linker, play a complex and not completely understood role in the enzyme inhibition. from the evaluation of inhibitory activity of the synthesized compounds on bace1, a moderate inhibitory activity of compound 105 was observed (inhibition of 28.69% and 13.97% at 50 and 10 µm, respectively). nevertheless, compound 105 did not show neuroprotective action on pc12 cells exposed to aβ [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] . then, in vivo evaluation of compound 105 using the morrison water maze method [162] was made and valuable results based on memory improvement in scopolamine-induced impairment were observed. similarly, rastegari and collaborators synthesized a series of 1,2,3-triazole-chromenone carboxamide derivatives and investigated their potential as anti-alzheimer's agents, in terms of inhibitory activity against ache, buche and bace1, besides their neuroprotective and metal-chelating properties [163] . the anti-ache activity of coumarin-3-carboxamide was already known [164] , as well as the ability of the 1,2,3-triazole moiety to enhance achei activity, if linked with tacrine or phenanthridinium derivatives and its bace1 inhibitory potential [165] . in vitro achei and buchei activities of the new compounds were evaluated, keeping donepezil as reference. higher activities were shown by compound 107, bearing 3,4-dimethylbenzyl connected to 1,2,3-triazole moiety and by compound 108, having 3-morpholinopropyl and 2-bromobenzyl moieties ( figure 41 ), even if they are much less active than donepezil (ic 50 = 0.027 µm). anti-bche activity was also modest and resulted to be affected by the type of amine connected to the amide moiety, that is, morpholine or piperidine and by the position and electronic properties of substituents on the benzyl group connected to 1,2,3-triazole ring. concerning the anti-buche activity, structure-activity relationship studies showed that cl and me substituents, as well as the methylene linker, play a complex and not completely understood role in the enzyme inhibition. from the evaluation of inhibitory activity of the synthesized compounds on bace1, a moderate inhibitory activity of compound 105 was observed (inhibition of 28.69% and 13.97% at 50 and 10 μm, respectively). nevertheless, compound 105 did not show neuroprotective action on pc12 cells exposed to aβ25-35. then, in vivo evaluation of compound 105 using the morrison water maze method [162] was made and valuable results based on memory improvement in scopolamine-induced impairment were observed. similarly, rastegari and collaborators synthesized a series of 1,2,3-triazole-chromenone carboxamide derivatives and investigated their potential as anti-alzheimer's agents, in terms of inhibitory activity against ache, buche and bace1, besides their neuroprotective and metal-chelating properties [163] . the anti-ache activity of coumarin-3carboxamide was already known [164] , as well as the ability of the 1,2,3-triazole moiety to enhance achei activity, if linked with tacrine or phenanthridinium derivatives and its bace1 inhibitory potential [165] . in vitro achei and buchei activities of the new compounds were evaluated, keeping donepezil as reference. higher activities were shown by compound 107, bearing 3,4-dimethylbenzyl connected to 1,2,3-triazole moiety and by compound 108, having 3-morpholinopropyl and 2bromobenzyl moieties ( figure 41 ), even if they are much less active than donepezil (ic50=0.027 μm). anti-bche activity was also modest and resulted to be affected by the type of amine connected to the amide moiety, that is, morpholine or piperidine and by the position and electronic properties of substituents on the benzyl group connected to 1,2,3-triazole ring. compound 107 was chosen for bace1 inhibitory activity evaluation, showing only modest results (ic50 = 21.13 μm compared to the reference om99-2 having ic50 = 0.014μm). also, neuroprotective potential of 107 was investigated by comparing 107-treated cells with intact (no intervention), quercetin+h2o2-treated (positive control) and h2o2-treated (negative control) cells. the mentioned compound showed moderate to good neuroprotective activity, not stronger than quercetin. finally, since ros, which are potentially involved in the neurodegenerative process of ad, may be generated from unregulated reaction of molecular oxygen with the redox active metals such as fe, cu and zn, the metal-chelating properties of 107 toward cu 2+, fe 2+ and zn 2+ were tested in methanol by means of uv-vis spectrophotometry. interaction between 107 and cu 2+ and zn 2+ was detected, whereas no interaction was observed between 107 and fe 2+ . compound 107 was chosen for bace1 inhibitory activity evaluation, showing only modest results (ic50 = 21.13 µm compared to the reference om99-2 having ic50 = 0.014 µm). also, neuroprotective potential of 107 was investigated by comparing 107-treated cells with intact (no intervention), quercetin+h 2 o 2 -treated (positive control) and h 2 o 2 -treated (negative control) cells. the mentioned compound showed moderate to good neuroprotective activity, not stronger than quercetin. finally, since ros, which are potentially involved in the neurodegenerative process of ad, may be generated from unregulated reaction of molecular oxygen with the redox active metals such as fe, cu and zn, the metal-chelating properties of 107 toward cu 2+, fe 2+ and zn 2+ were tested in methanol by means of uv-vis spectrophotometry. interaction between 107 and cu 2+ and zn 2+ was detected, whereas no interaction was observed between 107 and fe 2+ . de souza and co-workers designed and synthesized a series of 3-substituted-7-aminoalcoxycoumarin derivatives (109a-d, 110a-c, 111a-c, figure 42 ) whose achei/buchei activities and antioxidant properties were evaluated [166] . all the compounds showed good inhibitory activity on ache, with potencies in the nanomolar range, whereas their activity against buche was lower (ic 50 between 0.90 to 15.85 µm); 3-(4-(dimethylamino)phenyl)-7-aminoethoxycoumarin (111a) was considered a hit, showing an excellent inhibitory activity (ic 50 = 20 nm) and selectivity towards ache (ic 50 buche/ache = 354) compared to the reference drug donepezil (ic 50 = 6 nm, ic 50 buche/ache = 365). investigation of antioxidant properties showed that only compounds 111a-c presented activity in ferric ion reduction method (frap), whereas series 109a-d and 110a-c did not show significant results, suggesting that the dimethylaminophenyl moiety may be responsible for the antioxidant activity. de souza and co-workers designed and synthesized a series of 3-substituted-7-aminoalcoxycoumarin derivatives (109a-d, 110a-c, 111a-c, figure 42 ) whose achei/buchei activities and antioxidant properties were evaluated [166] . all the compounds showed good inhibitory activity on ache, with potencies in the nanomolar range, whereas their activity against buche was lower (ic50 between 0.90 to 15.85 μm); 3-(4-(dimethylamino)phenyl)-7-aminoethoxycoumarin (111a) was considered a hit, showing an excellent inhibitory activity (ic50 = 20 nm) and selectivity towards ache (ic50 buche/ache = 354) compared to the reference drug donepezil (ic50 = 6 nm, ic50 buche/ache = 365). investigation of antioxidant properties showed that only compounds 111a-c presented activity in ferric ion reduction method (frap), whereas series 109a-d and 110a-c did not show significant results, suggesting that the dimethylaminophenyl moiety may be responsible for the antioxidant activity. docking simulations showed that all the compounds were able to bind simultaneously the pas and cationic active site (cas) of the electric eel ache (eeache): the interaction with the cas took place by means of cation-π interaction between piperidinyl group and trp86. compounds 111a and 111c showed the ability to give a π-stacking interaction in the pas, with trp286. the smaller spacer of 111a allowed the coumarin moiety to be located properly in the gorge, achieving h-bonds with tyr337 and phe295. this is probably the reason for the most efficient binding mode (and consequently the best activity) to eeache of compounds with a short spacer. yang and his group synthesized a series of twenty-four 3-aryl coumarin derivatives and tested their cholinesterase inhibitory activity, monoamine oxidase (mao) inhibitory activity and antioxidant activity in vitro [167] . as far as cholinesterase inhibition is concerned, most of the mentioned compounds showed moderate activity. compound 117 ( figure 43 ) exhibited a strong activity (ic50 = 3.04 ± 0.32 μm), whereas compound 116 showed selectivity towards ache. compound 114 was more active against buche (ic50 = 2.76 ± 0.57 μm) than donepezil, whereas compounds 112, 113 and 115 showed selectivity towards buche. 3-arylcoumarins bearing hydroxy group both at r5 and r6 positions displayed higher activity respect to the mono-substituted counterparts, especially towards ache, whereas no significant impact on buche inhibition was observed. docking simulations showed that all the compounds were able to bind simultaneously the pas and cationic active site (cas) of the electric eel ache (eeache): the interaction with the cas took place by means of cation-π interaction between piperidinyl group and trp86. compounds 111a and 111c showed the ability to give a π-stacking interaction in the pas, with trp286. the smaller spacer of 111a allowed the coumarin moiety to be located properly in the gorge, achieving h-bonds with tyr337 and phe295. this is probably the reason for the most efficient binding mode (and consequently the best activity) to eeache of compounds with a short spacer. yang and his group synthesized a series of twenty-four 3-aryl coumarin derivatives and tested their cholinesterase inhibitory activity, monoamine oxidase (mao) inhibitory activity and antioxidant activity in vitro [167] . as far as cholinesterase inhibition is concerned, most of the mentioned compounds showed moderate activity. compound 117 ( figure 43 ) exhibited a strong activity (ic 50 = 3.04 ± 0.32 µm), whereas compound 116 showed selectivity towards ache. compound 114 was more active against buche (ic 50 = 2.76 ± 0.57 µm) than donepezil, whereas compounds 112, 113 and 115 showed selectivity towards buche. 3-arylcoumarins bearing hydroxy group both at r 5 and r 6 positions displayed higher activity respect to the mono-substituted counterparts, especially towards ache, whereas no significant impact on buche inhibition was observed. antioxidant activity in vitro [167] . as far as cholinesterase inhibition is concerned, most of the mentioned compounds showed moderate activity. compound 117 ( figure 43 ) exhibited a strong activity (ic50 = 3.04 ± 0.32 μm), whereas compound 116 showed selectivity towards ache. compound 114 was more active against buche (ic50 = 2.76 ± 0.57 μm) than donepezil, whereas compounds 112, 113 and 115 showed selectivity towards buche. 3-arylcoumarins bearing hydroxy group both at r5 and r6 positions displayed higher activity respect to the mono-substituted counterparts, especially towards ache, whereas no significant impact on buche inhibition was observed. as mentioned, mao-inhibitory activity was evaluated as well. in fact, mao is one of the enzymes responsible for oxidative stress, which is another factor involved in the neurodegenerative process characterizing ad and different studies identified various che/mao inhibitors as tools for ad treatment, showing positive results in clinical trials [168] . in yang's work, 3-arylcoumarins anti-mao activity was compared to that of rasagiline (a selective mao-b inhibitor): almost half of the compounds showed relevant activity [167] . among them, compound 117 was the most promising, with an ic 50 = 27.03 ± 0.50 µm, though weaker than rasagiline (ic 50 = 0.125 ± 0.005 µm). again, the presence of r 5 , r 6 hydroxy groups provided better mao inhibitory activity. finally, the antioxidant power of 3-arylcoumarins was studied by means of ferric ion reduction method (frap), using vitamin c as a reference. compound 117 resulted again the most active (frap value = 41.42 ± 0.35), also showing a certain activity in vivo, when tested on zebrafish, leading to an increase of total distance of zebrafish movement proportional to the concentration of compound used. in 2017, joubert and collaborators designed and synthesized a series of 7-substituted coumarins (118-146, figure 44 ), consisting in a coumarin motif (mao inhibitor) condensed with a benzyl-, piperidinyl-, n-benzylpiperidinylor p-bromo-n-benzylpiperizinyl moiety, which resemble the n-benzylpiperidine function present in donepezil (ache inhibitor), connected via an alkyl ether linkage at position 7 [169] . their biological activities were evaluated in terms of mao and che inhibitory potential. inhibition of hmao was achieved from all the designed compounds, which also showed selectivity towards mao-b. the benzyloxy series showed higher activity, in some cases even better than the reference selegiline (ic 50 = 0.008 µm), with ic 50 values in the nanomolar range (0.5-73 nm). piperidinyl-, n-benzylpiperidinyl-or p-bromo-n-benzylpiperizinyl moiety, which resemble the nbenzylpiperidine function present in donepezil (ache inhibitor), connected via an alkyl ether linkage at position 7 [169] . their biological activities were evaluated in terms of mao and che inhibitory potential. inhibition of hmao was achieved from all the designed compounds, which also showed selectivity towards mao-b. the benzyloxy series showed higher activity, in some cases even better than the reference selegiline (ic50 = 0.008 μm), with ic50 values in the nanomolar range (0.5-73 nm). . docking studies showed that 136 binds the substrate cavity through the coumarin ring, whereas the benzyl moiety occupies the entrance cavity. in addition, 136 was able to bind both the cas and pas of cholinesterase, which suggested that it could interfere with pas-induced β-amyloid aggregation. a different approach was followed by shi and collaborators, who designed four derivatives obtained by coupling two pharmacophores (carbazole and coumarin) to obtain potential multitarget agents for the treatment of ad [170] . the aim was to exploit the biological activities of both the mentioned moieties: on one hand coumarins are known to have inhibitory activity on ache, buche and mao, besides antioxidant properties; on the other hand, carbazole exhibits antioxidant activity [171] and the ability to inhibit aβ aggregation [172] . in addition, carbazole derivatives are reported to have inhibitory effect on ches, thus providing neuroprotection from β-amyloid toxicity [173] . thus, compounds 147a-d ( figure 45 ) were synthesized, characterized and their biological activities were evaluated: anticholinesterase activity was tested on eeache and on hache, using ellman's method [159] , keeping tacrine as reference drug. whereas all derivatives showed dose-dependent inhibitory activity on eeache (compound 147d resulted the best with an ic 50 of 3.75 µm), on hache they exhibited weak effects (147b ic 50 = 76.63 µm, 147d ic 50 = 70.51 µm). none of the tested compounds inhibited eqbuche or hbuche more than 30%, which means that compounds 147b and 147d showed selectivity for ache over buche. synthesized, characterized and their biological activities were evaluated: anticholinesterase activity was tested on eeache and on hache, using ellman's method [159] , keeping tacrine as reference drug. whereas all derivatives showed dose-dependent inhibitory activity on eeache (compound 147d resulted the best with an ic50 of 3.75 μm), on hache they exhibited weak effects (147b ic50 = 76.63 μm, 147d ic50 = 70.51 μm). none of the tested compounds inhibited eqbuche or hbuche more than 30%, which means that compounds 147b and 147d showed selectivity for ache over buche. as the alkyl linker length increased, the anti-cholinesterase activity improved, probably because a 5-methylene chain allowed the carbazole and coumarin moieties to bind both the cas and the pas, respectively. antioxidant properties were evaluated through the orac-fl method (oxygen radical absorbance capacity by fluorescein) [174] , using trolox (vitamin e analogue) as a standard; none of the tested compounds showed significant activity. among the tested derivatives, compound 147d resulted a promising lead candidate for the treatment of ad. epilepsy is a widespread neurological disorder, characterized by periodic and unpredictable attacks, involving seizures and/or transient behavioral changes. its pathogenesis has not been completely clarified yet; it is known, however, that an impairment between excitatory and inhibitory neurotransmission is involved [175] [176] [177] [178] [179] here, we report some recent advances in the use of coumarins as anticonvulsant compounds. abd-allah and collaborators recently studied the anticonvulsant activity of a series of coumarin derivatives, achieved by merging two or more pharmacophoric scaffolds in order to create new chemical entities with an improved biological activity [180] . the compounds here described possess all the necessary elements to exert anticonvulsant activity: a lipophilic aryl ring, a hydrogen-bonding domain and an electron-donor moiety [178, 179, 181, 182] . all the compounds were initially screened (phase i) using two standard animal seizure models, subcutaneous pentylenetetrazole (scptz) and maximal electric shock (mes) seizure tests using ethosuximide as reference drug. an assessment of the potential neurotoxicity was also done by means of rotarod test. phase ii consisted in the determination of ed50 value for compounds that conferred 100% protection in one or both tests. in the end, gaba level measurements were carried out in whole mouse brain for the most active compounds, using gabapentin as a reference drug. the results of phase i tests showed that all the tested compounds had protective activity against scptz-induced absence epilepsy (variable results in the range of 17-100% protection). among them, 148, 149 and 150 ( figure 46 ) were the most active (100% protection) at 0.238, 0.239 and 0.283 mmol/kg, meaning that the compounds are 1.49, 1.48, 1.25 folds more potent than ethosuximide, respectively. as the alkyl linker length increased, the anti-cholinesterase activity improved, probably because a 5-methylene chain allowed the carbazole and coumarin moieties to bind both the cas and the pas, respectively. antioxidant properties were evaluated through the orac-fl method (oxygen radical absorbance capacity by fluorescein) [174] , using trolox (vitamin e analogue) as a standard; none of the tested compounds showed significant activity. among the tested derivatives, compound 147d resulted a promising lead candidate for the treatment of ad. epilepsy is a widespread neurological disorder, characterized by periodic and unpredictable attacks, involving seizures and/or transient behavioral changes. its pathogenesis has not been completely clarified yet; it is known, however, that an impairment between excitatory and inhibitory neurotransmission is involved [175] [176] [177] [178] [179] here, we report some recent advances in the use of coumarins as anticonvulsant compounds. abd-allah and collaborators recently studied the anticonvulsant activity of a series of coumarin derivatives, achieved by merging two or more pharmacophoric scaffolds in order to create new chemical entities with an improved biological activity [180] . the compounds here described possess all the necessary elements to exert anti-convulsant activity: a lipophilic aryl ring, a hydrogen-bonding domain and an electron-donor moiety [178, 179, 181, 182] . all the compounds were initially screened (phase i) using two standard animal seizure models, subcutaneous pentylenetetrazole (scptz) and maximal electric shock (mes) seizure tests using ethosuximide as reference drug. an assessment of the potential neurotoxicity was also done by means of rotarod test. phase ii consisted in the determination of ed 50 value for compounds that conferred 100% protection in one or both tests. in the end, gaba level measurements were carried out in whole mouse brain for the most active compounds, using gabapentin as a reference drug. the results of phase i tests showed that all the tested compounds had protective activity against scptz-induced absence epilepsy (variable results in the range of 17-100% protection). among them, 148, 149 and 150 ( figure 46 ) were the most active (100% protection) at 0.238, 0.239 and 0.283 mmol/kg, meaning that the compounds are 1.49, 1.48, 1.25 folds more potent than ethosuximide, respectively. in the mes-induced seizures though, all the compounds failed in completely protecting the animals. the best profile was exhibited by compound 151 (figure 46 ) which was capable of a 50% protection at 2.1 mmol/kg. a quantitative determination of ed 50 values was carried out for compounds 148, 149 and 150, which were able to fully protect animals in the scptz test. compound 148 was found to be the most active with an ed 50 of 54.86 mg/kg (0.131 mmol/kg). consequently, it was chosen for further investigation to elucidate the mechanism of action, which was assessed through an evaluation of gaba levels in mice brain. as a result, the proposed mechanism for compound 148 is a gaba-mediated one, perhaps non-vesicular release of gaba, gaba a receptor activation or gaba b receptor inhibition; probably an enhanced synthesis or reduced metabolism of gaba are also involved. the most active with an ed50 of 54.86 mg/kg (0.131 mmol/kg). consequently, it was chosen for further investigation to elucidate the mechanism of action, which was assessed through an evaluation of gaba levels in mice brain. as a result, the proposed mechanism for compound 148 is a gabamediated one, perhaps non-vesicular release of gaba, gabaa receptor activation or gabab receptor inhibition; probably an enhanced synthesis or reduced metabolism of gaba are also involved. a similar bivalent drug approach was followed by mohammadi-khanaposhtani and collaborators, who synthesized a series of coumarin-1,2,4-oxadiazole derivatives in order to create a new chemical entity with better anticonvulsant profile than coumarin and oxadiazole alone [183] . in fact, different 5-member heterocyclic rings-containing compounds such as oxadiazoles, triazoles and thiadiazoles were reported to have good anticonvulsant activity [184] [185] [186] through benzodiazepine (bdz) receptor [187] . the activity of the new derivatives was tested using ptz-and mes-induced seizures in mice, keeping diazepam as a reference drug. most of the new compounds did not show activity against ptz-induced seizures, except for three of them, 152a, 152b and 152c (figure 47 ), being 152b the most active (25% of protection using 10 mg/kg). compounds 152d, 152e and 152f ( figure 47 ) showed a 100% protection against mes-induced seizures at the doses of 7, 40 and 20 mg/kg, respectively (it should be considered that diazepam shows 100% protection at 2 mg/ml) [188] . compound 152d, which showed the best activity, was characterized by the absence of substituents on position 4 of the coumarin ring and by a 4-chloroaryl group connected to the 1, 2, 4-oxadiazole ring. the most active compounds 152d and 152e were used to investigate the mechanism of action; to do so, the effect of flumazenil (a bdz receptor antagonist) on their activity was evaluated. flumazenil antagonized both 152d and 152e, confirming that these compounds act as bzs receptor agonists. finally, in vivo neurotoxicity of compounds 152d and 152e was assessed and the tested compounds gave less neurological deficits that the reference drug diazepam. a similar bivalent drug approach was followed by mohammadi-khanaposhtani and collaborators, who synthesized a series of coumarin-1,2,4-oxadiazole derivatives in order to create a new chemical entity with better anticonvulsant profile than coumarin and oxadiazole alone [183] . in fact, different 5-member heterocyclic rings-containing compounds such as oxadiazoles, triazoles and thiadiazoles were reported to have good anticonvulsant activity [184] [185] [186] through benzodiazepine (bdz) receptor [187] . the activity of the new derivatives was tested using ptz-and mes-induced seizures in mice, keeping diazepam as a reference drug. most of the new compounds did not show activity against ptz-induced seizures, except for three of them, 152a, 152b and 152c (figure 47 ), being 152b the most active (25% of protection using 10 mg/kg). compounds 152d, 152e and 152f ( figure 47 ) showed a 100% protection against mes-induced seizures at the doses of 7, 40 and 20 mg/kg, respectively (it should be considered that diazepam shows 100% protection at 2 mg/ml) [188] . compound 152d, which showed the best activity, was characterized by the absence of substituents on position 4 of the coumarin ring and by a 4-chloroaryl group connected to the 1, 2, 4-oxadiazole ring. the most active compounds 152d and 152e were used to investigate the mechanism of action; to do so, the effect of flumazenil (a bdz receptor antagonist) on their activity was evaluated. flumazenil antagonized both 152d and 152e, confirming that these compounds act as bzs receptor agonists. finally, in vivo neurotoxicity of compounds 152d and 152e was assessed and the tested compounds gave less neurological deficits that the reference drug diazepam. a series of 4-amino-3-nitrocoumarins (153a-e, figure 48 ) was synthesized and biologically evaluated by mokrov and co-workers [189] . the anticonvulsant activity was investigated using the mes mice model (grand mal seizures) and the corazole-antagonism test (modelling the so-called petit mal seizure). the only statistically significant result was given by compound 153a at doses in the range of 60-80 mg/kg, as it could increase the animal survival in mes test up to 60% (in the control group survival was 10%). corazole-induced seizures and animals' death were not prevented by compounds 153a, 153c and 153d. compound 153e exhibited good protection potential at 10-40 mg/kg and was able to prevent death in 50-63% of animals. the most active compound in the mes test was 153a, containing a coumarin ring with a 3-nitro group and a γ-aminobutirric acid fragment. the corresponding methyl-ester (153c) did not show any activity, as well as compound 153d, with an additional nitro group. compound 153e, bearing the methyl-ester of gaba pharmacophore and two nitro groups on the coumarin ring, showed the best profile in corazole-antagonism test. the corresponding free carboxylic acid 153d was found to be inactive, as well as compound 153c, having one less nitro group. compound 153a, bearing one nitro group and no substituent on gaba moiety, did not show any corazole-antagonist activity. therefore, it can be speculated that compounds 153a and 153e possess different mechanisms responsible for their anticonvulsant activity and they may be a starting point for further studies on coumarin derivatives as anticonvulsant agents. the recent medical history of anticoagulant drugs has been largely dominated by a simple class of molecules that has been discovered thanks to a mysterious livestock mortality and, later, employed as a powerful rodenticide: coumarins. the anticoagulant activity of coumarins was identified when in 1920′s seemingly healthy cattle of canada and north america died inexplicably for internal hemorrhages. the main cause of this decimation was attributed to a mold infestation of the damp hay, later known as the "sweet clover disease." however, it was not before 1940 that the responsible molecule was identified by karl link and his student harold campbell: it was 3,3'-methylenebis(4a series of 4-amino-3-nitrocoumarins (153a-e, figure 48 ) was synthesized and biologically evaluated by mokrov and co-workers [189] . the anticonvulsant activity was investigated using the mes mice model (grand mal seizures) and the corazole-antagonism test (modelling the so-called petit mal seizure). the only statistically significant result was given by compound 153a at doses in the range of 60-80 mg/kg, as it could increase the animal survival in mes test up to 60% (in the control group survival was 10%). corazole-induced seizures and animals' death were not prevented by compounds 153a, 153c and 153d. compound 153e exhibited good protection potential at 10-40 mg/kg and was able to prevent death in 50-63% of animals. the most active compound in the mes test was 153a, containing a coumarin ring with a 3-nitro group and a γ-aminobutirric acid fragment. the corresponding methyl-ester (153c) did not show any activity, as well as compound 153d, with an additional nitro group. compound 153e, bearing the methyl-ester of gaba pharmacophore and two nitro groups on the coumarin ring, showed the best profile in corazole-antagonism test. the corresponding free carboxylic acid 153d was found to be inactive, as well as compound 153c, having one less nitro group. compound 153a, bearing one nitro group and no substituent on gaba moiety, did not show any corazole-antagonist activity. therefore, it can be speculated that compounds 153a and 153e possess different mechanisms responsible for their anticonvulsant activity and they may be a starting point for further studies on coumarin derivatives as anticonvulsant agents. a series of 4-amino-3-nitrocoumarins (153a-e, figure 48 ) was synthesized and biologically evaluated by mokrov and co-workers [189] . the anticonvulsant activity was investigated using the mes mice model (grand mal seizures) and the corazole-antagonism test (modelling the so-called petit mal seizure). the only statistically significant result was given by compound 153a at doses in the range of 60-80 mg/kg, as it could increase the animal survival in mes test up to 60% (in the control group survival was 10%). corazole-induced seizures and animals' death were not prevented by compounds 153a, 153c and 153d. compound 153e exhibited good protection potential at 10-40 mg/kg and was able to prevent death in 50-63% of animals. the most active compound in the mes test was 153a, containing a coumarin ring with a 3-nitro group and a γ-aminobutirric acid fragment. the corresponding methyl-ester (153c) did not show any activity, as well as compound 153d, with an additional nitro group. compound 153e, bearing the methyl-ester of gaba pharmacophore and two nitro groups on the coumarin ring, showed the best profile in corazole-antagonism test. the corresponding free carboxylic acid 153d was found to be inactive, as well as compound 153c, having one less nitro group. compound 153a, bearing one nitro group and no substituent on gaba moiety, did not show any corazole-antagonist activity. therefore, it can be speculated that compounds 153a and 153e possess different mechanisms responsible for their anticonvulsant activity and they may be a starting point for further studies on coumarin derivatives as anticonvulsant agents. the recent medical history of anticoagulant drugs has been largely dominated by a simple class of molecules that has been discovered thanks to a mysterious livestock mortality and, later, employed as a powerful rodenticide: coumarins. the anticoagulant activity of coumarins was identified when in 1920′s seemingly healthy cattle of canada and north america died inexplicably for internal hemorrhages. the main cause of this decimation was attributed to a mold infestation of the damp hay, later known as the "sweet clover disease." however, it was not before 1940 that the responsible molecule was identified by karl link and his student harold campbell: it was 3,3'-methylenebis(4 the recent medical history of anticoagulant drugs has been largely dominated by a simple class of molecules that has been discovered thanks to a mysterious livestock mortality and, later, employed as a powerful rodenticide: coumarins. the anticoagulant activity of coumarins was identified when in 1920 s seemingly healthy cattle of canada and north america died inexplicably for internal hemorrhages. the main cause of this decimation was attributed to a mold infestation of the damp hay, later known as the "sweet clover disease." however, it was not before 1940 that the responsible molecule was identified by karl link and his student harold campbell: it was 3,3'-methylenebis(4-hydroxycoumarin), later known as dicoumarol. further studies by link's team brought to the discovery of warfarin in 1948 (so named from warf, wisconsin alumni research foundation, that financed the project), approved as a rodenticide in the usa in 1952 and for anticoagulation therapy in human in 1954, under the name of coumadin. currently, warfarin is one of the most widely used anticoagulation drug (in the uk it has been estimated that at least 1% of the population and 8% of people over 80 years are taking warfarin), together with other coumarin derivatives like dicumarol and acenocoumarol ( figure 49 ) [18, [190] [191] [192] . hydroxycoumarin), later known as dicoumarol. further studies by link's team brought to the discovery of warfarin in 1948 (so named from warf, wisconsin alumni research foundation, that financed the project), approved as a rodenticide in the usa in 1952 and for anticoagulation therapy in human in 1954, under the name of coumadin. currently, warfarin is one of the most widely used anticoagulation drug (in the uk it has been estimated that at least 1% of the population and 8% of people over 80 years are taking warfarin), together with other coumarin derivatives like dicumarol and acenocoumarol ( figure 49 ) [18, [190] [191] [192] . warfarin and other anticoagulant coumarins are vitamin-k antagonists (vkas). indeed, due to the structural similarity with vitamin-k, the compounds block the vitamin-k dependent pathways of coagulation, involving several factors (ii, vii, ix and x). despite the efficacy and the advantages of an oral therapy, warfarin is not devoid of side effects, mainly associated with bleeding and complications, like the narrow therapeutic range and interindividual genetic difference in pharmacokinetics, which requires a continuous monitoring of the patient [193, 194] . for this reason, the research of new safer and efficient compounds lead to the discovery of a novel vka, tecarfarin (ati-5923, figure 50 ), currently under development [195] . tecarfarin is active after oral administration and acts as a vitamin-k epoxide reductase (vkor) inhibitor; unlike warfarin, is not metabolized by the cytochrome p450 system but by human carboxylesterase-2 (hce-2) in hepatic microsomes. consequently, drug-drug or food-drug interactions are avoided, as well as genetic variability of cyp-450 system, providing a more stable anticoagulation effect compared to warfarin [196] . a detailed study on pharmacokinetics and pharmacodynamics of tecarfarin had been conducted on healthy patients by albrecht et al., together with the recent phase i study on its tolerability among patients with severe kidney disease [197, 198] . tecarfarin has the potentiality to be a valid substitute of warfarin in the oral therapy of thromboembolic disease. warfarin and other anticoagulant coumarins are vitamin-k antagonists (vkas). indeed, due to the structural similarity with vitamin-k, the compounds block the vitamin-k dependent pathways of coagulation, involving several factors (ii, vii, ix and x). despite the efficacy and the advantages of an oral therapy, warfarin is not devoid of side effects, mainly associated with bleeding and complications, like the narrow therapeutic range and interindividual genetic difference in pharmacokinetics, which requires a continuous monitoring of the patient [193, 194] . for this reason, the research of new safer and efficient compounds lead to the discovery of a novel vka, tecarfarin (ati-5923, figure 50 ), currently under development [195] . tecarfarin is active after oral administration and acts as a vitamin-k epoxide reductase (vkor) inhibitor; unlike warfarin, is not metabolized by the cytochrome p450 system but by human carboxylesterase-2 (hce-2) in hepatic microsomes. consequently, drug-drug or food-drug interactions are avoided, as well as genetic variability of cyp-450 system, providing a more stable anticoagulation effect compared to warfarin [196] . a detailed study on pharmacokinetics and pharmacodynamics of tecarfarin had been conducted on healthy patients by albrecht et al., together with the recent phase i study on its tolerability among patients with severe kidney disease [197, 198] . tecarfarin has the potentiality to be a valid substitute of warfarin in the oral therapy of thromboembolic disease. hydroxycoumarin), later known as dicoumarol. further studies by link's team brought to the discovery of warfarin in 1948 (so named from warf, wisconsin alumni research foundation, that financed the project), approved as a rodenticide in the usa in 1952 and for anticoagulation therapy in human in 1954, under the name of coumadin. currently, warfarin is one of the most widely used anticoagulation drug (in the uk it has been estimated that at least 1% of the population and 8% of people over 80 years are taking warfarin), together with other coumarin derivatives like dicumarol and acenocoumarol ( figure 49 ) [18, [190] [191] [192] . warfarin and other anticoagulant coumarins are vitamin-k antagonists (vkas). indeed, due to the structural similarity with vitamin-k, the compounds block the vitamin-k dependent pathways of coagulation, involving several factors (ii, vii, ix and x). despite the efficacy and the advantages of an oral therapy, warfarin is not devoid of side effects, mainly associated with bleeding and complications, like the narrow therapeutic range and interindividual genetic difference in pharmacokinetics, which requires a continuous monitoring of the patient [193, 194] . for this reason, the research of new safer and efficient compounds lead to the discovery of a novel vka, tecarfarin (ati-5923, figure 50 ), currently under development [195] . tecarfarin is active after oral administration and acts as a vitamin-k epoxide reductase (vkor) inhibitor; unlike warfarin, is not metabolized by the cytochrome p450 system but by human carboxylesterase-2 (hce-2) in hepatic microsomes. consequently, drug-drug or food-drug interactions are avoided, as well as genetic variability of cyp-450 system, providing a more stable anticoagulation effect compared to warfarin [196] . a detailed study on pharmacokinetics and pharmacodynamics of tecarfarin had been conducted on healthy patients by albrecht et al., together with the recent phase i study on its tolerability among patients with severe kidney disease [197, 198] . tecarfarin has the potentiality to be a valid substitute of warfarin in the oral therapy of thromboembolic disease. another approach reported in literature is the chemical modification of the coumarin scaffold by conjugation of 7-hydroxylcoumarin and 7-hydroxy-4-methylcoumarin with some derivatives of salicylic acid. among the compounds evaluated by bang and co-workers in 2019, derivatives 154 and 155 ( figure 51 ) showed high anticoagulant activity, with an increased prothrombin time (pt) of 10.88 ± 0.56 sec and 13.10 ± 3.56 sec, respectively. both compounds resulted 1.5 times more active than warfarin (pt 7.97 ± 1.93) [199] . structural modifications of the 4-hydroxycoumarin core was also the rational of montagut-romans et al., who in 2017 explored the potentiality given by modifications performed on c3 position by introducing a side chain (with at most one unsaturation) structurally related to vitamin-k cofactor [200] . the underlying premise was the sar study performed by gebaur in 2007, which pointed out that the activity of 4-hydroxycoumarin was enhanced only by structural modification for c3 position by isoprenyl motifs [201] . in this contest, 14 functionalized 4-hydroxycoumarins with alkyl chains of different length, both linear and branched, were synthesized and their activity was evaluated in vitro and ex vivo (phenprocoumon was included in the test as internal standard). the ability to inhibit vkorc1 in rat liver microsomes was evaluated in vitro and, except for two compounds, the c3-alkyl derivatives showed a sub-micromolar activity (from 20 nm to 200 nm) overcoming the benchmark compound phenprocoumon. further ex vivo studies assessed the ability to increase in vivo the prothrombin time (pt) and compounds 156a and 156b ( figure 52 ) showed a promising anticoagulant activity after 24 h. it is possible that the presence of the halogen atom protects the drug from liver metabolism. despite the interesting anticoagulant activity, follow-up studies on the liver metabolism are necessary to determine if these molecules are substrate of cyp2c9, to which is to attribute the variability in the dosage of oral vitamin-k antagonists, due to its polymorphism [202] . figure 53 ) presented a remarkable anticoagulant activity (pt = 41.2s and tt 128.5s) and no significant hepatic or renal toxicity when compared to warfarin (pt = 55.7s and tt 80.6s) [203] . although further studies are necessary to understand the mode of action of compound 157, it could be a promising anticoagulant agent for preclinical studies. structural modifications of the 4-hydroxycoumarin core was also the rational of montagut-romans et al., who in 2017 explored the potentiality given by modifications performed on c3 position by introducing a side chain (with at most one unsaturation) structurally related to vitamin-k cofactor [200] . the underlying premise was the sar study performed by gebaur in 2007, which pointed out that the activity of 4-hydroxycoumarin was enhanced only by structural modification for c3 position by isoprenyl motifs [201] . in this contest, 14 functionalized 4-hydroxycoumarins with alkyl chains of different length, both linear and branched, were synthesized and their activity was evaluated in vitro and ex vivo (phenprocoumon was included in the test as internal standard). the ability to inhibit vkorc1 in rat liver microsomes was evaluated in vitro and, except for two compounds, the c3-alkyl derivatives showed a sub-micromolar activity (from 20 nm to 200 nm) overcoming the benchmark compound phenprocoumon. further ex vivo studies assessed the ability to increase in vivo the prothrombin time (pt) and compounds 156a and 156b ( figure 52 ) showed a promising anticoagulant activity after 24 h. it is possible that the presence of the halogen atom protects the drug from liver metabolism. despite the interesting anticoagulant activity, follow-up studies on the liver metabolism are necessary to determine if these molecules are substrate of cyp2c9, to which is to attribute the variability in the dosage of oral vitamin-k antagonists, due to its polymorphism [202] . another approach reported in literature is the chemical modification of the coumarin scaffold by conjugation of 7-hydroxylcoumarin and 7-hydroxy-4-methylcoumarin with some derivatives of salicylic acid. among the compounds evaluated by bang and co-workers in 2019, derivatives 154 and 155 ( figure 51 ) showed high anticoagulant activity, with an increased prothrombin time (pt) of 10.88 ± 0.56 sec and 13.10 ± 3.56 sec, respectively. both compounds resulted 1.5 times more active than warfarin (pt 7.97 ± 1.93) [199] . structural modifications of the 4-hydroxycoumarin core was also the rational of montagut-romans et al., who in 2017 explored the potentiality given by modifications performed on c3 position by introducing a side chain (with at most one unsaturation) structurally related to vitamin-k cofactor [200] . the underlying premise was the sar study performed by gebaur in 2007, which pointed out that the activity of 4-hydroxycoumarin was enhanced only by structural modification for c3 position by isoprenyl motifs [201] . in this contest, 14 functionalized 4-hydroxycoumarins with alkyl chains of different length, both linear and branched, were synthesized and their activity was evaluated in vitro and ex vivo (phenprocoumon was included in the test as internal standard). the ability to inhibit vkorc1 in rat liver microsomes was evaluated in vitro and, except for two compounds, the c3-alkyl derivatives showed a sub-micromolar activity (from 20 nm to 200 nm) overcoming the benchmark compound phenprocoumon. further ex vivo studies assessed the ability to increase in vivo the prothrombin time (pt) and compounds 156a and 156b ( figure 52 ) showed a promising anticoagulant activity after 24 h. it is possible that the presence of the halogen atom protects the drug from liver metabolism. despite the interesting anticoagulant activity, follow-up studies on the liver metabolism are necessary to determine if these molecules are substrate of cyp2c9, to which is to attribute the variability in the dosage of oral vitamin-k antagonists, due to its polymorphism [202] . figure 53 ) presented a remarkable anticoagulant activity (pt = 41.2s and tt 128.5s) and no significant hepatic or renal toxicity when compared to warfarin (pt = 55.7s and tt 80.6s) [203] . although further studies are necessary to understand the mode of action of compound 157, it could be a promising anticoagulant agent for preclinical studies. figure 53 ) presented a remarkable anticoagulant activity (pt = 41.2s and tt 128.5s) and no significant hepatic or renal toxicity when compared to warfarin (pt = 55.7s and tt 80.6s) [203] . although further studies are necessary to understand the mode of action of compound 157, it could be a promising anticoagulant agent for preclinical studies. diabetes is a chronic metabolic disease characterized by high blood sugar levels and is generally caused by an insufficient production of insulin by β-pancreatic cells or by the inability of human body to use this hormone. the consequences of diabetes might be very serious: blindness, kidney failure, stroke, heart attacks, lower limb amputation [204] [205] [206] diabetes is a chronic metabolic disease characterized by high blood sugar levels and is generally caused by an insufficient production of insulin by β-pancreatic cells or by the inability of human body to use this hormone. the consequences of diabetes might be very serious: blindness, kidney failure, stroke, heart attacks, lower limb amputation [204] [205] [206] menteşe et al. synthesized a novel series of n -(2-(3,5-disubstituted-4h-1,2,4-triazol-4-yl)acetyl)-6/7/8 substituted-2-oxo-2h-chromen-3-carbohydrazides (158a-e, 159a-e, figure 54 ) [207] , merging the 1,2,4-triazole and the coumarin moieties, both characterized by a wide range of biological activities (including inhibition of α-glucosidases) and low toxicity profiles [208] [209] [210] [211] [212] [213] . then, their activity on α-glucosidases was studied, evaluating the enzyme inhibition in the presence of pnpg (p-nitrophenyl-α-d-glucopyranoside) as a substrate in the buffer (ph 6.8). among the new compounds, four molecules showed high inhibition activity, compared to acarbose (ic 50 = 8.85 ± 0.23 µg/ml): 158d (ic 50 = 4.28 ± 0.10 µg/ml), 158e (ic 50 = 0.96 ± 0.02 µg/ml), 159d (ic 50 = 6.75 ± 0.10 µg/ml) and 159e (ic 50 = 1.44 ± 0.06 µg/ml). diabetes is a chronic metabolic disease characterized by high blood sugar levels and is generally caused by an insufficient production of insulin by β-pancreatic cells or by the inability of human body to use this hormone. the consequences of diabetes might be very serious: blindness, kidney failure, stroke, heart attacks, lower limb amputation [204] [205] [206] menteşe et al. synthesized a novel series of n'-(2-(3,5-disubstituted-4h-1,2,4-triazol-4-yl)acetyl)-6/7/8 substituted-2-oxo-2h-chromen-3-carbohydrazides (158a-e, 159a-e, figure 54 ) [207] , merging the 1,2,4-triazole and the coumarin moieties, both characterized by a wide range of biological activities (including inhibition of α-glucosidases) and low toxicity profiles [208] [209] [210] [211] [212] [213] . then, their activity on α-glucosidases was studied, evaluating the enzyme inhibition in the presence of pnpg (p-nitrophenyl-α-d-glucopyranoside) as a substrate in the buffer (ph 6.8). among the new compounds, four molecules showed high inhibition activity, compared to acarbose (ic50 = 8.85 ± 0.23 μg/ml): 158d (ic50 = 4.28 ± 0.10 μg/ml), 158e (ic50 = 0.96 ± 0.02 μg/ml), 159d (ic50 = 6.75 ± 0.10 μg/ml) and 159e (ic50 = 1.44 ± 0.06 μg/ml). compounds 158e and 159e resulted the most active, probably because of the metoxy-group at position 8 of the coumarin ring. derivatives without substituents on positions 3 and 5 of the phenyl ring linked to the triazole nucleus resulted more active than compounds bearing a chlorine atom or a phenyl moiety on such positions. according to kinetic studies, the tested compounds inhibit αglucosidases in a competitive way. other studies focused on coumarins-mediated inhibition of αglucosidases were carried out by different groups. hu and collaborators synthesized through microwave radiation heating a new series of more than forty 3-arylcoumarins which were screened for antioxidant activity, α-glucosidases inhibition and advanced glycation end-products (ages) formation inhibition [214] . only eight of the synthesized compounds (160-167, figure 55 ) exhibited moderate to high inhibitory activity on α-glucosidase. compounds 158e and 159e resulted the most active, probably because of the metoxy-group at position 8 of the coumarin ring. derivatives without substituents on positions 3 and 5 of the phenyl ring linked to the triazole nucleus resulted more active than compounds bearing a chlorine atom or a phenyl moiety on such positions. according to kinetic studies, the tested compounds inhibit α-glucosidases in a competitive way. other studies focused on coumarins-mediated inhibition of α-glucosidases were carried out by different groups. hu and collaborators synthesized through microwave radiation heating a new series of more than forty 3-arylcoumarins which were screened for antioxidant activity, α-glucosidases inhibition and advanced glycation end-products (ages) formation inhibition [214] . only eight of the synthesized compounds (160-167, figure 55 ) exhibited moderate to high inhibitory activity on α-glucosidase. compound 165 was the most promising (ic50 = 1.37 ± 0.67 μm), with a α-glucosidase inhibitory activity slightly weaker than acarbose (ic50 = 0.050 ± 0.003 μm). from an extensive sar study, it emerged that the 7-hydroxy group is important for the inhibition of α-glucosidase. compounds 160, 163,164, 165 and 166 were then tested in vivo: none of them showed toxicity on mice (ld50 > 5000 mg/kg). the mentioned compounds were also tested on streptozocin-induced diabetic mice. compound 166 showed the best profile, exhibiting a strong reduction of glucose blood levels. oral daily administration of 166 ( compound 165 was the most promising (ic 50 = 1.37 ± 0.67 µm), with a α-glucosidase inhibitory activity slightly weaker than acarbose (ic 50 = 0.050 ± 0.003 µm). from an extensive sar study, it emerged that the 7-hydroxy group is important for the inhibition of α-glucosidase. compounds 160, 163, 164, 165 and 166 were then tested in vivo: none of them showed toxicity on mice (ld 50 > 5000 mg/kg). the mentioned compounds were also tested on streptozocin-induced diabetic mice. compound 166 showed the best profile, exhibiting a strong reduction of glucose blood levels. oral daily administration of 166 (30 mg/kg/day) restored glucose blood levels near normal values, showing an effect similar to that of the oral antidiabetic glibenclamide. asgari and co-workers synthesized a new series of biscoumarin-1,2,3-triazole derivatives ( figure 56 ) and evaluated their α-glucosidase inhibitory potential, using acarbose as a reference drug [215] . compound 165 was the most promising (ic50 = 1.37 ± 0.67 μm), with a α-glucosidase inhibitory activity slightly weaker than acarbose (ic50 = 0.050 ± 0.003 μm). from an extensive sar study, it emerged that the 7-hydroxy group is important for the inhibition of α-glucosidase. compounds 160, 163,164, 165 and 166 were then tested in vivo: none of them showed toxicity on mice (ld50 > 5000 mg/kg). the mentioned compounds were also tested on streptozocin-induced diabetic mice. compound 166 showed the best profile, exhibiting a strong reduction of glucose blood levels. oral daily administration of 166 (30 mg/kg/day) restored glucose blood levels near normal values, showing an effect similar to that of the oral antidiabetic glibenclamide. asgari and co-workers synthesized a new series of biscoumarin-1,2,3-triazole derivatives ( figure 56 ) and evaluated their α-glucosidase inhibitory potential, using acarbose as a reference drug [215] . here, again, two active moieties, both characterized by a wide range of biological activities, were merged together: the bis-coumarin and the 1,2,3-triazole moieties [216] . all the synthesized compounds showed excellent activities (ic50 between 13.0 ± 1.5 and 75.5 ± 7.0 μm) compared to acarbose (ic50 750.0 ± 12.0). compound 168c, bearing 2-chloro phenyl moiety, resulted the most active. the substitution of the chlorine atom with a methyl group or its shift on the c4 position caused a decrease in activity. moreover, the inhibitory activity seemed to depend importantly on the electron properties of the substituents. from further kinetic studies it emerged that compound 168c inhibits α-glucosidases in a competitive mode (ki = 11 μm). a different therapeutic approach may be the stimulation of insulin secretion. in this perspective, ahmed and collaborators extracted from the aerial parts of clutia lanceolata (a medicinal plant native to sub-saharan africa and the arabian peninsula) twenty-one coumarins, including methyltio-and methylsulfinil-coumarins, thirteen of which were reported for the first time [217] . the structures of these natural compounds were elucidated from 2d-nmr and ms spectra, whereas their anti-diabetic activity was tested measuring the glucose-triggered insulin secretion of freshly isolated murine islets. here, again, two active moieties, both characterized by a wide range of biological activities, were merged together: the bis-coumarin and the 1,2,3-triazole moieties [216] . all the synthesized compounds showed excellent activities (ic 50 between 13.0 ± 1.5 and 75.5 ± 7.0 µm) compared to acarbose (ic 50 750.0 ± 12.0). compound 168c, bearing 2-chloro phenyl moiety, resulted the most active. the substitution of the chlorine atom with a methyl group or its shift on the c4 position caused a decrease in activity. moreover, the inhibitory activity seemed to depend importantly on the electron properties of the substituents. from further kinetic studies it emerged that compound 168c inhibits α-glucosidases in a competitive mode (k i = 11 µm). a different therapeutic approach may be the stimulation of insulin secretion. in this perspective, ahmed and collaborators extracted from the aerial parts of clutia lanceolata (a medicinal plant native to sub-saharan africa and the arabian peninsula) twenty-one coumarins, including methyltio-and methylsulfinil-coumarins, thirteen of which were reported for the first time [217] . the structures of these natural compounds were elucidated from 2d-nmr and ms spectra, whereas their anti-diabetic activity was tested measuring the glucose-triggered insulin secretion of freshly isolated murine islets. the applications and properties of coumarin scaffold have remarkably wide boundaries. coumarin-based compounds have been exploited in numerous research and industrial sectors, as active pharmaceutical ingredients, pesticides, fragrances, dyes for several purposes from laser the applications and properties of coumarin scaffold have remarkably wide boundaries. coumarin-based compounds have been exploited in numerous research and industrial sectors, as active pharmaceutical ingredients, pesticides, fragrances, dyes for several purposes from laser technology to organic photoredox catalysis, cell imaging, photocleavable protecting groups and fluorescent biological probes [6, [218] [219] [220] [221] [222] [223] [224] [225] . in the following paragraphs, the most recent applications associated with the photophysical properties of coumarins have been reviewed. the development of a suitable formulation is a crucial step in order to achieve new functional therapeutics. the design of novel strategies aimed at selectively release the bioactive in a specific district at a determinate time to maximize efficacy and reduce off-target adverse effects represents an extremely active research frontline. so far, various stimuli-responsive systems have been considered in therapeutic approaches to regulate the release of the therapeutic cargo, including endogenous stimuli (e.g., ph, enzymes, redox reactions, etc.) and exogenous stimuli (e.g., light, magnetic field, ionizing radiations, etc.) [226, 227] . light-mediated therapies have shown excellent results in achieving on-demand therapeutics and optical tools for studying and controlling complex chemical and biological processes in localized areas, owing to their superior non-invasiveness and spatiotemporal precision upon applying a specific light-irradiation wavelength [227] [228] [229] . one method for the regulation of molecular processes with light is the use of photolabile "protecting" groups in key locations. ideally, this modification completely blocks the activity of any molecule and restores it only with light [230] . coumarins, particularly 4-hydroxymethyl derivatives, are known to undergo photolysis. keeping this concept in mind, several biomolecules of interest have been linked to the coumarin nucleus, mostly as acyl derivatives. then, under uv irradiation, the biomolecules can be released in biological systems. the photophysical parameters of the formed derivatives are determined by different factors as the mode of fusion, the chemical nature of additional rings and the presence of electron-donating and electron-withdrawing substituents [12] . fournier and co-workers in 2013 proposed a series of methyl-coumarins with redshifted absorption. in particular, three compounds (172-174, figure 58 ), were synthetically easily accessible and exhibited a significant action cross section for uncaging with blue-cyan light, whereas their uncaging ability in the uv spectral domain remained low in order to avoid their photoactivation when a properly tuned uv illumination is applied [231] . in the same year, fournier and co-workers further proved that compound 172 was a good blue-absorbing caging group, owing to its strongly donating substituent conjugated to the thiocarbonyl group. moreover, the research team demonstrated that this particular caging group could be used in zebrafish embryos in the context of development biology to perform chromatic orthogonal photoactivation of two biologically active species [232] . in 2017, gandioso and colleagues reported the development of green/red-absorbing chromophores based on coumarin scaffolds that could be useful as photocleavable protecting groups [224] . a series of coumarin derivatives in which the carbonyl of the lactone was replaced by a cyano(4-nitrophenyl)methylene moiety, by condensation of a thiocoumarin precursor with the corresponding arylacetonitrile derivatives, was synthesized and subsequently refined with the insertion of electro-withdrawing groups at the phenyl ring, leading to absorption in the green to red region (175, figure 58 ) [224] . the insertion of more than one electro-withdrawing group (such as -no 2 and -cn) decreased the fluorescence emission, whereas the mononitro-containing coumarin derivatives had a strong emission in the red region upon excitation with green light, as denoted by their significantly large stokes shifts. in order to demonstrate the utility of these new compounds as ppgs, a small collection of coumarin-based photocages of benzoic acid was prepared. thanks to photolysis studies with green light, it was demonstrated that the structure of the coumarin chromophore influenced the rate of the uncaging process. this observation gave the opportunity to exploit these new coumarin scaffolds as caging groups removable with visible light. on the other hand, bojtar and colleagues proposed water soluble red-shifted coumarin caging groups (176-178, figure 58 ), activated with green-light [233] . 58) [224] . the insertion of more than one electro-withdrawing group (such as -no2 and -cn) decreased the fluorescence emission, whereas the mononitro-containing coumarin derivatives had a strong emission in the red region upon excitation with green light, as denoted by their significantly large stokes shifts. in order to demonstrate the utility of these new compounds as ppgs, a small collection of coumarin-based photocages of benzoic acid was prepared. thanks to photolysis studies with green light, it was demonstrated that the structure of the coumarin chromophore influenced the rate of the uncaging process. this observation gave the opportunity to exploit these new coumarin scaffolds as caging groups removable with visible light. on the other hand, bojtar and colleagues proposed water soluble red-shifted coumarin caging groups (176-178, figure 58 ), activated with green-light [233] . the optical properties of coumarins as photo-responsive unites could be also applied to polymers that after a photochemical activation rapidly degrade into small molecules. in 2018, iturmendi and co-workers proposed that, through functionalization of polyphosphazenes with a coumarin-caged amino acid as a pendant group along the backbone, the sensitivity of the polymers to hydrolysis would be accelerated upon irradiation and effectively catalyze its own degradation [234] . coumarins possess a large electron-rich π-π conjugated system with charge transfer properties, reason why coumarin-based fluorophores are widely used for monitoring a variety of biologically important species and biochemical process in living cells, for example as diagnostic agent for detection of biothiols, enzymes, mitochondrial ph values, glucose and ions [3, 222, 235] . in particular, several coumarin scaffolds have been proposed and evaluated for the detection of ions in different fields, from cellular imaging to environmental waters. gong and co-workers based their work on an easily synthesized coumarin-based fluorescent probe (179, figure 59 ) that already was effective in the detection of glutathione (ghs) in the presence of cu 2+ ions, expanding its potentiality to the detection of hypochlorite ions with high selectivity and sensitivity. the probe showed a remarkable fluorescent intensity change in response to hypochlorite ions; moreover, this probe could be applied to detect cloin cells via intracellular fluorescent imaging [236, 237] . given the importance of hypochlorite the optical properties of coumarins as photo-responsive unites could be also applied to polymers that after a photochemical activation rapidly degrade into small molecules. in 2018, iturmendi and co-workers proposed that, through functionalization of polyphosphazenes with a coumarin-caged amino acid as a pendant group along the backbone, the sensitivity of the polymers to hydrolysis would be accelerated upon irradiation and effectively catalyze its own degradation [234] . coumarins possess a large electron-rich π-π conjugated system with charge transfer properties, reason why coumarin-based fluorophores are widely used for monitoring a variety of biologically important species and biochemical process in living cells, for example as diagnostic agent for detection of biothiols, enzymes, mitochondrial ph values, glucose and ions [3, 222, 235] . in particular, several coumarin scaffolds have been proposed and evaluated for the detection of ions in different fields, from cellular imaging to environmental waters. gong and co-workers based their work on an easily synthesized coumarin-based fluorescent probe (179, figure 59 ) that already was effective in the detection of glutathione (ghs) in the presence of cu 2+ ions, expanding its potentiality to the detection of hypochlorite ions with high selectivity and sensitivity. the probe showed a remarkable fluorescent intensity change in response to hypochlorite ions; moreover, this probe could be applied to detect clo − in cells via intracellular fluorescent imaging [236, 237] . given the importance of hypochlorite ions both in living systems (being one of the biologically most important reactive oxygens species) and in the environment (owing to its use as disinfectant), in 2019, shangguan and colleagues proposed another probe for this particular ion based on coumarin dye and malononitrile (180, figure 59 ). the fluorescence response of probe 180 at 459 nm towards hypochlorite ions gradually enhanced with the increase of clo − concentrations, resulting in 45-fold fluorescence enhancement. furthermore, probe 180 exhibited high accuracy for quantitative measurement of hypochlorite ions in real water samples and it can be used as a potential chemosensor for the detection of clo − in chemical environmental and biological systems [238] . in the same year, tang and co-workers worked on a coumarin based fluorescent probe (181, figure 59 ) able of rapidly discern hypochlorite and copper(ii) ions in water sample and biological systems. upon the reaction with clo − , the fluorescence wavelength of 181 displayed a strong blue shift along with the naked-eye visible changes from yellow to colorless. moreover, it exhibited an obvious fluorescence quenching behavior to copper(ii) with colorimetric analysis from yellow to luminous yellow [239] . because of the presence of the 7-diethylamino group and the 3-substituted lactone ring that are wellknown structural pattern accountable for the fluorescence properties. it was observed a different reactivity profile depending on the ph levels, probably due to the different reactivity of hypochlorite ions ascribable to the variation of the dissociation of the salt at different ph values. afterwards, a deeply investigation on the possible formation of chlorinated derivatives was conducted: hplc-pda-esi-ms analyses highlighted the presence of chlorinated derivatives and proved that the chlorination reaction was responsible for the linear fluorescence decays. the results suggest the possibility to exploit these coumarin ionic probes for the detection and quantitative determination of hypochlorite species in vivo. a coumarin fluorescent probe based on a nitro-3-carboxamide derivative for selective copper (ii) ions detection was reported by bekhradnia and colleagues. compound 185 (figure 60 ) showed the highest fluorescence intensity in presence of cu 2+ compared to a variety of other common heavy and toxic metal ions (for example pb(ii), co(ii), hg(ii)) and in aqueous solution at 320 nm [241] . another approach was attempted for the selective detection of copper (ii) ions by he et al. in 2018, which based the fluorescent probe on a coumarin-schiff base derivative (186, figure 60 ). this probe resulted to be particularly selective for cu 2+ even in the presence of several other ions [242] . saravana a noteworthy research on the mechanism of interaction between coumarin based ionic probes and hypochlorite ions had been conducted by starzak and collaborators [240] . first, the research team confirmed the linear decrease in the fluorescence emissions together with the increase in clo − concentration of three different coumarin derivatives, 182-184 (figure 59 ), which were selected because of the presence of the 7-diethylamino group and the 3-substituted lactone ring that are well-known structural pattern accountable for the fluorescence properties. it was observed a different reactivity profile depending on the ph levels, probably due to the different reactivity of hypochlorite ions ascribable to the variation of the dissociation of the salt at different ph values. afterwards, a deeply investigation on the possible formation of chlorinated derivatives was conducted: hplc-pda-esi-ms analyses highlighted the presence of chlorinated derivatives and proved that the chlorination reaction was responsible for the linear fluorescence decays. the results suggest the possibility to exploit these coumarin ionic probes for the detection and quantitative determination of hypochlorite species in vivo. a coumarin fluorescent probe based on a nitro-3-carboxamide derivative for selective copper (ii) ions detection was reported by bekhradnia and colleagues. compound 185 ( figure 60 ) showed the highest fluorescence intensity in presence of cu 2+ compared to a variety of other common heavy and toxic metal ions (for example pb(ii), co(ii), hg(ii)) and in aqueous solution at 320 nm [241] . another approach was attempted for the selective detection of copper (ii) ions by he et al. in 2018, which based the fluorescent probe on a coumarin-schiff base derivative (186, figure 60 ). this probe resulted to be particularly selective for cu 2+ even in the presence of several other ions [242] . saravana mani and colleagues designed in 2019 a coumarin hydrazine-based fluorescent probe for the detection of copper(ii), called benzepyr (187, figure 60 ), exploiting a reaction of condensation between 2-hydrazino benzothiazole and n,n -diethylamino-3-acetyl coumarin [243] . this particular fluorescent chemosensor could selectively detect cu 2+ among other disturbing metal ions, resulting particularly specific and highly responsive, with a visible colorimetric change of the solution, which turned from yellow to wine red. moreover, the limit of detection (lod) had been estimated to be 40 nm. benzepyr 187 was also tested for the fluorescence bioimaging of cu 2+ ions in hela cells using fluorescence microscopic analysis, resulting suitable for the exploitation as an ion marker in living cells. but also al ions and amino acids lys and arg [244] . in particular, the detection of lys and arg took place with a colorimetric (from yellow to colorless) and a fluorescent response (from a maximum absorption at 335 nm to 429 nm). at the same time the probe detected either way the presence of cu 2+ ions but could be used only for the fluorescent sensing of al 3+ . a further interesting exploitation of probe 188 (figure 60 ) was the fluorescent and colorimetric identification of cys, hcy and gsh when it was complexed with copper ions. despite the key role of chemical species like copper and hypochlorite, they are not the only ions valuable for detection. for this reason, a dual coumarin probe, fluorescent and colorimetric, was designed by chen and colleagues for the detection of palladium (ii) ions that can be used in living cells. this oxime-ether coumarin probe (189, figure 61 ) exhibited a strong green fluorescence with an emission peak at 500 nm. when palladium(ii) was added to the solution with compound 189, the fluorescence intensity at 500 nm decreased consequently, until 2 equivalents of pd 2+ were reached; at that point the fluorescence was almost completely quenched, which could clearly be observed with the naked eye. a linear fit between fluorescence and palladium (ii) concentration was observed in the range 0.0-8.0 μm, while the detection limit was measured to be 40 nm, which is far lower than the threshold for palladium content in drugs (5.0 ppm to 10.0 ppm -47.0 mm to 94.0 mm) specified by the world health organization [245] . it is also noteworthy the development of thioacetalised coumarin based fluorescent probes for the detection of mercury (ii), a hazardous ion both for human health and reproduction. cheng and co-workers exploited the known hg 2+ promoted deprotection reaction of dithioacetals to design two novel reactive fluorescent probes (190,191, figure 61 ) that showed a different behavior due to the different chemical structures: 190 displayed remarkable fluorescence quenching with the addition of another remarkable case of coumarin-based chemosensor had been presented by li and co-workers, who synthesized a multifunctional probe able to selectively detect not only copper (ii) ions but also al 3+ ions and amino acids lys and arg [244] . in particular, the detection of lys and arg took place with a colorimetric (from yellow to colorless) and a fluorescent response (from a maximum absorption at 335 nm to 429 nm). at the same time the probe detected either way the presence of cu 2+ ions but could be used only for the fluorescent sensing of al 3+ . a further interesting exploitation of probe 188 (figure 60 ) was the fluorescent and colorimetric identification of cys, hcy and gsh when it was complexed with copper ions. despite the key role of chemical species like copper and hypochlorite, they are not the only ions valuable for detection. for this reason, a dual coumarin probe, fluorescent and colorimetric, was designed by chen and colleagues for the detection of palladium (ii) ions that can be used in living cells. this oxime-ether coumarin probe (189, figure 61 ) exhibited a strong green fluorescence with an emission peak at 500 nm. when palladium(ii) was added to the solution with compound 189, the fluorescence intensity at 500 nm decreased consequently, until 2 equivalents of pd 2+ were reached; at that point the fluorescence was almost completely quenched, which could clearly be observed with the naked eye. a linear fit between fluorescence and palladium (ii) concentration was observed in the range 0.0-8.0 µm, while the detection limit was measured to be 40 nm, which is far lower than the threshold for palladium content in drugs (5.0 ppm to 10.0 ppm -47.0 mm to 94.0 mm) specified by the world health organization [245] . it is also noteworthy the development of thioacetalised coumarin based fluorescent probes for the detection of mercury (ii), a hazardous ion both for human health and reproduction. cheng and co-workers exploited the known hg 2+ promoted deprotection reaction of dithioacetals to design two novel reactive fluorescent probes (190, 191, figure 61 ) that showed a different behavior due to the different chemical structures: 190 displayed remarkable fluorescence quenching with the addition of hg 2+ ions while, in the presence of mercury ions, 191 displayed ratiometric fluorogenic and chromogenic response [246] . it should not be forgotten an on-off fluorescent probe for the tracking of iron (iii) ions based on 7-hydroxy-2-oxo-n-(pyridin-2-ylmethyl)chromene-3-carboxamide. in their work, warrier and kharkar demonstrated that compound 192 (figure 61 ) was selective towards fe 3+ ions and exhibited high fluorescence emission profile at 447 nm. the presence of other ions did not interfere with the detection of iron (iii) ions and the limit of detection was found to be 0.76 µm. moreover, cell imaging and mtt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay proved the potential utility of probe 192 as cell-permeable chemosensor of fe 3+ in living cells [247] . the approach of jiao and colleagues for the detection of fluoride ions was based on the linkage between the coumarin scaffold and fluorescein in order to obtain a highly selective and sensitive fluorescent probe. the mechanism of f − ions detection by compound 193 (figure 61 ) was explained and involved a desilylation reaction in the presence of fluoride ions. moreover, a linear relationship between the ratio of emission intensities at 532 and 465 nm and f − concentration over the range of 0-20 µm with a limit of detection of 0.025 µm was found [248] . differently, yao and co-workers exploited the capability of fluoride ions to form stable complex with ca 2+ to design a novel fluorescent sensor (194, figure 61) , synthesized from the combination of mandelic acid with 7-hydroxy-8-formylcoumarin through a hydrazine hydrate bridge, in order to selectively identify these two ionic species over other metal ions [249] . the fluorescence spectrum of compound 194 clearly increased when calcium ions were added to the solution with a limit of detection of 5.81 × 10 −7 m, while, once the complex between the probe and ca 2+ ions was obtained, the addition of fluoride ions to the solution lead to the turn-off of the fluorescence response with a limit of detection 4.28 × 10 −7 m. moreover, bio-imaging studies were performed in order to assure the possibility to exploit this novel chemosensor for the identification of ca 2+ and f − ions in vivo, with positive outcome. finally, reddy and choi designed and synthesized three dicyanovinylcoumarin probes as turn-on fluorescent sensor for the detection of cn − ions among other anions [250] . within the different synthesized probes only compound 195 ( figure 61 ) showed a remarkable increase of the fluorescence in presence of fluoride and cyanide ions with an interesting sensibility towards cn − ions: the limit of detection was up to 11.4 nm, lower than the maximum level in drinkable water according to who guidelines. high fluorescence emission profile at 447 nm. the presence of other ions did not interfere with the detection of iron (iii) ions and the limit of detection was found to be 0.76 μm. moreover, cell imaging and mtt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay proved the potential utility of probe 192 as cell-permeable chemosensor of fe 3+ in living cells [247] . the approach of jiao and colleagues for the detection of fluoride ions was based on the linkage between the coumarin scaffold and fluorescein in order to obtain a highly selective and sensitive fluorescent probe. the mechanism of fions detection by compound 193 (figure 61 ) was explained and involved a desilylation reaction in the presence of fluoride ions. moreover, a linear relationship between the ratio of emission intensities at 532 and 465 nm and f − concentration over the range of 0-20 μμ with a limit of detection of 0.025 μμ was found [248] . differently, yao and co-workers exploited the capability of fluoride ions to form stable complex with ca 2+ to design a novel fluorescent sensor (194, figure 61) , synthesized from the combination of mandelic acid with 7-hydroxy-8formylcoumarin through a hydrazine hydrate bridge, in order to selectively identify these two ionic species over other metal ions [249] . the fluorescence spectrum of compound 194 clearly increased when calcium ions were added to the solution with a limit of detection of 5.81 × 10 -7 m, while, once the complex between the probe and ca 2+ ions was obtained, the addition of fluoride ions to the solution lead to the turn-off of the fluorescence response with a limit of detection 4.28 × 10 -7 m. moreover, bio-imaging studies were performed in order to assure the possibility to exploit this novel chemosensor for the identification of ca 2+ and fions in vivo, with positive outcome. finally, reddy and choi designed and synthesized three dicyanovinylcoumarin probes as turnon fluorescent sensor for the detection of cnions among other anions [250] . within the different synthesized probes only compound 195 ( figure 61 ) showed a remarkable increase of the fluorescence in presence of fluoride and cyanide ions with an interesting sensibility towards cnions: the limit of detection was up to 11.4 nm, lower than the maximum level in drinkable water according to who guidelines. coumarins have found important applications also in the agri-food sector. in fact, the antimicrobial activity characterizing these natural compounds could be exploited for food coumarins have found important applications also in the agri-food sector. in fact, the antimicrobial activity characterizing these natural compounds could be exploited for food preservation or for the treatment of plant pathogens, infections in aquaculture or biofouling caused by eukaryotic organisms. in addition, because coumarin scaffold has been used in fluorescent probing, natural coumarins might be used in the detection of some substances in food samples. for instance, zhang and collaborators developed a new near-infrared probe constituted by a conjugated coumarin-indolium system, for rapid, colorimetric and ratiometric fluorescent detection of bisulfite and sulfite anions [251] . (bi)sulfite anions (hso 3 − /so 3 2− ) are widely used as preservative for foods and beverages in order to prevent oxidation, browning and microbial reaction during products' life cycle [252, 253] . unfortunately, high doses of (bi)sulfites can cause asthma or other allergic reactions. some individuals are very sensitive even to low levels of these anions [254] . in addition, sulfur dioxide (so 2 ) is one of the most distributed pollutants and it has a relevant impact on human health [252] , [253, 255] . an efficient tool to detect such molecules is provided by fluorescent probes; to date, different probes for hso 3 − /so 3 2− have been designed but many of them are intensity-based, which means that the signal output can be conditioned by different factors such as instrumental efficiency, probe concentration, environmental conditions. in addition, many of these probes show emissions only in the visible region and some of them need ultraviolet excitation, having limited biological applications. thus, new, more efficient probes are required. in zhang's work, a conjugated coumarin-indolium system with intramolecular charge transfer (ict) effect was developed, merging an electron-donating 7-diethylamino coumarin moiety and an electron-withdrawing positively charged indolium derivative, connected through an ethylene linker (figure 62 ). the whole system resulted in a typical 'push-pull' large conjugation dye system with ict properties. the so-designed probe showed nir fluorescence (667 nm) and a rapid, highly selective and sensitive detection process for hso 3 − /so 3 2− in aqueous solution under mild conditions; in addition, this probe exhibited significant colorimetric, nir fluorescent and ratiometric signal responses upon one excitation wave and a detection lower limit of ∼27 nm. this probe can be applied to detect hso 3 − /so 3 2− in real food samples, serum samples and living cells. when tested on real food samples (sugar, soft sugar, crystal sugar and wine in aqueous solution) this probe proved to be able to determine hso 3 − with good recovery. in addition, a paper test strip was developed, simply by wetting a strip of neutral filter paper with a solution of the probe in methanol (200 µm figure 62 ) can be used to develop a cheap, easy-to-prepare and easy-to-use paper test strip system for the detection of (bi)sulfites. and sulfite anions [251] . (bi)sulfite anions (hso3 − /so3 2− ) are widely used as preservative for foods and beverages in order to prevent oxidation, browning and microbial reaction during products' life cycle [252, 253] . unfortunately, high doses of (bi)sulfites can cause asthma or other allergic reactions. some individuals are very sensitive even to low levels of these anions [254] . in addition, sulfur dioxide (so2) is one of the most distributed pollutants and it has a relevant impact on human health [252] , [253, 255] . an efficient tool to detect such molecules is provided by fluorescent probes; to date, different probes for hso3 − /so3 2− have been designed but many of them are intensity-based, which means that the signal output can be conditioned by different factors such as instrumental efficiency, probe concentration, environmental conditions. in addition, many of these probes show emissions only in the visible region and some of them need ultraviolet excitation, having limited biological applications. thus, new, more efficient probes are required. in zhang's work, a conjugated coumarinindolium system with intramolecular charge transfer (ict) effect was developed, merging an electron-donating 7-diethylamino coumarin moiety and an electron-withdrawing positively charged indolium derivative, connected through an ethylene linker ( figure 62 ). the whole system resulted in a typical 'push-pull' large conjugation dye system with ict properties. the so-designed probe showed nir fluorescence (667 nm) and a rapid, highly selective and sensitive detection process for hso3 − /so3 2-in aqueous solution under mild conditions; in addition, this probe exhibited significant colorimetric, nir fluorescent and ratiometric signal responses upon one excitation wave and a detection lower limit of ∼27 nm. this probe can be applied to detect hso3 − /so3 2-in real food samples, serum samples and living cells. when tested on real food samples (sugar, soft sugar, crystal sugar and wine in aqueous solution) this probe proved to be able to determine hso3 -with good recovery. in addition, a paper test strip was developed, simply by wetting a strip of neutral filter paper with a solution of the probe in methanol (200 μm) . the result was a deep blue test paper ready for use: when a hso3 − solution is spotted on the test paper, rapid color changes can be observed, even if many other ions are present in the sample. different colors correspond to different concentrations of hso3 − . thus, probe 196 ( figure 62 ) can be used to develop a cheap, easy-to-prepare and easy-to-use paper test strip system for the detection of (bi)sulfites. a more recent example is a probe developed by nair and collaborators, able to selectively detect the amphiphilic bisulfate ion (hso4 − ) in edible plant foods, dog urine and drugs [256] . bisulfate consumption normally takes place through the ingestion of different edible plants, such as cabbage, broccoli, brussels sprouts, horseradish or seeds (black and white mustard, for instance). these plants contain glucosinolates [257, 258] that are hydrolyzed in our organism by the enzyme myrosinase, thus producing bisulfate ion [259] . in addition, bisulfate salts of many apis are currently on market, constituting another source of bisulfate ions [260] . when trying to evaluate the actual concentration of hso4 − , it is important to take account of the deprotonation equilibrium between hso4 − and so4 2-, a more recent example is a probe developed by nair and collaborators, able to selectively detect the amphiphilic bisulfate ion (hso 4 − ) in edible plant foods, dog urine and drugs [256] . bisulfate consumption normally takes place through the ingestion of different edible plants, such as cabbage, broccoli, brussels sprouts, horseradish or seeds (black and white mustard, for instance). these plants contain glucosinolates [257, 258] that are hydrolyzed in our organism by the enzyme myrosinase, thus producing bisulfate ion [259] . in addition, bisulfate salts of many apis are currently on market, constituting another source of bisulfate ions [260] . when trying to evaluate the actual concentration of hso 4 − , it is important to take account of the deprotonation equilibrium between hso 4 − and so 4 2− , using a highly selective probe able to discriminate between these two ions. nair and co-workers developed two fully water-soluble probes, coumarin-integrated glycine (cg) and coumarin-integrated alanine (ca) zwitterions, for the selective detection of hso 4 − at picomolar level (from 50 to 325 pm) ( figure 63 ). int. j. mol. sci. 2020, 21, x for peer review 41 of 83 using a highly selective probe able to discriminate between these two ions. nair and co-workers developed two fully water-soluble probes, coumarin-integrated glycine (cg) and coumarinintegrated alanine (ca) zwitterions, for the selective detection of hso4 − at picomolar level (from 50 to 325 pm) ( figure 63 ). glycine and alanine can interact selectively with the target through h-bond, due to their zwitterionic nature at physiological ph, whereas the 7-hydroxycoumarin moiety constitutes a good fluorophore probe, thanks to its biocompatibility, non-toxicity and water solubility. the cg/ca probes proved to be able to penetrate and stain living cell. when different unknown concentrations of clopidogrel bisulfate were added to a water solution of cg/ca, it was possible to precisely measure such concentration by measuring the emission intensity of each sample. confirmation of the experimentally observed values with the theoretically calculated ones supported the accuracy of the presented method. cg/ca probes were also tested on food samples: water extracts of cruciferous plant foods (cabbage, broccoli, mustard seeds, carrots) were added to aqueous solutions of the probes. again, titration of bisulfate ions was performed by emission measurements-addition of increasing volumes of aqueous food saps caused a growing reduction of probe's emission. cucumber and fenugreek were used as controls, as they do not contain bisulfates. eventually, bisulfate content was measured in pet urine samples (bisulfate is one of the common components of pet foods): urine samples were collected from two adult dogs and were treated with aqueous solution of cg/ca; a reduction of probe emission was noticed, revealing the presence of hso4 − (quenching was proportional to bisulfate quantity). in conclusion, cg and ca probes demonstrated the ability to detect bisulfate ions in aqueous solution at ph = 7.4, wherein hso4 − concentration was 10 5.4 lower than that of so4 2-, at concentration as low as 50 pm, even in presence of other ions. with regards to the antimicrobial activity of coumarin scaffold, some studies have been recently carried out, showing the potential of natural coumarins as food preservatives. yang and co-workers have studied the antimicrobial activity of eighteen natural compounds against r. solanacearum [261] , a bacterium responsible for the wilting of different plants such as tobacco, tomato, potato in (sub)tropical regions, causing significant economic losses [262, 263] . among them, four coumarins ( figure 64 ) showed an antibacterial activity stronger than that of thiodiazole copper treatment (antibacterial rate (mbc/mic) of 63.3%). daphnetin showed the highest activity, followed by xanthotol and esculetin (antibacterial rate 97.43%, 80.12% and 71.44%, respectively). antibacterial activity seemed to be enhanced by c6, c7 or c8 substitution, so hydroxycoumarins umbelliferone, esculetin and daphnetin were selected for further investigation of the mechanism of action. hydroxycoumarins were tested from 10 to 100 mg/l concentrations and from results it was clear that the good activity of umbelliferone can be enhanced by the additional hydroxylation of c6 position (esculetin), whereas even better results can be achieved by the dihydroxylation of c7 and c8 positions (daphnetin). tem images of r. solanacearum showed that daphnetin and esculetin caused irreversible damages to the cell membrane, whereas umbelliferone must follow a different path in inducing cell damage. it is worth noting that hydroxycoumarins showed very low cytotoxicity on human cells and have no effects on tobacco seeds' germination. furthermore, because r. solanacearum forms biofilm-like aggregations on host's roots, facilitating bacterial infection [264] , yang and his group speculated that hydroxycoumarins may interfere with biofilm formation. in fact, daphnetin, umbelliferone and esculetin (100 mg/l) reduced biofilm formation by 99.22%, 85.20% and 93.90%, respectively, probably influencing the swimming motility of the bacterium. thus, the glycine and alanine can interact selectively with the target through h-bond, due to their zwitterionic nature at physiological ph, whereas the 7-hydroxycoumarin moiety constitutes a good fluorophore probe, thanks to its biocompatibility, non-toxicity and water solubility. the cg/ca probes proved to be able to penetrate and stain living cell. when different unknown concentrations of clopidogrel bisulfate were added to a water solution of cg/ca, it was possible to precisely measure such concentration by measuring the emission intensity of each sample. confirmation of the experimentally observed values with the theoretically calculated ones supported the accuracy of the presented method. cg/ca probes were also tested on food samples: water extracts of cruciferous plant foods (cabbage, broccoli, mustard seeds, carrots) were added to aqueous solutions of the probes. again, titration of bisulfate ions was performed by emission measurements-addition of increasing volumes of aqueous food saps caused a growing reduction of probe's emission. cucumber and fenugreek were used as controls, as they do not contain bisulfates. eventually, bisulfate content was measured in pet urine samples (bisulfate is one of the common components of pet foods): urine samples were collected from two adult dogs and were treated with aqueous solution of cg/ca; a reduction of probe emission was noticed, revealing the presence of hso 4 − (quenching was proportional to bisulfate quantity). in [262, 263] . among them, four coumarins ( figure 64 ) showed an antibacterial activity stronger than that of thiodiazole copper treatment (antibacterial rate (mbc/mic) of 63.3%). daphnetin showed the highest activity, followed by xanthotol and esculetin (antibacterial rate 97.43%, 80.12% and 71.44%, respectively). antibacterial activity seemed to be enhanced by c6, c7 or c8 substitution, so hydroxycoumarins umbelliferone, esculetin and daphnetin were selected for further investigation of the mechanism of action. hydroxycoumarins were tested from 10 to 100 mg/l concentrations and from results it was clear that the good activity of umbelliferone can be enhanced by the additional hydroxylation of c6 position (esculetin), whereas even better results can be achieved by the dihydroxylation of c7 and c8 positions (daphnetin). tem images of r. solanacearum showed that daphnetin and esculetin caused irreversible damages to the cell membrane, whereas umbelliferone must follow a different path in inducing cell damage. it is worth noting that hydroxycoumarins showed very low cytotoxicity on human cells and have no effects on tobacco seeds' germination. furthermore, because r. solanacearum forms biofilm-like aggregations on host's roots, facilitating bacterial infection [264] , yang and his group speculated that hydroxycoumarins may interfere with biofilm formation. in fact, daphnetin, umbelliferone and esculetin (100 mg/l) reduced biofilm formation by 99.22%, 85.20% and 93.90%, respectively, probably influencing the swimming motility of the bacterium. thus, the expression of the regulating and structural flagellar genes flia, flhc and flhd in presence of daphnetin, umbelliferone and esculetin was evaluated and it turned out that the expression of flia and flhc was significantly repressed by the mentioned compounds. expression of the regulating and structural flagellar genes flia, flhc and flhd in presence of daphnetin, umbelliferone and esculetin was evaluated and it turned out that the expression of flia and flhc was significantly repressed by the mentioned compounds. another interesting biological activity ascribed to the coumarin nucleus is its antifungal activity, which can be useful not only in the medicinal field but also in the agricultural one. as far as the latter is concerned, there has been an increasing interest in the exploitation of coumarin scaffold for the design and synthesis of novel fungicides useful in the treatment of many plant diseases. many pathogenic fungi still cause massive death of crops, limiting production and causing significant financial losses. to date, farmers deal with this problem by using chemical fungicides but such solution comes with some drawbacks: the long and extensive use of these chemicals may cause environmental pollution, accumulation of pesticides residues in the plants and drug resistance by fungi. thus, there is an urgent need for alternative (and green) solutions. in 2019, ramìrez-pelayo and his collaborators studied the coumarin-based compounds contained in the peel of citrus grown in colombia [265] . six coumarins were isolated (197-202, figure 65 ) and their antifungal activity, in terms of mycelial growth and spore germination inhibition, was evaluated against the fungus causing anthracnose (colletotrichum sp.). the production of oranges, mandarins, grapefruit, lemons, limes is limited by the action of microorganisms able to proliferate in such fruits because of peculiar condition such as high nutrient content, humidity and low ph values. nevertheless, these plants can defend themselves from pathogens thanks to the production of antimicrobial compounds which can be constitutive (phytoanticipins) or induced (phytoalexins) [266, 267] . another interesting biological activity ascribed to the coumarin nucleus is its antifungal activity, which can be useful not only in the medicinal field but also in the agricultural one. as far as the latter is concerned, there has been an increasing interest in the exploitation of coumarin scaffold for the design and synthesis of novel fungicides useful in the treatment of many plant diseases. many pathogenic fungi still cause massive death of crops, limiting production and causing significant financial losses. to date, farmers deal with this problem by using chemical fungicides but such solution comes with some drawbacks: the long and extensive use of these chemicals may cause environmental pollution, accumulation of pesticides residues in the plants and drug resistance by fungi. thus, there is an urgent need for alternative (and green) solutions. in 2019, ramìrez-pelayo and his collaborators studied the coumarin-based compounds contained in the peel of citrus grown in colombia [265] . six coumarins were isolated (197-202, figure 65 ) and their antifungal activity, in terms of mycelial growth and spore germination inhibition, was evaluated against the fungus causing anthracnose (colletotrichum sp.). the production of oranges, mandarins, grapefruit, lemons, limes is limited by the action of microorganisms able to proliferate in such fruits because of peculiar condition such as high nutrient content, humidity and low ph values. nevertheless, these plants can defend themselves from pathogens thanks to the production of antimicrobial compounds which can be constitutive (phytoanticipins) or induced (phytoalexins) [266, 267] . another interesting biological activity ascribed to the coumarin nucleus is its antifungal activity, which can be useful not only in the medicinal field but also in the agricultural one. as far as the latter is concerned, there has been an increasing interest in the exploitation of coumarin scaffold for the design and synthesis of novel fungicides useful in the treatment of many plant diseases. many pathogenic fungi still cause massive death of crops, limiting production and causing significant financial losses. to date, farmers deal with this problem by using chemical fungicides but such solution comes with some drawbacks: the long and extensive use of these chemicals may cause environmental pollution, accumulation of pesticides residues in the plants and drug resistance by fungi. thus, there is an urgent need for alternative (and green) solutions. in 2019, ramìrez-pelayo and his collaborators studied the coumarin-based compounds contained in the peel of citrus grown in colombia [265] . six coumarins were isolated (197-202, figure 65 ) and their antifungal activity, in terms of mycelial growth and spore germination inhibition, was evaluated against the fungus causing anthracnose (colletotrichum sp.). the production of oranges, mandarins, grapefruit, lemons, limes is limited by the action of microorganisms able to proliferate in such fruits because of peculiar condition such as high nutrient content, humidity and low ph values. nevertheless, these plants can defend themselves from pathogens thanks to the production of antimicrobial compounds which can be constitutive (phytoanticipins) or induced (phytoalexins) [266, 267] . as already mentioned, from the methanol extracts of c. latifolia and c. aurantifolia peels six compounds were isolated-5-geranyloxy-7-methoxycoumarin (197) , bergamottin (198) , bergapten (199) , isopimpinellin (200) , limettin (201) and oxypeucedanin hydrate (202) and their activity was compared to that of umbelliferone, scoparone and scopoletin. compounds 197-201 were tested at 0.25, 0.50 and 1.00 mm, whereas 202 was tested at 1.00 mm concentration. carbendazin and thymol were used as reference compounds (0.5 mm). the results showed that compounds 197-202 significantly inhibited mycelial growth of colletotrichum sp., the activity being proportional to the dose used. the (201)) was investigated, each of them showing enhanced activity respect to both the individual compounds. furthermore, the activity of 199, 201 and their mixtures was compared to that of the phytolaxins scoparone, scopoletin and umbelliferone; the results showed that phytolaxins were just slightly more active than the isolated compounds, although the highest fungistatic activity was shown by the mixture of 201 (0.75 mm) and 199 (0.25 mm). this mixture, along with compounds 197, 199 and 201, was selected for the evaluation of the inhibitory effect on spore germination in comparison with scopoletin, scoparone and umbelliferone. compounds 201 and 199 exhibited very good activity, with 96.7% and 95.3% inhibition, respectively but these percentages rapidly decrease, being less than 5% after 24 h. again, the 199/201 mixture showed the highest activity causing a complete, long-lasting inhibition of spore germination, thus suggesting that there may be an additive or synergistic effect between these compounds. therefore, coumarins and furanocoumarins may be useful scaffolds in the design of new antifungal agents. in 2018, yu and co-workers designed a series of coumarin-3-carboxamide derivatives and evaluated their antifungal activity against botrytis cinerea, alternaria solani, gibberella zeae, rhizoctonia solani, cucumber anthrax and alternaria leaf spot [268] . these phytopathogenic fungi are all relevant in agriculture, because they can cause significant losses in crops and investments. in this work, two different scaffolds were merged in order to design a new category of more effective fungicides. in fact, the use of carboxylic acid amides (caa) in this context is well-established, because amide fungicides have been used for over 50 years [269] . more recently, new amide fungicides with high activity and broad spectrum of action were developed (e.g., fluopyram, bixafen, sedaxane, isopyrazam, penthiopirad and boscalid) [1, 270] . in addition, hydrazide scaffolds have been found to have interesting biological and pharmacological activities [271, 272] . similarly, the antifungal activity of coumarin nucleus is well known (paragraph 0). considering these elements, the authors decided to combine the coumarin and the carboxamide/hydrazide scaffolds with the aim of synthesizing high-performance fungicides. all the synthesized compounds were tested and their antifungal activities against the mentioned phytopathogenic fungi were evaluated. at 100 µg/ml concentration, most of the tested compounds showed poor antifungal activity. only compounds 203 and 204 ( figure 66 ) showed ec 50 values of 1.57 µg/ml and 1.65 µg/ml, respectively, against botrytis cinerea, proving to be equivalent to the reference boscalid (0.51 µg/ml), whereas 203 (ec 50 = 1.80 µg/ml) resulted more active than boscalid (ec 50 = 2.98 µg/ml) against rhizoctonia solani. as already mentioned, from the methanol extracts of c. latifolia and c. aurantifolia peels six compounds were isolated-5-geranyloxy-7-methoxycoumarin (197) , bergamottin (198) , bergapten (199) , isopimpinellin (200) , limettin (201) and oxypeucedanin hydrate (202) and their activity was compared to that of umbelliferone, scoparone and scopoletin. compounds 197-201 were tested at 0.25, 0.50 and 1.00 mm, whereas 202 was tested at 1.00 mm concentration. carbendazin and thymol were used as reference compounds (0.5 mm). the results showed that compounds 197-202 significantly inhibited mycelial growth of colletotrichum sp., the activity being proportional to the dose used. the highest inhibition was exhibited by compounds 199 and 201 (32% and 25%, respectively); therefore, the fungistatic (201)) was investigated, each of them showing enhanced activity respect to both the individual compounds. furthermore, the activity of 199, 201 and their mixtures was compared to that of the phytolaxins scoparone, scopoletin and umbelliferone; the results showed that phytolaxins were just slightly more active than the isolated compounds, although the highest fungistatic activity was shown by the mixture of 201 (0.75 mm) and 199 (0.25 mm). this mixture, along with compounds 197, 199 and 201, was selected for the evaluation of the inhibitory effect on spore germination in comparison with scopoletin, scoparone and umbelliferone. compounds 201 and 199 exhibited very good activity, with 96.7% and 95.3% inhibition, respectively but these percentages rapidly decrease, being less than 5% after 24 hours. again, the 199/201 mixture showed the highest activity causing a complete, long-lasting inhibition of spore germination, thus suggesting that there may be an additive or synergistic effect between these compounds. therefore, coumarins and furanocoumarins may be useful scaffolds in the design of new antifungal agents. in 2018, yu and co-workers designed a series of coumarin-3-carboxamide derivatives and evaluated their antifungal activity against botrytis cinerea, alternaria solani, gibberella zeae, rhizoctonia solani, cucumber anthrax and alternaria leaf spot [268] . these phytopathogenic fungi are all relevant in agriculture, because they can cause significant losses in crops and investments. in this work, two different scaffolds were merged in order to design a new category of more effective fungicides. in fact, the use of carboxylic acid amides (caa) in this context is well-established, because amide fungicides have been used for over 50 years [269] . more recently, new amide fungicides with high activity and broad spectrum of action were developed (e.g., fluopyram, bixafen, sedaxane, isopyrazam, penthiopirad and boscalid) [1, 270] . in addition, hydrazide scaffolds have been found to have interesting biological and pharmacological activities [271, 272] . similarly, the antifungal activity of coumarin nucleus is well known (paragraph 0). considering these elements, the authors decided to combine the coumarin and the carboxamide/hydrazide scaffolds with the aim of synthesizing highperformance fungicides. all the synthesized compounds were tested and their antifungal activities against the mentioned phytopathogenic fungi were evaluated. at 100 μg/ml concentration, most of the tested compounds showed poor antifungal activity. only compounds 203 and 204 ( figure 66 ) showed ec50 values of 1.57 μg/ml and 1.65 μg/ml, respectively, against botrytis cinerea, proving to be equivalent to the reference boscalid (0.51 μg/ml), whereas 203 (ec50 = 1.80 μg/ml) resulted more active than boscalid (ec50 = 2.98 μg/ml) against rhizoctonia solani. starting from these data it was possible to suggest some structure-activity relationships: the replacement of amide bond with a hydrazide bond led to an improvement in antifungal activity spectrum. furthermore, coumarins bearing amide group resulted to be remarkably more active against botrytis cinerea and rhizoctonia solani, whereas against alternaria solani, gibberella zeae, starting from these data it was possible to suggest some structure-activity relationships: the replacement of amide bond with a hydrazide bond led to an improvement in antifungal activity spectrum. furthermore, coumarins bearing amide group resulted to be remarkably more active against botrytis cinerea and rhizoctonia solani, whereas against alternaria solani, gibberella zeae, cucumber anthrax and alternaria leaf spot they showed less potency. it is noteworthy that compounds bearing an electron-withdrawing group did not show any activity, whereas compounds with an electro-donating group were active against alternaria leaf spot. the addition to the coumarin nucleus of cl/f-substituted phenylidrazine gave compounds active on cucumber anthrax. a similar approach is the one followed by yang and collaborators, who decided to exploit coumarin nucleus for the functionalization of chitosan, in order to create more effective chitosan-based fungicides [273] . in fact, chitosan is becoming more and more appreciated due to its antimicrobial activity, low toxicity, biodegradability, biocompatibility and film forming activity. however, its application as fungicide is limited by its low solubility and weaker activity respect to the on-market fungicides. in this work, four coumarin-functionalized chitosan derivatives (205a-d, figure 67 ), were synthesized and tested against the phytopathogens alternaria solani sorauer, fusarium oxysporum f.sp. vasinfectum and fusarium moniliforme by evaluating the mycelial growth rate in vitro. at 1.0 mg/ml, compounds 205a-d showed inhibitory activity against a. solani, following the sequence 205d > 205b > 205c > 205a (38.25%, 51.23%, 44.01% and 52, 78%, respectively). cucumber anthrax and alternaria leaf spot they showed less potency. it is noteworthy that compounds bearing an electron-withdrawing group did not show any activity, whereas compounds with an electro-donating group were active against alternaria leaf spot. the addition to the coumarin nucleus of cl/f-substituted phenylidrazine gave compounds active on cucumber anthrax. a similar approach is the one followed by yang and collaborators, who decided to exploit coumarin nucleus for the functionalization of chitosan, in order to create more effective chitosan-based fungicides [273] . in fact, chitosan is becoming more and more appreciated due to its antimicrobial activity, low toxicity, biodegradability, biocompatibility and film forming activity. however, its application as fungicide is limited by its low solubility and weaker activity respect to the on-market fungicides. in this work, four coumarin-functionalized chitosan derivatives (205a-d, figure 67 ), were synthesized and tested against the phytopathogens alternaria solani sorauer, fusarium oxysporum f.sp. vasinfectum and fusarium moniliforme by evaluating the mycelial growth rate in vitro. at 1.0 mg/ml, compounds 205ad showed inhibitory activity against a. solani, following the sequence 205d > 205b > 205c > 205a (38.25%, 51.23%, 44.01% and 52, 78%, respectively). these results suggested that the introduction of halogens caused an increase in activity, depending on the number and the type of halogens. for instance, the 3,5-dichloro derivatives were more active against a. solani than the correspondent 5-cl or 5-br derivatives, probably because of an increase of hydrophobicity. the four coumarin-chitosan derivatives showed higher activity than chitosan alone also against f. oxysporum and f. moniliforme. compound 205d showed an inhibitory index of 57.09% against a. solani, 77.24% against f. oxysporum and 66.12% against f. moniliforme. the great biological potential of coumarins might be even increased by considering the possibility to complex coumarin scaffold with other substances as, for example, some metals. in fact, it has been proved that it is possible to enhance the activity of a certain drug simply binding it to a metallic-element [274] ; moreover, by combining known active moieties with metals, it would be possible to improve the parent compound with a certain selectivity or even with a new mechanism of action. therefore, many groups have already started to explore this strategy, aiming to produce more active compounds, exploiting metals as copper, platinum, zinc or silver. in some cases, due to its intrinsic potential, coumarin scaffold was exploited in this approach. some examples are discussed below. the nature of coumarin-copper complexes had already been evaluated in 2001 by karaliota and co-workers, who synthesized and characterized a binuclear coumarin-3-carboxilate-copper (ii) complex (206) [275] . in this work, thanks to multiple instrumental characterization by means of ir, raman and nmr spectroscopy, the authors were able to identify the structure of the complex ( figure 68 ): a binuclear molecule [cu2(cca)4(h2o)2], where copper is coordinated by four carboxylic oxygens, one from each cca molecule and two water molecules. these results suggested that the introduction of halogens caused an increase in activity, depending on the number and the type of halogens. for instance, the 3,5-dichloro derivatives were more active against a. solani than the correspondent 5-cl or 5-br derivatives, probably because of an increase of hydrophobicity. the four coumarin-chitosan derivatives showed higher activity than chitosan alone also against f. oxysporum and f. moniliforme. compound 205d showed an inhibitory index of 57.09% against a. solani, 77.24% against f. oxysporum and 66.12% against f. moniliforme. the great biological potential of coumarins might be even increased by considering the possibility to complex coumarin scaffold with other substances as, for example, some metals. in fact, it has been proved that it is possible to enhance the activity of a certain drug simply binding it to a metallic-element [274] ; moreover, by combining known active moieties with metals, it would be possible to improve the parent compound with a certain selectivity or even with a new mechanism of action. therefore, many groups have already started to explore this strategy, aiming to produce more active compounds, exploiting metals as copper, platinum, zinc or silver. in some cases, due to its intrinsic potential, coumarin scaffold was exploited in this approach. some examples are discussed below. the nature of coumarin-copper complexes had already been evaluated in 2001 by karaliota and co-workers, who synthesized and characterized a binuclear coumarin-3-carboxilate-copper (ii) complex (206) [275] . in this work, thanks to multiple instrumental characterization by means of ir, raman and nmr spectroscopy, the authors were able to identify the structure of the complex ( figure 68 ): a binuclear molecule [cu 2 (cca) 4 (h 2 o) 2 ], where copper is coordinated by four carboxylic oxygens, one from each cca molecule and two water molecules. figure 69 ) which were tested on mcf-7 human breast cancer cells to evaluate their cytotoxicity [276] . in fact, the increase of ros levels might promote oxidative stressinduced cancer cell death, thus representing an alternative strategy for the treatment of some forms of tumors [277] [278] [279] . interestingly, it was found that the activity of such compounds depended also on their speciation; for example, 207, monomeric at neutral ph, showed valuable activity, whereas 212 -a dinuclear specie-resulted inactive. moreover, their cytotoxicity was not related to their ability to induce ros generation. such activity was showed only by complex 207, whereas 210 and 212 exhibited limited ros induction. 211 and 212 proved to be able to bind dna but none of the tested compounds showed significant nuclease activity. this means that imine-copper(ii) complexes showing cytotoxicity towards mcf-7 cells do not act by means of ros generation or nuclease activity, being these two the mechanisms of action previously ascribed to copper (ii) complexes [280] . in fact, complexes 208-210 acted as superoxide dismutase (sod) and catalase mimics. this is noteworthy, if we consider that in some cancer cells hypoxia induces an adaptive mechanism providing protection against damage and oxidative stress. in this context, copper (ii)-imine complexes acting as sod mimics may constitute a valid tool for the treatment of hypoxic tumors. in the same year, qin and collaborators investigated the potential of platinum (ii) complexes against cisplatin resistant human lung adenocarcinoma (a549/ddp) and hela cells [281] . cisplatin was used as reference. in this work, eleven complexes between platinum (ii) and quinoline-coumarin derivatives (214-224, figure 70) were designed, synthesized and tested. all the new complexes showed an improved antineoplastic activity on a549/ddp and hela cells when compared to cisfigure 69 ) which were tested on mcf-7 human breast cancer cells to evaluate their cytotoxicity [276] . in fact, the increase of ros levels might promote oxidative stress-induced cancer cell death, thus representing an alternative strategy for the treatment of some forms of tumors [277] [278] [279] . interestingly, it was found that the activity of such compounds depended also on their speciation; for example, 207, monomeric at neutral ph, showed valuable activity, whereas 212 -a dinuclear specie-resulted inactive. moreover, their cytotoxicity was not related to their ability to induce ros generation. such activity was showed only by complex 207, whereas 210 and 212 exhibited limited ros induction. 211 and 212 proved to be able to bind dna but none of the tested compounds showed significant nuclease activity. this means that imine-copper(ii) complexes showing cytotoxicity towards mcf-7 cells do not act by means of ros generation or nuclease activity, being these two the mechanisms of action previously ascribed to copper (ii) complexes [280] . in fact, complexes 208-210 acted as superoxide dismutase (sod) and catalase mimics. this is noteworthy, if we consider that in some cancer cells hypoxia induces an adaptive mechanism providing protection against damage and oxidative stress. in this context, copper (ii)-imine complexes acting as sod mimics may constitute a valid tool for the treatment of hypoxic tumors. figure 69 ) which were tested on mcf-7 human breast cancer cells to evaluate their cytotoxicity [276] . in fact, the increase of ros levels might promote oxidative stressinduced cancer cell death, thus representing an alternative strategy for the treatment of some forms of tumors [277] [278] [279] . interestingly, it was found that the activity of such compounds depended also on their speciation; for example, 207, monomeric at neutral ph, showed valuable activity, whereas 212 -a dinuclear specie-resulted inactive. moreover, their cytotoxicity was not related to their ability to induce ros generation. such activity was showed only by complex 207, whereas 210 and 212 exhibited limited ros induction. 211 and 212 proved to be able to bind dna but none of the tested compounds showed significant nuclease activity. this means that imine-copper(ii) complexes showing cytotoxicity towards mcf-7 cells do not act by means of ros generation or nuclease activity, being these two the mechanisms of action previously ascribed to copper (ii) complexes [280] . in fact, complexes 208-210 acted as superoxide dismutase (sod) and catalase mimics. this is noteworthy, if we consider that in some cancer cells hypoxia induces an adaptive mechanism providing protection against damage and oxidative stress. in this context, copper (ii)-imine complexes acting as sod mimics may constitute a valid tool for the treatment of hypoxic tumors. in the same year, qin and collaborators investigated the potential of platinum (ii) complexes against cisplatin resistant human lung adenocarcinoma (a549/ddp) and hela cells [281] . cisplatin was used as reference. in this work, eleven complexes between platinum (ii) and quinoline-coumarin derivatives (214-224, figure 70) were designed, synthesized and tested. all the new complexes showed an improved antineoplastic activity on a549/ddp and hela cells when compared to cisin the same year, qin and collaborators investigated the potential of platinum (ii) complexes against cisplatin resistant human lung adenocarcinoma (a549/ddp) and hela cells [281] . cisplatin was used as reference. in this work, eleven complexes between platinum (ii) and quinoline-coumarin derivatives (214-224, figure 70) were designed, synthesized and tested. all the new complexes showed an improved antineoplastic activity on a549/ddp and hela cells when compared to cis-pt(dmso) 2 cl 2 and the parent quinoline-coumarin derivatives, with ic 50 values ranging between 100 nm and 10. however, metal-coumarin complexes constitute an interesting tool not only in the pharmacological field but also in the chemical one. as an example of such application, we report here a work from aslkhademi and collaborators, who designed, synthesized ad characterized a new zn(ii) complex with a coumarin-hydrazone ligand (225, figure 71 ) [282] . in this case, the complex was evaluated as a catalyst for the synthesis of tetrazoles by means of 3 + 2 cycloaddition between a nitrile and an azide. to this purpose, many experiments were carried out using different nitriles and reaction conditions. in the end, the zn(ii)-coumarin-hydrazone complex resulted to be a suitable catalyst for the synthesis of tetrazoles; the most effective amount of catalyst to employ was 0.05 mmol and substrates bearing electron-donating groups reacted better than the ones with electronwithdrawing substituents. finally, it is worth mentioning the application of metal-coumarin complexes as constituents of sol-gel coatings. lately, sol-gels are gaining attention due to their chemical stability, homogeneity however, metal-coumarin complexes constitute an interesting tool not only in the pharmacological field but also in the chemical one. as an example of such application, we report here a work from aslkhademi and collaborators, who designed, synthesized ad characterized a new zn(ii) complex with a coumarin-hydrazone ligand (225, figure 71 ) [282] . in this case, the complex was evaluated as a catalyst for the synthesis of tetrazoles by means of 3 + 2 cycloaddition between a nitrile and an azide. to this purpose, many experiments were carried out using different nitriles and reaction conditions. in the end, the zn(ii)-coumarin-hydrazone complex resulted to be a suitable catalyst for the synthesis of tetrazoles; the most effective amount of catalyst to employ was 0.05 mmol and substrates bearing electron-donating groups reacted better than the ones with electron-withdrawing substituents. 100nm and 10.33 μm (75.02 ± 1.18 μm for a549/ddp or 12.09 ± 0.24 μm for hela). in addition, compounds 214-224 displayed selectivity towards the mentioned cancer cells over other tumoral cell lines and hl-7002 non-tumoral cell line. however, metal-coumarin complexes constitute an interesting tool not only in the pharmacological field but also in the chemical one. as an example of such application, we report here a work from aslkhademi and collaborators, who designed, synthesized ad characterized a new zn(ii) complex with a coumarin-hydrazone ligand (225, figure 71 ) [282] . in this case, the complex was evaluated as a catalyst for the synthesis of tetrazoles by means of 3 + 2 cycloaddition between a nitrile and an azide. to this purpose, many experiments were carried out using different nitriles and reaction conditions. in the end, the zn(ii)-coumarin-hydrazone complex resulted to be a suitable catalyst for the synthesis of tetrazoles; the most effective amount of catalyst to employ was 0.05 mmol and substrates bearing electron-donating groups reacted better than the ones with electronwithdrawing substituents. finally, it is worth mentioning the application of metal-coumarin complexes as constituents of sol-gel coatings. lately, sol-gels are gaining attention due to their chemical stability, homogeneity finally, it is worth mentioning the application of metal-coumarin complexes as constituents of sol-gel coatings. lately, sol-gels are gaining attention due to their chemical stability, homogeneity and technological applications (e.g., reservoir for the prolonged release of antimicrobial substances). in 2013, jaiswal and co-workers proposed the synthesis of silver-coumarin complexes useful as antibacterial agents in sol-gel coatings [283] . these complexes were based on coumarin-3-carboxylatosilver and its hydroxylated derivatives in 6, 7 and 8. antimicrobial activity was displayed even at concentration of 0.3% (w/w). the most active compound showed significative antibiofilm activity at 0.5% and 0.7% (w/w). these results are encouraging in the perspective of using ag-coumarin complexes as biomedical coatings. table 2 reports some of the coumarins mentioned above, summarizing the biological activity, the molecular target and the origin. and technological applications (e.g., reservoir for the prolonged release of antimicrobial substances). in 2013, jaiswal and co-workers proposed the synthesis of silver-coumarin complexes useful as antibacterial agents in sol-gel coatings [283] . these complexes were based on coumarin-3carboxylatosilver and its hydroxylated derivatives in 6, 7 and 8. antimicrobial activity was displayed even at concentration of 0.3% (w/w). the most active compound showed significative antibiofilm activity at 0.5% and 0.7% (w/w). these results are encouraging in the perspective of using agcoumarin complexes as biomedical coatings. table 2 reports some of the coumarins mentioned above, summarizing the biological activity, the molecular target and the origin. and technological applications (e.g., reservoir for the prolonged release of antimicrobial substances). in 2013, jaiswal and co-workers proposed the synthesis of silver-coumarin complexes useful as antibacterial agents in sol-gel coatings [283] . these complexes were based on coumarin-3carboxylatosilver and its hydroxylated derivatives in 6, 7 and 8. antimicrobial activity was displayed even at concentration of 0.3% (w/w). the most active compound showed significative antibiofilm activity at 0.5% and 0.7% (w/w). these results are encouraging in the perspective of using agcoumarin complexes as biomedical coatings. table 2 reports some of the coumarins mentioned above, summarizing the biological activity, the molecular target and the origin. and technological applications (e.g., reservoir for the prolonged release of antimicrobial substances). in 2013, jaiswal and co-workers proposed the synthesis of silver-coumarin complexes useful as antibacterial agents in sol-gel coatings [283] . these complexes were based on coumarin-3carboxylatosilver and its hydroxylated derivatives in 6, 7 and 8. antimicrobial activity was displayed even at concentration of 0.3% (w/w). the most active compound showed significative antibiofilm activity at 0.5% and 0.7% (w/w). these results are encouraging in the perspective of using agcoumarin complexes as biomedical coatings. table 2 reports some of the coumarins mentioned above, summarizing the biological activity, the molecular target and the origin. and technological applications (e.g., reservoir for the prolonged release of antimicrobial substances). in 2013, jaiswal and co-workers proposed the synthesis of silver-coumarin complexes useful as antibacterial agents in sol-gel coatings [283] . these complexes were based on coumarin-3carboxylatosilver and its hydroxylated derivatives in 6, 7 and 8. antimicrobial activity was displayed even at concentration of 0.3% (w/w). the most active compound showed significative antibiofilm activity at 0.5% and 0.7% (w/w). these results are encouraging in the perspective of using agcoumarin complexes as biomedical coatings. table 2 reports some of the coumarins mentioned above, summarizing the biological activity, the molecular target and the origin. and technological applications (e.g., reservoir for the prolonged release of antimicrobial substances). in 2013, jaiswal and co-workers proposed the synthesis of silver-coumarin complexes useful as antibacterial agents in sol-gel coatings [283] . these complexes were based on coumarin-3carboxylatosilver and its hydroxylated derivatives in 6, 7 and 8. antimicrobial activity was displayed even at concentration of 0.3% (w/w). the most active compound showed significative antibiofilm activity at 0.5% and 0.7% (w/w). these results are encouraging in the perspective of using agcoumarin complexes as biomedical coatings. table 2 reports some of the coumarins mentioned above, summarizing the biological activity, the molecular target and the origin. and technological applications (e.g., reservoir for the prolonged release of antimicrobial substances). in 2013, jaiswal and co-workers proposed the synthesis of silver-coumarin complexes useful as antibacterial agents in sol-gel coatings [283] . these complexes were based on coumarin-3carboxylatosilver and its hydroxylated derivatives in 6, 7 and 8. antimicrobial activity was displayed even at concentration of 0.3% (w/w). the most active compound showed significative antibiofilm activity at 0.5% and 0.7% (w/w). these results are encouraging in the perspective of using agcoumarin complexes as biomedical coatings. table 2 reports some of the coumarins mentioned above, summarizing the biological activity, the molecular target and the origin. knoevenagel condensation, wittig reaction, claisen rearrangement, heck reaction, pechmann condensation and perkin reaction are widely used methods for the obtainment of coumarin derivatives and have been widely reviewed over the years [284, 285] . the increasing interest in coumarins as potential biologically active compounds led to the development of innovative knoevenagel condensation, wittig reaction, claisen rearrangement, heck reaction, pechmann condensation and perkin reaction are widely used methods for the obtainment of coumarin derivatives and have been widely reviewed over the years [284, 285] . the increasing interest in coumarins as potential biologically active compounds led to the development of innovative knoevenagel condensation, wittig reaction, claisen rearrangement, heck reaction, pechmann condensation and perkin reaction are widely used methods for the obtainment of coumarin derivatives and have been widely reviewed over the years [284, 285] . the increasing interest in coumarins as potential biologically active compounds led to the development of innovative procedures and approaches aimed at the production of coumarins in high yield and in a sustainable manner. in the next paragraphs, we will focus on the most recent advances in coumarin synthesis. in the past two decades, flow chemistry emerged has a promising synthetic technology, due to the many advantages it offers compared to the traditional batch method. in fact, by using continuous flow reactors it is possible to control reaction parameters, such as temperature, stoichiometry, reaction time and others, very precisely, thus having the possibility to achieve better and more reproducible reactions. furthermore, the great surface area/volume ratio in continuous flow reactors often leads to better reaction yields. this new technology gives also the possibility to work with hazardous reagents and to perform superheated or pressurized reactions in safer conditions [286] [287] [288] . the applications of this technology are numerous and the improvement of chemical synthesis of natural compounds is one of them. in 2015, li and collaborators developed a two-stage synthesis of coumarins via o-acetylation of salicylaldehyde [289] . the reaction occurred via o-acetylation and intramolecular aldol-type condensation, followed by dehydration and it was performed using two heated coiled reactors (scheme 1). using this system, it was possible to reach a 120 g scale production. procedures and approaches aimed at the production of coumarins in high yield and in a sustainable manner. in the next paragraphs, we will focus on the most recent advances in coumarin synthesis. in the past two decades, flow chemistry emerged has a promising synthetic technology, due to the many advantages it offers compared to the traditional batch method. in fact, by using continuous flow reactors it is possible to control reaction parameters, such as temperature, stoichiometry, reaction time and others, very precisely, thus having the possibility to achieve better and more reproducible reactions. furthermore, the great surface area/volume ratio in continuous flow reactors often leads to better reaction yields. this new technology gives also the possibility to work with hazardous reagents and to perform superheated or pressurized reactions in safer conditions [286] [287] [288] . the applications of this technology are numerous and the improvement of chemical synthesis of natural compounds is one of them. in 2015, li and collaborators developed a two-stage synthesis of coumarins via o-acetylation of salicylaldehyde [289] . the reaction occurred via o-acetylation and intramolecular aldol-type condensation, followed by dehydration and it was performed using two heated coiled reactors (scheme 1). using this system, it was possible to reach a 120 g scale production. the use of immobilized reagents in organic synthesis is gaining ever more attention, due to the advantages offered by such type of reagents (for example, the simplification of product isolation and purification). this synthetic strategy has seen some applications in the production of natural scaffolds too. in 2018, mhiri and co-workers proposed a synthetic pathway for coumarin derivatives involving polymer supported reagents [290] . in the mentioned study, coumarin derivative a was prepared from 3-metoxy salicylaldheyde using reagents supported in a macroporous ion exchange resin, through the hydrolysis of the two intermediates iminocoumarin b and unsaturated nitrile c (scheme 2). the use of immobilized reagents in organic synthesis is gaining ever more attention, due to the advantages offered by such type of reagents (for example, the simplification of product isolation and purification). this synthetic strategy has seen some applications in the production of natural scaffolds too. in 2018, mhiri and co-workers proposed a synthetic pathway for coumarin derivatives involving polymer supported reagents [290] . in the mentioned study, coumarin derivative a was prepared from 3-metoxy salicylaldheyde using reagents supported in a macroporous ion exchange resin, through the hydrolysis of the two intermediates iminocoumarin b and unsaturated nitrile c (scheme 2). scheme 1. set-up for the synthesis of coumarins via o-acetylation of salicylaldehyde in a continuous flow reactor. the use of immobilized reagents in organic synthesis is gaining ever more attention, due to the advantages offered by such type of reagents (for example, the simplification of product isolation and purification). this synthetic strategy has seen some applications in the production of natural scaffolds too. in 2018, mhiri and co-workers proposed a synthetic pathway for coumarin derivatives involving polymer supported reagents [290] . in the mentioned study, coumarin derivative a was prepared from 3-metoxy salicylaldheyde using reagents supported in a macroporous ion exchange resin, through the hydrolysis of the two intermediates iminocoumarin b and unsaturated nitrile c (scheme 2). coumarin synthesis with polymer-supported reagent. coumarin synthesis with polymer-supported reagent. the first step provided the preparation of polymer supported phenate ions that then reacted with phenylacetonitrile, leading to the formation of iminocoumarin and unsaturated nitrile, which were finally deblocked from the resin with chloroform. the acid hydrolysis of the obtained mixture of compounds b and c gave coumarin derivative a in very good yield (95%). it is worth speaking about another emerging strategy in organic synthesis: photocatalysis. in 2015, metternich and gilmour utilized this approach to emulate coumarins biosynthesis, using (−)-riboflavin as a photocatalyst [291] . in this work, two discrete activation modes of (−)-riboflavin were sequentially exploited to induce the isomerization and cyclisation of e-cinnamic acids used as starting material (scheme 3), thus overcoming the use of phenol-derived and pre-functionalized aryl rings as starting materials. the first step provided the preparation of polymer supported phenate ions that then reacted with phenylacetonitrile, leading to the formation of iminocoumarin and unsaturated nitrile, which were finally deblocked from the resin with chloroform. the acid hydrolysis of the obtained mixture of compounds b and c gave coumarin derivative a in very good yield (95%). it is worth speaking about another emerging strategy in organic synthesis: photocatalysis. in 2015, metternich and gilmour utilized this approach to emulate coumarins biosynthesis, using (−)riboflavin as a photocatalyst [291] . in this work, two discrete activation modes of (−)-riboflavin were sequentially exploited to induce the isomerization and cyclisation of e-cinnamic acids used as starting material (scheme 3), thus overcoming the use of phenol-derived and pre-functionalized aryl rings as starting materials. similarly, in 2019, song and co-workers reported a one-pot photoredox-catalyzed protocol to achieve a series of 3-fluoroalkylated coumarins starting from ortho-hydroxycinnamic esters (scheme 4) [292] . ortho-hydroxycinnamic esters and brcf2cor' (or other commercially available perfluoroalkylated radical resources) were exposed to 30w blue leds irradiation under argon atmosphere for 12 h.; fac-ir(ppy)3 was found to be the best among the tested catalysts, whereas ch3cn, dmf, dmso and thf were all suitable solvents for this reaction. k2co3, et3n and dipea were all able to neutralize hbr produced during the process. this procedure gave 3-fluoroalkylated coumarins in good yields on milligram and gram scales. similarly, in 2019, song and co-workers reported a one-pot photoredox-catalyzed protocol to achieve a series of 3-fluoroalkylated coumarins starting from ortho-hydroxycinnamic esters (scheme 4) [292] . ortho-hydroxycinnamic esters and brcf 2 cor' (or other commercially available perfluoroalkylated radical resources) were exposed to 30w blue leds irradiation under argon atmosphere for 12 h.; fac-ir(ppy) 3 was found to be the best among the tested catalysts, whereas ch 3 cn, dmf, dmso and thf were all suitable solvents for this reaction. k 2 co 3 , et 3 n and dipea were all able to neutralize hbr produced during the process. this procedure gave 3-fluoroalkylated coumarins in good yields on milligram and gram scales. perfluoroalkylated radical resources) were exposed to 30w blue leds irradiation under argon atmosphere for 12 h.; fac-ir(ppy)3 was found to be the best among the tested catalysts, whereas ch3cn, dmf, dmso and thf were all suitable solvents for this reaction. k2co3, et3n and dipea were all able to neutralize hbr produced during the process. this procedure gave 3-fluoroalkylated coumarins in good yields on milligram and gram scales. another example is the bichromatic synthesis of coumarins proposed by eivgi and collaborators in 2017 [293] . in this work, 2-nitrobenzyl-protected 2-hydroxystyrenes and acrylates underwent a uv-a (380 nm) photoinduced cross metathesis (cm) reaction, in the presence of ruthenium as a catalyst. in this step a 1-pyrenecarboxaldheyde solution was used as a uv filter, without whom the catalyst would be permanently inactivated due to phenol deprotection and phenolate chelation to the ruthenium. after cm, the uv filter was removed and irradiation with uv-c light (254 nm) led to more complex structures, such as coumarins. in fact, irradiation with uv-c light started a threereactions chain-photodeprotection of the intermediate, e/z isomerization and cyclization to form the desired coumarins (scheme 5). another example is the bichromatic synthesis of coumarins proposed by eivgi and collaborators in 2017 [293] . in this work, 2-nitrobenzyl-protected 2-hydroxystyrenes and acrylates underwent a uv-a (380 nm) photoinduced cross metathesis (cm) reaction, in the presence of ruthenium as a catalyst. in this step a 1-pyrenecarboxaldheyde solution was used as a uv filter, without whom the catalyst would be permanently inactivated due to phenol deprotection and phenolate chelation to the ruthenium. after cm, the uv filter was removed and irradiation with uv-c light (254 nm) led to more complex structures, such as coumarins. in fact, irradiation with uv-c light started a three-reactions chain-photodeprotection of the intermediate, e/z isomerization and cyclization to form the desired coumarins (scheme 5). chemists usually carry out reactions in solution, following the aristotelian principle "corpora non agunt nisi fluida seu soluta" or in other and more contemporary words "compounds do not react unless fluid or if dissolved" [294] . however, this is not always completely accurate, because many solventless reactions proceed efficiently. an enhancement in kinetics, owed to the different concentrations of reactants given the lack of solvents, bring actually to a higher reactivity and, where necessary, milder experimental conditions. at the same time, the absence of solvents provides the chance to heat over the boiling point of the most common solvents and to exploit the benefits of microwave assisted irradiation. the occurrence of efficient solid-state reactions shows that the reacting molecules are able to move freely in the solid state, whereas monitoring of the reaction is possible thanks ir and uv spectra in the solid state [295, 296] . the most remarkable advantage of solvent-free reaction, in all probability, is the noteworthy cut in the waste production associated with solvents handling, resulting in a more effective and ecological chemical process. there are three solvent-free techniques: (1) reactions conducted on mineral supports, (2) reaction without any solvent, support or catalyst and (3) solid-liquid phase transfer catalysis [297] . the experimental conditions required for the classical synthesis of coumarin derivatives are, in some cases, drastic, as in the production of hymecromone via pechmann condensation where the final compound is obtain by stirring a mixture of resorcinol and ethyl acetoacetate in concentrated h2so4 for 12-24 h [295] . solvent-free reactions seemed to be a more ecological option. consequently, in 2001, sugino and tanaka proposed the synthesis of several coumarin derivatives both via pechmann and knoevenagel condensation reactions [298] . for example, p-toluensolfonic acid was employed as a catalyst and the reaction mixture was heated at 60 °c for 10 min. without solvent, chemists usually carry out reactions in solution, following the aristotelian principle "corpora non agunt nisi fluida seu soluta" or in other and more contemporary words "compounds do not react unless fluid or if dissolved" [294] . however, this is not always completely accurate, because many solventless reactions proceed efficiently. an enhancement in kinetics, owed to the different concentrations of reactants given the lack of solvents, bring actually to a higher reactivity and, where necessary, milder experimental conditions. at the same time, the absence of solvents provides the chance to heat over the boiling point of the most common solvents and to exploit the benefits of microwave assisted irradiation. the occurrence of efficient solid-state reactions shows that the reacting molecules are able to move freely in the solid state, whereas monitoring of the reaction is possible thanks ir and uv spectra in the solid state [295, 296] . the most remarkable advantage of solvent-free reaction, in all probability, is the noteworthy cut in the waste production associated with solvents handling, resulting in a more effective and ecological chemical process. there are three solvent-free techniques: (1) reactions conducted on mineral supports, (2) reaction without any solvent, support or catalyst and (3) solid-liquid phase transfer catalysis [297] . the experimental conditions required for the classical synthesis of coumarin derivatives are, in some cases, drastic, as in the production of hymecromone via pechmann condensation where the final compound is obtain by stirring a mixture of resorcinol and ethyl acetoacetate in concentrated h2so4 for 12-24 h [295] . solvent-free reactions seemed to be a more ecological option. consequently, in 2001, sugino and tanaka proposed the synthesis of several coumarin derivatives both via pechmann and knoevenagel condensation reactions [298] . for example, p-toluensolfonic acid was employed as a catalyst and the reaction mixture was heated at 60 • c for 10 min. without solvent, giving after the work-up 7-hydroxy-4-methylcoumarin in 98% yield (scheme 6). the experimental conditions required for the classical synthesis of coumarin derivatives are, in some cases, drastic, as in the production of hymecromone via pechmann condensation where the final compound is obtain by stirring a mixture of resorcinol and ethyl acetoacetate in concentrated h2so4 for 12-24 h [295] . solvent-free reactions seemed to be a more ecological option. consequently, in 2001, sugino and tanaka proposed the synthesis of several coumarin derivatives both via pechmann and knoevenagel condensation reactions [298] . for example, p-toluensolfonic acid was employed as a catalyst and the reaction mixture was heated at 60 °c for 10 min. without solvent, giving after the work-up 7-hydroxy-4-methylcoumarin in 98% yield (scheme 6). scheme 6. solvent-free synthesis of 7-hydroxy-4-methylcoumarin. scheme 6. solvent-free synthesis of 7-hydroxy-4-methylcoumarin. knoevenagel condensation was also studied under solventless conditions and efficiently brought to the production of both coumarins and benzocoumarin derivatives [298] . another methodology was proposed in 2012 by kalita and kumar, that exploited the pechmann reaction using trifluoromethanesulfonic acid functionalized zr-tms (zr-tms, zirconia-based transition metal oxide mesoporous molecular sieves) catalysts, under solvent-free heterogeneous catalytic conditions. the product selectivity was always found to be about 100% (scheme 7) [296] . knoevenagel condensation was also studied under solventless conditions and efficiently brought to the production of both coumarins and benzocoumarin derivatives [298] . another methodology was proposed in 2012 by kalita and kumar, that exploited the pechmann reaction using trifluoromethanesulfonic acid functionalized zr-tms (zr-tms, zirconia-based transition metal oxide mesoporous molecular sieves) catalysts, under solvent-free heterogeneous catalytic conditions. the product selectivity was always found to be about 100% (scheme 7) [296] . more recently, a novel series of hetero-annulated coumarins was obtained by ebrahimi and colleagues through a one-pot reaction under solvent-free conditions and evaluated as ache/buche inhibitors. as acidic catalyst nano-silica sulfuric acid was employed and the versatility of the system was assayed on appropriated benzaldehydes, dimedone and 4-hydroxycoumarin (scheme 8) [299] . also noteworthy is the application, once again, of a reusable heterogeneous catalyst, which contributed to the general environmental sustainability of the process thanks to the possible recovery and reuse [300] . scheme 8. solvent-free synthesis of hetero-annulated coumarins. another solventless procedure worth noting is the one presented by kour and paul, which exploited lewis acid grafted sulfonated carbon@titania composite as catalyst for the pechmann condensation. this particular kind of catalyst and its efficacy had been already studied by the team more recently, a novel series of hetero-annulated coumarins was obtained by ebrahimi and colleagues through a one-pot reaction under solvent-free conditions and evaluated as ache/buche inhibitors. as acidic catalyst nano-silica sulfuric acid was employed and the versatility of the system was assayed on appropriated benzaldehydes, dimedone and 4-hydroxycoumarin (scheme 8) [299] . also noteworthy is the application, once again, of a reusable heterogeneous catalyst, which contributed to the general environmental sustainability of the process thanks to the possible recovery and reuse [300] . knoevenagel condensation was also studied under solventless conditions and efficiently brought to the production of both coumarins and benzocoumarin derivatives [298] . another methodology was proposed in 2012 by kalita and kumar, that exploited the pechmann reaction using trifluoromethanesulfonic acid functionalized zr-tms (zr-tms, zirconia-based transition metal oxide mesoporous molecular sieves) catalysts, under solvent-free heterogeneous catalytic conditions. the product selectivity was always found to be about 100% (scheme 7) [296] . more recently, a novel series of hetero-annulated coumarins was obtained by ebrahimi and colleagues through a one-pot reaction under solvent-free conditions and evaluated as ache/buche inhibitors. as acidic catalyst nano-silica sulfuric acid was employed and the versatility of the system was assayed on appropriated benzaldehydes, dimedone and 4-hydroxycoumarin (scheme 8) [299] . also noteworthy is the application, once again, of a reusable heterogeneous catalyst, which contributed to the general environmental sustainability of the process thanks to the possible recovery and reuse [300] . another solventless procedure worth noting is the one presented by kour and paul, which exploited lewis acid grafted sulfonated carbon@titania composite as catalyst for the pechmann condensation. this particular kind of catalyst and its efficacy had been already studied by the team for the synthesis of 4h-pyrimido[2,1-b]benzothiazoles and benzoxanthenones under solvent-free conditions, so the one-pot synthesis of coumarin derivatives was the natural next step (scheme 9) [301, 302] . because the catalyst reusability contributes to reduce the cost of the practical application processes, the reusability of the catalyst was investigated in the pechmann condensation. the catalyst, another solventless procedure worth noting is the one presented by kour and paul, which exploited lewis acid grafted sulfonated carbon@titania composite as catalyst for the pechmann condensation. this particular kind of catalyst and its efficacy had been already studied by the team for the synthesis of 4h-pyrimido[2,1-b]benzothiazoles and benzoxanthenones under solvent-free conditions, so the one-pot synthesis of coumarin derivatives was the natural next step (scheme 9) [301, 302] . because the catalyst reusability contributes to reduce the cost of the practical application processes, the reusability of the catalyst was investigated in the pechmann condensation. the catalyst, recovered by filtration, after five catalytic runs had a mild decrease in the catalytic activity and exhibited almost the same tg curve as the fresh one, depicting that the present heterogeneous system showed high thermal stability after successive reaction cycles [302] . in order to obtain a different and ecological procedure for the synthesis of 3-substituted coumarins via knoevenagel condensation, ghomi and akbarzadeh developed a procedure starting from various salicylaldehydes and 1,3-dicarbonyl compounds. they used mgfe2o4 nanoparticles as an efficient catalyst under solvent-free condition and ultrasound irradiation (scheme 10). basically, ultrasound irradiation increased the dimension of the single bubbles together with the number of active cavitation bubbles. the resulting effect was expected to be a higher maximum collapse temperature and a faster synthesis of coumarin compounds by knoevenagel reaction. comparing ultrasound irradiation to conventional heating the research team demonstrated that the first method allowed to obtain higher yields in shorter times, probably because of the shock wave and microjet generated by the cavitation. furthermore, with this method, mgfe2o4 nanoparticles were dispersed in the reaction and provided more sites for the construction of cavity over their surface. the catalyst was recovered via magnetic separation and reused up to 6 cycles without relevant loss of activity [303] . the convenience given by the removal of the catalyst by means of an external magnetic field is a significant improvement in the work-up of a reaction mixture. consequently, another magnetic nano structured catalyst was employed for the synthesis of coumarin nucleus, this time through pechmann condensation. in 2019, pakdel and co-workers proposed the synthesis of coumarin derivatives by leveraging fe3o4@boehmite-nh2-coii nps as a catalyst, under solvent-free conditions that resulted the best choice comparing the results obtained with classic solvents. the catalyst could be recovered easily and reused up to six times. however, no desired coumarin derivatives were observed in the case of phenols bearing electron-withdrawing substituents (such as -cl and -no2), behavior attributed to the proposed mechanism of the reaction [304] . in the field of solvent-free reactions, the application of ionic liquids as catalyst for the synthesis of coumarin scaffolds deserves a stand-alone status. ionic liquids (ils) have become increasingly popular in the last decades as a safer alternative to classic solvent systems, in a perspective of more sustainable chemical processes. most ils possess a number of properties that made them appealing, proving to be non-flammable, to possess a negligible vapor-pressure, that means that solvent evaporation is eliminated and to dissolve a wide range of organic and inorganic compounds [305] . furthermore, since ionic liquids are low-melting salts, they are made at least from two components which can be varied (the anion and the cation), thus the solvents could be designed with a particular end use in mind or possessing a particular set of properties [306] . however, their use in large quantities is restricted by some limitations like biodegradability, toxicity and high costs [307] [308] [309] . mahato and colleagues in 2017 used ionic liquids as an acidic catalyst in the regioselective synthesis of pyrano [3,2-c] coumarins under solvent-free conditions. in particular, given the fact that brønsted acidic ion liquids (bails) acquired recognition in the field of catalysis, the research team reported the catalytic effect of 1-butane sulfonic acid-3-methylimidazolium tosylate, [bsmim]ots (bail-1), on the tandem reaction between 4-hydroxycoumarin and α,β-unsaturated carbonyl compounds for the formation of pyrano[3,2-c]coumarins (scheme 10) [310, 311] . in addition to the high yields and the regioselectivity, this procedure allowed to avoid column chromatography for further purification. lewis acid grafted sulfonated carbon@titania composite as catalyst for the pechmann condensation. in order to obtain a different and ecological procedure for the synthesis of 3-substituted coumarins via knoevenagel condensation, ghomi and akbarzadeh developed a procedure starting from various salicylaldehydes and 1,3-dicarbonyl compounds. they used mgfe 2 o 4 nanoparticles as an efficient catalyst under solvent-free condition and ultrasound irradiation (scheme 10). noroozizadeh and co-workers focused their efforts on the synthesis of bis-coumarins thanks to a different catalyst, acetic acid functionalized poly(4-vinylpyridinum) bromide (apvpb), in continuous of a previous work of the research team on the preparation of brønsted acidic ionic liquids and solid salts [312, 313] . the aim was the development of a novel and efficient procedure for the synthesis of bis-coumarins which lacked the classical drawbacks as the use of solvents, high costs and low yields [314] . temperature is a key parameter in almost the totality of chemical reactions involving both inorganic and organic compounds. generally, most organic reactions are performed heating by traditional heat transfer equipment like oil baths, sand baths and heating jackets. the drawbacks associated with these heating techniques are the slowness, the possible development of temperature gradient within the sample and consequent local overheating that could lead to the formation of byproducts. microwave technology started to emerge in inorganic chemistry since the late 1970s and almost a decade after attracted the attention of organic chemists [315] . compared to the traditional heating, microwave dielectric heating has several advantages: (1) higher heating rates, (2) no direct contact between the source of energy and the reacting chemicals, (3) possible selective heating, because reacting chemicals interact differently with the commonly used microwave radiations and (4) rapid increase of the temperature also above boiling point in pressurized systems [316] . microwave irradiation is often associated with other technologies, such as ionic liquids and neat reactions (already mentioned in the previous paragraph) [295, 317] . in the late 1990s progresses in the classic coumarins synthesis via microwave irradiation were reported by singh and co-workers, who confirmed a reduction in reaction time of the pechmann condensation and by saidi and colleagues, who demonstrated the improvement in the synthesis of pyranocoumarins and furanocoumarins through claisen rearrangement [317, 318] . ten years later, valizadeh and shockravi pushed further the opportunities given by microwave heating combining it with a task-specific imidazolium-based phosphinite ionic liquid (il-opph2) for the synthesis of e-cinnamates and coumarin derivatives via basically, ultrasound irradiation increased the dimension of the single bubbles together with the number of active cavitation bubbles. the resulting effect was expected to be a higher maximum collapse temperature and a faster synthesis of coumarin compounds by knoevenagel reaction. comparing ultrasound irradiation to conventional heating the research team demonstrated that the first method allowed to obtain higher yields in shorter times, probably because of the shock wave and microjet generated by the cavitation. furthermore, with this method, mgfe2o4 nanoparticles were dispersed in the reaction and provided more sites for the construction of cavity over their surface. the catalyst was recovered via magnetic separation and reused up to 6 cycles without relevant loss of activity [303] . the convenience given by the removal of the catalyst by means of an external magnetic field is a significant improvement in the work-up of a reaction mixture. consequently, another magnetic nano structured catalyst was employed for the synthesis of coumarin nucleus, this time through pechmann condensation. in 2019, pakdel and co-workers proposed the synthesis of coumarin derivatives by leveraging fe 3 o 4 @boehmite-nh 2 -coii nps as a catalyst, under solvent-free conditions that resulted the best choice comparing the results obtained with classic solvents. the catalyst could be recovered easily and reused up to six times. however, no desired coumarin derivatives were observed in the case of phenols bearing electron-withdrawing substituents (such as -cl and -no2), behavior attributed to the proposed mechanism of the reaction [304] . in the field of solvent-free reactions, the application of ionic liquids as catalyst for the synthesis of coumarin scaffolds deserves a stand-alone status. ionic liquids (ils) have become increasingly popular in the last decades as a safer alternative to classic solvent systems, in a perspective of more sustainable chemical processes. most ils possess a number of properties that made them appealing, proving to be non-flammable, to possess a negligible vapor-pressure, that means that solvent evaporation is eliminated and to dissolve a wide range of organic and inorganic compounds [305] . furthermore, since ionic liquids are low-melting salts, they are made at least from two components which can be varied (the anion and the cation), thus the solvents could be designed with a particular end use in mind or possessing a particular set of properties [306] . however, their use in large quantities is restricted by some limitations like biodegradability, toxicity and high costs [307] [308] [309] . mahato and colleagues in 2017 used ionic liquids as an acidic catalyst in the regioselective synthesis of pyrano [3,2-c] coumarins under solvent-free conditions. in particular, given the fact that brønsted acidic ion liquids (bails) acquired recognition in the field of catalysis, the research team reported the catalytic effect of 1-butane sulfonic acid-3-methylimidazolium tosylate, [bsmim]ots (bail-1), on the tandem reaction between 4-hydroxycoumarin and α,β-unsaturated carbonyl compounds for the formation of pyrano[3,2-c]coumarins (scheme 10) [310, 311] . in addition to the high yields and the regioselectivity, this procedure allowed to avoid column chromatography for further purification. moreover, given the mild experimental conditions, there were no by-products attributable to decomposition or polymerization. noroozizadeh and co-workers focused their efforts on the synthesis of bis-coumarins thanks to a different catalyst, acetic acid functionalized poly(4-vinylpyridinum) bromide (apvpb), in continuous of a previous work of the research team on the preparation of brønsted acidic ionic liquids and solid salts [312, 313] . the aim was the development of a novel and efficient procedure for the synthesis of bis-coumarins which lacked the classical drawbacks as the use of solvents, high costs and low yields [314] . temperature is a key parameter in almost the totality of chemical reactions involving both inorganic and organic compounds. generally, most organic reactions are performed heating by traditional heat transfer equipment like oil baths, sand baths and heating jackets. the drawbacks associated with these heating techniques are the slowness, the possible development of temperature gradient within the sample and consequent local overheating that could lead to the formation of by-products. microwave technology started to emerge in inorganic chemistry since the late 1970s and almost a decade after attracted the attention of organic chemists [315] . compared to the traditional heating, microwave dielectric heating has several advantages: (1) higher heating rates, (2) no direct contact between the source of energy and the reacting chemicals, (3) possible selective heating, because reacting chemicals interact differently with the commonly used microwave radiations and (4) rapid increase of the temperature also above boiling point in pressurized systems [316] . microwave irradiation is often associated with other technologies, such as ionic liquids and neat reactions (already mentioned in the previous paragraph) [295, 317] . in the late 1990s progresses in the classic coumarins synthesis via microwave irradiation were reported by singh and co-workers, who confirmed a reduction in reaction time of the pechmann condensation and by saidi and colleagues, who demonstrated the improvement in the synthesis of pyranocoumarins and furanocoumarins through claisen rearrangement [317, 318] . ten years later, valizadeh and shockravi pushed further the opportunities given by microwave heating combining it with a task-specific imidazolium-based phosphinite ionic liquid (il-opph 2 ) for the synthesis of e-cinnamates and coumarin derivatives via the one-pot horner-wadsworth-emmons-type reaction (scheme 11). moreover, given the mild experimental conditions, there were no by-products attributable to decomposition or polymerization. noroozizadeh and co-workers focused their efforts on the synthesis of bis-coumarins thanks to a different catalyst, acetic acid functionalized poly(4-vinylpyridinum) bromide (apvpb), in continuous of a previous work of the research team on the preparation of brønsted acidic ionic liquids and solid salts [312, 313] . the aim was the development of a novel and efficient procedure for the synthesis of bis-coumarins which lacked the classical drawbacks as the use of solvents, high costs and low yields [314] . temperature is a key parameter in almost the totality of chemical reactions involving both inorganic and organic compounds. generally, most organic reactions are performed heating by traditional heat transfer equipment like oil baths, sand baths and heating jackets. the drawbacks associated with these heating techniques are the slowness, the possible development of temperature gradient within the sample and consequent local overheating that could lead to the formation of byproducts. microwave technology started to emerge in inorganic chemistry since the late 1970s and almost a decade after attracted the attention of organic chemists [315] . compared to the traditional heating, microwave dielectric heating has several advantages: (1) higher heating rates, (2) no direct contact between the source of energy and the reacting chemicals, (3) possible selective heating, because reacting chemicals interact differently with the commonly used microwave radiations and (4) rapid increase of the temperature also above boiling point in pressurized systems [316] . microwave irradiation is often associated with other technologies, such as ionic liquids and neat reactions (already mentioned in the previous paragraph) [295, 317] . in the late 1990s progresses in the classic coumarins synthesis via microwave irradiation were reported by singh and co-workers, who confirmed a reduction in reaction time of the pechmann condensation and by saidi and colleagues, who demonstrated the improvement in the synthesis of pyranocoumarins and furanocoumarins through claisen rearrangement [317, 318] . ten years later, valizadeh and shockravi pushed further the opportunities given by microwave heating combining it with a task-specific imidazolium-based phosphinite ionic liquid (il-opph2) for the synthesis of e-cinnamates and coumarin derivatives via the one-pot horner-wadsworth-emmons-type reaction (scheme 11). scheme 11. imidazolium-based phosphinite ionic liquid (il-opph2) for the microwave assisted synthesis of coumarins. the same reactions were in parallel conducted with traditional heating and microwave irradiation: the former technique showed considerably longer reaction times (13) (14) (15) (16) the same reactions were in parallel conducted with traditional heating and microwave irradiation: the former technique showed considerably longer reaction times (13-16 h vs. 10-12 min) and lower yields (65-60% vs. 79-83%). furthermore, il-opph 2 worked at the same time as reaction medium and reagent as the research team already demonstrated in a previous study focused on the synthesis of coumarin nucleus via knoevenagel and witting reactions in ionic liquids [319, 320] . in 2016, fiorito and colleagues merged the advantages of solvent-free reactions with microwave heating for the synthesis of several coumarin derivatives. they started from variously substituted phenols and propiolic acids in the presence of ytterbium triflate hydrate 10% mol as catalyst [321] . during the last thirty years, triflates of metals belonging to the lanthanide series have been regarded as effective water tolerant recyclable lewis acids. these metals have been discovered to catalyze different carbon-carbon and carbon-heteroatom bond formation reactions, providing the desired products in excellent yields by means of a green chemical approach [322, 323] . furthermore, wang and colleagues already employed this particular catalyst for the synthesis of 4-methylcoumarins via pechmann condensation [324] . fiorito and co-workers developed for the first time a synthetic procedure to obtain different substituted coumarins in 2 min. under microwave irradiation in 91-98% yields [321] . moreover, the research team recovered the ytterbium triflate catalyst and demonstrated that no loss of activity occurred during several cycles. in the same year, bouasla et al. compared the efficacy of different heterogeneous acidic catalysts in the pechmann synthesis of 4-methylcoumarin and 7-hydroxy-4-methylcoumarin when coupled by microwave heating. the efficacy of different amberylst-type catalyst in the pechmann synthesis of 7-hydroxy-4-methylcoumarin was already pointed out by sabou and colleagues in 2005 [325] . therefore, the catalytic performance of amberlyst-15 was compared with zeolite h-β and sulfonic acid functionalized hybrid material ts-os-so 3 h, performing the reaction under solvent-free conditions, at 130 • c for 20 min in a microwave reaction tube. amberlyst-15 showed a higher catalytic activity (97% yield against 21-44% obtained with two others catalysts) due to its high density of acid centers [326] . it is also worth mentioning the work of konrádová et al., who in 2017 developed an interesting protecting-group-free method starting from commercially available aromatic aldehydes and using a microwave promoted wittig reaction of a stabilized ylide for the synthesis of different natural products, including coumarins. after the optimization of the experimental parameters and the development of a general synthetic methodology for the synthesis of different substitute coumarin derivatives (scheme 12a), the method was further developed to incorporate a claisen rearrangement step. in order to demonstrate the synthetic utility and versatility of the developed method, the research team performed the total synthesis of two coumarins, one of which being osthole, a natural coumarin derivative already mentioned in the anti-inflammatory paragraph (scheme 12b) [327] . and lower yields (65-60% vs 79-83%). furthermore, il-opph2 worked at the same time as reaction medium and reagent as the research team already demonstrated in a previous study focused on the synthesis of coumarin nucleus via knoevenagel and witting reactions in ionic liquids [319, 320] . in 2016, fiorito and colleagues merged the advantages of solvent-free reactions with microwave heating for the synthesis of several coumarin derivatives. they started from variously substituted phenols and propiolic acids in the presence of ytterbium triflate hydrate 10% mol as catalyst [321] . during the last thirty years, triflates of metals belonging to the lanthanide series have been regarded as effective water tolerant recyclable lewis acids. these metals have been discovered to catalyze different carbon-carbon and carbon-heteroatom bond formation reactions, providing the desired products in excellent yields by means of a green chemical approach [322, 323] . furthermore, wang and colleagues already employed this particular catalyst for the synthesis of 4-methylcoumarins via pechmann condensation [324] . fiorito and co-workers developed for the first time a synthetic procedure to obtain different substituted coumarins in 2 min. under microwave irradiation in 91-98% yields [321] . moreover, the research team recovered the ytterbium triflate catalyst and demonstrated that no loss of activity occurred during several cycles. in the same year, bouasla et al. compared the efficacy of different heterogeneous acidic catalysts in the pechmann synthesis of 4-methylcoumarin and 7-hydroxy-4-methylcoumarin when coupled by microwave heating. the efficacy of different amberylst-type catalyst in the pechmann synthesis of 7hydroxy-4-methylcoumarin was already pointed out by sabou and colleagues in 2005 [325] . therefore, the catalytic performance of amberlyst-15 was compared with zeolite h-β and sulfonic acid functionalized hybrid material ts-os-so3h, performing the reaction under solvent-free conditions, at 130 °c for 20 min in a microwave reaction tube. amberlyst-15 showed a higher catalytic activity (97% yield against 21-44% obtained with two others catalysts) due to its high density of acid centers [326] . it is also worth mentioning the work of konrádová et al., who in 2017 developed an interesting protecting-group-free method starting from commercially available aromatic aldehydes and using a microwave promoted wittig reaction of a stabilized ylide for the synthesis of different natural products, including coumarins. after the optimization of the experimental parameters and the development of a general synthetic methodology for the synthesis of different substitute coumarin derivatives (scheme 12a), the method was further developed to incorporate a claisen rearrangement step. in order to demonstrate the synthetic utility and versatility of the developed method, the research team performed the total synthesis of two coumarins, one of which being osthole, a natural coumarin derivative already mentioned in the anti-inflammatory paragraph (scheme 12b) [327] . a broad range of indolo [2,3-c] coumarins have been obtained in good yields (46-88%) by gu and co-workers in 2019 under microwave-assisted base-free intramolecular cross dehydrogenative coupling using palladium catalyst. the advantages of this methodologies compared to classic a broad range of indolo [2,3-c] coumarins have been obtained in good yields (46-88%) by gu and co-workers in 2019 under microwave-assisted base-free intramolecular cross dehydrogenative coupling using palladium catalyst. the advantages of this methodologies compared to classic synthesis of coumarin lactone ring were both atom economy and step efficiency, combined with c-h/c-h coupling without pre-functionalization [328] . the coumarin nucleus has been gaining increasing attention in the last years because of the variety of its applications both in medicinal chemistry and in agri-food sectors. in this review, we presented a collection of recent works that highlight the wide range of pharmacological activities ascribable to coumarins (antioxidant, antibacterial, antifungal, antiviral, anti-proliferative, anti-inflammatory, antidiabetics, anticoagulant and anti-neurodegenerative) and the possibility to easily modify and decorate this natural scaffold to perform structure activity relationships studies. the coumarin nucleus possesses some fundamental properties that ensure it an advisable role in the design of new biologically active derivatives, mainly due to its stability, low molecular weight and to the easiness to decorate it for increasing pharmacodynamic and pharmacokinetic properties. coumarin scaffold has been a valuable source of inspiration in the design of novel biologically active compounds and its role as a starting point is very attractive. this natural core is present in a number of currently available drugs used in the treatment of various diseases. for instance, the use of warfarin, acenocumarol and phenprocoumon in the treatment and prevention of thromboembolic diseases is now well established. other examples include the use of hymecromone as choleretic agent and that of the antihelminthic haloxon in veterinary medicine. even though the coumarin nucleus presents an impressive number of biological activities, its presence among marketed drugs is not widespread yet. further efforts are needed in order to achieve coumarin-based compounds with appreciable pharmacokinetic properties, along with high efficacy and a low toxicity profile. moreover, in this review we discussed coumarin photoproperties and their applications as photocleavable protecting groups and fluorescent probes. finally, we analyzed the latest updates in coumarin synthesis from both chemical and technological points of view. one of the most interesting approaches that emerged from this study is the design and use of coumarin-containing hybrid compounds. it is our hope that this review will contribute to further development in the investigation and exploitation of coumarins' potential. author contributions: conceptualization, a.p.; original draft preparation, f.a. and c.p.; writing-review and editing, s.d., a.p. and l.t.; supervision, a.p. and l.t. all authors have read and agreed to the published version of the manuscript. simple coumarins and analogues in medicinal chemistry: occurrence, synthesis and biological activity pharmacological and biochemical actions of simple coumarins: natural products with therapeutic potential coumarin compounds in medicinal chemistry: some important examples from the last year recent advances and therapeutic journey of coumarins: current status and perspectives nomenclature of organic chemistry pharmacological and nutritional effects of natural coumarins and their structure-activity relationships coumarin: a natural, privileged and versatile scaffold for bioactive compounds natural products as fungicide and their role in crop protection sustainable production 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on the antioxidant property of coumarin-fused coumarins coumarin-fused coumarin: antioxidant story from n,n -dimethylamino and hydroxyl groups significantly enhanced antioxidant activity of chitosan through chemical modification with coumarins the evaluation of antioxidant and antifungal properties of 6-amino-6-deoxychitosan in vitro synthesis and biological evaluation of novel coumarins with tert-butyl and terpene substituents the hallmarks of cancer discovering the structure-activity relationships of different o-prenylated coumarin derivatives as effective anticancer agents in human cervical cancer cells primers on molecular pathways lipoxygenases: their role as an oncogenic pathway in pancreatic cancer overexpression of 12/15-lipoxygenase, an ortholog of human 15-lipoxygenase-1, in the prostate tumors of tramp mice synthesis and sar studies of mono o-prenylated coumarins as potent 15-lipoxygenase inhibitors 5-farnesyloxycoumarin: a potent 15-lox-1 inhibitor, prevents prostate cancer cell growth kaseb-mojaver, n. 8-farnesyloxycoumarin induces apoptosis in pc-3 prostate cancer cells by inhibition of 15-lipoxygenase-1 enzymatic activity synthesis, in vitro cytotoxicity activity against the human cervix carcinoma cell line and in silico computational predictions of new 4-arylamino-3-nitrocoumarin analogues synthesis and antiproliferative activity of 3-and 7-styrylcoumarins styrylcoumarin 7-sc2 induces apoptosis in sw480 human colon adenocarcinoma cells and inhibits azoxymethane-induced aberrant crypt foci formation in balb/c mice synthesis and in vitro anticancer activities of diethylene glycol tethered isatin-1,2,3-triazole-coumarin hybrids pyrimidine derivatives as novel lsd1 inhibitors design, synthesis, cytotoxicity and mechanism of novel dihydroartemisinin-coumarin hybrids as potential anti-cancer agents triazole tethered isatin-coumarin based molecular hybrids as novel antitubulin agents: design, synthesis, biological investigation and docking studies azide-alkyne cycloaddition towards 1h-1,2,3-triazole-tethered gatifloxacin and isatin conjugates: design, synthesis and in vitro anti-mycobacterial evaluation discovery of fluorescent coumarin-benzo[b]thiophene 1, 1-dioxide conjugates as mitochondria-targeting antitumor stat3 inhibitors signal transducer and activator of transcription-3, inflammation and cancer: how intimate is the relationship? carbonic anhydrases: novel therapeutic applications for inhibitors and activators multiple binding modes of inhibitors to carbonic anhydrases: how to design specific drugs targeting 15 different isoforms? anhydrase inhibition and the management of glaucoma: a literature inhibition and the management of glaucoma: a literature antiglaucoma carbonic anhydrase inhibitors: a patent review anticonvulsant/antiepileptic carbonic anhydrase inhibitors: a patent review targeting tumour hypoxia: suppression of breast tumor growth and metastasis by novel carbonic anhydrase ix inhibitors interfering with ph 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nosocomial pathogens: no eskape 4-dihydropyrimidinone-coumarin analogues as a new class of selective agent against s. aureus: synthesis, biological evaluation and molecular modelling study microwave-assisted synthesis, computational studies and antibacterial/ anti-inflammatory activities of compounds based on coumarin-pyrazole hybrid bioassay techniques for drug develompent modulation of the antibiotic activity against multidrug resistant strains of coumarins isolated from rutaceae species isolation and antimicrobial activity of coumarin derivatives from fruits of peucedanum luxurians tamamsch novel coumarin-pyrazole carboxamide derivatives as potential topoisomerase ii inhibitors: design, synthesis and antibacterial activity hidden killers: human fungal infections gaffi fungal disease frequency|gaffi-global action fund for fungal infections how common are fungal diseases?-fungal infection trust nosocomial bloodstream infections in us hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study epidemiology of invasive candidiasis: a persistent public health problem synthesis and evaluation of a class of new coumarin triazole derivatives as potential antimicrobial agents 1,2,3-triazole incorporated coumarin derivatives as potential antifungal and antioxidant agents facile synthesis of novel coumarin derivatives, antimicrobial analysis, enzyme assay, docking study, admet prediction and toxicity study antifungal activity, mode of action variability and subcellular distribution of coumarin-based antifungal azoles antimicrobial activity of rhizomes of ferulago trachycarpa boiss. and bioguided isolation of active coumarin constituents coumarins reduce biofilm formation and the virulence of escherichia coli o157:h7 coumarin: a novel player in microbial quorum sensing and biofilm formation inhibition activity of coumarin against candida albicans biofilms susceptibility tests: diffusion test procedures microwave-assisted synthesis, biological evaluation and molecular docking studies of new coumarin-based 1,2,3-triazoles covid-19) events as they happen coumarin: an emerging antiviral agent. heliyon 2020, 6, e03217 plant derived antivirals: a potential source of drug development anti-viral activity of compounds from agrimonia pilosa and galla rhois extract mixture evaluation of antiviral effect and toxicity of total flavonoids extracted from robinia pseudoacacia cv. idaho in vitro and in silico study to evaluate the effectiveness of quercitrin as antiviral drug to dengue virus dehydrojuncusol, a natural phenanthrene compound extracted from juncus maritimus, is a new inhibitor of hepatitis c virus rna replication antiviral activity of resveratrol therapeutic potential of coumarins as antiviral agents bioactive prenylated coumarins as potential anti-inflammatory and anti-hiv agents from clausena lenis bioactive monoterpene indole alkaloids from nauclea officinalis novel tetrahydrofuran derivatives from trigonostemon howii with their potential anti-hiv-1 activities prenylated coumarins from the fruits of manilkara zapota with potential anti-inflammatory effects and anti-hiv activities evaluation of novel n -(3-hydroxybenzoyl)-2-oxo-2h-chromene-3-carbohydrazide derivatives as potential hiv-1 integrase inhibitors three new structures of the core domain of hiv-1 integrase: an active site that binds magnesium design and discovery of hiv-1 integrase inhibitors hydrazide-containing inhibitors of hiv-1 integrase chromone and chromanone derivatives as strand transfer inhibitors of hiv-1 integrase strand transfer inhibitors of hiv-1 integrase: bringing in a new era of antiretroviral therapy who -up to 650 000 people die of respiratory diseases linked to seasonal flu each year influenza pandemics of the 20th century new thiazolyl-coumarin hybrids: design, synthesis, characterization, x-ray crystal structure, antibacterial and antiviral evaluation synthesis and antimicrobial properties of some new thiazolyl coumarin derivatives synthesis, antimicrobial activity and advances in structure-activity relationships (sars) of novel tri-substituted thiazole derivatives bis coumarinyl bis triazolothiadiazinyl ethane derivatives: synthesis, antiviral activity evaluation and molecular docking studies regioselective ibx-mediated synthesis of coumarin derivatives with antioxidant and anti-influenza activities synthesis and structure-activity relationships of imidazole-coumarin conjugates against hepatitis c virus anti-hepatitis b virus activity of esculetin from microsorium fortunei in vitro and in vivo risposta del tessuto al danno origin and physiological roles of inflammation inflammation and the mechanism of action of anti-inflammatory drugs clinical perspectives of anti-inflammatory therapy in the elderly: the lipoxigenase (lox)/cycloxigenase (cox) inhibition concept cyclooxygenase-2 inhibitors: a painful lesson duration of treatment with nonsteroidal anti-inflammatory drugs and impact on risk of death and recurrent myocardial infarction in patients with prior myocardial infarction: a nationwide cohort study thiazole analogues of the nsaid indomethacin as selective cox-2 inhibitors synthesis, in vitro antiproliferative activity and in silico studies of fused tricyclic coumarin sulfonate derivatives molecular modeling, synthesis and screening of some new 4-thiazolidinone derivatives with promising selective cox-2 inhibitory activity synthesis and pharmacological evaluation of n-substituted 2-(2-oxo-2h-chromen-4-yloxy)propanamide as cyclooxygenase inhibitors synthesis and antiinflammatory activity of coumarin derivatives new coumarin derivatives as potent selective cox-2 inhibitors: synthesis, anti-inflammatory, qsar and molecular modeling studies 5-lipoxygenase development of 5-lipoxygenase inhibitors-lessons from cellular enzyme regulation synthesis, anti-inflammatory, analgesic, 5-lipoxygenase (5-lox) inhibition activities and molecular docking study of 7-substituted coumarin derivatives anti-inflammatory prenylated phenylpropenols and coumarin derivatives from murraya exotica the nf-κb signaling pathway in human genetic diseases phosphorilation meets ubiquitination: the control of nf-kb activity the in vitro and in vivo anti-inflammatory effect of osthole, the major natural coumarin from cnidium monnieri (l.) cuss, via the blocking of the activation of the nf-κb and mapk/p38 pathways anti-inflammatory effect of novel 7-substituted coumarin derivatives through inhibition of nf-κb signaling pathway alzheimer's disease: overview the cholinergic hypothesis of geriatric memory dysfunction butyrylcholinesterase inhibitors ameliorate cognitive dysfunction induced by amyloid-β peptide in mice mechanisms of disease: alzheimer's disease the b-secretase enzyme bace1 as a therapeutic target for alzheimer's disease antioxidant and anticholinesterase potential of ferulago cassia with farther bio-guided isolation of active coumarin constituents a new and rapid colorimetric determination of acetylcholinesterase activity novel tacrine-coumarin hybrids linked to 1,2,3-triazole as anti-alzheimer's compounds: in vitro and in vivo biological evaluation and docking study coumarins as cholinesterase inhibitors: a review novel tacrine-1,2,3-triazole hybrids: in vitro, in vivo biological evaluation and docking study of cholinesterase inhibitors design, synthesis and anti-alzheimer's activity of novel 1,2,3-triazole-chromenone carboxamide derivatives novel coumarin-3-carboxamides bearing n-benzylpiperidine moiety as potent acetylcholinesterase inhibitors multifunctional iminochromene-2h-carboxamide derivatives containing different aminomethylene triazole with bace1 inhibitory, neuroprotective and metal chelating properties targeting alzheimer's disease discovery of novel dual-active 3-(4-(dimethylamino)phenyl)-7-aminoalcoxy-coumarin as potent and selective acetylcholinesterase inhibitor and antioxidant synthesis and biological evaluation of 3-arylcoumarins as potential anti-alzheimer's disease agents dual inhibitors of cholinesterases and monoamine oxidases for alzheimer's disease synthesis and evaluation of 7-substituted coumarin derivatives as multimodal monoamine oxidase-b and cholinesterase inhibitors for the treatment of alzheimer's disease synthesis, characterization, crystal structure and evaluation of four carbazole-coumarin hybrids as multifunctional agents for the treatment of alzheimer's disease free-radical-scavenging effect of carbazole derivatives on aaph-induced hemolysis of human erythrocytes inhibition of beta-amyloid peptide aggregation by multifunctional carbazole-based fluorophores design, synthesis and biological evaluation of d-ring opened galantamine analogs as multifunctional anti-alzheimer agents aurone mannich base derivatives as promising multifunctional agents with acetylcholinesterase inhibition, anti-β-amyloid aggragation and neuroprotective properties for the treatment of alzheimer's disease cellular and molecular basis of epilepsy anticonvulsant profiles of certain new 6-aryl-9-substituted-6 design, synthesis and biological evaluation of new hybrid anticonvulsants derived from n-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide and 2-(2,5-dioxopyrrolidin-1-yl)butanamide derivatives synthesis, molecular modeling studies and anticonvulsant activity of certain (1-(benzyl (aryl) amino) cyclohexyl) methyl esters synthesis and docking study of pyrimidine-triazine hybrids for gaba estimation in animal epilepsy models design, synthesis and docking studies of novel benzopyrone derivatives as anticonvulsants design and synthesis of novel parabanic acid derivatives as anticonvulsants synthesis and pharmacological screening of pyridazinone hybrids as anticonvulsant agents design, synthesis, in vivo and in silico evaluation of new coumarin-1,2,4-oxadiazole hybrids as anticonvulsant agents -arylthiazol-4-yl)methyl]azoles as a new class of anticonvulsants: design, synthesis, in vivo screening and in silico drug-like properties new gaba-modulating 1,2,4-oxadiazole derivatives and their anticonvulsant activity design, synthesis and pharmacological evaluation of novel 2-[2-(2-chlorophenoxy) phenyl]-1,3,4-oxadiazole derivatives as benzodiazepine receptor agonists design, synthesis, pharmacological evaluation and docking study of new acridone-based 1,2,4-oxadiazoles as potential anticonvulsant agents anticonvulsant effect of satureja hortensis aerial parts extracts in mice synthesis and anticonvulsant activity of n-substituted 4-amino-3-nitrocoumarins pharmacogenetics of warfarin: current status and future challenges the future prospects of pharmacogenetics in oral anticoagulation therapy almost 60 years old and still causing problems acenocoumarol in thromoembolic disorders current and emerging direct oral anticoagulants: state-of-the-art current and emerging pharmacotherapy for ischemic stroke prevention in patients with atrial fibrillation tecarfarin, a novel vitamin k reductase antagonist, is not affected by cyp2c9 and cyp3a4 inhibition following concomitant administration of fluconazole in healthy participants pharmacokinetics and pharmacodynamics of tecarfarin, a novel vitamin k antagonist oral anticoagulant pharmacokinetics of tecarfarin and warfarin in patients with severe chronic kidney disease synthesis and in vivo evaluation of new coumarin conjugates as potential indirect-action anticoagulants synthesis and biological evaluation of c-3 aliphatic coumarins as vitamin k antagonists synthesis and structure-activity relationships of novel warfarin derivatives pharmacogenomics of 4-hydroxycoumarin anticoagulants coumarin derivatives from ainsliaea fragrans and their anticoagulant activity world health organization (who) a new oral therapy for diabetes management: alpha-glucosidase inhibition with acarbose use of the alpha-glucosidase inhibitor acarbose in patients with 'middleton syndrome': normal gastric anatomy but with accelerated gastric emptying causing postprandial reactive hypoglycemia and diarrhea synthesis and kinetics studies of n -(2-(3,5-disubstituted-4h-1,2,4-triazol-4-yl)acetyl)-6/7/8-substituted-2-oxo-2h-chromen-3-carbohydrazide derivatives as potent antidiabetic agents pharmacological significance of triazole scaffold 1,2,4-triazole: a review of pharmacological activities synthesis, spectroscopic characterization, reactive properties by dft calculations, molecular dynamics simulations and biological evaluation of schiff bases tethered 1,2,4-triazole and pyrazole rings synthetic heterocyclic candidates as promising α-glucosidase inhibitors: an overview synthesis, α-glucosidase inhibition and molecular docking study of coumarin based derivatives syntheses of new 3-thiazolyl coumarin derivatives, in vitro α-glucosidase inhibitory activity and molecular modeling studies synthesis and biological evaluation of 3-arylcoumarin derivatives as potential anti-diabetic agents biscoumarin-1,2,3-triazole hybrids as novel anti-diabetic agents: design, synthesis, in vitro α-glucosidase inhibition, kinetic and docking studies synthesis, in vitro evaluation and molecular docking studies of novel triazine-triazole derivatives as potential α-glucosidase inhibitors stimulation of insulin secretion by 5-methylcoumarins and its sulfur analogues isolated from clutia lanceolata forssk the in vivo dermal absorption and metabolism of [4-14c]coumarin by rats and by human volunteers under simulated conditions of use in fragrances a new coumarin laser dye 3-(benzothiazol-2-yl)-7-hydroxycoumarin lack of evidence for allergenic properties of coumarin in a fragrance allergy mouse model two novel aggregation-induced emission active coumarin-based schiff bases and their applications in cell imaging an injectable peg-bsa-coumarin-gox hydrogel for fluorescence turn-on glucose detection evaluation of antifungal activities and structure-activity relationships of coumarin derivatives development of green/red-absorbing chromophores based on a coumarin scaffold that are useful as caging groups application of coumarin dyes for organic photoredox catalysis the smart drug delivery system and its clinical potential light-triggered release of photocaged therapeutics-where are we now? photoactivated drug delivery and bioimaging beyond proof of principle light-controlled tools coumarinylmethyl caging groups with redshifted absorption a blue-absorbing photolabile protecting group for in vivo chromatically orthogonal photoactivation water-soluble red-shifted coumarin photocages coumarin-caged polyphosphazenes with a visible-light driven on-demand degradation a ratiometric two-photon probe for quantitative imaging of mitochondrial ph values a sensitive and selective fluorescent coumarin-based probe for detection of hypochlorite ion and its application to cellular imaging a sensitive and selective fluorescent probe for detection of glutathione in the presence of cu 2+ and its application to biological imaging a coumarin-based fluorescent probe for hypochlorite ion detection in environmental water samples and living cells a coumarin based fluorescent probe for rapidly distinguishing of hypochlorite and copper (ii) ion in organisms fluorescence quenching-based mechanism for determination of hypochlorite by coumarin-derived sensors novel coumarin-based fluorescent probe for selective detection of cu(ii) synthesis and application of a highly selective copper ions fluorescent probe based on the coumarin group coumarin based hydrazone as an ict-based fluorescence chemosensor for the detection of cu 2+ ions and the application in hela cells coumarin-based multifunctional chemosensor for arginine/lysine and cu 2+ /al 3+ ions and its cu 2+ complex as colorimetric and fluorescent sensor for biothiols a coumarin-based colorimetric and fluorescent dual probe for palladium(ii) ions that can be used in live cells thioacetalized coumarin-based fluorescent probe for mercury(ii): ratiometric response, high selectivity and successful bioimaging application highly selective on-off fluorescence recognition of fe 3+ based on a coumarin derivative and its application in live-cell imaging a novel fluorescein-coumarin-based fluorescent probe for fluoride ions and its applications in imaging of living cells and zebrafish in vivo a novel coumarin-based fluorescent sensor for ca 2+ and sequential detection of f − and its live cell imaging dicyanovinylcoumarin as a turn-on fluorescent sensor for cyanide ion a near-infrared fluorescent probe for rapid, colorimetric and ratiometric detection of bisulfite in food, serum and living cells a review of sulphites in foods: analytical methodologyand reported findings sulfites in foods: uses, analytical methods, residues, fate, exposure assessment, metabolism, toxicity and hypersensitivity clinical effects of sulphite additives health hazards from volcanic gases: a systematicliterature review estimation of bisulfate in edible plant foods, dog urine and drugs: picomolar level detection and bio-imaging in living organisms research on cruciferous vegetables, indole-3-carbinol and cancer prevention: a tribute to lee w. wattenberg crouciferous vegetables: cancer protective mechanism of glcisunolate hydrolisi products and selenium studies on the mechanism of myrosinase: investigation of the effect of glycosil acceptors on enzyme activity rosen's emergency medicines new insights into the antibacterial activity of hydroxycoumarins against ralstonia solanacearum molecular traits controlling host range and adaptation to plants in ralstonia solanacearum environmental cues controlling the pathogenicity of the plant pathogen ralstonia solanacearum needs aerotaxis for normal biofilm formation and interactions with its tomato host coumarins from the peel of citrus grown in colombia: composition, elicitation and antifungal activity secondary metabolites in plant innate immunity: conserved function of divergent chemicals two classes of plant antibiotics: phytoalexins versus phytoanticipins design, synthesis and antifungal activity evaluation of coumarin-3-carboxamide derivatives synthesis of new coumarin 3-(n-aryl) sulfonamides and their anticancer activity explosive properties of 1-hydroxybenzotriazoles synthesis and antitumor activities of a series of novel quinoxalinhydrazides synthesis, characterization and antibacterial activities of n-[1-(substituted phenyl) ethyl]-2-hydroxybenzohydrazide synthesis, characterization and antifungal activity of coumarin-functionalized chitosan derivatives principles of bioinorganic chemistry synthesis and characterization of a binuclear coumarin-3-carboxylate copper(ii) complex copper(ii) complexes of coumarin-derived schiff base ligands: pro-or antioxidant activity in mcf-7 cells? role of reactive oxygen species (ros) in therapeutics and drug resistance in cancer and bacteria the two faces of reactive oxygen species in cancer oxidative stress and lipid peroxidation products in cancer progression and therapy inhibition of landschutz ascites tumour growth by metal chelates derived from 3,4,7,8-tetramethyl-1,1 0-phenanthroline strong in vitro and vivo cytotoxicity of novel organoplatinum(ii) complexes with quinoline-coumarin derivatives synthesis, crystal structure and investigation of the catalytic and spectroscopic properties of a zn(ii) complex with coumarin-hydrazone ligand non-cytotoxic antibacterial silver-coumarin complex doped sol-gel coatings recent advances in the synthesis of coumarin derivatives via knoevenagel condensation: a review an overview on synthetic strategies to coumarins deciding wether to go with the flow: evaluating the merits of flow reactors for synthesis microreaction technology as a novel approach to drug design, process development and reliability continuous-flow technology-a tool fo the safe manifacturing of active pharmaceutical ingredients two-stage flow synthesis of coumarin via o-acetylation of salicylaldehyde synthesis of coumarin derivative using polymer supported reagents one photocatalyst, n activation modes strategy for cascade catalysis: emulating coumarin biosynthesis with (-)-riboflavin visible-light-driven, photoredox-catalyzed cascade of ortho-hydroxycinnamic esters to access 3-fluoroalkylated coumarins bichromatic photosynthesis of coumarins by uv filter-enabled olefin metathesis solvents and solvent effects in organic chemistry solvent-free coumarin synthesis via pechmann reaction using solid catalysts solvent-free reactions solvent-free coumarin synthesis hetero-annulated coumarins as new ache/buche inhibitors: synthesis and biological evaluation nano-silica sulfuric acid as an efficient catalyst for the synthesis of substituted pyrazoles preparation and characterization of lewis acid grafted sulfonated carbon@titania composites for the multicomponent synthesis of 4h-pyrimido[2,1-b]benzothiazoles and benzoxanthenones under solvent-free conditions a green and convenient approach for the one-pot solvent-free synthesis of coumarins and β-amino carbonyl compounds using lewis acid grafted sulfonated carbon@titania composite ultrasonic accelerated knoevenagel condensation by magnetically recoverable mgfe 2 o 4 nanocatalyst: a rapid and green synthesis of coumarins under solvent-free conditions fe 3 o 4 @boehmite-nh 2 -coii nps: an environment friendly nanocatalyst for solvent free synthesis of coumarin derivatives through pechmann condensation reaction ionic liquids-an overview ionic liquids. green solvents for the future when can ionic liquids be considered readily biodegradable? biodegradation pathways of pyridinium, pyrrolidinium and ammonium-based ionic liquids biodegradation studies of ionic liquids toxicity of ionic liquids: eco(cyto)activity as complicated but unavoidable parameter for task-specific optimization brønsted acidic ionic liquid-catalyzed tandem reaction: an efficient approach towards regioselective synthesis of pyrano[3,2-: c] coumarins under solvent-free conditions bearing lower e-factors novel brønsted acidic ionic liquids and their use as dual solvent-catalysts condensation of 2-naphtol with arylaldehydes using acetic acid functionalized ionic liquids as highly efficient and reusable catalysts tandem knoevenagel-michael-cyclocondensation reactions of malononitrile, various aldehydes and dimedone using acetic acid functionalized ionic liquid synthesis of bis-coumarins over acetic acid functionalized poly(4-vinylpyridinum) bromide (apvpb) as a green and efficient catalyst under solvent-free conditions and their biological activity microwave-assisted organic synthesis-a review dielectric parameters relevant to microwave dielectric heating acceleration of the pechmann reaction by microwave irradiation: application to the preparation of coumarins microwave promoted and improved thermal synthesis of pyranocoumarins and furocoumarins task-specific ionic liquid as reagent and reaction medium for the one-pot horner-wadsworth-emmons-type reaction under microwave irradiation one-pot wittig and knoevenagel reactions in ionic liquid as convenient methods for the synthesis of coumarin derivatives ytterbium triflate promoted coupling of phenols and propiolic acids: synthesis of coumarins microwave-assisted synthesis of xanthones promoted by ytterbium triflate ytterbium triflate catalysed meerwein-ponndorf-verley (mpv) reduction synthesis of coumarin by yb(otf) 3 catalyzed pechmann reaction under the solvent-free conditions synthesis of 7-hydroxy-4-methylcoumarin via the pechmann reaction with amberlyst ion-exchange resins as catalysts coumarin derivatives solvent-free synthesis under microwave irradiation over heterogeneous solid catalysts microwave-assisted synthesis of phenylpropanoids and coumarins: total synthesis of osthol synthesis of indolo[2,3-c]coumarins and indolo[2,3-c]quinolinones via microwave-assisted base-free intramolecular cross dehydrogenative coupling this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license funding: this research received no external funding. the authors declare no conflict of interest. key: cord-004879-pgyzluwp authors: nan title: programmed cell death date: 1994 journal: experientia doi: 10.1007/bf02033112 sha: doc_id: 4879 cord_uid: pgyzluwp nan it is widely held that all developmental cell death is of a single type (apoptosis) and that neuronal death is primarily for adjusting the number of neurons in a population to the size of their target field through competition between equals for target-derived factors. we shall draw on our research and on that of others to criticize these views and replace them by the following. at least three types of neuronal death occur, only one of which resembles apoptosis; a neuron can choose between several self-destruct mechanisms depending on the cause of its death. the purpose of the death is to regulate connectivity, not neuron number. competitors for trophic factors are unequal, and many losers have made axonal targeting errors. a neuron's survival and differentiation depend on multiple anterograde and retrograde signals. activity affects retrograde signals and some but not all anterograde ones. the pattern of activity is more important than the overall amount. in rodents, the period of naturally occuring cell death of motoneurons is followed by a period of supersensitivity to axonal injury. thus, in newborn rodents lesion of the facial nerve leads to a rapid degeneration of the injured motoneurons. we have tested whether overexpression, in rive, of the bcl-2 proto-oncogene was capable of preventing death of axotomized motoneurons. to address this question we used transgenic mice whose motoneurons overexpress the bcl-2 protein. one of the two facial nerves of newborn mice was transected on the 2nd-3rd post-natal day. seven days after the lesion, the morphology of the facial nuclei was analyzed. in control mice, and when compared to the intact nucleus, 70 to 80 % of axotomized motoneurons had disappeared. in contrast, in the transgenic animals, the number of motoneurons on the lesioned side remained unchanged when compared to the eontralateral nucleus. furthermore, their axons remained visible up to the distal lesion site. these experiments show that, in rive, motoneurons overexpressing the bcl-2 protein survive after axotomy, and suggest that, in rive, bcl-2 protect neurons from experimentally induced cell death and could be a target for treatment of motoneurons degenerative diseases. messmer s., mattenberger l., sager y., blatter-garin m-c., pometta d., kate a., james r.w. drpt de mrdeeine, drpt. de pharmacologie, div. de neurophysiologie clinique, facult6 de mrdecine, gen~ve. clusterin is a widely expressed glycoprotein, highly conserved across species. numerous functions have been postulated for this protein. the most important are roles in lipid transport, as elusterin is associated with apolipoprotein ai in hdl, complement regulation and tissue remodelling, in particular during cell death and differentiation. using cultures of rat spinal cord neurones (90% neurons and 5-10% non-neuronal cells), we have studied the expression of clusterin and ape e in glutamate-induced neuronal cell death to examine potential roles in lipid management. up-regulation of the two proteins was observed. clusterin and ape e appear in the conditioned medium respectively 15h and 7.5h after incubation with glutamate. control studies, in the presence of a noncompetitive nmda receptor agonist showed the secretion of clusterin and ape e to be diminished by >60%. no up-regulation of either protein was observed in complementary studies with exclusively non-neuronal cell cultures. the cellular origin of the 2 secreted proteins is presently under investigation. programmed cell death and tissue remodelling are consequences of hormonally induced restructuring of the rat ventral prostate after castration and the rat mammary gland after weaning. we used the "differential display"-method (liang and pardee, 1992, science 257:967) to detect and isolate edna fragments whose corresponding rnas are regulated either coincidentally, or in an organ specific fashion during mammary gland involution and postcastrational prostate regression. partial sequencing of 12 clones revealed high, but not absolute homology of 5 fragments with sequences, previously characterized in different biological contexts. these five encode functions which could be anticipated to be important for cell growth and/or programmed cell death, we are presently investigating the functions of several of these transcripts in cell culture and in rive. antisense oligos are being employed in vivo to determine whether these genes contribute to the phenotype of programmed cell death. b epitopes derived from the envelope gp52 glycoprotein (ep3) or from the viral superantigen of mmtv have been incorporated into inert or live vaccines. the inert vaccine consists of purified chimeric proteins which contain the b epitopes alone or fused to multimeric promiscuous t helper epitopes from tetanus toxin. mice were immunized subcutaneously with these chimeric proteins. the live vaccine consists of an avirulent strain of salmonella typhimurium which expresses the mmtv epitopes in the form of chimeric proteins fused to the nucleocapsid protein of hepatitis b virus. this vaccine is given to mice in one oral dose. the level, duration and isotype of the immune response generated by each vaccine have been measured and compared. the level of protection has been investigated by systemically challenging immunized mice with the relzovims. a reduced binding of oxytocin (ot) occurs with aging in some, but not all, areas of the rat brain (arsenijevic et al., experientia 1993, 49, a75) . the candate putamen showed the most impressive loss of ot receptors. two other regions, the hypothalamic ventromedial nucleus (vmh) and the islands of caueja (icj) had also an important deficit of ot binding sites. on the other hand, these two regions were known to be sensitive to sex steroids. in the present work, we treated from 20 month old rats during one month with testosterone propionate (2 #g/kg s.c., once every 3 days) dissolved in oil. three rats of the same age injected with oil only served as controls. we labelled ot receptors throughout the brain of old rats using a 125i-labelled ligand specific for ot receptors. analysis of autoradiograms by an image analyzer revealed that the testosterone treatment increased ot binding sites in the vmh, in the icj, and, to a lesser extent, in the bed nucleus of the stria terminalis, a region also sensitive to sex steroids, by contrast, in the caudate putamen, the disappearance of ot receptors was not compensated. in conclusion, the decrease of ot receptors occurring in vmh and icj with aging can be reversed by administration of gonadal steroids. in contrast, the loss of ot receptors in the striatum appears to depend on another mecanism. vasopressin (avp) receptors are expressed transiently in the facial nucleus during development (tribollet et el., 1991, dev. brain res., 58, 13-24) . avp may therefore play a role in the maturation of neuromuscular connexions in the neonate rat, and possibly in the restanration of these connexions after nerve lesion in the adult. in order to investigate the latter proposition, we have sectionned the facial nerve in adult rats and used quantitative autoradiography to look at avp binding sites in the facial nucleus at various postoperative times. we observed a massive and transient increase of avp binding sites on the operated side. the number of facial avp binding sites reaches a maximum about one week after nerve section, remains stable during 2-3 weeks, then begin to decrease towards control level. the induction of avp receptors is markedly delayed if the proximal stump of the nerve is ligated. to assess whether other motor nuclei would also react to axotomy by up-regulating the expression of avp receptors, we have sectionned the hypoglossal nerve and the sciatic nerve. in both cases, the binding of avp receptor ligand increases massively in the respective motor nuclei, with a time-course similar to that found in the facial nucleus. altogether, our data suggest that central avp could be involved in the process of nerve regeneration. cytotoxic t-cell mediated apoptosis schaerer,e, karapetian,o.,adrian,m. and tschopp,j. inst.de biochimie, univ.de lausanne, 1066 epalinges. an apoptotic cell death mechanism is used by cytolytic t cells (ctl) to lyse appropriate target cells. ctl harbor cytoplasmic storage compartments, containing the lytic protein perforin and serineproteases (granzymes), whose content is released upon target cell interaction. we show that these granules are multivesieular bodies and that degranulation releases these intragranular vesicles (igv) having granzymes, t-cell receptor and yet undefined proteins associated. isolated igvs and perforin induce dna breakdown in target cells within 20 minutes. microscopic analysis demonstrates that igv specifically interact with target cell via the t-cell receptor and that their contents is taken up by the target cell. already 15 min. after interaction, 3 distinct igv proteins are found in the nucleus of the target cell.one of the molecules has been identified to be granzyme a, previously reported to be involved in apoptosis. we propose that lymphocytes transfer apoptosisinducing proteins to the nucleus of the target cells using vesicles as vehicles for delivery. cytotoxic t cells kill their targets by a mechanism involving membranolysis and dna degradation (apoptosis). recently, two sets of proteins have been proposed as dna breakdown-inducing molecules in t cells: granzyme a, b and tia-i. in this study, we cloned and further characterized the tia-i mouse homologue. aa sequence comparison with the human tia-1 showed an overall identity of 93%. devoid of a signal peptide, tia is yet localized to cytotoxic granules, probably targeted via a gly-tyr-motif. as tia-i, its mouse homolcgue contains three rnabinding domains. expression of tia during development shows a very strong signal in the brain and weaker signals in thymus, heart and other organs. during embryonic development several structures that contribute to organogenesis form transiently and are later eliminated by apoptosis. this pattern of tia expression could indicate its involvement in apoptosis. prostate involution occurs after castration in rats and is associated with the death by apoptosis of a large fraction of the epithelial cells. we have isolated several genes from a prostate involution bacteriophage lambda library using differential screening methods. among these clones, one d~monstrated an especially strong signal when used as a probe against northern blots of prostate mlhna obtained before, and at different times after castration. this gene is down-regulated after castration by 40-fold within 5 days. intramuscular injection of a testosterone depot resulted in complete restoration of expression within 24 hours. upon sequencing it became apparent that this clone has a high degree of homology to a known ndah dehydrogenase encoded in mitochondrial dna. the clone failed to hybridize to any transcripts from rat organs other than prostate. we are now in the process of isolating the htm~n hc~olog to this gene for use as a biomarker in study of benign hyperplasia and developing carcinoma. this gene is a possible indicator for testosterone-independent cell populations or of cells lacking ftl~ctional testosterone receptor. during the first three postnatal weeks the rat lung undergoes the last two developmental stages, the phase of alveolarization and the phase of microvascular maturation. the latter involves a decrease of the connective tissue mass in the alveolar septa and a merging of the two capillary layers to a single one. speculating that programmed cell death may play a role during this remodeling, we searched for the presence of apoptotie cells in rat lungs between days 10 and 24. lung paraffin sections were treated with y-terminal transferase, digoxigenin-dutp, and anti-digoxigeninfluorescein-f(ab)-fragments, and the number of fluorescent nuclei was compared between sections at different days. while the number of apoptotie ceils was low until the end of the second week and at day 24, we observed an about eight fold increase of fluorescent nuclei towards the end of the third week. we conclude that programmed cell death is involved in the structural maturation of the lung. brunner, a., wallrapp, ch., pollack, i, twardzik, t. and schneuwly, s. lehrstuhl genetik, biozentrum universit~t w~rzburg, mutants in the giant lens (g/l) gene show a strong disturbance in ommatidial development. in the absence of any gene product, additional phetoreceptors, cone cells and pigment cells develop. opposite effects can be seen in flies in which the gene product of the giant lens gene can be ectopically expressed by heat shock. a second very typical phenotype is the disturbance of photoreceptor axon guidance. molecular analysis of gil shows that it encodes a secreted protein of 444aa containing three evolutionary conserved cystein-motives very similar to egf-like repeats. we propose that gil functions as a secreted signal, most likely a lateral inhibitor for the development of specific cell fates and that gil, either directly or indirectly, is involved in targeting photoreceptor axons into the brain. the decrease in cellularity during scar establishment is mediated through apoptosis desmouliere, a., redard, m., darby, i., and g. gabbiani department of pathology, cmu, 1 rue michel server, 1211 gen~ve 4 dudng the healing of an open wound, granulation tissue formation is characterized by replication and accumulation of fibroblastic cells, many of which acquire morphological and biochemical features of smooth muscle cells and have been named myofibroblasts (sch0rch et el., histology for pathologists, t992). as the wound evolves into a scar, there is an important decrease in ceuuladty, including disappearance of myofibroblasts. the question adses as to which process is responsible for myofibroblast disappearance. during a previous investigation on the expression of (z-smooth muscle actin in myofibroblasts, we have obsewed that in late phases of wound healing, many of myofibroblasts show signs of apoptosis end suggested that this type of cell death is responsible for the disappearance of myofibroblasts (darby et al., lab. invest. 63:21, 1990) . we have tested this hypothesis by means of electron microscopy and morphometry and by in situ end-labeling of fragmented dna (wijsman et al., j. histochem. cytochem. 41:7, t 993) . our results show that the number of apoptotic cells increases as the wound closes and suggest that this may be the mechanism for the disappearance of myofibroblasts as well as for the evolution of granulation tissue into a scar. (supported by the swiss national science foundation, grant n~ s01-16 r. jaggl, a. marti and b. jehn. universit~t bern, akef, tiefenaustr. 120, 3004 bern at weaning the mammary gland undergoes a reductive remodelling process (involution) which is associated with the cessation of milk protein gene expression and apoptosis of milk-produclng epithelial cells. this process can be reversed by returning the pups to the mother within 1 day. elevated nuclear protein kinase a (pka) activity was observed from one day post-lactation, paralleled by increased c-los, junb, ]und and to a lesser extent c-]un mrna levels. ap-1 dna binding activity was transiently induced and the ap-1 complex was shown to consist principally of cfos/jund. oct-1 dna binding activity and oct-1 protein were gradually lost from the gland over the first four days of involution, whereas oct-1 m_rna levels remained unchanged. comparing nuclear extracts from normal mammary glands with nuclear extracts from glands which had been cleared of all epithelial cells three weeks after birth revealed that pka activation, ap-1 induction and oct-1 inactivation are all dependent on the presence of the epithelial compartment. the increased fos/jtm expression and the inactivation of oct-1 may be consequences of the increased pka activity. when involution is reversed, both, pica activity and ap-1 dna binding activity (and fos andjun mrna levels) are reduced to basal levels. our data suggests a role for pka and ap-1 on progranlmed cell death of manlnmry epithelial ceils. bcl-2~ does not require membrane attachment for its survival activity c. borner*, i. martinout, c. mattmann*, m. irmler*, e. sch&rrer*, j.-c. martinou-j-, and j. tschopp*. * institute of biochemistry, university of lausanne, 1066 epalinges, 1 institute of molecular biology, glaxo inc.,1228 plan los ouates. 8cl-2(z is a mitochondrial or perinuclear-associated oncoprotein that prolongs the life span of a variety of cell types by interfering with programmed cell death. how it exerts this activity is unknown but it is believed that membrane attachment is required. to identify critical regions in bcl-2o~ for subcellular localization and survival activity, we created by site-directed mutagenesis, various mutations in regions which are most conserved between the different bcl-2 species. we show here that membrane attachment is not required for the survival activity of bcl-2o< a truncation mutant of bcl-2(z lacking the last 33 amino acids (t3) including the hydrophobic domain is soluble, yet fully active in blocking apoptosis of sympathetic neurons induced by ngf deprivation or l929 fibroblasts induced by tnfc~ treatment. we further provide evidence for a putative functional region in bcl-2 which lies in the conserved domains 4 and 5 upstream of the hydrophobic cooh terminal tail. the breakdown of nuclear dna is considered to be a hallmark of apoptosis. we previously identified the perinuclear membrane localized dnase i as the endonuclease involved in the formation of oligonucleosomal-sized fragments (dna ladder). it is not clear how the nuclease is activated and has access to the dna. we show that in thymocytes induced to undergo apoptosis, lamin breakdown preceded dna laddering. by transfeeting hela cells with a constitutively active cdc2 mutant, nuclear envelope breakdown and typical apoptotic features (ehromatin condensation) were observed. moreover, co-transfection with cdc2 mutant and dnase i led to dna degradation. we propose that apoptosis can be induced by wrongly timed and hence abortive mitosis leading to uncontrolled nuclear membrane disintegration. s02-01 s02-04 platelet-derived growth factor (pdgf) is thought to play an active role in fibrosing diseases. bronchiolitis obliterans-organizing pneumonia (boop) is a condition characterized by intraluminal proliferation of connective tissue inside distal air spaces. to evaluate pdgf expression in boop we performed immunohistoehemistry on lung biopsies from 20 patients and 10 controls free of fibrosis. sedal sections were stained with an antibody against either pdgf or the monoeyte/macrophage marker cd68, in both groups the pdgf ~9 cells were essentially tissue macrophages. using point counting to measure volume fraction (vv) , pdgf-pesitive cells represented 4.65+1.63% (mean+sd) of the volume occupied by lung tissue in the boop cases, and 2,12+0.65% in the controls (!0<0,001). similarily, 10.73+4.69% of the lung tissue was occupied by cd68 e~ macrophages in the boop cases, compared to 5.37:~3.73% in the controls (p<o.005). a positive linear correlation was found between the v v of pdgf ~ cells and the v v of cd68@ cells on the 30 cases (r=g.50 p<0.0o5). we conclude that boop is characterized by a recruitment of tissue macrophages and that a significant proportion of them express pdgf. as pogf can also induce the macrophage chemo-attractant mcp-1, we speculate that maerophages are involved in a positive feed back regulation, and may play a pivotal role in the pathogenesis of boop. barrier lesions in hydrostatic pulmonary edema d.wu, h.bachofen, u.gyger and e.r. weibel department of anatomy, university of bern, ch 3012, bern pulmonary edema induced in isolated perfused rabbit lungs by moderate elevation of perfusion pressure (14 tort ) results in the formation of characteristic bleb-like extensions of the thin cytoplasmic leaflet of alveolar epithelium ( bachofen et al. am rev resp dis. vo1.147. pp989-996,1993) . in this study, we show by means of electron microscopy and morphometry that the frequency of the blebs correlate with the distribution of alveolar edema fluid. we find that 5% of the epithelial surface is involved in the formation of blebs. three-dimensional reconstruction showed that blebs had near-spherical shape and that their opening toward the interstitium is irregular. these finding demonstrate a great plasticity of epithelial cell extensions when subjected to moderate pressure stress. at high pressure the cell linings demonstrate clear breaks. two major roles have been defined for no: cellcell communication mediated by the stimulation of cgmp synthesis, and cytotoxicity by direct interaction of the free radical no with cellular target. to investigate these effects in human epithelia, which constitute a major source of no in man, we introduced the murine inos sequence into sv40-t immortalized human bronchial epithelial cells (beas-2b). inos activity was higher than 100 pmol citrulline/min/ mg prot in the first passages of inos transfected cells, but it decreased by subculturing. no inos activity was detected in cells transfected with control vector. in inos transfected cells, no stimulated a cgmp synthesis and c-los expression which could be inhibited by addition of lnma. thus, in human epithelial cells no is able to significantly alter signal transduction pathway. partial pneumoneetomy, i.e. the resection of lung tissue, is known to initiate compensatory growth in the remaining lobes. under appropriate conditions this results in a complete restoration of lung parenchymal structure and function. unfortunately the molecular mechanisms controlling the onset of this compensatory growth are poorly understood. the aim of our study was to find and eharacterise genes which are up or down regulated during this time. for isolating such genes, we chose the differential display approach (p.liang and b.pardee 1992, science 257:p 967). therein a 3'end fragment of a subset of reverse transcribed mrnas is amplified by the polymerase chain reaction. the fragments can be separated on a dna sequencing gel. genes differentially expressed in pneumonectomised and sham-treated rats show different patterns on these gels. the fragments of such genes can be isolated from the gel and cloned into vectors in order to provide tools to characterise their gene. a set of such genes, some up-regulated and others downregulated following pneumonectomy, have been isolated, partially sequenced and characterised. time periods. our new plasma marker consisting of highly concentrated gold nanospheres was infused via a femoral vein catheter into the right atrium of anaesthetised and ventilated rabbits. five or 10 seconds after start of infusion, the blood flow was stopped and the lung was fixed by instillation. silver enhanced paraffin sections revealed labeled and unlabeled blood vessels. electron-microscopic sections showed various plasma gold particle concentration. morphometric analysis of electron micrographs showed an increase of the portion of marked capillaries from shorter to longer circulation times. based on our results, we conclude the existance of inhomogeneous plasma flow velocities within the pulmonary microvascular bed. (supported by snsf grant 31-30946.91) in schizosaccharomyces pombe pairing of meiotic chromosomes is not accompanied by the formation of a tripartite synaptonemal complex (sc), a structure which connects the homologs in most other eukaryotes. meiotic nuclei revealed novel structures ("linear elements") that might be related to the axial elements ($c precursors) of other organisms and function in meiotic chromosome pairing, recombination, and segregation (baler et al. (1993) jcb 121:241). to get further insight into the behavlour of meiotic chromosomes, we performed fluorescence in situ hybridizations on spread nuclei with probes from different chromosomal regions. interestingly, all centromeres and telomeres become clustered during meiotic prophase. the centl:omeres are paired already before meiosis, whereas the telomeres initiate pairing specifically during meiotic prophase, followed by pairing of interstitial regions. painting of whole chromosomes revealed that the homelogs occupy together a specific territory in the nucleus. an expression vector containing the vai gene read by rna polymerase ill, injected into xenopus zygotes, yields high levels of rna in virtually all embryonic cells. anti sense fina is produced from a segment of the xhoxla gene inserted in opposite orientation into the vai gene. immunological staining of whole mount embryos shows that target mrna level and xhoxla protein expression of xhoxla protein is diminished in about half of the antisense treated individuals. accordingly, nearly half of the embryos develop typical malformations in regions where the xitoxla gene is normally expressed. somites el the anterior trunk are heavily disorganized and mesodermal denvalives surrounding the intestinal tract are reduced or missing. antisense inhibition, through production of rna in situ from expression vectors, thus represents a useful tool to study the functional role of zygotic genes in xenopus development. cloning and characterization of the mouse homolog to s.cerevisiae sep1 encoding a dna strand exchange and homologous pairing protein vladimir bashkirov and wolf-dietrich heyer. institute of general microbiology, university of bern, baltzer-strasse 4, 3012 bern. homologous pairing and dna strand exchange are the central steps postulated in the current models of genetic recombination. to date no mammalian gene encoding a homologous pairing protein has been cloned. to get insight into the mechanism of recombination in higher eukaryotes we were interested to clone such genes. protein sequence comparison of the s.cerevisiae homologous pairing protein, sepi, and its homolog from s.pombe, p140 ex~ revealed several conserved stretches of amino acids in their nh2-terminal regions. using fully degenerate primers and mouse testes edna as a template for the polymerase-chain-reaction a product with the expected length has been amplified. this 360 bp dna was shown to encode a putative polypeptide with high degree of homology to both proteins from the two yeasts. the mouse pcr-product was used as a hybridization probe for screening a 1 gtlo mouse testis cdna library. after several rounds of successive screening a total of about 4 kb of mouse edna (msep1) was cloned and sequenced. the putative translation product revealed high homology throughout the entire sequence to its s.pombe and s.cerevisiae counterparts, 38 % and 41% of amino acid identity, respectively. the msep1 sequence was shown to be unique in the genome and the chromosomal gone is likely to contain many introns. we want to establish a functional full length edna clone of msepi and characterize the gone and protein product. we have earlier developed a cell free system to study recombinational repair of dna double strand breaks and deletions. the method allowed us to purify and characterize a high molecular weight multiprotein complex (rc-1), which catalyzes dna recombination. a dna polymerase and dna ligase as well as other enzymatic activities copurify with rc-1.we have commenced an investigation of homologous dna recombination in nuclear extracts which were prepared fzom normal and mutated mouse thymocytes. the scid mutation in mice causes an aberrant v(d)j joining reaction, an elevated sensitivity to ionizing radiation and a defect in recombinational repair. moreover, the normal mouse thymocytes were sorted into the cd4/cd4 double negative (dn), double positive (dp), and single positive (sp) subclasses and their nuclear extracts, and extracts from rag-2 -/-mice, were tested. only the scid mice and the cd4/cd8 sp thymocyte extracts were inactive in the recombinational repair reaction. this indicates a development stage specific activity and a scid specific defect of recombinational repair in thymocytes. restoration of the recombination activity was observed in thymocyte extracts derived from scid, which express a wansgene for the t-cell receptor i~ chain. a protein could be purified from normal thymus cells to near homogeneity which specifically rescues the recombination activity in scid extracts. this protein, srsp ~cid recombination stimulatory ~otein), migrates as a single band of app. 7 2 kda in sds polyacrylamide gel electrophoresis. the ade6-m26 mutation in the ade6 gene of the fission yeast schizosaecharomyces pombe gives rise to a hot spot for meiotic homologous recombination. to address whether transcription plays a role in m26 activity, the effect of the deletion of the ade6 promoter in combination with either the m26 and m375 mutations on recombination frequency was studied at the tetrad level. deletion in eis of the promoter abolishes m26 hot spot activity relative to the m375 control deletion strain. it is possible that deletion of the promoter has eliminated a sequence necessary for m26 hot spot activity, rather than lack of transcription being responsible for the loss of m26 activity. the role of transcription in m26 hot spot activity is currently being examined. we are analysing meiosis in a temperature sensitive top2 mutant. complementary temperature-shift experiments showed that topoisomerase ii is required for both meiotic divisions; early meiotic events are not impaired. in a meiotic time course, dapi staining revealed that the cells are blocked before the first meiotic division. interestingly, in meiotic spreads we found that the spindle pole body (spb) cycle is not affected by the block: the final arrest point showed 1 nucleus with 4 completely separated spbs. we want to analyse the number of spbs and spindle formation with immunofluorescence. we are planning to analyse the course of meiosis in a top2 rec7 double mutant (rec7 is recombination deficient) to see if topoisomerase ii activity is necessary for separation of recombined chromosomes. analysis of meiosis in a top2 rec8 double mutant (rec8 is recombination deficient and does not form linear elements) could give us a clue about the function of the linear elements. genetic recombination is able to induce cancer and to mediate its further progression. in cells from cancer predisposed individuals, mitotic recombination can lead to loss of heterozygous tumor suppressor genes as is e.g. observed in the hereditary form of retinoblastoma. one mechanism for gene loss is inter-or intrachromosomal recombination involving short duplicated sequences. our aim is to identify xenobiotics that are able to induce mitotic recombination and to elucidate the involved molecular mechanisms. for that purpose we have constructed transgenic d. melanogaster (1). rn.). cell lines. cells were stably transfeeted with an extrachromosomal dna element providing a putative recombination substrate+ "l%e constructed element contained two ta'uncated fragments of the neomycin resistance gene (nee r) interrupted by a hygremycin resistance marker (hygr). the hyg r insert was flanked on both sides by identical 352 bp fragments of nee r, different d. m. promoters shown to be constitutively active in various d. m. tissues were inserted 5' to both e. coil genes. restoration of a functional nee r gone may occur by deletion of the hyg r after recombination between the homologous regions. stably transfected schneider line 2 cells were obtained and preliminary results concerning chemical induction of genetic rearrangements will be shown. fleck, 0., sch~r, p. and kohli, j., institute of general microbiology, baltzerstr. 4, 3012 bern to detect mismatch-binding proteins of s. pombe we performed band shift assays with radiolabeled oligonucleotides containing a defined mismatch. we found two distinct mismatch-binding activities. one shows high affinity to most of the mismatches, but is absent for c/c. this protein might belong to the general mismatch-repair system, which is thought to be related to the bacterial muts dependent pathway. the second activity preferentially binds to those mismatches containing a cytosine and therefore represents a novel type of mismatch-binding protein. baur m., sch~r p. and kohli j., institute of general microbiology, baltzerstrasse 4, ch-3012 bern homologs of the salmonella typhimurium (mutl, s and 59 and streptococcus pneumoniae (hexa, b) genes involved in mismatch repair have been identified in several distantly related organisms. so far no mismatch repair gone is known in schizosaccharomyces pombe. in order to get more insight into the ~chanisals of mismatch repair in s. pombe we started the cloning of a mutl homolog. degenerated oligonucleotide primers based on conserved regions of mutl homologs were used in the polymerase chain reaction (fcr) to amplify a corresponding dna fragment from s. pombe. a dna fragment of about 220 bp was amplified whose deduced amino acid sequence shared a high degree of homology with paiql of saccharomyces cerevisiae and mutl, hexb. with the help of this dna fragment the gone was isolated from a genomic dna library by colony hybridization. sequence analysis of this mlhl gene (mlh = mut~ homolog) shows an orf of the expected size with three putative introns. while the deduced amino acid sequence of the 5' region shares strong homology with the procaryotic genes mutl, hexb and the eucaryotic gene pmsi, the amino acid sequenoe of the 3' region shows homology only to pmsi and hexb. the central region of the protein seems to be conserved only weakly. parisi s., and kohli j., institute of general microbiology, university of bern, ch-3012 bern rec7 and rec8 are supposed to be genes with major functions in meiotic recombination since mutations in these genes reduce the frequency of meiotic intragenic recembination ~1000 fold. these genes have been isolated and sequenced but no enzymological activity could be assigned to the protein. a first step toward enzymological and biochemical characterization of rec7 and rec8 is to make beth protein products available in sufficient quantities and in reasonable pure form. for this purpose both genes will be cloned in a s. /x~nbe over-expression system using the strong and inducible nmtl promoter.in parallel both genes will also be expressed in e. coli as gst fusion proteins in order to produce polyclonal antibodies in rats and rabbits. these antibodies can then be used for protein purification in s. pombe as well as for immunofluorescence experiments. induction of expression in e. coli produces a band of the expected size of 56kda for rec7 and of 73kda for rec8. to remove the gst carrier, the rec7 and rec8 fusion proteins can successfully be cleaved by thrombin or factor xa respectively. production of antibodies is in progress. d6bbeling, u., nobi, r,, berchtold, m.w. and kuenzle, c.c. institut fdr veterinarbiochemie, universit6t zurich, wintertharerstrasse 190, normally, only pre b-and pre t-cells can rearrange their immunoglobulin genes by v(d)j recombination, but when other cell types are transfected with the rag-i and rag-2 genes they also become able to perform v(d)j recombination. by transiently transfeeting nih 3t3 fibroblasts with both intact rag genes we found da recombination rate of 0.06%. no recombination was detected, when we transfected the intact rag-1 gene with a mutant rag-2 gene, which lacks 89 c-terminal amino acids, or the intact rag-2 gene with a rag-i gene containing a deletion in the topoisomerase iz homology region. these experiments show that these two regions of the rag genes are essential for v(d)j recombination. when the l4 fibroblast cell line, which has been rendered recombination competent by stable transfection with the rag genes was overtransfected with both wild type rag genes, we found in contrary to an expected increase of v(d)j recombination a reduction by a factor of 2-2.5. this decrease was not observed with the mutant rag genes.this result indicates, that there exists an optimal intracellular rag concentration for efficient v(d)j recombination. m. fischer, a. sailer, h. blieler, t. riilicke*, a. aguzzi +, p. autenried and c. weissmann; 1nstitut fiir molekularbiologie l universitfit ziirich, h6nggerberg, 8093 ziirich, *biologisches zentrallabor and + lnstitut fiir neuropathologie , universitdtsspital ziirich, 8091 zfirich, switzerland the infectious agent of transmissible spongiform encephalopathies such as creutzfeldt-jakob disease in man or scruple in sheep is thought to be prpsc, a posttranslationally modified form of the normal host protein prpc we have shown that mice devoid of prpc (prn-polo) are completely resistant to scrapie. here we report that also heterozygous prn-p o/+ mice with reduced levels of prp c show enhanced resistance to scrapie, as manifested by a long delay in the onset and slow progression of clinical disease. conversely, prn-po/o animals overexpressing prp transgeues exhibit shortened incubation times and accelerated progression of the disease. propagation of the scruple agent is completely abolished in prnpolo animals. high titers of infectivity, about 108 logld50 units/g of brain, are detected in prn-po/+ mice 33 weeks after inoculation (the earliest time point measured), 24 weeks or more before the animals die of scrapie. in contrast, wildtype animals survive no more than 16 weeks after reaching similar titers of infectivity. a practical implication of our findings is that moderate inhibition of prpc synthesis could mitigate disease progression in spongiform encephalopathies. the ruvb protein has been shown, biochemically and genetically, to be involved in the process of homologous recombination in e. coli. one of its functions is to promote branch migration within holliday junctions formed during the process of recombination. to investigate the mechanism by which ruvb promotes branch migration we have used conventional electronmicroscopy, cryo-electron microscopy, scanning transmission electronmicroscopy, and image analysis to study the interaction of ruvb protein with dna. we observed that ruvb forms multimeric rings on dsdna which encircle the dna. we also observed that such rings tend to localize at the holliday junctions. we propose that ruvb rings can actively move along dna using the energy of atp hydrolysis.this movement continues upon encountering a holliday junction, thus forcing branch migration along the dna. gschwind m. and huber g., pharma division, preclinical research, in some families with early onset aizheimer's disease mutations on the 8-app gene have been identified which cosegregate with the disease. so far all these pathogenic mutations have been identified on exons 16 and 17 in humans framing the sequence of the 8-amyloid itself.a genomic clone was isolated from mouse strain c57 black/6 containing the coding regions of the last three exons (16) (17) (18) according to the human sequence. sequencing of all three exons including the large 3' non coding region showed a 100% homology to the previously described balb/c mouse cdna. exon-intron boundaries were determined at the same positions as in human and rabbit genome and the flanking regions in the introns are also very similar, not only is the homology between the human and mouse f$-app very high (97.6%) but also the genomic organisation of their genes is nearly identical. therefore homologous recombination can be applied to introduce molecular 8-app changes and to study their functional consequences on the protein level. we systematically investigated the ability of reca protein to push the dna strand exchange reaction through heterologous regions. we observed that reca can push the strand exchange through substantial heteroiogy (up to 150 bp) when the heterologous insert was placed on the so called proximal end of the linear duplex. in the case of the medial beterology the strand exchange reaction could overcome heterologies of up to about 50 bp. heterology at the distal end was most difficult for reca to resolve, and already 22 bp long insert was completely blocking the progress of the strand exchange. these results, together with earlier electron microscopy studies, allow to propose a molecular mechanism by which reca can exchange strands between partially heterohigous dna molecules. institut for veterin~r-virologie, universit~t bern, ch-3012 bern bovine viral diarrhae virus is a positive-stranded rna virus of the pestivirus group. two biotypes, cytopathic and non-cytopathic in cell culture, can be distinguished. all cytopathic strains characterized so far carry insertions in their genomes. they probably result from a strand-switching activity of the viral rna polymerase during viral replication. to study the switching capacity of the viral enzyme, rna isolated from cells infected with two different strains of bovine viral diarrhea virus were analysed for homologous recombination in an rt-pcr assay. performing the assay we found that fragmented rna present during reverse transcription, the reverse transcriptase enzyme itself and viral rna present in the pcr reaction result in the production of artificial recombination during the assay. it was not possible to detect a homologous recombination activity of the viral polymerase above the background recombination frequency of the rt-pcr assay. our results strongly question the use of the rt-pcr assay for the study of recombination of rna viruses. transpositional rearrangements mediated by insertion sequence (is) elements represent an important source of genetic variation within populations 1. we analyze dna rearrangements at the level of the complete genome by rflp, using 7 is elements as probes for hybridisation to southern blots samples taken at different time points from 12 independent cuttures of e coli b grown by serial transfer for 10'000 generations 2 and stored at -70~ are used to study structure and evolution of genetic diversity within populations. the seven is elements contribute differently to genetic variation and lead to a succession of mutants affecting the genetic structure of the population. we now search for correlations between rflp patterns and the increase of relative fitness over time observed previously 2 naas t., 81o~ m, fitch w.m and arber w, (1993) the complete dna sequence of the locusta migratoria mitochondrial genome has been derived from four cloned mtdna fragments. the sequence is 15.2 kb in length, contains 13 peptide coding sequences, and genes for 22 trnas and two rrnas. the organisation of the genome shows a strong resemblance to drosophila, the gene order and orientation being the same except for the positions two trnas. this contrasts with the only other non-dipteran insect mtdna to have been sequenced, apls mellifera, which shows differences in the positions of ii trnas. this finding is discussed in relation to the at content of the different genomes. the sequences of the three mtdna genomes are also compared directly and the results related to existing knowledge concerning the evolution of insects. udem, new jersey medical school, newark, nj 07103-2714, usa nonsegmented negative strand rna viruses are so far not amenable to reverse genetics. the genomes must associate with nucleocapsid (n), phosphoprotein and polymerase proteins to form a functional rnp. as a step towards reverse genetics of measles virus (mv), plasmid vectors were constructed allowing synthesis of rnas corresponding precisely to a mv genome in which all coding and intercistronic regions are replaced by the chloram-phenicol acetyl transferase (cat) coding region, transfection of these negative polarity transcripts into mv infected hela or 293 cell lines gave rise to cat activity which could be serially transferred and massively amplified together with progeny helper virus in cell cultures. both expected mv/cat chimeric mrna and replication product were identified in the progeny. preliminary experiments indicate that only rnas in which the total number of nucleotides is a multiple of six replicate and transcribe efficiently, consistent with the data on natural and modified copyback di rna of sendal virus (calain and roux, j. virol. 67 4822 (1993) ). precise end to end encapsidation of rna, each n protein contacting six nucleotides, might be required. the embryonic tissue specificity of the human cytomegalovirus immediate-early (hcmv-ie) promoter/enhancer (-522 to +13) was examined by l~-galactosidase staining of transgenic mouse embryos carrying hcmv-ie regulated lacz genes. embryos of three transgemc lines were analyzed between 8.5 -13.5 dpc. for all lines, hcmv-ie transcription was primarily confined to subsets of neural and vascular cells. however, extents of transgene expression along the embryonic primary axis were slightly and greatly restricted in two of the lines relative to the third line at all stages analyzed. these differences most likely represent integration site-dependent chromatin constraints that affect levels of hcmv-ie-directed lxanscripfion. together, the three lines can be taken to define a graded potential for hcmv-ie transcription along the anteriorposterior axis of the embryo with greatest permissivity in mid-axial structures which include the dorsal neural tube at the level of the hindbraln and upper cervical spinal cord, the inner ear and vestibular acoustic ganglia, branchial arteries, aortic sac, and lung buds. the cell typespecificity and axially graded permissivity for hcmv-ie transcription in the mouse embryo provide a basis for understanding the pathology and relative frequencies of different types of birth defects caused by congenital hcmv infection in humans. stauffer, y., 0fford, e. and beard, p., swiss institute for experimental cancer research (isrec), ch-i066 epalinges, switzerland. hpvi8 is associated with genital carcinoma. like other human papillomaviruses, hpvi8 cannot be easily propagated in cell culture, because the viral growth cycle is dependent on the differentiation of its host cell, the keratinocyte. moreover, very little viral capsid protein is found in vivo in infected tissues. the hpvi8 l1 and l2 genes code for the major and minor capsid proteins, respectively. to obtain the viral capsid proteins we made constructs with the l1 and l2 genes for expression in e. coli (the pqe system) and in recombinant vaccinie virus (a modification of the phgs-i system) infected cells. the l1 and l2 proteins expressed in e. cell contained a histidine tag encoded by the vector. this allowed us to purify the proteins on a sickelaffinity column in order to raise antibodies. these will permit us to detect the presence of capsid proteins expressed from recombinant vaccinia viruses. we expect the l1 and l2 proteins expressed together from the vaccinia-based vector to selfassemble to form virus-like particles. to papain-like proteinases. the role of these putative catalytic residues in the activity of the proteinase was studied by site-directed mutagenesis. a minimal proteinase has also been constructed by n-and c-terminal deletion to determine the boundaries of the proteolytic domain, sequence analysis of the c-terminal cleavage product should indicate the site of cleavage. cell entry, uncoat~ng and nuclear transport of adenovirus z . u. f. greber and a. helenius. yale university school of medicine, new haven, ct, u.s.a. to enter cells, viruses must achieve three major goals: transfer their genome across the plasma membrane into the cytosol, target the genome to the replication site and decondense it for replication. adenovirus, an icosahedral nonenveloped particle with a double stranded genomic dna and ii to 15 structural proteins~ enters into human kb cells by a sequential disassembly program during receptor-mediated endocytosi~, acid-dependent penetration into the cytosol, and targeting to the nucleus. during internalization, the fibers, primarily responsible for virus binding to the cell surface, are detached. in early endosomes, the capsid-stabilizing proteins ilia and viii are released. fenton base, a vertexassociated protein implicated in penetration across the endosomal membrane, is removed in endosomes, if penetration is blocked with lysosomotroplc reagents. in the absence of inhibitors, a large fraction of penton base stays associated with the cytoplasmic capsid. the viral dna is disconnected from the shell after proteolysis of the internal protein vi~ a capsid and dna binding component. removal of protein ix, a capsid binding factor, further destabilizes the coat, and the remaining 'stripped-down' virus particles associate with nuclear pore complexes to release their dna-genomes into the nucleoplasm. this stepwise dissociation program is thought to provide the high efficiencies of the entry process needed to successfully deliver the viral dna across multiple barriers to the cell nucleus, the site of replication. ch. schmidt and w. arber; biozentrum der universit~t basel, abt. mikrobiologie, klingelbergstr. 70, ch-4056 basel bacteriophage p1, carried as a single copy plasrnid in e. col~, is widely known for its horizontal gene transfer ability (transduction) in enterobscteria. as a temperate phage, it can lysogenize its host and thereby provides it with accessory functions (restriction of foreign dna by ecop1, functions expressed by transposons carried on the p1 genome or integrative suppression). these properties reflect both symbiotic and evolutionary functions of pi. p1 regulates expression from its genes by two different mechanisms: trans(:ription of early regulatory genes is repressed by the phage encoded repressors c1 and bof, whereas transcription of its morphogenetic genes from p1 specific late promoters is induced by the early expressed gpl0. by site-directed mutagenesis the late promoter ps, composed of the -22 inverted repeat aagttactt and the -10 box, was functionally characterized. p5 and its derivatives compare well with several other late promoters of pt with regard to their sequence dependent strengths: the strong promoters ps, pdar and lpr2b all share the perfect inverted repeat around position -22, whereas the weaker promoters lpr91, lpr82a, lpr82b and lpr83 contain mismatches in their inverted repeats or the inverted repeat is positioned mere upstream relative to the transcription initiation site. homologies. however, the phages formed two immunity groups. the results may suggest a common genetic trait providing a selective advantage to lysogenic aa. by using a dna substrate with defined gap size we found that human immunodeficiency virus type 1 reverse transcriptase (hiv~rt) was able to perform strand displacement dna synthesis. this activity was not affected first by calf thymus proliferating cell nuclear antigen and replication factor c and second by escherichia coli single-stranded dna binding protein~ which together allow dna polymerase ~ to perform strand displacement dna syrlthesis (podust , v. and h~bscher, u. (1993) nucl. acids res. 21, . 3'-azido-2',3'-dideoxy-thymidine tripbosphate inhibited displacement completely indicating that dna synthesis is required for this reaction. the hiv-rt p66 polypeptide alone could perform limited strand displacement dna synthesis, whereas the hiv-rt p51 polydeptide was completely inactive, likely due to its inability to replicate extensively on a mi3 dna template, on the other hand the hiv-rt psl polypeptide e~hanced the strand displacement activity of the hiv-rt d68 subunit at a molar ratio of 4:1 mainly by chasing short products into longer ones. furthermore kinetic experiments after complementation of hiv=rt p66 with kiv-rt psl indicated that hiv-rt psl can restore rate and extent of strand displacement activity by hiv-rt p66 compared to the hiv-rt heterodimer d66/d51, suggesting a function of the 51 kda polypeptide, the mouse mammary tumor virus proviral dna contains an open reading frame in the 3' long terminal repeat which can code for a 36 kda polypeptide with a putative transmembrane sequence and five n-linked glycosylation sites. this gene is known to code for a superantigen which deletes a specific subset el co4 + t-lymphocytes in vivo, the superantigen encoded by the exogenous mouse mammary tumor virus of the gr strain acts specifically on v[314 bearing t-cells. we produced recombinant vaccinia viruses to express either the complete or a truncated orf protein after infection of primate cells in culture. the complete err gene in mammalian cells leads to the production of a 47 kda protein which is specifically detected with an anti-orf-peptide antiserum. the 47 kda protein can be labeled with d-[2-3h]mannose and its synthesis is inhibited by tunicamycin, an n-linked glycosylation inhibitor, indicating that it is a glycoprotein. the truncated orf protein beginning at the second atg of the open reading frame is also modified, but the c-terminal half of orf, starting at the fifth atg (orf5), has the expected size of the non modified polypeptide. pulse-chase experiments indicate that the 47 kda orf g}ycoprotein has a short half-life of about 1.5-2 hours in this system whereas the orf5 truncated protein is even less stable; its half-life is about 20 minutes. the cellular localization of the orf protein and the role it could play in vivo are currently under investigation. conclusion was based on a restriction fragment length polymorphism (rflp) with probes of is-elements. the mutants displayed different growth behavior. the evolutionary stability of 32 of these mutants was further tested by competing a mixture of them for ten days. polymorphism was maintained during this competition but two fitter mutants took over 60% of the population. implications of these results will be discussed in terms of generation and selection of genetic variants for the adaptation of microbial population. mouse mammary tumor virus (mmtv) is produced in the mammary gland of infected females and transmitted by the milk to suckling new-boms. the passage of mmtv from the gut to the mammary gland is still poorly understood, the nature of the tissue tropism of mmtv has not been elucidated, a single receptor present on mammary epithelial and lymphoid cells might serve for the entry of the virus. a cell-type specific entry mechanism could in part be responsible for this phenomenon. alternatively different surlace molecules might be involved in its uptake. we have studied the cell susceptibility to mmtv using a sensitive method of detection of newly acquired exogenous proviral dna sequences, prior to viral integration and transcription. thereby we confirm that mmtv infects mouse, rat and monkey cell lines of different tissue origin in vitro demonstrating the lack of a strict host range specificity in tissue culture as previously described. we have also observed the infection by mmtv of various human cell lines. furthermore various infection susceptibilities are observed for cell lines of a same species. r. cattaneo, p. spielhofer, k. kaelin, m.a. billeter mo/eku/arbio/ogie /, universit~t zorich, hsnggerberg, 8093 zorich subacute sclerosing panencephalitis (sspe), a rare, lethal disease is caused by clonally selected defective measles viruses (dmvs) characteristically mutated. this emerged from sequencing of five mv genes from sspe cases and by functional tests of the envelope glycoproteins hemagglutinin (h) and fusion protein if). these expressed h and f variants migrate poorly to the cell surface but maintain some cell fusion activity whereas the envelope associated matrix protein (m) appears dispensable. thus, the spread of dmv genomes through the brain seems to occur by membrane fusion at local cellular contacts, essentially hidden from the massive immune responses. in more than half of sspe cases, the propagated dmvs contain m genes in which up to 50% of u residues are replaced by cs. such "biased hypermutations" are likely due to the simultaneous conversion of many a residues to inosine in double stranded rna regions accidentally formed, mediated by an ubiquitously occurring adenosine deaminase. similar events at less spectacular degrees have now been observed in functional genes of several negative strand rna viruses. thus, this enzyme might be an important evolutionary driving force for mononegavirates. pull, i., wieland, s., nieder~st, b., keist, r., walter, e., and blum, h.e. department of medicine, university hospital z'tirich infection by hbv-positive organ donors remains a serious problem in transplant surgery. here we present a case of a 58-year-old woman who received a heart transplant from a hepatitis b surface antigen (hbsag) positive donor. the patient was negative for all hbv markers prior to transplantation. two months after transplantation she became hbsag positive and died from subacute liver failure about seven months later. cloning and sequencing of hbv dna revealed several hbv mutants in the serum of the recipient, each with a 108 bp in-frame deletion in the pre-s 1 region. transfection studies in huh-7 cells with the predominant pre-s 1 deletion mutant showed a higher replication competence compared to wild-type hbv. sequence analysis of pcr products from serum of the donor showed mainly wild-type hbv dna in the pre-s region without the pre-s 1 deletion. these data demonstrate that mutant viruses accumulate in immunesuppressed patients. specific mutants may play a critical role in the severe or fatal clinical course of hbv-infection in transplanted patients. in non-neuronal cells herpes simplex virus (hsv) establishes a productive lyric infection starting with transcription of its immediate early (ie) genes by corecruitment of a viral protein (vpi6) and a cellular factor (oct-l) onto characteristic oetamer-like sequence motifs ftaatgarat) in the promotors of the ie genes. in neurons, however, hsv establishes latent infections whereby transcription of the ie genes is prevented and the lyric program aborted. our efforts are focused towards determining which neuronal factors are involved in the regulation of hsv ie gene transcription and whether they may be involved in hsv latency or reactivation in neurons. in electrophoretic mobility shift assays (emsa) using mouse brain nuclear extracts we found that neuronal oct factors (noct-2, noet-3 and noct-4) bind to taatgarat sequences from the viral icp0 and icp4 promoters. an additional brain protein also binds to these sequences but not to a consensus octamer sequence (atgcaaat) as do neuronal oct factors. emsa assays with mutated taatgarat probes show that the taat sequence is crucial for this binding and we have tentatively named this brain protein taat-1. we are currently testing the noct factors in in vivo and in vitro transcription assays to study their involvement in hsv ie promoter regulation. biochemical and functional analysis of a vaccinia virus recombinant containing two copies of the p37k gene. schmutz, c., and wittek, r. institut de biologie animate, univei-sit~ de lausanne, ch-1015 lausanne. the most abundant protein of the vaccinia virus envelope is the palmitated 37k protein. this protein is required for envelopment and subsequent release of virus from infected cells. the amount of extracellular enveloped virus (eev) produced varies considerably depending on the virus strain and host cell line but remains in any case low (<30% of the virus production). if p37k is a limiting factor for envelopment, its overexpression might be a taeans to increase the production of eev. for this purpose a vaccinia virus recombinant containing a second copy of the p37k gene was enginee,'ed. western blot analysis clearly showed increased levels of this protein. titration assays revealed slightly lower yields of both intracellular naked virus (inv) and extracellular enveloped virus (eeu in ceus infected with recombinant virus. unexpectedly, with recombinant virus, the ratio of eev versus inv was significantly lower when compared to wild-type virus infection, despite the higher level of p37k expression. we are currently testing whether this is a consequence of a lower production, or a reduced infectivity of eev particles in cells infected with recombinant virus. hanspeter stalder, christian hertig and ernst peterhans institut f~r veterin~r-virologie, universit-st bern, ch-3012 bern bovine viral diarrhoea virus (bvdv) causes acute transient infection in animals exposed to virus postnataly and persistent infection in animals infected in utero in the early phase of gestation. the latter is associated with specific immunotolerance to the infecting viral strain. animals infected in utero may be born normal and remain healthy despite the persisting infection with a noncytopathic biotype of bvdv. mutation to the cytopathic biotype, or superinfection with an antigenically similar cytopathic biotype, results in lethal mucosal disease. we have pcr amplified and sequenced parts of the region coding for the surface glycoprotein e2 and p80, a nonstructural protein associated with enzymatic activities. over a time of one year, virus obtained from an immunotolerant persistently infected animal remained identical in its nucleotide sequence in the analyzed regions. contrasting with this remarkable evolutionary stasis over some 600-800 virus generations is the heterogeneity of viral isolates obtained from different persistently infected animals. we propose that the muitilineage distribution of bvdv is the result of antigenic drift in the population of acutely infected animals, combined with evolutionary stasis in immunotolerant animals. in 20 to 30% of naturally infected goats, the sevedty of disease in infected animals correlates with the antibody titers to the viral envelope protein (enw) and especially to the gp 38 transmembrane podion (tm). in order to analyze linear b epitopes of env, we produced a random library of caev env polypeptides fused to ~-galactosidase and expressed in e. coli. the complete env gene obtained from an infectious clone of caev-co was subcloned in a puc18 plasmid and dnase digested, random fragments of 200 bp were inseded in ~,gtl 1 dna and expressed in e. coil y1090. as the first step of b epitope mapping and in order to identify immunodominant group epitopes we used high titer sera from naturally infected swiss goats to screen the library. six immunoreactive clones were obtained and subsequently sequenced. all six mapped to the tm region of env. a fine mapping of these immunoreactive regions was performed using pepscan technology and elisa with synthetic peptides. four epitopes could be identified including the eysteine loop corresponding to an immunodominant epitope in other lentiviruses. these peptides were used in vitro to test the reactivity of sera from naturally and experimentally caev-infected goats and in rive to modulate the immune response of goats to tm. the minute virus of mice (mvm) has a 5kb linear single stranded dna genome. at both termini, short selfcomplementary sequences are folded into stable hairpin structures. the 5' (right) hairpin is a single stem structure with only three unpaired bases within the stem. these unpaired form a bubble structure. we examined the requirement in mvm lytic infection for the bubble structure in the 5' terminal hairpin of mvmp. mvmp rf dna containing the entire 3' extremity and extending to the hha i site downstream of the axis of symmetry of the 5' extremity was cloned into pgem-szf(+). this construct contains more than half of the 5' palindrome, however it lacks the sequence information required to form a bubble. murine fibroblast a9 cells were transfected with this dna and the 5' terminal sequence of resulting virus was analysed. a bubble was seen to be generated, however the sequence of nucleotides contributing to the bubble was of n0n-viral, plasmid origin. the nucleotides in question were removed from the original plasmid construct and the experiment repeated. virus was obtained as before. sequence analysis of the 5' end indicated that both flip and flop forms were present. the corresponding 5' hairpin deduced from these two forms contains no unpaired bases at the position where the bubble is normally located in wild type mvmp. the immediate early lie) gene promoters of herpes simplex virus type-1 share a similar sequence which in some cases overlaps with a binding site for octamer binding proteins. this sequence confers responsiveness to a viral transaetivator, vp16. on these promoters, vp16 combines with multiple cellular proteins to form an activating complex. we set up a reconstituted in vitro system consisting of hela nuclear extract and recombinant oct-1/pou domain protein (without the activation domain of oct-1 ) and vp16 where we showed that the activation of ie genes is not mediated by oct-i itself. rather, oct-1 serves to recruit vp16 that has no intrinsic dna binding capacity. vp16, however, possesses a stong acidic c terminal activation domain of -80 amino acids which is indispensable for the in vivo function of the protein. as this and other acidic activation domains possess only a relatively weak activating capability when we test them in in vitro transcription assays we are interested in investigating the role of this acidic activation domain in vitro i.e. under condition of cell free extracts and on naked template dna. we are using recombinant oct-1/pou domain protein and recombinant vp16 that has been truncated at the c terminus. additionally, we are also testing different truncations of vp16 in vitro that have been produced in cos cells. furthermore, we are also trying to see whether these different truncations already affect the assembly of the actvating multiprotein complex. we have studied the kinetics of synthesis of transcripts initiated from the vaecinia virus 7.5 kd gene early promoter. unexpectedly, after a first burst of rna synthesis early in infection, the promoter showed a second peak of activity in the late phase.reactivation of transcription was neither dependent on the presence of the sequences upstream of the early promoter, nor on the location of the promoter in the genome. reactivation seems to be dependent on virus assembly since it is prevented by rifampicin, that specifically inhibits an early step of viral morpho-g~nesis. analysis of several other early genes showed that reactivation is not unique to the 7.5 kd early promoter. analyzing, by in situ hybridization using digoxigenin-labelled riboprobes, the expression of the viral genome relative to that of an array of cytokines including tnf-(z; ifn-% il-i~, il-2, il-8 and gm-csf. initial experiments suggest that viral genome expression is restricted in the inflammatory infiltrates whereas certain cytokines, particularly tnf-oq are strongly expressed in the synovial membranes. overall, the results point to a prominent role of macrophage activation in the maintenance of chronic inflammation and possibly also in the development of arthritic lesions. the core protein of the hepatitis b virus (hbv) interacts with the viral rna pregenome and the reverse-transcriptase.jdna-polymerase to form a core particle which allows for replication of the viral genome. deletion analysis of the core protein revealed, that the arg-rich carboxyterminal domain is required for rna encapsidation and productive viral dna synthesis. we demonstrate, that the duck hepatitis b virus (dhbv) core protein can be derided into two domains comprising amino acids 1-67 (n-ter) and 67-262 (c-ter), respectively. the co-expression of these two core subdomains leads to the formation of replication competent core particles. furthermore, we demonstrate that the carboxy-terminal domain of dhbv core (c-ter) can be functionally replaced by the corresponding domain of the human hbv. the complete hbv core protein, however, does not form replication competent core particles with the dhbv pregenome and rt. these results imply that the carboxy-terminal part of the hbv core proteins define an exchangeable, functionally conserved domain. influenza ha mediates the adhesion and penetration of host cell membranes. acylation of three conserved cysteine residues in the membrane spanning region of several ha subtypes has been shown. the biological significance of this hydrophobic modification remains to be elucidated. a possible role for the membrane fusing activity has been controversially discussed in the past. removal of covalently bound fatty acid from protein by incubation with hydroxylamine is a commonly applied method. as we shall show for a number of different envelope viruses with acylated glycoproteins, this treatment leads to quite different consequences in a functional seiase depending on the virus used. as an alternative approach to study the biological significance of palmitoylation we expressed wild-type influenza ha and an hamutant lacking the fatty acid binding sites using the baculovirns system. both wild type and fa--mutant ha, after expression in insect cells bind to red blood cells, induce hemolysis, and fuse efficiently with fluoreseently labeled erythrocyte ghosts. hydroxylumine treatment of both recombinant wt-and mutant-ha leads to a loss of hemolysis and of fnsion activity. at least in the ease of fa--i-ia its inhibitory action must be related to some other effect than fatty acid cleavage. these results indicate that hydroxylamine treatment may not always be suitable for the generation of fatty acid free glycoproteius or virus particles for functional studies. gesemann and j.g. nicholls, biocenter univ. basel, 4056 basel, switzerland depolarization of leech neurons leads to growth cone collapse and neurite retraction. this response to depolarization is influenced by the substrata on which the cells are growing and is mediated by the influx of calcium (s. grumbacher-reinert and nicholls, 1992, j. exp. biol. 167:1-14; neely, 1993 , i. neurosci. 13:1292 -1301 . the morphologieal changes induced by depolarization of leech neurons growing on leech extracellular matrix extract is accompanied by a loss of microfilaments in the growth cones. leech neurons growing on a different substrate, the plant lectin coneanavalin a (coma), did not retract their neudtes or lose rnierofilaments, but continued to grow after depolarization. we attribute this to the reduced calcium influx during depolarization of neurons on cona (ross et al., 1988, pnas 85:4075-4078) . increasing the intraeellular calcium concentration by incubating neurons with the calcium-ionophore a23187, led to cessation of motility and disruption of microfilaments but not microtubutes in growth cones on coma. to investigate the mechanism by which calcium affects the organization of microfilaments we stained neurons with antibodies against gelsolin, a protein that severs microfilaments when it is activated with calcium. we have observed that these antibodies colocalize with microfilaments in leech growth cones, we are now analyzing the nature of this antigen that cross-reacts with the anti-gelsolin antibodies with the goal of establishing its potential role in the calcium-induced disruption of microfilaments in leech neuronal growth cones. this work was supported by a grant from the swiss nationalfond no. 3127814.89 to j.g. nicholls. activation of metabotropic glutamate receptors (mglurs) was studied in voltage-clamped ca3 cells in rat hippocampal slice cultures using the whole-cell pateh-clamp configuration. in saline containing 16 mm k +, 1s,3r-acpd (50 ~tm), a mglur agonist induced an inward current associated with an increase in membrane conductance. the reversal potential, assessed with a voltage ramp protocol from -80 to -60 mv and calculated by linear regression, was -15.7 â�¢ 18.7 mv, and was shifted to -53.4 â�¢ 6.4 mv when the concentration of external monovalent cations was halved. these results indicate that the conductance underlying this current is selective for cations (mainly k + and na+). the steady-state i/v curve for the 1s,3r-acpd-induced inward current showed a slight inward rectification. in most of the cells, this inward current was followed (or overlapped) by an outward current concomitant with a decrease in a k + conductance (ere v = -51.2 ~ 5.6 mv in [k+]o = 16 mm and shifted to -74.0 :t: 2.8 mv in [i4+]o = 8 ram). this k + current was voltage-independent in the membrane potential range of-120 to -20 mv. similar results were obtained with methacholine, a cholinergic muscarinic agonist. the non-specific cationic current, seen only in the presence of elevated extracellular k + concentration, could play an important role during intense neuronal activity. the c-myc protein has been implicated in the regulation of cell growth and differentiation, c-myc specifically interacts with max which in turn forms heterodimers with mad and mxil and homodimera with itself. we present evidence that this network o! proteins is regulated both at the level of relative protein concentration as well as by post-translational mechanisms. in the hematopoietic cell lines u-937, ml-1 and hl-6o e-myc mrna and protein are rapidly downregulated after induction o| differentiation whereas max expression is not significantly altered. in contrast the expression of mad is induced with properties similar to immediate early genes, taxi1 is induced to a lesser degree and with slower kinetics, in an effort to study the regulation of o-myc we have mapped all the major in vivo phosphorylation sites in both c-mye and max. two sites in the n-terminal transactivation domain are important for the regulation of the transforming potential el c-myc both in established cell lines as well as in primary rat embryo tibroblasts. however, no difference in the transactivating potential of corresponding mutants was observed. phosphorylatien of max at sites close to the basic region increases both on-and off-rates of either myc-max hetero-as well as max homodimers to dna. brain research institute, university of z~rich, ch-8029 z~rich, switzerland gaba b and adenosine receptors act via pertussis toxinsensitive g-proteins of the gi/g o class to mediate longlasting inhibition in the cns. this class of g-protein is negatively coupled to the enzyme adenylyl eyclase. our aim was to determine whether the inhibition of adenylyl nyclase mediated by these receptors has functional consequences for electrical signaling in hippocampal neurons. the calciumdependent k + current, i~, underlying adaptation of cell firing, was used as an electrophysiological bioassay for adenylyl cyclase activity, as it is very sensitive to intracellular levels of camp. accordingly, the ~-adrenergic agoniat iaoproterenol (5-500 nm), that activates g,-linked receptors, reduced the amplitude of i~ by 51.5 â�¢ 3.6% in voltage-clamped ca3 pyramidal cells in ttx (n=20). reduction of imm by isoproterenol was curtailed to 27.3 â�¢ 2.9%, (p < 0.001) in the presence of baclofen (i0 ~m), acting at gaba b receptors, or adenosine (50 nm). thus, the inhibition of adenylyl cyclase activity due to activation of gaba a and adenosine receptors can modulate responses to other neuretransmitters by an interaction occurring at the level of intracellular signal transduction. furthermore, stimulation of these receptors will tend to enhance i~, suppressing repetitive cell firing. this represents a further mechanism which will result in prolonged inhibition of hippocampal neurons. a study of the possible modulation by nitric oxide (no) of endogenous dopamine (da) release was performed in bovine retina in vitro. isolated half retinas were incubated in kkg at 37 ~ for 3 successive 30-rain incubations, and da was measured in the incubation medium by hplc with ec detection. when retinas were incubated in the presence of the no-generating compound hydroxylamine (300 ttm), a decrease in either basal or k +-(50 ram) stimulated da release was observed during the 2 nd 30-rain period. tested under similar conditions, dibutyryl cgmp (1 mm) was ineffective. the no synthase inhibitor l-ng-nitro-arginine methyl ester (l-name, 100 gm) increased basal da release during the 1 st and 2 nd incubation, whereas it did not affects the k+-stimulated da release. in addition, its effect were potentiated in calcium-free medium. finally, we have also shown in cell-free experiments that retinal no synthase activity was totally calcium-dependent and blocked by l-name. taken together, these results suggest that endogenous no regulates da release via a cgmp independent mechanism. division of endocrinology, university hospital, ch-1211 geneva 14 while the stimulation of aldosterone biosynthesis by angiotensin ii (angii) in bovine glomerulosa cells clearly depends upon a sustained influx of ca 2+, the pathway for ca 2 ⧠entry is not yet accurately defined. at least two mechanisms seem to be involved: 1) the opening of voltage-operated ca z ⧠channels, and 2) the stimulation of a "capacitative" ca 2+ entry regulated by intraocllular ca 2 ⧠stores; however the relative contribution of these pathways to the stimulation of steroidogenesis needs to be determined. two pharmacological tools were used: nieardipine, a blocker of to and l-type ca 2+ channels, and thapsigargin, which releases ca ~+ and therefore induces a capacitative ca ~+ influx. nicardipine (1 lam) completely blocked the cytosolic ca 2+ response to k +, which exclusively activates voltage-operated ca 2. channels, but only partially (22 %) the response to angli. in the presence of nicardipine, angll was unable to stimulate a ca 2+ influx aiter depletion of ca 2 ⧠stores with thapsigargin, demonstrating that angli principally stimulates a capacitative ca 2+ entry pathway. in addition, nicardipine completely blocked the steroidogenic response to k +, but only 30% of the response to angll. thapsigargin-induced aldosterone formation, as the response to angll, was markedly potentiated by low concentrations of k +. in conclusion, this study shows that in bovine glomerulosa cells, angli activates a sustained ca 2+ influx, principally through a capacitativc pathway, leading to aldostcrone formation. this finding could partially explain the poor efficiency of dihydropyridine ca 2 ⧠antagonists for preventing aldosterone secretion. harald holder and john m. lucocq*. institute of anatomy, university of berne, ch 3012 berne *depanment of anatomy and physiology, university of dundee, uk dundee dd1 4hn. subjection of hela cells to 45~ for 30 minutes leads to a complex activation pattern of map-kinases as revealed with renaturadon gels. exponentially growing hela cells show activallon of a myelin basic protein (mbp) phospborylating kinase migrating with an apparent molecular mass of around 60kda upon heat shock induction. in contrast, in cells arrested in go by serum starvation for 24h, essentially three different proteins phosphorylafing mbp are activated subsequent to heat shock treatment. these kinases migrate with apparent molecular masses of around 32kda, 44kda and 55kda. western blot analysis with antibodies recognizing the human boraologues of erkl and erk2 revealed that considerable pools of erkl and erk2 show retarded elec/rophnretic migration behavioar indicating that they are phosphorylated and probably activated in heat subjected and growth-arrested hela cells. down-regulation of protein kinase c (pkc) by a prolonged treatment with 12-o-tetradecanoyl-phothol-13-acetate (tpa) did neither change the major activation pattern observed upon separation of cell lysates on renaturation gels nor that one observed on western blots decorated with anti-erkl or anti-erk2 antibodies. these results suggest that heat shock induced activation of at least erkl, erk2 and the 55kda mbp-kinase is not mediated via activation of pkc. this work was supported by swiss nsf grant no. 31-27636.89. effects of dominant negative glucocorticoid receptor mutants in cell cultures and transgenic animals stefano brenz verca, stefan schneider, sandro rusconi, institut fiir molekularbiologie h der universitat zf~rich, winterthurerstr. 190, switzerland the glueocorticoid receptor (gr) is involved in a large number of developmental and biochemical processes. recently, we obtained a trans-dominant negative gr mutant (tdn) -called gr[aia] -by a frame shift of the cag-repeat of the rat gr (see poster by hug et al.) . the gr[ala]-mutant shows normal llgand-binding and dna-binding but cannot stimulate transcription (lanz et al., steroids; in press) . modulatable versions of the tdn-mutant have also been created by substituting the hbd of the grmutant with the hbd of sex-hormone receptors. these constructs have only been tested so far in transient cotransfection assays. therefore, we are currently trying to express them permanently in cell lines (rat hepatoma cell line fto-2b) in order to verify whether they can affect the transcription level of a known endogenous gr-dependent target-gene, like the tyrosine aminotransferase (tat) gene or a permanently transformed mtv-iacz reporter. so far no rapid test-system has been described, that allows the quantitative analysis of such effects in transient expression assays. we are about to set up such a rapid and generally applicable transient test-system. in the future we plan to express dominant negative gr mutants in a tissuespecific manner in transgenic mice. it will be particularly interesting to investigate their effects on the immune system or on liver functions. these studies investigated growth cone responses m brief local encounters with surface molecules implicated in neuronal pathfinding using chick drg neurons. we tested two interrelated hypotheses: 1) discrete points of information (guideposts) override sustained surface information and dominate growth cone behavior and 2) guidance molecules provide such information by activating second messengers instead of simply increase adhesivity. the potential guidance molecule lamialn was covalentiy coupled to polystyrene beads in order to mimic guidepost cells. encounters with laminin model guideposts significantly altered the behavior and morphology of growth cones advancing on fibronestin or polylysine. growth cones steered towards theses beads in a saltatory medea once a long lasting contact with a single filopodium had been established. subsequent to a pause at the bead, growth cones advanced with a significantly increased growth rate for the next llmin. the average increase in rate stimulated by bead contact was 5 times the basal rate on polylysine and 2.5 times that on fibronectin. positioning of multiple model guideposts along a path and within filopodial reach maintained this bead stimulated rate of outgrowth and directed growth cone advance. the laminin induced pattern of events was totally blocked in the presence of either h7 or sphingosine, two pkc inhibitors, or neomycin, a plc inhibitor. neither of these inhibitors affected neurite outgrowth on the substrata alone. our results demonstrate the necessity of filopodial contacts, the importance of spatially restricted stimuli and the activation of transducing pathways as key mechanisms in neuronal pathfinding. mechanisms of glucocorticoid receptor desactlvation by homopolymeric alanines martin hug, manuela h&fferer and sandro rusconi, institut fiir molekularbiologie 11 der universitat uz zart'ch, winterthurerstrasse 190, 8057 ziirich, switzerland the rat glucocorticoid receptor (gr) edna contains in its y-portion a (cag-repeat that encodes a siring of 22 gin residues interrupted by 1 arg. a mutant with the frame-shifted repeat which codes now for poly-ala residues (called gr[aia]) is completely trans-inactive, is more cytoplasmitally located in absence of ligund and exhibits dominant-negative properties in presence of ligand (lanz et al., steroids, in press) . we generated gr recombinants with increasing numbers of cag-triplets in three possible reading frames (oligo-gln, -ser or -ala). the gr-oligo[ala]~-~.23 mutants showed an activity comparable to wild type gr, whereas the groligo[ala]>_25 were inactive. the severity of the effect of oligo a.la su'ings seems to be dependent on the position of the repeat along the primary sequence (e.g. progressive decrease of the effects by moving toward more earboxy-position). we have also examined the effects of the oligo[ala]21, 23, 25, 27, 31 on gal4 chimeric factors and found that the inhibition by ala repeats is probably factor-specific. our results made us speculate that possible differences in post-translational modifications may be at the root of the negative effects by the ala repeats. we believe that these effects are due to transient association of the nascent gr[aia] mutant proteins with intraceuular membranes. these hypotheses are currently tested. the activation of steroidogenesls in calcium-clamped bovine glomerulosa cells and its modulation by angiotensin ii and camp, python cp, laban op, rossier mf, vallotton mb and capponi am division of endocrinology, university hospital, geneva, switzedand. the ca2+-messenger system plays a crucial role in the regulation of steroid secretion in adrenal zona glomerulosa cells, as it is known to mediate the action of angiotensin ii and potassium ion. in the present study, we used intact isolated glomerulosa cells in which the cytosolic free ca 2+ concentration ([ca2+]c) was clamped at various levels with the ca2+-ionophore, ionomycin, in order to locate the sites of action of ca 2+, to determine their sensitivity towards ca 2+ and the modulation by other physiological factors. by measuring simultaneously steroid synthesis and [ca2+]c, we show that ca2+-clamping (50-860 nm) induced a concentration-dependent increase in the production of both pregnenolone (506___70 % of the basal level) and aldosterone (255+27 % of the basal level, ec50 = 273 nm). by contrast, ca 2+ did not influence the conversion of 1 t-deoxycorticosterone into aldosterone at concentrations up to 600 rim, although at higher concentrations we observed a modest but significant inhibition. in addition, ca2+-clamping did not affect the formation of pregnenolone from a freely diffusible analogue of cholesterol, 25hydroxycholesterol, indicating that ca 2+ acts at a step upstream of cholesterol side-chain cleavage. both angiotensin ii and a membrane-permeant surrogate of camp, dibutyryl cyclic amp, potentiated the steroidogenic response to increases in [ca2+]c . in summary, these studies reveal an exquisite sensitivity of the glomerulosa cell steroidogenic pathway toward very small physiological changes in [ca2+]c. radtke, rainer heuchel, oleg georgiev, gerlinde stark#, michel aguet# & walter schaffner institut fiir molekularbiologie 11, universilat ziirlch, 8057 z'6rich # lnstitut rir molehdarbiologie i, universitiit ziirich, 8093 ztirich we have previously described and cloned a factor (mtf-1) that binds specifically to metal-responsive dna sequence elements in the enhancer/promoter region of metallothionein genes. mtf-1 is a protein of 72.5 kda that contains six zinc fingers and multiple domains for transcriptional activation. here we report the disruption of both alleles of the mtf-1 gene in mouse embryonic stem ceils by homologous recombination. the resulting null mutant cell line fails to produce detectable mounts of mtf-1. moreover, due to the loss of mtf-1 the endogenous metalinthionein-i gene is silent, showing that mtf-i is required for both basal and metal-induced transcription. accordingly, reporter genes containing either synthetic or natural metal-reslxmsive promoters were not transcribed after transfection into the null mutant ceils. cotramsfection of the mtf-i expression vector rescued both basal and heavy metal-induced transcription. these results demonstrate that mtf-1 is essential for normal metallothionein gene regulation. rat pheochromocytoma cells (pc12) were differentiated with ngf for 3-4 days, a time sufficient for the formation of growth cones and elongation of neurites. addition of the phosphatase inhibitor calyculin a (cl-a) (0.2.50.0 nm) led to a concentrationdependent collapse of growth cones and retraction of the neurites within 15 minutes. after the retraction, the cell bodies showed the appearance of a grape-like domain opposite to the cell nucleus. they recovered to normal shape within about 30-60 minutes if the inhibitor was washed out. the neurite retraction was further analysed using both low-light level fluorescence video-microscopy and fura-2 single cell microfluorimetry. the onset of retraction started in the central part of the growth cone prior to a complete collapse of filopodia. the basal intracellular free calcium concentration ([ca2+] i) remained constant during neurite retraction. as a measure for the viability of cl-a-treated cells, agonist-isduced responses of [ca2+] i were analysed. ca2+ entry across voltage-activated ca 2-~ channels was inhibited by 50% after 30 minute treatment with cl-a, while the atp-induced ca2+ entry via ca2*-permeable cation channels was not significantly reduced. however, the onset of the atp-induced rise in [ca2+]i started at the grape-like domain. the speed of the ca 2+ wave across the cell body was slower than in control cells. the addition of 0.5 gm okadaie acid, a different type of phosphatase inhibitor, caused no cell shape change or neurite retraction within 24 hours. taken the specificity of the two phesphatase inhibiters into account, the observed effects seem to be induced by inhibition of a ppl-class phosphatase. myosin in nematocytes of hydra michel nakano & robert p. stidwill dept. zoology, university of ztirich, switzerland the functions of nonmuscle myosins during cell locomotion are not clear. several models have been presented reaching from very active participation of the molecules in different parts of cells to only minor or no roles at all. there is increasing evidence that the functions of myosin ii (capable of forming bipolar filaments) are quite distinct from the ones of myosin i and generally that the different members of the growing family of myosin-like proteins may not all have identieal functions. we have attempted to demonstrate the occurrence and determine pattern of myosins in tissue of hydra and especially in migrating nematoeytes mainly for two reasons. first, nematocytes are fairly rapidly moving cells which can be studied in vitro and, second, the question is of interest from a evolutionary point of view. we have utilized several antibodies against vertebrate and invertebrate myosins for immunofluoreseenee (confocal laser scanning microscopy) and western blotting studies. in addition to these results findings on the screening of a ~.zap c-dna library are presented. the jaki protein tyrosine kinase is a member of a family of intracellular kinases characterized by having two kinase catalytic domains: a bona fide tyrosine kinase domain and an amino terminally positioned kinase-related domain. recently it has been shown that jaki and the other members of this family (jak2, tyk2) are intimately involved in cytokine and growth factor signalling. the presence of two putative nuclear localizing sequences in the amino terminus of the protein prompted us to examine the subcellular localization of this protein. immunohistochemical and biochemical analysis revealed that a part of jaki was localized to the nucleolus. metabolic labelling showed that (a) the jaki protein partitioned rapidly into the nucleus and (b) the nuclear jaki became smaller with time. the size difference was not due to either co-translational glycosylation or phospherylation. these results suggest that part of the jaki protein is rapidly translocated to the nucleoli where it becomes processed. revealed that this clone is expressed predominantly in spleen, mammary gland and thymus as two transcripts, a major species of approximately 5kb and a minor species of approximately 4kb. nucleotide sequence analysis revealed a 84% homology to the porcine syk gene. the syk gene is expressed as a major 3.0kb transcript and has been reported to be lymphoid specific. due to the discrepancy of transcript size and number, we suggest that our cdna clone represents a novel member of this family of ptks. we are currently isolating a full length sequence from a mouse spleen cdna library. in addition, we want to characterize which cell(s) in the mammary gland are responsible for the observed expression. expression and function of g-protein isoforms were studied in differenflaring (6c8) and nondifferentiaflng ((33) subclones of red-1 rauscher murine erythroleukemia cells. erythroid differentiaflon induced by the combined action of erythropoietin (epo) and dmso was associated with a selective loss (>70%) of immunoreactive gai3. simultaneously, a iruncated form of g~2 accumulated in the cytosol, while the membrane concentrations of gai2, gets and gaq/11 remained unchanged. nondifferenfiating (33 cells contained sigrtificanfly higher levels of most get subunit isoforms that remained unchanged upon epo/dmso treamaent. changes in adenylyl cyclase activity and in intracellular ca ion levels ([ca]i) resulting from activation of g-protein-coupled receptors were monitored for differentiation-dependent effects on transmembrane signaling. adp-mediated changes of [ca]i in native and differenflaflng 6c8 cells were consistent with p2t receptor coupling to gai3. thrombin receptors seemed to couple preferentially to gai in g3 cells and to gaq in 6c8 cells. our results document substantial alterations in g-protein-mediated signaling during erythroid differentiation. to investigate the involvement of protein tyrosine kinases (ptks) in the growth control of the mammary gland, we have used pcr based cloning to identify ptks expressed during normal mammary gland development. this approach led to the isolation of three members of the eph-related family of receptor ptks: myk-;, a novel member expressed predominantly in lung, heart and mammary gland; m-eck, the murine homologue of the human eck gene and mek5, the murine homologue of the chicken cek5. all three ptks were expressed in a distinct and narrow window of mammary gland development: puberty and estrus cycle. no expression was found either in late pregnancy or in the end differentiated state of lactation. 0vet-expression was found in the undifferentiated and invasive tumors of transgenic mice expressing the h-ras oncogene. in contrast, no elevated expression of all three genes was detected in the well differentiated, non-invasive mammary tumors characteristic of c-myc expressing transgenic mice. these results suggest that members of this family of ptks are involved in the control of proliferation of the mammary gland but are incompatible with its differentiation. np-tcii is a lymphocyte specific transcription factor (lattion a.-l. et el., mol. cell. biol., 1992, 12:5217-5227 a nucleotide receptor that mediates phosphoinositide and phosphatidylcholine hydrolysis by phospholipases c and d, respectively. here we report that atp and utp potently stimulate mesangial cell proliferation. both nucleotides stimulate phosphorylation and activation of map kinase and the up-stream map kinase kinase. activation of map kinase by both nucleotides is dose-dependently attenuated by the p2 receptor antagonist suramin. stimulation of protein kinase c (pkc) by phorbol ester increased map kinase phosphorylation and activation, and downregulation of pkc partially inhibited atp-and utp-induced map-kinase activation. inhibitors of pkc which display a selectivity for the ca2+-dependent isoenzymes (c~, [3, 7) , as compared to the ca2+-independent isoenzymes (5, e, ~, rl) such as staurosporine and the specific pkc inhibitor cgp 41251 did not inhibit nucleotide-stimulated map kinase phosphorylation, thus implicating the involvement of a ca2+-independent pkc isoform. in conclusion, these data suggest, that atp and utp trigger the activation of the map kinase signalling cascade in mesangial cells and this may be responsible for the potent mitogenic acitivity of both nucleotides. subcellular ion-gradients in ca signaling p. lipp & e. niggli, department of physiology, univexsity of bern recent experimental evidence has indicated an important role for submicroscopic concentration gradients during ca-signaling in various cell types. in heart muscle cells influx of ca via l-type ca channels triggers ca release from the sarcoplasmic reticulum (sr) and links electrical excitation to mechanical contraction (ec-coupling). we used a ratiometric confocal method (fluo-3 and fura-red) to follow the intraceuular ca-concentration while ha-and ca-currents (inn, ica) were elicited in the whole cell mode of the patch-clamp technique. both, icand in, were able to trigger ca release from the sr. kinetic differences between ini and ic -induced ca-transients, li-substitution experiments and the existence of a residual inn-induced ca transient in the absence of sr function suggested that these ca signals were mediated by the ha-ca exchange running in the ca influx mode. control experiments showed that ins-induced ca transients did not result from spurious activation l-type ca channels. the significant increase of the na concentration required to activate the ha-ca exchange can only occur provided cytosolic diffusion of na is restricted in the vicinity of the exchangers. this limited diffusion results in significant ha-gradients in the subsarcolemmal space. in conclusion, i~iinduced ca influx may represent a safety factor for ec-coupling while ic, ensures sustained ca-release and is the source for refilling the sr with ca. supported by snf. the regenerative ca-induced ca release (cicr) mechanism is an important amplifier in cardiac signal transducilon. the cicr contributes to the ca transient responsible for mechanical activity, but also generates ca waves propagating within the cytosol. we investigated subcellular ca signals in neonatal rat and adult guinea-pig ventricular myocytes using ratiometric confocal microscopy with a mixture of two fluorescent ca indicators. individual resting myocytes exhibited spontaneous ca release events from the sarcoplasmic reticulurn (sr) that were attributable to three distinct patterns: i) local ca release events without propagation; ll) planar ca waves propagating across the cell; iii) spiral ca waves spinning around a nucleus or shifdng along a subcellular region without entering it. transitions between these patterns were frequently observed, suggesting that a particular release pattern is not the property of an individual cell but reflects the functional state of the sr. the existence of focal release and refractory zones within a single cell indicates that the sr is not uniform on the subcellular level. the sr seems to he a network of elements with distinct functional properties. a suhcellular variability in the positive feedback of the cicr mechanism can account for the observed features of ca signaling. the degree of positive feedback exhibited by a single sr element may depend on its ca content. to characterise the intracellular signalling and regulatory properties of the cloned parathyroid hormone (pth) receptor we have stably transfected two renal epithelial cell lines with cdna's encoding the pth receptor (provided by dr g. segre). the proximal tubular, porcine (llc pk1 [clone 4]), and the distal tubular, madine darby canine (mdck) cell lines were transfected since the culture of either cell line on permeant filter supports leads to the development of polarised epithelial cell layers that mimic the epithelial organisation in vivo. transfection of the cloned pth receptor into each of these cell lines allowed us to examine, independently, the functional properties of the receptors expressed at the apical and/or the basolateral cell surface. while the basolateral application of pth produces a large increase in camp production in both cell lines, the relative efficacy of an apical application of pth differs between the cell types. the transfected llc pk1 cells exhibit a greater response to apical pth compared to the transfected mdck cells. pth-mediated regulation of intrinsic phosphate transport was also examined in polarised, transfected mdck cells. neither apical or basolateral application of pth affects intrinsic apical or basolateral ha-dependent or phosphate transport activities. similar experiments are being performed with the transfected llc pk1 cells. a. and preiss, a. biozentrum der universit&t basel, ch 4056 basel hairless, a recessive larval lethal mutation, causes dominant defects in sensory organs of adult drosophila flies.manifold genetic interactions indicate that hairless antagonizes neurogenic gene function suggesting an involvment also in neurogenesis. in order to gain insight into its function we have cloned the hairless gene which encodes an extremely basic, very serine rich protein of ii0 kd calculated molecular weight. no significant homologies to proteins of known function could be identified. therefore, we are concentrating by various means on a structure -function analysis of the hairless protein. firstly, we are testing a series of hairless deletion constructs for rescue capacity and generation of anti-hairless phenotypes after overexpression compared with the wild type gene. secondly, we are analysing hairless protein distribution throughout development, also at the subcellular level. heat induced hairless protein runs at ~ 150 kd, possibly due to phosphorylation which might be intrinsic to hairless function, a hypothesis we are currently scrutinizing. thy-1 and cd26 surface glycoproteins are both capable of delivering proliferative signals to t cells upon cross-linking with appropriate antibodies. the tyrosine phosphatase cd45 is a key regulator of t cell proliferation as it controls the activity of src family kinases p56/~k and p59fy n. associations of thy-1 with cd45, and of cd26 with cd45 have been reported after chemical cross-linking of intact cells and it is likely that such associations constitute part of the structural basis for the t-cell stimulating capacity of thy-1 and cd26 accessory molecules. we report that thy-1, cd26 and cd45 can be specifically crosslinked at the cell-surface and isolated as multimolecular complexes containing a 78 kda protein that is recognized by an anti-bip (grp78) antibody. in vitro kinase assays show the selective association of p56 ick and p59fy n with thy-1 and cd45 contained in multimolecular complexes. the multimolecular structure we describe could representa transducing unit incorporating surface receptors and regulators of tyrosine phosphorylation that are stabilized through interaction with the bip protein in the plasma membrane. structure-function-analysis of hairless, a gene involved in drosophila nervous system development maier, d., functional coupling of ppars and trs ijpenberg, a., wahli, w., desvergne, b., institut de biologie animale, 1015 lausanne we are interested in a possible functional coupling of thyroid hormone (tr) and peroxisome proliferator-activated receptors (pfar). to this end, we performed cotransfection experiments in nih 3t3 cells using cat reporter constructs containing the promoter of either the t3 responsive malic enzyme (me) gene, or the ppar regulated acyl coa oxidase (aco) gene. the me reporter construct showed very moderate response to the activated mouse ppar (mppar), whereas combination of try1 and unactivated mfpar potentialized the t3 induction. a putative ppar response element was identified within this promoter and will be further investigated. the aco reporter constructs were not responsive to trs. in constrast, a tr and t3 dependent inhibition of the fpar mediated activation was observed. the mechanism of functional coupling of these receptors, which may possibly involve the 9-cisretinoic acid receptor rxr, will be further investigated by molecular analysis. unliganded steroid receptors form a complex with hsp90 via their hormone binding domain (hbd). the receptor activation involves a hormone-induced release of hsp90. we genetically addressed the role of hsp90 in budding yeast by analyzing three kinds of hsp82 (the essential yeast homologue of hsp90) mutants. these mutations affect either the quantity of the protein (low versus normal levels), its integrity (deletion analysis) or its nature (hsp82 homologues from other species). hsp82 mutant are examined for their ability to complement a hsp82 deletion mutant. moreover, they are tested for functional interaction with a hbd. interestingly, a viable deletion of a charged region, suspected to be involved in hsp82-hbd interaction, only slightly interferes with hormonal activation of both estrogen or glucocorticoid receptors. furthermore, a short deletion in the last third of hsp82 leads to the production of a dominant negative mutant which prevents ceil growth at elevated temperature. the ability of various hsp82 homologues to palliate or not the absence of the yeast protein allows us to define, using chimeras, the regions of the protein which are necessary and sufficient to support cell life. zhang,h.,reynaud, s. and spohr,g. d~partement de biologie cellulaire, sciences iii, 30, quai ernest-ansermet, ch-1211 gen&ve 4 id cdnas expressed during early xenopus development have been isolated and sequenced. the encoded protein is homologous to the proteins described in higher vertebrates. northern blot analysis reveals that transcription starts soon after mid blastula and decreases to some extent after tailbud-stage. lower levels of transcription are detected also in adult frog tissues such as liver and heart. microinjection into oocytes of in vitro-synthesized myodor/and id-mrna, along with a cat reporter gene whose expression is under the controll of an a3-actin promoter supplemented with four e-boxes shows that myo d function is repressed by id. to investigate the importance of negative regulatory sequences we have isolated and characterised the id promoter. as a strategy for identifying cis-regulatory elements, chimeric genes consisting of different 5' elements of the promoter were attached to the cat coding sequences and microinjected into embryos. we are studying the tissue distribution of the rat peroxisome proliferators activated receptor (ppar~), a member of the nuclear hormone receptors superfamily, during development and in adults. high levels of ppar~ mrnas are detected in adult liver, kidney and heart. muscle, stomach, and intestine show moderate amounts of the ppar~, whereas brain, testis and lung present a very low expression. during postnatal development, we observe a continuous increase of the ppar~ expression in the kidney, in contrast to a steady level in the liver. anjard, c., groux, b ., gamboni, s. and reymond, c.d., institut d'histologie, unil, rue du bugnon 9, 1005 lausanne. the camp dependent protein kinase (capk) from dictyostelium is composed of a single catalytic (c) and a single regulatory (r) subunit. the c subunit we characterised, pkac is about twice the size of its mammalian counterpart. we showed that it possesses all properties of a catalytic subunit. it associates with the r subunit in absence of camp, it copurifies with capk activity and an increased activity is observed in cells over-expressing pkac. furthermore, we dissected its role during development and cell differentiation. overexpressing pkac under cell type specific promoters allowed to show the requirement for its activity during spore differentiation. pkac seems to play a more complex role in prestalk cells. we are now dissecting the functional regions of the pkac protein, making use of the known tertiary structure of the c subunits of mammalian enzymes. dna binding properties and stimulation of gene transcription of baculovirus expressed xppar~. hihi, a.k, mermod, n., medin, j., ozato, k. and wahli, w., inst. de biol. animale, uni lausanne, ch-1015 lausanne. lab. of mol. growth reg., nih, bethesda, md, 20892 usa. the peroxisome proliferators activated receptors (ppars) are members of the steroid/thyroid nuclear receptor superfamily. so far, they have been found in amphibians, rodents and humans. in xenopus laevis, 3 subtypes (xppara,~.y} have been isolated. in order to study the mode of activity of the ~ form, the xppar~ gene product was overexpressed using a baculovirus expression system. the nuclear protein obtained has the correct molecular weight of 46 kd. gel shift assays showed that nuclear extracts containing the recombinant protein are able to specifically bind the 'ppar response element' which is a direct repeat of the core aggtca motif. this dna binding activity is potentiated by another nuclear receptor, the mouse rxr~ and is inhibited by phosphatase, suggesting a role for phosphorylation in ppar binding to dna. a functional approach, using an in vitro transcription system, was chosen to define ppar and rxr action on transcription stimulation, as well as the role of their respective activators. arachidonic acid (0.5 -20 ram) induces various patterns of ca2+i signal in bronchial smooth muscle cells, as revealed by single cell dynamic video imaging of fura-2 loaded cells. most frequently, ca2+i oscillations were observed, although other patterns (a single ca2+i transient; a single peak followed by a sustained elevated level, or a sustained ca2*i elevation solely) were occasionally seen. the amplitude of ca2*i oscillations was related to the concentration of arachidonic acid. the frequency histogramm was noticeably shifted to higher frequencies by nordihydroguaiaretic acid (ndga, 0.2 rtm, a lipoxygenase inhibitor), whereas it was markedly shifted to lower frequencies by indomethacin (50 pm, a cyclooxygenase inhibitor) and by 5, 8, 11, 14-eicosatetraynoic acid (etya, 3 ~tm, a lipoxygenase and cyclooxygenase inhibitor). these observations suggest that: (1) arachidonic acid per se elicits ca2+ i oscillations in these ceils; (2) cyelooxygenase activity products modulate the frequency of these oscillations. promoter analyses of a growth factor regulated gene t. triib, m. kalousek, r. kessler, and r. klemenz dept. of pathology, university hospital, 8091 ziirich stimulation of quiescent cells to enter the cell cycle results in altered gene expression. some of the first genes to be induced (immediate early (i.e.) genes) encode transcription factors which are thought to pass on the mitotic signal to the delayed early (d.e.) genes. we have analysed the promoter elements required for growth factor mediated induction of the d.e. mouse gene t1 which encodes a secreted glycoprotein of the immunoglobulin superfamily. a 448 bp region located between 3.6 and 4.0 kb upstream of the transcription start site is sufficient for growth factor mediated inducibility. it contains an ap-1 binding site which is absolutely required for t1 gene expression. it is surrounded by 3 e-boxes. at least 2 of them are essential for efficient gene induction. thus the i.e. proteins encoded by the fos and jun gene family which form the ap-1 complex can activate the d.e. gene t1 in collaboration with helixloop-helix transcription factors binding to the e-boxes. the increase of radiolabelled inositol phosphates ([3h]ips) production in agonist-stimulated human airway smooth muscle cells (asmc) was determined by hplc. in order to observe the role of calcium, pkc and pla2 on plc activity during agonist stimulation, the effects of pharmacological agents (able to alter calcium, pkc and pla2) were tested on ip production elicited by a 5 sec stimulation with histamine. the inhibitor of pkc staurosporine increased, while pma (an activator) decreased the histamine-stimulated production of [3hi 1,4,5-ip3 and of its degradation products. an inhibition was also observed with the two pla2 inhibitors, quinacrine and p-bromophenacyl bromide. in calciumfree buffer, no difference in the ip increase was shown, but an inhibition was observed when thapsigargin, an inhibitor of the ca 2+, mg 2+-atpasc of the sarcoplasmic reticulum, was added in the same conditions. the calcium ionophore ionomycin increased the lp production in presence of external calcium, whereas it completely blocked the stimulation in calcium-free buffer. the results suggest that plc activity, in human asmc, is modulated by a positive feedback of calcium and by a negative feedback of pkc and pla2. regulation of hormone-stimulated calcium signals m. boolman, afrc, laboratory of molecular signalling, dept. zoology, cambridge, uk stimulation of cells with hormones that activate inositol 1,4,5-trisphosphate (insp~) formation, leads to an increase in the intracellular ca 2. concentration ([ca2+]~). these hormone-stimulated [ca2 ⧠signals have a complex temporal and spatial regulation, with the [ca2*]~ increase often taking the form of a series of repetitive spikes or oscillations, arising from a steady basal level. the spatial counterpart of a [ca2+]~ spike is a wave, where [ca2 ⧠is first elevated in a localised region of a cell, and then spreads throughout the cell in a regenerative manner. the [ca~*]~ spike frequency is modulated by several environmental factors including hormone concentration, extracellular calcium and thiol reagents. current evidence suggests that a cell can interpret these complex [ca2+]~ changes to regulate a diverse range of cellular activities. although many of the biochemical steps between hormonereceptor activation and the [ca2 ⧠response are known, the precise mechanism generating these complex [ca2+]~ signals is unclear. in order to understand this mechanism more clearly, we have investigated the regulation of insp~-mediated ca ~ ⧠release from intracellular stores in intact cells. our current data suggests that under certain conditions, the insp3-sensitive intracellular stores behave as functionally discrete units, and that during a ca 2 ⧠spike these stores become functionally coupled by the diffusion of ca z+ from one store to the next, triggering a regenerative ca 2+ release. phenylarsenoxide as a tool for identifying proteins involved in the secretory process wiedemann. c., schaefer, t.,*gitler, c., burger, m. m. friedrich-miescher institut, p.o.box 2543, ch-4002 basel "department of membrane research and biophysics, weizmann institute of science, rehovot 76110, israel phenylarsenoxide (pao) at 20btm blocks exocytosis in chromaffm cells of the bovine adrenal medulla. this inhibition can be reversed by small dithiois, such as dithiothrcitol or 2,3~limercaptopropanol. because trivalent arsenicals interact specifically with dithiol4containing proteins, we conclude that dithiol-proteins do play an important role in regulated secretion in chromalym cells. to identify dithiol-proteins putatively involved in the secretory process, we compare pao-binding proteins from chromaffm and pci2 cells and from fibroblasts as well as from sulx:ellular fractions by 2-dimensional gelelectrophoresis. the proteins are identified by radioactive pao bound to the dithiol in a complex that witltslands sds-page. affinity chromatography on pao-scpharose with various protein fractions is used to purify the dithiol-proteins of interest. enzymatic activity, e.g. kinase or phosphatase activity, can be measured in the eluates of such a column to give further hints at the possible function of dithiol-proteins. rac-pks (~elated to pka_ and pkcc protein kinnses) represent a serine/threonine kinase family whose catalytic domain is closely related to those of protein kinases a and c. they contain a ph (pleckstrin homology) domain n-terminal to the catalytic domain. in addition, rac-pk~ was shown to be a proto-oncogene. in order to investigate the role of rac-pk in cellular signallimg we undertook genetic and biochemical analysis of the drosophila homolog (drac-pk). the drac-pk gene encodes multiple classes of transcripts. antisera raised against the drac-pk recombinant protein specifically recognize 3 distinct molecular weight forms (68, 85 and 120 kda). all forms are expressed throughout development and are both maternally and zygotically regulated. the drac-pk gene is localized at 89b4-i0 region of the third chromosome, betwee~ the pannie~ and the stubble genes. universit6 de lausanne, rue du bugnon 9, 1005 lausanne when spinal meninges homogenates were incubated with reduced glutathione (gsh), a cofactor of prostaglandin (pg) biosynthesis, [14c] arachidonate (aa) was bioconverted into a major and a minor product which comigrated on tlc with pge2 and pgd 2 respectively. in absence of gsh: 1) pgd2 could not be detected; 2) the level of pge2 was dramatically reduced but surprisingly counterbalanced by accumulation of an unusual [i4c] aa metabolite which exhibits particular traits of migration on tlc and of retention time on hplc. this aa metabolite: 1) does not correspond to a degradation product of pge2; 2) crossreacts with pge 2 antibody; 3) fits the conditions required for a 15 hydroperoxy pge2 (chemical degradation into pge2 by hydroperoxyde reducing reagents such as snc12 or gsh-hemin). it is therefore inferred that depletion of gsh favours accumulation of hydroperoxyprostaglandins which would participate to oxidative injury. the ecdysteroid receptor (ecr) and ultraspiracle (usp) from both, chironomus tentans and drosoplu'la melanogaster, were expressed in e. coil as fusion proteins with glutathione-s-trsusferase. the identity of the expressed recombinant proteins was confirmed by western blot analysis. the drosophila ecr binds to its cognate hormone response element as a heterodimer with usp as a partner. we now want to compare the capabilities of the two receptor partners from both insect species in regard to dna binding and heterodimerization by means of gel mobility shift assays. the use of naturally occurring andartificial hormone response elements will allow to define a hormone response element consensus sequence for ecr-usp heterodimers and, if existing, for ecr and usp homodimers, respectively. for other members of this receptor family it was shown that the dna binding domain and adjacent sequences alone sufficiently defines response element specificity for both homo-and heterodimers. the dna binding domains of ecr and usp from the two species show a high similarity (92% and 85% identity, respectively). the corresponding sequences were selected for overexpression in e. coli. in combination with immtmoprecipimtion of receptor-dna complexes and pcr amplification steps, the expressed proteins will be used for the detection of naturally occurring hormone response elements within genomic dna. university of fribourg, ch-1700 fribourg. various agonist-induced cell responses in neutrophils and fibroblasts, such as chemotaxis and cytoskeletal rearrangements, have been shown to parallel the synthesis of ptdins(3,4,5)p 3. but the importance of this rise is not clear. we show here that wortmannin blocks pdgf-induced production of ptdins(3,4,5)p 3 in human foreskin fibroblasts with an ic50 of about 5 nm. a similar inhibition was observed in in vitro assays (ics0=lnm) with pi 3-kinase ii~nunoprecipitated by antibodies directed against the 85 kd subunit (p85). on the other hand, wortmannin did not affect pdgf-mediated phosphorylation of p85, indicating the correct interaction of p85 with the pdgf-~ receptor. the pl10/p85 complex of the pi 3-kinase remained intact in the presence of ~m concentrations of wortmannln. these results are consistent with a direct, specific inhibition of pi 3kinase by wortmannin at concentrations relevant for its previously reported effects on cellular responses. when stimulated with pdgf, human foreskin fibroblasts form circular membrane ruffles rich in filamentous actin. the fact that wortmannin inhibits these pdgf-mediated aotin rearrangements suggests the need of pi 3-kinase activity as a signal for this cell response. caicineurin is a calcium/calmodulin-regulated protein phosphatase which is comprised of a catalytic subunit a and regulatory subunit b. although calcineurin was discovered in brain, the calmodulin stimulated protein phosphatase which has caicineurin like structure has been described in other tissues. the protein structure show that the calcineurin b is very conserved in different tissues and species, using anti-calcineurin b monocicnal antiserum, the tissue extracts from rat have been explored. immunoreactivity was obverved in all tissues, although its intensity was different. the highest intensity was found in brain. spleen, heart and thymus had an intermediate level intensity. the results were supported by the quantitative measurement of calcineurin. the caicineurin concentration was 10 to 50 fold higher in brain than that in other tissues. furthermore, the distribution of the calcineurin activity in rat tissue was studied using dephosphorylaticn assay of calctneurin. the highest caicineurin activity was found in brain too, but was only 5 to 15 fold more than that in other tissues. the specific activity of calcineurin was different in tissues. these results indicate the caicineudn activity might be regulated in tissue specific fashion. burst and cytoskeleton arcaro a. and wymann m.p., institute of biochemistry, university of fribourg, ch-1700 fribourg chemoattractants like n-formyl-met-leu-phe (fmlp) stimulate in neutrophils a rapid and transient increase in the levels of ptdins(3,4,5)p3 (pip3) which is due to the activation of ptdlns(3)-kinase. pip 3 has been proposed to be a second messenger molecule, but its cellular targets are presently unknown.here we report that pretreatment of human neutrophils with wortmannin inhibits the fmlp-etimulated production of pip 3 in a dose-dependent manner (ic50 = 5 nm). furthermore, ptdins(3)-kinase activity immunoprecipitated from resting cells is totally abolished by i0 nm wortmannin (ic50 = 1 nm). these results show a potent and direct effect of wortmannin on ptdins(3)-kinase. wortmannin also inhibits the respiratory burst of neutrophils at nanomolar concentrations, which suggests that pip3 is involved in the signalling pathway controlling activation of the nadphoxidase.when pretreated with wortmannin and stimulated with fmlp, neutrophils display oscillatory changes in factin content that correlate with oscillations in cell ~hape. this, and the inhibition of ptdins(3)-kinase, suggest a modulatory role for pip 3 in cytoskeletal rearrangements.we are currently investigating the effects of pip 3 on the functions of gelsolinl a protein that is proposed to mediate actin polymerization and can be regulated by polyphosphoinositides and ca 2+. matthias gstaiger, walter schaffner and christopher hovens institut f'tir molekularbiologie ill der universitat z'tirich, ch 8057 ziirich the octamer motif atgcaaat plays a central role in mediating the activity of a number of both lymphoid specific and ubiquitous promoters and in the immunoglubulin heavy chain intronic enhancer. the activity of thils motif in lymphoid cells is presumably mediated by the lymphoid cellspecific factor oct-2a. when ectopically expressed in non-lymphoid ceils, oct-2a is not sufficient to mediate enhancer activity of a multimerized 50 bp enhancer core element derived from the immunoglobulin heavy chain intronie enhancer, a segment, which is however highly active in b-cells. this and other findings support the supposition that additional b-cell specific factor(s) can account for the specificity and extent of the octdependent transcription observed in lymphoid cells. to further our understanding of the molecular mechanisms involved in mediating lymphoid-specific expression, we have employed the yeast two hybrid system to identify human proteins capable of physically associating with human oet-2a. to this end, a oct-2a expressing yeast strain has been constructed which bears two integrated reporter genes, his3 and lacz under the control of the octamer motif. transformation of this strain with a cdna library has allowed us to isolate clones interacting with oct-2a. we are currently analysing these candidates to determine the veracity of these interactions in higher enkaryotic cell background. in general, untransformed nuclear hormone receptors are predominantly confined to either the nucleus or the cytoplasm of a cell. receptors of the latter class are translocated to the nucleus upon interaction with ligand. our studies of the use of the ecdysteroid receptor complex from insects, consisting of a heterodimer formed between ecr and usp, as a tool for target gene activation in vertebrate cells have revealed a novel mechanism of nuclear translocation of ecr that does not depend on hormone action. after transient transfection of respective expression plasmids into hela or cv-1 cells, the subeellular distribution of the individual receptor types was analyzed by indirect immanofluorescence staining. while ecr alone was detected in both cellular compartments, nucleus and cytoplasm, usp was predominantly found in the nucleus of either host cell. cooxpression of both ecr and usp resulted in an efficient transloeation of ecr into the nucleus. usp appears to trap ecr in the nucleus, presumably by the formation of a heterodimeric complex. thus, heterodimer formation of ecr and usp not only appears to be required for high affinity binding to cognate hormone response dements (and subsequently for transactivation of an adjacent target gene) and for binding to ecdysteroid but in addition for an enhanced translocafion of ecr into the nucleus. activation of rodent macrophages with cytokines or bacteria is accompanied by the induction of a ca2+-independent nitric oxide (no) synthase which plays a key role in antimicrobial and antitumoral activity. firm evidence for expression of a similar enzyme in other mammals or in man has been lacking. we now show that bovine bone marrow-derived macrophages produce nitrite in an l-arginine-dependent manner upon stimulation with heat-killed grampositive or gram-negative bacteria. homologous interferon-7 and tumor necrosis factor-~ induced little no 2-production, but primed macrophages for enhanced n0 2-production induced by salmonella dublin or lps. this is one of the first demonstrations of no 2-production by nonrodent macrophages and encourages a search for an involvement of reactive nitrogen species in the killing of parasites or tumor cells by macrophages from mammals other than rodents. lectins are used in many analytical methods to study the carbohydrate structure of glycoproteins. we report here a technique which combines the high resolution of two-dimensional gel electrophoresis (2-dpage) to separate plasma proteins, the specificity of lectins to bind carbohydrates and the sensitivity of chemiluminescence. this method is compared with that of a commonly used colorimetrio reaction. since the reference plasma protein map obtained by 2-d page is available (i), the technique described here allows a quick and specific identification of plasma glycoproteins. therefore any ma j or glycosylation modification which may happen in some diseases can be detected. (i) electrophoresis 1992; 13:707-714. this work was supported in part by fcar (quebec, canada). in our laboratory we use genetic and biochemical approaches to study translation initiation, in particular the initiation factor eif-4a in yeast. this factor from higher eukaryofic cells is known as an rna dependent atpase and functions as a rna helicase together with another initiation factor, elf-4b. it belongs to a family of putative rna helicases, the d-e-a-d proteins. to find new genes involved in translation initiation, suppressors of a temperature sensitive eif-4a mutant were isolated. one of the suppressors (stm1) is a single copy gene which encodes a protein resembling the human translation initiation factor eif-4b. disruption of the gene is not lethal to the cell, but affects growth. polysome analysis of strains with a deleted stm1 gene have shown that this new gene is also involved in translation initiation. it carries six repeated sequences of 25 amino acids of unknown function and has -like the human eif-4b -a rna binding domain. the second suppressor (stm2) is also a novel gene. it encodes a large protein which shows no homology to other proteins present in the data libraries. it contains high portion of charged amino acids and is rich in glutamines and serines. its function in translation initiation is currently under investigation. tobacco translationini~ationfactore~4aisencoded by a large anddiverse genefamily karl brander, therese mandel, isabelle lutziger, george owttrim and cris kuhlemeier, institute of plant physiology, university of berne, altenbergrain 21, ch-3013 berne using heterologous probes we isolated cdnas coding for translation initiation factor eif-4a from tobacco, eif-4a is encoded by a large multigene family of which at least nine members are expressed in leaves and most if not all other organs of the tobacco plant. while screening genomic libraries we found several additional genes. two of them code for genes highly homologous with eif-4a throughout the coding region, but with changes in the gkt, ptrela and dead-box motifs. a third gene is expressed exclusively in the developing male gametophyte. this eif-4a-related gene may have a role in the well-documented regulation of translation in pollen. in order to study the function of these genes we have begun altering their expression in transgenic tobacco plants. seauence reauirements for a non-aug protein initiaf~jon non-aug initiation codons are still infrequent and were initially observed in animal viruses, but a number of cellular examples have now also been reported. several years ago we reported the use of an acg in the p/c mrna of sendal virus (sen) to initiate the non-structurar c' protein. we recently described a c' protein in the human parainfluenza virus type 1 (hpiv1), which is initiated from a gug. in both cases, the c' protein is an n-terminally ebngated form of the c protein which is initiated at the second aug, in the +1 reading frame relative to the first aug. this aug is used to initiate the p protein, an essential component of the viral rna polymerase. unlike the acg in sen, the gug in hplvt is clearly not a weak start codon since c' is the most abundant protein expressed from this mrna both in rive and in vitro. it appears that not any upstream gug in an optimal context will function as a start codon. for instance, the p gene of hpiv3 cannot express a c' protein (the orf is closed by stop codons) but it does contain a gug in an optimal context upstream of the p protein aug, which could encode a p' protein. nevertheless, we have been unable to demonstrate that this gug functions as a start cedon. with the use of chimeric mrnas between hpivl and 3, we have shown that positions +5 and +6 (the first g of the gug triplet being +1) are the major determinants of the efficiency of gug as an initiaton codon for protein synthesis. we are currently investigating the possibility that these nucleotides may increase the initiation rate of weak non-augs such as the acg of sen. biological activity of the heterodimeric subunit srp9/14 of the signal recognition particle is retained in 2 fusion proteins fabrice bovia & katharina strub, d6partement de biologie cellulaire, universit6 de gen~ve, ch-1211 gen~ve 4 the targeting of secretory proteins to the membrane of the endoplasmie reticulum is mediated by a cytoplasmic ribonucleoparticle, the signal recognition particle. it is composed of 6 proteins and f rna molecule (srp rna). the 2 smallest proteins srp9 and srp14 are required for the translational control function of srp. it effects a specific arrest in the biosynthesis of er-targeted proteins. these 2 polypeptides bind to the srp rna exclusively as a heterodimer (srp9/14). we have constructed single-chain variants of this dimer using a short peptide linker of 17 aa to connect the c-terminus of the 14 kd subunit to the n-terminus of the 9 kd subunit (14-9) and vice versa (9-14). we found that both proteins bind srp rna with a similar efficiency as the wild type protein. glutaraldehyde cross-linking experiments and sedimentation analysis indicate that the 2 fusion proteins fold into a similar structure as srp9/14 and bind srp rna as a monomer. furthermore, they can functionally replace srp9/14 in the elongation arrest and translocation assay. since the 2 fusion proteins are circular permutations of each other, we can conclude from this results that n-and c-termini of both proteins have no essential role in folding, rna-binding and in mediating their biological activity. furthermore, the possibility to express an oligomeric protein as a single polypeptide facilitates the analysis of its functions as well as its structure. a24 s06-08 studer, r. and hunziker, p. e., 8iochemisches institut der universit~t z0rich, ch-8057 z0rich metallothioneins (mts) are small cys-rich proteins which are inducible by a variety of agents such as bivalent transition-metal ions, tumor promoters, cytokines and hormones. the biological role of mts is still unclear. initially postulated to serve in the sequestration of the toxic heavy metals such as cadmium and mercury, they are now believed to play a major role in the cellular metabolism of essential metals such as zinc and copper. to explore this function further we have now established a method to quantify basal mt production in several human cell lines. thus, cellular isomt contents were monitored by h.pj.c, and the rate of synthesis was followed by the incorporation of ~ss-cysteine. the results show that maximum mt synthesis and accumulation occurs when the cells enter the exponential growth phase. with increasing celldensity the mt content decreases progressively until confluence is reached. in correlation with preliminary measurements our results suggest a close relationship between the mt content, the cell growth rate and the intracellular abundance of zinc. vincent bernard, adrian etter, heinz tobler and fritz mtiller. institute of zoology, university of fribourg, 1700 fribourg (switzerland). the nematode ascaris lumbricoides undergoes chromatin diminution which causes a loss of dna in all somatic ceils. we have identified a ribosomal protein (rp) gene (coding for alep1 = ascaris lumbrieoides eliminated protein 1) which is located within the germqine specific material. alep1 shows strong homologies with the ribosomal protein s19 (rps19). the transcription of its gene takes place only in the germ-line. 2d-gel electrophoresis on germ-line and somatic purified ribosome fractions shows that the alep1 protein (rps19) is present only in the germ-line ribosome. does the alep1 germ-line specific protein have a homologue in the somatic ribosomes? we have cloned a homologue of alep1 from ascaris somatic tissue named rps19', we are currently studying the expression pattern of this rps 19'. these results indicate the existence of a developmentally regulated ribosome heterogeneity between the germ-line and somatic ceils. what is the function of rps 19? since ascaris is not suitable for genetic analyses, we isolated rps19 from the nematode caenorhabditis elegans. the c. elegans rps19 cdna shows only 60% homology at the nucleic acid level. rps19 is located on the c. elegans chromosome iv. the identification of a rps19 mutant in c. elegans will indicate its function. favre, d, l, pavlovic, j2 and michel, m.r. t 1 institute of medical microbiology, university of beme, ch-3010 berne. 2 institute for immunology and virology, ch-8006 ziirich we have successfully purified the recombinant c (recc) protein from spodoptera frngiperda (sf9) insect cells which were infected with acnpv-c (a). the recc protein retained the biological activities of native c protein obtained from wild type sfv (b). our results suggest that the c protein is responsible for the cellular shut-off of host protein synthesis by inhibiting the initiation of translation. we are currently investigating the molecular mechanisms involved in this shut-off. tif3 is a new eukaryotic initiation factor which shows 26% identity with the sequence of mammalian translation initiation factor eif-4b. the tif3 gene is not essential for growth; however, its disruption results in a slow growth and coldsensitive phenotype. in vitro translation of total yeast rna in an extract from a tif3 gene-disrupted strain is reduced as compared to a wild-type extract. the translational defect is more pronounced at lower temperatures and can be corrected by the addition of purified yeast tif3,or mammalian eif-4b. in vivo translation of ~galactosidase reporter mrnas with varying degree of rna secondary structure in the 5' leader region in a tif3 gene-disrupted strain show preferential inhibition of translation of mrna with more stable secondary structure. chloroplast protein synthesis requires the import of elongation factors ef-tu (tuf-gene) and ef-g (fusgene). it seems that the expression of both genes is light regulated. we recently cloned and sequenced a chloroplast specific nuclear fus gene in soybean (biochim.biophys acta 1174 : 191-194, 1993 . further analysis revealed that a second very similar (sequence, anatomy) nuclear fus-2 gene exists which, however, shows strong sequence diversion in the 3'trailer region. several cdna clones were partially sequenced but sofar only transcripts from the fusi gene were retrieved. we currently study the promoter regions of either gene to test possible differential expression of fus genes. comparative mapping and sequencing studies of the two genes in the 3"-region indicate that a major dna insertion occurred distal to the coding part of both fus genes what may also influence gene expression. the rna-binding domains in the 9kd and 14kd subunits of the signal recognition part~clehavea putative~-h~licalstructure. nazarena buiand katharina strub dpt de biologie cellulalre, science ill, universit4gen~ve the signal recognition particle (sr2), a cytoplasmic ribonucleoprotein is important for targeting nascent secretory proteins to the endoplasmic reticulum. srp9and srpl4are constituents of srpandare, together with the alu sequences of srp rna, required for the elongation arrest activity of the particle. srp9 and srpi4 form a heterodimer in the absence of the rna and bind the rnaexclusively as a heterodimer. the primary sequence of srp9 and srpi4 revealed that both proteins are highly basic and lack structural similarities to other characterized rna-binding proteins. we have therefore been interested in characterizing the molecular nature of rna-protein interactions. the analysis of n-and c-terminal deletion mutants of the proteins have shown that both proteins contribute to the formation of an rnabinding pocket, the rna-binding and dimerization domains in both proteins, are found in regions that have a predicted ~~helical structure. in sp~pi4, this u-helical region is located betwen aa 72and i00 at the c-terminus; it contains the rna binding and dimerization domains adjacent to each other. our results support the model that the hydrophobic face of the putative u-helix is implicatedinprotein-protein interactions and the positively charged face in rna-protein interaction. in sp~pg, the two rna-binding domains found n-and c~ terminally, also have a predicted amphipatic s-helical structure. it has been proposed that the decline in protein synthesis observed in aging organisms may result from a decrease in the protein synthesis elongation factor ef-la. john shepherd's finding that transgenic drosophila melanogaster carrying an additional copy of the ef-la gene have an extended lifespan further indicated that the ef-la gene may play an important role in determining longevity (shepherd et al., 1989) . in order to test this hypothesis, we have quantitated ef-la mrna, ef-la protein, as well as catalytic ef-la activity in john's flies. young and aging ef-hx transgenic (ef) and control lines ((2) were examined. because the the additional ef-lc~ copy is under the control of the hsp70 promoter, flies were aged either at 25~ or at 29~ the results show that transgenic ef flies do not express more ef-lct than the control flies, neither at the rna nor at the protein level. the question arises, whether the lransgene can be induced at all. this was tested by doing rnase protection assays. transgenlc message can be detected only in ef flies heat shocked at 37~ but not in ef flies grown at 29~ nor in control flies at 37~ from this experiment we conclude that the transgene is indeed inducible, but is not expressed in detectable amounts at 29~ we conclude that the difference in the lifespan does not result from the overexpression of the ef-la transgene. phenobarbital (pb) induces the expression of liver enzymes involved in the metabolism of drugs and other foreign compounds (xenobiotics). these enzymes include several cytochromes p450, nadph:p450 reductase, aldeyde dehydrogenase, epoxide hydrolase, udp-glucoronyltransferases and glutathione-s-transferases. many other proteins not yet recognized may however be induced by pb. our first approach therefore is aimed at identifying gene products which respond to pb treatment. as model systems we are using either chicken embryos (induction in ovo) or primary cultures of chicken embryo hepatocytes (induction in culture) (althaus et al., jbc 254 pp 2148 , 1979 . to differentially display mrna of induced vs non-induced cells we are using a rt-pcr technique with sets of random primers. electrophoretic separation of amplification products allows to identify mrna species which are induced (or decreased) following treatment. data obtained from these experiments and sequence information revealed from extracted pcr products indicate that pb treatment results in the transcriptional avtivation (or repression) of a variety of so far unknown genes. antithrombin -binding heparan sulfates from ovarian granulosa cells hosseini g., de agostini, a., clinique de st6rilit6, hcug, gen~ve. at the time of ovulation, several serine proteases of the plasminogen activator and coagulation cascades are activated in the ovary. these proteolytic activities are tightly controlled in time and space, and the heparin-activated serpins plaminogen-aetivator-inhibitor-1, protease nexin-1 and antithrombin (at) are present in the follicular environment. we have observed that granulosa cells produce a subset of heparan sulfate proteoglycans that bind and activate at. these at-binding heparan sulfates (ahspgs) endow the vascular endothelium with antithrombotic properties. we are now examining the role of ahspgs in the extravascular compartment formed by the ovarian follicle. using 125i-at binding assays, we have detected ahspgs on granulosa cell surfaces and culture media, in amounts comparable to those found for endothelial ceils. after 48h of stimulation by the gonadotropin fsh (1-100 ng/ml) granulosa cells increased the amounts of ahspgs they released in culture media by 2-6-fold, while keeping their cell surfaceassociated ahspgs constant. analysis of 35s-labelled hspgs revealed that heparan sulfates constitute about 40 % of the surface-associated and 10% of the soluble granulosa cell glycosamineglyeans. finally, using 125i-at autoradiography on rat ovary cryosections, we have localized ahspgs on the granulosa cell layers of large antral follicles, while ahspgs could not be detected in smaller follicles. these data suggest that granulosa cell ahspgs could be critically located to modulate proteolytic activities in the changing environment of the growing follicle. division, cibabasel, switzerland. the development of chelators which can mobilise excess storage iron (fe) and permit its excretion is necessary in the treatment of fe overloaded diseases. although oral administration of such a pharmaceutical is highly desirable, no orally active fe chelator is so far in regular clinical use. searches for orally active and safe fe chelators require appropriate animal models of fe overload in order to evaluate the potential of these drugs. an animal model is particularly useful for testing the capacity of new fe chelating ce~oounds in enhancing the mobilisation of clinically relevant storage iron. iron loading in animals was achieved by dietary supplementation with carbonyl fe, fe fumarate and 3,5,5-trimethylhexanoyl ferrocene (tmh-ferrocene). liver fe concentrations were increased 6-21 times above normal levels. tmh-ferrocene was the most efficient compound to induce stable liver fe overload. the orally given con'pounds induced a predominantly hepatocellular iron distribution and was found distributed among reticuloendothelial cells only at a later stage. this pattern of liver fe storage is similar to that observed in the early stages of primazy ha6~ochromatosis and late stage of transfusional hae~ochromatosis. eeeently, we and ethers have cloned and characterized novel transcription factors called peroxisome proliferator-actirated receptors (ppak). they belong to the superfamily of nuclear hormone receptors as the steroid, thyroid and retinoid hormone receptors. ppars are activated, beside xenobiotic peroxisome proliferators, by natural fatty acids and induce, together with the receptor for 9-cis retinoic acld (rxr), the transcription of genes involved in the ~-and ~-oxidation of fatty acids. ppar and rxr bind as heterodimers to the ppar response element (ppre), consisting of a direct repeat of aggtca-iike sequences with one intervening nucleotide. now, we show by gel retardation analysis and transient transfection assays that ppar/rxr heteredimers also bind to and transactivate gene transcription through estrogen response elements (ere). the ere is a palindrome with aggtca half-sites separated by three nucleotides. the selectivity and specificity of the transactivation through an ere was demonstrated by the testing of various related palindromic and inverted palindromic repeats of aggtca sequences, which were all inactive. the possible activation of estrogen responsive genes by fatty acids via ppars in vivo is discussed especially with regard to a debated role of fatty acids in breast cancer. the purpose of this study was to evaluate whether intermittent doxorubicin (dox) treatment in vitro selects for multidrug resistance in rpmi 8226 myeloma cells and, if so, to determine the underlying mechanism(s). cells were exposed to constant doses of dox for 4 days alternating with growth in dox-free medium for 17 days. after > 9 months of treatment, the cells developed an atypical mdr phenotype with 4-to 6-fold resistance to topo ii poisons. cross-resistance to vincristine and taxotere was < 2-fold, sensitivity to cisplatin and melphalan remained normal. efflux of mdr1 drugs was incresed with no effect of verapamit; subcellular dox distribution was normal. expression of topo ii a was found to be reduced by around 50 and 60-70% at the rna and protein level, respectively. minimum mdrt rna overexpression was detected when using rt-pcr; p-glycoprotein was not detectable. mrp rna expression was increased by 60-90% relative to the wild type cells without detectable p190. this data suggests that atypical mdr might play a role in acquired dox resistance in human myeloma. mucosal surfaces cover a vast area in the human body. they satisfy its need to communicate with its environment, e.g. in terms of nutrient uptake and sexual reproduction. however, at the same time they offer a vulnerable gate for invasion by pathogenic microorganisms. for immunoprotection of these regions, large amounts of iga antibodies are produced in the bloodstream and transported across the epithelial barrier to the lumen by means of polymeric ig receptor. there the receptor is cleaved, and its extracellular portion remains associated as secretory component (sc) to the secretory iga complex. in the context of a project aiming to mass production of secretory iga as a passive vaccine, we evaluated a series of eukaryotic expression systems for production of sc. the cdna encoding polymeric ig receptor was engineered in a way that only its extracellular domains corresponding to sc were expressed in yeast, in insect cells infected with recombinant baculovirus, and in mammalian cells infected with recombinant vaccinia virus. furthermore, a c-terminal poly-histidine tag was introduced for simple purification of the products. we will show optimization of expression levels for each of the systems as well as a quantitative and qualitative comparison of the products. recombinant vaccinia viruses (rvv) that express gone products from other organisms has become a popular and widely used laboratory expression system. our current interest is directed toward the production of secretory component (sc), a subunit of secretory iga antibody complex involved in mucosal immunity. toward this goal, we constructed rvv governing sc transcription under the control of two viral-based and the t7 bacteriophage promoters. expression of the protein was assessed in several mammalian cell lines, such as human tk-, human hela (grown in suspension or as a monolayer) and monkey cv-i. optimization of infection parameters and culture conditions were also performed. institut de biologic animale de runiversit~ de lausanne protection of mucosal surfaces is mediated by secretory iga ant/bodies (alga) constituted of dimeric iga and secretory component (sc) . toward the aim of reconstituting slga complexes in vitro and using them for passive oral immunization, sc has been produced in yeast, insect and mammalian expression systems. the gone encoding sc has been engineered so that a) the c-terminus of the protein carries a 6xhis tag and b) the newly synthesized polypeptide is released in the culture medium. culture conditions have been established to permit recovery of sc in serum-free medium. purification procedures based on nickel chelate and classical chromatographies have been optimized to yield sc under native conditions. finally, in vitro reassociation with purified dimeric igas obtained from hybddomas has been used to assess biological activity of recombinant sc. using a magnet-assisted subtraction-hybridisation technique (mast), 17 cdna clones were isolated that are expressed in nqrm~l l~nq %issue but n__d_i expressed in the corresoondina nsclc tissue, ii of the 17 edna clones are highly homologous to edna sequences for the following proteins: pulmonary surfactant proteins sp-a and sp-b; receptor for advanced glycosylated endproducts of proteins (rage); calmodulin-like protein; natural killer gone 5 (nkg5); matrix gla protein (mgp); glutamine synthetase; ubiquitin; vascular smooth muscle alpha-actin; cytoskeletal beta-actin; vimentin. the remaining 6 edna clones may represent parts of still unknown genes. the mast edna clones were derived from a patient who developed squamous adenocarcinoma. northern blot analysis of normal/tumour rna from three other nsclc patients showed that the lack of gone expression was independent of nsclc type and stage. enzymatic methylation of cytosines in mammalian dna is believed to play a fundamental role in basic gene regulation. methylation in the vertebrate genome is restricted to cpg dinucleotides at the c-5 position of cytosine. in vitro studies have shown that methylation can drastically influence the dna structure. cpg islands remain unmethylated in normal cells, whereas several immortalised, transformed cell lines contain partially methylated cpg islands. transcription of genes can be inhibited by methylation of cpg's. this modification contributes e.g. to the stable repression of genes on the inactivated x chromosome of females. the collagen vi genes show typical cpg islands in their promoter region and they were shown to be down regulated at the transcdptional level in src or myc transformed cells. given these facts we dee~ded to investigate the effects of methylation in the collagen vi gwene. e could demonstrate that the promoter activity of (x2(vi) chicken collagen gone was strongly diminished by methylation in vitro. furthermore dna methylation completely prevented binding of a novel transcription factor to the promoter, whereas binding of sp1 was not affected. to show evidence of methylation in vivo, we are currently iocalising the exact positions of methylated cpg's in normal and transformed cells by genomic sequencing. alterations of putative tumour suppressor genes in human non-small cell lung carcinoma (nsclc). r. shipman and c.u. ludwig, molecular oncology, lab 405 (zlf), basel, ch-4031 chromosomal subregion llp13 displayed non-random allelic loss in 61 nsclcs. llp13 contains the genetic loci for catalase (cat), the wilms' tumour gene (wti) and the follicle-stimulating hormone ~-chain (fsh~). 76% of the nsclcs were deleted at cat suggesting that loss of a gene(s) near cat may be involved in the progression of nsclc. yac clones containing the cat locus are being prepared for use in a direct selection procedure for cdnas encoded at this region. although 38% of the nsclcs were deleted at wti, no mutations were detected in the remaining wti allele. wti expression was subsequently demonstrated in fetal lung, fetal hematopoietic tissue, normal adult lung and nsclc. allelic deletion at 17p13, immuno-histochemical and sequence analysis of the p53 gene were also examined in our nsclcs. these analyses support the contention that aberrant expression of the p53 gene is a pivotal event in the progression of nsclc. $08-11 recently, a number of advances have been made which enable the experimentalist to study the effects of low levels of ionizing radiation. the results suggest a threshold to radiation damage exists and that above a critical level, recovery mechanisms in the cell are induced and the biological response falls. thus low levels of radiation cause greater than expected levels of biological response. this has been demonstrated in studies of mutations, cell survival and cancer induction. because the low dose-rates and the low doses per fraction result in the total doses being small, the magnitude of the biological effects induced is insufficient to warrant alarm. identification of genes associated with the revertion of the malignant phenotype of a rhabdomyosarcoma cell line. michele genini, sabina solinas toldo*, ruedi fries* and beat w. schafer, dept. of pediatrics, university of ziirich, steinwiesstr. 75, 8032 ziirich, and *institut for nutztierwissenschaften, eth ztifich, 8092 ztirich. the human rhabdomyosarcoma cell line rd is tumorigenic and differentiates very poorly despite the expression of the myogenic factors myf3 and myf4. therefore it can be regarded as natural mutant. to identify genes which are involved in suppression of tumorigenicity or enhance differentiation we applied two different approaches. since the development of human embryonal rhabdomyosarcoma has been associated with abnormalities of chromosome 11 band p15, in the first approach we transferred a normal human chromosome 11 into rd ceils via microcell fusion. the microcell hybrids did not show a higher degree of differentiation. suppression of tumorigenicity was observed in one clone but is probably independent of chromosome 11, as it was shown by chromosome 11 specific painting. ' in the second approach we applied a subtractive hybridization procedure between primary myoblasts and rd cells to find genes specific for the normal phenotype. these genes will be tested in vivo for induction of differentiation and/or tumorsuppression. deficient synthesis of the gpi anchor is the molecular basis of idiopathic paroxysmal nocturnal hemoglobinuria ("pnh") and a similar phenotype can accompany aplastie anemia cpnh-iike"). the population expressing cd48, a gpi-anehored membrane glycoprotein, was quantified by flow cytometry in samples from patient p.g. (pnh) and patient m.m. (pnh-like) and amounted to 90% (p.g.) and 78% (m.m.), respectively. t lymphocytes from both patients were isolated, expanded and cloned. from both patients, clones expressing the cd48 marker (cd48+) and cd48 deficient clones (cd48-) were obtained. takeda et al. (cell 73, 1993, 703-11) showed that in some (cd59-) bcell clones, a deletion of the p1g-a gene product is responsible for the expression of the pnh-phenotype. pcr analysis of t cell mrna from extracts of both cd48+ and cd48-clones of patient m.m. (pnh-like) using pig-a specific primers revealed a band pattern from which can he concluded that no deletion is present in the pig-a gene product. this result suggests that cd48-t lymphocytes of the pnh-like patient express a normal pig-a gene product; a disorder in regulation of pig-a expression or an unrelated biosynthetic defect may explain the cd48phenotype in these t cells. further work is aimed at defining the differences in biosynthetic defects of pnh and pnh-like cd48-t cell clones. supported by grant 31-30757-91 of the snsf to egb. w@ have recently employed an experimental approach to turn the function of "nuclear messengers" such as c-fos (and c-jun) on and off at will in polarized me/nmary epithelial cells. to this end we used constructs encoding the c-fos (and c-jun) genes fused to the hormone-binding domain of the human estrogen receptor, designated c-foser (and c-juner), we could show that short-term activation (30 mins.) of c-foser by estradiole (e2) led to the disruption of epithelial cell polarity within 24 hours, as characterized by the expression of apical and basolateral marker proteins. during the next 3-5 days, however, the cells fully regained their polarized phenotype. conversely, long-term activation (24-48 hours) caused the irreversible loss of epithelial cell polarity, leading ultimately to an epithelial-fibroblastoid cell transition. these cells underwent dramatic changes in gene expression including the complete loss or reduction of epithelial markers such as e-cadherin/uvomorulin, zo-i and cytokeratins, and the de novo expression of mesenchymal proteins like vimentin, type i collagen and fibronectin. expression of the v-ha-ras oncogene (acting upstream of c-fos) did not markedly influence epithelial cell organization when the cells were grown on plastic. however, when cultured within type i collagen gels in the presence of serum, or when injected into the mammary glands of nude mice, rapid cell growth and a clear epithelial-fibreblastoid cell transition was observed. growth properties determine the organ colonization ability of a metastatic murine melanoma cell line. the ability of metastatic tumor cells to specifically colonize distant organ sites strongly depends on the interaction of the metastatic cells with the local organ environment. we have selected a b16 routine melanoma cell fine (b16-ls9) which has an increased ability to colonize the liver, and compared its behavior with either its parental (b16-f1) or inng-specific (b16-f10) cell line. we have found that adhesion in vitro and organ lodgement in vivo were not different for ls9 and f10. in contrast, b16-ls9 grew at slower rates than the other lines both in vitro and in vivo after subcutaneous or intra-foot-pad injection. analysis of tyrosine-phosphorylated proteins indicated a higher phosphorylation activity in b16-ls9 cells, which might suggest an altered regulation of some kinase(s) in this cell line. intercellular contact with hepatocytes in vitro, or the liver in vivo could restore an efficient growth of b16-ls9, enabling thus these cells to colonize this organ much better than others. hepatocytes or liver plasma membrane (p.m.) extracts conserved the growth stimulatory activity towards b16-ls9 ceils. chromatographic fractionations of these extracts showed that a major growth sfimulatory protein in this fiver p.m. fraction turns out to be ttansferrin, which in the liver appears to exist in at least two different mature forms. protein import into mitochondria requires atp in two locations: outside the organelle, and in the matrix space. depletion of matrix atp arrests translocation of precursors across the inner membrane, but does not prevent precursors from crossing the outer membrane. as a result, proteins that reach the inner membrane or intermembrane space without passing through the matrix are often sorted normally in atp-depleted mitochondria. external atp is used to maintain precursors in a translocationcompetent state. however, some precursors can fold and still remain translocation-competent, and import of these precursors is independent of external atp. with the folded cytochrome b2 precursor, it is matrix atp rather than external atp that drives translocation across the outer membrane. thus matrix atp generates a pulling force that can drive both the translocation and the unfolding of precursor proteins. mitochondrial hsp70 probably functions as the atp-dependent import motor. arsenijcvic d, dulloo ag & girardicr l, dpt of physiology, cmu. geneva, ch. the relationship between body weight, daily energy expenditure (assessed by indirect calorimetry), and immune status (determined by lymphocyte proliferation tests) was investigated in swiss webster mice maintaining a stable body weight several weeks after infection with toxoplasma gondii(me49). following an initial period of weight loss (infection-induced cachexia), the surviving mice could be differentiated into two main groups: (i) those showing a partial regain in body weight (the infected gainers) and eventually stabilizing al a mean body weight 25% below a non-infected control group, and (if) those showing no weight regain (the infected non-gainers) and eventually stabilizing at 45% below control levels. immune function was found to be markedly suppressed in the infected non-gainers, whereas it was normal (and similar to control levels) in the group of infecled gainers. daily energy expenditure (and food intake), after normalisation for differences in body weight, was found to be the same in both infected gainers and non-gainers, but lower by 15% when compared to controls (p<0.01); thereby indicating that the infected groups were able to increase their overall efficiency of food utilization in response to the low food inlake. in conclusion, this study conducted during a weight stable phase of chronic infection shows a clear-cut association between the inability to regain body weight and impaired immune function. in contrast, no relationship was found between metabolic rate and body weight nor between metabolic tale and immune function, but the chronically infected mice exhibited the same phenomenon of enhanced metabolic efficiency characteristic of chronic underfeeding per se. identification and functional analysis of chaperonin 10, the groes homolog of yeast mitochondria. biozentrum, university of basel, klingelbergstrasse 70, ch-4056 basel, switzerland. phone ++41-61-2672171, fax ++41-61-2672148. the mitochondrial chaperonin system consists of chaperonin 60 (cpn60; also termed hsp60), which is homologous to e. cull gruel, and chaperonin 10 (cpnlo), which is homologous to e. coil groes. we have used a functional assay to identify cpnl0 of yeast mitochondria. when dimeric rubisco is denatured and allowed to bind to yeast cpn60, subsequent refolding of the enzyme is strictly dependent upon yeast cpnl0. in the presence of mgatp, yeast cpn60 and yeast epnl0 form a stable complex that can be isolated by gel filtration. the atpase activity of hsp60 is not inhibited by complex formation with cpnl0. a different result is obtained with the e, coil system, where groes is able to completely inhibit the atpase activity of gruel (1). to test the in vivo role of cpnl0, we have cloned, sequenced and disrupted the corresponding nuclear gene cpnio. this gene encodes a protein of 11,372 da that is imported into the mitochondrial matrix without detectable cleavage. haploid cells lacking a functional copy of cpnio fail to grow at temperatures between 23 ~ c and 37 ~ c (2). (1) rospert, s. et al. (1993) proc. natl. acad. sci. 90, in press. (2) rospert, s. et al. (1993) febs lett., in press. characterization of yeast mitochondrial heat shock protein 70 , l. bolliger, b. s. glick and g. schatz, biocenter, university of basel, 4056 basel. mitochondrial hsp70 (mhsp70) is thought to use the energy of atp hydrolysis to pull precursor proteins across the mitochondrial membranes. to facilitate the purification of yeast mhsp70, we used the cloned gene (a gift of dr. n. morishima) to modify mhsp70 with a six-histidine tag at its c-terminus. this construct supports growth of yeast cells lacking wild-type mhsp70. we developed a purification procedure that allows us to recover this tagged protein with a purity of about 95%. experiments are in progress to characterize the nucleotidedependent interaction of purified mhsp70 with unfolded proteins. in addition, we are looking for partner proteins that may modulate the action of mhspt0. we have copurified a protein of about 20 kd together with mhsp70. the 20 kd protein could be released from mhsp70 by atp or adp. when tryptic fragments of this protein were subjected to microsequencing, one fragment showed strong homology to the grpe protein of e.coli. the transmembrane electrical potential of mitochondria (awmit) can be assessed in single hepatocytes by using epifluorescence microscopy. for this end the rhodamine 123 fluorescence of a mitochondria-rich (fr) and a mitochondria-poor region (fp) are measured. the ratio of these signals depends on the awmit according to a modified nernst equation (reber b .f.x., somogyi r. & stucki j.w. (1990) , bba, 1018, 190-193) . to calibrate this method the cell membrane was permeabifized for monovalent cations with nystatin and gramicidin. then the cytosolie k + was replaced by na + from a k+-free bath solution. subsequent addition of valinomycin made the mitochondrial membrane permeable for k + ions. by the addition of different k + concentrations to the bath, the awmit could be clamped to defined values. the relation between the ratio fr/fp and the awmit could be well fitted to our modified nernst equation. however, the calibration curve flattens out at potentials higher than about 110 inv. therefore the detection limit of changes of a~mit within the physiological range is about 10-20mv. maximal stimulation of the na+/k + atpase by addition of nystatin to the na + containing bath medium caused a drop of awmit of about 20inv. subsequent addition of ouabain resulted in an almost complete reversal of this drop. this shows that changes in atp consumption can be assessed via changes of audmit. schneiter ph., di vetta v., j6quier e. and tappy l. institut de physiologie de l'universitg bugnon 7,1005 lausanne. the metabolic effects of an exercise performed in the fasting or fed state were studied in 8 subjects over a 8 hour period in a respiratory chamber. a mixed meal containing 13c labeled carbohydrates was ingested at 9:30 am and an exercise session (walking at 5 km/h 45 rain, slope 10 %) was performed at 8:15 am (fasting) or 11 am (postprandial). net carbohydrate (net cho ox) and lipid oxidation (indirect calorimetry) and oxidation of exogenous carbohydrates (exo cho ox, breath 13co2) were measured. glycogen breakdown was calculated as net cho ox -cho exo ox, and glycogen synthesis as cho in -cho exo ox. energy expenditure over 8 hours was similar but net cho ox was 16 % lower, lipid ox 61% higher and exo cho ox 39 % lower when exercise was performed in fasting vs fed state; moreover, glycogen synthesis was increased by 40 % (p<0.0001), while glycogen breakdown was increased by 12 % (p=0.06). in conclusion, a 45 min exercise in the fasting vs fed state a) increases utilization of endogenous lipids, b) enhances muscle glycogen turnover over a 8 hour period. hormonal regulation of e-abl tyroslne kinase by fusion to a steroid binding domain t. mattioni, o. 8chini hooft van huijsduijnen, p.k. jackson and d. picard d4partement de bielogie cellulaire, universit4 de geneve, ch-1211 geneve 4 the activities of a wide variety of proteins can be subjected to hormonal control by fusion to the hormone binding domain of steroid receptors. we show that this strategy can also be applied to a tyrosine kinase. in particular, transforming derivatives of c-abl become hormone-inducible oncogenes by fusion to a steroid binding domain. it is noteworthy that the hormone-inducible transformation of nih3t3 cells is completely reversible upon removal of the specific ugand. reversion experiments reveal another facet of abl: its ability to inhibit cell proliferation. 48 hours after hormone depletion, cells are not only morphologically reverted, but the cell cycle appears to be blocked in g1. a cytostatic effect of abl overexpression has been reported by several groups but, until today, it could only be examined by comparing different abl derivatives or the same derivatives in different cellular contexts. in contrast, our hormone-regulable system has the advantage that the two distinct functions of abl (transformation versus growth inhibition) can be studied using the same derivative expresed in the same cell line. we are using the hormone inducible system to investigate the mechanisms regulating the transtorming and the cytostatic effects of abl in a variety of cell lines. jiewu yang and herbert zuber institute for molecularbiology and biophysic, eth, 8093 z'tirich enzymes from thermophilic organisms show higher thermostability and temperature optima than those of mesophilic organisms. it has been shown that these properties are based on a specific structure of the protein. the primary structure of thermophilic (b. stearothermophilus) and mesophilic(b, megaterium) triosephosphate isomerase(tim) have been determined by cloning and sequencing of the tim genes. comparison of the primary structures of the thermophilic and rnesophilic enzyme showed specific amino acid substitutions, particularly in the c~-helices of the tim barrel, which could play a critical role with respect to thermostability. we have consmacted mutants by exchanging (x-helices between tim of b. stearothermophilus and b. megaterium. helix h1, h2 and h7 of tim from b. megaterium, corresponding to amino acid residues 13-36, 44-55. and 220-227 respectively, have been exchanged. the properties (thermostability and temperature optimum) of the hybrid-mutants were compared with the wild type enzymes. in a comparative investigation of structural and functional parameters related to the oxidative substrate metabolism in skeletal muscle cells of dogs and goats, a close relationship was found between intracellular lipid stores and mitochondria. as revealed in serial sections with subsequent em analysis, each lipid droplet was in direct contact to one or several mitochondria. quantitatively, 23% of the lipid droplet surface in goats and 40% in dogs were in direct contact to outer mitochondrial membranes or -in other terms -1% of outer mitochondrial membranes in goats and 2% in dogs were in direct contact to lipid deposits. these findings were in good agreement with the physiological data showing a 2 times higher oxidation rate of fatty acids drawn from intracellular stores for dogs than for goats. human muscle adapts to altered load with changes in the expression of enzymes involved in energy metabolism. endurance exercise in humans is known to increase the oxidative capacity of skeletal muscles (hoppeler, int. j. sports med. (1986), 7, 187) . at the ultrastructurai level, a higher mitochondriai content contributes to the higher oxidative capacity of skeletal muscles. little is known about the molecular mechanisms that lead to such adaptations in humans. the expression of mitochondriai components involves complex regulation of both mitochondriai and nuclear encoded genes. we have developed a pcr approach to quantify dna and rna from human skeletal muscle cryostat sections and addressed the question, whether the structural adaptations in endurance trained athletes are accompanied by concomitant changes in respective rna steady state levels. preliminary data indicate a higher concentration of coxiv and coxl (1.8 and 1.6 times, respectively) in trained subjects. no differences were observed for mtdna, despite a two fold higher mitochonddal content. human skeletal muscle contains three main fiber types which are based on the myosin heavy chain composition of the individual fiber, the isoforms of other contractile proteins within a given fiber type can vary leading to a continuum of different phenotypes. we are studying the fiber type specific expression of the mrnas of myosin alkali light clmins (mlc) using in situ hybridization, especially the rarer slow form mlc lsa. differences were found between shoulder muscles and leg muscles: m. deltoideus yielded stronger signals for mlc lsa and mlc lsb in the least glycolytic type i fibers (ia), weak but detectable signals for mlc lsb and mlc lf/f mrna in more glycolytic type i fibers (ib) and strong signals for mlc lf/3f in type ii fibers. in m. vast. lat. mlc lsb was strongly expressed in type ia as well as ib fibers, mlc lsa mrna was only found in some ia fibers. mlc isa has been shown to be expressed at a particular time point during muscle development and regeneration. in a biopsy of a becker dystrophy patient, we found very strong signals in some small fibers displaying the morphology of regeneralmg fibers. thus this probe could be useful to dete~t regenerative processes in pathological muscle samples. the aim of this study was to validate the complementarity of two new non-invasive methods for the assessment of autonomic regulations. reactions to a moderale exercise (75 w on a cycloergometer) were compared in cardiac transplant recipients (ctr) who modulate their heart rate (hr) by means of circulating calecholarnines, their heart being donervated, and in normal subjects where such exercise level doesn't stimulate the sympathetic system. the methods used were: 1) the spectral analysis of hr variability where the high frequency component (hf, above 0.15 t4z, related to the respiratory frequency) is a marker of the vagal activity, the low frequency component (lf; at about 0.1 hz) depends on both sympathetic and parasympathetic activities and the lf/rf ratio indicates changes in the sympatbo-vagal balance; and 2) a pure sympathetic index obtained from the amplitude of the t-wave of the ecg (twa) which is decreased by adrenergic stimulation of the myocardium. our results indicate that in both groups hr was increased by the exercise. in ctr, a net decrease in twa (-39_+ 10%, n=6) was measured. contrasting with this, in normal subjects, no change in twa (+2_+12%, n=6) but a decrease in beth hf (-67_+13%, n=7) and lf (-85_+5%, n=7) without change in lf/hf ratio (-19+_.26%, n=7) were observed. these results indicate that ctr subjects increase their hr by an adrenergic stimulation, whereas the normal subjects regulate their hr for such moderate exercise only by redudng their vagal tone. there was a significant relationship (r=0.79, p<0.0005) between ps and the ree. the slope of the regression line indicated a net cost of ps of 3.6 kcal/g. we conclude that obesity in children is associated with an absolute increase in whole-body protein turnover consistent with the increased ffm and ree. the "glucose paradox" in the anoxic and reoxygenated embryonic myocardium raddatz, e., tran, l., rochat, a.-c., kucera, p. and de ribaupierre, y. institut de physiologie de l'universitd, ch-1005 lausanne. the hearts of 4-day-old chick embryos explanted in vitro react rapidly, reversibly and reproducibly to anoxia and reoxygenation. this work deals with the effects of exogenous glucose on the mechanical activity of the hearts submitted to strictly controlled normoxia-anoxia-normoxia transitions. the anoxic periods lasted from 10 to 60 seconds. instantaneous heart rate, amplitude of contraction, velocities of contraction and relaxation of the ventricle wall were determined optically. in presence of 8 and 20 mmol/1 of glucose 1) the arrest of cardiac activity under anoxia was delayed by 60 and 110 %, respectively, 2) the duration of the postanoxic cardioplegia was reduced and 3) recovery was faster. paradoxally, these concentrations of glucose induced arrhythmias both during anoxia and reoxygenation. the incidences of such arrhythmias increased with the duration of anoxia. the absence of glucose or blockade of glycolysis suppressed arrhythmias. image analysis showed that the observed myocardial dysfunctions originated in the atrium and were sometimes associated with disturbances of propagation. the microinjeetion of okadaie acid into incompetent oocytas leads to the release of the prophase block with entry into m-phase. these g2/m-like alterations include chromatin condensation, germinal vesicle breakdown (gvbd), microtubules reorganization and formation of an abnormal (often multipolar) spindle of meiosis i, with chromosomes not aligned on the metaphase plate. the polar body is never extruded. moreover okadaic acid microiujection induces the emergence of phosphorylated cytolasmic foci as detected by the monoclonal antibody mpm-2, known to react with mitotic phosphoproteins associated with centrosomes. the kinetics of gvbd following mieroinjection is extremdy retarded (24-48hrs) when compared to spontaneous maturation (30ran-lhr) and depends on the oocyte diameter. interestingly, when inc~ompetent oocytes are cultured during 48hrs and then microinjeeted with okadaic acid meiosis resumes within a short delay (3-rhrs). mpm-2 epitopes are present on ecntrosomes only after okadaic acid injection. following gvbd one of the step depending on protein synthesis, the expression of tissue type plasminogen activator, is triggered. indeed oa-injected incompetent oocytes produce small amounts of this activator. altogether these results argue for an effect of okadaic acid favoring entry into mphase including microtubule organization, centrosomes phosphorylatiou and the recruitment of one of the masked mrnas (tpa). st~rillt~, ncb~, c~-121l g~. ~t4rs-inse~, monf~llier, france. meiotic reinitiation of the mouse oocyte is caracterized by a slow entry into metaphas~ i, b6~jinnin9 with germinal ve~ir break@own (gvbd) and ending with spindle formation. it is accompanied by a cascade of protein kinases and phosphatases increasir~g protein phosphorylation. the activation of histone hi kinase (maturation promoting factor, mpf) and of the 1342 hapk have been compared during spontaneous or okedalc acid (oa)-induced rhetoric reinitiation. in spontaneously maturing oocytes, hist6ne hi kinase activity increases before gvbd (2x) and is a~sociated with the disappearance of the upper (tyrosine phosphorylated) migrating form of p34 cdc2, following gvbd, histone hi klnase activity culminates (8x) in metaphase i. activation by phesphorylation of p42 mapk occurs after gvfld. in contrast, no increase of histone hi kinase is detec~cable in oocytes induced to reinitiate meiosis by a transient exposure %o oa, neither before gvbo nor during the following 7 hours of culture. the upper migrating form of p34 cdc2 is present for 8 houra. the activation of p42 mapk b~lins before gvbd. furthermore, when oa is applied on coclrces microinjected with p13 sucl, neither iocrease of histone hi kinase activity nor p34 cdc2 dephosphorylation are observed although gvbd is induced; p42 mapk is activated. altogether these results suggest that gvbd may or may not be associated with a detectable activation of histene hi kinase, depending on the experimental conditions. activation of ps4 cdc2 and p42 mapk are separable events. we propose that, in the mouse c~yte, oa might be able to aetivaf8 an alternative pathw~ leading to gvbd that is cdc2-independent and that involves p42 mapk activation ensuing mpf-independent phosphorylations. s 10-04 urich, k., 4002 basel, switzerland transformation of cells by polyomavirus is mediated by middle-t antigen. this viral protein is able to form complexes with src family tyrosine kinases (e.g. c-src), phosphatidylinositol-3-kinase (pi 3-k) and phosphatase 2a (pp2a). c-src associated with middle-t is constitutively dephosphorylated at tyrosione 527, a site negatively regulating the activity of this enzyme. this results in high tyrosine kinase activity in interphase and mitotic polyoma-transformed cells. middle-t is transiently hyperphosphorylated during mitosis as reflected by an increase in the apparent lvl, on sds acrylamide gels. two putative phosphorylation sites for a cyclin-dependent kinase present in middle-t, threonine 160 and threonine 291, are also phosphorylated by purified p34 ~a~ in vitro. we constructed mutant middle-t genes where individual phosphorylation sites were converted to alanine residues. mutants lacking threonine 160 were still able to associate with all cellular enzymes described above yet defective for gell transformation. interestingly, the defect of this mutation could be compensated by additional changes in the middle-t protein sequence introduced further downstream in a domain suspected to play a role in the targeting of this protein to intracellular membranes. $10-05 schmidt, s., fa~khauser, c., and viesturs, s. isrec, 1066 epalin~es, switzerland little is understood of the mechanisms which regulate cytokinesis, or how it is integrated with mitosis. we have cloned a number of genes from the fission yeast s. po~be which are required for the initiation of septation and are characterising them. defects in the cdc7 and cdcll genes result in continued nuclear division without cytokinesis, while cdcl6 mutants undergo multiple rounds of septum formation without cell cleavage. our analysis suggests that phosphorylation will play an important role in the regulation of cytokinesis and its integration with mitosis. a functional cdcl6 product is required for maintenance of p34 r activity in mitosis and the primary sequence of the cdc7 protein indicates that it encodes a protein kinase. data concerning the role of the cdc7, cdcll and cdcl6 genes will be presented. rfc was originally identified in human cell extracts as one of the cellular factors essential for the replication of sv40 origin-containing dna in vitro. it is a five subunit protein with one large subunit of 100-i40 kda and four small subunits of 36-40 kda. rfc binds specifically to primertemplate structures at the 3'-end of the primer. together with proliferating cell nuclear antigen (pcna) it serves as a primer recognition factor for dna polymerase ~ and ~. to show the invobement of rfc in cellular dna replication we are currently cloning s. cerevisiae rfc. the amino acid sequences of a number of tryptic peptides have been obtained. this information was used to amplify parts of the s. cerevisiae genes by per and to screen a genomic library with these probes. three genes were cloned so far. the large subunit, rfc1, codes for a 95 kda polypeptide that is 36% identical to the large subunit of human rfc (lirfc). the sequences for two of the small subunits were also obtained. rfc2, coding for a 40 kda polypeptide, is 50% identical to the hrfc 37 kda subunit. rfc3, coding for a 38 kda polypeptide, is 51% identical to the hrfc 36 kda subunit. there is also considerable similarity between the different subtmits. rfcj, rfc2 and rfc3, as well as the hrfc subunits, have consensus sequences for a atp/gtp binding site. all three genes are essential in s. cerevisiae. p53 is a major tumor suppressor gene which spefically interferes with the onset of e phase. we have therefore cxa~ined the possibility that p53 modulates the normal functions of enzymes and proteins directly involved in dna replication. we have shown that human wtp53 protein binds physically to calf thymus single stranded dna binding protein a, rp-a, we have also found that p53 blocks dna helicases in a typical strand displacement assay. this may reflect the fact that p53 is able to reanneal complementary dna single strands. since both wt and some mutant forms of p53 share this function, they are unlikely to be related to the p53 tumor suppressor activity. to our surprise, among the helicases which were tested we determined that the p53 inhibition could be released by rp-a in the case of cellular but not in the case of viral dna helicases. additionally, we found that p53 can partially inhibit dna polymerase ~ and s holoenzymes on singly primed mi3 dna. this inhibition, however, was only evident when e. coli ssb was substituted for rp-a. our data thus show that p53 exerts an inhibitory effect on several enzymes involved in dna replication and dna repair. the p53-rp-a interaction may function to protect replication enzymes from inhibition by p53 under certain physiological conditions. catalytic subunit showed that pol ~ is the most conserved replicative pol (cullmarm g., hindges r., berehtold m.w. and htibscher u., gene, in press). we synthesized pcr primers and cloned three fragments spanning the whole catalytic subunit. the resulting pcr products, called n-term, middle and c-term have a size of 1600, 1570 and 1280 bp, respectively. the primer for the n-term and middle fragments contained a histidin tag in order to purify the recombinant proteins by using a ni-nta column. these fragments were cloned into the expression vector pgemex174. because of the natural termination codon in the c-term fragment, the histidin tag was placed directly in the vector in front of the fusion protein, generating a new vector, prhh1s. all three fragments were expressed in e.coli. the fusion proteins could be purified in a single step and were used to raise polyclonal antibodies. finally, the entire pol 8 sequence was joined together and could be expressed. data on the characterisation of the antibodies and the proteins will be shown. hsp 70 from calf thymus can influence dna polymerase under heat shock conditions. we have generated antibodies against dnak protein, the hap70 homolog from escherichia coil, and used them for detecting of hsp70 protein during its purification from calf thymus tissue. the apparently purified protein has a molecular weight of 70kda, and has been recognized by antibodies against dnak and eucaryotic hsp72/73, it possesses a very week atpase activity, which can be stimulated by different poly-and oligopeptide substrates. results will be presented showing the influence of hsp70 on dna polymerase r from calf thymus under heat shock conditions. identification of a vertebrate cdc2 mutant which is unable to complete the g1/s transition. nicole sr and viestars simanis. chemin boveresses 155, 1066 epalinges,.isrec. s. pombe strains have been constructed which should permit the generic and molecular analysis of the events which occur in late gi when cells become committed to the mitotic cell division cycle and the initiation of dna synthesis. a number of mutants of the chicken cdc2 gene were eonsuueted during the course of analysis of phosphorylation sites some of which lead to the interesting phenotype of cold sensitivity when expressed in a cdc2 null background, when the only source of p34 cde2 is the chicken homologue of the gene, expressed from a muldcopy plasmid. in order to create a genetically tractable strain, the wild-type chicken cdc2 gone and one mutant into the s. pombe genome to create a strain in which cell cycle progression is dependent upon the chicken cdc2 gene. analysis of this strain indicates gnat the cells block predominantly before replication of dna. in order to determine whether the cells were arrested before or after the execution of the start control their ability to conjugate at the arrest point was assessed. consistent with a block before the traverse of start and contmim~ent to s-phase, cells were able to conjugate with high efficiency. further support for the view that this mutant is defective only for the g1-s transition is provided by the observation that it is able to complement a cdc2a2l mutation in trans. this mutant is defective only for (32 function and not traverse of start. tl~e double mutant is viable at the restrictive temperature of either of tile parents alld is no longer cold sensitive. further work will concentrate on cloning genes involved in the traverse of gi into s phase in fission yeast. (masson and kreis, 1993, j. cell biol. 123:357) where it is localized on a subset of microtubulea. when caco-2 cells are grown to confluency and become polarized, e-map-115 expression levels increase concomitant with a higher microtubule stability. transfection of fibroblastic cells (veto), which do not contain any detectable e-map-115, with cdna encoding this protein, results in stabilization of microtubules to nocodazole treatment. these data suggest that e-map-115 may be involved in the stabilization and reorganization of microtubules in polarizing epithelial cells. since e-map-115 is a stabilizing protein, its interaction with microtubulen should presumably be modified at the onset of mitosis to allow disassembly of the interphase microtubule network and formation of the mitotic spindle. indeed, immunolabeling for e-map-t 15 varies during mitosis. in early prophase, no e-map-115 can be detected on the microtubules of the forming mitotic spindle. the protein is observed on the spindle microtubules of metaphase cells and staining becomes bright on the new interphase microtubules of telophase cells. analysis of the protein in cells blocked in mitosis by low concentrations of nocodazole indicates that e-map-115 is hyperphosphorylated. this hyperphosphorylated form of e-map-115 is no longer able to co-sediment with microtubules in in vitro microtubule-binding assays. phosphopeptide map and phosphoamino acid analysis show the existence of novel phosphorylation sites in e-map-115 during mitosis compared to the protein during interphase, we are currently characterizing the kinases involved in the modulation of e-map-115 activity. little is known about the specific functions of calcium-binding proteins in epithelial cells ~md in particular in tumoral cells of epithelial origin. calretinin (cr) and parvalbumin (pv) are supposed to be involved in cell proliferation. we observed the endogenous expression of cr in several cell lines from human colon adenoearcinomas, and we obtained stably transfected cells for pv in a human ovarian adenocarcinoma cell line. we studied the cell cycle in correlation with the endogenous or ectopic expression of cr and pv, respeetiveiy. g1 and mitotic celts are strongly labelled with the cr-antiserum 7696, while pv immunoreactivity is dominant in interphasic, but not in mitotic cells. in cr expressing cells, the cell cycle seems to be normal and the rate of proliferation to be proportional to the percentage of positive cells. the cell cycle is inhibited at mitosis after the addition of cr antisense oligo nucleotides to the culture medium. the cell cycle is blocked in gi/s and g2 in the cells eetopically expressing pv. the results support the hypothesis that these two calcium.binding proteins, cr and pv, could intervene at different moments and probably with different mechanisms on the mechanism of the cell cycle in the tumoral cells studied. production of polyclonal antibodies against calretinin cr-22k gander, j.-ch., gotzos, v., cello, m.r. and schwaller, 8. institute of histology and gen. embryol., university; 1700-fribourg a mrna for an alternatively spliced form of calretinin, ealretinin-22k (cr-22k) was detected in widr cells (a cell line from a human adenocarcinoma). we therefore decided to produce antibodies specific for this protein. it is directed against the 14 amino-acids localized at the c-terminus of the truncated protein which are unique for this protein and not present in full-length calretinin. two different approaches have been chosen to obtain a specific antiserum. we have produced a chimeric protein containing the cterminal region of cr-22k and the (h)6(nanp)19 polypeptide as carrier. the fusion protein was expressed in e. coil and purified on a nickel chelate column. as a second method, a synthetic peptide (corresponding to the last 15 amino acids of cr-22k) was crosslinked with the carrier protein klh (keyhole limpet hemocyanin). the antigens were used to immunize two rabbits. after the first boost, the 2 sera were tested. we have observed that both antisera recognize besides the protein used for immunization also cr-22k (expressed in e. coil) in a western blot specifically. no other protein bands of either e.coli extracts or from eytosolic extracts of widr cells crossreact with the 2 antibodies. both antisera detected the protein cr-22k in immunohistochemieal stainings of widr cells confirming the presence of cr-22k in widr cells. the tctp (p23) is a growth related tumour protein which is expressed under translational control. homologous acidic proteins have been found in higher plants and in saccharomyces cerevisiae (ykl312) . the striking conservation of this polypeptide between these very divergent organisms suggests some important but still, unknown cellular function. to address this question, a null mutation was constructed in yeast, by deleting almost all the ykl312 structural gone. the deleted strain shows no detectable phenotype. therefore, we conclude that, in yeast, this gene is dispensable. the protein ykl 312has been overproduced in e. coil and injected to rabbits to raise antibodies. we are now characterizing the ykl312 expression at rna and protein level. ellenrieder,c., forrer,p., kosovsky,j., rischle, m., nueller, r. and jaussi, r., institut f~r medizinische radiobiologie, paul scherrer institut & universitgt zh, ch-5232 villigen radiation therapy of tumors could be improved by influencing cellular radiation sensitivity. yeast strains with defective cell cycle checkpoint genes (e.g. radl, rad3, radg) show increased radiation sensitivity. cross-hybridization of a probe from the tad9 gene on human mrna northern blots yielded a single prominent signal. experiments to clone the corresponding 4 kb cdna are underway. we have cloned a hamster cdna encoding the homologue of human cdk2. probably, this cdna encodes the p40 protein from hamster whose phosphorylation responds to radiation checkpoint signals and who does not bind to p13 sucl beads. treatment of cells with cdk2 antisense oligonucleotides is hoped to modulate the radiation sensitivity. proliferation of smooth muscle cells (smcs) is a major event in vascular development and atheromatous plaque formation. in order to characterize smc replicative potential, newborn rats have been injected with 3h-thymidine (3h-tdr) during their first week of life when most of smcs were proliferating; then, 3h-tdr labelling was evaluated in adult rats. depending on the number of mitosis that smcs had accomplished after the first week of life, four different smc subpopulations could be defined indicating that rat aortic smcs are heterogeneous in their replicative activity. 5-bromo-2'-deoxyuridine (brdu) incorporation after balloon induced endothelial denudation of rat aorta in adult rats showed that smcs entering into the cell cycle were mainly devoid of 3h-tdr labelling, this category could derive from two distinct smc subpopulations: smcs which were arrested in their proliferation before or just after birth or smcs which had actively replicated during development. thus, one (or two) subpopulation(s) of rat aortic smcs, characterized by a particular replicative activity during development, is (are) selectively activated after balloon induced endothelial denudation. the situation in vivo of dermal fibroblasts embedded within the connective tissue can be emulated in vitro by growing the cells in collagen gels. we have investigated how fibroblasts cultivated within a matrix react upon wounding of this dermal equivalent. human skin-derived fibroblasts (kd-cells) growing within attached collagen matrices were monitored over a period of 8 days. fluorescently labeled cytoskeletal elements (tubulin, vimentin, acfin) and fibronectin served as phenotypica/ markers. the 3d-arrangement of fibroblast microtubules, intermediate filaments and microfilaments, as well as of fibronectin was visualized by clsm and optical sectioning. cells at the wound margin, adjacent to the wound, and in non-wounded controls, up to a depth of about 100gm were analyzed. upon wounding, i.e. upon excision of a 1 mm 0 "punch biopsy" from the center of the collagen matrix, fibroblasts in the wound region oriented towards the matrix defect. layer-like structures of increased cell density evolved over time at the wound margin. the tissue defect was covered by fibroblasts, reminescent of the situation in vivo. global yeast genome expression analyzed by 2-d page after tctp homolog gene (ykl312) disruption. sanchez a translationally controlled tumor protein(tctp) was identified and microsequenced from a human liver sample. previous publications described the presence of a gene related to tctp in plants, mouse erythroleukemia and ascetic tumor, human mammary carcinoma and more recently in the yeast. tctp has no known function but its high degree of homology from plants to human underlines its probable crucial role in cell function. in order to study multifactorial chan~es in protein expression as a function of the ykl312 gene disruption m the yeast, we used high resolution two-dimensional gel electrophoresis combined with an efficient computer system (melanie). two groups of gels (ykl312 +(n=10) and ykl312 -(n=l 0)) were analyzed, compared and classified using correspondence analysis. there was no significant difference between the two populations of gels according to either qualitative and quantitative spots expression except for two spots which completely disappeared. the first one corresponded to ykl312 gsne product. the second one corresponded to a 28 kda peptide with an isoelectric point of 4.6. this ykl312 associated peptide still needs to be characterized and linked to the yeast genome. demyelinating and neurodegenerative diseases are associated with altered cellular responses including processes mediated by receptor protein tyrosine kinases. using a pcr cdna cloning approach we have identified a novel member of the eck/elk/eph genefamily in myelinating serum-free brain cell cultures. alternatively spliced cdnas encoding complete open reading frames for putative transmembrane proteins have been isolated from both rat and human brain. a recombinant protein derived from the rat cdna was demonstrated to have protein tyrosine kinase activity. an affinity purified antiserum to this protein was used to identify a glycoprotein of 120 kd molecular weight in brain cultures, and developping and adult brain tissue. the protein expression is most prominent during embryonal development and during the first three weeks of postnatal life. by in-situ mrna hybridisation the transcripts were found predominantly in restricted neuronal populations. in order to investigate systematically the effects of geometrically defhied abrupt tissue expansion on the characteristics of impulse propagation, we constructed expanding cellular structures in vitro using patterned monolayer cultures of neonatal rat heart cells (photolithographically patterned substrates.) the preparations, which consisted of narrow cell strands (40-80 tim wide) inserting into large reetangular cell areas (side lengths >1500 ~m), were stained with the voltage-sensitive dye di-g-anepps and were imaged onto a 12 x 12 photodiode matrix array (maximal spatial resolution 151.tin x 151am in the object plane). while impulse propagation was uniform in linear cell strands (average conduction velocity 0.35 m/s), a transient decrease in conduction velocity hi the transitional region was invariably observed in the suddenly expanded structures. this decrease was accompanied by marked distortions of the local action potential shapes due to bidirectional electrotonic interactions across the transition: upstream from the expansion, the repolarization phase was interrupted, a few ms after its inception, by a small secondary depolarization ("spike and dome" configuration), while, downstream, the action potential upstrokes were preceded by prominent prepotentials. in those cases where conduction failed at the tlansition, action potential durations upstream were dramatically abbreviated. these findings showed that phenomena similar to those recorded in intact purkinje-fiber-ventricular preparations can be observed in cell ensembles in vitro having comparable geometlins but consisting of cells with uniform active membrane properties. supported by swiss nsf grant 823a-028424 (sr) and usphs grant ns 16824 03ms). it is a noncollagenous glycoprotein with a molecular mass of 148 kda consisting of three identical subunits. cmp is selectively extracted with edta-containing buffer what indicates a divalent cation dependent anchorage of the protein in cartilage matrix. electron microscopy revealed the presence of three compact globular subdomains and sequence analysis indicated the presence of a coiled-coil c~-helical assembly domain formed at the c-terminal end of each subunit. the trimeric structure was retained after complete reduction under native conditions which reveals that the subunit structure of cmp is not only stabilized by disulfide bridges but also by the coiled-coil assembly domain. tissue distribution studies in mouse revealed that cmp is selectively expressed in some but not all cartilages. adult rat cardiomyoeytes (arc) in culture degenerate the myofibrillar apparatus and after attachment to the substrate they grow and reassemble new rnyofibrils. the appearance of new, well organized rnyofibrils can be observerd in several distinct regions. they appear close to the membrane proximal to the culture substrate and in the perinuclear region close to the substrate. previous studies have shown that embryonic cultured cells assemble new myofibrils which insert in adherens junctions at sites of cell-cell contacts and in sub-sarcolemmal adhesions plaques (saps) at sites of cell-substrate contacts. this saps are thought to function as the nucleation sites for myofibril formation. we have investigated the formation of this saps in cultured adult rat cardiomyocytes. using confocal light microscopy we can show that the interaction between rnyofibrils and membrans occurs close to the membrane proxirnal to the substrate and that this saps are flanking both nascent myofibrils and sfls, the latter structures being believed to serve as scaffold for myofibrillogenesis, additionally we have used electron microscopy to investigate the formation of this structures at higher resolution. $11-05 muscle satellite cells (sc) are quiescent myoblasts, ready to be activated and to regenerate new muscle fibers in case of muscle damage. in vitro, single human muscle sc, cultivated as clones, give rise in proliferating conditions to two subpopttiations of cells, cells expressing both ~-striated muscle actin and desmin (c~-sr+dsm+), and cells expressing desmin alone (dsm+). in culture conditions promoting differentiation, a clone .typically gives rise to a fusing progeny, yielding c~-sr+dsm + myotubes, and to non fusing myoblasts (nfmb). the latter are dsm +, a phenotype similar to quiescent sc found in situ. in order to determine whether nfmb are the in vitro equivalent of in situ quiescent sc, this first generation of nfmb were selectively collected and subcultured. in proliferating conditions, nfmb were able to resume proliferation and to give rise to a progeny with both c~-sr+dsm + and dsm + cells. when cultured in differentiating conditions, nfmb formed ct-sr+dsm + myntubes, and a new generation of dsm + nfmb. when nfmb of the second generation were again selectively collected and subculture& they resumed proliferation and ~re again able to yield both myetubes and a third generation of nfmb. nfmb of the first generation were also cultivated as clones, and 5 to 25% of them were able to resume extensive proliferation, yielding in their progeny both c~-sr+dsm + and dsm + cells, which differentiated into myotubes as well as nfmb. these results strongly suggest that sc have the stem cell property, of selfrenewal, yielding in their progeny both cells ready to differentiate into muscle and ceils similar to themselves. extra cellular matrix (ec~ regulates the expression of b-casein in cultured mouse mammary epithelial cells. we have developed a functional ~ epithelial cell strain, which expresses high level of milk proteins, forms alveolar-like structures when plated onto a reconstituted basement membrane and secretes casein unidirectior~lly into a it~aen. we have further shown that fflv~dependent regulation of b-casein occurs mainly at the transcriptional level and that 5' sequences play an inloortant role in this regulation. we have located a 160bp transcriptional enhancer (bcei) within the 5' flanking region of the b-casein gene. using functional assays, we show that bcei contains responsive elements for ~ and prolactin-dependent regulation. bcei placed upstream of a truncated inactive b-casein promoter reconstitutes a promoter even more potent than the intact prcmoter, which contains bcei in its natural context more than 1.5kb upstream. this small fusion promoter reconstitutes the nonaal regulation pattern. we show that bcei mediates f/24 dependent regulation even when linked to a het~rologous viral promoter. purified satellite cells (sc) were obtained from human skeletal muscle biopsies following cell sorting by flow eytometry. a chemiluminescent assay for acetylnholine (ach) revealed the presence of 1,3 nmoles/weu of 100.000 sc. in elonal cultures of proliferating sc, this intracellular level of ach was 0.l nmoles/well. when cells were cultured in the presence of an esterase inhibitor (10 ixm phospholine), the ach amount was enhanced 2 fold. conversely, cultivating the cells in presence of a potent inhibitor of choline acetyl transferase (2 gm bromoach), gave a 50% decrease of ach content finally, the release of ach was detected in the supernatant of cultured sc, following a 15 min incubation, and the measured level of ach was 3 pmoles/well/min. the presence of an ach-like compound in myogenic cells in both freshly isolated and in proliferating sc suggests that sc may contain ach in situ. in additiou, the fact that ach was present in the extracellular medium suggests that ach can be released spontaneously by the cultured sc. type xii collagen is an extracellular matrix protein associated with collagen fibrils in vivo. as observed for several other extracellular matrix proteins, the synthesis of type xii collagen by chick fibroblasts cultured in 0.1% fcs is stimulated by tgf-i~i. two splice variants of type xii collagen are known, with subunits of either 220 kda or 350 kda. by using antibodies specific for the large (350 kda) form of type xii collagen, we could show a more restricted distribution of the large variant compared to the smaller form in the embryo. while only the large variant carries chondroitin sulfate, both variants are identical in their collagenous domain. it is thought that via this domain, type xii collagen binds to collagen type i fibrils. we demonstrate here that type xii collagen can bind to collagen i fibrils also in vitro. by neutralizing acid soluble collagen type i, we could coprecipitate type xii collagen together with newly formed collagen type i fibrils. this interaction occurs in physiological saline but is highly salt dependent. in further studies we investigated wether 35s-methionine labeled type xii collagen treated with chondroitinase abc, ct-chymotrypsin, or collagenase can still be precipitated together with type i fibrils. in addition, we found that type xii collagen also affects the rate of type i collagen fibril formation. h.u. keller department of pathology, university of bern, ch-3010 bern locomoting blebbing cells colchicine (10-sm) can induce locomotion associated with blebbing in walker carcinosarcoma cells. blebs expand at a rate (about 2~m/sec) which is much faster than known rates of actin elongation. this suggest that the membrane is directly pushed forward by forces other than actin elongation, possibly by hydrostatic pressure. this interpretation is suggested by the observation that there is significant f-actin staining all along the cell membrane but not with the cytoplasmatic content the blebs. blebbing is suppressed by 0.5 m sorbitol. the finding that cytochalasin d suppresses blebbing shows that actin polymerization is nevertheless indirectly instrumental in bleb formation, possibly by generating a high intracellular pressure. a tentative model explaining locomotion of blebbing cells is presented. osteopetrosis encompasses a family of diseases with different causes and the common phenotype of impaired bone resorption. the osteopetrotic mouse mutant oo/oo is deficient in csf-1, the growth factor for the cells of the mononuclear phagocytic system. the phenotype of oo/ou mice is characterized by a low number of peritoneal macrophages and peripheral monocytes, and a virtual absence of osleoclasts, leading to the osteopetrotic phenotype. injections of csf-1 into op/oo mice reversed the osteopetrotic phenotype, proving the requirement for csf-1 during the process of osteoclastogenesis. by in situ hybridization, expression of csf-1 was demonstrated during bone development. osteoclast precursors and mature osteoclasts were identified as putative target cells for the growth factor, since these cells express csf-1 receptors, which is encoded by the proto-oncogene c-fm~. subsequently, specific binding of csf-1 to osteoclasts was shown. expression of c-fms parallels the expression of csf-1 in bone both in time and place, suggesting a local action of this growth factor in osteoclast formation, but also in the modulation of osteoclastic activity. different transcripts, raised by alternative splicing of a commmon nuclear rna precursor, have been found to encode a secreted and a membrane associated forms of csf-i. the secreted form can be modified by attachment of a glycosaminoglycan side chain, which serves as an anchor to integrate the peptide into the extracellular matrix. investigation of the role the different csf-1 forms play during recruitment of osteoclasts and regulation of their activity will provide further insights into the mechanisms governing these processes. comparative sequence alignments of known voltage-gated k + channels revealed two conserved cysteine residues in the putative transmembrane segments $2 and $6. if the cysteines were connected by a disttifide bridge, it would put a structural consllaint on possible channel models by placing $2 next to $6. we used site-directed mutagenesis to study systematically the potential roles of these invariant cysteines. fourteen of 17 substitutions in $2 (c232), and 7 of 19 substitutions in $6 (c393) maintained channel function in xenopus oocytes microinjected with mutant crna. therefore, the conserved cysteines are not essential for k + channel expression in xenopus oocytes. however, electrophysiological characterization of the cysteine replacement mutants revealed distinct roles for the two cysteines. inactivation, deactivation, and ion permeation did not considerably change in $2 mutants. in $6, in contrast, cysteine replacement by leucine, asparagine, glycine and valine accelerated inactivation and deactivation kinetics substantially, whereas serine and threonine showed opposite effects. furthermore, the voltage dependence of deactivation was differently affected. in summary, it appears that the side-chain at position 393 in $6 of kv2.1 is involved in channel gating by participating in the transitions from the open to the closed and inactivated state of the channel. (supported by grants from the nih and mda to rhj, and from the snf and the swiss foundation for medical-biological grants to rdz.) the segment between $5 and $6 of voltage-gatedk + channels, called h5 or p region, has been implicated to form part of the ion conduction pathway. little is known about the conforroation of this region although various models have been proposed including a j3 barrel-like structure formed by four adjacent antiparanel j3 sheets. to gain insight into the secondary structure of this region, we used cysteine substitution mutagenesis and sulfhydryl-specific, membrane-impermeant reagents to probe the accessibility of amino acid side chains in and around the p region of kv2.1 (drki). twemy-eighi positions from k356 to t383 were each mutated to a cysteine. after expression of mutant k + channels in xenopus oocytes, cysteine side chain accessibilities were probed by superfusion with ch3so2sch2ch2nme3 + (mtset), mtset can form a mixed disulfide with an accessible cysteine, and this may lead to current reduction if the covalently modified side chain is in or close to the ion conduction pathway. thus far, we have identified four mutations that showed k + current reduction after superfusion with 3 mm mtset. the mutants p361c, i379c, y380c, and k382c showed 87%, 99%, 39%, and 21% reduction in current amplitude, respectively. in coutrast, mutants $363c, a367c, t368c behaved like wild type kv2.1 with less than 5% current reduction. our results suggest that the side chains of p361,1379, y380, and k382 are directly accessible from the extracellular environment. taken together, these residues may, therefore, face the lumen of the ion channel pore. furthermore, the degrees of inhibition are in agreement with a model in which the ion conduction pathway narrows from residue k382 to y380 to i379. (supported by grants from nih and mda to rhj, and from the snf and the swiss foundation for medical-biological grants to rdz.) motejlek k., h,,iuselmann r., and liig:her b.; pharmakologisches institut der universitiit ziirich, winterthmtr. 190, ch-8057 zlirich comparison of 5' flanking sequences of three gabaa-receptor subunit genes (atl, ~, 8) revealed a novel conserved purine sequence dement with the consensus sequence gagaggggagaoga gagag(gg/aa)g. this element is present once each in the (zl and 72 subunit gene promoters and in seven tandem copies in the 8 gene promoter. a novel dna binding activity (bsf1) was identified that binds to various versions of this purine element. bsf1 was found ordy in brain but not in any other tissues tested. furthermore, the factor is distinct of beta, another brain-specific dna binding .protein with a purine-rich dna recognition sequence. the expression of bsf1 during differentiation of eerebeuar granule cells in vitro correlates with the expression of gabaa-receptor subunit genes. this correlation suggests a role for bsf1 as a transcriptional activator of neuronal genes. therefore, bsf1 may be important for cell ty~specific regulation of gabaa-receptor gone expression. we also found that bsf1 binds to wheat germ agglutinin, suggesting that this protein is glycosilated. taking advantage of this property, we are in the course of purifying bsf1, the question, whether bsf1 is indeed a transcription factor is being investigated in vivo and in vitro. to produce a simple method of estimating the free magnesium concentration ([mgzq) in solution, we have manufactured dip cast mg 2+ macroelectrodes using the neutral mg carrier eth 7025.we have used the elecmides to measure the dissociation constant (k~) for mgatp in a background solutions mimicking the intracellniar milieu. for the mcasmement of the big atp dissociation constant, the background solution contained 2 mmol/l n%atp. this solution was titrated with mgci 2 and the changes in [mg ~] above 0.05 retool/1 monitored with the maeroelectrode. during the titration the ph was maintained constant. fitting such titration curves with the standard hyperbolic binding equation, after correction for zero drift, gave the following mean+sd values for the ka (pmol/1 at 37~ ph 6.4, 103.3-+-25,3 (n=9); ph 7, 59.7:k-27.4 (n=6) and ph 7.4, 30.g~15.4 (n=7). elevation of the [naq to 50 m~l/1 and reducing the [kq to 1(30 mmol/l was without effect at ph 6.4 (n=6). reducing the temperature to 25~ increased the k a at 7.4 to 49.0:k21.6 (n= 5). mg =+ macroelcctrodes provide an easy way of measuring [mg z ⧠in solution. [mg ~] can also be measured in a background solution containing i mn~i/l ca, although the electrode response is reduced. interference by protein to date prevents their use in plasma. similarily to the block by zn 2+ and ca 2+, protons only partially block the channel. the ability for all mutated channels to. conduct na + current, and the partial block induced hy zn 2+, ca 2+ or h + indicate that y401 is not located deep in the pore but rather in the extracellular mouth of the channel. progesterone modulates the activity of glycine receptor expressed in colliculli neurons of neonatal rats. maury, k. & bertrand, d. dpt of physiology, cmu, 1211 geneva 11. steroids are potent narcotics and have been shown to act on ligand-gated channels activated by gaba or nicotine. however, little is known about their possible actions on other receptor types. for instance, while it was demonstrated that progesterone inhibits glycine receptors expressed by spinal cord neurons isolated from chick embryos, nothing has yet been reported for brain receptors. in this study, we have determined the properties of glycine channels expressed by brain neurons of neonatal rats using the whole-cell voltage-clamp technique. in inferior colliculi neurons, glycine elicited little-desensitizing inward currents which reversed around -40 mv. these currents were blocked by strychnine in the nanomolar range. surprisingly, however, we found that progesterone, in the micromolar range, potentiated the glycine evoked currents. these results, contrasting those obtained in chick spinal cord neurons, suggest that progesterone enhances or antagonizes glycine currents depending on the subtype of the receptor. the mammalian auditory organ (organ of corti) relies on two groups of sensory cell for its normal hearing: inner hair cell & outer hair cell. ihcs are mainly innervated by afferent fibers while the ohcs essentially receive efferent fibers. the ohcs possess unique electromofile properties which are thought to enhance the motion the basilar membrane and to refine the mapping of the sound frequency. we found, using whole ceil recordings, that 5-10% of the fleshly dissociated ohcs displays fast inward currents. the magnitude of peak currents which can be as large as 2na vary from cell to cell. further experiments have revealed that this current is sensitive to ttx and strongly reduced when extracellular sodium is replaced by choline. its kinetic is relatively slow compared to that of sodium current of typelspiral ganglion cell, and has a more negative inactivation. other voltage activated currents and ligand-gated currents are under inverstigation. these results will provide further insights in the hearing transduction mechanism. the formation and the properties of homotypic and heterotypic gap junctions were studied using two types of insect cells, c6/36 and sf9. two single cells were pushed against each other and the subsequent de novo formation of gap junction channels was assessed by means of the dual voltage-clamp method (bukauskas and weingart: pfl~g. arch. 423:152, 1993 it was symmetrical for homotypic junctions. the vj-sensitivity was less pronounced in sf9 cells. hence, the relationship for heterotypic junctions was asymmetrical. all pairs examined revealed a s-shaped relationship between gj(ss) and v, which was virtually superimposable (gj(ss) declined upon depolarization). each channel contains a vm-gate and two ~-gates. the vj-gates are operated independently. they close when their intracellular aspect is made positive. shaped curve compatible with a two-state boltzmann process. half maximal inactivation was reached at -77 mv and +66 mv, implying an asymmetrical gating behavior. in case of an asymmetrical protocol (~ and vewas stepped simultaneous, but in opposite direction), the q-dependent asymmetry disappeared (half maximal inactivation at -59 mv and +60 mv). the asymmetry in gj-gating, attributable to the pulse protocol, was also reflected in the time constants of ij inactivation (r177 cerebellar granule cell cultures were used to study the regulation of the nmda receptor expression. we have shown previously that chronic membrane depolarisation (25 him k +) or treatment with nmda {i00 ~m) promotes the functional expression of nmda receptors, as assessed by nmda-evoked 45ca2+ influx. we have now developed a rnase protection procedure for the quantitative determination of the mrna levels of the nmda receptor subunits known so far (nr-i,2a,2b,2c,2d). the growth conditions mentioned above failed to alter the mrna levels of the different subunits. at the protein level, nmda receptors were evaluated quantitatively by the labeling pattern obtained by the new photoaffinity ligand 125i-cgp55802a. the intensity of the phctolabeled bands, thought to represent nr-i and nr-2a subunits, was increased by treatment with high k + or nmda compared with control cultures. this result suggest an increase in receptor protein by high k + or nmda treatment. thus, posttranscriptional mechanisms seem to play an essential role in the modulation of the nmda receptor activity. the gene encoding the pore-forming subunit of the rat epithelial na + channel (~renac) was cloned recently and shown to belong to a novel gene family coding for cation channels (nature 361, p467-470 (1993) . transport of na + ions through these channels is the rate-limiting step of na + absorption and thereby controls osmotic balance of body fluids and secretions. in order to study the implication of ~renac in regulating sodium balance, blood volume and blood pressure and therefore its involvement in hypertension, we expressed ~renac under the control of the human cmv promoter in transgenic mice. several lines of mice showing expression in different tissues were obtained. progress in the analysis of these mice will be discussed. activation of metabotropic glutamate receptors increases the excitability of neurones in the lateral septum of the rat. to elucidate the mechanism(s) of this action, we used whole-cell patch-clamp recordings from coronal brain slices of young animals. in voltageclamped cells, the selective agonlst (is,3r)-acpd, at 1-50 /~m, had the following effects, a) it evoked a sustained inward current, which was resistant to ttx and to cadmi~am and which persisted in neurones loaded with bapta, a calcium chelator. this current displayed inward rectification and was reduced, or suppressed, when extracellular sodium was partially replaced with n-methyl-d-glucamine. b) it elicited a ttx-insensitive, voltage-dependent inward current, which activated at -50/-40 mv and which reversed at aound 0 mv. this current was suppressed in a low-calcium/high-magnesium perfusion solution and was undetectable in the presence of intracelhilar bapta. we suggest that acpd exerts a dual action on lateral septal neurones. it causes a steady depolarization by generating a sodium-dependent current and it triggers transient plateau potentials by inducing a calcium-dependent cationic current. interaction between these currents results in a powerful, self-reinforcing neuronal excitation. we have investigated the effects of protein kinase c (pkc) activators (4[~-pma, oag) and of phosphatase inhibitors (okadalc acid, calyculin a) on voltage-gated ca 2+ and k + channels in ngf-differentiated pc12 ceils. whole-cell ba 2+ and k + currents were recorded with the patch-clamp technique. by using specific ca 2+ channel blockers (co-conotoxin (cgtx), isradipine) we found 3 types of ba 2+ currents (iba): a) a ~cgtx-sensitive iba; b) an isradipine-sensitive iba; and c) a t0-cgtx plus isradipineresistant iba. 4[~-pma and oag specifically down-modulated the isradipine-sensitive iba. the inhibition of iba was prevented by staurosporine and pkc (19-31) (2 pk inhibitors). the delayed rectifier k + current was unaffected by pkc activators. applied externally, okadaic acid and calyculin a inhibited the total iba by affecting several components of the ba 2+ currents. however, the 0~-cgtx plus isradipine-resistant iba was unaffected by okadaic acid. in conclusion, our results suggest a differential modulation of voltage-gated ca 2+ and k + channels by the pkc signalling pathway in ngf-differentiated pc 12 cells. agrin, a protein isolated from basal lamina extracts of the electric organ of the marine ray, is thought to mediate the motor neuron-induced aggregation of acetylcholine receptors (achrs) in muscle fibers at their neuromuscular junction. recent cloning in the marine ray, rat and chick has revealed that agrin can be alternatively spliced at two sites in its c-terminal half. site a encodes either 0 or 4 amino adds and site b encodes either 0, 8,11 or 19 (8+11) amino acids. studies of agrin mrna expression indicate that early in synaptogenesis, chick motor neurons contain high levels of a4b19. in contrast, non-neuronal cells and muscle calls contain a4b0 and aob0 mrna. in the adult ray, electric lobe motor neurons that innervate the electric organ contain a4b8, whereas agrin mrna in the electric organ again lacks both sites (mcmahan et al. (1992) , curr. up, cell biol. 4:869-874). to investigate the functional properties of agrin isoforms in more detail, we have now compared their specific activity to aggregate achrs on cultured chick myotubes. heterologous expression of the c-terminal half in cos-7 and 293 cells revealed that chick agrin isoforms a4b8 and a4b19 were highly active, while a4bll was up to 40 fold lower in activity. no activity could be detected for the a4b0 and aob0 isoforms. in agreement with these findings the a4b8 isoform of the marine ray also showed high achr-clustering activity. therefore, we conclude that motor neurons throughout development synthesize highly active agrin isoforms while the postsynaptic target ceils do not play a primary inductive role in the formation of synapses. we have used whole-cell patch-clamp recordings and hypothalamic slices in order to characterize the effect of n-methyl-d-aspartate (nmda) on suprachiasmatic neurones. in ceils clamped at or near their resting membrane potential, nmda (50-100 #m) generated an inward current of 10-60 pa, which was insensitive to ttx and which reversed at about 0 inv. the nmda current-voltage (i-v) relation contained a region of negative slope conductance. the nmda current was reduced or suppressed by d(-)-2-amino-5-phosphonopentanoic acid (d-aps) or by mk-801. it was potentiated by reducing the extracellular magnesium concentration from 1 to 0.01 ram, or by adding glycine (10 /~m) to the perfusion solution. in a majority of neurones, lowering the extracellular calcium concentration from 2 to 0.01 mm caused a 1.5to 4-fold enhancement of the nmda current. i-v relations indicate that in the low-calcium solution, the region of negative slope conductance was attenuated. this effect was due to extraceuular calcium, since it persisted in neurones loaded with the calcium chelator bapta. we conclude that nmda channels present in suprachiasmatic neurones may be modulated by extraeellular calcium. institut, and abt. pharmakologie*, biozentrum, basel. during innervation of skeletal muscle, the myonuclei underlying the synapse begin to express acetylcholine receptor (achr} ~-subunit gene and they remain activated after the nerve is removed. our experiments show that this is due to a factor in the synaptic portion of the muscle fibre basal lamina (bl). one candidate for this factor is agrin, which regulates ache clustering in the synaptic membrane: i) unlike in untreated muscle, the level of s at synapses was strongly reduced in cultured rat muscle fibres after proteolysis of synaptic bl. 2) conversely, when rat myotubes were cultured on bl isolated from adult muscle, they preferentially expressed achr accumulations and ~-mrna at sites where they contacted the synaptic portions of the isolated bl. 3) when various substrates were impregnated locally with agrin a4big, an isoform expressed by motor neurones in fetal spinal cord, it locally induced in the myotubes the expression of s-mrna. art increase of functional nicotinic acetylcholine receptors precedes the fusion of cultured human muscle satellite cells. r. m. kranse, c.-r. bader and l. berrtheim. department of physiology and division of clinical neurophysiohigy, university medical center, 1211 geneva 4, switzerland nicotinic acetylcholine receptors (nactlr) are expressed on embryonic myoblast and acb may play a role in mechanism of fusion. our study focuses on the appearance of functional nachr in freshly isolated and cultured satellite cells (sc) from normal human muscle biopsies. sc cart be conditioned to either proliferate or fuse to form myotubes depending upon culture media. presence of ach-activated current was investigated using the whole-cell and single.channel patch-clamp technique. in freshly isolated sc (whose properties should be close to the quiescent in vivo sc/no nachr were observed (n=16). in the proliferating state, 54% (n=35) of the satellite ceils (which are also called myoblasts) displayed a small ach-activated current (10 pa/pf) and, as expected, after fusion of salellite cells into myotabes, 100% of the ceils displayed an ach-activated current (n=6; 26 pa/pf). to examine, whether the increased expression of nachr precedes sc fusion, sc were cultured in a medium which promotes fusion, but were prevented from fusing by keeping them at a low density. in this culture condition, 93% of the sc (n=15) displayed an ach-activated current and, in addition, these cells expressed a 4 times higher ach-current density. (40 pa/pf) than the proliferating sc. we also observed that, in high density cultures, a small population of sc do not fuse even atter 3 months in the medium promoting fusion. these non-fusing myoblasts (nfmb) had no nachr. our results suggest that the appearance of nachr may be related to the process of sc fusion as i) quiescent sc in vivo do not express nachll ii) nachr expression increases in conditions promoting fusion and iii) no nachr were observed in nfmb. the latter cells may be equivalent of quiescent sc in vivo. $12-20 we have investigated the sensitivity of neuronal nicotinic acetylcholine receptors (nachrs) of known subunit composition to various cholinergic antagonists. neuronal nachrs were expressed in xenopus oocytes after injection of pairwise combinations of (x2 or c~3 with either 1~2 or 1~4 crnas. the two-electrodes voltage-clamp technique was used to measure currents induced by rapid application of low concentrations of acetylcholine (ach) together with increasing concentrations of antagonists. the response of ct31~4 neuronal nachrs to ach was halfmaximally inhibited by following antagonist concentrations (ic50): hexamethonium (0.3 i.tm), mecamylamine (0.2 p.m), pentolinium (0.2 i.tm) and trimetaphan (1.2 ~tm). with (x3132 nachrs the ic50s of the same antagonists were about 10 times higher. finally, (+)-tubocurarine was a conventional competitive inhibitor of ach in ~3132 and t~2~2 nachrs. in contrast, low concentrations of (+)-tubocurarine increased the response to low ach concentrations in a3(34 and t~2~4 nachrs. these results further underline the importance of [l subunits in determining the functional properties of neuronal nachrs. supported by the hochschnlstiftung and nf grant 31.31018.91 to abc. previously, the role of the transglial tubular system was shown for the squid giant axon: in na + free (tris) solutions the series resistance increased in correlation with a decrease of the tubular opening density (tod). in the crayfish giant axon, which show similar tubules, we correlate the change in tod, measured in freeze-fracture preparations, witl] the conduction velocity. the control to'd was estimated to be 16 per #ms. after 30 and 75 rain tris-inkubation the tod decreased to 10,4 and 1,5, respectively. this reaction was reversible when these axons were reincubated in na~-c41ntaining solution. after 120 rain reincubatiou the tod was 11.8 per tam'z. to determine the influence of decreased tod on the impulse conduction velocity, the nerve was incubated for 30 min in tris solution followed by reincubation. impulse propagation then recovered in two phases. during an initial fast phase with a short time constant of 1-2 rain na + diffused back into the periaxonai space and allowed impulses to occur with a low conduction velocity. in the second phase with a long time constant of 7 min, conduction velocity recovered slowly to normal values. this time course was comparable to the recovery of tod, as estimated from recent freeze-fracture preparations after short reincubation. these results indicate that the normal tod is a necessary condition for the normal excitability of the axon and determines its conduction velocity. most spinal cord neurons respond to the inhibitory action of gaba and glycine, suggesting co-expression of gaba a-and glycine receptors 4n individual ceils. while glycine-receptors are exclusively found in post-synaptic densities, the cellular localization of gaba areceptors has not yet been characterized. in this study, the distribution of gabaa-receptors in the spinal cord was analyzed with an antiserum recognizing the (xl-subunit. double-and tripleimmunofluorescence staining were employed to identify synaptic receptors apposed to gabaergic terminals (immunoreactive for glutamic acid decarboxylase) and to assess their co-localization with glycine-receptors (visualized with an antibody to the 93 kda protein gephyrin). staining for the gabaa-receptor ctl-subunit decorated the soma and dendrites of numerous neurons in laminae iii-viii and x, revealing their morphology in clear detail. by contrast, laminae 1i and ix contained little immunoreactivity for these gabaa-receptors. most gabaa-receptor-positive cells also exhibited a prominent glycine-receptor immunoreactivity. both types of receptors had very similar distribution patterns and were frequently co-localized in sites apposed to gabaergic boutons. these results indicate that gaba aand glycine-receptors may co-exist within single post-synaptic densities, suggesting a possible synergism between gaba and glycine neurotransmission in spinal cord neurons. prenatal benzodiazepine (bdz) exposure changes both behavioral and neuiochemical parameters of developing iats. these changes are not a consequence of remaining bdz in brain tissue, but rather indicate alterations in bdz receptol binding and function. since the bdz binding site is located on the gaba~ receptor complex, it is possible that prenatal bdz treatment influences the expression of gaba~ receptor subunits. to evaluate effects of pienatal bdz exposure on mrnas expression specific for several gaba~. receptor subunits (~i, ~5, ~ & y2), we treated plegnant rats with diazepam (l.25mg/kg/d; s.c.) from gestational day 14 to 20. we then analyzed specific mrna levels in offspring at four diffeient developmental stages (gd20, pns, pni5 & adult) by in situ hybridization. pleliminary data flom optical density measurements indicate a deciease in expression of mrna in particular for ~-subunit of gab~ receptors in different brain reglons of plenatally diazepam-treated zats. processing of sensory information within the auditory system has been well characterized using histological and eleetrophysiologlcal techniques. however, relatively little is known about the neurotransmitters involved. the present study was performed to elucidate the possible role of excitatory (eaa] and inhibitory (i.e., gaba) amino acids in neurotransmission within the primary auditory cortex (all the main afferent to the ai, the ipsilatera} medial geniculate body (mgb), was electrically stimulated and evoked responses were recorded intracellularly in the ai region in halothane anesthetized cats. a seven-barrelled iontophoresis pipette glued alongside the recording electrode allowed localized application of eaaergic and gabaergic compounds onto the recorded neuron. in most ai neurons, mgb stimulation evoked a short latency, short duration epsp followed by a long-lasting ipsp. pattern, duration, and amplitude of synaptic potentia{s were highly variable and strongly dependent on stimulation intensity, reduction of gabaa receptor-mediated inhibition by iontophoretic application of either bicuculline or sr95531 markedly enhanced mgb stimulation-evoked epsps. only the late component of this enhanced epsp was reversibly blocked by the competitive nmda receptor antagonistl ap7 at currents which selectively blocked neuronal excitation induced by iontophoretic application of nmda, our results demonstrate that in the eat 1) nmda receptors in ai are activated following mgb stimulation and that 2) a dominant gaba~ receptor mediated inhibition usually masks this nmda receptor activation under our experimental conditions, a highly purified and specific cell wall degrading endo-l,5-u-l-arabinanase was isolated from an a. niger pectinase preparation by low and medium (fplc) pressure column chromatographic separation methods. the isolated enzyme was most active on linear 1,5-~-l-arabinans (-90 i.u./mg), whereas branched arabinans from sugar beets were degraded to a lesser extent (-14 i.u./mg). the enzyme was shown to be electrophoretically pure after silver staining (sds-page) and its identity was confirmed through specific binding to an antiserum directed against endo-l,5-~-l-arabinanase. the major physico-chemical characteristics of the enzyme were the following: mr 42'000, iep ~ 3.0, ph optimum 4.8, temperature optimum 55~ ph stability 3.5-8.0, temperature stability s 45oc, km = 0.205 mg/ml, vmax = 17.7.10 -3 ~unol/min. zn and hg showed potent inhibitory effects. 3.2) is a specific enzyme of the glyoxylate cycle, which plays a key role in the initiation of gluconeogenesis in germination oilseeds, this pathway is localized in glyoxysomes, single membrane organelles which are converted into peroxisomes when lipid reserves are depleted. the glyoxylate cycle enzymes have been the subjects of numerous conflicting reports typically addressing the problems of their synthesis (on free or bound ribosomes), targetting and suborganelar localization (membrane bound or matrix proteins). recent biochemical studies have shown that ms from germinating soybean cotyledons is an interconvertible enzyme (membrane bound, aggregated or soluble fomls) which is significantly affected by its ionic environment (henry et al., 1992, plant science 82:21-27) . microscopy studies have now been carried out using immunofluorescence staining or immunogold-silver staining for light microscopy, and immunogold labelling for electron microscopy. all results consistently characterize ms as a glyoxysomal mawix enzyme. chorismate mutase (ec 5.4.99.5) catalyzes the first step in that branch of the shikimate pathway which leads to the aromatic amino acids phenylalanine and tyrosine. we have isolated a cdna for this enzyme from the higher plant arabidopsis thaliana by complementing a yeast strain (aro7) with a cdna library from a. this is the first chorismate mutase cdna isolated from a plant. the a. thaliana chorismate mutase expressed in yeast revealed allosteric control by the three aromatic amino acids as previously described for plastidic chorismate mutase isozymes. an attachment of rubisco to chloroplast membranes under cu++-stress has been described for wheat (mehta et al., j. biol. chem. 267, 2810 -2816 . the displacement to the membranes has been discussed as a possible step in the degradation of this predominant stromal protein, e.g. during leaf senescence. in our recent experiments it became evident that such interactions are not restricted to rubisco. several other stroreal, and even a peroxisomal enzyme (glycolate oxidase), were also detected in the membrane fraction after 6 to 30 hours of oxidative stress induced in wheat seedlings by a treatment with 10 mm cuso 4. the velocity and the degree of membrane attachment varied for different enzymes. based on these results it was interesting to check the solubility of rubiseo and its previously described 45 kd fragment which accumulates during the incubation of bean leaf discs under oxygen deficiency (hildbrand and feller, experientia 47, a67) . neither rubisco nor the breakdown product were found to accumulate in the membrane fraction. thus, at present, the steps involved in rubiseo catabolism cannot be generalized. different mechanisms depending on the metabolic situation and on environmental conditions should be considered. overexpression of the four parsley phenylalanine ammonia-lyase (pal) isoenzymes in e. coli. characterization of the enzymes and kinetic analysis of the inhibition by 2-aminoindan-2-phosphonic acid. christoph appert, j/irg schmid, jerzy zorn* and nikolaus amrhein institute of plant sciences, eth-z/jrich, 8092 zijrich. *technical university wroclaw, 50-370 wroclaw, poland. all four phenylalanine ammonia-lyase (ec 4.3.1.5) isoenzymes of parsley (petroselinum crispum nym.) were expressed as glutathione s-transferase fusion proteins in e. coil after affinity purification, the glutathione stransferase moieties were cleaved off with factor xa and the phenylalanine ammonia-lyases were characterized. the proteins form enzymatically active tetramers, even as fusion proteins. the four isoenzymes have comparable km values for l-phenylalanine (15gm to 24.5/.tm), similar temperature (58~ and ph optima (8.5). the aminooxy-and phosphonic-analogues of l-phenylalanine are competitive inhibitors of the enzymes. 2-aminoindan-2-phosphonic acid (j. zo{a & n. amrhein, liebigs ann. chem. 1992,625) was found to be a potent slow-binding inhibitor of these and other phenylalanine ammonnia-lyases, both in vivo and in vitro. chlorophyll a fluorescence has been used to compare the drought resistance of two varieties of solanum tuherosum (avr/i)c-12st-19 and sibtema). fast fluorescence rise kinetics were measured during the first second of illumination with a time resolution of l0 ms and 1z bits in fluorescence intensity. the rise kinetics shows the typical steps called fo -j -i -p. with this values different indexes have been defind with the goal to compare the behaviour of these two varieties of potato. salicylic acid (sa) was proposed as a putative signalling molecule for the induction of systemic acquired resistance (sar) in infected plants. we studied its biosynthesis as follows. cotyledons of cucumber plants were injected with a solution containing pseudomonas lachr~ans and fed with radioactive sa precursors by injection of 14c-benzoic acid (14c-ba) or 14cphenylalanine (14c-phe). alternatively, leaf 1 of cucumber plants were inoculated with tobacco necrosis virus (tnv) and leaf disks were then vacuum-infiltrated using 14c-ba or 14c-phe. free and bound 14c-phenolies were quantified by hplc. incorporation of 14c into sa was found in the two plant-pathogen systems. i~c-ba gave mostly 14c-sa and an unknown polar compound. 14c-phe gave also some 14c-sa, in addition to 14c-labelled lignin precursors like ferulic and p-coumaric acids. the biosynthetic pathway of sa from phe through cinnamic acid and ba will be discussed. after infection with a necrotizing pathogen, systemic acquired resistance (sar) is often observed in plants. in cucumber, salicylic acid (sa) increases in the lower infected leaf as well as in the upper uninfected leaves. sa was also found in the phloem sap of infected cucumber plants and was proposed as a signalling molecule for the induction of sar. we intend to clarify whether sa is translocated from the site of infection to the upper leaf, or wether it is made in the phloem tissue upon the action of a primary systemic signal. one cotyledon of cucumber plants was first infected with pseudomonas lachrymans and then fed with 14c-sa, 14c-benzoic acid (ba) or 14c-phenylalanine. after different times, cotyledons and first leaves were collected and the radioactive ~henolics were analyzed by hplc. in some cases, 4c-sa was found in the first leaf. in addition, two unknown radioactive compounds were detected in leaf 1 after treatment with 14c-sa and 14c-ba respectively. we will discuss signalling processes for the induction of sar in cucumber. one modern approach of creating virus resistant grapevine plants is the introduction of resistance genes into existing grapevine varieties by genetic engineering. the goal of this work is to use the coat protein (cp) mediated strategy to induce resistance to nepovirus in vitis spp. as a first step, several chimeric nepovirus cp genes, were constructed by addition of various promoter regions upstream of the gflv and armv cp regions. their ability of conferring resistance to nepovirus infection in transgenic nicotiana benthamiana and n. tabacum plants will be presented. the best constructions will be used to transform several grapevine varieties in order to create nepovirus resistant grapevine plants. $13-12 the bctalains axe a class of natural pigments found only in plants of the order caryophyllales and in some fungi. the first step in betalain biosynthesis is the conversion of tyrosine to dopa. subsequently , dopa is transformed to betalamic acid, the bctalain chromophorr through the action of dopa-4,5-dioxygenase. we have purified and characterised a copper-containing enzyme of -38kda from amanita muscaria pileus that catalyses the hydroxylation of tyrosine to dopa. considering that the enzymatic activity was restricted to the coloured parts of the mushroom, we postulate that it is involved in betalain biosynthesis. the enzyme featured a broad substrate specificity, and also oxidised the diphenols to their corresponding ortho-quinones, a reaction typical for tyrosinases. the implications of our findings on betalain biosynthesis axe discussed. chlorophyll a fluorescence was measured under steady state conditions of pea and tomato leaves adapted to low light intensity (30 wm-2s -i) at different temperatures (havaux and strasser, z. naturforsch. 45c, 1133 -1141 , 1990 ). when leaves were exposed for a short period (i0 min) to heat (25 to 45~ in darkness, the level of variable fluorescence decreased. when the heat stress was imposed in presence of low light, the variable fluorescence was much less affected and virtually no effect of heat treatment was observed until 37~ this protecting mechanism by low light was absent in algae and mostly absent in submerged water plants but fully present in free floating plants on the water surface. we conclude that a mechanism has been developed for higher land plants during evolution which protects the plants against heat damage on warmer days. caryophyllales, e.g. portulaca grandiflora, and in a few fungal species, e.g. amanita muscaria. we analysed the similarity of a key enzyme of the pathway, dopa-4,5-dioxygenase, in plants and fungi both at the protein and the dna level. antibodies against purified dopa-4,5-dioxygenase from the fungus a. muscaria crossreacted with protein from p. grandiflora petals and two cdna clones were obtained from this plant. their similarity with fungal sequences and with other dioxygenases is discussed. kinetics of prollne hydroxylauonj intrucellnlur transport and c-terminal processing of the tobacco vacuolar cbltlnase. ernst frcydl, thomas boiler and jean-marc neuhaus botenisehes instimt, abt. pflanzenphysiologie, hehoistxasse 1, ch-4056 basel the vacuolar chitinase a of tobacco (nicotlana tabacum) is syndietlzed as a preproprotein with ma n-teaminal signal peptide which causes its synthesis in the er mad a c-termlnal extension, which has been idcmified as the vacuolar targeting peptide (vtp) (1) and whleh is cleaved off from the maybe chitinase. it has recently been shown that mature chltin:~e a contains several hydi'oxyprolines within the short peptide spacer that links the n-terminal cysteine-rieh chitln-bindlng domain to the catalytic domain (2). we received specific antibodies against mature chitinasr (a kind gift from dr. f.meins. f/vii) and raised antihodiea against a synthetic vtp. we performed pulse-chase experiments and cell [ractlonation with 1) stably transformed tobacco plants expressing chitinase a or mutants lacking either the chitin-binding domain and spacer (chitah) or the vacuolar targeting poptid 9 (chitavtp) and 2) transient expression of the same conswacts in nicotiana plumbaginifolia protoplasts. in both systems, proline hydroxylatino in chltinase a was detectable after 30 vain. and complete after 90-120 rain. the ehltinase intermediate forms were detected in the mlcrosomal and soluble fractions for up to 90-120 rain. the mature chitinase continuously accumulated only in the soluble fraction. in both systems chitah showed the same kinetics of intraceilular transport and processing. whereas the transport of cbitavtp seemed more rapid.these results indicate that in vivo cbitinase a is synthetized as a proprotein that is than modified by proline hydroxylation. this second intermedinte form is then transported to the oolgi apparatus and sorted to the vacuole. c-terminal processing occurs late in this pathway or in the vacuole. infection of bean roots with the soil-borne phytopathogenic fungus fusarium solani f.sp. phaseoli leads to a rapid increase of chitinase activity (~five-fold). part of this increase in enzyme activity is due to transcriptional activation of the basic class i chitinase isoenzymes. semi-native polyacrylamide gels stained for chitinase activity using the substrate glycol-chitin revealed the appearence of three additional chitinase isoenzymes that are absent from uninfected control roots. conventional protein purification techniques showed that fusarium solani infected bean roots contain two basic and two acidic chitinase isoenzyrnes. their physiochemical properties and possible biological function will be discussed. proteasomes are multicatalytic protease complexes that function as a major nonlysosomal proteolytic system. they exhibit multiple endopeptidase activities that promote the intracellular turnover of abnormal polypeptides and short-lived regulatory proteins. moss proteasome has been purified from protonemata by successive chromatography on macroprep q, bio-gel a-1.5, bio-gel a-5 and ha. the molecular mass was estimated to be 1,300 kd by gel filtration. gel electrophoresis of proteasome under nondenaturing condition gave a single band and two-dimensional gel electrophoresis identified the moss proteasome to be composed of 14 different subunits with molecular weight in the range 22-35 kd. electron microscopy in aqueous solution showed that it is a cylindrical particle composed of four stacked rings with a diameter of 9 nm and a height of 14 rim. end-on projection established a 7-fold symmetry of the outer disks. substrate specificity of proteasome indicates that it contains endopeptidase activity against substrates bearing hydrophobic, basic, acidic and glycine residues immediately preceding the cleavage site. antibodies raised against moss proteasome reacted with proteasomes of other eukaryotes, including human. $14-01 hunger r.e., hess m.w., laissue j,a,, mueller c. the nod (non obese diabetic) mouse, a widely used animal model for insulin dependent diabetes mellitus, shows in addition to mononuclear cell infiltration of the islets of langerhans, massive cellular infiltrates of the submandibular and lacrimal glands with considerable tissue damage. several lines of evidence suggest that both insulitis and sialadenitis represent autoimmune disorders, to investigate a possible role of tumor necrosis faetor-a (tnf-c0 and activated cytotoxic cells (nk cells, t cells) in the inflammatory process, tissue sections of nod mice were hybridized with radiolabeled rna probes specific for the detection of mrna for tnf-r and the serine proteases granzyme a and b (gra and gr8) and perforin which are expressed in activated cytotoxic cells. tnf-e expressing cells were mainly located in infiltrates and are absent in non affected glands, whereas gra, grb and perforin expressing cells were distributed over the whole section with highest densities in the zones adjacent to parenchyma. the finding that activated cytotoxic cells are present in early stage of disease development indicates that cell mediated cytotoxicity may play a crucial role in initial tissue destruction. the role of tnf-a in autoimmune diseases is not known yet. the appearance, however, of cells expressing this gene in the infiltrate indicates a possible role of this cytokine in the progression of the inflammatory reaction, e.g. by increasing the lymphocyte traffic to this organ. we further demonstrated that the induction of inos was at the transcriptional level. in order to understand the underlying mechanisms we characterized the promoter region of the rat nos gene. a 9enomic rat library was screened using a cdna-fragment coding for the if-end of the inos cdna (nucleotides 1-817). a 1,8 kb hinc-ii fragment containing the 5"-flanking region of the inos gene was characterized by dna-sequencing. the transcriptional start site was determined by primer extension. sequencing analysis revealed a multitude of possible cis-acting elements homologues to consensus sequences for the binding of different transcription factors including ifn~' response element (ire), nuclear factor-kb (nf-~b), tumor necrosis factor response element (tnf-re), 7f-activated sites (gas) and one x-box. nf-kb, a transcription factor involved in signaling and immediate early gene activation during inflammatory processes was induced by treatment of mesangial cells with 2 nm of il-113. this was shown by the appearence in nuclear extracts of the dna binding activity of nf-~b using electrophoretic mobility shift assays (emsas) with a 32p-labeled ~r dna probe. pyrrolidinedithiocarbamate (pdtq), an inhibitor of nf-kb, suppressed the expression of inos mrna in mesangial cells without affecting the dibutyryl-camp-triggered increases in nos mrna levels. this data suggest that the il-i ~ signalling pathway responsible for nos expression requires nf-k8 activation and is definetly different from the signalling cascade directed by camp. recent findings indicate that the multifunctional cytokine il-6, which plays a key role in irm-nune and inflammatory responses, also exerts specific effects in the central nervous system (cns). for example, il-6 promotes neuronal survival, induces differentiation and modulates neurotrophin production. using the very sensitive technique of reverse transcription combined with polymerase chain reaction the developmental profile of il-6 and its receptor mrnas was analyzed in various rat brain regions. our results indicate that both genes are expressed in a region-specific manner and are developmentally regulated. these findings support the hypothesis that il-6 is involved in differentiation and maintenance of neuronal subpopulation in the cns. interleuldn 2 (il-2) expression is strictly dependent on a signal transmitted by the t cell receptor upon antigen stimulation. this signal can be mimicked in vitro with phorbol esters and calcium ionophores. we have found that stimulation with ca-ionophore alone induces expression of il-2 mrna, but does not induce secretion of il-2. in polysome gradient fractionation of cells stimulated with phorbol esters and ionophore, the fractions containing il-2 rrtrna are found at the bottom of the gradient, bound to polysomes. however, in ionophorestimulated cells the fractions containing il-2 mrna are clearly shifted to the top of the gradient, and therefore are not lzanslated. this effect is specific for il-2, as other mrna's like 1~2-microglobulin are not regulated. this data suggests that il-2 gene is translationally regulated. in addition, in vitro experiments have shown that the lack of translation of il-2 mrna in ionophore-stimulated cells is due, most likely, to the presence of a translational regulator that specifically inhibits translation of il-2 mrna. assuming the presence of a translational regulator that specifically represses il-2 mrna translation, we can predict that, if the represser is in excess, even upon a second stimulus that is by itself able to induce secretion of il-2, there will be no translation of il-2 mrna. the resuhs of such an experiment confirmed our prediction. polysome gradients showed that after ionophore stimulation, il-2 mrna gets "stacked" with one ribosome, and no further ribosome loading takes place. if malaria is highly endemic, every febrile child is a presumptive malaria case, and there is no differential diagnosis based on clinical, immunological or parasitological grounds. we evaluated the diagnostic usefulness of interleukin-2 receptor (il-2r), tumor necrosis factor-receptors 55 (tnf-r55) and 75 (tnf-r75) . sera came from tanzanian pediatric fever patients and controls, all enrolled in the kilombero malaria project. we found that all 3 receptors marked pediatric fever episodes, whereas stability of observed levels was highest for tnf-r75 and lowest for tnf-r55. tnf-r55 levels reflected severity of the fever attack. regardless of the presence of a febrile illness, tnf-r75 levels were strongly associated with parasite density. immunological markers can be applied in rapid pre-and post-intervention morbidity assessments, validation of health interviews and monitoring of convalescence. we have recently shown that human eosinophils produce mrna for interlenkin (il)-8 when stimulated with calcium ionophom (eur. j. immunol. 23 (1993), 956). we now present evidence that human eosinophils contain preformed il-8 protein although they do not express mrna for il-8 as measured by rt-pcr. il-8 protein expression was determined using specific anti-human il-8 monoclonal antibodies by western blotting, facs analysis and immunohistochemistry. moreover, preformed il-8 was released into supernatant by 99% pure eosinophils after priming with gm-csf or il-5 and subsequent 25-min stimulation with paf, rantes, ionomycin or pma, however not with il-1 or il-2, as measured by elisa. this observation implies that activation of protein kinase c and/or inwacellular calcium mobilization am necessary events for il-8 release in human eosinophils. the determined amounts of preformed il-8 protein was higher in patients with asthma compared to normal individuals suggesting a role for eosinophil derived il-8 in asthma. since the eosinophil is the predominant cell in the asthmatic airways, we determined 1l-8 concentrations in bronchoalveolar lavage (bal) fluids from normal individuals and asthmatic patients. indeed, il-8 concentrations in bals from both groups were similar to those seen in the supematants released by activated eosinophils. the role for 1i-,-8 in asthma remains to be determined. to study the effect of canthaxanthin in vitro, the hydrophobic compound was introduced in vivo into chick high density lipoproteins (hdl) by canthaxanthin feeding. the effects of hdl and hdl associated canthaxanthin on two types of cultures have been tested: flat sedimented cell cultures of embryonic chick neuronal retina, retinal figment epithelium (rpe), brain and meninges, and in reaggregate cell cultures of the neuronal retina. at high canthaxanthin concentrations the formation of colored, birefringent entities were induced in the neuronal retina in vitro. in line with human data, parameters of cellular function and cell differentiation remained unaffected by canthaxanthin at these concentrations. by contrast, fidl was found to induce various cell type dependent effects. proliferation of meninges cells was decreased at low hdl concentrations as concluded from.light microscopic examination and indicated by protein content, and lysosomal and mitochondrial activity of the cultures (ic50:15-30 mg hdl apoprotein/l medium). these parameters, however, were increased in brain cell cultures, while they remained unaffected in cell cultures of neuronal retina and rpe. concentrations above 0.3 g. hdl apoprotein/l caused a reduction of glial cell differentiation. $14-08 tnf-c~ had close-dependent antiproliferative activity on hormone-dependent human breast cancer cells that represent an early stage of the disease, whereas hormone-independent cells representing a late stage were not affected. however, in the presence of 1000 u/ml interferon ?(inf), high concentrations of tnf-c((lnm) were able to inhibit the growth of hormone-independent cells. using specific monoclonal antibodies in ftow cytometry, no significant changes of either the p75 or the p55 tnf receptors were observed upon inf treatment of hormone-independent cells. this indicates that the increased responsiveness of these cells for tnf-c~ is not due to a change in available tnf receptors. the affinity of the receptors for tnf-r are one order of magnitude different for the two cell types. inf treatment shifted the ecs0 of hormone-independent cells towards the ecs0 of hormone-dependent cells. thus, the basis for the increased tnf-sensitivity of hormone-independent cells in the presence of inf might be the interconversion of tnf receptors from low-to high-affinity. the adhesion of tumor cells to endothelium is the first step in the processus of metastasis, and is frequently dependent of cytokinemediated activation of endothelial cells. cytokines are also involved in nitric oxide (no) production by various cells. these experiments were designed to evaluate no production during tumor cell adhesion to endothelium. rat brain-derived endothelial and rat colon carcinoma cell lines were used during these experiments. no was evaluated with the griess reagent. activation of endothelial cells with cytokines (tnf-d, and ifn-~ ), only slightly increased (10-20 %) the adhesion of tumors cells to endothelium. activation of endothelial cells with tnf-ol and ifn-~, induced a 4-8 fold increase of no production. however, addition of tumor cells to the endothelial monolayer, either directly or in a semi-permeable membrane, decreased the cytokine-induced no production. these results indicate that no production is modulated during the proeessus of adhesion of tumor cells to endothelium. is induced in rat mesangial cells by inflammatory cytokines such as interleukin 1 ]3 (il-1 ~) and requires bh 4 as a cotactor. 2,4-diamino-6-hydroxypyrimidine (dahp), a selective inhibitor of gtp cyclohydrolase i, the rate-limiting enzyme for bh 4 synthesis, potently suppressed il-i~induced nos activity, measured as nitrite production. inhibition of no synthesis by dahp was reversed by sepiapterin, which provides bh 4 via a salvage pathway. sepiapterin dose-dependently augmented il-1 i],-stimulated no synthesis, indicating that availability of bh 4 limits the production of no in cytokine-stimulated mesangial cells. n-acetylserotonin, an inhibitor of the bh 4 synthetic enzyme sepiapterin reductase, completely abolished il-lj~-induced no formation whereas methotrexate, which inhibits the pterin salvage pathway, displayed only a moderate inhibitory effect, thus suggesting that mesangial cells predominantly synthesize bh 4 tom gtp. in conclusion, these data demonstrate that bn 4 synthesis is an absolute requirement for cytokine induction of nos in mesangial cells. inhibition of bh 4 synthesis may provide new therapeutic approaches to the treatment of pathological conditions mediated by no. -1 [3) can induce a macrophage-type of nitric oxide synthase (inos). northern-blot analyses of inos mrna-levels and nuclear run-on experiments of control and il-1 ~ stimulated mesangial ceils revealed that the induction of inos was at the transcripuonal level. here we demonstrate that platelet-derived growth factor bb (pdgf-bb) can suppress the il-113 dependent inducuon of the inos gene. nortllern-blot analyses of cellular rna isolated from mesangial ceils that were coincubated with il-1 [3 (2nm) and pdgf-bb (10 ng/ml, 100 ng/ml and 300 ng/ml) revealed a dosedependent suppression of inos mrna-levels. using run-on experiments with nuclei from mesangial ceils after stimulation with il-i~ (2nm) and pdgf-bb (100 ng/ml) we demonstrate that the inhibition occurs at the transcriptional level. basic fibroblast growth factor (bfgf), on the other hand, potentiates the il-1 dependent stimulation of inos. coincubation of mesangial cells with il-113 (2nm) and bfgf (3ng/m[, 10ng/m[, 30ng/m[, 100ng/m0 dose-dependently superinduces inos mrna levels as shown by northern-blot analyses of total cellular rna form control and stimulated cells. run-on experiments with nuclei from mesangial cells stimulated with il-113 (2nm) and bfge (100ng/ml) revealed an enhanced transcriptional activity of the inos gene. thus, pdgf-bb and bfgf differentaity modulate the il-i~ dependent expression of the inos gene and are therefore an excellent model system to study the crosstalk between signal transductjon pathways. we report that ifnyr-/-mice have an increased resistance to lipopolysaccharide-induced toxicity (lps). lps-induced lymphopenia, thrombocytopenia and weight loss seen in wild type mice were attenuated in ifntr-/-mice. ifntr-/mice survived in the d-galactosamine-lps model when conditions were 100% lethal for wild type mice, correlating with serum tnf levels of up to 10 fold higher in wild type mice. bone marrow and splenic macrophages from ifnyr-/-mice had a 3 to 5 fold decreased lpsbinding capacity, with serum from these mice lowering macrophage lpsbinding by a further 50%. thus, depressed tnf synthesis, diminished expression of lps receptors and low plasma lps-binding capacity in the mutant mice likely combine to manifest in the resistant phenotype of ifny r-/mice to endotoxin. in a complementary approach we have screened the 5' portion (-7.8 kb/+4.1 kb) of the gene for tissue specific and/or inducible dnasei hypersensitive sites. in chromatin from fibroblasts or kidney epithelial cells this segment of the il2ra gene does not contain any dnasei hypersensitive sites. in contrast, in resting t-and b-lymphocytes, as well as in activated t cells, two sites (dhi, -0.05 kb; dh3, -5.8 kb) were found. a third site (dh2, -1.35 kb) is detectable in t cells that have been activated with concanavalin a and il2. this site maps to the same position as cisacting regulatory sequences that are required for the response of the il2ra gene to signals from the il2 receptor. interleukin-6 (il-6) production in cultured human dermal fibroblasts can be stimulated by interleukin-i (il-i} added to the culture medium. absence of fetal calf serum and of growth factors (insulin, egf, t3) reduced the stimulation by more than 50 %. egf and t3 and even more choleratoxin increased the il-6 production in absence of fetal calf serum. the effect was dose dependent. 2% human ab serum substituted for i0 % fetal calf serum. pretreatment of the cultures with serum or growth factors prior to stimulation with il 1 had a stimulatory effect (serum, t3, egf) or an inhibitory effect (hydrocortisone, choleratoxin). our results suggest that the il-i signal transduction to il-6 is modulated by serum components and growth factors as well as through c-amp produced by choleratoxin. interleukin-6 (il-6) is produced by a variety of cells and plays a central role in host defense mechanisms. its function include induction of il-2 and il-2 receptor expression, proliferation and differentiation in t-cells. in cocultures of human dermal fibroblasts and allogenic human t-cells a cell number dependent 10 to 100 fold increase of the il-6 production could be demonstrated after 24h and 48h. conditioned medium from tcells and from fibroblasts failed to induce il-6 secretion in fibroblast and t-cell cultures, respectively. no up-regulation of il-6 production was found in cocultures of fibroblasts with tcells killed by ethanol and in t-cell culture incubated with recombinant hll-lc~. after separation of the cells il-6 production in fibroblast and t-cells returned to precoculture levels. our results indicate that cell-cell contact more than soluble factors must be involved in the up-regulation of il-6 synthesis in cocultures of t-cells and fibroblast. using cocultures of autologes human t-cells and fibroblasts, we are currently investigating whether the cell contact essential for the il-6 production depend on immunolcical interactions of t-cells and fibroblasts. rapid growth of tumor often results in oxygen and nutritional deprivation leading to extensive necrosis, which is one of the characteristics of glinblastomas (gbl). neoplastic cells surrounding necrotic areas often present a peculiar arrangement of cells called "pseudo-palisading" -as a hallmark of this entity, together with abundant neovascularization. a special biological milieu is probably formed in these palisading areas by certain unknown cytokines and/or growth factors induced by the necrotic process. plate et al. (1992) reported the expression of vascular endothelial growth factor (vegf) in the palisading cells. we previously demonstrated that gbl cells produce il-8, which is known to be an angiogenic factor. the aim of this study is first to assess if il-8 is expressed in the palisading ceils, and second to determine if hypoxia can trigger il-8 gene expression. in rive observation using in siru hybridization and immunohistuchemistry showed that il-8 is highly expressed specifically in the palisading cells. we also demonstrated the mrna expression of il-8 receptor in the endothelial cells adjacent to necrotic area. to simulate the in vivo condition, two in-vitro models are currently developed to determine if il-8 is induced by hypoxic insult on cells. first, gbl cell lines are cultured in the absence of oxygen and il-8 expression is assessed by northern blot analysis. preliminary results have demonstrated the induction of il-8 by hypoxia. secondly gbl cells are grown in spheroids until development of central necrosis. the il-8 expression will be assessed by in situ hybridization combined with immunohistochemistry to determine if the ceils surrounding necrosis express il-8. it has been proposed by taniguchi and his colleagues that activation of the interferon (ifn)-i3 gene by virus or double-stranded rna involves induction and modification of irf-1. overexpression of irf-1 can induce expression of the ifn-b gene in certain cells. a role of irf-1 in the activation of ifn-a genes has also been proposed. furthermore, irf-1 has been claimed to play the role of a tumor suppressor gene. we have generated mice in which both irf-1 alleles were disrupted and found them to develop normally. no spontaneous tumors have been observed so far. injection of poly(i)-poly(c) resulted in the same levels oflfn in blood of wildtype and null mice, and equal ifn-ct mrna levels in spleen. induction of wild type and irf ~ embryo fibroblasts (mefs) with virus resulted in the same levels of ifn-a and ifn-i] mrna respectively. in the case of polyo)-poly(c) induction, the levels of both 1fn mrnas were higher in wild type than in mutant cells; this difference was abolished if the cells were first primed with type i ifn. we conclude that irf-1 does not play an essential role in the induction oflfn genes. mitsuhiro tada, annie-claire diserens, isabelle desbaillets, marie-france hamon, nicolas de tribolet, erwin van meir; chuv, lausanne there is increasing evidence that a variety of cytokines produced by glioblastoma (gbl) cells modulate the tumor growth by affecting the host's immune response and by stimulating neovascularization. cytokines such as il-i, il-6, il-8, mcp-i, il-10 and tgf-132, affect each other's production and receptor system and seem to form a cytokine network. among them, il-1 may play a pivotal role, since il-i can induce or increase the production of other cytokines (il-6,il-8,mcp-i,tgf-132) and modulate their receptor expression. to test this hypothesis, we investigated co-expression of cytokines and their receptors in 15 gbl cell lines and 15 gbl tissues using rt-pcr, northern blot analysis, immunnhistnchemistry and elisa quantification. a majority of the gbl cell lines and gbl tissues expressed il-i~, il-113, type i and type ii receptors, indicating the presence of an il-1 autocrine loop. il-1 stimulated growth of some of the cell lines. a positive correlation of il-6 and il-8 expressions with the presence of an il-i autocrine loop in the cell lines was found. immunohistochemical studies showed the in rive co-expression of the il-i family and the secondary cytokines (il-6, il-8) in gbls. antisense oligonucleotide to il-lc~ and/or il-113 partly suppressed this secondary cytokine expression. these results demonstrate that the il-1 autocrine loop plays a central role in the formation of the cytokine network in gbls. double-stranded rna-dependent protein kinase (pkr) is induced by type i interferon (ifn) and is implicated in the establishment of the antiviral state. upon activation, pkr phosphorylates the eukaryotic initiation factor eif-2, thereby throttling protein synthesis. it was suggested that pkr may be essential for the induction of ifn-13 gene expression by both virus and double-stranded rna and for the activation of at least some ifninducible genes. recently it was proposed that pkr is a tumor suppressor gene because 3t3 ceils overexpressing inactive human pkr gave rise to tumors in nude mice (koromilas et al., 1992; meurs et al., 1993) . we have produced homozygous pkr knockout (pkr ~176 mice and found that they develop normally and have no striking phenotype. induction of ifn-c~ and ifn-13 gene transcription by virus was unimpaired in fibroblasts derived from pkr ~ mice, as was the induction of several ifn-stimulated genes. so far, no spontaneous tumor formation was observed. pkr o/o embryonal stem ceils showed no increased malignancy in nude mice as compared to their wild type counterparts. we conclude that pkr does not play an essential role in viral induction of the ifn genes and that its tumor suppressing effect, if any, is redundant. tgf-13 plays an important role as a negative regulator of hepatocyte proliferation in liver regeneration. exposure of rodents to nongenotoxic carcinogens like cyproterone acetate (cpa), thioacetamide (ta) and phenobarbital (pb) causes abnormal hepatocyte proliferation and liver tumors after long term treatment. this suggests that these chemicals have either a direct mitotic activity or impair the negative regulatory system for growth. therefore the inhibitory activity of tgf-6 on dna-synthesis induced whith cpa was investigated in cultured rat hepatocytes. after a 48h exposure to cpa, a dose dependent increase in dna synthesis (3h-tdr incorporation) was observed. the maximum effect was attained with 12 pm cpa (up to 4.3 fold) which was stronger than with 10 ng/ml egf (up to 2.8 fold). tgf-81 and tgf-132 inhibited this response dose dependently (idso = 0.1 ng/ml) and reduced it to control levels at 1 ng/ml. these findings show that the tgf-6 mediated pathway for negative growth control in hepatocytes is still responding after cpa treatment. mouse mxl protein is an interferon (ifn) induced gtpase with intrinsic antiviral activity against influenza a viruses. it accumulates in the cell nucleus and inhibits the transcription of influenza virus genes, recently, we have shown that mxl exerts its inhibitory activity by interfering with the function of the viral polymerase subunit p82. pb2 is responsible for recruiting 5" ends of cellular mrnas as primers and for the elongation of nascent viral transcripts. to elucidate which pb2 dependent step of viral mrna synthesis is the target of mxl action, we examined the activity of recombinant mxl protein in an in vitro transcription system of influenza virus. first, we expressed wildtype and mutant mxl proteins, carrying a hlstidinetag at their n-terminus, in e. coil and purified them under nondenaturing conditions by ni-chelate affinity chromatography. mxl efficiently inhibited viral transcription in vitro, while mutant mxl proteins, lacking antiviral activity in vivo ,were inactive. moreover, the use of capped synthetic rna primers revealed, that mxl almost completely abrogated the elongation of viral transcripts, whereas the the cap-binding and endonuclease activity of pb2 was not affected. a50 $14-28 neutralization rates of tnf-a from eight animal species by a monoclonal antibody against human tnf: implication for receptor action? we have recently developed a sensitive bioassay for measuring porcine tnf-a based on homologous pk(15) cells. this bioassay is 100-to 1000-fold more sensitive than the widely used l929 bioassay, also, human tnf-a and human tnf-8 were detected with a slightly higher sensitivity in the new test compared to the l929 bioassay, we now show that also canine, ovine, bovine, equine and caprine tnf-a can be detected with the pk(15) bioassay. we tested a murine monoclonal anti-human tnf-a antibody for its capacity to neutralize tnf-a from eight different animal species. the action of this antibody revealed that neutralization of tnf bioactivity is a complex phenomenon, indicating species-specific differences in the interaction with tnf receptors. to study this differential neutralization, we are cloning the porcine tnf receptors for expression in a heterologous cell system. administration.of high dose r-tnf(~ in combination with ifn 7 in isolation and perfusion of the limbs in melanoma patients has shown to be very promising with a response rate greater that 80%. this observation prompted us to investigate the possible expression of the tnf receptors in melanoma cells using the monoclonal antibodies utr-1 specific for the tnf type a (55 kda) receptor and htr-9 specific for the tnf type b (75 kda) receptor. flow cytometric analysis of cultured melanoma cells showed the presence at low level of the tnf type a and to a slightly higher level of the type b receptor. similar results were obtained in vivo by immunohistochemistry on fresh tumor raateriai, treatmem of melanoma cells in culture with the-amp induced up to a 2 fold increase in the number of type a tnf receptors with only a minimal change in the number of type b receptors. this increased expression of tnf receptors is conflrmed by direct binding experiments using 125i-labeled tnftx. the number of cpm's bound on dbc amp treated cells was about 0.5-3 fold higher than on untreated control calls, likewise incubation of melanoma cell lines with ifny increased the specific binding of subsequently added 125i-labeled tnfct. from flow cytometric analysis using the two anti-tnf receptor antibodies it became evident that flint was able to increase the expression of both type a and b receptors depending on the cell line used. characterization of a murine tumor necrosis factor (x-lacz reporter construct tumor necrosis factor ot (tnfcq is a prominent mediator of a variety of different pathologies such as endotoxic shock syndrom and rheumatoid arthritis. it also plays a crucial role in host defence against bacterial and viral infection. up to now, only few data about signal pathways leading to tnfo~ induction are available. an easy to handle tnfc~ -lacz reporter assay will represent a useful tool to study signal transduction on the one hand and provide data about compounds with potential tnfo~ inducible activity on the other hand. in order to develop a routine macrophage cell line capable to easily report tnfc~ induction, different tnfe-lacz reporter constructs were assembled. data about functionality of the different constructs in routine macrophages will be presented in the context of tnfot expression. cloning of a novel protein binding to lymphokine, fos and myc mrna with unexpected enzyme activity j. nakagawa, h-p. waldner, s. meyer-monard, and ch. moroni inst. medizinische mikrobiolegie, univ. basel an au-rich sequence in the 3' untranslated region of lymphokines, c-los, and c-myc proto-oncogene mrna is responsible for their rapid decay. we have purified a 32 kd protein by an auuua affinity column from human brain. partial amino acid sequence was obtained and the corresponding edna has been cloned. unexpectedly the edna sequence exhibited significant homology to enoyl coa hydratase and bacterially expressed recombinant protein indeed showed the activity. while rat hydratase did not show rna binding activity, the recombinant protein bound to cfos, c-myc, auuua cluster of il-3 mrna, and adeno virus iv, but not to mutated ad-iv, nor irrelvant transcript. the binding was competed out by poly u. interestingly the protein seems to be processed from a larger precursor. we suspect that the protein plays a role in mrna turnover and perhaps in oncogenesis. institute for medical microbiology, university of basel in t cells, the immunsuppressive agent cea is known to inhibit ca 2+ dependent induction of lymphokine mrna transcription. in contrast, the immunsuppressive drug rapamycin inhibits the lymphokine induced proliferation. we have analysed the effect of these drugs on a v-h-ras induced autocrine mast cell tumor line which expresses il-3 constitutively. csa and rapa inhibited autocrine growth by different mechanisms,, because added il-3 could antagonize inhibition by csa, but not by rapa. whereas csa acted by downregulation of il-3 mrna expression, rapa blocked il-3 induced proliferation. cea also inhibited il-3 superinduction by ca-ionophore, whereas rapa did not. nuclear run on assay indicated that the mechanism is posttranscriptional. therefore, we have introduced il-3 transgenes with and without the au-rich region in the 3' utr of the mrna into a tumor line. in contrast to the wild type, the expression of the mutant construct was insensitive to csa. since the mutant lacks sequences known to regulate rapid mrna decay, we suggest that csa affects the regulation of il-3 mrna stability. some non-hematopoietic cell lines produce both scf and its receptor, the c-kit protein (c-kit). testing the hypothesis that scf may be involved in secondary growth of nb bone marrow metastasis, we found and previously communicated (beck d. et al, proc aacr, 34, 52, 1993) a low or absent expression of c-kit in nb cells. the mrna and surface expressions of scf gene in nb cell lines grown in chemically-defined medium were evaluated by northern blot analysis and flow cytometry. the scf antigenic concentrations in culture supernatants were determined by elisa and growth-inhibition experiments performed by pre-incubating nb cells with anti-scf antibodies prior to 3h-thymldine uptake assay. the scf mrna was expressed in 4 of 5 tested lines but no membrane-bound antigen could be detected on those cells. detectable levels of scf in the supernatants were found in 5/7 lines (range : 39-96 pg/ml). blocking experiments resulted in a decrease of dna synthesis in i out of 7 lines only. from these and previous results, we conclude that scf is synthesized and released by many nb cells in vitro but the functional ~ole of it remains undetermined since scf does not appear to be usually involved in autocrine or paracrine growth stimulation of nb cells (supported by swiss fnrs grant 3200-031005). expression of the v-h-ras oncogene in the il-3 dependent pb-3c mast cells leads to the generation of two classes of il-3 autocrine tumors in vivo. autocrine il-3 expression in class-1 tumors results from increased il-3 mrna stability due to the loss of negative trans-acting posttranscriptional control. this defect can be corrected by somatic cell fusion to the nontumorigenic parental pb-3c resulting in downregulation of oncogenic il-3 expression and concomitant tumor suppression. expression of the v-h-ras oncogene remains unaffected in class-1 hybrids. in contrast, class-2 tumors are characterized by transcriptional activation of the il-3 gene due to the insertion of an endogenous retroviral element (intracisternal a-particle) which cannot be overcome by ceil fusion (see hirsch et al, 1993, j.exp.medicine 178, 403-411) . although v-h-ras is required for generation of either class of tumors, expression of anti-sense ras constructs inhibited only the proliferation of class-i tumor cells. experiments are underway to characterize the target and the nature of the class-1 alteration. nitric oxide (no) promotes vasorelaxatlon of renal vessels in vitro. the purpose of the present study was to assess the role of no in regulating renal hemodynamics in vivo. renal blood flow (rbf) and glomerular filtration rate (gfr) were studied in anesthetized mechanicallyventilated newborn rabbits both before and after the administration of a no synthesis inhibitor, ng-nitro-larginine methyl ester (l-name), at a dose of 50 pg/kg followed by 10 jzg/kg x rain. such a dose of l-name did not modify mean arterial pressure or pulse rate. by contrast, the renal vascular resistance increased by 31 + 9% (p < 0.005) while rbf decreased by 20-+6% (p < 0.02). gfr and urine flow rate remained constant. the overall results suggest that in normal conditions no may play a role in decreasing the renal vascular resistance of the immature kidney, without altering systemic blood pressure. interleukin-4 (il-4) is a t-cell derived lymphokine which, in addition to its effects on the immune system, is also able to suppress the growth of certain tumor cells. il-4 reduced the growth rate of four human colon tumor cell lines up to 70% in a dose-dependent manner. three other cell lines showed no significant alteration of 3h-thymidine incorporation or colony formation in the presence of 100 u/ml il-4. responder and nonresponder tumors were immunopositive for il-4 receptor (il-4r) expression in flowcytometric analysis, using monoclonal antibodies raised against recombinant il-4r and bound biotinyated il-4. responsive tumors expressed more receptors. 11-4 binds with high affinity to a 130 kda transmembrane receptor (il-4r), through which the extracellular signal has been shown to be transduced. coimmunoprecipitation and chemical crosslinking studies have enabled us to demonstrate il-4r target proteins. tyrosine phosphorylation of various proteins between 70 and 170 kda appears to be initiated during il-4mediated signaling events in colon tumor cells. the cloning of human il-4r target proteins will contribute to the understanding of the il-4 signal transduction pathway at the initial stage. the renal effects of endothelin-1 were investigated in 16 anesthetized and meehanleally-ventilated newborn rabbits. each animal acted as its own control. in 8 newborn rabbits, a bolus injection of 5 nmol.kg "1 of endothelin-1 caused an initial fall in mean blood pressure (mbp) followed by a gradual but significant increase in mbp that lasted for 45 rain. the dramatic increase in renal vascular resistance (+ 28 -+ 4%) induced by endothelin led to a fall in glomerular filtration rate (-12 -+ 4%) and renal blood flow (-16 _+ 3%). in spite of the reduction of gfr and rbf, urine flow and sodium excretion rates increased significantly (+ 20 _+ 5% and + 49 _+ 9%, respectively). in 8 additional newborn rabbits, a bolus injection of lnmol.kgaof endothelin-1 -a dose that usually induces marked renal and systemic vasoconstriction in adult models -did not affect systemic or renal hemodynamics. in conclusion, endothelin induces renal and systemic vasoconstriction, and affects water and sodium homeostasis during the neonatal period. however, these effects occur under higher doses than those used in adult animals, possibly reflecting receptor immaturity and/or interference of high levels of counteracting hormones. ontogeny of the endocrine pancreas in xenopus laevis c, maake 1 , w. hanke 2 and m. reine~ke 1 , 1 institute of anatomy, university of z0rich, switzerland and department of zoology ii, university of kaflsruhe, f.r.g. the pancreatic islets of anurans show insulin (ins), glucagon (gluc), somatostatin (som) and pancreatic potypeptide (pp) containing cells. since only limited information exists on the ontogeny and on the presence of insulin-like growth factor 1 (igf-1), we studied the development of the gastro-entero-pancreatic (gep) system in xenopus laevis using immunohistochemical techniques. singular ins-immunoreactive (-ir) cells were observed in the pancreas as early as on stage 41. at stage 43, the first pp-ir cells were found in the gep system followed by som-ir and gluc-ir cells. the first pancreatic islets consisting of ins-ir and gluc-ir cells occurred around stage 50. between stage 54 and 60, i.e. after the onset of metamorphosis, the endocrine cells decreased in number and the islets partly disintegrated. starting with stage 61, the islets reformed and the amount of endocrine cells increased reaching the adult status at stage 63. around stage 52, igf-l-immunoreactions appeared. igf-1immunoreactivity was found in pp-ir and gluc-ir cells but not in ins-ir and som-ir cells. it is assumed that islet-derived igf-1 may p~ay an important role during metamorphosis in xenopus. t. cathomen and r. cattaneo, institut for molekularbiologie i, universit~t zorich, hfnggerberg, ch-8093 zorich the signals used for synthesis of the measles virus fusion (f) protein are poorly understood: a long (>570 bases) untranslated region is followed by three in frame augs at codons 1,4 and 15. f is a type i transmembrane protein with a postulated signal sequence of 31 amino acids. we confirmed that the 46 aminoterminal residues contain a signal peptide by transfering them to another protein. with the other envelope protein hemagglutinin, f protein induces virus-cell and cell-cell fusion. mutagenesis of the three augs indicates that proteins initiated at codon 1, 4 or 15 are all functional, but show slightly different fusion efficiencies. forms translated from aug 1 or 4 appear larger in electrophoresis than forms initiated at aug 15, suggesting that two different signal peptide cleavage sites are used. we are currently investigating whether both f protein forms are incorporated in viral particles. the production of protein isoforms with staggered amino-termini is common in cytoplasmic and type ii transmembrane proteins, but signal sequences usually preclude a similar organization of type i transmembrane proteins. beguin p., beggah a.t., rossier b.c., jaisser f. and geering k. institut de pharmacologie et de toxicologie de l'universit6, ch-1005 lausanne recent experimental evidence suggests that na,k-atpase might be a candidate for regulatory phosphorylation by protein kinases a and c (pka and pkc). to verify this hypothesis, we have mutated several serine and threonine residues in consensus phosphorylation sequences of the ~subunit of bufo marinus na, k-atpase. the mutants were expressed in x~nopus ooeytes and phosphorylation was studied in oocyte homogenates upon stimulation of oocyte, pka and pkc. our results indicate that a unique phosphorylation site for pka is located at serine 943 in the c-terminus of the ~-subunit but its phosphorylation can only be revealed in the presence of detergent. on the other hand, mutations of several serine and/or threonine residues located in consensus sequences for pkc phosphorylation did not or only partially abollsh phosphorylation by pkc. in particular, mutations close to the catalytic phosphorylation site of the ~-subunit influenced phosphorylation by pkc, suggesting that either this region contains a real phosphorylation site or is part of a conformational domain necessary for phosphorylation by pkc. the heat-stable antigen (hsa or mouse cd24) gene is differentially regulated but has a housekeeping promoter. roland h. wenger*, georges kshler and peter j. nielsen. max planck institut f(jr immunbiologie, st0beweg 51, d-79108 freiburg i. bsg. "present address: physiologisches institut der universit&t zi.)rich, winterthurerstrasse 190, ch-8057 z0rich, expression of the gpi-anchored murine glycoprotein heat-stable antigen (hsa) shows tissue-specific as well as developmental regulation. during the maturation of several hematopoietic lineages, hsa expression is generally high in immature precursor ceils and low or absent in terminally differentiated cells. we present evidence suggesting that this regulation of the hsa gene (cd24a) occurs at the transcriptional level. in addition, sequence and methylation analysis of the cd24a promoter revealed characteristics of both "housekeeping" and tissue-specific promoters, including a methylation-free, hpall tiny fragment (htf) island, multiple putative sp1 and ap-2 consensus binding sites and a tata box. functional analysis of a 0.6 kb dna fragment containing these elements fused to the cat reporter gone in transient transfection experiments showed activity in both hsa expressing and non-expressing cell lines with a strength similar to that of the hsv tk promoter. large fragments from the flanking region of the cd24a promoter did not influence the ubiquitous nature of this promoter, finally, the cd24a, cd24b and cd24c genes were mapped to mouse chromosomes 10, 8 and 14 respectively. we have cloned, sequenced and characterized the gone for the m__itochondriat nadh-cytochrome b5 reductase (mcri) of saccharomyces cerevisiae. surprisingly, this gone encodes two mitochondrial isoforms of the flavoenzyme: a 34 kda integral protein exposed on the outer surface of outer mitochondrial membrane (mcrlp34), and a 32 kda soluble protein of the intermembrane space (mcrlp32). the smaller intermembrane space isoform is generated from the larger form by the action of the inner membrane protease i (impi) on the outer surface of the inner membrane. this is the first demonstration that a single gone encodes proteins which are located in different compartments of the same organelle. with special interest in any with a mitochondrial localization. degenerate primers were designed on the basis of high homology between members of the abc family, and pcr was performed on yeast genomic dna. ten dna fragments bearing significant homology to the abc family were amplified, nine of which were previously unidentified. disruptions of five of the corresponding genes were performed. disruption of one gene led to markedly impaired growth on rich medium and cessation of growth on minimal medium. the gene was cloned and found to encode a protein of 694 amino acids, a "half-transporter" which likely forms a dimer. ibi~tlnofluorescence on yeast expressing the gene cmyc-tagged at the c-terminus reveals co-localization of the protein with porin and with mitochondrial dna, visualized by dapi staining. nycodenz-purified mitochondria are enriched for the protein by immunoblotting. additionally,the c-myc epitope is protease-protected in mitoplasts, indicating an inner membrane sub-localization and a probable orientation with the c-terminus in the matrix. the gene, termed atmi for abc transporter of mitochondria, thus encodes the first member of the large abc transporter superfamily to be localized to this organelle. present work is aimed at revealing the substrate of this transporter. cycle in cardiac myocyte. the molecular study of this protein has been difficult even after its over-expression was made possible by the cloning of the corresponding cdna. the chief problem is the lack of convenient domains which could be used in the purification of the molecule. we have previously reported the efficient expression of the cardiac na+/ca 2+ exchanger in mammalian cell lines using the t7 rna polymerase-hybrid vaccinia virus system. we have now constructed the recombinant virus for the exchanger with a 6xhis-tag at the c-terminal. this tag has enabled the purification of the protein through a ni-nta agarose column. the expressed tagged protein induced na-dependent ca uptake which was not different from that in intact cells, i.e., cells in which the exchanger did not have the tag, the most important aspects of the purification produced and those of the reconstitution of the expressed protein will be described. $15-08 the neural cell adhesion molecule l1 is involved in the formation and specification of cell contacts in the central and peripheral nervous system during development, and thus may also play a role in synaptic plasticity of the adult central nervous system, using extracellular recordings of evoked field potentials in the rat hippocampal slice, we found that locally applied polyclonal anti-l1 and fusion proteins containing the ig-domains i-vi of l1 specifically block the stabilization of ltp in the ca1 region. baseline synaptic transmission was not affected by the presence of the anti-l1 antibodies. the anti-ll-induced effect was dependent on the antibody concentration and anti-l1 antibodies had no effect on previously established ltp. ltp was also reduced by an antiserum against the recombinantly expressed ig-domains i-vi of l1, whereas controt antibodies against liver cell membranes, which bind to neuronal membranes in the hippocampus, exhibited no effect on ltp. the contribution of l1 to stabilization of ltp within the ca1 synapses suggests that members of the ig-supertamily of cell adhesion molecules are involved in the structural modifications which are associated with synaptic plasticity in the mammalian central nervous system. an increasing number of surface proteins are described as being attached to the membrane by a glycosyl-phosphatidylinositol (gp1) anchor instead of the conventional transembranous hydropbobic domain. they are initially synthesized with a coohterminal polypeptide extension which is cleaved in the e.r. and replaced by the preformed glycolipid. this processing event is directed by a signal, located in the c-terminal region of the protein, which requires a group of three small amino acids positioned 10-12 residues upstream of an hydrophobic c terminal sequence. we previously transformed the transmembfanous glycoprotein d of herpes simplex type i into a gpi-anchored molecule by deleting its cytoplasmic domain and thus creating a molecule, gdww63, which meets the requirements for anchoring via a gpi structure. we now demonstrate, using gpi-deficient cell lines and biochemical studies, that a hybrid protein consisting of the thy-i ectoplasmic domain and of the c-terminal region of gdww63 can be alternative[y expressed in a gpi-anchored or in a transmembranous form. service de p6diatrie, chuv, ch-1oll lausanne proteolipid protein (plp) is a major myelin protein in the central nervous system (cns). a number of x-linked dysmyelinating disorders have been identified as mutations in the pip gene. we have identified a novel plp mutation in paralytic tremor (it) rabbit, characterized by body tremor, spastic limb paresis and hypomyelination in the cns. reduced levels of plp protein and its mrnas were observed in the cns as well as in the pns. plp sequence analysis revealed a single nucleotide change in exon 2 which results in the substitution of a histidine by a glutamine at position 36. histidine 36 is localized on the boundary between the first transmembrane region and the intracellular part of the protein. therefore, its position can be crucial for the efficient plp interaction with other proteins and lipids, and correct incorporation of the plp molecule into the membrane. histidine 36 is also part of a short fragment of ten amino acids which is conserved in all species studied. the same amino acid is altered in another hypomyelinated mutant, shaking pup, stressing its functional importance. furthermore, the pt mutation affects the plp "premyelin" functions, including oligodendrocyte maturation. equilibrium constants for octamer dissociation as well as dissociation rates in vitro increased in correlation to the number of charged residues eliminated, e.g. mutant 4-7, in which all four charged residues in the n-terminal heptapeptide are substituted with uncharged amino acids, showed a 100-fold higher equilibrium constant for octamer dissociation and a 145-fold increased dissociation rate compared to wild type. this strongly suggests that the charged amino acids in the n-termlnal heptapeptide of mib-ck, and therefore an ionic interaction, play an important role in forming the oetameric molecule. gangliosides and neutral glycosphingolipids (gsls) cell surface molecules are involved in a variety of biological processes and are assumed to play an important role in the mechanisms of hiv entry into lymphoid and epithelial cells. the total lipid-bound sialic acid (lbsa) content and the composition of gangliosides and gsls were investigated on pbmn cells from hiv+ve and normal donors. lbsa level was quantified by a fluorimetric assay, while gangliosides and gsls were assayed with high performance thin layer chromatography (hf'tlc) after multistep lipid extraction of 100 0013 x g membrane fractions. in normal pbmn cells the lbsa content accounted for 2.6/~g/108 cells or 20 nmol/mg protein whilst in pbmn cells from hiv+ donors the lbsa level decreased to 1.6 ,ug/108 cells or 13 nmol/mg protein. the qaantitation of neutral gsls and gangliosides was carried out by scanning spots revealed on hptlc plates and the integrated areas computed with a dedicated software. our findings showed that the most relevant ganglioside present in normal pbmnc was gm3 (65-80 % of total gangliosides) followed by small amounts of od3 (5 -15 %) and other acidic glycosphingolipids. neutral gsls included gl1, gl2, gl3 and gl4-hexosylceramide. hiv+ pbmn cells were characterized by a decrease of neutral glycosphingolipids as well as gm3 content which represented only 55 % of the ganglioside fraction, indicating an alteration in the glycolipid composition in the plasma membrane of infected cells. the neuropeptide y (npy) is one of the most abundant peptides of the mammalian brain. upon stimulation of cell surface receptors, npy generates diverse physiologic responses. npy acts on the cardiovascular system. it is a vasoconstrictor by itself and in addition, it potentiates the action of vasoactive substances such as angiotensin ii or norepinephrine. npy also inhibits glucose induced insulin secretion and is a potent inducer of appetite. to facilitate the development of npy antagonists we are investigating the interaction of npy with its cell surface receptor.we first constructed a series of mutants in which negatively charged residues present in putative extracellular domains of the y1 receptor were systematically replaced by alanines. the mutant cdnas were transiently expressed in hela ceils using a vaccinia virus derived expression system and the abi}ity of the encoded proteins to bind npy was evaluated. the capacity of the mutant proteins to be recruited to the cell surface was assessed by confocal microscopy. we identified initially 4 spacially clustered extracellular acidic residues of the human y1 npy receptor essential for npy binding. subsequently, we mutated 36 additional residues. based on these data a detailed computer model of the interactions between npy and its receptor was built. functional activity to energy metabolism. l. pellerin and p.j. magistretti. institut de physiologie, universit6 de lausanne, switzerland. astrocytes play a central role in brain energy metabolism. for example glucose, the exclusive brain energy substrate, is avidly taken up by astrocytes (pnas 90:4042, 1993) . application of glutamate (glu), the main excitatory neurotransmitter, stimulates in a concentration-dependent manner 3h-2-deoxyglucose (2-19(3) uptake by astrocytes in primary culture with an ec50 of ~ 100 ~m. the effect is not receptor-mediated since it can not be reproduced by glutamatergic agonists such as (nmda, kainate, quisqualate, t-acpd) nor prevented by antagonists (apv, cnqx, ap3). instead, it involves glutamate uptake since the effect is blocked by dl-threo-j3hydroxyaspartate, a potent inhibitor of the glu transporter. replacement of na + in the medium by choline also prevents the effect, suggesting a na+ driven process. finally ouabain, a selective inhibitor of the na+/k + atpase, completely prevented the effect of glu. these observations suggest that when glutamatergic synapses are active, glutamate, taken up by astrocytes, stimulates 2-dg uptake by astrocytes whose end-feet are known to surround capillaries, i.e. the source of glucose. they also provide a simple explanation for the mechanism linking functional activity to energy metabolism. cloning and molecular characterization of the mouse astrocyte glycogen synthase. g. pellegri, c. rossier, s. van berchem, p.j. magistre~i and j.-l. martin. institut de physiologie, universit6 de lausanne, switzerland. in the brain glycogen is predominantly localized in astrocytes, although its presence has been detected in certain large neurons, in ependymal and choroid plexus cells. glycogen is synthesized by the enzyme glycogen synthase (glys) from udp-glucose. out of the different glycogen synthase isozymes, only the muscle and the liver forms of glys have been cloned. we have isolated and characterized from a mouse astrocyte ~.zapii cdna library, a cdna clone encoding the mouse cerebral cortical astrocyte glys. the 3.5 kb clone isolated contains an open reading frame of 2214 bp encoding a protein of 738 amino acids. the coding region of the mouse astrocyte glys cdna shares 87% and 86% identity with the nudeotide sequences of the human muscle and the rabbit muscle giys cdna respectively. the cellular distribution of the glys mrna studied by northern blot shows the presence of a single transcript of 4 kb in cultures of astrocytes and, to a lesser degree, of neurons. the distribution of glycogen phosphorylase, which was also examined by northern blot shows the presence of a 4.2 kb transcript both in cultured astrocytes and neurons with however higher levels expressed in astrocytes. the functional molecular size of the vacuolar h+-pyrophosphatase (h +-ppase ec 3.6.1.1) from maize (zea mays l.) roots was estimated by radiation inactivation, both for substrate hydrolysis and for proton transport. light microsomal vesicles enriched in tonoplast membranes were prepared from young (2 days) maize roots using sucrose step gradients. these vesicles were still competent for ppi-dependent proton transport after freeze-drying and rehydration of the membranes. frozen native or freeze-dried samples were irradiated with 6~ for various periods of time. after thawing the samples, the activities of glucose-6phosphate dehydrogenase (added as an internal standard) and h+-ppase (hydrolysis of pyrophosphate (ppi) and ppi-dependent proton transport) were tested. by applying target theory, the functional size for hydrolysis was found to be around mr 155 000 when the native or freeze-dried samples were irradiated. no ppi-dependent proton transport activity was measurable after irradiation of the native samples. on the contrary, the freeze-dried membranes were still pumping protons after irradiation and a target size of around mr 250 000 was measured for the transport activity. these experiments suggest that the native h+-ppase from maize roots may exist as a dimer (at least) of the mr 80 800 catalytic subunit. $15-20 the na,k-pump moves 3 na + out and 2 k + ions into the cells. the domains of the protein involved in ion translocation have not been determined. we have studied the role of the n-terminal region in the na + translocation by measuring the kinetics of charge movements under na/na exchange conditions in wild type (wt) and two n-terminally truncated forms of the bufo ~ subunit with deletions of the 31 (t31) and 40 (t40) first amino acids expressed in xenopus oocytes. we have developped a new technique to perform fast (< i ms) voltage clamp on whole oocyte. the membrane on one side of the oocyte is permeabilized by digitonin allowing to measure current flowing across the other half. we observed presteady state ouabain-sensitive currents similar to that reported by other techniques (j.gen. physiol. 101:117,1993) . the estimated forward charge translocation rate was lower in both n-truncated mutants (wt 430 â�¢ 14, t32 215 z 8, t41 124 z 3 s-l). the midpoint potential of the charge translocation (best fit to boltzman equation) was -79 , -45, and -28 mv in the wt, t31 and t40 groups, respectively. the highly charged nterminus of the q subunit has a significant role in the forward translocation of na + ions. deletion mutants of human small intestinal lactase-phlorizin hydrolase (lph) were constructed to determine the role of protein subdomains in the intracellular transport of lph. the mutant lph1646mact (236 aa deletions), which contains the membrane anchor (ma) and the cytoplasmic tail (ct), was transported to the cell surface in a fashion similar to wild type lph. by contrast, lph1646 lacking the transmembrane domain persisted as a mannose-rich polypeptidc. this suggests that the membrane anchor and/or cytoplasmic tail are implicated in the er-egress of lph. another mutant, lph1559mact (323 aa deletions), was not efficiently transported to the golgi apparatus. epitope mapping with monoclonai antibodies as well as protease-sens{tivity assays provided an evidence that the tertiary structure of lph1646mact is very similar to that lph1559mact. in view of the variations in the proportions of the complex glycosylated molecules of the two mutants we conclude that the domain between lph1646mact and lphi559mact may play an essential role along the secretory pathway of lph. containing tyrosine. hussein y. naim, and michael g. roth; dept, of biocherni~try, ut-southwestem medical center, at dallas, dallas,texas-usa we have investigated an artificial internalization signal created by site-directed mutagenesis of the short cytoplasmic sequence of the influenza virus hemagglutinin (ha). mutation of cysteine 543 to tyrosine converted ha from a protein essentially excluded from coated pits to one that internalized at rate similar to some cellular endocytic receptors. to determine whether or not the ha-y543 signal is a degenerate form of the internalization signal found in proteins such as the transferrin receptor and the mannose-6-phosphate/igfii receptor, we have mutated amino acid positions in the ha-y543 shown to be important for internalization of the two receptors. we have changed amino acids on either side of y543 to those that either fit, or break the pattern of aromatic and hydrophobic residues proposed as consensus sequence. our results indicate that the ha-y543 mutant contains a sub-optimum sequence for a tyrosine-based internalization signal similar to those found in the receptors for transferrin, ldl, and mannose-6-phosphate/igfil. however, amino acids with side chains having different chemical properties functioned well in positions that are important for the internalization signal, indicating that the consensus sequence for this signal is more variable than previously proposed. n-linked glycesylation is an essential protein modification occuring in all eukaryotic cells. core-oligosaccharides are transferred by the enzyme n-oligosaccharyl-transferase frc~ the donor glc man gicnac -dolichol to selected asparagine 9 2 . resldues of t~e nascent polypeptlde chazn. the donor is build up in a stepwise fashion at the er-membraneby the products of the alg-genes (asparagine-linked-~lycosylafion) mutants defective in this pathway are available, but often lack a phenotype suitable for isolation of the corresponding genes. we found that double mutants of alg5 or alg8 with a wbpl mutation (defective oligosaecharyl-transferase) are severely growth retarded at 30oc. the double mutant phenotype allowed the isolation of both alg5 and alg8 genes which have been refractory to cloning so far. both genes encode potential trarsmenbrane proteins and are able to complement the biochemical defects of the corresponding alg5 or alg8 mutants. the na,k-atpase produces the driving force for the regulated transport of na across epithelial ceils. it is composed of an c~-subunit which carries the catalytic and ouabain binding sites and a b-subunit. to test the role of the usubunit in transport regulation, we transfected a6 cells with the b. marinus al-subunit (txl-tbm). this subunit confers a 300-fold higher resistance to ouabain (k i = 50/~m) compared to the endogenous pump. taking advantage of this difference, stable transfectants were selected with ouabain. expression of the cd-tbm subunit was visualized by western blotting. approximately half of the positive cell lines showed a high electrical resistance similar to untransfected cells, when cultured on filters. na-transport activity, measured as isc, was lower even in the absence of ouabain, but the relative increase in response to aldosterone was maintained. functional pumps containing the exogenous or endogenous al-subunit were quantified by equilibrium ouabain binding. interestingly, cells cultured on plastic dishes had a large fraction of low affinity binding sites (txi-tbm), while most sites were of the high affinity type when the cells were grown on filters. we conclude that the ouabain-resistant na,k-atpase cd-subunit of b. marinus can be expressed in a6 cells and used as a selective marker. however, the results suggest that the expression of functional ouabain-resistant pumps containing the al-tbm subunit could be influenced by the degree of cellular differentiation. in infected cells fully functional na/pi-cotransport was found in a time dependent manner; up to a 50-fold stimulation of na/p;-cotransport compared to uninfected cells was observed aft6r 3-4 days. transport of phosphate by infected cells was highly dependent on the presence of sodium, exhibited a kmtd~ of around 0.16 mm and was dependent on extraceuular i~h'. in agreement with expressed transport of phosphate, infected cells produced an immunoreactive polypeptide of 66 kda that represents a non-glycosylated form of the 80 to 90 kda mature na/p;-cotransporter. we conclud$ that the protein napi-2/rat when expressed in sf9 cells exhibits na/p-eotransport with similar kinetic characteristics as observdd in prox ma tubular apical na/pcotransport. therefore the napi-2 protein as expressed h sf9 cells can be used for further structural and functional studies. talin interacts in vitro with actin, vinculin, integrins and bilayers of acidic phospholipids, and nucleates actin filament growth at the bilayer surface. it is therefore believed to be a key protein mediating aetin-membrane linkage. we have analyzed functional properties of proteolytic fragments of purified platelet talin. using limited proteolysis two fragments of 47 and 190 kd were generated. preliminary results, obtained using viscometry and f-aetin-nucleating assays, suggest that the 190 kd fragment contains the actin binding site. we also assessed the lipid-binding capacity of the fragments. when a mixture of the two fragments was centrifuged in the presence of large multilamellar liposomes containing phosphatidylserine, only the 47 kd fragment, but not the 190 kd domain, co-sedimented with the liposomes, suggesting the presence of a specific lipid-binding site in the 47 kd domain. interestingly, this fragment contains the n-terminus of talin which shows homologies to the membrane-binding regions of protein 4.1. we are now analyzing membrane-binding properties of talin domains in more detail using hydrophobic photolabelling. recent studies suggest a role of the neural cell adhesion molecules l1 and ncam in mechanisms of memory formation (doyle e, et al, j. neurochem. 59:1570 -1573 , 1992 scholey a.b. et al, neuroscience 55:499-509, 1993; lijthi a. et al. unpublished data and see usgeb 94) . in the present study we analyzed the effect of chronic intraventricular infusion of polyclonal antibodies against l1 (anti-u) or ncam (anti-ncam) on the performance of male wistar rats during the acquisition and retention of a spatial learning task. briefly, rats had to remember the position of a submerged escape platform in a water tank (morris water-maze). when the escape platform was removed after acquisition training (= retention test), the performance of animals chronically injected with anti-l1 showed an impaired search pattern when compared with that of salineqnjected animals (p>0.05, mann-whitney). whereas control animals spent up to 45% of their time searching for the platform in the correct (= training) quadrant, the time anti-l1 animals swam in the correct quadrant was closer to chance level (31%). although monitoring penetration of antkncam into the hippocampus was almost as efficient as that of anti-l1, the performance of the anti-ncam-group was highly variable and did not differ from the performance of controls. these results agree with recent findings on the involvement of neural cell adhesion molecules in other forms of learning. we have isolated cdnas from mouse and from xenopus encoding the ~-subunit of the gastric h,k-atpase, which is expressed in the parietal cells of the stomach mucosa. the gastric h,k-atpase transports h + into the stomach lumen with the counter transport of k +, all at the expense of atp hydrolysis. when xenopus oocytes are injected with crna encoding ~h,k from either species, as well as a gastric ~h,k subunit, the two subunit proteins are synthesized, assemble, and form functional pumps located in the plasma membrane as demonstrated by 86rb+ uptake. the sensitivity to the gastric h,k-atpase inhibitor sch28080 was determined, with the ki for beth species found to be in the low ~m range. spodoptera frugiperda ceils are often used to overexpress eucaryotle proteins using baculovirus vectors. the atomic force microscope (afm) is a device which allows the observer to obtain high resolution images of biological samples immersed in a fluid medium; this instrument has therefore the potential to visualize overexpressed proteins on the surface of living cells. however, the interpretation of the images is sometimes difficult and requires a good knowledge of the specimen topography, i.e. the "normal" plasma membrane of the cell used for the overexpressisn. on our poster we present afm images of the plasma membrane of both fixed and living spodoptera frugiperda ceils. in additon, we discuss the possibility of achieving high resolution in the imaging of dynamic changes which affect /iving systems. the deduced protein sequence consists of 295 amino acids with about 55% amino acid sequence identicy when compared to mammalian ~h,k-subunits. data from other species and from the structurally similar na,k-atpase has shown that the ~-subunit is necessary for the formation of a functional pump, which consists of an ~/~ heterodimer. following co-injection of xenopus oocytes with crna for both the ~-and ~subunits, we have observed by rb + uptake h,k-atpase activity in the plasma membrane. we are presently studying the role of the ~-subunit in the maturation and function of the h,k-atpase. the atomic force microscope (afm) is a new type of microscope which allows visualisation of inorganic and organic samples with a very high resolution. a sharp tip fixed at the end of a cantilever is used as a topographic sensor. by scanning the sample with this device, a computer records the minute deformations of the cantilever and computes the topography of the sample. the instrument allows also the measurement of the interaction between the tip and the sample as a function of the distance. the knowledge of these interactions allows us to compute several mechanical properties like indentation, elasticity and stiffness. on this poster we report the results of these measurements on cells (sfg, c131), bacteria (e. coli, b. subtilis) and proteins (actin). in addition we discuss the use of the afm as a sensor for probing the mechanical properties of biological systems at a nanometric scale. neonatal thymectomy of balb/c mice induces aig. we have cloned the ~-and ~subunits of the balb/c h,k-atpase and expressed the proton pump in crna injected oocytes. both and ~ proteins are made, form functional pumps, and are immunoprecipated using auto-immune sera. the ~-subunit can also be in~unoprecipated independent of the ~-subunit when oocytes are injected with ~ crna alone. balb/c mice will be immunized with oocyte membranes containing either the ~/~ or ~ antigens in order to study the role of each h,k-atpase subunit in triggering aig. the prion protein is expressed in both astrocytes and oligodendrocytes of the neonatal brain and is down-regulated in adulthood. m. moser, colello, r, , pott, u, and b. oesch institut for hirnforschung der universit~t zorich, august forel str. 1, 8029 zorich the infectious agent causing transmissible spongiform encephalopathies consists largely or entirely of prp sc, a modified isoform o| normal prp (prpc). prpc is most abundant in the brain. the development of the disease strictly depends on the presence of prpc. and incubation times are shortened by overexpression of prpc. the normal function of prpe is not known and its cellular iocalisation in the cns is poorly characterised, except for its evident presence in neurons. here we describe the expression of prp c mrna in both oligodendrocytes and astrocytes during neonatal development of hamster and rat. in adult animals, expression is down-regulated in gila but not in neurons. our findings provide a link to the previously described accumulation of prpsc in astrocytes and the apparently faster course of prion disease in neonatal hamster. our results argue for a major involvement of non-neuronal cell types in prion diseases. such structures we raised mab against membrane protein fractions. a ~lab against a vitronectin receptor containing fraction was found, which if applied in vitro to bi6fi mouse melanoma cells, influenced growth, adhesion and morphology. ~tnen bi6fi cells prior to tail vein injection were treated, 2 different outcomes were observed. short treatment (lh) resulted in 85% inhibition of lung colonization whereas long-ter~ treatment ( 2weeks) resulted in 10-15 times more lung colonies. a candidate molecule likely involved in these observed effects could be identified as a 150 kda cell surface protein. sequence information from peptides revealed in several cases 100 % homology to mouse centrosomin a, i.e.a 32 kda cytosolic protein involved in the organization of the spindle apparatus. these data suggest that we may have found a protein which may participate in a link between the extra-cellular space and the microtubular system. we are currently trying to clone this 150 kda protein from a bi6fi c-dna library. wahlberg, j.m., and spiess, m. dept.biochemistry,biozentrum, university of basel. signals for correct basolateral sorting of subunit hi of the human asialoglycoprotein receptor are located in the cytoplasmic tail, involving a tyrosine at position 5. removal of this residue results in nonpolarized transport of the protein to the surface in mdckii-celis. a deletion mutant of hi, lacking 30 of the normal 40 residues of the cytoplasmic tail, including the tyrosine, has been found not to be transported to the cell surface, but to be accumulated intracellularly. the mutant protein is complex glycosylated and sialylated, indicative of passage into or through the trans-golgi. immunofluorescence analyses show defined juxtanuclear, tubular structures, which do not colocalize with markers for er, early endosomes or lysosomes, but which show similar staining pattern as markers for late endosomes and tgn. to define the determinants responsible for the intracellular accumulation a series of deletions and fusions have been constructed. the results suggest that the length of the cytoplasmic domain is one important determinant for missorting of hi. salerr~ n., medilansld, j., pellegrinelli, n. and eder-colli, l. departement of pharmacology, cmu, 1211 geneva 4 in chollnergic neurons the enzyme choline acetyltransferase (chat) exists as cytosolic(80-90%) and membrane-bound activity (10-20%}. are these two activities encoded by a single mrna? is membranebound enzyme preferentially associated with a given cellular membrane? in order to investigate these questions we isolated a fulllength cdna (4.2 kb) encoding drosophila chat and used it to transfect xenopus oocytes, rat fibroblasts and human neuroblastoma cell lines. triton x-114 fractionation of transfected cells showed that hydrophilic and amphiphilic chat activity were produced. the sp act. of hydrophflic enzyme reached 2, 0.8 and 0.25 ~mol ach/h/rng protein respectively in oocytes, fibroblasts and neuroblastoma whereas the sp. act. of amphiphilic enzyme was 1.8, 0.4 and 0.125 hinol ach/h/mg protein, respectively. amphiphilic chat was less sensitive to inhibition by the product acetylcholine and to heat denaturation than was hydrophilic enzyme. similar results were observed for the native drosophila chat. amphiphillc enzyme sedimentation was affected by the type of detergent present in linear density gradients of sucrose. transfected oocytes were subjected to subfractionation. besides a large amount of soluble chat activity, a significant proportion of enzyme was found associated with a fraction enriched in endoplasmic reticulum (er). preliminary results using transfected mammalian cells indicate that apart cytosolic chat, plasma mernbranes+golgi enriched fractions as well as er-enriched fraction contain chat activity. we have characterized a drosophila melanogaster gene encoding a very hydrophobic protein. the amino acid sequence shows considerable homology (33-42% identity) to neurotransmitter transporters. the gene is, however, not expressed in neural tissue, but in the male germline. transcription and translation occurs in early spermatocytes. antibody staining data suggest the following pathway of the protein during spermatogenesis: the protein is synthesized in early spermatocytes and is transported into the nucleus during prophase of rt i. at meiosis it associates with the mitochondria and it stays associated while they fuse to the nebenkern. during individualization it is stripped off in the cystic bulge. inspection of the putative protein sequence shows that it possesses alle the necessary targeting signals to allow its transfer from the cytoplasm through the nucleus into the mitochondria: three nuclear targeting signals, a mitochondrial import signal and, moreover, two pest-sequences for rapid protein degradation are present. we shall present a, currently tested, working hypothesis. in hg-treated toad skins, water permeability is high or low depending on whether the major anion of the inner bathing medium is so~ or ci. we report here the results of experiments designed to determine if, in skins bathed with so4-ringer, net water flow (j~,) could be diminished by agents added to the outer medium. toad skins were mounted inbetween glass chambers and exposed to a standard osmotic gradient of 200 mosm. jw was continuously monitored by automatic, volumetric techniques. exposure to lmm hgclz or chzcihg (external side) led to the typical anion-induced j,, changes. re-exposure to hg caused a mild (10%), although significant (n=8, p<0.001), fall in jw. since cuso4 is a sh-reagent like hg, we looked at the effects of cuso4 (lmm). there was a 68% reduction of j, (n=7, p<0.001), an effect totally reversible by simple washing of the skins. likewise, addition of dithiothreitol (dtt, 5mm) in the presence of cu, caused a 90% reversal of the cu effect ; there was no change in jw if dtt was given before cu, or if both agents were added simultaneously. finally, in hg-treated, glutaraldehyde-fixed skins, lmm and 5ram cuso4 caused 51% and 92% jw decrements, respectively, that were totally reversible. in conclusion, cu causes a dtt-sensitive inhibition of jr, in hgtreated skins. further work is needed to establish if the cu effect is exerted on sh-groups of apical aquapories of toad skin. $15-44 the amino acid sequences of the pc-645 e-subunits and other cryptomonad biliprotein ~-subunits pose the most intriguing question about the phylogenetic origin of these unique chromopeptides. cryptomonad pc-645 ~-subunits seem not to be closely related to the known phycobiliproteins, linker polypeptides (lpps), bpe or any other light-harvesting chl polypeptides from the thylakoid. for most of them the number of identical amino acid residues was 15%-20%. sequence alignment of c-pe associated lpps with t-subunits from cyanobacteria and red algae and the cryptophytan ~-subunits however show that the identity between c-pe associated lpps and cyanobacterial x-subunits is still significant, whereas it reaches the limit of significance between cyanobacteril and eucaryotic x-subunits. nevertheless it has to be assumed that x-subunit derive from cyanobacterial c-pe associated lpps as well as cryptomonad ~-subunits may derive from t-subunits. whereas the the phycobiliprotein ~-and 8subunits remained structurally conservative during evolution (e.g. -70% identity between cyanobacterial and red algal pe-subunits) the lpps and t-subunits show drastical changes in their chromophore binding domains. $15-45 functional and evolutionary relationships of the components of the primary events in photosynthesis have been traced using amino acid sequence comparison. basically, the question was posed regarding the interrelationship of the apoproteins of different photosynthetic organisms which gather light and/or separate charges across membranes. do their protein-chemical structure and organisation decipher us certain clues and parts of the path by which they have been selected and derived from a possible primordial reaction centre polypeptide? within that context the light-harvesting-and energy transfer systems of purple bacteria offered as ideal model components. a data set of more than 70 apoprotein sequences has pointed to some keystructural elements which have been partially verified as such by limited proteolysis and genetic engineering. a careful inspection of the determined bacterial antenna-structures revealed some remarkable similarities to eukaryotic antenna and reaction centre apoproteins indicating possible basic structural motifs for complexing pigment molecules, like chlorophylls or bacteriochlorophylls in very distant representatives of phototrophs. a number of glycoproteins of the nervous and/or immune system that are involved in cellular recognition and/or adhesion share the common lz/hnk-1 carbohydrate structure. in a large proportion of patients with peripheral demyelinating neuropathy associated with paraproteinemic gammopathy (ppn), monoclonal igm antibodies (m-igm) react with the lz/hnk-1 determinant of mag and p0. we plan to test the hypothesis that anti-mag m-igm autoantibodies may alter the expression of hnk-1 bearing myelin proteins such as mag, p0, mog, ncam or pmp22. by using monoclonal antibodies to myelin proteins on tissue sections, preliminary results indicate that mbp protein content seems to be strongly downregulated, whereas galc and $100 protein do not appear to be affected by the loss of myelin in ppn nerves. gfap protein expression appear to be upregulated in demyelinating biopsy specimens from ppn patients with anti-mag m-igm gammopathy. in conclusion, the expression of some l2/hnk-1 negative proteins might be disregulated by the presence of circulating anti-mag m-igm in the serum of ppn patients. we have isolated a human liver na+-taurocholate cotransporting polypeptide (ntcp) by screening a human liver edna library with a rat edna probe derived from ntcp (pnas 88, 10629, 1991) . the cdna codes for a protein of 349 amino acids which shows 77% amino acid homology with the rat ntcp. expression of ntcp in x. laevis oocytes resulted in na+-dependent bile acid uptake which exhibited saturability with an apparent km for taurocholate of 6 ~m. ntcp mediated taurocholate uptake into oocytes was inhibited by all major bile acid derivatives, bumetanide and bromosulphophthalein. southern blot analysis of genomic dna from a panel of human/hamster somatic cell hybrids mapped the human ntcp-gene to chromosome 14. in conclusion, these studies supplement the previously suggested selective mammalian distribution of the ntcp gene family and provide the possibility for direct molecular characterization of human bile acid transporter genes. diverse cell surface molecules of the nervous system play an important role in specifying ceil interactions during development. with the aid of a polyclonal antibody against l1, a 1.skb edna clone closely related to l1 (ch-l1) was isolated from a ~. gtl 1 library derived from poly(a)* l~qa of day 8 mouse brain. using this edna as probe the remaining cdi'ia 5' sequences were cloned out of a plasmid library constructed from postnatal day 6-14 mouse brain poly (a)+ p, na. two independent full length clones of 4.2 and 4.4 kb encompassing the entire coding region of chl] were isolated. these represent putative alternatively spliced mp, nas. the deduced primary structure of chl1 reveals that, like the cell adhesion molecules (cams) mouse l1, chicken ng-cam, and nr-cam (bravo), chl1 is composed of six ig-like domains, a transmembrane domain, and a short cytoplasmatic region. in contrast to the other l1 family members, only four and a half instead of five fibronectin type hi-like repeals are found in chl1. the fibronectin type [h-like repeats were expressed in e. col[ and the protein fragment was used for immunization. as observed for li, ng-cam, and nr-cam, chl1 is susceptible to proteolytic cleavage. bands of 185kd, 165kd, 125kd, and 50kd could be detected bywestern blot analysis. by in situ hybridization, a predominant expression by neurons was found in the adult mouse brain. western blot analysis showed expression of chl1 protein in brain and heart, but not lung, kidney and intestine. in liver, a 50 kd immunoreactive band was found. the relationship of chl1 to molecules known to be involved in cell adhesion and neurite outgrowth, combined with its pattern of expression, suggests a role for this molecule in cell interactions dndng neural development. work from this laboratory revealed that in hg-treated, glutaraldehydefixed toad bladders, the water permeability coefficient (pf) is high in sod-ringer and low in ci-ringer ; in all these studies the osmolality of the serosal medium was 220mosm. as an attempt to determine the mechanism(s) underlying such pf changes, experiments were carried out with bladders whose serosal surface was exposed to [so-, hypo-or hyperosmotic media ; the outer surface was always exposed to a hyposmotic solution (22 costa). net water flows were measured continuously with automatic, volumetric techniques. after exposure to 1 mm chzcihg (10', outer medium) followed by fixation with 0.25% glutaraldehyde (5', serosal medium), the bladders were thoroughly washed. in sod-ringer, pf (/~m/sec) was as follows (n=6) we wanted to determine the molecular weight (mw) of ntcp and its topology in the plasma membrane (pm) of rat liver. a rabbit antiserum generated against the c-terminus of ntcp was used to determine its mw on western blots and its subcellular distribution in liver and in hepatocytes by immunofluorescene (if). site directed mutagenesis and deletion experiments were used to determine the n-linked glycosylation sites. ntcp encodes a 50 kd protein in rat liver pm vesicles. the basolateral localization of ntcp in liver was confirmed by if. ntop could only be immunostained in permeabilized hepatocytes suggesting an intracellular localization of the cterminus, cnda constructs showed the ntcp is glycosylated at positions 5 and ii. the data show a selective basolateral expression of ntcp in rat liver and they suggest that the two ends of ntcp are located on opposite sides of the pm. zymogen granule membranes (zgm) are known to contain proteins with nucleoside phosphatase activity, but their exact nature and function are unknown. the activities require ca ++ and mg ++ and are sensitive to atpase inhibitors but not to inhibitors of na/k-atpase activity. the aim of this study was to isolate individual proteins of pig pancreatic zgm, to attribute enzymatic activity to individual proteins and to characterize them with various substrates (gtp, atp, amp etc.), inhibitors (amp-cp, amp-cpp, gtpgs etc.) and lectins (maa, dsa, gna etc.). we have subjected highly purified zgm solubilized in chaps to ief on immobiline dry strips | (pharmacia) with s ph range 3--10.5 (ist dim.). these strips were then electrophoresed in a polyacrylamide gradient gel containing 0.2% chaps and tris/hci at ph 9.5 (2nd dim.). nucleoside phosphatase activity was detected histochemically in the 2d gel by incubation with substrate in the presence of lead nitrate. focussed strips were also subjected to sds-page for comparison. several proteins showed strong activity to gtp, atp and itp. the role of these phosphatases are discussed in the context of the role of gtp-binding proteins and the de-/phosphorylating events on the zgm in intracellular targeting and exocytosis. the disease-specific isoform of the priori protein (prp sc ) is an essential part of the infectious particle causing scrapie or bse. prp sc differs from prp of normal animals (prp c ) by its relative protease resistance. the physical nature of this difference is still unknown. here we analyzed the protease-resistance of prp sc quantitatively. prp sc was rendered completely protease-sensitive at alkaline ph or in guanidinium thiocyanate (>1.5 m). denaturation in 2m guanidinium thiocxanate (gdnscn) completely abolished protease resistance of prp bc within 15 min while denaturation in 6 m urea showed a much slower time course. denaturation cuwes were used to calculate the gibbs free energy (as d ) in dependence of gdnscn or urea at different concentrations. the linear relationship between agd and the denaturant concentration is suggestive of a two state model involving the conformational change of a single protein domain. this is also reflected in the small number of side chains (11,6) additionally exposed to the solvent upon conversion of prp sc to its proteasesensitive isoform. our results suggest that minor rearrangements of the structure of prp sc are sufficient to abolish its protease resistance. of physiology, university of zurich, winterthursrstr. 190, ch-8057 zt~rich in contrast to mammalian kidneys where inorganic phosphate (pi) is reabsorbed unidirectionally, flounder renal epithelial cells both re.absorb and secrete pi. in order to investigate on a molecular level the cellular pi handling we have cloned a re,absorptive nalp i transport system from flounder renal tubules. based on homology with the cloned na/p i cotransporter from rat we have isolated a edna clone (flounder napi-ii, 2424 bp) encoding for a protein of 637 amino acids. in the eight transmembrane spanning domains the protein is 78% identical with the corresponding system from rat kidney, in the hydrophilic regions only 30%. expression of in vitro transcribed rna in xenopus oocytes specifically stimulated na-dependent pi transport. binding properties of na and pi as well as the ph-dependency were similar to the ones found in mammalian systems (rat, human, ok cells). by northern analysis three transcripts, 1.9, 2.7, and 4.2 kb in length, could be detected. rna from flounder renal tubules contained all of the transcripts, intestinal rna only the two lower bands, and non-epithelial tissue from kidney expressed only the 1.9 kb transcript. mammalian systems share only the 2.7 kb transcript but not the related species. the close functional relationship of flounder napi-ii with the known najp i transport systems and the pronounced differences in primary structure provide the basis for structure function analysis of na/p i cotransport. the amyloid 13/a4 protein precursor (app) is a transmembrane protein expressed in different isoforms throughout the organism. our work has consisted in a detailed study of the structure and biological function of app. specifically, we have shown that the secreted form (sapp) of app-695 is involved in the growth regulation offibroblast% and also stimulates neurite extension in b 103 neurnblastoma cell line. functional mapping studies have shown that the domain responsible for both the growth-regulating and the neurotrophic activities of sapp-695 is the rerms site, arg-328 to ser-332. the presence ofsapp binding sites on the surface of b 103 ceils (through the active rerms site) indicate that the neurotrophic activity of sapp is mediated by a receptor, and the subsequent activation of a signal transduction mechanism. in addition, we have shown that the rerms site is also involved in the neuronal survival properties of sapp-695. finally, we have initiated an in vivo study of app biological function, and shown that a peptide representing the neurotrophic domain ofsapp-695 can strengthen memory retention in rat through stabilization of synaptic structures in the frontal cortex. this work was supported by grants from the swiss national science foundation (823a-028366), and nih (ag 05131). we previously showed that in smooth muscle cells, the (~i-ar is rapidly phosphorymted following exposure to agonist. to understand this biochemical event, we investigated the phosphorylation of the c(1b adrenergic receptor stably expressed in rat1 fibroblasts (3.3 pmol/mg prot.). the =(1b-ar was solubilized from 32p-labeled rat cells and immunoprecipitated with antibodies raised against the n-terminus part of the receptor. treatment of ceils with 100 ~.m epinephrine showed a rapid and significant increase in receptor phosphorylation above basal. treatment of cells with a specific pkc inhibitor (ro-318220) did not affect agonist.promoted phosphorylation while it completely abolished phorbol esterinduced receptor phosphorylation. this strongly suggests that pkc is not involved in agonist-induced c{1b-ar phosphorylation. the elb-ar contains several putative phosphorylation sites in both its third intracellular loop and c-terminus tail. to investigate the role of these domains, a trunoated receptor (lacking its last 147 amino-acids) was constructed and stably expressed in rat cells (1.3 pmol/mg prot). this receptor mutant displayed similar ligand-binding properties and abirity to activate phospholipase c as compared to the wild-type receptor. phosphorylation experiments revealed that this mutant is not at all phosphorylated, neither by agonist nor by phorbol esters. agonistinduced decrease in cell surface receptors, measured by binding of the antagonist 3h-prazosin at 4~ was however similar for both the wild-type and mutant receptor. this suggests that loss of phosphorylation sites does not affect receptor internalization. human intestinal lactase-phlorizin hydrolase (lph) is synthesized as a precursor molecule (pro-lph), which undergoes proteolytic processing during maturation. lph expressed in cos-1 cells was enzymatically active, the electrophoretic properties of lph-species synthesized by transfected cos-1 cells correspond to those in organ cultured human intestinal explants, in caco-2 ceils and in transfected mock ceils. they included the pro-lph and a proteolytically processed form, which had similar electrophoretic properties to the mature enzyme. the appearance of the putative mature form was inhibited by brefeldin a, ammonium chloride and chloroquine. in addition, only complex glycosylated pro-lph (pro-lphc) was inserted into the cell surface membrane. these data indicate that in cos-1 cells pro-lph is inserted into the cell membrane, is internalyzed and enters lysosomes where proteolytic events lead to the appearance of a mature-like enzyme. $15-59 assembly and transport of paba peptide hydrolase alpha and beta subunits in cos-1 cells eldedng, j.a., gr0nberg, j. and sterchi, e., institut for biochemie und molekularbiologie, universit&t bern. paba peptide hydrolase (pph) (ec 3.4.24.18), a metalloendopeptidase from epithelial cells of human small intestinal mucosa, consists of two subunits, ~ and 13, which form homodimers and oligomers, cdna cloning has revealed homologies with developmentally important proteins from different species and has led to the definition of a new family of metalloendopeptidases, the astacin family (j. biol. chem. 266, 21381, 1991) . here, we report on the expression of the cdnas of pphcz and pphj3 in cos-1 cells. when expressed on its own, pph(z is synthesized in a core glycosylated form only, suggesting that it is transport-incompetent and not able to exit the er. immune-fluorescence studies have confirmed that pph~ is not expressed on the surface of cos-1 cells. pphl3, on the other hand, matures and is transported to the cell surface. when the two subunits are coexpressed, ppho~ can also be detected on the cell surface, suggesting that a heterooligomeric assembly is necessary for the surface expression of pphtz. truncated forms of both pphc~ and pphi~ could be detected in the medium of transfected cos-1 cells. in our previous work, we demonstrated by immunocytochemical reaction that sensory neurons expressed nuclear triindothyronin receptors (nt3r) in embryonic as well as adult rats. in contrast, in sciatic nerve, schwann cells exhibited only transient nt3r immunoreactivity from e17 to postnatal day 10. in the present work, we attempt to demonstrate by radioautography that the detected nt3r are effectively the binding sites of [125i] labeled triiodothyronin. cryostat sections of dorsal root ganglia (drg) or sciatic nerve were incubated with 0.1 nm of l, 3,5,3'-[t25i]triiodothyronin 2200 ci/m mol (nen), while the controls were incubated with a large excess (llxm) of unlabeled trfiodothyronin (t3). in aduit rat drg, radioautographs revealed that silver grains were exclusively restricted to neuronal cell bodies, while the schwann cells ensheathing the axons were free of significant label. in newborn rat sciatic nerve, silver grains accumulated over schwann cells; in contrast in adult rat sciatic nerve, schwann ceils were free of label. in control drg or sciatic nerve sections incubated in large exceas of unlabeled "1"3, all the radioautographs exhibited only scattered silver grains. in conclusion, our results show that sensory neurons and schwann cells are able to express functional nt3r which bind thyroid hormones (snf 31-33671-92). characterization of the maturepreeursor glycophospholipid for glycosylphophatidylinositoi anchors of saccharomyces cerevisiae. many attempts to isolate the mature yeast precursor gpi glycophospholipid ready to be attached to newly translated proteins for gpi-anehoring have failed although such precursors were previously identified in mammalian cells and protozoa. here we show that metabolically labeled glycolipids of the expected structure can be observed if the incorporation, their accumulation and their extraction are optimized. two very polar, (3h)-mannose-labeled glyeolipids named cpi and cp2 were identified and purified. they were structurally characterized using phnspholipase treatments, partial deacylation with methanolie ammonia, hydrofluoric acid dephosphorylation, nitrous acid deamination, acetolysis, exoglycosidase treatments and combinations thereof to produce labeled fragments which could be analyzed by paper chromatography or thin layer chromatography. cp1 and cp2 both contain the identical core oligosaccharide mangl,2(x->po4-6)manal,2mamxl,6(y->)mana-glcn-lansitol, x and y being hydrofluoric acid-sensitive substituents (most likely ethanolamine and phospheethanolamine, respectively). the inositol is acylated although no acyllaositol is present on protein-bound yeast gpi-anchors. cp1 and cp2 can also be observed after lableling with (3h)myo-inositol, the lipid moieties of cp1 and cp2 can be completely removed by mild alcaline hydrolysis altough the protein-bound gpi-anchors made by the pmi210 cells under identical labeling conditions are completely mild base resistant. this finding reinforces the notion that the ceramide moiety typically found on the majority of yeast gpi-proteins are added only after addition of the gpi precursor glycolipid to proteins. the distributions of the mannose-6-phosphat e/igfll-recept or and a2,6slalyltransferase in hepg2 cells: no evidence for co-localization by confocal laser scanning microscopy. the organization of the trans golgi apparatus (ga) with respect to (recycling) man-6-p/igfii receptor (mpr) and (resident) c~2,6sialyltransferase (st) has been investigated by double immunofluorescence laser scanning confocal microscopy in hepg2 cells. they were chosen because biochemical evidence suggested sialylation of mpr upon recycling (duncan & kornfeld, j. cell biol. 1988, 106, 617-28) implicating colocalization of both proteins. in the steady state st was confined to the ga whereas total (endogenous) mpr was localized to coarse vesicles without evidence for colocalization by confoeal microscopy. endocytosis of surface-labeled mpr was monitored in presence and absence of brefeldin a (bfa): without bfa, early endosomes (2' of warming up) showed no, late endosomes (20') little if any colocalization with st. in presence of bfa, the st-compartment disappeared whereas late endosomes persisted with some tubular emanations. under similar conditions, in "absence of bfa, endogenous mpr concentrated in the ga region suggesting colocalization with st. in presence of bfa the mpr compartment formed a redculum whereas the st compamnem disappeared. in conclusion, within the limits of the techniques used, exogenous mpr was not detectable in the st compartment whereas endogenons mpr concentrated in the ga region but obvious co-localization was exceptional. supported by grant 31-30757.91 of the snsf to egb. cultures of dissociated striatal neurons prepared from fetal rats were grown in the presence of neurotrophin-4/5 (nt-4/5) as well as the other known neurotrophins, ngf, bdnf and nt-3. we found that acute administration of nt-4/5 to seven days old cultures stimulates the hydrolysis of phosphatidyllnositol, an event involved in neurotrophin signal transduction. growth of stdatal cultures in presence of nt-4/5 resulted in increased cell survival as indicated by elevations in total cell number, protein content and number of viable cells. nt-4/5 exposure increased gaba uptake and staining intensity in gaba immunocytochemistry in these cultures indicating atrophic action on gabaergic neurons. to further identify responsive cell populations we analyzed for calretinin, a calcium binding protein known to colocalize with gaba in a number of neuronal cells. nt-4/5 strongly increased the number of calretinin positive cells in cultures prepared from rats of embryonic day 15, as we]l as calretinin levels determined by western blot analysis. when cultures were prepared from embryonic day 18 rats, nt-4/5 very strongly increased the morphological differentiation of calretinin positive cells. while all effects produced by nt-4/5 were mimicked by bdnf with similar potency, nt-3 was found to be only marginauy effective, our findings identify nt-4/5 as a potent neurotrophie factor for striatal gabaergic neurons. fusion of cells and organelles is a general patho-biological phenomenon: muscle cells, transport vesicles, langhans cells and virus induced fusion from within (ffwi, during replication) and without (ffwo, by the particles). in vivo and after virus isolation only little fusion can be seen, only after cultivation fusing hsv can be detected; a selective pressure must be released. 6 syn loci are known on the genome; the syn 3 locus is located inside of the cell in the carboxy terminal part of glycoprotein b, all other fusion domains are outside the ceil. fusion inducing mutations are in aa 816, 853, 854, 856, 857 and 872 and were correlated to fusion from within and without as well as to selective cyclosporin a effects. a model is presented for the 3 syn locus. -by recombination the fusion capacity could be transfected to fusion negative viruses. -hsv penetrates the cellmembrane by fusion by 2 ligand-recepter interactions. ffwo+-strains penetrate at 0~ cell/cell fusion was analysed and found to need also 2 receptor-ligand interactions if induced by syn 3 mutations. timing analysis of fusion events and by certain inhibitors allow further conclusions. quenching of lhcii isolated from dark-adapted (lhcii-d) or preilluminated (lhcii-l) rye leaves was similar in both preparations, but reversibility of this process within two hours of darkness was slower in lhcii-d. no differences in fluorescence quenching and full reversibility was observed for both lhcii preparations in reverse micellar solution. xanthophyll/chl concentrations in lhcii were consi-derably decreased under this conditions. excitation difference spectra (before and after illumination) of lhcii in asolectin liposomes showed typical changes of energy transfer between carotenoids and chl. a model is proposed according the xanthophyll cycle controls reversibility of light-driven excitation quenching as well as the xa~a~q~_x~tentof ~,,~nchi~g i/~ lhc~z. myelinogenesis is a complex, developmentally regulated process involving coordinate expression of myellnafion-related proteins. the myelin forming cells are the oligodendrocytes in the central nervous system (cns) and the schwann cells in the peripheral nervous system (pns). we differentially screened a rat postnatal day 16 (p16) spinal cord edna library with probes derived from normal (plus probe) and from myelin-free (minus probe) p16 spinal cord mrnas. we succeded in the isolation of several novel edna clones whose corresponding mrnas were expressed either selectively by oligodendroeytes or by oligodendroeytes and sehwann cells. here we describe one of the edna clones (tentatively denominated ns 2) whose mrna is specifically expressed by oligodandroeytes and schwann cells. the 3.5 kd transcript is expressed at highest level during myellnation and at much lower levels in the adult as it is known for other myelin-specific mrnas. sequence analysis of the full length edna sequence showed a 50% identity to the rat liver udpglucuronosyltransferase family, involved in the detoxificafion system. the 541 amino acid open reading frame encodes a 62 kd protein with a putative signal peptide and two transmembrane domains, whether this ns 2 protein is involved in a detoxification reaction or in glycosilation of proteins and lipids with glueuronie acid is under investigation. neuroblastoma (n8) are sympathetic tumors of childhood characterized by mycn amplification in advanced stages. cd44 standard (cd44s) and splice variants (cd44v) represent a group of surface glycoproteins whose expression has been recently linked to metastasis. we analysed the expression of cd44s and cd44v on n8 tumors and cell lines by immunohistology and northern blot. results showed high levels of cd44s on 33/44 nb, 6/10 n8 cell lines and 4/4 ganglioneuromas (mature, benign sympathetic tumors). lack of cd44s staining was observed on stage iv, mycn amplified tumors only. in cell lines, expression was not detected on cells with a neuronal phenotype while high levels of cd44s were expressed by cells with a glial differentiation, independently of mycn amplification. up-regulation of cd44s was obtained with agents that induce a glial differentiation, while neuronal differentiation by retinoic acid was not accompanied by a change in cd44s expression. no gd44v were detected on tumors or cell lines. we conclude that cd44s expression is down-regulated in a subset of nb ceils and tumors and that mycn product and additional mechanisms responsible for control of differentiation are involved in this regulation. the glycoprotein gc of herpesviruses is known to be non-essential for virus replication. non-essential herpesvirus proteins are suggested to perform "luxury functions" which may influence the outcome of an infection. we are aiming to analyse the function(s) of ibc specified by bovine herpesvirus 1 (bhv1), bovine herpesvirus 5 hv5) and caprine herpesvirus 1 (caphv1). these viruses are closely related but differ in their pathogenic potential. in a first step we have cloned and sequenced the individual genes. the nucleotide and the deduced amino acid sequence homologies of two 8hv1 reference strains was found to be >98%. the sequence homologies between bhv1 and bhv5 were >75% and those between bhv1 and caphvl were >65%. comparisons on the amino acid sequence level revealed that the differences were most prominent in the n-terminal parts, whereby the potential signal sequence region might be concerned. the putative glycosylation sites, however, were not affected, and the transmembrane sequences were similar in size and location. in order to characterize the significance of these differences, we are constructing gc-deletion mutants and recombinant viruses carrying a foreign gc gene. the in vitro and in vivo behaviour of these viruses will be compared to the wildtype virus. $15-67 o. moullet and j.-l. dreyer, instimt de biochimie, unlversit6 de fribeurg, ch-1700 fribourg camp has been recently shown to promote a cell survival response by retarding apoptosis (berridge, m.v& el. (1993) exp. hematol. 21,269-276) . on the other hand quinones are widely distributed substances of often potential toxicological significance. we have tested the effects of quinones on adenylate cyclase. the enzyme is rapidly inhibited by pbenzoquinone (ic50=40-45 ktm) or dicnlorophenol-indopbennl (/6'50=200 ilm), but 2-substituted quinones are inactive. the lc50's are decreased 3-10 times after membrane soinbilization but are not affected by gtp, gdp or analogues nor by cholera and pertussis toxins. the inhibition is not mediated by a g-protein or by the activation of a defined receptor. quinone inhibition stoichiometrically competes with forskolin activation of adenylate cyclase, equimolar concentrations of beth quinone and forskolin restoring the enzyme activity to its basal value. the cytotoxicity of benzoquinone, tested in vivo on hep-g2, a human bepatocellular carcinoma cell line, could be prevented with forskolin. in plasma membranes quinones tightly bind to only one major and two minor proteins that were purified by page electrophoresis under native conditions. together these observations indicate that quinone action can be attributed to targetting to a limited number of proteins at tile plasma membrane in a highly selective way. by affecting adenylate eyclase, a modulation of the camp pool induces apoptosis as a result of the cytotoxicity via. t.congolense is an african,unicellular hemoflagellate, pathogenic for domestic animals such as cattle.within the host bloodstream,parasites adhere to the wall of the microvasculature,eausing severe organ damage.we have set up and characterized an in vitro assay in order to study the metabolic requirements,reeeptor-ligand interactions and the ultrastructure of this adhesion phenomenon.our findings suggest that adhesion is an active process requiring metabolic energy on part of the parasite, but is independent of the target cells.we also show that t.congolense possess a leetin-like domain localized at specific sites along the surface of the anterior end of the flagellum, which interacts with specific carbohydrate receptors, probably sialic acid and/or n-aeetylglueosamine residues,on the endothelial cell surface. this suggests that adhesion is likely to be mediated by a trypanosomal surface component distinct from the variable surface glycoproteins(vsgs). we also suggest that the cytoskeletal protein aetin is involved in this process. local immune response based upon intestinal parasitespecific t and b cells to giardia lamblia may be impomrant in parasite clearance. experimental infections qf mice (cr:nih:s) with g. lamblia (clone gem-h7 which e~presses a major 72kd antigen on its surface recognized b~ mab g10/4) have demonstrated that the parasite undergoes surface antigenic variation in rive. experimental i~fection of immu/%odeficient mice (nu/nu mice and sci~d mice) and oontrol nu/+ littermates provided insights into associated immunological mechanisms: dominant surfade epitope-specific slga plays a major role in modulatin6] surface antigenic variation, and thymus-dependent t-lyn~phecytes in the induction of selfcure. athymio nu/nu mioe (zu. icr-strain) were reconstituted with immune peyer:~ patch lymphocytes obtained from self-healed littermat~ nu/+ mice. intestinal b-cells from these nu/+ mice @s well as from reconstituted athymic nude mice synthesized in vitro parasite-specific iga. this iga showed a predominant immunoblet reaction with the major surface antigen (a mr 72,000 polypep-tide) characterizing the giardia clone in question. nu/nu mice reconstituted wit~ immune cells acquired the potential to decrease significantly their intestinal parasite mass. thus the hypothesis on the causative role of intestinal iga in tl~ control of intestinal g. lamblia infection has found further experimental support. stephan jentsch, heinz tobler and fritz m011er, institute of zoology, university of fribourg, p4rolles, "1700 fribourg the process of chromatin diminution in the parasitic nematode ascaris lumbricoides leads to the formation of somatic cells that contain less dna than the germ-line cells. during this process, which occurs between the third and the fifth embryonic cleavage divisions in five different blastomeres, the termini of the chromosomes are cut off and eventually degrade in the cytoplasm. to analyze this process at the molecular level we constructed a xzap somatic telomere library. the analysis of three cloned telomeres and their corresponding germ-line genomic regions revealed that chromosomal breakage takes always place within short specific chromosomal regions (cbrs) and is followed by the addition of the telomeric sequences (ttaggc)n to the breakage sites. the different cbrs do not crosshybridize to each other. a comparative nucleotide sequence analysis is now being performed to screen for the existence of conserved sequence motifs. in a parallel approach we analyze the chromatin structure of the cbrs and their flanking regions in developmental stages before and during the elimination process, putative recognition or regulation sequences important for the elimination process might be recognized as dnasel hypersensitive sites. once such sequences will be identified, it should be interesting to establish their role in the elimination process and to look for specific binding factors. chromatin diminution takes place in all presomatie ceils of the early embryo of ascaris lumbricoides and is followed by the addition of many repeats of the telomeric sequence ttaggc. chromosomal fragmentation is developmentally regulated and occurs within specific chromosomal regions (cbrs). one of these cbrs (cbr1) was analyzed in detail. agene located close to the cbr1 encodes a putative gtp-binding protein, whose promoter region is located within a distance of only 2 kb from the telomeric ttaggc repeats of the corresponding somatic chromosome. a homologous gene was isolated from c. elegans. most interestingly, the c. elegans gtp-binding protein gene is also located at a telomeric position, namely at the end of chromosome v. in ascaris, transcripts of this gene are present in all developmental stages, though they seem to be stronger expressed in oocytes and early embryos than in later developmental stages and somatic tissues. a high degree of sequence conservation of these genes between the two nematode species and man (a corresponding partial cdna clone was identified in a human heart cdna library) suggests an important biological function. currently, we are using genetic methods to determine the possible function of this gene in c. elegans and to test whether its telomeric localization in both nematode species has any functional importance. it encodes a nuclear protein which is associated with she chromatin together with other probeins of the poiyoomb group. the mechanism of gene regulation caused by pelycomb seems to be similar to that of the modifier genes of the position variegation effect which also encode structural proteins of the heterochromatin. one of these proteins is hpi which shares with polycomb a region of similarity at the amino terminus called the ~omatin ~rganisation m~difier domain (chromo domain). the particular function of the chromo domain is not yet understood, but it seems to be important for the protein-protein interactions in chromatin associated complexes. furthermore, chromobox cdntai~ing genes were found in several species, suggesting that they are widespread in the animal and plant kingdoms. here we show chat ~. clemens contains at least one chromobox containing gene (cdna: cm08h7). length and szrusture of the putative ~. elegs~ chromo domain protein are similar to those of the chromo domain containing prozeins of other species. the expression pattern of cm08h7 is s:udied with antibodies raised against the putative chromo domain protein and by injecting a 5-ga! fusion construct inns the germ line of ~. eleman$. chromatin diminution in the parasitic nematode ascaris lumbricoides is a complex molecular mechanism, involving cleavage of the chromosomes at specific breakage regions, degradation of the eliminated chromatin and new telomere formation in all presomatic cells during the early embryonic development. the aim of the present project is to reproduce this dna elimination process in vitro, step by step or all in one, by using cell-free extracts of a. lumbricoides eggs. therefore, 4-8 cell extracts were established and their quality was assessed by the ability to transcribe 5s rrna genes (pol iii) and sl rna genes (pol li). faithful extracts were then tested for cleavage activity on dna substrates derived from different chromosomal breakage regions. finally, preliminary results revealed a possible non processiv rnase sensitive telomerase activity in the in vitro extracts, capable of adding specific nucleotide sequences to an oligonucleotide primer containing four repeats of the telomeric sequence traggc. as de novo synthesis of several kilobases of somatic telomere is likely to require a strong telomerase activity, we assume that this a. lumbricoides in vitro system is a good tool to isolate the telomerase protein and its corresponding gene or other implicated factors, s16-07 university of berne, ch 3010 berne a common feature of all mycobacteria -whether pathogenic or not -is their richness and variety of lipids. among numerous lipids widely distributed, pathogenic mycobacteria exhibit a group of sulfated surface lipids thought to be determinants of virulence. therefore, we studied sulfolipid-bioslrnthesis in pathogenic and apathogenic (opportunistic) mycobacteria species by monitoring the kinetics of [35s]sulfate incorporation into cellular lipids, pathogenic mycobaeterla (two virulent m. tuberculosis strains) showed a marked sulfolipid-synthesis with highest rates during exponential growth, while apathogenic ones (an avirulent m. tuberculosis strain and an opportunistic species) manifested negligible incorporation of the label. the partial characterization of the [35s]-sulfated lipids of pathogenic mycobacteria by thin layer chromatographic separation, combined with autoradiographic detection, revealed the presence of several radiolabeled lipid components of different polarities and with altering expressionpatterns during growth. these results strengthen the hypothesis, that sulfolipids are involved in the pathogenesis of mycobacterial disease. maiada (1,2) . some features of human pf maiada are observed in mudne malaria, although the pattern of sequestration changes due to the different plasmodial species involved, in mice, we showed by immunohistocbemistry that the expression of vcam-1 and icam-1 is upregulated in brain, lung and heart of p/asmodium bergheiinfected baib/c and also in lps-pdmed 8cid mice. in $gid mice injected with labeled human erythrocytes (e), a higher rate of sequestration to brain was observed with pfe than with uninfected e. lps-pdming increased the retention of pfe in the brain and lungs significantly (t test), but decreased it in the spleen. we conclude that pfe express surface structures which allow them to adhere to the vasculature of the brain more efficiently than uninfected e. lps increases the number of adhesive molecules on the host endothelium. interestingly, sequestration in the 8cid mouse resembles that of pfe in man. f. roth, f. riniker, k. schopfer and t. burkart inst. med. microbiology, univ. of berne, ch 3010 berne it has recently been shown that bacterial lipids cancarry antigenic determinants. the study of antibody specificity to such lipid epitopes in vitro turns out to be difficult since hydrophobic antigens have to react with watersoluble molecules. we have developed a solide phase lipid antigen immunoassay (laia) that allows to quantify the antibody reactivities with an array of lipid antigens derived from mycobacterial cells. defined amounts of extracted lipids were immobilized as equidistant lines on a nitrocellulose sheet. small strips of nitrocellulose, each carrying the same sets of lipids were incubated with different human sera. bound antibodies were then reacted with [125-i]-iodine labelled antihuman antibodies and visualized by autoradiography. the reaction was quantified by densitometry. assay conditions were optimized for antigen and antibody concentrations and appropriate incubation times. in addition, the effects of different ingredients (salts, proteins, detergent) on antigen immobilization and epitope accessability were studied. the presented laia-system is rapid, sensitive and reproducible. it allows to compare the specificities of different bacterial lipid antigens and to quantify their reactivity with serumantibodies. the atomic force microscope is a very convenient tool for studying hard and flat samples with atomic resolution. biological objects, usually soft and rough, are sometimes difficult to image using this technique. in contrast bacteria, which are too small to be observed with high resolution using the optical microscope, are hard enough to be observed with an afm at a relative good level of magnification. this kind of microorganisms can be prepared for afm imaging using very rapid and simple techniques. this method could be a convenient tool to observe the effect of bioactive substances such antibiotics. we show in this poster an exemple with the action of penicillin on b. subtilis. within the family trypanosomatidae, leishmania is a protozoan pathogen producing a broad spectrum of human disease. its life cycle is divided in two stages. promastigote is the flagellated form which colonizes the gut of the sandfly vector, and amastigote is the non-flagellated form that is specialized for growth and survival in the vertebrate host macrophage. to understand gene regulation during the transition between the promasttgote and the amastigote stage, we were interested in the characterization of stageregulated genes. we isolated a gene, sw3, from leishmania major whose mrna is more abundant in amastigote compared to promastigote. structural analysis of this gene showed that an additional polypeptide could be encoded by the opposite strand of sw3. we confn'med the presence of an open reading frame on the opposite strand by in vitro translation of the sw3 antisense rna. in vivo, an amastigote sw3 antisense transcript and the corresponding polypeptide were also detected suggesting that the sw3 is transcribed in both directions and that stage specific transcripts and polypeptides could be produced. analysis of other gene segments tend to indicate that an intensive usage of the leishmania genome to produce various polypeptides could be a general phenomenon in trypanosomatidae. the four variants and / or posttranslational modifications of histone h1 of procyclic trypanosoma brucei brucei (protozoa, kinetoplastida) were purified by hplc reversed phase chromatograpy and characterized by their amino acid composition and partial amino acid sequence. differences in sequence of up to 45% as compared to histone h1 of higher eukaryotes exist. alkaline phospatase digestion and analysis of h1 by means of hpce was used to demonstrate the presence of phosphorylated modifications. the unique biochemical properties of h1 of t. b. brucei contribute to the structural differences seen in the chromatin of the parasite as compared to that of the higher eukaryote host. larvae of the parasitic wasp chelonus inanitus (braconidae, hymenoptera) develop inside embryonic and larval stages of the host spodoptera littoralis (noctuidae, lepidoptera). parasitism induces in the host a precocious onset of metamorphosis and developmental arrest in the precocious prepupa. the wasp injects, along with the egg, pdv and venom into the host egg. injection experiments with pdv and venom revealed that pdv play a crucial role in altering host development and in suppression of the host's immune system. the genome of the pdv of c. inanitus consists of at least i0 segments of double-stranded circular dna ranging in size from 7-30 kb. we analyzed the expression of viral genes in the course of parasitization with cdna made from mrna at various developmental stages of s.littoralis. we also investigated the localization of the expressed viral genes on the various segments. in addition, these bacteria were isolated and cultured from brain tissue. to identify the infectious agent we used, among other techniques, an atomic force microscope (afm). when the. isolation fluids derived from the cortex of three alzheimer's cases were examined with afm and scanning electron microscopes, we observed bacteria whose size and morphology fit to the order of spirochaetales. these images are presented and discussed on our poster. ~ottstein bruno, institute of parasitology, ch-berne. %lveolar echinococcosis ae is due to infection with the metacestode (larval stage) of echinococcus multilocularls. an immunodominant antigen em2 of e. multilocularis was characterized by mab-gii, respective studies revealed, that (a) antigen expression metacestode stage-sdeci-fic and (b} that expression was restricted to the laminated layer. the em2-antigen i s a lectin-binding carbohydrate linked to a lipid residue. the "em2positive" laminated layer proved to be a crucial factor in protecting the parasite from host immune-effector mechanisms: only e. multilocularis larval structures exp ressing em2 were able to induce secondary alveolar chinococcosis in rodents. subsequent experimental studies in resistant (c57bl/10) versus susceptible (c57bl/ 6j and akr) mouse strains showed that resistance was as-sociated with synthesis of anti-em2 igg 3 and igg i. sus-ceptibility of c57bl/6j-mice was associated anti-em2 s and no igg 3 . the parasite-specific in vitro lym-~hoproliferative i~une response remained stationary ~ith time after infection in c57bl/10 and decreased in r and aki< mice. day 30 p.i. cd4 ⧠-lymphoblast cells dominated in all meuse strains; day 90 p.i. an in-creased nu/0ber of cd4-/cd8 + and cd4+/cd8 + cells were observed. only susceptible mice exhibited a significantly ~eereased response of lymph node cells to cona-stimula-~ien with time p.i. in conclusion, subtypes of parasitespecific cellular and humoral host immune responses seem leishmania species are protozoan parasites causing a spectrum of clinical manifestations in man ranging from cutaneous lesions to morbidity and death. the insect stage (promastigote) and intracellular stage (amastigote) differ in terms of morphology, physiology, antigenicity and gone expression. it is clear that the amastigote stage is uniquely adapted for survival within the inimical environment of the macrophage phagolysosome and that mechanisms regulating these adaptations must be called into play during the early stages of infection when the parasite undergoes transformation. in an effort to elucidate these regulatory mechanisms, we have constructed a "subtracted" cdna library which is specifically enriched for parasite transcripts expressed during the first 7 hours of infection ("switch" genes). several clones have been characterized by sequence, northern, and southern analysis. three clones, sw3, sw44, and sw20, are expressed at a several-fold greater level in the amastigote stage and potentially encode proteins which interact with nucleic acids. sw3 predicted protein is homologous with histone h1 proteins and sw44 and sw20 potentially encode ribosomal proteins. analyses of transcripts from this library are yielding clues not only to mechanisms underlying the regulation of parasite transformation, but also insights into post-transcriptional and translational control of gene expression in leishmania. $16-17 microbiology, university of geneva, medical school, c.m.u., 9 avenue de champel, 1211 geneva 4. a viral rna representing the sequence of a defective interfering rna, naturally selected for efficient replication, i.e. only containing the replication promoter, was cloned under the control of the t7 rna polymerase promoter. the expressed 17 transcript was then replicated in rive by supplying in trans the replication functions, equally expressed from plasmids. this system, completely accessible to genetic manipulations, allowed us to define a basic rule directing sendal virus replication, the "rule of six" (calain and roux, j.virol. 67 : 4822-4830, ig93). a second defective rna, representing a shorter version of the viral rna, in that it contains not only the replication but also the transcription promoters, was then cloned and replicated in the same system, although with lower efficiency. the replication of this "transcribing" viral rna was found to obey the "rule of six" as well. replicationitranscdpfion promoters were then achieved to define the cissequence requirements needed for efficient replication or for replication /transcdption. results obtained with these chimerical viral rnas will be presented and their contdbufion to the comprehension of the paramyxovirus replication and transcription processes bovine t-cells that become infected with the intracellular parasite theileria parva undergo lymphoblastoid transformation and acquire the ability to proliferate continuously. they cease to require specific antigenic stimulation, become independent of exogenous growth factors and cause tumors in nude mice. the il2 and il2r genes are constitutively expressed and the transcriptional activator nfwd3 which regulates these two genes is continuously activated. these processes are all strictly dependent on the presence of the parasite in the host cell cytoplasm and cease upon the specific killing of the parasite by the theilericidal drug bw720c. we have shown that the presence of the parasite results in the activation of the protein tyrosine kinases fyn and ick, which can participate in early t cell receptor signalling, suggesting that the parasite may use this signal transducti0n pathway to induce continuous proliferation. cysteine proteinases of leishmania as targets for chemotherapy. jacques bouvier*t and judy sakanari*. *department of pathology, ucsf school of medicine, san francisco, ca, and tanimal health, ciba-geigy, ch1566 st. aubin, switzerland. the overall goal of the study is to apply the structure-based approach to develop lead compounds and design novel enzyme inhibitors as potential therapeutic agents against leishmaniases. in this regard, we have identified and characterized the cysteine proteinases of leishmania major, and showed that they consist of excellent targets for drug design. we have purified a membrane-boand cysteine pmteinase not previously described in other trypanosomatids or protozoans and obtained amino acid sequence information from the purified enzyme. in addition, we have also characterized and purified the soluble cysteine proteinases of the parasite. consequently, we have isolated two genes encoding the soluble and membrane proteinases. the soluble cysteine proteinase gene occurs in multiple copies of 2.9 kb with flanking regions of 1.2 and 2.2 kb and is localized on one chromosome of approximately 440 kb. the membranebound enzyme gene is 3.1 kb and occurs as a single copy on a chromosome of approximately 1100 kb. a three dimensional computer model of both genes wiu be generated and enable us to scan the vast chemical database for new lead componds. we akeady have identified several cysteine proteinase inhibitors that are effective compounds in killing both promastigote and amstigote stages of the parasite in the micromolar range in vitro without affecting the host cells. dollenn~ier, g., cassinotti, p. and weitz, m., institute for clinical microbiology and irananology, 9000 st .gallen/swit zerland hav is a ~r of the picornaviridae. its rna gencrne rf~y be divided into 3 coding regions (pi, p2 and p3), qhe p3 region contains a protease 3cp ro that generates mature proteins by cis-cleavage of the initial polyprotein precursor. catalytic activity of 3cp r~ has been d6~aonstrated in a vacciniavirus/t7 ~qa-polymerase hybrid expression system. (balouter aided ali~ts of hav 3cp r~ sequence with that of other picornaviruses imply that amino acid residues his-44 and/or h) the complete hav open reading frame, hav specific expression products were identified by radioj_rmmnoprecipitation and lyy sds-page. the analysis with an antibody specific for putative nonstructural protein 2b revealed a 27,5 kda peptide. this is in contrast to its predicted size (12 kda). however, analysis with anti-(putative)2a antiserum confirmed an upstream location of the 2b n-terminus. the new 2bpeptide was also identified in lyeates of hav infected mrc-5 cells. hence, recombinantly expressed hav polyprotein underwent authentic processing and the predicted p2 organization has to be modified. we are interested in the development of animal models for one of the two major pathological changes found in the brains of patients with aizheirnar~s disease, the formation of fleuroitbrillary tangles, a main component of which is abnormally phosphorylaled tau-protaln. (tau itself is a protoin associated with mlcrotubuli.) two fundamental questions may be answered with our models: first of nit we woald like to reproduce the formation of tangles, and secondly we hope to answer the question, whether the formation of these pathological structures is sufficient for the bevalopment of senile dementia ot the alzheimer type (sdat). at present there are only a tow putative candidate genes known which may be in-voned in the hyperphosphor~lation ot tau in v/vo, one of which is the map-kinase erk2. we have cloned this candidate kinase from rat txaln rna via an rt-pcr approach and expressed the cona under,the co.rat of tour different promoters in transgenis mice. these promoters were tested for maximal expression. a similar approach was chosen to express the human taut0 isofonn in the brain ot transganic mice. we have stated to analyze different tau an~ erk2 expressing lines, northern blot analysis has revealed high levels of expression for all transgenes (up to fnetoid {n comparison to endocjenous levels). the neuronal spectficry of the transgenio erk2expression was c~nfirmsd by in situ hybridization analysis. ir~ addition several tauexpi'esalog tines were analyzed in wealernblots to determine the relative amount of htau= protein in the brain. sections of the brain were stained with tau-sp~c~c ardib~iies to identify the neurons expressing tau. from intemrosses of tau-with erk2-expressing lines, we have already identified double-positive mice which we currer~y analyze by immunohistocbemistry and in western nots to examine the phosphorylaiton status of tau. we are well aware of the possibility that only animals trans~enic for both tau and erk 2 might beveiop tang{es, whereas single transgenics mlsht not. pairs of individual neurons were recorded with sharp electrodes or in whole-cell configuratio~ in hippocampal slice cultures of rat. synaptic connections were studied between pre-and postsynaptie cells in ca3 and ca1 hippneampal fields by eliciting single aetinn potential in the presynaptic cell. monosynapfic excitatory connections were highly probable between ca3 and ca1 pyramidal ceils (p=0.76, n=82) with almost no feed-forward inhibition (p=0.01). by contrast, disynaptic ipsps, blocked by cnqx or bieuculline were observed with a very high probability (9--0.6) between ca3 pyramidal cells. monosynaptic excitation was found ha 56% of cases between ca3 neurons (n=43) but only in 27% of cases (n=ll) between two ca1 cells. monosynaptie ipsps with very short lateneies (<lms) and blocked by bicuculline, but not by cnqx, were recorded within area ca3 (5/6) and within cai (2/3), but not between ca3 and cat fields (2/2). probability of transmitlzr release, studied in recordings where failures could be unambiguously distinguished from spontaneous or small psps, was found to be close to 100% at both excitatory and inhibitory synapses. specific inhibitors of presynaptie release at excitatory and hahibitory terminals (i.e. adenosine and opioid peptides, respectively) gradually reduced the anaplitude of the psps indicating that the release at excitatory and inhibitory unitary synapses is multiquantal. the cns of a neonatal opossum, monodelphis domestic& shows profuse growth of fibers across a lesion. as in other mammals, such repair is not seen in adults. experiments have been made to define the age of the animal and stage of development at which repair ceases to occur. the entire cns was removed from animals aged 3-6 days or 11-14 days after birth. the spinal cord was crushed and growth of fibers assessed 5 days later by electrical recording of impulses conducted through the crush. repair after lesions to spinal cords of younger animals occurred in 38% of the trials (45 animals, aged 3-6 days). in nervous systems from older animals (11-14 days after birth) the success rate was low (only 4 out of 38 preparations). similarly, the carbocyanine dye dil labeled numerous fibers in spinal cords of younger animals (12 out of 21), whereas only one preparation out of 15 showed scant outgrowth into lesions in the older animals. our results suggest that there is a critical period for neuronal repair; at about 12 days nerve fibers lose the ability to grow across lesions. it is an advantage that at this later stage the nervous system is still small enough to survive in vitro. features correlated with these changes are oligodendrocyte differentiation and myelin formation. with immunofluorescent staining as well as electron microscopy myelination becomes apparent at 8 days after birth. we are now using time lapse-videomicroscopy to analyze molecular mechanisms that could play a part in promoting or preventing repair. supported by grants to j.g.n from the swiss nationalfonds (31-3626292) and from the internat. res. inst. for paraplegia. i. impulse conduction in axons has integrative functions. the dorsal root ganglion (drq) of the embryonic rat put into culture forms a monolayer and is thus accessible to eonventionalmieroelectrode technique, which allows testing the above hypothesis. we have chosen a dual approach by correlating ext~rimental el~ctrophysiological data from the chlture with results obtained from a compartrhental computer model. ii. the safety factor for conduction was found to be low. thus failures of ap invasion of the drg cell soma could occur at sites of impedance mismatch when a second stimulus felt into the relative refractory period of the first or when the axon was repetitively stimulated. the electrotonic resiuues (e.rs) of the failed aps had discrete amplitude levels, suggesting that the failures were always caused at the same site along the axon. these sites of low safety factor were found to be the branch point in the unipglar drg cell and the entrance of the stem piece into the soma in both cell types, the bipolar as well as the unipolar. a systematic comparison of 15oth cell types showed that the ap conduction through the latter is more reliable.'the length of this stem piece is inversely' correlated to the delay caused at the branch point, as tile electrical load of the soma is more efficiently masked by a long stem piece. the dendrites of motoneurons (and subsequently many other neurons) have been assumed to be electrically passive since the work of coombs, ecc]es and fatt in the 1950's. however, direct evidence has been impossible to come by until recently with the advent of appropriate dyes and equipment for measuring very small and very fast optical signals. we examined the propagation of action potentials, both spontaneous and evoked, in the dendritic trees of spinal cord neurons using a 12 â�¢ 1 2 array of photodiodes and a ccd camera as weft as conventional microelectrode techniques. organotypic slice cultures of embryonic rat spinal cord were stained with a voltage-sensitive dye (di-8-anepps) or a calcium dye (fluo-3) and recordings were made from motoneurons identified by their location and morphology. the results indicate that there is in fact an increase in calcium in the dendrites that accompanies action potentials whether synaptically evoked or evoked by current injection at the soma. spikes in the dendrites up to 1 60 i~m from the soma are much larger than could be explained by models assuming passive dendritic trees. work is currently underway to test the possibility that sodium conductances might contribute to the propagation of action potentials in the dendrites. cat auditory cortex contains multiple cochlear representations in both hemispheres that are interconnected through the corpus callosum (cc). the distribution of auditory callosal axons was studied on the mediosagittal level after injections of biocytin at electrophysiologically identified locations. single injections were done in ai (2 cats), aaf, aii and paf; multiple injections in ai & aaf in one cat. an additional cat received 1 injection in sii. the mediosagittal image of cc was subdivided into 30 segments along a rostro-caudal axis. labelled axons crossing the midline were counted in coronal or horizontal sections. axons from auditory fields were found in the posterior 2/3 and those from the somatosensory cortex in the anterior third of cc. axons from a discrete injection (ca imm in diameter) spread over as much as i/5 of cc. the rostrocaudal distribution of axons at the midline reflected roughly the rostrocaudal position of the field of origin, but apparently not the tonotopic order of a given field. the major efferent projections of substantia nigra pars reticulata (snr) convey information to the thalamus, to the tegmentum, to the superior colliculus, and to the spinal cord. such a range of targets suggests a highly organized activity within snr and this study investigates the temporal organization of spike trains. in the portion of snr between +2.7 and +4.5 mm interaural levels, we recorded 40 single units along 6 electrode tracks in 4 young female wistar rats. most units (n=32) fired in a non-regular poisson process, although only a small number (7/32) was characterized by an average burst size with more than 3 spikes. crosscorrelograrns were computed for 25 pairs of units simultaneously recorded from the same electrode and for 26 pairs simultaneously recorded from distinct electrodes. most crosscorrelograms (27/51) showed a symmetrical peak centered on time zero, which indicates a tendency to synchronous firing. when units were recorded from the same electrode tip we observed that 1/8 to 1/40 of their spikes were synchronous. about half of the significant interactions were characterized by a relatively long duration (>250 ms) and are likely to correspond to a different mechanism of synchronization. these results support the hypothesis that snr cells receive common afferences of two different types and their fine temporally organized activity yields coordinated activity between the target structures. the neuronal microtubule-associated protein map2 binds to microtubules via a domain containing 3 or 4 repeats of an 18 amino acid sequence. we have previously shown that when cultured nonneuronal cells are transfected with map2 their microtubules become stiffer and are then capable of supporting process outgrowth when their cortical actin cytoskeleton is depolymerised. we hypothesise that this stiffening effect of map2 is caused by the 18 amino acid sequences binding to neighbouring tubulin subunits in the walls of the microtubules thus reducing their freedom of movement relative to one another. to investigate this further we have created map2 mutants in which 1 or 2 repeats of the tubulin-binding motif were deleted. these were tested by transfection and expression in cultured cells. the results confirm that the number of repeats affects the stability, stiffness and the cytoplasmic arrangement of cellular microtubules. we were previously able to demonstrate the presence ot kinin-like immunoreactive structures in the mouse brain. in order to characterize them, we have now performed a donble-immunofluorescence study using both polyclonai auti-srudykinin (bk) and mouse monocloual anti-neeron-specificenolase (nse) antibodies. the latter is considered to be a specific neuronal marker. after fixation with bonin-hollande solution, the brains were removed, embedded in paraffin and serially sectioned at 10 am. the sections were incubated with the antibodies, which were visualized by an indirect immunofluorescence technique using fitc for nse and by an avidin-biotin technique using texas red for bk. double-immunofluorescent cells were found in the thalamus ventralis, in the nucleus cechleuris ventralis and in the nucleus mesencephaficns nervi trigemini (v). bk-positive cells in the nucleus principalis v were nse-negative ih spite of their typical neuronal aspect. the other bk-positive structures (pia, ependyma, hnic~es) also were alwa~ nse-negatlve. most of the nsepbsifive bk-negative cells were seen in the cortex. the neuronal nature of many kinin-like immunoreacfive structures in the mouse brain is thus ascertained. in contrast to the amphibian optic nerve (on), on of adult mammals is unable to regenerate after injury. astrocytes disappear from and macrophages accumulate at the lesioned site (blaugrund et al., j. neurocytol, 118, 105, 1992) , we have followed the distribution of astrocytes and microglial cells in xenopus tadpole ons over 10 d after mechanical crush. astrocytes were stained for cytokeratin and vimentin, and microglial cells for griffonia isolectin b4. soon after crush, immunoreactivity for intermediate filament proteins was strongly increased. astroeytic processes extended into the lesion from both proximal and distal stump. at ,5 d, astrocytes had crossed the injured region. in the distal stump, some of them contained vacuoles indicating phagocytic activity. while in the normal on microglial cells were ramified and scarce, 2.5 d after crush they were roundish and vacuolized, and scarce within the lesion but numerous distal to it. at 10 d, their number was decreased and most of them had reacquired ramifications. conclusively, in the tadpole on, astrocytes are virtually never absent from the lesion site but rather extend rapidly into it. astrocytes may thus provide a guiding support for outgrowing axons, microglial cells that are frequent in the distal stump, do not accumulate at the lesion. the behaviour of the two cell types differs profoundly from that in mammals and is likely to be one of the factors that may contribute to successful neuronal regeneration of the amphibian on. ci4mence, j. f. 1, ranieri, j. p.2, aebischer, p.2, and sigrist, h. 1, 1institute of biochemistry, university of berne, ch-3012 berne; 2division autonome de recherche chirurgicale, chuv, ch-1011 lausanne. small peptidic sequences of laminin (yigsr, ikvav) are known to promote attachment and/or neurite outgrowth of various neuronal cell lines (pc12, nb2, ng108-15). new and established heterobifunctional photo crosslinkers were used to immobilize these peptides in a topically defined fashion onto biocompatible surfaces such as hydroxylated fluorinated ethylene propylene (fep-oh) and pely(vinymlcohol) (pva) . n-[m[(3-trifluoromethyl) diazirine-3-yl]phenyl]-4-maleimido-butyramide (mad) was thermochemically linked to the synthetic peptide cdpgyigsr via its n-terminal cysteine, leaving the bioactive part (yigsr) free for cell receptor interaction. mad-cdpgyigsr was radioactively labeled by reductive methylation and purified by reversed phase hplc. patterned (20 and 300 #m) peptide immobilization was attained by photocoupling onto pva and fep-oh and visualised by autoradiography. light-structured biomaterial surfaces with molecularly oriented nerve growth promoting peptides were investigated for spatially controlled neuronal cell attachment and differentiation. it is striking to observe that these inhibitory or stimulatory effects were corroborated by binduced decrease or increase in 2-deoxyglucose uptake, respectively. this gives b a putative role in the limbic regulation. the sheet-like processes of oligodendrocytes wrap themselves spirally around central axons, thus formtng the myelin sheath. a single oligodendrocyte may donate internodal segments of myelin to each of 20-30 or more adjacent axons. it is not known, if one oligodendrocyte picks out axons randomly or if it prefers axons with a certain diameter or with a certain chemical specificity. we have studied this last possibility by combining intracellular injection of a fiuorochrome, and immunohistological detection of calciumbinding proteins (cabp's), markers for the axons ef certain subpopulafions of nerve ceils. lucifer yellow was injected into oligodendrocytes of the optic nerve, of living rat brain slices. the oligodendrocytes were identified by their electrophysiologieal properties. slices containing the iniected cells were immunostained /or parvalbumin or calretinin using second antibodies labelled with texas red. using co.focal laser-scanning microscopy combined with a three*dimensional reconstruction programm, the relationship between oligodendrocyte processes and axons positive for one of the cabp's was determined. for ultrastructural confirmation, single, lucifer-yellow injected, oligodendrocytes were photoconverted, and the cabp's-positive axor~ were revealed by gold-labelled antibodies. the few oligodendrocytes injected show the classic morphology, that is of a small cell body and few processes running parallel to the course of the axons. the proximity between glial processes and positive axons can be easily appreciated in confocal laser-scanning images. a preferential assodation of oligodendrocyte processes with parvatbumin-positive axons has not as yet been found. thus, the confirmation of the hypothesis that oligodendrocytes choose their axonal targets according to chemical cues awaits further work including the injection of a lar~er number of these cells. to initiate a molecular genetic analysis of brain development in insects, we are characterizing the mechanisms of embryonic brain development in the fly drosophila. early in embryogenesis, a small number of molecularly diverse brain neuroblasts generates the neurons of the brain. subsequently, pioneering brain neurons initiate axonal outgrowth along glialbound brain compartments. these pioneering neurons establish the primary brain tracts. the pioneering brain axons express the cell-cell adhesion molecule fasciclin i dynamically during outgrowth and initial fasciculation; a subset of the pioneering axons expresses fasciclin ii. the axonal framework of the embryonic brain tracts is used for outgrowth and fasciculation by subsequently differentiating axons and, thus, prefigures the tracts of the mature brain. a comparison of early brain development in drosophila and in vertebrates reveals common mechanisms for brain development. supported by the swiss nsf. institute ot histology, university of fribourg, p~rolles, ch-1700 fribourg calretinin (cr), an ef-hand ca2+-binding protein, is expressed in cajai-retzlus cells during development of the rat cerebral cortex. these cells are located in the marginal zone and are the ear{iest cortical neurons to be generated. they have been consldered as unusual on account of their morphology and their fate during corticogenesis. the functional significance of cajai-retzius cells and their destiny in adulthood is still controversial. in order to approach these questions we have applied organotypic culturing methods. slices of neocortex from brains of 0-6 days-old rat pups were cultured for 10-21 days applying the interphase technique (stoppini etal, j. neurosci. methods 37, 1991) or the roller-tube technique (g&hwiler, j. neurosci. methods 4, i981) . the fixed cultures were immunolabe0ed with an antibody against cr. the obtained results showed that cr positive structures develop in vitro like in vivo. however, in organotyp[c cultures their distribution corresponded to a more mature stage than the situation on the day of the explantations. many cr-positive cells were seen in layer l they were horizontally oriented or had a pyriform shape. based on their morphology these ceils were identified as cajai-retzius cells. orpositive cells were numerous in layer i1-l[i and showed mostly a bipolar morphology. some muripo[ar cells could also be observed. a fine net of crpositive fibers and puncta was found in layers [ and iv. the demonstration that cajai-retzius celts are detectable in these cultures will allow us to follow the fate of these cells dynamically and [nte~ene in their function by applying exogenous factors. cervical primary afferent input to vestibule-spinal neurones projecting to the dorsal horn: a double labelling study in the rat. wheat germ agglutinin-horseradish peroxidase (wga-hrp) was injected by pressure into cervical spinal ganglia c2 or c3. in the same experiments fluorogold (fg) was iontophorefically injected into the ipsilateral dorsal horn of different cervical spinal cord segments. anterogradely labelled fibers (wga-hrp) were present mainly in the external cuneate nucleus, but also to a lesser extent in the caudal part of the medial and descending vestibular nuclei (mvn, dvn) . the fg injections, restricted to the cervical dorsal horn, revealed retrogradely labelled cells in the central part of the caudal mvn. a double exposure (fluorescent and polarisadon optics) under the microscope showed primary afferent fibers surrounding like baskets retrogradely labelled fg cells. the projection from cervical ganglia cells to the mvn represents a pathway for direct afferent information from neck receptors (in particular muscles) to vestibular nuclei and supplies the mvn with afferent information which could enable the vestibulospinal nenrones, projecting to the dorsal horn, to influence the local information processing. in the sciatic nerve, all myelinated and non-myelinated axons were snap-ir. in peripheral tissues, all nerve endings were positive. the preterminal schwann cells of motor axons were also snap-25-ir. after sciatic nerve section, many myelinating sehwann cells also expressed snap-25-ir. in conclusion, snap-25 is expressed by neurons in the pns. it seems to be also expressed by preterminal schwann cells and by myelinating schwann cells during regeneration. $17-19 repair of completely transected spinal cord of neonatal opossum in culture h.a. vischer, dept. pharmacology, 8iocenter, uni. of basel, switzerland woodward et al. (j. exp. biol. 1993 , 1993 have shown that fibers grow rapidly and profusely across a lesion made by crushing the spinal cord of the neonatal opossum monodelphis domestica. in such experiments careful controls must be made to establish that fibers were in fact broken by the lesion. tests have now been made to determine whether similar repair can occur after a more drastic lesion involving complete transection of the cord. after dissection of the entire gns from animals aged 3-8 days, the cord was cut into two with scissors at the c5 -c7 level. the two-ends were glued together with matrigel on a sylgard mold, which encompassed and held the cns. the rostral end was labeled with the fluorescent carbocyanine dye dil in order to visualize growing fibers. in 33 preparations fibers grew over the cut into the separated part of the spinal cord. such growth could extend to 500 ixm. fibers grew straight, branched and multidirectionally, or in fascicles. in another set of experiments cords were combined from different animals of the same or different ages. again, fibers could grow across the cut but they did so less frequently. these results set the stage for investigating fiber growth after rotating the two ends at a defined angle and for analyzing factors that influence the direction of growth. map la is a microtubule associated protein of 360 kd. in rat brain two monoclonal antibodies, a and bw6, recognized map la in neurons and their axonal and dendritic processes. in mouse cerebellum, monoclonal a recognized purkinje ceils and granule cells uniformly, as already described for rat brain. in contrast, monoclonal bw6 stained selectively some dendrites of purkinje cells, forming bands in the molecular layer. we compared this striation with that obtained with a monoclonal antibody, anti-zebrin ii, which recognized aldolase c. in the mouse vermis, zebrin stained in a striated pattern, but complementary to bw6. these results demonstrate that map la is present in forms which are differentially distributed in the mouse cerebellum. these observations may be explained either by differences in metabolic states of neurons, or by differences in their regional functions, or by differences in the regional stability of microtubules. this work was supported by grant no 31-33447.92 from the swiss national science foundation. serum free aggregating brain cell cultures prepared from 16 day old fetal rat telencephalon are used as a model to study brain development. in these cultures it is possible to distinguish ontogenetic events such as cell proliferation, differentiation and myelination, in the present study we examined synaptogenesis by analyzing the expression of synaptic proteins such as synaptophysin, snap-25, synapsin i, and brain spectrin. different developmental stages were analyzed by western blotting. immunoreactivity for synaptic proteins was detectable already at day 12 in culture, suggesting that the first synapses are already present at this early stage. the expression of synaptic proteins strongly increased between day 20 and day 30 in culture, reflecting a period of intense synaptogenesis. treatment of the culture with an elevated concentration of kci (30mm) increased the expression of synaptic proteins, suggesting a stimulation of synaptogenesis. these findings demonstrate the utility of this approach to study brain synaptogenesis, and possibly synaptic plasticity. drenhaus, u.i gunten, a.v., rager, g. ; institute of anatomy, university of ch-1700 freiburg the retina of tupaia comprises three types of ganglion cells (rgcs) analogous to x-, y-, and w-cells found in other mammals. these cells differ in size and in their distribution pattern: large rgcs are frequent in temporal, small rgcs in nasal retina. as the fiber diameter correlates with the size of its respective rgc, it can serve as a parameter for the topographic representation of rgcs in the nerve. to study this we analyzed the optic nerves of three tupaia. using the electron microscope we recorded the density, diameter, and the position of fibers in the nerves. we found, that fibers with the largest diameter are located dorso-temporally and close to the center of the nerve. they are surrounded by zones of smaller fibers. the diameter decreases gradually towards the periphery and is smallest in the ventro-nasal region of the nerve. since the fiber diameter distribution corresponds to that of the size of rgc-types in the retina, we assume that the topographic relationships in the nerve and the retina are similar. (supported by s 17-23 stoppini luc, s. duport, l. parisl, c. oropes~, departement of pharmacology, cmu, 1211 geneva 4 the micro environment of the central nervous system is important for neuronal function. in this context, the blood-brain (bbb) provides and maintains the extracellular medium that is compatible with normal neuronal and synaptic activity. due to the difficulties inherent using whole animal as an experimental model to study permeability and metabolic processes at the cellular level, major efforts have been engaged in recent years, in order to bring up suitable /n vitro models. we are presently developping an in vitro bbb by interfacing a coculture of endothelial cells monolayem and nervous organotypic cultures. the main idea of this study is based on the premise that the complex intercellular interactions between the different cell types pertaining to the nervous tissue and the neighbeurlng endothelial cell is of fundamental importance to promote a realistic bbb system. we expect that erganotypic culture of nervous tissue, combined to endothelial cell monolayers, will initiate and maintain a bbb phenomena/n vilro. we will test more specifically the hypothesis that neuronal activities [spontaneous or elicited) will influence gila] cell response which will in turn modify endothelial properties. preliminary results clearely show a good nervous tissue and endothelial cell survival. tight junction llke structures could be identified using freezefracture or conventional transmission electron microscopic thechniques. {work supported by chemodyne gent~ve ). the process of myelination is a prerequisite for the proper function of the brain since it enables rapid saltatory conduction of axons. in the central nervous system (cns) myelination is performed by oligodendrocytes. a differential screening approach was used to isolate new rat cdna clones that are expressed in this glial cell type. here we report the further characterization of two of these novel cdnas which appear to be expressed specifically in oligodendrocytes. the two characterized cdna clones (tentatively called cns1 and cop-25.1) share an approximately 420 bp region of sequence identity which suggests that they are dedved from the same gene by alternative splicing, within this area a putative open reading frame is located. we demonstrated by northern blot analysis and in situ hybridization that the two mrnas of the novel gene are expressed exclusively in oligodendrocytes. however, the mrnas show a different specific spatial localization within the cell. we compared mrna expression of myelin basic protein (mbp) and proteolipid protein (plp), with that of cns1 and cop-25.1 in brain and optic nerve during development. it appeared that the mrnas of the novel gene were delayed significantly as compared to mbp and plp mrnas. streit. phyaiologisches institut, btihlplatz 5, 3012 bern in organotypic slice cultures of the embryonic spinal cord, rhythmic spontaneous activity arises when the inhibitory synaptie transmission is blocked. the rhythmic activity consists of bursts of regular oscillations of activity at 4 -5 hz. this study investigates the origin of the regular oscillations. when stimulated electrically at 5hz, the synaptic transmission in the cultures showed strong depression by about 50% on average. the synaptie depression was highly variable among individual connections and tended do be less pronounced in frequently used connections. when random depression ratios with an average of 50% at 5hz were implemented in a computer model consisting of 100 randomly connected excitatory cells, regular oscillations of activity did not arise, even in the presence of synchronous pacemaker cells. however, when individual depression ratios were adapted according to the rate of activity of individual connections, regular oscillations at 4 -5 hz arose in the presence of few unsynchronized pacemakers. this finding suggests that the regular oscillations in the cultures originate in a network formed by the plasticity of synaptic depression. maier a., schlumpf m., beer h.f., sehubiger p.a. and lichtensteiger w., inst. of pharmacology, univ. of zurich, the ontogeny of expression of dopamine d 1 receptor mrna in the rat brain and binding to the receptor was studied at 12 time points from gestational day (gd) 13 to postnatal day (pn) 60 by quantitative receptor autoradiography and in situ hybridization.long evans rat pups and fetuses of time pregnant rats were frozen and sectioned coronally and sagitally at i0 um. d 1 recepto~o~inding , determined with the selective antagonist ~ji-sch 23982 was first noted on gd 18 in the developing striatum, basal ganglia and the olfactory tubercle, reaching adult levels at around pn 14. the expression of d i receptor ~na was studied by using a mixture of 3-~js-labelled oligonu-specific cleotides. on gd 13 messages were noted in the developing stiatum, olfactory tubercle and retina. this study shows a good regional correlation between the development of receptor binding and mrna, however, mrna precedes detectable binding to the receptor by several days. earlier work had shown that q~ replicase forms complexes with q~, plus strand rna via interactions at two internal binding sites, the s-and the m-site, while binding of the 3'-end is mediated by host factor. in contrast, the 3'-end of the minus strand appeared to be directly accessible by replicase. we have prepared a series of plus and minus strand variant rnas containing either internal deletions or terminal deletions extending from the 5'-end. the template activities of the variants were determined by single-round replication assays. for the plus strand we find that while deletion of the s-site remains without effect (as expected from previous results), deletion of the m-site reduces template activity to 25 % or less compared to wild-type rna; the residual activity shows a decreased dependence on the presence of host factor. the results agree with an important role of the m-site interaction both with replicase and with host factor for the formation of productive initiation complexes. the template activity of the minus strand was unexpectedly found to be strongly dependent on the presence of a segment between nt 76 and 210 from the 5'-end. this shows that recognition of the minus strand by rep(icase does not only involve interactions near the 3'-end but requires a previously unknown structural feature near the 5'-end of this template. $18-02 karsten graning and angela kr&mer d~partement de biologie cellulaire, universite de gen~ve, 30, quai emest-ansermet, 1211 gen~ve 4 splicing of nuclear pre-mrnas takes place within multicomponent complexes (spliceosomes) that are assembled by interactions between the pre-mrna, snrnps and protein splicing factors. we have purified two splicing factors that function in the formation of the pre-splicing complex. sf3a consist of three subunits of 60, 66 and 120 kda and corresponds to three proteins present in the functional 17s but not in the 12s u2 snrnp. it binds to the 12s u2 snrnp only in the presence of sf3b, suggesting a two step assembly pathway of 17s u2snrnp: binding of sf3b to the 12s u2 snrnp generates a 15s intermediate particle that is converted to the active 17s u2 snrnp by the subsequent association of sf3a. comparison of proteins enriched in the sf3b fraction with polypeptides found in the purified 15s u2 snrnp suggests that sf3b comprises at least five of the other 17s u2 snrnp specific proteins and together with sf3a promotes the u2 snrnp/pre-mrna interaction. by edna cloning, two of the subunits of sf3a have been identified as homologs of well characterized yeast proteins that function at the same stage of spliceosome assembly in yeast. splicing factor sf1, a heat-stable 75-kd protein, functions early during spliceosome assembly. tryptic peptides of sf1 were sequenced and cdna libraries were screened with degenerate oligonucleotides. the information obtained by comparing the sequences of three overlapping cdnas suggests the existence of at least three mrnas that could be derived from a common pre-mrna by alternative splicing. in agreement with this assumption mrnas of the expected sizes that are differentially expressed depending on cell line or tissue are detected by northern blotting. in theory, three polypeptides could be generated that differ by the presence or absence of 7 internal amino acids and in their ctermini. the deduced amino acid sequence is rich in prolines and contains several possible phosphorylation sites and a putative leucine zipper. proline-rich domains, which are also present in splicing factors psf and sap62, as well as leucine zippers have been found in transcription factors and could mediate protein/protein contacts, suggesting that sf1 could function during splicing by interaction with other spliceesomal proteins. pre-mrna splicing is catalyzed by a multicomponent complex (the spliceosome) that consists of small nuclear ribonucleoprotein particles (snrnps) and non-snrnp protein factors. the spliceosome is assembled in a stepwise fashion on the pre-mrna. chromatographic fractionation of hela cell nuclear extracts and subsequent reconstitution of splicing in vitro has allowed the separation and isolation of snrnps and several protein factors. sf4 triggers the transition between splicing complex b, which contains all spliceosomal snrnps but is not competent for catalysis, and complex c, the active spliceosome. this transition involves a conformational change that leads to altered base pairing interactions between snrnas and pre-mrna. the purification of sf4 has been impeded by its low abundance; however, a correlation between sf4 activity and a polypeptide of 50 kd could be established in the most purified fractions. we are currently investigating whether this protein represents sf4. interestingly an atp-dependent rna-helicase cofractionates with sf4 through several purification steps. whether or not this activity is relevant for the splicing process is under investigation. selection of iron-responsive element rnas with high affinity for iron regulatory factor henderson, b.r., menotti, e., bonnard, c. , and kith_n, l.c., isrec, ch-i066 epalinges iron regulatory factor (irf) post-transcriptionally regulates iron homeostasis via binding to mrna iron-responsive elements (ires). the ire loop sequence (5'-cagugn-3') and "bulge" cytosine are phylogenetically conserved. we prepared a pool of 16,384 ire molecules randomized at these 7 nucleotide positions, and employed in vitro selection to identify optimal sequences which bind human irf. we define two major classes of high affinity rna ligand, the optimal loop sequences of each are 5'-cagugn-3' (wild-type) and 5'-uaguan-3'. this novel finding predicts base-pairing within the loop between positions i and 5. all nucleotide substitutions in the "bulge" or at loop positions 2,3 and 4 decreased binding by 36% to 99%. in addition, binding specificity of rat irf differed with that of the related rat ire-binding protein, irf b. in vitro analysis of ire-like stem-loops identified by computer search has not yet revealed any new ire-containing genes. a73 $18-08 3' processing of histone pre-mrnas: a phylogenetic comparison reto kohler, petra duda and daniel schiimperli. zoologisehes institut der universtit/it bern, abteihing fiir entwicldungsbiologie, baltzerstr. 4, ch-3012 bern, switzerland. we are using a phylogenetic approach to study the biochemical reaction by which animal histone pm-mrnas are processed at their 3' ends. in mammals, the efficiency and specificity of this reaction is known to depend absolutely on the u7 snrna which interacts with conserved spacer sequences downstream of the proc~sing site. a highly conserved hairpin element which precedes the cleavage site serves as a binding site for an additional processing factor, but its importanea for efficient processing varies greatly between different historic genes and between extracts of different mammalian cell lines. to determine whether the relative importance of the hairpin and spacer dements have been conserved during evolution, we analysed the processing of histone genes from three different animal phyla in vitro, i.e. vertebrates (mouse), arthropods (drosophila melanogaster) and nematodes (c. elegans). in the mouse system, processing was strongly dependent on the presence of a mouse spacer element. in nuclear extracts of drosophila ke cells, processing occurred exclusively when a drosophila spacer element was present in the rna. processing efficiency was not reduced by foreign (mouse, c. elegans) hairpins. in whole cell extracts of ascaris lumbricoides embryos, an inverted situation was observed. 3' processing products were only produced by rnas that included an upstream segment derived from a c. elegans histone gene, irrespective of the spacer sequences present. these surprising results correlate with certain unique features in the c. elegans upstream element. we are currently in the process of cloning ascaris lumbricoides histone genes, which will allow us to verify our results in a homologous system. in xenopus laevls ooeytes, wild type mouse u7 rna gets assembled into functional u7 snrnps, both when transcr~ed from an injected gene or when injected as/n vitro synthesized rna. if the sm binding site of u7 rna is converted into the canonical sm sequence (u7 sm opt), the rna assembles into a particle which accumulates more efficiently in the nucleus but wbach is nonfunctional. this u7 sm off particle inhibits the function of preformed endogenous u7 snrnps, most likely by nonproductive binding to histone pre-mrnas, histone pre-mrna processing can be demonstrated in c/s if u7 rna is placed 3' of hlstone pre-mrna and injected into oocytes. only u7 sm wt sequences are active in this c/s processing. three proteins can be photoaffinity cross]inked to u7 sm wt rna, the common snrnp proteins g and b/b' and a u7-specific protein of 40 kd (50 kd in mouse). these crosstinks map to closely spaced positions within the sm binding site with the u7-specific crosslink being located most 3'. u7 sm opt rna does not become crosslinked to the u7specific protein and is also more tightly associated with sm proteins. we now investigate the in vitro assembly of u7 snrnps using these characterized u7 rnas and a preparation of highly enriched snrnp proteins. u7 sm wt and u7 sm opt rnas associate with common sm proteins in this system, but the interaction with the u7-speciflc protein could not be demonstrated. the double-stranded (ds)rna modifying enzyme, which can convert adenine to inosine, was first identified in xenopus laevis, but has since then been detected in different species and in mammalian cells. the enzyme is a specific adenine deaminase which uses dsrna as substrate and recently, it has been postulated to be responsible for a specific mrna editing reaction. we have purified this enzyme to homogeneity, approximately 400,000 fold from calf thymus whole cell extracts. the protein was purified primarily by ion exchange chromatography over six columns, with the final step being chromatography on a dsrna affinity column. the purified protein has a molecular weight of 115 kd and is the only protein present when enzymatically active fractions are visualized on an sds-polyacrylamide gel stained with silver. the dsrna modifying enzyme is a very low abundant protein. we are in the process of further characterizing the purified enzyme and of cloning cdnas coding for it. detailed mutational analysis of histone rna 3' end formation carmen spycher, andr6 furger, adrian streit, daniel albrecht, d. schiimperli, zoologlsehes iustitut der universtit/it bern, abteilung thr entwicklungsbiologie. baltzerstr. 4, ch-3012 bern, switzerland histone rna 3' ends are formed by cndonucleolytic cleavage resulting in poly(a)" mrnas with a highly conserved 3'-terminal hairpin loop structure. for the mouse h4-12 gene major and minor processing sites are located 3 and 5 nucleotides downstream of the hairpin, respectively. for the cleavage reaction, the u7 snrnp interacts with the spacer element of histone pre-mrna by base-pairing through the 5' end of its rna moiety, u7 rna. we have observed that this base-pairing potential extends further than previously recognised in either direction. we have made site-directed rnutagenesis in fltis potential hybrid region and around the processing site. rna processing and complex formation with the u7 surnp were analysed by incubation in nuclear extract from mouse k 21 cells. our results indicate that base-pairing with u7 rna both in the classical spacer element and downstream of it are very important for histone rna processing. in contrast, sequences upstream of the spacer element do not seem to be involved in base-pairing with u7 rna, but mutations in this region may affect processing by other mechanisms. systematic mutations of the highly conserved four nucleofides immediately following the hairpin show no qualitative or quantitative effects in the processing reaction. alteration of nucleotides 5 to 7 around the minor processing site, however, result in a dramatic decrease in processing efficiency and alter the specificity of the cleavage site. in further experiments we will introduce specific nuclcetidc modifications at the two processing sites, which should allow us to characterize potential cleavage factors by chemical crosslinking. evolution callaerts p., glardon s., j.,oosli f., quiring r. and gehring w. cell biology, biozentmm, basel. the pax genes encode a family of dna binding factors which share the paired-box and play an important role in the control of development. they can be subdivided into four different classes which differ in the presence or absence of other conserved sequence motifs coding for a paired type homeobox or an octapeptide. the pax-6 gene, which contains a paired-box and a homeobox, has been cloned from humans, mice, rats and zebra fish. recently, we have cloned the pax-6 homologue of drosophila melanogaster. the five genes share a very high degree of sequence identity both in the paired-domain and the homeodomain. similar to its mammalian counterparts, the drosophila gene is expressed in the eye primordia and the nervous system. in addition, mutations in pax-6 affect eye development: aniridia in humans, small eye in mouse and rat, and probably eyeless in drosophila. this would imply that the pax-6 homologues share a conserved function in eye morphogeuesis in both vertebrates and invertebrates. furthermore, this suggests that pax-6 may have been present in a common ancestor. therefore, we are now trying to isolate pax-6 homologues from other, more primitive organisms by means of the polymerase chain reaction, and to determine whether they are implicated in eye development. putative candidates have been identified from a number of species and are being characterized. their sequence similarity and some evolutionary implications will be discussed. keegan liam and gehrin~ walter j., biozentrum, university of basel, klingelbergstr. 70, ch-4056 basel, switzerland using an enhancer map screen we have identified two genes that may be direct targets of antennapedia involved in forming the adult leg. spalt major is repressed in the leg imaginal disc and tea, shirt is activated by antennapedia. we wish to determine whether the control of these genes by antennapedia is direct, spalt major is expressed in a ring in the antennal disc and is repressed by antennapedia in the leg disc. the antennal ring is repressed by ectopie antennapedia expression that transforms the antenna to a leg. we are defining an enhancer at spalt major that is required for antennal ring expression and repression by antennapedia. we are using various approaches to show that antennapedia binds to the antennal ring enhancer at spalt major. these include polytene chromosome banding studies using antibodies to antennapedia as well as in vitro dna-binding and genetic experiments. in search for novel regulators potentially involved in myogenesis, we identified a homeobox of the paired type in human muscle. subsequent cdna cloning revealed that we had cloned the full length coding region of human pax7. our human sequence extends the known mouse edna both on the 5' and the 3' end. as a first step in order to test for a functional role of pax7 in myogenesis, we analyzed its expression pattern during chicken development. transcripts are present in the neural tube and in the dermamyotome of the developing somites. therefore, the pattern of expression of pax7 is conserved between mouse and chicken. next, we assessed the expression of pax7 in different mouse and human cell lines. we found pax7 transcripts specifically in myogenic cells and not in any other cell types. interestingly, pax7 is already present at the myoblast stage. moreover, 10ti/2 fibroblasts converted to myoblasts by either transfection of myod or treatment with 5-azacytidine expressed pax7, whereas the parental cells did not. while myod was able to activate pax7 in 10t1/2 cells, expression of pax7 was not sufficient to induce the myogenic phenotype. nevertheless, our expression data are consistent with a role of pax7 during the mesodermal commitment to the myogenic lineage. the segmental organization of the drosophila head is achieved by a flow of positional information from maternal gene products to the zygotic gap genes. recent genetic analysis has identified three new gap genes involved in head development. one of these genes is the empty spiracles (ems) gene. mutations in eras cause severe defects in the head and the filzk~rper at the posterior end are missing. the eros gene encodes a putative transcription factor with a homeodomain. the eros rna is expressed in two phases of embryonic development. first expression is seen at the blastoderm stage in a single anterior band, correlating with its function as an anterior gap gene. later during embryogenesis eros is expressed in the posterior spiracles as well as lateral regions of each segment where the tracheal pits form and lateral neuroblasts originate. using g-gal fusions we could identify at least five different regulatory elements in the eros promotor region responsible for tissue specific expression of the gene. a 300 bp element responsible for the early expression which is dependent on the maternal gene bicoid, the key gene of the anterior system, and the terminal gene tailless was identified and studied in detail. this element contains two bicoid and two tailless binding sites. the effects of muations in these binding sites on eros expression will be discussed.this analysis should allow us to elucidate the molecular mechanisms by which the morphogen bicoid regulates subordinate target genes like ems in the drosophila embryo. the pax-6 gene of the mouse encodes a transcription factor with both a paired-and a homeodomain. pax-6 is affected by several allelic mutations in the small eye (sey) gene. sey mutations cause a reduction of the eyes in heterozygotes, and homozygotes lack both eyes and nasal openings and die as newborns. the corresponding mutation aniridia in humans affects eye and cranlofacial development in a similar manner. we have isolated a pax-6 homolog from drosophila (dpax-6), which shares more than 90% sequence identity in the paired-and the homeodomain with the mammalian genes. also outside of these domains we find considerable sequence conservation arguing for a true pax-6 orthologue. the drosophila gene spans ~16 kb and is expressed in the brain and the ventral nervous system of the embryo, and in the eye imagjnal discs and the brain of the larva. the gene was mapped to the eyeless (ey) locus. mutations at the ey locus cause either the partial or complete absence of the compound eyes or embryonic lethality. in one ey allele we have identified a transposable element in the first intron of dpax-6, which affects dpax-6 transcription in the eye discs, suggesting that dpax-6 is encoded by the ey gene. this implies that pax-6 (sey) in the mouse is homologous to ey in drosophila. thus, despite of the different modes of eye morphogenesis, the same gene seems to control eye development in insects and vertebrates. the drosophila homolog of the vertebrate serum response factor (srf) was isolated by low stringency-hybridization. nucleotide sequence analysis revealed that the drosophila srf homolog (dsrf) codes for a protein which displays 93% of sequence identity with human srf in the mads domain, the region required for dna binding, dimerization and interaction with accessory factors. the dsrf gene is expressed during several phases of embryonic development. both the rna and the protein are maternally provided to the egg and slowly decrease in their levels during gastrulation. after germ band retraction, high levels of zygotic expression were observed in a distinct subset of peripheral tracheal cells distributed throughout the embryo. many of these cells are at the tip of tracheal branches and are in direct contact with the target tissues these branches tracheate. the dsrf gene was mapped to position 60c on the second chromosome, and overlapping deficiencies which remove the gene were identified. analysis of tracheal development in embryos carrying these deletions revealed a degeneration of most of the major branches of the tracheal system. although the initial migration of tracheal cells was not affected in those deficiency embryos, many tracheal cells appeared not to maintain their formerly correct position and continued to migrate. thus, the dsrf gene might play a role in the proper formation and maintenance of the major branches of the trachea. s 19-08 our experiments would be expected to provide insight into such problems as the evolution and function of transcription factors with multiple dna binding domains. ideally, we would be able to mimic "gene splitting" which occurred during evolution. the poan gene plays an important role during drosophila neurogenesis. it is expressed in specific subsets of sensory mother ceils (smcs) and neuroblasts. the smcs that express poxn differentiate into polyinnervated external sense (p-es) organs. in the absence ofpoxn, smcs differentiate into mono-innervated rather than poly-innervated external sense organs. as the poxn gene contains a paired-box, it probably encodes a transcription factor. since the function ofpoxn in the central nervous system is not known and no po~n mutants are available, an ems mutagenesis screen was initiated to isolate such mutants. based on the assumption thatpoxn null alleles are lethal, we screened for lethals uncovered by the deficiency df(2r)wmg, which includes poxn, but survive over the deficiencies df(2r)xte-18 and df(2r)kl-32 flankingpoxn. in a further test, such lethals are screened for the absence of embryonic p-es organs. ifpoxn mutants are not lethal, they will escape this screen. therefore, we are inducing local hopping of p-elements that have inserted in the vicinity of poxn. the offspring of flies that display an altered eye phenotype are screened for p-element insertion into poxn. we hope that these approaches will generate mutants that will be crucial for future studies ofpoxn function in neurogenesis. chromatin structures and transcription of rdna in yeast saccharomyces eerevisiae reinhard dammaun, renzo lucchini, thee keller and jest m. sogo; institute of cell biology, eth-honggerberg, ch-8093 ziirich the chromatin structure of yeast ribosomal dna was analyzed in rive by cross-linking intact cells with psoralen. we found that in exponentially growing cultures the regions coding for the 35s rrna precursor fall into two distinct classes. one class was highly accessible to psoralen and associated with nascent rnas, characteristic for transcriptionally active rrna genes devoid of nucleosomes, whereas the other class showed a cross-linking pattern indistinguishable from that of bulk chromatin and was interpreted to represent the inactive rrna gene copies. by cross-linking the same strain growing in complex or minimal medium, we have shown that yeast ceils can modulate the proportion of active (non-nueleosomal) and inactive (nucleosomal) rrna gene copies in response to variations in environmental conditions which suggests that yeast can regulate rrna synthesis by varying the number of active gene copies, in contrast to the vertebrate ceils studied so far. whereas intergenie spacers flanking inactive rrna gene copies are packaged in a regular uucleosomal array, spacers flanking active genes show an unusual cross-linking pattern suggesting a complex interaction of regulatory factors and histories with dna. $20-04 r. felleisen & b. gottstein; institute of parasitology, university bern, bern, switzerland most patients suffering from alveolar echinococcosis (ae) respond to infection with a marked igg synthesis directed against e.multilocularis metacestode antigen ii/3, which represents a novel member of the family of cytovillin related proteins. although the respective gene basically is also present and expressed in e.granulosus, most of the cystic echinococcosis (ce) patients do not recognize the antigen. this phenomenon was tackled by generating cdna derived from full length ii/3 genes from both echinococcus species, performed by rt-pcr. sequence analysis revealed a very high degree of conservation of the primary sequence (98.6% homology), cdna fragments were expressed as recombinant proteins and were comparatively assessed in elisa respective to antibody-binding characteristics. sera from patients suffering from ce were showing no significant differences in reactivity with the antigens derived from both species. therefore, parameters others than minor differences in the primary sequence seem to be responsible for the lack of antigen recognition with respect to ce. $20-02 patrick rigoni, sandro rusconi, institut f molekularbiologie h der universitat zarich, winterthurerstr. 190, 8057 z~rich, switzerland. trinuclcotide repeats are often found in the coding sequence of transcriptional regulators in both mammals and yeast, however, their function is yet unclear and data collected in our laboratory on the gheocorticoid receptor even suggest that they may not have any, and that their presence is probably the result of a selfish replicative behavior. nonetheless, we believe that these motifs can be used as tags to identify flexible protein domains typical of modular proteins such as transcription factors. another kind or repeat (coding for a poly-glutamine/alanine) has been discovered in the yeast regulators gall1 and ssn6. from northern blots, we know that such caggcn repeats are present also in higher eukaryotes and we want to isolate them by constructing an enriched edna library using a 5' race protocol. so far, no mammalian protein bearing the motif glrdala has been characterized. among the positive clones we hope to find the homologous or paralogous of gall1 and ssn6 that have escaped so far conventional cloning techniques. meanwhile, we have been testing the effect of coexpressed yeast gall 1 on different reporter-activator combinations in mammalian cells. preliminary results show that galll but not the mutant form galllp can inhibit the activation properties of some (not all) gal4 chimeras. we are also producing antibodies directed against oligo-ala and oligo-gln and oligo-gln/ala motifs in order to better characterize natural proteins containing these repeats. we are interested in the early development of c. elegans, particularly in the contribution of genes whose homologues in vertebrates and d. melanogaster mediate positional infolzzations during development. therefore, we are working on ceh-13 which belongs to a cluster of at least 4 homeobox containing genes. this cluster is considered to be equivalent to the homeotie cluster of drosophila and to the hox clusters in vertebrates. by generating ~-galactosidase transgenie lines, we have observed that ceh-13 is expressed very early during embryogenesis, embryonic expression appears to be restricted to the posterior half of the embryo, which is rather surprising, since the ceh-13 hemologues in drosophila and vertebrates are anterior-specific. during larval stages, ceh-13 is expressed in neuronal cells. these results are now in the process of being confirmed by immunolocalization of the ceh-13 protein product with a polyclonal antiserum. in order to clarify the function of ceh-13 in the c. elegans development, we have isolated a ceh-13 mutant from a tcl insertion library, in collaboration with r. h. a. plasterk from amsterdam. from this worm, which looks wt, we are now trying to obtain a deletion derivative. previous studies have shown that bovine herpesvirus 1 (shv-1) infected cell pratein (bicp) 0 acts -depending on the promoter -as a strong transactivator or as a repressor in transient expression assays. the 81cp0 polypeptide contains a cysteine-rich zinc finger domain (c3hc4) which is conserved in a number of viral and cellular regulatory proteins including the 8icp0 homologs icr3 of herpes simplex virus type 1 and protein 61 of varicella zoster virus. this type of zinc finger (the so-called ring tinge0 was shown to bind zinc ions but functional requirement for zinc has not yet been demonstrated, a gap which we aimed to fill with the present study. transient expression assays were performed in oocytes which had been microinjected with thionein to chelate and deplete the intracellular pool of zinc. 81cp0-induced cat activity of a promoter-cat construct was 72-fold higher than the basal cat activity of the reporter plasmid, in the presence of thionein, bicp0-induced transaotivatlon was reduced 54old. with a set of control experiments we excluded that thionein might affect transcription and protein synthesis in general, to our knowledge this is the first demonstration of zinc-dependence for a member of the ring finger family. we have identified by pcr and edna cloning five pou genes expressed during early embryogenesis of zebrafislx four of these genes show extended homology to the brn-1 class of pou genes. preliminary evidence suggests that the brn-1like pou genes overlap with most of their expression domains.the gene studied in most detail so far (zp-50) is first expressed on the ventral side of the future fore-and midbrain. slightly later, expression is also found in rhombomeres 3 and 5 in the hindbraln. these rbombomeres were identified by the specific expression of the krox-20 marker using a novel in situ hybridization protocol which allows the simultaneous detection of two different transcripts using different color substrates. in the 24 hours old embryo a complex expression pattern is found involving a variety of brain structures and the spinal cord. the distribution of the transcript suggests that zp-50 is mostly expressed in glia cells. another pou gene we have identified (gp-9) defines a novel class of pou proteins. as a maternal mp, na it is initially ubiquitously present. after gastrnlation its transcript is found most notably in the developing hindbrain in rhombomeres 2 and 4 for which so far no good molecular markers were known. we are investigating the roles the identified pou genes may have in zebrafish hindbrain segmentation. we are currently attempting to manipulate gene expression in developing embryos by injection of rna or by the production of transgenic fish. we are also screening the zebrafish genome for new developmental marker genes. progress about these experiments will be presented. $20-07 willimann, t. and trueb, b. to gain insight into the regulatory mechanisms of collagen vi synthesis we have characterized the cis-acting elements of the chicken ctl(vl) collagen promoter. footprinting experiments with nuclear extracts from chicken embryos revealed three distinct elements, designated a, b and c, that were protected from dnase i digestion. the nuclear proteins that interact with the three sites were identified by gel retardation assays in combination with the use of various oligonucleotide competitiors as well as specific antibodies raised against well-characterized transcription factors. site a was found to be a target for transcriptional activator apf, whereas sites b and c were shown to be recognized each by two distinct nuclear proteins which belong to the spl multigene family. to address the question whether the three sites alone are able to direct transcription, a minipromoter construct was created in which the sequences of sites a, b and c were placed in front of a reporter gene. after transfection into chicken fibroblasts, this construct exhibited a high relative promoter activity when compared to a large genomic fragment containing the basic ctl(vi) collagen promoter. thus, the three sites are sufficient to induce transcription of this gene. octamer transcription factors (oct or otf) are a subset of the pou family of transcription factors which regulate expression of cellular and viral genes by binding to the octamer sequence atgcaaat. neurons and astroglial cells harbour, in addition to the ubiquitous oct-i factor, brain specific factors termed n-oct 2, 3, 4 and 5. in the present study we determined the chromosomal localization of the gene encoding the n-oct 3 transcription factor and characterized the structure of isolated n-oct 3 genomie clones. the chromosomal mapping was performed by analysis of somatic cell hybrid panels and radioactive and fluorescence in situ hybridization of human metaphase chromosomes. it is interesting that the 5' end of the n-oct 3 coding sequence contains repetitive cag and ggc residues. several disorders have been discovered to be related to expansion of trinucleotide repeats. we are presently investigating if the n-oct 3 cag or ggc clusters are hot spots for such mutation mechanisms and if so, which diseases could be associated with it. two dna regions upstream of the distal glucocorticoid receptor binding site interact with nuclear proteins that are tissue-specific (region a) or ubiquitous (region c). the characteristics of the dna-protein interactions have been studied in vitro. these sequences, in different combinations with the natural mmtv promoter, were tested in transfection assays of fibrcblast or mammary cell lines. we show that they are able to modulate the level of glucocorticoid-stimulated transcription. the proximal region of the mmtv promoter has binding sites for the transcription factors ctf/nf-i and oct-1. p~asmids with a deletion or mutations in the oct-1 site were tested in stable transfections, that are most representative of the state of proviral dna with respect to both number of integrated dna templates and chromatin organization. we show that the oct-1 site is important for the basal level of promoter activity. the data further indicate that the functional outcome may depend beth on the relative ratio of factors to dna templates and on the relative affinity of binding sites, as determined by oligonucleotide competition footprinting. besides the fact that telomeres represent specialised structures important for chromosome stability, the particular significance of cloned c. elegans telomeres is that they will contribute to the completion of the physical mapping of chromosomes and that they provide one set of elements for the construction of artificial c. elegans chromosomes. the cloning of c. elegans telomeres, however, is impeded by the fact that tandem repeats of the sequence ttaggc are not only located at the ends of the c. elegans chromosomes, but also at many internal chromosomal sites. therefore, we have developed a special protocol for the construction of a c. elegans endlibrary in 2~ zap. the resulting telomeric library was screened for recombinants containing ttaggc repeats and yielded about 70 positive clones. so far, 30 were analysed by partial sequencing and twelve of them satisfy all criteria required for telomeric clones. their ttaggc tandem repeats are located at the expected position, namely just adjacent to the blunt end cloning site in the polylinker. furthermore, the g-rich strand of the repeats is oriented 5' to 3' towards the end of the fragment, thus corresponding exactly to the defined orientation of eukaryotic telomeric sequences. finally, hybridisation data with one of these putative telomeric clones show that a subtelomeric fragment hybridises to bal31-sensitive fragments on a southern blot with c. elegans dna, indicating that this clone represents a c. elegans telomere. $20-09 to investigate whether chromatin structure or transcription can interfere with replication, derivatives of the yeast trp1ars1 minichromosome were constructed that contain either the ded1 or pet56 promoter firing against the origin of replication ars1. while the pet56 constructs transformed yeast at high frequencies and were maintained as high copy number minichromosomes, the ded1 constructs transformed at low frequency and the constructs were integrated in the genome suggesting that ars function was impaired. insertion of the h4-ars, a second origin of replication, rescued a ded1 construct as a minichromosome (yrpdh3).the chromatin structure mapped at low and high resolution of the ars1 region in yrpdh3 was indistinguishable to that of the pet56construct yrpft61. ananlysis of rna showed that transcription was going through ars1 in yrpdh3 and in yrpft61, but the levels of transcripts in yrpdh3 were much higher. we conclude that transcription through are1 interferes with replication and prevents extrachromosomal maintenance. dna sequence of a repeated dna segment on circular and linear plasmids of borrelia burgdorferi w.r. z%ckert and j. meyer, abt. pzmom, zahn&rztliches institut der universit~t basel a plasmid-associated repeated dna segment of b. burgdorferi strain b31 was cloned. at least two copies of this segment appear to be present on the 29 kb circular plasmid, whereas one copy is carried on the 50 kb linear plasntid of this strain. dna sequence analysis revealed a region containing a novel 906 bp putative open reading frame (orfl) on the 50 kb linear plasmid, orfl displayed extensive sequence homology (95%) to putative open reading frames present on the clones obtained from the 29 kb circular plasmid. heterogeneity is mainly caused by 3rd-base-wobbllng. flanking sequences share 85-95% homology, including the 5' end of an additional putative open reading frame irmaediately downstream of orfl. whether orfl and/or the related sequences are being transcribed and yield, in the case of orfl, an approximately 36.5 kd, lysine-rich polypeptide, is yet unknown. the repeated sequence seems to be specific for b. burgdorferi sensu lato and therefore may be useful in nucleic acidbased diagnostics of lyme disease. $20-16 similar to type ib oculocutaneous albinism in humans, overall production of pigment is greatly reduced in dark eyed albino and only obvious in eyes. we have studied the molecular basis of the c 44h mutation and show that expression of the tyrosinese gene is not affected. after sequencing tyrosinase cdna isolated from c44h/c44h homozygotes we uncovered a single base alteration from wild type leading to a serine to isoleucine exchange. the importance of this mutation was demonstrated by generating transgenic mice containing a mutated tyrosinase minigene. this showed that the single base change is sufficient to severely depress pigment production in transgenic mice. we therefore conclude that the point mutation is responsible and sufficient to generate the dark eyed albino phenotype. members of the pou-family of transcription factors are involved in developmental and differentiation processes. using a pcr-based cloning strategy with degenerated oligonucleotides we identified several pougenes from the lactating and involuting mouse mammary gland. three of these cdna clones which contained as yet unknown sequences were chosen for further investigations: clone 5.80 which has a high homology to pit-l, clone 5.54 which is related to the brn-3 gene and clone 5.48 which belongs to the class iii of pou-proteins. the expression levels were analyzed in different mouse organs and in several developmental stages of the mammary gland, including the postlactational process of involution. because of the low abundance of the specific transcripts we used a semiquantitative, reverse transcriptase-mediated pcr assay to investigate the mrna levels of the three novel pou-family members. distinct and specific expression patterns for all three members were obtained in the different investigated organs. interestingly, the expression of the pit-1 related cdna clone 5,80 was elevated in all developmental stages of the lactogenic-hormone dependent mouse mammary gland and in sceletal muscle, whereas its expression was low in other organs. repetitive sequences in dna can allow ectopic (outof-register) homologous recombination, which is often undesirable and can even lead to disease in humans. to prevent this, long homologies should often be interrupted during evolution. accelerated mutation of repetitive sequences by so-called ripping may be one of the mechanisms used to accomplish this in fungi. we are investigating if introns in vertebrate genes have an undiscovered role as interrupters of homology within and/or between genes, in addition to their established role in exon shuffling. by inserting homology-poor introns in an otherwise homology-rich region the genome could prevent ectopic homologous recombination. such a mechanism could be especially useful where there is an advantage in encoding highly repetitive protein sequences. it appears that within the collagen gene family there is indeed a correlation between repetitiveness and intron density. the influence of prenatal diazepam exposume (i,25 mg/kg/d, s.c.) from gestational day 14 to 20 on the development of the ~-opioid binding sites in striaturn, nucleus accumbens and midbrain of pn 14, pn 28 and 8 week old male and female rats was studied. 8 week old prenatally diazepam exposed male rats showed a significant decrease of k-opioid binding sites in the nucleus accumbens. a sex-dependent difference in k-opioid binding site densities could also be detected between pn 28 prenatally diazepam exposed male and female rats. we now investigate by in situ hybridisation whether changes in the level of mrna encoding fo~ prodynorphin, the precttrsor for various endogenous ~-opioid agonists, could be responsible for the decrease of ~-opioid binding sites in the nucleus accumbens of 8 week old prenatally diazepam exposed male ~ats. $21-04 distribution and morphology of nitric oxide-positive neurons in the marmoset cerebral cortex during pre-and postnatal development j.p. hornung* and j. schultz** *institute of anatomy, university of lausanne, 1005 lausanne, and **university of fdbourg, 1700 fribourg nitric oxide, synthesized by the enzyme nitdc oxide synthase (nos), is a neuroactive substance and a limited number of neurons were shown to express this enzyme either by histochemistry or by immunocytochemistry. both techniques were performed on brains of marmosets with ages ranging from 6 weeks before birth to adult. the morphology and distribution of nos-positive neurons were analyzed in all lobes of the cerebral cortex. the same pattern was revealed by histochemistry or immunocytochemistry. three populations of nos-positive neurons were revealed. first, a transient population of neurons in the molecular layer of the embryonic cortex, many having the morphology of the retzjus-cajal cells. a second population was made of large multipolar neurons in the deep layers of the cortex and the underlying white matter, which persisted from embryonic to adult life. a third, permanent, population was made of small, weakly reactive neurons in the supragranular layers appearing postnatally, this study demonstrates that no can exert its actions in the cerebral cortex through several pathways at different developmental stages. trimethyltin (tmt), a well-known neurotoxicant, was used to study the sensitivity of the different brain cell types (i.e. neurons, astrocytes, oligodendrocytes and microglial cells) to a neurotoxic insult. aggregating brain cell cultures of fetal rat telencephalon were treated during 10 days with tmt at different concentrations, ranging from 10 -10 m to 10 -6 m. microglia were found to be the most sensitive cell type, since already at 10-10m of tmt an increased number and clustering of griffonia simplicifolia-positive ceils could be observed. at 10 -8 m of tmt astrocytes showed increased staining for glial fibrillary acidic protein, characteristic of gliosis. neurons and oligodendrocytes appeared to be the least sensitive cells, since a decrease in the activity of cell type-specific enzymes was observed only at 10 -6 m of tmt. these results show that microglial cells and astrocytes respond to a toxic insult before any neuronal changes could be detected, and that the microglial reaction may be the first target of tmt. this reaction could then trigger the astrocytic response. vif is widely distributed in brain with suggested functions related to development. vip expression was studied during rat brain development using hybridization histochemistry with a 48met probe. vip mrna was found in thalamic nuclei on el7, later recognized as the ventrolateral and reticular nuclei after further maturation during prenatal period, vip mrna was found in hypothalamus, especially suprachiasmatic nucleus on el9 and its expression matured over the next days. few cortical neurons contained vip mrna on e21, they continuously increased in number and signal intensity over the perinatal period. vip gradually matured in the first three postnatal weeks and adult-like patterns were found on p 22, when cerebral cortex, ventrolateral and reticular thalamic nuclei, hypothalamus, esp. suprachiasmatic nucleus, were the regions with most prominent vip expression. these results demonstrate the relatively late appearance of vip gene expression in the rat forebrain, as compared with peptides like srif and cck, not suggesting a major role in earlv brain maturation. in primary cultures of mouse cerebral cortical astrocytes, a rapid glycogenolysis followed by a massive glycogen resynthesis ( six-to ten-fold over basal levels after 9 hr) are induced by vasoactive intestinal peptide (vip) or noradrenaline (na). both actions of these neurotransmitters are mediated by camp. since the induction of glycogen resynthesis triggered by vip or na is abolished by inhibition of transcription and translation, we applied the 2-d gel electrophoresis technique to search for astrocytic proteins induced by vip or na. the comparison of 35s-labeled proteins from primary astrocyte cultures treated or not with vip 1 ~tm revealed two newly synthesized proteins: a 36 kda protein (pi=5) and a 23/24 kda protein (pi=6). only the latter is visible on a silver stained 2-d gel, which is a prerequisite to consider its purification and microsequencing. induction of the 23/24 kda protein by vip was time-dependent with a maximum at -12 hr. preliminary results indicate a similar induction by na and isoproterenol. in humans, the central analgesic effect of tramadol (t) is only weakly reversed by the ~t opioid antagonist naloxone (nx) (br j clin pharmacol 1993; 35:73p). t analgesia may thus result from an action on monoaminergic pathways as suggested by in vitro and animal data. we therefore investigated the effect of ct2-adrenoeeptor antagonism by yohimbine (y) on t analgesia. according to a randomised double-blind crossover and placebo (p) controlled design, healthy volunteers (n=10) received t (100mg orally), followed (+ 3 h) by y (0.1mg/kg intravenously), and y + nx (0.8mg intravenously). analgesia was assessed over 8 h by subjective pain threshold (numerical scale -ns-) and objective pain threshold (rill nociceptive reflex -riii-) monitoring. peak analgesic effect was observed at ca. 3.25 h (riii +8.8 +semi.6 ma and ns +8.4 +1.8) vs p (rih -0.3 +1.2 and ns +2.3 +1.6, p<0.05) and lasted ca. 6 h. y reversed t analgesic effects during ca. 1 h (r/ii -2.9 +1.8, p<0.05 and ns +2.8 +1.5, p<0.05), whereas y + nx abolished t effects thoughout the study period (rill -1.9 4-1.1 and ns +0.6 +1.3, p<0.05), y alone tended to lower pain thresholds (rill -4.1 +1.5 and ns -1.9 +1.4, p>0.05 anova). yohimbine, an r antagonist, reverses tramadol effects, thus pointing to the major role of monoaminergic modulation in tramadol anfinociception. a monoclonal antibody (in-l)was raised against neurite growth inhibitory proteins present in rat cns myelin (caroniand schwab, neuron 1: 85, 1988) . in-1 neutralizes the inhibitory ettect of cns tissue in vitro and in vivo, thus permitting regeneration of certain lesioned cns fiber systems. we have used this antibody to immunostain cryostat sections of the nervous system of the rat. we found that in-1 stains white matter and myehnated fiber tracts in the cns. the staining pattern corresponds to that of the known myelin specific antigens mbp (myelin basic protein), mag (myelin associated glycoprotein) and mog (myelin oligodendrocyte glycoprotein). the staining of in-1 in the cns is found in regions of the adult nervous system which have been shown to be inhibitory for axonal growth. interestingly, the regions which show a high abundance of in-1 antigens are low in staining for gap-43, a marker for growth and plasticity in the developing and adult brain. in contrast, gap-43 is strongly expressed in unmyelinated fiber tracts or nuclei. prevention of oligodendrocyte development in rat spinal cord resulted in suppression of in-1 expression and elevated gap-43 levels. this suggests that inhibitory myelin proteins may negatively influence plasticity in the rat cns. to date, the existence of anp and npy in the adrenal medulla has been shown only for a few mammalian and anuran species. thus the present immunohistochemical investigation attempts to localize anp-like and npy-like peptides n the adrena gland of representative mammals, birds, reptiles, amphibia and bony fish by the use of antisera specific for mammalian anp and npy. furthermore, the catecholamine-containing cells were characterized by antisera against enzymes of catecholamine synthesis. anp-and npy-immunoreactivies were detected in adrenal chromaffin cells of all vertebrates studied while no immunoreactivities were observed in the adrenal cortex or in its homolog, the interrenal. the representatives of the different vertebrate classes exhibited marked differences in the proportions of anp-immunoreactive and npy-immunoreactive cells, in the presence of anp-and npy-immunoreactivities in noradrenalineand/or adrenaline-containing cells and in the amount of of cells containing both anp-and npy-immunoreactivities. our results give evidence for a long phylogenetie history of adrenal anp-and npy-like peptides. thus, they stress the physiological impact of these peptides as adrenal paracrine and/or endocrine hormones. human neuronal growth inhibitory factor (gie) is involved in the regulation of neuronal growth and is deficient in the brains of alzheimer's disease victims. this brain-specific metalloprotein consists of 68 amino acids out of which 20 are cys and has been found to contain copper and zinc. proteins with similar primary structure were isolated from bovine and equine brain. the amino acid sequence of these proteins shows about 70 % homology to those of mammalian metallothioneins (mt), with two conserved inserts of 1 thr and a glutamic acid rich hexapeptide in the n-and c-terminal regions, respectively. in contrast to mts, which usually contain only zn(i1), the presence of cu(i) and zn(ii) (6-7 moles total) in all gif isolations suggests the functional importance of both metals ions. to spectroscopically probe the native zn-binding sites, the zn(ii) ions were replaced by cd(ii). both bovine and equine cd/cu-gif derivatives exhibit similar absorption and cd spectra with features characteristic of cd-thiolate clusters. the release of 3h-arachidonic acid evoked by glutamate was characterized in cerebral cortical neurons in primary culture grown under conditions that prevent glial cell proliferation. in these cultures, 99% of the total ceils are immunocytochemicauy characterized as being positive for specific neuronal markers such as neuron-specific enolase and neurofilament. specific markers for astrocytes, oligodendrocytes or microglia were not detected. glutamate induces a concentration-dependent release of 3h-arachidonic acid with a ec50 of 3 pm. the pharmacological profile of this glutamate response shows the involvement of two receptor types: the nmda and the ampa ionotropic receptors. the glutamate-evoked release of 3h-arachidonic acid is phsensitive. when the incubation buffer is alkalinized from 7.25 to 7.65, both basal and glutamate evoked release of 3h-arachidonic acid are enhanced. the glutamate response is also enhanced as intracellular ph is alkalinized by pharmacological treatments: such as inhibition of c1-/hco3-antiport by dids. these results further stress the role of intracellular ph in glutamate-mediated neurotransmission. $21-09 multiceli culture system for the study of drug kinetics wechsler d., *schittny j.c. and honegger u. e., depts. of pharmacology and *anatomy, university of bern, ch-3010 bern a cell culture system was developed for the investigation of cellular drug kinetics in cultures with up to 4 cell types. it was used for the study of uptake, competitive uptake, release and redistribution of drugs. individual cell types (human skin fibroblasts, rat astrocytoma cells (c6) and rat hybfidoma ceils (oligodendrocyte x astrocytoma, roc)) were separately grown to confluency on glass circle sectors. in the same petri dish 4 sectors in various combinations were exposed to media containing radiolabelled chlorpromazine (cpz). single and repetitive uptake and release of cpz were measured in each cell type after individual exposure or exposure in any combination of cell types: in 2 hour competitive uptake studies fibreblasts reached 1.7 and 2.6 times the concentrations of c6-and roc-cells, :respectively. in redistribution experiments the exchange of cpz from preloaded to unloaded cells was tested. the exchange rate was dependent on cell type and loading period. it decreased with increasing time of exposure suggesting the formation of more stable cpz stores. we have previously demonstrated that certain neurotransmitters such as vip, noradrenaline (na) and adenosine promote glycogenolysis in mouse cerebral cortical astrocyte cultures (brain res. 563: 227-233, 1991) . the question that we then addressed was the nature of the metabolic substrate released by astrocytes following glycogenolysis. we therefore determined the presence of various metabolic substrated released from astrocytes under static conditions and in the superfusate of a laminar flow system developed in our laboratory. neither glucose nor any intermediate of the tficarboxylic acid cycle could account for the decrease in glycogen stores; astrocytes however were shown to release great quantities of pyruvate and lactate at a rate of 50 nmol/mg protein/minute. noradrenaline but not vip promotes a significant increase (42%) in lactate release. the effect of noradrenaline is mimicked by isoproterenol and inhibited by propranolol, suggesting a i~adrenergic mediation. when astrocytes are incubated in an isotonic solution containing no energy suhstrate, the glycogen stores are rapidly depleted and lactate, but not glucose, is released in the medium. in conclusion, lactate appears to play a major role in cerebral energy metabolism homeostasis, as substrate released by astrocytes to prey!de energy fuel for neurones and other neighboring cells. it is known so far that calcium-binding proteins (cabps: calbindin, pan, albumin and ca/retinin) are expressed in some cells of the pineal gland of laboratory mammals. the exact phenotype of these cells is unknown. nevertheless, according to recent studies, it seems that some of them are ganglion cells. therefore we studied the pineal gland of different species (gerbils, rats, goats, cows and humans) using calretiain (cr) antibody which is considered as marker for neurons (andressen & al, cell & tissue rss, 27t:181, 1993) , the cr-expressing cells were found in all oar species: in gerbils, rats, cows and humans they were scattered throughout the parenchyrna, whereas in goats they lined mostly the pericapillary spaces. according to our findings, it seems that most of the reactive ceils are rather neuron-like elements since morphological criteria for true neurons (axosomatic synapses, nissl bodies, bundles of neurofilaments) are missing. however one cannot exclude that among these cells some are intrapineal ganglion cells, because of ranvier's nodes found along the processes of some crpositive cells. thus, it is possible that these cells are involved in the modulatory control mechanism of the pineal neurohumoral activity and/er by accumulation of intracellular calcium in the formation of pincal calcifications. besides the excitatory amino acid transmitter glutamate, its sulfur containing analogue homocystcic acid (hca) could play a role in excitatory processes in the central nervous system. hca is present in and released from nervous tissue and is a potent neuronal excitant, predominantly activating n-methyl-d-aspartate receptors (do et al., 1992) . these properties are suggestive of a classic synaptic neurotrausmitter role. on the other hand, hca seems to be localized not in neurones but in glial cells (grandes et al., 1991; tschopp et al. 1992) . the efflux of hca is temporally delayed following activation of specific pathways (klancnik et at., 1992) ; these latter observations are not in accordance with the classic concepts of synaptically-mediated neurotransmission. a laminar flow superfnsian system of monolayer cultures was developed and the released materials from mouse cortical astrocytes, previously preincubated with [35s]-methioulne, were investigated. during application of either noradrenaline or the b-adrenergie agonlst isoproterenol, the extracellalar level of [35s]-methianine was decreased and that of labelled hca was increased. this effect was not observed with the -adrenergic agonist methoxamine, and could be blocked by the ll-adrenergic antagonist propanolol these results, combined with the delayed hca release, the glial localisatian of hca and its neuroactivity, strongly suggest a potential role of hca as a gliotransmitter, modulated by noradrenaline inputs. vasoactive intestinal peptide (vip) induces long lasting metabolic effects in the mouse cns. we have investigated a shorter neuromodulatory action of vip on the synaptic responses in coronal slices of adult mouse cingulate cortex by means of intracelhilar current clamp recordings. electrical stimulation of the underlying white matter evoked a multiphasic synaptic potential consisting of early epsp~ early ipsp, late epsp and late ipsp mediated by non-nmda, nmda, gaba a and gaba b receptors, respectively. vip (0.1 ~d~l) superfused for 5 rain. had no effect, whereas concentrations over 0.2 ixm selectively and reversibly depressed the nmda-medlated responses in 12/16 cells. in the presence of cnqx (10 [tm) and biencuuine (10 ~tm), the isolated nmda-mediated late epsp was still attenuated by 0.3 ~m vip in 8/10 cells (mean:-42% al~er 30 rain.). in 3 other cells vip induced a spreading depression (sd) and a strong, reversals inhibition of the late epsp (mean:-64 % after 10 min.).these results indicate that in the absence of gabaergic inhibition vip can induce two types of effects in the mouse cingulate cortex, a prolonged decrease of the nmdamediated syaaptic response and a striking sd. interaction between vip and glutamate on c-fos mrna expression. |.-l. martin, d. gasser and p.] . magistretti. institut de physiologie, universit4 de lausanne, lausanne, switzerland. the regulation of c-los mrna expression by vip and glutamate was examined in primary cultures of neurons originating from the mouse cerebral cortex. in primary cultures of cortical neurons, vip increases camp levels and stimulates c-los mrna expression. interestingly vip stimulation is completely blocked by mk-801, an nmda receptor antagonist but not by cnqx or ap3, which antagonize ampa/kainate and metabotropic receptors respectively, c-los expression stimulated by glutamate shares the same pharmacology. these results suggest an involvement of endogenously released glutamate in the stimulation of c-fos mrna expression evoked by vip. furthermore, when added together, vip and glutamate interact synergistically to increase cfos mrna levels. consistent with the pharmacology of vip and glutamate, the synergistic interaction between vii' and glutamate on c-fos mrna expression is also inhibited by mk-801. interestingly, this synergism is completely inhibited by h-89, a protein kinase a inhibitor, whereas staurosporin and kn-62 which block protein kinases c and ca++/calmodulin dependent respectively exhibit smaller inhibitory effects. presynaptic proteins involved in neurotransmitter release (i.e. synapsin, b-50) and some postsynaptie gaba a receptor subunits possess phosphorylation sites for camp-dependent protein kinase (pka) and/or protein kinase c (pkc). the functional consequences of phosphorylation by these kinases is not known. we have studied the action of specific activators of pkc and pka, phorbol ester (pdbu) and forskolin, respectively, on gabaergic synaptic transmission in area ca3 of hippocampal slice cultures. pdbu significantly enhanced the amplitude of evoked monosynaptic ipscs (in cnqx and ap5), and both pdbu and forskolin increased the frequency of spontaneous miniature ipscs (m[pscs) in the presence of cnqx, ap5 and ttx. this effect by pdbu was antagonized by staurosporin, a protein kinase inhibitor. pdbu and forskolin did not change the amplitude or decay of mipscs, suggesting that only presynaptic kinases are involved. furthermore, pdbu enhanced mlpsc frequency by a comparable amount in the presence of the ca 2+ channel blocker cd 2+, indicating that pdbu did not enhance gaba release from presynaptic endings by facilitating ca 2+ influx into axon terminals. valiunas, v., sc~ilinsky fluri, g. and weingart, r. arachidonic acid (aa) reversibly impairs the gap junction conductance (g~) in cardiac cells. now, we examined whether aaacts directly or via its catabolites. experiments were performed on pairs of neonatal rat myocytes using a dual voltage-clamp method. we used blockers which interfere with enzymes of various catabolic pathways. pretreatment with i0 ~mpoca (blocks carnitine acyltransferase i) did not prevent the decline in g9 by i0 ~m aa; i.e. intermediates of p-oxidation are not involved. similarly, preexposure to i0 ~m indomethacin (blocks the cyclooxygenase pathway) did not prevent aa from uncoupling; this rules out an involvement of prostaglandins and thromboxanes. exposure to 10 ~mndga (blocks the 5-1ipoxygenase pathway) per se produced a decrease in gj. likewise, exposure to i0 ~m etya (blocks the 15-1ipoxygenase pathway) also impaired gj. these effects cannot result from accumulation of endogenous aa because preexposure to 50 ~m 4bpb (blocks phospholipase a 2) did not prevent ndga-or etya-induced uncoupling. exposure to 75 ~m skf 525-a (blocks the epoxygenase pathway) also produced a decrease in gj. hence, ndga, etya and skf 525-a, compounds with different biochemical actions and of different chemical structure, act on g~ either directly or via an unknown mechanism. $21-19 potential changes associated with electrical activity in nonmyelinated nerve fibres a. robert and p. jirounek d~partement de pharmacologie, cmu, 1211 gen~ve 4 simuheneous measurements of the extracellular concentration of k + ([k+]e), and of the concomitant changes in the membrane potential (era) were measured in the rabbit vagus nerve during and after a period of activity. during a 15 hz stimulation there was a large increase in the [k+]e , accompagned by a change in era, which roughly followed the depolarization expected from the increase in [k+] e . at the end of the stimulation, although the [k+]e was still elevated, the nerve developed a posttetanic hyperpolarization (pth). the pth was generated by the electrogenicity of the na+-k + pump, but its amplitude and time-course were strongly affected by two depolarizing currents: (i) an inward ba2+-sensitive current of k + and (ii) an outward current of ci-. we hypothesize that during activity and during the early phase of recovery there was a ba2+-sensitive uptake of potassium by the schwann cells. the ci" current, by short-circuiting the na+-k ⧠pump, contributes to the control of the e m, and by this way to the driving force for the inward k ⧠current. overdose of ifosfamide has ooo/red in a canc~c patient (25g i.v. iy0/24 hours with 20 g as ~utectarfe). urir~ collece{on was obtaj/3ed in 24 hour ~ fcr t~o days. c~ was measured ~ a gas liquid d~romatogr~ method of the u5~ ~ (s~@pl. v, pag.2627-262g) ~sir~ a ~e~s ar~ ni~, sensitive ths~mi(~3ic d~. ~ u~s p~mt ~ ~ ~ ~ ~ ~ 1500 m~/24h and 207 r~/24h at day one ard day res~ec~vely. 9~ _h=~=mmtly ~ has also bsm foa~ in mti~s ~ mgh e~e ~ ~ (~/o~e). w~n ~ ~s am/nist~ to rats (i~ m~/~ ~c 200 m~f~ intraperit~neally), no ~ omild be detected in urine within 24 ht~r~ after drug administl-4(cicn. from these ~ data we conclude that c~ is extensi~_ly metabolized in rats and ~ mscbard_sm of ~ el ~mlnaticn in pati~rfc t=cj_ne m~cjlr[c reflect a flmicticrsl der~,~t of c~ m~t~0olisn in man to ifo~=~de ~tion. there are numerous reports of improved memory functions in rats following a dietary supplementation with choline. in some cases, this improvement can be related to enhanced cholinergic transmission (mack et al., behav neurosci., 103, 1234 -1241 , 1989 ). but it is not clear whether there is a general improvement in memory or whether specific aspects of learning and memory are affected. this work was aimed at analysing the effects of perinatal cholinergic supplementation on the development of spatial abilities and upon adult performance. choline supplementation (3.5 g./l. in 0.02 m saccharine solution in tap water) was maintained during two weeks before birth. additional supplementation was maintained from the 5th to the 1oth week postnatal. spatial learning capacities were studied at the ages of 26, 65 or 80 days in a cimular swimming pool (morris place navigation task) and at the age of of 7 months on a homing board. adult and aged rats were tested on a radial maze. selective aspects of the performance were markedly improved. this treatment had no specific effect upon spatial memory per se, but, instead, affected learning abilities by promoting efficient behavioural strategies hypothetically based on active representation and anticipation processes. the effects of the treatment remained evident up to 6 months following the supplementation. barcelona, e-08193 bellaterra females of both lines (n=b/grp) were injected sc daily between days 15-20 of pregnancy with vehicle (v), i(di) or 3(d3) mg/kg diazepam, or not(c). in these studies(a,b) their 5-7 mo old offspring were subjected to a 50-run session in a shuttlebox(a:12 males, 6 females of each line/condition) or a 6min session in a hexagonal tunnel maze with strategically-placed barriers and a lit central arena(b_:6-7 males of each line/condition). a: the freezing behavior of ld3 rats was reduced ~s lc, this behavior reverting more to escapes in males & avoidances in females, although v had an effect in the latter also. no within-line differences in inter-trial responses were seen, but pre-session activity was increased in female lvs and ldis, returning to lc levels for ld3s. no differences existed among the h groups. b: whereas h maze activity exceeded that of l, no wtthin-line differences appeared. hd3s entered the lit arena less than hcs or hvs, indicating an increased timidity after pnd, and a trend in the same direction existed for ldi/ld3 vs lv. two groups of abstinent female smokers, performing either a fixed rate or a subject paced version of a rapid information processing task were compared. both groups participated in two test sessions (in which the task was performed twice) to compare two different types of cigarettes which were smoked before and during the second task trial: a) the subject's habitual cigarette with a nicotine yield of at least 0.7 mg/cigarette and b) a nearly nicotine free test cigarette with a tar yield comparable to that of the habitual cigarette. reaction times to correct detections (series of three odd or even digits in a pseudorandom sequence of single digits) were longer with the subject paced version and not affected by the type of cigarette but shorter with the fixed rate version and even more decreased with the smoking of the habitual cigarette. on the other hand, the pre-to posttreatment increase in the subject paced processing rate was greater with the habitual than the test cigarette. it was concluded that the two task versions assess different cognitive functions. whereas the fixed rate version primarily assesses vigilance performance and seems to be more suitable to measure changes in reaction time, the subject paced version is more sensitive to changes in speed capabilities in rapid information processing. heart rate, physical activity, and cigarette consumption were continuously recorded under field conditions, whereas subjective craving was assessed six times per day and saliva cotinine was measured once daily. abstinence, habitual cigarettes (with a nicotine yield of at least 0.7 mg/cigarette), and nearly nicotine free test cigarettes but with a tar yield comparable to that of the habitual cigarettes were compare~) in a sample of twelve female smokers for two days each. whereas physical activity was similar for the three conditions, heart rate was highest with the habitual cigarettes, lowest on abstinence days and in between with the test cigarettes. cigarette consumption was similar for both types of cigarettes and subjective craving was higher on abstinence than on smoking days but no differentiation between the two cigarette types was obtained. saliva cotinine values were highest on the days with the habitual cigarettes but lower and similar with the test cigarettes and on abstinence days. it was concluded that heart rate and saliva cotinine depended only on the amount of nicotine absorbed, whereas subjective craving was reduced by smoking independently of the actual nicotine yield of the cigarette. (ptps) is the rate limiting enzyme in the synthesis of bh, in man, a cofactor for several hydroxylases involved in catecholamine and serotonin biosynthesis. the human and rat liver cdnas encoding the 16 kda subunit of ptps were expressed and the recombinant enzymes purified to homogeneity. the apparent k~ for the substrate, pi and heat stability of the re-combinant enzymes were similar to the native enzymes. the rat enzyme was crystallized and the x-ray structure showed a hexameric native form arranged as a sandwich of two trimers. three histidine, a glutamic acid, and -also shown by site-directed mutagenesis -a cysteine residue characterize the active center of the enzyme thought to be mg2+-dependent. according to the proposed ligands we replaced mg 2* by fe 2 ⧠or mn ~+ and observed an enhanced activity up to 100%. m. neerman-arbez, v. cirnlli and p. halban. laboratoires/eantet. centre m4dical universitaire, 1211 gen~ve 4. pci(3) and pc2, members of the mammalian family of pro-protein convertases homologous to the yeast kex 2, are both expressed in pancreatic islets of langerhans and are thought to be responsible for the conversion of proinsulin to insulin and c-peptide in beta cells. however, the insulin secreting beta ceils are not the only ceils present in these complex microorgans, prompting us to evaluate the expression of pc1 and pc2 in islet beta and non-beta cells. rat islet cells were sorted by autofluorescence activated flow cytometry to separate beta cells from non-beta ceils and conversion cndoprotease levels were analysed by western blotting. in beta cells, pc1 levels were higher than pc2 (pc1/pc2=2.6+0.2) whereas the opposite was found for non-beta cells (pc1/fc2=0.05â�¢ confirming that pc1 is important for proinsulin conversion while suggesting a role for pc2 in the conversion of proglucagon, prosomatostatin and propancreatic polypeptide. transformed murine cell lines (insulin-producing beta-tc and glucagonproducing alpha-tc) were found to faithfully reflect the primary rat cells in terms of their pc1/pc2 ratios. finally, post-translational modification of the convertases themselves was found to differ between cell-types, in particular, a precursor 75 kd form of pc2 accumulated in beta cells whereas only the fully processed 67 kd form was detected in the non-beta cells. the kringle (2+3) domains (k2+3) were successfully expressed in e. coil a n-terminal hexahistidine tag was fused to insure the isolation of k2+3 by affinity chromatography on a ni-2+-ntaagarose-column. to be able to remove the hexahistidine tag a factor xa cleavage site was introduced between the 6 histidine residues and the n-terminus of the kringle structures. the correct arrangement of the disulfid bonds was determined by amino acid-and sequence analysis. the lysine binding site was proven by affinity chromatography on iysine-bio-gel, as previously shown for k2. the dissociation constant of k2+3 was measured by intrinsic fluorescence titration with 6-aminohexanoic acid. a replication protein a copurifying dna helicsse from calf thymus anthl georgakl, narendra tuteja, blrglt sturzenegger and ulrich hobscher department of veterinary biochemistry university of z0rich-lrchel winterthurerstrasse f 90 ch-8057 z0rich, switzerland a dna helicase from calf thymus copurified with replication protein a through several steps of purification including deae-sephacel, hydroxyapatite and single stranded dna cellulose. it is finally separated from replication protein a on fplc mono q where the dna helicase elutes after replication protein a. we named this new calf thymus enzyme dna helicase f. characterization of the helicase f by affinity labeling with [a32p]atp indicated that the enzyme has a catalytic subunit of 72 kda. gel filtration experiments suggested that dna helicase f can exist both in a monomeric and an oligomedc form. the enzyme unwinds in the 5' ->3' direction in relation to the dna strand it binds. all eight deoxyribonucleoside-and ribonucleosidetriphosphates could serve as an energy source. testing a variety of dnndna substrates indicated that the dna heitcase f preferentially unwinds very short substrates and is slightly stimulated by a single stranded 3'-tail replication protein a allowed the dna helicase f to unwind longer dna substrates up to 400 bases suggesting that copurification of replication protein a with the dna helicase might be of functional relevance. 2,4,5,2',4',5'-hexachlorobiphenyl (6-cb) shows limited elimination and extensive storage in adipose tissue in vivo (lipophilie unmetabolizable compound) while chlorpromazine (cpz) is known for its lysosomal storage. uptake and release of 6-cb and cpz were studied in 3t3-preadipocytes (p) and 3t3-adipocytes (a). 3t3-cells were cultivated in dulbecco's minimal essential medium supplement with 10% fetal calf serum and grown to eonfluency. differentiation was stimulated by dexamethason and bezafibrate exposure for 48h. radiolabelled 6-cb (20~tm) or cpz (5~tm) was added to the medium. following a single dose 70-80% of cpz were taken up within an hour into p and a. in contrast, 6-cb reached an intracellular plateau of 30-50% of the dose after lh in p whereas dependent on the triglyceride content a accumulated up to 95% of the dose. 6-cb uptake into a was temperature dependent and not saturable with repetitive daily doses up to 1 week. 6-cb uptake into p was fully reversible while the release from a was limited to 10% of the accumulated drug. in contrast, the release of cpz was higher from p than from a as a consequence of the different lysosomal activities of the respective cells. the use of 3t3 cells allowed to show a remarkable difference in cellular handling of neutral and basic lipophilic drugs and may be helpful to further elucidate basic mechanisms involved. chronic granulomatous disease: an attempt to reconstitute the function of the x-linked form of the disease. chronic granulomatous disease (cgd) is a group of congenital immunodeficiencies that predispose patients to recurrent severe bacterial and fungal infections. the cause of the disease is lack or impairment of 02-production in phagocytic cells due to mutations in any of the four components of the superoxide producing system. approximately 70% of all cgd patients suffer from an x-linked form of the disease where the phagocyte oxidase gp91phox is affected. this project concentrates on the reconstitution of the deficient gene in phagocytic patient cells. expression of recombinant gp91 was assayed in vitro and in vivo. attempts to "tag" gp91 with a foreign epitope were unfortunately so far unsuccessful. the target cells for gene reconstitution are non-dividing monocytes. thus, we have chosen an adenovirus over other viral systems because of: a) its ability to infect nondividing cells, b) its genetic stability and, c) lack of human adenovirus related malignancies or side-effects. we constructed two recombinant adenoviruses: one recombinant expresses a lacz gene (control), another the gp9 lphox gene. we are trying the expression of recombinants adeno/lacz and adeno/gp91 in normal and patient-derived cells (for example ebvtransformed normal and patient b-cell lines), as well as the reconstitution of nadph oxidase function in peripheral blood monocytes of cgd patients. kinetics of molecular chaperone action schmid, d., gehring, h., *baici, a. and christen, p., biochemisches institut der universit&t z~rich, ch-8057 zorich; *rheumaklinik, universit&tsspital, ch-8091 z%rich molecular chaperones of the hsp70 type transiently sequester unfolded segments of proteins and promote their correct folding. target peptides labelled with an environmentally sensitive fluorophore allowed to follow their binding to the molecular chaperone dnak of escherichia coli in real-time. the two-step process is characterized by relaxation times z~ = 27 s and z2 = 200 s at 2 ~m dnak and 0.i ~m ligand at 25~ in the presence of atp, the formation of the complex is greatly accelerated and follows a single-exponential process with z : 0.4 s. the rate of dissociation of the complex was even more increased than that of association resulting in a decreased net affinity for ligands in the presence of atp. the binding-release cycle of dnak thus occurs in the time range of polypeptide chain elongation and folding and is too fast to be stoichiometrically coupled to the atpase activity of the chaperone (ko~ ~ = 0.13 min i at 30"c). supported by the olga mayenfisch stiftung, z%rich, and the hartmann m~ller-stiftung, z~rich. comparison of the activities of native bovine seminal ribonuclease and that secreted from e. coil schein, ch* (siat, technopark, pfingstweidstr. 30, ch8005 ziirich) and haugg, m (lab. org. chemistry, e.tm., . seminal ribonuclease (bs-rnase) differs from the pancreatic rnase a in that it forms a cysteine-linked dimer and has a relatively higher specific activity in the cleavage of double-stranded (ds-) rna substrates. we expressed bs-rn in a secretion system similar to that described previously (biochem. j. 283:377-344, 1992) using the signal sequence of routine pancreatic ribonuelease. about i mg bs-rn was isolated per liter culture supernatant. human and bovine recombinant interfcron-~ (ifn-r) inhibit the degradation of ss-and ds-rna by bovine pancreatic rnase while they activate the activity of bs-rn on the same substrates (febs letters, 270:229-332, 1990) . recombinant bovine or routine pancreatic rnase (produced in our secretion system) are inhibited by ifn-y, while the recombinant bs-rn or a cysteine-linked dimer of rnase a is also activated by ifn-y. ifn-y activates bs-rn even in the presence of inhibitory concentrations (0.1-1 ram) of mononueleotides. thus the mode of activation cannot be attributed to alleviation of product inhibition. kinetic studies using 300 bp ds-rna as substrate suggest that ifn-~ interacts directly with the bs-rn to increase the rate of highmolecular weight producffsubstrate release, as the apparent ks increases proportionately to the increase in kcat. significantly smaller movements were observed in the simulation with the liganded enzyme. the structural differences between the protein in the crystal and the ensuing calculated structures might arise from the absence of lattice forces in solution. evolutionary relationships among pyridoxal-5'-p-dependent amino acid decarboxylases sandmeier, e., hale, t.i. and christen, p. biochemisches institut der universit~t zgrich, 8057 z~rich. the pyridoxal-5'-p (plp)-dependent amino acid decarboxylases (de) can be subdivided into four apparently unrelated groups. group i is represented by glycine dc, group ii comprises glutamate, histidine, tyrosine and aromatic-l-amino acid dc, group iii procaryotic ornithine and lysine dc as well as the procaryotic biodegradative type of arginine dc, group iv eucaryotic ornithine and arginine dc as well as the procaryotic biosynthetic type of arginine dc and diaminopimelate dc. (n-l) profile analysis, a more stringent application of profile analysis [gribskov et al. (1990) methods enzymol. 183, 146-159] established the homology among the enzymes of each group. a search with the profile of group ii indicated a distant relationship with aminotransferases and thus with the ~ family of plp-dependent enzymes. no evidence was obtained that groups i, iii and iv are related with other plp-dependent enzymes or any other protein in the database. apparently, the amino acid dc are of multiple evolutionary origin, in some cases even if they have the same substrate specificity. a. lo russo, a.c. passaquin, u.t. r~eg~, ecole de pharmacie, %hiv. lausanne, c~-1015 lallsaraqe drug-induced local vasoccmstricticn appears to be respmnsible for the hypertensive side effect of the imn/nosti~pressant cyclosporin a (csa). we have therefore ex~rnir~ the [ca 2+] i increase caused by the vasoccr~trictor hormcme [ar~]vascpressin (avp) in vascular ameoth ~uscle cells (vsmc) treated with csa. [ca2+] i levels were m~nitored either with a fluoresc~qce analyser using fura 2 or by 45ca 2+ efflux. pretreatment of v~mc with csa increased the avp induced rise in [ca2+]i. a leftward and upwsxd shift of the ccncentraticm-respmnse curve of the avp-induced rise in 45ca2+ efflux was also observed after csa treatxr~%t. ilzg/cticn of csa dote~tiaticn w-as detectable after 3 rain, took 1 to 2 h to become fully established and was reversed after washout. csa potentiaticm was cc~centraticn-deperdent with a threshold at 10 -7 m. %]]is potentiating effect of csa may be the underlyin~ cause for csa-ind/ced hypert~3sicn. 92) barja, f. 1 and turian, g. i, ilab. gen. microbiology, 2dept. biochemistry, 3station exp. zoology, university of geneva, 30 quai ernest-ansermet, 1211 geneva 4 the actin has been found from the simplest forms of cell to the highly evolved ones. in higher organisms it is transcribed by multigene families but in yeast and several filamentous fungi there appears to be only one actin gene. it was therefore of interest to study the expression of actin gene in n. crassa in which three actin isoforms have been characterized (barja et al., 1991, fems left. 77:19-24) . after isolating genemic dna from wild type n. crassa a pcr was performed with primers corresponding to actin consensus sequences and an amplified fragment of the gene (verified by sequencing) was obtained. this fragment was 'used as a probe in a southern to assess the number of aetin gene(s). our results suggest that n. crassa has alsc only one actin gene. the blocking effect of the n-terminal decapeptide of msmooth muscle actin (sma) (ac.eeedstalvc) on the monoclonal antibody anti-asm-1 was compared with that of synthetic peptides modified by changing the n-terminal acetyl group or by substituting a single amino acid in position 1 to 5. using immunofluorescence or immunoblotting techniques, anti-c~sm-1 activity was abolished after incubation with the native peptide and peptides modified in position 1 (only when e~a) or 5. on immunoblots running bsa crosslinked peptides on denatured gels, only the natural peptide and peptides with substitution in position 1 or 5 were detected by the antibody. our results indicate that ac.(e)eed is the epifope for anti-c~sm-l. binding of anti-~sm-1 to sma increased actin polymedzation by decreasing the critical concentration. as control this action was not exerted on skeletal actin. this is the first example of the role of a precise n-terminal sequence in the polymerization of a single actin isoform. double immunofluorescence for msma and total actin on cultured aortic sm cells microinjected with the native peptide showed a selective disappearance of msma staining suggesting that this peptide traps a protein involved in msma polymerization. work is in progress to search for the putative actin-binding protein(s) physiologically interacting with the n-terminal sequence of o~-sma. ( within mitochondria, the mechanism of selective protein degradation is poorly understood. while the bulk of mitochondrial proteins are long-lived, some are degraded rapidly. the selective degradation of mitochondrial proteins is likely to be mediated by proteases similar to those found in bacteria; this is based on the hypothesis that mitochondria have evolved from bacterial endosymbionts, in conjunction with the finding that mammalian mitochondria contain an atp-dependent proteolytic activity similar to that in bacteria. one atp-dependent protease from bacteria, lon, is of particular interest because it is involved in regulated protein turnover. degenerate oligonucleotide primers corresponding to two regions of the bacterial lon gene were used to pcr amplify a 1 kb fragment from yeast dna that encoded an open reading frame with striking similarity to the bacterial lon protease. by screening a yeast cdna library, we isolated a 3.6 kb clone potentially encoding a protein of 1133 amino acids. haploids bearing a disruptedlon gene were unable to respire or to grow on ethanol/glycerol. fractionation of rnjtochondrial matrix from wild-type cells revealed a peak of aip-dependent proteolytic activity which was absent in the disruptant. furthermore, we have identified matrix proteins that are degraded with a half-time of ~60 min in intact wild-type ceils but are stable in the ion disruptants. electron microscopy of lon disruptants revealed the presence of many electron dense bodies in the mitochondrial matrix which are not found in lon + cells. hydrolysis of high molekular weight kin1nogen by plasma kallikrein benner, s. and duffer, h., laboratory for organic chemistry, eth h6nggerberg, ch-8093 z/inch high molecular weight kininogen (hmwk) is cleaved by the serineprotease plasma kailikrein to generate the hypotensive peptide bradykinin in human blood. we established a measuring system for the time course of the hydrolysis of hmwk in its native and deglycosylated form and in the presence or absence of c~-inhibitor. analysis of the bradykinin release and the yielded protein fragments lead to the three following results: 1. the hydrolysis did not follow plane michaelis-menten kinetics due to a hmwk-fragment operating as a competitive inhibitor. 2. addition of the ci-inhibitor stopped the hmwk-hydrolysis immediately, which is in contrast to the physiological role of plasma kallikrein in blood. 3. so far we have no evidence that the high content of o-glycosylated side-chains of the hmwk-light chain has an effect on the kinin-liberation as proposed in literature. ( tetrahydrobiopterin (bh4) is the essential cofactor for the hepatic phenylalaoine hydroxylase, but also for tyrosine and tryptophan hydroxylases that are responsible for the production of nettrofransmitter precttrsors. furthermore, bh4 is required for the deavage of giyceryl ethers aztd is i~vo[ved in the biosynthesis of nitric oxide by the nitric oxide synthase. a vazia~t type of hyperphenylalani~emia is caused by a deficiency of bh4. the most frequent form of this cofactor deficiency is due to lack of 6-pyruvoyl-tetrahydropterin synthase (ptps) activity. the human cdna for ptps was isolated, and the recombinant protein was found to be active when expressed in e. coil, thus allowing us to perform hmctional t e,3ting of mutant proteins. primary skin fibroblasts derived from ~everal ptps deficient patieilts were investigated for the molecular nature of this autosomal recessive disorder. all fibroblasts had f1"ps mrna expressed, and subsequent cdna sequence analysis revealed different mutatkons in the coding region of ptps (codon replacemenls, deletions, or nonseme codons) responsible for a lowered or no enzyme activity. in order to potentially correct ptps activity (and restore bi-i 4 biosynthesis) we are establishing a recombhtant rel~oviral-mediated gene transfer system to efficiently introduce the wild-type human ptps-cdna in these fibroblasts. the thylakoid membrane (tm) contains i0 molecular species of phosphatidylglycerol (pg). the relative amount of these species depends on the plant species and growth conditions. using phospholipase a2 (pla2) at 0~ to digest preferentially pg in the outer monolayer of spinach tm, we have shown previously that this monolayer was enriched in pg (ca 70~). the purpose of this investigation was to determine the transmembrane distribution of each pg molecular species. to this aim, we have studied the hydrolysis kinetics of each species in the presence of pla 2 in spinach and squash tm containing different relative proportion of molecular species. the hydrolysis rate and extent of the molecular species depended on the unsaturation degree of the fatty acid at the sn-i position as well as on the presence or absence of 16:1(3t) at the sn-2 position. for instance, the hydrolysis rate of pg ig:3/16:l(3t) was greater than that of pg 16:0/16:0. these results will be discussed in terms of the thylakoid transmembrane distribution of each pg molecular species. (supported by the snsf 3100-33693.92). from previous electrophysiological evidence, we concluded that metal ions (hgz+,cd2+,ag +) at low concentrations (i0-6m) increase rapidly (10-60 s) cation (na + , k + ) conductance at the apical membrane of epithelial cells. we investigated here the effects of these metals on [ca2+]i in mdck-i cultured cells, for a potential correlation between functional membrane changes and [ca2+]i . metals (10-4-10 -6 m) were added at the apical side of the epithelium, and [ca2+]i was measured with fura-2. hg 2+ induced a rapid (peak 5-10 s) and marked increase in [ca2+]i, whereas cd 2+ entailed a slower (peak 2-5 min) and marked response. the time-course of effects for both metals was similar to the electrophysiological changes. in contrast, the pattern of effects of ag + on [ca2+]i differed markedly from that on apical membrane conductance. thus, the effects of metals on [ca2+]i are conspicuous but not tightly associated with changes in membrane conductance. c. bulliard and l-l. dreyer, institut de biochimie, universit6 de fribourg, ch-1700 fribourg trans-plasma membrane oxydoreductases (pmo) play a significant role in energy transduction and post-receptor signal transmission, but the enzymes have not been characterized so far. we have achieved the purification of pmo from rat brain synaptic vesicles and from synaptic plasma membranes. the membrane enzymes were extracted by 1% lubrol xl tm, precipitated with ammonium sulfate (between 40 and 70% saturation) and purified by means of hydrophobic chromatography on butyl-agarose and gel f'fltration on superose-12, followed by page under native conditions. specific enzymatic staining of native page gels yields two homogenous diaphoraselike activities, pmo-i and pmo-ii . sds-page of the enzymes showed that pmo-i is made of two subunits of 52 kda and 40 kda whereas pmo-ii consists of a single sub-unit of 52 kda. the 52 ki)a subunits from pmo-i and pmo-[i are probably identical from their respective properties. partial n-terminal sequencing (13 aa) of the 40 kda subunit from pmo-i showed 84% homology with rat brain fructose-l,6-bisphophate aldolase. these data indicate that pmo is closely associated with the glycolytic enzyme that co-purifies in all steps under suitable conditions. the biochemical functions of pmo's remain to be established. ferredoxin:thioredoxin reductase (ftr) is the key enzyme in the ferredoxin/thioredoxin system, the light-dependent regulatory system in oxygenic photosynthesis. it is a nucleus encoded ironsulfur protein, composed of two dissimilar subunits, one of which is the catalytic subunit containing a redox-active disulfide bridge functional in the reduction of thioredoxins and a 4fe-4s cluster. using pcr we have isolated and characterized a cdna clone of 738 bp from spinach and a clone of 911 bp from corn coding for the catalytic subunits including the transit peptides. the first 31 (spinach) resp. 38 (corn) amino acids after the start exhibit typical features of chloroplast transit peptides. following the transit peptides are 113 (spinach) or 114 (corn) ami[~o acids representing the catalytic subunits. the primary structures are highly conserved between these two species with 91% similarity and 81% homology. seven of the eight cys residues found in the spinach subunit and important for the catalytic activity are at conserved positions. (snf 31-28811.90 and 31-37725.93) $23-20 r. zurbriggen and j.-l. dreyer, institut de biochimie. univarsit6 de fribourg, ch-1700 fribourg most mammalian cells display transplasma membrane oxidoreductase activity (pmo). the enzymes use intracellniar reduced pyridine nucleoticle to reduce an unspecific extracellular electron accepter as substrate. in order to set up a methodology for purifying, characterizing and quantifying pmo's, the plasma membrane from a neuroblastoma cell line, nb41a3, has been biotinylated. subsequently the biotinylated proteins were purified by immanoprecipitation with avidin, antiavidin-antibodies and insoluble protein a. the protein recovery of an immunopurified membrane preparation was <0.15% of the protein content in the cell extract. specific pmo activity was increased 15 to 20 fold compared to the activity in whole cells. this approach demonstrates the presence o1' pmo within the cell plasma membrane. this activity accounts for about one third of the total cellular diaphorase activity and cannot be attributed to an increased perraeabilization of the plasma membrane induced upon biotinylation nor to intracelluiar activity from lysed cells. pmo also promotes cell growth at low substrate concentrations (0.1-ll.tm). native gel electrophoresis of iarinobiotinylated and affinity purified plasma membrane extracts displays two diaphorase-positive bands, indicating that a homogeneous cell population may express several pmo activities at the plasma membrane. fluvastatin (fs) is a new hmg-coa reductase inhibitor. its affinity for 3 major human drug metabolizing p450 monooxygenases (cyp2c9, cyp2d6 and cyp3a4) was determined in liver microsomes. fs showed low affinity (ki > 50 ~tm) for cyp2d6 and cyp3a4 whereas cyp2c9 was selectively and competitively inhibited (ki = 0.1 ~tm). the potential for in vivo fs interactions with cyp2c9 substrates was therefore investigated in 14 volunteers using dicinfenac (df) as a probe (25 mg orally) on days 0, 1 and 8. df and 4'-ho-df kinetics were determined by hplc/uv. df cmax increased over time (0.28 [sd 0.12], 0.38 [0.20] and 0.45 [0.4] mg/l on day 0, 1 and 8, resp.). oral clearance was reduced on days 1 and 8 (14 % and 15 %, resp.). a time dependent decrease in urinary metabolic ratio (4'-ho-df/df) was noted (1.07 [0.34], 0.90 [0,23] and 0.70 [0.18] on day 0, 1 and 8, resp. (p < 0.0001)) only over the first 4 hours. fluvastatin therefore exhibits a two-phase interaction in vivo: an early transient and a late cumulative inhibition. the transient phase matches inhibition profiles predicted by quantitative models integrating in rive pharmacokinetics and in vitro inhibition data (q-dips). simple competitive inhibition by fs cannot explain the late phase. other mechanisms (i.e. fs or metabolite cumulation, mi complexation) are hypothesized. in some situations fs is expected to cause interactions with cyp2c9 substrates, which are partially predicted by quantitative modeling of in vitro data (snsf project n o 32-36600.92). 1211 gen~ve, switzerland. previously, a parvalbumin mutant, pvf~02w, was constructed with a unique reporter trp in the middle of the hydrophobie center. in the present study three new parvalbumin mutants, derived from pvft~w and containing alterations essential for ca2+-binding in either one, pv.câ�¢ and pv_m, or both, pv-co~, of the two ca2+-binding sites, were analyzed in order to study the metal binding properties of individual ca~+-binding domains and their contributions to the structure and stability of the protein. both, pv_ct, and pv.~, bind i ca ~ ⧠with affinity constants, kc,, of 1.1,107 and 3.2-106 m "~ in the absence of mg 2+. mg 2+ shifts the ca~+-binding isotherms of pv.co to higher free [ca z+] according to the competition equation with km~ = 2 9 103 m t. pv.m~ is much less effected by mg z* (kmg = 80 m "t) and can be considered as possessing a ca2+-specific site. nevertheless, in the absence of ca 2 ⧠both, pv.co and pv~, bind i mole of mg 2+ with much higher affinity than predicted from the competition studies. pv_cd;~ binds neither ca 2. nor mg 2+. trp-fluorimetry and uv-difference spectroscopy revealed that the ca~*-loaded conformations of pv_co, pv.ef and pvr~o~w are similar, with the trp-102 deeply buried in the hydrophobie core. however, striking differences between the mutants are found in the metal-free and mg~+-loaded forms. the conformation of pv~t~;~ is not affected by ca 2~ or mg z*. our results indicate that destroying the ca2+-binding of the ef loop, but not of the cd loop, strongly destabilizes the protein in the absence of ca z ⧠. biochemlsches institut der unlversitfit zfirich, switzerland, ch-8057. the sea urchin, strongylocentrotus purpuratus, metallothionein gene, mta, was constructed and expressed under the control of a heat shock promoter in a protease-deficient strain of e, coll. the nascent recombinant protein was stabilised by the addition of exogenous cd. the cd-containing protein was precipitated with ethanol and subsequently purified to homogeneity by gel permeation and ionexchange chromatography. the purified protein was identified as the desired gene product by tryptic peptide map analysis, sequence analysis and mass spectroscopy. typical yields of protein were 2mg per litre of culture. the presence of 7 cd ions per molecule was confirmed by the sh/cd ratio and by the existence of 7 h3cd nmr resonances. the apparent association constants of sea urchin mt with cd, which has a charge of -8, were found to be approximately 15 times weaker than for rabbit mt, at ph 7. (mt) are small metalloproteins displaying as their main feature clusters of d i~ metal ions bound to the thiolate ligands of the abundantly occurring cysteine residues (cys). from a set of over 85 sequences a general multialignment of 62 class i mt has been obtained on the basis of the needleman & wunsch algorithm. all cys are conserved in the man%mals and over 83% of them in other vertebrates and invertebrates. on the basis of similarity/identity scores we can isolate groups of sequences. evolutionary relationships between species and groups have been calculated with the maximum parsimony method of felsenstein and with the distance matrix method of fitch and margoliash. the divergences observed among the mammalian mts indicate a partition into four distinct subforms which all may have arisen before the separation of the species in evolution and which in part may differ in function. $23-28 bourque, a., singh, a., dykeman, a., macmahon*, a., and foster*, w. atlantic veterinary college, charlottetown, canada, and *reproductive toxicology section, environmental health centre, tunney's pasture, ottawa, canada. hexachlorobenzene (hcb) is a known environmental pollutant that is produced as an industrial by-product of certain chemicals. when administered in high dosage, hcb induces alterations in the rhesus monkey ovary. a purpose of the study was to determine a no observable adverse effect level (noael) for the compound. twenty, cynomolgus monkeys, 6-13 years old were placed in five groups. hcb was given orally in concentrations of 0.01, 0.1, 1.0, and 10 mg/kg b.w. in gelatin capsules, daily, for 13 weeks. one group of animals fed glucose only, served as the control. ovary specimens fixed in 2% buffered glutaraldehyde were prepared by conventional methods for transmission electron microscopy. ultrastructural alterations were revealed in the developing ova and follicular cells in all the ovaries from hcb-treated animals in a dose-related manner. clinical chemistry was unaffected by hcb. a noael for this reproductive toxicant is yet to be determined. a.b. was a recipient of the nserc undergraduate student award. an acidic haem-and zinc-containing protein has been isolated from bovine brain. native and sds-polyacrylamide-gel-electrophoresis reveal a monomeric protein with an apparent molecular weight of 47,2 kda. the absence of co-staining with tetramethylbenzidine implies that the haem group is non-covalently bound. the electronic absorption spectrum, with a soret-band absorption maximum at 412 nm and c~-and i~-bands at 540 and 575 nm, respectively, is similar to that of fe(ll)-myoglobin, consistent with the presence of haemiron in the ferrous oxidation state. in air the protein was easily oxidized to the fe(lll) form with a subsequent shift of the soretband maximum to 405 nm. atomic absorption measurements indicate approximately 1 mole of zinc and 1 mole of iron per mole of protein. the amino acid composition is similar to that of indoleamine-2,3-dioxygenase, although no such enzymic activity has been detected. in this study, we looked for this substance and its metabolites in young and old bovine lenses, because of their possible role in cataract formation. the substances were detected by hplc analysis. the kynurenine aminotransferase (kat) activity was determinated by the method of tobes (methods enzymol 1987~ 142:217) . the fluorescent substance formed from 3-hk was characterized by thin layer chromatography followed by reaction with rtinhydrin, uv and fluorescence spectrometry, and atom bombardment for molecular mass determination. 3-hk at concentrations of 0.08-+0.01, 0.2_+0.04, and 1_+0.4 btg/g of tissue was detected in iris/ciliary body, retina, and calf lens respectively. this substance is deaminated by kat. the activity of this enzyme was 2.6_+ 0.3, 3.7_+0.2, and 9.6+_2 ~tmol/g of tissue/hour in retina, iris/ciliary body, and lens respectively of old bovine eyes, but it was not found in calf eyes. the deamination of 3-hk resulted in the formation of a fluorescent substance which was identified as oxo-xanthurenic acid (oxa) with a molecular mass of 205 daltons. the accumulation of oxa acid possibly interacting with lens proteins could induce cataract formation. snf 32-36058.92, emdo, and hartmann-mtiller. the goal of covalent immobilization of oligonucleotide capture probes to solid supports has been accomplished by light-dependent, photolinker polymer mediated immobilization strategies. synthetic oligonucleotides were immobilized by facile layer coating procedures. the procedure does not require functionalization of the oligonucleotide probe or the matedal surface. oligonucleotides were linked to polystyrene with a biopolymer which was multiply substituted by photoactivatable diazirines. capture probe photoimmobilization (350 nm irradiation) was strictly light and diazirine dependent. using radiolabeled oligonucleotides as capture probes and polystyrene as material surface, the light dependent coupling efficiency was 40%. nonspecific oligonucleotide binding was below 2 %. photojmmobilized oligonucleotides retained the ability to form stable complexes with complementary dna strands, and target oligonueleotides of varying length were captured as well as dna fragments produced by pcr techniques. indoleamine 2,3-dioxygenase (ido), a superoxide radical scavenger, is present in transparent human lenses and produces 3-hydroxykynurenine, a uv-b filter. the synthesis of 3-hydroxykynurenine by ido and its degradation by kynurenine aminotransferase (kat) were measured in transparent lenses and cataracts. post-mortem eyes of elderly persons between 72 and 84 years of age with transparent lenses were obtained from the eye-bank of the department. fresh cataracts were obtained from cataract surgery patients. ido activity was measured by the method of yamazaki et al (biochem j 1985; 230:635) adapted for hplc. kat activity was measured by the method of tobes (methods enzymol 1987; 142:217) . ido activity found in post-mortem transparent lenses (cortex and nucleus) was 0.7_+0.3 nmol/mg protein/hour. in cataracts a low 1do activity of 0.3_+0.2 nmol/mg protein/hour was found in the cortex but not in the nucleus. kat activity, found in the eight cataracts examined, was 1.6_+0.9 nmol/mg protein/hour. inhibition of ido activity and induction of kat activity seems to be involved in human cataract formation snf 32-36058.92, emdo, and hartmarm-m011er photolinker polymer mediated covalent immobilization of antibodies and f(ab') fragments has been achieved by newly established lightdependent coupling procedures. anti alpha-l-fetoprotein monoclonal antibodies and f(ab') fragments derived from anti prostate specific antigen monoclonal antibodies were covalently linked to microplates. diazirine derivatized bovine serum albumin served as multifunctional light activatable linking agent. both immunoreagents, monoclonal antibodies and f(ab') fragments remained immunologically active after 350 nm irradiation (irradiance 0.7 mw cm -2 for 20 minutes). covalency of antibody binding was inferred from i) the photoreagent dependence, ii) the light dependence of the immobilization process, and iii) the reversibility of immunocomplexation after acid treatment. $23-33 two ~ of extracelluiar elecirical resistance in v~kwricular myocardil~ fleischhauer j.c., kl~ber a.g., dept. of physiol. bern in myocardial tissue, the electrical resistive properties of the extracellular space are important for local current flow during e~citation and therefore influence impulse conduction and the magnitude of the extracellular electrical field. to assess the r~tlti~t nature of the resistance of the extracellular space, electrical cable analysis was applied on an arterially perf~-sed rabbit papillary mascle. ~he resistivity of the intravascular space was changed (70 to 220~i~n) by variation of hematocrit frcrn 0 to 60% in the perfusate. in order to change the voltm~ and hence the electrical resistance of the interstitial space, colloidosmotic pressure in the perfusate was varied by changing dextran {m r 70000) concentration (i0 to 80g/l). altering the interstitial space volume had a marked influence on the extracellular resistance (ro) , conduction velocity and the magnitude of the extracellular field. variations of the resistive properties of the intravascular space had no significant influence on ro, conduction velocity and the magnitude of the extraeellular electrical field. our results suggest that the microvascular tree is insulated from the interstitial space and local electrical current flow during excitation in heart muscle is confined to the narrow interstitial clefts. s04-25, s 15-69 s05-18, s05-19 s 11-05, s11-06 bchini hooft van huijsduijnen s08-12 s05-30 s15-45 s08-13 s08-05, s08-06 s01-08 s15-67, $23-19 s13-03 nook, ek s01-15, s10-15 s05-02, s05-05 s 15-03, s15-29, s15-30 s19-03, s19-04, $19-05 s16-02, $16-15 s12-22 s15-48 s05-24 s03-01, s03-02, s03-03 s03-01 lrmler s05-28, s09-06 s03-07, s03-10, s03-11 s18-05 lipp, p. s05-26 so 1-02 s15-15, s15-16, $21-03, $21-11, $21-12, $21-14 $23-29,$23-30 s01-17 s 15-29, s15-30 s12-12, s12-24 molar s15-19 s08-05, s08-06 e s09-06, s09-07 s15-03, s15-29, s15-30 e. s03-05 s05-09, s05-10, s05-51 s05-52 s10-16 s08-14 s04-23, s04-29, s05-13, s05-53, $20-15 s10-16, s10-19 s09-01, s09-02 s15-68, s 17-24 stalder h. s03-19 s10-21 s09-09, s13-10, s13-13 s10-21 teheesen s01-10, s01-11, s01-18 s15-16 van dillewyn s05-32, s05-37, s05-38 s08-05, s08-06 s05-18, s05-19 huber d., ojha m. and turian g. laboratory of general microbiology, university of geneva, sciences iii, 30 quai ernest-ansermet, 1211 geneva 4, switzerland. ca2+-dependent protease in allomyces is predominantly localized in the apical region of the exponentially growing hyphae (huber and ojha, submitted) . many cytoskeletal proteins like actin and tubulin are also mainly localized in this growing region (heath, l~91). it is known that the ca2+-dependent protease proteolyzes a number of cytoskele~ ton proteins in order to regulate the plasticity of the cell (mellgren, 1987) . we have studied the proteolysis of some cytoskeletal proteins by this protease purified from the aquatic fungus allomyces erbuscula. actin, tubulin and desmin, were found to be rapidly proteolyzed in vitro at a high substrate to enzyme ratio. immunofluorescence studies of proteolyais made in situ in exponentially growing hyphae showed modification of apieally localized cytoskeletal proteins. this colocalization of the ca2+-dependent protease and cytoskeletal proteins in the same apical region is inferred in relation to hyphal elongation. jbiological databases grow exponentially. in order to provide access, as much data las needed and as little volume as possible shall be returned upon a query. the icurrentiy available dna and protein sequence databases are updated in a sophisticated network of procedures and expose a considerable amount of redundancy. research is performed on optimizing the creation of non-redundant data sets. the resulting data are disttibuted in switzerland, or made accessible to researchers who cannot utilize local resources. transparent access to the swiss resources, as well as paneuropean services, is provided with a job scheduling system which was developed in basel, the hierarchical access system for sequence libraries in europe ("hassle"), and various other computer programs which allow comprehensive browsing such as "gopher" and "www". training courses to use the resource effectively are running throughout the year. key: cord-006860-a3b8hyyr authors: nan title: 40th annual meeting of the gth (gesellschaft für thromboseund hämostaseforschung) date: 1996 journal: ann hematol doi: 10.1007/bf00641048 sha: doc_id: 6860 cord_uid: a3b8hyyr nan the variable molecular weight (mw) of vwf is due to differences in the number of subunits comprising the protein. it is assumed that endothelial cells secrete large polymeric forms of vwf and that smaller species arise from proteolytic cleavage. vwf has two main properties: it stabilizes factor viii protecting it from inactivation by activated protein c or factor xa, and it mediates platelet adhesion to subendothelium of the damaged blood vessel wall. each vwf subunit contains binding sites for collagen and for platelet giycoproteins gp ib and gp iib/i~a. multiple interactions of the multivalent vwf lead to extremely strong binding of platelets to subendothelial surface, that is capable of resisting high wall shear rate in the circulating blood. only the largest multimers are hemostatically active. lack of the largest vwf multimers was observed in patients with yon wiuebrand disease type 2a. unusually large molecular forms of vwf were found in patients with thrombotic thrombocytopenlc purpura. proteolytic enzyme(s) may be involved in the physiologic regulation of the polymeric size of vwf and thus play an important role in the pathogenesis of vwf abnormalities in some patients with congenital or acquired disorders of hemostasis. we have purified (-10,000-fold) from human plasma a vwf degrading proteas¢ using affinity chromatography and gel filtration. the proteolytic activity was associated with a high mw protein (mr -300 kd). vwf was resistant against the protease in a physiologic buffer but became degraded at low salt concentration or in the presence of 1 m urea. proteolytic activity had a ph optimum at g-9 and was not inhibited by serine protease inhibitors or sulfl~ydryl reagents. inhibition by chelating agents was best reversed by barium ions, the observed properties of the vwf degrading enzyme differ from those of all hitherto described pretenses. analysis of cleaved vwf showed that the peptide bond 842tyr-g43met had been cleaved -the same bond that has been proposed to be cleaved in rive. the endothelium releases the vasodilator nitric oxide (no) and the vasoconstrictor endothelin (et)-i. no is formed from l-arginine via the activity of constitutive nitric oxide synthase (cnos or enos). an inducible form of nos (inos) is activated by cytokines. no activates guanylyl cyclase in vascular smooth muscle and platelets, leading to the formation of cgmp which induces relaxation or platelet inhibition, respectively. in vessels, no is responsible for endothelium-dependent relaxations; in vivo it exerts a vasodilator tone which can be enhanced by shear forces and receptor-operated agonists such as acetylcholine, bradykinin, thrombin, atp and adp. infusion of no-inhibitors in vivo leads to vasoconstriction and increases in blood pressure and oral administration to hypertension in the rat. within the endothelium, no inhibits et gene expression and release of the peptide via cgmp. hence, no-induced hypertension is associated with increased plasma et levels. et, a 21-amino acid peptide, has potent vasoconstrictor properties via eta-and in part etb-raceptors on vascular smooth muscle. in endothelial cells, et activates etb-receptors linked to no and prostacyclin formation. under basal conditions, little et is formed, but is increased by thrombin, angiotensin ii, arginine vasopressin, cytokines and ox-ldl. et antagonists have been developed and allow to study the effects of et in vivo. et and no most likely play an important role in disease states such as hypertension, atherosclerosis, coronary artery disease, heart failure, pulmonary hypertension and subarachnoid hemorrhage. clinical trials to further define their role in these disease states are now under way. in summary, the endothelium is an important regulator of vascular tone and structure in vitro and in vivo. in disease states, their interaction is imbalanced leading to enhanced vasoconstriction, thrombus formation and structural changes of the blood vessel wall. pharmacological tools aiming to inhibit those changes are now being developed. j.m. harlan, r.k. w/nn, s. sharar, and n. vedder universit3, of washington, seattle, washington ischemia-reperfusion injury has been implicated in the pathogenesis of a wide variety of efinical disorders. [n preclinical models, tissue damage .clearly occurs during ischemia, but, parado.,dcally, may be exacerbated during reperfusion. this reperfusion injury appears to involve activation of the intlammato~, cascade with generation of complement 'components, lipoxygenase products, and chemokines as proximal mediators and neutrophils as final effectors of vascular and tissue damage. we have examined the role of leukocyte adhesion in reperfusion injury in two models -the rabbit ear as a model of isolated organ injury and hemorrhagic shock and resuscitation in the rabbit and primate as a model of traumatic shock and multiple organ failure. data regarding the efficacy, timing, and safety of leukocyte adhesion blockade using selectin-or integrindirected reagents in these models w/ll be presented. the current status of anti-adhesion therapy in other pre-clinical models and early clinical trials will be re~4ewed. an amidolytic assay for the determination of activated protein c (apc)-resistant factor va (fva) has been developed. this assay measures the cofactor activity of fva in diluted plasma samples via the rate of thrombin formation. the apc response is calculated from two fv determinations: one performed in the presence (apc-fv) and one in the absence of recombinant apc. the apc-fv activity is expressed as percentage of the initial fv activity and indicates the sensitivity of fva to apc. normal ranges were established by analysing plasma samples of 100 healthy individuals and an apc-fv activity above 60% was found to be indicative for apc-resistance (apc-r). in a control group of 34 patients the apc-r assay gave abnormal results in 15 patients. dna analysis confirmed heterozygous fv r506q mutation in all 15 patients and confirmed the non-carrier status in all of the 19 patients yielding normal results. an aptt-based apc-r assay performed on the same group of patients showed abnormal results in two of the non-carrier patients. one of these patients was diagnosed as positive for lupus anticoagulant, whereas the reason for the wrong positive result in the second patient remains unclear. eleven patients were analyzed before start of oral anticoagulation and during oral anticoagulant treatment. comparison of the assay results demonstrate a correlation of 96% indicating that the assay is independent of the activities of vitamin k-dependent clotting factors. the apc-r amidolytic assays allows specific and sensitive detection of fva-resistant to apc. the assay is performable in plasma samples of all persons in whom the diagnosis of apc-r may be indicated. in patients treated with oral anticoagulants or showing other clotting abnormalities affecting the aptt the apc-r amidolytic assay is helpful to establish the diagnosis of apc-r. dept of pediatrics, university hospitals kiel and mtinster, germany resistance to activated protein c (apcr), in the majority of cases associated with the arg 506 gin point mutation in the factor v gene is present in more than 50 % of patients < 60 years of age with unexplained thrombophilia. to determine to what exteut this relativdy common gene mutation affects the risk of thromboembolie events in infants and children, its occurrence was investigated in a population of children with unexplained venous or arterial thromboernbotism: thrombosis of the central nervous system (cns, n=4), vena portae (n=4), deep vein thrombosis (n=4), vena caval occlusion (n=4), neonatal renal venous thrombosis (rvi'; n--3), neonatal stroke (n=14), stroke (n=3), arteria femoralis ocdusion (n=2). four ont of these 38 patients showed a positive history of unexplained familial thrombophilia. apcr was measured in an activated thromboplastin time (afit) according to dahlbtick. the results were expressed as apc-ratios: clotting time obtained using the apc/caci2 -solution divided by dotting time obtained with cac12. concerning the special properties of the childhood hemostatic system, infants and children with apcr < 2 were considered to be apc-resistent only when the results were confirmed in a 1: l 1 dilution with factor v deficient plasma (instrumentation laboratory munich, germany). plasma of 268 healthy children served as controls. the arg 506 gin mutation of the factor v gene was assayed by amplification of the dna samples by pcr followed by digestion of the amplified products with the restriction enzyme mul i. results were confirmed by sscp -analysis or by direct sequencing of dna from patients with apcr. consistent with the ap'it based method 10 out of 19 children with venous (v) thrombosis and eight out of 19 patients with arterial (a) vascular insults showed the common factor v mutation. additional coagulation defects (autithrombin, protein c type i, enhanced antiphospbolipid igg, enhanced lipoprotein (a)) were found in 30% (v) and 28% (a). furthermore, we diagnosed exogenlc reasons (septicemia, postpastal asphyxia, fetopathia diabetica, central line and steroid/asparaginase administration) in six out of 10 (v) and three out of 8 (a) children with thrombosis and apcr. all four patients with a positive family history of thrombophilia (mothers only !) showed the common factor v mutation arg 506 gin. in the control group the prevalence of apcr was 5.1%. the high incidence of additional exengenic factors in children with apcr confirm literature data of previously described inherited coagulation disorders during infancy and childhood: an acquired risk of thromboembolic disorders masks the coagulation deficiency in the majority of patients with an inherited prethrombotic state. furthermore, the incidence of 42 % apc resistant children with arterial insults in this study challenge the view that apcr is associated with venous but not with arterial thrombo-st8. 12 activated protein c resistance and plasminogen deficiency in a family with thrombophilia m, zttger 1 , f. demarmels biasiutti 1, ch. mannhalter 2, m. furlan 1 , b.~e 1 1httmatologisches zentrallabor der universit~t, inselspital, ch-3010 bern 2klinisches institut fur medizinische und ehemische labordiagnostik, universit~t wien, a-1090 wien several hereditary defects of the proteins regulating blood coagulation have been associated with familial thrombophilia. since the recent discovery of activated protein c (apc) resistance due to the factor v rf06q mutation as a highly prevalent hereditary risk factor for venous thromboembolism (tel, evidence is accumulating that familial thrombophilia may be due to a combination of genetic defects. thus, protein c-or protein s-deficient patients having suffered from te seem to be more likely to carry the factor v r506q mutation than expected from its allelic frequency in the population. we report a family (see figure) in which plasminogen deficiency (0.60 u/ml) had been found in the propositus having had twice postoperative deep vein thrombosis (dvt) at ages of 29 and 31 yrs, respectively, as well as in 5 family members (0.53-0.69 u/ml). out of these 5 plasminogen deficient individuals, only the propositus' daughter had suffered from recurrent dvt at age <20yrs. reinvestigation of this family in 1995 showed factor v r506q mutation in the propositus, his daughter, an asymptomatic sister and a brother with postoperative pulmonary embolism (pe). his father had had postoperative pe; he is deceased and could not be examined. ~, [] plasminogen deficiency ~ ,rll factor v rf06q mutation /~ propositus ~¢ bistory of dvt and/or pe e superficial phlebitis j2~,,~ not investigated "1" deceased even though this family is small for establishing unequivocal association of te with known defects, the two most severely affected individuals with recurrent te at ages <30yrs had combined plasminogen deficiency and apc resistance whereas those with isolated plasminogen deficiency were asymptomatic. these data support the concept of multigenie interactions leading to familial thrombophilia. resistance to activated protein c (apc resistance) is the most common dsk factor for venous thrombosis (vt). in most cases apc resistance is caused by a single point mutation at position arg 506 in the factor v gene (factor v leiden). while ample data in hetarozygous patients have been published, reports in homozygous patients are limited. we studied 29 patients (12 males [m] , 17 females it]) in whom a homozygous mutation had been verified by dna analysis. the median age at the time of the study was 38.8 years (y) (range 9-83 y). twenty-five patients had experienced vt (10 m, 15 f). four patients were discovered dunng family studies and were asymptomatic, three were children (between 9 and 13 y) and one patient was a 62 y old man. in males the first thrombosis occurred at a median age of 38 y (range 21-82 y), in females this was at a significantly younger median age of 26 y (range 17-49 y). twelve of the 15 symptomatic females had taken oral contraceptives (oc, estregen content 0.02-0.ling) for 6 to 150 months (median 71 m) pdor to thrombosis. in 2 women vt occurred during pregnancy, in 1 female it was precipitated by hormone replacement therapy. in contrast, in 8,<10 males the thrombosis happened spontaneously, in 2 males it followed surgery. the sites of thrombosis were dvt in 9 males and 14 females, dvt and pulmonary embolism (pe) in 4 females and 1 male, dvt and caval vein thrombosis in 1 female and superficial thrombophlebitis in 4 males and 1 female. eight females had at least one pregnancy, in total 11 children and 5 abortions. two had thrombotic events during pregnancy and 2 after delivery. all homozygous patients showed apc ratios between 1.12 and 1.68 (mean 1.39 + 0.19). conclusion: patients with homozygous fv leiden have similar clinical symptoms as patients with deficiencies of antithrombin-, protein c or protein s deficiency. however, in contrast to these defects a very high dsk dudng oral contraceptive medication leading to an ealier manifestation in females can be observed. several synthetic (efegatran, argatroban, inogatran and napsagatran) and recombinant (hirudin, peg-hirudin and hirulog) antithrombin agents are in different stages of nlinical development for cardiovascular and thrombotic indications. while the specificity of these agents for thrombin is a concern, little has been done to study the effects of these agents on other serine proteases involved in coagulation and fibrinolytic processes. fihrinolytic compromise by site directed thrombin inhibitors has been reported recently (thromb res 74(3): 193-205, 1994) . while these agents have been shown to inhibit plasmin and related enzymes, little or no informatienentheir effects onthe generation and functionality of apc is available. sinceapc plays an increasingly important role both as an antienagulant enzyme, by inhibiting factors v and viii, end a pro-fibrinelytic enzyme, by stimulating the release of t-pa fi'om endothelial sites, an inhibition of apc may result in both pro-coagulant state and fibrinolytic deficit. representative thrombin inhibitors (dup 714 -a prototype boronic acid peptide derivative, efega~an, argatroban, hirulog, hirudin and feg-hirudin) have been compared for their ability to inhibit apc (american red cross). these bionhemically defined studies in which the remaining activity of apc after incubation with a thrombin inhibitor was determined speclrophotometrically with a ehromogenic substrate (s-2288, pharmacia, franklin, oh) , demonstrated that dup 714 and efegatran inhibit apc in a coneentrafinn dependent manner (ic~0=0.75 and 8,4 gm resp~tively), hirnlog inhibits apc weakly (60 p2¢i produced only 25% inhibition), while argatroban and ~ have no anti-apc activities, while hirulog, hirudin and argatroban produced no direct enti-apc activities, it is conceivable that they may inhibit thrombomodnlin-bound thrombin and thus prevent activation of protein c, resulting in a functional apc deficit and failure to improve clinical outcomes despite higher dosage. while initially it was thought that sole targeting of thrombin will provide monospacifin anticoagulant agents devoid of some of the adverse effects observed with heparin, the recent clinical trials clearly suggest that thrombin is not the only determinant of thrombogenesis. furthermore, potent antithrombin agents such as hirudin, hirulog and peptides, indirectly inhibit the generation of apc, by compromising thrombomodulin-bound thrornhin and such agents as efegalran and dup 714 also produce direct apc inhibition. endogenous inhibition of formed ape by thrombin inhibitors may therefore compromise the feedback regulatory funetiens of apc and may lead to thrombotic amplification in fully enticoagulated patients. these studies warrant prcelinlcal assessment of thrombin inhibitors to evaluate their relative inhibitory effects on apc. poor anticoagulant response to activated protein c (apc resistance) causes a significant portion of deep vein thrombosis (dvt) whereas its association with coronary artery disease (cad) and myocardial infarction (md is still controversial. therefore, we investigated 346 recently hospitalised patients suffering from cad with or without previous mi. the cad was proven by coronary angiography. apc resistance was analysed by using the ap'l-i'-based assay coatest apc resistance (chromogenix). eleven patients showed an apc sensitivity index below 2.0 viewed as apc resistance. using pcr technology, the factor v mutation causing apc resistance 11691 g "-) a) has been shown in nine of these eleven patients. this represents 2.6 % (9/346), compared to 8,5 % found in healthy blood donors (8/94). one homozygous carrier (male, age 76) was identified (apc sensitivity index 1.4) who suffered from dvt at age 59. recent angiography demonstrated diffuse cad, no thrombotic events were reported in his family. in contrast, multiple thrombotic manifestations (dvt, mi, stroke) occurred in the relatives of four heterozygous patients. we conclude that the prevalence of apc resistance is rather low in patients with cad. nevertheless, the natural history of coronary manifestation of apc resistance seems to vary, probably depending on the presence and severity of cardiovascular risk factors. resitanee to activated protein c (apc resistance) is the most cormnon hereditary cause of thrombophilia and significantly linked to factor v leiden pcr based methods are used to identify the crucial point mutation in the factor v g(me. we designed primers in order to identify factor v leiden by allele-specific pcr amplification. amplification specificity for factor v was ensured by the 3'primer fv1001, located at the introng/intronl0 border of the g~ae. one sense and two antisense primers were used in ~vo separate primer mixes specific for factor v arts06 (wildtypo) or factor v otn506 (factor v leiden) yielding 235bp products each. in each pcr reaction a pair of primers amplifying a fragment of the human growth hormone gene was included, fimctioning as an internal positive amplification control (429bp pcrfragment). after an initial denaturation step 10/.tl samples (100rig genomic dna) were subjected to 10 two-temperature cycles fouowed by 20 threetemperature cycles. for visualisation 8p3 of the amplification product were run on a 2% agarose gel presmined with ethidittm bromide. the presence or absence of specific pcr amplification allowed defiu/te allele assignment without the need for any postamplifieation specificity step. the in~ernal positive control primers it~cate a sucessf-u/pcr amplification, allowing the assignment of homozygosity. in a prospective study 126 p~e.ients with tlaromboembolic events were analysed using this technique and enmpared with pcr -rflp according to bertina et al. the concordance between these techniques was 100 %. in 27 patients a heterozygous factor v ohas06 mutation was detected, whereas one pa6ent with recurrent thrombcembolism was homoz-ygous. no false-positive or false-negative results worn observed in the homozygous as well as hcterozygous samples. in addition in 15 samples identified to carry the point mutation by al/ele-specifin pcr anxplification automatic :~equencing confirmed the heterozygous or homozygous point mutation. due to its time-and cost-saving features allele-specific amplification should be considered for screening of factor v leiden. background: an initial intravenous course of tmfractionated heparin ~ljasted on the basis of the activated partial thromboplastin time is the currmt standard treatment for most patients with venous thrombosis. low-molecularweight hqmrin pre~a~tious can be administered subcutaneously, once or twice daily, without laboratory monitoring. we compared the relative effic.~y and safety of low-molecular weight heparin versus anfractionated heparin for the initial treatment of deep venous thrombosis. methods: english-language reports of randomized trials vtta'e identified th~ a medline search (1984 through 1995) and a complementary extensive manual search. reasons for exclusion from the analysis were no hepada dosage adjustments, the lack of um of obj~tive tests for deep venous thrombosis, dos~ranging studies that used higher doses of low-molecularweight heparin than are ctareatly in use, and the failure to provide blind endpoint ~sossmeat. we assessed the incidence of symptomatic recurrent vinous thromboembolic disease, the incidence of clinically ii~t bleeding and mortality. results: twelve of the 21 identified trials satisfied the predetermined criteria. the relative risk reductions for symptomatic thromboembolic complicatious, clinically ~t bleeding, and mortality varied firom 20.60% aad were all statistically significantly in favor of low-molecular-wedght hqtmrin. coadusions: low-mol~ular--weight hoparim administered subcutaneously in fixed doses adjusted for body weight aml without laboratory nmaitori~ =re more effective and safea" tlum adjn~_-dose standard h~. sauce low molec~dar weight hqmrim vary in o~apositiou =ad pharma~ogical im)fil~ the benefits of each ~ shodd ~tabllsbcd separately. unfraetionated hcparin (uh) and low molecular weight heparin (lmwh) are widely used for the prevention and treatment of thrombotic disorders. uh and lmwh induce platelet aggregation in vitro. rgd peptides compete with fibrinogm for the binding to the glycoprotein receptor (gp lib-ilia) of platelets and inhibit platelet aggregation. to inhibit the heparin-indueed platelet ~tion and prolong the half-life in blood of rgd peptides, we linked ac-rgdv-ssggs-ahx-yk eovalently to lmwh in a ratio 1:1. the peptide is composed of three regions: a. rgd-gives the specificity for the receptor gp lib-ilia; b..ssggs-ahx-is the spacer between carrier and ligand, which should facilitate the intnraetion between the conjugate and the gp lib-ills receptor; c. -yk arc functional antino acids for iodination (y) and for covalent attachment (k) to the cattier lmwh. the aggregation achieved with different concentrations of lmwh, lmwh-eonjugate and lmwh/rgd-peptide mixture in a ratio 1:1 was mea.~ared after 20 rain.; maximum aggregation after platelet activation with 10 pm adp was set equal to 100%. platelet aggregation in normal human plateletrich eitrated plasma (prp; 220 000/p.l) was induced by lmwh in a dose ~ndent manner. heparin can induce antbodies which interact with platelets and endothelial cells. this causes thrombocytopenia and thromboembolic complications. hitpatients do need effective parenteral anticoagulation. we treated 82 patients (30m,52fm), median age 61 years (18-84) with laboratory prooven hit (hipa-test) with recombinant (r-)hirudin. as these patients had been preseleeted by their immunological response during heparin treatment and the treatment duration of the study was longer than in any other study using thlrudin, all patient samples were investigated for anti-r-hirudin antibodies himdin antibodies were screened by a sandwich elisa using r-hirudin fixed to the solid phase as antigen. all plasma samples were screened for anti-hirudin antibodies of the igg class, but solar only a suset of samples for lge anti-r-himdin antibodies. 38 of 82 patients (46.3%) developed anti-hirudin antibodies of the igg class. anti-hirudin antibodies were not detectable not before 6 days of r-hirudin administration solar no ige anti-hirudin antibodies were found. none of the patients devdoped thromboeytopenia or allergic symptoms. however, in a subset of patients the anti-hirudin antibodies enhanced the anticoagulatory effect of r-hirudin. in 5 patients the hirudin dosage had to be decreased by 2-3 fold to maintain a stable aptt level, in 3 patients, despite stable r-hirudin maintenance dose the aptt increased to values >100 see.. during the study 4 patients with anti-hirudin antibodies had to be reexposed to a second course of r-hirudin for parenteral anticoaguhtion none of these patients developed any allergic reaction. in conclusion we found a high proportion of anti-hirudin antibodies in hatpatients treated with r-hirudin for more than 7 days. these antibodies seem to have minor clinical relevance in regard to allergic reactions. however, one has to consider that these antibodies may influence the pharmacokinetics of rhimdin and thereby enhance its antieoagulatory potency. therefore, aptt must be monitored closely in patients receiying r-hirudin for more than 5 days a major concern in the use of hirndin, the most potent and specific thrombin inhibitor, is the risk of bleeding associated with the potential effect of this drug on hemostasis, particularly when the antithrombotic therapy is combined with invasivo procedures, fibrinolytic treatment, or patient's predisposition to abnormal bleeding. thus, availability of an antagonist to hirudin would be essential for instant neutralization of the antithrombotie action. however, thueh a hirudin antagonist is unknown in nature. to prepare an antagonist to hirudin, a mutant derivative of human prothrombin, in which active site aspartate at position 419 is replaced by an asparagine, has been designed, expressed in recombinant chinese hamster ovary cells, and purified to homogeneity. d419n-prothrombin was converted to the related molecules d419n-meizothrombin and d419n-thrombin by limited proteolysis by e. carinatue venom and o. scvutellatus venom, respectively. both d419n-thrombin and d419nmeizothrombin exhibited no thrombin activity and titration resulted no detection of the active site. however, binding to solid phase immobilized hirudin and fluorescence studies confirmed that the binding to the most specific thrombin inhibitor, hirudin, was conserved in both proteins, hi vitro examinations showed that d419n-thrombin and d419nmeizothrombin bind to immobilized hirudin, neutralize hirudin as well as in the purified system and in human blood plasma and re-activate the thrombin-hirudin complex. animal model studies confirmed that d419nthrombin and d419n-meizothromi.,in act as hirudin antagonist in blood cireulatlon without detectable effects on the coagulation system. while i.v. injections of hirudin in mice resulted in an increase in partial thromboplastin time, thrombin time and anti-thrombin potential, additional injections of d419n-thrombin and d419n-meizothrombin resulted in a normalization of these coagulation parameters. elevation of plasma homocysteine is a hereditary disorder of methionine metabolism associated with a high risk of arterial vascular disease. however, as yet relatively little attention has been directed towards the association between hyperhomocysteinemia and juvenile venous thromboembolism (vte). consequently the aim of our study was to evaluate the prevalence of hyperhomocysteinemia (hyper-hcys) and juvenile vte. patients: 85 patients (29 men, median age 42 ys; 56 women, median age 35 ys) who had at least one verified episode of vte before the age of 45 ys were investigated in regard to their total plasma hcys levels. none of the patients had renal or liver dysfunction or evidence of any autoimmune or neoplastic disease. methods: plasma total homocysteine levels were determined by hplc with fluorescence detection. hyperhomocysteinemia was defined as hcys levels exceeding the upper limit of the normal range obtained in our laboratory from 80 healthy control subjects (40 males, median age 25 ys, hcys 95% ci: 2.02-5.67 pmol/l; 40 females, median age 27.5 ys, hcys 95% ci: 2.99-5.40 ,gruel/l). resuits: 13 out of 85 patients had hyper-hcys, giving a prevalence of 15.3 %. of these 13 patients, 9 were male and 4 female, indicating that the relation between elevated plasma hcys levels and vte may not be as strong in woman as in men. discussion: according to previous reports, our study shows that there is a high prevalence of hyper-hcys in patients with juvenile vte. however, the mechanisms by which hyper-hcys can provoke vte and whether hcys is an exclusive risk factor or if it contributes to other existing predispositions, possibly working as a trigger factor is unknown yet. some authors suggest hcys-iaduced effect on factor v activation or inhibition of thrombomodalin-dependent protein c activation. in addition an influence on thrombocyte aggregation has been postulated. conclusion: measurement of hcys levels may be useful in the evaluation of patients with a history of juvenile venous thromboembolism and could be clinically important as hyper-hcys is easily corrected by vitamin supplementation. detailed determination of the pathogenesis of vte in patients with hyper-hcys should be the aim of further investigations. a deficiency of one of the coagulation inhibitors antithrombin (at), protein c (pc) or protein s (ps) and resistance to activated protein c (apc resistance) are established risk factors for venous thromboembolism (vte). in the majority of patients with apc resistance, the .tug 506 gin mutation (factor v leiden) is present. whereas deficiencies of one of the coagulation inhibitors are rare in the normal population, the allele frequency of factor v leiden is 2-7% in western europe. heterozygous individuals have a 3-7fold, homozygous an 80fold increased risk for vte the typical clinical features of all abnormalities are deep vein thrombosis, pulmonary embolism, superficial vein thrombosis and thrombosis at unusual sites, like mesenteric vein thrombosis or cerebral vein thrombosis. the thrombotic risk is low during childhood, but increases considerably after the 13th year of age. a retrospective study in adult patients out of families with a symptomatic deficiency of at, pc or ps revealed that around 30% of surgical interventions and traumas of the lower extremities were complicated by vte. therefore, these patients should receive thrombosis prophylaxis al~er surgery and trauma if their age is higher than 13 years. pregnancy is associated with a very high risk for vte in individuals with at deficiency and prophylaxis should be initiated already in the first trimester. after delivery, thrombosis prophylaxis is adviced for all females known to have an abnormality. oc increase the risk, especially in at deficient and in homozygous factor v leiden females and are therefore contraindicated in these individuals. oc do also increase the risk for vte in patients heterozygous for factor v leiden and females known to have this abnormality should be discouraged from taking oc or should at least be informed on their increased risk. university hospital-', jerusalem, israel, hospital bcan.iou ~. paris, france, increased frequency of thrombocmbolie events have been observed iu patients with b-thalassemia. our findings of shortened platelet survival and enhanced urinary excretion ofthmmboxanc a: metabolitcs (blood 77:1749 (blood 77: , 1991 suggested an increased platclet activation in tbese patients. we also fouud that isolated thalassemie rbc enhance prothronlbin activation, suggesting an increased membrane exposure of procoagulant phospholipids i.e, phasphatidylserine (am j. hematol. 44:33, 1993) . we now show that annoxin v, which has a high specificity and affinity for anionic phospholipids inhibits pmthrombm activation by factor xa, by binding to thalassemic rbc (ic~, = 0.32 nm). kerckhoff-klinik, bad nauheim ~, medizinische poliklinik bonn 2, institut for immunologie und transfusionsmedizin universit~ll greifswald a antibody-mediated intravascular platelet activation is believed to be the basis for both arterial and venous thrombosis in patients with hat. while the development of arterial thrombosis can explained sufficiently by intravascular platelet activation, it is a matter of discussion whether additional risk factors are involved in the pathogenesis of hat-related venous thrombosis. since resistance to activated protein c (apc) is the most common inherited risk factor for venous thrombosis described the frequency of apc resistance among a population of 68 hat patients has been studied. hat was diagnosed using the heparin-induced platelet aggregation assay and confirmed by the ~4c-serotoninrelease test. the diagnosis of apc resistance was established by two functional assays and genetic analysis. at time of diagnosis of hat, 38 patients showed venous thromboembolic complications. among these, 24 were found positive for apc-resistance. pulmonary embolism was diagnosed in 18 hat patients, 14 of them were apc resistance positive. none of the 18 hat patients who showed exclusively thrombocytopenia were apc resistance positive. early oral anticoagulation (oa) was initiated in 7 patients after the diagnosis of hat has been established. six of these patients developed serious thrombotic complications including skin necrosis. these results demonstrate that apc resistance is an additional and common risk factor for the development of hat-related venous thrombosis. early initiation of oa during an acute episode of hat dramatically increases the risk of thrombosis. therefore, oa in hat patients should be initiated only after platelet counts have been returned to baseline levels and effective parenteral anticoagulation is achieved. controlled trials for primary and secondary prevention of stroke g. de (3aetano, c. cerletti and v. bertel~ consorzio mario negri sud, santa maria imbaro, italy this presentation will review the antithrombotic treatments to prevent ischemic stroke that have been evaluated in controlled clinical trials. in two studies of aspirin therapy for pdmary prevention in male physicians there was no reduction in the incidence of stroke, while that of first myocardial infarction was significantly lowered. similar results were obtained in a prospective study in a large cohort of women taking aspirin daily. the incidence of vascular death was not modified by aspirin in any of these trials. this is possibly due .to an excess of strokes associated to aspirin treatment: indeed the four vascular events avoided in 1000 us physicians under aspirin prevention for five years would result from five myocardial infarction and one vascular death avoided and two additional strokes occurred. oral anticoagulant therapy decreases the relative risk of stroke in patients with non valvular atrial fibrillation. warfarin appears to be superior to aspirin, but the latter drug is a useful alternative when long-term anticoagulant therapy cannot be administered. a metanalysis of about 150 trials and over 100,000 patients with different vascular diseases treated with aspirin (at different doses) and/or other platelet inhibitors showed 25% overall reduction of vascular events including stroke. the optimal dose of aspirin for secondary stroke prevention could not be established. in patients with previous minor strokes or tia there was 22% reduction of vascular events and 23% of non fatal strokes. the avoidance of nine strokes of any cause among the 38 expected in 1000 patients at risk would result from the sum of 10 ischemic events avoided and a haemorrhagic one occurred in excess. ticlopidine was reported to reduce the risk of stroke in two large tdals (one in patients with major stroke), but there is no evidence that it is better or safer than aspirin. we compared the effect of the direct specific thrombin inhibitors, napsagatran (na) and rec. hirudin (rh) with unfractionated heparin (uh) on the further growth of preformed thrombi. as a model of thrombogenesls, an annular perfusion chamber exposing rabbit aortic subendothelium was perfused with native rabbit blood at an arterial wall shear rate (650/s). fibrin and platelet thrombi were allowed to form during a 10min perfusion period after which the test agents were given iv as a bolus and a continuous infusion (3 and 10pg/kg/min, n=6) and the perfusion continued for 20min. the control groups were perfused for 10 or 30 rain (n=6). fibrin deposited and platelet thrombi formed on subendothelium were evaluated by microscopic morphometry. the % surface coverage with fibrin was not reduced in the drug-treated groups since fibrin deposition was similar in the 10 and 30 min control groups (39+8% and 335:6%, respectively, mean:l:sem). platelet thrombus area (ta) in the control groups increased from 24+7pm2/pm after 10min to 97+32pm2/lim after 30rain perfusion. na at 1011g/kg/min reduced ta by 94% to values (6+_2ptm2/ ~m) lower than those of the 10min control group whereas rh at this dose reduced ta by 70% (30-j:141.tm2/i.tm). uh at both doses was ineffective. these findings show that in contrast to uh the direct thrombin inhibitors na and rh inhibit the growth of preexisting thrombi. these results could be explained by the higher potency of na and rh as compared to uh for inhibiting clotbound thrombin (gast et al., blood coagul fibrinol 1994, 5.' 879-87) and suggest that thrombus-bound thrombin is an important modulator of platelet thrombus growth and/or stability in this thrombosis model. platelet adhesion -the initial event of thrombosis -is believed to be completely prevented by intact endothelium. we challenged this theory by superfusing intact human umbilical vein endothelial monolayers with activated human platelet rich plasma utilizing the stagnation point flow adhesio-aggregometer (spaa). the spaa provides flow mediated contact of platelets with the superfused surface. heparinized (3.5 -4.0 u/ml) platelet rich plasma (prp) was obtained from healthy volunteers and activated by addition of adenosine diphosphate (adp 2"10 -6 m). platelet deposition was recorded on-line by video as well as by measuring scattered light. fixed samples were examined by phase contrast and electron microscopy, inhibition experiments were performed with either the tetrapeptide rgds, the non-peptide gpiib/llla-inhibitor ro-43-8857 or a monoclonal antibody directed against the gpilb/llla complex. stimulation with adp prompted platelets to adhere to intact endothelium single or as microaggregates of a diameter of up to 100 micrometer. adhesion was dependent upon convective transport resulting in platelet collision with the endothelial monolayer. infusion of rgds or ro-43-8857 into the flowing, adp-stimulated prp completely prevented platelet adhesion to the endothelium as well as subsequent aggregation. when the inhibitor inflow was stopped while adp stimulation persisted, adhesion and aggregation occurred immediately. re-establishing the inflow of the inhibitors -with still continued adp stimulation -led to disintegration of the adhering aggregates. when prp preincubated with the monoclonal antibody against gpllb/llla was superfused, platelet adhesion to the endothelium and aggregation were irreversibly blocked. our results suggest that convective transport and stimulation of platelets are prerequisites to overcome endothelial thromboresistance and that subsequent platelet adhesion to the endothelium is mediated via the platelet gpilb/llla receptor complex. prevent thrombus formation affer ptca i.p. 8tepanova t, g.v. bashkov 2, l.p.kapralova, 2 s.p. domogatsky ~ cardiology research center t and national haematology scientific cettter 2 russian academy of medical sciences, moscow, russia percutaneous transluminal coronary angioplasty (ptca) results in atheroselerotie plague rapture, vascular wall damage and thrombogenic collagen exposure. subendothelial collagen type i-lll is a very ~rong agonist of platelet-dependent thrombus formation in arteries. the anlithrombotic action of rabbit polyclonal antibodies to rat collagen type i-ill and their chemically synthetized conjugate with monoclonals to human recombinant two-chain/one-chain urokinase type plasminogen activator (rtcu-pa/rseu-pa), cross reacting with rat tcu-pa/scu-pa was studied both an in vifro and in vivo. anticollagen antibodies and bispecific conjugate inhibited human platelet adhesion, aggregation and formation of thrombi-like ~ructures induced by rat collagen immobilized with the polystiroi surface in a condition mimics the high shear rate in the large elastic-type arteries. the short-term treatment of the collagen-soaked silk thread by the collagen antibodies suppressed the platelet-dependent thrombus formation in the arterio-venous shunt in rats by 56_+4 % (p<0.001) as well as by the bispecific conjugate (44_+4%, p<0.001). the treatment of collagen-adsorbed conjugate by rtcu-pa did not increase the autithrombotic effect of bifunctional antibodies. the present date suggest, that the local administration of the anticollagen antibodies at the site of atherosclerotic plague rapture may tm the efficient tool for prophylaxis of platelet-dependent thrombus formation in arteries after ptca. increased levels of certain hemostatic factors have been shown to be related to an increased risk of cardiovascular events. hypercoagulability is suggested to predispose to arterial thrombosis and thereby to participate in atherogcncsis. we therefore assessed fibrinogen, prothrombin fragment 1+2 (fi+2) and yon willebrand factor (vwf) antigen in 80 consecutive patients (aged 59+5 years) with known coronary artery disease (cad) who all underwent coronary angiography. the extent of coronary artery disease was quantified according to modified criteria of the american heart association (total, proximal and distal "score"). furthermore the intima-media thickness (imt) was determined in the carotid and femoral arteries by standardized ultrasonographie measurement, vwf antigen was found to correlate positively with the total and proximal coronary score (r=029, p<0.05 and r=o.36, p< 0.01). while fi+2 showed no correlation with the coronary scores, it was significantly correlated with the imt values in the carotid arteries (r=0.27. p< 0.05). after differentiating tertiles of the parameters patients belonging to the upper tertile of fi+2 concentrations had significantly higher imt values of the carotid and femoral arteries (0.81_+0.11 mm vs. 0.72+_0.13 mm in the lower tertile, p<0.05:1.38_+0.44 mm vs. i. 15-+0.25 ram, p=0.05) whereas in patients belonging to the upper tertile of vwf antigen concentrations the proximal coronary artery score was significantly higher (!.71-+0.59 vs. 1.31+ 0.92 in the lower fertile, p<0.01). fro correlation of fibrinogen concentrations and extent of cad or imt values of the carotid and femoral arteries could be demonstrated. in conclusion procoagnlatory mechanisms as indicated by elevated concentrations of yon willehrand factor antigen and fi+2 may be contributing factors in atherogenesis. we have previously shown that pgei is a potent inhibitor of pdgf-ioducod proliferation of vascniar smooth muscle cells (vscm) and inhibits dna replication by a camp-related mechanism (grol~er et al, 1994) . the present study investigates of whether or not this aatimitogeni¢ activity of pget can be amplified by trapidil, a compound that has been shown recently to inhibit the incidence of restenosis of hmnan coronary arteries subsequenmt to ptca (maresta et al. 1994) , vsmc were prepared from coronary arteries of adult bovine hearts, passagod and kept under standard tissue culture conditions. cells of passage 4-9 wore incubated in serumfree medium for 24h in the presence of indomethacin (3 p.m). addition of pdgf-bb (10 ng/ml) under these conditions stimulated dna-replication as assessed from 'hthymidin lncm'poration, by 3.-4laid above control level. trapidil at 10 idvl caused a minor reduction of pdgf-induced mitogenesis whereas 10t) of the compound resulted in a marked reduction of dna replication by 69% (p < 0.05, n = 4). pgei at 0.3 nm diminished the incorporation rate by t 1% while the simultaneous administration of both pged and trapidil (100 idyll caused a significantly stronger response as seen from n reduction of ~h-thymidine incorporation rate by 82% (p < 0.05, n = 4). as a possible mechanism of action, trapidil might have inhibited phosphodiesterases. to establish this, we measured the camp-depcudont proteinkiaasc (pk) a activity in cell homogenates. trapfdil increased the basal fka-activity from 19% to 31% of the maximum response while the response to pget (10 am) amounted to 55%. coincubation of pgei with trapidil caused a 65% stimulation of pka activity, sugesting a small though detectable inhibition of vscm phosphodiesterases by trapidil at anttmitogenic concentrations. essentially similar results wore obtained when thrombin was used as the mitogenic agent. the data demonstrate a significant antimitogenic effect of trapidil at p.molar concentrations that are in the range of plasma levels after therapeutic administration of the compound in rive. at these concentratrations, pget induced inhibition of mitogenesis is markedly enhanced by trapidil. inc. i~enna, and ~cenlral itematnlogy laboroto~. , university hospital of bern pibrinogen (fg), yon willebrand factor antigen (vwf) and tissue-type plasminogeu activator antigen (l-pal have recently been shown in be independent risk factors for subsequent coronary events in patients with angina pectoris (nejm 1995; 332:635) although paul antigen has also been proposed as a risk factor, conclusive dam showing its predictive value is still lacking. furthermore, we have recently shown in a study investigating 200 survivors of myocardial infarction that not only are fg, t-pa and pai-i significantly increased in these patients when compared to a heahhy conlro[ group, but pci activity is also elevated (7hrornb. tfaemost. 1995;73:1137 abst.) , hi order to obtain cut.off points for the individual parameters, frequency histogram plotl; were transformed into straight line cumulative frequency (probit) plots (thromb i/aemost. 1995;74:718) . the cut-off valu~ for the four parameters were determined as follows: fg at 2.7 g/l, t-pa at 8.7 ng/ml, pal-i at 25 ng/ml and pc[ at 126% of a normal pooled plasma. utilising there cut.off points it was then possible to determine the accumulative discriminatow effectiveness of the parameters. when fg w;qs employed alone as the discriminatow factor, it was observed that 47% (941200) of the coronary heart dir, ease (chd) group eilher had the cul..off value or were below it aud 29% (29199) of the normal group were above the cut-off value, thus, resulting in 47% false ne$atives and 29% false positives. when a second additional risk factor, t-pa wa_~ introduced, the number of false negatives dropped to 21% [i.e. 79% (158/200) had two, risk factors elevated] and the number of false positives to 13% to investigate whether a third parameter could discriminate further, pai-i antigen was used to analyse the rcnudning false positives and negatives. an additional 4% could be detected, resuhing in 83% of the chd group having three risk factors elevated. similarly, the number of normal aubjecta with three parameters elevated dropped by 4% to 9% furthermore. when a fourth parameter was introduced, namely pci, it was round to discriminate a further 8% in the chd group, thereby increasing tile di~riminalion to 91%. the number of false positives dropped to 4%, additionally, determination of pci increased the discrimination of patienta having had multiple infarctions from 88°/= when thrce parameters were mcasured to 100%. from these results it can be concloded that determination of fibrinogen levels alone is not sumcicnt to separate patients from controls as t-pa adds significant discrimination. pai-i antigen which correlated strongly with t-pa did not significantly increase the discriminatory potential of both fg and i-pa. however, by employing pci as a fourth paramctcr, virtually complete separation between the chd and normal groups as well as rurthcr recoguitiou of' patients having had multiple infarctions could be obtained. to test the hypothesis that oral contraceptives (oc) enhance exercise-induced activation of blood coagulation we examined 11 women (27 + 3 (sd) years, bmi 20.4 + 2.0 kg/m 2, vozm.. 49 + 7 ml/kg/min) without oc between day 18 and 22 of the menstrual cycle and 10 women (24 + 2 (sd) years, bmi 20,6 ± 1,6 kg/m', vo2max 47 + 7 ml/kg/min) taking oc (150 mg desogestrel and 30 mg ethinylestradiol) between day 7 and 21 of drug intake. prothrombin fragment 1 +2 (ptf1 + 2) and fibrtnopeptide a (fpa) were measured before and after running for one hour on a treadmill at a speed corresponding to the anaerobic threshold. mean heart rate [191 ± 10 vs. 196 ± 12 min 1) and mean plasma lactate (3.3 ± 1.6 vs. 3.1 + 1.2 mmot/i) wera comparable during exercise between control and oc group, respectively. results for markers of thrombin and fibrin formation were: ptf1 +2 (nmol/i) fpa (ng/ml) control before 0,66 ± 0.19 1.0 + 0.2 after 0.73 + 0.23 2.2 + 1.2" oc before 0.82 + 0.31 1.0 + 0.2 after 1.11 + 0.48* + 5.8 -+ 6.0* + * p < 0.05 vs. baseline, + p < 0.05 between groups. we conclude that oral contraception with 150 mg desogestrel and 30 mg ethinylestradiol enhances exercise-induced thrombin and fibrin formation, our data suggest that exercise testing might be useful for evaluating the risk of thrombosis associated with different compositions of oc. a. haushofer +, wm. halbmayer +, j. radek +, m. dittel *, r. spiel *, h prachar *, j. mtczoch *, m. fischer + + zentraltaboratorium mit thrombose-und c~rinnungsambulanz -krankenhaus lai~: * 4. medizinische ab[eilung mtt ka~liolo$i¢ -krankenhaus lainz und ludwig bottzmann-lnstitut ftlr herzinfarktforsohung, wien fifty-one patients (age 61.6 ± 9.2a; 34 m / 171) implanted with 74 coronary stems 03 palm~-schatz, 27 gianturco-roubin, 14 micro stcnts) received a now antithrombotic treatment using a combination of ti¢lopidine (tic) 2 ×250 mg/d for 28 days and acetyl salicylic acid (asa) zoo mg/d for long-term treatment. patients (pat) only received 15000 tu standard hepartn as i.v. bolus immediately before stent implantation (day l ). side effects and changes in hematological (day i to 7, 14. 28 and 35 [= without t[cil liver and kidney parameters (day 7, 14, 28, 35) were monitored. thirty-eight pat (75%) came for the controls to our del~rtment and were additionally monitored by thromboelastograpy (teg) and bleeding time (bt) (day 2g and 35). the other pat were monitored externally, side effects were reported. thrombin geucration after stenting was monitored from day i to 7 by prothrombin fragment 1+2 (f 1+2) and thrombin-antithrombin-lll-comptex (tat). "k" of the "leg decreased (day 28 vs 35; p< 0.0l ). bt prolongation was negatively correlated with the bodysurf ace area (tic+asa: p< 0.05, asa: p< 0.0 l) and showed a reduction after withdrawal of tic (2 l0 sec, 180/ 300 so: [median, quartiles] vs. 120 sec, 881157 sec; p< 0.ix)01). f 1+2 and tat of day i (blood collection: 0, 2, 4, 6 h after intervention, f 1+2:0.84 nmol/i, 0.68/i.07 nmol/l: tat: 2.6 pg/1, 2.0/4.6 ilg/1) were lower compared to day 2 to 7 (f i +2: 1.31 nmol/l, ].08/i .62 nmol/l; tat: 4.8 pg/i, 3.2/7.2 ijg/1; p< 0.0001). tic scorns not to be a strong thrombin generation inhibitor. during stenting one pat (i.9%) sustained a non penetrating mci and one (1.9%) an ischaemic stroke. tic+asa were very effective, only with one pat (1.9%) stent thrombosis (acute) occurred. side effects: 8/15.7% gastrointestinal (one lead to hospitalization), 5/9.8% hematomas at the needle site in the groin (one surgical intervention), 5/9.8% leucopcnias (one agranulozytosis with hospitalization), 3/5.9% allergic skin reactions and 2/3.9% increased liver enzymes (got, gpt, "pgt, alkaline phosphatase; > 2 × of the j. ). with one pat with gastrointestinal disturbances and skin reactions tic had to be withdrawn and treatment was changed to oral anticoagulatlon + asa. one pat showed a combination of skin reactions, gastrointestinal distufl~aneas and on day 28 a heavy reaction of the liver enzymes ( j. after 5 weeks). a decrease of the white blood count (day 1:7.19 gh, 6.03/8.2 g/l, day 28:5.46 g/l, 4.63/6.47 g/l; p< 0.000 i) could be observed. the safety of the therapy with tic+asa should be elucidated and extensively discussed. the serpins c1 esterase inhibitor (cllnh), antithrombin iii (atiii), alantitrypsin (slat), and a2-antiplasmin (azap) are known inhibitors of coagulation factor xla (fxla). although initial studies suggested al at to be the main inhibitor of fxla, we recently demonstrated cllnh to be a predominant inhibitor of fxla in vitro in human plasma (wuillemin et el., blood 1995; 85:1517) . the present study was performed to investigate the plasma elimination kinetics of human fxla-fxla inhibitor complexes injected in rats. the amounts of complexes remaining in circulation were measured using elisas. the plasma tl/2 of clearance was 98 min for fxla-alat complexes, whereas it was 19, 1 8, and 1 5 min for fxla-cllnh, fxla-a2ap, and fxla-atiii complexes, respectively. thus, due to this different plasma tl/2, preferentially fxla-alat complexes may be detected in clinical samples. this was indeed shown in plasma samples from thirteen children with meningococcal septic shock (mss), a clinical syndrome which is complicated by activation of coagulation, fibrinolytic, and complement systems. fxla-fxla inhibitor complexes were assessed upon admittance to the intensive care unit. fxla-a 1at complexes were elevated in all patients, fxla-c11nh complexes in nine, fxla-atiii complexes in one patient, and no elevated fxla-a2ap complexes were found. we conclude from this study that, (1) although c1 inh is the predominant fxla inhibitor, fxla-alat complexes may be the best parameter to assess activation of fxi in clinical samples, (2) measuring fxla-fxla inhibitor complexes in patient samples may not help to clarify the relative contribution of the individual serpins to inactivation of fxla in rive, and (3) fxl is activated in patients with meningococcal septic shock. dudng the coagulation of plasma about 20 % of the (x2ap present is covalently crosslinked to fibrin by factor xiila (aoki und sakata 1980, thomb. res. 19: 149-155) . we investigated the binding of azap by factor xiila to soluble fibrin (desaabb-fibdno) whose polymerization was inhibited by an isolated fibrin ddomain named d=,,, (haverkate and tiemann 1977, thromb. res. 10: 803-812) . d==. is known to have an intact fibrin-polymerization site and is able to block the prolongation of the fibrin protofibrils at an early stage depending on its concentration. lateral association to fibrin fibers does not take place, since the inhibited protofibnls formed at the conditions used here do not reach a sufficient length (williams et el. 1981, biochem. j. 197: 661-668; hantgan et al. 1983, ann. n. y. acad sci. 408: 344-365) . material and method: soluble desaabb-fibrino was prepared by incubation of (lztl)-fibrinogen (0.48 mg/ml), d=1o (1.91 mg/ml; molar ratio d==o to fibrin 14:1) and 0.4 u/ml thmmbin for 45 min. then q2sl)-c~2ap (16 p.g/ml), faktor ×ill (2 ulml) and ca 2) (5 mmol/i) were added. the crosslinking reaction was stopped at different times of factor xiila-incubation by adding of urea/edtasolution. the suspension was analysed by ultracentrifugation on gradients containing saccharose, urea and sos. re~ultl: the elution profiles of the ultrecentifugation-gradients show the formation of cmsslinked fibrin oligomers of increasing size depending on the time of factor xiila-action. the crosslinked fibrin polymers contained about 80% of the fibrin initially added. although factor xiila acted well, crosslinking of azap in the fibrin oligomers could not be observed. conclusl0n: as we already demonstrated (kelach et el. 1994, ann, hematol. 70 (suppll) : a 60) the crosslinking of azap to fibnn clots depends on the structure of the fibdn network, especially on the degree of lateral association of the fibrin pmtofibdla. in desaabb-fibrino no lateral association of fibrin protofibnls takes place under the conditions chosen here. thus it is consistent with our theory that we did not observe any binding of aiap to the fibrin oligomers of desaabb-fibrino. human pci is a non-specific serpin that inhibits several proteases of the coagulation and fibrinolytic systems as well as tissue kallikrein and the sperm protease acrosin. it is synthesized in many organs including liver, pancreas, and testis. the physiological role of pci has not been defined yet. recently, we have cloned and sequenced the mouse pci gene (zechmeister-machhart etal., manuscript in prep.) . this enabled us to study pci gone expression in murino tissues using mouse pci edna and crna probes. by northern blot analysis, mouse pci tar.ha was exclusively found in the reproductive tract (testis, seminal vesicle, ovary), all other organs analyzed -including the liver were negative for pci mkna, indicating that in the mouse pci is not a plasma protein. to determine which cells of the reproductive tract synthesize pci, cellular localization was assessed by in situ hybridization of mouse testis and ovary sections. in testis, pci mrna was present in the spermstogonia layer and in leydig cells, while sertoli cells and peritubular myoid cells were negative. these results are consistent with the immunohistological localization of human pc1 (laurell et al,, 1992) . in the mouse ovary, stroma cells of the medulla and around the follicles were positive for pci mrna. no pci expression was detected in theca or granulosa cells. we also studied the regulation of mouse pci gone expression by steroid hormones in vivo. [n mature male mice castration caused an increase in pci mrna in seminal vesicles, which was reversible upon the administration of testosterone. in tissues of intact adult male and female mice, pci mrna levels decreased after injection of human chorionic gonedotropin (hcg), while in castrated male mice, hco had no effect on seminal vesicle pci mrna. progesterone and 17-b estradiol decreased ovarian pci mrna levels in immature female mice. these data suggest direct down-regulation of mouse pci by sex steroids. the different tissue specific pci-geoe expression in men and mice furthermore indicates a different biological role of this serpin in the two species. ctr. transgene technology, leuven "[' 1-tissue factor ('it) is a 47 kda glycoprotein mainly known a the primary cellular initiator of blood coagulation. whether tf expression may also play a role in development is unknown, but the lack of spontaneous viable mutations of the tf gene in rive leads to the speculation that its absence may not be compatible with normal embryonic development. to determine the significance of "if in ontogenesis, the pattern of tf expression in mouse development was examined and compared to the 'if distribution in human postlmplantation embryos and fetuses of corresponding gestational age. at early embryonic period of both murine (6.5 and 7.5 pc) and human (stage 5) development there is a strong tf expression in both ectodermal and entodermal cells. "if decoration was seen during ontogenetic development in tissues such as epidermis, myocardium, bronchial epithelium, and hepatocytes, which express "if in the adult organism. surprisingly, during renal development and in adult organism tf expression differs between men and mice. in humans maturing stage glomerali were "if positive whereas in mice glomeruli were negative and instead epithelia of tubular segments were tf positive. in ncuroepithelial cells there was a striking 'if expression indicating a possible role of'if in neumlation. moreover, there was a robust tf expression in tissues such as skeletal muscle, and pancreas, which do not express in adult. in contrast to tf, its physiologic ligand factor vii was not expressed in early stages of human embryogenesis, but was detectable in fetal liver, the temporal and spatial pattern of tf expression during murine and human development support the hypothesis, that 'if serves as an important mo~hojzenic factor darinz embrvozenesis. to serve as an anticoagulant, protein c (pc) must be activated by a complex formed between the enzyme thrombin (t) and its cofactor thrombomodulin (tm). therefore, downregulation of endothelial cell surface expressed tm, for example, triggered by an inflammatory stimulus, could become a critical factor in effective pc activation. in order to develop a recombinant (r) pc mutant which can be activated independently of the tkm-complex, a peptide sequence including p1-7 in the activation peptide of pc was modified to be identical to the factor xa (fxa)-cleavage site in prothrombin. the mutant was expressed in hu 293 cells, purified and its anticoagulant properties characterized. using purified fxa the mutant showed activation rates between 0.02 and 0.13 nmlmin at pc concentrations between 97 and 770 nm, while the rpc wild type was insensitive for fxa activation. the activation reaction is calcium-dependent reaching maximal activation rates at a calcium concentration of 3 mm and was enhanced to 3.8-fold by addition of anionic phospholipids (pl). in contrast to the wild type pc the rpc mutant was insensitive for activation by the t/i-m complex. addition of the mutant to normal human plasma induces a prolongation of tissue-factor and p-it-based clotting assays. using normal human plasma as a source for fxa the the activation rates of the mutant were found 5-fold higher than in the purified system if tissue factor was used to generate fxa. in conclusion, our data demonstrate that the rpc mutant is effectively activated by fxa in a purified as well as in a plasma system. interestingly, the activation rates are enhanced in the presence of pl and normal human plasma. fudher studies should clarify the potential use of this mutant as a novel anticoagulant. thrombln plays a pivotal role in thrombotic events. the time course of thrombln concentration in blood or plasma after activation is of special interest to answer a variety of questions. with a chromogenic assay developed by hemker et el. [thromb. haemostas, 70, 617, 1993] it became possible to measure the generation of thrombin in activated plasma continuously. inhibitors of clotting enzymes which are to be developed as anticoagulants should be able to inhibit thrombin generation or to immediately block generated thrombin. we have used a test based on hemker's thrombin generation assay to elucidate which potency and specificity an inhibitor of factor xa needs to efficiently block thrombin generation in human plasma. thrombin generation after extrinsic (tissue factor) or intrinsic (ellagic acid) activation was followed using the chromogenic substrate h-~ala-gly-arg-pna (pentapharm ltd.). a series of synthetic low molecular weight inhibitors as well as naturally occurring inhibitors of factor xa with different potency were investigated. because of the inhibition of activated factor x the generation of thrombin in plasma is delayed and the amount of the generated thrombin is reduced. the concentrations which cause a 50 % inhibition of thrombin generation (icso) correlate with the k~ values of the inhibitors. low molecular weight inhibitors with k~ values of about 20 nmol/i inhibit the generation of thrombin after extrinsic activation with icso in micromolar range. after activation of the intrinsic pathway tenfold lower concentrations are effective. the strongest inhibitory activity after extrinsic as well as intrinsic activation is shown by recombinant tick anticoagulant peptide (r-tap) with ic~o of 0.049 pmol/i (axtdqsic) and 0.037 pmo/i (intdnsic). in the compadson of synthetic low molecular weight inhibitors of thrombin end factor xa which have similar k= values for the inhibition of the respective enzyme (lowest i<1 20 nmol/i), factor xa inhibitors are less effective tn the thrombin generation assay. in contrast, the highly potent xa inhibitor r-tap shows a stronger inhibition of thrombin generation than the tight binding thrombin inhibitor hirudin. background: resistance to degradation of coagulation factor v by activated protein c is associated with a point mutation in which adenine is substituted for guanine at nucleotide 1691 in the gene coding for factor v. to date this specifc mutation appears to be the most common inherited abnormality which predisposes patients to venous thrombosis. for this reason a reliable, fast and automatable system for the diagnosis of the described point mutation is required. the conventional methods used to identify the mutation are based on allele-specific restriction enzyme site analysis or direct sequencing. these methods have disadvantages for a large scale dna diagnosis, which include the need for electrophoresis or a high cost and time consumption. methods: an alternative strategy of dna diagnosis, the allele-specific oligonucleotide ligation assay, was adapted for the diagnosis of tile point mutation of factor v. following pcr amplification of the target dna, tile procedure was performed completely automatically on a robotic workstation with an integrated elisa reader using a 96-well microtiter plate. allelespecific restriction enzyme site analysis was performed to confirm the genotypes. results: in 10 patients with the mutation and in 20 individuals without the mutation the genotypes determined with the conventional allele-specific restriction enzyme site analysis were in 100% concordance with the elisabased oligonucleotide ligation assay. discussion: the pck-oligonucleotide ligation assay applied as automated detection system for the identification of the coagul;mon factor v point mutation allows tile rapid, reliable, and large scale analysis of patients at risk for thrombosis. resistance to the asticoa=m~lant activity of activated protein c (apc resistance) has emerged as the most con'anon inherited thrombophilic state. patients lreterozygous for factor v leiden are more likely to suffer from thromboembolie events than controls. this risk is even more pronounced in homozygotes. due to the low sensitivity and speeifity of most coagulation tests some investigators suggest to examine patients for the presence of factor v leiden mutation by pcr-based methods. re e~tly we presented an aptt-based functional test (acceleria inactivation test ait): 1:20 diluted plasma (50bi) is mixed with factor v deficient plasma (50~tl) and aptt reagent (501.d), incubated at 37°c and then coagulation is induced by caci2 and a.pc (50~d). using a standard curve, the clotting time (see) is transferred in per cent accelerin inactivation (%ai). using this test, the widely used apc-ratio as well as pcr-based factor v leiden detection (confirmed by direct sequencing) we prospectively studied 172 consecutive patients with thromboembolic events. patients without the factor v mntation eonsitently showed more flazm 50% al with the exception of one patient with severe factor deficiencies (including factor v) due to hepatic failure and heterozygous for factor v-leiden resulting in 62*/. ai, there was a complete concordance between the pcrbased method and dysaseelerinemia detected by ait. due to these result a specifity and sensitivity of ait above 95 % was calculated. furthermore, a clear discrimination could be obsoved beween 34 heterozygotes (20%<ai<50%) and 8 (incl. 5 samples obtained from jeaa) homozygotes (ai<20%). in contrast a.pc-ratio resulted in a great overlap between patients homozygous for the wildtype , hcterozgous or homozygous for factor v leiden. in addition we confirmed thc di.~eulties nsing the apc-rauo for patients trader anticoagulation, or with different types of other d~rombophilie defects such as lupus anticoagulant or protein s deficiency. due to the time and cost-saving featttres the/kit should be considered for the rapid detection of apc resistance without the use of pcr-based techniques. in summary we found, well in accordance with previous reports, that apcr due to the fv-leiden mutation is • highly prevalent defect in vte pts. pts with the fv-leiden mutation were eharacterised by a higher incidence of vte's without recognised prothrombotic risk factors and more frequent positive family history; no higher incidence of recurrent vte's or other prothrombotie coagulation defects was found; the age at initial manifestation of thrombophitia was comparable. acquired a recently identified mechanism for famdial thrombophilia is characterized by a poor anticoagulant response to actavated protein c (apc), it is now welt recoginzed to be due to a genetic defect in the factor v gene, which results in the syn-,thesis of an abnormal factor v molecule and to be a frequent risk factor for thromb.osis, however apc resistenee can also be found in certain acquired conditions. since pregnancy ~s known to be associated with a hypercongulable state resulting in a prevalance of thrombosis up to 1% and thromboembolic events, we determined the prevalence of apc resistance during the course of pregnancy at the city hoslfital ruesselsbeim. germany. blood samples from pregnant woman were drawn at mmester 1, 2, 3 and at term. besides the routine coagulatton screening, protein c and viii:c, the apc resistance were evaluated. a modified aptt method utilizing human plasma fracnonated apc (amencan red cross. bethesda, maryland, ~usa) was used to assay apc resistance. this method relies on the determination of two aptt's in the patient plasma, one in the absence of apc and one in the presence of a fixed amount of apc. clotting tune was determined after mcubation using the aptt reagent ( we investigated the sensitivity of various species towards activated, human protein c (apc) and found an impaired response in the order monkey < horse < pig < dog < rabbit. we assume that this effect is mainly caused by differences in the apc cleavage region of factor v, as recently described for a factor v mutation in bumat~s: factor v-leiden. this was corroborated by the finding that addition of human factor v similar to the factor v added with the animal plasma had only minor influence on test outcome. coagulation assays dependent on apc, such as for protein c and protein s determination, in the standard apc assay, the more factor v-leiden specific apc assay and in the pcat, a new screening assay for the whole protein c system, are based on the inactivation of factor va via apc. thus, in all these assays, 5% of rabbit plasma added to human plasma mimicked a apc response as observed for heterozygous carriers of factor v-leiden and 20% as for homozygous carriers. we exploited this factor v-leiden like behaviour to prepare plasma standards for calibration of apc dependent assays by mixing rabbit plasma with human plasma~ as reference for these standards the 0% value was assigned to the clotting time in the absence of apc, while the 100% value was assigned to the clotting time in presence of apc as obtained with a human, normal plasma pool. then these calibrated standards were used for establishing reference curves. on automated coagulation analyzers, the results obtained were automatically reported in '% of normal' or '% sensitivity'• this not only eases evaluation, more importantly, this procedure also considerably improves the comparability of results obtained with different reagents and instruments. in order to establish the methods for activated protein c resistance and the faktor v leiden-mutation we investigated 204 healthy individuals (128 women, 76 men, median age 37 ys) using the assay "contest ® apc-resistance" (chromogenix, sweden). the mean apc-ratio was 2.65, the cut-off level for low response to apc was calculated as 2.15, 46/128 women were oral-contraceptive users. they showed a slight decreased apc-ratio (mean 2.46). 24 of the 204 individuals (11,8%) had a poor response to apc (mean ratio 1.94) as a apc-resistance. for detecting the factor v gene mutation we a used non commercial own modified easy to handle pcr-based test. in 14 of the 204 individuals (6.9%) the factor v-leiden mutation could be found as heterozygotes. in this group, the apc-ratio was significant lower (mean 1.87) as in the group with apc-resistanee and without deteetiun of the factor v mutation (mean 2.04). nevertheless in one individual with beterozygote factor v-leiden, the apc-ratio was found in the normal range (2.24) , also in a few individuals with very iow apc-ratio the factor v mutation could not he detected. finally this data suggests a high prevalence of apc-resistance and factor v-leiden mutation in the population of the southwest of germany. resistance to activated protein c (apc) has been reported to interfere in clotting assays for protein c (pc) and protein s (ps) from various manufacturers, resulting in decreased values or even apparent deficiency (type ii') of pc. or is. in our laboratory, when using functional, avit-based assay kits for pc., is, and apc sensitivity from behring (marburg, germany) on an automatic turbidimctric coagulation analyzer (fibrintiraer a, behring), the interference in the is assay has been observed in a very pronounced manner soon after the introduction of apc sensitivity testing into the standard laboratory procedure for patients with thrumbophilia. yet we were not aware of an interferenca caused by apc resistance in the pc test. we therefore evaluated our results for pc eed ps with respnet to apc sensitivity status in a retrospective analysis. exclusion criteria were: oral anticoagulant therapy with vitamin k antagonists, impaired liver function, borderline result in the apc sensitivity test, known analytical intedercnc* other than apc resistance (e.g. elevated factor viii:c). resu/ts: apparant pc activity was slightly (12 %) lower and apparent ps activity was grossly (45 %) lower in ape resistent patients than in patients with normal apc sensitivity: there was no case with a presumed du~l defect of both at~ rnsistaac¢ and pc deficiency. in addition, none of the few patients diagnosed with pc deficiency before the introduction of the apc sensitivity assay turned out to ix: apc resistent. much more of a problem is the known sensitivity of the pc test to an elevated factor viii concentration which is quite a common finding among hospital patients but rarely known in an individual case. by aeccelerating the clotting process, elevated factor viii can cause apparent pc deficiency that can not be confirmed in a chromogunie pc assay. ,clearly, when combined with apc resistance, this problem is exspacted to be more severe. on the other hand, the comhination of both apc resistance and apparent is deficiency was found on a regular basis with many decreased and some bordeflinc values for is but only few in the lower normal range and virtually none in the upper non~al range. also, several #eats previously diagnosed with is deficiency turned out to be pc resistent, and the diagnosis had to be corrected, but dual defects cannot be ruled out. conclusion: for, the interpretation of pc and/or 1:"3 values from clotting assays the apc sensitivity status must be considered. when apc resistance and an apparent deficiency of pc or is is found, a type i deficiency may be confirmed or ruled out using antigen assays, but at present, there seems to be no reliable way other than genetic analysis to address the question of dual defects with presumed type ii deficiency, especially in the case of ps. therefore, there is a clear need for analytical improvements and the development of functional assays that are less sensitive to interference by apc resistance. the most common inherited risk factor for venous thrombosis is the resistance to activated protein c (apc). the main reason for this resistance is a point mutation in the gene of dotting factor v (f v). a nueleotide exchange leads to a different amino acid sequence in the protein and thus to the loss of one of the cleavage sides which is important for the inactivation of the procoagulatory form off v. the imbalance in the coagalation cascade leads to a strong risk for venous thrombosis, which is thought to be inoreaced by factor 7 to 10 for heterocygotes and about 80 for homocygote individuals. the prevalence in patients with a history of thrombosis is about 20%, among apcresistant patients as high as 80%. prevalence-off v ,leiden" is reported to be 2% of the normal population in leiden/netherlands and as high as 15% in a certain city in the south of sweden. due to the obviously different geographic distribution and the highly increased risk of venous thrombosis in carriers of the mutation investigated the distribution of this mutation in the normal population of different regions in germany. first attempts to assess the prevalence off v mutation by molecular methods (polymerase chain reaction followed by restriction fi'agment length polymorphism analysis) revealed that in the area of wiirzburg 7.7% of blood donors carry the mutation, which is significantly different (fischer t-test, level of significance 0.05%) from published data observed in freiburg (3.4%). homburg/saar and harmover revealed 5.8% and 8.5%, respectively, and were not significantly different from wfwzborg. further investigations of blood somples fi:om other regions should give an overview of the geographic distribution of this mutation in germany and might finally help to draw a map era regional risk of venous thrombosis. the regular apc resistance test is not applicable to plasma from orally anticoagulated (oac) or heparinized patients due to decreased levels of vitamin k-dependent clotting factors and to thrombin inhibition by antithrombin, respectively. the former limitation is overcome by prediluting samples 1-1-4 with fv-defident plasma. for this purpose a special quality of fv-deficient plasma has been prepared, which also allows analysis of heparinized plasmas. this fv-deficient plasma has now been evaluated with the coatest apc resistance assay. a complete ape ratio discrimination for the fv q506 mutation is obtained for plasmas from oac and heparin patients as well as from non-treated individuals. ratios above 2.2 are obtained in normal genotype plasmas, whereas heterozygotes for the mutation yield ratios in the range 1.6-2.0. the basal apt times for 15 oacplasmas with widely spread inr-values were reestablished into the normal range. .the stability of the reconstituted fv-deficient plasma was found to be' at least 3 hours at room tempetature, indicating its suitability for .routin.e u.~. f .u~ .e~mv~., the influence of preanalytical procedures is rmm~ single or double cenuifugation as well as freezing of samples do not alter the apc ratios to any significant extent. our results prove the usefulness of the modified coatest apc resistance kit method as a reliable, simple and rapid tool when screening for the presence of the fv q506 mutation. hereditary resistance against the anticoagulatory action of activated protein c (apc-resistance, apcr) which was first described by dahlb~ick et al. (pnas, 90:1004 , 1993 was identified as a possible new thrombophilic factor in a high percentage (17-60%) of young adults with thrombotic events. a single missense mutation (r506q) due to a g/a transition (g1691a) in exon 10 of the factor v gene (bertina et al., nature, 369:64, 1994) is tightly linked with apcr and is regarded as the causative molecular defect. identification of this mutation by pcr-based methods is easy to perform and avoids preanalytie and analytic errors in the eoagulometric assay for apcr. since the impact of this mutation in children with thromboembolic disease has not been determined to date, we initiated a multi-center prevalence study on two pediatric populations, with and without thromboembolie events. we compared 125 pediatric patients with thrombosis, divided into 3 different age groups (0 to <0,5 years; >0,5 to <10 years; >10 to <18 years) with a normal population of 159 children. the mutation g1691a was found with an unexpected high prevalenee of 12% in our normal controls. however, the prevalence was significantly higher in the age groups: 0 to< 0,5 years (25%) and >10 to <18 years (30%). in patients between >0,5 to <10 years the overall prevalence was similar to the control (13%). however in patients of this age with spontaneous thrombosis apcr was also a significant risk factor (29%). our results emphasize the impact of apcr for thrombogenesis in children. however, the significance is agedependent and does possibly reflect the different physiology of haemostasis in our three age groups. activated protein c (apc)-resistanec is a newly reeognised risk factor for thrombosis. in at least 90% of the cases it is caused by a single point mutation in the factor v gene (g->a at nucleotide 1691), which predicts replacement of arginin 506 with ghitamin. one of the apc cleavage sites in factor va is located c-terminal of arginin 506, and mutated factor va (factor v leiden) is resistant to apc-mediated inactivation. from epidemiologic studies it is known, that this abnormality can be found in about one third of patients with thrombosis. apc-resistance is a major basis for venous thromboembolism and is prevalent in about 5.10% of the general caucasian population. recurrent spontaneous abortion (rsa) affects 1-5% of couples and represents a major concern for reproductive medicine. in spite of extensive endocrine, genetic, serologic and anatomic evaluation some 30-40% of rsa women remain unexplained. a frequent morphologic finding in placentae of aborted pregnancies is an increase of fibrin deposition within the intervitlous space. because of these findings we studied the prevalence of apc-resistance in women with rsa (more than 3 miscarriages) of unknown origin. in 2 of 35 cases we found a pathologic apc-resistance, both patients had a history of recurrent thrombosis and were heterozygous for factor v leiden. the prevalance of apc-resistance is 5,7% and thus equals the prevalence in the general population. our data do not support the hypothesis that apc-resistanee is a risk factor for recurrent spontaneous abortion. h~matologisches zentrallabor der universit~t, inselspital, 3010 bern resistance to activated protein c (apc) due to the mutation 506 arg --~ (]in of factor v (factor v leiden mutation) is the most frequent hereditary thrombophilic defect known today, with a prevalence of 20 -50 % in patients with idiopathic venous thromboembolism and of about 3 -5 % in the general population. with an allele frequency of 2 % the expected number of homozygous individuals is about 4 in 10000. homozygous and heterozygons individuals differ considerably with respect to the relative risk of thrombosis (80 -fold versus 7 -fold) as well as to the age of the first thrombotic event (31 versus 44 years). deficiency of the vitamin k dependent protein s (p$), an important cofactor of apc, is another hereditary thrombophilia which is, however, much rarer than apc resistance with a prevalence of 5 to 8 % in patients with venous thromboembolism. factor v leiden mutation as well as ps deficiency are associated with impaired anticoagulatory activity of apc, which is most pronounced in case of the combination of the two defects. the combination of ps deficiency (with an assumed prevalence similar to that of pc deficiency) with heterozygous or homozygous apc resistance can be expected with a probability of 1: ~ 5000 or 1: ~ 500000, respectively. it is well known that ps levels decrease towards the low normal or even subnormal range during pregnancy. moreovar, there is increasing evidence that the sensitivity of plasma to the antieoagulatory effect of apc decreases during pregnancy resulting in an acquired apc resistance. these pregnancy associated effects art obviously much more relevant in case of preexisting ps deficiency or apc resistance and should contribute to the elevated thrombotic risk during pregnancy in a subject with either of the two defects, and even more so for a woman who suffers from both defects. we describe a young woman with a combination of homozygens apc resistance ( apc ratio 1.10, normal range: 2.02 -3.73), pronounced ps deficiency (free ps 0.ll u/i, total ps 0.30 u/i, normal range: 0.55 -1.20 u/1 and 0.65 -lag u/i, respectively) and, moreover, impaired fibrinolysis (no change of euglobulin -lysis time after 10 rain venous occlusion) who developed deep vein thrombosis after cesarean section in her first pregnancy. examination of her familiy showed heterozygous apc resistance in her asymptomatie father (apc -ratio 1.99) , combination of heterozygous apc resistance (apc -ratio 1.60) and ps deficiency (free ps 0.30 u/i, total ps 0.58 u/i) in her nsymptomatic mother and no defect in her sister. considering the fact that the mother was still thrombosis free at the age of 49 one may assume that the thrombosis risk in the proposita was mainly influenced by the homozygnsity for apc resistance. s. ehrenforth, m. adam, b. zwinge, i. scharrer university hospital, dept. of angiology, frankfurt a.m., germany introduction: apc resistance has been shown to be the most commonly inherited defect which constitutes a risk factor for venous thrombosis (vt). however, most of the present epidemiological studies concerning apc-r prevalence in thrombophilia were derived from results of tests conducted onplasmas collected under various conditions. this may influence the great differences reported on the prevalence of apc-r among these patients. for example, it has been shown that freezing of plasma specimens prior to analysis of apc-r causes a significant decrease in the assay results.the aim of our study was to evaluate the influence of eentrifugation conditions on the results obtained with the chromogenic apc-r assay. patients and methods: blood was collected from 31 patients (t9 women, 12 men; fv gent.type: 13 r/r 506, 14 r/q 506, 4 q/q 506) through veinpuncture into trisodmm ciwat (1:9). platelet-rieh and platelet-poor plasma was obtained by immediately centrifugation at 4"c for 10, 20, 30, 40, 50, 60 rain at 1000, 2000, 3000, 4000, 5000 and 6000 rpm. additional, pnp obtained from 14 healthy individuals (7 male, 7 female without hormonal trealraent) was prepared equally. apc-response was determined within one hour after centrifugation using the coatest apc resistance kit from chromogenix. results: for both, pnp and sin/gle plasma samples, we observed continuous higher af'c-ratios after increasing cenwifugation intensity. for example, an increase from 1000 to 6000 rpm resulted m an increased apcratio from 2.7 to 3.26 (20 min), from 2.88 to 3.326 (60 rain) respectively. even though less distinctive, similar results were observed concerning the duration ol eentrifugation: when the duration was increased from 10 to 60 minutes we observed a continuous increase in apc-ratio, for example from 2.74 to 3.12 when using 2000 rpm and from 3.09 to 3.36 when using 4000 rpm. the decrease of the ratio after low eentrifugation is the eonse-9nence of the shortening of affft in the presence of apc, without a signhcant influence of basal al:rl~ without apc. conclusion: our results demonstrate that centrifugation conditions are important to consider for the interpretation of apc-r results. supporting our observations, recent studies from sidelmann et al. have shown that an increase in plasma platelet concentration, low eentrifugation respectively, causes a signficant decrease in the apc-response. however, so far the mechanism responsible for the significant effect of both on apc-r assay results is unknown. although technically simple, the biochemical cemplexitiy inherent in the chromogenic apc-r assay necessitates a standardized plasma handling procedure to secure a reproducible determination of apc-il compapjson of different assays for determination of apc-resistance with the geno'fyping factor v (arg -> glu) g. siegert*, s. gehrisch*, e. runge**. r. naumann**, r. kn6fler*** *institute of clinical chemistry, **clinic of internal medicine, *** childrens hospital resistance to apc diagnosed on the basis of prolongated clotting time in the aptt assay" is now considered a major cause of thrombophilia. in the majority apc resistance is ~ted with a point mutation in factor v molecule (arg506glu), but both are not synonym. protongated baseline aptt is a limitation of the assay. following the determination is not possible in risk groups of patients (factor)ill deficiency, lupus anticoagulan0 and in patients under anticoagulant therapy. in these causes a dilution of plasma in factor v deficient plasma is recommended. the immunochrom assay is based on the inactivation of factor villa by apc. the aim of the study was to compare different functional apc response assays with the result of the dna analysis. apc response was tested in 100 healthy probands, 81 thro~ patients and 16 family members using the lmmtmochrem assay, the contest (chromogeaix) and the contest with 1+ 4 dilution of the plasma in native factor v deficient plasma (immune). the dna analysis was performed as described by bertina. one patiem was homozygoas for factor v mutstion~ a hetemzygous result was obtained in 4 members of the control group, in 28 patients and in 6 family members. in all cases with factor v mutation the ratio of the immunochrom assay was lower than the laboratory own value, independent on anticoagulant therapy. pathological ratios in this assay were also obtained in one member of a family" with high thrombotic incidence (dna arg/arg) and in 17 patients under anticoagulant therapy ( two of this patients are one cloned twins). in the contest a ai~ response was diagnosed in all cases with factor v mutation without anticoagulant therapy and in 40 % of heterozygous patients under anticoaglant therapy. results of the test using the dilution in factor v deficient plasma showed a good agreement vath the results of the dna analysis but the method is obviously only sensitive for the factor v mutation. the reason for pathogical ratios in the lrnmunechrem assay in wildtype patients is unclear. the majority of this patients is treated with anticoagulants, a comparison with the contest is not possible. interestingly in one patient under heparin and low ratio in the immunochrom assay' after reduction of hepann the ratio of the coatest was also low. it seems necessary to investigate in which distance to the thrombotic events the apc resistance should be tested. following pathological ratios in ftmctional apc assays must be discussed: high levels of factor viii and or v wiuebrand antigen (acute phase reactien), other mutations in factor v and viii. the factor v dilution assay should be replaced by the dna analysis. due to their differing compositions, the "sensitivities" of various aptf reagealts differ not only with respect to factor depletions, heparin and fibrin-fibrinogen degradation products, but also with regard to pathological inhibitors. for lupus anticoagulants this means that "lupus-sensitive" reagents can be delineated from "lupus-insensitive" reagents. with a "lupus-insensitive" ai~ reagent there is no or only slight prolongation of the aptt in the plasma under investigation, whereas with a "lupus-sensitive" reagent marked prolongation is observed. for the meaninof~l use of aptr reagents it is necessary to know the extent to which they are influenced by lupus anticoagulants. the following 14 apti' reagents were tested: • ptt-reageaz, p'rta, ptta liquid, ptt-la, pti'-lt (boehringer/stago) • pathromtin, pathromfin sl, necthroratin (behring) • platelin s, piatehn excel ls (organon tekinka) • actin-fs, actin-fsl (dade) • aptt silica lye, aptt silica liquid (instntmentation laboratory) the material for investigation consisted of 20 plasmas from patients with lupus anticoagulants. a confirmatory test (lupus anticoagulant test, immune) was positive for all of the patients. measurements were made using the sta coagulation analyser (boehringer/stago). it can be seen from the results that in some instances very different prolongations were obtained in identical plasmas by using differing aptt reagents. low susceptibility to lupus anticoagulants was shown by actirt fs (dade), ptt-reagenz (bcehrlnger) and neothromfin (behring). high susceptibility was shown by platetin excel ls (organon teknika), ptt-la and pti'-lt (boehringer/stago). lupus anticoaguhant screening with the aptt reaction is promising when two aptr reagents differing as greatly as possible in their lupus anticoagulant sensitivity are used. the resistance to the anticoagulant response of activated protein c (apc) is a major cause of venous thrombosis. apcresistance is due to a single mutation in factor v gene, which predicts replacement of arg 560 in the apc-cleavage site with gln (factor v leiden mutation). in contrast to other known genetic risk factors for thrombosis, this factor v 1691 g-a mutation has a high prevalence in the common population of western europe (average 4-5 %). we have determined the prevalence of the factor v 1691 g-a mutation in a population of 700 probands of north-eastern part of germany. the mutation was found in 7 %. (heterozygoty were found in 48 subjects 1 person was homozygous.) the results are compared with our studies of populations from argentine and poland. me analysed the factor v 1691 g-a mutation in 71 patients with thrombosis from germany and hungary. this mutation has been found in about 60 % of these patients. in contrast, the frequency of this mutation was strongly reduced in a group of 47 patients with thrombosis and pulmonary embolism of argentine (3 heterozygotes in 47 patients; 6 %). the results of these different populations will be described and discussed. past medical history: venous thromboembolic events (re) at 18, 19 and 2i years; intermittent oral anticoagulation (oac) without te's. diagnosis of autoimmune disorder with elevated antinuelear-antibody-fiters and positive lupus-anticoagulant test. no other relevant illnesses; family history uneventful. two weeks prior to the referral to us -acute febrile illness with nausea, diarrhea, abdominal pain; hospitalisation, treatment with iv antibiotics and anticoagulation with fraetionated heparin; development of extensive deep vein thrombosis (dvt) of the right leg; initiation of full-dose unfractionated heparin; decline of platelet count from 121 to a nadir of 21 g/l; referral to our department. on admission an extensive coagulation screen yielded the following results (n/normal, t/elevated, i/reduced, +/positive, -/negative): pt t, aptt t, tr n, factor ii, v, viii n, factor vii, ix, xi, xii /,, fibrinogan t, atiii n, protein c, s *, activated protein c sensitivity ratio 1.92 ($), fv-leidenmutation pcr -, fibrinolytic system n, tat t, ft÷2 t, lupus anticoagulant +, heparin induced platelet antibodies +; no diagnosis of a specific autoimmuna disorder could be made. an immunosuppressive therapy with corticosteroids and anticoagulation with recombinant hirudin were init'~at~; no p~ogr~zsion of the dvt oeeured and normalisation of the platelet count was observed. during follow-up under oac ) and low-dose corticosteroids, the patient was well, the pathologic coagulat;.on results, including lupusanticoagulant and activated protein c resistance, have returned to normal; no further te's have been observed. in summary we present a case of a complex coagulation disorder as part of an autoimmune process, resulting in a clinically manifest prothrombotie dysbalance including lupus anticoagulant, acquired resistance against activated protein c and heparin induced thrombocytopenia (type ii), entering complete remission under combined immunosuppressive and anticoagulant therapy. in the last 30 years, a vast number of simplified analytical procedures have been developed for the diagnosis of haemostatic disorders. today the detection method have evolved from the mechanical hooking method or ball coagulometry to optical systems, which additionally can utilise chromogenic substrates or immunological methods. in these systems the clotting time is derived from algorithms (e.g. threshold or maximum of the first or second integral). we studied 50 healthy subjects, aged 21 to 65 years and 118 patients, aged 9 to 93 years using a new aptt reagent (pathromtin $l). the results were compared with those obtained with a routinely used reagent (pathromtin). the reference range, factor-, heparin-and lupus anticoagulant sensitivity were determined. analysis was performed using the behring fibrintimer a (bfa) with optomechanical clot detection, the behring coagulation timer (bct) with op-"dcal clot detection by threshold and the dw test and dw confirm for lupus anticoagulant diagnostic. our results showed that the new pathromtin sl reagent met the demands for a higher factor and lupus anticoagulant sensitivity. it is highly suitable for monitoring heparin therapy and gave comparable results with the optical and the optomechanical analyser systems, hence reagent c~n be used for both systems. restenosis following percutaneous transluminal angioplasty (pta) continous to be a major clinical problem. neoinfimal hyperplasia, being the major undedying cause, can not be sufficiently avoided. vadous plasmatic coagulation and fibrinolytic factors, have been associated with artedal restenosis. anticardiolipin antibodies (act_) have been established as dsk factors for venous or arterial thrombosis. methods: in a cohort of 75 patients (53 men and 22 women, age 68±13 years) undergoing pta of a peripheral artery we prospectively evaluated whether acl could influence 6 months restenosis rate. patients were clinically examined before, 3 and 6 months after pta. noninvasive grading of artedal stenosis was done by duplex scanning of jet peak velocities. restenosis was arbitrarily defined as more than 50% occlusion of the lumen at the site of dilatation 6 months after successful intervention. laboratory investigation at the same time included acl and other known atherosklerosis risk markers, such as fibdnogen (fbg), yon willebrand factor (vwf), homocystein (hcy), c-reactive protein (crp). thrombin generation markers, such as thrombin-antithrombin iii complexes and prothrombin fragments 1+2, as well as thrombomodulin (fm) as an endothelial activation marker, were also measured. results: 31/75 (41.3%) patients were considered to have developed restenosis after 6 months. 9/75 (12%) patients were found to have positive igg-(19-35 gpl) and/or igm-acl (14-103 mpl) at all three measurements. 1/75 was negative before but seroconverted (igm) 3 months after pta. 2/10 (20%) acl-positive and 29165 (44.6%) acl-negative developed restenesis at 6 months (chi-square p-value=0.14). all above mentioned coagulation parameters did not differ between acl-positive and -negative patients, measured before or 6 months after pta. some of them are shown below (values before pta): fbg ( basilar artery stenosis is a rare event in young children. risk factors are head or neck trauma with consecutive dissection of the vertebral artery, cardiac diseases or hypercoagulability. elevated lipoprotein (a) (lp(a)) serum levels in adults can mediate atherosclerosis. in addition, lp(a) might interfere with fibrinolysis. here we report on a 3 year old boy , who presented with acute brain stem symptoms. history revealed neither trauma nor infectious disease. conventional and mr angiography showed stenosis of basilar artery without ischemic lesions. laboratory findings were normal in routine blood and csf tests. global coagulation parameters as well as procoagulant and anticoagulant factors were normal. cardiac and autoimmune disease could be ruled out. lp(a) serum levels were significantly elevated to 104 mg/dl (normal range <30 mg/dl). analysis of other family members revealed a hereditary hyperlipoproteinemia (a) which might explain family history of an increased incidence of myocardial infarction and cve in elderly family members. clinically the patient recovered completely from brain stem symptoms after heparinization and subsequent oral anticoagulation with phenprocoumon. however, radiological signs of basilar artery stenosis were progredient. in a recently developed specific test, an elevated anti-phosphatidylserin antibody titer was detected one year after primary diagnosis. in conclusion, this is the first report on a child with stenosis of the basilar artery and elevated levels of lp (a). it is unclear, whether apa contributed to the onset of basilar artery stenosis or developed secondary due to endothelial defects after thrombosis and anticoagulation. apa, however, might increase the risk of further thrombotic events in this patient. in 110 patients with thrombotic events respectively patients with systemic lupus erythematodes antioardiolipin antibodies (aca) aund lupus anticoagulant (la) were ~ea~ured. for aca detecting we use the assays from elias for igg-and ig~}-antibodies. we use as sensitive methods for detecting la in our laboratory the testkits from diagnostlca stago (staclot la with hexagonal array of phospholipids, ,ptt-la a very sensitive pttmethod and staclot p~p-a platelet neutralization procedure) and the ptt from organon teknik~ (platelin excel ls with two incubation times, 1 and 10 minutes). i"~e results of this tests were compared with three new or~e on german market: specktin apot (aktlvated plasma clotting time), specktin aptt (aptt wlth purified soy extract) and 8pecktin la (phospholipid preparation in concentrations between 10 and 500 ~g/ml); all wak chemie. traditional aptt reagents were developed for the sensitive detection of factor vib an ix as a cause of hemorhage. high sensitivity against lupus anticoagulants, which also prolong aptt, was not required for this purpose, with increasing recognition of the importance of antiphospholipid antibodies as a risk factor for thrombembolism, more sensitive reagents were designed, which now reliable detect this condition. using such reagents as a screening test in a general hospital makes it necessary to distinguish both conditions quickly. we here report an algorhythm, by which we use an inhibitor (lupus anticoagulant) sensitive (sta aptt, boehringer) and an inhibitor insensitive reagent (actin fs, dade) to distinguish anticoagulants and factor deficiencies as a cause of prolonged aptt. citrate plasma from 33 patients with various diseases showed an unexpectedly abnormal inhibitor sensitive aptt (>40s). 13 plasmas with factor deficiencies remained abnormal with the insensitive aptt reagent. a regular correction of their defect occured on mixing with normal plasma. by measurement of single coagulation factors five patients with contact factor xii deficiency were found. this condition is associated with thrombosis and very rarly with bleeding. three patients with factor xi deficiency and two patient with factor ix deficiency were also identified. antiplatelets, of any kind, permits a secondary prevention of myocard ischemic lesions. there is no general consensus regarding secondary prevention of cerebral ischemic lesions. aspirin remains the most common substance, ticlopidlne also brings about prevention, but with important secondary effects. european stroke prevention study i has demonstrated that the combination of antiplatelets, in particular aspirin/dipyridamole (persantln), is also very active. to collect more information, esps 2 was organized and 6602 patients receiving either a placebo,either 50 mg aspirin,either 400 mg sustained release form of dipyridamole (persantin (r)), or the combination aspirin/dipyrldamole, were recruited. it ended march 31st 1995 with the following conclusions: i-aspirin, 50 mg a day, brings about a significant secondary reduction of stroke (18.z %), after a two year follow-up. notwithstanding the low dose of aspirin, haemorrhages remain important. 2-dipyridamole, at 400 mg a day, brings about a significant reduction of stroke (i6.3~), similar to that of aspirin. one could thus substitute 50 mg aspirin by 400 mg dipyridamole. 3-the combination of 50 mg aspirin and 400 mg dipyridamole brings about a significantly greater reduction of stroke (37.0 ~). esps 2 revealed that a low dosage of aspirin is active, that dipyridamole alone is also active, but that the combination of both gives far better results. the study of the primary end-points,the study of the survival curves, the factorial statistical analysis and the pairwise comparison analysis, led to these conclusions. the conclusions drawn from esp£ 2 underline that the combination aspirin/dipyridamole is a privileged choice for cerebral ischemia, the state of activation of circulating platelets in acute cerebral ischemia is controversial. activation of platelets on single cell level can be assessed by determining the shape change or the expression of antigens such as p-selectin (cd62). shape change is an early and rapidly reversible event in platelet activation whereas p-selectin is irreversibly expressed on the platelet surface upon stimulation. methods: we investigated 20 untreated patients within one day after cerebral ischemia, 20 patients months after stroke treated with warfarin, and 21 age and sex matched control subjects without vascular risk factors. venous blood was collected into a fixation solution blocking the metabolic processes in platelets within milliseconds. we determined the fraction of resting discoid platelets by phase contrast microscopy. the expression of p-selectin was measured by flowcytometry. results: the fraction of platelets expressing p-selectin was higher in patients with acute cerebral ischemia (7.8_+4.7%) than in control subjects (1.9_+1.1%; p<0.001, u-test). patients with stroke (n=15, 7.8+4.5%) and patients with transient ischemic attack (tia; n=5, 7.6-+6.1%) had similar values. patients months after stroke still had higher values (4.3+9,3 %, p<0.05) than control subjects. the rate of discoid platelets was not different between patients with acute ischemia (n=17, 85.6-+8.8 %), patients months after stroke (n=19, 85.7-+5.1%) and control subjects (n=21, 86.7_+5.8 %). platelet count was not significantly different between groups. conclusion: the elevated proportion of platelets expressing pselectin indicates strong platelet activation in acute cerebral ischemia and in a majority of patients months after stroke. assessment of pselectin revealed a higher sensitivity for platelet activation after stroke or tia than analysing the reversible shape change. further studies have to clarify if monitoring of platelet activation by flowcytometry is helpful as a prognostic tool and to evaluate therapeutic strategies after stroke. vascular smooth muscle cell (smc) proliferation and migration into neointima are the hallmarks of atherogenesis. the complexity of these processes and their concerted action and interaction of molecules are yet to be fully elucidated, one crucial molecule seems to be the urokinase-type plasminogen activator receptor (upar) recently also assigned as cd87 antigen, upar serves a dual function: (1) it directs upa proteolytic activity to a special location on the cell surface and (2) induces cellular signals leading to various phenotypic changes. we have investigated the signal-transducing capacity of upar in human smcs and provide here a molecular explanation for uparrelated cellular events. activation of these cells with upa (even with inactivated catalytic center) results in the induction of tyrosine phosphorylation, suggesting modulation of upar-associated protein tyrosine kinases (ptks) upon ligand binding. we obtained patterns of tyrosine-phosphorylated proteins with molecular masses of ~ 55-65 and 35-40 kd. using antibodies against different types of ptks as well as immunoprecipitation-and immunoblotting techniques the ptks involved in the upar-signalling complex were identified to be members of the src-ptk family. the cotocalization of upar and ptks at the cell surface of smcs was further confirmed by confocal microscopy studies. we conclude that the upar-ptk complex is most likely involved in this signal transduction pathway that provides the coordinated action of extracellular proteolysis, adhesion, and cell activation, which is required for cell migration. this mechanism may be crucial for the progression of atherosclerotic plaques. activation markers of haemostasis have been found elevated in relation to diabetic vascular lesions. simultaneous pancreas-and kidney transplantation (pkt) in type i diabetes has been shown to improve diabetic complications and long term survival. we measured haemostatic vascular risk factors and activation markers in plasma of 18 patients after successful pki', 17 patients after pkt and rejection of the pancreas graft and 5 patients after pkt and rejection of the renal graft. blood samples were taken during routine ambulatory visits, patients were free of any ongoing acute disorder or transplant rejection and under continuous immunosuppressive medication. despite individually adjusted insulin therapy hba1 plasma levels increased after pancreas rejection (5,37 vs 7,i2, p<0.001). platelet counts and plasma levels of fibrinogen, f1+2 fragment, tat-, app-complex and-fibrin monomer were found significantly elevated as compared to diabetic controls but not significantly different with respect to complete or partial successful pkt. one major reason of the increased activation state of haemostasis may be cyclosporin treatment given to all patients, t-pa and pal i plasma levels were within the normal range and significantly correlated to plasma triglyceddes (r.0.049; p<0.005). d-dimer plasma levels were significantly lowered after pancreas rejection (772(375) vs 324(232) nglml; mean(sem) p<0.005), which might reflect impaired fibrin degradation related to increased glycosylation of fibrinolytic factors. in conclusion, despite the marked improvement of glucose and lipid metabolism, plasma markers of activation of coagulation and flbrinolysis are not decreased to normal after simultaneous pancreas and kidney transplantation. according to the investigations of fowler et al. and pepe et al. the probability of an ards occurring with one risk factor is 5-8%, and in the presence of several risk factors, 25%. goris et al. and johnson et al. determined the level of severity with the aid of a fixed scale: the injury severity score. all these investigations are however not to be interpreted as typical following coronary surgery. these investigations demonstrated that the kallikrein and factor xii systems are of great importance as intraoperative risk factors. here the factor xii system plays a major role with direct or indirect activation of the kauikrein-kinin system with the splitting products alpha-factor xiia and bfactor xha respectively. all ards scores take the pmn-elastase into account. if the pmn-elastase values (1000 pg/l) are constantly high postoperatively then lung complications are to be expected. patients developing an ards displayed significantly lower alpha2-macroglobulin values. patients who developed a highly significantly raised kallikrein-like acdvity (>60 u/i) after the beginning of bypass and showed constantly high values during ecc are difficult to keep under control due to the blood pressure behaviour. the platelet pal also shows a significant rise and intraoperatively runs analogous to platelet factor 4, only antiparailel, since it attacks the endothelium. we were able to show that pai-1 is suitable as an indirect marker for a possibly developing restenosis. 85% of the patients investigated with lowered pai-1 values in the postoperative phase did not develop a restenosis. however, with patients showing significantly rising pa[-1 values from the 1 st. to 3rd. postoperative day 50% of all the cases had a restenosis. a further risk factor in this respect are significantly raised fibrinogen levels which lay over 800% at the end of surgery. if these fibrinogen values do not fall from the 1st. postoperative day onwards a raised risk of thrombosis must be reckoned with in the absence of therapeutic intervention. the following parameters represented haemostaseological risk parameters with significant behaviour within the framework of this study: 1) regards the blood pressure behaviour, the kallikrein-like activity (>60 u/i); 2) with regards to the lung complications, aipha2-macroglobulin and pmn-elastase (>900 g/i); 3) and final/y as a possible marker for a developing restenosis pai-1 and fibrinogen (>800%). resulting from numerous clinical studies homocysteinemia is found to be an almost independent risk factor of atherosclerosis including thrombotic complications as well as of venous thromboembolism. experimental investigations on the underlying mechanisms suggest endothelial cell damage accompartied by the development of an atherogenic and thrombogenic potential, increased platelet reactivity, oxidative modification of ldl, and enhanced affinity of lp(a) for fibrin. to our knowledge no results are published on the influence of homoeysteine on leukoeytes although these cells are deeply involved in pathological events within the vasculature. therefore, as a first approach different functional parameters of human polymorphonuclear leukozytes (pmnl) were followed under incubation with 60, 300, and 600 i.tm (final concentration) dl-homocysteine (hc) in isolated fractions or whole blood, respectively: l) spontaneous mobility of pmnl, measured as migration distance into micropore filters in a modified boyden-chamber, is found to be significantly enhanced by the two smaller hc concentrations. 2) chemotaxis induced by 0.1 i.tm formylmethionylleueylphenylaianine (tmlp) shows no significant differences. 3)monitoring of chemiluminescence signals (autolumat lb953, berthold) is complicated as hc influences the luminol-mediated indicator reaction. adjusting appropriate conditions the following results are obtained: spontaneous chemiluminescence and that induced by zymosane, tmlp, and the ca2+-ionophore a23187 are entranced by the two higher hc concentrations. there are, however, differences between the blood donors as a minority does not respond to hc in repeated measurements. with phorbol 12-myristate 13 acetate the signal is diminished by hc in all cases and with all concentrations. 4) phagoeytosis induced by zymosane (microscopic evaluation) as well as by opsonized e. coil (cytoflowmetric evaluation) is significantly increased by the two higher hc concentrations. conclusion: the activation of human pmnl is enhanced with respect to the majority of investigated stimuli by hc in concentrations reached under pathophysiological condititions. the effect of pysical exercise on hemostatic parameters was studied in 12 patients (male, mean age: 55 [range 36-68] yrs) with angiographically documented coronary artery disease (cad) and in 11 controls (male, 53 [43-62] yrs) both participating in an 1 hour group exercise session for cardiac rehabilitation. in each group relevant arteriosclerotic lesions in carotid, abdominal and leg arteries were excluded by doppler ultrasound examinations. patients were all under 6-blocking agents and aspirin. plasma levels of prothrombin fragment 1+2 (ptfi+2) and fibrinopeptide a (fpa) reflecting formation of thrombin and fibrin, respectively, were measured at rest and immediately after 1 hour of exercise consisting of jogging, light gymnastics and ball games. training intensity in both groups was comparable as indicated by the mean heart rate during exercise corresponding in patients to 80+6% (mean-+sd) and in controls to 79-+5% of the maximal heart rate previously determined on a bicycle ergometer. baseline values for ptf1 +2 were significantly lower in oatients (0.67-+0.15 nmol/i; mean-+sd) than in controls (1.01-+0.21; p<0.001 i. after exercise we found an increase of ptf1 +2 in controls to 1.14-+0.24 nmol/i (p<0.001) while in patients ptfi+2 remained unchanged (0.67-+ 0.14 after). accordingly, exercise induced r se of fpa was more pronounced in controls (from 0.62-+0.24 to 1.60-+0.62ng/mt; p<0.001) than in patients (from 0.63-+0.26 to 1.20+0.46ng/ml; p<0.00t). we conclude that in terms of thrombin and fibrin generation exercise training does not exert detrimental effects on hemostasis in patients with cad. lower baseline values and lack of exercise induced increases of ptf1 +2 in patients with cad might be attributed to medication with aspirin and/or b-blocking agents. periodontitis marginalis (pm) is an inflammatory oral disease that is caused by gram-negative bacteria and that has a high incidence in the second half of the life. clinical signs of pm are gingival bleeding, periodontal pockets, alveolar bone destruction and loss of teeth. recent epidemiologlcal studies have provided some evidence for an association between pm and atherosclerosis. in the present paper we will summarise some of the results that we have obtained in studies on patients with pm as well as on patients with hypercholesterolaemia (hc) and atherosclerosis. pm was frequently found to be associated with hc (90 % in rapidly progressive pm) and increased reactivity of peripheral blood neutrophils and platelets (e.g. generation of oxygen radicals and paf-induced aggregation). patients with hc and atherosclerosis had a higher frequency of severe pm when compared with data on the community periodontal health. the severity of pm was higher in patients with plasma cholesterol levels _> 6.5 mm when compared to those with plasma cholesterol < 6.5 mm. in patients with coronary atherosclerosis the severity of pm was significantly correlated with plasma cholesterol level, systolic blood pressure and the number of diseased coronary arteries. these results provide further evidence for an association between pm, hc and atherosclerosis. it can be speculated that hc is not only a risk factor for atheroscterosis but also a risk factor for pm and acts by increasing the reactivity of neutrophils and platelets. on the other hand, pm as a mild chronic inflammation could promote the development of atherosclerosis due to effects of endotoxins on vessel wall, blood cells and haemostatic factors. it has been also speculated that phagocyting leukocytes in the inflamed periodontal tissues could contribute to oxidative modification of ldl. so far, there is no evidence that atherosclerosis may contribute to the pathogenesis of pro protein z (pz) is a vitamin k dependant plasma protein synthesized in the liver. it promotes the association of thrombin with phosphorlipid surfaces. recently it has been shown that a deficiency of pz may lead to a bleeding tendency. in patients undergoing chronic hemodialysis, disorders of hemostasis are common. to examine if plasma levels of pz are altered in patients with end stage renal disease we determined pz in plasma of patients at the beginning of hemodialysis treatment. the results were compared with a group of 50 healthy controls. the difference of pz levels in plasma of patients with end stage renal disease with the control group was not significant. control group was 2900 + 487 ng/ml and in patient group was 3133 + -1394 ng/ml. one patient with marked bleeding tendency after hemodialysis pz was 1387 ng/ml. we concluded that patients with bleeding disorders pz determination may be helpful. the normal range of actin fs was reinvestigated in a multicentric approach. a protocol was developed which requests from each center to assess the aptt with one common and one variable lot of actin fs in 50 samples of suspected normals. inclusion and exclusion criteria based upon the results of clotting assays, liver enzymes and clinical data were defined. results: a total of 1056 results was obtained. the majority of centers in this study used the electra 1000 or 1600 c (mla). results for the electra group (n = 6) showed a precision for the common lot of actin fs with a common lot of a three level control from 1.5 % (level 1) to 1.1% (level 3) with an excellent accuracy between the 6 centers. clotting times with the variable lots of actin fs were very similar. the results from normals, however, showed a somewhat higher dispersion using the common lot of actin fs. 4 of 6 centers had almost identical mean values (range 26.6 to 27.2 sue) whereas one reported shorter and one longer clotting times (25.2 and 29.7 sec). results with the variable lots gave almost identical results as the common one. a total of 556 results of all lots gave a normal range of 23.5 to 31.7 (5 -95 % percentiles) on electra. mean values on acl (n = 200) were 26.3, on bct, 27.65 sec, on amga coagulometric, 29.4 sec, on amga turbidimetric, 26.6 sec (n = 100 each). all centers used sarstedt monovettes with 3.2 sodium citrate. discussion: the results of this study demonstrate the lot to lot consistency of all lots of reagents included in this study since the common and variable lots showed very consistent results. interestingly in the large group of electra users the normal ranges showed some differences, though the controls in all centers were almost identical. this confirms the recommendation that a normal range as stated from the manufacturer should be used for orientation only and that each laboratory should assess its own range. direct acting anticoagulant agents such as hirudin (r-h), argatroban (arg), efegatran (efe) and peg-hirudin (ph), represent specific and potent inhibitors of thrombin. blood samples collected in r-h (10 ~g/ml), arg (50 ~tg/ml), efe (25 ~tg/ml) and ph (10 ~tg/ml) do not clot for extended periods (>24 hours), thus allowing for the collection of plasma for analytical purposes. unlike heparin, these agents do not require any plasma cofactor for their anticoagulant effect. in contrast to citrate, oxalate, edta and heparin, these antithrombin agents do not alter the electrolyte or protein composition of blood. thus, blood collected in these agents may provide a physiologically intact (native) sample for clinical laboratory profiling. we have used all of these agents to prepare whole blood and plasma samples for various diuical laboratory measuroments. plasma samples collected with these agents are obviously not suitable for global clotting tests (pt, aptt, thrombin time, fibrinogen); however, these agents are optimal anticoagulants for the collection of samples for the molecular markers of hemostatie activation, such as fibrinogen/fibrin related degradation products, prothrombin fragment, protease cleavage products, tfpi, tnf and other protein mediators. electrolytes, blood gases, enzymes and protein profiling can also be satisfactorily measured on blood samples collected with these agents. antithrombin anticoagelatad blood used fur hematologic analysis showed equivalent blood count and differential results as that obtained with edta blood. unlike other anticoagulants, these agents do not interfere in the cell staining process. washed blood cells can also be prepared using antithrombin aents supplemented buffers for morphologic and fuuctional studies. thrombin inhibitors such as hirudin have also been used for flow cytometry and image analysis of blood cells and tissue exudates. our observations suggest that these anticoagulants can be used as suitable anticoagulants for clinical laboratory blood sampling. these agents can also be used as a flush anticoagnlant fur most automated instruments as these exhibit superior anticoagulant properties to heparin. furthermore, the hematologic parameters obtained in antithrombin anticeagnlated blood may be physiologically more relevant than those determined on blood collected in edta, citrate or heparin. antithrombin ul determination is one of the most popular method for in vitro diagnostic of number of different disorders. human fhrombin a~nity purified on heparin-rnodified silica-based sorbents was used for level of antithrombine lu determination by abilgaard method in blood of 40 patients with pregnancy pathology, acute leukemia, thrombocytopenia and anemia. it was founded, that antithrombin level is decreased to 50-60% of normal values in case of pregnancy pathology, to <50% -in case of acute leukemia and thrombocytopenia, to s0% -in case of anemia. obtained results show the strong relationships between named disorders and patient antifhrombin iii level. therefore anfifhrombine iii estimation may be used as simple and quick method for preliminary diagnosis of above named disorders. bm coasys 110 is a complete automated analyzer system for coagulation tests. it is well suited for routine coagulation testing in random access in a medium throughput laboratory environment. analytical performance and practicability were tested by a common evaluation program in five hospital laboratories. within run and day to day cv's were below 5 % in different samples (controls, patients) . comparison in different therapeutic ranges confirms the declaration of the isi-value for calculating inr-values. normal values for coagulation tests with results in pdmary units were checked in 390 samples and confirmed. due to the optical measuring principle of the bm coasys 110 there was a little tendency to shorter times with the thrombin reagent. in conclusion the performance of coagulation tests with the bm coasys 110 was rated as well or better compared to existing systems in the laboratories with advantages due to short timed familiarizing and easy handling. flexibility and stability of the system permit optimal integration and innovation into the w0rkflow of the routine laboratory. the purified thrombin and antithrombin iii (at iii) have a great interest in the clinical diagnostic and treatment practice, so their isolation methods are very important. molecules of these proteins have some fragments replying for interaction with native glycosaminoglycan, heparin. this interaction is used for isolation and purification of thrombin and at ul from native materials, blood plasma or its fractional products. we have done comparative studying these proteins purification on heparin sorbenfs, which contain heparin immobilized on sificagel, modified by glycidooxipropyl, gamma-aminopropyl and tosyl chloride groups, or on cellulose: heparin-epoxy-silica (1), heparin-gammapropyl-silica (2), heparjn-tozylsilica (3), and heparin-cellulose (4). we founded that thrombin binds with all sorbents, while at iii doesn't binds with sorbents 2 and 3. there wasn't any difference between silica and cellulose sorbents in thrombin desorbfion by t m naci. at iii binds more stronger with heparin-ceuulose t[~,~n with silica sorbents but specific activity and purity degree were approximately the same on both kinds of sorbents. thrombin specific activity and purity degree were approximately twice higher on sorbents 2 and 3 in comparison with sorbents ! and 4 (2250-3000 nih units/mg versus 1260-t350 nih unlts/mg). therefore, sorbents 2 and 3 can be used for isolation and purification of thrombin and sorbents t and 4 can be used for isolation and puriiication of at ii1. we used these sorbents for large scale purification of named proteins. purified thrombin was used for production of diagnostic kits for anfithrombine iii, fibrinogen, fibrin/fibrinogen degradation products and thrombin time determination. after an aerobic or anaerobic physical exercise various alterations of the hemostatic system were detected. numerous investigations of the hemostatic system exist of running and of bicycle ergometer exercise but not of swimming. young volunteers (n=54; median age 25 years) were investigeted~ there was an aerobic exercise (achieved heart rate 130 --10/min, lactate < 4 mmol; n= 27) and an anaerobic exercise (achieved heart rate 150 ~ lo/min, lactate > 4 mmol/1; n= 27). in both groups there was a significant shortening of the ptt. under anaerobic conditions hematocrit and quick significantly increased. factor viii activity rose significantly in both groups. indicating plasmatic clotting activation there was a significant increase in molecular markers tat and f1+2 only under anaerobic conditions (tat from 2,5 to 5,4 pg/1; f1+2 from 0,87 to 0,93 nmol/1). indicating activation of fibrinolysis t-pa activity increased significantly in the anaerobic group (from 0,1 to 0,4 iu/ml) but not in the other group. this findings indicate that there is e balance in the hemostatic system by activation of clotting as well as of fibrinolytic system in young volunteers during exercise by swimming dependend on the degree of exercise load. membranes as well as compact, porous disks are successfully used for fast analytical separations of biopolymers. as far as capacity, speed and performance of separation are concerned, the supports are as effective as other recently developed fast media for the separation of biopolymers °). so far, technical difficulties have prevented the proper scaling-up of the processes and the use of membranes and compact disks for preparative separations. in this report, the use of a compact tube made of poly(glycidyl methacrylate) for fast preparative separations of proteins is shown as a possible solution of these problems. the units have yielded excellent results, regarding performance and speed of separation as well as capacity. the application of compact tubes made of poly(glycidyl methacrylate) for the preparative isolation of the coagulation factors viii and ix from human plasma shows that this method can even be used for the separation of very sensitive biopolymers. in terms of yield and purity of the isolated proteins, this method was comparable to preparative column chromatography. the period of time required for separation was five times shorter than with corresponding column chromatographical methods. our measurements showed an excellent correlation of the two systems (r=0,99). the maximum amplitudes on the roteg were on average 2.2% higher than on the hteg, corresponding to a slightly lower reverse momentum of the measuring system in comparison to the hteg. we report first results out of the evaluation of sta compact (boehringer mannheim/diagnostica stack)). sta compact is designed for automated analyses of routine and special coagulation (chronometfic, photometric [405 nm] and turbidimetric [540 run]) tests. in addition, it does measure ,,derived" fihrinogen. tests as follows were evaluated: prothrombin time (pt), partial thromboplastin time (aptt), fibrinogen (clauss method), thrombin time, at iii (chromogen), hepato quick, as well as the factors ii, v, vii, x, and viii. results: within run cvs of the clotting tests were below 2% (calculated on the basis of seconds) in most cases, day to day cvs below 4% (not measured for factors, yet). at iii yielded within run cvs below 3% in the decision range. measuring ranges: at iii: 20-140 %; fibrinogen: 1.3-9.0 g/l (plasma -dilution 1/20), after rerun with other dilutions: from 0.3 g/1 (dilution: 1/5) to 18 g[l (dilution: 1/40). method comparisons, using sta as reference, yielded slopes close to 1.00 and negligible intercepts. throughput: with routine clotting tests about 100 tests/h, in a sample selective access mode. we conclude, that sta compact allows precise measurement of routine and special coagulation tests. it is also a reliable system for photometric tests and well suited for intermediate workloads as well as stat analyses. we did evaluate ptt lt, a new liquid, silica based ptt reagent. special attention was given to reference interval and heparin sensitivity. the new reagent is well suited for the measurement of intrinsic clotting factors and is reported to have high sensitivity for lupus anticoagulants (higher sensitivity than sta aptt [boehringer mannheim = bm]). it is stable for 4 days in the cooled compartment of the sta analyzer. methods: all experiments were made on sta. for comparison, we used three other ptt reagents (a lab. routine, silica based aptt, as well as sta aptt and sta ptt kaolin from bm). in addition, thrombin time (3 u/ml thrombin, sta thrombin reagent) and heparin (chromogenic xa test, rotachrom heparin) were measured. results: within mn imprecision (n=21) was below 0.7 % cv in the normal range and in two controls (mean values: 35 s, 76 s), and 1.4 % in a heparin plasma (mean: 81 s). between day imprecision (d=10) was below 3% in two controls ( mean values: 34 s and 58 s). the upper limit of the reference range is 40 s (97.5 th perc., median: 32 s; 90 patients with normal coagulation status [routine aptt, fib., pt], median age: 54 years); almost identical reference ranges were obtained with sta aptt and the routine ptt reagent, while sta ptt kaolin showed significantly lower values (97.5th perc.: 33 s, median 28 s). method comparison study: good agreement using plasmas from patients without heparin: (y= 0a + 0.98 x, n= 198, range of(x) from 25 to 79 s, r = 0.97; x = sta aptt). the median values from 54 patients under high dose heparin were: routine ptt: 81 s, sta aptt: 75 s, ptt -lt: 82 s0 sta ptt kaolin 54 s, thrombin time 37 s and heparin 0.5 iu/ml in conclusion, results of the new reagent compare well to our routine ptt and to sta aptt system reagent. it allows sensitive monitoring of high dose heparin therapy and is well suited for detecting abnormalities of the intrinsic clotting factor pathway. is a standard technique since many years. the interpretation of the thrombelastograms has been widely based on phenomenologic observations, while there is a lack of exact information concerning the coagulation mechanisms leading to the teg amplitude (a're~). the aa'ec is a measure for the mechanical stiffness of the clot and depends on: a) fibrin formation and adequate polymerisatiun of a 3-dlmensional network: measurements with nonrecalcified citrated blood activated with adp or epinephrine (both n=10) did not show any clot formation in the teg this relies on the need for a mechanical coupling between the teg pin and cup over a distance of 1 mm, which is accomplished by the fibrin network therefore, teg can only be performed under thrombin formation and thus under thrombin-activation of the platelets in the sample. factors, which inhibit platelet aggregation but don't limit thrombin-activation of platelets, cannot be monitored by teg. b) the attachment of the dot on the surface of the teg pin and cup. according to recent literature we suggest that the attachment of the clot in the teg relies exclusively on fibrinogen/fibrin adsorption to the surfaces of the pin and cup. interruption of this attachment can result in lower amplitudes or the so-called ,,stairway" phenomenon. we could show a complete interruption of the clot attachment by dipping the pin for one second in 30% albumine solution (n=10). c) the fibrinogen concentration (fg) and platelet count (pc) of the sample. in 50 volunteers we found only a poor correlation of the maximum amplitude (ma) with fg alone (r=0.40) or pc respectively (r=0.50), while there was a very good nonlinear correlation to the product of fg and pc. we suggest that the fibrin network forms the main structure of the clot while the thrombocytes enhance its stiffness in a concentration-dependent manner. this effect of the ptatelets can be completely reversed by gplibfllla antagonists. d) adequate coagulation activation: in nonactivated teg even small amounts of inhibitors can lead to a significant reduction of the ateg. conclusion: alterations in teg measurements can be judged more properly when the underlying mechanisms are understood. the consideration of the limitations of the method allows a more specific interpretation of the results. as a response on a customer request we did investigate the sample stability of blood samples for the aptt. the study was set up in a way that simulated the conditions of a large private laboratory in which the samples arrive several hours after blood collection. blood was drawn from 10 donors into 3.2 % sodium citrate and mixed well before it was divided into several aliquots which were kept at room temperature. the aliquots were centrifuged after ~ 0,5, 11, 23 and 29 h after venopuncture and the plasma was analyzed immediately with 3 different reagents on electra 1000. results: there was a clear difference in the change of the apttover time with these reagents. also f viii (determined with a chromogenic assay with complete and standardized activation) change considerably. 2 reagent a: ellagic acid, plant phospholipid, reagent b: sulfatide/kaolin, phopholipids, reagent c: ¢llagi¢ acid, plant and rabbit brain phospholipids the increase of aptt was apparently not a function of the decrease of fviii because the in vitro f viii sensitivity of reagent b. was inferior to reagent a though reagent b showed more prolongation of the aptt than reagent a. reagent c, however, showed only minor changes in the aptt. discussion: these data show that the sample stabifity of the aptt is reagent dependent and that it is not simply a function off viii sensitivity. other factors such as the buffer system but also the sensitivitiy towards other factors than f viii seem to contribute. a comparison of the technical principle of the roteg coagulation analyser and conventional thrombelastographic systems an. calatzis, p. fritzsche. al calatzis, +m. kling, +r. hipp, a. stemberger institute for experimental surgery and +institute of anesthesiology yechnische universit~t m0nchen thrombelastography (teg) was introduced by hartert in 1948 as a method for continous registration of the coagulation process. in 1995 we presented the roteg coagulation analyser, using a newly developed technical method. in teg systems according to i/artert the sample (blood or plasma) is placed in a cup which is alternately rotated to the right and left by 4,75 °. a cylindrical pin, which is suspended freely on a torsion wire, is lowered into the blood. when coagulation starts, the clot begins to transfer the rotation of the cup to the pin against the reverse momentum of the torsion wire. the angle of the pin is electromagnetically detected, transformed to the teg amplitude and continously recorded. in the roteg the pin is attached to a short axis, which is guided by a ball beating. thus all possible movement is limited to rpotation (r_oteg). the cup is stationary, and the pin is rotated alternately by 5 ° to the right and left by a feather system. when a clot is formed, it attaches to the surfaces of the pin and cup and starts preventing their relative movements against the reverse momentum of the feather. here the reduction of the rotation of the pin, which is detected optically, is tranformed to the teg amplitude. as can be shown by theoretical analysis and by control measurements, the roteg provides the same measuring capabilities as conventional teg systems. the main advantage is the solid guiding of the measuring system, which makes the roteg easily transportable and less susceptible to shock or vibration during measurement. yhrombelastography (teg) is a standard monitoring procedure for evaluation of coagulation. usually only nonactivated native blood teg measurements (nateg) are performed, which leads to a) a long time interval until coagulation and fibrinolysis parameters are available b) very high susceptibility of the measurement to inhibitors like heparin, which disturbes the judgement of other components of coagulation, c) unspecific results. our aim was to develop a coagulation monitoring system based on teg providing fast and specific information on the different components of coagulation. methods: the following measurements are performed in paralel using disposable pins/cups (haemoscope): a) extrinsic activated teg (exteg): 3551al whole blood (wb) + 5~tl innovin (recombinant thromboplastin reagent, dade). b) intrinsic activated teg (integ): 3551al wb + 5~tl kaolin (suspension 5g/l, behring). c) aprotinin teg (apteg): exteg + 20 kie aprotinin (trasylol, bayer). d) heparinase teg (hepteg) as decribed in (1). results: exteg and integ provide information on the extrinsic/intrinsic system within 5-10 min and information on the platelet/fibrinogen status within 10-20 min. because of the addition of potent activators integ and exteg can be performed when inhibitors like heparin are present in the circulation. fibrinolysis effects can be seen on exteg and integ and by comparison of exteg and apteg (apteg: invitro-fibrinolysis inhibition by aprotinin). if fibrinolysis is detected by exteg or integ and aprotinin-susceptibility is verified by apteg, aprotinin therapy will be initiated. heparin effects are revealed by hepteg. discussion: by the comparison of parallel teg measurements which have been differently activated, specific and fast information on the different aspects of the clinical coagulation status is provided. the presented tests can be easily performed bedside and only a small specimen of whole blood is needed (0,4-1,8 ml). introduction: a severely prolonged aptt (333s; normal: 40~os) was observed during preoperative screening for a planned splenectomy in a 71-year-old man with an 8 year history of osteomyelofibrosis. fellewing neer-normal~atien (71s) of the ap'ci" after 10 rain preincubation in a kaolin based aptt assay, pk deficiency was suspected and studies were performed to further investigate the nature of the pk deficiency as well as the mechanism underlying the normalization of the prolonged aptt by increasing the preincubation time. methods: the apl-r assay was peal'armed using kaolin/inesithin. high molecular weight kininogen clotting activitiy (hk:c), fxii:c end pk:c were measured by an aptt based assay using neothromtin ® (behnng) and 1 rain (pk:c) or 4 min (hk:c, fxli:c) preincubation. pk amidolytic activity (pk:am) was assayed using cosset pk ~ (chromogenix) and pk antigen (pk:ag) by quantitative immunoblotting. fxll and hk proteolysis dunng activation of plasma by kaolin (10mg/ml at 37=c) or ds (12.5~tglml at 4=c) was demonstrated by immunotilotting assays of fxii and hk following sds-page. assay pk:c pk:am pk:a~i fxfi: the propositus had pk:c<5%, pk:am=5% and pi~ag<2.5% as compared to normal pooled human p(asma (nhp). his son and two daughters had pk:c-50% and normal aptt values, incubation of the propositus' plasma with ds did not result in fxii or hk cleavage within 180 rain, whereas jn nhp detectable f×ii and hk proteolysis occurred after 5 rain and complete proteolysis was observed after -120 rain. in contrast, kaolin activation of propositus' plasma led to slow activation of fxii after 10 rain, presumably by autoactivation, and to fxlla-induced hk proteolysis. near-normalization of the propositus' aptt by prolongation of the preincubation time paralleled fxii autoactivation as evidenced by immunobletting. we describe a propositus with severely prolonged aptt due to hereditary, crm negative pk deficiency suffering from omf. activation with a particulate suspension of kaolin led to slow fxii autoactivation and hk proteolysis, whereas ds in solution did not induce fxii or hk cleavage. fxii autoactivation seems to be responsible for the normalization of the prolonged aptt in pk deficiency after prolonged preincubation times. in our study we compared a conventional bag with silicone tubing (a) for blood donation with 2 new ones (]3 from biotrans and c from baxter) with a newly developed y-shaped adapter. this adapter is integrated into the tubing and therefore provides the advantage for drawing blood samples in a closed system. the 3 systems were identical in amount and content of anticoagulant, i. e. 63 ml of cpd per bag resulting in approximately 14% of the final whole blood volume. the purpose of the study was to determine whether the different tubings can influence the quality of plasma products conceming the blood coagulation system. in plasma samples we measured several factors of the procoagnlatory and fibrinolytic systems. intralndividual control eitrated (.135 m) blood samples were initially drawn from the contralateral cubital vein from the same male donor (34 in each group). in all bag samples we found small but significantly higher levels of the global test parameters ap'it and ti" compared to controls, indicating a higher amount of anticoagulant. pt, however, revealed no differences, thus suggesting that factor activities were not altered (statistics according to mann-whimey). increase of procoagulatory activity measured as tat complexes showed elevated levels in bags a and c whereas prothrombin fragments fl+2 decreased only in a. conceming the fibrinolytic system, plasminogen a~tivators and pai-1 values were diminished in all three systems 03 < a < c) compared to controls. d-dimers were lowest in a followed by slightly higher values in c, controls and b. fibrin monomers did not reveal any significant differences: a < c < controls < b. in summary, the quality, of the 3 different blood sampling devices was comparable to the intraindividual controls as to factor activities measured by global tests. the activation of the procoagulatory and fibrinotytic systems was slightly but in most cases significantly higher in the two new devices than in the conventional one. all values, however, obtained from the plasma samples did not exceed the normal range of healthy blood donors. therefore we concluded that the two new closed blood drawing systems are favorable for blood donating procedures. in 20 patients with acute myocardial infarction (ami) and thromholytie therapy (13 patients with rt-pa, 6 patients with streptokinase and one with heparln) with ck, myoglobin and ekg criterions the patients were divided in two groups (reperfusion/no fellow two hours after starting the thrombolytic therapy) . blood samples were taken before, 30 rain, i h, 2 h, 4 h, 8 h,12 h after lysis and than every day till day 10. because of the central role of factor xii in activation of coagulation, fibrinolysis, kallikreln-kinin-system and complement cascade we investlgate the role of factor xila initiated by ami and the relation of factor xiia to the thrombolytie agent and reoeclusion rate. for the investigatlens we take the kits from shield diagnostics (xiia), behring diagnostica (c~-inactivator, pl~nogen, ~-antip]~n~n, pap), chromogefiilx ab (prekallikrein) and di~nostica stage (vile). the results: there is an increase of factor xiia immediately after starting the fihrinolysis (max. 30 rain after starting); the increase 5/i independently of the thrombolytie agent. parallel to factor xiia raises factor viia without significant changes of c1-1naotor and prekalllkrein. that means: activation of xiia and fibrinolytic pathway leads to relatively mild c.hanges in kallikrein system, hut to significant activation of extrinsic system by vila-tissue factor. in some patients is an additional rise in the system xiia -viia, when the fibrinolytic system is already in the normal range. there will need further investigations to define the risk of reocclusion as a result of activation of faktor viia by faktor >li ia. autoimmune thrombocytopenic purpura (aitp) is a frequent complication of chronic lymphocytic leukemia (cll] which developes on different stages of the disease and needs special treatment measure. mechanism of autoimmune disorders in cll remains uncleared. we investigated immunologic phenotype of blood lymphoid cells in 22 patients suffering from cll with aitp. in these patients we did not observe disorders in expression of b-lineage markers as compared with cll patients without immune complications (13 patients). but in the 1st group of the patients the greater number of b-celts expressed markers of activation. according to ig heavy chain expression, the lymphocytes in most cases of cll complicated by aitp had more mature phenotype. in all patients with k phenofype of cll lymphocytes we found immune disorders. the development of aitp was accompanied by lowered level of t cells and changed dis'flibution of their immunoreguiatory subsets: diminished number of cdz~cells and increased one of cd~'÷lymphocytes. the results of our investigations undirectly proved that malignant b-cells in cll are involved in production of autoantibodies against blood cells. dysbalance in t-cell system with functional disturbances of immunoregulation are significant in development of autoimmune complications in cll a24 in women with severe fvii deficency (<10%) hypermenorrhagia may cause life threatening blood loss. therefore, hysterectomy at a young age is reported frequently in the literature. a 12 year old girl without history for a bleeding disorder was transfered with hypermenorrhagia. the initial laboratory data revealed an abnormal quick-test of 30% due to fvll of 9,0%, normal platelet count and hemoglobin level of 7,2 g/dl. antifibrinolytic therapy (tranexamic acid 4x15mg/kg bw/d) and lynestrenol substitution were started to reduce the hemorrrhage. despite treatment the daily blood loss increased to a maximum of 290ml. therefore, substitution therapy with recombinant fvila (rfvila) (novonordisk) was started at a dose of 15 ilg/kg bw q 6 h. subsequently blood loss decreased to 30ml/d, but even with an increasing dose of rfvlla up to 35 i~g/kg bwq 4h (fvil activity max. 7400% 10 min after injection) and additional hormonal support with a lh-fsh-anatgonist some hemorrhage remained. a short .course of methergin was stopped due to severe pain. ultrasound of the uterus revealed a hypertrophic endometrium causing the persistent bleeding. it decreased slowly over several weeks and hemorrhage stopped completely after 40 d. the total rfvlla dose administered was 118 rag. no side effects were observed. no transfusions of blood products were necessary. currently, menstrual cycle is suppressed by estriosuccinate. conclusion: due to close cooperation with a specialised gynecologist, hypermenorrhagia was controlled and in this woman with severe fvll deficiency hysterectomy was avoided. in three male members aged between 27 and 52 years of a family suffering from inherited bleeding disorders the diagnosis of protein z deficiency was established. plasma protein z evaluated by elisa (asserachrom protein z, diagnostica stago, france) ranged between 200 and 300 ng/ml. the patients mostly suffered from moderate bleeding complications like prolonged bleeding secondary to trauma or invasive measures and also spontaneous hematuria. previous laboratory investigations revealed variable platelet function deficiencies and transitory boderline decrease of von-willebrand factor. spontaneous bleedings were rarely recognized, however, they occured more frequently when analgetics were taken. bleeding complications showed good response to hemostyptic measures and antifibrinolytic therapy. the use of pcc containing a high level of protein z in these patients is restrained to severe bleeding disorders or major surgery. defibrotide is a mammalian polydeoxyribonucleotide derived anti-ischemic and antithrombotic drug (crinos s.p.a., v"flla guardia, italy). while the drug is known to produce polytherapeutic effects owing to its multicomponent nature, the exact mechanisms of its anti-ischemic effects remain unknown at this time. since defibrotide is found to be effective in ischemic disorders such as paod, vod related occlusive disorders and related rnicroangiopathic conditions, we studied the effect of this drug on the contraction of dog and pig arterial strip/rings obtained from various sites. in vitro supplementation ofdefibrotide to the organ bath containing control dog and pig arterial rings did not modulate the serotonin and thromboxane (generated) contraction, however, tissues obtained from dogs treated with 10 mg/kg defibrotide iv exhibited a profound desensitization to the agonist induced contractile process. the time course of these effects was found to be much larger than the plasma half-life of defibrotide. this presentation will provide additional data on the effect of defibrotide on the contraction of vascular smooth muscles as a possible explanation for the anti-ischemic effects of defibrotide. a. wehmeier, a. popescu, w. schneider klinik for h,~matologie, onkologie und klinische immunologie der heinrich-heine-universit&t d0sseldorf in chronic myeloid leukemia (cml), evolution of blast crisis is the limiting factor of survival. however, as in other chronic myeloproliferative disorders, bleeding and thrombotic complications are a major source of morbidity but their incidence has rarely been analysed in larger patient groups. we retrospectively evaluated 182 patients with cml during chronic phase (170 cases), accelerated disease (58 cases), and blast cdsis (72 cases), and determined the incidence of thrombohemorrhagic complications in relation to the stage of the disease. in chronic phase, 28 patients had bleeding complications (8.4%/patient year) and 15 patients thrombotic episodes (4%/patient year). the incidence of bleeding increased significantly in accelerated disease (18 patients, 51.2%/patient year) and blast crisis (37 patients, 347%/patient year), and many patients had repeated complications. contrary to our expectations, the incidence of thrombotic complications also increased to 10.2%/patient year in accelerated phase and 39.8% /patient year in blast crisis, tn chronic phase, 3 patients died because of bleeding events. in accelerated phase, 5 patients died due to bleeding and 1 patient due to thrombotic complications. in blast crisis, bleeding was associated with 21 deaths, and pulmonary embolism with 2 deaths. analysis of the cause of thrombohemorrhagic complications revealed that in chronic phase, bleeding was often associated with uncontrolled busutfan therapy, whereas in blast crisis, severe bleeding occurred mainly when platelet counts were low and peripheral blasts increased. however, there was no obvious explanation for thrombotic complications. we conclude that bleeding and thrombotic complications are a major source of morbidity and mortality also in cml, and that the incidence of such complications increase in advanced stages of the disease. klinik for innere medizin °, klinikum schwerin patients suffering from primary or secondary amyloidosis may occasionally acquire a coagulation disorder characterised by isolated factor x deficiency. we report on a 60-years-old man who presented with lower gastrointestinal bleeding and prolonged prothrombin time (quick 50 %). amyloidosis was suspected and proven using biopsy of the rectum and histological analysis. in addition, a monoclonal gammopathy of undetermined significance was diagnosed by immunofixation (light chain, type x). detailed investigation of the prolonged prothrombin time led to the discovery of a pronounced factor x deficiency (residual activity 4 %). inhibitors of coagulation factors could not be demonstrated. the treatment of the patient consisted of red blood cell transfusion and infusion of prothrombin complex concentrates. due to the extremely rapid clearance of infused factor x, no increase of its activity was observed. chemotherapy of the monoclonal gammopathy was initiated (melphalan/ prednisone). over the following six months the frequency of major bleeding episodes gradually decreased. however, subclinical occult bleeding continued. the factor x activity was repeatedly found between 10 and 12 %. we support the suggestion from literature data that clinically relevant bleeding episodes are likely to occur in patients with amyloidosis-associated factor x deficiency if the residual activity is below 10 %. sepsis and septic shock is a disease entity which is characterized by inflammatory reactions (sirs), coagulation abnormalities (dic), organ failure (mof) and severe hemodynamic alteration frequently leading to death in a shock. the aim of our studies was to investigate the efficacy of antithrombin iii (kybernin ®) on ~he outcome of septic shock in a pig endotoxemic model. pigs, in this model respond to lps with elevated tnflevels, decreased leukocytes and platelets counts, increased tat and fibrin monomer levels, hypotension and in increase of the pulmonary arterial pressure (pap), indicating impaired lung function. a total number of 13 male castrated juvenile domestic pigs (25 -30 kg) were anaesthetized, ventilated mechanically and infused with saimonella abortus equi lipopolysaccharide (s. equ-lps) over three hours (0.5 ~g/kg * h). a swan-ganz-catheter was inserted into the pulmonary artery to measure the pap. animals were allocated to two groups,, the treatment group (n = 7) received antithrombin iii (at iii) according to the following regimen: 250 u/kg (t = 60 -30, i. v. infusion), 125 u/kg (i. v. bolus, t = 0) and 250 u/kg (t = 180 -240 rain, i. v. infusion). the placebo group ( n = 6) received the appropriate amount of human serum albumin: 50 -25 -50 mg/kg (same schedule as with at iii). main objective was defined as the mortality rate at six hours a_~er s. equ-lps infusion. whereas in the placebo group 4 out of 6 animal died (mortality rate: 66 %) all at iii-treated pigs survived the observation period of 6 hours (p < 0.05, x2-test). the at iii group was shown to have a lower pap than the control group, especially the second peak of hypertension was abolished by at iii. it is therefore concluded that at iii should be a useful tool for the treatment of severe sepsis and septic shock. in a nationwide monthly survey all childrens hospitals in germany (esped) were asked to clinical and therapeutical informations about children suffering from pmi. during july 94 till june 95 299 children were registered. from these, 87 had either ecchymoses and/or necroses related to an increased mordibity and mortality (20%), whereas 212 showed no bleeding signs except for petechiae. of these children one died. the therapeutic interventions concerning hemostasis are listed according to the defined two risk ~oups. from the patients with ecchymoses or necroses, 13/87 received combination therapy (compared to 5/212 with petechiae or no bleeding sign) of at iii, heparin and/or plasma. only t child received protein c concentrate. the data show that children with low risk did in part receive higher doses of heparin and/or at iii concentrate than did high risk patients, whereas plasma therapy was adjusted to severity of eoagnlopathy. furthermore, the wide range of given therapeutics allows no information about the different medications. therefore, controlled studies with respect to the different therapeutic interventions in children with high risk pmi is desirable. a fully automated procedure for the reptilase time assay y. schmitt (1) and h.j. kolde (2) (1) institute for laboratory medicine, st~dtisches klinikum, darmstadt, frg, (2) dade diagnostics, unterschlei6heim the reptilase time assay is a relatively simple technique for the detection of fibrinogen degradation products and fibrinogen deficiency or abnormality. the procedure is performed with citrated plasma and batroxobin reagent, a snake venom enzyme from bothrops atrox. this enzyme cleaves fibrinogen by releasing fibdno peptide a only but not fibdno peptide b. in contrast to the physiological enzyme thrombin that is readily neutralized by antithrombin iii and hepadn batroxobin is not inactivated by physiological inhibitors. at present this assay is mainly performed manually or on mechanical instruments. we have adapted this assay to the electra 1000 fully automated coagulation analyzer (medical laboratory automation, pleasantville, n.y.) using the thrombin clotting time procedure in the instrument software with batroxobin reagent (dade diathe clot formation is registrated turbidimetrically and the dotting time is pdnted. the within run precision (n= 10) of this procedure was tested with two plasmas from the daily routine and was between 2.8 and 3.4 %. in 25 normal samples we found clotting times from 10.5 to 12.8 sec. in 30 samples with liver disease (confirmed by pseudochlinesterase < 2000 u/ml) or on thrombolysis therapy with streptokinase or urokinase the fully automated assay on the electra was compared to the semiautomatic method using a kc 10 coagulometer (amelung, lemgo, germany) based on a rolling metal ball pdnciple and magnetic endpoint detection. the two assays agreed very well with a correlation coefficient of r = 0,948 and a regression line according to passing and bablok of y = 1.0 x + 1.7. these data show that the reptilase time can be performed with good precision and with good correlation to the manual technique on mechanical instruments on the electra 1000. introduction: disseminated intravasal coagulation (dic), due to a massive activation of the coagulation system, is frequently observed in intensive care patients suffering from severe underlying diseases. laboratory diagnosis of dic is based on different coagulation tests, but unfortunately the routine haemostaseological parameters react with latency in the course of acute dic objective: in four cases from a cohort of 43 patients with severe sepsis and dic we analysed special haemostaseological parameters (tat, f1-t2, d-dimers, human-leucocyte-elastese (file), catepsin g and heparin cofactor ii (hc ii)) and correlated them with a mof-score in order to test their predictability on the prognosis of these patients. results: all patients were substituted with at iii concentrate. l,1 the investigated patients median time of treatment with at iii concentrate was 8 (6-9) days and median time of dic-duration was 6 (4-8) days. none of the presented patients died during observation period. all analysed parameters, except d-dimers, showed a sufficient correlation with the evaluated mof-score (tat: r= 0,78; f1-f2: r= 0,84; hle: r= 0,71; catepsin g: r=-0,75; hc ii: 1"=-0,88). the d-dimers did not correlate with the mof-score, which is probably due to the delayed reactive hyperfibrinolysis in the course of dic. furthermore, the decrease of the tat-complexes, f1-f2, hle and catepsin g levels were followed by an increase of at hi and hc ii activity. conclusion: in general the analysed activation markers and coagulation parameters are sufficiently to describe the ongoing process of the dic. the hyperfibrinolytic activity of dic is sufficiently represented by the d-dimer test, but is of defered reactivity in the course of dic. unfortunately these parameters are not established in the routine monitoring of dic on intensive care units and therefore further studies are needed to investigate the practicability and reliability in the daily routine monitoring. we have previously reported that notoginsenoside r1 (ng-r1) has an effect on counteracting lipopolysaccharide (lps) induced upregulation of plasminogen activator inhibitor-1 and tissue factor expression in cultured human umbilical vein endothelial ceils in vitro and in mice in vivo [fibrinolysis 1994;8:(suppl 1)119]. in this study we investigated the effect of ng-r1 on prevention of lps induced lethal toxicity in mice. because mice are relatively resistant to lps when applied as a single agent, we sensitized them by simultaneous treatment with d-galactosamlne. the 80% lethality induced by lps (1.5 mg/mouse) plus d-galactosamine (8 mg/mouse) in c3hs-ie mice was reduced to 16% by simultaneous administration of ng-r1 (1.5 mg/mouse) with lps/galactosamine (p<0.05 by x 2 test). ng-r1 also significantly delayed lps/galactosamine induced lethal toxicity from 12 hours to 30 hours with all animals surviving beyond 30 hours. because lethality induced by lps involves the synergistic effect of multiple effector molecules such as tumor necrosis factor (tnf)-ct, interleukin (il)-i, interferon 3' etc., we also investigated the effect of ng-r1 on lps induced tnf-ct production from leukocytes in cultured human whole blood cells (hwbcs) ex vivo. the production of tnf--ct induced by lps (1 ng/ml for 24 hours) in the supernatant of hwbcs was inhibited by 46% and 22% respectively, when the cells were incubated 1 ng/ml or 10 ng/ml lps together with 100 i~g/ml ng-r1, respectively (tnf-ct concentration, 1 ng/ml lps treated cells: 297+192 pg/ml, i ng/ml lps plus 100 l.tg/ml ng-ri treated cells: 162+137 pg/ml, p<0.01; 10 ng/ml lps treated cells: 3094_+487 pg/ml, 10 ng/ml lps plus 100 pg/ml ng-r1 treated cells 2423+713 pg/ml, /'=-0.02). the present results suggest that ng-r1 can prevent the onset of lps toxicity as well as the lps induction of cytokines. therefor ng-ri may be effective in preventing the effects of septic shock in gram-negative infections. to elucidate the mechanisms by which coagulation is initiated in septic patients in vivo, coagulation measurements were prospectively evaluated in patients with severe chemotherapyinduced neutropenia. this group of patients was chosen because of their high risk of developing severe septic complications, thus allowing serial prospective coagulation testing prior to and during evolving sepsis or septic shock. 62 patients with febrile infectious events were accrued to the study. of these, 13 patients progressed to severe sepsis and an additional 13 patients to septic shock. at onset of fever, factor (f) vlla activity, f vii antigen and antithrombin iii (at iii) activity decreased from normal baseline revels and were significantly lower in the group of patients who progressed to septic shock compared to those that developed severe sepsis (medians: 0.3 versus 1.4 ng/ml, 21 versus 86 u/dl and 45 versus 95%; p < 0.001 ). the decrease of these variables in septic shock was accompanied by an increase in a marker of thrombin generation like prothrombin fragment 1 + 2 (medians: 3.6 versus 1.4 rim; p=o.05). these differences were sustained throughout the septic episode (p < 0.0001 ). f vlla and at ill levels of <0.8 ng/ml and <70%, respectively, at onset of fever predicted a lethal outcome with a sensitivity of 100 and 85%, and a specificity of 75 and 85%, respectively. in contrast, fxila-alpha antigen levels were not different between both groups at onset of fever and were only marginally higher further during the course of septic shock (p=o.001). thus, septic shock in neutropenia is associated with significant coagulation activation, presumably driven by the tissue factor pathway rather than the contact system. furthermore, in septicemia both f vlla and at iii measurements are sensitive markers of an unfavourable prognosis. hemostatic parameters in sepsis patients treated with anti-tnfct monoclonal antibodies c. salat 1, p. boekstegers 2, e. holler 1,3, b. reinhardt i, r. pihusch 1, k. werdan 2, m. kaul 4, t. beinert 2, e. hiller 1 med. klinik iii 1 und i 2, klinikum grosshadern der ludwig-maximilians-universitat monchen, h~imatologikum der gsf 3, knoll ag ludwigshafen 4 tumor necrosis factor et (tnfc~) is a central mediator in the pathogenesis of sepsis and septic shock. as administration of anti-tnfct monoclonal antibodies was able to protect animals from an otherwise lethal endotoxin challenge clinical studies were initiated in patients with sepis. tnfct exerts a procoagulant effect, e.g. by enhancing pai-i and activating thrombin as indicated by an increase in tat and pf 1/2 levels. therefore it may be involved in disseminated intravascular coagulation in sepsis. we determined tat, pf 1/2, d-dimers, tpa, upa, pai-i and vwf levels in 30 patients with sepsis or septic shock. 14 patients received the anti-tnfa monoclonal antibody mak 195f (knoll ag, ludwigshafen), whereas 16 patients served as controls. we found a significantly lower level ofupa in anti-tnfc~ treated patients. since the difference existed before onset of treatment it can not be attributed to tnfot antagonisation. all other parameters investigated did not differ significantly between the two groups throughout the study period. failure to detect modulation of hemostasis by anti-tnf~ might be explained by delayed initiation of treatment in clinical sepsis. in animal experiments it has been observed that the antibody prevented lethal endotoxin effects when given prophylactically or 30 minutes after endotoxin challenge, but not when it was administered 2.5 hours later. in addition, beneficial clinical and hemostatic effects of tnfet antagonisation might be observed only in subgroups of patients with hyperinflammatory sepsis. larger studies addressing this point are under way. protease receptors for thrombin and trypsin have been described for different cell lines. we investigated the ability of trypsin to activate human umbilical vein endothelial cells (huvec). cell activation was measured by the increase of intracellular free ca 2* (caff) with help of microscope fiuorometry (fura-2) and by the von willebrand factor release measured by a sandwich elisa. incubation of huvec with thrombin (1u/ml) or trypsin (10nm) showed a 2-10 fold increase of c~ff. a subsequent homologous stimulation after 80 s lead to a 2-5 fold lower concentration of ca~ 2÷ compared to the first stimulation. therefore cells have been desensitised by the first stimulation. inhibition of the proteolytical activity of trypsin by soybean trypsin inhibitor was followed by failure of trypsin inducing an increase of ca~ 2÷ concentration. in cross stimulation experiments with thrombin and trypsin, we could demonstrate, that cells first stimulated with thrombin showed a second maximal response by subsequent stimulation with trypsin. the same effect was measured with first stimulus trypsin and second stimulus thrombin. trypsin and thrombin induced a release of von willebrand factor (2-5 fold in comparison to unstimulated cells). we found a vwf release dependent on the concentration of trypsin similar to thrombin. an electrophoretic analysis of the released von willebrand factor showed a different multimeric composition of vwf between trypsin and thrombin stimulation. these results indicate, that there might be a protease receptor on huvec for trypsin being different from the thrombin receptor. clinical and laboratory findings of coagulopathy were investigated by an 1-year-survey to 320 children's hospitals. 291 meningococcal infections were evaluable. severe disease (characterized by need for mechanical ventilation, dialysis and/or catecholamines) was seen in 42 of these children; 29 of those survived and 13 died. clinical signs of severe coagulopathy were seen in 83 children: ecchymoses (n = 73) and skin necrosis (n = 36) were associated with increased mortality (16% and 20%, resp., compared to 4.5% overall mortality). five of 29 surviving children with skin necroses required surgical interventions (skin transplantation and/or amputations). petechiae were frequent (n = 156) and as isolated finding not related to severe disease or fatal outcome (6% mortaliy). platelet counts at admission were lower in non-survivors (10th-90th percentile: 30 -450.000/gl, median: 139.000/i.tl) than in survivors (10th-90th percentile: 140 -480.000/i.tl, median: 242.000/gl). at iii values showed no difference between survivors and non-survivors. protein c was available in few patients (n =14): in this subgroup, protein c was lowered in patients with limited disease (10th-90th percentile: 20 -105%, median: 48%) as well as severe disease (10th-90th percentile: 30 -75%, median: 60%). in conclusion, the findings "ecehymoses" and "skin necroses" were related to fatal outcome and therefore included in a prognostic score for severity of meningncoccal disease. the influence of irradiation on pai-i and vwf levels in human umbilical vein endothelial cell cultures k. fragiadaki, c. salat, r. pihusch, b. reinhardt, m penovici, e. hiller med. klinik iii, klinikum grosshadern der ludwig-maximilians-universitat monchen an elevation of pai-1 in bone marrow transplant recipients developing veno-occlusive disease (vod) of the liver has been described earlier. endothelial cell damage due to the preparative myeloablative radioehemotherapy is supposed to be an important step in the pathogenesis of the disease, which is characterized by an obstruction of small intrahepatic venules. in order to investigate a possible role of irradiation we studied the influence of several doses (0, 5, 15, 30 gy) on pai-1 and vwf levels in the supematant of human umbilical vein endothelial cell cultures (huvec). pai-1 antigen and vwf were determined by enzyme immunoassays. whereas pai-1 and vwf levels remained unchanged alter irradiation with 5 gy and in control cultures, a rise was observed one day after irradiation with 15 gy (mean day 0"-)day +1) in pai-1 (100,0% --)171,2 %) and vwf (100%--)159,7%) levels. the increase was more pronounced and reached levels of statistical significance after a dose of 30 cry (pai-1 100%--) 278,7% and vwf 100%--)168%). both pai-1 and vwf levels decreased on day 2 after irradiation with 15 and 30 gy. our results indicate that irradiation induces an increase of pal-1 and vwf in endothelial cells. nevertheless, this effect was observed only in doses above those ones used during conditioning when patients receive 3x4 gy. additional factors seem to be of significance. cytokines like tnfo~ enhance pai-1 and vwf in endothelial cell cultures and are known to be elevated in bmt-associated complications. it can be speculated that irradiation in concert with these factors may contribute to the development of veno-occlusive disease. disseminated intravascular coagulation is characterized by high consumption of coagulation factors, systemic elevation of fibrinolysis by tpa and concomitant elevation of pai-i secreted from inflamed endothelial cells. in an attempt to investigate the contribution of inflammatory cytokines, endothelial cells lines of microvascular origin were stimulated in vitro and pal-1 antigen was measured 2h, 4h and 24h after stimulation. in contrast to results published from experiments performed with macrovascular human umbilical vein cells (huve), our results obtained with 3 different microvascular endothelia isolated from skin, solid tumor tissue and bone marrow revealed that inflammatory cytokines reduced pal-1 antigen levels. in addition to tnf-a (25ng/ml) and lps (10pg/ml), we found that il-10 (100 u/ml) and gm-csf (100 u/rot) also reduced pai-i levels within the first 2h of incubation (from 120ng/ml to 80-110 ng/mll and the effect was even more pronounced after 4h and 24h (from 380 ng/ml to 250 ng/ml). il-1 (10 u/ml) and lps (10 pg/l) also reduced constitutive levels of pal-1 but the effect occured later than 4h after addition of the stimulator. the strongest synergistic effect was demonstrated with gm-csf plus il-1 resulting in pal-1 suppression of 50% after 2h and 30% after 24h. in contrast, g-csf (300 u/ml) induced the immediate (120 to 140 ng/ml after 2h and 380 to 420 ng/ml after 24h) upregulation of pal-1 antigen. stimulation of pat-1 levels was also observed with tgf-i~ (10 pg/ml), however not earlier than 18h of incubation. interestingly, both stimulatory cytokines, ie. g-csf and tgf-13, alone were able to counteract the decrease of pat-1 antigen by tnf-a but only a combination of g-csf plus tgf-g neutralized the effect by il-1. results indicate that inflammatory cytokines regulate pal-1 fibrinolysis in a synergistic and antagonistic fashion. we established the culture of human brain microvascular endothelial cells (hbmec) in order to investigate the pathophysiology of hu~man cerebral malada, which is still associated with a high mortality rate. it is widely accepted that among the reasons for the fatal outcome of cerebral malaria, the interaction of endothelial cells with cytokines and paras lites with subsequent changes in haemostaseological parameters is involved. the human microvascular endothelium may therefore play a deci §ive role in the pathophysiology of cerebral malaria. ery throcytes containing later stages of p. falciparum specifically bind to capillary ec in vivo (sequestration). tnf-cq il-1 and il-6 are considerably elevated in severe malaria. coagulation factors such as tissue factor and von willebrand factor are affected by malada suggesting the involvement of the hbmec in cerebral malada. so far, research on the involvement of the hbmec has been performed on ec cultured from human umblilical veins (huvec). the relevance of this model may be questioned on t, ,he grounds that the capillary endothelium probably plays a greater role than the endothelium of the large vessels. besides, some propertie.$ of the endothelioum seem to vary, upon the organ of origi/n. for the~ reasons, our laboratory has established the hbmec as a model to study the pathophysiology of human cerebral malaria. to demonstrate the relevance of this model in the context of malaria, hbmec were challenged with sera from different patients with severe p. falciparum malaria and with serum from a healthy donor. we can demonstrate that in cells challenged with malaria patient sera icam-1 and substance p were upregulated. on the other hand cells challenged with serum from a healthy donor expressed neither icam-1 nor substance p. these results strongly suggest the relevance of this model for vessel involvement in malaria. both, histamine and serotonin have been described as potent stimulators of yon willebrand factor (vwf) release from human umbilical vein endothelial cells (huvec). we performed experiments to differentiate the receptors for histamine and serotonin induced vwf release. absolutely unexpected we don't found any significant vwf release after the addition of serotonin to huvec or human artery endothelial cells (huaec) in concentrations from 0.1 ijm to 50 pm. in the case of histamine (0.1 pm -50 pm) we measured a vwf release 2-5 fold compared to unstimulated cells. this release was in the same order of magnitude as the release induced with 11u thrombin. to verify these results we measured the effect of histamine and serotonin on the intracellular ca 2÷ concentration (ca~ 2÷) in huvec and huaec. cells were labelled with fura-2 and the change in fluorescence after agonist addition was measured with a microscope fluorometer. using the same agonist concentrations as above we found an 5-10 fold increase of caj 2. with histamine or thrombin but no effect by addition of serotonin. this results indicate a similar activation of human endothelial cells by histamine and thrombin and that serotonin don't stimulate endothelial vwf release or increase of cay. activation and/or dysfunction of the endothelium can be triggered by cytokines (e.g. interleukin-2, tumor necrosis factor-alpha) or bacterial substances (e.g. endotoxins) and may contribute to shock and multi organ failure. pal-l and tm were assessed as parameters of activated endothelium following bsct in three to four days intervals from start of conditioning therapy through day +35. data were compared to the occurrence of sepsis, veno-occlusive disease (vod), capillary leakage syndrome (cls) and graftversus-host-disease (gvhd). patients with neither complication served as controls. no *days after stem cell tranplantation pai-1 and tm were increased in all patients with sepsis, cls~ vod and/or gvhd. pai-1 peaked at days 14 to 18 and the increase was highest in sepsis and lowest in cls. the increase in tm values was somewhat delayed (day +24) and was highest in vod and cls and lowest in gvhd. pai-1 and tm are sensitive markers of endothelial activation in sepsis, vod, cls, and/or gvhd, but they do not allow a differention between these complications. endothelin (et) is the most potent vasoconstrictor. it is known that et plasma concentration is correlated with a poor prognosis in patients with non ischemic cardiomyopathy (cm). the contribution of the heart to the production of et is still unknown. to investigate the pathogenetic mechanism in patients without coronary artery disease (cad), we examined 13 patients with hypertension ( . pulmonary capillary wedge pressure (pcwp) was measured in all patients. et and its precursor big-endothelin (bet) were determined at rest and after pharmacological stimulation with dipyridamole (0.5 mg/kg body weight), that increases coronary blood flow by factor 2 -4 on a non endothelial pathway. cardiac coronary et and bet concentrations were determined from the arterial blood samples, obtained from the aorta, and simultaneously from the coronary sinus (venous blood). blood samples were collected into ice chilled vacutainer tubes and stored after centrifugation at -70 *c. et and bet were analysed after extraction by a sepal< c 18 cartridge by radio immuno assay technique (immundiagnostik). it is concluded that et is increased with elevated filling pressures of the heart in patients with cm. it is not produced in considerable quantity by the heart neither at rest nor at increased blood flow. there4ore the lung has to be considered as the major organ for the production of et and bet in patients without cad. to characterize the incompatibility of blood with foreign surfaces valide in vitro methods especially in testing of platelet function are neceessary. it seems to be effective to use test systems which can also be helpful lateron in the clinic when foreign surfaces (e.g. venous catheters) are used and evaluated in so called phase-4-studies. we studied the influence of 21 reference polymers under standardized and controlled flow conditions on platelets in citrated blood specimen of healthy blood donors.the following tests were performed pre and post platelet-pol)aner contact: decrease of platelet count, platelet aggregation (wu-gmtemeyer index), analysis of platelet spreading capacity on standardized plastic surfaces by using a visual microscopic evaluation according to breddin and bfirck (1963) and an interactive computer-aided system (ibas, kontron gmbh, manchen, frg) by digitalizing the morphological picture of the platelet slides and area detection with a resolution of 512x512 pixels. results: platelet counts showed significant differences pre and post polymer contact, the wu-grotemeyer index demonstrated platelet activation only by blood contact with large volumes of polymeric material whereas both visual and computer-assisted evaluation of platelet spreading ability revealed a marked shift in the different classes of platelets: platelet activation results in a decrease of large structural elements and an increase of elements with spider threads. (pre contact (n=1000): 27:~-6 large forms of platelets, 700~-39 small forms and 275:l-41 spider forms; post contact (n=1000): 6-+-5 large forms, 510a:56 small forms and 484±58 platelets with spider threads). in some series there were significant differences between visual and computer-aided evaluation in the detection of small and spider forms. however, the relative increase of these nonspread spider forms could be stated with beth methods (wilcoxon test). we therefore conclude, that platelet morphometry with both methods is a sensitive and reliable ex vivo method to evaluate platelet interactions with artificial surfaces and can also be used lateron in phase-4-studies in patients. however, the ibas-system requires further maprovement in hard-and so,ware to reduce the high expenditure of this method. despite for the most part standardised methods such as hypothermia, cardioplegia the perioperative myocardial infartion rate is still high at approx. 6%. in cardiovascular surgery it is well known that various cardioplegic solutions are employed for myocardial protection during the ischemic phase. in order to evaluate the possible influence of these solutions we selected two of the most commonly used cardioplegic solutions for investigation in a randomised double-blind study: htk (group 1) and st. thomas (group 2). after randomisation each group consisted of 20 patients who had to undergo aortocoronary bypass surgery. aim of the investigation was to establish possible varying cellular changes during the reperfusion phase or in the early operative phase in order to be better able to apply reinforcing clinical measures. in the context of this study the classical enzyme-diagnostical methods ck,ck-mb and ldh as most useful, however not as convincing. still, we have in the meanwhile been able to show that the cardiac muscle troponin t proves a particularly sensitive parameter regards differentiated ischemic damage to the myocardium. ~his we were able to conflrm in extensive preliminary trials. cardiac troponin t was registered with a one-step lmmunoassay using two highly specific monoclonal antibodies directly via two different epitopes of cardiac troponin t. simultaneously the corresponding pre-and postoperative ecg was registered. further, within this context we investigated parameters that indicate cellular damage, such as platelet factor 4 (pf4), t-pa, interleukin-6 and pmn-elastase. in the reperfusion phase in group 2 there is a significant rise in tmponin t while in group 1 these values remain practically unchanged up to the 1st. postoperative day. of special importance is interleucin 6 since according to most recent studies the release of this substance leads to platelet activation via the arachidonic acid metabolism. this pathway must, further, be regarded within the context of free radical formation. on the 1st. postoperative day the 11 6 values in group 2 are significantly higher. the effects of membrane damage is also observed via pf4 and the pmn-elastase to be different in both groups. on the basis of this study we arrive at the conclusion that the htkcardioplegia is essentially less damaging than that of the st. thomas solution. (2) r. hetzer (2) (1) department of hematology and oncology, vimhow klinikum, humboldt university, berlin, germany (2) we investigated the influence of two different vad systems on these hemostatic changes. vads were implanted in 18 patients [11 bi-vad (berlin heart), 7 left vad (novacor n 100)] with end-stage heart disease who were awaiting heart transplantation. the following hemostatic parameters were measured during the first 51 days of bddging or until heart transplantation: thrombin-antithrembin iii (tat) complexes, prekallikrein, factor (f) xll, plasminogen, or2 -antiplasmin, and i?,thremboglobulin. results: during the first week of bridging, significantly higher tat levels were observed in novacor patients compared to berlin heart patients. prekallikrein activity levels were significantly lower in the berlin heart patients in the early bridging period. all other parameters were comparable in both groups throughout the entire observation period. differences in hemostatic parameters became apparent only in the early bridging period with more enhanced pmthrombin activation in the novacor group and more prominent contact activation in the berlin heart group. avoidance of the transmission of viral infections and saving in the use of blood products encouraged the use of apparatwe intraoperative autetransfusion techniques. patients and methods: arer randomization apparative intraoperative autotransfusion was performed in 5x7 patients during elective hip surgery using i-iaemonetics cell saver ill, haemonetics cell saver v, electromedics elmd, haemolite 3 and fresenius continuous autotransfnsion system (cats). at defined tmaes we detenmned a lab panel (clinical chemistry, lipids, proteolytic capacity, hemolysis, coagulation panel) at 9 determination points in the reservoir, the retransfused blood and in the patient. results: no significant differences concerning proteolytic capacity, prothrombin time, platelets, lipids, electrolytes. increased hemolysis (p<0.01) in the hcs iii group vs. the other groups (lo rain. after application of the retransfnsed blood). low heparin concentrations of retransfused blood in the hcs iii group( 0.32+-0.3 u/ml) vs. high concentrations in the cats group (0.47 +-0.3;p--0.01). parameters of thrombin generation were elevated in the hcs iii group vs. the other groups (p=0.02). conclusions: the use of 5 different apparative autotransfnsion systems dunng elective hip surgery results in dysturbances of hemocompatibility. the activation of the coagulation system during the collection and filtering is partly influenced by the elimination kinetics and the dose regime of heparin. however intraoperative autotransfusion must be roan~ged very carefully and possibly adverse effects of perioperadve heparin peak levels have to be considered. little information is available on the management of patients with factor viii deficiency who require cardiac surgery. we report the case of a 54 year old man with factor viii deficiency and combined severe aortic stenosis and incompetence and mitral incompetence who underwent a double valve replacement at our institution. he had a history of several bleeding episodes following minor surgery. previous factor viii levels were between 8 and 26%. using standard cardiopulmonary bypass, a double valve replacement with a 23 and 29 mm bileaflet prosthesis in aortic and mitral position, respectively, was performed. a high dose aprotinin regime was used (5.5 x 10 a iu). three doses of factor viii concentrate were given in the perioperative period, totalung 7000 1u until the 1st postoperative day. repeated measurements of the factor viii level were performed. the postoperative chest tube drainage was 350 rot. until the 4th postoperative day an additional dose of 3000 iu of factor viii was given to maintain a level of at least 30%. the obligatory anticoagulation was achieved initially with heparin i.v. in therapeutic dosage. due to a persistent 3rd degree av block a permanent pacemaker was inserted with additional 2000 iu of factor viii. on the 17th postoperative day warfarin was commenced aiming for an inr of 3.0 -3.5. the patient was discharged home therearer. he was trained to monitor his inr with a coagu chek device. no bleeding episode occurred during the first 3 months follow up. open heart surgery can be performed safely in patients with factor viii deficiency with the use of factor viii concentrates and monitoring of factor viii levels. coating of biomaterials was developed using synthetic polymers with incorporated anticoagulants. stents were coated with a thin layer consisting of a polylactide polymer containing peg-hirudin and a stable prostacyclin analogue. these materials were tested with a ,,human shunt model" using nonant/coagulated blood of healthy volunteers. within minutes uncoated stents were covered by fibrin and aggregated platelets, which could be seen macroscopically and by scanning electron microscopy; coated stents were free from coaguiation plugs. this observations were supported by analysis of coagulatiuon activation markers. unlike coated stents, uncoated stents revealed high levels (>detection limit) of tat complexes and prothrombin fragments (f1-2). in a series of experiments stents were tested in sheep. in 16 sheep stents (coated/uncoated patmaz-schatz stents) were ptaced by conventional techniques in the left anterior descending artery. anticoagulant therapy consisted of a heparin bolus and intravenously given aspirin before stent implantation. no ant/coagulation was given thereafter. existing data show hyperplasia in the area of uncoated stents which was reduced around coated stents (this study will be finished in january 1996). this coating technique with incorporated anticoagulants reduces thrombogenicity during the early and late phase of biomaterial implantation. studies concerning catheters, vascular prosthesis and oxygenators are in progress. the mechanical circulatory support (mcs) is a therapy for patients (pts) with endstage cardiac insufficiency. during mcs thrombeembolic events, due to the surface thrombogenicity of the implanted device, are feared complications. activated blood platelcts play a major role in this context. therefore, patient's platelet morphology was investigated. during the period of mcs, using the novacor left ventricular assist system n100, blood samples of 8 pts were observed by means of scanning electron microscopy (sem). blood was collected preoperatively and after implantation daily during the first week as well as weekly for the first 3 months. samples were drawn via an 18gauge cannula into caeodylic-acid buffered glutaraldehyde and platelets were prepared for morphological investigations. platelet alterations were classified as non activated, activated and aggregated, based on "shape change" morphology. additionally, the common blood coagulation parameters were evaluated. preoperatively, 15.0 + 4.6 % of activated platelets were found. within the first postoperative week, the mean level of activated platelets raised to 32.8 + 8.0 % (p<0.05). comparing short-(<30 days) vs. long-term (>30 days) mcs, a significant difference of activated ptatelets (overall mean values) could be seen (24.3 +_ 3.3 % vs. 34.8 _+ 3.4 %, p=0.004). during mcs a correlation between hemolysis and platelet aggregates, as well as the values of activated dotting time and activated platelets were observed. also, specific platelet deformations and damages appeared during mcs, which could not be found preoperatively. all pts with mcs showed alterations of their platelet morphology induced by the activation of the implanted synthetic material. with regard to the postoperative antithrombotic therapy, these observations should be taken into consideration. during extracorporeal circulation (ecc) the blood and its compenents are exposed to artificial surfaces and inflammatory respenses are activated, especially the complement, coagulation, fibrinolytic and kallikrein systems. furthermore leukocyte activation occurs and platelet function is impaired. these humoral and cellular systemic responses are known as the "pustperfusion syndrome" with clinical symptomes like lenkocytosis, increased capillary perraeability, accumulation of interstitial fluid and organ dysfunction. the impertance and even perhaps the existence of the damaging effects of cpb have been widely debated in the literature over the past 30 years. many efforts have been made to reduce traumatizing factors, e.g. the use of membrane instead of bubble oxygenators. recently, heparin-coated equipmen~ and tubings have been proposed to avoid excessive contact activation during cpb, the here presented study was designed to assess changes in coagulation and flbrinolytie activity in 20 patients undergoing cpb. in this regard we investigated coagulation parameters like fibrinogen, antithrombin, pmthrombin-fragments fl+2, thrombin-anthhmmhin complex, tissue-factor, fibrin-monomeres and parameters of the fibrinolytic system like tissue-plasminogen-activator, plasminantiplasmin-complex, d-dimers and plasminogen-activator inhibitor before, during and after cpb. the activation of the complement cascade was followed by measuring the concentration of c5a, c4 and c3c. the results demonstrate distinct alterations in above mentioned parameters. in spite of a high dose hepariulzation (act>450s) combined with an antifibrinolytic tw, atment an activation of the coagulation system was observed immediately after the onset of cpb followed by an activation of the fibrinolytic system. therefore further efforts should be done to develop new anticoagulatory regiments and improve the biocompatibility of materials used for cpb. during cardiopulmonary bypass blood is exposed to nonphysiologic conditions. the contact with artificial surfaces and mechanical stress result in a periopemtive response which includes activation of the complement, coagulation, fibrinolytic and kallikrein system, activation of nentrophils with degranulation and pmtease enzyme release, oxygen radical production and the synthesis of various proinflammatory cytokines. this so-called "pest-pump intlammatory response" has been linked to respiratory distress syndrome, renal failure and neurologic injmy. our goal was to investigate the time course of eytokine levels and the activation of leukozytes and platelets and to quantitate leucocyte subpepulatioas in 20 patients undergoing cpb. at different time points, pre, during and pest cpb, we determined the levels of interleukin (il) 113, il-2, il-4, il-6, il-8, il-10, tumor necrosis factor ¢z (tnf-a) and interferon "1' (ifn'--/) using elisa-techulques. lymphozyte subpepulations were characterized by flow cytometry and specific monoclonal antibodies against cd3 (pan t-cell marker), cd4 (surface antigen on t-helper cells), cd19 (surface antigen on b-cells), monocytes were determined by cd14 and platelets by cd41 (act. gpilb/llla) and cd42b (gp ib). single cell activation was analyzed using markers against cd25 (il-2 receptor), cd126 (il-6 receptor), hla-dr (mhc class ii), cd71 (transferrin receptor) and cd69 (activation inducer molecule), platelet activation was monitored with an antibody against cd62 (gmp-140). preliminary results revealed distinct increases in r,-6, il-8, and il-io following cpb whereas tnf-a and ifn--/levels were not significantly influenced. fttnhermore, activation of particular cell populations was observed. finally, our investigations should contribute to a better understanding of the complex humeral and cellular respenses induced by cpb and thus might help to develop new strategies to circumvent the negative impacts of cpb. optimal adjustment of anticoagulation in machine plasmapheresis is important for the quality of the prepared fresh frozen plasma (ffp) as well as for the safety of the donation. in the present study the suitability of prothrombin fragment ( ft+2 ) in the assessment of anticoagulation during plasmapheresis was investigated. matarlal and methods: 75 plasmapheresis procedures were performed on 25 donors (10 ~, 15o" ) using 3 different plasmapheresis machines (a 200, baxter; mcs 3p, haemonetics; pph 900, electromedics/medtronic). acid citrate dextrose formula a (acd-a) in a ratio to whole blood of 8 : 92 was used for anticoagulation. the concentration of fi+2 in the donor's blood was measured before and after plasmapheresis and in the prepared ffp. the actual acd-a volume used was also registered. results: there was a significant rise of the ft+2 -concentration in the donors blood after plasmapheresis with each of the three automatons: a 200:1.32 vs 1.14, p < 0.05; mcs 3p: 1.26 vs 0.98, p < 0.05; pph 900:1.20 vs 1.05, p < 0.05. the ffp prepared with each machine showed the following f~+2concentrations: 0.91± 0.18, 1.0:2 ± 0.17 and 0.93 ± 0.11 respectively. the difference between the groups was not significant. the elevation of the ft+2 -concentration in the donor's blood showed a negative correlation with the volume of the acd-a used. during 6 of the 75 procedures technical problems occurred (inadequate venous acces, occlusion of the citrate tube, reduced whole blood flow). after these procedures there was a marked elevation of f~+2 in the donors blood (2.74 ± 0.53), accompanied by an elevated f~+2 -concentration in the prepared ffp's. conclusion: these data show that ft+2 is a suitable parameter for the assessment of anticoagulation during plasmapheresis. several epidemiologic studies demonstrated that fibrinogen is an independent cardiovascular risk factor and should be considered for screening programs. prothrombin time derived fibrinogen (df) measurement combines the advantage of an established highly reproducible automated method with no additional reagents, except for calibration. several studies showed that the df values correspond well with the clanss method except in cases such as thrombolytic therapy in which the df results are higher. however, no results exist whether in patients with coronary heart disease with fibrinogen as a risk factor the df values are also comparable to the established clausss method. the aim of our study was to compare df values to clauss method results in cardiac patients, especially in patients before and after coronary bypass grafting (cab(]). measurements of df were performed on an acl 3000 (il) using the pt-fibrinogen-hs reagent. fibrinogen clanss method was done on the acl using fibrinogen c reagent (il) and on a kc4 (amelung) with fibrinogen a reagent (boehringer maanheim). for calibration we used the calibration plasma half volume (it.) with the fihrinogen concentration proposed by the manufacturer. plasma samples were obtained from 24 patients at admission before cabg and postoperatively up to 1 week, and from 23 healthy persons (staff). within assay imprecisious using normal and abnormal controls (il) were comparable with both methods showing cvs between 1.99 and 4.22 %. in normal healthy persons the medians of the df and the clanss method run on the acl were very similar (296 vs 302 rag/all), whereas kc4 values were about 10% lower (268 md/dl). in cabg patients at admission we found the same differences as in normals with the clanss method (acl: 363 vs kc4: 337rag/all), however the df values were siginficantly higher (median 418mg/dl). if we took a cutoff value of 320 mg/dl, as suggested by the results from the northwick park heart study, we would categorize into the high risk group 21 out of 24 patients using the df method, 20 with the clanss-acl method and 16 with the clanss-kc4 method, i.e. nearly 30% more patients were classified in the high risk group using the df method. postoperative samples showed the expected increases due to the acute phase response with the same magnitude of differences. because of its rapidity and reproducibility the df method is well suited for routine measurements, however, standardization remains an urgent task in order to avoid misinterpretation of results. for fibdnogen measurements in clinical laboratories, the two most widely used methods are the clotting time method according to clauss (cfib) and the sn called "derived" fibrinogen method (dfib) implemented in optical coagulometera with the fibrinogen concentration being derived flora the optical density of the fibrin clot in a standard prothrnmbin time (pt) assay. it is well known that under certain circumstances, e.g. in the presence of fibrin(ogen) degradation products (fdp), there is a discrepancy between the two methods with higher values for dfib than for cfib. yet the opposite discrepancy, i.e. fibrinogen values derived from the optical density of the clot grossly lower than values from dotting time assays, seems to be very rare and is poorly understood so far. the patient (male, 26 years) had ingested the esterase inhibitor parathion (e605) in an attempt o f suizide and was treated with high doses of atmpin. he had no clinical signs or history or family history of bleeding or thrombotic disorders. except for a very low pseudocholinesterase activity, all laboratory results were normal ineinding pt, afft, thrombin time, and factor xiii. pt and aptt did nnt differ between an optical coagulometer (electra 1000c, mla) and a mechanical one (kc..4, amelung). there was no evidence of disorders known to interfere with hemostasis like paraproteinemia or dyslipldemia. however, in all 7 blood samples received for dotting tests during a period of 7 days the macroscopic appearance of the fibrin clot was quite unusual (only slightly turbid/almost transparent) and there was a striking discrepancy between a very low or low dfib on the electra (pt reagent: thromboplastin is, dade) and a normal or high cfib (kc4; thrombin reagent, dade). on admission, values were 57 mgml (derived) vs. 275 mgldl (clauss). cfib rose to s42 mg]dl with dfib at 155 mg/dl in the last sample on day 7. ~ al! samples dfib was about 20 % (ls-23) of cf[b. when the patient's plasma was added m normal pooled plasma it caused, in a dose-dependent manner, values lower than predicted for dfib and values slightly higher than predicted for cfib. in the absence of data from additional (e.g. immunologic) methods the following principal possibilities (and combinations) have to be considered: 1) normal fibrinogen concentration and clot formation rate, but abnormal optical properties of the clot (cfib correct, dfib falsely tow); 2) normal optical properties of the clot, but accelerated clot formation and very low fibrinogen concentration (dfib correct, cfib falsely high). in either case, the molecular basis could be: a) a genetic or acquired molecular abnnrmality of fibrin/fibfinogen; b) an interfering substance. direct effects of the loxic agent parathion and/or the antidot drug atropin are not likely to be the cause since other patients, often with more severe parathion inmxicatian requiring higher doses of atmpin, showed normal optical density of the clot. we hope to perform a more in depth investigation of this abnormality in the future, including various methods, reagents, and instruments for fibrinogen measurement, a survey of the patient "s family, and studies of the molecular nature of the phenomenon. increased fibrinogen is known to be an independent predictor of subseqtmnt acut~ coronary syndromes. however. a multitude of methods for fibrinogen determination is available. there is a lack of standardisation among fibrinogen assays. in a family cohort study (patients'with combined hyperlipidaemia and f or hypemricaemia) fibrinogen was determined in plasma samples from 340 family members using a functional and an immunochemical assay. the fimctional assay according to clauss was performed on the analyser ca 5000 using the test fibrinogen a from boehringer. the immmmephelometric assay was performed on ~e behring nephelometer system using the reagent and standard from behring. a good similarity between both assays was obtained at low and high flbrinogen levels as well as in samples with increased c-reactive protein (crp). values obtained by both assays correlated similar with total cholesterol, ldl--cbelesterol and apolipeprntein b. the ratio functional fibrinagen / immlmochemial fibrinogen showed no dependence on cholesterol, t-pa, v wiuebrand factor and crp. release of two fibrinopeptides a from fibrinogen generates desaa-fibrin monomer, which rapidly aggregates, forming fibrin complexes. fibrin monomers can be detected in plasma samples after chemical desaggregation of fibrin complexes using thiocyanate by monoclonal antibody binding to the alpha-chain neo-n-termini generated by fibrinopeptide release. although postulated, an intermediate of fibrin formation, carrying one fibrinopeptide a and one fibrin alpha-chain neo-n-terminus has so far escaped analytical procedures. we have employed a monoctonal antibody specific for fibrin alpha-chain neo-n-terminus, mab 2b5, attached to magnetic microparticles, for isolation of fibrin-related material from plasma samples of patients with elevated soluble fibrin. the material was desorbed by sds-urea buffer and subjected to sds-page and immunoblotting. immunostaining with panspecific anti-fibrinogen and anti-fdp-e antisera showed a range of bands corresponding to fibrin monomers, and fibrin derivatives containing the fibrin e-domain. lmmunostaining with monoclonal anti-fibrinopeptide a antibody resulted in a doublet band corresponding in size to fibrin monomer. similar results were obtained with polyclonal antisera against fibrinopeptide a. for a more quantitative approach, desa-fibrin monomer was detected by an elisa procedure using mab 2b5 as capture and monoclonal anti-fibrinopeptide a antibody as tag. a sample with extremely high level of desaa-fibrin monomer, determined by elisa (enzymun®-test fm) was used for calibration, since reference material is not available. a correlation of r=o.g4 was found between desaa-fibrin monomer and relative desa-fibrin monomer levels. detection of desa-fibrin monomer required sample pretreatment with thiocyanate for desaggregadon of fibrin complexes. from these preliminary data it appears that desa-fibrin monomer accounts for a fairly constant proportion of soluble fibrin and is a polymerizing species. fibrinogen has been shown to be a major cardiovascular risk factor. especially for epidemiological studies, exact quantitation of fibrinogen in clinical plasma samples is of great imporance. fibrinogen levels are generally measured by clotting assay according to clauss, or by determination of derived fibrinogen values upon photometric measurement of prothrombin time (derfbg). the clotting assay has been shown to be influenced by high levels of soluble fibrin derivatives. the pt-derived fibrinogen levels appear rather convenient in clinical routine, since no additional reagents are needed. we have compared the clauss assay and derfbg with a turbidimetric fibrinogen assay using snake venom protease for fibrinopeptide release, performed in photometric autoanalyzers. d-direct antigen was measured in parallel using tinaqaant d-dimer lpia. results were correlated with total fibrinopeptide a release by thrombin, measured by elisa. a total of 484 samples were included, of which 29 samples (6 %) were recorded as above measurung range by derfbg. these samples encompassed a range of 5.90-10.40 g/l and 5.21-12.37 g/l in clauss, and turbidimetric assay, respectively. the range of values measured by derfbg assay was 0.72-9.14 g/i, corresponding to 0.26-11.00 gll and 0.24-10.48 g/1 in the clauss and turbidimetric assay, respectively. the correlation of derfbg with the clauss assay was re0.91, correlation with turbidimetric assay was r=0.92 for the values actually detected. the correlation between clauss and turbidimetric assay was r=0.93 for all values. there was no dependency of test results or inter-test variation upon d-direct. correlation graphs displayed a decreased test response of clauss assay in the high concentration range, resulting in an underestimation of fibrinogen concentration. the derfbg assay, in contrast, showed normal range values in samples from patients with fibrinotytic treatment and low fibrinogen levels in the other assays. correlation with fibrinopeptide a release was r=0.88 for clauss assay, r=0.89 for turbidimetric assay, and r=0.82 for derfbg. for clinical routine, derfbg appears to be applicable for all samples between 1.00 and 5.00 g/l with exclusion of samples from patients with fibrinolytic treatment or endogeneous hyperfibrinolysis. other samples may be analyzed by clotting assay or turbidimetric assay, although the latter appears to be more suited for measurement of high range samples. for inhibition of pk is 0.067 pmol/l the antifibrinolytic activity of the inhibitors was determined by measuring the lysis of radiolabelled human plasma clots• the compounds which inhibit plasmin and pk influence remarkably the streptokinase-induced clot lysis but not lysis induced by uk and tpa. surprisingly, inhibitors of uk and tpa do not influence clot lysis induced by uk or tpa. the structure-activity relationships for inhibition of ptasmin, uk, tpa and pk could help in the design of more potent inhibitors of fibrinotytic enzymes. uk inhibitors are of interest for the development of anti-invasiveness drugs, while plasmin/pk inhibitors could be prototypes of a "synthetic aprotinin". in the ecat angina pectoris study t-pa antigen was an indepcndem risk factor of subsequent acute coronary syndromes. pat indicates the risk bat depends on other known risk factors. it should be tested in 183 members of a family cohort study (patients with combined hyperlipidaemia and / or hyperuricaemia), if the active pal antigen or the whole pai antigen showed a stronger relation to t-pa and metabolic variables. the active pall antigen was determined using elisa actibind pat-1 (technoclone / lmmuno) , the whole pai-i antigen was measured using the f_lisa pat-1 (technoclone i immuno). t-pa activity was determined with the coaset t-pa from chromogenix, the tintelize tpa from biopool was the used test for determination of t-pa antigen. the active pat antigen showed a stronger correlation to t-pa activity and t-pa antigen than the whole pal antigen. circulating t-pa activity was influenced predominantly by the active pal antigen. both pat antigens were correlated in similar manner with metabolic variables, lipoproteins and b/vii. table: correlations of active and whole pal antigen (** p < 0,001) active pal antigen whole pal antigen active pat antigen 1,000 0,851 ** whole pal antigen 0,851 ** 1,000 t-pa activity -0,594 ** -0,492 ** bpa antigen 0,604 ** 0,497 ** body mass index 0,502 ** 0,462 ** triglycerides 0,452 ** 0,441 ** total cholesterol 0,252 ** 0,255 ** ldl-cholesterol 0,263 ** 0,264 ** hdl-cholesterol -0,357 ** -0,355 ** apolipoprotein b 0,428 ** 0,402 ** apolipoprotein a i -0,233 ** -0,211 ** the lower relationship of the whole pat antigen to t-pa is obviously caused by patient samples with high levels of whole pat antigen in contrast to normal values of active pat as well'as of t-pa. possibly, a high ratio of whole pai antigen / active pat antigen is caused by a raise of latent pal the main form of pat in the platelets. the clinical importance of an increased ratio whole pal antigen / active pal antigen remains under investigation. the cyclic antibiotics-polypeptides bacifracin a, bacilliquin from boci/lu~, licheniformis and gramicidin s from bocil/us brevis, var. (3. b., were used for investigation. we studied their influence on the fibrinoly~c and coagulation activity in vitro• me~hods. to solution of human plasmin (thrombin). containing 0.2 mg of protein (1 nih unff)/ml, the analyses' solution of antibiotics (0.1-8,0 mg) was added. then we defined the tlbrinolytlc activity of the mixes using azofibrin lysis, and fhrombin activity was determined according to the speed of fibrin clots formation from fibrinogen solution. results. in following table are submifled the results received in our laboratory {we also offer results of antibiotics influence on urokinase activity): ki, mm ki --the constant of inhibition. n. d. ~ in studied lirnils the inhibitor's activity was not observed. ---the inhibitor's activity was not define. i. --the inhibitor% activity was observed but ki not determined. +, +% +++ --effect of inhibffion (in rela*iive indexes). conc/us/on.~ the results received by us testify to the necessity of cautious approach to the use of antibiofics-polypeptides for various sorts of therapy in view of their possible influence on fibrinolytic and coagulation actlvlfy, of the organism. these results were used for preparation in our laboratory of biospeciflc sorbents containing c-ramicidin a, bacil}iquirt and gramicidin s.as ligands, they can reversibly bind thrombin, plasmin {plosminogen) and urokinase directly from crude exkacts. the enzymes are selectively eluted without substantial losses of specific activity in e yield of 60-90%. there is a great body of rather contradictory informations dealing with fibrinolysis in liver.. cirrhosis, which can be accelerated, normal or reduced, depending on the type of cirrhosis and investigation techniques (clot-lysis, fibrinolytic component measurements). our previous finding was, that in vitro plasma-clot lysis, induced by exogeneously added tpa or streptokinase proved to be reduced, and this had a good correlation with severity of the disease and the elevation of plasmatic yon willebrand factor levels. in vitro clo~/-[ lysis tests, induced by tpa were performed in 41 patients with alcoholic liver cirrhosis, utilising a microplate light-scattering assessment method. the tests were repeated using the same plasma samples in each patients with a microplate which was covered by cultured endothelial-cell monolayer (umbilical vein, huvec}. clot lysis speed proved to be 1.5-2 times slower with huvec milieu in the control group, while in the cirrhotic patients this inhibition was stronger and resulted in 5-fold reduction of lysis speed. our results suggest, that cirrhotic plasma is able to accelerate the release of fibrinolytic inhibitors from cultured endothelial cells, which phenomenon may also contribute to the complex alterations of in vivo fibrinolysis in cirrhotic patients. deep vein thrombosis (dvt) is a systemic disease with prolonged clinical manifectation. anticoagulation therapy in dvt is not completely effective. thrombolytic therapy may give rise to a systemic lytic state, the fibrinospesific agents (scu-pa and t-pa) have short half-lives in the circulation. we investigated the potency of the acylated plasminogen streptokinase activator complex (gbpg-sk) to deep vein clot dissolution as compared to well known sk and apsac both in v~tro and in vivo in the model of venous thrombosis in artherio-venous shunt in rats. it was shown in in vitro study that fibrinolytic activity of plasminogen activators mainly depends on their stability in plasma. stability studies carried out by incubating sk and pg-sk activator complexes in plasma with euglobulin precipitation . total fibrinolytic activity was measured by the fibrin plate method. gbpg-sk possessed the greatest stability in human plasma than apsac or sk because of its prolonged inactivation period (the deacylation half-life for gbpg-sk was 230 :e 21 rain in contrast with 73 -~ 6 min for apsac). the stability degree of two acylated thrombolytics (gbpg-sk and apsac) was in order to inverse proportion of their first order rate deacylation constants (2.9 • 10 -4 and 6.0 • 10-s sec-1 respectively). the fibrinolytic potency of sk, apsac and gbpg-sk was measured by 1251-labeled fibrin clot lysis in plasma and in vivo by lysis of the preliminary formed 1251-labeled fibrin clot inserted into the jugular vein. fibrinolytjc activity of acylated plasminogen activators gradually increased in time. under sk administration, the clot lysis came to the end by 2 hours while apsac and gbpg-sk haven't lost their activity for 5 -6 hours. gbpg-sk possessed significantly more prolonged fibrinolytic activity than apsac, the acyl-enzymes did not significantly influence on plasminogen,,.~2-anfiplasmin and fibrinogen levels in plasma according to their activity specific to fibrin-bound plasminogen. in opposite, sk produced a significant depletion of plasminogen, ~-2antiplasmin and fibdnogen levels in plasma. it seems, on the basis on in vitro and an animal experimentation, than apsac with its moderately fast deacylation rate is more suitable for rapid thrombolytic effect, but gbpg-sk with its slow deacylation rate is suitable for deep vein thrombosis, when the rapid thrombolysis is less critical. it's well known that the complete lysis of thrombi usually isn't observed at the thrombolytic therapy. at present study we have attempted to quantify the possible mechanism of fibrinolysis inhibition during the thrombolysis. 125i-labelled partially cross-linked fibrin clots of different volume (0.1-0.35 ml) were immersed in tris-hcl buffer (3 ml) containing plasmin (5-100 nm) at 37°c. the lysis rate was detected by counting of soluble fibrin degradation products (fdp). at all the eases lysis slowed down and stopped in 3 hs though clots dissolved up to only 60-85%. no irreversible inlaibition of plasmin caused by denaturation occur as was judged by the measurement of fibrinolytic activity at the diluted samples. however the increase of fdp concentration in surrounding buffer led to the reversible inhibition of fibrinolytic activity of plasmin up to 5% of baseline. the sds-page analysis under non-reduced conditions shown the acoumulation of high-molecular weight fdp at the surrounding buffer. the inhibition phenomenon could be connected with the specific binding of plasrnin with soluble fdp having exposed lysine residues and the subsequent removal of enzyme from fibrin surface. unexpectedly since the heterogeneous character of occurred reactions tile change of the clots surface area during lysis didn't affect the fibrinolysis kinetics in all the concentration intervals. to estimate the kinetic parameters the kinetic curves were linear in the coordinates [p1/t (l/t*ln(isl°{(lslo.lpi)). the obtained parameters were following: keat=l.36 min-l,km=l.33 ixm,kp=0.12 ~tm. the clinical trials have shown that fdp concentrations at the thrombolytic therapy of deep venous thrombosis and acute myocardial infarction usually was approximately in the range 0.05-0.2 ~tm. therefore the described phenomenon of fibrinolysis inhibition by formed fdp may take place during thrombolytic therapy. al. calatzis, an. calatzis, +m. klmg, +l. mielke, +r. hipp, a. stemberger institute for experimental surgery and +institute of anesthesiology technische universit~.t monchen thrombelastography (teg) is an established method for the detection of fibrinolysis. fibfinolysis is usually determined when the teg amplitude decreases by more than 15% atter the maximum amplitude is reached. this takes a considerable amount of time (more than 30 minutes). our approach bases on the understanding of fibrinolysis as a process which runs in paraue[ to coagulation and is not exclusively subsidiary to it. the effect of fibrinolysis on the growing clot in the teg is shown by the comparison of two parallely performed teg measurements: exteg: teg measurement with standardised activation of the extrinsic system. apteg: exteg with in-vitro-fibrinolysis inhibition via aprotinin. exteg-reagent (ex): 1:2 dilution of innovin (recombinant thromboplastin reagent, dade) with aqua dest. apteg-reagent (ap): 5 parts innovin, 2 parts trasy[ol (aprotinin, bayer, i0.000 kie/ml), 3 parts aqua dest. test procedure: l0 p1 ex or ap + 300 ~l citrated blood (cb) + 50 lal cacl2-solution 0,15 m. the only difference of the two reagents is the addition of 20 kie aprotinin in the apteg, leading to an in-vitro fibrinolysis inhibition. the usage of disposable pins and cups (haemoscope, illinois, usa/e.m.s., vienna) is recommended for ensuring standardised conditions for both measurements. results and discussion: when there is a better clot formation in the apteg (corresponding to a lower so-cafled k-value) than in the exteg, fibdnolysis can be suspected. this technique requires only commercially available reagents and is easy to perform on conventional teg systems. due to the standardised coagulation activation with a thromboplastin reagent, fibrinolysis can be detected also when inhibitors like heparin are present in the circulation. according to our experience using this technique during liver transplantation, clinical relevant fibrinolysis can be detected as described in less than l0 minutes. many thromboembolic (massive pulmonary embolism, proximal deepvein thrombosis, etc.) and coronary diseases (infarction, acute phase, etc.) require fibrinolytic therapy to early recanalizafion. the application of the well-known or new thrombolytic agents needs the use of specific, simple and reproducible methods for the determination of fibdnolyfic activity. we suggest new methods for measuring the blood plasma concentrations of plasmin, plasminogen, antiplasmins, and urine urokinase activity. these methods involve the employment of chromogenic substrafe azofibrin (human fibrin, covalently labeled with p-diazobenzenesulfonic acid). method~. 0.2 ml of studied solution was added to 0,8 ml of azofibrin suspension in certain buffer (5-10 mg/ml) and the mixture incubated at 37 oc for 10-60 rain. after the end of incubation the mixture was filtered, the volume of solution brought up to 4 ml by 0.02 m naoh and the optical density was determined at 440 nm. resuffs. azofibrin can be used for quantitative determination of proteinases activity in search of new fibrinolytic means. for comparison the results of our studies fibrinolytic activity of some proteinases with the use of azofibrin are presented: activity. with an increase of pal and ldl-and a decrease of hdl-cholesterol concentrations k is concluded that the increased cardiovascular risk in diabetes meilitus was partly caused by a down regulation of the fibrinolytic system, increase of erythrocyte aggregation and plasma viscosity. also disturbances of lipid metabolism an abnormal whr seems to be of an additional atherogenous factor in dm. plasma concentrations of thrombin-anfithrombin-iii (tat), alpha-2antiplasmin-plasmin (app) complexes and ddimer were investigated in 50 patients treated with thrombolytic therapy for acute myocardial infarction (ami) either with streptokinase (n=24), urokinase (n=16) or recombinant t-pa (rt-pa, n=10). all patients received an intravenous heparin bolus of 5,000 iu on admission, which was followed at once by an infusion of 1,000 iu/hr for the next three days titrated to maintain the partial thromboplastin time at twice control value. tat, pap and ddimer were measured by enzyme immunoassay on admission, 1, 2, 4, 6, 8, 12, 24 hours and on day 3 and 7 after admission. groups did not differ significantly in regard to age, sex, delay and infarct location. on admission, no marker differed significantly between groups. thereafter, tat levels increased significantly exclusively in rtpa treated group. from 2 to 6 hours after admission, tat were significantly higher in rtpa treated patients than in streptokinase and urokinase treated group (p<0.02). however, during continous heparin infusion, which was started immediately after stop of thrombolytic therapy, in each group tat concentrations decreased below admission values. app were significantly higher only 1 hour after admission in the rt-pa group (p=0.03). ddimer did not differ signifieanfly between groups. our results demonstrate, that rtpa induces a hypercoagulable state, which may contribute to reocclusion after successful reopening of the infarctrelated coronary artery. the significant tat decrease during continous heparin infusion supports the concomitant use of thrombin inhibitors as adjunctive therapy with thrombolytlc treatment for ami. thus, in acute myocardial infarction patients, thrombin generation is markedly influenced by the thrombolytic agent used and concomitant heparin therapy. endothelium derived relaxing factor-no (edrf-no) plays a major role in regulation of vascular tonicity and also exerts platelet inhibitory action~ however, due to the chemical nature of edrf-no few is known about its production and activity as a general index or marker of vascular function in human diseases. one way to achieve this can be measurement of nitrate/nitrite excretion in the urine, which seems to reflect vascular edrf-no production. in this report a self-developed elisa method is described, which was used for this perpose. nitrate/nitrite urinary exretion proved to significantly decreased in insulin dependent and in non-insulin dependent diabetes mellitus as well after a comparison of the excretion values to other markers of angiopathy (yon willebrand factod soluble thrombomodulin, beta -thromboglobulin) it seems to be acceptable, that urinary nitrate/nitrite excretion can be a useful indicate of diabetic vascular disorders. two major concerns still accompany the application of prothrombin complex concentrates (pcc). viral safety has to be guaranteed and therefore several measures for virus inactivation or elimination are taken during the manufacturing process. the inherent risk of thrombo-embolic side effects has to be considered. to minimize these risks and to achieve good clinical efficiency the quality criteria for pcc's are under pending discussion. it is generally accepted that a modem pcc-preparation should contain all of the four coagulation factors in a well balanced proportion and that it should also contain protein c and protein s. additionally, the concentration of activated coagulation factors should be kept at a minimum. a present pcc-produedon process mainly consists of a qae-sephadex extraction of cryopeer plasma followed by a solvent/detergent virus inactivation step. further purification is achieved by subsequent chromatography on deae-sephamse. the aim of this study was to improve product quality by avoiding f viiactivation without implementing major changes to the production process. at the same time, a second virus eliminating step was added to the production process. it could be shown that speeding up the chromatographical process by switching the deae-sepharose-chromatography from a classical axial column to a radial chromatography resulted in a significant reduction of f viia-genemtion. mainly the reduction of contact time, resulting from the highest possible flow rates, leads to the wanted effect. the relation between f vii/f viia was 10 : 1 or more. in order to investigate the feasibility of virus filtration the eluate of the deae-sepharose column was filtered through a virus removing ultipor vffilter. the analysis of the solution before and after fillration showed that the filtration had no influence on coagulation factors activity, protein content, proteolytic activity etc. preliminary studies showed significant virus reduction values. in the past few years the problem of expediency of the treatment aimed at developing immunological tolerance in hemophil;a patients by way of complete removal of inhibitor with high doses of factor viii has been discussed in literature. we observed 121 patients with hemophilia. inhibitors to factor viii:c were revealed in 32.7 % of patients with hemophilia a and fo factor ix --in 1.6 % of patients with hemophilia b. the level of an inhibitor was not higher than 87 befhesda u/ml, that is those patients were not regarded as "high responders". a high incidence of inhibifors in young patients [from 7 to 26 years of age, 51.9 %) compared with older patients (from 27 to 40 years of age, 11.2 %) testifies to the probability of inhibitors development during treatment with modern concentrated preparation of factor viii, ix. inhibitor development in patients (40.5 %] in the course of antihemophilic concentrates transfusions is an evidence of alloimmunization of patients with proteins. the investigations show that in the course of transfusion therapy patients develop secondary immunodeficiency due to chronic antigenic stimulation of immune system with high doses of allogenic proteins. against the background of immunodeficiency patients with hemophilia develop complications of immune character: infections complications --53.9 %, aufoimmune processes --44.9 %, secondary tumours --1.2 %. plasmapheresis is the most rational method of removing inhibitor in patients with low level of inhibitor ("low responders", < 10 bu/ ml) and in patients with mean response. thus it should be noted that the treatment of patients aimed at developing immunological tolerance is not only expensive and economically unprofitable but also not indifferent fo the organism. in a recent multicenter study 73 previously untreated patientens (pups) with severe hemophilia a were treated with a recombinant factor viii concentrate (rfviii, recombinate©). during fviii treatment 21 (29%) developed inhibitors, 6 high titer (>5 bethesda units (bu)/ml), 4 low titer (<5 bu/ml) and 11 transient inhibitors. plasma samples from before treatment and during treatment but before inhibitor occurrence were available in 12 inhibitor patients. these plasma samples were analyzed by a highly sensitive immunoprecipitation (ip) assay for the presence of anti-tviii antibodies. in 9 (66%) a significant increase of anti-fv]]i antibodies was seen indicating the development of a clinical relevant inhibitor titer. this immune response occurred after 2 to 17 (median 5) exposure days (ed). in the same period only 3 out of 15 inhibitor patients showed a decreased in vivo recovery. in 16 pups who developed no inhibitors plasma samples from the entire treatment period were available. an immune response to rfviii treatment was seen in 7 pups after 2 to 43 ed (median 24 ed). the immune response was later and less pronounced in comparison to inhibitor pups before inhibitor occurrence. with the ip method the detection of an early immune response is possible which might be predictive for a later inhibitor development. the inclusion of the lip method should be considered for future multicenter pup studies. in the past anaphylactie reactions to plasma and plasma components have been a common complication of replacement therapy in patients with hemophilia a and b. we report on 3 severe bleeding episodes in 2 patients with hemophilia a and b, respectively. both patients had a history of life threatening anaphylactic reactions after exposure to different plasma derived clotting factor concentrations including intermediate purity factor viii-and factor ix-concentrate, respectively. high purity factor concentrates were tolerated well without any allergic side effects. a 67 years old patient with a moderate form of hemophilia a (f viii 4 %) had a history of severe immediate reactions with skin manifestations and bronchospasm after exposure to fresh frozen plasma, ctyoprecipitate and 3 different plasma derived factor viii-concentrates of intermediate purity. in all episodes pretreatment with corticosteroids and antihistamines was unsuccessfull in avoiding severe bronchospasm. replacement therapy with two different recombinant factor viii concentrates was tolerated well without any side effects. a 12 years old haemophiita b patient developed hypersensitivity reactions to prophylactic factor ix substitution, which could be overcome by using a factor ix .concentrate with improved purity. a recent recurrence of hypersensitmty under this treatment was finally overcome by the use of highly purified (monoclonal antibodies) factor ix concentrate. we conclude from these findings that high purity of factor concentrates, possibly due to the absence of soluble hla-antigens, are advantageous in patients disposed to allergic reactions. introduction: antibody formation against factor (f) viii remains one of the most severe complications of repeatedly transfused patients with haemophilia a. as reported previously in our study about the incidence of fviii inhibitors, we have observed a high incidence of fviii inhibitors among our haemophilia a patients. it is still not clear why certain haemophiliacs develop antibodies and others do not. a number of previous studies suggest that there is a genetic predisposition for the fviii inhibitor development. thus, the purpose of our study was to examine, if there is a correlation between fviii antibody-formation and genetically determined histoeompatibility antigen (hla) patterns in our haemophiliacs. patients and methods: hla-class i (a, b, c) and hla-class ii (dr, dq) typing was carried out for 51 respectively 44 multi-transfused paediatric haemophilia a patients (fviii:c activity < 5%), including 22 who had developed an antibody to fviii: 19 were high responders (> 5 bu), 3 were low responders (< 5 bu). hla-typing has been performed by a standurcl two-stage microlymphoc~.ftotoxicity procedure (drk frankfurt) using antisera with defiend hla-specifity (biotest diagnostica). results: we found an under-representation of hla-a2 in fviii inhibitor patients when compared with the subgroup without inhibitor. in regard to the hla-b and hla-c antigen frequencies there are no apparent differences between the groups. among the class ii antigens there were higher frequencies of dr1, drw52 and dqwl in the non-inhibitor group. however, the reduction in hla-a1, hla-cw5, hla-dqw3 respectively hla-dr4 frequency for inhibitor patients as reported previously could not be confirmed in our study. conclusion: so far it remains unclear if there is a significant association of a certain hla allels with the development of fviii antibodies. recombinant factor sq (r-viii sq, pharmacia) is a b-domain-deleted recombinant factor viii. it is formulated without albumin (hsa). the product has been shown to have in vitro and in vivo biochemical characteristics similar to a plasma derived full-length protein (p-viii). the international clinical trial programme was initiated in march 1993. pharmacokinetic studies have shown that the b-deleted r-viii sq should be given according to the same dosage principles as a full length p-viii. at present, the product is being tested in previously treated patients (ptps) and untreated patients (pups) with severe haemophilia a (viii:c < 2 %), both during long-term treatment (on demand therapy or prophylaxis) as well as during surgery. the long-term study in previously treated patients in germany was started in january 1994. thirteen patients have been included in 8 centers. all patients are still on treatment with r-viii sq, most of them receiving prophylactic treatment. global treatment efficacy has in general been considered excellent or good. no serious clinical adverse events related to the study product have been reported, nor have any inhibiting antibodies to factor viii or antibodies to mouse-lgg or cho-cell components developed in the patients. further results such as data on efficacy, half-life, recovery and safety will be presented in detail at the meeting. nowadays it is not sufficient to regard hemophilia only as hemorrhagic diafhesis of coagulation genesis, caused by deficiency or molecular anomalies of coagulation factor, without taking into account the immunity state. on examination of 125 patients (pts) (hemophilia a --110 pts, hemophilia b --11 pts, willebrandt's disease u 4 pfs) the development of immune complications was revealed in 34.4 %. chronic persistent hepatitis (3.2 %), chronic active hepatitis (3.2 %), herpes simplex (1.2 %), chlamidiosis (1.2 %), bacterial infection (7.2 %} were regarded as infectious complications. bacterial infections have a routine course due to preserved phagocytic function of neufrophils. and viral infections, whose ability to resistance is connected with t -cell link immunity, take on a chronic persistent course, mechanism of the development of autoimmune processes (autoimmune thrombocytopenic purpura --2.4 % of pts, immunocomplex disease --4.9 % of pts, the appearance of immune inhibifors --34.4 % of pts} is connected with the impairment of immunological surveillance over b -cells aufoimmune clones as a result of dysbalance in the system of t -lymphocyfes immunoregulatory subpopulations. lymphadenopathy and splenomegaly (4.9%) develop due fo benign proliferation of lymphoid tissue as a result of impairment of regulatory function of t -lymphocytes system, or they may be an evidence of virus infection. we observed one episode of acute leukemia. immune complications in hemophilia patients develop against the background of secondary immunodeficiency caused by chronic antigenic stimulation of patients' immune system with high doses of allogenic proteins, which plasma preparations contain. in immune complications hemophilia patients develop hemorrhages, whose pathogenesis is quite different from that caused by coagulation factor, so it should be taken into account in the course of treatment. control of hemophilia therapy classically was based on four parameters: life span expectancy of patients, orthopedic status (normal zero), pettersson score and social integration. oren, however, these parameters described an irreversible status with permanent damage particularly of the joints, especially when patients were grown-up. in order to establish risk-adapted therapy protocols to prevent hemophllic osteoarthropathies, quality control programs have to he set-up that allow for early adjustment of dosage and substitution frequency. here bleeding frequency is one the main parameters, being a clear hint for the possible development of a target joint. since 1988 we have established a computer database (haemopat) that contains data from all patients treated in our center. tables and graphs allow for early detection of increased bleeding tendency in a given joint, and accordingly for adjustment of therapy. the results of 8 years of measuring reasons of joint damage and not documenting the orthopathies as such will be demonstrated. parallelly a new program (haemopat win 1.0) will he introduced allowing for easier handling of data and their evaluation. this program will be used as of december 1995. in combination with a substitution calender to be filled in by all patients, in which factor concentrates, lot numbers, dosage, and date of administration will he constantly recorded, this program will extend our existing database in order to follow closely clinical and orthopedic parameters of each patient, and consequently acts as strict control of therapy quality. additionally, it provides sufficient data to fulfil any documentation needs, requested by medical authorities. the program will be available for all those interested free of charge. 2) kinderklinik der westf. wilhelms univ. mttuster 3-6) biotest pharma gmbh, dreieich haemoctin® sdh; the fviii sdh (sdh = solvent detergent and dry heat = 100 °c, 30 rain) from biotest pharma is a high purity (specific activity ~ 100) fviii concentrate manufactured from large human plasma pools. virus validation studies have shown virus inactivation/reduction (log 10) during the manufacturing process for lipid coated vints~ such as: h]v-1 > 16.2; psr > 16.8; vsv > 14,5; bvdv > 15.7; hcv > 4.5* and non enveloped vimsas such as: parvo** = 2.7; reo > 5.3*** and hav > 13.9. more than 50 hemophilia a patients (ptps = previously treated patients), baseline fviii activity < 1%, were included in an international drug monitoring study to follow their fviii inhibitur status. the hemophilia centers included were three centers from hungaria (helm pal children hospital and the national inst. of haematology, budapest and regional blood transfusion center, debrecen) and four centers from germany (two from berlin, one fraukfurffmain and one monster). patients were enrolled in the drug monitoring beginning aug. 1993. at the entry none of the patients had a detectable inhibitor. at the end of sept. 1995 there were no side effects or adverse events in connection with the use of haemuetin®. before the haemoctin drug monitoring study, the patients were treated with cryoprecipitate, or purified fviii products. inhibitor testing was done on patients plasma samples using the bethesda method. repeated fviii recovery determination at one time (between 12 to 24 hrs) after haemoctin® application demonstrated the expected recovery and normal half life time. none of the hemophilia a patients, treated with haemuetin® sdh developed a clinical relevant inhibitor. at the beginning of the stud)', the clinical efficacy of haemuetin® was studied in 16 hemophilia a patients and shown to give an in vivo recovery of 71 + 15 % by one stage assay and 77 + 17 % by a chromogenic assay. t ½ values were 13 + 2.8 and 12.7 + 3.2 hrs respectively. the study for the clinical efficacy of haemoctin® sdh was repeated in a group of 6 patients approximately two years later. although cd4 lymphocyte counts are known as reasonable predictors of prognosis in hiv infection, the cd4 count is not in all cases an infallible indicator of prognosis. therefore several serological markers are used to predict disease outcome, including beta-2 microglobulin (132m), immunoglobulin a (iga), lymphocyte counts (lymph) and others. in this study we followed a cohort of 23 haemophiliacs (19 with haemophilia a, 4 with haemophilia b) and 2 patients with severe von willebrands disease over a period of 28 months (mean, range: 22-34). testing for l~2m, igg, iga, igm, cd4 and cd8 cell counts (abs. and relat.), cd4/cd8 ratio, and absolute resp. relative leucocyte and lymphocyte counts was performed at least 3 times a year. at the same time clinical examinations and review of history were undertaken. mean of laboratory tests for every quarter of a year and significant changes during time of observation were calculated and correlated with clinical data. 1-4 5-8 9-12 13-16 17-20 21-24 cd41 440+956 344+924 278+925 302.-1:240 273.+.220 166+125 cd8 ~ 1165+474 1171+523 1236+1187 1017+439 930±412 873+478 1~2m z 2.0+0.6 3.0+1.0 3.0+1.2 3.0+1.1 3.5±1.0 3.5±1.3 lymph ~ 1.98+0.6 1.83+0.6 1.72+0.7 1.67±0.6 1.46±0.6 1.31±0.5 means/pl ± standard deviation means mg/l ± standard deviation during time ef observation we found significant changes of cd4 (abs. and relat.), abs. cd8 counts, cd4/cd8 ratio, f~2m, leucocytes and lymphocytes. the abs. cd4 and cd8 counts correlated clearly with lymphocytes und leucocytes counts but not with 1~2m. the prognostic value of the tested parameters is discussed by calculation of correlations with clinical data, anti-retroviral treatment and treatment of haemophilia. the availability of high purity factor concentrates has recently encouraged clinicians to use perioperative continuous infusion of fviii or fix to prevent or reduce bleeding in patients with haemophilia. in conliast to repeated highdose bolus injections, the continuous infusion trealment regime maintains constant coagulation factor activity at a level necessary for hemostasis, reducing the total cost of treatment by about 20% and preventing possible side effects of bolus doses. the new application mode, however, requires stable products which tolerate slow passage through an infusion device. our objective was to test in vitro the fviii concentrate immunate (stim plus) and the fix concentrate immi.ynine (stim plus) at room temperature, under conditions of long-term contact with polypropylene tubing in an infusion pump. infusion rates were chosen to mimic clinical situation. the control samples were not infused through the pump but were otherwise treated identically. test samples were drawn before and at 1, 4, 8, 24 and 48 hours after the onset of each infusion run. fviii (one-stage, two-stage and chromogenic assay) and fix (one-stage) activity were measured using immuno reagents. presence of activated factors were measured by napt'i', while flla, fxa, plasmin and pre-kallikrein activator were detected with specific chromogenic substrates. the data showed equivalent results between test and control samples with no loss of fviii or fix activity. the potencies of both immunate (stim plus) and immunine (stim plus) remained within 100 + 20% of labeued values within 48 hours after onset of infusion. in conclusion, immunate (stim plus) and immunine (st1m plus) are suitable for contiuous infusion when using automatic infusion device within applied test criteria. in htanans, circulating half-lives of asparaginase enzymes from e. coli and erwinia chrysanthemi vary within a wide range. moreover, half-lives differ not only among different e. coli strains but also among commercial e. coli preparations. to investigate the possible influence of two different sources of e. coil asparagmase (asn) preparations on the fibfinolytic system of leukemic children a prospective randomized study was performed correlating asn pharmacokiuetics (asn activity, asparagine depletion) with fibrinolytic parameters (plasminogen (plas), o.2-antiphismin (ct2ap), tissue-type plasminogen activator (t-pa), tissue type plasminogen activator inhibitor 1 (pal 1), d -i)imer (i)-d)). together with prednisono, vincristine and an anthracycline 20 children received i0000 iu-/m 2 asn medae r (originally purchased: kyowa hakko, kyogo japan) and 20 children 10000 iu/m 2 crasintin r (bayer, leverkusen, germany). blood samples for pharmacokinetic and coagulation analysis were drawn before the first asn administration and every third day whilst on medication. the results are shown in the 0.05 asn activity shows a negative correlation (spearman: rho/p) to plas (-,637/0.0003) and ct2ap (-, 751/0.0001). a positive correlation was found between asn activity and d -dimer formation (0.475/0.01). t-pa and pal 1 showed no relationship to asn activity. all children showed complete aspamgiue depletion at a detection limit of 0.1 um during the course of asn admiatstration. two thrombotic events occurred in the kyowa group, one of the distinctions between the two e. coli asn preparations administered ill this stndy is the absence of cystine in the kyowa asn, which also has a lower isoelectric point and a longer half-life than the bayer type a asn. with respect to this observations this may lead to longer inhibition of protein synthesis, which then may be the cause of a bigher rate of side effects. along with studies on asn pharmacokinoties dose recommendations need to be tailored to the specific asn preparation employed to ensure optimal antineoplastic efficacy while minimizing the hazard of complications. different types of coagulopathy in hepatic veno-occlusive disease (vod) and capillary leakage syn-drome (cls) after bone marrow transplantation w. ntimberger, s. eckhof-donovan, st. burdaeh and u. g6bel department for pediatric hematology and oncology, heinrich heine university medical center, diisseldoff, germany it is generally accepted, that cls, coagulation activation and refractoriness to platelet transfusions are part of the syndrome of hepatic vod. we assessed patients with either vod or cls or both vod and cls, in order to analyze the influence of either syndrome on different aspects of hemostasis. vod was diagnosed according to jones et al. [transplantation 44 (1987) 778]. diagnosis of cls was >_3% increase of body weight in the past 24 hours and non-responsiveness to furosemide [niirnberger et al., ann hematol 17 (1993) 67] . patients with vod, cls or both were compared to control patients without either diagnosis. eight patients suffered from both vod and cls, 5 patients only from vod, and 8 only from cls. 61 patients had neither syndrome and served as control population. activation of the coagulation system was assessed by increase of tat-complexes and/or increased consumption of at iil the hemostasis patterns were as follows: no. introduction: lung cancer goes along with coagulation activation and increased thromboembolic risk. acute phase reaction in cancer patients leads to elevated levels of c4b-binding protein (c4b-bp) followed by a shift from free to c4b-bp-bound protein s. we tried to find out whether there is a correlation between alterations of c4b-bp, protein c protein s system and interleukin 6 (il-6), which is one of the most potent inducers of hepatic acute phase reaction. patients: i. 25 patients with lung cancer; 2. control group: 11 patients in complete remission after lung cancer. methods: clotting methods: protein c and s activity; elisa tests: protein c antigen, tat-complexes, prothrombin fragments f i+2, il-6. electroimmuno-diffusion (laurell): free and total protein s, c4b-bp. results: tat-complexes and f i+2 were elevated in cancer patients. c4b-bp levels were slighthly increased (129±19 % of n.), protein s activity was 89±33 % of n. (control group: 108±23 % of n.). il-6 in lung cancer patients was 37.2252.9 pg/l (control: 27.2±8.2 pg/l). conclusion: one source of the hypercoagulable state in lung cancer patients is decreased protein s activity due to elevated c4b-bp levels. this is probably caused by hepatic acute phase reaction which is triggered by increased il-6 levels. these plasma levels correlate with levels of the tumor marker ca 125 and with the stage of the disease but correlations with patient outcome (disease recurrence and overall survival) have not previously been shown. plasma levels of d -dimer and ca 125 (determined by sandwich elisa assays} were measured prior to treatment in 36 women with figo stage t to iii ovarian cancer and correlated with tumor stage, relapse and overall survival over a mean follow -up period of 28 months (range 16 to 40 months). levels in 71 healthy women and 27 patients with benign ovarian disease served as controls. the occurrence of deep vein thrombosis in the cancer patients was also determined by impedance plethysmography that, when positive , was confirmed by contrast venography. preoperative d -dimer and ca i25 levels in ovarian cancer patients were statistically signfficantly higher than in controls. preoperative cut off values were calculated for the prediction of cancer relapse and survival for both measurements. d -dimer levels above a cut off level of 2060 ng/ml were statistically significantly associated with the rate of relapse but ca 125 levels were not. deep venous thrombosis occurred in 33 % of cases but there was no difference between properafive levels of d -dimer in patients who subsequently did versus did not develop deep vein thrombosis. high levels of d -dimer are associated with more advanced disease and with poor prognosis in patients with ovarian cancer. the high levels of d -dimer are a biologic feature of the malignancy itself that may be attributable, at least in part, to increased conversion of fibrinogen to fibrin in the tumor bed with subsequent degradation of fibrin by the fibrinolytic mechanism. thus d -dimer levels may serve as a marker for overall tumor burden as well as "disease activity". a high incidence of deep vein thrombosis exists in the course of the disease in ovarian cancer patients but preoperative levels of d -dimer are not predictive of this occurence. yon tempelhoff georg -friedrich, michael dietrich, dirk schneider, lothar heilmann. dept. obstet. gynecol. city hospital of ruesselsheim. -germany. an increase of plasminogen activator inhibitor activity (pai act.) in the plasma of cancer patients has been recently discribed. we have longitudinally investigated pai act. in 136 patients with primary breast cancer and compared the results with the outcome of malignancy. patients with untreated primary breast cancer and without proof of metastasis (t 1-4 n0-2 m0) were eligible for this study. in all patients coagulation tests including fibrinogen {method according to clauss), d -dimer (elisa} and pal act. (upa dependent inhibition test) were performed prior to primary operation, 6 months thereafter and at the time of cancer relapse. seventy -two healthy women and 43 patients with benign breast disease served as controls. during a mean follow -up of 32 + 16 months 34 patients (25 %) developed cancer recurrence and 13 (9.6 %) patients died. in all cancer patients preoperative levels of fibrinogen and pai act. were significantly higher compared to healthy women and to patients with benign breast disease. preoperatively only pal act. was significantly higher in patients with vs. without cancer recurrence (4.52 _+ 1.67 u/ml vs. 3.4 1 + 1.55 u/ml; p = 0.002). in patients with later recurrence pai a~t. significantly dropped 6 months after operation (p = 0.02) and was again significantly increased at the time of cancer recurrence (4.90 _+ 2.89; p = 0.001). a preoperative cut off value (calculated via cox model) of pai act. above 3.52 u/ml was significantly associated with the rate of relapse (tog rank: p = 0.0005) and in 70 % of patients who died of cancer preoperative pai act. were also above this cut off. impaired fibrinolysis in patients with breast cancer is significantly associated with the outcome of cancer. a monoclonal heparin antibody (mab) has been raised against native heparin using a heparin-bovine serum albumin conjugate prepared by reductive amination. for further analyses tyramine, which was covalently bound to low molecular mass heparin by endp0int attachment (malsch r et al: anal biochem 1994; 217: 255-264) , was labeled with 125-iodine at the aryl residue. the tracer antibody complex was immunoprecipitated by goat anti-mouse immunoglobuline igg. the mab recognized specifically intact heparin and heparin fractions. the lower detection limit of heparin preparations was 100 ng/ml. no cross reactivity of the mab occurred with other glycosaminoglycans such as heparan sulfate, dermatan sulfate, chondroitin sulfate a and c. oversulfated heparin showed lower affinity to the antibody hl.18 than 2-0-and 6-0-desulfated beparin. the method established for the purification of the mab was ammonium sulfate precipitation with followed dialysis. sds-page and high pressure capillary electrophoresis prooved the high purity of the received antibody. the biological activity of mab was tested by the chromogenic assay $2222 and remained stabile while purified. in conclusion, the present abstract describes an purified igg 1 monoclonal antibody directed against heparin and heparin fractions, which can be used for biological measurements. the concentration of heparin and dermatan sulfate in biological fluids is usually measured using radiolabeling. for this purpose aromatic compounds are usually used to insert radioactive iodine labeling at the saccharide backbone of the glycosaminoglycan. we developed methods for the specific labeling of hepann and dermatan sulfate at the terminal residue. tyramine was bound by reductive amination to the 2,5 anhydromannitosyl end of heparin, produced by nitrous acid degradation and confirmed by 13c.nm r spectroscopy. (anal biochem 217: 254-264, 1994) this method was also used to produce a low molecular mass dermatan sulfate (lmmd)derivative after partial deacatylation. in order to choose the proper method for evaluating the specific anticoagulant activity in the row of chitosan polysulphate (cp) samples with different degrees of pol3~merization and sulphation we applied to pharmaeapea article (a~) when assessing the ability of direct anticoagulants to depress the coagulability of recalcificated sheep blood (using the 3rd international heparin standard), and to measuring such acti¢ity as per pharmacokinetic model (a2). the model admits the "kinetics of cp elimination be linear in ease of intravenous injection to rabbits, as it is observed in heparin: ct=co exp(-i~ x t), where ct is cp concentration at the time moment t; co is cp concentration at the moment of injection; i~ is the elimination constant. besides, it is assumed that there is a linear approximation of the anticoagulant effect on the dose, which finally makes it possible to calculate the specific actidty a2 : t=kt ct + tin, where t is the time of clot formation at different tlme intervals after of cp injection; t~, is the time of clot formation prior to cp injection. t value was assessed in two tests: in blood coagulation time (bct) and in activated partial thromboplastin time (aptt). no correlation was observed between a1 and a2. at the same time the values of ifm and the period of semieliminatinn (tvz) with the use of the original method that were obtained with the help of the quantitative determination of cp in rabbit's blood taken at different time intervals after injection, showed a close correlation (1"=0,94 p<0,05) between the same parameters, obtained with the help of the of the pharmacokinetic model in bct test. thus, experimentally it was proved that the assumption of the linear elimination and the effect-dose dependence was true, which is necessary for a2 calculation. we recommend to use intravenous injection of the samples to animals with further assessment of the results according to the pliarmacokinetic model to calculate the specific anticoagulant activity in the row of chemically related potential direct anticoagulants. in this investigation we compared the biological activity of a low-molecular-heparm (lmw-heparin, mono embolcx®) after intravenous, subcutaneous and oral application in rats. sprague-dawly rats were anaesthetized by ketamine/diazepam and the blood samples were taken from the retro orbital sinuus. 150 axa u/kg body weight of the lmw-heparin were injected intravenously and subcutaneously to 10 rats each. between 3 minutes and 10 hours after injection serial blood samples were taken. 200 mg/kg (20.000 axa u/kg) body weight of the lmw-heparin were applicated orally using a stomach tube. blood samples were taken between 1 and 24 hours after oral application. the antifactor xa and antithrombin activities of the plasma samples were measured, using ehromogenic assays and the substances s 2222 and s 2238 (kabi vitmm). after i.v. injection the maximum axa and alia activities were 2.8 axa u/ml and 0.8 aiia u/nil respectively. after s.c. application the antifactor xa activity of the lmw-heparin showed a maximum of 0.5 axa u/ml atter 120 minutes. the antithrombin activity exhibited an eatiier maximum activity of 0.2 alia u/nil 60 minutes after injection. after the oral application no increase of the axa or alia activities was measured. the lmw-heparin has a high antifaetor xa and antithrombin activity after i.v. and s.c. injection. after oral application no activity of the lmw-heparin was measurable. these results implicate that fractionated heparin is not absorbed after oral application or is inactivated in the gastrointestinal tract. to improve the activity after oral application modified hepatins have to be synthesized. in an in vitro study the effect of various heparin derivatives (calciparin, fraxiparin, cy 222, cy 231, astenose, hexasaccharide, ssh 14) on thrombin-and adp-induced platelet aggregation as well as on adpmediated platelet activation in whole blood was investigated. all heparin derivatives caused a concentration-dependent inhibition of thrombin-induced aggregation of washed platelets. calciparin and astenose were found to be the most effective compounds with ic5o values of 0.67 and 1.2 p, mol/l, resp.; higher concentrations (5-30 times) were required for the other compounds. furthermore, the heparin derivatives were studied with regard to their potentiating effect on adp-induced platelet aggregation. in a concenwation range from 1 to 10 u/nil calciparin, fraxiparin, cy 222 and astenose led to a potentiation of the adpinduced aggregation whereas cy 231, hexasaccharide and ssh 14 did not show this effect. the increase in aggregation was associated with an increase in thromboxane a2 lbrmation. in addition, the effect of calciparin, fraxiparin, cy 222 and astenose on adp-induced platelet activation in whole blood was investigated by flow cytometric analysis using monoelonal antibodies to platelet surface receptors opiiia (cd-61) and p-selectin (cd-62). at concentrations that caused a maximum potentiation of adp-induced platelet aggregation these substances led to a strong increase of adp-mediated activation of platelets in whole blood. the effect was most pronounced when the blood was anticoagulated with calciparin and astenose, resp. in conclusion, the results suggest that the aggregation-promoting effect of heparin derivatives included in this study is dependent on the molecular weight and the degree of sulfation and is in part due to the generation of thromboxane. heparins are negatively charged polysaccharides and bind protamine forming a stable complex. here we report on the properties of microbeads (4.5 pro) coated by protamine. protamine chloride (0.16 ijm) was covalently bound to 0.5 mg paramagnetic tosyl..activated microbeads m-450 (dynal). the covalent binding of protamine was from 1.0 to 13,0 mg/g beads. protamine-dynabeads were produced in a phosphate buffer at different ph (7,0; 7,5; 8,0 and 8,5). the protamine-dynabeads produced ph 7.5 showed the best properties for flow cytometry analysis. in saline solution they bound lmm-heparin-tyramine-fitc (lmmh-tyr-fitc) dose dependently from 0.001 to 2 u/ml, whereas in plasma and blood they bound lmmh-tyr-fitc from 0.05 to 2 u/ml. dependent on the binding protocol, the microbeads also bind proteins unspecifically, i.e bovine serum albumine and protamine to a lower extent.the adsorbed proteins, however do not bind lmmh-tyr-fitc dose dependently. the saturation of the proteins on the beads was determined as their relative fluorescence intensity (rfi). in saline solution the saturation was measured at 380 rfi, in human plasma at 325 rfi and in whole blood at 252 rfi. using flow cytometry erythrocyctes, lymphocytes, monocytes and granulocytes were not bound to protamine dynabeads. these data demonstrate that protamine-dynabeads can be used to measure the concentration of lmmh-tyr-fitc in saline solution, plasma and blood because they do not bind to human blood cells. the present study was designed to investigate the anticoagulant action of inhaled low molecular weight (lmw)-heparin in healthy volunteers. 3,000 iu (group t), 9,000 iu (group 2), 27,000 iu (group 3) or 54,000 iu (group 4) lmw-heparin were given to 20 healthy volunteers each at 4 weeks intervals. in group 1 tissue iactor pathway inhibitor (tfpi) antigen and activity, 82222 chromogenic factor xa assay, heptest, aptt and thrombin clotting time (tot) remained unchanged during the 10 days observation period. in group 2 tfpi antigen and activity, aptt, tct and the $2222 method remained uneffected. heptest coagulation times were 18.7 + 2.0 before, 26.1 + 5.2 sec. 6 hrs and to 20.5 + 1.9 sec. 24 hrs after inhalation. in group 3 tfpi antigen increased from 74.1 + 13.9 to 80.5 + 14.2 ng/ml 3 hrs after inhalation. tfpi activity remained unchanged. $2222 method increased from 0.01 to 0.08 + 0.06 iu/ml 6 hrs after inhalation. heptest coagulation values were prolonged up to 42 _+ 7.6 s ec after 6 hrs and returned to normal within 72 hrs after inhalation. aptt and tct remained unchanged. after inhalation of 54,000 iu lmw-heparin, the following changes were observed: tfpi antigen increased to 103 +_. 17.9 ng/ml and normalized within 24 hrs. -i'fpi activity increased to 1.14 _+ 0.23 u 3 hrs after inhalation and was normal after 24 hrs. antifactor xa activity, as measured by s2222 method, increased to 0.343 + 0.196 u/ml after 6 hrs and was normal after 72 hrs. heptest coagulation values increased to 77.5 + 11.8 sec 6 hrs after inhalation and normalized after 144 hrs. aptt and tct did not change throughout the observation period. the data demonstrate a resorption of lmw-heparin by intrapulmonary route in man. no side effects were observed. recently we developed a tritium-labelled arachidonic acid ([3h]aa) release test with high sensitivity to membrane-toxic agents. the assay performed in u937 cells is intended to evaluate ehemicals, drugs and biomatefials with regard to their eytomembrane toxicity [kloeking et at. (1994) , toxicology in vitro 8, 775-777]. local irritation reactions are described in patients receiving therapeurieat dosages of lmw heparin. this fact prompted us to examine the following lmw hepafins and heparinoids for their membrane toxicity in u937 cells: reviparin-sodium, enoxaparine-sodium, mueopolysccharide polysulphate (mps), pentosan polysulfate sodium (pps), polysulfated bis-lactobionic acid amide derivatives lw10082 (aprosulate) and lw10086. for this purpose, [31--1]aa labelled u937 ceils were incubated with different concentrations of lmw heparins and heparinoids at 37°c for 1 hour. compared with untreated cells, the [~h]aa release of cells treated with 5 mg of the drugs was two times higher with reviparin sodium, three rimes higher with bis-lactobionic acid amide lw10086, five times higher with pentosan polysulfate, 20 times higher with ertoxaparine-sodlum, but it was equal to the control with mucopolysaccharide polysulphate. the rate of araehidonic acid release in response to a test chemical may therefore be used to assess the membrane-toxic effect of this substance and to predict its the inflammatory potential in the skin. semi-synthetic glyensaminoglycans (gags) with antithrombotic properties can be prepared from the e. coli k5 polysaecharide by coupled chemical and enzymatic methods. the molecular weight of these semi-synthetic gags can be adjusted to obtain products mimicking the molecular profile of a low molecular weight hepatm. in order to compare the biochemical and pharmacologic properties of a semi-synthetic gag (sr 80486a, sanofi/choay) with a commereiany available low molecular weight heparin, fraxiparine (sanofi, paris, france), valid biooheanical and pharmacologic methods were used. the molecular profile of this agent as determined by hplc exhibited a comparable distribution profile (mr=5.7 kda) in comparison to fraxiparine (ma=5.1 kda) . the anticoagulant properties of sr 80486a were comparable to fraxiparine in the aptt and heptest. however, in the usp assay, this agent showed slightly weaker activity. sr 80486a also exhibi~d comparable affinity to atffl and hcii. in comparison to fraxiparine, it produced a much weaker response in the hit screening system. in~ viv0 studies, sr 80486a preduecd strong dose-dependent antithrombotic actions in both the iv and sc studies in the rabbit jugular vein stasis thrombosis model (ed50=i 5-60 gg/kg). additionally, it also produced antithrombotic aefiorts in a rat jugular vein clamping model. the hemorrhagic effects of this agent were comparable to those of fraxipafine as measured in a rabbit ear blood loss model. intravenous administration of sr 80486a also revealed a comparable pharmaeokinetie behavior to fraxiparine. no abnomaiitias of the clinical chemistry (change in liver enzymes) and hematology profile (thrombocytopenia and lencecytosis, etc.) were noted in primates. at a dosage of i and 2.5 mg/kg iv, this agent also caused a release of functional tfpi which was comparable to the observed responses of other low molecular weight heparins. these studies suggest that sr 80486a is capable of producing similar pharmacologic effects as other low molecular weight heparms, however, additional optimization studies are required for demonslrating product equivalence. limited information on the comparative pharmacoldnetics of low molecular weight heparin (lmwh) is available on the data obtained from aptt, heptest, anti-xa and antmia assays. since these drugs are currently used for therapeutic indications using relatively high dosages and intravenous administration. aptt, heptest and antmia test may be valuable in the assessment of their effects. in order to investigate the relative pharmacokinetics of lmwh using apt'i', heptest, anti-xa and anti-iia methods, certoparin (sandoz, basel, switzerland) was administered to individual groups of healthy male volunteers (52-70 kg) via intravenous (30 mg) and subcutaneous (50 nag) routes in a crossover study. blood samples were drawn at 0, 5, 15, 30, 60, 90, 180, 360, 540 and 720 minutes. using a baseline pool plasma obtained from the same volunteers, calibration curves for each of the individual tests were constructed to extrapolate circulating levels of certoparin. a non-compartmental model using trapezoidal technique was used to obtain pharmacokinetic parameters such as t 1/2, vd, and clsys. in the intravenous studies, the t 1/2 was found to be dosedependent for aptt, heptest, anti-xa and antm]a. the auc, however, was significantly different for each test and was dose-dependent following the order: aptt<heptest<anti-xa<anti-iia. in the subcutaneous studies, 1the t 1/2 was both test and dose-dependent. the auc was also dose-dependent and followed the order: anti-xa>heptest>aptt>antmia. the clsys of the antma was much faster in comparison to the other tests. the clsys of the aptt and heptest was independent of dose. however, anti-xa clsys by this route was lower than other tests. the apparent vd followed the order aptt>antmia>heptest>anti-xa. the bioavailability of the certoparin as measured by various tests ranged from 81-119%. these studies suggest that beside providing pharmacokinetic data, aptt, heptest and anti-iia assays may provide useful data on thier safety and efficacy at high dosages. the immunological type of heparin associated thrombocytopenla (hat ii) is a severe complication of heparin treatment and is associated with arterial and venous thrombosis. only patients with absolute thrombocytopenia have prompted suspicion of hat in clinical practice. we report on a 44 year old male, who developed thromboembolic episodes after coronary angiography like reinfarction and thrombotic episodes of a. brachialis. fibrinolytic therapy combined with i.v. unffactionated heparin treatment was the therapy of choice and was followed by severe fua~er thromboembolic adverse effects. besides an impaired fibrinolytic response and elevated antiphospholipid anitbodies, we diagnosed hat type ii in hipa and elisa (stago-boehringer, marmheim). this special patient had platelet counts within a normal range, when developing the thromboembolic episodes. it appears that the normal platelet count during the thromboembolic episodes reflect a relative thrombocytopenia. from a clinical point of view we recommend the use of a lab panel to exclude hat type ii in patients with thromboembolic episodes under therapy with fractionated or unfractionated hepafin. platelet counts within a normal range are no absolute exclusion criterion for hat ii. low molecular weight heparins (lmwhs) are now commonly used for the prophylaxis of post-surgical thromboembolic complications. in this indication, lmwhs are administered as a single or twice a day subcutaneous regimen. usually these agents are administered at 30-40 mg total dose which is equal to 3000-4000 anti-xa (axa) iu. newer methods such as ehromogenic substrate based axa methods and the heptest clotting time can be used to determine the effects of lmwhs during the initial phases of prophylactic therapy. this may be useful in the elderly and weight compromised patients where a fixed dosage may not be optimal and may produce bleeding effects. similarly in the overweight patients, a fixed dose may not be efficacious. thus, monitoring of lmwhs in these patients may be useful in the optimization of their therapy. lmwhs are also used in the treatment of deep vein thrombosis using both intravenous and subcutaneous protocols. high dosages of up to 200 mg sc/day and infusions of up to 30 axa iu/kg/hr have been administered. in these conditions, the monitoring of the circulating lmwh levels may be useful in optimizing the dosage. we have modified the aca heparin (do_pont merck, wilmington, de) method to measure the lmwh levels in the plasma of patients treated with both the prophylactic and therapeutic dosage. owing to the required turnaround time, simple operation and reliable results, this method was found to be of value in the monitoring of these agents. this presentation provides an overview of the clinical application of various lmwhs with particular reference to the need of monitoring for their effects to optimize the clinical outcome. a double-blind, multicentric, controlled trial was performed in order to compare the antithrombotic efficacy and safety of single daily doses of 5000 ie anti-xa of low molecular weight heparin (lmwh) sandoz (certoparin) and 5000 ie unfractionated heparin (ufh) tid. in 288 patients undergoing elective total hip replacement blood samples were drawn before the first subcutaneous injection of lmwh or ufh resp., two hours after administration on the first and 7th postop, day and on the last day of prophylaxis (day 10-14), anti-xaactivity was measured by chromogenic substrate assay, heptest and aptt by clotting assays and tissue factor pathway inhibitor (tfpi) and heparin-pf4-antibodies by elisa techiques. as expected, the anti-xa-activity and the heptest values were significantly higher in the lmwh-group at all time points after administration of the drugs; the mean values of heptest were 35 sec in the ufh-and 75 sec in the lmwh-group respectively, the aptt was not different in both groups. at the end of prophylaxis positive antibodies to heparin-pf4complexes were detected ~n both groups; this however was not correlated with clinical thrombocytopenia. a detailed correlation between patients with deep vein thrombosis (dvt) and positive antibodies has still to be done (all patients were screened for asymptomatic dvt between day 10-14 by bilateral phlebography. tfpi was markedly increased in the lmwh-and only slightly elevated in the ufh-group; the differences are statistically significant. summarizing it can be concluded that antibodies to heparin-pf4complexes may occur without clinical symptoms of hepafin-induced thrombocytopenia type ii and that tfpi may play a sigificant role for the antithrombotic efficacy of ufh and lmwh. unfractienated heparin represents one of the most severe and frequent causes of drug-induced thrombooytopenia. heparin-indueed thrombocytopeala (hit) occurring early in therapy is often mild and serf-limited, appearing to be caused by a direct aggregant effect of heparin on platelets (hit type i). hit type ii, however, is immune-related an may result in absolute thrombocytopenia (platelet count <i50/~l) or a relative decrease in platelet count (?_50% reduction from baseline values). unlike most other immune thrombecytopenias in which bleeding complications often predominate, hit type ii may results in severe arterial and/or venous thromboses. the management of patients with hit type ii and thrombosis is often difficult. heparin must be stopped immediately. antithrombotic, fibfinolytic, or antiplatelet agents have been used in these situations, as have anticoagulants other than unfraetionated hepafirl plasmapheresis, used in other immune-related disorders, has also been occasionally reported. at our institution this intervention appears to provide significant elininal benefit to hit patients. several recent reports have noted that the heparin-dependent antibodies in hit type ii are directed against a complex of heparin bound to platelet factor 4 (pf4) and an elisa-based test system for hepafin-pf4 eomplexinduced antibodies (hpia; stago) has been develuped~ we report our experience in 3 patients with hit type ii in whom plasmapheresis was instituted and the antibody levels in blood were measured pre-and post-treatment to identify wkether circulating antibody titers decreased thus providing some explanation for the clinical benefit. the patients developed profound absolute thrombocytopenia (range, 3-21 k/~tl) and positive heparin-induced platetet aggregation studies following treamlent with uofractionated heparin. thromh0cytopenia induced by hepariu (hat-syndrome) typically appears several days after initiation of heparin therapy. this phenomenon is caused by antibodies of the lgg type which are induced by a heparin-pf 4-complex and which also activate platelets. in a recent study (n engl j med 1995; 332:1330-5) the hat-syndrom was diagnosed in 2.7 % (9 of 332) patients who had received unfractionated heparin (ufh) and in none of those 333 patients who had been treated with low-molecular-weight heparin (lmwh). 8 out of the 9 patients treated with ufh presented with at least one thrombotic event (7 times a venous, once an arterial event). the incidence of heparin-dependent igg antibodies (7.8 %) was higher in the ufh treated group than in patients treated with lmwh (2.2 %). our patient was a male caucasian white, 62 years of age, otherwise in good clinical condition with no severe diseases in his pmh. both parents, however, had suffered several thrombotic episodes. the patient was admitted to the hospital in september "95 with clinical signs of a deep vein thrombosis (dvt) of the right leg. the diagnosis was phiebographically confirmed. after having given informed consent the patient was treated with 5 cycles of r-tpa (loco-regional application) and 3 cycles of uhsk as part of a clinical trial (uhsk vs. rtpa, boehringer/m). lysis was successful with complete recanalisation of the veins. after completion of lysis the patient was further anticoagulated with coumarin overlapped by heparin (ufh). as a complication the patient suffered from a retroperitoneal and an upper gi4ract bleeding from esophagitis grade ii to hi while being on coumarin prophylaxis. therefore the patient was put on lmwh and then discharged with this prophylaxis. after less than two weeks the patient presented again to the hospital with a relapse of dvt in the same leg. phiebographically nearly all deep veins were occluded to a higher degree than before, i. e. also with concomitant pelvic vein affection and with hea~ 3, swelling of the whole leg. blood count showed a marked decrease of platelets to 27/nl. the platelet aggregation test demonstrated a hat-syndrome which was confirmed by the hipa-assay. the heparinoid orgaran® was not reactive in the assay. having seen a previous bleeding episode in this patient, we did not see an indication for another b'sis therapy. therefore we decided to put the patient on an intermediate dose of coumarin overlapped by hiradin (hat study, behring) instead of orgaran®, since hirudin allows a much more precise dose adjustment. after 10 days the patient was discharged with no further complications. risk factors for dvt have not been detected until now. to our knowledge this patient is one of the first cases of hat-syndrome induced by lmveh. under routine conditions aptt is the most frequently used test method for the surveillance of patients undergoing hepann therapy. in literature a 1,5 fold to 2,5 fold prolongation of the initial value is recommended for individual therapy a~ixatian. in contrast to the control of the enmarin therapy by means of prothrombin time measurement the individual hupafin sensitivity of the ~ reagents of this standard value hardly receives notice. commercially marketed ~ reagents differ from one another method and concentration of the activator as well as their phosphelipids. when choosing a reagent the user must only take into consideration the sensitivity against its factors, heparin and lupus but also the adalxability of the reagent towards the given measurement system ( software variability towards incubation time, sodimentalion problems in reagents with high kaolin concentration). reagents offering a high heparin sensitivity already are showing a prolongation of the coagulation time at low heparin dosage (0.1 -0.3 u/ml). in the therapeutic range (0.4 -0.8 u/ml) very often no linear relationship between prolongation of the aptr and heparin concentration level is found_ therefore very often coagulation times above the routine measuring range (180 s) can be observed in this range. moreover, various medical measuring devices differ in their ability to recogniso the retarded entry of coagulation. coagulation limes with different apq't reagents in correlation with the heparin concentration level can be found in the adjoined table. significantiy prolonged coagulation ti.~es of aptt lead to a reduction of the hel~ dose in clinical practice. due to the discrepancy between heparin concentmon level and the prolongation of the afrt in reagents with a non-linear heparin sensitivity a reduction of heparin dosage leads to an inmt~cient anticeagulalion and the risk of further thrombosis. therefore the producers should be asked to include precise directions on the heparin sensitivity of the aptt reagent in the therapeutic range in order to enable an optimal surveillance of the heparin therapy. a coordination between clinic and laboratory should be compulsory for the choosing of the aptt reagent. introduction: hat is a rare but sometimes fatal complication of therapy with unfractionated (ufh) and low molecular weight beparins (lmwh). we report on our experience with the clinical coume and management in 12 patients (pts). in all cases a severe decline of platelet count and in 11 cases a positive hipa test (= heparin-induced platelet antibodies) were documented. patients, clinical course and outcome: 8 female and 4 male patients (median age 68 years, range 22-78) were analysed in this retrospective study. 9 pts were referred from outside hospitals. all pts had been treated with ufh (5 pts additionally with lmwh) for prophylaxis (n = 9. in 2 pts pedoperatively) or treatment (n = 3) of venous thromboembolism (v'te). in 11 pts at least one vte occured during heparin therapy, in all cases prior to hat-diagnosis (vte: n = 13; putmonary embolism: n = 6; artenal embolism n = 1; thrombosis of sinus transversus n = 1). hat was suspected 16.5 days (median; range 12 -48) after first heparin application. hepadn therapy was discontinued immediately, at that time the median plateiet count was 35 g/i (range 10 -117). 9 pts were treated with orgaran r, followed by recombinant hirudin (r-hirudin, behdngwerke ag: clinical study) in 4 cases; 3 pts were treated exclusively with r-hirudin. plat~let count normalized in 9 pts within 3 days (median; range 1-10). one patient in the orgaran" group died on recurrent pulmonary embolism. 11 pts recovered without any further thromboembolic complication. conclusion: in our small series of 10ts hat was complicated with vte in 11/12 cases. in many pts hat was diagnosed strikingly late. for early diagnosis careful monitonng of platelet counts are mandatory dunng hepadn therapy, both orgaran ~ and r-hirudin seem to be safe and effective in preventing further thromboembolism, p 179 to prospectively evaluate coagulation or fibrinolytic activation in children long-term anticoagulation with lmwh was administered a longitudinal study was perfomed. within a 30 months period 48 children and adolescents (6 -19 years) suffering from malignant bone tumors (ewing's sarcoma or osteosarcoma) received enoxaparin (clexane r/rhone-poulenc rorer, france) for primary propylams. the majority of patients received 0.6 mg/kg bw enoxaparin 12 hours before major surgery (limb salavage). in addition, ten neonates and children received enoxaparin in the same dosage for secondary prophylaxis. in both groups therapy was adjusted to 0.2 -0.4 iu/ml anti xa activity. median (range) duration of therapy was performed for l0 (6 -24) weeks. before ('1"1), one (t2) and 3 weeks (t3) within the context of the present study an investigation on the physiology of clotting was conducted in three groups each consisting of 12 patients, who had to undergo a total end-prosthetic hip implantation (teph) for the first time. criterion for allocation to a particular group was type of intraoperative transfusion the patient received : infusion of whole blood, erythrocyte concentrate or fresh frozen plasma. at eight fixed times the activity or concentration of the components of the kallikrein-kinin and inhibitory systems was determined. the statistic evaluation ensued with the spss software package. for all three patient collectives an activation of both systems was to be observed with the teph-implantation whereby there were no statistically striking differences between the three groups. thus in the case of the kallikrein-like activity an intraoperative rise of over 37% was registered, while at the same point in time there was only a 4% reduction in the prekallikrein activity. postoperatively a 10% rise in the prekallikrein activity was to be observed. as a reserve inhibitor alpha 2-macroglobalin showed no significant change in activity during the period of observation whereas the acute phase proteins alpha 1-proteinase inhibitor and cl-esterase-inhibitor yielded a postoperative rise of 135% and up to 100%. the beta-factor xllainhibition showed a rise of 121% during the reconvalescent phase. the 65 year old female caucasian patient presented here, was admitted to a nearby district hospital in december '94 with the acute symptoms of pulmonary embolism. the source of the embolus could not be detected. high dose heparin was administered. pmi-i revealed a coronary artery disease and a peripheral artery occlusive disease of the left lower leg, yet blood pressure values of the lower ex~emities were only slightly below brachial pressures. the first episode of pulmonary embolism was followed by a second one two weeks later for which the patient received heparin again. following this treatment the platelet count decreased significantly to 10/ni. with an acute thrombosis of the lower caval vein and with leriche's syndrome, i. e. occlusion of the abdominal antra from the bifurcation down to the ileofemoral arteries bilaterally, the patient was transferred to our hospital. on initial examination we saw a patient in no acute distress, no dyspnea, blood pressure 120/80 mmhg, pulse 94/rain, no peripheral edema or disturbances of sensitivity. neither femoral, popliteal, nor pedic pulses were palpable on either side. a heparin associated thrombopenia was detected with the platelet aggregation assay and confirmed by the heparin induced platelet aggregation assay (hipaa). the autoantibodies targeting the underlying pf4-heparin complex were determined later by an elisa method in serum samples of the patient (asserachrom hia, diagnostica st,ago). a temporary anth&~r filter system was inserted transbmchially into the lower eaval vein and placed jnst above the thrombus. in the following days, 2 cycles of lysis therapy with 9 mil u streptokinase were administered taking six hours for each cycle. heparin was given in the mean time via the filter system in therapeutic dosages in order to prolong values 1.5 to 2 times the individual base line level. at the end of the second cycle, pulses were palpable inguinally, two hours later both popliteal and the right pedic pulse were present. the left leg showed better microperfusion than before. computer tumoglaphy did not reveal an)-thrombotic material in the descending aorta and the iliac arteries. for continuous anticoagulation after lysis we used the heparinoid orgaran® to overlap the period until full anticoagulation was achieved by coumarin. the heparinoid was tested before us non-reactlve in the hipaa. with this prophylaxis the platelet count increased to normal values. hat ii related thrombosis is the most severe complication of heparin therapy, especially in patients with pre-existing thromoembolic disorders (dvt, dic). we present to our knowledge the first case of hat ii during thrombolytic therapy (it) with simultaneous hepatin administration. at admission a 16 year old girl showed complete occlusion of the pelvic veins of the right leg (r common and external iliac v., common and proximal superficial femoral v.). as in children no prospective studies on the thrornbolysis of dvt with rt-pa are available, we started tt according to the frankfurt/m. regimen: rt-pa (actilyse) bolus injection 0.4 nag/kg bw followed by continuous infusion of 1 mg/kg bw/d; simultaneous low dose heparin 100 -200 u/kg bw/d. doppler ultrasonographie control on day nine after the beginning of tr revealed a further distal growth of the thrombus into the popliteal vein. at the same time at ir was decreased to 70% and there was a dramatic drop in platelet count to <50% of the baseline value (216 to 86 gpt/1). the diagnosis hat ]i was confirmed by positive hipa-test. the only heparin preparation not cross-reacting with the antibody was the lmw heparinoid orgaran. heparin therefore was replaced by orgaran: bolus injection 1,500 u/h followed by infusion of 200 u/h. rt-pa-therapy was not discontinued. at ri substitution was performed at levels <70%. under orgaran administration platelet count rose from 77 to 239 gpt/1 within 5 days. on day 12 complete reperfusion was achieved and rt-pa was discontinued ,on day 14. the patient was discharged from the hospital on prophylaxis with phenprocomnon in good general condition at admission thrombophilia parameters (protein c and s, apc) were within normal range. heparin induced thrombocgopenia (hit) is a serious but rare side effect of treatment with unfractionated heparin. patients with hit present with a rapid decrease in thrombocyte count and occurence of venous or arterial thrombosis under heparin treatment. patients with arterial occlusive disease who need surgical intervention are of high risk to develop rethrombosis especially during the first few postoperative days while they are under i.v. heparin-treatment. in our study we investigated prospectively all patients who were admitted to a surgical unit because of arterial occlusive disease and received surgical intervention. 15 patients were screened on the 3rd and 6th postoperative day for heparin-antiplatelet 4-antibodies, decrease in platelet count and thromboembolic complications. 3 patients developed antibodies against heparin-platelet factor 4. one of the 3 patients developed a venous thrombosis in the operated leg in combination with a decrease in thrombocyte count. we conclude that heparin-platelet factor 4-antibodies are often produced in patients under i.v. heparin treatment. only few of these patients actually suffer from clinically s3anptomatic hit. medizinische universit~itsklinik, institut far klinische biochemie und pathobiochemie, d-97080 wtirzburg, germany the important physiological and pathophysiological role of platetets in health and disease is well established. platelets are also the primary target of many vasoactive hormones, factors and drugs. in addition, platelets provide a very important model system for studying agonist-and antagonist-mediated signal transduction events (1,2). platelet activation by agonists is associated with shape change, extension of filopodia, generation of intracellular messengers, secondary agonists and activation of adhesion receptors and integrins including the fibrinogen receptor gp iib/iiia. activation of protein tyrosine ldnases and calcium-dependent protein kinases appear to be of critical importance for the mechanism of platelet activation. in contrast, platelet inhibition by vasoactive factors and drugs is often mediated by the camp and/or cgmp signal transduction cascades. recent data form our laboratory suggest that the focal adhesion protein vasp, a major target of the intracellular no/cgmp and prostacyclin/camp effector systems, is an important link between signal transduction pathways and elements controlling cell motility (3-6). the implications of the recent advances in platelet signal transduction research for understanding the physiology, pathophysiology and pharmacology of human platelets will be discussed. domain. however, in vivo truncated "i'po is much less potent than full length tpo due to its rapid clearance. this suggest that the c-terminal glycosylated domain acts to stabilize circulating tpo. tpo stimulates both proliferation of progenitor megakaryocytes and their maturation to platelet producing megakaryocytes in vitro. in vivo tpo induces dramatic increa~ses in megakaryocyte number and platelet production in animals, indicating that tpo regulates both thrombopoiesis and megakaryocytopoiesis and is the primary regulator of physiological platelet production. this later notion is supported by the phenotype of ti~ and cmpl knockout mice in which platelet levels are only 10-20% of normal while other blood lineages are unaffected. in animal models of myetosuppresion ,and marrow transplant, tpo reduces the platelet nadir seen in the~se models and significantly accelerates platelet recovery, tpo also has profound effects on red cell recovery in these models. these results suggest that tpo will be clinically u~ful for the treatment of thrombocytopenia in caucer patients who undergo chemotherapy or bone marrow transplant. the haparg-study ( .~a.emostatic parameters as risk factors in healthy volunteers) was sponsored by the german ministry of research and started in 1984. a previous study ( pard, intern. angiology 5, 181-195,1986 ) had shown that spontaneously enhanced platelet aggregation as measured with the pat-ill-test and a high fibrinogen level were significant risk factors for new vascular occlusions in diabetic patients. it was the aim of the haparg study to establish if spontaneous plateletsggregation and other hemostatic variables are independent risk factors for vascular occlusions also in healthy volunteers. employees of hschst ag, frankfurt aged 40-65 years and personnel of the university of mainz, aged 30-60 y. entered this prospective study. besides anamnestic data on smoking, hypertension and diabetes blood pressure, the ankle/arm doppler -index and an ecg were recorded and serum cholesterol, hdl, ldl, triglycerides, uric acid and glucose were measured. 1885 men and 988 women entered the study and were followed for 4-6 years. during this period 53 vascular occlusions ocoured ( 36 coronary and 9 cerebral events and 8 peripheral vascular occlusions). only three of these events occured in women. besides age and gender as expected risk factors the multivariate logistic stepwise regression analysis revealed as significant risk factors slightly elevated levels of blood glucose (p=0.001), smoking (p=0.0013), an elevated diastolic blood pressure (p=0,018),followed by spontaneous aggregation (p = 0.017) and higher hdl-levels (p=0.034) as significant risk factors for men. higher fibrinogen levels just missed significance in this analysis (p=0.059). none of the other hemostatic parameters showed any significant correlation with the vascular events.to our knowledge this has been the first prospective trial in a large population of healthy individuals in which a platelet function parameter has been studied together with other possible risk factors. spontaneously enhanced platelet aggregation seems to be an independent risk factor and may be an indicator of an ongoing active atherosclerotic process. mild bleeding symptoms -petechia, prolonged bleeding after tooth extraction -were observed in a patient with normal coagulation tests but slightly disturbed phtelet function: decreased aggregation in response to adp, pal: and thrombin receptor-derived peptide (trap-6). platelet count, agglutination with both ristocetin and botrocetin and thrombininduced aggregation were in the normal range. an abnormal membrane glycoprotein detected in electrophoretic studies of the patient's platelet proteins was shown to be related to gp llxx by immunoblotting. the variant gp differed even from the largest normal polymorphic gp iba form a by an increase in molecular mass, platelets of the patient contain the variant and normal c form in equal amounts. the quantity of gp ib as detected by flow cytometry is normal. lmmunoblotting studies of proteolytic fragments of the abnormal gp indicated that the defect is located in the c-terminal part of the glycocalicin fragment. measurement of the length of glycocalicin molecules after glycerol-spraying rotary shadowing electron microscopy demonstrated two populations of molecules in the patient sample: a normal on with an average size of 470 angstr6m and a fraction with an average size 0f650 angstr6n~ preliminary results of molecular analysis of the gp iba gene in the patient confirm the localization of the genetic defect by protein studies. in summary, the patient is heterozygous for a hitherto undescdbed molecular variant ofgp ~a. barnes and co-workers have shown that simple triple-helical collagenlike peptides, comprising basically a repeat gly-pro-hyp structure, are highly platelet reactive in polymeric form. they additionally have shown that the activity does not involve gpla/lla (integdn c~z~l). platelets adhere under static conditions to these fibrillar peptides mainly in a bivalent-cation independent manner; the bivalent cation dependent adhesion can be inhibited by an antibody against gpflb/llla (integfin m~d33). however, platelets of a patient with type i glanzmann's thrombasthenia secreted their ¢z-granule-and dense body content just like normal controls while activated with the triple-helical gly-pro-hyp peptide. the lack of an essential involvement of cd36 was shown with cd36deficient platelets, which respond to the peptides as readily as normal platelets when fibdnogen binding and cd62 and cd63 expression were measured. to study the involvement of the yon willebrand factor (vwf) platetets from a patient with severe type 3 vwd (vwf not detectable in platelets and plasma) were investigated. the vwf deficient ptatelets responded quite normally to the fibritlar collagen-like peptide. addition of pudfied vwf to the vwf-deficient platelets before adding the paptide raised the platelet respond slightly we conclude that neither ~3~ integrin, nor m~ integdn, nor cd36, nor vwf are the pdmary platelet receptor for the collagen-like peptide. there is evidence that native low density lipoprotein (ldl) can be oxidatively modified in vivo and the oxidized ldl may contribute to thrombogenesis and atherogenesis by damaging endothelial cells, eliciting vasocontractiola and stimulating blood platelets. by means of biochemical methods, electron microscopy, reflection contrast microscopy and computer image analysis, we find that oxidized ldl, compared to native ldl, affects platelets at least in the following five aspects. 1) oxidized ldl alters platelet functional morphology in a time and dose dependent manner. 2) oxidized ldl induces secretion of serotonin from both morphological resting platelets and shape changed platelets. 3) oxidized ldl advances, accelerates and enlarges platelet adhesion. 4) oxidized ldl causes cytodamage in platelets and cultured endothelial cells. 5) oxidized ldl stimulates platelets and endothelium, which leads to a formation of microthrombi on the monolayer of human umbilical vein endothelial cells. further experiments show that oxidized ldl inhibits the activity of ca2+-atpase in purified plasma membranes of platelets, and therefore increases the cytosolic calcium level in platelets. these results clearly indicate that calcium signaling pathway is intimately involved in the mechanism of platelet activation induced by oxidized ldl. since oxidized ldl, a relevant platelet agonist, has been confirmed to be present in vivo, further studies on its role in platelet activation and plateletendothelium-interaction can help for better understanding thrombogenesis and atherogenesis. recantly physical parameters such as sheer stress, hypo-or byperoxic conditions and hyperthermia have been shown to modulate endothelial cell dependent fibrinolysis. in this study we investigated whether yet another physie~ stimulus namely hypothermia might have an impact on the expression of components of the fibdnolytic system of human endothelial cells in culture. confluent cultures of human umbilical vein endothelial cells (hlivecs) were exposed to gc ° for 10, 20, 40, 80, or 160 rain, respectively. control cultures were exposed to 37°c for the same time periods. thereafter medium was removed, fresh medium was added and the cells were incubated for 24 hours at 37°c conditioned media (cm) were harvested and plasminogen activator inhibitor-i (pal-i) antigen was determined by a specific elisa. our data showed that huvecs responded to exposure to 8°c with a time-dependent increase of pai-i in cm. maximum induction of pal-! antigen was seen after 20 minutes exposure to 8°c (pal-! antigen: 754_+.83 ng/105 cells for treated cells; 4202_17 ng/105 cells for control cells). the inereese in pal-1 antigen was also reflected on the level of specific mrna synthesis as evidenced by northern blotting which showed a twofold increase in pai-i specific mrna in huvecs exposed to 8°c as compared to control cells. in conclusion, our data gives evidence that hypothermic stress like several other forms of physiological or pathological stress, results in activation of endothelial ceils as evidenced by an increased expression of pai-i. our findings provide ~rther evidence that such activation of endothelial cells can be induced not only by biochemical mediators but also by physical stimuli. nitric oxide (no) is formed from intracellular l-arginine by two no synthase (nos) isoforms: a ca+ +-dependent constitutive form (cnos) and a ca+÷-independent inducible form (inos) which is expressed after challenge of cells with immunologic or inflammatory stimuli. endothelial cell derived no is an important signaling molecule inducing smooth muscle cell relaxation and platelet inhibition, however its role in cell proliferation is poorly understood. the present study investigates the pattern of nos isoforms and no production in actively proliferating low density human umbilical vein endothelial cells (ld-huvec) and in high density monolayers of quiescent cells (hd-huvec). ld-huvec but not hd-huvec expressed inos (measured as ca ++independent citrulline formation from l-arginine in cell lysates) with a maximal activity of 30-40 pmoles/min/mg protein, cnos (ca +÷dependent citrulline formation) was demonstrated in hd-huvec (0.02-0.2 pmoles/min/mg) but not measurable in ld-huvec lysates. lmmunoprecipitation of cell lysate with a mab against murine macrophage inos decreased inos activity of supernatant by 70% in ld-huvec. immunoprecipitation with an anti-ecnos mab did not modify inos activity in ld-huvec but abolished cnos activity in hd-huvec. no release (measured as nitrite + nitrate production in an 18 h incubation in the presence of 1 mm l-arginine) was 1.9 nmol/mg cell protein in the supernatant of hd-huvec whereas it was t0fold higher with ld-huvec. measurement of intracellular cgmp after stimulation of cells with i-2 #m ionomycin (a sensitive index of enos activity) showed a 400-800% increase of basal levels in hd-huvec but only 50-150% increase in ld-huvec. on the contrary, sodium nitroprusside-induced cgmp increase was similar in non-confluent and confluent cells. the expression of inos and the high no production in ld-huvec suggest a role of endogenous no in mediating cell proliferation. indeed, the nos inhibitors l-nmma and l-canavanine inhibited [~h]thymidine incorporation in ld-huvec by 60%. on the ++ nt no r uctlon ar x r d m other hand enos and ca -depende p od " e e p esse " differentiated huvec and correspond to the ability of the cells to regulate vascular tone and platelet activation. the processes of replication, maturation and motility of progenitor cells of different lineages in the bone marrow are tightly regulated, particulady by various cellular contacts and their modulation involving pericellular proteolysis. while mononuclear phagocytes and related cell lines are well characterized for surface localized plasminogen activation by receptor bound urokinase, megakaryocytes are poorly described in this respect. in the present study the expression of mrna of urokinase receptor in megakaryoblastic cell lines chrf-280, dami and meg01 was found at a low basal level and increased 5-10 fold after pma-treatment. urokinase receptor mrna levels remained differentiation dependent elevated for up to 5 days. functional analysis of upar expression on the cell surface was documented immunologically by facs-anatysis and on the functional level by direct binding of radio-labeled amine-terminal fragment of urokjnase and mirrawed the increase in urakinase recptar mrna level. as expected, antibody and ligand binding was sensitive towards treatment with phosphatidyl-inositol-specific phospholipase c, since the urokinase receptor belongs to the group of gpi-anchored receptors. in cultered megakaryocytes derived from bone marrow urokinase receptor mrna was demonstrated by rt-pcr, as well. these results indicate a differentiation-dependent increase of urokinase receptor expression on cells of megakaryoblastic cell lineage suggesting the participation of urokinase dependent events during maturation process and intravasation of these platelet precurors. regulation of am gene expression may be coupled to oxidative stress through redox sensitive transcriptional regulatory factors. the aim of this study was to investigate the influence of selected antioxidants on tnfa (100 u/ml, 6 h)-induced surface expression of elam-i, icam-i and vcam-i in human umbilical vein endothelial cells. am expression was determined by quantitative flow cytometry and immunohistochemistry (alkaline phosphatase anti-alkaline phosphatase method). the following antioxidants were tested: pyrrolidine dithiocarbamate (pdtc), n-acetylcysteine (nac), glutathionemonoethyl ester (gshmee), probucol and the estradiol-derived scavestrogens j824, j861, j901. additionally, 17fi-estradiol was examined. the used concentrations were 1-5 mm for nac and gshmee and 1-100 ~m for all other substances. according to their influence on tnf-induced am expression, the tested antioxidants can be divided into three groups: 1) pdtc, nac and gshmee inhibit the expression of all three am, 2) j861 and j824 reduce vcam-i and icam-i and increase elam-i, 3) j901 and probucol show no effect. 17fl-estradiol elicits a potentiation of tnfa-induced expression of all three am. tnfa-induced vcam-i expression was most effectively inhibited by ptdc and j861 (100 em: 68% and 78% inhibition, respectively). the best inhibitors of icam-i induction were pdtc (100 ~m: 51% inhibition) and j824 (100 t~m: 47 % inhibition). j824 and j861 most effectively increased the elam-t induction (100/~m: 85 % and 62 %, respectively). the results confirm that antioxidant-sensitive mechanisms play a role in tnfct-induced am upregulation. it is suggested that icam-1-and vcam-l-expression is regulated differently from elam-i. the diversity of antioxidant effects might be due to distinct levels of redox control of transcription with an antioxidant-inhibited and an antioxidant-promoted step. heamophiliacs who continue to produce tilers of f.v11i inhibitors no higher than 5 to 10 bethesda units (bu) when given continuous f,viii replacement therapy are classified as low responders. studies carried out on low responders to assess the effect of immune tole~lcc therapy flit) in these patients. it was found that elimination ofiahibitors in low responders could be achieved using f.vlii at 20 to too u/kg given every 2 to 3 days over a period of 6 weeks. we consider that 1tt is justified in this group of patients. prevents rebooster effects and reduces elimimfion time of f.viii inhibitors. it also reduces the enhanced mortality in patients with inhibitors and has been shown to be cost-effective. in view of the improved prognosis, we recontmend starting itt in low respor, ders soon after the diagnosis has been made in order to minimize f.vill exposure before isfi.tiating el'. itr in low responders has a better success rate an.,d requires shorter u'eatment than itt in high responders. w.schramm, med.klin~, innenstadt, lmu, mfinchen following replacement therapie~ in hemphilm t_be developmem of inhibimrs to the p*ficat's deficient factor is the most serious sequelae. the incidence of inhibitors varies between 15 and 35 % of persons with hemophilia a. inhibitor develop, mostly in paticm~ with sovere bemoph/lia a, early in life and afmr few exposure days (9 -12 days). using the bethesdamethod the inh~itom can be derided in low (< 5 bu) and high msponder (>5 bu) hemopb~iacs with high fitcrs have ~ually serious ~ problems. they are resistent to mg,,flary replacement therapy, the ~ goal in the treawnent is to control severn acum bleedin~ and to eradicate the inlu'bitor perrmnanfly and to induce tolea'ance. in the tream'tmt of acute blcedings in patients with hlhibitors factor viii inhibitor bypassing ag~ts like activated prothro~ complex concenuxtes (feiba) or prothrombin complex concentrates (pcc) arc mostly used. the meehani~n of aefiou of theses concentrates is net fully investigated. their effect is usually related to the high coment of activated clotting factors ~d phosphoupids. since some years acdwated recombinant factor vii (f vii a) is used to treat patients with inl'dbitocs successfully in several clinical situations including surgery. in addition porcine factor vii1 is widely used in particular in the uk for the treatment of factor v].ii inhibitor patients and could demonstrade good clir3cal results, in case of life threatening bleedings a temporary reducfic~ of inhihitors could be. ~hieved by using extem*,ivc plasma exchange (protein a adsorption) and immune suppression with cyclophosphamid (~alm6 protocol). follow~g the first description by h. bmc~'~mn some modifications for tlm induction of irmnune tolerance in hemophilia a patients have ~en propet'ed. these schedule, can be derided into high, intemxxfime and low dosage roglrmms di:ffea'jng in the dosage of factor viii infused. successful rates about 70 to go % can be obtained with ~ and high dose regimens. but is has to be co~sidered that the~ expensive trea.t~nt regimens have a great physical and p .syc.hosocial impact to the benx~-li~s and thch" farm~e& the different immu~ mler-a.~ze mg~-'~ predominantly used in high rcsponder inhibitor. most of the patients with low concentrations of inhibitors cm be managed with factor viii in increased dosage. this is in agreement with the consensus recorrnr~rdadons for u'eatlncnt hemophiliacs in germany fi'orn 1994. before vitamin k(vk) prophylaxis was generally accepted in japan, the incidence of infantile vk deficiency was 1:4000 both idiopathic and secondary types. since 1981, 4 nationwide surveys have been conducted. the current incidence rate is now about one-tenth that in early 1980. however, in a small number of eases, vk deficiency oceured despite prophylactic administration during the neonatal period. in order to clarify the absorption,excretion and transplacental transport of vk in the perinatal period,following studies were carried out. t)hepaplastintest(normotest) were performed on 65 women in the last stage of pregnancy and each coagulation factor was estimated as well. 2)correlations were made between mothers'and babies'hepaplastin test values. 3)transplacental transport of vk 2 was studied. the general activity of vk dependent factors in pregnant women was much higher than in non pregnant women. as far as the correlation between mothers'venous blood during delivery and cordvenous blood is concerned, in the group of mothers with hepaplastin test value of less than 120% of the normal adult value, the value of the hepaplastintest was less than 30 % of normal adult value in the cord venous blood° we also demonstrated that vk passed through the placenta but only in small qualities. 61hiv-negative patients (median age 4]yrs, 13-70}, formerly treated with non-virusinactivated coagulation products, underwent hepatologic examination, including afp screening and sonography. 31.suffer from severe, 20 from moderate or mild haemophilia a or b, 10 from other severe coagulation factor deficiencies. 52 had been treated with products of the swiss red cross (src) only (28 with small pool cryoprecipitate}, 4 with foreign products only, 5 with both src and foreign products. treatment intensity was variable with>20'000 iu/yr in 26,< 20'000 in ]6, < 1 treatment episode/yr in ]0, a total of only 1-3 treatment courses in 6 patients. 3 afibrinogenemic patients had prophylactic replacement therapy. hcv serology was positive in 56/6] patients (92%), in 47 with detectable hcv rna (77%). the 5 persons who escaped hcv infection, with normal alt-levels and without sonographic alterations, had low intensity treatment with small pool src preparations only. alt-levels were elevated in 33/56 anti-hcv positive patients (59%). 26/56 had abnormal sonographic findings (46%). there was a clear correlation between elevated alt-levels and abnormal sonographies: of 33 patients with elevated alt 23 had abnormal sonography, of 23 with normal alt 3 had abnormal sonography. 8 patients had liver cirrhosis (6 with clinically overt hepatopathy), 4 (4/56 = 1%!) with hepatocellu]ar carcinoma (hcc) with elevated afp-leveis. of these 4 patients 2 had intraarterial embolization with ]ipiodol-epirubicin; in 2 patients hcc diagnosis was made in a late stage. i patient with advanced liver cirrhosis underwent successful liver transplantation. 2 of the 6 patients with hepatopathy had severe haemophilia with temporary high alcohol intake, 4 had mild coagulatlon disorder with few treatment episodes. possible precipitating factors were coinfection with hbv, high alcohol consumation and first exposure to hcv contaminated blood products in an advanced age, but not intensive replacement therapy. very similar results for f vlll and vwf. since the factor viii level is kept steady above the level where there is an increased risk of haemorrhage, continuous infusion is haemostatically safer and more efficacious than bolus injections, another advantage is a progressive decrease of clearence during the first days after surgery which leads to a substantial reduction of factor concentrate consumption by avoiding the innecessary peaks of bolus injections. 22 children with severe form of haemophilia a undergoing elective surgery received continuous infusions with different plasma-derlved and recombinant f viii concentrates. before surgery, patients got bolus injections to raise the factor viii levels to more than 80 %. during continuous infusion factor viii levels were measured two to three times a day and the infusion rate of 4 to 5 iu/kg/h could be reduced on the second or third day to 2-3 iu/kg/h. the clinical efficacy was excellent with no bleeding events. in 5 children with vwd also undergoing elective surgery continuous infusions with humate pr were performed in the same way. no bleeding events were observed in these patients. none of the patients developed postoperative wound infections. the overall doses of f vtll concentrate 'were about 20-30 % lower than those required during replacement therapy with bolus doses. lg8 factor x frankfurt i : molecular and functional characterisation of a hereditary factor x defect (gla +25 lys) huhmann i., holler b., krinninger b., turecek p.l, richter g., scharrer i., forberg e., watzke h. univ. klinik f0r inhere med.i, abteilung for h~tmatologie und h~mostaseologie, w~en; immuno-ag, wien ; klinikum der j.w. goethe-univ. frankfurt am main, abt. f. angiologie. factor x (fx) is a vitamin k-dependent plasma protein which is activated either by fvila/tissue factor or ixaniila. fxa is the main enzyme for conversion of prothrombin to thrombin. the congenital fx-deficiency (stuart -prower-defect) being inherited as an autosomal recessive trait subsequently leads to bleeding diasthesis of varying severity. our propositus is a 24 year old patient presenting a mild bleeding tendency. his p'fi (36 sec) is within the normal range, the pt ( 73% of normal) is slightly reduced. the factor x antigen level is reduced to 55% of normal. molecular charactedsation of the genetic defect was performed by amplification of the eight exerts and exonintron junctions by pcr and subsequent direct sequencing of the products. in comparison to the normal sequence we could determine a single mismatch within exon ii resulting in the substitution of +25 gla (gaa) by lys (aaa). the mutation abolishes a naturally occuring mboll site in the dna sequence of exon ii. the status of the fx encoding alleles was determined in the propositus, his mother and one of his brothers by amplification of exon ii and restriction digest with mboll. these family members were heterozygous with respect to the mutation in exert ii. fx was isolated from plasma of the propositus by monoq ion exchange chromatography. performing clotting assays with purified fx frankfurt i we determined an activity of 89% of normal fx upon activation with rw, 77% upon intdnsic activation (aptt) and 81% upon extrinsic activation (pt). this compares well with the results obtained from the patient plasma ( pt 56%, ptt 55% and rw 57% of normal) when the reduced fx-ag-level of the plasma (55%) is taken into account. we therefore conclude that the substitution of gla +25 to lys results in a fx molecule which is severely defective in both the intrinsic and extrinsic pathway of blood coagulation. bleeding after cardiothoracic surgery is still a frequent, important and sometimes life-threatening complication. thus, the aim of this study was to examine routine parameters of hemostasis and their predictive values for severe bleedings. this prospective study included patients undergoing cardiopulmonary bypass surgery. blood samples were drawn preoperatively as well as 0, 6, 18 hours and 2, 3, 4, 5, 6, 7, 8 days after surgery. blood loss from drains, transfusion of blood products and other important clinical data were monitored apart from platelet count, hematocrit, thrombin time, thromboplastin time, aptt and levels of fibrinogen, atiii and c-reactive protein; soluble fibrin (sf) was measured via protamine sulfate aggregability and total fibrin(ogen) degradation products (ftdp) by an elisa from organon teknika. n= 109 patients were examined (age: 64__+9 y). they lost 750+__460 ml blood (mean+sd) into the drains within the first 18 hours after end of surgery. a severe bleeding was defined to exist, if the blood loss exceeded this range (> 1200 ml within 18 h). fibrin(ogen) split products proved to be a useful parameter in predicting the risk of severe bleedings : ftdp levels exceeding 12 mg/i at end of surgery (n = 105) had a negative predictive value of 94%, positive predictive value of 60%, specificity of 96% and a diagnostic efficacy of 91%. in contrast, soluble fibrin which correlated well with fibrinopeptidea (r>0.91, n= 20) did not correlate neither with degradation products nor with bleeding complications (n = 109). this observation does not match to the correspondence of sf with organ dysfunction during dic: sf reached a neg.predictive value near 95% and a diagnostic efficacy of >70% (pat. without antifibrinolytic drugs), which complies to findings from bredbacka (1994). other parameters were less predictive than ftdp and sf. therefore, further examinations are necessary to determine the value of soluble fibrin for a risk prediction of bleeding complications or dic. a differentiation of splits products deriving from either fibrinogen, fibrin or xl-fibrin will provide further insights into fibrin(ogen) metabolism. heparin induced thrombocytopenia represents a multicomponent syndrome associated with the use of heparin and related drugs resulting in not only thrombocytopenia, but also arterial thrombosis of varying magnitude. the initial diagnosis ofthis syndrome is usually made by clinical observation and a drop in platelet count. conventional diagnostic methods include platelet aggregation responses to patient's serum and ~4c serotonin release in response to patient's serum, aggregation/agglutination of patient's platdets in response to heparins and the detection of patients anti-heparin platelet factor 4 (hpf4-ab) ned-antibodies by using elisa methodology. several other individualized methods are also used to demonstrate platelet activation. to test the diagnostic validity of the platelet aggregation (pa) 14c serotonin release (sr) and the relevance of hpf~-ab 340 serum samples collected from patients with clinically eunfwmed eases of lilt syndrome were compared in parallel in various assay systems. the diagnostic efficacy of these tests varied from 60-74% with the pa test providing better results than others. when the pa test was compared with serotonin release, a poor correlation was noted (r=0.47). in contrast, the correlation between the pa and hp4-ab was somewhat better (r=0.66). in another study, blood samples collected from 50 patients treated with ahigh dose low molecular weight beparin for two weeks (80 mg o.d.) were tested. 20 of these patients showed a high titre of hpf4.ab without any decrease of platelet count. none of these patients were found to be positive in the 14c serotonin release assay. a third study included blood samples from dvt patients administered with iv heparin infusion, high dose sc lmw heparin (certoparin) and iv lmw heparin for the management of dvt. none of these patient groups (20-34) exhibited any hit responses, hmvever, the incidence of high hpf4-ab titre was found to be 53% in heparin, 36% in patients with lmw heparin iv and 26% in lmw heparin sc groups. pa and sr studies revealed 8% and 12% false positive ~ respeetively. these studies clearty suggest that the currently available ~ for laboratory diagnosis of hit syndrome are of limited value, and caution should be exercised in the interpretation of the results obtained with these tests. heparin-induced thrombocytopenia (hit) is one of the major severe side effects during treatment with heparin. in postoperative medicine clinical studies demonstrated the prevalence of hit with unfractionated over fractionated heparins. few data are available from the non-ope1"ative medicine and from patients without thmmboembolism before heparinization. in a controlled prospective randomized study the safety and efficacy of low-dose heparin was compared with a lowmolecular-weight (lmw) heparin over 10 days in bedridden medical inpatients (haemostasis, in press). 1968 patients were randomized and controlled for the development of thrombocytopenia. thrombocytopenia was defined as a platelet count below 40.000lid at day 10. 4 patients developed thrombocytopenia in the heparin group and no patient in the lmwheparin group (p<0.05). none of the patients with thrombocytopenia developed a thromboembolic complication. in a second prospective case control study 90 patients with side effects on anticoagulants were treated with lmw-heparin once daily subcutaneously for a period of 1 month to 9 years. platelet count was performed every 1 to 3 months. none of these patients developed thrombocytopenia during heparinization with lmw-heparin. it is concluded that hit is a very rare complication in nonoperated bedridden medical patients. a decrease of platetet count may occur in about 0.5% of patients receiving low-dose heparin. the incidence of hit with thrombosis during low-dose heparin and of hit during lmw-heparin in non-operated patients is manyfold lower and remains to be determined. terminology: instead of the term "hemorrhagic disease of the newborn (hdn)" the term vkdb should be used, since neonatal bleeding is often not due to vkdeficieacy and vkdb may occur after the neonatal period (i.e. after 4 weeks). definition: vkdb is a bleeding disorder caused by reduced activity of vkdependent coagulation factors which responds to vk. diagnnsis: in a bleeding infant a prolonged pt (inr > 3.5) together with normal fibrinogen and platelet count is almost diagnostic of vkdb. the diagnosis is proven, if vk shortens the pt (after only 30-60 minutes) and/or stops bleeding. classification: classification by age of onset into early (< 24 h~. classic fdav 2-7) and lale form (> i week <6 months), and by etiology into idionathic and ~ec0nd~'y. in secondary vkdb in addition to breast feeding other factors can be demonstrated, such as poor intake or absorption of vk and increased consumption of vk. vk-prophylaxis: benefits: oral and intramuscular (i.m) vk (one dose of i nag) prevents equally well the classic form of vkdb. lm. vk appears to be more effective in preventing the late form (times 15-> 50). the protection achieved by single oral prophylaxis (times 3-5) is improved by triple oral vk (times 15-30). risks: because of poten[ial ri~l~ associated with extremely high levels of vk and the possibility of injection injury, i.m. vk has been questioned as the prophylaxis of choice for normal neonates. since vk is involved not only in coagulation but 'also in carboxviation with multiple effects, excessive deviations from the low physiologic concentrations, which prevail in the fully breast-fed healthy mature infant should be avoided. proposal: repeated (daily or weekly) small oral doses of vk are closer to physiologic conditions than single i.m. bolus doses, which expose neonates to excessively high vk levels. the incidence of intracranial vkdb can be reduced if the grave significance of warning signs is recognized (i.e, icterns, failure to thrive, feeding problems, minor bleeding, disease with cholostasis). whether or not the more reliable absorption of the new mixed mieellar (mm~ nrenaral~i0n of vk can reduce the protective oral dose of vk-.prophylaxis has to be evaluated. before vitamin k(vk) prophylaxis was generally accepted in japan, the incidence of infantile vk deficiency was 1:4000 both idiopathic and secondary types. since 1981, 4 nationwide surveys have been conducted. the current incidence rate is now about one-tenth that in early 1980. however, in a small number of cases, vk deficiency occured despite prophylactic administration during the neonatal period. in order to clarify the absorption,excretion and transplacentel transport of vk in the perlnatal period,followlng studies were carried out. 1)hepaplastlntest(normotest) were performed on 65 women in the last stage of pregnancy and each coagulation factor was estimated as well. 2)correlatlons were made between mothers'and babies'hepaplastin test values. 3)transplacental transport of vk 2 was studied. the general activity of vk dependent factors in pregnant women was much higher than in non pregnant women. as far as the correlation between mothers'venous blood during delivery and cordvenous blood is concerned, in the group of mothers with hepaplastln test value of less than 120% of the normal adult value, the value of the bepaplastlntest was less than 30 % of normal adult value in the cord venous blood. we also demonstrated that vk passed through the placenta but only in small qualities. the point mutation g to a at nt 449 in exon v of the factor x gene (gin 102 to lys) has previously been found in two independent kindreds with fx deficiency. it occured in both families in an heterozygote state and was associated with two other genetic defect in the fx gene. we have identified another familiy in which this mutation occurs in a homozygote state. in this family the mutation is associated with the previously reported mutation gla 14 to lys which also occurs in a homozygote state. the pt and ptt of the proposita and her siter are markedly prolonged. the fx activity is reduced to <1% in the extrinsic system, to 30% in the intrinsic system and to 18 % after activation with rvv. the fx antigen is reduced to 20%. the coagulation profile of this family thus is identical with that of fx vorarlberg despite the fact that the fx vorarlberg kindred is only heterozygous for the mutation glal02 to lys. haplotype analysis could not rule out consanquinity with the fx vorarlberg kindred. these data suggest that the mutation at nt 449 which leads to a fairly dramatic amino acid change from glu to lys would indeed represent a polymorphism. to further address this question we cloned the fx gene in an expression vector (pcep 4) for transient expression in the human embryonic kidney cell line 293 and introduced the mutation at nt 449 by site directed mutagenesis. hereditary deficiency of factor ixa, a key enzyme in blood coagulation, causes hemophilia b, a severe x-chromosomelinked bleeding disorder; clinical studies have identified nearly 500 deleterious variants. the x-ray structure of porcine factor ixa shows the atomic origins of the disease, while the spatial distribution of mutation sites suggests a structural model for fx activation by phospholipid-bound flxa and cofactor villa. the 3.0 a resolution diffraction data clearly show the structures of the serine proteinase module and the two preceding epidermal growth factor (egf)-like modules; the n-terminal gla module is partially disordered. the catalytic module, with covalent inhibitor d-phe-pro-arg chloromethyl ketone, most closely resembles fxa but differs significantly at several positions. particularly noteworthy is the strained conformation of glu-388, a residue strictly conserved in known fixa sequences but conserved as gly among other trypsin-like serine proteinase. flexibility apparent in electron density together with modelling studies suggests that this may cause incomplete active site formation, even after zymogen activation, and hence the low catalytic activity of fixa. most hemophilic mutation sites of surface fix residues occur on the concave surface of the bent molecule and suggest a plausible model for the membrane-bound ternary flxa-fvilla-fx complex structure: the stabilizing fvilla interactions force the catalytic modules together, completing flxa active site formation and catalytic enhancement. factor x frankfurt i molecular and functional characterisation of a hereditary factor x defect (gla +25 ---, lys) huhmann i., holler b., krinninger b., turecek pi., richter g., scharrer i., forberg e., watzke h.. univ. klinik ftlr innere medi, abteilung for h~matoiogie und hamostaseologie, w~en; immuno-ag, wien ; klinikum der j.w. goethe-univ. frankfurt am main, abt. f. angiologie. factor x (fx) is a vitamin k-dependent plasma protein which is activated either by fvila/tissue factor or ixaniila. fxa is the main enzyme for conversion of prothrembin to thrombin. the congenital f×-deficiency (stuart -prower-defect) being inherited as an autosomal recessive trait subsequently leads to bleeding diasthesis of varying severity. our propositus is a 24 year old patient presenting a mild bleeding tendency. his ptt (36 sec) is within the normal range, the pt ( 73% of normal) is slightly reduced. the factor x antigen level is reduced to 55% of normal. molecular characterisauon of the genetic defect was performed by amplification of the eight exons and exonintron junctions by pcr and subsequent direct sequencing of the products. in comparison to the normal sequence we could determine a single mismatch within exon ii resulting in the substitution of +25 gla (gaa) by lys (aaa). the mutation abolishes a naturally occuring mboti site in the dna sequence of exon ii. the status of the fx encoding alleles was determined in the propositus, his mother and one of his brothers by amplification of exon ii and restriction digest with mboll. these family members were heterozygous with respect to the mutation in exon i1. fx was isolated from plasma of the propositus by monoq ion exchange chromatogrephy. performing clotting assays with purified fx frankfurt i we determined an activity of 89% of normal fx upon activation with rw, 77% upon intrinsic activation (aptt) and 81% upon extrinsic activation (pt). this compares well with the results obtained from the patient plasma ( pt 56%, ptt 55% and rw 57% of normal) when the reduced fx-ag-level of the plasma (55%) is taken into account_ we therefore conclude that the substitution of gla +25 to lys results in a fx molecule which is severely defective ip both the intrinsic and extrinsic pathway of blood coagulation. bleeding after cardioth~)racic surgery is still a frequent, important and sometimes life-threatening complication. thus, the aim of this study was to examine routine parameters of hemostasis and their predictive values for severe bleedings. this prospective study included patients undergoing cardlopulmonary bypass surgery. blood samples were drawn preoperatively as well as 0, 6, 18 hours and 2, 3, 4, 5, 6, 7, 8 days after surgery. blood loss from drains, transfusion of blood products and other important clinical data were monitored apart from platelet count, hematocrit, throm. bin time, thromboplastin time, aptt and levels of fibrinogen, atiii and c-reactive protein; soluble fibrin (sf) was measured via protamine sulfate aggregability and total fibrin(ogen) degradation products (ftdp) by an elisa from organon teknika. n= 109 patients were examined (age: 64+9 y). they lost 750+__460 ml blood (mean_+sd) into the drains within the first 18 hours after end of sur. gory. a severe bleeding was defined to exist, if the blood loss exceeded this range (> 1200 ml within 18 h). fibrin(ogen) split products proved to be a useful parameter in predicting the risk of severe bleedings : ftdp levels exceeding 12 mg/i at end of surgery (n = 105) had a negative predictive value of 94%, positive predictive value of 60%, specificily of 96% and a diagnostic efficacy of 91%. in contrast, soluble fibrin which correlated well with fibrinopeptide a (r>0.91, n= 20) did not correlate neither with degradation products nor with bleeding complications (n= 109). this observation does not match to the correspondence of sf with organ dysfunction during dic: sf reached a neg.predictive value near 95% and a diagnostic efficacy of >70% (pat. without antifibrinolytic drugs), which complies to findings from bredbacka (1994). other paramelers were less predictive than ftdp and sf. therefore, further examinations are necessary to determine the value of soluble fibdn for a risk prediction of bleeding complications or dic. a differentiation of splits products deriving from either fibrinogen, fibrin or xl-fibrln will provide further insighls into fibrin(ogen) metabolism. this study was conducted as a randomized parallel -group clinical trial comparing the safety and efficacy of a low molecular weight heparin {lmwh} -monoembolex sandoz and unfractionated standard heparin glfh) for the perioperative prevention of venous thromboembolie disease (dvt) following major surgms' in patients with gynecologic malignancy.. three hundred and twenty 4 women (six drop outsl werr randomized and received either 3 times daily 5000 [l" s.c. ul.'i-i (sandoz nuemberg germany] (n = 164) or once a day t5000 i~'v'. units s.c. monoembolex (n = 160) plus two placebo injections. heparin therapy was started the morning before opcrati(m and continued until the 7th postoperative day. up to the 10th poatop, day the incidence of dvt was 6.25 % (n = 10; incl. 7 pulmona~ embolisms pe) in the lmwh group and 6.10 % (n = 10; incl. 4 pe} in the ufh group. the overall incidence of clinically hemorrhagic wound complications was significantly decreased in the lmwh group 16.3 % (n = 2hi compared to the ufh group 26.8 % {n = 44; p < 0.0051. the incidence of major hemorrhagic episodes was 9.4 % in = 151 in the lmwh group and 14.0 %/n = 23) in the ufh group. this difference was not statisticauy significant. one case of fatal pe was observed in the lmwh -treated group. five women deaths in the lmwh group were observed during the study and 3 in the ufh group. this study demonstrates that the perioperative treaunent of low molecular weight heparins is more safety than standard heparins in gynecologic -oncologic patients undergoing major surge .ry. however, the incidence of thromboembohc complications is simmilar in both treatment regimes. to explore the effect of targeting an antithrombin to the surface of a thrombus, recombinant hirudin (hir) was covalently linked to the fab' fragment of fibrin-specific monoclonal antibody 59d8 (fab) resulting in a stable conjugate (hir-fab). in vitro, hir-fab was 9times more efficient than hir alone in inhibiting fibrin deposition on experimental clot surfaces in human or baboon plasma (p<0.01). to validate these results in vivo, hir-fab was compared to hir in a baboon model. the deposition of ill-in-labeled platelets onto a segment of dacron vascular graft present in an extracorporeal arteriovenous shunt was measured. blood flow rate was 40 ml/min. one hour local infusions of 4500 atu of either hir-fab or hir resulted in deposition of 0.16 x 109 and 2.17 x 109 plate!ets, respectively. equieffective dosages were 2000 atu hir-fab and 9000 atu hir resulting in deposition of 1.06 x 109 and 0.93 x 109 platelets, respectively. based on full dose response curves (n = 14), hir-fab was found to be > 4.5-fold more potent (based on activity) than hir. because of the small total amounts of antithrombins used and the short duration of these experiments, no significant systemic effects were observed. thus, fibrin-targeted recombinant hirudin prevents platelet deposition and thrombus formation more effectively than uncoupled hirudin in vitro and in an in vivo primate model. triabin, a 17 kda protein from the saliva of the assassin bug triatoma pallidipennis, is a new specific thrombin inhibitor (1). tt does not block the catalytic center but interferes with the anionbinding exosite of thrombin. the recombinant protein was produced with the baculovirus/insect cell system and used to study the inhibitory effect of triabin on thrombin-induced responses of human blood platelets and blood vessels. aggregation of platelets in tyrode's solution was measured turbidimetrically at 37°c. for the studies on blood vessels rings (2-3 mm) from small porcine pulmonary arteries were placed in organ baths for isometric tension recording. the integrity of the endothelium was assessed by the relaxant response to bradykinin. like hirudin, triabin inhibited the thrombin (0.1 u/ml)-induced aggregation of washed human platelets at nanomolar concentrations (ec50 = 2.6 nmol/l); whereas the adp-and collagen-induced aggregation were not suppressed. in pgf2c~-precontracted porcine pulmonary arteries, the thrombin (0.2 u/ml)-induced endothelium-dependent relaxation was inhibited by triabin in the same concentration range as found for inhibition of platelet aggregation. higher concentrations of triabin were required fo affect the contractile response of endothelium-denuded porcine pulmonary arteries to thrombin (1 u/ml). in all these assays, the inhibitory potency of triabin was dependent on the thrombin concentration used. these studies suggest that the new anion-binding exosite thrombin inhibitor triabin is one of the most potent inhibitors of the thrombin-mediated cellular effects. dept. of medicine, university hospital benjamin franklin, free university of berlin, dept. of medicine and dept. of surgery, heinrich-heine:university dusseldod after standardized training in home prothrombine estimation using the coaguchek system, 150 consecutive patients (p) who had st. jude medical aortic or mitral valve implantation were allocated to two random arms; 75 p were asked to control the inr themselves every third day. in the remaining 75 p anticoagulation was managed by the home physician without recommending an interval for these controls. all 150 p were monitored during the education period to a target therapeutic range of inr 3.5-4.0. p were asked to contact their home physician immediately if the inr was measured 0.5 below or above the target range (inr-corrider 3.0-4.5). all p had out-patient re-examinations every three months. thrombotic, thromboembelic and hemorrhagic complications were documented by the p using special documentation cards. the following findings were documented during the follow-up period: 4.49 0.9 0 the results of this randomized study demonstrate a significant improvement in the management of oral anticeagulation by home prothrombine estimation. significant (p<0.001) more inr measurements were found inside the target therapeutic range. moreover. bleeding and thromboembolic complications could be reduced (p = 0.038) in the study group with home prothrombine estimation. life-threatening thromboembolic and hemorrhagic complications were not observed in p who were on home prothrembine estimation, while three such events (2.72 %/year) were documented in group a. local vascular injury following ptca exposes circulating platelets to prodmmbogenic stimuli. by binding to platelet gp iiblliia fibrinogen crosslinks platelets, which represents the final common pathway of platelet aggregation. fradafiban (bibu52zw) is a non-peptide compound with effective, reversible inhibitory effects on fibrinogen binding to gp iib/ii/a on human platelets. in the first double-blinded, prospective phase ii study three escalating doses of bibu52zw as a continuous 24 h-i.v, infusion were tested in comparison to placebo in 65 patients with stable angina pectoris undergoing elective ptca. the mean receptor occupancy with 20rag, 40ms and 60ms per hour were 71.974, 84.5 % and 87.9% at 24 hours, respectively. as compared to placebo breeding time was significantly prolonged (7 vs 20rain) during fi-adaiiban infusion with a weak dose-dependency. platelet aggregation in platetet rich plasma ex vivo with collagen (2.0 and 4.0 gg/ml), adp (2.5 and 5.0 gmol/ml) or ca-ionophor a 23187 (2.5 and 5.0gg/ml) was significantly and dose-dependently inhibited as compared to placebo. using the two upper doses of fradafiban, we observed major bleeding complications in 8 patients requiring blood transfusions or vascular surreal repair. in these patients, too, maximal antiplatelet effects could be documented. these data sugest that bibu52zw is an effective fibrinogen receptor antagonist in patients. the requirement of ad hoc receptor occupancy determination or platelet function monitoring for safe and effective clinical use should be evaluated. in a placebo controlled interaction study 9 healthy volunteers were randomized to receive either a 24 hour infusion of peg-hirudin ( 0.02 mg/kg/h) after an i.v, bolus of 0.2 mg/kg + placebo, or 325 mg/day acetylsalicylic acid (asa) for three days followed by a placebo infusion or the peg-hirudin infusion + asa. each volunteer received all three treaments. there was a washout period of at least 14 days between the infusions. at short intervals aptt, activated clotting time (act), ecadntime (ect), alia-activity using the chromogenic substrate 2238, collagen-induced aggregation, platelet adhesion and platelet induced thrombin gene,ration time (pitt) were measured, bleeding time (simplate) was studied before drug administration, on day three before the infusion and 6 hours after start of the infusion.the infusion of peg-hirudin after 4 and 8 hours led to a mean hirudin plasma level of 1.8 pg/ml. asa markedly inhibited collagen induced aggregation as expected. the mean bleeding time was prolonged under the influence of peg-hirudin from 5.2 to 6.22 min, after asa from 5.8 -18.2 min and after the combination of peg-hirudin + asa from 5.4 -33.7 min. in each volunteer the bleeding time was longer under the combination than after asa alone. in two volunteers receiving peg-hirudin + asa the bleeding time measurement was stopped after 60 rain. none of the coagulation parameters or platelet function tests correlated with the prolongation of the bleeding time. however the bleeding time was excessively prolonged in those volunteers who had a marked prolongation under asa alone.the combination of hirudin at a higher dosage with asa probably is associated with a relative high risk of bleeding. either the hirudin dosage should be reduced if the combination seems feasabie or asa should be given after the end of hirudin treatment. fibrinogen with the sta/stago and the mla/dade systems correlated well, but neither system correlated well with the acl/il system. at iii, protein c, protein s, and anti-xa heparin assays using stago reagents performed as expected for normals and low abnormals on the sta. factor levels on the sta/stago system were less sensitive than factor levels obtained with the dade reagents on the mla or fibrometer. using the sta/stago system, thrombin time results correlated well with the aptt and heparin levels. the thrombin time was not associated with additional manipulation for assay preparation, nor any cross-contamination of reagent or sample, since on the sta reagents do not come into contact with tubing. the sta was not sensitive to hemolytic, icteric or lipernic samples for clotting assays artd showed the same sensitivity as the mla for chromogenic assays. the overall data comparisons, high throughput, minimal operator intervention for reagent/assay change and ease of operation warrant further evaluation of the sta hemostasis analyzer. a. wehmeier, d. s6hngen, c. rieth klinik for h#,matologie, onkologie und klinische immunologie der heinrich-heine-universit~it d0sseldorf hirudin selectively inhibits thrombin by direct interaction. because the effect of hirudin is independent of antithrombin iii and other factors, it seems an attractive alternative to current anticoagulants. however, it is uncertain whether hirudin influences plateletassociated thrombotic disorders and how it compares with conventional and lmw heparin. we investigated the effect of 2 recombinant hirudin preparations (rhein biotech, dt3sseldorf) on platelet function tests: in vitro bleeding time, adhesion to glass beads, aggregation in platelet-rich plasma and whole blood. hirudin was used in concentrations of 0.1-100 i.tg/mi, and was compared to trisodium citrate (0.38%), conventional heparin (50 iu/ml) or lmw heparin (fraxiparin, 500 iu/ml). both recombinant hirudins showed normal activity in thrombin neutralization tests, and prolongation of thrombin time and aptt. however, in vitro bleeding time was not prolonged by hirudin, but was more than doubled by addition of conventional and lmw heparins. platelet retention to glass bead columns was reduced by hirudin in a dose-dependent manner to about 40% but was more effectively reduced by both heparin preparations and citrate. hirudin had an inhibitory effect on p!atelet aggregation in prp induced by thrombin, collagen, and predominantly epinephrine but not adp and ristocetin. in whole blood, a small effect could only be observed with hirudin concentrations of >25 ~g/ml as compared to citrateanticoagulated blood. in summary, thrombin inhibition by recombinant hirudin has little effect on in vitro platelet function tests in comparison to heparins and calcium depletion. the role of endothelin (et), prostaglandins and the coagulation system in the pathogenesis of acute renal failure is still to be defined. in anaesthesized pigs the effects of i.v. infusion of et (3 /~g/kg) alone (group 1, n=6) and after pretreatment with the potent thrombin-inhibitor hirudin (0,5 mg/kg)(group 2, n=6) on haemodynamics, coagulation parameters (factor viii, antithrombin iii, precallicrein, fibrin monomers, aptt) and prostaglandins were investigated. plasma renin activity (pra)-, creatinine clearance-, urine volume-measurement and blood gas analysis were performed hourly. et-infusion caused an initial bp-reduction and marked hr-reduction followed by a transient bp-elevation and hr-reduction. activation of platelets can be directly measured by flow cytometry using monoclunal antibodies. in an in vitro study the effect of the thrombin inkibitors argatroban, efegatran, dup 714, recombinant hirudin and peghirudin on platelet activation induced by various agonists was studied in whole blood. blood was drawn from normal human volunteers using the double syringe technique without use of a tourniquet to avoid autoaggregatiun of platelets. for anticoagulation of blood the thrombin inhibitors mentioned above were used at a final concentration of 10 ~tg/ml each. blood samples were then incubated at 37°c either with saline, r-tissue factor (rtf), arachidonic acid (aa), adenosine diphosphate (adp) or collagen. at definite times (1, 2.5, 5, 10 rain) aliquots were taken and after various steps of fixative procedure the percentage of platelet activation was measured by means of fluorescent monoclonal antibodies to platelet surface receptors gpiiia (cd-61) and p-selectin (cd-62). the agunists used induced a platelet activation of 37.4 + 15.2 % (rtf), 65.1 + 12.1% (aa), 19.3 + 7.4 % (adp) and 27.1 + 12.8 % (collagen). flow cytometric analysis showed that all thrombin inlaibitors studied caused a nearly complete inhibition of r-tissue factor-mediated platelet activation. in contrast, after induction of platelet activation with the other agonists an increased percent cd-62 expression was found showing a strong platelet activation with a maximum at the same times as in non-anticoagulated blood. in conclusion, the results show that in whole blood thrombin inhibitors are effective in preventing platelet activation induced by r-tissue factor. the formation of active serine proteases including thrombin may be effectively inldbked by these agents. the observations further suggest that, while thrombin inkibitors may control serine proteases, these agents do not inhibit the activation ofplatelets mediated by other agonists. this work was supported by the grant bmft 07nbl01. animal experimental studies on the pharmacokinetics of peg-hirudin e. bucha, a. kossmehl, g. nowak max-pianck-gesellschaft e. v., arbeitsgruppe "pharmakologische h~imostaseologie", jena hirudin, when complexed with polyethylene glycol (peg), increases its molecular weight from 7 to 17 kda, thereby preventing extravasation of this drug. peg-hirudin is distributed almost only in the intravascular blood space. in addition, its increased molecular weight retards the renal elimination. the elimination half-life of hirudin in rats (58 + 12 min, as determined) is increased five-fold (244 ± 18 min). with the same hirudin dose applied, the blood level of hirudin is increased 19-fold, measured in the 13-elimination phase. in the urine of rats, 2 -3% of the hirudin activity were recovered following hirudin administration, but 48% could be detected after peg-hirudin had been applied. after subcutaneous administration of peg-hirudin, the trnaxwalue is reached at 380 rain (r-hirudin: 65 min); the cmax-value is increased 3-fold, compared to that of r-hirudin (2.5 pg/ml). 24 hours later, still one fifth of the maximum concentration (cma,) is present in the blood, and the renal elimination is still retarded. in the urine of rats, 12% of the hirudin activity applied were recovered in the 24-h urine sample. with intact renal function, following subcutaneous administration, peghirudin is abte to produce a constant blood level of hirudin over a long pedod. thrombin inkibitors such as r-hirudin (rh), argatroban (a), efegatran (e), and peghiradin (ph) are currently undergoing extensive clinical trials in such cardiovascular indications as ptca, ami, and treatment of unstable angina. a rapid assessment of the anticoagulant actions of these agents is, therefore, crucial to assure their efficacy and safety. currently, act and aptt are used to measure the anticoagulant effect of these agents. we have utilized a dry reagent technology based on the motion of paramagnetic iron oxide particles (plop) to measure the antithrombin effects of various thrombin inhibitors (cv diagnostics, raleight, nc). the heparin monitoring card has been modified to measure antithrombin agents in various anticoagulant ranges for (a)0 (e), (rh), and (ph). blood samples drawn from patients treated with (a) and (rh) have been evaluated and concentrations of these agents have been calculated using an external calibration curve. in the in vitro setting, citrated whole blood or citrated frozen plasma can be used to evaluate the anticoagulant effects of these agents. the results obtained are comparable to the act which is conventionally used for the monitoring of these agents. both (rh) and ( period. we would like to present a case of heparin-induced-thrombocytopenia (hit) in a 47 years old woman who underwend open heart surgery. she suffered from a combined aortic valve disease and leading stenosis. laboratory analysis showed constant low platelet counts (50/nl) without heparin application, so that an idiopathic thrombocytopenlc purpura was suspected. but platelets also decreased after heparin application. heparin-antibodies were found in the heparin induced platelet activation assay (hipaa). treatment with corticosteroids and immunoglobulines, respectively, showed no improvement but the patient unfortunately developed a pneumonia with legionetla pneumophila. therefore, the only suitable anticoagulant for the necessary aortic valve replacement was hirudin: a bolus injection of r-hirudin of 0,75 mg/kg b.w. was administered 10 min. bevore start of the extracorporal circulation (ecc), the heart-lung machine (hlm) was primed with 6 mg r-hirudin and another bolus of 5 mg of r-hirudin was administered. additionally 10 mg of r-hirudin was applicated to the cell-saver-reservoir. during the period of ecc ecarin clotting time and aptt values were taken every ten minutes for monitoring r-hirudin concentration. the postoperative anticoagulation was performed with a constant infusion of r-hirudin starting eight hours after the end of ecc and monitored by aptt. due to mechanical aortic valve the further anticoagulation was performed with phenprocoumon, starting 3 days postop. the therapy with hirudin showed no side-effects. hirudin, threrefore seems to be a suitable anticoagulant in patients with high risk for bleeding complications like this. doses fi:om 2-30 mg/kg gave similar post-op blood loss measurements without s dnseresponse (4-15 oc/kg) (less blood oozing than a historical heparin control but equivalent post-op blood loss; 10 q-3 ec/kg). doses >30 mg/kg showed more intra-op blood loss than the lowe~ doses, but equal post-.op blood loss. the bleeding time test was less elevated than for heparin. platelet counts and hematoerit did not vary except for hemodihition on pump. liver enzymes did not vary significantly pre-op to post. act values showed arg was eliminated (dose-dependently) by 1 hour post-op. dogs were hemodymamieally stable during the peri-operative period, and overall gave predictable responses to arg (as opposed to variable responses to heparin). in a substudy it was demonstrated that hypothermia did not affect the activity of arg, nor did varioos formnlations. this dose finding study strongly suggests that arg may be a safe and effective alternative to heparin for patients undergoing cpb. this is particularly important for the growing population of patients with hit who require cardiac surgery, for which no anticoagulant alternative is presently available. three recent clinical tdals with r-hirudin (timi 9, gusto 2 and hit) have shown that the risk of severe haemorrhagic side effects was strongly associated with high aptt-levels. the large intedndividual variability of the aptt and the lack of a linear dose-effect ratio, however, limits its value for reliable monitoring of the anticoagulant effect of hirudin since even severe overdosage due to impaired renal elimination may not be detected with this assay. we have therefore evaluated the ecadn clotting time (ect) as descdbed by nowak and bucha (thromb. haemost. 1993; 69: 1306) under conditions which allow conclusions on its reliability in the clinical situation.for this, citrated venous blood obtained from healthy volunteers, patients with unstable angina pectoris, and patients treated with marcumar was supplemented with different concentrations of peghirudin. measurements of aptt and ect were made in duplicate. in contrast to the aptt, the ect showed a close, linear relationship with peg-hirudin plasma concentrations in the range of 100 and 10 000 ng/ml. the lineadty of this relationship was not affected by the presence of unfractionated or low molecular weight hepadns in concentrations of up to 10 pg/ml. the ect was not affected by fibdnogen concentrations 60% below normal. a somewhat higher slope but no change in linearity was found in plasma from marcumar-patients with quick-values between 20 and 32%. no significant differences were found between values measured in citrated blood or plasma or using different coagulation timers. the most potent thrombin inhibitor containing a benzamidine moiety is napap (k i = 6 nmol/i). unfortunately, the pharmacokinetic properties (fast elimination by hepatic uptake and biliary excretion, poor enteral absorption) are unsuitable for the use of napap as an oral anticoagulant. the application of choice of a synthetic thrombin inhibitor would be the oral one, therefore, we looked for other lead structures. with the nc~-arylsulfonylated piperazides of 3-amidinophenylalanine we found a new group of derivatives which inhibit thrombin with ki-values in the nanomolar range. the piperazides exert anticoagulant activities with high selectivity, leaving activated protein c and components of the fibdnolytic system unaffected. in rats, the piperazides are rapidly eliminated from the circulation (tl/2 ~ 10 min) upon i.v. administration, too. after oral administration, the systemic bioavailability is low. upon intraduodenal administration of high doses widely varying blood levels were seen, depending on the mode of administration. to cladfy the importance of a possible hepatic first pass effect we studied in more detail the pharmacokinetics of the n~-(2naphthylsulfonyl)-3-amidinophenylalanine n'-acetylpiperazide in rats using hplc-analysis. like other benzamidines the piperazide is excreted via the bile to a high extent. enteral absorption rates of about 20 % are found after blocking the hepatic uptake and biliary excretion. hence, a hepatic first pass effect appears to be the main reason for low systemic bioavailability after orallenteral administration. at the same time, fast elimination from the circulation by hepatic uptake is the main problem for maintaining effective blood levels with benzamidines. therefore, the elucidation of the structural elements influencing the absorption and elimination processes of these types of inhibitors is necessary. the piperazides of 3-amidinophenylalanine bear the possibility to easily introduce a wide variety of substituents on the second nitrogen of the piperazine moiety. a 69-year-old female patient with diabetic nephropathy increasingly developed signs of allergisation combined with dyspnea, erythema, pruritus, and circulatory insufficiency two months after start of heparin-anticoagulated haemodialysis und initial surgical application of a double lumen venous catheter. in addition, growing thrombocytopenia was observed involving a drop in platelets by 50%, compared to the initial values. the haemodialytic efficiency was reduced by massive thrombosis of the dialyzer and subsequent repeated interruption of treatment. at the end of may 1995 heparin antibodies were detected and the hat diagnosis was confirmed. immediately afterwards, haemodialysis treatment was continued, applying hirudin as anticoagulant. using steam-stedlised haemophan dialyzers and 0.14 mg/kg r-hirudin (iketon, italy), the minimum therapeutic blood level of hirudin (0.4 pg/ml whole blood) was reached. this provided therapeutically relevant blood level conditions during a 4.5h haemodialysis. more than 80 regular haemodialyses were run without problems. in all hirudin-anticoagulated haemodialysis treatments the ecarin clotting time was used as the method of choice for bedside blood level and dosage control. after the 34th haemodialysis, the frequency was reduced from 3(4) to 2 haemodialyses a week. accordingly, the hirudin dose was increased to 0.2 mg/kg. the creatinine clearance increased continuously from initially 2.6 to 10.4 ml/min after the 13th week of hirudin-anticoagulated haemodialysis. platelet count and haemodialytic efficiency normalized. we could demonstrate that the regular use of hirudin as anticoagulant along with dialyzers impermeable to hirudin enables very good results in haemodialysis treatment in heparin-associated thrombocytopenia, hirudin is suited for use as anticoagulant in problem patients with hepadn-induced allergy when combined with a drug monitoring method fit for bedside use. capillary electrophoresis methods provide a fast measurement of proteins. thus we developed for pharmacokinetic measurements of r-hirudin and peg-hirudin capillary electrophoresis methods. for the measurement of r-hirudin we used fused silica capillary and a borate buffer. this buffer was used to detect r-hirudin, but could not be used to measure peg-hirudin. for simultaneous measurement we used a neutral capillary to prevent protein absorption to the capillary wall. the buffer was a 20 mm tricine buffer (ph = 8.0 field strength 500 v/cm). it resolved r-hirudin from peg-hirudin at 214 nm using reverse polarity. a linear correlation between the peak area and the concentration was found between 80 pgtml and 10 mg/ml for hirudin (r 2 = 0.99) and between 2,5 and 10 mg/ml for peg-hirudin (r2= 0.99) was found by coinspiking of human plasma and urine with r-hirudin and peghirudin the two proteins were completely resolved. a linear correlation between the peak area and the concentration was found. the method separates r-hirudin from peg-hirudin and may be applied to biological systems to measure the concentration of r-hirudin. triabin is a thrombin inhibitor from the saliva oft. pallidipennis structurally unrelated to any protease inhibitor known and which probably functions by an interaction with the anionbinding exosite of thrombin. we used sf9 insect cells infected with recombinant baculovirus to produce sufficient triabin for a detailed biochemical characterization. the activity of the protein purified from cell lysates was assessed in a fibrinogen clotting assay and was found to be similar to that of the natural protein. a 4-fold prolongation of thrombin-clotting time and aptt was achieved with 22 nm and 600 nm triabin, respectively. a kinetic analysis of the thrombin-catalyzed fibrinopeptide a release from fibrinogen showed that triabin is a tight-binding inhibitor. using the graphical method of dixon, the ki was determined to be 3 pm. introduction: thrombocytopenia is a common adverse effect of heparin therapy, in type ii hit platelet decrease induces severe complications. we here present two special cases of type ii hit. case report i: a 66 year old male patient with dvt of the left leg was treated with therapeutic doses of heparin. from the first to the 12th day of therapy, platelet count decreased from 122000 to 54000/ui. hit was confirmed by hipa-test, heparin therapy was s~opped and treatment with the heparinoid orgaran n was started. during the following days, arterial thromboses in the right a. femoralis occurred. several thrombe~tomies were not successful and although orgaran ~" was stopped because of suspected crossreactivity, amputation of the right leg could not be avoided. during the following days under hirudin-treatment platelet count normalized and no further complications occurred. case report 2: a 74 year old female patients suffering from hip fracture was treated by surgery with tep-operation and received prophylactic heparin treatment. after 6 days, platelet count decreased from initially 170000 to 8000/ul and dvt of the right leg was diagnosed. on the same day, severe bleeding into the left leg was observed and hemoglobin concentration was diminished to 7.8 g% (before surgery 16.0 g%). hit was confirmed by hipa te~t, heparin was stopped and treatment with orgaran started. thrombocyte count normalized and no further complications occured. conclusion: hit type ii can cause severe bleeding as well as thromboembolic complications. because of possible cross-reactivity between heparin and orgaran~,, hirudin should be given in hit patients. currently thrombin time (ti), aptt, activated clotting time (act) or anti ila -activity (alia), measured by a chromogenic substrate test are used to monitor hirudin treatment or prophylaxis. the "13" responds very sensitive to hirudin plasma levels end thus requires variable thrombin concentrations. aptt appears to be more adequate, however, it shows large interindividual variations and does not respond sensitive enough to higher hirudin concentrations. act is a simple whole blood clotting assay, but it is strongly influenced by the blood collection technique. the ecadn clotting time (ect) is a new clotting assay, recently described by nowak and bucha (thromb.haemost 1993, 69,1306) . it measures the clotting time of citrated blood or plasma after prothrombin activation by ecarin, a snake venom of echis carinatus. ec.t shows a linear dependence on different hirudin concentrations over a wide concentration range ( e.g. 0.1-5 pg/ml). in a clinical interaction study healthy volunteers were administered hirudin, asa or both. 15 male volunteers received an i.v. infusion of peg-hirudin (0.02 mg/kg/h) for 24 hours after an initial i.v. bolus of 0.2 mg/kg to compare the sensitivity and reliability of ect with aptt, l-r end act. the act was measured on the hemochron 801, usa, ect on a fibrin timer, aptf using the aptt lyophylized silica reagent by il and alia on an acl (il-milan) with the chromogenic substrate 2238. all tests were performed in duplicate. ect was more sensitive to different hirudin concentrations than aptt, or act. the ect results were better correlated with the alia-activity than ap'l"r and act. the lower detection range for ect is 0.05 pg/ml hirudin. ect is a very sensitive, simple and reliable test for the monitoring of hirudin treatment and prophylaxis. recombinant and synthetic inhibitors of thrombin such as hirudin, efegatran and argatroban are currently in various phases of clinical trials in several surgical and medical indications. the therapeutic effects of these agents are usually monitored by aptt whereas in cardiovascular indications, cefite act and hemotech® act are used. the reliability of both aptt and the act tests in predieting the safety of various thrombin inhibitors has been heavily debated. furthermore, some of these inkibiturs are administered simultaneously to heperinized or coumadinized patients and the obtained aptt and act results do not lady refleot the effects of these agents. fcafin is a snake venom derived fi'om echis carinatus which converts prothrombin into mesothrombin, targeting the arg~3-ile tm bond between the a and b chains of prothrombm. while thrombin inhibitors are capable of inhibiting mesothrombin, atiii/beparin complex does not have any effect. using purified ecarin, nowak and bucha (1993 thromb haemost 69:1306) proposed to assay hirudin. since thrombin inhibitors exhibit similar mechanisms of thrombin inhibition, ecarin clotting time (ect) was evaluated to test its diagnostic efficacy in various experimental and clinical settings. lyphilized eoarin was obtained from knoll ag, ludwigshafen, germany). concentration dependent clotting times for himdin, efegatran and argatroban were obtained in a range of 0-15 p.g/ml. all of the antithrombin agents produced a concentration dependent prolongation of ect and showed va~angpotendies inthe order ofefegatran> argatreban> hirudin, on a gravimetriebasis. on a molar basis, the anticoagulant order of potency was found to be hirudin> afegatran> argatroban. utilizing the ect, the effect of these inhibitors on patients undergoing bolus or infusion therapy, resulting in a concentration level of ~ 10 gt g/rnt, have been measured. unlike such global tests as pt and aptt, patients receiving simultaneous heparin or oral anticeagulants can be monitored for antithrombin specific prolongation ofthe ect. plasma samples from heparinized (aptt 45-90 sec) or coumadinized ('pt 15-25 see) patients, supplemented with argatroban or hiredin did not show any differences m the ect. a medified ecarm act comparable to the celite act has also been developed. initial results demonstrate that this test is not affected by aprotinin, heparin and reduction of the prothrorabin complexes in the inr range of 1.5 -3.0. these results indicate that ecarin based clotting times provide slx~etlie ~lts of circulating levels of thrombin inhibitors, which can provide reliable information to optimize their safe(y and efficacy. r-hirudin is a highly potent and selective inhibitor of the serine proteinase thrombin. after intravenous administration, r-hirudin is eliminated exclusively with the urine. its plasma half-life is very short, 1-2h. peg-hirudin is a derivative produced by coupling polyethylene glycol (peg) to a specially designed recombinant hirudin mutein. peg-coupling results in a considerable prolongation of the plasma half-life of peg-hirudin, compared to r-hirudin. after intravenous administration of r-hirudin into rats, a very small amount of ,,hirudin-like" activity (2 -4% of applied activity) was recovered in the urine. in contrast, after peg-hirudin had been administered, more than 30% of the applied activity could be recovered in rat urine. these results suggest differences in the renal metabolism of peg-hirudin and r-hirudin. within the scope of pharmacokinetie studies in rats we investigated the appearance of biologically active metabolites of peg-hirudin after kidney passage in urine. affinity chromatography on immobilised thrombin was used as a quick and gentle method in searching for biologically active hirudin metabolites in rat urine. but it had to be completed by anion-exchange and/or reversed-phase chromatography to ensure that all active metabolites were detected. the isolated biologically active metabolites were purified by reversed-phase hplc and were biochemieally characterized. in previously reported studies we found a hlrud n derivative consisting of the amino acids 1-50 as the main metabolite in rat urine following intravenous administration of r-hirudin. this metabolite was not detected in the urine after administration of peg-hirudin, confirming the suggestion of a different renal metabolism. carrageenans are high molecular weight sulfated polygalactans of plant origin (derived from red algae) with anticoagulant properties. in previous studies we investigated the anticoagulant activity of lambda-carrageenan, a highly sulfated type of carrageonans. unlike heparin, lambda-earrageenan exerts its anticoagulant activity primarily through direct inhibition of the serine proteinase thrombin. only a part of its antithrombin activity is indirectly mediated through antithrombin iii. to investigate relations between molecular weight and biological activities, tambda-carrageenan has been hydrolysed and fractionated. the molecular weight has been determined with the aid of size exclusion hplc using dextrans as molecular weight standards. the degree of sulfation has been determined by anion-exchange hplc. we have obtained low molecular weight lambdaearrageenans ranging from 10,000 dalton to 100,000 dalton with degrees of sulfation of 13 -17% and 33 -38%. the anticoagulant and antithrombin activity of low molecular weight carrageenans have been determined using coagulation assays and purified systems, and we have compared their activities with those of heparin and other sulfated polysaecharides. further, we have investigated the ability of lambda-carrageenan and its low molecular weight derivatives to inhibit the activity of human blood phagocytes. the activity has been determined by measuring the cellular chemiluminescence in a mieroplate himinometer using a himinol-dependent assay and zymosan as phagocytosis activating agent. we have used an assay in human whole blood and assays with isolated human mononuclear and polymorphnuclear cells. the anticoagulant activity and also the ability of carrageenans to inhibit the activity of human macrophages decrease with decreasing molecular weight and decreasing degree of sulfation. the natural ocouring, yellow pigment curcumin is the major component of tumeric and is commonly used as a spice and food-coloring agent. since curcumin has been reported to have anti-tumorpromoting, antithrombotic and anti-inflammatory properties, we studied, whether curcumin acts on the transcription factors ap-l(jun/fos) and nf-~:b in cultured endothelial cells (ec). when ec were cultured in the presence of curcumin, electrophoretic mobility shift assays (emsa) demonstrated, that binding of endogenous ap-1 to its dna recognition motif was suppressed. inhibition was due to direct interactions of curcumin with the dna-binding motif for ap-i. enhanced ap-1 binding, induced after tnfa stimulation of ec, was decreased in cells pretreated with curcumin. this resulted in reduced transcription and expression of tissue factor, known to be controlled by ap-f and nf-~b. nuclear run on assays proofed, that curcumin directly reduced the tnfa mediated transcription of genes, regulated by ap-1, as tf, endothelin-1 and c-jun. thus, curcumin did not only suppress apl(jun/fos)-binding, but also inhibited tnfa induced jun transcription, transient transfections with tissue factor promotor plasmids confirmed, that inhibition by curcumin was dependent on intact ap-i sites. beside its effect on ap-l-binding, curcumin reduced the radical dependent activation of nf-kb due to its antioxidant properties, however, this inhibition was indirect and less prominent. the relevance of the in vitro data was confirmed in vivo in mice bearing meth-a-sarcoma. when mice received curcumin before tnfa was injected, tumors showed reduced ap-1 activation. simultanously fibrin/fibrinogen deposition decreased, most probably due to reduced tissue factor expression. thus, curcumin inhibits ap-t activation and expression of endothelial genes controlled by ap-t in vitro and in vivo. (jung, 1991) . additionally, haemorhenlogical parameters (plasma viscosity, erythrocyte aggregation) were measured. in all patients aptt, bleeding time, platelet adhesiveness, von wiuebrand f~ctor and factor viii concentration and activity were determined. the patients with von willebrand disease showed characteristic morphological changes of capillary geometry. tortuosity of nailfold capillaries was markedly increased as well as the diameter of capillariez on the arterial and venous side. plasma viscosity was significantly low. multiple parameter analysis concerning to galen and gambino (1983) and using the parameters ,,plasma viscosity below 1.25 mpas", ,,torquation index higher than 5", ,,erythrocyte column diameter bigger than 16,9 gin" showed a positive predictive value of 100 %. capillary diameter and capillary tortuosity have a positive predictive value of 91,2 %. additionally, a reduction of the vasomotorie reserve and/or a decreased erythrocyte velocity in the capillaries below the reference range was found in most of the yon willebrand patients. it was quite remarkable, that 16 of 40 of the yon willebrand patients showed significant capillary bleedings. these findings confirm some former observations (e.g. o'brian 1950) and preliminary reports of our group (koscielny 1994). polymerase chain reaction (pcr)-based quantitation of mrna transcripts is an important tool in the investigation of the underlying molecular defects in inherited platelet disorders, such as the bernard-soulier syndrome. however, for the exact quantitation of mrna a number of methological requirements has to be met. first, a standard (s) mrna must be synthesized which is able to undergo the same processing as the target wild type (wt) mrna. secondly, the quantitation step following the pcr must differentially recognize standard and target dna, and thirdly, the assay must be precise with respect to both inter-and intraassay variability. in order to satisfy these requirements we constructed a s-gpib mrna which is identical to the wt-gpib mrna except a 13 bp long primer recognition site at its 5" end allowing differentiation between the pcr amplified wt-or s-gpib cdna through incorporation of a fluorescein or biotin labelled 5" primer. both standard and w[ gpib mrna showed identical amplification kinetics in the pcr reaction. the amplified dna was quantified using an dna binding assay. in this assay binding of amplified dna to gcn4 fusionprotein-coated microtiterplates is measured. since the gcn4 binding motif is incorporated into the wt-and s-gpib cdna through an identical 3" primer, competition between s-and wt-cdna during amplification has been analyzed. at a given concentration of 250 nm of gcn4. primer no competition between the sdna and wt-dna for the primer was observed during 25 pcr cycles. the sensitivity limit of the assay performed in this way was 250 amol wt-gpib~, dna, and intraassay variability reached from 1.66% to 6.72% calculated for 100 fmol and 5 fmol dna, respectively. to sum up, combination of rt-pcr with the amplified dna binding assay and usage of an internal standard mrna allows sensitive and accurate quantitation of gpiba mrna in human platelets. since upa and thrombin are main conrtibutors to the process of proliferation and migration of vascular smooth muscle cells (vsmc), which is part of the pathogenesis of atherosclerosis. we are currently assessing the role of spatial expression of upa and thrombin receptor (tr) on cells with human carotid artery plaques (n=10). we have used a double immunolabeling approach, combining anti-upa and anfi-tr antibodies. to identify the different cell types, we used the following antibodies: anti a-smooth muscle actin (a-sma) for smooth muscle cells, ulex europaeus agglutinin i (uea i) for endothelial cells, inflammation cell cocktail (cd68+cd45) for monocyte/macrophage and lymphocytes and an anti-proliferation cell nuclear antigen antibody (pcna) to stain proliferating cells. in the carotid atherosclerotic plaques, upa immunostaining was distributed focally, preferentially in the fibrous cap and some cells of the foam cell rich region (fcrr). it was present in distinct patterns: cytoplasmic staining. tr staining was distributed similar to upa staining. with double staining combining anti upa antibodies with anti-tr antibodies, cellular co-localisation of both upa and tr was demonstrated. these cells were identified as smooth muscle cells by -sma. inflammatory cells were mainly localized within the fcrr, they only stained for upa. in conclusion: our data demonstrates that upa and tr are coexpressed in vsmcs in human carotid artery atherosclerotic plaque tissue. we therefore conclude, that the mitogenic activity of upa is associated with the thrombin signalling pathway. in the proficiency test of the ,,deutsche gesellschaft flir klinische chemie" (dgkc) 1/95, 5 lyophilised plasma samples (immuno ag) were sent to participants: a normal plasma and 4 plasmas from persons under oral anticoagulation (oac-plasmas. inr 1.9 to 3.7). the participants (n=552) returned the pt times obtained and in most cases (n=355) also the isi value for the thromboplastin used (isi of pack insert). the inr was calculated using the pt of normal plasma and the isi of pack insert (method i). two additional methods for inr calculation were compared with method i. according to the concept of calibrated plasmas (houbouyan et al., t993), a calibration curve was constructed using the normal plasma and the 30ac-plasmas. the inr calculated using the pt •fn•rma¿ plasma and the laboratory-specific isi value given as 1/slope of the calibration curve (method ii) or was read off directly (method hi). for inr values, calculated by the 3 methods from the participants data (n=355), outlier elimination (2sd, iterative) was performed. the inr mean values for all 3 calculation models remain in a narrow range. using calibrated plasmas (method 1i and m), less outlier were eliminated and cv's obtained were smaller than using the conventional procedure ( i ). obviously, the inr inherited problems, such as accurate isi value, pt value of normal plasma and instrument/laboratory influences on isi, can be reduced using calibrated oac-plasmas. practical approach and educational considera-tions of home prothrombin time estimation a. bernardo, a. bernardo, c. halhuber herz-kreislauf-klinik, bad berleburg, germany specific training is necessary for the patient to achieve reliable and reproducible results in prolhrombin time measurement. the training scheme is based in many respects on experience with similar training courses for home control and management of diabetes and asthma. the education program is divided into a theoretical and a practical part. the theory part has group sessions of twenty patients of a time. the practical course is reduced to a maximum of five patients. the sessions are conducted by a medical doctor and by specialized medicaf/technical assistants. on average eight hours of theoretical education and two hours of practical training are sufficient. the contents of the theoretical lessons are: • need for anticoagulation after heart valve replacement, • potential interaction between anticoagulants and other medication, • accurate recording of the measured prothrombin time results, • techniques of prospective determination of the necessary amount of anticoagulant, • calculation of the individual doses, • potential pitfalls and mistakes, • corrections in case of over-and under-dosage, • early recognition of thromboembelic and/or bleeding complications. an alternative is a full-day intensive course which can be held during the weekend. our recently reported (1) observation that oral anticoagulant treatment causes an increase of heparin cofactor ii (hc ii) activity in plasma is now confirmed by a more extensive study. in 43 thrombophilic patients who were on vitamin k antagonist therapy (marcumar r) we found a median hc ii level of 142 % as compared to 119 % for 72 thrombophilic patients without any therapy (p < 0.002" ) and 104 % for 59 healthy controls (p < 0.001" ). moreover we observed that the increase of hc ii level was significantly correlated with increasing inr-values (r = 0.63, p < 0.001). follow-up observations on some patients showed, however, clear differences in the levels of hc ii activity after onset of vitamin k antagonist therapy. thus, some patients responded rapidly with a significant increase in activity ("strong responders") while others showed only slight changes ("weak responders"). in conclusion, the determination of hc ii activity may result in an improved estimation of the risk of bleeding, especially in high intensity treated patients (inr > 3.5). after intracoronary stent implantation an aggressive oral anticoagulation (oac) therapy is mandatory. to find out whether coagulation activation occurs after coronary stent implantation during high dose oac therapy markers of plasmatic coagulation and d-dimer were measured. patients 5 male patients (average age 57 years) were examined. blood samples were taken before and right after stent implantation and during the following week. patients got 30 mg phenprocoumon during the first three days and additionally heparin and acetylsalicylic acid (asa) were given. methods ptz, aptt, tz, protein c, tat-complexes, fi+2 and d-dimer were measured. results d-dimer levels increased steadily between day 0 and day 7. tatcomplexes showed a slight increase from day 0 (2.4 bg/i) to day 3 (15.3 ~tg/i). on day 7 tat levels were down again (2.0 p,g/l). fl+2 (day 0:1.0 ng/ml) also showed a slight increase on day 3 (1.3 ng/ml). protein c decreased steadily from day 0 (108%) to day 7 (15%). conclusion during the initial phase of oac therapy a coagulation activation is reported but no significant elevation of tat or fl+2 was found. this result shows that additional heparin and asa therapy was sufficient to avoid systemic coagulation activation. the increase of d-direct should be interpreted as a si~=m of local fibrinolytic reaction due to stent implantation. three methods for the determination of prothrombin time from capillary blood in patients under oral anticoagulation have been investigated. two methods were run on coaguchek® monitors (boehringer mannheim) from capillary whole blood. after fingerpuncture the first drop of blood was applied to the well of a coaguchek® test strip directly from the finger-tip, whereas the second drop was sucked into a non-anticoagulated plastic capillary (hirschmann) and immediately applied to the test strip -and vice versa to eliminate any influence of first and second drop of blood. the third method was hepato quick (boehringer mannheim) which was determined out of citrated capillary blood from an earlappuncture. 66 specimen of patients under oral anticoagulation were investigated. the method comparisons between each of the coaguchek® methods and the laboratory method show good results and the correlation between the coaguchek® methods is excellent. mean differences to the lab methods are -0.1 inr in both cases. no mean deviation was detectable between the coaguchek® methods. scattering of coaguchek® versus hepato quick was +/-0.6 inr in the range 1 to 4 inr except for three outliers and one patient with fluctuating results in the lab method which could not be resolved. introduction: haemorrhagic coumarin skin necrosis is a severe complication during initial phase of oral anticoagulant therapy. histological examination shows thrombotic occlusion of small vessels, but little is known concerning the pathophysiologic background of the bleeding component. recently, we described protein z deficiency in patients with bleeding complications of otherwise unknown origin. thus, we were prompted to measure protein z in patients with coumarin skin necrosis. patients: 4 patients (i man, 4 women; age: 35±10 years) suffering from haemorrhagic coumarin skin necrosis were examined. all patients had normal liver protein synthesis function, none was under oral anticoagulant treatment during this study. method: protein z antigen test, diagnostika stago, france. results: 4 out of the 5 patients examined had diminished protein z levels ( 700, 820, 1080, 1700 ug/l) in comparison to normals (2900 ug/l). in one of our patients, protein z was normal (3020 ug/l). conclusion: low protein z levels are additional risk factors for haemorrhagic coumarin skin necrosis. oral anticoagulant therapy is the treatment of choice in patients with need for long-term anticoagulation. since oral anticoagulants interfere with the function of vitamin k, it is not clear whether stable oral anticoagulation can be achieved in patients with need for continous substitution of fat-soluble vitamins including vitamin k. we report about a 59-year-old man who had experienced progressive hypertrophic obstructive cardiomyopathy over the preceeding 21 years. atrial fibrillation has been first diagnosed 18 years ago. latter on, recurrent ischemic attacks and embolism of the right arteria iliaca occurred. in 1993 the patient received extirpation of the ileum and subtotal amputation of the jejunum because of mesenteric infarction. the resulting short bowel syndrome requires continous substitution of fat-soluble vitamins. since vitamin k free preparations of fat-soluble vitamins for parenteral use are not available, prophylaxis of thrombosis has been performed with unfractionated hepadn. as a consequence of the longterm treatment with hepadn the patient developed severe osteoporosis. therefore, the decission 1:o discontinuate heparin therapy and initiate oral anticoagulation has been made. because of its shorter halflife warfarin (coumadin) was used instead of dicoumarol. over a 4 weeks lasting induction phase inr values were controlled daily. a dosage regime starting with '10 mg warfarin at the day of vitamin application (day 1) followed by 3.75 mg on day 2 and 1.25 mg on days 3, 5, and 6, respectively, was found to be optimal to maintain inr values within the target range (inr: 2.0-3.0). in order to minimize the risk of hemorrhage the vitamin administration was changed to the subcutaneous route. during an observation period of 6 months neither any bleeding or thrombotic complications nor a vitamin deficiency occurred. these data indicate that stable oral anticoagulation can be achieved despite extreme variation of vitamin k plasma levels. portable monitors for home monitoring of inr are well established for adults on oral anticoagulants. patient's compliance is improved as well as long term outcome. experience concerning accuracy of the procedure in children is limited. 32 inr determinations were performed in parallel from venous and capillaryblood samples of an infant on phenprocoumon, starting at the age of 4 months. the coaguchek® monitor from boehringer mannheim was used. choosing an arbitrary range of agreement of ,qnr 0.5 for both determinations, 81% of the measurements were within the defined range. 5/6 outliers were due to low inr resulting from difficulties in capillary blood sampling. the degree of agreement increased when the procedure was performed at least once a week. in conclusion: inr determination with a portable monitor may be helpful in home monitoring oral an.ticoagulant therapy in young children. a dose adjustment should be done only on the base of inr determination of venous blood -if it is considered the gold standard -to avoid over-anticoagulation. a stable anticoagulation is one of the most difficult tasks in attending patients with heart-valve-prosthesis. if prothrombin times are out of the therapeutic range, the risk of bleeding or thromboembolism increases disproportionately. for this reason any improvement in anticoagulant control and/or management can have far reaching consequences in decreasing complications, in extending longevity and in improving quality of life. for the first time a clinical trial was started in 1986 and continues until today at the cardiac rehabilitation center bad berleburg, germany with patients mainly after heart valve replacement. the patients were trained to measure their own prothrombin time and to adjust their own dosage of the oral anticoagulant. within six years 600 patients were trained: 216 patients could be followed up with regard to their selfdetermined prothrombin times. the results were within the therapeutic range in 83.1% of the measurements (n=14.812) taken by the patients themselves. on average, the patients who determine their prothrombin time themselves did so at a weekly interval. neither major bleeding nor thromboembolic complications could be observed in the 205 patient-years of home prothrombin estimation. it is to be hoped that the usual rate of complications can be reduced when patients determine their prothrombin time themselves at a close interval, resulting in more constant values in the therapeutic range and slight corrections of the anticoagulant dose. home prothrombin estimation promises better quality of life and has a considerable potential to achieve this goal. circulating plasma thrombomodulin (tm) is a novel endothelial cell marker, which may reflect endothelial injury. tm acts as thrombin receptor which neutralises the fibrin-forming effect of thrombin, and also accelerates the formation of the anticoagulant protein c/s pathway. tm therefore belongs to the anticoagulant defence system against thrombosis. increased tm levels have been described in various diseases such as ards, thromboemboembolic diseases, ttp, diabetes, le and cml reflecting alterations of the vascular system at the endothelial level. to find out to what extent cardiac catheterisation imtates vascular endothelium, tm concentrations (stago, asnieres, france: x 10 3 iu/ml) were investigated prospectively in 58 infants and children (three days -16 years). blood samples were drawn before the intervention, immediately at the end and 24 h later, snap frozen (-70 °c) and investigated serially in dublicate six weeks -3 months later. the results (median and range values) are shown in the enhanced tm concentrations immedately after the operative intervention, followed by normalisation within 24 h, indicates that cardiac catheterisation in pediatric patients rather leads to a short lasting irritation of the vascalar endothelium than to severe irreversible endothelial damage. recently in an al=wl" based method dahlb~ick et al described in vitro resistance to the anticoagulant effect of activated protein c (apc) in thrombophilic adult patients. apcr is in the majority of cases associated with the arg 506 gin point mutation in the factor v gene. concerning the special properties of the neonatal hemostatic system (low vitamin k dependent coagulation factors, physiological prolongation of the pt and aptf) we adjusted this ap'it based method (chromogenix, m~,lndal, sweden) to neonatal requirements: apcr was measured in 120 healthy infants according to dahlb~ck. the results were expressed as apc-ratios: clotting time obtained in a 1:1, 1:5 and 1:11 dilution with factor v deficient plasma (instrumentation laboratory munich. germany) using the apc/caci2solution divided by clotting time obtained with cac12 in the same i:1, 1:5 and 1:11 dilution. in addition, plasma of 24 neonates with septicaemia were investigated and data of 18 infants aged birth -three months with arg 506 gin +/-were shown. the arg 506 gin mutation of the factor v gene was assayed by amplification of the dna samples by pcr followed by digestion of the amplified products with the restriction enzyme mnl i. results were confirmed by sscp -analysis or by direct sequencing of dna from patients with apcr. results are shown in the 1.6(1.4-1.95) neonates and infants were considered to be apcr when the aptt ratio was < or = 2. concerning the special properties of the neonatal hemostatic system, our data show concordance with the pcr method in neonates and infants only, when the aptt based method was performed in the i: 11 plasma dilution. case report: we report on an 8-year old boy with severe hemophilia b and frequent screaming at night. eeg showed spike wave activity, starting from the temporal lobe, but generalizing within seconds. complex partial seizures were diagnosed and therapy with carbamazepine was initiated. as no improvement was seen nmr was performed. this revealed lesions within the right frontal cortex. higher doses of carbamazepine were not succcssfull as was therapy with phenytoin and pfimidone respectevely. the patient is now treated with carbamazepine and valproate. he still suffers from one short seizure per day. because of his seizures we started prophylactic replacement therapy with 600 i.e. factor ix twice per week. discussion: in 1992 wilson et al. first detected brain abnormalities in 25 of 124 children and adolescents with hemophilia a or b who were negative for immunodeficieney virus (1). the most common findings (14/25 patients) were small, focal, nonhemorrhagic white matter lesions of high signal intensity on t2weighed images. similar lesions have been reported in children with sickle cell cerebral infarction (2) . only three of these 14 patients had seizures, all of those having a documented history of intracranial hemorrhage. our patient has similar lesions as those described by wilson et al. but no history of intracranial hemorrhage is documented. even if tuberous sclerosis might be a differential diagnosis, we think that the abnormalities are related to hemophili a or its treatment, because the patient has no further signs of this disorder. conclusions: 1. in patients with hemophilia and seizures nmr might be useful as a high sensitive method for the detection of gray and white matter changes. 2. further studies should be initiated to determine the prevalence of pathological conditions in the brain of hemophiliac patients. disseminated intravascular coagulation (dic) is a rare, but foudroyant disease occuring in gram-negative sepsis like meningococcal septicemia. despite the avallibility of potent antibiotics, mortality in mertingococcal disease remains high ( about 10 % ), rising to 40 % in patients presenting with severe shock and consecutive dic. as the clinical course and the severity of manifestations of systemic meningococcal infections varies there is a need for early diagnosis of the infection and stage of coagulopathy in order to reduce the high mortality rate. few and rapidly available parameters are needed to classify the wide spectrum of clinical and laboratory findings in patients with dic. the parameters include partial thmmboplastin time, pmthmmbin time, plasma levels of fibrinogen, fibrin monomers and dimers, fibrin degradation products and the thrombocyte count. monitoring the course of hemostaseologicai findings in 26 pediatric patients with systemic meningococeal infections we observed a change of coagulation parameters as early as in the first stages of the infection: a prolongation of partial thromboplastin time to an average of 69. 1 sec (range 22 -150 sec, norreal 30 -45 sec), a decrease of prothrombin time to 45.7 % (range 13 -71 %, normal 70 -100 %) and of antithrombin iii to an average level of 16. 8 u/ml (normal 20 -29 u/ml ) was found 1 to 4 (-6) hours after admission. the consecutive development of hemostaseological parameters mentioned above permitted to define the stage of coagulopathy and thus to induce a stage related therapy. primary treatment consisted in control of shock by liquid substitution, compensation of metabolic acidosis, correction of clotting disorders ( at iii and heparin in stage of pre-dic ; at iii and fresh frozen plasma in case of advanced dic ) and treatment with g-lactam antibiotics ( e. g. cefotaxime or ceftriaxone ). an early assessment of the coagulation disorders in meningococcal disease can be based on few coagulation parameters, thus an appropriate treatment may be arranged to prevenl the patient from a fatal outcome of meningococcai septicemia and protect him from the development of a waterhouse-friderichsen-syndrome. this study was designed to prospectivdy evaluate coagulation and flbrinolyfie activation in 60 children (neonate -16 years) during cardiac catheterisation with low dose flush heparin (10 iu/ml saline). aptt (instrumentation laboratory: see), anti xa activity (xa; chromogenix: iu/ml), prothrombin fragment ft.2 (f1.2; behring werkc marburg: nmol/l) and d -dimer formation (d-d; bnhring werke/vhrburg: ug/l) were investigated before (t1), at the end (t2) and 24 h after cardiac catheterisation (t3). in addition, to evaluate the influence of inherited thrombophilia in all patients resistance to activated protein c (apcr), protein c, protein s and antithrombin were investigated. during catheterisation median (range) hepadn was administered in a total dose of 60 (17-206) iu/kg bw. in addition infants < 6 months of age (arterial catheterisatiun only) or patients with known thrombophilia received 300 -400 iu/kg hepafin for fmther 24 hours. the results (median and range) are shown in the ft.2 was sigificanfly elevated above the pediatric boundary immediately after the intervcation and nearly reached baseline values 24 h later. in contrast no cfinically relevant fibrinolytic activation was seen: d -dimer formation increased within the pediatric boundary immediately after the catheter and returned to basdine levels 24 h later. three children showed resitance to apc. tn one child stroke occurred before. not knowing the result of apcr in the remaining two patients only one neonate received further prophylactic heparin. the third neonate without heparin prophylaxis suffered from venous occlusion within two days after the intervenfon~ in addition, no protein c, protein s or antithrombin deficiencies were found. although administration of low dose flush heparinisation during cardiac cathetefisation could not prevent short -term coagulation activation, no thrombotic events occurred in children without inherited thrombophilia. if fnrther prophylactic hepariuisation in children with a~r, protein c, protein s or antithrombin deficiencies may prevent vascular occlusion requires a more intensive study. a.sandvoss, w.eberl, m.b0rchert introduction: capillary leakage, edema and hypovolemia are common complications in preterm infants especially if birth weigth is below 1.500 g. septicemia, asphyxia and immaturity seem to be most important risk factors. to determine the influence of c 1-esterase inhibitor (cilna) in preventing contact phase and complement activation we investigated c11na concentrations in normal and symptomatic preterm infants. methods: activity of cilna were measured by chromogenic substrate method (behringwerke), cilna concentration with radial immunodiffusion (behringwerke,germany). results: cllna-activity in asymptomatic preterm infants (n= 14) was 65+/-15% of normal at birth. healthy newborns showed activities of 80+/-20%. cilna reached normal adult values 2-4 days after birth. preterm infants with respiratory distress syndrome(n = 14) showed lower activity on day 2-5, patients with additional septicemia (n=15) had decreasing c1 ina-activities in the first three days of life. individual course of cllna-activity and thrombocyte count correlated in the group with irds with and without septicemia. in children with capillary leakage onset of diuresis went parallel with raising cllna-activity. markers of contact phase (f xlla) and complement activation (c 5al were investigated in single cases and evidence for involvement of both systems was found. conclusion: contact activation and complement system play an important role in capillary leakage in preterm infants. cilna regulates both systems. activity of cilna correlates with clinical course, substitution therapy is possible and may improve outcome of these critical ill patients. antiphospholipid antibodies (apa) interfere with hemostasis probably by inhibition of protein c or prothrombinase complex. thereby, apa might lead to thrombosis or increased bleeding. however, incidence and clinical importance of apa has not yet been investigated in children. therefore, we assayed plasma samples of 220 children, aged 0,1 to 19 years (mean 7 years) by elisa detecting igg-and lgm-antibodies directed against eardiolipin, phosphatidyl serine and phosphatidic acid. in patients with increased bleeding, thrombophilia or prolonged clotting tests a detailed coagulation analysis was performed. according to their diagnosis children were devided into 5 groups: i. autoimmune diseases, ii. infections, iii. metabolic diseases, iv. other diseases, v. healthy children. results: apa were found in 69/220 patients. in the respective groups we demonstrated apa in the following proportions: 1. lgg-isotype: activitiy of c1 esterase inhibitor (c11na) is reduced in preterm infants especially if birth weigth is below 1.500 g and respiratory distress syndrome and/or septicemia is present. capillary leakage with generalized edema, hypovolemia and hypotension is resulting in imbalance between inhibition and activation of contact phase and complement system. iln four patients we investigated seven courses of substitution ;with commercial c1 esterase inhibitor preparation (berinertr,behringwerke), case reports are given. all patients had clinical symptoms of capillary leakage, all had septicemia accompanied by either respiratory distress, disiseminated intravascular coagulation or mutiple organ failure. jefficiacy of substitution therapy is dose related, supranormal iactivities of cilna are necessary, reflecting raised consumption of inhibitor in ongoing disease. clinical effects on diuresis, catecholamine need and especially on thrombocyte counts are demonstrated. or arterial thromboembolic event in children e. lenz, c. heller, w. schr6ter*, w. kreuz johann w. goethe-universit~itskinderklinlk, frankfurt a. main, germany * georg-augast-universit/itskinderidinik, g/3ttingen, germany venous thrombosis as well as arterial thrombo-occlusive events are rarely observed in childhood, but can lead to life-threatening situations and longterm sequelae in these patients. after the initial stage of treatment (thrembolysis or thrombectomy) the pediatrician has to decide how to efficiently prevent re-thrombosis in the individual patient. anticoagulation after venous thrombosis is generauy recommended for 6 months after the event; if an underlying thrombophilic condition has been detected in the patient anticoagulation has to be considered lifelong. when evaluating antithrombotic therapies for children it is of importance to consider whether the anticoagulatory effect is mainly necessary in the venous or arterial vessel system. the hemorrhagic risk and side effects of the different anticoagulatory preparations have to be taken into account, especially when treating small children. only limited experiences exist concerning the suitability of the preparations for long-term anticoagulation in children and general recommendations on the ideal dosage in pediatric patients are still missing. we want to disscuss different types of anticoagulants (such as coumarins, unfractionated heparin, low molecular weight heparin (lmwh) and inhibitors of platelet aggregation) their mode of action, their suitability for pediatric patients and their side effects and relevance of these side effects especially in children. from the experience in our own pediatric patients, we would like to report on the indications, which can be given to administer these different preparations, the dosage regimen we recommend and the laboratory tests to monitor save and efficient re-occlusion prophylaxis in our patients. in this context we would like to present our data on 8 patients with either thrombosis or arterial infarction due to a thrombophilic condition, who had all contraindicatioas to oral anticoagulation by coumarins. because prophylaxis for re-thrombosis was mandatory in these patients, lmwh was given for long-term anticoagulation in a dally subcutaneous dosage of 100-150 anti-xa u/kgbw. monitoring was done by anti-xa-test (0,4-0,8 anti-xa u/ml). under this regimen none of the patients developed re-thrombosis or bleeding complications. alopecia was seen as a side-effect. this study was designed to prospectively evaluate coagulation and fihrinolytic activation after cardiopulmonary bypass with aprotinin (2x17000 u/kg bw) in 42 infants and children aged 0.1 -15 years, and to correlate these findings to the clinical outcome. prothrombin fragment f 1.2 (f1.2; behring werke marburg: nmol/l), antithrombin-serinesterase -complex (atm; stago: ng/ml), d -dimer formation (d-d; behring werke marburg: ug/l), tissue-type-plasminogen activator ag (t-pa; chromogenix: ng/ml), plasminogen activator inhibitor 1 antigen (pai; chromogenix: ng/ml) and cl-inhibitor (c1; behring werke marburg: x 10-3 g/l) were investigated before the operation (t1), at the end of the operation (t2), and on postoperative days 1 (t3), 4-6 (t4) and 7-9 (t5), respectively. the results are shown in the table (median and median absolut deviation): t1 t2 t3 t4 "1"5 nv fi.2 0.9 +/-0.5 1.7 +/-0.9 1.4+/-1 1.8+/-0.8 1.6+/-0. the platelet (pl) function defect induced by thrombolytic agents has been attributed either to the degradation of pl surface receptors or to the anti-aggregatory effect of fgdps. in contrast to other plasminogen activators scu-pa is intimately inked with pl: they can rapidly incorporate exogenous seu-pa, release it upon stimulation and bind the proenzyme. recently we have reported that exposure of prp to recombinant scu-pa (2.5-t00 um) in timed interval 1-30 min resulted in dose-dependent inhibition of pl aggregation. timecourse changes of the process were followed by the biexpotential kinetics: a rapid initial inhibition during the first 3-5 rain with the moderate suppression of pl aggregation in the 30 min period. when tcu-pa (25-100 nm) was exposed to prp in the same conditions dose-and time-dependent inhibition of pl aggregation was also observed. since the effect was obtained no earlier than t0 min after exposure of tcu-pa to prp, and the threshold dose was higher. comparable inhibition of pl aggregation was obtained with 25nm of scu-pa versus 100nm of tcu-pa and the llbrinogen depletion by the end of the 30 min period was 2% and 30% respectively. it's likely that tcu-pa and its precursor have different mechanisms of action on the pl aggregatory function. in a recent study we have shown that recombinant rscu-pa inhibits platelet (pl) aggregation in prp. to exclude the possible influence of rscu-pa/plasma interfere on this process the aggregation of washed pls was under the investigation. pls were washed according to modified mustard's method, suspended in buffer and adjusted to 250,109/1. the resuspended pls were exposed to 5-100 nm of rscu-pa for 30 min at 370(;. at time points 3, 5, 15 and 30 min the aggregation with 0.6 iu/ml of thrombin was measured. it was found that the exposure of pls to rscu-pa (20-100 nm) for 3 man resulted in marked inhibition of their aggregation. since after 15-30 man of incubation with 20-50 nm of rscu-pa the inhibitory effect on pl aggregation became less pronounce or even disappeared. when 5 nm of rseu-pa was used the inhibition of pl aggregation became significant only by 15 rain of exposure period and didn't change for 30 man of investigation. the observed results may be cormeeted with uptake of rscu-pa by pls from surrounding buffer as well as with individual variations of pl response to the same concentration of rscu-pa. loss of glycosylation may result in a reduced platelet (p) survival and perhaps altered function. we analyzed the structural and functional effect of specific deglycosylation (combinations of n/o-glycosidase and neuraminidase treatment) of p and isolated p gpib. washed and formaldehyde-fixed p were digested as follows: 1) with neuraminidase (0.125u/ml) + o-glycosidase (3.1mu/ml) + n-glycosidase (1.25u/ml), 2) with neuraminidase alone (0.2u/ml), 3) with n-glycosidase (2u/rnl) and 4) with neuraminldase (0.2u/ml) + o-glycosidase (5mu/ml). all reactions were performed in the presence of protease inhibitors (pmsf, leupeptin, sbti), after washing x2 the p and identically treated controls were analyzed by flowcytometry with the antibodies 6di (mab: a-gpib), 7i-l2 0vlab: a-gpiiia), and the lectins wheat germ agglutinatinln (wga, for neunac) and peanut agglutinin (pna, for [3dgal(1-3)-galnac) which confirmed effective and specific deglycosylation by the respective enzymes (but gave only minor differences with 6di and 7h2). the botrocetin (13) and ristocetin (r)induced agglutinations showed arer treatment 1) (all enzymes) a full inhibition of r-induced agglutination but only a mildly reduced b-induced agglutination (70% of normal). treatment 2 and 3 (neuraminidase alone, and n-glycosidase alone) affected both agglutinations only mildly (70-80% of normal).treatrnent 4) (o-deglycosylation) however showed a major inhibition of r-agglutination down to 30%, while b-agglutination interestingly was almost fully retained. the results of the rotary shadowing electron microscopy of purified gpib suggested a collapse of the normally stretched, glycosylated, gplb, not only after the treatment with all three glycosidases, but also .after o-deglycosylation alone. we conclude that oglycosylation is most important for ristocetin-induced platelet-von willebrand factor-interaction and responsible for the typical stretched shape. the phenomenon of in vitro platelet aggregation and consequent pseudothrombocytopenia (ptcp) in the presence of calciumchelatization by na-edta and sodium-citrate was studied in blood samples of a patient. initial platelet counts electronically measured were 20000/ul blood anticoagulated with na-edta and sodium-citrate. normal platelet counts were found in heparin-anticoagulated blood and in capillary blood. immunoglobulines of the igg and igm subclass were identified in the patients plasma. by incubation of the patient's serum with platelets of healthy individuals, platelet-clumping occurred in the presence of na-edta and sodium-citrate but not in the presence of heparin. the platelet membrane glycoproteins (gp) hb/llia, ix and iiia/vnr g-chain were involved in the antigen antibody reaction as demonstrated by specific antibodies and flow-cytometry. on platelet surface permanent calcium-exchange and -replacement is dependent on external calcium concentration. calcium depletion induced by calcium chelators as na-edta and sodium-citrate might conformationally change platelet surfaces and induce formation of neoantigens. the decrease of gp llb/illa platelet surface antigen to 10% (normal >75%) indicated the important role of the gp iib/iiia receptor at ptcp. the saliva of tdatoma pallidipennis, a triatomine bug, was found to contain a protein called "pallidipin", that specifically inhibits collageninduced platelet aggregation but not adhesion or shape change. to investigate the mechanism of action of recombinant pallidipin the influence on platelet fibdnogen binding after activation by collagen type i in different concentrations was measured by flow cytometry. the same concentrations of pallidipin that inhibited the couagen-induced platelet aggregation completely did not cause any inhibitory effect on fibdnogen-binding in the prp from the same donor measured contemporaryly. collagen type i-induced platelet aggregation of cd36-deficient platelets from two different unrelated blood donors was inhibited by the same concentration of pallidipin that inhibited aggregation of control platelets. there was no inhibition of collagen-induced fibdnogen-binding in the cd36-deficient platelets as well. pallidipin did not cause inhibition of collagen-induced membrane expression of cd62 and cd63 of control and cd36-deflcient platetets as measured by flow cytometry. however eadier studies had shown an inhibition of collagen-induced atp and {3tg secretion by pallidipin. therefore we compared the effect of pallidipin in unstirred and stirred prp samples. while pallidipin had no effect in unstirred samples it showed strong inhibition of ptg secretion in stirred samples. we therefore conclude that pallidipin does not act on collagen-induced aggregation through cd36 and that the inhibition is a post fibdnogenbinding event. pallidipin does not influence the first steps in secretion, which are independent from cytoskeleton and platelet-platelet contact, but inhibits the following steps. 17-hydroxy-wortmannin does not inhibit the transport of 1nm-gold labelled fibrinogen in resting platelets. e. morgenstem, b. kehrel and k.j. clemetson medical biology, saarland univ., homburg, germany, haemostasis research, univ. muenster, germany and theedor-kocher-lnstitut, univ. bern, switzerland. wortmannin, an inhibitor of phosphoinositide 3-kinase and of myosin light chain kinase blocks reactions of the activated platelet. to obtain informations about the role of the contractile cytoskeleton in receptor-mediated transport of resting platelets, the effect of 17-hydroxy-wodmannin (hw) on the endocytosis of fibrinogen from the surface of resting platelets was studied. gel filtered platelets (gfp) were incubated for 10 min at 37°c with hw (3x10-6m) or with iloprost. controls and gfp preincubated with hw or ilopmst were incubated with 1.4nm-gold labelled fibrinogen molecules (fg-au; final concentration 40p.g/ml) at 37°c. the experiments were stopped after 5 or 30 min by rapid freezing. after freeze substitution in acetone with 4% osmiumtetroxide, sedal sections were prepared. the sections were examined after incubation with ascorbic acid (5% in h20) for 30 rain at 20°c (to reduce metallic osmium) and silver-enhancement using danscher's (1981) method (to visualize the fg-au). examination of adp stimulated platelets in the presence of 40fg/ml fg-au shows that the ligand is able to mediate aggregation. the examination reveals, that fg-au was present in a low density on the platelet surface, in higher density in the surface connected system (scs), in coated pits and vesicles and separated smooth vesicles (representing endosomes?) as well as in the matrix of alpha-granules. after 30rain, the number of labeled granules was increasing. labels on the surface and on the mentioned cytoplasmic membranes were observed during the whole period of incubation. hw or iloprost did not alter the resting gfp and the mentioned qualitative ultrastructural findings in both preparations did not show differences to the controls. we conclude from the results with hwthat the regular contractile function of the cytoskeleton is not necessary to transport the fg-au in resting platelets. methods: edta anticoagulated whole blood was incubated with thiazole orange and analyzed with a flow cytometer. young platelets were defined by having a high fluorescence from thiazole orange (normalized to platelet size). platelets were also incubated with fluorescent antibodies to gpib, gp lib/ilia and gmp-140 (two colour method). results: surface expression of gpib was the same in young and older platelets. results for gp lib/ilia and gmp-140 (in resting and activated platelets) will be presented. conclusion: young platelets can easily be detected using thiazole orange and flow cytometry. there is no differential expression for gpib. further results will be presented. the influence of erythrocyte and thrombocyte content on the release of atp by different agents in whole blood specimens was tested. the measurement had been performed in the lumi-aggregometer using the principle of the luciferin-luciferase reaction. altogether 39 blood samples were diluted gradually before induction of the release reaction by arachidonic acid (1,25 mmol/i final concentration), adp (30 ijmol/i) and collagen (1,0 and 5,0 tjg/ml). the peak of the obtained curves was transformed into percent values of the maximal deflection by the undiluted sample (= peak in relation) and into atp concentrations (= absolute peak) after testing the atp standard in parallel for each dilution step separately. the peak in relation increases by increasing dilution with all inducers. it was identic with the atp standard and with collagen, somewhat lower with arachidonic acid and much higher by adp. a luminescence-optical effect may influence all these results. the absolute peak decreases by dilution under arachidonic acid and collagen as it was expected by the decreasing thrombocyte content of the samples. under induction by adp no decrease of the absolute peaks by increasing dilution of the samples was abserved. this can be explained only by liberation of atp from the erythrocytes. the atp standard is essential for the quantification of the release reaction. adp doesn't suit for it. collagen with a final concentration of 1 pg/ml was proven as the best inducer. platelet aggregation induced by several agents has been photometrically investigated in disc shaped rotating cuvettes coated with vessel wall tissues obtained from human umbilical cord, either endothelium or smooth muscle cells or extracellular matrix or combinations of them. in addition, effects of endothelium incubated with several cytokines on platelet aggregation have been studied. endothelial cells strongly inhibited aggregation depending on their cell count and the concentration of the inducer. smooth muscle cells showed the same effect but very less marked. in presence of extracellnlar matrix spontaneous aggregation occured. endothelium could inhibit this spontaneous aggregation when present in the same cuvette, smooth muscle cell could not. incubation of endothelium with several cytokines increased its anti-thombotic properties. for example, at a platelet count of 3x105/id in the prp, 10 -6 m adp led to maximal aggregation in uncoated cuvettes, in presence of 5,5x106 endothelial cells aggregation was completely abolished, in presence of 2,75x10 "6 cells aggregation was decreased to 40%. smooth muscle cells diminished the aggregation effect of 0,1 nih thrombin to 67% when only one side of the cuvette was coated and to 63% when both sides were coated. endothelium could not inhibit aggregation induced by 2,5 x 10 -6 m adp but endothelium incubated with 500 u/ml tnf-a or 30 u/ml intedeukin-lfl or lmm l-nitro-arginin for 24 h did completely inhibit aggregation. platelets become sticky and adhere to surfaces or to another without contracting and secreting. during maturation of megakaryocytes finally platelets lost their genomic nuclear message. only mitochondrial dna of platelets can be identified. we focused our attention on the impact of mitochondrial dna and the mitochondrial transscriptive mechausisms during platelet activation in normals. materials and methods: leucocyte free (nagentte chamber, flow cytometric analysis) platelet rich plasma or platelet concentrates a_~er hemapheresis were filtered by pall 100 leucocyte filters. the influence of different anticoagulants (commercially available sarstedt tubes containing citrate, heparim edta and 500 atu/ml hirudin wacker) was examined. activation was due to a 60 nun. hemapheresis procedure ( 3-5fold increase of cd 62, cd 63) and ex rive stmaulation due to 4 niy u/ml thrombin, 0.025 m cac12 or combmatious. the guanidiurn method for total rna preparation were used according to t. brown: current protocols in molecular biology 4.21-4.9.14,1991. different primers of mitochondrial genome (e.g. cytochrome b and atpase) were prepared using pcr and mitochondrial transscription was examined using northern-blot-technique. results: 1., there is less activation of mitochondrias using hirudin anticoagnlation, but a 2fold increase of mitochoindrial rna content in heparinized samples. 2., stimulation with thrombin leas to an increase to 5.5 e-l0 rna btg/platelet, compared to 4.7 -4.8 e-10 rna ~tg/platelet under unstimulated conditions.. conclusion: there is evidence for the importance of platelets mitochondrial dna and mitochondrinl transsefiption in regulation of cytosceleton and platelet activation. thrombospondin-1 (tsp-1) is a large homotrimeric glycoprotein originally identified as a platelet alpha-granule component. the investigation of its putative role in a variety of pathophysiologies like haemostatic disturbance, malignancy and wound healing requires specific laboratory reagents. monoclonal antibodies are one of the most powerful of these reagents. therefore, we purified human tsp-1 from thrombin-stimulated platelets using affinity chromatography to generate monoclonal antibodies in mice. a subclass igg 1 monoclonal antibody designated 48.42 was purified from ascitic fluid and further characterised. western blot experiments demonstrated that this antibody reacted only with the unreduced molecule whereas the tsp-1 subunit chain was not recognised. no cross-reactivities with human fibrinogen, fibronectin, vitronectin and von willebrand factor were found. preliminary results indicate that the monoclonal antibody 48.42 can be used to investigate tsp-1 function in several assays including immunocytochemistry and cell adhesion as has been demonstrated for hl-60 cells. in addition, a sandwich enzyme immunoassay was developed using goat-antihuman tsp-1 igg and derivatised monoclonal antibody 48.42 (peroxidase, biotin) as a sensitive method for detection of tsp-1 in human body fluids. in the following study the expression of the platelet antigen (cd62p) and the leukocyte antigen (cdllb) were measured in whole blood, in addition to platelet-leukoeyte adhesion (rosette formation) by means of multicolour fluorescent labelling (cd45, cd14, cd42a). the measurements were carded out both in freshly drawn whole blood which had been antieoagulated with different agents, and in stirred samples of whole blood under controlled conditions (37°c, 1000 rpm, different stirring times). the results are presented as the percent positive events in each gate (platelets, leukocytes -pmnl, monocytes, lymphocytes and rosettes -plateletpositive events in the pmnl, monocyte and lymphocyte gates), whose mean fluorescence is given in addition to an index comprising the product of the percent positive events and their mean fluorescence. stirring (max 15 rain) induced an increase of cd62p on the platelet surface of ca. 10%, without any change in the mean fluorescence. under these conditions increased cdllb on pmnl and monoeytes could be detected. an increase in the rosette formation could also be measured (greater index), in that the percent of monocytes which were platelet-positive increased with no change in the mean fluorescence of the positive events, whereas pmnl showed an increased mean fluorescence, but not an increased number, of platelet-positive events. the time-dependent changes in rosette formation on stirring could be further increased by addition of adp. these results show that it is possible to measure rosette formation, and also the influence of effector agents (inhibitors or activators of platelets or leukocytes) on rosette formation, in whole blood using flow eytometry. 17 itp patients undergoing splenectomy were observed after 1-30 years following operation and divided into 2 groups. first group consisted of 8 patients with normal platelets count and absence of haemorrhagic syndrome. second group was formed of 9 itp-patienfs with episodes of thrombocytopenia recovery following certain time period after splenectomy. in the aim to study the cellular immunity there were carried out immunophenotypical investigations of blood samples using immunofluorescence method with monoclonal antibodies application. the increase of b-cells, expressing cd22, cd37, hla-dr-antigen has been revealed in the 2nd group. quantity of srfc, cd3 +, cd5 + cells in the blood of recovered patients was lower than in patients of the first group. this group was also characterized by statistically significantly increased level of cd4 + cells while the cd4/cd8 ratio was equal to 1.0 :i: 0.3 % (0,5 + 0,1% in patients of the second group, respectively, p>o,05}. also the relatively high expression of activating antigens in patients with thrombocytopenia recovery after splenectomy was stated. among infectious complications in all patients observed were predominantly found various types of throat infection, mainly with unsatisfactory treatment possibilities. we have observed the opsi-syndrome in 2 patients, being featured with marked tiredness, breath loss, intolerance of hard physical working, diminished ability to maintain physical activity. extracellular matrix (ecm) produced by human endothelial cells closely resembles the vascular subendothellal basal lamina in its organization and chemical composition. thus it contains collagens, fibroneetin, von witlebrand factor, thrombospondin, fibrinogen, vitronectin, laminin and heparin-sulphate. platelets carry different receptors on their membrane surface with specific binding capacities for one or more of these extracellular matrix proteins, such as glycoprotein (gp) iibiiia, gp ib/ix and gpiiib. incubation of platelets with ecm results in platelet adhesion, degranulation, prostaglandin synthesis and aggregation. we studied patients whose platelets showed either a receptor defect in gpiibiiia or gpiiib or a storage pool disease. adhesion experiments were performed using siliconised glass, collagen coated surfaces, immobilized fibrinogen as well as human subendothelial matrix. platelet adhesion of patients with thrombasthenia glanzmann (receptor defect of gpiibiiia) resulted in a total lack of binding to silieonised glass and immobilized fibfinogen. adhesion to collagen was almost normal in spite of the fact that only single platelets sticked to the surface and no microaggregates were observed. the adhesion to ecm was diminished and also no aggregates were detected. patients with a receptor defect in gpiiib showed normal platelet adhesion to siliconised glass and immobilized fibrinogen but binding to collagen and ecm was markedly reduced, while platelets with a storage pool defect sticked to siliconised glass but failed to adhere to ecm. by centrifugation of citrate blood (250 x g, 10 min) erythrocytes and leucocytes go to the bottom, whereas plasma and thrombocytes stream in the upper part of the probe. so the thrombocyte count doubbles in the platelet rich plasma in contrast to the platelet count in the whole blood volume. if the thrombocytes are more or less activated, they adhaere on erythrocytes, leucocytes or aggregate end are not able to stream upwards. the quotient between thrombocyte counts in prp and whole blood is a measure for thrombocyte activation. we chequed the value of this screening in different groups of patients with arterial occlusions disease (aod), chronical venous disease (cvd), diabetes mellitus (dm] and in healthy control persons (control). variation coefficient of the method is 3.7 (prp) and 4.4 (tc) respectively (coulter counter). differences to the control group are significant. changes in the patient groups in dispensaires follow up 5 years are also significant. nicardipin -induced immunthrombocytopenia p. eichler 1, c. hinrichs 2 , g greinacher l i.institut fur immunologic und transfusionsmedizin, ernst-moritz-arndt-universitat greifswald, 2. deister-s0ntel-klinik, bad m0nder drug-dependent immune-thrombocytopenias are a rare but clinically important variant of immune-thrombocytopenias. patients are at risk to suffer from severe bleeding complications. especially in patients receiving multiple drugs, diagnosis of drug-dependent immune-thromboeytopenia is often difficult. we report the case of a 71 year old male patient who received allopurinol, captopril, digitoxin, furosemid, and nieardipin. the patient presented with hematomas (pit. count < 10 g/l) and later developed bone marrow dysplasia. in an elisa using whole platelets and patient serum, a weak reactivity in the presence of furosemid, but a stronger reactivity in the presence of nicardipin (antagonil, ciba-geigy) could be demonstrated. the reaction pattern is given in the the enzyme-immunological determination of soluble fibrin (sf) proved to be highly sensitive and specific. this sf-elisa detected fibrin hacking fibrinopeptide a (fpa) via the monoclonal antibody 2t35 specific for the neoepitope generated on the aa-chain after the split of fpa. lill et al. recently introduced a new assay modification which utilizes the same antibody as the old one but takes advantage of a pretreatment of plasma specimens with kscn. this strong chaotropic ion is used to dissociate the various fibrin complexes possibly hiding fibrin epitopes. it was the aim of this study, therefore, to compare the two sf-elisa modifications (with and without kscn-pretreatment of specimens) . in order to examine the dynamics of thrombin-induced fibrin(ogen) metabolism we made course observations in patients with a certain form of septicemia. both assay modifications detected fibrin(ogen) derivatives which differed considerably in kinetics (n= 160 samples from 10 courses). the former sf-elisa (no kscn) correlated well with prothrombin fragments, thrombin-antithrombin !11 -complexes and with the release of fibrinopeptide a ( r > 0.96, n= 151). results of the new sf-elisa with kscn pretreatment of patients' plasma, however, correlated conspiciously well with d-dimer levels (r > 0.94) but distinctly less with the markers of thrombin generation (-0.12 < r < 0.29). this good correlation with d-dimer levels was unaccountable since the d-dimer maximum occured significantly later than the peak of markers of thrombin generation (p < 0.05). therefore, kscnpretreatment of fibrin specimens seems to lead to a change in the specificity of the fibrin assay despite usage of the same catching antibody. different half-iifes of differently composed fibrin complexes should be considered in trying to explain the findings. nevertheless, the results of the former assay without kscn-treatment correlated much better with the well-known dynamics of thrombin-induced fibrin generation during hemostasis activation than the data from the new assay modification. consequently, further examinations are necessary to specify the effect of kscn on soluble fibrin complexes and the resulting assay specificity. a rapid assay for the determination of the primary hemostasis potential (php) of whole blood has been developed (kundu et al, 1995) from the original method of kratzer and born. the new system employs a disposable test cartridge which holds the sample (citrated whole blood) and all components for the tests at the same time. the test procedure is very simple. the cartridge is loaded with -500 p.l citrated whole blood and is inserted into the platelet function analyzer (pfa 100aaw). the test is started automatically after a preincubation phase of 2.5 rain. the reaction starts with the contact of the whole blood and the capillary which is connected with a collagerdephinephrin coated membrane with a small aperture inside the test cartridge. under constant negative pressure the sample is aspirated and through the contact ofplatelets and vwf with collagen adherence and aggregation begins. the adhesion and aggregation process leads to the formation of a platelet plug which obstructs the flow through the aperture. the result of the php is reported as closure time (ct). additional parameters such as bleeding volumes are possible as well. first results show good reproducibility, normal values in the range of up to 150 sec. and a good discrimination of healthy donors from patients with congenital or acquired platelet dysfunctions. the system detects aspirin induced thrombocyte function defects and von willebrand disease. in ease of an abnormal result in the collagerdepinephrin system a second type of cartridge with a collagerdadp coating can be employed. in the majority of cases aspirin induced dysfunctions are normalized and could thus detect aspirin use. the proposed system may be a valuable tool for routine assessment of the primary hemostasis potential in a routine citrate blood sample laboratory. inducing mental stress in 20 young healthy male volunteers aged 20 to 40 ),ears with no previous history of thmmbophilia or a hemorrhagic diathesis was performed by a first time parachute descent from an altitude of 1000 meters. the purpose of this investigation was to find out whether there are any changes in the corpuscular and plasmatic fractions of peripheral blood. we were especially interested in elucidating changes in the procoagulatory and/or fibrinolysis systems. venous blood samples were obtained directly before and directly after the jump. flight time from the departure of the airplane to the landing of the parachutists was approximately 20 minutes. the maximum time that elapsed between the two blood withdrawals were 45 minutes. in a preliminary study with different voinnteem, certain fluid imbalances had been observed. absolute numbers of leukoeytes (6.9 vs. 9. l/n0, erythrocytes (4.6 vs. 5.1/pl), and platelets (246 vs.276/nl) significantly increased (p < .001), as well as the hemoglobin concentration from 145 to 156 g/l (p < .018). even though fluid imbalances before and after the jump had practically been excluded by measuring nearly identical hematoerit values (.41 vs..42), we noticed a marked drop in aptr (27 vs. 23 sec) and a significant increase in factor viii ~tivity. as a direct stress response, we found a rise in fibrinogen concentration (2.4 vs. 2.8 g/l) which is one of the shortest acting acute phase proteins. concerning reactive fibrinolysis, d-dimers showed an increase in concentration from 115 lag/l to still normal values of 192 lag/l, which was not significant due to low numbers of values (p = .086). we observed similar changes in fibrin monomers and prothrombin fragments fl+2. from other investigations on the kinetics of the activation of the procoagulatory system we know that maximum activil7 is not reached until 24 hours after initiation of activation.these investigations studied perioperative changes in different kind of operations which served as a control group concerning the degrees of tissue damage and resulting coagulation disturbances. to better understand these phenomena we plan to induce mental stress in a laboratoq' environment to further exclude unknow~a influences on the mechanisms which can activate the procoagulatory and fibrinolytic systems. triodena (t) 30/40/30 ug ee, 50/70/100 ug gestodene) were tested for their effect on hemostatic parameters. three groups (n=20) of healthy female volunteers were treated for 6 months with one of these oc. blood was taken before treatment (day 24-28 of pretreatment cycle, 0) and on days 18-22 of the 3 ~ (i) and 62 (ii) treatment cycle. indications of an activation of blood coagulation and fibrinolysis were detected as the plasma levels of prothrombin fragment f i+2 and of fibrin split product d-dimer and plasmin antiplasmin complexes were found elevated during treatment. the following main regulatory components of blood coagulation, activators and inhibitors, were investigated: factor vii antigen fviiag, fvii clotting activity fviie, circulating activated factor vii cfviia and antithrombin 3 at3 activity, total protein s antigen tps-ag, free protein s antigen fps-ag, protein s activity psact, circulating thrombomodulin etm fviiag, fviie and cfviia significantly increased during treatment; cfviia: 0: c 32.4 mu/ml a prethrombotic condition characterized by elevated levels of circulating soluble fibrin has been claimed to be a predisposing factor for accumulation of coronary thrombotic material in acute myocardial infarction. the present study includes 161 patients with clinical suspicion of myocardial infarction. blood samples were drawn by the primary care physician, upon arrival in the hospital, and after 2, 6, 12, and 24 hours of hospital stay. patients with myocardial infarction were identified by typical course in 12 lead ecg, and upon sequential determination of troponine t, myoglobin, ck, and ck-mb. patients with primary cpr were excluded from evaluation. soluble fibrin was measured by enzymun®-test fm (boehringer mannheim). patients with acute myocardial infarction display soluble fibrin levels within the normal range (< 5 ~tg/ml) during the initial two hours after onset of symptoms. there was no significant difference between patients with myocardial infarction and patients with coronary heart disease without myocardial infarction. slightly elevated levels were found in patients with atrial fibrillation, reflecting intracardiac fibrin formation. in patients without fibrinolytie treatment, a slight increase of soluble fibrin levels with a maximum after approximately 8 hours is observed. most patients with fibrinolytic treatment display a considerable increase in soluble fibrin, with maximum levels immediately after infusion of the fibrinolytic agent. four patients with pulmonary embolism showed soluble fibrin levels in the range of 40-300 [.tg/ml, which remained in the same range during the entire observation period. in conclusion, circulating soluble fibrin is not increased in patients with acute myocardial infarction and does not appear to be a predictor of acute coronary events. high levels of soluble fibrin in patients with fibrinolytic therapy may reflect release of fibrin from thrombotic material, but also de novo generation of fibrin due to release of active thrombin from thrombi not necessarily located in the coronary vessels. detection of elevated levels of soluble fibrin in patients with acute chest pain should result in careful examination for signs of pulmonary embolism or aortic aneurysm. the possibility to determine activated coagulation factors opens the question if data provide evidence of an activated coagulation or fibrinolysis and if this has a prospective value. we investigated patients with confirmed thrombosis, postsurgical septieaemia and also after liver transplantation. in all patients factor viia, xii, xiia and also the fibrinolytic parameters t-pa, pai-1, pap, plasminogen and a2-ap were determined. in addition, f1+2 and apc-resistance with heterocygote factor v-leiden-mutation and confirmed thrombosis. we found increased factor viia which showed partly also an increased fl+2. patients with other pathological results such as a reduced t-pa and/or increased pai-1 showed a low incidence of elevations in factor vii or f1+2. the activation of factor xii seems to be of minor importance in patients with thrombosis. a different picture is found in septic and transplanted patients. obviously factor xii-activation is of major importance in this group. a deterioration of the clinical symptoms is correlated with an increased factor xiia which is paralleled by a decrease of factor xiiactivity. the investigation of fibrinolysis parameters such as pai-1 and pap demonstrate a fibrinolytic disturbance of the balance. statistically significant are differences in septicaemic patients both in the surgical and in the internistical group in contrast to polytrauma patients. in patients with liver transplantations significant changes are apparently related to rejection of the transplanted organ together with a deterioration of the clinical picture. the possibility to detect activated coagulation factors may be a tool to detect changes in the hemostasis system at an early stage and to use this for an improved therapy. control of long-term oral anticoagulation is usually performed by serial determinations of the prothrombin time. however, the assessment of effective anticoagulation versus the potential risk of bleeding complication is difficult to achieve. molecular markers of blood coagulation activation might add valuable information in individual cases. we investigated 48 patients with thromboembolic manifestations (deep vein thrombosis n=22, pulmonary embolism n= 13, myocardial infarction n= 13) for one year beginning with admission to the hospital. tat, prothrombin fragments f 1 +2, d-dirner and fibrin monomer concentrations were analysed. all markers were significantly increased at the time of initiation of anticoagulant therapy thus reflecting a prethrombotic situation. patients suffering from venous thromboembolism demonstrated higher concentrations of tat and f 1 +2 in comparison to myocardial infarction (34.6 vs 12.3 pg/1, p=o.009; 2.8 vs 1.3 nmol/i, p=0.0025). f 1 +2, tat and d-dimer concentrations decreased gradually over the first 14 days of anticoagulant therapy reaching values within the established normal ranges in all cases. f 1 +2 and tat concentrations reflect the activity of the coagulation system during long-term anticoagulation whereas analysis of fibrin monomer yielded partly controversial results. we conclude that f 1 + 2 and tat appear to be superior to fibrin monomer for the individual control of oral anticoagulant therapy. the influence of thyroid failure on haemostasis is controversial. mainly hypoceagulable states have been described in clinically overt hypothyroidism. since hypothyroidism has been associated with an increased risk of atherosclerosis, we studied a wide range of haemostatic factors in untreated female patients with subclinical (b, n=42, age 59+13) or overt (c, n=8, age 55-zcj) hypothyroidism, as well as in hypothyroid women under 1"4 treatment (d, n=8, age 57+9) and euthyroid controls (a, n=80, age 50+14). simple screening tests (prothrombin time, activated partial thromboplastin time, fibdnogen), procoagulant factors (fvii, fviii, von willebrand factor), coagulation inhibitors (antithrombin ill, hepadn cofactor ii, protein c, protein s) and fibdnolytic factors (plasminogen, antiplasmin, plasminogen activator inhibitor, tissue plasminogen activator) were measured. results factor vii activity (vii:c), factor vii antigen (vii:ag) and their ratio were found increased in hypothyroid patients. factor viii activity showed the same tendency, whereas von willebrand factor ramained unchanged, as did all other parameters with exception of free protein s, which declined in overt hypothyroidism and in t4 treated subjects. these differences tended to diminish after exclusion of 26 women with estrogen replacement therapy for menopause, but the ratio vii:cnii:ag, as well as fvii:c still remained significantly higher in hypothyroid patients. conclusions: subclinical and overt hypothyroidism are associated with significantly higher levels of factor vii:c and vii:ag. the disproportionate increase in vii:c compared to vll:ag, as shown by their ratio, might reflect the presence of activated factor vii (vila), which in turn indicates a hypercoagulable state. this pattern becomes more pronounced with the concomitant estrogen replacement after menopause. exocytosis following platelet activation leads to translocation of cd62p (p-selectin), cd63, and thrombospondin, from cytoplasmic granules to the cell surface membrane, where these molecules, serving as activation markers, can be detected by flow cytometry. we here report detectability of these molecules preformedprior to platelet activation -inside the cytoplasm of resting platelets. two different methods are compared, i. e. using either methanol or the fix&perm kit (an der grub) for cell membrane permeabilization. in addition, interleukin(il)-ice is shown to be present in platelet cytoplasm after methanol treatment, but not after permeabilization using fix&perm. whenever cell surface positivity for a specific marker coincides with intracellular presence, blocking of the surface membrane sites prior to membrane permeabilization is required in order to obtain fluorescence intensity attributable to cytoplasmic staining. our data demonstrate the feasibility of the methods presented for the detection of intracellular platelet molecules. this technique should also provide a means for estimating the relative quantity of intracellular platelet antigens, provided the permeabilization procedure does not lead to antigen leakage or destruction. physical exercise activates the clotting as well as the fibrinolytic system as indicated in numerous investigations of exercise by running and by bicycle ergometer but not by swimming. the positive effect of an endurance training in coronary sport groups is induced also by influences on the hemostatic system. the influences are suppression of the clotting activation by the acute exercise and by an increased fibrinolysis response. different hemostatic parameters, therefore, were analyzed before and after swimming of male coronary patients (n=33; median ag~ 61 years, achieved heart rate: 68/min). indicating plasmatic clotting activation there was a significant increase in molecular markers tat and f1+2 among the coronary patients (tat from 2,1 to 3,4 pg/1; fi+2 from 0,92 to 1,1 nmol/1). the degree of clotting activation among the coronary patients was less than that observed in a group of young volunteers in a former investigation. this must be explained by existence of the coronary heart disease or by the higher age in the patient group. indicating an activation of fibrinolysis t-pa activity increased significantly in coronary patients (from 0,14 to 0,5 iu/ml) resulting in an unchanged balance between coagulation and fibrinolysis. from this findings of the hemostatic systems no increased risk of the coronary patients by swimming can be derived. a prerequisite, however, are precautions l±ke to devoid exercise in the anaerobic range, exclusion of major heart failure and of cardiac arrhythmias before begirming of the swim training. the principle of the fontan operation consists in anastomosing the right atrium to the pulmonary arteria, thus bypassing the right ventricle and using the only functional single ventricle as a pump for the systemic circulation. there are only few data about the influence of the changes in hemodynamics on coagulation and fibrinolysis. we investigated the coagulation system in 20 children and young adults aged 4 to 21 years in a general examination 4 to 61 months after fontan procedure. besides other abnormalities of the coagulation system, there were significantly increased values for the thrombin-antithrombin-iii-complex (tat) in 12 patients (60%). as a marker for an activation of the fibdnolytic system we found elevated plasmin-alpha2-antiplasmin-(pap-) levels in 14 patients (70%). less frequently, the concentrations for the prothrombin-fragments 1 and 2 (f1 and 2) (7 patients, 35%) or the d-dimer (2 patients, 10%) were increased. we didn't find significant differences in a clot-lysis-assay between fontanoperated patients and an age-matched control group. there was no significant correlation between activation of coagulation and clinical situation or diameter of the pulmonary arteria. whether the present data can help to estimate the risk for a thrombo-embolic complication following fontan procedure, still has to be investigated. the results of the clot-lysis-assay suggest, that for lysis of thrombi the same dose of rt-pa should be used as for other patients. a 2nd generation functional protein s assay p. van dreden* and e. adema** * serbio, gennevilliers france, ** boehringer mannheim, tutzing germany a second generation protein s test was developed with improved sensitivity to protein s and better reagent stability. the test result was found to be unaffected by apc-resistence (10 patients, heterozygote for the mutation with a apti' + apc ratio between 1.4 and 1.9), heparin up to 2 iu/ml and f viii activity between 1 and 250%. in the test, diluted sample is mixed with protein s deficient plasma, activated factor v, activated protein c, phospholipids and an intrinsic pathway activator. this mixture is incubated for 3 minutes. during this time, the activated protein c inactivates part of the f va. the extend of f va inactivation depends on the protein s concentration. after 3 minutes caci2 is added and the time untill clot formation is measured. the clotting time is a linear function of the protein s concentration between 10 and 140% protein s. for the three preproduction lots the difference in dotting time between 10 and 100% protein s was 43-54 seconds. this compares to 30-40 seconds typically obtained with the old test. within run precision (n= i0 on sta) is cv= 2 -7% on the basis of protein s. day to day precision (n=10 on sta) was found to be cv= 4 -11%, again calculated on the basis of protein s concentration. the cv of 11% was obtained for an avk plasma with 13% protein s; it corresponds to a standard deviation of only 1.5% in protein s. the insensitivity to interferences, in particular apc-resistence and better precision and stability are expected to improve the quality/reliability of a protein s determination. in this study we evaluated the use of hormonal contraception on the parameters protein c, protein s and pal. samples from 71 women with, without hormonal contraception and in menopause were assayed by coagulometric (protein s clotting test (behdngwerke, marburg, frg) or chromogenic methods (protein c activity test and pal reagent from behringwerke, marburg, frg) in double determination and were compared with the reference ranges. in addition thromboplastin time (thromborel s reagent) and fibrinogen (multifibrin) from behringwerke, marburg, frg, and aptt (actin fs reagent from dade corp., unterschlei6heim, frg) were determined. in women using hormonal contraceptives (p<0,01) and in menopause (p<0,05) protein s activity was significantly reduced compared to other women (<45 years) while protein c acitivity did not change. in menopausal women a higher susceptibility to thrombosis was supported by an increase of aptt (p<0,05) and fibronogen (p<0,01). while there was no change for pal, plasminogen was significantly lower in women using hormonal contraceptives and in menopause (p<0,05). we could not observe a higher turnover of coagulation and fibdnolytjc system with hormonal contraception. noteworthy was the occurence of low (<200 mg/dl) and borderline fibrinogen (max. 220 mg/dl) in 40,9% of women res. in 22,8% of women (together with borderline aptt) who had an individuell risk for arterial disease. protein s protein c fibdno~en aptt plasminog~ without hcc 109,1-+13,6 78,3-+14,1 255,0-+14,0 36, [2] [3] [4] [5] 4 24, [0] [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] 2 with hcc 85, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] 2 78, 5±12, 0 253, 8.+24, 1 35, [2] [3] [4] 8 14, 9 menopause 90, 3+97, 6 87, 4±41, 4 307, 05:57, 6 39, [8] [9] [10] 0 12, 6 hcc= hormonal contraception hemostatic parameters in a patient undergoing bone marrow and subsequent liver transplantation due to veno-occlusive disease c. salat 1, , e. holler t,3, hi. kolbl, 3, b. reinhardt l, r. pihusch 1, p. g0hring 2, s. poley 2, e. hiller 1 l=med. klinik iii, 2 = institut flit klin. chemie, klinikum grosshadern der ludwig-maximilians-universit~tt mfinchen, 3=h~tmatologikum der gsf a 40 year old patient suffering from all received allogeneic bone marrow transplantation (bmt). after an uncomplicated early posttransplant period the patient was dismissed after 4 weeks. a bilirubin rise with subsequent liver failure was observed during the following weeks. according to biopsy proven hepatic veno-occlusive disease (vod) liver transplantation was performed on day 79. unfortunately the patient died on day 140 due to aspergillosis. we monitored levels of protein c (pc) and s (ps) as well as pall during the pre-and posttranspiant period. pal1 level was normal (<43 ng/ml) during the first 4 weeks after bmt but increased with the manifestation of vod (317.5 ng/ml on day 47). it reached its peak immediately before liver transplantation (547.6 ng/ml) and returned to normal levels within the next few days. pc levels which were normal before bmt decreased prior to clinical diagnosis of vod and were normal after liver transplantation. ps levels lay within the normal range at all timepoints. vwf was elevated before bmt (240%) and remained relatively stable during the whole investigatonal period ranging from 170 to 260%. it is assumed that vod is initiated by an endothelial cell injury -possibly due to radiochemotherapy -and subsequent hypercoagulability. our results indicate that the "endothelial cell marker" vwf is not helpful in predicting vod. the kinetics of the investigated parameters underline the significance of pc and pai-1 as described by others and our group earlier, whereas ps does not seem to play a role in the pathogenesis of vod. the budd-chiari syndrome (bcs) is characterized by hepatic venous outflow obstruction that may be caused by the precipitation of a thrombus. it frequently coseggregates with other major diseases like myoloproliferative diseases or defects in the haemostatic system (antiprotein c and protein s deficiencies e.g.). only recently, the factor v leiden mutation (fvlm) has also been associated with bcs. we hypothesized that defects in the thrombo-modelling associated anticoagulant pathways (tmaap) are a major risk factor for the precipitation of bcs. we screened our cohort of 27 patients (pts) with bcs for the presence of defects in the tmaap and identified 3 pts with protein s deficiency (psd). these pts were screened for the three point mutations in exon 1 (codon-25; ins t), exon 15 (codon 636; a-->t) and in intron 10 (g-->a + 5) of the ps alpha-gene that have been demonstrated by bertina et al to coseggregate with psd. restriction enzyme analysis and confirmation-sensitive gel electrophoresis for the detection of single-base differences in doublestranded pcr-products were employed. all living family members of the indicator pts were also screened for heterogeneties in the three point mutation as described. no single abnormality in these genes despite presence of pbd in those family members was found. in addition, pts and family members were also screened for fvlm. one pt and two of his family members, in addition to psd, were subject to fvlm. the other two lots and their family members were not subject to fvlm. in contrast to the first family, despite psd, those two pts suffered from morbus crohn and acute myeloid leukaemia as risk factors for bcs. we conclude: psd is one major risk factor for the precipitation of bcs. to precipitate this disease, one additional risk factor is required. psd may be caused by genomic defects in the protein s gene other than those described by bertina. only a few publications describe a thromboembolic disease due to dramatically reduced protein s levels being associated with viral or bacterial infections, autoimmune mechanisms are suspected but the aetiopathogenesis is still under discussion. we report on a 5 year old boy who developed purpura fulminans of the left leg during varicella infection. on the fourth day of infection the disease started with pain and haemorrhagic efflorescence localized at the left taft. on admission the boy suffered from a purpura fulminans with central necrosis measuring 15x8 era. suspecting a hereditary thrombophilic disease we started therapy with protein c concentrate and recombinant tissue type plasminogen activator. the fellowing coagulation investigation showed a severe deficiency of protein s (total protein s-antigen < 5 u/ml, free antigen not measurable) in combination with factor v leiden mutation. other thrombophilie and coagulation parameters did not show deviation from normal range. after 4 weeks we saw a slight improvement of the total protein s antigen up to 50 u/ml. the free protein s antigen was still undetectable. during the following weeks the patient recovered slowly and the protein s activity and antigen normalized. because of skin necrosis thromboembolie prophylaxis was initiated with low molecular weight heparin (fragmin®, 100 ie/kgbw/die) and continued for 6 months. under this therapy there were no further thromboembolic events. these results suggested an autoimmune protein s deficiency in a patient suffering from chickenpo×. an analyses of autoantibodies at the time of diagnosis showed a slight increase of the antieardiolipin antibodies (igg 16,1 iu/ml, igm 15,1 iu/ml) which normalized during hospitalisation. we suspect an antibody to protein s probably caused by similar presented viral antigens. we suppose that autoimmune mechanism during different infections in combination with a heterzygous apc-resistance may be a potential risk factor for developing thrombotic disease. in the central nervous system mrna encoding for prothrombin and thrombin receptor is present and astroglial cells in culture process and secrete thrombin. moreover, effects of thrombin on brain cells including change of neudte outgrowth and astrocyte shape are described, but the molecular mechanisms are unclear. we investigated the effects of human <z-thrombin and a six amino acid thrombin receptor activating peptide (trap-6, sfllrn) on [ca2+]i, phosphoinositide hydrolysis and protein kinase c activation in the rat glioma c 6 cell line which is a widely used in vitro model for studying glial functions. stimulation of c 6 cells with both c{-thrombin and trap-6 resulted in transient [ca2+]i mobilization, [3h]inositol phosphate response and activation of the pkc isoenzymes c{,i],7,5, and s. these results suggest that ~-thrombin and trap-6 activate at least partially the same intraceltular signaling pathways in rat glioma c 6 cells which is an evidence for involvement of "tethered ligand" receptor in thrombin induced signaling in glioma c 6 cells. further investigations in this field are in progress including both receptor binding studies and more detailed analysis of thrombin receptor mediated signaling network and the cellular response of thrombin induced transmembranal signal transduction pathways in c 6 glioma. thrombin is a potent mitogen in vascular smooth muscle cells (smc). the mitogenic effect of bovine thrombin and human thrombin receptor-activating peptide (hutrap-14) was investigated in bovine coronary artery smooth muscle cells. confluent cells (passage 4-8) were incubated for24 h in serum-free medium with thrombin (10 nm) or trap (10-100 pm). the mitogenic response was assessed using [sh]thymidine incorporation. results demonstrated that thrombin induced a three-to fourfold increase in [3h]thymidine incorporation compared to the control, thus being as active as the potent mitogen pdgf-bb (20 ng/ml). the mitogenic effect of thrombin was dependent on its proteolytic activity. when the active site of thrombin was irreversibly inhibited by d-phe-pro-arg-chloromethylketone (ppack) no increase in [°h]thymidine incorporation was observed even at the high concentration of 250 nm ppackthrombin. thrombin-induced [3h]thymidine incorporation was significantly reduced by the reversible direct thrombin inhibitor nc£-(2naphthylsulfonylglycyl)-4-amidinophenylalanine piperidide (napap, lpm) . in contrast to thrombin trap-14 at a concentration up to 100 pm did not increase [3h]thymidine incorporation in the absence or presence of the aminopeptidase inhibitor amastatin (10 ijm). in order to demonstrate whether hutrap caused a stimulation of the receptor and signal transduction in bovine smc the activation of protein kinase c (pkc) was measured as translocation of the cytosolic to the membrane bound fraction. it was found that both thrombin (10 nm) and trap (10 pm) induced a translocation of pkc after 20 rain. in conclusion, thrembin and trap activated pkc in coronary smooth muscle cells but only thrombin acted as a mitogen. endothelial cells once stimulated with thrombin are resistant to subsequent stimulation directly after the first. but after a recovery period of about 90 min the cells are sensitised again for activation by thrombin. in our experiments this resensitisation couldn't be inhibited by actinomycin d or cycloheximide nor by the phosphatase inhibitor ocadaic acid. therefore an intracellularly pool of thrombin receptors has been proposed possibly co-localised with golgi vesicles. brefeldin a (bfa) has been used extensively to investigate the intracellular sorting of proteins because of its dramatic alteration of the structural and functional organisation of the golgi apparatus. because of this we examined the effects of bfa on the regeneration of the thrombin receptor response in human umbilical vein and artery cells. the vwf release from weibei-patade vesicles was used as physiological parameter of activation of thrombin receptors by thrombin. the addition of bfa (20 pg/ml -20 ng/ml) to endothelial cells blocked the receptor response regeneration, whereas the first stimulation was not influenced by bfa at the concentrations used. these results give further evidence that the proposed storage pool of thrombin receptors is iocalised in vesicles of the golgi apparatus. randomized comparison of double bolus reteplase (r-pa) and front-loaded alteplase (rt-pa) in patients with acute myocardial infarction (rapid ii) c. bode, rw. smalling, j. kalbfleisch, g. berg, dj. odenheimer, e. bshm, wd weaver and the rapid ii investigators. med. klinik iii, university of heidelberg, germany the study was undertaken to assess the efficacy of a double bolus regimen of 10 + 10 megaunits of reteplase, a recently developed deletion mutant of tissue-type plasminogen activator, relative to front-loaded (gusto-regimen) alteplase. there was no age limit and patients were recruited up to 12 hrs after onset of symptoms. the primary endpoint "patency at 90 minutes" was centrally assessed in a blinded fashion. infarct related coronary artery patency (timi grade 2 or 3) and complete patency (timi grade 3) at 90 min for reteplase vs. alteplase were 83.4% vs. 73.3% (p=0.03) and 59.9% vs. 45.2% (p=0.01), respectively. at 60 minutes, the incidence of both, patency and complete patency was also significantly higher in retep ase vs. aiteplase treated patients: 81.8% vs. 66.1% (p=0.03). and 51.2% vs. 37.4% (p=0.01). reteplase treated patients required fewer additional coronary interventions within the first 6 hrs of treatment (13.3% vs. 26.5%, p=0.01). there were no significant differences between reteplase vs. alteplase regimens in 35 day mortality (4.1% vs. 8.4%), severe bleedings (12.4% vs 9.7%) • or hemorrhagic stroke (1.2% vs. 1.8%). reteplase, when given as a double bolus of 10+10 mu to patients with acute myocardial infarction, achieves significantly higher rates of early patency and requires fewer acute coronary interventions than front-loaded alteplase without an apparent increased risk of complications. lysis therapy with uhsk of deep vein thrombosis (dvt) of the lower extremity commoniy results in a relatively high patency rate. in our patients, patency was achieved in approx. 70-80%. these results were supported by several studies. in dvt of the pelvic veins, however, this kind of aggressive therapy is excluded due to the risk of iatrogenically induced pulmonary embolism. on the other hand, the need for an effective therapy is evident because of the usually occtnring complete occlusion of the pelvic veins and the following severe clinical symptoms 0phlegnmsia cerulca dolens etc.). until september "95, 44 consecutive patients (16 males, mean age 41 ranging from 18 to 63 years) with dvt of pelvic veins were included in this study. after clinical and phiebographic/computer tomographic (ct) diagnosis, a temporary filter system was placed intravenously either by a wambmchial (34 pat.) access route, lransfemorally (6 pat_) or wansjngularly (4 pat.) under mdiological control. the open filter was positioned above the thrembus in the lower caval vein: 26 times an antbeor system, 11 times an anglocor and 10 times a cook filter were used. in the following three days up to 3 cycles of lysis therapy with 9 mli u of streptokinase were administered, each cycle lasted six hours. heparin was given simultanously via the filter system in therapeutic dosages in order to prolong alrit values 1.5 to 2 times the individual base line level. effective lysis was controlled by a second phlebography/ct, and the filter system removed through the introducer sheath valve. after lysis therapy all patients were put on coumarin with overlapping heparinization. successful lysis (complete recaanlisation without remaining thrombi and lysis of more than 50 per cent of the original thrombus) could be achieved in 62% (29 patients). in two cases a fatal pulmonary embolism occurred. 5 times great parts of a thrombus were captured by the filter and could be removed with the system. due to an insufficient boparin therapy we detected a newly developed thrombosis of the lower cavul vein twice after lysis. this complication could be resolved by additional administration of streptokinase. in nearly all cases we found extensive hematomas on the catheter insertion sites. 4 patients with transfemoral applications had to be transfitsed with red packed cells due to retroperitoneal hematoma. one filter system was broken after use. lysis of dvt of the pelvic veins with uhsk and with the protection of a removable temporary filter system situated in the lower caval vein enables us to carry out an effective therapy. the filter systems, however, have to be ameliorated in order to better prevent fatal pulmonary embolism. heparin should be administered at least in the remainder of the day of lysis. the insertion site should be the enbital vein because compression for bleeding complication is easily feasible at this location. we report about seven of our patients (pts.) with bcs treated by fibrinolytic therapy (5 female, 2 male, age 27, 6 years + 8). only one case was of unknown origin while in four pts. myeloproliferative syndromes, in one protein c deficiency and in another one morbus behcet was detected in five pts. chronical and in two acute bcs was diagnosed. as complications were found: in three pts• thrombosis of vena iv.) cava, in one ofv. porta and v. lienalis, in one more of v• porta and v• cava. we treated just one patient with urokinase (3 x 800.000 iu/d for 18 days). four pts. were treated with rt-pa in concentrations of 25 to 50 mg/d (for a period of up to 14 days) and continuous heparin infusion. high doses of rt-pa (80 to 120 mg/d for one or four days) were given in one case of acute and one of chronic bcs. partial (after high-dose rt-pa nearly complete) recanalisation of thrombotic hepaticel veins was only achieved in the two acute cases of bcs. no significant recanalisation was proven in hepatical veins of chronic bcs. three times complete fibrinolysis of v. cava thrombosis (two after highdose therapy) and one partial lysis could be attained. in one patient partial lysis of v. porta and in another one of v. lienalis was found. furthermore complete recanatisation of v. porta followed local rt-pa lysis during surgical intervention. in our two pts. with acute bcs significant improvement of hepatical venous flow could be measured after fibrinolytic therapy• in chronic bcs hepatical venous flow did not improve while the complication of v. cava thrombosis could eventually be treated successfully. probably due to fibrinolytic therapy one case of esophageal varix bleeding occurred. the benefit of flbrinolytic therapy of bcs seems to be influenced by the dur~ion of the disease• according to our results fibrinolytic therapy should be considered in cases of acute bcs as well as in the above mentioned thrombotic complications• recent studies suggest that transport of thrombolytic agents through the clot can play a major role in determining the reperfusion time needed for thrombolysis. as the mechanisms of this phenomena are rather unclear we studied fibrin (0.4-1.5 ~tm) clot lysis by plasmin (30 nm) presented outside or inside the clot in purified system (tris-hc1 buffer, 37°c). the clots was formed by addition of cac12 (30 mm), thromboplastin (1 u/ml) and human thrombin (6 nih/ml)t the lysis degree was determined either by counting of radiolabelled fibrin degradation products or by time of total lysis. obtained kinetic parameters of this reaction: kest=99.4 rain -1, km=i.9 ~tm (plasmin inside clot); kcat=l.36 rain -1, km=i.3 lzm (plasmin outside clot). this data indicate that eatalytie efficiency of plasmin from inside the clot is 50-fold higher than from outside as measured from kcat/km. furthermore we studied plasma clot (0.25 ml) lysis in original plasma (3 ml) containing sk (5-100 nm) and seu-pa (5-100 rim) in the final concentrations from inside or outside the clot. the results are presented in the the similar results have been got when the fibrin clots were formed with batroxobin instead of thrombin. it may be concluded that fibfinolysis is a surface-dependent process. it's likely that the higher level of plasminogen activators in blood would prevent thrombus formation, while physiological clots would stay resistant. for the improvement of the thrombolytic therapy in children more clinical data are needed. therefore we report on our experiences with rt-pa presenting the clinical course in 11 patients (age range: 7 days to 16 years) with venous thrombosis (n=9) and purpura fulminans by meningococcosis (n=2). the patients were treated with rt-pa (actilyse r) for 10,5 hours to 13 days. the venous thromboses were localized in lilac/femoral veins (n=4), brachiocephalic/jugular/subclavian veins (n=3) and the cava superior vein (n=l). central venous catheters were causative in 2 cases. the dosage range was between 0.2 to 0.5 mg/kg for the initial dose and 0.8 to 2.0 mg/kg/d for the subsequent continous infusion. ten patients received simultaneously heparin. complete clot dissolution occured in 6 cases, partial clot dissolution in 1 patient. in 4 patients with a history of the first clinical signs of thrombosis of 3 weeks or more the lysis was not successful. concerning the side effects only small bleeding episodes on former venipuncture sites were observed in 5 patients. systemic effects such as the decrease of the plasma flbrinogen concentration were rare too. in conclusion, the thrombelysis with rt-pa presents an effective and safe therapy in children at: the rt-pa dosage used. in case of a history of the thrombotic event of more than 3 weeks a thrombotytic therapy should not be carried out. d.dimer is widely used in clinical situations associated with thrombotic diseases. the most popular application is the exclusion diagnosis of deep-veinthrombosis and pulmonary-embolism and the assay is performed in emergency. d.dimer is also frequently measured for evaluating the thrombotic risk in the post-operative period. quantitative, flexible and sensitive assays are required. we have developed a new automatic method which works on the coagulation walk-away instrument, sta. this assay was developed with two monocloual antibodies specific for d.dimer coated on microlatex particles and it uses an immuno-turbidimetric technology. absorbance is measured at 540 nm and is a direct relationship of d.dimer concentrations. the assay works with 50 ~tl undiluted plasma and is performed in less than 10 minutes. the dynamic range is from 0.2 to 16 [~g/ml and no prozone effect is observed up to concentrations higher than 100 ~tg/ml. the detection threshold is of 0.2 [lg/ml. the intra-assay reproducibility is below 5% and the inter-assay reproducibility is below 6%. recovery studies of purified d.dimer added to normal plasma were between 94 and 105%. no interference of heparin, bflirubine, lipids or of rheumatoid factor up to concentrations of 100 u/ml is observed. there is no effect of high fibrinogen concentrations (> 10 g/l). when compared with conventional elisa techniques (asserachrom ddi), the assay demonstrated a correlation coefficient of 0.97 on 131 samples from normal individuals and hospitalised patients with elevated d.dimer concentrations. slope was of 0.97 and intercept was of -0.07. this new assay offers a full flexibility for individual testing as the calibration curve is stable for at least one week on the instrument. it is then well adapted for all the applications of d.dimer measurements in coagulation laboratories. 16 children between an age of 3 days and 11 months ( median 6 weeks ) with thrombotic or embolic occlusion of major vessels were treated with rt-pa for thrombolysis. the affected vessels were both sided renal veins or one sided renal vein and v. cava inf. in 8 cases, the v. cava superior in 3, the v. cava inf. plus renal veins plus aorta in 1, the left ventdcle in 1, the aorta in 1, the a. femoralis in 1 and the v. portae in 1 case. 10 out of 16 occlusions were associated with an indwelling catheter. underlying dieseases were sepsis (4), prematudty (3), vitiurn (2), asphyxia (1), short bowel syndrome (1), hus (1), diabetes (1), cmv (1), exsiccosis (1) and m. hirschsprung (1). thrombolysis was performed with an bolus of rt-pa (0.1-0.2 mg/kg) followed by continuous infusion (0.8-2.4 (-9) mg/kg/24h, median 1.8 mg/kg/24h). low dose hepadn (100 ie/kg/24h) was given dudng full dose hepadn (aptt 1,5-2 times normal) after the thrombolysis. in 5 pts. rt-pa was administered locally through the catheter and in 11 cases systemically. in 13 patients the vessels could be recanalised completely, in 2 partially, in 1 patient the therapy had to be discontinued. in 2 vessels a reocclusion occurred. bleedings were noted in three patients, all from recent venous puncture sites. the results encouraged us to start a multi-canter trial which has been approved by the ethical committee and is open for recrural. the aim is to compare efficacy and safety of rt-pa with urokinase, the only recommended standard in the management of critical major vessel obstruction in newborns and infants. the design is a randomised, notblinded trial with a cross-over option after three days in cases without success. study end points are recanalisations, major bleedings and number of cross-overs. inclusion criteria are age under 1 year, lifethreatening vessel obstruction, age of thrombus up to 10 days, no precaeding fibdnolytic therapy. exclusion cdteda are cerebral hemorrage, pedventricular leukomalacia, surgery dudng the last 7 days and cns injuries during the last 2 months. although our knowledge on inherited thrombotic coagulation disorders has greatly expanded within the last years, there are still man}, patients with recurrent venous thrombosis in whom no obvious predasposition can be identified.thus we decided to include also so-called rare defects associated with thrombosis in our routine thrombophilia screening programme, such as fxii deficiency. fxii is an important element m the intrinsic pathway of fibrinolysis and there is evidence for an insufficient fibrinolytic activity in fxii deficient pts..up to date only few and controversial data exist about the frequency of fxii deficiency in pts. with thrombophilia. cons~uently the aim of our study was to evaluate the association between fxii deficiency and juvenile venous thrombosis in a great population. patients and methods: 1554 pts. (851 female, 703 male, aged i to 61 ys, median age 38.2 ys) with venous thromboembolism before the age of 45 ys were studied. one-stage clotting activity assay of fxii (fxii:c) was performed on acl using fxii deficient plasma from instrumentation laborato~. fxii antigen concentration (fxii:ag) was measured by electroimmundiffusion using reagents from behfingwerke, enzym research respectively. the normal ranges are tl~. routine reference values obtained m our labratory from 80 healthy subjects (40 males, 40 females, median age 26.2 ys); 95% range: fxii:c 53-135%, fxii:ag 57-132%). results: 122/1554 pts.were classified as fxi1 deficient (f 60, m 62), giving a prevalence of 7.8%. severe fxii deficiencies with fxii:c below 1% were observed in 7 pts..ll5 pts= proved to have moderate fxii deficiency with fxihc ranging lrom 2 to 51% and fxii:ag ranging from 1 to 53%. in none of them inherited deficiencies of other well established thrombophila risk factors could be detected. none of the fxii deficient pts. had positive lupus anticoagulant tests. familial fxii deficiency was found m 9 cases. discussion and conclusion: the precedences of fxii deficiency amongpts, with venous thromboembolism was previously described to be 7.5-10%. supporting these data, we have shown a praevalence of fxii deficiency of 7.8 %. in comparison to the frequency of other well established thrombophila risk factors we consequently have observed a relatively high prevalence of fxii deficiency m our study group.these data, from the largest such study reported, strongly indicate that fxii deficiency may not be a rare deficiency and may be more frequently associated with thrombosis than currently suspected. we describe a family with an exceptionally rare, i.e. plasminogen, deficiency, combined with subnormal activities of coagulation factor xii (hageman factor). the first thromboembolic event, pulmonary embolism in the proposita was diagnosed at age 35. since that time, 'spontaneous' venous thromboembolic events verified by phlebography and perfusion/ventilation lung scan recurred once every year despite oral coumarin therapy, whose intensity varied over an exceptionally wide range despite tight control the patient was repeatedly given succesful thrombolytic therapy with streptokinase or recombinant tissue plasminogen activator. her plasma plasminogen chromogenic activity was 51-59 % compared to a normal plasma pool (reference range 70-130 %), plasminogen antigen was diminished to the same extent. the patient's factor xii exhibited only 28-55 % activity in a factor-deficient plasma assay as compared to a normal plasma pool. other known risk factors for recurrent venous thromboembolism were not present : no evidence of malignancy, no obvious precipitating events, normal values of antithrombin iii, protein c, protein s, fihrinogen, thrombin time, platelets, lupus-like anticoagulant, aptt prolongation after addition of activated protein c. the proposita's mother had died at age 60 from pulmonary embolisnt no coagulation studies are available. the proposita's sister was first diagnosed deep leg vein thrombosis at age 17, since that time recurrent episodes of venous thromboembolism have been diagnosed also in an other hospital. this sister's plasminogen activity was 120%, but factor xii activity was reduced to 55 %. three brothers of the proposita were examined, too, all in their 3rd decade of life. none of them recalled symptoms of or treatment for thromboembolic disease. in one brother, factor xii activity was normal (100-105 %), but plasminogen only about 50 %. in the 2nd brother, factor xii was very variable (64, 42 and 94 %), plasminogen was in the lower normal range, in the 3rd brother, factor xii was about 50 % (repeatedly), plasminogen was normal. current knowledge about the risk of thromboembolism with both enzymes is limited, the optimal management remains controversial. msrgit serbsn,maria cucuruz,dan madras,carmen petrescu, natalie rosiu,rodica costa iii rd psediatric clintc,universtt v of medicine, the unsatisfactory efficiency of entihepetitis b vaccination in our haemsphiliscs suggested the control of the immune status in 52 hiv negative patients,by establishing through flowcitomstrie with monoclonsl antibodies the lymphocyte subsets (cd3,cd4,cds,cd&/cd8 ratio and cd19) and by seric tmmunoglobulins levels; the immunological parameters have been correlated with the serological markers of hepatitis infections (hay, hbv,hcv ebd hdv) as well as on dependence with the treatment (blood,plasma,crysprecipitate,fector viii/ix concentrate) and the quantity of their consumption (ui/k9 weight/yesr).the interpretation of the results pointed out • significant lower level of cd3,cd& (p<o,o01) st the group of hsemophiliscs treated with blood,plasma and cryopraclpitate; sdditionsly,in this group of patients it was found s significant drop of the cd19 counts (p< 0,001) without an immunoglobulin-deficiency. the same behaviour was found at the group of haemophiliacs carriers of hb~ and hcv markers.the superpositisn of hepatitis infections on the treatment with blood and native blood products in most of the cases has let unanswered the question related to the mechanism of the immunodeficiency: the hepatitis b or c infections or the negative impact of the itarative important alloantigenlc load ? illumination with l~tm methylenblue (mb) is used for inactivation of enveloped viruses in human plasma. via a photooxidative mechanism viral structures as well as plasmaproteins especially fibrinogen can be damaged. fibrinogen is an important mediator in the cell-cell interaction of platelets. the four rgd sequences (arg-gly-asp) of fibrinogen can be recognized by the activated fibrinogenreceptor gphb/iiia . after binding to gpiib/iiia one molecule fibdnogen can link two platelets. the aim of this study was to investigate the influence of mb treatment on the fibrinogen activity in human plasma. furthermore we were interested to demonstrate if altered fibrinogen inhibit platelet aggregation by blocking the gpiib/]iia receptor. human plasma was treated with 1-50~m mb. after illumination fibdnogen was isolated by fl-alanin precipitation. purified fibdnogen was analysed by one-and twodimensional gelelectrophoresis. influence of modified fibrinogen on platetet aggregation was measured with gelfiltrated platelets. possible receptorblockade were examined in an 125j-fibrinogen competition assay. densitometric scanning of reduced sds-pages showed a decrease of the a-subunit as well as an increase of high molecular aggregates with increased mb concentrations. this effect was not detectable for fibrinogen isolated from plasma with 1bm mb. this results were confirmed by two-dimensional gelelectrophoresis. ibm mb treatment showed no receptor blockade in aggrement with normal platelet aggregation after stimulation with adp and fibrinogen. proposed validation of a quantitive quality-assured pcr method f. dorner, j. eibl, g. zerlauth immuno ag we have developed a highly sensitive and reliable quantitative method, based on the polymerase chain reaction (pcr) and on the reverse transcriptase polymerase chain reaction (rt-pcr), to screen for the nucleic acids of human immunodeficiency virus type 1 (hiv-i), hepatitis b virus (hbv) and hepatitis c virus (hcv) in plasma and plasma products. the detection of pcr products is carried out by laser induced fluorescence, the procedure is therefore termed "laser induced fluorescence pcr" (lif-pcr). the procedure allows the precise quantitation of genome equivalents due to the use of two calibrators that are co-amplified by the same set of primers as the target template in one and the same test tube. a high degree of reliability is achieved by co-precipitation of the extract, co-amplification and co-determination of the calibrators together with the genome equivalents to be determined. in this report we present validation data for the lif-pcr and discuss them in the light of the recently published ich-guidelines on the validation of analytical procedures. taking into account the pecularitles of the replacement therapy of haemcphiliacs in romania the study aimed at evaluating the dimension and profile of blood-born infections in these patients; 142 hsemophlllacs,elasslfled in 3 groups: a-ulth no exposure to blood or bloodproducts, b-treated ~ith factor-concentrates and c-ulth plasma,cryoprecipitate + factor-concentrates in their therapy have been investigated (elisa method) for serological markers of hepatitis a,b,c end d, cvtomegalovirus (cmv) and hiv i and hiu 9 infection. the infections profile of our haemophiliacs~hes some peculsrities. the hepatitis infection is the most extended,lnvolving more than 75% of the patients. the frequency is for hepatitis -bz,z~ , u -"/~,~ , g -bb,7% end o -24% : the multiple infection was diagnosed in more than 50% of cases. the prevalence uas significantly hlgher in the group c. we studied blood count and relevant coagulation system parameters of autologous blood donors. indications for autologous blood donation were divided in 4 categories: a group of diverse (a, n=13), reduction mammoplasty (b, n:5), prostatectomy (c, n=io) and total hip replacement (d, n=74). we performed bleod count (bc), thromboplastin time (tt), partial thromboplastin time (aptt), fibrinogen (fib), f vhi act, vwf rco, vwf ag, f xiii, at iii, d-dimer-fragments (dd) and fibrinmonomers (fm). all parameters were analyzed as initial value, bc, tt, aptt, fib, f xiii and at iii were measured additionally at each donation. the percentage of initial pathologic data was (1) were included in the study. the inclusion criterittm was: an endegen(y.~ red cell reserve (ercr) of less than 400 ml in men and less than 540 ml in women. ercr is the volume of blood that can be withdrawn up to tim hematocrit of 0.34. the selected dosages of rhepo (200, 400 and 800 iu/kg body weight biweekly over 4 weeks) were based on the calculated blood volume and the ercr. a unit of whole blood, separated into an autologens red cell concentrate and one unit of fresh frozen plasma, was collected when ]mnoglobin concentration was at least 11.5g/100 ml (or hematocrit was at least 0.34) up to an autologons predeposit of 800 ml red cell volume (rcv). additionaly, platelet concentrations and serum ferritin levels were measured~ results: at the initial clinical examination the ercr was 333 ml in women and 307 in men. this resulted in a weekly dosage of 2x32.000 iu rlaepo in women and 2x25.000 iu rhepo in men. 7 patients donated 5 red cell each (858 ml rcv on an average) within 19.1 days. the targeted rcv could not be reached in 3 patients because the operation was reschednled. the collection from one patient had to be aborted because of angina pcetoris complains. tic following side-cffczts occurred: episodic hypcrtejlsion (2 tim~), chest pain (5), fafigne (8), inflnenza-lihe syndrome (3). platelet count increased from initially 203 4-68 x 10'//d (i00 %) during therapy to a maximum level of 260 + 93 10v~ (128 %) without any clinical signs of thrombeembolism. in the control group (autologous blood donation weekly 3 or 4 times without erythrupoiedn stimulation, n=12) platelets increased from initially 230 __+ 73 10'/fi (100%) to 255 + 71 10v#i (110%) i1]aximlzm colldttsions: the adlninist/'ation of lhepo in the above described manner to autologous donors undergoing heart surgery is well tolerated. it allows to collect a sufficient number of autologous blood units and the preparation of a predeposit at short notice. essential thrombocythemia is a chronic myeloproliferative disease with a benign clinical course that is mainly complicated by microcirculatory or thrombotic vascular disorders. it is at present uncertain which patients need (lifelong) therapy and whether cytoreduction is necessary. we retrospectively evaluated vascular complications in 128 patients (33 men, 95 women) with a median age of 61 years in relation to therapy. three major treatment groups were analysed: 17 patients treated with acetylsalicylic acid (ass) alone, 29 patients given alkylating agents, and 62 patients treated with interferon alpha (ifn). there was no significant number of patients treated with hydroxyurea. ifn-treated patients were younger (median age 54 years) than patients in the other groups, but platelet counts before therapy were similar (median 0.84-1.13 x 1012/i). before therapy, 47% of patients treated with ass, 45% treated with alkylating drugs, and 32% treated with ifn had bleeding events, microcirculatory or thrombotic complications. dudng therapy, median platelet count was 0.9 x 1012/i in patients on ass, 0.68 x 1012/i in patients treated with alkylants, and 0.48 x 1012/i in ifn-teated patients. the rate of patients with complications during therapy was 18% (9.5%/patient year) for ass, 14% (6.3%/patient year) for patients on alkylating drugs, and 15% (6.2°/dpatient year) for ifn-treated patients. we conclude that all three treatment regimes seemed to have a beneficial effect on the incidence of vascular complications in essential thrombocythemia. the results give a rough estimate of complication rates to be expected and may form the basis for randomized clinical trials. adhesion of activated platelets to leukocyte* has been shown to be mediated by at least two molecules on the platelet surface: p-selectin (cd62) and fibrinogen (fg) bound to the gp iib/iiia complex. interaction of platelets with teukocytes via cd62 is believed to stimulate the production of reactive oxygen species (ros) in leukocyte*. the role of platetet-bound fg as a signalling molecule in platelet-leukocyte interaction is controversially discussed: stimulatory as well as inhibitory effects on leukocyte ros production has been described. in the present study we measured ros production in undiluted samples of citrated whole blood (wb) as well as in suspensions of erythrocytes and leukocytes in platelet-rich plasma (platelet-rich wb) or platelet-poor plasma (ptatelet-poor wb). ros production was determined by measuring luminol-enhanced chemiluminescence (cl). stirring wb samples at 1,000 rpm for 10 min resulted in an activation of leukocyte* (increase of cl, upregulation of cd1 lb) and platelets (platelet aggregation, upregulation of cd62). stirring-induced cl was significantly higher in platelet-poor wb when compared to platelet-rich wb. higher cl in plateletpoor wb than in platelet-rich wb was also found when adp (moderate stimulation of platelets and leukocyte*) or fmlp (strong stimulation of leukocyte*) but not when pma (strong platelet and leukocyte stimulation) was added. dextran sulphate that is known to block cd62-mediated interaction ofplatelets with leukocyte* caused a dose-dependent inhibition of stirring-induced cl in wb samples. in contrast, when binding of fg to the platelet surface was blocked by rgds or the fg receptor antagonist gr144053, stirring-induced cl, but not cl in unstirred samples, was significantly enhanced. gr144053 also reduced adhesion of platelets to neutrophils, but did not affect cd62 exposure. the results indicate that interaction of platelets with leukocyte* in whole blood via cd62 and fg may have opposite effects on leukocyte ros production: stimulation by cd62 and inhibition by platelet-bound fibrinogen. late reocclusive processes represent one of the most commonly seen serious adverse events after coronary angioplasty. the pathophysiology of both of these processes is multifunctional and represens complex time dependent pathophysiologic events involving both the humeral and cellular processes. to test the effects of low molecular weight heparin (certoparin, sandoz ag, numberg germany) against late occlusive processes, four groups (groups a-d) of rats (7-10) were anesthetized and the external common carotid arteries were exposed. all groups received a 200 u/kg of unfraetionated heparin through the femoral vein. a 2f balloon emboleetomy catheter was inserted into the left carotid artery and passed throug the left common carotid artery leading to the aortic arck vascdar injury was induced by repeatedly (x3) inflating the catheter balloon and withdrawing it to the bifurcation. following this procedure, two hours after, equlgravimetric amounts of various agents were administered via sc route to each of the individual groups. group a was administered with certoparin od for 21 days. group b received a lower low molecular weight heprin (mr 3.4 kda). group c received unfraetionated heparin and group d served as a control. on day 21, perfusion fixation was performed on all animals. the injured and contralateral carotid arteries were excised for histelogic examinatior~ in comparison to the saline control, varying degrees of the inhibition of myointimal proliferation following arterial injury was noted in all groups. however, the degree of inhibition followed the order: heparin<certopann<llmwh_ in contrast to heparin treated group (c), the other groups a, b, and d did not exhibit any hemorrhagic lesions. however, degree of perivascular fibrosis, hemorrhage and inflammation were observed at the infusion site. in comparison to heparin, certoparin and its lower low molecular weight derivatives exhibit better efficacy in reducing balloon angioplasty induced restenosis. the observed efficacy of these agents may be due to better bioavailability and sustained duration of action. in the present paper the binding of heparin to granulocytes of human and animal species was analyzed and compared with the binding to monocytes and lymphocytes. a low-molecularmass heparin (lmmh) was covalently bound to tyramine (tyr) and subsequently tagged with fluorescein-5-isothiocyanate (fitc) by endpoint attachmenc. binding to leukocytes was quantified using flow cytometry analysis. the leukocyte populations were identified by their light scatter properties in man by cd4, cd13, and cd14 antibodies, in mice by cd 4 and gr 1 antibodies, and in rat by cd 45, ed 9, and rp 3 antibodies. granulocytes, monocytes and lymphocytes of all species bound dose-dependently lmm-heparin. the binding of lmmh-tyr-fitc ~to granulocytes was higher in human, rat, rabbit, dog, and pig as compared to monocytes and lymphocytes. mouse and sheep granulocytes did not bind more heparin than monocytes or lymphocytes. binding of lmmh-tyr-fitc was reversible in the presence of unlabeled heparin or lmmh. more than 99% of human, rat, rabbit, dog and sheep granulocyte populations could be'distinguished from monocytes and lymphocytes by their fluorescence intensity after incubation with lmmh-tyr-fitc. this separation was not obtained for mouse and pig granulocytes. the data present evidence of a specific heparin binding to granulocytes of many species and indicate the significance of fluorescent labeled lmm-heparin for biological investigations. background and purpose: ptca is an effective treatment for restoring coronary artery pat~cy; however, acute thrombotic rencclusion is a drawback of the procedure. currem prevention of rencclusion relies on antiplatelet agmts (aspirin) and antithrombin agents 0aeparin). in the present study we examined the time course of changes in blood coagulation and fibrinolytic function following ptca in patients with ischaemic heart disease. 20 patients were randomly assigned to receive either standard therapy (250 nag aspirin p.o.; 5.000 iu hepafin i.e. bolus, and 5-10.000 iu heparin intra-proeadurally)(n=10), or adjunctive therapy (aspirin, hepann; 100 mg sodium pmtosan polysulphate, spp intra-cortmarily and subcutaneously) (n=10). the sensitive markers of thrombin activation (thrombin-amithrombin iii complex ('rat), prothrombin fragmem f 1+2 (pl+2), d-dimer) and of fibrinolytic activation (plasmin-antiplasmin complex (pap), tissue type plasminogan activator (t-pa) and plasminogen activator inhibitor (pal)-i activities) were measured before, immediately after, and 6 hs after ptca. results: we found elevated mean levels of tat and fl+2 in both groups during the procedure, while plasma coneentrafiuns of d-dimer and pap remained in normal range. activity of pai-i was not elevated compared to normal comrol. the t-pa activity obtained immediately post-ptca showed significant increase in patients given spp. primary angiologic success was achieved in 80% of both groups. one patient in each group had recurrent ischaemia and emergency interventions (angioplasty, stent, bypass surgery) were performed. one patient receiving adjunctive therapy died in cardiogenic shock within 24hs arer ptca. major bleeding was observed in one patient given spp. conclusion: no significant intracoronary haemostatic activation occurred in the majority of patients during and up to 6hs after ptca. treatment with spp was associated with enhanced fibrinolytic activity. the benefits and risks of adding spp to heparm and aspirin should be evaluated by further studies, in july 19,¢1 the paul ehdich institute became responsible for quality and safety of bt~d and blood products. since ~his time, batch m~se of ppsb and factor ix prapamtions were carded out regularly, whereas ihe obligatop i testlng of all other products derived frem pooled plasma will be starling in janualy 1996. the european pharmacopoeia does .or contain directions for the quality control of ppsb preparations. therefore, ifua ~ coneenning factor ix clotting concentrates are applied, ircludmg the non activated ptt and the classical tkrombln test these m~ods are not su'rlable for reliable exclusion of ituombogenk~b/ ,of pp,sfl, because vlla actlvry is not d~. a hllhedo unsolved problem is the high sensitivity of file classical thrombin test often not correlating with ~ data of thrombogenlci~. in ordm td es~abl~ an effective quality conlrd protocol, different methods for the qual~ative detennlna~on of ila, vlla. ixa, and xa (chrornogenic, fluorege~c clolfing reactions) wera instaued. in agreement with the [deralure, considerable differences were found among the ppsb pr~ons registered in germany. cedain amounts of activated do~ng racers may be well tole~ted in redp/ents. ~no are in a steady state condition. however, in severely ill patienls, eg. post operatively, tissue factor may be expressed which pofenllatesthe dsk of ~aclor vlta. in the case of a certain ppsb preparation hlgh ¢onceatratio~ of acthrated cto~ng factors, especially ~vated factor vii, cotmlaled with fatal comlffcal~ns, evaluating ~ expe~ concenlralbn limits for adjvated prothrombin co~np~.x factors as prerequisite for ~e batch r~ease have to be applied. aprotinin is used as a hemostatic agent in cardiopulmonary bypass surgery and other indications where hemostatic compromise results in hemorrhagic manifestations. the mechanism by which this agent produces hemostatic effects is not known, however, inhibition of serine proteasas such as plasmin, kallikrein and dastase and modulataion of platelet receptors have been considered as possible target sites. to determine the effect of aprotinin on tissue factor mediated activation of human platelets, whole blood samples were drawn at baseline and 24 hours aider aspirin (650 mg po) ingestion (n=5). blood samples were supplemented with saline and aprotinin (40 ug/ml) and tissue factor activation was studied at 200 pg/ral. platelet activation was investigated using gtycoprotein ffla and gmp 140 specific antibodies on a flow cytometer (epic coulter). tissue factor was found to produce the activation of both the baseline and asp' "mnized platelets. aprotinirt augmented the tissue factor activation of platelets in both the control and aspirin supplemented groups. however, the relative increase in the aspifinized group was much monger (25% of the baseline). these results suggest that aprotinin augments the tissue factor mediated activation of platelets in both the normal and aspirinized individuals. while these effects may result in hemostatic restoration in aspirin compromised patients, in normal individuals the clinical implications of this effect remain to be clarified. , 10-20 (group 2) or >20 years (group 3) duration. anticoagulated whole blood was incubated with fluorescent antibodies to gpib and gmp-140 (two colour method) and analyzed with a flow cytometer. thrombomodulin, f1+2, protein s, 13-thromboglobulin were measured according to standard procedures. results: surface expression of gmp-140 was not different in groups 1 to 3, however, there was a tendency to higher acitvation in group 1 (<10 years iddm). results for thrombomodulin, f1+2, protein s, 13-thromboglobulin will also be presented. conclusion: though it did not reach statistical significance, platelet acitvation seems to be more important during early diabetes. this wilt be correlated with endothelial and plasmatic activation markers. in our clinic four patients with hiv-related thrombocytopenia were treated with a lot of gammagard (93f21abllf), which later turned out to be hcv contaminated. before infusion all patients were negative for hcv antibodies and hcv rna. 2 to 8 months after infusion 2/4 patients, who suffered from arc at the time of hcv infection with cd4 counts >100/pl, seroconverted, whereas in the two other patients, who suffered from aids with cd4 counts below 100/pl, there was no seroconversion. in all cases hcv rna was found. genotyping with inno-lipa (innogenetice) showed hcv genotype l(b) in all patients. liver enzymes and hcv rna copies were measured repeatedly over a period of one year after infection. the 2 patients with arc showed a strong increase of hcv rna titre during the first 3 to 4 months after infection, followed by a rapid decrease within the next months. in the patients with aids hcv rna copies increased moderately within the first 4 to 6 months, followed by a slow decrease. elevation of liver enzymes was mild in the aids patients and seems to be independent from the hcv rna titre. in the arc patients liver enzymes changed parallel to hcv rna titers with a delay of 2 to 3 months. the course of hiv infection was only slightly influenced by the acute hepatitis c as measured by cd4 counts, i%2microglobulin and hiv rna copies. introduction:mechanisms underlying ischemia/reperfusion injury have been thoumughly investigated in experimental models. leucocytes appear to play a main role through production of cytokines and overexpresssion of adhesion molecules. in experimental animals, administration of monocional antibodies (mab) recognizing cd18 can reduce organ injury following ischemia/repedusion. no data, however, have been reported concerning clinical ischemia situations. patients and methods:we investigated expression of cdt8, cd1 la, cdf l b and cd1 lc in granulocytes, monocytes and lymphocytes from peripheral blood of five patients undergoing elective hand surgery. the tourniquet was applied on the upper arm and heparinized samples from cubital veins were obtained before and at the end of ischemia. control samples were drawn from the nonischemic contralataral arm with the same timing, duration ot ischemia ranged between sixty and one hundred minutes (80~16). whole blood samples were incubated with specific, fluorochmme labelled antibodies and analyzed by fluorocytometry (facscan, becton dickinson, san jose, ca). mean fluorescence intensity (mfi), quantitatively reflecting surface expression of the indicated markers was evaluated for the individual cell populations. data were compared by the paired student's t-test, p<0,05 was evaluated as significant. results:mfi for all markers was comparable in all cell populations in samples obtained before ischemia from both arms. in contrast, expression of cd18 was significantly enhanced in granulocytes (321_+50 vs. 189_+38), monocytes (653-+54 vs. 426+122) and lymphocytes (299_+45 vs. 228-+36) from samples derived from the ischamic arm, as compared with the nonischemic arm, as measured at end of ischemia. at the same time, an increase of cdf lb on granulocytes (500~_342 vs. 213+150) and monocytes (533+359 vs.237-+206) but not on lymphocytes was found, no modifications of cdlta and cdttc expression could be observed. there was no correlation between duration of ischemia and quantitative expression of these markers, conclusions:our data indicate that relatively short ischemia periods induce an increased expression of ~2" integrins adhesion molecules on leucocytes. these results suggest, at close similarity with findings from expodmental models, that overexpression of adhesion molecules might play an important role in the induction of ischemia/reperfusion injury, in humans. in patients suffering from chronic inflammatory bowel diseases, such as morbus crohn and colitis ulcerosa, we observe massive, sometimes barely staunchable bleedings. hereby, the deficiency of coagulation factors, especially of factor xiii in plasma is established. ttowever the influence of factor xiii on the pathomechanism of the underlying disease is still under discussion. therefore we studied the f xiii content in the intestinal mucosa. an immunohistochemicat method was developed using commercially available antibodies against f xiii subunit-a, the detection of mucosal factor xiii depends on the amount of chromogen bound to the antibody-horseradish-peroxidase complex. with this method, it is possible to locate but not to quantify f xili in the intestinal tissue. therefore we developed an elisa-metbod in homogenized intestinal tissue, using commercially available antibodies. its precision was validated using a standard curve with commercially available factor xiii preparations (fibrogemmin®). the detection limit of this method is > 0.05 i.u. f xiii/ml of tissue solution. freezed dried intestinal tissue (lmg) was homogenized in 1 ml buffer using a potter. specimens of the large bowel revealed f xiii values of 0,21 + 0,0038 i.u. (x __+ sd), tissue solution. with this method it is possible to quantify tissue-bound faxtor xiii. studies are in progress to elucidate the content of f xiii in the intestine of patient's suffering from infammatory bowel diseases in order to contribute data to the pathomechanisms of f xiii deficiency. in a previous double-blind, controlled trial we were able to show that aprotinin administration has significantly contributed to reduce periand postoperative bleeding complications without increasing the risk of thromboembotic complications. the question arises whether this beneficial effect may be associated with its effects on intraoperative fibrinolysis. therefore, 20 patients were treated with or without aprotinin (2 million kiu loading dose over 15 minutes followed by 500,000 kiu per hour), and citrated blood samples were obtained at the following time points: before operation, after induction of the anesthesia, at the beginning of operation, intraoperatively when the femur shaft was implanted, and 24 hours postoperatively. the determinations of plasmin/antiplasmin-complexes, d-dimers, thrombin/antithrombin iii-complexes, and prothrombinfragments 1 +2 were performed by means of test kits from behring, germany (enzygnostrpap micro, enzygnost r d-dimer testkit, enzygnost r tat micro and enzygnost r f 1 +2 respectively). -all markers of activated fibrinolysis and blood coagulation were significantly increased in the groups with and without aprotinin treatment, the highest activities to be seen when the femur shaft was implanted. however, the values of pap and d-directs of the aprotinin group were below the values of the control group until the end of operation. the markers of activated coagulation showed the opposite effect, however the differences between the two groups were not significant. as expected, the aptt was significantly prolonged in the aprotiningroup. the aprotinin treatment was also associated with a significantly lower blood loss in these patients. -concluding it can be said it is not clear whether the blood saving effect of aprotinin may be exclusively attributed to its antiplasmin activity since the differences of the fibrinolysis parameters were not statistically significant. further blood samples should be analysed between the implantation of the femur shaft and the end of operation. in our laboratory large amounts of human prothrombin are required (30-50 mg/week). as we try to produce meizothrombin and meizothrombin-des-fragment-1 from human prothrombin and to apply it as an antidote for hirudin, the classical adsorption to barium sulphate or aluminum hydroxide from human plasma cannot be used. commercially available human prothrombin is expensive and of an unacceptable quality for our applications. in most of these batches we found small amounts of factor x and prothrombin activation products. we now developed a procedure to isolate prothrombin from "prothrombin complex concentrates" (ppsb-250-bulk, drk-blutspendedienst nds.). the concentrate also contains fac-tor vii, factor ix, and factor x. the prothrombin had to be separated from these factors. the concentrate we used contained amounts of other proteins and activation products of prothrombin (e.g. prethrombin-1) as well. for the preparation of prothrombin from ppsb we used anion exchangechromatography (resource-q ®) on an fplc ®. we applied dissolved ppsb directly or after buffer exchange on sephadex g-25 onto the column at room temperature. the prothrombin was eluted with an naci-gradient in trisodium citrate buffer, ph 7.0. the buffer conditions are similar to the conditions used in the preparation of ppsb. the quality of the prothrombin so obtained was sufficient for most of our experiments. a second purification step on ion-exchange resulted in a 99% pure product devoid of contaminating factor activities and activation intermediates as examined with coomassie and silver stained sds-page electrophoresis and assays for factor x. this prothrombin contained full enzymatic activity and its activation by specific snake venom prothrombin activators showed the known activation products. we are now able to isolate the amounts of pure prothrombin required for preclinical investigations. most of the commercially available lmwhs such as enoxaparin, fraxiparin, and fragrnin are prepared by chemical methods which can result in desulfation and other chemical modifications of the internal structure leading to differences in the pharmacologic effects. on the other hand, tiactionated lmwhs retain their native characteristics and are structurally similar to heparin. in addition, the oligosaocharide sequence responsible for atiii binding is not modified. physical methods such as gamma irradiation (~co) have been used to fi'agment sulfated glycosaminoglyeans yielding fragraents without chemical modifications (deambrosi et at. in : biomedical and biotechnological advances in industrial polysaccharides, pp. 45-53). utilizing this technique, depolymerized heparius exhibiting different molecular weights can be obtained. this communication reports on the biochemical and pharmacologic effects of several such depolymerized heparins to demonstrate the molecular weight dependence on biologic activity. fragments exhibiting molecular weights of 5, 7, 8, and 9 kda were prepared by exposing concentrated heparin solutions to a rectilinear gamma ray beam at intermittent doses of 2.5 to 25 mrad under controlled temperatures. unlike the chemically depolymerized heparins, these fractions did not exhibit any decrease in charge density or atiii affinity. in routine assays for heparin, a clear cut molecular weight dependance on the anticoagulant and antiprotease actions was observed. on a gravimetric basis, these agents produce superior antithrombotic actions in comparison to chemically depolymerized derivatives. these studies suggest that gamma irradiation can be used to prepare lmwhs which retain their molecular integrity and therefore may prove to exhibit a more comparable biologic profile to hepari~ futthermore, lmwhs produced by gamma irradiation lack the usual double bond fommtion which requires the use of additives which can alter the product profile. university hospital, dept. of angiology, frankfurt a.m., germany introduction: thromboembolic disease constitutes a major clinical problem and among others a defective fibrinolytic system has been suggested as a predisposing factor for the development of thrombosis. the plasma fibrinolytic system can be impaired by inherited deficiencies of plasminogen defective release from the wessel wall tissue plasminogen activator (t-p'a) or by high ptusma levels of regulatory proteins, such as plasmino-8en. activator inhibilors (pal). the aim ....... of the present study w~s to eshmate the prevalence of decreased fibnnolyl~c actwlty m young pls. with thrombophilia. patients: a great population of 884 pts. (fenmle 478, male 406; age 21-61 ys median 39.8 ys) with venous thromtx~emolism before the age of 45 years were investigated in regard to their plasma fibrinolytie system. in none of them well established thrombophilia risk factors could be identified previously. methods: plasminogen ~behdngwerke), pai-1 activity (ehromogenic assay, biopool), pal-i anugen coneentration (elisa, biopool), t-pa activity (chromogenic assay, biopool) and antigen concentration (elisa, biopool) were measured before and after venous oeclusion.vo was performed z 12 month after the last thromboembolic epi~xle. 24 healthy subjects (median age 24.7 ys) served as controls. results." 24 pts.(2.7%) were classified as plasminogen deficiencies (activity and antigen). 142 pts.(16%) had significantly elevated levels of pal activity (up to 120 u/ml) and pal antigen (up to 90 ng/ml). none of the pts. with high pal levels had laboratory signs of acute phase reaction. low t-pa activity could be demonstrated and confirmed in 121 pts., aecordingto a prevalence of 13.6% (range: 0-2.7 u/ml; reference limils: 2.8 -21.8 u/ml). however, there was a significant negative correlation between t-pa activity and pal values. in 67 pts. (55.4%) the low t-pa activity was associated with increased pal levels whereas the t-pa antigen concentration was normal. a parallel reduction of t-pa activity and t-pa antigen (range: 0.35-3.5 ng/ml; reference limits: 3.6 -21.0 ng/ml) were determined repeatedly in 54 pts. (f 23, m 31, median age 39 ys). thus, the prevalence of a defective t-pa release was 6.1% in our study group. conclusion." in comparison to the frequency of inherited deficiencies of other well established thrombophila risk factors we have observed a relativel~ high prevalence of diminished t-pa activity, elevation of pal respectively in our study group. our data strongly indicate that besides t-pa and pal acuvity, antigen concentration for both parameters should be determined in pts. with thrombophilia. the antithrombotic and anticoagulant effect of the supersulfated low molecular weight heparin ssh 14 was studied after i.v. and s.c. administration in rats. thrombus formation in the jugular vein was induced by i.v. injection of activated human serum and following stasis for 20 rain and was assessed by a thrombus score ranging from 0 (no thrombus formation) until 3 (complete thrombus formation). ssh t4 injected either 10 min (i.v.) or 30 rain (s.c.) before thrombus induction caused a dose-dependent antithrombotic effect in a range from 0.25 to 2 mg/kg i.v. and 1 to 4 mg/kg s.c. there were clear differences in the antithromboric effectiveness between female and male animals, i.e, in female rats antithrombotically effective doses were lower than in male rats (edh0 after i.v. injection in females 0.35 mg/kg, in males 0.9 mg/kg). the sex differences were confirmed in studies on the time course of the antithrombotic effect. after i.v. injection of fully effective doses (2 mg/kg i.v. and 4 mg/kg s.c., resp.) the antithrombotic effect disappeared after 8 h in female or after 4 h in male rats. for studies on the anticoagulant action blood was drawn from the femoral artery and after centrifugation global clotting assays were performed in plasma. similar to its antithrombotic action ssh 14 also caused doseand sex-dependent anticoagulant effects. the most sensitive assays were the aptt and the heptest; thrombin time and prothrombin time were less or not influenced by ssh 14. in conclusion, ssh 14 was found to be an effective anticoagulant and antithrombotic agent in experimental studies in rats. at present there is no explanation for the clear sex differences found in this species. venous thromboembolic disease is the most frequent complication in patients undergoing total knee replacement therapy. patients and methods: after informed consent 3x30 patients were included in an open randomized clinical study and the incidence of venous thromboembolisrn was examined using different regimes for heparin prophylaxis (30 patients received fraxiparin 36 rag once daily, 30 patients clexane 40 once daily and 30 patients 7500 u calciparin twice daily). there were no differences between the groups concerning age, sex, body weight, risk factors, surgeons, decrease in hemoglobin~ and requirements for blood products. pre surgery, day 1, day 5-7 phiebograms were performed and also tat, dimers, fl+2 prothrombin fragments were examined. results: 1., dvt in 26 patients (28.9%). dvt in 5/30 patients under calciparm prophylaxis, 8/30 patients under fraxiparin and 13/30 patients under clexane treatment. 2., low speciflty (3.4%) of dimers and tat (24%) for detecting a dvt in these special patients undergoing knee replacement therapy, elevated fi+2 fragments in the dvt group at ti and t2 vs the patients without dvt (t1 dvt: 3.24+-1.8 vs. 1.6+-0.3 -p= 0.0042). 3, only 8/26 patients (31%) with dvt had clinical signs of thrombosis. conclusions: 1., there is an increase of thrombin gneeration measured by tat and dimers after knee replacement therapy. there are further studies with more patients necessary to confirm that fl+2 prothrombin fragments can discriminate between patients with and without dvt from a clinician's point of view. 2., phlebographicauy confimled dvt in almost 30% of our patients demonstrate the high thromboembolic risk in these patients. von willebrand's disease (vwd) type 2 is characterized by absence of high molecular weight muitimers. qualitative changes in the structure of the molecule might be associated with enhanced binding of von willebrand factor (vwf) to platelet glycoprotein lb. therefore in some patients vwd type 2 is associated with severe thrombocytopenia. here, we report on a 9 year old boy who presented with severe purpura and platelet counts about 20000/gl at the age of 2 years. thrombocytopenia did not respond to corticosteroids. a normalized platelet count of short duration was observed after high-dose immunoglobulins. in addition, increase of platelets was seen after anti-d treatment. thus, although platelet associated antibodies were not detected, thrombocytopenia seemed to be caused by an autoimmune mechanism. despite platelet counts above 50000/gl, the patient experienced severe bleedings with a significant decrease of hemoglobin levels. therefore, he needed several transfusions. coagulation analysis revealed vwd. application of ddavp lead to a normalization of partial thromboplastin time (ptt) and an increase of factor viii with subsequent cessation of bleeding symptoms. recently, vwd was typed 2 by lack of high molecular weight multimers. in conclusion, we report a case with vwd type 2 responding to ddavp. however it is unclear, whether thrombocytopenia is part of the vwd type 2 or of autoimmune origin. since autoimmune antibodies have not been detected, the effect of immunoglobulin treatment might be explained by blockade of enhanced binding of vwf to glycoprotein lb. von willebrad disease (vwd) with a prevalence of 0,8% (ruggeri 1994, rodeignere 1987) seems to be the most frequent inherited hemostatic disorder. • the diagnostic criteria for vwd are clinical picture, family hostory, laboratory findings: bleeding time, partial tromboplastine time (ptt), level of factor viii:e, vwf, vwf:ag, ristocetin induced platelets aggregation (ripa) and multim~-analysis.the diagnosis ofvwd is occasionally difficult, especially in early childhood because the laboratory data may vary due to time of investigation, as well as abnormalities may not be present in all sub-types the aim of this study was the evaluation of diagnostic approach to vwd in childhood and diagnostic reliability of all available laboratory tests. all previously mentioned laboratory tests have been done on our own material (51 child who satisfied all criteria for vwd, 23 boys and 28 girls, 1-9 years old) except mulfimer analysis which was unavailable in some cases. majority of laboratory tests proved to be highly specific and necessary for diagnosis. however, the diagnostic reliability of fviii:c and adhesion of platelets is much lower in mild cases in comparison to total sample, while ptt is an unvaiied test. the most specific screening test for vwd is vwf which diagnostic reliability is almost 1,00. the optimal strategy to establish general diagnosis of mild forms ofvwd is use of vwf and vwf:ag plus ripa if necessary and multimer analysis to classify variant types. we report on a new multimeric structural defect of vwf detected in a german family (two sisters and their three children): all members of the family who presented to our outpatient clinic had an increased spontanous bleeding tendency (moderate or strong hematoma, epistaxis, menorrhagia). prolonged bleeding could be observed after surgical procedures (adenotomia, tooth extraction) and after trauma (laceration). wound heeling was impaired in two cases. clotting assays showed slightly prolonged apti" and a mild decrease of f viii:c, vwf:ag and vwf:rcof levels. collagen binding activity was within normal ranges. bleeding time (simplate i) was slightly prolonged. the analysis of the multimeric structure in plasma showed quantitative and qualitative abnormalities: all multimers were detectable; the structure of vwf was reproducably abnormal in all family members so that the defect must be caused genetically. the thmmbocytic vwf showed neither qualitative nor quantitative alterations. minirin@ (ddavp) was administered as a test dose of 0,3 ~tg/kg bw in 100 ml 0,9% nacl-solution i.v. to evaluate efficacy and tolerance: clotting assays showed normalization of a_vrt, f viii:c, vwf:ag, vwf:rcof in plasma and shortening of bleeding time in three cases. an insufficient rise of vwf:ag and vwf:rcof levels could be observed in one case. one patient had no rise of f vm:c but a corrected bleeding time. multimeric analysis showed no structural change. the administration of ddavp was well tolcrated in all cases. the existance of all multimers in plasma and the normal collagen binding activity suggest that the structural abnormalities of vwf in this family does not cause functional defects so that the defect could be classified as a type i vwd. the response to ddavp was only partially effective. mild von willebrand disease (vwd) is far the most frequent congenital bleeding tendency. its diagnosis is very helpful in pre-operative check-up in order to avoid bleeding complications during surgery. following post-operative periods or monitoring the management of haemorrhagic episodes in vwd patients is also strongly recommended. current methods involve complex technologies, are time consuming and require large series. these assays lack the expected flexibility for rapid individual testing in patients. a new and flexible assay which works on the fully automatic walk-away coagulation instrument, sta, has been developed for these applications (liatest vwf). the technology is an immuno-turbidimetric method using mierolatex particles coated with rabbit polyelonal antibodies specific for vwf. the assay has a dynamic range from 2 to 420% yon willebrand factor (vwf) concentration, it works with a 2 fold dilution of tested plasma (50 td) and it offers a calibration established with the nibsc international standard. the total assay time is of less than 10 minutes and the detection threshold is of 2% there is no prozone effect up to concentrations higher than 1,000% vwf. intra-assay reproducibility is < 4% and inter-assay one < 5%. in dilution studies a mean recovery of 98% was obtained. in a study on 55 plasma samples from norma~ individuals, patients with high vwf concentrations, and vwd, comparison with the elisa technique demonstrated a correlation coefficient of 0.997 with a slope of 0.978 and an intercept of 3.30. in the low assay range too, a good agreement was obtained with the elisa. we conclude that liatest vwf is a reliable, flexible, sensitive, and rapid automated assay which fits well the vw'f assay applications in coagulation laboratories. fibrinolysis, the process during which the active enzyme plasmin is generated in a regulated and localised way, is -in a classical understanding -responsible for the dissolution of blood clots formed in a vessel. for this activity, t-pa is generally assumed to be the most important plasminogen activator and its activity, is regulated by enzyme kinetic mechanisms dependent on the presence of fibrin. with this background t-pa is used for thrembolytic therapy with great success. however, data from t-pa knockout mice indicate that t-pa might not be responsible for inhibiting the spontaneous development of intravsacular thrombi but only for dissolution of fibrin formed upon a coagulation challenge. in contrast, u-pa, generally assumed to be important for extravascular proteolytie activity on activated or tumour cells, seems to lead to the development of spontaneous fibrin formation in a mouse knockout model. on the other hand, the major plasminogea activator inhibitor pal-i seems not only to regulate intravascular fibrinolysis but seems to also be important for the progression of vascular diseases (neointima formation is e.g. increased in a pai-1 knockout model, but increased levels of pai-1 seem to predict reocclusion after angioplasty). in addition to their functioning as enzymes and inhibitors, components of the fibrinolytic system seem also to be involved in signalling processes in tumour and other cells. the u-pa/u-pa-receptor system could be shown to function as a chemotactic system and to elicit a migratory and mitogenle response in monoeytes and tumour ceils as well as in vascular cells. for such a response activation of tyrosine kinases of the sre-family might be responsible in some cell lines, but other signal transduction pathways e.g. involving caveolae and the starprotein can not be excluded. there seems to be a further important role of components of the fibrinolytic system which involves serine protease inhibitors (serpins): serpins have homologies to hormone binding proteins and cleavage of serpins by their target enzymes not only leads to inactivation of the enzyme but also to a possible release of bound hormones from the serpins. from these data clearly the relevance of any regulation of the fibrinolytie, system depends on the specific function of the system to be dealt with. in addition to "fibrin binding", "receptor mediated" and "genetic control" (e.g. 4g vs. 5g in the pai-i promotor) also "signal transduction" and "hormone delivery" are distinct functions of the system with specific regulation. plasmatic for both, healthy persons as well as for patients with angina pectoris it could be shown that increased values of plasma fibrinogen, factor viic and vwf:ag are significantly associated with the risk to suffer an acute myocardial infarction or cardiac sudden death. the same holds for tpa:ag. however, a group analysis in quintiles reveals that particularly low tpa:ag values are connected with a particularly low coronary risk. unexpectedly also the acute phase protein crp is positively associated with increased coronary risk. for clinical purposes these factors have already been included into coronary risk scores in order to improve the individual risk prediction in combination with lipids and other risk factors. the assessment of the pathophysiological significance of these observations remains at dispute. 4 pathways are discussed: 1. the assumption that increased plasma values of those factors indicate increased coagulation activity could so far not be established in prospective studies. 2. both vwf:ag and tpa:ag are produced in endothelial cells. an increase of their plasma level could therefore indicate increased endothelial cell functions which accompanies progressive atheromatosis. the risk association of the two acute phase proteins crp and fibrinogen could be interpreted analogously. 3. first prospective studies favour the assumption of a genetic determination to an increased production of coagulation proteins in persons at particular coronary risk. it could also be shown that there is a certain dependance of the gene-polymorphism for co-and 13-fibrinogen chains from the coronary risk. 4. even slightly elevated concentrations of fibrinogen and/or vwf:ag may influence the quality of a coronary thrombus both by increased physical stability and by reduced fibrinolytic lysibility. this could mean that an early coronary clot under these conditions could more readily develop to a stable, occlusive thrombus. a newborn with pronounced bleeding tendency had a prothrombin (prth) deficiency below 2.8% in a clotting assay. both parents had activities of 71% and 69%, respectively. however, the immunological determination ofprth by elisa revealed normal concentrations in all family members (93%-101%). furthermore, thrombin generation as investigated by a chromogenic assay using ecarin for activation of prth was normal as well. activation of prth by fxa was investigated by reealcificafion of the plasma samples and further analyzed for prth and its derivatives produced. although clotting times still were different, finally, normal levels of fl+2 and tat were generated as determined by elisa. western blot analysis using polyclonal (rabbit) antibodies to prth and a monclonal antibody specific to human thrombin, revealed different patterns of prth degradation products. tat was only weakly visible in the serum of the mother and nearly absent in the child.the mobility of prothrombin and thrombin was different compared to normals indicating a lower molecular weight. after reduction of disulfide bridges a higher molecular weight of thrombin was observed compared to normals indicating an insufficient cleavage ofprth and formation ofprethrombin 2. these observations let suggest that prothrombin marburg is a deletion mutant lacking the cleavage region arg 320-ile321. upon cleavage by factor xa only prethrombin 2 is formed under liberation of fl+2. this prethrombin 2 is able to cleave chromogenic substrates in the ecarin assay. probably, prethrombin 2 forms a complex with atiii which is detected by elisa, but unstable under denaturing conditions as in the western blot. as a major complication of haemophilia a treatment, up to 30% of the severely affected patients develop antibodies to substituted factor viii. investigating 133 patients and considering the data of further 231 patients of the haemophilia database, we could show, that risk of inhibitor developement depends on the patient's mutation type. patients with more severe gene defects, like intron 22 inversions, stop mutations or large deletions had a risk of about 35% for inhibitor developement, which was about 7 times higher than for missense mutations or small deletions. besides an influence of mutation type, we investigated other parameters e. g. immune response genes (i-ila-genotype) and clinical aspects (treatment onset and frequency, type of concentrate) that might also affect inhibitor formation. to exclude any effect of mutation type, we focussed on 72 patients with an intron 22 inversion. hla-typing showed that some t-ila-alleles (dqb0602, bt) occurred more otten and others (dqa103, dqb603, dr13, c2) less frequent in inhibitor patients. treatment onset, frequency and type of concentrate apparently do not affect inhibitor incidence. the results presented here, prove that inhibitor development is considerably influenced by the mutation type. this supports the hypothesis that patients with severe molecular defects have no endogenous factor viii protein and that substituted factor viii represents a foreign protein, leading to an immune response, e. g. the production of alloantibodies. in addition, the immune response seems to be modified by the hla-genotype. however oar findings (in terms of genotype and treatment parameters) can only explain part of the inhibitor pathogenesis. it is still unsolved why substituted factor viii does not lead to a recognizable immune response in 2/3 of the patients with severe molecular factor viii gene defects. consequently other factors, probably concerning the antenatal phase, must be involved. viia in the treatment of patients with inhibitors against factor viii or ix: a german/swiss/austrian multi~center trial d. ellbriiek*, i. scharrer**, j. dethling***, and the rfviia study group *section h~mostaseology, university ulm **dept. of angiology, jwg-university hospital frankfurt a.m. ***novo nordisk, mainz administration of activated recombinant factor vii (rfviia) can by-pass the fvnlwlx pathway and offers an alternative treatment for patients with antibodies (inhibitors) against these factors. from november 1994 to october 1995, a total of 25 bleeding episodes and 10 surgical interventions in 18 patients were treated with rfviia in a phase iiib multicenter trial. diagnosis was hemophilia a (n = 15) or b (n=l) with inhibitor, and acquired inhibitor against factor viii (n=2). various serious bleeds, from complicated joint and gingival bleeds to lifethreatening psoas bleeds, have been treated. operations have been tooth extractions, radiosynovectomy, implantation and explantadon of porth-acaths and one adenotomy. dose regimen was 90-120/zg/kg bw every two to three hours until clinical improvement, with subsequent dose reduction. results: for bleeding episodes, response to rfviia after 24 hours was effective in 72%, partially effective in 12 9"0, ineffective in 129"o and not evaluable in 1 (4%) of the patients. two of the three treatment failures were associated with very long dosage intervals of rfviia. the third patient was in a critical situation with artificial high pressure respiration and polytransfusion because of a hematothorax, and suffered a terminal intracerebral bleed. the efficacy of rfviia for surgery was very good. response to treatment was independent of antibody titer. no signs of dic or activation of coagulation were noted. conduslon: in our experience, rfviia is an efficient and safe treatment for inhibitor patients with acute bleeding episodes. it should be investigated, whether rfviia can be an alternative treatment also for the hometreatment situation. successful immunetolerance therapy of f vih-inhibitor in children after changing from high to intermediate purity f vih concentrate w. kreuz, j. joseph-$teiner, d. mentzer, g. auerswald*, t. beeg, s. becker zentrum der kinderheilkunde, j. w. goethe-universit~itj frankfurt am main *professor hess kinderklinik bremen introduction: inhibitor to f viii is the most severe complication in treatment of patients with haemophilia a. the incidence of f viii inhibitors is estimated to range between 15-33%. several authors reported that the immunetolerance therapy (itr) of f viii-inhibitors can be induced with high dose f viii concentrate. objective: this presentation will show data of four children with haemophilia a and f viii inhibitor (high responder), who had an unsuccessful lit with high dose f viii concentrate (high purity) in the first step. f viii concentrate was changed to an intermediate purity product (haemate hs®) in the subsequent course of h't. all patients received bleeding prophylaxis with an activated-prothrombin-complex-concentrate (feiba®). results: median age was 13 (9-18) months, when the inhibitor was first detected. in all four patients the f viii inhibitor titre increased under immunetolerance treatment with f viii concentrate (high purity) in the first step of therapy. after changing the f viii concentrate (intermediate purity) the inhibitor titres decreased continuously after a rebooster effect to 0be within months. median duration of f viii inhibitor elimination time (until first testing of 0 be) was 3 (2-5) months. in all patients the f viii inhibitor was successfully eliminated. until now all patients are under prophylactic treatment with f viii concentrate and had no positive inhibitor testing since. median observation time since the first testing of 0 be is 14 (4-60) months. conclusion: different studies concerning immunetolerance treatment have been successful with f viii concentrates of different purity. according to our experience in these four presented patients, we assume that probably not the purity of the f viii concentrate is important for the induction of immunetoleranee, rather than the type of f viii presentation in the used concentrate. the used preparation (haemate hs®) is a f viii concentrate with high concentration of vwf, which is known to be important for the protection of f viii against degradation by proteases. this may be a mechanism for a prolonged antigen presentation to the immunesystem and thus may have a positive impact on the outcome ot itr. long scale trials are needed to prove the above assumptions. thrombasthenia glanzmann is a disease affecting platelet function because of a partial or total lack of glycoprotein (gp) ilbllla expression or a modification of this complex. since the receptor dysfunction goes along with reduced or absent platelet aggregation and adhesion, it causes bleeding complications in case of injury. here we report about a 60 years old women, who suffered since early childhood from a severe bleeding disorder. life threating bleeding complications occured after tooth extraction and after abdominal surgery. analysis of the patients platelets revealed normal values for the platelet count, whereas their volume showed to be increased (11 fl). clot retraction was diminished to 17%. platelet adhesion to siliconised glass and human subendothelial matrix was reduced, as was the spreading of the platelets. adp (i#m) induced platelet aggregation was inhibited, while collagen-, ristocetin-and thrombin-induced aggregation showed to be normal. cross immunelectrophoresis resulted in an atypical peak of gpiibllla with reduced electrophoretic mobility. in the electroimmunoassay according to laurell 14% of gpiibllla was detected. moreover we observed a markedly diminished 125j-fibrinogen binding. sequence analysis of the gpiib and gpiila cdna after pcr amplification unraveled a g2508 --, a transition in gpiib, substituting gly805 --* glu. the structure/function relationship of this mutation has still to be investigated. we report two new abnormal fibrinogen variants, denoted as bem iv and milano xi, both having an exchange of arginine to histidine in position 16 of the ac~-chain. routine coagulation studies revealed prolonged thrombin and reptilase clotting times, low plasma fibrinogen concentrations determined by a functional assay but normal fibrinogen levels measured by the immunological assay. the onset of turbidity increase following addition ofthrombin to purified fibrinogen was markedly delayed in both variants. release of fibrinopeptide b by thrombin, measured by reversed phase hplc, was normal whereas only one half amount of normal fibrinopeptide a was released. in addition to normal fibrinopeptide a, an abnormal fibrinopeptide a* was cleaved from both dysfunctional fibrinogens. the structural defect was determined by asymmetric pcr and direct sequencing of a gene fragment coding for the nh2-terminus of the aachain. both variants were found to be heterozygous for the transition g to a at nucleotide position 1203, leading to the substitution actl6 arg-->his, resulting in a delayed fibrin polymerization. the simple assay permits detection of the most common amino acid substitutions occuring in the nh2-terminus of the ac~-chain of the functionally abnormal fibrinogen variants. protein c inhibitor (pci) a member of the serpin family is also known as plasminogen activator-3 (pal-3). pci was first described as a component of human plasma, regulating the activity of activated protein c and other sedne proteases of the human coagulation and fibdnolysis system. since then pci was found to be present in extra-plasmatic systems also. high concentrations of pci were detected in human seminal plasma suggesting a role for pci in human fertility. significant concentrations of pci mrna and antigen were located in lysosomes of proximal tubular kidney cells suggesting an intracellular function for pci in this environment. in this study we present evidence that pci is also present in human pancreas. rna from human pancreas was reverse transcribed and pcr amplified. the resulting pci cdna was identical with pci cdna from human liver. ~p labeled antisense rna probes used in in situ hybridization experiments with human pancreas tissue sections showed that pci rna was located in the acinar ceils. pancreatic fluid was analyzed by sds-page and immunoblotting. using monospecific antibodies directed against human plasma pci, a 57 000 mw protein band was observed which comigrated with purified human plasma pci. our results show that pancreas cells contain a significant concentration of pci mrna. this message is localized in the secretory acinar cells. therefore we conclude that pci antigen found in pancreatic fluid is likely to originate in the pancreas. the role of pancreatic pci is unknown at present. however, since thrombosis and systemic hypercoagulable states are known complications of pancreatic diseases our results and in vitro experiments by others showing that pci can inhibit pancreatic enzymes such as chymotrypsin and trypsin indicate that pci may be part of the inhibitor potential which protects pancreatic tissue from auto degradation. these inhibitors normally prevent the release of active pancreatic proteases into the vasculature or microcirculation where destabilization of the coagulation balance and subsequent thrombus formation could occur. institute for clinical chemistry and laboratory diagnostics and *clinic for cardiology, universi w of duesseldorf p-selectin (cd 62p, the former granule membrane protein 140 or gmp 140) is an integrated membrane protein of platelets and endothelial cells. under inactivated conditions it is stored in the alpha granules of platelets and in the weibei-palade bodies of endothelial cells. endothelial cells covering atherosclerotic plaques show an increased expression of p-selectin. 13-thromboglobulin (13-tg), which is also expressed from the alpha granules of platelets during adhesion or aggregation, is regarded as a marker of platelet activation in vivo. coronary thrombosis plays a central role in the pathogenesis of acute coronary syndromes. we therefore analysed cd 62p and 13-tg in acute coronary syndromes, healthy subjects (hs, n=l i), patients with stable angina pectoris (sap, n=20), unstable angina pectoris (uap, n=l 2) and acute myocardial infarction (ami, n= 12). plasma samples were obtained by using ctad vacutainer tubes (0.109 m na~-citrate, theophylline, adenosine dipyridamole). patients with cad showed significantly increased plasma concentrations of cd 62p (hs: 98+20 versus sap: 133+38ng/ml, p<0.05; versus uap: 128+28 ng/ml, p<0.01; versus ami: 144+72 ng/ml, p<0.05) independent of the severity of clinical symptoms. in comparison only patients with ami showed significant higher 8-tg concentrations compared with hs (hs: 30+20 versus ami: 39+14ng/ml, p<0.05). although the cd 62p plasma concentrations showed no relationship to the clinical severity, hence there was a positive correlation between cd 62p (r=0.47; p< 0.001; n=55) to the severity of cad classified as i, 2, 3 vessel disease. it is concluded that elevated cd 62p concentrations are correlated with the severity of cardiovascular disease. cd 62p is not suitable for differential diagnosis of acute coronary syndromes, because it is elevated independently of the clinical status of the patients. the involvement of platelets in the pathogenesis of acute myocardial infarction may be indicated by the increased 13-tg concentrations. iklinik nr herz-, thorax-und herznahe gef&schirurgie und 2institut x~tr klinische chemie und laberatodumsmedizin der universint regensburg an increased blood loss following surgery with extracorporeal circulation (ecc) contributes to the morbidity and mortality. postoperative haemorrhage following ecc has been related to a platelet function defect and the activation of the blood dotting and fibrinulytic system. we investigated platelet surface antigen expression and parameters indicating activation of the clotting and fibrinolytic cascade to assess the predictive potential of these variables for increased blood loss after ecc. g0 patients referred for coronary bypass gra~ing with no history of a bleeding disorder and normal routine clotting tests were included. on the day prior to surge~ and immediately upon arrival on the intensive care unit blood samples were drawn. the surface expression of glycoprotein (gp) lib-ilia, gp lb, and p-selectin was meamred with and without in vitro stimulation with adenosine diphosphate (adp) using whole blood flow cytomet~y. platelet counts and platelet factor 4 (pf4), as well as, routine clotting tests were performed. activation of the clotting and fibrinolytic system were judged from thrombin-antithrombin-iii complex fiat), fibrinogen fig), d-dimers (dd), cc2-antiplasmin (tz2a), prothrombin fragment 1+2 (fl+2),and tissue plasm~ activator (t-pa). blood loss fxom chest tubes was measured hourly until removal of drains. following ecc the levels of pf4, tat, dd, o~2a, fl+2, and dd were sigulticnatly increased (p<0.0001) compared to baseline values. gp iib-iila, gp ib, p-selectin, platelet count, and fg were significantly reduced (p<0.0001). analysis of variance (anova) revealed that postoperative values of gp ib (p<0.0001), dd (p<o.05), fg (p<0.01),and platelet count (10<0.05) had a significant influence on blood loss. none of the preoperative data was significantly associated with increased blood loss. a subgroup of patients who bled more than the mean + 1 sd (14 cases) was formed_ in logistic regression analysis gp ib expression (p=0.01) and dd (p=0.007) were of value in identifying bleeders. the sensitivity of the equation was 56%, the stx~ificity 97% and the positive predictive value 82%. although the reasons for an increased blood loss following ecc are multifactorial, we were able to identify a small number parameters with a significant infl~nco. the combination of the expression of yon willebrand receptor on the platelet su_rfaco and d-dimer levels in plasma was successful in predicling an increased blood loss following heart surgery. this may be helpful to define indications for platelet transfusion and flesh frozen plasma. the most widely distributed receptor for the fc-region of igg is the fcreceptor iia (fcyriia). for this receptor a functionally relevant polymorphism of amino acids argmhis is known. we previously showed hit-antibodies to bind to platelets via feyriia and now whether clinical manifestation of hit is associated with the fe'/riia polymorphism. therefore, we developed a rapid two step allelle-specific pcr (asp) method for genotyping the foyriia. with control samples the asp was compared with the time consuming and complicated allele-specific oligonucleofide hybridization technique, the phenotype expression of fctriia (using the moabs iv.3 and 41h16) and the monocyte stimulation assay. complete concordance between the assays was found. we used the asp to determine the allelic distribution of the inst. physiol. chem., marburg; *)univ. of virginia, charlottesville, the functional heterogeneity of blood platelets is clinically significant and may contribute to the pathogenesis of cardiovascular and atherosclerotic diseases l). in order to understand the biochemical basis of the functional heterogeneity of platelets, we have investigated the signal transducing pathway of platelet activation a) in terms of guanylate cyclase and phosphodiesterase activities and b) at the level of protein phosphorylation and the receptor-effector coupling via g i-and gs-proteins in functional heterogeneous blood platelet subpopulations separated according to their densities. furthermore, we analyzed the adp-ribosylation of rhoa, which is a lowmolecular weight gtp-binding protein and is thought to be involved in the platelet "shape change" reaction and aggregation. aggregation was enhanced in low-density platelets (sp i) and less inhibited when cgmp was increased by no, compared to high-density platelets (spiii). sp i possessed a lower basal level of cgmp and exhibited a smaller increase in cgmp after stimulation with no. cyclic amp-dependent phosphodiesterase activity was higher in sp i compared to spiii, whereas cgmp-dependent phosphodiesterase activity was similar in sp i and sp iii. sp i, when compared to sp iii platelets, showed a higher degree of prephosphorylation of proteins in the range of 47, 32-34, and 20-22 kda. after thrombin stimulation, the phosphorylation of these proteins was similar in the subpopulations. pertussis toxin,induced adp-ribosylation of the 40-41 kda gi-protein was highest in sp i, whereas cholera toxin induced adpribosylation of the gs-protein and botulinum c3 exoenzyme-induced adpfibosylation of rhoa were highest in sp iii. the findings suggest that a) the higher aggregability in sp i compared to sp iii may be caused by a poorer ability of guanylate cyclase to form cgmp and a higher activitiy of campdependent phosphodiesterase and that b) the adp-ribosylation of g-protein subunits and rhea may contribute to the functional heterogeneity in the subpopulation leading to the greater ability of sp i for aggregation. 1) opper et al., atherosclerosis, 113, 211-217, 1995. causes of accelerated atherogenesis in non-insulin-dependent diabetes mellitus (niddm), known to be associated with increased plasma activity of plasminogen activator inhibitor type 1 (pal-l) as well as with increased plasma concentrations of proinsulin and insulin, remain enigmatic. this study was designed to determine whether proinsulin and insulin increase pal-1 in vivo thereby potentially accelerating atherogenesis. to simulate hyper(pro)insulinemia seen with niddm, we infused equimolar concentrations of insulin (1 iu/kg), proinsulin, c-peptide, or placebo, all endotoxin-free, i.v. over 1 h in 53 conscious rabbits maintained euglycemic. plasma pal-1 did not increase with placebo (n=16) or c-peptide (n=4), but did with proinsulin and insulin peaking in 3 h at 3.5-and 3.7-fold (n=16 and n=17, p<0.o02 for each) in parallel with pal-1 protein quantified by reverse fibrin autography. the increases were specific as reflected by unchanged activities of o~-antiplasmin and t-pa. pat-1 gene expression (pal-1 mrna) increased with proinsulin and insulin by 2.1-and 2.1-fold in 3 h in aorta, 1.9-and 2.4-fold in liver (p<0.05 for each), but not in heart, lung, spleen, or kidney and returned to baseline in 24 h (n=4 each). in situ hybridization localized the increased pal-1 mrna in aorta to endothelium (n=3 each). thus, both proinsulin and insulin directly augment gene expression of pal-1 in arterial endothelium in vivo, thereby increasing plasma pal-1 activity, attenuating endogenous fibrinolysis, and potentially accelerating atherogenesis. the sarco-endoplasmic-retikulum ca+÷atpases (serca) that sequester calcium into intracellular stores, represent the most important mechanism to maintain cytosolic calcium levels low and to return to a resting level after activation. recent studies in platelets and rat endothelial cells have identified two forms belonging to the serca 2b and serca 3 types. the monoclonal antibody pl/im 430 raised to human platelet intracellular membranes has been shown to recognise the serca 3 ÷+ isoform and to inhibit ca sequestration in human platelets. the aim of this study was to identify ca atpase distribution in human umbilical vein endothelial cells (huvec) and human polymorphonuclear leukocytes (pmnl). polyclonal antibodies with known specificity towards serca 2b (r 2/88) and serca 3 (89/3) in addition to pl/im 430 were used in histochemical studies of permeabilised cells and in western blotting experiments using cell lysates and mixed membranes. using the alkaline phosphatase anti-alkaline phosphatase (apaap) method r 2/88 and 89/3 reacted strongly with proteins in permeabilised huvec and pmnl, however pl/im 430 showed good staining only with pmnl. in western blotting experiments r 2/88 recognised proteins with molecular sizes of 100 kda (known to be the parent ca++atpase) and 70 kda (which may represent antibody recognised degradation product), in both huvec and pmnl. 89/3 ÷+ recognised bands at 100 kda (parent ca atpase), 74 kda and 46 kda in both huvec and pmnl membranes. in agreement with histochemical results pl/im 430 recognised proteins (100, 74 and 46 45 + + kda) only in pmnl membranes. in ca transport studies pl/im 430 ÷+ inhibited ca uptake in membranes prepared from pmnl but not in huvec. the distinct reaction of pl/im 430 with pmnl and huvec suggests the possible presence of distinct sub-isoforms of serca 3 with the pmnl and platelet proteins being similar and different to the serca 3 isoform present in huvec. microvascular endothelial cell (ec) lines with a limited life span of 2-3 months in vitro were established from human bone marrow, skin or solid tumor tissue. homogeneity of the lines was verified by flow cytometric analysis and cytokine expression patterns. the cultured ec constitutively expressed hla-class i antigens, cd44, procollagen type i, cd62e, cd34, and receptors for granulocyte colony stimulating factor (g-csf). endothelial cells constitutively produced il-6 and il-8 as well as tpa and pai-i as determined by elisa. in the presence of 5 iu/ml of exogeneous il-la, g-csf was secreted at high amounts (lng/ml) and gm-csf at low amounts (15pg/ml) in confluent monolayers and during over night incubation. these ec potently bound to isolated neutrophilic granulocytes. when granulocytes were stimulated by either 10~ 4 or 10 .7 m formyl-methionyl-leucyl-phenylanaline (fmlp) to produce oxygen radicals, the preincubation with a single cell suspension of ec caused oxygen radical scavenging. a ratio of 1 : 100 ec to neutrophilic granulocytes resulted in a 50 __+ 10% inhibition of fmlp-induced and luminol-enhanced chemiluminescence in vitro. this effect was probably due to the constitutive nitrogen oxide (no) production by ec. unexpectedly, preincubation of ec with 50 iu/ml of interferon-y (ifn-y) to increase no-secretion did not alter the extent of the oxygen scavenging effect measured at different ratios of ec to neutrophilic granulocytes. according to the surface antigen expression pattern, identical ifn-f treatment upregulated adhesion receptors on ec. concomitantly, binding of neutrophils to ec was significantly increased. thus, the net oxygen-scavenging effect by ec appears to be stable under the condition of ifn-y stimulation which indicates maintenance of ec function and effective neutralizition of non-specific oxygen radical damage. moreover, the established test system can be useful to investigate the selective contribution of other inflammatory cytokines, adhesion molecules, macrophages and platelets on ec function in vitro. atherosclerosis is characterized by intimal migration and proliferation of smooth muscle cells (smcs), by macrophage infiltration and extracellular matrix remodeling. circumstantial evidence suggests that urokinasetype plasminogen activator (upa) may be relevant in these processes. this study examines upa expression and upa antigen localization in human coronary arteries with ,carious degrees of atherosclerotic lesions. representative segments of macroscopically normal areas (mnas), early lesions (els) and fibrous plaques (fps) were processed in parallel for in situ hybddization and immunohistochemical analysis. in mnas, upa was detected both in the luminal endothelium and in celocalization with ~-actin-positive smcs throughout the tunica media. in els, upa mrna and antigen were substantially reduced in the luminal endothelium. however, cellular upa expression was detected in the subendothelial area in colocalization with tissue-infiltrating cd68positive macrophages. in the deeper intimal and in the medial layers strong upa expression was observed in association with ~-actinpositive smcs, with most of the upa-positive cells being located in areas with a high proliferative activity as detected by pcna staining. in fps, upa mrna was widely expressed in macrophage-containing areas within the fibrous cap and at the periphery of the necrotic core of the lesions. in addition, upa antigen was identified in acellular areas of the necrotic core regions. compared to the diseased intima of these lesions, the adjacent media stained only weakly for upa. in conclusion, this study demonstrates an enhanced expression and synthesis of upa by intimal smcs and macrophages in advanced atherosclerotic lesions of human coronary artedes these findings suggest a role for upa in the progression of the coronary atherosclerosis. adenosine produced by vascular cells, platelets and other tissues has been proposed to be a vasodilator, inhibits platelet aggregation, stimulates proliferation and migration of endothelial cells as well as exhibits antiinflammatory effects. adenosine of endothelial origin intra-and extracellularly formed and highly concentrated at the luminal surface may constitute an important antiaggregatory mechanism, which is part of the well-known antithrombogenicity of an intact endothelial lining. it was the aim of this study to investigate whether adenosine could influence the fibfinolytic potential of endothelial cells. when confluent human umbilical vein endothelial cells were incubated with adenosine (0.01-10mm) a significant dose dependent decrease in plasminogen activator inhibitor type-1 (pai-i) antigen production by this cells was seen as compared to control. this effect by adenosine was also time dependent and seen after 2 hours of incubation with adenosine. as determined by elisa, pai-i antigen in conditioned media of endothelial cells incubated with 10mm adenosine decreased to 57% as compared to control. pai-i antigen in extracelullar matrix of these cells was also reduced by incubation with adenosine. these results were also reflected on the level of specific mrna as determined by northern blotting. pai-i mrna decreased in endothelial cells after incubation for 4 hours with adenosine (10mm) to 77% (2.2kb) and 85% (3.2kb) of control. in conclusion our data demonstrate that adenosine can downregulate the expression of pai-1 in cultured endothelial cells thereby increasing the profibrinolytic capacity of these cells. this effect of adenosine might contribute to its antithrombotic potential in addtion to its known antiaggregatory effect. sowohl die effizienz als such die komplikationen einer antikoagulantien-dauerbehandlung sind in erster linie yon der gore und engmaschigkeit der gednnungskontrellen abh~ngig seit 1986 wird die schulung zur gerinnungs-selbstkontrolte in der herz-kreistauf-klinik bad bedebarg in gruppen eingeobt. die theoretische und praktische anleitung erfolgt in 8 stunden durch eine in der patientenschuiung erfahrene arzthelfedn (oder krankenschwested-pfleger) und einen arzt. die praktische schulung wird durch einen patientennahen theoretischen tell erg~nzt. verglichen mit konventionell ~berwachten oral antikoagulierten patienten (ca. 50% der tpz-werte liegen im theapeutischen bereich) erreichten 216 quickwert-selbstbestimmer, daf$ 83,1% yon 14 812 tpz-werten im therapeutischen bereich lagen (tabelle 1 ). schwerwiegende thromboernbolische ereignisse oder blutungskomplikatienen wurden nicht beobachtet. durchschnittlich kontrolliert der quickwert-selbstbestimmer seine gerineungswerte w6chentlich, itmgstens im zweiwochen-abstand. ob sich blutungs-und thremboembolierisiko langfristig senken lassen bei gleichze~iger verbesserung yon prognose und lebensqualit~it, bedarf des nachweises im rahmen einer randomisierten, kontrollierten und prospektiven studie. tabelle i verteilun~l der selbstbestimrnten gednnur ~swerte zeitraum 1986 zeitraum -1988 zeitraum 1986 zeitraum -1990 zeitraum 1986 zeitraum -1992 to evaluate the potential benefit of an optimized oral anticoagulation management with inr measurements performed by the patients themselves, two prospective randomized studies have been completed. the first study (n=60) was performed to evaluate the accuracy of inr self-testing (inr-st) compared with standard laboratory controls as well as the improvement of anticoagutation monitoring by inr-st, e.g. the percentage of measurements found inside the target therapeutic inr-range. the 231 parallel measurements revealed a high accuracy of inr-st with a 4.5% median difference between parallel controls of single measurements. in addition, inr-st resulted in a significant increase of measurements inside the target therapeutic range (from 54 to 80%) in the entire group. however, inr-st performed every 4 weeks did not result in a significant improvement, while selfcontrols every week (from 44 to 84%) and every 2 days (from 51 to 84%) did. in a second study (150 patients) the incidence of bleeding and thromboembolic complications were evaluated in patients who either had standard anticoagulation measurements o[ performed inr-st. during a median follow-up of 1.5 years in the group with standard oral anticoagulation management an incidence for bleedings of 10.9% per year and for thromboembolic complications of 3.6% per year was found, while in the group of patients with inr-st bteedings occured with an incidence of 4.5% per year and thromboembolisms with 0.9% per year. it can be concluded that inr-st can be performed with high accuracy by the patients themselves. inr-st results in more frequent controls of the inr and consequently in significantly longer time periods where the patients are inside the optimal target therapeutic inr range. the more accurate anticoagulation management resulted in our study group in a significantly lower incidence of thromboembolic and hemorrhagic complications. the cell biology of tissue factor (thromboplastin) h. prydz, the biotechnology centre of oslo, norway tissue factor (tf,thromboplastin) is the most potent trigger of blood clotting knuwn.lts presence in tissue extracts has been known since before the turn of the century.erwin chargaff (of dna fame) first described that tf consisted of two parts (a protein and a lipid moiety), a fact that was independently rediscovered in the early sixties by deutsch and lechner in vienna and by myself in oslo. in 1973 tf was first purified and shown to be an integral membrane protein of mr about 50 kda (this was a slight overestimate,recent studies indicate 46-48 kda). in 1987 four groupes independently cloned tf edna, later also genomic dna. the structure of tf suggested that it was a membrane spanning receptor, with an extracellular part of about 219 amino acids folding into two domains remotely like the domains of the ig-superfamiliy, and more specifically the eytokine receptors. tf is synthesized to a varying degree in many tissues, but not normally in tissues in contact with blood. however, during acute inflammatory situations and under the influence of a number of agents (immune cnmplexcs, lectins, cytuklnes,bacterial components, complement components etc) monucytes and vascular endothelial cells are induced to synthesize tf. another possible way tu get tf into contact with blood is thruugh trauma, surgical ur otherwise. the fact that tf is inducible has created a lot uf interest in its cell biology. we reported recently that binding of factor viia tu tf un the cell surface elicited a marked increase in intracenular ca spikes, thus rendering the final proof that tf not only luoks like a receptor but really is one, since all the three essential criteria are fulfilled: tf has a transmembrane segment and a cytoplasmic tail tf has a high specificity/high affinity ligaud (factor vii and factor x) tf induces an intracellular signal upon ligand binding recent results regarding tf as a receptor and regarding its intracellular traffic will be reported. both f.ix and f.x are activated by the tf/f.viia complex. we have used a tf-initiated model system to examine the roles played by f.ixa and f.xa in initiating coagulation. the in vitro model contained a cellular tf source; plasma concentrations of f.ii, v, viii, ix and x, tissue factor pathway inhibitor and antithrombin iii. we tested the ability of exogenously added f.ixa or f.xa to substitute for tf/viia activity in initiating platelet activation and thrombin generation. f.xa was 10 times more potent than f.ixa in initiating platelet activation, but f.ixa was more potent in promoting iia generation. in the presence of tf/viia, zymogen f.x was required both for platelet activation and iia generation, while f.ix was only required for iia generation. thus, tf/viia-activated f.ixa and f.xa have distinct physiological roles. the main role of f.xa is to activate platelets by producing an initial small amount of iia. the process of platelet activation is much more efficient if this initial iia is produced on the tf-bearing surface. f.ixa, by contrast, enhances iia generation on platelets by providing f.xa for platelet surface prothrombinase assembly. we conclude that activation of both f.ix and f.x by tf/viia is essential for efficient initiation of coagulation. furthermore, f.x activated on the surface of a tf-bearing cell isnot functionally equi.valent to f.x activated on the platelet surface. therefore, f.x activation by tf/viia does not compensate for a lack of f.ix or viii in hemophilia because the f.xa activated by tf/viia is located on the wrong surface for efficient thrombin generation. our results emphasize that the location, of an activated coagulation factor is a critical determinant of its role in hemostasis. the x-ray crystallographic structure of the fviia-tf complex located the areas within fviia in contact with tf to the first egflike and the protease domains. high-affinity binding of fviia to tf depends on ca2+ and previous studies suggest that ca2+ binding to a site in the protease domain is required. however, using surface plasrnon resonance we have shown that removal of the n-terminal gla domain and hydrophobic stack (hs) from fviia results in a decreased affinity for tf. in search for the mechanism by which regions in fvua not in direct contact with tf affect the affinity, various techniques have been employed. a plausible model is one in which the gla domain and hs optimize the interaction with tf by affecting regions that mediate the association with tf. using gel filtration we demonstrated that the hydrodynamic radii of des(1-38)-fviia and, especially, fviia decreased upon ca2+ binding, whereas des(1-44)-fviia was virtually unaffected. this suggested that the gla domain and the hs interact in a ca2+-dependent manner with other regions in fviia, presumably the protease domain, making fviia more globular. using cd spectroscopy, we could detect ca2+-induced changes in the secondary structure of fviia, all assignable to an increased helical content of the gla domain. the amidolytic activity of fviia in the absence of tf has a ca2+ dependency that suggests involvement of the gla domain in obtaining full activity. this activity was very sensitive to guanidine hydrochloride (c50<0.5 m) and its quenching corresponded to unfolding of the ca2+-loaded gla domain as measured by changes in the trp fluorescence of fviia. based on these results it is conceivable that the gla domain and perhaps the hs of fviia interacts with the protease domain and that this globular form mediates the initial contact with "it. several studies have indicated a profound role for fvii(a) in arterial thrombogenesis. in the present study we quantified the inhibitory effect of dansyl-glutamyl-glycyl-arginyl-chloromethyl ketone recombinant fviia (degr-rfviia) on acute thrombus formation at medium-sized artery blood flow conditions characterized by a wall shear rate of 650 s -1. native human blood was drawn directly from an antecubital vein by a pump into a heparin coated mixing device where degr-rfviia (0.9 -880 nmol/l final plasma concentration) or buffer were mixed homogeneously with the flowing blood. subsequently, the blood entered a parallel-plate perfusion chamber in which a thrombogenic surface consisting of immobilized tf and phospholipids was positioned. fibrin deposition, platelet-fibrin adhesion and platetet thrombus volume triggered by this surface were measured by morphometry. degr-rfviia inhibited these thrombus parameters in a dose dependent manner with ic50 values between 0.5 and 0.7 nmol/l the plasma levels of thrombin-antithrombin m complexes were inhibited at an ic50 of 0.6 nmot/l since the concentration of fvii and fviia in normal plasma is about 10 and 0.1 nmol/l, respectively, theic50 values indicate that degr-rfviia is a very efficient inhibitor of thrombus formation in this model. thus, inhibition of the extrinsic pathway of coagulation may represent and entirely new and effective arterial anti-thrombotic approach. the anticoagulant agent from medicinal leeches, hirudin, is presently gaining popularity as an antithrombotic drug. the design of recombinant forms of the minipretein and their availability led to the clinical use of this naturally occurring thrombin inhibitor. extensive studies in experimental animals have shown that due to its pronounced and specific antithrombin effect hirudin is an antithrombotic agent of high quality. apart from its anticoagulant effect, hirudin is pharmacodynamically inert and is well tolerated in vivo. brain abnormalities in male children and adoleescents with hemophilia: detection with mr imaging mri of sickle cell cerebral infarction in addition, portions of the preparations were incubated with 1.4nm-gold labelled fibrinegen molecules (fg-au; final concentration 4gp_g/ml) for 10 see at 37°c. the expenments were stopped al~er 3 or 10 rain by glutaraldehyde fixation. serial sections were prepared and examined after silver-enhancement using danscher's (1981) method to visualize the fg-au. hwdid not alter the resting gfp. after .3,rain, activated hw-platelets changed their shape moderately but in contrast to the controls did not aggregate. moreover, the conatdcting cytoskeleton that centralizes platelet organelles was not formed. fg-au was found on the surface of the control platelets, in high density in the contact spaces between the aggregated platelets and internalized in the surface connected system. only a very small number of hwtreated cells had fg-au on their surface and showed internalization. 10 rain after adp most of the platelets in both preparations had retumod to a discospherecytic shape it is confirmed that hw inhibits the platetet aggregation and in addition, 1) the formation of the contractile cytnskeleton as well as 2) the binding and interalization of the used ligand fibrinogen santoso 2 l.institute for immunology and transfusion medicine, emst-moritz-arndt-university greifswald; 2. institute for transplant vasculopathy in cardiac allografts is the leading limitation to long-term survival of cardiac transplant recipients. activation of the coagulation mechanism by adherent monocytes expressing tissue factor (tf) is expected to be involved in the development of transplant atherosclerosis. in this in vitro study the effect of the immunosuppressant cyclosporine a (csa) on monocyte tf expression and the regulation of the tf gene transcription was analysed. csa was shown to inhibit the induction of monocyte procoagulant activity (pca) by decreasing lipopolysaccharide (lps)-induced tissue factor (tf) expression in monocytes. csa exerted a dosedependent inhibitory effect on monocyte tf expression at concentrations (1,5 txmol/l csa: 50 % inhibition) not decreasing the cellular protein synthetic rate. addition of csa during lpsstimulation prevented activation of the nuclear factor (nf) ~b as demonstrated by electrophoretic mobility shift assay. western blot analysis confirmed reduced nuclear translocation of nf-~b in the presence of csa. inhibition of the lps-induced nf-~b activation by csa resulted in a reduced tf gene transcription as demonstrated by rt-pcr-analysis. thus, cyclosporine a prevents tf expression by inhibiting the induction of tf gene transcription in activated monocytes. csa may therefore provide a therapeutical tool acting not only as a direct immunomodulating agent, but also by influencing local coagulation activation. the n-glyean pattern of recombinant human coagulation factors ii (rf-i~ and ix (rf-ix), derived from both transfected chinese hamster ovary (cho) cells and african green monkey (vero) cells, and produced at industrial scale were analyzed by binding to carbohydrate specific lectins and were compared with the glycan structure of human plasma-derived coagulation factors. human plasma-derived coagulation factors 1i (hpf-i1) and ix (hpf-ix) exhibited complex-type glycan structures with carbohydrate chains capped with c~(2-6)sialic acid. terminal galactose-b(1-4)-n-acetylglucosamine units were detected in hpf-ix. both cho cell-derived rf-]i and rf-ix exhibited complot-type glycosylafion and contained ~2-3)-sialio acid in addition to terminal galactose-b(1-4)-n-acetylglucosamine. vero cell-derived if-ix exhibited a complex-type glycan structure sinailar to that of cho cell derived rf-ix. in contrast, rf-li produced by vero cells exhibited a glycan microheterogeneity composed of hybrid-type glycosylation containing "highmarmose" structures and complex-type glycosylation containing et(2-3)-sialie acid. galaetose-b(1-4)-n-acetylglucosamine structures and a low concentration of ct(2-6)-sialic acid were detected in both micro-heterogeneity fractions of veto cell-derived rf-li. although different in the'tr carbohydrate structures, coagulation factors ii and ix obtained recombinantly from both transformed cho-cells and vero-cells exhibited coagulation activities comparable to the plasma-derived proteins. exposure of tissue factor (tf) to flowing blood, as a consequence of vascular injury, leads to tf/f.viia-initiated coagulation which may play a key role in triggering intravascular thrombosis. in order to evaluate the potential advantages of tf pathway blockade over existing therapy, the antithrombotic and the anticoagulant .effects of a monoclonal anti-rabbit tf antibody (ap-1) and heparin were compared in a rabbit arterial thrombosis model. thrombosis was induced by a local injury to the carotid artery and the recurrence of thrombus formation was followed by monitoring the cyclic flow variations (cfv) of the arterial blood flow. rabbits received intravenously either ap-1, heparin or vehicle, and cfv were monitored on line for 120 min. activated partial thromboplastin time (aptt), and activated clotting time (act) were measured at the end of the experiment control ap-i heparin <lmg/kg* 0.2mg/kg/hr 0.8mg/kg/hr intraabdominallretroperitoneal, cns) on a named-patient basis. later also mild/moderate bleeds, predominantly joint bleeds were included.in 80-90% of the bleeds novoseven was found to be hemostatically efficacious. also in major surgery (hip-and knee-replacement including a bilateral total condylar arthroplasty, herniotomy) hemostatic efficacy has been found to be 94% since rfvlla is proteotytically active only in complex with tf the risk for systemic activation of the coagulation system should be minimal. also very few side-effects have been seen.doses between 75-120 tjg/kg have been used, which initially have to be given at 2 hours intervals. to achieve satisfactory hemostasis both an initial dose high enough to induce immediate hemostasis and an appropriate dose interval ensuring a plasma level of fvli:c well above the hemostatic lower level for a period of time, the length of which is dependent on the type of bleeding, are important. accordingly, the highel the initial dose the more robust with regard to dose interval will the treatment be. acute myocardial ischemia is usually triggered by coronary thrombosis. heparin (hep), along with aspirin, is commonly used for the treatment of unstable angina (ua) and myocardial infarction (mi). hirudin (hiq is a potent antitbrombin agent which may offer therapeutic advantages over standard hep for the treatment ua and mi. the oasis phase i pilot study conducted in 24 canadian centres, has evaluated the safety, feasibility and efficacy of hir (hbw 023, behringwerke ag, marburg) compared to hep in 601 patients with mi or suspected non q-wave mi. patients (95 % received aspirin) were randomized to a 72 hr infusion of either hep (5000 u bolus followed by 1200 u/hr; n=253), or low dose hir (0.2 mg/kg bolus then 0.10 mg/kg/hr; n=175) or medium dose lair (0.4 ms/ks bolus then 0.15 mg/kg/hr; n=173). at seven days of follow-up the incidence of major bleeds was 0.4 % in the heparin group, 0.6 % low dose hit, 1.2 % medium dose lair (p=ns), minor bleeds were 10.3 %, 17.1% and 19.1% respectively (p---0.008 hep vs med dose); there were no hemorrhagic strokes. the incidence of the primary outcome of cardiovascular death, new mi and severe recurrent ischemia (refractory or severe angina) was 17.0 %, 15.4 % and 8.7 % respectively (p---0.014 hep vs reed dose). there was also a trend towards lower rates of coronary artery bypass surgery in reed dose hir compared to hep. in a 200 patient substudy, levels of thrombin antithrombin complex, fibfinopeptide a and d-dimer were all suppressed to a greater extent by mr compared to hep. these results indicate that hirudin may be a more effective agent for the treatment of acute coronary syndromes, but more information on safety and efficacy is needed from larger randomized trials. full clinical and coagulation resuks will be presented. hat is caused by an immunologic response to heparin. affected patients need effective parenteral anticoagulation. we treated 82 patients (30m,52fm), median age 61 years (18-84) with laboratory confirmed hat (hipa-test) with r-hirudin (behringwcrke ag, marburg). treatment regimens were: ai acute hat: 0.4 mg/kg b.w., continuous infusion 0a5 mg/kg b.w/h (51 patients); a2 acute hat with thrombolysis: bolus 0.2 mg/kg b.w., continuous infusion 0.1 mg/kg b.w./h (5 patients); b: proph, treatment: continuous infusion 0. i mg/kg b.w.pa (18 patients); c: during cardio-pulmunary bypass. primary objective was: increase in platelet counts or maintenance of normal baseline values during effective anticoagulation. secondary objectives were the incidence of new thromboembolic complications (tecs), major hemorrhage, limb amputations and deaths. results were compared with a historical control of 120 ix-at patients, all confirmed by the hipa test, but treated before rhirudin was available (danaparoid, marcumar, no anticoagulation). primary efficacy analysis: respunders ai: 74%o, a2: 60%, b: 44%, c: 50%, total: 65%. all adverse events beside bleeding complications had been judged to be caused by the underlying disease and not directly related to r-hirndin. proportion of patients with event in the obse~wation period; death: a1 5.9%, a2 0%, b 16.774, c 0%, total: 7.3%, during rhirudin treatment: 1.2%; new tec: ai 5.9%, a2 0%, b 27.8%, c 0%, total 9.8%, during rhirudin: 2.4%; limb amputation: ai 3.9%, a2 0%, b 5.6°/0, c 0%, total: 3.7%, during rbirudin: 3.7% eaapoiat we conclude, that r-hirndin is an effective anticoagulant in hat typa ii patients allowing rapid re, coved" of platelet counts. compared to a historical control the r-hirudin group had a strongly reduced incidence of combined endpuint of mortality, limb amputation and new tecs. major bleedings occurred slightly more frequent than in the historical control. however, the dosage used in regimen b might have been too low, as flds group revealed an effective anticoagulatiun and increase in platelet counts in only 43% and concomitantly had the higl)est incidence of new tecs. key: cord-023209-un2ysc2v authors: nan title: poster presentations date: 2008-10-07 journal: j pept sci doi: 10.1002/psc.1090 sha: doc_id: 23209 cord_uid: un2ysc2v nan the boron neutron capture therapy (bnct) based on the interaction between 10 b isotope and thermal neutron has been highly noted in recent years as one of promising techniques for treatment of cancers. p-( 10 b)borono-l-phenylalanine (l-10 bpa), in which boron atom is enriched with 10 b isotope, is now using clinically as an effi cient 10 b carrier for treatment of patients in particular with malignant brain tumor and melanoma (1) . in order to develop a practical method for the synthesis of l-10 bpa, we have recently examined the enantioselective method utilizing the negishi reaction based on the coupling of 4-( 10 b)boronoiodobenzene derivative with 3-iodo-l-alanine derivative (2) . from the standpoint of diagnosis of cancers, the magnetic resonance imaging (mri) is noted as one of common techniques. in particular, mri based on the measurement of 19 f atom is becoming a remarkable one. to create practical materials utilizing as not only the 10b carrier but also mri probe, we had already synthesized the compounds containing both 10 b and 19 f atoms in a single molecule such as [4-( 10 b)borono-2,6-difl uorophenyl]-dl-alanine [dl-10 bpa(2,6f 2 )] 3., [4-( 10 l-proline is conformationally unique among coded amino acids in that its torsion angle is blocked (-65 ± 10) by its characteristic fi vemembered pyrrolidine ring structure and the preceding torsion angle (tertiary amide) can undergo cis (0) <-> trans (180) isomerization much easier than the secondary amides of the usual peptide bonds. in addition, its torsion angle is commonly found either in the right-handed 3 10 -/helical region (-30 ÷ -50, or cis' conformation) or in the left-handed, semiextended, region [-150 ±10, or trans', or poly-(l-pro) n conformation]. methylation at the c -position of a pro residue was suggested to block the preceding tertiary amide ( ) torsion angle of the resulting ( me)pro to the trans disposition and to restrict the , surface to the single region where the 3 10 / -helices are found. we have synthesized a large set of n -blocked, ( me)pro-containing, dipeptide n'alkylamides having the general formulas p-d-( me)pro-xxx-nhipr and p-xxx-d-( me)pro-nhipr, where p is ac or boc and xxx is d-ala, l-ala, aib, gly, d-( me)pro, or l-( me)pro. the results of the present x-ray diffraction analysis clearly show that the region of the conformational map overwhelmingly preferred by ( me)pro is indeed that typical of 3 10 / -helices, but the semi-extended, type-ii poly(pro) n helical region can exceptionally be explored by this extremely sterically demanding c -tetrasubstituted -amino acid. in addition, the known high propensity for -turn formation of the pro residue is even enhanced in peptides based on its c -methylated derivative. to complete the picture of the preferred conformation of ( me)pro, the synthesis and crystal-state investigation of a series of terminallyprotected homo-peptides from d-( me)pro are currently in progress in our laboratory the interest in guanidine-containing compounds is due to their important role in biological recognition processes. five-member cyclic n-amidinoamino acids represent hybrid structures, combining both proline rigidity and high positive charge of arginine guanidine group. structurally similar n-amidino-proline, n-amidino-pyroglutamic acid and cyclocreatine (2imino-1-imidazolidine acetic acid) were chosen as objects of our study. the problem of their incorporation into peptide structure requires use of substituted derivatives or effective guanidylation technique. recently we have presented the synthesis of mts-protected n-amidinoproline. nevertheless, its application in n-termini modifi cation of peptides shows slow condensation rate in classical as well as solid phase synthesis for short peptides (2-5 residues) or completely fails in case of longer ones (10 residues). on the other hand, polymer supported guanidylation of proline by bis-boc-protected carboxamidine-1hbenzotriazole seems to be preferred route to n-amidino-proline containing peptides. three methods of n-amidino-pyroglutamic acid synthesis have been investigated and model dipeptide has been acquired on wang polymer by the cyclization of guanidine-glutamic acid. for selective boc-deprotection on wang resin the literature technique has been utilized. however, in this case esi-ms and nmr analyses evidence for side-reaction of triethylamine alkylation by polymer linker fragment. hplc analysis shows n-amidino-pyroglutamyl-phenylalanine stability at acidic and physiological ph but fast ring opening in water solution at ph 9. for cyclocreatine application in peptide synthesis we suggest the use of its p-toluenesulfonate having good solubility in dmf and showing effective coupling in cl-hobt/dic condensation. the examples of namidino-amino acids practical application in peptide synthesis will be presented. tryptophan is often a key pharmacophore which determines the affi nity of peptide ligands for their receptors. cyclic analogues of tryptophan which introduce local constraints and reduce the fl exibility of the indol moiety are very valuable tools to probe the bioactive conformation of the peptide ligands. one of the possibilities to freeze indol moiety of tryptophan is the synthesis of the additional 6-member ring by the formation of 1,2,3,4-tetrahydro--carbolines. we report the synthesis of 1,3-disubstituted 1,2,3,4-tetrahydro--carbolines via the pictet-spengler reaction. methyl ester of tryptophan or dipeptides with n-terminal trp (trp-ala-ome, trp-leu-ome) were used as arylethylamine substrates and -amino aldehydes derived from l and d-amino acids were used as carbonyl components. we determined that there were no differences of the stereoselectivity of the pictet-spengler reactions in the case of trp-ome or trp-dipeptides. we also investigated the conformation of the newly created 6-membered ring in cis and trans diastereomers. the roesy spectra were used to assign the more stable conformation for each isomer. the conformations of cis isomers derived from tryptophan and dipeptides were the same and substituents on c-1 and c-3 were in both cases equatorial. the conformation of trans isomers were depended on the carboxyl part of trp. for methyl ester of tryptophan the ester group was axial, whereas for dipeptides we observed the opposite conformation with equatorial substituent on c-3. our results show that the pictet-spengler reaction can be successfully performed for amino acids and peptides and the conformation of the newly created 6-membered ring depends on the substrates and the size of c-1 and c-3 substituents. helical-screw handedness of peptides composed of diastereoisomeric cyclic amino acids nagano 3 helical-screw handedness in proteins is believed to result from thecarbon chiral center of l--amino acids. recently we have reported that the helical-screw sense of oligopeptides can be controlled without a chiral center on the peptide-backbone but by chiral centers at the side chain. that is to say, we designed and synthesized an optically active cyclic , -disubstituted amino acid (s,s)-ac(5)c(dom), in which the -carbon atom is not a chiral center but chiral centers exist at the side chain. conformational analysis of the (s,s)-ac(5)c(dom) peptides revealed that the hexapetide formed left-handed 3 10 -helices both in solution and in the solid state, and the octapeptide assumed a left-handed -helix. herein we designed new two diastereoisomeric cyclic , -disubstituted amino acids; (1s,3s)-and (1r,3s)-1-amino-3-(methoxy)cyclopentanecarboxylic acid (ac(5)c(om)) having chiral centers both at the -carbon atom and at the side chain. the amino acids (1s,3s)-and (1r,3s)-ac(5)c(om) were synthesized starting from l-(-)-malic acid. that is to say, at fi rst, the malic acid was converted to diiodide compound, and bisalkylation of dimethyl malonate with the diiodide gave a cyclic diester. monohydrolysis of the diester, followed by curtius rearrangement produced separable mixtures of (1s,3s)-and (1r,3s)-ac(5)c(om). we prepared both diastereoisomeric (1s,3s)-and (1r,3s)-homooligomers by solution-phase methods, respectively, and studied their preferred secondary structures using 1 h nmr, ft-ir, cd and x-ray crystallographic analysis. zobel, ansgar; gaus, katharina; wollschläger, katrin; nieß, anke; juodaityte, jovita; sewald, norbert organic and bioorganic chemistry, bielefeld university, germany novel highly active and specifi c dna binding molecules have a considerable potential particularly with regard to applications in medical science. the major and minor grooves of the dna serve as recognition areas. it is of high interest for chemical biology to control dna-binding abilities of synthetic molecules. a possible modulator is light in combination with photoswitchable molecules. triostin a is a natural bisintercalator which belongs to the quinoxaline antibiotics originally isolated from streptomyces s-2-210. it consists of a bicyclic depsipeptide with n-methylated amino acids and a cystine bridge. its heteroaromatic quinoxaline moieties are able to intercalate gc-specifi cally into the dna inducing a change in conformation and therefore inhibit the transcription by blocking spezifi c enzymes. tandem is the n-unmethylated derivative of triostin a which binds at selectively to the dna due to the change in the hydrogen bonding pattern. the exchange of the cystine bridge of tandem with substituted azobenzene units leads to photoswitchable triostin a analogs. the synthesis of the depsipeptidic basic structure which contains the quinoxaline moieties is introduced as well as the coupling with azobenzene amino acids. the double cyclization step is accomplished under pseudo high dilution conditions. in order to differ between cisand trans-confi gurations of the molecule, nmr-and ir-spectroscopy was carried out as well as the photoswitchability of different analogues was tested by irradiation and rp-hplc analysis. for synthetic peptide vaccine prototype development: (pc) , which has an heteroatom in its structure and their bioconjugates obtained by microwave and carbodiimide methods were explained. for pc obtaining, the synthesis of the monomer, 1azabicyclo[4.2.0]octane, (c), was carried out by organic methods which then will be used in polymer synthesis [1] [2] . consequently, polymers having different molecular weights and their water soluble bioconjugates were synthesized and the characterization of these polymers and conjugates were done by different methods such as uv, atr ft-ir, sec with four detectors and zeta sizer. for the chemical modifi cation of pc, bromoacetic acid was used and a water soluble polyampholyte synthesis was achieved with the quaternization of polymer. atr ft-ir spectra of pc was recorded and molecular weights, polidispersity values of polymers, mark-houwink constants and molecular diameters of the polymers were measured with size-exclusion chromatography with four-detectors (light-scattering, refractive index, viscosity and uv). with zeta sizer, size-analysis of polymer chains were done and their zeta potentials were measured. it is determined that molecular weights of the polymers were affected by the change in the amount and the type of initiators used and also from the polymerization media. analysing of having biodegradable characteristics of the synthesized polymers has been being reviewed. after synthesis and modifi cation of pc, its conjugates with antigenic peptides like avian infl uenza hemagglutinin (ha 98-106) ypydvpdya and rgdsggc cell receptor peptide were obtained. their characterization was also performed. japan; 3 in alzheimer fs disease research, sparing water-solubility and uncontrolled self-assembly of amyloid peptide (a ) 1-42 are signifi cant obstacles to establish an experimental system that clarifi es a pathological mechanism of a 1-42. to solve these problems, we herein disclose water-soluble "click peptides" based on an o-acyl isopeptide method. these peptides contain an o-acyl instead of n-acyl residue at the gly 25 -ser 26 of a 1-42, and are converted to a 1-42 via an o-n intramolecular acyl migration triggered by phchange (ph-click) or photo-irradiation (photo-click). these peptides had remarkably higher water-solubility than a 1-42. in addition, these peptides clearly adopted monomer state with a random coil structure, which were verifi ed by various physicochemical assays. importantly, we could establish an in situ system predominantly comprised of monomer a 1-42 as a result that the monomer click peptide was converted to a 1-42 quickly and quantitatively in accordance with ph-change. both selfassembly and conformational change of the produced a 1-42 in situ were observed with time. similarly, the photo-triggered click peptide afforded a 1-42 by uv-irradiation. because the in situ production of intact a 1-42 from the click peptides could overcome the handling problems of a 1-42, this strategy would provide a reliable experimental system for investigating a pathological function of a 1-42 in alzheimer fs disease. the antioxidant effect of introducing phenylpropenoyl moiety in either in n-terminal group of analgesic oligopeptides or in c-terminal end of opioide active amino acid, modifi cated with polyamines have been evaluated. the series of hydroxycinnamoyl peptide and amino acid amides were synthesized by standard method used in peptide chemistry. obesity is a major public health problem associated with morbidity and mortality and continues to increase worldwide. the gastrointestinal tract and the pancreas release hormones regulating appetite and body weight. obestatin is a recently described 23 amino-acids peptide derived from preproghrelin. it was identifi ed by bioinformatic prediction (1) . obestatin decreases food intake and body weight gain, decelerates gastric emptying, promotes sleep in rat, inhibits water drinking. it has been described that obestatin effects on feeding behavior and an u-shaped dose-response relationship was found: low doses (0.01-3 nmol/kg) and high doses (1-3 nmol/kg) were ineffective. the 23 amino-acid carboxy-amidated human obestatin nalapropheaspvalglyilelysleuserglyvalglntyrglnglnhissergln alaleunh 2 and corresponding rat obestatin hpheasnalapropheasp valglyilelysleuserglyalaglntyrglnglnhisglyargalaleunh 2 were synthesized by different schemes. synthesis was developed on the basis of solid phase synthesis of protected peptide fragments followed by their assembly into the fi nal product. fragments 1 -8, 9 -13, 1 -13, 14 -20, 14 -22 were built on the acid-labile 2-chlorotrityl chloride resin using the orthogonal fmoc/tbu strategy. fragment 21-23 (hcl•hargalaleunh 2 ) was obtained in solution. the assembly of the full-length peptide was carried out by fragments coupling in solution or using solid phase method. the free peptide was obtained by treating the protected peptide with mixture of tfa: h 2 o:tis (95: 2.5: 2.5). the peptide was purifi ed with hplc and isolated by lyophilization with 95%+ purity according to analytical reversed-phase hplc. the mass of molecular ion determined by maldi-tof spectrometry was in fi ne agreement with calculated value. the inverstigation of biological actions of obestatins is in progress. straightforward synthesis of enantiopure tfm-amino acids from chiral cf 3 brigaud, thierry; chaume, grégory; huguenot, florent; caupène, caroline university of cergy-pontoise, france trifl uoromethylated amino acids (tfm aas) are current synthetic targets due to their unique properties and their synthesis in enantiopure form remains a challenge. we will report that chiral 2-trifl uoromethyl-1,3oxazolidines (fox) are highly versatile synthons for the stereoselective synthesis of various functionalized -trifl uoromethylamino compounds such as -amino nitriles , -and -amino acids, diamines and amino alcohols. 1, 2 moreover, trifl uoropyruvate-based oxazolidines proved to be very valuable building blocks for the stereoselective synthesis of both enantiomers of -trifl uoromethyl proline and -tfm-pyroglutamic acid, in enantiopure form. 3 moreover, the synthesis of the (s)--tfm--allylglycine and the novel (s)--tfm norvaline were achieved in a few steps from this starting material. we will also report a recent straightforward synthetic route to tfm-dihydroxyprolines in enantiopure form from trifl uoropyruvate-based chiral oxazolidines. chitinases catalyse the hydrolysis of chitin, the natural homopolymer of (1,4)-linked n-acetyl-d-glucosamine. chitin is a key structural component of the cell walls, exoskeletons, and eggshells of pathogenic fungi, insects, and nematodes, respectively, which all rely on the ability to hydrolyse chitin at specifi c points in their life cycles. chitinase inhibitors are now attracting considerable interest as novel fungicides and insecticides, as well as chemical tools to study human diseases as diverse as asthma and malaria. in this context, the cyclic pentapeptide natural products, argifi n and argadin, are two exciting inhibitors which pose some interesting synthetic challenges. the argifi n structure includes two sensitive -linked asp residues, as well as an unusual carbamoylated arg side chain, while argadin contains a unique aspsemialdehyde residue, that is cyclised to the peptide backbone to generate a potentially labile hemiaminal. we will describe improved routes to both compounds that allow us to avoid signifi cant side reactions such as aspartimide formation and homoserine-mediated backbone cleavage that are observed in our previously reported syntheses. [1, 2] this is achieved by carrying out the assembly and cyclisation of both peptides, including key side chain derivatisation steps, entirely on solid phase. the application of the strategies devised to the automated synthesis of argifi n and argadin and related natural products will be described. during the last two decades combinatorial chemistry has become an important tool for the quick synthesis of large numbers of small molecules used, for example, in the generation of the new lead structures for medicinal applications. additionally, it allows rapid access to diverse chemical libraries with novel structures and properties. small molecules libraries are generally prepared on solid support simplifying tedious purifi cation steps of the intermediates and facilitating the entire synthesis. piperazines and keto-piperazines are amongst the important backbones in today's drug discovery. the piperazine framework has been defi ned in medicinal chemistry as a "privileged scaffold". it is a molecular backbone with versatile affi nity properties representing a frequently-occurring binding motif, and providing potent and selective ligands for a wide range of biological targets. the high number of positive hits revealed in biological screens with the piperazine scaffold urged chemists to develop plenty of different synthetic methods that allow fast and effi cient building of these heterocyclic systems on solid support as well as homogenous chemistry. however, the majority of these methodologies is not stereospecifi c and the complex mixtures of the stereoisomers are generated. in order to preserve important chiral centers in potential hits we propose to use pipearzine backbone, initially prepared in optically pure form, bearing various tethers with orthogonally protected groups applicable via solid phase organic chemistry (spoc). we will describe a novel synthesis of keto and diketopiperazine building blocks. these chiral building blocks are applied in "around-the-scaffold" modifi cation strategy by spoc, introducing valuable physico-chemical properties in 3 independent diversity points. solid-phase strategies for constraining peptide structures can serve for elucidating the relationships between conformation and activity. for example, the systematic substitution of natural amino acids by their aza-amino acid counterparts has been accomplished using a fmoc strategy featuring couplings with aza-amino acid chlorides to provide insight into the biologically active conformers of the melanocortin receptor agonist ac-his-d-phe-arg-trp-nh2 as well as the calcitonin gene-related peptide antagonist [d31, p34, f35]cgrp27-37 (1) (2) (3) . employing cyclic sulfamidates, we have now developed fmoc strategies for the effective solid-phase introduction of alpha-and beta-amino gamma-lactam constraints into peptides. since their pioneering use in a somatostatin analog with about 9 fold greater activity (4), lactam restraints have been used to study a variety of relevant targets (5, 6) . our strategies now provide effective means for performing lactam scans of biologically active peptides as demonstrated using the growth hormone secretagogue ghrp-6, and an allosteric modulator peptide antagonist of the il-1 receptor. our presentation will focus on recent developments in the solid-phase chemistry for synthesizing such constrained peptide analogs and the relationships between peptide conformation and biology, elucidated by these novel methods. references: 1 barcelona science park, university of barcelona, 08028-barcelona, spain; 2 linaclotide is a 14-residue peptide currently undergoing phase ii clinical trials for the treatment of gastrointestinal diseases such as chronic constipation (cc). linaclotide, which can be administered orally, is an agonist of the guanylate cyclase type-c receptor found in the intestine. from a structural point of view, this small peptide presents a constrained structure with the presence of three disulfi de bridges between cys1-cys6, cys2-cys10, and cys5-cys13. h-cys( 1)-cys( 2)-glu-tyr-cys( 3)-cys( 1)-asn-pro-ala-cys( 2)-thr-gly-cys( 3)-tyr-oh in order to reach the large amounts required for a marketed peptide, an effi cient synthesis needs to be attained. to optimize the synthesis, its fundamental limitations need to be determined and addressed. in the case of linaclotide, the key points are related to the numerous cys (some of them consecutive) present in the peptide, for two reasons: the potential risk of racemization upon assembling the linear chain, and the misfolding of the three disulfi de bridges. for that, the concourse of different protecting groups and folding conditions as well as the analysis of the disulfi de bridges in the fi nal folded peptide has been studied and will be discussed in this presentation. balalaie, saeed 1 ; arabanian, armin 1 ; mohammadnejad, mahdieh 1 ; gross, juergen h. 2 1 university, iran (islamic rep.); 2 university, germany gnrh analogues have been used for the treatment of steroid-dependent tumors, such as prostate and breast cancers. the development of more potent gnrh analogues depended largely on the important made in the science of peptide chemistry. ugi-4mcr reaction is known for the synthesis of amide bond. we wish to report herein an effi cient method for the synthesis of some gnrh analogues based on ugi reaction using fourcomponent reaction of n and c-terminus peptides, aromatic aldehydes and isocyanides. this ugi-4mcr could describe to build up novel gnrh analogues deriving from triptorelin and gonadorelin. all of the products were purifi ed using preparative hplc and the structures were assigned according to maldi mass spectrometry data. peptide synthesis is based in a proper combination of protecting groups and in the right choice of the right coupling method. nowadays, almost all peptide bond formed are carried out in the presence of 1hydroxybenzotriazole (hobt) or its derivatives (hoat, cl-hobt). thus, hobt derivatives are used in combination with a carbodiimide or another coupling agent or built into a standalone reagent such as immonium ( thus, a replacement of hobt should be found for preparation of peptides for research purposes and, more important, for the production of peptide based apis. herein, several alternatives to hobt will be discussed taking into account the explosivity properties. furthermore, a new family of immonium salts, which incorporates an proton acceptor in its carbocation skeleton. the novel proton acceptor coupling reagent has shown superiority to the described previously. an oxygen in the carbocation moiety confers to the reagent more solubility, enhances coupling yields and decreases racemization, allowing the use of just one equivalent of base. examples on the use of the non-explosive replacement for hobt together with carbodiimides or built into phosphonium and immonium salts, with the proton acceptor, will be discussed. thiocarbamate-linked peptides by chemoselective peptide ligation using phenylthiocarbamate chemistry peptide chemical ligation chemistries, which allow the chemoselective coupling of unprotected peptide fragments, are useful tools for synthesizing native polypeptides or unnatural peptide-based macromolecules. native chemical ligation (ncl) (1), staudinger ligation (2) lead to the formation of a native peptide bond at the ligation site. other methods result in the formation of unnatural covalent bonds such as oxime (3), thioester (4) linkages. these methods are of great interest when native peptide bonds are not absolutely required. in this context, we examined the potential use of the phenylthiocarbamate group in ligation chemistry. the phenylthiocarbonyl group can be easily introduced into peptides on alpha or epsilon amino group using phenylthiochloroformate and standard solid-phase method (5) . it reacts chemoselectively with cysteinyl peptides to give an alkylthiocarbamate bond. s,n-shift of the alkylaminocarbonyl group from the cys side chain to the alpha-amino group did not occur. the method was used for linking two peptides chain through their n-termini, for the synthesis of a cyclic peptide or for the synthesis of di-or tetravalent multiple antigenic peptides. synthetic peptide derivatives which contain secondary amide moiety at c-terminus are inhibitors of serine and cysteine proteinases and thus could be effi ciently used as ligands in affi nity chromatography in order to isolate these enzymes. there is a number of chemical syntheses of amidous peptide derivatives in the literature, but the reaction yields are usually low and reaction conditions and purifi cation procedures are too complicated. it is also known that the application of enzymatic catalysis allows to obtain high reaction yields with simultaneous simplifi cation of synthetic and purifi cation procedures, and at the same time preserves the optical purity of target compound. subtilisin 72 sorbed on silochrome could be effi ciently used as catalyst of acylation of secondary amides by the esters of acylpeptides. subtilisin's wide substrate specifi city allows to carry out the syntheses with peptides which contain hydrophilic, hydrophobic, dicarbonic-and diamino-acids at c-terminus. piperidine, morpholine, indole, diethylamine and other secondary amines are used in the syntheses as the amino-components. the reaction yield equals about 60-80% in dependence of the nature of c-terminal amino acid of acylating peptide, pka of amine and the hydrophobicity of the fi nal compound. the distribution of the reaction product in liquid and solid reaction phases is investigated, the optimal reaction conditions for enzymatic acylation of secondary amines are established. all the compounds obtained are characterized by its nmr and mass spectra. the inhibition constants for some proteolytic enzymes with the compounds of research are defi ned. 1 unnatural amino acids are becoming increasingly important substrates in modern drug design, synthesis and discovery research. in particular, arylhistidines naturally occur in the active site of the heme-copper oxidases as well as in cytotoxic and antifungal marine peptides (1) . a method of choice for the preparation of unsymmetrical biaryl systems is the suzuki-miyaura cross-coupling of an aryl halide with an arylboronic acid. it has been shown that microwaves signifi cantly enhance this reaction leading to higher overall yields and purities as well as shorter reaction times. although a great variety of biarylic compounds have been prepared following this approach, so far, it has not been applied to the arylation of the histidine imidazole ring. initially, we studied a methodology for the synthesis of 5-arylhistidines via a microwave-assisted suzuki-miyaura cross-coupling reaction in solution (2) . taking into account the advantages of the synthesis on solid support, such as the avoidance of tedious work-up which are particularly valuable for palladium-catalyzed reactions, we studied the application of the above methodology on solid-phase. here, we report the suzuki-miyaura reaction between a 5-bromohistidine and an arylboronic acid on solid support. the reaction conditions were optimized by varying several parameters such as the solvent, the reagent concentrations and the reaction time. the optimized conditions were subsequently applied to the synthesis of peptides containing a 5-arylhistidine residue. this work constitutes the fi rst suzuki-miyaura coupling involving the imidazole ring of a histidine on solid support. microwave-assisted attachment of fmoc-amino acids to resins via triazine "superactive esters". the substitution level of the new functionalized resin was calculated following a standard protocol based on the spectroscopic measurement of the soluble chromophore piperidine-dibenzofulvene. coupling reactions to the resin proceeds at room temperature in 3 hours. moreover we carried out the time-consuming attachment of fmoc-amino acids to the resin by an automatic monomode microwave (mw) instrument (liberty, cem), in fact microwave is proposed as a valid alternative to enhance effi ciency of coupling reactions and it has been widely applied to spps. novel peptide-hetorocycle conjugates: derivatives of 3-(benzimidazol-5-yl)alanine (1) . considering the biological activity and complexing abilities of benzimidazoles we developed a direct solid-phase synthesis of benzimidazole-peptide conjugates, expecting these new compounds to express novel biological properties (2) . the peptide-heterocycle conjugates are obtained by on-resin reaction between aldehydes and peptides containing a specially designed -(3,4-diaminophenyl)alanine residue (3) . the stoichiometric amount of aldehyde leads to 2'-substituted 3-(1h-benzimidazol-5-yl)alaninecontaining peptides, the increase in aldehyde content results in 1`,2`disubstituted derivatives. the reaction with dialdehydes gives novel amino acid residues containing tricyclic systems, in the case of ophthalic aldehyde -the pyrido-[1,2-a] benzimidazole. the compatibility of our method with the fmoc solid phase peptide synthesis protocols was proven by the synthesis of analogues of immunosuppressory fragments of ubiquitin and hla-dq [4, 5] . the biological activity of the modifi ed oligopeptides was compared to that of original fragments 6. as well as to similar quinoxaline-peptide hybrids. the collision-induced dissociation of substituted benzimidazole-peptide conjugates leads to characteristic fragmentation ions, which may be used as a diagnostic tool in the fragmentation of the benzimidazole-peptide hybrids. cardona, valérie; oswald, benoit genzyme pharmaceuticals, switzerland dehydroamino acids are an important class of compounds due to their presence in many biologically active natural products including the antrimycins, tentoxin, phomopsin a, or the phosphatase inhibitors like microcystin and nodularin. in the last decade an increasing interest in -branched dehydroamino acids has been developed based on their importance as commodity chemicals and value as tools in structure relationship studies. the incorporation into a peptide of such unsaturated amino acids introduces an element of conformational rigidity, as well as changes in reactivity, allowing for the development of high affi nity ligands for receptors. a variety of methods exist for the synthesis of dehydroamino acids. some of these approaches rely on thermodynamic control to dictate the alkene geometry. if there is no strong thermodynamic preference, or if the desired product is not the thermodynamically favoured isomer, the existing synthetic methods are often ineffective. we describe here an effi cient stereoselective method for the synthesis of , -branched dehydroamino acids (iii) from -aryldehydroamino acids (i). the -aryldehydroamino acids (i) were prepared from commercially available z/boc-phosphonoglycine trimethylester and aldehydes using the schmidt protocol. the transformation of (i) into their -bromodehydroamino acid derivatives (ii) is performed by a hoerrner reaction in a very good yield giving the trans derivatives. suzuki cross-coupling on (ii) with a boronic derivative generated the high value building blocks (iii). a variety of side chains can be incorporated using this type of reaction. the stereochemistry of these compounds has been determined using noe enhancement experiments. we report here a scalable and high yielding synthesis of stereospecifi c , -aryldehydroamino acid derivatives. versatile methods for synthesizing acyl-tetramic acids peptide analogs. davidov, gali; mozes, tamar; khandadash, raz; byk, gerardo bar ilan university, israel acyl-tetramic acid derivatives have been identifi ed as potential antiviral and antibacterial compounds. in this work we have improved their synthesis starting from tetramic acid lactams using peptide coupling reagents such as bop under microwave heating for generating the carbon-carbon bond of the acyl-tetramate. using this procedure we have synthesized a number of new tetramic acid derivatives in solution and generated a series of acyl-tetramic acid building blocks that were introduced into peptides using conventional spps methods. additionally, we present here a new solid phase microwave assisted synthesis of acyltetramates on pre-synthesized peptides and amino acids. antibacterial and antiviral activity of the products will be presented. peptide sequence and include all side-chain groups. in practice this is diffi cult to achieve. the task of making a turn mimetic can be regarded from the perspective of the backbone dihedral angles -to make a beta turn mimetic the phi and psi angles of two consecutive residues have to be controlled. limiting the dihedral angle space is most effectively accomplished with a cyclic constraint -one or more may be used. in this case a single medium ring cyclisation from the (i) carbonyl to the (i+3) amine -in place of the classical hydrogen bond -forces the four backbone dihedral angles into a turn conformation. due to the polyfunctional nature of peptide systems the introduction of unnatural constraints can be synthetically challenging. in the present case a number of side reactions were encountered and had to be overcome. some were well known such as diketopiperazine formation and others involving transannular interactions were unexpected and gave rise to interesting byproducts. ultimately the side reactions were overcome by choice of cyclisation position and synthetic modifi cations. auto-assembling antimicrobial cyclic pseudopeptides including aza-3 -amino acids antibiotic resistance of pathogens against conventional antibiotics is increasing at a rate that far exceeds the pace of new development of drugs. so, antimicrobial peptides, both synthetic and from natural sources, have raised interest as potential useful drugs in the future. however, due to proteolytic degradation, peptides are not ideal candidates for pharmaceutical development. that is why numerous researches try to develop non natural peptidic analogues for enhancing metabolic stability, bioavailability, and biological absorption. in this class of peptidomimetics, pseudopeptides consisting exclusively or including aza 3 -amino acids have emerged as a promising new class of compounds that favour hydrogen bond formation and can enhance biological activities when compared to natural parent peptides (1) . we have designed "mixed" cylic pseudopeptides composed of -and aza3 -amino acids that target bacterial cell wall and induce the death of the pathogens. particularly, some of these cyclic pseudopeptides have broad spectrum antibactericidal activities on gram positive and gram negative bacteria with low minimum inhibitory concentrations (mic). on the other hand, this type of molecules is not haemolytic and cytotoxic at antimicrobial activity levels(2) now, we try to explain the mechanism of action of our pseudopeptides that act on the microbial membranes. with nmr studies we have demonstrate that in solution cycles auto-associate at high concentrations and we investigate their behaviour in presence of small unilamellar lipidic vesicules (suv) (3) . this phenomenon of auto association seems to be facilitated at the lipid interface. liu, hongqiang 1 ; pattabiraman, vijaya r. 2 ; vederas, john c. 1 1 university of alberta, canada; 2 lantibiotics are a class of antimicrobial peptides containing lanthionine (1) and/or -methyllanthionine (2) residues in cyclic moieties, and are currently used for food preservation. they also have potential as human therapeutics. lacticin 3147 is a two-peptide lantibiotic produced by l. lactis subsp. lactis dpc3147, and its components (a1 and a2) are active against a wide range of gram-positive bacteria by a synergistic mechanism in sub nm concentrations. however, the oxidation of the thioether of lanthionine (1) and -methyllanthionine (2) is a major source of lantibiotic instability and loss of activity. to investigate structure-activity relationships and determine whether sulfur can be replaced, an analogue of lacticin 3147 a2 in which oxygen atoms replace sulfur was synthesized by solid phase peptide synthesis. utilizing the stereochemicaly pure oxa-lanthionine (3) and oxa--methyllanthionine (4) with orthogonal protecting groups, the conformationally constrained tricyclic moiety in oxa-lacticin 3147 a2 was constructed. the results of biological evaluation of the oxidatively stable analogue will also be described. controlling -helical secondary structure of oligopeptides and its use as a chiral catalyst replacement of -hydrogen atom of -amino acids results in ,disubstituted amino acids. as an , -disubstituted amino acid, achiralaminoisobutyric acid (aib) is well-known, and widely used to construct helical secondary structures of oligopeptides. the helical secondary structures constructed by using aib usually show 3 10 -helices, but nothelices in the case of short oligopeptides. recently we designed and synthesized a chiral cylic , -disubstituted amino acid (s,s)-ac 5 c dom , in which the -carbon atom is not a chiral center but chiral centers existing at the side chain. homooctapeptide composed of (s,s)-ac 5 c dom formed a left-handed -helix both in solution and in the solid state. furthermore, in the case that the (s,s)-ac 5 c dom was incorporated into l-leuhexapeptide, the hexapeptide cbz-{l-leu-l-leu-(s,s)-ac 5 c dom } 2 -ome preferentially formed a right-handed -helix in the crystal state, whereas the hexapeptide cbz-{l-leu-l-leu-aib} 2 -ome formed a right-handed 3 10 -helix. the fi nding that the propensity of cyclic amino acid ac 5 c dom is to form -helix over 3 10 -helix, stimulated us to use the -helical oligomer containing the cyclic amino acid as an asymmetric catalyst. we prepared several l-leu-oligopeptides containing , -disubstituted amino acids, analyzed their preferred secondary structures, and studied a enantioselective reaction of prochiral substrate using -helical oligopeptides as a chiral catalyst. fbp28 domains: spps using pseudoproline and depsipeptide approaches, stability and structure of glutamine-rich analogs (nature biotech., 2008) . one peptide will soon be tested in clinical studies. the success depends on effi cient synthetic access to large amounts of the peptide. systematic modifi cations of the lead structure hbvpres/2-48 have to be performed resulting in a panel of peptide variants to be studied with respect to their applicability, pharmacokinetics and serum stability. we selected a series of peptides carrying deletions, point mutations, d-amino acid exchanges and sequence permutations. the syntheses of 40 derivates were performed by fmoc-solid-phase synthesis on a rink amide am resin using hbtu/dipea activation on an applied biosystems 433a peptide synthesizer. after completion of the peptide sequence, stearic acid was attached to the n-terminus. the products were deprotected and detached from the resin by tfa treatment and subsequently purifi ed by hplc. the stability of the peptides was determined in human serum. we were able to synthesize all hbvpres lipopeptides in acceptable yields (7-40%). preparative hplc was used to obtain intended compounds in high purity as determined by hplc and esi mass spectrometry. the serum stability studies revealed exceptionally long biological half-lives (t1/2 > 24 h for all lipopeptides) mainly due to the fatty acid residue. the peptides belong to a class of intrinsically unfolded peptides and are therefore not prone to aggregations within the synthesis. consequently solid phase peptide synthesis provides a suitable access to the peptides described. it can be speculated that the peptides are protected against degradation due to an association via their lipophilic end to serum proteins. sugar derivatives for self-assembling -cyclic peptides brea last years, numerous inorganic and organic nanotubes have been developed. self-assembling peptide nanotubes (spn) made from cyclic peptides have structural and functional properties that may be suitable for various applications in biology and material science. recently, our group have reported , -cyclic peptide ( , -cp) that forms highly stable homo-and/or heterodimers with partial hydrophobic cavities. in the present communication we will describe the design, synthesis and applications of a new class of self-assembling -cyclic peptides containing sugar derivatives that modify both the inner and the outer surfaces of the resulting supramolecular entities. peptido2.rotaxanes: can a tetramide macrocycle travel to a station by wrapping up around a helical peptide thread? we are currently expanding this fi eld by synthesizing and studying the properties of new sets of peptido2.rotaxanes. the initial set examined includes symmetrical, achiral compounds with a fmoc stopper at each terminus, threads built up with a central fumaric diamide station and two helical aib ( -aminoisobutyric acid) homo-peptides of different lengths (from 1 to 5 residues), and an aromatic tetramide macrocycle. these supramolecular systems have been characterized spectroscopically and two of them by x-ray diffraction as well. the fundamental interactions between macrocycle and thread (the same interactions offering the major contribution to the template-directed preparation of this family of molecules) are intercomponent h-bonds comprising the four amide groups in the ring and the two amide bonds in the fumaric derivative. the second, more complex, set of peptido2.rotaxanes examined is represented by non-symmetrical compounds involving a thread based on a central helical -(aib) 6 -peptide linker and two stations of opposite chirality (a -d-leu-gly-gly-tripeptide at the n-terminus and a fumaric diamide-l-leu moiety at the c-terminus). as stoppers and the macrocycle, we selected two diphenylacetyl groups and the usual aromatic tetramide, respectively. as spectroscopically assessed, the macrocycle initially positions on the fumaric diamide-l-leu station. subsequently, by using photons as stimuli to induce the fumaric<->maleic equilibrium, we were able to switch partially the relative macrocyclebinding affi nity in favor of the -d-leu-gly-gly-tripeptide station. this is the fi rst example of a rotaxane where the ring makes a journey to one of the stations by wrapping up around a helical peptide thread. synthetic novel n phenyl and bi-phenyl tetracarboxamides bis peptides. an approach to dna threading intercalators as expected potential pharmaceutical carriers for catatonic agents. naphthalene diimides were reported to bind to dna opposite grooves via the threading intercalation mode (1, 2) . on the other hand, peptides constitute an excellent class of molecules for rapid drug discovery and lead optimization. the title bis dipeptides may, consequently, offer signifi cant dna biological probes. potential anticancer drugs or drug carriers could thus be presumed. herein, the title bis peptides a and b were suggested and synthesized as new prototype candidates of such class compounds. pre-assembled and purifi ed peptides via the conventional methods of peptide synthesis are, subsequently, coupled to either 1,2,4,5-benzene tetra-carboxylic dianhydride or 1,4,5,8-naphthalene tetra-carboxylic dianhydride. the expected structures of obtained a and b as preliminary candidates were confi rmed via the chemical, chromatographic and spectroscopic methodologies. the chemistry of both a and b will be discussed. synthesis of other candidates, the corresponding biological, pharmacological and physicochemical investigations are under realization. x = phenyl or bi-phenyl ring [compound a and b respectivel 63 the fragment pth(1-34) is suffi cient to bind and activate the pth type i receptor (pth1r). the molecular mechanisms by which pth binds to and activates the pth1r have been extensively investigated. recent investigations focusing on the interaction of n-terminal modifi ed fragments pth(1-11)nh2 with pth1r showed that certain modifi cations can increase signalling potency and that enhancement of the -helicity in the pth(1-11) sequence yielded potent analogues of pth(1-11)nh2. the design of cyclic analogues represents a widely used strategy to increase peptide stability and potency. the structural constraint induced by cyclization reduces conformational fl exibility and may enhance potency, selectivity, stability and bioavailability as well as membrane barrier permeability. initially, the work was concentrated on conformational constrains in n-terminal such as the introduction of ctetra-substituted amino acids. global restrictions in the conformation of a peptide are possible by limiting the fl exibility of the peptide strand through cyclization. to this purpose, the amino acid side chains that are not involved in receptor recognition are connected together or with the peptide backbone. previous works on the role of side chains of aa determined through d-scan, analogues which maintained better -helical structure, contained d-gln in position 6 and 10. recently gardella and co-workers reported that an analogues of pth(1-11) cyclized between 6 and 10 residues exhibited almost the same activity of linear analogues. so, in this work two new potent cyclic analogues in position 6 and 10 were synthesized directly on spps, using side chains of lys and glu or ser. then they were biologically tested and analyzed by cd, according to our previous work. boudreau, marc; vederas, john university of alberta, canada the neopetrosiamides a and b are two diastereomeric tricyclic peptides that inhibit amoeboid invasion of human tumor cells. they were isolated by anderson and co-workers from the marine sponge neopetrosia sp. collected in papua new guinea, with their subsequent structure elucidation by the same group in 2005. the peptides are 28 residues in length and contain three disulfi de bonds, as well as the unusual amino acid methionine sulfoxide at position 4. the two peptides differ only by being epimeric at this sulfoxide functionality. we report the total synthesis of neopetrosiamides a and b as well as an analog wherein the methionine sulfoxide has been replaced by norleucine, the carbon analog of methionine. the strategy involved solid phase peptide synthesis to generate the linear species, and selective formation of the disulfi de bonds from orthogonally protected cysteine residues. synthesis of mimetics of the antibody 82d6a3: a new class of antitrombotics. in the lab for tromboses research of the kulak (belgium) an antitrombotic antibody (ab) called 82d6a3 has been characterized (1) . the ab inhibits the interaction between the plasma peptide von willebrand factor (vwf) and by injury exposed collagen, a prerequisite interaction in thrombusformation in arteries (high blood shear). it has been shown in vivo that the ab 82d6a3 has an antitrombotic effect without showing the bleeding complications that known antitrombotics exhibit. this makes the ab an attractive lead for antithrombotic drug design. by x-ray and mutagenesis studies, the paratope (active site) of the antibody has been determined. 6 amino acids, discontinuous in the primary structure of the antibody but a quasi linear unit in space, play an important role in the interaction. incorporation of the functional groups of the amino acids and fi xation of the secondary structure of the paratope will be the aim in the design of the paratope mimetics. with this rational design we will try to develop an orally available, synthetic paratope peptidomimetic with the same antitrombotic effect as the antibody. the development (solid phase and solution peptide synthesis) of the mimetics is a stepwise process. in each step conformational restrictions are introduced. in this we hope to divert away from the peptide and go to a drug like molecule. 1 peptide ligands for sh2-and ptp domains containing phosphotyrosine are of great interest to infl uence the activity of kinases, phosphatases and other functional proteins. backbone cyclization can help to stabilize these ligands against proteolytic degradation and to form their bioactive conformation. till now backbone cyclization was performed with bifunctional and in few cases with trifunctional amino acids but not with phosphotyrosine. the assembly of such peptides requires preformed building units. because the necessary reductive alkylation of phosphotyrosine derivatives leads only to very low yields we used n-functionalized pseudodipeptides with the unprotected phenolic hydroxyl group. for fragment condensation we synthesized building units of the common structures: fmoc-aaø[co-n(x)-(ch2)n-nh-alloc)tyr(oh)-oh and fmoc-aaø[co-n(ch2)m -cooall)tyr(oh)-oh. depending on the steric hindrancy of the n-terminal amino acid these pseudodipeptides were synthesized in solution or at sasrinresin. they were purifi ed by fl ash chromatography and analytically characterized by hplc, esi-ms and nmr. we tested our strategy on the synthesis of an octapeptide-ligand for the n-terminal sh2-domain of the phosphatase shp-1: glu-gly-leu-asn/abu-ptyr-nle-asp-leu-nh2. couplings of the dipeptide units were performed with pybop, stepwise coupling to the peptide fragments with unprotected phenolic hydroxyl group with pentafl uoro phenylesters. after fi nishing the assembly the obtained polymer bound octapeptides were consecutively cyclized, phosphorylated, removed from the resin and purifi ed by hplc. based on elucidated side reactions the synthetic strategy was optimized. the obtained backbone cyclic and phosphorylated ligands were tested for their infl uence on the phosphatase activity of shp-1. the found enzymatic activities are correlated to size, direction, hydrophobicity and conformational fl exibility of the lactam bridges. biology and medicine. in this context, we have devised a new family of compounds named « polyamide amino acids » (paas), constituted by a pna (peptide nucleic acids) backbone mimicking rna sugarphosphate backbone, onto which aminoacid residues are linked. therefore, these paas could constitute a new type of rna ligands, liable to specifi cally interact with an rna target through an original interaction mode. to assess the ability of paas to be potential rna binders, we fi rst prepared 8 tetra-paas (t1-t8) via solid-phase synthesis, starting from 4 paa monomers deriving from alanine, phenylalanine, lysine and arginine residues. interactions of the 8 tetramers with a hiv-1 tar rna fragment, taken as a target model, was investigated by fl uorescence spectroscopy and circular dichroism. to give insights about specifi city, binding affi nities to tar were also assessed using an excess of a trna mixture. results showed kd values varying from 0.08 to 2032 m, indicating the importance of the aminoacid side chains in the interaction. thermodynamic analyses revealed that even if electrostatic interactions play a part in the complex formation, the binding is enthalpy-driven, highligthing the importance of non-electrostatic interactions in the recognition process. these results are of special interest since rna/ ligand association specifi city is typically assumed to be due to shortrange non-electrostatic interactions. moreover, t1-t4 were shown to be specifi c to tar in the presence of an excess of trna. all together, these results are encouraging and they highlight the potential of paas as rna ligands. indeed, the use of only four paa monomers as building blocks leads to tetra-paas displaying both affi nity and specifi city for their rna target. studies on the solid phase synthesis and selective detection of peptide derived amadori products by mass spectrometry stefanowicz mass spectrometric analysis of glycation products of proteins and peptides attracts increasing attention (1) . peptide-based amadori products could be used as markers of diabetes mellitus, which makes them the subject of interest in clinical chemistry. recently, several procedures of siteselective synthesis of amadori-modifi ed peptides has been published [2, 3] . in this communication we will present the synthesis of a new, fully protected derivative of glycated lysine which was successfully applied as a building block for incorporation of the glycated lysine moiety into peptide chain according to the standard fmoc-solid phase synthesis protocol. we will also present a new and straightforward method of selective detection of peptide-derived amadori products by esi-ms basing on characteristic neutral losses in the sugar moiety. the proposed approach in contrast to the neutral loss scanning, which requires triple quadrupol mass spectrometer, can be performed on the instruments with higher resolution and sensitivity, like q-tof . new apa inhibitors interacting with the s1 subsite bring new insights in the apa substrate specifi city mediated by the calcium ion. 3 aminopeptidase (apa, ec 3.4.11.7) is a membrane-bound zinc metallopeptidase involved in the maturation of brain angiotensin iii, a peptide which exerts a tonic stimulatory action on blood pressure in hypertensive animals (1) . therefore, developing inhibitors of this enzyme should result in new antihypertensive agents with possible new application in the treatment of certain forms of hypertension (2) (3) . we and others have previously reported the design of such inhibitors (4) (5) . nevertheless, the improvement of the affi nities of the inhibitors is relatively impaired since the three dimensional (3d) structure of the enzyme is not available. with the aim of getting insight into inhibitors optimization, a 3d model of apa was constructed in our group as an alternative (6) . furthermore, it is well established that apa substrate specifi city and enzymatic activity are calcium dependent. in order to render this model more accurate and so on to identify the amino acids residues involved in calcium binding, we have designed new inhibitors able to explore the apa s1 subsite to better understand its specifi city. we will report the synthesis of these new inhibitors, as well as an inversion of the specifi city of apa s1 subsite in absence of calcium. these results and the identifi cation of the amino acids implicated in calcium binding will be discussed on the basis of site-directed mutagenesis studies. one of the designed inhibitors, ni 926 (ki = 70 nm) is to date the more potent inhibitor of apa activity measured without calcium. altogether, these data should allow the improvement of apa inhibitors by structure aided design. synthesis, resolution, and absolute confi guration of bpaib, a benzophenone-containing c -tetrasubstituted -amino acid photoreactive amino acids with benzophenone side chains, the prototype of which is bpa (4-benzoyl phenylalanine), have found numerous applications as photo-probes for covalent modifi cation of enzymes and receptors, as well as in intramolecular quenching by a nitroxide free radical in trichogin peptide analogs. however, the remarkable fl exibility of the bpa side chain may question the extrapolation of results of photo cross-linking experiments and photophysical data to protein mapping and intramolecular distances, respectively [m. saviano et al., chembiochem, 2004, 5, 541-544 and references cited therein]. to overcome this problem, we designed a new "constrained bpa" amino acid, bpaib, belonging to the sub-class of the ci <->ci cyclized, c -tetrasubstituted -amino acids (strong -turn and helix inducers in peptides). racemic boc-bpaib-oh was prepared by bis(alkylation) of ethyl isocyanoacetate under phasetransfer conditions with 1-benzoyl-3,4-(bis)bromomethyl benzene as alkylating agent, followed by acidic hydrolysis, n -boc protection, and saponifi cation of the ester function. resolution was achieved through poster abstracts the terminally-blocked dipeptide bz-bpaib-l-phe-nhchx with chromatographic separation of the diastereomers and acidic hydrolysis. x-ray diffraction analysis of a crystal of a z-bpaib-l-phe-nhchx diastereomer allowed the assignment of the absolute confi guration of the bpaib enantiomers. photo-crosslinking studies with this residue are currently in progress. n-methylation of n -acetylated, c -ethylated, fullyextended homo-peptides: synthetic and conformational aspects moretto peptides characterized by single or multiple n-methylated, ctrisubstituted (protein) -amino acids are one of the subject of increasing interest in medicinal chemistry. several naturally occurring peptides, remarkably stable to proteolytic attacks, are based on n-methylated peptides. n methylation of the -conh-function is a useful tool for discriminating solvent exposed from intramolecularly h-bonded secondary amide groups in peptides. we are currently extending this reaction to linear peptides based on c -tetrasusbtituted -amino acids. after having investigated synthesis and conformation of the n-methylated homo-peptides from the c -methylated, helicogenicaminoisobutyric acid and c -methylnorvaline residues [a. moretto et al., biopolymers (pept. sci.) 2006, 84, 553-565], in this work we examined the n-methylation reaction on homo-peptides from c , -diethylglycine (deg), known to overwhelmingly adopt the fully-extended, multiple c5, conformation. we studied the following peptide series: ac-(deg) n -n(et) 2 , with n = 1-5. under the experimental conditions used, only monomethylation (on the n-terminal, acetylated residue) takes place. our ft-ir absorption, nmr, and x-ray diffraction analyses support the view that the fully-extended conformation preferred by the original peptides is dramatically perturbed in all of the derivatives mono-methylated at position 1. computational study on secondary structure of oligopeptides containing chiral , -disubstituted -amino acids we have shown mcmm conformational search method and amber* force fi eld using macromodel is useful to predict secondary helical structures ( -helix, 3 10 -helix) of oligopeptides prepared from ,disubstituted -amino acids. moreover, we have studied conformational analysis of oligopeptides containing chiral , -disubstituted -amino acids to predict the helical screw sense of helical structures. we calculated , -disubstituted peptide using mcmm conformational search with various force fi elds (amber*, mmff, opls) . in the case of using amber* force fi eld the results were in agreement with those of x-ray and were most stable conformation evaluated by 3-21g level molecular orbital calculation. these results indicated that computational simulation using conformational search calculations with amber* force fi eld is most useful for conformational analysis of oligopeptides containing , -disubstituted -amino acids. a new colorimetric test for solid-phase amines and thiols. simple colorimetric tests still remain the most convenient tests for providing information on the course of solid-phase reactions. a colour test, which indicates the presence or absence of a functional group by visual detection, is a simple, practical tool to monitor the completeness of the reaction. although the variety of colour tests for amines, there is still a need for a reliable and sensitive test in some synthetic approaches. here, we wish to report a new simple, fast and sensitive colorimetric assay for the visual detection of solid-phase bound primary, secondary amines and solid-phase bound thiol groups using 1-alkyl-2-aryl-imidazo[1,2-a]pyrimidinium salts. in the search for a new synthetic approach to polysubstituted 2-aminoimidazoles (1), we have reported a new procedure, employing substituted 2-aminopyrimidines and -bromocarbonyl compounds. the intermediate imidazo [1,2a] pyrimidinium salts in the synthesis of substituted 2-aminoimidazoles appeared to give a strong colour when reacted with primary and secondary amines. due to the strong colour change of amino functionalised resins after reaction with the "desc" reagent (2), a protocol for the detection of resin bound primary and secondary amines has been developed. the "desc" test is a new, practically, simple, quick and reliable test for the visual detection of resin bound primary and secondary amines in a sensitive way. the "desc" reagent is accessible from the cheap starting materials and can be prepared in two steps. tetrafl uoroborates of chiral n-triazinylammonium salts were found stable and useful in enantioselective (ee up to 98%) peptide synthesis from racemic amino acids in solution. several chiral n-triazinylammonium tetrafl uoroborates were obtained as stable l and/or d selective coupling reagents (1) . broad range of common amines (strychnine, brucine, sparteine etc.) were found useful as a chiral auxiliary, opening access to amino acids of both required confi gurations. we have attempted to expand the scope of application of this family of reagents expecting their effi ciency in enantiodifferentiating reactions in solid phase peptide synthesis (spps). tetrafl uoroborates of chiral n-triazinylammonium salts were obtained by treatment 2-chloro-4,6-dimethoxy-1,3,5-triazine with tetrafl uoroborates of appropriate tertiary amines in the presence of sodium bicarbonate (2) . enantiodifferentiating reagents were used in synthesis on wang resin under classic condition for triazine based coupling reagents (3) . the most important advantage of application of chiral n-triazinylammonium tetrafl uoroborates is the predictability of confi guration and repeatability of enantiomeric purity of incorporated amino acid. even if only threefold excess of racemic carboxylic acid, enantiomeric purity of coupling products in spps reached ee 98-99. the demand for diversity of non-proteinogenic amino acids, willingly used as new building blocks, is severely restricted by laborious procedures leading to enantiomerically homogeneous individuals. typical sources such as isolation from natural products, biotechnological methodology, even the asymmetric syntheses posses a limited value because complex or tedious synthetic procedures or limited access to the pool of chiral auxiliaries. herein we present the novel, general approach allowing enantioselective incorporation of any enantiomer of amino acids directly from racemic substrate. according to our procedure, the chiral auxiliary is used only at the stage of enantioselective activation. the departure of chiral auxiliary yields in the traceless way, the activated derivative of n-protected amino acid identical with those which is obtained with the well known, classic, achiral triazine coupling reagent (1) . therefore, the traceless chiral coupling reagents can be successfully applied directly in the coupling stage without additional studies of their reactivity. several chiral n-triazinylammonium tetrafl uoroborates were obtained as stable l and/or d selective coupling reagents. broad range of common amines (strychnine, brucine, sparteine etc.) were found useful as a chiral auxiliary, opening access to amino acids of both required confi gurations. the most important advantage of application of chiral n-triazinylammonium tetrafl uoroborates is the repeatability and predictability of enantiomeric purity and confi guration of incorporated amino acid. freeman, noam s.; hurevich, mattan; gilon, chaim hebrew university jerusalem, israel azapeptides are peptide analogues in which one or more of thecarbons, bearing the side chain residues, has been replaced by a nitrogen atom. azaamino acid residues conserve the pharmacophores necessary for biological activity while inducing conformational changes and increased resistance to proteolitic degradation. these properties make azapeptides an attractive tool for structure-activity relationship studies and drug design. a general approach for solid phase synthesis of azapeptides has been developed based on the in-situ activation of n-2-(3,5dimethoxyphenyl)propan-2-yloxycarbonyl (ddz), n €™-substituted hydrazines, with phosgene, followed by introduction to n-terminus of resin-bound peptide. the ddz-aza-amino building units include aliphatic, aromatic and functionalized side chains, protected for synthesis by the fmoc strategy. solid-phase azapeptide synthesis is demonstrated including selective mild deprotection of ddz with mg(clo 4 ) 2 and coupling of the next amino acid with triphosgene. the mild ddz deprotection is also orthogonal with boc chemistry. we describe the synthesis of n €™-substituted ddz protected hydrazines which have wide applications in the synthesis of azapeptides as well as in general synthesis of substituted hydrazines and aza containing peptidomimetics. ddz protected hydrazines offer the possibility to construct novel and unique drug-candidate structures such as branched and cyclic azapeptides. fast improved synthesis of insulin-like peptides barlos the a-and b-chains of insulin-like peptides were synthesized by various methods. the best results were achieved by dividing the sequences of the a-chain into three protected fragments and that of the b-chain into two fragments. the fragments were prepared on 2-chlorotrityl resin and/ or 4-methoxybenzhydryl resin using fmoc-amino acids. condensation of the fragments was carried out either by solution phase or solid phase techniques. the joining of the a and b chains was also studied using either biomimetic or chemoselective oxidative folding methods. a new approach for amides and peptides chemical synthesis by means of phosphonic acid/alkylene oxide chemistry few different derivatives of l-phe were synthesized, using originally discovered method of phosphonic acid/alkylene oxide chemistry. this method provides the successful synthesis of variety amides and dipeptides without preliminary protection of alfa-nh2 group of lamino acids. the supposed mechanism displays the formation of nphospholane carboxyanhydride (1-oxy-3-aza-2-phospholane-5-one), or an o-hydroxypropyl h-phosphonate salt of an amide of l-phe. the nphospholane carboxyanhydride is protected at the n-terminus, as well as is activated at the c-terminus. this pathway favors the desired reaction with a variety of nucleophiles from the n-to c-terminus. this method also allows the synthesis of different esters of amino acids employing alcohols as nucleophiles previously described by us (1). formation of disulfi de bonds in synthetic peptides is one of the more challenging transformations to achieve in peptide chemistry, in view of possible formation of oligomeric by-products and other side reactions, as well as occasional solubility problems in aqueous oxidizing media. the recently introduced polymer-supported oxidant, clear-ox™, has proven to be a very valuable tool in the preparation of disulfi de-bridged peptides. [1, 2] oxidations using clear-ox were carried out at ph ranging from 4 to 7 in water/acetonitrile solutions, with concentrations 10-15 times higher than comparable solution oxidations. the latest progress in the preparation of various monocyclic and bicyclic peptides will be presented. the amyloid -peptide (a ) is believed to play a causal role in alzheimer's disease (ad). one of the hypotheses of a neurotoxicity is that it induces the generation of reactive oxygen species (ros) and hydrogen peroxide (h 2 o 2 ) formation by binding to metals such as copper and iron. it is hypothesized that the sole tyrosine residue plays an important role in a -mediated toxicity, due to its ability to form a dityrosine cross-link from tyrosyl radicals generated in the highly oxidative environment in the brain. hence, studies of the dityrosine cross-linked a peptide dimers would increase our understanding of the oxidative alteration and dimerisation of a in amyloid formation and ad related neurodegeneration. we are investigating the synthesis of the a peptide dimers containing the dityrosine cross-link. dityrosine, suitably protected for solid-phase peptide synthesis (spps), has been prepared from iodotyrosine using a miyaura borylation-suzuki coupling method. studies on the synthesis of dityrosine-linked peptide dimers through incorporation of dityrosine in spps are underway. initial model studies employing 2,6-diaminopimelic acid (dap) have validated this approach. preparation of the model daplinked a peptide dimers will be discussed, as well as progress towards the dityrosine-linked a peptide dimers. nepomniaschiy, natalia; brik, ashraf ben-gurion university of the negev, israel the staudinger reaction, discovered nearly a century ago, occurs between a phosphine and an azide to form an aza-ylide. this transformation was further explored and developed, leading to several reactions of highly synthetic importance. traceless staudinger ligation is one example of such modifi cations, in which an ester moiety is placed within the phosphine structure to capture the nucleophilic aza-ylide, by intramolecular cyclization, leading to a stable amide bond. while the aza-ylide intermediate is known to be stable in organic solvents, it tends to hydrolyze rapidly under aqueous media to furnish the primary amine product. in the traceless staudinger ligation, however, the reduction of the azide to amine is a competing side reaction, as it would reverse the capture step leading to two peptide fragments. we have found that such reduction step would be benefi cial if an electrophile and an azide (rather than the phosphine) are placed within the switch peptide system. investigating various reducing reagents led us to the discovery that tris(2-carboxyethyl) phosphine hydrochloride (tcep) is excellent azide reducing reagent. both the reduction and the acyl transfer steps are rapid and occur in a few minutes. applying the tcep-mediated triggering in switch peptides, derived from the a to understand the currently unclear processes of pathological folding, self-assembly, and aggregation of amyloid peptide will also be reported. fast fmoc-deprotection reagent for peptide synthesis diffi cult sequences have long been known in solid phase peptide synthesis, theses sequences can experience sever aggregation both during synthesis and under purifi cation. the aggregation is believed to depend mainly on -sheet formation or/ and hydrophobic properties of the peptide. now a days there are several strategies to circumvent theses problems, one of the most successful is to incorporate a backbone protective group as the hmb group, develop by johnson and co-worker(1), and thereby prevent aggregation due to -sheet formation. an additional charge will increase the solubility of peptides towards aqueous solutions and thereby facilitate the purifi cation by hplc (using acetonitrile and water system) and analysis (by maldi-tof mass spectrometer) of the peptide. here we present the nmec (n-methyl-n-[2-(methylamino)ethyl] carbamoyl) group(2) that is a orthogonal protective group for tyrosine side chain and the hydroxyl moiety of the hmb group. the nmec group is fmoc compatible and is protected during the synthesis with a boc group. the fi nal cleavage from the resin with tfa renders the amine of the nmec group protonated and will increase the solubility of the peptide during purifi cation and analysis. the nmec group can be easily removed with a mild alkaline treatment by a cyclization elimination reaction. the nmec group has been employed in the synthesis of diffi cult and hydrophobic peptides as the membrane spanning sequence of the calciton receptor, amyloid (25-35) and amyloid (1-42) with success. [3, 4] . for example, infl uenza a virus-related peptide (h-gilgfvftl-h) with a diffi cult sequence was synthesized using o-acyl isodipeptide unit. analysis of the crude peptide revealed high purity of the product with no by-product derived from the diffi cult sequence or epimerization. using ch 2 cl 2 as solvent in coupling the isodipeptide unit, a 1-42 was also synthesized with almost no major side reaction 5.. racemization-free segment condensation method was developed by employing n-segments possessing a c-terminal urethane-protected o-acyl ser/thr residues 6.. the synthesis of long peptides/proteins by racemization-free segment condensation has thus become possible at ser/thr residues and not only c-terminal gly/pro residues. growing interest to peptide substrates, inhibitors and other peptidomimetics required new highly effective synthetic techniques. the important goal of enzymatic peptide bond formation is the optical purity of the peptide, which facilitates the product isolation. thus using enzymatic condensation as a last step in peptidomimetics production is preferable. fixing of enzymes on/in suitable insoluble supports has many advantages: high operational stability, ease of separation, possibility of recycling and improved activity in low water media. chitosan, natural hydrophilic polysaccharide, was used as a matrix as it has distinct advantages over the other supports due to (a) its renewable nature -it is available in large quantities as a waste product from fi shing industry and (b) its excellent fi lm-forming ability, allowing attachment to reactor walls for advanced processing. serine proteases subtilisin and chymotrypsin, and cysteine protease papain were immobilized onto chitosan. the fi lms of biocatalysts were prepared by drying the mixed solution of chitosan and enzyme in acetate buffer ph 5.6. treatment with glutaraldehyde was found to give material with high stability and good mechanical properties. the obtained biocomposites showed high amidase activity against specifi c chromogenic peptide substrates and protein substrate azacasein. immobilized subtilisin and chymotrypsin also possess esterase activity against p-nitrophenyl acetate. the longterm storage in aqueous buffer and acetonitrile had a little effect on the hydrolytic activity of subtilisin-based biocomposite. the dependence of subtilisin/chitosan hydrolytic activity on temperature and ph was studied. obtained samples possessed high synthetic activity and were capable to catalyze peptide bond formation in dmfa/mecn mixture in reaction zaalome+fpna zaalfpna for subtilisin, zaaome+lpna zaalpna for papain. the product yield reached 60-100% in 24 h. acknowledgement: this work was supported by rfbr 06-03-33056 69 synthetic chemistry or on solid phase and b) the chemical ligation of the [cys12(acm)]-(1-25)-thioester with the [cys48(acm)]-(26-68) segment, both deprotected and hplc purifi ed. the required intermediate peptides were prepared by optimized fmoc-based methods either stepwise or by convergent synthesis. for the formation of the two disulfi de bridges a two step procedure was investigated, involving a dmso oxidation step to form the cys26-cys45 linkage, followed by iodine oxidation to form the cys12-cys48 bond. native chemical ligation at valine haase, christian; rohde, heike; seitz, oliver humboldt-universität zu berlin, germany native chemical ligation is perhaps the most useful technique for peptide segment coupling in water. (1) a c-terminal peptide thioester reacts with an n-terminal cysteine. the requirement for this rare amino acid represents the major bottle neck to the synthesis of native proteins. several strategies have been developed to overcome this limitation. in the extended ligation, n-terminally attached auxiliaries allow access to other ligation junctions by mimicking the cysteine-thiol moiety. the ligation-desulfurization approach employs -thiol amino acids for fragment coupling followed by sulphur removal. herein, cysteine acts as precursor of the abundant alanine(2) and phenylalanine can be obtained by using its -mercapto derivative. (3) . we demonstrate the application of penicillamine in the generation of valine junctions employing the ligation-desulfurization approach. the , -dimethylcysteine building block is commercially available with various protecting group patterns suitable for routine solid phase synthesis of peptides. the ligation at penicillamine proceeded surprisingly fast despite the steric demand at the thiol group. even leu-val ligation sites, which appear in hydrophobic peptide segments, are accessible. we also present an improved method for achieving metal-free desulfurization and show applications in the synthesis of valine-containing peptides.(4) references: 1. p. e. dawson the application of n-alkyl cysteine (nac)-assisted thioesterifi cation reaction to the synthesis of polypeptides by the thioester method azide as a protecting group for lysine side chains on the solid phase peptide synthesis oriented toward the peptide condensation by the thioester method peptide ligation chemistry has been developed based on the use of peptide thioester as a building block in the thioester method (1) and native chemical ligation (2) . we found that a cysteine-containing peptide is transformed into the corresponding s-peptide (peptide thioester) by the n to s acyl shift reaction (3). on the other hand, in 1985, zanotti et al. reported that a diketopiperazine thioester, cyclo(-cys(coch2ph)-pro-) (1) was formed when a dipeptide p-nitrophenyl ester, phch2co-cys(st-bu)-pro-onp (2), was treated under reductive aqueous conditions (4). thioester 1 would be formed via intramolecular n-s acyl shift reaction followed by diketopiperazine formation. based on these observations, we designed a cysteinyl prolyl ester (cpe) autoactivating unit for preparation of the peptide thioester and for the peptide ligation [5, 6] . a peptide containing the cpe unit, peptide-cpe 3, was transformed into a peptide thioester of diketopiperazine, cyclo(-cys(peptide)-pro-) 4, then thiol-thioester exchange reaction with an external thiol produced the peptide thioester 5 and a diketopiperazine, cyclo(-cys-pro-) (6) . the poster abstracts peptide-cpe 3 was also able to ligate with a cysteinyl peptide 7, via the thioester in one-pot, to give a polypeptide 8. (1) has enabled the synthesis of entire proteins including those carrying posttranslational modifi cations. one of the most frequently encountered modifi cation of eukaryotic secretory and cell surface proteins is the attachment of oligosaccharides to asparagine residues (n-glycosylation). despite many efforts in this fi eld the function of nglycosyation is poorly understood, which is mainly caused by the lack of pure glycoproteins. since purifi cation of natural glycoproteins is quite tedious due to heterogeneity in the sugar part, the total synthesis of homogeneous glycoproteins has become an attractive target (2) . we have addressed this topic by choosing rnase c as a model glycoprotein. instead of the oligomannosidic type rnase c is containing a complex type n-glycan. for synthetic reasons ligations had to be conducted in a sequential manner (3) by fi rst reacting the recombinant 40-124 cys-segment (4) with an n-terminally protected and glycosylated thioester 26-39 5.. selective deprotection of the ligation product 26-124 and ligation with synthetic thioester 1-25 in a one pot manner was accompanied by unexpected side reactions. these drawbacks were fi nally overcome leading to full-length glycosylated rnase c displaying enzymatic activity. a new pseudo-native ligation: multiple, successive azidealkyne cycloadditions. (1) the reaction has become a classics. as triazoles are stable to acid and basic hydrolysis, and reductive and oxidative conditions, the reaction has been widely used in chemistry and biochemistry, proving to be an useful tool in combinatorial, bioconjugate or medicinal chemistry. the reaction has been applied in the peptide fi eld as an easy way to synthesize cyclic, labelled and side chain modifi ed peptides including effi cient synthesis of pseudo-glycopeptides. conformational and structural studies prove that the triazole ring is an excellent trans-amide surrogate and thus, can be considered as a new pseudo-native linkage. (2) in order to explore the potential of the reaction as a way to successively assemble several peptide chains to generate mimics of proteins, we have elaborated a new strategy for consecutive triazole formation based on a semi-orthogonal alkyne protection scheme. (3) so far, we have improved our fi rst one-pot successive cycloaddition approach performing the fi rst three successive cuaac in mild conditions thanks to a highly selective deprotection of two different alkyne groups. the application of this strategy represents a new promising method for the chemoselective pseudo-native ligation dedicated to the synthesis of protein chimera or for the decoration of molecular templates. solid phase synthesis using lightly crosslinked polystyrene supports has proved to be a successful route to the manufacture of peptides. the methodology introduced by merrifi eld nearly fi fty years ago has remained largely unchanged. during the last twenty years or so, it has been argued that incorporating a hydrophilic polymer within the conventional hydrophobic polystyrene matrix is benefi cial. others have suggested that polyamide or polyether-based resins may prove to be superior to polystyrene. despite this, large-scale peptide manufacture has generally relied on traditional polystyrene supports. this is due in part to the diffi culty in producing composite supports economically in large volumes, but also due to the handling diffi culties that are often associated with very high swelling polar polymers. other problems arise from the fact that many of these types of resin are relatively low loading and so yields are greatly diminished. in this poster we offer a solution to these problems: a polystyrene derived support which is modifi ed to create economical, amphipathic resins suitable for large scale peptide synthesis. attachment of appropriate handles or linkers enables both peptide acids and peptide amides to be produced. the synthesis of a variety of peptides is demonstrated. peptide synthesis in water using boc-amino acids nanoparticles reactants. here, we studied in-water solution-phase method using water-dispersible nanoparticulate boc-amino acids, which are the most common building blocks but are diffi cult to use for peptide synthesis in water. water-dispersible boc-amino acid nanoparticles were prepared by pulverization using a planetary ball mill in the presence of peg, and the size of the resulting water-dispersible nanoparticles was determined by dynamic light scattering analysis. the scanning electron microscopy image of water-dispersible boc-phe-oh nanoparticles also revealed nanosize particles. we studied in-water coupling reaction using waterdispersible boc-amino acid nanoparticles, and leu-enkephalinamide was successfully synthesized in water according to the boc chemistry. an alternative way for conopeptide formation kádár, kinga 1 ; panyi, györgy 2 ; tóth, gábor k. 1 1 university of szeged, hungary; 2 university of debrecen, hungary the chemistry used to oxidize the free thiol bonds to the corresponding disulfi de bond in a controlled fashion remains a signifi cant challenge in spite of many advances in peptide chemistry. the primary reason of this lies in the diffi culties involved in the formation of multiple regioselective disulfi de bonds. in this work we focused on the elucidation of synthetic strategies for the preparation of multiple disulfi de containing peptide venoms regulating the ion channels of immune cells-anuroctoxin and tc32-, and a neuropeptide with regulatory functions-orexin a. for the synthesis of these naturally occuring cys-rich peptides we had chosen the oxidative folding being the most simple of the methods available. in the case of anuroctoxin we isolated the native form of the peptide toxin with high selectivity and we optimized the folding conditions, so within an hour the majority of the linear peptide folds in the cyclic form with all four disulfi de bonds in the correct form. the regioselectivity of the isolated isomer was verifi ed with biological measurements. for the synthesis of orexin a we optimized the folding conditions and, using a polymer-supported oxidant-the clear-ox resin-, within two hours we could isolate the correctly folded isomer as the major reaction product. the correct structure was proven by coelution of the isolated isomer and the commercially available orexin a. but oxidative folding does not always give the correctly folded isomer. the best proof for this is the tc32 scorpion toxin which albeit our tryings always gave the misfolded isomers if we used the linear, unprotected peptide. therefore a new chemical synthesis using different side-chain protection needs to be taken into consideration. the synthesis of orthogonally protected tc32 and its use for the preparation of the correctly folded peptide to be underway. poster abstracts have fundamental importance in biological recognition processes. one of the most challenging task among of them the rational preparation of the glycosylated peptides especially having oligosaccharide moieties. there are two main strategies for the synthesis of glycopeptides: the synthon and global (convergent) method. both of them can be implemented in liquid or solid-phase. since the glycosylation could appear on o and n atoms of the amino acid side-chain, due to the different reactivity of the glycosidic linkage different chemical strategies will necessitate. in this presentation we compare several chemical strategies for the preparation of two model peptides (leu-lys-asn*-gly-gly-pro, gly-val-glu-asp-ile-ser*-gly-leu-pro-ser-gly,*site of glycosylation). as glyco-part several mono, di and trisaccharide including chitobiose, galactosilxilose, mannosil-n-acetyl-glycosil-n-acetyl-glucosamine were used and several of the used strategies led to successful preparation of these glycoconjugates. site-specifi c pegylation of human igg1-fab using a rationally designed trypsin variant in the present contribution we report on a novel, highly selective biocatalytic method enabling c-terminal modifi cations of proteins with artifi cial functionalities under native state conditions. the approach is based on the fourfold trypsin variant k60e/n143h/e151h/d189k which was generated by site directed mutagenesis and initially specifi ed in terms of selectivity and activity using a peptide library of the general structure bz aax aa x bb x cc aag-oh. the trypsin variant was found to bear a restricted proteolytic activity towards the rare recognition sequence yrh with an occurance of less than 0.5% in native proteins. the specifi city is zinc ion dependent due to an artifi cial metal binding site in the s2´-subsite (1). placing of the recognition sequence at the cterminal region of respective proteins via standard mutagenesis protocols provides suitable precursor targets for site-specifi c modifi cation via a transamidation reaction. due to the great demand for polymer-modifi ed antibody fragments for pharmaceutical purposes we evaluated the function of the approach on example of the c-terminal pegylation (peg...polyethylene glycol) of the human igg1 fab fragment. the fragment itself was expressed with the recognition sequence and an additional strep-fusion enabling effi cient purifi cation. the derivatization of the antibody fragment with 40 kda peg via the trypsin variant (mw ~ 24 kda) proceeds effi ciently with a total yield of isolated modifi ed protein of 40%. interestingly, no undesired cleavage reactions could be detected leading to fully active modifi ed antibody fragment. references: 1. willett, w.s., brinen, l.s. et al. (1996) . "delocalizing trypsin specifi city with metal activation." biochemistry 35 (19): 5992-8. 2-chloro-4,6-bis-(2,2,2-trifl uoroethoxy)-1,3,5-triazine and n-4,6-(2,2,2-trifl uoroethoxy)-1,3,5-triazin-2yl)ammonium tetrafl uoroborates as highly effi cient coupling reagents jastrzabek, konrad; kolesinska, beata; kaminski, zbigniew, j. we expected that modifi cation of substituents in the triazine ring improve activity, stability and solubility of new triazine based coupling reagents. in order to increase activity we prepared 2-chloro-4,6-(2,2,2trifl uoroethoxy)-1,3,5-triazine by treatment of cyanuric chloride with 2,2,2-trifl uoroethanol. taking advantage of modular structure of triazine coupling reagents the entire family of n-(4,6-(2,2,2-trifl uoroethoxy-1,3,5-triazinyl-1)ammonium tetrafl uoroborates have been obtained. effi cient coupling reagents should be useful for amide bond formation between broad range of substrates, work in stoichiometric quantities, be soluble and stable in most of the solvents. it should function effi ciently in solution as well as in spps, to get high purity crude products and minimize racemization of the products. we found n-(4,6-(2,2,2trifl uoroethoxy-1,3,5-triazinyl-1)ammonium tetrafl uoroborates useful for activation of carboxylic components. the participation of triazine "superactive ester" as intermediate in the condensation has been proved in the model experiments. utility of reagents n-(4,6-(2,2,2trifl uoroethoxy-1,3,5-triazinyl-1)ammonium tetrafl uoroborates were confi rmed by peptide synthesis in solution in high yield. in our study we focused our attention on the performance (in terms of purity of the crude and extent of racemization) testing the solid phase synthesis of acp(65-74) and model peptides with aibaib fragment, which are a good example of diffi cult peptide sequence. purifi cation of chemically synthesised proteins by use of cleavable imac-based n -terminal protein protecting groups (tags) (1), there remains a challenge in terms of time and cost, for the separation of impurities from the chemically synthesised product in spps. this is particularly acute in the case of protein synthesis where the crude product mixture contains capped (acetylated) truncates of comparable size and structure to the protein product. such a common situation results in extensive, and expensive, purifi cation techniques such as large scale hplc. to this end we designed a hydrophobic tag, tbfmoc (2) , which incorporated the base-labile characteristics of the fmoc (3) group. the tbfmoc group causes retention on hydrophobic columns and hence separation from untagged impurities. although this was effective, we were of the opinion that a complementary tag was required which would have the characteristics of metal-complexation and cleavage by -elimination for subsequent removal of the tag from the protein product after imac. considerations implicit in the design of such an n -terminal protecting group, or tag, will be discussed , and applications to the chemical synthesis of chemokines will be dealt with in the presentation. in last few decades, acridines which are known as anticancer, antimicrobal and antiviral agents have been in the center of interest of scientists. the heterocyclic molecules demonstrate a noteworthy group of compounds which interact with biological targets e.g. topoisomerase i, ii [1, 2] . we wish to pay our attention to a new group of tuftsin-acridine conjugates. tuftsin offers a wide range of biological activities such as supporting of immune system, bactericidal, tumoricidal activity. however tuftsin is unstable in plasma which reduces its effi cacy. that is the reason for search of new analogues that were more resistance to proteolysis degradation (3). tuftsin-acridine conjugates were synthesized using a fmoc solid phase strategy. the elongation of peptide chain was based on twostep procedure: deprotection and coupling reaction with dic and the additive of hobt in dmf/dcm/nmp mixture. tuftsin analogues were modifi ed at -amino group of lysine via introduction of the simple amino acids to obtain isopeptide bond. tuftsin analogues were conjugated to the acridine molecule via fl exible linkers. the carboxylic group of linker was connected to n-terminal group of peptide-resin using standard method for amide bond formation. the coupling reaction was performed with peptide-resin and the 4-fold excess of 1-nitro-acridine derivative using tbtu, hobt in the presence of diea in dmf for 24h. simultaneous deprotection of peptide side chain and cleavage from resin was done with the tfa cocktail. final products were purifi ed by spe and characterized by elemental analysis, ms and 1 h-nmr spectroscopy. the effect of microwave irradiation under controlled temperature conditions on the solid-phase synthesis of peptides was investigated. for optimization studies a model peptide (h-gly-ile-leu-thr-val-ser-val-ala-val-oh) was selected which suffers from poor synthetic effi ciency under standard spps conditions. synthesis of the nonapeptide was performed using various combinations of solid supports (polysterene, tentagel, chemmatrix) and solvents employing fmoc/but orthogonal protection strategy. applying controlled microwave heating, the reaction times were signifi cantly reduced while maintaining a high purity of crude product with no racemization being observed. the optimized microwave synthesis method was succesfully applied for longer, aggregated peptides. microwave coupling and cleveage were accomplished in a dedicated reactor setup that allowed accurate internal reaction temperature measurment using a fi ber-optic probe system. comparison studies between microwave-and conventionally heated reactions will be presented. during the last few years microwave assisted peptide synthesis has became popular and it was reported to be useful in some cases. there is also an ongoing discussion about an additional "microwave effect". based on this background we have synthesized several model peptides on a microwave synthesizer and compared with the synthesis on conventional batch and continuous fl ow synthesizers with and without microwave irradiation. during this investigation we have also compared reaction rates and purity of the peptides synthesized under the infl uence of microwave irradiation or conventional heating on this different synthesizer systems by various conditions. in no case we can fi nd any additional postulated "microwave effect". the results of this investigation will be presented and discussed. conotoxins form a large family of peptide toxins from cone snail venoms that act on a broad spectrum of ion channels and receptors. the subgroup -conotoxins specifi cally and selectively bind to subtypes of nicotinic acetylcholine receptors (nachrs), which are targets for treatment of several neurological disorders. the aim of this work is to develop an improved method able to generate conotoxins in high yield and purity. this will overcome a key barrier currently preventing the effi cient synthesis of small focused libraries in order to investigate the structureactivity relationship (sars) of those peptides. the development of general synthetic strategies for the preparation of conotoxins and analogues are essential to effi ciently approach important questions within the area of neurobiology and for the development of novel drugs for treatment of various neurological diseases. a new and highly effi cient synthetic strategy for the synthesis of alpha-conotoxin-mii has been developed. this strategy combine solid phase synthesis with microwave assisted heating to produce the two disulfi de bonds peptide in high yield and purity. we fi rst report here the use of microwave assistance in order to form a disulfi de loop. this technique demonstrates the advantage of preparing the fi rst disulfi de bridge while the peptides are resin bound. this step is critical to provide the dicyclic native peptide, that was performed by a followed classical in solution oxidation by iodine strategy. an enhanced total procedure for the synthesis of ctxmii is recommended, which can be effi cient applied at several small disulfi de rich peptides of biological importance. solid phase peptide synthesis in aqueous environment using microwave assistance heating galanis, athanassios s. 1 since the fi rst reports on the use of microwave heating more than 20 years ago, microwave-assisted organic synthesis (maos) has become an important tool for rapid and effi cient synthesis of organic molecules. microwave assisted heating has been further applied to peptide synthesis in order to accelerate the rate of synthesis and to improve the yields for the synthesis of diffi cult sequences. as far as water as solvent is concerned, numerous recent publications report the combination of water as an environmentally benign solvent for chemical transformations with the use of microwave irradiation as an effi cient heating method. we report herein, the combination of the microwave-assisted heating and the use of water as solvent for solid phase peptide synthesis. a variety of common amino acids derivatives and coupling reagents have been studied in order to optimize coupling reactions in water by microwave-assisted heating. we also describe the total synthesis of a small peptide using water as solvent. the solid-phase synthesis using the environmental friendly aqueous medium dramatically reduces the cost of the synthesis and could be broadly applied in research or in industrial production of peptides. vanier, grace; collins, jr., michael; singh, sandeep; collins, jonathan cem corporation, united states the application of microwave energy for solid phase peptide synthesis (spps) represents a major breakthrough for overcoming incomplete and slow reactions typical of conventional spps. microwave energy has been applied successfully in a manual and automated approach for enhancing synthesis of peptides and peptidomimetics. common side reactions such as racemization and aspartamide formation have been studied and shown to be easily controllable with optimized methods that can be applied routinely. we will present the latest research on improving the synthesis of diffi cult peptides with microwave energy. synthetic antifreeze glycopeptides and analogues: synthesis, structural analysis, and functional studies norgren, anna s. 1 glycosylated peptides are involved in various biological processes such cell adhesion and differentiation. in contrast to mucin-type oglycan peptides which are present on the membrane of mammalian cells antifreeze glycopeptides (afgps) are a little investigated example of glycosylated peptides containing similar components. afgps usually consist of a varying number of repeating units of (ala-ala-thr) with minor sequence variations and the threonine hydroxyl oxygen glycosylated with the disaccharide -d-galactosyl-(1-3)--n-acetyl-d-galactosamine. antifreeze activity has been proven by different experimental observations like suppression of recrystallisation and ice nucleation, thermal hysteresis and change of the crystal habitus. although it is known that the n-acetyl group at the c2 position of the galactosamine, the -confi gured glycosidic bond to the threonine hydroxyl group and the -methyl group are essential, the adsorption mechanism is not yet understood. (1) . in contrast to fragment condensation, solid phase peptide synthesis gives the possibility to prepare one defi ned product and to introduce structure inducing amino acids such as proline. the disadvantage of spps with the bulky glycosylated amino acids is the low coupling effi ciency. this problem was overcome by using more active coupling reagents and microwave-enhanced methods during coupling leading to suffi cient coupling effi ciency without having to apply great excess of the glycosylated amino acid. after purifi cation the peptide structure was examined by cd and nmr in water and dmso at different temperatures. additionally, the substances were microphysically analysed according to their recrystalisation inhibition activity and their infl uence on the crystal habitus. microwave technology applied to spps has been recently proposed as valid support to the enhancement of coupling rates. we also used microwave energy in conjugation process between polyelectrolytepeptide bioconjugation with edc, hbtu. the use of peptide epitops of viruses particles in the composition of polymeric conjugates as vaccine has several potential advantages over whole viral or bacterial preparation. recently, our group report a novel approach to a totally synthetic vaccine which consists of fmdv (foot and mouth disease virus) vp1 peptides, prepared by covalent conjugation of peptide biomolecules with membrane active carbochain polyelectrolytes in the present study, peptide epitops of vp1 protein both 135-161(p1) amino acid residues (ser-lys-tyr-ser-thr-thr-gly-glu-arg-thr-arg-thr-arg-gly-asp-leu-gly-ala-leu-ala-ala-arg-val-ala-thr-gln-leu-pro-ala) and triptophan (trp) containing on the n terminus 135-161 amino acid residues (trp-135-161) (p2) were synthesized by using the microwave assisted solid-phase methods. synthesis of peptides were performed by microwave assisted spps. peptides characterized by lc-ms and purifi ed by rp-hplc. bioconjugation between polyelectrolytespeptide were synthesized by two different microwave assisted method. the fi rst one is classical carbodiimid activating method. the second one is hbtu activating method. the second method is a novel and effective method for bioconjugation process. peptides and polyelectrolytespeptide bioconjugates analysed and comparised by gpc system with four dedector (uv. refractive index, light scattering, viscosity). microwave-assisted solid-phase synthesis of peptide probes to detect specifi c biomarkers: shifting off limitations affecting conventional synthetic strategies biomarkers play a key role in development of diagnostic/prognostic tools. in fact, since their involvement in several diseases such as autoimmune diseases (i.e., multiple sclerosis, rheumatoid arthritis) and neurodegenerative diseases as alzheimer's disease, biomarkers represent a great promise for the development and set up of tuned therapies. for autoimmune diseases, we proposed the development of synthetic posttranslational modifi ed peptides as antigenic probes for characterization of specifi c and high affi nity autoantibodies as biomarkers. by an innovative "chemical reverse approach" we propose optimisation of antigenic probes by a statistically signifi cant screening of sera guided by autoantibodies circulating in patients' biological fl uids (1) . spps via the building block approach is the principal strategy leading to modifi ed peptides. high purity is a condicio sine qua non for effi cient biomarker detection. these syntheses can present several diffi culties as result of internal aggregation of resin-bound peptides during elongation steps, reducing reagents penetration, and signifi cantly decreasing reaction rates in both acylation and deprotection steps. such events strongly affect purity of the crude peptides and therefore, fi nal yield. we demonstrated that application of microwave (mw) energy in spps of modifi ed complex peptide probes exhibits several advantages improving coupling rates, possibly because of the decrease of chain aggregation during the synthesis (2) . we report the mw-assisted synthetic conditions of diffi cult peptides (i.e. glycosylated, citrullinated, multiple antigenic, amyloidogenic peptides, etc.) by which we strongly improved preparation of diagnostic/prognostic probes in terms of crude purity, fi nal yield, and time-consuming compared to conventional protocols. fidan, zerrin; volkmer, rudolf charité berlin, institute for med. immunology, germany the alpha-helical coiled-coil structure is a versatile protein-interactiondomain, in which the coiled-coil is composed of at least two righthanded amphipathic alpha-helices, which are coiled up around each other into a left handed supercoil. this widespread structural motif is involved in many biological procedures like transcription, scaffolding or signalling. in addition, the most characteristic and important identifying feature of a coiled-coil is the recurrence of a periodic heptad repeat sequence. coiled-coils are able to form complexes up to heptamers of different orientation. based on this signifi cant occurrence and also their structure coiled-coil peptides have the ability to function as molecular recognition molecules. our goal is to use self-associating coiled-coil sequences as molecular building blocks (lego brick). the ligation of desired functionalities on coiled-coil sequences opens up the opportunity to add different functionalities on artifi cial complex peptide molecules that stick together via coiled-coil moieties. this makes coiled-coil sequences to useful tools during complex oligopeptide assembly. we want to present novel chimeric oligopeptides which are build up by two segments, one responsible for association the other for functionality. as a model for the association segment several gcn4-leucine zipper mutants (1) and as functionality segments amongst others ww domains were used. both segments were linked by the native chemical ligation approach. we will present in detail the synthesis of a gcn4-leucine zipper mutant and also the following c-terminal thioester formation for the subsequent ligation step. furthermore, we will present the synthesis of the functional device, a ww-domain with a n-terminal cystein residue and also the native chemical ligation of both synthesized peptide fragments supported by hplc and mass-chromatography. dialysis-related amyloidosis, a disease arising from long-term dialysis is characterised by gradual accumulation of 2-microglobulin ( 2m) amyloid fi brils in bones and ligaments. although 2-m is known to form amyloid fi brils in vitro under acidic ph conditions, seeding of preformed amyloid fi brils or as an effect of ionic strength, the mechanism underlying aggregation of soluble 2-m into insoluble fi brils under physiological conditions is largely unknown. interestingly, eakin and co. recently showed that the chemical basis of the amyloidogenesis process is a backbone isomerisation of the conserved pro 32. our aim is to understand the molecular mechanism associated with 2-m amyloidogenesis using fourier transformed infrared (ftir) spectroscopy and site-directed-isotope labelling, a method in which the vibration of the labelled residue is shifted from its original position in the spectrum. we already demonstrated that ftir spectroscopy along with site directed-isotope-labelling is a promising approach to obtain information on the microenvironment of tyrosine side chain residues.2 to use this approach in the case of 2-m amyloidogenesis, we decided to synthesise an isotopically labelled 2-m at crucial residues such as pro 32 that should undergo drastic variations in their microenvironments during the misfolding. with its 99 residues, 2-m is over the limits of a reasonable synthesis using spps but the two suitably positioned cysteines in its sequence offer the possibility of a chemical synthesis using native chemical ligation. thus, we were able to realise the chemical synthesis of an isotopically labelled 2-m in a good yield using a three segments strategy with disconnections at the two cysteines and thiazolidine as a masked cysteine for the middle segment. the synthetic 2-m obtained was in full agreement with the characteristic 2-m ftir spectra. a novel cyanophycin synthetase from thermosynechococcus elongatus bp-1 catalyzes non-primer-dependent cyanophycin synthesis. cyanophycin (multi-l-arginyl-poly[l-aspartic acid]) is synthesized by cyanophycin synthetase (cpha). it was believed that cyanophycin synthetase requires asp, arg, atp, mg 2+ and primer (low-molecular mass cyanophycin) for cyanophycin synthesis and catalyzes the elongation of low-molecular mass cyanophycin. despite extensive studies of cyanophycin, the mechanism of primer supply is still unclear. in the present study, we searched for a cyanophycin synthetase that synthesizes cyanophycin from asp and arg without added primer in vitro. cyanophycin synthetase from thermosynechococcus elongatus bp-1 (tlr2170 protein) was produced by an escherichia coli geneexpression system as a c-terminal his-tagged protein. we found that tlr2170 protein synthesized cyanophycin without added primer. the tlr2170 protein had strict substrate specifi city and used only asp and arg as substrates. the optimal ph was 9.0, and mg 2+ or mn 2+ was essential for cyanophycin synthesis. atp could not be substituted by gtp, ctp, or ttp. the molecular mass of the tlr2170 protein as estimated by gelfi ltration chromatography was 400 } 9 kda. thus, the tlr2170 protein appeared to be a homo-tetramer of 100-kda subunits including the his-tag sequence. the tlr2170 protein had thermal stability and fully retained its activity after a 15-min incubation at 60 ‹c. additionally, we examined cyanophycin synthesis at 30 ‹c, 40 ‹c, 50 ‹c, and 60 ‹c. sdspolyacrylamide gel electrophoresis showed that the molecular mass of cyanophycin increased with increased reaction temperatures. synthesis of peptide thioester by fmoc chemistry through hydroxyl side chain anchoring barta , 2005) . azapeptides are usually synthesized on solid phase. a method, permitting the convergent synthesis of azapeptides starting from unprotected fragments, would offer the possibility to study aza amino acids effect in complex polypeptides or proteins. we have focussed our study on ligation reactions leading to the formation of an azagly residue at the ligation point, as gly is a frequent amino acid in peptides or proteins. the fi rst synthetic strategy relies on the reaction between a peptide thioester and a n-terminal azaglycine peptide. experimental conditions were found which permitted the chemoselective formation of azapeptide but with racemisation of the c-terminal amino acid of the thioester fragment. alternately, a synthetic strategy based on the chemistry of the phenylthiocarbonyl group, permitted the successful synthesis of azapeptide without racemisation. (2), native glycoforms are of therapeutic interest. our retrosynthetic strategy implies sequential native chemical ligation (3) and the split of il-6 into three fragments. an n-terminal fragment il-6 (1-42) thioester and the cysteine fragment il-6 (49-183) were expressed in e. coli. the carbohydrate carrying fragment il-6 (43-48) was synthesized via spps. to obtain the recombinant fragments specifi c termini were required. the n-terminal il-6 (1-42) was fused between two inteins and after expression and refolding from inclusion bodies the target peptide was released as a c-terminal thioester after dual intein cleavage. the cysteine fragment il-6 (49-183) was expressed as a single intein fusion also leading to inclusion bodies. this enabled the release of il-6 (49-183) with an n-terminal cysteine after intein cleavage of the refolded fusion protein. the central segment il-6 (43-48) contains a sugar residue attached to the n-glycosylation-site at asn44. this fragment features both a c-terminal thioester and an n-terminal cysteine protected as a thiazolidine. sequential ligations leading to the full length il-6 glycoprotein will be presented. synthetic chemistry syntheses of shorter fragments which are fi nally linked together (4) . this "small building block approach" should also simplify synthesis of modifi ed prion protein. in our plan of mouse prion (moprp) synthesis, we have employed consecutive chemical ligations. we have started with moprp(203-231) in which it is possible to study native chemical ligation between cysteine in the position of 213 and methionine in the position of 21(2) several moprp(203-212) peptide thioesters with aryl and alkyl thiols were prepared for further study of the native chemical ligation of moprp(213-231) and moprp(203-212). the optimal ligation conditions were found by a kinetic study which was carried out in a range of ph and with various thiols. infl uence of electron withdrawing and electron donating groups was studied in both aromatic and aliphatic thiols and it was found that it is possible to affect the ligation by choosing an appropriate thiol. ligation optimal conditions, kinetic study results, and suitability of various thiols for the ligation are discussed. low-cost industrial chemo-enzymatic synthesis of pharmaceutical, nutraceutical and diagnostic oligopeptides the production costs for oligopeptides and derivatives thereof by chemical synthesis are extremely high. therefore dsm pharmaceutical products embarked on a research programme on chemo-enzymatic peptide synthesis. important advantages of this technology are that no expensive stoichiometric coupling reagents nor side-chain protection are required and that no racemisation occurs. focus is on elongation in the n ¨c terminal direction using novel enzymatic c-protecting group interconversion methods. one of the preferred building blocks are amino acid amides, but selective deprotection of peptidic c-terminal amides is a challenge. we discovered 1 that using a peptide amidase 2 c-terminal peptide amides can be directly converted to methyl esters in almost pure methanol. thus, separate enzymatic hydrolysis and reactivation steps are no longer required. other versatile building blocks are amino acid t-butyl esters. we discovered that peptide c-terminal t-butyl esters can be transformed, using commercially available proteases, to activated alkyl esters such as methyl-and benzylesters which can be directly used in another protease-mediated coupling reaction. 3 furthermore, we found that using commercial enzymes side chain unprotected peptides with a free c-carboxyl terminus can be directly transformed to cterminal thioesters 4 and c-terminal aryl amides 5 in the presence of the corresponding thiol and aniline, respectively. finally, a real-life example of large-scale industrial chemo-enzymatic peptide production will be shown. it is known that chiral amino acids, as well as their dipeptides, may catalyze the asymmetric condensation of glycolaldehyde in water. on the basis of the particularly large erythrose enantiomeric excesses (ee) obtained when utilizing the chiral l-val-l-val catalyst and given the possibility of an abundant delivery of other types of amino acids to the early earth, we have studied the catalytic effect on this synthesis of the peptides based on c -methylated -amino acids, such as iva (isovaline or c -methyl, c -aminobutyric acid) and c -methylvaline, ( me)val, that are abundant in meteorites. results of the catalysis experiments showed the all c -methylated peptides to the tetramer level exhibit signifi cant chiral infl uence on the synthesis of tetroses and mimic the effect of the l-val-l-val catalyst in having a larger erythrose ee than threose ee, as well as in their confi guration relationship with the sugars (the product erythrose acquires ee of confi guration opposite to that of the catalyst in case of peptides, while it is the same for amino acids). interestingly, the largest ee (45% for erythrose) was obtained with the iva homo-tetrapeptide under mild conditions. the homo-dipeptides of both iva and ( me)val also produced a signifi cant ee (41% for erythrose) that appears to increase with time. because c -methylated amino acids are non-racemic in meteorites, do poster abstracts not racemize in aqueous environments, and are known to be (3 10 )-helix formers in peptides with as few as four residues, these results suggest that meteoritic, c -methylated, -amino acids may have contributed to molecular evolution upon delivery to the early earth by catalytically transferring their asymmetry to other prebiotic molecules. synthesis' and application of peptide adhesives for wound healing natural polymers with repeating peptide sequences, like spider silk and mussel glue have interesting properties that can be used for biomedical applications. however, the isolation from their natural sources is rather diffi cult and the desire to introduce modifi cations necessitates the development of novel methods to synthesize such polymers with repeating peptide sequences. recently, we have shown that the microwave-assisted copper(i)-catalyzed 1,3-dipolar cycloaddition can be used in the polymerization of the model dipeptide azido-phe-alapropargyl amide. by varying the reaction conditions we could either obtain small cyclic oligomers (4-20 aas) or linear polymers which consisted up to 300 aas residues (1). to broaden the scope of this polymerization reaction, two novel biodegradable monomers, azido-phe-ala-lys-propargyl amide and azido-phe-ala-glyc-lys-propargyl amide were polymerized. both monomers are water-soluble and contain recognition sites for the proteases trypsin and chymotrypsin; moreover, the tetrapeptide can be chemically hydrolyzed depending on the ph of the solution. the molecular weight of the polymers could be tailored between 4 -14 kda (33 to 100 aas residues). the enzymatic degradability of the polypeptides was monitored by a ninhydrin-based colorimetric assay and by maldi-tof. the results showed that the peptidic polytriazoles were smoothly degraded by trypsin and chymotrypsin. from these data we can conclude that the microwave-assisted copper(i)-catalyzed 1,3-dipolar cycloaddition reaction is an effective tool to synthesize biodegradable functional polymers which provides new opportunities to design (novel) biomedical materials. details of monomer synthesis, polymerization reactions and degradation studies will be presented. peptide synthesis on cellulose membranes, known as spot synthesis, is an ideal tool to determine biological interactions and/or key positions in proteins. in general we can differ in two kinds of synthetic peptides with different possibilities: over the past 15 years synthetic peptide library techniques have emerged as powerful approaches to determine t-cell epitopes and to specify peptide binding to mhc-i and/or mhc-ii molecules. the sequence-based approach is a straightforward method to directly identify t-cell antigens belonging to a specifi c target (e.g. a virus of interest) without the need of any further deconvolution steps. the entire protein sequence is presented as a set of overlapping peptides (peptide scan), which are subsequently screened for t-cell stimulation. several cd-8 t-cell epitopes in selected cmv proteins have been identifi ed using standard spps techniques. however, the peptide sequences synthesized by standard spot synthesis can only be cleaved without authentic c-termini (ß-alanine or glycine as c-terminal amino acid). here, we summarized three different established methods to generate cleavable peptides with authentic c-termini in adequate amounts, with suffi cient purity and within a justifi able time-scale. the standard spot synthesis to generate membrane bound peptides use glycine or ß-alanine membranes to map epitopes of b-cells, allergenic proteins, antibodies etc. furthermore, we have also established the "method of inverted peptides" to screen protein domains requiring peptidic ligands with free c-termini such as pdz-or 14.3.3 domains. another highlight of peptide libraries is the screen of posttranslational modifi cation in proteins by phosphorylation at serine, threonine, and tyrosine residues which plays a role in eukaryotic cell cycle regulation, cell differentiation, apoptosis, or cytoskeletal regulation. labeled peptide libraries, consisting of -helices, -loops and -sheets peptides, have been successfully prepared for use in peptide arrays that act as a protein-detection system and have been applied for proteinrecognition studies [1] [2] [3] . it is known that more than half the proteins in a mammalian cell are post-translationally modifi ed by such processes as phosphorylation, glycosylation, and acetylation. such modifi cations play an important role in various bio-recognition, for instance glycoproteins are involved in cell-adhesion, infection, and biological protection. hence the designed peptide library used for arrays has been expanded to include fatty acid attached peptides and glycopeptides. fatty acids were easily introduced by conventional fmoc-spps, while the synthesis of glyco-peptides was not easy. in the present paper we describe the effi cient construction of o-glycoside structured and labeled peptides by fmoc-spps using a building block strategy. glycosylated threonine has been used as a building block and was prepared in large amounts. glucose, mannose, galactose and lactose were acetylated and coupled to the hydroxyl group of fmoc-thr in the presence of bf3oet2. the resulting protected sugar-amino acids were manually assembled on to the peptidyl resin containing a fl uorescent dye using a peti-syzer® (hipep laboratories). after construction of the glycol-peptides the acetyl groups on the sugar were removed by hydrazine hydrate, and the peptides were then cleaved and purifi ed. the resulting glycopeptides were characterized by lcms. the present protocol has been used to prepare ca. one hundred glycopeptides that have been tested against various carbohydrate-binding proteins. parallel small scale peptide synthesis meets a fast, lowcost purifi cation method for the production of high quality peptide microarrays to analyze dna/protein interactions herein, a parallel synthesis in a small scale (0,1 mol) which takes place in 384-micro well plates is demonstrated. with regard to a fast but yet effi cient, low-cost purifi cation in a parallel manner a ''fmoc-on'' purifi cation method, which is integrated into the synthesis process, was developed. peptides were cleaved from the resin with their fmoc-group still on. the crude products were transferred by extraction into another 384-micro well plate fi lled with purifi cation material. purifi cation takes place due to the high affi nity of the terminal fmoc-group to this material. hplc measurement shows a high recovery (98%) and purity (90%). cleavage of the fmoc-group during the purifi cation process is also possible. here, hplc measurement demonstrated a recovery of 93% and a purity of 92%. a fully automated synthesis of more than 300 peptides in a 384-micro well plate combined with a parallel ''fmocon'' purifi cation is shown. the peptides were used for the production of microarrays to analyze dna/protein interactions. the "fmoc-on" purifi cation allows easy and cost-effi cient purifi cation of peptides for all kind of biological applications. is an antibiotic-macrolide related to a group of tylosin (tyl) which structure is based on 16-member lactone with carbohydrate substitutes attached. macrolides are well known translation inhibitors, they bind to ribosomal tunnel (rt) in a way that their lactone ring is located orthogonally to the long axis of the rt, covering most of its cleft; hence, the mechanism of protein synthesis inhibition by macrolides relies on the mechanical obstruction they provide to the passage of nascent polypeptide chain through the rt. recently we have designed and synthesized a number of peptide derivatives of macrolides where the peptide part modeled the growing chain, while the antibiotic served as an "anchor" for positioning the peptide at the specifi c site of rt. these derivatives are of interest both as antibacterial agents and as potential probes for investigation of the interactions of nascent peptide chain with the specifi c sites of the rt and their infl uence on the translation. now we report a new type of peptide -macrolide conjugates in which peptide binds by its -amino function through a spacer to the 4'-hydroxyl group of the mycaminose residue of des. two proline-containing peptides are chosen: gp and ggp. proline residues of these peptides are supposed to interact with the peptidyl transferase center region when the complexes of these 4'-peptidyldesmycosins with bacterial ribosomes are formed. the fi rst step of the synthesis was acetylation of 2'-, 2"-, 4"and 4"'-oh-groups of tyl by ac2o in pyridine following by hydrolysis in 1n sulphuric acid resulted in des with all protected oh-groups except 4'-oh-group of mycaminose. this free hydroxyl group was used for reaction with succinic anhydride leading to formation of reactive carboxyl group. the next step was a reaction between this carboxyl function with -amino groups of peptide methyl esters. the structures of new 4'-peptide derivatives of des were proved by ms maldi and nmr-spectroscopy. synthesis .0]octane, (c), was carried out by organic methods which then will be used in polymer synthesis [1] [2] . consequently, polymers having different molecular weights and their water soluble bioconjugates were synthesized and the characterization of these polymers and conjugates were done by different methods such as esi lc-ms, uv and atr ft-ir spectroscopy. for the chemical modifi cation of pc, bromoacetic acid was used and a water soluble polyampholyte synthesis was achieved with the quaternization of polymer. it is determined that molecular weights of the polymers were affected by the change in the amount and the type of initiators used and also from the polymerization media. analysing of having biodegradable characteristics of the synthesized polymers has been being reviewed. atr ft-ir spectra of pc and pc-hemagglutinine peptide was recorded. for peptide synthesis we have used microwave assisted solid phase peptide synthesis method. lc-ms was used to determinate of molecular weight of hemagglutinin. after we have obtained m/z values of peptide we used preparative hplc for purifi cation of hemagglutinin. after synthesis and modifi cation of pc, its covalent conjugates with hemagglutinin (ha 98-106) were obtained by carbodiimide activation. its characterization was also performed. the peptide coupling reagent fi eld has clearly evolved in the last decade from carbodiimides to onium (phosphonium and uronium) salts. the era of industrial coupling reagents began in 1955 with the introduction of dicyclohexylcarbodiimide (dcc), which at that time was already known and well studied, as a reagent for the formation of amide bond. unfortunately, carbodiimides did not comply with the concept of ultimate coupling reagents because its high reactivity provokes racemization and side reactions during the coupling reaction. at the beginning of the 70's, 1-hydroxybenzotriazole (hobt) was proposed as an additive to dcc to reduce racemization and from then on other benzotriazole derivatives such as 1-hydroxy-6-chlorobenzotriazole (cl-hobt) or 1-hydroxy-7azabenzotriazole (hoat) have also been used. the obt active esters are less reactive than the o-acylisourea, but are more stable and less prone to racemize. the use of the most reactive aminium salt, hatu , is inconvenient because of the price, which makes its use detrimental for industry. hctu/tctu, based on cl-hobt, are a good alternative to hbtu/tbtu, because of the presence of the chlorine atom that stabilizes the structure, hence, making these reagents less hazardous. herein, pyclock, the phosphonium salt of the cl-hobt is introduced p20100-001 n-methylation of biologically active peptides is of interest because it results in increased conformational integrity and stability against enzymatic degradation. furthermore, n-methylated peptides have a decreased ability to form h-bonds to water molecules and, consequently, a better ability to cross biological barriers. this is exemplifi ed by the naturally occurring peptide cyclosporine which is orally active. in an effort to improve the blood-brain barrier permeability of the cyclic enkephalin analogues h-dmt-c[d-cys-gly-phe-d(or l)-cys]nh 2 (dmt = 2',6'-dimethyltyrosine), we prepared analogues that were n-methylated at phe 4 and/or cys 5 . n-methylated cys derivatives were prepared either by direct methylation (ch 3 i)/nah) or by using oxazolidinone chemistry. single n-methylation of the two cyclic peptides at phe 4 or d(or l)-cys 5 produced four analogues that all showed very high mu and delta opioid agonist potencies in the guinea pig ileum and mouse vas deferens assays. the two analogues n-methylated at both phe 4 and d(or l)-cys 5 also retained high agonist activity at both receptors. results from molecular mechanics and molecular dynamics studies on the conformational behavior of these peptides will be presented. during the last years the number of deaths due to hemostatic impairments such as coronary angioplasts, coronary thromboembolisms, myocard heart attack, pulmonary embolism etc. has become equal to those caused by cancer formations. haemostasis is a key process whose correct functioning is an important defence mechanism of the human organism. it is a blood coagulation process activated in case of injury of the blood system. if it is functioning correctly vascular-motor and cell reactions are triggered and the blood coagulation cascade is activated. one of the most important enzymes in the blood coagulation cascade is factor xa. that's why its inhibitors are promising alternative against thrombotic disorders. in our previous work, we reported the synthesis of hybrid structure between isoform 2 and 3 of antistasin and the active sequences d-phe-pro-arg; d-arg-gly-arg; phe-ile-arg and tyr-ile-arg (1). beside the analogues with c-terminal cooh group, the peptide d-phe-pro-arg-pro-lys-arg-nh2 was synthesized. the biological activity of the last one was 60 times bigger than natural isoform 3 of ats and some times more active than all other synthesized analogues. in the current work we described synthesis and biological activity of c-terminal amide analogues of all early synthesized peptides in order to deduce the structure-activity relationship. the anticoagulant activity according to the aptt and ic50 was determined. the neo-angiogenesis is the formation of new blood vessels from a pre-existing vasculature which enables the supply of the nutrients and oxygen necessary for tumor growth. consequently, inhibiting the blood vessels sprouting in order to starve the tumour constitutes an attractive anti-tumoral therapy. the vascular endothelial growth factor or their receptors (vegfr-1 and 2) are key mediators of neo-angiogenesis and constitute nowadays validated targets for anti-angiogenic therapies (1) . despite this fact, strategies aiming to develop pure receptors antagonists and not inhibitors of the tyrosine kinase activity remain scarce. we have previously designed antagonists that interact with the extra-cellular domain of vegf receptor 1 and thus prevent vegf binding (2) . these antagonists were proved to be potent and able to interfere with the vegf signalling pathway. because these compounds were rationally designed by using the co-crystallized structure of vegf with the immunoglobulin like fragment 2 of vegfr-1, we raise the following question: do these antagonists really target the immunoglobulin like domain 2 of vegfr-1? in order to respond to this question but also with the future aim of optimizing our antagonist in a rational way we need to co-crystallize at least one of our antagonists with the d2 fragment of the vegfr-1. furthermore, since human vegfr-1 d2 is a potential target in the development of angiogenesis modulators, the availability of this protein domain, and mutants, should be useful in the screening and development of novel specifi c ligands. up to now, the 101-amino acid polypeptide chain of vegfr-1d2 has been only produced by gene expression (3) . here, we report the fi rst solid phase peptide synthesis of the vegfbinding domain of the vegf receptor 1 and the in vitro experiments which permitted to verify its biological activity. chung, nga n. 1 different types of turns are important elements of secondary structure in peptides and proteins. the most common ones are different kinds of -turns involving four consecutive amino acid residues. the dipeptide unit containing the second and the third residues of such turns exists in the cis-conformation. our cispeptide bond motif 4-aminopyroglutamic acid promotes the -turn type vi/vi', and can be treated as a hybrid of glycine and alanine. this feature prohibits its utilization as a replacement for any possible dipeptide unit. in order to convert our compound into a more valuable tool for probing the existence of a particular peptide bond in the cis-conformation, we have elaborated the synthesis of nmonoalkylated derivatives of 4-aminopyroglutamic residue through a reductive alkylation reaction performed on solid support. following this idea, we have obtained analogues with n-benzylated and n-(phydroxy)benzylated 4-aminopyroglutamic residues as scaffolds for the phe-ala and tyr-ala dipeptides units, respectively. only one of 12 enkephalin analogues, [n(bzl)-(r,r)-apy4-5]-enkephalin amide, possesses similar agonist potency as leu-enkephalin in the gpi assay, indicating that the c-terminal part of this peptide may assume the cisconformation upon binding to the receptor. this is the fi rst active opiod analogue containing a 4-aminopyroglutamic acid residue. supported by kbn (poland) grant 2 p05f 00129 and nih (u.s.) grant da-04443 mif-1 (l-prolyl-l-leucyl-glycine amide, plg) is a brain peptide, presented in hypothalamic tissue, as well as the c-terminal tripeptide of the neurohypophiseal hormone oxytocin and inhibits the release of melanocyte-stimulating hormone (msh) in some systems. recently, some chemical modifi cations of mif-1 to enhance opiate agonist/ antagonist actions as well as binding activity of analogues have been reported. on the other hand, the concept of structural modifi cation in peptide fragments to confer them specifi c properties is of current interest in the study and design of new bioactive targets. a well known example is the incorporation of unnatural amino acids into the molecule of natural biologically active peptides leading to analogues with signifi cant theoretical and practical importance. with this idea in mind we studied possibilities of introducing unnatural amino acids canaline (can), norcanaline (ncan), canavanine (cav), nor-canavanine (ncav) and slys into mif-moiety in order to achieve a better analgesic effect. to obtain the peptide mimetics, both fmoc-and boc-based spps approach were used. analgesic activity was determined by the paw-pressure (pp) and hp tests. the experiments were carried on male wistar rats. the changes in the mechanical nociceptive threshold of the rats were measured by the randall-selito paw pressure test using analgesimeter (ugo basile). it was found that substitution of leu in position 2 of mif-molecule by unnatural amino acids increased the pain threshold. the analgesic effect with cav-substitution was highest, whereas parent mif-1 showed only a minute increase of pain-threshold. the compound m6 had the strongest effect on learning and memory and the compound p6 showed the strongest and fastest analgesic effect. the compound m6 and p6 were also able to modify the effect of the model cns-drugs (hexobarbital and pentylenetetrazole). theoretically and experimentally determined octanol/water logp values of the compounds correlate with their cns-effects. m6 and p6 had higher logp than m3 and p3 and showed better antinociceptive and anticonvulsant activity in vivo. compounds possessed also chelating activity towards fe ions. the stronger chelating activity of the compounds with the 6-spacer clearly correlated with their better neuropharmacological activity in vivo (in comparison to the compounds with the 3-spacer). the position isomery may also contribute for the variations in their pharmacological activity. the limited number of the compounds does not allow derivation of well-defi ned structure-activity relationships, however, their 3d models show possibility for many low energy conformers with different atoms involved in formation of fe chelating complexes. a major challenge in opioid peptide chemistry is the synthesis of novel compounds mimicking the endogenous peptide ligands. these new peptidomimetics should be biologically active and more stable against enzymatic degradation than their parent ligands. one of the possibilities is the introduction of -amino acids into the peptides sequence. monosubstituted -amino acids ( 2 -or 3 -) due to their similarity in structure to -amino acids, moreover their tendency to give folded structures even in short peptides and to the stability towards mammalian peptidases may be very useful in the creation of the new potentially active compounds. we have developed simple and effi cient two step conversion of the cyanoacetate into fully protected 2 -amino acids. the procedure involves knoevenagel condensation of the methyl cyanoacetate and aromatic aldehydes (for aromatic path) or alkylation of the methyl cyanoacetate with various alkyl halides (for aliphatic path) at fi rst then reduction and boc-protection (performed in one pot) of the resulting fi rst stepproducts. we focused our attention on applying above method to the synthesis of different 2 -amino acids (as homologues of -amino acids) as elements for the synthesis and structure -activity relationship study of endomorphin analogues. small library of analogues has been created in which -amino acids in every position with exception of pro were substituted by their respective 2 -analogues. as templates, endomorphin-1 (tyr-pro-trp-phe-nh 2 ), endomorphin-2 (tyr-pro-phe-phe-nh 2 ) and its d-ala2-analogue (tapp) have been used. in this communication the pharmacological consequences of such modifi cation in endomorphins will be discussed. solid-phase synthesis and effects of amino acid and peptide analogues of non-protein amino acid canavanine on nociception non-protein amino acids have been widely used as components of peptides to enhance biological activity, proteolytic stability, and bioavailability. it is well known that unnatural amino acids with guanidine functionality exhibit diverse pharmacological effects when introduced in biologically active systems. our previous efforts were focused on the preparation and the characterization of unnatural amino acids, particularly those containing a basic functionality in the side chain. we have synthesized numerous unnatural amino acids, structural analogues of arginine and lysine, which demonstrated certain biological effects. recently, as part of our ongoing research focused on the search of novel arginine mimetics, we developed an effi cient approach for solid-phase synthesis of unnatural amino acids nor-canaline (ncan) and nor-canavanine (ncav). next we studied the possibilities of introducing ncan and ncav into the molecule of biologically active peptides. we also studied their antinociceptive effects using the paw pressure (pp) and hp tests. in the last few decades, considerable attention has been devoted on the potential role and activity of certain environmental pollutants (edcs) in increasing anomalies that involve the endocrine system of wild species and man. (1)the development of a fast high-throughput detection system for the quantitative analysis of edcs requires the development of a novel sensitive solid phase edcs extraction method. because of the high affi nity of edcs with the estrogen receptor, this has been greatly investigated. this study has leaded the recognition of the amino acids located in the ligand binding domain of the receptor which interact with edcs. use of a dipodal scaffold molecule and different combination of the crucial amino acids, will allow the generation of a library of small to medium size biomimetic receptors. in this communication, we will disclose our fi rst results in the preparation of a small library of biomimetic receptors, with and without the dipodal scaffold, to evaluate their affi nity towards edcs and the role played by the scaffold structure. romeralo tapia, rosa 1 ; van der eycken, johan 2 1 ghnet university, belgium; 2 ghent university, belgium endocrine disrupting chemicals (edc's) are an important class of pollutants, which have in common that they show affi nity for the hormone binding domain of the estrogen receptor, thus disturbing the endocrinal system. detection in waste water has not been succesful due to their low concentration. therefore, new sensitive screening methods are highly demanded. a possible solution is the use of estrogen receptor mimics for affi nity chromatography. to this end, scaffold 1 will be synthesized and used for building a tetrapodal peptidomimetic library. amino acids known to be important for the estrogen-receptor interaction will be preferably incorporated. first of all, we set out to prepare one dipodal peptide library member 6. the orthogonally protected scaffold 2 was synthesized in one single step starting from the commercially available 3amino-5-nitrobenzoic acid.2 using fmoc solid phase chemistry on wang resin as the solid support, this dipodal scaffold allowed the attachment of two different oligopeptide chains.employing this methodology the synthesis of a small library will be performed, and the affi nity of the different peptidomimetics for edc's will be investigated. on-resin microwaves-assisted ring closing metathesis for the synthesis of octreotide dicarba-analogues di . octreotide is an antitumoral agent used mainly as a carrier of radionuclides for cancer diagnosis and therapy. it shows the same disulphide bridge of the parent srif that is prone to be opened by oxidizing and reducing agents. this prompted us to search a more stable tether bridging the active motif of the cognate molecule. the premier reaction of rcm was performed in an oil bath under severe experimental conditions i. e. anhydrous argon atmosphere and long reaction times [3;4] . the microwaves assisted version was, instead, effi cient for the cyclopeptides yield and required very short reaction times. we also evaluated the effi cacy of different grubbs catalysts in the microwaves assisted rcm, operating in different conditions of temperature and time. in the search for potential antimicrobial drugs, we have been dealing with two microbial enzymatic systems: n á -succinyl-l-diaminopimelic acid desuccinylase (dape 1, 2 ) and n á -acetyl-l-ornithine deacetylase (arge 3, 4 ), which might be promising targets for potent and selective enzyme inhibitors based on the modifi cation of n á -succinyl-diaminopimelic acid (dap) and n á -acetyl-ornithine (orn). the inhibition of both the enzymes would possibly interrupt the pathways leading to development of bacteria due to a role of meso-dap as essential component of peptidoglycan based bacterial cell walls and orn, as well, being a component of biosynthetic pathway for arginine that can serve as a source of both the carbon and nitrogen in microorganisms. our effort was focused on the synthesis and characterization of two series of n á -substituted derivatives of dap and orn, potentially interfering with the hydrolytic action of dape and arge in the processes of bacterial growth. in this introductory study, the compounds prepared were also assayed against bacterial strains escherichia coli and bacillus subtilis and inhibitory activity of orn derivatives was found with regard to differences in the structure of n á -substituent. the fi rst report on practically effective bradykinin (bk) antagonists for b 2 receptors was published in 1984. the key to conversion of bradykinin into an antagonist was replacement of pro 7 with an aromatic d-amino acid; d-phe was fi rst used. however, our studies demonstrated that the d-amino acid residue at position 7 of the bradykinin antagonist, until recently considered to be necessary for b 2 antagonism, can be replaced by suitable l-amino acid or achiral residue or, together with the amino acid occupying position 8, by a sterically restricted dipeptide unit. having all this in mind we synthesized and bioassayed two new analogues of bradykinin. the peptides were designed by substitution of position 7 of bradykinin b 2 receptor antagonist ([d-arg 0 ,hyp 3 ,thi 5,8 ,d-phe 7 ]bk), previously described by stewart's group, with structural isomer of proline: 2 -iso-pro or its homologue: 3 -homo-pro. it's worth emphasizing that position 7 in bradykinin molecule is occupied by proline residue. our previous results demonstrated the importance of the position in the peptide chain into which the sterically restricted 1-aminocyclohexane-1-carboxylic acid residue (acc) was inserted. these fi ndings prompted us to investigate how introduction of l-pipecolic acid residue (l-pip) in position 7 or 8 of stewart's antagonist will affect pharmacological properties of resulting compounds. in comparison to the acc residue, the ring of l-pipecolic acid also consists of six atoms, but includes the nitrogen atom. bearing in mind that acylation of the n-terminus of several known b 2 blockers with a variety of bulky groups has consistently improved their antagonistic potency in the rat blood pressure assay, the aforementioned four analogues were also synthesized in the n-acylated form with 1-adamantaneacetic acid (aaa). the activity of eight new analogues was assessed in isolates rat uterus and in rat blood pressure test modifi cations of the myelin basic protein epitope mbp87-99 divert th2 to th1: immune responses in peripheral blood mononuclear cells (pbmc) from multiple sclerosis patients. postranslational modifi cations (citrullination, phosphorylation, deamidation, methylation, glycosylation) are common biological processes that alter specifi c parts of a protein after synthesis. nearly all known proteins undergo some form of postranslational modifi cation and almost all amino acids can be altered by one or more of these processes. the modifi ed protein thus, contains new or rare amino acids or new specifi c side groups that can have critical infl uence on the structure and function of the protein molecule. conversion of arginine to citrulline, an important postranslational modifi cation, was fi rst described by fearon. arginine residues in proteins can undergo this modifi cation and the resulting citrulline remains part of the protein in the position of arginine. citrulline is not a natural amino acid in proteins, and may induce immune responses. such responses have been recently implicated in the pathogenesis of autoimmune/infl ammatory diseases such as ms and rheumatoid arthritis 1. herein we investigated cytokine secretion in peripheral blood mononuclear cells (pbmc) of 7 ms patients and 7 controls, and attempted to correlate cytokine polarization with the nature of the antigenic stimulus. we synthesized peptide analogs that map to the myelin basic protein ( . analogs p4 and p5 resulted from the citrullination of the 91 and 97 arginine residues in epitopes p2 and p3. we then tested ms and control pbmc with various concentrations of the peptides and investigated cell proliferation by the brdu proliferation assay and cytokine secretion by elisa. we suggest that citrullination of self-antigens maybe an important step in triggering disease in susceptible individuals. incorporation of aza-3-amino acid into 26rfa(20-26), the endogenous ligand of gpr103 : structural analysis. aza-3-peptides, mixing -and aza-3-amino acids (the aza analogs of 3-amino acids), represent a novel and exciting type of peptidomimetics.1 in particular, we have shown that aza-3-amino acid induces a n-n or hydrazino turn, stabilized by an eight-membered-ring intramolecular hydrogen bond between the carbonyl acceptor group of the residue i-1 and the amide proton of the residue i+1. interestingly, this n-n turn promotes a well-defi ned -turn formation (hydrogen bond between the co of the residue i-2 and the hydrazidic proton of the aza-3-moeity) when an -amino acid is foregoing.2 26rfa, a novel neuropeptide of the rfamide superfamily, exhibits high affi nity for gpr103 and induces a potent orexigenic effect in mice. 3 in biomimetic environment, 26rfa encompasses an -helix between pro4 and arg17 residues and a canonical -turn centered on ser23. 26rfa(20-26), whose sequence is strictly conserved across species, is about 100 times less potent than 26rfa. this heptapeptide shows important distortions of the -turn that may be responsible for its weak potency. the aim of this study was to restore the -turn formation in 26rfa(20-26) (ggfsfrf-nh2) by the presence of an aza-3-amino acid. for this purpose, we have (i) synthesized the aza-3-counterpart of each residue of 26rfa(20-26), (ii) individually incorporate the surrogate into the heptapeptide and (iii) investigated the 3d structure under nmr restraints of the hybrid peptides. the results will be presented with a particular attention on the serine position. the analysis of the structure-antithrombotic activity correlation for peptide receptors of adhesive glycoproteins with the general formula arg-xaa-asp gribovskaya, olga; martinovich, vera; golubovich, vladimir institute of bioorganic chemistry, belarus structural analogues of the arg-gly-asp sequence with the general formula arg-xaa-asp, where xaa -ala, d-ala, -ala, -abu, -ak, pro, d-pro, asn-trp, were synthesized using classical methods of peptide chemistry in solution, the levels of their antithrombotic activity were determined and stable conformations were calculated using a pairwiseadditive approximation method. interatomic distances in the obtained conformers were calculated between various atoms in the functional groups, including carbons in the carboxylic groups, nitrogens in the -aminogroups, amino and imino nitrogens in the guanidine group, 16 distances in total. the analysis of correlation of the calculated interatomic distances and measured values of antithrombotic activity was carried out using statistical methods. we show that the distance between the amino nitrogen of the guanidine group of arginine and the -carboxyl carbon of aspartic acid determines the antithrombotic activity. it has the optimum value in the arg--ala-asp peptide, which was the most active among the synthesized analogues of the arg-gly-asp sequence (ic50 -10.6 mkm, adp, 1.5 mkm). microbial proteases with narrow specifi city as an instrument of peptide chemistry kotlova the problem of selective removal of short n-terminal peptides from the gene-engineered proteins is actual by many reasons. at fi rst it is connected with gene-engineering method of protein preparation in form of its precursors. the problem of n-terminal formyl-met removal from gene-engineered proteins, which are produced by bacillacae may be solved by introduction of specifi cally-cleaved insert after formyl-met. subsequent specifi c removal of such short n-terminal peptide results in mature protein. moreover, the analysis of cleaved short peptides could characterize the process of mature protein formation. we suggest microbial enzymes with narrow specifi city to solve the mentioned problem. for this aim we have isolated the trypsin-like enzyme and postproline-specifi c endopeptidase from aspergillus sp. and glutamyl-specifi c protease from b. intermedius. these enzymes have high specifi city approved by hydrolysis of short synthetic peptides and long polypeptides. for example, specifi city of postproline protease was established by hydrolysis of mellitin. the fi nal peptide mixture was analyzed by rp-hplc and mass-spectra. the use of enzymes with narrow specifi city is demonstrated in the analytic method of detection of formyl-met presence at the n-terminus of gene-engineered -interferon. trypsine hydrolysis of -interferon leads to more than 14 peptides are formed including the 12-membered n-terminal peptide. being hydrolyzed with glutamyl-specifi c enzyme -interferon is converted to 6 peptides, including 41-membered n-terminal peptide. application of postprolinespecifi c enzyme for -interferon hydrolysis leads to formation of 1 short 4-membered peptide and 4 long fragments. experimental data show that in case of analytic control of -interferon maturity the utilization of postproline-specifi c protease is preferable. in other cases the enzyme selection is ruled by the features of protein primary structure. bimodal action of cystatin related epididymal spermatogenic (cres) protein and its reactive site loop derived s-s bridge bicyclic peptides on proprotein convertase-4 (pc4) activity several precursor proteins found on sperm surface and reproductive tissues were proposed or confi rmed as substrates of pc4. these include adam proteins, growth factors proigf1 and 2 and hormonal protein pacap. lack of pc4 leads to impaired fertility in mice, suggesting its important role in reproduction. pc4 is thus considered an important target for development of nonhormonal contraceptive agents. pc4-inhibitors are expected to fi nd therapeutic, clinical and biochemical applications. a lot of interest has grown to develop specifi c pc4 inhibitors. recently natural inhibitor of serpin family have been described for pc1, 2, and furin, but not for pc4. however, a new serpin, cres, has been reported from epididymis fl uid, where pc4 may be found. so far, cres has only been shown to inhibit pc2, which is not present in reproductive tissues. we propose that cres may represent a natural regulator of pc4, based on localization and other studies. in this study we generated recombinant human cres protein (139 aa), and its reactive-site loop (rsl) derived acyclic and cyclic 43-mer peptides with various s-s linkage combinations. cres inhibited pc4 with ic50 ~50um, while rsl-peptides were found to be more potent with ic50 50-250nm, depending on the s-s linkage locations. furthermore, we noted that at lower concentrations, cres and its peptides fi rst enhanced pc4 activity before any inhibition occurred. this bimodal behavior may be due to cleavage. molecular modeling showed strong interactions between cres and pc4 via some of their key residues. overall, we noted that "rsl" is the most crucial element required for pc4 inhibition by cres. funds were from from cihr (ab). the de novo design of peptides and proteins has assumed considerable interest didehydroresidues, in particular alpha, in the recent years. alpha, beta-beta-didehydrophenylalanine (deltaphe) are being considered important conformational constraints inducing tools in de novo peptide design. deltaphe is a noncoded, achiral residue, an analog of the naturally occurring phenylalanine amino acid with a double bond between calpha and cbeta atoms. introduction of deltaphe in peptide sequences is known to induce conformational constraint, both in the peptide backbone as well as the side chain, and to provide the peptide with increased resistance to enzymatic degradation. in small peptides containing eta turn structure, and in peptides containing a single deltaphe, a type ii b more than one deltaphe residues, helical structures are stabilized. a number of deltaphe peptides varying in length, content and position of deltaphe residues have been found to contain 310 helices of both screw senses. following these design principles, we were able to design, synthesize and characterize super secondary structural motifs like helix-turn-helix, helical bundle and glycine zipper. we have extended this work to the de novo design of peptides with antibiotic and anti-fi brillization activity. a series of cationic peptides containing deltaphe residues have shown remarkable antibiotic activity and are being developed further. deltaphe containing peptides may have longer in vivo half life owing to their ability to resist enzymatic degradation. more recently, we have observed that small peptides containing deltaphe self-assemble in nanotubular and nanovesicular structures. these nanostructures have also been used to entrap small drug like molecules. we have fully characterized these systems and are exploring their potential as delivery agents in biological systems. the synthesis and design of peptidomimetic oligomers that adopt designed, compact conformations (foldamers) have become a challenging task in recent years. among them, -peptide foldamers exhibit rich diversity of secondary structures. [1, 2] a relationship has been established between the backbone chirality pattern and the prevailing secondary structure, which underlines the role of stereochemical control in the -peptide foldamer design.(3) an important challenge is to introduce proteinogenic side-chains to generate diverse anchor points on the molecular surface promoting their application in drug discovery. it has been proved that stable helices can be obtained when sequences were coupled with -amino acid enantiomeric pair motifs in a lego approach.(3) in this work, -amino acids and open chain functionalized -amino acids were inserted between the enantiomeric pair design element for 1 and 2, respectively. confi gurations in the backbone were designed to promote helix formation. nmr and ecd measurements augmented with ab initio calculations revealed that 1 forms a h9-12 helix and 2 forms a h14/16 helix. these helices are unprecedented in the literature. to prove that the stereochemical patterning is crucial in the folding, we perturbed the confi guration motifs of the backbones by swapping the sequence of the central -amino acids. the peptides with exchanged residue order could not fold into helices. these novel structures can be useful scaffolds for biomedical applications. introduction small length synthetic peptides, based on sp c-terminal fragment, increase the secretion of tnf-and prevent the proliferation of several cancer cell lines. we have already shown the antiproliferative activity of tri-and tetra-peptoids in breast and prostate cancer cells. the aim of this study was the synthesis of hexa-peptoids, analogs of sp c-terminal region, containing the residues d-trp and tic and the peptoid ones nhn(r)ch2co, nphe and nala in their sequence and their evaluation against cancer cells proliferation. methods and results all the syntheses were carried out stepwise using the fmoc/ but methodology on the solid support 2-cltr resin and dic/hobt as coupling reagent. all analogs were purifi ed (hplc) and identifi ed ( cyclotheonamides constitute a family of structurally related cyclic pentapeptides of marine origin that inhibit trypsin-like serine proteases. their binding mode has been elucidated by the x-ray structure of cyclotheonamide a in complex with trypsin. an extended peptide conformation which is stabilized by macrolactamization allows to address in a substrate-like manner beside the s1 pocket also the s1' and s2 pocket. the s1 ligand, (s)-3-amino-6-guanidino-2-oxo-hexanoic acid, interacts via its guanido function with asp 189 at the bottom of the s1 pocket. in addition, the ketone covalently modifi es the gammaoxygen of ser 195 by hemiketal formation (1) . recently, two novel cyclotheonamides have been isolated from a marine sponge of the genus ircinia. one of them, cyclotheonamide e4, is a potent inhibitor of human beta-tryptase (2) . in this study, cyclotheonamide e4 was modifi ed at two positions: (i) the s1 ligand was replaced by beta-homolysine or as betahomoarginine to obtain reversible acting tryptase inhibitors, and (ii) the alpha amino function of (s)-2,3-diamino propionic acid, which is not part of the cyclic backbone, was used as anchoring point for basic p3 residues to exploit interactions with the negatively charged glu 217 of tryptase. these analogs were synthesized by a combination of solid phase and solution phase chemistry 3.. synthetic details as well as the inhibitory profi le of these novel cyclotheonamide e4 analogs will be discussed. oostatic peptides containing d-amino acids: activity and degradation in the fl esh fl y neobellieria bullata bennettová deteriorating effect of c-terminus truncated 4p and 5p analogues [1, 2] of decapeptide h-tyr-asp-pro-ala-pro 6 -oh (3) on ovarian development (i.e. oostatic effect) of insect species diptera, orthoptera and hemiptera has stimulated an analysis of metabolic degradation of corresponding peptides after application to the insect body [4] [5] [6] [7] . radiolabeling in different positions of the peptide chain allowed determination of the degradation decisive steps -the fast splitting off the c-terminal pro from the 5p followed by successive cleavage of tyr and asp from the nterminus. after introduction of corresponding d-amino acids into peptide chain of 5p, we could see the same or even stronger oostatic effect of the analogues in comparison with parent peptide after application on the fl esh fl y neobellieria bullata. in the degradation assay, an elimination of enzymatic cleavage of peptide bonds pointing from the central labeled pro residue to either d-ala or d-asp residues in the neighbourhood was observed, resulting in total increase of the analogs stability. structure-activity relationships( sar) studies of camk ii inhibitors. in fact, in vitro it can phosphorylate up to 40 proteins, including enzymes, ion channels, transcription factors and a number of these proteins appear to be physiological substrates. for example, cam-kii is highly concentrated in the postsynaptic density of glutamatergic synapses where it phosphorylates and potentiates current through the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor ion channel (ampa-rs). in fact, this family is encoded by four genes ( , , and ), whereas the and isoforms are expressed in diverse tissues and , isoforms are most prominent in neural tissues. the identifi cation of camk ii inhibitors is important to better defi ne its physiological. in literature is reported a 79-aa brain-specifi c protein that and potently inhibited kinase and bound the catalytic domain of cam-kii activity with an ic50 of 50 nm. the inhibitory protein (cam-kiin), and a 27-residue peptide derived from it (camkiintide, krppklgqigrakrvvieddriddvlk), was highly selective for inhibition of cam-kii with little effect on cam-ki, cam-kiv, cam-kk, protein kinase a, or protein kinase c. cam-kiin interacted only with activated cam-kii (i.e., in the presence of ca2+/cam or after autophosphorylation). here we report a structure-activity relationships study using as template the camkiintide. we synthesised a peptide contains all 28 amino acids present in camkiin-tide, however, in random sequence (camkiin-tide scramble). then we operated a progressive deletion of amino acids from c-terminal and dall'n-terminal to detect minimum active sequence. moreover camkiintide and cankiintide scrumble was made cell-permeable by n-terminal addiotion of an antennapedia (rqikiwfqnrrmkwk). here we report the biological results of our study. ptprj: a receptor-type protein tyrosine phosphatase as a target of new peptides with antitumoral activity to be signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. it has been demonstrated that ptprj (also known as hptpeta, dep-1, cd148) is able to inhibit cell growth promoted by specifi c ptk some of which are over-expressed in certain types of cancer. moreover a role for ptprj was also assessed in vasculogenesis; infact its reduced activity in enhanced vegf-induced vegfr2 activity leads to increased cellular responses, supporting a ptprj-vegfr2 interaction. on these basis it is important the identifi cation of molecules with agonist activity which are able to stimulate the function of residual ptprj in malignant cells and stopping the proliferation and possibly trigger programmed cell death. using as a template a peptide, already identifi ed, with agonist activity against ptprj(h-[cys-his-his-asn-leu-thr-his-ala-cys]-oh), here we report a structure-activity study carried out through endocyclic modifi cations (ala-scan, d-substitutions, single residue deletions, substitutions of the disulfi de bridge) and the preliminary biological results of this set of compounds. faulty of chemistry, university of gdansk, poland; 2 faculty of chemistry, university of gdansk, poland isolated in 1999 from the sunfl ower seeds trypsin inhibitor sfti-1 is up to date the smallest naturally occurring peptidic proteinase inhibitor and therefore is an excellent starting structure to design peptidomimetic serine proteinase inhibitors. peptomeric library consisting of 360 monocyclic analogues of sfti-1 was designed and synthesized by the solid phase method with the intension of select chymotrypsin and cathepsin g inhibitors. all peptomers contained in positions 5 and 12 nbenzylglycine (nphe) that mimics proteinogenic phe. in the synthesized library different peptoid monomers were introduced in the segment 7-10. this is a turn region that makes an important contribution to structural integrity and rigidity of sfti-1. deconvolution of the library against both proteinases by the iterative method in solution revealed that the highest chymotrypsin inhibitory activity displayed analogue with n-[4-(2-aminoethyl)morpholyl]-glycine (naem) in position 8. this analogue was even more active than the one with pro in this position which is absolutely conserved in bownan-birk inhibitors. while, deconvolution carried out against cathepsin g indicated that analogue with npiperonylglycine (npip) in position 8 and 9 and with n-butylglycine (norleu) in position 10 presents the highest inhibitory activity. acknowledgements: this work was supported by ministry of science and higher education (grant no. 2889/h03/2008/34). 3 a platform for the design and optimization of new antimicrobial peptides effective against specifi c pathogens has been set up. the main features expected for these peptides are low environmental impact, broad spectrum of activity, reasonable bacterial selectivity, and low eukaryotic cytotoxicity. our approach also includes the use of a design of experiments protocol in order to fi nd peptide sequences that fi t these features. the obtained peptides would represent an alternative to currently used antibiotics or pesticides. this work focused on fi nding new control agents against economically important plant pathogenic bacteria such as erwinia amylovora, pseudomonas syringae and xanthomonas vesicatoria for which the available methods are not suffi ciently effective. nowadays, their control is mainly based on copper compounds and antibiotics. although antibiotics are highly effi cient, they are not authorized in several countries and resistance has been developed on plant pathogens. we have synthesized combinatorial libraries of cyclic decapeptides and linear undecapeptides. these libraries have been screened for antibacterial activity and eukaryotic cytotoxicity, and have led to the identifi cation of peptides with mic values of 1.6-12.5 microm. notably, cyclic peptides active against e. amylovora have been found, constituting the fi rst report of this type of peptides with activity towards this bacteria. the best peptides are bactericidal, display a low eukaryotic cytotoxicity at concentrations 30-120 times higher than the mics, and show a low susceptibility towards protease degradation. best peptides have been tested in vivo by evaluating their preventive effect of inhibition of p. syringae, x. vesicatoria and e. amylovora infections. the most active peptide is slightly less effective than streptomycin, currently used in fi eld. therefore, the best analogues can be considered as good candidates for the development of antibacterial agents for use in plant protection. selection of chromogenic and fl uorogenic substrates of neutrophil serine proteases using combinatorial chemistry approach human serine neutrophil and mastocytes proteases such as cathepsin g, neutrophil elastase, proteinase 3 and -tryptase are involved in several physiological processes. unwanted activity of those enzymes yields to severe pathological states like infl ammation, wegener granulomatosis or various types of cancer. therefore monitoring of the activity of these proteases is crucial for the proper therapeutical treatment. the simplest method for determination of protease activity is the use of synthetic substrates. they are also useful for characterization of the enzyme specifi city. in this work we report selection of chromogenic and fl uorogenic substrates of human serine neutrophil and mastocytes proteases applying combinatorial chemistry approach. peptide libraries were synthesized by the portioning-mixing method. deconvolution of synthesized libraries was performed using iterative approach in solution. 5-amino-2-nitro benzoic acid attached to the c-termini of synthesized peptides served as a chromophore released upon the interaction of peptide with enzyme. additional introduction of 7-methoxy-4-coumaryl acetic acid or 2-amino benzoic acid on á-amino groups of the chromogenic substrates converted them into fret displaying compounds. the most active substrates were subjected to further modifi cation applying non-proteinogenic amino acids. as a result, one of the most selective substrates of these proteases was obtained. we also attempted to construct a simple tools to detect activity of human serine neutrophil and mastocytes proteases by immobilization, through amide and peptoid based linkers, of selected substrates on solid phase. angiogenesis modulation: identifi cation of tetrameric tripeptide as inhibitor of vegfr-1 by the screening of peptide combinatiorial libraries 5 vascular endothelial growth factor receptor-1 (vegfr-1, flt-1) and their ligands are involved in complex biological processes associated to severe pathological conditions, like angiogenesis, infl ammation and metastasis formation (1). thus, the search for antagonists of flt-1 has recently gained a growing therapeutic interest. in order to identify new molecules able to selectively bind flt-1 and neutralize its activity, a screening of a combinatorial tetrameric tripeptide library built with nonnatural amino acids has been carried out by a competitive elisa-based assay. the library has been designed on a branched tetrameric structure in order to obtain molecules with a high recognition surface and, using 30 building blocks, a complexity of 27.000 different peptides has been achieved. peptide mixtures composing the library have been utilized as competitors of the flt-1/plgf (placental growth factor) interaction and the most active components have been isolated following an iterative deconvolution procedure. the selected most active hit shows a selective binding to flt-1 over kdr and inhibits in vitro its interaction with both plgf and vegf-a. the peptide is fully stable in biological environments, prevents flt-1 phosphorylation and blocks huvec capillary-like tube formation stimulated by plgf or vegf-a. in vivo the peptide inhibits the vegf-induced neoangiogenesis in chicken embryo chorioallantoic membrane assays and also stimulates cornea neovascularization (cnv) by displacing vegf from a complex with sflt-1. all data suggest that the compound has potential applications in diseases characterized by pathological angiogenesis, such as tumor growth and ischemic retinopathy. heinlein, christian; unverzagt, carlo bioorganische chemie, universität bayreuth, gebäude nw i, 95440 bayreuth, germany since the purifi cation of homogeneously glycosylated glycoproteins remains a diffi cult task the synthesis of entire glycoproteins is a fi eld of emerging interest (1) . especially the synthesis of the required protein fragments in high purity and good yields demands for effi cient methods. fragment condensation of protected peptides can reduce deletions in the crude product and thus facilitates purifi cation. condensation of peptide fragments in cspps (2) is however subject to epimerization upon cterminal carboxyl activation. to overcome this problem the use of cterminal gly or pro is usually performed. peptides containing c-terminal pseudoprolines can also be coupled without stereomutation because the serine or threonine residue has been reversibly protected as a prolinelike oxazolidine (3). this concept facilitates the synthesis of long peptides by epimerization-free fragment condensation at c-terminal ser/ thr residues in addition to gly/pro residues thus increasing the number of safe condensation sites. after solving several problems associated with the generation of peptide acids with c-terminal pseudoprolines the synthesis of the glycosylated rnase fragment was carried out as a racemization-free fragment condensation. tuftsin is liberated from fc-domain of the heavy chain of igg by two specifi c enzymes. being the tetrapeptide of biological origin is extremely important product because it can activate a few elements of immune system such as granulocyte and macrophage. tuftsin indicates not only immunological stimulating factor but also antibacterial, antivirial and antitumor properties. in spite of its wide range of activity, the peptide is unstable in plasma and it has become the aim of the formation of novel analogues more resistant to proteolysis degradation. the introduction of the additional residue at -amino group of lysine caused that new bond became stronger than peptide bond in central chain [1] [2] [3] . we synthesized linear tuftsin derivatives that were prepared on the solid phase using a fmoc/tbu procedure. the method of elongation of peptide chain was based on two-step procedure: deprotection and coupling step. segment coupling reaction was carried out with tbtu, hobt and in the presence of diea in dmf/dcm/nmp mixture. the introduction of the simple amino acid (ala, -ala, val, ile or gly) at -amino group of lysine let us obtain the isopeptide bond. the modifi cation was achived by introducing lysine residue, protected at -amino group with mtt. the selective removal of mtt group was caused by 2% tfa treatment. peptides were cleavage from resin and purifi ed. tuftsin derivatives were confi rmed by ms, amino acid analysis, elemental analysis and rp-hplc analysis. peptides were sent to assay their microbiological properties and the results will be described as a structure-activity relationship. the synthesis and structural study of iso-aß(1-42) the aß(1-42) peptide is diffi cult to synthesize, because of its high tendency for aggregation during the synthesis. both the couplings and the fmocremoval can be troublesome, due to the steric hindrance. the incorporation of the ester-bond disturbs the structure, thus ease the synthesis after the 25 th residue. if the peptide would be synthesized using boc-chemistry the removal of the -amino protecting group would be less problematic, because the 50% tfa/dcm mixture solubilizes well the peptide chain. thus a boc-strategy was devised for the synthesis of iso-aß(1-42). the purifi ed peptide was studied with cd-spectroscopy and dynamic light scattering. these structural studies revealed that the isopeptide has some ß-sheet content even in a ph 2 solution. it was realized also that small aggregates are present in the precursor peptide. both techniques mentioned above showed, that after altering the ph to 7.4, the ß-sheet content of the peptide increases and aggregation takes place. with the use of the isopeptide, aß oligomers and -with prolonged incubation -fi brillar structures can be formed for biological studies. continuing our program of syntheses of muramyl dipeptide (mdp) and nor-muramyl dipeptide (nor-mdp) conjugates as potential immunomodulators, we designed novel conjugates of mdp or nor-mdp with tuftsin derivatives containing isopeptide bond between -amino group of lysine and carboxylic group of simple amino acids such as alanine, glycine and valine. the synthesis of a greater number of conjugates will enable structure-activity relationship studies. tuftsin analogues containing isopeptide bond showed increased chemical resistance and activity in relation to tuftsin. the introduction of the additional residue at -amino group of lysine by nhco-formation caused that isopeptide bond became stronger than peptide bond in central chain. the protected pentapeptides (h-thr-lys(y)-pro-arg(no 2 )-obn, y= ala,gly,val) were synthesized by the conventional chemical procedure using mixed anhydride method. acylation of the thr amino group of partially protected pentapeptides by 1-benzyl-mdp or 1-benzyl-nor-mdp was performed using the mixed anhydride method with isobutyl chloroformate and n-methyl-morpholine (nmm) in dry dmf. the protected conjugates were isolated and purifi ed with a preparative tlc. the identities of the protected products were confi rmed by high resolution 1 h-nmr (500 mhz, cosy, tocsy, roesy, ghsqc, ghmbc) spectroscopy. the fi nal products were hydrogenated with h 2 / pd/c in 50% methanol-acetic acid and purifi ed with preparative tlc. the identities of the conjugates were confi rmed by tlc qualitative amino acid analysis, and elemental analyses. finally, the combined use of muramyl peptides with other immunomodulators, e.g. such as tuftsin, retro-tuftsin other chemotherapeutics is promising in the therapy of different infections, autoimmunological diseases and anticancer therapy. effi cient microwave-assisted synthesis of myelin epitopes mog35-55 and mog97-108 using cltr-cl resin fmoc/tbu methodology. unlike conventional heating, microwave energy directly activates any molecule with a dipole moment and allows for rapid heating at the molecular level. the protected peptides were synthesized using the cem liberty automated microwave peptide synthesizer in 18 and 13.5 hours respectively, with a 3-minute fmoc deprotection (25% piperidine solution in dmf) and 5-minute coupling reactions using dic/hobt in dmf solution. the maximum temperature reached during both the deprotection and coupling reactions was 80 °c except for the coupling of fmochis(trt)oh (50 °c). the fi nal crude products were of high purity as identifi ed by analytical rp-hplc. siah up-regulates the hif-1alpha hypoxic response pathway: siah binding peptides coupled to cpps inhibit siah activity and demonstrate proof of concept that siah is a viable anti-tumor drug target vegf, a secreted downstream component crucial for angiogenesis, has been successfully targeted clinically using antibody therapy (avastin). the hypoxic response, however, activates other pathways that stimulate tumor growth, suggesting that inhibitors of upstream components in the pathway may be useful to give a broader spectrum of inhibition. we have focused on the siah proteins that regulate hif-1ƒñ levels by ubiquitylation and degradation of the prolyl hydroxylases (phds) immediately upstream of hif-1ƒñ in the hypoxic response pathway. in a proof-of-principle study, we have shown that siah inhibition by expressed protein fragments (from a drosophila high affi nity interacting protein, phyllopod) can inhibit the stabilization of mammalian hif-1ƒñ during hypoxia and limit tumor growth in a mouse tumor model. a 23 amino acid peptide sequence (phyl) from the phyllopod protein has been identifi ed as the interaction site with siah. the aim of this work was to show that a small peptide could mirror the activity of the transfected recombinant phyllopod protein. to achieve this, phyl was covalently attached to cell penetrating peptides (cpps) and tested for its ability to inhibit hif-1ƒñ stabilization and hypoxic response in human u2os cells. both the tat sequence and penetratin were utilized as cpps and attachment was via disulfi de, maleimide or through a pro10 spacer sequence. the cpp¡vphyl constructs were found to have varying activities but a penetratin-pro10-phyl was found to be inhibitory in the u2os cell-line hif-1ƒñ stabilization assay. this result demonstrated proof of concept that siah inhibition could be attained by targeting a restricted and specifi c protein-protein interaction site. (1) . the principal event in the development of prion disease is the transformation of the normal cellular prion protein (prpc), which is highly helical in nature, into the pathogenic isoform prpsc which is insoluble and has an extensive ƒò sheet structure. the events surrounding this transformation are very poorly understood, but the mechanism of the detailed steps involved in this process is fundamental to the understanding of prion disease pathogenesis. in an endeavour to study the structure of polypeptide component of the n-terminal section of prpc, synthesis of a range of prp polypeptides were assembled on a cem liberty microwave synthesiser. these fragments ranged in length from 20 to 111 amino acids and span the protein sequence from position 1 to 144. standard cem synthetic coupling cycles were used, except when peptides were greater than 30 amino acids in length, whereby longer coupling cycles were employed. a number of purifi cation strategies were employed, such as c4 and c18 rp-hplc at either 25c or 60c and size exclusion chromatography. the purifi ed peptides were characterised by rp-hplc and esi-ms. in addition, they were analysed for secondary structure by cd spectrometry, and evaluated for cell toxicity and fi bril forming ability with tht. the synthesis of a 60mer peptide for investigating the mechanism of action of the hiv fusion inhibitor t20 however, the mechanism of action of t20 is still in debate. it is believed that peptides derived from gp41 chr region may share a common mechanism, by binding to gp41 nhr coiled coil and preventing formation of the fusogenic gp41 core-six helix bundle (6hb), thereby inhibiting fusion between the virus and target cell membrane. we have synthesized a 60-mer nhr peptide-n60-covering all the binding sites for any length of the chr-peptide. synthesis of the polypeptide was carried out using conventional as well as microwave assisted solid phase synthetic methods. details, including comparison of the synthetic approaches will be presented. using c34, another anti-hiv chr peptide containing the pocket-binding domain, as a control, we analyzed the activity of t20 to interact with n-60 to form 6hb and to inhibit the 6-hb formation between n-60 and c34 by cd spectroscopy and elisa. we found that t-20, unlike c34 could neither form a stable 6hb with n60, nor inhibit the 6-hb formation of the fusogenic 6hb core. our results thus suggest that t-20 and c-34 peptides inhibit hiv fusion by different mechanisms of action. the oligomerisation equilibrium of ts is modulated by a fi ne tuning; shifting this equilibrium towards the monomeric form would cause ts inactivity and low translation, overcoming resistance mechanisms encountered for (co)substrate-like inhibitors as the inactive monomer regulates its own expression. our group designed some small ligands to interfere with thymidylate synthase dimerisation, including short peptides taken from the interface sequence that represent the natural ligand for this region, mimicking the other subunit without forming a functional dimer. interesting biological data are arising from the activity tests but a rapid screening assay is necessary to prove they are really interfering with ts oligomerisation. fret is a spectroscopic phenomenon whose intensity depends on the distance between two fl uorophores; when this value varies due to conformational changes of the protein the probes are linked to, consequent fret variation can be used to sense the state of the protein(s) ( stromal derived factor-1 (cxcl12) is a 68 amino acid cxcchemokine with critical role in homing, migration and guiding of different cell types including hematopoietic progenitor cells (hpc), stem cells, tumour cells and neuronal cells during embryogenesis and in adults (1). these various functions in physiological as well as pathophysiological processes make this small protein interesting for regenerative medicine. to apply this chemoattractant in medicine, it is needed to form spatially and temporally controllable concentration gradients of active sdf-1 in response to a non-tissue damaging trigger like visible or near uv-light. after irradiation of an inactive prodrug dramatic change in conformation or lack of sterical hindrance should then lead to fully biological activity under physiological conditions within a few minutes. to prove the principle and assess its potential in photodynamic therapy of neuronal injuries a water-soluble, photosensitive sdf-1 analogue has been developed. for this the expressed protein ligation (epl) approach has been used, in which one segment has been recombinantly expressed and purifi ed using the impact ® -system to yield the corresponding peptide thioester (2) . the modifi ed peptide fragment has been chemically synthesized on solid phase using fmoc-strategy. the photocleavable moiety, the 6-nitroveratryloxycarbonyl (nvoc) protecting group, has been introduced at a side chain amino group. furthermore, the analogue has been characterised by physico-chemical methods and has also been tested in vitro on transfected cos-7 cells in order to determine its biological activity after activation. sdf-1 is a chemokine that plays a major role in traffi cking of hematopoietic stem cells (hsc). thus it enables the formation of bone marrow during embriogenesis and later in adult life it supports retention and homing of these cells in the bone marrow. furthermore it is involved in organogenesis and regeneration, respectively. 1 due to these promising features the subform sdf-1 could serve as a therapeutic target. for studies on the small protein concerning its molecular properties as well as its therapeutic potentials, it needs to be modifi ed chemically. in order to reach these goals, the n-terminus sdf-1 1-49 has been cloned and expressed recombinantly in e. coli er 2566 as a thioester, while the c-terminus sdf-1 50-68 has been synthesized via solid phase peptide synthesis. modifi cations are thereby introduced at the c-terminus at lys56. up to now carboxyfl uorescein has been coupled to the -amino group of the lysine residue. the two fragments then have been ligated via expressed protein ligation (epl), a subform of the native chemical ligation (ncl). 2 activity studies and fl uorescence microscopy on hek293 cells transfected with the sdf1-specifi c g-protein coupled receptor cxcr4 have been conducted. stueber, werner; lewandrowski, peter; frisch, juergen; weinschenk, toni; singh, harpreet immatics biotechnologies gmbh, germany ima901 is a multiple peptide vaccine for the treatment of renal cancer (rcc). the tumor-associated peptides (tumaps) contained in ima901 were identifi ed by immatics directly from primary renal cells (= primary rcc tumor tissue samples), selected regarding their over-expression in rcc and proven to be immunogenic using in vitro t-cell assays. ima901 consists of 10 individual peptides (10 tumaps) and nonactive ingredients which are used as excipients of the pharmaceutical presentation of ima901. all 10 peptides are synthesized by conventional fmoc chemistry. the sequences of the peptides will be presented and technical issues will be discussed. in the fi nal formulation of ima901 578 g of each peptide plus excipients are fi lled into glass vials and lyophilized. the challenges of the production of multi peptide drugs will be discussed. such challenges comprise the synthesis, the production of the formulation as well as the analyses of the fi nal presentation of ima901. results of the phase 1 trial in 28 vaccinated rcc patients showed that (1) ima901 was safe, (2) multiple t-cell responses to vaccinated peptides correlated with favourable clinical outcome and (3) patients with a lower percentage of regulatory t cells (tregs) were more likely to develop a vaccine-induced multiple t-cell response. novel peptides -msh analogs with high candidacidal activity in an attempt to improve the candidacidal activity of -msh and to better understand the peptide structure-antifungal activity relations, we designed and synthesized novel peptide analogs. because previous data suggested that the peptide [dnal-7, phe-12]--msh(6-13) has greater candidacidal activity than -msh and is the most potent of the analogs tested in the past, this compound has became our lead (1, 2) . from this lead compound we have synthesized a new library of peptides where we have replaced the glycine in position 10 with unconventional amino acids. here, we report new analogs with a strong antimicrobial and candidacidal activity. the obtained results are very encouraging in that they show the great potential of these peptides as a truly novel class of candidacidal compounds. derivative were modifi ed by the same amino acid to establish infl uence of the adjacent amino acid on antimicrobial activity of the compounds studied. the activity of all obtained peptides was screened against model gram-positive (bacillus subtilis) and gram-negative (escherichia coli) bacteria whereas antifungal activity was tested against yeast pichia pastoris. all tests were performed using antibiogram method whereas the minimal inhibitory concentrations were determined using two-fold serial dilution technique. huang, yen-hua 1 ; colgrave, michelle l. 2 the cyclotides are a family of naturally occurring macrocyclic peptides that combine the unique features of a head-to-tail cyclic backbone and a cystine knot motif, which impart extraordinary stability to this peptide family. a recent study demonstrated that the prototypic cyclotide kalata b1 possesses signifi cant activity against two economically important sheep nematodes haemonchus contortus and trichostrongylus colubriformis. an alanine scan of the molecule highlighted the residues critical for activity. in this work, we explore the relative importance of positively charged residues in different regions of the molecule to aid the understanding of the structural and biological basis of its nematocidal activity. a lysine scan has been conducted, in which each of the non-cys residues in this 29 amino acid peptide has been successively replaced with lysine and the suite of peptides have been assayed against the two sheep nematodes. substitution of residues in loop1, v10, g12, n15, t16, w23, v25, l2, p3, and v4 decreased or completely abolished the activity, suggesting that these residues are critical to the nematocidal activity of kalata b1. on the other hand, incorporation of a positive charge in positions g18, t20, t27, n29, and g1 signifi cantly enhanced the anthelmintic activity of the grafted peptides, up to fourfold, compared to native kalata b1. these increases in activity after lysine incorporation into the kb1 scaffold raise the possibility of being able to engineer greater anthelmintic activity into the peptides, further highlighting their potential as anthelmintic agents. ivanova, v.p. 1 interaction of cells with extracellular matrix (ecm) affects many aspects of cell behavior including growth, morphology, migration and differentiation. integrins are known to mediate cell adhesion to proteins of ecm. ligand-binding properties of integrins depend not only on composition of ecm ligands and level of integrin expression in cell, but also on spectrum and activity of soluble factors indirectly infl uencing cell-matrix contacts. interaction of cells with substratum may be divided into cell adhesion and spreading. following an initial cell attachment event, cell may or not spread depending on cell type and the nature of the molecular signals they receive. oligopeptides released from different proteins during their proteolysis in or out of cells may act as the such short-time existing signals. in the present study we investigated the effect of multiply repeated peptide fragment in different collagen types on cell spreading. murine embryonic fi broblasts (200000/ml) were allowed to adhere for 45 min at 37° c with or without peptide (in various concentrations) to plastic surface pre-coated or not with gelatin. it was shown that the synthetic peptide increased the number of spread cells and caused shape changes in cells spread on different substrata. a 30-min pretreatment of cells with the peptide (before conducting of cell spreading assay) resulted in inhibiting of cell spreading on gelatin. our results suggest that the peptide regulation of cell spreading could be related to its effect on re-distribution of integrin receptors in sites of cell contacts with substratum. bioactive peptides from cyanobacteria cyanobacteria are versatile source of small peptides from which vast majority are cyclic. cyanobacterial culture collection in university of helsinki contains over 1000 strains and this collection is used for screening of new bioactive compounds. cyclic heptapeptides, microcystins are the best known cyanobacterial peptide family. over 80 structural variants have been described from which most are strong hepatotoxins. our research group have participated to the determination of the structure of many novel microcystins and other cyclic and linear cyanobacterial peptides. in recent years we have found highly toxic and novel microcystins from lichen associated cyanobacteria (1) and from anabaena strains of baltic sea (2). in the structural analysis of new peptides from the known peptide families we have used liquid chromatography ion trap mass spectrometry which have proven to be very effective method and is in many cases the only method needed in the verifi cation of a new structure. with this lc-itms method we have found many new structural variants from anabaenopeptin, anabaenopeptilide and spumigin peptide families. in collaboration with many research groups we have found cyanobacterial compounds/extracts which inhibit protein kinase c activity, inhibit/activate boar sperm motility, disintegrate cell membranes or are antidotes for microcystin toxicity. structures of the compounds are under study except the microcystin antidote which structural analysis showed that it belongs to a rare peptide family containing imino bond in cyclic skeleton. the results of n-terminal modifi cation of arginine vasopressin with cis-1-amino-4-phenyl-cyclohexane carboxylic acid. the highly potent oxytocin receptor antagonists arginine vasopressin (avp) is a cyclic nonapeptide with multiple functions. the main peripheral physiological roles of avp are the regulation of water balance, the control of blood pressure, and the release of adrenocorticotropin hormone (acth). moreover, avp also exhibits to some extent typical oxytocin (ot, a closely related neurohypophyseal peptide) activities such as the galactogogic and the uterotonic effects. all peptides were tested for the pressor, antidiuretic and uterotonic in vitro activities in the rat. cis-apc 2 modifi cation at position 2 of avp is suffi cient to change the pharmacological profi le of the peptides. analogues i -iv were moderately potent antidiuretic agonists with prolonged action. in regard to the uterotonic activity, all peptides with cis-apc were highly potent antagonists, except compound i. it supports our earlier hypothesis that an amino acid residue in position 2 has signifi cant impact on pharmacological activities. the incorporation of unnatural non-proteinogenic -amino acids into peptides has emerged as a novel and promising approach in peptide modifi cation. the conformationally restricted amino acid derivatives are of particular interest. this thesis are also supported by our already 12-years research focused on the effects of steric restriction and the presence bulky substituent in the n-terminal part of avp molecule on biological properties of the resulting analogues. all peptides were tested for the pressor, antidiuretic and uterotonic in vitro activities in the rat. all the analogues were devoid of the pressor potency and exhibited only negligible antidiuretic activity. interestingly, in regard to the uterotonic activity, the new compounds exhibited moderate (i) or high (ii -iv) antioxytocic potency. it should be point out that single substitution e.g. replacement of tyr in avp molecule with igl results in moderately potent and highly selective antagonists of oxytocin (i). our new igl 2 substituted peptides proved that the presence of the sterically restricted amino acid residue may result in high active and selective antiuterotonic agents. these in our opinion interesting fi nding demonstrates the usefulness of our approach in the design of new highly active and selective analogues with desired pharmacological properties. arginine vasopressin (avp), a neurohypophyseal hormone and neuromodulator, is a cyclic nonapeptide with a disulfi de bridge between cys residues at positions 1 and 6. as a hormone avp exerts its biological effects upon binding to three receptor subtypes termed: v 1a , v 1b (v 3 ), and v 2 . furthermore, avp to some extent can interact with the oxytocin receptor (ot). biological activity of peptides is determined by their structure and conformation. conformational restriction of bioactive peptides is therefore a well-established strategy to change their pharmacological profi le. peptide fl exibility can be restricted by a local constraint imposed, e.g. by introducing amino acids with limited conformational freedom, that has an impact on specifi c orientations of the peptide backbone and the side chains. in this work, we decide to check the infl uence of the bulky ( design, synthesis and biological activities of temporin a and temporin l analogues. temporins a (ta) and l (tl) are antimicrobial peptides isolated from the skin of red european frog "rana temporaria". temporins are active against a broad spectrum of microrganism: ta (flpligrvlsgil-nh2) is preferentially active against gram-positive bacterial strains; tl (fvqwfskflgril-nh2) has the highest activity against fungi, and bacteria, including resistent gram-negative strains, but it shows haemolytic activity too. ta exerts its antimicrobial activity by its ability to form a transmembrane pore via a 'barrel-stave' mechanism or to form a 'carpet' on the membrane surface via the 'carpet-like' model. recently we investigated the preferential conformation of tl and ta in sds and dpc solutions which mimic bacterial and mammalian membranes, respectively. in sds, the peptides prefer a location at the micelle-water interface; in dpc, they prefer a perpendicular location to the micelle surface, with the n-terminus imbedded in the hydrophobic core. tl shows higher propensity, with respect to ta, in forminghelical structures in both membrane mimetic systems and the highest propensity to penetrate the micelles (1). on these results we designed and synthesized new ta and tl analogues and found interesting differences in their effi cacy against microbial species, and fi nding a new potent antimicrobial agent without haemolytic activity. arginine vasopressin (avp), a neurohypophyseal nonapeptide hormone [cycle1-6 (h-cys 1 -tyr 2 -phe 3 -gln 4 -asn 5 -cys 6 -pro 7 -arg 8 -gly 9 -nh 2 )], elicits a variety of responses both centrally and peripherally by acting on three distict g-protein coupled receptors: v 1a (vascular), v 1b (pituitary) and v 2 (renal). it also binds to the oxytocin (ot) receptor. in addition to its well-known antidiuretic activity, avp has also complex cardiovascular actions and adrenocorticotropic hormone (acth) releasing activity. binding of avp to the v 1a receptor subtype also stimulates glycogenolysis in the liver and promotes platelet aggregation. it is generally accepted that the conformation of the n-terminal part of neurohypophyseal hormones analogues is important for their pharmacological activity. in continuing our work aimed at the design of selective avp analogues, we synthesized twelve new analogues of avp containing mercapto propionic acid ( we also studied the effect of modifi ed c-terminal amide on biological potency of the new avp analogues. the analogues were synthesized by fmoc/bu t solid phase methodology and were tested for their rat uterotonic in vitro activity, rat pressor activity and antidiuretic activity using conscious rats. the modifi cations performed had a signifi cant impact on pharmacological activities of the analogues. acknowledgements: we thank the european social fund (esf), operational program for educational and vocational training ii (epeaek ii), and particularly the program pythagoras i, for funding the above work. it was also supported by the research project no. z40550506 of the academy of sciences of the czech republic. three-dimensional structure and mechanism of action of an antifungal peptide generated from hemocyanin cleavage in a penaeid shrimp an antifungal peptide, pvhct, which corresponds to the 23 amino acid c-terminal sequence of the shrimp respiratory protein hemocyanin, has been previously identifi ed in the plasma of the penaeid shrimp litopenaeus vannamei (1) . it is generated by proteolytic cleavage in response to a microbial challenge. similarly, a c-terminal fragment of hemocyanin displaying antimicrobial activity has been isolated from crayfi sh plasma (2) . these peptides are believed to contribute to the crustacean defence. the phenomenon of in vitro antimicrobial peptide generation from a respiratory pigment already observed with hemoglobin thus appears not to be restricted to mammals (3). pvhct displays a broad spectrum of antifungal activity with minimum inhibitory concentrations (mics) in the range 3-50 m (12.5 m against the shrimp pathogen fusarium oxysporum). its activity would be based on the inhibition of spore germination (1) . to contribute to the elucidation of the mechanism of pvhct antifungal activity, we determined its three-dimensional structure by circular dichroism (cd), nmr and molecular modelling and examined its effects on the f. oxysporum spore ultrastructure by transmission electron microscopy (tem). cd and nmr data indicate that pvhct is unfolded in an aqueous environment but adopts a similar helical structure in methanol solution and in dodecylphosphocholine (dpc) micelles used to mimic biological membranes. the structure consists of an amphipathic -helix spanning residues 8 to 18. tem shows that pvhct induces structural changes of the plasma membrane accompanied by a disorganization of the cytoplasm and a signifi cant decrease of lipid bodies. glucagon-like peptide-1 (glp-1), glucagon (gcg), and oxyntomodulin (oxm) are highly homologous peptide hormones derived from posttranslational processing of the preproglucagon gene. the sequence of oxm in particular, is identical to the sequence of gcg, with an 8amino acid extension at the c-terminus. pharmacological doses of oxm activate both the glp-1 receptor (glp1r) and the glucagon receptor (gcgr) albeit with lower affi nity compared to glp-1 and glucagon, respectively. in order to understand the origin of this dual specifi city, we synthesized a number of oxm analogs in which one or more of the native amino acids were replaced. first, a chimera was produced in which all the residues differing between glp-1 and gcg were grafted into the oxm native sequence, yielding an analog with the same pharmacologic profi le as glp-1. further studies showed that surprisingly, the switch from glp1r/gcgr co-agonism to glp1r-selective agonism could be obtained by a single amino acid substitutions at position 3 of oxm. in particular, the analog with gln3 substituted by glu (oxm-q3e) showed the same activity as native oxm on glp1r, but complete loss of activity on gcgr. oxm and oxm-q3e were compared in a hyperglycemic clamp study performed in diet-induced obese (dio) mice. due to the short half-life of the peptides, both were infused intravenously at ~16 g/kg/ min in chronically catheterized mice. the amount of exogenous glucose required to maintain the hyperglycemic level was 2-fold greater with the selective glp1r agonist oxm-q3e than with the glp1r/gcgr coagonist oxm, showing that abolishment of gcgr activity signifi cantly improves the glucose-lowering effect. we believe that these fi ndings could be useful for the development of a peptide therapeutic for the treatment of type 2 diabetes. hospitals across europe and north america have lately been plagued with infections caused by clostridium diffi cile, a diffi cult to treat bacterium due to its resistance to many commercially available antibiotics. recently, we isolated a two-component bacteriocin, thuricin, produced by bacillus thuringiensis that exhibits activity against c. diffi cile. we found that these peptides, called trn and trn , operate together in a synergistic fashion to inhibit bacterial growth. the peptide combination was found to be highly active against a wide range of c. diffi cile strains, including the virulent epidemic strain of the o27 ribotype, as well as most other clostridia and some bacilli and listeria species. maldi mass spectrometry revealed that both peptides have molecular weights that are lower than those predicted from their genetic sequences, indicating that they are post-translationally modifi ed. sequencing of the mature peptides through tandem mass spectrometry showed that each peptide has three modifi ed amino acid residues near its c-terminus. these residues were found to be two units lighter than their expected natural amino acid masses, suggesting a loss of two hydrogen atoms through dehydrogenation or oxidation. in order to confi rm these proposed modifi cations, we are investigating the production of 13 c-and 15 n-labeled trn and trn for structure elucidation by nmr. isolation of labeled peptides will be achieved by growing b. thuringiensis on defi ned media containing [u-13 c] glucose and ( 15 nh 4 ) 2 so 4 . subsequent nmr experiments will enable us to determine the three-dimensional solution structures of trn and trn , individually and bound together. by elucidating thuricin's structure, we aim to better understand its mechanism of action against c. diffi cile. the most potent peptidic human bradykinin (bk) b 2 receptor antagonist is hoe-140 (icatibant). in this study we present the synthesis of nine new analogues of hoe-140 substituted at position 7, 8 or 9 with chosen d-amino acid residues and/or nonproteinogenic amino acid residues, e.g. n-cyclohexylglycine (nchg), 1-aminocyclohexane-1-carboxylic acid (acc), octahydroindole-2-carboxylic acid (oic), piperidine-3-carboxylic acid (nip) and 4-phenylpiperidine-4-carboxylic acid (ppc). in the next nine peptides we combined the above mentioned modifi cations with the placement of 1-adamantane acetic acid (aaa) at position 0. all new analogues were tested on human umbilical vein for their antagonistic potency. only three compounds containing nchg 8 , nchg 9 or ppc 9 exhibited noticeable antagonistic activities on human bradykinin receptors thus still less potent then original hoe-140 sequence. each of them with aaa 0 showed lower activity then parent analogues. peptides: d-arg-arg-pro-hyp-gly-thi-ser-d-phe-nip-arg and aaa-d-arg-arg-pro-hyp-gly-thi-ser-d-phe-nip-arg, although strongly potent antagonists against bk-induced blood pressure lowering responses in rats, did not show noticeable antagonistic activity on bk-induced contraction of the isolated human umbilical vein, a well-established b 2 r bioassay system, suggesting a species dependent activity of the compounds. due to advances made in the peptide fi eld during the last few years, peptides as therapeutics have continued to gain more interest. the interest in peptide therapeutics generally comes from their high specifi city to targeted sites, their diversity and their usually low toxicity. pt-141, also known as bremelanotide, and mt-ii are potential future drugs and both are heptapeptides with a 23-membered cyclic monomeric lactam bridge. they are melanocortin receptor agonist and an analog of alpha-melanocyte stimulating hormone (a-msh). the pt-141 molecule has a c-terminal acid group while mt-ii has a c-terminal amide function. both neuropeptides have been tested in treating male sexual and erectile dysfunction as well as female sexual arousal disorder. pt-141, patented by palatin technologies, new jersey, usa, is the only known synthetic aphrodisiac and unlike other erectile enhancers like viagra, it does not act upon the vascular system. instead, it directly increases sexual desire and is used nasally as a spray. a scalable method for the synthesis of both peptides using fmoc-chemistry and the problems involved during the synthesis process will be discussed in details. tateaki, wakamiya; takahiro, nishimaru; kiyoe, mori; yoshihiro, yamaguchi kinki university, japan l-glutamate (glu) is known to be major excitatory neurotransmitter not only in the mammalian central nervous system but also in the ganglia of arthropods. binding of spider toxins to glutamate receptors (glurs) results in the inhibition of glu-mediated neurotransmission. in order to elucidate the mode of binding between glu and glurs, we focused on the visualization of glurs by complex formation with fl uorescentlabeled analogs of nptx-594 (1), a spider toxin with the structure of n 1 -(2,4-dihydroxyphenylacetyl-l-asparaginyl)-n 12 -l-lysyl-4,8-diaza-1,12-dodecanediamine [dhpa-asn-dada(12lys)]. in the present study, the modifi ed nptx-594, i.e., dhpa-asn-dada(12abg) (2) in which the lys residue of 1 was replaced with the n-(4-aminobutyl)glycine (abg) residue, was employed as a template compound to create the fl uorescentlabeled analogs of nptx-594, since the biological activity of 2 is three times higher than that of 1. we thus carried out the modifi cation of dhpa in the analog 2, i.e., 1) dhpa was replaced with the coumarintype acyl residues (type-1); 2) the phenylacetyl residues having alkyne side chains that can be converted into suitable fl uorophores based on the click chemistry (type-2); and 3) the coumarin-type acyl residues having the mercapto group to form disulfi de bond with the cys residue in glurs (type-3). this paper presents the synthesis and biological activity of various nptx-594 analogs to adopt as probes for visualization of glutamate receptors. an investigation of the functional requirements of apidaecin ib c-terminal fragment by means of peptoidpeptide hybrids by acting on one or more intracellular targets, without damaging the cytoplasmatic membrane. their particular killing mechanism and their low toxicity against mammalian cells make them attractive for the development of new antibiotics. structure-activity relationships studies have been carried out on short pro-arg rich antimicrobial peptides isolated from insects and the characterization of various natural isoforms of the 18-residues peptide apidaecin ib allowed to identify an evolutionary conserved region in the c-terminal part of the molecule. even a single point mutation in this region results in reduction or loss of antimicrobial activity. we recently described the synthesis of some apidaecin ib peptoid-peptide hybrids in which each arginine was replaced by the corresponding n-alkyl glycine residue (1). the afforded modifi cation made the resulting peptoid-peptide hybrids more resistant to proteolysis but moving the [narg]residue from the n-to the c-terminal end of the molecule progressively reduced the antibacterial activity. here we report the synthesis of a series of novel analogues containing a n-homoarginine or n-norarginine residue in position 4, 12 or 17. the effect of the size of the side-chain of the peptoid residue on the antimicrobial activity is also reported. in order to strengthen the peptide resistance to proteolysis and by considering that enzymic cleavage of the arg 17 -leu 18 peptide bond yields a fully inactive compound, we also prepared the [nleu 18 ]-apidaecin analogue. the conformational properties of the resulting peptoid-peptide hybrid, which is devoid of any antimicrobial activity, will be compared to those of apidaecin ib and the other [narg]peptoid-peptide hybrids. a wide variety of organisms produce antimicrobial peptides as part of their fi rst line of defense. however, antimicrobial activity is not the main function of some of these peptides. in the cuttlefi sh sepia offi cinalis we observed an antibacterial activity for the neuropeptide : h-alsgdaflrf-nh 2 (1). this decapeptide belonging to the fmrfamide family involved in regulation of reproduction and chromatophore function and is able to inhibit the growth of marine bacteria. circular dichroism studies have revealed for this amphiphilic peptide a helical structuration in sds micelles and in 50% tfe. to improve this antimicrobial activity, we fi rst introduce a lysine residue instead of aspartic residue, the antimicrobial activity of this new peptide has been improved by augmentation of the positive net charge (+1 to +3). moreover preliminary results have shown that the incorporation of aza-3 -amino acids analogues could create original hybrid pseudopeptide with superior activity . the natural peptide is not effi cient against staphylococcus aureus whereas the pseudopeptidic analogue revealed a minimum inhibiting concentration (mic) between 8-16 m. therefore some -amino acids were replaced by aza-3 -amino acids. we will show in this communication that depending on the substituted residue and on the structuration, these modifi cations could lead either to no activity or to a drastic enhancement of the antimicrobial activity, demonstrating that aza-3 -amino acids can facilitate the burying in lipidic bilayer of antimicrobial peptides that acts on bacterial membranes(2) references: isolation and structural characterization of capistruin, a lasso peptide predicted from the genome sequence of burkholderia thailandensis e264 the poor overall fragmentation behavior in ms n studies suggested a branched cyclic peptide with a rigid lasso structure. optimization of the fermentation conditions increased the production by 200-fold and subsequent nmr structural studies proved the lasso structure of the peptide that was named capistruin. heterologous production of the lasso peptide in e. coli showed that the identifi ed genes are suffi cient for the biosynthesis of capistruin, which exhibits antimicrobial activity against closely related burkholderia and pseudomonas strains. to our knowledge, this is the fi rst rational based identifi cation of a novel lasso peptide and the presented approach should be advantageous for the isolation of further lasso peptides in the future. endogenous antimicrobial peptides (amps) are the earliest molecular factors in the evolution of innate immunity. marine invertebrate animals have no acquired immunity with a system of antibodies diversifi cation. they are presumed to use an amps-based system as principal defense against potential pathogens. we have discovered a new family of small (21-residue) amps, termed arenicins, in coelomocytes of marine polychaeta lugworm arenicola marina. these amps exhibited activity against gram-positive, gram-negative bacteria and fungi. complete amino acid sequences were determined for each isoform. arenicins have one disulfi de bond (cys3-cys20). arenicins have no structure similarity to any previously identifi ed antimicrobial peptides. a novel 40-residue antimicrobial peptide, aurelin, exhibiting activity against gram-positive and gram-negative bacteria, was purifi ed from mesoglea of a scyphoid jellyfi sh aurelia aurita. complete amino acid sequence of aurelin was determined. aurelin has 6 cysteines forming three disulfi de bonds. the total rna was isolated from the lugworm coelomocytes and from the jellyfi sh mesoglea, rt-pcr and cloning were performed, and cdnas were sequenced. a 202-residue preproarenicin contains a putative signal peptide (25 amino acids) and a long prodomain. a 84residue preproaurelin contains a putative signal peptide (22 amino acids) and a propiece of the same size (22 amino acids). aurelin reveals partial similarity both with defensins and k+ channel blocking toxins of sea anemones and belongs to shkt domain family. overlapping of biological properties of marine animal amps and toxins along with their sequence homology might be a consequence of divergent evolution from a common ancestor. antimicrobial peptides from marine organisms could afford design of new antibiotics manifesting broad-spectrum antibacterial activity. cyclic enkephalin analogs containing two alkylurea units we shall present some structure-activity results for 8 enkephalin analogs, derived from the exhaustive combinations of d-lys and d-orn in position 2 with lys, orn, dab and dap in position 5, both positions coupled ¦ø-¦ø¡¯ by means of the urea bridge. accordingly, they all are restrained by 14-18-membered rings. in addition, we introduced the -nh-ethylurea unit instead of -nh2 of amide group in previously published analogs [1] [2] [3] . their in vitro activities were determined in the gpi and mvd assays. the peptides are more active than enkephalin in the gpi while have similar activities to the latter in the mvd assay. the effect of the introduction of ethylurea unit at the c-terminus on the activities is also discussed. chemical shifts of the peptides in water were fully assigned and their sequences confi rmed. several cross-peaks between the protons of amidoalkylurea unit and preceding residues have been observed in each case, suggesting that the unit may be involved in specifi c interactions between residues 4 and 5. understanding the structure/activity relationships of hepcidin iron is an essential element for nearly all living organisms and plays a key role in a range of processes including oxygen transport and storage, catalysis of redox reactions, production of metabolic intermediates and host-defence (1). until recently, little was known about the regulatory elements involved in the control of iron uptake and distribution within the body. the recently discovered peptide hepcidin has been shown to be a key regulator of iron metabolism within the body in response to a range of conditions, including infl ammation, hypoxia and anaemia (2) . this talk will focus on work towards elucidating structure/activity data for hepcidin with the aim of gaining a better undertstanding of the interaction between hepcidin and its receptor, ferroportin. we hope that this information will facilitate the design of synthetic agonists or antagonists of ferroportin to be used as potential drug leads. three-dimensional structures of investigated analogues were determined using two-dimensional nmr spectroscopy and molecular dynamics simulations with time-averaged restraints. the analysis of structural differences exhibited by different modifi cations provides the basis for understanding conformation -activity relationships and thereby the mechanism of interactions of the analogues with receptors. nmr structure of the micelle-bound 26rfa and 43rfa, two peptide ligands of the gpr103 receptor a novel rfamide peptide, named 26rfa and with no meaningful similarity with other members of this family, has been recently characterized. its precursor encompasses several potential cleavage sites and thus may generate various mature peptides including an nterminally extended form of 26rfa, termed 43rfa. both peptides act as endogenous ligands of the g-protein coupled receptor gpr103. this receptor has been recently implicated in the bone metabolism regulation the determination of the 3d structure of such peptides is essential for the elucidation of their structure/function relationships and for the design of potent agonists or antagonists. although structure elucidation of a ligand in the absence of the target receptor can deliver limited insight into the bioactive conformation, there is emerging evidence that interactions with the cell membrane is a key step required for receptor recognition. in this context, we have investigated the solution conformation of 26rfa and 43rfa by cd, nmr and molecular modelling in different media, in particular in one miming the "free" form of the molecule (methanol or mixture tfe/water) and in a cell membrane mimetic medium (dpc micelles). in an organic solvent, both peptides adopt the same conformation, i.e. an amphipathic alpha-helical structure, fl anked by two n-and c-terminal disordered regions. when bound to dpc micelles, the n-terminus remains fl exible and an helix is present at the same position as in the "free" form for both molecules. in contrast, the cterminal extremity becomes structured adopting an inverse gamma-turn conformation. these data represent the fi rst step for the rational design of new molecules that might be used in the treatment of osteoporosis. acknowledgements: supports were obtained from inserm and ifrmp 23. the nmr spectrometers are supported by grants of the conseil régional de haute-normandie. nmr and molecular modelling facilities are provided by the centre de ressources informatiques de haute-normandie. antimicrobial activity of analogues of a peptide isolated from venom glands of social wasps polistes major major inhabiting the dominican republic recently we have described isolation and biological activities of several new peptides from the venom glands of social wasps polistes major major found in dominican republic. we have also reported the synthesis of their analogues in order to investigate structure-activity relationship with respect to the antimicrobial and hemolytic activities (1). here we report the activities of a few further analogues of one of the peptides called pmm (h-ile-asn-trp-lys-lys-ile-ala-ser-ile-gly-lys-glu-val-leu-lys-ala-leu-nh2). the parent sequence or its truncated analogues were modifi ed on the n-terminus with 6-aminocaproic acid, glycolic acid, palmitoic acid, and 9-acridinyl and 9-(1,2,3,4-tetrahydro)acridinyl groups. the new analogues were tested for their antimicrobial activity (determination of minimal inhibitory concentration values -micusing broth dilution method) and hemolytic activity (determination of the ic50 value using suspension of rat erythrocytes). the palmitoylation unfortunately did not enhance antimicrobial activity against the tested microorganisms (bacillus subtilis, staphylococcus aureus, escherichia coli and pseudomonas aeruginosa). substitution of amino acids ser8 or glu12 subsequently for alanine, serine or lysine did not infl uence the activity of the peptides signifi cantly. gramicidin s, gs, c-(val-orn-leu-d-phe-pro)2, was isolated from bacillus brevis. it forms a two-stranded antiparallel -sheet fl anked by two ii' -turns. it was found that the distribution of hydrophobic and hydrophilic residues on the opposite sides of the sheet is a structural feature required for gs antimicrobial (am) activity. despite its wide gram+ and gram-antimicrobial activity gs is useless in therapy because of its high hemotoxicity in humans. it was found, however, that the analogues of gs-14 (gs with lys-leu inserted into each strand) got more am selective, when their amphipatic moments were perturbed by swapping adjacent lys leu/val or confi guration reversal at lys (1) . here, we report on effects of similar perturbations put on gs original c-decapeptide, using the following examples: c-(val-lys-leu-d-his-pro)2, 1, 1. as the mother compound, and its three analogs, viz. c-(val-d-lys-leu-d-his-pro-val-lys-leu-d-his-pro) -lys2 converted to d, c-(lys-val-leu-d-his-pro-val-lys-leu-d-his-pro) -val1-lys2 swapped, and c-(val-leu-lys-d-his-pro-val-lys-leu-d-his-pro) -lys2-leu3 swapped, 2-4; all having reduced ring-sequence symmetry. the peptides were synthesized by solid-phase methods using 9-fl uorenylmetoxycarbonyl (fmoc) methodology. having solved their structures by 2d-nmr and having tested their activities/selectivities, we confi rmed that only 1 had relatively favorable bio-profi le, as already published (1) cereulide, a foodborne peptide highly toxic towards the insulin producing beta-cells of the pancreas cereulide is a heat stable cyclic, lipophilic (log kow 5.96) peptide (1152 g mol/1) produced by certain strains of bacillus cereus, a bacterium connected to emetic food poisonings. it is insoluble in water, soluble in ethanol, methanol, dmso, food oils. cereulide exposure caused collapse of mitochondrial membrane potential in all tested human cells at low (ng/ml) exposure concentration (nk cells, t lymphocytes, caco2, calu3, neural paju, hela). toxicity is caused by its action as ion carrier with a selectivity of k + : na + = >1000 : 1. in various foods connected to human illness, concentrations of 0.01 to 3 g/g of cereulide were measured. in a case where the remains of a meal that had caused acute serious illness of two adult persons, were obtained for analysis, 1.3 g of cereulide was found /g of food. we undertook to explore the effects of purifi ed cereulide, and cereulide containing bacterial extracts, on porcine pancreatic islet cells in culture. foetal porcine islet cells were exposed to heat killed extracts from food-borne b. cereus strains producing or not producing cereulide and to purifi ed cereulide. effects were assayed using viability staining with fl uorochromes and cellular contents of dna and insulin. exposure to 1 ng/ml of purifi ed cereulide caused necrotic cell death of the islet cells impairing their insulin content within 2 days. cell extracts of cereulide positive b. cereus strains connected to food poisoning or isolated from food items were toxic, corresponding to their measured cereulide content. extracts of b. cereus strains producing or not producing the b. cereus diarrhoeal toxin, but not cereulide, were tolerated by the porcine islet cultures up to concentrations 1000 fold higher compared to extracts from strains containing cereulide, produced substance toxic towards porcine fetal langerhans islets and beta cells. application of non-sequential pharmacophore concept for design of antimicrobial peptide dendrimers unique structure of dendrimeric compounds consisting of a central core and several generations of branches provides opportunity of multiple practical solutions in the area of medicine. location of a high number of functional groups at the surface, allows to present multiple pharmacophoric units to the receptors, with immediate application in the design of a new generation drugs or vaccines, tools for studying autoimmune diseases or understanding gene delivery mechanism. here we present another possible application of dendrimeric compounds -preparation of drug molecules, which mimic active conformations of various macromolecular ligands. we focused on low molecular weight basic dendrimeric peptides, which mimic active conformations of recently discovered natural antimicrobial peptides. structurally, natural compounds are linear cationic peptides consisting of 10-50 amino acids that kill a broad spectrum of microbes destabilizing ordered structure of their cell membrane. it is generally accepted that positive charge and an induced amphipathic conformation are necessary for their antimicrobial activity. the project is related to multi-drug resistance of numerous bacteria against conventional antibiotics and involves de novo design of 1-2 generation dendrimeric peptides, which mimic sequencerelated active conformations of natural compounds (non-sequential pharmacophore concept). apparently, several groups of small peptide dendrimers were synthesized and structurally characterized (nmr, cd). they are potent antimicrobials, active against broad spectrum of species including mrsa and esbl strains. interactions between model membranes and peptide dendrimers of various structure will be discussed. seawater desalination is most commonly done today by reverse osmosis (ro) using thin-fi lm composite membranes. a major problem in ro desalination is biofouling, caused by adhesion and growth of bacteria to form biofi lm on the membrane surface. recently we proposed a new approach to reduce biofi lm formation on ro membranes that is based on immobilization of antimicrobial peptides (amps) onto the membrane surface. in this study we screen amps capable of reducing biofi lm growth under conditions simulating seawater desalination, and search for mode of binding to the membrane without affecting the peptides bioactivity. a specifi c bioassay was developed for screening peptides activity in high salinity conditions in order to evaluate the inhibition of biofi lm growth, based on growing biofi lmforming bacteria in a 96-wells microtiter plate. we prepared various amps known from the literature by solid phase peptide synthesis (spps) using fmoc-chemistry. the bactericidal activity of amps was examined in fresh water and compared to high salinity water. most amps lost their activity in high salinity conditions; yet, few peptides possessed their bactericidal activity and were used in subsequent experiments. searching for mode of linkage was performed by evaluating the bactericide activity of amps modifi ed with numerous types of linker molecules. based on literature studies that showed no decrease in bactericidal activity of amps upon n-terminal modifi cation, we prepared the corresponding peptides with modifi ed spacers on their amino-terminal and evaluated their antimicrobial activity in solution. indeed, we obtained gly 3 -spacers that retained activity, which were used subsequently as linkers to ro membranes. the mode of binding, as well as bactericide activity of the peptides and of the membranes will be presented and discussed. this study will lay the bases for a novel approach to decrease biofi lm formation on the surface of ro membranes during ro desalination. bioactive tripeptides ile-pro-pro and val-pro-pro protect endothelial function in vitro in normotensive and hypertensive rats milk drink containing casein-derived bioactive tripeptides isoleucylprolyl-proline (ipp) and valyl-prolyl-proline (vpp) has been shown to decrease blood pressure both in animal models and clinical studies. this effect can be attributed to the tripeptides. it has been suggested that one possible blood pressure lowering mechanism of the tripeptides could be angiotensin-converting enzyme (ace) inhibition. however, not all studies support this fi nding. the effect of tripeptides ipp and vpp on vascular function was investigated in vitro using rat mesenteric arteries. superior mesenteric arteries isolated from male wistar-kyoto and spontaneously hypertensive (sh) rats were incubated in krebs solution containing 1 mm of the peptide (either ipp or vpp) in +4 ºc for 48, 24, 12 or 1 h. after incubation mesenteric artery rings were mounted in an organ bath chamber and extensive vascular reactivity measurements were performed. acetylcholine-induced endothelium-dependent relaxation was better preserved (p < 0.05) in mesenteric arteries of both strains incubated with ipp or vpp compared to the control. clear differences were not observed in sodium nitroprusside-induced endotheliumindependent relaxation. the ace-inhibitory activity of ipp and vpp was studied by measuring the response to a single administration of angiotensin i and ii in organ chambers. proportioned to kcl-induced contraction, no clear reduction in angiotensin i -contraction was seen. thus, ace-inhibition may not be the main mechanism for the long-term effects of ipp and vpp in the protection of endothelial function. we suggest that the tripeptides do not affect smooth muscle but they protect endothelium during incubation indicated as preserved acetylcholine-induced endothelium-dependent relaxation. and directly and modulate other parts of host innate immunity. today, more than 800 cationic peptides have been identifi ed. they all have certain conserved physical features including a net positive charge, contain approximately 50% hydrophobic amino acids and have sizes ranging from 12 to 50 amino acids. however, virtually any -sheet, loop including -helix, type of secondary structure can arise including -turn and extended. the multitude of cationic peptide sources, structures and a spectra of activity is matched by a number of complex and controversial models attempting to describe and explain their modes of action. little is known about the sequence requirements of short host defense peptides like bactenecin (12mer). with help of our novel technique using a artifi cially created luminescence producing gram negative bacteria and peptide synthesis on cellulose we can investigate the sequence requirements of such peptides. hundreds of peptides are tested for their ability to kill pseudomonas aeruginosa. complete substitutional analyses of different bactenecin variants as well as a semirandom peptide library with about 2000 members were measured. the complete substitutional analysis will give us information about the importance of each single position whereas the peptide library will give us broader information which composition of amino acids results in an active antimicrobial peptide. the data will be analyzed using the quantitative structureactivity relationship approach (qsar) to identify sequence patterns that discriminate between superior activity cf. equivalently active and inactive. this will give us mechanistic cues for a better understanding of the mode of action of the short antimicrobial peptides. the results of these measurements and analyses will be discussed in detail. here we propose a simple and rapid fl ow cytometric method to assess internalization of the peptides in bacteria. the method is based on the use of fl uorescently-labeled peptides and of the extracellular quencher trypan blue to discriminate between a cell surface and cytoplasmic localization of the tested molecules. to his aim, we used bodipylabeled peptides showing different modes of action. these included some fragments of bac7, a proline-rich peptide known to penetrate bacterial and eukaryotic cells without membrane damage [1, 2] , and polymyxin b, a peptide antibiotic that binds to lps and to the cell membranes. by using this approach coupled to fl ow cytometric analysis, we showed that the fl uorescence intensity of e. coli and s. typhimurium cells treated with sub-inhibitory bodipy-bac7 concentrations did not decrease despite extensive washing and addition of the quencher trypan blue. in contrast, the fl uorescence of cells treated with bodipy-polymyxin b, as well as that of bacteria treated with a fl uorescein-labeled anti lps antibody, were promptly and almost totally quenched by addition of trypan blue, indicating their accessibility on the bacterial surface. these results confi rm the suitability of this method to rapidly infer the localization of labelled molecules in an accessible or inaccessible compartment of the treated bacterial cells. hp (2-20) is an antimicrobial peptide derived from the n-terminus of helicobacter pylori ribosomal protein l1 (rpl1). in our previous study, several analogues of hp (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) , with amino acid substitutions that increased or decreased net hydrophobicity, were designed and showed that an analogue, a3 designed by substituting gln and asp with trp at positions 17 and 19, respectively, caused increased antibacterial activity in minimal inhibition concentration (mic) and minimal bactericidal concentration (mbc) without having hemolytic activity. the peptide a3 acted also synergistically with known antibiotics including chloramphenicol against bacterial cells. fluorescence activated fl ow cytometry showed that a3-treated cells had higher fl uorescence intensity than untreated cells, similar to that of melittin-treated cells. the peptide a3 showed a strong antimicrobial activity against antibiotic-resistant pseudomonas aeruginosa from otitis media, clinically isolated mrsa and vrsa including biofi lm-forming bacteria in vitro and in vivo. the ototoxicity of a3 was studied in vivo by topical application to the middle ear in guinea pig model. twenty guinea pigs (5 groups, each group n=4) were each injected by transtympanic approach with 20ul of 8ug/ml,16ug/ml,32ug/ml, 128ug/ml of a3, and 0.4% gentamicin sulfate was instillated as a control. auditory brainstem responses (abr) to click were measured between 1st and 7th days after injection. histologic investigation of cochlea was performed by scanning electron microscope and light microscope. the results showed that topical application of a3 to the middle ear is well tolerated without cochlear damage. the present study, therefore, demonstrates that the usefulness of antimicrobial peptides for multi-drug resistant bacteria including as a new ototopical agent for crpa otitis media. structure-activity relationship study of kiss-10: identifi cation of an antagonist of gpr54 in the absence of ccda, ccdb inhibit the cell division and can kill bacteria by a mechanism that involves the dna gyrase. bacterial dna gyrase is unique among the type ii topoisomerase with ability to negatively supercoil dna. the enzyme consists of two subunits, a (gyra) and b (gyrb) and operates as an a 2 b 2 heterotetramer. the mechanism of the inhibition of the gyrase activity by ccdb is still an object of many debates, but is clear that r462 residue of gyra and the c-terminus of ccdb (w99-i101) play a crucial role in the gyrase-ccdb interactions. as an approach for a better understanding of this mechanism as well as for development of new gyrase inhibitors, we have synthesized peptide analogues of the ccdb protein and studied its activity by supercoiling assays and bacterial growth. five fragments (ccdb et1 , ccdb et2 , ccdb et3 , ccdb et4 and ccdb ss1 ) of the natural ccdb were designed and synthesized by spps. for the design, we considered the 13 residue c-terminal -helix (residues e87 to w99), the loop that connects two strands of the wing sheet (residues r40 to l50) and an n-terminal region that includes the fi rst of the fi vestranded antiparallel -sheet. all peptides, except ccdb et4 , showed inhibition of the supercoiling activity of the dna gyrase, especially ccdb et2 with a mic = 15 m. free peptides not showed antimicrobial activity, but when encapsulated in liposome (suv) were able to inhibit the bacterial growth in liquid culture medium. the growth inhibition in vitro for ccdb et2 was about 70% for gram negative bacteria. our fi ndings revealed a novel synthetic inhibitor of dna gyrase and ccdb et2 analogue is a good starting point for the development of a new and specifi c class of antibacterial agents based in the dna gyrase inhibition. acknowledgements: support: fapesp and cnpq synthesis and neuroprotective properties of short peptides consisting solely from glycine and proline residues now appears more and more data on biological activity of short peptides consisting solely from glycine and proline residues. it is suppose, that these peptides, named as "glyprolines" (gps), can be formed from collagen, åñì and related proteins. however an effect of these peptides on cns is not yet understood. the aims of this work were to improve a methodology of gps synthesis and to study cytoprotective properties of some new gps in culture of neuronal cells. gps with a common structure (gp)n, (pg)n, and (pgp)n (n=1-3) were synthesised by consecutive growing of peptide chain and fragment's condensation. synthesised peptides were characterised by hplc, mass-spectrometry, element analysis etc. cytoprotective activity of gps was assessed on an increase of survival of cultivated ðñ12 cells after í 2 î 2 -induced oxidative stress. it was shown, that from all tested peptides only (gp)n and pgp reveal cytoprotective activity. at the concentration 100 m these peptides reduced an amount of damaged cells 1.7-2.0 times in comparison to the control. thus preliminary data received show that some gps demonstrate a strong cytoprotective activity and therefore it is advisably to put them on further study on in vivo models. in preliminary investigations we found that all the peptides inhibited to a high degree the replication of hsv-1 in vero cells. moreover, these compounds did not show any cytotoxic activity against the vero cells. sexual dimorphism of hldf-6 peptide neuroprotective action in alzheimer's models in vivo and vitro we established that hldf-6 peptide, biologically active fragment of human leukemia differentiation factor (hldf), restores ability for learning, and also prevents loss of long-term memory and decrease in the exploratory behavior of wistar rat-males with experimental alzheimer's disease induced by intrahippocampal injection of betaamyloid peptide a (25-35) and ibotenic acid. the hldf-6 peptide was shown to render protective infl uence directly on primary culture of hippocampal and cerebellar neurones, isolated from newborn male brain, under conditions of the beta-amyloid toxicity. protective action of hldf-6 peptide on newborn rat-male neurons is connected with poster abstracts its ability to reduce 5-alpha reductase mrna expression in more than 30 times, blocking testosterone metabolism into dihydrotestosterone (dht) and thus interfering hyper activation of nmda receptors in ca1 areas of hippocampus. the protective action of hldf-6 peptide was investigated also and on female-rats with experimental alzheimer's disease. it was shown, that in contrast to males at which both forms of memory are broken: and long-term and working ones, in the case of females, injections of a (25-35) + ibotenic acid result in infringement of only working memory, at safety of long-term memory. introduction of hldf-6 peptide restored the broken working memory at females as effectively, as at males. however the mechanism of protective action of peptide on primary culture of hippocampal and cerebellar neurones, isolated from newborns female-rat brain, is connected not with the decrease in hyper activation of nmda receptors, but with abad (17 beta-hydroxysteroiddehydrohenase of the 10th type) mrna expression enhancement which is a target of beta-amyloid peptide action and blocking of progesterone conversion into 5 alpha-dihydroprogesteron due to 5-alpha-reductase mrna expression inhibition. in vitro and in vivo studies of p-19, an antimicrobial peptide active against multidrug resistant gram positive cocci we have designed octapeptide based on the sequence of sepecin b, an antibacterial protein of sacrophaga peregrina, effective against gram + bacteria. we systematically incorporated internal hydrophobic residues for a cationic, helical amphipathic structure effective for its high antimicrobial activity. it is also modifi ed c-terminally by a dehydro leucine residue effective for its structural stability. the synthesis of dehydro amino acid was done by solution phase and other amino acids were coupled by solid phase peptide synthesis method. anti-bacterial activity of the peptide was done by standard micro broth dilution technique against clinical isolates of gpc including methicillin resistant s. aureus (mrsa), methicillin sensitive s. aureus (mssa), hlar, group a and group b streptococci cultured from pus, wound and throat swab. all these isolates were known to be responsible for nosocomially acquired infections. s. aureus atcc 25023 was used as quality control strain. invivo effi cacy of this peptide was also tested in a mouse model with epidermal lesions caused by mrsa. the mic obtained for the peptide was 10 g/ml (average) for the tested strains. the peptide showed no hemolytic activity against human red blood cell. in the invivo studies the healing was induced early (after 72 hrs total healing was seen) in the experimental animals. this novel peptide has a potential to evolve as a therapeutic option in infections caused by resistant gpc. , an adhesive protein, is recognized by the activated platelet integrin á ééb 3 through the arg-gly-asp (rgd) sequence resulting to platelet aggregation and thrombus formation. inhibition of this process can be achieved by rgd peptide analogues that bind to á ééb 3 receptor. however, this class of inhibitors upon binding to receptor cause an outside-in signaling which induces a further activation of the platelet. in previous studies we presented cyclic (s,s)-cdc-containing compounds (ic 50~2 ìm) and á ééb derived sequences (y 313 mesradr 320 , á ééb 313-320 ) (ic 50~2 50ìm ) that exhibit a non-rgd-like inhibitory activity [1, 2] . this interesting aspect could be the basis for the design and development of a new class of anti-platelet agents that could overcame the drawback of platelet activation through the outside-in signaling. to this aim we designed, synthesized and tested for their inhibitory potency various á ééb 313-320 hybrid analogues incorporating the (s,s)-cdc-motif. cyclization reduces the allowed conformations, of both the backbone and the side chains, and possibly induces a favourable for the biological activity orientation of the charged side chains. the inhibition assays on the adp induced platelet aggregation revealed that incorporation of the (s,s)-cdc-motif considerably increases the inhibitory activity of the á ééb 313-320 analogue. the unique toxic effect of acrebol, a novel peptide from acremonium exuviarum a novel peptaibol, named acrebol, was isolated and purifi ed from the fungal strain bmb4 found in water-damaged wood-based indoor building material. the strain bmb4 was identifi ed as acremonium exuviarum based on morphology, its sequence, and cycloheximide resistance. ms/ms analysis showed that acrebol is a mixture of two almost identical peptaibols composed of 16-17 amino acid residues with masses 1726 and 1740 da, acephe-iva/val-gln-aib-ile-thr-leu-aib-pro-aib-gln-pro-aib and acephe-iva/val-gln-aib-ile-thr-leu-val-pro-aib-gln-pro-aib, respectively. the c-termini of the peptaibols was seroh however the sequence (mass of 216 da) between b13 and seroh could not be interpreted. both isoforms of acrebol had a strong toxic effect on boar spermatozoa, feline fetus lung cells, murine neuroblastoma, and mouse insulinoma min 62 cells. we found that, unlike other peptaibols, acrebol in toxic concentrations did not increase the ionic and solute permeability of membranes of isolated rat liver mitochondria. acrebol did not disturb the ionic homeostasis and osmotic balance of mitochondria and induced no release of apoptogenic proteins (cytochrome c) from the intermembrane space of mitochondria. acrebol strongly inhibited complex iii of the respiratory chain (ic 50 ~ 110 ng/ml), presumably, the outer quinone-binding center and, similarly to myxothiazol but in contrast to antimycin a, decreased the production of superoxide anion in the outer compartments of mitochondria. in the boar spermatozoa, acrebol, blocking the respiratory chain, caused the atp depletion due to the oligomycin-sensitive reversion of the reaction of atp synthesis, which resulted in the inhibition of progressive movement. acrebol induced necrosis-like death of mouse insulinoma min 62 cells whose energetic metabolism is strongly dependent on oxidative phosphorylation in mitochondria. thus, acrebol is a unique peptaibol with a specifi c pattern of the toxic effect. delta sleep inducing peptide (dsip), its analogues and deltaran®: biological activity and mode of action for the last decade we have been engaged in studies on the endogenous neuromodulator dsip (waggdasge) and a large group of its derivatives in respect of both their physiological activity and mechanism of action. a wide range of evidences confi rmed benefi cial effects of dsip and some active analogues under experimental stress models. dsip has emerged as promising and potentially effective therapeutic agent due to strong and unique adaptive and stress protective activity revealed during the study. dsip related drug deltaran® registered in russia has also showed the signifi cant effi ciency in animal test models and clinic. the peptides of dsip family often do not demonstrate any effects under normal and comfortable conditions or even cause slight prooxidative and stress promoting effects. these properties and established wide profi le of biological activities of dsip complicate the work on dsip mode of action. cellular and subcellular effects of this peptide still remain poorly studied. previously we investigated some biochemical events underlying the stress protective effi ciency of dsip and its derivatives. in continuation of these studies we have attempted to evaluate the putative dsip infl uence on classical cellular processes utilizing stressprotective heat shock proteins (hsps) and apoptosis. effect of dsip on the level of hsp70 expression in human erythroleukemia cell line k562 was detected.. we have found that dsip down regulates the increase of intracellular hsp70 level during incubation of cells in high density cell culture. according to our preliminary data dsip increased hsp70 level in murine t-cell line ctll-2 similar to -and ß-adrenergic receptor agonists. in murine thymocytes dsip increased both apoptosis and hsp70. we propose that registered effects of dsip are mediated through adrenergic receptors. cellular mechanisms of dsip and related peptides action are under way. identifi cation, chemical synthesis, and antimicrobial activity of tbd-1 -the fi rst -defensin isolated from reptiles antimicrobial peptides (amps) and proteins have been discovered in single and multicellular organisms indicating the importance of this peptide class for the innate immune system. amps are active against bacteria, fungi and viruses by affecting either the microbial cytoplasmic membrane, thus increasing its permeability or interacting with specifi c targets. defensins form one of the major subfamilies of amps, among which -and -defensins play a signifi cant role in bridging innate and adaptive immunity in mammals. the cationic -defensins are cysteine-rich and vary in length from 36 to 44 residues. the -stranded structure of -defensins is stabilized by a characteristic arrangement of three conserved disulfi de bonds between cysteines 1-5, 2-4, and 3-6. although -defensins lyse the bacterial membrane at higher concentrations it has been shown that they modulate also the immune system, e.g. being chemotactic for t-cells. we have isolated a novel 40mer -defensin called tbd-1 from leukocytes of the european pond turtle and deduced its complete sequence de-novo by combining different tandem mass spectrometry techniques (maldi, esi; cid and etd) and edman degradation. it was also possible to identify the disulfi de pattern in a tryptic digest by maldi-mass spectrometry. the deduced peptide sequence was afterwards confi rmed by solid phase peptide synthesis. thus two fragments were synthesized by standard fmoc/ t bu chemistry using three orthogonal cysteine protecting groups (trt, acm, tbu) to selectively form the right disulfi de bridges. after native chemical ligation of the two peptide fragments the fi rst two cysteines were oxidized on air followed by iodide oxidation for the second and dmso oxidation for the third disulfi de bridge. in antimicrobial activity assays, tbd-1 synthesized in -conformation was active against gram-positve, gram-negative bacteria, and fungi similar to the native peptide. trichogin ga iv (trga), an antimicrobial peptide of the lipopeptaibol family, continues to reveal peculiar properties since the time of its former identifi cation by rebuffat and coworkers. x-ray diffraction studies showed that trga is folded in a mixed 310/a-helix conformation, while nmr, cd and ir absorption experiments proved that this structure is predominantly populated in solution, although more disordered structures contribute to the conformational landscape of trga. we have recently shown by time-resolved experiments, that an equilibrium between helical conformers and more compact, folded conformers takes place in solution, with interesting transition dynamics in the microsecond time scale. in our conformational studies on trga we realized that the 3d geometry of the peptide chain reproduces the structural environment of the ion coordination site of calcium-binding proteins. this fi nding urged us to investigate the binding properties of trga with respect to ca(ii), gd(iii) and tb(iii), because lanthanide ions have been widely employed in biochemical studies as best substitutes of ca(ii). the binding of ca(ii), gd(iii) and tb(iii) to trga gives rise to a conformational transition, monitored by cd spectra at different ionpeptide molar concentration ratios. at high r values, the cd curves of all the ion-peptide complexes show a positive maximum at ~214nm, typical of type ii turns, suggesting the population of bent structures. the quasi-isodichroic point found for all systems between 198 and 202 nm, indicates that an equilibrium between extended helical and bent conformations actually takes place. fluorescence experiments on the tb(iii)/trga adduct have shown that, upon ion binding, the fl uorescence quantum yield of tb(iii) markedly increases, due to the release of water molecules from the ion coordination inner shell. molecular dynamics calculations on the peptide/ca(ii) adduct were also performed to obtain structural and dynamical information. antibiotic resistant bacterial strains represent a global health problem with a strong social and economic impact. thus, there is an urgent need for the development of antibiotics with novel mechanisms of action. castros group isolated and determined the sequence of the peptide hy-a1 (ifgailplalgalknlik) of skin secretion from the frog hypsiboas albopunctatus which showed antimicrobial activity. the aim of the present work was evaluated 4 analogues to supply information poster abstracts about the relationship structure-biological activity. the peptides were synthesized by spps using the fmoc chemical approach. the biological activities were assayed by measuring growth inhibition of two types of gram-positive bacteria and others two types of gram-negative. the synthesis and purifi cation of peptides by hplc was effi cient and a high purity level (96%) was obtained. the peptide containing trp in position 6 (for fl uorescent studies) replacing leu presented mic values comparable to wild type sequence: 32 um, 32 um, 8 um and 2 um for e. coli, p. aeruginosa, s. aureus and b. subtilis, respectively. two peptides with this modifi cation but containing at the n-terminal region one group acetyl or a residue of asp showed mic values of 128 um for e. coli and p. aeruginosa, although 4 um for gram-positive bacteria. different results were observed when the residue added was lys. in this case, the activity against whole bacteria was sustained or increased. conformational properties were investigated by cd techniques in water, tfe and in zwitterionic micelles (lpc). the cd experiments demonstrated that in water, the peptides have a random structure, but in tfe and lpc solutions they acquired an ordered structure, composed mainly by -helix. however, these data there is no relationship between the structure and activity against bacteria gram-positive. these results showed that the n-terminal region of the peptide hy-a1 develop key roles in its antibacterial action different types of bacteria. 4 pexiganan is an antimicrobial peptide remarkably effective against bacteria causing skin infections, and has been commercially developed as a topical cream to treat infected diabetic foot ulcers. [1] [2] [3] as peptide drug-based therapy often suffers with low drug stability in vivo, suitable delivery systems must be developed. with this purpose in mind, we have designed a pexiganan-chitosan conjugate to combine the exceptional bioadhesion and tissue regenerating abilities of chitosan [4] [5] [6] with the excellent antibiotic properties of pexiganan. we herein wish to report our fi rst results on the successful synthesis, ft-ir and amino acid analysis of a pexiganan-chitosan conjugate prepared by covalent attachment of a cys-containing pexiganan analogue to the chitosan's amino groups, by means of the heterobifunctional cross-linker sulfo-emcs.7. overexpression of e. coli oligopeptidase b confers resistance to the proline-rich antibacterial peptides scocchi, marco; de gobba, cristian; mattiuzzo, maura; gennaro, renato university of trieste, italy the proline-rich antimicrobial peptides (pramps) are a large group of cationic peptides isolated from mammals and insects, which show a spectrum of antibacterial activity limited to gram-negative bacteria and a remarkably low cytotoxicity towards eukaryotic cells. pramps are thought to act in a permeabilization-independent manner, via energydependent internalization into bacteria followed by recognition and inactivation of internal molecular targets. with the aim of investigating their mode of action, we have isolated a number of e. coli clones showing increased resistance to the bovine proline-rich peptide bac7 after transformation with a dna library from bac7-resistant mutants. among the recombinant plasmids responsible for resistance, some of them harboured the gene coding for the oligopeptidase b (opdb), a serine peptidase belonging to the prolyl oligopeptidase family (pop) broadly distributed among unicellular eukaryotes and gram-negative bacteria, which has emerged as an important virulence factor. opdb was cloned and expressed under the control of an inducible promoter and the transformants tested for susceptibility to a panel of antimicrobial peptides, including pramps. olipopeptidase activity of purifi ed opdb was then tested in vitro against the same peptides. the results indicate that the clones overexpressing opdb are more resistant to the pramps and that the degree of resistance correlates with the expression level of opdb. in addition, in vitro incubation of pramps with the purifi ed peptidase, followed by mass spectrometry analysis, showed that it promptly hydrolyzes all the peptides assayed to short, inactive fragments. these results suggest that opdb may contribute to cleavage and inactivation of antimicrobial peptides that are internalized into the target cells and support the notion that opdb is a novel virulence factor. guan, shuwen; huang, lei; li, pengfei; wang, liping; li, wei college of life science, jinlin university, china great progresses have been made in the research on identifying molecules of therapeutical potential for delaying aging. using caenorhabditis elegans, we had tested the effects on stress resistance and life span of treatment with dhhp-6 (deuterohaemin-alahisthrvalglulys), synthetic mimetics of the antioxidant peroxidases, which neutralizes peroxide. to further test the mechanisms of dhhp-6 on life span extension, exogenous protein sod and catalase levels were measured. we show that dhhp-6 is able to elevate in vivo sod and catalase activity levels after two days administration. treatment with exogenous dhhp-6 affected endogenous protein sod and catalase levels and elevated the expression of sod-3::gfp in the head and vulva. on the other hand, dhhp-6 can extend life span of c. elegans lacking sod-3 or ctl-2 gene expression by rnai. this suggested that the antioxidant enzyme in c. elegans may not the only target affected by dhhp-6. du, haidong; yan, yumei; wang, liping; li, wei college of life science, jinlin university, china high concentration of hydrogen peroxide (h2o2) induces nuclear dna fragmentation, lipid peroxidation and has been implicated in many diseases including heart failure, parkinson's disease, and cancer. in a systematic attempt to develop an effective scavenger of h2o2, we have successfully synthesized an artifi cial microperoxidase, deuterohaemin -alahisthrvalglulys (dhhp-6) as core catalytic center to which 6 amino acid peptide was covalently attached. dhhp-6 exhibited potent peroxidase activity, favorable membrane permeability and thermal stability.two experimental models of oxidative injury were established in order to investigate the anti-oxidative effect of dhhp-6: one was induced by hypoxia-reoxygenation in cultured heart-derived h9c2 cells bioactive peptides and another was caused by h2o2 in cultured neonatal rat ventricular myocytes. dhhp-6 could protect cells against oxidative injury by determining mtt cell proliferation assay, ldh leakage and ca2+-atpase activity. furthermore, dhhp-6 repressed the apoptosis gene expression, such as p21, hsp70 and heme oxygenase¢ñ, that proved dhhp-6 had protective effect in cultured neonatal rat ventricular myocytes . taken together, this small microperoxidase exhibited excellent ¡°druggable¡± properties, and could be a promising agent for ros-associated diseases with unmet needs. trichogin ga iv is the most extensively investigated member of the class of lipopeptaibols that are linear peptide antibiotics of fungal origin, characterized by the presence of a variable, but remarkable, number of aib residues, a fatty acyl group at the n-terminus, and a 1,2-amino alcohol at the c-terminus. several analogues of trichogin ga iv with amino acid substitutions or deletions were designed which allowed determination of the minimal inhibition concentration against gram-positive and gram-negative bacteria and various pathogenic fungal cells. the natural peptide exhibits a specifi c activity against s. aureus and only a marginal hemolytic effect. interestingly, trichogin ga iv is active also against several methicillinresistant s. aureus strains. studies on synthetic analogues demonstrated that substitution of the c-terminal leucinol by leu-ome, or substitution of one aib residues by the epr label toac do not perturb signifi cantly the biological activity of the peptide. on the other hand, removal of 3 or 7 n-terminal residues eliminated any antibacterial activity. finally, studies of proteolytic degradation on trichogin ga iv and analogues where the 3 aib residues are replaced by leu demonstrated that the presence of several non-coded aib residues endows the natural peptaibol with remarkable resistance to proteolysis. the present results indicate that trichogin ga iv is a promising lead compound for the development of new, selective and protease-resistant, antibacterial drugs. the siderophore microcin family: from the genetic systems to the antimicrobial peptides vassiliadis, gaëlle; peduzzi, jean; rebuffat, sylvie muséum national d'histoire naturelle-cnrs, france microcins are low molecular weight antimicrobial peptides secreted by enterobacteria and involved in microbial competitions within the intestinal tract. they are synthesized by the ribosomal pathway. we have isolated the fi rst siderophore peptide (1), a post-translationally modifi ed form of the chromosomally encoded microcin e492 (mcce492) from klebsiella pneumoniae, having a potent bactericidal activity mainly directed against escherichia coli. the post-translational modifi cation consists of a glycosylated catechol-type siderophore linked to the c-terminus. we have recently identifi ed the genes responsible for the acquisition of this modifi cation and proposed a model for its biosynthesis (2) . in order to identify novel siderophore peptides, we analyzed the genetic systems of several microcinogenic strains. based on the genetic organization of the microcin gene clusters, three microcins which had preparation of a close mimic of the n-terminal part of the c5a receptor (c5ar) by selective introduction of sulfated tyrosine residues (2) and chips in complex with our sulfated c5ar-model by multi-dimensional nmr techniques. based on these interaction data and the solution structure of the complex, chips-based c5ar inhibitors for use in anti-infl ammatory therapy might be designed. insulin-like peptide 5 (insl5) was fi rst identifi ed through a search of the expressed sequence tags (est) databases. primary sequence analysis showed it to be a prepropeptide that is predicted to be processed in vivo to yield a two-chain sequence (a and b) containing the insulin-like disulfi de crosslinks. the high affi nity interaction between insl5 and the receptor rxfp4 (gpcr142) coupled with their apparent co-evolution and partially overlapping tissue expression patterns strongly suggest that insl5 is an endogenous ligand for rxfp4. given that the primary function of insl5/rxfp4 pair remains unknown, an effective means of producing suffi cient quantities of this peptide and its analogues is needed in order to systematically investigate its structural and biological properties. a combination of solid phase peptide synthesis methods together with regioselective disulfi de bond formation were used to obtain insl5. both chains were identifi ed as being ¡ §diffi cult sequences¡¨ and were unusually resistant to standard synthesis protocols including those mediated by microwaves. the a-chain was also prone to signifi cant aspartimide formation. the b-chain, in particular, required the use of the strong tertiary amidine, dbu, for more effective nƒñ-deprotection during its assembly. following chain combination and sequential disulfi de bond formation, the resulting synthetic insl5 was obtained in good overall yield and shown to possess a similar secondary structure to human relaxin-3 (h3 relaxin). the peptide was able to inhibit camp activity in sk-n-mc cells expressing the human rxfp4 receptor with a similar activity to h3 relaxin. in contrast, it had no activity on the human rxfr3 receptor. novel cyclic bacteriocin-like peptides from strains of anabaena (cyanobacteria) leikoski (3) and microcystis aeruginosa (4) . the genome sequence of fi lamentous and diazotrophic cyanobacterium anabaena 90 revealed a putative gene cluster (acy) encoding a bacteriocin-like peptide. one of the acy genes encoded a prepeptide, which showed n-terminal homology to the known cyanobacterial prepeptides but the mature peptide product in anabaena 90 could not be predicted. we sequenced prepeptide genes from several anabaena strains to fi nd a prepeptide containing either cysteine or methionine, since sulphur containing amino acids enable the detection of the mature peptide product through 34s-labelling and lc-ms. the nucleotide sequence of the prepeptide genes revealed enormous variety in closely related anabaena strains. one strain, anabaena 844b contained a methionine in the prepeptide, and a cyclic heptapeptide product corresponding to the precursor was discovered in this strain. since the cleavage sites were found to be conserved in all anabaena strains the products could be predicted from the amino acid sequence and identifi ed by lc-ms. in addition, the biosynthesis of the decapeptide anacyclin in anabaena 90 was verifi ed by heterologous expression of the acy genes in e. coli and the structure of the peptide was confi rmed with a synthetic reference peptide. altogether, new cyclic peptides were found in 19 strains of anabaena with very little sequence conservation. cyanobacteria seem to be versatile producers of peptides using both ribosomal and non-ribosomal biosynthetic pathways. it is well established that n-terminal fl ank of corticotropin-releasing factor molecule is crucial for its biological activity. little, however, is known about in vivo effects of crf-derived peptides. this study was aimed to investigate possible actions of a tripeptide crf fragment 4-6 pro-pro-ile (ppi). tripeptide ppi was found to exert effects similar to fullsize crf molecule after central administration. the tripeptide induces behavioral activation in home cage, but inhibits behavioral activity under stressful conditions. ppi increases blood pressure and heart rate, blood glucose level and body temperature, decreases pain sensitivity, increases eeg amplitude and large doses of ppi induce seizures. ppi inhibits sexual motivation and performance in mating tests in males. crf antagonist -helical crf 9-41 abolishes ppi infl uence on circulatory system, glucose metabolism, thermoregulation and pain sensitivity. adrenalectomy does not interfere with hyperglycemic and hyperthermic actions of ppi, while pancreatectomy prevents hyperglycemic effect. nonselective beta-adrenergic blocker obsidan prevents hyperglycemic and decreases hyperthermic effects of the tripeptide and ganglionic blocker hexamethonium abolishes both effects. taken together effects of ppi correspond to stress-reactions, involving corticotropin-releasing factor and evidence that ppi is either 1) a part of a crf molecule active site, or 2) physiologically active crf derivative, realizing its effects through activation of crf receptors, or 3) an independent regulator, affecting crf-ergic neurons. acknowledgements: the work was supported by rfbr grant 06-04-49-620-à huggins, kelley; andersen, niels university, united states amyloid fi bril formation is associated with at least 17 human diseases; among these, the human-amylin(ham)-derived deposits in type ii diabetes were the discovery system and ham aggregation is one of the more thoroughly studied systems. to date, inhibitors of beta-aggregate and fi bril formation have been polyphenols, mutants of ham, or short peptide related to the ham(22-29) sequence, nfgailss. we now report that stable beta-hairpin scaffolds displaying trp and tyr residues are effective inhibitors, delaying the onset of both the cd changes associated with beta structure formation and the nucleation time and net enhancement of the fl uorescence observed with added thiofl avin-t (tht). under our test conditions (8 microm ham, 2% hfip in 5mm phosphate buffer, ph 7), ham begins to display an increase in beta structure by cd at 40 min with a constant maximal value from 85 -220 min. tht fl uorescence also indicates a circa 50 min onset time with a rapid (<20 min) rise to the full response. inhibition (delayed onset and reduction in the maximal fl uorescence enhancement) has been observed with a number of hairpins; those with two trp residues on a single face of the hairpin are more potent. of these, our best inhibitor to date, kkltvwipgkwitvsa (p = d-pro), increases the onset time more than 2-fold at equimolar concentrations. at 4 molar equiv., the onset time is greater than 320 min and tht fl uorescence levels out at < 40% of the control value. in analogy to the report by prof. ghosh (jacs 2006, p. 14456) that a beta-sheet protein with added tyr and trp residues inhibits fi bril formation by the alzheimer-related abeta peptide; we expect that designed beta-hairpin scaffolds will be more generally applicable and will afford new insights into the recognition phenomena of amyloidogenesis. baabur, hemda; ashkenasy, gonen ben gurion university, israel the main advantage in utilizing proteins for the development of sensors and receptors, over the more often used approach that exploits small molecules, is the ability to manipulate large recognition surfaces, which expose toward the bulk solution number of different functional groups. we search for stable artifi cial proteins that can be utilized as 'universal' recognition entities. to that end, modular chemical syntheses are exploited for the preparation of repeats-proteins. leucine rich repeat proteins (lrrs) are 20-29 residue sequence motifs present in several proteins with diverse functions. internalin b (inlb), a surface lrr protein of the human pathogen listeria monocytogenes, promotes invasion into various host cell types by inducing phagocytosis of the entire bacterium. consensus design uses statistical analysis of sequence alignments of families of homologous proteins for protein engineering. we show here that using consensus design, series of protein mutants that differ in the recognition surfaces are synthesized and will be probed for their ability to bind different natural and non-natural ligands. comparative structural studies of potent neuroprotective peptides of the humanin family although the rescue activity of hn peptides has been linked to a number of signaling pathways and receptors, their mechanism of action remains unknown. in this work cd and nmr data on the above peptides are presented and compared in an effort to defi ne structural characteristics related to their function. evaluation of our fi ndings in combination with existing structure-function relationship data for this class of peptides, brings forth fl exibility as an important structural feature that may facilitate interactions with functional counterparts of the neuroprotection pathway. the ability to adopt a partial helical conformation in the presence of low concentrations of tfe is another common structural feature that may be defi ning the interactions of these peptides in the environment of cell membranes. desleu-aga(c8r)hng17 and cl display a more complex behavior shifting from -helical to -sheet conformations depending on ph, peptide concentration, and % of tfe present in solution. this fact may be related to their high potency since in hn literature evidence for self-association ability has been linked with neuroprotection. our cd and nmr experimental data combined with theoretical modeling will hopefully provide important clues for the elucidation of the mechanism of action of the hn family of peptide the process of molecular recycling describes a dynamic mechanism by which individual amyloid fi brils are continuously dissolving and reforming (1) . the present work aims at the study of the dynamic properties of the -amyloid, a (1-42), amyloid fi brils. since the formation of a aggregates has been suggested as a key process in the pathology of alzheimer's disease, the study of the dynamic nature of a ( the coiled coil structure is widely distributed in natural proteins, such as transcription factors, receptor proteins and enzymes. this motif has been recently used by chemists for the design of functional synthetic proteins. specifi c coiled coil sequences can be utilized as templates for self replication processes (1, 2) . the stability and the activity of these replicators depend on the characteristics of the amino acid residues in the recognition interface. it has been suggested that it is possible to control protein structure and the replication process by external triggers such as light, and that the light can also be used to monitor the conformational changes and/or to follow the protein functionality(3). we show here our ability to control the folding stability of coiled coil peptides and their reversible or irreversible self replication effi ciency by light, and we demonstrate the possibility to exploit fret couples to facilitate in-situ monitoring of the folding and reactivity. we use 'caged' mutants with a photo-switchable molecule in the recognition interface of the peptidewhich disrupts its coiled coil structure -as inactive species. deprotection of the caged proteins is used as a mechanism to restore the self replication process. the ligation is followed by monitoring the changes in fl uorescence of either the donor or acceptor of the fret couple. we will describe synthesis and structural characterization of caged and cage-free peptides and measurements that show, as expected, that the cage free peptides are more stable as coiled coils and better catalyst for their own formation than the caged analogs. moreover, we discuss the reversible replication process and how we can shift and control its equilibrium staes. , a member of the rfamide peptide family, has been reported to have pronociceptive and analgesic activities as well as pro-and anti-opioid effects. these contradictory effects seem to result from npff capacity to bind two different gprotein coupled receptors (npff1 and npff2). although the exact role of npff1 and npff2 receptor is still unclear, this complex system appears to play an important role in pain regulation. structure-activity relationship (sar) studies using analogs of npff and derived peptides have shown that the c-terminal part of the molecule (i.e. pqrf-nh 2 ) is crucial for affi nity and activity. however, some of the essential features for ligand recognition by npff receptors are still missing to design highly selective molecules. in order to provide further insight into ligand recognition, we have investigated the solution conformation of npff in different media using circular dichroism and nmr spectroscopy. our results showed that (1) in the presence of methanol or trifl uoroethanol, turn-like elements are present in npff structure, and (2) are not yet established, although genetic and animal models have shown a causal role of amyloid -peptide (a ) in ad. however, recent debate has focused on whether amyloid fi brils or soluble oligomers of a are the main neurotoxic species contributing to neurodegeneration and dementia. one approach for preventing aggregation would be the conversion of the peptide conformation. prior investigations indicate that polymeric nanoparticles offer strong advantages in modulating the secondary structure of the peptide (1, 2) . these results have encouraged us to extend our work on polymeric nanostructures for conformational transformations contributing to the development of new therapies for these diseases. complexes of polyampholytes and dodecanoic or perfl uorododecanoic acid were prepared (2) resulting in nanoparticles with hydrodynamic diameters ranging from 3 to 5 nm. the fl uorinated nanoparticles induced -helix rich structures in a peptide, whereas their hydrogenated analogues were less effi cient leading in most cases to aggregation orsheet formation, as determined by circular dichroism spectroscopy. the degree of fl uorination, the hydrophilic balance and the charge density of the fl uoropolymers, as well as the size of the nanoparticles in aqueous solution, are decisive for the interactions. the impact of these structures on the a -induced toxicity in cultured neurons was studied. we report that the fl uorinated nanoparticles increased the a -mediated mtt (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazoliumbromide) reduction, which indicates a higher cell viability. the anti-apoptotic effect of the complexes was evaluated by determining the activation of caspase-3. this assay also confi rms the decrease of a -mediated cytotoxicity in the presence of fl uorinated biocompatible complexes. alternative strategy to investigate enzymatic activity using peptides containing toac spin probe (1), the present work extended the investigation of the specifi city of angiotensin i-converting enzyme (ace, ec 3.4.15.1), a dipeptidyl carboxypeptidase which cleaves the c-terminal dipeptide from angiotensin i (angi, drvyihpfhl) to produce the potent vasoconstrictor angiotensin ii peptide (angii). the use of paramagnetically labeled ai analogues attaching the toac (2,2,6,6tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) probe (2) advantageous allows the monitoring of their conformation and its enzymatic hydrolysis specifi city through the epr and fl uorescent methods, the latter due to the quenching effect induced by the stable free radical toac probe upon the tyr4 residue of angi. the study of toacattaching ai analogues at positions 0, 1, 3, 5, 8, 9 and 10 indicated that the fi rst four analogues are substrates for ace in the decreasing order 0 ~ 1 > 3 > 5, thus confi rming that greater the proximity of the unnatural probe to the cleavage site (8-9) of the sequence, the smaller are the substrate specifi city of analogues. otherwise the quenching effect of tyr4 fl uorescence by toac decreased with increasing distance between both residues, thus suggesting overall fl exible structures for most of analogues. these fi ndings were also corroborated in a combined cd and epr studies although some differences were detected among the derivatives either in the variation of ph or amount of the structuring tfe studies. finally, differences between epr spectral lineshapes of some labeled analogues and their corresponding cleavage products seems to allow a real time monitoring of the enzymatic reaction. the effect of the so called " -sheet breakers" (bsbs) on a 1-42 aggregates inhibition of aggregation of amyloid -peptide seems to be a critical step in the therapeutic approach to prevent amyloidosis in alzheimer's disease. the therm " -sheet breaker" (bsb) had been introduced by soto c. et. al. as a 5-residue peptide in 1998, that inhibits amyloidprotein fi brillogenesis. we synthetized several derivatives of the "soto peptide" as well as a big number of peptidomimetics. their mechanism of action has been studied by nmr spectroscopy, cd and transmission electron microscopy (tem). all of these methods showed that the soto peptide lpffd and the similar peptides can not prevent a aggregation. these compounds bind to the surface of a aggregates and decrease bioactive peptides the specifi c surface area of a which accessible for the cell-membranebound receptors, can lead to a decreased toxicity. the -sheet breaking effect does not work up to an a peptide-small peptide ratio of 1:10. as a consequence, these compounds are not -sheet breakers, only they modify the surface of a fi brils and rather speed up the formation of -sheet structure. designing trehalose-conjugated peptides for the inhibition of alzheimer's a oligomerization and neurotoxicity. interactions between cationic or anionic porphyrins and polypeptide templates with charges that are opposite to those of porphyrins have been extensively investigated for their possible applications in biomedicine and photodynamic therapy (pdt). the infl uence of porphyrin on the conformation of the peptide part of the complexes is studied in this work. non-covalent interactions of cationic tripeptide l-lysyl-l-alanyl-lalanine (kaa) with anionic meso-tetrakis(4-sulfonatophenyl)porph yrin (tpps) and its copper(ii) (without axial ligand), iron(iii) (one axial ligand), and manganese(iii) (two axial ligands) derivatives were investigated in aqueous solutions by vibrational (vcd) and electronic circular dichroism (ecd) spectroscopies. although both the cd spectroscopies are sensitive to conformation, particularly vcd is extremely sensitive to subtle conformational changes. the vcd spectra of pure kaa in the amide i' (c=o stretch vibration) region showed the spectral patterns typical for left-handed polyproline ii helical conformation (ppii). interaction of kaa with non-metallated tpps was accompanied by change of the amide i' vcd patterns -loss of vcd intensity and arising of a new negative band at ~1630 cm -1 -that was interpreted as a partial change of ppii into less compact conformation as extended helix or -sheet segment. in case of cu(ii)-and fe(iii)-tpps, the loss of the amide i' vcd intensity of kaa was observed only. for mn(iii)-tpps having two axial ligands, the vcd pattern was unchanged compared to the pure tripeptide indicating that this derivative is not able to change the conformation of kaa in peptide-porphyrin complex. suitable models of biologically important small-sized proteins -histons -located in the chromosomes of eucariotic cells. they are able to interact with negatively charged functional groups of many biologically important molecules as dna, polyuronic acids, and porphyrins and thus infl uence a broad range of biological functions. in this work, the solution conformation of synthetic oligotripeptides (llysyl-l-alanyl-l-alanine) n [n = 1, 2, 3] was investigated at the different temperatures and ph using combination of vibrational (vcd) and electronic circular dichroism (ecd) spectroscopies. vcd spectra of all the oligopeptides measured at room temperature show a negative couplet (positive to lower frequency) in amide i' region. this spectral pattern is indicative of left-handed polyproline ii helical conformation (ppii), which is stable at wide range of ph. the temperature dependence of the vcd spectra indicates that ppii conformation of all the oligopeptides remains stable even at 90°c, independently on length of oligopeptide chain. these results were confi rmed by temperature dependent ecd experiments, where the characteristic negative and positive bands at about 195 and 220 nm, respectively, were observed. taken together, vcd and ecd results suggest that ppii conformation of (lys-ala-ala) n sequence is the dominant conformation within the range of temperature from 20 to 90°c. all the results including spectral data analysis are discussed in detail. acknowledgment: the work was supported by the research grant 203/06/p371 from the grant agency of the czech republic. we thank miroslava žertová, msc (iocb prague) for the oligopeptide synthesis. -peptides are probably the most thoroughly investigated peptidomimetic oligomers. to extend the fi eld of -peptides towards the construction of possible new secondary structures, the replacement of the c and c atoms of the -amino acid with heteroatoms could be an attractive modifi cation, for example c -atom of -peptides by an nr moiety, leading to hydrazine peptides. in the literature, there are only a few studies about hydrazine peptides [1] [2] [3] , and hydrazine peptides with cyclic side-chain have not been studied yet. in order to determine the secondary structure preference of h-[(cis or trans)-acpc-2s-aza-acpc] 3 -nh 2 peptides (figure 1 ), their potential energy hypersurface were probed at the ab initio b3lyp/6-311g** level. the cis (1r,2s) formed 10/12-helices, while the opposite acpc enantiomer resulted 6 strand. the trans (1s,2s) formed 8-strand, while the opposite acpc enantiomer resulted 12-helix. the hybrid-peptides in question were synthetized on solid support, and their high-resolution 3d assignments were made by using nmr, which was supported by ecd, vcd, qls and tem methods. the hydrazino modifi cation resulted in better water solubility than that of the acpc homooligomers. this result is very important concerning the further biological applicability. mazur, adam; katarzy ska, joanna; zabrocki, janusz; jankowski, stefan technical university of lodz, poland cyclolinopeptide a, (cla, 1), a cyclic nonapeptide cyclo(-pro 1 -pro 2 -phe 3 -phe 4 -leu 5 -ile 6 -leu 8 -val 9 -), possesses strong immunossupresive and antimalarial activity as well as the ability to inhibit cholate uptake into hepatocytes [1, 2] . the mechanism of cyclolinopeptide a activity is similar to that of cyclosporine a. the object of our investigations are six cla analogues with pro 1 and pro 2 residues replaced by 2 -isoproline or 3 -homoproline. the immunosuppressive activity of these new cla analogues was evaluated on the basis of the mouse splenocyte proliferation assay 3.. nmr spectra analysis (chemical shifts assignment and structural constraints) was based on 1d and 2d nmr experiments at 700 mhz. long (300 ns) molecular dynamics calculations were carried out using gromacs program (gromos g53a6 force fi eld) for isomers of at least 30% content. in woolley, andrew university of toronto, canada thiol-reactive azobenzene-based cross-linkers provide a straightforward means for introducing a photo-isomerizable unit into a peptide or protein structure. we have shown that the conformational response of the peptide in such systems is critically dependent on the manner of attachment of the cross-linker as well as the cross-linker structure. cross-linkers can be introduced in which trans-to-cis photoisomerization leads of formation of helical structure, or conversely to loss of helical structure. these effects can be understood in a semi-quantitative manner by calculating the degree of mismatch between the steric requirements of the linker and the conformational ensemble of the peptide. kinetic studies reveal that in general photoisomerization of the linker is fast (several ps) whereas subsequent peptide folding or unfolding occurs over hundreds of microseconds. such cross-linkers are thus potentially useful phototriggers for studying protein folding processes, as well as for controlling the equilibrium stability of different conformational states. applications to photo-control of bioactive peptides and proteins will be discussed. short corticotropin-like peptides with stress-protective activity it was synthesized 87 linear è 9 cyclic corticotropin-like peptides, which contained from 13 to 2 amino acid residues. the ability of each of the synthesized peptides to inhibit the specifi c binding of tritium-labelled corticotropin (11-24) to the adrenal cortex membranes of rat in vitro was investigated. on the base of the obtained results 12 peptides with the highest inhibitory activity were selected for in vivo tests. the infl uence of the selected peptides on the level of 11-oxicorticosretoids and catecholamines in the adrenals and blood of rats in the experiments on acute hemorrhage and hypobaric hypoxia, cold and heat shock and low doses of g-radiation were studied. it was established that intravenous injection of 5 short peptides at the dose of 1 g/kg could correct disturbance of 11-oxycorticosretoids-catecholamines system in the adrenals and plasma of rats that were subjected to hemorrhagic shock and hypoxia. the rest of investigated peptides possessed lower stress-protective activity. it was also shown that under cold or heat shock, thrice-repeated intranasal injection of these peptides into rats at the dose of 10-20 g/animal abolished temperature induced changes in the level of 11-oxycorticosretoids and catecholamines in the adrenals as well as the content of free histamine and the activity of diaminoxydase in the myocardium. it has been shown that the stress-protective activity of corticotropin-like peptides is mediated by the corticotropin receptor in cortex of the adrenals. a study of immunobiological activity of the wrnwdyyk octapeptide the evaluation of the effect of japanese herbal in many cases, japanese herbal (kampo) medicines have been used in the empirical treatment of chronic hypofunction. however, the kampo medicines consist of several herbs, whose pharmacological mechanism is not clear. in western medical science, medical doctors usually treat patients according to disease diagnosis. in eastern medical science, treatment is based on diagnostics called gsho h, which is a unique concept in kampo medicines and quite different from diagnosis. the concept of gsho h is diffi cult for non-professionals to understand, furthermore, there are responders and non-responders when nonprofessionals prescribe kampo medicines. the concept of gsho h focuses on individuals, not diseases, therefore, it is diffi cult to gain a given effect for everyone by general clinical trials. in this study, we investigated the effects of prokinetic kampo medicines on plasma levels of gut-regulatory peptides (somatostatin, motilin and gastrin) and compared with that of dopamine receptor antagonists, the western prokinetics. the fi ve kampo medicines, including pinelliae tuber and zingiberis rhizoma, three dopamine receptor antagonists or placebo was orally administered. venous blood samples were taken before and till 240 min after administration. plasma peptide levels were measured using a sensitive enzyme immunoassay. the dopamine receptor antagonists and kampo medicines caused signifi cant increase of plasma gut-regulatory peptide levels compared with placebo group. in recent years, chronic hypofunction without mechanical problem, such as non-erosive refl ux disease and functional dyspepsia, is diffi cult to cure using western medicines. the preliminary study indicated the plasma somatostatin levels of patients with any symptoms are high and plasma motilin levels of them are low compared with those of healthy subjects. to evaluate kampo medicines using bioactive peptides as biomarkers, it may be possible to cure diseases that is diffi cult to treat by western medicines. bapst, jean-philippe; calame, martine; eberle, alex n.; tanner, heidi university hospital basel, switzerland radiolabeled -msh analogs are potential candidates for melanocortin-1 receptor (mc1-r)-mediated melanoma targeting. several short -msh peptides carrying a dota (1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid) metal chelator were designed and evaluated as potential diagnostic (e.g. with 111in, 67,68ga) or therapeutic (e.g. 90y, 67cu) radiopharmaceuticals. the analogs tested to date showed high affi nity for the mc1-r in vitro, excellent internalization into the tumor cells, as well as a good incorporation in tumor xenografts and a low uptake in normal tissues in vivo, except the kidneys where considerable uptake is observed. our current studies attempt to infl uence the pharmacokinetic parameters in order to address specifi c uptake (i.e. by melanoma) versus non-specifi c uptake (i.e. by the kidneys). as glycosylation had been shown to improve tumor-to-kidney ratios in the case of somatostatin and to reduce peptide re-uptake by the tubular system of the kidneys in general, we investigated glycosylated analogs of [nle4, asp5, d-phe7]--msh4-11 (napamide). carbohydrate moieties such as glucose, galactose and maltotriose were introduced at various positions on the msh peptide carrying the metal chelator dota for labeling with 111in. the peptides were evaluated in vitro in both murine and human cell lines for mc1-r binding and cellular localization, and in vivo in b16f1 tumor-bearing mice for tissue distribution. the tumor-to-kidney ratio for gal-napamide (4-48 h aucs) was superior to any of the previously published msh peptides. other glycopeptides showed very good binding affi nities but lower selectivity in vivo. in additional, a series of non-glycosylated dimeric derivatives, bearing one or two moieties of the chelator complex, were developed which displayed excellent receptor affi nity but tended to result in higher kidney accumulation. by contrast, at least one negatively charged dota-napamide showed excellent tumor-to-kidney ratios. there is a critical lack of validated early biomarkers for most conditions and diseases. early diagnosis does enable treatment of less severe disease states, the use of less invasive techniques and could potentially reduce the costs of healthcare systems. however, it is most likely that peptides in tissue or blood -or their modifi cations -can be specifi cally associated with different disease states. the discovery and verifi cation/ validation of such disease markers are two distinct workfl ows. during the discovery phase, a relatively small number of samples with a high number of potential biomarker candidates are screened. the highthroughput provided by the itraq™ reagent strategy allows for simultaneous analysis of such samples. once biomarker candidates have been identifi ed with initial statistical signifi cance, these have to be validated. this validation workfl ow involves analyzing a large number of samples with a relatively small number of candidates to establish the biological signifi cance of the biomarker candidates. rather than switching to immunological techniques for this validation step, we suggest a mass spectrometry based approach. this orthogonal strategy is a novel targeted, high throughput quantitative multiplexed multiple reaction monitoring (mrm) approach. the approach relies on assay development using a combination of mrms to target specifi c peptides identifi ed in discovery, followed by ms/ms to confi rm that the quantitative mrm signal results from the target peptide. in addition to being a quantitative method, this validation approach is extremely specifi c and sensitive. several published examples of the application of this mrm-based approach utilizing the actual or hypothetical physical properties of peptides in complex mixtures will be presented. infl uence of the charge on the in vivo behavior of radiolabeled bombesin analogues prostate and breast cancers are the second leading cause of cancer death in men and women, respectively. the side effects related to the treatments that are available today have a great infl uence on the patients' quality of life. therefore, the development of new diagnostic and therapeutic strategies may have an important impact in the outcome of these cancers. gastrin-releasing peptide (grp) receptors are present in high quantities in a variety of cancers, prostate and breast tumors among them. targeting of over-expressed grp receptors with radiolabeled bombesin (bbs) analogues would offer an interesting tool for tumor imaging and therapy, depending on the radionuclide used. some analogues of bbs, based on the fragment 7-14, were functionalized with the (n his)chelator for labeling with the 99m tc-and 188 re-tricarbonyl-core. despite an increased metabolic stability, these analogues showed very low tumor uptake. additional insertion of a ala-ala linker led to increased tumor uptake but still unfavorable in vivo properties. in order to further improve the biodistribution, novel polar linkers with different charge were introduced in the molecule. a positive charge resulted in increased kidney uptake, whereas one single negative charge led to a signifi cant increase in the tumor uptake and also signifi cantly higher tumor-totissue ratios. co-injection with natural bbs importantly inhibited the uptake in the tumors and receptor-expressing tissues, which confi rmed the specifi city of the in vivo uptake. moreover, imaging of the tumor xenografts by spect/ct was also much clearer with the analogues bearing one negative charge. additional negative charges, however, resulted in a loss of binding affi nity and internalization, and unfavorable biodistribution. in conclusion, bbs analogues with one single charge in the linker hold a greater potential for imaging and therapy of grp receptor-overexpressing tumors peptide-macrolide conjugates as novel instruments for protein biosynthesis study bacterial ribosomes are targets of numerous therapeutic agents. macrolide (ml) antibiotics prevent nascent polypeptide growth by blocking the ribosomal exit tunnel (rt). availability of high-resolution structures of complexes of ribosomal 50s subunits with ml enabled developing novel ideas concerning their inhibitory activity of translation. formerly we obtained peptide derivatives of ml in which the peptide part modelled a growing chain, while an antibiotic moiety served as an "anchor" for positioning the peptide in the rt. the goal of this study was to synthesise tryptophan-containing peptide derivatives of 5-omycaminosyltylonolide (omt) to monitor location of the specifi c trp binding site that modulate the activity of the peptidyl transferase centre of the ribosome. the peculiarity of compounds designed in this study lies in that of a trp containing peptide fragment, connected to the primary hydroxyl group in position c23 of omt, is oriented in the rt towards the hypothetical trp binding site. the following peptides were used: boc-l-trp-gly-oh, boc-l-trp-ala-oh, boc-l-trp-abu-oh, boc-l-trp-ape-oh. variable distance between trp residue and macrolide ring was achieved by introduction of glycine, -alanine, -aminobutyric acid and -aminovaleric acid as c-terminal amino acids of the dipeptides. the peptides were obtained from boc-l-trp-oh and ethyl esters of the corresponding amino acids by condensation with bop followed by saponifi cation of the peptide esters. the reactions between peptides and omt were produced using dcc and dmap as condensation agents. as a result peptide-macrolide derivatives were obtained. all compounds were purifi ed by column chromatography on silica gel and characterized by hplc, mass-spectrometry and nmr. it was shown by means of chemical probing that trp containing omt derivatives specifi cally bound to rt of the ribosome. moreover these compounds displayed an antibiotic activity in testes with several staphylococcus strains. zwanziger, denise 1 ; neundorf, ines 1 ; schatzschneider, ulrich 2 ; beck-sickinger, annette g. 1 1 university of leipzig, germany; 2 university of bochum, germany npy is a 36 amino acid peptide amide that belongs to the pancreatic polypeptide-hormone family (1) . it is the most abundant neuropeptide in the brain and triggers a number of central activities, such as regulation of food intake as well as stress induced reactions [2] [3] [4] . npy forms a selective interaction with at least three receptors, which are called y1, y2 and y5. in the past it was shown that y1-receptors are overexpressed in >90% of all breast tumors as well as in 100% of the derived metastases. normal breast tissue expresses y2-receptors, while the neoplastic tissue expresses y1-receptors 5.. as a result, the npy-y1-receptor system can be used for tumor targeting and therapy. in the fi rst step we synthesized the truncated and modifi ed npy analogue [pro30,nle31,bpa32,leu34 ]npy(28-36) (nle-norleucine; bpa-benzoyl-phenylalanine) by solid bioactive peptides phase peptide synthesis using fmoc/tbu strategy. the npy analogue was characterized with respect to in vitro binding affi nity and selectivity at y1-receptor expressing human breast cancer mcf-7 cells and the metabolic stability in human blood plasma, respectively. then we coupled different potential chelators to [pro30,nle31,bpa32,leu34]np y(28-36), which was n-terminally modifi ed by two ß-alanines as spacer between the peptide sequence and the chelator. next, we determined their ability of metal conjugation of diverse metals, as cu2+ and re3+. metal conjugated peptides were characterized by maldi-tof-ms (matrix assisted laser desorption/ionization mass spectrometry), rp-hplc (reversed phase high performance liquid chromatography) and ir-spectroscopy (infrared). after optimization of the metal conjugation fi rst binding affi nity studies were performed. 2 chain is mainly expressed in skeletal muscles and peripheral nerves, and interacts with cell surface receptors such as integrin, dystroglycan, and heparan sulfate proteoglycans. biological functions of the laminin 2 chain n-terminus including integrin binding and heparin/heparan sulfate binding were found previously. here, we focused on the n-terminal region of the mouse laminin 2 chain (position 1-1566) and screened the biologically active sequences using 142 peptides. the synthetic peptides were generally 12 amino acids in length and overlapped with neighboring peptides by 4 amino acids. cell attachment activity of the peptides was evaluated using a peptide-coated plastic plate assay and a peptide-conjugated sepharose bead assay using ht-1080 human fi brosarcoma cells. eleven peptides showed cell attachment activity on plate assay and fi ve peptides showed cell attachment activity on beads assay. previously we screened active sites on the laminin 1 chain and identifi ed several active sequences. when we compared with active sequences of the 1 and 2 chains, a2-31 (yydetvasrnlsln) and a2-112 (ggklkyaiyfea) are unique active peptides only within laminin 2 chain. these results suggest that the biological activity of a2-31 and a2-112 are 2 chain specifi c and are useful for investigating the 2 chain specifi c functions of laminin 2 chain. novel peptide biopharmaceuticals by using phage display technology štrukelj, borut; lunder, mojca; bratkoviè, tomaž university of ljubljana, faculty of pharmacy, slovenia libraries of random peptides displayed on the surface of bacteria, mammal cells or bacteriophages are an essential tool that enables systematic study of target molecule interactions. the identifi cation of ligands from large biological libraries by phage display has now been used for almost 15 years. in a last few years several improvements have led to numerous high affi nity peptide ligands that express various biological activities. phage-displayed peptide libraries have been used successfully to isolate peptide ligands directed to a functional site for which the natural ligand is or is not a protein or peptide. by using a modifi ed, in-house developed selection proctocol, we successfully selected several phage clones with high affi nity to pancreatic lipase, ghrelin and cysteine protease cathepsin k as target proteins. based on their deduced animoacid sequences, twenty heptapeptides with the highest affi nity to target proteins were synthesized and characterized for their capacity to inhibit enzyme or acceptor function. the most succesful peptide candidates inhibited pancreatic liapase with the apparent inhibition constant of 16 um, and cathepsin k with the apparent inhibition constant of 0,10 um. a set of 22 peptidomymetic compounds were sinthesyzed based on the aminoacid sequence of selected peptides and their inhibitory activity was determined. the most potent candidates were selected for further development of peptide biopharmaceuticals aganist obesity (inhibitors of pancreatic lipase and ghrelin) and in the prevention of osteoporosis (cathepsin k inhibitors). novel approach to non-specifi c elution in phage display using ultrasound phage display is used to select and optimize peptides or protein domains binding to virtually any protein and sometimes even non-protein targets. several rounds of screening are performed, until the increase in phage output, or binding assays performed with phage pools, indicate that the population of binding phages has been adequately enriched. in cases where ligands of particular target are not known or available, targetbound virions are released by non-specifi c elution for example with acidic buffer, or competitively with free target molecule, or by addition of bacterial host directly to the target-bound phages. we used streptavidin, immobilized by adsorption, as a model target protein for affi nity selection of peptides from phage display library. the effi ciencies of a number of well-known typically used non-specifi c elution strategies in selecting and retrieving a phage clone displaying the tripeptide biotin mimetic (hpq) from a streptavidin coated surface were compared. all the commonly used elution strategies have failed to elute and select high affi nity hpq-bearing phage clones bound to streptavidin. the failure was shown to be due to the inability of eluants to break the interaction of high affi nity clones with the target, which is thus likely to be the cause for failed selection with other targets also. to surmount this, we have introduced a new elution strategy, combining low ph elution buffer with sonication which, in addition to loosening the peptidetarget interaction, also serves to detach the target molecule from the immobilization surface. this ultrasound-based method enabled single step selection of a high affi nity peptide from a library of diversity greater than 10 9 , and thus represents a dramatic improvement in searching for novel, specifi c peptide ligands. apoptosis to various tumor cells but not in normal cells. using fmoc solid phase synthesis, cellulose membrane-bound octameric peptide library of trail scan was prepared and cell viability assay was directly performed on peptide disk with jurkat cells. six peptide sequences that could induce cell death were found, and particularly rnscwskd (trail227-234) peptide was shown the strongest effects. then, peptides of stronger effects were found through amino acid substitution, and the cnscwskd peptide induced >90% cell death in treated cells. features of apoptosis, such as dna fragmentation, activation of caspase, phosphatidylserine externalization, chromatin condensation, and competition with trail for binding to the death receptor (dr) 4 or dr5 were observed, suggesting that this peptide is a trail mimic. caspase-3 activation was observed in various tumor cells treated with this peptide as well as with trail, while no activation was observed in human normal fi broblasts. the cnscwskd peptide is a potential candidate for use in cancer therapy. the being an incretin mimetic, exendin-4 exerts the same action as glp-1 including the glucoregulatory and the insulinotropic effects through glp-1 receptor. exendin-4 may be preferable to glp-1 on the aspect of stability. however it was comprised with 39 amino acids and was not suitable for developing as an oral drug. recently, there was an upsurge in the development of exendin-4 mimetic as potential therapy for type 2 diabetes. in this study, a receptor-binding region on the surface of rinm5f cell was used as the target to screen peptide ligands for the receptor in a phage 12-mer peptide library. dna sequencing revealed a group of closely related peptides from the fourth round of selection. through the activity of decreasing blood glucose assay in vivo, the cell proliferation assay and capability against dppiv in vitro, one highest bioactivity peptide (qpsvgmkpsprh, ex-12pa) was acquired. the bioactivity of ex-12pa was almost identical with exendin-4 on the promoting cell proliferation in a dose-dependent manner. the decreasing blood glucose effect of ex-12pa was up to 45 min after administration. the stability against dppiv of ex-12pa maintained good performance that 35% parent peptide remained after 48h. in summary, ex-12pa as a shorter glp-1 receptor agonist mimicked the action of exendin-4 and built a good foundation for designing the oral diabetes drug. key words: exendin-4, mimetic, phage display peptide library, screen over the last decades we have witnessed the emergence of bacterial resistance to virtually all clinically important antibiotic types. therefore, continuous development of antibacterial agents with completely novel modes of action accompanied by rationalization of chemotherapeutic prescription is the key strategy to adopt in a long-run struggle against the growing problem of pathogen resistance. numerous indispensable antibiotics interfere with peptidoglycan cell wall biosynthesis making this unique metabolic pathway a well validated target for antimicrobials. while nearly all of these antibiotics inhibit late stages of murein synthesis occurring on the extracellular side of plasma membrane, initial cytoplasmic steps have not been extensively exploited as drug targets. we have performed affi nity selections from random linear and conformationally constrained (disulphide cyclized) peptide libraries displayed on bacteriophage particles against two essential bacterial enzymes murd and mure, involved in the cytoplasmic synthesis of peptidoglycan monomer precursor. selected peptides were found to inhibit respective targets in an in vitro assay with ic 50 values of 140 m to 1.5 mm. reported inhibitory peptides should be regarded as templates for design of low-molecular-weight peptidomimetics. resulting murd and mure inhibitors with improved potency and/or physicochemical characteristics (especially membrane permeability) have the potential to act as broad spectrum chemotherapeutics. fidan, zerrin; ljeskovica, nizama; portwich, michael; volkmer, rudolf charité-universitätsmedizin berlin, germany coiled coil (cc) sequence motifs are common structural motifs and versatile protein interaction modules. the cc is composed of at least two right-handed amphipathic a-helices that wrap around each other into a left-handed supercoil such that their hydrophobic surfaces are in contact. cc can associate up to heptamers, form homomeric and heteromeric complexes at different stoichiometries, and be aligned parallel and antiparallel. a characteristic of all coiled coils is the presence of heptad repeat sequences [abcdefg]i, where i denotes the heptad number. although cc motifs can be predicted with a high degree of confi dence, predicting the association states and topologies is still a great challenge. we will report on synthetic peptide arrays useful to study cc associations at the amino acid level. in contrast to rational design, which mostly depends on short model peptides, our approach relies on the full-length homodimeric gcn4 -and the heteromeric cjun/cfos coiled-coil domain formation. the infl uence of amino acid substitutions on association is tested without restrictions and presumptions and the stoichiometry is examined by biophysical methods. furthermore, we generate arrays comprising hundreds of (putative) cc sequences and subsequently probe them for association to a set of several native cc sequences. an interactome can be drawn for each of the investigated cc sequences, enabling one to interpret the biological functions. we will present: (i) association analyses of all single substitution variants of the gcn4-, cfos and cjun leucine zipper probed with the associated native leucine zipper; (ii) the exposure of a heteromeric cc-network deduced from gcn4 variants and the determination of the networkstoichiometry; (iii) the cc-interactom of several native coiled coils like the cc sequences of akap 18 , pqbp1 18, orai1 and others. almost two decades ago we used the spatial screening technology to optimize activity and receptor subtype selectivity among rgd recognizing integrins. this culminated in the avb3 selective superactive pentapeptide c(-rgdfv-) which gave rise to a number of peptidomimetic modifi cations. among them we studied all retro-, inverso-and retroinverso peptides of this structure. only one retro-inverso peptide exhibits full activity: the peptide c(-dgrvf-) strongly inhibits avb3/vitronectin binding (4 nm) but not aiibb3/fi brinogen binding (>10 000 nm). all investigated retro-sequences show very low affi nity for avb3. recently it was discovered that the ngr sequence in the fi bronectin domain fi5 after rearrangement into iso-asp-g-r exhibits high binding affi nity for integrin avb3. this surprising result stimulated us to investigate cyclic peptides containing the iso-asp-gly-arg sequence3.. after optimisation we obtained peptides which bind with high activity to integrin a5b1 and lower, but signifi cant activity for avb3. in the ongoing work we investigate if receptor modelling can help to understand these results. selectivity and activities for these two integrins have been obtained recently in our group using two different peptidomimetic scaffolds bioactive peptides (tyrosine based and diacylhydracine based) using a homology model of the integrin a5b1 for which no x-ray structure is known. binding of integrin ligands involves binding of a carboxyl group to the metal ion (ca or mn) in the so-called midas region of the integrin. so far all integrin ligands contain a carboxyl group and any attempt to substitute this group by an isoster failed. we recently found that hydroxamic acids (-conhoh) allow for the fi rst time a substitution of this carboxyl group. the activities and selectivities for integrin subtypes avb3, a5b1 and aiibb3 have been explored and give interesting results. development of gnrh-iii-antracycline conjugates as multifunctional drug delivery systems for targeted chemotherapy targeted chemotherapy based on the cell specifi c or overexpressed receptors on tumors might be an effi cient therapeutic approach for the treatment of cancer. expression of gonadotropin-releasing hormone (gnrh) receptor was identifi ed on different types of tumors.1 it has been shown that gnrh-iii (glp-his-trp-ser-his-asp-trp-lys-pro-gly-nh2) isolated from see lamprey has antiproliferative activity on numerous tumor cells, and signifi cantly less potency on releasing gonadotropin hormones (lh, fsh); therefore, it is a more selective antitumor agent than the human gnrh derivatives.2 in our work, gnrh-iii was used as targeting moiety for the preparation of multifunctional drug delivery systems for targeted cancer chemotherapy. daunomycin (dau) and doxorubicin (dox) as antineoplastic agents were attached to the side chain of 8lys of gnrh-iii through amide, oxime, hydrazone or ester bonds, either directly or by insertion of an enzyme cleavable tetrapeptide spacer. stability studies of the conjugates were performed in human serum, as well as in the presence of chymotrypsin. the effect of chemical structure of the conjugates on in vitro antitumor activity was studied using different cancer cell lines (mcf-7 human breast, ht-29 human colon and c26 murine colon cancer cells). oxime bond linked dau-gnrh-iii conjugate was selected for in vivo experiments using c26 colon tumor bearing mice. the conjugate showed similar antitumor activity as the free drug, but less toxic side effect and longer survival of the animals was determined in the case of the application of the conjugate. guillon, g. 1 (1) . it was also shown to be highly selective for the human v1b receptor (1) . we now report the synthesis and some pharmacological properties of three fl uorescent hv1br ligands (a,b,c), based on modifi cations of the lys8 residue in d[leu4,lys8]vp, with alexa 488 (a), alexa 647 (b) and antraniloyl (atn) (c). the fl uorescent peptides a, b and c exhibit the following affi nities for the hv1br: (a) ki = 1.2 nm, (b) ki =186 nm and (c) ki = 0.65 nm. peptides a and c exhibit moderate affi nities for the hotr and very weak affi nities for the hv1ar and for the hv2r. on v1b-transfected att20 cells, they activate plc coupling as evaluated by stimulation of ip3 levels. they also induced receptor internalization visualized by accumulation of fl uorescence in endosomial vesicles. one or more of these new fl uorescent hv1br ligands promise to be useful tools for studying human v1b receptor localization or traffi cking. distribution of prolyl oligopeptidase in the mouse wholebody sections and peripheral tissues prolyl oligopeptidase (pop) is a serine endopeptidase that hydrolyses proline-containing peptides shorter than 30-mer, including many bioactive peptides. the distribution of pop in the brain has been studied but little is known about the distribution of peripheral pop. we used immunohistochemistry to localize pop in mouse whole-body sections and at the cellular level in peripheral tissues. furthermore, we used a pop activity assay to reveal the associations between pop protein and its enzymatic activity. the highest pop protein densities were found in brain, kidney, testis and thymus, but in the liver the amounts of pop protein were small. there were remarkable differences between the distribution of pop protein and activity. the highest pop activities were found in the liver and testis while kidney had the lowest activity. in peripheral tissues, pop was present in various cell types both in the cytoplasm and nucleus of the cells, in contrast to the brain where no nuclear localization was detected. these fi ndings support the proposed role of pop in cell proliferation in peripheral tissues. the dissociation of the distribution of pop protein and its enzymatic activity points to nonhydrolytic functions of pop and to strict endogenous regulation of pop activity. the rapid cytoplasmic entry of cationic cell-penetrating peptides in the past few years, our understanding of the cellular import of cellpenetrating peptides has evolved rapidly. the initial concept of cell entry by direct permeation of the plasma membrane, was followed by endocytosis as a major route of import at least for most cellpenetrating peptide-cargo conjugates. more recently, we and others observed a rapid cytoplasmic delivery of fl uorescein-labeled analogs of the cationic cpps nonaarginine and tat-peptide at subtoxic lower poster abstracts micromolar concentrations (1). this import originates from spatially confi ned zones of the plasma membrane and depends on the presence of heparan sulfate proteoglycans. these molecules have been proposed to interact polyvalently with the guanidinium groups of the arginine residues. moreover, the threshold for the induction of this import could be lowered by inhibition of endocytic routes of entry. recently, it was reported that the absence of serum in the medium lowers the threshold for this uptake process (2) . in summary, these observations suggest that the concentration of peptide associated with the plasma membrane is a decisive trigger for this import process. currently, it is not known, to which degree this cytoplasmic entry of labeled conjugates at higher concentrations is based on similar molecular mechanisms as the direct permeation of unlabeled conjugates that has also been observed at lower concentrations (3). here we summarize the current knowledge on direct transport across the plasma membrane of cationic cpps and present new data on the molecular events involved in this uptake process. plasma membranes have numerous essential functions for maintaining cellular homeostasis. however, the membranes are also barriers to intracellular delivery of various therapeutic molecules. for improvement of their translocations, we developed a novel method using gala peptide/cationic lipid complexes. gala, a 30-residue amphipathic peptide with a repeat sequence of glutamic acid-alanine-leucine-alanine, was designed to mimic the function of viral fusion protein sequences that mediate escape of virus gene from acidic endosomes to cytosol (1) . when attached with bioactive cargoes, the gala peptide may thus serve as intracellular vector bearing effi cient endosomal escape function. however, because of negative charges from glutamic acids (7-residues) in the gala sequences, access of the peptide on negatively charged cell surface would be not so effi cient. to overcome this problem, cationic lipid was employed as an gadhesive h for pasting the gala peptide onto cell surface to accomplish effi cient cellular uptake. we examined the ability of gala peptide as a delivery vector using fitc as a model of membrane-impermeable low-molecular weight drugs. when fitc-gala (1 ƒêm) was administrated to hela cells, co-addition of cationic lipid, lipofectamine 2000 (lf2000), drastically increased the effi ciency in uptake of fitc-gala. in a time-dependent manner, the fitc-gala escaped from endosomes, and diffuse fl uorescent signals were observed in both cytosol and nucleus. also the gala/cationic lipid system was applied for the intracellular delivery of fitc-avidin protein (68 kda). when fitc-avidin was mixed with biotinylated-gala/ lf2000 complexes, fitc-avidin (250 nm) was internalized into cells effectively. in the absence of these complexes, internalization of fitcavidin was poor. these results suggest the usefulness of our approach for intracellular delivery using gala peptide and cationic lipid. the membrane repair response masks membrane disturbances caused by cell penetrating peptide uptake even though cell-penetrating peptides are able to deliver cargos of different sizes into cells, their uptake mechanism is still not fully understood and needs to be elucidated in order to improve their delivery effi ciency. recent studies have suggested that there might be a direct penetration of peptides in parallel with different forms of endocytosis. however, the direct penetration of hydrophilic peptides through the hydrophobic plasma membrane is highly controversial. three proteins involved in target cell apoptosis -perforin, granulysin and granzymes share many features common in uptake of cell-penetrating peptides e.g. they bind proteoglycans on the plasma membrane. the uptake of perforin activates the membrane repair response, a resealing mechanism triggered in cells with injured plasma membrane, due to extracellular calcium infl ux. upon activation of the membrane repair response, internal vesicles are mobilized to the site of the disrupted plasma membrane resealing it within seconds. in this study we present evidence that the membrane repair response is able to mask damages caused by cell-penetrating peptides when they internalize cells, thus preventing leakage of endogenous molecules out of the cell. the hypothalamic decapeptide gonadotropin-releasing hormone (gnrh-i; gnrh symmetric dimer derivatives ([ in vitro (cellular uptake and antiproliferative effect of the dimer derivatives) and in vivo (comparison of per os and intraperitoneal administration) antitumor effect of these symmetric dimers were investigated. the cellular uptake of dimer derivatives were studied by fl ow cytometry (bd lsr ii) on mcf-7(human breast cancer) and ht-29 (human colon carcinoma) cell lines using carboxifl uorescein labeled symmetric dimer derivative. the cells were treated with different concentration of synthetic dimers and after the treatment were analysed by fl ow cytometry in order to investigate their cellular uptake. the antiproliferative effect of symmetric gnrh-iii dimers were studied on mcf-7, ht-29 and t47-d (human breast cancer) cell lines. ht-29 xenograft was applied for studying in vivo antitumor effect of gnrh-iii dimers in different administration routes. the cellular uptake and the antiproliferative effect are cell type dependent as it can be found in the literature. we found that symmetric dimer derivatives of gnrh-iii signifi cantly decreased the tumor volume in vivo (40%). iib 3 receives intracellular signals (inside-out signaling) that allow cytoplasmic proteins to interact with the cytoplasmic domains of iib 3 subunits, resulting in platelet aggregation. the aim of this work is to inhibit platelet thrombus formation by specifi cally disrupting the insideout signalling pathway using synthetic peptides based on the cytoplasmic region of 3 subunit, residues 743-762. peptide analogues derived from 118 3 743-762 region ( 3 743-750, 3 743-750(ptyr747), 3 743-756, 3 755-762 and 3 749-756) were synthesized in their free, palmitoylated and/or tagged with the tat(48-60) signaling sequence and carboxyfl uoresceinlabeled in order to investigate their membrane permeability, as well as their inhibition potency on the platelet aggregation. from the biological assays in prp and washed platelets we concluded that the modifi ed peptides that carry either palmitoyl-group or the tat(48-60) signaling sequence penetrate platelet membrane and inhibit human platelet aggregation, in contrast to the corresponding free peptide analogues. acknowledgements: the gsrt (epan yb/88) and e.u. for the fi nancial support. ; aib = -aminoisobutyric acid) is a potent mitochondriotoxic analogue of mp that demonstrates a signifi cant intracellular co-localization with mitochondria. through co-operation with a protein of the mitochondrial permeability transition pore vdac, mitoparan specifi cally promotes apoptosis. in contrast, cytc 77-101 , a cryptic fragment of human cytochrome c, is a moderately potent apoptogenic cpp that demonstrates a strong propensity for colocalization within the endoplasmic reticulum. using a sychnologic modular design, we have synthesized and evaluated a broad range of chimeric constructs combining apoptogenic cpp (message) and peptidyl address motifs. the overriding objective of these studies was to enhance cytotoxicity and/or develop target-selective drug delivery vectors. address motifs that target both plasma membrane and nuclear envelope protein structures included i) the integrin-specifi c rgd sequence, ii) a fas ligand mimetic wewt, iii) heptagastrin (aygwmdf) that targets a novel membrane binding site on high grade astrocytoma and, iv) a mimetic of fg nucleoporins (nups). incorporation of peptidyl address motifs, by simple n-terminal acylation or via a fl exible aminohexanoic acid (ahx) linker, produced chimeric constructs with enhanced cytotoxic potencies. most signifi cantly, ld 50 values readily achievable in vivo were demonstrated by the modular apoptogenic cpp z-gly-rgdf-mitoparan, (ld 50 = 1.4 m) and ac-nfkfglss(ahx)cytc 77-101 (ld 50 = 1.0 m). in conclusion, targetspecifi c modular design of apoptogenic cpp is a promising strategy for the study and therapeutic induction of apoptosis. phage display screening of geminin-binding peptide and validation of its therapeutic use in tumors yoshida, kenichi meiji university, japan dna replication is controlled by the stepwise assembly of a pre-replicative complex (pre-rc). geminin is a component of the pre-rc and plays a role in preventing incorporation of the minichromosome maintenance protein complex into the pre-rc via binding and inhibiting cdt1 function. it may be possible to design low-molecular-weight chemicals that would bind to geminin to suppress the aberrant cellular proliferation in tumor cells. a random 12-mer peptide phage library was used for the selections. phage was selected by panning on immunotubes coated with recombinant geminin. positive clones were identifi ed by a screening phage produced from single colonies for specifi c binding to gst-geminin in elisa. fluorescent-labeled peptide linked to the sv40 nuclear localizing signal was synthesized by solid-phase synthesis, using fmoc chemistry. by detecting fl uorescein fl uorescence to mark specifi c transfected cells, we examined the effects of the peptide transfection on the synthesis of new dna in hct116 cells. by screening a random peptide phage library, we identifi ed a certain peptide sequence bound to geminin. using a series of mutant geminin, elisa test revealed the amino acid residues 31-111 are responsible for the peptide binding. we found fewer brdu positive cells following transfection of the geminin-binding peptide than following that of the control peptide. this study suggests that the chemical peptidomimetics of this peptide might form the basis for the development of drugs that could be used to prevent tumor progression. acknowledgements: this study was supported by kakenhi. it has been previously demonstrated that n3-(4-metoxyfumaroyl)-l-2,3diaminopropanoic acid (fmdp) is a strong inhibitor of glucosamine-6phosphate synthase, a potential target for antimicrobial chemotherapy. fmdp is transported into the cells when incorporated in a peptide chain and hydrolyzed by intracellular peptidase releasing free inhibitor as the "warhead" component, which can react with the target enzyme. the concept of utilizing of peptide transport system for delivery of toxic amino acids into the microbial cells is characterized by authors as "illicit transport". unfortunately, peptides are not stable in physiological fl uids, due to activity of peptidases. analogs with modifi ed amide backbone are more resistant towards enzymatic degradation. it has been shown that enzymatic hydrolysis of endothiopeptides is often signifi cantly slower than natural peptides. in our studies we synthesized nva[csnh]-fmdp, an analog of nva-fmdp, which is a strong antimicrobial peptide. because of the fact that the "warhead" component in this peptide contains an amide backbone, fi rstly we decided to synthesize free fmdp with thioamide backbone. the results of enzymatic kinetics studies showed that in this way modifi ed compound is much weaker inhibitor of glucosamine-6-phosphate synthase than fmdp. it was the reason why we planned and carried out a multi-step synthesis in order to obtain a potential more resistant towards enzymatic degradation, antimicrobial peptide containing a thioamide backbone only between nva and fmdp. activity of nva[csnh]-fmdp nva-fmdp against selected microorganism in medium containing blood serum was determined. neuroprotective effects of cortexin and cortagen in rats with focal brain ischemia and brain trauma there is a growing body of evidences that natural peptides and their smaller synthetic analogues have good outlooks for medical use. three compounds were studied in this research -cortexin, cortagen and nîleylcortagen. cortexin is a balanced combination of oligo-peptides isolated from calf/porcine cortex. the number of non-clinical and clinical studies confi rmed cortexin effi ciency as nootropic and neuroprotective drug. in spite of benefi cial results the natural source of the drug implies some diffi culties for it general use. to overcome this issue a number of putative cortexin synthetic analogues were developed. among them is tetrapeptide cortagen. in order to improve the peptide transport to the brain and lipophility the hydrophobic oleyl radical was added to the peptide chain. the aim of research was to study the effects of these peptides on recovery of conditional refl ex in active avoidance paradigm after heavy brain trauma and neurologic defi cit dynamics induced by focal brain ischemia in rats. 170 males albino rats were involved in experiment. closed craniocerebral trauma was modeled by weight-drop method. focal cerebral ischemia was induced by cortical phototrombosis. experimental therapy started in 0.5 hours after mechanical action and continued for 7 days. synthetic peptides were administrated i.p. once-a-day in dose of 10 -1000 mcg/kg. cortexin was used at the same schedule in dose of 150 mcg/kg. cortagen and cortexin administration in dose of 10 and 150 mcg/kg respectively resulted in pronounced neuroprotective effect in rats with focal cerebral ischemia. the observed effects were proved by limb-placing test. cortexin experimental therapy resulted in earlier restoration of conditional refl ex starting from the 3rd day after brain trauma. nîleylcortagen had weaker benefi cial effect with signifi cant improvement in conditioned response recovery by the 4th day. probiotics such as lactobacillus rhamnosus gg provide health benefi ts beyond their mere nutritive value, and are useful in the treatment and prevention of several diseases. isolation and use of the anti-apoptotic factor(s) would ultimately culminate in the development of novel therapeutic agents in enteropathy resulting from chemo-and radio-therapy in the treatment of cancer patients, which could in turn be administered in a consistent and pharmacologically sound manner. in present study, intestinal epithelial cells were isolated from wistrar rats (n=12), and were grown in dmem medium supplemented with 5% foetal bovine serum at 37°c in a humidifi ed 5% co 2 atmosphere. after 72 h, these cells were treated with bioactive peptides isolated from lgg (10-100 ìmol/ lit) for 2 h, and were incubated with camptothecin (20 ìmol/ lit) for 2 and 4 h, and cell viability was measured by mtt assay. caspase activity assay was carried out to determine caspase 3 and caspase 9 activity. moreover, cell lysates were also pretreated with caspase 3 inhibitor (200 ìm). dna fragmentation assay was carried out to determine the increase in dna fragmentation. bioactive peptides signifi cantly prevented camptothecin induced apoptosis in rat intestinal epithelial cells. the treatment of intestinal epithelial cells with camptothecin for 4 h resulted in fi ve-fold increase in dna fragmentation, and resulted in three-fold increase in caspase 3 activity, compared with controls. pre-treatment with bioactive peptides (10 ìmol/ lit) signifi cantly attenuated the camptothecin-induced dna fragmentation by 29% (p<0.001), and signifi cantly inhibited caspase 3 and caspase 9 activity by 35% and 39%, respectively, after 4 h of camptothecin exposure (p<0.001). hence, it was concluded that novel bioactive peptide from lgg were found to be potential anti-apoptotic agents and hence could be administered as therapeutic molecule for treatment of enteropathy resulting from chemo-and radiaton-therapy. cisplatin belongs to the most powerful and useful anticancer agents. it binds strongly to dna in regions containing several guanine units, forming pt-dna links within strands. through disrupting base-pairing guanine to cytosine cross-links lead to unwinding of the dna. as a result cisplatin works against both types of cells, destroying cancer and normal type ones. therefore more selective delivery system of platinum to cancer cells is still needed. based on the evidence of the presence of -and -opioid receptor types in carcinoma cells we proposed to use opioid peptides as selective carriers for delivering platinum ions to the cancer cells. we designed hybride molecules which combine two fragments. one part of the molecule contains the opioid pharmacophore and the other fragment is designed to form a complex with platinum ion. such molecule can serve not only as carrier for platinum, but also give a strong analgesic effect. as a result such hybride molecule should express analgesic properties and provide anticancer activity. we will present synthesis of a few opioid peptide-platinum(ii) complexes, the binding affi nity at the opioid receptors and effect on the proliferation of the human glioblastoma cells. peptides derived from the c-terminal heptad repeat region of the hiv fusogenic protein gp41 are potent inhibitors of viral infection, and one of them, t20 (enfuvirtide), is used for the treatment of therapy-experienced aids patients. the mechanism of action of these peptides is to interfere bioactive peptides with the fusion of the viral and target cell membranes, and it is known that hiv entry takes place in membrane microdomains ('lipid rafts') enriched in cholesterol and sphingolipids. therefore we explored the advantage of targeting a peptide fusion inhibitor to membranes by addition of a lipid moiety, specifi cally a cholesterol group. since derivatization with lipids is also known to improve the half-life of peptide therapeutics, we chose the antiviral peptide c34, which is more potent than t20 in cell-based assays, but unlike t20 has a very short half-life in vivo. we show here that attachment of cholesterol to c34 (c34-chol) dramatically increases its antiviral potency against a panel of hiv strains, including primary isolates: for strain hxb2, ic50 = 4 pm for c34-chol, versus 205 pm for c34, and 692 pm for enfuvirtide). consistent with the anticipated mechanism of action, c34-chol accumulates at the site of action, since washing of the target cells after incubation with the peptide, but prior to triggering fusion, increases ic50 only 5-fold, relative to 400-fold for c34. moreover, cholesterol must be strictly positioned at the c-terminus of c34, in line with the need of an antiparallel orientation of the c-peptide relative to n-peptide trimeric coiled-coil, present in the fusion-active conformation of gp41. in addition to boosting antiviral potency, derivatization with cholesterol has the expected benefi cial effect on the peptide pharmacokinetics. we believe that these fi ndings may be of general utility for viruses, which share with hiv the dependence on a type i fusogenic machinery. multiple sclerosis (ms) is an autoimmune demyelinating disease mediated primarily by cd4+ t cells of the th1 subset. the design of peptide mutants of disease-associated myelin epitopes to alter immune responses offers a promising avenue for the treatment of ms. we designed and synthesized a number of peptide analogues by mutating the principal tcr contact residue based on mbp83-99 epitope. the synthesis of the linear peptide agonist mbp83-99, as well as of the cyclic analogue was carried out by the fmoc/tbu methodology, utilizing the 2-chlorotrityl chloride resin. cyclization was achieved using obenzotriazol-1-yl-n,n,n',n'-tetramethyluronium tetrafl uoroborate (tbtu) and 1-hydroxy-7-azabenzotriazole, 2,4,6 collidine allowing fast reaction and high yield cyclization product. the purifi cation was achieved using hplc reversed-phase chromatography and the peptide purity was assessed by analytical hplc and by mass spectrometry (esi-ms). agonist and antagonist (linear and cyclic) peptides were conjugated to reduced mannan. immune responses were diverted from th1 to th2 in sjl/j mice and generated antibodies which did not cross react with native mbp protein. (1) . due to its high effi cacy and specifi city, hn represents a potential lead to new therapeutic approaches of ad. however, the molecular mechanism(s) of hn function(s) are not yet fully elucidated. wild-type hn and hn-derivatives were synthesized by spps and amino acid sequences and homogeneities of the rp-hplc purifi ed peptides ascertained by esi-and maldi-mass spectrometry (ms). the complex formation between hn and a (1-40) was studied by affi nity-chromatography and high resolution ms (fticr-ms), as well as by immunoanalytical (elisa) techniques. the binding sites between the two peptides were identifi ed by applying proteolytic epitope extraction/excision procedures [2, 3] integramide a, an effi cient inhibitor of the coupled reaction of hiv-1 integrase, is a 16-mer linear peptide characterized by 9 c -methylated -amino acids (5 iva, isovaline, and 4 aib, -aminoisobutyric acid, residues) that was isolated from fungal extracts of dendrodochium sp. the amino acid sequence was fully elucidated by the merck group a few years ago (s. b. singh et al., org. lett. 2003, 4, 1431-1434) . on the other hand, the chiral sequence was only partially determined. in particular, the precise stereochemistry of the iva 14 -iva 15 dipeptide (known to contain one d-and one l-residue) near the c-terminus was not reported. to solve this unsettled issue and to assess integramide a primary structure-bioactivity relationship we performed by solution methods the total chemical independent syntheses of both l-d and d-l 16-mer diastereomers and compared their properties with those of the natural inhibitor. for an unambiguous, complete stereochemical assignment of integramide a we relied heavily on hplc and nmr techniques. our results clearly indicate that the chirality sequence of the iva 14 -iva 15 dipeptide of the natural product is l-d. the two integramide a diastereomers were also evaluated as inhibitors of hiv-1 integrase in the coupled reaction of proviral dna into the host cell dna. . a prototype peptide, r11-29, spanning rantes residues 11 to 29, contains two hydrophobic clusters of fundamental biological importance connected by a nonessential hydrophilic linker. r11-29 has been rationally modifi ed to improve its ccr5 binding and its antiviral potency. nmr studies on the modifi ed peptides revealed important similarities and differences with the three-dimensional organization of rantes. although these peptides are consistently more active as dimers, an increase in anti-hiv-1 activity of the monomeric forms was observed in parallel with their molecular evolution. in addition, no tertiary interactions could be detected by nmr, indicating an autonomous folding of the monomers also in the context of dimeric peptides. the most potent peptide designed so far, rmax, shows anti-hiv-1 activity in the low nanomolar range (vangelista et al, in preparation). strikingly, a similar potency was observed in the same assay with t20, an hiv-1 gp41-derived peptide currently licensed for use in aids therapy. these results provide a rationale for the use of rantes-derived peptides as new candidates for treatment and prevention of hiv-1 infection. 10% of the population older than 65 years and 40% exceeding the age 80 years are affected by alzheimer's disease (ad).(1) a factor in the pathogenesis of ad is the cerebral deposition of amyloid fi brils as senile plaque. bace1 initiates the pathogenic processing of app by cleaving at the n-terminus and the resulting c99 membrane bound c-terminal peptide can then be hydrolyzed by -secretase to form apeptide (a 40 or a 42). bace1 is not only a promising target cause it is a key player in formation of a but also because bace1 knock-out mice are viable and free of the gross phenotypic changes. our group was recently able to shown that the phosphino dipeptide (pdp) isostere is a suitable replacement of the hydroxyl ethylene isostere in om00-3(2) resulting in pseudo peptidic inhibitors of about same potency. [2, 3] in our search for conformationally restrained pdp isostere bace1 inhibitors we speculated that the p1 and p3 cyclization would lock the active conformation of the cyclic linear pseudo peptidic inhibitor in the n-terminal region. first the detail analysis of the crystal structure of om00-3 bound to bace1(2), revealed that the ideal macrocyclus should consist of a 13-membered heterocycle. on this basis we developed a macrocyclic inhibitor with p1 and p3 cyclized side chains containing a pdp isostere with improved serum stability. specifi c tumor-targeting, via tumor-associated antigens (taa), selectively expressed or over expressed on tumor cells, is the goal of modern cancer therapy aimed at overcoming non-specifi c toxicity of most anticancer drugs. the expression of taa varies among different tumors and patients, resulting in highly variable response to tumor targeted therapies. therefore, diagnosis should provide information on the expression of the targeted antigen in each patient, thus allowing to predict possible effi ciency of a therapy mediated by targeting agents directed to the same tumor antigen. in this approach, the molecule used for tumor cell tracing should be as close as possible to that used for therapy. the use of peptides as tumor targeting agents was envisaged years ago with the fi nding that receptors for different endogenous regulatory peptides are over-expressed in several primary and metastatic human tumors, and can be used as tumor antigens (reubi jc. j nucl med 1995;36:1825-35). in previous works, we demonstrated that peptides synthesized in a branched form, result in molecules that are resistant to proteolytic activity and can retain (or even increase, through multivalent binding) peptide biological activity. a branched peptide that targets neurotensin (nt) receptors, known to be over-expressed in a number of tumors, including colon, pancreas and prostate carcinoma (reubi jc. endocr rev 2003;24:389-427) and was conjugated to effector units and proved to be stable and active in vivo (falciani c, et al. mol cancer ther. 2007;6:2441-8). here, we created new nt-based molecular tools conjugated to different fl uorophores and chemotherapeutics and demonstrated that branched peptides can be modulated either as tracers for measuring the presence of the specifi c target in primary tumor or metastasis on human surgical resections, or as specifi c drug-carriers, which use the same target to enter and eventually kill tumor cells in vitro and in vivo. aquaculture has become an increasingly important activity, as an immediate source of animal protein required for several countries growing population. vaccination of fi sh against bacterial and viral diseases is one of the best strategy for controlling certain infectious bioactive peptides diseases in aquaculture worldwide, thus decreasing the need for antibiotics, and increasing cost-effectiveness and net profi ts. infectious pancreatic necrosis virus (ipnv) is a pathogen of farm fi sh with a worldwide distribution. peptides derived from ipnv protein 2 (vp2), a major structural protein, could be used for development of effective vaccine against ipnv, according to a search of vp2 antigenicity we selected two peptide sequences, k 19 pyvrledetpqg 42 (vp2-19-42) and n 91 fslaeqpanetk 103 (vp2-91-103), that are expected to be highly immunogenic. these peptide sequences were synthesized in their n-terminus iodoacetylated form and conjugated to ac-(lys-aib-cys) 4 -nh 2 , a cell penetrating sequential carrier (cpsc). conjugation of four copies to cys side chains of the carrier was realised by the chemoselective ligation method. the resulted constructs were purifi ed by hplc and characterized by esi-ms. immunizations are in progress in order to test their immunoprotective potency. acknowledgements to the gsrt (greece-egypt project 266-å) for the fi nancial support. for more then two decades, the rgd sequence is known to bind integrins, e.g., 5 1, v 3, and iib 3 among many others. it is present in the natural ligands for these integrin receptors, as in fibronectin, vibronectin, or fibrinogen. recently, it was shown that fibronectin in which the rgd sequence has been mutated into an rge sequence (which is known not to be recognized by integrins) still has the ability to interact with its receptor. however, it forms fn fi brils only with a slightly different phenotype than wild-type fibronectin (1). a hypothetical model for these unexpected results was proposed by curnis et al. (2) . the ngr (asn-gly-arg) sequence, present at four positions in the fi bronectin molecule, is able to undergo a rearrangement to isodgr (isoasp-gly-arg), which shows activity on v 3 and -with less potency -on 5 1. the mechanism of asn-deamination is already known for a long time and has widely been considered to be a process of degradation, acting as a biochemical clock that limits protein lifetimes in vivo (3). curnis et al. were the fi rst to show that the deamination process increases protein function instead (2). these fi ndings stimulated us to create a library of cyclic peptides containing the isodgr sequence. a screening of this library showed various new peptides with high activity and selectivity towards the integrin receptor 5 1. the correlation between the intensities of muramyl peptides adjuvant effect and nod2 activation. it is well known that essential activity of muramyl peptides -minimal structures of bacterial cell wall -are adjuvanticity. the comparison of the adjuvant effect of these compounds with the ability to activate nf-kb pathway through nod2 was examined. the adjuvant activity of di, tetrasaccharide peptides and stearoyl containing derivatives has at least two peaks in dose-response curves and greater of them correlates with respective dose-response data for nf-kb stimulation through nod2. introduction of stearoyl moiety, with the aim of improving muramyl peptide interaction with the cell membrane and subsequent intracellular delivery, infl uenced the corresponding activities in vitro, but did not correlate with improved effects in vivo experiments. the comparison of the adjuvanticity in vivo and the nod2 activation in vitro revealed clear correlation between two responses. these fi ndings confi rm the view that nod2 pathway activation should account, at least in part, for the adjuvant effect of these compouds. the correlations of the nf-kb pathway induced by muramyl peptides with the aim of enhancing their adjuvant activity were investigated. the thymus gland plays an important role in overall immunomodulation. the studies in early 1960s by several authors have established that thymus is necessary for the normal development of immune response. it is thought to be responsible for the development and regulation of tcell immunity, acting through endocrine mechanism (1). it is known that thymus peptides play an important role in the development, maturation, differentiation, and activation of t-lymphocytes. investigation of the function and properties of this gland shows that the thymus contains pharmacologically active components with immunological properties. when the immune system is challenged, thymic peptides seem to regulate the expression of various cytokine and monokine receptors on t-cells and induce secretion of il-2, interferon alpha, and interferon gamma. up to date several thymic extracts have been isolated using different biochemical methods of extraction, homogenization and purifi cation. these are, usually, semipurifi ed aqueous and lipid calf thymus extracts. the goal of this study was to determine the biological activity of peptide components, isolated from the calf thymus. extract of calf thymus was prepared and fractioned into lipid and nonlipid (peptides) fractions. the nonlipid fraction was isolated and characterised by biuret, ir , nmr and hplc methods. analyses of ir and nmr spectra indicated the presence of charasteristic bands and peaks for peptides. results estimated from hplc analyse showed that molecular masses of isolated peptides were below 1500 daltons. biological activity was accesed by using proliferation of thymocites and splenocites in vitro in the presence of mitogen, and obtained results showed signifi cant imunomodulatory effect. keywords: thymus, peptides, immunomodulators, thymocites, splenocytes, proliferation according to contemporary knowledge about proteins, every protein can play the role of a precursor of biologically active peptides. bioactive peptides isolated from food proteins display various activities, for example: antihypertensive, antithrombotic, immunomodulating, opioid and antibacterial. the database of protein and bioactive peptide sequences designed in chair of food biochemistry (www.uwm.edu.pl/biochemia) contains information on proteins which are precursors of bioactive peptides. the database, biopep, enables an evaluation of food proteins according to the following criteria: the frequency of the occurrence of fragments with the given activity in a protein chain, a potential activity of protein fragments, and profi les of a potential biological protein activity i. e. the type and location of a bioactive fragment in the protein chain. the biopep database was used to determine the profi les of potential biological activity of food proteins and classify them into families and subfamilies. results that we obtained indicate that the greatest number of bioactive peptides can be released from milk proteins. we also analysed structural properties of bioactive fragments encrypted in protein chains which are predicted to be accessible for endopeptidases. the application of biopep and msblast software enabled us selection of a fragments with high degrees of identity to the celiac-toxic peptides in food proteins sequences. based on the biopep, we are able to design the processes of the release of bioactive peptides from protein sequence and with the use of mass spectrometry -to identify released peptides. the detailed results of authors in silico food proteins analysis will be presented. the computational methods and the above-mentioned tools can be applied for designing food with the special designed and desired properties (functional food) as well as production of nutraceuticals i. e. food with therapeutic properties. neurotensin analogs with high affi nity and selectivity at human neurotensin receptor 1. neurotensin (nt), pglu 1 -leu 2 -tyr 3 -glu 4 -asn 5 -lys 6 -pro 7 -arg 8 -arg 9 -pro 10 -tyr 11 -ile 12 -leu 13 -oh, exerts numerous physiological actions in the central nervous system and in the periphery. studies in animal models have suggested its involvement in the modulation of dopamine transmission, hypothermia, analgesia, locomotor activity, cardiovascular function, and others. at present, three receptors are known to bind nt; two of them are g-protein coupled-receptors (ntsr1 and ntsr2). the physiological effects of ntsr1 have been extensively studied but the functions associated with the nt binding to ntsr2 are less well defi ned. the c-terminal fragment of nt, arg 8 -arg 9 -pro 10 -tyr 11 -ile 12 -leu 13 -oh, has been long recognized as an essential and suffi cient segment for the effective interactions of nt with the receptors; a short peptide, arg 8 -arg 9 -pro 10 -tyr 11 -ile 12 -leu 13 -oh, designated nt(8-13), displays binding affi nity similar to that of the full-length nt at both ntsr1 and ntsr2. in this study, the role of arg 8 in the interactions of nt(8-13) with the human ntsr1 and ntsr2 was examined through ligand structure-function studies. the side chain of arg 8 was determined to not be critical for binding to hntsr1 but essential for binding to hntsr2. several analogs of nt(8-13) are reported which are high affi nity hntsr1 ligands (ic 50 = 0.1 to 3 nm) and more than 500-fold selective versus hntsr2. human umbilical vein endothelial cells (huvecs) form tubular networks when cultured on top of the matrigel basement membrane preparation, refl ecting the ability of the cells to form blood vessels. using this model, we have previously shown that psa (also known as klk3) inhibits endothelial cell tube formation(1), indicating reduced angiogenic potential. furthermore, we have shown that the antiangiogenic activity of psa is related to its enzymatic activity [1, 2] . we have developed peptides that stimulate the enzymatic activity of psa towards a small chromogenic substrate. these peptides also enhance the anti-angiogenic activity of psa both in vitro and in vivo. this supports our hypothesis that enhanced psa-activity by our peptides could be used to reduce tumor angiogenesis and, thus, to reduce tumor growth. the most extensively studied tads are those that contain acidic tads and one extremely important protein is the human tumour suppressor protein p53. given the presence of repetitive stretches of acidic amino acids tads are generally disordered in the free state and this has also been demonstrated for p53tad. using a combination of nmr spectroscopy, isothermal titration calorimetry and site-directed mutagenesis studies we have recently characterized the interaction of the p53tad with the pleckstrin homology (ph) domain of the tfb1/ p62 (yeast/human) subunit of tfiih. the nmr structure of the tfb1/ p53tad complex demonstrates that p53 forms a short alpha-helix in complex with tfb1 (1). this structure is an important step towards developing a sequence code for acidic tad binding to the ph domain of tfb1/p62. such detailed structural information is essential to design molecules that modulate transcription activators such as p53. we will present the structure of the p53/tfb1 complex, structural and biophysical characterization studies with peptides designed to mimic acidic tads and compete with p53 for binding to tfiih. integrins are a family of transmembrane cell surface receptors, which mediate cell-cell and cell-matrix adhesion. the binding of integrins to their natural ligands is the molecular basis of physiological processes such as cell adhesion, migration and signal transduction of cells, as well as of patho-physiological processes. thus, small molecules capable of interfering with this integrin-natural ligand binding process have pharmacological potential in the therapy of cancer and infl ammatory diseases. the amino acid sequence rgd, present on many of the natural ligands, is a prominent recognition motif of integrin ligands. synthetic rgd-containing peptides are an excellent starting point for the identifi cation, synthesis and development of selective integrin ligands. the affi nity and selectivity of the peptide ligands towards different integrins depend strongly on the secondary structure of the sequence and the overall three-dimensional shape. cyclization is frequently used as a method to reduce the accessible conformational space. additionally, the incorporation of non-natural conformationally constrained amino acids can greatly affect the secondary structure of the peptide, in such a way that the synthetic ligands prefer to adopt a particular conformation. the aim of this investigation are small cyclic peptides containing the rgd motif and constrained amino acids (such as -methylated amino acids, -amino acids or dehydroamino acids) that exhibit well-defi ned conformational properties. the present communication describes the synthesis of different cyclic rgd peptides with the general sequence c-(-arg-gly-asp-xaa-yaa-) and the evaluation of their activity as ligands for the v 3 and 5 1 integrins, present on human cells. acknowledgments: this work is supported by a marie curie intra-european fellowship from the 6th framework programme. in recent years there has been increasing interest in the design of chemical agents capable of inducing dna interstrand crosslinking (isc), which, shutting down the dna replication process, represents by far the most cytotoxic of all the alkylation events. quinone methides (qms) are interesting compounds that have been proposed as intermediates in a large number of chemical and biological processes. the asymmetry introduced by the presence of two electronically different substituents, carbonyl and methylidene, on the cyclohexadiene ring imparts a strong dipolar character to quinone methides not found in benzoquinones and quinodimethanes. qms have been successfully used to accomplish nucleoside alkylation and dna isc by photochemical and fl uorideinduced activation. apart from their involvement in biological processes, o-qms react with a variety of nucleophiles (michael addition), resulting in substituted hydroquinones. they also function as effi cient heterodienes in diels-alder reactions and undergo a variety of self dimerization reactions. here we present the synthesis of a new series of synthetic o-qms (bis-napthalene type) conjugated with rgd analogue peptides. the biological activity of these compounds is under investigation. aknowledgements: we thank the european social fund (esf), operational program for educational and vocational training ii (epeaek ii) and particularly the program pythagoras ii for funding the above work. platelets aggregation causes clotting in the blood vessels during the blood circulation due to several reasons. factor viii (fviii), a blood coagulation glycoprotein, is a key component of the blood coagulation system. fviii in its activated form (fviiia) acts as a cofactor to the serine protease fixa in the conversion of the zymogen fx to the active enzyme (fxa). the role of fviiia is to increase the catalytic effi ciency of fixa in the activation of factor x (fx). the target of this research is the synthesis of biologically active peptides, which are expected to inhibit selectively the maximisation of thrombin production depended on factor ix (fix) and accordingly the additional activation of platelets. these peptides are based on the regions in which the fviii interacts with fix. glycoprotein fviii is composed of three distinct domain types in the arrangement, a1-a2-b-a3-c1-c2. the sequence 558-565 of a2 subunit is the following: ser558 -val-asp-gln-arg-gly-asn-gln565 this work covers the synthesis and biological evaluation of linear and cyclic head to tail peptides, analogs of the loop sequence 558-565 of the a2 subunit, aiming at the inhibition of interaction of fviiia with fixa. substitutions have been taken place at asn564, which are related to the pharmaceutical groups at the side-chain, like asp(r)564. all the synthesized analogs are purifi ed (rp-hplc) and identifi ed (esi-ms). the synthesized peptides analogs were investigated for their inhibitory activity and tested for clotting defi ciency by measuring their activated partial thromboplastin time (aptt) in vitro. these results will be discussed in relation with their biological activity against the fviiia factor of blood coagulation. nerve growth factor (ngf) is an homodimeric protein that binds two different cellular receptors: 1) the transmembrane tyrosine kinase receptor trka, a member of tyrosine kinase family; and 2) p75, a member of the tumor-necrosis-factor receptor superfamily. both receptors are involved in the activation of different intracellular signal transduction cascades. small peptides retaining the most essential elements of ngf may be useful either as agonist or competitive antagonist in the treatment of several neurodegenerative desease and nerve injuries. the n-terminal fragment of ngf was previously demonstrated to be an important determinant for affi nity and specifi city in the binding to trka. crystal structure of ngf-trka complex, site-directed mutagenesis studies and substitution of individual amino acids, contributed to identify within the ngf molecule the most relevant domains for its biological activity in the loop-1 (29-35), loop-4 (92-98) and in the nterminal region (3-18). we synthesized twenty peptides mimicking the loop1 and 4 linked together with aminoacid-based spacers (n glycines) or with two unit of 8-ammino-3,6-dioxaottanoic acid with or without the n-terminal region. l1l1 and l4l4 homodimeric loops, moreover l1 and l4 were also synthesized, as single loop, and their biological properties were compared to l1l4 and n-l1l4 activity . the n-l1l4 (hpifhrgefsvadsvsvwvgdctdikgkctgacdgkqc) and l1l4 (ctdikgkctgacdgkqc) peptides showed a good ngf agonist activity at concentration as low as 3 um. both were able to induce differentiation of chick dorsal root ganglia (drg) and to stimulate the tyrosine phosphorylation of trka but not of trkb receptor. in addition the l1l4 peptide was able to induce the pc12 cells differentiation into sympathetic-like neurons. moreover the l1l4 peptide was shown to reduce neuropathic behaviour and restore neuronal function in a rat model of peripherical neuropathic pain, thereby suggesting a potential therapeutic role for this peptide. short proline-rich peptides interact with sh3 domains, exhibiting little or no secondary structure before their binding to the cognate proteintargets. under these conditions the binding process of a proline-rich peptide with the sh3 domain shows unfavorable binding entropy, likely resulting from a loss of rotational freedom on the formation of the ppii helix. with the aim of stabilizing the ppii helix conformation in sh3 binding motifs, in the previous years, we replaced the proline residues of the hpk1 proline-rich decapeptide, ppplppkpkf (p2), either with 4-r-(fp) or with 4-s-(fp) fl uoro-l-proline at different i, i+3 positions. the interactions of the fl uoroproline-peptides with the sh3 domain of cortactin protein were analyzed quantitatively by non-immobilized ligand interactions assay by circular dichroism (nilia-cd), whereas cd thermal transitions were measured to correlate their propensity to adopt ppii helix with their affi nity for sh3. results show that although the introduction of the fp residue stabilizes the ppii helix conformation of peptides in a position-dependent manner, the induction of a stable peptide conformation does not increase the ligand affi nity towards the sh3 domain of cortactin. to explore the effect of electron-withdrawing substituent in the pro residue, the fp residues of p2 were replaced by the natural amino acid 4-r-hydroxy-proline (hyp). unexpectedly, nilia-cd and cd thermal transitions results showed that the hyp-containing peptides exhibit a stable conformation in aqueous buffer and k d values lower than the corresponding fp peptide-analogues. in particular, hyp3 peptide containing hyp residues at i, i+3 and i+6 positions adopts the greater percentage of ppii helix conformation over the entire studied temperature range and shows a k d value comparable to that of the parent p2 peptide. we are now searching for inhibitory peptides able to reduce survivin function in breast cancer cells. we take advantage of the observation that survivin forms homodimers in vivo and have derived short peptides representing the dimerization domain. to enable intracellular expression and visualization of these short peptides, they will be fused to a fl uorescent carrier protein. alternatively, the peptides comprising the dimerization domain will be inserted into a scaffold protein. this allows the presentation of the peptides in a constrained and stabilized conformation. we will analyze the effects of lentiviral expression of these fusion peptides in cancer cells expressing high levels of survivin. the peptides will be analyzed for their ability to inhibit proliferation, cell-cycle progression and/or to induce apoptosis in cancer cells. cd11c/cd18 ( x 2) is a member of the leukocyte integrin (cd18 or 2 integrin) subfamily of adhesion molecules and it is expressed on monocytes, macrophages and granulocytes. its ligands include fi brinogen, ic3b, lps, collagen type i and denatured proteins as well as intercellular adhesion molecules icam-1 and icam-4. icam-4 is a red cell specifi c membrane glycoprotein that was fi rst described as anerythrocyte blood group antigen lw (landsteiner-wiener) and later it was found to belong to the family of intercellular adhesion molecules. the fi rst reported receptors of icam-4 were leukocyte integrins cd11a/cd18 and cd11b/cd18. later it has been reported to bind to several other integrins as well. latest reported interaction is to cd11c/cd18. the binding of icam-4 to integrins expressed in macrophages might clarify some of the controversy concerning the recognition and uptake of senescent red blood cells in spleen. another function of icam-4/ macrophage integrin interactions could be in retaining the maturing red cells in bone marrow until they are ready to be released in the circulation. adhesion between icam-4 and integrins have also been found to be important in different pathological situations and inhibition of these adhesion events could be of important therapeutic value. we have studied the interaction between icam-4 and cd11c/cd18 using peptides derived from both binding partners using pepspot method and synthetised peptides. the inhibitory or activating role of these peptides could beof clinical importance in pathological conditions where abnormal red cell adhesion is observed. the regulatory effects of relaxin in mammals realize via receptors of the serpentine type. in this work we studied the relaxin activating effect on the adenylyl cyclase (ac) activity in the rat tissues and muscle tissues of invertebrates using a peptide strategy. the strategy involved the synthesis of the peptides 619-629 and 615-629, as well as the palmitatemodifi ed peptide 619-629, all corresponding to the c-terminal region of the third intracellular loop (c-icl3) of the human type 1 relaxin receptor lgr7. the peptides 615-629 and 619-629-lys(palm) had a dose-dependent activating effect on the ac activity and gtp-binding in myocardium, brain and, to a smaller extent, skeletal muscles of rat. the 619-629-lys(palm) had a stronger ac effect than the longer peptide 615-629, because of the possibility to be anchored in the membrane by the hydrophobic radical and, hence, to interact with the g protein more effectively. in mollusks and earthworm muscles, the stimulatory effects of both peptides were weak. competitive inhibition of the stimulating effects of relaxin by the lgr7-derived peptides indicates that the relaxin signal in myocardium and brain is transmitted to ac via lgr7 receptor. in skeletal muscles of rat and muscle tissues of invertebrates, the inhibition of ac and gtp-binding stimulating effects of relaxin by peptides was almost indiscernible. it can be concluded that relaxin controls the ac of these tissues via the receptor different from the relaxin receptor lgr7. the stimulating effects of lgr7-derived peptides also decreased in brain and myocardium in the presence of ñterminal peptide 385-394 of mammalian g s -subunit and after cholera toxin treatment. thus, relaxin stimulates ac via lgr7 receptor and g s protein in myocardium and brain, and the coupling between receptor and g s protein is mediated by the interaction of receptor ñ-icl3 and ñ-terminal segment of g s . acknowledgements: supported by rfbi (grant 06-04-48809) and «russian science support foundation». normal tissue function depends on adequate supply of oxygen through blood vessels. angiogenesis is a fundamental process by which new blood vessels are formed and which is highly regulated in healthy individuals. however, many diseases are driven by unregulated angiogenesis. excessive angiogenesis is associated with cancer, rheumatoid arthritis, psoriasis, while insuffi cient angiogenesis results in ischemia or artherosclerosis. many new angiogenic modulators have been developed in the last years, mostly to inhibit angiogenesis but only few peptide-based angiogenic stimulators have been reported. there are data in the literature suggesting roles of small peptides or basichexa peptides as angiogenic modulators like ringseis et al. reporting effects on endothelial cell function (such as ec proliferation) and fazekas et al. describing effects for the latter. endostatin, an endogenous inhibitor of angiogenesis, among its proteolitic fragments contains both, an inhibitor and a stimulator of angiogenesis. we considered it possible that fragments of basic heptapeptide d-phe-cys(-)-tyr-d-trp-lys-cys(-)-thr-nh 2 (tt-232), a strong antitumor agent, could also act as angiogenic modulators. the heptapeptide was developed in our laboratory and has been recently in a clinical trial (phase ii). we synthesised, characterized and tested partially protected di-and tripeptide fragments of it such as boc-d-phe-cys(acm)-tyr-ome, d-phe-cys(acm)-tyr-ome, boc-tyr-d-trp-cyclohexilamide and h-tyr-d-trp-2-adamanthylamide. the biological activity of the compounds was tested in vitro using an immortalized kaposi sarcoma cell line to determine their pro-anti-angiogenic character. to test the angiogenic potential of the best compound, the aorta ring assay was used, which by using intact vascular explants reproduces more accurately the environment in which angiogenesis occurs. in this study we report the synthesis and angiogenesis modulating effects of the peptides. somatostatin is a neuropeptide that regulates several functions of the endocrine and exocrine systems. it also affects cell proliferation and neurogenic infl ammation through a family of g protein-coupled receptors. neurogenic infl ammation plays signifi cant role in the pathogenesis of numerous infl ammatory diseases (such as asthma, arthritis, allergy and migraine). inhibitory effect of somatostatin on infl ammation is well known but the pharmaceutical use of the native peptide is limited due to its broad spectrum of anti-secretory effects and short plasma half-life time. tt-232, a heptapeptide analogue of somatostatin (developed by our research group and it is in clinical phase ii trials) has selective antitumour and anti-infl ammatory effect without regulating other endocrine or exocrine processes. receptor-ligand binding experiments with various analogues of the somatostatin as well as tt-232 [d-phe-c(cys-tyr-d-trp-lys-cys)-thr-nh 2 ] verifi ed that the side chains of tyr, d-trp and lys are important pharmacophoric groups for somatostatin-like biological activity. linear, cyclic and branching derivatives were designed and synthesised using above amino acids to selectively inhibit infl ammatory actions. unnatural moieties also were applied to enhance their enzyme resistance. linear and cyclic peptides consist tyr and d-trp, while ring closed ones contain lys too. branching peptidomimetics have the same, fl exible core [tris(2aminoethyl)amine] and three protected or unprotected amino acids situated in equal positions. the biological activity of the compounds was evaluated by in vitro assay of substance p release and in vivo assay of plasma protein extravasation. the most potent agents strongly inhibited substance p release by 90 -95 % and one of them showed strong anti-infl ammatory activity when administered orally. the structure of the novel compounds and the relationship between their structure and biological activity will be discussed. urotensin ii (u-ii), a potent vasoconstrictor, is found in diverse species, including human. several biological studies indicate that u-ii is the most potent mammalian peptide vasoconstrictor reported to date, and it appears to be involved in the regulation of cardiovascular homeostasis and pathology. in order to elucidate the importance of trp residue for receptor interaction and biological activity recently we have designed, synthesized new analogues where trp7 was replaced with constrained analogues ltpi or dtpi (1,2,3,4-tetrahydronorharman-3-carboxylic acid). the tpi residue was replaced in both agonist p5u and antagonist urantide sequences. on these new ligands we performed biological and nmr conformational studies. the new ligands will be used in further biological investigations of the ut receptor. gnrh analogs as carriers for targeted suicide gene delivery suicide gene therapy represents one of the promising approaches to the cancer treatment. an application of herpes simplex virus thymidine kinase (hsvtk)/ganciclovir system possesses additional advantage due to bystander effect on neighboring cancer cells. overexpression of gnrh receptors in the case of most adenocarcinomas creates the basis of gnrh analogs use as carriers for targeted suicide gene delivery. we investigated different manners of gnrh molecule modifi cation; their infl uence on peptide/dna complex formation and its penetration into cancer cells. analogs, containing nls from large antigen of sv40 were synthesized using combination of boc-and fmoc-chemistry. depending on peptide structure (agonist or antagonist) nls was attached via position 6 or 1 of the natural molecule. the competition experiments demonstrated that internalization of peptide/ dna complex into hepg2 cells is mediated by specifi c receptor binding. moreover, nls/dna complexes, lacking gnrh moiety were unable penetrate cellular membrane. subsequent studies permit to identify the infl uence of analog structure on in vitro effi ciency of suicide gene therapy, followed by acyclovir treatment. it was shown that application of reference peptide gene delivery system damaged about 50% of tumor cells. use of cationic peptide conjugated with rgdf sequence provides high effi ciency of treatment, however can not ensure selective action on cancer cells. gnrh analogs completely suppress tumor growth in vitro due to specifi c interaction of peptide/dna complex with correspondent receptor. the effi ciency of suicide gene therapy depends on peptide structure and is in favor of agonists as compared to antagonists. preliminary data of experiments in vivo on laboratory animals demonstrated practical utility of tested peptides in the course of intravenous administration. thus, it was shown that gnrh analogs containing nls moiety represent promising candidates for the delivery of hsvtk gene into the cancer cells. the structural-functional study of putative grape (vitis vinifera) uncharacterized protein sequences was performed. we developed a special method of computer analysis (1) for this. this method has allowed to reveal new potentially active regulatory oligopeptide sequences yet not investigated experimentally. information on grape amino acid sequences of public databases [2, 3] , computer database erop-moscow (endogenous regulatory oligopeptides) 4. containing the information on structure and functions of known natural oligopeptides and specially created computer programs were used for this. protein amino acid sequences were compared with all known oligopeptide sequences in this method. as a result several tens of grape oligopeptide sequences were elucidated. the similarity of their sequences with the known oligopeptide structures of other biological species was the basis for the prediction their potential functional properties. it has been shown that grape contain putative regulatory oligopeptides possessing functions of antibacterial and antifungal agents, enzyme inhibitors, calmodulin binding structures, rapid alkalinization factors, etc. the primary structure similarity of grape sequences was found not only with plant species but with bacteria, fungi, and animals also. cyclotides are plant derived mini-proteins with compact folded structures and exceptional stability. their stability derives from a headto-tail cyclised backbone coupled with a cystine knot arrangement of the three-disulfi de bonds. taking advantage of this stable framework we developed novel vegf-a antagonists by grafting a peptide epitope involved in vegf-a antagonism onto the stable cyclotide framework. antagonists of this kind have potential therapeutic applications in diseases where angiogenesis is an important component of disease progression, including cancer and rheumatoid arthritis. a grafted analogue showed biological activity in an in vitro vegf-a antagonism assay at low micromolar concentration and importantly the in vitro stability of the linear epitope was markedly increased using this approach. in general, the stabilization of bioactive peptide epitopes is a signifi cant problem in medicinal chemistry and in the current study we have shown the cyclotide framework is ideally suited for such stabilization. cystine rich scaffolds are emerging as valuable templates in drug design and in the current study we have shown that the cyclotide scaffold, with the advantageous features of a knotted disulfi de core and a cyclic backbone, has signifi cant potential in stabilizing a wide range of bioactive peptide epitopes. gonadotropin releasing hormone (pglu-his-trp-ser-tyr-gly-leu-arg-pro-gly-nh2, gnrh) plays a signifi cant role in the controlling of gonadotropins and steroids hormones. a large number of linear gnrh analogues has been synthesized and tested for several medical uses. leuprolide acetate (pglu-his-trp-ser-tyr-(d)leu-leu-arg-pro-nhet, lpa) is a potent gnrh agonist and is used to treat a wide range of sex hormone related disorders, including prostatic cancer, endometriosis and precocious puberty. despite its widespread use, only limited information based on spectroscopic evidence regarding the solution conformation of leuprolide are known. moreover, non crystallographic data is available for the receptor of gnrh (g protein-coupled receptor). the aim of this study was to characterize the conformation of leuprolide and its modifi ed linear analogue (pglu-his-trp-ser-tyr(ome)-(d)leu-leu-arg-aze-nhet) in dmso solution (which simulates better the receptor environment) using nuclear magnetic resonance (nmr) and molecular modeling techniques. by using both nmr and molecular modeling we have characterized the secondary structural preferences of these gnrh analogues. structural determinants of binding to the mu-opioid receptor -an important target in analgesia -attracts great scientifi c attention. many natural and synthetic peptides and peptidomimetics were shown previously to bind to the mu-opioid receptor selectively but there is no consensus about what structure is responsible for such biological activity. no high resolution structure of this receptor is available and the binding site of ligands is not exactly known despite numerous site-directed mutagenesis studies. this suggests that the determination of structural aspects of mu-opioid activity should focus on the ligands. mu-opioid ligands with similar affi nity and selectivity should possess at least one common structural feature in which they differ from other ligands of different affi nity and selectivity. comparative structural analysis of such ligands, considering adequate representation of binding conditions may reveal key features of bioactivity. in this study ten mu-opioid receptor ligands, damgo, tyr-w-mif-1, morphiceptin, endomorphin-1 and 2 and their analogues, possessing different affi nity and selectivity were examined using molecular dynamics. conformational preference of these molecules was determined in aqueous and dmso media which were meant to model different possible binding environments. no structural trend, correlating with previously measured bioactivities was observed in aqueous media. in dmso it was found, that a preference for trans orientation of the tyr1 side chain and gauche (-) orientation of the third aromatic side chain, the free rotation of the phe4 side chain and a high propensity of bent backbone structure is favorable for high affi nity binding to the mu-opioid receptor, while deviations from these criteria results in variable loss of bioactivity. constellation of these four key conformational parameters may be a guiding principle in the future for the design novel mu-opioid receptor ligands. in 5 cui-catalyzed azide-alkyne 1,3-dipolar huisgen's cycloaddition -prototypic "click reaction"-is a recently developed synthetic procedure to obtain cyclopeptides carrying, as a rigid linking unit, the lactam bioisoster, 1,4-disubstituted [1, 2, 3] triazolyl ring (1). we have recently reported the synthesis and conformational analysis of the 1,4-disubstituted[1, 2, 3] triazolyl containing cyclopeptide derived from the sequence of the potent i-to-i+4 side chain-to-side chain lactam-containing antagonist of parathyroid hormone-related peptide (pthrp) 2.. the conformational properties of triazolyl-containg peptide were compared to those of the corresponding lactam analog. cd and nmr studies revealed that, despite a slight difference of the backbone arrangement, triazolyl containing cyclopeptide and lactam-containing cyclopeptide share a common orientation of the side chains (3). here we present the structural study of a new library of disubstituted[1, 2, 3] triazolyl containing cyclopeptides designed to obtain different cycle dimensions and different triazolyl-ring positioning. cd and nmr analysis in different solvent systems shows that both, the dimension of the cycle and the specifi c positioning of [1, 2, 3] triazolyl ring are critical to fully resemble the conformational properties of the potent lactamcontaining antagonist of parathyroid hormone-related peptide (pthrp). the somatostatin (srif) is a cyclic tetradecapeptide, which exerts inhibitory effects on the secretory processes in the endocrine and exocrine systems, and the cell proliferation through somatostatin receptors (sstr1-5). sstrs are distributed throughout human body, not only in normal cells but also in tumor cells. most of the somatostatin analogues developed for clinical use, such as octreotide which act longer than somatostatin are being used in the diagnosis and treatment of endocrine tumors. the use of these analogues as antitumor agents has been limited because of their antisecretory effects and poor oral bioavailability. tt-232 [d-phe-c(cys-tyr-d-trp-lys-cys)-thr-nh 2 ], was reported by keri et al. to have potent antiproliferative activity without antisecretory action 1.. based on the above, we aimed to design and synthesize somatostatin analogues with more potent antiproliferative activity and high oral bioavailability. we synthesized pyrazinone ring containing cyclic peptides (2) and linear peptides which are substituted at the c-terminus lys with hydrophobic and rigid groups. our focus was on the active sequence: tyr-d-trp-lys. we also examined their antiproliferative activity and found that boc/h-tyr-d-trp-1-adamantylamide exhibited the most potent antiproliferative activity higher than that of tt-232. furthermore, on the best analogues we studied dna fragmentation by facs analysis and cellular morphology. the results demonstrated that these somatostatin analogues induced cell death by apoptosis. 6 kobe gakuin universit, japan background and aims: endomorphin-2 (em-2: h-tyr-pro-phe-phe-nh 2 ) has high affi nity and selectivity for the mu opiod receptor (1). we focus on the pro residue, which imposes strong restraints on the conformation of the peptide chain or induces cis-trans isomerization of x-pro bonds. we substituted the pro with 2-azetidinecarboxylic acid (aze) or piperidinecarboxylic acids (pip) to increase or decrease the conformational fl exibility. in this paper, we deal with the synthesis of em-2 analogues containing aze and pip and the evaluation of the biological functions of the analogues on the opioid receptors. methods: the synthesis of peptides was achieved according to the procedure of okada y. et al. (2) the fi nal products were identifi ed by maldi-tof mass spectrometry and elemental analyses. the receptor binding activity of peptides was assessed by radio-ligand receptor binding assay using mu and delta opioid receptors from cos-7 cell membranes expressing each opioid receptors. for the evaluation of the biological function of peptides, the gpi and the mvd tests were performed. the new analogue of csf114(glc) 1., [pro 7 ,asn 8 (glc),thr 10 ]csf114 is characterized by a type i -turn around the minimal epitope asn(glc), and it was demonstrated to show the highest antibody affi nity in competitive elisa in multiple sclerosis (ms) patients' sera and thus it appears as a promising tool for the detection in patients' sera of specifi c autoantibodies. in previous studies, we synthesized and tested different fragments of this new glucosylated peptide, [pro 7 ,asn 8 (glc), thr 10 ]csf114, identifying the shortest sequence [pro 7 ,asn 8 (glc),thr 10 ] csf114(5-11) able to detect antibodies by elisa in ms patients' sera. this heptapeptide is characterized by thr in position 10 generating a characteristic n-glycosylation consensus sequence. according to the results obtained with the linear heptapeptide in ms, we applied the backbone cyclicization method to develop two backbone cyclic libraries of glycopeptides based on the sequence of the linear hepta active peptide. backbone cyclization is a method that allows obtaining cyclic peptides without changing the natural sequence or the chemical character of the amino acid residues, in order to enhance activity, stability to metabolic degradation, selectivity and bioavailability (2) . the fi rst library contains a gly building unit at the c-terminus and is connected to the n-terminus by linker of various lengths (n=2, 3, 4, 6) . in the second library, his 9 of the heptapeptide is replaced by gly building units with various alkyl chains (n=2, 3, 4, 6) [fmoc-n (n alloc(n-alkyl))gly-oh] that is connected to the n-terminus by linker of various lengths. twenty cycloglycopeptides were synthesized, and screened by competitive elisa s on ms patients' sera to select the most bioactive cyclic glycopeptide. peptide nucleic acids have become, arguably, one of the most interesting of dna mimics. owing to their high chemical stability and resistance towards nucleases and proteases, they are very attractive as antigene/ antisense agents, molecular biological tools and for genetic diagnosis. the lack of charge and polar groups in the backbone decrease their solubility in aqueous environment and their ability to cross cell membranes, reducing their performance in in vivo applications. in order to overcome these problems -to improve solubility, increase affi nity and specifi city of binding, a number of analogues were synthesized. this study describes the synthesis of pna-monomers on the base of non-protein amino acids analogues of basic amino acids lys and arg. nucleobases are the second residue we have selected. studies will include replacement for example of the uracil, with the fl uorinated analogue. growing evidences indicate that n-glycosylation is a co-translational modifi cation that, either native or aberrant, may play a fundamental role in a large number of biological events. in particular among postand co-traslational modifi cations glycosylation plays a crucial role in the immune system. in fact, almost most of all the key molecules involved in the immune response are glycoproteins. there are growing evidences of defects in glycosylation with diseases that assets to pathway oligosaccharides as code words. in previous studies, we demonstrated that the presence of a -dglucopyranosyl moiety on an asn residue at position 7 of csf114(glc) is fundamental for auto-ab recognition resulting the fi rst multiple antigenic synthetic probe (msap) able to detect autoantibodies in ms patients' sera. up to now we have investigated the carbohydrates infl uence and specifi city in ms antibody recognition introducing several glycosilated building blocks in the msap sequence (i.e. glc, man, glc glc, gal, glcnac on the side chain of ser, thr, asp, glu, hypro) 1.. due to microheterogeneity and the extremely high specifi city of carbohydrateprotein interaction we included in our library screening, the ribose. we report the synthesis of new asn-derivatives bearing on the side chain ribose rib and rib linked by an n-glycosidic bond and protected for spps.these building blocks introduced in the csf114 -turn scaffold lead to the new ribosylated peptides contributing to the library of glycopeptides to fi shing out families of autoantibodies specifi c for different autoimmune diseases. fraczyk, justyna; kujawska, nina; kaminski, zbigniew, j. we designed and prepared supramolecular structures formed from nlipidated oligopeptides immobilized in the regular pattern on the cellulose surface which are able to specifi c binding of ligand molecule. due to the conformational fl exibility of the fragments forming the supramolecular structure, the shape and prosperities the binding cavities are adjusted the most effectively to requirements of the guest molecules. the previous studies documented that process of binding guest molecules is highly selective, reversible and competitive. therefore, we supposed that under favorable circumstances the structures could operate as catalysts if suitable molecular fragment are included inside the binding pocket. in order to verify this hypothesis we prepared library of supramolecular hosts with catalytic triade: his asp(glu) ser, incorporated into the binding pocket 1.. for the fi rst generation library the rate of hydrolysis of p-nitophenyl esters of n-protected amino acids was measured by spectrophotometric determination of liberated p-nitrophenole in buffered, aqueous methanol and compared with appropriate data obtained in the absence of catalytic structures. the most active catalyst were selected from the library and their stability, selectivity and ability for re-use was studied. for the second generation of library the stereoselectivity of artifi cial esterase we present here a new technique for identifying very small quantity of peptide mixtures that are selected out from a peptide library in solution, by using multi-component fl uorescence labeling. the technique is basically a modifi cation of positional screening method associated with a 1:1 correspondence between the amino acid at the i-th position and the type of the fl uorescence lebel at the n-terminal. 2-dimensional fl uorescence spectroscopy was employed for identifying the fl uorescence labels in the mixture of peptides that bound to target cells or target proteins. the results of the new screening method will be presented for peptides that specifi cally bind to human cancer cells. show increased biological activity in a dimeric form1. in recent years there have been signifi cant efforts to obtain minimized versions of naturally occurring proteins such as dimeric dna binding proteins which retain their function. the gcn4 basic region peptides were connected trough a disulfi de bond to give a dimer which specifi cally bound the ap1-dna sequence. dimeric peptides and proteins were obtained also by non covalent interactions.2 in this work we propose a strategy for obtaining by expressed protein ligation (epl), one pot protein homodimers covalently connected at the c-terminus. the synthetic strategy was extended also to the synthesis of heterodimers. epl is a protein engineering tool for the chemo and region-selective modifi cation of proteins based on the use of intein containing constructs.3 in this work dimers were obtained by reacting a new bi-functional linker with carboxyl-activated polypeptides. we synthesized a linker containing two cysteines in a n-terminal-like position, separated by an ethylendiamine spacer, and obtained thioester proteins by intein mediated splicing reactions. this strategy affords chemically stable dimeric proteins. the linker can be easily modifi ed at need, changing the lenght and rigidity of the spacer between the cysteines. this strategy has potential in biochemical and bioorganic applications, for obtaining minimized and/ or modifi ed natural proteins and for joining two different proteins at the c-terminus position. this technique will be extended to the synthesis of dimeric proteins mimicking the transcription factor ap-1. 1 previous studies showed that csf114(glc), 2,3 a designed glycopeptide characterized by a -d-glucopyranosyl moiety, can detect and isolate specifi c autoabs in sera of a signifi cant number of ms patients. this synthetic ag could be considered a mimetic of aberrantly glycosylated myelin proteins triggering autoimmunity in ms. myelin oligodendrocyte glycoprotein (mog) is considered a putative autoag in ms. 4 our aim is to obtain mog properly glycosylated to characterize the molecular mechanisms of ab-mediated ms and to design new antigenic probes to detect autoabs as biomarkers. production of specifi c glycoproteins may benefi t from a chemical approach, such as expressed protein ligation (epl) a protein engineering strategy useful to introduce noncanonical amino acids and biological probes into proteins. 5 epl allows synthetic and recombinant polypeptides to be chemoselectively and regioselectively joined together. the recombinant rmog ed (1-97), obtained as c-terminal thioesther by protein splicing, will be ligated to the peptide fragment [gly 103 ,a sn 104 (glc)]mog ed (98-117) bearing a cys residue at the n-terminus. an alternative strategy exploits the selective reaction between a glucosyl iodoacetamide derivative and the cys free thiol of a protein. 6 a site directed mutation has been performed on rmog to introduce a cys residue at its native site of glycosylation. the semi-synthetic proteins will be tested by elisa using ms patients' sera. automation is an identifi ed goal in the peptides r&d at lonza. this approach would facilitate and accelerate peptide production. this is highly desired within peptides r&d, where the need exists for rapid synthesis of peptides to fulfi l iso and gmp projects requirements. automated peptide synthesisers are available on the market but many have limitations which make them inappropriate for lonza (e.g., low scale, coupling systems limitation, pre-activation procedure at low temperature). furthermore, there is no obvious standard instrument for scalable spps equipped with pat. ge healthcare provides scalable and complete solutions for automated solid-phase synthesis of oligonucleotides from small research amounts to full commercial production (1 mol-1mol) based on the äkta, oligopilot 400 and oligoprocess™ platforms. the unicorn™ software provides control and monitoring of the processes. ge healthcare synthesisers are designed around fl ow-through column reactors, giving faster kinetics and lower solvent and reagent peptide biotechnology and diagnostics consumption compared to batch synthesisers. based on experience with scale-up of oligonucleotides, ge healthcare and lonza are confi dent that the fl ow through-column technology can be scaled up for peptides to a cost effi cient process. this is a strong argument that the potential of the äkta platform as a peptide synthesiser should be explored. ge healthcare was approached by lonza to develop a peptide synthesiser based on the äkta platform instrument to compete with the state of the art in the peptide fi eld. the aim is to develop the fl uidics, programming and chemistry methods of the äkta system to lonza's needs for routine production of 0.4g to 2g of crude peptide, and later scale up to 2 kg. this synthesiser, controlled by the unicorntm software, has the capability to perform synthesis using on-line mixing or pre-activation at different times and temperatures of amino acids, coupling reagents, solvents and additives. results from peptides will be presented. neuropeptides are produced from precursor proteins by selective cleavage at specifi c sites. classical biosynthetic cleavage occurs at basic residues due to the activity of a small number of well-known proteases. however, with the discovery and characterization of new neuropeptides, a new non-classical pathway has been described with cleavage occurring at tryptophane, leucine and other residues. neuropeptide-processing peptidases involved in this new non-classical pathway are completely unknown but essential for correct processing of certain neuropeptides. therefore, we are interested in identifying proteases involved in the non-classical pathway using activity-based proteomics. for this purpose, we fi rst designed and synthesized some peptides based on the sequence the mature neuropeptides described in the literature but with an active functional group able to bind the active site of the proteases of interest. bound peptides were labelled with biotin or rhodamine by click chemistry, and the brain and pituitary proteome was then characterized by in-gel analysis and multidimensional nlc-ms/ms. several proteases that may be involved in neuropeptide processing have been identifi ed in this experiments. further studies concerning cloning, protein expression and activity assays will confi rm their role in biological tissues. the incidence of autoimmune diseases continues to increase. although an enormous research effort has been directed in understanding this increment, etiology of human autoimmunity remains enigmatic. primary biliary cirrhosis (pbc) is a chronic cholestatic liver disease characterized by destruction of bile ducts and the presence in the serum of antimitochondrial antibodies (ama positive in 90-95% of patients), directed against the e2, which is one of the three component enzymes of the 2-oxo acid dehydrogenase multienzyme complex family chiefl y pyruvate dehydrogenase complex (pdc-e2). environmentally induced co-or post-translational modifi cations of autoantigens are hypothesized to break immune tolerance leading to self reactivity in pbc 1.. in this context it is possible to take advantage of a unique technology that allows the monitoring of peptide epitope modifi cations that will lead to the identifi cation of altered autoantigens that the human host will recognize as foreign, similarly to what recently reported in multiple sclerosis (2) . our approach will lead to new diagnostic tools that can be used in earlier stage patients and possibly to monitor disease activity. herein we report the synthesis of lipoamide and glyco-peptides characterized by a -hairpin structure with the modifi cation on the tip of the -turn as peptidomimetic of native antigens involved in pbc. in order to identify the best synthetic antigens and to clarify the role of the -turn in exposing the modifi cation we compared the antibody recognition in pbc sera by elisa using the modifi ed peptides in comparison with the proposed autoepitope of pdc-e2 [pdh (44-63) ]. the synthetic antigens were able to detect by elisa autoantibodies in 30% of ama negative sera of pbc patients confi rming that ptm-peptides are useful diagnostic/prognostic tools. alzheimer fs disease is characterized by the abnormal accumulation of amyloid peptide (a ) into extracellular fi brillar deposits known as amyloid plaques. a can self-assemble into soluble oligomers, protofi brils, and amyloid fi brils, and all of these aggregated forms contain signifi cant -sheet structure. the core of a (14-23 residues) containing hydrophobic region is a key to promoting a aggregation. in the previous study, we constructed green fl uorescent protein (gfp) variants which have the core part of a sequence on the surface -sheets. it has been demonstrated that the gfp variants inhibit a aggregation 1.. in this study, we have utilized a small protein, insulin-like growth factor ‡u receptor domain 11 (igf2r-d11) as a scaffold, and a part of a sequence was incorporated into the -sheet surface of igf2r-d11. igf2r-kk and igf2r-ka were designed by substituting two a derived sequences for some amino acids in igf2r-d11 as parallel and anti-parallel -sheet models, respectively, of the a aggregates. these insoluble proteins expressed by e coli. were solubilized by denaturing buffer, and the denatured proteins were refolded in conditions permitting formation of native proteins. after refolding, the proteins were purifi ed as monomers by size exclusion chromatography. we have investigated the interaction between a and the designed protein variants by surface plasmon resonance studies. as a result, igf2r-kk bound tightly to a more than igf2r-ka. inhibitory activities of a fi brillization in the presence of igf2r-kk, igf2r-ka, or wild type igf2r-d11 were evaluated by thiofl avin t (tht) binding assay. it was demonstrated that igf2r-kk inhibited a fi brillization effectively. because of their high structural fl exibility. on the other hand, a 2stranded -structure is presumed to be more stable than a single strand as a fi bril forming intermediate. therefore, the aggregation mechanism of prion protein remains to be further studied on their precursor structures. recently, we have established a novel strategy to determine the amino acid sequence for amyloid formation, based on their essential interactions. a number of fi bril forming peptides at positions from 160 to 230 in the amino acid sequence were obtained by our calculation method. in order to confi rm whether aggregates of the candidate peptides consist of the 2-stranded -structure, a series of peptides consisting of g10 residues-turn-10 residues h were prepared. several candidate peptides, of which regions are 166-187, 168-189, 170-191, and 178-199 , showed the typical enhanced fl uorescence intensities in the thiofl avin t-binding assay, suggesting the amyloid formation. ir spectra of these peptides showed the typical bands corresponding to the -structure. in addition, the peptide (178-199) exhibited a shoulder band at 1654 cm -1 in ir spectrum, refl ecting a turn structure. these results revealed that the amyloids of the peptide (178-199) are constructed with the 2-stranded -structure, which may be formed by intra-molecular hydrogen bonds. in conclusion, the results obtained here indicate that the sequence from 178 to 199 could be a key region for the transition from a normal to an abnormal structural state. single-molecule force spectroscopy provides a powerful tool to investigate biomolecular interactions. a different approach measures forces required for breaking a bond in a differential format by comparison with a known reference bond of dsdna (1). here we apply this molecular force balance to the integrin v 3, which is over expressed in ovmz-6 cells. this protein interacts with ligands containing an -arg-gly-asp-(rgd) motive. ligands containing the rgd sequence were made by peptide synthesis and linked to a gcn4-peptide using a peg-spacer. for detection carboxyfl uoresceine was introduced to a lysine side chain between both peptides. the gcn4-peptide can be recognized by specifi c anti-body fragments (2) and can act as a reference bond. these force balances was linked to a pdms-surface, whereas the reference bond is attached to the surface and the rgd-peptide is accessible to the integrin. when the pdms-stamp gets in contact with the ovmz-cells, the interaction of ligand and receptor can occur. by applying a force at the stamp the force balance is stretched and the weaker bond brakes. investigation of the fl uorescence-level on stamp and cells enables the localization of the balance construct and thus an estimation of the bond force. first stamp-experiments on living cells showed that the gcn4anti body interactions are too strong to act as a reference system. since the binding forces of dna are well investigated by afm and small dna-molecules were already used as force sensor, dna can act as a more sensitive reference. thus in a further approach the rgd-peptide was linked to a biotinylated peg. this enables the application of short double-stranded dna as a reference bond via a fl uorescence-labelled streptavidin. using this system the force balance could be transferred to the v 3 integrines located in the cell membrane of ovmz-cells. nanogaps, which allow making electrical contact to structures on the nanoscale, are increasingly used for the preparation of biosensors. the positioning of the synthetic or biological species inside the nanogap must be controlled to reach optimal electrical or detection properties. (1) (2) (3) in this context, the chemical properties of the layer between the electrodes is of prime importance, since they will impose the imbibition of the nanogap and permit the formation of chemical bonds between the molecules of interest and the substrate. thin fi lms can be characterized by a variety of physical or chemical methods. however, the characterization of the chemical properties of nanogaps is complicated because of the small size of the substrate delimited by the electrodes. in this context, novel experimental tools are needed for probing rapidly the chemical reactivity of nanogaps. we show here for the fi rst time that a specifi c functional group in a 30-90 nm nanogap can be detected by combining peptidecapped gold nanoparticles and electrical detection.(4) a semicarbazide layer and semicarbazone chemoselective ligation was used in this proofof-concept study, which thus required the preparation of stable peptidecapped gold nanoparticles modifi ed by aldehyde groups and control gnps derivatized by amide groups. the chemoselective insertion of gold colloids into the nanogaps led to current increases from 2 to 4 orders of magnitude, in accord with the number of gold nanoparticles in the nanogaps detected by scanning electron microscopy. 3 we present the electrical detection of immunoglobulin g (iggs) from human serum using a nanogap-based biosensor. the detection method is based on the capture of iggs by a probe immobilized between gold nanoelectrodes of 30 to 90 nm spacing. the captured iggs are further reacted with secondary antibodies labelled with gold nanoparticles (gnps). insertion of gnps into the nanogap resulted in increasing the conductance through the nanogap. the use of a chip with ninety nanogaps enabled the calculation of a quality factor for the detection which, coupled with a non-linear regression analysis of the data, easily discriminated specifi c and differential capture of human antibodies by arrayed probes. we obtained a 500-fold higher quality factor with protein a compared to goat anti-murine antibodies. this method can be applied, through these proof-of-concept experiments, to the detection of protein-protein interactions in biological samples. in the past several decades, hundreds of peptides have been identifi ed which have specifi c biological activity and are highly potent in in vitro assays but lack the prerequisite pharmacokinetics to become effi cacious human therapeutics. we have developed a novel antibody-based platform technology that provides an improved pharmacokinetic profi le for biologically active peptides, resulting in a long duration of action. one such mimetibody™, cnto 528, is a novel erythropoietin (epo) receptor agonist. although cnto 528 bears no sequence homology to erythropoietin, it is a potent erythropoietin receptor agonist, rescuing epo dependent cells from apoptosis in vitro and stimulating erythropoiesis in vivo. studies were done in normal rats to explore the pharmacodynamics and pharmacokinetics of cnto 528 in normal rats and to demonstrate its effi cacy in rat models of anemia. in vitro, cnto 528 was approximately 10 fold less potent than rhepo in stimulating the growth of ut-7epo cells. despite this lower in vitro potency, when compared to rhepo and darbepoietin in normal rats, a single subcutaneous dose of cnto 528 resulted in a longer-lived reticulocytosis and longer-lived increase in hemoglobin. also, cnto 528 caused only minor changes in red cell distribution width (rdw) or mean cell volume (mcv) and led to the release of mature reticulocytes. we have also shown that cnto 528 was effi cacious in rat models of anemia and in a rat model of pure red cell aplasia. taken together, our data show that cnto 528 is a novel stimulant of erythropoiesis in rats. this platform has been applied to other biologically active peptides as well and has proven to be a robust platform for enhancing the pharmacokinetics of peptides that would otherwise be rapidly cleared. cell penetrating peptides (cpps) have been recognized as promising tools for the delivery of different therapeutic molecules. previous studies in our laboratory have shown that the s4(13)-pv peptide accumulates inside cells very effi ciently through a rapid, dose-dependent and nontoxic process. formulations based on the s4(13)-pv cell penetrating peptide presented great potential for the delivery of plasmid dna, which may prove useful for gene-based therapies. in the present work, we aim to 1) investigate the relevance of the dermaseptin-derived sequence and of the nuclear localization signal to the effi ciency of the overall process of plasmid dna delivery by the s4(13)-pv and related peptides; 2) compare the potential of the s4(13)-pv peptide to mediate plasmid dna delivery with that of the extensively studied tat cpp. a comparative analysis of the transfection effi ciency mediated by the systems based on the s4(13)-pv, reverse nls and scrambled peptides was performed in tsa and hela cells. in general, for both cell lines, the reverse nls peptide mediated transfection at effi ciencies comparable to those observed for the s4(13)-pv peptide. however, transfection mediated by the scrambled peptide was signifi cantly less effi cient than that obtained for the s4(13)-pv and reverse nls peptides. to compare the biological activity of the s4(13)-pv with that of the tat peptide, we transfected tsa cells with various s4(13)-pv-and tatbased formulations. our results have shown that both peptides enhanced the activity of cationic liposome-based systems. above a threshold peptide/cationic liposome/pdna charge ratio (10/1/1), the enhancing effect was independent of the peptide used, although for the lowest charge ratios, this effect seemed to be more relevant in the case of the s4(13)-pv peptide. higashi, nobuyuki; kawamura, yoko; koga, tomoyuki doshisha university, japan the aim of tissue engineering is to replace failed organs with new functional tissue and organs. to realize this, materials are needed, which can direct the growth of cells to generate new tissue. to control and direct cell behavior, a defi ned biomimetic environments is needed, which surrounds the cells and promotes specifi c cell interactions. the rgds sequence has been recognized as the cell attachment site of the natural extracellular matrix. the purpose of this study is to fabricate rgdscarrying nanoscaffords towards cell adhesion using a self-assembling technique. here we synthsize two types of rgds-based materials, one of which is an amphiphilic triblock peptide composed of leu and lys (rgds-l4k8l4) that has been revealed to self-assemble into ƒà-sheet nanofi ber under specifi c conditions 1., and another one is a rgdsended polystyrene (pst-rgds) that is a typical artifi cial polymer. these peptide and peptidomimetic were prepared by solid phase synthesis using fmoc-chemistry and by coupling fmoc-chemistry and atom-transfer radical polymerization (atrp) method, respectively. rgds-l4k8l4 was found to self-assemble into ƒà-sheet-based nanofi ber at ph 9.6, and by lowering ph the conformational change from ƒà-sheet to random coil structure was induced, giving no aggregation of the peptide. when mouse nih/3t3 cells were seeded on the rgds-l4k8l4 nanofi bercoated plate, successful cell adhesion and spreading were observed, but not for the rgds-l4k8l4 random coil-coated plate, indicating the importance of the rgds sequences densely and regularly located at the nanofi ber surface. the utility of pst-rgds nanofi lms will be discussed, in comparison with that of nanofi bers. the role of deiminated protein antigens in the diagnosis of rheumatoid arthritis autoantibodies directed against citrulline-containing peptide (acpa) have high specifi city of rheumatoid arthritis (ra). citrullinated proteins are formed by posttranslational modifi cation, namely by deimination of arginine residues in protein sequences by peptidylarginine deiminase enzymes (padi). autoantibodies to deiminated (citrullinated) proteins are the most specifi c serological markers of rheumatoid arthritis. rheumatoid arthritis symptoms develop gradually, and it is diffi cult to precisely date the beginning of the disease. these antibodies are detectable already years before the fi rst clinical symptoms of the disease. the aim of our study was to identify the epitopes of vimentin and fi laggrin derived-peptides targeted by ra specifi c antibodies to provide further information about the nature of the initial autoantigenic substance. we used conventional solid-phase peptide synthesis (fmoc strategy) carried out on "multipin ncp" (chiron mimotopes peptide system) non-cleavable kit. identifi cation of epitope structure of antigenic proteins represents one of the major applications of these technologies. the peptides were prepared in duplicates. citrullinated peptides and the nonmodifi ed counterparts containing arginine instead of citrulline residues were synthesized in order to compare their respective reactivities. in the "indirect" elisa experiments the presence of acpa was determined using serum samples of ra patients and healthy blood donors. this series of experiments effi ciently identifi es citrullinated epitopes of fi laggrin and vimentin as a potential antigenic target for ra specifi c antibodies. the determination of epitopes of these proteins could be important for the development of appropriate diagnostics in this most frequent human systemic autoimmune disease. at the present time the human lysosomal enzymes cathepsins b and l, cysteine proteinases of c1 family, attract great attention. they not only take part in protein degradation but also appear to play role in other important physiological processes, for example, antigen presentation, caspase-independent cell death and involved in different diseases such as osteosarcoma, acute pancreatitis, tumor invasion and metastasis. but selective detection of these enzymes is diffi cult because determination of their enzymatic activity is carried out using substrates also correspond to specifi city of trypsin-like enzymes. the chemo-enzymatic synthesis of selective substrates of cysteine proteinases of c1 family was developed. these compounds are chromogenic glp-phe-ala-pna (i), glp-val-ala-pna (ii) and fl uorogenic substrates abz-phe-ala-pna (iii), glp-phe-ala-amc (iv). peptides were obtained in preparative quantities and were characterized by the data of amino acids analysis, hplc, mass-spectrometry, spectrophotometry (i and ii) and fl uorimetry (iii and iv). the specifi c activities of cathepsins b, l and similar enzymes of plants papain, bromelain and fi cain were determined using synthesized substrates and commercial available substrates z-phe-arg-pna, z-arg-arg-pna and bzl-arg-pna. the specifi c activities of enzymes of other classes -serine (chymotrypsin, trypsin and subtilisin), aspartic (pepsin) and metalloproteinases (thermolysin) -were also defi ned with these substrates. it was shown, that glp-phe-ala-pna, glp-val-ala-pna and glp-phe-ala-amc are not detectable cleaved by enzymes of other classes. acknowledgements: this work has been supported by rfbr grant ¹ 06-03-33056à. to implement a new electrochemical biosensor for autoantibody detection in ms we used csf114(glc) analogues, properly modifi ed at n-terminus with ferrocenyl and ferrocenyl-thiophosphine derivatives, as "electrochemical probes" in cyclic voltammetry (2) . the electrochemical properties of ferrocene, coupled to thiophosphine ability to build simple monolayers on gold surfaces, allow peptides to be anchored on the working electrode used for detection. in particular, 4-fcphp(s)abu organometallic amino acid was specifi cally designed to be used directly in solid phase peptide synthesis. the organometallic moiety introduced in the new glycopeptides did not affect autoantibody recognition as demonstrated both in sp-elisa and in inhibition experiments. an electrochemical monitoring was able to detect interactions of the modifi ed glycopeptides with isolated antibodies from ms patients' sera. we demonstrated a detection sensitivity comparable to elisa method. therefore, the new electrochemical probes can be proposed to characterize autoantibodies as biomarkers of multiple sclerosis by a simple, rapid, and reproducible cyclic voltammetry-based diagnostic methodology. troponin is a structural protein complex, located on the thin fi lament of the contractile apparatus. it is composed of three protein subunits: troponin i (24kda), troponin c (18kda), and troponin t (37kda) and exists as isoforms specifi c to the cardiac and skeletal muscle cells, respectively. cardiac troponins are released in the peripheral blood during irreversible cardiac muscle damage in a time-specifi c manner. rapid troponin elisa assays based on the production of specifi c antibodies against the whole complex or individual subunits have been shown to possess suffi cient sensitivity and specifi city for use in the emergency departments. however, their usefulness sometimes is limited by various factors, such as the selection of epitopes for antibodies production, derived from cardiac troponins representing high homology against the skeletal isoforms, interfering blood factors etc. aiming to contribute in the fi eld of developing highly sensitive and specifi c reagents for the detection and isolation of cardiac troponins in the sera of patients with cardiovascular diseases, we selected epitopes derived from the cardiac isoforms for production of antibodies, mainly based on their predicted immunogenicity, the minimum homology against the skeletal isoforms and the lack of interferences of the produced antibodies with various blood factors. the selected sequences were conjugated to the tetrameric sequential oligopeptide carrier (soc 4 ), either by the classic solid phase step-by-step methodology or by chemoselective ligation reactions and the resulted conjugates were used as immunogens for releasing anti-troponin specifi c antibodies. the performed elisa experiments revealed the high affi nity and specifi city of the produced anti-troponin antibodies against the native protein. a chemical reverse approach for autoimmune diseases diagnosis alcaro an increasing number of individuals throughout the world is affected by autoimmune diseases, a large and diverse group of disorders that are categorized by tissue injury or pathology. although the incidence and prevalence of individual autoimmune diseases are not high, the population burdens of the disease are large and underestimated. thus, reliable diagnostic/prognostic tools are necessary for an early diagnosis and for monitoring disease activity. in this scenario, we proposed a 'chemical reverse approach' based on the use of patients' sera to screen synthetic modifi ed peptides. we showed that this approach could lead to the effective identifi cation of specifi c probes able to characterize highly specifi c autoantibodies as disease biomarkers of highly relevant autoimmune diseases such as multiple sclerosis and rheumatoid arthritis [1, 2] . toscana biomarkers is a r&d company involved in the application of the "chemical reverse approach" to different autoimmune conditions for the development of innovative diagnostic/prognostic tests. as a number of autoimmune diseases have been associated with posttranslational modifi cations, which alter the function and immunogenicity of protein/peptide antigens, we are synthesizing and screening focused peptide libraries based on post-translational modifi ed amino acid, conformational and minimal epitope diversity. lead compounds can be thus used as antigenic probes for specifi c recognition of autoantibodies as biomarkers of diseases in the set up of diagnostic/prognostic assays (3). autoimmune diseases are considered now as a plague. in fact some autoimmune diseases previously considered rare are actually increasing their frequency because of an earlier diagnosis (e.g. celiac disease). possibly also an environmental factor (bacterial and/or viral infection) should contribute to autoimmune diseases. the idea we have been investigating for years and more recently proposed by others, is that aberrant post-translational modifi cations, i.e. glycosylation, deimination etc., create neoantigens triggering autoantibodies. this could explain why proteins (both recombinant and isolated), components of target organs or tissues, are failing. anyway, it is evident that one single biomarker will never enable to reach successful diagnostic & prognostic tools. on the contrary, synthetic peptides specifi cally modifi ed (with sugars, citrulline, lipoyl moieties, etc.) are interesting tools to fi shing out of patients' sera these autoantibodies. we have recently reported that this can be effi ciently done following a "chemical reverse approach" 1.. we successfully applied this strategy in the development of the fi rst multiple sclerosis antigenic probe [msap] : an n-glucosylated peptide characterised by a -hairpin structure exposing at the best the minimal epitope asn( -glc) involved in antibody recognition (2) . a wider application of our sap is obtained by its citrullinatation and/or galactosylation (3) useful for rheumatoid arthritis or lipoylation for investigating primary biliary cirrhosis. in addition to being able to show the viral infection from a serum sample within a few minutes, at the point of care, the poc assays are inexpensive, easy-to-perform and do not require special equipment or laboratory. as antigen in the poc assay we used a 24-amino acid peptide in four branches of a lysine core, with the kyvtgin sequence in the middle. the peptide antigen was conjugated to gold particles and absorbed to a fabric ribbon and dried. in the test, a serum sample is added together with buffer, and the solution dissolves the conjugate. a positive result is obtained when the antibodies together with the gold conjugate bind to anti-human-igg on the nitrocellulose, and form a specifi c coloured line which can be detected in the test window. serum samples were collected from patients with acute b19 infection, and control samples were drawn many years after infection; additional control sera came from subjects devoid of b19 antibodies. the assay was shown to be stable in accelerated stability study. the conditions for industrial scale manufacturing were evaluated and the sensitivity and specifi city were addressed, whereby the assay proved to be highly specifi c for acute b19 infection. alzheimer's disease (ad) is a chronic neurodegenerative disease characterized by a progressive loss of memory and cognitive decline, for which the aggregation and plaque-formation by the -amyoild (a ) polypeptide has been identifi ed as a key event. recently, unpaired variable domains of llama single chain antibody (vhh) fragments against a have been found to exert considerable therapeutic potential for ad. vhh represents the smallest antigen-binding unit with a molecular size of ~15 kda, compared to ig-heavy and light chain variable domains, fab fragments and complete igg antibodies. human cystatin c (hcc) is a cystein protease inhibitor present in all human body fl uids which has a propensity to co-associate with a -plaques/fi brils, and has plaque-inhibitory properties. using proteolytic extraction and excision of the llama-vhh-a (1-40) immune complex (e.g., trypsin, glu-c protease) in combination with electrospray ionization (esi)-and maldi-mass spectrometry, the a -binding epitope was identifi ed at the middle-carboxyterminal domain of a , a (17-28). an analogous mass spectrometric approach was employed for the identifi cation of the binding epitopes of the hcc-a -complex, using immobilized hcc and a -affi nity matrices. an almost identical minimal epitope to that of the vhh-anti-a -antibody ( a (17-24) ) was found, which binds to a specifi c c-terminal domain of hcc, hcc(101-117) 1.. the identifi ed hcc epitope peptide was found to specifi cally inhibit a -oligomerization in vitro, in agreement with the a -epitope domain interefering with the a -aggregation. the identifi ed a and hcc epitopes represent new lead structures for designing neuroprotective inhibitors of the a -aggregation process, and for molecular ad diagnostics. using peptide arrays to reveal mechanisms of apoptosis aspp2 is a pro-apoptotic protein that stimulates the p53-mediated apoptotic response. the c-terminus of aspp2 contains ankyrin repeats and an sh3 domain (aspp2 ank-sh3 ), which mediate its interactions with apoptosis-related proteins such as p53 and bcl-2. we have used a combination of membrane-bound peptide arrays and biophysical methods to study the protein-protein interactions of aspp2 at the molecular level in order to reveal their possible role in apoptosis. using peptide arrays, we have mapped the binding interfaces of aspp2 with its partner proteins such as bcl-2, nf-b and other proteins that are involved in apoptosis. we then applied the peptide array results to study profoundly the interactions of aspp2 with proteins from the anti-apoptotic bcl-2 family. we found that aspp2 ank-sh3 binds to bcl-2, bcl-xl and bcl-w at two homologous sites in all three bcl proteins tested: (i) the conserved bh4 motif (ii) a binding site for pro-apoptotic regulators. quantitative biophysical analysis of the interaction with the free peptides revealed that the binding was selective, and the bh4 domain of bcl-2 binds tightest to aspp2. we propose a mechanism in which aspp2 induces apoptosis by inhibiting functional sites of the anti apoptotic bcl-2 proteins. the array screening results also served as a basis for docking studies that resulted in binding model for the complex between the full length protein bcl-2 and aspp2 ank-sh3 . we conclude that the use of combinatorial methods such as peptide arrays, combined with quantitative biophysical techniques, can signifi cantly contribute to better understanding of biological pathways. micron-sized monodispersed polystyrene for synthesis, tagging and biological screening of small molecules peptide arrays are useful tools to characterize antibodies, to determine sequence specifi cities of enzymes (e.g. kinases) or to fi nd interaction partners to given peptide sequences. one popular format for such arrays is a cellulose sheet with hundreds of synthetic peptides bound to it. these spot-arrays have been used successfully in a broad range of applications since their invention 15 years ago 1.. a drawback is the use of large reagent volumes and the limited throughput with only one copy of the library. celluspots™ represent a new method [2, 3] that allows the production of hundreds identical peptide arrays from a single synthesis run on individual membrane disks. the peptides are synthesized on a modifi ed cellulose support which is dissolved in a cleavage-mixture after the synthesis. resulting solutions of peptide-cellulose-conjugates are then spotted onto coated microscope slides by conventional spotting techniques. the identical arrays are useful tools for large, parallel sera screening projects. there is a great importance in biological macromolecules integration within electronic devices. this is since these molecules are expected to be suitable both as the active components of electronic devices, or as guides for bottom-up assembly of hybrid structures. the goal of this research is to study the assembly and electronic properties of novel devices that exploit synthetic protein molecules. here we show the fabrication of de-novo protein based diode-like devices. we have chosen coiled coil protein structures, which can adopt two conformational states that differ in their internal molecular dipole. the proteins have been equipped with surface binding groups, typically cysteine thiols, on both ends. dithiol bridges at one side facilitated self assembly on gold surfaces. protected cys residues were placed on the other end that after deprotection will allow successive binding processes in order to complete the device assembly process. characterization of the correct folding in solution has been achieved by hplc and cd. the dependence of the dipole in the dimeric protein has been shown using large scale kelvin probe and high resolution kelvin force microscopy (kfm) measurements. we believe that this work will contribute to the understanding of electronic processes in proteins and may serve as foundation for their exploitation in device confi gurations by making use of the ability to trigger conformational changes in order to control device activity. synthesis of new tetracyclic rgd peptides for specifi c tumor cell targeting. the integrin family of adhesion molecules participate in important cell-cell and cell-extracellular matrix interactions in a diverse range of biological processes. the avb3 integrin is overexpressed in several types of cancer cells and play an important role in angiogenesis as well as in tumour cell migration by interacting with vitronectin on the extracellular matrix mainly through the recognition of the tripeptide sequence rgd. the search for highly selective ligands to target the endothelial cell integrin avb3 is currently the focus of many research groups as they may represent new therapeutics or valuable diagnosis in a number of areas such as metastasis, angiogenesis, arthritis and retinopathy. to date, the cyclic (-rgdfx) peptides and related n-methylated analogues developed by kessler's group are among the most active and selective compounds for the avb3 integrin receptors. for instance, the use of c(-rgdf(nme)v-) is actually evaluated in several clinical trials. therefore, we designed a new class of cyclic tetrapeptides. rgdk peptides were fi rst synthesized by solid phase synthesis then cyclized in solution through a urea bond using n,n'-carbonyldiimidazole. using skmel28 and a549 cells, expressing and non-expressing avb3 respectively, we demonstrate that one of our peptide showed a better internalization than the reference peptide crgdfe. our strategy allowed the coupling of the best cyclic recognition peptide through its extracyclic acidic group to any molecular moieties containing an amino group that makes him a great cadidate for easy multimerization. along this line, different applications are currently developped in our group. design in this study, we described a simple method for aminocylation of unprotected saccharides with mildly activated amino acids esters as potential ace inhibitory activity. as a model reaction, we investigated esterifi cation of sucrose -" royal carbohydrate" with cyanomethyl ester of benzyloxycarbonyl-phenylalanine.the difference of reactivity between the eight hydroxyl groups is a key factor in chemical exploration. in polar solvent as dmf or dmso the primary positions might react faster than secondary ones if the reaction is essentially sensitive to steric interactions. on the other hand, the reaction might be more sensitive to the activity of the alcohol functions. in this respects, 2g-oh has been shown to be the most reactive among eight hydroxyl groups of sucrose. after removal of benzyloxycarbonyl group, the products possess groups which can accommolated in the hydrophobic s1 and s2 subsites of angiotensin i converting enzyme. the free amino group in the amino acid-carbohydrate esters can also serve as good ligands for zn2+ in the ace active site. carbohydrates possess both hydrophobic and hydrophilic groups in their structure and could also bind with enzyme subsites. the measurements of ic50 value of newly amino acyl esters of carbohydrates are in progress. synthetic polyelectrolytes (pe) have been widely used to modify proteins via complex formation and covalent attachment, increasing (or reducing) the immunoreactivity and/or immunogenecity of originally antigenic proteins and improving their in vivo stability with prolonged clearance times. such conjugates seem to be great importance for medicine and immunobiotechnology in particular with respect to drug delivery and vaccine innovation. synthetic peptides are promising candidate vaccines for the control of viral diseases. previous studies with foot-and-mouth disease virus (fmdv) have identifi ed fragments of isolated vp1 protein and synthetic peptides from vpi which stimulate antibody production, albeit of poor neutralizing activity, or are recognized by antivirus antibodies. fmdv is an attractive model with which to study the potential of peptide-based synthetic vaccines. in this study, we sought to evaluate the immunogenecity of a candidate containing 135-161 synthetic peptide epitops of vp1 capsid protein of fmdv. the immunogenic properties of the conjugates were also investigated and the relationship between immunogeneticity and structure formation in the solutions is analyzed. a new high immunogenic protein-polymer complex and conjugates with antibody production, processing relatively prolonged times was obtained. it was obtained that a single immunization of mice with pepeptide bioconjugates without classical adjuvant increased the primary and secondary peptide-specifi c immune response to fmdv. polyacrylic acid (paa) is a well known bioactive polymer (bioadhesive nano-and microparticles, ph-sensitive paa grafted poly(vinylidene fl uoride) membrane bags, ultrafi ne cellulose fi ber surfaces grafted with paa for enzyme immobilization, paa-polyvinylpyrrolidone biopolymeric systems for the treatment of the dry eye, etc. paa, their alkyl-esters and non-toxic copolymers with vinyl pyrrolidones are strong adjuvants for primary and secondary responses and that they are promising alternatives to the mineral oil-based adjuvants presently used in various veterinary vaccines. in this study, the interaction between peptide epitops of vp1 protein of foot-and mouth disease (fmdv) and polyacrylic acid (paa) will be discussed on the basis of the experimental results obtained by the size-exclusion chromatography (sec) with online quadruple detection system: uv absorption (uv), refractive index (ri), right angle light scattering (ls) and viscosity (vis) detectors and the binding coeffi cient, i.e., the binding ratio of peptide to polyacrylic acid. human igg-specifi c binding peptides to distinguish normal and abnormal conformers: applications for igg purifi cation and detection in recent years, human immunoglobulin g (igg) attracts attention as protein of the main format of the antibody medicine. in the purifi cation of igg, protein a originated from bacteria is frequently used as a affi nity ligand, but several problems such as the contamination of endotoxin and the deterioration of protein a by the repeated use were pointed-out in the use of protein a. on this account, the development of low molecular ligands and mimic peptides which can be used instead of protein a has been performed. we have searched for the peptides which specifi cally bind to human igg using a t7 random peptide phage library and, as a result, succeeded in isolation of two kinds of peptides called type i and type ii. type i peptide recognize normal structure of human igg and can be used for the purifi cation and the detection of igg. however, it not only binds to human igg but also to mouse and rabbit igg with comparatively high affi nity. on the other hand, type ii peptide is extremely specifi c only for human igg. a more important thing is that type ii peptide does not bind to igg in serum, but get possible to bind to igg antibody purifi ed by a protein a column. from this result, we elucidated the abnormal structure of igg is generated by acid condition used in the elution from a protein a column and type ii peptide recognizes this specifi c structure. in this presentation, we report the results that the type i peptides functioning as an affi nity ligand instead of protein a can be applied for the purifi cation system of the antibody by improvement by amino acid substitutions and chemical conversion. furthermore, we also report the generation condition of an abnormal conformer of igg structure by ph and the temperature and the effective removal of the generated specifi c conformer by the type ii peptideimmobilized column. acknowledgements: this research was partially supported by a grant of practical application research from japan science and technology agency. the isolation and detection of low abundant enzymes and proteins is one of the most challenging tasks in bioanalytical and pharmacological fi elds, especially if information about their state of activity is required. for this purpose tailored solutions for addressing the members of a protein family are required. peptide chemistry provides established methods to assemble building blocks to construct such molecular probes. we chose matrix metalloproteinases (mmp) for validation of such an approach. this protein family processes and degrades various extracellular matrix proteins and possesses a highly conserved catalytic site with a zinc ion in its centre. most of the known and potent inhibitors are peptidomimetics containing zinc chelating hydroxamate groups (e. g. marimastat). although it binds reversibly, it is potent and active against a wide range of metalloproteinases. it was chosen as synthetically available binding group to target the protein in its active state. it was modifi ed by peptide chemistry in order to introduce multiple functions. depending on the purpose, different reporter groups, photoreactive or cleavage sites were chosen. the probes were tested positively to inhibit various human recombinant mmps using activity assays. mmp were isolated using streptavidin coated magnetic beads and a biotinylated probe. furthermore a photoreactive group was introduced to enhance the binding to the target protein. a covalent interaction was achieved by irradiation of a probe protein mixture, isolation with magnetic beads and cleavage from the beads. pyclock superior coupling reagent for biosensors construction based on peptides coupling onto solid phase polymer atias, danit; abu-rabeah, khalil; marks, robert s. electrochemically biosensors are valuable analytical tools for the monitoring of various biological/ chemical molecule levels, and tremendous research effort has been put into the development of such accurate analytical devices. one crucial aspect in the fabrication of a biosensor is the deposition of biological macromolecules in high amounts with retention of their specifi c activity. among the various deposition methods of biomolecules, such as direct adsorption onto the surface, cross-linking, covalent attachment by carbodiimide chemistry is one of the few methods that allows a stable deposition into a biocompatible environment. hence the urgent need for proteins coupling with high yield to solid surface in the aim of biosensors construction. however the use of carbodiimide as peptide coupling agents was found to be limited in various aspects. thus, during the activation of hindered carboxylic components, such as those involved in bulky protein coupling reactions to solid phase fi bers pyclock was found to be very effi cient for slow coupling reactions and it can be used in excess to assure a complete activation of the carboxylic function. in this study 6-chloro-benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafl uorophosphate coupling reagent (pyclock) was shown to have better performance than 1-ethyl-[3-(dimethyl¬amino)propyl]-3ethylcarbodiimide (edac) and hydroxysulfosuccinimide (nhss) in peptides large sequence coupling to solid phase fi bers. cantel, sonia; valmalle, charlene; subra, gilles; enjalbal, christine; martinez, jean general approaches used in protein identifi cation and characterization involve consecutive purifi cation steps. then the desired protein extract is submitted to mass spectrometry analysis (lc-ms/ms) after enzymatic digestion. technical diffi culties are involved in determining the pmf of a protein particularly in relative low abundance. a typical protein will give rise to at least twenty to thirty peptides after trypsin digestion. not all of these peptides will appear in maldi analysis. one factor that is believed to cause incomplete detection is competition for protonation during the ionisation process inducing ion discrimination. we have recently developed a new technology allowing specifi c labeling of lysine residues in proteins and easy maldi-ms detection and identifi cation of labeled peptides following protein hydrolysis (1) . nhydroxysuccinimide ester of -cyano-4-hydroxycinnamic acid (chca) was used as a labeling reagent to increase maldi signal of lysinecontaining peptides in cytochrome c proteolytic mixture. this original approach enables to discriminate labeled peptides of interest among other abundant peptides. herein, we report the optimization process to investigate the limits of this tool. a defi ned receptor-binding domain (rbd) on the viral spike protein (s) mediates the attachment of sars-coronavirus to its cellular receptor, angiotensin-converting enzyme 2 (ace2). we have synthesized peptide libraries for identifi cation of rbd binding epitopes by surface plasmon resonance (spr) and saturation transfer difference (std) nmr spectroscopy. three dodecapeptides were identifi ed to have signifi cant affi nity to ace2; the best of them had a kd = 85 m . further refi nement yielded a hexapeptide from y438 to l443 (ykyryl) of s that bound ace2 at kd= 46 m. std nmr spectroscopy reveals close contacts of the aromatic tyrosine residues to the receptor. the peptide was also analyzed for antiviral activity using an in vitro assay that measures sars-cov infection of vero cells. at a concentration of 7 mm virus replication was reduced about 100 fold. no replication occurred at peptide concentrations above 10 mm. the peptide blocks the binding site on ace2 that is necessary for the virus to infect the cells. it can be used to design peptidomimetic compounds as entry inhibitors for sars-cov. to exclude other effects that were due to unspecifi c inhibition of viral replication, the peptide was tested during an alpha virus infection of vero cells. no signifi cant inhibition was observed. however, for the human corona virus nl63 that causes severe colds in humans and that uses the same receptor a clear inhibition could be observed comparable to the results obtained for sars-cov. additionally, we have synthesized a hexapeptide library with amino acid substitutions of the chemical lead ykyryl and measured their binding affi nity to ace2 via spr to analyze the importance of the individual amino acids. spr studies indicate an important role of r441. prostate cancer is one of the most common malignancies in men and is responsible for more deaths than any other cancer, except for lung cancer. in the last decade we have developed bradykinin antagonist peptides (b10234, b10238), peptide dimer (b9870, suim-(d-arg-arg-pro-hyp-gly-igl-ser-d-igl-oic-arg)2, suim: suberimidyl; hyp: trans-4-hydroxyproline; igl: -(2-indanyl)glycine; oic: 2s,3as,7asoctahydro-1h-indole-2-carboxylic acid) and four generations of our small molecule, bkm-570 which showed high inhibition against lung cancer in vitro and in vivo. some of the compounds also showed inhibition against prostate cancer. it is well known that prostate cancer metastasizes into bones. while prostate cancer itself has many treatment options, no drugs currently exist for the treatment of bone metastatic prostate cancer. we modifi ed our potent anti-cancer small molecules by incorporating an aminobisphosphonate group to target these compounds to bone. bkm-570, f5c-oc2y-atmp (f5c: 2,3,4,5,6pentafl uorocinnamoyl; oc2y: (o-2,6-dichlorobenzyl)-tyrosine; atmp: 4-amino-2,2,6,6-tetramethylpiperidine) is the fi rst generation of our small molecules. this compound consists of three parts: a, an acyl group; b, a tyrosine amino acid residue and c, an amide group. we introduced aminobisphosphonate or aminobisphosphonate derivatives at position c, and the new n-(bis-phosphonatoalkyl)amide small molecules had anti-cancer activity against prostate cancer metastases in mice. one of these compounds has been shown to be effective against the growth of pre-established human prostate tumors in mouse skeleton through the induction of apoptosis and blockade of survivin expression. the synthesis of these aminobisphosphonate small molecules, the structure relationship studies and the biological activity of these compounds in cultured human prostate cancer cells and animal models will be presented. hayouka novel, potent and selective angiotensin iv short analogues the hexapeptide angiotensin iv: h-val-tyr-ile-his-pro-phe-oh (ang iv) mediates a wide range of physiological actions, including control of blood fl ow and cognitive enhancement. it exerts its effects by binding to at4 receptors, which are widely distributed across tissues. the at4 receptor has been identifi ed as the insulin-regulated aminopeptidase or irap. it has been proposed that ang iv exerts its action by inhibiting the catalytic activity of this enzyme. we have reported that the -homo amino acid containing analog h-2 hval-tyr-ile-his-pro-3 phe-oh (al-11) is a potent, selective and stable ang iv antagonist, in which the 2 hval is responsible for stability and the 3 hphe for selectivity. 1 in this study we report the new ang iv short analogues. the incorporation of erythro-mephe 6 or tic 6 resulted in analogues which have great ability to inhibit the hydrolysis of leu-p-nitroanilide by irap or by ap-n. our data showed that the full ang iv sequence is not necessary for high potency. peptides with pro deletion containing tic or erythro-mephe were even more potent than full ang iv sequence. moreover a peptide design and synthesis of a non -peptide par1 thrombin receptor antagonist, using cyclohexane as template receptors that mediate thrombin action are attractive drug discovery targets because of their involvement in cardiovascular pathophysiology (dysregulation of platelet aggregation and endothelial cell function). the cellular actions of thrombin are, in large part, caused by the activation of proteinase-activated receptors (pars) 1, 3 and 4 (pharm. rev. 54:203). the serine proteinase thrombin cleaves and activates cellular par1 in many pathophysiological settings associated with hemostasis, tissue injury and the proliferation of vascular smooth muscle and tumor cells. in the present study, we synthesized a novel non-peptide par1 mimetic, based on a conformational analysis of the s 42 fllr46 tethered ligand sequence of par1 in order to inhibit the cellular actions of thrombin. the rational design, based on nmr constraints and molecular dymamics, led to compound 1 (fig.1) containing the spatially closed key pharmacophoric guanidyl and phenyl groups, attached to cyclohexane as a template. compound 1, inhibited both tfllr-amide (10 m) and thrombin (0.5 and 1 u/ml)-mediated calcium signaling in a cultured human hek cell assay (j pharm. exp ther. 288:358). aboye, teshome leta 1 ; clark, richard j 2 ; craik, david j. 2 ; göransson, ulf 1 1 biomedical centre, sweden; 2 institute for molecular bioscience, australia the cyclic cystine knot motif, as defi ned by the cyclotide peptide family, is an attractive scaffold for protein engineering (1). however, to date the utilization of this scaffold has been limited by the inability to synthesize members of the most diverse and biologically active subfamily, the bracelet cyclotides. here we describe the synthesis and fi rst direct oxidative folding of a bracelet cyclotide, cycloviolacin o2, and thus provide an effi cient method of exploring the most potent cyclic cystine knot peptides (2) . the linear chain of cycloviolacin o2 was assembled using fmoc solid phase peptide synthesis and cyclized by thioester-mediated native chemical ligation, and the inherent diffi culties of folding bracelet cyclotides were successfully overcome in a single step reaction. the folding pathway was characterized and included predominating fully oxidized intermediates that slowly converted to the native peptide structure. angiogenesis, the process of new blood vessel formation, is important in both physiologic and pathologic situations, and its inhibition can be useful in the fi ght against several diseases, such as tumor development, diabetic retinopathy, etc. one of the key factors in promoting angiogenesis is the vascular endothelial growth factor (vegf), which exerts its biological activity through interaction with specifi c receptors, vegfr-1 (flt-1), vegfr-2 (kdr, flk-1) and vegfr-3 (flt-4) . vegf is over-expressed in all examples of pathologic angiogenesis, and its effects on tumor growth are mainly mediated by kdr. our approach to novel anti-angiogenic drugs is centered on the search for inhibitors of vegf-kdr interaction. directed mutagenesis studies allowed the identifi cation of a surface of vegf recognized by kdr, with residues arg 82 , ile 83 , lys 84 , his 86 and glu 89 , located in a -hairpin of loop 3, identifi ed as essential for the interaction. most residues in this region are also located in the interface of interaction between vegf and some none-humanized phage-displayed antibodies with potent antiangiogenic activity, suggesting a binding to vegf similar to that of vegf receptors. starting from vegf 81-91 fragment (mrikphqgqhi), we have designed different hydrocarbon-bridged analogues to preserve the -hairpin native structure. this communication will describe the solid-phase synthesis of olefi n-bridged (c=c) peptides and their saturated (c-c) analogues. considering that the -turn of native vegf 81-91 is slightly distorted, due to the presence of an extra amino acid residue, in addition to the bridged undecapeptides, two series of decapeptide analogues have also been prepared, just by removing the residues glu 87 or gly 88 . the conformational behavior of linear and bridged-peptides has been analyzed by nmr (noe, 1 h and 13 c chemical shifts). the ability of these peptides to adopt the native vegf -hairpin structure will be compared with their anti-angiogenic activities. integrins constitute a family of heterodimeric, transmembrane cell adhesion receptors which connect cells to the scaffolding proteins of the extracellular matrix. the pioneering observation that integrins -especially v 3 and 5 1 -are hallmarks of metastatic cancer and seriously involved in the process of tumor angiogenesis turned them into attractive targets for cancer therapy. out of that, the inhibition of integrin function is a major challenge in medicinal chemistry. potent ligands are currently in different stages of clinical trials for the antiangiogenic therapy of cancer and age-related macula degeneration (amd). especially the subtype 5 1 has recently been drawn into the focus of research due to its genuine role in angiogenesis.(1) in here, we describe the rational design and the synthesis of high affi nity 5 1 binders and the optimization of their activity and selectivity against v 3 by means of extensive sar-studies and docking experiments.(2) starting from a tyrosine scaffold(3) we succeeded in getting compounds with affi nities in the low and even sub-nanomolar range and selectivities of 400 fold against v 3. the insights about the structure-activity-relationship gained from the tyrosine based ligands could then be successfully transferred to ligands bearing an aza-glycine(4) scaffold to yield 5 1 ligands with affi nities of even sub-nanomolar range and selektivites exceeding 10.000 fold. modeling studies and biological activities of a nonpeptide at 1 receptor angiotensin ii antagonist the octapeptide angiotensin ii (h-asp-arg-val-tyr-ile-his-pro-phe-oh) is the major factor of the renin-angiotensin system (ras) and plays a signifi cant role in the regulation of arterial blood pressure. in the present study, we have modeled a losartan analogue, the non-peptide angiotensin ii at 1 antagonist, 5-butyl-1-hydroxymethyl-1-{[2'-(1htetrazol-5-yl)biphenyl-4-yl]methyl} imidazole (v8). structure activity relationship (sar) and molecular modeling studies indicate close proximity of hydroxymethyl and tetrazole pharmacophoric groups of antagonist v8 and losartan. conformational analysis was performed using a grid scan search in order to derive all the possible conformations from which six energy local minima (syn and anti) were extracted after a cluster analysis. furthermore, these different conformations were superimposed with losartan, where a spatial correlation among the pharmacophoric groups is observed. antagonist v8 showed similar potency with losartan in our in vivo model with anesthetized rabbits and in vitro binding studies to at 1 receptor. at specifi c concentrations compound v8 showed higher affi nity compared to losartan indicating that reorientation of butyl and hydroxymethyl groups on imidazole template allows a better binding to at 1 receptor. 1h-benzotriazolium 1-[bis(dimethylamino)methylene]-5-chloro-,hexafl uorophosphate (1-),3-oxide (hctu) is a non-toxic, non-irritating and non-corrosive coupling reagent [1] [2] . seven biologically active peptides (ghrp-6, 65-74 acp, oxytocin, g-lhrh, c-peptide, hamylin 1-37 , and -amyloid 1-42 ) were synthesized with reaction times reduced to deprotection times of 3 minutes or less and coupling times of 5 minutes or less using hctu as the coupling reagent. no expensive coupling reagents or special techniques were used. total peptide synthesis times were dramatically reduced as much as 42.5 hr (1.8 days) without reducing the crude peptide purities. it was shown that hctu can be used as an affordable, effi cient coupling reagent for fast fmoc solidphase peptide synthesis. human sdf-1 contains sixty-eight amino acids and is a member of the chemokine family of peptides. this long peptide was synthesized step-wise using our quality control conditions in 51 hours. the reaction times were then reduced to deprotection times of 2 x 2 min and coupling times of 2 x 2.5 min, resulting in a total synthesis time of 22 hours. the effect of different resins, resin substitutions and deprotection reagents on the crude peptide purities were compared. a small portion of crude peptide was purifi ed using an rp-hplc column and the mass of the fi nal product was confi rmed with maldi-tof mass spectrometry. references: 1. steven l. kunkel and nuria godessart, chemokines in autoimmunity: from pathology to therapeutics, autoimmunity reviews, 1, 313-320 (2002). pedersen, søren l.; sørensen, kasper k.; jensen, knud j. despite the development of new coupling reagents and solid supports, spps is still often faced with diffi culties in the assembly of long or "diffi cult" sequences, e.g. due to aggregation and steric hindrance giving rise to incomplete reactions. the use of convenient and precise heating with microwaves for spps has gained in popularity as it for many syntheses has provided signifi cant improvement in terms of speed, purity, and yields, maybe especially in the synthesis of long and "diffi cult" peptides. thus, precise microwave heating has emerged as one new parameter for spps, in addition to coupling reagents, resins, solvents etc. we have previously reported on microwave heating to promote a range of solid-phase reactions in spps. here we present a new, fl exible semi-automated instrument for the application of precise microwave heating in solid-phase synthesis. it combines a slightly modifi ed biotage initiator microwave instrument, which is available in many laboratories, with a modifi ed semi-automated peptide synthesizer from multisyntech. a custom-made reaction vessel is placed permanently in the microwave oven, thus the reactor does not have to be moved between steps. mixing is achieved by nitrogen bubling. washing steps are automated, however the activated amino acid derivatives have to be added manually. first, we developed optimized protocols for short cycle times in semi-automated spps with general fmoc chemistry. we utilized a microwave-compatible temperature probe for exact temperature measurements during microwave heating. then we developed protocols for on-resin reductive amination for anchoring of the fi rst amino acids to a bal handle. finally, we used the new instrument and the optimized protocols to assist in the synthesis of a range of diffi cult and long sequences. we believe that these successful syntheses demonstrate that this semi-automated instrument and the methods developed for it, can be an effi cient starting point for spps with microwave heating. investigation of polyelectrolyte-antigenic peptide conjugates by fl uorescence spectroscopy in proteins or peptides, it is possible to localize the interaction between protein and pe at certain protein domains. we investigate covalent binding mechanism of synthetic peptides which include tryptophan residue in the peptide sequence with copolymers of acrylic acid and n-vinylpyrolidone (vp\aa) depending upon the weight concentration ratio of components by fl uorescence method. antigenic peptides are synthesized by microwave assisted solid phase peptide synthesis method. characterization of these peptides are performed with lc-ms. subsequently these crude peptides are purifi ed by preparative hplc system. peptide-polymer covalent conjugation are performed in organic and pbs media. covalent conjugation are carried out by carbodiimide method. carbodiimide is used for the activation of carbonile groups of synthetic polymer which is the binding area of peptides that includes free amino groups. from the analysis of peptide-pe conjugates, it is possible to discuss the mechanism of the conjugate formation and structure of forming particles. according to the fl uorescence analysis results, free peptide which containing trp residue, gives fl uorescence spectrum. however, after the conjugation reaction it is observed that products max decreased and it is characterized as blue shift of max. this indicates that when conjugates formed, trp is completely isolated from aqueous solution. antiangiogenic thrombospondin type 1 repeat analogs: synthesis, structure, and biological activity the thrombospondin type 1 repeat (tsr) has been shown to inhibit angiogenesis and tumor growth in a number of in vivo models of cancer, but the details of this activity, including structure-function relationships, are not well understood. we explore these tsr elements in further detail via structural and biological evaluation of a series of tsr analogs. the tsr domain has a characteristic fold consisting of a two-stranded -sheet and a third 'rippled' strand. three tryptophans on the 'rippled' strand form cation-stacking interactions with two arginines on the adjacent sheet, forming an extended cation-network. in addition its structural importance, it has been postulated that the surface formed by this interaction is a key site for protein-protein recognition. a synthetic approach to the generation of novel tsr analogs, utilizing spps and native chemical ligation, has allowed us to rationally introduce unnatural amino acids in an effort to probe the structural and functional landscape of this clinically relevant protein domain. we have synthesized analogs of the second tsr domain of human thrombospondin-1 (tsr2) designed to probe the importance of the cation-stack. the arginine residues have been replaced by ornithine and citrulline, and thermal denaturation experiments by circular dichroism have been used to estimate stability differences between the mutant tsr2 and the native. taken as a set, these thermodynamic measurements provide insight into the stability afforded by a cationinteraction in the context of a native protein fold. synthetic access to the tsr2 domain also provides the opportunity for the introduction of various modifi cations, including the introduction of aminooxy groups as chemical handles, pegylation for in vivo stability, and biotinylation to create an affi nity caputure reagent for identifi cation of protein-protein interaction partners. the tetradecapeptide bombesin (bbs) has a high affi nity for the gastrin-releasing peptide (grp) receptor. these receptors are overexpressed in human tumors such as breast and prostate cancers. therefore they can serve as targets for in vivo imaging and therapy of these tumors with 99m tc-and 188 re-radiolabeled bbs analogues, respectively. for the radiolabeling, a chelator is attached to the n-terminus of the bbs analogues. our group developed the (n his)ac chelator, which is easy to synthesize and has a very high affi nity for the 99m tc-and 188 retricarbonyl complexes. however, an important part of the injected radiolabeled bbs analogues accumulated in healthy organs, such as liver and kidneys. to improve the pharmacokinetic properties, a bbs analogue was glycated via the lys side chain of the spacer using the maillard reaction. unfortunately, during glycation, overalkylation on the secundary amine of the chelator was observed. therefore, two alternatives for this glycation were examined. the fi rst one was based on the chemoselective reaction between a hydroxylamine (incorporated into the spacer) and an aldehyde (glucose). the second alternative was the cu(i) catalyzed cycloaddition between an alkyne (incorporated into the spacer) and an azide (azido-glucose). these approaches for carbohydration circumvented the problem encountered during glycation via the maillard reaction. glycation by the cu(i) catalyzed cycloaddition was, by far, the easiest way to incorporate the glucose moiety. moreover, after 99m tc labeling, this analogue showed the best biodistribution and the best diagnostic properties of all glycated analogues. an alternate approach to improve the pharmacokinetics consisted of including different polar spacers such as ³hglu, ³hasp, ³hlys, ³hser and -nh(ch 2 ch 2 o) 2 co-between the (n his)ac chelator and the bombesin sequence. in particular the analogues with a negative charge ( ³hglu, ³hasp) in the spacer showed a favorable effect on the pharmacokinetics. determination of binding ratio of hydrofobic peptidepolymer conjugates by using fl uorescamine assay budama battal, yasemin; derman, serap; mansuroðlu, banu; mustafaeva, zeynep yildiz technical university, bioengineering department, turkey non-fl uorescent 4-phenylspiro-[furan-2(3h),1-phthalan]-3,3'-dione (fl uorescamine) reacts readily with primary amines in amino acids, peptides and proteins to form stable, highly fl uorescent compounds (fl uorophors). fluorescamine has been used to detect free amino groups on peptides or completion of coupling reactions in solid phase peptide synthesis and not only in aqueous solution but also in organic solvents and on solids. in this study, peptide epitops of vp1 capsid protein of foot-and-mouth-diseases virus 40-60 amino acid residues (val-lys-ile-asn-asn-thr-ser-pro-the-his-val-ile-asp-leu-met-gln-thr-his-gln-his-gly) were synthesized by sigma. we synthesized the covalent conjugate of 40-60 amino acid residues with copolymers of acrylic acids and nvinylpyrolidone (vp\aa) at different ratio of components (npeptide/ npolymer) and investigated the mechanism of condensation reaction by using different physicochemical analyses as hplc, fluorescence spectroscopy and fluorescamine assay. for determination of binding ratio of hidrofobic peptide-polymer congutages, fl uorescent measurements were performed in the presence of fl uorescamine at the wavelengts of excitation 390 nm and emission 475 nm. when conjugation reaction completed, the amount of free amino groups had decreased and observed that fluorescence intensity had decreased and the estimated degree of primary amino group after conjugation, calculated from fl uorescence spectrums. (4-phenylspiro-[furan-2(3h),1-phthalan]-3,3'-dione) which heterocyclic dione is a reagent for the detection of primary amines on peptides or completion of coupling reactions in the picomole range. its reaction with amines is almost instantaneous at room temperature in aqueous media, organic solvents ect . fluorescamine reacts with primary amines (in peptide, protein ect.) to form highly fl uorescent product (fl uorophors) whereas the reagent and its degradation products are nonfl uorescent (1). this is the basis of a fl uorescent protein assay [1, 2] . fluorescamine is used in many sensitive detection methods, e,g., characterization of poly-l-lysine (pll)/dna complexes post-modifi ed with a multivalent hydrophilic polymer (3), or synyhetic peptide-polymer conjugates. in this study, foot-and-mouth-diseases virus vp1 capsid protein's synthetic peptide epitope which containing tryptophane (trp), 135-161 (p1) amino acid residues (try-ser-lys-tyr-ser-thr-thr-gly-glu-arg-thr-arg-thr-arg-gly-asp-leu-gly-ala-leu-ala-ala-arg-val-ala-thr-gln-leu-pro-ala) were synthesized by using the solid-phase methods. we synthesized the covalent conjugate of copolymers of acrylic acids and n-vinylpyrolidone (vp\aa) with 135-161 amino acid residues at different npeptide/npolymer ratio and investigated the mechanism of condensation reaction by using fluorescence spectroscopy and fluorescamine assay. in the fl uorescamine assay the conjugate solution was measured on a pti qm-2003 steady state fluorescence spectrometer with an excitation wavelength of 390 nm and emission at 475 nm. after the conjugation reaction, the amount of free amino groups had decreased because of binding carboxyl group and observed that fluorescence intensity had decreased. determination of free amino group degree after conjugation, calculated from fl uorescence spectrums. newly developed high strength and chemically stable silica gel based preparative reversed phase packing materials kuriyama, naohiro; shoji, noriko; morishita, kiyoshi; omote, masakatsu ymc co., ltd., japan a new high strength silica gel and a bonding technology based on preparative bulk packing materials for hplc have been developed to provide improved recovery, selectivity, and longer life time for the preparative peptide separations. the novel preparative silica particle was successfully prepared by the new generation process, which allows the higher gel density than typical silica gel and the particle size distribution would be practically mono-dispersed character. for the effective reversed phase peptide separations, pore size and pore volume of these new particle were optimized depending on the molecular weight of peptides. to enhance chemical stability and selectivity under the typical peptide purifi cation conditions, the combination of chemical bonding method and functional group density was optimized for maximum performance. by repeated packing and unpacking of this synthesized gel with large dynamic axial compression column, it was demonstrated that no fi ne has appeared and no back pressure increasing has occurred comparing to commercially available packing materials. also cost effective peptide purifi cation with high loadability, productivity, and recovery was achieved with signifi cant small and large peptides. calcitonin gene-related peptide (cgrp) is a 37 amino acid neuropeptide produced by tissue-specifi c alternative mrna splicing of the calcitonin gene. the cgrp peptide signals through a seven transmembrane g protein-coupled receptor (gpcr) belonging to the secretin receptor family. co-expression of the calcitonin-like receptor (clr) with receptor activity modifying proteins-1 (ramp1) forms a mature cgrp receptor on the cell membrane surface. cgrp is widely distributed in the peripheral and central nervous systems. in the later, cgrp is expressed in trigeminal ganglia nerves and when it is released has potent dilator effects on cerebal and dural vessels. through this mechanism, cgrp is involved in the regulation of blood fl ow to the brain and painsensitive meninges. the pathology of migraine has been associated with the vasodilation effects of cgrp on cerebal circulation. it has been reported that the cgrp peptide consist of an alpha-helical n-terminal region, a fl exible center region, and two putative c-terminal beta-turns. truncation of the fi rst seven residues in cgrp results in antagonists of the cgrp1 receptor (ic50 4 nm), however, cgrp(8-37) is rapidly degraded in plasma. here, we report the iterative design and synthesis of new high affi nity cgrp antagonists with signifi cantly increased plasma stability, and higher affi nity for the human cgrp1 receptor. cordopatis, paul; pappa, eleni; zompra, aikaterini; magafa, vassiliki; diamantopoulou, zoi; lamari, fotini; katsoris, panagiotis university, greece mammalian gonadotropin releasing hormone (gnrh-i) and the sea lamprey gonadotropin releasing hormone type iii (gnrh-iii), belong to the class of conserved gonadotropin releasing hormone peptides. in addition to the classic hypophysiotropic action of gnrh-i, it has been shown that many malignant cells, such as prostate cancer cells, secrete gnrh-i and express the gnrh-i receptor/s. gnrh-iii has no endocrine activity in mammals even at high doses, but has been shown to suppress directly the growth of breast and prostatic cancer cells in vitro. in a continuation of our previous work and in order to study the effect of modifi cations in positions 4 and 6 of leuprolide, an agonist of gnrh-i, on prostate cancer cell proliferation, we synthesized ten new conformationally restricted analogues. d-leu6 of leuprolide was substituted by d-lys, d-or l-glu and ser4 by n-me-ser. we also synthesized fi ve new analogues of gnrh-iii and studied their effect on prostate cancer cell proliferation. asp in position 6 of gnrh-iii was substituted by asn and glu, pro9 was substituted by á-aminoisobutyric acid (aib) and trp3 and/or trp7 by d-trp. peptides were synthesized by the solid phase methodology and fmoc/but chemistry in high yields. results show that the inhibitory effect of gnrh-i analogues on the proliferation of human prostate cancer cells depends on the nature of the substituted amino acid in position 6. incorporation of d-trp in positions 3 and 7 of gnrh-iii preserved its antiproliferative activity, whereas all other modifi cations reduced its activity. mastoparan (mp) is an antimicrobial cationic tetradecapeptide with following primary structure inlkalaalakkil. this amphiphilic -helical peptide was originally isolated from the venom of wasp paravespula lewisii. however some mp analogues (tetradecapeptides with different amino acids sequence) have been also isolated from the venom of hornets, yellow jackets and paper or solitary wasps. they are rich in hydrophobic amino acids such as leu, ile, ala and commonly posses lys residues. mp shows a variety of biological activities such as inhibition of the growth of gram-positive bacteria, activation of mast cell degradation and histamine release, activation of phospholipase a2 and c or erythrocyte lysis. mp is also turned out to enhance the permeability of artifi cial and biological membranes and activate gtp-binding regulatory proteins by a mechanism analogous to that of g protein-coupled receptors. nowadays many mastoparan analogues have been synthesized. some of them contains in primary structure the mastoparan sequence but they loss some of the properties of mp, such as transportan (a cell penetrating peptide, consisting n-terminal fragment 1-12 of galanin and mastoparan at the c-terminus linked with lys residue) or its truncated analogue, transportan-10. in present study we have designed and synthesized several new chimeric mastoparan analogues composed of mp and other biologically active peptides (e.g. galanin, rnaiii inhibiting peptide) and containing natural or unnatural structures. next we examined each of these hybrid constructs as well as natural peptides for they antimicrobial activities. acknowledgement: this work was supported by the university of gdansk grand bw 8000-5-0133-8 cinnamic acid derivatives as esters, amides and glycosides are known to have antibacterial, antiviral, antiinfl ammatory, antiproliferative, immunostimulatory etc. properties. some of them (amide and ester analogues of caffeic acid with natural amino acid esters) exibit stronger antioxidative activity. in particular thiazole, oxazole and imidazole amino acids that may play a key role in biological activities of unusual peptides are also important intermediates for natural product synthesis and peptidomimetics. here we report the synthesis of novel cinnamic acid amides with oxazole containing glycyl-methyl ester as potential antioxidants. the amides were synthesized from sinapic, coumaric, ferulic acids and the corresponding glycyl-methyl ester hydrochloride form using n-ethyl-n-(3-dimethylaminopropyl) carbodiimide hydrochloride (edc) and 4-n,n-(dimethylamino)-pyridine (dmap). their antioxidant activity was evaluated by 2,2-diphenyl-1-picrylhydrazyl dpph (1,1diphenyl-2-pycrylhydrazyl) test. the venoms of marine cone snails contain a complex mixture of peptide neurotoxins known as conotoxins (1) . they are a diverse class of biomolecules that exhibit exquisite selectivity and potency for a wide range of pharmacological targets in the central nervous system, making them valuable tools for studying the mechanisms of neurotransmission and pain. despite their diversity, conotoxins have evolved from a relatively small number of rigid disulfi de bonded frameworks that give rise to a series of intervening loops of amino acids that project outwards from the framework and interact with the receptor binding site. receptor targets are generally determined by the shape of the framework, with the amino acids within the framework infl uencing receptor subtype specifi city. this work aims to use positional scan libraries (2) based on 4/3 -conotoxin frameworks to study the interactions of amino acids with the binding site of neuronal nicotinic acetylcholine receptors, and to discover new ligands that possess greater potency and selectivity for different subtypes these receptors. the disulfi de bond frameworks were formed by oxidizing each mixture in dilute aqueous buffer and their correct formation monitored by cd spectroscopy. an á7 nachr functional assay was used to screen each mixture to determine the activity of each mixture and the results of this assay were used to design a series of individual candidates for use in further structure activity relationship studies. melittin effect on the sensory neurons prelevated from double transgenic vs. normal mice melittin is a 62 aa peptide. it is the active compound of bee venom and a powerful stimulator of phospholipase a2. its antimicrobial effect was extensively studied in the literature, but little is known about its neuroactive properties. melittin increases the cell membrane permeability in excitable and non-excitable tissues, as a result of its interaction with the negatively charged phospholipids. our interest was focused on the algesic properties of melittin and on its potency against the electrophysiological parameters of sensory neurons from dorsal root ganglia. in our study, we have performed patch-clamp studies, by the whole-cell confi guration. primary cell cultures were obtained from double transgenic mice tcr-ha+/ins-ha+ with type i diabetes. this animal model is well-suited for the study of neuropathic pain and it enables us to test the analgesic effects of melittin (0.1-5 m). our recordings indicate that the active-peptide modifi es the algesic profi le of the sensory neurons, in particular by acting against the capsaicinactivated ionic currents. in addition, we have monitored the action of melittin on the i h currents, na + voltage -dependent currents, delayed rectifi er k + currents. a particular interest was focused on the recordings of ca 2+ voltage-dependent currents. the maximal effect was obtained at a dose of 1 m melittin, and at higher doses the active-peptide strongly disturbs the lipid membrane order. in conclusion, the algesic profi le of the sensory neurons from double transgenic mice is signifi cantly modifi ed by the neuroactive melittin in comparison with the profi le of neurons from balb/c mice. these electrophysiological data are important, and are very well corroborated with the increased thermal and mechanical hypersensitivity induced by melittin itself and the bee venom extract, that have been proved their effi ciency in behaviour tests . the toxic effects of amps tested on mammalian cell cultures the amps (antimicrobial peptides) is a class of molecules that belongs to the innate immunity. the ability of some of these peptides to penetrate the microorganism's external membrane and to induce their death suggests that this class of molecules may represent a new generation of antibiotic pharmaceuticals. in our work, we focused on the amps side effects on mammalian cells. the target was to characterize the toxicity of several amps evaluated on in vitro cellular cultures. the following amps were used in our experiments: melittin, magainin i and ii, cecropin a and mastoparan. the viability of cells in cultures was assessed by mts test on chinese hamster lung fi broblasts v79 and on human lymphocytes primary cultures. concentrations in the range of 0.1 -100 m were used. the ic50 value for each type of amp was evaluated from the viability versus concentration curves. haemolysis assays were also performed for these active-peptides. based on ic50 values, melittin has a stronger citotoxicity than magainin i and magainin ii. on the other hand, magainins appears to posses higher haemolytic properties than melittin. the less cytotoxic peptide seems to be mastoparan. these types of results are very useful to characterize the ability of amps to induce toxic effects in human body during the treatment. acknowledgements: financial support by the cnmp research grant pnii number 61-016/2007. the melanocyte-inhibiting factor (mif-1), which is the tripeptide pro-leu-gly-nh2, was isolated in the conventional way from bovine hypothalamus tissue. mif-1 represents a class of naturally occurring opiate antagonists with varying activities in independent situations. the purpose of the present study was to investigate the analgesic activity of mif-1 and its analogues modifi ed at position 2 with unnatural amino acids cav, slys, sleu, sile and snle. the experiments were carried out on male wistar rats (180-200 g). the changes in the mechanical nociceptive threshold were measured by the paw-pressure test using an analgesiameter (ugo basile). mif-1 and analogues (all in dose 1mg/ kg) were administered intraperitoneally (i.p.). mif-1 exhibits a weak analgesic effect and selective affi nity for the -opioid receptor. mifanalogues -mif-cav, mif-sleu, mif-ile and mif-nle were found to have a naloxone-reversible analgesic effects. pacap (pituitary adenylate cyclase-activating polypeptide) exists in two biological isoforms, i.e a 38-amino acid peptide and a shorter form of 27 residues corresponding to the n-terminal part of pacap38. previous structure-activity relationships studies revealed that the n-terminal segment is essential for the activation of the specifi c and selective type i pacap receptor (pac1). in order to clarify the molecular requirements for pac1 activation, we initiated structure-activity investigations of the n-terminal part of both pacap isoforms. in particular, an important library of analogs focusing on the fi rst seven residues (his1-ser2-asp3-gly4-ile5-phe6-thr7) was rationally developed and pharmacologically evaluated for their capacity to bind the pac1 receptor and to induce ca2+ mobilization in chinese hamster ovary (cho) cells stably expressing the human pac1 receptor. ala scan demonstrated that the carboxylic acid function of residue asp3 and the benzyl group of phe6 are essential feature for proper pac1 receptor activation. the inversion of chirality of residues ile5, phe6 and thr7 caused a loss of affi nity whereas the incorporation of d-amino acids in positions 1, 2 or 3 did not affect the binding properties of pacap. moreover, using a n-methyl scan, we showed that the n-terminal domain of pacap is not tolerant to back-bone modifi cations. however, replacement of ser2 by pro did not decrease signifi cantly the potency of the peptide while substitution of this same residue by d-pro totally inhibited the biological activity of pacap. furthermore, other structural constraints reduced the potency of pacap, suggesting that the n-terminal domain needs fl exibility to bind and activate the pac1 receptor. finally, residues 28-38 of c-terminally extended peptides played a favorable role for the binding affi nity towards the pac1 receptor as pacap38 analogs demonstrated highest binding affi nity compared to their pacap27 counterparts. neutrophil elastase-dependent synthetic host defence pro-peptides for the treatment of bacterial infections in cystic fi brosis patients chronic infection and infl ammation play a major role in the pathophysiology of lung disease in cystic fi brosis (cf). besides pseudomonas aeruginosa, there is increasing evidence that other multidrugresistant organisms, such as methicillin-resistant staphylococcus aureus (mrsa) are implicated in the activation of the infl ammatory response in cf patients. cationic host defence peptides, multifunctional mediators of the innate immune system, have been recognised as promising candidates for the development of novel antimicrobial agents (1). most of these macromolecules are produced as inactive prepropeptides and are proteolytically activated to release a c-terminal cationic peptide with antimicrobial activity. we thought to mimic this natural mechanism to target cf pathogens with synthetic host defence propeptides. saltresistant, all-d cationic antimicrobial peptides (2) were conjugated at their n-termini to a polyglutamic acid sequence to compensate the net positive charge of the parent peptide, through a linker selectively degraded by a disease-associated enzyme (neutrophil elastase). in vitro effects of the all-d p18 peptide candidate on planktonic and biofi lm forms of pseudomonas aeruginosa and staphylococcus aureus clinical isolates were assessed. reactivation studies of the propeptide were performed in presence of purifi ed neutrophil elastase and in physiologically relevant conditions, using bronchoalveolar lavage fl uids from cf patients. the nmr structures of the propeptide and active p18 sequences were also compared. these studies confi rmed that the propeptide remains inactive in the absence of neutrophil elastase and that the latter enzyme can potentiate its antimicrobial activity. synthesis and biological activity of a series of aza 3pseudopeptides related to 26rfa, the endogenous ligand of gpr103 26rfa, a novel neuropeptide of the rfamide family, is the natural ligand of the previously orphan receptor gpr103. both 26rfa and gpr103 mrnas are highly expressed in hypothalamic nuclei of rodents, and icv injection of 26rfa induces a potent orexigenic effect in mice. recently, it has been shown that gpr103-defi cient mice suffer from osteopenia. analysis of the 26rfa precursor reveals that it may generate several additionnal rfa-peptides including an n-terminally extended form (43rfa) and a truncated form (26rfa (20-26) ). 26rfa and 43rfa increase dose-dependently [ca 2+ ]i in gpr103-transfected cells while 26rfa(20-26) is about 100 times less potent than 26rfa. molecular modeling under nmr constraints of 26rfa shows that the n-terminal region encompasses an -helix and the c-terminal region adopts a -turn in dpc micelles. the c-terminal peptide 26rfa(20-26) exhibits major distortions of this turn that may be responsible for its weak potency. the aim of this work was to introduce an aza 3 residue in 26rfa(20-26) in order to stabilize the -turn. sequential aza 3-counterpart substitution of amino acids at positions 20 and 21 enhanced by 2 and 7 folds the potency of 26rfa(20-26), respectively. the aza 3-phe 22 analog was 2 times less potent than 26rfa(20-26). replacement of the native ser 23 by the aza 3 surrogate of (ho)homothr (aza 3-hth) to favor hydrogen bonding, generated an analog that was 2 folds more potent than 26rfa(20-26). in contrast, substitution of residues 24 to 26 led to analogs totally devoid of effect on calcium mobilization. as the -turn is centered on the ser 23 of 26rfa, we can assume that the aza 3-hth 23 moiety partially mimics the -turn in the 26rfa(20-26) sequence. these data constitute the fi rst step towards the development of new gpr103 analogs that could prove useful for the treatment of feeding disorders and/or osteoporosis. acknowledgements: supported by inserm (u413 and pnr-re) and the région haute-normandie. olm is recipient of a fellowship from mesr. it is well known that microtubule targeting agents (mtas) are used in clinical treatments as anticancer agents. recently, it has become clear that some mtas also function as gvascular disrupting agents (vdas), which induce tumor-selective vascular collapse. as one of such candidates, a natural cyclicdipeptide, phenylahistin (plh) exhibiting colchicine-like anti-microtubule activity, has been our focus. we have succeeded in synthesizing plh, and performed the structure activity relationship study of its derivatives. from the biological evaluations, according to these results, a highly potent derivative npi-2358 (ic50 = 15 nm, ht-29) was selected, which is now in phase i clinical trial as an anticancer drug in the us. furthermore, we have established the synthetic route of npi-2358 and its derivatives. about 100 analogs were prepared so far and screened by ht-29 citotoxicity assay. as a result, several highly potent derivatives ware developed. although npi-2358 and its derivatives are believed to be recognized around the colchicine binding site on tubulin, the three-dimensional structure of npi-2358 could not be superimposed over that of colchicine. in order to understand the precise binding mode of npi-2358, we developed a highly potent cytotoxic derivative, kpu-244 (ic50 = 3.9 nm, ht-29 cells) with a benzophenone structure. because benzophenone is recognized as a photo-reactive group, we synthesized biotin-tagged kpu-244 derivative as a photoaffi nity probe. since this probe has biological activity enough to function to tubulin, photoaffi nity labeling was performed to analyze the binding site. as a result, irradiation-time-dependent labeling towards tubulin was observed. the labeling was also dose-dependently inhibited by colchicines addition, suggesting that the probe specifi cally recognizes around the colchicine binding site on tubulin. chemical biology study for determining the precise binding site is now in progress. conformational analysis of the new temporin analogues: gln3ta and pro3tl. temporins are antimicrobial peptides (amp €™s) isolated from the skin of red european frog rana temporaria. they are active particularly against gram-positive bacteria, candida species, fungi. they have the ability to bind and permeate both artifi cial and biological membranes. we have recently investigated by spectroscopic means two members of this amp family temporin l (fvqwfskflgril-nh2) and temporin a (flpligrvlsgil-nh2) (1). based on the nmr results, we developed two new analogues of these peptides named pro3tl (fvpwfskflgril-nh2) and gln3ta (flqligrvlsgil-nh2). biological data indicate that pro3tl has a higher antimicrobial activity and a lower hemolytic activity than the native peptide tl. in contrast, gln3ta more hemolytic than the parent peptide ta. the conformational behavior of the new analogues was investigated in membrane mimetic environment (sds and dpc micelles) by spectroscopic and computational methods. diagnostic nmr parameters observed for glu3ta and pro3tl indicate a conformational propensity toward helical structure. for glu3ta, bturn structures are also observed along the n-terminal fragment of the peptide. we are currently studying the conformational behavior of the four peptides also by molecular dynamics simulation in explicit solvated dodecylphosphocholine and sodium dodecylsulfate systems. these simulations would provide a realistic picture of the interactions between the peptide and models of bacterial and mammalian membranes. where xaa was selected form a collection of cyclic and acyclic nonaromatic amino acids. the peptides were prepared by standard spps methods using either the fmoc or boc strategy and tested in vitro for their agonistic potency and effi cacy at the v1a and the related receptors. unlike avp, which has aromatic amino acids in positions 2 and 3, these analogues contain only non aromatic residues, but are still potent v1a agonists. compounds with cyclic aliphatic residues (e.g. cha) in position 2 were found to be generally more potent v1a agonists than the ones containing acyclic residues. the ala 2 analogue ([ala 2 ,ile 3 ,dab 8 ]vp) was totally inactive (no signifi cant v1ar agonism up to 1000 nm) as pharmacology and medical peptide chemistry reported previously for [ala 2 ]avp (2). the cha 2 containing peptides are slightly less potent as v1ar agonists than their phe 2 counterparts, but the overall in vitro pharmacological profi le of the two classes of compounds is similar. selected new analogues were also tested in vivo in a rat pressor model. the compounds were found to be effective in raising arterial blood pressure and appeared to be longer acting in vivo when compared to avp and related compounds, as demonstrated by slower disappearance of their pressor effect at equieffective doses. detailed experimental procedures, the results of biological testing, and sar discussion will be presented. the impact of lithium cations on the peptide bond lithium ions play an important role in some biological processes, e.g. in the treatment of neuronal dysfunctions and some metabolic pathways. however, the mechanisms for these actions are not known up to now. recently it has been shown that lithium cations infl uence the isomerisation state of peptide bonds which is essentially pronounced in the case of the amino acid proline. such an isomerisation goes hand in hand with conformational changes possibly resulting in altered folding and structuring. thus, lithium cations might essentially infl uence the biological function of peptides and proteins. to shed light on the underlying mechanism we carried out detailed studies on several model peptides. thus, the isomerisation as well as the kinetic properties of the peptides have been determined employing various techniques of nmr spectroscopy. besides, quantum chemical studies on li + -peptide complexes were performed to get insight into the structural aspects of the cation-peptide interaction. enhanced screening and understanding of hypolipidemic peptides assisted by informatics hypolipidemic peptides targeting the bile acid to inhibit cholesterol absorption in intestine has been indicated by several naturally obtained short peptides. these bile acid-binding peptides are known to disrupt the micellar formation of bile acid for capturing intestinal cholesterol, and lead the aggregates pass the intestine without absorbance. such effect also disrupts the hepatic circulation of bile acids, which accelerates the conversion of stored cholesterol into lacking bile acids. however, traditional extraction procedure of peptides form natural products is time-consuming. therefore, to enhance the screening of such functional short peptides, we have combined the array-based screening strategy with bioinformatics strategy, to design effective experiments by prediction of physicochemical peptide structures. as a result, we resulted in signifi cantly higher screening effi ciency and high bile acid affi nity compared to random screening. we hare report the introduction of one of supervised learning algorithms, fuzzy neural network, and unsupervised algorithm, hierarchical clustering scheme for such functional peptide screening. analogues of the kinin b1 receptor antagonist r-954 bearing n-terminal lipid moieties. adrenomedullin (am) is a 52-amino acid peptide that shares structural similarities with regulatory peptides belonging to the calcitonin family (calcitonin, cgrp and amylin). am is known to produce a potent vasodilation via the coupling to the calcitonin receptor-like receptor (crlr) associated with a chaperone protein, the receptor-activity modifying-protein (ramp). binding studies performed in mammalian tissues revealed that the largest distribution of am binding sites was found in the heart and lungs. in fact, the pulmonary blood vessels display a vast abundance of am binding sites that act as clearance receptors. pulmonary arterial hypertension (pah) is a condition associated with obliteration of pulmonary arterioles which carries a very poor prognosis, in part because of delayed diagnosis due to the lack of specifi c noninvasive diagnostic tools. likewise, there currently exists no molecular imaging agent to diagnose pulmonary embolism, a pathological condition that occurs when a blood clot blocks a lung artery. therefore, due to the high density and incidence of am receptors in the cardiorespiratory system, am could represent a key-target for diagnosis with radiolabeled am-related drugs. in this study, we looked at structureactivity relationships and synthesized various am fragments exhibiting high binding specifi city for am receptors but reduced biological activity. moreover, we introduced different chelating moieties into these am peptides to evaluate the possibility of labelling those molecules for imaging purposes. using cell binding assays, we observed that a cyclic moiety combined with the am(22-52) sequence is able to maintain a good affi nity for am receptors. furthermore, we showed that the incorporation of a 4-amino acid sequence into the peptide chain was the best chelating moiety that allows high effi ciency labelling with 99mtc. hence, our study strongly suggests that am derivatives are promising leads for lung specifi c imaging. 99m tc-labeled analogs with pansomatostatin properties may lead to clinically useful radiotracers and further search for analogs displaying improved biological profi le is warranted. and for the rat (r) sst 2 by competition binding assays in ar4-2j cell membranes with [ 125 i-tyr 3 ]octreotide as radioligand. after 99m tc-labeling, radiopeptide internalization was studied in ar4-2j cells. biodistribution of [ 99m tc]demotates was studied in healthy swiss albino mice and for [ 99m tc]demotate 1, 2, 3 and 6 in ar4-2j tumor bearing mice. results: demotate 1 showed the highest affi nity binding for the hsst 2 and the rsst 2 , while introduction of one asp linker(s) at the n-terminus diminished the affi nity for the hsst 2 and the rsst 2 and led to lower internalization rates. substitution of thr 8 by asp 8 in demotate 6 resulted in good affi nity for the rsst 2 and lower affi nity for the hsst 2 . single (d)phe 1 -substitution/5 or thr 8 substitution by asp 8 combined with asp introduction at the nterminus/7 led to inferior binding affi nity, poor internalization capacity and poor uptake of the respective radiopeptides in target-organs and, in the case of [ 99m tc]demotate 1, 2, 3 and 6, in ar4-2j tumors in mice. conclusion: introduction of asp residues in the original [ 99m tc]demotate 1 chain exerted a negative effect on receptor affi nity, internalization capacity and targeting of somatostatin binding sites in mice precluding their use as hydrophilic pharmacokinetic modifi ers. the family of secreted aspartic proteinases (saps), which are encoded by ten distinct genes, is an important virulence factor of the human pathogen candida albicans.(1) due to an increase in the number of candida strains that are resistant to the drugs currently used in therapy, these proteinases are highly promising new drug target candidates. based on the knowledge of sap2-substrate specifi cities(2)and x-ray structural studies of sap2 in complex with inhibitors,(3) we designed and synthesized a series of sap inhibitors by modifying the structure of the pentapeptide pepstatin a, which is a potent yet non-selective aspartic proteinase inhibitor. these inhibitors were synthesized manually via a spps methodology using a 2-cl-tritylchloride resin and fmoc-protected amino acids. in addition, the key residue of the designed peptide inhibitors, the -amino acid statine (sta), which was prepared according to a slightly modifi ed literature procedure,5. was further protected at its hydroxyl group as a silyl ether for the purpose of avoiding competitive side reactions during amino acid couplings. we prepared 9 new inhibitors by varying the pepstatin a structure at the p3, p2, or p2' position. all variants showed effi cient inhibition when screened against the isoenzymes sap1, sap3, and sap6, and some of them exhibit ic50 values similar to or even lower than that observed for pepstatin a. peptide library is a general technique for screening specifi c peptides that bind to a target protein. but, in a standard screening method, peptide libraries need to be fi xed on large carriers like phage, beads and so on. the large carriers would affect the binding between peptides and the target protein. in this work, we develop a new method for screening peptide libraries without carriers. we attached a variety of fl uorescent amino acids to peptide libraries and the peptides that bind to target proteins were identifi ed through 2-dimensional fl uorescence spectroscopy in aqueous solution. in this presentation, 36-different fl uorescent amino acids were synthesized or purchased. of those amino acids, we newly synthesized 30 fl uorescent amino acids. 2-d fl uorescence spectra of those amino acids were measured. they were different in fl uorescence excitation/emission wavelengths. excluding those of weak fl uorescence intensities, 15 fl uorescent amino acids are selected. those 15 fl uorescent amino acids are mixed in methanol/water (ph7.4) (=1/1(v/v)) and the 2-d fl uorescence spectrum was measured. then, concentrations of the component amino acids were evaluated by a linear least-squares method. the results suggested that all fl uorescent amino acids can be quantifi ed. the set of fl uorescent amino acids combined with the 2-d fl uorescence spectroscopy technique will be a powerful tool for screening peptides and other drug compounds without using any carriers. n-methyl phenylalanine-rich peptides as potential blood brain barrier shuttles malakoutikhah, morteza; teixidó, meritxell; giralt, ernest institute for research in biomedicine, barcelona, spain several peptide families containing n-methylated amino acids were designed and synthesized using solid-phase peptide synthesis (spps). the permeability of these compounds was studied by parallel artifi cial membrane permeability assay (pampa) and immobilized artifi cial membrane chromatography (iamc) so as to select the best peptides in terms of length, terminal groups and amino acid replacement to be used as carriers that pass through a model of blood-brain barrier (bbb) by passive diffusion for non-permeating agents. furthermore, their enzymatic stability in human serum and their cell viability were tested by mtt assay. these peptide families showed great stability and nontoxicity. the three best peptides were coupled to levodopa and assessed. these peptides transferred levodopa through an artifi cial membrane and transformed it from a non-passive permeating drug into a compound able to cross the membrane by passive diffusion. the structure-activity study of insulin-like peptide 3 (insl3) requires the design and synthesis of various analogues. in order to test these for their receptor binding affi nity, a high-throughput receptor binding assay is needed. we have therefore developed an effi cient solid phase synthesis protocol to prepare specifi cally mono-labeled human insulin-like peptide 3 (insl3) for the study of its interaction with its g-protein-coupled receptor, rxfp2. a commercially available chelator, diethylene triamine pentaacetic acid (dtpa), was coupled to the n-terminus of solid-phase bound insl3 a-chain and then a coordination complex between eu 3+ and dtpa chelator was formed. after combination of the purifi ed aand b-chains together with sequential formation of the three insulinlike disulfi de bonds, the labeled peptide was purifi ed at high yield using high-performance liquid chromatography with high ph buffer to prevent the liberation of europium from chelator. saturation binding assays were undertaken to determine the binding affi nity (pk d ) of labeled insl3 for rxfp2 in hek 293 stable cell line expressing rxfp2. the binding affi nity of dtpa-labeled insl3 (9.05 ± 0.03) was comparable to that of 125 i-labelled insl3 (9.59 ± 0.09). the effi cient solid-phase synthesis has provided a novel lanthanide-coordinated, dtpa-labeled insl3 with excellent sensitivity, stability and high specifi c activity and which is superior to the traditional 125 i-insl3. this labeled peptide can be used in a high-throughput screening of insl3 analogues in structure-activity studies. multimodal tumor imaging using mri and pet/spect provides comprehensive diagnostic information as it combines anatomical information (by mri) and functional information (by pet or spect). development of dota-derived imaging probes is advantageous for multimodal imaging as it accommodates many metal ions employed in different imaging modalities including gd(iii) for mri. however, high relaxivity and enhanced specifi c up-take at the targeted site is important for the gd-based mri contrast agents. the dota-based prochelators required for the multivalent vectorization of targeting ligands were synthesized by functionalizing cyclen or do2a-tertbutyl ester with modifi ed glutamic acid. multivalent conjugation of bombesin peptides, which specifi cally target tumors expressing gastrinreleasing peptide (grp) receptors, yielded the corresponding mono-, di-, and tetravalent bombesin analogues. the conjugates showed excellent chelating properties with gd(iii) (for mri applications) and also with 111 in, 177 lu and 68 ga (for radiopharmaceutical applications). the 111 in and 177 lu labeled divalent conjugates showed rapid internalization and slower externalization rate compared to their corresponding monovalent analogues as studied with prostate tumor cell lines. the gadolinium complexes of monovalent and divalent conjugates showed signifi cant relaxivities at 60 mhz ranging from 9.3 to 19.2 mm-1s-1 at 25°c. the values represent the 2 to 4 fold enhancement of relaxivity compare to clinically employed dotarem® (gd-dota). further, the relaxivities increased signifi cantly from monovalent to divalent conjugates. this is highly promising as we would expect much higher relaxivities in case of tetravalent conjugates, which are currently under investigation. in conclusion, the work demonstrates the development of high relaxivity multivalent bombesin analogues, which could be employed for the multimodal imaging such as pet/mri or spect/mri with improved tumor targeting capabilities. a new highly potent dota-conjugated bombesin antagonist for grpr-positive tumor targeted imaging peptide receptors are very promising targets for tumor imaging. the somatostatin receptors were successfully targeted with peptides labeled to -emitters ( 111 in, 67 ga and 99m tc) and positron emitters ( 18 f, 68 ga, 86 y) for diagnostic imaging. for tumor targeting, radiolabeled agonists were developed as they usually trigger the internalization of the peptidereceptor complex. we have recently shown that somatostatin-based radiolabeled antagonists may not only have a higher tumor uptake than equipotent agonists but also a longer lasting tumor uptake. among the most promising receptors to be targeted, the bombesin receptors are of great interest as they are overexpressed in major human tumors such as prostate and breast. the aim of this study was to develop a radiolabeled bombesin-based antagonist conjugated to the macrocyclic chelator dota which allows effi cient and stable labeling with a variety of two-and three-plus charged radiometals for imaging (pet, spect). we compared its in vitro pharmacologic properties and biodistribution in the pc-3 mouse model side-by-side with the potent agonist [ nat,111 in]-do3a-ch 2 co-g-4-aminobenzoyl-q-w-a-v-g-h-l-m-nh 2 (amba). in(iii)-amba was shown to be a potent agonist by ca 2+ fl ux and immunofl uorescence studies and in(iii)-rm1 was effi ciently antagonizing the activity of in(iii)-amba. the pharmacokinetics showed a distinct superiority of the radioantagonist with regard to the high tumor uptake as well as to all tumor to normal tissue ratios. [ 68 ga]-rm1 showed similar pharmacokinetic than [ 111 in]-rm1 with a lower initial kidney uptake and a faster wash out from the kidney. the pet/ ct scintigraphic studies of [ 68 ga]-rm1 in the animal model confi rm the signifi cant high tumor uptake and tumor to background ratio. as we found for somatostatin receptor-targeting radiopeptides,bombesin-based radioantagonists also appear to be superior to radioagonists for in vivo imaging and potentially also for targeted radiotherapy of grpr-positive tumors cell penetrating peptides (cpp) are a special class of peptides that possess the property to traverse the formidable barrier of the plasma membrane and deliver cargos into cells. using cpp as vectors and dna, mrna or proteins/enzymes as potential intracellular targets, a new generation of intracellular contrast agents (cas) can be developed. these agents have prospective use for molecular imaging (both optical and magnetic resonance imaging) by targeted labeling of cells. aiming to image the presence of specifi c mrnas or enzymes, two mrna targeting (contains a pna sequence antisense or non-sense to the target mrna of dsred) and one enzyme targeted (contains a unit cleavable by -galactosidase) cas were tested for their activity in the presence and absence of respective targets. the antisense targeting ca, their nonsense derivative and the enzyme targeted ca were taken up effi ciently into cells by an exclusively endosomal mechanism as observed by fl uorescence microscopy. cell free binding assays proved a specifi c interaction with a synthetic target for the antisense but not for non-sense ca. magnetic resonance studies showed a higher uptake in transgenic dsred expressing cells than the parent cells. however, no difference was observable for antisense versus non-sense ca in dsred cells, due to the vesicular entrapment which is preventing the specifi c interaction between ca and cytosolic target. since a comparable cellular distribution was visible for the enzyme targeted agent, a specifi c accumulation in -galactosidase containing cells is also unlikely. the results show that even though the designed cas were effi ciently taken up into cells, they can interact specifi cally with the target only if colocalization is achieved. however, a lack of specifi city is caused by the endosomal entrapment. further modifi cations are required to achieve the release from endosomes or a direct uptake into the cytosol. the infl uence of pegylation on the tumor accumulation of frop-1, a tumor specifi c peptide identifi ed by phage display the pool of natural peptides suitable for the tumor targeting is limited and therefore novel lead structures identifi ed by screening of phagedisplayed libraries are highly warranted. however, transfer of the novel peptide sequences to clinical application is often diffi cult. we had shown that the coupling of dota to frop-1, a peptide identifi ed in phage display libraries resulted in a fundamentally improved in vitro binding capacity. however, a biodistribution study revealed that the slow binding kinetics frop-dota (h-glu-asn-tyr-glu-leu-met-asp-leu-leu-ala-tyr-leu-lys(dota)-cys-nh2) allowed the excretion to forestall a signifi cant tumor accumulation. the aim of this study was to investigate whether the conjugation of peg to frop-dota results in a derivative with a prolonged residence time in the blood. frop-1 bearing a c-terminal dota residue to allow labeling with in-111 and a cysteine to attach a maleimido-modifi ed 2000 da peg oligomer was obtained by solid phase synthesis. the conjugate was purifi ed by size exclusion chromatography. several attempts to couple different peg derivatives on the solid support did not proceed satisfactorily and therefore the peg residue was attached in solution. the breast cancer cell line mcf-7 showed a relative low accumulation of the pegylated peptide in the cells. in contrast, biodistribution studies of the labeled conjugate in mice bearing human fro82-2 showed a time dependent increasing uptake of the pegylated peptide with a high retention (at 24 h p.i. 76% of the maximal activity concentration persisted in the tumor). the highest uptake values were determined at 120 min. p.i. reaching 2.3 %id/g tumor as compared to 0.06% %id/g observed for the non-pegylated derivative at 135 min p.i. apparently pegylation provides a substantially improved stabilization in the circulation which allows a stable tumor accumulation. in vivo spect/ct imaging of intracranial human glioblastoma xenografts with 111indium-labeled homing peptide. huhtala, tuulia* 1 ; enbäck, julia* 2 ; rautsi, outi 1 ; weisell, janne 1 ; laakkonen, pirjo* 2 ; närvänen, ale* 1 *equal contribution. 1 university of kuopio, finland; 2 university of helsinki, finland phage display libraries provide a powerful tool to identify new biologically active peptides that bind specifi cally to their target molecules. different tumors express a distinct range of molecular markers on their vasculature providing a possibility to use peptides for targeting. tumor-homing peptides offer an opportunity to target, image and destruct the target tissue. glioblastomas represent the most aggressive form of brain tumors. we have identifi ed a novel peptide, coop, using an ex vivo / in vivo phage display screen. this peptide homes specifi cally to the early stage astrocytoma model. in addition, the peptide homes to the u87 human glioblastoma xenografts (enbäck and laakkonen, unpublished data). we have studied the biodistribution and tumor homing properties of indiumlabelled peptide in mice bearing intracranial human u87 glioblastoma tumors using spect/ct imaging. coop peptide was conjugated with the dtpa (diethylenetriamine pentaacetic acid) via amino terminus and labeled with 111indium. imaging was performed in four different time points (15 minutes, 1 hour, 2 hours & 24 hours), and the organ and tissue specifi c radioactivity was measured after the scarifi cation of the animals. intravenously injected 111in-labeled coop peptide accumulated in the u87 braintumors. the amount of the peptide increased with time and a clear radioactive accumulation was seen in 1 /3 animals at 1 hour and in 5 / 6 animals at 2 hours after injection. due to the small size of the dtpa-peptide conjugate the majority of the molecules were secreted via the kidneys during the fi rst 15 minutes. at 2 h timepoint the tumorto-brain tissue ratio was 11,5. we report here that the coop peptide, which specifi cally homes to the brain tumor vasculature and tumor cells, strongly accumulated in vivo to the xenografted human glioblastoma tumors in mice. this is the fi rst time when a synthetic peptide has been used in spect imaging of brain tumors in animal mode. failure of the development of a novel peptide tracer for molecular imaging of cancer therapy response 343-9 (2008) have shown that fl uorescently labeled derivatives of the hvggssv motif accumulated in tumors undergoing therapy. this peptide motif had been identifi ed by the in vivo screening of phage-displayed peptide libraries. the ability of the peptide to differentiate between responding and nonresponding cancers after treatment could be utilized for the development of promising tracers to monitor cancer therapy. the goal of this study was to investigate whether radiolabeled derivatives of the hvggssv motif can be used for the noninvasive determination of therapy response in vivo. therefore three different conjugates were synthesized by solid phase synthesis using fmoc chemistry. the fi rst peptide was n-terminally modifi ed with the chelator dota (dota-ggghvggssv-conh2) to allow radiolabeling with metallic radioisotopes such as 111in or 68ga. a glycine linker was placed at the n-terminus to separate the peptide motif from the chelator. in order to obtain derivatives accessible to radioiodination a tyrosine was introduced at the n-terminus (h2n-yggghvggssv-conh2) of the second peptide. the third peptide was biotinylated at the n-terminal amino group and bound to radioiodinated streptavidin (sa-biotin-ggghvggssv-conh2). the biodistribution was monitored by scintigraphy in mice bearing a431-tumors treated with a vegf receptor-specifi c inhibitor (su5416). this study revealed that the 111in labeled hydrophilic dota conjugate shows a rapid renal clearance, the iodinated peptide accumulates mainly in the liver and kidneys. all of the peptide derivatives do not specifi cally accumulate in treated tumors after i.v. injection. in contrast to the promising results shown by han z. et al. our study shows that the hvggssv motif can not be easily adapted to radiolabeling approaches with clinical relevance. in conclusion, the peptide motif is not attractive for further clinical evaluation as new diagnostics for the imaging of cancer response. a novel approach to improve cellular delivery of 5-aminolaevulinic acid: new ala-containing peptide prodrugs for photodynamic therapy. photodynamic therapy (pdt) is a binary therapeutic modality which is currently under investigation for the treatment of several kinds of malignancies. it relies on the interaction of two individually harmless components: a photosensitiser and an external radiation. the interaction of the photosensitiser with light of the appropriate wavelength and molecular oxygen results in the generation of cytotoxic species, namely singlet oxygen and/or radicals, and localized destruction of tumours or infected tissue. in 5-aminolaevulinic acid photodynamic therapy (ala-pdt), exogenous administration of ala is employed to generate elevated intracellular levels of the natural photosensitiser protoporphyrin ix (ppix), via metabolism through the haem biosynthetic pathway. however this approach suffers from several drawbacks associated with ala's lack of stability at physiological ph and its highly hydrophilic nature, which prevent it from crossing biological membranes and hence limit tissue penetration. ala delivery with synthetic peptide prodrugs is a promising way to address these problems. in this work we report the synthesis and characterisation of a series of peptide prodrugs of general structure ac-xaa-ala-or, where xaa is an alpha amino acid, chosen to provide a prodrug with appropriate lipophilicity and water solubility. the uptake of the compounds and metabolism to ppix in pam212 keratinocytes, relative to ala is evaluated by fl uorescence spectroscopy, and further quantifi ed by recovery and chemical derivatisation of intact/ partially metabolised prodrugs. in a parallel study, we have also explored the possibility of coupling of ala and ala prodrugs to cell penetrating peptides (cpp). the conjugation of one or more molecules of ala to a cpp sequence represents an interesting approach to enhanced topical delivery of ala. preliminary results of these studies are reported herein. acknowledgements: thanks are due to biotechnology and biological sciences research council (grant bbd0127831) bioshuttle as a carrier for temozolomide transport into prostate cancer cells if metastatic prostate cancer gets resistant to antiandrogen therapy, there are few treatment options, because prostate cancer is not very sensitive to cytostatic agents. temozolomide (tmz) as an oral applicable chemotherapeutic substance has been proven to be effective and well tolerated with toxicity especially for brain tumors. unfortunately tmz was ineffi cient in the treatment of symptomatic progressive hormone-refractory prostate cancer. this may have different reasons like the short plasma half-life of tmz, a non adapted application schema and as a result, an insuffi cient bioavailability. to improve the specifi city, we built our so called tmz-bioshuttle-construct with cathepsin b (ctsb) mrna specifi city. this complex combines, a transmembrane transporter molecule connected via a disulfi de bridge to an antisensepeptide nucleic acid (pna) against a docking site in exon 1 of the ctsb mrna. furthermore this part is connected via a ctsb cleavable peptide substrate to a nuclear localization sequence coupled with tmz. inside the target-cell, the pna recognizes the cytoplasmic ctsb mrna and after annealing (the pna/rna hybride is not a substrate for rnase h) results in a cell-specifi c retention of the tmz in the cytosol especially of ctsb-expressing cells. then, after the cathepsin b-mediated cleavage in the cytoplasm, the nls-sequence is separated and activated for an ran/importin-mediated transport of the tmz-cargo into the nucleus of the target cells. this bioshuttle-mediated tmz transfer could be a step forward to a successful therapy with higher specifi city, avoiding adverse reactions and circumventing the previous therapy limiting situation. the intracellular delivery of protein segments and other bioactive molecules using membrane -permeable peptides has been investigated in multiple aspects. most of the currently recognized cell penetrating peptides (cpp) are of cationic nature and derived from viral, insect or mammalian proteins endowed with membrane translocation properties. these peptides enter the cell by a receptor independent mechanism, which is poorly understood and carry only one epitope. our target was to achieve a cell-penetrating carrier able to transport more than one bioactive molecule. to this aim we designed a cell penetrating sequential carrier (cpsc) formed by the repetitive -lys-aib-cysmoiety, which incorporates the cysteine residue for anchoring the bioactive molecules through a thioether bond. the lysine free side chain possesses the cationic nature of the construct while the á-amino isobutyric moiety could induce a 3 10 helicoid peptide backbone with amphipathic characteristics. to test the ability of the cpsc to penetrate the cell membrane we synthesized the carboxyfl uorescein -labelled cpsc, cf-[lys-aib-cys(-ch 2 conh 2 )] 4 -nh 2 , while the cf-tat-cys(-ch 2 conh 2 )-nh 2 analogue, which is a small basic peptide that has been shown to deliver a large variety of cargoes into the cells, was used as a positive control. the results indicated that cpsc had a homogeneous distribution into the cytoplasm suggesting that cpsc may provide a new powerful biological tool for drug transportation. acknowledgements: the gsrt and eu (pened 03åä629) for the fi nancial support. amphipathic pro-rich peptides as intracellular drug delivery systems pujals, silvia; fernandez-carneado, jimena; giralt, ernest institute for research in biomedicine, spain shared among many of the known cpps. in 2004 our laboratory described a novel group of cpps: amphipathic proline-rich peptides. the principal advantages of these compounds are non-cytotoxicity, nonviral origin and high solubility in aqueous media. the best candidate for cpp among the amphipathic pro-rich peptides was sap (sweet arrow peptide), (vrlppp) 3 .(1) derivatives with hydrophobic moieties, such as fatty acids or silaproline, have shown highly improved internalisation effi ciency; an all d-amino acid version of the cpp sap was shown to be completely protease resistant and was evaluated in a preliminary in vivo study. cd and tem studies regarding the self-assembly properties of this family of peptides highlight the possible role of aggregated species in the internalisation process. finally, these cpps have shown to be internalised via caveolae or lipid-rafts mediated endocytosis, which circumvents the lysosomal route of degradation. in order to test a challenging cargo for sap, gold nanoparticles were conjugated to sap. thereafter, the transport of au np by sap in hela cells has been studied by tem. while np alone are not internalised, np-c-(vrlppp) 3 are clearly uptaken. studies with qds (quantum dots) and egfp as sap cargo are currently being done in our laboratory. 1. pujals, sílvia; giralt, ernest. proline-rich, amphipathic cell-penetratingpeptides. addr (2008), 60, 473-484. novel cysteine-rich cell penetrating peptide: effi cient uptake and cytosolic localization introduction: crossing the plasma membrane is a prerequisite for intracellular targeted drug delivery. cell penetrating peptides are actively used as the delivery tool for intracellular delivery of various cargos. however, confi nement of biomolecules into endosomes limits their use for intracellular targeting. therefore, there is a need for vectors capable of transferring cargo molecules directly into the cytoplasm. herein, we focus on the development of a novel cpp (derived from polypeptide crotamine 1.) which shows an effi cient uptake at low concentrations ( 2.5 m) and cytosolic distribution along with vesicular uptake. methods: series of peptides were synthesized by fmoc strategy, introducing mutations in cro (27-39) (proposed cpp sequence in crotamine). all were n-terminally labeled with fl uorescein isothiocyanate for optical imaging. structure activity relationship (sar) studies were done by substitution and/or deletion of amino acid residues in the sequence observing the uptake behaviour by fl uorescence spectroscopy and microscopy. results: amongst 60 synthesized peptides, one of shorter length showed the best intracellular delivery and cytosolic distribution at lower concentration (2.5 m) when compared to other cpp. replacing or deleting cysteines had negative impact on internalization. results also displayed the involvement of tryptophans in cellular uptake indicating, along with cationic amino acids, the importance of each residue in this optimized sequence. conclusions: sar studies identifi ed a novel cell penetrating peptide showing, besides of endosomal uptake, also an effi cient delivery into the cytoplasm at low concentrations. thus, this peptide might prove useful for effi cient transmembrane delivery of agents directed to cytosolic targets. peptide nucleic acid (pna) is a dna mimic consisting of the four common bases of dna on a pseudopeptide backbone that makes it extremely stable in biological fl uids. antisense pna is targeted against mrna in cytoplasm in a sequence specifi c manner. however, the main hindrance to the effective use of pnas has been their relatively poor uptake by cells. endosomal release or direct uptake into cytosol of agents is mandatory for attaining mrna based targeting. there are reports on the cell penetrating peptide (cpp) based delivery system. it has also been reported that conjugates of cholesterol and sirnas facilitate cellular import (1) . the aim of this study was to synthesize different sequences of cholesterol coupled antisense pna and to compare its uptake characteristics with a cpp-pna conjugate. the synthesis of pna (anti-dsred pna (agcgcctgtacc), specifi cally targeted to mrna of dsred, a red fl uorescent protein) conjugated to cpp (d-tat) or cholesterol was performed in fully automated synthesizer (prelude, protein technologies, inc.) using continuous solid phase chemistry. to increase the solubility in water, linkers (aeea) and additional charged amino acids were coupled or the sequence of peptide, pna and cholesterol was changed. all compounds were labelled with fitc to confi rm the cellular uptake by fl uorescence microscopy and spectroscopy. cell uptake studies showed that the cpp bound pna was located predominantly in vesicles indicating an endosomal uptake mechanism and subsequent entrapment in vesicles. cholesterol bound pna was also effi ciently internalized. however, it was also located inside vesicles without detectable cytosolic distribution. pna-cholesterol has fewer synthetic steps than pna-cpp. however, it was also located inside vesicles restricting its applicability for mrna targeting. the effi cient uptake might make it a promising cellular delivery agent after further improvements. opioids are gold standards in pain treatments. unfortunately, fast development of tolerance and dependence creates limitations in application of these drugs in chronic pain treatment. over twenty years ago we have proposed development of multitarget medicines as a new avenue of drug discovery. identifi cation of numerous endogenous components that participate in the formation, transmission, modulation and perception of pain signals offers various strategies for the development of new analgesics. neurotensin is an endogenous neuropeptide that play important regulatory role of pain transmission. therefore, it was interesting to develop chimeric analogues that hybridize opioid and neurotensin active components. in such chimeric compounds the possible structural interference may signifi cantly infl uence on interaction of both components with target receptors and/or biological barriers transport systems. in this communication we present synthesis, pharmacological profi le and structural analysis of new potent chimeric compounds. acknowledgements: presented studies have been supported in part with eu grant normolife (lshc-ct-2006-037733) artifi cial ribonucleases based on short peptides. the synthesis of molecules that are capable of nonrandom rna cleavage has found a variety of important applications in molecular biology. for instance, such molecules are used as structural probes for nucleic acids in solution, or in rational design of novel anti-infectives, since rna is the genetic material of many pathogenic viruses. here we represent design and synthesis of peptide-like molecules mimicking the catalytic site of natural ribonucleases (a and t1): series f aa1 -aa2 -aa3 -phe -oalkyl; aa1 -glu/lys, aa2 -thr/ser/lys, aa3 -glu/ lys/thr/arg; series r glu -x -arg -gly -oalkyl; series k glu -x -lys -gly -oalkyl; -gly, -ala, 4-aminobutyric acid, 6aminohexanoic acid, p-aminobenzoic acid; series l aa3 -aa2 -aa1 -l1 (l2)-aa1 -aa2 -aa3 , l1 -4,9-dioxa-1,12-dodecanediamine, l2 -1,12-diaminododecane; aa1-aa3 -glu, lys, ser, arg. ability of artifi cial rnases to rna cleavage was shown in experiments with 96mer rna hiv-1. the effi cacy of rna cleavage depends on artrnases structure (for example, on location of negative and positive charged amino acids (glu, arg lys) in peptide). the effi cacy of the most active compounds was 60-98 % at 18 h incubation. anti-infl uenza activity in vitro of 12 such artrnases was investigated by inhibition of reproduction virus a/hong kong/1/68 (h3n2) in tissue cultures of chorioallantoic membranes (ccm) of 12-14 days age chick embryos. acknowledgements: this work was supported by rfbr (07-04-00990a, 08-04-90038-bel_a), pharmamed ruxo-008-n -06, the grant of novosibirsk region government for the best young scientists. (ziagen)-(1s,cis)-4-[2-amino-6-(cyclopropylamino)-9hpurin-9-yl]-2-cyclopentene -1-methanol is a synthetic carbocylcilic nucleoside analogue with inhibitory activity against hiv. serious and sometimes fatal hypersensitivity reactions have been associated with abacavir. a possible way to increase the side effects is by modifying the known antiviral drugs with various amino acids. the aim of this study was to design and to synthesize of new amino acids and peptide (gly, gly -gly) and thiazole containing (gly, gly -gly) esters prodrugs of abacavir and to explore their activity on the hiv. chemical stability of some purine analogues in the search of new prodrugs effective against herpes simplex virus, series of acyclovir-9-[(2-hydroxyethoxy)methyl]guanine (acv) esters with peptidomimetics, as well as abacavir-(1s,cis)-4-[2-amino-6-(cyclopropylamino)-9h-purin-9-yl]-2-cyclopentene-1-methanol derivatives have been synthesized and tested for antiviral activity. the chemical stability of some of them is studied at ph 1 and 7.4 and temperature of 37°c. a high-performance liquid-chromatografi c (hplc) method was developed for quantifi cation of the unchanged ester concentration. the promelittin represents a readymade cancer pro-toxin; the prodomain inhibits cytolytic activity and the sequence of the prodomain could be manipulated into a substrate for activating proteases. here we present the development of a novel promelittin pro-toxin that can be activated by the serine protease fi broblast activation protein (fap). fap is over expressed on the surface of reactive stromal fi broblasts present in the stroma of human epithelial tumors. fap is not expressed by tumor epithelial cells or by fi broblasts in other tissues, thus making fap a pantumor target for pro-toxin development. peptides containing truncated pro-domain sequences were tested on erythrocytes to determine the optimal prodomain length for inhibiting cytolytic activity. once the length was optimized, modifi ed promelittin peptides were generated that contained previously identifi ed fap substrate sequences in the prodomain. these peptides were digested with fap and tested in vitro against fap+ and fap-cell lines in order to determine their fap cleavage potential and toxicity. our lead promelittin peptide was found to be effi ciently activated by fap and selectively toxic to fap+ cell lines with an ic50 value in the low micromolar range that is similar to that of melittin. pharmacology and medical peptide chemistry region of highly hydrophobic and the antigenic part of hbsag by using microwave assisted solid phase peptide synthesis (spps). tryptophan at the n terminus of the sequence was added by our research group for fluorescence dedection analysis. this peptide was conjugated with synthetic polyanions. different conjugates comparised each other. the synthesized bioconjugates analysized by choromatographic and fl uorometric methods. the fi nal product peptide was characterized by lc-ms and purifi ed by rp-hplc. the bioconjugates were synthesized using different activation mechanisms and the different initial molar ratios (npeptide/npolymer = 1, 3, 5, 7, 9) of the polyanions and the peptide. also physicochemical properties of the bioconjugates were investigated. the structure and characterization of the synthesized conjugates was analyzed by various choromatographic and fl uorometric methods such as hplc, gpc and fluorescence spectrometer. the reaction of high temperature solid-state catalytic isotope exchange (hscie)(1) between insulin and spillover tritium was studied. hscie reaction with the solid mixture containing 1 mg recombinant human insulin was performed for 10 min at 120o c using 5% pd/baso4 catalyst. the product of the reaction was purifi ed by hplc on waters delta-pak c4 300a 3.9 x 150 mm column. tritium labeled insulin with specifi c radioactivity 40 ci/mmol and radiochemical purity of more than 98% was obtained. performic acid oxidation of cysteine residues and subsequent acid hydrolysis were used to analyze the distribution of tritium. it was demonstrated that specifi c radioactivity of à and b chains amounted to 8.2 and 31.8 ci/mmol respectively. all the amino acids contained tritium and its content in his residues of the b chain was equal to 45%. experiments for the evaluation of the biological activity of tritium labeled insulin were performed on cd-1 6-8-week-old awake male mice. activity of labeled insulin was compared with the activity of standard insulin. insulin was injected subcutaneously at a dose of 0.8 u/kg. twofold statistically signifi cant lowering of glucose level was observed 40 min after injection (4,9 + 0,4 and 4,3 + 0,3 mmol/l for standard and tritium labeled insulin, respectively). lowering of the glucose level in animals obtaining labeled insulin was the same, as in animals obtaining standard insulin. thus, hscie reaction of insulin with tritium doesn't change its hypoglycemic activity. cortexin is polypeptide (up to 10 kda) brain extract accepted for clinical use in several countries including russia due to its positive effects on memory, attention, and cortical processes. a synthetic peptide analog of cortexin, cortagen (ala-glu-asp-pro), was recently developed. the heptapeptide semax (met-glu-his-phe-pro-gly-pro) is an analogue of the acth(4-10) fragment, which is completely devoid of any hormonal activity associated with the full-length acth molecule. both cortexin and semax are currently effectively used for treatment of brain hypoxia and ischemia with comparable clinical benefi ts. however, the cellular and molecular mechanisms underlying the action in the brain are mainly unknown. in present work we studied effects of cortagen, cortexin, semax, its analogue pro-glu-pro (pgp) on glutamate-induced neuronal death and intracellular ca2+ homeostasis deterioration in cultured cerebellar granule cells. peptide drugs gain increased interest as a new supra-group of therapeutics between the classic-organic, small-molecule drugs and the large biotechnology-derived bio-drugs. they are used in different therapeutic areas like allergy, anti-infection, oncology, obesity, etc… due to their particular structure and biochemical origin, pharmaceutical development of a peptide drug poses special challenges which will be discussed here, exemplifi ed by own research as well as literature data. currently, there are about a dozen classical peptides described in pharmacopoeia, excluding the derived peptidomimetics like ace-inhibitors. these pharmacopoeial peptide-drugs, together with the approved marketing authorisations of new peptidic entities, are a good starting point to look at the desired characteristics. last, the regulatory developmental guidelines are in a general way seldom taken peptide drugs into account, leaving an interpretational gap between the two current main supragroups of therapeutics. after the initial active pharmaceutical ingredient (api) synthesis, analytical characterisation is aimed at integrity and purity evaluation of the api, which is also required for biomedical experiments. in this analytical characterisation, sample treatment issues like solubility and adsorption are considered as well. the chemical and plasma-metabolic stability, a critical parameter for peptides, is to be assessed to obtain kinetic and mechanistic information. functionality is tested in vitro using cell-and organ-based protocols, including ligand binding studies, as well as in vivo encompassing adme and target-organ confi rmation like brain. the pharmaceutical drugability information thus obtained allows further development decisions including required api modifi cations and proof-of-principle drug delivery formulations. lysine dendrimers and starburst copolymers as new carriers of anticancer drugs based on the complexes of platinum and gold. though the complexes of metals such as platinum and gold are very promising compounds for the treatment of different types of cancer, the low solubility complicates their application as medicines. in this work a series of lysine dendrimers of different structure and starburst copolymers of lysine and glutamic acid was studied as solubilizing carriers of anticancer drugs based on the complexes of platinum (cisplatin) and gold. cd analysis showed the small changes in the structure of the polymers upon metal binding. the esem study revealed that the presence of the amino groups on the surface of the carriers is crucial for the metal ligation. the highest content of metal bound was found in the samples with high amount of lysine residues. while the content of gold was rather low in all the samples (about 2%), the copolymer of lysine and glutamic acid with the ratio 2:1 as well as lysine dendrimer of fourth generation had 7 to 10% of platinum linked to the carrier. the absence of the bromine in the samples in the case of gold complex allowed to assume the covalent bond between metal and polymer. in the same time all the conjugates were highly soluble in water. the peculiarities of the synthesis and the anticancer activity in different cell lines will be discussed. acknowledgements: the work was supported by the "russian science support foundation". prospects for the development of synthetic peptide vaccines against hepatitis c no vaccinoprophylaxis of this disease or cardinal treatment means has been developed till now because of the problems produced by high genetic variability and heterogeneity of hepatitis c virus (hcv) isolates, lack of both hcv infection models and effi cient expression systems for the virus and its components. the problem of anti-hcv vaccine development can be solved via a principally new approach, the creation of artifi cial synthetic immunogenic constructs with the help of bioinformatics and high-throughput screening technologies. two approaches exist towards the development of anti-hcv peptide-based vaccines: one is to develop vaccines that stimulate cytotoxic t-cell response (peptides loading dendritic cells; the so-called "cell vaccines") and the second is to construct immunogenic peptides raising protective antibodies. however, the fi rst approach may lead to the massive hepatocyte death and the development of the heavy hepatic injury during hcv infection. in order to develop peptide immunogenic constructs able to raise antibodies against whole native hcv envelope proteins analysis of amino acid sequences of these proteins belonging to different hcv genetic variants has been performed. this analysis has allowed to choose the most conserved and presumably functionally important protein fragments. immunogenicity of these fragments in the whole protein molecules has been determined and interaction sites for one of the putative hcv receptor, heparansulfate, have been revealed with the help of peptide scanning. despite of the low immunogenicity of the most fragments inside the whole protein, antipeptide antibodies against them have been obtained via immunizing by conjugates of the peptides with certain carriers. two synthetic peptide immunogenic constructs have been developed that have raised antibodies against whole hcv envelope proteins. synthetic immunoactive fragments of endogenous proteins: selection and application for diagnostics and immunotherapy we have selected and synthesized the immunoactive fragments of four endogenous proteins: nucleophosmin, survivin, prion and alpha-7 subunit of acetylcholine receptor (achr). nucleophosmin and survivin are overexpressed in tumor cells and exhibit an antiapoptotic activity. their detection in tumor cells could be used for tumor differential diagnostics and selection of optimal chemotherapy scheme. accumulation of pathogenic isoform of prion protein in brain causes the neurodegenerative prion diseases and antiprion antibodies as known could be used for immunotherapy of prion disorders. alzheimer's disease (ad) development is accompanied by an accumulation ofamyloid peptides ( a) in brain and destruction of neurons . one of the ad development hypotheses assumed that a when binding to the alpha-7 achr forms a complex that penetrates into the cells, resulting in the neurons death. we have proposed that alpha-7 achr could be a new target in the ad therapy and antibodies against alpha-7 subunit could prevent its binding to a. selected synthetic fragments of these four proteins were able in a free nonconjugated to a protein carriers state induce antibody response in experimental animals. obtained rabbit antibodies against fragments of nucleophosmin and survivin were affi nity purifi ed. antibodies against nucleophosmin detected monoand oligomeric forms of this protein in immunoblotting of hela cells lysates. antibodies against survivin were able to detect this protein in immunohistochemical test of the breast cancer tumor samples. rabbit antiprion antibodies interfered with patigenic prion isoform in scn2a neuroblastoma cell culture. it was shown that synthetic alpha-7 achr fragments induced the antibodies formation in mice with symptoms of ad which penetrated the blood brain barrier, regenerated the spatial memory and normalized the beta-amyloid level in animal's brain. acknowledgements: supported by ras program mcb and rfbr grants 06-04-48710, 06-04-48388. investigation peptide-based cyclin a inhibitors: new tools to understand and to regulate the cell cyle-apoptosis signalling pathway the knowledge of the aetiology of cancer has increased considerably in the last two decades. the objectives have moved from unspecifi c cytotoxic chemotherapeutics to the identifi cation of small molecules (and antibodies) with a well defi ned molecular target. proliferation of eukaryotic cells is under control of a series of concerted molecular mechanisms defi ned as the cell division cycle whose progression is tightly governed by members of the cyclin-dependent kinase family. the protein-protein complexes formed between different cyclins and cdks (cdks) are central to cell cycle regulation. these complexes have been object of extensive research in cancer programs. considerable effort has been focused on the development of atp-competitive small molecule inhibitors of cdks. however, in general, these compounds have several alternative protein targets that compromise their demanded selectivity. cdk-2 was recently suggested to be dispensable for cell proliferation, although this does not appear to be the case for cyclin a, which therefore is defi ned as a more appropriate target for drug design. we have identifi ed an hexapeptide (nbi1) that inhibits the kinase activity of the cdk2-cyclin a complex through selective binding to cyclin a (1). the characterization of the inhibitory mechanism revealed that the hexapeptide does bind neither to the atp site nor to the cyclin recruitment site. a cell permeable derivative of the nbi1 peptide induces apoptosis and inhibits proliferation of tumor cell lines. we believe that the current structural and mechanisms of action studies on nbi1 will be useful for characterizing and controlling the important relation between cell cycle and apoptosis signalling pathways. the average size and size distributions of protein, polymer and peptideprotein or polymer-protein mixtures (complexes) and conjugates was investigated using photon correlation spectroscopy with a zetasizer nano zs instrument. the zeta potential measurements of this complex and conjugates were carried out in the folded capillary cell of the zetasizer nano zs instrument the same as used for dynamic light scattering measurements. in this study we investigate that size and zeta potential of synthetic peptide-carrier protein covalent conjugates. synthetic peptides are small antigenic molecules and not strong immunogens because of their small size for this reason, synthetic peptide must be coupling to a suitable carrier proteins or polymers (bsa, klh, ova) for increasing their immunogenity. most of the vaccine development studies are focused on the preparation of a good effective adjuvant. in this study we prepare carrier-protein (bovine serum albumin protein)-synthetic peptide conjugates whit carbodiimide method by using 1-ethyl -3-(3dimethylaminopropyl) carbodiimide hydrochloride (edc). for one protein molecule we studied with different ratios of peptides (npeptid/ nbsa) and synthesized the conjugates at these ratios. conjugates are purifi ed by using different column systems. purifi ed conjugates are characterized and the mechanism of the binding process was investigated comparatively with synthetic peptide, bsa and bsa-synthetic peptide physical mixture by using zeta-sizer nano zs depent on the time. an analysis of deletion mutants of the pld1 d4 domain defi nes short regions within the pld1 interacting with ped/pea15: implications for the development of peptides-specifi c antagonist. (1) . several studies have revealed that it regulates cellular functions by binding components of intracellular transduction pathways. recent reports also evidenced that it binds to phospholipase d (pld1) and enhances its stability, resulting in increased intracellular levels of diacylglycerol(2), deregulating protein kinase c signalling and generating resistance to insulin action on glucose transport (3) . thus, disrupting the interaction between these proteins by a cell-penetrating compound represents a novel strategy for improving insulin sensitivity in target cells. the expression of d4 domain, (the shortest ped-interacting region with pld1) in l6 skeletal muscle cells stably overexpressing ped, reduces the interaction of this protein with pld1 (4), suggesting that this region could bind ped preventing its interaction with the whole pld1 and restoring insulin action. aim of this work is the identifi cation of d4 crucial residues for ped interaction and the development of specifi c antagonists. we expressed three soluble truncated d4 domains, determining whether binds to ped like the d4 wild type, by carrying out dose-response elisa. only one of these regions exhibited an effi cacy similar to d4-wt and functional cellular data support this evidence. to further investigate the d4 residues involved in ped binding, we prepared a set of overlapping peptides covering the d4-region identifi ed. our results suggest that the n-terminus region of d4 encompassing residues 762-801 is involved in ped/pea15 recognition and, currently, cellular experiments on peptides are underway. ew-peptide family. one family, two drugs. two dipeptide analogs -l-glu-l-trp (1) and -d-glu-d-trp (2) -had been registered in russia as immunomodulator thymogen (1) and immunosuppressor thymodepressin (2). these two drugs possessed reciprocal activities in vivo [1, 2] . the individual peptides were initially separated by preparative hplc from the crude thymus homogenate and sequenced. dipeptide l-glu-l-trp was the most active in the majority of in vitro and in vivo tests. in the process of sar studies, the "signal" role of l-glu-l-trp in the immune response was discovered, as well as the critical role of manifestation of in vitro effects as well as the infl uence of precise chemical and optical structures on biological activity of different ewpeptide analogs will be discussed. the experiments were conducted on blood neutrophiles, monocytes, thymic epithelial cells, lymphocytes, thymocytes, endothelial cells of human blood vessels and newborn blood cord. we evaluated the peptide effects on the phagocyte activity of cells, on the expression of functionally important membrane molecules and cell activation markers, and on the capacity of cells for mutual adhesion. the dose dependence data of 1 and 2 on such activities as cytokine production, the processes of stimulation and suppression of apoptosis and proliferation of immunocompetent cells will be presented. ion exchange chromatography (iec) is widely used for analysis and purifi cation of biomolecules. we have newly developed polymer-based iec column, named ymc-biopro, specially designed for separation of proteins, peptides and nucleic acids. ymc-biopro iec columns are based on 5 micron @porous and non-porous hydrophilic polymer beads with low nonspecifi c adsorption, and they show higher binding capacity and higher recovery of biomolecules compared to conventional iec columns. the completely spherical and monodispersed beads, with optimal packing technology, provide high theoretical plate number and symmetrical peak shape. excellent resolution is achieved from the high column effi ciency coupled with the excellent selectivity of qa and sp ion exchangers. in this poster, we will show benefi ts of ymc-biopro iec columns and some example cases of superior separation of important biomolecules, such as monoclonal antibody and dna. the analysis of proteins and peptides by rp-hplc is important in the development of well-characterized biotechnology pharmaceuticals. since polypeptides interact only slightly with the stationary phase after desorption, short columns are suited for fast separations. also, by decreasing the particle size of hplc packings, column effi ciency is increased. we demonstrate the use of short hplc columns packed with 1.5um, large pore c18 silica for ultra-fast biomolecule analysis. due to the short column format, high fl ow rates and optimal separations are possible, with moderate backpressures (< 3000 psi), using a conventional high-pressure gradient, binary pump system. narrow-bore lc and lc-ms analyses were performed on a binary or a quaternary hplc system, with detection by uv or ms (ab/mds sciex q trap). the solvent system comprised of acn/water or n-propanol/water plus one or two ion pairing agents (formic acid, tfa, hfba) at 0.01 to 0.2%. a fl ow rate of 0.8 ml/min (1200 cm/h), 4x higher than typical for a 2.1 mm id column, allowed for ultra-fast one minute separations of proteins with broad physicochemical characteristics. closely related insulin variants (bovine, sheep, human) may be separated in under 1.5 minutes. while the typical hplc run time for the separation of hemoglobin chains on conventional columns ranges from 25 to 60 minutes, runs under two minutes may be realized with the large pore, sub-two micron column. accordingly, fi ve different hemoglobin samples (from different animal species) may be compared after a total of only 14 minutes (includes run and equilibration time). 10 highly reproducible runs of a synthetic peptide mixture can be completed in < 20 minutes, including equilibration with 20 column volumes between runs. intact igg in sheep serum may be separated from albumin in < 4 min. using a n-propanol/water solvent system containing 0.1% tfa. new peptides and triterpene derivatives as dimerization inhibitors of hiv-1 protease protein-oligopeptide fragmentomics zamyatnin the substantiation and defi nition of the term "fragmentomics" is given. within the framework of this scientifi c direction the theoretical structural-functional analysis of all possible fragments of protein molecule can be performed with the purpose of determination of its sites which could be potential sources of the regulatory oligopeptides. the data on the primary structure of proteins from public protein databases, information from the erop-moscow database (1) that contains data on the structures and functions of the natural oligopeptides, and special computer program complex were used for it. as a result the natural regulatory oligopeptides both representing exact structures of protein fragments and containing protein fragments were found. the method has allowed also to reveal new potentially active sites of protein amino acid sequence yet not investigated experimentally. it was shown that different fragments of the food protein molecules are involved in amino acid sequences of many natural antimicrobial oligopeptides, toxins, neuropeptides, and hormones. these results confi rmed deep relationship between the basic regulatory systems. in connection with the obtained data, the process of oligopeptide biogenesis, the possibility of natural formation of regulatory oligopeptides from different protein molecules, formation of exogenous oligopeptides pool, and correspondence of the obtained results with the conception of oligopeptide continuum are discussed. a possible practical importance of active fragments of proteins in regulatory processes of a living organism is noted. the binding of peptides containing tyrosine residue to -cyclodextrin cyclodextrins form inclusion complexes with various organic molecules. aromatic amino acid residues bind to -cyclodextrin with deep penetration of the cyclodextrin cavity. in case of oligopeptides binding depends on the peptide conformation. the formation ofcyclodextrin inclusion complexes with the tyrosine residues within three peptides was investigated using steady-state fl uorescence spectroscopy and molecular dynamic simulations. the free energy along the reaction pathway delineating the inclusion of tyrosine's aromatic ring into -cyclodextrin was computed using umbrella sampling molecular dynamic simulation. the association constant and the corresponding association free energy were derived by integrating the potential of mean force over a representative ordering parameter. the three peptides studied consist of eighteen amino acids and the tyrosine residues are located at the position 1, 2 or 4. selected sequences are fragments of notch receptors. (notch1=ykieavqsetveppppaq, notch2 =tlsyplvsvvsesltper, notch3=pyplrdvrgepleppeps). out of three peptides only notch3 binds strongly to -cyclodextrin with binding constant similar to that of actyrnhme. the binding of cyclodextrins with phenolic compounds involves nonspecifi c van der waals and hydrophobic interactions and depends on accessibility of tyrosine sidechain. role of carboxyl groups in the secondary structure and function of sturgeon gonadotropin free negatively charged carboxyl groups were selectively modifi ed (neutralized) in sturgeon (acipenser güldenstädti br.) gonadotropic hormone (gth) and subunits. 11 carboxyl groups, 3 in and 8 in subunit, were neutralized by the reaction with glycine ethyl ester. investigation of re-associated -dimers (recombinants) comprising one or both modifi ed subunits showed that specifi c hormonal activity was completely lost while immunoreactivity was lowered in comparison with that of the standard -dimer. cd-spectroscopy of the modifi ed subunits did not indicate considerable changes in their spatial structure. conclusion was made that free cooh-groups of gth are important as bearers of the negative charge necessary for the hormone activity on the level of the hormone-specifi c membrane receptors. wagner, nathaniel; dadon, zehavit; yishay, eliya; ashkenasy, gonen ben gurion university of the negev, israel living cells can process rapidly and simultaneously multiple extracellular input signals through the complex networks of evolutionary selected biomolecular interactions and chemical transformations. recent approaches to molecular computation have sought to mimic or exploit various aspects of biology. a number of studies have adapted nucleic acids and proteins to the design of molecular logic gates and computational systems, while other works have affected computation in living cells via biochemical pathway engineering. we described recently the graph structure and experimental analysis of a self-organized synthetic peptide network (1-3). the system was designed rationally to operate in neutral aqueous solutions based on sequence selective auto-and cross-catalytic template-directed coiled-coil peptide fragment condensation reactions. here we show that such de novo designed synthetic networks can also mimic some of the basic logic functions of the more complex biological networks. consequently, we describe experimental and simulation studies that highlight the possibility of segments of the networks to express all sixteen two-input boolean logic functions (4, 5) . many studies deal with the folding of double helices in nucleic acids. in contrast, much lesser attention is given to double helices in peptides. nevertheless, the investigation of peptides with alternating l-and d--amino acids, like gramicidin a, shows various double helix patterns with antiparallel and parallel arrangements of the strands. in this study, we want to give a systematic overview on the possibilities of double helix formation in -peptides on the basis of ab initio mo theory. according to general principles, double helices with characteristic intermolecular hydrogen bonding patterns were generated and verifi ed as minimum conformations at the hartree-fock level employing the 6-31g* basis set. the calculations show several types of antiparallel and parallel double helices with different stability, which is strongly infl uenced by backbone substituents. the dengue virus causes a spectrum of clinical symptoms, ranging from mild dengue fever to the severe forms of dengue hemorrhagic fever and dengue shock syndrome. there are four dengue virus serotypes (den1-4). the dengue virus ns3 protease is an attractive target for development therapeutics against dengue. the aim of the study was to investigate the prime side specifi city of dengue virus ns3 protease substrates using proteochemometrics, a new technology for drug target interaction analysis. a set of 48 internally quenched peptides were designed using statistical molecular design (smd), synthesized and assayed with proteases of four subtypes of dengue virus for michaelis (k m ) and cleavage rate (k cat ) constants. the obtained data were subjected to proteochemometrics analysis, concomitantly modeling all peptides on all the four dengue proteases, which yielded highly predictive models for both activities. the interpretation of the models suggested that considerably differing physico-chemical properties of amino acids (aa) contribute to k m and k cat activities. it was found that for k cat activity only p1' and p2' prime side residues played important role, while for k m all four prime side residues, p1'-p4', were important. high cleavage rate (characterized by k cat ) was obtained for peptides having small aa (ser, gly, ala) at the p1' position and small or fl exible aa at the p2' position. for high affi nity (low k m ), at the p1' position aa should be small and moderately hydrophilic, while acidic residue is highly unfavorable. for the p2' position, the most favorable was trp; however, this position allowed a broader diversity of aa. for the p3' position, cys was more benefi cial than the aa present in native cleavage sites of the dengue polyproteins. for the p4' position, the best affi nity would be obtained with ala, gly or cys. the models may be used to modify each p' substrate position to optimize separately substrate affi nity and cleavage rate for den1-4 proteases. identifi cation of novel bioactive peptide sequences from human proteins for the development of potential therapeutics many protein-protein interactions are facilitated by the binding of peptides recognition modules to short linear motifs within the target proteins's primary sequence. therefore, synthetic peptides based on parent sequences of human proteins containing functional motifs are useful tools to uncover protein signaling and interactions. to date, peptide studies typically derive sequences from a single identifi ed protein or (at the other extreme) screen random combinatorial peptides, often without knowledge of the signaling pathways targeted. the objective of the novel bioinformatic approach presented here was to determine whether rational design of oligopeptides specifi cally targeted to potentially signaling-rich juxtamembrane regions could identify modulators of human platelet function (1) . the aim of this project was to identify peptides that span residues strongly conserved in the corresponding proteins in other species (orthologues), but that differ from those in related human proteins (paralogues). synthetic selection rules to avoid unfavorable amino acid combinations and excessively hydrophobic peptides were devised to reduce the risk of by-products during synthesis, postsynthetic degradation and solubility problems. high-throughput in vitro platelet function assays of fatty acid-modifi ed cell-permeable peptides corresponding to these regions identifi ed many agonists and antagonists of platelet function. the combined bioinformatic and experimental screens of human protein subsequences can therefore be used to validate functions of candidate proteins and provide templates for the development of potential therapeutics. denessiouk, konstantin; denesyuk, alexander; johnson, mark s. åbo akademi university, finland many small molecule ligands (or parts thereof) with important roles in the biochemistry of the cell are recognized by different proteins with different cellular functions. these proteins do not necessarily share a common fold since their three-dimensional structures can differ completely such that they are considered to be unrelated proteins. surprisingly, we see on numerous occasions that similar ligands are often recognized in a very similar way by means of a common structural motif formed from main-chain elements of several consecutive amino acids within the ligand-binding site of the proteins. these short polypeptide segments can be found in many protein folds, and variations in their structure can often explain different elements of protein function. in particular, we have characterized the presence of a nucleotide binding motif present in more than 30 protein folds that function in part to recognize the adenine ring system in many essential biological ligands (e.g. atp, nad(p), fad, fmn, coa, etc.). furthermore, a study of unrelated folds among pyridoxal phosphate dependent enzymes led to the characterization of a c nn structural motif for protein recognition of phosphate ions common to 104 fold-representative protein structures that belong to 62 different folds. interestingly, amino acid side chains play little or no role in ligand recognition, explaining the evolutionary independence of such binding mechanisms (i.e. the motifs are found in unrelated folds), while possibly refl ecting the types of interactions that were characteristic of the earliest protein-ligand interactions during early stages of molecular evolution. bioinformatics meets medicine: structure-based peptide design as a basis for drug development here, we present different structure-based approaches on the design of peptides mimicking the whole surface, a specifi c region of a protein or even tumor markers. the potential of such methods stretches from the development of peptide-derived drug candidates to immunological screenings. mimicry of binding sites: we have developed superficial, a program that proposes peptide libraries representing the entire surface or just regions of proteins. these peptides can be synthesised, e.g. using the spot-synthesis technique, and tested for their ability to mimic linear as well as non-linear binding sites. as a proof of principle, a library of peptides representing the surface of lysozyme was generated starting from a crystal structure of a lysozyme-hyhel-5 complex. adjacent sequence segments were linked by spacers to conserve local conformations. disulfi de bonds were generated to tether the peptides. binding assays against the hyhel-5 antibody identifi ed several peptides representing the non-linear recognition site of the lysozyme. translation of a tumor marker into peptides: the tumor-associated carbohydrate antigen gd2 is an established target for immunotherapy in neuroblastoma. we proposed peptidic mimics of this ganglioside for active immunisation against the tumor marker gd2 using molecular modelling. the ability of these peptides to mimic the carbohydrate moiety was verifi ed with in silico docking experiments. in a next step they were successfully tested as mimotopes in a mouse model. design of switchable peptides: photo-switchable compounds are becoming increasingly popular for a series of biological applications. goal is the reversible photo-control of structure and function of biomolecules. a required death domain for the binding of proapoptotic proteins (e.g. bak) to the hydrophobic groove of anti-apoptotic proteins is the bh3 helix. inserting the photo-reactive compound hemithioindigo into this short peptide, stabilization towards proteolytic degradation is achieved. the identifi cation of peptides that bind to antibodies is an important step in characterizing antibody specifi city in order to study molecular recognition. numerous approaches have been developed to select peptides that bind with desired specifi city and affi nity to antibodies of interest. in our study peptide arrays produced by spot technology were used to develop and optimize binders to antibody derived by mutations from the anti-p24 (hiv-1) single chain fv antibody, cb4-1. three mutations were introduced in the complementarity determining region 3 of the light chain being in close proximity with epitope-homologous peptide. this mutated antibody had completely lost its affi nity for the epitope-homologous peptide. a substitutional analysis of epitopehomologous peptide was performed on cellulose, in which all positions of the peptide sequence were substituted by each of the 20 naturally occurring amino acids. the peptides representing the binding activity in spot membranes were synthesized using solid phase synthesis and their binding activity was confi rmed by fl uorescent polarization method. a substitutional analysis indicated that binding to mutated antibody could be restored and improved by combining favourable substitutions at the n-and c-terminal residues of the epitope-homologous peptide. this approach has allowed us to generate binders from non-binders to mutated antibody in high micromolar range. it is well known that physiological regulatory peptides that act as hormones and neurotransmitters are produced by the specifi c cleavage of their precursor proteins that per se have no biological functions. during these processes, many fragmented peptides are also produced from the same precursor proteins. it is expected that they may have various unexpected biological activities, but their biological functions have not been investigated in depth. the neutrophil-activating peptides we recently purifi ed turned out to be the peptides that are cleaved from mitochondrial proteins by proteolysis, suggesting that fragmented peptides produced by maturation and degradation of functional proteins may also have various biological functions [1, 2] . therefore, we named such functional cryptic peptides hidden in protein sequences cryptides, and those cryptides that are derived from mitochondrial proteins "mitocryptides" in particular (3). we then identifi ed many mitocryptides by the combined investigation with "dry" and "wet" experiments, i.e., the sequences of mitocryptides that activate g proteins were predicted based on the distribution of charged and hydrophobic residues and their activities were examined on granulocytic cells. receptors for these peptides were also characterized by the direct cross-linking experiments between peptides and their targeted proteins. moreover, it is demonstrated the presence of a novel signaling mechanism involving mitocryptides. the comprehensive identifi cation of cryptides is expected to lead to the elucidation of various cryptic signaling mechanisms. the 4-benzoylphenylalanine (bpa) residue is widely used as a photoaffi nity label for the study of intermolecular (peptide) ligand-protein (receptor) interactions, where it is thought to function by hydrogenabstraction from met residues followed by covalent c-c bond formation of the resulting radical pair. we are carrying out detailed studies of this reaction in a series of fi ve, structurally rigid, hexapeptides of general sequences boc-u x bu y mu z -ome and boc-u x mu y bmu z -ome, where b = l-bpa, u = aib, m = l-met, and u x +u y +u z = 4, with a view to determining the effects of spacer length (u y = 1-3) on the rate of the intramolecular excited state reaction and the chemical and 3d-structures of the resulting products. the triplet state lifetimes of the bpa residues in the compounds, determined in a deoxygenated, dilute, acetonitrile solution by laser fl ash photolysis, vary in the following order: ubu 2 mu, = 60 ns; umu 2 bu, = 190 ns; ubumu 2 , = 350 ns; ubmu 3 , = 430 ns; and ubu 3 m, = 920 ns. in addition to the information these data provide on the structural requirements for intramolecular excited state quenching in the molecules, they also serve to defi ne the conditions necessary for optimal intramolecular reaction in preparative photolysis experiments. accordingly, the products resulting from ubu 2 mu and ubumu 2 have been prepared in high yields, isolated, and structurally characterized by hplc, mass spectrometry, nmr, and cd techniques. for each of the two photoinduced macrocyclizations, two diastereomers, arising exclusively from the bpa diradical regioselective attack on the met s-methyl group, were found. gattin, zrinka; van gunsteren, wilfred eth, switzerland during the past decades, the dependence of the secondary structure of peptides on their amino-acid sequence has been the focus of numerous investigations. in addition to naturally occurring peptides based onamino acids, peptides based on -amino acids, have raised particular interest because of their potential for pharmaceutical use. these peptides often have high folding propensities and it has been found that peptides with as few as four residue may fold into a stable secondary structure. -peptides offer the possibility to investigate the folding-unfolding equilibrium in the context of very small systems, therefore they have also been the subject of a number of investigations based on atomistic molecular dynamics (md) simulations which revealed that the foldingunfolding equilibrium typically occurs on time scale of nanosecond to tens of nanoseconds. how the backbone bound heteroatoms (oh, nh 2 ,f) infl uence the structure of a peptide is little known by now, both experimentally as well as theoretically. we performed several md studies on the conformational behavior of centrally placed hala( -met) fl uoro and hydroxy analogues, hala( -f) and hala( -oh), as function of chirality and level of substitution to investigate the infl uence of these factors on secondary structure formation and stability. all -peptides were simulated in methanol solution at temperature of 340 k and a pressure of 1 atm using the gromos96 biomolecular simulation software and the gromos 45a3 force fi eld. the ensembles of trajectory structures were analyzed in terms of conformational space sampled by the peptide, folding behavior, structural properties such as hydrogen-bonding and in terms of the level of agreement with the available experimental nmr data, noe's and 3 j-coupling constants. the nitroxide spin-labelled 2,3 -cyclic amino acid poac was synthesized, resolved and its absolute confi guration assigned (k . wright et al., tetrahedron, 2008, 64, 4416-4426 ) in order to be used as a spin-probe to evaluate the 12-helicalpeptide secondary structure. a series of -hexapeptides, b o c -a c p c -p o a c -p o a c -a c p c -a c p c -a c p c -o m e , b o c -a c p c -p o a c -a c p c -p o a c -a c p c -a c p c -o m e , boc-acpc-poac-acpc-acpc-poac-acpc-ome, and b o c -a c p c -p o a c -a c p c -a c p c -a c p c -p o a c -o m e , based on the (3r,4r)-poac enantiomer, combined with (1s,2s)-acpc for confi gurational homogeneity of the amino acid components, was designed. in these hexapeptides two poac residues are incorporated at positions i, i+n (n = 1-4) to observe conformation-related spin-spin interactions. the peptides were synthesized by n-to -c chain elongation of n -boc protected peptide segments in solution. their conformational analyses, performed by cd, ft-ir absorption and esr spectroscopic techniques, will be also described. computational design has been successful in creating new proteins. we focus on the design of a -sandwich protein which is challenged by general problems as h-bonds between neighboring strands being dependend on a tightly packed hydrophobic core and aggregation. an improved version of our program propac was tested to repack the backbone of several natural protein structures with high reproducibility. starting with backbone coordinates we found amino acid sequences by packing amino acid side chains with defi ned conformations (rotamers) into the core of -sandwich proteins. in a fi rst approach we have assembled four synthetic -hairpins on a cyclic peptide template (tasp) to form a -sandwich with two identical four-stranded antiparallelsheets. improvement of surface residues reduced the aggregation of the proteins. spectroscopic analysis shows a fold with a free energy near -25 kj/mol and confi rms the -structure by cd and ftir. we improved the design to a partial asymmetric -sandwich assembled from three different -hairpins. the synthesized molecule was the fi rst -sandwich molecule showing a defi ned fold in 2d-nmr. this data and results from the symmetric betabellins (1) suggest that symmetrical sheets do not fold into defi ned structures. to synthesize completely asymmetric sheets we simplifi ed the synthesis of -sandwiches by directed coupling of two four-stranded antiparallel -sheets. the computed proteins were selected for tight packing of the hydrophobic core and analyzed for a stable fold during dynamic simulations. one of our goals is to fi nd a correlation between the experimentally determined protein stability and the parameters used for the selection of the residues in the hydrophobic core and at the surface. biocatalyst-catalyzed peptide synthesis using inverse substrates as acyl donor sekizaki previously we reported that the p-amidinophenyl esters behave as specifi c substrates for trypsin and trypsin-like enzymes. in these esters the site-specifi c group (charged amidinium) for the enzyme is included in the leaving group portion instead of being in the acyl moiety. such a substrate is termed an inverse substrate (1) . inverse substrates allow the specifi c introduction of an acyl group carrying a non-specifi c residue into the trypsin active site without recourse to a cationic acyl moiety characteristic of conventional substrates. we also showed in an earlier study that inverse substrates were applicable to enzymatic peptide synthesis. therefore, a general method for the preparation of a variety of inverse substrates would be valuable. we designed two series of new type inverse substrates, p-(amidinomethyl)phenyl and m-(amidinomethyl)phenyl esters derived from n-(tert-butyloxycarbonyl)aminoisobutylic acid, were prepared. we also analyzed the kinetic behavior of trypsin towards these synthetic esters (2) . they were found to be readily coupled with an acyl acceptor such as l-alanine p-nitroanilide to produce dipeptide. the optimum condition for the coupling reaction was studied by changing the organic solvent, ph, and acyl acceptor concentration. the optimum standard condition was selected as follow: acyl donor (inverse substrate), 1 mm; acyl acceptor (l-alanine p-nitroanilide), 20 mm; enzyme, 10 m; 50% dmso-mops (50 mm, ph 8.0, containing 20 mm cacl2); 25 ž. an -aminobutyric acid containing dipeptide was obtained in high yield. streptomyces griseus trypsin was a more effi cient catalyst than the bovine trypsin. it was found that the enzymatic hydrolysis of the resulting product was negligible. peptides derived from nk-2 selective for phosphatidylserine as novel cancer agents chemotherapeutic agents, commonly used as anti-cancer drugs, have severe side effects, also affecting healthy human cells. natural antimicrobial peptides, and their derivatives, have gained interest as potential anti-cancer agents. under normal conditions, due to the asymmetric distribution of plasma membrane lipids across the bilayer, mammalian cells comprise phosphatidylserine (ps) only in the inner leafl et. in the case of malignant transformation inner leafl et ps can move to the outer leafl et and act as a surface marker. the surface exposure of negatively charged ps on various tumour cells makes these cells susceptible to killing by cationic membranolytic peptides such as nk-2 (1).the aim of this study is to develop short peptide sequences still acting selectively towards ps exposed on cancer cells without damaging healthy cells. in order to use this new strategy with ps-specifi c peptides it is necessary to analyze the lipid composition of mammalian cancer and non cancer cell membranes. further as a basis for peptide activity studies the biophysical characteristics of cancer cell membranes and healthy counterparts with respect to lipid composition were determined by investigation of liposomal mimics composed of phosphatidylcholine and/or phosphatidylserine by dsc and x-ray. these model systems were also used to screen the activity of a series of peptides derived from nk-2. fluorescence spectroscopy was applied to test the release of fl uorescence marker molecules from liposomes composed of solely ps, pc or pc/ps mixtures in the presence of various concentrations of peptides. data revealed that some nk-2 derived peptides have a high affi nity towards ps causing signifi cant leakage of liposomal content, whereas healthy mammalian cell mimicking pc liposomes were not affected. optimized peptides resulting from these experiments will be used for in-vitro studies on prostate cancer cell lines. in most moths, the sex pheromone production is regulated by pheromone biosynthesis-activating neuropeptide (pban), a 33-34 amino acid neuropeptide that is released from the subesophageal ganglion into the hemolymph in response to physiological and environmental cues. pban exerts its pheromonotropic effects by binding to pbanr, a member of the rhodopsin-like family of g protein-coupled receptors (gpcr) that is predominantly expressed in the pheromone-producing cells of the female pheromone gland. the structure-activity relationship studies for bombyx mori pban have revealed that the shortest peptide with pheromonotropic activity is the c-terminal pentapeptide-amide, pban(29-33)-nh 2 (fsprl-nh 2 ), and that the c-terminal amide group is required for the pheromonotropic activity of pban. in this study, we have analyzed the solution structures of the c-terminal decapeptides derived from bombyx mori pban with an amidated and a free c-termini by two-dimensional nmr. the pheromonotropically active decapeptide-amide, pban(24-33)-nh2 (srtryfsprl-nh2), and its inactive counterpart, pban(24-33)-oh (srtryfsprl-oh), were dissolved in three kinds of solvents: (1) 50 mm sodium phosphate buffer (ph 6.0)/100 mm nacl/0.02% nan3 in 90%(v/v) h2o/10%(v/ v) d2o (buffer a), (2) 500 mm dodecyl phosphocholine (dpc)-d38 in buffer a, and (3) 30%(v/v) 2,2,2-trifl uoroethanol (tfe)-d 3 in buffer a. the nmr data indicated that these decapeptides did not adopt specifi c conformations in buffer a, but they adopted specifi c conformations in the presence of dpc micelles or tfe. these peptides exhibited different chemical shifts for some protons in all the solvents used, and they took similar but different conformations in the presence of dpc micelles. the conformational difference between these peptides may refl ect their difference in pheromonotropic activity. structuring and membrane interactions of the human antimicrobial cathelicidin ll37 cathelicidins are a family of vertebrate host defence peptides with both a direct capacity to inactivate microbes and to modulate components of the innate and adaptive immune systems. they are characterized by a conserved pro-region carrying individual, highly variable antimicrobial sequences, that become active only after proteolytic release, and with quite different structural and aggregational features that lead to differential biological effects on prokaryotic or eukaryotic cells. ll-37 is the only human cathelicidin, and displays a broad-spectrum, medium sensitive antimicrobial activity in vitro that is accompanied by some cytotoxicty towards eukariotic cells at antimicrobial concentrations, and a strong capacity to modulate host immune and healing processes at lower concentrations. these activities likely all involve interaction with biological membranes at some point, and are related to its amphipathic, helical structure and strong tendency to aggregate in specifi c conditions. the latter is an evolved feature absent in some primate orthologues, whose activities appear more limited to a direct antimicrobial action. the structural and aggregational behaviours of ll37 and selected primate orthologues were systematically investigated by biophysical and biochemical methods. these included cd spectroscopy in different buffers, sds micelles or anionic or zwitterionic luvs, transmission ftir spectroscopy, dye release from liposomes, and atr-ftir spectroscopy on supported lipid monolayers, followed by atomic force microscopy to probe morphological effects on lipid order. data was also collected from antimicrobial, cell lysis and cytofl uorimetric assays, giving a more complete picture of the possible mode of action of these helical peptides, and highlighting the importance of biochemical parameters such as structuring and dimerization/oligomerization processes in the selective interaction with biological membranes, leading to cytotoxic or immunostimulatory effects. oligomeric structure of fowlicidin-1, an antimicrobial/ anti-endotoxic peptide from the family of cathelicidin, in lipopolysaccharide bilayer a recurring theme in the structure-function correlation studies of the cationic antimicrobial peptides (amps) is that the formation of oligomeric structures in lipid environments by amps. self-assembly of amps may result disruption of the membrane (outer/inner) structures of the microorganisms by forming pores or ion channels. however, high-resolution oligomeric structures of amps in lipid environments are diffi cult to obtain. to-date, oligomeric structure at atomic resolution of any antimicrobial peptide has not been reported. secondary structures of amps are usually obtained in perdeuterated sds or dpc micelles, as a mimic to the inner cytoplasmic membrane, by nmr spectroscopy. cathelicidins comprise a major family of host-defense antimicrobial peptides in vertebrates. these peptides are synthesized as a part of large precursor proteins containing a well conserved cathelin domain and an extremely variable, in terms of length and amino acid compositions, cterminal region. the c-terminal part of the cathelicidins is bestowed with antimicrobial and lps neutralizing activities. here, we report a tetrameric structure of a 22-residue active fragment of fowlicidin-1 or vk22, one of the fi ve cathelicidins found in chicken, in lipopolysaccharide by nmr spectroscopy. the tetrameric structure of the vk22 determined from trnoe is highly helical. a large number of trnoe cross-peaks were observed connecting residues far apart in the sequence. calculated structures reveal an anti-parallel arrangement of four helices. the interface of the tetramer appears to be rich in polar or charged residues delineating a plausible pore or channel. most of the non-polar residues are found to be exposed indicating plausible interactions with non-polar fatty acyl chains of lps or with membrane in general. the oligomeric structure of vk22 peptide may be useful to understand structure/activity relationship, in particular pore formation, by the helical antimicrobial peptides. the interaction of the antimicrobial peptide nk-2 with different lipid systems antimicrobial peptides are important components of the natural defense system of most living organisms against invading pathogens. these are relatively small (below 10kda), cationic and amphipathic peptides of variable length, sequence and structure. in this study, the antimicrobial peptide nk-2, which is a 27 amino-acid residue derivative of the cationic core region of nk-lysin, has been used. the used membrane mimetic model systems have different dimensionality: two-dimensional (monolayers at the air-liquid interface) as well as three-dimensional (vesicles, micelles) systems of different phospholipids. the aim of this study was to investigate the infl uence of peptide-lipid interactions on the lipid structure and vice versa on the secondary structure of the peptide. the peptide secondary structure was measured by cd (circular dichroism spectroscopy) in bulk and irras (infrared refl ection-absorption spectroscopy) at the air-liquid interface. the structure of lipid langmuir monolayers has been examined by numerous techniques such as pressure-area isotherm measurements, fl uorescence and brewster angle microscopy and x-ray techniques. charged as well as zwitterionic phospholipids have been used to study the adsorption of nk-2. the peptide adsorbs at the air-buffer interface due to its amphiphilic character. it also inserts into uncompressed phospholipid monolayers. the peptide reorients from random coil in bulk to -helix lying fl at at the interface. the incorporation of nk-2 into a dppg monolayer leads to a different orientation (either -helix with an oblique orientation or random coil) and to the fl uidization of the aliphatic chains. nk-2 infl uences also the structure of ordered dppg domains. to assess the location of the peptide nk-2 in the lipid matrix, specular x-ray refl ectivity studies were carried out. wadhwani, parvesh; reichert, johannes; buerck, jochen; ulrich, anne s. karlsruhe institute of technology, germany antimicrobial peptides (amps) constitute an essential part of innate immunity and act against an external microbial invasion where as cell-penetrating peptides (cpps) can carry a biologically functional molecule through the cell membrane and are relevant in drug delivery developments. fusogenic peptides (fps) are active on membraneinterfaces and are instrumental in fusion of membranes which is vital for various fertilization and viral processes. membrane fusion properties of short amps and cpps have been seldom tested and have therefore eluded the attention of most investigators, instead only their cytotoxicity is investigated. there is general lack of understanding if these peptides are fusogenically active. we have investigated the ability of amps and cpps to trigger membrane fusion. as an example, hiv-1-fusion peptide fp23 is used as benchmark to compare fusion activity of various amps and cpps. most fps are believed to be unstructured or conformationally fl exible ( -helix or -sheet); therefore the secondary structure of the peptides before and after the fusion reaction is investigated and correlated to their respective ability to execute membrane fusion. our results show that various amps and cpps which are capable to switch their secondary structure are also able to promote fusion to an extent that is even higher than the known hiv-1 fusion peptide fp23. these results will be presented in the poster. interaction of the minimal active peptide sequence of human growth hormone releasing factor with negatively charged liposomes zschörnig, olaf; thomas, lars; weigelt, heiko; köhler, guido university of leipzig, germany the most bioactive peptides such as hormones and neurotransmitters do not exist in an ordered structure in aqueous solution. the conformation of the peptide changes from this fl exible unordered structure into an inherent one, only when it reaches a biomembrane. that's why it is important to investigate the such peptides like growth hormonereleasing factor (ghrf) in the presence of lipid bilayers. human ghrf is an amidated peptide consisting of 44 amino acids residues. a lot of studies has show that the residues n-terminal part are the active core of the peptide. we studied the interaction of human ghrf (1-29) with phospholipid membranes. the interaction of ghrf (1-29) with the liposomes was investigated fl uorescence spectroscopy. to detect a fl uorescence signal the peptide were labelled at fi rst, 15th, 24th and 29th position with dansyl. the fl uorescence intensity of ghrf (1-29) at different lipid-peptide-ratios were analysed using a model, which includes the hydrophobic and the electrostatic interaction between the peptide and the phospholipid membrane. the hydrophobic binding constant of ghrf(1-29) and its effective charge were determined using this model. further the peptides position in the membrane were investigated using spin labelled phospholipids, which are able to quench fl uorescence over large wavelength arrays. the quenching effi ciency will decrease by increasing the distance between fl ourophore and spin probe. the use of 15 mol% tempo-pc, 5-doxyl-pc, 10-doxyl-pc and 16-doxyl-pc in the phospholipid composition of the liposomes allows the determination of the peptides membrane penetration depth. all results indicate, that the binding of ghrf (aa 1-29) to phospholipid membranes is increased strongly by increase of the surface charge density of the liposomes. the peptide is arranged parallel to the membrane surface and is localized in membrane-water-interface of the liposomes. folding propensity and biological activity of selected antimicrobial peptides bozzi, argante 1 ; di giulio, antonio 1 ; aschi, massimiliano 1 ; rinaldi, andrea c. 2 1 university of l'aquila, italy; 2 university of cagliari, italy the innate immunity of multicellular organisms relies in large part on the action of antimicrobial peptides (amps) to resist microbial invasion. crafted by evolution into an extremely diversifi ed array of sequences and folds, amps do share a common amphiphilic 3-d arrangement. this feature is directly linked with a common mechanism of action that predominantly (although not exclusively) develops upon interaction of peptides with cell membranes of target cells. it is generally agreed that amps are essentially unstructured in the aqueous phase and fold upon contact with the membrane, adopting an amphiphilic fold. this favours absorption of peptides onto lipid bilayer and their subsequent integration into the membrane with expansion of the outer leafl et, which in turn leads to membrane thinning and permeabilization. however, recent observation suggest that things might be more complicated than previously believed. indeed, md simulations coupled to cd spectroscopy, studies with model membranes, and antimicrobial assays, have shown that, at least for some peptides, a signifi cant correlation exists between the conformation adopted by the peptide in solution, i.e. before the interaction with membranes, and its antimicrobial activity. the linear peptides we have studied following this approach include two members of the frog skin-derived temporin family, namely temporin a and temporin l, and two members of amphibian bombinins h, i.e. bombinins h2 and h4. we observed that the presence of a partially folded structure in water solution may facilitate, both thermodynamically and kinetically, the peptide folding in the microbial membrane, and thus favour biological activity. looking for such built-in conformational characteristics could well help to rationalize the different spectrum and level of activity recorded for cationic alpha-helical amps on membraneenveloped targets, and assist the design of improved analogs and biomimetic synthetic peptides with antibiotic properties. investigation and computational modelling of antimicrobial peptides linser the uprising resistance of pathogenic bacteria against treatments with conventional antibiotics emerged an acute search for alternatives. one class of promising alternatives are naturally occurring antimicrobial peptides. we present a comparative study of computational modelling of peptide properties with structural characterization of the interaction and antibacterial or haemolytic activity of three peptides (nk-cs, nkcs-[lp] and nkcs-[aa]). we compared computational interaction models with measurements of antibacterial and haemolytic activity, small angle x-ray and surface plasmon resonance data, and structure predictions by circular dichroism (cd). all peptides were active against escherichia coli (gram negative) and staphylococcus carnosus (gram positive) bacterial cultures, but the haemolytic properties against human red blood cells were found to be poor and indicated the peptides' selectivity. cd studies of the peptide secondary structure confi rmed the prediction of peptide helicity. the antibacterial activity can be correlated with a change of the hexagonal phase transition temperature of 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine (pope) as determined by small angle x-ray scattering (saxs). the calculated peptide membrane affi nity is not related in linear way with the antibacterial activity. the reason for this might be aggregation as shown by surface plasmon resonance. the inverse hexagonal phase transition temperature was increased by the peptides and this promotes a positive curvature of the membranes. we assume that this curvature fi nally leads to the disruption of the model membranes. in summary an over all helical structure, electrostatic and hydrophobic parameters as well as strong amphipaticity are good measures to describe antibacterial peptide interaction. kier, brandon; andersen, niels university, united states over the last decade, -hairpins have emerged as excellent model systems for probing the intrinsic stabilities of portions of -sheet structure and for studying the sequence dependence of the folding dynamics of -strand association and alignment. for many decades, it has been established that peptide helices can be substantially stabilized (3 -7 kj/mol) by starting the sequence with an n-cap (acetyl, asp or sede). no comparable strategy has appeared for reducing the end-fraying of -hairpins. fully folded models of hairpins have typically been constructed by conversion to a cyclic system: inserting another turn favoring sequence (e.g. d-pro-gly, pg) at the end of the hairpin strands. we now report a set of favorable through-space interactions that can serve to cap -hairpins. the discovery followed from nmr studies of ac-w-ixgk-wtg (x = p, n). these studies indicated a favorable face-to-edge (fte) w1/w6 interaction which also allows for a g8-hn to w6 indole ring h-bond and the sequestration of the thr hydroxyl by h-bonding to the acetyl. these interactions produce spectroscopic diagnostics: a cd exciton couplet together with extreme upfi eld shifts for he3 of the c-terminal trp (1.5 -2.1) and hn of the c-terminal gly (2.7 -3.6 ppm). these feature disappear when x = l-pro and the gly-hn shift returns to its coil value (as in ac-wtg) upon removal of the n-terminal ac. in its most favorable application, ch3ch2co-wipglwtgps, we were able to establish that dgu at 280k was 8.9 kj/mol (97.8% folded) by backbone hn h/ d exchange protection measures. we now report that these interactions are retained in longer hairpins. peptides of the general formula, ac-w-(zz)ningk(zz)n-wtg (n = 1, 2, or 3) display enhanced fold stability. in each case, the ac is essential to prevent end-fraying and to elicit the full set of chemical shift diagnostics of the " -cap". the unique structural features of polyproline peptides to adopt an extended, relatively rigid polyproline ii (ppii) backbone conformation in aqueous solution, has led to their widespread application as a "molecular ruler" in biological and biophysical investigations. in the left handed ppii helix the peptide bonds show the all-trans conformation resulting in an interterminal distance, which increases by 2.8 to 3.1 å per proline residue. the same backbone conformation has been identifi ed in important proline rich protein-protein recognition motifs involved in the regulation of physiological processes like transcription, cell growth, cytoskeleton rearrangement and postsynaptic signal transduction via modular sh3, ww or evh domains. to investigate the structural features of polyproline and proline rich sequences by fl uorescence resonance energy transfer (fret) and other spectroscopic methods, these peptides were labelled n-and c-terminally with different fl uorescent dyes. for synthesis of homopolymeric polyproline peptides with a sequence length of more than 20 amino acids a fragment condensation strategy has been applied to prevent the occurrence of shorter side products which can perturb distance measurement by fret. spectroscopic analysis of these peptides indicated that even for short polyproline sequences (< 5 residues) there is a remarkable deviation from the expected ppii conformation. fret measurements revealed that polyproline peptides show an increase of the mean interterminal distance of about 1.7 å per proline residue. single molecule fret measurements of polyproline peptides with a length of 21 amino acids point to a heterogeneous ensemble of different conformations caused by randomly distributed cis conformations resulting in diverse species with different interterminal distances. similarly the structural consequences of the integration of non-proline amino acids in a polyproline sequence have been investigated by fret. the unusual helix stability of a vegf mimetic peptide understanding helix stability and formation is a prerogative to elucidate mechanism of protein folding and design helix peptide with specifi c activity. peptide helix is a simple model system in which various contributions to helix formation can be dissected and understood qualitatively. many strategies have been pursued to design peptide helices and notable results have been achieved even with very short sequences, but mainly these methods rely on the use of non natural amino acids or introducing constraints. in this communication, we report the stability characterization, via cd, nmr and md studies, of a designed, -helical, 15-mer peptide, composed only of natural amino acids, which activates the vegf-dependent angiogenic response (1). this peptide shows an unusual thermal stability whose structural determinants have been determined. two factors, the n-terminal region and an hydrophobic interaction i, i+4, are found as playing a mayor role of this remarkable stability (2) . these results could have implication in the fi eld of protein folding and in the design of helical structured scaffolds for the realization of peptides to be applied in chemical biology. adiponectin, a cytokine secreted by adipose tissue, which has been shown to affect lipid and glucose metabolism, attracts interest because it is a target for therapeutics in the metabolic syndrome. the molecule has a tendency to associate to form various multimers. although this multimer formation could modulate its biological function, the details of this mechanism are still unclear. thus studies on this system from the aspect of molecular assembly are interesting. the molecule consists of three domains, i.e. a variable (v), a collagen-like (c) and a globular (g) domain. the structure of the g domain determined by x-ray crystallography showed that it exists as a trimer but the remaining c and v domains have not been well characterized. we synthesized various parts of the molecule, c, g, vc, cg, and full length vcg in e. coli. the cd spectra of cg and vcg showed a positive peak around 230nm which is the hallmark of collagen structure. this may be attributed to the repeats of typical collagen type triplet, x-y-gly. the temperature dependency of these cd spectra showed the three states conformational changes of these peptides. the lower transition, where the positive peak disappeared corresponds to the melting of collagen like structure and the higher one corresponds to that of globular structure of the g domain. the apparent molecular weights determined by ultra-centrifugal analysis showed that these peptides exist in trimeric form at the intermediate state. however, neither c nor vc showed such transitions. these results showed that, contrary to our expectations, the contribution of the triplet of x-y-gly is not the dominant one for stabilizing the trimeric state. in order to explore the stabilizing factor, we are carrying out various experiments, such as mutation analysis, titration of ca ++ , redox reaction of disulfi de bonds and so on. one result is that ca ++ was shown to be a factor for increasing the thermal stability of the trimer. are v57 mutants of amyloidogenic protein -human cystatin c more or less resistant to denaturation conditions? jankowska, elzbieta; orlikowska, marta; radulska, adrianna; szymanska, aneta university of gdansk / faculty of chemistry, poland cystatins are natural inhibitors of cysteine proteases -enzymes widely distributed in animals, plants and microorganisms. human cystatin c (hcc) has been also recognized as an amyloidogenic protein directly involved in formation of pathological fi brillar aggregates which deposit in the brain arteries of elderly persons causing cerebral amyloid angiopathy (1). our studies were performed to explore possibilities of preventing this lethal disease. the overall 3d architecture of monomeric human cystatin c is not known but its fold could be anticipated from the structure of the dimer which has been already determined (2) . the dimeric cystatin c is created through the exchange of 'subdomains' between two molecules (3d domain swapping) and consists of two identical subunits in great extent reconstituting the fold of the monomeric chicken analogue of hcc. it is possible that similar mechanism is involved in cystatin c oligomerization and fi brilization process. the most signifi cant structural changes during dimerization process are observed in the l1 loop (55-59, qivag). this loop occurred to be a hinge region in the 3d domain swapping event. with the aim to check implications of greater or decreased stability of this loop for dimerization and aggregation propensity of human cystatin c, we designed and construct hcc l1 mutants with val57 residue replaced by asp, asn or pro, respectively. the structural studies of these mutants and their thermal denaturation process have been performed by means of cd and ftir spectroscopies. results of these studies will be presented. azobenzene-mediated photomodulation of a collagen triple helix monitored by ir spectroscopy (1), our most recent efforts were addressed to the design and synthesis of a photoswitchable collagen triple helix. by replacing in suitable positions of the ac-(gly-pro-hyp) 7 -nh 2 a pro and hyp residue with (4s)mercaptoproline, respectively, a side chain-to-side chain crosslinking of the collagen peptide was afforded with a purposely designed azobenzene derivative. as expected from modeling studies, self-association of the modifi ed collagen model peptide into a stable triple helix was observed with the trans-azobenzene clamp, while its photoisomerization to the cis isomeric state leads to unfolding processes as well assessed by nmr structural analysis (2) . unfolding pathways can be studied by comparing ftir difference spectra induced by temperature or light. we found the photomodulation of the triple helix to be reversible and the effi ciency of the photoisomerization increased with temperature reaching a maximum value shortly below the melting point. ir spectroscopy was thus used to identify the optimal temperatures required for structure destabilization at suffi cient extents to enable unfolding by the weak driving force of the azobenzene clamp. the results confi rmed the correctness of the design and the ability of the azobenzene switch to photocontrol this complex tertiary structure, thus allowing for time-resolved monitoring of the triple-helix unfolding process. studies on various collagen model peptides (x-y-gly) 10 where x and y are often imino acids, pro or hyp r (4(r)-hydroxyproline), have shown that hyp r at the y position plays an important role in stabilizing the collagen triple helix. however substitution of non-natural 4(s)-hyp (hyp s ) at both positions decreases the thermal stability of triple helix as (pro-hyp r -gly)10 exists in a stable triple helical state, whereas (hyp s -pro-gly)10 and (pro-hyp s -gly)10 are in a single coil state. similar effects on the stabilities of triple helices are provided by the substitution of fl uoroproline. however there is an exception that (fpro s -pro-gly)10 takes a triple helix at 4 c whereas the counterpart, (hyp s -pro-gly)10, is in a single coil state. although the difference could be explained by the steric hindrance of hydroxyl group in s confi guration, it is still controversial how much this hindrance could obstruct the triple helix formation. therefore, even though apparently the tripeptide with the hyp s -pro-gly sequence is not capable of triple helix formation, we could expected that (hyp s -pro-gly) n forms a triple helix as n increases. the transition temperature of (pro-pro-gly) n was shown to have the chain length dependency. here, we synthesized (pro-pro-gly) n and (hyp s -pro-gly) n (n=10,15) and investigated their thermal stabilities. the cd spectra of (hyp s -pro-gly)15 showed the conformational transition from collagenlike triple helix to random coil with the melting temperature at 21 c. the apparent molecular weight, determined by the sedimentation equilibrium method, showed that (hyp s -pro-gly)15 exists as trimer at 4 c and as monomer at 37 c. the melting profi le was also clearly detected by dsc. @thus, it is concluded that the existence of hyp s at the x position is not essential to interfere the triple helix formation. we are on course to investigate the stabilizing mechanism of the collagen structure. the infl uence of o-glycosylation on the folding and stability of -helical coiled coil peptides glycosylated proteins have been shown to play a key role in many biological events like cell-cell communication, immune response, cell adhesion, intracellular targeting, protease resistance, and many other processes. recently, there is a growing interest in the effect of glycosylation on the secondary structure of proteins, because of the association with the so called conformational diseases that arise from the dysfunctional aggregation of proteins in not native conformations. in this study we used -helical coiled coil based peptides as model systems to investigate the effects of serine-linked -galactose on both the secondary structure and amyloid formation tendency. thehelical coiled coil structural motif consists of two to seven -helices which are wrapped around each other with a slight superhelical twist. its simplicity and regularity have made it an attractive system to explore fundamentals of protein folding.(1) the importance of the coiled coil motif is obvious with the amount of 5% of all amino acid residues in the protein data bank being part of a coiled coil motif. at fi rst we examined to which extent and at which positions a glycosylation is possible without destroying the coiled coil structure. therefore, we systematically incorporated one to six serine-linkedgalactose units into several solvent exposed positions of a 26 amino acid long coiled coil peptide. the hydrophobic dimerization face was not modifi ed. the preformed l-ser(ac 4 --d-gal)-oh building blocks were introduced by convenient solid-phase synthesis following the fmocstrategy. folding and stability of the glycopeptides were monitored by cd spectroscopy. the amyloid formation tendency was investigated by a tht fl uorescence assay. aging, associated with decreasing protein homeostasis (proteostasis) capacity and increasing oxidative stress, is a prominent risk factor for amyloid diseases. alzheimer fs disease (ad), which is one of the most common amyloid diseases, involves intra-and extracellular amyloid formation by the amyloid bata peptide (abeta). however, how abeta can aggregate in vivo even though its physiological concentration (pc) is much lower than its critical concentration (cc) for aggregation is a mystery. we have proposed that covalent modifi cation of abeta by small molecule oxidation products can explain how abeta can form amyloid at pc. the aldehyde-bearing cholesterol oxidation product 1(2), which can modify abeta by schiif-base formation, is an example of the products that could affect ad onset. however, signifi cant questions about the modifi cation of abeta by 1(2) persist, including: does modifi cation by 1(2) lower the cc of abeta aggregation into the pc range? and, is abeta modifi ed by 1 (2) able to aggregate at low concentrations on a biologically relevant time scale? in this study, these questions are answered by studying chemically synthesized analogs of abeta that are site-specifi cally modifi ed by 1(2) at asp1, lys16, or lys28. modifi cation at the different sites has a similar effect on the thermodynamic propensity for aggregation. in contrast, the effect of metabolite modifi cation on aggregation kinetics depended strongly on the modifi cation site. abeta modifi ed at lys16 formed amorphous aggregates fastest and at the lowest concentrations. in contrast, the appearance of thiofl avin-t positive aggregates at higher concentration was fastest for abeta modifi ed at asp(1). the infl uence of modifi cation site on the nature of the aggregates suggests that amorphous aggregation and fi brillization place different conformational demands on abeta. furthermore, these studies may partially explain how abeta can aggregate at nm pcs when the cc of unmodifi ed abeta is in the ƒêm range. code, christian; domanov, yegor; kinnunen, paavo k.j. antimicrobial peptides are ubiquitous in nature and consist of several families of cationic amphiphilic peptides. they partition into lipid membranes and promote the segregation of anionic phospholipids (2, 3) . we have shown several antimicrobial peptides, e.g. temporin b and l, indolicidin, and magainin 2 to activate secretory phospholipase a 2 (pla 2 ) (1, 4) and we concluded that this could represent synergistic action of these peptides/proteins in defense against microbes. the fact that the sequences of these amps are very different suggest rather non-specifi c mechanism to be involved. to pursue the latter in more detail we used förster-type resonance energy transfer (fret) between labeled pla 2 and temporin b. interestingly, fret coincides with concentration dependant activation of pla 2 hydrolysis of dipalmitoylphosphatidylcholine (dppc) liposomes. accordingly, temporin b and pla 2 interact forming a supermolecular complex terminating into amyloid-like fi brils (4). homo-oligomeric fi bers are formed on a slower time scale when pla 2 interacts alone on dppc liposomes (5) . a general mechanism of peptide induced pla 2 activation forming heterooligomeric cofi brils is suggested. human islet amyloid polypeptide forms lipid-encased amyloid fi brils on supported lipid membranes domanov, yegor; kinnunen, paavo university of helsinki, finland islet amyloid polypeptide (iapp) forms fi brillar amyloid deposits in the pancreatic islets of langerhans of the patients with type 2 diabetes mellitus and its misfolding and aggregation are thought to contribute to -cell death. increasing evidence suggests that iapp fi brillization is strongly infl uenced by lipid membranes and, vice versa, the membrane architecture and integrity is severely affected by amyloid growth. we performed direct fl uorescence microscopic observations of the morphological transformations accompanying iapp fi brillization on the surface of supported lipid membranes. within minutes of application in submicromolar concentrations, iapp caused extensive remodelling of the membrane including formation of defects, vesiculation, and tubulation. the effects of iapp concentration, ionic strength, and the presence of amyloid seeds on the bilayer perturbation and peptide aggregation were examined. growth of amyloid fi brils was visualized using fl uorescently labelled iapp or thiofl avin t staining. two-colour imaging of the peptide and membranes revealed that the fi brils were initially composed of the peptide only, and vesiculation occurred in the points where growing fi bres touched the lipid membrane. interestingly, after 2-5 hours of incubation iapp fi bres became "wrapped" by lipid membranes derived from the supported membrane. progressive increase in molecular level association between amyloid and membranes in the maturing fi bres was confi rmed by förster resonance energy transfer (fret) spectroscopy. the possible role of lipid wrapping in stabilization of iapp amyloid in vivo is discussed. fibrinogen-derived peptides that mediate cell adhesion: structure and activity studies interaction of human islet amyloid poly peptide with phospholipid membrane vesicles dannehl, claudia; zschörnig, olaf university of leipzig, germany amylin, also known as human islet amyloid polypeptide (hiapp), is a 37-residue peptide, suspected to play a major role in the malfunction of insulin secretion in diabetes mellitus type ii. co-secreted with insulin in the beta-cells, hiapp, in higher rates destroys the barrier function of the beta-cells, leading to a failure in insulin production. because of its amyloidogenity, aggregates of fi brils can be observed in the islands of langerhans to indicate its overexpression. we studied the physico chemical properties of hiapp by observing changes in its structure depending on time and the surrounding media using maldi-tof-ms, atr ft-ir-spectroscopy. to understand the process of penetration and toxicity to cells, we performed leakage measurements of carboxyfl uoresceine containing phospholipid large unilamellar vesicles by means of fl uorescence spectroscopy. moreover we determined membrane binding of dansyl-labeled hiapp. at physiological ph value, hiapp is positively charged and thus negative charges at the phospholipid membrane surface accelerate the process of peptide folding. being random coil as initial state, a mixture of anti-parallel betasheet and alpha-helices emerges in time. in the presence of negatively charged phospholipids, hiapp aggregates can be seen within a few minutes after titration. also in absence of any free charges, as seen in water, fi brils grow and after an incubation for 24 hours at 37°c, some alpha-helices are twisted and after two weeks, no random coil is detected anymore. titration of hiapp to carboxyfl uoresceine fi lled liposomes, showed different results concerning equilibrium time and maximal extent depending on age and preparation of the peptide. in particular the composition of the vesicles seems to determine their stability in the presence of hiapp. nanoparticle induced folding and fi bril formation of a coiled coil peptide nanoparticles present large surface areas and are capable to catalyze fi bril formation of peptides. (1) . one assumes that the nature of surface controls which of the peptides will interact with the nanoparticles and that an enhanced local peptide concentration reduces the lag-time for aggregation. in previous studies, we reported the design of a coiled coil peptide that can adopt a random coil, -helical structure, and -sheet folding in dependence of ph and peptide concentration. (2) here, we expose this peptide to au nanoparticles that are either negatively or positively charged. the interaction of peptide and nanoparticle was investigated by cd and uv/vis spectroscopy, gel electrophoresis, and transmission electron microscopy. we found that electrostatic interactions with au nanoparticles can affect the peptide folding resulting in more than one secondary structure, namely, a competition between the -helical and -sheet structures. the latter folding is very likely a consequence of the high local peptide concentration on the surface of nanoparticle that facilitates a -sheet fi brillation of peptide. moreover, several factors such as ph, peptide concentration, and size of the nanoparticle have a strong infl uence on the nanoparticle-mediated folding. these results provide valuable information on pharmaceutical applications of nanoparticles and will help to reduce possible adverse effects. antimicrobial peptides are synthesized by all living organisms as a part of their innate immune system. those molecules are endowed with a broad spectrum of activity against pathogens. they can be divided into two classes differing in the mechanism of killing: membrane disruptive antibiotics cause a dysfunction of the membrane and subsequent cell lysis; non-membrane disruptive peptides are focused on the intracellular targets. in both situations the initial stage consists in the interactions with the cytoplasmic membrane, in most cases without the exploitation of any receptors. a non-receptor type of interactions decreases the possibility of development of microbial resistance and makes peptides a very potent alternative to conventional antibiotics. in the face of the reduced effi ciency of traditional drugs there is an urgent need for the design of new therapeutic agents with the optimized activity. monte carlo simulations of peptide-membrane interactions can be one of the strategies. that method takes in the account biophysical properties of a membrane as well as structural and physicochemical nature of peptides. in our work we are focused on the development of new analogues of nkcs, a derivative of potent antibiotic peptide nk-2. in the present study the outcome of monte carlo simulations is compared to experimental results of antibacterial tests performed against escherichia coli. the insight into the molecular mechanism of peptides activity is obtained in vitro using saxs method and artifi cial systems mimicking a bacterial cytoplasmic membrane. the results indicate that monte carlo modelling is a good tool to predict the peptide -membrane interactions and can be very useful for the design of novel antibiotics. a 16-35 peptide: structural features in membrane mimicking systems amyloid (a ) peptides, the hallmark of alzheimer disease, depending upon conditions, undergo conformational transition from soluble monomers to the highly toxic -sheet oligomers which form the mature fi brils. conformational and biological analyses were carried out on several different a fragments, to understand the role of the single residues in the fi brillation process. a 25-35, is considered the shortest fragment exhibiting large -sheet aggregates and retaining the toxicity of the full-length peptide. we recently reported the conformational analysis of the synthetic a 25-35 under several solution conditions, and analyzed the modulation of the conformational behaviour of a 25-35 peptide, by interaction with nicotine-like molecules. a 16-35 is the 20-mer a peptide, composed of the a 25-35 fragment, and additional n-terminal residues, known to be endowed with fi bril disaggregating activity. a 16-35 encompasses part of hydrophobic (1-28) and hydrophilic (29-35) a -regions. due to the amphipathic character of this fragment, common to the full a 1-42 and a 1-40, a tossicological mechanism involving a -peptide-membrane interaction may be hypothesized. on these bases we decided to perform the structural investigation of the fragment a 16-35 in membrane mimicking systems including micelle and vesicles aggregates, differing for composition and structural complexity. high resolution three dimensional structure was solved by nmr spectroscopy in negatively charged sds and in dpc/ sds mixed micelles. fluorescence and epr spectroscopies were used to monitor the peptide lipid interaction. the data agree on the critical role played by the membrane composition on the stabilization of different conformers, potentially driving to different oligomeric and/or polimeric toxic species. machan, radek; jurkiewicz, piotr; benda, ales; hof, martin j.heyrovsky institute of physical chemistry, czech republic charge and hydrophobicity are important determinants of membrane activity of antimicrobial peptides. the model peptide lah 4 whose charge can be easily controlled by ph of the medium (1) is a very convenient tool to study the relationship between physical properties of peptide molecules and their membrane activity. it was shown that the changes in the charge of lah 4 molecules result in changes in their orientation with respect to phospholipid bilayers (1). in the present study, effects of lah 4 on the properties of phospholipid bilayers at different ph values are studied by several methods. its infl uence on the lateral mobility of lipids within an spb and on the stability of suspensions of large unilamellar vesicles were characterized by fl uorescence correlation spectroscopy, showing evidence of peptide induced aggregation of lipids at basic ph. changes of hydration and local viscosity within the bilayer following changes in orientation of peptide molecules were measured by solvent relaxation technique. the effect of phospholipid charge was also taken into account in each case. several bioactive peptides, such as antimicrobial, cell-penetrating or fusogenic peptides, exert their biological function by interacting with cellular membranes. therefore, structural data on the location of these molecules inside lipid bilayers are very important for a detailed understanding of their mechanism of action. fluorescence spectroscopic methods are particularly suited to the study of peptide-membrane association, but give only low-resolution information on peptide position in the lipid bilayer. molecular dynamics simulations, on the other hand, can provide a very detailed picture of the peptide-membrane interaction, but need to be validated by quantitative comparison with experimental data. we applied several fl uorescence approaches, together with md simulations, to the investigation of two antimicrobial peptides: the lipopeptaibol trichogin ga iv, and pmap-23, a member of the cathelicidin family. to perform the spectroscopic studies, a variety of peptide analogues containing a single fl uorophore were synthesized. fluorescence spectra, depth-dependent quenching experiments, and peptide-translocation assays were employed to determine the location of the two peptides inside lipid bilayers, in particular as a function of peptide/lipid ratio. molecular dynamics simulations were performed by a "minimum bias" approach, starting from a random mixture of water, lipid and peptide, and following the spontaneous self-assembling of the lipid bilayer. the fi nal membrane-bound 3d-structure is in quantitative agreement with the position of the fl uorescent labels determined by depth-dependent quenching experiments. for both peptides investigated, the atomic details of md simulations provide new insights on the mechanism of membrane destabilization. acknowledgements: with the support of the ministry of education, university and research, and of foreign affairs of italy. cavaco-paulo, artur university of minho, portugal surfactant proteins (sp-) are found in lungs of mammalians. most surfactant proteins have a carbohydrate binding domain (crd) and neck domain. crd have defense function in mammalian mucosa's, by eliminating microbes, due to strong binding to sugars in the cell walls. neck domains are found to order phospholipids allowing the exchange of oxygen between the alveoli and blood. x-ray structures indicate that neck domains are alpha-helixes, but circular dicroism indicates that in the presence of phospholipids, those peptides present a disordered structure. neck domains with 20 residues from natural sequences of sp-a, sp-b, sp-c, sp-d have been tested to be delivered in lipophilic media. only disordered structures would have the ability to cross the lipid barriers. the results obtained here indicated that neck domains possibly can be used as carriers to deliver molecules across skin and hair. fraternal twins ! -peptides and oligoureas are isosteric, isostructural foldamers endowed with yet distinct biomolecular recognition properties. in the fi eld of peptidomimetics, there has been a sustained interest towards the design of non-natural oligomeric backbones with new folding patterns. over the past 12 years, the amide linkage has become the quintessential motif to elaborate folding oligomers. aliphatic and aromatic oligoamides (peptoids, -, -peptides) have provided numerous helical-folded structures, many of which have shown interesting biological activities (1). interestingly, substituting urea for the ch 2 -co-nh units in the 4 -peptide backbone represent a spectacular case of isosteric and iso-structural replacement. detailed nmr studies of the resulting oligoureas revealed a helical fold very similar to that reported for the cognate 4 -peptides. defi nitive confi rmation of this isostructural relationship came with the recent x-ray structure determination of the canonical 2.5-helix of oligoureas. how such isosteric and isostructural oligoamide and oligourea backbones compare in biomolecular recognition events is an interesting question that we attempted to address in the present work. notably the two systems were compared for their antimicrobial activity, membrane interaction and disruption properties. both -peptides and oligoureas designed to mimic globally amphiphilic alpha-helical host-defense peptides have been synthesized and tested. the results showed a surprising dichotomy in bactericidal activity between the two isostructural systems and enlightened the unique antibacterial of amphiphilic oligourea helices. to question whether this functional difference results from differential membrane disruption activities, we have undertaken detailed physicochemical investigations using negatively charged phospholipid membranes as model systems. understanding of the cellular uptake of an amphipathic cell penetrating peptides complexed with sirna konate, karidia; divita, gilles; heitz, frédéric crbm umr5237 -cnrs, france the effi ciency of delivery of macromolecules into living cells is very important for therapeutic purposes. the discovery of a class of peptides, known as cell-penetrating-peptides (cpps), with the ability to mediate translocation of various cargoes both in vitro and in vivo has provided new perspectives in the fi eld of delivery. we have designed a new secondary amphipathic cpp, caddy, which can transfect sirna. many studies have tried to elucidate cpp internalization mechanisms and there is currently still much controversy between endocytosis phenomena and direct interaction with membranes. however, elucidating the interactions between peptides and lipid membranes is essential to understand how caddy delivers cargoes in cells. to highlight these interactions, we have applied biophysical method to phospholipids monolayer and bilayer as model plasmic membranes. regarding the increase of the phospholipids monolayer surface pressure in presence of caddy, it is obvious that this peptide have high affi nity with lipids. while cholesterol presence does not induce anything in peptide insertion on a phospholipids monolayer, the cargo and a widely studied partner of the internalization: heparan sulfate of the gag' s family, do not induce the same behaviour. the intrinsic probe of caddy, tryptophan residues, and the fitc probe bound to the cargo allowed to follow and identify interactions between these two entities by fl uorescence spectroscopy. adding a bilayer vesicular solution unsettle these interactions, tryptophan residues interact with phospholipids while fitc probe are less embarrassed by peptides. caddy alone in solution is not structured but cd measurements show a typical spectrum of helical conformation in presence of phospholipids vesicles. and when the peptide complex its cargo, cd spectrum show a contribution of the sirna relevant of a conformational change inside both partners interacting together. high membrane coverage as the basis of antimicrobial peptide activity the interaction of the antimicrobial peptides (amps) omiganan (h-ilrwpwwpwrrk-nh 2 ) and bp100 (h-kklfkkilkyl-nh 2 ) with model bilayers was characterized. the activity and selectivity of these peptides could be attributed to a strong preference towards anionic membrane model systems, which mimetize bacterial membranes. regarding the interactions with bacterial membrane models, there were marked differences in the interaction patterns, as well as in functional properties of the peptides at high peptide:lipid ratios. these differences occurred for both peptides, despite their being unrelated in sequence and in occurrence in nature. such events at high membrane coverage could represent the equivalent at the molecular scale of the conditions at which the antimicrobial activity of the peptides is triggered. although the lipid: peptide ratios at these transitions are lower than 10 phospholipids per peptide molecule, the plausibility of this hypothesis was demonstrated taking into account an estimate of the amount of lipid per bacterium, and the bacterial concentration in minimum inhibitory concentration (mic) assays. according to the partition constants obtained towards bacterial membrane models, these peptides are expected to reach, at the mic, precisely those high concentrations in the membrane. in addition, surface charge neutralization was shown to occur in these conditions. activity at high membrane coverage is thus likely not only for these peptides but also for any peptide displaying high membrane affi nity and micromolar mics, which is common amongst amps. biosensors based on artifi cial membrane system that permits the functional reconstitution of transmembrane receptors have been studied for the past decade. immobilized liposome encapsulates intracellular chemicals in the internal water cavity and provides a potential advantage over supported planar lipid membrane. to prepare a stable liposome adlayer, we have proposed an immobilizing method based on small-peptide modifi cation of solid surface. in the present study, we investigated kinetics of liposome adsorption on the surface-bound synthetic peptides which have several alanine, lysine and tryptophan residues as amphiphilic segment with a cysteine at terminus. the quartz crystal microbalance studies showed that the peptide sequences can be divided into two groups according to liposome-size dependence of langmuir adsorption constant. while the initial adsorption processes could be satisfactorily described by simple langmuir adsorption kinetics, the amounts of liposome adsorbed irreversibly were increased as time advances. the results from afm reveal that most of the liposomes are bound to peptidic surfaces as single fl attened particles without fusion together for several hours. we next performed the detection of ganglioside gm1-lectin interaction on the liposome surface. all the binding constants and binding amounts, as observed by qcm, were fairly consistent with each other and and showed the effectiveness of our approach. herce, henry rensselaer polytechnic institute, united states recently we have proposed theoretically an energy independent pathway for the uptake of cell penetrating peptides (cpps) that challenges fundamental concepts associated with protein membrane interactions, h. d. herce and a. e. garcia, pnas, 104, 20805 (2007) . this mechanism involves strong interactions between cpps peptides and the phosphate groups on both sides of the lipid bilayer, the insertion of charged side chains that nucleate the formation of a transient pore, followed by the translocation of cpps peptides by diffusing on the pore surface. this mechanism explains how key ingredients, such as the cooperativity among the peptides, the large positive charge, and specifi cally the arginine amino acids, contribute to the uptake. we will describe the details of this mechanism and present novel experimental results that directly validate the model. a comparative studies on lipid affi nity of cell penetrating peptides in presence or absence of cargo a growing number of natural and /or synthetic peptides with cell membrane penetrating capability have been identifi ed and described in the past years. these molecules have been considered as targeting structures for the delivery of bioactive compounds into various cell types. although the mechanism of uptake is still unclear, it is reasonable to assume that the relative contribute of each proposed mechanism could differ for the same peptide, depending on experimental protocol and cargo molecule composition. in this work we try to connect the capability to interact with model lipid membrane of cpp and their structural and chemical characteristics in order to obtain a biophysical classifi cation that predicts the behavior of cpp-cargo molecule in cell system. indeed, the interaction with cell membrane is one of the primary step in the interaction of cpp with cells, and consequently the studies on model membrane could become important for understanding peptide-membrane interaction on a molecular level, explaining how cpps may translocate a membrane without destroying it and how this interactions come into play in shuttling cpps via different routes with different effi ciency. we analyzed by fl uorescence spectroscopy the binding properties of six different cpps (kfgf, antp and tat derived peptides, and oligoarginine peptides containing 6, 8 or 10 residues) in absence or presence of the same cargo peptide (the [392-401]ptyr 396 fragment of hs1 protein). the binding properties were correlated to the conformational and chemical characteristic of peptides, as well as to the cell penetrating properties of the cpp-cargo conjugate. results show that even if certain physico-chemical properties (conformation, positive charge) govern cpp capability to interact with the model membrane, these cannot fully explain cell-permeability properties. great deals of data show that alzheimer's pathology lead to the loss of neuronal functionality and synaptic plasticity. these effects seem to be the consequence of a toxic effect of a peptides related to their ability to modify cell membrane homeostasis. in particular, a peptides, as full length or in fragments, being in oligomeric or polymeric form, could alter membrane fl uidity and compromise its functionality. we have previously demonstrated that the 25-35 fragment of a peptide a (25-35) is able to penetrate into the outer leafl ets of the membrane. furthermore, it is able to alter membrane fl uidity changing membrane response to cholesterol, and thus affecting its ability to form low fl uid membrane regions named lipid rafts. a (25-35) is considered the shortest fragment exhibiting large -sheet aggregates and retaining the toxicity of the full-length peptide. flavonoids are a group of naturally occurring, benzo--pyrone derivatives, ubiquitous in plants. they are endowed with tumor prevent activity and they act as antioxidants through a membrane mediated molecular mechanism involving the membrane ion transport. on the basis of the common ability of fl avonoids and a peptide of altering membrane fl uidity, we carried out an epr spectroscopic analysis of several fl avonoids -specifi cally quercetin, naringenin, rutin and naringin -in 1, 2-dioleoyl-sn-glycero-3-phosphocholine (dopc) vesicles. the modifi cations of the dopc physio-chemical environment were analyzed in presence of fl avonoids and beta-amyloid fragment a (25-35). our results indicate that the addition of fl avonoids induces a decrease in membrane fl uidity. this effect is retained in presence of a (25-35). thus fl avonoid compounds could be able to antagonize the effect induced by amyloid peptide on membrane fl uidity and in this respect they could be therapeutically useful as neuroprotective agents. analyse lipid peptide interactions of p59 and lipo-p59 with membrane mimicking represented by micelles and vesicles. nmr structures of p59 and lipo-p59 in mixed sds/dpc micelles are reported; the positioning of p59 and lipo-p59 peptides with respect the lipidic surface is analyzed using 5-doxyl stearic and 16-doxyl stearic acid. epr analysis is carried out in the zwitterionic dimyristoyl phosphatidylcholine and the anionic dimyristoyl phosphatidylglycerol vesicles using several different lipid spin labels. both peptides bind to lipid bilayers and trp residues and lipidic chain of lipo-p59 show a important role in the process of peptide adsorption onto the membrane, to exert its antiviral activity. studies on human cell membranes were necessary to further establish the role of membranes in these peptides mode of action. this interaction was assessed by evaluating the effects that these peptides have on the membrane dipole potential of human erythrocytes, using the fl uorescence probe di-8-anepps. in the presence of enfuvirtide or t-1249, a decrease in the di-8-anepps fl uorescence excitation ratio dependent of peptide concentration was observed. these results show that t-1249 has ten times more affi nity to the erythrocyte membrane than enfuvirtide, a factor that can be associated with the adsorption of t-1249 on cholesterol rich membranes observed on the studies with membrane model systems. moreover, as a fraction of hiv associates with erythrocytes in vivo, these cells can have a role in delivering these peptides to the viral surface. the improved clinical effi ciency of t-1249 relative to enfuvirtide may be related to its higher partition coeffi cient and ability to adsorb to rigid lipid areas on the cell surface, where most receptors are located upon membrane fusion. moreover, adsorption to the sterol-rich viral membrane helps to increase the local concentration of the inhibitor peptide at the fusion site. the platelet receptor iib 3 plays a critical role in the process of platelet aggregation and thrombus formation. upon platelet activation its conformation changes leading to an increased affi nity for fi brinogen, which forms bridges between adjacent platelets and assembles them into an aggregate. the iib 3 activation is regulated by "outside-in" and "inside-out" signaling. among the protein-protein interactions, which contribute to «inside-out» signaling, that of talin with the 3 cytoplasmic tail is the most important. it has been recently suggested that talinmediated iib 3 activation relies on the cooperative interaction of the membrane proximal (mp) and the membrane distal (md) 3 regions with talin f3 domain and that the n 744 ply 747 motif of 3 , which can be phosphorylated at y 747 , plays a critical role in this process. to evaluate the interaction of talin with the 3 tail of integrin we designed and synthesized two peptides corresponding to the md and mp parts of 3 in their carboxyfl uorescein-labeled form (md: cf-r 736 akwdtannplyke 749 and mp: cf-k 716 llitihdrke 726 ). emission and anisotropy fl uorescence spectroscopy was used to quantitatively assess the affi nities of these peptides for talin. furthermore, to challenge the role of the y 747 phosphorylation in talin-iib 3 interaction we also studied the binding of talin to the modifi ed analogue of md, cf-r 736 akwdtannpl(ptyr) ke 749 . our experiments revealed that the md and mp parts of 3 bind tightly to talin and that y 747 phosphorylation has an inhibitory effect on this binding. finally, circular dichroism studies of all peptides in aqueous solutions and mixtures with tfe were also performed in order to characterize structure-affi nity relationships. acknowledgements: gsrt and eu (epan yb/88) for fi nancial support. the infection of human cells with hiv requires two initial steps: binding of the viral glycoprotein gp120 to the receptor cd4 followed by the interaction between the v3 loop of gp120 and a seven helix transmembrane coreceptor (ccr5 on macrophages or cxcr4 on t cells). the n-type glycosylation at asn301 of the v3 loop is assumed to play a crucial role in this process. in order to understand the interaction in detail, it is important to analyze the binding of v3-glycopeptides to the membrane integrated coreceptors on an atomic scale. here, we present binding studies between multiple v3-peptides and -glycopeptides and the coreceptor ccr5 by stdd nmr and surface plasmon resonance (spr). spr experiments were carried out by immobilizing the (glyco)peptides on an spr chip by amide coupling. several concentrations of ccr5 overexpressing human osteosarcoma (hos-r5) cells were passed over the sensor surface. the role of individual amino acids in the binding process to ccr5 can be analyzed by using different v3-peptides and -glycopeptides. additionally, the observation of interactions between the glycopeptide ligands and the receptor proteins embedded in liposomes is possible by using the saturation transfer double difference (stdd) nmr technology. stdd nmr allows removal of all unwanted signals resulting from native binding processes in cells. (1) . we used ccr5 liposomes derived from hos-r5 cells. substraction of an std spectrum of ccr5 liposomes from an std spectrum of the same concentration of liposomes incubated with a ligand allows recording of clean stdd nmr spectra presenting only signals of protons of the ligand interacting with the transmembrane receptor.(2) k d values of the ligand with respect to ccr5 have been determined in series of experiments with varying ligand concentrations. we could show that the binding epitope between hiv and ccr5 is formed by the carbohydrate at asn301 as well as the peptide. this knowledge is important for the design of new inhibitors. temperature dependant methionine proximity assays highlights conformational variations occurring through the mechanism of peptidergic gpcr activation arsenault, jason; clément, martin; leduc, richard; guillemette, gaétan; lavigne, pierre; escher, emanuel university of sherbrooke, canada g protein coupled receptors are invaluable for cell signal transduction mediated by external stimulus. pharmacological efforts towards these targets are of primordial importance albeit few efforts have resulted in structural characterisation, although rational drug design necessitates such information. recent advances in crystallisation of the -adrenergic receptor have been highly insightful and such a structure corroborates our results on the human angiotensin ii type 1 receptor (hat1) using the methionin proximity assay (mpa) in identifying ligand receptor contact points. unfortunately, physical methods such as crystallography are still far from routine procedures and are limited to a static picture of a given receptor. in the present contribution we propose a more accessible method that permits analysis conformational variations of different receptor states through an energy landscape, spanning from low energy conformations to conformations at physiological temperatures. photolabelling, mpa mutants constructed on the wt receptor, the constitutively active receptor (cam) and a corresponding non-activable mutant across the temperature range reveal activation-status dependent labelling patterns. as an example, position 256 becomes signifi cantly less accessible in the cam receptor compared to the wt receptor. ligand accessibility can be classifi ed from easily accessible (low temperature photolabeling) to less accessible residues (higher temperature photolabeling). this labelling pattern can be associated to the activation status of the receptor and may allow quantifying and identifying structural changes occurring during receptor activation. such structural understanding is crucial for future endeavours in rational drug design. capillary electrophoresis for diffi cult characterization of hardly soluble polypeptides recently, we have designed and synthesized polypeptides able to stimulate the natural plant defenses. these homopeptides were obtained by ring opening polymerization of n-carboxyanhydride (nca) with several initiators. ratio nca/initiator is determinant for the length of the polymers. on the bases of elicitor activity, we identifi ed a leader, called lapp6, which results from l-ala-nca polymerization initiated by alaninol. the mixture of poly(alanine) with different lengths is diffi cult to analyze considering important solubility problems. malditof confi rmed polymerization despite limitation due to molecular discrimination during ionization. nmr in deuterated tfa was possible but only afforded the number-average degree of polymerization (dp). to obtain more detailed characterizations, we developed separation by capillary electrophoresis in new solvents mixtures based on hexafl uoroisopropanol (hfip) and water. this technique allowed us to separate the oligomers with baseline resolution. different parameters infl uencing electrophoretic separation were investigated and optimized conditions were established. this technique enabled a full characterization of the polymer distribution, with determination of number-average dp, weight-average dp and polydispersity index. the pertinence and the reliability of capillary electrophoresis for characterization of non-water soluble polypeptides have been confi rmed, with analysis of short and long peptide chains. weakly polar interactions support polypeptide structures although the sequence of a polypeptide appears to determine the secondary and tertiary structure, weak inter-residue interactions such as between aromatic side chains and other aromatic side chains, aliphatic side chains and the peptide backbone may contribute signifi cantly to the stability of the fi nal folded structure. recent advances in structural bioiformatics and computational chemistry made it possible to study precise strurctural features and energetics of these interactions in model peptides and miniproteins such as tc5b, app and c-vhp. the interaction energies of the weakly polar interactions are of the same order as of the hydrogen bonds which occur in biopolymers (15 -65 kj/ mol). furthermore, these interactions not only stabilize local secondary structures but also entire tertiary folds of miniproteins. acknowledgements: this work was supported by the nih-inbre grant (p20 rr016469) and the carpenter endowed chair in biochemistry, creighton university trusova, valeriya; gorbenko, galyna v.n. karazin kharkov national university, ukraine a number of so-called conformational diseases including neurological disorders (parkinson's, alzheimer's and huntington's diseases), type ii diabetes, spongiform encephalopathies, systemic amyloidosis, etc., are associated with the deposition in tissue of highly ordered aggregates of specifi c peptides and proteins. despite the main structural elements of amyolid fi brils (particularly, cross--structure) are well-characterized, the mechanisms of fi brillogenesis remains poorly understood. accumulating evidence substantiates the idea that formation of fi brillar structures can be initiated and modulated by peptide/protein-lipid interactions. membrane-related determinants of fi brillization are thought to involve conformational changes of the peptide/protein, increase of its local concentration at lipid-water interface, specifi c orientation of aggregating species, neutralization of the peptide/protein surface charges by anionic lipid headgroups, particular arrangement of the inserted and solvent exposed segments of the peptide/protein molecule, etc. the present study was undertaken to explore the formation of lysozyme (lz) amyloidlike fi brils in the model lipid-protein systems. lipid component of the model system was represented by liposomes prepared from zwitterionic phosphatidylcholine (pc) and anionic phosphatidylglycerol (pg) lipids in the molar ratio 4:1. fluorescence microscopy studies performed with fl uorescein-labeled and rhodamine-labeled lz showed the presence of long lz fi bers (length >80 ìm). the to elucidate the nature of the events preceding lz self-assembly into amyloid-like structures, the protein adsorption onto pc:pg vesicles was examined by monitoring fl uorescence changes of fl uorescein-labeled lysozyme. the observed sigmoidal shape of the adsorption isotherm is strongly suggestive of oligomerization of membrane-bound protein. it seems highly probable that such oligomers serve as nuclei in the membrane-assisted lysozyme fi brillogenesis structure and dynamics of photosystem ii lightharvesting complex revealed by high-resolution fticr mass spectrometric proteome analysis structure and dynamics of membrane-bound light-harvesting pigmentprotein complexes (lhcs), that collect and transmit light energy for photosynthesis and thereby play an essential role in the regulation of photosynthesis and photoprotection, were identifi ed and characterized using high-resolution fticr mass spectrometry. lhcs from photosystem ii (lhcii) were isolated from the thylakoid membrane of arabidopsis thaliana leaves after light stress treatment using sucrose density gradient centrifugation, and separated by gel fi ltration into lhcii subcomplexes. tsekova, daniela university of chemical technology and metallurgy, bulgaria formation of fi brous supramolecular complexes from l-val derivatives and their arrangement in spherulites was studied applying spectrophotometric approach and electron-microscopic observations. experiments show that boiling one and the same compound with different initial concentrations in water to completely dissolving and posterior cooling to room temperature lead to formation of stable supramolecular complexes with different sizes and properties. they behave like polymer molecules which specifi c numbers of monomers depend on the initial concentration of the boiling solution, moreover at higher supersaturations they crystallize in sperulites which is typical for crystallization of polymer molecules. metal -organic gels based on the self-assembly of peptidomimetics and cu (ii) ions peptidomimetics constructed from l-val and nicotinic/isonicotinic acid are good low molecular weight gelators and their supersaturated solutions in a number of solvents turn into gels. they make complexes with some metal ions. mixing of pure solutions of the peptidomimetic and some cu (ii) salts lead to immediate gel formation in some solvents. this kind of compounds, named metal-organic frameworks (mofs), are new class of nanoporous materials and are very promising ones for applications in catalysis, pharmaceutical industry, etc. the stability and molecular structure of these complexes in some solvents is in the process of investigation. peptide metalloconstructs often possess particular conformations and hence display increased activities and metabolic stabilities. we investigated new rgd peptide analogs cyclized through oxorhenium / oxotechnetium coordination. the rgd sequence is known to bind specifi cally to 10 of the 25 known integrins, a family of integral proteins that plays an important role in tumor neoangiogenesis, development and proliferation. several cyclic rgd pentapeptides bearing an exocyclic tc-99m oxotechnetium core have been proposed for molecular imaging, however few of them have displayed attractive selectivities and metabolic stabilities. structure, biological activity and metabolic stabilities of metallated peptides cannot be predicted. therefore, we preferred a combinatorial approach to generate a panel of tracers that may be evaluated by tumor imaging in mice. tracers were constructed from a rgd model of general formula : ns2-x1-x2-x3-rs where x1,2,3 are respectively arginine, glycine and aspartic acid analogs and r is a series of linkers that feature various lengths and geometries (ns2 is a n-bis(ethylthio) moiety). first attempts for synthesizing these peptides by the versatile ugi multicomponent reaction did not yield the peptides with suffi cient purities. a representative library of 64 peptides was obtained by standard parallel peptide synthesis and purifi cation of all members. peptides were metallated either with rhenium or technetium using standard procedures. their resistance towards mice serum, glutathione and tumor extracts was evaluated and showed that compounds containing an aminoethanethiol linker displayed higher stabilities. infl uence of the chemical environment on isomers ratios of the metallated peptides was also investigated. finally, oxorhenium peptide coordinates were assayed as specifi c ligands of integrins. their oxotechnetium equivalents were evaluated for tumor imaging in mice. degradation products of desmopressin in phosphate/ citrate buffer it shows, that introduction of unsaturated residue to the peptide chain could be useful tool to design bioactive compounds with desirable structure [8] [9] . therefore full knowledge about relation between presence of dehydroamino acid and peptide's conformation is necessary to predict biological proper and to design newdrug. for that reason we have undertook conformational investigations of numerous peptides containing two dehydroamino acid residues ( z phe, e phe, ala) in peptide chain, in different position. the investigations were based on nmr measurements (standard 2d techniques and 1d experiments, typical for detection of hydrogen bonding) and theoretical calculations. conformational preferences of investigated systems were obtained on base of roesy and noesy experiments and calculations by use of x-plor and quantum chemical calculation is concentration overload or volume overload the best strategy for synthetic peptide purifi cation? as the complexity of synthetic peptides increases so the demands on the synthesis and purifi cation increase. whilst improvements in synthesis resins and techniques enables higher purity peptides to be produced, 100% purity is not achieved. for many applications post-synthesis purifi cation is still required. methods for the purifi cation at the mg level can be relatively straightforward but where there is a need to develop methods which may be used for larger scale production there are a number of additionly considerations. the method developed must be scaleable and the economics of the process must be compatible with the fi nal product costs. when looking to develop a purifi cation method the loading will be critical to the throughput and fi nal production costs. the selection of the purifi cation media and the purifi cation conditions are two of the major infl uences on loading as these determine capacity and resolution. however, it is also important to consider the the physical properties of the pepitide -especially its solubility. as part of the purifi cation method development a loading study must be performed -increasing the amount of peptide loaded onto the column. the most common way of doing this is to increase the concentration of the sample and keep the injection volume constant, concentration overload. but this does require that the peptide is readily soluble in a solvent compatible with the hplc purifi cation conditions. alternatively volume overload can be used where the concentration of the peptide solution is kept constant and the volume purifi ed increases. the most commonly used method is concentration overload. the work presented in this poster compares the two overload strategies, concentration overload and volume overload, for the purifi cation of synthetic peptides. the suitability of the two strategies for large scale manufacture will be explored. expansion of human stem cells by passive transmembrane transfer of homeoproteins . these results clarify the effect of hoxb4 in the early stages of human lymphopoiesis, emphasizing the contribution of this homeoprotein to the intrinsic lympho-myeloid differentiation potential of defi ned lymphoid progenitor subsets. finally, this supports the potential use of hoxb4 for hsc and hpc expansion in a therapeutic setting, by means of direct transmembrane protein transfer. tumor selective delivery by cell-penetrating peptides systemic delivery of therapeutic agents used for cancer chemotherapy today lead to undesired side effects and toxicity. increasing the selectivity of the anti-cancer agent will not only facilitate lower dosage for equal therapeutic effect, but also decrease the frequency of drug resistance. we have studied two different targeting approaches both based on cellpenetrating peptides. the fi rst approach is a chimera between cancer homing peptides and a cpp the other is a enzyme activated prodrug design. the cyclic peptide ccpgpegagc (pega) is a homing peptide that has previously been shown to accumulate in breast tumor tissue in mice. pega peptide does not cross the plasma membrane per se; however, when attached to the cell-penetrating peptide pvec, the conjugate is taken up by different breast cancer cells in vitro. additionally, the homing capacity of the pega-pvec is conserved in vivo, where the conjugate mainly accumulates in blood vessels in breast tumor tissue and, consequently is taken up. matrix metalloproteinases (mmps) are over-expressed in a variety of tumor tissues and cell lines, and their expression is highly correlated to tumor invasion and metastasis. to exploit these characteristics, we designed a tumor cell-selective prodrug, by constructing modifi ed version of the already shown to be functional mtx-yta4 conjugate. it is an inactive pro form of a cell-penetrating peptide (nope) conjugated to the cytostatic agent metothrexate (mtx), selectively cleavable and thereby activated by mmps. characterization of hantavirus envelope structure hepojoki, jussi; strandin, tomas; vaheri, antti; lankinen, hilkka university of helsinki / haartman institute, finland hantaviruses are zoonotic viruses carried by different rodent species and if transmitted to man cause two severe diseases, the hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome to which there is no specifi c therapy. the hantavirus envelope consists of spike structures formed by surface glycoproteins gn and gc which anchorage to the viral lipid bilayer. the quaternary complexes formed by glycoproteins have not been solved. to begin with, it is important to understand the structure of virus particle in order to treat infection for example by prevention of membrane fusion. using pepspot technique the interaction site between gn and gc proteins was mapped to peptide level. to interpret interaction mapping results three dimensional (3d) models of gc protein were created. the structure of hantavirus was also investigated in this study by cryo-electronmicroscopy (cryo-em). according to our results, hantavirus spike consists of either four or eight glycoprotein units and the spike would consist of equimolar associations of both glycoproteins. overall it seems likely that the glycoproteins of hantavirus form a heterodimeric base unit analogous to semliki forest virus e1-e2 glycoprotein complex, where the interaction between e1-e2 proteins hides the fusion peptide in e1 protein and thus prevents premature fusion. isolation of a novel 24 kda protein from ginger rhizomes having anti-fungal and anti-proliferative activity gill medicinal properties of ginger have been known for long. ginger has been used in traditional indian and chinese medicine and is effective on a wide range of ailments including diarrhea, nausea, respiratory disorders, infl ammatory diseases, arthritis etc. a number of constituents and active ingredients are present in ginger. recent studies have shown the role of ginger extract in the modulation of biochemical pathways involved in chronic infl ammation and thus providing evidences for the anti-infl ammatory role of ginger. mainly the medicinal properties and anti-proliferative activity of the ginger is because of the presence of certain pungent vallinoids, viz. 6.-gingerol and 6.-paradol, as well as some other constituents like shogaols, zingerone etc. we have identifi ed and purifi ed a novel anti-fungal and anti-proliferative protein with a molecular mass of 24 kda from the crude extract of ginger rhizobium (zingiber offi cinales), belonging to the zingiberaceae family. the isolation procedure involved ion exchange chromatography using deae-cellulose and affi nity chromatography using affi -gel blue gel. crude extract was loaded on the deae-cellulose column equilibrated with 10 mm tris-hcl buffer (ph 7.0). the unadsorbed protein fraction from deae-cellulose was further loaded on affi -gel blue gel column equilibrated with 10 mm tris-hcl buffer (ph 7.0), from which the elution of adsorbed protein was done with the same buffer. the purifi ed protein of 24 kda exhibited a potent anti-fungal activity against the mycelial growth in different fungal species, for example aspergillus fumigatus. in addition, the antifungal activity is also seen against important fungi, viz, fusarium and candida species. further, the purifi ed protein also showed 60 % inhibition of cell proliferation at 10 m concentration. the anti-proliferative activity was checked on human oral cancer (kb cells). search for native interacting partners of fl uorinated amino acids using phage display the introduction of fl uorine has proven to be a successful concept for improving the biological and pharmaceutical properties of drug candidates. the unique properties of fl uorine as well as its absence from the pool of canonical amino acids make fl uorinated amino acids a promising tool in the development of peptide based drugs.(1) however, the application of fl uorinated amino acids for rational protein design requires a comprehensive knowledge of their properties within a native protein environment. our research focuses on the effects caused by single substitutions by fl uorinated amino acids within a polypeptide environment.(2) based on a parallel, heterodimeric -helical coiled coil peptide we applied phage display technology (3) to screen for preferred interaction partners of fl uorinated building blocks within the pool of the twenty canonical amino acids. three fl uorinated amino acids were introduced either at an a-or a d-position into one of the coiled coil monomers. the second coiled coil monomer was randomized at the four positions that represent the direct interaction partners of the fl uorinated amino acid within the dimerization domain. coiled coil pairing selectivity was used to determine the best binding partner out of the library. using the acylation method, fatty acyl chains are covalently linked to the free amino residues forming a stable amide bond. in this study, a novel method for acylation of proteins was developed using in situ prepared fatty acyl chloride dispersion in aqueous acetonitrile solution, which allows protein modifi cation under very mild conditions. chicken cystatin, a reversible 13 kda inhibitor of papain-like cysteine proteases, was selected as a model protein due to its high potential to inhibit intracellular cathepsins. the protein was modifi ed using fatty acyl chlorides with 6, 8, 10, 12, 14, 16, and 18 carbon atoms. based on the cell culture assays, we examined the transport properties of fatty acylated cystatin, the effectiveness of its internalization and effi ciency to inhibit intracellular enzymes, which was measured indirectly by cathepsin b inhibition. the experiments showed that acylated cystatin quickly internalized into the cells and effectively inhibited cathepsin b. in contrast, non-acylated cystatin didn't cause inhibition as it was unable to enter the cell. the permeability enhancement effect was shown to depend on the length of the attached fatty acyl chain as the strongest inhibition was caused by cystatin acylated with 18 carbon atoms long stearoyl chloride. additionally, chemical modifi cation did not infl uence the protein's immunogenicity. the results of our study provide clear evidence that fatty acylation greatly improves membrane permeabilization properties of proteins. daisogel wing takes your process separation to a higher level. custom made solution for your process peptide purifi cation problem can be easily delivered using the daisogel wing polymer grafted silica based platform. "silica or polymer?"-it used to be an evergreen debate when it came to choose the adequate stationary phase for process scale separation or purifi cation. silica based stationary phases feature much higher mechanical strength and stability, better controlled porosity and as a result higher separation effi ciency, while polymers boast wider ph range. the attempts to bring the good aspects of these opposite methods together failed so far, or failed to provide fl exible solutions. the new daisogel wing platform for silica surface polymer grafting preceding chemical modifi cation is presented. the revolutionary new technique offers high versatility: most variations of the base silica (different pore sizes or particle sizes of choice) can be combined with your choice of familiar chemical surface modifi cations to tailor make the perfect stationary phase for your given chromatography challenge. the polymer graft on the silica surface provides extreme shielding effect, the produced stationary phase displays outstanding ph stability and durability. any of your preferred and trusted regular silica surface modifi cations can be done on the top of the grafted polymer layer. you may enjoy the usual high performance separation, excellent effi ciency, high plate numbers, mechanical strength of silica based stationary phases with a dramatically extended ph range you expected so far only from polymer resins. you have the full range of choices of particle and pore sizes with the full choice of desired bonding chemistries. finally daisogel wing is here to deliver you the most versatile polymer grafted silica hybrid platform to provide the best solution for your demanding process scale peptide separations. przybylski, józef; rogala, piotr; siemaszko-przybylska, krystyna melanin, a natural compound formed in the tyrosine metabolic process. according to occurrence, melanin is divided into that present in true skin and in internal organs. the melanin synthesis and secretion process takes place in melanocytes under the control of two neurohormons: msh -melanocyte stimulating hormone and mch -melanocyte concentrating hormone. research has shown the signifi cant role of melanin in the process of breeding and storing, in regulating metabolism of fat tissue, and in carbohydrates regulation. melanin biopolymer fi nds application in it technology as neurotransmitters for non cellular intelligence. biologically active collagen constitutes the architectonics of all tissues and organs. due to its piezoelectric and dielectric properties it also provides storage and relay functions. for the needs of medical practice, transplants, pharmacy, cosmetology and information technology the key issue is to obtain a compound of melanin with biologically active collagen. this compound we named melanocollagen type i. melanocollagen type i is formed in result of protonating collagen amino acids with melanin, which yields coloured gel ranging: grey, graphite and black. /melanin (h+) + collagen melanocollagen type i/. the therapeutical capacity of biologically active collagen type i with melanin is an ideal formulation inhibiting aging and mitigating related neurovegetative and dementia symptoms, and in vivo transplantology. freeze dried melanocollagen type i retains the features of both collagen type i and melanin. it can be a formulation on its own or a supplement for in vivo and in vitro tissue engineering, biotechnology, information technology, pharmaceutical industry and cosmetology. a patent application for melanocollagen was placed with the polish patent offi ce on 05.08.2004 as p-369439, entitled: melanocollagen type i -method of obtaining. [ 1, 2 ] . 1,4-dhp lipid structure was calculated with ab initio quantum mechanics to obtain the charges for molecular dynamics with amber 8.0 force fi eld. dhp-lipid molecules were subjected to molecular dynamics from the initial structure of a periodic lipid bilayerwater box, with a small amount of excessive water on the lipid edges to ensure the mobility of lipid molecules. after 14 ns of md simulation the lipid molecules with the fatty acid tails started to squeeze from one bilayer layer to another one. after 35 ns few lipid molecules turned with their charged heads to the side of the lipid bilayer and after 100 ns a profound tubular micelle structure began to form. the tubular micellae structure becomes more perfect during the course of simulation of 300 ns. conclusion is that one of the gene transfection agent 1,4-dhp lipid structures is a tubular micellae, and we could expect that such the micellaes are capable to form lipoplex for the dna transfection or peptide delivery. introduction a newly developed hydrophilic polymer-based ion exchange chromatography column for biomolecules. ion exchange chromatography (iec) is a widely used for analysis and purifi cation of biomolecules. we have newly developed polymer-based iec column, named ymc-biopro series, specially designed for separation of proteins, peptides and nucleic acids. the completely spherical and monodispersed porous / nonporous beads (5 m), optimally packed with advanced technology, provide high theoretical plate number and symmetric peak shape. excellent resolution is achieved from the high column effi ciency coupled with the excellent selectivity of qa and sp chemistries. in this paper, we will show features and benefi ts of ymc-biopro series. james p. tam, school of biological science, nanyang technological university, 60 nanyang drive, singapore 637551. viral entry requires fusion of the viral and cellular membranes. in all class-1 envelope viruses including hiv-1, fusion is mediated by envelope glycoprotein which has a homotrimeric quaternary structure which forms a hairpin-like assembly of six-helix bundle (6hb) during the fusion event. the 6hb employs a 3-on-3 locking mechanism in which 3 hrc (heptad repeating-carboxyl region) chains crosslink 3 hrn (heptad repeating-aminal region) chains to enable membrane fusion. this locking mechanism confers avidity due to multi-chain interactions and a high genetic barrier to mutations in the viral entry strategy despite the high mutation rate of their envelope protein. this mechanism also provides inspiration to our approach in designing mutation-resistant entry inhibitors of hiv-1. t20/enfuvirtide, a synthetic hrc-peptide monomer designed to interrupt membrane fusion, is the fi rst and only fda-approved entry inhibitor against hiv-1. however, t20 becomes ineffective in 20% of aids patients due to acquired resistance to t-20 resistant during the treatment course. our inhibitor design is based on a novel quaternary protein mimetic approach. key elements include a covalent-link 3parallel-chain construct mimicking the quaternary structure of gp41 in its pre-fusion state and an inhibition mechanism mimicking the multimeric interactions found in the highly conserved 6hb formation. our hypothesis is that covalent-linked, 3-chain quaternary mimetics (called 3 mimetics) of hrc peptides can confer multi-chain binding to the hrn region in a multi-chain locking mechanism that may lead to mutation-resistant hiv fusion inhibitors. we also extend our design to 2-chain hrc mimetics (called 2 mimetics) which may bind to the hrn as a 2-on-3 locking mechanism. in contrast, t20 and other single-chain peptide inhibitors with a 1-on-3 binding mechanism lacks the advantages offered by the quaternary mimetics and is susceptible to gp41 mutations. this report will describe all three types of inhibitors as a model to further our understanding of gp41 mutations and viral fusion mechanism in developing mutation-resistant entry inhibitors. references: 1. lain et al. cancer cell costentin, beauvillain, vaudry, proc. natl. acad. sci pnas 2001, 98, 944. 1080504. references: 1. a.a.zamyatnin. fragmentimics of proteins and natural oligopeptides. biofi zika the fi rst genome sequence of an elite grapevine cultivar (pinot noir vitis vinifera l.): coping with a highly heterozygous genome the grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla the erop-moscow oligopeptide database press references: 1. alcaro galactosylated peptides, their preparation and use in autoimmune disease diagnosis new coupling reagents: development and industrial aspects assessment of new 6-cl-hobt based coupling reagents for peptide synthesis. part 1: coupling effi ciency study fast conventional synthesis of chemokine sdf-1 (1-68) on the symphony® references: 1. pardridge, w. m. drug discov.today alberto 2 ; rondina, maria 3 ; rosato maura 2 ; quintieri synthesis and biological activity of new linear and cyclic shp-1 n-sh2-ligands the sequence specifi city for most sh2 domains is dictated by the amino acids surrounding the py-residue (position 0) and the c-terminal positions relative to py (2). in contrast, the sh2 domains of shp-1 in addition to py+1 and py+3 depend on position py-2 (3). it was further demonstrated that beyond this minimal consensus sequence the positions py+4 and py+5 also signifi cantly affect the binding affi nity and specifi city of the shp-1 sh2 domains (4). for the investigation of shp-1 mediated signaling pathways, inhibitors of this phosphatase are of great interest. therefore, we focused our research on linear and cyclic peptide ligands based on the previous studies [3-5]. the new ligands were c-terminally prolongated according to the recognition determinants for py+4 and py+5. except, an additional motif designed to occupy a basic gap on the surface of the ptp-domain was introduced. the latter was predicted to impair the n-sh2-ptp dissociation process design of a directed molecular network restructuring artifi cial peptide networks by external triggering the road to non-enzymatic molecular networks boolean logic functions of a synthetic peptide network systems chemistry: logic gates, arithmetic units and network motifs in small networks peptides: the wave of the future references: 1. d'andrea l.d.; iaccarino g.; fattorusso r.; sorriento d.; carannante c.; capasso d.; trimarco b.; pedone c proc. natl. acad. sci agnieszka 1 ; gofman nir 3 ; willumeit, regine 1 1 gkss research centre, germany; 2 hasylab/desy, germany urtti novel cationic amphiphilic 1,4-dihydropyridine derivatives for dna delivery dioleoyl phosphatidylethanolamine and peg-lipid conjugates modify dna delivery mediated by 1,4-dihydropyridine amphiphiles masako 1 ; kuriyama, naohiro 2 poster abstracts lacking his-pro residues and stabilized by introduction of 2 hval resulted in an analog h-2 hval-tyr-ile-tic-oh (al-35) that was very selective for irap versus ap-n and at(1) . the targeting properties of cell penetrating peptides cell penetrating peptides (cpps) offer the ability to penetrate across the plasma membrane of mammalian cells and to carry cargoes such as proteins, oligonucleotides and liposomes. due to this, cpps have gained high attention to improve the cellular uptake of the delivery of 'biologicals'. however, most of the research that has been performed with cpps restricted to in vivo studies. preclinical investigations are rare and the clinical validation of potential of cpps is required. a series of cpps were synthesized by solid phase peptide synthesis (spps) on an abi 433a synthesizer. the stability of these peptides in human serum was determined. subsequently, the uptake was studied in the following cell lines: sw 1736, pc-3, mh wt, hno in summary, the in vitro uptake studies did not reveal great differences that might have revealed a tumor type specifi city. in vivo studies revealed neither a distinct tissue uptake or tumor specifi city. design and synthesis of a tripartate paclitaxel prodrug for melanoma therapy therapy of melanoma continues to be a challenge since, regardless of the treatment used, long-term survival is quite uncommon. in an attempt to improve the effectiveness and decrease the toxicity of anticancer chemotherapy, one useful approach might be to administer a prodrug that specifi cally releases the active cytotoxic drug at the tumor site. in this work, we describe the synthesis of a peptide conjugate of paclitaxel potentially useful in the treatment of human melanoma, and characterized by the simultaneous presence of three functional domains: a "targeting domain", an "activation sequence", and the antitumor drug paclitaxel. the "targeting domain" of the prodrug is represented by an rgd-containing cyclic peptide, able to bind selectively to alpha-v beta-3 integrin, which is known to be highly over-expressed by both metastatic human melanoma cells, and endothelial cells of tumor vessels. the "activation sequence", responsible for the selective release of the drug, is a short peptide which is cleaved specifi cally by cathepsin b, a protease highly up-regulated in malignant tumors. the results of nmr conformational studies, as well as those of biological experiments aimed at evaluating the plasma stability of the prodrug and its ability to inhibit alpha-v beta-3-mediated tumor cell adhesion to vitronectin, will be also presented. angiogenesis is a remodeling process characterized by the sprouting of new blood vessels from pre-existing ones. it occurs during embryogenesis and to a limited extent in the adult. vegf is a homodimeric protein and has been characterized as a prime regulator of angiogenesis and vasculogenesis; when cells lose the ability to control the synthesis of vegf, angiogenic disease ensues (1) . in vitro studies show that vegf is a potent and specifi c angiogenic factor involved in the development of the vascular system and in the differentiation of endothelial cells (2) . vegf biological function is mediated through binding to two receptor tyrosine kinases: the kinase domain receptor (kdr) and the fms-like tyrosine kinase (flt-1), which are localized on the cell surface of various endothelial cell types. this binding activates signal transduction and can regulate both physiological and pathological angiogenesis (3) . vegf and its receptors are overexpressed in pathological angiogenesis, making this system a potential target for therapeutic and diagnostic applications (4) . the extracellular portion of vegf receptors is comprised of 7 immunoglobulin-like domains; deletion studies have shown that the ligand binding function resides within the fi rst three domains of flt-1 and in domains 2 and 3 of kdr. actually, no structural data are known on the extracellular portion of these receptors except for the second domain of flt-15.. so, our aim is the cloning and the expression of part of extracellular domains of both vegf receptors for structural characterization and to be used in interaction studies with peptide ligands or small organic molecule. we are interested in an activation mechanism of receptor tyrosine kinases (rtks), especially, how the transmembrane (tm) and the intracellular juxtamembrane (jm) region couple ligand binding to tyrosine phopsphorylation. one of the rtks that we are working on is neu. neu (erbb2) is one of the four members of erbb receptor family. this rtk with a mutation in the transmembrane region (v664e) has been recognized as a potent transforming oncogene product. structure of the tm region dimer and its structural difference between the wild type and v664e mutant have been reported by smith et.al. (1) , providing structural constraints for modeling the tm dimer. recently, we have performed a structural study on a tm-jm region of egfr (erbb1) showing that the tm helix breaks at the membrane interface and the unfolded jm region binds electrostatically to the membrane. we also have shown that ca2+ complex with calmodulin (ca2+/cam) binds to the positively charged jm region and pulls this portion off the membrane. these results are consistent with an electrostatic engine model, postulated by mclaughlin and coworkers (2) , for the autoinhibition and activation of the egfr. in this research, we are revisiting the structure of tm-jm region of neu to see if we can apply the electrostatic engine model to neu and to see if there is a structural difference between the wild type and the mutant tm-jm sequence. here we describe structural studies on the tm-jm sequence, neu(647-693), reconstituted into bilayer vesicles. a combination of solid state nmr, infrared and fl uorescence measurements are used to draw conclusions that the unfolded jm binds electrostatically to the membrane and binding of ca2+ -calmodulin to the positively charged jm sequence can, under some conditions, reverse its charge and release it from the membrane. @the collagen triple-helix consists of three polypeptide chains, each of which takes the poly-l-proline ii form in a left-handed helix and undergoes a transition to a single coil state as the temperature increases. this characteristic structure is ascribed to the unique amino acid sequence x-y-gly, where x and y are commonly pro or 4(r)-hydroxyproline (hyp r ). @so far, various nmr studies have been done to explain the mechanism of folding and the thermal stability of the triple helical structure by using model peptides rich in imino acids. however, the complexity of nmr spectra imposed severe limitations on detailed analysis. the spectrum is complicated because of a large number of conformational isomers due to cis/trans isomerization around each imino acid causing various chemical shifts with small differences in 1h-nmr spectra. it has been demonstrated that the site directed 15n enrichment allows real time nmr monitoring of the folding of residues at specifi c locations of collagen model peptides (1) . @to approach this problem, we have applied 19f-nmr study on various collagen model peptides containing 4(r)-fl uoroproline (fpro r ). compared to 1h and 13c, 19f chemical-shifts are extremely sensitive to small environmental changes around the nuclei and show a wide dispersion of chemical shifts. as a result, we not only distinguished the signals from the triple helix and unfolded states, but also differentiated the signals at intermediates states in 1d 19f-nmr spectrum of (pro-fpro r -gly) 7 . @here, in this study, a combination of 2d nmr experiments including exsy, hoesy, and tocsy has been used to investigate detailed equilibrium properties of collagen model peptides. especially, in the 2d 19 f-exsy spectra, we successfully observed many exchange cross peaks at high temperature. the 19 f-nmr method shown here provides a clue to analyze the conformational transition, dynamics and stability of collagen triple-helix. by contrast to well-folded natural peptides, oligomers consisting of nonnatural building blocks, so-called foldamers, are highly stable towards proteolytic degradation which is a key feature in the development of peptide-based drugs. aside from the fact that foldamers consisting ofand -amino acid building blocks are able to display different secondary structures, the combination of different homologous building blocks to hybrid peptides has recently shown some promising results. [1, 2] the ab initio mo theoretical studies have shown that / -hybrid peptides composed of alternating -and amino acid building blocks might be very well suited to mimic helical conformations. (3) here we report the fi rst examples of -helical coiled coil formation with synthetic foldamers. based on the computational studies, we synthesized heterogeneous peptides by automated solid phase peptide synthesis (spps). purifi cation was carried out by hplc, and products were confi rmed by esi-tof-ms. furthermore, temperature-dependent cd spectroscopy was applied to investigate the stability of hetero-oligomers formed between, either / -or / / -hybrid peptides with a naturalhelical coiled coil peptide. in addition, the interaction potential with their natural -helical counterparts in aqueous solution were investigated by cd, and förster resonance energy transfer (fret). using reversed phase high performance liquid chromatography and two-dimensional gel electrophoresis, the lhcii proteins, lhcb 1-6 and fi brillins were effi ciently separated, and identifi ed by fticr-ms proteome analysis. some of the lhcii subcomplexes were shown to migrate from photosystem ii to photosystem i as a result of short term adaptation to changes in light intensity. in the mobile lhcii subcomplexes, decreased levels of fi brillins and a modifi ed composition of lhcii protein isoforms were identifi ed compared to the tightlybound lhcii subcomplexes. in addition, fticr-mass spectrometric analysis revealed several oxidative modifi cations of lhcii proteins (1) . a number of protein spots in 2d-gels were found to contain a mixture of proteins, illustrating the feasibility of high resolution mass spectrometry to identify proteins that remain unseparated in 2d-gels even upon extended ph gradients. phosphinic analogue of the homophenylalanyl-phenylalanine dipeptide was demonstrated to be a potent, competitive inhibitor of cytosolic leucine aminopeptidase (lap, e.c.3.4.11.1), mimicking the transition state of the reaction catalysed by the enzyme. exhibiting ki value at low nanomolar range it was ranked among the most potent inactivators of lap reported so far (1) . recently, the compound has been also successfully employed to inhibit leucyl aminopeptidase of p. falciparum, a potential target protease for the development of new antimalarials (2) . thus, its structure represented an attractive lead for the design and construction of next generations of modifi ed analogues. they were targeted towards both lap as well as a related metalloprotease -microsomal leucine aminopeptidase (apm, e.c.3.4.11.2). these achievements, including recent results of studies on synthesis and activity, will be summarized here. we recently described the synthesis of some cyclic tetrapeptides bearing imidazole side chains and we analyzed, in detail, their copper(ii) binding properties in aqueous solution. 1 the copper(ii) species obtained are of interest in relation to copper-protein active-site biomimetics. in particular, a 13-membered ring cyclic tetrapeptide c(lys-dhis-ala-his) (dk13) was synthesized by the solid-phase peptide synthesis method and its copper(ii) coordination properties were studied. 2 surprisingly, all collected data strongly support the presence, at alkaline ph, of a stable peptide/copper(iii) complex that is formed in solution by atmospheric dioxygen oxidation. in order to clarify the mechanism of the copper oxidation through the average cu-n bond distance and to get information on the local geometry around copper, depending on the peptide sequence, we have collected experimental xanes and exafs spectra. the investigated cyclopeptide/copper complex shows pre-edge peak energy position and integrated intensity consistent with those of cu 3+ model compounds. also, edge energy is consistent with the presence of trivalent copper. then, calculations performed on the exafs measures should give information on the local geometry, confi rming the square planar geometry proposed by us for this dk13/cu(iii) complex. improved expressed protein ligation method for consecutive coupling of polypeptide fragments in the post-genomic era, to support the structural and functional studies of proteins, there is a growing demand for novel methods with which a wider range of selective protein modifi cations achievable. expressed protein ligation (epl) can be the method of choice for the preparation of unique protein derivatives not available by recombinant methods when the protein fragments extend the size accessible by solid phase peptide synthesis, and when extra chemical information (i.e. a special covalent modifi cation) is introduced into a well-defi ned part of the sequence. however, this method is not generally applicable, especially in the case when the target protein derivative is assembled from three or more polypeptide fragments. we demonstrate here an improved epl method that facilitates the effective ligation of large fragments in a consecutive way. the method employs a novel protection-deprotection scheme. supported by otka f049222 and jános bolyai fellowship (cs.t.). protein ligation using protein trans-splicing , in order to facilitate protein ligation using synthetic peptides. highly effi cient fl uorescent labeling of proteins has been demonstrated in a site-specifi c manner by protein ligation using the newly designed intein. this approach could be applied for sitespecifi c modifi cation of proteins in vivo because protein trans-splicing is an autocatalytic process requiring no additional cofactor. moreover, protein trans-splicing approach could be extended to dual site-specifi c modifi cation using an additional intein. la doppel (dpl) is a glycosylphosphatidylinositol-anchored protein that exhibits 26% sequence homology with prion protein but lacks the octarepeat region. the role of prion is not yet completely known but prpc seems to be involved in cu (ii) traffi cking from synaptic clefts and in preventing neuronal oxidative damage. doppel is expressed in heart and testis and has been shown a regulator of male fertility. therefore, despite a high sequence homology and a similar three-dimensional fold, it's possible to hypothesize that the function of the two protein is not correlated. in the literature is reported that both prpc and of dpl are able to bind cu (ii) but the characterization of the complex species is well defi ned only for prpc. several binding sites of dpl are involved in the complexation with cu (ii). [1, 2] in the present work we have focused our attention to the third alpha-helix of the dpl which is the preferential binding site for the metal ion. we have synthesized two peptide fragments relative to the 122-140 sequence of the dpl. the fi rst peptide has the native sequence whereas in the second fragment the asp 124 was replaced by a asn residue. this substitution was performed to understand the role played by the carboxylate group of the asp residue in the complexation of cu (ii). cd ad nmr spectra were carried out on the two peptides in absence and presence of cu (ii) to investigate conformational features upon cu (ii) binding. finally, the complex species formed were completely characterized by using spectroscopic (nmr, cd, uv-vis, epr) and thermodynamic measurements (potentiometric titrations). three-dimensional (3d) domain swapping of proteins is a phenomenon associated with formation of oligomers and fi brils of several amyloidogenic proteins (1) . the propensity of a particular protein to undergo domain swapping depends on factors like changes in environment or presence of specifi c mutations, but also on some topological determinants. it was proposed that domain swapping-prone proteins have some common "hot spots" in their structures that facilitate the process. one of these motives are so called "hinge loops" -turns showing enhanced propensity for unfolding due to conformational constraints (2). cystatin c (hcc), main inhibitor of cysteine proteases in human body was shown to dimerize (3) and oligomerize (4) through domain swapping. hcc contains highly conserved throughout the cystatin family loop l1, connecting two beta strands which, together with the only helix in the protein fold create a structural unit that undergoes the 3d process. in order to confi rm important role of distortions in l1 loop, which are centered around valine residue in position 57 in the dimerization/ oligomerization process of hcc, we designed point mutations which should inhibit or promote aforementioned processes. as it was expected, substitution of val57 with either asparagine or aspartic acid residues abolished dimerization process almost completely. in contrast, v57p mutant shows enhanced dimerization propensity. above observations are in good agreement with theoretical studies, performed on entire hcc and its fragment encompassing the hairpin structure centered on loop l1. the expansion of the cag trinucleotide that produces polyglutamine (polyq) segments in several proteins is responsible for at least eight neurodegenerative diseases. a possible therapeutic approach would be to inhibit the polyq self-assembly process using disrupting agents. although, screening studies have identifi ed several polyq aggregation inhibitors, their mechanism of action remains unknown. this is due to the poor aqueous solubility and fast aggregation of uncharged polyq peptides, which complicate their study. the most common strategy to resolve these problems is to produce polyq stretches fl anked with charged residues. however, this strategy was found to modify the aggregation behaviour of polyqs, their aggregate structure and their affi nity for disrupting molecules. to circumvent these problems, polyq peptides containing morpholine moieties, whose charge is ph-dependent, were produced using fmoc-based chemistry on spps. following an acid treatment, the designed peptides were soluble in acidic aqueous solutions and their aggregation rate could be controlled by ph variations and followed by dynamic light scattering. furthermore, aggregation initiated at physiological ph provided uncharged fi brils allowing the evaluation of the effects of charged disrupting agents. the actions of known polyq aggregation inhibitors such as polyq-binding peptide 1 (qbp1), trehalose and congo red were studied. peptide-protein and protein-protein interactions play key roles in most biological processes, and therefore represent valuable targets for drug discovery. an approach in the search of new chemical entities able to interfere with these interactions is the use of small molecules able to mimic or to induce precise aspects of specifi c peptide secondary structures. among the secondary structure elements, reverse turns have been shown relevant in many biomolecular recognition events. thus, different efforts have been directed to the design of turn mimetics or inducers. in this sense, 2-substituted azetidine-2-carboxylates, synthesized by our group through a versatile procedure from amino acids, possess the dihedral angle restricted to approximately 70º or -70º, depending on the absolute confi guration at the asymmetric carbon. these values are similar to those reported for the central residues of main types of -and -turns, and in fact, recent studies have shown the ability of the 2,2-disubstituted azetidines to induce -turns when incorporated at the i+1 position of model peptides. to know if this conformational behavior is distinctive of these restricted amino acids, we now investigate the infl uence of the incorporation of these amino acids at the i+2 position of model peptides. with this aim, we have synthesized a series of simplifi ed tetrapeptide models, rco-l-ala-azx-nhme, in which the rco and nhme groups are simplifi cations of the i and i+3 residues of the turn, respectively. for comparative purposes, dipeptide derivatives incorporating azetidine-2-carboxylate, pro and -mepro have also been prepared. the turn inducing capacities of all these model tetrapeptides have been analyzed by molecular modelling, ¹h rmn, ft-ir and x-ray methodologies. the results have shown the importance of the , -disubstitution at the heterocyclic amino acid for stabilizing reverse turns, and the infl uence of the ring size on the induced turn type infl uence of position of dehydroamino acid in peptide chain on conformation of dehydropeptides containing two dehydroamino acid residues it is known that presence of c -c double bound in dehydroamino acid infl uences on dramatic limitation of conformational space, not only side-chain but also main-chain [1] [2] . according to the peptide's length and neighboring amino acid residues, dehydroamino acid exerts s-shaped, -turn or helical conformation in peptides [3] [4] [5] [6] [7] . angiotensin-i converting enzyme (ace) somatic form bears two catalytic domains with two zn metal ions, while the testis form bears remarkable similarity to the aminoacid sequence of ace c-catalytic domain and only one zn metal ion. however, both exhibit their hydrolytic activity to vasoactive peptides such as angiotensin i (angi) and bradykinin (bk), though with different effi cacy. in order to study experimentally the possible interaction and binding events between the ace active site(s) and known ace substrates or inhibitors, we constructed peptide-based catalytic site maquettes (csm). 46-residue peptides were synthesized through solid-phase peptide synthesis having the aminoacid sequence that correspond to the ace val380-ala425 n-domain segment and to the val978-ala1023 c-domain segment and enzyme's active sites were reconstructed through addition of zinc metal ion in solution. the resulting complexes have the metal ion bound in a native-like mode, as manifested by previously reported nmr data. the reconstituted peptides were titrated separately by captopril and angiotensin-i with a molar ratio ace:captopril/angi 1:15 and 1:5, respectively. titration was followed by 1 h nmr spectroscopy and spectral changes (chemical shifts, line broadening, etc.) were recorded and analyzed. after the end of angi titration the solution of interacting peptides was titrated with captopril and the titration was followed by 1 h nmr spectroscopy. at the end of each titration 2d homonuclear 1 h-1 h tocsy and noesy spectra were recorded. analysis of high-resolution nmr data probes the conformational variation of ace csm and peptide substrates upon interaction, and in the presence of captopril in ace-angi solutions. contemporary development in nanotechnology is fabrication of nanostructured materials using the peptide-based self-assembly. importance of peptide as monomeric components is their capacity to be engineered to mediate spontaneous supramolecular self-assembly and their inherent chemical nature that facilitates chemical and biological recognition process. tubular self-assembled peptide nanostructures are of special interest since these can serve in various applications including 'bottom-up' fabrication of molecular scaffolds, nanoelectromechanical systems and nanomachines. an important discovery by gazit group revealed that the simple dipeptide molecule of phenylalanine which is core recognition motif of the alzheimer's -amyloid polypeptide effi ciently self-assembles into well-ordered nanotubular structure (1) which could be controlled by relatively simple chemical modifi cations at its termini. based on these observations and our interest in the synthesis of softmaterials for nanotechnology, we have successfully synthesized novel monomeric units of di-peptides with ureido functional group at nterminal of various hydrophobic l-amino acids. these derivatives have been prepared by using solid phase peptide synthesis protocol with cost effective process retaining the chirality of molecule. these ureido dipeptide formed nanostructures with high yield under mild and controlled condition in aqueous media. to fully understand the molecular and supramolecular mechanism guiding the formation of nanostructures we have selected a multidisciplinary approach by combining spectroscopic studies with advanced electron microscopic techniques. the morphology of nanostructures can be controlled by using variety of l-amino acids. these novel ureido di-peptides nanotubes can be used in fabrication of biocompatible peptide-based nanostructures and they will undoubtedly have nanotechnological applications. biomolecules are excellent in hierarchically organizing a characteristic structure by self-assembly. the spontaneous assembly of biomolecules has advanced a bottom-up approach for fabrication of nanostructured materials. we have developed fabrication of controlled nanofi bers through self-assembly of simple and short de novo designedsheet peptides with unique sequences [1, 2] . the single straight nanofi ber with high regularity constructed by -sheet fi peptide (sequence: pkfkiiefep) was functionalized with proteins by biotinylated peptides (3) and using functional anchors that have binding and functional groups. conjugation of self-assembled peptide nanofi bers with biomolecules such as peptides and proteins will expand their potentialities of the nanofi bers for various applications. to identify peptides binding to the self-assembled fi peptide nanofi ber, a phage display combinatorial screening using a random peptide library was performed. after fi ve rounds of biopanning, phage pools with highly specifi c affi nities to fi nanofi bers were screened. as a result of dna sequencing after phage cloning, 12 phage clones were identifi ed. binding affi nities of these clones were quantitatively investigated by an enzyme-linked immunosorbent assay. these clones showed specifi c affi nities to fi nanofi bers, as compared with ff and vi nanofi bers having slightly differences of amino acid sequences. affi nities and specifi cities were dependent on the sequence of each peptide, indicating that different amino acid sequences contributed to peptide interactions with nanofi bers. infrared spectroscopy studies by using chemically-synthesized peptides showed that the peptide binds specifi cally to the fi nanofi ber with structural transitions. owing to their diversity in size and shape, easy access, and biocompatibility, peptides represent versatile units for the construction of h-bonded tubular assemblies and other biomimetic materials with potentially useful applications. so-called peptide nanotubes (pnts) have been obtained through multiple and complementary approaches (1). originally designed from -peptides made of d-and l-amino acids (2) , fl at macrocyclic systems forming cylindricalsheet like assemblies have diversifi ed to include oligoamides made from higher amino acid homologs as well as peptide hybrids (e.g. ,peptides). tubular sheet-like assemblies however are not restricted to oligoamides. the urea group which shares a number of features with the amide linkage, i.e. rigidity, planarity, polarity, and hydrogen bonding capacity is an interesting surrogate. we and other have demonstrated that macrocyclic biotic and abiotic n,n'-linked oligoureas have a unique propensity to self-organize into polar h-bonded nanotubes (3). partial peptide backbone n-methylation has been introduced as a general strategy to generate truncated stacks (i.e. h-bonded dimers) useful to gain access to the thermodynamics of nanotube formation (1). herein, we describe biotic macrocyclic amide/urea hybrids with partially nalkylated backbones as new candidates for the formation of h-bonded dimers. a cysteine branched pga (polyglutamic acid) hydrogel is being investigated for therapeutic peptide and protein controlled delivery. entrapment of hgh as a model protein in this hydrogel has been carried out in order to check its later release in different aqueous buffered media and stability. different physico-chemical analysis (by maldi-tof, sec-mals-ri, 1h-nmr, cd, …) of the released hgh showed no effect on destabilization of the protein. controlled release systems of proteins involve important challenges such as maintaining the protein integrity. researchers need an exhaustive understanding of protein stability issue in drug delivery formulations. common degradative reactions in proteins involve: oxidation, deamidation, stability of disulfi de bonds and aggregation, which may affect secondary and tertiary structure of proteins and thus, their biological activity. the high water content which imbibes the polymer network of hydrogels enables a good accommodation for proteins. polyamino acids have been already studied in some biotechnological applications, however, in spite of their potential, they have barely been taken into account for the development of controlled release microparticles. we show that proteins could be easily loaded into cys cross-linked pga hydrogels. the protein hgh has been loaded and released preserving its physico-chemical integrity. thus, these hydrogels are promising for their use as therapeutic protein delivery systems. totally synthetic collagen-like gels by intermolecular folding of designed peptides our goal is to develop artifi cial collagens that can use as safer and functional biomaterials. here, we report the development of collagenlike gels by means of the intermolecular folding of chemically synthesized peptides. the peptides are disulfi de-linked trimers of collagenous gly-x-y triplet repeats with self-complementary shapes. the self-complementary peptides are able to form elongated triple-helix through spontaneous intermolecular folding. upon cooling of the peptide solutions, hydrogels of peptide supramolecules formed. the gel-sol transition was appeared to be reversible, and the transition temperatures were found to be tunable by the design of the peptides. our strategy for the totally synthetic collagen will offer possibilities for the development of innovative biomaterials. recent advance in biotechnology enables us to fi nd the peptides with affi nity for nonbiological materials and with function of mineralizing inorganic materials. the use of the functional peptides is attracting a growing interest for bottom-up fabrication approaches of nanoscale devise. zinc oxide (zno), a semiconductor with a wide direct band gap, possess unique optical, acoustic, and electronic properties, so that it is one of most widely studied metal oxides for solar cells, ultra violet nanolaser, blue light-emitting diode and so on. this wide variety of applications requires various fabrications of morphologically and functionally distinct zno nanostructures. here, the peptides which are selected from the phage-displayed peptide library with a 12-mer on the surface, can bind zno particle but not other metal oxides particle, and further, the zno-binding peptides play an important role on the crystal growth of zno in its synthesis. the anti-zno peptide can assist the synthesis of zno nanoparticles from a zn(oh) 2 solution even at 4 ºc, and further, the peptide leads to the self-assembly of synthesized zno particles to fl ower-type morphologies. we describe the biomimetic zno synthesis using the artifi cial peptide with affi nity for zno. the design of nanoparticles suitably functionalized for applications in biology or medicine is an ongoing challenge for both chemists and biologists. nanoparticles such as quantum dots, gold nanoparticles or carbon based-materials offer, in addition to their remarkable physical properties, the possibility to be functionalized by biomolecules, making them suitable for sensing, detection, diagnostic, and/or therapeutic applications. recently, quantum dots have been designed for biological imaging owing to their unique size-dependent fl uorescence properties. however, their plausible toxicity still remains a major concern for in vitro and in vivo applications. nanodiamonds, (nds) are also candidates for biomedical applications considering their intrinsic or induced fl uorescence and biocompatibility. the work will describe: i) the functionalisation and the characterization of 35 nm non-fl uorescent cnds and ii) their use in toxicity and transport studies in living cells after the grafting of a fl uorescent model peptide. we chose to introduce a nonpermeant fl uorescent peptide for the tracking of the nanoparticles on or inside the cells. nanodiamonds (cnds, 35 nm) coated by silanisation or with polyelectrolyte layers have been grafted with a fl uorescent thiolated peptide via a maleimido function, leading to aqueous colloidal suspensions stable for months. for each step of the preparation, the diameter of the nds measured by dynamic light scattering (dls), the zeta potential, the amount of amino groups grafted onto the surface, as well as the stability of the different nds suspensions no cytotoxicity was observed up to 72 hours incubation with cho cells with any of the prepared (40 g/ml). their capacity to enter mammalian cells, and their localisation inside cho cells, have been ascertained by confocal microscopy, refl ected light and fl uorescence. hepatitis b virus is one of the most common problem and potential danger of all over the world and also in our country. it is known that the use of proteins and peptide antigens as vaccines has several potential advantages over whole viral or bacterial preparations. to elicit the maximum immunogenic response from synthetic peptide antigens ,it is generally necessary to bind the peptide to carrier protein. moreover, to realize the full potential of synthetic peptide antigens protein-peptide conjugate will have to be used with an adjuvant. we have developed new approches for obtaining highly immunogenic peptide conjugatessynthetic polyelectrolytes (pe) were used for the conjugation with peptide molecules in which pe carry out the carrier and adjuvant roles simultaneously. this concept drive us to create new immunogenic bioconjugates of hepatitis b surface antigenic polypeptides (hbsag) with synthetic pe. in this study we used the region 95-109 of the s gene. the synthesis of peptides was performed by explorer pls ® automated microwave synthesis workstation (cem), by using f-moc chemistry. copolymers of acrylic acid with different monomers were used as a pe for the conjugation with polypeptides. pe-peptide conjugates was synthesized by microwave assisted method. composition and structure of bioconjugates werw characterized by hplc, spectrofl uorometry and different spectrophotometric methods. protein-protein and protein-ligand interactions are among the few essential cell processes whose understanding provide us insights into fundamental events of the life cycle. aside from in silico methods, natively folded proteins are an absolute prerequisite in all current biotechnological tools used for the study of these interactions. however, the recently developed ianus peptide array has a potential to evolve in to a protein-free method for detection of protein-protein interaction sites. using this assay, protein-protein interactions could be represented by and investigated as peptide-peptide interactions using peptide pairs immobilized on a solid support.(1) . two main obstacles had to be overcome for successful implementation of this array: (i) the library size and (ii) identifi cation of interacting peptide pairs. if, for example, interactions between two small proteins of only approx. 100 amino acids each should be analyzed, a library of ca. 2500 peptide pairs would be needed to cover all possible combinations of overlapping peptides derived from these proteins. although libraries of several thousand peptides were already successfully synthesized, the library size could be reduced drastically if the binding pocket of one protein is already known or if the protein-ligand interactions are studied. up to date, two methods for the identifi cation of interacting peptides in a library have been developed in our group. both methods will be demonstrated in studies of protein-protein and protein-ligand interactions. the role of the non-helical tailpiece in myosin ii assembly the hebrew university of jerusalem, israel self-assembly of macromolecules into large complexes is often mediated by folding of disordered regions. we used peptides to investigate the role of the non-helical tailpiece from non-muscle myosin iic (nm-iic) in its self-assembly process. nm-iic is a motor protein composed of a globular motor domain in the n-terminal region and a coiled-coil rod domain in the c-terminal region, which terminates with a non-helical 47-residue tailpiece (residues 1954-2000) . nm-iic molecules undergo self-assembly into fi laments that are necessary for its contractile activity. the tailpiece has an unfolded character and participates in the assembly process of nm-iic. the n-terminal region of the tailpiece (residues 1954-1968) has a net positive charge of +3 while its c-terminal region (residues 1969-2000) has a net negative charge of -10. we designed peptides that correspond to the entire tailpiece and to its negative and positive regions. these peptides were used to study interactions with the rod, their structures in the free and bound states, and structural changes they undergo upon binding to residues 1296-1854 of nm-iic rod. fluorescence anisotropy binding studies showed that a peptide corresponding to the positive region of the tailpiece (residues 1947-1968) bound the nm-iic rod (residues 1296-1854) with an affi nity of 13 m. circular dichroism studies showed that the positively charged peptide became folded upon binding the rod, indicated by a shift of the spectral minimum from 200 nm to 220 nm upon binding. a peptide corresponding to the negative region of the tailpiece (residues 1969-2000) did not bind nm-iic (residues 1296-1854). based on our results, we suggest that the positive region of the tailpiece is required for ordering and aligning neighboring molecules by electrostatic attraction, leading to fi lament formation. our results provide molecular insight into the role of the structurally disordered tailpiece of nm-iic in the selfassembly process. stefanidakis, michael; karjalainen, katja; pasqualini, renata; arap, wadih; koivunen, erkki md anderson cancer center, united states acute myelogenous leukemias (aml) are characterized with non-random patterns of medullary and extramedullary invasion. we hypothesized that a supramolecular complex, the leukemia cell invadosome, which contains certain integrins, matrix metalloproteinases (mmps) and other as yet unidentifi ed proteins, is essential for tissue invasion and may be central to the phenotypic diversity observed in the clinic. here we show that the specifi c binding of mmp-9 to leukocyte surface ám 2 integrin is required for pericellular proteolysis and migration of amlderived cells. an effi cient antileukemia effect was obtained by peptide inhibitors that prevented prommp-9 cell surface binding, transmigration through an endothelial cell layer, and extracellular matrix degradation. notably, the functional protein anchorage between ám 2 integrin and prommp-9 described in this study does not involve the enzymatic active sites targeted by any of the existing mmp inhibitors. taken together, our results provide a biochemical working defi nition for the human leukemia invadosome. disruption of specifi c protein complexes within this supramolecular target complex may yield a new class of anti-aml drugs with anti-invasion (rather than cytotoxic) attributes. the molecular mechanisms of action of the polycationic lysine homoand heterodendrimers on functional activity of adenylyl cyclase signaling system (ac system) in the myocardium and the brain of rats were studied. the lysine homodendrimers of the third (i), the fourth (ii) and the fi fth (iii) generations, as well as the lysine heterodendrimers of the fi fth generation -[(nh 2 ) 64 (lys-glu) 32 (lys-glu) 16 (lys-glu) 8 (lys-glu) 4 (lys-glu) 2 lys-ala-ala-lys(clac)-ala-nh 2 ] (iv), [(nh 2 ) 64 (lys-ala) 32 (lys-ala) 16 (lys-ala) 8 (lys-ala) 4 (lys-ala) 2 lys-ala-lys(clac)-ala-ala-nh 2 ] (v) and [(nh 2 ) 64 (lys-gly-gly) 32 (lys-gly-gly) 16 (lys-gly-gly) 8 (lys-gly-gly) 4 (lys-gly-gly) 2 lys-gly-gly-lys(clac)-ala-ala-nh 2 ] (vi) stimulated by receptor-independent mechanism the activity of heterotrimeric g proteins, preferably of inhibitory type, and interacted with c-terminal regions of their -subunits. the homodendrimers ii and iii and heterodendrimer v were more effective g protein activators. the treatment of the membranes with pertussis toxin (inactivating g i protein), but not with cholera toxin, led to a decrease of g protein activation effect of the dendrimers. the lysine dendrimers disturbed the functional coupling of the receptors of biogenic amines and peptides hormones with g i proteins and, to a smaller extent, with g s proteins. it was illustrated by the decrease of regulatory effects of the hormones on ac activity and g protein gtp binding and by the decrease of receptor affi nity to agonists in the presence of the lysine dendrimers, as result of receptor-g protein complex dissociation. it was shown that the molecular mechanisms of the action and the g protein selectivity of the polylysine dendrimers are similar to those of mastoparan and melittin, natural toxins of insect venom, which also preferably activate g i/o proteins and inhibit g i/o -coupled signaling. acknowledgements: supported by rfbi (grant 06-04-48809) and «russian science support foundation». 4 genetics, bielefeld university, germany dna-protein interactions are a key element in the regulation of cellular processes. transcription factors are able to recognize their cognate dna sequences and regulate the expression of proteins. as a model system, the specifi c dna binding of the transcription factor phob from e. coli is investigated in single molecule experiments. structurally, phob belongs to the family of winged helix-turn-helix proteins. it is composed of a transactivation domain (amino acids 1-127) and a dna binding domain (amino acids 123-229). after phosphorylation of the transactivation domain, the protein binds to specifi c dna sequences containing a tgtca consensus sequence. different c-terminally modifi ed protein epitopes representing parts of the dna binding domain of phob were chemically synthesized using microwave assisted solid phase peptide synthesis. the binding contributions of these molecules are compared to the complete dna binding domain (127-229). this protein was purifi ed using intein mediated protein splicing, an additional cysteine was ligated to the protein by intein mediated ligation. performing single molecule force spectroscopy experiments, kinetic off-rates were obtained, and sequence specifi c dna-binding of both peptide and protein was proven in competition experiments. alanine scans of strategic residues revealed the contributions of single amino acid residues for peptides and proteins. structural investigations of the peptides, the proteins and dna/protein complexes were performed using circular dichroism measurements. this method revealed structural differences of the peptides, proteins and dna upon complex formation. furthermore, the protein/dna or peptide/dna interaction was determined by surface plasmon resonance experiments and electrophoretic mobility shift assays. acknowledgment: this project was supported by dfg (sfb 613). reversed-phase chromatography is important in the development of well-characterized peptide pharmaceuticals. several factors infl uence sensitivity, resolution, and retention for lc and lc-ms applications: bonding chemistry, pore size, particle size, column confi guration, and solvent composition. by decreasing the particle size of hplc packings, column effi ciency is increased. we demonstrate the use of short 10-mm length hplc columns packed with 1.5 m, 500 å c18 silica for ultrafast, one-minute peptide analysis with moderate backpressures (< 3000 psi) using a conventional hplc system. for more complex peptide samples, such as protein digests, on conventional hplc, a novel column confi guration packed with 1.5 m, 100 å c18 is described for 10-to-20min. separations that traditionally take 30 to 80 minutes. we also discuss fast two-minute separations on an ultra-high pressure system using a variety of 1.5 m, small pore columns with unique selectivity. "unknown-genome" proteomics-based identifi cation of a new nadp-epimerase/dehydratase from desulf. phosphitoxidans by inverted-pcr, edman-sequencing and high resolution mass spectrometry protein identifi cation in proteomics is generally based on the availability of genomic data. using amenable databases, identifi cation in "bottom-up" proteomics is often straightforward but is highly complex in the absence of genome data, which typically requires "de novo"-identifi cation approaches. we present here a new approach for identifi cation of proteins from a bacterial strain, desulfotignum phosphitoxidans, with unknown genomic background, using a combination of (i), inverted pcr of degenerate primers derived from n-terminal edman sequencing, and (ii) high resolution maldi-fticr mass spectrometric peptide mass fi ngerprint-proteomics of expressed proteins. desulfotignum phosphitoxidans is an anaerobic sulfatereducing bacterium from marine sediment in which phosphite oxidation was found crucial for energy metabolism. culturing under different growth conditions provided 4 specifi cally expressed proteins with molecular masses of ca. 40 kda in the presence of phosphate, which were subjected to 2d-gel electrophoretical separation for soluble and membrane fractions using pdquest comparative analysis. n-terminal sequences of the proteins, determined by edman analysis were used for inverted pcr of degenerate primers, and provided a series of orf candidates, one of which coded for a putative nad-dependent dehydratase. in a complementary approach, protein spots from the 2d-gels were excised, digested with lys-c protease, and digestion mixtures analysed by maldi-fticr-ms. the accurate peptide masses unequivocally matched to identify a new nad-dependant epimerase/ dehydratase. the detailed functional characterization of the new protein, and further development and application of this approach to proteome analysis with unknown genomic background, are currently in progress. based on specially treated large pore silica and enhanced with a proprietary bonding process, vydac ms reversed-phase (rp) hplc columns offer superior performance for peptides and proteins. the deamidation of human growth hormone (hgh) has been monitored for many years by rp using vydac columns. the vydac ms c4 column provides the best overall performance characteristics (recovery, resolution, and peak symmetry) for the common important assay of hgh and desamido hgh. although hydrophobic membrane proteins are particularly diffi cult to separate, the vydac ms c4 column provides better separation and recovery (up to 86% higher vs. other leading columns) for a reptilian reovirus p14 protein and myristolyated form, a component of a potentially new vaccine delivery system. separation of the trypsin digest of fetuin, a glycoprotein, exhibits improved selectivity for peptide mapping on a vydac ms c18 column compared to other c18 columns, revealing some peaks otherwise not seen. the improved selectivity for peptides on the vydac ms columns results in better primary structure defi nition and easier identifi cation of degradation products and other protein characteristics. hemoglobin peptides in mammalian tissues: facts or artifacts?yatskin, oleg; karelin, andrei; ivanov, vadim shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, russian federation comparative rp-hplc and ms analysis of peptide composition of rat brain, heart, lung and spleen acidic extracts was performed. the extracts were prepared from snap-frozen organs both in the presence and in absence of protease inhibitors (10 -6 m pepstatin a, 10 -4 m pmsf, 2 mm edta) which stabilize the peptide composition of tissue acidic homogenates. the absence of inhibitors led to appearance of novel peptides and did not affect the concentration of predominant peptide components detected in samples prepared with protease inhibitors. therefore, the latter were considered as present in the tissues prior to extraction, i.e. as endogenous. the majority of predominant peptide components were found common in the studied tissues. we believe that the presence of predominant (> 100 pmol/g) peptide components in the protease-inactivated tissue extracts can be reliably extrapolated to their in vivo presence in the tissues. the endogeneity of minor (< 10 pmol/g) components requires a separate consideration. generally, the results of peptidomic studies strongly depend on sample preparation procedures involved, making diffi cult the straightforward comparison of the results obtained by different research groups. urine is a promising source of endogenous peptide biomarkers, because the protein concentration is 1000-fold lower than in plasma but the peptide concentration is equal. urine contains small metabolites that interfere with peptide analysis and urine fl ow varies between subjects and within a day. these variations pose a challenge to urine sample preparation, which, in case of serum, has been shown to be critical for reproducibility. we aim to optimize the enrichment method for urine peptides, to characterize these and to fi nd potential biomarkers. all methods resulted in good peptide recovery if the peptide concentration was normal and metabolite content low. slightly different peptides were obtained with different methods. the df preferentially recovered hydrophilic peptides, while sec-spe recovered high molecular weight peptides better than the df-method. sec-spe provided the best peptide enrichment especially for samples with high metabolite concentration. normalization was critical especially with very dilute samples. without normalization the number of peptides detected was highly dependent on urine concentration. normalization against specifi c gravity proved the most reproducible results. we have identifi ed several peptides found in all samples and some differences between cancer and control samples. validation of these differences is currently under way. jalili, pegah 1 ; ball, haydn 2 1 sigma-aldrich, united states; 2 in order to improve the detection of phosphorylated peptides/proteins, we developed a novel protocol that involves the chemical derivatization of phosphate groups with a chemically engineered biotinylated-tag (biotintag), possessing three functional domains; a biotin group for binding to avidin, a base-labile 4-carboxy fl uorenyl methoxy-carbonyl (4-carboxy fmoc) group, and a nucleophilic sulfhydryl moiety on the side-chain of cysteine. using this approach, the derivatized, enzymatically digested peptides were selectively separated from unrelated sequences and impurities on immobilized avidin. unlike previously published phosphopeptide enrichment procedures, this approach upon treatment with mild base, liberates a covalently bound gly-cys analog of the peptide(s) of interest, exhibiting improved rp-hplc retention and ms ionization properties compared to the precursor phosphopeptide sequence. the results obtained for a model peptide akt-1 and ovalbumin protein digest, demonstrated that the method is highly specifi c and allows selective enrichment of phosphorylated peptides at low concentrations of femtomoles/ l. in search of physiological substrates of brain prolyl oligopeptidase. prolyl oligopeptidase (pop) is a serine protease, which cleaves small peptides at the carboxyl side of an internal proline residue. substance p, arginine-vasopressin, thyroliberin and gonadoliberin are proposed physiological substrates of this protease. pop has been implicated in a variety of brain processes including learning, memory, and mood regulation, as well as in pathologies such as neurodegeneration, hypertension and mood disorders. however, there is no defi nite information about the physiological substrates of pop in brain. we have determined previously that some pop inhibitors, when administrated orally or intraperitoneal, are able to cross the blood-brain barrier and inhibit pop ex vivo. in this work also inhibition duration according to the doses were determined. using this system we studied the natural occurring peptide profi le in brain tissues from inhibited animals and compare it with the profi le in control tissue. we were able to design a method in which the background peptide level was importantly decreased which allowed us to identify, with high degree of confi dence, the peptides which were changed in rat hypothalamus upon pop inhibitor treatment. conclusions about the physiological substrates of pop are discussed. [1, 2] . in the present work, we further studied the dependence of peptide production by the cells on their functional state. using rat hepatocytes as a model, we compared peptide sets generated by: (1) primary hepatocytes in a differentiated state; (2) dedifferentiated primary hepatocytes with fi broblast-like phenotype; (3) rat hepatoma cells. peptides were extracted from the cellular lysates and supernatants by solid-phase extraction and then separated by rp-hplc. peaks were analyzed with maldi-tof and/or maldi-tof/tof. acknowledgements: this work was supported by ras presidium grant "molecular and cellular biology"; russian president grant "scientifi c schools" # 5737.2008.4; rfbr grant # 08-04-00550-a. triostin a is a naturally occurring quinoxaline depsipeptide antibiotic originally isolated from streptomyces s-2-210. with its two quinoxaline moieties it bis-intercalates into dna in a gc selective manner. triostin a contains n-methylated l-valine and l-cystein. the unmethylated analog tandem binds at selectively due to a different hydrogen bonding pattern. substitution of the disulfi de bridge of tandem by an azobenzene moiety leads to photoswitchable analogs. their photoswitchability was investigated and quantifi ed using uv, cd and nmr spectroscopy as well as hplc. in order to determine the three-dimensional structure, conformational analysis by nmr spectroscopy in combination with molecular dynamics calculations was carried out. in this process, distance restraints were obtained from nmr spectra measured in dmso-d 6 and applied in distance geometry/simulated annealing followed by molecular dynamics calculations. as it is possible to distinguish between cis-and transisomer in nmr spectra due to the difference in the chemical shifts, structures of both isomers can be investigated. dna binding studies were carried out using an optical tweezers setup with -dna. furthermore, uv melting curves were recorded for the tandem derivatives in complex with dna. cd spectroscopy was applied to observe changes in dna structure upon formation of the complex. in addition to structural investigations, the antibiotic activity of the tandem derivatives was investigated in minimal inhibition concentration assays against gram-positive bacillus subtilis. acknowledgment: this work was supported by the dfg (sfb 613). k. gaus gratefully acknowledges the fi nancial support (phd grant) by the international nrw graduate school of bioinformatics and genome research. cyclic peptide nanotubes are formed by self-assembly of cyclic peptides with alternating l-and d-amino acid. however, peptide nanotube selfassembly phenomenon had not yet been made cleared. intermolecular electrostatic interaction is one of the important driving forces to assemble cyclic peptides to peptide nanotubes. here in, a series of cycilc hexapeptides, cyclo(-l-lys-gly-) 3 , cyclo(-l-glu-gly-) 3 , cyclo(-l-lys-d-ala-) 3 , cyclo(-d-glu-l-ala-) 3 , cyclo(-l-trp-d-lys-) 3 , and cyclo(-l-trp-d-glu-) 3 , were designed and synthesized by solid-phase peptide synthesis using fmoc strategy. turbidity study revealed that the abilities of self-assembly formations of the 1:1 mixtures of cyclo(-l-trp-d-lys-) 3 and cyclo(-l-trp-d-glu-) 3 in aqueous solution are extremely higher than the abilities of each individual positively or negatively charged cyclic peptides. transmission electron microscope (tem) experiments showed that mixture of cyclo(-lys-xxx-) 3 and cyclo(-glu-xxx-) 3 formed fi brous structure. tem images indicated that, the diameters of the nanotubes were approximately 2-5 nm. diameter of similar cyclic peptides was found to be 1 nm approx. these results also supported that the bundle formation of peptide nanotube. conformations of monomer cyclic hexapeptides were calculated based on nmr observation. estimated formation of peptide nanotube is consisted with the bundle structure. koèevar, nina; obermajer, nataša; kreft, samo faculty of pharmacy, slovenia therapeutic proteins offer a promising potential as effective drug compounds. however, proteins are generally poorly transported across biological membranes due to hydrophilicity. fatty acylation with reactive fatty acid derivatives represents one of the basic methods for increasing the proteins' hydrophobicity and improving membrane permeability. key: cord-006230-xta38e7j authors: nan title: deutsche gesellschaft für experimentelle und klinische pharmakologie und toxikologie e.v. date: 2012-02-22 journal: naunyn schmiedebergs arch pharmacol doi: 10.1007/s00210-012-0736-0 sha: doc_id: 6230 cord_uid: xta38e7j nan nucleoside diphosphate kinases (ndpks) are multifunctional enzymes involved in a variety of cellular processes including cancer metastasis and heart diseases. the plasma membrane content of the three major ndpk isoforms ndpk a, b and c is increased in human heart failure. we have previously shown that the ndpk b isoform regulates camp levels and cardiac contractility through a receptor-independent gprotein activation involving direct g protein β subunit phosphorylation. the precise role of ndpk c in the heart is unknown and was the object of this study. ndpk c function was assessed in neonatal (nrcm) and adult (arcm) rat cardiomyocytes with real-time pcr, immunoblotting, and quantification of camp content. heart failure was induced by chronic treatment with isoproterenol (iso, 2.4 mg/kg/d 4 days) via minipumps. chronic iso increased mrna levels of ndpk c by 9.4±1.9-fold and its protein levels by 2.1±0.11-fold. immunoprecipitation of the g protein β subunit resulted in coimmunoprecipitation of ndpk c and the stimulatory gαs subunit: iso enhanced this interaction. upon iso stimulation, ndpk c translocated from the cytosol to the plasma membrane within 3 hours in both nrcms and arcms. adenoviral overexpression of ndpk c in nrcms caused a 1.5-fold increase in basal and iso induced camp synthesis, whereas sirna mediated knockdown of endogenous ndpk c decreased camp levels by ~50%. our results establish ndpk c as a novel and critical regulator of camp synthesis and gs signaling in the heart. the up-regulation of ndpk c and the increased responsiveness to iso in failing hearts point to ndpk c as a potential counterregulatory factor in the onset of heart failure. uptake and metabolism of methylated myricetin derivatives: studies in cell culture and c. elegans ackermann d. 1, 2 , büchter c. secondary plant compounds like flavonoids that are ubiquitary present in fruits and vegetables are believed to exert health protective effects in terms of lowering the incidence of widespread diseases such as cardiovascular diseases and cancer. besides their antioxidative effects, flavonoids may also modulate cell signaling pathways and thereby performing their disease-protective actions. though this class of dietary polyphenols has become increasingly popular as dietary supplements, only little is known about their metabolic fate in vivo. therefore, we investigated the absorption and metabolism of several flavonoids such as myricetin and its methylated derivatives laricitrin, syringetin, and myricetin-3',4',5'-trimethylether in the human colon carcinoma cell line hct116 and the human hepatoma cell line hepg2 as well as the model organism caenorhabditis elegans. all flavonoids were rapidly taken up by both cell lines as shown by hplc analyses. the intracellular amount of myricetin and laricitrin did not increase with time and was only half of that of syringetin and myricetin-3',4',5'-trimethylether. interestingly, no metabolites of these flavonoids could be detected which might at least in part be due to their low intracellular amounts. absorption as well as intracellular distribution was also evidenced by using the fluorescent dye "naturstoff reagent a" nsra) . fluorescence microscopy indicated a predominant cytosolic distribution of the employed flavonoids. in the model organism caenorhabditis elegans, the flavonoids were exclusively distributed in the intestine as visualized by nsra. the antioxidative capacity of the four flavonoids (measured by using the cell free teac assay and the h 2dcf-da assay in hct116 cells) decreased with increasing methyl groups in the b-ring. myricetin-3',4',5'-trimethylether (three methyl groups) was the least effective radical scavenging flavonoid in both the cell free system and in hct116 cells compared to myricetin (no methyl groups). in conclusion, for exerting their biological effects, uptake and distribution as well as metabolism of flavonoids in certain organs such as liver and gut are important and more research in that field is warranted. munich heart alliance, münchen, germany signaling through g protein-coupled receptors is affected by receptor polymorphisms, yet the molecular basis for the functional differences of individual receptor variants is unclear. to investigate the impact of the frequent gly389arg variant of the β1-adrenergic receptor (β1ar) on receptor conformation we used β1ar-sensors capable of fluorescence resonance energy transfer (fret). these sensors retained the pharmacological and functional characteristics of the native receptors. upon stimulation of the sensors we determined the activation characteristics of the polymorphic receptors in real time and in living cells. we found the β1ar variants to behave similar upon a single stimulation with an agonist, but to differentially respond with a change of their activation kinetics during subsequent stimulations. while the arg389-β1ar did not show altered activation kinetics after prestimulation, the gly389-β1ar became slower compared to the initial stimulation suggesting that β1ars possess a memory of previous activation. we then permeabilized β1ar-sensor-expressing cells with saponin to remove soluble cytosolic factors. upon permeabilization the β1ar variants did not display receptor memory, suggesting that the β1ar memory depended on the interaction of the receptors with soluble cytosolic factors upon their initial activation including the phosphorylation of agonist-bound receptors by protein kinase a or g protein-coupled receptor kinases. our findings suggest an intrinsic, polymorphism-specific property of βars that alters activation kinetics upon continued stimulation and that might account for individual drug responses. micro-rna replacement therapy: nanoparticle-mediated in vivo delivery of mirna-145 or mirna-33a exerts antitumor effects in colon carcinoma xenograft mouse models weirauch u. 1 micro-rnas (mirnas) control the expression of various genes, and under pathological conditions several mirnas are up-or downregulated. previous in vitro studies have established a pro-apoptotic and anti-proliferative role of mir-145, which shows decreased levels in colon carcinoma. in contrast, while mir-33a is only weakly expressed in several tumors as well, its role in cancer has not been analysed so far. in this study, we demonstrate the tumor-relevance of mir-33a and identify the protooncogenic kinase pim-1 as a target of mir-33a. pim-1 harbours a highly conserved mir-33a binding site within its 3'-utr, and seed mutagenesis of this target sequence abolishes the mir-33a-mediated downregulation of pim-1. the knockdown of pim-1 by rnai or mirna transfection inhibits proliferation in leukemia and in colon carcinoma cells by decelerating cell cycle progression, thus establishing a tumor inhibitory function of mir-33a. we furthermore introduce polyethylenimines (peis) for the therapeutic application of mirnas in vivo, which is critically dependent on the development of appropriate delivery tools. peis are able to form non-covalent complexes with mirnas, leading to mirna protection after systemic application in combination with an attractive biodistribution profile and the efficient uptake in target organs/cells. therapeutic effects of pei-mediated mirna delivery were demonstrated in subcutaneous colon carcinoma xenograft mouse models. the in vivo application of mirna-145 through systemic or local injection of pei/mirna complexes resulted in efficient mirna delivery and in antitumor effects, based on the concomitant repression of erk5. likewise, tumor growth inhibition was observed upon treatment of tumor-bearing mice with pei-complexed mir-33a. this is due to the mir-33a-mediated downregulation of pim-1 expression and resembles the pim-1 knockdown through rnai / pim-1 sirnas. taken together, in tumor xenograft mouse models we establish mirna replacement therapy through the pei-complexation of mirnas as a novel therapeutic strategy and demonstrate that mir-145 and mir-33a may be promising mirnas in colon carcinoma therapy. md 288, a hybrid of chloroquine and primaquine, is a potential drug against infectious diseases such as malaria. since one moiety of the hybrid, the known antimalarial drug chloroquine, is a known intercalator, the potential of md 288 to intercalate into dna was determined. due to the ability of intercalators to cause frame shift mutations, the mutagenic potential of md 288 was also investigated. the potential of md 288 to intercalate into dna was investigated by means of fluorescence based micro plate assay using ethidium bromide (eb) and isolated calf thymus double stranded dna. as a positive control chloroquine was used. the potential of md 288 to cause gene mutations was determined using the hypoxanthine-guanine phosphoribosyltransferase (hprt) test in chinese hamster v79 lung fibroblasts (v79 cells). v79 cells were treated with 0.4 µm, 0.8 µm and 1.5 µm md 288 or the positive control, the direct mutagen 4-nitroquinoline-n-oxide (nqo, 1 µm) for 24 h. on day 6, mutants exhibiting loss of hprt function were selected with 6-thioguanine . whereas at 1 µm chloroquine, a 38% decrease in fluorescence intensity of eb (indicating dna intercalation) was observed, 10 µm md 288 were needed to observe a similar decrease (28%) in fluorescence intensity of eb. therefore, md 288 is 10fold less potent to intercalate into dna than its moiety chloroquine. the frequency of spontaneous 6-tg resistant mutants per 10 6 colony-forming cells was 9 ± 2. as expected, 1 µm nqo caused a significant increase in the mutant frequency (mf, 168 ± 9) . in contrast, mf was not significantly affected by treatment with md 288 at both noncytotoxic (0.4 µm: 5 ± 4) and cytotoxic (0.8 µm: 9 ± 7) concentrations. in conclusion, md 288 is a less potent intercalator than the known antimalarial drug chloroquine. furthermore, md 288 does not cause gene mutations in the hprt test. since current studies show that various metabolites of md 288 are formed in vitro, their mutagenic potential is currently under investigation as well. -conducting channels but depends critically on the membrane potential. trp channels form cation entry channels thereby either contributing to ca 2+ entry or depolarisation. recently, we showed that trpm4 acts as a ca 2+ -activated non-selective cation channel and critically determines the driving force for ca 2+ influx in mast cells following fcεri-stimulation (1) . in addition to trpm4 we also identified the expression of other trp transcripts in bone marrow derived mast cells (bmmc) including those encoding trpc2, trpc3, trpc5, trpc6 and trpm7. in peritoneal mast cells (pmc), rt-pcr indicated expression of trpc1, trpc4, trpc5, trpc6, trpm2, trpm4 and trpm5. to identify the functional role of those trp channel proteins for mast cell activation we analysed ca 2+ signaling using microfluorimetry in bmmcs and pmcs after stimulation with substances known to activate trpc6 channels in other cell systems such as the diacylglycerol analogue oag, the hyperforin analogue hyp-9 and flufenamic acid (ffa), but could not evoke a rise in the [ca 2+ ]i in both pmc and bmmc. sphingosine 1phosphate and lysophosphatidylcholine, which were reported to activate trpc5 channels, induced only minor rise in [ca 2+ ]i in bmmcs, respectively. here, we will present our analysis of ca 2+ signaling following stimulation of the fcεri receptor and application of secretagogues that are supposed to affect ca 2+ -dependent mast cell activation such as adenosine, endothelin-1, substance p and compound 48/80 in bmmcs and pmcs derived from mouse lines with inactivation of trpc1, trpc3, trpc4, trpc5 or trpc6 since specific antagonists are still lacking for these trp channels. the α2a-ar is the main ar in the central nervous system and it plays a crucial role in regulating norepinephrine (ne) release from nerve terminals via presynaptic feedback inhibition. it is also associated with a number of physiological effects, including hypotension, pain perception, sedation and modulation of mood. ne, once released in the synaptic space, binds to α2a-ars and induces a rearrangement of the receptors from the inactive state into an active conformation. this allows the binding and activation of the cognate gi-protein and, hence, the transduction of the transmembrane signal to the downstream effectors. generally, α2a-ar activation has been deduced from the stimulation of a receptor-mediated biological response that could be easily followed experimentally. however, most of these approaches do not employ living cells and are normally applied under equilibrium conditions that need prolonged incubation periods incompatible with the physiological temporal dynamics of ne. here, we monitored the ne-mediated α2a-ar and gi-protein activation by using a fluorescence resonance energy transfer (fret)-based approach in living cells. to examine the effects of increasing concentrations of ne on the speed and extent of α2a-ar activation with very high temporal resolution, we took advantage of the previously described α2a-ar flash/cfp sensor [1] . the results indicate that in our system the efficacy of ne in eliciting α2a-ar flash/cfp activation increases in a time-dependent way and reaches the maximum with a half-life of ~ 70 ms. the ec50 values decrease in an exponential manner and arrive at ~ 2 µm with a half-life of ~ 330 ms. next, we analyzed the ability of increasing concentrations of ne to trigger a downstream intracellular response after α2a-ar stimulation by monitoring the kinetics and amplitude of gi activation in living cells. we applied the previously well characterized gi cfp/yfp sensor [2] . the results show that both the efficacy and the potency of ne in inducing gi activation reach the steady state slower compared to receptor activation (half-life ~ 700 ms and ~ 3,000 ms respectively). in conclusion, we were able to monitor ne-mediated events occurring in the millisecond time scale and reaching the equilibrium in a time interval compatible with physiological conditions. sphingosine-1-phosphate (s1p) is an immune modulator produced by sphingosine kinase 1 (sphk1) and sphingosine kinase 2 (sphk2) and de-phosphorylated or degraded irreversibly by s1p phosphatases and a lyase, respectively. we recently showed that tlr4-induced il-12p70 is selectively counter regulated by sphk1, s1pr1 and its extracellular ligand s1p. on the other hand, spiegel et al. have demonstrated that specific, sphk1-dependent, binding of s1p to traf2 enhances the tnf-alpha signaling. therefore we were interested whether the tlr/tir and tnf-alpha-signaling pathways are interfered with each other and are modulated by s1p. in a first approach we focused our investigations on sphk1 effects on both traf2 and traf6 stimulatory signals and cytokines produced downstream. experimentally, with gm-csf expanded, bone marrow-derived dcs we first desensitized the lps-tlr4 or cpg-tlr9 signal by a defined time period of costimulation with tnf-alpha. the initial results showed a partial decrease of il-12p70 secretion in tnf-alpha-co-stimulated dcs in contrast to lps stimulation alone. this might indicate that traf2 activated via tnf-alpha interacted with the traf6 pathway to reduce il-12p70. further series with dcs derived from sphk1-deficient mice confirmed our former results that il-12p70 in contrast to other cytokines is specifically sensitive to sphk1-s1p feedback, but did not change the effects of tnf-alpha on wt dcs il-12p70 release. in comparison, cpg-tlr9-induced il-12p70 release reached only 20% of lps-induced il-12p70 levels and was less sensitive to tnf-alpha costimulation. however, sphk1-deficiency strongly augmented cpg-dependent il-12p70 production. in ongoing experiments we started to analyze the details of traf2/rip1 and traf6/tak1 activation by ubiquitination blots in wt, sphk1-and s1plyase-deficient dcs. in conclusion, we hope to unravel possible mechanisms of the observed differential effects of s1p and its enzymes on inflammation and cancer-relevant cytokines. identification of the kh type splicing regulatory protein (ksrp) as a new important mediator of the anti-inflammatory effects of resveratrol art j. 1 , besche v. 2 , bros m. 2 , li h. 1 , handler n. 3 , bauer f. 3 , erker t. 3 , behnke f. 4 , mönch b. 5 , förstermann u. 1 , dirsch v. m. 6 , werz o. 5 , kleinert h. 1 , pautz a. university of vienna department of pharmacognosy, althanstr. 14, 1090 wien, austria resveratrol, a polyphenol derived from different plants, possesses multiple pharmacological functions such as anti-oxidative, anti-diabetic, cardioprotective, anticancer, neuroprotective and anti-inflammatory properties. many of these effects have been attributed to its anti-oxidative activity but resveratrol also modulates signal transduction pathways like the p38 mapk pathway or the activity of different transcription factors like nf-κb redox-independently. moreover, the histone deacetylase sirtuin 1 (sirt1) is an important mediator of resveratrol effects. nevertheless the direct molecular target of resveratrol remains unclear. in target fishing experiments we identified the rna-binding protein ksrp as direct resveratrol binding partner. ksrp is an rna-binding protein that controls proinflammatory gene expression on the post-transcriptional level by modulation of mrna stability. moreover, it is involved in the biogenesis of mirnas. resveratrol treatment of human dld-1 cells resulted in a decreased mrna expression of a number of well known ksrp target mrnas and enhanced mirna-155 function. downregulation of ksrp expression by sirna prevented the mrna destabilizing effect of resveratrol. as the activity of ksrp is mainly regulated on the post-translational level by phosphorylation of different serine and threonine residues we analyzed whether resveratrol changes ksrp activity by altering the phosphorylation of the protein. indeed, our immunoprecipitation experiments demonstrated that resveratrol reduces the p38 mapk-mediated phosphorylation of threonine residues in the ksrp protein and thus leads to an increase of ksrp activity. interestingly, resveratrol does not block p38 mapk activation or activity. in addition we have evidence that sirt1 is not involved in the resveratrol mediated activation of ksrp. so we believe that activation of ksrp by resveratrol is the major mechanism mediating the anti-inflammatory effects of resveratrol. in vitro testing of oecd reference nanomaterials (nm-series) in rat precision cut lung slices aumann a. 1 the oecd has defined reference nanomaterials (nm) to be tested in different endpoints concerning human health and environmental safety (1) in order to evaluate if the toxicity of nanomaterials can be linked to their physico-chemical properties. for nanomaterials, inhalation presents the major exposure route of concern and can be assessed using acute inhalation toxicity studies in rodents. however, these in vivo studies are resource intensive and animal consuming. the oecd working party on nanomaterials has named several alternative methods as being of particular interest for testing of nanomaterials; among them is the precision-cut lung slices model (pcls) to estimate respiratory toxicity. we have tested all 16 nm in pcls measuring cytotoxicity, apoptosis, oxidative stress and inflammatory response of the tissues as well as observing them histological. for in vitro exposure of pcls the test material was dispersed in medium. since it is the nature of these materials to change their surface characteristics and agglomeration state in different environments, a standardized dispersion method (nanocare) using bovine serum albumin as a stabilizing agent, was used. particle size-distributions of the nanomaterial dispersions were characterized via analytical ultracentrifugation and found the nanomaterials well dispersed. silver and zinc oxide but none of the other nm showed cytotoxicity to the lung tissue in the tested concentrations. however, differences in cytokine profiles among the nm were observed and showed several correlations to the results obtained in in vivo inhalation or instillation studies. universität des saarlandes institut für molekulare zellbiologie, gebäude 61, 66421 homburg, germany tmem2 proteins show similarities in their primary sequence to motifs that are conserved amongst various members of the trp protein family. based on hydropathy analysis these proteins exhibit 6 to 10 membrane spanning domains. in contrast to trp channels there is no evidence that these proteins form ion channels in the plasma membrane following overexpression of their cdna in hek293 cells. tmem2 -/mice are viable and show no obvious signs of disease, but exhibit increased pancreatic amylase and lipase plasma levels. microfluorimetric measurements using fura-2 revealed that the elevation of the cytosolic ca 2+ concentration after stimulation with carbachol and the cholecystokinin analogue caerulein is unchanged in tmem2-deficient acinar cells. tmem2 is expressed in several cell types including pancreatic acinar cells, cardiac myocytes, cardiac fibroblasts, but their subcellular localization is still unkown. we generated several constructs encoding tmem2 fusion proteins with fluorescence protein tags by fusing eyfp, mcherry and tagrfp-t to the n-and c-terminus of the protein, respectively. based on western blot experiments and expression in hek293 cells the tmem2-eyfp construct was most suitable for further colocalisation analysis and generation of viral vectors including adenovirus and semliki forrest virus. in contrast to the prediction by the psort ii algorithm tmem2-eyfp could not yet be identified in the plasma membrane of fibroblasts, cardiac myocytes or acinar cells but showed a vesicular subcellular localization pattern. we localized tmem2-eyfp in acidic compartments and predominantly in lysosomes (pearson coefficient (pcc) 0,79 ± 0,03, n= 4 using lysotracker ® dye). analysis of subcellular localization with independent tmem2 fusion constructs and additional vesicular markers will be presented as a framework to get insights towards the cellular function of tmem2 and to reveal the mechanisms underlying increased amylase release from acinar cells of tmem2 -/mice. the small molecule bcl-2/mcl-1 inhibitor tw-37 shows single-agent cytotoxicity in neuroblastoma cell lines bachmann h. s. 1 , akdeli n. high-risk neuroblastoma (nb) remains a therapeutic challenge in paediatric oncology. pro-survival bcl-2 family proteins critically regulate apoptosis, and may represent important therapeutic targets in nb. primary nb tumours heterogeneously express mcl-1 or bcl-2, with high expression correlating to high risk phenotype. co-expression can be detected in approximately 10% and is correlated to reduced survival. recent studies with two inhibitors that predominantly target bcl-2 and other proteins, but not or to a lesser extend mcl-1, elucidated the importance of mcl-1 inhibition for cytotoxicity in nb. tw-37 is a small molecule inhibitor that showed almost equal affinity to bcl-2 and mcl-1. to explore the effect of combined bcl-2/mcl-1 inhibition on neuroblastoma cells, four cell lines (sk-n-as, imr-5, sy5y and kelly) were treated with tw-37 and changes in growth properties were determined. furthermore, nude mice with kelly (human neuroblastoma cell line) xenografts were treated with tw-37. using sirna, we investigated the functional relevance of mcl-1 and bcl-2 in kelly cells. for in vitro cell viability we observed ic50 values of 0.59 ± 0.39 µmol/l. on treatment with 1 µmol/l dose of tw-37, all neuroblastoma cell lines analyzed showed significantly reduced proliferation and increased apoptosis rates. bcl-2 as well as mcl-1 knockdown induced apoptosis in kelly cells. interestingly, tw-37 was able to reduce, but not to abrogate growth of kelly neuroblastoma xenografts in nude mice. in conclusion, combined inhibition of bcl-2 and mcl-1 using tw-37 exhibits strong single-agent antitumor activity on human neuroblastoma cells in vitro, but limited single-agent activity in vivo. therefore, inhibition of bcl-2/mcl-1 may represent an interesting therapeutic strategy, most likely in combination with conventional chemotherapy and other specific inhibitors. localization and functional characterization of membrane transporters for sulfated steroid hormones in the human testis bakhaus k. 1 , wapelhorst b. circulating sulfated steroid hormones like estrone sulfate (e1s) or dehydroepiandrosterone sulfate (dheas) are delivered to the testis via membrane uptake carriers such as the sodium-dependent organic anion transporter (soat). inside the cell these sulfated steroids can be metabolized to active steroid hormones by the catalytic activity of the steroid sulfatase (sts), which shows high enzymatic activity in the testis ("sulfatase pathway"). in addition to soat, other candidate carriers like the organic solute carrier protein 1 (oscp1) and the organic anion transporting polypeptides oatp6a1 and oatp1c1 are predominantly expressed in the human testis and demonstrate transport activity for sulfated steroids. we aimed to evaluate the cellular expression of soat and the other steroid sulfate carriers and their co-localization with the steroid sulfatase (sts) in human testis. furthermore we want to perform functional transport studies with the steroid sulfate carriers in stably transfected hek293 cells. we detected soat by rt-pcr and western blot analysis in the human testis. single cell analysis and in situ hybridization revealed pachytene primary spermatocytes to express the soat mrna. soat expression in specimens showing maturation arrest at the level of early round spermatids seems to be severely reduced or absent. sts mrna was detected by rt-pcr in testis homogenates. preliminary immunohistochemical data showed that sts may be expressed in germ cells and interstitial leydig cells. hek293 cells stably expressing the soat carrier protein showed significant transport activity for dheas. this was demonstrated by using a radiolabeled [ 3 the hepato-intestinal induction of the detoxifying enzymes cyp3a4 and cyp3a5 by the xenosensing pregnane x receptor (pxr) constitutes a key adaptive response to oral drugs and dietary xenobiotics. in contrast to cyp3a4, cyp3a5 is additionally expressed in several, mostly steroidogenic organs, which creates potential for induction-driven disturbances of the steroid homeostasis. using cell lines and mice transgenic for a cyp3a5 promoter we demonstrate that the cyp3a5 expression in these organs is noninducible and independent from pxr. instead, it is enabled by the loss of a suppressing yin yang 1 (yy1)-binding site from the cyp3a5 promoter which occurred in haplorrhine primates. this yy1 site is conserved in cyp3a4, but its inhibitory effect can be offset by pxr acting on response elements such as xrem. taken together, the loss of yy1 binding site from promoters of the cyp3a5 gene lineage during primate evolution may have enabled the utilization of cyp3a5 both in the adaptive hepato-intestinal response to xenobiotics and as a constitutively expressed gene in other organs. our results thus constitute a first description of uncoupling induction from constitutive expression for a major detoxifying enzyme. they also suggest an explanation for the considerable tissue expression differences between cyp3a5 and cyp3a4. serum albumin adducts as biomarkers for systemic bioavailability of active metabolites of various glucosinolates in animal models and humans barknowitz g. 1 , engst w. glucosinolates (gls) are natural pesticides of brassicales, which comprise many important food and feed plants. upon physical damage to the plant, the enzyme myrosinase can convert gls to reactive metabolites (e.g. isothiocyanates). the same reaction can also be catalyzed by enzymes of the intestinal microbiota. modification of sensor proteins (e.g. keap-1) by some gls metabolites leads to adaptive responses, resulting in enhanced detoxification of reactive metabolites and other protective reactions. at least in experimental models, this mechanism can be exploited for chemoprevention of carcinogenesis induced by various chemical carcinogens. however, own research revealed that certain reactive gls metabolites can covalently bind to dna in vitro and in vivo, involving possible genotoxic and carcinogenic risks. animal models are useful for studying beneficial and adverse effects of gls. however, it has to be taken into account that the toxicokinetics of gls may differ between rodent and humans and that the active metabolites are short lived and thus difficult to detect and quantify. likewise, exposure of humans to gls and their breakdown products may enormously vary depending on (i) food preferences, (ii) cultivars, growth conditions and preparation of plants consumed and (iii) variations in human xenobiotic metabolizing system and composition of intestinal microbiota. in order to estimate individual levels of systemic exposure to reactive gls metabolites, blood protein adducts may be useful. we have developed lc-ms/ms methods for quantifying serum albumin adducts formed by glucoraphanin, glucotropaeolin and neoglucobrassicin. the method involves digestion of the protein to amino acids and the usage of isotope-labelled amino acid adducts as internal standards. serum albumin adducts were detected in mouse models after feeding broccoli and pak choi respectively, as well as after administration of purified gls and breakdown products. likewise, gls adducts were detected in human blood plasma after consumption of broccoli or cress. this work was financially supported by the bundesministerium für bildung und forschung (grant 0315370d) . barlow s. harrington house, 8 harrington road, brighton, bn1 6re, great britain values for cancer endpoints for the application of the ttc approach have been derived by linear extrapolation of results from animal carcinogenicity studies to calculate "virtually safe doses" (vsds). a vsd is defined as an exposure that represents an estimated upper bound increase in risk of 1 in a million of developing cancer during a lifetime. in 1995, from consideration of a range of vsds for carcinogens, the us food and drug administration (fda) proposed and adopted a value of 0.5 micrograms/kg of diet (0.5 ppb) as a threshold of regulation to be used for substances present in food contact materials. the fda considered that if exposure to a substance in the diet was below this value, consumers would be protected "with reasonable certainty of no harm" and no toxicological data on the substance need be submitted. the value of 0.5 ppb is equivalent to 1.5 micrograms/person per day, assuming that 1500 g of food and 1500 g of fluids diet might be consumed daily. this value was subsequently incorporated into the ttc approach to be used for assessment of substances without a structural alert for genotoxicity. in 2004, kroes and colleagues further explored cancer as an endpoint and recommended a lower ttc value of 0.15 micrograms/person per day for substances with a structural alert for genotoxicity and exclusion from the ttc approach of certain groups of high potency carcinogens (with vsds below this value). in this presentation, the data underpinning these ttc values will be discussed from the perspective of their reliability for risk assessment of substances with low exposures, for which there are no toxicity data, but which may in fact be genotoxic or non-genotoxic carcinogens. stable conjugates which are recognized by the immune system. in the current investigations, the ability of chemical pre-treatment to interfere with antibody-protein binding has been investigated using ovalbumin (ova) as the model protein and naturally occurring anti-ova igg from healthy human donors. preparation of conjugates: ova (1 mg/ml) was dissolved in sodium borate buffer (0.1m, ph 9.4) . for chemical treatment various amounts (50-200 mg) of 1-fluoro-2,4dinitrobenzene (dnfb; sensitizer) or 2,4-dichloro-1-nitrobenzene(dcnb; non-sensitizer) was added and stirred for 2 hrs at room temperature. unbound compound was removed by consecutive dialysis against phosphate buffered saline (pbs) and distilled water. inhibition elisa: plates were coated with 100 µg/ml ova and blocked with 10% fcs in pbs. ova samples (native or conjugated; 0.6 -10 µg/ml) were pre-incubated with polyclonal antibodies (ab) from pooled normal human serum for 30min and then added to the plates. human anti-ova igg abs were detected by colorimetric analysis using ortho-phenylendiamine as a substrate. the concentration of soluble native ova or conjugate required to displace 50% of ab to plate-bound ova (ic50) was calculated (minimum of n=3 independent experiments). treatment of ova with the chemical sensitizer and protein reactive dnfb resulted in increased ic50 value, whereas mock treatment resulted in comparable ic50 values to native ova. treatment with dcnb showed that the presence of a chemical per se (and possible denaturation) was not sufficient to alter the ic50 value; conjugation of the compound to the protein was required. the analysis is not test chemical specific (specific ab against compound-protein conjugates are not required) and the colorimetric analysis is unaffected by absorbance of the compound itself. thus, this method may have utility for the identification of chemical sensitizers which are directly protein reactive. 2´-deoxy-camp in human cell lines: another second messenger? beckert c. 1 , hinz c. we have shown that 2´-deoxy-camp (dcamp) is synthesized by recombinant human soluble adenylyl cyclase (sac). here, we report that dcamp can be detected and quantified by liquid chromatography coupled to mass spectrometry (lc-ms) in various human cell lines. in most cells a ratio of camp : dcamp of ~10 was observed. as a remarkable exception, in hl-60 promyelocytic leukemia cells, the dcamp concentration exceeded the camp concentration more than 3-fold. a differential regulation of camp versus dcamp was determined upon replacement of the incubation medium (proliferating condition with serum / serum-free resting condition). for example, camp was dramatically reduced in hek293 cells after 24 hours under resting conditions whereas dcamp was significantly increased. in cellular subfractions of hek293 cells ac assays (mn 2+ /forskolin-or mn 2+ /bicarbonate-stimulated) with either atp or datp as substrate revealed that comparable amounts of camp and dcamp accumulated. in addition to sac, membranous acs such as ac v were capable of forming dcamp with vmax and km values for datp comparable to those for atp. we also analyzed the substrate-specificity for several human phosphodiesterases. pde3 and pde4 hydrolyzed dcamp more effectively than camp. taken together, these data point to a putative second messenger role of dcamp in human cells. we are currently investigating the regulatory role(s) of camp and dcamp in apoptosis of hek293 cells. as a novel tool for these studies, we will use the cellpermeant dcamp-acetoxymethylester which penetrates the plasma membrane and releases dcamp intracellularly. the biogenic amine histamine is recognized by target cells via four different histamine receptors subtypes (h1r -h4r), which all belong to the family of g-protein-coupled seven-transmembrane receptors. histamine plays a crucial role in allergic reactions such as rhinitis or conjunctivitis and also in allergic asthma. previously, we showed an interaction of the effects of antagonists at the h1r and h4r in a mouse model of allergic asthma. however, not much is known about the signaling pathway activated by murine h1r and h4r. in order to analyze these signaling pathways, we established a cellular model using transfected hek 293 cells which stably express recombinant mh1r or mh4r. proper expression of the receptors was verified by western blot analysis and flow cytometry. in functional assays we demonstrated that histamine stimulation results in the increase of intracellular ca 2+ concentration ([ca 2+ ]i) in cells expressing either of both, mh1r (pec50 = 8.2) or mh4r (pec50 = 6.9). as a second readout, we analyzed the modulation of forskolin-induced camp-accumulation. in mh1r-expressing cells the intracellular camp concentration was increased by stimulation with histamine, while in mh4r-expressing cells forskolin-induced camp accumulation was reduced. the histamine-induced effects in h1r-expressing cells were blocked by the h1r antagonist mepyramine ([ca 2+ ]i: pkb = 8.6) and those in the h4r-expressing cells by the h4r antagonist jnj7777120 ([ca 2+ ]i: pkb = 8.8) or by pertussis toxin, which selectively blocks receptor gi-protein coupling. jnj7777120, which behaves as a partial mh4r agonist in the steady-state gtpase assay using membranes of infected sf9 cells, was without effect on [ca 2+ ]i and forskolin-induced camp-accumulation in the mh4r-expressing cells. currently, we are investigating mitogen-activated protein (map)-kinase pathways activated by the h1r and the h4r. using a phospho-map-kinase array, histamine dependent phosphorylation of erk1/2, p38, jnk, creb, pkb (akt), and mkk 3/6 were detected in cells expressing either of both, mh1r and mh4r. in summary, the hek 293 cell lines stably expressing selective histamine receptors are very useful tools to investigate hxr signaling pathways in-vitro. enhanced fibroblast motility in the absence of the β3 regulatory subunit of voltage-activated calcium channels belkacemi a. 1 cavβ subunits of voltage-activated ca 2+ channels are required for trafficking the poreforming cavα1 subunit to the plasma membrane and modulate the kinetics of its current. mouse embryonic fibroblasts (mefs), acutely isolated cardiac fibroblasts (cfs) and nih 3t3 fibroblasts do express cavβ2 and cavβ3 subunits, but we could not detect any voltage-activated ca 2+ influx. whereas in mouse cardiomyocytes or hek 293 cells coexpressing cavβ3 and cavα1 subunits a dihydropyridin-sensitive voltage-activated ca 2+ influx was readily detectable. apparently, cavβ subunits serve functions in fibroblasts unrelated to voltage-activated ca 2+ influx. among the proteins potentially interacting with cavβ3 are the inositol 1,4,5-trisphosphate receptors (ip3rs) [1, 2] . we therefore coexpressed mouse cavβ3 and mouse ip3r type 1, 2 or 3 in cos-7 cells and found coimmunoprecipitation of ip3rs using an antibody for cavβ3 and vice versa. to study the release of ca 2+ from ip3-sensitive stores we performed fura-2 measurements on fibroblasts isolated from wild type and cavβ3-deficient mice either in the presence of thapsigargin or after stimulation of gq-coupled receptors by par-1, lpa or bradykinin. receptor-activated ca 2+ release was more pronounced in β3-deficient mefs and cfs, whereas thapsigargin-induced ca 2+ release was the same in cells from both genotypes. in addition, ip3 production measured by a radioreceptor assay was already increased in β3-deficient cells under basal conditions. fibroblasts are migrating cells and involved in various physiological and pathophysiological processes. we therefore started in vitro assays for proliferation, migration and angiogenesis as well as in vivo assays for skin wound healing. angiogenesis and proliferation were apparently not different in both genotypes but migration (measured as transwell migration and in scratch assays) and wound healing were affected in different ways. fluorescent staining of cytoskeleton and quantification of the f-actin/g-actin ratio show similar results in both genotypes, suggesting that the increased migration rates and wound repair in β3 knockout may result, in part, from the increased amount of ip3-releasable ca 2+ . [1] berggren, yang, murakami, et al., removal of ca channel β3 subunit enhances caoscillation frequency and insulin exocytosis. cell 119, (2004) 273-284. [2] müller, haupt, bildl, et al., quantitative proteomics of the cav2 channel nanoenvironments in the mammalian brain. pnas 107, (2010) 14950-14957. bender-sigel j., closs e. i. universitätsmedizin mainz institut für pharmakologie, obere zahlbacher straße 67, 55101 mainz, germany human cationic amino acid transporters (hcat) are a family of multimembrane spanning proteins that mediate the transport of cationic amino acids through the plasma membrane. our earlier results have demonstrated that activation of either protein kinase c (pkc) by pma or cdc42 by egf leads to an internalization of these transporters. in addition, in a recent collaboration with the group of alexander sorkin (university of colorado denver) we found that ubiquitination and clathrin-dependent endocytosis are necessary for the down regulation of hcat-1-mediated arginine transport by pma (vina-vilaseca et al, j biol chem 2011 286:8697) . this mechanism requires nedd4 e3 ligases, but hcats do not contain a ppxy motif to bind the ligases, suggesting that an adaptor protein takes part in this process. however, an involvement of the adaptor protein beta-arrestin in this mechanism could be excluded. using sirna against pkc alpha we now show that pkc alpha is the major isoform that induces the reduction of arginine transport in human u373 glioblastoma cells overexpressing hcat-2a-egfp. in addition, sirna-mediated knock down of cdc42 prevented the decrease of hcat-2amediated transport induced by pma. taken together pkc seems to negatively influence the constitutive cycling of cats by activation the ubiquitination machinery and clathrinmediated endocytosis. cdc42 is part of this pathway. converging of the classical mitochondria-related pathway in parkinson and nuclear dna-repair signaling? scherr a. -l. 1 parkinson disease is the second most neurological disorder worldwide. despite the fact that most cases are idiopathic and only few can be traced back to specific genes, general progression between both tracks of the disease is comparable. the variety of clinical symptoms in motor control like tremor, rigor and postural problems all originate from loss of dopaminergic neurons in the substantia nigra pars compacta of the brain. several proteins mutated in pd are involved in surveillance pathways, monitoring functionality and integrity of proteins and organelles either by the proteasome degradation machinery or by clearance of mitochondria via autophagy (mitophagy). disturbed calcium-and redox-homeostasis seems to play a major role in susceptibility to cell death signals in dopaminergic neurons, but if this is a preceding or successive event in cell death related to pd progression is not known. on the other hand, experimentally elicited parkinsonism by oxidative stress inducers like paraquat or rotenone and mptp (inhibitors of mitochondrial complex i) lead to damage in nuclear dna and activation of the dna repair protein poly(adp-ribose) polymerase 1. by consuming substantial amounts of its substrate nad + , this enzyme can drastically decrease energy levels and disturb the redox balance within a cell, also sensitizing it to stress induced cell death. inhibition of poly(adp-ribose) polymerase 1 has been proven to be beneficial to some extent in cell culture models as well as in experimental pd in mice. our research focuses on a putative crosstalk mechanism we recently discovered between the two pathways of experimentally induced cell death in culture models, i.e. mitochondrial signaling and parp1-dependent poly(adp-ribosyl)ation. both converge on two mitochondrial chaperones, mortalin and trap1. whereas mutations in mortalin have been reported recently to be responsible for some parkinson disease cases in humans, trap1 is a specific target of the kinase pink1 (pten induced putative kinase), which is often mutated in autosomal-recessive forms of the disorder. pink1 is a central regulator of the mitophagy process, tagging mitochondria with dissipated membrane potential for destruction. we could show now that both chaperones bind to short-chain poly(adp-ribose) specifically synthesized by poly(adp-ribose) polymerase 1. we will present our most recent findings about regulation of these two chaperones after application of parkinson-inducing toxins. aldrich). the effect was reversible within ten minutes when cells were re-incubated in regular cell culture medium. stimulatory effects were not due to osmolarity or cell stress due to medium exchange. analysis of different components of both media (table 1) revealed that bicarbonate stimulates accumulation of ccmp and cump besides cgmp and camp in a time-and concentration-dependent manner. bicarbonate is known to activate soluble adenylyl cyclase (sac) and particulate guanylyl cyclase g (pgc-g), regulating sugar metabolism, sperm motility and olfaction by synthesis of camp and cgmp, respectively. in order to identify a responsible cyclase for ccmp and cump generation after bicarbonate treatment, we are currently analyzing transiently and stably transfected hek293 cells overexpressing various known adenylyl and guanylyl cyclases (sac, mac1, mac3, mac9, soluble guanylyl cyclase, pgc-g, pgc-a, and pgc-d) for their pyrimidinyl and purinyl cyclase activity in vivo and their regulation by bicarbonate. in addition, cell fractions will be analyzed for the detection of specific cyclase compartments. question: long term ventricular pacing, especially at the right ventricle (rv), results in left ventricular (lv) failure. there are several lines of evidence that disturbed ca 2+ homeostasis is involved in the pathophysiology of human heart failure. in this study we examined if ventricular pacing affects the na + -and ca 2+ -channels and the expression of ca 2+ -handling proteins and investigated if there is a differential effect between right ventricular free wall (rvfw) pacing and left ventricular apex (lva) pacing. methods: after av-node ablation 14 minipigs underwent ventricular pacing at 120 beats/min (ddd mode) for one year. 7 minipigs were paced from the rvfw and 7 minipigs from the lva, respectively. 7 minipigs with normal sinus-rhythm served as control group. patch-clamp-experiments were studied to measure na + -and ca 2+ currents. western-blots were carried out to investigate the expression of the ca 2+handling proteins l-type ca 2+ -channel, serca2 and phospholamban. results: both rvfw-and lva-pacing led to significant decreased ca 2+ -currentdensities in cardiomyocytes of the lv compared to the control group. the plateau phase of the action potential was significantly shortened after ventricular pacing in relation to control minipigs. furthermore cardiomyocytes of rvfw-and lva-paced minipigs had significant lower na + -current-densities than control minipigs. the action potential amplitude was significantly decreased after rvfw-and lva-pacing whereas the diastolic potential remained unchanged. the expression of the l-type ca 2+ -channel was significantly reduced after ventricular pacing, regardless of the pacing site. in contrast rvfw-and lva-paced minipigs showed significant increased serca2-expression. the expression of phospholamban remained unchanged after rvfw-and lva-pacing compared to control minipigs. conclusion: in a chronic animal model ventricular pacing leads to remodeling of ionchannels and ca 2+ -handling-protein-expression, regardless of the pacing site. investigation on metabolic competence of dermal systems: native human skin, in vitro skin models and keratinocytes blatz v. 1 the implementation of reconstructed human skin equivalents (rhes) as an alternative method for dermal toxicity testing became very prominent in the last decades. their advantages are e. g. the human cell origin and an organ-like 3d structure. already regulatory accepted methodologies are widely in use for testing the skin corrosion (oecd 431) and irritation (oecd 439) within rhes. but there are still some questions open, one of them the metabolic competence of such dermal systems. in this context, enzyme activities of oxidizing (cyp; fmo; adh; aldh) and conjugating enzymes (nat; ugt) were investigated in subcellular fractions of in vitro systems such as keratinocytes and rhes (epidermis model epiderm tm (mattek), full-thickness skin models epiderm tm ft (mattek) and phenion ® ft (henkel ag)) and compared to those of native human skin. activities of cyp 1a, 2b and 3a isoenzymes were measured fluorometrically by oxidative desalkylation of alkoxyresorufines. fmo 1/3 activities were evaluated by hplc/fld detection of n-oxygenated product of benzydamine [1] . adh and aldh activities were investigated by photometrical detection of nadh generation during ethanol (adh) [2] or propanal (aldh) oxidation [3] . nat1 activity was followed by hplc/uv detection of acetylated p-aminobenzoic acid. ugt1 activity was quantified fluorimetrically by glucuronidation of methylumbelliferone [1] . during the course of this study the following results were observed: (loq = limit of quantification) since the metabolic competence of rhes is confirmed, these in vitro systems are estimated as suitable for further toxicity tests (e. g. genotoxicity by comet assay), where metabolic activation of substances may play a crucial role. however, for the data assessment, the determined metabolic profiles should be taken into account. we acknowledge bmbf funding this project (0315226d). signalling pathway indicates that in b104 cells the adenine receptor couples to a gqprotein followed by activation of phospholipase c pathway. these findings represent a new signalling pathway of the adenine receptor and allow the assumption that different adenine receptor subtypes exist in the rat brain. in the scope of the project lexukon ("foodborne exposure to environmental contaminants -data analysis to support and standardise exposure assessments based on nvs ii") exposure to the heavy metals cadmium (cd), lead (pb) and mercury (hg) via food consumption has been assessed for the german adult population based on the national nutrition study ii ( the updated intake assessments show that especially foods regularly consumed such as vegetables and grain contribute mainly to exposure of cd that is about 1.5 µg/kg body weight (bw) per week for average consumers over all food groups. this corresponds to 58% of the tolerable weekly intake (twi) of 2.5 µg/kg bw for cd defined by the european food safety authority (efsa) in 2009. beverages and vegetables are the food groups most relevant for exposure to pb. about 3.7 µg pb/kg bw is taken up by average consumers that is below the benchmark dose for renal toxic effects (4.41 µg/kg bw) defined by efsa for the weekly pb intake. for hg the intake amounts for all population groups examined were significantly below the toxicological reference values. for average consumers the weekly intake of hg is 0.49 µg/kg bw that is primarily taken up by eating fish and fishery products. however, individual population groups and high consumers reach and/or exceed the toxicological reference values for the daily intake amounts for cd and pb. high consumers almost reach the twi for the cd with 94%. for pb a weekly intake of 5 µg/kg bw was estimated for high consumers that exceeds the benchmark dose for renal toxic effects for the weekly pb intake. the results show that data collection should also focus on highly consumed and not only on highly contaminated foods. further, uncertainties in concentration levels should be reduced e.g. by lowering and standardizing the analytical limits. it's recommended to consider further measures in view of the reduction of contents of environmental contaminants in foods. however, other sources can also contribute to the intake of the mentioned heavy metals (e.g. smoking). major cell biological processes are regulated by rho-gtpases, actin-mediated processes in particular. amongst others, rho-gtpases are stimulated by the receptormediated activation of gα12/13 and gαq via specific rhogefs. the p63rhogef is activated by gαq and plays a major role in the acute response of vascular smooth muscle cells to angiotensin ii treatment. the aim of the present study was to establish a fret-assay between gαq-cfp and venus-p63rhogef and characterize the dynamics of p63rhogef-gαq-interaction in single living cells. the fusion of p63rhogef with venus resulted in a functional gαq-regulated p63rhogef-protein as determined by means of rho-luziferase-assays. whereas no specific fret signal was observed between the two interaction partners in the absence of receptor stimulation, a robust and rapid fret signal developed in response to stimulation of histaminergic h1and cholinergic m3-recptors. the onset of this signal after rapid application of agonist paralleled gαq activation kinetics. similar to the kinetics of gαq-protein deactivation the dissociation of p63rhogef and gαq after withdrawal of agonist was slow (tens of seconds). the specificity of the fret signal between gαq-cfp and venus-p63rhogef was verified by introducing point mutations rendering p63rhogef unable to bind to active gαq. furtehrmore we observed a robust acceleration of the dissociation of p63rhogef and gαq upon cotransfection of rgs2, suggesting a very short lifetime of the p63rhogef-gαq-complex or the ability of rgs2 to bind to p63rhogef-associated gαq. taken together, fret-based imaging of the interactions between p63rhogef and gαq revealed fast interaction kinetics closely resembling g-protein activation kinetics, both of which can be regulated by rgs2. toxicity of silver nanoparticles in intestinal cells boehmert l. 1 the rapid development of nanotechnology has been accompanied by an increased concern for the safety of nanomaterials. especially silver nanoparticles are used in many manufacturer identified consumer products including silver coated food contact materials or hydrosol silver supplements. these products lead to an intentional or unintentional oral uptake of silver nanoparticles and hence to a contact with the intestinal barrier. the human cell line caco-2 is a well established model system in studying effects on human enterocytes. although these cells are colon carcinoma cells and exhibit typical features of cancer cells when they are kept sub confluent, these cells have the capability to differentiate into polarized cells with morphological and biochemical properties of small enterocyte cells. we investigated the effects of silver nanoparticles on these colon carcinoma (proliferating) and small intestinal epithelium like (differentiated) caco-2 cells. the silver nanoparticles agpure were commercially available from rent-a-scientist gmbh. the behaviour of these silver nanoparticles in cell culture medium were characterised using asymmetric flow-fild flow fractionation (a4f), small ankle x-ray scattering (saxs) and dynamic light scattering (dls). we investigated the particle toxicity on both cell states using cell titer blue assay, xcelligence impedance measurements, annexin-v and caspase measurements, diclorofluorescein assay and antioxidant pre incubated cells. the agpure stock solution is an aqueous suspension of silver particles with a metal core radius of 7.2 nm stabilised with tween-20 und polyoxyethylenglycerol trioleate. agpure silver nanoparticles were toxic for proliferating as well as differentiated caco-2 cells in a time and concentration depending manner. the presence of foetal calf serum in the incubation medium has a minor influence on the toxicity. prior to cell death, morphological abnormal adherence characteristics and morphological changes in the cells were observed using microscopy and quantified by xcelligence impedance measurement. it is concluded that cell death is caused by an oxidative stress related mechanism rather than apoptosis. the release of sphingosine-1-phosphate from human platelets during acute coronary syndrome is attenuated by aspirin böhm a. 1 , polzin a. in addition to atherosclerosis ttp knock out mice develop more cardiovascular dysfunctions. in tail vein bleeding assays we monitored a significant difference in the bleeding times of ttp deficient mice in comparison to wildtype mice, triggered by a stronger granulopoeisis. our results leed us to the assumption that the chonic inflammation seems to be more improtant for the development of cardiovascular diseases in ra patients than the traditional risk factors. differentially expressed cardiac genes in a mouse model with heart-specific overexpression of pp2a bollmann p., makarova e. a., gergs u., neumann j. institute for pharmacology and toxicology medical faculty, magdeburger str. 4, 06112 halle (saale) , germany in transgenic (tg) mice with cardiac myocyte-specific overexpression of the catalytic subunit of protein phosphatase 2a (pp2a) reduced cardiac protein phosphorylation, cardiac hypertrophy and impaired cardiac contractility were noted compared to wild type (wt) littermates. the hearts of tg mice also suffered from ventricular dilatation and a diminished response to β-adrenergic stimulation. analyses of mrnas expressed in tg and wt hearts (n=3) using affimetrix mouse genome microarray chips resulted in several candidate genes possibly differentially regulated. in this study, we focussed on verifying the mrna data of selected genes important for stress response and signal transduction on protein level in cardiac homogenates by western blotting. hearts from wt littermates were used as control. compared to wt heat shock protein 25 (hsp25) and calcium calmodulin dependent protein kinase type ii (camkii) mrnas were upregulated in tg but only hsp25 protein was increased (p<0.05, n=7-8) but not camkii (p>0.05). protein phosphatase type 5 (pp5) and superoxide dismutase (sod) were downregulated on mrna level in tg but on protein level this could be found only for sod (p<0.05, . in contrast, pp5 protein was upregulated (p<0.05, in tg compared to wt. for comparison the regulatory a-subunit of pp2a and hsp90 were studied. both genes were unchanged on mrna level in tg: western blotting revealed the same results for the corresponding proteins. in summary, mrna expression data could only partially be confirmed on protein level elucidating the importance of western blotting studies. these data indicate that increased pp2a activity is associated with modified gene expression in tg hearts possibly affecting stress response and regulation of cell signalling. (supported by the deutsche forschungsgemeinschaft) center for regenerative therapies, technische universtität, dresden, germany cell therapy in the form of beta cell replacement to cure diabetes has been practiced for decades without become a routine clinical therapy. more widespread clinical application is hindered by the scarcity of suitable organ donors, a dramatic loss of transplanted cells within the first days post-transplant, the requirement of long term immunosuppression to maintain graft survival, and despite this, a loss of graft function from a recurrence of autoimmunity in some patients. research is currently dedicated to overcome each of these limitations. additional beta cell sources investigated include embryonic stem cell derived insulin producing cells, human insulin producing cells lines, and xenogeneic beta cells. parallel to these efforts are the development of encapsulation devices to protect these sources from immune and inflammation mediated destruction, and transplantation into new sites such as the muscle and bone marrow to infuse beta cells. additional therapy to reduce immune suppression includes the infusion of t regulatory cells to control autoimmune and alloimmune response, and cytokine and chemokine receptor directed compounds aimed at blocking early inflammation or autoimmunity. these efforts are likely to lead to an expansion of clinical activity to replace beta cells in diabetes, and to novel pharmaceutical therapies that may be more generally applicable in patients with diabetes. pdgf-bb induces the h2s producing enzyme cystathionine-γ-lyase via a rosdependent mechanism in rat renal mesangial cells boosen m. 1 , hassan m. there is increasing evidence that hydrogen sulfide (h2s) that is endogenously produced in several cell types serves as a potent gasotransmitter in a wide variety of physiological processes involving vascular homeostasis and inflammation. in the present study we investigate the expression and the regulation of the hydrogen sulfide synthesizing enzyme cystathionine γ-lyase (cse) in cultured rat renal mesangial cells. as demonstrated by qpcr and western blot experiments, mesangial cells showed a marked time-and dose-dependent upregulation of cse mrna and protein levels after treatment with platelet-derived growth factor (pdgf-bb). the cse upregulation by pdgf-bb is accompanied by a marked increase in reactive oxygen species (ros) formation. interestingly, co-administration of the ros scavenger n-acetylcysteine, the glutathione peroxidase mimetic ebselen and the nadph oxidase inhibitor diphenylen iodonium chloride (dpi) drastically reduced pdgf-induced cse expression, indicating a role for endogenously produced ros in mediating regulation of cse. as demonstrated by electrophoretic mobility shift (emsa) experiments pdgf-bb induces binding of the redox-sensitive transcription factor nf-e2-related factor 2 (nrf2) to a consensus antioxidant response element and this effect was also diminished by co-administration of antioxidants (dpi, nac, ebselen) . furthermore, lps/ifnγ-as well as pdgf-bb-induced cse upregulation was nearly completely abolished in nrf2 -/spleen macrophages and mesangial cells, respectively. as a consequence of the elevated cse levels we could demonstrate increased h2s levels and a higher cse enzyme activity in mesangial cells after stimulation with pdgf-bb by using the colorimetric methylenblue method and a cse activity assay. importantly, in a rat model of anti-thy-1-induced proliferative glomerulonephritis we observed a marked upregulation of cse protein during the course of the disease paralleled by a stabilization of nrf2 protein. from our data, we hypothesize that pdgf-bb-mediated regulation of cse via a redox-mediated activation of nrf2 may constitute a protective mechanism during glomerular inflammatory disease. rac1 knockout protects from acute hepatic damage following doxorubicin treatment bopp a., wartlick f., fritz g. heinrich-heine-universität institut für toxikologie, universitätsstr. 1, 40225 düsseldorf, germany rac1 belongs to the best characterized members of the ras-homologous (rho) family of small gtpases, which are key regulators of the actin cytoskeleton. furthermore, rac1 is part of the activation of the nadph oxidase, which produces reactive oxygen species and regulates the activity of stress kinases (e.g. sapk/jnk) and transcription factors such as nf-κb and ap1. anticancer drugs cause dna damage, which in turn stimulates the dna damage response (ddr) regulating dna repair, cell cycle progression and, in case of non-repairable dna damage, triggers apoptosis. so far, a role of rac1 in the ddr has not been reported. based on its exceptional function as a regulator of transcription and because of its recently found ability to translocate to the nucleus, we hypothesize that rac1 may be involved in the ddr. to study the in vivo function of rac1 we used an inducible cre-based knockout mouse model (rac1 flox/flox/mxcre ). mice were treated with different doses of doxorubicin for different periods of time. we monitored gh2ax foci formation as a marker of dna strand breaks, used the masson-goldner staining for the detection of collagen accumulation, analyzed phosphorylated histone 3 as a marker of mitotic events and performed a tunel assay to detect apoptotic cells. in the absence of rac1 the basal mrna expression of pro-fibrotic ctgf was decreased. collagen levels were increased and mmp1 mrna expression was reduced in the liver of rac -/animals as compared with rac1 proficient animals. in addition we found more apoptotic cells in rac1 -/mice. 96 hours after treatment with the anthracycline derivative doxorubicin the number of gh2ax foci in rac1 -/animals was reduced in comparison to rac1 +/+ animals. we also found lower level of ctgf mrna expression and reduced amount of collagen in rac1 -/mice. none of these protective effects resulting from rac1 deficiency could be detected after administration of three consecutive doxorubicin injections over a time period of 21 days. there were no significant differences in the number of gh2ax foci or collagen accumulation. the mrna expression of ctgf was even higher in rac1 -/animals. furthermore the number of mitotic events was almost two times higher in the rac1 -/mice compared to the rac1 +/+ mice. summarizing, our findings show that impaired hepatic expression of rac1 protein is hepatoprotective against acute damage following doxorubicin exposure, but does not protect against doxorubicin-induced subacute toxicity. in vitro cytotoxicity of tbhq (tert-butyl-hydroquinone) braeuning a., vetter s., schwarz m. institut für experimentelle und klinische pharmakologie und toxikologie toxikologie, wilhelmstrasse 56, 72074 tübingen, germany at high concentrations, tert.-butyl-hydroquinone (tbhq), a phenolic antioxidant frequently used as a food preservative, exerts cytotoxic effects, which are closely linked to its ability to form reactive oxygen species as a consequence of redox cycling processes. here we describe that treatment of murine 3t3 cells with tbhq in 96-well culture plates induces the death of untreated cells in neighboring wells on the same plate. the mechanisms underlying that effect were investigated. death of the seemingly untreated neighboring cells was caused by a more toxic and volatile tbhq oxidation product which was formed in a non-enzymatic process involving metal ions and oxygen. the unexpected perturbation of cytotoxicity testing by the volatile tbhq metabolite shows that not only metabolic processes, but also non-enzymatic mechanisms have to be considered as important parameters for in vitro assays. furthermore, our data show that even cells several wells distant from the site of treatment do not necessarily constitute proper "untreated" controls when cells are treated with tbhq, e.g. in assays aimed to analyze the activity of the tbhq-inducible nrf2 pathway. s14 056 angiotensin ii causes oxidative stress and dna damage in mouse kidneys via the angiotensin ii type 1 receptor brand s. 1 , amann k. 2 , schupp n. 1 1 universität würzburg institut für pharmakologie und toxikologie, versbacherstr. 9, 97078 würzburg, germany 2 universität erlangen-nürnberg institut für pathologie, krankenhausstraße 8, 91054 erlangen, germany angiotensin ii (ang ii), the reactive peptide of the renin-angiotensin-system, causes vasoconstriction and, in higher levels hypertension, which is connected with an increased cancer risk in the kidney. treatment of male c57bl/6 mice with ang ii results in the formation of superoxide radicals and dna damage in the kidney as well as in the heart. to answer the question if the dna damage is caused by hypertension or by elevated ang ii concentrations, mice were treated with different compounds: the angiotensin-converting-enzyme blocker ramipril, the ang ii receptor blocker ramipril, the ang ii receptor candesartan, the antioxidant tempol and the vasodilator hydralazine. the effect on blood pressure and renal function of ang ii-treated c57bl/6 mice was examined. treatment with ang ii led to a significant increase in blood pressure. candesartan and hydralazine led to a decrease, whereas intervention with ramipril and tempol had no effect. equal conditions could be found by examining renal function regarding the excretion of urinary albumin, which was ameliorated by candesartan and hydralazine. in addition, histopathological changes were investigated. there was significant glomerular damage and tubulointerstitial damage in ang ii-treated animals compared to control animals, which was significantly improved by candesartan and tempol. hydralazine and ramipril mitigated the observed renal damage but were less effective than candesartan. furthermore, the ang ii-induced formation of superoxide radicals in the kidney and the heart was slightly affected by all interventions. genomic damage, in the form of double strand breaks was prevented by the ang ii receptor antagonist candesartan and the antioxidant tempol. to sum up, the results from this study show that ang ii induces the elevation of markers of kidney failure and dna damage, which is prevented by substances lowering blood pressure like candesartan, showing the receptor responsibility for the induction of dna damage. actually by substances not lowering blood pressure like tempol, the oxidative stress and dna damage was ameliorated, showing the involvement of reactive oxygen species. optimization of the balb/c-3t3 cell transformation assay by coupling a drug metabolizing system brauneis m. d., steinberg p. stiftung tierärztliche hochschule hannover institut für lebensmitteltoxikologie und chemische analytik, bischofsholer damm 15, 30173 hannover, germany the analysis of the carcinogenic potential of chemicals plays an important role in toxicology. up to now the acquisition of such data requires a large amount of animal experiments. the aim of this study is to reduce the number of experimental animals being used by further optimizing the balb/c-3t3 cell transformation assay, an already well-established in vitro method. this method, which is also well suited for high throughput screening applications, allows a quantitative analysis of the aforementioned carcinogenic potential. the incubation of balb/c-3t3 cells (murine embryonic fibroblasts) with mutagenic compounds leads to a loss of contact inhibition between these cells, which results in the development of so-called foci. these foci can be distinguished by characteristic changes in cell growth behaviour, a result of the treatment with carcinogenic compounds, and their number is therefore directly related to the genotoxic potential of the latter. a major disadvantage of the "classic" balb/c-3t3 cell transformation assay is that a number of compounds initially require a metabolic transformation to gain their full genotoxic potential. hence, without prior metabolic transformation many chemicals are not detected as carcinogenic in the abovementioned test system. to overcome this drawback the balb/c-3t3 cell transformation assay has been coupled to a drug metabolizing system, in this case the so-called liver s9. in a first step the well-known genotoxic agents benzo[a]pyrene, aflatoxin b 1 and nnitrosodimethylamine were tested in this assay. all three compounds led to a concentration-dependent increase in the number of foci formed, whereby this concentration-dependent increase was observed in a non-cytotoxic concentration range. in a next step the balb/c-3t3 cell transformation assay will be coupled to further drug metabolizing systems as well as to the soft agar assay. this study is being financially supported by the stiftung set and the doerenkamp-zbinden foundation. adenylyl cyclases (acs) synthesize the second messenger camp. the family of acs consists of nine membranous and one soluble isoforms with ac5 and ac6 being the predominantly expressed isoforms in the heart. in the heart, acs integrate β-adrenergic (β-ar) signaling as the main physiological mechanism to improve cardiac performance. although ac5 and ac6 share high sequence homology, opposing effects on cardioprotection have been reported, where disruption of ac5, as well as overexpression of ac6 both exerting beneficial effects in heart failure. prospective pharmacological treatment of heart failure on the level of ac is under investigation. our study explored the impact of ac5 ko on ac-activities in the heart at a functional level. complementary, mrna expression studies of the β-ar-g-protein-ac signaling cascade were performed to detect possible compensatory alterations. hearts from 16-20 week old homozygote ac5 knockout and wild-type male littermates were examined in this study. ac activities where measured in cardiac membrane preparations from left ventricles. ac activities were assessed under β-ar and g-protein (g s) stimulation by isoproterenol, guanosine 5'-triphosphate (gtp) and 5'-o-(3thiotriphosphate) (gtpγs) as well as for direct activation by forskolin. relative mrna expressions for ac1-9, gs-, gi-a and β1-, β2-ar where measured by quantitative realtime pcr. surprisingly, assessment of basal, β-ar and g-protein-mediated ac-stimulation as well as direct activation by forskolin revealed no changes in ac activities. besides from detection of the ac5 knockout, mrna expressions analysis of ac1-9, gs-, gi-a and β1-, β2-ar did not detect any compensatory alteration. these findings suggest that proximal adrenergic signaling in the heart does not necessarily require ac5. whether physiological integration of beta adrenergic signaling in the heart is mediated by both isoforms ac5 and ac6, or can be attributed to one main isoform remains to be elucidated. melanocortin-promoted pka activation decreases ampk activity via erk-1/2 and lkb-1 in hypothalamic gt1-7 cells breit a., ellen d., gudermann t. goethestrasse 33, 80336 münchen, germany α-melanocyte stimulating hormone (α-msh)-induced activation of the melanocortin-4 receptor (mc4r) in hypothalamic neurons increases energy expenditure and inhibits food intake. intrahypothalamic injection of melanocortins decreased food intake due to the inhibition of amp-activated protein kinase (ampk) that has recently been reported to enhance food intake in rodents. until now, it is not clear if α-msh affects ampk via direct intracellular signaling cascades or if the release of paracrine factors is involved. herein, we used a murine, hypothalamic cell line (gt1-7 cells) and monitored ampk phosphorylation at thr 172 which has been suggested to increase ampk activity. we found that α-msh dephosphorylated ampk at thr 172 and consequently decreased phosphorylation of the established ampk substrate acetyl-coa-carboxylase at ser 79 . inhibitory effects of α-msh on ampk were blocked by specific inhibitors of protein kinase a (pka) or extracellular-regulated kinases-1/2 (erk-1/2), pointing to an important role of both kinases in this process. since α-msh-induced activation of erk-1/2 was blunted by pka inhibitors, we propose that erk-1/2 serves as a link between pka and ampk in gt1-7 cells. furthermore, down-regulation of liver kinase b-1 (lkb-1), but not inhibition of calcium-calmodulin-dependent kinase kinase-β or transforming growth-factor-beta-activated kinase-1 decreased basal phosphorylation of ampk and its dephosphorylation induced by α-msh. thus, we propose that α-msh inhibits ampk activity via a linear pathway including pka, erk-1/2 and lkb-1 in gt1-7 cells. given the importance of the melanocortin system in the formation of adipositas detailed knowledge about this pathway might help to develop drugs targeting obesity. autism spectrum disorder (asd) is a complex neurodevelopmental disorder with dysfunction of social interaction and communication. a hitherto unknown complexgenetic principle of origin probably underlies asd. so far, more than 100 candidate genes were identified in literature. the patients affected with the monogenic timothy syndrome show multiorgan dysfunction including lethal arrhythmias, immune deficiency, skeleton-dysplasia, syndactylia and autism. this single gene disorder serves as a model disease for asd, giving insights in a possible pathophysiology. here, a point mutation in a highly-conserved region of the pore-forming subunit of the voltage-dependent calcium channel (ca v) cav1.2 gene (cacna1c) results in incomplete inactivation of the l-type calcium currents (splawski et al., cell 2004; 119:19-31) . functionally similar biophysical effects can be induced by structural variation β1-and β2-subunits of the voltagedependent calcium channels (herzig et al., faseb j. 2007; 21:1527-38; jangsangthong et al., pflugers arch. 2010; 459:399-411) . supported by findings in a meta-analysis of linkage data of asd patients (trikalinos et al., mol psychiatry. 2006; 11:29-36) , we are investigating a function-based candidate gene hypothesis linking the β2 subunit gene (cacnb2) with asd. we performed a case control study sequencing all exons and flanking intronic regions of cacnb2 in 155 patients with asd. we found three rare missense mutations in asd patients, but not in 259 unaffected controls. all three mutations occur at highly conserved positions and might alter protein function; additionally results one amino acid substitution highly probable in a post-translational modification by phosphorylation. so far, we characterized two of these mutations and also a phosphorylation-mimicking mutant in electrophysiological studies. all variants show a decelerated and incomplete time-dependent inactivation of the co-transfected cav1.2 subunit. furthermore, two variants exhibit a significant increased slope factor of voltage-dependent steady-state inactivation. we here present mutations in the β2 subunit gene of asd patients that result in a retardation of inactivation behavior, thus phenocopying the monogenic timothy syndrome mutations of cav1.2. β2 subunit mutations may influence neuronal function or development in some asd patients. jangsangthong, w., kuzmenkina, e., khan, i.f., matthes, j., hullin, r., and herzig, s. (2010) . inactivation of l-type calcium channels is determined by the length of the n terminus of mutant beta (1) subunits. pflugers arch 459, 399-411. herzig, s., khan, i.f., grundemann, d., matthes, j., ludwig, a., michels, g., hoppe, u.c., chaudhuri, d., schwartz, a., yue, d.t., et al. (2007) . mechanism of ca(v)1.2 channel modulation by the amino terminus of cardiac beta2-subunits. faseb j 21, 1527-1538. splawski, i., timothy, k.w., sharpe, l.m., decher, n., kumar, p., bloise, r., napolitano, c., schwartz, p.j., joseph, r.m., condouris, k., et al. (2004) . ca(v)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. cell 119, 19-31. trikalinos, t.a., karvouni, a., zintzaras, e., ylisaukko-oja, t., peltonen, l., jarvela, i., and ioannidis, j.p. (2006) . a heterogeneity-based genome search meta-analysis for autism-spectrum disorders. mol psychiatry 11, [29] [30] [31] [32] [33] [34] [35] [36] background: brain serotonin (5-ht) has been implicated in the regulation of food-intake. the ingestive effects of 5-ht are mediated by a number of different receptor subtypes under which the 5-ht1a-receptor plays a central role. former in vivo studies have shown an increased intake of food, elicted by 5-ht-receptor agonists. the aim of this behavioural pharmacologic project was to determine if the hyperphagic effect is mediated by presynaptic 5-ht1a autoreceptors in the raphe nuclei or by postsynaptic 5-ht1a heteroreceptors in serotonergic terminal structures. methods: the effect of the 5-ht1a receptor agonist 8-oh-dpat (0.1, 0.5 or 1.0mg/kg) was investigated on feeding behaviour in non-food-deprived young-adult and adult nmri and transgenic l35 mice. l35 mice are characterized by an overexpression of postsynaptic 5-ht1a receptors. results: the administration of the 5-ht1a receptor agonist induced hyperphagia in all groups of mice, except for the adult transgenic mice which showed no drug effect. conclusion: the results confirm a key role of the 5-ht1a receptor in food intake. further, we make the assumption that the hyperphagic effect of 8-oh-dpat is mediated by presynaptic 5-ht1a autoreceptors in the raphe nuclei which decreases 5-ht function in the central nervous system. it can be speculated that the aberrant feeding behaviour of the adult transgenic mice refers to a possible opposite role of the postsynaptic 5-ht1a receptors. these receptors might affect the release of neuropeptides in the hypothalamus. the efflux transporter abcc2 (mrp2) expressed at different compartment barriers is important for the elimination of various endogenous and exogenous compounds. with some evidence inflammatory processes regulate abcc2 expression and cause changes of absorption, distribution and clearance of a number of xenobiotics. the investigation of the influence of interleukin (il) 1β on abcc2 mrna and protein expression in various cell lines representing specific tissues is the aim of our study. a further aim is to characterize the signaling pathways regulating abcc2 expression while inflammation. three different cell lines a) hepg2 cells (liver tissue) and b) caco2 (colon tissue) both without naïve il1β expression and c) skhep1 cells representing physiological liver tissue with naive il1β expression, were stimulated with different concentrations of il1β (range 10 pg/ml to 10 ng/ml). over a period of 48h samples were taken at defined time points. abcc2 mrna and protein expression were quantified by qrt-pcr and western blot analysis, respectively. by using small molecule kinase inhibitors for signal transduction proteins (p38 mapk, akt, erk1 and jnk) we analysed the signal transduction pathways associated with il1β-mediated transcriptional abcc2 regulation. on abcc2 mrna level an up-regulation in caco2 cells (1, 35 fold) and hepg2 cells (3, 6 fold) within the first hour after stimulation with 1 ng/ml il1β was shown. in contrast skhep1 cells demonstrated a decreased abcc2 mrna expression (0,59-0,62 fold) in comparison to unstimulated controls. the abcc2 protein expression exhibited a time and il1β dependent regulation as well. the analysis for the signal transduction showed for p38mapk a moderat time dependet down regulated phosphorylation (15%) in hepg2 cells whereas it showed no effect in caco2 cells. concluding, the expression of abcc2 is regulated moderately by il1b in a concentration and time-dependent manner. interestingly, the effects are strongly tissue-dependent concerning abcc2 expression and signal transduction pathways and show partly contradictory results. the regulation of the different signaling pathways is currently subject of ongoing investigations. introduction: despite the remarkable success of imatinib treatment of chronic myeloid leukemia (cml), therapy resistance emerged as a major clinical problem. the aim of this study was to identify micrornas, which may serve as biomarkers for therapy response or predict pathways involved in pharmacoresistance of imatinib treatment. methods: blood was collected from 21 cml-patients, ten of whom responded to imatinib therapy. after rna extraction from leukocytes, we performed a taqman low-density array screen to determine the expression of 667 micrornas. statistical analysis using the 2 -∆ct method was performed. micrornas showing a p-value<0.01 and a fold change>2 were considered to be significantly differently expressed. in addition, by using microrna target prediction databases (targetscan 1 , mirdb 2 , pictar 3 , microcosm 4 , diana microt 5 ), selected putative target genes were further functionally investigated by the david bioinformatics database 6 . results: comparing treatment-naïve responders and non-responders four micrornas were identified to be deregulated that were predicted to target 97 genes, especially transcription regulators (21%). pathway analysis showed that six of the predicted genes are relevant in cancer pathways, four of which play a role in cml (smad4, nras, rb1, raf1). when comparing patients' expression profiles before and under treatment, seven micrornas were identified to be deregulated in responders and five micrornas in nonresponders. ninety-nine targets of the latter include transcription regulators (19%), but also cellular transporters (18%, especially uptake transporters of the slc-family). most target genes are involved in mapk signalling or endocytosis pathways. conclusion: analysis of microrna expression profiles revealed four micrornas involved in imatinib-response and 12 micrornas deregulated during imatinib treatment. predicted target genes code mainly for transcription factors as well as oncogenes relevant for cml and are involved in transporter expression and endocytotic processes. dissociations in the effects of beta2-adrenergic receptor agonists on camp formation and superoxide production in human neutrophils brunskole i. 1 , buschauer a. activation of the β2-adrenergic receptor (β2ar), a classically gs-coupled receptor, in neutrophil granulocytes results in an inhibition of inflammatory responses [1] , which could be further therapeutically exploited. the aim of the present study was to evaluate the effects of various β2ar ligands on cyclic adenosine 3',5'-monophosphate accumulation (camp assay) and n-formyl-l-methionyl-l-leucyl-l-phenylalanine (fmlp)induced superoxide anion production (o2 •assay) in isolated human neutrophils, which are a physiologically relevant native test system. camp concentration in neutrophils was determined by hplc/tandem mass spectrometry, and o2 •formation was assessed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. (-)-isoproterenol, (-)-adrenaline, salbutamol and dobutamine were more potent in inhibiting fmlp-induced o2 •production than in stimulating camp accumulation. (-)-ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the camp assay, but could partially inhibit fmlp-induced o2 •production at higher concentrations. moreover, (-)-adrenaline and dobutamine were equi-efficacious in both assays whereas the efficacy of salbutamol was more than two fold higher in the o2 •assay. this suggests that salbutamol is able to stabilize a different receptor conformation than the other two ligands. thus, ligand-directed signaling via β2ar can also occur in human neutrophils. in addition, differences between the data from neutrophils and recombinant test systems [2, 3] were noticed, pointing to the problem of insufficient comparability of effects in recombinant and native test systems. the investigation of β2ar antagonists on neutrophil granulocytes is subject of ongoing work, in order to find out whether pkb values of β2ar antagonists in the camp assay and the o2 •assay are different. such differences were previously reported for β2ar antagonists in other test systems [4] . moreover, studies with protein kinase a inhibitors should give deeper insight into the signaling events in neutrophils that result in inhibition of fmlp-stimulated o2 •production and clarify how camp increase interferes with this events. agonist-selective internalization of the human 5-ht2a receptor buchborn t., kahl e., höllt v., koch t. otto-von-guericke-universität magdeburg institut für pharmakologie und toxikologie, leipziger straße 44, 39120 magdeburg, germany the serotonin 2a (5-ht2a) receptor is a g protein coupled receptor and the molecular target of lsd-like hallucinogens. downregulation of 5-ht2a receptors is an adaptive process considered relevant for the therapeutic action of diverse serotonergic antidepressants, such as ssris. since the antidepressant targeting of 5-ht2a receptors, however, is largely restricted to indirect agonists and/or antagonists, little is known about the mechanisms and implications of their regulation by direct agonists. in the present study we, therefore, investigated the capacity of various agonists to regulate the human ha-tagged 5-ht2a receptor by internalization. using immunocytochemical techniques in stably transfected hek293 cells, we show that agonists differ in their capacity to internalize the receptor. serotonin, quipazine and doi are the agonists most efficaciously internalizing the receptor, dmt and methysergide, on the other hand, hardly internalize; other agonists like psilocin, ergotamine and lsd induce low to intermediate internalization. the specificity of the agonistic effect was demonstrated by the 5-ht2a selective antagonist ketanserin, which blocked the agonist-induced internalization. in additional experiments, we show that the internalized 5-ht2a receptors colocalize with a488-labelled transferrin receptors, and that the internalization can be blocked by high molar sucrose; these results are indicative of a clathrin associated sequestration of 5-ht2a receptors in recycling endosomes. also, we demonstrate that the proteinkinase c activator pma efficaciously induces 5-ht2a internalization in the absence of an agonist, and that the doi-induced internalization can be blocked by the proteinkinase inhibitor staurosporine. we, thus, confirm previous findings that the activation of proteinkinases seems to be necessitated for the 5-ht2a internalization to occur. overall, we conclude that the internalization of the human 5-ht2a receptor is agonist-selective, and employs a proteinkinase (possibly pkc) dependent, clathrincoated endosome associated pathway. as there is recent evidence that the regulation of 5-ht2(a) receptors by agonists might have antidepressant (-like) properties, knowledge about the agonist-selective processing of 5-ht2a receptors could help to identify agonists most promising for future (pre-)clinical research. non-clinical safety assessment of homeopathic medicinal products: criteria for establishing a first safe dilution buchholzer m. -l., werner c., knoess w. bfarm bundesinstitut für arzneimittel und medizinprodukte zulassung 4, kurt-georg-kiesinger-allee 3, 53175 bonn, germany like all human medicinal products the homeopathic medicinal products for human use must demonstrate adequate safety. in general, they are regulated according to the analogue non-clinical safety principles (points to consider on non-clinical safety of homeopathic medicinal products of botanical, mineral and chemical origin, adoption by hma 2007). one particular approach is the recently introduced concept of a first safe dilution (fsd; introduction to the list of first safe dilutions, adoption by hma 2010) . this contribution summarizes the first experiences in establishing fsds of a selection of given homeopathic preparations by bfarm. for a given preparation the major toxicological concern and available data set is identified. this determines the safety assessment route: food regulation, permitted daily exposure (pde), threshold of toxicological concern (ttc) or lowest human recommended dose (lhrd/100). finally the acceptable amount/tolerable daily intake is derived and the respective fsd is calculated. for example the draft evaluation for reserpinum (ph. eur. method 4.1.1) and for atropine (ph. eur. method 3.1.1 or 4.1.1) based on lhrd leads in each case to a suggested fsd of d7 related to 10 g of preparation. furthermore, the draft evaluation for potassium iodide (ph. eur. method 3.1.1 or 4.1.1) based on food legislation emerged a proposed fsd of d6 related to 10 g of preparation. the concept of fsd combines a scientific and at the same time pragmatic approach in differentiated risk assessment of homeopathic medicinal products. impact of myricetin and its methylated derivatives laricitrin, syringetin and myricetin-3`,4`,5`-trimethylether in c. elegans büchter c. 1 , ackermann d. polyphenolic compounds ubiquitously present in herbal food are discussed to contribute to the health beneficial effects of a diet rich in vegetables and fruits. additional to a strong antioxidative activity of various flavonoids, most of these substances display a variety of other pharmacological properties. we investigated the flavonoid myricetin found in several species of berries, as well as the methylated derivatives laricitrin, syringetin and myricetin-3`,4`,5`-trimethylether. in this study caenorhabditis elegans was used as a model to explore the impact of myricetin and its methylated derivatives in vivo and to investigate molecular modes of action. myricetin (100 µm) caused an increase in mean and median adult lifespan of c. elegans. this longevity effect was associated with a decrease of the aging marker lipofuscin as well as a decrease in ros induction, measured by using the h 2dcf-da assay. however, myrictin failed to improve heat stress resistance, an attribute often associated with longevity in c. elegans. the methylated myricetin derivatives (100 µm) showed a decrease in lipofuscin accumulation and ros induction and they further improved the heat stress resistance. in order to elucidate the basis of the life prolonging action of myricetin, we investigated its influence on factors known to have important functions in stress response and the regulation of aging, namely the foxo homologue daf-16, the nad + -dependent protein deacetylase sir-2.1 and the heat-shock transcription factor hsf-1, respectively. lifespan extension by myricetin disappeared in daf-16 and sir-2.1 loss of function mutant strains, showing the effect is at least partially dependent on these signaling molecules. by using a hsf-1 loss of function mutant strain of c. elegans, it was further shown that the life prolonging effect of myricetin is independent of hsf-1. in conclusion, our results indicate that the life prolonging effect of myricetin is at least in part dependent on daf-16 and sir-2.1, probably due to a modified expression of target genes. stimulatory and inhibitory control of phospholipase c-gamma 2 bühler a., walliser c., becker l., gierschik p. universität ulm institut für pharmakologie und toxikologie, albert-einstein-allee 11, 89081 ulm, germany activation of phospholipase c-γ2 (plcγ2) upon b cell antigen receptor (bcr) stimulation has been implicated to be a critical step in the bcr-mediated calcium signaling. therefore it is important to understand the mechanisms of how the activity of plcγ2 is stimulated and inhibited. the mammalian plcs are divided into six subfamilies, designated β, γ, δ, ε, ζ, and η. within the plcγ subfamily, the two plcγ isoforms share a number of features that are distinct from those of the other plc subfamilies. the most striking difference is the insertion of additional domains between the catalytic subdomains x and y. this specific array (sa) contains a second, split pleckstrin homology (spph) domain, consisting of two halves separated by two src homology 2 (sh2) domains and one src homology 3 (sh3) domain. there is abundant evidence in the literature that plcs are autoinhibited in their basal state by structural elements within their x/y linker, pointing to a conserved role of the x/y linker in autoinhibitory regulation of plc isozymes. data from our group show that plcγ 2 is also regulated by autoinhibitory elements within its specific array (walliser et al., 2008; everett et al., 2011) . our recent data demonstrated that plcγ2 is negatively regulated by its sa. specifically, within the sh domain tandem, the c-terminal sh2 (sh2c) and the sh3 domain in combination, but not either one alone, cause the strongest autoinhibitory control of plcγ2. plcγ2 has been shown to be phosphorylated at tyrosine residues 753 and 759 upon bcr stimulation (kim et al., 2004) . both tyrosines are located in the linker between the sh2c and the sh3 domain, which we have shown to be the major elements involved in autoinhibitory regulation of plcγ2. interestingly, a novel phosphorylation site in plcγ2 was found in non-small cell lung cancer (nsclc) tissue which is located at tyrosine residue 733 (rikova et al., 2007) . in this work, we demonstrate, for the first time, the activation of plcγ2 by phosphomimetic mutations in these three positions and the functional interplay of the three tyrosine phosphorylation targets. most interestingly, mimicking phosphorylation of tyr733 is critical to fully activate the enzyme. the results not only point to a crucial role of plcγ2 in pulmonary tumorigenesis, but also prompt and stimulate the search for the protein kinase involved in phosphorylating plcγ2 at tyr733. molecular characterization of hepatotoxic effects of perfluorooctanoic acid (pfoa) buhrke t., scharmach e., lampen a. bundesinstitut für risikobewertung (bfr) lebensmittelsicherheit, max-dohrn-str. 8-10, 10589 berlin, germany perfluorooctanoic acid (pfoa) is an industrial chemical that is used for the fabrication of numerous products with oil-, dirt-and water-repellent properties. pfoa is resistant to chemical, thermal and biological degradation and has become a global contaminant of soil, water, air and food in the meantime. the toxicological data of pfoa give cause for concern as the substance was shown to damage the liver of rodents and to impair embryo development. currently, the hazard potential of pfoa for humans is controversially discussed. in this study the human liver cell line hepg2 was employed to analyse the hepatotoxic effects of pfoa on the cellular and on the molecular level. pfoa was shown to stimulate cellular proliferation at concentrations in a range between 5 µm and 25 µm. at concentrations higher than 25 µm the substance was cytotoxic to the cells (ic 50 47µm). cytotoxicity was not due to apoptotic mechanisms as no increase of caspase activity was detected up to a level of 100 µm pfoa. on the molecular level pfoa is known to act as an agonist of the peroxisome proliferator-activated receptor alpha (pparα), and the observed hepatotoxic effects in rodents are associated with pfoa-mediated pparα activation. here we show that pfoa has the capacity also to activate the human isoform of pparα. additional human nuclear receptors were tested for activation by pfoa, and pparγ as well as the pregnane x receptor (pxr) were shown to be activated at high concentrations of pfoa whereas pparδ and the liver x receptor alpha (lxrα) were insensitive to activation by pfoa. notably, we observed a significant inhibition of the activity of the hepatocyte nuclear factor 4α (hnf4α) by incubating the cells already with moderate concentrations of pfoa at a level of about 1 µm. these findings indicate that additional, pparα-independent mechanisms may contribute to the observed hepatotoxicity of pfoa. the elucidation of novel modes of action of pfoa is relevant for the ongoing risk assessment of the substance. s17 070 human breast stem cells as a toxicological model for endocrine disruptors, such as soy isoflavones stempin s. 1 , bumke scheer m. (kao et al., 1995) . two daughter cell lines were developed from m13sv1 after x-ray irradiation (m13sv1 r2) and an additional transfection with a mutated erbb2 oncogene (m13sv1 r2-n1), resulting in high and low tumorigenicity respectively and showing a change in estrogen response after growth in minimal media (wang et al., 2010) . isolated isoflavones are currently widely used in the treatment of postmenopausal symptoms of women. according to the stem cell theory of carcinogenesis, breast stem cells are the ideal target for the proposed research. however, the epidemiological data to the effect of isoflavone intake on breast cancer is contradictory. therefore, we want to develop a toxicological model using these human breast stem cell lines to test the effect of endocrine disruptors, such as soy isoflavones in a human relevant model. in the present study we analyzed the effect of the phytoestrogen genistein, the most intensively studied soy protein, on the differentiation of the 3 hbec lines. the expression of different luminal epithelial cell markers, estrogen receptors and stem cell markers was measured on the mrna level by quantitative real time pcr. the analysis of several of these markers was also performed on the protein level using western blot. additionally, a broad number of genes related to breast cancer and estrogen receptordependent signal transduction were studied using a commercial pcr-array. in parallel we are also analyzing the changes on the protein level using 2d gel electrophoresis. we want to use this panel of different markers to establish a toxicological model that can be used in the future to analyze a wide range of different endocrine disruptors. kao cy, nomata k, oakley cs, welsch cw, chang cc. (1995) bronchial asthma is a common inflammatory disease of the airways whose occurrence has increased dramatically over the past decades. histamine plays an important role in mediating the inflammatory response leading to characteristic symptoms like wheezing, coughing, chest tightness, and shortness of breath. since antagonizing the histamine h1-receptor (h1r) shows no ameliorating effects on asthmatic symptoms, h4r antagonists may be new drugs for asthma therapy. in addition to the h3r-and h4rselective antagonist thioperamide, the selective h4r antagonist 1-[(5-chloro-1h-indol-2yl)carbonyl]-4-methylperazine (jnj7777120) is used in pharmacodynamic studies. a correct interpretation of the collected data requires the detailed knowledge of the pharmacokinetics of the applied substances. for this reason, we developed a fast and robust method based on high performance liquid chromatography coupled to tandem mass spectrometry (hplc-ms/ms) which allows the simultaneous quantitation of thioperamide and jnj7777120 as well as the selective h3r antagonist 1-({4[3-(piperidin-1-yl) propoxy]phenyl}methyl)piperidine (jnj5207852) in murine plasma and lung tissue. the treatment of plasma samples based on protein precipitation performed with a mixture of methanol and 0.2 m znso4 using 30 µl of plasma. analyte extraction from lung tissue was achieved by treating 150 -200 mg of tissue with a mixture of ethanol and water followed by rigorous mixing using a fastprep-system. ten µl of the extracted samples were transferred to a synergy polar-rp 80a mercury column (10 x 2mm; 4µm) connected to a polar-rp security guard. chromatographic separation was performed via a gradient using an acetate buffer (ph 5) and methanol at a flow rate of 0.4 ml/min. the analytical run-time was 5 minutes. for plasma samples the assay was linear over a concentration range of 0.078 -40 µg/ml for jnj7777120 and jnj5207852, and 0.313 -40 µg/ml for thioperamide, respectively. in tissues, thioperamide could be quantified in a concentration range of 0.008 -2 µg/sample, jnj7777120 and jnj5207852 in a range of 0.004 -2 µg/sample. our results show that the developed hplc-ms/ms method is suitable for the quantitation of all tested histamine-receptor ligands in murine plasma and lung tissue. the functionality of the heart greatly depends on strict homeostasis and interplay of a range of signalling cascades. deregulation of either one is always harmful and eventually detrimental for life. some of the most relevant signals in the adult heart are triggered by the stimulation of g protein-coupled receptors such as adrenergic or angiotensin receptors. those in turn are modulated by a small subset of kinases, the g protein-coupled receptor kinases (grks). interestingly, grks, which for the longest time were believed to regulate only g protein-coupled receptors were shown to modulate also non-receptor-mediated signalling pathways. by now it is well documented that grk5 plays important roles in both the physiological as well pathological setting of the adult heart. in spite of the important functions of grks in the adult heart, it must be assumed that grk5, one of the two main cardiac grks, may also be involved in signal modulation in the course of heart development. deregulation of grk5-dependent pathways may very well be causal for impaired cardiac development up to congenital heart disease. in fact, grk5 is already expressed during embryogenesis and we can detect it in the developing heart. however, grk5 has not been studied yet for a potential function during embryonic development in general or heart formation in particular. we have established zebrafish as a very time and cost efficient vertebrate model to investigate the role of grk5 on cardiac signalling and development. tools for grk5 specific loss-as well as gain-of-function analyses have been developed in our lab. we revealed an unexpected role of grk5 in the development of left-right asymmetry in zebrafish. clinically, this has been associated with disorders such as heterotaxy and other syndromes linked to ciliary dysfunction. many of those disorders are known to affect proper heart development resulting for example in septum defects or in detrimental translocation of the outflow tract. precisely, depletion of the close homolog of human grk5 in zebrafish mirrors the human syndrome called heterotaxy by displaying randomized placement of inner organs, aberrant heart looping and disrupted valve formation in the heart. in addition, loss of zebrafish grk5 results in a lower heart rate as well as dilatation of the embryonic heart at later stages of development. therefore, we believe that grk5 may potentially serve as a candidate gene for congenital heart disease. identification of a hcn3 interacting protein in mouse brain cao-ehlker x., hammelmann v., zong x., fenske s., biel m. department of pharmacy, center for drug research ludwig-maximilians-universität, munich center for integrated protein science cipsm, butenandtstr. [5] [6] [7] [8] [9] [10] [11] [12] [13] 81377 münchen, germany hyperpolarization activated cyclic nucleotide-gated cation channels (hcn) pass a depolarizing current (ih) that is involved in cardiac pacemaking and the control of numerous basic functions in neuronal circuits. the four hcn channel types (hcn1-4) display specific expression pattern in brain suggesting that each channel fulfills a distinct physiological function. while hcn1, hcn2 and hcn4 channels have been studied in quite some detail there is only little information on the particular role of the hcn3 channel. as an important step towards achieving a better understanding of hcn3 function we set out in this study to identify proteins that are assembled with hcn3 in brain tissue. to this end we performed a yeast two hybrid screen with a mouse cdna library using the hcn3 c-terminal domain as bait. several proteins were obtained and confirmed for interaction with hcn3 using heterologous coexpression in hek293 cells. here, we provide an in-depth analysis of the functional interaction between hcn3 and one of the identified interacting proteins (hip3.1). we show that hip3.1 physically binds to hcn3 channels in vitro and in vivo. still several open issues remain to be clarified i.e. the precise function of tpc1 and its tissue-specific and subcellular distribution. therefore we established a mouse model with a general deletion of tpcn1 and generated a series of tpc1 antibodies. using these tools we investigate the closer molecular and vesicular environment by different biochemical approaches i.e. affinity purification from native tissue derived from wild-type and as a control from knock-out mice, density gradient based vesicle separation, fluorescence activated organelle sorting (faos), total internal reflection fluorescence (tirf) and confocal microscopy. so far, we confirmed the tpc1 knock-out model by our self-generated tpc1 antibodies. tpc1 knockout mice are viable and do not show any obvious deficits. to isolate tpc1 containing vesicles or protein complexes, tissue or cell culture derived material was prepurified by sucrose density gradient centrifugation. for a subsequent mass spectrometric analysis this preparation was taken as a source material for coimmunoprecipitation or faos respectively. in another approach the migration pattern of tpc1 containing endosomes on linear density gradients was compared with a series of endolysosomal markers i.e. different rab-, er-, golgi-and lysosomal antibodies. potentially interesting markers were then in turn analyzed for their co-localization with tpc1 by confocal microscopy/tirf. by combining these results with that from mass spectrometric analysis of faos samples we collect data to get detailed information on the precise endolysosomal distribution pattern of tpc1. foxos are involved in a wide spectrum of cellular functions, including cell proliferation, apoptosis and regulation of oxidative stress. in order to identify novel target genes of foxo transcription factors and to achieve further insight into their role in cancer cells, dna microarray analysis was performed using wild type mcf-7 breast cancer cells and mcf-7 cells overexpressing foxo. we found that several genes involved in the tnf receptor/nf-κb pathway were differentially regulated. one of the genes that was identified to be up-regulated in foxo4 overexpressing cells was a20 a negative regulator of nf-κb signaling pathway. at both mrna and protein level foxo4-dependent up-regulation of this ubiquitin modifying enzyme was confirmed. to determine whether a20 is a direct target of foxo4, a luciferase reporter containing a 1.2 kb of the a20 promoter was co-transfected with different amounts of foxo4 wild-type expression construct. foxo4 induced a dosedependent increase in a20 promoter activity, supporting the assumption that a20 is a direct transcriptional target of foxo4. overexpression of foxo4 led to decreased activity of nf-kb signaling pathway as confirmed by reporter gene and nf-kb specific elisa assays. in addition, mcf-7 cells can be sentisized to tnf-α mediated cytotoxicity which is assocciated with a dimineshed activation of nf-κb. altogether, we identified a20, an ubiquitin modifying enzyme, as a novel foxo4 target gene. our data implicate that sustained foxo4 expression may be involved in regulation of tnf receptor/nf-κb pathway and leading to reduced cell survival. trpm7 is a bi-functional protein consisting of a transient receptor potential ion channel segment linked to an α-type protein kinase domain. trpm7 is essential for motility, proliferation and cell growth. up-regulation of trpm7 function is involved in anoxic neuronal death, cardiac fibrosis and tumor cell proliferation. recently, we have demonstrated that the recombinant trpm7 channel is inhibited by the known modulators of sk1-3 channels such as antimalarial plant alkaloid quinine, cyppa, dequalinium, ns8593, ska31, ucl 1684. the most potent of these compounds, ns8593 (ic50 1.6 µm), interferes with the regulation of trpm7 by cytosolic mg 2+ . here we show that ns8593 (10 µm) fully and reversibly inhibits native trpm7-like currents in hek 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes. furthermore, we examined whether targeting of the native trpm7 currents by ns8593 would impact cellular processes known to be affected by a genetic inactivation of trpm7. we found that ns8593 (10-30 µm) suppressed motility of hek 293 cells without a detectable effect on cell viability. taken together, our findings indicate that ns8593 is a potent and reversible inhibitor of endogenous trpm7 currents and may be a good candidate drug for pharmacological targeting of trpm7. sulfur mustard (2,2'-dichlorodiethylsulfide; sm) is a highly toxic and mutagenic warfare agent classified as a weapon of mass destruction. as soon as sm was first used as a warfare agent, research aimed at the development of an effective antidote was launched. early studies with first-generation inhibitors of poly(adp-ribose) polymerases (parp) have revealed promising therapeutic potential in sm-induced skin injury, but the underlying mechanism remains elusive. the current renaissance of parp inhibitors in cancer chemotherapy has revived the discussion on their use for treatment of sm injury. thus we established a comprehensive study aiming the elucidation of the role of parp in sm pathology based on model substance 2-chloroethyl ethyl sulfide (cees), which is not classified as warfare agent. we have recently demonstrated that parp becomes rapidly activated in living human keratinocytes (hacat) after treatment with cees. the maximal parp activity was observed 10 minutes after treatment with 3 mm cees. the activation was transient and dose dependent. to our knowledge this is the first demonstration of parp activation after treatment with mustards in the context of live cells. an important question is how parp-1 becomes activated upon treatment with mustards. parp-1 is a first-line protein involved in the cellular response to dna strand breaks. however, mustards do not directly induce large numbers of such lesions. one possibility is that parp-1 is activated by dna breaks incorporated as base excision repair (ber) or nucleotide excision repair (ner) intermediates. thus, we performed knockdown experiments of ape1 and ercc1, i.e. endonucleases involved in ber and ner, respectively. the reduction of ape1 expression had no effect on parp activity. surprisingly, the knockdown of ercc1 almost completely abolished the cellular par production after cees treatment. the functional consequence of the errc1-parp cross-talk with regards to adduct removal is under investigation. however, our present data indicate that parp activity is not obligatory for the survival of cells upon cees treatment, as revealed by the lack of effect of the potent parp inhibitor abt-888. expression and activity of g protein coupled receptor kinase 2 (grk2) are elevated in several conditions of compromised heart function. although grk2 inhibition has been characterized as a promising therapeutic strategy in heart failure, a specific grk2inhibitor is not available. raf kinase inhibitor protein (rkip) inhibits raf1 but it also acts as a physiological inhibitor of grk2 upon phosphorylation by pkc at serine153. a detailed understanding of the rkip/grk2 interaction may help to identify inhibitory compounds for grk2. since phosphorylation often induces homo-oligomerization of proteins, we investigated whether this could be implicated in switching rkip from a raf1-into a grk2-inhibitor. co-immunoprecipitation assays showed that rkip self-association was substantially increased after pkc-mediated phosphorylation of rkip. rkip mutants either lacking or mimicking s153 phosphorylation confirmed that this phosphorylation is indeed a prerequisite for rkip/rkip association. cross-linking experiments with myc-tagged rkip in living cells or with purified rkip revealed that rkip phosphorylation by pkc promotes rkip dimers -not oligomers. to test whether dimerization is a critical step for the association of rkip with grk2, we generated a peptide to inhibit rkip dimerization. intriguingly, the peptide did not only prevent rkip dimerization but also attenuated rkip/grk2 association. this implicates, that dimerization of rkip is essential to bind grk2. to determine whether rkip dimers consequently inhibit grk2 activity, we established rkip mutants with high tendency to form dimers. subsequent functional analyses demonstrated that enhanced dimerization of rkip indeed translates into increased grk2 inhibition. we conclude that pkc-mediated phosphorylation of rkip is important for dimerization and that these dimers are essential for grk2 binding and inhibition. our results reveal new insights in the molecular mechanism of rkip/grk2 interaction and will help to develop specific grk2 inhibitors. expression and function of trpm3 ion channels in epithelial mdck2 cells dembla s., meiser j., philipp s. university of saarland institute for experimental and clinical pharmacology and toxicology, kirrberger str. 1, 66421 homburg, germany trpm3 proteins build ca 2+ permeable cation channels [1] activated by steroids [2] and sensitive to increased temperatures [3] . trpm3 channels are expressed in pancreatic ßcells as well as neurons of the dorsal root ganglion, where they act as mediators of insulin release [2] or as nociceptors of noxious heat, respectively [3] . however, northern blots and in situ hybridization experiments revealed that trpm3 is also expressed in epithelial cells of the choroid plexus and the ciliary body [1] as well as in the kidney. pcr analysis of different epithelial cell lines indicated that trpm3 is also expressed in madin-darby canine kidney2 (mdck) cells. quantitative analysis of trpm3 expression by qrt-pcr revealed a ~ 5 fold upregulation in mdck2 cells grown in confluency compared to well separated, proliferating cells. in contrast the level of expression of trpm7, a related ion channel described as regulator of proliferation in other cell types, remained constant. hek293 cells overexpressing trpm3 channels did not proliferate in the presence of the trpm3 agonist pregnenolone sulphate. however, as indicated by impedance analysis, the proliferation of mdck2 cells in the presence pregs was only slightly affected. when we analysed the transepithelial resistance (ter) of mdck2 epithelial cells in transwells as a measure for the formation of tight junctions, we found that the ter of cells grown in the presence of pregs was reduced. interestingly, ca 2+ imaging experiments using fura2 revealed that pregnenolone sulphate induces ca 2+ entry in well separated mdck2 cells but not in cells growing in confluency. we hypothesize that trpm3 might act as a regulator of cell proliferation and/or the formation of tight junctions in mdck2 cells. inhibition of grk2 by rkip improves cardiac contractility and structure in a transgenic mouse model of heart failure denzinger s., schmitt j. p., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. 9, 97078 würzburg, germany the raf kinase inhibitor protein (rkip) has been identified as a physiological inhibitor of g-protein coupled receptor kinase 2 (grk2). grk2 initiates g protein coupled receptor (gpcr) desensitization. since expression and activity of grk2 are upregulated in human heart failure, it has been proposed that grk2 inhibition may resensitize badrenergic receptor activity in heart failure patients. in this study, we evaluated chronic grk2 inhibition by rkip as a potential strategy to improve cardiac function in heart failure. to analyse the effect of rkip on heart failure, rkip transgenic mice were crossed with mice carrying a mutation in phospholamban (pln r9c ). pln r9c causes severe heart failure and premature death in humans and transgenic mice. cardiac function was significantly improved in the presence of rkip as shown by left ventricular catheterization and echocardiography. expression of heart failure marker genes anf and bnp was indistinguishable between wild-type mice and mice co-expressing rkip and pln r9c . in line with these findings, the life span of double transgenic mice was significantly prolonged compared to pln r9c transgenic mice. slow calcium transport into the sarcoplasmatic reticulum was characterised as cause for dilatated cardiomyopathy of pln r9c transgenic mice. since western blot analyses of rkip transgenic heart lysates showed increased phosphorylation of important regulators of cardiomyocyte relaxation, we analysed calcium transients and contractility of isolated cardiomyocytes as possible mechanism of the rkip mediated rescue. in the presence of rkip, calcium reuptake into the sarcoplasmatic reticulum was accelerated and cardiomyocyte relaxation improved. furthermore, coexpression of rkip significantly attenuated pathological cardiac remodelling. interstitual fibrosis and apoptotic cells were quantified in histological sections after sirius red-and tunel-staining. this study revealed a protective function of rkip in a genetic mouse model of human dilated cardiomyopathy by improving cardiac contractility and attenuating interstitial fibrosis and apoptosis. a detailed understanding of this rescue may help to find a new therapeutic strategy to improve cardiac contractility in heart failure. gαi-proteins comprise a group of three highly related members characterized by specific expression patterns. based on previous work of gi-mediated signaling pathways in cardiomyocytes and platelets, we checked gαi expression in mouse heart and platelets. the analysis revealed the presence of gαi2 and gαi3 with gαi2 as the predominant isoform. gene-targeted mice lacking either gαi2 or gαi3 were analyzed to unravel the physiological role of gαi-proteins in the cardiovascular system. extraordinarily prolonged bleeding times in gαi2-deficient animals were an obvious phenomenon. detailed analysis using isolated platelets gαi2-deficient mice exhibited reduced platelet activation and attenuated aggregation in response to stimulation by various agonists accompanied by reduced thrombus formation and diminished stability on a collagen-coated surface. employing in vivo injury/thrombosis models revealed abrogated thrombus formation selectively in gαi2-deficient mice. comparable results were obtained in experiments using mice with megakaryocyte/platelet-specific gαi2deficiency. to assess the pathophysiological consequences of platelet gαi2 function, we challenged these mice in experimental models of myocardial and cerebral ischemia. the results clearly show that platelet-gαi2-deficient mice were protected from both, myocardial and cerebral ischemia. in contrast, conventional gαi2-deficient mice subjected to the heart ischemia/reperfusion model exhibited a significantly increased susceptibility to ischemic injury as compared to wild type controls. in contrast, gαi3deficient mice were strongly protected from injury. thus we suggest that gαi2 and gαi3 play distinct roles in major cardiovascular disorders pointing to specific, non-redundant functions of these two highly related gαi isoforms. the cgmp signaling pathway is activated by nitric oxide (no), natriuretic peptides (anp, bnp & cnp), and cgmp-elevating drugs. it regulates several physiological functions such as smooth muscle relaxation, platelet inhibition, and cell growth and differentiation. recent studies indicate that cgmp signaling might also play a role in tumorigenesis, but the cellular and molecular mechanisms of cgmp's potential pro-and/or anti-tumor activities are not well understood. this study has examined the expression and function of components of the cgmp pathway in melanoma cells of murine and human origin. we have found that mouse b16 melanoma cells specifically express the alpha isoform of the cgmp-dependent protein kinase type i (cgkialpha) but not the beta isoform. treatment of intact cells with the membrane-permeable cgmp analog 8-br-cgmp induced the phosphorylation of cgki substrates, vasodilator stimulated phosphoprotein and phosphodiesterase 5. anp and cnp, ligands of the membrane-bound guanylyl cyclase gc-a and gc-b, respectively, did also activate the endogenous cgmp/cgki pathway. however, b16 melanoma cells did not respond to dea-no, which stimulates no-sensitive soluble guanylyl cyclases. interestingly, activation of cgmp/cgkialpha signal transduction was associated with an increase in erk1/2 and p38 phosphorylation, growth and migration of b16 melanoma cells. similar results were obtained with wm1205 human melanoma cells. in conclusion, we have identified a gc-a/gc-b/cgmp/cgkialpha pathway in melanoma cells, which stimulates tumor cell growth and migration in vitro. pharmacologic inhibition of cgmp signaling may offer a promising strategy for the treatment of melanoma. an increasing body of evidence supports important roles for voltage-gated calcium channels in idiopathic generalized epilepsies (iges), however which calcium channels participate in ige pathogenesis and how has yet to be fully understood. recently, it has been proposed that cav2.3 (r-type) and t-type calcium channels jointly contribute to oscillatory bursting in the reticular thalamus (rt) 1 , which is associated with absence epilepsy. cav3.2 is one of the two t-type calcium channels known to be expressed in the rt 2 . it has been demonstrated that ablation of either cav2.3 or cav3.2 reduces susceptibility to experimentally induced epilepsy 3;4 and in addition that both channels share several pharmacological properties [5] [6] [7] . to gain further insight into interacting mechanisms of these two channels in epilepsy, we tested cav2.3(-|-), cav3.2(-|-) and cav3.2(-|-)xcav2.3(+|-) mice side-by-side in the kainic acid model of epilepsy. we provide first in vivo data supporting a synergistic mode of action for cav2.3 and cav3.2 calcium channels in epileptogenesis. the deubiquitinase cyld regulates mechanisms of rip1/rip3-dependent necroptosis in neuronal cells diemert s. 1 , krieg s. vivo model of cerebral ischemia, we found, that cyld -/-mice exhibit significantly reduced infarction volume compared to control littermates. overall, these data reveal a role for cyld in rip1/3-dependent mechanisms of necroptosis in a model of glutamate toxicity in neuronal cells and further suggests cyld-mediated mechanisms of neuronal cell death as a potential therapeutic target after acute brain injury in vivo. cyanamide-mediated inhibition of n-acetyltransferase 1 dierolf d., bonifas j., blömeke b. university trier department of environmental toxicology, universitätsring 15, bldg. n, 54286 trier, germany cyanamide has been used for decades for medical purposes in the treatment of alcoholism and for agricultural purposes as a plant growth regulator and bud-breaking agent. its therapeutic effect is mediated by reversible inhibition of aldehyde dehydrogenase and it was reported to be metabolised in vivo mainly via coenzyme a dependent n-acetylation by n-acetyltransferases. reported to be a substrate for n-acetyltransferases, cyanamide has a different molecular structure to arylamines and hydrazines, the preferred substrates for nacetyltransferases. therefore a more detailed investigation of its interrelations with nacetyltransferases was performed. we analysed nat1 enzyme activities after incubation of thp-1 cells with cyanamide for 24h, and found that the metabolic conversion of the classic substrate para-aminobenzoic acid was significantly reduced at physiologically relevant concentrations. in detail a significant dose-and time-dependent nat1 protein inhibition was observed for 100 and 1000 µm cyanamide using over-expressed human recombinant nat1 (insect cell cytosol containing recombinant human nat1*4). however, no inhibition was found in the presence of recombinant nat2*4. as we also provide evidence that cyanamide is not metabolised via coenzyme a dependent nacetylation in vitro by human nat1 or nat2 cytosol, by thp-1 cells or by human liver cytosol, we can conclude that this inhibition is not based on substrate-dependent downregulation of nat1. further mechanistic and kinetic studies indicated that cyanamide reacts with the active site cysteine residue of nat1, leading to its rapid inhibition. since the presence of the reduction agent dithiothreitol did not reverse the results it could be that it is possibly not caused by oxidative processes. in sum these data indicate that cyanamide is able to interact with cysteine residues of human nat1, which causes its inhibition but not by a substrate-dependent mode of action. taken together our results show, that cyanamide is not n-acetylated by human nats, but might modulate nat1 dependent detoxification and activation of arylamines. dissecting the signal transduction pathway of acute hypoxic vasoconstriction (hpv) in precapillary pulmonary arterial smooth muscle cells ( low levels of oxygen in the pulmonary airways induce acute hypoxic pulmonary arterial vasoconstriction (acute hpv) redirecting blood flow to normoxic areas of the lung to assure optimal uptake of oxygen during ventilation. acute hpv lasting several minutes occurs predominantly in the precapillary region of the pulmonary vascular tree [1] . therefore, precapillary pulmonary arterial smooth muscle cells (pasmc) have been suggested as sensor as well as effector cells and trpc6 a member of the classical transient receptor potential (trpc) ion channel family was identified to be essential for the initiation of ca 2+ influx and the subsequent contraction of pasmc [2] . however, the underlying oxygen sensor and the exact signal transduction pathway(s) in pasmc have not been fully elucidated yet. by using gene-deficient mouse models as well as downregulation of potential candidate proteins by specific small interfering rnas (sirnas), we aim to dissect signaling cascades of trpc6 channel activation in acute hpv. for pasmc isolation and culture from mice we use a technique based on magnetic separation of intrapulmonary arteries originally developed in rats [3] . trpc-expression in freshly isolated and passaged pasmc cultured in low (5%) and high fetal bovine serum (25%) was analyzed. interestingly higher passage numbers resulted in a significant down-regulation of trpc1 and trpc6 the most predominantly expressed channel monomers in pasmc, while different serum concentrations resulted in no significant changes in their expression rates. sirnas were designed, transfected and successfully tested in hek293 cells and pasmc. initial results of the dissection of the signal transduction pathway activating trpc6 and inducing acute hpv in pasmc will be presented. references [1] staub, n. c. (1985) . site of hypoxic pulmonary vasoconstriction, chest 88, 240s-245s. [2] weissmann, n. et al. (2006) . classical transient receptor potential channel 6 (trpc6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange, proc. natl. acad. sci. u.s.a. 103, 19093-19098 trpv5 is a highly selective calcium channel expressed in various tissues amongst others in placenta. the channel may be involved in transcellular calcium transport in epithelial tissues thereby playing some role in calcium homeostasis of the body. in the placenta trpv5 is assumed to contribute to the maternal-fetal calcium transport. most probably trpv5 is part of a multiprotein channel complex but most of the components of this complex are unknown so far. our aim is to find interaction partners of the trpv5 protein in the placenta that might contribute to the regulation of the trpv5 protein function. therefore we expressed the intracellularly located n-and c-terminal parts of trpv5 (aa 1-330 and 571-723) as trpv5-gst (glutathione-s-transferase)-fusion proteins and used the purified recombinant proteins for pulling down proteins from human placenta cell extracts. the proteins pulled down by this approach were analysed by mass spectrometry. we identified several potential trpv5 interacting proteins which were not associated with the gst protein only. one of the proteins which was highly enriched with the n-terminal part of the trpv5 protein is calpain 6. in contrast to the classical calpains (calcium activated cystein proteases), calpain 6 is unique in that it lacks the cysteine residue in the active site. calpain 6 is mainly expressed during embryogenesis and is reported to be involved in cytoskeleton stabilisation but with unknown function in placenta. the interaction between trpv5 and calpain 6 was confirmed in reciprocal pulldown experiments and the trpv5 binding region for calpain 6 was narrowed down by using overlapping n-terminal trpv5-gst fusion proteins. after injection of trpv5 crna into xenopus laevis oocytes calcium uptake into the oocytes was measured; this uptake was largely reduced after co-injection of the calpain 6 crna. in further experiments we want to study potential regulatory effects of the trpv5 protein on the calpain 6 function in cell culture models. comparative studies on the effects of the human carcinogen inorganic arsenite and its recently identified thiolated metabolite thio-dma v on human urothelial cells ebert f., leffers l., unterberg m., schwerdtle t. universität münster institut für lebensmittelchemie, corrensstr. 45, 48149 münster, germany it has been demonstrated that chronic ingestion of 50-200 µg/day inorganic arsenic is associated with an increased risk for cancers of the skin, the lung and the bladder, but until now the underlying toxic modes of action are still under debate. in this context, in the last five years one main focus has been given to the role of human inorganic arsenic metabolism and nowadays it is generally accepted that human biomethylation contributes to inorganic arsenic induced carcinogenicity. due to further improvements in arsenic speciation techniques recently a new thiolated arsenite metabolite, the thio analogue of the well known metabolite dimethylarsinic acid (dma v ), the so called thiodimethylarsinic acid (thio-dma v ), has been discovered in human biological samples. after synthesizing and analytically characterizing this metabolite (bartel et al. 2011 , j toxicol. 2011 we investigated its toxic effects in direct comparison with ias iii in human urothelial cells. thereby cell cycle studies revealed a g2/m-and s-phase arrest as well as subg1 peak formation in case of thio-dma v . moreover, thio-dma v induced apoptosis (subg1, caspase 3 activity) at lower concentrations and earlier time points as compared to ias iii . most likely this is partly due to the higher cellular bioavailability of thio-dma v (aas/icp-ms). regarding genotoxicity, a generation of dna single strand breaks (alkaline unwinding technique) as well as an increased formation of reactive oxygen species (ros, dcfh-da-fluorescence) occurred only at high cytotoxic concentrations. however, thio-dma v strongly increased h2o2 induced ros formation at very low nanomolar concentrations, which might result in cogenotoxic effects. since our earlier studies have shown a strong inhibition of h2o2 induced poly(adp-ribosyl)ation especially by trivalent methylated arsenic metabolites, actual studies investigate the impact of thio-dma v on cellular poly(adp-ribosyl)ation, parp-1 gene expression, protein level and cellular cleavage, which might hopefully give further hints regarding the mode of action behind thio-dma v induced apoptosis. mitochondrial dysfunction in models of alzheimer´s disease eckert a. universitäre psychiatrische kliniken basel neurobiology laboratory, wilhelm klein strasse 27, 4012 basel, switzerland the histopathological characteristics of alzheimer's disease (ad) are amyloid-ß (aß) containing plaques and neurofibrillary tangles (nfts) as well as neuronal and synaptic loss. until today, the underlying mechanisms of the interplay of plaques and tangles remained unresolved. there is increasing evidence that mitochondrial dysfunction might be a possible link, as revealed by studies in several app and tau transgenic mouse models. recently, we examined mitochondrial function in a novel triple transgenic mouse model (pr5/app/ps2) -triplead mice -that combines both pathologic features of the disease in brain. using comparative, quantitative proteomics (itraq) and mass spectroscopy we found a massive deregulation of 24 proteins, of which one-third were mitochondrial proteins mainly related to complexes i and iv of the oxidative phosphorylation system (oxphos). remarkably, deregulation of complex i was related to tau, whereas deregulation of complex iv was aß dependent, both at the protein and activity levels. the triplead mice showed synergistic effects of aß and tau already at the age of 8 months, resulting in a depolarized mitochondrial membrane potential. at 12 months, the strongest defects on oxphos, synthesis of atp and reactive oxygen species were exhibited in the triplead mice, again emphasizing synergistic, ageassociated effects of aß and tau in impairing mitochondria. evidences from ad post-mortem brain as well as cellular and animal ad models indicate that aß and tau protein trigger mitochondrial dysfunction through a number of pathways, such as impairment of oxidative phosphorylation, elevation of reactive oxygen species production, alteration of mitochondrial dynamics, and interaction with mitochondrial proteins. moreover, recent reports indicate that aß may also interact directly with intracellular proteins such as the mitochondrial enzyme abad (aß binding alcohol dehydrogenase) in executing its toxic effects. mitochondrial dysfunction occurs early in ad, and aß's toxicity seems to be in part mediated by inhibition of abad. in total, a vicious cycle as well as several vicious circles within the cycle, each accelerating the other, can be drawn emphasizing the synergistic deterioration of mitochondria by tau and aß. olesoxime is a novel mitochondrial-targeted compound that is orally active and crosses the blood brain barrier. the cholesterol-oxime targets proteins of the outer mitochondrial membrane and represents a promising drug candidate for neurodegenerative diseases 1 . we evaluated olexoxime's neuroprotective effects against mitochondrial dysfunction in an animal model for alzheimer`s disease (ad). dissociated brain cells (dbc) and mitochondria were isolated from brains of c57/bj6-thy1-appsl (ad-mice) mice that were fed with 600 mg olesoxime/kg feed for 3 months. drug plasma levels reached approx. 600 ng/ml. respiration of isolated mitochondria were significantly diminished in ad-mice due to reduced complex i, i+ii and cox activities. consequently, mitochondrial membrane potential (mmp) was significantly reduced in dbc from ad-mice. olesoxime normalized respiration chain complex activities and the mpp. to further evaluate the beneficial effects of olesoxime on complex i activity, we challenged dbc with rotenone ex vivo and observed that olesoxime treatment was protective. to further clarify the mode of action, we analyzed the ability of olesoxime to prevent opening of the mitochondrial permeability transition pore (mptp) in vitro using energized brain mitochondria by measuring ca 2+ -and atractyloside (atr) induced swelling. the opening of mptp precedes apoptosis and can be induced by mitochondrial dysfunction due to calcium overload, oxidative stress, elevated phosphate concentrations or adenine nucleotide depletion. olesoxime prevented ca 2+ -as well as atr induced mitochondrial swelling. atr inhibits the adenine nucleotide translocase (ant) that requires appropriate membrane properties to mediate mitochondrial permeability transition (mpt). since cholesterol (cho) and its derivates represent potent modulators of membrane viscosity, we related the effects of cho and olesoxime on mptp opening to membrane properties. both, cho and olesoxime reduced the flexibility of membrane acyl-chains in energized mitochondria and prevented atr induced mptp opening. however, cho didn`t prevent ca 2+ -induced mptp opening, indicating a different mode of action for olesoxime. our data confirm olesoxime as drug candidate against mitochondrial dysfunction, which is considered to play a pivotal role in neurodegenerative diseases. the work was supported by the european union under the 7th framework program for rtd -project mitotarget -grant agreement health-f2-2008-223388. several inflammatory glomerular kidney diseases are accompanied with a massive production of reactive oxygen species (ros) that may attack the glomerular filtration barrier by affecting podocyte function and may contribute to apoptotic or necrotic cell death of mesangial cells. otherwise, ros also trigger fine-tuned signaling processes that may result in cell proliferation or cell migration. to define such redox-driven signaling devices, we performed a non hypothesis-driven proteomic approach, to identify homo-or heteromeric protein complexes induced by ros. to this end, protein lysates of human podocytes were treated with or without hydrogen peroxide (250 µm) for 10 min. thereafter, the cell lysates were subjected to diagonal 2d gel electrophoresis and putative redox-affected proteins were analyzed by ms/ms-analysis. by this approach, we could identify a series of proteins that form interprotein-disulfide bonds in a redoxdependent manner. one of those proteins could be characterized as the regulatory subunit of protein kinase a (r-subunit of pka), which belongs to the family of serine/threonine kinases. to evaluate whether ros is capable to activate pka also in a more physiological setting, we treated rat mesangial cells with pdgf-bb to induce ros formation and we could demonstrate that pdgf-bb induces dimerization of r-subunits in a redox-dependent manner. to demonstrate whether pdgf-bb induces pkadependent pathways, we analyzed the effects of pdgf-bb on phosphorylation of serine 157 of vasodilater stimulated protein (vasp) a classical target of pka. in fact, pdgf-bb induced vasp phosphorylation independently of intracellular camp levels. moreover, elevating camp levels via activation of adenylate cyclase with forskolin did not change the dimerization state of r-subunits. pdgf-bb-induced dimerization of the r-subunits and subsequent phosphorylation of vasp was blocked by diphenyljodonium (dpi), indicating activation of a nadph oxidase is essential for pka activity. taken together, we demonstrate a redox-dependent activation of pka by pdgf-bb and this may hint also for a probably protective role of ros in rat mesangial cells. testing the potential sensitizing capacity of chemicals is currently done by using the murine local lymph node assay (llna). animal welfare and eu cosmetics directive demands alternative methods to animal tests. the purpose of this study was to establish an in vitro assay for the prediction of skin sensitizers. based on the finding that the majority of skin sensitizers are electrophilic or have the potential to be metabolized to electrophilic substances, it is assumed that they can activate the nrf2-keap1-antioxidant response element (are) regulatory pathway. here, we report the results obtained from the lusens assay that detects electrophilic chemicals using the nrf2 pathway. the cell line lusens was derived from immortalized keratinocyte hacat cells and carries a luciferase reporter gene under the control of an are-element from the rat nadph quinone reductase nq1. the lusens assay was in house validated with a panel of 54 chemicals and cosmetic ingredients including the 22 performance standard substances of the local lymph node assay. the predictivity of this assay was compared to the predictivity of the murine llna and to human patch test data and can be considered as reliable screening approach (accuracy of 83% compared to human data). however, in order to cope with the complex multi-step mechanism of skin sensitization, an integrated approach of in vitro assays mimicking several steps was designed; thereof, the are-dependent gene activation represents one module. time-resolved fluorescence ligand-binding assays for parathyroid hormone receptors emami-nemini a. 1 ligand-binding studies represent essential tools for pharmacological research on g protein-coupled receptors. in recent years, time-resolved fluorescence gained significant relevance as readout for ligand binding studies. however, ligand-binding assays for parathyroid hormone receptors (pthrs) utilizing fluorescent parathyroid hormone (pth) were missing. therefore, we generated various fluorescent pth analogues which exhibit properties of native pth in terms of affinity, potency and internalization. for the purposes of academic and commercial research, we utilized labeled pth to set up three time-resolved fluorescence assay formats: (i) classical separation binding assay, based on time-resolved fluorescence and suitable for native receptors; (ii) homogeneous timeresolved fluorescence resonance energy transfer (htrf) based on tag-lite technology for high through-put screening; (iii) htrf based on antibodies, a synergistic approach using htrf with minimized receptor modification. this work will facilitate the development of new drugs directed to the pthr as well as fundamental research on the pthr. anandamide production in eosinophilic granulocytes is independent of il-5 and eotaxin stimulation engeli s., reinke j., zörner a., tsikas d., jordan j. medizinische hochschule hannover institut für klinische pharmakologie, carl-neuberg-straße 1, 30625 hannover, germany introduction: some animal and in vitro studies suggest that endocannabinoids exert anti-inflammatory effects. specifically, inhaled anandamide reduced the obstructive effect of leukotrien d4 in airways, and a specific cannabinoid receptor agonist significantly reduced pulmonary inflammation in guinea pigs. we have recently shown that segmental bronchial allergen challenge is associated with significant increases of anandamide concentrations in bronchoalveolar fluid of patients with asthma. the concomitant increase in eosinophilic counts, eotaxin and il-5 concentrations in bronchoalveolar fluid led us to hypothesize that anandamide is produced by eosinophilic granulocytes in response to chemotactic stimuli. peripheral eosinophilic granulocytes were isolated from whole blood by means of percoll gradient centrifugation and magnetic separation employing cd16antibodies conjugated to magnetic beads. isolated cells were counted and anandamide measurements were typically performed in whole cell lysates of 1.5x10 6 eosinophils. we stimulated eosinophils with varying concentrations of il-5, eotaxin-1 (ccl11), eotaxin-2 (ccl24), and eotaxin-3 (ccl26). to prevent anandamide degradation, a specific fatty acid amide hydrolase (faah) inhibitor (oloxa) was employed. anandamide concentrations and faah activity were determined by stable isotope dilution using lc-ms/ms protocols. results: first, we confirmed the ability of eosinophilc granulocytes to synthesize anandamide. however, cellular anandamide content could only be measured when faah was effectively blocked with oloxa, and strong faah activity was demonstrated in eosinophils. with oloxa, typical anandamide concentrations were in the range of 2-4 pm/1.5x10 6 eosinophils. neither il5 (50-500 pg/ml), nor any of the eotaxins (50 ng/ml either alone or in varying combinations) did stimulate anandamide production after 30min of incubation. our results suggest that chemotactic molecules like eotaxin and il-5 are not responsible for increased anandamide formation in eosinophils during allergen challenge. in a next step, the effects of anandamide on eosinophils and bronchial epithelial cells need to be determined. the suprisingly high faah activity in eosinophils may point to an alternative pathway facilitating prostaglandin and leukotriene synthesis by production of arachidonic acid. screening methodology for estimatation of dermal absorption in vitro fabian e., goth c., guth k., mehling a., van ravenzwaay b., landsiedel r. basf se experimentelle toxikologie, carl-bosch-strasse 38, 67056 ludwigshafen, germany dermal absorption is used in the evaluation of the effectiveness of pharmaceutical or cosmetic formulations, but often dermal absorption is a critical parameter in risk assessment of pesticides or chemicals. therefore, knowledge of dermal absorption is e.g. helpful in formulation development. skin absorption is routinely measured in vivo or in vitro following oecd tg 427 or 428. however, these tests are complex, time consuming and expensive. therefore, a method was developed to allow simple and rapid screening. the experiment uses dermatomized skin in modified franz type diffusion cells. 10 µl of test substance preparation are applied to the skin preparation. after 6h, the skin is washed and the amount of penetrated substance is quantified. the receptor fluid and the washing solutions are optimized for subsequent analyses by lc-ms. we performed dermal absorption screenings in parallel to our routine guideline studies and demonstrated a good correlation of the results of both study types: the total recovery found in the screening studies is somewhat lower than in the corresponding guideline studies but is always in an acceptable range above 80%. the efficacy of the skin washing procedure is lower than under routine conditions, most probably due to the change to an lc-ms-compatible washing solution. overall, the dermal absorption screening is an easy, fast and cost-effective screening method for the estimation of dermal absorption of a wide variety of test substances and formulations. the p53 tumor suppressor protein is frequently inactivated in human cancers by diverse mechanisms. owing to its fundamental role in the maintenance of genomic stability and cancer prevention, p53 is an attractive target in cancer therapy and several approaches were pursued to restore p53 function in tumor cells. polyamidoamine (pamam) dendrons are positively charged molecules with a systematically branched structure that interact with the negatively charged cell membrane, inducing cellular uptake via endocytosis. in the present study, biotin-labeled p53 protein was attached to a dendronized streptavidin nanocarrier to facilitate its internalization into different tumor cell lines. first, biotin-substituted pamam dendrons were conjugated with streptavidin to allow formation of the dendronized streptavidin nanocarrier. the nanocarrier displayed uptake into hela cervix carcinoma and a549 lung adenocarcinoma cells without detectable cytotoxicity. biotin-labeled p53 was then conjugated to the dendronized streptavidin, preserving its specific dna-binding in vitro. immunoblot analysis revealed efficient internalization of biotin-p53 into hela cells in the presence of dendronized streptavidin. in line with this finding, specific cellular uptake of biotin-p53 was observed by confocal microscopy, which showed a cytoplasmic and peri-nuclear localization in hela, a549 and saos osteosarcoma cells. the internalized biotin-p53 also partially co-localized with early endosomes. importantly, the delivery of biotin-p53 into p53-deficient saos cells resulted in impaired cell viability and upregulation of caspase 3/7 activity, demonstrating its biological functionality. this study intriguingly demonstrate the efficient delivery of functional biotin-p53 into different tumor cell lines using the novel streptavidin nanocarrier, which can be further modified to allow cell-type specific targeting and combined with cytotoxic drugs such as doxorubicin. identification of novel ahr target genes in rat liver oval cells faust d. 1 , vondracek j. the aryl hydrocarbon receptor (ahr) is a transcription factor involved in physiological processes, but also mediates most, if not all, toxic responses to 2,3,7,8tetrachlorodibenzo-p-dioxin (tcdd), some polycyclic aromatic hydrocarbons (pahs) and dioxin-like polychlorinated biphenyls (pcbs), such as pcb126. activation of the ahr by these ligands leads to its dimerization with arnt and transcriptional activation of several phase i and ii metabolising enzymes. while it is generally accepted that many pahs are thereby transformed to genotoxic metabolites, this classical signalling pathway so far failed to explain the tumour promoting effects of the nongenotoxic compounds tcdd and pcb126. thus, there is an urgent need to define genetic programmes orchestrated by ahr to unravel its role in physiology and toxicology. we have recently shown that treatment of rat liver oval cells with tcdd or pcb126 leads to a release from contact-inhibition involving activation of the ahr, elevation of jund protein levels and transcriptional activation of cyclin a (1, 2) . loss of contact-inhibition is one hallmark of tumour promotion. to better understand ahr-driven pathways we identified the transcriptional programme using high density microarrays in response to pcb126. already 6 h after treatment, 69 genes were found to be upregulated and 76 genes downregulated indicating that these are direct ahr-dependent target genes. david analysis revealed that these genes are involved, for instance, in drug and lipid metabolism, cancer pathways, tgf-b signalling and cell-cell communication. ten of the 69 genes were selected for confirmation by semi-quantitative rt-pcr. using the ahr inhibitor ch-223191 and sirna directed against ahr and arnt, we further demonstrated that ahr-and arnt-function is required for transcriptional activation of the selected genes. finally, we identified the transcription factor foxq1as a novel ahr target protein in rat liver oval cells. although the function of foxq1 is poorly understood, it has been shown very recently that foxq1 is overexpressed in colorectal cancer and is involved in epithelial-mesenchymal transition in breast cancer cells. its function in ahrmediated tumour promotion, however, remains to be determined. the practical relevance of histamine h1 and h3 receptors in the brain can be easily deduced since h1 receptor antagonists of the first generation have a sedative effect and an inverse h3 agonist, pitolisant (close to its introduction to the market), is active against excessive daytime sleepiness associated with narcolepsy. in this context the question arises whether also h2 and h4 receptors possess a practical relevance in the brain. to this end, we examined whether the electrically evoked 3 h-noradrenaline release in superfused human cerebral cortex slices is affected by agonists at the above receptors. the h2 agonist impromidine 10 µm failed to affect noradrenaline release in human cortex slices although it facilitated noradrenaline release in guinea-pig cortex slices; the maximum extent of facilitation was 50 %, the pec50 was 6.9 and the pa2 of the h2 antagonist ranitidine against impromidine was 7.2. with respect to h4 receptors there is some controversy in the literature whether they occur in the brain at all. however, we were able to detect h4 receptor mrna in the human and mouse cortex by the reverse transcriptase polymerase chain reaction. in cortex slices of either species, noradrenaline release was not affected by the h4 agonist 4-methylhistamine 10 -30 µm but inhibited by histamine 10 µm via h3 receptors by 28 and 55 %, respectively. in mouse cortex membranes, 4-methylhistamine 30 µm also failed to affect 35 s-gtpγs binding although r-α-methylhistamine 3 µm, acting via h3 receptors, increased it by 20 %. in conclusion, h2 receptors facilitating noradrenaline release are detectable in the isolated guinea-pig but not human cortex. despite the presence of h4 mrna in the brain, functional readouts of this receptor, i.e. modulation of noradrenaline release (humans, mice) and modulation of 35 s-gtpγs binding (mice), could not be shown. murine cx40 promoter activity is dependent on the transcription factor creb fels b., nunes f., schmitz w., müller f. u. westfälische wilhelms-universität münster institut für pharmakologie und toxikologie, domagkstraße 12, 48149 münster, germany connexin 40 (cx40) is a gap junction protein expressed in atrial myocytes and the ventricular conduction system, mediating the electrical intercellular communication in the myocardium. alterations in cx40 function were linked to the pathophysiology of atrial fibrillation and heart failure. in the heart, camp dependent gene transcription is regulated by members of the creb/crem/atf family which bind to camp responsive elements (cres). similar to the human cx40 gene promoter, the murine promoter contains one cre. cardiomyocyte-specific overexpression of a crem-isoform (crem-ib∆c-x) led to cx40 down-regulation, suggesting that creb related transcription factors are involved in cx40 gene regulation. in order to study the functional role of creb in the regulation of the cx40 promoter we have generated a luciferase based murine cx40 promoter reporter gene construct and monitored its activity in a permanent cell line upon overexpression of constitutive-active creb (cacreb) and a non-phosphorylatable dominant-negative creb (dncreb) isoform. surprisingly, overexpression of cacreb and dncreb both lead to a reduction of cx40 promoter activity (cacreb 46% ± 5% vs control 100% ± 3%; p<0.05 vs control , n=15), dncreb 45% ± 2% vs control 100% ± 3%; p<0.05 vs control, n=18). the activity of the murine connexin 40 promoter is modulated by creb. both cacreb and dncreb led to cx40 down-regulation, which could be explained by induction of inhibitory transcription factors creb/crem/atf1 transcription factor family, which in turn could suppress cx40 promoter activity. (supported by the dfg) results: fxa increased par-2 mrna, protein and cell-surface expression and augmented par-2-mediated mitogenesis. par-1 expression was not influenced. the regulatory action of fxa on par-2 was concentration-dependent and mimicked by a par-2 selective activating peptide. the thrombin inhibitor argatroban or par-1 gene silencing did not influence fxa-stimulated par-2 expression. fxa increased oxidative stress and expression of the nadph oxidase subunit nox-1 in smc. nox-1 gene silencing prevented fxa-stimulated par-2 regulation, as did ebselen and catalase. exogenous hydrogen peroxide increased par-2 expression and mitogenic activity. fxa induced nuclear translocation and par-2 dna binding of nuclear factor kb (nfkb). inhibition of nfkb prevented fxa-stimulated par-2 expression. in separate studies, fxa promoted par-2 mrna stabilisation through increased human antigen r (hur)/par-2 mrna binding and cytoplasmic shuttling. hur gene silencing abolished fxa-stimulated par-2 expression. conclusion: expression and mitogenic activity of vascular par-2, but not par-1, is upregulated by fxa. this action involves transcriptional and post-transcriptional mechanisms mediated through nox-1-containing nadph oxidase and its downstream effectors hydrogen peroxide, nfkb and the mrna stabilising protein hur. continued generation of fxa by the mural thrombus, and the autoregulatory feedback control of par-2 may maintain the inflammatory and proliferative state of the injured vessel, thereby promoting vascular remodeling. the mrna stabilising factor hur is a critical regulator of human proteaseactivated receptor 4 aim: we recently reported that functional expression of par-4 thrombin receptors is induced in human saphenous vein smc exposed to high glucose. this effect could be attributed in part to transcriptional mechanisms mediated through nfkb but the contribution of post-transcriptional effects such as mrna stabilisation is not known. this study explored the potential role of the mrna stabilising factor human antigen r (hur) in the regulation of par-4. methods: human saphenous vein smc were serum-deprived prior to study. gene expression was determined by realtime pcr, protein expression by western blotting. gene silencing utilized commercially available sirna. hur binding to par-4 mrna was determined by immunoprecipitation ("pull-down") pcr. results: high glucose (25 mm vs 5.5 mm) slowed par-4 mrna degradation in the presence of actinomycin d. par-1 mrna decay was not affected. hur binding to par-4 mrna and nucleo-cytosolic shuttling was enhanced by high glucose, total hur protein expression was not affected. hur sirna abolished the high glucose-stimulated induction of par-4 mrna. hydrogen peroxide (h 2o2) also induced cytosolic hur shuttling and increased par-4 mrna and total protein expression. the role of endogenously generated h2o2 in the regulatory effect of high glucose on par-4 expression was investigated with the nadph oxidase inhibitors apocynin/diphenyliodonium (to prevent h2o2 generation) and cell-permeant catalase (to degrade cellular h2o2). both approaches prevented the stimulatory effect of high glucose on par-4 expression. cyclic amp has been reported to suppress hur activation and in the present study, the cyclic amp stimuli forskolin and cicaprost (prostacyclin analog) suppressed basal hur shuttling and par-4 transcript stability. cicaprost also attenuated high glucose-induced hur binding to par-4 mrna and as a consequence normalised par-4 expression and inflammatory signalling in high glucosetreated cell. conclusion: the regulation of par-4 thrombin receptors in human vascular smc is critically dependent on the mrna stabilising actions of hur. through activation of hur, high glucose and other hur stimuli such as ang ii and exogenous h2o2, increase par-4 expression, while cyclic amp agonists such as prostacyclin oppose this effect. such interactions could potentially represent a fine-tuning mechanism to control par-4 expression and ultimately also the mitogenic and inflammatory actions of thrombin in the vessel wall. nucleoside diphosphate kinase b (ndpk b) is a member of a family of ubiquitously expressed enzymes required for nucleoside triphosphate synthesis. thus, they are involved in the regulation of a variety of cellular processes, e. g. g protein mediated signal transduction. however, whether ndpk b has a specific role in the regulation of angiogenic processes in endothelial cells is unknown. therefore, we studied the function of ndpk b in the vasculature in a developmental, an ischemia-induced and an in vitro model of angiogenesis. firstly, depletion of ndpk b expression was achieved by morpholino-mediated knockdown in zebrafish embryos in which the developing vasculature can be visualized by egfp expression in the endothelium. 72h post fertilization, ndpk b knockdown larvae showed a dramatic inhibition of intersegmental and dorsal longitudinal anastomatic vessel formation compared to control injected fish. this phenotype could be rescued by early re-expression of ndpk b. secondly, ischemia driven angiogenesis was studied in ndpk b-depleted mice and wildtype littermates after excision of the left femoral artery. hind limb blood flow was assessed by laser doppler perfusion imaging immediately before and after ligation (day 0) and on postoperative days 3, 7, 14, 21, 28, and 35 . a significant reduction of recovery was observed in the ndpk b depleted mice at days 3 and 7. thirdly, in vitro-sprouting angiogenesis was analyzed in human umbilical vein endothelial cell (huvec) spheroids with and without sirna-mediated ndpk b knockdown. vascular endothelial growth factor (vegf)induced sprouting was significantly attenuated by ndpk b knock down by more than 50% in comparison with control transfected huvec. we conclude from these results that ndpk b is an essential regulator of angiogenesis. the loss of ndpk b may specifically interfere with the vegf-induced migration and proliferation during endothelial sprouting. ethylene oxide in blood of ethylene-exposed volunteers ethylene (et) is a commercially important high volume industrial chemical. inhaled and endogenous et is metabolized in mammals to ethylene oxide (eo), which is carcinogenic in rats and mice. until now, no data on the oxidation of et in et-exposed humans has been published. in the present study, we investigated the formation of eo in four male adult volunteers exposed for 4 hours to constant atmospheric et concentrations of 5, 20, or 50 ppm by means of a breathing mask. during exposure, et concentrations were measured in inhaled and exhaled air by gc/fid and eo concentrations in venous blood by gc/ms. rates of et metabolism were obtained from the product of the differences in the et concentrations with the pulmonary ventilation. in each subject, linear correlations were found between the et exposure concentration and the rate of et metabolism or the eo concentration in blood. mean rate of et metabolism was 5.5 ± 1.4 nmol/h/ppm/kg body weight. steady-state concentrations of eo in blood differed by a factor of 2 between the volunteers. these inter-individual differences likely reflect the polymorphism of glutathione s-transferase theta, the main eo metabolizing enzyme in human liver. mean eo concentration in blood at steady state was 1.5 ± 0.08 nmol/l blood per ppm of et. the data will be used for validating a physiological toxicokinetic model which will describe the et related eo tissue burdens in rodents and humans. the model predictions will support risk evaluations of et. financially supported by the lower olefins sector group of cefic. in vitro effect of stw11 on human dendritic cells fink c. 1 , bonaterra g. a. extracts of echinacea (purple coneflower) are used in the prevention and therapy of infectious diseases. the medicinal product stw 11 contains the extract from purple coneflower, and in addition, extracts of monkshood, venom of honey bee and bushmaster snake in homeopathic dilutions. previous studies showed a stimulation of the cellular and humoral immune response. dendritic cells (dcs) are antigen presenting cells that act at the interface of the innate and adaptive branches of the immune system. during stages of dc differentiation, the ability to internalize antigens varies and decreases during maturation. in this study, we determined the influence of stw11 on the expression of maturation related genes (cd1a, cd83), cytokines (tnfα, il-4, il-12), chemokines (ccr7), major histocompatibility complex ii(mhc-ii) and toll-like receptors (tlr2, tlr4). in mature (mdc) and immature dc (idc) using real-time rt-pcr. peripheral blood mononuclear cells (pbmcs) were isolated from buffy coats of human volunteers by densitygradient (ficoll ® ) and seeded in 6 well plates. non-adherent cells were eliminated. to induce idc development, 50 ng/ml il-4 and 50 ng/ml granulocyte macrophagecolony stimulating factor (gm-csf) were added. at day 6, maturation was induced by addition of lipopolysaccharide (lps) at a final concentration of 1µg/ml and cultured for additional 3 days. after incubation with different concentrations (0. in idc, compared to control, we found a significantly increased expression of cd83 (2.1-2.6 fold) and tnfα (2.6-3.3-fold) genes after treatment with 0.1-5% stw11, respectively, but no effect was found on the expression of cd1a, il-4, il12, adam19, cd11c, cd40,tlr2, tlr4, mhc-ii and ccr7. in summary, these data demonstrate a stimulatory effect of stw 11 in idc and especially in mdc, concerning an increase of various genes related to maturation (cd83), immunomodulation (tnfα, cd40), adhesion (ccr7) and antigen presentation (mhc-ii) and are in accordance with the therapeutic use in infectious diseases. waterproofing sprays are widely used consumer products containing for example fluorinated polymers or silicon based compounds dissolved in alcohols or volatile petroleum distillates. there have been repeated reports on cases of severe acute respiratory disorders especially when using products that newly entered the market. it is hypothesized that impairment of the pulmonary surfactant by deposition of inhaled respirable particles of the active compound is one of the main causes of the acute lung injury. since the inhalation toxicity cannot be predicted a priori based on the physical and chemical properties of the formulation, proper test strategies are required to ensure consumer safety. we propose to combine screening tests addressing both, exposure and acute lung toxicity. the exposure potential of the spray product is characterized by determining the release fraction of the active compound in the respirable particle size range under conditions relevant for the product application. this is carried out by spraying defined quantities of the product into a control volume and measuring the concentration of health related size fractions. this procedure takes into account spray ageing, especially size reduction of the droplets due to solvent evaporation. the isolated perfused lung is used as a model for testing acute toxicity. ventilated rat lungs are exposed to aged aerosols with proper particle size of approximately 1 µm mmad generated from the liquid spray formulation. lung compliance and lung resistance are continuously monitored during exposure. dose dependent deviations from the normal values (without exposure) are used as read-out parameters. using the combined procedure, different sprays could be ranked according to their realistic exposure risk and, most importantly, sprays with known lung toxicity could be uniquely distinguished from those that have been shown to be safe. in its current stage of development the simple test method is recommended for screening of substances only. induction of oxidative damage in calf thymus dna by the fusarium mycotoxin zearalenone after metabolic activation with liver microsomes fleck s. c., pfeiffer e., metzler m. kit -institute of applied biosciences chair of food chemistry, adenauerring 20a, 76131 karlsruhe, germany zearalenone (zen) is an estrogenic mycotoxin produced by fusarium species. the adverse effects of zen and its reductive metabolite zearalenol (zel) are often compared to those of 17-beta-estradiol (e2) and estrone (e1). these endogenous estrogens are associated with an increased risk for cancer, which may be mediated by two mechanisms, i.e. (i) hormonal activity and (ii) genotoxic effects by p450-catalyzed metabolic activation to catechols (wang et al., chem res toxicol 23, 1365 . like e2 and e1, zen and zel exhibit marked estrogenicity and also undergo aromatic hydroxylation to catechol metabolites (pfeiffer et al., mol nutr food res 53, 1123 . the aim of the present study was to examine the formation of catechol metabolites of zen by liver microsomes of various species and their potential for redox cycling. catechol metabolites are frequently associated with the generation of reactive oxygen species and subsequent oxidative damage of dna, for which 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxo-dg) is a common biomarker. the propensity of the catechol metabolites of zen and zel to cause the formation of 8-oxo-dg in isolated calf thymus dna was determined using a lc-esi-ms/ms method. to this end, zen was incubated with microsomes from human, rat, mouse, bovine and porcine liver as well as with human cyp1a2, and the incubations were extracted with ethyl acetate. the extract was analyzed with lc-ms and then added to a solution of calf thymus dna in the presence of copper(ii) ions and nadph. the formation of 8-oxo-dg could be demonstrated with each extract. the levels of 8-oxo-dg correlated directly with the extent of catechol formation, which increased from steer to swine to human to mouse to rat microsomes. 15-hydroxylated zen/zel, which is the major catechol, was more reactive than 13-hydroxylated zen/zel to form 8-oxo-dg. in conclusion, our study has shown that the catechol metabolites of zen are highly reactive and give rise to oxidative dna damage in vitro. in addition, recent research from our laboratory revealed that the catechols of zen are less efficiently inactivated by catechol-o-methyl transferase than the catechols of e2 and e1. the genotoxic potential of zen may constitute another biological activity in addition to the well-known estrogenicity. supported by deutsche forschungsgemeinschaft (grant me 574/32-1) and "food and health" of kit. thrombin regulates expression of sphingosine kinase-1 (sphk-1) in human vascular smooth muscle cells -inhibition by dabigatran reduces vascular sphk-1 expression and atherosclerotic burden in vivo flößer a. 1 results: thrombin induced a time-and concentration-dependent (1-100 nmol/l) increase in sphk-1 mrna and protein expression in human saphenous vein smc, n=6-7. this was mimicked by a synthetic par-1 ligand. inhibition of sphk-1 attenuated thrombin-induced smc proliferation but not smc migration (n=5). the regulatory action of thrombin on sphk-1 expression was suppressed by sirna against the mrna stabilisiserhur. in thrombin-stimulated smc, hur binding to sphk-1 mrna and subsequent nucleo-cytosolic shuttling was enhanced. accordingly, thrombin induced sphk-1 mrna stabilisation in smc in the presence of actinomycin d. in apoe-deficient mice, long-term treatment with the direct thrombin inhibitor dabigatran significantly reduced aortic sphk-1 expression by 50% (n=5) and plaque size by 35% compared to control animals (n=10). conclusions: thrombin induces sphk-1 expression and s1p synthesis in vascular smc via the mrna stabilising protein hur. this leads to increased smc proliferation. inhibition of thrombin by dabigatran treatment in vivo attenuates progression of plaques possibly by reducing sphk-1 expression. mycotoxin contamination and cytotoxicity of grain mill products typical grain mill products from north-rhine westphalia, i.e. grains, flour, wholemeal flour and bran (from wheat, rye and spelt) as well as typical by-products (outsourced fractions) from the milling process were analysed for their mycotoxin content by lc-ms/ms. the cytotoxicity of sample extracts with known mycotoxin composition was then assessed in v79 cell cultures by means of the neutral red uptake assay, in parallel with pure reference mycotoxin mixtures. extracts from flour and wholemeal flour samples with low levels of deoxynivalenol and enniatin b (from not detectable to 0.4 µg/g don and 0.5 µg/g ennb) induced no measurable cytotoxicity. on the other hand, although mycotoxin contamination levels were also rather low in bran, these samples induced strong cytotoxicity: extracts of bran derived from rye and spelt were more cytotoxic than those of wheat bran. the cytotoxic effects of the bran samples cannot be related to their mycotoxin content as comparable concentrations of pure mycotoxins and mycotoxin combinations tested in parallel were not cytotoxic. by-products from certain stages of the milling process (sorting and waste fractions) were found to contain mycotoxins at rather high levels: enniatin b was detected in nearly all samples, and also t-2 toxin, ht-2 toxin, ergotamine, ergocornin and deoxynivalenol were present. waste sample extracts with notable mycotoxin levels (up to 6 µg/g don, 7 µg/g ennb, 16 µg/g ergotamine, 50 ng/g ht-2 toxin) exert pronounced cytotoxicity in v79 cells. the cytotoxicity of these samples was somewhat stronger than expected when compared with mixtures of reference mycotoxins tested in parallel. in summary, the tested flour and wholemeal flour extracts contained only low levels of mycotoxins and were not cytotoxic. in contrast, bran samples showed cytotoxicity which cannot be explained solely by their mycotoxin content. this unexpected observation in real samples and combination effects of mycotoxin mixtures require further studies. the ahr is a ligand-activated transcription factor that mediates the toxicity of dioxins and related compounds. upon ligand binding the ahr translocates into the nucleus and dimerizes with arnt to modulate gene expression, e.g. of cyp1a1. recently, we have shown that uvb irradiation of human keratinocytes results in activation of the ahr and associated egfr signaling leading to an enhanced expression of cyp1a1 and proinflammatory cox-2, respectively. the initial step is the uvb induced intracellular formation of the tryptophan photoproduct 6-formylindolo [3,2b] carbazole (ficz), a high affinity ahr ligand. thus, the ficz activated ahr is an important mediator of the dna damage independent part of the uvb response. our current study aims to identify further aspects of ahr mediated uvb responses. therefore, we analysed changes in protein expression, proliferation and apoptosis in ahr+/+ and ahr-/-keratinocytes (nctc 2544) by western blot, flow cytometry and brdu incorporation. uvb exposure of nctc cells led to a dose-dependent increase in apoptosis. compared to ahr+/+ cells, ahr-/-cultures exhibited an increased amount of apoptotic cells. this finding was confirmed in irradiated ahr+/+ cells, pretreated with the ahr antagonist 3'methoxy-4'-nitroflavone. moreover, the proliferation of sham as well as uvb irradiated ahr-/-cells was significantly decreased. in ahr-/-cells we found a reduced expression of checkpoint kinase 1 (chk1), an important cell cycle regulator that arrests the cell in g2/m upon dna damage. interestingly, uvb exposure led to a higher net phosphorylation of chk1 in ahr-/-cells, indicating that this pathway is responsible for the observed ahr-dependent differences in proliferation and apoptosis. further expression analyses of chk1 client proteins emphasize our hypothesis. in conclusion our study identifies the ahr as an anti-apoptotic player in uvb irradiated human nctc cells. therefore, we propose that the ahr is a suitable target to prevent uvb induced skin diseases. synthetic progestins exert divergent effects on thrombosis in a murine model of atherothrombosis background: medroxyprogesterone acetate (mpa), a synthetic progestin often used in postmenopausal hormone replacement therapy, has previously been described to be pro-thrombotic in a murine model of accelerated atherosclerosis. however, nothing is so far known about effects of progestins with receptor profiles different from mpa (i.e. agonism or antagonism of mineralocorticoid-or androgen-receptors), such as drospirenone, levonorgestrel and norethisterone acetate. methods: apo -/mice were bilaterally ovariectomized (ovx) and substituted subcutaneously with mpa, drospirenone, levonorgestrel and norethisterone acetate as well as the respective placebo pellets for 90 days on a western-type diet. subsequently, thrombosis was induced by photochemical injury to the right carotid artery using rose bengal and a green light laser. results: compared to placebo, animals substituted with mpa showed significantly shortened times to occlusion of the right carotid artery (placebo mpa: 50.0 ± 5.8 min. vs. mpa: 33.4 ± 4.2 min., n = 6 -9, p < 0.05). in contrast, drospirenone, levonorgestrel or norethisterone acetate did not alter thrombotic responses. however, at least drospirenone (placebo drospirenone: 53.7 ± 5.4 min. vs. drospirenone: 47.5 ± 4.8 min., n = 7 -8) and levonorgestrel (placebo levonorgestrel: 47.0 ± 3.2 min. vs. levonorgestrel: 41.8 ± 2.1 min., n = 6) showed a trend towards shorter times to stable occlusion. furthermore, analysis of aortic gene expression revealed that in aortas of mpa-treated mice expression of matrix-metalloproteinase 9 (mmp-9) was induced as compared to placebo-treated mice. conclusion: mpa, a progestin with glucocorticoid effects, exerts a pro-thrombotic effect that is either progesterone-or glucocorticoidreceptor-dependent while progestins with receptor profiles different from mpa do not show a significant pro-thrombotic effect. furthermore, the pro-thrombotic effect exerted by mpa may be associated with increased expression of mmp-9, a metalloproteinase being known to destabilize atherosclerotic plaques and make them more prone to rupture. rapid screening for mitochondrial toxicity in vitro using an oxygen-sensitive phosphorescent probe freyberger a. bayer healthcare bph gdd ged toxikologie -p & cp, aprather weg 18, 42096 wuppertal, germany impaired mitochondrial function has been implicated with disease, aging, and druginduced toxicities. analyzing mitochondrial respiration (mr) rates is one of the most informative ways to assess mitochondrial function as it provides information on the the bioenergetic capacity of a tissue, however, previously measurements using polarography (clark electrode) were cumbersome with only low throughput. in this work we explored luxcel's water-soluble phosphorescent oxygen-sensitive probe mitoxpress tm for the assessment of mitochondrial toxicity in freshly isolated male rat liver mitochondria (rlm) in a 96-well plate format using glutamate/malate (20 mm/0.5 mm) and succinate (25 mm) as respiratory substrates. inhibition of mitochondrial complexes i to iv, adenosine triphosphate synthetase and the adenosine diphosphate (adp) / adenosine triphosphate (atp) antiporter by rotenone, thenoyltrifluoracetone (ttfa), antimycin a, potassium cyanide, oligomycin, and atractyloside was readily detected in adp-stimulated rlm. use of the two different substrates in parallel allowed to discriminate complex i inhibition by rotenone from complex ii inhibition by ttfa, whereas downstream of these complexes inhibition by the other model inhibitors occurred independent of the substrate used. decoupling of mr from oxidative phosphorylation by carbonylcyanid-p-trifluormethoxyphenylhydrazone (fccp) was detected best in the absence of adp. compared to polarographic measurement, the use of an oxygen-sensitive probe is superior with regard to assay cycle time and sample throughput and offers new opportunities to characterize and screen for mitochondrial toxicity, but also to support studies on mitochondria-mediated modes of action of new chemical entities. the murine local lymph node assay (llna) and the guinea pig maximization test (gpmt) have been used to study the sensitization potential of a series of unsaturated compounds by kreiling et al. (2008) . we have examined the same substances in the loose-fit coculture-based sensitization assay (lcsa), developed by our working group (schreiner et al., 2007) . eight unsaturated compounds [oleic acid (oa), linoleic acid (la), linolenic acid (lna), undecylenic acid (ua), fumaric acid (fa), maleic acid (ma), squalene (sq), 1-octyn-3-ol (oc)] and succinic acid (sua) were investigated using a coculture of keratinocytes and dendritic cell-related cells (dcrc). sensitization potential was quantified by flow cytometry measuring the increase of cd86 expressed on dcrc (ec50 = half maximal effective concentration). a pronounced induction of cd86 at low concentrations was seen with la, lna and oa (ec50: 3, 5 and 5 µmol/l, respectively). ua exhibited an intermediate response (ec50: 307 µmol/l). with oc and ma, we observed effects at higher concentrations only (ec50: 1340 and 2293 µmol/l). no significant increase of cd86 was observed with fa, sua and sq. because of poor solubility, sq could not be studied adequately. induction of cd86 was generally observed at concentrations which did not cause a major impairment of cell viability. our results show a high degree of concordance with those obtained by the gpmt, except for oa. in comparison with the results of the llna, those compounds which showed a strong effect in the llna (oa, la, lna) also induced an increase of cd86 at low concentrations, whereas those with low stimulation indices in the llna induced no significant increase of cd86 (fa, sua) or only at higher concentrations (ua). we observed a discrepancy between the tests with ma and oc, causing a strong stimulation of the murine lymph nodes, while the expression of cd86 was increased at high concentrations only. we assume that ma and oc might be false-positives in the llna, because they were also negative in the gpmt. background: the first step in elimination of many cationic drugs is their uptake from the blood into hepatocytes and renal proximal tubular cells by the organic cation transporter 1 (oct1) and oct2, respectively. the pivotal role of octs in the excretion of cationic drugs raises the possibility of drug-drug interactions in which one drug reduces octmediated elimination of a second drug. although many psychoactive drugs are cationic at ph 7.4 and some of these have already been recognized as oct inhibitors, a systematic screen of this class of compounds is missing. methods: we screened a drug library of 50 most frequently prescribed psychoactive drugs (inpatient prescriptions in germany, at least 4 million ddd each) for their inhibitory interaction with oct1 and oct2. human embryonic kidney (hek) cells stably overexpressing oct1 or oct2 and the prototypical oct substrate 1-methyl-4phenylpyridinium (mpp+) were used as a test system. cells transfected with the empty vector were used as controls. results: at 20 µm, 59% and 51%, respectively, of the tested compounds significantly decreased oct1-and oct2-mediated uptake of mpp+. the most potent inhibitors (inhibition >75%) of oct1 were chlorprothixen and clomipramine, whereas olanzapine, clomipramine and doxepin were the most potent inhibitors of oct2. in contrast, neither at 20 µm nor at 200 µm carbamazepin, haloperidol, lithium, moclobemide and valproic acid did significantly inhibit mpp+ uptake into hek-oct1 or hek-oct2 cells. there was a significant correlation between the degree of oct1 and oct2 inhibition (p conclusions: our results demonstrate that inhibition of oct function by psychoactive drugs has to be considered as a potential mechanism underlying drug-drug interactions. considering estimated peak sinusoidal concentrations e.g., of chlorprothixen and clomipramine between 30 and 80 µm in humans, inhibitory interactions of these compounds with hepatic oct1 have to be taken in account. our data will help to create a chemoinformatic model to predict potential oct-dependent interactions of psychoactive drugs with the hepatic or renal elimination of coadministered drugs. this project is supported by the german federal ministry of education and research (bmbf), project grant no. 01 ex1015b. cardiac gene expression is altered during the development of hypertrophy and heart failure compared to the healthy heart. the molecular mechanisms controlling gene expression in cardiac failure are only partially known. dna methylation is one epigenetic mechanism that regulates long-term changes in gene-expression. to elucidate whether dna methylation is altered during the development and progression of chronic heart failure, genome-wide dna methylation profiles were determined in myocardial biopsies from control patients and patients with cardiac hypertrophy or failure. cardiac biopsies were obtained from patients with aortic aneurysm who served as control and did not show clinical signs of chronic heart disease (ef: 58±3 %, n=3) and from patients with aortic stenosis. the latter group was subdivided according to the ejection fraction into hypertrophic (ef: 63±7 %, n=3) and failing patients (ef: 28±1 %, n=3). after bisulfite conversion of extracted dna, the methylation status of genomic dna was quantified using the infinium® humanmethylation450 beadchip (illumina). this microarray allows analysis of more than 485,000 methylation-sites throughout the whole genome at single-base-pair resolution. these experiments identified 1280 cpg sites in hypertrophic samples and 1365 cpg sites in failing samples which were differentially methylated compared to control specimens (delta >15%; p<0.05). 523 cpg sites were significantly altered in both aortic stenosis groups compared with control hearts. from these cpgs, 496 sites were altered concordantly in hypertrophic and failing samples. analysis of regions harbouring distinct cpg densities revealed that most changes occured in shelf regions of cpg islands whereas the methylation status in the cpg islands was more stable. further analysis showed that differences in methylation were most frequent in gene body, enhancer and 3`utr regions. specifically 9 probes spanning a cpg-island at the promotor region of the muscle-specific serine kinase 1 (srpk3) showed diminished cpg-methylation in hypertrophic (-11.5±0.02%) and failing (-11±0.02%) as compared to control biopsies. remarkably, no alterations of dna-methylation were observed in loci of classic marker genes of chronic heart failure like nppa, serca, ctgf, myh6 or myh7. these results indicate that dna methylation is specifically altered in chronic heart disease but does not affect classic marker genes of chronic heart failure. gliomas are the most abundant type of primary brain tumor in the central nervous system in adults. the current standard of glioblastoma multiforme (gbm) therapy is surgery followed by radiotherapy and chemotherapy. however the morbidity and mortality of gbm remain very high and the median survival period is only 15 months even with treatment. therefore it is important to identify novel drugs to reduce gbm cell proliferation. purine-analogues (pa) are well known for their anti-proliferative effects on eukaryotic cells. in this study novel pa were synthesized and the library of substance-derivatives was tested using different gbm cell lines namely ln18, u87-mg and gl261. the effect on proliferation and viability was assessed by using brdu and resazurin assays. using these in vitro methods we were able to identify several compounds with cytotoxic and anti-proliferative effects in vitro showing ic50 values in the deeper µm range. cytotoxicity of selected compounds was further analyzed by assessment of caspase 3 and propidium iodide based cell cycle facs analysis to discriminate between apoptosis and cell cycle arrest. based on these data purine-derivatives might inhibit proliferation and induce apoptosis in glioma cells. as a result we hypothesize that these compounds could be potentially interesting for the drug-development of gbm therapy and therefore a clue for chemical modifications. further studies are required to identify the exact underlying mechanism of action of the tested purine-analogues. the biological role of adenosine receptors in brown adipose tissue gnad t. 1 brown adipose tissue (bat) is responsible for basal and inducible energy expenditure in mammals. bat contains large amounts of mitochondria and is highly vascularized. bat lipolysis and thermogenesis are stimulated by sympathetic neurons. importantly, recent findings indicate that adult humans possess metabolically active bat 1 . here, we analyzed the expression and function of adenosine receptors in bat. adenosine receptors (ador) are members of the superfamily of g protein-coupled receptors. there are four subtypes of adors in humans referred to as adora1, a2a, a2b and a3. they are widely expressed in tissues and mediate a variety of cellular functions, mostly due to their regulation of camp levels within cells. interestingly, it has been shown that adenosine can either inhibit or stimulate lipolysis in white adipocytes through adora1 or a2a, respectively 2 . however, the role of adenosine in the differentiation of brown preadipocytes to adipocytes and in bat function is not clear. to analyze the role of adors in bat, we use preadipocytes isolated from bat of newborn mice and subjected them to a differentiation protocol. 3 abundance of adora1, a2a, a2b and a3 mrna was measured using qpcr. all four receptor subtypes are present in preadipocytes with adora2b being the most abundant. adora1, adora2a and adora3 are significantly transcriptionally upregulated -albeit at varying degree -during differentiation. adora1 is upregulated 4.4 fold (+/-0.3 fold) and 11 fold (+/-0.49 fold) at day 4 and at day 7, respectively, as compared to preadipocytes (n=5). adora2a is 23 fold (+/-1.96 fold) upregulated at day 4 and 57 fold upregulated (+/-2.48 fold) at day 7, respectively (n=4). adora3 was found upregulated 3.6 fold (+/-0.46 fold) at day 7 (n=4). in contrast to this, ador2b was downregulated to 0.87 fold (+/-0.09 fold) at day 4 and to 0.69 fold (+/-0.04) day 7 compared to preadipocytes (n=5). to investigate the functional role of ador in bati cells, we analyzed lipolysis in mature cells after acute treatment with specific agonists and antagonists. we observed that adora2a activation by cgs21680 significantly increased lipolysis by 88% (+/-0.33%) compared to untreated control. moreover, adora1 antagonist psb36 increased lipolysis by 35% (+/-0.05%) (n=4). in conclusion, ador are highly regulated during brown fat cell differentiation. lipolysis of mature brown fat cells is significantly increased by adora2a agonist or adora1 antagonist, respectively. munich heart alliance, münchen, germany activation of the sympathetic nervous system and the subsequent activation of βadrenergic receptors (βars) through catecholamines represents the strongest mechanism to increase cardiac function. however, long-term activation of cardiac βars is clearly detrimental and β-blockers have been introduced as an effective treatment modality in cardiac failure. despite their central role in cardiac physiology and disease, our knowledge about the intracellular mechanism of βar stimulation is confined to a few targets and is likely incomplete. here, we report a functional proteomics approach to directly assess the entire phosphoproteome of βar-stimulated mouse hearts in vivo. to identify proteins that are phosphorylated in response to β-adrenergic stimulation in vivo, we treated mice with isoproterenol or, as a control, with propranolol. after lysis of hearts and tryptic digest, phosphopeptides were enriched by tio 2 or immobilized metal ion affinity chromatography (imac). subsequent analysis of eluated peptides by tandem mass spectrometry (ms/ms) mapped several phosphopeptides to cardiac proteins, among which known mediators of βar signaling such as phospholamban, troponin i and myosin binding protein c. we then employed multiple reaction monitoring (mrm) as a quantitative approach to assess changes of phosphorylation after βar stimulation. using this combination of ms approaches, we identified 39 peptides with pka consensus phosphosites that were more abundantly detected under βar stimulation. among those, we found myozenin-2 (myoz2) and g protein signaling modulator 1 (gpsm1, also termed ags3) as proteins previously not related to βar signaling. we validated the βar-dependence of phosphorylation at these sites in isolated cardiomyocytes by in vivo labelling or phosphoepitope-specific antibodies. current efforts aim at the functional characterization of these novel candidate mediators of βar signaling in the heart. taken together, we report the β-adrenergic phosphoproteome of the mammalian heart in vivo. we have identified several new targets of βar signaling that may represent essential factors in cardiac physiology and disease. background: drug measurement in autopsy material is normally used to investigate the cause of death. in our study it was possible to measure concentrations of drugs that were part of a regular treatment without connection to the cause of death. metamizole is used as an analgetic and spasmolytic agent. the active metabolite maa (4-methyl-aminoantipyrin) is metabolized by the liver and eliminated by the kidney. hepatic and renal dysfunction can therefore influence maa clearance. methods: maa concentrations were measured in different samples of the autopsy material (heart blood, venous blood, urine, liver, kidney and brain) using an hplc-ms/ms method. information about the dosage and time of drug application as well as information about existing renal or hepatic disorders were taken from the corresponding patient records. because of the low number of cases an explorative single-case study was necessary. results: 10 cases with oral intake of metamizole in a customary continuous dosage could be indentified. the maa distribution into body liquids and organs depended on the time between last oral intake and death. in two cases without renal or hepatic diseases maa blood levels were below 10 µg/ml. five cases with combined renal and hepatic disorders showed either increased blood levels of 40-50 µg/ml or prolonged maa elimination half-life of up to 12 hours. in one case with manifest hepatic insufficiency an maa concentration of more than 200 µg/ml was measured in venous blood. two cases with renal insufficiency alone had maa venous blood levels of less than 10 µg/ml. (pet) . pet detects the positron emission of neutron-deficient radioactive nuclides and allows their external localization in vivo. fet, a modified amino acid, is not incorporated in proteins but accumulates in glioblastomas. one pathway responsible for its accumulation is the preferential transport into the tumor cells, probably via amino acid transporters. we investigated in more detail (a) which individual, cloned amino acid transporters accept fet as substrate and (b) which transporter is responsible for the major fet transport into glioblastoma cells. studies with xenopus laevis oocytes, expressing individual human amino acid transporters, revealed that system l, y + l and b 0+ amino acid transporters recognize fet as substrate (lat1 and 2, y + lat2, and b 0+ at, respectively). in contrast, y + lat1 and atb 0,+ did not transport fet. rna expression studies using qrt/pcr revealed that lat1 is the dominant amino acid transporter in all glioblastoma cells investigated (ln229/u373/u87mg/u251/a172/t98g). a strong lat1 expression was also shown on the protein level. to find out whether lat1 is the main transporter responsible for fet accumulation, we first studied transport of the parent amino acid l-tyrosine in ln229 glioblastoma cells. [ 3 h] tyr uptake was completely na + -independent and inhibited by leu, phe and trp, but not by arg, pro or ser. sirna-mediated down-regulation of lat1 in ln229 cells led to a concomitant decrease of lat1 mrna and tyr transport (down to 3% and 20%, respectively). these results indicate that tyr is exclusively transported by lat1 in ln229 cells. we are currently performing transport studies using [ 18 f]fet to investigate whether fet transport is also exclusively mediated by lat1 in glioblastoma cells. a further question is if lat1, a sodium-independent transporter, can be responsible for the accumulation of fet observed in glioblastoma cells. if true, other amino acid derivatives that are lat1 substrates might also proof useful in cancer diagnosis. telmisartan reduces adipose tissue inflammation and biglycan accumulation in diabetogenic ldl-receptor knockout mice grandoch m., nagy n., fischer j. w. institut für pharmakologie und klinische pharmakologie, universitätsklinikum der heinrich-heine-universität düsseldorf, moorenstraße 5, 40225 düsseldorf, germany in addition to lowering blood pressure some of the angiotensin ii at1 receptor antagonists (arb) such as telmisartan have additional beneficial effects on the onset of type 2 diabetes mellitus and obesity. this was contributed mainly to peroxisome proliferator activated receptor (ppar)γ modulating activity. hyaluronan (ha), a high molecular weight polysaccharide and the small leucine rich proteoglycans, decorin and biglycan, are known to be involved in atheroprogression. mechanistically these matrix components contribute to inflammatory processes via toll-like receptor-signalling and are supposed to modulate lipid retention. the aim of this study was to elucidate the effects of telmisartan in comparison to valsartan, an arb without pparγ activity, on extracellular matrix remodelling and inflammation in atherosclerosis and the interrelationship with adipose tissue inflammation using the ldlr-/-model of accelerated atherosclerosis. male ldlr-/-mice were fed either a diabetogenic diet alone or in combination with telmisartan (10 mg/kg), valsartan (25 mg/kg) or valsartan (50 mg/kg) from 8 weeks of age for 17 weeks. all treatment groups except of the lower valsartan dose showed significant effects on reducing the aortic plaque score. the content of ha, collagen and decorin in the aortic root were not changed. however, telmisartan reduced the content of biglycan in the aortic root significantly in contrast to valsartan. in addition, a trend towards decreased mac2-positive macrophages in abdominal adipose tissue was detectable after telmisartan treatment as well as a strong reduction in the adipose tissue mrna expression of biglycan. finally, telmisartan reduced the expression of hyaluronan catabolizing enzymes potentially leading to an increase of high molecular weight ha in the adipose tissue, which is thought to be homeostatic and antiinflammatory. in summary, the results of this study underline the pronounced anti-inflammatory capacity of telmisartan on atherosclerosis and adipose tissue inflammation in comparison to valsartan and strongly suggest that biglycan might be an additional target of telmisartan not only concerning matrix composition of atherosclerotic lesions but also concerning the structure of adipose tissue and metabolic effects of the compound. human primary malignant cancer cells derived from peritoneal effusions of a patient with colorectal carcinoma, as assessed by comet assay. the primary cancer cells were more efficient in dsb repair than ht-29 cells, and their doxorubicin ic50 was four times higher. comparative protein expression levels showed that the primary cells had less rad 51 and 52 as well as less topoiiα, while ku70 and 80 levels were similar. another very interesting protein is the mrn (mre11-rad50-nbs1) complex that initializes the phosphorylation of atm and thereby starts the signalling cascade. the newly described mrn-atm pathway inhibitor mirin interrupts mrn activity by inhibiting the exonuclease activity of mre11. the toxicity of mirin in ht-29 cells was measured using a luminescence-based assay detecting the amount of atp, which is correlated with cellular viability. mirin did not show any toxic effects up to a concentration of 100 µm and incubation times of 24 hours, indicating that mirin can be used under these conditions without detrimental effects. we are currently investigating the effect of mirin on the toxicity of topoiiα inhibitors. the inhibition of dna repair may be a valuable strategy to enhance the effect of dnadamaging anticancer drugs. since tumours (even of the same entity) are not only heterogeneous, but also polyclonal, a broad selection of response modifiers of anticancer drugs would be helpful to individually enhance chemotherapeutic effectiveness. evidence has been provided that diet and environmental factors directly influence epigenetic mechanisms associated with cancer development in humans. epigenetics play an important role in the control of gene expression. epigenetic mechanisms comprise modulation in dna methylation, histone modification and non-coding rna. several polyphenols have been reported to possess histondeacetylase (hdac) inhibitory properties [1] . histone deacetylation is generally linked to transcription repression. furthermore, hdac belongs to the group of small ubiquitin-related modifier (sumo) substrate proteins. sumoylation of hdac is associated with a modulation of its biological activity [2] . little is known so far about the mechanism by which hdac sumoylation mediates inhibition of gene transcription. we addressed the question whether sumo e1 and hdac 1 expression and whether potential hdac-sumoylation will be affected by polyphenols such as chlorogenic acid, genistein and (-)epigallocatechin-3-gallate (egcg). chlorogenic acid, genistein and egcg decreased sumo e1 protein level in the human colon carcinoma cell line ht29 after 24h of incubation measured with western blot analysis. egcg exhibited the most pronounced effect at concentrations ≥ 50 µm. hdac 1 expression was also affected by these polyphenols. the direct impact of polyphenols on the hdac sumoylation is detected by co-immunoprecipitation experiments with the respective antibodies against hdac-1 and sumo e1. these experiments are still under investigation. in conclusion, chlorogenic acid, genistein and (-)-epigallocatechin-3-gallate influenced the sumo and hdac expression in vitro. in further studies the direct impact on subtract-sumoylation will be investigated. these studies contribute to a better understanding of potential chemopreventive effects of dietary polyphenols on specific epigenetic alterations may provide chemopreventive strategies for reducing cancer risk. the no/cgmp cascade is thought to be essential for penile erection. within the smooth muscle of corpus cavernosum, nitric oxide activates the no-sensitive guanylyl cyclase (no-gc) which raises the intracellular concentration of cgmp. this second messenger activates the cgmp-dependent protein kinase i (pkgi) and subsequent phosphorylation of target proteins leads to relaxation of cavernosal smooth muscle. knock out of key enzymes of the no/cgmp cascade has led to discrepant results: the deletion of pkgi in the mouse has been shown to lead to erectile dysfunction whereas mice lacking neuronal no synthase are fertile. to investigate the role of the no receptor in fertility we have generated mice lacking no-gc (gcko), a bottleneck enzyme of the no/cgmp cascade. we have shown that lack of no-gc resulted in arterial hypertension concomitant with a totally abolished no responsiveness of vascular and gastrointestinal smooth muscle. in addition, we generated a mouse line in which no-gc was specifically deleted in smooth muscle cells (sm-gcko). using these ko strains we here examined the role of no/cgmp signaling with regards to the smooth muscle relaxation of corpus cavernosum. no failed to affect corpus cavernosum from gcko in organ bath experiments: neither exogenously produced no by no donors nor endogenous no release from neurons induced by electrical field stimulation led to relaxation. similar results were observed in the corpus cavernosum of sm-gcko mice. to our surprise, the gcko animals were fertile and produced offspring albeit at a reduced rate compared to wt animals. our data show that interruption of no/cgmp signaling results in complete absence of no-induced relaxation of penile corpus cavernosum in mice and reduces the ability to produce offspring but does not abolish fertility. novel modes of invasive cell motility regulated by the formin class of actin nucleators khan j., grosse r. philipps-universität marburg, pharmakologisches institut, karl-von-frisch-str. 1, 35034 marburg, germany pathological invasive cell migration essentially reqires actin polymerization. formins are the largest group of rho-gtpase effectors involved in actin nucleation and assembly as well as microtubule dynamics. here we studied the role of formins in cytoskeletal regulation during homotypic cancer cell invasion. we identified the actin-dependent steps and structures involved for this process. using live cell analysis we characterize the distinct actin dynamics controlled by formin-like 2 and rho function. the specific involvement of this signaling module will be discussed. formin-driven nuclear actin assembly controls mal/srf activity baarlink c., wang h. polymerization of actin in the cytoplasm is tightly linked to transcriptional activation of the srf cofactor mal (also known as mrtf-a) through release of actin/mal interactions and subsequent nuclear accumulation of mal. formins directly promote assembly of actin filaments thereby efficiently regulating mal-dependent transcription for cell shape, adhesion and motility. here we show that formins assemble f-actin and promote mal activation inside the mammalian nucleus. the rho-gtpase effector mdia2 rapidly enters the nucleus in a signal-dependent fashion and an active mdia confined to the nucleus potently promotes release of g-actin from mal to specifically activate srf. live cell imaging reveals formin-mediated nuclear actin dynamics. moreover, using actin assembly assays we find that inhibition of endogenous mdia formins controls f-actin turnover in isolated nuclear extracts. thus, formin activity is dynamically compartmentalized to the mammalian nucleus to potently regulate actindependent mrtf function. in women the placenta becomes the main source of maternal estrogens during pregnancy. placental estrogen biosynthesis is located in the syncytiotrophoblast, a syncytium that builds the main part of the placental barrier and limits the transfer of substances between the fetal and maternal compartment. since the human placenta is unable to convert cholesterol into 17-oh-pregnenolone, the placenta tissue highly depends on the supply of c-19 steroids for their conversion into c-18 estrogens. in contrast to lipophilic unconjugated steroids that penetrate the cell membrane passively via diffusion, circulating sulfated steroid hormones are delivered to the placenta via carrier-mediated transport, followed by their reactivation via the catalytic activity of the steroid sulfatase (sts). dheas of maternal and fetal origin contributes about equally to the placental formation of estrone (e 1) and estradiol (e2), while 16αoh-dheas supplied by the fetus contributes to over 90% of placental estriol (e3) synthesis. soat, a member of the slc10 family with highest expression in hormone-responsive tissues such as testis, placenta, and mammary gland has been shown to transport the sulfoconjugated steroid hormones dehydroepiandrosterone sulfate (dheas), estrone sulfate (e1s), and pregnenolone sulfate (pregs) [1] . aim of this project is to investigate the role of soat for placental estrogen synthesis by means of the choriocarcinoma cell line jeg-3 as in vitro model for the human syncytiotrophoblast. therefore, we characterized a jeg-3 cell line that transformed dhea into e2 and 16αoh-dhea into e3. by qrt-pcr we found expression of sts and aromatase, both essential for estrogen synthesis in these cells. upon transient transfection of soat the carrier was located in the cell membrane of transfected jeg-3 cells. currently we investigate the transformation of dheas of these soat-jeg-3 cells by lc-ms-ms. we could demonstrate transport of 16αoh-dheas for stably transfected soat-hek293 cells. in situ hybridization and immunohistochemistry showed coloured syncytiotrophoblasts and vascular endothelial cells in late term placenta. in conclusion, soat-mediated transport of sulfated steroids could play a pivotal role for placental estrogen synthesis from sulfated steroid hormones. developing non-animal test systems for evaluation of toxicity was important in the past and will remain essential in the future. here we present a toxicity test using the chicken yolk sac area vasculosa (cav) of fertilized white leghorn chicken eggs [1, 2] and compare it to hen's egg test on chorioallantoic membrane (het-cam) [3] for polymer toxicity testing. fertilized chicken eggs were incubated and after 72 h explanted shell less into sterile petri dishes. test substances were applied on the cav and the appearance of different effects (vascular lysis, haemorrhage, aggregation of blood components, lethality) was determined by light microscopy after 1 -48 h (fig.1 ). these effects were combined to a cav test score based on the irritation score calculation used for het-cam evaluation. different polymers like poly(ethylene glycol) (peg; neutral), poly(ethylene imine) (pei; cationic) and dextran sulphate (ds; anionic), as well as guideline-conform (recommended het-cam protocol from the interagency coordination committee on the validation of alternative methods) negative (0.9 % nacl) and positive controls (1 % sodium dodecyl sulphate (sds) and 0.1 n naoh) were investigated. additionally ld 50 values for different cationic polymers have been determined. within the selected incubation times (1 -48 h) , effects such as vessel lysis and blood component aggregation could be detected. additionally to het-cam, lethality as well as recovery of the cav could be observed. differences between neutral, positively and negatively charged polymers were obtained. pei showed strong vessel lysis and aggregation of blood components whereas ds and peg showed none of these effects. lethality was found to increase from peg < ds < pei and is concentration and time dependent. the results demonstrate that differences, regarding the toxicity of the used polymers, can be shown with this test. these findings in the cav test can be well correlated with already existing data. in summary, the cav test provides same data (testing control substances) and more information (recovery and lethality) compared to het-cam and could be a suitable model for toxicity testing of polymers. risk characterisation of chemicals consists of three steps (1) hazard identification and characterisation, based on substance-specific toxicological hazard data, (2) estimates of the level of exposure toward the substance and (3) the comparison between the toxicologically safe level and the exposure level. in contrast to the classical risk assessment approach, the threshold of toxicological concern (ttc) approach is developed as a tool to assess the risk of substances without toxicity data. its application requires (1) information on human exposure, for which it is essential that exposure is fully captured and (2) knowledge of the chemical structure to assess whether the chemical is not excluded from the application of the ttc concept. instead of chemical specific no observed (adverse) effect levels (noels/noaels), the ttc approach utilises knowledge on the empirical distribution of several hundreds of noels/noaels, originally 613, based on toxicological testing in animals (munroe et al., 1996) . with the basis on noaels, the ttc concept builds on the fundamental principle of toxicology, that toxicity is a function of dose and that a dose exists, below which no adverse effects of the substance can be detected. it is assumed that exposures below this level will not result in health risks. three separate ttcs were derived (munroe et al., 1996) by classifying the chemicals into three toxicity classes using a decision tree based on a series of 33 questions related to chemical structure, and on natural occurrence in food and in the body (cramer et al., 1978) . the ttc values are derived from empirical distribution of the noels/noaels in the class taking the 95th percentiles and dividing them by the default uncertainty factor of 100. it is assumed that the probability is very low that the unknown noael of a not tested chemical will be lower than the value of the 95th percentile in the distribution of the known noels/noaels. hence, at exposures below the ttc values, the probability of adverse effects on human health is considered to be very low. introduction: kibra, mainly expressed in kidney and brain tissue, is involved in brain development and memory formation as a postsynaptic scaffold protein. in podocytes, kibra is proposed to regulate cell motility as a linker between components of the cytoskeleton and polarity protein complexes (duning et al, jasn 2008) . furthermore, kibra has been identified as key regulator of the hippo pathway, which is involved in organ size control and tumorigenesis. in the current study, we focused on the identification of kibra gene expression regulation and functional promoter characterization. methods: serial promoter deletion constructs were generated by cloning 3627 bp of the 5'flanking region of kibra into the pgl3-vector system. deletion constructs were transiently transfected into human neuroblastoma cells (sh-sy5y) and immortalized human kidney epithelial (ihke) cells. potential transcription factors (tfs) were investigated in cotransfection experiments. transcriptional start sites (tss) were determined by rapid amplification of 5'cdna ends (5'race) . tss utilization between cell lines was assessed by semiquantitative pcr. transcriptional activity (ta) of the kibra promoter p1 was separated by ~1060 bp into two distinct regions, promoter p1a and p1b. deletion constructs harbouring promoter p1b were transcriptionally active only in ihke cells. 5'race revealed two alternative tss in both cell lines upstream of the annotated tss (nm_015238). exclusively in ihke cells, two additional tss were detected in intron 1, resulting in two alternative exons. deletion constructs harbouring the putative regulatory regions (p2 and p3) of both exons were transcriptionally active only in ihke cells. overexpression of full length tcf7l2 (transcription factor 7-like 2 [t-cell specific, hmgbox]) resulted in a ~3-fold increase of promoter p1a and intron promoter p2 ta. kibra gene expression is driven by a complex alternative promoter system comprising the constitutional promoter p1 and three alternative promoters p1b, p2 and p3. the tss utilization is cell type-specific. subsequent usage of an alternative translation start site within exon 3 could result in truncated kibra protein isoforms. tcf7l2 is involved in the differential kibra gene expression regulation. resulting kibra protein isoform and their cellular function will be assessed in further studies. skin absorption in vitro based on the study of human/animal skin ex vivo or reconstructed human epidermis, respectively, is an alternative method which is accepted by the oecd. guideline tg 428 and a corresponding technical guidance document (gd 28) give technical guidance how to perform valid experiments 1, 2 . the requirements include integrity evaluation tests for the skin samples. different tests are proposed to ensure an exclusively use of undamaged skin. to decide which test suites best to our routine test strategy, we investigated the correlation between integrity test results and absorption profiles of various penetrants (logp range: -0.07 -6.2). finite dose experiments using rat and human skin were performed with 14 c-labeled testosterone, caffeine, mcpa (4-chloro-2-methylphenoxyacetic acid) and its 2-ethylhexyl-ester mcpa-2ehe. for each experiment at least three of the five following integrity tests were conducted: transepidermal electrical resistance (teer), transepidermal water loss (tewl), transepidermal tritiated water flux (³h2o), transepidermal absorption of methylene blue (blue) , transepidermal absorption and flux of a ³h-labeled internal standard (istd); ³h-testosterone or ³h-mannitol was used as istd. the applied radioactivity of the ³h-istd was selected to show no analytical interference with the 14 c-penetrants. teer, tewl and ³h2o represent pre-study, istd concurrent and blue post-study tests. calculated maximal permeability constants (kp) and absorbed doses (ad) of the penetrants were compared to the results of the integrity tests. individual linear regression analysis was used to evaluate the correlation the correlations varied over a wide range for all five methods and four penetrants. the best correlations in average were achieved with the istd. no inverse correlations were obtained for the istd, but partly for tewl, teer, ³h2o and blue. in conclusion, the istd represents best the achieved absorption profiles of the test compounds and is based on that the most suitable integrity test for our dermal absorption studies. we will further confirm its effectiveness and generate a sufficient historical database in order to include the istd in our routine test protocol. investigation of mirna expression and dna methylation in focal and non-focal brain tissue of therapy-resistant epilepsy patients haenisch s. 1 background: resistance to anticonvulsants affects one third of all epilepsy patients. limited bioavailability of the drug at the target site caused by increased expression of efflux transporters on the blood brain barrier or alterations of target genes are potential mechanisms for therapy resistance. however, these mechanisms alone cannot completely explain the observed resistance and it is likely that multifactorial alterations lead to pharmacoresistance. there is increasing evidence that expression of micrornas probably caused by dna modifications is deregulated in many neuronal diseases. we hypothesize that mirna regulation of target genes is involved in drug resistance in epilepsy. methods: hippocampal focal and cortical non-focal brain tissue samples from 13 patients diagnosed with mts (mesial temporal sclerosis) who underwent neurosurgery have been screened for mirna expression using taqman low density arrays. in silico approaches for both a hypothesis-based (efflux-transporter and target gene) as well as a hypothesis-free approach were used to identify potential phenotype-relevant target genes. using the program r (bioconductor) a mann-whitney-u test was performed to compare mirna expression between brain regions. pyrosequencing was performed to investigate methylation status 5'-upstream of dna regions encoding for selected candidate mirnas. results: out of 754 mirnas, 150 were detected in both tissue types. the expression of one mirna was 7.2 fold higher (q=0.01) and another was 3.8 fold lower (q=0.01) in the hippocampus relative to the cortex. evidence could be found that down-regulation of the latter is possibly caused by hypermethylation of 5'-flanking region of its encoding dna locus. bioinformatic analysis has identified eight genes important for neuronal regulation and signal transmission (e.g. sox11, mecp2, bsn), as well as one abc effluxtransporter, as potential targets for these differentially regulated mirnas. conclusion: differential regulation of two mirnas could contribute to an altered function of several genes resulting in an imbalance between neuronal excitation and inhibition that is independent from mechanisms presently targeted by anticonvulsants. this work was supported by a fellowship from dfg and nih grant gm61390. recently, it has been reported that human b cells express and secrete the cytotoxic protease granzyme b (grb) after the combined stimulation of the il-21-and the b cell receptors. grb produced by b cells is enzymatically active and b cells deliver grb to sensitive cancer cell lines, thereby inducing apoptosis. to date, there is little experimental evidence on the mechanisms involved in grb expression, or its function in b cell biology. as experimental transgenic murine systems should enable us insights into these issues, we assayed for grb in c57bl/6 b cells using an extensive array of physiologically relevant stimuli, but were unable to detect either grb expression or its proteolytic activity, even when antigen specific transgenic b cell receptors were cross-linked. similar results were also obtained with b cells from dba/2, cba or balb/c mice. in vivo, infection with either influenza virus or murine γ-herpesvirus induced the expected expression of grb in cytotoxic t lymphocytes, but not in b cell populations. we also investigated a possible role of grb on the humoral immune response to np-klh, but grb-deficient mice produced normal amounts of antibody with typical affinity maturation and heightened secondary response, demonstrating conclusively the redundancy of grb for antibody responses. our results highlight the complex evolutionary differences that have shaped the immune systems of mice and humans and demonstrate the need to develop novel in vivo systems to study human humoral immune responses. investigations of the cholinergic neurotransmitter system in dyt1 mice hamann m. 1 early-onset torsion dystonia is an autosomal dominant inherited movement disorder associated with the dyt1 gene defect with deletion of a glutamic acid residue in the protein torsina. despite the gene defect, the pathophysiology is poorly understood. animal models can help to understand the underlying mechanisms and thereby to develop new therapeutic strategies. sharma et al. (2005, j. neurosci. 25 [22] , 5351-5355) initially described a transgenic mouse model (dyt1 mice) with overexpression of mutant torsina. previous studies in these mice pointed to alterations in the cholinergic system. to investigate the functional relevance of these in-vitro findings, we carried out pharmacological in-vivo experiments and determined the density of striatal cholinergic interneurons as well as the expression of choline acetyltransferase in different brain regions. the acute intraperitoneal administration of the cholinomimetic drug pilocarpine (75, 100 and 125 mg/kg) as well as a long-term treatment over 21 days (100 mg/kg/d) did not induce pronounced effects in dyt1 mice compared to wildtype controls. the higher incidence of epileptic seizures in dyt1 mice compared to controls after repeated local striatal applications of pilocarpine (25 and 50 µg/0.5 µl/hemisphere) let presume an altered synaptic plasticity in dyt1 mice. the immunohistochemical investigations revealed a moderately reduced density of striatal cholinergic interneurons in the dorsomedial subregion of dyt1 mice compared to wildtype controls, while significant differences in other striatal subregions were not detected. western blot analysis did not show clear differences in the expression of choline acetyltransferase between dyt1 and wildtype control mice. these results indicate that the cholinergic system seems not to play a key role in this line of dyt1 mice. ongoing receptor autoradiographic analysis of binding to different muscarinic receptors subtypes have to further clarify the existence of possible alterations within the cholinergic system of these dyt1 mice. inhibitors direct against cell cycle-regulatory kinases are being tested in clinical trials as anti-proliferative agents. thus, the atp-competitive kinase inhibitor pd332991 which inhibits cdk4 and cdk6 is currently tested in patients with solid tumors such as glioma. we found that pd332991 suppressed il-1-induced expression of il-8 suggesting that cdk4 or cdk6 may have unknown anti-inflammatory properties. to study the effects of cdk6 on the il-1-signaling network, we established a bidirectional doxycyline-inducible system to express a constitutively active mutant of cdk6, cdk6 s178p, in asynchronized hela cells. cdk6-expressing cells were identified by gfp which was expressed from the same promoter, isolated by laser-microdissection and analysed for mrna expression using a down-scaled rt-qpcr assay. compared to the uninduced state, cdk6 s178p enhanced il-1-induced il-8 and il-6 mrna expression. moreover, shrna-mediated suppression of endogenous cdk6 confirmed a role of this kinase in regulation of maximal il-1-induced gene expression of il-8. these data also revealed that the contribution of cdk6 to inflammatory gene expression is highest in g1, when activity of endogenous cdk6 is activated by d-type cyclins. these findings were corroborated in hela cells expressing fluorescent ubiquitin-dependent cell cycle indicator (fucci) proteins. hela-fucci cells from g1, g1/s, g2 or mitotic states were isolated by laser-microdissection and analyzed by rt-qpcr for tnf-inducible gene expression. stable knockdown of cdk6 in hela fucci or inhibition by pd332991 suppressed inducible il-8 expression. microarray experiments identified many additional genes that required active cdk6 for maximal il-1-or tnf-inducible gene expression. we also found that cdk6 co-immunoprecipitated with p65 nf-κb, colocalized with p65 in the nucleus and was recruited together with the p65 subunit to the proximal il-8 promoter as assessed by chip and re-chip experiments. collectively, these results suggest an unexpected control of inflammatory gene expression through a classical cell cycle regulatory pathway. these results also imply that pharmacological targeting of cdks may have effects and side-effects on the immune system in addition to inhibition of cell cycle progression. trp channels form a heterogeneous family of calcium-permeable channels, which play major roles in physiological functions ranging from sensory reception to cellular signal transduction. members of the trpc subfamily (classic transient receptor potential channels) are downstream targets of hormone receptors. of particular interest is the biological role of trpc6 channels. they are directly activated by diacylglycerol due to phospholipase c-driven signalling pathways which are involved in smooth muscle contractility, neuronal plasticity, keratinocyte differentiation and renal function. their impact in renal function became evident from analyzing patients suffering from familial forms of focal segmental glomerolusclerosis (fsgs) which could be linked to trpc6 mutations. since the first descriptions at least 17 different pathogenic mutations have been identified in humans to cause fsgs. in order to study the underlying pathophysiological mechanisms of trpc6 mutations, we have analysed all mutated trpc6 channels known to date heterologously expressed cells. one set of mutations showed a gain-of-function phenotype which has been previously suggested to cause an increased intracellular calcium load and subsequent cell death. hence, gain of function mutations fit to the current paradigm of fsgs pathophysiology. however, another set of mutations found in the patients showed a loss-of-function phenotype. our results enable a change in the current paradigm for the role of trpc6 in renal pathophysiology and may provide a basis for our understanding of the pathophysiology of loss-of-function mutations in familial focal segmental glomerolusclerosis. karlsruher institut für technologie (kit) institut für angewandte biowissenschaften, abteilung lebensmittelchemie und toxikologie, adenauerring 20a, 76131 karlsruhe, germany risk assessment for genotoxic carcinogens is an important challenge in toxicology. even though manifold attempts have been made to substitute carcinogens and to reduce exposures, their complete elimination appears to be not possible. thus, low concentrations of known or suspected genotoxic carcinogens are present at workplaces, in the environment and in food. in order to deal with this situation and to set priorities for risk management, different concepts have been established such as the alara principle (as low as reasonably achievable) and the margin of exposure (moe), based on the ratio between concentrations being carcinogenic in experimental animals and the actual exposure of humans for example via foodstuff. while usually linear doseresponse-relationships have been used as default assumption, analytical methods are now available to assess the induction and repair of dna lesions on low exposure conditions, including environmental background exposure, and to relate the extent of exposure-induced dna lesions to endogenous dna damage. this may be an important prerequisite to establish health-based limit values for selected genotoxic carcinogens. within this workshop, different examples will be discussed and research need will be identified. dendritic cells from h4r-deficient mice lose their ability to properly stimulate t lymphocytes hartwig c., seifert r., neumann d. mhh pharmakologie, carl-neuberg str. 1, 30625 hannover, germany the incidence of allergic airway diseases is increasing throughout the world, especially in western countries. although histamine (ha) is found at high concentrations in asthmatic lungs, a role for ha in bronchial asthma is still a neglected topic in clinical research. in particular, the capacity of ha to modulate the underlying immune reaction is far from being understood. the histamine h4-receptor (h4r) is involved in acute inflammation and th2 cytokine production. consequently, we intended to analyze the role of h4r in a murine th2 lymphocyte transfer-based model of asthma. specifically the ability of h4r expressed on dendritic cells (dcs) to modulate t cell function was analyzed. ova-specific cd4 + t cells were polarized in vitro under th2-favoring conditions with ova peptide-pulsed dcs, obtained either from wild-type or h4r -/mice. analysis of the polarized t cells after in vitro restimulation revealed a marked decrease of il-4 production in t cells polarized in the presence of h4r -/-dcs compared to those polarized in the presence of wild-type dcs. thus, on dcs, the h4r is essential for proper stimulation of spleen t cells and for directing their polarization towards a th2 phenotype. the transfer of in vitro polarized t cells into recipient mice and subsequent provocation elicited an asthma-like disease. the h4r on dcs not only affects in vitro polarization of t cells, but also the in vivo function of the obtained polarized t cells. a parameter indicating allergic inflammation is the enhanced influx of inflammatory immune cells into the lung tissue, mainly driven by eosinophils, which are virtually absent in non-asthmatics. when analyzing the number of eosinophils, a dramatic difference due to the polarizing conditions of t cells occurs. in bal fluids of mice that received t cells polarized in the presence of wild-type dcs, about 40% eosinophils were detected. in contrast, the transfer of t cells polarized in the presence of h4r -/-dcs yielded only about 10-20% eosinophils in bal fluids. in summery, the h4r on dcs plays an important role for t cell polarization and consequently affects the allergic reaction during sensitization. since the lack of the h4r on dcs reduced their ability to stimulate proper th2 polarization of cd4 + t cells, we conclude that ha via the h4r significantly affects the manifestation of asthmatic inflammation. antioxidant polyphenols and their effects on nrf2 (skn-1) signalling in a cell culture system and the model organism c. elegans havermann s., wätjen w. heinrich-heine-universität düsseldorf institut für toxikologie, p.o. box 101007, 40225 düsseldorf, germany oxidative stress has been connected with a variety of diseases, (e.g. alzheimer`s and parkinson´s disease), cancer and ageing over the last years. certain polyphenols were shown to have an antioxidant capacity as well as being able to activate the protective nrf2 signalling pathway. compared to direct radical scavengers modulators have the advantage of building up a permanent defense against oxidative insults whereas scavengers do not protect any more after consumption or may even cause stress due to redox cycling. we have employed cell culture based assays (dcf, western blot, gfp reporters) to analyse the effects of polyphenols. further we tested the coumpounds in vivo in the nematode c. elegans where skn-1 is the nrf2 homologue. baicalein and caffeic acid phenethylester (cape) protected cells and the nematode from ros accumulation after application of stress (shown by dcf assay). activation of nrf2 signalling is correlated with translocation of the transcription factor into the nucleus. in both systems nrf2::gfp accumulation in the nuclei could be observed after incubation with baicalein (fluorescence microscopy). but while cape is a potent activator of nrf2 in cells, it has no effect on skn-1 localisation. further the effect on the nrf2 protein amount was investigated by western blot analysis. the expression of target genes can be investigated by differing means: while pcr methods and western blotting are standard for in vitro studies, the vast number of available gfp reporter strains offers opportunities for research using c. elegans. we have performed congruent assays in a cell culture system and the model organism c. elegans to compare antioxidative capacity and effects of polyphenols on nrf2 signalling. therefore, depending on the substance tested, c. elegans is a suitable model system to investigate effects of natural compounds in an organism. being associated with adverse health effects, the human exposure to dehp is subject to concern. quantifying the population's exposure and determining the contributions of different exposure routes is a key task of environmental health risk assessment. the study presented comprises a review of the available data on dehp levels in foods, consumer products, and house dust. extensive survey data, e.g. from the current national nutrition survey ii and the eu rapex system were processed for modeling the exposure by the oral, inhalative and dermal path of the population in germany. the study also included analytical analyses of dehp levels in selected foods and consumer goods (incl. migration rates for mouthing). probabilistic techniques allowed elucidating the exposure's variation and the relevance of different routes. mean exposure estimates for german children and adults to dehp are 36 and 26 µg/(kg d), resp. for children, food accounts for 36% of the total exposure, followed by mouthing (30%) and house dust (19%). the adult exposure is almost entirely (82%) due to food. as dietary exposure is a result from concentration and consumption, foods exhibiting high contamination e.g. butter (8%) and dressings (mayonnaise) (14 %) as well as highly consumed foods e.g. bread and bakery (16%) and vegetables (6,8 %) contributed significantly. the mean estimate of children's dehp exposure via mouthing revealed 0,9 µg/(kg d). high exposures were estimated (95th percentile) up to 10,8 µg/(kg d). on average people in germany are exposed to dehp below the current tdi of 50 µg/(kg d). however, individual exposures exceeding the tdi still cannot be excluded. current data on dehp and other plasticizers in foods are scarce, which warrants broader monitoring. our findings highly facilitate further exposure modeling focusing on dehp substitutes and risks of combined exposure. this study was funded by the federal ministry for the environment, nature conservation and nuclear safety in the frame of the environmental research plan (umweltforschungsplan, förderkennzeichen (ufoplan) 3707 61 201). on the basis of the available measurements of dehp, the exposure assessment has been focused on 37 food categories characterising a selection of the most important food groups covering all major food classes of the german population. based on an extensive literature survey, the analysis considered the available data of dehp measurements in food, as well as the official german food control data taken from the national food consumption survey. the high amount of considered data allowed the consideration of several exposure assessment tiers (deterministic and probabilistic by using monte carlo simulation). a quantitative evaluation of the uncertainties of the estimate of the 37 food categories groups has been performed by means of a sensitivity analysis by using the methodology proposed within the 2008 who ipcs guidance document of characterising and communication uncertainty in exposure analysis. qualitative uncertainty analysis (tier 1) was applied to determine the most important sources of uncertainty, i.e. concentration of dehp in all food categories. the probabilistic monte carlo simulation (tier 2) was then used to rank the cumulative probability distributions of the exposure assessments of 37 food categories on the basis of the food categories that appear to dominate. sensitivity analyses were applied to prove the impact correlation of food groups for uncertainties. by a scenario based concept, the aggregation of the 37 food groups to 10 groups has been evaluated, as well as the sensitivities by characterising particular scenarios. for this purpose, particular "meals" have been described as fixed combined scenarios and. the aggregation leads to a considerable higher exposure estimate which can be explained by the combination of high contaminated foods with others of high consumption. the evaluation confirms the considerable role of possibly high contaminated foods e.g. fats, or mayonnaise. the evaluation shows that quantitative probabilistic sensitivity analysis is a suitable and pragmatic tool for uncertainty analysis in exposure assessment. the transcription factor camp response element (cre)-binding protein (creb) plays a critical role in regulating gene expression in response to activation of the campdependent signaling pathway, which is implicated in the pathophysiology of heart failure. we observed creb knock-out cardiomyocytes to be larger than wildtype cardiomyocytes (cell area in µm 2 ; mean±sem; creb-ko 4871±258 vs. wt 3396±171; n=68/69 cells, n=4 mice, p<0.01 vs. ctr.). the nuclear factor of activated t-cells c3, nfatc3, is another transcription factor involved in the development of heart failure and also a known positive regulator of hypertrophy. hence, we investigated whether inhibition of the cre-dependent transcriptional activation has an impact on the nfatc3 signaling pathway. we first studied the effects of an overexpression of a dominant negative creb mutant (dncreb) or of nfatc3 on the activity of a nfat-dependent model promoter in a permanent cell line. overexpression of dncreb evoked an 8.0±1.5 fold increase of the nfat model promoter activity (n=36; n=6 transfections), but had no impact on kv4.2 promoter activity which is known to be regulated by nfatc3 (1.2±0.1 fold; n=18; n=3; p<0.05 vs. ctr.). nfatc3 overexpression led to a 4.5±0.7 fold increase of the nfat-dependent model promoter activity (n=12; n=2) and to an inhibition of kv4.2 promoter activity (0.8±0.1 fold; n=18; n=3; p<0.05 vs. ctr.). we conclude that creb is a negative regulator of nfat-mediated gene transcription and that activation of nfatc3 might contribute to the observed hypertrophy of creb-ko cardiomyocytes. neuroleptika der perazin-klasse sind potente modulatoren des p2x7-rezeptors -perazine-type neuroleptic drugs are potent modulators of p2x7 receptors hempel c., nörenberg w., urban n., sobottka h., schaefer m. universität leipzig rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße 16-18, 04107 leipzig, germany p2x7 receptors belong to a family of atp-gated, non-selective cation channels, which play an important role in immune cell activation, inflammatory hyperalgesia and neuropathic pain. they differ from other p2x family members by the low atp affinity, and by the ability to form or recruit dilated pores in the sustained presence of atp. owing to its involvement in many diseased states, p2x7 is a promising target for pharmacological intervention. accordingly, p2x7 blockers are currently tested in phase ii clinical trials. in an attempt to identify p2x7-modulating properties of approved drugs or natural compounds, we performed a medium-throughput screen, using an appropriate compound library (spectrum collection) and a stably transfected hek293hp2x7 cell line. with ic50 values of 1-2 µm, the tricyclic antipsychotics prochlorperazine and trifluoperazine showed a high potency and efficacy to block the atp (1 mm)-triggered increases in the intracellular ca 2+ concentration ([ca 2+ ]i) that was mediated by human p2x7 (hp2x7). the closely related phenothiazine-class neuroleptic drugs, such as chlorpromazine or triflupromazine did not have an appreciable effect on hp2x7mediated ca 2+ influx. whole-cell inward currents, measured at -60 mv, were blocked by more than 60% by 3-10 µm prochlorperazine. the inhibitory effects of perazines developed within about 200 ms, hinting to a direct mode of action by binding to the p2x7 protein. prochlorperazine added intracellularly via the patch pipette did not substitute for the extracellularly applied drug, indicating that its binding site is accessible from the extracellular side. in addition, both compounds blocked yo-pro-1 uptake when preincubated before p2x7 stimulation with 1 mm atp or when applied subsequent to the agonist. interestingly, when added to a hek293 cell line expressing the rat p2x7, perazines potentiated the atp-induced increase in [ca 2+ ]i. measurements in human monocyte-derived macrophages confirmed the ability of prochlorperazine and trifluoperazine to inhibit atp-evoked increases in [ca 2+ ]i, changes in yo-pro-1 permeability and whole cell currents. taken together, we conclude that perazine-type neuroleptics impede on p2x7 activity in a species-specific manner, presumably by binding to an extracellularly accessible binding site of recombinant or natively expressed p2x7. similarly, pre-treatment with lov also lowered dox-induced stabilisation of p53 and phosphorylation of chek1 and sapk/jnk. while lov had no influence on ir-induced initial dna damage formation in huvec and rat cardiomyoblasts (h9c2), it decreased dox-and eto-induced phosphorylation of histone h2ax, which is a surrogate marker of dna-double strand breaks. this indicates that lov specifically protects against the genotoxicity of topoisomerase type ii poisons. in an acute and subacute balb/c mouse model lov protected from ir-induced toxicity. this effect rested on inhibition of pro-inflammatory and pro-fibrotic processes as measured via quantification of mrna levels of il6, ctgf and tnfα. the same was true for dox-induced toxicity, i.e. heart and liver damage. similar to the in vitro experiments, dox-induced hepatic dna-damage was attenuated by lov treatment. overall, liver and heart toxicity were reduced by lov as mirrored by the serum levels of gldh/gpt and ctn-i, respectively. both in liver and in heart we observed collagen rich perivascular areas following dox treatment. under situation of lov-co-treatment these areas occurred more rarely and were less pronounced, pointing to a lowered level of fibrosis. pcr-array-based mrna analyses showed inhibitory effects of lov on dox-triggered expression of genes involved in oxidative stress response, drug transport, dna repair, cell cycle progression and cell death. for instance, up-regulation of p21, wee1, cjun/fos and hmox-1 following dox administration was attenuated by lov. altogether, we suggest that including lov in current cancer therapeutic regimen might widen the therapeutic window of anticancer therapeutics by lowering normal tissue damage. the p values. in addition, array data underwent cluster analysis for identification of substantial differences of gene regulation among the three different types of biopsies. results: of particular interest in our study was the expression of genes coding for metabolism and transport proteins. therefore 42 genes from the 156 significant differentially regulated genes, were selected for the qrt-pcr analysis. genes coding for abcb1 and abcg8 transport proteins showed higher expression in the jejunal tissue one year after surgery compared to the duodenal tissue (fold change 1.80 and 1.73). moreover, cyp7a1 mrna involved in metabolic processes is higher expressed in postoperative jejunum than in the jejunum tissue taken during the surgery (fold change 1.9). in conclusion roux-en-y gastric bypass operation leeds a change of mucosal gene expression profile in the jejunum during one year. there was also a significant differential gene expression between the original duodenum and jejunum one year after surgery. these results give strong evidence that jejunum not exposed to pancreatic but only to gastric fluids may change its gene regulation. background. numerous genome-wide association studies (gwas) identified polymorphisms located in transporter genes such as slc2a9, abcg2, npt1, and urat1 as predicitive for the serum levels of urate 1 . these genes encode membrane proteins expressed in the apical membrane of human kidney proximal tubule cells and are assumed to facilitate tubular exchange of urate 2, 3, 4, 5 . importantly several single nucleotide polymorphisms (snp) located in vicinity of slc2a9 have been identified as highly associated with serum urate levels. little is known about the transcriptional regulation of slc2a9. therefore, the aim of our study was to investigate which sequences in the slc2a9 gene harbour ciselements and regulate its gene expression. we also asked whether intronic snps influence gene expression at the transcriptional level. methods and results. performing dual luciferase reporter gene assays we found gene regulating modules in the slc2a9 gene. dna from human kidney samples was then genotyped for rs6449237 being part of this region. next total slc2a9 mrna-expression levels of the samples were determined using real-time quantitative rt-pcr assay. male samples with two minor alleles of snp rs6449237 showed lower slc2a9 mrna levels than samples with the wild type alleles. the effect was not seen in females. reporter gene constructs with either minor or major allele of rs6449237 were then used in luciferase assays, however showing no significant difference in activity. furthermore mrna-expression levels of other urate transporter genes were determined in kidney samples. after linear regression a positive correlation of mrna-expression of slc2a9, urat1, npt1, and oat10, respectively was observed. conclusion. our data suggest that the slc2a9 snps rs6449237 and rs74794351 might influence slc2a9 mrna-level without controlling the transcriptional activity. it needs to be elucidated whether those snps alter mrna stability. however, the mrna coexpression of slc2a9 and other urate transporter might be attributed to a common gene regulating pathway of an "transportosome" controlling urate homeostasis. sulfotransferases mediate the bioactivation of methyleugenol to a reactive sulfate ester binding to dna in vitro and in vivo herrmann k. 1 methyleugenol (me) is a secondary metabolite occurring in many herbs and spices. although me is hepatocarcinogenic in rodents, standard genotoxicity tests were negative. this may be due to the lack of critical activating enzymes responsible for the terminal bioactivation of me to a genotoxicant. me is initially hydroxylated by cytochrome p450 enzymes yielding 1´-hydroxymethyleugenol (1´-ohme). this alcohol can be further activated by sulfotransferases (sults) to an electrophilic sulfate ester that can be easily attacked by dna. the dna adducts formed could lead to mutation and further carcinogenicity observed in animals. the aim of the present study was to clarify whether individual human (h) and murine sult forms are involved in the activation of me to a genotoxicant. in order to identify critical sults, mutagenicity tests including bacteria expressing different sult forms were conducted. (±)-1´-ohme (separated into its enantiomers) served as test compound. we could show that hsult1a1, standing out due to its high expression level in many tissues, can efficiently activate both enantiomers even at low concentrations. furthermore, dna adduct formation in hsult1a1-proficient and sult-deficient bacteria was examined after incubation with 7 µm of (+)-or (-)-1´-ohme. for selective detection and quantification of me-derived 2´-deoxyadenosine (da) and 2´-deoxyguanosine (dg) adducts we developed a sensitive tandem mass spectrometry method including stable isotope dilution analysis. adduct formation was only observed in bacteria expressing hsult1a1. the concentration dependence of adduct formation in hsult1a1-proficient bacteria was examined for (+)-1´-ohme. both adducts turned out to be concentrationdependent. to check the extent and organ specificity of adduct formation in vivo we administered 54 mg/kg bw (±)-1´-ohme (i.p.) to mice carrying the hsult1a1/1a2 gene cluster. mice getting only the vehicle served as controls. animals were sacrificed and dna from eight organs was extracted. by means of tandem mass spectrometry adducts were measured and quantified. da and dg adduct formation was observed in all tissues studied, but not in untreated animals. furthermore, adduct levels were higher than in experiments using wild-type mice. altogether, we herein could show that sulfo conjugation leads to bioactivation of me to a dna-binding intermediate in vitro and in vivo. this work was financially supported by bundesinstitut für risikobewertung. objective: the soluble adenylyl cyclase (sac) activates the na + /k + -atpase in renal epithelial collecting duct cells. nuclear sac constitutes a functional complex with camp response element binding protein (creb), suggesting a more general role of sac in overall gene regulation. we determined the chromatin binding capacities of sac at cre sequences and its influence on genes, which play a role in aldosterone signalling. furthermore, we functionally characterised expression relevant promoter portions of sac and the influence of aldosterone and camp mediated signalling pathways on sac gene regulation. design and methods: in vascular endothelial cells (ea.hy926) and in human kidney cell lines (hek293t; ihke), we performed chromation immunoprecipitation (chip) assay with antibodies against sac and creb. we conducted transfection with a cre luciferase reporter vector and sac promoter constructs, following treatment with sac inhibitors and aldosterone. total rna of ea.hy926 cells, which were treated with sac inhibitors and aldosterone, was isolated and subsequently analysed by real-time pcr for expression of genes involved in aldosterone signalling. in vivo binding of sac at cre motifs was shown using cre consensus sequences in chip experiments. specific pharmacological inhibition of sac led to a significant decrease of transcriptional activity of the cre control vector in endothelial and kidney cell lines. furthermore, we were able to show the different effects of sac on the expression of downstream targets of aldosterone signalling, e.g. mineralocorticoid receptor and na + /k + -atpase alpha1 and beta1 and sac itself. regulation of sac itself is mediated by two different promoter portions, which are influenced by aldosterone and inhibition of sac and differentially accessed in kidney and endothelial cells. sac has transcriptional trans-acting properties as it interacts with cre sites and potentially influences the expression of genes, which play a role in aldosterone signalling. transcription of sac is regulated via aldosterone and camp. the location of promoter ta is cell type-and stimulation-specific. the role of the sodium-calcium exchanger (ncx1) in cardiac pacemaking herrmann s., stieber j., ludwig a. friedrich-alexander-universität erlangen-nürnberg institut für experimentelle und klinische pharmakologie und toxikologie, fahrstrasse 17, 91054 erlangen, germany the mammalian heart is driven by the sinoatrial node, the primary cardiac pacemaker. the unique feature of sinoatrial node (sn) cells is the ability to generate a spontaneous diastolic depolarization that periodically initiates action potentials which set the heart rhythm. the molecular origin of this cardiac pacemaker activity is still a matter of debate. recent findings point to a coordinated interplay between intracellular ca 2+ -cycling processes and plasma membrane-localized ion channels which determines the origin, periodicity and rate modulation of pacemaker potentials. in this study, we investigated the contribution of the cardiac sodium-calcium exchanger (ncx1) to pacemaking. ncx1 is a key sarcolemmal protein for the maintenance of calcium homeostasis in the heart. it was speculated that the membrane depolarizing current incx, whose activity is dependent on intracellular ca 2+ -fluctuations, represents a main determinant of the spontaneous diastolic depolarization. we used an inducible and sinoatrial node-specific cre-transgene to delete ncx1 in the murine pacemaker system. the successful creation of a cardiac pacemaking and conduction system specific ncx1 knockout (cpncx1ko) was demonstrated by transcript quantification as well as immunofluorescence experiments. telemetric ecg recordings of cpncx1ko displayed a distinct cardiac phenotype. mutant animals were deeply bradycardic and lost their capability of maintaining a stable heart beat as demonstrated by various ecg abnormalities like sn arrhythmia, sn pauses, av block and ventricular tachycardia. analysis of the spontaneous activity of isolated sn preparations showed a slower and arrhythmic contraction rate of the mutant tissues strips confirming that the bradycardia and arrhythmia induced by deletion of ncx1 results from a slower and arrhythmic intrinsic pacemaker activity. a battery of experiments using different heart rate lowering as well as increasing drugs revealed an altered heart rate modulation in cpncx1ko animals as compared to controls. in conclusion, these initial results establish ncx1 as a major contributor to cardiac pacemaking. a wide variety of contaminants are ingested through food, among them the procarcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (bp) which is resorbed and partially metabolized in the enterocytes of the small intestine. previous in vitro studies revealed that bp phenols are excreted as phase ii metabolites including bp glucuronides and bp sulfates. this export is mediated by the breast cancer resistance protein (bcrp/abcg2). the ultimate carcinogenic phase i bp metabolite anti-bp-7,8dihydrodiol-9,10-epoxide (bpde) can be detoxified by glutathione conjugate formation catalyzed by various glutathione s-transferases. in the present study, differentiated human intestinal caco-2 cells were used as a model for the human small intestine to investigate the detoxification of bpde and the subsequent transport of the stereoisomeric glutathione conjugates in the presence of an inhibitor (acivicin) of the glutathione-cleaving enzyme gamma-glutamyl transpeptidase (ggt) at the surface of the cells. the results indicate that the glutathione conjugates of bpde are formed and excreted mainly to the apical and to a minor extent to the basolateral side of the polarized caco-2 monolayer. to stimulate the transport rate several inducers known to enhance gene expression of xenobiotic-metabolizing enzymes as well as transport proteins were used (quercetin, oltipraz, butyrate). however, solely oltipraz substantially increased the efflux of bpde glutathione conjugates after inhibition of ggt. inhibition studies revealed that the multidrug resistance-associated proteins (mrps/abccs) are involved in the transport of the bpde glutathione conjugates. stable abcc1, abcc2 and abcc3 knockdown cell lines were generated allowing to demonstrate that abcc1 mediates the basolateral, abcc2 the apical excretion of the bpde glutathione conjugates. in conclusion, the ultimate carcinogen bpde is detoxified via glutathione conjugation and subsequently excreted by caco-2 cells in both apical and basolateral directions. .this finding is equivalent to a transport into the feces as well as blood system in the in vivo situation. signaling via irag regulates store operated calcium entry (soce) in aortic vsmc hieke b., hüttner j., schlossmann j. university of regensburg department of pharmacology and toxicology, universitätsstr. 31, 93053 regensburg, germany the mechanisms involved in the activation of store operated calcium entry (soce) through depletion of intracellular stores and their regulation are not yet fully understood. we examined the effect of inositoltriphosphate-receptor associated cgmp-kinase substrate (irag) on soce. aortic vascular smooth muscle cells (vsmc) from wild type (wt) and irag-knock-out (ko) mice were loaded with the calcium indicator fura 2-am and soce was measured as a change in the intracellular calcium concentration. in experiments with vsmc from wt mice soce was attenuated by the application of 8-br-cgmp. this effect was not observed in vsmc isolated from irag-ko mice. these differences in the strength of the soce-signal were abolished by the replacement of extracellular sodium with n-methyl-d-glucamine. the observed sodium dependence of the soce regulation via irag suggests, that an alternated sodium conductance might be responsible to some extent for the differences detected in wt and irag-ko vsmc. as a change in sodium conductance might result in a changed membrane potential we tried to track these changes with the flipr membrane potential assay kit while executing the soce protocol with and without 8-br-cgmp. no significant differences in membrane potential could be detected in the various stages of soce. in conclusion, our results indicate that irag exhibits a dual action on calcium regulation as it inhibits not only the intracellular calcium release but also the extracellular calcium influx through soce. induction of the icer promoter in vascular smooth muscle cells hildebrandt i. 1 tokyo metropolitan institute of gerontology, tokyo japan several transcription factor isoforms are encoded by the crem (camp response element modulator) gene. one prominent isoform is the inducible camp early repressor (icer), which acts as a transcriptional repressor on so called camp responsive elements (cres) in its target gene promoters. the icer mrna expression is regulated by an intronic promoter of the crem gene. in vascular smooth muscle cells (vsmcs) crem/icer is involved in the regulation of cell proliferation and apoptosis with physiological consequences in vivo. for instance crem-knockout mice, in which none of the known isoforms can be expressed, exhibit an increased neointima formation after carotid ligation as well as an increased atherosclerotic plaque formation after high fat diet on an apoe background. these observations were associated with an increased proliferation rate in isolated crem deficient vsmcs. on this background we wanted to clarify the specific role of icer isoforms in the vasculature. in first experiments we examined the inducibility of icer in primary vsmcs and smooth muscle cell lines. reporter luciferase assays showed that the activity of the icer promoter is induced at the maximum of fourteen fold after 4 hours of stimulation with forskolin (fsk) in immortalized rat vsmcs (13.7 ± 0.93; n=12 from 3 isolations). in a7r5 rat smooth muscle cells the icer promoter showed a maximum stimulation of 3.2 ± 0.16 fold after two hours of fsk stimulation (n=20 from 4 isolations). these pilot experiments showed that the icer promoter is inducible in vsmcs by camp dependent pathways. further experiments have to be carried out to elucidate the role of icer in the vascular system for example by stimulation of primary vsmcs and analysis of icer knockout mice. (supported by the dfg) overexpression of transmembrane channel-like proteins (tmcs) uncouples receptor-mediated calcium mobilisation hill k., urban n., straub i., schaefer m. universität leipzig -universitätsmedizin rudolf boehm-institut für pharmakologie und toxikologie, härtelstr. 16-18, 04107 leipzig, germany the family of transmembrane channel-like proteins (tmcs) consist of 8 members (tmc1-tmc8) all tmc genes are predicted to encode transmembrane proteins with at least six membrane-spanning helices. mutations of tmc1 cause deafness in human and mice whereas tmc6 and tmc8 (also referred to as ever 1 and 2) are linked to epidermodysplasia verruciformis (ev), a skin disorder, which is characterised by an enhanced susceptibility towards cutanous infections by human papillomaviruses. the cell biological and physiological functions of tmc proteins still remain elusive. we have overexpressed tmc6 and tmc8 in hek293 cells to get insights into their physiological function. all tmcs were located within the endoplasmic reticulum (er) after overexpression of yfp or cfp-tagged constructs. ratiometric calcium imaging revealed that after overexpression of tmc6 or tmc8, stimulation of gq-coupled receptors with carbachol and atp resulted in a greatly reduced amount of calcium release from the er. moreover, challenging tmc6-or tmc8-expressing cells with the serca pump inhibitor thapsigargin was also not followed by a release of er-based calcium within the cell. to test whether the lack of calcium release was caused by a reduced calcium content within the er, we investigated calcium dynamics within the er using an er-targeted fret-based calcium indicator (d1er cameleon). the experiments revealed that the amount of calcium within the er was reduced upon overexpression of tmc6 or tmc8. recently, it has been reported that tmc6 and tmc8 might influence intracellular zinc distribution in human keratinocytes. we could confirm the presence of tmc6 and tmc8 mrna in a human keratinocytes cell line (hacat). upon overexpression of tmc8, hacat cells revealed the same phenotype as described above for the hek293 cells with an uncoupling of the receptor-mediated calcium mobilisation due to a depletion of the er calcium store. the mechanism by which overexpression of tmc proteins causes a reduced calcium concentration within the er remains unclear. considering that the presumed topology of the tmc proteins distantly resembles those of other ion channel superfamilies such as anoctamins, one might speculate that a conductance through the tmc protein itself leads to a calcium leak from the er. terahertz radiation is defined as radiation between 0.1 thz and 10 thz. a number of applications are currently being developed using radiation in this frequency range. these applications will lead to exposure of the general public, making it very important to study potential effects on biological systems. historically, only a few studies on effects caused by terahertz radiation have been conducted because of the lack of suitable generators and detectors. during the last decade, a number of studies on effects caused by radiation with frequencies around 100 ghz have been published. the present study investigated the genotoxic potential of terahertz radiation at three different frequencies, 0.106 thz, 0.380 thz and 2.520 thz. two skin cell types were used, primary human dermal fibroblasts (hdf) and a keratinocyte cell line (hacat). the cells were irradiated applying different exposure times and different power intensities. two genotoxicity tests were applied: the comet assay quantifies dna strand breaks as well as alkali-labile sites whereas the micronucleus test quantifies chromosomal damage. all experiments were performed and evaluated under blinded conditions as three independent replicate experiments. positive (mms-treated) and negative (untreated, sham-exposed) controls were included. in the comet assay no dna damage was observed as a consequence of the exposure under all experimental conditions. the same was true for the chromosomal damage investigated with the micronucleus test. the latter finding was particularly interesting for the experiments at 0.106 thz, because this type of radiation had been reported to cause mitotic disturbances. therefore these experiments were extended, applying higher power intensities and longer exposure periods. also with these modifications, no genomic damage was observed in the form of micronucleus formation. all in all, terahertz radiation did not induce genomic damage under the applied experimental conditions. this result is in line with published findings on genotoxicity of low-frequency terahertz radiation around 0.1 thz. the question, why the reported mitotic disturbances do not lead to manifest genomic damage remains open and requires further research. introduction: tea flavonoids derived from camomile and green tea such as apigenin and epigallocatechin gallate (egcg) can inhibit intestinal neoplasia. recurrences of adenomas and cancers were reduced in patients with resected colorectal cancer by treatment with tea bioflavonoids after tumor operation [1] . to clarify the biomolecular pathway for suppression of neoplasia we investigated the anti-inflammatory effect of a nutritional supplement flavo natin® (fn) which had been used in the clinical study on tertiary tumor prevention and of egcg in a colon tumor cell line. the aim of our study was to investigate if tea flavonoids are capable to suppress the inflammatory markers produced by tumor cells after cytokine stimulation. method: we studied the cytotoxicity of fn in the colon cancer cell line t-84 by resazurin fluorescence and compared it with the placebo supplement. additionally, the t-84 cells were incubated with fn, egcg or placebo and stimulated with tnf-alpha, if-gamma and il-1-beta. after the cytokine stimulation the mrna expression of ip-10, il-8 and tnf-alpha was measured by quantitative real-time pcr (qrt-pcr). results: stimulation of t-84 cells increased the expression of ip-10 (gamma-interferon inducible protein 10), tnf-alpha and il-8. by preincubation with fn at 10 µm the mrna expression of ip-10 was strongly reduced (log2-ratio -14). the tnf-alpha mrna was also but less decreased by fn. egcg displayed an inhibition pattern similar to fn. placebo did not influence the mrna expression of the chemokines and tnf-alpha. discussion & conclusion: clinically useful dietary tea bioflavonoids inhibit the expression of inflammatory genes in a colon cancer cell line. down-regulation of inflammatory gene products could be achieved in vivo by botanicals without clinically relevant side effects. [1] h. hoensch, b. groh, l. edler, w. kirch (2008) . prospective cohort comparison of flavonoid treatment in patients with resected colorectal cancer to prevent recurrence. world j gastroenterol, 14, 2187-2193. the cxcr7 c-terminal domain mediates efficient cxcl12 uptake and degradation hoffmann f., müller w., schütz d., schulz s., stumm r. universitätsklinikum jena institut für pharmakologie und toxikologie, drackendorfer str. 1, 07747 jena, germany cxcl12-signaling mediated by the g protein-coupled cxcr4 receptor plays a key role during embryonic development and disease states including cancer and inflammation. the second cxcl12-receptor cxcr7 modulates cxcl12/cxcr4-signaling by acting as a cxcl12-scavenger. given the distinct functions of cxcr4 and cxcr7, we hypothesized that trafficking and receptor stability are differently regulated by the distinct c-terminal domains. here, we examined epitope-tagged wild type and c-terminal mutant receptors expressed in human embryonic kidney cells (hek293) with respect to trafficking, stability, 125 i-cxcl12 radioligand degradation, and g protein-coupling. we found that the 24 c-terminal residues of cxcr7 were sufficient for cxcr7 to undergo rapid spontaneous internalization. replacement of the cxcr7 c-terminal domain with that of cxcr4 (cxcr7-4tail mutant) abolished spontaneous internalization but permitted ligand-induced internalization in conjunction with c-terminal phosphorylation. conversely, replacement of the cxcr4 c-terminal domain by that of cxcr7 caused ligand-independent internalization of cxcr4. receptor-mediated 125 i-cxcl12-uptake, release of 125 i-cxcl12-degradation products, and degradation of the receptor protein itself were accelerated with receptors bearing the cxcr7 c-terminus. while the cxcr7 c-terminus was sufficient to abolish g protein coupling in the cxcr4-7tail mutant, replacement of the cxcr7 c-terminus, cxcr7 second intracellular loop or both domains with the corresponding cxcr4 domain did not generate a g protein-coupled cxcr7 chimera. taken together, we provide evidence that the cxcr7 c-terminal domain influences the ligand-uptake/degradation rate, g protein-coupling, and stability of the receptor. this suggests that heterologous regulatory pathways targeting the cxcr7-c-terminal domain may effectively control cxcr7 functions. soluble guanylyl cyclase is a key mediator of brown adipocyte differentiation hoffmann l. s. 1 brown adipose tissue (bat) uses energy to produce heat by inducible thermogenesis. recent studies show that active bat is present in adults and involved in human energy balance, suggesting that the energy consuming property of bat might be exploited to fight obesity and related diseases. the no/cgmp signaling pathway is a key player in diverse physiological processes. recently, we have shown in bat that cgmp signaling is connected with insulin signaling and abrogation of cgmp signaling leads to impaired bat differentiation and function (haas, b. et al., sci signal, 2009 ). here we investigated the role of the cgmp generating enzyme soluble guanylate cyclase (sgc) in bat differentiation in vitro. mesenchymal stem cells isolated from bat of newborn sgcβ 1 -/mice and wt littermates were differentiated in vitro into brown adipocytes in the presence or absence of cgmp. abundance of sgc isoforms was determined by qrt-pcr and western blotting. bat differentiation was assessed by redo staining of accumulated intracellular lipids, measurement of triglyceride (tg) content, determination of expression of bat marker proteins pparγ, c/ebpα, ap2 and bat marker genes ucp1, pgc1α, cidea. the α2 and β1 isoforms of sgc were highly expressed in bat whereas α1 sgc could not be detected. redo staining of wt brown adipocytes showed basal differentiation which was increased upon addition of 8-pcpt-cgmp. in contrast, staining was lower in sgcβ1 -/cells compared to wt under control conditions and increased in the presence of cgmp. tg measurement showed that sgcβ1 -/brown adipocytes contain approximately 50% less lipids than wt cells under basal conditions. addition of cgmp doubled tg content in both genotypes. similar results were observed for marker protein expression. deletion of sgc resulted in 56-40% decrease in c/ebpα, pparγ and ap2 expression compared to wt. again, cgmp roughly doubled protein expression in sgcβ1 -/and wt cells compared to control. under basal conditions, bat marker gene expression was decreased by approximately 80% in sgcβ1 -/cells compared to wt cells. this decrease was prevented by addition of cgmp. these results show that sgc deletion leads to dysfunctional bat differentiation and emphasize the central role of cgmp signaling in bat differentiation. further investigation of sgc/cgmp signaling in bat might reveal new drugable targets bringing bat-dependent pharmacological therapy to treat obesity and related disease into closer reach. comparative inhalation toxicity of carbon-nanomaterials (multi-wall carbon nanotubes, graphene and carbon black) hofmann t. 1 carbon black is a spherical carbon anomaterial whereas multi-wall carbon nanotubes (mwcnt) are cylindrical and graphene is a laminar allotrope of carbon. processing and handling as well as abrasion processes can set free inhalable cnt particles. results of rodent studies collectively show that regardless of the process by which cnts were synthesized and the types and amounts of metals they contained, cnts were capable of producing inflammation, epithelioid granulomas, fibrosis, biochemical and or toxicological changes in the lungs (lam et al. 2004 , muller et al. 2005 , ma-hock 2009 , pauluhn 2010 . graphene possess similar physical properties as cnt but may different toxicological property. we performed short-term inhalation studies in rats to compare the toxic potency of four different cnt, two graphenes and one carbon black. the materials are characterized thoroughly according to the oecd list. the four mwcnt caused morphological changes as descriped above. several biochemical and cytological parameters in the broncho-alveolar lavage fluid were strongly increased consistent with the histological findings. two mwcnt exhibited a higher toxic potency than two other mwcnts and findings caused by one graphene typ were even less severe. the graphene with lower surface area as well as low surface area carbon black did not cause any adverse effects up to 10 mg/m 3 . the short-term inhalation studies were able to descriminate different toxic potencies of carbon-based nanomaterials and is hence used for the selection of less toxic materials for further product development as well as to define and prioritize higher-tier toxicological testing of nanomaterials. 165 synthesis and analytical assessment of possible dna adducts formed after activation of the tobacco alkaloid myosmine högg c., zwickenpflug w., gudermann t. walther-straub-institut abt.: toxikologie, nußbaumstraße 26, 80336 münchen, germany myosmine represents one of the minor tobacco alkaloids and its effective uptake from smokeless tobacco or tobacco smoke, as well as by consumption of food is not understood in detail. myosmine can be activated by peroxidation and n-nitrosation yielding 4-hydroxy-1-(3-pyridyl)-1-butanone (hpb) which is well known as reactive intermediate during activation of a variety of tobacco specific n-nitrosamines (tsna). therefore, myosmine may be a potential candidate for possible mutagenic or carcinogenic risk to human health. furthermore, myosmine n-nitrosation yields the tobacco specific nitrosamine n-nitrosonornicotine (nnn), which is classified as carcinogenic to humans. considerable efforts have been undertaken, especially in organic synthesis, to verify and elucidate the significance of the hpb precursor the pyridyloxobutyl (pob) intermediate and its dna-adducts, which were analysed only in animal experiments till now. the formation of 3-pyridylmethanol was observed under myosmine peroxidation and identified as a metabolite in rat urine after application of myosmine to rats. the formation of the 3-pyridylmethanol intermediate, might provide for the reactive electrophilic picolinium ion which could interact with dna. these possible adducts might be of special interest to elucidate the role of myosmine concerning its uptake by smokers and passive-smokers in contrast to non smokers ingesting the substance by consumption of food. dna adducts may help to obtain more information about possible risk assessment of myosmine and to differentiate between the activation from the other nicotinoids and tsna. the specific dna adducts, 5-methyl-1,3-bispyridin-3-ylmethyl-1h-pyrimidin-2,4-dion and 3-(3''-picolyl)thymidine have been synthesised as reference substances. the former was prepared by addition of diisopropylazodicarboxylate (diad) to a mixture of 3-pyridylmethanol, thymine and triphenylphosphine. the reaction product was identified using nmr ( 1 h, 13 c, cosy, hmqc, hmbc). for synthesis of 3-(3''-picolyl)thymidine the hydroxyl groups of thymidine were initially acetylated using acetic acid anhydride and 4dimethylaminopyridine (dmap). the existence of this thymidine adduct was confirmed using nmr. this adduct was labelled with 5-([4,6,-dichlorotriazin-2-yl]amino)fluorescin (dtaf) to enhance the sensitivity using hplc-fluorescence chromatography and used as reference substances for analysis of dna samples from biological tissue. supported by dfg grant (ty 81/1-1). cancer and cardiovascular diseases such as atherosclerosis are the most important causes of death in western societies. common to both diseases is a deregulation of cell death, with significant contribution of inflammatory processes. enhanced oxidative stress plays a dominant role in such events as it forms a vicious cycle with inflammation and controls multiple forms of cell demise. therefore, anti-oxidative enzyme systems gained considerable interest since control of reactive oxygen species (ros) has the capacity to regulate cell death in either direction. the human enzyme family of paraoxonases consists of three members, known as pon1, pon2 and pon3. while pon1 is found predominantly in the circulation, pon2 and pon3 are intracellular enzymes with established anti-oxidative functions. it has been shown that both pon2 and pon3 are protective against atherosclerosis. underlying mechanisms of their protective and antioxidative functions however remained elusive. here we demonstrate that both enzymes locate to the endoplasmic reticulum (er) and mitochondria where they fulfill vital functions in the control of ros generation. in particular, pon2 and pon3 were shown to interact with coenzyme q10 which diminishes mitochondrial ros formation. as a consequence, these enzymes reduce execution of mitochondrial apoptosis, such as cardiolipin peroxidation, cytochrome c release and caspase activation. moreover, pon2 and pon3 reduced er stress-triggered cell death, i.e. by diminishing jnk signaling and chop expression. while these results elucidate their protective role in cardiovascular diseases, it also establishes a relevant function in survival of tumor cells. in accordance, we demonstrate that both enzymes are frequently found overexpressed in various tumors. in cancer cell culture studies, overexpression of both enzymes granted considerable resistance against chemotherapeutics. in turn, knock-down of pon2 caused spontaneous apoptosis of several cancer cell lines. finally, our analyses also revealed that pon2-knockout mice show severe alterations of the hematopoetic stem cell compartment, which implies a significant role in leukemias where these enzymes are frequently found overexpressed. together, our results propose pon2 and pon3 as new putative anti-tumor candidates and demonstrate the efficacy of interventions targeting cellular redox-balance. steigerwald arzneimittelwerk gmbh wissenschaftliche abteilung, havelstr. 5, 64295 darmstadt, germany stw 5 (iberogast ® ), a multi-component herbal drug, is successfully used in the therapy of functional dyspepsia and irritable bowel syndrome (ibs). previous studies revealed effects of stw 5 on disturbed motility and inflammatory processes. although the antiinflammatory properties of stw 5 are well examined, the contribution each of the individual herbal constituents to the anti-inflammatory effect remains unclear. therefore, we studied the effects of stw 5 and its components on inflammation-induced cell death and on the release of the pro-inflammatory cytokine tnf-α. the aim of these investigations was to analyse additive or synergistic effects of the components. the experiments were carried out on caco-2 cells after stimulation with lps (10 ng/ml) for 2 hours. cytotoxicity was evaluated using a commercially available ldh (lactate dehydrogenase)-assay. furthermore, the release of tnf-α after lps (100ng/ml) stimulation of differentiated thp-1 cells was measured using a commercially available elisa. stw 5 (62.6 -500.5 µg/ml) reduced lps (10 ng/ml)-induced cell death in a concentration-dependent manner with a maximum inhibition of 50.0 %. the herbal components in equivalent concentrations contributed to the inhibitory effect of stw 5 to different extents. the maximum inhibition differed in a wide range between the components. stw 5 (500.5 µg/ml) reduced significantly the release of tnf-α by 87 % in lps (100 ng/ml)-stimulated differentiated thp-1 cells while having no effect in untreated cells. in concentrations equivalent to stw 5 caraway, milk thistle, lemon balm and greater celandine had no effect on the lps-induced increase in tnf-α release. bitter candytuft, peppermint, chamomile, liquorice and angelica reduced the tnf-α release, though less pronounced as compared to stw 5, indicating a possible synergistic effect. our results indicate a multi-target effect of stw 5. the anti-inflammatory effect may be due to a reduction of the cytotoxic effect on intestinal mucosa cells and to an inhibition of the release of the pro-inflammatory cytokine tnf-α from immune cells. the individual herbal components seem to contribute by different mechanisms of action to the overall effect of stw 5. immune cell-induced local steroidogenesis in the lung: implications for asthmatic disease and therapeutic intervention hostettler n. 1 , brunner t. glucocorticoids are steroid hormones with potent anti-inflammatory properties. synthetic glucocorticoids are frequently used for the therapeutic treatment of inflammatory disorders, such as asthma. endogenous glucocorticoids are predominantly produced in the adrenal glands in response to emotional, physical and immunological stress. recent years, however, revealed several alternative sources of these immunoregulatory steroid hormones. thus, we have found that the intestinal epithelium is a rich source of glucocorticoids and intestinal glucocorticoids contribute to the maintenance of local immune homeostasis (1) . as the intestinal and the pulmonary epithelium have much in common, i.e. barrier functions and transport of nutrients, resp. gases, we wondered whether the lung mucosa is also capable of synthesizing immunoregulatory glucocorticoids in response to immune cell activation. the murine lung was found to expresses all enzymes required for the synthesis of corticosterone from cholesterol. while most enzymes where expressed in a constitutive manner, cyp11a1, encoding p450ssc, was strongly induced in response to immunological tress. treatment of mice with t cell-activating anti-cd3 antibody of macrophage-activating lipopolysaccharides induced strong local glucocorticoid synthesis, which was effectively blocked by the corticosterone synthesis inhibitor metyrapone, indicating that glucocorticoids measured were produced bona fide in the lung tissue. surprisingly, allergen-induced allergic airway inflammation failed to trigger local glucocorticoid synthesis despite the massive infiltration of neutrophils, eosinophils and t cells. in contrast to that in the intestinal epithelium local glucocorticoid synthesis in the lung was found to be dependent on the presence of adrenal glands as adrenalectomy abolished pulmonary steroidogenesis. in line with the notion that the lung metabolizes steroid precursors we found that ex vivo cultured lung tissue metabolized 3 hduring the last decade small rna molecules has been identified as important regulators for gene expression. these micrornas (mirna) are single-stranded transcripts, which are expressed in many cell types, where they modulate rna stability and translation and, therefore, controlling various cellular mechanism and tissue development. against this background, in the present study the mirna expression and potential target genes were studied in the human placenta as a tissue demonstrating various developmental changes in a limited period of time. taqman®array microrna cards for profiling of 365 mirnas in placentas of different gestation times revealed a significant expression of 277 mirnas by comparing placentas of early gestation (<10.week), preterm (<30.week) and term (>37.week). when comparing the analyzed groups, 106 of these mirnas expose a continuous up-(including mir-20a, mir-379) or downregulation (including mir-9, mir-200a). while comparing placentas of early gestation to term placentas, 30% (e.g. mir-9, mir-429) were up-and 70% (e.g. mir-126, mir-373) were downregulated. by focusing on the latter group with early preterm placentas the ratio is opposed. here, 60% of mirnas showed higher (e.g. mir-107) and 40% lower expression (e.g. mir-627, mir-191). with emphasize on these mirnas, a computational prediction algorithm using the mirò database predicted a potential interaction between corticotropin-releasing hormone (crh) and the mirnas mir-9 and mir-429. specific expression analyses validate an inverse expression between mirnas and target with a reduced expression of mir-9 (93%) and mir-429 (94%) in term placentas, while crh is upregulated 114fold. this interaction was verified on functional level by reporter gene assay. a significantly suppressed luciferase activity of the reporter plasmid containing the 3´-utr sequence complementary to crh was exhibit for the predicted mir-9 binding site. here, the mirna-mrna-interaction reduces the luciferase activity by ~40%, whereas the decreased luciferase activity for the predicted mir-429 binding site is not significant. the results demonstrate a gestation dependent expression of placental mirnas, which may help to explain gestational changes in gene expression and highlight the potential of mirnas as biomarker for pregnancy-related pathologies. since homo sapiens era wound treatment by early civilizations was based on the use of local flora. scilla indica knuth liliaceae (s.i) is a perennial herb with a pear shaped, tunicated bulb, bearing fibrous roots, white flowers and leaves on the stem resembling u. maritima. it is used as cardiac tonic, against inflammation, ulcers and sinus diseases. traditionally the powdered bulb is used topically for warts treatment, while roasted and crushed bulbs are applied to corns of the feet soles. the plant contains steroid glycosides (bufadienolides 1-3%), glucoscillarene a, proscillaridine a, scillarene a, scillcyanoside ,scilliglaucoside ,mucilage and alkyresorcinol derivatives exerting skin healing properties similar to wheat bran products. the study is focused on the investigation of the restoration quality of a skin dorsal incision wound in wistar rats in three groups, control (a1) and experimental (a2,a3 ) of rats weighing 350-450g local application of dichloromethane extract of s.i in vehicle olive oil preparations of 50 and 100mg were used. animals were anaesthetized ( ether pro narcosi ), trauma 2 cm long and 2mm deep was performed by lancet on depilated dorsal area skin until the muscular aponeurosis and were treated for 4 days with 60λ of each preparation. group a3 showed increased remodelling of trauma area (limited width). granulomatous tissue was more pronounced in length, width and surface in group a2. the macroscopic ulcus dimensions were better in group a3 while the microscopic view were similar in a2 and a3 and better in comparison to control a1. a tendency to trauma remodelling was observed, without statistical significance (t =0.3) in recent years, it has been suggested that nanoparticles generated from combustion processes (e.g. diesel engine exhaust particles), may contribute to the pathogenesis of neurodegenerative diseases such as alzheimer's disease (ad). the aim of our current study was to investigate the effects of subchronic exposure to diesel engine exhaust (dee) in the 5xfad mouse model, which is characterised by progressive behavioural deficits as well as amyloid plaque formation and neuron loss. ten weeks old female 5xfad mice and their nontransgenic littermates were exposed by whole body inhalation to diluted dee (~1 mg particles/m 3 ) or clean air (controls) for 3 or 13 weeks (5 days/week and 6 hour/day). subsequently, all animals were subjected to a series of behavioural tests. at ten days post-exposure, mice were sacrificed to investigate lungs and brain tissues for pathological and biochemical and molecular-biological changes. in line with the expectations, the 5xfad mice displayed typically age-dependent behavioural deficits and amyloid plaque formation in cortex and hippocampus. a significant dee exposure-related effect was observed for the string suspension test, representing a measure of motor coordination/grip strength. dee exposure was also associated with mrna expression changes of markers of inflammation and oxidative stress in specific brain regions, including the olfactory bulb. histopathology of plaqueload in cortex and hippocampus (from a limited number of animals investigated so far) did not reveal clear evidence for increased plaque formation due to the dee exposures. further research is needed to evaluate the effects of long term exposure to nanoparticles in the central nervous system. this work is supported by funds from the research committee of the medical faculty of the university of düsseldorf (9772-365), the dfg graduate school grk1033 and the rivm -centre for environmental health, bilthoven, netherlands. mono-glucosylation of (h/k/n)ras by clostridium sordellii lethal toxin (tcsl) blocks critical survival pathways, including the pi3k/akt, the ralgef/ral, or the raf/erk, and results in apoptosis. in this study, ras glucosylation is presented to result in expression of the cell death-regulating small gtpase rhob based on transcriptional activitation. rhob expression depends on k-ras inhibition, as sirna-mediated knock-down of specifically k-ras (neither h-ras nor n-ras) provokes rhob expression. rhob expression further depends on inhibition of pi3k/akt, as activation of pi3k/akt using a pi3k activator prevents rhob expression downstream of inactivated ras. newly synthesized rhob is gtp-loaded and rapidly degraded in a proteasome-and a caspase-dependent manner, providing first evidence for caspase-dependent degradation of a rho family protein. although often characterised as a pro-apoptotic protein, rhob suppresses caspase-3 activation in tcsl-treated fibroblasts. conclusions: 1. rapid and efficacious ras inactivation by tcsl turns out to be particularly useful in the characterisation of ras inactivation-induced rhob expression as an immediate-early gene response. 2. the finding on the cytoprotective activity of rhob in tcsl-treated cells re-enforces the concept that rhob exhibits cytoprotective rather than pro-apoptotic activity on cellular background of inactive ras. the activity of the rhob promoter is suppressed by rhoa (through a not yet identified pathway or ras (through the pi3k/akt pathway. ras glucosylation by tcsl results in de-suppression of the rhob promoter and rhob expression. the calcium-binding protein annexin a4 (anxa4) is involved in diverse cellular processes including e.g. vesicular transport, ion channel regulation and transcriptional regulation. upon ca2+ binding anxa4 undergoes conformational changes, which lead to oligomerization of anxa4 to homotrimers and cause an increased affinity for membrane phospholipids, which in turn provokes the translocation from the cytosol and the nucleoplasm to plasma and nuclear membranes. in order to examine the effect of anxa4 on cre-and creb-(camp response element-binding protein) dependent transcription, a series of transient transfections using a luciferase reporter gene driven by the creb target promoter of the inducible camp early repressor (icer) was carried out. when the luciferase reporter construct was co-transfected with anxa4, there was significant reduction of basal (dmso) luciferase activity ( the implantation of drug eluting stents (des) after coronary artery intervention was an important step treating coronary artery disease. indeed, cytotoxic compounds like sirolimus used on des are responsible for reduced in-stent restenosis in comparison with bare metal stents. pimecrolimus, a potent anti-inflammatory drug has also been investigated for its efficacy on des. preclinical studies in pigs revealed promising antiproliferative effects of pimecrolimus on neointima formation. however, in humans, pimecrolimus coated stents exerted adverse effects. we hypothesize that compared to the highly active sirolimus, pimecrolimus may influence additional cellular processes leading to the worse outcome. in order to identify those processes we conducted in vitro studies in human coronary artery endothelial cells (hcaec) and smooth muscle cells (hcasmc). brdu in vitro assays of hcaec treated with pimecrolimus examined an ic50 value of 6.787 µm [5.745 to 8 .017] which is much higher than ic50 values of sirolimus described in literature [0.1nm to 1nm]. genechip array analysis comparing gene expression in pimecrolimus and sirolimus treated hcaec and hcasmc showed significant induction of several genes involved in interferon signaling. in detail, the expression of ifnβ activated genes like irf9 and ifitm1 was up regulated in cells treated with pimecrolimus while no or oppositional effects were observed with sirolimus. gene regulatory effects were validated by real time pcr. incubation with ifnβ itself showed similar effects in up regulation of genes involved in interferon signaling. furthermore, we were able to demonstrate a significant increase of ifnβ secretion in hcasmc and hcaec treated with pimecrolimus. however, comparison of ifnβ and pimecrolimus on proliferation of hcaec and hcasmc revealed different cellular responses. while ifnβ significantly decreased hcasmc and increased hcaec proliferation, treatment with pimecrolimus lead to anti-proliferative effects on both cell types. in conclusion, pimecrolimus activates pathways involved in interferon signaling but exerts different pharmacological effects, compared to the endogenous compound suggesting that infβ secretion is not the major factor contributing to the difference in pimecrolimus function. identification of novel phospho acceptor sites of the mu opioid receptor regulating receptor internalization illing s., just s., doll c., schulz s. uniklinikum der fsu jena pharmakologie und toxikologie, drackendorfer straße 1, 07747 jena, germany the opioid alkaloid morphine is among the most potent clinically used analgesics. however, the clinical utility of morphine to treat chronic pain is limited by its rapid development of tolerance and dependence. the stimulation of the mu opioid receptor (mor) with damgo or morphine results in different patterns of receptor phosphorylation and trafficking. so far, the three major phosphorylation sites of the mu opioid receptor namely serine 363 (s363) threonine 370 (t370) and serine 375 (s375) have been identified using phosphosite-specific antibodies. however, mutations of these three residues to alanine (s363a/t370a/s375a) did not prevent agonist-dependent internalization. in the present study, we have constructed a series of phosphorylationdeficient mutants showing that mutation of at least six residues (s363a/t370a/s375a/t376a/t379a/t383a) was required to completely block agonistdriven endocytosis. consequently, we generated phosphosite-specific antibodies to t376 and t379 which enabled us to provide direct evidence for agonist-dependent phosphorylation of these sites. our analysis of time-and dose-dependent phosphorylation of mor revealed that s375 was the primary phosphorylation site, whereas t370, t376 and t379 were secondary sites. moreover, the partial agonist morphine induced only the phosphorylation of s375 but not of t370, t376 of t379. our results show, that mor phosphorylation occurs in a hierarchical and agonist-selective manner directly regulating receptor sequestration. assessment of mda effects from toxicity studies with regard to endocrine modulation jäger r. 1 methylene dianiline (mda; cas no. 101-77-9) is on the usepa list 2 for endocrine disruption screen testing. in a yeast androgen screening (yas) assay (in vitro assay on receptor binding or blocking) mda revealed a significant anti-androgenic activity at a concentration of 0.01 mm. to assess the biological relevance of this observation under in vivo conditions the existing data from animal toxicity studies with mda were reviewed for indications of possible endocrine modulating (em) effects (weight-of-evidence approach). in addition, literature was searched for relevant studies on mda and structural analogues using the american chemical society scifinder client-server software. structural analogues indicated several drivers for em activity, notably the diphenolic ring structure. however, in vitro receptor binding assays showed mda had no androgenic or anti-estrogenic activitiy. one report described a weak estrogenic binding at 0.2 mm mda, while another described no effect at similar concentrations and a rat estrogen receptor binding study found no effect at 0.2 mm. overall it is concluded that mda does not bind to the estrogen receptor. endocrine related effects of mda were investigated in several species and dose routes in unvalidated research-type protocols. guideline subchronic and chronic toxicity studies with rodents revealed neither adverse organ weight changes nor histopathological alterations of sex organs. the main systemic effects from the oral 13-week studies were body weight reduction and histopathological changes of the thyroid and bile duct (lowest loael: 7.5 mg/kg bw). mda was carcinogenic to rats and mice (thyroid and liver) after oral administration over 2 years. non-neoplastic effects in the thyroid (rats and mice) were observed (lowest loael for systemic toxicity: 9 mg/kg bw). mda inhibits iodide oxidation which with concomitant decreased thyroid hormone formation is known to induce thyroid tumors. in summary, in vitro screening tests revealed no consistent endocrine related effects of mda. in vivo, there was an effect on the thyroid gland, possibly by enzyme inhibition. there were no histopathological changes of gonads and accessory sex organs and no evidence of sex hormone related em. the evidence from the full dataset on mda does not indicate androgenic or estrogenic effects. overall, based on a weight of evidence assessment there are insufficient alerts for em activity to suggest further testing should be done. the gtpase arfrp1 controls the assembly of apoa-i to and the lipidation of chylomicrons in the golgi of intestinal epithelium jaschke a. 1 background: the uptake and processing of dietary lipid by the small intestine is a multi-step process that involves luminal digestion, cellular uptake of fatty acids by the mucosa, and subsequent synthesis and export of chylomicrons. the gtpase adpribosylation factor related protein 1 (arfrp1) is a member of the arf-family and controls the arf-like 1 (arl1)-mediated golgi recruitment of grip domain proteins which in turn bind several rab-gtpases. the aim of the study was to define the role of arfrp1 in intestinal nutrient absorption. methods: for the generation of intestine-specific null mutants arfrp1 flox/flox mice were crossed with mice expressing the cre recombinase under the control of the intestinespecific villin promoter (vil-cre) and arfrp1 expression was suppressed by sirna in caco2-cells. the phenotype in respect to lipid absorption and chylomicron production in the intestinal epithelium and in caco2-cells, respectively, was analyzed. results: arfrp vil-/mice were viable but showed an early postnatal growth retardation (mean body weight of arfrp1 vil-/was 43.3±5% lower than that of control mice at the age of 28 days) arfrp1 vil-/mice displayed reduced triglycerides, free fatty acids and glucose plasma levels indicating that the growth retardation is the result of a malabsorption. uptake of glucose and amino acids were unaffected by the deletion of arfrp1. in contrast, lipid uptake as elucidated by oral fat tolerance tests was impaired in arfrp1 vil-/mice. arfrp1 vil-/enterocytes as well as arfrp1 mrna depleted caco-2 cells absorbed fatty acids normally but secreted chylomicrons with a markedly reduced triglyceride content. in addition, while the release of apolipoprotein a-i (apoa-i) was dramatically decreased apoa-i accumulated in the arfrp1 vil-/epithelium and was predominantly colocalized with rab2. our results demonstrate that the growth retardation of arfrp vil-/mice is a consequence of impaired intestinal fatty acid absorption. we suggest that arfrp1 is required for the assembly of aopa-i to the chylomicrons and for the further lipidation of chylomicrons in the golgi of intestinal epithelial cells. this finally leads to an secretion of chylomicrons with a markedly reduced triglyceride content. rhoa influences adhesion and spreading of cardiac fibroblasts via complex regulation of cytoskeletal proteins jatho a. 1 the monomeric gtpase rhoa is thought to be involved in the pathology of heart diseases, however, its role in cardiac cells is not well defined. therefore we intended to analyze the effect of rhoa in cardiac fibroblasts by using a lentivirus-based knockdown approach. by doing so, we could show that the knockdown of this gtpase by about 50% resulted in a massive change in cell morphology, but displayed no effect on cell viability. the appearance of the cells in the 2d-culture changed from a fibroblast-typical stretched morphology with intracellular stress fibers to a more epithelial-like cell morphology. by morphometrical analyses we demonstrate that fibroblasts with reduced rhoa expression display an increase in cell surface by 2.2-fold and in perimeter by 1.5fold. moreover, the depletion of rhoa significantly influences the adhesion velocity, as within the first hour after cell detachment about 20% of the rhoa knockdown cells reattach, but only 5% of the respective control cells. based on these findings, we investigated the distribution and composition of different cytoskeletal proteins by immunofluorescence stainings and immunoblot analysis. we found, that the amount of b-actin is not reduced in rhoa knockdown cells, however, the distribution is markedly changed. in these cells internal star-shape bundles of actin could be found instead of the commonly appearing stress fibers in control cells. in contrast, the cortical actin fibers, mainly consisting of g-actin, were not affected. in addition, smooth muscle-actin, which is characteristic for myofibroblasts, was clearly reduced in rhoa knockdown cells compared to control cells by 33%. this reduction might be responsible for the more relaxed cell shape. in summary, the knockdown of rhoa influences cell adhesion and the morphological characteristics of cardiac fibroblasts, without obviously affecting cell proliferation and viability. using site-specific fluorescence labeling to study uptake of pasteurella multocida toxin into eucaryotic cells jehle d., aktories k., orth j. institut für experimentelle und klinische pharmakologie und toxikologie abteilung 1, albertstraße 25, 79104 freiburg, germany pasteurella multocida is an opportunistic pathogenic bacterium living in the nasal pharyngeal space of animals. p. multocida is of particular importance in the livestock management of pigs. under special conditions infection of pigs with p. multocida leads to an atrophic rhinitis, which is characterized by the atrophy of nasal turbinate bones accompanied by a shortening and twisting of the snout. the causative agent of the atrophic rhinitis was found to be the bacterial protein toxin pmt (pasteurella multocida toxin). after entering the cell the 146-kda toxin activates various signal transduction pathways by stimulating heterotrimeric g proteins of the gαq, gα13 and gαi family. the underlying mechanism of the activation of heterotrimeric g proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the g protein. the uptake of pmt into cells is not comprehensively understood. therefore, we utilized a recently described technique, called "sortagging" (popp mw et al. nat chem biol. 2007; 3: 707) , to specifically couple fluorescence tags to the n-or c-terminus of pmt. the enzyme sortase a (srta) from staphylococcus aureus attaches proteins to the bacterial cell wall. the substrates are recognized by an lpxtg motif. srta cleaves the peptide bond after the threonine and adds a glycine-containing nucleophile. we introduced these motifs into pmt to express srta-recognized toxin and coupled the toxin with fluorescence tags, respectively. fluorescently labeled pmt was used to study the uptake of the toxin into eucaryotic cells by laser scanning microscopy. munich heart alliance, münchen, germany cells within the myocardium communicate by secreted factors and this has been suggested to contribute to cardiac remodeling. to identify novel factors secreted by the myocardium, we have previously reported a genetic yeast screen which led to the identification of protease inhibitor 16 (pi16). here we report the generation of a mouse line where pi16 can be deleted globally or conditionally using the cre/loxp system. after electroporation of the pi16 floxneo targeting vector in embryonic stem cells and injection into murine blastocysts we gained a mouse line that carried the targeted modification of the pi16 allele. global pi16 deficiency (pi16 lox/lox ) per se did not lead to a cardiac phenoytpe (hw/bw (mg/g): pi16 +/+ = 5.4 ± 0.2 (n = 6), pi16 lox/lox = 5.9 ± 0.7 (n = 4), p > 0.05; fibrosis (%): pi16 +/+ = 3.3 ± 0.2 (n = 7), pi16 lox/lox = 3.9 ± 0.8 (n = 5), p > 0.05; fractional shortening (%): pi16 +/+ = 29.8 ± 0.7 (n = 7), pi16 lox/lox = 26.4 ± 2.5 (n=5), p > 0.05). in addition we carried out an immunohistochemical analysis of pi16 expression using pi16-deficient mice as negative controls. pi16 localized to cardiac fibroblasts, to the epididymis and the trachea. in the failing heart we detected accumulation of pi16 preferentially in fibrotic areas. we are currently applying cardiac stress models to gain a broader understanding of the function of pi16 and its potential as a therapeutic target molecule. enantioselective determination of r-and s-hyoscyamine in mammalian plasma and urine samples john atropine (atr) is the racemic mixture of the tropane alkaloids s-and r-hyoscyamine (hyo). s-hyo acts as a competitive acetylcholine antagonist at the muscarinic receptors (eutomer) inducing mydriasis, excitations, hallucinations, coma and ultimately death, whereas r-hyo does not (distomer). atr is used for clinical intervention of poisoning with organophosphorus (op) pesticides or nerve agents. despite well known differences in pharmacological behavior, individual pharmacokinetics of both atr enantiomers have rarely been addressed in the literature [1, 2, 3] . therefore, we initially developed a nonchiral liquid-chromatography-electrospray tandem mass spectrometric method (lc-esi ms/ms) that allows quantification of atr and additional natural and synthetic tropane alkaloids from plasma after simple deproteinization [4] . to discriminate both atr enantiomers the sample preparation step was expanded by an enzymatic pretreatment. samples were incubated either with diluted human serum (not containing atropinesterase, atre, procedure a) or with diluted rabbit serum (procedure b). rabbit serum contains atre (ec 3.1.1.10) which is suitable for stereospecific hydrolysis of shyo into tropine and tropic acid while r-hyo remains unaffected. after sample precipitation, hyoscyamines were quantified by the lc-esi ms/ms method. following procedure a the concentration of total hyo and following procedure b remaining r-hyo were determined. s-hyo was calculated by the difference between these concentrations [3] . the impact of potential matrix ingredients that may appear in samples from oppoisoned patients under atr therapy were evaluated (oximes, op agents, carbamates) [3] . the assay was applied to diverse toxicological and pharmacological samples. i) measurement of natural s-hyo in an extract of atropa belladonna leaves as well as in plasma and urine of a female patient who was poisoned after ingestion of such leaves revealed that no biotransformation to r-hyo occurred. ii) analysis of plasma obtained from an op-poisoned female patient under atr therapy revealed faster elimination of shyo when compared to r-hyo [3] . iii) in contrast, no enantioselective differences were obvious in healthy anaesthetized swine after intravenous injection of atr [5] . data indicate that the enzymatic enantioselective procedure represents a useful tool to characterize in vivo behavior of r-and s-hyo allowing to reveal individual kinetics. failing hearts are unable to adequately supply the body with blood and oxygen. common therapeutic strategies interfere with cardiac remodelling, reduce cardiac preand afterload or aim at direct improvement of cardiac contractility. cardiac contractility is mainly controlled by β1-adrenergic receptors. resensitization of b-adrenergic receptors by inhibition of g-protein coupled receptor kinase 2 (grk2) is discussed as a potential strategy to treat heart failure. we characterized raf kinase inhibitor protein (rkip) as a physiological inhibitor of grk2 and found rkip to increase contractility of neonatal cardiomyocytes. the present study evaluated the role of rkip in heart failure. we assessed its effects on cardiac function in pressure overload induced heart failure and determined the expression patterns of rkip in failing hearts of humans and mice. transverse aortic contriction (tac) was performed on 7-week-old c57/bl6 mice to induce heart failure by pressure overload of left ventricles. after three weeks of tac, echocardiography showed distinct signs of decreased cardiac function in wild-type mice. fractional shortening was reduced and left ventricular diameters were increased. histological analyses revealed increased interstitial fibrosis, caspase-and tunelassays indicated myocyte apoptosis. western blot analysis showed significant upregulation of rkip expression in failing hearts compared to non-banded control hearts. interestingly, this upregulation of rkip expression was also detected in failing human hearts and in samples of patients with aortic valve stenosis but not in healthy control samples. to assess the effects of rkip overexpression on heart failure, we analysed heart function and structure of rkip transgenic mice and wild-type mice 3 weeks after tac. while left ventricular hypertrophy was increased to similar extents in wild-type and rkip trangenic mice, rkip mice did neither develop dilatation of the left ventricle nor a decrease in fractional shortening. in contrast to wild-type mice, the expression of the heart failure markers bnp and anp was not upregulated in banded rkip transgenic mice after 3 weeks of tac. taken together, cardiac overexpression of rkip prevented left ventricular dilatation and loss of contractile function in a mouse model of pressure overload induced heart failure. therefore, increased rkip expression may be an interesting target to prevent detrimental effects from increased left ventricular pressure. the main focus of this study was the chromatographic separation. the most frequently prescribed antibiotic drugs clarithromycin, erythromycin, clindamycin, cefuroxim, doxycyclin, amoxicillin, levofloxacin, and ciprofloxacin were selected for the screening method. method: a relatively short operation time and a sufficient separation were reached by column, eluent, and gradient optimization with poplc (phase optimized liquid chromatography). in the first step columns with the five stationary phases c18 eps 2, c18 sh 2, phenyl 2, cn 2, and c30 were used to determine the retention times of the drugs in an isocratic mode. the stationary phases, the column length and the retention times were fed in the poplc software and the optimal column was calculated. this column contained different stationary phases and was compared with customary columns. results: using the optimal column a sufficient chromatographic separation of the eight antibiotic drugs was reached. that was not possible with the customary columns. with the optimal column the time of measurement was too long. using a mobile phase gradient the measuring time could be reduced. discussion: with lc-ms/ms a complete chromatographic separation of all analytes is not necessary. but when measuring many transitions in a biological matrix two problems should be excluded: ion suppression and a too small number of measurement points per peak. especially when using positive and negative ionization in the ms a good separation is mostly necessary. to determine only the eight antibiotic drugs an optimized column is not necessary, but for a screening method with more than twenty drugs the poplc system is very helpful. we have investigated the uptake mechanism of cdt and in particular the intracellular membrane translocation of cdta. our data indicate that cdt requires acidification of the endosomal lumen for translocation of cdta across endosomal membranes into the cytosol. bafilomycin a1, an inhibitor of endosomal acidification protects vero (african green monkey kidney) cells from intoxication with cdt. consistently, translocation of cdta was observed when the acidic conditions of the endosomal lumen were mimicked at the cytoplasmic membrane of intact cells. next, we tested whether host cell factors are involved in membrane translocation of the toxin. radicicol, a specific pharmacological inhibitor of the chaperone heat shock protein hsp90 as well as cyclosporine a, an inhibitor of cyclophilins delayed the intoxication of cells with cdt but not with toxins a and b [1] . this result was confirmed by analyzing the adp-ribosylation status of actin from such cells in the presence or absence of the inhibitors. in addition, we excluded that the inhibitors of hsp90 and cyclophilins have any effect on receptor binding, endocytosis or enzyme activity of cdt. the data strongly suggest that the participation of hsp90 and cyclophilin is crucial for translocation of cdta into the cytosol. comparable results were obtained for the related binary iota toxin of c. perfringens. in vitro purified immobilized hsp90 and cyclophilin a specifically bound to the enzyme components of cdt and iota toxins. in conclusion, the results imply a common hsp90/cyclophilin a-dependent translocation mechanism for the family of binary actin-adp-ribosylating toxins. our current investigations focus on the participation of fk506-binding proteins (fkbps), another group of peptidyl-prolyl cis/trans isomerases in the membrane translocation step of these toxins. [1] kaiser, e., kroll, c., ernst, k., schwan, c., popoff, m. r., fischer, g., buchner, j., aktories, k. and barth, h. (2011) . complex multicellular in vitro coculture models represent a promising tool regarding e. g. cellular interactions with nanoparticles, since they more closely mimic the cellular composition of the body. therefore, we used our developed coculture model of the alveolar-capillary barrier composed of lung epithelial cells (nci h441) on top and microvascular endothelial cells (iso-has-1) on the bottom side of a filter-membrane to study nanoparticle cytotoxicity and cellular uptake. with a coculture period of about 10 days the cells achieve a more differentiated and polarized state and develop a tight barrier, which can be measured via ter (transepithelial electrical resistance). regarding cellular uptake of fluorescently labeled amorphous silica nanoparticles (asnps, 30 nm) the coculture took up much less asnps than conventional monocultures. besides, we could not verify a specific uptake mechanism (e. g. clathrin-, caveolae-mediated endocytosis) via immunofluorescence staining of the cells. however, we detected asnps incorporated in flotillin-1 and -2 labelled vesicles. former studies concerning cytotoxicity (lactate dehydrogenase assay) of amorphous silica nanoparticles (asnps, 30 nm) revealed that our coculture behaved much more robustly compared to conventional monocultures. however, regarding inflammatory responses (e. g. sicam, il-8 increase) the coculture responded more sensitively than conventional monocultures. in a further development we added a third cell type, the alveolar macrophage (am), to our coculture. since ams embody the front-line of alveolar defense against inhaled pathogens and particles, they play a central role in regulating lung immunity. as model we applied the human acute monocytic leukemia cell line, thp-1 (prestimulated with 8 nm pma for 4d) apically to the epithelial monolayer of the coculture. our preliminary studies concerning inflammatory responses of the tripleculture (h441/iso-has-1 with thp-1) revealed a higher sensitivity of the triple-culture compared to the double coculture. the triple-culture responded with an increased il-8 release upon lps or tnf-a stimulation. in conclusion, this triple-culture model offers a promising prospect to mimic more closely realistic cell interactions with nanoparticles in the distal lung. ethanol is a component of many herbal fluid preparations [1] , since it is an excellent extraction solvent for the phytochemical components of herbal drugs and contributes to the stability of these medicines. toxicological and pharmacokinetic evaluations [2] have shown that the small amounts of ethanol applied with therapeutic doses are safe even in children. despite that these medicines have been used safely since many decades, they have occasionally been subject of discussion in the public, triggered by the increasing problem of recreational abuse of alcoholic beverages by children and young persons [3, 4] . therefore, there is a growing need of a systematic evaluation of pharmacovigilance data on these medicines. for evaluating the experience gained from the therapeutic use of these medicines, 16 pro-and retrospective studies with 10 herbal medicinal products containing ethanol at doses of 40 to 240 mg per single dose, depending on the age group, have been analyzed, covering 49 816 patients of 0-12 years of age. in these studies, altogether 15 adverse drug effects have been described, none of which was attributable to the ethanol content of the medicines. in a survey of the worldwide use of these and other 6 herbal medicinal products it was shown that during the past few years, more than 764 mio daily doses have been sold, corresponding to more than 33 mio of patients (data obtained from manufacturers; figures available partly from 1993 onwards, partly from 2003/4 onwards). from the packages sold in germany in the years between 2005 and 2009, 48.1 mio were attributable to self-medication, 10.8 mio to prescriptions reimbursed by health insurance (ims, frankfurt). as non prescription medicines are reimbursed in germany only in children, at least the latter part of the prescriptions can be attributed mainly to children. all of these medicines are registered or licensed by regulatory authorities. adverse effects are covered by the pharmacovigilance system, and no adverse effects attributable to the ethanol content have been reported. this set of data supports the conclusion drawn from the experience of a safe use over decades, i.e. that the ethanol content of herbal medicinal products does not give any causes for concern regarding their safety even in children. dedication: this contribution is dedicated to prof. dr. hilke winterhoff, institute for pharmacology and toxicology, university of münster, who died on 9 may 2010. she has initiated this work. prucalopride was introduced as a new selective 5-ht4 receptor agonist that is approved for treating obstipation. whereas one could expect -due to the fact that 5-ht4 receptors are functionally expressed in the human heart -in clinical trials prucalopride did not show cardiac effects. this is quite surprising because other 5-ht4 receptor agonists have been withdrawn from the market just for that reason. in this study we used prucalopride for in vitro studies with atrial preparations from transgenic (tg) mice with cardiac myocyte-specific overexpression of the human 5-ht4a receptor. isolated electrically driven (1 hz) left and spontaneous beating right atria of tg mice were compared with those of wild type (wt) littermates. moreover, we used isolated electrically driven (1 hz) human right atrial preparations from patients undergoing cardiac surgery. finally gr113808, a 5-ht4 receptor antagonist, was added. prucalopride exhibited a dose dependent positive inotropic effect in left atria and a positive chronotropic effect in right atria of tg mice with a logec50 of -6.8 and -7.1, respectively (p<0.05 vs. wt, n=3). in human atrial tissue prucalopride also acts as an agonist, leading to an inotropic effect. all effects could be antagonized by 10 µm gr113808. we could demonstrate that prucalopride acts as an agonist at the 5-ht4 receptor in our transgenic mice model and also in human right atrial preparations. these findings suggest that tachycardia and arrhythmias are possible side effects, which should be carefully looked for. the involvement of psychological factors, especially stress, are known to play an important role in functional gastrointestinal diseases (1, 2) probably by affecting the brain-gut axis. based on the good correlation between stress & functional dyspepsia (fd), many animal models for fd have been developed where animals are subjected to various kinds of psychological stress either during the neonatal period or in adulthood. this stress was found to induce gastric motor dysfunction resembling symptoms of fd. two models for stressinduced fd were performed in order to choose the more adequate one for studying sensitivity changes of the fundus to various mediators as well as changes in some relevant hormone levels in the blood for subsequently testing the efficacy of treatment with stw 5 (a 9 component herbal preparation of proven clinical efficacy in fd and in irritable bowel syndrome (3, 4) ). in one model, maternal separation (5) was performed on weanling rats starting from postnatal day 2 for 3 h each day for 3 weeks. rats were then allowed to mature to an adult age. the other model was that of restraint stress (rs) (6, 7) . adult animals were restrained for 90 min/ day for 1 week. the animals of both models were eventually sacrificed, the stomach fundus was isolated and its sensitivity in vitro to carbachol, potassium chloride, serotonin and adrenaline was tested. blood samples were taken to assess levels of ghrelin, corticosterone releasing factor (crf) and corticosterone. the sensitivity of fundus strips from restrained rats towards the agents tested, partly representing autonomic responsiveness, was more depressed than those from maternally separated ones. levels of ghrelin, crf and corticosterone were also more elevated in the rs model. that model was therefore chosen to test the efficacy of stw 5 in restoring the deranged parameters. a group of animals received stw 5 orally once daily starting treatment 1 week before exposing them to rs and continuing treatment for a further week during subjection to rs. stw 5 was effective in normalizing the depressed stomach fundus responses exhibited by animals subjected to rs and to normalize to a large extent the deranged blood levels of ghrelin, crf and corticosterone. the findings lend further evidence to the role of the brain-gut axis in fd and gives supportive evidence for the first time for the clinical usefulness of stw 5 in this condition. there is good evidence that oxidative damage to dna leads to down-regulation of transcription of affected genes and epigenetic gene silencing in a mechanism dependent on the 8-oxoguanine dna glycosylase (ogg1), which generates harmful repair intermediates [1, 2] . we have recently shown that the magnitude of inhibition of transcription of an egfp reporter gene by single 8-oxo-7,8-dihydro-deoxyguanosine (8-oxodg) varies between the opposing dna strands of the gene [3] . we now have addressed the question, to which extent the transcription inhibitory potential of 8-oxodg depends on its position in the gene and on the dna microsequence surrounding the modified nucleobase. to investigate the effect of position, we produced plasmid vectors containing single 8-oxodg or dg (underlined) in the second position of an agc trinucleotide. measurements of egfp expression in transfected mammalian host cells showed that a single 8-oxodg caused a strong inhibition of gene transcription. the magnitude of this effect for 8-oxodg situated in the transcribed dna strand of the gene was the same as in the non-transcribed dna strand. similarly, there was no quantitative difference between the effects of 8-oxodg present in the 5′-versus 3′-utr of the gene. the results thus indicate that inhibition of transcription by this base modification does not depend on the position in the gene. further comparisons were done between the effects of 8-oxodg nucleobases localised in the same dna strand but within different sequence contexts. gene expression analyses in the repair proficient host cells showed that the degree of inhibition of transcription caused by single 8-oxodg was dependent on the neighbouring nucleotides. among three tested sequence motifs, the minimal effect on the gene transcription was found to correlate with a significantly less efficient base excision by the purified human ogg1 protein. the results thus support the initiatory role of ogg1 in the mechanism of transcriptional repression. in addition, the finding of the effect of dna sequence on the base excision activity of ogg1 suggests that repair rates of single base modifications in genome could also be heterogeneous. allgayer j., kitsera n., epe b., khobta a. johannes gutenberg university of mainz institute of pharmacy and biochemistry, staudingerweg 5, 55128 mainz, germany interference of dna base modifications with gene transcription is an important biological consequence of genotoxic damage [1] . an efficient method for incorporation of a single 8-oxo-7,8-dihydroguanine (8-oxog) at a defined position in the egfp gene in a plasmid vector was recently developed in our lab. the method relies on the availability of adjacent sites for a sequence-specific nicking endonuclease, which allow the insertion of a synthetic oligonucleotide containing 8-oxog in a chosen position. we further showed that this single lesion inhibits transcription after excision by ogg1 [2] . in order to determine to which extend the observed effect depends on the position of the modified base in the gene, we constructed several new plasmid vectors which allow incorporation of the same dna oligonucleotide containing 8-oxog or g in different positions in different dna-strands. dna sequence cassettes were designed to contain a 5′-cattgcttcgctagcacg nucleotide sequence in different orientations, which was flanked by two unidirectional bsrdi recognition sites (5′-gcaatgnn). adapters containing the restriction sites for directional cloning into the 5′-or the 3′-utr of the plasmid-borne egfp gene were added. the produced plasmid vectors thus allow the insertion of a single 8-oxog in the same sequence context into opposing dna strands in the 5'utr and in the 3'utr. keratinocytes are the major cell type of epidermis and are responsible for the formation of an outer barrier, the statum corneum, to protect an organism against harmful environmental influences. for generation of this barrier, keratinocytes pass through a complex differentiation program that is accompanied by synthesis of lipids, like cholesterol and ceramides. finally, the differentiation of keratinocytes leads to apoptosis. another function of keratinocytes is to sense environmental factors, some of which are decoded by members of the transient receptor potential (trp) ion channel family. trpv3, for example, is predominantly expressed in keratinocytes, and decodes different chemical and physical stimuli like the terpenoid-derived ligands camphor and thymol or temperatures above 33°c. less is known about the influence of cholesterol on trpv3 signalling. we modified the cholesterol content of hek293 cells stably transfected with trpv3 and performed flipr-based calcium measurements. these experiments revealed that cholesterol enrichment robustly potentiates trpv3 by sensitizing it to lower agonist concentrations. we verified these results with whole-cell patch-clamp measurements. in contrast, trpv2, another heat-sensing channel, was not affected by cholesterol modification. since former studies showed a defective formation of epidermal barrier in trpv3 -/mice, our results imply that a cholesterol-regulated trpv3 signalling may contribute to the progression of differentiation or initiation of apoptosis of keratinocytes. ischemia-reperfusion injury causes severe problems in the early period after lung transplantation. since transient receptor potential (trp) channels are important regulators of vascular permeability and tone, we investigated the influence of a trpc6 blocker on pulmonary function after simulated transplantation, using an ex vivo model of isolated perfused and ventilated rabbit lungs. to this end, heart-lung blocks were excised and mounted in an artificial thorax chamber. negative pleural pressure ventilation was initiated, and lungs were perfused with albumin-containing tyrode-solution (100 ml min -1 ). after equilibration in a stable perfusion and ventilation mode for 30 min, lungs were flush-perfused with perfadex ® solution, followed by an ischemic storage for 4 h on ice. subsequently, ventilation and perfusion were re-initiated to simulate a transplantation situation. in the oxygenated group, pulmonary artery po2 was adjusted to 120 mmhg, in the deoxygenated group, the perfusate inflow was gassed with nitrogen to achieve a pulmonary artery po2 of 50 mmhg. both transplantation conditions were conducted in the absence or in the presence of the trpc6 blocker larixol acetate (5 µm). hemodynamic and ventilatory parameters were continuously monitored. the weight of deoxygenated lungs steadily increased during 2 h of reperfusion from 22.1 ± 1.32 g to 35.4 ± 4.23 g. this weight gain was inhibited by supplementation of the trpc6 blocker (from 21.4 ± 0.91 g to 27.4 ± 2.42 g 2 h after reperfusion). in contrast, oxygenated lungs showed no marked weight gain after reestablishment of perfusion (from 17.5 ± 1.51 g to 21.5 ± 2.29 g 2 h after reperfusion), and the trpc6 blocker had no additional effect (initial 19.5 ± 1.28 g, 2 h reperfusion 21.3 ± 1.22 g). we conclude that a trpc6-blocking compound to the lung perfusate during simulated transplantation counteracts the endothelial permeability increase and the resulting post transplant weight gain. the results indicate a role for trpc6 in the regulation of pulmonary vessel permeability and support the concept that perfusion of donor lungs with trpc6 blockers may prevent edema formation caused by ischemiareperfusion injury shortly after lung transplantation. regulation of human inducible nitric oxide synthase (inos) expression by noncoding rnas (ncrnas) kleinert h., schmitz k., koch k., hahn s., pautz a. universitätsmedizin der johannes gutenberg-universität mainz institut für pharmakologie, obere zahlbacher str. 67, 55101 mainz, germany the transcriptome analyses of human cells showed that additionally to the 30.000 protein coding sequences the human dna codes for much more (450.000 ?) non-coding rnas 1 . beside the ribosomal, and transfer rnas (rrna and trna) involved in the mechanism of translation there are also short (snrna, sorna, mirna) and long noncoding rnas (ncrnas) implicated in regulation of gene expression (splicing, translation, chromatin packaging etc.). matsui et al. described regulation of il-1β-induced inos expression by an antisense rna (as-3-utr-rna) in rat hepatocytes 2 . a promoter located on the antisense strand 3' to the last exon (exon 27) of the rat inos gene drives the expression of an as-rna complementary to the 3'-utr of the rat inos mrna. using different sense primers with homology to the 3'-utr sequences of the human inos gene for specific rt-pcr reactions (detecting only an as-rna) we were not able to detect such an as-3-utr-rna in human cells. in addition, transient transfection analyses using constructs containing a 2.7 kb fragment of the 3'-flanking genomic sequences (as used by matsui et al. in the rat system) of the human inos gene in front of a luciferase reportergene into dld-1 cells revealed no promoter activity of these sequences. korneev et al. described the expression of a 2 kb anti-inos-ncrna in different brain tumors and showed reciprocal expression to the inos mrna in human embryonic stem cells 3 . analyzing the expression of this anti-inos-ncrna in cytokine-treated dld-1 cells also showed a basal expression of this as-rna and an enhancement of the expression by cytokine-treatment. downregulation of the anti-inos-ncrna by sirnas reduced whereas overexpression enhanced cm-induced inos expression in human dld-1 cells. this indicates that this anti-inos-ncrna regulates cytokine-induced inos expression in a positive manner. rna 14, 2030 rna 14, -2037 rna 14, (2008 . using directed evolution to improve functional expression of class b g-protein coupled receptors klenk c., scott d. j., plückthun a. universität zürich institut für biochemie, winterthurerstrasse 190, 8057 zürich, switzerland the class b of g-protein coupled receptors (gpcrs) comprises 15 peptide hormonebinding receptors which regulate important endocrine and neuroendocrine functions of the human body. several of these receptors are implicated in the pathogenesis of severe diseases such as diabetes, osteoporosis, growth disorders and depression, which makes them attractive targets for drug therapy. to develop new compounds targeting these receptors a detailed understanding of the molecular structure is required which has not been succeeded to date. structural studies of proteins by x-ray crystallography or nmr spectroscopy generally require large and homogenous quantities of protein. for gpcrs this prerequisite is difficult to achieve as the vast majority of gpcrs exhibits low endogenous expression and is very unstable in solution. therefore, improved expression conditions are necessary for the efficient characterization of new gpcr structures. here we present a method to optimize class b gpcrs for improved heterologous expression and increased thermostability by means of directed evolution. libraries of class b gpcrs were obtained by random mutagenesis and were expressed in a heterologous expression system in which functional gpcr is targeted to the inner membrane of e. coli. mutants that display increased receptor expression levels and ligand binding were selected by flow cytometry using fluorescently labeled ligands. repetitive cycles of randomization and selection allow to gradually increasing the level of protein expression and stability. with this evolutionary approach key residues within the receptor sequence can be identified rapidly that are responsible for improved biophysical properties without affecting the pharmacological features of the receptor. such gpcr mutants will become a valuable tool on the way to express high quantities of stable receptor protein for subsequent structural studies. pasireotide (som230) is currently under clinical evaluation as a successor compound to octreotide for the treatment of acromegaly, cushing's disease and carcinoid tumors. whereas octreotide acts primarily via the sst2 somatostatin receptor, pasireotide was designed to exhibit octreotide-like sst2 activity combined with enhanced binding to other somatostatin receptor subtypes. somatostatin and octreotide stimulate the complete phosphorylation of at least six carboxyl-terminal serine and threonine residues namely s341, s343, t353, t354, t356 and t359, which in turn leads to a robust endocytosis of the sst2 receptor. surprisingly, pasireotide failed to phosphorylate the four threonine residues and induced only a detectable phosphorylation of s341 and s343. somatoprim and ke108 are recently developed somatostatin analogs capable of binding to four of five somatostatin receptor subtypes. here, we performed an in vitro study comparing the effects of pasireotide, somatoprim and ke108 on sst2 somatostatin receptor binding, phosphorylation, internalization and signaling. further somatostatin, octreotide, pasireotide, somatoprim and ke108 were tested for functional selectivity at sst5 receptor mutants, which possess a carboxyl-terminal sst2tail. this approach allows detection of receptor activation by phospho-specific sst2 antibodies. compared to octreotide, somatoprim activates sst2 but has a higher activity on sst5. ke108 and pasireotide are partial agonists at the sst2 receptor. pasireotide, ke108 and somatoprim show comparable effects on sst5 receptor. however, none of these new pan-somatostatin analogs behaves like natural somatostatin on the sst5 receptor. cadmium modulates ahr-associated gene expression in the rat intestine after oral exposure kluxen f. m. 1, 2 , höfer n. cadmium has been shown to mimic steroid estrogen effects in vivo and in vitro. we have recently identified cross-talk of estrogen receptor (er) and aryl hydrocarbon receptor (ahr) in the rat uterus where 17beta-estradiol (e2) and cdcl2 modulate ahrassociated genes via er after i.p. injection in a similar fashion (kluxen et al., arch toxicol doi 10 .1007/s00204-011-0787-x). however, the predominant route of exposure to cadmium in non-smokers is via diet. moreover, uterus expresses mainly the receptor subtype eralpha, whilst small intestine and colon express mainly erbeta. thus, we now investigated by real-time rt-pcr the effects of cadmium (2mg/kg b.wt cdcl2 (cd2)) or steroid estrogen (0.1 mg/kg b.wt. 17alpha-ethinylestradiol (ee2)) on ahrassociated gene expression (i.e., ahr, cyp1a1, gsta2, nqo1) in the small intestine of rats after oral exposure (3 days gavage). the animals were also co-treated with cd2 and pure anti-estrogen (2.5 mg/kg b.wt zk191703 (zk)) or ee2, to asseess whether ermediated processes are involved. we also measured cyp1a1 mrna expression in two estrogen receptor negative colon cancer cell lines (ht-29 and caco2) treated for 5 days with cdcl2 (1µm) and e2 (0.01µm). the dose-dependency of cadmium induced ahr target gene modulation was studied in a second animal experiment, with administration of cadmium in drinking water for 28 days (0.4-9 mg/kg b.wt cdcl2 equivalent to 5, 50, and 150 ppm) and ee2 (0.08 mg/kg b.wt) as steroid reference. in summary we present two major results: the metalloestrogen cadmium modulates dose-dependently the ahr-associated gene expression in the intestine after oral exposure. yet, since the cadmium induced modulation of ahr target genes was not antagonized by anti-estrogen in the small intestine in vivo and was also found to occur in er-negative colon cells in vitro, we propose that er-independent mechanisms might play a role in this effect. meg) is the most cytotoxic lesion. if not repaired by o 6 -methylguanine-dna methyltransferase (mgmt), o 6 meg/t mismatch is recognized by the mismatch repair system (mmr) that performs futile repair cycles. during this process secondary lesions (i.e. dna single-strand brakes) are formed, which block dna replication in the next replication cycle, leading to dna double-strand breaks (dsbs). these dsbs eventually signal to apoptosis and other genotoxic endpoints. here, we wished to address the question whether autophagy is part of the cellular response triggered by o 6 meg. we also assessed whether autophagy influences apoptosis induced by tmz in glioma cells. we show that tmz induces autophagy in u-87 mg and ln-229 glioma cell lines. the maximum amount of autophagy was observed several days (96 h) after tmz treatment and mgmt proficient cells did not display significant autophagy. thus, the data show that mgmt protects against tmz induced autophagy, pointing to o 6 meg as the critical lesion responsible for induction of autophagy. using colon cancer cell lines proficient and deficient in mmr, we show that mmr is required for tmz-induced autophagy. because dsbs, which emerge during the processing of o 6 meg, are repaired preferably by homologous recombination (hr) ln-229 cells stably down-regulated for hr were tested for autophagy induction. the data indicate that dsbs are involved in tmz-induced autophagy. because autophagy following tmz treatment occurs earlier than apoptosis we hypothesize that autophagy protects glioma cells against apoptosis. using an early stage autophagy inhibitor 3-methyladenin we have shown, that autophagy inhibition sensitized glioma cells to tmz-induced apoptosis. taken together our data point out that tmz induces autophagy in glioma cells and that autophagy protects glioma cells against tmz-induced apoptosis. o 6 meg is the lesion responsible for autophagy induction. furthermore, the data also shows that mmr and dsbs are involved in the induction of autophagy after tmz treatment. work was supported by bmbf (02nuk016). retinitis pigmentosa (rp) is a severe human retinal disease characterized by a progressive degeneration of rod photoreceptors and a secondary loss of cone function. in most cases, rp finally leads to legal blindness. mutations in the regulatory subunit of the rod cyclic nucleotide-gated (cng) channel (cngb1a) have been found in patients suffering from rp. we used cngb1-deficient (cngb1 -/-) mice to establish a gene replacement therapy as a potential treatment for rp by means of recombinant adenoassociated viral (raav) vectors. the packaging limitations of raav vectors required a capacity-optimized vector of the large cngb1a cdna (approx. 4 kb). therefore, we replaced regulatory elements within the expression cassette and used a short mouse rhodopsin promoter element for rod-specific expression. after injection of therapeutic raavs (serotype 8) into the subretinal space of 2-week-old cngb1 -/mice, we assessed the restoration of vision by analyzing i) protein expression and localization, ii) retinal function and morphology and iii) vision guided behavior. we found that treated cngb1 -/mice expressed full-length cngb1a and cnga1, which were previously downregulated. both proteins co-localized in rod outer segments and formed regular cng channel complexes in the treated area of the cngb1 -/retina. using electroretinography (erg) we observed a distinct rescue of rod-driven responses. moreover, cngb1 replacement significantly preserved outer segment morphology and delayed retinal degeneration. finally, treated cngb1 -/mice performed significantly better than untreated mice in a modified water maze task designed to test for rod-mediated, vision-guided behavior. in summary, this work provides a proof-of-concept for the treatment of rod channelopathyassociated retinitis pigmentosa by raav-mediated gene replacement. most endocrine disruptors interact with hormone receptors or steroid biosynthesis and metabolism, thereby modifying the physiological function of endogenous hormones. here, we present an alternative testing paradigm for detection of endocrine modes of actions that replace and reduce animal testing through refinement. receptor mediated endocrine effects were assessed using the yeast based receptor mediated transcriptional activation yes/yas assays and effects on steroid hormone biosynthesis were assessed using the human cell line h295r in the steroidogenesis assay. in our testing paradigm we propose to complement the in vitro assays with a single in vivo repeated dose study in which plasma samples are analyzed for their metabolome profile. the combination of these methods does not only contribute to refinement and reduction of animal testing, but also has significantly increased the efficient allocation of resources and allows for a sound assessment of the endocrine disruption potential of compounds. thus, this proposal constitutes a potentially attractive alternative to epa's endocrine disruptor screening program. data on 14 reference substances for which the in vitro yes/yas and steroidogenesis assays and the in vivo metabolome analysis were performed to assess their putative endocrine mode of action is presented here. the bovine corneal opacity and permeability (bcop) test has been adopted by oecd for the identification of corrosive and severe ocular irritants (ghs category 1) for single component substances and multi-component formulations. eye irritation tests (eit) using human reconstructed tissue models (such as epiocular) have been described to predict ocular non-irritants (ghs no category). thus the ultimate repaltement of the draize rabbit eye irritation test (oecd tg 405) by a combined or tiered testing strategy could be possible. the purpose of this study was to evaluate whether the bcop with additional corneal histology together with the eit could be used to predict eye irritancy of agrochemical formulations according to different classification schemes including un ghs and epa systems. we have performed the bcop (plus histology) and the eit of 50 agrochemical formulations for which in vivo eye irritation data were already available (for registration purposes). using the oecd tg guideline evaluation scheme for opacity and permeability in the bcop was not predictive for the agrochemical formulations assessed here, while corneal histology grades and the epiocular tissue viabilities were useful predictors of eye irritancy potencies and could be applied for the different classification schemes. the nanomaterials offers extraordinary opportunities. the nano-structure can change the physical and chemical properties, and often also alters the biological effects. hence the toxicity of a nanomaterial can differ from its larger-scale material; but as of today, no new quality of a general nano-specific toxic effect has been observed. therefore the established testing methods are generally suitable. it is, however, difficult to apply the nanomaterials to in vitro test systems, since it is the nature of these materials to change their surface properties and agglomeration stateand the uptake and distribution in the body may differ from their larger-scale materials. while the methods for topical effects may be used for nanomaterials without further modification, the in vitro methods for genotoxicity testing require the dispersion in culture media. the use of reproducible and well-documented dispersion protocols andthe characterization of its particle size distribution is de rigueur . [1] . for many nanomaterials published genotoxicity studies did not give a consistent picture [2] and therefore there are rather effects of individual nanomaterials than nano-genotoxicity per se. modern toxicology is based on the insight into toxic pathways. for nanomaterials a testing strategy will include testing for their primary effects (which might be only a handful: particle effects, catalyzing the formation of reactive molecules and ion release) and their uptake, distribution and clearance. the use of dermal penetration studies in vitro for the risk assessment of sunscreen nanomaterials has been demonstrated [3] . in vitro methods for specific effects (such as inflammation, pulmonary toxicity, sensitization) are currently awaiting validation (for both chemicals/molecules as well as nanomaterials). in the meantime alternative short-term in vivo studies with optimized biological readouts can deliver information on the toxic pathways as well as the biokinetics and dose-response relation of nanomaterials in the body. a short-term inhalation test for nanomaterials has already been used successfully [4] . testing strategies based on those methods engage less animals and provides more significant data than classical testing. moreover data from these methods will serve as a benchmark and a validation for the in vitro models under evaluation. nanoparticles (np) are increasingly used in various field of industry which necessitates evaluation of their safety. also in the food industry, nps have gained strong interest, for example as food additives or to improve food packing. however, the potential risks of ingested np have been rarely investigated. inhalation studies have revealed that inflammation and oxidative stress may represent unifying mechanism for the induction of adverse health effects of toxic np. in the present study, a co-incubation model of human polymorphonuclear neutrohils (pmns) and caco-2 human intestinal epithelial cells was used as a model to address potential genotoxic effect of np during intestinal inflammation. oxidative dna damage induction (measured by the fpg-modified comet assay) was induced in the caco-2 cells by activated pmn and this effect increased with increasing pmn to caco-2 cell ratio. the crucial involvement of the phagocyte nadph oxidase complex could be demonstrated using treatment of caco-2 cells with bone marrow-derived pmn from nadph oxidase deficient mice. dna damage by pmn as well as h 2o2 was increased in buthionine sulphoximine (bso) pre-treated caco-2 cells, illustrating the importance of the cellular glutathione (gsh) status in these target cells. gsh depletion in caco-2 cells could also be shown upon treatment with various types of np. our data suggests that ingested np may increase the susceptibility of the colon mucosa to genetic damage during the occurrence of intestinal inflammation. the ingestion of seafood contaminated by acute toxic doses of the marine toxin okadaic acid (oa) is responsible for diarrhetic shellfish poisoning. it is recently known that both the rat and the human hepatic cytochrome p450 monooxygenases (cyp) are able to metabolize this toxin. currently, there is a lack of data about the toxicity and mode of action of oa after xenobiotic metabolism. the aim of our study was the measurement of the toxicity and oxidative stress status in hepg2 cells incubated with oa in the absence and presence of s9 mix. pure oa, as well as oa pre-activated with liver homogenisates (s9 mix) were used to treat human hepg2 cells that have nearly undetectable levels of functional cyp but express phase ii enzymes. the experiments were performed with both human and rat s9 fraction plus cofactors of phase i enzymes. the cell viability was measured after 4 h using mtt-test and xcelligence real time cell monitoring system. furthermore, levels of intracellular reactive oxygen species (ros) were detected by 2´,7´-dichlorofluorescein diacetate and additionally by measuring the intracellular glutathione content. in the presence of both human and rat s9 mix oa showed a higher toxicity than the parental substance. oa pre-incubated in rat s9 mix was toxic at 75 nm oa. strong effects could be observed when oa was pre-activated with human s9-mix at a concentration of 50 nm oa. pure oa was non-toxic in that concentration range. we could also detect an increase of oxidative stress in hepg2 cells treated with oa in the presence of all investigated s9-mix. these results suggest that oa is activated after oxidative xenobiotic metabolism into metabolites which possess a higher cytotoxic activity and increase the amount of intracellular ros in hepg2 cells. ballast water treatment -emerging health risks werschkun b., banerji s., krätke r. bundesinstitut für risikobewertung, max-dohrn-strasse 10, 10589 berlin, germany the introduction of invasive marine species into new environments by ships' ballast water, via ships' hulls and other vectors has severe impacts on the oceans. in 2004 the international maritime organisation (imo) launched the international convention for the control and management of ships ballast water and sediments which requires ballast water to be treated in order to eliminate alien aquatic species. ballast water treatment may include mechanical, physical or chemical measures. any ballast water management system using active substances needs imo approval. therefore identification of active substances, relevant chemicals and submission of specified datasets on their physical, chemical and toxicological properties is required in order to assess the safety for the aquatic environment and for human health. the bfr is the german federal agency responsible for health risk assessment and has been involved in more than twenty assessment and approval processes so far. the majority of imo approved systems are based on oxidative principles such as chlorination and ozonation. these methods can generate disinfection by-products (dbps), which are a mixed group mostly of halogenated organic substances like trihalomethanes, haloacetic acids and haloacetonitriles. the formation of dbps is well known from the disinfection of drinking water. some dbps are regulated under drinking water directives because of their long-term toxicity but many are unregulated and have unknown toxicological properties. the formation of dbps may vary significantly depending on the treatment system as well as on environmental parameters like temperature, ph and composition of the organic matter within the aquatic environment. in sea water, sources for dbp formation besides ballast water treatment are aquaculture and the cooling systems of coastal power plants. in order to address possible health and environmental risks from dbps formed during ballast water treatment a conference on emerging risks from ballast water treatment was held at bfr in october 2011. here we summarise the main conference findings and identify areas for future research. two presentations corroborated that significant amounts of dbps can be formed in sea water and a presentation on the toxicological properties of dbps pointed out that many have genotoxic properties. accordingly, the determination of dbp species and generated concentrations under different ballast water treatment conditions was seen as a mayor task. different approaches for health and environmental risk assessments were also discussed. appropriate human exposure scenarios and methods for exposure assessment, taking into account common approaches used in risk assessment were presented during the conference. a suitable approach based on derived pec-values for exposure quantification was proposed in order to improve the procedure available for risk assessment of chemical agents used for ballast water treatment. agonist-selective signaling of µ-opioid receptors in t lymphocytes kraus j., börner c., lanciotti s., koch t., höllt v. inst. für pharmakologie und toxikologie, leipzigerstr. 44, 39120 magdeburg, germany opioids are the most potent analgesics and irreplaceable for the treatment of severe pain. in addition to their central effects, opioids modulate a great variety of immune effector cell functions, which may result in unwanted side effects during opioid treatment. the effects of most of the commonly used opioids are mediated by µ-opioid receptors, which belong to the superfamily of g protein coupled receptors. recent data support the concept that g protein coupled receptors function as dynamic entities, which may occupy multiple conformations and activate multiple signaling pathways in a ligand-dependent manner. consequently, different ligands activating the same receptor may have different cellular effects, which has been termed "agonistselective signaling". little is known about agonist-selective signaling of µ-opioid receptors in immune effector cells. in a first attempt to understand if and why such different profiles among different opioids occur we investigated effects of different opioids in human jurkat t cells. we report that the µ-opioid receptor ligands fentanyl, methadone, loperamide and betaendorphin induce internalization of a µ-opioid receptor-green fluorescent reporter construct, whereas morphine and buprenorphine did not induce internalization. the internalization was dependent on p38 mapk and phospholipase d2. in line with this, we observed marked phosphorylation of p38 mapk and activation of phospholipase d2 induced by the internalizing opioids, but no or little such activity by morphine and buprenorphine. as a physiological result, fentanyl, methadone, loperamide and betaendorphin treatment of primary human t cells and jurkat t cells resulted in a strong, up to 100 fold induction of il-4, which was dependent on p38. in contrast, morphine and buprenorphine only showed a weak, approximately one order of magnitude lower induction of il-4. by inducing il-4 opioids significantly modulate the t helper cell balance into the type 2 direction, which influences various immune responses, e. g. the antiviral, t helper cell type 1-mediated response. considering the vital necessity of opioid use in humans, it is an intriguing goal to identify analgetically feasible opioids that have little or no immunosuppressive or -modulatory effects. modulation of cgmp signals by phosphodiesterases in smooth muscle cells krawutschke c., koesling d., russwurm m. ruhr-universität bochum pharmakologie und toxikologie, universitätsstrasse 150, 44780 bochum, germany within the cardiovascular system, cgmp mediates smooth muscle relaxation and inhibition of platelet aggregation. cgmp is formed by particulate guanylyl cyclases and nitric oxide-sensitive guanylyl cyclases, that are activated by natriuretic peptides or nitric oxide (no), respectively. besides the cgmp-forming enzymes, the cgmp-degrading phosphodiesterases strongly determine amplitude and shape of cgmp signals. in vascular smooth muscle cells, three phosphodiesterases are considered to be responsible for cgmp degradation: pde5, the cgmp-specific phosphodiesterase is activated directly by cgmp binding to its gaf domains; this activation if further stabilized by cgmp-dependent protein kinase-mediated phosphorylation. pde1, the ca2+/calmodulin-stimulated pde constitutes the majority of cgmp-hydrolyzing activity in smooth muscle cells at least in the presence of high intracellular ca2+ concentrations. and lastly pde3, the cgmp-inhibited pde displays some cgmp-degrading activity, although cgmp binding to its catalytic domain is primarily thought to inhibit the campdegrading activity of pde3. cgmp signals measurable in radioimmunoassays require stimulation with cgmpincreasing vasodilator concentrations that are orders of magnitudes higher than those required for relaxation. thus, we developed fluorescent sensors for real-time measurement of cgmp signals in single cells. by using these indicators, we analyzed the contribution of different cyclic nucleotide-degrading phosphodiesterases to the modulation of cgmp signals elicited by physiologically relevant vasodilator concentrations. hyaluronan (ha) is a major component of extracellular matrices and is thought to control cellular phenotypes such as proliferation and migration. therefore, ha synthesis may play an important role in the pathophysiology of atherosclerosis. there are three major ha-synthase isoenzymes (has1-3). the has3 gene is alternatively spliced. has3 transcript variant 2 (has3v2) encodes the smallest has isoenzyme which has a different c-terminus and contains only two transmembrane domains compared to has3 transcript variant 1. the aim of the present study was to investigate whether has3v2 is expressed by vascular cells, how it is regulated and where it is localized in cells and whether it indeed synthesizes ha. has3v2 mrna expression was monitored by quantitative real-time rt-pcr. protein expression was determined by western blotting using a polyclonal antibody that was raised in rabbit. an n-terminal eyfp-has3v2 fusion protein and a ddk-tagged has3v2 construct were expressed for subcellular localization studies and co-immunoprecipitation (co-ip). endogenous has3v2 mrna was expressed in both vascular smooth muscle cells (vsmc) and endothelial cells. furthermore, western blotting revealed has3v2 protein expression in vsmc and platelets. in vsmc has3v2 mrna expression was strongly up-regulated in response to interleukin-1β (il-1β, 10 ng/ml) whereas stimulation with interleukin-10 (10 ng/ml), platelet-derived growth factor-bb (10 ng/ml), transforming growth factor β (10 ng/ml), tumor necrosis factor α (10 ng/ml) and interferon-γ (10 ng/ml) had no effect. transfection of hek cells with eyfp-has3v2 fusion protein revealed localization to the endoplasmic reticulum but not to the plasma membrane. furthermore, co-ip experiments showed that tagged has3v2 proteins were precipitated together suggesting formation of multimeric has3v2 complexes. transfection of has3v2 did not cause increased secretion of ha into the cell culture supernatant in hek cells. in conclusion, has3v2 is present in vascular cells and responds to inflammatory cytokines such as il-1ß. because of the intracellular localization and the lack of ha secretion in has3v2 transfected cells, has3v2 may serve intracellular functions apart from ha synthesis. the tubulin antagonist pretubulysin shows strong vascular-disrupting properties in vitro several epidemiological studies indicate a correlation of human exposure to ultrafine particulate air pollution caused by incomplete combustion processes and an increase in the incidence of pulmonary immune diseases like asthma. as a possible mechanism behind this pathological phenomenon, the adjuvant effect of lung inflammation induced by poorly soluble environmental particles has been hypothesised. the aim of our study was to investigate the causal link between carbon nanoparticle-induced lung inflammation and modulations of immune cell populations during processes leading to sensitization, and allergic immune responses of the airways. therefore mice were treated with ovalbumin (ova) alone or in combination with carbon nanoparticles (cnp) by pharyngeal aspiration. the induction of inflammation and the immune adjuvant activity were studied in the lungs and lung draining peribronchial lymph nodes (pbln) at the level of sensitization, and at the level of the immune response. ova-specific ige antibodies were measured in blood serum, and the development of allergic airway inflammation was studied after ova challenge. results at the level of sensitization showed that cnp-induced immediate airway inflammation had immune adjuvant activity resulting in an increase of specific cell populations in pbln and in a stimulation of asthma-specific th2 cytokines. a specific reduction of the neutrophilic lung inflammation by application of the compatible solute ectoine significantly reduced the adjuvant effects of cnp. in ova-sensitized mice, application of cnp 12 hours prior to allergen challenge, led to a significant increase in inflammatory cell infiltrate and respective cytokines in broncho-alveolar lavages. coapplication of 1 mm ectoine together with cnp reduced the particle-induced effects. our data show a link between neutrophilic lung inflammation and adjuvant effects of cnp. a specific reduction of neutrophils by the application of ectoine attenuated this np induced adjuvant effect, indicating that particle-induced lung inflammation rather than the direct interaction of nanoparticles with immune cells is the critical step in environmentally modulated pulmonary immune diseases like asthma. introduction: drug-eluting stents (des) are commonly used in the treatment of acute artery occlusion. however, even if released cytotoxic drugs reduced neointimal proliferation significantly there is still the risk of in-stent thrombosis. it is presumed that this is due to reduced reendothelialization. it has been suggested that coating the stent with biomolecules may provide a new approach to circumvent the lack of healing of the endothelial layer. one approach would be the use of biomolecular signals, such as (poly)peptides and growth factors. rgd and redv are peptide motifs, known to enhance cell attachment and spreading. aim: the aim of our study was to evaluate the efficacy of proteins, derived from elastin-like proteins (elp) and artificial modified by incorporating with the amino acid motifs rgd,redv and p15, in terms of endothelial healing on stents and other cardiovascular devices. results: in this work, we generated vectors encoding for different biopolymers consisting of various bioactive signal molecule sequences. the peptides, e.g. based on the elastinlike matrix (vpgig)2-vpgkg-(vpgig)2, were synthesized using heterologous expression. after optimizing culture conditions and extraction procedures their biological activity was assessed using human umbilical vein endothelial cells (huvec). elastin like proteins with differently incorporated bioactive signals (redv, rgd and a small p15 peptide) were linked covalently via carbodiimide coupling to poly(l-lactide) (plla) films. huvec growth was determined on these modified surfaces using the brdu assay (cell proliferation) and resazurin assay (cell viability). the chemically modified plla surfaces conferred higher cell viability after 1 h adhesion (60%) and an enhanced proliferation (63%, 1 h adhesion, 24 h cultivation) in comparison to the unmodified plla. these results indicate that the synthesized elp incorporated with amino acid motifs promote an accelerated endothelialization of the biodegradable stent material plla. discussion: in summary, we were able to generate elastin-like proteins modified by bioactive sequences. those sequences enhanced endothelial cell proliferation and adhesion. further studies are warranted to determine the activity on smooth muscle cells of these peptides. (1) the failing heart is characterized by excessive extracellular matrix production by myofibroblasts (myocfs) causing fibrosis and myocardial stiffening. myocfs represent phenotypically transformed cardiac fibroblasts (cfs) and are characterized by the expression of contractile proteins like a-smooth muscle actin (α-sma) and enhanced secretion of growth factors (e. g. ctgf). identification of intracellular enzymes that modulate this transformation process is desired to therapeutically modulate pro-fibrotic progression in heart failure. we show that pde2a, a phosphodiesterase isoform, that is able to hydrolyse cgmp and camp, is markedly upregulated in failing hearts from patients with end-stage heart failure (2-3-fold, p<0.05, n=8). notably, pde2a protein abundance is 4-fold higher in myocfs compared to cardiomyocytes from neonatal rat hearts (p<0.05, n=7). to this end we tested whether pde2a modulates the transformation of cfs isolated from neonatal rat hearts to myocfs. indeed, as assessed by immunoblotting and fluorescent microscopy (α-sma, phalloidin, dapi), adenoviral pde2a overexpression induced α-sma expression (3.2-fold p<0.05, n≥8) and to a lower extent ctgf synthesis (1.5-fold, p<0.05, n≥5). mechanistically, pde2a showed preferential subsarcolemmal localisation with diminished total cgmp levels (-56%, p<0.05, n≥5). consistently, parallel stimulation with atrial natriuretic peptide (anp), a selective activator of membrane-bound guanylyl cyclase, normalized ctgf synthesis indicating that pde2a controls cgmp in a discrete subdomain near the plasma membrane. moreover pde2a overexpression diminished the protein levels of vasodilator-stimulated phosphoprotein, a membrane cytoskeletal component (-60%, p<0.05, n≥5). these data implicate pde2a-dependent subsarcolemmal cgmp regulation in myofibroblast formation and potentially cardiac fibrosis. therefore, targeting pde2a may lead to regression of the fibrotic remodeling associated with heart failure. several anorganic nanoparticles (np) causedhigher inhalation toxicity than the corresponding coarse particles (oberdoerster et al. 2005) . we examined an organic pigment and a polymer dispersion each as nanomaterial and as the chemical identical coarse material in short-term inhalation studies in malerats. the polymer was an anionic acrylic ester copolymer containing free carboxylic groups. three different particle sizes were synthesized by varying polymerization conditions: 12, 80 or 250 nm. although polymeric acrylic ester was reported to be irritating to the respiratory tract at 3 mg/m3, all three tested polymers -including the np (12 and 80 nm) -did not cause any changes in lavage fluid and in histopathology at 10 mg/m3. the organic pigment was a poorly soluble pyrrol with an intense orange color. the np (10 to 50 nm width and 30 to 400 nm length) and the coarse pigments (70 to 200 nm width and 0.3 to 3 µm length) are both needle-like. they were tested at 1, 3, 10 and 30 mg/m3 for the np and 3, 10 and 30 mg/m3 for the coarse materials. mild and partly reversible morphological changes were observed in lung and lymph nodes at the highest concentrations, but the more pronounced effect were found in rats exposed to the coarse material. likewise there was an increase of lavage parameters in rats exposed to thecoarse material but not to the np. these data demonstrate that inhalation of finer np is not necessarily associated with higher toxicity compared to the coarse material. the results were obtained with two organic particles of rather different size and composition but are in contrast to the more severe effects seen with several anorganic np when compared to the corresponding coarse particles. within the nanocare project a standard short-term inhalation test to examine the toxicity of inhaled aerosols from nanomaterials has been developed. the inhalation toxicity of nano-andpigmentary materials was studied: baso4, zno, ceo2, al-doped ceo2, zro2, amorphous silica, surface-coated amorphous silica, titania, carbon black and three multi-wall carbon nano tubes, all with complete phys-chem-characterization as planned for the oecd sponsorship program. quartz dust tio2 and zno were tested as pigmentary materials. rats were exposed nose-only to three concentrations of one of these materials, 6 h a day for five consecutive days. positive controls were exposed to quartz dust or pigmentary zno, negative controls to clean air. a wide range of endpoints for pulmonary toxicity were evaluated immediately after the last exposure and after 3 days and 3 weeks after the last exposure. among these parameters, polymorphnuclear granulocyte count in bronchoalveolar lavage fluid is the most sensitive early parameter indicating inflammation process in lung, while histological examination reveals the type and localization of inflammation. among these substances, we identified baso4 as having the lowest toxicity. all mwcnts were most potent in producing progressive inflammation in the lung; granulomas in lung and lung associated lymph nodes were observed without indication for fibrosis. the noaecs of the 16 substances ranged between < 0.1 and >50 mg/m 3 . generally the material was only found in the lung (surface and macrophages) and in the draining lymph nodes. surface modified amorphous silica was also found in the spleen. the data demonstrate that the method is able to differentiate the toxic potential of different nanomaterials and to indicate regression or progression of the effects effects. moreover the lung burden and potential translocation to other tissues was detecable. comparing the material properties and effects of the 16 materials, no general relationship between the toxicity and either particle size, specific surface area or aerosol particle number concentration was found. hence we must not expect to find a gerneral "nanotoxicology" or a unifying dosimetry for all nanomaterials. we must rather be prepared to test individual nanomaterials for their effects. and to develop grouping concepts not only based on material properties but also on biopersistence, biokinetics and biological effects. part of this studes has been sponsored by bmbf (nanocare). endpoint-centric search for toxicological information and data to support the information retrieval for regulatory programs landsiedel r. 1 , wächter t. the eu reach regulation no 1907/2006 requires industry to ensure the safety of chemical use and manufacturing. all substances manufactured or imported in quantities above one tonne per year must be registered. information requirements for the dossiers increase with increasing tonnage or once hazards are suspected. searching for substance specific literature and the compilation of hazard data for safety assessments are highly challenging procedures. the novel web-based search engine go3r, accessible free of charge at www.go3r.org, has been created to allow quickly finding relevant hazard information and data. go3r provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information. furthermore, go3r specifically highlights information on animal testing alternatives. search results are presented automatically linked to an "intelligent table of contents" which enables the user to sort the literature listed in pubmed or the toxicology data network (toxnet) in a fast and comprehensive manner. retrieved documents are automatically organized in categories relating to the iuclid 5 chapters. hereby, the user can browse directly through the entire 21 million documents without even having to start the search with an initial query. the semantically enriched platform supports the user during query formulation, allows for bibliographic analysis, and specifically highlights information related to the replacement, reduction, and refinement of animal experiments. search results in go3r are shown in an dynamic table of contents (left) making them browsable for the contained information on animal testing alternatives and toxicologogical endpoints. towards a differentiation therapy of acute myelogenous leukemia with histamine h2-receptor agonists laue s., burhenne h., seifert r. hannover medical school institute of pharmacology, carl-neuberg-str. 1, 30625 hannover, germany acute myelogenous leukemia (aml) is a devastating malignancy characterized by a differentiation block of myeloid progenitor cells. recently, histamine dihydrochloride in combination with interleukin 2 has been approved as orphan drug for the consolidation treatment of aml (1) . it is assumed that histamine exerts its effects by activating the histamine h2-receptor (h2r) in human neutrophils, resulting in improved anti-tumor function of t killer cells by inhibiting nadph oxidase-catalyzed superoxide formation. previous studies had shown that histamine also induces myeloid differentiation (2) . considering the fact that all-trans-retinoic acids constitutes a powerful differentiation therapy of acute promyelocytic leukaemia, a specific subtype of aml (3), we initiated a study to explore the possibility that h2r-mediated myeloid differentiation provides an alternative or complementary strategy to treat leukemias associated with differentiation blocks. as model system, we used hl-60 cells. in hl-60 cells, histamine and various h2-receptor agonists induced concentrationdependent increases in camp levels. interestingly, ligands differentially increased cytosolic calcium concentration and extracellular receptor kinase (erk) pathways, indicative for ligand-specific h2r conformations. h2r activation resulted in myeloid differentiation as assessed by enhanced formyl peptide receptor-mediated increases in cytosolic calcium concentration. h2r agonists showed no signs of cytotoxicity. intriguingly, following h2r activation, the majority of the formed camp was exported into the extracellular space via multi-drug resistance protein (mrp) 4, indicating that export is a more important pathway for signal termination than cleavage of camp by phosphodiesterases. despite effective camp export, even a short-term exposure (30 minutes) of cells was sufficient to induce expression of functionally active formyl peptide receptors. these data indicate that in contrast to previously held dogma, induction of myeloid differentiation does not require continuous presence of a camp signal. from a therapeutic point of view this is very important since "spike" therapy with campincreasing substances may be sufficient to induce a therapeutic effect in aml, thereby also reducing toxic side effects. currently, we are systematically exploring the effects of h2r agonists on signal transduction pathways and differentiation in various myeloid cell types to identify highly efficacious compounds. introduction. activation of gαq/11 protein-coupled receptors of postsynaptic neurons can elicit the production of endogenous cannabinoids (endocannabinoids), which in turn inhibit transmitter release from axon terminals by activating presynaptic cb1 receptors. the aim of the present experiments was to study the mechanism of the endocannabinoid production. specifically, we wanted to clarify the role of ca 2+ release from intracellular stores in triggering endocannabinoid production. methods. patch-clamp-and ca 2+ imaging experiments were performed on purkinje cells in mouse cerebellar brain slices. glutamatergic excitatory postsynaptic currents (eepscs) were elicited by electrical stimulation of parallel fibers. the gαq/11 proteincoupled metabotropic glutamate receptor 1 (mglur1) was activated by superfusion of (rs)-3,5-dihydroxyphenylglycine (dhpg) or -more physiologically -by burst stimulation of the parallel fibers. results. both dhpg superfusion and burst stimulation of parallel fibers elicited an increase in intracellular ca 2+ concentration in the postsynaptic purkinje cells. dhpg superfusion and burst stimulation suppressed eepscs, and this suppression was abolished in the presence of the mglur1 antagonist cpccoet. the suppression of the eepscs was also sensitive to the cb1 receptor antagonist rimonabant, pointing to involvement of endocannabinoids and cb1 receptors. the suppression of the eepscs was attenuated after depletion of the endoplasmic reticulum ca 2+ stores by thapsigargin, cyclopiazonic acid and ip3. the results indicate that after activation of the gαq/11 protein-coupled metabotropic glutamate receptor 1 (mglur1) of the postsynaptic neuron ca 2+ is released from the endoplasmic reticulum. this ca 2+ release significantly contributes to the production of endocannabinoids. the endocannabinoids diffuse in the synaptic cleft retrogradely to the terminals of afferent axons and inhibit transmitter release there through presynaptic cb1 receptors. the guanine nucleotide exchange factor dock9 controls reelin dependent cdc42effects on radial migration pichler m. 1 the regulation of blood glucose levels is under tight control of a complex system including hormone and neurotransmitter signalling. many of these cellular signalling pathways are initiated by binding of the ligand to a g-protein coupled receptor (gpcr), e.g. noradrenaline inhibits insulin secretion upon binding to a gi-coupled receptor. upon gpcr activation the heterotrimeric g-protein is activated and both the α-subunit and βγdimers are released and interact with their specific target proteins. by the usage of bordetella pertussis toxin (ptx) as a common gαi inhibitor gαi-dependent signalling pathways are interrupted which leads to increased insulin secretion, and significantly improves glucose tolerance. since the gαi-isoform specific roles in the regulation of glucose homeostasis are still debated we studied the glycemic control in gαi2-deficient mice. surprisingly and in contrast to the ptx data, glucose tolerance was unchanged in the gαi2-deficient mice compared to wild type controls. however, the plasma insulin levels were significantly reduced upon glucose challenge. these findings point to disturbed islets function and improved peripheral insulin sensitivity. analysing gαi2deficient islets we show that islet size and number of nuclei are reduced. nevertheless, in vitro insulin secretion is improved at low (3 mm) and high (16 mm) glucose concentrations and can be further stimulated upon ptx-treatment. these data indicate that gαi2 proteins influence islet development and inhibit insulin secretion. in addition, these findings support our hypothesis that gαi2-deletion influences peripheral insulin sensitivity. therefore, we investigated glucose homeostasis and pakt-levels after two hours feeding ad libitum in gαi2-deficient mice. under feeding conditions no differences in plasma insulin levels were visible although blood glucose levels were significantly reduced in gαi2-targeted mice. pakt-levels of liver and skeletal muscle were unaltered, whereas akt phosphorylation in white adipose tissue was significantly increased, indicating improved glucose uptake of adipocytes. in conclusion, gαi2 is a negative regulator of both insulin secretion and peripheral insulin sensitivity and important for the maintenance of glucose homeostasis. 11689, 1992) in a radioligand binding test and to determine their functional effects with a membrane potential test using the dye r7260 (molecular probes, 0.125 mg/ml, excitation 505 nm, emission 530 nm). the affinity of compounds in radioligand binding was slightly higher in sur2b than in sur2a-type channels, but the enantiomeric ratio in sur2a channels matched that one determined for the sur2b-type indicating some conformity of the binding pockets of sur2a and sur2b-proteins. surprisingly, however, the membrane potential tests revealed that the (3r,4s)-enantiomer acted as agonist (a) whereas the (3s,4r)-enantiomer acted as antagonist (b): (3r,4s)-bms-191095 induced membrane hyperpolarisation whereas (3s,4r)-bms-191095 repolarised cells prestimulated with submaximally effective concentrations of diazoxide. concluding, bms-191095 is not selective for sur2a as compared to sur2b-type k atp channels. its enantiomers activate and block sur2-type katp channels in a stereospecific manner. thieno-thiadiazine derivatives with full agonistic activity at sur2b-type katp channels act as partial agonists at cardiac sur2a-subtypes oldenhage c., grittner d., schmidt c., lemoine h. heinrich-heine universität, inst. für lasermedizin, mol. wirkstoff-forschung, universitätsstr. 1, 40225 düsseldorf, germany new potassium channel openers (kco) of the thieno-thiadiazine(ttd)-type initially developed as agonists for the sur1-type katp channels (nielsen et al., j med chem 45: 4171, 2002) were characterized in sur2b-type katp channels as agonists and antagonists, if r contains a quaternary (methyl-cycloalkyl) and a tertiary (r = cycloalkyl) carbon, respectively (lemoine et al., this journal 375, r45, 2007) . to investigate the selectivity of ttd-derivatives for myocardial katp channels the membrane potential actions of compounds were tested in hek 293(kir6.2/sur2a)-cells and compared to hek 293(kir6.1/sur2b)-cells as a model for smooth muscle-type katp channels. membrane potential was measured by fluorescence (excitation 505 nm, emission 530 nm) using 0.125 mg/ml of the dye r7260 (molecular probes). standard-kco induced hyperpolarisation with ~10-fold smaller potency (pec50) in sur2a. ttd-compounds with ch3-cycloalkyl residues not only lost potency but also intrinsic activity for channel activation (emax) in sur2a. possibly, this loss of emax would be much greater in native heart cells with a normal channel density. in contrast, ttd-compounds with cycloalkyl residues acted as antagonists of cells pre-hyperpolarized with diazoxide with similar affinity in sur2a and sur2b-type katp channels. concluding, selectivity of kco for katp channel-subtypes cannot only be achieved by a different affinity but also by a selective stimulation of the channel of interest. small-conductance calcium activated potassium (kcnn/sk/kca2) channels maintain neuronal calcium homeostasis, shape synaptic functions and prevent excitotoxic neuronal death. so far, little is known about the function of kca2 channels in nonneuronal cells. the aim of this study was to investigate the expression of kca2 channels in microglial cells and their potential function in microglial activation and maintenance. expression of kca2 channel subtypes in microglial cells was assessed by mrna analysis, western blots and immunocytochemistry. lipopolysaccharide (lps)-induced microglial proliferation was evaluated by the xcelligence impedance-based system and mtt assays, and immunogenic activation of microglia was determined by measuring cytokines and nitric oxide (no) release into the cell culture medium. the kca2.2 and kca2.3 channel activator cyppa (25 µm) and specific inhibitory peptides (50 µm) were applied to distinguish effects mediated by the kca2 channel subtypes. all kca2 channel subtypes were detected on mrna and protein levels in resting and in lps-activated microglial cells. xcelligence real-time measurements and mtt assays demonstrated that lps (200 ng/ml) induced microglial proliferation. the kca2.2/kca2.3 channel activator cyppa reduced lps-induced microglial proliferation in a concentration-dependent manner. specific peptide inhibitors of kca2.3 channels, but not of kca2.2 channels, reversed the cyppa-effects on lps-induced microglial proliferation. cyppa alone did not alter the production of tnf-alpha or il-6, but strongly reduced the lps-dependent cytokine production. interestingly, chelation of extracellular calcium by edta induced differential cytokine kinetics by decreasing lps-dependent il-6 production while tnf-a production was not affected. moreover, using inhibitory sk3/kca2.3 channel peptides, we demonstrated that sk3/kca2.3 channels modulate lps-induced cytokine il-6 production in a calcium-dependent manner, while the tnf-a release was independent of extracellular calcium. in summary, the present study revealed that kca2.3 channel stimulation reversed microglial activation. thus, kca2.3 channels may serve as a therapeutic target for reducing microglial activity and related inflammatory responses in cns diseases. (3r,4s)-(3s,4r)intracellular amyloid beta (aß) oligomers and extracellular aß plaques are key players in the progression of sporadic alzheimer disease (ad). still, the molecular signals triggering aß production are largely unclear. we asked whether mitochondria-derived reactive oxygen species (ros) are sufficient to increase aß generation and thereby initiate a vicious cycle further impairing mitochondrial function. complex i and iii dysfunction were induced in a cell model using the respiratory inhibitors rotenone and antimycin resulting in mitochondrial dysfunction and enhanced ros levels. both treatments lead to elevated levels of aß. presence of an antioxidant rescued mitochondrial function and reduced formation of aß demonstrating that the observed effects depended on ros. conversely, cells overproducing aß showed impairment of mitochondrial function such as comprised mitochondrial respiration, strongly altered morphology, and reduced intracellular mobility of mitochondria. again, the capability of these cells to generate aß was partly reduced by an antioxidant indicating that aß formation was also ros-dependent. moreover, mice with a genetic defect in complex i, or ad mice treated with a complex i inhibitor, showed enhanced aß levels in vivo. several lines of evidence show that mitochondria-derived ros result in enhanced amyloidogenic amyloid precursor protein processing, and that aß itself leads to mitochondrial dysfunction and increased ros levels. we propose that starting from mitochondrial dysfunction a vicious cycle is triggered that contributes to the pathogenesis of sporadic ad. comparison of methods to derive health-based guidance or limit values for chemicals licht o., voss j. -u., mangelsdorf i. fraunhofer item chemikalienbewertung, nikolai-fuchs-str. 1, 30625 hannover, germany health-based guidance or limit values are derived for chemicals to compare measured or estimated exposure concentrations with these values. if the exposure is below the limit value, adverse effect for human health can be regarded as negligible, e.g. the exposure is expected to be tolerable. in germany such values have been derived since years for chemicals that can be found in soil, water and air as well as human blood and urine (biomonitoring). in a research project sponsored by the german umweltbundesamt several methods used by the agency are compared to the method laid in reach guidance document r.8 to derive a derived no effect level (dnel). the aim was to identify possibilities for standardization as well as to figure out specific elements in individual methods. in addition to extrapolation factors the public availability of guidance and specific derivations as well as procedures for consensus on the limit value were evaluated. the comparison of extrapolation factors revealed that, although they are named differently such as extrapolation, safety or assessment factors, they are used in a comparable manner. factors for interspecies and intraspecies extrapolation are presented in more detail. the standard factor for such extrapolation is 10 in most cases. in the reach guidance this factor consists of a part for allometric scaling and remaining differences. other factors are only defined in some methods, like a factor for extrapolation from loael to noael, data quality or data gaps. a factor for data quality is not laid down in the basic scheme for setting of indoor air guidance values, but is used in some of the recent derivations of limit values. also the who guidelines for drinkingwater quality use comparable factors to account for adequacy of studies or database and nature and severity of effect. a transparent and documented derivation is necessary for acceptance of the value. the derivation methods as well as the evaluation document on a specific substance are available through publications or the internet in nearly all cases. for the dnel only the numeric value is available at the echa website, but not any information on starting point and extrapolation factors. although all guide or limit values are derived in a comparable way, differences, however, exist in some details. in most cases detailed explanation is lacking when deviating from standard or default assumptions. often such deviation is based on expert judgement. hepatocellular carcinoma (hcc) is the fifth most common cancer in the world and has a poor prognosis with limited therapeutic options. up to now, no curative systemic therapy exists emphasizing the high clinical importance of new therapies for hcc. therefore, the identification and characterization of novel drugable targets is a relevant goal. cyclin-dependent kinase 5 (cdk5) is well characterized for its function in cns development and disease. recently, few reports indicate functions of cdk5 in cancer. cdk5 was shown to regulate tumor growth, and our group discovered that cdk5 regulates angiogenesis. since hcc is a highly vascularized tumor and anti-angiogenic treatment (sorafenib) has shown some therapeutic benefit, we hypothesize that cdk5 is an interesting target for hcc therapy. the aim of this study was to characterize the function of cdk5 in hcc. histology of tissue micro arrays indicates an increased expression of cdk5 in human hcc tissue in comparison to healthy liver tissue of the same patient. to investigate the function of cdk5 in hcc, we analyzed the impact of both pharmacological inhibition of cdk5 and specific downregulation of cdk5 with rna interference on hcc cells. pharmacological inhibition of cdk5 with the small molecule roscovitine (r-roscovitine, seliciclib) decreased proliferation and clonogenic survival, induced g2/m cell cycle arrest and cell death, and reduced motility of huh7 and hepg2 cells. transient downregulation (sirna) and stable knockdown (shrna) of cdk5 also reduced proliferation, clonogenic survival, migration and invasion of huh7 cells. in a subcutaneous hcc xenograft model, treatment with roscovitine reduced tumor growth and angiogenesis, indicated by decreased tumor weight and volume, and reduced vessel density. moreover, cotreatment of hcc cells with roscovitine and tumor necrosis factor related apoptosis inducing ligand (trail) resulted in an over-additive additive effect on the induction of apoptosis. this coincided with reduced phosphorylation and activity of the anti-apoptotic transcription factor stat3 at ser727 that is directly phosphorylated by cdk5, and tyr705. in line with this, the expression of the antiapoptotic protein mcl-1 is reduced by inhibition of cdk5. our results point to an important function of cdk5 in hcc and suggest cdk5 as an interesting pharmacologically druggable target for hcc therapy. delivery of mono-biotinylated rnasea into macrophages with streptavidinconjugated clostridium botulinum c3 toxin lillich m. 1 , chen x. clostridium botulinum produces the adp-ribosyltransferase c3, which modifies and thereby inactivates exclusively the small gtp binding proteins rho-a,-b and -c. recently, we discovered a specific endocytotic internalization of c3 toxin in macrophages and myeloid leukaemia cells, but not in epithelial cells [1] . thus, c3 toxin provides a tool to target cells of the monocyte/macrophage lineage, which are involved in various diseases and are of great clinical interest. we used a biochemical crosslinking approach to design a delivery system based on an enzymatic inactive c3bot mutant (c3mut) and streptavidin. the c3 portion mediates uptake of the transporter into monocytes/macrophages and streptavidin allows for binding of biotinylated cargo molecules to the transporter. in vitro, the generated c3mut-streptavidin bioconjugate showed specific and concentration dependent binding to biotinylated oligonucleotides as demonstrated by electrophoretic mobility shift assay. cell fractionation experiments indicated an uptake of the bioconjugate into the cytosol of j774a.1 macrophages. in the next step, mono-biotinylated bovine pancreatic ribonuclease a (rnasea) was used as a model cargo for the delivery of macromolecules by the bioconjugate. rnasea is a highly stable, well studied protein which catalyzes the degradation of rna. mono-biotinylated rnasea interacts in a specific and concentration dependent manner with the c3mut-streptavidin bioconjugate in vitro as analysed with dot blot technique. the c3mut-streptavidin bioconjugate efficiently mediates the internalization of biotinylated rnasea into j774a.1 macrophages as analyzed with laser scanning microscopy in fixed cells. this finding was also confirmed by live cell imaging. furthermore, cell fractionation showed a cytosolic delivery of biotinylated rnasea in the presence of c3mut-streptavidin. as expected we could not observe a cytotoxic effect of biotinylated wild-type rnasea on j774a.1 macrophages, which is attributable to the presence of ribonuclease inhibitor protein in mammalian cells. in summary, the c3mut-streptavidin bioconjugate mediates the efficient internalization of biotinylated (macro)molecules into macrophage like cells, and therefore represents a useful tool for the transduction of exogenous molecules into macrophages. in addition, cytotoxic rnasea mutants are available and will be used in further studies. organometal compounds such as cisplatin or the second generation complexes carboplatin and oxaliplatin have become more and more important as antitumor agents. nevertheless there is still an increasing demand for novel metal-based compounds. this is necessary due to severe side effects and the occurence of resistent tumour cells. in this context we investigated the cytotoxic effects of imidazole-based phosphane gold(i) complexes as potential agents for cancer treatment. initially we have used the mtt-assay to examine the toxic potential of the gold complexes in h4iie rat hepatoma cells. in this context cw60 (a diphosphane ligand with azoyl substituents r2p(ch2)2pr2, r= thiazol-2-yl) turned out to be the compound with the highest cytotoxic potential with an ic50 value of 6,5mm (24h incubation). further investigations revealed that cw60 induced an apoptotic cell death in h4iie demonstrated by the activation of caspase 3/7 (48h incubation with 10mm cw60). in addition the induction of apoptosis was confirmed by the dna ladder formation (24h incubation with 5mm cw60). in connection with the molecular mechanisms of apoptosis induction we used the comet assay to analyse the generation of dna strand breaks as well as the dcf-assay to detect the formation of reactive oxygen species. however neither dna strand breaks nor increased levels of reactive oxygen species were detected after 1h of incubation. furthermore we analysed if the compound influences intracellular signalling pathways such as the jnk pathway and the pi3k/akt but after 24h of incubation neither pakt nor jnk were influenced. the imidazole based phosphane gold (i) complex cw60 shows strong toxic effects in h4iie cells and turned out to be a promising compound as a potential agent for cancer treatment. the high and inappropriate intake of loop diuretics in hypertensive elderly reported in former studies has again been confirmed. remembering that inappropriate intake of loop diuretics can lead to exsiccosis and electrolyte loss especially in elderly, better medical education has to follow these alarming results to improve the pattern of diuretic prescription. furthermore, our results lead us to assume a high estimated number of unreported cases of torasemide use in uncomplicated arterial hypertension in elderly. this loop diuretic agent shows a longer duration of action compared with furosemide (elimination half-life: 3-4 hrs vs. 1 hr) and is effective in decreasing blood pressure in subdiuretic doses. it must be pointed out that loop diuretics are still frequently inadequately prescribed because current guidelines recommend loop diuretics only in complicated arterial hypertension. the role of cgmp/cgki signaling and trpc channels in regulation of vascular tone loga f., domes k., hofmann f., wegener j. pharmakologie und toxikologie for 923, biedersteiner str 27, 80802 münchen, germany signaling by intracellular cgmp and cgmp-dependent protein kinase i (cgki) is the major pathway in vascular smooth muscle, by which endothelial no regulates vascular tone. recent evidence suggests that trpc channels are targets of cgki in smooth muscle and mediate, at least partially, the relaxant effects of cgmp. we tested this concept by investigating the role of cgmp/cgki signaling on vascular tone and peripheral resistance using cgki-, trpc6-, and trpc3-, and trpc3/c6-double knock-out mice. we found larger contractile responses to α-adrenergic stimulation in intact aorta from cgki-, trpc6-, and trpc3/c6-double knock-out mice as compared to aorta from ctr and trpc3-knock-out mice indicating a functional link between cgki and trpc6 channels. no differences were found if the vasodilator tone, provided by the no generation in the vascular endothelium, was inhibited by l-name. likewise, no differences were observed in the increase in peripheral resistance by α-adrenergic stimulation using the hind limb perfusion system. activation of cgki by 8-br-cgmp diminished aortic tone and peripheral resistance to a similar extent in control, trpc6-, trpc3-, and trpc3/c6-double knock-out mice. no effect of 8-br-cgmp was observed in preparations from cgki -/mice. to test the co-localization of cgki and trpc channels, we performed immunocytochemistry on isolated smooth muscle and endothelial cells from aorta of ctr, trpc3-, and trpc6-knockout mice. trpc3 could be detected in both smooth muscle and endothelial cells whereas trpc6 was only detected in endothelial cells. the results suggest that absence of cgki or trpc6 impairs the vasodilator tone induced by endothelial no production but that cgki and trpc6 channels are not functionally coupled in vascular smooth muscle. we thank profs birnbaumer (nih) and freichel (homburg) for providing us with trpc6 -/-, and trpc3 -/mice and prof. flockerzi (homburg) for the antibodies against the trpc channels. whole genome microarray analysis of the effects of tcdd and pcb 153 in human hepatic cell models lohr c. 1 , neser s. after the treatment with tcdd, however, a total of 281 genes were more than 2-fold up regulated in hepg2 e.g. cytochrome p450 1a1 (cyp1a1) (32-fold) a sensitive marker for ahr activation. additional up regulated genes in hepg2 were; arylhydrocarbon receptor repressor (ahrr) 15-fold, aldehyde dehydrogenase 3 a1 (aldh3a1) 11-fold, and cytochrome p450 1b1 (cyp1b1) 10-fold. 44 genes were more than 2-fold down regulated in hepg2 cells e.g. -proprotein convertase subtilisin/kexin type 9. markedly different findings were obtained in hheps, i.e., 117 genes were up regulated, the highest up regulated gene was cyp1b1 with a 95-fold increase in gene expression, followed by cyp1a1 (41-fold) and aldh3a1 (21-fold). only a small group of genes were significantly down regulated (17 in total), e.g., solute carrier family 2 (facilitated glucose transporter). comparing both human cell types, there was an unexpected small overlap of genes being up or down regulated. interestingly, in both cell types, only 30 in common genes were up regulated, including cyp1a1, cyp1a2, cyp1b1 and aldh1a3. only platelet-derived growth factor receptor, beta polypeptide, was down-regulated in both hepg2 and hheps. in conclusion, our data indicate pronounced differences in the patterns of tcdd-regulated genes between hepg2 cells and hheps. detection of redox modified proteins in nociceptive processing lorenz j. e. 1 , kallenborn-gerhardt w. recent data indicate that redox modifications of proteins induced by reactive oxygen species (ros) contribute to sensitization of pain pathways during persistent pain. however, little is known about the targets of ros in pain processing, because the relatively unstable nature of many reversible protein oxidation states hampers the reliable detection and identification of modified proteins. here, we used the quantitative thiol trapping technique termed oxicat to identify proteins which are redox modified during nociceptive processing. we investigated spinal cords of untreated mice, after zymosan injection into a hindpaw (inflammatory pain model) and after spared nerve injury (neuropathic pain model). we identified several proteins with marked changes in their redox states after nociceptive stimulation. our results show that the oxicat method is an efficient method to detect redox modifications in proteins and that redox modifications seem to play a role in pain processing. supported by the deutsche forschungsgemeinschaft (sfb 815/a14). additive antinociceptive effects of a combination of vitamin c and vitamin e after peripheral nerve injury lu r., kallenborn-gerhardt w., geisslinger g., schmidtko a. pharmazentrum frankfurt/zafes institute of clinical pharmacology, goethe university, frankfurt am main, germany accumulating evidence indicates that increased generation of reactive oxygen species (ros) contributes to the development of exaggerated pain hypersensitivity during persistent pain. in the present study, we investigated the antinociceptive efficacy of the antioxidants vitamin c and vitamin e in mouse models of inflammatory and neuropathic pain. we show that systemic administration of a combination of vitamins c and e inhibited the early behavioral responses to formalin injection and the neuropathic pain behavior after peripheral nerve injury, but not the inflammatory pain behavior induced by complete freund's adjuvant. in contrast, vitamin c or vitamin e given alone failed to affect the nociceptive behavior in all tested models. the attenuated neuropathic pain behavior induced by the vitamin c and e combination was paralleled by a reduced p38 phosphorylation in the spinal cord and in dorsal root ganglia, and was also observed after intrathecal injection of the vitamins. moreover, the vitamin c and e combination ameliorated the allodynia induced by an intrathecally delivered ros donor. our results suggest that administration of vitamins c and e in combination may exert synergistic antinociceptive effects, and further indicate that ros essentially contribute to nociceptive processing in special pain states. -206, -213 and -214) replaced by leucine residues. both amino acids are comparable in terms of hydrophobicity, volume and the preference for forming α-helices, but only methionine is oxidizable to a sulfoxide, in contrast to leucine. in the present study we examined the protein-protein interaction (ppi) of recombinant ac 1, expressed in sf9 insect cell membranes, with cam, cam-206, -213, -214 and -215 by measuring the catalytic activity of ac 1. cam-mutants show a 3-4-fold lower potency than cam, but they are more efficacious than cam. most prominently, cam-215 was 133 % more efficacious than cam. such striking differences between cam and cam-mutants have not yet been observed for other mammalian effector proteins. as a result of the exchange of all methionine against leucine residues in cam-215, it is more hydrophobic than cam and this leads to a better ppi with ac 1. in future studies we will examine the effects of cam inhibitors, antidepressants and antipsychotics on cam/ac 1 interaction. furthermore we will analyze the effects of oxidized cam and cam-mutants on the catalytic activity of ac 1. because oxidative stress is of great importance in aging, it is important to know more about the abovenamed interaction in view to the demographic change. taken together, our data point to a unique cam/ac 1 interaction that may be selectively targeted by small molecules. in particular, enhancers of these interaction could be useful to improve memory and learning. gender differences in fat distribution and diabetes prevalence in nzo mouse lubura m., scherneck s., zucker a., schürmann a. deutsches institut für ernährungsforschung experimentelle diabetologie, arthur-scheunert-allee 114-116, 14558 potsdam, germany background: excessive fat accumulation in visceral but not subcutaneous fat depots as well as ectopic fat storage in liver, skeletal muscle and pancreas are associated with an increased risk for the development of type 2 diabetes in humans. in this study we aimed to examine the influence of early fat distribution on onset of type 2 diabetes in mice. methods: nzo mice are regarded as insulin resistant model in which only males become diabetic. we used male and female mice fed with high-fat and standard diet. we determined fat distribution by computed tomography for three times and conducted oral glucose tolerance tests on two different time points. besides we assessed body weight and blood glucose levels on weekly basis. results: contrary to previous findings, we observed that not only male nzo mice on high-fat diet develop diabetes. blood glucose levels at the 16 th week of age and total pancreatic insulin content indicated diabetes prevalence of 68% in males and 25% in females these results lead to the conclusion that high-fat diet counteracts protective action of estrogens against diabetes. inversely to the findings in humans, female mice tend to store more fat in abdominal region than males. there was no relationship between early accumulation of fat in abdominal region and onset of type 2 diabetes. however, visceral fat was associated with liver fat in males as well as in females. furthermore, at the age of ten weeks hepatic fat content correlated with blood glucose levels (r² = 0.69) indicating that the early hepatosteatosis is a predictor for hyperglycemia. however, there was no correlation between hepatic insulin sensitivity (indicated by quantitative insulin sensitivity index-quicki) and amounts of hepatic fat we conclude that early hepatosteatosis does not predict for glucose intolerance in nzo mice. in the nzo mouse, the amount of liver fat but not the early fat distribution predicts for the later onset of type 2 diabetes. further experiments are needed to examine the gender dependent differences in the diabetes prevalence of this mouse strain. with a prevalence of about 20-30% non-alcoholic fatty liver disease (nafld) represents the most common liver disorder in europe. nafld manifestation ranges from steatosis through steatohepatitis (nash) to fibrosis and cirrhosis, followed in some cases by liver failure and hepatocellular carcinoma. fatty degeneration of liver cells, increased oxidative stress with concomitant lipid peroxidation and an induction of pro-inflammatory cytokines are proposed as possible causes for developing inflammation and fibrosis, but the exact pathogenesis of the progression of nafld into nash is still unknown. thus, besides life style modifications and weight reduction interventions, no established pharmacological therapy exists so far. to gain further insights into the pathogenesis of nafld and nash and to develop new therapeutic strategies, appropriate animal models are essential. thus, in the present study three different dietary animal models for nafld were evaluated and compared to the biochemical and metabolic alterations seen with nafld and nash in man. male adult lewis rats were given standard food or one of three different diets: fatty liver diet [fld] , methionine/choline deficient diet [mcd] or methionine/choline deficient plus high fat diet [mcd+hf] . after 1, 2, 4, 6 or 12 weeks of treatment, animals were sacrificed and body and liver weights, laboratory parameters (asat, alat) as well as histopathological changes in the livers and different parameters indicating oxidative stress or representing the biotransformation capacity of the livers were analyzed. with fld and mcd+hf a normal body weight gain was observed, whereas with mcd body weight gain was strongly impaired. liver weights were mainly increased after mcd+hf. elevation of asat and alat values and hepatic steatosis were more pronounced after mcd and mcd+hf than after fld. all three diets caused an increase in the oxidative stress in liver tissue, but especially with mcd a tremendous elevation in the hepatic levels of lipid peroxidation products was seen. with regard to liver biotransformation capacity, with all three diets mainly an induction of the cytochrome p450 2e1 and 4a1 isoforms expression and activity was observed, which was most pronounced after mcd and mcd+hf. in summary, the changes induced by mcd or mcd+hf most closely resemble the alterations described in literature for nafld in man and thus should be preferred over fld in future investigations on nafld and nash. ep3 receptors for prostaglandin e2 convey stimulatory and inhibitory effects. e.g., their stimulatory effect leads to vasoconstriction in the human pulmonary artery and their inhibitory activity to reduction of neurotransmitter release from neuron endings. the aim of our study was (1) the pharmacological characterization of ep3 receptors in human pulmonary arteries and (2) the examination of the involvement of these receptors in the regulation of the neurogenic tachycardia in pithed rats. l-826266 served as the ep3 antagonist. experiments were performed in human pulmonary arterial rings isolated from patients undergoing lobectomy during resection of lung carcinoma and in pithed and vagotomised rats. the ep1/ep3 agonist sulprostone (1 nm -100 mm) concentrationdependently contracted human pulmonary artery rings (pec50 and emax; 6.89±0.12 and 106.5±5.2%, relative to the contraction induced by kcl 60 mm). the concentrationresponse curve of sulprostone was not affected by the ep1 antagonist sc 19920 (10 µm) but shifted to the right by l-826266 (10 µm) (apparent pa2 6.22). extending the exposure time to l-826266 from 0.5 to 3 h increased its antagonistic potency to 7.39 (schild plot-based pa2; concentrations 0.1, 1 and 10 µm). in pithed rats electrical stimulation (0.66 hz, 1 ms, 50 v for 5 s) of the preganglionic sympathetic nerve fibers or intravenous isoprenaline (0.15 nmol/kg) increased heart rate (hr) by 55 beats/min. sulprostone (10 -1000 nmol/kg) did not affect the isoprenaline-induced increase in hr but inhibited the neurogenic tachycardia dose-dependently, maximally by 80%. l-826266 (3 µmol/kg) diminished the inhibitory effect of sulprostone 1000 nmol/kg on the neurogenic tachycardia by 20%. in conclusion, ep3 receptors (1) located postsynaptically strongly contract human pulmonary arteries and (2) located presynaptically on sympathetic nerve fibres supplying the heart of rats strongly inhibit the neurogenic tachycardia. -5-bromo-n[3-(5voltage-gated ca 2+ channels of the central nervous system control a multitude of ca 2+ dependent processes such as neurotransmitter release, neuronal excitability, neurite outgrowth, synaptogenesis, plasticity and neuronal survival. the cav2.1 ca 2+ channelalso known as p/q-type channel -belongs to the subfamily of high voltage activated ca 2+ -channels. ca 2+ influx via cav2.1 ca 2+ channels located at presynaptic nerve terminals triggers vesicle fusion and transmitter release at brain synapses and at the neuromuscular junction. thus, cav2.1 ca 2+ channels play a crucial role in synaptic transmission. the global cav2.1 knock-out phenotype is characterized by severe ataxia, dystonia and lethality during the first postnatal weeks and is therefore an unsuitable model to analyze the importance of cav2.1 ca 2+ channels for learning and memory. therefore, we crossed a floxed cav2.1 mouse line with nex-cre transgenic mice to establish a viable, forebrain specific knock-out mouse line (fbko-mice). results from western blot analysis confirmed an efficient knock out of cav2.1 in hippocampal and cortical preparations, whereas the expression level in the cerebellum was not altered. to investigate the specific role of cav2.1 channels in hippocampus and neocortex dependent behavior, we performed tests for motor functions and sensory abilities and in particular learning and memory tasks. mice with a forebrain specific cav2.1 knock-out show significant deficits in spatial learning & reference memory and a significant reduced recognition memory as revealed by the morris water maze and an object recognition task. the fbko-mice exhibit no obvious locomotor deficits during behavioral tasks in the open field test and elevated plus maze. some fbko-mice demonstrate episodes of seizures in the morris water maze and during different rotarod tasks. to assess motor-function of fbko-mice in a stress reduced environment, we performed home cage based running-wheel motor-learning tasks. in summary, the diverse phenotypes of the forebrain specific knock-out mouse line emphasize the critical importance of cav2.1 for learning and memory. helicobacter hepaticus-infected rag2 -/mice emulate many aspects of human inflammatory bowel disease (ibd), including the development of colitis and colon cancer [erdman et al., 2009 , pnas 106: 1027 -1032 . toward the goal of elucidating mechanisms of inflammation-induced carcinogenesis and developing biomarkers of inflammation, we undertook a comprehensive analysis of macromolecular damage products during disease progression in h. hepaticus-infected rag2 -/mice. infected mice developed severe colitis and hepatitis, accompanied by infiltration of myeloperoxidase-positive neutrophils and f4/80-positive macrophages, by 10 wks postinfection (pi), progressing into colon carcinoma by 20 wks pi. qpcr array-based gene expression profiling revealed that pathophysiological changes were associated with characteristic alterations in the expression of genes related to inflammation, dna repair, and oxidative stress response. to study inflammation-related macromolecular damage, colon and liver tissues were analyzed by isotope-dilution chromatography-coupled mass spectrometry to quantify a battery of 16 different dna, rna and protein damage products thought to represent the full spectrum of inflammation-related chemistries. our data revealed a significant predominance of chlorinated dna-, rna-, and protein damage products by 20 weeks pi. in contrast, levels of damage products arising from oxidation, nitration and nitrosation changed only modestly or remained unchanged. our analyses also revealed higher levels of damage products in rna than in dna and demonstrated organ-specific differences of oxidative damage products, such as 8-oxo-dg and its oxidation products spiroiminodihydantoin and guanidinohydantoin. collectively, these results suggest that neutrophil and myeloperoxidase-induced chlorination chemistry may serve as a biomarker of ibd and may play important roles in the pathophysiology of ibd and colitis-associated cancer. characterization of a membrane protein expressed in mouse heart and brain mannebach s. 1 recently, a novel membrane protein in drosophila was shown to be localized in presynaptic vesicles. it appears to mediate a ca influx after vesicle fusion with the plasma membrane. disruption of the corresponding gene leads to endocytic defects in drosophila [1] . apparently, this protein plays a role in exo-and endocytosis and could serve as a ca channel supplying ca required for endocytosis. we have identified a protein in mouse, c90rf7, which shares 26,3% amino acid sequence identity with the drosophila protein. it covers 171 amino acid residues. using rt-pcr the full length transcripts could be identified in brain, kidney, pancreas, heart, spleen, thymus and mast cells. coexpression of c90rf7 and the ca v2.2 channel in xenopus oocytes reduced the amount of the α1b and cavβ3 subunits of the ca 2+ channel in the plasma membrane but did not affect the gating properties of the cav2.2 channel. expression of c90rf7 alone did not yield any channel activity. we therefore started to produce recombinant protein using the his-sumo-prokaryotic expression vector. the protein was efficiently expressed as his-sumo-c9orf7-fusion in e.coli (yield 2mg at 1mg/ml). we are currently preparing the c9orf7 part of the his-sumo-c9orf7fusion protein by ulp1-protease digestion followed by various chromatographic steps. the purified recombinant protein will be used to immunize rabbits to get antibodies. in parallel we generated antisera by immunizing rabbits with peptide fragments derived from the c9orf7 sequence. we could not identify any homologues of c9orf7 in the mouse genome and to analyze its function we are currently generating c9orf7 deficient mouse lines by gene targeting. we have chosen a strategy for conditionally inactivation of the gene with the option to study the cellular localization of c9orf7 by expression of the bgalactosidase gene under the control of the endogenous c9orf7 promoter. by southern blot analysis we´ve already identified 210 homologous recombinant embryonic stem cell clones out of 300 analyzed ones and we will proceed with blastocyst injection to get chimeric mice and finally mice carrying the introduced mutations in the c90rf gene. parps are involved in various biological processes such as regulation of dna repair, cell cycle progression, and cell death. consequently, several parp inhibitors are currently in clinical development as chemo-and radiosensitizers as well as monotherapeutic agents following the concept of synthetic lethality. pharmacological and toxicological studies call for an accurate analysis of parp activity in terms of a detailed knowledge of the structure of par and a reliable method for its quantification. we have developed a sensitive, precise, and accurate bioanalytical method based on liquid chromatography coupled to electrospray tandem mass spectrometry (lc/ms-ms) to characterize and quantify par with femtmol sensitivity: par is extracted from cells and hydrolysed to specific monomeric units, i.e., ribosyladenosine, which is characteristic for linear par, diribosyladenosine, which is characteristic for branching points, and adenosine, which represents the terminal part of the polymer. using this method, we are currently analyzing par levels in different cell lines and in primary human peripheral blood mononuclear cells (pbmcs) both under physiological conditions as well as upon genotoxic stress and in the presence of potent parp inhibitors. we expect that after completing method validation this assay will be useful for a wide range of applications in pharmacology and toxicology. gene mutagenic potential and metabolite profile of 17β-estradiol in cultured v79 cells expressing human cytochrome p450 1a1 martínez jaramillo d., lehmann l. university of wuerzburg, institute of pharmacy and food chemistry section of food chemistry, am hubland, 97074 wuerzburg, germany oxidative metabolism of the female sex hormone 17β-estradiol (e2) is considered to play a major role in the initiation of hormone-induced carcinogenesis. in extrahepatic tissues, e2 undergoes metabolic activation by cytochrome p450-dependent monooxygenase (cyp) isozyme 1a1 to 2-hydroxy-(2-ho) and to a lesser extent to 4-ho-e2. if not conjugated, these catecholestrogens (ce) can further oxidize to electrophilic quinones (q), which may react with dna and induce thereby mutations. conjugation of these ce in extrahepatic tissues is mainly catalyzed by catechol-omethyltransferase. in order to identify possible mutagenic metabolites (i) the induction of gene mutations by e2 was determined in male chinese hamster lung fibroblasts (v79 cells) expressing human (h) cyp1a1 and (ii) the metabolite profile of e2 in these cells was analyzed via gas chromatography/mass spectrometry after solid phase extraction of the cell suspension in the culture medium. (i) gene mutations were assessed using the hypoxanthine-guanine phosphoribosyltransferase assay. the promutagen benzo[a]pyrene (bap) served as positive control requiring metabolic activation by hcyp1a1 and dimethylsulfoxide as solvent control. v79 hcyp1a1 were treated with 100 nm e2 for 3 weeks and the resulting 6-thioguanine (6-tg) resistant mutants selected at weeks (w) 2 and 3. the frequency of spontaneous 6-tg resistant mutants per 10 6 colony-forming cells ranged from 5 ± 1 (w2) to 9 ± 4 (w3). as expected, 0.25 µm bap induced a significant increase in mutant frequency (mf, 266 ± 13, w2 and 132 ± 46, w3) . treatment with 100 nm e2 resulted in a 3-fold (17 ± 2, w2) and a 2-fold (23 ± 4, w3) increase in mf, suggesting slight mutagenic activity. in culture medium of v79 hcyp1a1 treated with 100 nm e2, 2-ho-e2, 2-methoxy-(meo)-e2, 3-o-methyl-2ho-e2 and 4-meo-e2 (suggesting intracellular formation of 4-ho-e2) were detected. while 4-meo-e2 concentration remained constant over the exposure period, the concentration of the other metabolites increased in a timedependent manner. the maximum concentration increase was reached at w3 for methylcatechols and at w2 for 2-ho-e2, correlating with the maximum increase in mf, observed after 2 weeks as well. in conclusion, e2 possessed a slight mutagenic potential after hcyp1a1-mediated activation to 2-, 4-ho-e2 and their corresponding methylcatechols. cumulative effects of three triazole fungicides in a broad dose range in vitro rieke s., kneuer c., bumke scheer m., lampen a., hirsch-ernst k., marx-stoelting p. bundesinstitut für risikobewertung chemikaliensicherheit, max-dohrn-str., 10589 berlin, germany consumers are exposed to multiple residues of different pesticides via the diet. this raises questions concerning potential cumulative effects, especially for substances causing toxicity by a common mode of action. the aim of this work was to investigate potential combination effects of the three triazole fungicides epoxiconazol, tebuconazol and flusilazol for selected parameters in a broad dose range in vitro. parameters investigated were cytotoxicity, hormone synthesis (17-β estradiol, progesterone and β-hcg), expression of a panel of androgen-or estrogen-responsive genes in the human placental choriocarcinoma cell-line jeg-3 and transactivation via estrogen receptors α and β in stably-transfected hek 293 cells. the ability to inhibit steroidogenesis was analysed by measuring the concentrations of 17β-estradiol and progesterone in cell culture supernatants of jeg-3 cells. additionally, the placental peptide hormone β-hcg was measured. while no change in β-hcg and 17β-estradiol concentrations were observed, all triazoles induced a dose-dependent decrease in progesterone concentration and a cumulative effect was observed implying dose additivity at individual doses of >1.7 µg triazole/ml. significant activation of erβ by the three triazoles, especially by flusilazol, was observed at 5 µg triazole/ml and combined exposure showed additive effects, while no significant activation of erα was observed. based on the data, our findings suggest dose-additivity of triazole pesticides with the same mode of action for selected parameters in vitro. no significant effects were observed at lower doses [1ng -1µg triazole/ml] neither for substances applied individually nor in combination. transient receptor potential channels as mediators of catecholamine release mathar i. 1 trp proteins form cation channels that are regulated through strikingly diverse mechanisms. recently, genetic association studies identified many trp genes including trpm4 as risk factors for disease states such as arrhythmias, hypertension and cardiomyopathy. the melastatin trp channels trpm4 and trpm5 have distinct properties within the trp channel family; they form non-selective cation channels activated by intracellular calcium ions and are expressed in heart, aortic endothelial cells, kidney and adrenal gland. disruption of the trpm4 gene in mice leads to increased basal blood pressure without evidence for impairment of endothelium-or smooth muscle-dependent regulation of contractility of peripheral resistance vessels, the renin angiotensin aldosterone system, basal cardiac output or body fluid homeostasis. instead, trpm4-deficient chromaffin cells exhibit increased acetylcholine-induced exocytosis of catecholamines which is associated with elevated level of epinephrine in the plasma and its metabolites in the urine. this indicates that trpm4 serves as an inhibitory regulator of exocytotic catecholamine release, at least in chromaffin cells. whether catecholamine release is also regulated by trpm4 in other cells of the sympathetic nervous system such as perivascular neurons still needs to be clarified as well as the molecular mechanism underlying how trpm4 regulates catecholamine release. besides trpm4 we recently identified transcripts encoding additional trp channels including trpc5 and trpc6 in chromaffin cells isolated by laser capture microdissection but their functional role in these cells is still unknown. measurements of the time course of the intracellular calcium concentration before and during acetylcholine stimulation (10µm) of catecholamine release as well as the analysis of the number of released vesicles in chromaffin cells relvealed no changes in trpc1/c5/c6 triple knock out mice compared to wildtype controls. although it seems that these trpc proteins are not directly involved in catecholamine release from chromaffin cells induced by acetylcholine application in our hitherto existing experiments, their contribution to the modulation of catecholamine release by agonists of gq-coupled receptors still needs to be analysed. aims: sulfonylureas (sus) are among the most widely used oral hypoglycaemic drugs that stimulate insulin secretion. in addition, sus have pleiotropic effects on other tissues. regarding the effects of sus on adipocytes conflicting findings were reported. we have now investigated the actions of glimepiride and glibenclamide (=glyburide) in primary human adipocytes. methods: primary cultured human white pre-adipocytes were differentiated in vitro according to a standard protocol. lipid accumulation was assessed by oil red o staining and determination of triglyceride content; gene expression was measured by real-time pcr and western blotting. results: we initially characterized the genes regulated during human preadipocyte differentiation by a global microarray analysis. treatment with glimepiride and glibenclamide caused a strong accumulation of lipid droplets and an increase in triglyceride content. genes involved in lipid metabolism were induced, chemokine expression was decreased. interestingly, the effects of sus were over all qualitatively and quantitatively similar to pioglitazone. in direct comparison glibenclamide was more potent than glimepiride in respect to the induction of fabp4 (ec 50 0.32 vs. 2.8 µm), an important adipocyte marker gene. su-induced differentiation was virtually completely blocked by the pparγ-antagonist t0070907 but not affected by diazoxide, indicating pparγ activation by sus. repaglinide, causing insulin liberation like sus but being structurally different, had no effect on adipocytes. conclusions: in primary human pre-adipocytes, glibenclamide and glimepiride strongly induced differentiation, apparently by activating pparγ . thus, sus but not repaglinide may be used to influence insulin resistance beyond their effect on insulin liberation. the role of at1a and at1b receptors as mechanosensors in myogenic vasoconstriction blodow s. 1 , schneider h. arterial myogenic tone denotes the intrinsic property of vascular smooth muscle cells to constrict in response to an elevated intraluminal blood pressure. this physiological reaction is more distinct in small resistance arteries than in large conduit arteries. understanding the underlying mechanisms should provide useful information for the treatment of diseases like anaphylactic shock and systemic hypertension in which this reaction is altered. whereas the underlying signaling cascade has been extensively studied, the molecular identity of the mechanosensory elements still remains elusive. recent studies at the cellular level suggest a sensory function for a subgroup of gprotein coupled receptors (gpcrs) coupling to gq/11-proteins. by determining mrnaexpression levels of selected gpcrs in consecutive pairs of resistance and conduit vessels, we could identify a subset of gq/11-coupled receptors such as angiotensin ii at1b, vasopressin v1a, endothelin eta and etb and α1a adrenoceptor significantly enriched in resistance vessels. by pharmacological blocking of those highly expressed gpcrs by different antagonists and inverse agonists, we evaluated their influence on the formation or the intensity of myogenic tone, as measured in isolated murine mesenteric arteries ex vivo. while blocking of v1a receptor and α2a and α2ab adrenoceptors showed no differences of myogenic tone, blocking of at1a and at1b receptors by losartan and candesartan, eta receptor by bq123 and α1a adrenoceptor by prazosin caused significant reductions of the vascular response. analyzing the myogenic response of at1a -/mice with and without additional blocking of at1b receptors by candesartan suggested that especially at1b receptors play a dominant role for mechanosensitivity in mice. this was further supported by investigating the myogenic response of at1b -/mice. these findings suggest that mechanosensitive gq/11-protein coupled receptors, especially at1b receptors, play a dominant role for the development of myogenic vasoconstriction. trpm3 ion channels are activated by steroidal compounds and noxious heat and are considered to be involved in insulin secretion and pain perception. the expression of the trpm3 gene generates a variety of different transcripts which arise by alternative splicing and the use of different promoters [1] . they encode a substantial variety of isoformes and so far we have identified more than 20 distinct trpm3 proteins in mouse and rat each varying in exons 1, 2, 8, 13, 15, 17 and 24 . these variants differ enormously in their biophysical properties. for example splicing within exon 24 affects the channel pore and causes significant changes of the ionic selectivity of trpm3 channels [2] , whereas splicing of 54 nucleotides encoded by exon 13 leads to dormant trpm3 proteins. however, the frequency of these different isoformes in trpm3 expressing tissues is completely unknown. to get insight into the significance of the different trpm3 isoformes we investigated the abundance of alternative trpm3 transcripts in different tissues and cell types by reverse transcription quantitative pcr (rt-qpcr). we found that the frequency of splicing within exon 13 ranges from 5 up to 20 % in different cell types and tissues. furthermore we analyzed the trpm3 transcriptome in the choroid plexus of the brain and the pituitary gland, tissues in which trpm3 transcripts are most abundant. for that purpose we sequenced more than 120 clones, each. corresponding to our rt-qpcr result, we found a significant number of transcripts lacking exon 13. in cells of the choroid plexus nearly all (124 /126 clones) carried the short ca 2+ permeable pore. furthermore, we identified seven variants spliced in exon 20 encoding truncated trpm3 proteins. however, the composition of the trpm3 transcriptome in the choroid plexus and pituitary gland differed enormously, indicating the importance of alternative splicing for trpm3 function in different tissues. the concept of "thresholds of toxicological concern" (ttc) defines tolerable dietary intakes for chemicals without toxicity data and is widely applied to chemicals present in food in low concentrations such as flavorings. based on a statistical evaluation of the results of many toxicity studies and considerations of chemical structures, the ttc concept derives a maximum daily oral intake without concern of 1800, 540 or 90 µg/person/day for non-genotoxic chemicals depending on the allocation to so-called cramer classes i, ii or iii. for substances with a structural alert for genotoxicity a ttc value of 0.15 µg/person/day might be used. recently, it has been investigated, whether the ttc values, which were derived based on mostly chronic oral dietary rodent studies would cover all relevant toxicities (neurotoxic, repeated dose, reproductive and developmental, immune effects and endocrine-related effects). several authors using different specific databases have confirmed that the ttc values derived using cramer classes are also covering immunotoxic, neurotoxic, reproductive and developmental effects. a respective decision tree is going to be presented, also considering substances or substance classes which shall be excluded from the ttc approach. there are several areas in which the ttc concept is already used, or a ttc approach is considered useful, to assess low-level human exposures, or help in prioritizing toxicological testing; as for example the assessment of plant metabolites and degradates of pesticide active substances, feed and food additives, chemicals with a low exposure profile under reach, residues, metabolites and impurities in plants, chemicals, plant protection products or pharmaceuticals. if no structural alert for genotoxicity is given or standard genotoxicity tests are negative the cramer class iii value of 90 µg/person/day, which corresponds to a dose of 1.5 µg/kg bw is considered to represent a chronic tolerable daily intake of the test substance. examples for current and future uses of the ttc concept in regulatory toxicology are presented. objective: hyaluronan (ha), synthesized by three ha-synthases (has1, -2, -3), is a prominent matrix component of atherosclerotic lesions. the aim of the present study was to identify the has isoenzyme that is associated with ha-matrix remodeling in inflammatory regions of atherosclerotic plaques. furthermore the underlying regulatory pathways were determined and functional aspects of this regulation in vascular smooth muscle cell (vsmc) were addressed. methods and results: during atherosclerosis in apoe deficient mice the peak of macrophage invasion at 14 weeks coincided with ha deposition and induction of has3 in aortic root plaques. in human symptomatic carotid artery plaques has3 was by far the most prominent has isoenzyme as determined by quantitative real time rtpcr. in vitro, in human vascular smooth muscle cell (vsmc) has3 was specifically induced via activation of nfkb by interleukin-1β (il-1β) and tumor necrosis factor alpha (tnfa) as shown by chip assay and utilization of nfkb inhibitor bay 11-7082. has3 was also upregulated in a co-culture system by activated macrophages via paracrine release of tnfa and il-1β as verified by neutralizing antibodies. in human atherosclerotic lesions nfkb positive vsmc were frequently detected in close proximity with ha and f4/80 positive macrophages as shown by immunohistochemistry. to study the effects of has3 mediated ha synthesis in human coronary vsmc, lentiviral overexpression and knockdown of human has3 were employed. overexpression of has3 resulted in increased migration and proliferation whereas knock down had the opposite effect. the effects of has3 were mediated by both pi3k signaling and mapk signaling via hyaluronan receptors cd44 and rhamm. conclusion: the present results suggest that has3-dependent ha synthesis is induced in human vsmc by inflammatory cytokines released from activated macrophages. moreover, has3-mediated ha production induced phenotypic activation of vsmc. pulmonary inflammation and airway remodeling are major features of chronic obstructive lung disease (copd). in addition, pulmonary hypertension is a common comorbidity, which is associated with a poor prognosis of the disease. recent studies in a guinea pig model of allergic asthma have shown that increased arginase activity, which converts larginine into l-ornithine and urea and competes with nitric oxide synthases for the common substrate, contributes to allergen-induced airway inflammation, hyperresponsiveness and remodeling. there is evidence that cigarette smoke and lipopolysaccharide (lps), both involved in the pathogenesis of copd, increase the expression of arginase, however, its role in the pathogenesis of copd is currently unknown. this study aimed to investigate the role of arginase in pulmonary inflammation and remodeling, using a guinea pig model of lps-induced copd. to this aim, guinea pigs were instilled intranasally with lps or saline twice weekly for 12 weeks and were pretreated by inhalation of the arginase inhibitor (2)s-amino-boronohexanoic acid (abh) or pbs. repeated lps exposure increased lung arginase activity, resulting in increased lornithine/l-arginine and l-ornithine/l-citrulline ratio's. both ratio's were reversed by abh treatment. repeated lps exposure also induced increased il-8 levels, neutrophils, goblet cells, hydroxyproline and airway collagen content in the lung, which were all abrogated by abh. moreover, repeated lps exposure increased right ventricular mass, indicative of pulmonary hypertension, which was similarly prevented by abh. in conclusion, increased arginase activity contributes to pulmonary inflammation, airway remodeling and right ventricular hypertrophy in a guinea pig model of copd, indicating that arginase inhibitors may have therapeutic potential in the treatment of this disease. (supported by msd). behavioral abnormalities in hcn3-deficient mice michalakis s., schöll-weidinger m., mader r., cao-ehlker x., fenske s., wahl-schott c., biel m. center for integrated protein science munich (cipsm) department of pharmacy -center for drug research, ludwig-maximilians-universität münchen, butenandtstr. 5-13, 81377 münchen, germany hcn3 encodes a hyperpolarization-activated and cyclic nucleotide-gated channel, which is expressed in various brain regions including thalamic, hypothalamic and habenular nuclei as well as brain stem and olfactory bulb. in this study we performed a comparative analysis of hcn3 -/and hcn3 +/+ mice using a battery of behavioral tests and telemetric biopotential measurements to evaluate a potential role of hcn3 in central nervous system function. in general, the knockout mice showed normal motor function as assessed by the rotarod and open field tests. telemetric home cage activity and core body temperature measurements confirmed a normal circadian behavior, but revealed a lower basal activity that concurred with decreased body temperature during the light phase and the light-dark transition phase. hippocampus-dependent spatial learning was normal. by contrast, hcn3 knockout mice showed more immobility than control mice on day two of the porsolt forced swimming test, which could reflect increased depressionlike behavior. however, center exploration in the open field test as well as performance in the light-dark transition and the elevated-plus maze tests was normal in hcn3 -/mice. this suggests that general anxiety was not changed in the knockout mice. in addition, hcn3 knockout mice were less active on the second day of the open field test, which supports a habituation phenotype. finally, hcn3 -/mice had higher burying scores in the marble-burying test, which is a test for certain aspects of obsessive compulsive disorder in rodents. taken together, genetic deletion of hcn3 in mice results in distinct behavioral abnormalities related to behavioral despair and expression of repetitive behaviors in response to mild stressors. mielke h. 1 , gundert-remy u. alcohol consumption when breast feeding is discussed controversially. some groups recommend breast pumping before alcohol consumption and feeding the stored milk instead of breast feeding after drinking alcohol. this study was performed to simulate the blood concentration in the breastfed baby and to assess the health impact. method: we established a physiologically based kinetic model. its parameters were calculated (partition coefficients tissue/blood ; schmitt, 2008) silva et al.,1993) . we simulated 1. the alcohol concentration in a breastfed neonate and a 3-month-old suckling infant after the nursing mother had consumed alcohol,2. the alcohol concentration in utero/fetal compartment during pregnancy assuming the identical alcohol consumption of the pregnant woman 3. the alcohol concentration during infant´s treatment of bloating by an approved herbal drug containing alcohol. results: peak maternal alcohol concentration was 0.59 ‰ after consuming 0.25 l of wine, peak concentration was 0.0033 ‰ in the newborn, 0.0038 ‰ in the 3-month-old infant and 0.38 ‰ in the utero/fetal compartment. the peak concentration after herbal drug treatment was 0.015‰ in the neonate and 0.015‰ in the 3-month-old infant, respectively. we discuss the results of the simulations and compare it with doses and published concentrations measured in experimental animals or in vitro studies. conclusions: we conclude that the recommendation "1 to 2 glasses of wine on occasion" (agence nationale d'accréditation et d'évaluation en santé, assante 2002) is in accordance with the simulation results presented here whereas stricter rules are not scientifically sound. (2002) http://www.has-sante.fr/portail/upload/docs/application/pdf/ breastfeeding_guidelines.pdf da silva et al. (1993) adenylyl cyclases (ac) mediate physiological responses in virtually all cells, where their regulation through receptors and g proteins results in the modulation of camp. in the present study we focused on the kinetics of interactions between the alpha-subunit from inhibitory g protein type 1 (gαi1) and adenylyl cyclase type v (ac5). these proteins were labeled with cfp and yfp, respectively. the dynamics of their interactions was monitored by means of high temporal resolution fret imaging in hek cells expressing unlabeled a2a-receptor and gβg subunits. to activate the signaling pathway, we applied agonist using a rapid superfusion device. application of norepinephrine resulted in the development of a fret signal, indicating interaction between gai1-cfp and yfp-ac5. after withdrawal of agonist the fret signal recovered with a remarkably slow time course compared to the deactivation kinetics of gi proteins reported previously (bünemann et al. 2003) . to further analyze the properties of the dissociation between gai1 and ac5 we measured in parallel the offset kinetics of the interaction between gai1-yfp and gβg-cfp (gi1-fret) after agonist withdrawal under comparable conditions. in addition we tested to what degree the coexpression of rgs4 accelerated the deactivation of gi proteins and the dissociation of gai1-cfp from yfp-ac5. these experiments revealed that in the absence of rgs4 the dissociation of gai1 from ac5 after agonist withdrawal takes about 3 times longer than the deactivation of gi proteins. in the presence of rgs4 this difference is even larger due to the pronounced acceleration of g protein deactivation. the dissociation of gai1 from ac5 was only marginally accelerated by rgs4. these observations lead us to hypothesize, that ac5 might trap activated g protein-subunits and thereby affect the g protein cycle by shifting the equilibrium towards activated g proteins. if this hypothesis is true, it should result in a left-shifted dose response curve compared to g protein activation dose response. in support of this hypothesis we found that the concentration response curve for gai1-ac5 interaction was several-fold leftward-shifted compared to the concentration-response curve of gi-protein activation under very similar conditions. influencing the dynamics of the g protein cycle by effectors may represent a novel and powerful mechanism for finetuning the sensitivity of receptor evoked responses in an effector-specific manner. obesity, the excessive accumulation of white adipose tissue (wat), has reached pandemic dimensions. the factors that determine fat mass are not fully understood, but adipocyte hypertrophy and adipokine secretion are thought to be important. in present study, we investigated the role of the cyclic gmp (cgmp) signaling pathway focusing on cgmp-dependent protein kinase i (pkgi) in white adipocytes. pkgi is expressed in wat, preadipocytes and differentiated adipocytes as demonstrated by real-time pcr, western blot and immunochemistry. differentiation of pkgifl/fl preadipocytes, using an optimized protocol, resulted in an enhanced lipid accumulation as evidenced by oil red o staining. deletion of pkgi in pkgifl/fl adipocytes infected with a cre lentivirus (lv-cre, pkgi0/0) exhibited reduced differentiation. analysis of the triglyceride (tg) content revealed a significant decrease of tg levels by 65% ± 1% in pkgi0/0 as compared to pkgifl/fl adipocytes. western blot analysis of white adipocytes showed a significant decrease of c/ebpalpha (19% ± 4.8%), ppargamma (66% ± 2.9%) and ap2 (37% ± 10.6%) expression in pkgi0/0 cells as compared to pkgifl/fl. treatment of 3t3-l1 cells with cgmp resulted in increased lipid accumulation and enhanced expression of fat marker genes. lentiviral overexpression of pkgi further increased differentiation. importantly, pkgi significantly induced mitochondrial biogenesis in 3t3-l1 cells. concomitant activation of pkgi in 3t3-l1 preadipocytes and treatment with the demethylating agent 5-aza-deoxycytidine significantly increased expression of uncoupling protein-1 (ucp-1) -a unique protein of brown fat cells. we found rhoa as major target of pkgi signaling with increased phosphorylation of rhoa at ser-188 in pkgi overexpressing cells. moreover, pkgi-dependent phosphorylation counteracts the effects of rhoa on insulin signaling as well as adipokine expression. taken together, pkgi is a key player in white adipocyte differentiation that regulates cell size and has an anti-inflammatory effect. pkgi decreases the secretion of proinflammatory adipokines via inhibition of rhoa signaling. in addition, activation of pkgi can establish a brown fat cell like phenotype during white adipocyte differentiation if the ucp-1 promoter is accessible. the rag gtpases, raga, ragb, ragc, and ragd form a subfamily gtpases of the ras-related superfamiliy. rag proteins are characterized by a modified ras-like gtpbinding domain and a unique c-terminal region lacking a lipid modification motif. interestingly, rag proteins have been proposed to function as heterodimeric complexes consisting of raga or ragb associated with ragc or ragd. rag gtpases have been implicated in the control of mammalian target of rapamycin (mtor) function, in particular in regulation of the nutrient-stimulated and/or hormone-regulated mtor activity. the protein kinase mtor is found as the catalytic subunit of two larger protein complexes referred to as mtor complex 1 and 2, mtorc1 and 2. under amino-acidrich conditions, activated mtorc1 promotes protein synthesis and inhibits autophagy, while under starvation autophagy inhibition is released. increasing evidence suggests that activation of rag gtpases contributes to mtorc1 function. thus, rag proteins were found to be associated with a protein complex termed ragulator, a major regulatory protein of mtorc1 function and guanine nucleotide exchange of rag gtpases within the rag-ragulator-complex were described to promote mtorc1 translocation to its functional lysosomal compartment. however, the guanine nucleotide exchange properties of rag proteins are poorly characterized, and it is currently unknown, how amino acids promote rag proteins to facilitate the formation of the active, raptor-binding state of the rag heterodimers. to characterize the guanine nucleotide exchange properties of the rag gtpases as momomers or heterodimers in more detail, recombinant rag proteins were expressed in bacteria and purified from this source to near homogeneity. first, the parameters of gdp/gtp exchange of each of these proteins were compared using the non-hydrolysable gtp analogon gtpgs. the results showed that the rag isoforms are distinct in their guanine nucleotide exchange activities. in particular, nucleotide exchange on raga and ragc, but not on ragb and ragd, was only observed at low concentrations of gdp and mgcl 2 in extraction and assay buffers, i.e. conditions favoring the gdp/gtp exchange. these findings may indicate that guanine nucleotide exchange on raga and ragc is controlled by guanine nucleotide exchange factors and suggest specific functions of the individual rag gtpases within individual rag heterodimers. in in-vitro studies on rat and canine mast cells and human mast cell leukemia cells hmc1.2 bz at micromolar concentrations inhibited mediator release which appeared to be related to an inhibition of the intracellular camp pathway. in order to identify potential targets on/in mast cells at which bz may cause an inhibitory effect on mast cell activation, the 1,4-bz flunitrazepam (flu), clonazepam (clo) and 4chlorodiazepam (4-cd) were selected because of their different affinity and selectivity to/for the gaba-a-receptor and the translocator protein (tspo): flu and clo bind with nanomolar affinity to gaba-a receptors, whereas 4-cd is a selective ligand at tspo with nanomolar affinity to tspo but only micromolar affinity to gaba-a receptors. flu also possesses nanomolar affinity to tspo, whereas clo has no or only micromolar affinity to tspo. after incubation of hmc1.2 cells with 4-cd, flu and clo for 1, 6 and 24 hours up to 712 genes were significantly differently expressed in a substance-specific and timedependent manner. comparison of the genes differently expressed at 6 hours revealed that the expression of 217 genes was regulated by both flu and clo but only 8 genes were regulated by both 4-cd and flu suggesting that flu and clo induce gene expression by acting at a target site different from that of 4-cd. the difference between the gene regulation by flu and clo on the one hand and that of 4-cd on the other hand is also reflected in pathway analysis. since it was conceivable that the beneficial effects of the 1,4-bz could be mediated by the recognition sites targeted by the 2,3-bz, i.e. the gria2-encoded ionotropic glutamate receptor ampa2, we investigated by quantitative pcr whether hmc1.2 cells express gria2, tspo, the genes encoding the subunits of the gaba-a receptor and the gaba-forming enzyme glutamic acid decarboxylase. tspo, gabra3, gabrb3, gabre and gabrd were moderately expressed. in addition, there was a week or very week expression of gabra2, gabra4, gabrb2, gabrg3 and gabrr2. expression of gria2 was not detectable. taken together, it cannot be decided yet from our data whether the inhibitory effect of benzodiazepines on mast cell activation is due to an action at tspo or at gaba-a receptors of a novel subunit composition. monien b. h., glatt h. german institute of human nutrition (dife) department of nutritional toxicology, arthur-scheunert-allee 114-116, 14558 nuthetal, germany 5-hydroxymethylfurfural (hmf) and furfuryl alcohol (ffa) are common constituents of foodstuffs in which they are formed by heat-and acid catalyzed reactions from carbohydrates. hmf and ffa have been reported to induce the formation of hepatocellular adenomas in female mice and renal tubule neoplasms in male mice, respectively. we studied whether the carcinogenic effect of these hydroxymethylsubstituted furans may originate from sulfotransferase (sult)-catalyzed formation of electrophilic esters. hmf was inactive in in vitro mutagenicity tests using standard activating systems. in contrast, it was mutagenic in v79 cells genetically engineered for expression of human sult1a1 suggesting that hmf is converted into the reactive 5sulfooxymethylfurfural (smf). following incubation of mutagenic smf with porcine liver dna in vitro, specific methylfurfural adducts were detected using liquid chromatography tandem mass spectrometry (lc-ms/ms), i.e., n 6 -((5-formylfuran-2-yl)methyl)-2'deoxyadenosine (n 6 -ffmda) and n 2 -((5-formylfuran-2-yl)methyl)-2'-deoxyguanosie (n 2 -ffmdg). these adducts were also detected in dna from v79-sult1a1 cells incubated with hmf. in order to determine sulfo conjugation of hmf in mice in vivo, we conducted pharmacokinetic measurements showing that about 500 ppm of the hmf dose was converted to smf and reached the circulation. like hmf, ffa was negative in the standard ames test and various other in vitro genotoxicity tests. we showed that ffa is mutagenic in salmonella typhimurium ta100 engineered for expression of human sult1a1. the putative mutagen 2-sulfooxymethylfuran was synthesized and incubated with porcine liver dna, in which various nucleoside adducts were found. the main adducts, -mfda were detected in dna of ffa-exposed salmonella strain ta100-sult1a1 and in dna of liver, lung and kidney of fvb/n mice that had received about 390 mg ffa/kg body weight per day via the drinking water for 28 days. in summary, both furan derivatives form mutagenic sulfate esters in vitro and in vivo. in the future, we will use genetically engineered mice to characterize the role of single murine and human sult forms in the bioactivation of the furan derivatives and the contribution to tumor induction. background: micrornas are small non-coding rnas that can negatively regulate gene expression on a post-transcriptional level and have been shown to interact with epigenetic mechanisms like dna methylation. mecp2 (methyl cpg binding protein 2) is a protein that binds methylated dna cpgs in the promoter region of genes and can thus regulate their expression. otherwise, mecp2 is known to be a target gene for several micrornas including the cluster mir-132/212 in the brain. recently, our group could show that mecp2 expression is downregulated in human heart failure suggesting that mecp2 might be involved in cardiac pathogenesis. the aim of this project is to study the upstream regulation of mecp2 by the cluster mir-132/212 in the heart during cardiac hypertrophy in-vitro and in-vivo. methods and results: to test whether hypertrophic stimuli can induce mir-132/212 expression, we treated cultured nrcms with 40 µm norepinephrine for 72 hours. this induced cardiomyocyte hypertrophy and expression of the hypertrophy marker nppa, but also of mir-132 (1.79 ± 0.14-fold of untreated cells, p<0.01) and of mir-212-3p (1.73 ± 0.26-fold of untreated cells, p<0.05) and downregulated mecp2 mrna and protein levels (0.80 ± 0.04-fold of untreated cells, p<0.05). to check whether mecp2 downregulation also occurs by direct mir-132/212 activation we increased levels of mir-132 and mir-212 in cardiac myocytes by transfecting precursor mir-132 and mir-212-3p molecules. again, we observed nrcm hypertrophy, nppa mrna upregulation and mecp2 mrna and protein downregulation (0.75 ± 0.05-fold of control, p<0.05) after mir-132 overexpression. similar results were obtained by overexpression of mir-212-3p. to test the effects of adrenoceptor activation on the mir-132/212-mecp2 axis in-vivo, wild-type mice received isoprenaline and phenylephrine via osmotic minipumps (30 mg/kg/day each). after 7 days, cardiac ventricles were analyzed. nppa gene expression (2.81 ± 0.32 -fold of control animals), mir-132 and mir-212-3p levels (3.67 ± 0.5 and 2.62 ± 0.4 -fold of control animals, p<0.01 and p<0.05, respectively) were increased while mecp2 protein levels decreased to 77% (p<0.05) conclusion: these results suggest that in-vitro and in-vivo adrenoceptor stimulation leads to the activation of mir-132/212 expression and to downregulation of mecp2 in cardiac myocytes in-vitro and in-vivo. leopold-franzens-universität, innsbruck, austria at-1 receptor antagonists block the angiotensin ii-enhancing effect on noradrenaline release from sympathetic neurons. in a cell-free assay the binding affinity of the at-1 receptor antagonists telmisartan and valsartan to the gamma peroxisome proliferatoractivated receptor (pparγ) is close to that of the pparγ selective agonists thiazolidinediones (tzds). we tested whether the tzds rosiglitazone and pioglitazone would also modify the prejunctional facilitatory effect of angiotensin ii. left ventricular slices of rats were incubated with tritiated noradrenaline, perifused and electrically stimulated. the negative logarithm of the drug concentration that caused a 30% increase of control (pec30%) was calculated. angiotensin ii caused a concentration-dependent increase of tritium overflow induced by electrical stimulation [pec30%=8.6±0.2 (mean±sem, n=18); maximum increase=110±8%]. neither rosiglitazone nor pioglitazone (0.3-3 µm) had a direct effect. the concentrationresponse to angiotensin ii in the presence of fixed concentrations of rosiglitazone was shifted to the left with increase of the maximum (pec30%=8.8±0. 2, 9. 2±0.2 and 9.3±0.3; maximum increase=118±14%, 146±13% and 148±16%, in the presence of 0.3, 1 and 3 µm of rosiglitazone, respectively, n=4-6, each). in contrast, pioglitazone in concentrations up to 3 µm had no effect on the release-enhancing effects of angiotensin ii. results show that rosiglitazone but not pioglitazone potentiates the noradrenalinerelease enhancing effect of angiotensin ii. this action might contribute to the risk for myocardial infarction from rosiglitazone use but not from pioglitazone use. deleted in liver cancer 1 (dlc1) is a tumor suppressor whose allele is lost in 50% of liver, breast, lung and 70% of colon cancers. despite its significance, the molecular mechanisms that drive cancerous transformation upon dlc1 loss remain unclear. we found that the transcriptional coactivators megakaryoblastic leukemia 1 and 2 (mkl1/2) are constitutively localized to the nucleus in hepatocellular and mammary carcinoma cells that lack dlc1. moreover, dlc1 loss and mkl1 nuclear localization correlated in primary human hepatocellular carcinoma. nuclear accumulation of mkl1 in dlc1-deficient cancer cells was accomplished by activation of the rhoa/actin signaling pathway and concomitant impairment of erk-mediated mkl1 phosphorylation. dlc1 loss led to constitutive activation of the mkl-dependent, tumor-relevant target genes ctgf, cyr61, myl9 and myh9. furthermore, we identified a novel target gene, integrin a5, with a key role in cell migration and metastasis, that exhibited a dlc1-and mkldependent regulation. depletion of mkl1/2 suppressed not only cell migration, but also cell proliferation and anchorage-independent cell growth induced by dlc1 loss. our data provide insight into the mechanism by which dlc1 loss initiates tumorigenesis. as mkl1 and 2 have a key role in this process, this pathway may provide promising pharmacological targets for cancer therapy. universität bonn, pharma-zentrum bonn pharmazeutisches institut, pharm. chemie i, an der immenburg, 53121 bonn, germany membrane receptors activated by purine and/or pyrimidine nucleotides ("p2 receptors") are widely distributed in the body and constitute novel (potential) drug targets. they are subdivided into g protein-coupled p2y receptors (p2y1, 2, 4, 6, 11, 12, 13, 14) , and homo-or heterotrimeric ligand-gated ion channel or p2x receptors (subunits: p2x1-7). we have been interested in the identification and development of potent and subtype-selective ligands -as tool compounds and potential drugs -for the various p2y and p2x receptor subtypes. our strategy involves (i) establishment of a proprietary compound library consisting of synthetic small molecules and natural products; (ii) development of screening assays suitable for medium throughput screening; (iii) careful analysis of structure-activity relationships at each target and systematic optimization of the lead structures; (iv) pharmacological evaluation of selected compounds. this approach has led to new biological tools for several targets, including p2y and p2x receptors [1] [2] [3] [4] [5] [6] . fine particles in particulate matter (pm) are effective vehicles to transport toxicants into the lung; depending on their size, smaller particles may reach the bronchiolar or alveolar space. in recent years the pm fraction pm2.5 has especially been correlated with both pulmonary and cardiovascular diseases. in order to better characterize pm emission and distribution of environmental tobacco smoke (ets) from cigarettes (reference cigarette (rc), brand cigarette (bc)) we have developed an ets emitter to simulate human smoking emission and measured pm2.5 concentration in a telephone booth (1,75 m3 volume) as an example for small indoor spaces like cars. fine particulate matter was measured using an aerosol spectrometer with 6 sec time resolution; laser scatter allowed a size resolution from 0,25 µm to 32 µm. for the pm2.5 concentration the following values were calculated: cumulative pm2.5 concentration as auc-pm2.5 (µg/m3/sec), peak pm2.5 concentration as cmax-pm2.5 (µg/m3) and average pm2.5 concentration cmean-pm (µg/m3). in closed door condition both cigarettes produced particulate auc-pm2.5 values of 59 000 ± 15 000 µg/m3/sec (rc) to 85 000 ± 31 000 µg/m3/sec (bc after myocardial infarction (mi) inflammatory cells and cardiac fibroblasts (cf) determine the remodeling response. interleukin-6 (il-6) is induced in the ischemic myocardium and is known to stimulate the differentiation of fibroblasts to myofibroblasts. hyaluronan (ha) is an extracellular matrix component synthesized by ha-synthase isoenzymes (has 1-3) and is also known to control fibroblast phenotypes. however, it is presently unknown whether il-6 participates in the remodeling of the ha-matrix or whether the ha-matrix modulates the responses to il-6. therefore, the aim of the present study was to elucidate whether il-6 regulates the expression and function of ha-matrix in cfs. cells were isolated from c57bl/6j mice and used during passage 2-3 for experiments. cfs were stimulated with il-6 or hyper-il-6 which is a fusion protein of il-6 and soluble il-6 receptor (sil-6r). after 10 and 30 min signal transducer and activator of transcription 3 (stat3) was phosphorylated in response to hyper-il-6 but not in response to il-6. rt-pcr revealed rapid upregulation of has 1 (5.94 ± 2.49 fold of unstimulated control, 1h) in response to hyper-il-6. has2 was induced to a lesser degree (2.04 ± 0.55 fold of unstimulated control, 1h) whereas has3 was not responsive (1.49 ± 0.42 fold of unstimulated control, 1h). in contrast, il-6 had no effect on transcript levels of has isoenzymes. in turn, expression of has1 and has2 in response to hyper-il-6 was inhibited by ag490, which indicates the involvement of stat signaling. interestingly, despite induction of has1 and has2 the amount of secreted ha as determined by an elisa-like assay was not affected by hyper-il-6. this may indicate that il-6 regulates the cell surface associated ha-matrix of cfs. in conclusion, the present data demonstrate that cardiac fibroblasts respond to il-6 trans-signaling (hyper-il-6) via the soluble il-6r and subsequent stat3 signaling with increased ha-synthesis. the fact that il-6 had no significant effect suggests that the expression of the non-signaling membrane-bound il-6 α-receptor (il-6r) in cultured murine cardiac fibroblasts is not sufficient to induce has 1 and -2 gene expression. therefore, il-6 trans-signaling mediated by il-6 and the circulating sil-6r might be necessary to mediate the il-6-induced has expression in vivo. mrgprd receptor endogenously expressed in dorsal root ganglia: evidence for an activation by 3-aminoisobutyric acid müller s., hoffmann k., von kügelgen i. universität bonn institut für pharmakologie und toxikologie, sigmund-freud-straße 25, 53127 bonn, germany the gpcr mrgprd (mrgd) is highly expressed in small diameter dorsal root ganglion (drg) neurons and has been implicated to play a role in nociception. the receptor was previously shown to respond to β-alanine. in the present study we searched for agonistic activity of structural analogues of β-alanine. for further characterization of the receptor we used fura-2 fluorimetry, a nfat luciferase reportergene assay and the determination of the inhibition of forskolin-induced camp production ([ 3 h]-camp affinity assay). first, we confirmed the activation of the receptor by β-alanine and gaba. in reportergene experiments we then identified 3-dlaminoisobutyric acid as an agonist, with similar potency but weaker affinity when compared to β-alanine (ec50 165 µm). fura-2 fluorimetry showed an increase in intracellular ca 2+ levels by 3-dl-aminoisobutyric acid (300 µm). moreover, 3-dlaminoisobutyric acid reduced the forskolin-induced camp production by up to 65 % (ec50 195 µm). in addition to 3-dl-aminoisobutyric acid, we identified 3-dl-aminobutyric acid as a weak agonist acting at the mrgprd. other closely related substances failed to show significant responses. next to the agonists we further characterized antagonists inhibiting the response to βalanine mediated by mrgprd. chlorpromazine shifted the concentration-response curve of β-alanine to the right with an apparent pkb of 4.9 (nfat assay), thioridazine with an apparent pkb of 5.4 (nfat assay) and 5.3 (camp assay) and rimcazole with an apparent pkb of 5.2 (nfat assay) and 5.2 (camp assay). in conclusion we show for the first time that 3-dl-aminoisobutyric acid is an agonist at the mrgprd and that the structure-activity relationship of agonists at mrgprd is very close. the sdf-1-chemokine receptor cxcr4 plays a key role during embryogenesis and regulates functions of immune and stem cells in adult life. furthermore, cxcr4 is involved in disease states including inflammation and cancer. it is well established that sdf-1-stimulated cxcr4 receptors activate gi protein-dependent signal transduction pathways and undergo c-terminal phosphorylation and internalization. because the cxcr4 c-terminal domain contains 15 serine and 3 threonine residues, it is incompletely understood which of the potential phosphorylation sites contribute to homologous and heterologous regulation of cxcr4. here, we analyzed the phosphorylation pattern of cxcr4 at 3 c-terminal sites after stimulation of the receptor with sdf-1 and after pma-induced activation of the pkc pathway as a model for heterologous receptor phosphorylation. using phospho-specific antibodies against s324/325, s338/339 and s346/347 in immunoblot analyses, we showed that the 3 sites were phosphorylated after stimulation with sdf-1 or pma. stimulation with egf or forskolin did not induce phosphorylation at these sites. sdf-1-induced phosphorylation at s324/325, s338/339 and s346/347 was reversible after wash out of the ligand. time course analyses revealed that phosphorylation occurred first at s346/347 and then at s324/325 and s338/339. taken together, these results indicate that the c-terminus of cxcr4 is phosphorylated at multiple sites by homologous and heterologous pathways and that phosphorylation at the different sites may be hierarchically organized. human milk represents the best form of nutrition for infants early in life. however, it can also contain toxic contaminants that may adversely affect infant's development. the nephrotoxin ochratoxin a (ota) is present in human milk (tab. 1 in [1] ), but information on transfer from maternal blood to milk is scarce: published data [2] indicate that levels of ota in milk are roughly one tenth (0.1) of those in blood. but, the efficiency of the ota-transfer at various stages of breastfeeding may vary since studies in animals revealed that transfer of ota is apparently time-and dose-dependent. thus, the aim of this study was to assess the ota transfer from blood to milk at different stages of breastfeeding in humans. in a small chilean cohort, 18 lactating women were asked to provide blood and milk on the same day. these samples were collected on four different occasions within the first months after delivery and analyzed using hplc with fluorescence and/or tandem mass spectrometric detection [1] . the transfer of ota from blood to milk was quantitatively assessed by measuring the milk to plasma ratio (m/p). the average ota level in blood plasma was 235 ± 129 ng/l, and no major variations were observed over time (p = 0.19). on the other hand, ota levels found in colostrum (85 ± 60 ng/l) were higher than in mature milk (p < 0.05). in line with these data, higher m/p ratios (table) were obtained with samples collected in the first six days after delivery. this study showed that the transfer of ota from blood to milk was more efficient with colostrum (m/p 0.43 ± 0.26) than with mature milk. thus, a higher exposure to ota can be expected for neonates than for infants at later stages of breastfeeding. moreover, the lactating women have lower average ota levels in plasma than non lactating women from chile [3] , indicative of milk as additional excretion route. acknowledgement: this work has been supported by a stipend from conicyt/daad to km. exposure of infants to ochratoxin a (ota) deserves particular attention since ota is nephrotoxic, and one of the most potent rodent renal carcinogen studied to date [1] . moreover, infants may be more vulnerable to the toxic effects of contaminants than adults. ota-levels in plasma of infants are indicative of an early exposure in life [2] . but blood sampling is an invasive method not readily applicable for breastfed infants. thus, the aim of this study was to implement a non invasive biomonitoring method to assess ota-exposure in this group. to assess the exposure to ota, breast milk and infants' urine specimens were collected, from two different cohorts: chile (n= 28) and turkey (n=10, only urine). analysis of the samples was performed using enzymatic hydrolysis prior to extraction and hplc-ms/ms [3] . the magnitude of infants' exposure was assessed by calculating the ota-daily intake with human milk and relating it also to urinary ota levels. calculations of the daily intake with human milk [4] showed that infants may be exposed to ota at high levels, exceeding the tolerable daily intake (tdi) of 5 ng/kg-bw/day set for adults [1] . in both cohorts, most of the urine samples tested positive for ota (chile 72%, turkey 80%). ota levels observed in urine samples from the turkish infants (range: 116 -1,361 ng/l) were 10 fold higher than levels found in chilean samples (range positive samples: 17 -320 ng/l). further analysis of phase ii metabolites in urine confirm the excretion of ota as conjugate (glucuronide) in highly exposed infants. in conclusion, ota exposure of infants early in life was documented. given that otaintake by several infants exceeded the tdi for adults, further biomonitoring in this vulnerable group is advised including also suitable biomarkers of effect. a mixture of (e)-and (z)-clomiphene citrate is the first line therapy of female infertility. however, up to 30% of patients do not respond. (e)-clomiphene is structurally closely related to another selective estrogen receptor modulator, tamoxifen which is frequently used for the treatment of hormone receptor-positive breast cancer. like tamoxifen, clomiphene is extensively metabolised by the cytochrome p450 system. using the estrogen receptor response assay (e)-4-hydroxyclomiphene and (e)-4-hydroxy-n-desethylclomiphene (ec50: 2.5 and 1.4 nm, respectively) turned out to be 100 times more active at the er compared to the parent drug isomers and de-ethylated metabolites. using recombinant expressed human cyp isoforms and inhibitory antibodies, cyp2d6 revealed to be the major isoenzyme involved in the formation of 4-hydroxlated metabolites. n-deethylation was catalysed by cyps 3a4/5, 2d6, 2c19 and 2c8. rates of 4-hydroxylation in microsomes from 30 human liver donors correlated with the number of functional cyp2d6 genes. these in vitro results were confirmed in a pharmacokinetic study with female healthy volunteers receiving a single dose of clomiphene. in carriers of two non-functional cyp2d6 genes (poor metabolizers) cmax of (e)-4-hydroxyclomiphene and 4-hydroxy-ndesethylclomiphene was 8 and 12 times lower, respectively, when compared with subjects with at least one fully functional cyp2d6 allele. in contrast, half-life of (e)-clomiphene and (e)-n-desethylclomiphene was 10 and 50-fold higher, respectively, in poor metabolizers. our data provide first evidence of a pharmacogenetic rational for the variability in the response to clomiphene treatment. among the tested compounds, compound 1 proved to be the most active derivative, showing a significant toxicity at a concentration of 2,5 µm. compounds 2 and 3 showed significant toxic effects at a concentration of 5 µm. the compound 4 showed no toxicity up to a concentration of 100 µm. all derivatives 1, 2 and 3 have a ec50 between 10 and 15 µm. we further proved the induction of apoptosis by apo-one assay (caspase 3/7 activity) and life/dead-assay (fluorescence microscopy). in conclusion, these gold complexes exhibit an example of interesting potential candidates for future anticancer pharmaceuticals due to relatively high cytotoxicity. gene regulating effects in mouse liver subsequent to treatment with selected dioxin-like compounds and pcb 153 using whole genome microarray analysis neser s. 1 , lohr c. 1 , van ede k. i. 2 , andresen k. 3 interaction with the aryl hydrocarbon receptor (ahr), with 2,3,7,8-tetrachlorodibenzo-pdioxin (tcdd) being the most potent congener amongst the ahr agonists. recent risk assessments have employed the toxic equivalency factor (tef) concept. the current eu-project systeq aims at developing, validating, and implementing human systemic tefs as indicators of toxicity for dl-compounds. at present, the best known parameter of ahr mediated effects is the induction of cytochrome p450 isoenzymes (cyps), i.e., cyp1a1, 1a2, and 1b1. one of the major objectives of the systeq project is the identification of novel quantifiable biomarkers. in a three day study, female c57bl/6 mice were treated with single doses of six dl-congeners (tcdd, pcb 118, pcb 126, and pcb 156) , and the 'non-dioxin-like' (ndl) pcb 153. quality tested (agilent ® bioanalyzer) mrna isolated from livers was analyzed using the agilent ® mouse whole genome array (4x44k) system. the quantity of genes affected (≥ 2fold) was highly heterogeneous amongst the dl-compounds. whereas tcdd-treatment upregulated 89 genes, and down-regulated 66, 4-pncdf-treatment had impact on 3208 (up), and 2396 (down). treatment with pcb 118 led to marginal numbers of 16 up and 6 down-regulated genes. with 64 (up), and 38 (down) genes shared, the most extensive overlap occurred between tcdd-and 1-pncdd-treatment. no overlap was found due to treatments with the ndl pcb 153 (21 up, 1 down) and tcdd. when comparing the effects of all dl-congeners, minor numbers of genes of 19 up, and 5 down-regulated remain, most of them being related to drug metabolism. while pcb 118 regulated only genes involved in drug metabolism, omission of pcb 118-regulated genes resulted in consistently 51 (up), and 24 (down) regulated among dl-compounds. in conclusion, our findings suggest that the pattern of gene regulation in mouse liver elicited by pcb 153 was strictly different from tcdd, while a very limited coincidence of genes was found even among dl-compounds. comparison of these 'core' genes with data from human models is required with respect to determination of novel biomarkers. introduction: proper use of antibiotics is essential with regard to effective treatment of bacterial infections. providing adequate information for patients can contribute to achieve this aim. materials and methods: data was collected from the relational database of the drug information service at dresden university of technology. the patients, who used the service, were interviewed concerning socio-demographic characteristics, reason for enquiry, number and kind of drugs taken, and diseases. possible contact paths were phone, e-mail or letter. in the present evaluation, all enquiries from the years 2009 and 2010 were analyzed descriptively focussing primarily on systemic antibiotics as reason for the enquiry. results: in the evaluated period, 4454 enquiries were registered in total. in 4.7% of those enquiries systemic antibiotics were named with a total number of 283 drugs. 50.9% of those antibiotics were found to be the direct reason for the enquiry. most common information requested by patients corresponded to adverse drug reactions (50.7%), diagnosis/treatment (35.4%), drug application (29.2%) and drug-druginteractions (19.4%). the majority of the requesting patients (61.5%) was born before 1970. a correlation between incidence of enquiries especially concerning antibiotics and quarterly statistics could not be detected. conclusion: mainly patients aged 40 years or more seem to need or search for further information about antibiotic medication. advice is required especially regarding adverse drug reactions and diagnosis or treatment. in order to this, the advisory service can help patients to lose their insecurity and to gain more confidence in handling antibiotic drugs. colon cancer is one of the most frequent cancers in the industrialized nations. epidemiological studies show a correlation between highly processed meat and the development of colorectal tumours. it is assumed that the risk of developing colorectal cancer, among various different factors, is related to the uptake of toxic substances contained in food such as heterocyclic aromatic amines that arise during the processing of fish and meat. phip is the most abundantly formed heterocyclic compound, and therefore has the biggest impact. in a previous study, we measured the absorption of phip in different intestinal segments of the rat. in the present study we focussed on the potential mechanisms by which phip is reabsorbed. the unidirectional phip transport from the mucosal to the serosal compartment (j ms) and in the opposite direction (jsm) was examined using the ussing chamber technique and 14 c-phip as a radiotracer. the proximal jejunum and distal colon of male fischer 344 rats in short-circuit current chambers was clamped, so that mucosal and serosal compartments were built. the phip flux rates were determined at defined intervals over 120 min. the experimental conditions were selected in such a way that negative net flux rates (jnet = jms-jsm) were indicative of an active secretion. both intestinal segments showed large differences. while in the jejunum jms and jsm of phip were not significantly different, there was an active secretion in the colon. in a next step the transport proteins involved in this process should be examined. introduction: human organic anion transporter 2, oat2 (slc22a7), is abundantly expressed in kidney and liver and mediates the sodium-independent uptake of clinically relevant drugs like 5-fluorouracil, paclitaxel, bumetanide, tetracycline, and zidovudine. while immunohistochemical studies have localized human oat2 to the basolateral membrane of kidney proximal tubules, its hepatic localization is currently unknown. we, therefore, firstly determined oat2 localization in human liver. because interindividual variability of oat2 expression may affect hepatobiliary drug uptake and elimination, we next systematically investigated the influence of genetic and non-genetic factors on hepatic oat2 expression. methods: an expression profile of oat2 for 20 human tissues was determined by realtime quantitative polymerase chain reaction (taqman). oat2 mrna expression was analyzed in well-characterized human liver samples from 150 caucasians that were accompanied by detailed demographic and clinical data. oat2 was localized in human liver cryosections using a commercial rabbit polyclonal antibody and hepatic oat2 protein levels were determined. resequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and genome-wide single nucleotide polymorphism microarray technology served to genotype variants in the slc22a7 gene region. results: oat2 mrna was expressed in several human tissues, including liver. moreover, a new alternatively spliced variant of oat2 was identified in human liver. hepatic expression of full-length oat2 mrna and oat2 protein varied 37-fold and 41fold, respectively. oat2 mrna and protein levels did not correlate with each other. oat2 was localized to the sinusoidal membrane of human hepatocytes. no novel variants in the 9 exons, the 5'-flanking region, or the 3'-untranslated region of the slc22a7 gene were identified. univariate analysis showed that oat2 mrna is reduced in patients diagnosed for cholestasis (p=0.0012) and is affected by genetic variants. whereas the influence of genetic variants on hepatic oat2 expression appears to be limited, cholestasis significantly contributes to the variable interindividual oat2 expression. this indicates consequences for hepatic drug elimination of and response to oat2 drug substrates such as paclitaxel or tetracycline. the life threatening toxicity of organophosphorus (op) nerve agents is caused by the inhibition of the acetylcholine esterase (ache). oximes were shown to be potent reactivators of inhibited ache, but in poisoning by some compounds, e.g. soman, they have only a small therapeutic effect. for such cases, an alternative new strategy may be the intervention at nicotinic acetylcholine receptors (nachr). previous studies with the bispyridinium non-oxime mb327 demonstrated therapeutic effects against soman in vitro and in vivo which was partly attributed to its direct interaction with nachrs [1] . we investigated the interaction of mb327 and several structure analogous at the orthosteric binding site of human α7 nachr (hα7 nachrs), a subtype which appears to be widespread in the human body, and compared the results with data obtained from torpedo-nachrs, which show a high degree of homology with human muscle-type nachrs. interaction of compounds with the orthosteric binding site of hα7 nachrs were investigated with radioligand binding experiments performed as high-throughput method [2] . membrane preparations of gh 4c1 cells stably expressed hα7 nachrs were incubated with the nachr agonist [³h] epibatidine and appropriate concentrations of the unlabelled competitors e.g. bispyridinium compounds. after incubation, bound and free [³h] epibatidine were separated by rapid vacuum filtration. ki values of the competing compounds were calculated with nonlinear regression. three bispyridinium compounds, mb442, mb456 and mb770 exhibited ki values at micromolar concentrations while three other compounds, mb327, mb583 and the pharmacological inactive mb424 (negative control) did not show any interaction with the orthosteric binding site of hα7 nachrs. with torpedo-nachrs, ki values were in similar orders of magnitude -except mb442 which indicated significant subtype selectivity. interestingly, the affinity of monomeric pyridinium derivates did not correlate with their bispyridinium structure analogues. obviously, no correlation between the affinity to the orthosteric binding site and the functional improvement of neuromuscular transmission exists, although species-related differences cannot be excluded. in this study, we analysed the cytotoxic and clastogenic effects of the anticancer drug nimustine (acnu) in cells deficient in repair proteins involved either in homologous recombination (hr) or non-homologous end-joining (nhej). we show that hr mutants are extremely sensitive to acnu as measured by the induction of apoptosis and colony formation as well as the induction of chromosomal aberrations. the nhej mutants were slightly sensitive to acnu and differed in their sensitivity, with the ku80 mutants being moderately sensitive and the dna-pkcs mutant resistant, comparable to the wild-type (wt). cell death was mostly executed via the caspase-dependent apoptotic pathway with involvement of caspase-3 and -7, and necrosis was also induced. further, we investigated the kinetics of dna double-strand break (dsb) formation that resulted from the repair of acnu-induced interstrand cross-links by means of γh2ax and 53bp1 foci analysis in wt and mutant cells. cells mutant in hr did not repair dsb and went into the apoptotic or necrotic pathway, whereas wt cells were able to repair most of the dsb. cells deficient in ku80 formed at early times after acnu treatment less γh2ax and 53bp1 foci compared to the corresponding wt, which might be due to a reduced capacity of recognising dsb. at later times after treatment, ku80 mutant cells show foci levels similar to the wt indicating restitution of h2ax phosphorylation. we also analysed whether dsb formation after acnu treatment was replication-dependent using synchronised cells. we determined the formation of γh2ax and other dsb marker in wt cells that passed through the first cell cycle after demecolcine synchronization. the level of γh2ax foci increased significantly in the s-phase and remained at a high level during g2 where a fraction of cells remained arrested. rad51, atm, mdc-1 and rpa-2 foci were also formed and shown to co-localize with γh2ax. these foci were ameliorated significantly in s-and g2-phase, which was similar to the time course of γh2ax foci formation. in western blots, we confirmed a higher phosphorylation level of atm and chk2 and less phosphorylation of chk1 in hr mutants. the data indicate that acnuinduced dna cross-links give rise to cyto-and genotoxicity via the formation of dsbs that activate the cellular dna damage response. the endocannabinoid system has been established as a mediator of numerous central and peripheral biological functions. cannabinoids have emerged as attractive alternatives or supplements to therapy with opioids for chronic pain states. however, in human the activation of cannabinoid receptors is associated with side effects. for clinical exploitation of the analgesics properties of cannabinoids, a major challenge is to devise strategies that reduce or abolish their adverse effects on cognitive, affective and motor functions without attenuating their analgesics effect. in animal studies, the anti-nociceptive efficacy of cannabinoids has been unequivocally demonstrated in several models of inflammatory and neuropathic pain. however, there are marked inconsistencies between different reports with respect to the locus of these pain-protective effects. we are working towards establishing the contribution of cb1 receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia. using cb1 globally knock-out animal as background, we induce the expression of cb1 specifically in nociceptive neurons localized in the peripheral nervous system and test the analgesic effects of cannabinoid systemical delivery in these mice. our results support the development of peripherally acting cb1 analgesic agonist with reduced central side effects. furthermore, we are utilizing proteomics approach to identify protein complexes that interact with cb1 receptor which hold promise in understanding cannabinoid signaling in health and disease. most chemoattractants, including chemokines, complement c5a, fmlp, and leukotriene b4 are signaling through heterotrimeric g proteins of the pertussis toxin (ptx)-sensitive gi family. the functional inactivation of all gαi proteins with ptx leads to a fulminant decompensation of the immune system, whereas the constitutive inactivation of a single gαi coding gene results in mild phenotypes in mice. we are mostly interested in the nonneuronally found gαi2 and gαi3 isoforms and their redundant and specific roles in immune function and infection. for this purpose cellular in vivo and ex vivo models and in vivo infection model with listeria monocytogenes are being used. macrophages were isolated from the peritoneal cavity of wild type (wt) and gαi-deficient mice 4 days after i.p. injection of 4% thioglycolate that induces peritonitis in vivo. we confirmed previous observations that in gαi2-deficient mice the migration of macrophages into the peritoneal cavity was reduced after induction of peritonitis. regarding the expression levels of gαi and gβ isoforms in the lavage samples, the predominant gαi isoform gαi2 was upregulated in gαi3-deficient macrophages. vice versa gαi3 was upregulated in gαi2-deficient macrophages. concerning gβ isoforms, both gβ1 and gβ2 were strongly reduced in the gαi2-deficient macrophages which resulted in a reduced total amount of gβ. surprisingly, the gαi3-deficient macrophages showed reduced gβ1 protein levels only which caused a change in the gβ2/ gβ1 quotient in favour of gβ2. we are currently establishing an in vivo infection model with l. monocytogenes in gαi2and gαi3-deficient mice. our previous in vitro infection studies in mice embryonic fibroblasts provided us with information about possible distinct roles of these two isoforms as far as the uptake of l. monocytogenes in the cells is concerned. challenging the immune system of gαi-deficient mice with this pathogenic organism will give us new insights into the systemic immune response in these mice upon bacterial infection. our data indicate that we may surmount the redundancy between these two isoforms and focus on their distinct and specific roles in pathogen defense. fret-based β-arrestin2 biosensors reveal conformational changes upon binding to the β2-adrenergic receptor in real time and living cells nuber s., zabel u., ziegler n., hoffmann c., lohse m. j. institut für pharmakologie und toxikologie pharmakologie, versbacherstr.9, 97078 würzburg, germany β-arrestins are multifunctional adapter proteins that regulate seven transmembranespanning receptor (7tmr) signaling and initiate also alternative signaling pathways. studies have shown that β-arrestins undergo conformational changes upon receptor stimulation, which are thought to be necessary for its downstream actions. to investigate these conformational changes in living cells we constructed fret based biosensors of β-arrestin2, in which cfp was fused to the c-terminus and the flashbinding motif (ccpgcc) was inserted to different positions within the n-or c-domain of β-arrestin2. upon β2-adrenergic receptor (β2ar) stimulation we observed a decrease of the intramolecular fret signal between cfp and flash at the n-domain (β-arrestin2flash2), indicating a conformational change moving the c-terminus and the ndomain of β-arrestin2 relative to each other. kinetic analysis revealed that this conformational change immediately follows β-arrestin2/β2ar interaction on a timescale of seconds. a β2ar mutant that was previously shown not to interact with β-arrestin2 was utilized as control and did not induce a conformational change in the β-arrestin2 molecule. our data provide evidence that β-arrestin2 changes it`s conformation upon binding to the activated β2ar in living cells. the β-arrestin2flash2 sensor could serve as universal biosensor for gpcr activation. studies on the physiological role of annexin a4 in the heart nunes f. 1 the calcium binding protein annexin a4 has been examined in the context of heart failure in the past. annexin a4 expression level was found to be elevated in ventricles of human failing heart in comparison to expression levels in non-failing ventricles. furthermore the intracellular localization pattern in atrial cardiomyocytes was found to be altered in the failing human heart (moravec and matteo, cardiovasc res 2000). in order to gain insight into the possible physiological significance of these findings we utilized an annexin a4 gene trap model (gt) in which the annexin a4 protein content was not detectable in ventricles and atria. measurements of sarcomere shortening and calcium transient kinetics in isolated ventricular cardiomyocytes revealed a prolonged calcium transient decay at stimulation frequencies of 0.5 hz, 1hz and 2 hz as well as an increased sarcomere shortening at 1 hz and 2 hz in anxa4 gene trap animals in comparison to wild type (wt) ( the effects of the β-adrenoreceptor agonist isoprenaline (iso) on the shortening of ventricular cardiomyocytes was increased in gt as compared to wt (0,2±0.013 vs. 0.17±0.012, *=p<0.05 vs. wt; n=14-18/5). western blot analyses indicated that the expression of the sarcoplasmic reticulum (sr) ca 2+ -atpase (serca2a) and the phosphorylation status of its regulator protein phospholamban (plb) did not differ between groups (n=7). however, co-immunoprecipitation experiments suggest, that anxa4 is able to interact with hax1, which acts as a repressor of serca2a (n=4). we performed force measurements in isolated and electrically stimulated left atria in response to rising isoprenaline concentrations (10 -9 m-10 -5 m). the positiv inotropic effect of isoprenaline was significantly increased in gt atria (rel. force at 10 -4 m iso [%]: wt: 433±76; gt: 699±63 *= p<0.05 vs wt; n=8-10). in conclusion, annexin a4 contributes to the regulation of cardiomyocyte contractility. the anxa4 up-regulation might therefore contribute to diminished cardiac performance in heart failure. matteo rg, moravec cs. immunolocalization of annexins iv, v and vi in thefailing and non-failing human heart. cardiovasc res. 2000 mar;45 (4) background: pregnane x receptor (pxr) is considered the most important sensor of natural and anthropogenic xenobiotics in vertebrates. in contrast, the amphibian ortholog is involved in neural development and irresponsive to xenobiotics. instead, the xenopus laevis constitutive androstane receptor (car) was recently found to possess pxr-like properties, featuring low basal activity and a pronounced ligand spectrum. thus a structural and functional characterisation of x. laevis car may provide further insights into human car basal and ligand-induced activity. methods: the time-point of origin of car genes was determined by macrosynteny analyses of car, pxr, and vdr (vitamin d receptor) gene loci, which form the nr1i subfamily of nuclear receptors. based on a 3-dimensional protein model of xenopus laevis car, docking studies with structurally diverse agonists were conducted. proteinligand-interactions as well as sequence comparisons were performed in order to select amino acids to be mutated towards human car. the organ response to car activators was determined in xenopus laevis using rna microarrays. results: car emerged together with pxr and vdr from an ancestral nr1i gene in early vertebrates via two whole-genome duplications. this was followed by losses of car from the fish lineage and of pxr from sauropsida (reptiles and birds). amino acids important for ligand binding were identified. structural features responsible for the pronounced basal activity in human constitutive androstane receptor are not present in x. laevis car. in human pxr the inter-helical loop in front of helix 3 is part of the ligandbinding pocket and supposed to be responsible for the wide substrate spectrum. in amphibian car this inter-helical loop plays no role in ligand binding. car agonists resulted in a pronounced induction of antimicrobial peptides in the ovary. conclusions: car emerged already in early vertebrates and it is conserved in land vertebrates, whereas xenosensing pxr is found only in the fishes and mammals. we provide a comprehensive modeling and mutational analysis of this first reported amphibian xenosensor. the induction of antimicrobial peptides by car activators suggests a link between xenosensing and innate immunity. the latter one may play a previously unrecognized role in the amphibian reproduction. background: retigabine belongs to a novel class of potent anticonvulsant drugs and is currently being investigated in clinical routine. the therapeutic range of retigabine serum concentration is unknown. a therapeutic drug monitoring (tdm) is used for most other anticonvulsant drugs. the aim of this study was to develop a method for the determination of retigabine in serum of patients and to compare the effect and the side effects of retigabine with the blood levels of the drug. method: a hplc method with tandem mass spectrometric detection for the sensitive determination of retigabine was developed. solid-phase extraction (spe) of 250 µl serum with oasis hlb cartridges allowed a reliable quantification down to 5 ng/ml. in order to develop an assay with high sample throughput and to obtain maximum response for the analytes we required the shortest possible retention time. to implement the determination of retigabine in a second step in the routine tdm of anticonvulsant drugs the corresponding hplc method was selected: a purospher rp18 column (55 mm x 2 mm; 4 µm, merck) and a mobile phase with a steep acetonitrile gradient. results: the great advantage of having analytes with different molecular masses and similar retention times in combination with ms/ms detection enabled us to aim at a minimum separation that might remove some salts or matrix components that can suppress or interfere with the analyses from the target components, while maintaining good sample throughput. the method was validated. the assay is precise, accurate, fast, sensitive, and selective. discussion: the developed method is suitable for therapeutic drug monitoring of retigabine. the correlation of the serum concentration and the effect of the drug and thus the necessity of tdm have to be tested. targeting inflammatory t lymphocytes with conditional chemokine receptor antagonist expression for a tissue-specific therapy of chronic inflammatory disorders ogrissek n., giegold o., pfeilschifter j., radeke h. h. uniklinikum der goethe-universität pharmazentrum / zafes, theodor-stern-kai 7, 60590 frankfurt am main, germany chemokines and their receptors are known to be involved in the pathogenesis of chronic inflammation and autoimmune diseases. several approaches tried to use chemokine receptor antagonists as therapeutics to reduce exagerrated immune response, however, due to compensation and systemic side effects clinical trials often failed. in previous experiments our group identified three promising antagonists. cxcl11(4-79) has antagonistic function for cxcr3, cxcl12(p2g2) is able to inhibit cxcr4 and the herpesvirus 8 encoded protein vmip-ii interferes with ccr1, -2 and -5 as well as with cxcr3, -4 and cx3cr1. their expression and secretion was confirmed in pichia pastoris and antagonistic function has been proven by a reduction of t cell migration. the aim of this project is to develop a cell-based therapy for chronic inflammation with a treatment that is based on the collective effect of cxcl11(4-79), cxcl12(p2g2) and vmip-ii. with targeting of stable transduced memory t cells these antagonists should be conditional expressed and secreted directly in the centre of inflammation, resulting in inhibition of further inflammatory t cell accumulation. to realize this project we first cloned constitutive lentiviral constructs containing these antagonists and optimized transduction of t cells, such as the ova-specific memory th-1 cell clone if12 with the potential to initiate antigen specific nephritis in scid mice. next we investigated expression and secretion of cxcl11(4-79), cxcl12(p2g2) and vmip-ii with pcr, western blot and elisa. at the moment we want to measure the inhibition efficiency of t cell migration in vitro with chemotaxis and flow chamber assays. construction of an inducible lentiviral vector plasmid to ensure expression of the antagonists only upon t cell activation, is also part of our current work. finally we would like to test the chemokine receptor antagonists in vivo in two relevant mouse models of type-1-diabetes and contact dermatitis. small heterodimer partner 1 (shp-1) is a member of the superfamily of nuclear receptors (nrs). in contrast to other nrs this orphan receptor lacks the dna binding domain. however, shp-1 is known to inhibit activity of several nrs by direct proteinprotein interaction. importantly several of the interacting nrs have been shown to directly regulate shp-1 expression, suggesting that shp-1 is involved in negative feedback loops of various metabolic pathways, such as cholesterol-, bile acid-and drug metabolism and glucose homeostasis. recently binding sites for nrs were identified in the promoter region of shp-1, including hnf4α, lrh1, lxr, fxr, srebp1c and pparγ. the aim of our study was to identify single nucleotide polymorphisms (snps) in the promoter region of shp-1 and to determine their impact on the transactivation of shp-1. 120 dna samples from subjects of the population based cohort study of health in pomerania were analyzed by sanger sequencing, thereby we identified four snps namely -594t>c (rs71636795), -413g>c, -423 c>t (rs78182695) and del-195ctga (rs145613139). subsequently those polymorphisms were tested for their functional consequence performing cell based reporter gene assays testing all above mentioned modulators (lrh1, lxr, fxr, srebp1c and pparγ) of shp-1 expression. only the transactivation by hnf4α was decreased in the presence of the -423 c>t polymorphism to 69% and the -413g>c polymorphism to 75%. in conclusion we described snps with impact on transactivation. it will be aim of future studies to determine the potential impact on physiological processes or disease development. autosomal recessive polycystic kidney disease (arpkd) is a rare genetic disease, afflicting about 1 in 20.000 individuals. arpkd is characterized by cystic fusiform dilatations of the renal collecting ducts leading to massive enlargement of the kidneys and ultimately loss of renal function. in addition, the patients suffer from congenital hepatic fibrosis (chf), possibly leading to portal hypertension and liver enlargement. so far, there is no cure for arpkd. therapy is focussing on controlling the disease symptoms [1] . mutations in the pkhd1 gene cause arpkd. more than 300 different mutations in this gene have been reported, all leading to the same phenotype, though there are differences regarding the severity of the disease [2] . in animal models of autosomal dominant polycystic kidney disease (adpkd) as well as arpkd elevated levels of camp were shown [1] [2] [3] . in isolated kidney cells camp stimulates cl-secretion and activates the b-raf /mek/erk pathway. these both are important factors for cyst development and disease progression [2, 3] . intracellular camp regulation is based on conversion of atp to camp by adenylyl cyclases (acs) and degradation by phosphodiesterases . referring to this, we asked the question if there are differences in the activation and expression pattern of acs in pck rats, an animal model of arpkd [4] and in sprague dawley rats. therefore, we examined membranes in a radioactive ac activity assay using various stimulatory compounds, e.g. forskolin, a direct ac activator, or hormones like glucagon and vasopressin to characterize acs. furthermore, we examined ac isoform expression on the mrna level via rt-pcr. we observed that in pck rats ac activity was decreased in general in comparison to sprague dawley rats. in future experiments we are aiming to obtain further knowledge about the influence various hormones exhibit on pck rat acs and to biochemically characterize acs. the major pathogenicity factors tcda and tcdb from clostridium difficile monoglucosylate and thereby inactivate small gtp-binding proteins of the rho subfamily after entering host cells via receptor-mediated endocytosis. although the intracellular mode of action of the toxins is well understood, far less is known about binding structure and internalization pathway of tcda and tcdb. since antibodies directed against the c-terminal located clostridial repetitive oligopeptides (crops) are able to neutralize toxin cytotoxicity the crop domain is acknowledged to mediate receptor binding. however, we recently demonstrated that crop deletion mutants of tcda (tcda 1-1874 ) and tcdb (tcdb 1-1852 ) enter host cells and exhibit full cytopathic potency though lacking the proposed receptor binding domain. we therefore refute the accepted opinion of a solely crop-mediated toxin uptake and re-evaluate the role of the crops in toxin endocytosis. tcda 1-1874 and tcdb 1-1852 induced time and concentration dependent cell rounding and rac1-glucosylation. however, depending on the cell line, truncated toxins exhibit up to 10-fold reduced potency towards host cells compared to the respective full length toxin. the observed difference in toxin potency might reflect the recognition of different receptor structures or the use of various endocytotic routes. interestingly, pre-incubation of cells with the isolated crop domain enhances binding as well as cytotoxicity of subsequent applied truncated tcda indicating that the crops primarily determine toxin uptake. in fact, competition experiments revealed that tcda and tcda 1-1874 predominantly use different receptor structures corroborating the notion of alternative internalization processes utilized by tcda. different routes for cellular uptake might enable the toxins to enter a broader repertoire of cell types leading to the observed multifarious pathogenesis of c. difficile. thus, characterization of alternative endocytotic pathways used by the c. difficile toxins might therefore be the basis to investigate the opportunity of toxin uptake inhibition as therapeutic option. in neurodegenerative diseases, such as alzheimer´s disease and parkinson´s disease, mitochondrial pathways of apoptosis are considered as major features of the underlying neuronal cell death. such mitochondrial mechanisms of apoptosis are mediated by the bh3-only protein bid, a member of the bcl-2 family that triggers mitochondrial permeabilization and the subsequent release of death-promoting proteins into the cytosol. the pivotal role of bid in apoptotic cascades of neuronal cells has been shown in our previous studies showing a neuroprotective effect of bid sirna and small molecule bid inhibitors such as bi6c9 in vitro. in vivo, however, the available bidinhibitors failed to protect brain tissue likely because the compounds were not bioavailable or did not cross the blood brain barrier. therefore, chemical modifications of bi-6c9 were generated resulting in new structures and molecules with different pharmacophors. the aim of the present study is to identify novel potent bid inhibitors available for applications in model systems of brain damage in vivo. for the first screening of 80 compounds we used a model of glutamate toxicity in immortalized mouse hippocampal neurons (ht-22 cells). in this model system, glutamate induces a decrease of intracellular glutathione levels resulting in lipoxygenase activity and enhanced formation of toxic reactive oxygen species (ros). to investigate the compounds' ability to prevent glutamate induced cell death, we first analyzed the cell viability by the mtt assay. in addition, we examined the cell survival by using real time monitoring of cell impedance (xcelligence system) to determine the neuroprotective potency of the new structures. using these assays, we identified 10 novel molecules that significantly prevented glutamate-induced toxicity in ht-22 cells. further we were able to express and to purify recombinant bid in a high amount. in the ongoing study the purified bid protein will be used for co-crystallization with the identified neuroprotective structures for further optimization of novel bid inhibitors for therapeutic applications in experimental models of neurodegenerative diseases in vivo. polymorphic enzymes, urinary bladder cancer risk and structural change in the local industry ovsiannikov d. 1 , selinski s. in the 1990s, an uncommonly high percentage of glutathione s-transferase m1 (gstm1) negative bladder cancer cases (70 %) was reported in the greater dortmund area (golka et al., 1997) . the question arose whether this uncommonly high percentage of gstm1 negative bladder cancer cases was due to environmental and occupational exposure decades ago. thus, 15 years later, another study on bladder cancer was performed in the same area after the coal, iron and steel industries had finally closed in the 1990s. in total 196 bladder cancer patients from the st.-josefs-hospital dortmund-hörde and 235 controls with benign urological diseases were investigated by a questionnaire and genotyped for gstm1, gstt1 and the n-acetyltransferase 2 (nat2) tag snp rs1495741. the frequency of the gstm1 negative genotype was 52 % in bladder cancer cases and thus much lower, compared to a previous study performed from 1992-95 in the same area (70%). nat2 genotypes were distributed equally among cases and controls (63% slow acetylators). less gstt1 negative genotypes were present in cases (17%; controls 20%). apoptosis inducing factor (aif) has been identified as a key factor in intrinsic pathways of caspase-independent neuronal death in model systems of acute brain injury and neurodegenerative diseases, such as alzheimer's disease and parkinson's disease. aif is a mitochondrial intermembrane flavoprotein with the capacity to translocate to the nucleus where it induces chromatin condensation and large-scale dna fragmentation. previous studies revealed that aif deficiency leads to protective effects in different models of neuronal death in vitro and in vivo. however, aif also plays an important physiological role for the integrity and function of the mitochondrial respiratory chain. thus, aif deficiency may significantly alter mitochondrial functions and metabolic homeostasis thereby preconditioning the cells to tolerate subsequent stress stimuli. the present study addresses this hypothesis and investigates whether neuroprotection by aif depletion was attributed to a preconditioning effect, i.e. protecting mitochondrial function and integrity. as model system we use glutamate induced oxytosis in immortalized mouse hippocampal ht-22 neurons. silencing of aif expression by sirna (20nm) protected mitochondrial morphology and integrity against glutamate induced damage. microscopy analysis of the mitochondrial morphology revealed that aif sirna prevented mitochondrial fission. furthermore, facs analysis confirmed that mitochondrial membrane potential was stable in cells with aif silencing. this protection of mitochondrial morphology and integrity by aif depletion was associated with preserved atp levels and inhibition of cell death as detected by an mtt assay. pronounced formation of lipidperoxides as another indicator of mitochondrial damage was also attenuated in cells preconditioned by aif sirna. these protective effects of aif sirna were highly similar to effects obtained with low doses of rotenone (20nm), which was applied as an inhibitor of complex i and mediated comparable preconditioning effects in the ht-22 cells. overall, these findings support the conclusion that aif depletion mediates a preconditioning effect protecting neuronal cells from a subsequent glutamate toxicity. in order to link these preconditioning effects to complex i functions, protein expression and functional analysis of complex i are being analysed to identify the molecular mechanisms of aif dependent control of neuronal life and death. -dependent inactivation and display very slow voltage-dependent inactivation. both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained ca 2+ influx through cav1.4 channels is required to couple slowly graded changes of the membrane potential with tonic glutamate release. mutations in the gene coding for cav1.4 cause severe impairment of retinal circuitry function and have been linked to congenital stationary night blindness type 2a (csnb2), aland island eye disease (aied) and cone-rod dystrophy type 3 (cordx3). the clinical phenotypes of these eye diseases vary substantially regarding the ratio of rod to cone functional impairment. the reasons for this variability are not known. to gain more insights into the pathophysiology caused by loss of cav1.4 function we analyzed the visual phenotype of cav1.4-deficient mice. to this end, we combined immunohistochemistry, electroretinography (erg) and vision-dependent behavioral testing. immunohistochemical analysis using synaptic and postsynaptic markers revealed severe synaptic defects in cav1.4-deficient mice. heterozygous cav1.4 mice showed mosaic synaptic defects most probably caused by random x-chromosomal inactivation of the healthy allele. electroretinography revealed a loss of scotopic and photopic photoreceptor function. this loss of retinal network function resulted in impaired performance of cav1.4 knockout mice in a water maze-based behavioral test of rod and cone function. in conclusion, loss of cav1.4 channels strongly impairs rod and cone retinal function and vision in mice. lsr is the host cell receptor for clostridial iota-like toxins papatheodorou p., aktories k. albert-ludwigs-universität freiburg institut für experimentelle und klinische pharmakologie und toxikologie, albertstr. 25, 79104 freiburg, germany the human enteric pathogen clostridium difficile is the most serious cause of antibioticassociated diarrhea and pseudomembranous colitis. hypervirulent strains of the pathogen, associated with more severe disease and increased death rates, produce the binary actin-adp-ribosylating toxin cdt (c. difficile transferase) in addition to the rho glucosylating toxins a and b. cdt is member of the family of clostridial iota-like toxins, including c. spiroforme toxin (cst) and the eponym c. perfringens iota toxin. the toxins induce depolymerization of the actin cytoskeleton and the formation of microtubulebased cell protrusions that increase adherence and colonization of clostridia. using a haploid genetic screen, we identify the lipolysis-stimulated lipoprotein receptor (lsr) as the target molecule for entry of cdt into host cells. in addition, we present evidence that lsr is shared as a cell entry point by all members of the iota-like toxin family. identification of the toxin receptor provides a most valuable basis for antitoxin strategies. bisphenol a (bpa) is a chemical of high interest due to its endocrine activity. controversy exists concerning the blood concentration due to normal exposures. some authors claimed to have measured concentrations in the ng/ml range which is in contrast to kinetic properties of bpa. bpa is excreted in the urine as glucuronide and sulfate metabolites. recently, data on the in vitro metabolism of bpa by recombinant udpglucuronyltransferase 2b15 enzymes (ugt2b15) revealed that ugt2b15.2 and ugt2b15.5 had markedly lower intrinsic clearance as compared to ugt2b15.1 (hanioka et al., 2011) . using the in vitro metabolism data, we scaled the kmand vmaxvalues in an established human physiologically based toxicokinetic (pbtk) model (mielke and gundert-remy, 2009, mielke et al., 2011) to the values of the variants. for oral doses at relevant exposure levels, the maximum blood concentration (cmax) for the ugt2b15.2 variant (v2) was 5 fold and those of the ugt2b15.5 variant (v5) was 7 fold higher than that of the ugt15.1 variant. with dermal exposure at a relevant exposure level, the cmax values were 1.4 (v2) and 1.6 fold (v5) of ugt15.1 variant. a combined exposure of oral and dermal exposure, an exposure scenario, which occurs in daily life, resulted in 2.4 fold (v2) and 3.2 fold (v5) higher cmax values as compared to ugt15.1 variant. the values for the area under the blood concentration time curve (auc) were for a relevant oral dose 5.7 fold (v2) and 8.6 fold (v5), for relevant dermal exposure 1.4 fold (v2) and 1.6 fold (v5), and for combined exposure 1.9 fold (v2) and 2.5 fold (v5) of ugt15.1 variant. from the results we conclude: (1) polymorphism of udpglucuronyltransferase (2b15.2 and 2b15.5) has an impact on the blood concentrations which, however, is less than 10 fold for cmax and for auc. the effect is more pronounced for oral as compared to dermal or combined exposure. (2) polymorphism of metabolism does not explain the blood/plasma concentrations in the ng/ml range measured by some authors. hanioka n, oka h, nagaoka k, ikushiro s, narimatsu s. effect of udpglucuronosyltransferase 2b15 polymorphism on bisphenol a glucuronidation. arch toxicol. 2011, 85(11) :1373-81. mielke h, gundert-remy u. bisphenol a levels in blood depend on age and exposure. toxicol lett. 2009 8; 190(1) :32-40. mielke h, partosch f, gundertthe heart responds to maladaptive pro-hypertrophic stimuli by stimulating intrinsic signals that contrast and dampen the onset and development of hypertrophy. cyclic guanosine monophosphate (cgmp) and its downstream effector cgmp kinase i (cgki) have been suggested to be an important anti-hypertrophic signaling pathway (1) . intracellular levels of cgmp can be raised by the action of nitric oxide (no) and natriuretic peptides (anf, bnf), or by inhibiting cgmp-degrading phosphodiesterases (pde). a growing body of evidence suggests that the pde5 specific inhibitor sildenafil (sil) prevents and reverses hypertrophy and chamber remodelling in the heart of mice subjected to thoracic aorta constriction (tac) by elevating cgmp levels and cgki activation (1) . in contrast, using a mouse model that lacks cgki expression in every cell type except smooth muscle cells (βres mice; see ref. 2), we recently showed that the absence of this kinase does not alter the onset of hypertrophy induced by tac or isoproterenol infusion (2) . sil is believed to increase cardiac cgmp levels, although it is unclear, if its target (pde5) is expressed in cm (2). sil may act on other pdes, such as pde1c which is abundant in cms. it is also unclear if sil effects are mediated by other cardiac cell types, in particular by cardiofibroblast. to answer these questions, we are currently investigating whether sil is able to prevent hormone induced cardiac hypertrophy in the absence of cgki in cm. preliminary results on βres mice show that even in the case of chronic angii infusion, lack of cgki in cm does not alter the induction of hypertrophic response, at least in the initial phase (7days of angii infusion at 2mg/kg/day). interestingly, βres mice showed impaired cardiac function, as indicated by decreased fractional shortening. sil was able to partially block the onset of cardiac hypertrophy in wt animals, but not in βres mice, indicating a requirement of cgki in this process. in particular, sil was able to block the transcription of pro-fibrotic genes such as tgfβ, ctgf, collageni and fibronectin. the overexpression of the somatostatin receptors sst2 and sst5 in neuroendocrine tumors provides the molecular basis for therapeutic application of the stable somatostatin analogs octreotide and pasireotide. whereas the phosphorylation of the carboxyl-terminal region of the sst2 receptor has been studied in detail, little is known about the agonist-induced regulation of the human sst5 receptor. here, we have generated phosphosite-specific antibodies for the carboxyl-terminal threonines 333 (t333) and 347 (t347), which enabled us to selectively detect either the t333-or the t347-phosphorylated form of sst5. we show that agonist-mediated phosphorylation occurs at t333, whereas t347 is constitutively phosphorylated in the absence of agonist. we further demonstrate that the pan-somatostatin analog pasireotide and the sst5-selective ligand l-817,818 but not octreotide or ke108 were able to promote a clearly detectable t333 phosphorylation. however, none of these compounds was able to stimulate t333 phosphorylation and sst5 internalization to the same extent as the natural somatostatin. agonist-induced t333 phosphorylation was dose-dependent and selectively mediated by g protein-coupled receptor kinase 2 (grk2). like that observed for the sst2 receptor, phosphorylation of sst5 occurred within seconds. however, unlike that seen for the sst2 receptor, dephosphorylation and recycling of sst5 were complete within minutes. we also identify protein phosphatase 1g (pp1g) as sst5 receptor phosphatase. together, we provide direct evidence for agonist-selective phosphorylation of carboxyl-terminal t333. in addition, we identify grk2-mediated phosphorylation and pp1g-mediated dephosphorylation of t333 as key regulators of rapid internalization and recycling of the human sst5 receptor. termination of signaling of activated g protein-coupled receptors (gpcrs) is essential for maintenance of cellular homeostasis. although the regulation of agonist-induced phosphorylation has been studied in detail for many gpcrs, the molecular mechanisms and functional consequences of receptor dephosphorylation are far from understood. recent studies have shown that phosphatase inhibitors, such as okadaic acid and calyculin a, can block the dephosphorylation of a number of gpcrs including the ß2 adrenergic receptor, d1 dopamine receptor, parathyroid hormone receptor 1, thromboxane a receptor and the vasopressin receptor 1. however, a specific phosphatase has not been identified so far. in present studies, we have examined the mechanism and function of receptor dephosphorylation using the sst2a somatostatin receptor and the µ-opioid receptor (mor) as models. within those analyses, we have identified protein phosphatase 1beta (pp1ß) as the phosphatase for the cluster of phosphorylated threonines (353ttetqrt359) within the sst2a somatostatin receptor carboxylterminus using sirna knock down screeening. those phosphorylation sites mediate ß-arrestin binding. we have also identified protein phosphatase 1gamma (pp1γ) as mor phosphatase that catalyzed t370 and s375 dephosphorylation at or near the plasma membrane within minutes after agonist removal. here, we show the different activated phosphatases with functional selective mutants. we examined tailswap mutants which specify the different phosphatase activities. therefore we produced a mor-rsst2a chimera with the rsst2a c-terminal tail and a rsst2a-mor chimera with a mor c-terminal tail. detoxification by conjugation of glutathione? formations of dna adducts of patulin activated by glutathione pfenning c., lehmann l. university wuerzburg, institute of pharmacy and food chemistry section of food chemistry, am hubland, 97074 wuerzburg, germany as a frequent contaminant in apple juice, the mycotoxin patulin (pat) has shown mutagenic potential in cultured mammalian cells at concentrations which are equivalent to those found in marketable foods. this fact is in contrast with the assumption that conjugation to the major intracellular nucleophile glutathione (gsh) leads to detoxification of the electrophile pat. although pat reacts readily with gsh, previous studies showed that co-incubation of pat with model thiols and amine compounds increased the reactivity of pat towards amines forming mixed-type adducts. thus, we hypothesise that the potential to react with dna bases after being activated by gsh might contribute to the mutagenicity of pat. adduct formation of dna bases (adenine, guanine or cytosine) with pat in the presence and absence of gsh was studied under neutral conditions. liquid chromatography coupled with electrospray ionization tandem mass spectrometry was applied for identification and structure elucidation of putative adducts. besides published as well as hitherto unknown pat-gsh adducts, several pat-dna base adducts were formed both in presence and absence of gsh. in addition, with each of the three dna bases one product exhibiting a mass to charge ratio and fragmentation pattern suggesting a mixed thiol-amine adduct was detected. based on the fragment ions of adducts formed with gsh and chemically modified derivatives, we postulate a cyclic structure of the pat-gsh-dna base adducts, resulting from the reaction of the α-amino group of the glutamic acid residue with the c7-carbonyl function of pat. the exocyclic amino group of the dna base is linked to c1 of the pat backbone by an amid bond. thus, the present study demonstrates the reactivity of pat towards dna bases and the participation of gsh in adduct formation. the postulated structures of dna adducts could be used as biomarkers for the determination of the internal exposure to pat in humans. prescribing information detailed in the summary of product characteristics (spc) forms the officially approved basis for safe prescribing of drugs. in a project funded by the german federal ministry of education and research (bmbf) we aimed to derive an internationally valid data set for safe prescribing of psychiatric drugs and therefore analyzed and compared the content of internationally available prescribing information. a team of pharmacists and clinical pharmacologists performed an in-depth comparison of the german, swiss, british and us-american spcs of 10 top prescribed psychiatric drugs. for 7 drugs (of identical pharmaceutical form) the spcs from the same manufacturer were available for all countries, whereas for three drugs spcs were only available from different companies. in these cases the most recent prescribing information from each country was included in the comparison. in 40 spcs 2220 individual data points (55.5±17.4 per individual spc) were compared. between countries the timeliness of prescribing information for an individual drug varied by a median of 18.5 (range: 6-134) months. the respective spcs covered on average 71.4±30.3% (range: 12.5-100%) of all mentioned indications and 70.1±24.4% (range 15.4-100%) of all mentioned contraindications. the warnings and precautions section of an individual spc covered on average 59.5±17.1% (range: 12.5-93.3%) of all mentioned warnings and precautions for that drug. the variation observed was only marginally improved when restricting the analysis to the 28 spcs of the 7 drugs available in all four countries from the same manufacturer. across countries, the summary of product characteristics of individual psychiatric drugs show substantial variation in crucial prescribing information. as different manufacturers are unlikely to explain much of the observed variation, these data argue for a better international cooperation and standardization of the content of summary of product characteristics. this project is supported by the german federal ministry of education and research (bmbf), project grant no. 01 ex1015b. protein kinase ck2 (former name 'casein kinase 2') is a highly conserved serine/threonine kinase, which acts as a component of regulatory networks implicated in many cellular processes but is also linked to various types of human cancer [1, 2] . elevated ck2 activity has been associated with aggressive tumor behavior and results in growth advantage, enhanced survival and dynamic adaption to stress of cancer cells [3] . ck2 is a heterotetramer consisting of two catalytic subunits (ck2α) attached to a dimer of regulatory subunits (ck2β) [4] . ck2β stabilizes ck2α against denaturation and modulates the substrate specificity of the catalytic subunit [5] . due to the relatively small and hydrophobic ck2α-ck2β interface (832 å²) [4] , low molecular weight inhibitors are able to interfere with the ck2 subunit interaction and thus affect the kinase activity [6] . such inhibitors might exhibit an increased specificity in comparison to those compounds interacting with the conserved atp binding site [3] . we have developed an elisa-based ck2α-ck2β binding assay using recombinant human ck2 subunits. different blocking reagents were analyzed to minimize nonspecific binding. the optimized binding assay was then applied to screen for inhibitors of the ck2 subunit interaction. primary hits were further characterized by determination of the parameters ic50 and ki as well as by comparing the results from the binding assay with literature-known or recently obtained crystal structures. numerous epidemiological studies have shown associations between exposure to ultra fine particles and an increase in cardiovascular diseases such as atherosclerosis, coronary heart disease and myocardial infarction. ultra-fine particles have an aerodynamic diameter of < 0.1 µm and are highly diverse with impurities of transition metals and organic compounds (e.g. polycyclic aromatic hydrocarbons). posttranslational modification (ptm) of proteins, particularly phosphorylation, is a key element in the regulation of cell function and any disturbance can lead to multiple diseases. the present study focused on the proteomic-based identification of phosphorylated proteins to understand the mechanism behind ultrafine particle exposure and cardiovascular disease development. as one of the major sources for ufp emissions are diesel exhaust, therefore to mimic the diesel particles, carbon black (cb) and benzo[a]pyrene loaded carbon black (cb+) were used in the present study. cells of the endothelial cell line ea.hy926 were exposed for 14 days to 100 ng/ml cb and cb+. phosphoprotein extraction of whole cell lysates was carried out by the method developed in our lab 1 . the obtained proteins were then separated by two-dimensional gel-electrophoresis followed by maldi-tof-ms (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) analysis of differently expressed proteins. to further validate the results invasive potential of cells were monitored by plating exposed cells for 24 hrs on top of matrigel-coated inserts. differential expressions of 20 phosphoproteins were found in cb-treated cells while an altered expression of 33 phosphoproteins was observed in cb+-treated cells. the maldi-tof analysis revealed proteins involved in the regulation of the endothelial permeability and the cellular plasticity such as vimentin, actin and transitional endoplasmic reticulum atpase. further, the invasion assay supported these results as the cb-exposed cells showed a high invasive potential as compared to control. [1] pink m. et. al. , precipitation by lanthanum ions: a straightforward approach to isolate phosphoproteins, j.protomics, 75, 2011, [375] [376] [377] [378] [379] [380] [381] [382] [383] 302 application of the ttc approach in cosmetics platzek t. bundesinstitut für risikobewertung, max-dohrn-straße 8-10, 10589 berlin, germany regulatory toxicologists in europe have been discussing the ttc approach since more than a decade, e.g. the previous scientific committee on food in 1996. since then, the concept was further developed and is now applied in the eu for the assessment of flavouring substances by efsa. two committees are discussing possible applications: the efsa scientific committee prepared an opinion exploring options for the application in food and feed, e.g. for impurities of food additives, thermal reaction products, food contact materials, contaminants etc. in addition, an eu non-food expert committee consisting of members of sccs, scher and schenir discussed the ttc concept in general as well as additional possible fields of application with the focus on cosmetics and an opinion was already published for public consultation. the proposal to apply the ttc approach also for cosmetic ingredients was introduced by a paper published in 2007 by a colipa (the european cosmetics association) supported working group. major aspects to be considered are the following: 1. applicability domain. the chemical space of the ttc dataset (> 600 compounds) has to be compared with that of cosmetic ingredients (> 10 000 compounds). route to route extrapolation. since no adequate dermal toxicity database is available both data on oral intake used in the ttc approach and on dermal exposure to cosmetic ingredients have to be transfomed to internal exposure figures. gastrointestinal and dermal bioavailability as well as route specific differences in metabolism have to be integrated. exposure. the reliability of exposure estimation is the second pillar of the ttc approach. compared to food, data on exposure to substances in cosmetics and consumer products is scarce. a pragmatic step forward is the comparison of ttcs and noael-derived safe exposure levels for cosmetic ingredients. this work was already done with substances in food contact materials and chemicals from the elincs list. for cosmetic ingredients a similar european project is ongoing. further refinement of the ttc approach is needed taking into account the up-to-date toxicological knowledge. with cosmetics specific problems may arise in praxi: according to the new eu cosmetic legislation the safety of cosmetic products available on the market has to be assessed by the manufacturer or importer and also assessors with limited toxicological experience may apply the ttc approach, e.g. by running the toxtree software. plöttner s., marczynski b., käfferlein h. u., welge p., groth h., engelhardt b., schmitz k., erkes a., brüning t. institut für prävention und arbeitsmedizin der deutschen gesetzlichen unfallversicherung -institut der ruhr-universität bochum (ipa) toxikologie, bürkle-dela-camp-platz 1, 44789 bochum, germany polycyclic aromatic hydrocarbons (pahs) comprise several hundred compounds with different carcinogenic potentials, typically occurring in complex mixtures. due to a lack of data risk estimations for pah mixtures are usually based on those for benzo[a]pyrene (b[a]p). the aim of our present study was to explore the suitability of a permanent human lung cell line as tool for future studies on genotoxicity of pah mixtures. in this pilot study we investigated the time-and concentration-dependent generation of specific anti-benzo[a]pyrene -7,8-diol-9 ,10-epoxide (anti-bpde)-dna adducts as well as cytochrome p450 (cyp) 1a1/1b1 enzyme activities after b[a]p-incubation in vitro. we used metabolically competent a549 lung carcinoma cells which display several characteristics of alveolar epithelial type ii cells. after 24 h and 48 h incubations with different b[a]p-concentrations cytotoxic effects were assessed with the neutral red assay and cyp1a1/1b1 activities using luminescent tests. the formation of specific anti-bpde-dna adducts was determined by hplc with fluorescence detection. a time-and concentration-dependent formation of anti-bpde-dna adducts was observed with maximum rates of 340.5 ± 39.1 and 599.6 ± 132.4 anti-bpde/10 8 nucleotides after incubation with 1 µm (24 h) and 3 µm b[a]p (48 h), respectively. however, the mean adduct rates decreased at higher b[a]p-concentrations. the reduction was more pronounced after 24 h than after 48 h. increased cyp1a1/1b1 activities were observed at > 0.1 -1 µm (24 h) and > 0.1 -3 µm (48 h). a clear decrease of enzyme activities was observed at higher concentrations for both incubation times. in the neutral red assay no more than 10% cytotoxicity in relation to the negative control were found after 24 h incubation with ≥ 10 µm b[a]p and after 48 h with ≥ 1 µm b[a]p. overall, incubation of a549 cells with b[a]p resulted in a time-and concentration-dependent increase of cyp-activities and anti-bpde-dna adducts. this clearly shows that a549 cells are able to generate mutagenic dna-adducts. thus, the in vitro model used in the present work appears suitable for genotoxicity studies of individual pahs and pah mixtures, and therefore may be a useful tool for research on syncarcinogenesis. the β subunit of the cav1.2 channel complex has been shown to play a key role in cav1.2 channel trafficking and channel characteristics like opening probability. furthermore, the last exon of the cacnb2 gene coding for cavβ2, exon 14, contains several potential phosphorylation sites, e.g. for protein kinase a or ca 2+ /calmodulin dependent camkii. pka-dependent phosphorylation mediates β-adrenergic stimulation of cav1.2. potential phosphorylation sites are ser478 and ser479 (in the β2 subunit in rat, perez-reyes et al. 1992 a ) and ser1928 in the c-terminus of the poreforming α1c subunit (lemke et al. 2008 b ) . in cardiomyocytes camkii regulates ca 2+ release and reuptake from and into the sr and is involved in the facilitation of the calcium channel. potential interaction sites between the cav1.2 channel complex and camkii are thr498 in the β2 subunit (in rat, grueter et al. 2006 c ) and ser1512 and ser1570 in the cterminus of the α1c subunit (blaich et al. 2010 d ) . however, the exact pathways remained widely unclear up to now. to clarify these mechanisms and to identify the relevant phosphorylation sites for pka and camkii we established a mouse line carrying a stop codon in exon 14 after aa pro501. this mutation prevented translation of the cavβ2 c-terminus containing the corresponding potential phosphorylation sites mentioned above (cavβ2stop mouse). these mice were viable, showed unaltered expression of the truncated of cavβ protein and unchanged ecg and echocardiography. electophysiological analysis of isolated cardiomyocytes showed no differences in current density, the effect of isoproterenol, the time course of inactivation and the facilitation property when compared to cells isolated from littermate controls. for further investigations we bred the cavβ2stop mice with s1928a mice (lemke et al. 2008 b ) lacking the pka phosphorylation site s1928 in the α1c subunit (s1928aβ2stop mouse) or with sf mice (blaich et al. 2010 d ) lacking the camkii phosphorylation sites s1512 and s1570 in the α1c c-terminus (sfβ2stop). both mouse lines were viable and showed unchanged echocardiography recordings compared to their control littermates and unaltered ecg for s1928aβ2stop mice. electrophysiological investigations on cardiomyocytes of s1928aβ2stop mice showed unchanged β-adrenergic stimulation with isoproterenol compared to littermate controls. these results suggest that β adrenergic regulation of the cardiac cav1.2 channel is not mediated by these phosphorylation sites. introduction. chronic atrial fibrillation (caf) is marked by increased fibrosis which contributes to the perpetuation of the disease. in addition to the role of fibrosis in structural remodeling of cardiac tissue, fibroblasts can couple with cardiomyocytes via gap junction thereby altering the electrophysiological properties of the later and potentially participating in atrial electrical dysfunction. in order to understand the importance of fibroblasts in the pathophysiology of caf, we compared the electrical properties of atrial fibroblasts isolated from patients in sinus rhythm (sr) and caf. methods. fibroblasts were isolated by outgrowth culture from right atrial biopsies and cultivated in medium containing 10% fetal calf serum. we used whole-cell patch clamp techniques to investigate ion currents and membrane potential. results. sr and caf fibroblasts showed similar capacitance (sr: 43.6 ± 4.6 pf, n=33; caf: 54.7 ± 5.1 pf, n = 17) and membrane potential (sr: -21.0 ± 4.3 mv, n = 14; caf: -27. 4 ± 4.8 mv, n = 16) . in both groups, we observed fast activating outward currents with a mean threshold at -20 mv. interestingly, current amplitude was significantly larger in sr than caf cells (sr: 23.8 ± 4.2 pa/pf, n = 15; caf: 6.1 ± 1.0, n = 6; p < 0.05). when maintained in culture for 3-5 weeks, cells from both groups developed na + currents. surprisingly, the fraction of caf cells displaying such currents was larger than the sr counterpart (caf: 38%; sr: 15%). furthermore, na + current amplitude was significantly larger in caf fibroblasts (sr: 6.1 ± 2.0 pa/pf, n = 5; caf: 17.4 ± 4.4 pa/pf, n = 6; p < 0.05). na + currents were not altered by 100 nm tetrodotoxin (ttx), but 10 µm ttx reduced current amplitude to 42% of control, suggesting that the channel involved is the cardiac ttx-resistant isoform nav1.5. conclusion. in the context of caf, fibroblasts undergo electrophysiological changes which need to be thoroughly described. understanding whether those changes contribute to the af substrate might provide new therapeutic targets for the treatment of caf. the initial recruitment of gi to the alpha2a-adrenergic receptor is affected by gprotein-coupled receptor kinases and arrestins prokopets o. s., krasel c., 35043 marburg, germany most g-protein-coupled receptors undergo homologous desensitization after agonist stimulation. in this process, agonist-activated receptors are phosphorylated by gprotein-coupled receptor kinases (grks), followed by binding of arrestins to the still agonist-occupied, phosphorylated receptors. it is assumed that arrestin competes with heterotrimeric g-proteins for the receptor molecule and thereby causes desensitization of g-protein-mediated responses. we tested this idea by investigating the effect of grks and arrestins on the recruitment of g-proteins by the alpha2a-adrenergic receptor. hek293t cells were transfected with yfp-tagged alpha2a-adrenergic receptor and the three subunits of gi1. the g(beta) subunit was cfp-tagged. upon stimulation with noradrenaline, a very rapid, robust increase in fret between the receptor and the g(beta) subunit was observed, confirming previous observations that the alpha2aadrenergic receptor recruits gi with a half-life of around 150 milliseconds. when arrestin3 and grk2 were also co-transfected, the half-life of gi recruitment was substantially delayed, increasing to around 2 seconds. there seemed to be no effect of grk2+arrestin3 cotransfection on a second stimulation; neither the kinetics nor the extent were altered compared to the first stimulation. interestingly, grk2 alone seemed to cause a similar but slightly less pronounced delay of g(beta) recruitment to the alpha2a-adrenergic receptor. in corresponding experiments, we measured the recruitment of arrestin3-cfp to yfp-tagged alpha2a-adrenergic receptors in the presence of grk2. upon stimulation with noradrenaline, we observed a robust increase in fret between the receptor and arrestin3, confirming previous observations that the alpha2a-adrenergic receptor recruits arrestin3. co-transfection of the three subunits of gi1 had no effect on the kinetics of arrestin3 binding to the alpha2a-adrenergic receptors. based on previous results with other receptors, an attenuation of gi recruiting to the alpha2a-adrenergic receptor is quite unexpected. our data suggest that the relation between receptors, g-proteins, grks and arrestins may be more complex than previously postulated. introduction: human urinary bladder expresses mrna of the three known badrenoceptor (b-ar) subtypes (b1, b2, b3) in detrusor and urothelium. we have shown previously that only b3-ar is involved in human detrusor relaxation. to investigate the urothelium-induced modulation of b-ar-mediated relaxation, we have examined systematically whether other b-ar subtypes are involved. human detrusor tissue samples were obtained from patients undergoing radical cystectomy for the treatment of bladder cancer. detrusor strips were studied with and without an intact mucosa layer. muscle strips were precontracted with 1 µm carbachol and relaxation was studied in response to the b-ar agonist ne. a-ar mediated processes were blocked with the a-ar antagonists phentolamine and prazosin. selective b-ars antagonists were used to investigate b-ar mediated relaxation. at the end of each experiment 10 µm forskolin was used to determine maximum camp-mediated relaxation. the presence of intact urothelium reduces potency but not effectivity of ne indicating involvement of urothelium-mediated processes not only during detrusor contraction but also during relaxation. ne-mediated detrusor relaxation is mediated through b3-ar in the absence of urothelium. but in intact detrusor strips b2-ars seem to have an additional inhibitory effect. affinity of the selective b3-ar antagonist l748,337 is unchanged, therefore the intact urothelium does not interact with the function of b3-ars. aldosterone causes oxidative stress and dna damage independent of blood pressure in vivo queisser n., schupp n. universität würzburg institut für toxikologie, versbacherstr. 9, 97078 würzburg, germany background: an inappropriate increase of the mineralocorticoid aldosterone (ald) can be induced by a stimulated renin-angiotensin-aldosterone system. epidemiological studies exploring the connection between hypertension and cancer found higher cancer mortality and an increased risk to develop kidney cancer in hypertensive individuals. we recently showed that ald produces oxidative stress, activates transcription factor nf-kb and is genotoxic in kidney tubule cells. objectives: this study investigated the capacity of ald to induce oxidative/nitrosative stress, dna damage, dna repair, apoptosis, cell proliferation and the activation of nf-κb in rat kidneys. methods: mineralocorticoid-dependent hypertension was induced by ald/salt in sprague dawley rats. dna damage and oxidative/nitrosative stress markers were detected immunohistochemically. results: ald/salt treatment caused increased blood pressure compared to untreated rats. tempol, an antioxidant, and hydralazine, a vasodilator acting independent of the renin-angiotensin-aldosterone system, could lower the blood pressure, while the mineralocorticoid receptor (mr) antagonist spironolactone was administered in a subtherapeutical dose not lowering the blood pressure. ald/salt treatment caused oxidative and nitrosative stress, structural dna damage, double strand breaks, dna repair and nf-κb activation. spironolactone decreased these markers significantly. tempol was also able to reduce these markers, while hydralazine had no effect. ald/salttreated kidneys showed a tendency to lower apoptosis and to increased cell proliferation compared to control rat kidneys. discussion: this study provides a first hint of blood pressure-independent effects of ald. the mr and the production of ros seem to be crucial for the damaging effects of ald. an aberrant or long-term activation of nf-κb by persistently high ald levels could support resistance to apoptosis and the survival of cells with damaged dna, and increase cell proliferation. these actions could contribute to the increased cancer incidence in hypertension by initiating carcinogenesis. grant support by the dfg is gratefully acknowledged. creb regulating transcriptional coactivator 1 (crtc1) is a transcriptional coactivator of the transcription factor creb. we have recently shown its expression in cardiomyocytes and its activation by beta-adrenergic signaling. beta-adrenergic signaling contributes to the pathogenesis of cardiac hypertrophy, leading to heart failure, as evidenced by the therapeutic success of the beta-adrenoceptor antagonists. in order to investigate if crtc1 is involved in this process, we investigated the expression of crtc1 in hypertrophied myocardium from mice and humans. methods: protein lysates from mouse and human samples were investigated for crtc1 protein expression. we distinguished between an acquired and an inherited form of cardiac hypertrophy. acquired cardiac hypertrophy is an adaptation of the heart to an increased cardiac workload and can be found in patients with an aortic valve stenosis. in mice this kind of hypertrophy can be evoked by transverse aortic constriction (tac). the inherited form of cardiac hypertrophy is caused by mutations in genes coding for proteins of the sarcomeric apparatus and is referred to hypertrophic cardiomyopathy (hcm). as a model for hcm, transgenic mice with a mutation in the mybpc3 gene, coding for the sarcomeric protein cardiac myosin-binding protein c (cmybp-c), were used. these transgenic mice were characterized by a hypertrophied heart. in case of human samples we distinguished between patients with an acquired form of hypertrophy due to an aortic valve stenosis, and patients with a hypertrophic obstructive cardiomyopathy (hocm), a special form of hcm. results: in the tac mice and in the myppc3 mutant mice the expression of crtc1 was significantly higher than in the controls (2.1-and 1.9-fold, respectively; n=3). in both forms of human hypertrophy, we found a significantly 5-fold upregulation of crtc1 protein level in comparison to human samples from non-failing myocardium or samples from patients with a dilatative or ischemic cardiomyopathy (n=7-10 for each group). conclusions: crtc1 is upregulated in cardiac hypertrophy with acquired or geneticbased origin. the evaluation of the role of crtc1 in the heart may help to elucidate the role of beta-adrenergic signaling in the development of cardiac hypertrophy. microarray gene expression profiling reveals up-regulation of the cardiac lipid metabolic process at the onset of heart failure fu x., abd alla j., quitterer u. eth zürich molekulare pharmakologie, winterthurerstrasse 190, 8057 zürich, switzerland atherosclerosis and chronic pressure overload are major cardiovascular risk factors for the development of heart failure in patients. to mimic those risk factors in experimental models we used atherosclerosis-prone apolipoprotein e (apoe)-deficient mice, and chronic pressure overload was imposed by abdominal aortic constriction (aac). cardiac function was monitored by echocardiography. severe atherosclerosis in aged apoedeficient mice or chronic pressure overload induced signs of heart failure as evidenced by a significantly reduced cardiac ejection fraction (<30%). to investigate pathomechanisms underlying the development of heart failure, microarray gene expression analysis and qt-rt-pcr were performed of failing heart tissue relative to age-matched controls. gene ontology analysis of the microarray data revealed that the onset of heart failure, in two different experimental models, was characterized by a strong up-regulation (≥2-fold) of the cardiac lipid metabolic process and lipid overload. lipid metabolism genes were involved in lipid synthesis, storage and oxidation. the major palmitate-synthesizing enzyme, fatty acid synthase, was causally related to the development of cardiac dysfunction by enhancing cardiomyocyte apoptosis. taken together the data support that the onset of experimental heart failure is characterized by a dysfunction of the cardiac lipid metabolism promoting cardiomyocyte death. at1-receptor blockers (arbs) are established for the treatment of high blood pressure and new onset of diabetes is reduced by arbs. in the past years evidence increased that body weight may also be lessened particularly in rats and mice. however, less data are available whether arbs still reduce weight, when treatment was initiated not until animals became obese by diet. prior to drug treatment, spontaneously hypertensive rats were fed for 6 months with chow but also with a cafeteria diet (cd) to develop obesity. controls received only chow (conchow). cd-fed shr were treated for 3 months with telmisartan (tel 8mg/kg/d) or amlodipine (aml, 12 mg/kg/d), whereas controls received vehicle (concd). systolic blood pressure (sbp), feeding behaviour, body weight, abdominal fat mass (by mrt) and energy expenditure (by indirect calorimetry) was monitored. leptin sensitivity was assessed by measuring energy intake and expenditure after repetitive injections (s.c.) of leptin. insulin sensitivity was functionally determined by glucose and insulin tolerance tests. due to cd feeding body weight was increased after 6 months by more than 60 g. tel normalized sbp whereas it remained >200 mmhg in concd and conchow. tel additionally reduced cd-induced increase of body weight and abdominal fat mass. food intake was diminished during the first 4 weeks, but raised beyond control levels during the last 4 weeks of treatment. the shift of the respiratory index to lower levels indicated improved energy expenditure. in response to exogenous leptin, the food intake of concd was higher compared to conchow, indicating a leptin resistance. this assumption is further supported by high triglyceride concentrations of concd. after tel, leptininduced food intake was reduced and energy expenditure was increased compared to concd, indicating that leptin sensitivity was at least partially restored. accordingly, triglycerides were reduced. compared to concd, the insulin sensitivity was improved by tel since maximal increases in plasma concentrations of glucose and insulin in response to glucose challenge were reduced, but glucose response to insulin challenge was diminished. even though reduction in blood pressure was almost similar between tel and aml, metabolic and antiobese efficacies of aml were markedly attenuated. we conclude that telmisartan reveals wide efficacies in improving all symptoms of the metabolic syndrome. the pleiotropic effects are not related to the hypotensive action of tel. a β2-adrenoceptor -camp mediated, immediate stimulation of β2-adrenoceptor gene expression in human lung fibroblasts is opposed by a delayed up-regulation of inhibitory factors racké k., lamyel f., kämpfer n., schütz i., warnken m. univ. bonn dept. pharmacol. &toxicol., 53105 bonn, germany based on their bronchodilatory effects, β2-adrenoceptor agonists constitute essential elements in the treatment of bronchial asthma and copd. however, treatment with long-acting β2-adrenoceptor agonists has been associated with possible worsening of airway hyper-reactivity, possibly because of loss of β-adrenoceptor function. therefore, the molecular regulation of β2-adrenoceptor expression was addressed here. mrc-5 human lung fibroblasts were cultured for up to 48 h in absence or presence of test substances, followed by β2-adrenoceptor mrna determination by qpcr. β2-adrenoceptor mrna decreased with a half-life of 25 min after inhibition of mrna synthesis with actinomycin d (30 µm), but increased by 333±85%, 502±52% and 640±165% (means±sem) within 1.5, 4 and 6.5 h, resp. after inhibition of protein synthesis by cycloheximide (30 µm). the β2-adrenoceptor agonists formoterol and olodaterol (1-100 nm) induced a rapid increase in β2-adrenoceptor mrna (maximally within 1 h by 100±19% and 110±19% at 10 nm, resp.). however, after 4 h exposure to 10 nm formoterol or olodaterol a reduction in β2-adrenoceptor mrna by 59±8% and 58±6%, resp., was observed. both, the stimulatory and inhibitory effects of β2adrenoceptor agonists were mimicked by forskolin (10 µm, increase by 88±14% and inhibition by 49±4%) and cholera toxin (5 ng/ml, increase by 76±12% and inhibition by 77±7%). the formoterol-induced up-regulation of β2-adrenoceptor mrna was blocked by actinomycin d, but not by cycloheximide. moreover, in presence of cycloheximide, β2adrenoceptor agonist induced inhibition was converted into a marked stimulation. in conclusion, expression of β2-adrenoceptors in human lung fibroblasts is highly regulated at transcriptional level. the observations with cycloheximide indicate that the β2-adrenoceptor gene is under strong inhibitory control of short-living, not yet identified suppressors. although both, the time-dependent up-and down-regulation of the β2adrenoceptor gene expression by β2-adrenoceptor activation appears to be mediated via adenylyl cyclase -camp signalling, only the stimulatory effect appears to be a direct action on the β2-adrenoceptor gene. et-1 appears to be involved in the pathogenesis not only of pulmonary hypertension, but also in fibrotic remodeling associated with chronic obstructive airway diseases. since human lung fibroblasts (hlf) are a source of et-1 and have been shown to be controlled by muscarinic receptors and β-adrenoceptors, a possible muscarinic and βadrenergic modulation of et-1 expression in hlf was explored. mrc-5 hlf were cultured for up to 24 h in absence or presence of test substances, followed by prepro-et-1 (ppet-1) mrna determination by qpcr. the muscarinic agonist oxotremorine (10 µm) induced an increase in ppet-1 mrna by 180%, an effect prevented by 10 nm tiotropium. the β2-adrenoceptor agonist olodaterol (up to 100 nm) caused a reduction of ppet-1 mrna expression by 45%. the effect of 10 nm olodaterol was prevented by ici 118,551 (1 µm), but not affect by cgp 20712 (3 µm) . the pka agonist 6-bnz-camp (500 µm) caused a reduction in ppet-1 mrna expression by 65%, whereas the epac agonist 8-cpt-2'-o-me-camp (100 µm) caused only a marginal inhibition by 22%. olodaterol (10 nm) strongly opposed the stimulatory effect of 10 µm oxotremorine. an increase in ppet-1 mrna expression by 185% caused by 0.3 ng/ml tgf-β was effectively opposed by 10 and 100 nm olodaterol, resulting in an inhibition comparable to that in absence of tgf-β. however, the increase in ppet-1 mrna caused by a maximally effective concentration of tgf-β (1 ng/ml, increase by 620%) was not significantly affected by 10 or 100 nm olodaterol. likewise, the pkaagonist 6-bnz-camp (500 µm) opposed the increase in ppet-1 mrna expression caused by 0.3 ng/ml tgf-β, but not that caused by 1 ng/ml tgf-β. tgf-β caused, with an ic 50 of 0.3 ng/ml, a marked down-regulation of β2-adrenoceptor mrna expression, maximally by 90% within 6 h. et-1 expression in hlf is stimulated by muscarinic receptors and inhibited by β2adrenoceptors. the effect of β2-adrenoceptors may be mediated via pka. et-1 expression in hlf is markedly up-regulated by tgf-β, but only effects of sub-maximally effective concentrations of tgf-β are opposed by the β2-adrenoceptor -pka pathway, in part because of tgf-β-induced down-regulation of β2-adrenoceptors. since et-1 can promote pro-fibrotic features in hlf, inhibition of et-1 expression could contribute to long-term beneficial effects of long-acting β2-adrenoceptor agonists such as olodaterol and long-acting muscarinic antagonists such as tiotropium. cardiac hypertrophy leads to up-regulation of dipeptidyl aminopeptidase-like proteins in human and rat radicke s., hutschenreuther a., schaefer m. universität leipzig rudolf-boehm-institut für pharmakologie und toxikologie, härtelstr. cardiac hypertrophy is a major risk factor for heart failure and associated morbidity and mortality. functional down-regulation of k + currents is a prominent feature of cells isolated from failing ventricles. a marked decrease in the transient outward potassium current ito has been shown in various models. changes in the k + channel expression differ depending on the species, and the mechanism of induction of heart failure. to study the regulation of ito channel subunits we compared the hypertrophic responses in human ventricular tissues from failing hearts with doxorubicin-induced hypertrophy in rats and in h9c2 embryonic rat cardiac cells. specifically, we quantified mrna expression of the pore-forming subunits kv4.3 and kv4.2, the cytosolic β-subunit kchip2 and the transmembrane subunits dpp6 and dpp10 using rt-pcr. treatment with doxorubicin (2 µm) induced hypertrophy and increased the mrna expression of the hypertrophy marker genes anf, bnp and beta-mhc in h9c2 cardiac myoblasts. while kv4.3 was detected in h9c2 cells and hearts from human and rat, kv4.2 mrna was only expressed in adult rats. during hypertrophy kv4.3 was downregulated in human tissue as well as in doxorubicin-treated h9c2 cells compared to the controls. in rat hearts kv4.2 expression was increased after doxorubicin treatment. interestingly, kv4.2 was also found to be up-regulated in rat heart tissues and h9c2 cells after treatment with doxorubicin. as previously shown, kchip2 mrna expression was significantly reduced in tissues of failing hearts. in contrast, kchip2 mrna was upregulated in hypertrophic rat hearts and h9c2 cells. the expression of dpp6 and dpp10 was observed only in human hearts. but mrna levels of both were significantly increased in failing tissues. dpp6 and dpp10 were not expressed in the adult rat heart or h9c2 cells, whereas in rats with doxorubicin-induced cardiac hypertrophy and in doxorubicin-treated h9c2 cells, the mrna of dpp6 and dpp10 was up-regulated. h9c2 cells showed almost identical hypertrophic responses to those observed in human ventricles and rat hearts. this finding validates h9c2 cells as a model for in vitro studies of cardiac hypertrophy. in further studies we will investigate the consequences of a knock-down of dpp6 and dpp10 in doxorubicin-induced hypertrophic h9c2 cells. in preliminary experiments specific short-hairpin rna, targeting dpp6 and dpp10, has been designed and tested in heterologous expression systems. the nonsynonymous c.521t>c germline genetic variation in the liver-specific organic anion transporter slco1b1 is associated with methotrexate pharmacokinetics in pediatric acute lymphoblastic leukemia radtke s. 1 background: methotrexate (mtx) plasma concentration is related to its clinical effect. transport proteins, such as abcc2, slco19a1, and slco1b1, have been implicated in the disposition of mtx. here we investigated whether common reduced-function variants in abcc2, slco19a1, and slco1b1 contribute to the interindividual variability in methotrexate pharmacokinetics in children with acute lymphoblastic leukemia (all). we analyzed mtx pharmacokinetics (mtx plasma concentration at the end of infusion c24h, mtx auc24-48h, and mtx clearance cl) in an unselected population of 419 children with all from the all-bfm 2000 trial (clinicaltrials.gov: nct00430118) who received 1676 courses of mtx at 5 g/m 2 as 24 h infusions. the contribution of genes (genetic component, rgc) to the interindividual variability in mtx pharmacokinetics was estimated according to the method of kalow et al. (1998) . abcc2 c.-24c>t (rs717620), slco19a1 c.80g>a (p.his27arg, rs1051266), slco1b1 c.521t>c (p.val174ala, rs4149056) and slco1b1 388a>g (p.asn130asp, rs2306283) genotypes were analyzed by taqman polymerase chain reaction. there was substantial interpatient variability in average (± sd) mtx c24h (50.95 ± 24.15 µmol/l), auc24-48h (57.44 ± 37.52 h*mg/l), and cl (390.72 ± 223.95 ml/min/m²). the rgc values of c24h, auc24-48h, and cl ranged from 0.61-0.71 suggesting that variation in mtx pharmacokinetics has a substantial genetic component. after adjustment for age and sex by multiple regression, the slco1b1 c.521t>c snp was significantly associated with c24h (p<0.001), auc24-48h (p<0.001), and cl (p=0.011) of mtx. compared with the wildtype genotype, in patients with the tc genotype c24h and auc24-48h increased by 18% (p=0.009) and 28% (p=0.003), respectively, whereas cl significantly decreased by 15% (p=0.012). pharmacokinetic variables significantly changed with increasing number of variant c.521t>c alleles (p<0.02, jonckheere-terpstra), suggesting a per allele effect consistent with a co-dominant model of association. in contrast, the abcc2 c.-24c>t, slco19a1 c.80g>a, and slco1b1 388a>g polymorphisms did not show an association with mtx pharmacokinetics. conclusions: the nonsynonymous c.521t>c polymorphism in slco1b1 contributes to the variability of mtx pharmacokinetics in this study of high-dose mtx in pediatric all. this project is supported by the johannes und frieda marohn-stiftung. the antitumorigenic mechanism of the selective cyclooxygenase-2 (cox-2) inhibitor celecoxib is still a matter of debate. using human lung cancer cell lines (a549, h358, h460), the present study investigates the contribution of cox-2 and peroxisome proliferator activated receptor γ (pparγ) to apoptosis elicited by celecoxib. celecoxib was found to cause apoptotic cell death in a concentration-dependent manner (10 -50 µm), whereas structurally-related cox-2 inhibitors (etoricoxib, rofecoxib, valdecoxib) were inactive in this respect. apoptotic cell death by celecoxib was suppressed by preincubation of tumor cells with the selective cox-2 inhibitor ns-398, the pparγ antagonist gw-9662 and by sirna targeting cox-2 or pparγ. celecoxib-induced apoptosis was paralleled by a time-and concentration-dependent upregulation of cox-2 and pparγ at both mrna and protein level. using an established cox-2 activity assay monitoring immediate conversion of exogenously added arachidonic acid to the respective prostaglandins (pgs), ns-398 was shown to suppress celecoxib-induced cox-2 activity when added prior to arachidonic acid. among the cox-2-dependent pgs analyzed, pgd 2 and its dehydration product 15-deoxy-∆ 12,14 -pgj2 were found to induce cytosol-to-nucleus translocation of pparγ as well as pparγ-dependent apoptosis. celecoxib-elicited translocation of pparγ was inhibited by preincubation of cells with ns-398 which itself did not alter celecoxib-induced total pparγ protein expression. finally, a cox-2-and pparγ-dependent proapoptotic mechanism of celecoxib was confirmed in primary tumor cells obtained from brain metastases of two lung cancer patients. together, our data demonstrate a proapoptotic mechanism of celecoxib involving initial upregulation of cox-2 and pparγ and a subsequent nuclear translocation of pparγ by cox-2-dependent pgs. uncoagulated ppp contained 40-60 nm thrombin throughout. fxa was initially measured in clots at 10 nm. this level declined over time while clot-conditioned pbs accumulated fxa. exposure of human aortic smc to clots or native unclotted ppp for 24h only marginally influenced smc apoptosis but increased mitogenesis over 15-fold. this was reduced by all 4 inhibitors. clot-stimulated induction of tnfα and interleukin-6 mrna was also attenuated by the inhibitors. denatured ppp (no protease activity) increased smc mitogenesis to a level seen in smc exposed to clot and combined hirudin + dx9065a, reflecting the well-known mitogenic actions of serum alone. conclusion: coagulation of human plasma generates nanomolar amounts of thrombin and fxa, sufficient to stimulate the proliferative and inflammatory properties of adjacent smc. our observations validate the use of purified thrombin and fxa at nanomolar concentrations for in vitro studies, and support the individual and coagulationindependent roles of these proteases in cell proliferation and inflammation. antithrombotic therapy with argatroban or rivaroxaban may limit the cellular effects of clot-derived thrombin and fxa, while normal anti-platelet therapy would not. this aspect should be considered in the clinical use of these agents, specifically in healing processes after vessel injury. rhogef17 mediates cgmp/cgk induced adherence and relaxation of vascular smooth muscle cells rauch j. 1 , stephan-schnatz k. the guanine nucleotide exchange factor rhogef17 is the only gef known so far to be directly activated by cgmp-dependent kinase. it is expressed in various types of smooth muscle cells and has been shown to play a role in the regulation of cell integrity. in a previous investigation we showed that the knockdown of rhogef17 by a shrna approach caused a loss of actin stress fibers and a subsequent change of smooth muscle cell morphology that finally resulted in cell rounding. we now provide evidence that the expression level of rhogef17 influences the re-attachment of cultured rat aortic smooth muscle cells (rasmc) to a surface after detachment. although rhogef17 depleted rasmc were still able to adhere and spread, their cell surface area remained considerably smaller than that of control cells in the first 24 hours after seeding. cell counting revealed that 6 to 12 hours after seeding the percentage of adherent cells was significantly lower in the rhogef17 knockdown group compared to the control group. these data indicate a delay in attachment. interestingly, the knockdown of rhogef17 was paralleled by a loss in rhoa and cadherine expression. as rhogef17 mediated a cgmp-induced activation of the small gtpase rhoa in rasmc, we studied the effect of a stable cgmp analogon (8-pcpt-cgmp) on the adhesion process. in accordance with previously published data, cgmp treatment accelerated the attachment of rasmc to the surface within the first 12 hours. in contrast, the adhesion of rasmc after rhogef17 knockdown was no longer stimulated by cgmp. as these data indicate that rhogef17 mediates cgmp-dependent signalling in a physiological process we wondered whether this protein might also play a role in the regulation of cgmpdependent relaxation of vascular smooth muscle cells. thus, we performed a collagenbased contraction assay with rhogef17 depleted rasmc. while the contraction in response to serum was not affected by the depletion of rhogef17, we observed a slight decrease in basal contractility. interestingly, cgmp was not able to counteract the serum-driven contraction of rhogef17 knockdown cells. there was no cgmp-induced relaxation in these cells. we conclude that rhogef17 is involved in the cell adhesion of vascular smooth muscle cells and likely promotes the expression of specific proteins. its activation is required to mediate cgmp-induced signalling in terms of vascular smooth muscle cell adherence and relaxation. human eosinophil and neutrophil granulocytes are cells of the innate immune system. they both express formyl peptide receptors (fpr) und histamine h2 receptors (h2r). activation of fpr leads to a release of reactive oxygen species (ros). h2r activation results in an increase of intracellular 3'-5'-cyclic adenosine monophosphate (camp) concentration and inhibition of fpr-mediated ros release via adenylyl cyclase activation. in this study we compared the effects of various h2r ligands on camp accumulation and formyl peptide n-formyl-l-methionyl-l-leucyl-l-phenylalanine (fmlp)induced ros release in isolated eosinophils and neutrophils. camp concentration was determined by hplc/tandem mass spectrometry, and ros release was assessed by monitoring superoxide dismutase-inhibitable reduction of ferricytochrome c. in eosinophils, histamine, amthamine and 5-methylhistamine exhibited similar potencies and efficacies with regard to camp accumulation and inhibtion of ros release. in marked contrast, in human neutrophils, we observed dissociations in potencies and efficacies of ligands at increasing camp accumulation and inhibition of ros production. our data suggest that in human eosinophils, but not neutrophils, camp mediates inhibtion of ros production. in a broader context our data provide a compelling example of the context-dependency of the pharmacological properties of g-proteincoupled-receptors. specifically, one has to be cautious when extrapolating experimental observations from one cell type to another, even when they are very closely related to each other. comonomers and monomers are used as dental restorative materials (e.g. in dental composites). unconverted compounds can be released from dental composites and can enter the body in humans. comonomers can induce various side effects in humans. this study was evaluated to qualify and to quantify eluted compounds from various dental composites. following composites were tested (producer in parentheses): els extra low shrinkage (saremco), synergy duo shade (coltène), grandio (voco), tetric evo ceram (vivadent), venus (kulzer), gradia (g.c.), and premise (kerr).polymerized composites (100 mg) were incubated in gc vials with 1 ml dest. water or 1 ml methanol, each at 37 °c for 72 hours. aliquots were taken, and eluted compounds were analyzed with the method of gas chromatography/mass spectrometry (gc-ms) and liquid chromatography/mass spectrometry (lc-ms).from all composites 18 different compounds were found. following comonomers were quantified (µg/ml; mean ± s.d.; n=4)( fig. 1 ).following range of the eluted and detected comonomers from dental composites was found (dest. water; decreasing elution): venus > gradia > synergy duo shade > tetric evo ceram premise > grandio > els extra low shrinkage. * n.d. = not detectable (below limit of detection). triphenylstibane was detected in tetric evo ceram (5 ± 2 µg/ml). reimann c., lupp a., schulz s. institut für pharmakologie und toxikologie universitätsklinikum jena, drackendorfer str. objective: cxcr4 is a plasma membrane chemokine receptor which is involved e.g. in organogenesis, hematopoesis and inflammation. in the adult organism cxcr4 is physiologically expressed on various cell types, in particular on lymphocytes. with respect to neoplastic tissues, in the current literature an over-expression of cxcr4 is described in different types of tumors, especially in breast and prostate cancer. additionally, an involvement of cxcr4 in tumor metastasis is discussed. subsequently, detection of cxcr4 expression in a given human tumor sample would provide a valuable predictive information on disease prognosis and possible therapeutic intervention. however, previous attempts to localize cxcr4 using poorly characterized mouse monoclonal or rabbit polyclonal antibodies have yielded predominant nuclear and occasional cytoplasmic staining, but did not result in the identification of cell surface receptors. thus, the aim of the present study was to reassess the cxcr4 expression in a panel of formalin-fixed and paraffin-embedded human normal and neoplastic tissue samples by means of immunohistochemistry using the well characterized novel rabbit monoclonal anti-cxcr4 antibody umb-2. methods: in comparison to negative and positive control samples (cxcr4-knockout and wild-type mouse embryos) the extent of staining in the different normal and neoplastic tissue specimens was scored from zero (no expression) to three (high expression). results: cxcr4 was found to be expressed in all neoplastic tissue entities analyzed. in many cases, the receptor was predominantly localized at the plasma membrane of the tumor cells. however, in all cxcr4-expressing tumor entities a huge interindividual variability both in the percentage of positive cells and in the intensity of staining was noted which strongly differed also between the various types of cancer. the most intense (score three) staining was found in the samples of (small cell) lung cancer, ovarian cancer and of pheochromocytoma. additionally, lymphatic organs such as lymph nodes, spleens and tonsils were cxcr4 positive, with mainly b cells displaying a distinct staining of the plasma membrane. the rabbit monoclonal antibody umb-2 may prove to be of great value in the assessment of cxcr4 expression in different human tumor entities and of the mechanisms underlying the formation of metastases, thus helping to find new targets and strategies in cancer therapy. link between β2-adrenoceptor-mediated inhibition of formyl-peptide-induced o2 β2-adrenoceptor (adrb2)-agonists are in daily use for asthma therapy. although most cases of asthma are controlled by standard medication, a subpopulation of asthmatics remains difficult to treat. the adrb2 gene contains a total of 49 polymorphisms. this variability could cause part of the ~70 % genetically-determined differences in therapy response. genetic and corresponding functional data on adrb2 can help to understand the complex disease and, in cases of severe asthma, optimize therapy with adrb2agonists for each individual. (ortega et al. 2007; chung et al. 2011) our present study connects sequence data with pharmacological data of prototypical adrb2-ligands, namely, (r)-isoproterenol, (r,r)-and (s,s)-fenoterol, (r)-and (s)salbutamol and (r,r)-formoterol. as a pathophysiologically relevant cell type, we analysed human neutrophils from peripheral blood of healthy volunteers. formyl-peptide-induced o2 .production and its inhibition by the agonists are examined in a 96-well cytochrome-c assay. characteristic pharmacological values (pic50, emax) are obtained for each individual. the data-set for each individual is supplemented by a differential blood cell analysis and an asthma-related questionnaire. most importantly, each volunteer's adrb2-sequence variant is determined by sanger-sequencing. complete determination of a 1,490 bp sequence, including the entire adrb2 exon and part of the flanking 5'-and 3'-untranslated regions, allows mapping of the most common, but also of new or rare polymorphisms. first data demonstrate the inter-day and intra-individual robustness of the functional data. in the next step, we will link sequence variants and functional differences within a population of sixty volunteers with sufficient statistical power. collectively, this study represents a straightforward approach to link functional and genetic data of a clinically relevant receptor. cyclic nucleotide phosphodiesterases (pdes) are classified into eleven families and are essential for second messenger metabolism in human cells. 1 recently, we have shown that several human pdes possess a much broader substrate-specificity than previously assumed, being capable of hydrolyzing not only the purine nucleotides cyclic adenosine 3',5'-monophosphate (camp) and cyclic guanosine 3',5'-monophosphate (cgmp), but also pyrimidine nucleotides such as cyclic uridine 3',5'-monophosphate (cump). 2 these data were obtained using a highly sensitive hplc mass spectrometric assay which is quite expensive and whose technical requirements are available only in few laboratories. in our present study we developed a fluorimetric pde activity assay using 2'-o-(n'methylanthraniloyl) (mant)-substituted cyclic purine and pyrimidine nucleotides that can be used more broadly in the scientific community. human pde3a is important for the regulation of platelet aggregation, oozyte maturation, vascular smooth muscle relaxation and contractility of cardiac myocytes. 1 moreover, this pde shows a broad substrate-specificity, hydrolyzing camp, cgmp, cump and cyclic inosine 3',5'-monophosphate (cimp). 2 using this enzyme, here, we demonstrate that various mant-substituted cyclic nucleotides are substrates of pde3a and undergo a significant change in fluorescence whilst being hydrolyzed, thus allowing a quantitative analysis of catalysis via fluorescence detection. in fact, not only native cump but also mant-cump is a substrate of pde3a. this finding is consistent with data published by hardman and sutherland 3 who described a cump-degrading pde activity in homogenates from beef and dog heart, leading to the assumption that the pde activity described there could be attributed to pde3a. as cump has furthermore been proven to be present in mammalian cells 4 , to differentially activate camp-and cgmp-dependent protein kinases 5 and to be synthesized from utp by mammalian soluble guanylyl cyclase 6 , our present study supports the hypothesis that this cyclic nucleotide could play an important role in cell metabolism. the newly established fluorescence assay with mant-cump facilitates future studies on pde3a and the assumed second messenger function of cump. rhoa is reportedly involved in stat-dependent transcription. however, the pathway connecting the gtpase and stat signaling has not been characterized. we made use of bacterial toxins, which directly activate rho gtpases to analyse this pathway. cytotoxic necrotizing factors (cnfs) are produced by pathogenic escherichia coli strains and by yersinia pseudotuberculosis. they activate small gtpases of the rho family by deamidation of a glutamine, which is crucial for gtp hydrolysis. we show that rhoa activation leads to phosphorylation and activation of stat3 and identify signal proteins involved in this pathway. rhoa-dependent stat3 stimulation requires rock and junkinase activation as well as ap1-induced protein synthesis. the secretion of one or more factor/s activate/s the jak-stat pathway in an auto/paracrine manner. we identify ccl1/i-309 as an essential cytokine, which is produced and secreted upon rhoa activation and which is able to activate stat3-dependent signaling pathways. the knowledge about the connection between rhoa and stat signaling is crucial for understanding several deseases, especially cancer. acid sphingomyelinase-deficient mice are protected from the lethal cardiovascular effects in tnf-induced septic shock reiss l. k. 1 christian-albrechts universität institut für immunologie, michealisstrasse 5, 24105 kiel, germany introduction: the cytokine tumor necrosis factor (tnf) is a mediator of septic shock. sepsis is a major cause of acute respiratory distress syndrome (ards), a heterogeneous lung disease with a mortality of about 50%. the present study was designed to investigate the effects of high systemic tnf-levels on the lung and on the systemic circulation in wildtype and acid sphingomyelinase-deficient (asm -/-) mice. the enzyme acid sphingomyelinase generates the signaling molecule ceramide that plays a critical role in edema formation and vasodilatation. material and methods: asm -/and wildtype mice were ventilated mechanically at vt=8ml/kg and f=180min -1 with fio2=0.3 and peep=2cmh2o while lung mechanics were followed. half of the mice received 50µg of murine tnf intravenously. blood pressure was stabilized by intra-arterial fluid support and body temperature was kept at 37°c to prevent lethal shock and to allow investigation of blood gases, lung histopathology, pro-inflammatory mediators and microvascular permeability 6 hours after tnf application. results: tnf induced septic shock in wildtype mice, as indicated by metabolic acidosis, high serum levels of the sepsis marker procalcitonin, decreasing blood pressure and reflex tachycardia. interestingly, asm -/mice were protected from the tnf-induced cardiovascular effects and mortality. in the present study, circulating tnf failed to cause lung injury. lung mechanics stayed stable during ventilation in all groups and also pulmonary histopathology, cytokine levels and microvascular permeability were unaffected. conclusion: circulating tnf alone is not sufficient to cause acute lung injury. we conclude that the cardiovascular effects in tnf-induced septic shock are partly mediated by acid sphingomyelinase. cyclophilin a sirna provides mitoprotection and prevents aif-dependent neuronal cell death reuther c. 1 cyclophilin a (cypa) is a peptidyl-prolyl-cis-trans isomerase which is localized in the cytosol. recent data suggested that neuronal cell death involved cytosolic cypa translocation to the nucleus, where it formed a pro-apoptotic complex with apoptosis inducing factor (aif). this cypa-aif complex induced caspase-independent chromatin condensation, dna degradation and cell death in various paradigms of apoptosis. on the basis of these data, the selective inhibition of the aif-cypa complex was proposed as a potential strategy to prevent aif-dependent cell death in neurons. therefore, the aim of this study was to determine effects of cypa silencing in a model of glutamate toxicity in immortalized hippocampal ht22 neurons. first, we addressed the interaction of aif and cypa by immunoprecipitation and their translocation to the nucleus by immunohistochemistry and confocal fluorescence microscopy. after exposure of ht-22 cells to glutamate the translocation of aif and cyp a occurred prior to cell death. cypa sirna attenuated glutamate-induced cell death as detected by the mtt-assay, impedance measurements (xcelligence system), and by facs analysis after annexin v/ propidium iodide staining. most intriguingly, cypa sirna also preserved the mitochondrial membrane potential as shown by facs analysis after tmre staining. further, confocal microscopy showed that cypa silencing prevented mitomorphology alterations and blocked the release of mitochondrial aif to the nucleus. the inhibition of the aif translocation to the nucleus was also shown by western blot analysis. in summary this study demonstrates that silencing of cypa prevents mitochondrial disruption and attenuates glutamate toxicity in vitro. thus, cypa is a promising target for mitoprotection as a basis for novel strategies of neuroprotection. up to now the question is unresolved how the ingested dose influences the absorption and metabolism of chlorogenic acids (cga) from food. so far no studies have been performed on the impact of the dose on cga absorption, circulation and excretion. recently we performed a dose-response study in a randomized, double-blinded, crossover design with five ileostomy subjects. in three trials the volunteers consumed after a two day polyphenol free diet coffee with varying cga content (high 4525 µmol; medium 2219 µmol; low 1053 µmol). the cga concentrations in plasma, ileal effluent and urine were subsequently identified and quantified by hplc-esi-ms, hplc-esi-ms/ms and hplc-dad. the results showed that the consumption of higher cga concentrations lead to a faster ileal excretion measured in the ileal effluents. this corresponded to the renal excretion of 8.0 ± 4.9% (high), 12.1 ± 6.7% (medium) and 14.6 ± 6.8% (low) of total cga and metabolites. we found that cgas with a caffeic acid moiety are predominantly sulphated and those with a ferulic acid moiety are predominantly conjugated via glucuronidation prior renal excretion. furthermore, in the ileal effluents, sulphation of both structural units dominated. in plasma samples (after enzymatic deconjugation) the auc values were determined by the major cga classes in coffee, the caffeoylquinic acids: 551.5 ± 93.8 nm*h -1 (high); 299.3 ± 79.6 nm*h -1 (medium) and 222.8 ± 91.3 nm*h -1 (low). no major differences in the metabolic pattern were observed. additionally, we were able to identify new metabolites of cga in urine and ileal fluids. we conclude that the consumption of high concentrations of cga via coffee might influence the gastrointestinal transit time and consequently affect cga bioavailability. this study was supported by the nestlé research centre (lausanne, switzerland). interaction of antagonists with the atp binding pocket at the human p2x3 ion channel helms n., riedel t., illes p. rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße 16-18, 04107 leipzig, germany the homomeric p2x3 receptor (p2x3r) is a rapidly activating and desensitizing cation channel, gated by extracellular atp. it consists of three homomeric subunits. this representative of the p2x receptor family is highly expressed on sensory afferent neurons and plays a significant role in chronic pain, bladder reflexes and taste sensation. therefore, the development of selective antagonists for p2x3 receptors and knowledge about the binding of these antagonists are of great significance for future pain therapy and therapy of urge incontinence. to simulate the shape of the rapidly desensitizing agonist-induced current responses via p2x3 receptors, we created a specific markov model to describe the binding of agonists and competitive antagonists. this model can be used to prove the competitive character of inhibition and to calculate the association and dissociation constants of the antagonists. furthermore we use this model to fit current responses at p2x3 wild type receptors and their mutants to α,βmethylene atp in the presence of different antagonists. whole-cell patch-clamp recordings were performed on hek 293 cells, heterologously expressing the human p2x3 receptor, to determine the concentration-response relationship of different antagonists. by applying increasing concentrations, differences of antagonist potency could be observed at the wild type receptor. afterwards, we chose amino acid residues for replacement by alanine, which seem to be important for agonist binding and should be so for competitive antagonist binding as well, based on our homology model, developed from the zebrafish p2x4r crystal structure and previous mutagenesis studies. we intend to identify those amino acids which are important for competitive antagonist binding by monitoring the altered antagonist potency on the mutated receptor when compared with the wild-type receptor. analysis of the p2x3 agonist binding site by double mutant cycles riedel t., wiese s., illes p. rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße 16-18, 04107 leipzig, germany purinergic p2x receptors belong to the family of ligand-gated ion channels. they are non-selective cation channels, activated by extracellular atp. one of the seven members of the p2x receptor family, the p2x3 receptor, is localized at the plasma membrane of sensory neurons and is involved in pain perception. therefore, this receptor is a possible target for new drugs in pain treatment. the development of such drugs can be supported by an exact knowledge of the receptor structure and function. there are many hints to the atp binding site, but the interaction of the p2x receptor with its agonists and antagonists remains still unknown. in this study, we investigated the effects of single alanine substitutions of amino acid residues in the supposed atp binding site of the homomeric human p2x3 receptor on the effect of nucleotide analogues. the mutant receptors were expressed in hek293 cells and the nucleotide effects were measured by means of the whole-cell patch-clamp method. modifications in the receptor binding site changed the concentration-response dependency as well as the current kinetics during fast pulsed agonist applications. based on this fact, we were able to distinguish binding from gating, conductance, and desensitisation, using a markov model that describes the complete channel behaviour by a matrix of rate constants. the results were also checked for consistency with a structural hp2x3 model that we developed from the known zebra fish p2x4 crystal structure in the closed state. voltage-dependent modulation of alpha2a adrenergic receptor signaling rinne a., birk a., bünemann m. philipps-universität marburg institut für pharmakologie und klinische pharmazie, karlvon-frisch str. 1, 35034 marburg, germany g protein-coupled receptors (gpcrs) are proteins that regulate numerous signaling pathways by activation of intracellular g proteins. gpcrs are activated by extracellular stimuli, such as light, hormones and neurotransmitters. recent evidence suggests that some gpcrs exhibit voltage-sensitivity leading to a modulation of their activity by the membrane potential (vm). we used a fret-based biosensor of the α2a adrenergic receptor to analyze receptor activation at defined membrane potentials in hek 293 cells by means of voltage-clamp recording. the biosensor was stimulated either with the partial agonist clonidine or with the full agonist norepinephrine (ne) and receptor activation was measured as decrease in the ratio of acceptor-/donor-fluorescence. receptor stimulation by ne was inhibited at depolarizing membrane potentials but enhanced by hyperpolarization. inhibition of ne activated receptors was strong at low concentrations (500 nm: 60 % inhibition) but almost absent at saturating agonist concentrations (100 µm: 9 % inhibition). both agonist-induced and hyperpolarizationinduced receptor activation exhibited a similar monoexponential time course and speed of activation was primarily dependent on agonist concentration for both activation modes. the latter indicates that depolarization lowers the apparent affinity of the ne receptor interaction and thus causes receptor deactivation by means of ne release. application of clonidine (1 µm, vm=-90 mv) resulted in a fret response that was inhibited by 40 % at +60 mv. in contrast to ne, strong receptor inhibition at +60 mv was present even at super-saturating concentrations of clonidine (100 µm), suggesting that voltage alters the equilibrium between active and inactive conformations of the receptor. voltage-dependence of the a2a adrenergic receptor also modulated downstream receptor signaling: g protein activation or the recruitment of arrestins, which we determined in fret assays that directly detect gαi protein activation or receptor-arrestin interactions, were both substantially inhibited at vm = +60 mv. therefore we conclude that negative membrane potentials promote active conformations of the a2a adrenergic receptor, increase affinity of full agonists and enhance receptor signaling. in conclusion the present data show that scc cells extrude more ha, possibly related to increased levels of has3, in comparison to keratinocytes. increased amounts of ha appear to be essential for the uvb induced tumourgenesis of sccs in mice. this effect might be related to the pro-proliferative property of high molecular weight ha. furthermore biological active ha fragments derived from ha degradation by hyaluronidases (hyal1,2) are thought to be pro-angiogenetic, anti-apoptotic and proinflammatory, thus possibly also promoting tumour growth and malignancy. estradiol induced paracrine release of egf from keratinocytes protects the dermal hyaluronan/versican matrix during photoaging röck k. 1 , meusch m. hyaluronan (ha) and versican are key components of the dermis and are responsive to uvb induced remodeling. the aim of the present study was to investigate the molecular mechanisms of estrogen (e2) mediated effects on ha-rich ecm during actinic aging. 10 weeks of uvb irradiation (3 x 1 med (80 mj/cm 2 ), weekly) of hairless skh-1 mice caused a marked decline of dermal ha, which was aggravated by ovariectomy (ovx). subcutaneous substitution of estrogen (e2) by means of controlled release pellets abolished these effects confirming the stimulatory role of e2. the increase of dermal ha correlated with induction of ha synthase has3 by e2. in addition the ha-binding proteoglycan versican was induced by uvb and further increased by e2. however in cultured skin fibroblasts e2 reduced the expression of versican and had no effect on has3. therefore, direct upregulation of has3 and versican in fiborblasts by e2 was excluded. however, e2 increased the expression of egf in uvb irradiated skin in vivo and in keratinocytes in vitro. egf in turn upregulated the expression of has3 and versican in dermal fibroblasts. furthermore the supernatants of estradiol treated keratinocytes led to the same effects in dermal fibroblasts, which could be abolished by previous treatment of the supernatant with neutralizing egf antibody or treatment of the fibroblasts with egf receptor blocker erlotinib. functionally, dermal ha and versican induction by e2 correlated positively with proliferation and negatively with accumulation of inflammatory macrophages in the dermis. collectively these data suggest that e2 treatment increases the amount of dermal ha and versican via paracrine release of egf which may be implicated in the pro-proliferative and anti-inflammatory effects of e2 during photoaging. differential pro-and eukaryotic toxicity of silver released from nanocomposite surfaces increases the therapeutic window of silver in antibacterial treatments röhl c. 1 , hrkac t. silver has been used since ancient times as antimicrobial agent. recently, silver gained new attention due to its higher effectiveness in its nanoform. this led to new developments of silver nanomaterials, e.g., for medical devices and consumer products. though, it is generally assumed that silver is less toxic for eukaryotes than for prokaryotes, concern is raised, if nanosilver at the same time might also increase mammalian cytotoxicity. in our study we examined the toxicity of silver released from nanocomposite surfaces with that of silver from agno3 solutions for adherent bacteria and mammalian cells. therefore, we established an in vitro reference system which enabled us to compare the therapeutic window between prokaryotic toxicity and eukaryotic integrity in both exposure settings. we focussed especially on the comparability of the bacterial and mammalian cell systems and the development of characterized ag/tio2 nanocomposite coatings with well-defined silver filling factors and silver surface release, which could be varied over a wide concentration range. as reference cells the e. coli sar 18 strain and human dermal fibroblasts, which are of special relevance in the context of medical devices like implants or wound dressings, were chosen. bactericidal effects were determined by direct growth visualization of the gfp-producing e. coli strain by epifluorescence microscopy. mammalian cell growth and toxicity was determined by the mtt assay, protein measurements and phase contrast microscopy. the ag/tio2 samples were prepared by sputter co-deposition from two separate magnetron sources. the silver surface concentration release was determined by xps and the silver release by icp-ms. in solution a concentration-dependent constant silver concentration could be determined between 2 and at least 72 hours at the surface. while lowest bactericidal and cytotoxic concentrations of ag + from agno3 solutions with 0.64 and 0.95 mg/cm 2 , respectively, differed only slightly, the therapeutic window increased significantly if ag + was released from the nanocomposite surface. while the toxicity on the fibroblasts was unchanged the bactericidal potency increased at least one order of magnitude. taken together, it can be concluded that local exposure factors i) can be modulated by silver nanocomposites and ii) play an important role for the differential toxicity of surface silver on bacteria and mammalian cells. charite -universitätsmedizin institut für integrative neuroanatomie, funktionelle zellbiologie, philippstr. 2, 10115 berlin, germany c3 exoenzyme (c3bot) a clostridial adp-ribosyltransferase does not possess a cellbinding/-translocation domain. nevertheless, c3 is able to efficiently enter intact cells, including neuronal cells but the mechanism of uptake is not yet understood. in the present work, binding of c3bot to the hippocampus-derived ht22 cell line was characterized by means of binding and blot overlay assays as well as mass spectrometry analysis to identify binding partners of c3bot. the binding assays established that c3bot bound in a concentration-dependent manner to ht22 cells. in the overlay assay we detected one clear band of 55 kda. to elucidate whether glycosylation is important for the c3bot-protein interaction, ht22 cells were incubated with glycosidase f resulting in a decreased binding of c3 to the 55 kda band. to explore the involvement of phosphorylation in the binding of c3 to the putative binding protein, blot was pre-treated with cip (calf intestinal phosphatase) before overlay with c3bot. pretreatment greatly reduced the c3bot-protein interaction. moreover, inhibition of dephosphorylation by vanadate before in intact cells showed an increased level of c3botprotein interaction in the following overlay. thus, interactions between c3bot and ht22 cell proteins may require phosphorylation. to further characterize the 55 kda band as binding target of c3bot, the 55 kda band was digested with trypsin and then subjected to lc-orbitrap mass spectrometry analysis. from this 55 kda single gel band 141 proteins were identified. further analysis of the identified proteins will provide a possible interaction partner of c3bot. in sum, protein overlay assays revealed that phosphorylation and glycosylation are critical for efficient c3bot-protein interaction. s76 335 solvent effects on enzyme kinetics in vitro rokitta d., pfeiffer k., gerwin h., streich c., fuhr u. uniklinik köln institut für pharmakologie, gleueler str. 24, 50931 köln, germany kinetic parameters provide essential quantitative information for characterisation of drug metabolising enzymes. such enzymes are located in an a partially queous environment, but to solve potential lipophilic substrates for in vitro measurements organic solvents are regularly needed. to preserve the enzymes from denaturation and other solvent related effects, the concentration of these solvents must be kept low. data on nature and extent of such solvent effects is sparse. in this study, we investigated the effects of methanol, ethanol, acetonitrile and dimethylsulfoxide (1% to 4%) on the assessment of k m, vmax and clint with regard to the 1-hydroxylation of midazolam via cyp3a4 and the cyp1a2 catalyzed metabolism of caffeine to paraxanthine in vitro. the presence of acetonitrile showed the highest vmax value for paraxanthine formation but the lowest values for 1-hydroxymidazolam formation. the km value for midazolam showed no systematic effects of organic solvents, while for caffeine km was up to eightfold lower for solvent free samples compared to solvent containing samples. the present example suggests that the presence of organic solvents may considerably influence enzyme kinetic parameters beyond a mere change in apparent activity. these effects are differing between enzyme-substrate systems and solvents. it remains to be determined to which extent such effects compromise in vitro -in vivo extrapolations, and which solvents are most appropriate. atrial remodeling and arrhythmia induced by the transcription factor er81 rommel c. 1 , rösner s. introduction: the transcription factor er81 (ets related 81) belongs to the large family of ets-transcription factors that are involved in developmental processes and in the pathogenesis of cancer. er81 is activated by gq-and gs-coupled receptors leading to a phosphorylation of the transcription factor by map-kinases and protein kinase a, respectively. cardiac er81 mrna expression is increased in failing human hearts. however mechanical unloading by a left ventricular assist device leads to normalization of er81 expression. thus, the aim of the present study was to investigate the cardiac function of er81 in genetically modified mouse models. we previously generated transgenic mice overexpressing er81 under control of the cardiomyocyte-specific α-myosin heavy chain gene (αmhc) promoter by pronuclear injection and established independent transgenic lines. electrocardiography (ecg) was assessed in mice at day 5 after birth (p5) and in adult mice (3 months) during isoflurane anesthesia and by ecg telemetry in awake mice, respectively. ecg analysis revealed no differences between the genotypes at day 5 after birth. however, we found a decreased heart rate, a replacement of regular p-waves by an undulating baseline and frequent supraventricular extrasystoles in adult er81 αmhc transgenic mice. next, isometric contractile force measurements on isolated left atria were carried out in organ baths. while wt left atria responded to increasing concentrations of isoprenaline, nkh477 and calcium with an increase in contractility, the maximal positive inotropic responses to these substances were severely blunted in er81 αmhc atria. we performed western blots to identify potential aberrations of calcium handling and regulatory proteins. phosphorylation of serine 16 of phospholamban (pln) was reduced in er81 αmhc mice. in addition, protein phosphatase 1 (pp1) expression was significantly increased in er81 αmhc mice, which is consistent with the increased dephosphorylation of phospholamban. furthermore, we found a decreased expression of calsequestrin and serca2a protein in er81 αmhc atria. electron microscopy revealed the significant structural remodeling of er81 αmhc atria at 3 months of age. conclusion: increased cardiac expression of the ets-transcription factor er81 leads to structural and electrical remodeling of the atria. thus, er81 may play an important role in the pathogenesis of cardiac arrhythmias in chronic heart failure. signal transduction pathway of atp and utp in neonatal rat cardiac myocytes rothkirch d., gergs u., neumann j. institute for pharmacology and toxicology medical faculty, magdeburger str. 4, 06097 halle (saale), germany extracellular atp and utp can be released from the heart during pathological conditions such as ischemia or hypoxia. in humans, atp and utp levels are increased during myocardial infarction. atp and utp can act via p2-purinoceptors which are further divided in p2x1-7 and p2y1-14-receptors. as previously shown atp and utp can induce inotropic effects in cardiac preparations of mice and man. for rat articular chondrocytes and human intestinal cells it has been demonstrated that the mapk cascade can be activated by atp and utp. therefore, the cardiac effects of atp and utp on force of contraction probably occur via the mapk pathway. to investigate the signal transduction pathway involved, we studied the effects of atp and utp on mapk phosphorylation in isolated neonatal rat cardiac myocytes using phosphorylation-specific antibodies. 100 µm atp as well as utp transiently increased phosphorylation of erk 1/2 and p38 mapk with a maximum effect at 5 to 10 minutes after application of atp and utp in neonatal cardiac myocytes (n=3 preparations each). the maximum phosphorylation of p38 increased with atp to 284% ± 68% (p<0.05) at 10 minutes and with utp up to 204% ± 21% (p<0.05) at 5 minutes. the phosphorylation with erk 1/2 mapk increased with atp to 234% ± 46.5% (p<0.05) at 5 minutes and to 337% ± 27% (p<0.05) with utp at 10 minutes of basal values, respectively. after 20 minutes, predrug values of mapk phosphorylation were reached again. in summary, we noted an atp-and utp-induced phosphorylation of erk 1/2 and p38 mapk in isolated neonatal rat cardiac myocytes. the involved receptor subtype(s) and the link between mapk phosphorylation and inotropic effect of atp and utp need to be elucidated. hameel: use of elearning in teaching pharmacology and toxicology -the halle experience rulf k. 1 , gergs u. introduction: during the past three years, our faculty has started to integrate items of elearning into the standard curriculum of a classical medical school: the "hallesches medizinisches elearning -hameel". our hypothesis was that these new elearning tools would improve the willingness of students to spend more time into learning and this would lead to an improved outcome (in multiple choice tests). methods: hence, we offered medical students (5 th or 10 th semester) additional learning environments. the courses for students (experimental pharmacology and toxicology or clinical pharmacology) were existed of a weekly lecture and in addition tutorials (problem-based-learning style, paper cases) or classical seminars. furthermore, we offered the possibility to use an online multiple choice quiz (involving 8 -12 previously used tests) and/or an online module on heart failure each week. we used the learning management system ilias software in combination with the content management system stud.ip. all students were subjected to an introductory test (to assess knowledge prior to our teaching section and allowing us to exclude a conceivable bias due to previous knowledge, involving basic items from prior teaching opportunities), a mid-term test and a final test to assess gain of knowledge. a maximum of 60 points could be obtained as a sum of both tests. results: in the means 40% of students used the new elearning tools (quizzes, heart failure module). however, there was no association between the use of self-assessment quizzes and examination results. the usage of the online quizzes increased in the periods before the exams. however, usage of the heart failure module was accompanied by significantly increased scores in exams. moreover, in a formalized evaluation system, students positively commented on our elearning efforts. conclusions: while usage of our quizzes did not improve test marks, another more sophisticated clinically oriented elearning module seemed to be improving test outcomes marginally. targeting of erk thr188 phosphorylation attenuates cardiac hypertrophy but preserves the anti-apoptotic effects of erk1/2 ruppert c., vidal m., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. 9, 97078 würzburg, germany background and aims: the extracellular regulated kinases 1 and 2 (erk1/2) play an important role in cardiac hypertrophy and cell survival. erk1/2 are phosphorylated at the so-called tey motif, which in turn activates erk1/2. hypertrophic stimuli lead to an additional autophosphorylation threonine188 (thr188). this autophosphorylation of erk1/2 stimulates activation of nuclear erk targets, which are known to induce hypertrophy. the aim of this study is to investigate whether specific targeting of erk thr188 phosphorylation affects both erk functions -erk mediated hypertrophy and cardioprotective cell survival. methods and results: for the analysis of cardiomyocyte hypertrophy in vitro, we stimulated cardiomyocytes with phenylephrine and measured the incorporation of tritiated isoleucine. cardiac hypertrophy was assessed by echocardiography before and after transverse aortic constriction (tac). for analysis of cell survival, caspase activity and dna fragmentation was determined upon hydrogen peroxide stimulation in vitro and in response to tac in vivo. to differentiate between inhibition of erk1/2 activity and prevention of erk thr188 phosphorylation, we either inhibited erk activity with pd98059 or overexpressed a mutant of erk2, which cannot be phosphorylated at thr188 phosphorylation. while inhibition of overall erk activity with pd98059 attenuated cell survival and hypertrophy in vitro, specific targeting of erk thr188 phosphorylation by overexpression of the phosphorylation deficient mutant (erk2 t188a ) attentuated phenylephrine induced hypertrophy, but preserved the anti-apoptotic effects of erk. cardiac overexpression of erk2 t188a significantly reduced tac-induced hypertrophy compared to wild-type erk2 overexpressing mice. in line with the in vitro experiments, erk thr188 inhibition only prevented hypertrophy in the tac model without promoting apoptosis. conclusions: these results show that blockade of erk thr188 phosphorylation attenuates cardiomyocyte hypertrophy but preserves anti-apoptotic effects of erk1/2. therefore, specific targeting of erk thr188 phosphorylation might be a promising strategy for the treatment of pathological hypertrophy. intracellular camp levels are determined by interplay of camp formation by adenylyl cyclases and camp degradation by phosphodiesterases (pde). eleven families of pdes are known. one of the most recently identified pdes is pde10, a pde in principle capable of hydrolysing camp as well as cgmp. pde10 contains a tandem of so called gaf domains in its n-terminal regulatory domain that mediate activation by camp. because current knowledge about the tissue distribution of pde10 was mostly based on the analysis of mrna distribution, we generated antisera against pde10 to analyze tissue distribution of the protein level. using these antibodies, we found a prominent occurrence of the enzyme in testis and in brain, where it was confined to the striatum. thus, pde10 displays a comparably restricted tissue distribution which is in contrast to that of many other pdes. low camp levels in so called medium spiny neurons of the striatum have been implicated in schizophrenia. furthermore, studies using the nonspecific pde10 inhibitor papaverine as well as specific pde10 inhibitors suggest pde10 as a target for the treatment of schizophrenia. here we set out to analyze the contribution of pde10 to camp degradation in striatum, to identify the physiological pathways pde10 is involved in and to clarify the functional impact of the proposed phosphorylation of the enzyme. identification of cgmp-dependent kinase i substrate complexes salb k., schlossmann j. universität regensburg lehrstuhl pharmakologie, universitätsstrasse 31, 93053 regensburg, germany the cgmp-dependent kinases (cgks) are components of the no/cgmp/cgk-signalling pathway and have a great physiological importance in a multitude of tissues and organs such as smooth muscles and platelets. two isoforms of the cgki and the cgkii are known. cgkiα and cgkiβ differ only in their first ~ 100 amino acids which constitute the leucine zipper and the autoinhibitory domains. the n-terminal leucine zipper domains mediate homodimerization of the kinase and the interaction with diverse substrate proteins. since cgkiα and cgkiβ express different n-termini they interact with different substrates. the cgkiβ isoform is assembled in a macrocomplex at the endoplasmic reticulum (er) with the intracellular calcium release channel inositoltrisphosphate receptor i (insp 3r-i) and the inositol-trisphosphate receptor associated cgmp kinase substrate (irag). we investigated, whether irag also interacts with the insp3r-ii and the insp3r-iii in murine platelets and tissues. additionally, we analyzed the interaction between the 52 amino acid peptide phospholamban (plb), which is also located at the er and regulates the er calcium reuptake by the sarco/endoplasmic reticulum ca 2+ -atpase (serca), and the two cgki isoforms. we performed cgmp-agarose experiments with murine wt and irag-ko platelets to examine the irag-insp3r interactions. the insp3r-ii isoform was neither bound to cgmp-agarose nor detected in the anti-irag immunoprecipitate. on the other hand, insp3r-iii from wt but not from irag-ko platelets was bound to cgmp-agarose. hence, insp3r-iii interacts directly with irag but not with cgkiβ in murine platelets. however, in colon smooth muscle lysate, insp3r-iii not only interacted with the irag protein but was also detected in the anti-cgkiα-immunoprecipitate. phospholamban from wt and irag-ko platelets was also bound to cgmp-agarose. subsequent immunoprecipitation experiments with the respective antibodies against the two cgki isoforms revealed that plb interacted both with cgkiβ and cgkiα. these results were supported by analysis of colon smooth muscle tissue from wt and irag-ko mice. in conclusion, irag interacts with insp3r-i and insp3r-iii but not with insp3r-ii in murine platelets and colon smooth muscle tissue. moreover, phospholamban is an interacting partner of both the cgkiα and the cgkiβ isoform. the human immunodeficiency virus type 1 enhancer binding protein 1 (hivep1) is regulated by proinflammatory stimuli and statins salomon a. 1, 2 , schmitz b. objective: hivep1 binds nf-ĸb and other proinflammatory consensus sequences, and is suggested to be involved in inflammatory processes. we recently identified two tagging snps, one positioned 90 kb upstream (rs169713) and another in exon 4 (rs2228220) of the hivep1 gene, to be replicatively associated with venous thrombosis in gwas and follow-up studies (ajhg, 2010; plos one, 2011) . methods: total rna isolation was performed after treatment of vascular endothelial cells (ea.hy926) with proinflammatory cytokines or statins (24h). serial hivep1 promoter deletion constructs were cloned into the pgl3-basic vector, a potential enhancer fragment, harbouring rs169713c/t, into the pgl3-promoter vector. in ea.hy926 cells and thp1 monocytes, reporter gene assays were performed by transient transfection and overexpression of transcription factors. chip and bandshift assays were performed to identify candidate transcription factors. results: in ea.hy926 cells, endogenous hivep1 expression was increased by proinflammatory cytokines tnfα and il-1β. simvastatin (1.2 and 2.4 µm) and atorvastatin (9 µm) -but not pravastatin or aspirin -both dose-dependently decreased basal and tnfα-stimulated hivep1 expression. the construct harbouring rs169713t exerted significantly higher transcriptional activity (ta) compared to rs169713c (p<0.001). for an intronic modulator, reporter gene assays demonstrated a regulatory effect on hivep1 expression in ea.hy926 and thp1 cells. cotransfection of sp1 and egr1 led to an increase in ta, while wt1 exclusively upregulated ta of constructs comprising the intronic modulator. chip and bandshift assays combined with specific antibody detection revealed binding of sp1 to the 5'-flanking region and the intronic modulator of hivep1. conclusion: increased hivep1 expression during inflammatory conditions can be repressed by simvastatin and atorvastatin, and not by pravastatin or aspirin. basal hivep1 expression is regulated by sp1 combined in a transcription factor module with egr1 and wt1 under basal and/or inflammatory conditions. the rs169713 site harbours potential activational capacity for hivep1 gene transcription and may communicate with the sp1/egr1/wt1 module. to date, the treatment of various movement disorders of the central nervous system is still insufficient. in most cases this is due to the sparse knowledge of the pathophysiology. l-dopa-induced dyskinesias (lid) represent a severe complication of long-time pharmacotherapy in parkinson's disease that deserves novel therapeutics. an increased activity of striatal projection neurons, which express kv7.2/3 channels, seems to be involved in the pathophysiology of these spontaneous involuntary dystonic and choreatic movements. previous studies demonstrated an antidyskinetic effect of the kv7.2-7.5 channel opener retigabine after acute and chronic treatment in a rat model of lid. in order to clarify if this effect was based on the modulation of kv7.2/3 channels, we examined the acute effects of the preferred kv7.2/3 channel opener ica 27243 on lid in this animal model. four weeks post 6-ohda lesioning of the left forebrain bundle, dyskinesia was induced by chronic treatment with 10 mg/kg l-dopa and 15 mg/kg benserazide for 20 days. three subtypes of dyskinesia (limb, axial and orolingual) were rated according to a score system from 0 to 4 over 180 min. for drug testing, ica 27243 (5, 10 and 15 mg/kg) was administered intraperitoneal additionally to l-dopa (or vehicle). effects of drug action in comparison to vehicle controls were detected by adding up the severity scores of each observation time. additionally, effects on parkinsonian symptoms were examined 20 min after drug administration using the block and the stepping test. ica 24273 reduced the severity of dyskinesia significantly at all doses while no negative impact on the antiparkinsonian effect of l-dopa was observed. whereas the antidyskinetic effect was restricted to the first 20 min after the application of 5 mg, it lasted up to 110 min in rats treated with 10 mg ica 27243. a higher dose of 15 mg did not further enhance the antidyskinetic effect. the results of our study suggest that the antidyskinetic effect of the k v7 channel opener retigabine was based on its action on striatal kv7.2/3 channels. in line with the results of previous studies with retigabine, this action does not seem to interfere with the antiparkinsonian effect of l-dopa. this study was supported by the micheal j. fox foundation. background: skeletal muscle toxicity is the major side effect of hmg-coa-reductase inhibitors (statins) and can be simulated in engineered skeletal muscle. statins are known to exert "pleiotropic" effects, e.g. reducing endothelial dysfunction by inducing no synthases and no production. the role of no synthases in skeletal muscle under statin treatment is largely unknown. interestingly, some skeletal muscle pathologies (e.g. duchenne muscular dystrophy) may be exacerbated by increased inos activity. here we tested whether or not statin-induced skeletal muscle toxicity would be associated with enhanced no synthesis. we generated engineered skeletal muscle (esm) from rat skeletal muscle cells, matrigel and collagen. esms displayed typical skeletal muscle properties (differentiated muscle fibres, tetanic contractions). under baseline conditions esm expressed enos most abundantly, followed by inos and nnos (n=4-5). myotoxic cerivastatin (0.01, 0.1, 1 µm for 5 days) caused a concentration-dependent decrease of contractile force (p<0,05, n=17-20) paralleled by an increase in inos transcript (mean±sem: 0.01 µm 4±0.9-fold, n=3 p<0.05; 0.1 µm 9.3±2.8-fold, n=3 p<0.05) and protein (0.01 µm 5.1±2-fold, n=4 p<0.05; 0.1 µm 7.8±0.8-fold, n=3 p<0.05). mevalonic acid fully prevented the inos increase suggesting that the induction is hmg-coa reductase-dependent. to test whether inos may contribute to the decrease in contractile force we co-treated esm with 1400w, a specific inos inhibitor. we applied 5 µm of 1400w, a concentration found to potently reduce lipopolysaccharide (lps)induced no-production in cultured myotubes. however, we did not observe a rescue effect (n=9-15). also, l-name (10 mm), an unspecific nos inhibitor, did not improve contractile function, instead we observed increased myotoxictiy (n=6-13, p<0.05). to further investigate the role of no for muscle function we treated the esms with increasing concentrations of the no-donor snp. only high concentrations of snp (10 µm) caused a reduction of contractile force. combined treatment with cerivastatin and 0.1 µm snp showed a tendency towards improved force development in esm. conclusions: statins increase inos activity in our skeletal muscle model (esm). however, this does not seem to functionally contribute to myopathy in esm. increased production of no may in fact be a protective measure. esm may help to dissect clinically relevant functional changes in statin myotoxicity. characterization of primary skin fibroblasts of patients with 3m syndrome and mutations in the cul7 gene meyer k., hieber m., engelhardt s., sarikas a. technische universität münchen institut für pharmakologie und toxikologie, biedersteiner str. 29, 80802 münchen, germany 3m syndrome is an autosomal-recessive disorder characterized by pre-and postnatal growth retardation (< -4 sd), facial dysmorphism and skeletal anomalies. the majority of patients harbor missense mutations of the cul7 (76%) or obsl1 (16%) gene, respectively. cul7 constitutes an e3 ubiquitin ligase that is involved in the regulation of the insulin-like growth factor 1 (igf-1) signaling pathway via ubiquitin mediated degradation of insulin receptor substrate 1 (irs-1). to investigate the role of cul7 mediated irs-1 degradation in the pathogenesis of 3m syndrome. primary skin fibroblasts of seven 3m syndrome patients (six with cul7 mutations, one with a obsl1 mutation) and control fibroblasts were analyzed for proliferation rate (cell counter), cell cycle profile (facs), cell morphology and cellular senescence (histochemistry), irs-1 protein concentrations and activation of the igf-1 signaling pathway (western blot). the proliferation rate of 3m patient fibroblasts was significantly increased when compared to control cells. in contrast, irs-1 protein levels and activation of the pi3k/akt and erk mapk pathway were only increased in a subset of 3m cells that carried cul7 mutations, but not in cells from a patient with the obsl1 mutation. no significant differences in cell cycle profile, cell morphology or cellular senescence were observed in 3m patient fibroblasts when compared to control cells. to determine the pathogenetic contribution of increased irs-1 levels to the observed phenotype, human imr90 fibroblasts were stably transfected with retroviral vectors encoding irs-1. despite 20-fold overexpression of irs-1 compared to empty vector controls, no significant effect of igf-1 stimulation on proliferation rate or pi3k/akt and erk mapk signaling was observed. skin fibroblasts of 3m patients with cul7 mutations displayed an increased proliferation rate and enhanced activation of the igf-1 signaling pathways. despite accumulation of irs-1 in fibroblasts from a subset of 3m patients with cul7 mutations, no pathomechanistic role for irs-1 could be demonstrated. collectively, our data indicate that a dysregulated igf-1 signaling may contribute to the pathogenesis of 3m syndrome, yet in an irs-1 independent manner. pharmacases.de -a student-centered elearning project of clinical pharmacology zollner b., berg c., gros n., muß n., oestreicher d., engelhardt s., sarikas a. technische universität münchen institut für pharmakologie und toxikologie, biedersteiner str. 29, 80802 münchen, germany pharmacases.de is a novel e-learning website of clinical pharmacology that presents clinically relevant aspects of pharmacology and toxicology in an interactive and multimedial manner. the aim of the project pharmacases.de was to develop an innovative concept for creating high quality elearning content that i) integrates and promotes the theoretical and cooperative skills of final year medical students and ii) is easily adoptable by cooperating institutes and hospitals. a peer-teaching concept was developed in which final year medical students with the elective pharmacology (pj wahlfach pharmakologie) independently researched and wrote elearning lessions ("pharmacases"). subject-specific expertise was acquired by consulting elective students of other disciplines. at present (11/2011) , this "peer network" consists of elective students of nine cooperating institutions (pathology, microbiology, radiology, cardiology, psychiatry, dermatology, neurology, ophthalmology, pediatrics) at the technische universität münchen. the average time for the generation of one elearning lession by the peer network was 10 days. to date, the website consists of 49 pharmacases that are available to all students online (http://www.pharmacases.de). the website also contains a discussion forum and evaluation form for direct feedback. on average, pharmacases.de has 1000 visitors per month with the following evaluation results: "excellent": 76%, "good": 15% and "satisfactory": 9% (n=33). the didactic concept of pharmacases.de enabled the efficient generation of high quality elearning content in a student-centered and interdisciplinary manner. the peer-teaching approach supports the collaborative skills of final year medical students and facilitates the transfer of theoretical pharmacological knowledge into clinical practice. improved glucose tolerance, less chronic adipose tissue inflammation and reduced adipose tissue mass in mice with adipocyte-specific loss of tak1 sassmann a., offermanns s., wettschureck n. max-planck-institut für herz-und lungenforschung pharmakologie, ludwigstr. 43, 61231 bad nauheim, germany tgf-β activated kinase 1 (tak1) is known to be involved in numerous inflammatory processes by linking receptors for inflammatory stimuli like lps, interleukin-1 or tnfa to ikk, p38 and jnk activation. chronic inflammation of white adipose tissue is one of the major causes for the development of insulin resistance and impaired glucose tolerance in states of obesity. to investigate the role of tak1 in white adipose tissue, we crossed the tamoxifen-inducible white adipocyte-specific cre mouse line adipoqcreer t2 with animals carrying floxed alleles of the tak1 gene. adipoqcreer ; tak1 fl/fl animals and cre negative control littermates are viable and fertile and do not show any developmental defects. after tamoxifen induction and high fat diet feeding adipocytespecific tak1 knockout mice show improved glucose tolerance and lower fasting insulin levels compared to control animals. in line with this, serum levels of the adipose tissuespecific hormone resistin are reduced in adipocyte-specific tak1 knockout mice. these findings are accompanied by a lower state of chronic inflammation of adipose tissue as indicated by a dramatic reduction of adipose tissue macrophage number and lower serum levels of tnfα and interleukin-6. stimuli like tnfα, interleukins and tgf-β released from macrophages and adipocytes are known to promote obesity-related adipose tissue inflammation. when stimulated with these substances tak1 deficient adipocytes show reduced activation of jnk and p38 which both play an important role in the development of insulin resistance. interestingly, we observe a lean phenotype in adipocyte-specific tak1 knockout mice when fed a high fat diet which reflects a reduction of white adipose tissue mass. currently we are investigating the molecular mechanisms underlying the reduced adiposity and lower state of chronic inflammation in adipose tissue. growth of small cell lung cancer (sclc) cells is regulated via the autocrine stimulation of g protein coupled receptors (gpcrs), i. e., neuropeptide and muscarinic acetyl choline (ach) receptors. the activation of gq/11 and calcium-dependent gpcr pathways results in the stimulation of erk signaling which is necessary for the mitogenic effects of neuropeptides or ach on sclc cells. in contrast, the role of calcium-independent gpcr signaling and its interplay with gq/11-regulated pathways in sclc cells are less well defined. the aim of our studies was to characterize the molecular make-up and the interaction of these pathways, and to delineate the phenotypic effects of calciumdependent and -independent signaling cascades in sclc cells. using a panel of sclc cell lines, we found that the stimulation of neuropeptide receptors led to an increase of calcium which was independent of extracellular calcium and could be prevented by depleting internal calcium stores. this calcium increase was sufficient to activate the tyrosine kinase pyk2 and subsequently the erk1/2 cascade. the role of pyk2 for the growth of sclc cells was further supported by the fact that inhibition of pyk2 using a sirna approach or a novel specific inhibitor, pf431396, exerted pronounced cytotoxic effects on sclc cells, whereas non-sclc cells were less sensitive. interestingly, the inhibition of g 12/13 signaling by sirna-mediated g(alpha)12 or g(alpha)13 knockdown also markedly reduced the growth of sclc in vitro or in subcutaneous tumor xenografts, and increased the sensitivity of sclc cells towards certain cytostatics. to further define the role of calcium-dependent signaling via pyk2 versus the role of calcium-independent signaling via g12/13, we tested the effect of pyk2 inhibition in cells with impaired g12/13 signaling. notably, pyk2 and g12/13 double inhibition led to an even increased proliferation. thus, we propose that dysbalanced g protein signaling favoring either pyk2 activation or g12/13-dependent cascades inhibits the growth of sclc cells, whereas the parallel inhibition of both pathways restores again the balance and the growth capacity in this tumor entity. dendritic cells (dcs) are essential for the initial immune response and for the defence against inhalated pulmonary toxins and carcinogens in lung. to differentiate dcs, the cell line thp-1 were used for 7 days and stimulated with various cytokines (il-4, gm-csf, tnf-a, ionomycin). the dcs were characterized by flow cytometry with different typical dendritic cell markers (for example cd11c, cd209, cd83) and by immunfluorescence compared to monocytes. the bronchial tract contains up to 800 dcs per mm² and therefore we established a triple culture model to mimic the situation in vivo. the triple culture consists out of primary human epithelial cells from small bronchi (hbec) and lung fibroblasts which are cultured under air-liquid conditions on filter membranes for 4 weeks and dcs which were added after the differentiation phase of the bronchial cells. during the cultivation time the hbec formed an epithelial layer expressing both tight and adherens junctions. they also produced mucus, formed functional cilia with a beat frequency of between 16 to 20 hz and the transepithelial resistance values were stable between 600 to 800 ω·cm². pathomechanisms of pulmonary toxicity in vivo are difficult to investigate, so the tripleculture model is the basis for investigations of the toxic effects at cellular level. lungtoxic substances such as organophosphates are usually absorbed through inhalation. organophosphates are dangerous nerve agents for the human organism. at high concentrations organophosphates damage in the coculture without dcs the cell-cell contacts of the epithelial layer. in the triple culture dcs firstly respond to inhaled organophosphates and seem to compensate effects on the other cells. in summary, it is very important to understand the pathogenic mechanisms of lung injury in relation to the role of dendritic cells in lung. they could play an essential role in therapy against damage of organophosphates in the lung. co-purification of arf gtpase-activating protein git1 and cavb3 schalkowsky p., wissenbach u., fecher-trost c., flockerzi v. universität des saarlandes institut für experimentelle und klinische pharmakologie und toxikologie, kirrbergerstraße, 66421 homburg, germany high-voltage activated ca channels are assembled from pore-forming α1 subunits and two distinct types of auxiliary subunits, cavβ1-β4 and, maybe, α2δ1-δ4. by a cavβ3-specific antibody based affinity chromatography the cavβ3 protein was highly enriched from rat brain microsomal membranes. proteins associated with cavβ3 were identified by mass spectrometry (lc-esi-ms/ms) and include α1-subunits, α2δ-subunits and β-subunits. in addition to these expected interacting proteins additional proteins were co-purified with the cavβ3 protein, including the g protein-coupled receptor kinase-interactor 1 (git1). the 770aa git1 is a ubiquitously expressed multidomain protein which may serve as a scaffold to bring together molecules to form signaling modules controlling, for example, vesicle trafficking, cytoskeletal organization and cell migration. in rat brain lysates the git1 and cavβ3 proteins were co-immunoprecipitated by the antibodies for cavβ3 and git, respectively. we cloned the git1 cdna from mouse brain and co-expressed it with the cavβ3 subunit in hek cells. like in brain lysates the git protein was retained by cavβ3 precipitated by the antibody for cavβ3 and cavβ3 was retained by the git1protein precipitated by the antibody for git1. both proteins, cavβ3 and git1 are endogenously co-expressed in mouse embryonic fibroblasts (mef). we could not observe potassium-induced voltage-activated ca influx in these acutely prepared cells. accordingly, mefs can be used as a model system to study the impact of cavβ3-git1 interaction in the absence of functional cav channels. in addition, using mefs from cavβ3-deficient mice enables us to control the impact of cavβ3 on git1 function. vice versa down-regulation of git1 by specific sirnas might allow to control the impact of git1 on cavβ3 function. as read-outs we use cell migration assays and monitor receptor-dependent and receptor-independent calcium signaling in these cells. effects of sphingosine-1-phosphate and fty720 on epidermal hyperproliferation and inflammation in an imiquimod induced mouse model of psoriasis the sphingolipid sphingosine 1-phosphate (s1p) is a mediator that modulates various physiological functions of skin cells. s1p has distinct direct effects on keratinocytes as it diminishes proliferation and induces differentiation which is a classical goal of psoriasis therapy. furthermore, s1p modulates the function of various immune cells, mainly to an anti-inflammatory direction. thus, the strategy of targeting immune cells with locally acting s1p was explored in an experimental animal model of psoriasis vulgaris, the recently established imiquimod induced psoriasis mouse model and in the mouse tail test. topical administration of imiquimod onto back and ear skin led to a distinct inflammatory response characterized by epidermal hyperproliferation, scaling and redness which was scored with a modified pasi (psoriasis area and severity index). the positive control diflorasone diacetate and s1p, but not fty720 reduced the epidermal hyperproliferation by topical administration onto ear skin, indicating a mode of action for s1p via the s1p2 receptor, which is not activated by fty720. there was also a moderate reduction of inflammatory cell influx and edema formation in ear skin by s1p treatment, which was even more pronounced by treatment with diflorasone diacetate. the pasi determined on back skin was, however, only significantly reduced by diflorasone diacetate. the discrepancy between outcome on ear and back skin remains elusive. in the mouse tail assay, the influence of s1p in stratum granulosum formation (orthokeratosis) was tested compared to the positive control calcipotriol. whereas topical administration of calcipotriol led to the expected significant increase of stratum granulosum in mouse tail epidermis, s1p lacked such an effect, indicating a different mode of action in epidermal differentiation. taken together, these results imply that topical administration of s1p might be a new option for the treatment of mild to moderate psoriasis lesions. inhalation of toxicants such as sulphur mustard (sm), an alkylating chemical warfare agent, cause pulmonary complications like respiratory failure, pulmonary edema and secondary pneumonia. in order to investigate pathomechanisms of pulmonary toxicity, an in vitro alveolar-capillary co-culture model has been established recently by our group. in this model the human lung adenocarcinoma epithelial cell line (h441) is mimicking the epithelial site of the alveoli while the human hemangiosarcoma cell line (iso-has) represents the endothelial site. acute respiratory injuries are accompanied by disruption of the alveolar-capillary barrier that can be detected by the use of biochemical markers (e.g. ldh) and electrochemical indicators (e.g. transepithelial resistance). sm-mediated pulmonary injury is characterized by the increased secretion of proinflammatory mediators (e.g. il-6). a shortcoming of this model is the missing inflammatory component in the lung. aim of the present project is the addition of macrophages to the established co-culture model to improve the model and to investigate the relevance of inflammatory processes in toxic lung injury. the effect of sm on this triple-culture model is characterized with special regard to the interaction of epithelial cells and macrophages. the human acute monocytic leukemia cell line (thp-1) was stimulated to allow differentiation into macrophages. validation of the cellular differentiation was checked by specific clusters of differentiation (e.g. cd206) using flow cytometric analysis. after successful differentiation into macrophages, these inflammatory cells were added to the co-culture model before and after exposure with sm, respectively. the cytotoxicity of sm on the triple-culture model was evaluated by xtt assays and ter measurements. furthermore, immunohistochemical staining of tight junction proteins (e.g. zo-1) and of adherens junction proteins (e.g. e-cadherin) was conducted to enhance the knowledge of the function of the intercellular junction in injured and rejuvenated regions as well as the interaction of epithelial cells and macrophages. for the contact allergen para-phenylenediamine (ppd) we showed that concentrations above 50 µm are accompanied with inhibition of nat1 activity in human keratinocytes [1] . in the following we investigated the impact of ppd on nat1 activity in antigenpresenting cells using dendritic cell-like cells, namely the monocytic thp-1 cells. measured nat1 activity of thp-1 was comparable to those found in primary keratinocytes. a 24h treatment of thp-1 cells with physiologically relevant concentrations of ppd (10-200 µm) led to a 47% reduction of nat1 activity. comparable results were found for mono-acetylated ppd (mappd) whereas di-acetylated ppd demonstrated no inhibition. time-dependent studies found a significant decrease in enzyme activity already 8h after application of ppd or mappd while nat1 mrna levels were not modified. these results are indicative for a substrate-dependent inhibition. further investigations concentrated on the restoration of nat1 activity after treatment with ppd or mappd. here we found that n-acetylation capacities were restored after 24h cultivation of the treated cells in fresh medium. independent of the enzymatic activity, certain compounds are known to oxidise the catalytic cysteine or form adducts with the nat1 protein. therefore we studied whether ppd and/or oxidised ppd including the trimer bandrowski´s base interact additionally with recombinant nat1 protein itself in the absence of acetyl-coenzyme a. we found that all compounds but mappd bind to nat1 protein after 2h. the greatest inhibition was found for oxidised ppd (up to 50%). due to the greater inhibition by oxidized ppd we propose that oxidation products interact with the protein whereas ppd itself modulates nat1 enzyme activity in a substrate-dependent mode of action. overall we demonstrated that ppd can inhibit nat1 in two different ways. the work was partially financed by federal office of public health (foph), switzerland and stiftung zur förderung begabter studierender und des wissenschaftlichen nachwuchses objective: fabry's disease is a rare progressive multisystem disorder resulting from deficiency of the lysosomal enzyme alpha-galactosidase a (gla, ec 3.2.1.22). we hypothesize that genetic gla variants, especially those in its promoter region are of pathophysiological relevance for the development and progression of fabry's disease phenotypes. this study focuses on the characterization of the gla promoter, identification of functional genetic variants and impact of transcription factor eb (tfeb), a regulator of lysosomal genes. we screened 4011 bp of the 5'-flanking region of gla in 60 patients with fabry's disease and 60 controls for genetic variants. serial promoter deletion constructs for reporter gene assays were designed and identified genetic variants were introduced by site-directed mutagenesis. constructs were transiently transfected into immortalized human kidney epithelial (ihke) cells and human vascular endothelial cells (ea.hy926) to determine transcriptional promoter activity (ta). sequencing of patients' dna revealed five genetic variants in the 5'flanking region of gla, significantly more frequent in fabry's patients compared to control group (rs2071225; rs3027580; rs3027579; rs59647857; rs3027575; all minor alleles p<0.0027). we identified two regions, a proximal one between -110 and -425 and a distal region between 1106 and -1421 with significant ta, in both cell lines. cotransfection with tfeb activated ta of both regions significantly up to 5.3-fold (p<0.001). in ihke cells, insertion of the minor t allele (rs2071225) significantly enhanced basal ta of the proximal promoter region (p=0.0006), while insertion decreased basal ta (p<0.0001) of the distal promoter portion. the combined insertion of the minor c alleles (rs3027580; rs3027579), which were in complete linkage disequilibrium, significantly increased basal ta of the distal promoter region (p=0.0037). our results indicate that three genetic variants, overrepresented in fabry's patients, are located within transcriptionally active regions, possibly altering tf binding sites and therefore, affecting gla expression. future analysis will assess the impact of gla promoter variants and gla regulation by tfeb with respect to fabry's phenotypes. multiple sclerosis (ms) and its animal counterpart experimental autoimmune encephalomyelitis (eae) have a major inflammatory component that drives and orchestrates both diseases. ceramides (cer) are known as mediators of inflammatory processes, but until now their role in ms was not elucidated. we measured the ceramide levels in the cerebrospinal fluid of ms patients and control patients using lc-ms/ms. interestingly, the c16:0-cer levels were 1.9 fold increased in ms patients. this translates into the finding that c16:0-cer levels were also significantly elevated in the lumbar spinal cord of eae mice. the raised c16:0-cer levels in the lumbar spinal cord were caused by a transiently increased expression of ceramide synthase (cers) 6 in macrophages. nitric oxide (no) and tumor necrosis factor alpha (tnf-α) secreted by interferon gamma (infγ ) induced macrophages play an essential role in the development of ms. astonishingly, rnai experiments reveal that cers6 and its product c16:0-cer are mediators of inf-γ induced no/tnf-α release in raw macrophages. moreover, treatment of eae mice with l-cycloserine prevented the increase of c16:0-cer and of inos/tnf-α expression and caused a remission of the disease. in summary, cers6 plays a critical role in the initial phase of ms, most likely by regulating the no and tnf-α synthesis. this let us speculate, that a substance designed to inhibit cers6 and therefore to limit the inflammatory effects of c16:0-cer may represent a new drug in ms therapy. role of cgmp-dependent protein kinase i for kidney fibrosis schinner e. 1 cgmp is synthesized via nitric oxide-or natriuretic peptide-stimulated guanylyl cyclases and exhibits pleiotropic regulatory functions also in the kidney. hence, the integration of cgmp signaling via cgmp-dependent protein kinases (cgk) might play a critical role for renal physiology. both isozymes were detected in arterioles, mesangium and within the cortical interstitium. in contrast to cgkiα, the β isoform was not detected in the juxtaglomerular apparatus and medullary fibroblasts. here, we focused on the function of cgki in the renal interstitium emphasizing a functional differentiation of both isoforms. the interstitium exists mainly of fibroblasts playing a prominent role in the interstitial fibrosis. accordingly, cgki could also be involved in this pathophysiological process. therefore, we studied whether cgki influences renal fibrosis which was induced by unilateral ureter obstruction (uuo). at first we analysed the role of the no/cgmp signaling by application of cgmp increasing yc1 or isdn. thereby we detected antifibrotic effects of these substances. subsequently we tested whether these effects are mediated by cgki by using mutant mice. on the one hand we examined αsm-rescue mice (expressing cgkiα only in smooth muscle under the control of the sm22 promotor with a cgki-ko background) and cgki-ko mice (expressing no cgki). on the other hand we used tgtg mice expressing more cgkiα in smooth muscle than wt mice (transgenic cgkiα under the control of the sm22 promotor). due to the steeply increased use of nanomaterials for commercial and industrial applications, toxicological assessment of their potential harmful effects is urgently needed. moreover, the continuous development of novel materials requires the implementation of hazard-predicting models to prevent potential health effects resulting from human exposure. in the present study, we studied the toxic potential of a set of nanoparticles (np) with varying physicochemical properties in human a549 lung epithelial cells, hepg2 liver epithelial cells and hk-2 proximal tubule epithelial cells. the used nanomaterials incorporated five tio 2 samples, two zno samples (i.e. uncoated and coated), two multi-walled carbon nanotube (mw-cnt) samples and a nanoparticulate ag sample. cells were treated with np at doses ranging from 0.3 to 80 µg/cm 2 for cytotoxicity and from 06 to 40 µg/cm 2 for genotoxicity. dna damage was evaluated using the alkaline comet assay while concurrent cytotoxicity was determined by the wst-1 assay. marked contrasts in cytotoxic and dna damaging properties were observed among the different materials. the overall strongest responses were observed with the uncoated zno-np sample and with ag-np, although effects were found to depend on the cell type. notably, the dna damaging effect of ag-np could at least partly be attributed to its dispersant. present results form part of a growing data set which are generated in the framework of the eu fp7 project enpra (fp7-nmp) to establish dose-response relationships and a mathematical model to predict the hazard of nanoparticles. increased spontaneous hprt mutant frequency in v79 cells expressing human cytochrome p450 1b1 schlechtweg a., esch h. , martínez jaramillo d., lehmann l. university of wuerzburg/institute of pharmacy and food chemistry section of food chemistry, am hubland, 97074 wuerzburg, germany the hypoxanthine-guanine phosphoribosyltransferase (hprt) assay in chinese hamster v79 lung fibroblasts (v79 cells) represents a widely-used mammalian test system to detect gene mutations. since v79 cells do not express any cytochrome-p450dependent monooxygenase (cyp) isozymes, usually an activating system has to be added. therefore, v79 cells expressing human (h) cyp isozymes have been commercialized. to test these v79 cells for their use in the hprt test, v79 h1a1 and h1b1 cells were characterized regarding (i) spontaneous frequency of 6-thioguanineresistant clones per 10 6 clonable cells (smf), (ii) the stability of which over 4 weeks (w), and (iii) the mutational spectrum (ms) of cdna from mutant clones. ms of cdna was determined by isolation of total rna, reverse transcription/amplification of the coding region by polymerase chain reaction and sanger sequencing of the amplification product. activity of cyp isozymes was verified by ethoxyresorufin-o-deethylase (erod) assay. (i)/(ii) whereas the smf of v79 cells (w2:13±4; w4:2±1) and v79 h1a1 (w2:17±4; w4:3±0) only varied within the range of historical controls, smf of v79 h1b1 increased continuously over time (w2: 36±6; w4: 68±13). (iii) although the smf of v79 and v79 h1a1 were similar, the mutational spectrum of v79 cells was characterized by as many transversions as transitions and deletions of exon 4 or exon 4+5, whereas the mutational spectrum of v79 h1a1 was characterized exclusively by transversions and deletion of exon 7+8. surprisingly, with 57 out of 59 cdnas derived from v79 h1b1 mutant clones, no amplification product was detected. first results indicate that there is at least one gene mutation in the untranslated region before and behind the coding region precluding amplification with the original primers. (ii)to reduce the smf of v79 h1b1, cells with wildtype hprt activity were cloned and one clone with an erod activity which did not differ significantly from the original cell population was further characterized. initially, smf of the clone varied between 0.7±0.6 and 1.2±0.0. yet its smf was unstable reaching up to 104±6. in conclusion, the mutational spectrum differed between the v79 cell lines. furthermore, h1b1 expression seemed to enhance smf in v79 cells. even though a temporary reduction of the smf by cloning was possible, smf of v79 h1b1 cells was unstable. we wanted to investigate the possible antithrombotic effects and elucidate the chemical identity of the active principles involved in inhibitory effects against adp-induced aggregation of human platelets by wild garlic, allium ursinum l. method: bioassay-guided isolation procedure was used followed by spectrometric identification of pure active compounds. for the bioassay, blood was taken from healthy human volunteers and platelet rich plasma (prp) was prepared for turbidimetric platelet aggregation tests. prp, stimulated with 20µm adp, was treated with extracts of different polarities, fractions and isolated single compounds from allium ursinum. the extracts were investigated by thin layer chromatography, hplc, mass spectroscopy, esi-ms and 1d/2d 1h/13c-nmr spectroscopic techniques. for references the adt-antagonist mes-amp was used. result: fresh allium ursinum leaves were extracted with ethanol, which was the potent form that effectively inhibited adp-induced aggregation of human platelets. this ethanolic extract was subjected to liquid-liquid partition. whilst the aqueous phase containing the moiety of cysteine sulphoxide and thiosulphinate derivatives showed only weak activity on platelet aggregation, the ethyl acetate and especially the chloroform partitions showed highest aggregation inhibiting potency. thus, in our bioassay effects of alliins/allicins could be neglected. the chloroform phase, possessing the strongest activity, was separated into 28 fractions by gradient elution open cc on silica gel. the most active fractions 11-17 were separated again yielding 10 subfractions. this afforded 1,2-di-o-α-linolenoyl-3-o-β-d-galactopyranosyl-sn-glycerol and β-sitosterol-3-o-β-dglucopyranoside, the structures of which were determined by esi-ms and 1d/2d 1h/13c-nmr spectroscopic techniques. furthermore, the diminutive amounts of volatile oil of a.ursinum leaves obtained by steam distillation according to ph.eur. could be evaluated as a third aggregation inhibiting principle. conclusions: at the first time two active, non-sulphur-containing constituents of wild garlic, namely a galactolipid and a phytosterol, could be identified exhibiting inhibitory action on adp-induced aggregation in human blood platelets. as a major constituent, the galactolipid 1,2-di-o-α-linolenoyl-3-o-β-d-galactopyranosyl-sn-glycerol, not yet found in allium spec., appears as a new, highly useful marker substance for a.ursinum drugs, or their pharmaceutical preparations. in recent years, public attention focused more and more on risk factors which may impair sperm quality and thereby human reproduction. in this context, for example pesticides, alcohol, cigarettes, and even mobile phones are discussed. a variety of parameters exists including sperm counts as well as sperm motility, which are considered to be two of the most important parameters to evaluate sperm quality in animal models with the final aim to assess human risk. in recent years computer assisted sperm analysis (casa) devices mostly replaced the formerly used manual counting and manual motility assessment. however, although casa offers multiple opportunities and can allow for an objective and more detailed evaluation, several pitfalls exist which can alter the results profoundly and consequently compromise the quality of the data and ultimately the validity of a study. the aim of the present study was to establish and validate the casa device tox ivos sperm analyzer from hamilton thorne and thereby to gain detailed knowledge about the practical advantages but also intricacies which may alter the obtained results. in this regard healthy adult male rats (10-12 weeks old) were used. ultrasonic sound resistant sperm heads were isolated from the testis and in addition, sperms were isolated from the cauda epididymis. testicular sperm head counts and sperm motility were assessed using different isolation procedures and/or instrument settings. results different instrument settings modulate both -sperm motility and testicular sperm counts. in this regard, a wide range of results including slight changes as well as false positive/negative results were obtained. in addition, the modification of the isolation procedure can lead to variable results especially for sperm motility. conclusion isolation procedures as well as instrument settings can alter the results. consequently, in an experimental setting, potential adverse effects can be confounded with methodologically mediated apparent findings exerted via inappropriate use of the device -depending on the respective conditions in the test laboratory. this study demonstrates the relevance of standardization of testing conditions adopted for computer assisted sperm analysis and the need for a robust validation prior to use in experimental settings. orai and stim proteins have been identified as central components of the highly ca 2+ selective, store-operated current in immune cells (icrac). the molecular basis of selective orai-mediated activation of the calcineurin/nfat pathway and the crosstalk with other channel and scaffold molecules of the trpc family are still incompletely understood. using patch clamp recordings complemented by fluorescence and tirf microscopy we investigated interactions between orai1 and trpc3 in plasma membrane microdomains of rbl-2h3 mast cells. orai1-mediated crac currents, activated by passive store depletion, were found significantly reduced by over-expression of trpc3. this negative impact of trpc3 on icrac was independent of channel function as the trpc3 pore dead mutant (e630k) inhibited icrac to a similar extent as wild type trpc3. importantly, despite a reduction in icrac, nfat translocation in trpc3 overexpressing rbl cells remained unchanged, or was even slightly promoted. store depletion-induced nfat translocation in rbl cells was as well unaffected by trpc3e630k but substantially reduced by trpc3 mutants with either i) eliminated fkbp12/calcineurin binding (p704q) or ii) deficiency in pkc phosphorylation (s712a). moreover, inhibition of pkc phosphorylation by (gfx109203x; 3 µm) strongly suppressed nfat signaling. we suggest trpc3 as a scaffold that links orai-mediated ca 2+ -entry to nfat/calcineurin signaling within plasma membrane microdomains. neurally-induced bronchoconstriction in human and guinea pig precision-cut lung slices schlepütz m. 1 , rieg a. d. introduction: precision-cut lung slices (pcls) are well suited to study peripheral airway responses in different species. airway tone is under close control of the autonomic nervous system and dysregulation may contribute to airway hyperresponsiveness as observed in human lung diseases such as asthma. hence, the aim of the present study was to characterize neurally induced bronchoconstriction (bc) in guinea pigs (gp) and to compare the results with those in human pcls. methods: pcls were prepared from gp or human lung tissue. nerve endings in pcls were activated by electric field stimulation (efs) or capsaicin addition. cholinergic nerve responses were proven by atropine. capsaicin was used to show excitatory nonadrenergic non-cholinergic (enanc) responses. ruthenium red or skf96365 were used to confirm transient receptor potential (trp) channel contributions upon enanc activation. results: gp and human pcls were both sensitive to efs and airways contracted to 39±26% of the initial airway area (%-iaa) and 63±21%-iaa, respectively. in frequency response curves half maximal response was found at 7.0±1.2 hz for guinea pig pcls and 8.1±0.8 hz for human pcls. efs-induced bc was inhibited by atropine in both species. capsaicin contracted gp to 18±15%-iaa. 60% of human pcls were responsive to capsaicin and airways contracted to 72±10%-iaa, respectively. in gp ruthenium red and skf96365 blocked capsaicin-as well as efs-induced bc. conclusion: gp and human pcls contain atropine sensitive cholinergic and capsaicin sensitive enanc nerve endings. since gp pcls were sensitive to trp channel inhibitors, the involvement of those channels can be characterized with respect to lung diseases. in conclusion, gp pcls resemble the human distal lung innervation and represent a useful model to study neural airway pharmacology. the erk1/2-pathway is involved in pkc-induced nox4 up-regulation schlufter f., xia n., förstermann u., li h. universitätsmedizin mainz institut für pharmakologie, obere zahlbacher straße 67, 55131 mainz, germany nadph oxidases (nox) are major producers of reactive oxygen species in the vascular wall and nox4 is the most abundant nox isoform in human endothelial cells. we have previously shown that treatment of human ea.hy 926 endothelial cells with phorbol 12myristate 13-acetate (pma) for 48 h leads to an up-regulation of nox4 expression. this effect of pma is mediated by protein kinase cα, because it is preventable by the pkc inhibitor gö 6983 and by pkcα-sirna. the present study is aimed to investigate the signal transduction cascade downstream of pkcα. pma-induced nox4 up-regulation can be attenuated by pd 98,059 (an erk1/2 inhibitor), but not by sp 600125 (a jnk inhibitor), indicating in the involvement of erk1/2. consistently, pma treatment leads to a sustained activation of erk1/2, and sirnamediated knockdown of erk1/2 markedly reduces the pma-induced nox4 up-regulation. h89, an inhibitor of the mitogen-and stress-activated protein kinases (msks) has no effect on the pma-stimulated nox4 expression, indicating that msks are not the target molecules of erk1/2 in this scenario. on the contrary, knockdown of the transcription factor elk-1 by sirna significantly reduces the pma-induced nox4 up-regulation. in conclusion, erk1/2 and elk-1 are involved in the pkcα-induced nox4 up-regulation. determination of spontaneous mutation frequencies in normal human mammary gland tissue using the random mutation capture technique schmalbach k., lehmann l. university of wuerzburg section of food chemistry, am hubland, 97074 wuerzburg, germany annually, over 57,000 women develop breast cancer in germany. the accumulation of genetic mutations in mammary gland tissue during lifetime may be reasonable for developing breast cancer. in particular mutations in tumor suppressor genes, e.g. p53, seem to play an important role in developing cancer. up to now, lack of a method sensitive enough to determine the expected very low spontaneous mutation frequency (smf) in normal mammary gland tissue precluded the investigation of the role of spontaneous mutations acquired in the p53 gene in epidemiological studies. the only test with the potential to determine low smfs was the random mutation capture (rmc) assay, a genotype selective method which detects mutants that render the mutational sequence non-cleavable by the taqi restriction enzyme after accumulation of the target sequence. therefore, the suitability of the rmc assay to determine smf in p53 gene in normal human mammary gland tissue was evaluated. thus, the rmc assay was optimized concerning (i) dna isolation, (ii) pcr conditions, and (iii) amount of mammary gland tissue. (i) genomic dna from normal human mammary gland tissue, obtained from healthy women who underwent mamma reduction surgery for cosmetic reasons, was isolated using an extended proteinase k digestion prior to chloroform extraction. (ii) the target sequence in intron 6 of p53 gene was captured by hybridization with a complementary uracil-containing dna-probe synthesized via polymerase chain reaction (pcr), followed by magnetic separation from the remaining genomic dna. the copy number of the target sequence was quantified by competitive pcr. the number of mutants was detected after cleavage of the target dna with taqi by means of pcr with a primer set flanking the restriction site. (iii) with 2 g of normal mammary gland tissue a smf of 2.2±1.4x10 -7 per base pair was determined indicating the rmc assay suitable for smf determination. in conclusion, the smf in the p53 gene in normal human mammary gland tissue was determined for the first time, enabling the future investigation of factors influencing the smf during breast cancer development. cigarette smoke-induced release of pro-inflammatory cytokines including interleukin-8 (il-8) from inflammatory as well as structural cells in the airways, including airway smooth muscle (asm) cells, may contribute to the development of chronic obstructive pulmonary disease (copd). despite the wide use of pharmacological treatment aimed at increasing intracellular levels of the endogenous suppressor cyclic amp (camp), little is known on its exact mechanism of action. we report here that next to the β2-agonist fenoterol, direct and specific activation of either exchange protein directly activated by camp (epac) or protein kinase a (pka) reduced cigarette smoke extract (cse)-induced il-8 mrna expression and protein release by human asm cells. cse-induced iκbαdegradation and p65 nuclear translocation, processes that were primarily reversed by epac activation. further, cse increased extracellular signal-regulated kinase (erk) phosphorylation, which was selectively reduced by pka activation. cse decreased epac1 expression, but did not affect epac2 and pka expression. importantly, epac1 expression was also reduced in lung tissue from copd patients. in conclusion, epac and pka decrease cse-induced il-8 release by human asm cells via inhibition of nf-κb and erk, respectively, pointing at these camp effectors as potential targets for antiinflammatory therapy in copd. however, cigarette smoke exposure may reduce antiinflammatory effects of camp elevating agents via down-regulation of epac1. polycyclic aromatic hydrocarbons (key marker substance benzo[a]pyrene (bap)) have been assumed to play a role in the development of bladder cancer. the objective of the present study was to unravel cellular and in particular cytoskeletal response to bap. to follow the sequential steps of chemical carcinogenesis the differential proteomic profile was analyzed at early and late time points. the study was carried out in a superficial human bladder cancer cell line (rt4) exposed to 0.5 µm bap, a subacute concentration based on results of proliferation (brdu) and dna damage (tunel) tests. cells of a human bladder cancer cell line (rt4) were exposed to 0.5 µm bap for 24 h (n=5), 4 wk (n=7) and 8 wk (n=3). proteins of whole cell lysate were separated by twodimensional electrophoresis (fig. 1) . differentially expressed proteins were identified by matrix-assisted laser desorption/ionization-time of flight analysis. cortactin, actin and tubulin were immunohistochemical stained. changes in migration and colony forming ability were assessed by scratch wound-healing assay and soft-agar colony formation. results: by using several databases (uniprot, reactome, panther) the identified proteins were categorized into different functional classes such as mrna processing, translation, protein metabolic process, or several areas associated with the organization of the cytoskeleton. 45 % of the differentially expressed proteins after 24 h of treatment are involved in the processing of pre-mrna (20 %) and protein metabolism (25 %). this pattern changed after 8 wk of treatment. then, 48 % of the proteins affected the cytoskeleton whereas still 26 % were categorized to protein metabolism and only 7 % to pre-mrna processing. in the immunhistochemical staining, the treated cells appeared after 8 wk of exposure larger and rounder predominantly due to the modifications of the actin cytoskeleton. merged images of actin and cortactin revealed that both proteins colocalized in invadopodiae. after 8 wk, a higher number of treated cells moved toward the centre of the wound and they formed more soft-agar colonies compared to vehicle conditions, suggesting a transformation of the cells. in conclusion, bap exposure causes in an early phase an activation of the spliceosome which can led to an epithelial-tomesenchymal transition. two coordinators of a cell-type-specific splicing program, epithelial splicing regulatory proteins 1 and 2, are currently being validate by pcr. fused master gel : representative 2-de gel of rt4 cells exposed to 0.5 µm bap for 8 wk. protein spots which were differentially expressed compared to control and identified were marked. cannabinoids stimulate mesenchymal stem cell migration via a mitogen-activated protein kinase pathway schmuhl e. 1 , ramer r. mesenchymal stem cells (mscs) are known to be involved in various regenerative processes such as cardiac, ocular, skin and bone tissue healing. however, little is known about the pharmacotherapeutical options aiming at tissue healing steps such as the mobilization and homing of mscs. here, we show that cannabidiol (cbd), a nonpsychoactive cannabinoid, stimulates the migration of human adipose-derived mscs in both boyden chamber and in vitro scratch wound assays. in boyden chambers cbd (0.01 -3 µm) was shown to promote cell migration in a time-and concentration dependent manner. this promigratory action was inhibited by am-630 (cb2 receptor antagonist) and by o-1602 (g protein-coupled receptor [gpr] 55 agonist). moreover, cbd activated the mitogen-activated protein kinase (mapk) pathway as evidenced by increased phosphorylation of extracellular signal-regulated kinase (erk) 1/2. blockade of erk activation by pd98059 prevented cbd-stimulated msc migration, whereas inhibition of p38 mapk by sb203580 was inactive in this respect. furthermore, am-630 and o-1602 were found to attenuate cbd-induced erk activation. an erk-dependent promigratory action was likewise demonstrated for the phytocannabinoid ∆ 9 tetrahydrocannabinol and for the hydrolysis-stable anandamide analogue r(+)methanandamide. we conclude that cbd promotes msc migration via receptordependent erk activation, possibly contributing to tissue healing. the duffy antigen receptor for chemokines (darc) binds promiscuously many inflammatory chemokines without showing intracellular signal transduction. it is mainly expressed on endothelial cells of postcapillary venules and on red blood cells, where it acts as a transendothelial transporter of chemokines and as a chemokine sink, respectively. surprisingly, as shown for human and mouse brain, darc is also expressed at high density in the cerebellum. however, nothing is known about the function of darc in this location. we addressed this question by subjecting c57bl/6 wildtype and darc-deficient mice to a series of behavior experiments including morris water maze-, elevated plus maze-, rotarod-and actometer tests. while the results from the water maze experiments are ambiguous, elevated plus maze trials show a strong aversion of darc -/mice to walk to the end of the open arm, which is consistent with anxiety-like behavior. moreover, darc -/mice show greatly reduced locomotor activity, which is at least partly caused by episodes of reduced mobility occurring more frequently than in the corresponding wildtype controls (elevated plus maze, actometer). finally, darc -/mice spend a significantly reduced time on the rotating rod compared to c57bl/6 wildtype controls, which may indicate an impaired cerebellar function. we conclude that darc in fact modulates brain function. surprisingly, this appears to be happening under homeostatic conditions, although darc binds for the most part to inflammatory chemokines. it remains to be elucidated, how this effect can be caused by a non-signaling chemokine receptor. it may be an indirect consequence of altered brain chemokine concentrations or of as yet unknown signaling pathways activated by darc. transporter gene expression in human head-neck squamous cell carcinoma and epigenetic regulation mechanisms schnepf r. 1 hals-nasen-ohren-klinik, kopf-und halschirurgie, friedrich-alexander-universität erlangen-nürnberg, waldstraße 1, 91054 erlangen, germany background: membrane transporters may affect the disposition and thereby treatment efficiency of anticancer drugs in human head-neck squamous cell carcinoma (hnscc). the gene expression profile of transporters in hnscc, however, is unknown and was evaluated in this study. moreover, we evaluated mechanisms by which transporters are regulated in hnscc. we focused on the role of the nuclear pregnane x receptors (pxr, nr1i2) and epigenetic mechanisms. methods and results: real-time rt-pcr revealed a significantly increased mrna expression of slco1a2 and slco1b3 and a significantly decreased expression of transporters such as slco2b1, slco2a1 and abcc3 in human hnscc tissue samples compared to adjacent normal mucosa. moreover, an association between slco2b1 mrna levels in tumor tissues and five-year survival of hnscc patients was observed (χ 2 =6.59; p=0.010; n=34). bisulfite sequencing revealed that promoter cpg islands of abcc3 and slco2a1 were not methylated and thus these genes were not epigenetically silenced in hnscc tissues. in the hnscc-derived umscc-1 and scc-15 cell lines, transcript expression of transporters (e.g., abcc3, slco2a1; p<0.001) and pxr (nr1i2; p<0.001) was markedly induced by the dna methyltransferase inhibitor decitabine. cotreatment with the prototypical pxr activator rifampicin significantly reversed decitabine-induced abcc3 and slco2a1 expression. conclusions: transporter expression profiles significantly differed between hnscc and normal mucosa and expression levels of slco2b1 may serve as a marker for prognosis. modulation of the epigenome with the anticancer drug decitabine substantially affects transporter expression in umscc-1 and scc-15 cells, suggesting epigenetic regulation mechanisms. moreover, interactions between epigenetic and nuclear receptor-mediated mechanisms in transporter regulation occur. this work was in part supported by the johannes und frieda marohn foundation. the role of hcn2 in neuropathic and inflammatory pain schnorr s. 1 , eberhardt m. the pacemaker current ih is carried by hyperpolarization-activated cyclic nucleotidegated cation channels (hcn1-4) and contributes to cellular excitability in the heart and the nervous system. hcn1 and hcn2 are the two most abundant hcn subunits in peripheral sensory neurons with hcn2 being the prevalent isoform in nociceptive small sized c-fibre dorsal root ganglion (drg) neurons. we examined the role of hcn2 for peripheral sensitization and spontaneous neuronal activity in neuropathic and inflammatory pain. we generated a conditional deletion of hcn2 by using a nociceptor specific cre-transgene driven by the nav1.8 promoter. the nociceptor-specific knockout of hcn2 in drg neurons (nosphcn2ko) was confirmed by quantitative rt-pcr and western blot. immunohistochemical staining revealed that the deletion of hcn2 was mainly restricted to small sized drg neurons. the conditional loss of hcn2 resulted in a significant reduction of ih positive small diameter drg neurons pointing to a central role of this isoform to the hcn current in nociceptive neurons. behavioral studies showed that the lack of hcn2 did not influence basal pain responses but led to a significant reduction in mechanosensation in both neuropathic and inflammatory pain models. however, thermosensation of the mutants was only decreased in neuropathic pain conditions. in wild-type animals, intraperitoneal, intraplantar and even intrathecal injection of the hcn channel blocker zd7288 nearly eliminated tactile allodynia caused by inflammation in contrast to thermal hyperalgesia which remained unaffected. in contrast, pain thresholds in nosphcn2ko mice did not significantly increase after pharmacological block of ih. additionally, experiments revealed that the inflammatory condition induced an upregulation of hcn2 protein in the spinal dorsal horn compared to saline injected mice. our results suggest that hcn2 might be a new target in the treatment of neuropathic and inflammatory pain. the proper functioning of the central, as well as the peripheral nervous systems is of outstanding importance to the survival and well-being of humans. yet, the integrity of neuronal systems is constantly challenged by a plethora of environmental and occupational toxins. some of these toxins preferentially target neural cells. these neurotoxins can exert their devastating effects by very different modes of action. neurotoxins may induce apoptosis or necrosis of neurons, or interfere with axon growth and elongation. these processes can be identified by specialized in vitro tests. furthermore, neurotoxins have been described to alter glial function which may compromise the viability of surrounding neurons. as another important mode of action, several neurotoxins act on neurotransmitter receptors, thereby altering signal propagation within neuronal networks. countless natural and synthetic substances have been characterized for their effects on neurotransmitter receptors and today can be used for detailed studies of receptor function. however, environmental toxins of anthropogenic origin and occupational toxins that both represent constant sources for human exposure are still poorly studied with respect to their effects on neurotransmitter receptors. thus, the need for a better understanding of the susceptibility of neurotransmitter systems for toxic effects exerted by these substances is of outstanding importance for the protection of human health. here, we introduce an imaging-based approach for the screening of the effects of potential and known neurotoxins on neurotransmitter receptors of intact cells in vitro. different neuronal cells were tested for their sensitivities for classical neurotransmitters using life-cell imaging experiments. in more detail, we examined the proportion of responding cells and determined the ec50 values for the most prominent neurotransmitters in cell lines widely used for in vitro neurotoxicity studies on the one hand, namely sh-sy5y and lumes cells, and primary mouse neurons on the other hand. with these data at hand, we are now able to identify and characterize the effects of neurotoxins on receptor function in chronic, as well as acute exposition paradigms. the use of an in vitro imaging-based physiological test system is at the interface between non-functional in vitro approaches and in vivo toxicity tests, thus, giving mechanistic insight into neurotoxic processes without requiring animal experiments. apomorphine acts on trpa1 channels scholze a., schaefer m., hill k. universität leipzig -universitätsmedizin rudolf boehm-institut für pharmakologie und toxikologie, härtelstr. 16-18, 04107 leipzig, germany apomorphine is a non-narcotic derivative of morphine which acts as a dopamine agonist and is clinically used to treat "off-states" in patients suffering from parkinson´s disease. adverse effects of apomorphine treatment include dopaminergic effects such as nausea, but also ulceration and pain at the injection site. we wanted to test whether an activation of trp (transient receptor potential) channels in sensory neurones contributes to the perception of pain after apomorphine injection. while the warm/heat receptors trpv1, trpv2, trpv3, and trpv4 and the cold receptor trpm8 were insensitive towards apomorphine treatment, trpa1 could concentration-dependently be modulated by apomorphine. low micromolar apomorphine concentrations potently activated heterologously expressed trpa1 channels in a stably transfected cell line (hek293-trpa1), as well as natively expressed trpa1 in cultured dorsal root ganglion neurones. on the other hand, when using higher concentrations of apomorphine, we observed inhibition of trpa1 activity. previous studies have shown that subcutaneously administered apomorphine produces a biphasic dose response relationship in rats, inducing hyperalgesia at low doses whereas high doses of the substance cause antinociception. from our studies we conclude that such in vivo effects of apomorphine are presumably mediated by activation/inhibition of trpa1 expressed in sensory neurones agonist binding to a g protein-coupled receptor (gpcr) induces a conformational change of the receptor protein, which results in the activation of receptor-associated heterotrimeric g proteins [1] . in radioligand binding studies, conducted to investigate ligand binding to specific gpcrs, receptors are usually probed with radioantagonists. as in other gpcrs [2] , agonists of the muscarinic m2 receptor exhibit biphasic kinetics and biphasic competition curves with radioantagonists, indicating a more complex situation probably caused by g protein interactions. here, we present a detailed study of the binding of agonists to muscarinic m2 receptors including the novel super-high affinity agonist iperoxo and a differential chemical knockout of g proteins. in addition to membrane homogenates living cells were employed. we demonstrate that the high affinity fraction in biphasic curves does not differ between selected full agonist and is sensitive to pertussis toxin, thus indicating that this receptor population is associated with gi proteins. however, despite promiscuous signalling properties of m2 receptors, the low affinity fraction is not associated with any other g protein, since low affinity binding is insensitive to high concentrations of guanylnucleotides and cholera toxin. moreover, high affinity agonist binding appears solely in membrane homogenates but not in experiments conducted with living cells, probably due to their high intracellular concentration of guanylnucleotides. taken together the chemical knock-out of g proteins revealed that the high affinity binding of agonists in membrane homogenates is associated with the interaction of the muscarinic m2 receptor with gi proteins. the low affinity binding cannot be related to another g protein, although the muscarinic m2 receptor exhibits promiscuous g protein signalling properties. interestingly data obtained with living cells do not reveal any high affinity binding of agonists. prolonged stress leads to a dysregulation of the hypothalamus-pituitary-adrenal (hpa)axis and may affect the sensitivity of pain perception. however, it is not yet known whether the alterations of hpa-axis and increased pain sensitivity are related. to create a long lasting stressful situation, male wistar rats were exposed to a restraint-stress for 1 h daily over a period of two weeks. the effect of stress on the hpa-axis was determined by adrenal morphology and stress hormone levels, the influence on mechanical pain sensitivity was evaluated by the randall-selitto paw pressure test. on day 15 the animals exhibited a significant mechanical hyperalgesia. they also showed increased acth and corticosterone plasma levels and an enlarged zona fasciculata of the adrenal gland, indicating a dysregulation of the hpa-axis. for testing the correlation of hpa-axis dysregulation and hyperalgesia a persistent increase in plasma corticosterone in wistar rats was generated by the administration of corticosterone via the drinking water for two weeks. these animals also showed an increased mechanical nociceptive sensitivity with an accompanied decrease of the adrenal glands and reduced acth levels. the results show that chronic stress leads to a dysfunction of the hpa-axis with an accompanied mechanical hyperalgesia which can be mimicked by oral administration of corticosterone. thus, this in-vivo test system may provide a new animal-friendly pharmacological model for stress-related pain disorders. the alternaria mycotoxins aoh and ame induce cyp1a1 and apoptosis in murine hepatoma cells dependent on the aryl hydrocarbon receptor mycotoxins are secondary metabolites of fungi including the genus alternaria (black mold). alternaria fungi are known to infest different types of foodstuffs and produce diverse toxins amongst them the mycotoxins alternariol (aoh) and alternariol methyl ether (ame) which are potential carcinogens. as planar compounds, aoh and ame are preferentially metabolized by cytochrome p450 (cyp) 1a1 and 1a2. the most prominent regulator of cyp1a1 is the dimeric transcription factor complex ahr/arnt, which is activated by planar ligands. therefore we studied the activation of ahr/arnt by aoh and ame and monitored cyp1a1 induction in murine hepatoma cells (hepa-1c1c7). indeed, aoh and ame enhanced the levels of cyp1a1 in hepa-1c1c7 cells but not in cells with inactivated ahr (hepa-1c1c12) or arnt (hepa-1c1c4). furthermore, we studied the cytotoxicity of aoh and ame. by using a fluorescence-based microscopic readout we measured effects on cell counts, apoptosis, senescence and micronuclei formation. both aoh and ame reduce the cell number and the cell nuclei show drastic morphological changes e.g. enlargement after aoh treatment or micronuclei formation. the observed effects where, except for the induction of apoptosis, independent of ahr/arnt. in summary, aoh and ame activate the ahr/arnt pathway to induce cyp1a1 expression and apoptosis. however, the predominant cytotoxic effect of aoh and ame in hepatoma cells is a profound reduction in cell numbers, which is independent of the ahr/arnt pathway. special purpose databases are the first place for researchers in the life sciences to obtain expert curated data. naturally, such resources are limited in terms of timeliness and comprehensiveness. the literature database pubmed alone lists more than 20,000,000 scientific abstracts, and 700,000 are newly added every year. the protein sequence database uniprotkb stores over 10,500,000 sequences, a hundred times more than ten years ago. turning these data into meaningful information and making it accessible to both humans and computers have become an essential part of biological discovery and biomedical research. text mining techniques have proven useful to extract the missing links in areas such as drug-target and drug-disease prediction, the extraction of mutation-phenotype associations, or the prediction of protein-protein and protein-ligand interactions. by systematically extracting information from available literature, reports or patents, text mining techniques can help to refine existing categorical knowledge stored in databases, and hence will support drug repositioning, the discovery of novel cancer biomarkers, or help to understand causes of hereditary diseases. in the area of regulatory toxicology we developed go3r, the first knowledge-based search engine for alternative methods to animal experiments. the system not only helps retrieving information on the availability of alternative methods that allows for replacing, reducing or refining animal experiments, but also provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information and data. the up-to-date taxonomicstructured "table of contents" provided by go3r allows for search in the literature listed in pubmed or the toxicology data network (toxnet) in a fast and comprehensive manner. the semantically enriched platform supports the user during the query formulation, allows for bibliographic analysis, and reveals existing relations to replacement, reduction, and refinement of animal experiments. impaired cardiac excitation-contraction-coupling in mice with complete inactivation of the crem gene schulte j. s., tekook m., schmitz w., müller f. u. westfälische wilhelms-universität institut für pharmakologie und toxikologie, domagkstraße 12, 48149 münster, germany the structurally related transcription factors camp response element binding protein (creb) and camp response element modulator (crem) mediate a regulation of gene transcription in response to camp and are expressed in the heart. mice with complete inactivation of crem display impaired cardiac contraction and relaxation, decreased expression of serca and down-regulation of β1-adrenoceptors. to elucidate the underlying functional mechanisms on the cellular level we here investigated cellular electrophysiology and ca 2+ -cycling in ventricular cardiomyocytes from crem ko mice (ko). adult ventricular cardiomyocytes were isolated from ko and wildtype (wt) mice (age 16-20 weeks) and subsequently used for experiments within 6 hours after isolation. action potentials (aps) were recorded from ventricular cardiomyocytes (perforated patch, whole cell current clamp inactivation of crem seems to have no consequences for ap duration and possibly associated ion channels but leads to impairment of ca 2+ cycling and sarcomere shortening under basal conditions well explaining the previous findings in vivo. our results show that crem is essential for a regular excitation-contraction coupling in the mouse heart. (supported by the izkf münster) new mechanistic insights in no/cgmp actions in the vasculature schulte k., koesling d., universitätsstr. 150, 44781 bochum, germany hypertension, a major risk factor for cardiovascular diseases, is associated with vascular changes resulting in increased vascular contractility und vascular peripheral resistance. a prominent factor in the development and maintenance of hypertension is the reninangiotensin-aldostrerone system. angiotensin ii (ang ii) is the main mediator of this system and a powerful vasoconstrictor. ang ii increases the intracellular ca 2+ concentration thereby activating myosin light chain (mlc) kinase, which enhances mlc phosphorylation and subsequent vascular contraction. opposite to angii-induced vascular contraction, no/cgmp pathway promotes vascular relaxation by decreasing ca 2+ concentration and lowering mlc phosphorylation. responsible for mlc dephosphorylation is the mlc phosphatase (mlcp), whose activity is regulated by different phosphorylations. phosphorylation of mlcp via rhoa-activated rho-kinase enhances phosphatase activity while phosphorylation via the cgmp-dependent protein kinase has been proposed to decrease enzymatic activity. to investigate the interplay of angii with the no/cgmp pathway, we treated wild-type and ko mice lacking the cgmp forming no receptor, no-gc1, with angiotensinii (1.44 mg / kg bw / d) for two weeks. in addition to various cardiovascular parameters, physiological changes in vascular reactivity of aortic rings of angii-treated wt and no-gc1 ko mice were assessed in organ bath experiments and correlated with biochemical parameters as the phosphorylation state of mlc, mlcp and rho-kinase activities examined by immunoblot analysis. analysis of cgmp levels revealed that angii treatment decreased cgmp in wt mice to levels comparable to those of the ko mice which were unaltered by the treatment. our study will provide further mechanistic insights in the molecular interactions between constrictor and dilator stimuli in the vasculature. nanomaterials are already used today and offer even greater use and benefits in the future. the progress of nanotechnology must be accompanied by investigations of their potential harmful effects. for airborne nanomaterials, lung toxicity is a major concern and obviously the particle size is discussed as a critical property directing adverse effects. while standard toxicological test methods are generally capable of detecting the toxic effects, the choice of relevant methods for nanomaterials is still discussed. we have investigated two genotoxic endpoints -alkaline comet assay in lung tissue and micronucleation in polychromatic erythrocytes of the bone marrow -in a combined study 72 hours after a single instillation of 18 µg gold nanoparticles (np) into the trachea of male adult wistar rats. the administration of three test materials differing only in their primary particle size (2-, 20-and 200-nm) did not lead to relevant dna damage in the mentioned tests. the measurement of clinical pathology parameters in bronchoalveolar lavage fluid (balf) and blood indicated neither relevant local reactions in the animals' lungs nor adverse systemic effects. minor histopathology findings occurred in the lung of the animals exposed to 20-nm and 200-nm sized nanomaterials. in conclusion, under the conditions of this study the different sized gold np tested were non-genotoxic and showed no systemic and local adverse effects at the given dose. platelet dense granule secretion mediates platelet-dependent enhancement of tumor cell transmigration and formation of metastases schumacher d., strilic b., wettschureck n., offermanns s. mpi für herz-und lungenforschung offermanns, ludwigstr. 43, 61231 bad nauheim, germany tumor cell metastasis to distant organs is the primary cause of mortality in cancer patients. tumor cells leave the primary tumor, intravasate, survive in the circulation and extravasate through the endothelial cell layer to grow in the target organ. it has long been known that blood platelets play an important role in tumor cell survival and dissemination, but the mechanism by which platelets promote metastasis remained unclear. given that platelets are found closely associated with tumor cells shortly after vascular arrest, we explored whether platelets can facilitate the transmigration of tumor cells through the endothelium and thereby promote extravasation of tumor cells into the organ parenchyma. the ability of various mouse and human tumor cells like lewis-lung carcinoma cells (llc1), b16f10 melanoma cells or human neuroblastoma cells (sh-sy5y) to transmigrate through an endothelial cell layer was strongly enhanced by seeding tumor cells together with mouse or human platelets onto the endothelial cell layer. this indicates that platelets facilitate tumor cell transmigration in vitro. we found that platelet granule secretion is involved in this process as supernatant from platelets incubated with tumor cells but not from resting platelets was sufficient to enhance tumor cell transmigration. additionally, no platelet-mediated increase of tumor cell transmigration was observed in dense granule secretion-defective platelets of munc13-4 deficient mice. thus, dense granule secretion is required for platelet-dependent tumor cell extravasation in vitro. while the growth and weight of primary tumors after subcutaneous injection of llc1 and b16 cells was indistinguishable between wild-type mice and animals lacking munc13-4, the number of metastases in the lung was strongly reduced in munc13-4-deficient animals. the strong decrease in formation of metastases in munc13-4 deficient mice was also observed after i.v. injection of llc1 and b16f10 cells. thus, platelet dense granule secretion plays a critical role in tumor cell metastasis by enhancing tumor cell transmigration through the endothelial cell layer. formation of dna adducts in mouse tissues by the brassica ingredient 1methoxy-3-indolylmethyl glucosinolate and its break-down product 1-methoxy-3indolylmethyl alcohol schumacher f. 1 , engst w. glucosinolates are secondary metabolites present at substantial levels in cruciferous vegetables, such as broccoli and cabbage. after injury of plant tissue they are activated by the enzyme myrosinase to form various electrophilic degradation products like isothiocyanates. we previously showed that 1-methoxy-3-indolylmethyl (1-mim) glucosinolate (or neoglucobrassicin) is a potent genotoxicant in bacterial and mammalian cells after activation by myrosinase. the induction of mutations could be correlated with the formation of dna adducts [1] . we have identified and synthesized the major dna adducts n 2 -(1-mim)-dg and n 6 -(1-mim)-da. moreover, we developed a highly sensitive uplc-esi-ms/ms method for selective mrm quantification of these adducts using stable-isotopic labeled adducts as internal standards. while the plant enzyme myrosinase is probably almost completely inactivated after cooking the vegetables, the glucosinolates reach the gut mostly intact due to their good heat and ph stability. enzymes of individual intestinal bacteria are able to cleave the glycosidic bond of the glucosinolates, which leads to the formation of reactive metabolites within the gut lumen. we were able to detect significant levels of n 2 -(1-mim)-dg and n 6 -(1-mim)-da in a dose-dependent manner in the large intestine of mice treated orally with isolated 1-mim glucosinolate. the peak levels of n 2 -(1-mim)-dg and n 6 -(1-mim)-da in the murine large intestine were reached 8 h after a single administration of 600 µmol 1-mim glucosinolate/ kg body weight. the oral application of the relatively stable metabolite 1-mim alcohol to mice led to the formation of identical dna adducts. this benzylic alcohol can be activated by sulfotransferases to an electrophilic sulfo conjugate. in contrast to the intact glucosinolate the orally administered 1-mim alcohol generated significant levels of n 2 -(1-mim)-dg and n 6 -(1-mim)-da not only in the large intestine but also in other tissues, such as the liver, of mice. [1] h. glatt, c. baasanjav-gerber, f. schumacher, b. h. monien, m. schreiner, h. frank, a. seidel, w. engst, chem.-biol. interact., 192 (2011) human pregnane x receptor genotype of the donor but not of the recipient is a risk factor for delayed graft function after renal transplantation schwab m. 1, 2 , schaeffeler e. delayed graft function (dgf) is an important immediate complication in renal transplantation significantly contributing to decreased long-term allograft survival. in addition to donor-and recipient-related risk factors altered renal excretion of xenobiotics by membrane transporters may influence dgf as well. using recipients' and donors' dna, we assessed the impact of genetic variants on dgf for the transporter proteins, pglycoprotein (abcb1) and multidrug resistance protein 2 (abcc2), and the nuclear pregnane x receptor (pxr/nr1i2), regulating the transcription of drug metabolizing enzymes and membrane transporters. in our local cohort of transplant patients (n=178) dgf occurred in 27.5 %. logistic regression analysis using four different genetic models (i.e. co-dominant, dominant, recessive and log additive) indicates that only the donor's pxr rs2276707 8055tt genotype was significantly associated with dgf (recessive model: or, 9.0; 95%ci, p=0.004 unadjusted) , even after correction for multiple testing (p=0.035 holm-adjusted). when we performed multivariate analysis including genetic and 16 clinical co-variates (i.e. age, gender, hla mismatches, panelreactive antibodies, immunosuppression using cni, t cell-depleting agents, anti-il-2 receptor antibody and steroids, cold or warm ischemia time, living vs deceased donors or graft loss) again dgf was significantly associated only with the pxr rs2276707 tt genotype of the donor (recessive model: or, 16.08; 95% ci, 2.0-129.46; p=0.0046 unadjusted) which held true after correction for multiple testing (p=0.04). for abcc2 variants only the donor rs17222723 3563t>a genotype correlated with dgf by univariate (or, 4.65; 95%ci, p=0.005 unadjusted) as well as by multivariate analysis (or, 5.5; 95%ci, p=0 .01; table 4 ) but not after correction for multiple testing (p=0.12). for variants in the abcb1 gene no significant associations with dgf were detected for both the donor's and the recipient's genotype. in summary, our findings suggest for the first time that pxr may be a risk gene for the development of dgf independently from previously identified risk factors. supported by the robert-bosch foundation, stuttgart, germany, the bmbf grant 03is2061c (berlin, germany) and by the ferdinand eisenberger grant of the german society of urology (id krs1/fe-10). formation, morphology and structural requirements for formation of microtubule protrusions by clostridium difficile toxin cdt schwan c., kruppke a. s., nölke t., aktories k. institut für experimentelle und klinische pharmakologie und toxikologie i, albertstr. 25, 79104 freiburg, germany clostridium difficile is an anaerobe, gram-positive pathogen. it causes antibioticassociated diarrhoea and pseudomembranous colitis by production of the rho gtpaseglucosylating toxins a and b. recently emerging hypervirulent clostridium difficile strains additionally produce the binary adp-ribosyltransferase toxin cdt (clostridium difficile transferase). cdt is taken up via receptor-mediated endocytosis after binding to the lipolysis stimulated lipoprotein receptor (papatheodorou et al., pnas 2011) . in the cytosol, cdt adp-ribosylates actin at arg 177, thereby actin polymerization is blocked, resulting in disruption of the f-actin network. cdt and other binary actin-adp-ribosylating toxins induce redistribution of microtubules in the cell interior and formation of long (>150 µm) microtubule-based protrusions at the surface of intestinal epithelial cells which increase bacterial adherence (schwan et al., plos pathog 2009 ). the clostridial actin-adp-ribosyltransferases influence the dynamicity of microtubules and their capture at the cell cortex by indirectly affecting different microtubule regulating proteins like clasp2 and acf7. besides the influence of cdt on microtubule regulatory proteins, the formation of protrusions depends on plasma membrane composition. depletion of cholesterol, the breakdown of sphingomyelin or inhibition of sphingolipid-synthesis reduce the formation of microtubule-based protrusions. surprisingly, most of the cdt-induced processes contain membrane-tubules derived from the endoplasmatic reticulum (er). the remodeling of the er is microtubule dependent and is mainly mediated by stim1 that usually functions as a calcium sensor in the er and activates the store operated orai1 calcium ion channels in the plasma membrane. the data suggest that toxin-induced changes of the microtubule system including alterations of the er, may affect trafficking and er-dependent signalling. bilobalide is a neuroprotective constituent of ginkgo biloba with an unknown mechanism of action. in the present study, we first used microdialysis in mice to evaluate changes in the extracellular fluid of the brain during and after stroke. microdialysis probes were implanted into the striatum of cd-1 mice, and dialysates were obtained while a monofilament was inserted for 60 min via the common carotid artery (cca) to block perfusion through the middle cerebral artery (mca). while glucose levels dropped immediately upon middle cerebral artery occlusion (mcao), glutamate concentrations in the microdialysates -as measured with a cma 600 analyzer -rose extensively during ischemia to more than 2000% of baseline level. both glucose and glutamate levels recovered rapidly when mcao was terminated after 60 min. when bilobalide (10 µm) was perfused into the striatum through the microdialysis probe during mcao, glucose levels dropped but the neurotoxic rise of glutamate was significantly attenuated and reached only 500% of baseline level (p<0.01). in the following experiments, we investigated the activity of mitochondria in ischemic brain. ischemia was induced by mcao, and ischemic as well as "healthy" tissue from the opposite hemisphere was obtained. mitochondria were isolated and mitochondrial respiration was monitored using the oroboros ® oxygraph. significant deficits of respiration were observed after ischemia. in the healthy hemisphere, the respiratory states (leak i+ii, complex i+ii+iv, oxidative phosphorylation (oxphos) and electron transport system (ets) capacity) showed a decrease of oxygen consumption to 60-70% of sham-operated mice. in the ischemic hemisphere, several values were lower at 40% of sham-operated mice (leak i+ii, complex ii+iv,oxphos and ets) whereas complex i showed a remarkably low respiratory capacity of 16% of baseline. direct addition of bilobalide (10 µm) to post-ischemic mitochondria caused a 2-fold increase of complex i activity in vitro. pretreatment of mice with bilobalide (10 mg/kg i.p.) one hour before mcao caused a significant, 3-fold improvement of complex i respiration when measured ex vivo. these data clearly indicate that bilobalide targets mitochondrial processes within the respiratory chain, preserving complex i function during ischemia. this action likely explains its neuroprotextive activity in vivo. unreacted resin monomers such as 2-hydroxyethyl methacrylate (hema) are environmental stressors released from dental composites after incomplete polymerization. the production of reactive oxygen species (ros) is a major response of cells to monomer exposure. moreover, adverse effects of monomers including delayed cell differentiation or mineralization processes, dna damage or apoptosis are associated with increased ros production. the intracellular redox homeostasis is controlled by the major non-enzymatic antioxidant glutathione (gsh), and antioxidant enzymes. here, we hypothesized that cells exposed to hema responded by a differential expression of antioxidant enzymes such as superoxide dismutase (sod-1), catalase (cat) or glutathione peroxidase (gpx1/2). raw246.7 mouse macrophages were exposed to hema (0-8mm) for 24h, and protein expression was analyzed by western blotting. to study the influence of intracellular gsh on enzyme expression, gsh synthesis was reduced by the inhibitor buthionine sulfoximine (50µm bso), or enhanced by 2-oxothiazolidine-4-carboxylate (5mm otc) and n-acetyl cysteine (10mm nac). expression of sod-1 found in untreated cultures was decreased in the presence of hema and even further reduced by bso. in contrast, nac counteracted hemainduced inhibition of sod-1 expression. cat expression was not detected in untreated cells, however, the enhanced expression of cat in cells exposed to hema indicated the decomposition of abundant levels of hydrogen peroxide. the minor influence of bso or otc showed that expression of cat was independent of gsh levels while a decrease of hema-induced cat expression in the presence of nac indicated reduced oxidative stress. gpx1/2 was expressed in untreated cultures, and its down-regulation by bso indicated that this enzyme was primarily responsible for h2o2 decomposition. the inhibitory effect of hema on gpx1/2 expression was enhanced by bso but counteracted by otc or nac. these findings indicate that h2o2 is the predominant reactive oxygen species generated in the presence of dental resin monomers like hema. abundant h2o2 production leads to the activation of cat expression and a feed-back inhibition of sod-1 expression. the hema-induced reduction of gpx1/2 expression is most likely a consequence of reduced gsh levels because of the formation of glutathione disulfide (gssg) or by gsh-hema adducts. the life-threatening effects of certain organophosphorus compounds such as soman or fenamiphos cannot be antagonized adequately by the treatment with atropine and oximes. alternative approaches are necessary. since the adequate restoration of disturbed muscle function is considered to be life-saving, a model is needed for screening of potentially therapeutic substances. an established model for the development of such new therapies is the diaphragm preparation. however, this model requires a large number of animals and experimental available time frame is limited to some hours. here, the organotypic nerve-muscle co-culture may be an appropriate alternative, because a large number of specimens with low numbers of animals and a long period of investigation over several days is possible. in the present study, the restoration of vx paralysed muscle function with obidoxime was investigated by using both models. slices of spinal cord and muscle tissue were dissected from mice embryos (e 14-15) , fixed to coverslips and incubated in roller tubes for about 2-4 weeks. spontaneous muscle activity was recorded by video microscopic techniques and was quantified offline. muscle force production in mice diaphragm preparations was elicited by indirect field stimulation technique in a 12 chamber organ bath and quantified as time-force diagrams (auc) that were expressed as relative changes of the muscle force compared to the control data. application of the nerve agent vx (0.75 µm) resulted in a strong reduction of muscle activity in the co-culture and of muscle force production in the diaphragm muscle. after obidoxime (10 µm) was added spontaneous muscle activity in the co culture recovered from 0.45 ± 0.24 hz to 1.67 ± 0.24 hz (control 1.79 + 0.22 hz) . muscle force remained stable over the next days. the vx-blocked muscle force of diaphragm was restored to 69.9 ± 21.3 % by obidoxime compared to control. muscle force production after indirect stimulation was stable for 6 hours only. our results suggest that the organotypic nerve-muscle co-cultures may be an appropriate tool for the screening of new therapeutic approaches in restoration of blocked neuromuscular transmission. moreover, the model offers an additional advantage as long-term experiments may be performed and pre-and postsynaptic effects may be assessed directly. additionally, the number of experimental animals could be reduced. the modulation of gene expression by the transcription factor crem (camp responsiveelement modulator) represents a fundamental mechanism of gene control in response to elevation of intracellular camp levels. in vascular smooth muscle cells (vsmcs) crem is involved in the regulation of cell proliferation and apoptosis supporting its relevance for vascular proliferative diseases. mice with a global inactivation of crem (cko) showed a significant increase in neointima formation after ligation of the carotid artery and an increase of atherosclerotic plaque formation after high fat diet on an apoe knockout background compared to wildtype controls (wt). on the cellular level a crem deficiency was associated with a 1.3 fold increased proliferation rate of primary vsmcs after stimulation with the platelet-derived growth factor (pdgf; n=10 from 6 isolations). microarray analysis and subsequent realtime-rt-pcr validation revealed that the alpha-type platelet-derived growth factor receptor (pdgfra) the regulator of g-protein signaling 5 (rgs5) and peptidylprolyl isomerase a (ppia) were 1.5-2.5 fold upregulated in pdgf treated cko vsmcs compared to wt vsmcs (n=6). transcripts of rgs5 (2.6 fold, ) and ppia (1.8 fold, were also upregulated in the carotid artery of cko mice in comparison to wt mice (n=10-12). we conclude that crem deficiency is associated with transcriptional changes of rgs5, pdgfra, ppia, which might explain the increased proliferation rate in cko vsmcs and the increased responsiveness to pathophysiological conditions. (supported by the izkf münster). the role of non-catalytic p101 and p87 subunits in regulating phosphoinositide 3kinase γ by gβγ and h-ras shymanets a. 1 phosphoinositide 3-kinase γ (pi3kγ) controls a plethora of cellular responses. pi3kγ, a heterodimer formed by non-catalytic p101 or p87 and catalytic p110γ subunits, is regulated by gβγ and ras. earlier we speculated that p101 binds to gβγ to translocate cytosolic pi3kγ to the plasma membrane, enabling direct activation of p110γ (brock et al., j. cell biol. 2003) . however, the p87 subunit does not function as gβγ adapter (kurig et al., pnas 2009) . since the impact of each non-catalytic subunit in regulating p110γ by gβγ and ras still remains elusive, we studied their role in detail. gβ1γ2 variants harbouring mutations in positions involved in interaction with gα subunit were purified from sf9 cells and tested for their ability to activate pi3kγ. we observed that p101, but not p87, was able to rescue the stimulatory activity of gβ1γ2 mutants incapable to activate p110γ (shymanets et al., biochem. j. doi:10 .1042/bj20111664). to further study the functional impact of the non-catalytic subunits on pi3kγ regulation, we have designed phospholipid vesicles containing similar amounts of recombinant pi3kγ variants. although p87/p110γ exhibited stronger sensitivity to gβ1γ2 than p110γ, the activity of vesicles-associated p101/p110γ was significantly higher as compared to vesicles-associated p87/p110γ or p110γ in the absence and in the presence of gβ1γ2. to study an effect of ras proteins on pi3kγ variants, recombinantly expressed h-ras was purified from sf9 cells. the posttranslational processing and lipidation of the protein was verified by mass spectrometry analysis. the impact of h-ras on regulation of p87/p110γ and p101/p110γ differed, which may explain integration of pi3kγ variants in different signalling pathways. taken together, p87 and p101 subunits implement discrete functions in respect to (i) membrane recruitment of pi3kγ and (ii) regulation of enzymatic activity by gβγ and h-ras. preparation of consolidated exposure scenarios for mixtures under reach sica m., dorn s., mostert v. dr. knoell consult gmbh, marie-curie-str. 8, 51377 leverkusen, germany under reach, formulators of mixtures need to include substance-related information into extended safety data sheets (esds), if mixtures are classified as dangerous according to the dangerous preparation directive (directive 1999/45/ec). one way to add information on substances into esds of mixtures is to generate exposure scenarios (ess) for mixtures. in order to fulfil this task, two approaches have been developed for the identification of the risk-determining substances (lead substances) in the mixtures: the critical component approach (cca) relies on dnels and pnecs for all substances, their concentrations in the mixtures as well as substance and use-specific availability parameters (echa, 2008) . in contrast, the dpd+ method is based on the legislation for classification of preparations (directive 1999/45/ec). the dpd+ method defines a lead substance for each route of human exposure and for the aquatic environment (cefic, 2010) . however, each of these methods has certain limitations. the aim of the present work is to improve the preparation of consolidated ess for a number of mixtures and provide information about their safe use. to this end, we first adopted information on risk management measures (rmms) and operational conditions (ocs) of the lead substances using the dpd+ methodology. at the same time, we considered the specific conditions of use of the mixtures (e.g. spraying, brush painting). we then conducted risk assessments by deriving dnels for the mixtures and using exposure modelling tools recommended under reach (e.g., ecetoc tra, riskofderm, art). we compared the outcome of these assessments with results obtained from the application of the dpd+ methodology. the work presents the results of application of dpd+ approach and the cca and indicates possible improvements for the risk assessment of mixtures. to check for seasonal and weather dependent influences in the prescription rate of drugs used to treat cardiovascular and respiratory diseases (atc codes c and r) a survey covering all prescriptions of a specimen german general regional health insurance (aok plus, data for saxony, largest health insurance service, approx. 50 % of all saxonian citizens are inscribed to this service) for the years 2005 to 2007 was analysed on a monthly basis. the number of prescriptions for cardiovascular drugs changed approximately +/-20 % around the mean for the different month without a clear seasonal pattern. for respiratory drugs only the systemic anticholinergics and drugs used to treat obstructive lung disease displayed a distinct seasonal pattern with a 50 -70 % above average prescription figure during spring time (february to may) and a 25 to 40 % trough in late summer/autumn (july to october). the data have to be analysed for further cofounders (e.g. influenza prevalence, environmental conditions etc.) to fully understand the fluctuations observed. the prescription rate for cardiovascular drugs and respiratory drugs seems to be influenced by multiple factors aside seasonal influences. pasteurella multocida toxin prevents osteoblast differentiation by activation of heterotrimeric g proteins siegert p., aktories k., orth j. institut für experimentelle und klinische pharmakologie und toxikologie abt. i, albertstr. 25, 79104 freiburg i. br., germany pasteurella multocida toxin (pmt) is a major virulence factor of pasteurella multocida causing pasteurellosis in man and animals and is responsible for atrophic rhinitis in pigs. the toxin modulates various signaling pathways by acting on the heterotrimeric g proteins gαq, gα12/13 and gαi. pmt activates gq to increase inositol phosphate production via phospholipase cβ and alteration of gene expression via the jak/stat pathway. the toxin also activates rhoa via gαq and gα12/13 family proteins. we showed that the underlying mechanism of the activation of heterotrimeric g proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the g protein. because pmt is the causative agent to induce progressive atrophic rhinitis in pigs, which is characterized by loss of nasal turbinate bones leading to a twisting and/or shortening of the snout, we studied the effect of pmt on bone cells. here we studied the effect of the toxin on osteoblast differentiation in st-2 cells and in primary osteoblasts from rat calvaria. st-2 cells are stromal derived cells, which can be differentiated into osteoblasts or adipocytes. the toxin inhibits the differentiation of st-2 cells into osteoblasts studied by determination of specific osteoblast markers. additionally, pmt represses the induction of transcription factors essential for osteoblast differentiation. moreover, the principal pathways activated by pmt to induce these effects were investigated. ventilator-induced lung injury (vili) is a serious problem in intensive care medicine. its mechanisms are only incompletely understood, although it is widely accepted that ventilation-induced inflammation (biotrauma) makes an important contribution. the isolated perfused mouse lung (ipl) is a valuable tool to investigate the mechanisms of vili. several studies have shown considerable differences between various mouse strains with respect to lung mechanics and inflammatory responses. therefore, we hypothesized that the pulmonary responses to mechanical ventilation differ between c57bl/6 and balb/c mice. in addition, this study introduces the novel half lung technique that allows to obtain lung tissue from the same mouse at two different time points. isolated perfused mouse lungs from c57bl/6 or balb/c were subjected to high (25cmh 2o) or low pressure (8cmh2o) ventilation for 240 minutes. after 180 minutes the left lung was removed and used for western blot analysis. the right lung was ventilated for another 60 minutes. by the end of experiment the right lung was removed and qrt-pcr performed. during the whole experiment perfusate sample were taken from the venous catheter and used for protein quantification by elisa. it was possible to remove half of the lung and to further ventilate the other half without acute changes in lung mechanics. in both strains high pressure ventilation elicited a significantly higher cytokine release than low pressure ventilation. c57bl/6 mice showed higher tnf, il-1β and amphiregulin levels after high pressure ventilation, whereas balb/c exhibited increased production of cxc chemokines (cxcl-1, cxcl-2) and il-6. kinase activities (jnk, akt, erk1/2, p38 map kinase) were increased in high pressure ventilated animals, but were strain independent. the novel half lung technique builds on the well established ipl method. it permits to separately analyze the left and the right lung at different time points during continual ventilation. this method reduces animal numbers by 50% and allows statistical within subject analysis. using this method, the present study showed that inflammatory response to mechanical ventilation differ between c57bl/6 and balb/c. these findings show that the biotrauma response in mice depends on the strain that is studied. macrophages play an important role as an integral part of the first line of immune defense. two different macrophage populations have been described. m1 macrophages produce proinflammatory cytokines and are involved in inflammatory processes. by contrast, m2 macrophages release anti-inflammatory cytokines and extracellular matrix components. they can enhance wound repair and angiogenesis, but they can also promote tumor progression. recently, industrial nanoparticles have raised concerns because of their putative toxic effects. on the other hand, specifically designed nanoparticles can be used as clinical diagnostics and as drug carriers for pharmacotherapy. thus, investigations on the interactions of engineered nanoparticles with living cells and organisms are of great importance. macrophages as phagocytosing cells scavenge nanoparticles circulating in the bloodstream. therefore, we analyzed how nanoparticles with different surface functionalization might affect functions of human macrophages. monocytes were isolated from buffy coats and differentiated to macrophages with macrophage colony-stimulating factor. carboxy-(ps-cooh) and amino-(ps-nh 2) functionalized polystyrene nanoparticles were produced by the miniemulsion polymerization process and the average particle size, the polydispersity index and their zeta potential were determined by dynamic light scattering. the macrophages were cultured in the presence or absence of different concentrations of ps-cooh and ps-nh2 nanoparticles for up to 6 days. analysis of cell viability revealed that ps-nh2 but not ps-cooh concentration-and time-dependently reduced the macrophage viability. by annexin v/propidium iodide double staining we could show that ps-nh2 trigger apoptosis in macrophages. we further polarized macrophages to either m1 or m2 using ifn-γ and lps or il-4, respectively. these macrophage populations were characterized by their expression of extracellular markers by flow cytometry and their production of cytokines by elisa. the effects of functionalized polysterene nanoparticles on the cytokine production and surface marker expression of m1 and m2 macrophages were analyzed. our data indicate that surface functionalization is a critical parameter in the nanoparticle-induced toxicity in human macrophages. this work was supported by the dfg spp1313. loos c., lunov o., syrovets t., simmet t. ulm university institute of pharmacology of natural products & clinical pharmacology, helmholtzstr. 20, 89081 ulm, germany nanoparticles are currently used for various medical applications including imaging, diagnosis and drug delivery. due to particle size and surface area, their fundamental properties differ significantly from those of corresponding bulk materials. nanoparticles circulating in the blood are mainly sequestrated by the reticuloendothelial system that consists predominantly of phagocytic macrophages. macrophages express a variety of cellular receptors for sensing and internalizing particular material like viruses, microorganisms, and foreign particulate matter including nanoparticles. therefore, a detailed understanding of the intracellular fate and processing of the nanoparticles by macrophages is indispensable for controlled biomedical applications of nanoparticles. introducing distinct surface modifications, one might control nanoparticle uptake by different cell types and thereby target specific tissues and cellular compartments. tumor cell lines are frequently used as models for primary cells to analyze the effect of nanoparticles on cells. here we show that carboxy-(ps-cooh) and aminofunctionalized (ps-nh 2) polystyrene nanoparticles of ~100 nm in diameter are internalized by human macrophages, thp-1 monocytic leukemia cells, and by pmadifferentiated thp-1 cells via different mechanisms. in buffer, macrophages and thp-1 rapidly internalize both types of nanoparticles, yet, the carboxy-functionalized particles were taken up to a higher extent. the uptake of both nanoparticles was drastically reduced in media containing serum. using pharmacological and antisense in vitro knockdown approaches, we showed that the specific interaction between cd64 receptors and the particles determines the macrophage uptake of particles by phagocytosis, whereas particle internalization by thp-1 cells occurred via dynamin iidependent endocytosis. by contrast, pma-differentiated thp-1 cells took up the particles via macropinocytosis. in line with the in vitro data, more intravenously applied ps-cooh particles accumulated in liver tissue, whereas ps-nh 2 were preferentially targeted to tumor tissue. these data show that the amount of particle internalization, the uptake mechanisms, and kinetics differ significantly among primary cells and model tumor cells, whether differentiated or not, and that they are further critically dependent on the particle opsonisation by serum proteins. this work was supported by the dfg spp1313. specifically designed and functionalized nanoparticles hold great promise for a variety of biomedical applications. to ensure their safe application, such particles require a rigorous analysis of their effects on cell functions. here we demonstrate that aminofunctionalized polystyrene nanoparticles (ps-nh2) of ~100 nm in diameter in contrast to carboxy-(ps-cooh) and nonfunctionalized (ps) particles induce an nlrp3 inflammasome activation and the subsequent release of il-1β in human macrophages. amino-functionalized ps nanoparticles induced time-dependent lysosomal destabilization followed by release of lysosomal enzymes. this resulted in mitochondrial damage and formation of reactive oxygen species. accumulation of mitochondrial reactive oxygen species was accompanied by oxidation of thioredoxin, a protein playing a central role in maintaining the cellular redox balance. upon oxidation, thioredoxin dissociated from the thioredoxin-interacting protein (txnip). liberated txnip, in turn, interacted with the nlrp3 protein resulting in a conformational change of the pyrin domain of the nlrp3 protein as predicted by molecular modeling. txnip interaction with nlrp3 led to assembly of the nlrp3 inflammasome complex, to caspase-1 activation, and release of il-1β. using an in vitro knockdown approach, we showed that ps-nh 2 induced activation exclusively of nlrp3, whereas other inflammasomes remained unaffected. treatment of macrophages with n-acetyl-l-cysteine, a scavenger of reactive oxygen species, abolished both, the caspase-1 activation and the subsequent release of il-1β caused by ps-nh2 nanoparticles. these data reveal a novel mechanism of the nlrp3 activation induced by amino-functionalized nanoparticles and provide a strategy as to how such an effect can be functionally antagonized by supplementation with a radical scavenger. this work was supported by the dfg spp1313. the semi-permeable barrier of the endothelial cell lining of the blood vessels has important synthetic and metabolic functions including transport of cells and biomolecules, regulation of vascular smooth muscle tone, and control of hemostasis. plasmin is a serine protease, which is generated from its zymogen plasminogen under physiological and pathological conditions. small amounts of plasmin are produced in the context of contact activation during inflammation. consistently, increased generation of plasmin has been reported during atherosclerosis. we have shown previously that plasmin, in addition to its role in fibrinolysis, could induce proinflammatory activation of various cells including monocytes, macrophages, and dendritic cells. therefore, we analyzed how plasmin might affect the functions of endothelial cells, which could be relevant during inflammation and atherosclerosis. using flow cytometry, western immunoblotting and fluorescent microscopy, we show that endothelial cells of different origin express the plasmin receptor complex composed of annexin a2 and s100a10. addition of plasmin to human umbilical vein endothelial cells (huvec) induced timeand concentration-dependent cytotoxic effects in the cells. in addition, within 30 min plasmin triggered a rapid and prolonged expression of free radical oxygen species (ros) in endothelial cells as analyzed by microscopy and fluorometry using the rossensitive dye carboxy-h 2dcfda. the ros production in endothelial cells was accompanied by cell detachment. fluorometric and western blot analysis of caspase 3 activation in the cells treated with plasmin showed that plasmin induced apoptotic cell death in endothelial cells, which was evident already several hours after exposure to plasmin. thus, plasmin might induce production of ros in endothelial cells, their detachment and apoptosis, events which might be relevant for the development of atherosclerosis. this work was supported by the dfg. sesquiterpene lactones (stl) comprise a large group of secondary plant metabolites that constitute the active principle of a number of traditional anti-inflammatory phytomedicines. specifically helenalin and parthenolide have recently gained considerable attention as lead compounds or putative therapeutics for the treatment of inflammation and possibly cancer. both compounds have been shown to interfere with the signal transduction through inhibition of the nuclear factor κb (nf-κb). whereas the inhibitory effects of the stl on nf-κb are undisputed, their molecular mechanism of action remains a matter of debate. surface plasmon resonance (spr) analysis allows label-free measurement of molecular interactions. yet, analysis of the interaction of immobilized recombinant proteins with small molecular ligands remains a technically challenging task. in the present study we used spr technology to investigate the molecular interaction of the stl helenalin with putative intracellular target proteins such as the nf-κb protein p65/rela, the catalytic subunits of the ikk complex, namely ikkα and ikkβ, and the intracellular antioxidant glutathione (gsh). at physiological ph 7.4, helenalin interacts with rela (k d = 4.8 µm), yet it failed to bind either ikkα or ikkβ. hence, when dna with nf-κb binding consensus sequence was immobilized on sensor chips, the binding of rela was inhibited by helenalin with an ic50 of 5.0 µm. moreover, we provided several lines of evidence that stl may modify rela on cysteine 38 by a michael-type addition. this interaction was confirmed by molecular docking that identified the best matching interaction between rela and helenalin with predicted hydrogen bonding interactions between helenalin and residues arg35, lys37, gly44 and ile118 of rela. consistent with our hypothesis that helenalin interacts with sulfhydryl groups at ph 8.0, helenalin was also able to interact with reduced, but not oxidized, glutathione with a kd of 24 µm, though no significant interaction was observed at ph 7.4. thus, we showed that the sesquiterpene lactone helenalin interacts with the nf-κb protein rela but not with ikkα or ikkβ. moreover, at physiological ph, helenalin does not interact with glutathione to any significant extent. direct interaction of helenalin with rela leading to inhibition of rela-dna binding and transactivation might present the molecular mechanisms underlying the anti-inflammatory effects of stls. although nanosized materials are quickly taken up by macrophages, our understanding of the involved processes is still rather limited. therefore, we analyzed the uptake of diagnostically used carboxydextran-coated iron oxide nanoparticles of two different sizes, superparamagnetic iron oxide nanoparticles of 60 nm (spio) and ultrasmall superparamagnetic iron oxide nanoparticles of 20 nm (uspio), by human macrophages. by pharmacological and in vitro knockdown approaches, the principal uptake mechanism of macrophages for both particles was identified as clathrin-mediated, scavenger receptor a-dependent endocytosis. further, we created a mathematical model of the nanoparticle uptake by macrophages that permitted determination of key parameters of endocytotic process, such as the uptake rate, the mean uptake time, the number of particles taken up by a cell, and the correlation between the number of internalized particles and their extracellular concentration. the model also provided information on the individual and collective wrapping time of the nanoparticles and described the relation between biophysical parameters such as cytoskeletal forces, membrane elasticity, and the uptake time. finally, we gained information on the minimal linear spacing between simultaneously acting neighboring endocytotic pits that contain single nanoparticles and govern the collective uptake process. the calculated parameters were further confirmed experimentally using spinning disc confocal microscopy. thus, the new model provides important insights into the biophysical processes involved in endocytosis of nanoparticles by human macrophages. this work was supported by the dfg spp1313. prostaglandins (pg) are hormones which are formed during inflammatory processes from arachidonic acid by cyclooxygenases and prostaglandin synthases [4] . in the subsequent metabolism, in which the five-membered ring is dehydrated, α,β-unsaturated carbonyl compounds are generated [2, 3] . these come along with mercapto groups of amino acids in a michael addition reaction associated with activation of cellular enzyme cascades [1] that potentially contribute to their possessed antiinflammatory, antineoplastic and antiviral effects [5] . however little is known so far about possible adverse health effects.we addressed the question whether selected cyclopentenone prostaglandins (cypg) exhibit potential mutagenic and genotoxic properties in the hamster lung fibroblast cell line v79. induction of dna damage was investigated by single cell gel electrophoresis assay (scge). the impact of cypg on cellular redox status was detected by total glutathione (tgsh) assay. the induction of micronuclei and apoptosis was determined by staining with 4',6-diamidino-2-phenylindole (dapi). furthermore the hypo-xanthine-guanine phosphoribosyltransferase (hprt) assay was used for mutagenicity testing. , followed by prostaglandin a2 (pga2), showed the most distinctive genotoxicity, i.e., induction of micronuclei, and apoptotic effects. furthermore, the 15dpgj2 and pga2 -induced significant decrease in the tgsh level in v79 may contribute to the observed increase in oxidative dna-damage. however, none of the tested cypg exhibited mutagenic properties in the hprt assay. in conclusion, a potential in vitro genotoxicity of cypg has been observed which may be involved in carcinogenesis associated with chronic inflammation. parabens and methylisothiazolinone are used as preservatives in personal care products. sensitization to parabens and methylisothiazolinone is relatively rare considering their wide use in cosmetics, but only few quantitative or clinical data exist. therefore, we have tested methyl-, ethyl-, propyl-, isopropyl-, butyl-, isobutyl-, pentyl-, phenyl-and benzylparaben , and methylisothiazolinone in the loose-fit coculture-based sensitization assay (lcsa) developed by our working group. the coculture of primary human keratinocytes and allogenic dendritic cell-related cells (dc-rc) in this assay emulates the in vivo situation of the human skin. sensitization potential of the test substances was determined by flow cytometric analysis of the dc-rc maturation marker cd86. determination of the concentration required to cause a half-maximal increase in cd86-expression (ec 50) allowed a quantitative evaluation. the irritative potential of the substances was assessed by 7-aad (7-amino-actinomycin d)-staining. the concentration required to devitalize 50 % of the examined cells compared to a zero control was termed ec50%. parabens exhibited weak (methyl-, ethyl-, propyl-and isopropylparaben) or strong (butyl-, isobutyl-, pentyl-and benzylparaben) sensitizing potential, phenylparaben was found to be a moderate sensitizer, with ec50-values ranging from 16.67 µmol/l (pentylparaben) to 325.36 µmol/l (methylparaben). due to a pronounced cytotoxicity (ec50% = 70.94 µmol/l), we could not estimate an ec50-value for methylisothiazolinone. sensitization potential of parabens correlated with side chain length. parabens showed no (methyl-and ethylparaben) or weak irritative potential (propyl-, isopropyl-, butyl-, isobutyl-, phenyl-and benzylparaben) , only pentylparaben was rated to be irritative. apart from phenyl-and benzylparaben, irritative potential also correlated with side chain length but did not correlate strictly with the sensitization potential. overall, we were able to demonstrate and compare the sensitizing potential of parabens in this in vitro test. it was weak for methyl-and propylparaben, the most commonly used parabens. furthermore, we showed an irritative potential for most of the perservatives. thus the lcsa is a useful in vitro test to compare the sensitizing potential of xenobiotics. phosphorylation of the neurodegeneration-related septin 4 by protein kinase dyrk1a at serine 107 affects protein stability soppa u. 1 septins are gtp-binding proteins forming heterooligomeric complexes and filaments by interactions of the 13 family members. these complexes have important functions by building scaffolds for proteins involved in cell cycle or cell polarity but their subcellular distribution as well as their regulation remain largely unclear. septin 4 (sept4) was found in neurodegeneration related protein aggregates and is associated with migration of cortical neurons. here we first describe a potential mechanism for regulation of sept4 by phosphorylation via dual specificity tyrosine-phosphorylation-regulated kinase 1a (dyrk1a). dyrk1a is overexpressed in down syndrome and supposed to be involved in neurodevelopment and neurodegeneration. by site directed mutagenesis of flag tagged mouse sept4 and overexpression in hela cells we identified serine 107 as the major phosphorylation site of dyrk1a and generated a phosphospecific antibody. transient coexpression of sept4 and dyrk1a in hela cells increased phosphorylation of serine 107 by 50% in relation to basal phosphorylation. in contrast, cotransfection of the kinase deficient dyrk1a mutants k188r and d287n did not increase serine 107 phosphorylation. moreover we could show, that inhibition of kinase activity by the dyrk1a inhibitor harmine reduced phosphorylation of exogenous sept4 at serine 107 about 25% in hela cells. furthermore, down regulation of dyrk1a by rna interference lead to decreased phosphorylated serine 107. these results indicate that endogenous dyrk1a contributes to sept4 phosphorylation in hela cells. finally we analyzed protein stability of wild type sept4 compared to the phosphorylation resistant s107a mutant in hela cells by inhibition of translation with cycloheximide. we found that in living cells the sept4 s107a mutant is more stable than wild type sept4. in summary, our results suggest phosphorylation at serine 107 by dyrk1a as a novel mechanism to regulate sept4 stability and indicate a possible link of these proteins in cellular processes. is formaldehyde a good example for a "genotoxic carcinogen" with a threshold mode of action? speit g. institut für humangenetik, universität ulm, 89069 ulm, germany formaldehyde (fa) induces toxic and genotoxic effects in directly exposed cells (site of contact). several studies in which fa was administered to rats by inhalation showed evidence of tumor induction in nasal epithelium. there is also some epidemiological evidence that fa causes nasopharyngeal cancer in humans. although fa is a known mutagen, it is still a matter of discussion whether carcinogenesis is primarily mediated via a mutagenic mode of action. there is evidence that cytotoxicity and induced proliferation are the main causes for tumor formation. however, a decisive role of mutagenesis cannot be excluded and a mutagenic mode of action has to be considered for risk estimation. the basic assumption is that mutagens have a non-threshold mode of action. a threshold mode of action for a chemical is likely when a substance with a known mutagenic potential does not induce mutations at low concentrations due to a specific type of reaction with the genetic material and / or physiological protective mechanisms. because fa is a directly acting dna-reactive substance, a threshold mode of action may only be considered because of physiological protective mechanisms. after inhalation, pre-lesion protection occurs by unspecific binding to mucus, cellular proteins and glutathione. furthermore, fa is efficiently inactivated by enzymatic pathways. if fa reaches and damages the nuclear dna, dna repair mechanisms act as efficient postlesion protection mechanisms. in vivo inhalation studies with rats indicated that fa induces primary dna damage in the nasal epithelium but increased mutation frequencies were not measured. data are now available to show the relative distribution of endogenous versus exogenous dna adducts in different locations of the nose and other organs. considering the fa concentrations present in every living cell and the background levels of endogenous dna adducts, appropriate risk assessment and the identification of practical thresholds for fa-induced genotoxicity become feasible. fraud and misconduct in clinical trials steffen c. formerly federal institute for drugs and medical devices clinical trials unit, kurt-georg-kiesinger-allee 3, 53175 bonn, germany plagiarism in scientific publications has been the subject of public ("guttenplag") and scientific debate. plagiarism violates, however, "only" the intellectual property of its authors. in clinical trials, misconduct may also endager the health of patients. the suppression or falsification of data in clinical trials may mislead patients and doctors to use worthless treatments. clinicians may be provoked to repeat these trials, thereby wasting time and money. in clinical trials, misconduct includes everything from suppression or their repeated publication, the "correction" of unwanted results to the complete invention of the data, their intentional or negligent misinterpretation leading to the publication of biased conclusions from otherwise correctly performed clinical studies. the peer review system cannot protect against fraud, as few reviewers will have the means and the time to reevaluate original data. they will have to rely on these data, the calculations and statistics that are presented to them. some kinds of fraudulent behavior, such as double publications, can be found in the review process, but this is also time-consuming and depends on a helpful librarian. other kind of fraud, as the suppression of patient data in clinical trials (the deletion of "non-responders", according to cinderella: the good ones go into the pot, the bad ones go into your crop) can only be detected by the national competent authorities performing an inspection according to good clinical practice. even if the results of such an inspection become public as in the case of ukrain (gansauge et al. 2002) , there is no institution that will further analyze and publish the misconduct or initiate the retraction of the incriminated paper. a german agency similar to the us office of scientific integrity could foster good clinical practice in germany. although it is sometimes very difficult to decide whether improper results arise from fraud or error, a bias for the source of funding is obvious. trials funded by for-profit organizations were significantly more likely to recommend the experimental drug as treatment of choice (als-nielsen et al. 2003) . medicinal products with disputed efficacy such as orally applied enzymes for systemic action, bacterial preparations for irritable bowel syndrome and food supplements are to be reviewed with special attention. further examples from the author's experience will be presented. ]i favors further kca opening, kca may establish a feed-forward regulation of ca 2+ influx. in the present study we analyzed whether kca channels of sk4 and bk type play a role in sustaining ca 2+ oscillations at g1-to s-phase transition of primary mouse tumor cells and in human breast cancer cells, aiming towards a better understanding how kca modulate tumor cell proliferation. methods: kca expression was quantified by qpcr in human breast cancer biopsies, transgenic mmtv/c-neu + mouse mammary tumors and primary tumor cells derived thereof. the identity of the tumor cells was verified by gliolan staining. proliferation of the primary mammary tumor cells in the presence or absence of bk and sk4 modulators was tested using a real time cell monitoring system. the cellular dna content as a measure for cell ploidity was determined by propidium iodide staining and flow cytometry. changes in [ca 2+ ]i oscillations and peak amplitude were determined using ca 2+ indicator fura-2am. results: sk4, but not bk, expression is detectable in human and mouse breast cancer biopsies and in primary tumor cells derived from the mmtv/c-neu + mouse model. sk4 inhibition by tram-34 dose-dependently (0,1 to 10 µm) inhibits the growth of primary mammary tumor cells probably by a g1 cell cycle arrest. ca 2+ oscillations in proliferating mmtv/c-neu + tumor cells were ablated upon pharmacologic inhibition of sk4 channels. -dependent cell cycle progression is dependent on sk4 activity. blocking sk4 disrupts a feed-forward loop that coordinates ca 2+ influx via trp or crac channels in tumor cells. the consequences of sk4 inhibition in mammary tumors in vivo will be discussed. synthesis of a triphenylphosphonium substituted derivative of 5-hydroxymethyl-5-methylpyrroline n-oxide stolze k. 1 esr combined with spin trapping is a well-known analytical approach to detect free radicals formed in various biological systems, e.g. superoxide, hydroxyl and a series of carbon-centered free radicals, which are involved in oxidative stress. our aim was to modify the established spin trap 5,5-dimethyl-pyrroline n-oxide (dmpo) with a functional side chain, which can be used further as anchor for moieties enabling the spin trap to penetrate mitochondrial membranes, such as the positively charged triphenylphosphonium substituent. several synthetic routes were tested to introduce a 4-carboxybutyltriphenylphosphonium-substituent to the spin trap 5-hydroxymethyl-5-methylpyrroline noxide (hmmpo). while the activation of the carboxy group via the corresponding chloride was not successful, the use of a mixed anhydride with acetic acid appeared to be a promising way, although the reaction is considerably slower. preliminary spin trapping experiments have been performed with model systems generating superoxide, hydroxyl-, and carbon-centered radicals. othman e. m., stopper h. universität würzburg toxikologie, versbacher str. 9, 97078 würzburg, germany type 2 diabetes mellitus (dm2) is a growing health problem affecting more than 150 million people worldwide. it is associated with severe acute and chronic complications that negatively influence both the quality of life and survival of affected individuals. epidemiological studies clearly indicate that the risk of several types of cancer (including pancreas, liver, breast, colorectal, urinary tract and female reproductive organs) is increased in diabetic patients. diabetic patients are exposed to oxidative stress which plays a pivotal role in the pathogenesis of both micro-and macro-vascular complications. this is due to a decreased antioxidant capacity and chronic exposure to increased levels of reactive oxygen species (ros). since the insulin resistance in dm2 leads to hyperinsulinemia we studied the cellular consequences of the elevated insulin level and showed that it generates superoxide anions (o 2-) and dna damage by a nadph oxidase dependent mechanism in cultured cells. in addition, we found elevated genomic damage in the lymphocytes of diabetic patients as well as oxidative stress and genomic damage in kidneys of diabetic rats. this effect of insulin may contribute to the pathogenesis and progression of dm2 complications including the elevated cancer risk. the classical transient receptor potential (trpc) channel subfamily is regarded as nonselective, calcium permeable cation channels involved in a wide range of physiological events that require calcium signaling. until now, the specific roles of trpc channels in neuronal function are still elusive. given that trpc1 is able to form receptor-operated heterotetrameric channel complexes with other trpc channel subunits, we investigated the role of trpc1 for receptor-operated calcium influx in the heterologous expression system as well as in neurons. for this electrophysiological whole-cell measurements, fluorimetric calcium measurements, mn 2+ quenching and qpcr analysis were applied. furthermore, the effect of trpc1 knock-down on neuronal migration was monitored performing scratch assays, videomicroscopy and g-actin/f-actin assays. employing these techniques, we found that recombinant trpc1 was not able to function as a homomeric channel. instead, trpc1 subunits formed functional receptor-operated heteromeric channel complexes with trpc3, 4, 5, 6, and 7. heteromers containing trpc1 subunits showed significantly decreased calcium permeation in heterologous cell systems. mutation of amino acids in the putative pore forming region of trpc1 further reduced calcium permeability. in gnrh neurons endogenously expressing trpc1, 2, 5, and 6, downregulation of trpc1 by shrna resulted in increased basal cytosolic calcium concentrations and elevated calcium permeability. trpc1 was not involved in store-operated cation influx in gnrh neurons. moreover, trpc1 suppressed the migration of gnrh neurons without affecting cell proliferation. these findings suggest a novel regulatory mechanism relying on the expression of trpc1 and the subsequent formation of heteromeric trpc channel complexes with reduced calcium permeability, thereby fine-tuning neuronal migration. the transient receptor potential melastatin-3 (trpm3) is a calcium permeable nonselective cation channel that can be activated by the neurosteroid pregnenolonesulfate (pregs) or heat. trpm3 is expressed in various tissues, including insulin-secreting βcells and a subset of sensory neurons from dorsal root (drg) and trigeminal ganglia. the ability of pregs to evoke trpm3-like currents in pancreatic β-cells and to induce insulin secretion indicated its involvement in blood glucose regulation. however, trpm3 -/mice show so far no metabolic deficits but further investigations are recommended to evaluate its function in insulin secretion. further studies showed that trpm3 is a nociceptor channel involved in sensing heat and inflammatory thermal hyperalgesia. we performed a calcium-based screening of a compound library (spectrum collection) that identified several natural compounds as trpm3 blockers. the most potent blockers were the citrus fruit flavonoids hesperetin and naringenin as well as ononetin, a chalcon from ononis spinosa. the ic50 values of the substances are in the low micromoles ranges. electrophysiological whole cell measurements as well as calcium measurements confirmed the potency of the trpm3 blockers. furthermore, we could show that these blockers are effective on endogenous trpm3 in drg neurons from mice and isolated β-cells. by drinking grapefruit juice naringenin could be consumed in concentrations that are sufficiently high enough to block trpm3 activity in vivo. in sensory neurons, trpm3 may exert similar functions as trpv1. thus, trpm3 blocker could bear a therapeutic potential for analgesic treatment. xtt-based cell viability assay was used to determine the half-maximum effect concentration (ec50) for the investigated composite components in hgf. following concentrations of substances were used to determine the induced double strand dna breaks (dsbs): 1 /25× ec50, 1 /10× ec50 , 1 /3× ec50, and 1× ec50. each experiment was performed at least four times. hgf were incubated with various concentrations of substances for a period of 6 hours. induced dna double-strand breaks (dsbs) were tested by the γh2ax focus assay, which is a direct marker for dsbs using anti γh2ax antibodies. for quantitative γh2ax analysis foci in cell nucleus were counted by eye down using a fluorescence microscope. each experiment was performed at least four times. in the xtt test following ec50 values of substances were found (mmol/l;mean +/-sem): tmp(eo)9ta a, b 0.087 ± 0.011; 1,6-hddma b, c 4.500 ± 0.700; etma a, c ; 12.000 ± 1.100; a significantly (p < 0.05) differently to 1,6-hddma, b significantly (p < 0.05) differently to etma, c significantly (p < 0.05) differently to tmp(eo)9ta. after six hours of exposure with tmp(eo)9ta at 0.00348 mm there were induced 0.55 γ-h2ax foci-formations in hgfs, at 0.00870 mm 0.67 foci, at 0.02900 mm 0.86 foci and at 0.08700 mm 0.97 foci. after exposure with 1,6-hddma at 0.180 mm there were induced 0.45 γ-h2ax foci, at 0.450 mm 0.64 foci, at 1.500 mm 0.94 foci and at 4.500 mm 1.32 foci. after exposure with etma at 0.48 mm there were induced 0.43 γ-h2ax foci, at 1.2 mm 0.50 foci, at 4.0 mm 0.61 foci and at 12 mm 0.71 foci. the negative controls dmso and medium cultures displayed 0.31 -0.34 γ-h2ax foci/cell. it was found that the induction of foci/cell were concentration-dependet for all xenobiotics in the order of: 1,6-hddma > tmp(eo)9ta > etma. these results show that dental composite components can induce dsbs in primary oral cells and therefore these substances demonstrate a genotoxic potential. effects of antioxidants on the dna-toxicity of dental (co)monomers in human gingival fibroblasts styllou p. 1 , scherthan h. unreacted (co)monomers can be released from restorative dental materials and may show biologic activity after ingestion in the human organism. in previous studies the mutagenic/carcinogenic effect of dental monomers/co-monomers (e.g. methacrylates) on the human dna was demonstrated. in this study the effects of the antioxidants vitamin c and n-acetylcysteine on the dna toxicity of the (co)monomers triethylenglycol-dimethacrylate (tegdma) and 2-hydroxyethyl methacrylate (hema) was investigated. the induction of dna double-strand breaks with (co)monomers alone and in combination with antioxidants was investigated in human gingival fibroblasts (hgf). hgf were incubated with substances without or with antioxidants for a period of 6 hours. induced dna double-strand breaks (dsbs) were tested by the γh2ax focus assay, which is a direct marker for dsbs using anti γh2ax antibodies. for quantitative analysis of the γ-h2ax test, foci were counted by the same investigator by eye down the fluorescence microscope. each experiment was performed at least four times. the halfmaximum effect concentration ec 50 (mmol/l) of triethylenglykol dimethacrylat (tegdma) and 2-hydroxyethyl methacrylat (hema) was taken from of a previous study after using xtt-based cell viability assay. tegdma induced significantly (p < 0.05) higher dsbs compared to hema (1.91 ± 0.04 vs 1.66 ± 0.02). the mean number of cells scored and the standard deviation (sd) were calculated. when cells were exposed to tegdma in combination with the antioxidant vitamin c an increase of dsbs was observed (2.02 ± 0.06), compared to tegdma alone. when cells were exposed to hema in combination with vitamin c an increase of dsbs was observed (1.89 ± 0.07), compared to hema alone. when cells were exposed to tegdma in combination with the antioxidant nacetylcysteine a decrease of dsbs was observed (1.64 ± 0.04), compared to tegdma alone. when cells were exposed to hema in combination with n-acetylcysteine a decrease of dsbs was observed (0.76 ± 0.02), compared to hema alone. these results show that dental (co)monomers can induce dsbs in primary oral cells. it also shows for the first time that the genotoxic potential may be reduced by the addition of the antioxidant n-acetylcysteine. purpose: we aimed to investigate the role of superoxide and peroxynitrite generated by genetically destabilized enos for the development of endothelial dysfunction and vascular remodelling. methods: a mutant of bovine enos in which cys 101 was replaced by ala (c101a) resulting in destabilization of enos has been generated (enos-c101a). transgenic mice carrying c101a were generated on a c57bl/6 background using the endotheliumspecific tie-2 promoter. by breeding these mice with enos knockouts (enos-ko), mice that express enos-c101a (enos-ko/enos-c101a-tg) exclusively in the endothelium were obtained. unilateral common carotid artery ligation experiments were performed in c57bl/6, enos-ko, and enos-ko/ enos-c101a-tg to study a role of destabilized enos for vascular lesion formation. results: western blot analysis confirmed the expression of enos in enos-ko/enos-c101a-tg in aorta (37.1±8.4%, n=9), skeletal muscle (45.4±5.3%, n=10) and myocardium (17.4±4.9%, n=7) and revealed an increased phosphorylation of enos on ser1176/79 (470±47%) as compared to c57bl/6 (p<0.05, n=8). endothelium-specific overexpression of destabilized enos induced a large increase in superoxide and peroxynitrite formation in the aorta and the heart of enos-ko/enos-c101a-tg (p<0.05, n=5-8), which was abolished by nos-inhibitor l-nitroarginine (l-na) suggesting enos-c101a as a source of elevated radical generation. endothelium-specific introduction of enos-c101a at ~ 35% of c57bl/6 level almost completely restored aortic endotheliumdependent relaxation. experiments with l-na, soluble guanylyl cyclase inhibitor odq, peg-catalase and no-scavenger fe(detc)2 indicated that endothelium-dependent relaxation in enos-ko/enos-c101a-tg is nos-and cgmp-dependent and nomediated. four weeks after the carotid artery ligation, neointima formation, media thickening and luminal narrowing were observed in the ligated arteries of all studied genotypes (p<0.05, n=4-7). consistent with vasoprotective roles of enos, neointima formation was accelerated in enos-ko (n=4-7, p<0.05). despite significantly higher vascular levels of nitrotyrosine and peroxynitrite, neointima formation in enos-ko/enos-c101a-tg was substantially lower then in enos-ko and tended to be similar to c57bl/6. conclusions: increased vascular superoxide and peroxynitrite formation caused by destabilization of enos does not induce endothelial dysfunction in healthy mice and has negligible effect on neointima formation. fenton reactivity as a determining parameter for the interaction of manganese oxide nanoparticles with lung epithelial cells sydlik u. 1 , bieschke c. nanoparticles consisting of manganese oxide have been suggested for several innovative technological approaches, including the use in nanomedicine and diagnostics. therefore, the interaction of such nanoparticles with human target cells is of particular interest for the success of nanomedical approaches but also with regard to unintended side effects. to address this problem, we tested different kinds of manganese nanoparticles (mnnp) in an in vitro system which we earlier evaluated for proliferative, apoptotic, and pro-inflammatory endpoints induced by carbon nanoparticles (cnp). mnnp were synthesized by hydrothermal treatment of manganese salt solutions. the particles were subsequently characterized by scanning electron microscopy and dynamic light scattering. biological and toxic effects of the generated particles were studied in comparison to carbon nanoparticles (cnp) in experiments with rat and human lung epithelial cells (rle-6tn and 16hbe14o-). cytotoxicity was determined as measures of membrane damage (lactate dehydrogenase release) and metabolic activity (water soluble tetrazolium conversion). the oxidative capacity of the particles as well as the generation of intracellular oxidative stress was monitored using dichlorofluorescein diacetate in cell free experiments and flow cytometry assays (facs), respectively. the particle-specific phosphorylation of src family kinases (sfk) and mitogen activated protein kinases erk1/2 were investigated using western blot techniques. after physico-chemical characterization, a set of three mnnp consisting of mn3o4 or mno2 with significant differences in size and shape were selected. according to the different oxidation stages of manganese, the particles showed significant differences in fenton reactivity in the cell free system. these data did not reflect the capacity of the particles to induce intracellular oxidative stress. the characteristic to trigger membranedependent signaling processes, however, was correlated to the intrinsic oxidative capacity of mnnp than to the ability to induce intracellular ros. furthermore, the metabolic activity (wst) was negatively correlated with intracellular ros, indicating a link between mitochondrial activity and ros generation. none of the particles had effects on the membrane integrity of the cells. the data demonstrate that mnnp, unlike other poorly soluble nanoparticles (e.g. cnp), mainly trigger adverse health effects through ros production via the fenton reaction. acute ozone induced airway inflammation does not effect resting human sympathetic nerve traffic tank j. 1 numerous mediators released in inflammatory and neuropathic pain states activate gprotein-coupled receptors (gpcrs) and modulate nociception via activation of gs, gi/o, g12/13, or gq/11 g proteins. each of the g protein-coupled receptor pathways is involved in nociceptive modulation and pain processing, but the relative contribution of the individual signaling pathways in vivo has not yet been worked out. the gq/11 signaling branch is of particular interest in pain research because it leads to the activation of phospholipase c, protein kinase c, and the release of calcium from intracellular stores. using a conditional gene-targeting approach we generated double-deficient mice lacking gaq and ga11 selectively in nociceptors to investigate the contribution of the entire gq/11signaling pathway in nociceptors towards the regulation of pain. we observed that mice lacking gq/11 in nociceptive neurons show normal development of the nociceptive circuitry. the nociceptor-specific loss of gq/11 results in reduced pain hypersensitivity following paw inflammation or spared nerve injury. surprisingly, our behavioral and electrophysiological experiments also indicated defects in basal mechanical sensitivity in gq/11 deficient mice, suggesting a novel function for gq/11 in tonic modulation of acute nociception. patch-clamp recordings revealed changes in voltagedependent tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels in nociceptors upon a loss of gq/11, whereas potassium currents remained unchanged. our results indicate that the functional role of the gq/11 branch of g-protein signaling in nociceptors in vivo not only spans sensitization mechanisms in pathological pain states, but is also operational in tonic modulation of basal nociception and acute pain. provocation of arrhythmic events in single primary isolated adult mouse ventricular cardiomyocytes tekook m., fehrmann e., schulte j. s., schmitz w., müller f. u. westfälische wilhelms-universität institut für pharmakologie und toxikologie, domagkstraße 12, 48149 münster, germany ap duration and ca 2+ cycling are altered in cardiomyocytes of different genetic mouse models. here, we systematically tested various protocols to study the inducibility of arrhythmic events in mouse cardiomyocytes. adult ventricular cardiomyocytes were isolated from wildtype (wt) mice by enzymatic digestion and subsequently tested to trigger arrhythmic events within 6 hours after isolation. in patch clamp experiments (perforated patch, whole cell current clamp) aps were stimulated for 1 second (5hz; 10hz; 5hz + s2-stimulus after 50-80ms) followed by a 4s rest period. the resting-membrane-potential (rmp) was observed over 30 cycles. we observed rmp-fluctuations of different length (amplitude <5 mv; % of 13 observed cardiomyocytes, mean events/cell; <1s: 100%, 17.1; <3s: 92%, 11.5; >3s: 69%, 5.8), spontaneous depolarizations (>5 mv; 92%, 5.1) and spontaneous aps (62%, 1.9). intracellular ca 2+ transients and sarcomere shortening were measured after loading cardiomyocytes with indo-1/am. after preconditioning (10 min/1 hz) cells were measured under basal and continuous isoprenaline (10 -6 m) stimulation (iso). a 4 min pacing period was followed by a 1 min interval of no pacing. pacing frequency was reduced after each cycle (1 hz, 0.5 hz, 0.25 arrhythmic events were provocable with stimulation/rest protocols both by field stimulation and direct stimulation via patch pipette. however, low stimulation frequencies seem to lead to distinct destabilization of cardiomyocytes probably due to ca 2+ overload. we conclude that the tested stimulation protocols are able to provoke arrhythmic events even in wt single adult mouse ventricular cardiomyocytes and may serve as a tool to test for the relevance of potential proarrhythmic substrates in mouse models. ruhr-universität bochum pharmakologie ma nord1, bochum, germany cgmp is a second messenger involved in many (patho-)physiological processes such as smooth muscle relaxation, platelet inhibition, and the development and plasticity of the nervous system. however, it is not fully understood how cgmp regulates these and other processes on a mechanistic level. in particular, the existence and functional relevance of global and local cgmp signaling domains is not clear. recently, highly specific genetically-encoded optical biosensors for cgmp have been developed. these cgmp indicators are either based on fluorescence resonance energy transfer (fret), with cgmp-binding domains sandwiched between fluorescent proteins with overlapping spectra, or they consist of a single fluorescent protein fused to cgmp-binding domains. with these cgmp indicators, the spatiotemporal dynamics of cgmp signals, which result from the interplay between cgmp-producing guanylyl cyclases, cgmp-binding effectors, and cgmp-degrading phosphodiesterases (pdes), can be monitored in living cells. here, we report the generation of transgenic mice expressing the fret-based cgmp indicators cgi500 and cgi6000 with apparent cgmp affinities of 500 nm and 6000 nm, respectively. one mouse line expresses cgi6000 driven by a cmv promoter in neural cells. fret experiments were performed with isolated cerebellar granule neurons, hippocampal neurons, and astrocytes. we observed nitric oxide (no)-induced cgmp transients and analyzed the capability of other agents (natriuretic peptides, glutamate) to induce cgmp responses. in another mouse line, the sm22alpha promoter directs cgi500 expression specifically to smooth muscle cells (smcs). fret experiments have been performed with smcs isolated from aorta, bladder and colon, as well as with intact vessels in the retina and cremaster muscle of transgenic animals. in primary smcs we studied responses to no, atrial and c-type natriuretic peptide (anp,cnp). in different smc types we observed differences in the overall ability to react to these stimuli and in the kinetics of the induced cgmp transients. we also studied the effects of pde inhibitors on the no-, anp-, and cnp-induced cgmp signals. importantly, we were able to detect cgmp transients upon no stimulation in intact vessels of the retina and cremaster. we conclude that the cgi transgenic mouse lines are valuable tools to visualize cgmp signals in living cells in vitro and, possibly, also in vivo in the intact animal under physiological and pathophysiological conditions. current research data dealing with pharmacotherapy of α-ama intoxication shows a particularly high variability regarding the protective effect of silibinin. the aim of this study was therefore to evaluate the influence of the frequently used clinical antidotes benzylpenicillin, silibinin and their combination in human hepatocyte culture intoxicated with α-ama. cytotoxicity and apoptosis testing were performed after two and five days of simultaneously exposure to α-ama and/ or tested antidotes. to quantify apoptosis, necrosis and cell viability, we used cell death detection elisa plus®, toxilight® bioluminescence assay and cell proliferation kit ii (xtt). furthermore, we analysed the ways of apoptosis by using immunohistochemistry (differential detection of caspase 3, 8 and 9, activated caspase 3, and aif). exposure of hepatocytes to α-ama at concentrations of 0,2 µm, 0,5 µm and 1 µm resulted in disorder of cell cultures, apoptosis and reduction in cell viability compared with unexposed hepatocytes. in hepatocyte cultures treated with benzylpenicillin at concentrations of 30 µm and 1mm, silibinin at 50 µm and 100 µm and a combination of both (30 µm benzylpenicillin and 50 µm silibinin, 1mm benzylpenicillin and 100 µm silibinin), toxilight® values in the supernatant and xtt values were not significantly different from untreated cultures. simultaneous exposure to α-ama (at all tested concentrations) and benzylpenicillin, silibinin or combination of both showed higher cell viability and lower values of necrosis compared to the cultures exposed to α-ama alone (exept 50 µm silibinin at 0,2 µm α-ama); however, in both groups dosed with benzylpenicillin the highest hepatocyte viabilitiy was observed. this protective effect was particularly revealed at high α-ama concentrations (0,5 µm and 1 µm). in conclusion, our data suggest that benzylpenicillin in monotherapy is more effective than in combination with silibinin or silibinin alone. glucocorticoids (gcs) are important hormones in the regulation of metabolic homeostasis. synthetic gcs, such as dexamethasone (dex), play a fundamental role in the treatment of inflammatory diseases. there are numerous side effects of a dex therapy, e.g. the development of hypertension. in the pathogenesis of hypertension oxidative stress is a crucial factor. glucocorticoid-induced hypertension has been shown to be associated with an imbalance between nitric oxide (no) and superoxide. however, the source of this elevated superoxide production is unknown. we hypothesize that an uncoupling of the no synthase (enos), a key mediator of vascular homeostasis, may contribute to dex-induced oxidative stress. incubation of human endothelial cells (ea.hy 926) with dexamethasone led to a decrease in enos expression at mrna and protein levels. this effect of dex was timeand concentration-dependent. since the major cause of enos uncoupling is a deficiency of its co-factor tetrahydrobiopterin (bh 4), we analyzed the amount of bh4 in ea.hy 926 by hplc. a concentration-dependent reduction of bh4 and also bh2 (dihydrobiopterin) could be demonstrated in response to treatment with dexamethasone. bh4 can be synthesized endogenously by two different pathways -the de novo pathway (from gtp with gtp cyclohydrolase i, gch1, acting as the rate-limiting enzyme) and the salvage pathway (conversion of sepiapterin to bh4 involving dihydrofolate reductase, dhfr). treatment of ea.hy 926 cells with dex decreased mrna and protein expression of both gch1 and dhfr. because bh4 is the major "coupling switch", an enos uncoupling is likely to occur in dex-treated cells. in summary, we showed that dex treatment led to a reduced availability of the important co-factor bh4 which could lead to enos uncoupling. the uncoupled enos may possibly contribute to glucocorticoid-induced vascular oxidative stress. the cellular oncoprotein c-fos is a major component of the heterodimeric transcription factor ap-1 and has been commonly found over-expressed in tumors and cancer cells of different origin. previous work showed that mouse cells lacking the immediate-early gene c-fos are hypersensitive to ultraviolet (uvc) light. here we demonstrate that in human telomerase-immortalized vh10tert foreskin fibroblasts (behaving like primary cells) and sv40-immortalized gm637 fibroblasts, uvc-triggered induction of c-fos protein is a delayed and long-lasting event. sustained up-regulation of c-fos went along with transcriptional stimulation of the nucleotide excision repair (ner) gene xpf, carrying an ap-1 binding site in the promoter. c-fos mrna was induced in a biphasic manner. an immediate c-fos mrna expression (30-90 min after exposure) was not translated into the protein, the second wave of transcription (4-24h after uvc exposure) resulted in c-fos protein expression, 18-48h post-uv. the stress-activated/mitogen-activated protein kinases (jnk, p38k and erks) were immediately induced upon uvc exposure and stayed active for at least 24h. inhibitor experiments revealed that c-fos was phosphorylated by erks and jnk. the activation of c-fos preceded re-synthesis and the induction of xpf mrna, which was observed 24-40h post-uvc, resulting in the increased expression of the xpf protein. cells over-expressing c-fos showed an accelerated induction of xpf mrna, and consequently a faster repair of cyclobutane pyrimidine dimers (cpds). sirna-mediated silencing of c-fos (transient c-fos knockdown) resulted in abrogated uvc-triggered induction of xpf, attenuated repair of cpds and increased apoptosis. finally, we observed that the removal of cpds but not of photoproducts was significantly faster when cells were pre-exposed to a low uvc dose, indicative of an adaptive response to dna damage. the work was financed by deutsche forschungsgemeinschaft (dfg ch 665/2-1). the addition of clopidogrel to aspirin reduces ischemic events in patients with acute coronary syndrome and in those undergoing percutaneous coronary intervention (pci). however, recurrent ischemic event occurrence during dual antiplatelet therapy remains a major concern. variability in the pharmacodynamic response to clopidogrel is well recognized, and patients with higher platelet reactivity while receiving clopidogrel are at increased risk of ischemic cardiovascular events. clopidogrel is an inactive prodrug requiring biotransformation to form the platelet inhibiting metabolite. interindividual differences in clopidogrel metabolism are the major source of variability in antiplatelet response. polymorphically expressed cytochrome p450 (cyp) enzymes play a critical role in the metabolism of clopidogrel. these findings gave rise to the concept of personalized antiplatelet therapy -i.e. individual platelet function testing and correction of insufficient platelet inhibition to reduce ischemic events in patients with high on-clopidogrel platelet reactivity (hcpr). gravitas was the first study to test this concept by comparing double-dose clopidogrel to standard-dose clopidogrel in patients with hcpr. gravitas failed to correct hcpr consistently in the study arm, which coupled with a low overall event rate precluded demonstrating a substantial benefit from improved platelet inhibition. the trigger-pci trial tested the effectiveness of the more potent thienopyridine prasugrel versus clopidogrel in patients with hcpr after elective pci with implantation of drug-eluting stents (des). switching from clopidogrel to prasugrel in patients with hcpr afforded effective platelet inhibition. however, given the low rate of adverse ischemic effects using contemporary des after pci in stable ischemic heart disease, the clinical utility of this strategy could not be demonstrated and the study was terminated prematurely for futility. multiple studies have shown that both heterozygotes and homozygotes for loss-offunction cyp2c19 alleles have higher rates of adverse cardiovascular events as compared with noncarriers on approved maintenance dosing of clopidogrel (75mg qd), albeit carriage of cyp2c19 loss-of-function alleles accounted for only a minor proportion of the variability in on-clopidogrel platelet reactivity. results of ongoing studies with antiplatelet treatment stratified by cyp2c19 genotyping are awaited to assess the clinical benefit of this approach. the organic cation transporter novel type 2 (octn2/slc22a5) represents a high affinity uptake system for carnitine. besides metabolic disease like severe system carnitine deficiency, genetic variants within the slc22a5 gene have been associated with inflammatory diseases like colitis ulcerosa. against this background, we characterized octn2 expression in peripheral blood cells thereby identifying its expression in all cell types. in the present work we studied octn2 expression in monocytes and thp-1 cells as an in vitro model for this cell type. in addition we examined transcriptional regulation of the carnitine transporter in lps activated thp-1 and investigated the effect of carnitine and its analog mildronate on the respective cytokine response. octn2 expression was characterized on monocytes and thp-1 cells on mrna and protein level. transporter mrna expression could be shown in both cell types by realtime pcr. however, the protein expression was analyzed by western blot, flow cytometry and immunofluorescence microscopy demonstrating octn2 specific signals as well as a localization in the plasma membrane. following thp-1 cells were activated using lps (10ng/ml) for up to 6h, thereby indicating the expected cytokine response as demonstrated by increased tnfα (24fold induction) and il-1β (37fold induction) mrna levels. in addition, octn2 expression was analyzed identifying an initial reduction of around 60% compared to untreated cells. in parallel activated thp-1 cells were coincubated with increasing concentrations of the octn2 substrate carnitine or its analog resulting in reduced cytokine release as shown by elisa for tnfα. here, the tnfα effect was diminished by 64% in the presence of 50mm carnitine. this effect does not rely on a direct neutralization of lps by carnitine since it was also present in cells only preincubated with carnitine. in the present work we could show that thp-1 cells represent a useful model to study octn2 expression and function. in addition, we demonstrate immunosuppressive effects of octn2 substrates like carnitine. further experiments will be necessary to identify the underlying mechanism of this observation. castor oil has been used for more than 3000 years for its laxative effects and also to induce labor in pregnant women. despite its wide-spread use, the mechanism of action remained unknown. the active metabolite of castor oil is ricinoleic acid which is released from castor oil by intestinal lipases. we have found that exposure of meg-01 cells to ricinoleic acid caused an increase in [ca 2+ ]i, an effect which was dose-dependent and abolished by pretreatment of cells with pertussis toxin, suggesting the involvement of a g-protein coupled receptor. to search for a putative receptor, we determined ricinoleic acid-induced [ca 2+ ]i increases in cells transfected with a sirna library directed against human gpcrs. in this way, we identified prostaglandin e2 receptors ep3 and ep4 as mediators of ricinoleic acid-induced effects. to test if ep3 and ep4 receptors mediate pharmacological effects of castor oil in vivo, we analyzed laxative effects induced by castor oil in wild-type (wt) mice, ep3-deficient (ep3 -/-) or ep4-deficient mice (ep4 -/-). while ep4 -/mice responded similarly to the wt mice, ep3 -/animals were totally insensitive to castor oil-induced laxation. moreover, mice lacking the ep3 receptor only in the smooth muscle cells did not respond to castor oil, in contrast to mice which lack ep3 receptor only in epithelial cells of the intestinal mucosa. similarly, ricinoleic acidinduced contractions of isolated ileal segments were absent in segments lacking ep3, consistent with a preferential expression of the ep3 receptor in the longitudinal muscle layer of the intestine. also, ricinoleic acid-induced contractions of isolated uteri were dependent on the expression of ep3 receptor in the myometrium. these findings identify the cellular and molecular mechanism underlying the effects of castor oil and indicate a role of the ep3 receptor as a pharmacological target to induce laxative effects. introduction: patients seek health information from various sources. they are facing the challenge to differentiate between reliable and untrustworthy sources and at the same time identify the best drug therapy for them. furthermore generalised health information confuses more than they benefit or rather unsettle. patients are not necessarily qualified to assess the evidence of statements properly. there is thus a need for providing competent drug information, which is offered by the independent drug information service at the institute of clinical pharmacology in dresden, germany. for the present descriptive evaluation we selected 5 drugs (arimidex ® , cipralex ® pentalong ® , onbrez ® and pradaxa ® ), that were affected by new referenceprice formation, generic registration, warnings or directions in 2011. in specified time frames we assessed the increase in and the cause of enquiries. deductively we draw conclusions for a perspicuous presentation of patient information. since generic registrations of the aromatase inhibitor arimidex ® enquiries on side effects of this drug were stable, but 17 additional consultations were held on generic changeovers. the antidepressant cipralex ® as well as the long-acting β-agonist onbrez ® were assigned to reference-price groups, which resulted in an 8-times (cipralex ® : 5 → 40) and 5-times (onbrez ® : 2 → 10), respectively, increase in enquiries. main aspect was to give background information on reference prices and point out therapeutic alternatives (cipralex ® : 34 of 40; onbrez ® : 10 of 10). an additional amount of 22 conversations were carried on the fictive registered drug pentalong ® after health insurance companies advised practitioners to avoid recourse by not prescribing this organic nitrate. notable insecurity was aroused by media reporting on lethal bleeding after taking pradaxa ® for anticoagulation. every tenth enquiry in the evaluation period was focussing on these instigative reports (21 of 227). patients are confronted by current changes, but often do not get enough background information from their health care providers to become acquainted with the tidings. health seekers may find eligible data from media coverage. however the individual assessment as well as the risk-benefit-relation may not be feasible for them. the drug information service for patients is a convenient helpline to reduce lack of knowledge and uncertainties and therefore support shared decision making. insulin effects on hyaluronan production -a possible link between diabetes and cancer? twarock s., fischer j. w. institut für pharmakologie und klinische pharmakologie, universitätsklinikum der heinrich-heine-universität düsseldorf, moorenstraße 5, 40225 düsseldorf, germany background: epidemiological studies have shown an elevated incidence of certain tumor entities in diabetes type 1 and type 2 patients. to reveal the underlying mechanisms we focused on the effects of increased glucose uptake in cancer cells with respect to matrix production. abundant production of hyaluronic acid (ha) in the vicinity of gastrointestinal cancer cells is a hallmark in tumor development. esophageal cancer is a rare but severe kind of gastrointestinal cancer which is differentiated in adenocarcinoma and squamous cell carcinoma (scc) of the esophagus. we studied the effects of increased glucose levels on ha production in an scc cell line (osc1). in starving and full media, elevated glucose concentrations increased the production of ha secreted to the medium in 24h as measured by an ha-binding protein linked assay (starved, 0g/l glucose: 100±4.7%; 1g/l: 185.7±44.8%; 4g/l: 362.6±58.3%; full medium 100±15.2%; 155.3±32.3%; 422.7±32.0%). surprisingly, total ha concentrations were about 2.2-2.8 fold higher under starved conditions. to investigate whether this effect might be due to insulin actions, starved cells were treated with 10 mg/l insulin for 24h. we observed a dosedependent decrease in ha production following insulin treatment (control vs insulin, 1g/l glucose: 100±6.5% vs 67.83±4.4%; 4g/l: 100±2.2% vs 71.8±6.6%). this finding might suggest that insulin directs glucose usage to the glycolytic pathway thereby diminishing ha synthesis. a premise to this assumption is the ability of osc1 cells for insulin independent glucose uptake. to verify this thesis, mrna expression levels of insulinindependent and insulin-dependent glucose transporters (glut1, glut4) were analyzed by qrt-pcr. the relative abundance was 24.32±3.49 in favor of glut1 indicating the presence of insulin-independent glucose transport. conclusion: in osc1 the absence of insulin actions caused increased ha production which might be due to diminished insulin driven glycolysis, thus leading to the use of early glucose metabolites for ha production instead of energy gain. this finding could be important in the context of diabetes type 2, where insulin actions are also diminished because of insulin resistance. since increased ha production is of critical importance for cancer growth and spread, the cellular shift in glucose usage from glucose catabolism to ha anabolism could therefore indicate a possible link between diabetes type 2 and cancer progression. schwarz m., unterberger e. universtität tübingen, institut für klinische und experimentelle pharmakologie und toxikologie abteilung toxikologie, wilhelmstraße 56, 72074 tübingen, germany chemical hepatocarcinogenesis is a multi-stage process triggered by an intitiating mutation in a gene encoding an important cell-regulatory protein. tumour initiation may be caused by genotoxic substances which directly interact with the dna, causing mutations. cells carrying permanent mutations experience clonal expansion which may be accelerated by exposure of the experimental animals to tumour promoters during the following step of tumour promotion. it has been shown that substances which constantly activate certain nuclear receptors act as tumour promoters in rodent liver, such as the model tumour promoter phenobarbital which, amongst others, activates constitutive androstane receptor (car). since these tumour promoters do not seem to directly interact with dna causing mutations they can be regarded as non-genotoxic carcinogens. however, the molecular mechanisms of non-genotoxic carcinogenesis are still widely unknown which also poses a major problem in preclinical drug-development. the aim of the marcar (biomarkers and molecular tumour classification for nongenotoxic carcinogenesis) project is to establish early biomarkers for non-genotoxic carcinogenesis by creating a comprehensive molecular profile of tumours generated by a regimen including model tumour promoters such as phenobarbital. the ultimate aim is to differentiate spontaneous liver tumours from tumours generated by non-genotoxic carcinogens. this molecular profile includes mutational analyses, immunostaining for known tumour-specific markers, phospho-proteome analyses, genome wide and promoter-specific dna methylation analyses, as well as mirna analyses. mutation analyses were carried out with mouse and rat tissue from phenobarbital promoted liver tumours to identify mutations which phenobarbital provides a growth advantage for. furthermore, real time pcr measurements show that the expression of a particular non-coding rna and mirna precursor is up-regulated in tissue isolated from phenobarbital promoted mouse liver tumours. additional in-situ-hybridisation experiments demonstrated the localisation of this transcript in ctnnb1-mutated tumours. larch-derived diterpenes are potent and selective trpc6 blockers urban n. 1 , kübler w. the transient receptor potential channel trpc6 is a poorly ca 2+ -selective cation channel that is activated by the membrane-resident second messenger diacylglycerol (dag). consistent with the major sites of trpc6 expression, its activation has been implicated in pulmonary and renal diseases, such as pulmonary hypertension, lung edema, chronic obstructive lung disease, allergic airway disease, and focal segmental glomerulosclerosis. amongst various plant extracts, conifer oils and resins are traditionally used to treat pulmonary ailments. therefore, we reasoned that they may contain constituents with a biological activity to modulate trpc6 activity. the true turpentines, oils and resins of various coniferous genera were tested with respect to a possible inhibition of dag-or receptor-induced activation of trpc6 and trpc3. indeed, turpentines and resins, but not coniphere oils blocked trpc6 and trpc3 in a concentration-dependent manner. interestingly, the larch-derived turpentine exerted a trpc6-prevalent inhibition. we identified larixol and its mono-and diacetates as the specific compounds that are contained in larch resin and give rise to a trpc6-selective block. larixol acetates displayed an ic50 towards the dag-or receptor-stimulated trpc6 activity of about 0.3-1 µm, but did not strongly inhibit a number of other trp channels, including trpv1, trpm2, trpm3, trpm8, or trpa1. selectivity for trpc6 compared to its closest relative, trpc3, was about 30-fold. unlike conipherous oils, which contain toxic pinenes, the resin constituent larixol ant its acetates exerted no significant cellular toxicity at concentrations that are required to block trpc6. electrophysiological analysis confirmed the highly potent block, which was voltageindependent and reversible. in a murine hypoxia-induced pulmonary vasoconstriction (hpv) model, larixol acetate abrogated the euler-liljestrand mechanism and, thus, mimicked the phenotype of trpc6 -/mice. we conclude that trpc6 blockers and, more specifically, larixol-related derivatives may provide novel therapeutic strategies to treat or prevent pulmonary diseases. dioxin is an environmental contaminant, believed to affect basic biological equilibria such as calcium and iron homeostasis. however, the molecular mechanisms underlying these effects are still largely unknown. this strongly hampers the estimation of the hazard to humans associated with dioxin exposure and necessitates further studies aimed at the clarification of these mechanisms. it has been suggested that nearly all biological and biochemical processes are mediated by protein complexes. the most commonly used technology for monitoring changes in the expression of complex protein mixtures is still 2d gel electrophoresis, but this method suffers from poor expression of low or moderately abundant proteins. blue native page and subcellular fractionation form an ideal partnership when it comes to enrichment and analysis of intracellular organelles and low abundant multiprotein complexes. the aim of the study is to identify and characterize multiprotein complexes by blue native page to elucidate the network of protein-protein interactions that regulate protein function after dioxin exposure. sample preparation and subcellular fractionation rt4 cells were cultured in mccoy's 5a medium. cells at confluence were harvested and fractionated into cytosolic, membrane/organelle and nuclear fraction by using the proteoextract subcellular proteome extraction kit. first dimension (bn-page) 50 mg of protein sample was mixed with 5% of coomassie blue g-250 (cbb g-250) and loaded in each lane of 4-15% polyacrylamide native gradient gels. the lanes from the first dimension were cut into individual strips and were placed into a 12% sds gel. the gels were stained with coomassie and the spots were picked up for mass spectrometry. bn/sds-page combined with ms led to the identification of proteins involved in the regulation of both calcium and iron homeostasis in dioxin-exposed cells. these results demonstrate for the first time that dioxin exposure simultaneously affects calcium and iron metabolism. since important iron and calcium requirement changes occur during the regulation of cell growth, the protein expression changes observed in our study may be associated with dioxin-dependent cell-fate decisions. the murine protease inhibitor serpina3n inhibits mechanical allodynia in a model of neuropathic pain vicuna l. 1, 2 , simonetti m. several chronic diseases are accompanied by strong, long-lasting pain. a majority of chronic pain diseases are not well understood yet and cannot be controlled by conventional analgesics or non-pharmacological approaches. therefore, there is a major need to develop novel therapeutic principles. using a genetic screen, we identified serpina3n, a serine protease inhibitor, which is homologous to human a1-antichymotrypsin, to be a determinant of low neuropathic pain. we found that serpina3n is expressed in the dorsal root ganglia (drg) and spinal cord and that it is upregulated in these tissues in mice developing neuropathic pain. importantly, we observed that spinal delivery of recombinant serpina3n inhibits mechanical allodynia in a mouse model of neuropathic pain. we identified a novel serine protease substrate for serpina3n, which is upregulated in the spinal cord in mice undergoing neuropathic pain ('enzyme e'). recombinant enzyme e delivered intrathecally to the spinal cord of mice elicited rapid and long-lasting allodynia, which was fully blocked by concomitant administration of serpina3n. our results suggest that serine protease-serpin signaling modulates spinal neuronal and glial cell networks involved in processing pain and that activity-induced spinal release of serpina3n constitutes an endogenous defence mechanism against establishing chronic pain hypersensitivity. these data have important implications for the pathophysiology of pathological pain and potentially hold therapeutic relevance. gβγ subunits are involved in β-adrenergic receptor induced cardiac hypertrophy vidal m., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. 9, 97078 würzburg, germany introduction. activated β1-adrenergic receptors and their g protein gαs induce the development of cardiac hypertrophy. however, the hypertropic effects of direct activation of downstream effectors, such as adenylyl cyclase, camp or pka, are controversely discussed. recently, a hypertrophic pathway involving a g protein βγ subunit induced phosphorylation of the mitogenic kinases erk1/2 at threonine 188 (erk thr188phosphorylation) has been described to mediate erk-induced hypertrophy. this study aims to investigate whether erk thr188 -phosphorylation is involved in cardiac hypertrophy triggered by β-adrenergic receptors. -phosphorylation was detected in hek cells overexpressing β1-receptors, murine hearts and neonatal rat cardiomyocytes after isoprenaline treatment. we performed [ 3 h]-isoleucine incorporation assays to assess cardiomyocyte hypertrophy in vitro. neonatal rat cardiomyocytes (nrcms) overexpressing wild-type erk2 showed a significant increase in [ 3 h]-isoleucine incorporation after isoprenaline treatment. in contrast, nrcms transfected with erk thr188 -phosphorylation deficient mutants (erk2 t188a and erk2 t188s ) or pretreatment with the erk inhibitor, pd98059, significantly attenuated cardiomyocyte hypertrophy. for in vivo studies, isoprenaline was given subcutaneously for 14 days to wild-type mice and transgenic mice overexpressing either wild-type erk2 t188t or erk2 t188s . echocardiography and histological analyses revealed that erk t188s mice developed less left ventricular hypertrophy than control mice. hypertrophic target proteins of erk (e.g. elk1) are located in the nucleus. western blot and confocal microscopy analyses showed that overexpressed erk2 t188a or erk2 t188s are retained in the cytosol and prevented elk1-phosphorylation after isoprenaline stimulation. co-immunopreciptation assays in hek cells and nrcms underlined the direct involvement of g protein βγ/erk interaction upon isoprenaline stimulation. in line with this finding, direct activation of adenylyl cyclase by forskolin did not lead to gβγ induced erk thr188 -phosphorylation. conclusion. taken together, gβγ-subunits participate in β1-adrenergic receptor mediated hypertrophy by enhancing erk thr188 -phosphorylation. these findings add important insight to the molecular signaling of g proteins in cardiac hypertrophy. the protein tyrosine kinase src and its role upon alpha-toxin stimulation of human platelets vogel k. 1 , burke m. introduction: alpha-toxin, a 34 kda calcium pore forming exotoxin, is a major virulence factor in the pathogenesis of staphylococcus aureus infections. alpha-toxin affects human blood cells such as platelets and induces aggregation that is accompanied by multiple changes in platelet protein tyrosine phosphorylation and dephosphorylation (1). in the present paper, we focused our interest on the protein tyrosine kinase src, the most abundant member of the src-family kinases present in platelets (2) . by the use of various inhibitors, we studied src and its role in α-toxin-induced platelet aggregation. methods: isolated human platelets from healthy volunteers were stimulated with α-toxin in the presence or absence of the src-family member inhibitors pp1, pp2 or su6654 (referred as src inhibitors). src and autophosphorylation of src were analyzed by sds-page and western blotting using specific antibodies against src and tyr-416-phospho-src from calbiochem and cell signaling, respectively (3). furthermore, calpeptin, an inhibitor of the calcium-dependent protease calpain, was used. platelet aggregation was measured by the method of born. staphylococcal α-toxin induced platelet aggregation in a concentration-dependent manner (0.18 -3.0 µg/ml of toxin). pre-incubation with 3 src inhibitors (pp1, pp2 or su6654) reduced α-toxin-induced platelet aggregation by about 50%. similar inhibitory effects have been observed by the use of calpeptin that acts as an inhibitor of the src degrading protease calpain. with respect to src itself, a-toxin induced autophosphorylation at tyr-416 followed by a fast and complete dephosphorylation within 10 min. while calpeptin modified the time course of dephosphorylation, only little effect of the src inhibitors has been seen on tyr-416 phosphorylation/dephosphorylation. the typical calpain-dependent degradation of src can be blocked by calpeptin (1µm), but also by depletion of extracellular calcium indicating that it is a calcium-dependent process. conclusion: taken together, our data demonstrate that α-toxin of staphylococcus aureus induces platelet aggregation accompanied by src degradation and autophosphorylation at tyr-416 typically observed in activated platelets. inhibition of the cellular tyrosine kinase src as well as the protease calpeptin reduces aggregation indicating an important role of src and/or other src-family members in α-toxin-induced platelet stimulation. the five subtypes of muscarinic acetylcholine receptors belong to the superfamily of gprotein coupled receptors. the even-numbered subtypes m2 and m4 prefer coupling to gi proteins, whereas the odd-numbered receptors m1, m3 and m5 prefer coupling to gq proteins. with respect to ligand binding and m2 receptor activation, the conserved epitope trp 7.35 at the beginning of tm7 displays remarkable functional features. it is located at the junction between the orthosteric and the allosteric binding site of the m2 receptor [1] . in the inactive m2 receptor, it provides subtype-independent baseline affinity for allosteric antagonists [1] . in the active receptor, m2 trp 7.35 affords binding affinity for the full agonist acetylcholine and intrinsic efficacy for the partial agonist pilocarpine [2] . to study the role of trp 7.35 for m3 receptor activation, agonist-induced formation of dmyo-inositol-monophosphate was measured in cho-cells transfected with the respective human receptor-cdna. surface receptor expression measured by radioligand binding was similar in hm3 wild-type-cells and hm3 trp 7.35→ala-cells, amounting to 0.07 and 0.11x10 6 receptors per cell, respectively. the intrinsic efficacy of acetylcholine was not influenced at m3 trp 7.35→ala relative to m3 wild-type, whereas potency was reduced about tenfold. these findings resemble those made previously in m2 and the corresponding mutant. in the case of pilocarpine, replacement of trp 7.35 by alanine in m3 did not reduce intrinsic efficacy. this finding is in contrast to m2, where the corresponding mutation induced a loss of pilocarpine's intrinsic efficacy. the potency of pilocarpine was diminished about tenfold at the m3 trp 7.35→ala mutant relative to m3 wild-type. this finding is also in contrast to m2, at which pilocarpine's potency was not sensitive to the trp 7.35→ala mutation. taken together, the diverging sensitivity of pilocarpine to the trp 7.35→ala mutation between the m3 and the m2 receptor suggests that the role of this epitope for receptor function may differ between even-and odd-numbered muscarinic acetylcholine receptors. in vivo experiments for inhalation toxicity are time and animal consuming. thus several in vitro methods aim to replace or reduce and refine the in vivo experiments. human 3dtissue models are commercially available reconstructed from different donors (normal, smokers, chronic obstructive pulmonary diseases), which show a normal human bronchiole tissue that reveals a pseudostratified epithelial structure, numerous microvilli and cilia on the apical surface of the cultures. the presence of tight junctions and mucus secretion has also been confirmed comparable to the in vivo situation. these 3d-models are cultured on a porous membrane as air-liquid interface. test substances can be applied apically, either as solution or with an aerosol-inducer. in our in house validation to test the strengths, handling and reproducibility of such 3dmodel systems as well as determining the correlation between in vivo inhalation data, we have assessed the epiairway tm model from mattek, usa. a set of 20 substances were selected with known in vivo toxicity data and mode of action. the substances were tested in the epiairway model an in parallel, in 3t3 and a549 cell lines to assess putative unspecific cytotoxic effects of the test substances. a comparison of toxicity data from the 3d-model and the in vivo data revealed, that the model is only predictive of respiratory toxicity in vivo for a subset of substances with specific modes of action. the epiairway tm model has proven to be robust, showing high reproducibility between pre-and main-tests as well as in the concurrent controls but it will need a strict definition of its applicability domain or further development of the test protocol to achieve a wider applicability. remodeling of intracellular ca 2+ handling and cyclic amp-dependent signaling in atrial myocytes from patients with chronic atrial fibrillation. voigt n. 1 background: in atrial myocytes ca 2+ entry through l-type ca 2+ channels (ica,l) triggers a larger ca 2+ release (ca 2+ transient,cat) from the sarcoplasmic reticulum activating contractile myofilaments. reduced ica,l is a hallmark of atrial remodeling in chronic atrial fibrillation (caf) and is supposed to contribute to action potential shortening and contractile dysfunction. however, the coupling efficiency between ica,l and cat and its regulation by camp-dependent signaling in caf patients are unexplored. methods: ica,l (voltage-clamp) and cat (fluo-3) were measured simultaneously in rightatrial myocytes from sinus-rhythm (ctl) or caf patients. a saturating concentration of the non-selective β-adrenoceptor (ar) agonist isoprenaline (iso, 1µm) and the nonselective phosphodiasterase (pde) inhibitor 3-isobutyl-1-methylxanthine (ibmx, 10µm) were used to increase cellular camp content. camp content was assessed with immunoassay. results: in caf amplitudes of ica,l (3.3±0.3pa/pf, n=12/6 [myocytes/patients] vs 6.2±0.8 pa/pf, n=15/10, p<0.01) and cat (182.2±26.8nm vs 307.5±30.9nm, p<0.01) were lower than in ctl myocytes, whereas diastolic [ca 2+ ]i levels were unchanged (caf, 312.3±56.7nm; ctl, 305.3±43.6nm). the coupling efficiency between ica,l and cat was similar in ctl and caf. application of iso increased ica,l amplitude to 10.4±2.0pa/pf (n=7/4) in caf and to 14.3±1.7pa/pf (n=9/7) in ctl. the corresponding cats increased to 493.0±132.3nm in caf and to 545.0±79.0nm in ctl. although the amplitudes of ica,l and cat also increased after pde inhibition with ibmx, the magnitude of these increases was smaller than the iso-induced enhancements. both iso and ibmx had no effect on diastolic [ca 2+ ]i and coupling efficiency. however, the relative iso-induced increases in ica,l (caf, +202.3±47.3% vs ctl, +98.4±16.1%, p<0.05) and cat (caf, +215.4±57.8% vs ctl, +101.9±20.3%, p=0.06) were significantly higher in caf compared to ctl myocytes and a similar tendency was found for ibmx. basal camp levels were higher in caf compared to ctl (caf, 9.9±1.5pmol/mg, n=6 vs ctl, 5.0±0.6pmol/mg, n=7, p<0.05), pointing to an increased camp-dependent signaling in caf patients. conclusions: these data point to remodeling of camp-dependent signaling in caf patients which likely contributes to the stronger relative increases of ica,l and cat amplitudes after β-ar stimulation and pde inhibition. remodeling of camp-dependent signaling might be a novel contributor to af pathophysiology. direct visualisation of g-protein-coupled receptors and heterotrimeric g-proteins using single-molecule microscopy wagner j. 1 g-protein-coupled receptors (gpcrs) form the largest family of membrane-bound receptors and mediate the effects of several extracellular stimuli. although the basic mechanisms of gpcr signalling have been extensively studied, a full characterization of the involved protein-protein interaction is still missing, largely due to technical limitations. in this study, we developed new methods for labelling gpcrs and g-protein subunits based on snap-and clip-tags and visualise them with single-molecule sensitivity. the snap-tag is a mutant of the dna repair protein o 6 -alkylguanine-dna alkyltransferase that reacts with fluorescent benzylguanine derivatives, whereas the clip-tag is reacting specifically with o 2 -benzylcytosine derivatives. these tags allow labelling proteins directly in living cells with very high specificity and low background. snap/clip-tagged receptors and g-proteins were covalently labelled with small organic fluorophores and visualised by total internal reflection fluorescence microscopy, which allows to selectively illuminate only fluorescent molecules located on or immediately underneath the cell surface. particles were automatically analysed with previously published as well as newly developed algorithms. the results indicated that both receptors and gproteins, although diffusing with high speed on the cell surface (diffusion coefficients: receptors ~ 0.05 mm 2 /s, g-proteins ~ 0.1mm 2 /s), can be visualised and correctly tracked. a variable fraction of receptors and g-proteins are immobile or show hop movements, possibly suggesting their interaction with cytoskeletal or other membranebound proteins. our data also suggest the feasibility of performing two-colour analyses with snap-and clip-tagged proteins aimed at directly visualizing transient interactions between receptors and g-proteins or among g-protein subunits. in-vitro screening systems are particularly well suited to preclinical toxicology testing at an early stage of drug development as they have the advantage of being fast and requiring only a small amount of test substance. the demands for in-vitro screening assays for systemic toxicity are multiple and include the need of organ specific cell systems, the use of optimal cell numbers, cell passages and incubation times. even minimal changes in the conditions of the test system may lead to significant changes of the biological system. therefore a reliable normalization compensating biological variability is crucial prior to any interpretation of results generated from a biological system. basf has developed an in-vitro metabolite profiling assay and a subsequently tuned normalization strategy allowing the prediction of specific organ toxicity. the in-vitro assay consist of exposing cells lines to test substances and to determine the metabolite profile using chromatography coupled to mass spectrometry systems. herein, we compare five different normalization strategies referring to their suitability in the application to in-vitro metabolite profiling data. the strategies comprise statistical approaches, approaches referring to reference values from each individual sample or samples generated in dependent batches. best results were achieved by an individual strategy using a new reference value correlating well over a large range of cell counts previously used for generating corresponding cell extracts. statistical analysis revealed the normalization based on the new reference value greatly improved the quality of the results compared to non-normalized samples as well as to all remaining strategies. generation and application of this new reference value and the corresponding normalization strategy will be presented the first time. validation will be featured on the basis of extracts of the human hepatocellular carcinoma cell line hep g2. molecular mechanisms of the inhibitory function of rhoh in phospholipase cmediated signalling walliser c., löschmann y., ziegler v., kühne e., schilling p., rasonabe z., bühler a., vatter p., gierschik p. universitätsklinikum ulm institut für pharmakologie und toxikologie, albert-einstein-allee 11, 89081 ulm, germany rho gtpases are a subfamily of ras gtpases regulating diverse signalling pathways, for example those regulating the reorganisation of the actin cytoskeleton. among them, rac2 and rhoh show an expression restricted to the hematopoietic lineage. rhoh is constitutively active, because it carries mutations in two positions (s13 and n62) known to be important for gtp hydrolysis. hence, rhoh is controlled on the level of protein expression and, possibly, by tyrosine phosphorylation. rhoh has been implicated in human malignancies, since the gene is subject to somatic hypermutation in its noncoding regions and to translocation to the gene encoding laz3/bcl6 or to other genes in human b-cell lymphomas. furthermore, rhoh is overexpressed in primary human chronic lymphocytic leukemia (cll) cells. these findings suggested that rhoh is involved in the initiation and/or progression of cll. we previously showed that rhoh acts as a potent inhibitor of both rac2-mediated phospholipase c-β 2 (plcβ2) and plcγ2 activation in intact cells. the aim of this study was to elucidate the molecular mechanisms of the inhibitory effect of rhoh on plc activity. the results showed that rhoh directly inhibited the activity of constitutively active variants of plcγ2, plcβ2, and plcδ1, but that it had little or no effect on the activity of plcγ1 and plcε. the amino acid residues s13 and n62, likely to be the cause for the gtpase-deficiency of rhoh, are not required for the inhibitory function of rhoh. furthermore, the switch-i and switch-ii regions of rhoh are not necessary for the inhibitory effect of rhoh, since rhoh mutants carrying switch-i or switch-ii regions of rac2 caused inhibitory effects on rac2-mediated plcβ2 and plcγ2 stimulation indistinguishable from wild-type. interestingly, rhoh seems to interact with regions of plcγ2 distinct from those which are necessary for rac2 interaction, as the split pleckstrin homology domain of plcγ2, which is essential for its interaction with activated rac2, is dispensable for the inhibitory effect of rhoh. in summary, our results indicate, that rhoh acts as a plc-isozyme-specific negative regulator of the activity of plcβ2 and plcγ2, both of which are specifically expressed in hematopoietic cells. these findings suggest a novel mechanism of plc isozyme regulation by rhoh. the results also suggest that rhoh plays an important role in b cell maturation, function, and leukemogenesis by modulating b-cell-receptor-mediated plcγ2 activation. effect of rac1 inhibition on doxorubicin mediated cell response wartlick f., fritz g. heinrich-heine-universität düsseldorf institut für toxikologie, universitätsstrasse 1, 40225 düsseldorf, germany background: the small gtpase rac1 is a well characterized member of the rashomologous (rho) family. rac1 is not only a key regulator of the actin cytoskeleton but also regulates the activity of nadph oxidase, stress kinases and transcription factors (e.g. nf-κb, ap1). furthermore, rac1 can translocate into the nucleus and interacts with topoisomerase type ii (topo ii). yet the general nuclear function of rac1 is still unclear. here, we address the question how rac1 influences the genotoxicity of the topo ii poisons doxorubicin and etoposide. methods: to study the function of rac1, human hepatoma cells were pretreated with the rac1-inhibitor eht 1864 before they were exposed to doxorubicin, etoposide or, for control, ionizing radiation (ir). to check the influence of rac1 inhibition on the outcome of genotoxin treatments, cell viability and cellular stress response were analyzed by the wst-assay, western blot (wb), co-immunoprecipitation experiments, facs-analysis and the alkaline comet-assay. results: as compared to the control, cells that have been pretreated with the rac1 inhibitor showed a higher viability, less phosphorylation of h2ax (s139) and a reduced dna damage formation (measured by alkaline comet-assay) after treatment with doxorubicin and etoposide but not after treatment with ir. furthermore inhibition of rac1 resulted in a reduced phosphorylation of topo iiα (s1106) and an increased interaction of topo iiα with hsp90 in doxorubicin treated cells. the data indicate that inhibition of rac1 protects human hepatoma cells against topo ii poisons due to interference with topo iiα function. the presence of drugs or other potential toxic substances in milk has enormous toxicological and nutritional consequences for consumers of dairy products. the atpbinding cassette (abc) transport protein breast cancer resistance protein (bcrp; abcg2) is expressed in alveolar epithelial cells of the mammary gland in cows, sheeps and goats. bcrp is known to play a major role in the active secretion of a variety of xenobiotics into human milk. so far there is little information about the transport activity and substrate specificity of dairy bcrp. therefore we aimed to establish a mdck cell in vitro model expressing bcrp of dairy animals. bcrp mrna was isolated from bovine, caprine and ovine mammary gland. full-length clones were generated using race (rapid amplification of cdna ends) pcr. the final full-length bovine, ovine and caprine abcg2 cdna-clone sequences were submitted to the ncbi genebank (eu570105, gq141082 and gq241418). stable transfection of bcrp in mdck cells was performed and the subcellular localization of bcrp at the apical plasma membrane was identified by confocal laser scanning microscopy. bcrp-mediated transport of the substrate hoechst 33342 was measured and the selectivity was determined by the bcrp inhibitor ko143. inhibition studies using hoechst 33342 identified various drugs including the antibiotic enrofloxacin or anthelmintic agents like oxfendazole as substrates of bovine, caprine and ovine bcrp. to further characterize bcrp carrier activity, bidirectional transport studies were performed with transwell® filter inserts that allow studying drug transport between an apical and basolateral compartment. cell monolayer integrity was checked by measuring teer values as well as by measuring the paracellular flux marker atenolol by lc/ms. bidirectional transport studies with enrofloxacin were performed to characterize the bcrp transporter activity. our results may contribute to increase the understanding of carrier associated drug transport into the milk of dairy cattle and therefore enlarge consumer protection. acrolein and acrylamide: excretion of mercapturic acids after consumption of potato chips watzek n., scherbl d., berger f., feld j., eisenbrand g., richling e. technische universität kaiserslautern fachbereich chemie; fachrichtung lebensmittelchemie & toxikologie, erwin-schrödinger-str. 52, 67663 kaiserslautern, germany acrolein (ac) and acrylamide (aa) may be formed from food constituents during heating of food. ac is supposed to be generated via heat induced formation from glycerides/glycerol, aa is known to arise during the maillard reaction from asparagine and reducing carbohydrates. ac also has also been suggested to be formed by endogenous metabolism as a side product of carbohydrate and/or amino acid turnover or by oxidative desamination of polyamines [1] . as an α,b-unsaturated aldehyde, ac forms 1,4-michael-adducts with biomolecular nucleophiles, such as sulfhydryl and amino groups. in the organism, ac and aa are preferentially conjugated to glutathione and are excreted as mercapturic acids (ma), n-acetyl-s-(3-hydroxypropyl)-cysteine , n-acetyl-s-(carboxyethyl)-cysteine (cema), (n-acetyl-s-(2-carbamoylethyl)-cysteine (aama), and (n-acetyl-s-(2-hydroxy-2-carbamoylethyl)-cysteine (gama). data on human exposure to ac and its occurrence in the diet are scarce. in general, contents in heat treated foods are considered to be in the low ppb range (µg/kg) [2] . nevertheless, in a pilot study in humans urinary 3-hpma excretion of 14 non-smokers was reported to be about three fold higher, as compared to aama [3] . in the present human intervention study we monitored the excretion of mas in five healthy volunteers (male) after ingestion of commercially available potato chips (175 g), equivalent to an uptake of 44 µg aa (absolute amount), together with an as yet unknown amount of acrolein [4] . urinary ma contents were monitored by hplc-ms/ms following solid phase clean-up of urine for up to 24 h after test meal uptake. the results demonstrated kinetics of 3-hpma and cema excretion in human urine to be clearly related to ingestion of the potato chip meal. on the basis of auc values, total excretion of 3-hpma plus cema exceeded that of aama plus gama by a factor of about four. the results confirm earlier findings on urinary mas, suggesting markedly higher human exposure to dietary ac / potential ac precursors than to aa. it is an as yet unresolved question, whether and to what extent concomitant substantial ac exposure may influence toxicology of such dietary heat-induced toxicants. [1] stevens, j.f. and maier, c.s. (2008) molecular nutrition & food research 52; 7 [2] osorio, v. m. and de lourdes cardeal, z., (2011) journal of chromatography a 1218; 3332 [3] schettgen, t., musiol, a., and kraus, t., (2008) rapid communications in mass spectrometry 22; 2629 [4] ewert, a., granvogl, m., and schieberle, p., (2011) lebensmittelchemikertag 2011 450 protective effects of increased nad + levels in human peripheral blood mononuclear cells exposed to dna damaging agents weidele k., beneke s., bürkle a. university of konstanz molecular toxicology group, department of biology, jacob-burckhardt-str.31, 78457 konstanz, germany the dna damage-activated enzyme poly(adp-ribose) polymerase 1 (parp-1) acts as a nick sensor and modifies target proteins by covalent attachment of poly(adp-ribose) [par] using nad + as substrate. the intracellular levels of par and nad + are important parameters for biological responses to genotoxic stress and influence diverse cellular functions including dna repair or maintenance of genomic stability. notably, loss of genomic stability is a hallmark of both carcinogenesis and the ageing process. here we analysed the impact of elevated nad + levels in human blood peripheral mononuclear cells (pbmc) with regard to (i) poly(adp-ribose) formation, (ii) cell death, (iii) initial dna damage and subsequent repair, as well as the influence on (iv) genomic stability under genotoxic stress. after ex vivo supplementation of pbmc with low concentration of nad + precursor nicotinic acid (na) intracellular nad + level significantly increased up to 2 fold in unstimulated [1] and 1.5 fold in mitogen-stimulated cells. after dna damage infliction, parp activity was dramatically increased in supplemented cells, necrotic cell death was reduced and dna strand break repair was significantly affected. furthermore the frequency of micronuclei decreased significantly after irradiation damage, emphasizing the fundamental role of adequate nad + levels in maintaining genomic integrity. the cyclic purine nucleotides adenosine 3':5' monophosphate (camp) and guanosine 3':5' monophosphate (cgmp) are well-examined second messengers with many proven biological functions. in a recent study, using a highly sensitive and specific mass spectrometry method, we have shown that cyclic 3':5' cytidine monophosphate (ccmp), a pyrimidine nucleotide, is naturally occurring in several mammalian cells [1] . ccmp activates both camp-and cgmp-dependent protein kinases with low potency [2] but the physiological function of ccmp is still very poorly understood. in an effort to delineate the function of ccmp, we analyzed expression of the early response gene egr1. we chose this gene because it is regulated by numerous stimuli including camp [3] . in our first study, we showed that dibutyryl-ccmp and ccmp failed to increase egr1 gene expression levels after stimulation of kb cells under various experimental conditions using real-time pcr (taqman®). we have now changed the experimental set-up using hela cells and the new ccmp analogue, ccmp-acetoxymethyl ester (ccmp-am), still focusing on gene expression of egr1. esterification of the negatively charged cyclic phosphate of ccmp allows better transport of the nucleotide across the cell membrane, thus augmenting possible intracellular effects. hela cells were stimulated in cell culture medium with extracellularly applied ccmp-am (3, 10, 33, 100 µm) over 15 to 240 min 24 h after seeding. analysis of real time pcr (taqman®) experiments, using β-actin as a housekeeping gene, showed a significant increase of egr1 expression in a time and concentration dependent manner. these effects were specific for stimulation with ccmp-am but not the control phosphate trisacetoxymethyl ester. hela cells were also cultured in serum free resting medium (mcdb 153, sigma) that induces growth arrest, one to eight hours prior to stimulation. here, even higher egr1 expression levels through ccmp-am stimulation could be seen. these results suggest that ccmp could function as a second messenger just as camp and cgmp do. studies are in progress to further examine the mechanisms of the ccmp-am effects on egr1 expression in hela cells. methyl-cpg-binding protein 2 (mecp2) recognizes methylated dna, it is involved in chromatin remodeling and it acts as a transcriptional repressor or activator. we have previously shown that expression of mecp2 is diminished in murine and human heart failure. prevention of mecp2 downregulation in transgenic mouse models aggravated cardiac hallmarks of heart failure. in patients with rett syndrome, which is caused by mutations in the mecp2 gene, mitochondrial function was found to be altered in the central nervous system. as the impact of mecp2 on mitochondrial function in the heart is unknown, the aim of the present study was to characterize the significance of mecp2 of cardiac mitochondria in mouse models with cardiac myocyte-specific expression or ablation of mecp2. in order to investigate the cardiac function of mecp2, two genetically modified mouse models were previously generated, including mice with inducible transgenic expression of mecp2 in cardiac myocytes under control of the tetracycline-system (mecp2-tg) and mice with targeted ablation of mecp2 in myocytes (mecp2 mlccre ). these mice were analyzed under basal conditions and after chronic transverse aortic constriction (tac). at baseline, cardiac-specific overexpression of mecp2 did not cause any difference in cardiac function as compared to control mice using millar catheterization. isolated interfibrillar mitochondria showed a decrease in citrate synthase activity. after chronic pressure overload, the decrease in cardiac mecp2 expression could be completely prevented by the mecp2 transgene. cardiac contractility and relaxation were significantly decreased in mecp2-tg animals. upon electron microscopical investigation, transgenic mecp2 expression was associated with a significant reduction of interfibrillar mitochondria, clustering of mitochondria in the perinuclear region and smaller mitochondrial cross sections as compared with control specimens. in contrast, cardiac myocyte-specific ablation of mecp2 caused a rightward shift in the size distribution of mitochondria as compared with mecp2-tg hearts. epigenetic processes, including the recognition of dna methylation by mecp2, may play an important role in the control of mitochondrial gene expression, structure, subcellular localization and function in the heart. thus, precise control of mecp2 expression and function is essential to prevent deterioration of metabolic function during chronic heart failure. normalisation of blood pressure does not prevent angiotensin ii-induced dna damage in kidney and heart of ren2 rats weissenberger s., hey v., lau d., schupp n. universität würzburg institut für pharmakologie und toxikologie, versbacher strass 9, 97078 würzburg, germany increased activity of the renin angiotensin system (ras) with enhanced levels of angiotensin ii (angii) leads to oxidative stress with endothelial dysfunction, hypertension and atherosclerosis. epidemiological studies revealed a higher cancer mortality and an increased kidney cancer incidence in hypertensive patients. we could show in vitro and in vivo that angii causes structural dna damage dose-dependently in kidney cells and in the kidney. elevated angii levels therefore might contribute to carcinogenesis of the kidney. in a model of high angii organ levels, the transgenic ren2 rat, carrying an additional renin gene, dna damage in the kidney was analysed in animals of 13 and 32 weeks. untreated ren2 rats exhibit increased blood pressure from the age of 8 weeks on. therefore, the line is kept on angiotensin i converting enzyme inhibitor therapy, which normalizes blood pressure and kidney function to values of control sprague dawley rats. despite this normalized blood pressure of the ren2 animals, a significant higher superoxide production could be observed in kidneys already in 13 week old animals. also a higher frequency of structural dna damage and double strand breaks could be detected in the comet assay and with an antibody against the double strand break marker γ-h2ax in kidneys. further, fittingly, an increased dna repair activity exists in kidneys of ren2 rats compared to control rats. as another organ affected by hypertension the heart of the ren2 animals was analysed for oxidative dna damage. although only a marginal increase of superoxide production could be found, also in the heart a significant higher frequency of dna double strand breaks and cells positive for dna repair activity could be observed. our data let us conclude that normalization of blood pressure in a state of activated ras is not sufficient to prevent angii-induced genotoxicity. this further implies that also patients with treated hypertension still might suffer from endorgan-damaging effects of elevated angii levels. the d541a pore mutation leads to complete inactivation of trpv6 channels in epididymis weißgerber p. 1 , kriebs u. replacement of aspartate residue 541 by alanine (d541a) in the pore of trpv6 channels in mice disrupts ca 2+ absorption by the epididymal epithelium resulting in abnormally high ca 2+ concentrations in epididymal luminal fluid and in a dramatic but incomplete loss of sperm motility and fertilisation capacity raising the possibility of residual activity of channels formed by trpv6 d541a proteins (sci signal 4, ra27, 2011) . it is known from other cation channels that introducing pore mutations even if they largely affect their conductivity and permeability can evoke considerably different phenotypes compared to the deletion of the corresponding protein. to gain insights whether the trpv6 d541a pore mutant still contributes to residual channel activity and/or channelindependent functions in vivo, we compared important fertility-parameters between trpv6 -/and trpv6 d541a/d541a mice: the fertilization rate observed in permanent matings, the in vivo fertilization rate as judged by the rate of embryos isolated from plug positive females of matings with males homozygous for either trpv6 mutation as well as the motility, in vitro fertilization capacity and viability of sperm were reduced to the same extent in both genotypes. also, no differences were observed in copulatory behavior between trpv6 -/and trpv6 d541a/d541a males. the profound reduction in ca 2+ uptake by the epididymal epithelium was identical in trpv6 -/and trpv6 d541a/d541a males. this direct comparison of these parameters indicate that deleting trpv6 does not further aggravate the phenotype observed in trpv6 d541a/d541a mice, and -in our opinion -allows the conclusion that the d541a pore mutant of the trpv6 protein leads to complete inactivation of the trpv6 channel activity or channel-independent scaffolding functions in epididymal epithelium. characterization of a naturally occurring c-terminal mutation (n996i) on herg channel function in hek293 cells sellmaier v. 1 , moretti a. long-qt-syndromes (lqts) are acquired or inherited disorders which predispose patients to cardiac arrhythmias and sudden death. in affected individuals, the electrocardiogram shows a prolongation in the qt interval, due to an unstable repolarization of the action potential. acquired forms of lqts are often the result of treatment with medications that block cardiac potassium channels, such as class iii antiarrhythmic drugs or antihistamines. inherited lqts are caused by mutations in cardiac ion channels. congenital long qt syndrome 2 (lqt2) is caused by loss-offunction mutations in the human ether-á-go-go-related gene herg (also known as kcnh2 or kv11.1). herg encodes the pore-forming α-subunit of the rapid delayed rectifier potassium current i kr, whose physiological role is to repolarize the late phase of cardiac action potentials. herg channel α-subunits exist as 2 isoforms (1a and 1b) that are identical except for structurally divergent n termini. native cardiac ikr channels are tetraheteromers containing 2 of each α-subunit types. a loss-of-function can be due to either defects in a) channel opening (gating), b) ion permeation or c) protein maturation and trafficking. we have identified a so far uncharacterized dominant missense mutation in the herg1 gene (n996i) in a patient with lqt. both herg1a and herg1b subunits were cloned from a human heart cdna library and the specific n996i mutation introduced by site directed mutagenesis. hek 293 cells were transiently transfected with equal amounts of mutated herg1a and wild type (wt) herg1b cdnas and the resulting potassium current compared to herg1a/b wt. whole-cell patch-clamp analysis showed similar current densities for wt versus mutated channels. also the voltage-dependence of activation was unchanged with a halfmaximal activation at -27 mv for wt and -29 mv for the mutated channel assembly. differences were found for the deactivation and inactivation kinetics. the deactivation was faster in the mutated channels with t fast = 62 ms and tslow = 480 ms versus tfast = 90 ms and tfast = 620 ms in wt channels, determined from the tail current at -40 mv after a 5-second pulse to + 50 mv. the half-maximal steady-state inactivation of the tail current was shifted by 20 mv to the depolarizing direction in the mutated channel compared to the wt. a defect in channel gating appears to be the most likely explanation. background: abcc2 is adjacent to p-glycoprotein the most important efflux transporter for various endogenous and exogenous compounds and is expressed at several compartment barriers. by increasing evidence it is shown that the abcc2 polymorphisms are of clinical significance. the aim of our study is to analyze the epigenetic regulation of distinct abcc2 haplotypes by the influence of micrornas. methods: abcc2 cdna clones containing five distinct haplotypes were generated by site-directed mutagenesis, cloned into pires-zsgreen expression vectors and transfected into different cell lines for further functional analysis. one modified vector set contained a short 3'utr sequence of abcc2 whereas the other contained the full length (fl) sequence. mirnas potentially interacting with abcc2 were identified form an mirna array after rifampicin stimulation of hepg2 cells. results: we could demonstrate that there is no difference in the basal protein expression level comparing the two vector types (fl 3'utr vs. mod. 3'utr) concerning the -24c/1249g/3972c (cgc) abcc2 wt in hepg2 cells. using the fl vector construct, the expression level of cac haplotype protein was increased (136,15% ± 20%). transfection assays with the mir-397, which was identified as a candidate mirna targeting abcc2 mrna, and the two different vector constructs harboring the cac or the cgc (wt) haplotype, confirmed that mir-379 was able to down regulate the abcc2 protein expression. there was no significance in downregulation for one abcc2 haplotype, respectively (modified 3'utr: cgc 22-34%; cac 20-40%, fl 3'utr: cgc 20-25%; cac 17-44% down regulation compared to mir-negative control). discussion: differences of abcc2 protein expression level are due to the epigenetic regulation of abcc2 haplotypes. to further characterize the effect of mir-379 on tcg, tgt and cgt abcc2 haplotypes, transfection assays are currently performed using cell cultures as well human primary leukocyte cultures. sex differences affect the pathophysiology and pharmacology of leukotriene biosynthesis werz o. friedrich-schiller-university jena, institute of pharmacy, philosophenweg 14, 07743 jena, germany inflammatory diseases affect more females than males. thus, women suffer more often from asthma, rheumatoide arthritis, alzheimer´s disease, and many autoimmune diseases than men. of interest, sex differences also exist in drug responses, with respect to both pharmacodynamics and pharmacokinetics. we have recently discovered a sex bias in the biosynthesis of pro-inflammatory leukotrienes (lts) due to testosterone, which may represent a molecular basis for gender differences in inflammation and asthma. interestingly, testosterone downregulates lt biosynthesis and also causes a sex bias in the efficiency of lt synthesis inhibitors, which demands for the clinical evaluation of a gender-tailored therapy with anti-lts. we found that certain inhibitors of lt biosynthesis were more efficiently in females than in males, and that androgens are responsible for these gender differences. in fact, the flap inhibitor mk886 effectively reduced ltb 4 pleural levels in female but not in male rats treated with carrageenan, and mk886 increased survival only of female mice in the lt-related disease model of paf-induced lethal shock. administration of testosterone to female mice abolished the protective effects of mk886. in view of the current active development of lt synthesis inhibitors as therapeutics in respiratory and cardiovascular diseases, our data prompt for consideration of gender issues in the development and use of such drugs, in order to optimize medical therapy both for men and women. considering the complexity of deposition and kinetics of air-borne nanomaterials in the lung, potential pulmonary toxicity of biopersistent nanomaterials should be evaluated by inhalation studies. those studies demand special equipment and large quantities of test material. intratracheal instillation appears as a simple and low substance-consuming alternative, although bolus dosing and the more central distribution of the particles in the lung are a well known trade-off. we compared the inflammatory response of the lung to amorphous silica (as) after instillation and inhalation. for inhalation the established short-term protocol for nanomaterials was employed (ma-hock et al. inhalation toxicology, 21:102, 2009 ): male wistar rats were exposed to the test items for 6 h/day on five consecutive days. the lungs were evaluated by analysis of bronchoalveolar lavage fluid (balf) and by histopathology three days and three weeks after the end of the exposure. in a parallel study, rats were intratracheally instilled and equally evaluated three days after instillation. assuming a deposition rate of 10%, the instilled dose corresponded to the aerosol concentration of 10 mg/m 3 used for inhalation. results show that inhalation and instillation of nominally equal mounts of amourphous silica elicit different results in the lung with inhalative treatment being less harmfull. this difference may be due to the bolus effect inevitable linked to instillation. instillation stuldies with amorphous silica may, therefore, be of limited value with respect to doseresponse assessment. sunscreen products containing uv filters protect consumers from the harmful effects of uv exposure. pigmentary grades of metal oxides like zno result in an opaque whiteness as a result of scattering visible light, whereasnanoparticles result in transparent products for better consumer acceptance and thus improved protection of human skin against uv-induced damage. in addition scatter uv light is most efficiently reflected at a nanosize of 60-120 nm. in the last 2 years the toxicological properties of nanozno in comparison with pigmentary zno were examined, the results of these comprehensive studies are presented. all tests were performed according to oecd guidelines, which were modified, especially in regard of substance preparation where appropriate. nanosized zno showed no acute toxicity after dermal application, in the bcop assay as well as in the epiderm assay it showed no corrosion / irritation potential. nanosized zno does not penetrate the intact as well as the sunburned skin. a dermal absorption test in rats (oecd 427) with 65 zn-labelled test item as well as penetration tests in weanling pigs after uv radiation did not show a penetration of the zno nanoparticles through the skin. genotoxicity was tested in vitro in the ames test, in the chromosomal aberration test in v79 cells, both showing negative results whereas the mouse lymphoma mutation test / l5178y/tk+/-cells was positive. in vivo no mutagenic effect was detected in two mouse micronucleus tests, on with intraperitoneally application and another after repeated inhalation. nanosized zno was tested in 5-days, 14-days and 90-days inhalation studies, in all studies the predominant effects were reversible local inflammatory changes in the nasal cavity and lungs, with a noaec of 1.5 mg/m3 in the 90-day study. in a prenatal developmental toxicity study according to oecd tg 414, with repeated inhalation exposure to female wistar rats from gestation day 6 through 19, maternal toxicity was observed by increase lung weights and inflammations in the lungs. but no substance-related effects on reproductive parameters (conception rate, corpora lutea, implantation sites, preimplantation loss, postimplantation loss, resorptions, dead fetuses) and no increase in external and soft tissue malformations and variations could be detected. the overall result of all the toxicological studies with nanosized and pigmentary zno is that the toxicological profile of both is very similar. studies were sponsored partly by cefci lri and partly by basf se. use of reach registration data for improving thresholds of toxicological concern (ttc) wieneke n., dorn s., jakupoglu c., schäfer c., sica m., wiegand c., mostert v. dr. knoell consult gmbh, marie-curie-str. 8, 51377 leverkusen, germany the threshold of toxicological concern (ttc) concept is utilised to identify human exposures that are so low that in-depth toxicological investigations are expendable. this is called "exposure-based waiving". exposure-based waiving serves to focus available resources on substances with relevant human exposure potential. important work into establishing ttc values has been published by munro et al. (1996) . the initial report used a database of 613 organic substances compiled from publicly available sources. in total, 2941 noels were collected in this fashion. the munro concept used the cramer classification to categorise substances according to their hazard potential. we broadened the ttc database by including noaels published on the echa website as per 3 november 2011, containing data for more than 3900 registrations. only nongaseous mono-constituent substances with oral noaels were included in the ttc database. organophosphates and genotoxic substances were excluded from the database as well as noaels obtained for surrogate substances. noaels for all systemic endpoints (general toxicity, developmental toxicity, fertility, neoplasia) were taken into account. where appropriate, default assessment factors of up to 6 were used to establish chronic noaels for each substance. for every eligible substance, we collected the published clp category for acute oral toxicity as a potential predictor of overall hazard potential. this gives rise to five categories of acute oral toxicity. a ttc is calculated from the 5 th percentile of noaels in each of these categories using the reach rules for establishing dnels for workers and the general population. this poster presents the preliminary results for more than 1000 substances. the results indicate that the ttc concept becomes more robust when using the very broad echa database. it also suggests that acute oral toxicity categories can be used as a predictor for the overall hazard potential of a substance. comparison of different in-vitro models for inhalation toxicology with respect to the effects of cigarette smoke total particulate matter wiese j. 1 , schumann b. b-and l-moc can be cultivated up to 70 days without loss of viability, as determined by resazurin-assay. viability of cell cultures was determined by mtt-assay after incubation with increasing doses of tpm. exposure of h322 to tpm (30 mg/l) reduced viability to 95% or 83% after 24 or 72h, respectively. in a549 viability was 67% after 72h with tpm (30 mg/l). the same dose of tpm lead to a decrease in viability to 82% (24h) or 25% (72h) in nhbec and to 74% (24h) or 28% (72h) in plc. as a marker of oxidative stress the level of intra-cellular glutathione (gsh) was determined by hplc. in both the tumor cell lines analysed gsh-level was increased by tpm (5 mg/l). in h322 the induction was 1.6 and 1.2 fold after 24 or 72h, respectively. while in a549 it was 1.3 (24h) and 1.4 fold (72h). in nhec and plc tpm (5 mg/l) did not have a significant effect on gsh-levels after 72h. in n-moc tpm (5 mg/l) also did not modulate gsh after 24h, but it diminished gsh-level by 0.5 fold upon prolonged contact (72h). in b-moc gsh concentrations also decreased to 0.6 or 0.8 fold the level of controls after 24 or 72h incubation. the results presented show that in-vitro models of varying complexity and origin within the respiratory tract clearly differ in their response to tpm, which was used a model inhalative toxicant. the tumor cell lines used seem to be better adapted to chemical stress, while the models closer to the in-vivo situation are more vulnerable. the nongenomic effects of the mineralocorticoid receptor in transgenic mouse heart winter s. 1 , schreier b. within the renin-angiotensin-aldosterone system (raas), the mineralocorticoid receptor (mr) is important for the regulation of fluid and electrolyte balance in the kidney, salivary glands, sweat glands and colon. however, survival of patients with severe heart failure is increased when mr blockage is combined with standard therapy suggesting aldosterone, the mr ligand, as a key factor in the development of cardiovascular diseases, but the mechanism is not yet fully understood. in recent years, evidence accumulated that besides its function as a hormone-activated transcription factor the mr also functions via nongenomic pathways. to investigate the function of the nongenomic effects of the mr in cardiovascular dysfunction, we generated a transgenic (tg) mouse model expressing a truncated human mr lacking the dna-binding site (hmr def ) under control of the cardiac specific α myosin heavy chain promoter (αmhc), a model for nongenomic effects of the mr in the heart. in this mouse model no enhanced mortality could be observed. body weight (bw), heart weight and relative heart weight were not different compared to wild type (wt) while left atrial weight/bw was increased by 20 % (wt 0,25 ± 0,01 mg/g vs. tg 0,30 ± 0,02 mg/g, p<0.05, n=12). compared to wt mice neither surface electrocardiographic experiments nor echocardiographic experiments revealed modified parameters for tg mice under basal (i.e. unstimulated) conditons as well as under β-adrenergic stimulation by isoproterenol (iso, 100 µl 1 mm iso intraperitoneally applied). to uncover the role of aldosterone in the development of cardiovascular diseases treatment with aldosterone and high-salt diet (1%) was performed. after 28 days cardiac function and heart dimensions were analyzed, surface electrocardiographie uncovered increased p duration (16 ± 0.5 ms vs. 14 ± 0.7 ms, p<0.05) and qtc interval (61 ± 2 ms vs. 50 ± 3 ms, p<0.05) in tg (n=7) compared to wt (n=5) animals. these findings probably indicate more sensitive conduction pathways to aldosterone in tg mice. oligomerization is important for regulation of phospholamban activity wittmann t., lohse m. j., schmitt j. p. institut für pharmakologie und toxikologie, versbacher straße 9, 97078 würzburg, germany phospholamban (pln) is a heart specific protein located in the membrane of the sarcoplasmic reticulum. it inhibits the ca 2+ -atpase serca2a, thereby decelerating cytosolic ca 2+ clearance during diastole of the cardiac cycle. upon phosphorylation the inhibitory activity of pln on myocyte ca 2+ transport is attenuated. further, it is believed that phosphorylation favors the formation of (rather inactive) pentamers and that pln pentamers itselves were an inferior substrate for phosphorylation compared to monomers. this would suggest an important role of pln oligomerization in the regulation of pln activity. to prove this hypothesis, we are investigating the patterns and kinetics of pln phosphorylation in the context of alterations in pln structure. the introduction of specific point mutations into the transmembrane region of pln yielded mutants that are purely monomeric (l37a, i40a and c41f) or favor pentamer formation (i45a, v49a). transfected hek293 cells expressing these mutants or wildtype pln were stimulated with forskolin to induce pln phosphorylation before lysis of cells and western blot analysis using antibodies directed against phosphorylated pln. surprisingly, phosphorylation was increased for both monomeric and pentameric pln after stimulation with 0.25µm forskolin for less than one minute. at increasing forskolin concentrations phosphorylation signals increased in parallel for monomers and pentamers. for measurement of phosphorylation kinetics stimulation of cells with 2.5µm forskolin was stopped at different time points. we found phosphorylation of both pln monomers and pentamers within seconds of stimulation. differences in phosphorylation patterns became more pronounced when assays were performed at low temperature (14°c). intriguingly, preliminary analyses suggest that pka dependent phosphorylation occurs first in pentamers and that phosphorylation of monomers may catch up only after pentamer phosphorylation is almost complete. our data suggest that both pln pentamers and monomers are suitable substrates for pka dependent pln phosphorylation. unlike the prevalent assumption, kinetics of pentamer phosphorylation seem to be at least as fast as that of pln monomers in transfected hek293 cells suggesting an important role of pln pentamers in the regulation of pln activity. regulation of cardiac contractility by nucleoside diphosphate kinases in zebrafish wolf n. m. 1, 2 , abu-taha i. in the heart, nucleoside diphosphate kinases (ndpks) can interact with heterotrimeric g proteins, thus regulating camp synthesis in a receptor independent manner and thereby influencing contractility in cardiomyocytes. we further investigated the interaction of ndpk isoforms with heterotrimeric g proteins in the heart in vivo and in vitro using zebrafish embryos and embryonic fibroblast from ndpk a/b double knockout mice (ndpk a/b ko mefs). in zebrafish the morpholino-mediated knockdown of ndpk a did not lead to an obvious phenotype, although the total ndpk activity was reduced. depletion of ndpk b caused a cardiac phenotype characterized by severely impaired atrial and ventricular contractility and insufficient blood flow. the depletion of ndpk b was associated with a significant decrease of protein levels of the heterotrimeric g protein subunits gβγ, gα s and gαi. the knockdown of ndpk c led to a more restricted cardiac phenotype with markedly reduced pumping function of the ventricle, while the atrium was unaffected. in accordance to the reduced cardiac pumping function, camp levels were significantly diminished in the ndpk b and ndpk c morphants. similar findings were obtained in ndpk a/b ko mefs. the absence of ndpk a and b resulted in a decrease of the plasma membrane content of gβ and gαs and a significant reduction in camp synthesis. the protein expression of the isoform ndpk c was also significantly reduced in the ndpk a/b ko mefs. the re-expression of ndpk b but not ndpk a rescued the basal camp production and the membrane content of g proteins. interestingly, the overexpression of ndpk c led to a 5-fold enhancement of the camp level and a significant increase of the membrane content of gβ and gα, and thus rescued the knockout phenotype. our data indicate, that the ndpk isoforms b and c are essential for cardiac contractility, most likely by forming a signaling complex at the plasma membrane including ndpk b, ndpk c and heterotrimeric g proteins. the isoform ndpk c, with its n-terminal hydrophobic region, might serve as a membrane anchor for the ndpk/g protein complex. induction of apoptosis via pka-dependent and pka-independent pathways by cyclic purine and pyrimidine nucleotides in mouse lymphoma cell lines wolter s. 1 camp is a second messenger that plays an important role in intracellular signal transduction of various hormones and neurotransmitters. a major function of camp in eukaryotes is the activation of camp-dependent protein kinase a (pka). pka is involved in the control of a variety of cellular processes. pka exists as an inactive tetramer of a dimeric regulatory (r2) and two catalytic (c) subunits that releases the active c-subunits upon binding of camp. stimulation of the mouse t-lymphoma cell line s49 wild-type (wt) with dibutyryl (db)-camp induces apoptosis by an intrinsic, mitochondria-dependent mechanism. apoptosis induced by db-camp occurs via a pka-dependent mechanism, since s49 kincells lacking the catalytic subunit of pka are resistant to db-camp-mediated cell death. db-camp is cleaved by esterases into the biologically active compound n 6 -mb-camp and into 2'-o-mb-camp. other cyclic nucleotides (cnmps) in addition to camp, like ccmp and cump can also function as second messengers and activate pka and cgmp-dependent protein kinase (pkg) 1 . therefore, we investigated the effects of a series of membrane-permeable analogues of camp, cgmp, ccmp and cump in s49 wt und s49 kincells on apoptosis. stimulation with db-ccmp or db-cgmp induced neither apoptosis in s49 wt nor in s49 kincells. interestingly, we observed apoptosis in s49 wt and s49 kin cells after incubation with membrane-permeable nucleotide acetoxymethyl ester (am)-analogues of cgmp, ccmp, cump and also camp. induction of apoptosis occurs via pkadependent and also pka-independent pathways. a potential role of pkg and of the exchange protein activated by camp (epac) in the induction of apoptosis is unsolved and will be explored by specific pkg-and epac-activators in this system. investigations are on the way to identify the targets, the involved signal transduction pathways and the mechanisms of pro-apoptotic actions mediated by cnmp-ams. (1) wolter s, golombek m, seifert r (2011) differential activation of camp-and cgmpdependent protein kinases by cyclic purine and pyrimidine nucleotides. biochem biophys res commun. in press apoptosis in s49 cells : induction of apoptosis in s49 wt and in s49 cells lacking the catalytic subunit of pka (s49 kin-) with a) db-cnmps and b) cnmp-ams for 72h. nebivolol reduces vascular inflammation in spontaneously hypertensive rats wu z., xia n., förstermann u., li h. institut für pharmakologie, obere zahlbacher str. 67, 55131 mainz, germany nebivolol is a third generation β1 receptor blocker with additional effects on endothelial nitric oxide production. the aim of the present study is to investigate the antiinflammatory effects of nebivolol in vivo. 48 spontaneously hypertensive rats (shr) were divided into two groups: control or nebivolol treatment group. nebivolol treatment (5 mg/kg/day for 10 days) significantly reduced the blood pressure and the heart rate in shr. the drug had no effect on coagulation. aorta from nebivolol-treated rats showed significantly improved endothelial function. nebivolol did not change the expression levels of aortic nf-kb, but significantly reduced its dna binding activity. furthermore, nebivolol decreased the expression of adhesion molecules (e.g. icam-1, vcam-1) and pro-inflammatory cytokines (e.g. il-6). in conclusion, nebivolol reduces vascular inflammation in experimental hypertension. it inhibits nf-kb activity, decreases the expression of adhesion molecules and pro-inflammatory cytokine, and improves endothelial function. characterization of the cellular activity of pde4 inhibitors using two novel pde4 reporter cell lines wunder f., quednau r., barg m., tersteegen a. bayer pharma ag lead discovery wuppertal, aprather weg 18a, 42096 wuppertal, germany cyclic nucleotide-specific phosphodiesterases (pdes) play an essential role in cellular signal transduction by regulating the intracellular levels of camp and cgmp and, therefore, are important pharmacological targets. we report here the generation and pharmacological characterization of two novel pde4 reporter cell lines. plasmid constructs encoding human pde4b1 or pde4d3 were stably co-transfected with the beta1-adrenoceptor in a parental camp reporter cell line expressing a cyclic nucleotide-gated (cng) cation channel, acting as the biosensor for intracellular camp. in this reporter cell line, camp levels can be monitored in real-time via aequorin luminescence stimulated by calcium influx through the cng channel. by using different pde4 and non-pde4 inhibitors, we could show that our novel pde4b1 and pde4d3 reporter cell lines specifically monitor pde4 inhibition with high sensitivity. pde4-selective inhibitors alone did not increase basal luminescence levels in this experimental setting. however, these inhibitors induced concentration-dependent luminescence signals in combination with the adrenoceptor agonist isoproterenol. in contrast, in a stable beta1-adrenoceptor reporter cell line with no recombinant pde4 expression, pde4 inhibitors had no effect on isoproterenol-stimulated luminescence signals. we compared the cellular activity of different pde4 inhibitors with the in vitro inhibition of full-length and truncated (catalytic domain) pde4d3 from cell lysates. two different groups of pde4 inhibitors could be identified. the first group, including the allosteric inhibitors pmnpq and d159153, showed high cellular activity and much better inhibition of full-length versus truncated pde4d3. the second inhibitor group, including classical competitive inhibitors like roflumilast, cilomilast and piclamilast, showed comparably lower cellular activity and similar inhibitory activity on full-length and truncated pde4d3. the results imply that these novel pde4 reporter cell lines are well-suited for the characterization of the cellular activity of pde4 inhibitors and may also support a better understanding of the complex pde4 pharmacology. plexin-b2 is required for kidney regeneration after acute renal failure xia j. 1 , gröne h acute renal failure is a common clinical problem with unsatisfactory therapeutic options and high mortality in humans. therefore, unraveling the mechanisms that promote kidney regeneration and repair may provide new therapeutic strategies for acute renal injury. plexin-b2 belongs to a family of transmembrane receptors which mediate the cellular effects of semaphorins. while plexins have first been described in the context of axon guidance, several recent studies have established them as key regulators of organogenesis, the immune system and cancer. we have recently found that plexin-b2 is highly expressed in the adult kidney, particularly in tubular epithelial cells which are most sensitive to acute ischemic injury. to study the role of plexin-b2 during kidney regeneration we generated mice lacking plexin-b2 specifically in tubular epithelial cells. under physiological conditions, these mice displayed normal kidney morphology and function. in contrast, following ischemia/reperfusion injury, plexin-b2 conditional knockout mice exhibited severely impaired kidney regeneration. while the renal function of control mice fully recovered within 3 weeks after injury, plexin-b2 knockout mice had strongly elevated serum creatinine and urea levels associated with increased morbidity and mortality. this was accompanied by hyperproliferation of tubular epithelial cells and obstruction of tubular lumina. we conclude that plexin-b2 is required for regeneration after acute ischemic renal injury and that pharmacological interventions activating plexin-b2 might represent a new therapeutic strategy in acute renal failure. the nadph oxidase enzyme complex consists of two membrane-bound catalytic subunits (a nox protein and p22phox) and several cytosolic regulatory components including p47phox, p67phox, p40phox and the small gtpase rac1. we have previously shown that treatment of apolipoprotein e knockout mice with resveratrol led to a downregulation of nox2 and nox4 in the heart. our recent data demonstrated that resveratrol also reduced the enzymatic activity of cardiac nadph oxidase. because activation of nadph oxidase enzyme complex is induced by translocation of the regulatory subunits, we studied whether the reduced enzymatic activity is due to an inhibition of such a translocation. indeed, resveratrol treatment prevented rac1 membrane translocation from cytosol. resveratrol is known as an activator and expression enhancer of the longevity gene sirtuin 1 (sirt1). we then wanted to find out whether the effect of resveratrol on rac1 was mediated by sirt1. sirt1 is a histone/protein deacetylase. in vitro incubation of rac1 with sirt1 led to a reduction of lysine acetylation. deacetylation of rac1 on lysine 166 could be identified by mass spectrometry analyses. the lysine 166 lies within the p67phox-binding region of rac1. consistently, in vitro incubation of rac1 with sirt1 markedly reduced its binding activity to p67phox. in conclusion, we provide evidence that rac1 is a direct target molecule of sirt1. sirt1 deacetylates rac1 on lysine 166 and thereby inhibits its interaction with p67phox. this is a novel mechanism of nadph oxidase inhibition by sirt1/resveratrol. mutational analysis of the effects of the cardioprotective drug dexrazoxane on topoisomerase ii beta in vitro yan t., deng s., frensch i., gödtel-armbrust u., wojnowski l. universitätsmedizin der johannes gutenberg-universität mainz institut für pharmakologie, obere zahlbacher straße 67, 55101 mainz, germany dexrazoxane (drz, icrf-187) is the only approved drug shown to protect against anthracycline-induced heart failure. the protection is usually ascribed to the ironchelation by drz, which is thought to reduce anthracycline-induced oxidative stress. however, similarly to anthracyclines, drz is also an inhibitor of the anthracycline target topoisomerase ii (top2). we hypothesized that the cardioprotective effects of drz are mediated by the prevention of the anthracycline-induced dna damage mediated by top2b, the dominant cardiac top2 isoform. this was investigated using top2b mutants resistant to drz, which were expressed in cells depleted of wild-type top2 isoforms. top2b-mediated double-strand dna breaks were assessed as γ-h2ax. the levesl of dsb generated by the top2b mutants in response to the anthracycline doxorubicine (dox) was indistinguishable from that mediated by a wild-type top2b. preincubation with drz depleted wild-type top2b and this was accompanied by a decrease in the dna damage following a subsequent exposure to dox. in contrast, neither top2b depletion nor the reduction of dsb by drz was seen in drz-resistant top2b mutants. furthermore, the cardially ineffective drz analog icrf-161, capable of iron chelation but not of top2 binding, affected neither the stability of top2b, nor the dox-induced dna damage mediated by this enzyme. these results indicate that drz may exert its cardioprotective effects by reducing the dna damage mediated by doxpoisoned top2b rather than by iron chelation. they also suggest a cardioprotective function of top2b, which is currently under investigation using cardiomyocyte-specific top2b mouse knockouts. aminoglycosides are important antibiotics in the treatment of life-threatening infections, especially those caused by gram-negative bacteria. their nephrotoxic and ototoxic potential is well-known, but little is known about the effects of aminoglycosides on the male reproductive system. we studied the effects of four aminoglycosides on sertolicells in vitro. rat sertoli-cells from the cell line serw3 were cultivated for 3, 6, and 9 days in dmem supplemented with three different concentrations of amikacin, streptomycin (30 mg/l, 100 mg/l, 300 mg/l), gentamicin or tobramycin (10 mg/l, 30 mg/l, 100 mg/l). we determined the expression of two junctional proteins (connexin 43, ncadherin) and one protein of the cytoskeleton (vimentin) by western blot. cells were solubilized in lysis buffer. lysates were separated by sds-page and electroblotted on a pvdf-membrane. after incubation with primary antibodies overnight and horseradish peroxidase-conjugated secondary antibody the visualization was achieved by a chemiluminescence-detection system. in addition, proteins were detected by immunohistochemistry. after three days in culture amikacin caused the most pronounced effect. at the lowest concentration tested (30 mg/l) connexin 43 and n-cadherin were reduced to 55±19% and 92±10% of the controls (n=6). no change was recognized for vimentin (102±16%). effects obtained with streptomycin were less pronounced for these these proteins (68±16%, 96±7%, and 102±13%, respectively). similar, but less pronounced effects were observed with gentamicin and tobramycin at a concentration of 10 mg/l (connexin 43: 88±15% and 81±15%; n-cadherin: 95±18% and 103±11%; vimentin: 81±12% and 102±19%) and 30 mg/l (connexin-43: 84±19% and 78±18%; n-cadherin: 98±19% and 103±13%; vimentin: 102±8% and 115±15%). after incubation for 6 and 9 days the effects occurred in the same range. the substances showed no influence on the viability of serw3 sertoli-cells up to 300 mg/l in the mtt assay. by immunohistochemistry we showed that the localisation of the proteins -connexin 43 and n-cadherin at the cell membrane and vimentin in the cytoplasm -was not influenced by the aminoglycosides. large conductance calcium-and voltage-gated potassium (bk) channels play an important role in controlling membrane potential and calcium influx, and are strongly modulated by protein kinases at multiple sites. the stress-regulated exon (strex) adds to the bk channel c terminus a cysteine-rich insert of 59 amino acids that inverts the channel regulation by protein kinase a (pka) from excitatory to inhibitory. here we investigated the mechanisms by which the strex insert influences bk channel regulation by protein kinase c (pkc). activity of bk channels without strex insert (bk-zero), transiently expressed in hek cells, was inhibited by pkc in inside-out membrane patches (~ 50% inhibition). bk channels with strex insert (bk-strex), however, were insensitive to pkc. phosphomimetic mutation of a pkc phosphorylation site (s700e) in bk-strex, resulted in a ~50% reduction of basal channel activity, whereas the s700a mutant retained normal activity. to examine whether palmitoylation, and thus association of the strex domain with the plasma membrane, prevents pkc inhibition of bk channel gating, palmitoylation was abolished by either site-directed mutagenesis (c12:13a) or by pharmacological inhibition of palmitoyl transferases with 2-bromopalmitate (2-bp). both, mutation and pretreatment with 2-bp resulted in the expression of bk-strex channels which were sensitive to pkc (~ 50% inhibition of channel activity). no inhibitory pkc effect was observed in patches of the bk-strex s700a channel mutant pretreated with 2-bp. in a clonal rat somatomammotroph pituitary cell line (gh3b6), in which pcr products without (zero) and with the 174 bp strex exon could be identified, the pkc activator pma blocked channel activity by ~25 %. this inhibition was increased to over 50% when gh3b6 cells were pretreated with 2-bp, indicating that both channel isoforms were functionally active. in summary, the present study demonstrates that palmitoylation of strex prevents bk channel regulation by pkc, which is mediated by phosphorylation of ser700, probably by steric hindrance. our results provide further evidence for a cross-talk between palmitoylation and phosphorylation as a crucial mechanism underlying the dynamic regulation of ion channels. human pleural mesothelial met-5a cells are a limited in vitro model system in determining potential asbestos-like genotoxic effects of multiwall carbon nanotubes ziemann c. 1 , reamon-büttner s. multiwall carbon nanotubes (mwcnt) are nanomaterials with important technological impact. depending on their diameter, length, and biopersistence, however, some mwcnt seem to exhibit a fiber-like cytotoxic and genotoxic potential, similar to asbestos. thus, a project funded by the german federal ministry of education and research (bmbf contract no. 03x0109a) focuses on potential adverse biological effects of different types of mwcnt to enlarge the knowledge base about toxicity determining parameters. this project comprises both in vitro (rat) and in vivo endpoints with long amosite asbestos as a positive control. as mesothelial cells are target cells for adverse effects of asbestos, in particular mesothelioma development, the human sv40transformed, non-malignant pleural mesothelial cell line met-5a was chosen as the main in vitro model in this project. in the present study part, met-5a cells were characterized concerning their usefulness as an in vitro model to study potential asbestos-like cytotoxic and genotoxic effects of different mwcnt varieties. using an mwcnt-optimized lactate dehydrogenase liberation assay and proliferation parameters derived from cell counts, concentration-dependent cytotoxicity of long amosite asbestos (2, 10, and 20 µg/cm 2 ) was demonstrated in met-5a cells. cells also showed asbestosinduced increase in dna-strand breaks and oxidative dna-damage in the hogg1modified comet assay. thus, met-5a cells were responsive to asbestos treatment. owing to asbestos potential to induce aneugenic effects and spindle fiber damage, micronucleus induction, determination of numerical chromosome aberration, and altered meta-, ana-, and telophase morphology were planned as in vitro endpoints. met-5a cells were thus initially characterized in this regard and were found to exhibit highly variable chromosome numbers with lower than 10% cells exhibiting a normal diploid chromosome set, an up to twentyfold higher spontaneous micronucleus frequency, as compared to polychromatic bone marrow erythrocytes in rodents, and a profound number of aberrant meta-, ana-and telophases with bridges, lagging chromosomes and multipolar divisions. in conclusion, met-5a cells are of only limited value as an in vitro model system to study potential asbestos-like effects of mwcnt and also biopersistent fibers. the cells are indeed responsive to asbestos, but unfortunately demonstrate marked genomic instability and thus limited significance concerning genotoxic effects. waixenicin a inhibits cell proliferation through magnesium-dependent block of trpm7 channels zierler s. 1 transient receptor potential melastatin 7 (trpm7) channels represent the major magnesium-uptake mechanism in mammalian cells and are key regulators of cell growth and proliferation. they are abundantly expressed in a variety of human carcinoma cells controlling survival, growth and migration. these characteristics are the basis for recent interest in the channel as a target for cancer therapeutics. we screened a chemical library of marine organism-derived extracts and identified waixenicin a from the soft coral sarcothelia edmondsoni as a strong inhibitor of overexpressed and native trpm7. waixenicin a activity was cytosolic and potentiated by intracellular free magnesium (mg 2+ ) concentration. mutating a mg 2+ -sensitive site on the trpm7 kinase domain reduced the potency of the compound, whereas kinase deletion enhanced its efficacy independent of mg 2+ . waixenicin a failed to inhibit the closely homologous trpm6 channel and did not significantly affect trpm2, trpm4, and crac (calcium release activated calcium) channels. therefore, waixenicin a represents the first potent and relatively specific inhibitor of trpm7 ion channels. consistent with trpm7 inhibition, the compound blocked cell proliferation in human jurkat t-cells and rat basophilic leukemia cells. based on the compound's ability to inhibit cell proliferation through mg 2+ -dependent block of trpm7, waixenicin a or structural analogs may have cancer-specific therapeutic potential, particularly since certain cancers accumulate cytosolic mg 2+ . release of metals from different sections of domestic drinking water installations zietz b. p., richter k., laß j., suchenwirth r., huppmann r. governmental institute of public health of lower saxony division of environmental medicine and environmental epidemiology, roesebeckstraße 4-6, 30449 hanover, germany different metals were used as important piping materials in the drinking water supply for a long time. due to corrosion metals can leach into the tap water. of special importance is the toxic element lead. however other heavy metals in drinking water such as copper, nickel and cadmium can also give reason for health concerns. in this study it was investigated in which amount relevant metals were released from different parts of domestic installations into the cold water. for the spatial allocation of the emission sources a sequential water sampling protocol was used after three hours of stagnation time representing the first 5 litre of the water column. after stagnation ten sample volumes were collected in series. existing facilities of domestic installations constructed with different plumbing materials were examined predominantly from residential buildings. the elements al, as, cd, cr, cu, fe, mg, mn, ni, pb, sb, se, u and zn were detected by means of icp-ms. in total 16 water pipe strands of 11 domestic installation systems were examined. they comprised 379 single water samples and 5306 single parameters. depending upon the type of plumbing different courses and concentration ranges of the elements could be measured in the tap water samples. terminal taps or installation parts were frequently responsible for a release of nickel and in several cases of cadmium. the concentration courses of the element zinc proved as a good indicator for the allocation of the emission source to a brass containing section of the installation (zinc as an alloy component of brass). one can conclude that an investigation by means of a sequential water sampling protocol and multi-element detection can be a valuable non-destructive method for drinking water-hygienic investigations of domestic installations. novel interaction partners of the murine trpc4 protein zimmermann j., beck a., flockerzi v. universität des saarlandes experimentelle und klinische pharmakologie und toxikologie, kirrberger str. 1, 66421 homburg, germany in this work novel interaction partners of the murine protein transient receptor potential canonical 4 (mtrpc4) were identified. the trpc4 protein is the major subunit of a cation channel, residing in the plasma membrane. it comprises six trans-membrane domains and cytosolic amino and carboxyl termini. two major splice variants of the trpc4 gene exist, trpc4a (974 aa) and trpc4b (890 aa), trpc4b lacks aa 781 to 864 of the trpc4a variant. both trpc4 variants are co-expressed in endothelial cells, intestinal smooth muscle and brain. to identify trpc4-interacting proteins a yeast two-hybrid system, cytotrap®, which allows identification of protein-protein interactions within the cytosol was used. a premade mouse brain cdna library was screened by the cytosolic amino and carboxyl terminal parts of mouse trpc4a (aa 1 to 324; aa 622 to 974). for the carboxyl terminal part fourteen proteins were identified. to independently prove the interaction, the fulllength cdnas of all fourteen proteins were cloned, fused to a flag-tag and coexpressed with trpc4 in hek 293 cells. co-immunoprecipitations were performed for all candidates using both the anti-flag-antibody and the antibody for trpc4. in addition, changes of cytosolic calcium were monitored and trpc4 currents were recorded in hek 293 cells expressing the candidate cdnas and stably expressing the trpc4a or trpc4b and the muscarinic receptor type 2 cdnas. the tarbp2 protein, one of the candidates shown to interact with trpc4, changed calcium influx when coexpressed with trpc4. in order to identify the domains of trpc4 responsible for its interaction with the tarbp2 protein, six trpc4-gst-fusion proteins covering the carboxyl terminal 353 aa of trpc4a were expressed in e. coli and used for pull-down experiments. by this approach two domains of trpc4 could be identified to interact with tarbp2. one of these domains is well conserved within the trpc5 protein, corresponding to the result, that trpc5 and tarbp2 effectively co-immunoprecipitate, too. the tarbp2 protein has been shown to be a component of the risc loading complex, also known as the micro-rna loading complex which is composed of dicer1, eif2c2/ago2 and tarbp2 (chendrimada et al. 2005) . by its interaction it may link trpc4 to pre-microrna processing. increased levels of angiotensin ii provoke dna damage and have influence on dna repair in mouse kidneys zimnol a., brand s., schupp n. universität würzburg institut für toxikologie, versbacherstr. 9, 97078 würzburg, germany the renin-angiotensin system (ras) plays a crucial role concerning the blood pressure, electrolyte balance and cardiovascular homeostasis. angiotensin ii (ang ii), the active hormone of the ras, in higher concentrations leads to vasoconstriction, oxidative stress and hypertension. hypertensive patients have an increased risk to develop cancer, especially kidney cancer. we have shown in vitro and in vivo, that ang ii is capable to cause an elevation of blood pressure as well as dna damage dose-dependently. to investigate whether the high blood pressure or the enhanced levels of ang ii are responsible for dna damage, male c57bl/6-mice were equipped with osmotic minipumps, delivering ang ii in a concentration of 600ng/kg · min during 28 days. additionally they were treated with ramipril, an angiotensin-converting-enzyme blocker, with the ang ii receptor antagonist candesartan, the vasodilator hydralazine, and the antioxidant tempol. dna damage was analysed with the comet assay. we measured the base excision repair (ber)-related dna repair in the kidney with a comet-based in vitro repair assay. furthermore, the distribution and expression of the ang ii-type 1 (at1) receptor in the kidney was analyzed by immunohistochemistry. treatment with ang ii led to a significant increase of blood pressure, whereas the medication with candesartan decreased the systolic blood pressure. the intervention with hydralazine lowered the blood pressure only for a short time. the other substances had no effect at all on the blood pressure. genomic damage, quantified with the comet assay, was augmented by ang ii and improved by all interventions, particularly by candesartan and tempol. beyond that, ang ii showed a tendency to reduce dna repair. treatment with candesartan, hydralazine and tempol increased the repair capacity. furthermore, ang ii tended to result in a downregulation of the at1 receptor in kidney tubule cells. candesartan and ramipril, especially were able to augment the expression of the at1 receptor, whereas hydralazine achieved the opposite. these results demonstrate that ang ii leads to dna damage in the kidney independent of blood pressure. apparently elevated levels of ang ii affect dna repair and expression of at1 receptor. to confirm these findings we are going to examine more precisely the manifestation of other enzymes, which are implicated in dna repair. regulation of hcn channel activity by cyclic cytidine 3´, 5´-monophosphate zong x., krause s., chen c. -c., gruner c., cao-ehlker x., fenske s., wahl-schott c., biel m. lmu münchen, department pharmazie, pharmakologie für naturwissenschaften center for integrated protein science munich (cipsm), butenandtstr. [5] [6] [7] [8] [9] [10] [11] [12] [13] 81377 münchen, germany hyperpolarization-activated cyclic nucleotide-gated (hcn) channels play a key role in controlling cardiac pacemaker activity and are essential for normal function of neuronal circuits. hcn channels are principally gated by voltage but are coactivated by the cyclic nucleotides camp and cgmp which directly bind to a c-terminal binding domain. recently, cyclic cmp (ccmp) was shown to be present in various cell lines and tissues at concentrations that are comparable to cellular cgmp levels. moreover, there is recent evidence that ccmp can activate camp-and cgmp-dependent protein kinases in vivo. here, we examined whether ccmp exerts effects on hcn channels. to this end, we recorded hcn channel-mediated currents (i h) in hek 293 cells that stably express hcn1, hcn2, hcn3 or hcn4, respectively. currents were measured either with a standard patch-clamp setup or by employing the planar patch-clamp technology. in hcn2 and hcn4 channels, ccmp shifted the membrane potential for half maximal activation (v0.5) to more positive values. in addition, ccmp accelerated activation while it slowed down deactivation kinetics. the ec50 for ccmp was 30 µm which is about 5 times higher than the ec50 of cgmp. cyclic cmp is a partial agonist of hcn channels since it activates only 65 % of the maximal current obtained with camp or cgmp. to identify in vivo effects of ccmp we recorded ih of murine sinoatrial node (san) cells in the presence and absence of 1 mm ccmp. like for heterologously expressed hcn channels, ccmp shifted v0.5 of ih by about +5 mv. importantly, the steepness of the diastolic depolarization of san pacemaker potentials which is mainly determined by the amplitude of ih was profoundly increased by ccmp, compared to control conditions. as a consequence of the upregulation of ih the frequency of san pacemaker potentials was increased by about 20 % in the presence of ccmp. our results suggest that ccmp is a physiological regulator of hcn channel activity. a 3d actinic keratosis like construct for assessment of innovative tumour therapeutics zoschke c. 1 the incidence of actinic keratosis (ak) has increased dramatically in the last decades and it is considered the most frequent carcinoma in situ today. especially immunosuppressed patients are at high risk to develop invasive squamous cell carcinoma (scc) [1] which asks for most efficient and well-tolerated ak therapy. yet, current measures do not fit with these demands. nucleotide analogues, recently identified by molecular modelling [2, 3] , outperformed the current standard for the therapy of actinic keratosis, 5-fluoruracil, when tested in the tumour cell line scc25, in normal human keratinocytes and fibroblasts [4] . as next step in pre-clinical drug assessment, we aimed to characterise the effect of the most selective nucleotide analogue oxbu in reconstructed human tumour skin. based on the 3d construct of scc developed by höller and co-workers [5] for start we introduced several adaptations with respect to keratinocyte, fibroblast and scc12 seeding to grow an aklike construct with scc12 cells forming nests in particular in the epidermis. in addition, first experiments with the oxbu on the ak like constructs showed promising results for an efficient treatment of actinic keratosis. efficacy was derived from immunohistology (marker for proliferation: ki-67, marker for scc: cytokeratin-10, axl, marker for invasion: mmp2, marker for apoptosis: caspase-7, nuclei were stained with dapi) as well as the effects on the secretion of cytokeratin-18 and its caspase-induced cleavage product into the culture medium following drug exposure for up to 7 days. efficacy of a 0.05% oxbu solution proved close to or even better when compared to both a 0.1% 5fluorouracil and 0.025% aphidicolin solution. the former being the gold standard of current ak therapy, the latter is a frequently used inhibitor of human polymerase alpha and delta, however, failed to be introduced into clinical use. -in fact, in monolayer cultures aphidicolin proved most toxic for normal human keratinocytes which was not true with oxbu [4] . -therefore, these 3d tumour constructs offer a new approach to pre-clinical drug assessment and may be added to other 3d models of skin diseases currently gaining increased interest as test platforms. ima910, a multi-peptide cancer vaccine for advanced colorectal cancer, induces multiple cd8+ and cd4+ t-cell responses associated with improved survival walter s. 1 , kuttruff s. ima910 is a novel peptide-based vaccine consisting of 10 hla-a*02 and 3 hla-dr binding synthetic peptides that were identified based on natural presentation on human colorectal cancer (crc) samples. ima910 was characterized in a phase i/ii trial in advanced/metastatic crc patients being at least clinically stable after 12 weeks of first-line oxaliplatin-based therapy. all patients received a single application of low-dose cyclophosphamide for immunomodulation. this was followed by repeated ima910 vaccinations in combination with low-dose gm-csf (cohort 1; n=66) or ima910 / gm-csf plus topically applied imiquimod (cohort 2; n=26). before and post vaccination, patients were analyzed by hla-multimer assay and intracellular cytokine (ics) assay for cd8 + t-cell responses and by ics assay for cd4 + tcell responses. as immune status biomarkers, 6 phenotypically defined myeloid derived suppressor cell populations (mdsc1-6) were analyzed prior to immunotherapy. tumor status of patients was monitored repeatedly by ct/mri according to recist and corresponding tumor scans were reviewed centrally. clinical assessment included disease control rate (dcr), time to progression (ttp), progression-free survival (pfs) and overall survival (os). ima910 overall was immunogenic in 73/81 (90%) evaluable patients, with 43% and 65% of patients mounting multiple cd8 + and cd4 + t-cell responses, respectively. patients that received the immunomodulator imiquimod presented with significantly more multiple cd8 + cell responses as detected by ics (p=0.016). multiple cd8 + and multiple cd4 + responses were individually associated with significantly better clinical outcome. the association was most pronounced for patients with both multiple cd8 + and multiple cd4 + responses. these patients had significantly higher dcr at 6 months (p=0.002), improved ttp (p=0.006) and improved pfs (p=0.009) than other patients. most importantly, a trend for prolonged os was also observed in these patients (p=0.088, hazard ratio 0.53). in the study population, levels of 5 different mdsc phenotypes were significantly increased as compared to age/gender matched controls. high mdsc levels were associated with fewer immune responses and for mdsc4 and mdsc5 high frequencies were associated with shorter os (p=0.007 and p=0.019, respectively). to summarize, both hla-a*02 and hla-dr restricted peptides in ima910 were immunogenic. a significantly better clinical outcome of multi-tumap responders in comparison to patients with one/no tumap response strongly indicates clinical activity of ima910. acrylamide (aa), a genotoxic carcinogen (iarc class 2a) is formed in food by thermal treatment from different precursors. after oral ingestion, aa is metabolically epoxidized in the liver by cyp450 2e1 into glycidamide (ga). ga binds to dna, forming covalent adducts, primarily at n7 of guanine (n7-ga-gua). both, aa and ga undergo conjugation to glutathione (gsh) to be excreted via urine as mercapturic acids (ma), namely nacetyl-s-(2-carbamoylethyl)-cysteine (aama), and n-acetyl-s-(2-hydroxy-2carbamoylethyl)-cysteine (gama). in a dose response study, encompassing the dosage range from human dietary exposure levels up to 10 mg/kg bw, female sprague dawley (sd) rats on a diet devoid of detectable aa content were gavaged with single doses of aa. formation of urinary mas and of n7-ga-gua dna adducts in liver, kidney and lung was measured 16 h after application, a time point where cmax of n7-ga-gua was reached. the untreated control group was found to excrete about 0.8 nmol (aama plus gama) in the urine (16 h), indicating a background of endogenous aa formation. compared to untreated control, the lowest dosage of 0.1 µg aa/kg bw neither resulted in significantly enhanced ma excretion, nor in a detectable n7-ga-gua adduct levels in any organ tested (limit of detection, lod, 0.2 adducts/10 8 nucleotides). at the tenfold higher dose (1 µg/kg bw), adducts were found in kidney (about 1 adduct/10 8 nucleotides) and lung (< 1 adduct/10 8 nucleotides), but not in liver. at 10 and 100 µg/kg bw, adducts were found in all three organs, at levels not significantly different to those found at 1 µg aa/kg bw (about 1-2 adducts/10 8 nucleotides). the results of this in vivo study and of further recent research on aa toxicology will be discussed with respect to risk assessment. exposure of rats to single doses of aa in the range of human dietary exposure (0.1-10 µg/kg bw ) leads to n7-ga-gua adduct levels in the tissues monitored obviously not exceeding the range of steady state background dna lesions associated with endogenous/exogenous exposure to various genotoxic electrophiles. thus, the question of significant impact on human background dna damage resulting from exposure to a given genotoxic carcinogen, and on potentially ensuing biological consequences may become a highly relevant issue in risk assessment. pharmaco-economic impact of price, volume and demographic development böcking w., kirch w. institut für klinische pharmakologie, medizinische fakultät der tu dresden, fiedlerstr. 27, 01307 dresden health insurance costs in germany have grown by 3% p.a. over the last ten years and amount to approx. 280 bn eur in 2009. while costs for stationary treatment as the largest cost category have been intensely analyzed over the past years, pharmaceutical expenses have been analyzed in less detail, mostly focusing on the statutory health insurance side, even though pharmaceutical expenses have grown almost twice as much as costs for ambulant treatments. therefore, the question was asked how pharmaceutical expenses in a large german private health insurance company are allocated with respect to age and indication groups, and how those have developed from 2007 to 2011. the data of a private health insurance company with more than 600.000 customers was split into price and volume effects per age group to understand if price or volume drives the cost development. additionally, the two largest indication groups are analyzed in detail. as a result, both price and volume effects drive an overall cost increase. these effects are even stronger in older age groups. this cost increase is not sustainable for the german health insurance system over a longer period of time and will even further increase due to the ageing of the german population. a novel animal replacement system for the detection of endocrine disruptive capabilities in sexual development scheider j. 1, 2 , winter p. alternatives to animal testing for prediction of local toxicity and genotoxicity have been recently established. however, currently these methods are not suitable for measuring endocrine effects in developing organs such as e.g. embryonic gonads. here we present a phenotypic anchoring of a comprehensive study on sex-specific gene expression analysis accompanied by histological analysis of endocrine disruption in chicken embryo gonads, having the potential for an animal replacing system for endocrine disruptive toxicologic and ecotoxicologic examinations of chemicals. chicken embryos were inoculated with different amounts of tributyltin (tbt) and bisphenol-a (bpa). embryos were incubated and their gonads analyzed histologically 2 d prior to hatching. from identically treated embryos right and left testes and ovaries were separated and genome-wide transcription profiles generated using supertag digital gene expression (st-dge, supersage) profiling. male and female gonadal tissues both revealed histological aberrations in response to tbt and bpa. female gonads became masculinized in response to tbt and, viceversa, bpa-treated male gonads underwent feminization whereas in female gonads clearly visible structural aberrations occurred. in both chemicals mortality increased especially in the most affected sex (tbt: females, bpa: males). the expression profiles of more than 60 million mrnas revealed massive effects of both chemicals, tbt and bpa, on important cellular signaling pathways. gene expression differences were most pronounced in the phenotypically most affected sex. our results demonstrate that endocrine disruptive chemicals exert their effects on several levels including but not restricted to known hormone-based pathways. together with an ongoing study of gene expression differences in very young life stages and different chemicals these data will form the base for a blow-by-blow analysis of sexspecific gene expression of embryonic development. the project builds on already existing and further to generate data with the aim of the development of an in vitro method for testing chemicals at chicken eggs for 1) replacement of tests on juveniles and (sub-) adult rodents, 2) stages with impossibility of sensation of pain in the individuals, 3) highly sensitive prospects of modes of action of chemicals, which 4) might show consequences in the next generations. 3 channels are critical for oscillatory burst discharges in the reticular thalamus and absence epilepsy differential distribution of three members of a gene family encoding low voltage-activated (t-type) calcium channels hippocampal seizure resistance and reduced neuronal excitotoxicity in mice lacking the cav 2.3 e/r-type voltage-gated calcium channel transcriptional upregulation of cav3.2 mediates epileptogenesis in the pilocarpine model of epilepsy structure and functional expression of a member of the low voltage-activated calcium channel family a molecular determinant of nickel inhibition in cav3.2 t-type calcium channels histidine residues in the is3-is4 loop are critical for nickel-sensitive inhibition of the cav2.3 calcium channel substrate recognition and translocation by polyspecific organic cation transporters proton pump inhibitors inhibit metformin uptake by organic cation transporters (octs) structural determinants of inhibitor interaction with the human organic cation transporter oct2 (slc22a2) functional characterization of the human organic cation transporter 2 variant p.270ala>ser extra-adrenal glucocorticoid synthesis in the intestinal epithelium: more than a drop in the ocean? local glucocorticoid production in the mouse lung is induced by immune cell stimulation biomimetic materials in tissue engineering biomaterials offer cancer research the third dimension synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering injectable self-assembling peptide nanofibers create intramyocardial microenvironments for endothelial cells directed growth of fibroblasts into three dimensional micropatterned geometries via selfassembling scaffolds novel pcl-based honeycomb scaffolds as drug delivery systems for rhbmp-2 tissue engineering spatio-temporal vegf and pdgf delivery patterns blood vessel formation and maturation presentation of rgds epitopes on self-assembled nanofibers of branched peptide amphiphiles controlling mammalian cell interactions on patterned polyelectrolyte multilayer surfaces langmuir avintegrins as receptors for tumor targeting by circulating ligands heparin binding nanostructures to promote growth of blood vessels tirrell endothelial cell adhesion to the fibronectin cs5 domain in artificial extracellular matrix proteins design and bioproduction of a recombinant multi(bio)functional elastin-like protein polymer containing cell adhesion sequences for tissue engineering purposes stimuli-responsive thin coatings using elastin-like polymers for epac as a novel effector of airway smooth muscle relaxation ) camp inhibits airway smooth muscle phenotype modulation functional roles of epac and pka in human airway smooth muscle phenotype plasticity assessment of the sensitizing and irritative potential of preservatives by loose-fit coculture-based sensitization assay (lcsa) sonnenburg a nsc-631570 (ukrain) in the palliative treatment of pancreatic cancer. results of a phase ii trial association of funding and conclusions in randomized drug trials: a reflection of treatment effect or adverse events a general method for the covalent labeling of fusion proteins with small molecules in vivo robust single-particle tracking in live-cell time-lapse sequences correlation of structural class with no-observed-effect levels: a proposal for establishing a threshold of concern trbp recruits the dicer complex to ago2 for microrna processing and gene silencing index a 009, 019, 035 011, 014 003, 044 objective: hypertension and arterial stiffness is influenced by environmental and genetic factors. high plasma sodium concentration leads to mechanical stiffening of endothelial cells resulting in endothelial dysfunction and elevated blood pressure. here we investigated whether endothelial cell stiffness of ex vivo preparations of human arteries is linked to plasma sodium concentrations and functional genetic variants of the mineralocorticoid receptor (nr3c2), rs2070951 modulating blood pressure, renin, and aldosterone levels, and rs5534, which alters a mirna binding site. design and methods: twenty patients were enrolled after a vein stripping procedure and collateral arterial blood vessels were prepared for atomic force microscopy (afm). plasma sodium concentration was routinely determined and dna for genotyping was extracted from edta blood samples. sodium levels >140 mmol/l were defined as 'high'. after application of 5 µm amiloride, a specific blocker of the endothelial sodium channel (enac) changes in endothelial cell stiffness, were defined as 'weak' (≤10%), or 'strong' (>10%). statistical analyzes were performed by anova. results: in ex vivo artery preparations of patients with high sodium levels (n=12), mechanical stiffness of endothelial cells was tend to increase (∆ amiloride) (p=0.06). both nr3c2 variants were associated with a change >10% in endothelial stiffness after amiloride treatment. the rs2070951 c allele was significantly associated with a strong amiloride response (p=0.024), while the rs5534 a allele only showed a trend towards stronger amiloride effects (p=0.06). conclusion: our findings indicate that high plasma sodium concentration results in an increased endothelial amiloride response and thus influencing mechanical stiffness, modulated by functional nr3c2 variants. our novel approach linking patients' sodium levels and genetic status to endothelial stiffness by afm will be further evaluated in larger clinical settings. protein expression changes in bap-exposed human bladder cancer cells from spliceosome activation towards redistribution of the cytoskeleton after long-term exposure to subacute concentration schmitz-spanke s., pink m., jeske e., stempelmann k., rehn s., verma n., rettenmeier a. w. universitätsklinikum essen institut für hygiene und arbeitsmedizin, hufelandstr. 55, 45122 essen, germany deregulation of the β-catenin signaling pathway plays an important role in the development of hepatocellular tumors. activating mutations in ctnnb1 (encoding β-catenin) are frequently observed in murine and human liver tumors (e.g. human hepatoblastomas). activation of β-catenin signaling induces an overexpression of several cytochrome p450 (cyp) enzymes, including cyp2e1. cytotoxicity of acetaminophen (aap) is based on its cyp2e1-catalyzed metabolism to the electrophilic compound n-acetyl-p-benzo-quinone imine, which forms covalent adducts with cellular macromolecules if depletion of glutathione occurs. treatment with aap should therefore lead to a selective damage of cyp2e1-overexpressing ctnnb1mutated hepatoma cells. mice were injected with a single dose of the liver carcinogen n-nitrosodiethylamine (den) and subsequently treated with the tumor promoter phenobarbital to select for ctnnb1-mutated tumors. administration of a single dose of aap (300 mg/kg of body weight) followed the tumor promotion protocol. two days after treatment immunohistological analysis of the livers showed about 90% necrotic tissue in the larger tumors which were positive for glutamine synthetase (gs), a marker for ctnnb1-mutated tumor cells. by contrast, gs-negative tumors remained unaffected. at later time points we observed regeneration processes with infiltration of the necrotic tissue by inflammatory cells followed by fibrotic cells. proliferation of normal hepatocytes surrounding the damaged areas could also be observed. however, repopulation of parts of the former tumor areas by remaining gs-positive tumor cells was also detected. these results suggest that treatment with aap might serve as a future therapeutic possibility to selectively poison cyp2e1-overexpressing hepatoma. release of 5,6-epoxyeicosatrienoic acid (5,6-eet) upon neuronal activity induces trpa1-dependent mechanical pain hypersensitivity sisignano m. 1 , epoxyeicosatrienoic acids (eets) are cyp-epoxygenase (cyp450) derived metabolites of arachidonic acid (aa) which act as endogenous signaling molecules in multiple biological systems. we investigated the specific contribution of 5,6-eet to transient-receptor potential-(trp)-channel activation in nociceptor neurons, and its consequence for nociceptive processing. we found that during capsaicin-induced nociception 5,6-eet-levels increased in the drg and it is released from activated sensory neurons in vitro. 5,6-eet potently induced a calcium flux [10 nm] in cultured drg-neurons which was completely abolished when trpa1 was deleted or inhibited. in spinal cord slices 5,6-eet dose-dependently enhanced the frequency, but not the amplitude of spontaneous excitatory postsynaptic currents (sepsc) in lamina ii neurons that also respond to mustard oil (aitc), indicating a presynaptic mechanism. furthermore, 5,6-eet-induced enhancement of sepsc frequency was abolished in trpa1 null mice, suggesting that 5,6-eet pre-synaptically facilitates spinal cord synaptic transmission via trpa1. finally, intrathecal injection of 5,6-eet caused mechanical hyperagesia in wild type but not trpa1 null mice. we conclude that 5,6-eet is synthesized upon acute activation of nociceptors and leads to mechanical hypersensitivity via trpa1 at central afferent terminals in the spinal cord. sisnaiske j. 1 , hardelauf h. introduction: neurite outgrowth and plasticity of neuronal networks are essential processes e.g. during brain development and learning. thus, morphological readouts of neuronal connectivity are thought to be important endpoints to assess neurotoxic effects of environmental chemicals as well as when discovering new drugs. to analyze neurite outgrowth and connectivity level rapidly and easily in vitro we developed the network formation assay (nfa) (pct/ep2010/002811). this platform requires a spatially standardized hexagonal array for culturing neuronal networks with no need to fix or stain the cells to visualize neuritic processes. methods: to demonstrate the feasibility of the nfa we performed experiments in which we disrupted mature neurite networks or inhibited generating networks of human sh-sy5y cells with different concentrations of acrylamide (acr). we also observed the counteracting effects of brain-derived neurotrophic factor (bdnf) and calpeptin in these systems. to create the hexagonal array we used a poly(dimethylsulfoxide) bilayer stencil comprising through holes for adhesion spots and interconnecting tracks. plasma stencilling a peg-coated glass substrate produces adhesive nodes for the neurons and micron-scale-tracks for guiding neurite outgrowth and connectivity. results: in both systems, the developing and mature network, we found not only a concentration dependant effect of acr and bdnf but also a time dependant effect with a limited capability of the developing system to regenerate, even in the presence of acr. the co-treatment of the cells showed that inhibition of calpains by calpeptin might reduce the effect of intracellular elevated ca2+, a known neurotoxic mechanism of acr. moreover, the neurothrophin bdnf acts via trkb receptors on pathways stimulating neurite outgrowth and thereby counteracting the adverse effect of acr. conclusion: with the nfa we provide a rapid and simple way to analyze neurite outgrowth and connection formation in real time. by spatially standardizing the array we provide assay coordinates to streamline the analysis process and bring it towards high throughput testing. furthermore preliminary data showed that modification of the surface with biomolecules allows cell adhesion of other neuronal celltypes (e.g. primary mouse neurons) and extracellular matrix proteins (e.g. laminin) stimulate neurite outgrowth via integrins. transcriptional regulation of nox4 by histone deacetylases siuda d. 1, 2 , zechner u. 3 , prawitt d. 4 nox4 is a member of the nadph oxidase family, which represents a major source of reactive oxygen species (ros) in the vascular wall. nox4-mediated ros production mainly depends on the expression levels of the enzyme. the present study is aimed to investigate the regulation mechanisms of nox4 transcription by histone deacetylase (hdac). in cultured human ea.hy 926 endothelial cells, treatment with the pan-hdac inhibitors (scriptaid, trichostatin a, tsa, and suberoylanilide hydroxamic acid, saha) leads to a drastic decrease in nox4 mrna expression. a similar down-regulation of nox4 mrna expression can be achieved with sirna-mediated knockdown of hdac3. hdac inhibition in endothelial cells is associated with enhanced histone acetylation in the human nox4 promoter region, with no significant changes in dna methylation. consistently, scriptaid-treated cells show increased chromatin accessibility in nox4 promoter. in addition, we provide evidence that c-jun plays an important role in controlling nox4 transcription. knockdown of c-jun with sirna leads to a marked downregulation of nox4 mrna expression. in response to scriptaid treatment, the binding to c-jun to the nox4 promoter region is reduced despite the open chromatin structure. in parallel, the binding of polymerase iia to the nox4 promoter is significantly inhibited as well, which may explain the reduction in nox4 transcription. in conclusion, hdac inhibition decreases nox4 transcription in human endothelial cells by preventing the binding of transcription factor(s) and polymerase(s) to the nox4 promoter. this is very likely because of a hyperacetylation-mediated steric inhibition. cyclopentenone prostaglandins induce oxidative dna-damage in hamster lung fibroblast v79 cells solecki g. m. 1 key: cord-001835-0s7ok4uw authors: nan title: abstracts of the 29th annual symposium of the protein society date: 2015-10-01 journal: protein science doi: 10.1002/pro.2823 sha: doc_id: 1835 cord_uid: 0s7ok4uw nan c-terminus glutamine-rich sequence deleted) to elucidate the role of metalloprotein hpn-like by fluorescence resonance energy transfer (fret) (figure 2 ) [2] . we found the selective coordination of ni(ii) and zn(ii) to the purified sensors and in e. coli cells. surprisingly, specific interaction between the fret sensors and bi(iii) was observed. our fret analysis confirmed the role of hpnl for ni(ii) storage and revealed the potential association of hpnl with bi-based antiulcer drugs in cells. pb-006 rna fate is controlled by highly-regulated rna binding proteins molecular mechanism that links the mrna-degradation pathway with extracellular signaling networks through the reversible unfolding of a rna binding domain (rbd). rna binding is also controlled by ph conditions. this finding becomes relevant for rbps such as t-cell intracellular antigen 1 (tia-1), which shuttles between two cellular compartments (nucleus and cytoplasm) with slightly different ph values. in fact, rna binding by tia-1 is modulated by slight environmental ph changes due to the protonation/deprotonation of tia-1 histidine residues [3, 4] . the ph dependence of the tia-1/rna interaction provides a new insight into the function of tia-1 in recognizing new rna targets [5] , like the 5' terminal oligopyrimidine tracts (5tops) of translationally-repressed mrnas. along with tia-1, the rbp hu antigen r (hur) is involved in the assembly/disassembly of cytoplasmic stress granules (sg), which arise as a protective mechanism by preventing mrna decay under stress situations. despite wide acceptance that rbps harboring aggregation-promoting prion related domains (prds), such as tia-1, stimulate rapid self-association and formation of sgs, we propose that scaffolding sgs may be driven by rbds, since prd-lacking rbps, like hur, often form oligomers [6, 7, 8] and are included in sgs. under continuous stress, the transition from the physiological to pathological aggregation of rbps in sgs may depend on post-translational modifications of rbds. rna-binding proteinopathies, characterized by the nucleation of irreversible sgs, are often found in neurodegenerative diseases. altogether, resulting insights into rna biology suggest that highly-regulated rbps determine mrna fate from synthesis to decay. a threat to 70 million people in underdeveloped nations around the world, african trypanosomiasis (sleeping sickness) is a neglected tropical disease (ntd) caused by the protozoan parasite trypanosoma brucei (t. brucei). t. brucei is transmitted to humans via the tsetse fly, and replicates in the blood before crossing into the brain, causing death for the infected individual. current treatments that are available for african sleeping sickness are highly toxic and usually difficult to administer past the blood-brain barrier. it is our belief that coupling less toxic compounds with efficient drug delivery systems will contribute to the development of the most effective drug against african sleeping sickness. our goal was to determine a novel and effective chemical inhibitor with the potential to prevent the replication of t. brucei in the human body. the enzyme target for inhibition studied in this research was 6-phosphogluconate dehydrogenase (6pgdh), a cytosolic enzyme in the pentose phosphate pathway (ppp) of t. brucei. 6pgdh is essential in the ppp due to its ability to oxidize 6-phosphogluconate into ribulose-5-phosphate, which is essential for the formation of nucleotides. primer overlap extension polymerase chain reaction (pcr) was used to synthesize the coding dna sequence of the 6pgdh gene, which was then cloned into a pnic-bsa4 inducible expression plasmid with an n-terminal 6 histidine tag, by way of ligation independent cloning. the protein was then expressed in bl21 (de3) escherichia coli (e. coli) cells and purified via nickel column affinity and size exclusion fast protein liquid chromatography (fplc) to perform inhibition assays. through virtual screening, various ligands obtained from the chembridge library and nih clinical collection) were docked into the active site of the crystal structure of tb6pgdh (pubchem identification 1pgj) using gold molecular docking software. the top scoring compounds were selected by utilizing parameters such as hydrophobic interactions, hydrogen bonds, and van der waals forces. the compounds with the best scores that also satisfied lipinski's rule of 5 criteria for druggability were then tested in spectrophotometric enzyme inhibition assays monitoring the absorbance of nadph at 340 nm. compounds that show inhibitory activity in the assays will be taken to higher levels of testing to determine their effect on t. brucei in other organisms. nmr studies of the structural influence of phosphopantetheinylation in nonribosomal peptide synthetase carrier proteins and impact on binding affinities andrew goodrich 1 , dominique frueh 1 1 nonribosomal peptide synthetases (nrpss) are modular enzymatic systems responsible for the production of complex secondary metabolites in bacteria and fungi. each module is comprised of (at least) three core domains whose combined action leads to the selection, activation, and incorporation of a single small molecule into a growing peptide. central to each module is the carrier protein (cp), which is first primed via attachment of a 4'-phosphopantetheine moiety (ppant arm) to a conserved serine to generate the active holo form. an adenylation (a) domain then covalently attaches an amino or aryl abstract acid onto the ppant arm via formation of a thioester. the cp then shuttles activated monomers and growing peptides between the active sites of catalytic domains in both the same and adjacent modules. during cp priming and peptide elongation, a cp thus exists in multiple different post-translational states and interacts with numerous catalytic domains. understanding how nrpss are able to efficiently orchestrate this series of sequential protein-protein interactions between a cp and its partner catalytic domains is key to unraveling the molecular mechanism of nrp synthesis. using a combination of isothermal titration calorimetry and nuclear magnetic resonance (nmr) titrations, we found that converting a cp from the apo to holo form alters its affinity for its partner a domain. this change in binding suggests a means by which directionality in protein-protein interactions is achieved in nrpss. however, we also found that a domain binding affects the same subset of residues in both the apo and holo forms. in order to identify the molecular features underpinning this difference in affinity, we solved the nmr solution structures of the apo and holo forms of the cp. here, we present the solution structures of an apo and holo cp and discuss them in light of their differential binding to an a domain. functional analysis of of conditional analog-sensitive alleles of essential protein kinases in the fission yeast schizosaccharomyces pombe. juraj gregan 1, 2 1 mfpl/imp, 2 the genome of the fission yeast schizosaccharomyces pombe encodes for 17 protein kinases that are essential for viability. studies of the essential kinases often require the use of mutant strains carrying conditional alleles. to inactivate these kinases conditionally, we applied a recently developed chemical genetic strategy. the mutation of a single residue in the atp-binding pocket confers sensitivity to small-molecule inhibitors, allowing for specific inactivation of the modified kinase. using this approach, we constructed conditional analog-sensitive alleles of 13 essential protein kinases in the fission yeast s. pombe. i will present the functional analysis of these mutants during meiosis. peptide conjugates: from self-assembly towards applications in biomedicine ian hamley 1 1 university of reading, dept of chemistry self-assembling peptides and their conjugates offer exceptional potential in nanomedicine. i will present some of our recent work on nanoscale assembled peptides and their conjugates, focussing on lipopeptides [1, 2] and peg-peptide conjugates [3] . pegylation is an important technique in the development of conjugates for applications in therapeutics. it is found to greatly influence self-assembly of peptides and proteins -one example from our own work is a peptide which itself forms twisted fibrils but when peg is attached, self-assembly of the conjugate leads to spherical micelles [4] . the conjugate can be enzymatically degraded using alpha-chymotrypsin, releasing the peptide. this nanocontainer delivery and release system could be useful in therapeutic applications. thermoresponsive telechelic peg/peptides with hydrophobic dipeptide end groups (di-tyrosine or di-phenylalanine) were developed, one of which shows a de-gelation transition near body temperature and which may be useful in bioresponsive delivery systems [5] . examples from our recent work on self-assembling lipopeptides will also be outlined. our focus is to investigate potential relationships between self-assembly and bioactivity, in particular in the fields of regenerative medicine [6] [7] [8] [9] [10] , antimicrobial systems [11, 12] and immune therapies [13] . been shown to become derivatized with argpyrimidine, a prominent nem that occurs on arginine residues [6] , in certain human cancer tissues and cell lines [7, 8] . this nem was linked to the elevated antiapoptotic activity of the protein [7, 8] , whereby modification of arg-188 appeared to be of particular significance [7] . in this work, hsp27 homogeneously modified with argpyrimidine at position 188 is generated for the first time. using expressed protein ligation [9] , the first semisynthesis of the unmodified protein is achieved as well. our approach, which combines organic chemistry, peptide synthesis and protein synthesis, enables complete control over protein composition and thus can provide previously unattainable insight into the properties of this vital chaperone following nonenzymatic modification. the synthesis of argpyrimidine-modified hsp27 and the progress towards structural and functional characterization of the protein will be presented herein. kunitz-type protease inhibitors belong to a widespread protein family present in many plant species and play an important role in plant defense against insect pests and pathogens. members of this family are typically inhibitors of proteases of serine class. interestingly, a few members were identified as inhibitors of proteases of cysteine class, however, they have not been functionally and structurally characterized. our study is focused on kunitz-type inhibitors of cysteine proteases (pcpis) from potato (solanum tuberosum). a series of 20 kda pcpis was purified using a multi-step chromatographical protocol, and two most abundant and effective isoinhibitors named pci 1-5 and pci 3 were characterized in detail. they were screened against a broad panel of model cysteine proteases and digestive cysteine proteases from herbivorous insects. pci 1-5 and pci 3 exhibit different inhibitory specificity pattern and potency up to the nanomolar range. both isoinhibitors were crystallized and their spatial structures were solved and refined at 1.5 å (pci 1-5) and 1.7 å (pci 3) resolutions. a position of reactive sites against cysteine proteases on the conserved b-trefoil fold scaffold was proposed. the work provides the first analysis of pcpis with respect to the structure-function relationships and evolution within the kunitz-type inhibitor family. role of the abcc2 transporter in the mode of action of the bacillus thuringiensis cry1ac toxin in the diamond back moth plutella xylostella 1 protonation pattern influence actively properties of molecules and play an essential role in biochemical mechanisms. for an accurate determination of the protonation equilibria, the absolute proton solvation free energy needs to be known. the determination of this energy represents one of the most challenging problems in physical chemistry. this is particularly difficult for protons solvated in water, where the solvation is dynamically performed by different water clusters and the proton is not attached to a single solvent molecule. the proton solvation is notably important in order to quantify mechanisms of proton transfer and such processes have been investigated for a long time based on different approaches, often leading to contradictory conclusions. a rigorous and accurate protocol for computing proton solvation in solvents of different nature is of prime importance for applied (pharmaceutical and material science) and fundamental sciences. in this study, proton affinities, electrostatic energies of solvation and pka values of a reference set of organic molecules are computed in protic and aprotic solvents. proportional to the free energy of proton dissociation, the pka value calculation is therefore strongly dependent on the free energy of proton solvation. such energy is then determined in acetonitrile (acn), methanol (met), water and dimethyl sulfoxide (dmso) in order to obtain the best possible match between measured and computed pka values. the computation of these values is based on a combination of quantum chemical (qc) and electrostatic approaches by using a thermodynamic cycle connecting gas-phase and solvent-phase of proton dissociation. the computed proton solvation energies in acn, met, water and dmso of the present study are very precise (rmsd much lower than 1 ph value). they will be a basis for better understanding of proton solvation and help to predict pka values of organic compounds in different solvents more precise. biochemical characterization of two evolutionary distant ten-eleven translocation enzymes and their utility in 5-methylcytosine sequencing in the genomes at single-base resolution subtypes leading to an inability to perceive pain and painful neuropathies, respectively. however, as nav ion channels are intimately involved in almost all aspects of physiology, only the most selective inhibitors would be suitable as drug leads. disulfide-rich venom derived mini-proteins from cone snails and spiders are being actively pursued as novel therapeutics for pain, because of their high selectivity and potency at human ion channels, including sodium channels (nav). two main strategies of inhibition have been identified; blocking the pore and interacting with the voltage-sensor domains (vsd) surrounding the pore. the ion-conducting pore is highly conserved between all sodium channel subtypes whereas the voltage-sensor domain binding sites are less conserved. therefore, inhibition of a specific nav isoform is more achievable using inhibitors that modulate vsds than with pore blockers. gating modifier toxins from spider and cone snail venom inhibit nav1.7 and nav1.8 by interacting with the vsd. they appear to reach their target by partitioning into the lipid membrane surrounding the ion channel, thus enabling access to the vsd. toxin pharmacology may therefore not only be driven by the peptide-ion channel interactions, but also including the lipids surrounding the channel protein, a feature that is very much under explored. it is therefore apparent that peptide-lipid interactions in combination with peptide-channel interactions need to be considered when designing potent inhibitors. using a range of biophysical techniques, including surface plasmon resonance and nuclear magnetic resonance, we are studying the interactions underpinning the mechanism of action between toxins and membranes and toxins and ion channels. initial results show that the lipid composition surrounding ion channels play a major role in terms of toxin:lipid interaction and that these interactions can be used in combination with traditional structure-activity relationship studies to design selective and potent nav inhibitors, which will be discussed. we believe that our studies will ultimately delineate what drives toxin pharmacology and nav subtype selectivity and will lead to improve rationally engineering of novel therapeutics for the treatment of pain. micelles promote aß42 assembly into pore-forming oligomers montserrat serra-batiste 1 , mariam bayoumi 2 , margarida gair ı 3 , mart ı ninot-pedrosa 1 , giovanni maglia 2 , nat alia carulla 1 1 institute for research in biomedicine (irb barcelona), 2 biochemistry, molecular and structural biology section, university of leuven, 3 the formation of amyloid-b peptide (ab) oligomers at the cellular membrane is considered to be a crucial process underlying neurotoxicity in alzheime rs disease (ad). 1-2 therefore, it is important to understand how oligomers form within a membrane environment. using solution nuclear magnetic resonance (nmr) spectroscopy, combined with size exclusion chromatography (sec), we have studied the two major ab variants-ab40 and ab42, the latter having a more prominent role in ad than the former-under carefully selected micelle conditions intended to mimic a membrane environment. our results indicate that after an incubation period, ab42, but not ab40, assembles into oligomers with specific structural properties, which we have named stabilized micelle oligomers (smos). smo complexes incorporate into lipid bilayers as well-defined pores, a feature linked to neurotoxicity. these results have important implications in the ad field as they provide a new perspective on how ab oligomers cause neurotoxicity. indeed, our findings constitute a first step towards the establishment of a new therapeutic target for ad. dimer formation. it should be noted that this nb peptide contains the autophosphorylatable ser-11 associated with phk activation, and phosphorylated nb; peptide was considerably less effective in promoting b-dimer formation than non-phosphorylated peptide. these results suggest a role for ser-11 autophosphorylation in mediating homodimeric b subunit interactions within the phk complex, and augment previous studies on the activation of phk by phosphorylation in which changes at the nterminus of b are critical in the activation of the catalytic g subunit. summing these results leads to a new model of activation. in this model, in the inactive state, the nonphosphorylated n-terminus of b interacts directly or indirectly with the regulatory c-terminal domain of the g subunit, inhibiting catalytic activity. upon phosphorylation of the n-terminus of b, three important events occur: 1) the interaction between b and g is disrupted, 2) the b subunits of the holoenzyme self-associate, and 3) the catalytic domain is activated. thus, we envision that the n-terminus of b acts as an allosteric switch, with activation triggered by phosphorylation of this region, causing disruption of its previously inhibiting interactions with g and promotion of b b dimerization to stabilize the activated conformation of g . the research was supported financially by the university of kansas medical center biomedical research training program and nih grant dk32953. pb-032 hssb1 is involved in the cellular response to oxidative dna damage christine touma 1 , nicolas paquet 2 , derek j. richard 2 , roland gamsjaeger 1,3 , liza cubeddu 1,3 1 school of science and health, university of western sydney, 2 queensland university of te chnology, 3 school of molecular bioscience, university of sydney cellular dna is subject to oxidative damage in the presence of reactive oxygen species. the 7,8-dihydro-8-oxoguanine (8-oxog) adduct is the most common form of oxidative damage and results in g:c to t:a transversions; these lesions are normally processed by the base excision repair (ber) pathway. singlestranded binding (ssb) proteins of the oligonucleotide binding domain family are heavily involved in dna repair processes, which involve the detection of dna damage and recruitment of repair proteins to the site of damage. using immunofluorescence we demonstrate that hssb1 (a novel human ssb) levels increase in response to oxidative damage (h202). cells depleted of hssb1 are hypersensitive to oxidative damage and are also unable to efficiently remove 8-oxog adducts. we show that hssb1 forms dimers and tetramers under oxidative conditions and that this oligomerisation is likely mediated by inter-domain disulfide bond formation. furthermore, using surface plasmon resonance, we also show that oxidised hssb1 binds to 8-oxo-g damaged ssdna with higher affinity than non-damaged ssdna, indicating a direct role for oxidised hssb1 in the recognition of 8-oxo-g lesions. as oxidative stress is associated with aging, cancer and alzheimer's disease, understanding the molecular mechanisms of how cells repair oxidative dna damage will be crucial in the development of potential therapeutic treatments. epidemic typhus, which is caused by the bacterial pathogen rickettsia prowazekii, is a menacing disease world wide that the nih lists as one of america's greatest biological weapons threats. this research seeks to find novel inhibitors of b-ketoacyl-acp-reductase (fabg), an enzyme that catalyzes one of the reactions in the fatty acid synthesis type ii system in bacteria. this pathway is essential for survival in bacteria. the fabg enzyme uses nadph as a substrate, which facilitates the binding of the second substrate, acetoacetyl-acp into the active site. the acetoacetyl-acp is subsequently reduced into b-hydroxyacyl-acp. the coding dna sequence for the rpfabg protein was cloned into a pnic vector and transformed into e.coli bl21(de3), then the protein was expressed and purified using metal affinity and size exclusion chromatography methods. high throughput molecular docking software (gold) was used to screen a commercial library of ligands against the acetoacetyl-acp region of the active site. the ligands with the best gold scores were selected to be tested in vitro. spectrophotometric enzyme inhibition assays were performed to determine whether the drugs could inhibit rpfabg activity. chlorogenic acid, a previously known inhibitor of homologous fabgs, was tested along with the other potential drugs, and was determined to have moderate inhibitory effects on rpfabg. loop modeling using icm software was performed in order to create a prediction of the complete rpfabg structure, including the disordered loops that are not a part of the 3f9i pdb structure. co-crystallization of rpfabg with both substrates was carried out in order to obtain a structure, but only nondiffracting crystals resulted. further inhibition assays and crystallography trials are being performed in order to continue the search for a novel inhibitor of rpfabg and ultimately a treatment for epidemic typhus. 1 the university of hong kong bioconjugation of proteins has emerged as a useful tool in the study of biological systems. there is an increasing need to develop new synthetic technologies for the bioconjugation reaction of proteins, and metal-catalyzed site-selective modification of proteins has attracted considerable interest in recent years. we have developed a ruthenium glycosylated porphyrin-catalyzed carbenoid transfer reaction for the site-selective modification of proteins. we firstly applied the catalysis to the selective modification of the n-terminus of peptides. by using ruthenium glycosylated porphyrin as catalyst, the n-terminus of a number of peptides can be modified through carbenoid n-h bond insertion in aqueous media with moderate to excellent conversion. the reaction is highly selective, for example, the reaction with ytsssknvvr, which contains various types of oxygenhydrogen and nitrogen-hydrogen bonds possibly available for carbenoid insertion, catalyzed by the ruthenium glycosylated porphyrin gave the n-terminal-modified product with >99% conversion and without the formation of other modified peptides including doubly modified and oxygenhydrogen bond insertion products. we next extended the n-terminal modification method to proteins. eventually success was attained in the modification of rnase a and insulin. the reaction of rnase a with a diazoacetate mediated by ruthenium glycosylated porphyrin gave corresponding n-terminal-modified protein with 65% conversion. we also achieved a bioconjugation to ubiquitin via ruthenium glycosylated porphyrin-catalyzed alkene cyclopropanation in aqueous solution in two steps: (1) incorporation of an alkenic group by the reaction of n-hydroxysuccinimide ester with ubiquitin and (2) cyclopropanation of the alkene-tethered lys6 ubiquitin with the fluorescent labeled diazoacetate in the presence of a catalytic amount of ruthenium glycosylated porphyrin. the corresponding cyclopropanation product was obtained with 55% conversion based on maldi-tof mass spectrometry. in conclusion, we developed a ruthenium porphyrin-catalyzed siteselective modification of peptides and proteins in aqueous media. the method provides an entry to new bioconjugation reactions for protein modifications using metalloporphyrins as catalysts. uridine monophosphate synthase: architecture versatility in the service of late blight control francisco tenjo castaño 1,2 , manuel garavito 1,2 , leonor garc ıa 1,2 , silvia restrepo 2 , barbara zimmermann 1 1 biochemistry and molecular biology research group, universidad de los andes., 2 mycology and plan pathology laboratory, universidad de los andes uridine monophosphate synthase (umpase), a bifunctional enzyme in the de novo pyrimidine biosynthetic pathway, is a protein comprised of orotate phosphoribosyl transferase (oprtase) and orotidine monophosphate decarboxylase (odcase). different fusion orders of the two domains have been documented to exist in nature. in some organisms oprtase and odcase are monofunctional proteins, and act as a complex. here, umpase from solanum tuberosum (potato) and from phytophthora infestans (an oomycete) were examined. p. infestans causes late blight disease in s. tuberosum, destroying crops and increasing production costs. since pyrimidines are fundamental cellular components, we have proposed that umpase could serve as a target to control p. infestans infection. the enzymes from p. infestans and s. tuberosum differ in their fusion order of oprt and odc. the study of these two umpase could facilitate the design of species-specific inhibitors, and might shed light on the effect of fusing umpase domains in one order or the other. to this end we carried out bioinformatic and biochemical characterization of the enzymes. sequence analyses showed 20 residue differences among the p. infestans umpase sequences from three strains: 4084, 1306 and t30-4. strain t30-4 was found to have a duplicated umpase, but neither sequence corresponded to the ones predicted previously from the genome. a recombinant umpase from 4084 strain was expressed in bacteria and purified but it showed low solubility and was inactive in vitro. the recombinant umpase from the 1306 strain complemented both oprtase and odcase deficient e. coli strains. a soluble, active, recombinant protein was expressed and purified in the presence of high salt and the product ump (specific activity 0.2 lmol min-1 mg-1). the sequence skq was found at the c-terminus of the p. infestans umpase sequences and resembles a peroxisome signal peptide (skl). the predicted hydrophobicity of this umpase and its architecture (oprt at the c-terminus and odc at the n-terminus) resembles that of the umpase from leishmania donovani, which has been localized to the peroxisome. we suggest that p. infestans umps could also be located in this organelle. in contrast to the oomycete enzyme, s. tuberosum umpase is highly soluble, and has a higher specific activity (vmax5 8.8 lmol min-1 mg-1). we measured the kinetic parameters km(orotate)5 16.2 lm, km(prpp)5 25.5 lm, and found that it exhibited product inhibition by pyrophosphate. in conclusion, the different architectures of the two umps might be related to distinct biochemical characteristics, further supporting this protein as a good candidate for p. infestans control. we present computer simulation studies of three different antimicrobial peptides we have been studying by md computer simulation in collaboration with experimentalists. the first is daptomycin, a potent lipopeptide currently licensed to treat infections caused by multi-drug-resistent bacteria. the mechanism of action of daptomycin is currently not completely understood. we have solved the nmr structure of this molecule, and attempted to determine the size of its oligomer by small angle neutron scattering (sans) supported by computer simulation. feglymycin is a 13-amino-acid peptide with a high percentage of unusual amino acids such as 4-hydroxyphenylglycine and 3,5-dihydroxyphenylglycine. feglymicin inhibits mura and murc enzymes which are involved in bacterial peptidoglycan synthesis, while also displaying anti-hiv activity by interaction with the viral envelope protein gp120. a previous x-ray structure shows the molecule forming a dimer. here, the molecule was studied by nmr in water and dmso. in water, the molecule is clearly at least a dimer, while in dmso it is a monomer. we have performed noe refinement simulations in order to elucidate a structure, however, due to a lack of long-range noe contacts, a unique structure cannot be determined. labyrinthopeptin a2 is a lantibiotic that contains labionin, a unique carbacyclic posttranslationally modified amino acid that links the protein backbone in three different locations. labyrinthopeptin a2 has shown promising activity as a pain killer. starting from the x-ray structure, we present results from the first md simulation studies of this unique peptide. because of the extensive cross-linking, this peptide is observed to be highly rigid in its native form. simulation results of mutants are also presented. antibiotics with new mechanism of action are urgently required to combat the growing health threat posed by resistant pathogenic microorganisms. here we report the discovery of a new peptidomimetic antibiotic (l27-11), which is active with a minimum inhibitory concentration (mic) in the low nanomolar range, only against pseudomonas sp., and with a non-membrane-lytic mechanism of action. a drug target identified both in a forward genetic screen for resistance determinants and by photoaffinity labeling is the ß-barrel protein lptd, which plays an important role in lps transport and the outer membrane biogenesis. the x-ray structure of lptd in complex with lpte from shigella flexneri shows a 26 stranded b-barrel linked to a periplasmatic n-terminal jelly-roll domain. interestingly the homology model structure for lptd from pseudomonas shows a significant difference: an insertion of around 100 amino acids in the n-terminal domain. the results of our attempts to purify and characterize this large outer membrane protein and to determine the binding site of the peptidomimetic antibiotic will be shown. the theory of how life on earth begun still remains unclear. nevertheless, according to some theories, at the beginning level proteins did not emerge as a complex globular forms as know today. at the times, when solely rna molecules stored both genetic information and catalyzed the chemical reactions in primitive cells, peptides acted as a proteins nowadays [1, 2] . literature postulate that the possible role of primordial short peptides was to catalyze reactions in rna-world, as they possess an excellent ability to self-assemble into well-ordered nanostructures [3, 4] . elementary functional loops (efls) can be considered as a small structures (blocks) having specific signatures and providing functional residues important for binding/activation as well as principal chemical transformation steps of the enzymatic reaction [5] . p-loop efl is a widespread structure across vast majority of protein families such as motor domains, aaa1, reca, pepck and many others. sequential alignment of these protein families reveals existence of a conserved p-loop motif, that is able to bind atp molecule. we investigated the structure and atpase activity of peptides, which sequences possessed strongly conserved gxgk[t/s] motif from ploop. the goal of our work was to check if peptides corresponding to the most conserved p-loop motif fragment are able to bind and hydrolyze atp molecule. all peptides under study were chemically synthesized and their structures was investigated by nmr spectroscopy. the ability to bind atp molecules was analyzed by using hplc chromatography. results of our study show, that peptides with conserved p-loop motif have a suitable structures to promote binding of the molecules with phosphate group, but cannot accelerate pyrophosphate hydrolysis process. conference participation for w. _ z. supported by the fp7 project mobi4health (grant agreement no 316094). computational resources were provided by the informatics center of the metropolitan academic network (ic man task) in gdansk, poland. ck2 is a ubiquitous serine/threonine protein kinase, being one of the most pleiotropic of all protein kinases1. ck2 plays a key role in cell growth, differentiation, cell death and survival, and become the therapeutic target in cancer treatment, since its level is significantly increased in cancer cells2. halogenated ligands have been widely developed as potent inhibitors of protein kinases. among them 4,5,6,7-tetrabromobenzoteriazole (tbbt) is one of the first potent and selective inhibitor of ck2a, directed towards the conserved atp binding site3. to assess contribution of electrostatic interactions to the specificity and strength of binding of multi halogenated inhibitors by a protein kinase, we have studied interaction between ck2a and nine benzotriazole derivatives, representing all possible patterns of halogenation on the benzene ring. herein, we present results that support existence of two alternative regions that are involved in ligand binding. aspartic acid 175 is known for its function in coordination of a mg21 ion, which is required for atp binding 4. asp175 has been identified in crystal structure of ck2:tbbt complex (pdb1j91, fig. 1 ) as the charged residue closest to tbbt. there is also lys68 proximal to tbbt, interaction with which may favor anionic form of ligands5 (pk for tbbt <5), however it is involved in the intramolecular salt bridge, and thus its mutation may significantly change stability of the protein. crystal structure of tbbt complexed with ck2 (pdb:1j91). residues with a distance to tbbt (magenta) shorter than 5a are shown. red residue is negatively charged, blue ones are protonated. abstract comparison of kdiss values determined for ligands at ph 8 and at ph 7 shows that strength of the complex significantly varies upon deprotonation of the triazole ring. this confirms former hypothesis that a negatively charged ligands cluster at the atp binding site region proximal to lys685, which is beneficial both to the specificity and to strength of the binding. we have also observed for the tested ligands variations in their binding to either wild type protein and its d175n mutant (with less negative charge distributed over atp binding site). all ligands displaying higher pka for dissociation of the triazole proton bind to the mutant visibly weaker than to the wild-type protein. altogether reveals the predominance electrostatic intermolecular interactions. although, negatively charged ligands most probably cluster at the atpbinding site proximal to lys68, beneficial for the strength of binding, the less dissociated forms are favored due to unfavorable interactions of the anionic form of ligands with asp175. 1 there are many virulence factors produced by these strains, many of which are encoded on mobile genetic elements. 2 psms are of specific interest because these virulence factors are encoded on the core genome of the bacteria and therefore all strains of staphylococci bacteria produce some variation of psms with a variety of biological functions. 2 the specific mechanism by which psms act as virulence factors has been poorly understood until recently. biological functions of psms include cell lysis, biofilm formation and the ability to kill neutrophils after phagocystosis.1 these toxins are of special interest to our research group due to their genetic similarities to certain bacteriocins, namely leaderless bacteriocins. 3 both groups of peptides are ribosomally synthesized with a n-terminal formyl methionine and secreted from the bacteria by atp-binding cassette (abc) transporters without any leader sequence or signal peptide. abc transporters may also play a role in immunity towards psms and leaderless bacteriocins. these similarities led our group to investigate the solution structure of these peptides through nuclear magnetic resonance (nmr). isolating psms from the producer organisim, s. aureus, typically involves lengthy extractions and low yields. 4 for these reasons, we opted to chemically synthesize the desired peptides using solid phase peptide synthesis (spps). utilizing a variety of spps techniques, psm a1 and psm a3 were successfully synthesized, however, due to the hydrophobic nature of psm b2, an alternate genetic approach was devised to isolate psm b2. formation of a fusion protein between psm b2 and the small ubiquitin like modifier (sumo) protein allowed for heterologous expression. upon cleavage of the fusion protein with sumo protease, and subsequent purification and isolation of the cut peptide, psm b2 was obtained. as previously reported, the psms were found to be alpha-helical in structure inducing solvents. 5 a series of 2 dimensional (2d) nmr experiments were ran to determine chemical shift assignments and to obtain noe data. importing the chemical shift assignments and noe data into the structure calculating software, cyana, we were able to elucidate the solution structure of psm a1 and psm a3 and we are currently working towards the elucidation of psm b2. the synthesis, isolation, characterization and solution structures of the aforementioned psms will be discussed here. 1 transition metals are critical for enzyme function and protein folding, but their excess can mediate neurotoxic oxidative processes [1] . as, energy production involves oxidative phosphorylation, a process requiring a continuous flow of electrons, mitochondria are particularly vulnerable to oxidative damage [2] . as such, mitochondria are the major sites of reactive oxygen species (ros) generation, which are produced as byproducts of the electron transport chain. since free iron and certain ros can engage into potentially deleterious processes such as fenton reaction, mitochondrial iron homeostasis must be tightly controlled, and dysregulation of iron metabolism in this organelle has been associated with various diseases, including friedichs ataxia (fa), alzheimer's, and other neurodegenerative disorders [3] . engineering an efficient mitochondriatargeting, cell-permeable vector is a challenge due to the fact that mitochondrion is impermeable to a wide range of molecules. the development of delivery vectors has been made possible by a greater understanding of mitochondrial structure and chemical features of molecules that selectively localize within this organelle. from these findings, two generalized requirements for mitochondrial localization are delocalized positive charge and lipophilicity [4, 5] . targeting iron in this organelle is proposed as a means to ameliorate fa symptoms. desferrioxamine (dfo) is a bacterial siderophore with high affinity for iron, but low cell penetration. we prepared conjugates of dfo with mitochondria penetrating peptides and studied their iron-binding characteristics in vitro. the lipophilic and charged peptides tat49-57 (h-arg-lys-lys-arg-arg-gln-arg-arg-arg-oh) [6] , 1a (h-cha-arg-cha-lys-cha-arg-cha-lys-nh2) [6] , ss-02 (h-dmt-arg-phe-lys-nh2) [7] and ss-20 (h-phe-arg-phe-lys-nh2) [7] , are known to permeate cytosolic and mitochondrial membranes. they were prepared and conjugated to dfo in solid-phase [8] , an alternative synthetic route. once detached from the resin, fully deprotected, purified and characterized by means of lc/ms and aminoacid analysis, it was observed that the dfo-conjugated peptides displayed iron-binding abilities identical to the free chelator dfo. dfo-conjugated peptides were also able to quench the iron-catalysed oxidation of ascorbate (a model of oxidative stress in plasma of iron-overloaded patients), as probed by a high throughput fluorimetric method [9, 10] . these results indicate that our synthesis and conjugation strategy were successful in preserving the iron-binding moiety and the antioxidant ability of the free chelator dfo. the proteolytic activity and oligomerization status of the human htra3 protease functioning as a tumor suppressor of an n-terminal domain not required for proteolytic activity, a central serine protease domain and a cterminal pdz domain. the latter serves as a substrate or regulator binding domain and may participate in oligomerization. htra3s, its short natural isoform, lacks the pdz domain which is substituted by a stretch of 7 c-terminal amino acid residues, unique for this isoform. down-regulation of htra3 in tumors, shown by other groups and us, suggests htra3s involvement in oncogenesis [1] . htra3 acts as a proapoptotic protein and is suggested to function as a tumor suppressor. it promotes cytotoxicity of etoposide and cisplatin in lung cancer cell lines [2, 3] . to date, htra3 has been poorly characterized from the biochemical point of view, mainly due to the fact that it is difficult to purify recombinant htra3. we were able to express in bacterial system and purify htra3 in quantities sufficient to perform structural studies. the aim of this study was to characterize and compare the proteolytic properties and quaternary structure of the htra3 isoforms. both studied isoforms lacked the n-terminal domain. htra3 with the pdz domain removed (htra3-dpdz) and htra3s (htra3s) were fully active at a wide range of temperatures and their substrate affinity was not impaired. this indicates that the pdz domain is dispensable for htra3 activity. as determined by size exclusion chromatography, htra3 formed stable trimers while both htra3-dpdz and htra3s were monomeric. this suggests that the presence of the pdz domain, unlike in other human htras (htra1 and htra2), influences htra3 trimer formation. the unique c-terminal sequence of dn-htra3s appeared to have little effect on activity and oligomerization [4] . cyclodextrins (cds) are cyclic oligosaccharides that have been recognized as useful pharmaceutical excipients. in aqueous solution cds are capable to form complexes with various ligands, hosting inside their cavity either a whole molecule, or part of a ligand. inclusion complexes with cds offers a variety of physicochemical advantages over the biologically active ligands, including the improved aqueous solubility, solution stability or an increase of bioavailability. ck2 is an ubiquitous, highly pleiotropic and constitutively active ser/thr protein kinase. halogenated benzotriazoles have been developed as potent and selective inhibitors of this enzyme. the interaction of the catalytic domain of human protein kinase ck2 with a series of brominated ligands, which represent all possible patterns of halogen substitutions to the benzene ring of benzotriazole, was previously studied by microscale thermophoresis (mst) [1] . this method alloweddetermination of binding affinities for seven ligands, all of which were found consistent with the values determined independently by isothermal titration calorimetry (itc). however, a very limited aqueous solubility of some brominated benzotriazoles may decrease their bioavability, thus affectingtheir apparent activity [2] . to overcome this limitation, the aqueous solubility of halogenated benzotriazoles in the presence of cyclodextrins has been tested. the formation of inclusion complexes with b-cyclodextrin (b-cd), hydroxypropylb-cyclodextrin (hp-b-cd) and g-cyclodextrin (g-cd) in aqueous solutions, followed by uv-vis spectroscopy, substantially improved the solubility of tbbt and its derivatives. the interaction between protein kinase ck2 and cyclodextrins, and also with their inclusion complexes with halogenated benzotriazoles, was followed with the aid of the microscale thermophoresis. the results obtained clearly demonstrate that the binding of halogenated benzotriazoles by ck2 is only moderately affected by cyclodextrins. oligonucleotide-based molecular circuits offer the exciting possibility to introduce autonomous signal processing in biomedicine, synthetic biology, and molecular diagnostics. here we introduce bivalent peptide-dna conjugates as generic, noncovalent, and easily applicable molecular locks that allow the control of antibody activity using toeholdmediated strand displacement reactions. employing yeast as a cellular model system, reversible control of antibody targeting is demonstrated with low nm concentrations of peptide-dna locks and oligonucleotide displacer strands. introduction of two different toehold strands on the peptide-dna lock allowed signal integration of two different inputs, yielding logic orand and-gates. the range of molecular inputs could be further extended to protein-based triggers by using proteinbinding aptamers. insights of a novel kind of cell wall binding domain that cleaves the peptidoglycan muropeptide: the cw_7 motif noem ı bustamante 1,3 , manuel iglesias, noella silva-mart ın, isabel uson, pedro garc ıa, juan hermoso, marta bruix, margarita men endez 1 institute of physical-chemistry 'rocasolano', csic, 2 institute of physical-chemistry 'rocasolano', csic, 3 ciber of respiratory diseases (ciberes), 4 center of biological research (cib), csic, 5 enzybiotics constitute a hopeful alternative to current treatments to fight against bacterial infections. phage endolysins are consider as enzybiotics due to their capacity to cleave the peptidoglycan (pg) of gram-positive bacteria in a generally species-specific manner and kill bacteria when exogenously added (1, 2) . the cpl-7 endolysin, a lysozyme encoded by the pneumococcal cp-7 bacteriophage, is a remarkable exception among all the pg hydrolases produced by streptococcus pneumoniae and its bacteriophages due to its capacity of degrading pneumococcal cell walls containing either choline or ethanolamine (3, 4) . this fact confers to cpl-7 the advantage of displaying a broader microbicide spectrum comparing to choline binding proteins (5) . this behavior results from the acquisition of a cell wall binding module (cwbm) made of three identical repeats of 48 amino acids each (cw_7 motifs), with unknown specificity and totally unrelated with the choline-binding motives present in pneumococcal hydrolases. interestingly, cw_7 repeats have been identified in many putative proteins potentially involved in cell wall metabolism (pfam entry: pf08230) from different species of gram positive and gram negative bacteria, and some bacteriophages (6) . preliminary studies of thermal stability in presence of a small cell wall structural-analogue (glcnac-murnac-l-ala-d-isogln) point to the muropeptide as the cell wall target recognized by cw_7 motifs (7) . in this communication we have gone in depth in the characterization of cw_7 repeats. we present the first crystal structure of the cw_7 motif, which reveals a three-helical bundle folding. using std_nmr spectroscopy the epitope of binding of the disacharide dipeptide to this repeats has been identified. interestingly, the b anomer of the murnac moiety, the form present in the peptidoglycan, seems to be preferentially recognized with respect to the a anomer. finally, a docking model of the complex cw_7/gmdp compatible with std results was built allowing to identify the major contacts between the protein and the muropeptide and to propose the relevant role of a conserved arginine residue in this interaction. 1 energy-dependent aaa1 proteases carry out regulated proteolysis to ensure protein quality control and post-translational regulation of many cellular processes. control of proteolysis occurs primarily at the level of substrate recognition, which can be modulated by adaptor proteins. the clps adaptor protein enhances and inhibits degradation of different classes of substrates, and thus triggers a specificity switch in clpa. whereas the mechanism for substrate delivery by clps has been described in detail, the inhibition mechanism is poorly understood. we show that clps inhibits ssra substrate recognition and processing, instead of simply preventing substrate binding. we demonstrate that clpa engagement of the clps n-terminal extension (nte) is necessary, and may even be sufficient, for inhibition. in addition, we find that inhibition of substrate processing requires a longer nte, as compared to inhibition of substrate recognition. interestingly, the nte length required for inhibiting substrate processing is also necessary for suppression of the clpa atpase rate. furthermore, preliminary data suggests that clps slows down substrate translocation. these results support a model where there is an ssra•clpa•clps inhibitory complex in which the clpa pore engages the clps nte. this engagement of the nte causes suppression of atpase activity, and therefore slower substrate translocation and processing. this model illustrates how an adaptor protein can inhibit recognition of one type of substrate while efficiently promoting degradation of a different substrate. single-molecule assay development for studying human rna polymerase ii promoter-proximal pausing rna polymerase ii (polii) pausing has been shown to play a significant role in transcription regulation of elongating polii complexes in a large number of metazoan and mammalian genes (1) . the traditional understanding of transcription regulation in mammals involved controlling polii recruitment to promoters and controlling initial steps at the promoter, including pre-initiation complex formation and promoter escape. most works investigating promoter-proximal polii pausing have employed chromatin immunoprecipitation followed by sequencing to determine polii localization or in vitro transcriptional assays using nuclear extracts analyzed with radio-active gel electrophoresis. in order to gain greater mechanistic insight into the regulation of promoter-proximal polii pausing, we have been developing a diffusion-based single-molecule method using alternating laser excitation on the micro-second timescale (msalex). the method detects rna transcripts generated by a reconstituted human polii system in vitro using complementary doubly dye-labeled single-stranded dna (ssdna) probes. the human gene hspa1b for heat shock protein 70 (hsp70) is used as a model system due to its extensive characterization in drosophila. the method would provide a rapid, sensitive and robust avenue to screen for protein factors regulating promoter-proximal polii pausing. controlling of the pic composition using the reconstituted system allows for dissection of the functional roles of different pic components in facilitating regulation of polii pausing. we have demonstrated the hybridization of double dye-labeled ssdna probe to complementary ssdna mimicking rna transcripts and to transcripts generated with bacterial rna polymerase. also, a functional reconstituted human polii system has been verified using radioactive polyacrylamide gel electrophoresis of transcripts from in vitro transcription assays. malaria is a major global health problem. in 2013, there were an estimated 128 million case of malaria and 584 000 deaths, most of them children under 5 years old [1] . among the 5 malaria species that affect humans, plasmodium falciparum is the most deadly form. since no efficient vaccine is available yet, the fight against malaria includes vector control, protection from mosquito bites and artemisinin combined therapy. however, resistances to all known treatments have been observed. therefore, new antimalarial strategies involving novel targets and new mechanisms of action are needed. during its life cycle, in erythrocytic stage, which causes all the malaria symptoms, plasmodium falciparum relies on phospholipids to build the membranes necessary for daughter cell development. approximately 85% of parasite phospholipids consist of phosphatidylcholine (pc) and phosphatidylethanolamine (pe) synthesized by the parasite through the de novo kennedy pathways. in the pathway of phosphatidylcholine biosynthesis, the second step catalyzed by ctp:phosphocholine cytidylyltransferase [ec 2.7.7.15] is rate limiting and appears essential for the parasite survival at its blood stage [2] [3] . we are focused on the structural characterization of this enzyme, the identification of effectors by fragment-based drug design approach (fbdd) and then their optimization to eventually design a lead. the first reported crystal structure of the catalytic domain of the enzyme target (pfcct) has been solved at resolution 2.2 å, 3 enzyme-substrates complexes (cmp-, phosphocholine-and choline-bound forms) at resolutions 1.9-2 å and an enzyme-product (cdp-choline) complex structure at resolution 2.4 å that give detailed images of binding pocket, demonstrate conformational changes between apo-and holo-protein forms and provide the information about the mechanism of the catalytic reaction at atomic level. the fbdd method uses a library of small molecules (fragments) with molecular weight that does not exceed 300 da to explore target binding sites. although fragments often have too low affinities to evoke a biological response, their probability of binding is high because they are small enough to prevent unfavorable interactions with target protein-binding sites. moreover, they represent more attractive and synthetically tractable starting points for medicinal chemistry compared to more complex compounds. as the affinity is low, fragment screening usually depends on detecting binding rather than inhibition. screenings of a fragment library (300 molecules) has been performed by fluorescence-based thermal shift assay and nuclear magnetic resonance saturation transfer difference (nmr std) [4] . this combination of techniques identified so far 4 fragment hits that are currently evaluated for their binding modes and affinities. co-crystallization of the protein-fragments complexes is carrying out to provide accurate information on the molecular interactions. topology of interactions will be used to rationally monitor every iterative round of the optimization process allowing subsequent rational design. [1] world health organization, world malaria report (who press, geneva, switzerland), http://www. who.int/malaria/publications/world_malaria_report_2014/wmr-2014-no-profiles.pdf?ua51 protein scaffolds play a crucial role in signaling pathways by generating signal specificity and increasing signal efficiency and amplitude. engineered protein scaffolds can be used as key regulators for signal transduction in artificial signal transduction cascades where they can regulate in-and output of the network. in this research a 14-3-3 protein scaffold is developed which induces dimerization of proteins mediated by the small molecule stabilizer fusicoccin. as proof of principle caspase 9 is used to constitute proximity induced dimerization. dimerization of caspase 9 leads to its activation and consecutively initiates the caspase cascade involved in the programmed cell death pathway. caspase 9 does not naturally bind to 14-3-3 proteins, therefore the caspase 9 monomer is conjugated to a 14-3-3 binding motif which is known to bind into the binding grooves of a 14-3-3 dimer. this interaction can be stabilized by the small molecule fusicoccin. we showed that upon addition of small molecule fusiccocin caspase dimerization is induced, resulting in caspase activity which is measured using a synthetic caspase substrate. moreover the biphasic effect of the 14-3-3 scaffold could be proven. additionally, the activated caspase 9 is also able to cleave its natural substrate caspase 3, downstream in the caspase cascade. these results indicate that the 14-3-3 platform is a versatile small molecule induced dimerization platform which can be used as tool for engineering of a synthetic signaling network. the g308e variant of the apoptosis inducing factor, responsible of a rare encephalopathy, is hampered in nad1/h binding luca sorrentino 1 , laura rigamonti 1 , mirvan krasniqi 1 , alessandra calogero 1 , vittorio pandini 1 , maria antonietta vanoni 1 , alessandro aliverti 1 1 the apoptosis inducing factor (aif) is a highly conserved mitochondrial flavoprotein known to play two opposite roles in eukaryotic cells: in mitochondria it is required for efficient oxidative phosphorylation (oxphos), while, when released into the cytoplasm, it triggers caspase-independent apoptosis (1) . the mechanism of aif-induced apoptosis was extensively investigated, whereas its mitochondrial role is poorly understood. there are many evidences of aif importance for mitochondrial correct morphology and functions and recently the discovery of its direct interaction with chchd4, a key regulator of respiratory complexes subunits import and folding in mitochondria, was reported (2) . a unique feature of aif, probably pivotal for its vital function, is the ability to form a tight, air-stable charge-transfer (ct) complex with nad1 and undergo dimerization. although some aspects of aif interaction with nad1/ h have been analyzed, its precise mechanism is not fully understood. we investigated the effect of the pathogenic g308e replacement, associated with oxphos defect and neurodegeneration (3) , to understand how it could alter aif properties at the molecular level. to do so, we analysed how the wild type and the g307e forms of murine aif interact with nad1/h and nicotinamide mononucleotide (nmn1/ h), finding that the pathogenic replacement resulted in a dramatic and specific decrease of the rate for ct complex formation and consequent protein dimerization only in the case of the physiological ligand. our results demonstrate that the adenylate moiety of nad1/h is crucial for the ligand binding process and that the g307e replacement causes an alteration of the adenylate-binding site of aif that drastically decreases the affinity for and the association rate of the ligand. in addition, we shed new light on the mechanism of the dimerization process, demonstrating that fad reduction rather than nad1/h binding initiates the conformational rearrangement of aif that leads to quaternary structure transitions. taken together, our results contribute to define how aif works at the molecular level in binding nad1/h and undergoing dimerization and also point out that the g308e replacement, responsible of a rare neurodegenerative disease, has the selective effect of slowing down the formation of aif dimeric ct complex. dipartimento di bioscienze, universit a degli studi di milano, 2 dipartimento di scienze veterinarie e sanit a pubblica, universit a degli studi di mical, from the molecule interacting with casl, indicates a family of conserved cytoplasmic multidomain proteins that catalyze a nadph-dependent f-actin depolymerization activity through their essential n-terminal fad-containing monooxygenase-like domain (mo) in response to semaphorin signaling [1] . this domain is followed by calponin homology (ch) and lim domains, proline-and glutamate-rich regions and a c-terminal coiled-coil motif that mediate the interaction with various proteins (e.g: crmp, casl, plexin, g proteins, ndr) [1] . to contribute to establish the catalytic properties of mical mo and their modulation by the additional domains and by the interacting proteins, we have produced and are characterizing the human mical1 (mical-fl) and forms containing the mo [2] , mo-ch and mo-ch-lim domains. all mical forms contain stoichiometric amounts of fad in the mo domain and 2 zn11 ions in the lim domain. mical-mo catalyzes a nadph oxidase (h2o2-producing) activity. the ch, lim and c-terminal domains lower its catalytic efficiency (kcat/km, nadph) mainly due to an increase of km for nadph. the kcat is similar for all forms excepted for mical-fl where a 7-fold drop is observed, in agreement with the proposed autoinhibitory function of the c-terminal domain [3] . the ph dependence of the kinetic parameters of mo, moch and mochlim is complex suggesting that it does not reflect the ionization state of individual groups, but rather the overall protein charge. mical-mo, -moch and -mochlim catalyze a nadph-dependent f-actin depolymerization with a similar apparent km for actin. f-actin (but not g-actin) stimulates the rate of nadph oxidation by increasing kcat and lowering knadph. the extent of nadph oxidation exceeds total f-actin which is in contrast with the proposal of specific modification of actin met44 or met46 reported for drosophila and mouse moch [4] [5] , but it suggests that f-actin stimulates the nadph oxidase activity or a case of substrate recycling. accordingly, with hmical mo and moch several actin residues are oxidized beside met44 and met46. thus, the ch and lim domains do not seem to be important for the mical-actin interaction and actin modification may be mediated by in situ h2o2 production. in hek293t and cos-7 cells mouse collapsin response mediator protein-1 (mcrmp1) interacts with mical1 inhibiting h2o2 production [3] , suggesting that crmp1 could be a hydroxylatable substrate of mical-mo. we have produced the same mcrmp1 form (8-525 aa) and we have shown that under conditions that limit non specific interactions a mild stimulation (up to 20%) of nadph oxidation is observed. f-actin reversed the effect of mcrmp1 suggesting their competition for mical. these results suggest that crmp1, a major microtubules regulator, is not the substrate of the mo domain, but actin and microtubules cytoskeleton components may be linked through the formation of crmp-mical complex in response to semaphorin-plexin signaling. experiments are in progress to complete the characterization of mochlim and full length mical forms. green fluorescent protein (gfp), owing to its genetically encoded strong fluorescence, has become one of the most important tools in modern biology [1] . enhanced gfp (egfp, f64l/s65t-gfp), frequently used variants of this protein, is thermodynamically more stable and 35-times brighter than gfp [2] . due to the improved fluorescent properties, egfp is commonly used as a fluorescent intracellular marker in bio-imaging in vitro and in vivo. despite sustained interest of the scientific community and numerous practical applications, the actual biological role of gfp remains elusive. recent reports put forward a hypothesis of antioxidant and photo-protective functions of gfp [3] . in this study, we focused on the photo-protective role of egfp against reactive oxygen species (ros) photo-generated by visible light in water suspensions of nano-particular nitrogen-doped titanium oxide (n-doped nano-tio2), that is in the system: 'n-doped nano-tio2)/visible light'. n-doped nano-tio2 (sumitomo tp-s201) was chosen as a photo-catalyst, since it is widely accepted that nitrogen doping enhances visible light photoactivity of tio2. 4-hydroxy-2,2,6,6-tetramethylpiperidine-n-oxyl (tempol), a paramagnetic water-soluble compound, belonging to the nitroxide class o superoxide dismutase (sod) mimetics, was used as a target for photo-generated ros. a solar simulator, with the flux output intensity of 1 kw/m2, was used as a visible light source. electron spin resonance (esr) was employed to monitor the changes in the paramagnetic signal of tempol exposed to the action of ros in the absence and presence of egfp. in the absence of egfp and after 50 min of illumination, due to a combined action of superoxide (o2•-) and hydroxyl (oh•) radicals generated by the system 'n-doped nano-tio2)/visible light, the esr signal of 100 um tempol decayed by 20%. moreover, the growth of a new signal, interpreted as 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (tempone), resulting from the attack of oh•radicals on tempol, was also observed. in contrast, in the presence of egfp (7.5 um) , the ros-induced decay of the esr signal of tempol was markedly smaller, not exceeding 5%. concomitantly, the growth of the esr signal of tempone was also partially inhibited (30% smaller amplitude), as compared to the process performed in the absence of egfp. in summary, our results point to a significant inhibition of the photodecomposition of tempol in the presence of egfp and support the hypothesis of the protective role of this fluorescent protein against ros generated by the system 'n-doped nano-tio2)/visible light'. school of chemistry, national university of ireland galway, 2 school of biochemistry and immunology, trinity college dublin by studying a variety of anionic ligands and their interactions with cationic cytochrome c, we are building knowledge of protein recognition geared towards regulating activity. in previous work it was shown that psulfonatocalix [4] arene selectively binds to, and encapsulates, three lysine side chains on cytochrome c 1. here, the binding of two small molecule ligands to cytochrome c was investigated. nmr spectroscopy was used and in one case, a crystal structure of the complex was obtained (fig 1) . the calixarene bound to cytochrome c, reveals a crystal packing assembly that suggests it is a key mediator of crystal formation. nmr data analysis indicates the calixarene's binding site on cytochrome c. the pillararene, a relatively new class of compound, is a symmetrical arrangement with a p-rich cavity 2, related structurally to calixarenes. this suggests good host-guest complexation properties. previously, the carboxylatopillararene showed selective binding to arginine, lysine and histidine 2. with this ligand, an interaction with cytochrome c was observed and a complex formed. additionally, biphasic binding behaviour was observed through analysis of the chemical shift perturbations. this may indicate more than one binding event taking place. the data from these studies indicate that recognition is occurring and again that lysine side chains play an essential role. the enzyme dihydrofolate reductase (dhfr) is necessary for the growth and development of all organisms. 1 the structure and function of escherichia coli dhfr have been characterized in buffer. however, dhfr exists in living cells, where the protein concentration can exceed 300 g/l. 2 we know that weak, non-specific chemical interactions with cytosolic proteins alter protein conformation and dynamics,3,4 both of which are expected to influence dhfr catalysis. investigators have examined steady-state enzyme kinetics under crowded conditions, but conclusions can be conflicting. 5, 6 here, the effects of crowding on e. coli dhfr catalysis are assessed through specific activity measurements in solutions of synthetic polymers. these kinetics studies are complemented by in-cell and in vitro 19f nmr data from fluorinated tryptophan residues. preliminary results suggest that the effects of polymeric crowders on dhfr activity are non-monotonic, which may arise from the polymer's transition from the dilute to semi-dilute regime. the data suggest that synthetic polymers are not a valid representation of the cellular interior. biotechnology department, university of verona calcium (ca21) is one of the most important second messengers in eukaryotes. ca21 binding proteins can be subdivided into two categories: "ca21 buffers" that modulate ca21 ion concentrations in cells, and "ca21 sensors" that decode ca21 signals in a wide array of physiological processes in response to external stimuli. calmodulin (cam) is the prototypical example of ca21 sensor proteins in both animals and plants. in addition to conserved cam, plants possess a unique family of 50 cam-like proteins (cmls). many of these cmls still remain uncharacterized and the investigation of their biochemical and biophysical properties will provide insight into ca21 signalling in plants. herein, a detailed characterization of arabidopsis thaliana cml14 is reported. cml14 is a protein of 148 amino acids with a theoretical molecular weight of 16,579 da and 50% amino acid sequence identity with atcam2. cml14 is predicted to have one functional ca21 binding site despite the presence of three ef-hand motifs (prosite). we overexpressed cml14 in e. coli and analyzed its biochemical and biophysical characteristics, i.e. calcium affinity and stoichiometry and eventual changes in conformation, thermal stability and proteolytic susceptibility upon ca21 binding. isothermal titration calorimetry (itc) and nuclear magnetic resonance (nmr) spectroscopy identified one ca21 binding site in cml14 and showed that ca21 and mg21 compete for the same binding site. the kd values determined by itc established that cml14 has higher affinity for ca21 than for mg21. our data were consistent with the sequence based prediction of one functional calcium binding site. differential scanning calorimetry (dsc) showed that ca21 and mg21 have the same stabilizing effects on protein folding. apo-cml14 undergoes two thermal unfolding transitions, but in the presence of ca21 or mg21 only one unfolding event at an intermediate temperature occurs. limited proteolysis experiments showed that ca21 binding affords protection against cml14 digestion by trypsin. surprisingly, cml14 exhibits very few conformational changes upon calcium binding, which were evaluated by ans fluorescence and stokes radius measurements in the apo-and ca21 bound-forms. these results suggest that cml14 does not show the characteristics of a classical ca21 sensor protein. to better understand the physiological role of cml14 in plants, in vivo analysis will be performed. pb-068 fbp17 controls the hepatocyte morphology through rho signaling jun zhang 1 , mingming ling 1 , qianying zhang 1 , yunhong wang 1 , deqiang wang 2 1 the department of cell biology and genetics, 2 the formin-binding protein 17 (fbp17) widely expressed in eukaryotic cells was previously identified to play a role in morphological maintenance in hepatocyte, but the molecular mechanism keeps still unclear so far. in the present investigation, it was found that rho family proteins cdc42/rac1 signaling was involved in the morphological regulation controlled by fbp17. knockdown of endogenous fbp17 expression with rnai technique or dominant negative mutant of fbp17 could trigger the cell morphological remodeling from the epithelioid to fibroid following the significant down-regulation of cdc42/ rac1 activities and dephosphorylation of paxillin. while the rho protein specific activator could restore the cdc42/rac1 activities, and in turn abrogated the silence effect. overexpression of wild type fbp17 could not result in any of the morphological transition. furthermore, withdrawal of the silence could induce morphological recovery when the fbp17 expression, cdc42/rac1 activities and paxillin phosphorylation were restored to the normal level. the experimental evidences strongly indicated that fbp17 was implicated in morphological control probably via rho signaling pathway in hepatocyte. key words: fbp17; rho signaling; paxillin; morphological control; hepatocyte this work was supported by a grant from national natural science foundation of china (nsfc, no. cytochrome c oxidase (cco) is the final enzyme in the respiratory chain of mitochondria but also an integral part of the metabolism of many types of bacteria. in a complex, stepwise redox-reaction, cco catalyzes the reduction of molecular oxygen to water and utilizes the resulting free energy to pump protons across the membrane thereby creating an electrochemical gradient [1, 2] . to investigate proton pumping spectroscopically it is possible to label the entrance of the proton entrance channel with fluorescein, a ph sensitive dye, which allows determining time resolved local changes in proton concentration at the cytoplasmic cco surface and related properties. it has already been shown that the redox state of copper and heme centers affects such properties at the cytoplasmic surface. [3] this study is a theoretical approach to investigate changes of pka values of the fluorescein label at the entrance of the k-channel for different protonation pattern in both oxidized and reduced cco by performing molecular dynamics (md) simulations. further work is based on calculations of pka values of the fluorescein using software karlsberg1 [4, 5] . methods for genetically and synthetically manipulating protein structure enable a greater flexibility in the study of protein function. we have shown that using inteins as traceless, cleavable purification tags enables the separation of full length unnatural amino acid (uaa) containing proteins from their corresponding truncation products. this method has been used to incorporate uaas in previously unattainable positions in a variety of proteins using a myriad of uaas, inteins, and purification tags. in other applications, we have used e. coli aminoacyl transferase (aat) to selectively modify the n-termini of proteins with uaas in denaturing conditions and conditions that maintain folding. applications of particular interest include overcoming the need for an n-terminal cys residue in expressed protein ligation, transfer of reactive handles for "click" chemistry labeling of proteins, and transfer of fluorogenic molecules for photophysical experiments. we have found that aat can transfer protected cysteine, homocysteine, and selenocysteine to expressed proteins. after ligation, these residues can be converted to met or ala, making the ligation traceless. we continue to develop variants of aat to broaden the substrate scope of both its transferred substrate and n-terminal recognition element. in addition, expressed protein ligation is being used to incorporate backbone modifications, such as the thioamide, into various positions in the protein calmodulin to determine how these modifications can impact the structure and function of an ordered protein. in general, by working at the interface of several protein modification technologies, we have made beneficial discoveries that might be missed by more focused approaches. function and modularity of cw_7 motives in the c-terminal region of the endolysin cpl-7 encoded by the cp7 pneumococcal bacteriophage manuel iglesias-bexiga 1,2 , noelia bernardo-garc ıa 3 , rub en mart ınez-buey 4 , noem ı bustamante 1,2 , guadalupe garc ıa 1,2 , marta bruix 1 , juan hermoso 3 , margarita men endez 1,2 1 dept. of biological physical-chemistry, iqfr-csic, 2 ciber of respiratory diseases (ciberes), 3 department of crystallography and structural biology, iqfr-csic, 4 bacteriophage lytic murein-hydrolases have been proposed as enzybiotics, an efficient way to fight bacterial infections. however, the use of these enzymes is normally restricted to gram-positive bacteria since the outer membrane of the gram-negative bacteria hampers the access of the hydrolases to the peptidoglycan substrates. all the murein hydrolases reported in the pneumococcal system, both from host or phage origin, depend on the aminoalcohol choline to be fully active. there is only a unique exception to this rule, the cpl-7 lysozyme. this hydrolase is encoded by the lytic pneumococcal phage cp-7 and, instead of the common cell wall binding module (cwbm) that recognizes choline, cpl-7 harbors a completely different cell wall binding structure. recent studies have revealed that reducing the net charge of the cwbm, from 214.9 to 13.0, leads to an improvement in the antibacterial activity of cpl-7 (1) . the cwbm of cpl-7 is composed by three identical repeats of 48 amino acids, the cw_7 motives, and it folds both in the presence and in the absence of the n-terminal catalytic module (2) . this module shows the capacity of recognize the glcnac-murnac-l-ala-d-isogln muropeptide (gmdp), structurally related with the peptidoglycan basic unit (3) . here, we report the high resolution structure of the cell wall binding module of the cpl-7 endolysin. each cw_7 repeat is composed of a bundle of three a-helices with a highly negative electrostatic charge at the surface. the strong inter-repeat interactions and the high ionic strength used in the crystallization conditions allow them overcoming the electrostatic repulsions inducing a closed-packed structure with a three-fold symmetry. the module dimensions (49 x 38 x 34 å) and the repeat arrangement in the crystal structure are inconsistent with the gmdp binding characterization, the activity displayed by cpl-7 truncated variants with one or two cw_7 repeats, or the experimental determined hydrodynamic properties. using the small angle x-ray scattering (saxs) technique and the atsas computational platform (4), a different arrangement of the cw_7 repeats is envisaged in solution (fig. 1) , whose rather opened structure (70 x 44 x 46 å) is consistent with the experimental data. additionally, employing the saxs-based structure and the honeycomb structure proposed for the peptidoglycan, a model, where each cw_7 repeat of the cell wall binding module fit in adjacent glycan chains, has been derived. in 2000, the protein structure initiative (psi) was started as to determine three-dimensional structures of proteins within every family. once solved, structures are deposited into the protein data bank (pdb) and termed structural genomics (sg) proteins. as of june 2015, there are over 13,300 sg proteins deposited in the pdb and most of them are of unknown or uncertain biochemical function. in addition, many of these sg proteins have a putative functional assignment based on their sequence and structural similarities with proteins of known function; such comparisons can be made against large databases using programs such as blast or dali. however, these putative functional assignments are often incorrect. this project analyzes members of the crotonase superfamily (cs). the cs consists of five diverse functional subgroups that are well characterized structurally and functionally, representing different types of reactivity, including hydrolase, isomerase, hydratase, and dehalogenase activities. this superfamily also contains at least 70 sg proteins, so it is ideal to test predictions of protein function. our approach is based on local structure matching at the computationally predicted active site. first, partial order optimum likelihood (pool) is used to predict the functionally important residues of each sg protein and of the proteins of known function in the superfamily. next, structurally aligned local sites of activity (salsa) is used to align the predicted catalytic residues of the well-characterized members in the superfamily. from this analysis we generate chemical signatures for each functional subgroup and compare them to the sets of catalytic residues predicted for the sg proteins. we demonstrate based on these computational methods that the majority of the putative annotations in the cs superfamily are likely incorrect. currently, biochemical assays are being used to test these predictions. preliminary biochemical results show that one sg protein, thermus thermophilus q5sls5_thet8, classified as a probable enoyl-coa hydratase, possesses hydrolase activity as predicted by our methods. the outcomes of this project will be to successfully classify the biochemical functions of sg proteins based on their local structure at the predicted active sites and to provide a conceptual framework for the functional classification of the remaining sg proteins within the pdb. this work is supported by nsf-che-1305655. directly observing the synergistic dynamics in f-actin and microtubule assembly jun zhang 1 , deqiang wang 2 1 the department of cell biology and genetics, 2 key laboratory of molecular biology on infectious disease although important in cellular activities, little attention was paid to the synergistic effects of actin and microtubule cytoskeleton assembly. with the time-lapse atomic force microscope (tl-afm), we directly observed the large-scale dynamic structure of actin filaments formed in the presence or absence of microtubulin in solution. in absence of microtubulin, the g-actin could be polymerized into ordered filamentous structures with different diameter from the slimmest filament of single f-actin to giant filament in tree-like branched aggregates. the polymerized actin filaments, to which our most intense attention was attracted, was discretely arranged and showed obvious polymorphism in structures completely distinct from those in the presence of microtubulin. the supra-molecular complex structures of the latter were mainly composed of single f-actin and/or multifilaments clearly consisting of several single f-actin and regularly cross-linked with the assembled microtubular bundles. the experimental results demonstrated that the f-actin dynamics could be coordinated by microtubule assembly. further analyses implied that the interactions between f-actin and microtubule could prevent the emergence of structural polymorphism of f-actin alone, and give rise to organization of specific complex structures instead. it was suggested that dynamic synergy between the f-actin and microtubule would be implicated in living cells. the adaptor protein 14-3-3 is found in a diverse range of pathologically relevant protein-protein interactions (ppis). as 14-3-3 is a hub protein with very diverse interactions, it is able to influence the intracellular localization of their binding partners and they are key regulators of signal transduction processes as well as regulators of cell cycle functions.nevertheless, there are only few examples of 14-3-3 acting extracellularly. one of the extracellular targets for 14-3-3 is aminopeptidase n (apn). apn is an extracellular trans-membrane enzyme that acts as a receptor for 14-3-3. binding to apn, 14-3-3 excreted by keratinocytes can upregulate the excretion of matrix metalloproteinase-1 (mmp1) in fibroblasts. mmp1, by breaking down collagens, is key in the remodeling of the extracellular matrix. modulation of the 14-3-3/apn interaction thereby may play a crucial role in the fundamental understanding and ultimately treatment of wound healing, respiratory diseases and tumor growth. in the eukaryotic cell, the 14-3-3 dimer operates as an adapter platform for binding partners. a wide range of classes of (small) molecules, natural products and peptides has been used to modulate the ppis, providing either stabilization or inhibition of the interactions of 14-3-3 with its binding partner. binding partner fragments or peptides are known to bind to the 14-3-3 binding groove via arecognition motif containing a phosphorylated serine or threonine. making use of the dimeric structure of 14-3-3, novel small-molecule inhibitors may be tethered to exploit the bivalent effect. from a large virtual screening and experimental validation, a scaffold containing a phenyl phosphonic moiety was identified, showing inhibitory properties for 14-3-3 ppis. potent derivatives of this scaffold were bridged by polyethylene glycol (peg) linkers of varying lengths, thereby facilitating the compound to reach both binding sites of the 14-3-3 dimer and concurrently increasing the compound's solubility in aqueous solution. similar bivalent inhibitors have been proven to synergistically increase their efficacy. biophysical evaluation by means of fluorescence polarization (fp) inhibition competition assays, revealed an increase of the half maximal inhibitory concentration (ic50) from approximately 81 lm for the monomeric phenyl phosphonate to approximately 1.8 lm for the bivalent inhibitor with a 60å linker. this demonstrates a 45-fold increase of inhibitory effect towards 14-3-3 and its binding partner peptide mimic. extensive thermodynamic, kinetic and structural analysis of the interaction is in progress.phosphonic moieties have been shown to pass the cell membrane poorly, due to their highly charged character. by being able to specifically inhibit the extracellular interaction between 14-3-3 and apn, these inhibitors are prevented from interfering with the extensive intracellular 14-3-3 interactome. hence, these bivalent phenyl phosphonate inhibitors provide a promising strategy towards extracellular application. the mre11 complex is an oligomeric assembly comprising of dimmers of mre11 and rad50 proteins in archea and additionally nbs1 subunit present in eukaryote. it is the central player in the dna damage response -a functional network comprising dna damage sensing, signal transduction, cell cycle regulation and dna double strand breaks (dsbs) repair [1] . recent structural studies revealed that rad50 hinge domain is rather a short kink in the coiled-coil region and adopts unusual dimerization mode by intermolecular coordination of zn(ii) and formation of so-called zinc hook domain [2] . to date, very limited structural data on the zinc hook domain have been reported, the only known structure was resolved for rad50 homologue from hyperthermophilic archaeon -p. furiosus. unusual zn(ii) coordination mode in zinc hook domain raises question of how zinc hook domain assembles to form interprotein zinc binding site with sufficient stability to function at low intracellular free zn(ii) concentrations [3] . our study on minimal zinc hook domain fragment (14 aa) indicated low femtomolar affinity towards zn(ii) [4] . extended zinc hook domain fragment (45 aa) reveals even zeptomolar affinity. therefore, our main goal was to probe the thermodynamic and structural effects that are hidden in the small interprotein interface and are responsible for the dimerization of the large and critical protein machinery. probing of those effects was achieved by detailed biophysical characterizations (including potentiometry, nmr, hdx ms and cd spectroscopy) of 18 protein fragments of zinc hook domains with a number of point mutations. we showed that extremely high stability of zinc hook domain from p. furiosus is achieved by the formation of hydrogen bond network in b-hairpins and interprotein hydrophobic core. eindhoven university of technology dna-based molecular circuits have become a very attractive tool in molecular imaging, synthetic biology, molecular diagnostics and biomolecular computing. the highly modular and predictable nature of watson-crick base pairing allows the construction of complex circuits using a limited set of logic gates and building blocks. however, the lack of generic approaches to interface dna-based molecular circuits with protein activity limits their application in biomedicine and molecular diagnostics. here we present a new, highly modular approach to control the activity of a reporter enzyme based on the dna-directed assembly and disassembly of a complex between tem1-b-lactamase and its inhibitor protein blip. both proteins are conjugated to a unique oligonucleotide, allowing the assembly of the enzyme-inhibitor pair and inhibition of enzyme activity by the addition of a complementary template strand. addition of an oligonucleotide that is complementary to a loop sequence in the template results in the formation of a rigid dsdna spacer that disrupts the enzyme-inhibitor complex, restoring enzyme activity. using this noncovalent approach allowed easy tuning of the template and target sequences with only a single set of oligonucleotide-functionalized enzyme and inhibitor. to show the modularity of the system, a panel of 8 different template sequences were selected. only in the presence of their complementary viral dna sequences restoration of enzyme activity was observed. in addition to this excellent specificity the system showed to by higly sensitive towards its target, since the presence of as little as 2 fmol of target resulted in an observable increase in enzyme activity. the use of a stable and well-characterized enzyme-inhibitor pair, complemented by the modular design of our reversible dna-directed protein switch make it an attractive system to implement in dna-based molecular circuits. several studies demonstrated important roles of human carbonic anhydrases (hcas) in a variety of physiological and pathological processes. consequently, in recent years the 12 catalytically active hca isoforms have become an interesting target for the design of inhibitors with biomedical applications [1] . derivatized sulfonamides of type r-so2nh2 represent the class of ca inhibitors (cais) mostly used and best characterized. the large number of crystallographic studies so far available on these molecules clarified the main factors responsible for the binding of the sulfonamide moiety to the ca active site. 1 in particular, it has been highlighted that even though these molecules generally behave as very potent cais, they do not show selectivity for the different isoforms. indeed, the sulfonamide moiety plays a predominant role in the interaction with the enzyme, while any change in the nature of the r substituent has generally a rather marginal effect on the enzyme-inhibitor affinity. these characteristics make difficult the design of sulfonamide derivatives selective for the different ca isoforms. consequently, much efforts were dedicated in last years to the development of new inhibitors that, although presenting lower affinity for the ca active site, would be able to be more selective toward the different isoforms. carboxylic acids have been recently investigated as cais, showing that these molecules can adopt different binding modes to the enzyme active site. in particular, they can coordinate directly to the zinc ion or be anchored to the zinc-bound water molecule. however, the structural reasons responsible of this peculiar behavior have not been clarified yet. in a general research project aimed at providing insights into the binding mode of these molecules to cas, we have undertaken the characterization of two carboxylic acids, namely an ortho-substituted benzoic acid [2] and a saccharine derivative, by means of kinetic, crystallographic and theoretical studies. exploring the mechanism of fibril formation using fluorescently labelled human lysozyme variants ana bernardo gancedo 1 1 'exploring the mechanism of fibril formation using fluorescently labelled human lysozyme variants' human lysozyme is a widely characterised protein whose mutational variants misfold into fibrils that are associated with systemic amyloidosis (1) . although the process of aggregation for human lysozyme has been well studied, the details of early events within this process are not fully characterised. single molecule fluorescence microscopy has been used to determine the oligomeric distributions present in the aggregation process of a number of disease-related intrinsic disordered proteins (idps) (2) . recent advances in site-specific labelling of human lysozyme (3) have made this protein amenable to these single molecule fluorescence studies. we have introduced alexa-fluorophores into the i59t variant of human lysozyme and have demonstrated that the process of in vitro fibril formation is not significantly altered. using these fluorophore-labelled proteins we can apply single molecule fluorescence to study the early aggregation events within this system, allowing us to compare protein aggregation in a globular protein and with the aggregation process of idp's. abstract protein structure, folding and function, while specific interactions with lipid molecules can also contribute towards the biological activity of some membrane proteins. improving understanding of the interactions has resulted in the development of artificial lipid systems that allow the bilayer properties to be rationally manipulated in vitro to control protein behaviour. the bacterial transporter lacy is a well known integral membrane protein from the major facilitor superfamily, responsible for the protondriven uptake of d-lactose in e. coli. with a high resolution structure available and considerable understanding of mechanistic detail, and with observed changes to both structure and function in different bilayer environments, lacy is a good model system for examining the behaviour of a major class of membrane proteins in these lipid systems. purified lacy has been reconstituted into liposomes and droplet interface bilayer systems of varying lipid composition and the effect on protein function and bilayer properties examined. targeting abeta oligomers by trehalose-conjugated peptides: a molecular dynamics study alzheimer's disease (ad) is currently one of the most common and devastating forms of dementia correlated with beta-amyloid peptide (abeta) accumulation in human brain tissue [1, 2] . inhibiting abeta selfoligomerization in brain tissue remains one of the main strategies to prevent or treat this disorder. as a consequence, in recent years much efforts have been spent in the understanding of the amyloid fibril growth process and its modulation by putative drug molecules. an interesting class of compounds able to prevent abeta fibrillogenesis, is represented by beta-sheet-breaker (bsb) peptides [3] . although these molecules are thought to recognize in a self-complementary manner the abeta hydrophobic core region, however their precise mechanism of interaction is still unclear. in this context, we have studied the structural basis underlying the inhibitory effect of abeta(1-42) fibrillogenesis explicated by two promising trehaloseconjugated bsb peptides (ac-lpffd-th (thct) and th-succinyl-lpffd-nh2 (thnt)) [4] using an all-atom molecular dynamics (md) approach [5, 6] . the pentameric nmr structure [7] of abeta has been used to model amyloid protofibril, and the two protofibril ends have been investigated as putative binding sites. our simulations suggest that the interaction with the two protofibril ends occurs through different binding modes. in particular, binding on the odd edge (chain a) is guided by a well defined hydrophobic cleft, which is common to both ligands (thct and thnt). moreover, targeting chain a entails a significant structure destabilization leading to a partial loss of b structure and is an energetically favoured process, as assessed by mm/pbsa calculations. a significant contribution of the trehalose moiety to complexes stabilities emerged from our results. the basic structural unit of chromatin is the nucleosome, which is composed of histone proteins forming a scaffold with about 150 base pairs of dna wrapped around. chromatin compacts eukaryotic genomes and regulates gene activity, which is mediated in part by posttranslational modifications (ptms) on the n-terminal tails of the histones. uncovering the detailed relationship between histone tail modifications and gene activity is a major topic of biomedical sciences and general techniques for generating nucleosomes with defined modification patterns in large numbers would greatly facilitate such investigations. to this end we are establishing a chemical toolbox for designer chromatin with defined histone ptm patterns. a protein semysinthesis approach is used that bases on "ligation-ready nucleosomes" with truncated histone h3 that can be ligated with the corresponding synthetic histone tail. we resorted to sortase-mediated ligation as chemoselective ligation method. here we report our recent developments in establishing the envisioned chemical toolbox for designer chromatin. evaluating cation-pi and pi-pi interaction in proteins using various biophysical methods in proteins the aromatic residues phenylalanine (phe), tyrosine (tyr), and tryptophan (trp) can be involved in aromatic interactions known as cation-pi and pi-pi interactions (dougherty 2000). compared to other non covalent interactions in proteins, like h-bonds, dipole-dipole, or van der waals interactions, relatively little is known about the pi-pi and the cation-pi interactions. the strength of both aromatic interactions is dependent on the pi-electron density in the aromatic residues. a lowering of electron density can be created by introducing strong electron-withdrawing substituents like fluorine atoms in the aromatic ring (dougherty 2000). in this way a nearly isosteric change in the aromatic system results in a marked change in electron density. substitution with methyl groups is known to slightly increase the electron density. the response to low cellular oxygen levels in humans and other animals is induced by the hypoxia inducible transcription factors (hifs). these transcription factors are regulated by hypoxia inducible factor prolyl hydroxylases (phds), which act as 'oxygen sensors' by hydroxylating hifs, thus leading to the proteomic degradation of the transcription factors. over the last years, there have been multiple reports that describe additional phd substrates other than hifs. among them are the large subunit of rna pol ii, several transcription factors, and components of signalling pathways. validating these reports is of major medicinal relevance given that phd inhibitors are now in the late stage phase 3 clinical trials. in order to investigate the selectivity of phds, the reported proteins have been tested as substrates for hydroxylation by mass spectrometry, and as binders or competitors of the phds. initial work on peptides that contain the putative hydroxylation sites has indicated that the phds are much more selective for their well-established substrate hif. however, in ongoing work these initial results are going to be validated on protein level by co-expressing phds with the reported substrates. additionally, peptides of reported substrates were screened for their ability to alter the kinetics of hif-hydroxylation by phd2. an inhibitory effect of at least two different peptides on phd2 was observed, suggesting that there is an interaction between the prolyl hydroxylase and these peptides. in order to investigate the mode of binding and inhibition, nmr studies have been carried out and binding of the two inhibitory peptides on phd2 has been shown. altogether, these results indicate that, although phds might be more selective for hif as a substrate as it was initially thought, the enzymatic activity of the prolyl hydroxylases is possibly influenced by a number of other proteins that can directly bind to phds. non-natural aminoacids via the mio-enzyme toolkit alina filip 1 , judith h bartha-v ari 1 , gergely b an oczy 2 , l aszl o poppe 2 , csaba paizs 1 , florin-dan irimie 1 1 biocatalysis and biotransformation research group, department of chemistry, ubb, 2 department of organic chemistry and technology an attractive enzymatic route to enantiomerically pure to the highly valuable a-or b-aromatic amino acids involves the use of aromatic ammonia lyases (als) and aminomutases (ams). all these enzymes have in common an auto-catalically formed 5-methylene-3,5-dihydroimidazole-4-one (mio) electrophilic prosthetic group, and show high structural and sequence similarities. the recent advances in improving the functional properties of these enzymes increased both their biocatalytic and therapeutic applications. we aimed to create a library of recombinant mio-enzymes consisting of the pals and pams with large substrate promiscuity in order to provide access to various non-natural aminoacids through enzymatic ammonia addition and/or ammonia elimination reactions of the substrate library already available in our researchgroup. the developed complementary substrate and enzyme library would provide the mio-enzyme toolkit useful for the synthesis of nonnatural aminoacids. the synthetic gene of the enzymes (pcpal, rtpal, avpal, papam) were cloned into pet19b_j906 expression vector using xhoi and bpu1102i cloning sites. the plasmid dna was transformed to several e.coli host strains (rosetta, bl21, origami 2) in order to optimize the expression yields. the enzymes containing an n-terminal his10-tag were purified with affinity chromatography, followed by ion-exchange or/and size-exclusion chromatography, obtaining pure and homogenous proteins, in their tetrameric, presumably native fold. the enzyme activity and the kinetic parameters of the purified enzymes was determined towards the natural substrate l-phenylalanine, as well as towards novel bulkier aromatic substrates (heteroaryl alanines, styryl alanines, biphenylalanines). furthermore to enhance their biocatalytic applicability we covalently immobilized the enzymes to carboxylated single-walled carbon nanotubes (swcnt cooh) using linkers with different lengths, and tested the activity and recycling of the immobilized enzyme. antibodies that bind protein antigens are indispensable tools in biochemical research and modern medicine. utilizing a phage display selection strategy, we have obtained synthetic antigen binders (sabs), based on a fab fragment of igg, to a wide array of proteins as distinct as membrane proteins, structural proteins, scaffold proteins and nuclear targets. here we demonstrate the applicability of the sabs towards the native, full-length proteins in cells. we show that the generated sabs are able to pull-down endogenous proteins from mammalian cell extracts along with their natural binding partners. we developed a method of utilizing our high affinity and specificity binders as fluorescently labeled tools to visualize target proteins in their native environment in the cells without the need of secondary antibodies or blocking reagents. our system also includes a method of efficient delivery of generated antibodies to living cells, where they can perform their function. the sabs have been successfully used for altering biological processes in a controllable manner. in vitro evolution from pluripotent peptide libraries with natural neurotoxin scaffolds to target receptors, proteases and trophic factors tai kubo 1 , mohammed naimuddin 1 , seigo ono 1 1 national institute of advanced industrial science and technology (aist) in vitro evolution from pluripotent peptide libraries with natural neurotoxin scaffolds to target receptors, proteases and trophic factors small molecule natural products are precious resources for drug discovery. during millions of years of evolution, natural products must have been exposed to various selection pressures and have been refined in structure and function to obtain the present features. in some peptide neurotoxins, however, the basic molecular scaffold mainly configured by disulfide (s-s) bridges and/or alpha/beta structures, is strictly conserved within each family even under the evolution pressure. on the other hand the loop regions, which are not heavily involved in scaffold formation, are highly diverged. this mode of molecular evolution named 'accelerated evolution', is reasonable to quickly adapt to the vigorous change of the environment. the evolutionally selected scaffold is compact harboring both rigidity and flexibility in nature, and it may support a topology appropriate for target recognition and selective interaction. inspired by the system, we designed random peptide libraries from the peptide neurotoxins of the accelerated evolution. a three-finger (3f) shaped snake neurotoxin consists of huge family evolved by accelerated gene evolution. we prepared a 3f-peptide library by introducing random sequences in each fingertip. another random peptide library with an ick (inhibitor cystine knot) motif was prepared based on a neurotoxin gtx1-15 from spider; originally identified as a t-type ca21 channel modulator. each library was subjected to in-vitro evolution directed to specific target molecules. for the 3f-peptide library cdna display method was applied to select binders. when interleukin-6 (il-6) receptors were targeted, the selected 3f peptides showed binding affinities (kd 100 nm) comparable to the native ligand il-6. when trypsin was targeted, peptides with serine protease inhibitor activities similar to sti and bpti (ki 30 nm) were isolated. specific binders to a trophic factor vegf were also generated from the 3f library. to target membrane proteins, we developed a unique in-vitro evolution system, and named it as the periss (intra periplasm secretion and selection) method. in the system, target membrane proteins are expressed in inner membrane of e. coli and peptides are secreted to the periplasmic space, in between the inner and outer membranes; and the space is served for interaction and selection. the periss method enabled us to identify a peptide specific to muscarinic receptor m2 subtype from the ick peptide library. in conclusion, it was proved that the library designed from the scaffold of peptide toxin, which evolved in the mode of accelerated gene evolution, has pluripotency in target recognition, interaction and even bioactivity. phenylalanine ammonia lyase from petroselinum cripsum (pcpal) belongs to the class of enzymes containing 4-methylideneimidazole-5-one (mio) as a prostetic group and it is responsible for the conversion of l-phenylalanine into trans-cinnamic acid. this reaction is reversibile under high ammonia concentration. 1 we analyzed several factors that can influence the enantioselective synthesis of nitrophenylalanine mediated by whole cells as well as purified mio-containing and mio-less pcpals. first we investigated the behaviour of the enzymes depending on the ammonia concentration. we also inspected the influence of the ph on the pcpal catalyzed biotransformations. based on our results, we concluded that variation of ammonia concentration and the ph leads to decrease of enantioselectivity, suggesting that pcpal is able to catalyze the formation of both l-and d-enantiomers of electron-deficient structures. all microbial cellulase appears to have a conserved 'sg' amino acid sequence at an identical position in the n-terminal domain. the properties of the n-terminal 15 amino acid sequence were also predicted computationally. this analysis showed that n-terminal sequence of the enzyme is unstable. the nterminal sequence also showed potential cleavage sites by different proteases which may contribute to its instability. the secondary structure analysis showed that the n-terminal sequence has 40% of the 15 a.a. sequence in extended strand and 60% in random coil conformation. the n-terminal sequence was also analyzed for potential phosphorylation sites. while no potential serine and threonine sites were predicted, two tyrosine phosphorylation sites were predicted in the n-terminal sequence. the n-terminal sequence was also examined for the presence of kinase specific phosphorylation sites. the results showed the presence of one potential site which may be phosphorylated by pkc at position 1 of the n-terminal sequence. the analysis for the prediction of the presence of oglcnac sites revealed that two such sites may potentially be present in the sequence. we have also predicted the ligand binding site in the n-terminal sequence of the protein. protein arginine methylation catalyzed by protein arginine methyltransferases (prmts), is a pivotal protein post-translational modification involved in a growing number of physiological and pathological processes including signal transduction, proliferation, differentiation and malignancy. prmt1 accounts for the majority of protein arginine methyltransferase activity in mammalian cells and, in consistence, a large amount of cellular substrates have been identified. several studies have reported that the activity of prmt1 changes upon stimulation in various cellular processes. in mammalian cells, prmt1 exists in a high molecular weight complex. the interacting partners of prmt1, such as antiproliferative proteins btg1 and btg2, protein phosphatase 2a, the orphan receptor tr3, and ccr4-associated factor 1(hcaf1) are shown to play a role in modulating the methyltransferase activity and the substrate selectivity of prmt1. due to the pivotal roles of prmt1 in physiological and pathological conditions, intensive efforts have been put on the search of small synthetic chemical molecules which can efficiently modulate the activity of prmt1 for the potential development of therapeutics. in light of this, the intracellular small molecules that either transmit extracellular stimulation or act as cofactor to dictate the activity of prmts in cells are still poorly understood. our study focused on examining how cellular ions might affect the activity of prmt1 and found that divalent and monovalent ions differentially modulated the catalytic activity of prmt1 toward different substrates. oligomerisation properties of light-dependent protochlorophyllide oxidoreductase prothoracicotropic hormone (ptth) is one of the most important neuropeptide regulators for insect molting and metamorphosis. however, preparation of its recombinant protein has hardly been successful, because it is a homodimer protein with very complicated disulfide-bond structure. for example, silkworm ptth has three intramolecular disulfide bonds in its 109-residue polypeptide chain, and the two chains are further linked by an additional intermolecular disulfide bond to form the homomeric dimer. although the recombinant silkworm ptth was previously expressed in escherichia coli, the product was obtained only in precipitation fractions, and refolding of the precipitated protein provided the active dimer ptth in very poor yield. under such reductive conditions as in cytosol of the e. coli cells, formation of the correct disulfide-bond arrangement must be difficult. alternatively, for the heterologous expression of the silkworm ptth, we employed brevibacillus choshinensis (formally referred to as bacillus brevis), which has achieved good results in expression of various disulfide-bond-containing proteins. in this study, the silkworm ptth was expressed in the brevibacillus cells with an additional his6-tag sequence at the c-terminus, for easier detection and purification. first of all, since the brevibacillus bacteria are equipped with a secretory system of the expressed proteins, a secretory signal sequence to be attached before the silkworm ptth was carefully selected. among four candidates in a commerciallyavailable kit, a signal sequence derived from an intrinsic cell-wall protein mwp gave better results in expression levels of the protein. second, incubation time of the cells was optimized, because an oligomerization state of the secreted ptth in the cell culture medium changed with the time. in the medium, various ptth oligomers including a monomer and a dimer were initially observed, but higher oligomers became a major portion of the secreted product after longer incubation than 48 h. incubation for 24-36 h may be suitable for obtaining the native dimer form of the silkworm ptth. to remove the undesired monomer and higher oligomers, which mostly retained free sulfhydryl groups, the secreted proteins were treated with maleimide-peg2-biotin. in the purification using a ni1-nta column, the dimer of the his6-tagged silkworm ptth was eluted with an imidazole gradient, separately ahead of other biotinylated proteins, probably due to interaction of the peg2 spacer with the ni1-nta groups of the resin. after the reversed-phase hplc purification, the final product showed a single band on the nonreductive sds-page, and it had adequate ecdysone-releasing activity from isolated silkworm prothoracic glands. the brevibacillus bacteria are most promising host cells for the heterologous production of the insect ptth. role of the disulfide bridges in the transmembrane region of the insect prothoracicotropichormone receptor, torso torso is an insect cellular-membrane protein, which was recently identified as a receptor for prothoracicotropic hormone (ptth). although ptth is one of the important regulatory molecules in insect molting and metamorphosis, activation mechanism of torso by the ligand has not been elucidated yet. in this study, an oligomerization manner of the silkworm torso was examined, using heterologous expression in drosophila s2 cultured cells, because torso is a single-polypeptide receptor tyrosine kinase (rtk), and activation of such rtks is often triggered by the ligand-induced receptor dimerization on the cellular membrane. when activated with silkworm ptth, dimerization of the silkworm torso in the s2 cells was observed, using a cross-linking reagent bs3, and the subsequent receptor autophosphorylation and downstream erk phosphorylation were also detected. surprisingly, however, the torso dimerization was revealed to occur even without the ligand stimulation, while the autophosphorylation and the erk phosphorylation were held in response to the stimulation. when fractionated by non-reductive sds-page, the silkworm torso showed an obvious dimer band, in addition to a faint monomer band, both with and without the ptth simulation, even though the receptor was not treated with the cross-linking reagent. this indicates that the torso protein is expressed originally as a disulfide-bond-linked dimer. in addition, by examining oligomerization states of several truncation and substitution mutants, cysteine residues in the transmembrane region were found to participate in the intermolecular disulfide bridges, linking the two receptor molecules in the dimer. when all of the three cysteines in the transmembrane region were replaced by phenylalanines, the disulfide-bond-linked torso dimerization was not observed, but spontaneous, ligand-independent association of the torso molecules was detected using the crosslinker bs3. this spontaneous dimerization caused the apparent torso autophosphorylation, but it could not induce the downstream erk phosphorylation. consequently, without the intermolecular disulfide bridges, torso loses its responsiveness to the ptth stimulation. in conclusion, the disulfide bridges in the transmembrane region may play a role to preserve suitable relative position between the two torso molecules, which could induce ligand-dependent autophosphorylation leading to activation of the downstream signaling pathways in the cells. the yeast enzyme neutral trehalase (nth1, ec 3.2.1.28) from saccharomyces cerevisiae hydrolyses the non-reducing disaccharide trehalose which serves as an energy source and a universal stress protectant in many different organisms. enzymatic activity of nth1 is enhanced by the yeast 14-3-3 protein (bmh1 and bmh2) binding in a phosphorylation-dependent manner. nth1 activity is also regulated by ca21 binding to the ef-hand-like motif containing domain of nth1 [1] .the native tbe page and analytical ultracentrifugation show that nth1 forms very stable complexes with bmh1 and bmh2 [1] . to study the structure of nth1 alone and its complex with the 14-3-3 protein we used circular dichroism, h/d exchange coupled to mass spectrometry, chemical cross-linking [2] and small angle x-ray scattering (saxs) [3] . at the same time protein crystallography of nth1 alone and its complex with bmh1 is performed.the low resolution structure of pnth1:bmh1 protein complex revealed that binding of bmh1 induces a rearrangement of the whole nth1 molecule and that the region containing the ef-hand motif forms a separate domain which interacts with both bmh1 and catalytic domain of nth1. we proved that integrity of the ef-hand motif is crucial for the bmh1 mediated activation of nth1 and ca21 binding. our data suggest that the ef hand-like motif functions as the intermediary through which bmh1 modulates the function of the catalytic domain of nth1. these structural changes probably enable the substrate entry into the enzyme active site [3] . our study of 14-3-3 protein complex with the fully active enzyme nth1 offers a unique structural view of nth1 activation enabling us to better understand the role of the 14-3-3 proteins in regulation of other enzymes. the assembly of self-regulating synthetic biochemical pathways in vitro has great potential as alternative catalysts for the high-yield production of low value/high volume commodity chemicals from biomass. high yields of low-value/high volume compounds that are required for economic viability is particularly difficult via traditional in vivo metabolic engineering of microbes due to competing biochemical pathways and toxicity. we have developed an alternative approach, called synthetic biochemistry, where the glycolysis pathway of central metabolism is reconstituted in vitro with an anabolic pathway that can produce useful compounds at high yield. in the specific synthetic biochemistry system described, reducing equivalents, atp, and carbon from glycolysis are funneled through the anabolic mevalonate pathway to produce the monoterpene limonene from glucose. the successful implementation of the in vitro pathway required development of a molecular purge-valve consisting of an nad1 and nadp1 specific reductase (ie wild-type and mutant pyruvate dehydrogenase), and nadh oxidase, noxe, to maintain proper nadp1/nadph cofactor balance while allowing continuous carbon flux. we find that the purge-valve concept is readily transportable to other nad(p)h generating steps in central metabolism and can be used to convert glucose to limonene at high yield. chitinases (ec 3.2.1.14) are enzymes that randomly hydrolyze b-1,4 glycosidic bonds of chitin and produce n-acetylchitooligosaccharide ((glcnac)n) that has various physiological functions such as immunostimulatory activity. most of fish takes crustacean such as shrimp and crab as food. therefore, the fish has chitinase in the stomach to chemically disrupt the chitinous envelope of crustacean. four chitinase isozymes (42-60 kda), pachia [1] and pachib [2] , and ptchia and ptchib, [3] were purified from the stomach of silver croaker pennahia argentatus and threeline grunt parapristipoma trilineatum, by ammonium sulfate fractionation and column chromatographies, respectively. all the chitinases were stable and showed activity in the acidic ph range (ph3-5). pachia and ptchia preferentially degraded the second glycosidic bond from the non-reducing end of (glcnac)n and pachib and ptchib had a preference for the third glycosidic bond of those. all the chitinases showed different substrate specificity toward insoluble long substrates. moreover, chitinase cdnas (pachi-1 and pachi-2) encoding pachia and pachib, and cdnas (ptchi-1 and ptchi-2) encoding ptchia and ptchib were obtained by cdna cloning using the rt-pcr and race method. the deduced amino acid sequences of all the chitinase cdnas contained n-terminal signal peptide, gh family 18 catalytic domain, linker region, and chitin-binding domain. phylogenetic tree analysis of vertebrate chitinase revealed that fish stomach chitinases form unique chitinase isozyme groups, acidic fish chitinase-1 (afcase-1) including pachia and ptchia, and acidic fish chitinase-2 (afcase-2) including pachib and ptchib, which was different from an acidic mammalian chitinase (amcase) group. [3, 4] the previously reported purified fish stomach chitinases [5] can also be classified into two chitinase isozyme groups, afcase-1 and afcase-2, by the n-terminal amino acid sequence. this study suggested that fish have excellent chitin degrading enzymatic system in which two different chitinases isozyme groups, afcase-1 and afcase-2, with different degradation patterns are expressed in the stomach. recently, the enzymes produced by psychrophilic organisms have gained huge interest especially in the studies of temperature adaptation of the protein. previously, a cold-adapted yeast, glaciozyma antarctica pi12 was isolated from a marine environment in antarctica and the yeast was known to produce lipolytic and proteolytic enzymes. a gene encoding a unique recombinant bifunctional enzyme (lippi12) with cold active lipase with protease activity was successfully expressed, purified and characterized. temperature profile of the bifunctional lippi12 enzyme showed that the lipase functions optimally at 208c whereas the protease was more active at 408c. ph profile showed that both lippi12 lipase and protease were active at near neutral condition. activity of lippi12 lipase and protease were also activated in the presence of cacl2 but its protease counterpart seemed to be more active in the presence of zncl2. effect of surfactants showed lippi12 lipase was activated by tween 80 and sls and in contrast, lippi12 protease was almost deactivated in all surfactants tested. the presence of organic solvents did not affect both the lipase and protease activities. the lipase was more stable at solvents with higher log p value whereas the protease was slightly activated at low log p value particularly with dimethylsulfonyl. inhibitor studies revealed that lippi12 lipase was partially inhibited with edta and pmsf whereby the lippi12 protease was inhibited by pepstatin, edta and pmsf. lippi12 enzyme was successfully crystallized via vapour diffusion method. crystal of lippi12 enzyme was diffracted via synchrotron radiation. the three-dimensional structure of cold-adapted pi12 provided insight into cold adaptation and better understanding of the structural properties of lippi12 enzyme. the bifunctional properties of the enzyme could be potential candidate for low temperature industrial application. conformation-specific antibodies as enhancers and inhibitors of phosphatase activity of dep 1 malgorzata nocula-lugowska 1 , mateusz lugowski 1 , anthony a. kossiakoff 1 1 the university of chicago dep-1 (cd148/ptp-h) is a transmembrane receptor-like protein tyrosine phosphatase (ptp) that has been implicated in the density-dependent regulation of cell growth, differentiation and transformation. it counteracts protein kinases by dephosphorylating a number of their substrates as well as the kinases themselves, thus potentially controlling the specificity of signals. for example egfr, vegfr 2, met, pdgf b receptor have been shown to be dephosphorylated by this phosphatase. dep-1 has been shown to act as a tumor suppressor and it has been proposed as a molecular target in antiangiogenesis therapy. as a result, both enhancers and inhibitors of dep-1 activity have the potential of elucidating pathways responsible for abnormal cell behavior. we generated synthetic antibodies against intracellular catalytic domain of dep-1 that act as modulators of the enzyme's phosphatase activity. by applying a combination of selection pressures an array of antibodies has been raised from phage display libraries of fab fragments which are capable of either enhancing or inhibiting dep-1 activity. in phosphatase assays with catalytic domain of dep-1 the antibodies demonstrate non-competitive or mixed kinetics. the crystal structure of dep-1-inhibitor complex shows that this antibody binds to the part of the protein that is distant from the active site and acts by locking the enzyme in the nonnatural catalytically inactive state by hindering the closure of the wpd loop which is crucial for the reaction to occur. by contrast, as judged from the crystal structure of a complex of dep-1 with the antibody that enhances its phosphatase activity, this antibody seems to act by stabilizing the naturally found active state of dep-1 with wpd loop in the closed conformation. the antibodies are also able to recognize dep-1 in cells, as they stain dep-1 in immunofluorescence experiments. to test the applicability of raised antibodies in cells the activator was additionally used to pull down full-length endogenous dep-1 after being delivered to live cells. inhibition and enhancement of dep-1 activity by locking the enzyme in conformations which are either natural or imposed by allosteric binding of antibodies seems to be a mechanism that can be utilized to modulate activity of other tyrosine phosphatases. investigating acinetobacter baumannii pathogenesis: crystal structure of wbjb epimerase from a polysaccharide biosynthesis cluster oxygen homeostasis is regulated by hypoxia inducible factor, a transcription factor. when the oxygen level becomes too low (hypoxia), hypoxia-inducible-factor 1 (hif-1a) activates the expression of over a hundred genes, associated with angiogenesis, erythropoiesis, vegf (vascular endothelial growth factor), cell migration, and energy metabolism etc. hif-1a cellular level is highly dependent on oxygen concentration and regulated by oxygen sensor enzyme, hif prolyl hydroxylase (phd2 plant sulphite reductase (sir) forms an electron transfer complex with ferredoxin (fd) for the reductive conversion of sulphite to sulphide. although previous studies have highlighted electrostatic interactions between oppositely-charged residues of the two proteins, detailed thermoenergetics of the intermolecular interaction for the complexation remains unknown. we herein carried out isothermal calorimetry of fd:sir complex formation at various nacl concentrations. driving force plot constructed from calorimetry showed that the complex was thermodynamically stabilized by both enthalpy and entropy through favourable electrostatic and non-electrostatic interactions. increasing nacl concentrations weakened interprotein affinity and contribution of the negative enthalpy changes became decreased, while no such significant decrease was found in the contribution of positive entropy changes. furthermore, a negative heat capacity change obtained from the enthalpy changes at distinct temperature indicated a contribution of hydrophobic interactions. these findings suggested that both electrostatic and nonelectrostatic interprotein interactions were energetically important for the complex formation. fddependent sir activity assay revealed a bell shaped activity curve with a maximum under a certain nacl concentration, while the methyl viologen-dependent assay of sir exhibited a profile of saturating curve, suggesting that an optimized interprotein interaction is a crucial factor in control of fd-dependent-sir activity. a residue-based nmr measurement of 15n-labeled fd upon complex formation with sir revealed that charged and non-charged residues were differentially contributed in the complex formation depending on nacl concentrations. we proposed that non-electrostatic forces were also critical for forming the fd:sir complex, and an optimized complex conformation for maximum enzymatic activity was achievable by a delicate balance among non-covalent intermolecular forces. these results may be extended for understanding of complexation between redox proteins containing biased charge clusters. ornithine transcarbamylase has a spatially extended active site as computationally predicted lisa ngu 1 , kevin ramos 1 , nicholas delateur 1 , penny beuning 1 , mary jo ondrechen 1 1 understanding how an enzyme catalyzes a reaction is a fundamental problem in protein science. biochemical experimentation has revealed catalytic mechanisms of many enzymes; however these studies have focused almost exclusively on amino acid residues in direct contact with the reacting substrate molecule(s). here we report on the computational prediction and experimental verification of the importance of distal residues in enzyme catalysis, using e. coli ornithine transcarbamylase as an example. partial order optimum likelihood (pool), developed at northeastern university, is a machine learning technique that only requires the tertiary structure of a protein to predict important catalytic residues, based on computed, residue-specific electrostatic and chemical properties. pool has been shown to predict accurately the catalytic residues and to discern between compact and spatially extended active sites. dynamic conformational changes during catalysis and strong electrostatic interactions give rise to significant coupling between remote residues and the canonical active site residues of an enzyme. this suggests that at least some enzyme active sites are spatially extended, with second-and third-shell residues playing significant roles in catalysis. in this project, we focus on ornithine transcarbamylase (otc), for which dynamic processes are believed to play a role in its catalytic mechanism. otc is reported to undergo induced-fit conformational changes upon binding carbamoyl phosphate, which affects the subsequent binding of ornithine. residues predicted by pool to be catalytically important include five in direct contact with the substrate, r106, h133, d231, c273 and r319. pool also predicted remote residues to form a spatially extended, triple-layer active site. guided by computational predictions and using site-directed mutagenesis and kinetics assays of asp140, his272, glu299 and arg57 variants, we show that these pool-predicted remote residues, located in the second and third layers, are important for catalysis. alternative energy is a major focus of current research efforts. biodiesel, a mixture of fatty acid alkyl esters, is one of the most versatile alternative fuels currently in use. this is due to the fact that it is similar to gasoline and compatible with diesel engines found throughout the existing global infrastructure. biodiesel precursor lipids are abundant in cultivated feedstock organisms such as algae and bacteria. however, the standard process for converting oil to biodiesel is heat-intensive and requires complete removal of water, reducing the overall net energy gained in its production. our work constitutes an attempt to explore enzymatic synthesis of biodiesel from lipids such as those derived from emerging fuel crops. previous literature describes fatty acid alkyl ester formation in human patients with mrsa staphylococcus aureus wound lesions. these esters are formed by partially characterized esterase activity from an unidentified source. we have identified two mrsa enzymes responsible for this activity by using a combination of size exclusion chromatography, gas chromatography-mass spectrometry, and mass spectrometric protein sequencing. these two highly similar enzymes in the glycerol ester hydrolase (geh) family of proteins catalyze the synthesis of fatty acid alkyl esters in aqueous conditions at or near room temperature. we have demonstrated that other non-staphylococcal lipases do not exhibit this behavior. we have expressed these staphylococcal esterases in e. coli, and shown via gas chromatography that the expressed proteins catalyze the formation of fatty acid alkyl esters. based on sequence similarity to homologous proteins that have already been crystallized, we have predicted a structure for these enzymes and have engineered mutant fusions with higher rates of catalysis. our design hypothesis is that increased avidity for substrate molecules will yield a higher substrate concentration in the vicinity to the enzyme. to increase substrate concentration we have designed and expressed one of the enzymes as a chimeric fusion with the drosophila melanogaster alcohol-binding protein lush. gc-ms determination of biodiesel production rate indicates that the chimeric fusion has a lower-order rate constant with respect to ethanol. in other words, the fusion enzyme is less dependent on substrate concentration and is a superior catalyst at low ethanol concentrations. this result indicates that the rationally designed modification of binding avidity constitutes a potential avenue for improving the ability of enzymes to catalyze reactions with low-concentration or low-solubility substrates. functional elements of a human antizyme essential for binding and inhibiting human ornithine decarboxylase proteases are ubiquitous enzymes that catalyze the hydrolysis of peptide bonds within protein substrates; they have served as key model enzymes for studying the molecular basis for catalytic power and specificity. protease substrate specificity is most often defined in terms of linear sequence motifs that flank the cleavage site; however, the natural substrates of proteases are proteins with 3-dimensional shapes and complex conformational dynamics that are not well represented by 1-dimensional sequence alone. these structural and dynamical properties can impact recognition and binding of substrates by proteases, as well as the efficiency of catalysis itself. in this study, we explore the importance of substrate structure and dynamics for proteolysis using as our model the cleavage of the kunitz-bpti family of canonical serine protease inhibitors by mesotrypsin. bovine pancreatic trypsin inhibitor (bpti), an archetypal serine protease inhibitor of the kunitz family, has a high affinity interaction with trypsin, yet its peptide bond hydrolysis is many orders of magnitude slower than other peptide substrates. mesotrypsin, a trypsin variant, has been shown to hydrolyze kunitz family inhibitors at accelerated rates; this is especially true of human kunitz domain inhibitors. amyloid precursor protein inhibitor (appi) and amyloid precursor like protein-2 (aplp2), two human kunitz domain family members, are hydrolyzed by mesotrypsin several hundred times faster than bpti. here, we present a new, unpublished crystal structure of a cleavage intermediate aplp2 bound to mesotrypsin, refined to 1.4å resolution, revealing a dramatic substrate conformational change we hypothesize to be required during cleavage of a kunitz domain. using this structure along with published structures of appi and bpti complexes, we have modeled acyl-enzyme intermediates of mesotrypsin, and we have carried out molecular dynamic simulations that explore the transition of the initially formed native-like acyl-enzyme through the conformational transformation that allows the progression of the hydrolysis reaction. we further identify a specific hydrogen bond, present in bpti but not appi, which forms a stabilizing feature of the bpti scaffold. using site directed mutagenesis, we probe the contribution of this bond to the proteolytic stability of bpti. collectively our data for these highly structured substrates show that proteolysis rates are limited by a necessary conformational change in the substrate as the reaction progresses. rigid substrates possessing stabilizing features that render them highly resistant to this conformational change are proteolyzed more slowly than more flexible substrates of similar structure. lpmos are copper metalloenzymes that carry out the oxidative cleavage of the b-1,4-glycosidic bond, generating new chain ends that can subsequently be processed by cellulases, boosting the cellulose degradation. lpmos have a b-sandwich conformation with a flat binding surface, allowing for the enzyme to bind to crystalline cellulose. the cu21 ion, required for activity, is located in a so-called "histidine brace", in which the n-terminal histidine is highly conserved. regioselectivity according to the carbon atom being oxidized, 3 lpmo types are identified: type 1 and type 2 oxidizing at the c1 and the c4 respectively, type 3 lpmos oxidizing both the c1 and the c4 adjacent to the glycosidic linkage. we were able to express a type-1 lpmo (phanerochaete chrysosporium gh61d) and a type-3 lpmo (trichoderma reesei cel61a) in p. pastoris. this has proven to be very challenging, as lpmo activity requires a perfect cleavage of the signal sequence. after activity assays on pasc, characteristic hpaec-pad traces were obtained which will serve as a reference for engineering experiments. enzyme engineering using the 3dm database, a structure based multiple sequence alignment tool, it is possible to identify residues specifically conserved in subsets of protein sequences. by defining a subset for each lpmo type, we were able to identify residues contributing to regioselectivity. these positions are now being rationally engineered in subsequent rounds of mutagenesis, using trcel61a as a template. the effect of the mutations will be determined by analyzing the hpaec-pad trace released from pasc. the main goal is to investigate the possibility of deleting the c4 specificity in a type 3 lpmo. folding topology determines substrate binding order in the ribokinase superfamily alejandra herrera-morand e 1 , victor castro-fern andez 1 , madrid, españa ribokinase superfamily comprises three enzyme families: the adp-dependent sugar kinases family, the atpdependent coenzyme kinases family and the atp-dependent sugar kinases family. in all these families there is a large domain composed by a rossmann motif but only the atp-dependent enzymes have a b-meander motif in the c-terminal end. interestingly, these enzymes display an ordered kinetic mechanism where the substrate that will be phosphorylated binds first to the enzyme. the adp-dependent enzymes present a topological re-ordering of the secondary structural elements which produces an equivalent tertiary structure, which can be thought as a non-circular permutation (ncp) of the bmeander region. these enzymes also display an ordered kinetic mechanism but with an inversed order being the nucleotide the first substrate to bind to the enzyme. as this b-meander region of the proteins constitutes almost entirely the nucleotide binding site, and given that the permutation is the major structural difference between adp and atp-dependent kinases, it could the responsible for the nucleotide specificity. to test this hypothesis we introduce, by permutation, an atp-dependent topology in the homologous adp-dependent glucokinase from t. litoralis (pergk). size exclusion chromatography and circular dichroism spectra show that both the wild type and the permutated enzyme eluted as monomers with similar hydrodynamic behavior, and have the same secondary structure content. kinetic assays employing atp or adp as substrate demonstrate that even in the presence of 10 mm atp, the pergk enzyme is not able to carry out the phosphoryl transfer. to test if the ncp has an impact in the kinetic constants and substrate binding order we determine the kinetic mechanism through classical protocols, involving initial velocity studies, product inhibition and dead end inhibitors. the results demonstrate that the pergk enzyme presents an altered substrate binding order compared to the wild type enzyme, where glucose was the first substrate to bind to the enzyme and glucose-6-p the last product to be released. also, ligand-induced conformational changes were determined in the crystal structures. the apo, the enzyme-glucose and enzyme-glucose-adpbs structures were determined at 2.14 å, 1.95 å and 2.44 å resolutions, respectively. structure analysis reveals that glucose binding provokes major conformational changes in the pergk enzyme, whereas adp binding does not cause further changes in the conformation of the protein. the results show that although the permutation has no effect on the nucleotide preference it provokes a change in the substrate binding order that correlates well with that those observed in the crystal structures. also, they demonstrate that during the evolutionary history of the ribokinase superfamily folding topology dictates the substrate binding order (fondecyt 1150460). background: human ceruloplasmin (cp) is a circulating copper-containing glycoprotein produced in the liver and first described as a component of alpha2-globulin fraction of human plasma. cp belongs to the multicopper oxidase family and it is nowadays regarded as a "moonlighting" protein, because it changes its function according to substrate, localization and expression. cp plays a key role in copper transport and iron metabolism and it is also a potent inhibitor of leukocyte myeloperoxidase (mpo) (kd5130nm), a major source of oxidants in vivo. the protein is extremely susceptible to proteolysis. in fact, cp is a structural homolog of coagulation factors v and viii, that are physiological substrates of thrombin (fiia). interestingly, thrombin participates in both haemostatic and inflammatory responses: in some focus of inflammation, such as rheumatoid arthritis (ra), the high activity of fiia has been documented. it was demonstrated that fiia can promote the chemotaxis of neutrophils and monocytes and their adhesion to endothelial cells, to increase vascular permeability. all these effect are mediated by par-1 interaction, that are abundantly expressed in inflamed rheumatoid synovial tissues. aims: in this study the interaction of cp with thrombin was investigated to confirm the participation of fiia in "spontaneous" proteolytic degradation of cp. in fact, in vivo the integrity of cp is essential for its role in the transport or metabolism of copper. results: our results indicated that thrombin cleaves cp in vitro at 481arg-ser482 and 887lys-val888 bonds, generating a nicked species that retains the native-like fold and the ferroxidase activity of the intact protein, whereas the mpo inhibitory function of cp is abrogated. analysis of the synovial fluid of 24 ra patients reveals that cp is proteolytically degraded to a variable extent, with a fragmentation pattern similar to that observed with fiia in vitro, and that proteolysis is blocked by hirudin, a highly potent and specific thrombin inhibitor. we demonstrate that fiia has intrinsic affinity for cp (kd 5 60-270 nm), independently of proteolysis, and inhibits cp ferroxidase activity (ki 5 220 6 20 nm). mapping of thrombin binding sites with specific exosite-directed ligands (i.e. hirugen, fibrinogen gamma-peptide) and thrombin analogues having the exosites variably compromised (i.e. prothrombin, prethrombin-2, alpha-thrombin), reveals that the positively charged exosite-ii of thrombin binds to the negative upper region of cp, while the protease active site and exosite-i remain accessible. these results suggest that thrombin can exacerbate inflammation in ra by impairing via proteolysis the mpo inhibitory function of cp and by competitively inhibiting cp ferroxidase activity. an artificial pathway for isobutene production by direct fermentation: combining metabolic engineering and protein engineering benoit villiers 1 , franc¸ois stricher 1 1 the purpose of global bioenergies is to develop innovative metabolic pathways for the production of light olefins from renewable resources, by direct fermentation. light olefins (ethylene, propylene, linear butylene, isobutene and butadiene) are the core of the petrochemical industry. however, microorganisms do not naturally produce light olefins and no bioprocess to convert renewable resources to these molecules has been industrialized so far. global bioenergies has developed an artificial metabolic pathway including all the necessary enzymatic reactions from feedstock to isobutene. the metabolic route leading to isobutene can be divided in three parts, the first one being the use of natural reactions occurring in the host microorganism. second, heterologous natural reactions were introduced into the same host microorganism. finally, in contrast with most former approaches, non-naturally occurring reactions as enzymatic key steps were used, for example the decarboxylation of hydroxyisovaleric acid into isobutene. such non-natural critical steps were made possible by taking advantages of the natural catalytic and substrate promiscuity of exogenous enzymes. candidate enzymes are then evolved using systematic, random and semi-rational approaches in successive rounds in order to reach the desired catalytic efficiency. since all these reactions are enzymatic, isobutene can be obtained by direct fermentation, e.g. a process wherein all the chemical transformations are carried on by the host microorganism. the scale-up of this process began in november 2014 in a pilot plant installed in pomacle-bazancourt, france, with an annual capacity of 10 tons of oxidation-grade isobutene. importantly, production of a volatile compound such as isobutene (and other light olefins) by direct fermentation presents two major advantages: first, the product is spontaneously removed from the culture broth, which alleviates the limitations linked with titer issues. second, the purification process is considerably easier and cheaper since no energy consuming methods such as distillation or phase separation are necessary to purify the end product. for the first time, batches of industrially produced isobutene from renewable resources have been obtained in the first half of 2015. this isobutene has been in turn converted into isooctane, an additive currently used to improve gasoline quality, which could also be used as a standalone fuel. a demonstration plant is planned in leuna, germany, with an annual capacity of 100 tons of polymer-grade isobutene and ibn-one, a joint venture with cristal union (4th european beet processor), has been formed to build and operate the first plant in france converting renewable resources into isobutene. finally, while the isobutene process is progressing towards industrial scale, global bioenergies is also developing new artificial metabolic pathways enabling direct bio-production of butadiene and propylene. the development of a coupled enzyme assay to detect isochorismate pyruvate lyase activity protein folding is typically defined in terms of the spatial arrangement of structural elements, i.e. helices, sheets and loops. we have, however, been developing an alternative and complementary paradigm based on conserved hydropathic interaction networks within proteins. these networks can be viewed as environments comprised of a mixture of polar and hydrophobic interaction fields, and may be the most important factor driving protein folding. this concept applies even to the lowest structural level within a protein: the sidechain conformations (or rotamers). exhaustive statistical analysis of existing crystallographic structures of proteins showed rotameric preferences and led to the creation of rotamer libraries frequently used in multiple aspects of structural biology, e.g., crystallography of relatively low-resolution structures, homology modeling and biomolecular nmr. however, little is actually known about the forces and factors driving the preference or suitability of one rotamer over another. in our study, tyrosine was analyzed since its sidechain has a comprehensive set of hydropathic properties that made it ideal as a proof of concept residue. construction of 3d hydropathic interaction maps of tyrosine residues in our dataset, reveals the environment around each, in terms of hydrophobic (p-p stacking, etc.) and polar (hydrogen bonding, etc.) interactions. after partitioning the tyrosines into backbonedependent bins, a map similarity metric based on the correlation coefficient was applied to each mapmap pair to build matrices suitable for clustering. notably, the first bin representing 631 tyrosines, reduced to 14 unique hydropathic environments with most diversity arising from favorable hydrophobic interactions with many different residue partner types. polar interactions for tyrosine include ubiquitous hydrogen bonding with the phenolic oh and somewhat surprisingly a handful of unique environments for the tyrosine backbone. all but one of the 14 environments are dominated by a single rotamer, the exception being an environment defined by a paucity of interactions with the tyrosine ring and as a consequence its rotamer is indeterminate. this is consistent with it being composed of mostly surface residues. each tyrosine residue attempts to fulfill its hydropathic valences and thus, structural water molecules are seen in a variety of roles throughout these environments. alanine was analyzed using the same protocol as well. having the smallest sidechain (and small hydropathic interaction maps), alanine allowed us to investigate a significantly larger database, permitting us to examine the correlation between hydropathic maps and various structural features. in conclusion, the analysis of hydropathic environments strongly suggests that the orientation of a residue in a three-dimensional structure is a direct consequence of its hydropathic environment, which leads us to propose a new paradigm, interaction homology, as a key factor in protein structure. it is not the surrounding residues that direct sidechain conformations, but rather the hydropathic "field" of the surrounding atoms. folding studies of independent domains of lysine, arginine, ornithine binding protein (lao) protein folding problem has been addressed from the past 50 years until nowadays, however, we still can not explain how proteins acquire their native structure from their amino acid sequence. different approaches has been taken in order to study protein folding, for example, the comparative study of folding mechanism between homologues proteins with high identity of sequence and structure, and the study of independent regions within a single protein. previously in our laboratory, thermodynamic and kinetic folding properties of lysine, ornithine, arginine binding protein (lao), a 238 amino acid periplasmic binding protein (pbp), composed by two rossmann fold domains (one continuous and the other discontinuous) attached by a hinge region, has been studied. even there is a functional research about binding characteristics of histidine binding protei ns (his j) domains of when expressed independently (chu, b. 2013 ); there are no folding studies in these conditions for this or another pbps. it should be noted that his j shares 70% of sequence identity and tertiary structure (rmsd 1å) with lao. in order to know the folding effect of encoding different domains in the same poly peptidic chain, as well as its influence in function, we are studying the thermodynamic and kinetic characteristics of folding of independently expressed lobes of lao, and comparing with those of native protein. by now, we expressed and purified the discontinuous domain. circular dichroism (cd) and fluorescence intensity spectra show that this independent domain has primary and tertiary structure. thermal denaturation has a single cooperative transition, which indicates this domain is folded. thermodynamic analysis of temperature and urea-induced experiments suggest that lao's folding characteristics are not just the addition of those from independent domains. furthermore, folding and refolding kinetics suggest the presence of a burst phase intermediate. a hypothesis to reconcile the physical and chemical unfolding of proteins a comprehensive view of protein folding is crucial for understanding how misfolding can cause neurodegenerative diseases and cancer. when using physical or chemical perturbations, nmr spectroscopy is a powerful tool to reveal a shift in the native conformation toward local intermediates that act as seeds for misfolding. high pressure (hp) or urea is commonly used to disturb folding species. pressure favors the reversible unfolding of proteins by causing changes in the volumetric properties of the proteinsolvent system. however, no mechanistic model has fully elucidated the effects of urea on structure unfolding, even though protein-urea interactions are considered to be crucial. here, we provide nmr spectroscopy and 3d reconstructions from x-ray scattering to develop the "push-and-pull" hypothesis, which helps to explain the initial mechanism of chemical unfolding in light of the physical events triggered by hp. in studying mpnep2 from moniliophthora perniciosa, we tracked two cooperative units using hp-nmr as mpnep2 moved uphill in the energy landscape; this process contrasts with the overall structural unfolding that occurs upon reaching a threshold concentration of urea. at subdenaturing concentrations of urea, we were able to trap a state in which urea is preferentially bound to the protein (as determined by nmr intensities and chemical shifts); this state is still folded and not additionally exposed to solvent [fluorescence and small-angle x-ray scattering (saxs)]. this state has a higher susceptibility to pressure denaturation (lower p1/2 and larger dvu); thus, urea and hp share concomitant effects of urea binding and pulling and water-inducing pushing, respectively. these observations explain the differences between the molecular mechanisms that control the physical and chemical unfolding of proteins, thus opening up new possibilities for the study of protein folding and providing an interpretation of the nature of cooperativity in the folding and unfolding processes. zinc: a promoter or inhibitor for iapp aggregation? feng ding 1 , praveen nedumpully-govindan 1 1 zinc ions have been found to play an important and yet complex role in human islet amyloid polypeptide (hiapp) aggregation, which is associated with b-cell death in type-ii diabetes (t2d). both concentration-dependent promotion and inhibition of iapp aggregation by zinc ions have been observed in vitro. similarly, at the population level, both positive and negative correlations were reported between the activity of a b-cell specific zinc transporter and t2d risk. zinc ions are able to bind a single histidine in hiapp and coordinate the formation of zinc-bound hiapp oligomers. we hypothesize that the relative zinc/hiapp concentration determines the population of zinc-bound hiapp oligomers with different molecular weights. we have applied molecular dynamics (md) simulations to systematically study the structure and dynamics of a range of zinc-coordinated hiapp oligomers, including monomers, dimers, trimers, tetramers, and hexamers. our computational results suggest that different zinc-bound oligomers have distinct aggregation propensities. high-molecular weight oligomers (2 peptides) have higher aggregation propensity than zinc-free and zinc-bound hiapp monomers at 2 mm concentration in silico. therefore, our results provide a molecular insight into the complex role of direct zinc binding on hiapp aggregation. at low zinc/hiapp stoichiometry, zinc binding promotes aggregation. as the stoichiometry increases and zinc ions bind to single hiapp peptides, the aggregation of hiapp is inhibited due to electrostatic repulsion between the charged zinc ions. our computational study sheds light on the complex role of zinc on hiapp aggregation and t2d development. biomolecules function in the densely crowded and highly heterogeneous cell, which is filled up to a volume of 40% with macromolecules [1] . often, artificial macromolecular crowding agents are used to mimic these conditions in vitro and the excluded volume theory is applied to explain the observed effects [2] . however, recent studies emphasize the role of further contributions aside from a pure volume effect including enthalpic and solvent effects [3, 4] . we study cosolute effects at high molecular and macromolecular concentrations via a thermodynamic analysis of the thermal unfolding of ubiquitin in the presence of different concentrations of cosolutes (glucose, dextran, polyethylene glycol, potassium chloride) [5] . in contrast to the excluded volume theory, we observed enthalpic stabilization and entropic destabilization forces for all tested cosolutes. the enthalpic stabilization mechanism of ubiquitin in macromolecular polysaccharide solutions of dextran was thereby similar to the effects observed in monomeric glucose. further, it remains unclear how such cosolutes reflect the physicochemical properties of the complex cell environment as a characterization of the in-cell crowding effect is lacking. thus, we developed a fret-based macromolecular crowding sensor to study the crowding effect in living cells [6] . the averaged conformation of the sensor is similar to dilute aqueous buffer and cell lysate. we find that the in-cell crowding effect is distributed heterogeneously and can change significantly upon osmotic stress. the presented method allows to systematically study in-cell crowding effects and understand them as a modulator of biomolecular function. the stability of biomolecules under co-solvent conditions is dependent on the nature of the co-solvent [1] . this can alter a protein's properties and structural features through biomolecular interactions between its functional groups and the co-solvent molecules. ionic liquids (ils) represent a rather diverse class of co-solvents. the design flexibility of these molten salts is an attractive feature, allowing the properties of the il to be tuned to meet the requirements of different applications [2] . particularly, the modulation of reaction pathways between folding states, offering possibilities to control irreversibility in non-native protein aggregation [2] . this has led us to investigate the impact of ils as co-solvents with the well-known protein denaturant urea. urea is considered to be a non-ionic chaotrope disturbing considerable the grid of hydrogen bonds with the protein backbone. urea interacts preferentially with the protein surface, mainly apolar residues and that dispersion, rather than electrostatic interactions, is the main energetic contribution to explain the stabilization of the unfolded state of the protein and the irreversibility of the unfolding process in the presence of urea [3] . a large body of multidomain protein folding work has been devoted to study monomeric proteins. how do multidomain multimeric protein fold, avoiding accumulation of stable intermediate is yet to be studied in detail. our present study is focussed on understanding the folding and assembly of the domains of a homodimeric l-aspraginase from a hyperthermophile pyrococcus furiosus (pfa). each monomer of pfa consists of distinct n-and c-terminal domains (npfa and cpfa, respectively), connected by a linker. the folding mechanism of each domain with respect to full length protein was studied by mutating one out of two tryptophans, one in each domain. domains were purified and studied individually to obtain parallel account of the folding of each domain in isolation. subunit assembly was studied by analytical size exclusion chromatography (sec), multiangle light scattering and functional activity. through far uv cd, intrinsic trp fluorescence and sec, we demonstrated that domain folding and subunit association were intimately linked in full length pfa. interestingly, en route to its folding there was complete absence of hydrophobic intermediates as probed by ans fluorescence. folding of npfa was highly cooperative and, it provides interacting surfaces for cpfa to fold and also facilitates subunit assembly. the folding cooperativity of isolated domains was very less compared to the folding cooperativity of their full length counterparts, as indicated by equilibrium m values. to our surprise, during ph induced denaturation, at ph 2 and 13, the dimer dissociates into highly hydrophobic folded monomers which readily underwent amyloidogenesis. we showed that at such extreme conditions, cooperativity in folding process in multidomain multimeric protein is not solely governed by the folding of individual domains, rather by concomitant folding and association of domains directly into a quaternary structure. in other case, where subunit folding occurred prior to association, protein readily underwent extensive aggregation. groel assisted folding of multiple recombinant proteins simultaneously over-expressed in e.coli megha goyal 1 , tapan kumar chaudhuri 1 1 aggregation prone recombinant proteins very often form inclusion bodies and also exhibits poor yield of functional protein during in vitro refolding process from chemically denatured form. bacterial chaperonin groel provides folding assistance to several proteins, when over-expressed with one of the recombinant proteins. there are instances that groel in presence of few other co-expressed chaperones like dnaj, dnak etc provides better yield of folded protein during homologous and heterologous expression. considering the ongoing events in the cells, it is known that molecular chaperone groel assists in the folding of various proteins in the cytoplasm. hence attempt to fold multiple recombinant proteins over-expressing simultaneously with the co-expression of chaperones can be worth trying. this approach may cut down various complexities in the functional recombinant protein preparation, including time and effective cost. keeping this view in mind, folding of two simultaneously expressed aggregation prone proteins, 69 kda e.coli maltodextrin glucosidase (malz) and 82 kda yeast mitochondrial aconitase have been investigated with the co-expression of groel and groes in e.coli cytosol. it has been previously reported that both the chosen proteins undergo co-expressed groel-groes assisted folding in e.coli cytosol, when they over-express alone. in this study we have optimized the overexpression of malz and aconitase simultaneously in e.coli. further optimisation was carried out to coexpress groel along with malz and aconitase. based on the basic philosophy that soluble protein mainly contains folded fraction, the event of groel/es assisted folding of simultaneously overexpressed proteins, malz and aconitase was monitored through the attainment of soluble proteins under various sets of conditions such as temperature. the major outcome of the present study is that, with the groel-groes assistance, the yield of soluble proteins (malz and aconitase) together constitutes higher percentage of folded protein in contrast to the percent yield when a single protein was overexpressed. significance of this type of study relies on the fact that the cells can over-produce higher amount of recombinant proteins, when multiple over-expression takes place. not only pushing up cell's capability of over-expression, co-expression of groel and groes efficiently assists in the folding of multiple proteins simultaneously over-expressed in e.coli. amyloid fibrils associated with serious diseases including alzheimer's, parkinson's, and prion diseases promoted the challenge of studying protein misfolding, leading to the development of amyloid structural biology. amyloid fibrils form in supersaturated solutions via a nucleation and growth mechanism. although the structural features of amyloid fibrils have become increasingly clearer, knowledge on the thermodynamics of fibrillation is limited. furthermore, protein aggregation is not a target of calorimetry, one of the most powerful approaches used to study proteins. here, with b2-microglobulin, a protein responsible for dialysis-related amyloidosis, we show direct heat measurements of the formation of amyloid fibrils using isothermal titration calorimetry (itc). the spontaneous fibrillation after a lag phase was accompanied by exothermic heat. the thermodynamic parameters of fibrillation obtained under various protein concentrations and temperatures were consistent with the main-chain dominated structural model of fibrils, in which overall packing was less than that of the native structures. we also characterized the thermodynamics of amorphous aggregation, enabling the comparison of protein folding, amyloid fibrillation, and amorphous aggregation. in order to obtain general thermodynamic properties of protein aggregations, we further investigated aggregation of glucagon and insulin, two of the most famous amyloidogenic peptide hormones, using itc. we also observed characteristic heat of spontaneous amyloid fibrillation of both proteins after a lag time. taken all together, we showed that thermodynamic studies on amyloid fibrillation and amorphous aggregation were indeed possible by means of itc-based qualitative and quantitative calorimetric analyses. itc will become a promising approach for clarifying the thermodynamic properties of protein aggregates. the more case studies are required toward the establishment of thermodynamics of protein misfolding and aggregation when hydrophobic proteins are, for any reason, exposed to the cytosol they are rapidly captured by protective complexes which shield them from the aqueous surroundings and decide their fate (by either targeting them to their correct membrane homes or marking them for degradation by the ubiquitin/proteasome system). the bag6 holdase is a heterotrimeric protein complex, comprising bag6, ubl4a and trc35, which works closely with the cochaperone sgta to triage hydrophobic proteins and pass them along the appropriate pathway. sgta also interacts with viral proteins and hormone receptors and is upregulated in numerous cancer types. these functions require further investigation to determine the scope of sgta as a therapeutic target. our lab has solved the solution structure of the n-terminal dimerization domain of sgta and characterised its interaction with two different ubiquitin-like (ubl) domains in the bag6 holdase (one from ubl4a and the other from bag6 itself) using nmr chemical shift perturbation data and other biophysical techniques including isothermal titration calorimetry and microscale thermophoresis. at this meeting i will report on the progress we have made in structurally characterising further key players that participate in this quality control, with the aim of clarifying the intricate network of molecular interactions that governs these processes in health and disease. ensemble, ribbon and electrostatics spacefill views of the sgta dimerization domain structure. the final panel shows the structure overlaid with its yeast homologue. alpha synuclein is a small protein (14 kda) expressed at high levels in dopaminergic neurons. fibrillar aggregates of a-synuclein inside the dopaminergic neuron are the major components of lewy bodies and lewy neuritis inclusion, which are considered as potential hallmark of parkinson's disease (pd). both in vitro as well as in vivo studies suggest that the soluble, oligomeric forms of a-syn are the more potent neurotoxic species, responsible for neuronal injury and death in pd. therefore, molecules that inhibit the toxicity of oligomers either by reducing their formation or by converting their more toxic oligomeric state to less-toxic fibrillar state would be effective agents for the drug development against pd. curcumin is one of the asian food ingredients which has shown a potential role as therapeutic agent against many neurological disorders including pd. however, the instability and low solubility makes it less attractive for use as potential therapeutic agent. the present work focuses on screening of the compounds similar to curcumin but having better effects on the morphology and toxicity of oligomeric and fibrillar assemblies of a-syn, which could be used as therapeutic agent preferentially over the naturally occurring curcumin. we synthesized and analyzed the effects of nine compounds, which are structurally similar to curcumin, on different stages of a-syn amyloid aggregation. here, we showed that curcumin and its analogs accelerate a-syn aggregation to produce morphologically different amyloid fibrils in vitro. however, there is no significant effect of curcumin and its analogs on the secondary structure of preformed a-syn fibrils. furthermore, these curcumin analogs showed differential binding affinities with the preformed a-syn aggregates, possibly due to difference in their chemical structures. the present data suggest the promising role of curcumin analogs in the treatment of a-synucleinopathy disorders. in vitro folding mechanisms determine the forces applied during co-translational folding there is currently much debate as to whether experiments conducted in vitro describe the folding of proteins in vivo. in particular, it is often suggested that the co-translational folding of nascent protein chains is dominated by the presence of the ribosome and associated chaperones, and that folding mechanisms will be affected by the vectorial nature of translation. here we use an arrest peptide assay to investigate the co-translational folding of a number of all-a spectrin domains that exhibit a range of thermodynamic stabilities and in vitro folding rates. our unexpected finding is that that the force exerted on the ribosome by these domains is not related to either the thermodynamic stability of the domain, or to the folding (loading) rate, but rather to the in vitro folding mechanism. we infer that the in vitro folding mechanisms of these domains are unaffected by the presence of the ribosome -even when part of the nascent chain is retained within the ribosome exit tunnel. there has been much work to date investigating the intermediates present in stalled translation complexes -but now, for the first time, we can begin to directly explore the rate limiting transition state in the co-translational folding of homologous proteins. can the structure of a protein (h3.1) depend on the treatment of a solvent medium (explicit vs effective) in a coarse-grained computer simulation? ras pandey 1 , barry farmer 2 1 university of southern mississippi, 2 air force research laboratory solvent medium plays a critical role in orchestrating the structure and dynamics of a protein. in computer simulation modeling of protein structure in a solvent medium, explicit, implicit, effectivemedium, approaches are often adopted to incorporate the effects of solvation. because of the complexity in incorporating all atomic and molecular details, the multiple components, reaching the large-scale, etc. implicit solvent or effective medium approach is generally more viable than the explicit solvent methods. some of the pertinent characteristics such as excluded volume of the solvent constituents, its concentration, and the underlying fluctuations which may be important in probing some issues are generally ignored in effective medium or implicit solvent approaches. using a coarse-grained approach, we investigate the structure and dynamics of a protein (a histone, h3.1) in the presence of both effective as well as explicit solvent media over a range of temperatures with the monte carlo simulations. the protein is represented by a coarse-grained chain of residues whose interactions are described by knowledge-based residue-residue and hydropathy-index-based residuesolvent interactions. in effective medium approach, each empty lattice site around the protein structure acts as a solvent. only a fraction of lattice sites are occupied by mobile solvent constituents along with the protein chain in explicit solvent medium. large scale simulations are performed to analyze the structure of the protein for a range of residue-solvent interactions and temperature in both explicit and effective solvent media. we study a number of local (e.g. solvation and mobility profiles) and global (radius of gyration and structure factor) physical quantities as a function of temperature. we find that the response of the radius of gyration of the protein in explicit solvent is different from that in effective medium solvent. thus, the presence of fluctuations in explicit solvent approach have considerable effects on the structure and dynamics of protein h3.1. differences due to type of solvent on the response of some of these quantities as a function of temperature as well as general similarities will be presented. single-molecule vectorial folding and unfolding through membrane pores david protein folding and unfolding in vivo is frequently vectorial. for example, proteins are synthesized at the ribosome and emerge n-terminal first. as the polypeptide chain emerges from a 2 nm wide pore is free to fold, interact with partners or misfold1. in another example, proteins are unfolded at the proteasome by pulling from either the n or c terminus against a 1-2 nm wide pore, applying a tension on the residues surrounding the terminus of the protein2. under this conditions, proteins may behave differently than when unfolded/refolded with temperature or urea. this may have important implications, as protein folding and unfolding in vivo is related to both function and disease. we noticed that vectorial folding is inherently linked to nanometer size pores. making use of nanopore technology we developed a method to monitor protein unfolding during membrane translocation at the single-molecule level3. briefly, an oligonucleotide attached at either end of a protein threads a single protein nanopore inserted in a lipid membrane. in response to an applied membrane potential, the oligonucleotide pulls the protein through the pore and as it is forced to translocate it unfolds. analysing the ionic current we obtain the unfolding pathway and information on the polypeptide sequence. this methodology has shown that proteins unfold with different kinetics when pulled from one terminus or the other4. remarkably, it is also possible to say whether the protein has been phosphorylated or not, and where5. we have recently advanced our model system to study protein folding after translocation at the singlemolecule level6. a single-protein molecule was translocated through a pore and forced to translocate back at predetermined times. we measured the stability of the refolded state at different times and we obtained the vectorial folding pathway of the protein. further, we observed that the protein was capable of co-translocational folding and that this premature folding contributed to the complete translocation of the protein. our results show that nanopore technology applied to proteins can be used to describe the vectorial folding and unfolding of proteins, providing insight to how these processes may work in vivo. further, single-molecule protein sequencing is a possibility that could revolutionise our knowledge on biological processes. thermodynamics studies of oligomeric proteins, which are the dominant protein natural form, have been often hampered because irreversible aggregation and/or slow reactions are common. there is not a single report on the reversible equilibrium thermal unfolding of proteins composed by (b/a)8 barrel subunits, albeit this "tim barrel" topology is one of the most abundant and versatile in nature. the eponymous tim barrel, triosephosphate isomerase (tim) is a ubiquitous glycolytic enzyme that catalyzes the isomerization of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. the unfolding of several tims, mainly of eukaryotic organisms, has been extensively studied. regarding thermal unfolding, eighteen tims, mainly from eukaryotes, as diverse as amoebozoa, euglenozoa, ascomycota and chordata, have been studied. even though a full thermodynamic characterization has been hampered by irreversible aggregation and/or the presence of hysteresis in all of them, the activation parameters that describe the kinetic control of five eukaryotic tims have been reported. we characterized the structure, catalytic properties, association state and temperature-induced unfolding of the eponymous tim barrel, triosephosphate isomerase (tim), belonging to five species representative of different bacterial taxa: deinococcus radiodurans (drtim), nostoc punctiforme (nptim), gemmata obscuriglobus (gotim), clostridium perfringens (cptim) and streptomyces coelicolor (sctim). irreversibility and kinetic control were observed in the thermal unfolding of nptim and gotim, while for drtim, sctim and cptim, the thermal unfolding was found to follow a two-state equilibrium reversible process, a behavior not observed previously for others tims. shifts in the global stability curves of these three proteins are related to organismal temperature range of optimal growth and modulated by variations in maximum stability temperature and in the enthalpy change at that temperature. reversibility appears to correlate with low isoelectric point, the absence of residual structure in the unfolded state, small cavity volume in the native state structure, low conformational stability and a low melting temperature. furthermore, the strong coupling between dimer dissociation and monomer unfolding may reduce the possibility of aggregation and favor reversibility. it appears that there is a delicate balance between several contributions whose concerted interplay is necessary to achieve thermal reversibility in oligomeric enzymes. furthermore, the finding that the three reversible proteins come from organisms from different phyla suggests that unfolding reversibility may be more common than what is currently known supported by a critical step in the late phase of human immunodeficiency virus type 1 (hiv-1) infection is targeting of the virally encoded gag proteins to the plasma membrane (pm) for assembly. prior to assembly, the hiv-1 gag polyprotein adopts a compact "folded over" conformation and exists in the monomeric or low-order oligomeric states. whereas it is established that the nucleocapsid domain of gag specifically recognizes motifs in the viral rna genome for packaging, there is compelling evidence that the myristoylated matrix (ma) domain also binds to cellular rna to prevent premature gag targeting to intracellular membranes. upon transport of gag to the pm, the interaction of ma with rna is exchanged for an interaction of ma with pm components. this molecular switch induces an extended conformation of gag, leading to formation of high-order gag oligomers on the pm. because gag is anchored and therefore captured by its interaction with the available phospholipids, the intracellular targeting of gag is likely to be determined by the relative strength of its interaction with the dominant lipids composing each membrane subcompartment. the key to understanding this essential molecular switch is elucidating at the molecular level the interaction of ma with specific pm components. for over two decades, biochemical, in vivo, in vitro and genetic studies have focused on factors that modulate binding of retroviral gag proteins to membranes but only recently the structural and molecular determinants of gag assembly have begun to emerge. in addition to the electrostatic interactions between a highly conserved basic region of ma and acidic phospholipids, it is now believed that the hydrophobicity of the membrane interior represented by the acyl chains and cholesterol also play important roles. we employ nmr methods to elucidate the molecular determinants of gag binding to the membrane. our structural studies revealed that phosphatidylinositol-4,5-bisphosphate (pi ( the production of functionally antibodies depends on the transition of immature b cells to mature plasma cells and is tightly linked to several "quality control" check points. during b cell development, the pre-b cell receptor (pre-bcr) is the first checkpoint which determines the viability and proliferation of the pre-b cell. the pre-bcr is composed of an immunoglobulin (ig) heavy chain molecule associated with an ig light chain-like molecule called the surrogate light chain (slc). the slc is composed by two proteins k5 and vpreb which possess a unique region at the n-or c-terminus, respectively. vpreb lacks a b-strand which is provided by the k5 protein allowing the non-covalent interaction essential for formation of the slc heterodimer. our understandings of the molecular mechanism of slc function and assembly are still at an early stage. in particular, we do not know how the slc associates and forms the pre-bcr for the selection of all heavy chains (hcs). our study focuses on dissecting the "fab fragment" of the pre-bcr to study the effect of the unexpected structural features of the slc to gain insight in hc selection. the analysis of the assembly of the slc revealed a significant difference between the single domains and the complexes in terms of stability and assembly. the folding behavior of the ch1 domain in the presence of the slc is key for the first quality control mechanism in the endoplasmic reticulum (er) prior to surface expression. our results show that the slc interacts with ch1 domain in a similar manner to the cl domain. thus, the folding of the naturally disordered ch1 domain upon interaction with the slc releases the hc retention in the er by bip. taken together, our study provides new insights into the folding and assembly of the "fab fragment" of the pre-bcr and paves the way for a detailed mechanistic understanding of hcs selection by the unique slc. though the 22-29 (sfgailss) region of human islet amyloid polypeptide (hiapp) has long been known to be crucial for amyloid fiber formation, lack of b-ordering of this region in structures of the final fiber as determined by both nma and x-ray has been puzzling. new evidence now suggests that the fgail region forms ordered b structures only in early intermediates. we present new 2dir studies on the fgail region of hiapp, with uniformly 13c 18o labeled amides, along with spectral and kinetic modelling. evolution of the peak frequency and 2d lineshape of the labeled region clearly present a transition from random coil to a stable b sheet, a conclusion which is substantiated by simulation of the 2d ir spectra. as determined from kinetic modeling, the fgail b-sheet creates a free energy barrier that is the cause of the lag phase during aggregation. these findings help to rationalize a broad range of previous fragment and mutation studies as well as provide a mechanism for fiber formation that has self-consistent kinetics and structures. the temperature dependence of protein stability in living cells studies addressing the consequence of crowding that exist in the interior of cells have reached an interesting stage. experimental data so far, predominantly from, small to medium sized proteins are indicating that, in general, natively folded proteins including, intrinsically disordered, gain structure and stability under conditions mimicking cell interior. however, on the other hand, a few studies on small proteins indicate destabilization of the native state. in very few instances, crowding resulted in compaction and aggregation of the unfolded and partially folded states. experimental data on the consequences of cell-like crowding situation on relatively large proteins with complex folding free energy landscape are absent. alpha subunit of tryptophan synthase, a 29 kda tim barrel protein, provides a unique opportunity to address the consequence of crowding on the structure and stability of the native state and also on a partially folded state stable equilibrium intermediate populated in its (un)folding reactions. in the presence of increasing amounts the most commonly used crowding agent, ficoll-70, a non-monotonous increase in the far uv-cd is observed for the native state. a steady increase up to 250mg/ml ficoll followed by a decrease in far-uv cd region is observed, indicating loss of structure at increased concentrations of the crowding agent. 1h-15n hsqc nmr and fluorescence (fl) spectra confirm the of loss of structure at higher concentrations of ficoll-70. loss of native base line in the urea induced unfolding reaction monitored by cd and fl clearly confirms the destabilization of the native state. similar to the structural changes observed for the native state, for the equilibrium intermediate state maximally populated at 3 m urea also, non-monotonous changes in the far uv cd and fluorescence spectra are observed. the highly populated equilibrium intermediate shows an initial steady increase in the far uv cd signal followed by a sudden decrease. our results suggest that the structure of both native and partially folded states may be affected under crowding conditions. alpha-1 antitrypsin (aat) is a 44-kda serine protein inhibitor (serpin), which acts as an inhibitor of neutrophil elastase within the lungs. during inhibition, the protein undergoes a dramatic conformational change in which its exposed reactive centre loop (rcl) is cleaved and inserts into the central a-sheet as an extra beta-strand. this highly dynamic protein is also susceptible to mutations, resulting in misfolding and the accumulation of ordered polymers as intracellular inclusions within the endoplasmic reticulum of hepatocytes, where aat is synthesized. despite much knowledge of the folding and misfolding properties of aat as an isolated protein, very little is understood of how aat acquires its structure during biosynthesis. like all proteins, the biosynthesis of aat takes place on the ribosome, and protein folding occurs in a co-translational manner as the nascent polypeptide chain emerges from the ribosome's exit tunnel. this study aims to develop the biochemical and nmr structural strategies to characterize the co-translational folding characteristics of aat as it is being synthesized on the ribosome. for these studies, we have designed a series of secm-stalled ribosome nascent chain complexes (rnc) of aat of different lengths, which mimics the "snapshots" of the protein synthesis, capturing the folding process of the nascent chain during its emergence from the ribosome. using this library, we have recently developed a strategy to produce large quantities of the rncs both in vitro and in vivo within e. coli, a prerequisite for detailed biochemical and structural studies. using the aat-rncs, we are developing a suite of biochemical strategies to probe the capacity for aat nascent chains to adopt native structure on the ribosome. we have combined protease inhibition assays, western blot and native-page analysis to demonstrate that aat can fold while bound to the ribosome. in addition, we have employed a cysteine-based modification "pegylation" assay to probe lowresolution structural information of aat-rnc and this will guide our structural studies by nmr spectroscopy to provide a detailed understanding of aat folding on the ribosome at high resolution. thermodynamic properties of proteins vary with the environmental solvent condition (temperature, ions, ph, denaturants, etc.). although the effect of each environmental factor on proteins has been well studied, the complex effect of more than two environmental factors was not studied thoroughly. in this study, we investigate the simultaneous effect of urea denaturation (disruption of non-covalent bonds in proteins) and acid denaturation (titration of protein residues) on the nature of the folding transition for 2cyu protein. we performed the molecular dynamics simulations of bbl (pdb code: 2cyu) protein in various urea concentration at 300k. we calculated ph-dependent free energy landscape using the extended munoz-eaton model and described the phase diagram for the folding transition of bbl at various ph value and urea concentration. we mapped out the phase diagram of the folding transition of 2cyu, which clarifies the condition with which it undergoes the cooperative folding transition or the barrierless folding transition. biophysical analysis of partially folded states of myoglobin in presence of 2,2,2-trifluoroethanol paurnima talele 1 , nand kishore 1 1 the protein folding process involves one or more distinct populated intermediates. one such partially folded structure of particular importance observed during protein folding pathway is molten globule state. the properties of a molten globule state are intermediate between those of native and unfolded protein molecules. the importance of studying equilibrium molten globule is in its greater stability and flexible structure which has been shown to bind a variety of substrates and play a definite role in certain human diseases via aggregation, misfolding or some other mechanism. a protein must assume a stable and precisely ordered conformation to perform its biological function properly. the stability of a protein under specific conditions depends on its interactions with the solvent environment. therefore it is essential to understand protein folding intermediates, protein solvent interactions and protein stabilization. we have made attempts to thoroughly investigate the formation of stable molten globule state of the protein induced by alcohol using combination of calorimetric and spectroscopic techniques. the presentation will cover the topic on biophysical studies on partially folded states of myoglobin in presence of 2,2,2-trifluoroethanol. the thermal denaturation of myoglobin was studied in the presence of 2,2,2-trifluoroethanol (tfe) at various ph values using differential scanning calorimetry and uv-visible spectroscopy. the most obvious effect of tfe was lowering of the transition temperature with increasing concentration of tfe up to 1.5 mol•dm-3, beyond which no thermal transitions were observed. the conformation of the protein was analyzed by a combination of fluorescence and circular dichroism measurements. at ph 5.0 and 11.0, partially folded states of myoglobin were confirmed by cd spectroscopy. quantitative binding of ans to the tfe induced molten globule state of myoglobin was studied by using isothermal titration calorimetry (itc). the results enable quantitative estimation of the binding strength of ans with the molten globule state of myoglobin along with the enthalpic and entropic contributions to the binding process. the results also suggest occurrence of common structural features of the molten globule states of proteins offering two types of binding sites to ans molecules which has been widely used as a fluorescence probe to characterize partially folded states of proteins. modules. each cbr comprises a b-hairpin core followed by a short linker sequence. choline molecules are bound between two consecutive repeats through hydrophobic and cation-p interactions with aromatic side chains. apart from its biotechnological applications as an affinity tag for protein immobilization and purification, clyta is useful as a model for understanding the folding and stability of repeat proteins. in this sense, we proposed to get minimal peptides encompassing the sequence of a single cbr or even only its b-hairpin core able to maintain the native fold and the ability to bind choline. to that end, we first proceeded to analyze the peptide comprising the third b-hairpin core, denoted as clyt3. based on cd and nmr data we demonstrate that the peptide clyt3 conserves its native bhairpin structure in aqueous solution, but forms a stable, amphipathic a-helix in detergent micelles and as well as in small lipid vesicles [1] . considering the great differences in the distribution of hydrophobic and polar side chains shown by clyt3 b-hairpin and a-helix, we propose that amphipathic structures are stabilized in micelles or lipid vesicles. this "dual" behavior is the only up-to-now reported case of a micelle-induced conformational transition between two ordered peptide structures. to check whether other cbr repeats also undertake b-hairpin to a-helix transition in the presence of micelles, so that it represents a general tendency ascribed to all pneumococcal choline-binding modules, we will show new experimental evidences based on cd and nmr structural studies on peptides derived from the bhairpin cores of other clyta repeats, as well as in modified clyt3 peptides. continuing our studies of the effect of like-charged residues on protein-folding mechanisms, in this work, we investigated, by means of nmr spectroscopy and molecular-dynamics simulations, two short fragments of the human pin1 ww domain [hpin1(14-24); hpin1(15-23)] and one single point mutation system derived from hpin1(14-24) in which the original charged residues were replaced with non-polar alanine residues. results, for both original peptide fragments of hpin1 demonstrate the presence of ensembles of structures with a tendency to form a b-chain reversal. understanding the biology of huntington's disease via the pathogenic huntingtin monomer huntington's disease (hd) is caused by an abnormal extension of the polyglutamine (polyq) region within exon 1 of the protein huntingtin from typically 25 glutamines to over 36. disease onset correlates with the huntingtin misfolding and causing the formation of aggregates, however recent studies have postulated that pathogenic huntingtin monomer may form compact structures that are responsible for neuronal toxicity in hd. we sought to examine the conformation of huntingtin monomers, how polyq sequence length affects monomer structure and which protein-binding partners in the cell may exert a gain-of-toxic mechanism in pathology. hydrogen-deuterium exchange mass spectrometry was used to measure the degree of structure in both non-pathogenic (25q) and pathogenic (46q) huntingtin, with results showing that both forms exchanged 79% of potential nh hydrogen bond donors within 30 seconds (n53), with little to no further exchange over the following ten minutes. this result suggested that the pathogenic conformations are not stabilized by slow exchanging hydrogen bonds. binding partners to the monomer were assessed in neuro2a cell culture by immunoprecipitation and quantitative ms/ ms proteomics approaches after depletion of aggregates by pelleting. proteins that more prevalently co-precipitated with pathogenic huntingtin included fused in sarcoma (fus), glycine-trna ligase (gars), peroxiredoxin 6 (prdx6), phosphatidylethanolamine-binding protein 1 (pebp1/rkip), and histone subunit hist1h4a, all of which were significantly enriched by two-fold or greater. rna-seq analysis indicated that none of these proteins had altered expression levels, suggesting that the binding interactions are not due to changes in background abundance. overall we found that the conformational differences are subtle, yet are sufficient to generate several specific proteome interactions that offer clues to a toxic gain-of-function mechanism in pathology. work is ongoing to probe the more subtle changes in conformation and the importance of these interactors to mediating mechanisms of dysfunction. hereditary tyrosinemia type i is an autosomal recesive disorder caused by deficiency of fumarylacetoacetate hydrolase (fah) enzyme. deficiency of fah leads to cellular accumulation of toxic metabolites which include mainly, succinylacetone (sa), maleylacetoacetate (maa) and fumarylacetoacetate (faa) in many body tissues. fah is mainly expressed in hepatocytes and renal proximal tubular epithelium. therefore, liver and kidney are the two primary organs affected by this disorder, and development of hepatocellular carcinoma is the major symptom. missense mutations leads to a loss of enzymatic efficiency which, in a high number of mutations, correlates with loss of kinetic and thermodynamic stability of the enzyme. in our ongoing project, we are trying to elucidate the molecular basis of tyrosinemia by means of biophisical and structural characterization of fah wild type along with its mutations. this knowledge should help us design new therapies based on the identification of pharmacological chaperones that could restore the altered enzymatic stability of the enzyme. human fah wild type and 19 selected mutants were synthesized and inserted in an expression vector for e. coli. the proteins were purified in a fplc and, their thermodynamic and kinetic stability investigated using circular dichroism. our preliminary results confirm the loss of termodinamic stability of different mutants and its variability compared to wild type protein. repulsion between net charges of subunits during ferritin assembly daisuke sato 1 , hideaki ohtomo 1 , atsushi kurobe 1 , satsuki takebe 1 , yoshiteru yamada 2 , kazuo fujiwara 1 , masamichi ikeguchi 1 1 department of bioinformatics, graduate school of engineering, soka university, 2 jasri/spring-8 the organisms have a lot of spherical shell-shaped supermolecules consisting of identical or distinct subunits (e.g., ferritin, virus capsid, lumazine synthase and encapsulin). such multimeric proteins spontaneously assemble into their native structures from the subunits to acquire the specific functions. however, the assembly mechanism of such supermolecules has not been understood in detail. hence, to investigate the assembly mechanism is biologically important. escherichia coli non-heme ferritin (ftn) consists of 24 identical subunits, which are assembled into a spherical shell-shape with 4/3/2 symmetry. ftn is able to store iron inside cavity. the subunit includes a-d helices forming 4-helix bundle, a long bc-loop between b and c-helices and a short e-helix at the c-terminal. ftn dissociates into dimers at acidic ph. the dimer was shown to maintain the native-like secondary and tertiary structures by circular dichroism spectra and small angle x-ray scattering (saxs). the acid-dissociated ftn is able to reassemble into the native structure when ph increases. to clarify ftn assembly mechanism, we performed the stopped-flow time-resolved saxs (tr-saxs) experiments. the saxs profiles could be acquired every 15 ms after the initiation of reassembly. the initial velocity calculated from the forward scattering intensity increment was proportional to the square of the protein concentration, implying that the reaction is second-order. we propose the sequential bimolecular reaction, in which two dimers bind to form tetramer, then another dimer attaches to the tetramer to form a hexamer, and so on. the assembly rate depended on ph and ion strength, indicating that the electrostatic interaction plays an important role in the assembly reaction. the assembly rate decreased with increasing ph in the range from 6.0 to 8.0 and increased with increasing nacl concentration. this indicates that there are repulsive electrostatic interactions between assembly units and that they increases with increasing ph from 6.0 to 8.0. a possible interaction is the repulsion between net charges of dimers since pi of ftn is expected to be 4.6. to test this possibility, we made several mutants with different net charges. as mutational sites, we selected charged residues that are far from the subunit interface. selected sites were glu5, glu8, glu12, glu85 and glu89. we constructed the mutants with one, two, three or four glu -> gln substitutions of selected sites. the structures of those mutants were similar to that of wild-type ftn. if aforementioned hypothesis is correct, the assembly rate is expected to increase with increasing the number of substitution. the result agreed well with this expectation and strongly suggested that the electrostatic repulsion between dimers is an important factor determining the assembly rate of ftn. improved modeling of protein unfolding rates and pathways through solvation and modeling of beta-barrels benjamin walcott 1,2 , lu ıs garreta 3 , christopher bystroff 1,2,4 1 department of biology, rensselaer polytechnic institute, 2 center for biotechnology and interdisciplinary studies, 3 department of computer science, universdad del valle, 4 department of computer science, rensselaer polytechnic intitute an understanding of the folding and unfolding pathways of proteins is integral to improving our ability to associate the structural impact of point mutations and disease etiology. information gained here can also be used for protein structure prediction and design. to model unfolding pathways in proteins we utilize a computational method called geofold. this approach uses recursive hierarchical partitioning of protein structure and finite elements simulation. geofold considers three types of partitioning operations: translational motion (break), single point revolute joints (pivot), and rotation around two points (hinge). from these operations, a directed acyclic graph (dag) is constructed where nodes correspond to the substructures created by these operations and the edges represent the operations. for each operation in the dag, its dissociation and reassociation rates are determined as a function of solventaccessible surface area, hydrogen bonds, voids, and conformational entropy. finite element simulations are carried out to simulate the kinetics of unfolding. this model accurately predicts changes in unfolding pathways due to disulfides in a four-protein case-study, but it fails to produce a realistic pathway for b-barrel proteins such as green fluorescent protein (gfp). to better model these barrel proteins, a new partitioning operation is introduced involving the breaking of all contacts between an adjacent set of b-strands, called a seam. in addition, to improve the accuracy of kinetic modeling, several updates have been made to the energy function, including an improved solvation model and a contact-orderbased estimation of the reassociation rates. the predicted unfolding rates and pathways using this improved geofold are compared with experimentally measured values in kineticdb for proteins with multi-state unfolding kinetics, point mutations, circular permutations, and engineered disulfides. the presence of multiple domains in a protein can result in the formation of partially folded intermediates, leading to increased aggregation propensity. this can be reduced by cooperative, all-or-nothing folding of the multi-domain protein. in good agreement with ensemble folding experiments, a coarsegrained structure-based model of e. coli adenylate kinase (ake) folds cooperatively. ake has three domains, nmp, lid and core. we examine the role of the interfaces between these domains in facilitating folding cooperativity in ake. mutants in which these interfaces are deleted exhibit similar folding cooperativities as wild-type ake. on closer inspection, we observe that unlike a typical multi-domain protein in which one domain is singly-linked to its adjacent domain, nmp and lid are inserted into core, i.e. they are both connected to core by two linkers each. we create circular permutants of ake in which the inserted domains are converted to singly-linked domains, and find that they fold less cooperatively than wild-type ake. domain insertion in wild-type ake facilitates folding cooperativity even when the inserted domains have lower stabilities. the n-and c-termini of nmp and lid are constrained upon the folding of core and this facilitates their folding. thus, nmp and lid which undergo large conformational changes during catalysis can be smaller with fewer stabilizing interactions. in addition, inter-domain interactions need not be optimized for folding, and can be tuned for substrate binding, conformational transition and catalysis. analysis of protein domains using structural bioinformatics suggests several examples of multi-domain proteins in which domain insertion is likely to facilitate folding cooperativity. tuning cooperativity on the free energy landscape of protein folding pooja malhotra 1 , jayant udgaonkar 1 1 national centre for biological sciences, tata institute of fundamental research the mechanism by which a protein explores the free energy landscape during a folding or unfolding reaction is poorly understood. determining whether these reactions are slowed down by a continuum of small ( kbt) free energy barriers or by a few large (> 3 kbt) free energy barriers is a major challenge. in this study the free energy landscape accessible to a small protein monellin is characterized under native-like conditions using hydrogen exchange in conjunction with mass spectrometry. cooperative and noncooperative opening processes could be directly distinguished from the mass distributions obtained in the ex1 limit. under native conditions, where the native state is maximally stable, the unfolded state is transiently sampled in an entirely non-cooperative and gradual manner. under conditions which stabilize the unfolded state or destabilize the native state of the protein, the slowest structure opening event becomes cooperative. the present study provides an understanding of the relationship between stability and folding cooperativity. it suggests that the cooperative transitions observed in unfolding reactions maybe a consequence of the changes in the stabilities of the unfolded state and the transition state. it also provides rare experimental evidence for a gradual unfolding transition on a very slow timescale. role of electrostatic repulsion between unique arginine residues on the assembly of a trimeric autotransporter translocator domain eriko aoki 1 , kazuo fujiwara 1 , masamichi ikeguchi 1 1 haemophilus influenzae adhesin (hia) belongs to the trimeric autotransporter family. the autotransporter consists of an n-terminal signal peptide, an internal passenger domain and a c-terminal translocator domain. the signal peptide directs to export across the inner membrane via the sec system and is cleaved, the passenger domain is a virulence factor, and the translocator domain (hiat) is embedded in the outer membrane. the crystal structure of hia translocator domain (hiat) has shown that hiat forms a transmembrane b-barrel of 12 b-strands, four of which are provided from each subunit. the b-barrel has a pore that is traversed by three a-helices, one of which is provided from each subunit. the protein has a unique arginine residue at 1077. arg1077 side chains from three subunits protrude from the b-strand toward the center of the barrel and are close to each other. these residues seem to have an unfavorable electrostatic effect on the assembly and decrease the trimer stability. to investigate the role of this residue on the trimer assembly and stability of hiat, we replaced this arginine with the neutral amino acid, methionine (r1077m) or the positively charged residue, lysine (r1077k), and properties of these mutants were investigated. hiat and two mutants were dissociated by formic-acid treatment, and they were able to reassemble in the presence of the detergent. to measure the time course of trimer reassembly, amounts of reassembled trimer and monomer were quantified by sds-page at different assembly times. although the neutralized mutation increased the rate of reassembly, the final amount of reassembled trimer decreased, especially at higher protein concentration. these suggest that the neutralized mutation cause the incorrect oligomer formation. the far-uv cd spectrum of reassembled wt hiat was nearly identical with that of the native wt hiat. however, the spectrum of the reassembled r1077m mutant was more intense that of the native r1077m mutant, although the proportion of trimer was much lower than that of the wt hiat. this suggests that the incorrect oligomer has a secondary structure different from the wt hiat. r1077k mutant showed assembly properties similar to those of the wt hiat. therefore, the repulsion between positively charged residues seems to be important for preventing hiat from misassembly. similar proximity of arginine residues is observed for hiv capsid protein, carboxysome shell protein, lumazine synthase and so on. the electrostatic repulsion between arginine residues may be a general mechanism for protein assembly. department of veterinary pathobiology, kagoshima university, 2 institute for food sciences, hirosaki university, 3 faculty of fisheries, kagoshima university, 4 department of veterinary histopathology, kagoshima university, 5 veterinary clinical training center, kagoshima university, 6 department of veterinary anatomy, kagoshima university, 7 sakamoto kurozu inc., 8 the united graduate school of agricultural sciences, kagoshima university kurozu is a traditional japanese rice vinegar. during fermentation and aging of the kurozu liquid in an earthenware jar over 1 year, solid residue called kurozu moromi is produced. in the present study, we evaluated whether concentrated kurozu or kurozu moromi could ameliorate cognitive dysfunction in the senescence accelerated p8 mouse. senescence accelerated p8 mice were fed 0.25% (w/w) concentrated kurozu or 0.5% (w/w) kurozu moromi for 4 or 25 weeks. kurozu suppressed cognitive dysfunction and amyloid accumulation in the brain, while kurozu moromi showed a tendency to ameliorate cognitive dysfunction, but the effect was not significant. we hypothesize the effect is caused by the antioxidant effect of concentrated kurozu, however, the level of lipid peroxidation in the brain did not differ in senescence accelerated p8 mice. dna microarray analysis indicated that concentrated kurozu increased hspa1a mrna expression, a protein that prevents protein misfolding and aggregation. the increase in hspa1a expression by kurozu was confirmed using quantitative real-time pcr and immunoblotting methods. therefore, the suppression of amyloid accumulation by concentrated kurozu may be associated with hspa1a induction. however, concentrated kurozu could not increase hspa1a expression in mouse primary neurons, suggesting it may not directly affect neurons. young-ho lee 1 although amyloid fibrils are associated with a number of pathologies, their conformational stability remains largely unclear. we herein investigated the thermal stability of various amyloid fibrils. a-synuclein fibrils, freshly prepared at 37 c at neutral ph, cold-denatured to monomers at 0-20 c and heat-denatured at 60-110 c. meanwhile, the fibrils of b2-microglobulin, alzheimer's ab1-40/ab1-42 peptides, and insulin exhibited only heat denaturation, although they showed a decrease in conformational stability at low temperature in the presence of chemical denaturants. a comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in the fibril cores contributed to the cold denaturation of a-synuclein fibrils. reinforced electrostatic repulsion at low temperatures may promote cold denaturation, leading to a unique thermodynamic property of amyloid fibrils. we propose that although cold-denaturation is common to both native proteins and misfolded fibrillar states, the main-chain dominated amyloid structures may explain amyloid-specific cold denaturation due to the unfavorable burial of charged side-chains in fibril cores. key structural differences between tbtim and tctim revealed by thermal unfolding molecular dynamics simulations angel piñeiro 1 , miguel costas 2 , andrea guti errez-quezada 2 1 dept of applied physics, university of santiago de compostela, 2 lab. of biophys. chem., dept of physical chemistry, fac. of chemistry, unam the thermal unfolding pattern obtained by differential scanning calorimetry for trypanosoma cruzi and trypanosoma brucei triosephosphate isomerase (tcim and tctim) are significantly different although the crystal structure of both proteins is almost indistinguishable and the sequences are highly homogolous. in order to explain these differences at molecular level a set of molecular dynamics simulations were performed at different temperatures between 400 and 700 k. the obtained trajectories were analyzed in detail and the residues that showed to be key in the unfolding pathway of each species were identified. a set of residues that behave significantly different between both proteins were selected and proposed for mutations. the general aim is to identify the minimum amount of residue mutations that allow providing tbtim with the behaviour of tctim and vice versa. experimental complementary work is also being performed on the same protein. repositioning som0226 as a potent inhibitor of transthyretin amyloidogenesis and its associated cellular toxicity salvador ventura 1 , ricardo sant'anna 1 , maria ros ario almeida 2 , nat alia reixach 3 , raul insa 4 , adrian velazquez-campoy 5 , david reverter 1 , n uria reig 4 1 universitat aut onoma de barcelona, 2 instituto de biologia molecular e celular, icbas, 3 the scripps research institute, 4 som-biotech, 5 universidad de zaragoza transthyretin (ttr) is a plasma homotetrameric protein implicated in fatal amyloidosis. ttr tetramer dissociation precedes pathological ttr aggregation. despite ttr stabilizers are promising drugs to treat ttr amyloidoses, none of them is approved by the food and drug administration (fda). repositioning existing drugs for new indications is becoming increasingly important in drug development. here, we repurposed som0226, an fda-approved molecule for neurodegenerative diseases, as a very potent ttr aggregation inhibitor. som0226 binds specifically to ttr in human plasma, stabilizes the tetramer in vivo and inhibits ttr cytotoxicity. in contrast to most ttr stabilizers, it exhibits high affinity for both ttr thyroxine -binding sites. the crystal structure of som0226-bound ttr explains why this molecule is a better amyloid inhibitor than tafamidis, so far the only drug in the market to treat the ttr amyloidoses. overall, som0226, already in clinical trials, is a strong candidate for therapeutic intervention in these diseases. neurometals as modulators of protein aggregation in neurodegenerative diseases s onia s. leal 1 , joana s. crist ovão 1 , cl audio m. gomes 1 1 protein misfolding and aggregation is a hallmark across neurodegenerative diseases such as alzheimer's disease and amyotrophic lateral sclerosis (als). since these diseases are mostly sporadic, the formation of protein amyloids in the nervous system depends of chemical and biological triggers within the neuronal environment, such as metal ions [1] . in this communication i will overview the metallobiology of neuronal calcium, zinc and copper, which are key players in brain function and have altered homeostasis in most neurodegenerative conditions. our recent work will illustrate how this allows establishing molecular mechanisms in neurodegenerative diseases [2] [3] [4] [5] [6] . in the pursuit of this goal, in the last years we have been investigating superoxide dismutase 1 (sod1), a cu/zn metalloenzyme that aggregates in the fatal neurodegenerative disorder als, as a model. in sod1-als cases, this ubiquitous protein selectively aggregates in motor neurons, implicating a local biochemical factor in the process: interestingly, zn21 and ca21 levels are upregulated in the spinal and brain stem motor neurons of als patients, and increased ca21 triggers multiple pathophysiological processes which include direct effects on the sod1 aggregation cascade [2, 3] . recently we established that calcium ions promote sod1 aggregation into non-fibrillar amyloid, suggesting a link to toxic effects of calcium overload in als [4] . we showed that under physiological conditions, ca21 induces conformational changes on sod1 that increase sod1 b-sheet content and decrease sod1 critical concentration and nucleation time during aggregation kinetics. we also observed that calcium diverts sod1 aggregation from fibrils towards amorphous aggregates. interestingly, the same heterogeneity of conformations is found in als-derived protein inclusions. we thus hypothesized that transient variations and dysregulation of cellular ca21 and zn21 levels contribute to the formation of sod1 aggregates in als patients [4, 5] . in a follow up study we combined experimental and computational approaches to show that the most frequent ligands for ca21 are negatively-charged gatekeeper residues located in boundary positions with respect to segments highly prone to edge-to-edge aggregation. calcium interactions thus diminish gatekeeping roles by shielding repulsive interactions via stacking between aggregating b-sheets, partly blocking fibril formation and promoting amyloidogenic oligomers such as those found in als inclusions. interestingly, many fals mutations occur at these positions, disclosing how ca21 interactions recreate effects similar to those of genetic defects, a finding with relevance to understand sporadic als pathomechanisms [6] . the amino acid proline is well-known by its disorder promoting and helix breaking properties. prolines can be accommodated within transmembrane (tm) alpha-helices and participate in important biological tasks like signal transduction, ligand binding and helix-helix packing. x-ray crystallography and nmr indicate that proline residues in membrane proteins induce distortions of the helix geometry to different extents ranging from small bends to severe kinks. however, such studies provide essentially a static snapshot of membrane-embedded helices. therefore, the link between proline dynamics and function is not completely understood. in this work we have used singlemolecule f€ orster resonance energy transfer (smfret) and fluorescence correlation spectroscopy (fcs) to probe the structure and dynamics of the tm domain of human glycophorin a (gpa), a widely used model membrane protein for oligomerization studies. a fluorescent dye pair has been attached to both ends of the membrane-spanning region of gpa, which allowed monitoring the average distance and distance fluctuations between the attachment points. site-specifically double-labeled gpa has been reconstituted into two membrane-mimetic systems: sds micelles and phospholipid bilayers assembled into nanodiscs. using proline-scanning mutagenesis we have systematically evaluated the impact of proline residues in different positions along the membrane normal on transmembrane helix length and lateral packing. furthermore, we have investigated the distance distribution in tm helices containing native prolines, namely the insulin receptor and the nesprin protein. our results shed light into the relation between proline dynamics and the folding and function of tm helices. thermodynamic contributions of specific mutations of l30e protein in the rna: protein interface region measured by analytical ultracentrifugation and gel shift assay bashkim kokona 1,2 , sara kim 1 , margaret patchin 1 , britt benner 1 , susan white 1 1 in saccharomyces cerevisiae, ribosomal protein l30e acts as an autoregulator by inhibiting the splicing of its pre-mrna and translation of its mrna. the l30e protein-rna binding site has been previously studied, revealing a rna kink-turn motif, which is characterized by a sharp bend in the phosphodiester backbone due to unpaired nucleotides and internal tertiary interactions. l30e structural flexibility at the rna-binding interface makes such interaction an excellent model to explore the energetics of rna protein binding. we made l30e k28a, f85a, and f85w mutants to quantify the thermodynamic contributions of such interactions to the protein-rna complex. we used analytical ultracentrifugation sedimentation equilibrium (se) and sedimentation velocity (sv) to investigate conformational changes and protein-rna binding free energy changes due to mutations. our computed changes of binding free energy based on the sedimentation equilibrium experiments were consistent with the gel shift assay results. in addition, sedimentation velocity experiments on the l30e wild type indicate that protein-rna interaction is highly dynamic and involves conformational changes of the kink-turn rna induced by l30e protein. our results provide new insights on understanding the binding between ribosomal proteins and their rna molecules counterpart, which can be used to complement the x-ray structure. role of a non-native a-helix in the folding of equine b-lactoglobulin takahiro okabe 1 , toshiaki miyajima 1 , kanako nakagawa 1 , seiichi tsukamoto 1 , kazuo fujiwara 1 , masamichi ikeguchi 1 1 equine b-lactoglobulin is a small globular protein (162 residues). although elg adopts a predominantly b-sheet structure consisting of nine anti-parallel b-strands (a-i) and one major a-helix in the native state, it has been shown that a non-native a-helical intermediate accumulates during the burstphase of folding reaction from the unfolded state in the concentrated denaturant. to ask whether the non-native helix formation is important for acquiring the native b-sheet structure, we determined first where the non-native a-helix is formed. a stable analogue of the burst-phase folding intermediate was observed at acid ph (a state). the amide hydrogen exchange experiment and proline-scanning mutagenesis experiment have shown that the non-native a-helix is formed at the region corresponding to the h strand in the a state. to investigate the role of this non-native a-helix on refolding reaction of elg, we constructed several mutant proteins, which were designed to destabilize the nonnative a-helix in the folding intermediate without perturbation on the native structure. a mutant, a123t, fulfilled this requirement, that is, a123t showed a native structure similar to that of the wildtype protein, and largely reduced cd intensity in the a state. then, the refolding kinetics were investigated by the cd and fluorescence stopped-flow method. a123t mutation resulted in reduction of the burst-phase cd intensity, which confirmed that the non-native a-helix is formed around the h strand region. subsequent to the burst-phase, four kinetic phases were observed for a123t and the wildtype protein. importantly, the folding rate constants of the four kinetic phases were similar between both proteins. furthermore, interrupted refolding experiments demonstrated that the native state was formed in the two parallel pathways in the two slower phases of the four kinetic phases. the relative amplitudes of the two pathways were similar between a123t and the wild-type protein. these results clearly showed that the formation of the non-native helix has little effect on the folding rates and pathways, and suggested that the non-native helix formation may not be a severe kinetic trap for protein folding reaction. impact of the chaperonin cct in a-synuclein(a53t) amyloid fibrils assembly ahudrey leal_quintero 1 , javier martinez-sabando 1 , jose mar ıa valpuesta 1 , begoña sot 1 1 centro nacional de biotecnolog ıa (cnb/csic)., 2 centro nacional de biotecnolog ıa (cnb/csic)., 3 centro nacional de biotecnolog ıa (cnb/csic)., 4 centro nacional de biotecnolog ıa (cnb/csic) and fundaci on imdea-nanociencia cct is a eukaryotic chaperonin that uses atp hydrolysis to encapsulate and fold nascent protein chains. moreover, it has recently been shown that cct is able to inhibit amyloid fibers assembly and toxicity of the polyq extended mutant of huntingtin, the protein responsible of huntington disease. although this opens the possibility of cct being also able to modulate other amyloidopathies, this has not addressed yet. the work presented here intends to determine the effect of cct in the amyloid fibers assembly of a-synuclein(a53t), one of the mutants responsible of parkinson disease. it is demonstrated that cct is able to inhibit a-synuclein(a53t) fibrillation in a nucleotide independent way, suggesting that this effect is based on binding rather than on active folding. furthermore, using deletion mutants and assaying the interaction of cct with monomers, soluble oligomers and fibres, it has been possible to unravel the mechanism of this inhibition: cct interferes with fibers assembly by interacting with a-synuclein(a53t) nac domain once soluble oligomers are formed, thus blocking the reaction before the fibers start to grow. amyloid-like aggregation of nucleophosmin regions associated with acute myeloid leukemia mutations daniela marasco 1 , concetta di natale 1 , valentina punzo 1 , domenico riccardi 1 , pasqualina scognamiglio 1 , roberta cascella 2 , cristina cecchi 2 , fabrizio chiti 2 , marilisa leone 3 , luigi vitagliano 3 1 department of pharmacy, cirpeb: centro interuniversitario di ricerca sui pepti, 2 section of biochemistry, department of biomedical experimental and clinical scie, 3 institute of biostructures and bioimaging nucleophosmin (npm1) is a multifunctional protein involved in a variety of biological processes and implicated in the pathogenesis of several human malignancies. npm1 has been identified as the most frequently mutated gene in acute myeloid leukemia (aml) patients, accounting for approximately 30% of cases (1). the most frequent human npm1 mutations lead to variants with altered c-terminal sequences of the c-terminal domain (ctd) that, in its wild form, folds as a three helix bundle. aml modifications lead to (a) an unfolding of the ctd in the mutated protein and (b) its accumulation in the cytoplasm due to the loss of nuclear localization sequences with mutations of trp290 (mut e) and also of trp288 (mut a) (2) . to gain insights into the role of isolated fragments in npm1 activities we dissected the ctd in its helical fragments. here we describe the unexpected structural behavior of the fragments corresponding to the helices h2 and h3 in both wild-type and aml-mutated variants. h2 region shows a remarkable tendency to form amyloid-like assemblies while only the muta sequence of h3 region is endowed with and b-sheet structure, under physiological conditions, as shown by circular dichroism, thioflavin t and dynamic light scattering. the aggregates of h2, are also toxic to neuroblastoma cells, as determined by using the mtt reduction and ca21 influx assays (3) . furthermore the effects of the local context on the different tendencies to aggregate of h2 and h3 were investigated and appeared to influence for the aggregation propensity of the entire ctd. since in aml mutants the ctd is not properly folded, we hypothesize that the aggregation propensity of npm1 regions may be implicated in aml etiology. these findings have implications to elucidate the pathogenesis of aml caused by npm1 mutants and aggregation phenomena should be seriously considered in studies aimed at unveiling the molecular mechanisms of this pathology. we report a resume of our study regarding the effects of microwaves in the range 900-1800 mhz on a typical protein, myglobin. previous literature have concerned the effects on living and in vitro organic systems induced by high frequencies electromagnetic fields. we have focused our attention on a typical protein, myoglobin, because proteins are the simplest organic systems that are fundamentals in organic functions of livings. myoglobin is a protein found mainly in muscle tissue of vertebrates, consisting of a single protein chain with 153 amino acids and one heme group that stores oxygen in the muscle cells. the physiological importance of myoglobin is mainly related to its ability to bind molecular oxygen. in particular, we focused our attention on the secondary structure of this protein in order to highlight whether exposure to microwaves unfold the protein producing transitions from a-helix component to b-sheet features. to this aim fourier transform infrared (ftir) spectroscopy have been used. the importance of this study is related to previous literature which indicated that transition from a-helix to b-sheet structure in a protein can be responsible for aggregation mechanisms that can lead to neurotoxicity and neurodegenerative disorders that can be considered as the first step to some pathologies [1] [2] [3] . the aggregates consist of fibers containing unfolded proteins with a prevalent b-sheet structure termed amyloid [4] . in our studies myoglobin in deuterium oxide (d2o) solution was exposed for 3 h to mobile phone microwaves at 900 and 1800 mhz at a power density of 1 w/m2. ftir spectra were recorded by a spectrometer vertex 80v from bruker optics, following the protocol accurately described in [5] [6] [7] . ftir spectroscopy analysis evidenced an increase in intensity of b-sheet structures and a significant shift to lower frequencies of about 2.5 cm-1 of the amide i vibration after exposure [8, 9] . these results led to conclude that mobile phone microwaves induce proteins unfolding and formation of aggregates [10, 11] . membrane proteins play a vital role in many biological processes, and yet remain poorly understood as they are frequently unstable in vitro. the goal of this project is to investigate the insertion and folding of membrane proteins into lipid bilayers, using a cell free expression system. we have used both e.colibased cell extracts (s30), and commercial translation systems (purexpress) in combination with synthetic liposomes of defined lipid composition. these studies will aid understanding of cooperative folding, folding intermediates, and the effects of the lipid bilayer on folding and insertion. model e.coli proteins have been investigated, as they can offer important insights into other proteins, and thus facilitate the further study of more biologically relevant proteins. it has been found that the rhomboid protease glpg spontaneously inserts into liposomes without the aid of an insertase such as secyeg. this spontaneously inserted glpg is functional, and is able to cleave bodipy-labeled casein, yielding a fluorescent product. the major facilitator superfamily (mfs) transport proteins lacy, galp and glpt have also been found to insert spontaneously into liposomes. it has been shown that the lipid composition of the liposomes has an effect on the amount of protein inserted into the bilayer, with all proteins tested to date preferring liposomes containing at least 50 mol% dopg. ongoing and future work will involve the use of rare codons to alter the rate of translation, to investigate the effect this has on the final folded structure of the protein. preliminary work is also currently being done into whether the two domains of the mfs family transporters fold cooperatively or independently, thus aiding understanding into the folding and stability of membrane transport proteins. frederic greco 1 , audrey toinon 1 , nadege moreno 1 , marie claire nicola€ ı 1 rabies remains an important worldwide health problem that causes a fatal encephalomyelitis [1] . currently, rabies in humans is under control in europe and north america following the use of efficient vaccines for dogs and wild animals. however, it still kills more than 55,000 people every year mainly in africa and asia [2] . human vaccination prevents infection with very high efficacy. the vaccine contains an inactivated rabv produced on vero cells. rabv is an enveloped, negative single stranded rna virus which encodes five proteins, namely the nucleoprotein (n), the phosphoprotein (p), the matrix protein (m), the glycoprotein (g), and the viral rna polymerase (l) [3] . the viral envelope is covered by trimer spikes of g-glycoprotein which is the most significant surface antigen for generating virus-neutralizing antibodies. here we illustrate the use of dsc (differential scanning calorimetry) to identify structural domains or proteins involved in thermal transitions. the dsc thermogram for intact beta-propiolactone inactivated rabv samples in pbs buffer reveals two major thermal transitions with a tm respectively at 618c and 718c. we have initially focused our investigations on one of the major proteins encode in rabv, glycoprotein g [4] . glycoprotein g contains disulfide bridges on the ectodomain [6] , is sensitive to bromelain cleavage [5] and shows reversible conformation changes at low ph [7] . considering these characteristics, our results provide evidence on the identity of one thermal transition observed by dsc. keywords: rabies virus, differential scanning calorimetry, protein unfolding domain swapping of the dna-binding domain of human foxp1 is facilitated by its low folding stability exequiel medina, sandro l. valenzuela, crist obal c ordova, c esar a. ram ırez-sarmiento and jorge babul departamento de biolog ıa, facultad de ciencias, universidad de chile, santiago, chile protein folding and dimerization (or oligomerization) are biologically relevant processes when reaching the quaternary structure is required for function. proteins that form dimers by exchanging segments or domains of their tertiary structure with another subunit, the so-called domain swapping phenomenon, are examples where folding and dimerization are tightly concerted processes. previous studies on domain swapping proteins, such as p13suc1 and diphtheria toxin, have shown that, in general, a high kinetic barrier separates monomers and domain swapped dimers, and that this barrier can be lowered by promoting protein unfolding and refolding at high protein concentrations, thus favoring the swapped oligomer. recent crystal structures of the dna-binding domain of several human forkhead box (fox) proteins have shown that the p subfamily of these transcription factors (foxp) can form swapped dimers. the human foxp proteins are interesting models of domain swapping, because mutations of the dna-binding domain of these proteins are linked to diverse inherited disorders in humans, such as ipex and language deficits, and some of these mutations are located in the hinge region that connects the exchanged segment with the rest of the protein. moreover, foxp1 and foxp2 have been described to reach monomer-dimer equilibrium in solution after hours of incubation, suggesting that a low kinetic barrier separates both species. using foxp1 as a model of domain swapping, we analyzed the temperature and protein concentration effects on the dimer dissociation, obtaining the free energy change and enthalpy of the process by van't hoff analysis (dh8 of 23.1 kcal•mol-1, ds8 of 0.082 kcal•mol-1•k-1 and dg8 at 258c of 20.95 kcal•mol-1). these results indicate that the monomer-monomer association is an example of an enthalpy-driven process. to understand how foxp1 domains swap without protein unfolding, we performed equilibrium unfolding experiments using gndhcl as denaturant, showing that the wild-type protein has a low stability (dgu 5 6 kcal•mol-1, cm 53.5 m at 258c), in contrast to other domain swapping proteins with high kinetic barriers. we further explore the domain swapping mechanism of foxp1 through biased targeted molecular dynamics simulations, showing that the exchange process can occur by specific local destabilization and unfolding of the hinge region and helix h3. to further corroborate that the low stability of wild-type foxp1 facilitates its domain swapping, we engineered a monomeric version of foxp1 through a single-point mutation in the hinge region, which has been previously described in the literature, and used this protein to visualize the effect of monomer stability in the dimer formation. comparison of the folding stability of the monomeric mutant a39p and wild-type foxp1 shows that ddgu (mutant-wild-type) is 2.5 kcal/mol, concluding that the ability of foxp1 to domain swap rapidly can be explained through its low monomer stability and local unfolding of the exchange region. funding: fondecyt 1130510 and 11140601. determining the coupled interactions that stabilize the structural framework of the ß-propeller fold loretta au 1 , david green 2,3,4 1 department of statistics, the university of chicago, 2 department of applied mathematics and statistics, stony brook university, 3 graduate program in biochemistry and structural biology, stony brook university, 4 laufer center of physical and quantitative biology, stony brook university b-propeller proteins are a highly evolved family of repeat proteins that are involved in several biological pathways, such as signal transduction, cell-cycle modulation and transcription regulation, through interactions with diverse binding partners, despite having a similar fold. as for all repeat protein families, there is a consistent pattern in secondary structure for each repetitive region, in addition to the entire family. typically, four to ten propeller blades (each containing four anti-parallel b-sheets) are arranged in a toroidal shape, thus providing a large binding surface for ligands or other proteins. about 1% of known proteins adopt this distinctive fold, and although the requirements for tertiary structure and protein function are fundamentally encoded in primary structure, this relationship is not fully understood, and addressing it could provide insight on why the b-propeller fold is common. many techniques in comparative sequence analysis can successfully identify amino-acid conservation between closely related proteins, but molecular interactions between amino acids are often neglected, and further experimentation is still needed to determine the reasons underlying conservation. to explore how primary structure can dictate fold and function, we devised a computational approach to perform large-scale mutagenesis, by adapting the dead-end elimination and a* search algorithms (dee/a*), and also leveraged the structural conservation of each repeating region to understand how sequence variation influences protein fitness, defined here as a combination of stabilizing and binding interactions. dee/a* can evaluate low-energy protein sequences and their corresponding three-dimensional structures, and we used the bsubunit of a g-protein heterotrimer (pdb: 1gp2, gia1b1g2) as a model system to demonstrate: (1) how the multiple roles of individual amino acids in protein fitness can be deconvolved, and (2) how epistatic interactions between them can contribute to structural stability. in doing so, we were able to identify important patterns in sequence complementarity between repeating regions that cannot be found using sequencebased methods alone. these results suggest that computational approaches can be used to determine important protein interactions, and help elucidate the prevalence of b-propeller proteins in biology. temperature induced conformational changes of the villin headpiece miniprotein stanislaw oldziej 1 , wioletta _ zmudzi nska 1 , anna hałabis 1 1 the c-terminal subdomain of the actin-binding protein villin called hp35 (villin headpiece) has been used as a model protein in a number of studies of protein folding kinetics and protein folding mechanism [1, 2] . the hp35 is a 35 residue miniprotein with an alpha-helix bundle three-dimensional fold. the goal of our work was to determine conformational ensemble of polypeptide chain of the investigated miniprotein at a wide range of temperatures to get detailed information about how protein structure is influenced by temperature. 2d nmr spectra of the title miniprotein were registered at 278, 293 and 313 k. the three-dimensional structure of the hp35 based on restraints derived from nmr spectra registered at 278 k is almost identical with structure deposited in the pdb database in the record 2f4k [2] . at higher temperatures (293 and 313 k) the general shape of the protein remains unchanged, with well packed hydrophobic core. however, with temperature increase alpha-helices start to melt. at 313 k structure of the protein remains compact and in general shape similar to structure observed at 278k, but none of the alpha-helices could be observed. results obtained for hp35 protein are in agreement with previous observation for the trp-cage miniprotein [3] , that with temperature increase regular secondary structure elements melt first before the break-up of the hydrophobic core of the protein. biological membranes provide a selective and chemically sealed barrier for cells. transport of ions and small molecules across the membrane is mediated by transporter proteins and the breakdown of a cell's ability to produce functionally folded membrane transport proteins can lead to dysfunction and has been implicated in many diseases1. however little is known about the processes that govern the misfolding of a-helical integral membrane proteins, taking into account that these proteins fold and maintain functional structures within membranes of various organelles. the neurotransmitter sodium symporter (nss) protein family is an example of a-helical transporter proteins. the nss family encompasses a wide range of prokaryotic and eukaryotic ion-coupled transporters that regulate the transport of neurotransmitter molecules whose dysfunction has been implicated in multiple diseases and disor-ders2. we have investigated the folding processes of prokaryotic homologue of the nss family leut responsible for the transport of neurotransmitters and amino acids to the sodium electrochemical gradient. previously folding processes of membrane transporters have mainly been characterised within detergent micelles. however, detergent micelles are not an accurate depiction of the environment of the membrane bilayer, with this in mind we have also attempted to investigate folding processes within a bilayer pd-051 nmr investigation of ph-induced unfolding of b domain of an escherichia coli mannitol transporter ii mannitol in the bacterial phosphotransferase system kim gowoon 1 , yu taekyung 1 , suh jeongyong 1 1 the bacterial phosphotransferase system (pts) mediates sugar phosphorylation and translocation across the cytoplasmic membrane. cytoplasmic b domain (iib mtl) of the mannitol transporter enzyme ii mannitol, a pts family protein, delivers a phosphoryl group from a domain to an incoming mannitol that is translocated across the membrane. iib mtl is comprised of a four-stranded ß-sheet and three helices, representing a characteristic rossmann fold. we found that the iib mtl of escherichia coli unfolded at a mildly acidic condition. we made iib mtl mutants to investigate the mechanism of the ph-induced unfolding using nmr spectroscopy. we monitored backbone amide groups and side chain imidazole groups of histidine residues using 2d hsqc nmr, and pointed out a potential histidine residue that might be responsible for the unfolding. histidine residues may be generally important to the folding stability in response to environmental ph changes. can site-directed mutagenesis shed light on the refolding pattern of human glucose 6-phosphate dehydrogenase (g6pd)? nurriza ab latif 1,2 , paul engel 1 1 conway institute, univerversity college dublin, 2 faculty of biosciences and medical engineering, universiti teknologi malaysia human glucose 6-phosphate dehydrogenase (g6pd) is the first enzyme involved in the pentose phosphate pathway (ppp). this oligomeric enzyme catalyses the reaction of glucose 6-phosphate to form 6phosphogluconolactone with concomitant reduction of nadp1 to nadph. in erythrocytes nadph is important mainly for protection against oxidative stress. in connection with its role as the sole source of nadph, g6pd deficiency commonly causes haemolytic disease and is known as the most common human enzyme deficiency globally. protein folding problems and instability are believed to be the major defects in the deficient enzymes. in this study, we employed site directed mutagenesis with hope to give more information on the role of -sh groups in the refolding of human g6pd. two mutants were created: 1) one in which all 8 cys residues were replaced by ser and 2) one in which only c13 and c446 were retained. the refolding of recombinant human g6pd has been studied primarily by measuring the enzyme activity after refolding. we also used a combination of intrinsic protein fluorescence, ans (8-anilino-1-naphthalenesulphonic acid) binding and limited proteolysis to look at the conformational change during the refolding. the results showed that gdnhcl-denatured recombinant human g6pd wild type could be refolded and reactivated by rapid dilution technique. even though, as recombinants in e. coli, the mutants were well expressed and active, they remained inactive after attempts were made to refold them in vitro. the methods we applied may have provided some insights on the refolding pattern of this oligomeric protein, albeit qualitatively rather than quantitatively. a single aromatic core mutation converts a designed 'primitive' protein from halophile to mesophile folding connie tenorio 1 , liam longo 1 , ozan s. kumru 2 , c. russell middaugh 2 , michael blaber 1 1 department of biomedical sciences, florida state university, 2 department of pharmaceutical chemistry, university of kansas experiments in prebiotic protein design suggest that the origin of folded proteins may have favored halophile conditions. these results are consistent with salt induced peptide formation which shows that polymerization of amino acids is also promoted by high salt concentrations. as a result of various origin of life studies, a consensus on which amino acids likely populated early earth has emerged. these residues were synthesized by abiotic chemical and physical processes from molecules present in the surrounding environment. the properties of the consensus set of common prebiotic amino acids (a,d,e,g,i,l,p,s,t,v) are compatible with known features of halophile proteins, meaning these proteins are only stable in the presence of high salt concentrations. the halophile environment, thus, has a number of compelling aspects with regard to the origin of structured polypeptides. consequently, a proposed key step in evolution was, movement out of the halophile regime into a mesophile one commensurate with biosynthesis of "phase 2" amino acids -including the aromatic and basic amino acids. we tested the effects of aromatic residue addition to the core of a "primitive" designed protein enriched for the prebiotic amino acids (a, d, e, g, i, l, p, s, t, v) that required halophilic conditions for folding. the subsequent results show that the inclusion of just a single aromatic residue was sufficient for movement to a mesophile folding environment. thus, the inclusion of aromatic residues into the codon table could have conferred key stability to early proteins enabling adaptive radiation outside of a halophile environment. contact prediction methods that rely on sequence information alone, such as evfold, can be used for de novo 3d structure prediction and identification of functionally important residues in proteins. large multiple sequence alignments of protein families consisting of evolutionarily related and plausibly isostructural members reveal co-variation patterns that can be used to identify interactions between pairs of amino acids. we use a global probability model to disambiguate direct and indirect correlations. specifically, we use a maximum entropy approach called pseudo-likelihood maximization (plm) to distinguish causation (residue interactions) from correlation (correlated mutations) and compute evolutionary couplings (ecs). the inferred set of residue interactions can then be interpreted as physical contacts and used in de novo 3d structure prediction. furthermore, the interactions that are inferred can help guide experiments that measure the phenotypic consequences of protein substitutions, making the method useful for functional studies. the present work can be divided into three areas: (i) methodological improvements related to alignment, folding procedure, structure refinement and ranking; (ii) folding of proteins of known structure for benchmarking and prediction of proteins of unknown structure; and (iii) focused exploration of specific cases of interest. developing shuffle as a platform for expression and engineering of antibodies na ke 1 , alana ali-reynolds 1 , bryce causey 1 , berkmen berkmen 1 1 shuffle is a genetically engineered e.coli strain that allows disulfide bond formationin its cytoplasm with high fidelity. many proteins containing disulfide bonds have been successfully expressed in shuffle. in this study, we expressed, purified and characterized full-length monoclonal antibody igg in shuffle. for the first time, a fulllength igg can be functionally expressed in the cytoplasm compartment of an e.coli strain. in order to improve the folding and assembly of igg, we have investigated the expression of igg in various formats and vectors; we have co-expressed chaperones and other helper proteins with igg. several-fold increase in the yield of fulllength igg was observed. we characterized the shuffle produced igg and found it comparable to hybridoma produced igg. optimization of fermentation conditions for a large-scale production is in progress. we aim to develop shuffle as an easy, fast, robust platform for antibody engineering, screening and expression. experimental and computational studies of the effects of highly concentrated solutes on proteins: insights into the causes and consequences of quinary protein structure and cytoplasmic organization most studies of protein structure and function focus on pure, diluted samples; however, real-world biochemistry and typical biotechnological applications of proteins take place in complex media with very high concentrations of solutes (100-400 g/l) of varied size and chemical nature. on one side, this has recently fostered the study of proteins in vivo, in cell, or at least in media mimicking the native conditions. on the other hand, physical chemistry has for a long time studied the general effects of crowded and viscous conditions on proteins, looking mainly at coarse traits like diffusion and stability. but the general effects on traits relevant at atomic/residue resolutions have been less studied, and one fundamental issue remains unsolved: to what extent are proteins forced into interactions with highly concentrated solutes, and with what direct consequences? i will present here our ongoing efforts to dissect the fine effects of high solute concentrations and macromolecular crowding on proteins, based on nmr experiments and md simulations, two complementary techniques of high spatial and temporal resolutions. our results show that smaller solutes are prone to extensive interactions with proteins when at high concentrations while large solutes act chiefly through excluded-volume effects. overall, we observe location-specific perturbations of a protein's surface, its internal dynamics and internal dielectrics, and its hydration, all very dependently on the solute's size and chemical nature. our results support the growing notion that proteins should be studied in native-like media, adding that not only macromolecular crowders but also small molecules should be considered in these studies. last, the fact that high-concentration conditions affect far more than a protein's diffusion rate and stability suggests critical consequences of quinary protein structure and cytoplasmic organization on the regulation of proteins within cellular biochemistry. aldona jeli nska 1 , anna lewandrowska 1 , robert hołyst 1 1 we developed an analytical technique for the study of interactions of ligands (e.g. cefaclor, etodolac, sulindac) with most abundant blood protein (e.g. bovine serum albumin) using the flow injection method. the experiments were conducted at high flow rates (31 cm/s) in a long (>15m), thin (250mm) and coiled capillaries. the compound of interest (10 ml) was injected into carrier phase, which moved by the poisseule laminar flow. at the detection point we measure the concentration distribution of the analyte. the width of the final profile of the analyte concentration is inversely proportional to the effective diffusion coefficient of the analyte. from the differences between the widths of the concentration distribution of free and bound ligand we can determine value of the association constant. carbohydrate binding modules (cbms), which are defined as contiguous amino acid sequences within a carbohydrate-active enzyme, have been found in both hydrolytic and non-hydrolytic proteins and are classified into 71 families, according to their primary structure similarity. the characterization of cbms by different methods has shown that these modules concentrate enzymes on the surface of polysaccharide substrates. it is thought that maintaining the enzyme in proximity with the substrate leads to more rapid degradation of the polysaccharide. therefore, the study of these kinds of modules or domains is relevant, since they are involved in multiple processes in organisms, like signaling, defense and metabolism; and some of them are involved in allergenic responses. in the present work we studied two different models: the first one is a cbm of the family 26 from lactobacillus amylovorus (lacbm26) that binds starch these domains are present in a a-amylase like a repetitive tandem of five modules that are consecutive and do not present connectors. by means of itc and using a single recombinant lacbm26 domain we determined a ka 5 2.31x104 m-1 for b-cyclodextrin and a ka 5 8.54x104 m-1 for acyclodextrin. when the number of consecutive recombinant modules increased to three or five tandem modules, the ka values increased to 106 m-1; however, these constants did not show an additive or a synergic effect. for these experiments we fitted the isotherms to different models and used different algorithms. additionally, we used circular dichroism in the uv-far region to determine if there existed conformational changes upon binding of the cyclodextrin molecules to the different tandem modules. we could only observe slight changes in a positive band centered around 220-240 nm, which has been explained in terms of p-p; interactions of the aromatic residues at the binding site. these cbms have been used as carriers for in vivo vaccine delivery and affinity tags. the second model is a hevein-like cbm of the family 18 present in a chitinase-like protein from hevea brasiliensis (hbcbm18). hevein is a lectin from h. brasiliensis that shows a 63% identity with hbcbm18. these cmbs are connected to the catalytic domain, in proteins such as chitinases, by a linker of approximately 10 residues. in these experiments we used fluorescence techniques to determine the affinity constants for chitotriose. we previously reported a ka 5 2.08x106 m-1 when using a hbcbm18 that has a met residue at the nterminal region. besides the aromatic residues at the binding site, the met residue also interacts with the ligand, as determined using crystallographic and docking techniques. the mutant hbcbm18-r5w that does not have the met residue showed a ka of 2.8x104 m-1 with chitotriose, similar to the value reported for hevein using itc (ka 1.42x104 m-1). interestingly, there exists an isoform of the hbcbm18 that has a connector between one cbm18 and a half cbm18 (1.5xhbcbm18). this protein has a ka of 6.7x105 m-1 with the same ligand. initiating vesicle formation at the golgi complex: auto-regulation and protein interactions govern the arf-gefs gea1 and gea2 margaret gustafson 1 , j. chris fromme 1 1 molecular decision-makers play critical roles in the effort to maintain efficient and accurate cellular functions. in the case of vesicular traffic at the golgi complex, the decision to initiate vesicle formation is made by a set of guanine nucleotide exchange factors (gefs) that activate the small gtpase arf1, which is the master controller for the recruitment of cargos and coat proteins. saccharomyces cerevisiae possess three golgi arf-gefs, gea1, gea2, and sec7, which work at distinct sub-compartments of the golgi to activate arf1 only when and where appropriate. in the case of sec7 at the trans-golgi network (tgn), this requires a positive feedback loop in which active arf1 relieves autoinhibition of sec7, as well as recruitment to the golgi membrane and catalytic stimulation by signaling rab gtpases. we know far less about the decisionmaking process for gea1 and gea2, which are responsible for retrograde traffic within the golgi and to the endoplasmic reticulum. i have found that both gea1 and gea2 can bind membranes weakly in vitro, an ability which is counteracted by their c-terminal hds3 domains. in addition, i have discovered membrane recruitment in vitro is aided by the rab gtpase ypt1. however, these interactions cannot fully explain the distinct localization patterns of gea1, gea2, and sec7, as all three have been shown to be recruited by ypt1, which is found throughout the golgi. my work has revealed that in addition to the well-established distinct localization from sec7, gea1 also occupies different golgi compartments from gea2, so specific signals must exist which help the gefs decide where to go. my current efforts focus on understanding the roles of the other domains of gea1 and gea2, identifying the signals which send them to different parts of the golgi, and unraveling the different roles they play in vesicle trafficking pathways. sequence variation in archaea through diversity-generating retroelements sumit handa 1 , blair g paul 2 , kharissa l shaw 1 , david l valentine 2 , partho ghosh 1 1 department of chemistry and biochemistry, university of california san diego, 2 marine science institute, university of california protein diversification is an essential tool for the survival and evolution for various species. diversitygenerating retroelements (dgr) in bacteria is known to generate massive variation in dna through an error prone reverse transcriptase and retrohoming, which leads to variation in protein sequence. recent discovery of dgrs in intraterrestrial archaeal systems have opened an opportunity to study this massive sequence variation in third domain of life (paul bg, et al. nat. comm.) here, we present the first crystal structure of variable protein from archaea with ligand-binding pocket is surface exposed. also, it has conserved c-type lectin (clec) fold, as shown by previous work on variable proteins, major tropism determinant (mtd) and treponema variable protein a (tvpa) which bind ligands through the clec fold. despite weak sequence identities (10-15%) among these variable proteins, clec fold was found to be conserved. this variable ligand-binding site for archaea variable proteins can potentially generate 1013 variants. protein synthesis is a dynamic process mediated by a variety of proteins and enzymes. recent studies have shown that hydroxylation is a key post-translational modification involved in translation termination. in particular, the fe(ii)-and 2-oxoglutarate-dependent oxygenase, jumonji domaincontaining 4 (jmjd4), regulates translation termination via the carbon 4 hydroxylation of an invariant lysine residue, k63, of the eukaryotic release factor, erf1. in eukaryotes, translation termination is mediated by a release factor complex that includes erf1. erf1 is comprised of three domains, and it is responsible for recognizing stop codons in mrna transcripts before triggering polypeptide release from the ribosome. the lysine residue hydroxylated by jmjd4 falls within the n-terminal domain and more specifically within the highly conserved niks motif. this motif has been identified by cross-linking and mutagenesis studies to play an essential role in stop codon recognition. while hydroxylation of k63 by jmjd4 has been found to increase translational termination efficiency, the exact molecular mechanism by which hydroxylation influences termination remains unclear. this work aims to understand how hydroxylation of erf1 affects translation termination by exploring the effect of hydroxylation on the structure, dynamics, stability, and binding of the n-terminal domain of erf1 (erf1-n) using mass spectrometry, protein nmr spectroscopy, circular dichroism and differential scanning fluorimetry. in our efforts to understand the effect of hydroxylation, an additional jmjd4-catalyzed modification, characterized by a 130 da mass shift on k63, was identified in vitro. the effect of this modification on erf1 was similarly explored. our findings suggest that hydroxylation has no effect on the in-solution nmr structure of erf1-n, which experiences chemical shift changes localized to the target lysine residue. correspondingly, there are no significant differences in secondary structure content between wild type and hydroxylated erf1-n. hydroxylation was also found to have no effect on protein stability or dynamics. interestingly however, the 130 da modification appears to cause more significant chemical shift changes dispersed beyond the niks motif. this suggests a more global effect on the in-solution nmr structure despite the little differences observed in protein dynamics and secondary structure content. the 130 da modification was also found to have a destabilizing effect on erf1-n. neither hydroxylated nor 130 da modified erf1-n exhibited differences in rrna binding. while hydroxylation of erf1 was found to have little effect on protein structure, dynamics, stability, or binding, the 130 da modification has marked effects on protein structure and stability. such differences suggest that this modification has the potential to play an important role in translation. functional and structural analysis of a gh20 ß-n-acetylglucosaminidase from the marine bacterium vibrio harveyi piyanat meekrathok 1 , arthur t. porfetye 2 , marco b€ urger 2 , ingrid r. vetter 2 , wipa suginta 1 1 biochemistry-electrochemistry research unit, suranaree university of technology, 2 max planck institute of molecular physiology vibrio harveyi b-n-acetylglucosaminidase (so-called vhglcnacase) is a new member of the gh20 glycoside hydrolase family responsible for the complete degradation of chitin fragments, with nacetylglucosamine (glcnac) monomers as the final products. however, the 3d structure of glcnacase is still unknown. in this study, crystal structure and function of glcnacase were investigated based on protein crystallography. size-exclusion chromatography and the native-page were employed to verify the protein state of glcnacase in a native form and the acidic active-site residues were mutated using sitedirected mutagenesis method. the effects of mutations on the binding and hydrolytic activities were studied by enzyme kinetics. to provide a structural basis of glcnacase, the wild-type enzyme was crystalized at 293 k using a solution containing 0.1 m sodium acetate ph 4.6 and 1.3 m sodium malonate and recorded x-ray data. the wild-type enzyme was crystallized within 3 days in the monoclinic crystal form, belonging to space group p21, with unit-cell parameters a 5 90.2, b 5 130.7, c 5 98.5 å. the crystal structures of v. harveyi glcnacase were solved and refined to highest resolution of 2.4 å. structural investigation revealed that glcnacase comprises three distinct domains, designated as the n-terminal carbohydrate-binding domain, the a1b topology domain and the tim-barrel catalytic domain. the substrate binding groove of glcnacase is a small pocket, which is suitable to accommodate a shortchain chitooligosaccharide. kinetic analysis revealed that a group of the adjacent d303-h373-e438 showed a significantly decreased activity as compared with the wild-type enzyme, and these residues might be important for enzyme catalysis. silencing the molecular timekeeper in human cancer alicia michael 1 , stacy harvey 1 , patrick sammons 1 , amanda anderson 2 , hema kopalle 1 , alison banham 2 , carrie partch 1 1 university of california -santa cruz, 2 university of oxford the circadian clock coordinates temporal control of physiology by regulating the expression of at least 40% of the genome on a daily basis.1 disruption of circadian rhythms through environmental stimuli (e.g. light at night) or genetic means can lead to the onset of diseases such as: diabetes, cardiovascular disease, premature aging and cancer.2-5 the circadian clock orchestrates global changes in transcriptional regulation via the bhlh-pas transcription factor clock:bmal1. pathways driven by other bhlh-pas transcription factors have a homologous repressor that modulates activity on a tissue-specific basis, but none have been identified for clock:bmal1. we discovered that the cancer/testis antigen pasd1 fulfills this role to suppress circadian rhythms. pasd1 is evolutionarily related to clock and interacts with the clock:bmal1 complex to repress transcriptional activation. furthermore, deletion of one region, highly conserved with clock exon 19, alleviates repression by pasd1 to suggest that it utilizes molecular mimicry to interfere with clock:bmal1 function. structural and biochemical studies of the direct interaction of pasd1 with the clock:bmal1 complex using recombinant protein expression and biophysical techniques are currently underway. as a cancer/testis antigen, expression of pasd1 is natively restricted to gametogenic tissues but can be upregulated in somatic tissues as a consequence of oncogenic transformation. reducing pasd1 in human cancer cells significantly increases the amplitude of transcriptional oscillations to generate more robust circadian rhythms. our work suggests that mechanisms to suppress circadian cycling can be hard-wired in a tissue-specific manner and our data show that they can be co-opted in cancer cells to attenuate clock function. the scaffolding protein iqgap1 participates in various cellular functions such as cell-cell adhesion, cell polarization and migration, neuronal motility, and tumor cell invasion by binding to target proteins, including rac1 and cdc42, two members of the rho family. to better understand the molecular basis of these interactions, we utilized in this study a novel time-resolved fluorescence spectroscopy to determine individual rate constants for iqgap1 interaction with fourteen different rho proteins. the results indicated that iqgap1 binds among rho proteins selectively to rac-and cdc42-like proteins only in a gtp-dependent manner. moreover, the interaction of rho proteins with the c-terminal half of iqgap1 (grd-c), shorter fragment contains grd-gbd, only the grd and also grd-gbd with single and double phosphomimetic mutations s1441e and s1443d was performed. obtained results showed that, when both grd and gbd are existing, fluorescence changes is detected but for grd alone or in the case of s1443d or s1441e/s1443d no change was observed, suggesting that gbd and specifically, cysteine 1443 is critical for this interaction. furthermore, fluorescence polarization results showed that the grd-c interact with cdc42 and rac1 but not with rhoa, and interestingly the grd domain showed similar behavior, but with 10 to 15 folds lower affinity as compared with the grd-c. consistent with this, a gdp-bound form of cdc42 showed interaction with both grd and the grd-c in quiet comparable affinities. at last, competition experiments utilizing interacting partners of rac1, e.g. tiam1, p50rhogap, plexin-b1, p67phox, pak1 and rhogdia, along with structural analysis, revealed two negative charged areas on the surface of rho-and rnd-like proteins, which might explain their inaccessible interaction with iqgap1. the overlapping binding site of cdc42 and rac1 on the surface of iqgap1 together with the kinetic details of the selective interaction of iqgap1 with rac-and cdc42-like proteins suggests that these interactions are most likely mediated via the same mechanism. ing4 dimerizes through its n-terminal domain, with a symmetric antiparallel coiled-coil structure3, making it a bivalent reader of the h3k4me3 mark. ing5 is highly homologous with ing4, but forms part of a different histone acetyl transferase complex1. here, we show that ing5 is also a dimer and thus a bivalent reader of the h3k4me3 mark. however, the crystal structure of the n-terminal domain of ing5 shows an asymmetric dimer, different from the homologous ing4 domain. our nmr data (backbone assignment and paramagnetic relaxation effects) and saxs data indicate that the structure of the n-terminal domain of ing5 in solution is similar to ing4, suggesting that the crystal structure of ing5 is likely a crystallization artifact. three point mutations in the n-terminal domain of ing5 have been described in oral squamous cell carcinoma: q33r, i68v, and c75r4. we have found that the n-terminal domains of the three mutants are dimeric coiled-coils but with different stability, as measured by thermal denaturation. while the q33r mutant is as stable as the wild type, the i68v and c75r mutants are strongly destabilized, suggesting a role in cancer development at least for these two mutants. efforts so far, to combat alzheimer's disease (ad) have focused predominantly on inhibiting the activity of enzyme(s) that are responsible for the production of the main causative beta amyloid forming peptide. however, the inherent complexity associated with the network of pathways leading to the progress of the disease may involve additional targets for designing effective therapies. recent experimental findings have identified abelson's tyrosine kinase (c-abl), a non-receptor kinase involved in a variety of cellular functions as a new target for ad. in the present study we employed energy optimized multiple pharmacophore modeling strategy from multiple c-abl structures bound with ligands in the inactive atp binding conformation. virtual screening followed by docking of molecules from chembridge_cns database, and maybridge databases resulted in the identification of 15 best scoring molecules. based on docking score and selectivity assessment and druggability parameters, four out of the 15 molecules are predicted to show increased specificity for c-abl in comparison to closely related kinases. given the implied role of c-abl not only in ad but in parkinson's disease, the identified compounds may serve as leads to be developed as effective neurotherapeutics. rafael palomino 1 , glenn millhauser 2 , pietro sanna 2 1 university of california santa cruz, 2 the scripps research institute the central melanocortin system is recognized as a key regulator of energy balance and appetite. the hypothalamic melanocortin receptor, mc4r, is a g-protein coupled receptor that is antagonized by the peptide ligand, agouti-related peptide (agrp), leading to increased feeding and weight gain. while much research has gone into how this ligand exerts its effects at the receptor, less is known regarding nonmelanocortin components of the pathway. syndecan-3, a heparan sulfate proteoglycan, has previously been implicated in potentiating agrp antagonism, however details of this interaction are unclear. this work aims to investigate the role of syndecans at both a molecular level and in vivo. we hypothesize that agrp binds the glycosaminoglycan (gag) components of syndecans, and that this interaction increases the local concentration of the peptide near mc4r. furthermore, we have previously shown that designed positive charge mutations to agrp lead to increased in vivo efficacy that is independent of mc4r activity, and we hypothesize that this is due to greater affinity for the negatively charged gags. using isothermal titration calorimetry we have shown tight binding between agrp and heparan sulfate, the major gag component of syndecan-3, and this affinity is strengthened by additional peptide positive charge. through nmr, we see that both positively charged and polar residues are necessary for binding various heparan sulfate polymers. these data implicate a specific region of agrp that is not required for mc4r binding as being necessary in its role as a heparan sulfate binding protein. expanding on these findings, we are now using a syndecan knockout mouse line to explore the mechanism of differential feeding in our designed mutants. preliminary results indicate a reduction in weight gain in knockouts compared to their wildtype littermates post peptide administration. collectively, these data show that the physiologically relevant form of agrp, previously considered unable to interact with syndecans, is indeed a heparan sulfate binding protein. furthermore, our designed mutants have differential affinities for gags, with increased affinity correlating to increased feeding potency. finally, as the mc4r pathway is thought to be a viable target for wasting disorders such as cachexia, we are interested in leveraging this data to improve the potency and stability of our designed agrp mutants. taken together, this work aims to develop new insights and probe the therapeutic potential of a critical metabolic pathway. evidence of a proteolytic phenomenon in the starch binding domain of the a-amylase from lactobacillus amylovorus zaira esmeralda s anchez cuapio 1 , alejandra hern andez santoyo 2 , sergio s anchez esquivel 1 , romina rodr ıguez sanoja 1 1 instituto de investigaciones biom edicas, universidad nacional aut onoma de m exico, 2 instituto de qu ımica, universidad nacional aut onoma de m exico a-amylases are glycoside-hydrolases that catalyze the hydrolysis of internal a-1,4 glycosidic bonds in starch and glycogen generating smaller oligosaccharides (1). these multidomain proteins contain a catalytic barrel (b/a)8 and, in some cases, one or more non-catalytic domains whose function is generally described as carbohydrate binding module (cbm) and particularly as starch-binding domains (sbd). the sbd can bind granular starch increasing the local concentration of substrate at the active site of the enzyme and may also disrupt the structure of the starch surface (2) . the a-amylase from lactobacillus amylovorus has a structure that consists of a catalytic domain (cd) and an unusual carboxy-terminal starch-binding domain with 5 identical cbms (belonging to family 26) in tandem (3). each repeat acts as an independent fixing module with an additive or synergic effect between the units (4). when we stored pure sbd from l. amylovorus we found multiple forms of low molecular weight with a constant pattern, which does not correspond to random degradation. interestingly, when the protein is stored at ph close to 5 and edta is added, such proteolysis appears to decrease. so far, there is little information about the proteolytic process of amylases and the nature of it. here we show that divalent ions induce a proteolytic cleavage of the sbd, raising the possibility of an autoproteolytic activity. acknowledgments: this work is supported by grants papiit in222113-3 and conacyt 131149. s anchez cuapio z is supported by a personal grant from consejo nacional de ciencia y tecnolog ıa, m exico. unnatural amino acid and related methods provided a special mechanism to implement site-specific spectroscopy active probe incorporation in a specific membrane protein in cells. the site specific incorporation resulted in a single signal during acquisition, resulting in unambiguous signal assignment. the protein specific labeling makes it possible for in situ membrane protein analysis using nmr or fluorescence detection. the 19f containing unnatural amino acid incorporation has been applied for dynamic studies of transporters in native lipid membrane, and the phosphorylation quantification analysis for tyrosine kinase in native lipid membrane with the aid of lipodisc. the fluorescent unnatural amino acid incorporation enabled the site-specific channel responses analysis upon ligand binding in a single cell. heather wiebe 1 , noham weinberg 1,2 1 department of chemistry, simon fraser university, 2 department of chemistry, university of the fraser valley the mechanism by which conformational changes, particularly folding and unfolding, occur in proteins and other biopolymers has been widely discussed in the literature. molecular dynamics (md) simulations of protein folding present a formidable challenge since these conformational changes occur on a time scale much longer than what can be afforded at the current level of computational technology. transition state (ts) theory offers a more economic description of kinetic properties of a reaction system by relating them to the properties of the ts, or for flexible systems, the ts ensemble (tse). the application of ts theory to protein folding is limited by ambiguity in the definition of the tse, although the experimentally observed first-order kinetics for folding of small single-domain proteins lends itself to interpretation by this theory. the pressure dependences of the folding rate constant can be used to obtain activation energies and activation volumes, which are rationalized as the properties of the folding tse. the large amount of activation volume data in the literature has gone largely uninterpreted at the quantitative level. we propose to utilize this data in conjunction with md-calculated volumetric properties to identify the tse for protein folding. the effect of pressure on reaction rates is expressed in terms of logarithmic pressure derivatives, known as activation volumes. according to ts theory, activation volumes can be identified as the difference in volume between the ts and reactant species: activation volumes dv ‡ have been experimentally determined for the folding of several proteins. the concept of activation volume can be extended to that of a volume profile, dv(y), which describes how the volume of a system changes along reaction coordinate y. if the position y ‡ of the ts along the reaction coordinate is unknown, it can be found by locating dv ‡ on the volume profile: such volume profiles can be built using our recently developed md-based displacement volume method.* using this method, volumes of single molecules can be calculated by taking the difference between the volume of pure solvent and solvent containing the desired solute. this method takes into account the strength and type of solvent-solute interactions as well as the geometrical configuration of the solute. in this work, we present the successful application of this method to several conformationally flexible systems. structure of the p15paf/pcna complex and implications for clamp sliding on the dna during replication and repair to the canonical pip-box binding groove on the pcna front face. in contrast to other pcna interacting proteins, however, p15paf also contacts the inside of, and passes through, the pcna ring. the mostly disordered p15paf chain termini thus emerge at opposite faces of the ring, but remain protected from degradation by the 20s core proteasome. we also unveil a novel dna binding activity of p15paf, both free and bound to pcna, which is mainly mediated by its conserved histone-like n-terminal tail. molecular modeling shows that a ternary complex with a duplex dna inside the pcna ring is energetically feasible and our electron micrographs show increased density inside the ring. we propose that p15paf acts as a flexible drag that regulates pcna sliding along the dna, and may facilitate the switch from replicative to translesion synthesis polymerase binding upon dna damage. acknowledgements: this work has been mainly sponsored by mineco grant ctq2011-28680 and juan de la cierva-2010 contract to alfredo de biasio. metabolic syndrome (mets) is one of the leading causes of the death worldwide; however, exact pathophysiological mechanisms of mets remain largely unknown. growing evidence suggests that the increased availability of glucocorticoids at the tissue level play an important in mets development. one of the major determinants of glucocorticoid local action seems to be the enzyme 11b-hydroxysteroid dehydrogenase 1 (11b-hsd1). this enzyme is a well-known member of the short-chain dehydrogenase/reductase (sdr) superfamily. it is an important carbonyl reducing enzyme that, besides its role fine-tuning of glucocorticoids actions, is involved in the biotransformation of drugs and in the development of lung cancer through metabolism of the tobacco specific carcinogen nnk. the phylogenetically closest relative of 11b-hsd1 is dhrs7 enzyme from the same superfamily. unlike 11b-hsd1, dhrs7 is poorly characterized however it can be supposed at least partially overlapping function to 11b-hsd1. moreover its possible association with similar pathological conditions in human as 11b-hsd1 has already been indicated by several studies. the aim of this study is the basic biochemical characterization of dhrs7. the enzyme is a member of cluster 3 of "classical" sdr; such members are considered to be retinoid and steroid metabolizing enzymes, so characterization the enzyme was based on this assumption. dhrs7 was prepared in recombinant form in the sf9 cell line. it was proved that this enzymes is an integral membrane-bound enzyme localized in the endoplasmic reticulum with luminal orientation, similarly to 11b-hsd1. known substrates of 11b-hsd1 and related enzymes were tested also as substrates of dhrs7. it was proved that dhrs7 is nadph-dependent reductase with important substrates as steroid hormones cortisone and androstene-3,14-dione, all-transretinal and also xenobiotics as 1,2-naphtoquinone or carcinogen nnk at least in vitro. for better understanding of the catalytic function of dhrs7 its structural model was prepared and it is used also for the identification of additional substrates by ligand virtual screening. dhrs7 enzyme is expressed in several human tissues as adrenals, liver, prostate, small intestine and kidney. these brand new initial results point to the possible involvement of dhrs7 in important cellular processes that deserve further investigation. these results will lay the foundation for an understanding of dhrs7 role in human physiology resp. pathophysiology. this project was supported by grant agency of charles university (677012/c/2012) and unce 204026/2012). structure-based functional identification of helicobacter pylori hp0268 as a nuclease with both dna nicking and rnase activities bong-jin lee 1 , ki-young lee 1 1 hp0268 is a conserved, uncharacterized protein from helicobacter pylori. here, we determined the solution structure of hp0268 using three-dimensional nuclear magnetic resonance (nmr) spectroscopy, revealing that this protein is structurally most similar to a small muts-related (smr) domain that exhibits nicking endonuclease activity. we also demonstrated for the first time that hp0268 is a nicking endonuclease and a purine-specific ribonuclease through gel electrophoresis and fluorescence spectroscopy. the nuclease activities for dna and rna were maximally increased by mn(21) and mg(21) ions, respectively, and decreased by cu (21) ions. using nmr chemical shift perturbations, the metal and nucleotide binding sites of hp0268 were determined to be spatially divided but close to each other. the lysine residues (lys7, lys11 and lys43) are clustered and form the nucleotide binding site. moreover, site-directed mutagenesis was used to define the catalytic active site of hp0268, revealing that this site contains two acidic residues, asp50 and glu54, in the metal binding site. the nucleotide binding and active sites are not conserved in the structural homologues of hp0268. this study will contribute to improving our understanding of the structure and functionality of a wide spectrum of nucleases. high-fidelity recombinant protein production in a silkworm bioreactor sungjo park 1 , in-wook hwang 1 , tatsuya kato 2 , enoch park 2 , andre terzic 1 1 center for regenerative medicine, mayo clinic, 2 laboratory of biotechnology, shizuoka university the domesticated silkworm, bombyx mori, is an attractive host naturally equipped with a proficient posttranslational modification machinery adequate to fulfill stringent demands of authentic recombinant protein production. silkworm-based protein expression has originally relied on a prototype baculovirus vector system that employs silkworm as a bioreactor in place of more traditional cell lines. recent development of the silkworm trophic b. mori nucleopolyhedrovirus (bmnpv) bacmid launches a second generation of silkworm-based protein production technology. introducing the recombinant bacmid dna into silkworms expedites heterologous protein expression by eliminating prior virus construction and amplification steps. salient examples of heterologous eukaryotic proteins produced in silkworms are acetyl-coa carboxylase 2, malonyl-coa decarboxylase, spot14/mig12 heterodimer and a2,6-sialyltransferase with consistent high levels of protein expression. thus, equipped with a fail-safe post-translational modification machinery, eukaryotic proteins are readily bioengineered using a silkworm-based protein expression platform. studies exploring potential applications of synthetic antifreeze proteins in the frozen food industry ho zee (charles) kong 1 , conrad perera 1 , ivanhoe leung 1 , nazimah hamid 2 , viji sarojini 1 1 school of chemical sciences, the university of auckland., 2 school of applied sciences, auckland university of technology in nature, certain species of plants, insects and fish produce a group of antifreeze glycoproteins and polypeptides which enable them to survive the freezing temperatures of their natural habitat. these naturally occurring antifreeze proteins (afps) were first discovered in polar fishes such as antarctic notothenioids and winter flounder. these afps have the ability to bind to ice crystals and restrict their size and morphology; decrease the freezing point of water and inhibit the ice recrystallization processes. ice crystal formation is of primary concern to the frozen food industry, as ice crystal formation during freezing can be disruptive to and cause damage to the cellular structures in food. the unique properties of afps can be developed into a potential solution to minimize freeze-thaw damage to frozen food. a number of tailor made synthetic analogues based on the naturally occurring afps were successfully designed and synthesized. antifreeze activity studies of the afps were carried out using the clifton nanoliter osmometer attached with a microscope. the afps exhibited thermal hysteresis as well as modification of ice crystal morphology, confirming their antifreeze activity in vitro. the ability of these synthetic afps in preserving the texture and structure of frozen food was evaluated using the techniques of scanning electron microscopy. the afps showed great potential to preserve the cellular structures of frozen food samples during freeze-thaw process. additionally, secondary structure analysis of the afps was carried out using circular dichroism. this presentation will summarize our current results on the design, synthesis and anti-freeze activity analysis of the synthetic afps. invasive fungal infections remain a leading cause of death in immunocompromised patients. current antifungal agents have a host of issues including limited efficacy, host toxicity and an alarming increase in resistance. current research in our laboratories is focused on targeting the calcineurin signaling pathway that has been shown to be required for fungal pathogenesis. calcineurin is a highly conserved serine-threonine-specific ca21-calmodulin-activated phosphatase important in mediating fungal pathogenesis and stress responses. it is a key regulator of a signal transduction network required for survival of the most common pathogenic fungi in humans, making it an ideal target for fungal drug development. calcineurin is a heterodimer of a catalytic (a) and regulatory (b) subunit. phosphatase activity requires association of the two subunits. calcineurin is also the target of the immunosuppressant fk506, which functions as an inhibitor by first complexing with the peptidyl-prolyl cis-trans isomerase immunophilin, fkbp12. the fkbp12-fk506 complex subsequently binds to calcineurin in a groove between the a and b subunits and inhibits its activity. although fungal calcineurins are targeted by fk506, it also targets mammalian calcineurin and is thus immunosuppressive in the host. in order to improve therapeutic efficacy, we have undertaken a unique effort that utilizes both structural biology and molecular mycology in an effort to overcome the fungal versus human specificity barrier. the nmr studies to be presented here have been focused on determining the resonance assignments and solution structures for the fkbp12 proteins from the pathogenic fungi candida albicans, candida glabrata and aspergillus fumigatus. notably, the x-ray crystallography structures of the wild-type candida albicans and aspergillus fumigatus fkbp12 proteins revealed an intriguing intermolecular interaction involving four residues in the 80's loop including pro104 (in c. albicans) and pro90 (in a. fumigatus) which are stabilized in the cis conformation. these data suggest that the protein might use itself as an enzyme substrate. in efforts to establish if this interaction remains in a solution environment, we have determined the nmr structure and measured the t2 relaxation rates for the wild-type a. fumigatus fkbp12 protein and for the p90g mutant variant that adopts a dramatically different orientation of the 80's loop and does not form an intermolecular interaction in the crystal structure. the nmr chemical shift data indicate that, while the remainder of the protein structure remains unchanged, the 80's loops in the two variants are indeed different. in addition, the t2 relaxation rates of the residues in this region are dramatically dissimilar in the two variants, but remain identical throughout the rest of the protein. we have also begun inhibitor binding studies of all of the fkbp12 proteins from each of the pathogens by titrating the fk506 inhibitor into native and mutant fkbp12 proteins in order to examine conformational changes associated in the protein upon complex formation. using this approach we plan to determine the relative kd values for binding of each inhibitor to the fkbp12 protein from each pathogen for comparison of binding proclivities. lupin (lupinus angustifolius l.) b-conglutin proteins: structure functional features, catalytic mechanism modeling and cross-allergenicity identification using protein threading and molecular docking methods lupin is an important pulse, which displays a wide range of benefits in agriculture, particularly these involved in possible plant pathogen suppression. furthermore, lupin seed proteins promote different positive health aspects, preventing cardiovascular disease, and reduction of glucose and cholesterol blood levels. "sweet lupine" seeds seem to be promising as a source of innovative food ingredients due to averaged protein content similar to soybean and an adequate composition of essential amino acids. thus, lupin seeds may be important source of proteins for human and animal consumption. however, and as drawback feature, the number of allergic people to lupin seed proteins is rising, becoming a serious and a growing problem in the western world, because of the rapid introduction of lupin seeds as new ingredients in traditional and novel foods. the goals of this study are the characterization the structure-functional properties of lupinus angustifolius l or narrow leafed lupin (nll) b-conglutin proteins, with a focus in its catalytic mechanism, and its molecular cross-allergenicity with other legumes, i.e. peanut, by extensive analysis using different computer-aided molecular approaches covering (i) physicochemical properties and functional-regulatory motifs, (ii) sequence analysis, 2-d and 3d structural (threading) modeling comparative study and molecular docking, (iii) conservational and evolutionary analysis, (iv) catalytic mechanism modeling, and (v) sequence, structure-docking based b-cell epitopes prediction, while t-cell epitopes were predicted by inhibitory concentration and binding score methods. b-conglutins (vicilin-like or 7s proteins) are seed proteins typically found in reserve tissues (endosperm and cotyledon). they belong to the cupin superfamily of proteins, containing a globular domain constituted by a conserved b-barrel. two barrels were found in all b-conglutin protein isoforms and an additional mobile n-terminal arm constituted bye a-helices. molecular modeling analysis has shown that one of this barrel contain a semi-conserved metal binding motive (hyx. . .r), typically found in oxalate oxidase (oxox) enzymes. interestingly, our results revealed considerable structural differences between b-conglutin isoforms, particularly affecting 2-d elements (loops and coils), and numerous micro-heterogeneities are present in fundamental residues directly involved in epitopes variability, which might be a major contributor to the observed differences in cross-reactivity among legumes. we also identified multiple forms of b-conglutins polypeptides ranging from 15-80kda, with ige-binding characteristics in atopic patients. thus, b-conglutins might be considered as major allergen in different species of lupin, including the "sweet lupin" group, since several of these polypeptides were recognized by human iges, having the potential to trigger an immune response leading to allergy symptoms. 1 influenza virus is one of the most prevalent pathogens causing respiratory illness which often leads to serious post influenza complications such as pneumonia and myocarditis. some viruses, as the avian influenza h5n1, are especially dangerous and draw special attention of who. this highly pathogenic virus spreads quickly among domestic poultry and wild birds resulting in high mortality. what is more distressing, the h5n1 virus may be transmitted to humans. because of antigenic drift it is impossible to deliver an effective vaccine against all subtypes of the h5n1 virus. moreover, traditional egg-based production of influenza vaccines is time-and cost-consuming, what makes it inadequate in case of a pandemic. hence, we have developed an efficient production process of influenza vaccine based on a recombinant hemagglutinin antigen (rha). recombinant vaccines underlay strict regulations and quality requirements. the purpose of this work was to develop a battery of analytical methods that allow to evaluate key quality attributes of rha on each stage of production. at first, we have focused on rha structure as a crucial issue for its activity. the primary structure of rha was confirmed by peptide mapping and tof/tof fragmentation (hplc, maldi tof/tof). furthermore, ftir analysis was used to evaluate the secondary structure of the protein. the disulfide bonds, which stabilize the tertiary structure, were assigned by peptide mapping. additionally, free thiols were measured using ellman's reagent. moreover, we have employed rp-hplc, sec-mals and dls to explore oligomerization of rha. these techniques appeared to be useful not only to confirm existence of native oligomers, but also to find and discard misfolded fraction, aggregates and truncated forms. in addition, two analytical methods (rp-hplc and cge) were developed to assess the purity of rha as required by ich guidelines. we also have determined isoelectric point and heterogeneity of rha by cief. afterward, developed methods were applied in the stability studies that provide a valuable insight into a chemical degradation process and conformational changes of rha during storage. this work was supported by innovative economy operational program, grant no. wnd-poig.01.01.02-00-007/08-00 as a part of project "centre of medicinal product biotechnology. package of innovative biopharmaceuticals for human and animal therapy and prophylactics." muscle cell atrophy via hsp gene silencing was counteracted by celastrol-mediated hsp overexpression molecular chaperone heat shock proteins (hsp) are known to assist protein quality control under various stresses. although overexpression of hsp70 was found to promote muscle mass retention in an unloading state, it is unclear whether muscle atrophy is induced by suppression of hsp expression and is counteracted by active hsp overexpression. in this study, we pre-treated hsp70 sirna to rat l6 cells for the hsp gene-silencing, and determined myotube diameter, hsp72 expression and anabolic and catabolic signaling activities in the absence or presence of triterpene celastrol (cel), the hsp70 inducer. relative to a negative control (nc), muscle cell diameter was reduced by 11% in the sirna-treated group, increased 1.2-fold in the cel-treated group and remained at the size of nc in the sirna1cel group. hsp72 expression was decreased 65% by sirna whereas the level was increased 6-to 8-fold in the cel and sirna1cel groups. expression of foxo3 and atrogin-1 was increased 1.8-to 4.8-fold by sirna, which was abolished by cel treatment. finally, phosphorylation of akt1, s6k and erk1/2 was not affected by sirna, but was elevated 2-to 6-fold in the cel and sirna1cel groups. these results suggest that hsp downregulation by hsp gene-silencing led to muscle cell atrophy principally via elevation of catabolic activities. such anti-atrophic effect was counteracted by cel-mediated hsp overexpression. the centers for disease control and prevention report that at least 2 million people in the united states will become ill due to antibiotic resistant pathogens leading to 23,000 deaths each year. in order to circumvent these resistance mechanisms, it is essential to quantitatively understand how the function of the protein(s) involved relates directly to resistance. integral membrane efflux pumps are known determinants of single-drug and multi-drug resistance in a wide variety of pathogenic organisms. these transporters are proteins whose characterization typically requires reconstitution in an artificial membrane. subsequently, these important proteins are difficult to characterize by traditional in vitro studies. my project aims to determine the physicochemical parameters of the efflux pump tetb utilizing molecular biology and mathematical modeling. tetb is composed of 12 transmembrane (tm) alpha-helices and is found within the inner membrane of gram-negative bacteria. this protein allows for the efflux of tetracycline (tet), doxycycline (dox), and minocycline (mcn) antibiotics from the cytoplasm into the periplasm. these tetracyclines are a bacteriostatic class of antibiotics that inhibit protein synthesis by binding to the 30s ribosomal, therefore, blocking the binding of aminoacyl-trna. for cells grown in tetracyclines, the efflux mechanism of tetb decreases the cytosolic antibiotic concentration allowing for the rate of protein translation to increase. i have inserted a tet(b) expression system into the chromosome of an escherichia coli lab strain and have determined its growth profile under various concentrations of tet, mcn, and dox using a high-throughput 96-well plate format. the growth rate profiles correlate with tetb pumping rates for each drug. tetb more readily pumps out tet compared with dox and mcn and we observe that cells expressing tetb can grow at higher tet concentrations compared with dox and mcn. the shapes of the growth rate profiles produced in the different drugs give insight into the physicochemical mechanism of tetb. we have built a preliminary mathematical model that can simulate these growth profiles and predict efflux pump physicochemical parameters. we are currently working on understanding how efflux expression effects bacterial growth by testing ribosome binding site (rbs) sequences of varying strengths in our tet(b) expression system. future work is geared toward modeling more complex efflux pumps such as the tripartite pumps which traverse both bacterial membranes and cause multi-drug resistance. collectively, this project aims to build an in vivo system which will allow for the characterization of a variety of efflux pumps without the arduous tasks of protein purification and subsequent reconstitution. (2007) identified a small transmembrane region of both kcne1 and kcne3 that are essential for their unique modulation of the kcnq1 channel. by swapping a triplet motif in the transmembrane region of kcne1 and kcne3, we can flip the primary function of these two proteins. while the key for kcne1 and kcne3's unique modulating is believed to lie in this triplet motif, the mechanism and structural changes involved in this modulation is not fully understood. by using nmr spectroscopy, biochemical studies, and computational docking, we aim to look at the structural and conformational differences between kcne1 and the triple mutant kcne1 substituted with the three essential kcne3 residues. we have expressed and purified 15n-labled kcne1 triple-mutant in sufficient quantities for nmr studies in lmpg detergent micelles and other membrane mimetics, and we have collected 2d nmr spectra using a trosy-based pulse sequence. partial backbone assignments of kcne1 triple mutant have been determined by aligning and transfer assignments of the wt kcne1 previous determined in our lab. with the structure of kcne1 triple mutant determined, we aim to computationally dock the triple mutant into a model of the full-length kcnq1 channel in the open and closed state. lastly, we will compare the known structure of kcne1 docked to a model of kcnq1 to that of the kcne1 triple mutant to determine key interactions, significant structural and conformational changes, and how the triple motif region gives rise to its specific structural and functional differences. with this information, we can begin to understand the mechanism of the functional diversity of the kcne family on kcnq1 potassium channel. biochemical characterization of brassica napus diacylglycerol acyltransferase 1 and its regulatory domain .a) expressed in saccharomyces cerevisiae. purified bnadgat1 in n-dodecyl-b-d-maltopyranoside (ddm) micelles behaves as dimers, which can associate further to form tetramers. the acyl donor preference of the major dimeric form with sn-1,2-diolein as acceptor follows the following order: a-linolenoyl-coa > oleoyl-coa 5 palmitoyl-coa > linoleoyl-coa > stearoyl-coa. the first 113 residues of bnac.dg-at1.a corresponding to a soluble regulatory region was expressed in escherichia coli and purified. truncation of this soluble domain reveals that the dimeric interface is located within residues 49-113, while the first 48 residues allow formation of tetramers. this n-terminal region was implicated as an allosteric exosite for acyl-coas as revealed by previous lipidex-1000 binding studies. in the current study, circular dichroism spectroscopy and isothermal titration calorimetry were used to probe the binding kinetics and thermodynamics. dgat1 appears to shift between two oligomerization states, a phenomenon that may be related to regulation of enzyme activity and mediated by the n-terminal domain. alteration of lysine and arginine content as a strategy to modify such an interaction was found to increase the activity of rdrp in vitro. further, deletion of c terminal 43 amino acid residues also resulted in increase in the polymerase activity that was comparable to the full length rdrp-p10 complex. it was proposed that the conserved c terminal disordered domain of rdrp was responsible for interaction with p10 and modulation of the activity. in the present study, role of the c terminal disordered domain was further investigated by determining the oligomeric status of the complex and the c terminal deletion mutants of rdrp and also by quantitating the rdrp-p10 interaction using surface plasmon resonance. size exclusion chromatography revealed that rdrp eluted in the void volume of the column whereas a significant fraction of the rdrp-p10 complex eluted at a position corresponding to the size of the 1:1 complex of rdrp and p10 (77kda). activity measurements indicated that the heterodimeric complex was more active than the aggregate eluting in the void fraction. interestingly, the c terminal deletion mutants of rdrp (c del 43 & c del 72 rdrp) were also found to be less aggregated as compared to full length rdrp and some of the protein eluted at a position corresponding to the respective monomers. these monomers were also more active than the aggregate fractions. these results demonstrate that the increase in activity observed either upon interaction with p10 or deletion of the c terminal domain could be due to the change in the oligomeric state of rdrp. in order to further analyze the interaction of rdrp with p10 surface plasmon resonance was used. rdrp and its deletion mutants were immobilized on biacore sensor surface and p10 protein was used as an analyte. full length rdrp and c del 43 rdrp were shown to interact with p10 with kd values of 0.6 and 1 um respectively. however, c del 72 and c del 85 rdrp did not show any binding with p10. these results suggest that the region 43-72 from the c terminus of rdrp is essential for the interaction with p10. further, the c del 85 rdrp was inactive although c del 72 rdrp continued to be active suggesting that residues 72-85 from the c terminus are crucial for rdrp activity. further studies are in progress to identify the residues within these motifs that may be essential for the activity or interaction with p10. aggregation of androgen receptor in spinal bulbar muscular atrophy is a multistep process spinal bulbar muscular atrophy (sbma) is a member of the polyglutamine (polyq) expansion diseases, like huntington disease, and it is caused by a genetic expansion of the polycag tract in exon 1 of androgen receptor (ar) that codes for the polyq region. sbma is a late onset disease, which involves a progressive degeneration of the motor neurons and consequent muscular atrophy. there is still no treatment available for this disease. ar is a nuclear receptor that responds to testosterone and that regulates the expression of the masculine phenotype. it is composed of an intrinsically disordered nterminal domain (ntd) that bears the polyq tract, a dna binding domain and a ligand binding domain. aggregates of ar protein with an extended polyq are observed in the motor neurons of sbma patients. in vitro studies showed that aggregation of androgen receptor takes place only in presence of testosterone1 and that the cleavage of the protein by caspase 3 is a crucial event for cytotoxicity. however, there is no clear knowledge of the mechanism of aggregation, for this protein. an increasing body of evidence supports the hypothesis that the aggregation of these proteins is controlled by regions flanking the polyq tract, by regulating the rate of aggregation depending on their secondary structure. we have applied nuclear magnetic resonance (nmr) and circular dichroism for generating information on the secondary structure of the n-terminal cleavage product of ar by caspase 3 and we have studied its aggregation with a set of biophysical methods, like dynamic light scattering, an hplc sedimentation assay and transmission electron microscopy. we have found that the polyq tract of ar presents a high degree of helicity. we attribute this conformation to the n-terminal flanking region, characterized by high helicity and we have tested this hypothesis by performing mutations. we have also observed that the rate of the first step of oligomerization is not dependent on the number of glutamine repeats, but instead is due to self interactions of a region n-terminal to and far from the polyq. its progression to fibril is dependent to the number of glutamines in the tract. we have therefore identified two steps in the aggregation process of ar, where a motif far from the polyq at its n-terminal drives the early oligomerization, followed by the interaction of the polyq chains that stabilize it and determine the progression to fibrils. these findings shed a light for possible interventions on the ar oligomerization process, thus suggesting a different strategy to study the onset of the disease in sbma patients. destabilizing the transient helical conformation of islet amyloid polypeptide hastens peptide self-assembly and potentiates cytotoxicity carole anne de carufel 1 , phuong trang nguyen 1 , alexandre arnold 1 , isabelle marcotte 1 , steve bourgault 1 1 amyloidogenic polypeptides can be divided into two different structural classes: those that are intrinsically disordered and those that show a well-defined structure in their monomeric soluble state. natively folded proteins, such as transthyretin, have to unfold (or misfold), at least partially, to form amyloids. in contrast, intrinsically disordered polypeptides, such as the islet amyloid polypeptides (iapp) and abeta peptide, need to undergo conformational rearrangements allowing the formation of locally ordered structure(s) to initiate the amyloidogenic process. studies have shown that iapp and abeta adopt an alphahelix conformation in the initial steps of amyloidogenesis. this intermediate is believed to be on-pathway to fibril formation, although this hypothesis is still the matter of debate. in this study, we designed human iapp (hiapp) derivatives in which alpha-helix destabilizing substitutions were incorporated into the putative helical segment of iapp to probe the initial structural event in amyloid formation. using trifluoroethanol titration, we observed by cd spectroscopy that strategic incorporation of d-amino acids at positions 15 and 16 leads to an iapp derivative (diapp) that cannot fold into a helix. in homogeneous solution, hiapp and diapp show similar kinetics of fibrillization, as measured by thioflavin t fluorescence. although their amyloid fibrils display different characteristics by afm, iapp and diapp are able to self-associate to form amyloids when mixed together and when seeded with one another. studies in heterogeneous environment, notably in presence of glycosaminoglycans and model membranes of dopc/dopg (7:3), showed a helical intermediate for hiapp while only a beta-sheet secondary structure was apparent for diapp. while the rate of amyloid fibril formation was increased for both peptides, diapp was drastically affected by these anionic biomolecules with an absence of lag phase. the incapacity of adopting a transient helical conformation accentuates cell toxicity, supported by the caspase 3/7 activation level and the increase in intracellular calcium level. overall, this study indicates that the helical intermediate is offpathway to iapp amyloid formation and offers novel mechanistic insights for the development of molecular identities modulating peptide self-assembly and iapp-induced cytotoxicity. for an organism to survive, its proteins must adopt complex conformations in a challenging environment where macromolecular crowding can derail even robust biological pathways. the situation is perilous: many diseases arise from improper folding of just a single protein. to cope, cells employ a repertoire of molecular chaperones and remodeling factors that usher unfolded proteins into active conformations, sequester them, or target them for degradation. yet, not all aggregated proteins are the result of mis-folding. yeast prions are self-templating protein-based mechanisms of inheritance that rely upon chaperones for their propagation. the best studied of these is the prion domain (nm) of sup35, which forms an amyloid that can adopt several distinct conformations (strains) that produce distinct phenotypes. using genetic, biochemical, spectroscopic, and solid state nmr techniques, we investigated the structural and dynamic underpinnings of sup35 amyloids and found that prion strains differ in both their atomic structure as well as their dynamic motions. interestingly, these mobility differences correlate with differences in the interaction with molecular chaperones in vivo. limitations on the specificity and sensitivity of biophysical techniques typically restrict structural investigations to purified systems at concentrations that are orders of magnitude above endogenous levels. therefore, i developed an approach to apply a sensitivity-enhancement technique for nmr, dynamic nuclear polarization (dnp), to investigate interactions between sup35 and molecular chaperones at endogenous concentrations in their native environments. critically, i found that the cellular environment induced structural changes in a region of sup35 that is intrinsically disordered in purified samples but known genetically to influence prion propagation from one generation to the next. this approach enables structural and mechanistic investigation of proteins in biologically relevant contexts. genetic instability within regions encoding repetitive proteins as a driver of adaptation stephen fuchs 1 1 more than ten percent of all eukaryotic proteins contain within them a region of repetitive amino acid sequence. these repetitive domains range from short stretches of a single amino acid to multiple copies of longer, heterogeneous amino acid sequences and generally show lack of defined structure. they play diverse roles in cells including acting as structural proteins, promoting cell-cell interactions, and mediating the assembly of molecular machines. tandem repeat proteins are known to be variable in length within cellular populations although the mechanisms dictating this variability have not been elucidated. here we describe work uncovering specific features within the coding sequences of repetitive proteins that contribute to tandem repeat instability in yeast. furthermore, we demonstrate that cells will expand and/or contract repetitive regions in order to adapt to environmental stresses and describe a role for dna repair proteins in this process. lastly, we demonstrate how these mechanisms are likely conserved in higher eukaryotes, including humans. this study uncovers the molecular basis for an important aspect of natural protein evolution and describes a novel mechanism for adaptation in response to environmental changes. a proline-tryptophan turn in the intrinsically disordered domain 2 of ns5a protein is essential for hepatitis hepatitis c virus (hcv) nonstructural protein 5a (ns5a) and its interaction with the human chaperone cyclophilin a (cypa), a peptidyl-prolyl cis-trans isomerase (ppiase), are both targets for highly potent and promising antiviral drugs that are in late stage of clinical development [1, 2] . despite its high interest in the development of drugs to counteract the worldwide hcv burden, ns5a is still an enigmatic multifunctional protein poorly characterized at the molecular level. ns5a is required for hcv rna replication and is involved in viral particles formation and regulation of host pathways. thus far, no enzymatic activity or precise molecular function has been ascribed to ns5a that is composed of a highly structured domain 1 (-d1), as well as two intrinsically disordered domains 2 (-d2) and 3 (-d3). ns5a-d1 structure has been solved by x-ray crystallography and ns5a-d2 and -d3 have been characterized by nmr spectroscopy. these two last domains do not adopt a stable 3d structure but rather exist as an ensemble of highly dynamic conformers. using nmr spectroscopy, hcv ns5a-d2 has been shown to establish a direct interaction with the human cypa and to be a substrate for the enzymatic ppiase activity of cypa [3] . the cypa interaction site in ns5a-d2 is composed of nearly 15 residues that correspond to the most conserved region of the domain, with 3 proline residues being strictly conserved among all hcv genotypes. whereas ns5a-d2 is mainly disordered, some of its nmr resonances, corresponding to residues in the cypa binding site, display unexpected 1h and 15n nmr chemical shifts for an intrinsically disordered domain. thus we have further characterized this region by nmr spectroscopy. a short structural motif in the disordered ns5a-d2 has been identified and we solved its nmr structure. in a cellular assay, we showed that this structural motif, a minimal pro314-trp316 turn, is essential for hcv rna replication. we demonstrated that this pro-trp (pw) turn is required for proper interaction with the host cypa and influenced its enzymatic ppiase activity on residue p314 of ns5a-d2. this work provides a molecular basis for further understanding of the function of the intrinsically disordered domain 2 of hcv ns5a protein. in addition, our work highlights how very small structural motifs present in intrinsically disordered proteins can exert a specific function. [1] [2] . this 27-residue peptide also shows toxicity towards mammalian cells but at higher concentrations, suggesting its possible usefulness as a treatment for trypanosomiasis. here we present the peptide's relative cytotoxicity for bloodstream and procyclic forms of t. brucei and for mammalian cells, the fate of the peptide in t. brucei using fluorescently-labelled bt-6, and its three dimensional structure using nmr spectroscopy.minimum inhibitory assays confirmed the peptide's selective toxicity towards both bloodstream and procyclic forms of t. brucei, demonstrating its potential to serve as a starting point for a trypanocidal drug. fluorescence spectrophotometric experiments, carried out using fluorescein labelled bt-6, show that the peptide is released from the external surface of the parasite into the suspending medium under de-energized conditions but retained in energized cells. heteronuclear and homonuclear biomolecular nmr experiments (tocsy, noesy, 1h-13c-hsqc,1h-15n-hsqc, etc) folowed by structural calculations (chemical-shift based as well as simulated annealing techniques) in the free state indicate that this peptide is mostly unstructured in aqueous solution, suggesting that there is a major conformational change upon binding to t. brucei that is required for uptake. we suggest that the evolutionary pressure that selected for the intrinsically disordered structure of this peptide was the advantage it conferred upon the host to bind to many different surface structures throughout the microbiological world. physikalische biologie, heinrich heine university, 2 structural biochemistry (ics-6), research centre j€ ulich, 3 chemistry and biotechnology, swedish university of agricultural sciences (slu) the misfolding and amyloid formation of proteins featuring intrinsically disordered regions is a pathological hallmark of several neurodegenerative diseases, including alzheimer's disease and parkinson's disease. engineered binding proteins targeting amyloidogenic proteins aid in the elucidation of the aggregation mechanism and suggest therapeutic strategies. we have constructed phage display libraries enriched in binders to amyloidogenic intrinsically disordered proteins, using zab3, a protein with high affinity for the amyloid-beta peptide, as a scaffold. binding proteins selected from these libraries are termed beta-wrapins (beta-wrap proteins). the beta-wrapins as69 and hi18 exhibit nanomolar affinity for monomeric alpha-synuclein or islet amyloid polypeptide, respectively. as69 and hi18 potently inhibit in vitro amyloid formation and toxicity at substoichiometric concentration ratios, indicating that they interfere with the nucleation and/or elongation of amyloid fibrils. the nmr structures of the betawrapin:target complexes reveal beta-hairpin motifs in alpha-synuclein and islet amyloid polypeptide which are stabilized by coupled folding and binding. in the case of alpha-synuclein, the beta-hairpin is formed in the sequence region 35-59 which contains the beta-strand segments b1 and b2 of amyloid fibril models and most disease-related mutations. we show by disulfide engineering, biophysical techniques, and cell viability assays that intramolecular tertiary interactions between the b1 and b2 segments of alpha-synuclein interfere with its aggregation, and moreover inhibit aggregation of amyloid-beta peptide and islet amyloid polypeptide. our results reveal a common preference of different amyloidogenic proteins for formation of beta-hairpin motifs and demonstrate a critical role of hairpin conformers in the control of amyloid formation. interaction profiling through proteomic peptide phage display cecilia blikstad 1 , moon-hyeong seo 2 , norman davey 3 , roland arnold 2 , sachdev s sidhu 2 , philip m kim 2 , ylva ivarsson 1 1 department of chemistry -bmc, 2 donnelly centre a considerable part of the human proteome is intrinsically disordered. the disordered regions are enriched in short motifs serving as docking sites for peptide binding domains. domain-motif interactions are crucial for the wiring of signaling pathways. these interactions are typically transient and difficult to capture through most conventional high-throughput methods. we therefore developed a novel approach for the large-scale profiling of domain-motifs interactions called proteomic peptide phage display (prop-pd) (1). in prop-pd we combine bioinformatics, oligonucleotide arrays, peptide phage display and next-generation sequencing. this allows the interrogation of domain-motif interactions on a proteome-wide scale and the de novo motif discovery.in our pilot experiment we generated two distinct phage libraries, one displaying all human c-terminal sequences and one displaying c-termini of known virus proteins. we used the prop-pd libraries to identify interactions of human postsynaptic density 95/discs large/zonula occludens-1 (pdz) domains. we successfully identified novel pdz domain interactions of potential relevance to cellular signaling pathways and validated a subset of interactions with a high success rate. recently, we created a prop-pd library that displays peptides representing the disordered regions of the human proteome. we validate our disorderome library against a range of peptide binding domains, which provides novel insights into their binding preferences and suggest interactions of potential biological relevance as will be presented here. prop-pd can be used to uncover protein-protein interactions of potential biological relevance in high-throughput experiments and provides information that is complementary to other methods. prop-pd is scalable and can be developed to any target proteome of interest. phosducin is a 30 kda phosphoprotein that regulates visual signal transduction by interacting with the gtbg; subunit of the retinal g-protein transducin. the function of pdc is regulated by phosphorylation at ser54 and ser73 in a process that involves the binding of phosphorylated pdc to the regulatory 14-3-3 protein, but the molecular mechanism of the regulation by 14-3-3 protein is still unknown. pdc was also suggested to be involved in transcriptional control, the regulation of transmission at the photoreceptorto-on-bipolar cell synapse, and the regulation of the sympathetic activity and blood pressure [1] [2] [3] . here, the solution structure of pdc and its interaction with the 14-3-3 protein were investigated using small angle x-ray scattering, circular dichroism, quenching of tryptophan fluorescence, analytical ultracentrifugation, hydrogen-deuterium exchange coupled to mass spectrometry and nuclear magnetic resonance. we show that the 14-3-3 protein interacts with and sterically occludes both the n-and c-terminal gtbg binding interfaces of phosphorylated pdc, thus providing a mechanistic explanation for the 14-3-3depedent inhibition of pdc function. the 14-3-3 protein dimer interacts with pdc using surfaces both inside and outside its central channel. the n-terminal domain of pdc, where both phosphorylation sites and the 14-3-3 binding motifs are located, is intrinsically disordered protein which remains likely highly flexible when bound to 14-3-3 indicating the fuzzy-like character of this complex. in addition, it has been speculated that the 14-3-3 protein binding decreases the rate of pdc dephosphorylation after a light stimulus through its interaction with phosphorylated ser54 and ser73, thus lengthening the time that pdc remains phosphorylated after a light exposure. pdc is dephosphorylated in vivo by protein phosphatases known to cause neurodegenerative disease in a polyglutamine-length dependent manner. despite intense study, the molecular basis of polyq toxicity in hd or any of the other diseases has only partially been elucidated and potential routes to therapeutic intervention are sparse. the use of genetically tractable model organisms to identify the cellular pathologies caused by mutant huntingtin expression is essential to our understanding of the disease pathology in humans. in eukaryotes, many of the protein folding homeostasis pathways are highly conserved and yeast cells expressing a glutamine-expanded fragment of huntingtin exon 1 exhibit a polyq length-dependent toxicity that recapitulates many of the basic protein folding defects associated with polyq diseases in neurons. taking an unbiased approach, we screened an overexpression library of the entire yeast genome for suppressors and enhancers of polyq toxicity and identified seven proteins with prion-like, q-rich domains that are strong suppressors in yeast. intriguingly, the q-rich domains of these proteins, and several other q-rich domains, suppress toxicity when expressed in isolation. these suppressors are also efficacious in mammalian cells and, strikingly, one suppressor was independently shown to alleviate polyq-expanded ataxin-3 toxicity in a drosophila model. in yeast, the suppressors co-aggregated with an otherwise highly toxic 103glutamine expanded huntingtin exon 1 protein (htt103q), resulting in a non-toxic aggregate and eliminating populations of diffusible oligomeric species. using a transcriptional sensor for protein coaggregation, we determined that yeast and human proteins that normally co-aggregated with htt103q did not co-aggregate with these hetero-aggregates. thus, these q-rich domains may suppress htt103q toxicity by two complementary mechanisms: trapping potentially toxic oligomers in larger aggregates and by limiting the interactome of the larger htt103q aggregates. structuring disorder: the case of the intrinsically disordered unique domain of c-src mariano maffei 1 1 about two thirds of eukaryotic proteins contain large intrinsically disordered regions. they represent a change of paradigm from "structure-function" to "information-function" (uversky, 2011; babu et al., 2011). structured proteins are information rich, but the current challenge is to discover how information is stored in disordered protein. regulation of c-src activity, the first discovered oncoprotein, by its intrinsically disordered n-terminal region has been recently demonstrated (perez et al., 2013). functional studies have revealed that mutations in the ulbr cause strong phenotypes when introduced in fulllength c-src and expressed in xenopus laevis oocytes (perez et al., 2013) or in human sw620 colorectal cancer cells (unpublished). however, the connection with the classical regulatory mechanisms is still missing. c-src domain structure consists of four "src-homology" domains: sh4, sh3, sh2 and sh1, arranged in this order from the n-terminus to the c-terminus, with the intrinsically disordered "unique" domain separating the sh4 and sh3 domains. classically, the sh3 and sh2 domains are involved in regulation and the sh4 domain is the membrane anchoring site. we will present our recent results showing that the unique domain is part of a long loop closed by the interaction of the sh4 and sh3 domains (maffei et al., 2015). the conformational freedom of this disordered region is further restricted through direct contacts between the rt-loop of the sh3 domain and, primarily, residues located within the recently discovered unique lipid binding region (ulbr). the interaction between the unique and sh3 domains is allosterically modulated by a poly-proline ligand binding to the canonical binding site of the sh3 domain (maffei et al., 2015) . these results demonstrate a direct connection between classical c-src regulation involving the sh3 domain and the new regulation mechanisms involving the intrinsically disordered regions and provide new evidence of the functional importance and the underlying mechanism behind regulation of signalling pathways by intrinsically disordered domains. in mammalian cells, the golgi reassembly and stacking proteins (grasp55 and grasp65) are involved in the stacking of golgi apparatus cisternae and in the formation of the golgi ribbon. since grasps have been identified in many organisms, other roles for grasps have already been pointed out, such as chaperoning and transport of other proteins, involvement in cell apoptosis, cell migration, unconventional secretion, and in mitosis. in saccharomyces cerevisiae, it is observed that only 40% of the golgi cisternae are in stacks and do not form ribbon structures. this build yeast contains a single grasp, called grh1, that is analogue to grasp65. the structural differences of the golgi apparatus and the functional repertoire of grasps suggest a structural dynamic of these proteins. here, we used a combination of biophysical/biochemical methods to investigate the behavior of grh1. bioinformatics and circular dichroism (cd) analyses of grh1 indicated a high percentage of either flexible regions or extended loops. the partial unfolded grh1 structure in solution folded into more ordered structures under temperature increasing, dehydration onto a surface and nonaqueous solvents as reported also by cd. hydration of the dehydrated folded protein is a reversible process that is accompanied by unfolding. furthermore, grh1 showed slow migration in sds-page, high susceptibility to proteases and low cooperativity of the chemical-induced unfolding process. fluorescence of trp residues along with cd data showed grh1 preserves a considerable amount of residual secondary structure, and the unfolding transition monitored by trp presented higher cooperativity. another cooperative transition was also reported by the extrinsic hydrophobic fluorescence probe ans upon chemical denaturation. these set of experiments indicate that grh1 behaves as a protein containing intrinsically disordered regions (idrs), characterized by unstructured regions of high polypeptide mobility experiencing many conformations. these findings suggest that an idp-like behavior may be the solution found by nature to account for grh1 functional need for interactions with several different partners in the cell. conformational changes governing dengue virus capsid protein function and its inhibition by pep14 23 andr e f. abstract dengue virus (denv) infection affects millions of people and is becoming a major global disease for which there is no specific treatment available. the interaction of denv capsid (c) protein with host lipid droplets (lds) is essential for viral replication. pep14-23, a peptide designed based on a denv c intrinsically disordered conserved region, inhibits this crucial interaction. combining bioinformatics and biophysics we determined pep14-23 structure and ability to bind different phospholipids, in the context of denv c function. pep14-23 becomes a-helical upon binding to anionic phospholipids. structure prediction of denv c n-terminal intrinsically disordered region reveals orientations that alternatively shield or expose denv c hydrophobic pocket, supporting a novel autoinhibitory role for this region. these findings pave the way for similar studies to understand disordered proteins and improved peptidomimetics drug development strategies against flaviviruses. topics intrinsically disordered proteins protein-lipid interactions pf-015 developing mechanistic insight into modulators of tau aggregation eri nakatani-webster 1 , hannah baughman 1 , shaylin higgins 1 , abhinav nath 1 1 the pathological self-association of microtubule-associated protein tau is implicated in a range of neurodegenerative disorders collectively called tauopathies, perhaps the most prominent of which are alzheimer's disease (ad) and chronic traumatic encephalopathy (cte). tau aggregation in vitro shares many features in common with fibril formation by other amyloid-forming proteins: a nucleationdependent polymerization reaction progressing via oligomeric intermediates into b-sheet-rich fibrillar aggregates, characterized by a distinctive sigmoidal kinetic. over the years, many investigators have advanced our understanding of how these time-courses might best be characterized and interpreted. in particular, elegant analytical and numerical approaches have been developed that supersede the empirical sigmoidal equations typically used to fit fibril formation traces. these modern approaches have enabled more rigorous insight into the mechanism of amyloid formation, and into how small molecules, protein chaperones, and other binding partners can modulate the process. an understanding of a modulator's effects on amyloid formation mechanism is necessary in order for us to predict and engineer its effects on amyloid pathology in a biological context. a given modulator may affect rates of primary or secondary nucleation, elongation, or fibril fragmentation to different extents. each of these perturbations, individually or in combination, can alter the kinetics of aggregation, the final state of the amyloid fibrils, and the sampled ensemble of oligomeric intermediates. unfortunately, fitting of mechanistic models to amyloid formation kinetics is an example of an "ill-posed problem", in that dramatically different combinations of elementary parameters can nevertheless generate very similar sigmoidal kinetic traces. this has typically necessitated global analysis of amyloid kinetic traces collected over a broad range of protein concentrations -a substantial expenditure of time, effort and material that must then be repeated in the presence of a modulator in order to gain insight into its effects. we propose an alternative approach: to fit amyloid formation traces to a large distribution of parameter sets, and determine how various aggregation modulators affect the distribution of parameters. this socalled "parameter distribution analysis" enables the inference of mechanistic effects from measurements at a single protein concentration. parameter distribution analysis based on numerical modeling has been made tractable by advances in computer hardware and software, and can be easily extended to include additional mechanisms or phases relevant to a protein or modulator of interest. here, we illustrate how parameter distribution analysis, complemented by fluorescence correlation spectroscopy (fcs), electron microscopy (em) and other biochemical techniques, can shed light on fundamental aspects of tau amyloidogenesis. we examine the disparate effects that natural products, pharmacotherapies and protein chaperones can have on the mechanism of aggregation, and also discuss the effects of heparin (widely used as an inducer of tau aggregation). these insights demonstrate the value of parameter distribution analysis as applied to amyloid formation and other ill-posed biochemical problems. new insights into amyloidogenesis of tau protein induced by enantiomers of polyglutamic acid amyloidogenesis of tau protein leads to the formation of amyloid fibrils (ordered fibrillar protein aggregates) which are accumulated in neurons of central nervous system during the course of neurodegenerative diseases called tauopathies. studying tau (a typical intrinsically disordered protein) amyloidogenesis has been challenging for many reasons. positive charge on the tau molecule must be compensated (e.g. in the presence of polyanions) in order to initiate the process. heparin (glycosaminoglycan) has been the most intensively studied charge-compensating agent in this context. on the other hand induction of tau aggregation by polyglutamic acid is poorly characterized. mechanisms responsible for the propagation of tau conformations has become an interesting research objective. prion-like features of tau amyloid can be studied in vitro also in the seed-induced regime of aggregation. tau amyloid seeds can act as nuclei for amyloidogenesis. such seeds can be obtained by fragmentation of amyloid fibrils by means of sonication. given that amyloidogenesis can proceed through various assembly pathways resulting in distinct amyloid 'strains' (self-propagating structural variants of amyloid) we have used poly-l-glutamic acid (plga) and poly-d-glutamic acid (pdga) to direct tau onto different amyloidogenic pathways. we have hypothesized that the chirality of the inducers could lead to fibril polymorphism. in our studies, we have used a recombinant human 2n4r tau isoform. we have been using transmission electron microscopy (tem), sedimentation and kinetic measurment. firstly, we have characterized unseeded plga-/pdga-induced tau aggregation to find out that corresponding kinetics were significantly different. secondly, we have used sonicated fibrils to characterize the kinetics of seeded processes. both plga-/pdga-induced amyloid seeds were able to efficiently seed tau aggregation in the presence of plga, whereas in the presence of pdga the aggregation was much less effective. surprisingly, we found that pdgainduced amyloid seeds were able to catalyze fibrillogenesis of tau more clearly in the presence of soluble plga than in the presence of pdga -the primary inducer. we could not induce aggregation of tau in the absence of polyglutamic acids which indicates that positive charge on tau molecules must be unconditionally compensated in order to promote amyloidogenesis. thirdly, using tem we have characterized different morphologies of tau amyloid fibrils generated in unseeded and seeded processes. finally, to further characterize properties of the fibrils we have performed sedimentation experiments. fibrils induced by plga, pdga and heparin revealed different sedimentation properties. heparin-induced fibrils underwent sedimentation more readily than pdga-induced fibrils, whereas plga-induced fibrils remained in the supernatant. these results indicate distinct physicochemical properties of these fibrils. we believe that our findings will contribute to the current understanding of the molecular dynamics of tau amyloidogenesis. self-organizing structures of alpha-synulceins and its aggregates by a coarse-grained monte carlo simulation ras pandey 1 , peter mirau 2 , barry farmer 2 1 alpha-synuclein (asn) consisting of 140 residues, an intrinsically disordered protein, is linked to such neurodegenerative diseases as parkinson's disease (pd) and alzheimer disease via toxic clumping into abstract amyloid fibrils. we investigate the structure and dynamics of an asn chain as a function of temperature by a coarse-grained approach where a residue is represented by a node. in our coarse-grained approach, a residue is represented by a node. the basic idea is borrowed from the 'united atom' approach in polymer chain modeling that has been used extensively where the benefits and pitfalls of the method is explored for decades. such coarse-grained method has also been used protein chain modeling in recent years (e.g. aip advances 5, 092502 (2015)). although the atomic scale structural resolution is sacrificed its specificity is captured via a set of unique knowledge-based residue-residue interactions matrix (e.g. classic miyazawa-jernigan matrix, macromolecules 18, 534 (1985)). a number of local and global physical quantities are analyzed such as contact map, neighborhood and mobility profiles, mean square displacement of protein, its radius of gyration and the structure factor. based on the mobility profile, we are able to identify three distinct segment of asn along its contour, i.e. sluggish nterminal (1-60) and c-terminal (96-140, least mobile) separated by the central region (61-95), the nonamyloid component (nac) with higher mobility. contact profile shows that the probability of intrachain residue aggregation (clumping) is higher in the n-terminal region than the c-terminal with least aggregation in the nac region. we find that the radius of gyration (rg) decays monotonically with the temperature, consistent with the finding of allison et al. (jacs, 131, 18314 (2009) ). from the detail analysis of the structure factor we are able to predict the variation of the spatial mass distribution with the temperature as the residues in asn chain organize and disperse by evaluating its effective dimension d. we find the protein conforms to a globular structure (d3) at the low temperatures and to a random coil (d2) at high temperatures which is consistent with the estimates of uversky et al. (j. biol. chem. 277, 11970 (2002)). in addition, we provide the estimates of d (3 d 2) for the intermediate structures as the protein chain makes a transition from globular to random coil. questions under-investigation includes what are the effects of mutations (e.g. b-and g-synuclein), how does the structure of an isolated asn chain change in presence of many interacting protein chains, and how do they organize over the multiple length scales? attempts will be made to address some of these issues as the data become available. tear down the wall: dismantling the biofilm scaffold of e.coli cesyen cedeno 1 , nani van gerven 1 , wim jonckheere 1 , imke van den broek 1 , han remaut 1 , peter tompa 1 1 csga is the major subunit of the so-called curli fiber system. this is an amyloid structure formed in the outer membrane on e.coli and acts as a scaffold for the biochemical machinery/matrix in the extracellular milieu (biofilms). extracellular matrices of this nature are robust platforms helping bacteria colonization; in this context csga becomes a key target in order to break the architecture within bacterial biofilms. chaperones are molecular machines able to stabilize misfolding prone proteins or even retrieve proteins trapped in non-physiological states. here we show how erd14 acts as a molecular chaperone inhibiting the formation of csga amyloid fibers in vitro. this work illustrates an alternative approach towards biofilm treatment at a molecular level. coupled folding and binding of transcription factors sarah shammas 1 , alexandra travis 1 , jane clarke 1 1 intrinsic protein disorder is ubiquitous in transcription, particularly within transcription factors, which frequently fold into structures upon binding to partner molecules (dna or protein). the coupled folding and binding reactions that take place between individual transcription factors and the key hub co-activator proteins are crucial in determining the expression profile of the cell, and hence its phenotype. these interactions have been well studied by structural and equilibrium methods. here we present mechanistic insights into the process, gained through complementary kinetics experiments, for the binding of five separate transcription factors to a single prototypical co-activator (cbp kix). the transcription factors investigated belong to cellular (cmyb, mll, creb, e2a) and viral (htlv-1 blz) classes. these reactions are remarkably fast; after removing the effect of long-range electrostatic rate enhancement the association rate constant is still approximately 2 x 10 7 m-1s-1, which is just above the typically quoted upper limit for diffusion-limited reactions between pairs of proteins (10 5 -10 6 m-1s-1), and is also the highest such value we have found reported. this, combined with the apparent insensitivity of the association rate to residual structure within the unbound state, indicates that binding preceeds folding (induced fit mechanism). interactions between kix and its transcription factors are additionally modulated by allostery between its two binding sites. we investigate the basis for this, finding it to be mediated by changes in protein flexibility. alternative hit finding strategies for intrinsically disordered proteins, exemplified by forkheadbox transcription factors harm jan (arjan) snijder 1 , maria saline 1 , tomas jacso 1 , frank janssen 1 , mattias rohman 1 , tyrrell norris 1 1 astrazeneca r&d, discovery sciences, se-431 83,pepparedsleden 1 forkhead box o (foxo) proteins are emerging as key transcription factors in insulin and glucose metabolism, regulation of immune responses, and to balance cell proliferation, apoptosis and senescence. foxo proteins are predicted to be intrinsically disordered proteins (idps); idps are largely unstructured and often function as hubs mediating multiple interactions. idps are considered to be largely evasive from classical small molecule interference and lead-generation approaches, as they lack defined binding pockets. the available methods for addressing these targets have been lagging behind and needs to be developed to assess tractability of this target class. here we have evaluated the tractability of fragment screening on various domains of a forkhead box o member. we could confirm the intrinsically disordered character of foxo and used nmr screening to identify fragments that interact with foxo. one of these fragments was subsequently confirmed as a direct foxo binder in 2d hsqc-nmr spectroscopy and this fragment showed an effect in a foxo reporter gene assay. these results demonstrate that fragment screening may be a valuable approach for intrinsically disordered proteins although challenges remain to expand these fragments into more potent hits in the absence of detailed structural data. the characterization of amyloid-beta peptide (abeta) oligomer samples is critical to advance in the field of alzheime rs disease (ad). here we report a critical evaluation of two methods used for this purpose, namely sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), extensively used in the field, and electrospray ionization ion mobility coupled to mass spectrometry (esi-im-ms), an emerging technique with great potential for oligomer characterization. to evaluate their performance, we first obtained pure cross-linked abeta40 and abeta42 oligomers of specific order. analysis of these samples by sds-page revealed that sds affects the oligomerization state of abeta42 oligomers, thus providing flawed information on their order and distribution. in contrast, esi-im-ms provided accurate information, while also reported on the chemical modifications and on the structure of the oligomers. our findings have important implications as they challenge scientific paradigms in the ad field built upon the sds-page characterization of abeta oligomer samples. coarse-grained simulation of protein association: application to rate prediction and implication for association mechanisms yinghao wu 1 , 1 the kinetics of protein binding is of paramount importance for understanding cellular functions. for instance, the binding kinetics between membrane receptors and their ligands control the speed of signal transduction after cells are exposed to stimulation. the experimentally measured association rates of protein binding span ten orders of magnitude, a range that was divided into two regimes. it was proposed that a fast association regime is limited by protein diffusion, while the other side of the spectrum is controlled by conformational changes. consequently, all previous simulation methods neglected conformational changes when calculating the association rate of a diffusion-limited regime. however, the most updated theory of protein binding suggests that a protein remains in a pre-existing equilibrium of unbound conformations. binding shifts the equilibrium toward its bound state. this highlights the importance of conformational factors for regulating protein binding. enlightened by this conformational selection model, we hypothesize that the conformational flexibility of protein structures regulates association more widely than previously anticipated. we develop a new coarse-grained model to simulate the process of protein association via the kinetic monte carlo (kmc) algorithm. each residue in this model is represented by its ca atom and a side-chain functional site. a simple physically based potential is used to guide the relative diffusion of two interacting proteins. given the size of the simulation box and the length of the simulation, the association rate constant can be derived by counting the frequency of dimerization among a large number of simulation trajectories. we further designed a prediction strategy that accounts for both the conformational and energetic factors of binding. our method is able to predict rates of protein association that are highly correlated with experimentally measured values. due to the coarse-grained feature, our model was further applied to several special cases of protein association. in one example, we studied the binding kinetics of proteins with flexible linkers. the interaction between thrombin and its functional inhibitor, rhodniin, was used as a testing system. we captured the conformational changes of flexible linkers from the all-atom molecular dynamic simulations. we found that the association with full-length flexible rhodniin was faster than its two individual domains and that their dissociation was more difficult, supporting a "flycasting" mechanism in which partial structures of an intrinsic disordered protein (idp) dock to the target first, while the remaining segments undergo conformational searches and sequentially coalesce around the target. in another example, we studied the binding kinetics of membrane receptors from cellular interfaces. the interaction between membrane proteins cd2 and cd58, cell adhesion molecules known to mediate the activation of t cells and natural killer cells, was used as a testing system. the diffusive properties of these proteins on lipid bilayer were captured from all-atom molecular dynamic simulations. we showed that both 3d and 2d association rates could be simulated quantitatively with our method. the calculated values were close to the experimental measurements. we also provided detailed analysis of how molecular diffusions and membrane fluctuations affected 2d association. pf-023 (un)structure-function relationships on the ureg enzyme in the nickel-dependent urease system barbara zambelli 1 , francesco musiani 1 , stefano ciurli 1 1 urease is an essential enzyme for many pathogens and soil microorganisms. its activity relies on the presence of nickel in the active site (1) . the incorporation of this metal ion into the enzyme requires the formation of a supra-molecular chaperone involving four accessory proteins, named ured, uref, ureg and uree. uree is a metallo-chaperone involved in nickel binding and delivery into the enzyme active site. ureg is a gtpase essential for providing energy to the process of nickel site assembly. uref and ured form a complex that regulates the gtpase activity of ureg. the present work focuses on ureg, which exists in solution as an ensemble of inter-converting conformations (2) . this observation made this protein the firstly discovered natural enzyme with an intrinsically disordered behavior, possibly allowing it to interact with different protein partners, such as uree (3, 4) and uref (5) and cofactors, such as metal ions (6), in the urease activation network. ureg folding was studied perturbing protein conformation with temperature and denaturants, and investigating its folding response using circular dichroism, nmr and fluorescence (7). a combination of light scattering, calorimetry, mass spectrometry, and nmr spectroscopy shed light on the effect of metal ion binding onto the conformational equilibrium of ureg ensemble (8) . the results suggest that metal binding and solution conditions modulate affect the protein-protein interactions and enzymatic activity of ureg. nuclear inclusion protein a-protease (nia-pro) is a protease involved in processing of pepper vein banding virus (pvbv) encoded polyprotein to generate various intermediates and mature proteins at different stages of the viral life-cycle. nia-pro has two domains-n-terminal viral protein genome linked (vpg) and the c-terminal protease domain (pro).vpg belongs to the group of proteins that are intrinsically disordered, but attain stable structures upon interaction with other globular proteins. such proteinprotein interactions have a regulatory role on the function of the interacting partners. previously, the influence of vpg domain on the activity of pro was studied and it was shown that there was a substantial increase in the protease activity upon interaction with vpg (both in cis and in trans). in the present investigation, several deletion mutants of vpg and nia were constructed with a view to delineate the domain of vpg involved in interaction with pro. it was observed that deletion of residues from nterminus of vpg resulted in a decrease in the activity of pro in cis and in trans probably because of the abrogation of interaction between the two domains. interaction studies using spr (surface plasmon resonance) and elisa confirmed that the n-terminal 22 residues of vpg are important for interaction with pro. the n-terminal 22 residues of vpg are a part of the disordered region of vpg and their deletion resulted in the change in the secondary structure of the vpg and its oligomeric state. the ser 129 and trp 143 residues of pro domain were shown to be important both for the interaction of the two domains and for the activity of protease by mutational analysis earlier. these residues were identified to be a part of wc loop (w143-c151) which relay the conformational changes to the active site catalytic triad (his 46, asp 81 and cys 151) leading to activation. however, mutations of these residues did not completely abolish the protease activity as well as the interaction with vpg. therefore, in the present study h142 and h167 which are observed to interact with trp143 and c151 (via non-covalent interactions) were mutated to alanine and the h142a and h167a mutants showed a drastic reduction in the activity of protease. molecular dynamics simulations of the wild type pro and the mutants revealed that trp 143 -his 142 -his 167 -cys 151 interaction pathway of the wild type pro was disrupted in the mutants and additional residues were involved in the interaction pathway, such alterations in the network of interactions could be responsible for the loss of activity. however, a change in the oligomeric status of these mutants was also observed as compared to the wild-type pro, suggesting that these residues are important for both the structural and functional integrity of pro and its interaction with vpg. thus, these results provide a molecular insight into the vpg-pro interactions and the modulation of their structure and function upon mutation of residues that are part of the interaction interface. transthyretin (ttr) is one of many proteins that are capable of forming amyloid fibrils in vivo. this protein is associated with two distinct amyloidosis: familial cardiac amyloidosis (fca) that causes a restrictive cardiomyopathy and familial amyloid polyneuropathy (fap) that affect peripheral nerves, they are hereditary and caused by mutations in the ttr gene. the non mutated protein can also aggregate in cardiac tissue in advanced age patients. the diagnosis was established at university hospital since 2008 due to a collaborative between our group and the center of amyloidosis antônio rodrigues de mello (ceparm). the only mutation found in brazil was v30m in 3 patients diagnosed in france. our group discovered 5 new mutation not described in brazil and a novel mutation not described yet a19d. the diagnosed patients are registered in transthyretin amyloidosis outcomes survey (thaos). the novel mutation a19d causes a severe restrictive cardiomyopathy that is certainly related to a higher profile of aggregation observed for this mutant if compared to others amyloidogenic mutants of ttr. structural predictions using a bioinformatics tool called foldx showed that the insertion of the mutation cause a electrostatic clash that facilitates the dissociation and aggregation of protein. this mutant was purified heterologously and biophysical studies revealed that this protein is a dimer and not a tetramer as commonly the ttr structure. the crystallographic structure indicates that this mutant is structurally identical to wild type. biophysical studies revealed that this protein is a dimer and not a tetramer as commonly the ttr structure. the thermodynamic stability of a19d is lower than the wild type ttr. the aggregation profile showed us that this protein can aggregate in a higher manner and with a fast kinetic to that observed for others amyloidogenic mutants of ttr, forming fibers in two hours of aggregation. heterotetramers of a19d and wt are able to aggregate in the same fiber structure. the analysis of interface interaction of this mutant using the pdbsum showed modifications in the profile of hydrogen bonds and non bonded contacts. in addition the oligomers of a19d are toxic for primary culture of cardiomyocytes from murine heart. the amyloidogenic profile displayed by this new mutant can be directly correlated with the aggressiveness observed in the disease developed by the identified patient. furthermore the recent consolidation of ttr diagnosis in our university hospital led to the identification of the rare a19d variant in a brazilian patient, suggesting that other new, uncharacterized mutants could be identified in the coming years. multiple cellular proteins interact with ledgf/p75 through a conserved unstructured consensus motif [1] . the ledgf/p75-mll1-menin complex was structurally characterized, but only partially [2] . using nmr spectroscopy, we identified and mapped a novel mll1-ledgf/ p75 interface. colony forming assays in mll1-af91 leukemic cells expressing mll1 interactiondefective ledgf/p75 mutants revealed that this additional interface is essential for leukemic transformation. interestingly, the newly defined interface overlaps with the binding site of known ledgf/p75 interactor, the hiv integrase [1] . while the pathophysiological interactions of ledgf/p75 are intensively studied, its physiological role remains unclear. since ledgf/p75 contributes to hiv integration and leukemic transformation and has become a new therapeutic target for drug development, it is crucial to study its physiological interactions. in addition to hiv in and mll1-menin, the ledgf/p75 integrase binding domain (ibd) also interacts with several other proteins [3, 4] . our recent data (manuscript accepted in nat. commun.) revealed structural details of ledgf/p75 interactions with physiological binding partners. the interaction with the ledgf/p75 ibd is maintained by an intrinsically disordered ibd-binding motif (ibm) common to all known cellular partners. based on the knowledge of this motif, we identified and validated iws1 as a novel ledgf/p75 interaction partner. naturally occurring single mutants, i56t, f57i, w64r and d67h of lysozyme in human, have been known to form abnormal protein aggregates (amyloid fibrils) and to accumulate in several organs, including liver, spleen and kidney, resulting in familial systemic amyloidosis. these human pathogenic lysozyme variants are considered to raise subtle conformational changes compared to the wild type. here we examined the effects of the aberrant mutant lysozymes i56t, f57i,w64r and d67h, each of which possesses a point mutation in its molecule, on a cultured human cell line, hek293, in which the genes were individually integrated and overexpressed. western blot analyses showed lesser amounts of these variant proteins in the medium compared to the wild type, but they were abundant in the cell pellets, indicating that the modified lysozyme proteins were scarcely secreted into the medium but were retained in the cells. immunocytochemistry revealed that these proteins resided in restricted regions which were stained by an endoplasmic reticulum (er) marker. moreover, the overexpression of the mutant lysozymes were accompanied by marked increases in xbp1s and grp78/bip, which are downstream agents of the ire1_ signaling pathway responding to the unfolded protein response (upr) upon er stress.rnai for the mutant lysozymes' expression greatly suppressed the increases of these agents. next, we addressed the interaction between amyloidogenic lysozyme and grp78/bip as the former proteins were obtained by immunoprecipitation with the latter protein as well as colocalization of both proteins in the er. lysozyme composes of a-domain rich in helices and b-domain rich in sheet. two helices of a1 and a2 in the n-terminal region arrange in parallel and face to face where hydrophobic amino acids at the 3f, l8, l12, l15, l25 and l31 allocate with equal interval there. in the back of dock, there is a core region of amyloid fibril formation, of which the side chain of i56 is exposed on the protruding. probably, these hydrophobic amino acids might be crucial for lysozyme folding. although mutated lysozymes undergo folding by grp78/bip in such environment, the dissociation of the grp from lysozyme by failure of folding is likely inhibited and both proteins remain bound to, resulting in staying to the er. a part of aberrant lysozymes seem to remain bound to grp78/bip during folding and insolubilize with aggregation, thus accumulate in the er accompanied with er stress. lysozyme amyloidosis might be caused by long-term accumulation in the endoplasmic reticulum of the abnormal protein. structural characterization of toxic oligomers that are kinetically trapped during alpha-synuclein fibril formation the accumulation of abnormally aggregated proteins within the body is a common feature of several medical disorders, such as alzheimer's disease, parkinson's disease and diabetes mellitus type 2. while the specific protein found to be the major component of such deposits varies from one disease to another, the formation of the pathological aggregates seems to occur via a common process of misfolding and self-assembly of a normally soluble polypeptide chain into a series of oligomeric intermediates and, ultimately, into insoluble amyloid fibrils that accumulate within specific organs and tissues. increasing evidence indicates that certain oligomeric protein species generated during the self-assembly of specific proteins into ordered fibrillar aggregates can be highly cytotoxic and are likely to be key players in the initiation and spreading of neurodegenerative diseases. however, little detailed structural information is currently available for these oligomeric species due to their often transient nature and, more importantly, because of their variability in terms of size and structure. we report here the isolation and detailed characterization of an ensemble of stable toxic oligomers of alpha-synuclein, the protein whose deposition is the hallmark of parkinson's disease. by defining and minimizing the degree of heterogeneity of these isolated alpha-synuclein oligomers which have accumulated during the process of amyloid formation, we have identified distinct subgroups of oligomers and determined their structural properties and three-dimensional molecular architectures. this characterization has been achieved by the application of a set of complementary biophysical techniques, including a variety of spectroscopic techniques along with analytical ultracentrifugation, atomic force microscopy, and electron microscopy. although these oligomers exist in a range of sizes, with different extents and nature of beta-sheet content and exposed hydrophobicity, all the oligomeric subgroups possess hollow cylindrical architectures with marked similarities to amyloid fibrils. this suggests that these types of oligomers are kinetically trapped during protein self-assembly and that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their beta-sheet structures. our findings reveal the inherent multiplicity of pathways of protein misfolding and the key role the beta-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the rates of structural conversions, and thus the kinetic stabilities and pathological nature of different amyloid oligomers. the results of this study provide the basis for a more complete understanding of the nature of the self-assembly of polypeptides into beta-sheet rich amyloid aggregates, and potentially contributes to efforts to identify specific targets for drug discovery. fish otoliths and mammalian otoconia, biominerals composed of calcium carbonate and organic matrix, are involved in the functioning of the inner ear, the sensory organ that plays an important role in hearing and balance [1] . however, their developmental origins, growth, and the role of the matrix, especially the protein component, are still poorly understood. it has been shown that proteins involved in the formation of biominerals are usually very acidic. they often belong to the group of intrinsically disordered proteins (idps), a class of proteins devoid of a rigid tertiary structure [2, 3] . the shape and polymorph selection of calcium carbonate otolith in danio rerio is controlled by the starmaker (stm) protein [4] . recently, a gene was identified encoding the starmaker-like (stm-l) protein from oryzias latipes, a putative homologue of stm. it has been suggested that stm-l has a similar function as stm, although there is no sequence similarity between stm and stm-l [5] . several methods, such as size exclusion chromatography, cd spectroscopy and analytical ultracentrifugation demonstrated that stm-l is an coil-like idp, with the tendency to form locally ordered structures [6] . because stm-l was suggested to play a crucial role in calcium carbonate mineralization, it is possible that calcium ions may influence its conformation, as was previously shown for stm [7] . however, other ions may also be involved in this process. the aim of this study was to investigate the effect of mono and divalent metal ions on the conformation of stm-l. we used single molecule f€ orster resonance energy transfer (smfret) and fluorescence correlation spectroscopy (fcs), which have shown that calcium ions compacts the proteins most efficiently, followed by magnesium and the monovalent ions. the difference in the effect of monovalent and divalent ions on the protein dimensions is likely to result from the different properties of the ions, like charge density and radius. cd experiments have shown that a high excess of calcium ions caused the formation of ordered secondary structure in stm-l, which may be crucial for the formation of calcium carbonate crystals, when the ratio of building ions to protein is high. it has been demonstrated that dmp1 is proteolytically processed into fragments, including 37k n-terminal region and 57k c-terminal region. as many proteins characterized to be engaged in biomineralization, dmp1 and its fragments belong to the group of intrinsically disordered proteins (idps). it has been suggested that dmp1 and its fragments can take a part in otoconia mineralization, as the protein is present in mouse otoconia, but the role of dmp1 and its fragments in the mineralization of calcium carbonate has not been examined until now. to determine the influence of the dmp1 fragments for otoconia development, 57k dmp1 protein was expressed in bacterial expression system, purified and used in in vitro biomineralization test of calcium carbonate. in particular, immobilized metal anion affinity chromatography (imac) was applied as a first step of purification procedure. because of high content of acidic amino acids, ion exchange chromatography with a mono q column was used as a next step. the development of insects is regulated by the combined action of ecdysteroids and juvenile hormones (jh). pulses of 20-hydroxyecdysone (20e) initiate each step of metamorphosis, while jh modulates its action and prevents precocious differentiation. the biological and molecular mechanism of 20e action is well described. in contrary, the way of the jh activity is still poorly understood. in 1986 wilson and fabian [1] reported that drosophila melanogaster mutants lacking met are resistant to toxic doses of jh and its analogue methoprene. it has been proved, that met binds jh at physiological conditions. therefore met is believed to be a putative jh receptor. met may also be involved in a cross-talk between two hormonal signalling pathways, involving 20e and jh. the detailed structure of met is still unknown. therefore our main aim is to characterize structural properties of met. in silico analysis performed on a full-length met suggested, that n-terminal part of met contains three conserved domains characteristic for bhlh-pas transcription factors, whereas c-terminal part is most probably unstructured. 2010)). capitalizing on self-and cross-amyloid interactions, we designed highly effective, peptide-based inhibitors of amyloid self-assembly of abeta and iapp. due to their favourable properties the designed peptides are promising leads for targeting protein aggregation in ad, t2d or both diseases while the inhibitor design strategy should be applicable to other amyloidogenic polypeptides and proteins as well. apoptosis, the process of programmed cell death, must be carefully regulated in multi-cellular organisms to ensure proper tissue homeostasis, embryonic development and immune system activity. the bcl-2 family of proteins regulates the activation of apoptosis through the mitochondria pathway. dynamic interactions between pro-and anti-apoptotic members of this family keep each other in check until the proper time to commit to apoptosis. the point of no return for this commitment is the permeabilization of the outer-mitochondrial membrane (omm). translocation of the pro apoptotic member, bax, from the cytosol to the mitochondria is the molecular signature of this event. molecular interactions and conformational changes associated with this event have been difficult to obtain due to challenges associated with taking subtle measurements in the complex environment of live cells. to circumvent these challenges, we developed a novel method to reliably detect f€ orster resonance energy transfer (fret) between pairs of fluorophores to identify intra-molecular conformational changes and inter-molecular contacts in bax as this translocation occurs in live cells. in the cytosol, our fret measurements indicated that the c-terminal helix is exposed instead of tucked away in the core of the protein. this coincided with measurements using fluorescence correlation spectroscopy (fcs) that showed that cytosolic bax diffuses much slower than expected, suggesting possible complex formation or transient membrane interaction. we propose that this exposed helix allows for this contact to occur. cross-linking the c-terminal helix (a9) to helix a4 reduced the instances of these interactions while at the same time yielded fret measurements that are consistent with the a9 helix tucked into the core of the protein. after translocation, our fret measurements showed that bax molecules form homo-oligomers in the mitochondria through two distinct interfaces involving the bh3 domain (helix a2) and the c-terminal helix. these findings provide insight into the molecular architecture that may involve possible contacts with other bcl-2 proteins to permeabilize the omm, which would also be necessary for the regulation of apoptosis. abstract spatial resolution is especially advantageous for bacterial cells because of their small sizes. in the past few years the spatial organization and dynamics of a variety of bacterial cellular structures and protein macromachineries have been revealed with unprecedented details. as the field matures, it is now time to focus on the functional aspect of the observed spatial organizations and dynamics. are they essential in carrying out a specific cellular function? do they play a regulatory role in controlling the on and off of a certain cellular process? in this work i will present a few examples from our laboratory that examine the spatial and functional organization of macromolecules involved in bacterial cell division. transcription factors (tf) exert their function by interacting with other proteins and binding to dna. the nucleus is a compartmentalized space, and the spatial organization of tfs and their partners represents other step of gene expression regulation. we used the glucocorticoid receptor (gr) as a model of tf's mechanism of action. gr is a ligand-activated tf with a relevant role in physiology and a great variety of effects. it can be recruited to specific response elements on dna or interact with other tfs. also, the activity of gr is modulated by different co-regulators, e.g. tif2/grip1. gr and tif2 do not distribute homogeneously within the nucleus but accumulate in distinctive clusters. the functional role of this particular intranuclear organization remains unknown. we used advanced fluorescence microscopy techniques to study the dynamics of gr and tif2 in the nucleus of living cells with high spatial and time resolution. gr and tif2 fused to fluorescent tags were transiently expressed in newborn hamster kidney (bhk) cells and visualized by a confocal microscope. fluorescence correlation spectroscopy (fcs) experiments were carried on to measure the intranuclear mobility of both proteins. the method is based on the analysis of fluorescence intensity fluctuations due to the movement of fluorescent molecules in and out the confocal volume. the data could be fitted with a model that considers a free diffusion of tif2 and gr in the nucleus and their binding to fixed targets. we also studied the dynamics of different gr mutants in the presence of different ligands and our results suggest that the binding depends on dna. both gr and tif2 autocorrelation curves reveal an increase in the bound population upon gr activation by its agonist dexamethasone (dex). a cross-correlation analysis showed that, as expected, dex-stimulus increases the population of gr-tif2 complexes. without hormone, gr shows a homogeneous distribution and tif2 forms large clusters in the nucleus. upon dex-binding, gr accumulates in the nucleus, is rapidly recruited to tif2 foci and there is an important re-distribution of both proteins, that co-localize in the same pattern of small intranuclear clusters. the dynamics of gr and tif2 molecules at these clusters were studied by performing orbital-scanning measurements, tracking the clusters position in silico and analyzing the intensity fluctuations of the clusters along time. a positive cross-correlation between both channels indicates that dex-bound gr and tif2 interact at these foci and dissociate from them forming tif2-gr hetero-complexes. in conclusion, advanced fluorescence microscopy methods allowed obtaining a dynamical map of gr distribution and function in the nucleus of mammalian living cells. assembly of membrane pores as a mechanism for amyloid cytotoxicity by the bacterial prionoid repa-wh1 cristina fern andez 1 , rafael n uñez-ramirez 1 , mercedes jimenez 1 , germ an rivas 1 , rafael giraldo 1 1 amyloid fibril formation is associated with human neurodegenerative diseases. prefibrillar oligomers formed during the fibril assembly process, rather than mature fibrils are known to be central to disease abstract and may be responsible for cell damage. a commonly proposed mechanism for the toxicity of small oligomers is their interaction with the lipid bilayer of cell membranes, leading to loss of membrane integrity [1] . recent studies from our laboratory have shown that repa-wh1, a winged-helix domain from a bacterial plasmid replication protein, can assemble into amyloid fibrils in vitro. when expressed in escherichia coli repa-wh1 functions as a cytotoxic protein that shares features with the mammalian amyloid proteinopathies. these features have proved repa-wh1 to be a suitable synthetic model system to study protein amyloidosis [2, 3, 4] . in this work, using the repa-wh1 bacterial model system, we have studied the interaction between the protein and model membranes (large and giant unilamellar lipid vesicles, luvs, and guvs respectively). repa-wh1 shows association and aggregation to membranes composed of anionic phospholipids. protein association in guvs did not result in lysis of the vesicles, suggesting the assembly of discrete protein pores as the mechanism for repa-wh1 membrane damage. to investigate the formation of pores we analyzed by electron microscopy the aggregation of repa-wh1 in the presence of a pre-formed e. coli lipid monolayer. the em images show the presence of pore-like particles on the monolayer. amyloid pores formation explains the permeabilization effect of repa-wh1 in vesicle models and is in agreement with observations for human amyloidogenic proteins. the approaches presented here provide a deeper insight into amyloid cytotoxicity towards membranes and will make possible the assay of inhibitors and effectors of amyloidosis under controlled conditions. references: b2-adrenergic receptor (b2ar) is a member of g protein-coupled receptors, which represent the single largest family of cell surface receptors involved in signal transduction. b2ar recognizes a variety of ligands and communicates with cytoplasmic g-proteins by transmitting signals through the cellular membrane. thus, investigation of communication pathways for b2ar may give important insights for understanding its allosteric mechanisms and identifying new target sites for more specific and efficient drug molecules to be used in the treatment of pulmonary and cardiovascular disease. in this study, various conformations from 2 ms molecular dynamics (md) simulations and available crystal structures of human b2ar were investigated to reveal alternative signaling pathways between its extra and intracellular regions. specifically, shortest communication paths connecting key residues (more than 35 å apart) at the orthosteric ligand binding site (d113, s203, t286, f289, n312) to either l266 or s329 located near the g-protein binding site were investigated. the conformers from previous md simulations [1] include the intracellular loop 3 (icl3), which especially affects the transmembrane collective dynamics but is lacking in x-ray structures. the protein was described as a graph composed of nodes linked by edges. nodes were placed at the alpha-carbon atoms and the edges were calculated based on the number of atom-atom interactions within a cut-off distance 4.5 å for each residue pair. twenty shortest pathways were revealed using k-shortest path algorithm [2] on the coarse-grained network. our results indicated that distinct signaling paths progressed most frequently on tm6 but alternative paths were also present, which passed partially through tm5, tm7, tm3 or tm2 depending on the conformation. among the critical residues that transmitted the signal between distant sites, f282 and n318 were detected, whose functional roles were reported in previous experimental studies. pathway shifting was observed depending on the open-to-closed transition of icl3 during md simulations. the sulfonylurea receptor 1 (sur1) is an atp binding cassette (abc) protein that forms the regulatory subunit in katp channels found in the pancreas and the brain. mgatp binding and hydrolysis at the two cytosolic nucleotide binding domains (nbd1 and nbd2) in sur1 control gating of the katp channel pore. 1,2 proper regulation of katp channel gating by sur1 is critical. 2 over 100 mutations that lead to diabetes, hyperinsulinism and developmental delay have been identified in different domains of sur1, including the nbds. 3 therefore, molecular-level understanding of the structure and function of the nbds is essential for designing improved treatments for sur-related diseases. here we present biophysical and biochemical studies aimed at understanding the effect of disease-causing mutations on the conformation and nucleotide binding of sur1 nbd1. specifically, we are investigating sur1 nbd1 mutations that cause neonatal diabetes (r826w and h863t) or congenital hyperinsulinism (c717 d, g716v, r824g, r837 d and k890t). 3 our nuclear magnetic resonance (nmr) data shows that the hyperinsulinism mutation k890t causes chemical shift changes throughout the spectrum of nbd1, implying overall changes in protein conformation that may affect mgatp binding and inter-domain interactions with other domains in the sur1 protein. size-exclusion data show that the other hyperinsulinism mutations (c717 d, g716v, r824g, r837 d) produce mostly aggregated protein, likely as a result of misfolding of nbd1. misfolding of nbd1 may be the underlying cause of reduced katp trafficking seen with these mutations and hence decreased katp channel gating observed in hyperinsulinism. in contrast to the k890t mutations, the congenital diabetes-causing mutations (r826w and h863t) cause few nbd1 nmr spectral changes. however, the congenital diabetes mutation r826w decreases the affinity of nbd1 for mgatp, which is unexpected for congenital diabetes mutations. our fluorescence, circular dichroism and microscale thermophoresis data corroborate the results that we have obtained by nmr spectroscopy. our data provide molecular-level details on the effects of disease causing mutations in human sur1. egfr increased stability: rmsf of the ca atoms during the md simulations suggest that glycosylation is associated with dampened motions, suggesting that the glycans stabilize the structure. subdomain iii is the most stabilized while subdomain i is stabilized largely in the proximity of the ligand. both dimer interfaces including the dimerization arm from domain ii and the tip of domain iv fluctuate less upon glycosylation. hydrogen bonding; persistent interactions seen for protein-glycan: in the disaccharide-containing system, we observed three highly occupied hydrogen bonds between the glycans and domain iii and iv of egfr. hydrogen bonds of domain iii involve the residue asp323 in which a sidechain oxygen interacts with oxygen atoms of the n-acetylglucoseamine linked to asn328. in domain iv a hydrogen bond is seen between the cys 515 backbone amide and the oxygen atom of n-acetylglucosamine linked to asn 504. in the oligosaccharide-containing system hydrogen bonds observed between the glycan attached to asn 172 and domain ii. these hydrogen bonds form between the gln193 sidechain oxygen atom and cys 191 backbone oxygen atom and the mannose linked to asn 172. the reduction in the mobility of these amino acids suggests that hydrogen bonds impart stability to both the sugars and to the interacting egfr. insects possess a complement-like immune response utilizing thioester-containing proteins, or teps. the only arthropod tep of known structure is anopheles gambiae tep1, which is a key component in the natural immunity of this mosquito to malaria parasites (genus plasmodium). unlike vertebrate complement factors, agtep1 does not contain an anaphylatoxin domain which acts to regulate a massive conformational change accompanying activation of the protein. the mechanism of agtep1 must therefore involve an alternative mechanism for allosteric regulation of thioester activation. in place of a small internal domain, a large, heterodimeric complex of two leucine-rich repeat (lrr) proteins, lrim1 and apl1c, have been shown to specifically bind and stabilize the active conformation of agtep1. i will present my group's most recent work in this area. we have shown that different alleles of tep1, which are known to influence the vectoral capacity of wild mosquitoes, differ significantly in their susceptibility to thioester hydrolysis. allelic variation is centered on residues at the protein-protein interface within tep1 containing the thioester bond. the lrim1/apl1c heterodimer is shown to form an extended and flexible ensemble in solution. two closely-related genes to apl1c, apl1a and apl1b, can also form a complex with lrim1, and apl1b lrr domain can form a homodimer. we propose that a flexible and heterogeneous group ensemble of lrim1/apl1 dimers interact with the active conformation of tep1, thereby producing an array of immune complexes to protect mosquitoes from a diverse set of pathogens. human flap endonuclease-1 (hfen1) is an essential metallo-nuclease involved in okazaki fragment maturation and long-patch base excision repair. during these processes, bifurcated nucleic acid intermediates with ssdna 5'-flaps are generated by polymerase strand displacement synthesis and then cleaved one nucleotide into the downstream duplex by fen1 to create a nicked-dna that is a suitable substrate for ligase. until recently, how hfen1 achieves tremendous catalytic power (rate enhancements >10exp17) and exquisite selectivity for the scissile phosphate had been understood poorly (1) . in 2011, the grasby and tainer labs solved the structures of hfen1 in complex with product and substrate. this study revealed that scissile phosphate selectivity is largely due to the substrate dna undergoing a novel di-nucleotide unpairing (dnu), which places the scissile phosphate diester in contact with the requisite divalent metal ions. in addition, by comparing the structures of hfen1 alone (2) and in complex with substrate and product dnas (3), grasby and tainer proposed a model, whereby protein conformational changes occur upon binding substrate resulting in placement of key basic residues that position and/or electrophillically catalyse hydrolysis of the scissile phosphate diester. further work using a cd-based assay showed that metals are absolutely required for dnu, whereas the key basic residues in the active site are not. surprisingly, perturbations to the protein structure that are much more distant from the fen1 active site (i.e., helical cap) prevent dna unpairing, implying that the fen1 protein actively participates in the unpairing process (4,5); however, how it does remains a mystery. the maximal multiple turnover rate of hfen1 reaction is rate-limited by enzyme product release, whereas hfen1 kinetics under substrate-limiting conditions ([e]<[s]<km) suggest that the enzyme is approaching catalytic perfection (i.e., almost diffusion controlled -10exp7 m-1s-1) (6) . previous work has shown that a paralogue of hfen1 (i.e., t5 fen1) is not ratelimited by chemistry under single turnover (st) conditions, but rather some physical limitation such as conformational change (6) . to ascertain whether the protein and/or dna conformational changes of hfen1 mentioned above are rate limiting under st conditions, we have initiated kinetic and nmr studies to determine the role of conformational dynamics in the catalytic cycle of hfen1. owing to the latest advance in the computational technologies of microprocessor, high-speed inter-processor connection, and parallelization algorithm, all-atom molecular dynamics (md) simulations of microsecond time scale are getting popular to study the pharmaceutical target proteins. an antibody binds to its antigen with structure changes. a small molecule moves around the target protein and finds an entrance to get into the binding site. these phenomena can be observed in microsecond simulations, but the vital issue is whether the adopted force field is enough accurate to correctly describe the phenomena. we are developing a force field (fuji) based on general amber atom types and amber94 van der waals parameters in order to describe arbitrary organic molecules in a unified manner including proteins and nucleic acids. we use the first principle theoretical method to refine the force field in contrast to common empirical fittings to experimental data. the dihedral torsion parameters of protein backbone were determined to agree with the torsion energy profiles calculated by high-level quantum mechanical theory for the model systems of protein backbone. conformational preferences of dipeptides in water were measured by vibrational spectroscopy. comparing with the experimental distribution of ramachandran angles of dipeptides, how accurately molecular calculations predict the conformation distribution of dipeptides were investigated for various force fields and semiempirical quantum methods. fuji force field gave the best prediction score among the various methods (tzanov et al, 2014) . in this work we examine dynamical structure behaviors of pharmaceutical target proteins by microsecond md simulations with fuji force field. epiregulin (epr) is a transmembrane protein with 140 residues belonging to the epidermal growth factor family. mature epr (46 residues) binds to epidermal growth factor receptor (egfr), which stimulates the proliferative signaling in cancer cells. our epr antibody has a proline at the residue 103 in the third complementarity-determining region (cdr) of the heavy chain, which has cis peptide bond in free state and trans one in the bound state with epr according to x-ray crystallography analysis. in order to clarify the structure changes in binding we performed extensive md simulations of our antibody (fab; 436 residues) and epr. the total simulation time was about 240 microseconds. we also performed md simulations for a protein kinase and a small molecule. we show how the complex system reaches thermal equilibrium states in simulations from the stiff x-ray crystal structure. the calculations are compared with experiments such as x-ray crystal structures and thermodynamic quantities measured by isothermal titration calorimetry (itc) and surface plasmon resonance (spr). this research has been supported by mext spire supercomputational life science (hp130006, hp140228) and first kodama project in japan. structure-based recombination of drug resistance enzymes: structural and functional tolerance to new dynamics in artificially-evolved enzymes our understanding of the contribution of protein dynamics to function is still emergent. in a protein engineering context, do we need to take into account the dynamics in order to maximize the fitness and function of the resulting proteins? using high resolution crystal structures, nmr relaxation dispersion and ms molecular dynamics simulations, we compare two naturally evolved homologous class a b-lactamases, tem-1 and pse-4 which share a high degree of structural and functional conservation. we observed a conservation of dynamics on a catalytically relevant timescale. this is consistent with dynamics being an evolutionarily conserved feature. however, laboratory-engineered chimeric enzymes obtained by recombination of the two homologs exhibit striking dynamic differences, despite the function and structure being conserved. the laboratory-engineered chimeras are thus functionally and structurally tolerant to modified dynamics on the timescale of the catalytic turnover. this tolerance of blactamases to dynamic changes could be linked to the high fitness of the naturally evolved proteins and implies that maintenance of native-like protein dynamics may not be essential when engineering functional proteins. conformations solution. an immense advantage of our recombinant system is the possibility to perturb it by introducing unnatural amino acids for subsequent labelling with fluorescent dyes or to omit subunits from the multisubunit rnap. comparing free, nucleic acid-and factor-bound rnap complexes we found that the position of the clamp is modulated during the transcription cycle in a fashion exceeding the simplified closed-open picture drawn from crystallographic studies. we show that the clamp adopts at least two distinct conformations in both initiation and elongation complexes. moreover, we were able to link conformational states to the catalytic activity of the rnap. transcription factors tfe and spt4/5, which both bind to the rnap clamp domain and stimulate the activity of the enzyme, shift the equilibria towards one of the main conformations suggesting that both prompt an allosteric switch that influences the structure of the rnap. hence, our data provide a mechanistic rationale for the function of these factors and provide new insights into the complex dynamic behaviour of the transcriptional machinery. solvent models for protein simulations -the good, the bad and the applications duy hua 1 , amitava roy 1 , he huang 1 , carol post 1 1 the solvent environment plays a crucial role in determining the structure, dynamics and function of a biomolecule. in order to utilize molecular dynamics (md) simulations to examine the structural changes abstract of biomolecules, it is imperative that the solvent environment be accurately modeled. implicit solvent models (isms), in which water molecules are absent and the solvent effect is estimated using an energy function, offer a low-cost approach to describe the solvent environment in md simulations. isms have been used for a variety of biological studies; yet, the performance of implicit solvation methods in capturing the structural characteristics of proteins has not been rigorously demonstrated. in this work, three isms, namely gbmv ii, facts, and scpism, were evaluated for their abilities to emulate the solvent environment and its effect on the structures, dynamics and electrostatic interacthioredoxins (trx) are ubiquitous proteins that play a key role in redox state regulation of the cell. they interact with multiple targets in the cell. our group solved the structure of s. cerevisiae (ytrx1) and measured its dynamics in different timescales (pico to seconds). it is well established that the asp24 is the proton acceptor in the reduction process of the target protein. we proposed that asp24 also works to couple hydration and motion of the interacting loops. we studied conformational motions of the residues of water cavity and interacting loops. the water molecules in water cavity of the protein play a vital role in the redox activity of thioredoxin. the degree of mutational frustration explains in a great deal the multiple timescales observed in the protein dynamics in ytrx1 and the mutant d24a. the ytrx1 displays a complex equilibrium between the ground state and several excited hydration states. this excited states are more permeable to water and it is key to understand the proton transfer and activation of cys33. in this current study, we examined the mutant d24a, and observed that this small hydrophobic residue induces conformational exchange in residues exposed to the water cavity, such as cys33. along with molecular dynamics simulations and calculation of mutational frustration levels, we were able to describe conformational details of the water cavity. it is formed by three independent contiguous lobes which we called lobes a, b and c. lobe b is the central lobe that contains the catalytic important residues cys33 and asp24. it closed upon mutation d24a. lobe a and c remains open upon mutation, meaning that their hydration are independent. the nmr structures for the oxidized and reduced state of the mutant d24a has been studied. in-vitro and in-silico studies of ligand binding to the nuclear receptor ppargamma using fret and md narutoshi kamiya 1 , gert-jan bekker 1 , takuma shiraki 2 , haruki nakamura 1 1 ipr, osaka university, 2 kinki university the nuclear receptor ppargamma is a ligand-dependent transcription factor which regulates gene expression related to glucose homeostasis and insulin sensitization. it is a potential therapeutic target for metabolic syndrome, cancer, and inflammatory diseases. crystal structures have revealed that its ligands bind deep inside the pocket. however, how the ligand recognizes ppargamma and penetrates inside the pocket remains unclear. we have measured the binding kinetics using the fret method and have calculated potential dissociation paths via molecular dynamics (md) simulations. the binding kinetics were measured using fret between the ligand, c8-bodipy, which is a fluorescent pigment structurally similar to one of its agonists 15-oxo-eicosatetraenoic acid, and cyan fluorescent protein (cfp) which is fused to ppargamma. fluorescence of c8-bodipy is observed when it is close to cfp when cfp is excited by light at 410nm. the ligand was simultaneously added to the ppargamma-cfp solution using the stopped-flow method. time-resolved fluorescence signals of c8-bodipy and cfp were also monitored. two time constants were observed by the fret experiment, which correspond with the landing and docking processes. we used our new md program, psygene-g [1] , which utilizes the gpu for acceleration of the non-bonded interactions, such as electrostatic interaction which uses our original zero-dipole summation method [2, 3] . previously we were able to attain similar thermodynamics with respect to the particle mesh ewald method in membrane proteins and dna-water-ion systems [4, 5] and have applied it to the thermodynamic study of dynein [6] . we have introduced random accelerated md (ramd) into the psygene-g program to predict a ligand's dissociation path from the initial crystal structure (pdb id: 2zk6). in ramd, an external random-directional constant force is added to the ligand. we executed 96-ramd simulations with different random seeds for the initial pull directions. twelve ramd trajectories managed to dissociate within 1 ns, while the remaining simulations did not manage to disassociate within this time-frame. in all twelve cases the ligand went past r288, which is located at the surface of ppargamma. interestingly, a somatic mutant (r288h) is related to colon cancer. we concluded that r288 plays a role of a gate keeper to assist ligand binding. sumoylation is a post-translational modification involved in various cellular processes, such as nuclearcytosolic transport, transcriptional regulation, apoptosis, protein stability, response to stress, and progression through the cell cycle. sumoylation is a conjugation of glysine residue of sumo protein with lysine residue in target(rangap1) protein through the influence of enzyme e2(ubc9) and e3(ranbp2). sumo proteins modify the function of target protein in the cell. but the kinetic mechanism of sumoylation is not well-understand. we traced out the full kinetic process of sumoylation of the sumo1-ubc9 -rangap1-ranbp2 complex in atomic detail via molecular dynamic simulation. we uncovered the kinetics of sumoylation mechanism and verified the important residues which play a key role in the sumoylation process. the comparison of our results with the experimental one are also discussed. human potassium channel kcnq1 is expressed in several tissues including inner ear, heart muscle, lung, intestine, and stomach, each requiring a unique current profile for proper function. to tune its output current, kcnq1 complexes with several accessory proteins from the kcne family, each kcne family member modulating kcnq1 distinctly: kcne1 causes the channel to delay opening and become more conductive in the open state, kcne3 causes the channel to be constitutively conductive, and kcne4 closes the channel. the purpose of this study is to uncover the atomic-scale, mechanical mechanism of how kcne3 modulates kcnq1. to do this we modeled the interaction between kcnq1 and kcne3 with a hybrid experimental-computational approach. our strategy is to determine the nmr structure of kcne3 alone in bilayer-mimicking bicelles, build a homology model of the open-state kcnq1, and dock the structure of kcne3 onto the model of kcnq1 using electrophysiology-based restraints to validate and refine the resulting model complexes. paramagnetic relaxation enhancement, residual dipolar coupling and sequence analysis suggest the single span transmembrane helix of kcne3 is significantly curved. hydrogen-deuterium exchange, nuclear overhauser effect cross peaks, and amber simulation suggest kcne3 carries a significant amount of water in the c-terminal side of the transmembrane helix. the 1h-15n two dimensional nuclear magnetic resonance spectra of a known phenotype-changing mutation in the center of the transmembrane helix, v72t, suggests the helix adopts multiple conformations when the extra side-chain hydroxyl group is present. a docking funnel corroborates a binding pocket suggested by the in vivo electrophysiology data where kcne3 wedges between a helix near the potassium pore and two helices in the voltage sensitive element of the channel. within this pocket, we believe the conformational sampling and structural rigidity-or flexibility, in the context of the v72t mutant-of the accessory protein kcne3 directly influence the modulation profile. watching conformational changes in proteins by molecular dynamics simulations kresten lindorff-larsen 1 1 proteins are dynamical molecules and their ability to adopt alternative conformations is central to their biological function. examples include motions that underlie allosteric regulation or ligand binding, or protein dynamics in enzymes that can modulate the overall catalytic efficiency. protein motions can often be described as an exchange between a dominant, ground state structure and one or more minor states. the structural and biophysical properties of these transiently and sparsely populated states are, however, difficult to study, and an atomic-level description of those states is challenging. in an attempt to determine how well molecular dynamics simulations can capture slow, conformational changes in protein molecules we have studied two different protein systems which are known to undergo conformational exchange on the millisecond timescale, and for which structural information is available for both major and minor states. using enhanced-sampling all-atom, explicit-solvent molecular simulations, guided by structural information from x-ray crystallography and nmr, we show that current force fields and sampling methods allow us to sample experimentally-determined alternative conformations with surprisingly high accuracy. in particular, we find that we can reversible sample both the ground state and minor state, and that the simulations capture the structure of the minor states also. our simulations enable us to calculate the conformational free energy between the two states, and comparison with relaxation dispersion nmr experiments demonstrates a high accuracy. thus, we show for two distinct proteins that we can map the free energy landscapes and that both the structure and energetics are in excellent agreement with nmr experiments. our simulations provide insight into the structural and biophysical properties of transiently populated minor states, and help reinterpret previous experimental measurements. further, our results demonstrate that, at least in the two cases we have studied, modern simulation methods enable us to examine these otherwise "invisible" states of proteins and describe their structural, functional and thermodynamic properties. our results in this way help demonstrate how simulations can now add to our knowledge of transiently formed, "hidden" states of proteins. cell signaling is a fundamental process for all living organisms. g protein-coupled receptors (gpcrs) are a large and diverse group of transmembrane receptors which convert extracellular signals into intracellular responses primarily via coupling to heterotrimeric g proteins. in order to integrate the range of very diverse extracellular signals into a message the cell can recognize and respond to, conformational changes occur that rewire the interactions between the receptor and heterotrimer in a specific and coordinated manner. by interrogating the energetics of these interactions within the individual proteins and across protein-protein interfaces, a communication network between amino acids involved in conformational changes for signaling, is created. to construct this mapping of pairwise interaction energies in silico, we analyzed rhodopsin gpcr coupled to a gai1b1g1 heterotrimer. the structure of this g protein complex was modeled in the receptor-bound and unbound heterotrimeric states as well as the activated, monomeric ga(gtp) state. from these tertiary structural models, we computed the average pairwise residue-residue interactions and interface energies across ten models of each state using the rosetta modeling software suite. here we disseminate a comprehensive analysis of all critical interactions and create intra-protein network communication maps. these networks represent nodes of interaction necessary for g protein activation. structure and dynamics of the polymyxin-resistance-associated response regulator pmra in complex with the promoter dna yuan-chao lou 1 , yi-fen kao 1 , tsi-hsuan weng 2 , yi-chuan li 2 , chwan-deng hsiao 2 , chinpan chen 1 1 institute of biomedical sciences, academia sinica, 2 institute of molecular biology, academia sinica pmra, an ompr/phob family response regulator (rr), takes part in the two-component system that manages genes for polymyxin resistance through a phosphorylation-dependent regulation. phosphorylation of ompr/phob rr induces the formation of a two-fold symmetric dimer in the n-terminal receive domain (rec), promoting 2 c-terminal dna-binding domains (dbds) to recognize tandem repeat dna sequences on the promoter to elicit adaptive responses. recently, narayanan et al. presented the complex structure of active-like kdpe in complex with dna, which reveals a unique asymmetric rec-dbd interface that is necessary to form stable complexes for transcription activation. in this study, we report the 3.2 å resolution crystal structure of bef3-activated pmra in complex with promoter dna as well as its dynamics in solution by nmr. unlike the case of kdpe, pmra-dna complex structure reveals a different asymmetric rec-dbd interaction. interestingly, nmr assignments and dynamics study suggest that rec and dbd do not form stable contacts in solution; instead, 2 domains tumble separately in the absence or presence of dna. relaxation dispersion experiments on methyl groups further show that several rec-dbd interfacial residues exhibit similar slow dynamics in the presence of dna. it is hence highly possible that the slow dynamics observed on these interfacial methyl groups are related to the formation of asymmetric rec-dbd interaction, which reduces the mobility of pmra-dna complex and promotes crystallization. in solution, two domains tumble independently and have diverse orientations, which together with the dbd-dbd interface may facilitate searching best interactions with rna polymerase holoenzyme for transcription activation. time-resolved x-ray observations of nano-scale protein assembly networks yufuku matsushita 1 , hiroshi sekiguchi 2 , noboru ohta 2 , keigo ikezaki 1 , yuji goto 3 , yuji sasaki 1,3 1 the university of tokyo, graduate school of frontier science, advanced materials science, 2 spring-8, 3 osaka university, institute for protein research protein aggregation and network formation in diverse protein species had been studying in various experimental approaches such as absorbance spectrophotometry, dynamical light scattering and x-ray scattering. however, these conventional methods are only able to observe averaged information on bulk conditions and also these techniques have a limitation of time resolved observations. recently, development of single molecular observation techniques are remarkable. diffracted x-ray tracking (dxt) is one of the notable single molecular observation methods to capture detailed intramolecular dynamics of an individual single protein molecule by the labeling x-ray method. in this study, we present an innovative method for observing the protein assembly networks of nano-scale size on the bulk solution using a dxt that possess pico-meter scale positional accuracy and micro-second time resolution. this method is founded by detecting angular rotational displacement of a coexisted and dispersed single gold nanocrystal (approximately 100 nm) on target solution. for target sample, we choose a crystal precursor metastable state of 20 mg/ml lysozyme solution (hen egg white lysozyme: 14331.20 g/ mole on 0.2 m sodium acetate 3 -4 w/v nacl, ph 4.7 buffer) and 10 mg/ml of lysozyme solution such as stable condition. for reference sample, we prepare 20 mg/ml and 10 mg/ml of ribonuclease a solution (ribonuclease a (bovine): 13708.40 g/mol, 0.2 m sodium acetate 3 -4 w/v nacl, ph 4.7 buffer). the experiment was carried out at spring-8 bl40xu. from dxt analysis, we obtained logarithmic rotational velocity distribution of 20 mg/ml, 10 mg/ml lysozyme solution and 20 mg/ml, 10 mg/ml of ribonuclease a solution during 1000 ms and we processed regression analysis of gaussian fitting in each distribution. from this result, 10 mg/ml of lysozyme solution (stable) and 20 mg/ml, 10 mg/ml (reference) have definitely regressed by single peak normal gaussian distribution that are positional peaks at 3.66 mrad and 1.4 mrad and 1.2 mrad, respectively. in contrast, 20 mg/ml of lysozyme solution consisted of double peak logarithmic distribution at 3.42 and 9.54 mrad. this peak separation tendency from dxt are typically observed in a supersaturated condition of the sodium acetate solution which coexisting nano-scale solute networks (dxt and small angle x-ray scattering (saxs) experiment). from this study, dxt measurement results and detailed analysis process for a protein solution such as crystal precursor metastable state of lysozyme solution are concerned that this technique is a powerful tool for observing nano-scale protein network's existence and its local dynamics in bulk solution. at the present time, we will demonstrate the detailed analysis and processing from dxt raw data and the conformity of the results from another experiment confirmation such as saxs and darkfield microscopy. finally, we present a detailed protein network model of lysozyme metastable solution from dxt result. increasing evidence suggests that conformational fluctuations experienced by proteins play a key role in promoting function. however, the experimentally derived dynamic behavior of discrete protein systems has yet to provide clear evidence on the evolutionary conservation of motional events between structural and functional protein homologues. in this work, we used a statistical coupling analysis (sca) approach to analyze the role of co-evolving amino acids in protein function and flexibility. sca facilitates the identification of distinct residue sectors and provides an understanding of the correlation between amino acid co-evolution and biological function. as a model system, we used the ribonuclease a (rnase a) family, which includes canonical members that are structurally similar, but perform diverse activities such as angiogenesis, anti-pathogenicity and neurotoxicity, while preserving the common ribonucleolytic function. analysis of 1922 sequences with sca 6.0 allowed the determination of five independent components (ics) corresponding to amino-acid sectors with distinct functional, structural and/ or dynamical roles within the family. while most ics correspond to residues critical for protein stability and catalytic function, ic4 correlates with residues experimentally determined to be highly dynamic on the catalytic timescale in several rnase a homologues, as we confirmed by nmr 15n-cpmg experiments. additionally, ic5 residues form a hydrogen-bonding network shown to allosterically modulate and coordinate catalytically productive motions. these results provide a direct observation between the role of residue sectors in stability and motions relevant to function in the rnase a family. this study highlights the role of concerted action of multiple residues towards a common function, which can guide experiments for engineering proteins to enhance function such as catalysis. halophilic archea are a type of extremophiles that thrive in hypersaline conditions. in order to avoid the osmotic shock, their cytoplasm is maintained with higher ionic strength up to 4-6m. such a high ionic strength of intracellular environment protects the cells from desiccation or salting out. however, one wonders how such high salt concentrations keep the cell functional and active throughout all the cellular reactions (i.e. enzyme catalysis). the answer lies in the evolution of the halophiles that has left the halophilic proteins with a certain preference for amino acids composition [1] (prefers more acidic residues and overall decrease in hydrophobic side-chains). it's also known that the salt concentration can affect the activity and the underlying mechanism [2] of the halophilic enzymes. in order to further understand the haloadaptation, we have chosen to study a model system, dihydrofolate reductase (dhfr) from haloferax volcanii (hvdhfr). dhfr is a very well understood enzyme and known to bound variety of ligands that can modulate the dynamics and the energy landscapes of the enzyme [3] . we have probed the ls-ms dynamics of hvdhfr bound to the cofactor nadph (holoenzyme) and other substrates (dhf, thf) using relaxation dispersion nmr. in order to understand the salt contribution to the dynamics and catalytic mechanism, the experiments were done in the increasing order of salt concentration, shedding light on the conformational ensembles involved in the catalytic mechanism. the nmr relaxation dispersion analysis of different enzyme complexes in the presence of 3m and 2m kcl has shown more conformational fluctuations than compared to the enzyme with 1m kcl. this result suggests that the enzyme preferably is highly active & samples more flexible conformers in the hypersaline environment. further, quantification of these conformers/invisible-states and their salt-dependence will provide new insights into the mechanism of the enzyme-catalysis. also, it will facilitate designing novel enzymes that can be used in the bioprocess industrial set-up under extreme conditions. 1. tadeo we have identified the extracellular protein hbps in the cellulose degrader streptomyces reticuli and shown that it specifically binds iron ions, heme and aquo-cobalamin. based on 3d crystal structures, structural alignments, sequence comparisons, mutagenesis, and comparative biochemical investigations, we identified the coordination sites for iron, heme and aquo-cobalamin, and binding kinetics were elucidated [1, 2] . hbps interacts with the membrane-embedded sensor kinase sens. while under nonstressed conditions hbps inhibits sens autophosphorylation, under oxidative-stressing conditions activates it. sens in turn phosphorylates the response regulator senr that activates the transcription of anti-oxidative genes [3] . we crystallized hbps and solved its 3d crystal structure that revealed an octomeric assembly which is required for interaction with sens [4] . using mutagenesis, fret, cd spectroscopy, fluorescence spectroscopy and site-directed spin labelling combined with pulse electron paramagnetic resonance spectroscopy, we demonstrated that ironmediated oxidative stress induces both secondary structure and overall intrinsic conformational changes within hbps. we additionally showed that hbps is oxidatively modified, leading to the generation of highly reactive carbonyl groups and tyrosine-tyrosine bonds [5] . we concluded that these molecular events are responsible for the hbps-mediated activation of the sensor kinase sens. this presentation will focus on the hbps structure and dynamics within the protein. indian institute of technology bombay for20 (fop-related protein of 20 kda), a centrosomal protein, is conserved across all ciliated eukaryotes. it has been shown to be involved in ciliogenesis in rpe cells and in the s-phase progression in hela cells. it localizes to the pericentriolar satellite and at the centrosome. the localization of for20 at the centrosome has been found to be throughout the cell cycle. in paramecium, for20 is involved in docking of the basal body to the cell membrane and in the assembly of the transition zone. we found that for20 colocalizes with ciliated microtubules in nih3t3 cells. since, the localization of for20 to the pericentriolar satellite is dependent on microtubules and the cilium itself is a microtubule-based structure; we sought to investigate the role of for20 in microtubule assembly. the over-expression of gfp-for20 in hela cells led to the depolymerization of microtubules while only the gfp expressing hela cells showed typical microtubule network. in addition, the microtubules of gfp-for20 expressing hela cells were found to be more sensitive towards nocodazole, a microtubule-depolymerizing agent, suggesting that for20 destabilizes microtubules. the effect of for20 on the polymerization of tubulin was monitored by light scattering, sedimentation assay and electron microscopy. in vitro, for20 inhibited the rate and extent of polymerization of tubulin. for example, 5, 10, 15 and 20 mm for20 inhibited the polymerization of purified tubulin by 11, 20, 39 and 52%, respectively. further, the sedimentation assay suggested that for20 inhibited the polymerization of tubulin. in addition, electron microscopic analysis of the assembly mixture showed that for20 inhibited microtubule formation in vitro. the binding of for20 to purified tubulin was monitored by several complimentary techniques. using size exclusion chromatography, for20 was found to be co-eluted with tubulin indicating that for20 binds to tubulin. further, the interaction between for20 and tubulin was analyzed by surface plasma resonance technique and the result showed that for20 binds to tubulin with a modest affinity. the data together suggested that for20 regulates microtubule assembly through a direct interaction with tubulin. amide hydrogen exchange (hx) is widely used in protein biophysics even though our ignorance about the hx mechanism makes data interpretation imprecise. notably, the open exchange-competent conformational state has not been identified. based on analysis of an ultra-long molecular dynamics trajectory of the protein bpti, we propose that the open (o) states for amides that exchange by subglobal fluctuations are locally distorted conformations with two water molecules directly coordinated to the n-h group. the hx protection factors computed from the relative o-state populations agree well with experiment. the o states of different amides show little or no temporal correlation, even if adjacent residues unfold cooperatively. the mean residence time of the o state is 100 ps for all examined amides, so the large variation in measured hx rate must be attributed to the opening frequency. a few amides gain solvent access via tunnels or pores penetrated by water chains including native internal water molecules, but most amides access solvent by more local structural distortions. in either case, we argue that an over-coordinated n-h group is necessary for efficient proton transfer by grotthuss-type structural diffusion. differences in redox reactions with nadp1/h between ferredoxin-nadp1 oxidoreductases from bacillus subtilis and rhodopseudomonas palustris daisuke seo 1 , hidehiro sakurai 2 , pierre s etif 3 , takeshi sakurai 1 1 graduate school of natural science and technology, kanazawa univ., 2 research institute for photobiological hydrogen production, kanagawa university, 3 ferredoxin-nad(p)1 oxidoreductase (fnr) is a ubiquitous enzyme that catalyze the redox reaction between soluble small iron-sulfur protein ferredoxin and nadp1/h. fnrs from photosynthetic green sulfur bacterium chlorobaculum tepidum (ctfnr), low-gc content gram-positive bacterium bacillus subtilis (bsfnr) and purple non-sulfur bacterium rhodopseudomonas palustris (rpfnr) are homodimeric proteins containing one fad prosthetic group per subunit. crystal structure analyses of ct-and bsfnrs have revealed their significant structural homology to nadph-thioredoxin reductase from eschelicia coli, which is distinct from the monomeric fnrs from plastids of higher plants, cyanobacteria, g-proteobacteria and putidaredoxin reductase. previous studies on steady-state reaction of bs-and rpfnrs with nadp1/h by diaphorase assay have demonstrated that nadph oxidation rates of bsfnr and rpfnr were comparable to those of the fnrs from plastid and cyanobacteria. but affinities toward nadp1/h and rates for oxidation of nadph differed significantly between bsfnr and rpfnr, despite their high amino acid sequence homology. in this work, we report pre-steady state reactions of bsfnr and rpfnr with nadp1/h by a stopped-flow spectrophotometry. mixing oxidized bsfnr with nadph yielded a rapid formation of charge transfer complexes (ctcs) followed by a reduction of the enzyme. bsfnr was almost fully reduced at equilibrium. mixing photochemically reduced bsfnr with nadp1 also rapidly provided an absorption spectrum of ctc but followed reoxidation of reduced bsfnr was very slow. the amount of oxidized bsfnr after equilibrium depended on nadp1 concentration. kinetic analyses indicated that the rate-determining steps were the hydride-transfer reactions in both directions and the rate for the forward direction was much faster than that for the reverse direction. mixing oxidized rpfnr with nadph exhibited a rapid ctcs formation followed by a slower reduction of the enzyme. increase in nadph concentration reduced the observed rate, suggesting redox potential of rpfnr was similar to that of nadp1/h couple in the presence of excess nadph. mixing photochemically reduced rpfnr with nadp1 rapidly provided ctcs. the decay corresponding to the reoxidation of reduced rpfnr with nadp1 involved two distinctive kinetic phases, which would be due to the similarity in hydride transfer rates of forward and reverse directions, and the presence of excess amount of nadp1. kinetic analyses indicated that the rate-determining steps were the hydride-transfer reactions in both directions and the rate for the forward direction was close to that for the reverse direction. obtained data suggested bsfnr and rpfnr are optimized for different direction of the reaction and may play different physiological roles. stephan k€ ohler 1,2 , friederike schmid 1 , giovanni settanni 1,3 1 institute of physics, johannes gutenberg university mainz, germany, 2 graduate school materials science in mainz, 3 fibrinogen is a serum multi-chain protein which, when activated, aggregates to form fibrin, one of the main components of a blood clot. fibrinolysis controls blood clot dissolution through the action of the enzyme plasmin, which cleaves fibrin at specific locations. although the main biochemical factors involved in fibrin formation and lysis have been identified, a clear mechanistic picture of how these processes take place is not available yet. this picture would be instrumental, for example, for the design of improved thrombolytic or anti-haemorrhagic strategies, as well as, materials with improved biocompatibility. here, we present extensive molecular dynamics simulations of the fibrinogen complex which reveal large bending motions centered at a hinge point in the coiled-coil region of the complex. this feature, likely conserved across vertebrates according to our analysis, suggests an explanation for the mechanism of exposure to lysis of the plasmin cleavage sites on fibrinogen coiled-coil region. it also explains the conformational variability of fibrinogen observed during its adsorption on inorganic surfaces and it is supposed to play a major role in the determination of the hydrodynamic properties of fibrinogen. in addition the simulations suggest how the dynamics of the d region of fibrinogen may contribute to the allosteric regulation of the blood coagulation cascade through a dynamic coupling between the a-and b-holes, important for fibrin polymerization, and the integrin binding site p1. membrane curvature -the assembler of proteins mijo simunovic 1,2 , gregory voth 1 , patricia bassereau 2 1 chemistry department, the university of chicago, 2 physico-chimie curie, institut curie many biological phenomena require the membrane to change its shape. this process is often mediated by curvature-generating proteins, most notably by those containing one of many bar domains. at the same time, membrane curvature controls the way proteins interact with one another and so it acts as a vital signaling mechanism in the cell. we combine theoretical modeling with microscopy imaging techniques to study the driving force underlying the reshaping of biological membranes induced by n-bar proteins. in particular, we employ a combination of coarse-grained molecular dynamics with field-theoretical simulation methods to study the assembly of proteins on the membrane at molecular resolution. this approach allowed us to elucidate the precise mechanism by which cell membranes rapidly and from large distances recruit proteins to membrane-reshaping sites. it also let us identify a surprising role of membrane tension in directing the strength and geometry of protein-protein interactions. by complementing our simulations with fluorescence and atomic force microscopies, we study the way molecular assembly of proteins affects the membrane morphology at a more macroscopic level. we demonstrated how largescale ordering of the proteins on the membrane is mediated by a strikingly long-range interaction that is driven by membrane fluctuations. in sum, our combined theoretical and experimental approach gives vital clues on how the mechanical properties of the membrane may regulate protein dynamics in living cells. adnan sljoka 1 , alexandr bezginov 2 1 kyoto university, 2 university of toronto understanding how a protein functions depends in critical ways on predicting which parts are rigid and which are flexible. using rigidity-theoretical techniques, a number or programs such as first, proflex or abstract kinari can rapidly decompose a protein into flexible and rigid regions. in this study we extend this technique and develop a novel computational approach for detecting protein allosteric interactions. it is widely believed that the binding of a ligand at the allosteric site triggers a conformational change that is transmitted through the protein to cause a rearrangement and alteration of the shape of the active site. allostery was first described more than 50 years ago; however, the underlying allosteric mechanism is still not well understood. we will introduce a rigidity-based allosteric mode of communication together with an algorithm which can detect transmission of rigidity and shape changes between two (or more) distant binding sites in allosteric proteins. our algorithm is also used to predict and identify regions in the protein that are critical for the coupled communication between distant sites (i.e. allosteric pathways). starting with a set of known gpcr 3-dimensional structures, we apply our methods and show how binding of an activating ligand (i.e. agonist) triggers small rigidity changes which propagate to the critical g-protein binding regions. in contrast, in the inactive gpcr structures no such rigidity allosteric communication is observed. detailed predictions and analysis on activated (agonist-bound) and inactive adenosine receptors is discussed and results are compared with experimental evidence. these results show that rigidity-based allosteric model and algorithm is a powerful new tool for detecting allostery in gpcrs. comparing the intrinsic dynamics of multiple proteins using elastic network models reveals global similarities based on their overall shape sandhya tiwari 1,2 , nathalie reuter 1,2 1 with increasing protein structural data, we find ourselves with the opportunity to study the dynamical similarities within large ensembles in order to understand the role of protein dynamics at various levels. is there evolutionary pressure to conserve dynamics? is this necessary to retain functionality, as it has been suggested? we performed strategic comparative analysis using the elastic network model, which has proven to be highly efficient and informative (fuglebakk et al., bmc bioinformatics, 2014) to investigate how the dynamics of related proteins change when their functional or oligomeric state shifts, and if it is conserved within protein families. in our work, we have found that proteins with different ligandbinding states, as in adenylate kinase (tiwari et al., bmc bioinformatics, 2014), or oligomeric states, as in the pyrr family (perica et al., science, 2014), can be classified based on the global similarity of their intrinsic dynamics. moreover, our recent analysis on functionally distinct protein families with the tim barrel fold indicates that the overall similarity in shape dictates similarity in intrinsic dynamics regardless of sequence and functional conservation or evolutionary links. we suggest that since shape dictates a large part of dynamical similarity in proteins, the changes in local flexibility play a strong role in differentiating various functional states. oxygen-affinity and cooperativity of hemoglobin introduction: the widely-held mechanism of allostery of hb has been that the changes from the r-quaternary/tertiary structures of oxy-hb to the t-quaternary/tertiary structures of deoxy-hb exert certain constraints on the coordination structure of the heme group, leading to a lower o2-affinity of the hemes and thus that of hb [1] . however, we found that there is no causal correlation between the static t/r-quaternary structures and the low/high o2-affinities of hb, respectively [2] . we explore an alternative mechanism of the regulation of the ligand-affinity and cooperativity in hb. results and discussion: the o2-affinities of free fe[ii]-protoporphyrin ix-nitrogenous base complexes in organic solvents are very low (p50 > 102 103 torr), whereas the apparent o2-affinities of these metalloporphyrins, which are incorporated in apo-myoglobin, apo-hb, serum albumin, etc., increase substantially to p50 < 10-1 101torr, though their coordination structures are apparently unchanged [3] . such substantial increases in the apparent ligand-affinities of metalloporphyrin-containing proteins are accomplished by preventing/inteferring with the dissociation of the ligand by protein matrix, since the interior of globin is nearly fully packed by protein matrix. in hb, the dissociation process of the ligand proceeds through the "caged" state [4] [5] [6] , which can be produced by cryogenic photolysis of the ligated-states at 4.2k and in which the metal-ligand bond is broken and the un-bonded ligand is trapped near the bonding site within the globin moietiy. this "caged" state has spectral features distinct from those of either deoxyor ligated states of the respective hemoproteins. the apparent ligand-affinities of hb are regulated by heterotropic effectors without detectable changes in either static quaternary/tertiary structures of the globin moiety or the coordination/electronic structures of the metalloporphyrin moiety and thus the ligand-affinity of the metalloporphyrins themselves [7] [8] [9] . the reduction of the apparent ligand-affinities of hb may be caused by increases in the migration rate of ligands through globin matrix from the "caged" state to solvent, resulting from the effector-linked, enhanced high-frequency thermal fluctuations which increase the transparency of the globin matrix toward small diatomic ligands [7] [8] [9] . conclusion: the ligand-affinity of hb is regulated through protein dynamics by heterotropic effectors, rather than static quaternary/tertiary structural changes. thus, the "caged" state of hb acts as a critical transition state in regulation of the affinity for small diatomic ligands in hb [9] . the role of metal ions in the regulation of life processes is extremely important. they act as signal transducers, protein configuration stabilizers, enzymatic cofactors, oxygen transport supporters and many others. for example, subtle perturbations in calcium homeostasis may lead to mental disabilities and are linked to diseases such as autism spectrum disorders (asd). in this study we focus on complex protein systems, mainly those present in the brain. we search for dimers mediated by the presence of metal ions, and determine the impact of the presence or absence of the latter on the structure and energetic properties of the complex in the protein-protein interface. we investigate ions' influence on the interface stability using classic molecular dynamics methods (md), including steered md. moreover, we apply a novel suite of enhanced md-based methods recently developed by our team (rydzewski & nowak) to explore ion diffusion pathways in protein fragments of the synapses. finally, we describe specific inter-protein ion binding motifs with the most important interactions, collating them with various structures deposited in the protein data bank [1] . the binding of integrins to collagen plays a critical role in numerous cellular adhesion processes including platelet activation and aggregation, a key process in clot formation. collagen is an unusually shaped ligand, and its mechanism of recognition and role in selectivity and affinity are unique, and at this stage not well understood. the i-domain of the integrin protein binds to collagen specifically at multiple sites with variable affinities, however the molecular mechanism of integrin i-domain (ai) regulation remains unknown. using nmr, along with isothermal titration calorimetry, mutagenesis, and binding assays we are developing a novel integrated picture of the full recognition process of the integrin a1i binding to collagen. the adhesion of the a1b1 integrin receptors to collagen is cation-dependent with collagen binding a mg(ii) ion that is located at the top of the extracellular integrin a1i-domain (a1i). our results show evidence for a regulatory effect of the mg(ii) ion on a1i affinity, by inducing allosteric ms-ms motions of residues distant from the binding site. we propose a novel model of a1i recognition to collagen, comprising a two-step mechanism: a conformational selection step, induced by mg(ii) coordination, and an induced-fit step caused by collagen binding. hydrogen-deuterium exchange experiments show that the induced-fit step is facilitated by the reduced local stability of the c-terminus. we propose that the conformational selection step is the key factor that allows discrimination between high and low affinity collagen sequences. cytochromes p450 (cyp) are heme containing enzymes involved in the metabolism of endobiotics and xenobiotics, such as drugs or pollutants. [1] in humans, cyps are attached to the biological membranes of endoplasmic reticulum or mitochondria by n-terminal transmembrane anchor and they are partially immersed by their catalytic domain to different level. [2] generally, the composition of lipid membrane may significantly affect behavior of protein embedded in respective membrane e.g. the cholesterol in membrane alters membrane properties such as: thickening of the membrane, changing the stiffness or enhancing ordering of the membrane. furthermore, the increasing amount of cholesterol in membrane may also alter interaction with membrane proteins and affect solute partitioning between membrane and water molecules. [3] cholesterol is also known to noncompetitively inhibit the most typical drugmetabolizing cyp -cyp3a4, [4] however the mechanism was unknown. for this reason, we prepared the set of simulations of cyp3a4 embedded in dopc lipid bilayers with various cholesterol concentrations (0, 3, 6, 20 and 50% wt; figure 1 ) and the 200ns1 long md simulations were carried out. md simulations showed the formation of funnel-like shape of the lipids close to the catalytic domain of cyp. in addition, the cholesterol molecules have tendency to accumulate in the vicinity of membrane-attached f/g loop. the catalytic domain sunk deeper into the membrane with cholesterol and also the number of amino acids in contact with membrane was bigger than in the pure dopc bilayer. in contrast, the presence of higher amount of cholesterol affected the pattern of channel opening effectively blocking the access to the active site from the membrane, which in turn may affect the substrate preferences and catalytic efficiency. [5] finally, we study the effect of different lipid types on membrane-attached cyp3a4. anti-(4-hydroxy-3-nitrophenyl)acetyl (np) antibodies are one of the most widely analyzed type of antibodies, especially with respect to affinity maturation [1] [2] [3] . affinity maturation is a process in which b cells produce antibodies with increased affinity for the antigen during the course of an immune response, and is like "evolution" in term of increasing antigen-binding affinity. during the course of affinity maturation, the structural dynamics of antibodies, which are closely correlated with the binding function, can change. to analyze the structural dynamics at atomic resolution and the single-molecule level, we tried to express and purify single-chain fv (scfv) antibodies against np. using scfv antibodies, we can also analyze the effects of key residues on affinity maturation via site-directed mutagenesis. as the first step, we have succeeded in generating a sufficient quantity and good quality of scfv of affinity-mature anti-np antibody, c6, with a linker composed of four repeats of gggs. the scfv protein was expressed in the insoluble fraction of e. coli, and solubilized using 8 m urea, followed by refolding by step-wise dialysis to decrease the urea concentration. the final step of purification using an antigen column indicated that approximately 2% of the solubilized protein was correctly refolded and possessed antigen-binding ability. the analytical ultracentrifugation (auc) analysis showed that the purified c6 scfv exists in the monomeric state with little oligomeric contamination. the secondary structure and thermal stability of c6 scfv were analyzed using circular dichroism (cd). the far-uv cd spectra of c6 scfv indicated typical b-sheet-rich structures. upon antigen binding, the far-uv cd spectrum remained unchanged, but the thermal stability increased by approximately 20oc. the antigen-binding function of c6 scfv was analyzed using a surface plasmon resonance (spr) biosensor, biacore. the binding affinity and kinetics of c6 scfv for np conjugated to bovine serum albumin immobilized on the sensor chip were similar to those of intact c6. taken together, the results of auc, cd, and spr indicated that c6 scfv could be refolded successfully and would possess its functional structure. next, to analyze the structural dynamics of c6 scfv in the absence or presence of antigen, experiments involving diffracted x-ray tracking (dxt) were performed [4] . c6 scfv with an n-terminal his-tag was immobilized on substrate surfaces using tag chemistry, and au-nanocrystals were labeled on the surface of scfv as tracers. the motions of c6 scfv were analyzed in two rotational directions representing tilting (u) and twisting (v) mean square displacement (msd) analysis from more than 200 trajectories showed that the slope for c6 scfv without antigen, especially in the u direction, was greater than that for c6 scfv with antigen, suggesting that the motion of scfv was suppressed on antigen binding. the antibiotic resistance enzyme aph(2'')-ia confers antimicrobial resistance to aminoglycoside antibiotics in staphylococci and enterococci. this kinase phosphorylates aminoglycosides such as gentamicin and kanamycin, chemically inactivating the compounds. we have determined multiple structures of the enzyme in complex with nucleoside and aminoglycoside substrates and cofactor magnesium. introduction of aminoglycoside to crystals of aph(2'')-ia induce gross conformational changes in crystallo, illustrating several important stages of the catalytic cycle of the enzyme. an interaction between nucleoside triphosphate and an amino acid residue on a conserved loop has also been identified that appears to govern a conformational selectivity and modulates the enzyme activity when no substrate is present. comparisons between multiple protein molecules both within and between crystal structures allow us to infer functional states of the enzyme as it carries out catalysis. these structures collectively highlight an enzymatic flexibility that not only allows the binding of diverse aminoglycosides, but also appears to transition from a stabilized, inactive enzymatic state to a catalytically active enzyme with an active site geometry identical to distantly-related eukaryotic protein kinases. mechanistic insight gained from these studies begin to demystify a widespread staphylococcal resistance factor, and provide a starting point for the development of anti-infectives toward this important antimicrobial resistance machine. ryan godwin 1 , william gmeiner 2 , freddie salsbury 1 1 wake forest university -department of physics, 2 wake forest university health sciences -department of cancer biology the zinc-finger of the nf-jb essential modulator (nemo) is a ubiquitin binding domain, and an important regulator of various physiological processes including immune/inflammatory responses, apoptosis, and oncogenesis. the nominally functioning 28 residue monomer (2jvx) is represented by a bba motif, with a cchc active site coordinating the zinc ion. here, we investigate the effects of a single point mutation that has been linked to the disease states associated with ectodermal dysplasia. the single mutation of the last binding cysteine (residue 26) to a phenylalanine (2jvy) distorts the available conformation and dynamics of the protein, as shown via microsecond, gpuaccelerated molecular dynamics simulations. we examine these two proteins in various states of zinc-binding and coordinating cysteine protonation. in addition to destabilization of the alphahelix induced by the cysteine to phenylalanine mutation, prominent conformations show the bsheets turned perpendicular to the alpha-helix, providing a possible mechanism for the induced disease state. , catalytic (51-220 aa) and c-terminal (220-270 aa)) were expressed in e. coli. several truncated in variants containing amino acids 1-160, 1-220, 51-160 and 51-280 were also prepared. a full-size ku70 with a gst-tag on its n-terminus was purified from e. coli. all the experiments performed showed that neither n-terminal nor c-terminal domains of hiv-1 in are essential for its binding with ku70 despite a weak binding capacity retaining to the c-terminal domain. the catalytic core (51-220 aa) as well as the mutant lacking c-terminal domain (1-220) both demonstrated affinity to ku70 comparable to the affinity of the full-size in, whereas its truncated variant (51-160 aa) bound to ku70 protein only weakly. we also expressed a c-terminal ha-tagged full-length in and its 1-220 variant in hek 293t cells together with a wt ku70-3flag and showed that both in variants are stabilized by co-expression with ku70 by approx. twofold. we hypothesize that the binding surface within in lies in the region from 160 to 230 a.a. that is a long a-helix. we have shown that a homologous integrase from prototype foamy virus that lacks this structural element does not bind to ku70. it is worth noting that ku70 does not affect the interaction of in with its major cellular partner -ledgf/p75 as well as its interaction with the dna substrate. this work was supported by an rfbr grant 14-04-00833 and by an rscf grant 14-14-00489. the nadph-dependent cytochrome p450 oxidoreductase (cypor) is large 677 amino-acid long microsomal multidomain enzyme responsible for electron donation to its redox partner cytochrome p450 (cyp) involved in drug metabolism. electron transfer (et) chain is mediated by two riboflavin-based cofactors -flavin mononucleotide (fmn) and flavin adenine dinucleotide (fad) within their respective domains and nicotinamide adenine dinucleotide phosphate (nadph). during this electron transfer cypor undergoes several structural changes in open and closed state of both domains in different degree of contact. in spite of the fact that cyp-cypor complexes play a key role in drug metabolism, the atomistic mechanism of structural rearrangements during complex electron transfers is still lacking. here, we present the results of our study on structural changes during cypor multidomain complex movement between individual electron transfers using classical molecular dynamics (md) and metadynamics (mtd) simulations with cofactors of nadph, fad and fmn in resting state. homology model of human cypor in both forms (opened and closed) were embedded into pure dioleoylphosphatidylcholine (dopc) bilayer. after system equilibration (figure 1 ), structural changes of protein, anchor and cofactor movement were studied. we were able to select possible cypor-membrane orientation which would allow interaction with cytochrome p450. in addition, spontaneous closing of open cypor was observed. however structural changes between crystal structures and structures obtain from md simulations lead us to the use of metadynamics in order to speed up the process. fmn and fad cofactor remained in close van der waals contact during the 100-ns long simulation stabilized by p stack interaction of fad with trp676, whereas continual movement of nadph continually weakens its p stack interaction with fad. after 100 ns of classical md additional metadynamics simulations were performed in order to investigate internal motion of cofactors during electron transfer. atoms c4n (nadph) and n5 (fad) which are responsible for et were able to move closer to the distance of 3 å after adding biasing potential. this distance is more than sufficient for electron transfer to occur. after switching back to classical md cofactors got into resting positions (8 å) again. our results show that cypor undergo several structural changes and internal motions of cofactors in order to transfer electrons to its redox partner -cyp. research & utilization div., jasri/spring-8, 2 grad. school frontier sci., univ. tokyo, 3 grad. sch. sci., univ. hyogo, japan, 4 national institute of advanced industrial science and technology, japan, 5 pentameric ligand-gated ion channels (plgics) are a major family of membrane receptors that open to allow ions to pass through the membrane upon binding of specific ligands. plgics are made up of five identical (homopentamers) or homologous (heteropentamers) subunits surrounding a central pore. structural information about their multiple allosteric states, carrying either an open or a closed channel, has become available by recent studies by x-ray crystallography. however, dynamic information are needed to understand their mechanism of gating, notably the long-range allosteric coupling between the agonist binding site and the ion channel gate. here we used the diffracted x-ray tracking (dxt) method (1) to detect the motion of the extracellular and transmembrane domain two plgics: the nicotinic acetylcholine receptor (nachr) and a proton-gated bacterial ion channel from gloeobacter called glic. dxt is a powerful technique in biological science for detecting atomic-scale dynamic motion of allosteric proteins at the single molecular level and at tens of micro seconds timescale resolution. the dynamics of a single protein can be monitored through trajectory of a laue spot from a nanocrystal which is attached to the target protein immobilized on the substrate surface (2,3). dxt detects two kinds of rotational motions of nanocrystal, tilting and twisting, based on x-ray incident beam axis. dxt analysis with 0.1ms/f time resolution showed that tilting motion of the transmembrane domain of glic and both tilting and twisting motions of the extracellular domain of glic and nachr were enhanced upon application of agonists (lowering the ph for glic, and binding of acetylcholine for nachr). the detailed dynamic information, including size effect of gold nanocrystal to the motion of them, is discussed. [ proteins possess unique structure-encoded dynamics that underlie their biological functions. here, we provide experimental evidence for an evolutionary mechanism driven solely by long-range dynamic motions without significant backbone adjustments, catalytic group rearrangements, or changes in subunit assembly. crystallographic structures were determined for several ancestral gfp-like proteins that were reconstructed based on posterior sequence predictions, using members of the stony coral suborder faviina as a model system. the ancestral proteins belong to the kaede-type class of gfps, a group of proteins that undergoes irreversible green-to-red photoconversion and is therefore frequently employed in superresolution microscopy. surprisingly, we find that the structures of reconstructed common green ancestors and evolved green-to-red photoconvertible proteins are very similar. therefore, we analyzed their chain flexibility using molecular dynamics and perturbation response scanning. we find that the minimal number of residue replacements both necessary and sufficient to support lightinduced color conversion provide for increased fold stiffness at a region remote from the active site. at the same time, the allosterically coupled mutational sites appear to increase active site conformational mobility via epistasis. these data suggest that during evolution, the locations of fold-anchoring and breathing regions have been reversed by allosteric means. therefore, we conclude that the green-tored photoconvertible phenotype has arisen from a common green ancestor by migration of a knob-like anchoring region away from the active site diagonally across the beta-barrel fold. based on titration experiments, we estimate that at ph 6, 0.1% of the protein population harbors neutral side chains for his193 and glu211, residues that form an internal salt bridge near the chromophore. we propose that this reverse-protonated subpopulation constitutes the catalytically competent state. in the electronically excited state, light-induced chromophore twisting may be enhanced, activating internal acid-base chemistry that facilitates backbone cleavage to enlarge the chromophore. in this way, a softer active site appears to be coupled to a mechanism involving concerted carbon acid deprotonation and betaelimination. dynamics-driven hinge migration may represent a more general platform for the evolution of novel enzyme activities by tuning motions in the active site. the binding of an agonist to a gpcr causes a conformational change in the receptor that leads to its activated functional state. rhodopsin, the membrane receptor responsible for photoreception in the vertebrate retina, is a prototypical gpcr and has been extensively used in structural, biochemical and biophysical studies of this class of receptors. different small molecules have been described to be capable of binding to rhodopsin. in addition, mutations in rhodopsin have been associated with retinal diseases and efforts have been carried out in order to find potential ligands that can offset the effect of these mutations. cyanidins, a group of flavonoids within the larger family of polyphenols, have been reported to stimulate chromophore regeneration of rhodopsin by means of the formation of regeneration intermediates. the aim of the current study was to evaluate the effect of the flavonoid quercetin on the conformational properties of both native bovine rhodopsin and heterologously expressed recombinant rhodopsin. rhodopsin was purified from bovine retinas by immunoaffinity chromatography, and photobleaching, thermal stability, metarhodopsin ii decay and chromophore regeneration assays were carried out in the absence or in the presence of 1mm quercetin. for recombinant rhodopsin, a plasmid encoding wild-type opsin was transfected into mammalian cos-1 cells, in the absence or in the presence of 1mm quercetin, harvested, regenerated with 11-cis-retinal, or 9-cis-retinal, and subsequently purified in dodecyl maltoside solution. no differences in photobleaching behavior, upon illumination, could be detected in the purified quercetin-containing samples compared to those in the absence of this flavonoid. in the case of rhodopsin, and the recombinant wild-type protein regenerated with 11-cis-retinal, quercetin did not significantly alter the thermal stability and rate of regeneration of the purified proteins under our experimental conditions. however, a two-fold increase in the thermal stability and a 40% increase in chromophore regeneration were observed for the recombinant wild-type protein regenerated with 9-cis-retinal in the presence of quercetin. in contrast, the presence of quercetin did not alter the electrophoretic and basic spectroscopic properties of rhodopsin, or those of the recombinant wild-type protein, suggesting no important structural alterations as a result of quercetin binding to the receptor. the positive effect of quercetin on the stability, and chromophore regeneration of rhodopsin, could be potentially used to counteract the effect of naturally-occurring misfolding mutations in rhodopsin. thus, quercetin could help stabilizing rhodopsin mutants associated with retinal diseases such as retinitis pigmentosa. furthermore, docking of the ligand, carried out on the crystallographic structure of rhodopsin (entry 1gzm), reveals several favorable sites for quercetin binding. one of this would be compatible with 9-cis-retinal suggesting a complementary binding to the receptor of this isomer which would not be compatible with 11-cis-retinal binding. identification of prospective allosteric sites of p38 by computational methods protein function is intrinsically associated with structural flexibility, so that understanding the functional properties of proteins requires going beyond the static picture produced by x-ray diffraction studies. structural flexibility can also be interpreted as a dynamic exchange between different conformational states with low energy barriers at room temperature. allosterism is a mechanism to regulate protein function associated with the plasticity exhibited by proteins. allosteric sites can be considered transient cavities that can be occupied by a small molecule with the subsequent modulation of the protein plasticity. occupation of these sites may modify the affinity of the protein for its native substrate that can be positive when the affinity increases or negative when the affinity decreases. allosterism can be used for the design of non-competitive ligands as new therapeutic agents. this mechanism of activity modulation is particularly interesting for those targets that use a common substrate for activation, like in the case of kinases to search for selective compounds. proteins can be viewed in solution as an ensemble of diverse energy accessible conformations. binding of an allosteric ligand produces a redistribution of the population of the diverse conformational states, which at the end modulate the affinity of the native substrate. allosteric sites can be characterized using computational methods by ensemble docking. it consist of characterize a set of structures that represent the accessible conformations of a protein that can then be used to perform virtual screening. in the present work we have studied prospective allosteric sites of p38 using computational methods. the protein is a member of the mitogen-activated protein kinases (mapks), a highly regulated group of enzymes that control a variety of physiological processes, including mitosis, gene expression, apoptosis and metabolism movement among others. the conformational profile of p38 was assessed using a 4 us trajectory of accelerated molecular dynamics as sampling technique in explicit solvent. we used as starting structure the apoform of p38 in its inactive conformation (entry 1p38). the conformational features of the protein were assessed through the analysis of the variance of the most flexible regions of the protein using principal component analysis. the snapshots of the trajectory were projected onto the two principal components. subsequent cluster analysis permitted us to select a few structures for further studies. specifically, prospective biding sites were identified using a hydrophobic probe as implemented in the sitemap program. the results show previously described regulatory sites and some new prospective ones. hydrogen/deuterium exchange-mass spectrometry provides clues on the mechanism of action of min e maria t. villar 1 , kyung-tae park 2 , joe lutkenhaus 2 , antonio artigues 1 1 cell division in most bacteria is initiated by the formation of the z ring, an essential cytoskeletal element that serves as a scaffold for the cytokinesis machinery, at the mid body of the cell. in e coli the spatial location of the z ring is regulated by the min protein system, comprised by three major proteins: minc, mind and mine. the dynamic interaction between these proteins results in the formation of an oscillating protein gradient between the poles of the cell. this oscillation determines the position of the formation of the z ring. many aspects of this simple mechanism are beginning to be understood. in particular, the conformational changes associated with the interaction of the three min proteins between them and with the cell membrane, are of especial interest. hydrogen/deuterium exchange mass spectrometry (hdx ms) is a sensitive technique for the detection of changes in protein conformation and dynamics. the main advantages of this methodology are the ability to study native proteins in solution, the requirement for low protein concentrations, the potential to discriminate multiple coexisting conformations, and the lack of an upper limit to the size of protein to be analyzed. here we use hdx ms to analyze the dynamics of the wild type mine and of its inactive double mutant d45a d49a. our results show significant differences in the rates of exchange and in the total amount of deuterium exchanged at the end of the reaction between these two forms of mine. the wild type protein exchanges most of the amide hydrogen during the first few seconds of initiation of the exchange reaction. on the other hand, the mutant protein exchanges only 50% of the total amide hydrogen atoms during the first seconds of initiation of the exchange, and the remaining 50% amide hydrogen atoms are exchanged more slowly during the next few minutes of the reaction. our data are consistent with the existence of a highly flexible structure for the wild type protein and the coexistence of at least two rigid conformations for the double mutant that are undergoing a cooperative transition. interestingly, the central b-sheet forming the interface between the two subunits is protected against exchange on both proteins. these results provide insights into the conformational changes that mine undergoes during its interaction with mind. biased signalling and heteromization of the dopamine d2 receptor in schizophrenia and parkinson's disease pablo herrera nieto 1 , james dalton 1 _ , jes us giraldo 1 _ 1 universidad aut onoma de barcelona biased signalling and heteromization of the dopamine d2 receptor in schizophrenia and parkinson's disease as a significant component of dopamine signalling in the brain, the dopamine d2 receptor (d2r), a member of the class a gpcr family, is an important target in the treatment of neurological conditions such as schizophrenia and parkinson's disease. d2r shows a variety of signalling pathways through g proteins, including adenylyl cyclase inhibition, gbgpotentiation of adenylyl cyclase 2, and erk kinase activation, in addition to b-arrestin recruitment,. these pathways are differentially activated by some agonists and it has been suggested that d2r ligands with gai/o antagonist and b-arrestin agonist activity may have anti-psychotic behavioural activity with reduced extra-pyramidal side effects. d2r has also been found to form homodimers or higher-order hetero-oligomers with other gpcrs, which may modulate d2r conformation and activity, thus constituting an additional form of allosteric receptor regulation. based on these findings, we have computationally modelled the full-length structure of d2r, including its long intracellular loop 3 (icl3) that is 1301 residues in length and absent in all homologous gpcr crystal structures. using state-of-the-art tools, such as rosetta for ab initio protein folding and acemd for micro-second1 molecular dynamics (md) simulations we have successfully de novo folded icl3, which primarily consists of extensions to transmembrane helices (tmh) 5 and 6 and an intervening disordered histidine/proline-rich region, which is highly flexible. the latter is observed to interact with other receptor intracellular loops (icl1 and icl2) and appears to restrict access to the g-protein binding-site. in addition, we have docked a structurally diverse collection of 14 ligands (biased agonists, antagonists and allosteric modulators) into our d2r model and observed characteristic binding patterns suggestive of different biased signalling mechanisms. finally, through protein-protein docking with rosettadock, we have generated a complete heterodimer model of d2r with the adenosine a2a receptor (aa2ar), where a mutual interface is formed between their respective tmhs 4 and 5, as well as an association between the c-terminus of aa2ar and icl3 of d2r. this may be a particularly relevant biological complex in the treatment of parkinson's disease where antagonists of aa2ar have been shown to ameliorate disease effects, potentially through direct interaction with d2r. bis-ans as a tool to monitor conformational changes upon assembly of binary and ternary complexes of eif4e, 4e-bp1 inhibitory protein, and the mrna 5'cap specific recognition of the mrna 5' terminal cap structure by the eukaryotic initiation factor eif4e is the first and rate-limiting step in the cap-dependent translation. small 4e-binding proteins, 4e-bp1, 4e-bp2, and 4e-bp3, inhibit the translation initiation by competing with eif4g initiation factor for the same binding site, and by blocking the assembly of the translation machinery [1] . our recent studies revealed intricate cooperativity between the cap and 4e-bp1 binding sites of eif4e [2] . here, we applied a fluorescent dye, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate (bis-ans) to investigate conformational changes upon assembly of binary and ternary complexes composed of human eif4e, 4e-bp1, and the mrna 5'cap analogue, m7gtp. the fluorescence quantum yield of bis-ans increases significantly upon binding to hydrophobic sites of proteins, making the probe a convenient tool to determine the accessibility to hydrophobic surfaces, and to monitor structural reorganisation of macromolecules [3] . we characterised the interaction of bis-ans with eif4e and 4e-bp1 by fluorescence titration. the association processes takes up to several hours until the saturation of the fluorescence signal is achieved, reflecting high flexibility of the protein structures. the association constants kas of eif4e/bis-ans complexes are very high for the non-specific interaction. the kas values for eif4e/bis-ans and eif4e/4e-bp1/bis-ans are similar (10 7 m 21 ), whereas the presence of m7gtp results in ca. 5-fold weaker binding of the probe to eif4e. the affinity of bis-ans for 4e-bp1 is 10-fold lower than that for eif4e. we found no effect of either m7gtp or 4e-bp1 on the fluorescence of bis-ans in complex with eif4e, thus indicating lack of conformational changes around the probe on eif4e/m7gtp or eif4e/4e-bp1 complex formation. it also testifies that bis-ans does not bind to the cap-binding site, despite the hydrophobic nature of this eif4e region. on the contrary, addition of m7gtp to the eif4e/4e-bp1/bis-ans complex causes an increase of the probe fluorescence, which indicates differences in the structural reorganisation in the binary, m7gtp/eif4e, compared with the ternary, m7gtp/eif4e/4e-bp1, complexes, and confirms the spatial cooperation between the cap and 4e-bp1 binding sites. we also observed an increase of fluorescence for bis-ans bound to 4e-bp1 in the presence of eif4e, pointing out that 4e-bp1 partially folds upon association with eif4e. in summary, our results provide a deeper insight into the structural aspects of the molecular interaction at early stages of the cap-dependent translation. acknowledgements: this work was supported by the bst 170000/bf project from university of warsaw background: beta2-glycoprotein (b2gpi) is a protein abundantly present in human plasma and highly conserved in all mammals. b2gpi has been identified as the major antigen in the antiphospholipid syndrome (aps), a severe thrombotic autoimmune disease. despite its importance in the pathogenesis of aps, the physiological role of b2gpi is still elusive. in a previous work we have demonstrated that b2gpi significantly prolongs the clotting time in fibrin generation assays, and inhibits aggregation of gel-filtered platelets (ic5050.36um), either isolated or in whole blood, by inhibiting cleavage of par1 on intact platelets (ic5050.32um) and in solution. importantly, b2gpi does not alter the ability of thrombin (fiia) to generate the anticoagulant protein c, with or without thrombomodulin added. hence, we concluded that b2gpi inhibits the key procoagulant properties of fiia, without affecting its unique anticoagulant function. we also proposed that b2gpi, together with other more efficient anticoagulant pathways such as thrombomodulin-fiia -protein c and antithrombin iii-fiia, may function as a mild anticoagulant in vivo especially in those compartments were the efficacy of thrombomodulin is limited, as in the large vessels, or is even absent, as in the brain vasculature. aims: lacking the threedimensional structure of b2gpi-thrombin complex, the aim of this work is to identify the peptide regions either on thrombin and b2gpi involved in complex formation. results: data obtained by fluorescence and surface plasmon resonance (spr) indicated that b2gpi interacts whit fiia whit physiological affinity (kd543 6 4nm). kd values calculated by reverting the interacting systems are very similar to each other (kd598 6 9nm), suggesting that b2gpi in the mobile phase has a conformation which is competent for the binding to immobilized fiia. the affinity of fiia for immobilized b2gpi is markedly decreased by increased ionic strength (i.e. kd increases by 50-fold going from 0.1 m to 0.4 m), suggesting the electrostatic interactions play a key role in fiia -b2gpi recognition. filling/inactivation or perturbation of fiia active site does not alter the affinity of fiia for immobilized b2gpi, confirming that the active site is not involved in the interaction. mapping of thrombin binding sites with specific exosite-directed ligands (i.e. hirugen, gpibalpha, hd1 aptamer) and thrombin analogues having the exosites variably compromised (i.e. prothrombin, prethrombin-2, alpha-thrombin), reveals that the positively charged exosite-ii of fiia plays a key role in b2gpi binding. from the docking model of the bb2gpi-thrombin complex, we identified a highly negatively charged segment 219-232 in domain v of b2gpi interacting with positively charged pathes in thrombin exosite ii. the synthetic peptide b2gpi(219-232) was able to bind to fiia with an affinity (kd538 6 9nm) comparable to that of full-length b2gpi, deduced from fluorescence or spr measurements and to compete in spr measueremnts with the binding of full-length b2gpi to thrombin. hence, combining experimental and theoretical data, we obtained a reliable model of the b2gpi-thrombin complex. metalloproteases are one of the most diverse types of proteases, presenting a wide range of folds and catalytic metal ions. in the case of the merops ma clan, where most of the known metalloproteases are grouped based on the consensus hexxh sequence motif, a single catalytic zinc ion and common fold architecture [1] . despite these common features, members from distinct families present distinct domain composition and topology. given our interest in developing new tailor-made metalloproteases for bioengineering applications, an in-depth understanding of the factors governing their function is required. protein internal dynamics includes the space of functionally-relevant structural changes occurring during an enzymatic reaction, and there is an increasing understanding on how it relates with protein sequence and structure evolution. therefore, we have recently assessed how the structural heterogeneity of metalloproteases relates with the similarity of their dynamical profiles [2] . first, the dynamical profile of the clan ma type protein thermolysin, derived from the anisotropic network model, was evaluated and compared with those obtained from principal component (pc) analysis of a set of 112 crystallographic structures and essential dynamics (ed) analysis of a 20 ns molecular dynamics simulation trajectory [3] . a close correspondence was obtained between normal modes (nm) derived from the coarse-grained model and experimentally-observed conformational changes (rmsip between nm1-nm3 and pc1 of 0.81), corresponding to functionally-relevant hinge bending motions that were shown to be encoded in the internal dynamics of the protein (cumulative overlap of ed1-ed3 and pc1 of 0.85). next, dynamics-based comparison methods that employ a related coarse-grained model (b-gaussian elastic network model) was made for a representative set of 13 ma clan members [4] , allowing for a quantitative description of its structural and dynamical variability. although members are structurally similar (87% pairs with dalilite z-score > 2.0), they nonetheless present distinct dynamical profiles (69% of pairs with aladyn p-value > 0.02), with no identified correlation between structural and dynamical similarity. for cases where high dynamical similarity was observed, the respective modes corresponded to hinge-bending motions encompassing regions close to the active site. further inspection of the produced alignments indicates that for ma clan metalloproteases, conservation of internal dynamics has a functional basis, namely the need for maintaining proper intermolecular interactions between the protein and respective substrate. previously unnoticed dynamical similarity between clan members botulinum neurotoxin type a, leishmanolysin and carboxypeptidase pfu was also found. together, these results suggest that distinct selective pressure mechanisms acted on metalloprotease structure and dynamics through the course of evolution. this work shows how new insights on metalloprotease function and evolution can be assessed with comparison schemes that incorporate additional information of protein dynamics. glucokinase from antarctic psychrotroph pseudoalteromonas sp. as-131 (psgk) has a higher specific activity at low temperatures and a higher thermal stability than its mesophilic counterpart from e. coli (ecgk). in order to elucidate the structural basis for cold-adaptation and thermal stabilization of psgk, we have determined the crystal structure of psgk at 1.69 å and compared it with the ecgk structure. psgk is a homodimer of the subunit of 328 amino acid residues. each subunit consists of two domains, a small a/b domain (residues 7-125 and 314-328) and a large a 1 b domain (residues 126-313). the active site is located in a cleft formed between the two domains. the identity of amino acid sequence between psgk and ecgk was 36%, but three dimensional structures of them are very similar to each other, having the conserved catalytic residues and substrate-binding residues. the analysis of the mainchain temperature factors revealed that the regions of small domain and the hinge region connecting two domains of psgk showed higher temperature factors with a lower number of intramolecular hydrogen bonds and ionic interactions than the corresponding regions of ecgk. however, the large domain regions of psgk showed lower temperature factors with a higher number of intramolecular hydrogen bonds than ecgk. furthermore, the atomic temperature factors of catalytic asp112 on the small domain were higher, but those of glucose-binding glu169, his172, and glu199 on the large domain were lower than ecgk. these results suggest that highly flexible hinge region and the catalytic residue on the small domain of psgk may contribute to its cold-adaptation, namely higher activity at low temperatures, whereas a more rigid structure of the large domain of psgk stabilizes its overall structure more strongly than ecgk. nowadays non-waste technologies in synthetic chemistry become more and more popular. such processes are often carried out using different enzymes. dehydrogenases represent the large group of enzymes, which are widely used in synthesis of chiral compounds and other useful molecules. such enzymes need nadh or nadph as a cofactor and due to high cost of reduced coenzymes a cofactor regeneration system is an obligate part in such kind of processes. it was shown that formate dehydrogenase (fdh, ec 1.2.1.2.) is one of the best enzymes for nad(p)h regeneration. fdh catalyses the reaction of formate oxidation to carbon dioxide coupled with reduction of nad(p)1 to nad(p)h. the main advantages of fdh are the irreversibility of catalyzed reaction, low price of formate ion and wide ph optimum of activity. our laboratory has the largest collection of formate dehydrogenases from different sources. many fdh genes from bacteria, yeasts and plants were cloned and enzymes were expressed in active and soluble forms. mutant formate dehydrogenases from bacterium pseudomonas sp.101 show the highest thermal stability as well as activity in comparison with other reported formate dehydrogenases. now we have focused on eukaryotic genes. the recombinant enzymes from soya glycine max (soyfdh), arabidopsis thaliana (athfdh), moss physcomitrella patens (ppafdh) and yeast ogataea parapolymorpha (opafdh) were obtained by genetic engineering methods. it was revealed, that soyfdh has the best michaelis constants among all known fdhs, but it's less thermally stable compared to other fdhs. new mutant forms of soyfdh with excellent catalytic characteristics and high thermal stability were obtained by protein engineering. other enzymes (athfdh, ppafdh and opafdh) are comparable in their stability with majority of bacterial enzymes (but not with psefdh), so all the new obtained fdhs can be successfully used for cofactor regeneration. marmara university, 2 wellesley college, 3 antibiotics are essential therapeutic drugs widely used in the treatment of bacterial infections. unfortunately, misuse of these drugs resulted in the development of bacterial defense mechanisms. blactamase synthesis is among these mechanisms that renders b-lactam antibiotics ineffective. understanding the dynamic behavior of this enzyme is an important step in controlling its activity. in a former study, the importance of highly conserved w229 in modulating the hinge type h10 motion was reported. in the light of this information, mutant tem-1 b-lactamase enzymes with w229a, w229f and w229y substitutions were constructed. wild-type and mutant tem-1 b-lactamases purified with ni21affinity chromatography were subjected to enzyme assay using centa as the substrate. with w229f and w229y mutations, the remaining activity was approximately 10% of the initial activity. however with the w229a mutation, activity was totally lost. structural studies of the w229a mutant with cd and florescence spectroscopy indicated that there was no major change in the overall structure. however this mutation disrupted the interactions of w229 which resulted in an increase in the flexibility of this region of the protein. this project was supported by t € ub _ itak project no 113m533. light-switchable zn21 binding proteins to study the role of intracellular zn21 signaling stijn aper 1 , maarten merkx 1 1 zn21 plays an important catalytic and structural role in many fundamental cellular processes and its homeostasis is tightly controlled. recently, free zn21 has also been suggested to act as an intracellular signaling molecule. to get increased understanding of the signaling role of zn21 we are developing light-switchable zn21 binding proteins to perturb the intracellular zn21 concentration using light. these protein switches consist of two light-responsive vivid domains and the zn21 binding domains atox1 and wd4, linked together with flexible peptide linkers. in the dark, zn21 is tightly bound in between the two zn21 binding proteins. light-induced dimerization of the vivid proteins disrupts this interaction and thus results in zn21 release. the fluorescent proteins cerulean and citrine were attached to the vivid domains to allow the different conformational states of the protein switch to be monitored using fret. zn21 titrations revealed a 3-fold decrease in zn21 affinity going from dark-to light-state for the initial design, which was further improved to 10-fold by optimizing the linkers between the protein domains. in addition, the zn21 affinities of both states were tuned to be optimal for intracellular applications. switching between the high affinity dark-state and the low affinity lightstate was found to be reversible for at least two light-dark cycles. following the in vitro characterization, we are currently assessing the performance of this genetically encoded 'caged' zn21 in mammalian cells. proteins as supramolecular building blocks: engineering nanoscale structures 5 school of biological sciences, university of auckland, 6 school of biological sciences, victoria university proteins hold great promise in forming complex nanoscale structures which could be used in the development of new nanomaterials, devices, biosensors, electronics and pharmaceuticals. the potential to produce nanomaterials from proteins is well supported by the numerous examples of self-assembling proteins found in nature. we are exploring self-assembling proteins for use as supramolecular building blocks, or tectons, specifically the n-terminal domain of a dna binding protein (nterm-lsr2) and a typical 2-cys peroxiredoxin (hsprx3). non-native forms of these proteins have been designed undergo selfassembly into supramolecular structures in a controllable manner. self-assembly of nterm-lsr2 is initiated via proteolytic cleavage, thereby allowing us to generate supramolecular assemblies in response to a specific trigger. we will show that the degree of oligomerisation can be controlled by variations in environmental conditions such as ph and protein concentration. furthermore, via protein engineering, we have introduced a new "switch" for oligomerisation via enteropeptidase cleavage. the new construct of nterm-lsr2 can be activated and assembled in a controlled fashion and provides some ability to alter the ratio of higher ordered structures formed. hsprx3 has been shown to oligomerise into dimers, toroids, stacks and tubes in response to specific triggers such as ph and redox state. in this work we have utilised the histidine tag to further control the assembly of this versatile protein tecton. we will show that minute variations in ph can induce oligomersation of hsprx3 toroids into stacks and tubes. furthermore, by utilising the histidine tag as a ligand we can bind divalent metals to these supramolecular structures. this not only drives the formation of higher ordered oligomers but also provides a facile route which may facilitate the functionalisation of these protein nanoscale structures after they have been assembled. danielle basore 1,2 , rajesh naz 5 , scott michael 6 , sharon isern 6 , benjamin wright 3 , katie saporita 1 , donna crone 1 , christopher bystroff 1,2,4 1 biological sciences, rensselaer polytechnic institute, 2 cbis, rensselaer polytechnic institute, 3 chemical and biological engineering, rensselaer polytechnic institute, 4 computer science, rensselaer polytechnic institute, 5 obstetrics and gynecology, west virginia university, 6 unintended pregnancy is a worldwide public health concern, with 85 million pregnancies being classed as unintended in 2012 . the magnitude of this number clearly indicates an unmet need in terms of contraception. methods that are currently available are effective, but exhibit many problems. side effects, ease of use, cost, and availability are all concerns. we propose a contraceptive vaccine that would be safe, effective, long-lasting, cheap, and reversible. our vaccine would prevent pregnancy by targeting sperm with antibodies raised in the woman's body. several approaches have been taken to developing a contraceptive vaccine in recent years. the most successful so far has been using human chorionic gonadotropin (hcg), a hormone produced during pregnancy, as an antigen . the hcg vaccine progressed to phase 2 clinical trials, but only displayed an 80% efficacy, which is insufficient for a contraceptive. our lab uses a structure based approach to the design of an anti-sperm antigenic protein. we believe this will raise a more vigorous immune response that will produce a longer lasting titer. the catsper complex is a heterotetrameric calcium channel found in the tail region of sperm . each subunit of the complex contains an exposed loop known as the p-loop. the p-loop is unique on the surface of sperm because it is not glycosylated, allowing antibodies to potentially recognize and bind it. ylp12 is a twelve residue peptide that mimics the glycans in the glycocalyx of sperm . ylp12 is a member of the flitrx library, and in mice, produced protective titers that were reversible both voluntarily and involuntarily. our designs will introduce these two potential antigens into a loop of the l1 protein of human papilloma virus. l1 spontaneously assembles into virus like particles, and will aid in the production of a robust immune response. protein carriers for passage of the blood-brain barrier sinisa bjelic 1 1 medical solutions that help protein therapeutics accumulate into the brain are crucial for future treatment of neurological disorders. biodrugs have a tremendous potential to treat disorders of the nervous system, but their efficiency has been severely restricted. to reach the brain all drugs must traverse the blood-brain barrier (bbb) -a permeable wall that separates blood from the brain -whose main function is to protect the nervous system from environmental influences of bacteria and toxins. unfortunately the bbb is also the culprit that effectively blocks access to therapeutics required for treatment of neurological diseases. a way to boost exposure of therapeutics across the bbb is to piggyback onto the transferrin receptor, a multidomain protein anchored in the membrane, which is involved in the physiological facilitation of iron uptake. here i present research that aims at successfully developing potent protein carriers for transferrin receptor-mediated passage of the bbb by using computational protein design in combination with yeast display methodology for hit validation and optimization. the longterm goal is to couple therapeutics -as for example drugs against alzheimer's -to the designed carriers to increase the brain uptake and cure neurological disorders. medium-throughput multistep purification of coagulation factor viia jais r. bjelke 1 , gorm andersen 1 , henrik østergaard 1 , laust b. johnsen 1 , anette a. pedersen 1 , tina h. glue 1 1 there is a need of medium-to-high throughput purification of low-titre recombinant protein variants for screening to identify the final biopharmaceutical lead. such proteins include coagulation factors to be used for treatment of haemophilia and other bleeding disorders. at novo nordisk we have established a platform for production of recombinant coagulation factor viia variants, which include a spectrum of single-point mutations to large domain insertions. the variants were produced using transiently transfected hek293f, hkb11 or choebnalt85 (qmcf technology) suspension cells. harvest cultivations were typical in the range of 0.3-to 1l. a 3-step continuous, multistep purification method was implemented on € aktaxpress systems (ge healthcare). the interlinked process steps include capture using an immunoaffinity column, polish, concentration and buffer exchange using an anion-exchange column and proteolytic activation of the zymogen variant forms using a coagulation factor xaimmobilized column. buffers were designed such that elution from the capture column was aligned with binding conditions on the polish column to avoid a desalting step in-between. the following and final enzymatic activation was optimized with regards to flow rate to ensure full conversion while minimizing unwanted secondary cleavages in factor viia. the final products were fractionated in sharp chromatographic peaks ready for characterization. hplc and sds-page analyses showed a solid quality of the produced variants and more than 800 variants have been produced in sub mg scale using the outlined method. biomimetic sequestration of co2: reprogramming the b1 domain of protein g through a combined computational and experimental approach esra bozkurt 1 , ruud hovius 1 , thereza a. soares 2 , ursula rothlisberger 1 1 ecole polytechnique f ed erale de lausanne, 2 federal university of pernambuco protein engineering is a powerful tool to generate highly specific enzymes for biomimetic production of chemicals. among many applications, the development of enzymes to accelerate carbon dioxide fixation is a possible route to limit co2 emission. in this project, we are inspired by the ancient enzyme carbonic anhydrase which efficiently catalyzes the reversible hydration of carbon dioxide in the presence of a zinc ion active site.1 to create an efficient biocatalyst, the engineered gb1 domain2 containing a his3cys zn (ii) binding site was used as a starting point.3 in subsequent work, b1 domains comprising of his3wat zn (ii) binding sites have been rationally designed to produce carbonic anhydrase mimics. the re-engineering was accomplished through a series of mutations to orient the zinc bound reactive species to form a hydrogen bond network in the active site while retaining the native secondary structure. we performed classical molecular dynamics (md), quantum mechanics/molecular mechanics (qm/ mm) simulations and metadynamics, with the aim to explore potential catalytic roles of the reengineered b1 domains and to elaborate the reaction mechanism. briefly, we introduced novel zn (ii) binding sites into thermostable b1 domain. in parallel, experiments are underway. wild-type protein was expressed and purified. structural and mutagenesis studies are ongoing. the results emphasize the power of theoretical work to enable the mimicking of nature's enzymes for desired catalytic functions. the roles of entropy and packing efficiency in determining protein-peptide interaction affinities diego caballero 1,2 , corey o'hern 1,2,3,4 , lynne regan 2,5,6 1 physics, yale university, 2 integrated graduate program in physical and engineering biology, yale university, 3 mechanical engineering and materials science, yale university, 4 applied physics, yale university, 5 molecular biophysics and biochemistry, yale university, 6 chemistry, yale university despite many recent improvements in computational methods for protein design, we still lack a quantitative and predictive understanding of the driving forces that control protein stability, for example, we do not know the relative magnitudes of the side-chain entropy, van der waals contact interactions, and other enthalpic contributions to the free energy of folded proteins. in addition, we cannot reliably predict the effects of point mutations on enzyme specificity or sequence tolerance in ligand binding sites. the tetratricopeptide repeat (tpr) motif is a common and versatile protein system that has been used as a model to study protein-protein interactions. for example, recent studies have experimentally measured the binding affinity and specificity for different tpr binding pockets and peptide ligands and generated a ranking of the protein-peptide pairs with the highest affinity. to gain a fundamental understanding of the interplay between atomic close packing and fluctuations of side-chain conformations in protein-peptide binding pairs, we performed all-atom langevin dynamics simulations of key residues near the binding interface of tpr proteins and their cognate peptides. the langevin dynamics simulations enabled us to calculate the entropy and potential energy of side chain conformations in the presence of backbone fluctuations for each protein-peptide pair. we compile rankings of the stability and affinity of mutant tpr-peptide structures to those obtained from experimental studies. this research has enhanced our ability to rationally manipulate protein-peptide interfaces. advances from this research will enable the design of tpr modules that specifically recognize biologically important proteins. monitoring protein-protein interactions using tripartite split-gfp complementation assays protein-fragment complementation assay (or pca) is a powerful strategy for visualizing protein-protein interactions in living cells. previously described split-gfp based sensors suffer from the poor solubility of individual pca fragments in addition to background signal originating from their spontaneous selfassembly (1). we developed a new encoded genetic reporter called "tripartite split-gfp" for visualizing protein-protein interactions in vitro and in living cells. the assay is based on tripartite association between two twenty amino-acids long split-gfp tags, gfp10 and gfp11, fused to interacting protein partners, and the complementary gfp1-9 detector. when proteins interact, gfp10 and gfp11 selfassociate with gfp1-9 to reconstitute a functional gfp (2). using coiled-coils and frb/fkbp12 model systems we characterize the sensor in vitro and in escherichia coli. we extended our studies to mammalian cells and examine the fk-506 inhibition of the rapamycin-induced association of frb/fkbp12. the small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence and for screening modulators of complex formation in cell-based assays. aldehyde dehydrogenases (aldhs) catalyze the oxidation of aldehydes to their corresponding acids using nad(p)1 as coenzyme. these enzymes are responsible for the detoxification of lipid peroxidation products, which have been involved in the etiology and pathogenesis of different diseases involving increments in oxidative stress. recent data from our group, showed that aldh3a1 is resistant to inactivation by lipid peroxidation products, even at concentrations 50-100 times higher than those required to inactivate aldh1a1 and aldh2. the amino acids sequence of the aldehyde-binding site of the three enzymes was analyzed, and it was found that the enzymes susceptible to the effect of lipid peroxidation products (aldh1a1 and aldh2), have cys residues flanking the reactive cys (position 302), based on this criteria and considering that these aldehydes react preferentially with cysteine, a mutant of aldh2 was generated changing the cys residues adjacent to cys302. the mutant aldh2-cys301thr-cys303val, was resistant to the inactivation by acrolein and 4-hne, even at concentrations 1000-fold higher than those required to inactivate aldh2. however, the mutant presented values of km 2, 5 and 50-fold higher for acrolein, propionaldehyde and acetaldehyde, respectively, compared to the wild type enzyme, but showed a catalytic efficiency similar to the parent enzyme. these data revealed that cys residues near to the reactive cys in aldh2 are important in the inactivation process induced by lipid aldehydes, but also participate in determining the specificity for the substrates in this enzyme. small molecule-assisted shutoff: a widely applicable method for tunable and reversible control of protein production h. kay chung 1 , conor jacobs 1 , yunwen huo 2 , jin yang 3 , stefanie krumm 4 , richard plemper 4,5 , roger tsien 0 , michael lin 3 1 department of biology, stanford university, 2 department of pediatrics, stanford university, 3 department of pharmacology, university of california san diego, 4 department of pediatrics, emory university, 5 institute for biomedical sciences, georgia state university, 6 department of chemistry and biochemistry, university of california san diego, 7 howard hughes medical institute, university of california san diego, 8 the ability to quickly control the production of specific proteins would be useful in biomedical research and biotechnology. we describe small molecule-assisted shutoff (smash), a technique in which proteins are fused to a self-excising degron and thereby expressed in a minimally modified form by default. degron removal is performed by a cis-encoded hepatitis c virus (hcv) protease, so that applying clinically available hcv protease inhibitors causes degron retention on subsequently synthesized protein copies and suppresses further protein production. we find that smash allows reversible and dosedependent shutoff of various proteins with high dynamic range in multiple cell types, including yeast. we also successfully use smash to confer drug responsiveness onto a rna virus for which no licensed drug inhibitors exist. as smash does not require permanent fusion of a large domain, it should be useful when control over protein production with minimal structural modification is desired. furthermore, as smash only uses a single tag and does not rely on modulating protein-protein interactions, it should be easy to generalize to multiple biological contexts. top, a protein of interest is fused to the smash tag via a hcv ns3 protease recognition site. after protein folding, the smash tag is removed by its internal ns3 protease activity, and is degraded due to an internal degron activity. bottom, addition of protease inhibitor induces the rapid degradation of subsequently synthesized copies of the tagged protein, effectively shutting off further protein production. vaccine development has emerged, epitope-focused immunogens, but in the past these have failed to deliver the expected outcome. here, we employed a new computational design methodology (rosetta fold from loops or ffl) to design epitope-focused immunogens. ffl was devised to insert structurally defined functional sites into protein scaffolds. throughout the ffl stages the structure of the scaffold is folded and its sequence designed to stabilize the desired functional conformation of the inserted site. we used ffl to design epitope-focused immunogens for the respiratory syncytial virus (rsv), for which despite the intense research we are still lacking an approved vaccine. we designed three-helix bundles harboring an rsv epitope, that was previously co-crystallized with the neutralizing antibody motavizumab. the designs were thermodynamically stable (tm > 100˚c) and showed extremely high affinities to motavizumab (kd 30 pm). structural characterization through x-ray crystallography of antibodybound and unbound scaffolds showed good agreement to the computational models in the overall structure (rmsd -1.2 å) and exquisite mimicry of the epitope region (rmsd -0.4 å), when compared to the peptide-epitope in complex with motavizumab. the designed immunogens were used to immunize non-human primates (nhp), and approximately 75% of the cohort developed rsv neutralizing activity, in some instances with high potency. to evaluate the therapeutic relevance of the elicited neutralization activity, we compared the nhp neutralization titers to those of human sera after natural rsv infection, which generally yields protective levels of antibodies. the neutralization potency of the best nhp responders was comparable to that of the human sera. to better understand the features of the antibodies elicited, we isolated several rhesus monoclonal antibodies (rhmabs) from the animal that exhibited the most potent neutralization. two of the rhmabs bound to the immunogen with very high affinity (kd 3 pm) and were potent rsv neutralizers. interestingly, these rhmabs were approximately 10 fold more potent than the fda-approved prophylactic antibody palivizumab. our results provide the first proof-of-principle for epitope-focused vaccine design, and demonstrate the power of the ffl figure 1 . schematic of nucleotide binding, exchange and hydrolysis in tubulin, and its coupling to mt assembly. exchange of gdp (orange) for gtp (magenta) at the e-site in b-tubulin (blue) happens in the unpolymerized dimer (left). the active, gtpbound tubulin dimer adds to a growing mt (right). interaction of the incoming a-tubulin (green) with the e-site nucleotide at the plus end of a mt (with b-tubulin exposed) results in gtp hydrolysis. the mt cartoon (bottom right) shows an oversimplified representation of a gtp cap as it first grows by tubulin addition and then shrinks by polymerization-coupled gtp hydrolysis (here b-tubulin that is bound to gtp is shown in red and that bound to gdp is shown in blue). cryo-em density map (emdb-6349) and atomic model (pdb: 3jak) for an eb3-decorated mt bound to gtpgs. a-tubulin, b-tubulin and eb3 are colored green, blue, and orange, respectively. computational methodology. we anticipate that ffl will be useful for a variety of other challenges in the computational design of functional proteins. designed repeat proteins as templates for photoactive molecules and fluorescent nanoclusters sara h. mejias 1,2 , antonio aires 1,2 , javier l opez-andarias 3 , pierre couleaud 1,2 , begoña sot 1,2 , carmen atienza 3 , nazario mart ın 1,3 , aitziber l. cortajarena 1,2 1 imdea nanoscience, c/faraday, 9, ciudad universitaria de cantoblanco 28049, 2 cnb-csic-imdea nanociencia associated unit "unidad de nanobiotecnolog ıa", 3 departamento de qu ımica org anica i, facultad de qu ımica, universidad complutense self-assembly of biological molecules into defined functional structures has a tremendous potential in nanopatterning, and the design of novel bionanomaterials and functional devices. molecular selfassembly is a process by which complex three-dimensional structures with specified functions are constructed from simple molecular building blocks. we present first the study and characterization of the assembly properties of modular repeat proteins, in particular designed consensus tetratricopeptide repeats (ctprs), and their application as building blocks in order to generate functional nanostructures and biomaterials. ctpr proteins can be assembled into self-standing thin films,1 and thin nanometer fibers in solution.2 in this work, we show the use of the designed consensus repeat proteins as scaffolds to template: (1) photoactive organic molecules, and (2) fluorescent nanoclusters. 1.we explore the potential of ctpr proteins to arrange donor-acceptor pairs for electro-active materials. in particular, porphyrin rings arranged by ctprs in a defined distance and orientation for favoring face-to-face orientation which should lead to an improvement in the optoelectronic properties. our results confirm the successful ability of ctpr proteins to be used as scaffold for ordering organic chromophores, while preserving their structure. the unique self assembly properties of ctpr scaffolds have been exploited to generate ordered conductive films of the protein-porphyrin conjugates. these results open the door to fabricate hybrid protein-based solid devices. 2.we show results on the ability of ctpr to encapsulate and stabilize fluorescent gold nanoclusters. we investigated the influence of the protein sequence in the final properties of the nanoclusters. the structural and functional integrity of the protein template is critical for future applications of the protein-cluster complexes. therefore synthetic protocols that retain the protein structure and function have been developed. as a proof of concept, a ctpr module with specific binding capabilities has been successfully used to stabilize nano clusters. biohybrid photoelectrochemical cells have been developed by functionalizing the hematite photoanode with the light-harvesting cyanobacterial protein c-phycocyanin (pc) yielding a substantial enhancement of the photocurrent density. photoelectrochemical cells combining light-harvesting proteins and inorganic semiconductors have potential for the use in artificial photosynthesis. in this work we present processing routes for the functionalization of hematite photoanodes with pc, including in situ co-polymerization of pc with enzymatically-produced melanin and using a recombinantly produced pc 2. moreover, recombinant forms of the light-harvesting protein c-phycocyanin from synechocystis sp. pcc6803 were engineered to carry a peptide with affinity for hematite. similarly, a bacterial laccase was engineered to acquire affinity for hematite. results obtained from the different approaches to hematite functionalization and the advantages offered by protein engineering will be presented. minimizing a suitable free energy expression is arguably the most common approach in (ab initio) protein structure prediction. the achieved accuracy depends crucially on the quality of the free energy expression in use. here, we present corrections to existing free energy expressions which arise from the thermal motion of the protein. we (i) devise a term accounting for the vibrational entropy of the protein, and (ii) correct existing potentials for 'thermal smoothing'. (i) vibrational entropy is almost always neglected in free energy expressions as its consideration is difficult. this practice, however, may lead to incorrect output because distinct conformations of a protein can contain very different amount of vibrational entropy, as we show for the chicken villin headpiece explicitly [1] . for considering vibrational entropy, we suggest a knowledge based approach where typical fluctuation and correlation patterns are extracted from known proteins and then applied to new targets. (ii) at ambient conditions, timeaveraged potentials of proteins are considerably smoothened due to thermal motion where the strength of this effect varies strongly between atoms. distinguishing these inhomogeneities by introducing new atom species regarding their locale environment can therefore increase the precision of time-averaged potentials [2] . extraction of general principles from the continually growing protein data bank (pdb) has been a significant driving force in our understanding of protein structure. atomistic or residue-level statistical potentials, secondary-structural propensities, and geometric preferences for hydrogen bonding are among the classical insights that arose from observations in the pdb. given the magnitude of structural data available today, it is likely that many quantitative generalizations remain to be made. here we hypothesize that the pdb contains valuable quantitative information on the level of local tertiary structural motifs (terms), with term statistics reflecting fundamental relationships between sequence and structure. we define a term to be the structural fragment that captures the local secondary and tertiary environments of a given residue, and put our hypothesis through a series of rigorous tests. first, we show that by breaking a protein structure into its constituent terms, and querying the pdb to characterize the natural ensemble around each, we can estimate the compatibility of the structure with a given amino-acid sequence through a metric we term "structure score." considering submissions from recent critical assessment of structure prediction (casp) experiments, we find a strong correlation (r 5 0.69) between structure score and model accuracy, with poorly predicted regions readily identifiable. this performance exceeds that of leading atomistic statistical energy functions. next, we show that by considering the terms of a structure that are affected by a given mutation, and mining the pdb to characterize sequence statistics associated with each, we are able to predict mutational free energies on par with or better than far more sophisticated atomistic energy functions. finally, we ask whether term statistics are sufficient to enable the design of proteins de-novo. we demonstrate that given a native backbone conformation, term considerations alone with no input from molecular mechanics correctly predict roughly the same fraction of amino acids from the corresponding native sequence as state-ofthe-art computational protein design methods. knowledge-based energy functions have already put pdb statistics to good use by parsing structural environments into geometric descriptors, generally assuming their conditional independence. our results suggest that it may now be possible to instead consider local structural environments in their entirety, asking questions about them directly. if this is the case, then the pdb is an even larger treasure trove of information than it has been generally known to be, and methods of mining it for term-based statistics should present opportunities for advances in structure prediction and protein design. comprehensive understanding of a protein fold is intertwined with successful design. recent advances in designing de novo structures have shown that proteins can be designed for a few globular and helical folds. however, designing all-b structures and barrels remains challenging because loops and intricate long range interactions that are important in these topologies are difficult to control. for designing novel catalysts, the (a/b)8 -barrel (or tim-barrel) fold is one of the most important examples, for it is the most common topology for enzymes. for almost 30 year, attempts in designing de novo tim barrel structures have all resulted in poorly folded proteins. here we describe the successful design of a 4-fold symmetrical (a/b)8 barrel directly from geometrical and chemical principles. 22 designed variants with a wide range of stabilities from being molten globules to cooperatively folded proteins were experimentally characterized, and the results revealed the importance of sidechain-backbone hydrogen bonding for defining the characteristic a/b-barrel. the 184 residue tim barrel structure is among the smallest tim-barrels and has a fully-reversible melting temperature of 888c. the x-ray crystal structure shows atomic-level agreement with the design model. despite this structural similarity, psi-blast searches do not identify sequence similarities to known tim-barrel proteins. more sensitive profile-profile searches suggest that the design is sufficiently distant from other native tim-barrel superfamilies to be in a superfamily of its own, further implying that nature has only sampled a subset of the sequence space available to the tim-barrel fold. the ability to de novo design tim-barrels opens new possibilities for custom-made enzymes. 3 university of texas southwestern medical center, 4 biofrontiers institute, university of colorado creation of new molecular sensors and actuators based on fluorescent proteins relies on methods for identifying complex photophysical phenotypes and subsequently performing separations on cell populations. we developed a microfluidic flow cytometry approach tailored to interrogating the performance of genetically-encoded fluorophores and present the results of studies employing this technology. the system screens cell-based libraries on the basis of multiple photophysical parameters relevant to imaging, including brightness, photostability, and excited-state lifetime (i.e. a proxy for fluorescence quantum yield) at a rate of up to 180 cells/sec. in a first generation of experiments, molecular dynamics-guided design was used to create a library of mcherry mutants that was screened with this system, resulting in the identification of a variant with a higher stability b-barrel and improved photostability but with a decreased brightness due to reduction in the fluorescence quantum yield. to avoid inadvertent decreases in this important performance criterion, subsequent rounds of selection were performed on the basis of both photostability and excited-state lifetime as sorting criteria. in these second generation selections, mutations were designed to target pathways of oxygen access through the bottom of the bbarrel in addition to a position that directly interacts with the chromophore. furthermore, subsequent rounds of screening were used to improve folding and maturation. the multiparameter sort identified multiple clones with up to 8-fold improved photostability and up to double the excited-state lifetime of the parent mcherry fluorescent protein. the best mutant we identified produces one order of magnitude more photons before photobleaching compared to mcherry, at excitation conditions characteristic of confocal fluorescence microscopy. our results demonstrate the utility of combining moleculardynamics-guided library design with technology for photophysics-based selections. we anticipate that the new fluorescent proteins obtained in this work will find use in low-copy-number and long-duration imaging live cell imaging applications in cell-lines created by genomic editing techniques. targeted protein degradation achieved through a combination of degrons from yeast and mammalian ornithine decarboxylase rushikesh joshi 1 , ratna prabha c. 1 1 the maharaja sayajirao university of baroda targeted protein degradation achieved through a combination of degrons from yeast and mammalian ornithine decarboxylase targeting the over accumulated protein in the cell for degradation using specific degrons is an emerging research area. the degradation of the vast majority of cellular proteins is targeted by the ubiquitin-proteasome pathway. but in the case of ubiquitin independent protein degradation, odc/az system is more effective in achieving targeted protein degradation than other types of degradation 1. ornithine decarboxylase (odc) is key regulatory enzyme in the biosynthesis of polyamines. the protein has two domains namely, n terminal a/b barrel domain and c-terminal b-sheet domain. degradation of odc is mediated by polyamine inducible protein, antizyme (az). antizyme interacts with odc on n-terminal region, which results in degradation of odc by proteasomes. in mammalian odc the c-terminal has an unstructured tail of 37 residues, which pulls odc into proteasome for degradation. it was reported earlier by coffino's group that the unstructured tail acts as a degron in chimeric fusion with gfp 2. in yeast, same function is achieved by n-terminal 44 residues 3. present study focuses on accomplishing targeted protein degradation in saccharomyces cerevisiae by adding these two degradation signals or degrons of yeast odc and mammalian odc as tags to a reporter protein. we have selected two degrons namely, n terminal a/b barrel domain of yeast odc and c-terminal 37 residues of mouse odc and grafted them to n and c-terminus of the reporter protein yegfp. degradation of yegfp and yegfp fusion with degrons of odc (degron-yegfp) were monitored by western blot using anti-gfp antibody and fluorescence spectroscopy. initially, the amount of degron-yegfp fusion protein was very low compared to control yegfp. it means that the chimeric protein underwent rapid degradation in the cells. after inhibition of proteasome, increase in the level of degron-yegfp was observed, confirming that the degrons cause rapid degradation of reporter protein through proteasome. earlier, we have also tagged ubiquitin from yeast with last 37 residues of modc and observed enhanced degradation of ubiquitin in saccharomyces cerevisiae. therefore, both the degrons of odc alone and in combination are capable of decreasing stability of reporter protein in the cells. however, the combination of degrons is more effective than either of them in isolation. enzymes fold into unique three-dimensional structures, which underlie their remarkable catalytic properties. the requirement that they be stably folded is a likely factor that contributes to their relatively large size (> 10,000 dalton). however, much shorter peptides can achieve well-defined conformations through the formation of amyloid fibrils. to test whether short amyloid-forming peptides might in fact be capable of enzyme-like catalysis, we designed a series of 7-residue peptides that act as zn21dependent esterases. zn21 helps stabilize the fibril formation, while also acting as a cofactor to catalyze acyl ester hydrolysis. the fibril activity is on par with the most active to date zinc-protein complex. such remarkable efficiency is due to the small size of the active unit (likely a dimer of 7-residue peptides), while the protein is at least 15-fold larger in molecular weight. the observed catalytic activity is not limited to ester hydrolysis. we have designed copper binding peptides that are capable oxygen activation. these results indicate that prion-like fibrils are able to not only catalyze their own formation -they also can catalyze chemical reactions. thus, they might have served as intermediates in the evolution of modern-day metalloenzymes. these results also have implications for the design of self-assembling nanostructured catalysts including ones containing a variety of biological and nonbiological metal ions. rational design of the cold active subtilisin-like serine protease vpr with improved catalytic properties and thermal stability abstract proteinase vpr, from a psychrophilic vibrio species and its thermophilic structural homologue, aqualysin i (aqui) from thermus aquaticus, we set out to design a mutant of vpr which would be more thermostable, but would retain the high catalytic activity of the wild type enzyme. our starting protein template was a previously stabilized mutant containing two inserted proline residues close to the nterminus of vpr (n3p/i5p). this vpr_n3p/i5p mutant was shown to have a significantly increased thermal stability but displayed a concomitant tenfold loss of catalytic efficiency. from our previous studies we selected two mutations, one which increased catalytic activity (q142k) of the enzyme significantly and another which stabilized the protein against thermal denaturation (n15d). the n15d mutation had been shown to introduce a salt bridge into the structure of the cold adapted proteinase, yielding higher stability but without negative effects on activity. the q142k exchange had been shown to double the turnover number (kcat) to that of the wild type enzyme. insertions of these selected mutations into the vpr_n3p/i5p mutant were according to predictions; the q142k increased the kcat tenfold, and the n15d mutation increased the thermal stability. in the combination mutant, vpr_n3p/i5p/n15d/q142k, thermal stability was increased by 88c and 108c, in terms of tm and t50%, respectively. furthermore, the catalytic activity of the mutant was somewhat higher than that of the wild type enzyme. critical peptide stretches may not serve as faithful experimental mimics for protein amyloidogenesis bishwajit kundu 1 , dushyant garg 1 1 certain amino acid stretches are considered critical to trigger the amyloidogenesis in a protein. these peptide stretches are often synthetically produced to serve as experimental mimics for studying amyloidogenesis of the parent protein. here we provide evidence that such simple extrapolation may be misleading. we studied the amyloidogenesis of full length bovine carbonic anhydrase ii (bcaii) and compared it with those formed by its critical amyloidogenic peptide stretch 201-227 (pepb). under similar solution conditions and initial monomeric concentrations, we found that while amyloid formation by bcaii followed aggregation kinetics dominated by surface-catalyzed secondary nucleation, pepb followed classical nucleation-dependent pathway. the afm images showed that bcaii forms short, thick and branched fibrils, whereas pepb formed thin, long and unbranched fibrils. atr-ftir revealed parallel arrangement of cross b sheet in bcaii amyloids, while pepb arranged into antiparallel b sheets. amyloids formed by bcaii were unable to seed the fibrillation of pepb and vice versa. even the intermediates formed during lag phase revealed contrasting ftir, far uv cd signature, hydrophobicity and morphology. we propose that for any polypeptide, the sequences flanking a critical region are equally effective in modulating the initial nucleation events, generating prefibrillar and finally fibrillar species with contrasting characteristic. the results have been discussed in light of amyloid polymorphism and its importance in the design of therapeutic strategies targeting such toxic regions. aksana labokha 1 , ralph minter 1 1 all approved biological drugs target extracellular proteins and not the majority of the expressed human genome, which resides within intracellular compartments. included in the latter category are many important, disease-relevant targets which cannot be easily addressed by small molecule approaches, such as the oncology targets c-myc and k-ras. although bacteria and viruses have evolved strategies to deliver biological material to the cell cytoplasm and nucleus, our ability to engineer recombinant proteins to replicate this is somewhat limited by (i) our nascent understanding of protein uptake and trafficking pathways and (ii) the ability to easily quantify cell delivery to the cytoplasm and cellular organelles. the aim of my project is to address these challenges by developing an effective assay for cytoplasmic uptake and then using it to measure the delivery efficiency of recombinant proteins which mimic natural delivery strategies e.g. cell penetrating peptides fusion, exotoxin mimics, and supercharged proteins (proteins with high surface charge which can enter cells). i also intend to explore the influence of the rab superfamily, which are the master regulators of protein trafficking, to influence and control both the kinetics and final subcellular destination of exogenous proteins. protein engineering: what's next? with the growing industrial need for engineering enzymes for the deconstruction and transformation of plant biomass in biorefineries, there is a want for the development of new approaches for designing special purpose biocatalysts. techniques, such as directed evolution, which mimic the natural selection process by evolving proteins towards the improvement of a given property, have unquestionably demonstrated their value and are routinely used in large industrial companies. nevertheless, the brute force employed in these methods, could significantly gain from an all-atom description of the underlying catalytic mechanisms, to center the efforts on more limited areas of the protein. in the last years, we have developed computational tools, which combine the electronic structure description of qm/mm methods with the potential to model long time scale processes of pele,1 to study the details of a variety of reactions. examples, which will be discussed, include rationalizing the selective oxyfunctionalization of steroids using fungal enzymes2 and the study of the effect of point mutations on the oxidation efficiency of laccases.3 these methods have shown their potential not only at the descriptive level but, more importantly, through their high predictive capability that opens many opportunities for their use in biotechnology. in this talk, we will show how recent advances in in silico approaches are setting new grounds for future computer guided directed evolution. several orthogonal bioreactions take place simultaneously within membrane bound organelles in eukaryotes and proteinaceous microcompartments in bacteria. these subcellular structures contain sets of enzymes co-involved in metabolic pathways. towards the goal of creating artificial protein microreactors, we seek to develop an artificial organelle that emulates the metabolic activity of the carbon fixating organelle of autotrophic bacteria, the carboxysome. here, we show that the two key carboxysomal enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) and carbonic anhydrase (ca), can be efficiently co-encapsulated using our previously reported encapsulation system which is based on a bacterial capsid formed from the protein lumazine synthase (aals-13). our preliminary results suggest that the enzymes can act in tandem and that the co-encapsulation of ca with rubisco in the capsid is necessary for enhanced rubisco activity in vitro. we attribute this observation to the high local concentrations of the rubisco substrate, co2, produced by ca within the capsid. we are developing a theoretical model of a minimal carboxysome using the kinetic rate constants of our rubisco and ca variants and aals-13 as the shell to complement these experiments. next, we will incorporate our minimal carboxysome within an expression host such as e.coli, opening up the possibility of further optimization through directed evolution. in the past targeting and engineering of chemokines has led to several interesting drug candidates. [1] amongst them, met-rantes, a met-ccl5 with high g protein-coupled receptor (gpcr) affinity but no subsequent signal transduction, as well as mutants addressing the interaction with the so-called glycosaminoglycans (gags) seem to be the most promising candidates. both, gag knockout as well as gag affinity matured chemokine isoforms have been considered as anti-inflammatory drug candidates, out of which an il-8 mutant with 5 modifications reached clinical phase 1 where it was profiled for acute neutrophil-related exacerbation in copd. [2] cxcl10 (ip-10) is a proinflammatory chemokine released by various cells following stimulation by interferon g (ifn-g) . it is therefore considered as a late chemokine being responsible for the attraction of different lymphocytes. [3] any therapeutic indication is consequently related to chronic and multiple applications. we have therefore engineered cxcl10 very conservatively at positions to ultimately generate dominant-negative mutants with a mildly improved gagbinding affinity and an entire knock off gpcr activity. the first steps of our engineering approach were in silico modelling of the mutants and the establishment of a suitable upstream-and downstreamprocessing protocol. next we generated a fluorescently engineered cxcl10 variant for our fluorescence-based affinity studies which was subjected to biocomparability investigations relative to the native, non-fluorescent protein. compared to the wild type, the fluorescently engineered mutant exhibited similar biological, chemotactic and gag-binding properties. next we started to produce sufficient amounts of the members of our nascent mutant library which were tested with respect to their biophysically behavior as well as to their knocked out chemotactic potency on cells. these experiments included gel electrophoresis and western blot analysis to determine identity and purity; circular dichroism (cd) and chaotrope-induced unfolding to approximate structure; isothermal fluorescence titration (ift); surface plasmon resonance (spr) and isothermal titration calorimetry (itc) to quantify gagbinding affinity and boyden chamber experiments to determine the chemotactic activity. our results show that we are able to tune the gag binding strength along with the gpcr activity of human cxcl10 which could lead to therapeutic applications in the future. nanodiscs are composed of a nanometer-sized phospholipid bilayer encircled by two a helical, amphipathic membrane scaffold proteins (msps). these particles provide a unique detergent free lipid bilayer model enabling biochemical and biophysical characterization of membrane proteins in a physiologically relevant medium. previously, the largest diameter reported of a nanodisc assembled using msps was about 16-17 nm. here we present a method to create large nanodiscs (up to 80nm in diameter) assembled with covalently circularized msps (cmsp). we can observe the homogeneity in nanodiscs diameter as a narrow distribution using negative-stain em. using our method, we have created 50 nm nanodiscs and used them to study poliovirus (35 nm diameter) entry and rna translocation. a 50 nm nanodisc is sufficiently large to accommodate multiple copies of the cd155 receptor (also known as the poliovirus receptor), and has enough surface area to act as a surrogate membrane for the rna translocation complex during viral uncoating. the 50 nm nanodiscs functionalized with the his-tagged ectodomain of poliovirus receptor, cd155, were generated by adding lipids derivatized with a nta nickelchelating head group to the lipid mixture during nanodisc assembly. cd155 receptor was added to the already assembled nanodiscs and incubated for 30 minutes at room temperature. the receptordecorated nanodisc complex was purified by size exclusion chromatography. the purified complex was then incubated with poliovirus for 5 minute at 4 c, and then heated to 37 c for 15 minutes to initiate receptor-mediated viral uncoating. virus binding to nanodisc-cd155 complex and subsequent insertion of viral components into and across the membrane were confirmed by negative-stain electron microscopy (figure 1c) . to obtain a high-resolution structure for the rna translocation complex we conducted single-particle cryo-em studies using a polara f30 microscope. unlike liposomes, generating a reconstruction of samples containing nanodiscs is less complicated since the nanodiscs are more homogenous in size, and allow for thinner ice. also, the viral rna can be visualized more easily. the method for making large nanodiscs as well as the negative stain and cryo-em data will be will be presented and discussed. parametric design of alpha-helical barrels and pore-like assemblies with very high thermodynamic stabilities computational design of novel protein structures and enzymes with new functions is a promising tool to create superior biological materials with tailor-made properties, new pharmaceuticals, complex fine chemicals or renewable fuels. it also challenges our understanding of protein folding, protein evolution, molecular recognition and catalysis. here we present a procedure for designing proteins with backbones produced by varying the parameters in the crick coiled-coil generating equations [1] . combinatorial design calculations using the software suite rosetta identify low energy sequences for alternative helix supercoil arrangements. after that, loop modeling is applied to connect the designs with lowest energy. the extent to which the designed sequences encode the designed structures is evaluated using large-scale structure prediction calculations, as well as symmetric and asymmetric protein-protein docking calculations. subsequently, synthetic genes are generated for sequences that converge strongly on the designed structure for experimental characterization. we applied this approach to monomeric three and four helical bundle structures as well as a pentameric five-helix bundle structure using idealized coiled-coil geometries [2] . recently we expanded this approach to higher complexity backbones, which resulted in the de-novo design of monomeric, antiparallel six-helix bundles with untwisted, left-and right-handed geometries. circular dichroism (cd), size-exclusion coupled multi-angle light scattering measurements (sec-mals), negative stain electron micrographs (em) and small angle x-ray scattering (saxs) of these designs suggest that they indeed form the designed structures. in addition, we used rosetta protein-protein interface design functionality to computationally design oligomers out of our previously published three and four helix bundle structures to generate self-assembling pore-like structures with the potential use as channels or transporters. again, experimental validation of these designs by cd, sec-mals, em and saxs show that the designs are correct. we are currently undertaking further structural investigation of all these designs by x-ray crystallography. the designs described above can act as templates for protein or small molecule binding, holding a catalytic machinery or for scaffolding enzymes in reaction cascades. some of these applications are currently under investigation, including a self-sufficient redox system employing two copper-centers, binding of heme-moieties as a prosthetic group and tailoring the pore-like geometries to be used in nanopore sequencing. university of washington, 2 university of california, san francisco, 3 repeat proteins are an example of how evolution proceeds by building on existing structures and functions, but also a source of modular protein scaffolds for molecular recognition and biomaterials. however, it is unclear whether the limited number of folds and families that we know today is the result of the intrinsic limitations of polypeptide chains or the consequence of the path followed by evolution. we explored this hypothesis by computational design of repeat proteins based on modular units formed by two alpha helices and two loops of variable lengths, without relying on information from available repeat protein families. the automated sampling of the conformational space resulted in a large number of architectures from which 83 de novo designs were selected for experimental characterization. 66% of the proteins were stable up to 958c and monodisperse and 42 designs were structurally validated by small angle x-ray scattering. crystal structures were solved for 15 of them, with root mean square deviation from the models between 0.7å and 2.5å. the designs differ from known proteins both at the sequence and structure levels and cover a broader range of geometries than observed in naturally occurring repeat protein families, indicating that existing architectures represent only a small fraction of what can be achieved. our results show that it is possible to expand the range of repeat protein architectures beyond the naturally occurring families, and that computational design can provide new scaffolds and enable the design of proteins tailored for specific applications. the serpin family of proteins consists of over 1500 members, all with a highly conserved native structure that is metastable (1). serpins use this metastability to control the activity of proteases, via a specific inhibitory process. the serpin binds to its target protease through specific residues within the reactive centre loop, the protease cleaves the loop and results in a large conformational change causing the protease to become distorted and catalytically inactive whilst the serpin becomes much more stable (1, 2, 3) . the metastable nature of aat is therefore required to facilitate the rapid and gross conformational changes required for its inhibitory function (2, 3) . several disease-causing mutants of aat have been identified, the most common of them being the z-variant (4). the z-variant has an increased propensity to polymerize in the endoplasmic reticulum of hepatocytes leading to cell death and liver damage (4) . during the past fifteen years, many groups have unsuccessfully screened a number of serpins and a vast range of solution conditions to identify a combination of serpin and conditions that will enable the folding reaction of a serpin to be characterized. we have now taken an alternative approach and designed a synthetic "model" serpin that folds reversibly to its native state. in order to do this, we used a consensus design approach, analysing a sequence alignment of 212 serpin sequences and determining the prevalent amino acid residue at each position, we termed this serpin conserpin (consensus serpin). here we present the structural, biophysical and functional characterisation of conserpin. combined crystallographic and folding studies reveal the characteristics of conserpin that likely dictate its unique stability and folding behaviour, whilst retaining activity as a serine protease inhibitor. the development of enhanced protein binding scaffolds is a key for engineering protein inhibitors and biosensors with advanced characteristics. utilizing the structural variability and designability of repeat proteins offers a means for designing protein binders where the overall shape is customized to optimally match a target molecule. we developed a computational protocol for the design of repeat proteins with a predefined geometry. by combining sequence optimization of existing repeats and de novo design of capping structures, we designed leucine-rich repeat (lrr) proteins where the building blocks assemble into a novel structure. the suggested design procedure was validated by engineering an artificial donut-like ring structure, which is constructed from ten self-compatible repeats. characterization of several designed constructs further suggests that buried cysteines play a central role for stability and folding cooperativity in certain lrr proteins. this effect could provide a means for selectively stabilizing or destabilizing specific parts of an lrr-based protein binder. the computational procedure may now be employed to develop repeat proteins with various geometrical shapes for applications where greater control of the interface geometry is desired. engineering apobec3g enzymes for altered specificity and processivity louis scott 1 , muhammad razif 1 , aleksandra filipovska 1,2 , oliver rackham 1,2 1 harry perkins institute of medical research, 2 school of chemistry and biochemistry, the university of western australia apobec3g (a3g) is a host-encoded protein involved in the defense against hiv-1 and other retroviral infections. a3g is a cytidine deaminase with a 3' to 5' processive nature, causing targeted c to t mutations along a dna strand. the catalytic and processive activity of a3g leads to the hypermutation of nascent retroviral cdna, resulting in premature termination codons and dysfunctional proteins. ultimately, the action of a3g inhibits viral replication. the ability of a3g to jump and slide along a dna strand, deaminating at targeted sequences, makes it an interesting candidate for protein engineering. engineered a3g enzymes for increased activity, altered specificity, and altered processivity are attractive options for expanding the dna modifying enzyme toolbox. mutation of catalytic residues, residues thought to affect its processive nature and those thought to be involved in target recognition, can create novel a3g enzymes. using structure guided selection, residues in key functional sites that are amiable to mutation will be chosen. individuals from the resulting libraries of mutants will be selected by directed evolution for desired characteristics. the resulting a3g enzymes will be examined for the relationship between their structure and function. such engineered a3g enzymes could be targeted to catalyse the reversion of deleterious genetic mutations. furthermore, engineered a3g enzymes could be used in mutational studies that call for targeted deamination along a dna strand, or mutational studies that call for unspecific and high throughput dna deamination. engineering porous protein crystals as scaffolds for programmed assembly thaddaus huber 1 , luke hartje 1 , christopher snow 1 1 a key motivation for nano-biotechnology efforts is the creation of designer materials in which the assembly acts to organize functional domains in three dimensions. crystalline materials are ideal from the validation perspective because x-ray diffraction can elucidate the atomic structure. relatively little work has focused on engineering protein crystals as scaffolds for nanotechnology, due to the technical challenges of coaxing typical proteins into crystallizing, and the likelihood of disrupting the crystallization process if changes are made to the monomers. we have circumvented these limitations by installing guest protein domains within engineered porous crystals (13 nm pore diameter) that have been rendered robust using covalent crosslinks. the retention of the scaffold structure despite changes to the solution conditions and macromolecule uptake can be validated through x-ray diffraction. we have engineered scaffold crystals for the non-covalent and covalent capture of guest macromolecules. by controlling the reversible loading and release, we can prepare "integrated" crystals with spatially segregated guest loading patterns. as assessed using confocal microscopy, such host-guest crystals are highly stable. ultimately, the resulting crystals may serve as a robust alternative to dna assemblies for the programmed placement of macromolecules within materials. engineering ultrasensitive protein probes of voltage dynamics for imaging neural activity in vivo francois st-pierre 1,2 , michael pan 1,2 , helen yang 3 , xiaozhe ding 1,2 , ying yang 1,2 , thomas clandinin 3 , michael lin 1,2 1 department of bioengineering, stanford university, 2 department of pediatrics, stanford university, 3 nervous systems encode information as spatiotemporal patterns of membrane voltage transients, so accurate measurement of electrical activity has been of long-standing interest. recent engineering efforts have improved our ability to monitor membrane voltage dynamics using genetically encoded voltage indicators. in comparison with electrophysiological approaches, such protein-based indicators can monitor many genetically defined neurons simultaneously; they can also more easily measure voltage changes from subcellular compartments such as axons and dendrites. compared with genetically encoded calcium indicators, voltage sensors enable a more direct, accurate, and rapid readout of membrane potential changes. however, several challenges remain for in vivo voltage imaging with genetically encoded indicators. in particular, current voltage sensors are characterized by insufficient sensitivity, kinetics, and/or brightness to be true optical replacements for electrodes in vivo. as a first step towards addressing these challenges, we sought to develop new voltage indicators that further improve upon the performance of the fast voltage sensor accelerated sensor of action potentials 1 (asap1). in asap1, voltage-induced conformational changes in a natural voltage-sensing domain perturb the fluorescence emission of a covalently linked green fluorescent protein (gfp). using a structurebased approach to guide mutagenesis, we discovered several amino acids that tune the kinetics and voltage sensitivity of asap1. these residues are not only located in the voltage-sensing domain, but also in the fluorescent protein and in the linkers bridging sensing domain and gfp. our most improved variant, asap2, exhibits improved sensitivity to voltage transients such as neuronal action potentials and subthreshold depolarizations. we sought to characterize the ability of these new voltage sensors to monitor neural activity in vivo using laser-scanning two-photon microscopy, a technique that allows imaging with lower autofluorescence and deeper tissue penetration. we report that asap sensors were able report stimulus-evoked voltage responses in axonal termini of the fly visual interneuron l2. asap sensors enabled voltage imaging with dramatically improved temporal resolution compared to three recently reported calcium and voltage sensors. overall, our study reports novel voltage indicators with improved performance and highlights how specific amino acids can tune the performance of a proteinbased fluorescent sensor. we anticipate that these results will pave the way for further engineering of voltage sensing proteins, and that our new sensor asap2 will facilitate current and future efforts to understand how neural circuits represent and transform information. assembly of armadillo repeat proteins from complementary fragments erich michel 1 , randall watson 1 , martin christen 1 , fabian bumback 3 , andreas pl€ uckthun 2 , oliver zerbe 1 demonstrated that complementary fragments of a designed consensus armadillo repeat protein (armrp) recognize each other [1] . the two fragments ym2: ma, in which y, m and a denote the n-cap, internal repeats and the c-cap, respectively, form a 1:1 complex with a nanomolar dissociation constant, which is essentially identical to the crystal structure of the continuous ym3a protein. we further demonstrate that structurally intact armadillo repeat protein complexes can be reconstituted from fragments obtained at various split sites -essentially after every repeat but also within repeats. the fragments display variable affinities towards each other, depending on the split site. the low affinity of some complementary pairs can be dramatically increased upon addition of peptide ligands. while a number of proteins are known that can be reconstituted from fragments we believe that the fact that armadillo repeat proteins can be reconstituted from various complementary fragments is novel and opens new interesting perspectives and applications in biochemistry. a reliable method for generating optically controllable proteins would enable researchers to interrogate protein functions with high spatiotemporal specificity. we recently engineered a tetrameric fluorescent protein, dronpa145n, that undergoes light-induced monomerization, then developed a general architecture for lightinducible proteins based on this light-induced transition. we created proteins whose active sites were blocked by fused dronpa145n domains in the dark, but would become unblocked by light. here we present further two extensions to this concept that together enabled the generalization of this method to additional classes of proteins. first, we engineered a photodissociable dimeric dronpa (pddronpa) with tunable affinity, faster photoswitching speed, and decreased level of protein aggregation, enabling better performance of fusion proteins. second, we introduce the concept of caging a protein active site by insertion of dronpa domains into loops rather than strictly at the protein termini. we use the pddronpa system to impose optical control on kinases and the cas9 endonuclease. the resulting light-inducible mek1 kinase, raf1 kinase, and cas9 endonuclease showed high caging efficiency of protein activities in the dark, and robust protein activation upon light illumination. we believe that our efforts on further improving and generalizing this method would bring the power and benefits of light control to a broad community of biologists. exploring the evolution of folds and its application for the design of functional hybrid proteins saacnicteh toledo patiño 1 , birte h€ ocker 1 1 the structural diversity of proteins may appear endless, nevertheless even large protein complexes can be decomposed into protein domains and smaller sub-domain sized fragments. only recently, we could identify such fragments employing sequence-based comparisons of different folds, as the tim-barrel and the flavodoxin-like fold (farias-rico et al., 2014) . as an extension of this work, we compared all a/b proteins and identified several fragments shared by different folds illustrating how nature may have achieved structural and functional diversity from a reduced set of building blocks. inspired by this combinatorial concept, we searched for homologous fragments bearing active sites to engineer a functional fold-chimera. we extracted the vitamin-b12 binding part from methylmalonyl coa mutase, which belongs to the flavodoxin-like fold (fl) and used it to replace the corresponding fragment in uroporphyrinogen iii synthase, which belongs to the hemd-like fold (hdl). the new hybrid resulted in a stable and well-folded protein whose structure was determined by x-ray crystallography. moreover, cobalamin-binding function was successfully transferred to the new protein from the fl parent, which shows the advantage of using this approach for the design of new functional proteins. in addition, profile alignments revealed sequence and structural evidence that suggested an evolutionary path for hdl from fl by gene duplication. to test this hypothesis, we expressed a modified c-terminal half of uroporphyrinogen iii synthase and solved its structure by nmr spectroscopy, thereby confirming the predicted fl architecture. altogether, our approach facilitates the detection of common ancestry among different folds contributing to our understanding of protein development. furthermore, our results show how new complex proteins can be designed using fragments of existing proteins that serve as building blocks in a lego-like manner. we believe that combining fragments containing existing properties will provide a successful method for the design of novel functionalities in the future. [2] . the active site cysteine plays a key role in the reaction mechanism and we investigated this residue in more detail by exchanging this moiety with selenocysteine (sec) and homocysteine (hcy). the sortase mutants were generated by semisynthesis using expressed protein ligation (epl). the resulting cys-, sec-and hcy-sortase enzymes were characterized and showed a moderate 2-3-fold reduction of activity for sec-sortase. the activity of hcysortase was barely detectable with less than 1% of wildtype activity. the alkylation efficiency of the active site nucleophiles correlated with the expected pka values of sec, cys and hcy. analysis of the ph dependency of the transpeptidation reactions showed that the activity optimum of sec-sortase was shifted towards more acidic conditions. these investigations provide further insights into the reaction mechanism of sortase a and the semisynthetic enzymes may provide new tool for further biochemical studies. propanediol oxidoreductase from escherichia coli (fuco) uses nadh/nad1 as cofactors to catalyze the conversion of s-lactaldehyde to s-1,2-propanediol and vice versa. fuco is an attractive enzyme in the search for possible biocatalysts producing a-hydroxy aldehydes, which are important for the synthesis of natural products and synthetic drugs. enzymes catalyzing these types of reactions are unique in catalytic power and stereoselectivity. the usage of fuco in synthetic industry is limited by the restricted substrate scope, which makes fuco inactive with larger phenyl-substituted alcohols. we used reengineering and directed evolution to enable fuco to catalyze the regio-and enantioselective oxidation of arylsubstituted vicinal diols, such as phenylpropanediols, into a-hydroxy aldehyde products. we mutated amino acids considered to restrict the entry into the active site, and modeled the mutants that were most active with the substrates phenylacetaldehyde and s-3-phenyl-1,2-propanediol and performed docking studies with them. as expected, our experimental and in silico results show that the mutations enlarge the active site cavity and enable the mutant enzymes to accommodate the new substrates. we also found specific amino acids in the active site, which need to be conserved to allow the substrates to make stabilizing interactions. interestingly, an asparagine residue makes the mutant enzymes able to discriminate between phenylacetaldehyde and s-3-phenyl-1,2-propanediol. in conclusion, we successfully re-engineered the specialist enzyme fuco to accept also bulkier molecules as substrates, thereby making it more useful for industrial purposes. one way to gain insight into the sequence-structure-function relationship in proteins is to de novo design artificial proteins. despite impressive successes in de novo protein design, designing a folded protein of more than 100 amino acids still remains a challenge. using this approach, an idealized (beta/ alpha)8 fold protein was designed leading to the production of a protein of 216 amino acids (octarellin v). this protein showed a low solubility and stability. through directed evolution we produced a soluble variant, octarellin v.1. the biophysical characterization of octarellin v.1 shows a well folded monomeric and thermostable protein with a tm over 908c. however, after several screenings, we could not find crystallization conditions for this protein. as an alternative, we decided to co-crystallize octarellin v.1 with a protein partner that helps the crystallization process. we used 2 protein partners: alpha-reps and nanobodies. the first one is characterized to interact through a large surface contact, whereas the second is characterized to recognize an specific small epitope. crystallization of both complexes was performed successfully by vapor diffusion and the structures were solved. the experimental structures correspond to the first for an artificial protein of this size and it will allow to criticize the computational design of the octarellin v. generation of synthetic antibodies against membrane proteins in nanodiscs for use in structural biology methods. here, we describe a robust strategy for generating a class of high performance antibodybased affinity reagents that have proven useful in determining the structures of relevant functional states of membrane proteins. these reagents are fab fragments that are generated by phage display from fully synthetic libraries and are called synthetic antibody fragments, or sabs. we have developed phage display sorting strategies that can trap a desired conformational state, making it accessible to structural analysis, or target a particular epitope on the protein surface. however, to maximize this technology for membrane proteins, several limitations of phage display sorting in detergent formats had to be overcome, the greatest being that using detergents can produce non-native conformational biases. we sought to address these limitations by embedding membrane proteins into nanodiscs, soluble lipidfilled discoidal particles, to better mimic the native membrane environment. nanodiscs stabilize the membrane protein and allow it to respond to conformation-inducing stimuli such as ligands, ions and ph during phage display selections. we have established and validated an improved protocol using two membrane protein systems: 1) mj0480, an archaeal membrane protein of unknown function, and 2) cora, a pentameric magnesium ion channel. using mj0480, we compared the nanodisc protocol with the standard method performed in detergent, and as an important byproduct, we characterized the influence of the membrane protein environment on the apparent affinity of sabs to their cognate antigen. using cora, we developed a more sophisticated sorting strategy resulting in a variety of sabs specific to either the open or closed conformation of the channel. finally, using sabs as crystallization chaperones we obtained the structure of mj0480 at 3.5å resolution, and crystallized cora in several new conditions. lipocalin-type prostaglandin d synthase (l-pgds) is a member of the lipocalin superfamily, and binds a large variety of small hydrophobic molecules. using this function of l-pgds, we have already reported the feasibility of l-pgds as a novel drug delivery vehicle for the poorly water-soluble drugs [1] . sn-38, 7-ethyl-10-hydroxy-camptothecin, is a semi-synthetic analogue of anti-cancer alkaloid camptothecin that targets dna topoisomerase i. despite of the potent anti-tumor activity, however, sn-38 was not used directly in a clinical practice due to its poor water solubility. thus, irinotecan hydrochloride (cpt-11), which is the water-soluble prodrug of sn-38, is used for the cancer treatment. however, cpt-11 shows approximately 0.1% cytotoxic activity of sn-38 against the various cancer cell lines in vitro, and its metabolic conversion rate is 10% of the original volume of cpt-11. here, we show the development of the drug delivery system utilizing l-pgds, which enables a direct clinical usage of sn-38. first, we investigated the effect of l-pgds on the solubility of sn-38. in the presence of 2 mm l-pgds, the concentration of sn-38 was 1.7 mm, which was 1,130-fold as compared with that in pbs. then, we carried out isothermal titration calorimetry measurements to investigate the detailed binding mode of sn-38 to l-pgds. as a result, it was revealed that l-pgds binds three molecules of sn-38, and the dissociacontrol over the sensitivity with which artificial biomolecular receptors respond to small changes in the concentration of their target ligand is critical for the proper function of many cellular processes. such control could likewise be highly useful in artificial biotechnologies in which highly responsive behavior is of value, such as biosensors, genetic logic gates, and "smart" materials and delivery devices. in nature, the control of molecular responsiveness is often achieved using "hill-type" cooperativity, a mechanism in which sequential binding events on a multivalent receptor are coupled such that the first enhances the affinity of the next, producing a steep, higher-order dependence on target concentration. here we use an intrinsic-disorder-based mechanism that can be implemented without requiring detailed structural knowledge to rationally introduce this potentially useful property into several normally noncooperative biomolecules. to do so we fabricate a tandem repeat of the receptor that is destabilized (unfolded) via the introduction of a long, unstructured loop. the loop spatially separates the two sets of the two halves of the binding sites, preventing a complete binding site that enables target molecule binding without prior closure of the loop. thus, the first binding event requires the energetically unfavorable closing of this loop, reducing its affinity relative to that of the second binding event, which, in contrast occurs at a pre-formed site. using this approach we have rationally introduced cooperativity into three unrelated aptamers, achieving in the best of these a hill coefficient experimentally indistinguishable from the theoretically expected maximum. the extent of cooperativity, and thus the steepness of the binding transition, are, moreover, well modeled as simple functions of the energetic cost of binding-induced folding, speaking to the quantitative nature of this design strategy. essential and non-essential amino acid species for an ancestral protein satoshi akanuma 1 1 the translation system is an essential element for life because it links genetic information embedded in genes to functional molecules, proteins. the modern genetic code, which encodes the standard 20 amino acids (and three terminations) using 64 triplet codons, is shared by most of the extant organisms on the earth. a number of theories have been proposed for the origin and evolution of the genetic code, and these theories suggest that only a fewer amino acids were used in primitive proteins and later the amino acid repertoire gradually increased up to 20 through the course of evolution. if so, one would wonder how many number of and which types of amino acids were involved in the primitive proteins. i have begun to address this issue experimentally. i first resurrected several ancestral proteins and then restricted the amino acid usage of one of the resurrected proteins. i targeted nucleoside diphosphate kinase (ndk) that catalyzes the transfer of a phosphate from a nucleoside triphosphate to a nucleoside diphosphate. ndk may have arisen early because at least one gene that encodes ndk is present in most extant organisms. the first step in the reconstruction of ancestral ndk sequences is to prepare multiple amino acid sequence alignments using homologous sequences of ndk from extant species. then, phylogenetic trees were built. ancestral sequences of ndk that represent the last common ancestors of archaea and of bacteria were reconstructed using the information contained in the predictive phylogenetic trees. the reconstructed ancestral kinases are extremely thermally stable [akanuma et al., 2013] . then, using the most thermally stable ancestral ndk, arc1, as the starting molecule, i restricted its amino acid usage. arc1 does not contain any cysteine residue and therefore consists of 19 amino acid species. i completely replaced one of the 19 amino acid species by other amino acid species and thus created 19 proteins each of which consisted of 18 amino acid species. then, i evaluated the stabilities and activities of the resulting 19 arc1 variants to assess the individual contributions of the 19 amino acid species. as the result, i found that the 19 amino acid species do not equally contribute to the stability and activity of arc1 and that some amino acid species can be easily lacked but others are important or essential for its stability and function. the result clearly shows that the full amino acid species are not necessarily essential and supports the hypothesis that proteins in the early stage of evolution were made from a reduced amino acid set. the protein surface recognition for protein-protein interactions (ppi) is involved in signal transduction, immune reaction, and creation of the nanostructures in living cells. the methods for rational designing of ppi that could provide non-antibody scaffolds and nanostructured materials are required for the therapeutic and nanotechnological applications. although there have been some successful rational designs with computational methods, it is still difficult to design freely the ppi onto arbitrary proteins. the reason for this limitation is decreased solubility in the designed protein due to the additional hydrophobic residues in order to drive ppi. another reason is a limited set of design modes by which proteins can interact, because the target proteins have individual surface structures. therefore, many methods of constructing an interface for numerous target scaffold proteins without loss of their solubility are necessary. surface exposed a-helices are often observed in natural globular proteins. moreover, there are many examples for naturally occurring oligomeric proteins where an a-helix from each subunit interacts to form an intermolecule coiled coil. further, the works related to designing of artificial helical bundle reported by the several other groups have provided information about how to generate and tune the interaction between a-helices. therefore, a surface exposed a-helix would be a good target for designing a de novo interface onto the scaffold protein. here we engineered two different proteins, sulerythrin and cys-larfh, to form the cys-larfh-sulerythrin dimer-cys-larfh heterotetramer via an intermolecular helix-helix interaction. wild-type sulerythrin forms a dimeric eight-helix bundle. cys-larfh is a designed monomeric protein that forms four-helix bundle containing interhelical s-s bonds. both sulerythrin and cys-larfh are extremely thermostable. to design protein-protein interfaces onto the individual proteins, we first introduced six leucines to the two a-helices of sulerythrin and three leucines to a a-helix of cys-larfh. as expected, the introduction of the hydrophobic amino acids reduced their solubilities. to recover the solubility, we then introduced six aspartates or glutamates around the hydrophobic surface of the sulerythrin (hereafter referred to as 6l6d or 6l6e). similarly, three arginines were introduced around the artificial hydrophobic surface of the cys-larfh (hereafter referred as iv-3l3r). the solubilities of the mutants with the hydrophobic interface and additional charged residues were recovered their solubility. in addition, the sulerythrin mutants 6l6d and 6l6e exist mainly as dimer. the cys-larfh mutants iv-3l3r, also exists as monomer. we then examined the interaction between 6l6e or 6l6d and iv-3l3r. a pull-down experiment, in which co21 beads bound to either his-tagged cys-larfh and iv-3l3r were used to pull down wild-type sulerythrin, 6l6d, or 6l6e, demonstrates that 6l6d or 6l6e specifically interacts to iv-3l3r. furthermore, when analysed by size exclusion chromatography, the dominant peaks of the mixture of 6l6d and iv-3l3r appeared at the volume expected for the heterotetrameric complex. thus we successfully created the de novo ppi by using a very simple concept involving hydrophobic interaction in combination with charge interactions. in vitro selection of liposome anchoring peptide by cdna display naoto nemoto 1 , ryoya okawa 1 , yuki yoshikawa 1 , toshiki miyajima 1 , shota kobayashi 1 1 a liposome-anchoring peptide (la peptide) was selected against liposomes composed of dioleoyl-snglycero-3-phosphocholine (dopc) by in vitro selection using cdna display method. the selected peptide la peptide consists of the n-terminal region (hydrophobic) and the c-terminal region (basic) in a characteristic manner. thus, la peptide was synthesized chemically and the interactions between la peptide and particular types of liposomes were investigated and confirmed by confocal laser scanning microscopy. designing of a novel platinum-binding amino acid sequence on a protein surface asumi kaji 1 , hiroya niiro 1 , satoshi akanuma 2 , tetsuya uchida 1 , akihiko yamagishi 1 1 designing of a novel interaction between a metal and a protein is a key to create hybrid materials between organic and inorganic materials. for example, in a glucose biosensor, which is widely used for measuring glucose concentration in blood, glucose oxidoreductase molecules are immobilized on a platinum electrode by polyacrylamide gel. a metal-binding tags that is added to the n-or cterminus of a protein is also used for fix the protein to a metal. however, a technique to create a metal binding site on a desired position of a protein has not been invent. if such a technique would be established, the technique would contribute to developing and improving biosensors and to producing new bionanoelectronic materials. in this study, we created a platinum-binding site on a loop located at a protein surface. we used an artificial protein, larfh, that had been synthesized by connecting four identical alpha helices originated from the c-terminal segment of the escherichia coli lac repressor with three identical loops. we randomized the ser, gly, gln, gly, gly, ser sequence within one of the inter-helical loops and then selected for binding to platinum by a t7 phage display system. most of the selected larfh variants contained the tyr, lys, arg, gly, tyr, lys (ykrgyk) sequence in the randomized segment. we then evaluated the affinity of the larfh variant to platinum by means of quartz crystal microbalance analysis. we found that the variant binds to platinum more strongly than does the original larfh. in the annual symposium, we will also report about the affinity of the isolated ykrgyk sequence to platinum and about the crucial role of the first tyrosine in binding to platinum. engineering of an isolated p110a subunit of pi3ka permits crystallization and provides a platform for structure-based drug design pi3ka remains an attractive target for development of anticancer targeted therapy. a number of p110a crystal structures in complex with the nsh2-ish2 fragment of p85 regulatory subunit have been reported, including a few small molecule co-crystal structures, but the utilization of this crystal form is limited by low diffraction resolution and a crystal packing artifact that partially blocks the atp binding site. taking advantage of recent data on the functional characterization of the lipid binding properties of p110a, we designed a set of novel constructs allowing production of isolated stable p110a subunit missing the adapter binding domain (abd) and lacking or featuring a modified c-terminal lipid binding motif. while this protein is not catalytically competent to phosphorylate its substrate pip2, it retains ligand binding properties as indicated by direct binding studies with a pan-pi3ka inhibitor. additionally, we determined apo and pf-04691502 bound crystal structures of the p110a (105-1048) subunit at 2.65 å and 2.85 å respectively. comparison of isolated p110a (105-1048) with the p110a/p85 complex reveals a high degree of structural similarity, which validates suitability of this catalytically inactive p110a for iterative sbdd. importantly, this crystal form of p110a readily accommodates the binding of non-covalent inhibitor by means of a fully accessible atp site. the strategy presented here can be also applied to structural studies of other members of pi3kia family. identification of structural determinants involved in the differential conformational changes of ef-hand modules emma liliana arevalo salina 1 , joel osuna quintero 1 , humberto flores soto 1 , gloria saab rinc on 1 1 instituto de biotecnolog ıa, universidad nacional aut onoma de m exico identification of structural determinants involved in the differential conformational changes of ef-hand modules calcium signals are regulated by several proteins, most of which belong to the ef-hand superfamily. the ef-hand motif is formed by a helix-loop-helix that binds calcium through its loop1. these motifs occur in adjacent pairs, forming a single globular domain which is the basic structural and functional ca21 binding unit. the proteins in this family can be classified as calcium sensors or modulators, according with their function. the first group undergoes a major conformational change upon calcium binding, while the second one remains practically unchanged1,2. to explain the biophysics behind the different behavior of these proteins upon ca21 binding, we have sought to identify structural determinants that could account for these features, especially for the difference in the conformational change. we examined the primary structure from two ef-hand motifs: a sensor ef-hand from chicken troponin c (sciii) and a modulator ef-hand from bovine calbindin d9k (clbn). the main differences were in the binding ca21 loop and a group of charged residues in the h2 helix of the modulator ef-hand. then, we constructed chimeric clbn motifs containing the loop or the loop and h2 from sciii motif (h1clbnsciii and h1h2clbnsciii). these constructs were analyzed using a reporter system that discriminates ef-hand-sensor motifs from signal-modulators at the single-motif level. this reporter is based on the fusion of genes codifying for the ef-hand and the prephenate dehydrogenase from e. coli (tyra), a protein which is active only as a dimer. isolated ef-hand motifs have the ability to homo-dimerize and in the fusion can stabilize and activate tyra. the sensor motif exhibits a conformational change by binding calcium and in doing so, destabilizes the dimeric conformation of tyra and virtually eliminates its activity. in the modulators, on the other hand, the rather small conformational change only gives rise to a decreased tyra activity. both constructed chimeric ef-hand fusions showed a loss of activity upon ca21 binding, indicating that the 12 residues connector of the sensor ef-hand from sciii is sufficient to confer the conformational change. in addition we used cd and extrinsic fluorescence spectroscopies to analyze any conformational change in the h1h2clbnsciii and h1clbnsciii isolated modules, not finding any difference between the ca21 free and ca21 bound chimeras, suggesting that the change in activity of the reporter protein is due to a change in the orientation of the helices in the ef-hands induced by calcium. the effect of ca21 binding of the chimeras in the context of the entire calbindin d9k protein is under investigation. mapping side chain interactions at the n-and c-termini of protein helices nicholas e newell, independent researcher interactions involving one or more amino acid side chains near the ends of protein helices stabilize helix termini and shape the geometry of the adjacent loops, contributing to supersecondary structure. side chain structures that have been identified at the helical n-terminus include the asx/st n-caps, the capping box, and hydrophobic and electrostatic interactions. at the cterminus, capping is often achieved with main-chain polar groups, (e.g. the schellman loop), but here also particular side chain motifs clearly favor specific loop geometries. key questions that remain concerning side chain interactions at helix termini include: 1) to what extent are helix-terminal motifs that include multiple amino acids likely to represent genuine cooperative interactions between side chains, rather than chance alignments? 2) which particular helix-terminal loop geometries are favored by each side chain interaction? 3) can an exhaustive statistical scan of a large, recent dataset identify new side chain interactions at helix termini? in this work, three analytical tools are applied to answer the above questions for both n-and c-termini. first, a new perturbative least-squares 3d clustering algorithm is applied to partition the helix terminal structures in a large (25,000 example), low-redundancy pdb dataset by loop backbone geometry. the clustering algorithm also generates a set of structural exemplars, one for each cluster, that is used to represent the most important loop geometries at each terminus. next, cascade detection (newell, bioinformatics, 2011), an algorithm that detects multi-amino acid cooperativities by identifying overrepresented sequence motifs, is applied to each cluster separately to determine which motifs are most important in each loop geometry. finally, the results for each motif are displayed in a capmap, a 3d conformational heatmap that depicts the distribution of motif abundance and overrepresentation across all loop geometries by projecting these quantities onto the structural exemplars generated by clustering. the capmap reveals the loop conformations most favored by a motif. actual structures from the clusters corresponding to these favored conformations are then examined in a structure browser to characterize the side chain interaction associated with the motif. this work identifies a 'toolkit' of side chain motifs which are good candidates for use in the design of synthetic helix-terminal loops with specific desired geometries, because they are used in nature to support these geometries. highlights of the analysis include determinations of the favored loop geometries for the asx/st motifs, capping boxes, big boxes, and other previously known and unknown hydrophobic, electrostatic, h-bond, and pi-stacking interactions. a goal of future work is to make these results available in a structurally-addressable database that would enable researchers to immediately retrieve the side chain interactions most compatible with a desired loop geometry. generation of fluorescent protein-tagged gp120 mutants to analyze the intracellular distribution of hiv-1 envelope protein shuhei nakane 1 , zene matsuda 3 1 green earth research center, green earth institute co., ltd., 2 res ctr for asian infect dis, inst of med sci, the univ of tokyo, 3 lab of struct virol and immunol, institute of biophysics, cas hiv-1 is a causative enveloped virus of aids. its envelope protein (env) has two non-covalently associated subunits, gp120 and gp41, which are proteolytically processed from a gp160 precursor. the gp120 subunit is a surface protein and gp41 is a transmembrane protein. the gp120 and gp41 subunits are responsible for the receptor recognition and membrane fusion, respectively. the cytoplasmic tail (ct) of gp41 is about 150 amino acids long and is believed to play a critical role in intracellular trafficking of env. to visualize dynamic trafficking, the c-terminus of gp41 has been tagged with fluorescent proteins such as gfp. however, tagging of ct may cause a concern to affect the interactions between the ct and cellular proteins that are involved in intracellular trafficking. to avoid this problem, here we tried to insert gfpopt, a gfp variant, into five variable regions of gp120. we have analyzed the phenotypes of env mutants, such as the cell surface expression, processing of gp160, membrane fusion activity, and virion incorporation. among 5 variable regions of gp120, the v3 region was most sensitive to insertion. v1/v2 region was less sensitive than v3. consistent with the recently revealed structure, exteriorly located v4 and v5 were highly tolerant to insertion. we used the mutant with the gfp insertion in the v5 region to analyze the intracellular distribution of env with and without ct. we found that deletion of ct increased the presence of vesicles colocalized with late endosome markers. this is consistent with the hypothesis that the ct region contains a motif regulating intracellular trafficking. our results showed that env with gfpopt insertion in its gp120 subunit is a useful tool for the study of intracellular dynamics of hiv-1 env. these mutants would also be useful to trace the fate of virus particles during infection. pi-055 ngs-guided phage panning: comparison to conventional panning strategy buyung santoso 1 , dorain thompson 1 , john nuss 1 , john dwyer 1 1 phage display is a powerful tool for generating binders to a target protein. multiple rounds of panning with conventional phage display strategies typically result in a number of hits, which are then individually screened using in vitro assays. clones screened at this stage are a combination of specific binders, sequences that are selected due to amplification bias, and non-specific binders. if the number of specific clones is low relative to the non-specific sequences, a larger number of clones have to be screened to ensure sufficient diversity of early leads. with the advent of next generation sequencing (ngs) technology, we aim to test whether we can increase the diversity of specific hits and decrease the number of non-specific sequences. in our experiment, four rounds of conventional panning produced ten peptide binders to target protein. ngs analysis after two rounds of panning was done in parallel, yielding more than ten thousand sequences, ranked by abundance. all ten binders from conventional panning were found in the top 150 most abundant ngs hits. more importantly, additional hits were found in ngs analysis but not in conventional panning, highlighting this strategy as a promising alternative for hit discovery with the significant upside of more diverse and higher affinity leads. numerous processes in pharmaceutical development, including construct screening, structural genomics, protein engineering and expression optimization among others, require the use of higher throughput plasmid dna purification. the majority of issues encountered in mini, midi, and maxiprep purification kits involve flocculate removal following alkaline lysis, and there is currently no easy way to produce large amounts of plasmid dna without the addition of complicated and time consuming clarification steps. the existence of a hassle-free automated system that is not restricted by sample size would significantly help in cutting time and costs during the initial processing steps of plasmid purification. the autoplasmid mea instrument provides a fully automated solution to traditional problems faced in plasmid purifications, allowing mini, midi, and maxiprep plasmid purifications to be performed on a single instrument. the data presented here on plasmid yield, purity, and suitability for sequencing and transfection/transformation illustrate a new strategy for automated plasmid preps. by eliminating traditional clarification methods, cell culture volumes between 1 -120 ml can be processed leading to yields ranging from 3 -1000 lg. this flexible system was developed in order to satisfy a wide variety of concentration and yield requirement, while eliminating the time consuming steps previously needed to obtain similar results. the ability to perform fully automated mini, midi, and maxi plasmid preps on one instrument allows for a customized all-in-one purification system that is not restricted by traditional clarification methods, eliminating manual intervention, and streamlining the purification process. the modular nature of protein architectures suggests that proteins have evolved through duplication and fusion to give rise to modular, often symmetric forms, which later diversified under the influence of evolutionary pressure. we have developed a computational protein design method termed reverse engineer evolution (re3volution) to create symmetrically self-assembling protein building blocks. we have used this method to design a perfectly symmetric b-propeller protein called pizza. subsequently, we have engineered a metal binding site into this pizza protein. this new pizza variant carries two nearly identical domains per polypeptide chain, and forms a trimer with three-fold symmetry. the designed single metal ion binding site lies on the symmetry axis, bonding the trimer together. two copies of the trimer associate in the presence of cadmium chloride in solution, and high resolution x-ray crystallographic analysis reveals a nano-crystal of cadmium chloride, sandwiched between two trimers of the protein. this nano-crystal, containing seven cadmium ions lying in a plane and twelve interspersed chloride ions, is the smallest reported to date. our results indicate the feasibility of using rationally-designed symmetrical proteins to biomineralize nano-crystals with applications in bionanotechnology. bacillus licheniformis trehalose-6-phosphate hydrolase structures suggest keys to substrate specificity chwan-deng hsiao 1 , min-guan lin 1 , long-liu lin 2 , yuh-ju sun 3 1 institute of molecular biology, academia sinica, 2 department of applied chemistry, national chiayi university, 3 depaertment of life science, national tsing hua university trehalose-6-phosphate hydrolase (trea) of the glycoside hydrolase family 13 (gh13) catalyzes the hydrolysis of trehalose-6-phosphate (t6p) to yield glucose and glucose-6-phosphate. products of this reaction can be further metabolized by the energy-generating glycolytic pathway. here we present the crystal structures of bacillus licheniformis trea (bltrea) and its r201q mutant complexed with p-nitrophenyl-a-d-glucopyranoside (r201q/ ppng) at 2.0 å and 2.05 å resolution, respectively. the overall structure of bltrea is similar to other gh13 family enzymes. however, detailed structural comparisons revealed that the catalytic groove of bltrea contains a long loop adopting a different conformation from those of gh13 family members. unlike the homologous regions of bacillus cereus oligo-1,6-glucosidase (bcogl) and erwinia rhapontici isomaltulose synthase (nx-5), the active site surface potential of bltrea exhibits a largely positive charge, contributed by the four basic residues his281, his282, lys284 and lys292. mutations at these residues resulted in significant decreases of bltrea enzymatic activity. strikingly, a 281hhlk284 motif and the lys292 residue played critical roles in bltrea substrate discrimination. crystal structure of engineered lrrtm2 synaptic adhesion molecule and a model for neurexin binding anja paatero 1 , katja rosti 1 , alexander shkumatov 2 , cecilia brunello 3 , kai kysenius 3 , prosanta singha 1 , henri huttunen 3 , tommi kajander 1 1 institute of biotechnology, university of helsinki, helsinki, finland, 2 dept of pharmaceutical and pharmacological sciences, ku leuven, leuven, belgium, 3 neuroscience center, university of helsinki synaptic adhesion molecules are key components in the development of the brain, and in the formation of neuronal circuits, as they are central in the assembly and maturation of the chemical synapses. several families of neuronal adhesion molecules have been identified such as ncams, neurexins and neuroligins, and in particular recently several leucine rich repeat protein families, e.g. netrin g-ligands, slitrks and lrrtms. the lrrtms form a family of four proteins. they have been implicated in excitatory glutamatergic synapse function, and were specifically characterized as ligands for neurexins in excitatory synapse formation and maintenance. in addition, lrrtm3 and lrrtm4 have been found to be ligands for heparan sulphate proteoglycans. we report here the crystal structure of a stability-engineered mouse lrrtm2, with a tm 308c higher than the wild type protein, while retaining its function. we localized the neurexin binding site to the concave surface based on protein engineering, sequence conservation and prior information on the ligand interaction with neurexins, allowing us to propose a tentative model for lrrtm:neurexin interaction compex. cell culture studies and binding experiments show that the engineered protein is functional and capable of forming synapse-like contacts. small angle x-ray scattering data suggests that the wild type protein forms transient dimers, which may have importance for the function. the structural and functional data presented here provide the first structure of an lrrtm protein, and a model for molecular mechanism of lrrtm function in adhesion. computational design of phenylalanine binder olga khersonsky 1 , gil benezer 1 , sarel fleishman 1 1 recently, abdesign algorithm was developed in our lab for de novo design of antibodies (1). it is guided by natural conformations and sequences, and exploits the modular nature of antibodies to abstract generate an immense space of conformations, which can be used as scaffolds for design of stable highaffinity binders. we have used abdesign to design a binder of phenylalanine. 30,000 antibody scaffolds were obtained by splicing h3 and l3 fragments into a template (pdb id 2brr), and subsequent optimization of vh and vl orientation. phenylalanine binding site, based on native phenylalanine binders, was introduced into the scaffolds with rosettamatch (2), and the sequences were subsequently optimized by rosetta enzyme design protocol (3) . 30 designs were experimentally tested by yeast display for binding of biotinylated phenylalanine ligand. several designs were found to bind the ligand, and we plan to further characterize this affinity and improve it using directed evolution techniques. in collaboration with the group of prof. johnsonn, the resulting phenylalanine binder will be incorporated in a bio-luminescent (lucid) sensor for phenylalanine (4) . phenylalanine monitoring device would be of primary importance for patients with phenylketonuria, a genetic disease with phenylalanine metabolism problem. cold-adapted enzymes are interesting because of their higher catalytic activity compared to mesophilic and thermophilic homologues. alkaline phosphatase (ap) from a psychrophilic vibrio marine bacteria (vap) has an unusual large surface loop that extends from each of its monomers to stabilize a homodimeric structure (1). in many cold-adapted enzymes, the loop regions are longer compared to proteins of mesophilic organisms and our aim was to study the functional and structural role of this loop. three substitutions (r336l, y346f and f355y) were introduced within the large surface loop as directed by 1microsecond molecular dynamics (md) simulations. with the r336l mutation, two hydrogen bonds were broken that connect the loop to residues on the adjacent subunit, and further two hydrogen bonds broken with the adjacent q334. as a consequence, r336l displayed a 25% higher kcat compared with wild-type and a slight decrease in the km value. overall, the catalytic efficient improved by 45%. the global heat stability (tm) and the active site sensitivity to heat (t50%) were reduced by 68c and 138c, respectively. md simulations showed that hydrogen bonds to arg336 are important for longrange communication to the active site. certain rotamers of two important residues in the catalytic site, ser65 and arg129, were favored, presumably toward states more competent for catalysis upon the replacement of arg336 with leu. in the y346f variant, removal of one hydrogen bond between the loop and the other subunit caused a small drop in stability parameters, whereas both kcat and km were reduced by about half, giving similar kinetic efficiency (kcat/km) to that of wild-type. finally, we changed a residue at the root of the large loop (f355y) such that one new intersubunit hydrogen bond could form. this variant maintained the wild-type characteristics. in conclusion, removing hydrogen bonds connecting the major loop of one subunit to the protein surface of the other subunit in vap produced higher catalytic activity and this shows functional connections between loop mobility and the active site. our study also demonstrates that interactions between residues in the large disordered loop and the opposite subunit in the dimeric vap are determinants of its stability. thus, we managed to show that loosening of interface contacts between the two vap subunits by replacement of crucial residues provides a way to orchestrate structural and kinetic dynamics in a productive way. the de novo design of artificial proteins arises as a stringent test of our understanding of the relationship between sequence, structure, and function. examples include the design of a four a-helix bundle, a new protein topology called top7, and a series of artificial (ba)8-barrels called octarellins. however, de novo design has proven difficult for larger proteins with more than 100 amino acids. here we present two methods to generate the backbone and to perform the de novo design of (ba)8-barrel proteins through the use of the software rosetta; both have different advantages and limitations. the first method for generating the backbone is knowledge-based, with a first analysis of a non-redundant database of natural (ba)8-barrel proteins in order to obtain statistical analysis on preferred secondary structure element length and amino acidic propensities. with this information we use the rosetta cm software to create more than 1000 models which are then ranked in term of rosetta energy. the second method is performed with the parametricdesign package of rosetta, in which only geometrical information are requested (number of strands and helices, radius of the b-and a-barrels, degree of inclination, orientation of the side chains, among others). both methods contain a step of loop refinement and multiple steps of sequence design with the package rosetta design, in order to find low scoring amino acid sequences for each of the starting backbone conformations. thousands of models will be generated by both methods and then analyzed in term of sequence similarity, secondary and tertiary structure prediction, and stability by molecular dynamics simulations. the 30 best candidate sequences will be selected for the experimental verification. in order to identify a putative successfully design, we added a metal binding site during the design step. all the proteins will be expressed in e. coli. the solubility of the designed proteins inside bacteria will be determined thanks to the fusion to green fluorescence protein (gfp). solubility, stability, secondary structure, and cooperativity of folding will be assessed for each protein before determination of their three-dimensional structure. construction of protein capsule possessing drugs controlled release ability shota shimizu 1 , masatoshi nakatsuji 1 , keisuke yamaguchi 1 , yuya sano 1 , yuya miyamoto 1 , takashi inui 1 1 most compounds that exhibit anti-tumor activities are water-insoluble, thus limiting their clinical use. chemical modification of these compounds and the use of solubilizing agents such as organic solvents, surfactants and ph modifiers improve their solubility. however, chemical modification of compounds decreases their potency, and the use of solubilizing agents causes toxicity in many cases. thus, drug delivery systems (dds) for poorly water-soluble anti-tumor drugs which exploit liposomes, cyclodextrins, and lipid nanoparticles have been studied intensely. in these dds, the controlled release of drugs from the delivery vehicle is one of the most important functions. selective release in target cells leads to adequate therapeutic efficacy with few side effects. in our laboratory, we have already demonstrated that lipocalin-type prostaglandin d synthase (l-pgds), an intravital transporter protein, is a novel and valid drug delivery vehicle for sn-38, a poorly water-soluble anti-tumor drug. in this study, we generated l-pgds-based protein capsules with a controlled-release function by introducing a disulfide bond into the upper part of the drug-binding cavity of l-pgds. the intracellular concentration of glutathione (0.510 mm) is known to be substantially higher than the extracellular concentration (2 mm). therefore, it is expected that in the extracellular oxidative environment the disulfide bonds in the protein capsule remain stable, avoiding premature release of the internal drugs during circulation of blood, after reaching the target cells, the disulfide bonds are cleaved in the intracellular redox-environment, and then the internal drugs are released. we generated three kinds of protein capsules which have disulfide bonds in different positions, w54c/w112c, k58c/h111c, k58c/w112c, based on tertiary structure information of human l-pgds (pdb id: 3o2y). firstly, we performed circular dichroism (cd) measurements to confirm the structure of each capsule. the cd spectra of three protein capsules were similar to that of wild-type l-pgds in the far-uv region. therefore, the secondary structures of three protein capsules were not changed from wild-type l-pgds by introducing the mutations. quantitative analysis of the free thiol group in the protein capsule by dtnb assay revealed that the intermolecular disulfide bond was formed by h2o2-induced oxidation and cleaved by dithiothreitol-induced reduction. in addition, to investigate the solubility of sn-38 in the presence of protein capsules, we mixed the protein capsule of reduced-form with sn-38 suspension, and stirred at 37 c for 48 hours. the resulting concentrations of sn-38 in pbs with 1 mm w54c/w112c, k58c/h111c, and k58c/w112c were 374 mm, 194 mm, and 349 mm, respectively. these values were approximately 200-fold higher than without protein capsules. sds-page analysis showed that the bond formation decreased in a time-dependent manner, and that new intermolecular disulfide bond was not formed in the protein capsules after 48 hours' incubation. from the above, we succeeded in generating drug delivery vehicles possessing openable and closable lids that are responsive in an oxidation-reduction environment. takaaki miyamoto 1 , mai kuribayashi 1 , satoshi nagao 1 , yasuhito shomura 2 , yoshiki higuchi 3,4 , shun hirota 1 1 graduate school of materials science, nara institutte of science and technology, 2 graduate school of science and engineering, ibaraki university, 3 department of life science, graduate school of life science, university of hyogo, 4 domain swapping has been of interest as a mechanism of protein oligomerization, where a secondary structural region or a domain of one protein molecule is replaced with the corresponding region or domain of another protein molecule. we have previously shown that c-type cytochromes and myoglobin form oligomers by domain swapping.1,2 in this study, we show that a four-helix bundle protein cyt cb562, in which the heme of cyt b562 is attached to the protein moiety by insertion of two cys residues, forms a domain-swapped dimer. dimeric cyt cb562 was more stable than dimeric cyt b562 at 48c, showing that attachment of the heme to the protein moiety stabilizes the domain-swapped structure. absorption and cd spectra of dimeric cyt cb562 were similar to the corresponding spectra of the monomer, showing that the active site and secondary structures were similar between the dimer and monomer. the redox potential of dimeric cyt cb562 was also similar to that of its monomer. the dissociation temperature of dimeric cyt cb562 was 508c, and its dh on dissociation to monomers was 213.3 kcal/mol (per dimer). according to x-ray crystallographic analysis, dimeric cyt cb562 exhibited a domain-swapped structure, where the two helices in the n-terminal region (helices 1 and 2) in a protomer and the other two helices in the c-terminal region (helices 3 and 4) of the other protomer interacted between each other. the heme coordination structure of the dimer was similar to that of the monomer. we have previously shown that domain-swapped oligomers of horse cyt c form through intermolecular hydrophobic interaction between the n-and c-terminal a-helices at the early stage of folding.3 it has been suggested that helices 2 and 3 form first at the initial stage of folding in wild-type apo cyt b562.4 therefore, we propose that cyt cb562 forms a domain-swapped dimer when helices 2 and 3 interact intermolecularly at the initial stage of folding, whereas the intramolecular interaction of helices 2 and 3 results in formation of a monomer. a highly buried and conserved tryptophan residue close to the dimer interface in a cold-adapted phosphatase is phosphorescent and important for activity. jens g. hj€ orleifsson and bjarni asgeirsson. department of biochemistry, science institute, university of iceland, dunhagi 3, 107 reykjavik, iceland. alkaline phosphatase (ap) from vibrio g15-21 is a cold-adapted dimeric enzyme with one of the highest catalytic efficiency reported for known aps. it contains five intrinsic tryptophan (trp) residues and one additional trp located on the c-terminal streptag used for expression and purification. in this study, we made several single trp-substitutions to determine the role of each of the trp in the fluorescence emission spectrum. we also determined their solvent exposure by acrylamide fluorescence quenching. the results indicate that trp301, trp460 and trp475 are mostly responsible for the fluorescence emission. quenching experiments with acrylamide indicated that all the trp residues were about equally accessible for quenching, except trp460 which was shown to be highly buried in the core of the protein. interestingly, the enzyme was found to be highly phosphorescent at 108c, having two phosphorescence lifetimes. the longer lifetime is due to trp460. trp460 is located close to the dimer interface and points towards a helix in the active site where his277 binds an active-site zinc ion. in other aps, an aromatic amino acid is conserved in the location occupied by the trp460 residue. in most cases for cold-adapted aps it is indeed a trp. interestingly, the mutation of the trp460 to a phenylalanine affected both stability and activity of the enzyme. kcat/km was 10-fold lower than for wild-type. overall, this study reveals that trp460 can be used as a phosphorescent probe of local dynamics and could possibly also serve to study the dimer-monomer equilibrium due to proximity to the dimer interface, an area clearly crucial for enzyme activity and stability. modulating protein-protein interaction with a molecular tether helen farrants 1 , oliver hantschel 1 , kai johnsson 1 1 ecole polytechnique f ed erale de lausanne (epfl) high-affinity scaffolds for protein-protein interactions, such as monobodies and darpins can be engineered in vitro to bind to protein targets. we speculate that the affinity for the target protein can be modulated by incorporating these evolved scaffolds and a synthetic intramolecular tether into protein switches, in a protein construct of composed of snap-tag, a monobody and a circular permutated dihydrofolate reductase. the tether, attached to the construct via snap-tag, was composed of a linker and trimethoprim, which interacts reversibly with the circular permutated dihydrofolate reductase. we have investigated the affinity between the n-sh2 domain of the phosphatase shp2 and an evolved monobody in such a protein construct using a fret assay. when the intramolecular tether was bound the circular permutated dihydrofolate reductase ("closed" conformation), there was an increase in the affinity of the construct to the target n-sh2. in the presence of a small molecule competitor ("open" conformation) the affinity of the monobody construct to its target was reverted to the value reported in the literature. the intramolecular tether in these protein constructs combined with engineered scaffolds for protein-protein interactions may be a general approach towards protein switches. because most proteins are long polymers of amino acids with twenty or more chemically-distinct sidechains, there are an enormous number of potential protein sequences. here, we report the construction of biologically active proteins with minimal chemical diversity. transmembrane domains of proteins can specifically interact with other transmembrane domains to modulate the folding, oligomerization, and function of transmembrane proteins. for example, the bovine papillomavirus e5 protein is a 44-amino acid transmembrane protein that transforms fibroblasts to tumorigenicity by binding directly to the transmembrane domain of the platelet-derived growth factor b receptor (pdgfbr), resulting in ligandindependent receptor activation and cell transformation. these studies showed that a free-standing transmembrane domain could fold properly in cells and act in trans to modulate the activity of a larger transmembrane protein target. because of the relative chemical simplicity of transmembrane domains and this ability to act even when not linked to more complex soluble protein domains, we reasoned that short transmembrane proteins could be used to define the minimal chemical diversity sufficient to construct biologically active proteins. to accomplish this, we infected cultured mouse cells with a retroviral library expressing 26-amino acid proteins consisting of an initiating methionine followed by a randomized sequence of leucines and isoleucines, two hydrophobic amino acids that differ only by the position of a single methyl group, and selected rare proteins with transforming activity. we isolated numerous proteins consisting of diverse sequences of leucine and isoleucine that cause morphologic transformation, escape from contact inhibition and focus formation, and growth factor independence. genetic and biochemical analysis of these proteins indicate that like e5 they interact with the transmembrane domain of the pdgfbr to specifically activate the receptor and transform cells. mutational analysis of individual proteins identified specific leucines and isoleucines required for transforming activity, and insertion of a single isoleucine at a particular position in a stretch of leucines is sufficient for activity. these proteins identify the minimal chemical diversity required to generate a biologically active protein and have important implications for biochemistry, protein evolution, protein engineering and synthetic biology. yusuke azuma 1 , donald hilvert 1 1 virus-like particles that are precisely loaded with functional cargo are an important tool to study the effect of spatial confinement and create novel entities with application in biotechnology and medicine. by genetic fusion to a positively supercharged green fluorescent protein (gfp(136)), an enzyme retroaldolase (ra) was efficiently targeted to the negatively charged lumen of an engineered protein cage, aquifex aeolicus lumazine synthase variant 13 (aals-13). the encapsulation is quantitative under mild aqueous condition up to a mixing ratio of 45 guest enzymes per host cages. the chromophoric tag is used for precisely quantifying the enzyme concentration, which allows detailed characterization of the effect of encapsulation on the enzyme activity. the generality of the encapsulation system was examined with 8 structurally different enzymes. introduction and purpose: in the immune system, high affinity antibodies are generated by selection of b cells activated by antigen-stimulation followed by additional optimization through somatic hyper mutation of antibody genes. in the artificial antibody libraries, such as phage libraries, selection of specific antibody clones from the library is performed by in vitro selection process called biopanning and the subsequent binding screening. however, in spite of high efficiency of enrichment in biopanning, there is a possibility that we overlook the minor antigen-specific clones in the screening because of the limitation of the number of clones employed for screening. in recent years, high-throughput analysis of dna sequences by the next-generation sequencer (ngs) has become available not only for genomic analysis of organisms but also repertoire analysis of antibodies. in this presentation, we report the successful isolation of a variety of antigen specific antibodies from patients-derived antibody phage library by a combination method of high throughput sequence analysis on ngs and biopanning. method: we constructed two kinds of human single chain fv (scfv) antibody libraries from pooled mrna of five cancer patients and of a wheat allergy patient, respectively. after biopanning against a cancer antigen or wheat allergy antigen "gluten", the phagemid vector dna prepared from the pooled phages before or after biopanning was used for pcr amplifications of vh genes, adding the index and adapter sequences for ngs analysis. the high throughput sequencing was performed on miseq (illumina) using miseq reagent kits v3. after discarding the short sequences and low quality data, 5'-and 3'-reading sequences were unified by a merge program. the frequencies (%) of all vh sequences were evaluated using a program based on usearch 8.0 clustering software and the changes of the frequency (%) of each sequence between before and after panning were assigned as amplification rate. results and discussion: vh sequences at each round of pooled phages after biopanning against cancer antigen were analyzed on ngs. after three rounds of biopanning, three clusters of antibody sequences were specifically enriched suggesting these are specific binders. to check this, scfv gene were regenerated by pcr using h-cdr3 specific primers and scfv-displaying phages reconstructed were subjected to binding analysis. all three phages showed a clear specific binding to cancer antigen in elisa. subsequently, to test the usefulness of this method, we applied it to identify allergen-specific scfv from allergy patient-derived antibody phage library. the phylogenetic tree analysis of vh sequences which showed the amplification rate higher than 2.5 by a single round of biopanning elucidated total eleven clusters of vh sequences. the vh sequences in the two clusters with the highest amplification factor were selected and the regenerated scfv-displayed phages were tested for binding analysis. the prepared scfvdisplayed phages and also scfv proteins showed a clear binding ability to allergen. thus, it is suggested that the analytical method of vh sequences on ngs before and after biopanning is very useful to isolate a variety of disease related antigen-specific novel antibodies quickly with high degree of certainty. biochemical analysis of the recognition helix of z-dna binding proteins: roles in conformational specificity yang-gyun kim 1 , xu zheng 1 , so-young park 1 1 conversion of right-handed b-dna into left-handed z-dna is one of the dramatic structural transitions in biological processes including gene regulation and chromatin remodeling. z-dna binding motif, zalpha (za), was first discovered from human adar1. subsequently, with sequence and structure similarity to the hzaadar1, families of proteins including viral e3l, interferon-induced protein dai (zbp1) and pkz has been identified to have za domain(s). interestingly, the za domain of the e3l protein from vaccinia virus (vvzae3l) was confirmed to have the ability of z-dna-binding, but it does not have the b-to-z conversion activity. here, we showed that the replacement of the a3-helix of vvzae3l (vvzae3l-a3) with that of hzaadar1 results in acquiring the ability to converting b-dna to z-dna. the detailed biochemical analysis of the a3-helix mutants of vvzae3l further suggested that the contribution of positively charged residues in the c-terminal part of the a3-helix is crucial during the b-to-z transition. in addition, hydrophobic residues of the n-terminal part of the vvzae3l-a3 also influence on the b-to-z conversion activity, possibly through forming a tightly-packed structure. in conclusion, our results revealed the previously-unknown contribution of amino acid residues existed in the a3-helix of the za domains to the b-to-z conversion. moreover, it strongly implies that such residues may play important roles in initiating conformational changes of dna structure during the b-to-z conversion event. the ability of switching the activity of proteins at will is of great interest from an application point of view. one promising approach utilizes a protein modification with an organic photochromic molecule. linking two protein side chains with the photochrome that undergoes a light induced conformational change, protein secondary and tertiary structure can be stabilized or destabilized and thus the structure dependent activity can be switched "on" and "off" by light irradiation. for this the photochrome must fulfil several requirements. foremost, it must possess two states of comparable stability that differ significantly in their geometry. it must further be water soluble and non-toxic, and should not experience fatigue phenomena upon multiple irradiations. there are two classes of molecules that fulfil those requirements: azobenzenes and spiropyrans. we are pursuing two different strategies for the design of photoswitchable proteins. in the first approach we attach an azobenzene compounds to side chains of the alpha-helical antifreeze protein type i. the end to end distance of the photochromic molecule is sterically compatible with the folded helix only in one form, photoisomerization therefore switches the folding state between an active helical state and an inactive unfolded form. in a second, more general approach we use the trp-cage domain as a switching unit. the trp-cage is the smallest known folded protein (20 amino acids). its folding is induced by hydrophobic interactions of a tryptophan side chain in a short helical segment. after modification with a photochromic molecule in appropriate positions, its structure is rendered sensitive to the state of the chromophore. by creating protein chimera of such a trp-cage and biologically active peptides with helical propensity, we aim at conferring the light-dependent fold of the cage to the attached peptide moiety. salt-bridges are electrostatic interactions between groups of opposite charges. net interaction energy (ddgnet) of a salt-bridge is partitioned into bridge (ddgbrd), desolvation (ddgdsolv) and protein (ddgprot) energy-terms of which estimation of ddgdsolv and ddgprot are only possible by computational means. thus, general purpose poisson-boltzmann equation solver: "delphi" (in commercial package of insight-ii) and "apbs" (open-source) are popularly used to determine these energy-terms. nevertheless, the computation-method is highly involved one than other uses of these solvers. moreover, protein-specific saltbridges, grid-points, center, hydrophobic-isosteres-mediated mutation-files of original charge-radius file and others are to be worked out prior to the computation. this might answer as to why only limited numbers of structure files (2% of crystal-structure-database) are worked out till date. at this juncture, an efficient fully automated all-in-one-procedure that could analyze large dataset in a single run would be useful. to the best of our knowledge, such procedure is truly lacking in public domain. at this end, our fully automated all-in-one procedure: adsetmeas (available freely at http://sourceforge.net/projects/adsetmeas/along with detailed documentation) uses "apbs" method to compute component as well as net energy-terms of salt-bridges and redirect compact output in excelformat. further, micro-environments of salt-bridges are also been reported based on the presence of polar, dipolar, acidic, basic and hydrophobic side-chains in their proximity. the procedure provides versatility to users in choosing a] model for computation of energy-terms to-date available in the literature and b] method (default or advanced) for parametric optimization in "apbs" calculations. it works in unix like environment including cygwin. it processes all proteins present in the working directory with any number of salt-bridges in them. a pre-released version of the procedure was successfully applied for energy-terms on 220 salt-bridges from 22 halophilic proteins. overall, our adsetmeas provides intricate details on salt-bridge energetic from crystal structures and find application in the field of computational structural biology. these and other results will be discussed in the conference. next generation analgesics -targeting ion channels with antibody-drug conjugates (adcs) anna wojciechowska-bason 1 , clare jones 2 , chris lloyd 3 1 postdoctoral fellow, adpe, medimmune, cambridge, 2 ria, medimmune, cambridge, 3 adpe ion channels are common targets for chronic pain therapies. small molecule analgesics are widely used therapeutically, but due to poor specificity they often cause a wide range of side effects. as a result, efficacy of existing treatments is very limited. we believe that to achieve the required specificity and efficacy, a novel and innovative approach is required that would combine the potency of the small molecule with the selectivity of an antibody. therefore, we propose to apply antibody-drug conjugates (adcs) to deliver small molecules or peptides to ion channels in order to specifically modulate pain signalling pathways. voltage-gated sodium channel nav1.7 has a well characterised role in the perception of pain. here we present the activity of the peptide huwentoxin-iv (hwtx-iv) and small molecule inhibitors ptc-a, ptc-b and ptc-c on voltage gated sodium channels nav1.7 and nav1.6. in novel findings, we report that these inhibitors show little selectivity between the voltage-gated sodium channel family members, nav1.6 and nav1.7, and that the ic50 values and the impact on channel biophysics (voltage-dependence of activation and fast inactivation) of the inhibitors are largely similar for both channel types. therefore, the use of hwtx-iv and other small molecule inhibitors of nav1.7 for pain therapy could be dose-limited due to side effects mediated by the inhibition of channel nav1.6. in conclusion, we propose that hwtx-iv and the investigated small molecule inhibitors could be used for the treatment of pain as part of a nav1.7 antibody-drug conjugate (nav1.7-adc), establishing nav1.7 specificity and minimising side effects. maria antonietta carillo 1 , daniel varon silva 1 1 malaria is one of the most infectious diseases caused by plasmodium species parasites. the merozoite surface protein 1 (msp1) is the most abundant protein on the surface of the plasmodium species merozoite stage, which plays an important role during the erythrocytes invasion process [1] . msp1 is synthesized as a 200-kda glycosylphosphatidylinositol (gpi) anchored protein precursor which is processed at the end of the schizogony into four different fragments. the primary processing step produces a complex of four fragments that are present on the merozoite surface. the secondary processing step at erythrocytes invasion results in the detaching of the complex from the surface, except for the cterminal 19-kda domain (msp119), which remains anchored to the parasite surface by the gpi moiety. in human malarial infections, the gpi is considered to be a toxin that causes the expression of various host genes and induces a pro-inflammatory immune response, making it a valuable candidate for the development of anti-malarial drugs. in order to study the function of the gpi and evaluate the effects, msp119 fragment has been expressed, purified and anchored to the synthetic gpi molecule using protein trans-splicing strategy based on the split intein method [2] . the role of the gpi moiety will be studied through protein folding experiments and the effect of the anchored protein will be evaluated in vitro in order to understand the function of the gpis. assessment of uch-l3 substrate selectivity using engineered ubiquitin fusions with varying linker lengths peter suon, mario navarro, john love 1 san diego state university, 2 san diego state university, 3 assessment of uch-l3 substrate selectivity using engineered ubiquitin fusions with varying linker lengths peter suon, mario navarro, and john j. love san diego state university the ubiquitin proteasome system (ups) is a complex system composed of multiple structural and functional elements that play key roles in cellular processes such as signal transduction, cell cycle regulation, apoptosis, and protein degradation. proteins destined for degradation are first tagged with the protein, ubiquitin, which is covalently attached to internal lysine residues. once the target has be degraded by the proteasome; the enzyme ubiquitin carboxy hydrolase l3 (uch-l3) is believed to prepare ubiquitin for additional rounds of ubiquitination by cleaving small peptides and chemical adducts from the ubiquitin c-terminus. previously in our laboratory, protein substrates of uch-l3 were engineered and used to characterize uch-l3 substrate selectivity. the engineered substrates consisted of n-terminal monoubiquitinated test variants derived from streptococcal protein g (protein gb1) and staphylococcal protein a (spab). the thermal denaturation temperatures (tm) of the fusion proteins were measured using circular dichroism and span a range of over 608c. more importantly, the rate of hydrolysis for the fusion proteins is demonstrated to be directly correlated to the tm of the test variant fused to the c-terminus of ubiquitin. previously, the engineered substrates were designed to emulate natural ubiquitin fusions and thus did not contain any 'linker' residues between the c-terminus of ubiquitin and the n-terminus of the test protein. to explore the effects of linker length on uch-l3 hydrolysis we are engineering new uch-l3 substrates that contain an unstructured 12 amino acid linker between ubiquitin and the test protein. to further explore the catalytic efficiency of uch-l3 we will revisit diubiquitin (ub-ub), which is not hydrolyzed by uch-l3, and will make mutations in the hopes of generating a hydrolysable substrate. using rational design, the new variants will be engineered to destabilize the c-terminal ubiquitin to determine if this results in hydrolysis of the new ub-ub construct. the thermal stability of these new fusion protein substrates will be measured using circular dichroism spectroscopy (cd) and uch-l3 hydrolysis rates will be characterized using existing assays. our goal is to continue the use of engineered substrates to further explore the catalytic properties of uch-l3 activity and the potential role in protein trafficking and degradation within living cells. we present a biophysical study of a suite of helical proteins that have been modified to contain 12and 17-amino acid additions on their termini that impart increased resistance to degradation in e. coli abstract recombinant expression systems. the b domain of staphylococcal protein a (ab) and the homeobox dna-binding domain from d. melanogaster engrailed (en) are small 3-helix bundles. these domains do not appreciably accumulate in the e. coli bl21 (de3) cytoplasm when expression in a pet vector is chemically induced. this is likely due to host protein degradation/recycling factors that function to efficiently degrade these two proteins. addition of sequences encoding either of two amino-terminal beta-hairpins to either the n-or c-terminus of ab and en results in the accumulation of large amounts of these new chimeric proteins. additionally, destabilization of the ab or en sequence does not abolish the expression enhancement effect of the beta-hairpin addition. we have investigated the biophysical origins and effects of the beta-hairpin additions using circular dichroism (cd) spectroscopy, and have determined that the added sequence does not significantly perturb the secondary structure of ab or en, nor does it significantly influence the unfolding temperature (tm). while investigation into the origin of the accumulation effect is ongoing, we hypothesize that the addition of the sequence is disruptive to recognition events in the native protein degradation machinery in e. coli. thus, this approach represents both a biotechnological tool for expressing helical peptides recalcitrant to expression, as well as a system well-suited to probing mechanisms of protein recycling and homeostasis. a special class of these proteins are lipidated proteins containing a glycosylphosphatidylinositol (gpi) glycolipid moiety at the c-terminus. the lipid chains of the gpi anchor molecule are responsible for the membrane association of the attached protein. a unique feature of gpi-anchored proteins is that after isolation they can be reinserted into the membrane of recipient cells with the retention of the biological function. accordingly, the exogenous introduction of fluorescent gpi-anchored protein analogues into cell membranes is a useful method for visualizing the cellular traffic of membrane associated proteins and for engineering cell surfaces. we have recently shown that cholesterol can be applied for anchoring proteins to the plasma membrane of live cells without perturbing the membrane. in order to introduce proteins containing covalent modifications that are not genetically encoded, an enzymatic method was considered and fused with the c-terminal cholesterylation method. the usefulness of the method is demonstrated via the preparation of multimeric model proteins of 40 kda monomers, that is an appropriate representation of the ligation of domain size proteins. transmembrane domain dimerization drives p75ntr partitioning to lipid rafts irmina garc ıa carpio 1 , marc¸al vilar 1 1 sociedad de biof ısica de españa. sbe p75 neurotrophin receptor (p75ntr), is best known for its role in mediating neuron cell death during development or after injury but it also regulates cell proliferation, axon guidance or survival. the key to understand its signaling could rely in its structure and conformational states. it has been described that p75 forms disulfide-linked dimmers through the cys257 in the transmembrane domain which are essential for its ngf mediated signaling. previous studies have shown that p75 is present in lipid rafts, where it interacts with intracellular adaptors to activate different signaling pathways. we design several p75 mutants in the tm domain that impairs dimerization and study the role of tm domain dimerization in lipid rafts recruitment. our analysis suggests that p75 tm domain dimerization influences lipid raft partitioning. these results could be a key role to understand its signaling and processing pi-079 bioluminescent sensor proteins for therapeutic drug monitoring of the monoclonal antibody cetuximab martijn van rosmalen 1 , remco arts 1 , brian janssen 1 , natalie hendrikse 1 , dave wanders 1 , maarten merkx 1 1 therapeutic drug monitoring (tdm) -adapting the drug dosage scheme to the individual patient's pharmacokinetic and pharmacodynamic characteristics -is still uncommon for therapeutic monoclonal antibodies, despite preliminary studies showing its potential benefits. one of the factors impairing tdm implementation is the lack of equipment and trained personnel to regularly measure drug concentrations in patients receiving treatment. point-of-care diagnostic devices which could be used by patients themselves or by their general practitioners would greatly advance the feasibility of tdm. here we present a biosensor for the therapeutic monoclonal antibody cetuximab. we developed a series of cyclic peptides that specifically recognize cetuximab, covering a fourfold range of affinities, and incorporated these cyclic peptide sequences into a set of luminescent sensor proteins. the sensors translate cetuximab concentrations into a change in emission color that can be read out using a mobile phone camera. together, these sensors can quantify cetuximab levels within the relevant therapeutic concentration range and we propose that they can be used for therapeutic drug monitoring applications. genetically encoded biosensor for cell permeability of inhibitors of the p53-hdm2 interaction silvia scarabelli 1 , thomas vorherr 2 , kai johnsson 1 1 ecole polytechnique f ed erale de lausanne, 2 the evaluation of the permeability across the cellular membrane is a key step in the development of therapeutics, since it affects the distribution and the efficacy of the latters. reliable and versatile techniques for the determination of structural permeability determinants of molecules and information about the entry kinetics are still missing. we introduced in the past a class of semi-synthetic ratiometric sensor proteins (snifits) that has been shown to be suitable for the measurement of intracellular metabolites concentrations. here we describe a totally genetically encoded sensor based on the snifits modular design for the assessment of the cell permeability of small molecules and peptides inhibitors of the protein-protein interaction between p53 and hdm2. we show that our sensor detects the presence of hdm2-binding stapled peptides in vitro, and, when expressed in mammalian cells, it responds to the perfusion of the known small molecule hdm2 inhibitor nutlin-3a. moreover, experiments made with an automated microscope show that the sensor is suitable for measuring and comparing the kinetics of entry of different kinds of inhibitors in the cytosol of living cells. in parallel, we are developing an hcaii-based sensor protein for the sensing of sulfonamides and eventually their peptide derivatives. we show that the sensor responds to the presence of different kinds of hca-inhibitors in vitro and in perfusion experiments. this second sensor would broaden the range of molecules and peptides whose permeability can be studied with our tools beyond the family of the hdm2-binders. our sensors overcome the limitations of the already existing techniques for measurements of permeability while offering a simultaneous measurement of the cell permeability and of the binding efficiency of small molecules and peptides of interest. archer: predicting protein function using local structural features. a helpful tool for protein redesign. jaume bonet 1 , javier garcia-garcia 1 , joan planas-iglesias 2 , narcis fernandez-fuentes 3 , baldo oliva 1 1 structural bioinformatics lab, grib, upf, 2 division of metabolic and vascular health, university of warwick, 3 the advance of high-throughput sequencing methodologies has led to an exponential increase of new protein sequences, a large proportion of which remain unannotated. the gap between the number of known proteins and those with assigned function is increasing. in light of this situation, computational methods to predict the function of proteins have become a valid and necessary strategy. here we present archer, a server that exploits archdb's hierarchy of super-secondary structures to map go and enzyme functions upon protein regions and, thus, infer the function of a protein. the server relies on either the sequence or structure of the protein of interest and returns the mapping of functional subclasses extracted from archdb. moreover, it computes the functional enrichment and significance of each subclass, combines the functional descriptors and predicts the function of the query-protein. combining the functional enrichment analysis of the super-secondary structures with the structural classification of archdb, users can select variants of the target sequence that swap the region of a supersecondary structure by another that putatively fits in the same scaffold minimizing the effect on the global tertiary structure. only variants that modify the predicted function are offered for selection, thus providing a rational, knowledge-based, approach for protein design and functionalization. the archer server is accessible at http://sbi.imim.es/archer. phytochromes are natural photoreceptors known to regulate photosynthesis in plants, fungi and bacteria. phytochromes found in bacteria share common architecture and consist of a pas-gaf-phy photosensory core and a c-terminal output module, responsible for biological function. a bacterial phytochrome, bphp1, from rhodopseudomonas palustris undergoes reversible conversion from the farred absorbing state (pfr) to the red-absorbing state (pr) followed by the conformational change upon 740 nm light irradiation. as most of bacterial phytochromes, bphp1 forms a dimer. it was shown that 740 nm light causes a protomer swapping between the bphp1 dimers; and likely, the output module is involved in this process. however, the mechanism of the light-induced swapping is poorly studied. we tested an ability of the protomer swapping between bphp1 dimers using pull-down biochemical assay. for this, strep-tagged bphp1 was immobilized on strep-tactin sepharose beads in the presence of untagged bphp1 fused to mruby2 at different concentrations. after incubation, the proteins were eluted and visualized in sds-gel using a zinc-induced fluorescence assay. an amount of the bound to beads protein was estimated by densitometry. it was found that more than 75% of heterodimers (streptagged-bphp1 and bphp1-mruby2) form within 2.5 h of incubation under 740 nm light at 8-fold excess of one of the interacting partners. in darkness, the swapping was much slower. in the similar setup we checked the amount of heterodimers after 15, 30 and 120 min of incubation. no difference was observed for different time points, suggesting that the protomer swapping is relatively fast process. next, a role of the c-terminal effector domain of bphp1 in the light-induced interaction was studied. for this, kinetics of the pfr-to-pr transition was analysed by measuring of absorbance at 680 nm and 740 nm for full-length bphp1 and a bphp1 mutant with the deleted c-terminal domain. while full-length bphp1 showed the normal pfr-to-pr transition, absorbance of the mutated bphp1 at 680 nm did not raise. however, 740 nm absorbance changes were similar for both proteins; and surprisingly, the similar dark relaxation kinetics was observed. we propose that the impaired pfr-to-pr transition is caused by restricted pr conformation in the mutant rather than by fast pr-to-pfr relaxation. understanding the mechanisms of the bphp1 light-induced structural changes and the protomer interaction should advance engineering of bacterial phytochromes into fluorescent probes and optogenetic tools. antibody detection is an integral part of many diagnostic strategies, most crucially so when infectious diseases are involved. currently used assays, such as elisa or spr, enable detection of antibodies in the laboratory with high sensitivity, yet a translation of these technologies to an application outside of the laboratory setting is far from trivial. problematically, the burden of disease for many infectious diseases is carried precisely by those countries where access to laboratory facilities is severely limited. we therefore developed a novel, one-step assay that allows the detection of antibodies directly in solution using a luminescent sensor protein. our strategy is based on the use of a bright luciferase, nanoluc, tethered to a green fluorescent protein (mneongreen) via a semi-flexible linker containing two epitope sequences. crucially, two small helper domains were fused to the protein termini. these domains keep nano-luc and mneongreen in close proximity in the absence of antibody, enabling efficient bioluminescence resonance energy transfer (bret). binding of antibody to the epitopes in the sensor proteins linker domain pulls the bret partners apart, effectively changing the color of emission from green to blue. the assay allowed the detection of picomolar amounts of anti hiv1-p17 antibodies directly in solution, both under optimized buffer conditions and in blood plasma. in principle. the modular sensor architecture should allow detection of any antibody with a well-defined epitope of sufficient affinity. to demonstrate this, the hiv-epitopes were substituted for two ha-tag epitopes, yielding a sensor that enabled the detection of picomolar amounts of anti-ha antibodies. the simple optical readout provided by the sensor system allowed us to record the emitted signal with a conventional mobile phone camera. a simple software application that analyzes the image based on rgb values sufficed to interpret the recorded image vis-a-vis the presence of antibody. bearing in mind the eventually envisioned application in a point-of-care diagnostic setting, this combination of sensor recording and interpretation using nothing more than a mobile phone and a software application holds considerable diagnostic potential. beyond point-of-care diagnosis of infectious diseases, a simple assay to detect and quantify antibodies directly in solution could also have a substantial impact in other fields. antibodies are ubiquitous in biotechnology, and this is reflected by the plethora of potential sensor applications, which range from a role in microfluidic circuits or monitoring the biotechnological production of antibodies, including validation of bispecificity, to veterinary applications, diagnosis of autoimmune diseases and monitoring the success of vaccination campaigns. the continually growing protein data bank (pdb) has been a key resource for general principles of protein structure. for example, parsing structural observations in the pdb into simple geometric descriptors has given rise to statistical energy functions. here we present a novel strategy for mining the pdb on the basis of local tertiary structural motifs (term). we define a term to be the structural fragment that captures all local secondary and tertiary structural environments of a given residue, and query the pdb to obtain quantitative information for each terms. first, we show that by breaking a protein structure into its constituent terms, we can describe its sequence-structure relationship via a new metric we call "structure score." using submissions in recent critical assessment of structure prediction (casp) experiments, we find a strong correlation (r 5 0.69) between structure score and model accuracy -a performance that exceeds leading atomistic statistical energy functions. next, we show that querying terms affected by point mutations enables the quantitative prediction of mutational free energies. our simple approach performs on par with state-of-the-art methods fold-x and popmusic on 1300 mutations, and provides superior predictions in certain cases where other methods tend to fail. in all, our results suggest that the data available in the pdb are now sufficient to enable the quantification of much more sophisticated structural observations, such as those associated with entire terms, which should present opportunities for advances in computational structural biology techniques, including structure prediction and design. exploiting natural sequence diversity for protein crystallization sergio mart ınez-rodr ıguez 1 , valeria risso 1 , jos e m sanchez-ruiz 1 , jos e a. gavira 2 , 1 departamento de qu ımica-f ısica, universidad de granada, 2 laboratorio de estudios cristalogr aficos, iact-csic-ugr granada during the last decade, different rational and high-throughput approaches have been successfully applied in the protein crystallography field to widen thejjso-called "protein crystallization bottleneck" [1, 2] . despite the enormous efforts carried out by our community, the statistics presented by structural biology consortiums [3] suggest that so far only the easy-to-pick fruit has been attained; thus, new approaches are necessary to further expand the crystallization limiting step to relevant targets. on the basis of previous hypothesis suggesting that the difficulties found in protein crystallization might be a result of evolutionary negative design [4] , we have used two different protein engineering approaches exploiting natural sequence diversity using beta-lactamase as toolbox: i) ancestral reconstruction and ii) consensus approach [5] . both approaches resulted in hyperstable and promiscuous ancestral derivatives. furthermore, our initial crystallization results also suggest that both approaches increased the crystallizability of the resulting enzymes when compared to the extant tem-1 beta-lactamase. the adipocyte-derived hormone adiponectin has become a key player for the understanding of overweight related diseases like obesity, diabetes, atherosclerosis or the metabolic syndrome. one of its abstract major functions are the insulin sensitizing effects, which are mediated by the activation of ampk, p38-mapk and ppara (1). furthermore adiponectin is involved into glucose regulation and fatty acid oxidation. recently, three adiponectin receptors adipor1, adipor2 and t-cadherin have been described while an unknown fourth receptor is hypothesized (2) . for only two of them (adipor1 and adipor2) the signaling transduction via adiponectin has been confirmed (3). in order to find new binding partners or co-receptors, we cloned and expressed full length adiponectin as a fusion protein with a c-terminal intein and a chitin binding domain (cbd) as well as an n-terminal his10-tag. by using the impactsystem, the fusion protein was cleaved to form the corresponding thioester. to separate the starting materials as well as the cleaved intein chitin binding domain, the purification was performed with chitin beads. furthermore, the product was concentrated by ni-nta-affinity chromatography. accordingly, the obtained adiponectin thioester was reacted with a tamra-or a biotin labeled peptide, respectively, to receive the corresponding ligation product. finally the functionalized adiponectin was purified by size exclusion chromatography. further studies will allow screening for interacting molecules in cell and tissue derived samples. departamento de quimica fisica, facultad de ciencias university of granada, 2 dpto. de quimica fisica biologica. instituto de quimica fisica rocasolano, 3 departamento de quimica organica, facultad de ciencias university of granada, 4 rational design of non-natural enzyme activities has proved challenging. here, we report the introduction of catalysis of the kemp elimination (a model of proton abstraction from carbon) in scaffolds corresponding to precambrian nodes in the evolution of the antibiotic resistance protein b-lactamase. we used a single-mutation, minimalist approach based on chemical intuition, and obtained catalysis levels similar to those reported in the literature for computational kemp-eliminase designs involving multiple mutations. remarkably, the approach was unsuccessful when performed on modern b-lactamases. we provide experimental evidence that enhanced conformational flexibility contributes to the success of the minimalist design in the ancestral scaffolds. this work has implications for the understanding of function emergence in protein evolution and demonstrates the potential of ancestral protein resurrection in enzyme engineering and design. exploring the importance of dimerization for dj-1 function through engineered domain fusions sierra hansen 1 , jiusheng lin 1 , mark wilson 1 1 parkinson's disease is a progressive neurodegenerative disease that affects approximately 6.3 million people worldwide and is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. dj-1 (park7) is one of several genes that are mutated in rare forms of familial parkinsonism. dj-1 is a dimeric cytoprotective protein that defends against oxidative stress and preserves mitochondrial function. dimerization of dj-1 is thought to be essential for this function, as some diseaseassociated mutations cause poor folding and disrupt the dj-1 dimer. however, recent reports suggest that dj-1 may be functional as a monomer. to test this, we have engineered a non-dissociable dj-1 dimer that is a fusion of two human dj-1 domains. this construct cannot dissociate into monomers and thus will provide a stringent test of the importance of monomeric dj-1. our engineered construct is modeled on plant dj-1 homologs, which feature naturally occurring duplicate dj-1 domains separated by a small (19 amino acid) linker region. using x-ray crystallography, we confirmed that this engineered non-dissociable human dj-1 dimer has identical structure to the naturally occurring dimeric protein. we have investigated the influence of enforced dimerization of the pathogenic effects of the parkinsonian l166p and l10p mutations. cd spectroscopic analysis reveals that single and double l166p mutations in the non-dissociable dj-1 dimer maintain a higher degree of structure than l166p mutations in the native protein. additional characterization of the protective capacity and subcellular trafficking of this non-dissociable dj-1 dimer is underway. the purification, crystallization and preliminary characterization of sdre from s. aureus the purification, crystallization and preliminary characterization of sdre from s. aureus staphylococcus aureus (s.aureus) is an important human opportunistic pathogen which colonizes about 20% of the human population persistently [1] . surface proteins of s.aureus can excretion a kind of sortase, which represents a surface organelle responsible during the pathogenesis of bacterial infection the host circulation [2] . sdr proteins were a component of cell wall anchored family proteins, including sdrc, sdrd and sdre [3] . sdre could combine with the complement regulatory protein factor h to escape the alternative pathway of complement [4] . to further investigate the functions of sdre, we have expressed and purified the adhesive domain (residues 141-'06:15), and crystallized the recombinant protein. in addition, we also constructed the mutant s.aureus, and the cell experiments confirmed that sdre gene participate in the bacteria invasion. bacterial microcompartments (bmcs) are proteinaceous organelles that sequester key metabolic reactions to increase enzymatic efficiency or to prevent the loss of volatile or toxic intermediates. there is an increasing desire to engineer bmcs for non-native enzymatic processes. it is thought this will increase multi-enzyme pathway efficiency and allow the expression pathways that may produce toxic or volatile intermediates in bacteria. the mechanisms of small molecule transport and retention of toxic intermediates by bmcs remain poorly understood. better understanding of the bmcs pores critical to engineer bmcs for these non-native pathways. in order to better understand the bmc pore we have undertaken structure-guided modifications of the the hexameric pdua shell protein of the 1,2-propanediol utilization microcompartment (pdu mcp). these modifications include pore mutations in an attempt to alter substrate specificity and permutations of pdua to allow more drastic alterations to the structure of the protein. crystal structures of pdua pore mutants, solved to atomic resolution (2-3.3å) provide evidence of the pore residues that confer specificity. further, a pdua permutation (pduap) has resulted in a closed icosahedral cage. this novel pduap cage shows a ph and salt dependent assembly and may serve as a reaction vessel or be utilized for cargo delivery. (1, 2) . anm-mc is used to identify targeted transition pathways and intermediates between open and closed states of proteins. at each step of this iterative technique, the protein is deformed along the collective anm mode showing the best overlap with the target direction and its energy is minimized via short mc run. in this work, optimization of simulation parameters (number of mc moves and their perturbation strength, anm deformation factor in each cycle and force constant for backbone bonds) was performed in order to increase the efficiency of this technique. as a result, this technique can now be applied to much larger systems and conformational changes. the transition pathway between apo and dna-bound conformations of the yeast rna polymerase, which is a hetero-10-mer with more than 3500 residues, will be presented here. moreover, the pathway intermediates for more than 10 diverse proteins were analyzed in terms of changes in local strain energy and backbone torsional angles during apo-to-complex transitions. certain residues interacting with the ligand are detected to exhibit large changes with respect to any of these two parameters for more than half of the proteins in our dataset. department of chemistry and chemical biology, harvard university, 2 howard hughes medical institute, harvard university, 3 transgenic crops have radically reshaped the agricultural landscape. since their introduction in the late 1990s, transgenic crops have affected economic gains greater than us$110 billion globally due to reduced production costs and increased yield gains. crops modified to produce biological insecticides derived from the soil bacterium bacillus thuringiensis (bt) are among the most robust methods of pest control. bt toxins offer many advantages over traditional insecticides, chiefly their inability to affect human biology and exquisite selectivity for defined pest species. however, the evolution of resistance to bacillus thuringiensis oendotoxins (bt toxins) in insects has been widely observed in the field, and greatly threatens the use of this mechanism of pest control in the future. we developed a phage-assisted continuous evolution (pace) platform for the rapid generation of high-affinity protein-protein interactions and validated the system by evolving known high affinity antibody mimetics in <5 days of pace. we applied this system to the evolution of the bt toxin protein cry1ac to recognize a non-cognate cadherin-like receptor from trichoplusia ni, a pest for which bt toxin resistance has been observed in both the laboratory and the field. the resulting evolved cry1ac variants exhibits high affinity for the target receptor, and kill insect cells more potently than wild-type cry1ac. our findings establish that the directed evolution of novel receptor recognition in bt toxins can be used to target resistant pests, and has far-reaching implications for biological reagents and therapeutics. optimization of a designed protein-protein interface brian maniaci 1 , collin lipper 2 , john j. love 1 1 san diego state university, 2 university of california protein-protein interactions play key roles in practically every biological process. protein-protein interactions vary with composition, affinity, and lifetime of the complex. studying designed protein-protein interactions will provide insight into the underlying principles of complex assembly and formation. computational protein docking and amino acid sequence design were used previously to generate protein dimers from monomeric proteins. the normally monomeric b1 domain of streptococcal protein-g (gb1) was computational docked to itself, followed by optimization of the interfacial side chains. two variants, monomera and monomerb, were computationally derived as a result of a designed protein-protein interface. these designed proteins were characterized using analytical ultracentrifugation and heteronuclear nmr techniques. this design resulted in a pair of protein monomers that formed a heterodimer of modest binding affinity. a tetrahedral metal-templated interface design strategy was implemented in an attempt to strengthen the monomera-monomerb complex by introducing cross-monomer metal coordination. another advantage of using the metal-templated interface is the ability to control the protein-protein interaction both temporarily and spatially. a number of newly engineered variants of monomer a and monomer b with metal coordination sites were designed, produced, and tested for increased affinity of the protein-protein complex. while the generation of a metal-templated monomera-monomerb complex was unsuccessful, we were able to obtain monomera variants that form a homodimer assembly only in the presence of zinc (ii) ions. the crystal structures of metal-templated monomera variants in the presence of zinc provide an explanation for the observed dimer formation. the crystal structure indicates that the protein-protein interaction is not driven by the designed protein interface, but rather non-specific association via edge-strand interactions. new variants were designed with the goal of engineering a high affinity homodimer in a helix-to-helix orientation as the originally designed protein-protein interface. current evaluation of monomera variants for self-association via metal coordination are being evaluated using size exclusion chromatography with a multi-angle light scattering detector for oligomerization state quantification. the results of this protein design project should lead to a greater understanding of the biophysical parameters that drive natural protein-protein interactions. continuous evolution of site-specific recombinases with highly reprogrammed dna specificities the ability to precisely modify the genome of human cells has enormous potential as a novel therapy and a powerful research tool. in contrast to reprogrammable nucleases, such as talens or a cas9/ sgrna pair -which specifically cleave dna but then rely on stochastic host cells processes to effect gene insertion -site specific recombinases directly catalyze genomic integration with high efficiency. a major limitation of this approach is that recombinases, such as cre, natively bind with high specificity to long dna target sequences (loxp in the case of cre) that do not exist in the human genome. previous attempts at evolving cre resulted in modest changes to its specificity, or required hundreds of rounds of manual protein evolution. we developed and validated a phage assisted continuous evoluiton (pace) selection for rapidly altering the dna specificity of cre recombinase towards a site present in a human genomic safe harbor locus. the pace experiments resulted in cre variants capable of recombining a substrate with nearly 50% of the nucleotides altered compared to loxp. we successfully used one of these variants to integrate exogenous dna into the genome of unmodified human cells. we are currently using sequencing methods to determine the specificity of the new recombinase clones. aleardo morelli 1 , burckhard seelig 1 1 generation of comprehensive deletion libraries mediated by in vitro transposition analysis of protein enzymes and ribozymes from nature, and from in vitro evolution, revealed that deletions of up to dozens of amino acids (or nucleotides) can be structurally tolerated. furthermore, shortened variants can exhibit better stability and increased catalytic activity. in order to investigate the effects of deletions, we developed a new procedure based on in vitro transposition to build libraries of more than 10,000 deletion mutants in three to four days. we tested our procedure on dna sequences coding for an artificial rna ligase called ligase 10c. we used the generated library for an mrna display selection, and isolated two active mutants containing 18 and 13 amino acids n-terminal deletions. structural characterization of ppsc, a multi-domain polyketide synthase from mycobacterium tuberculosis using a fragment-based approach alexandre faille 1 , nawel slama1 1 , anna grabowska 1 , david ricard 1 , annaik qu emard 1 , lionel mourey 1 , jean-denis pedelacq 1 1 polyketide synthases are of great interest in numerous scientific fields. they are composed by multiple domains, each having a different role to play in the catalysis of sequential reactions including condensation, reduction and esterification. their reaction products, named polyketides, represent a large variety of chemical compounds, from antibiotics to immunosuppressors or even anticancer drugs. ppsc is a 231 kda polyketide synthase, organised into six catalytic domains (ks-at-dh-er-kr-acp) with singular functions. along with other type i polyketide synthases, ppsc is responsible for the biosynthesis of an essential polyketide for the virulence of mycobacterium tuberculosis (mtb) and thus is a target of choice for the design of inhibitors. to date, no structural information of any type i polyketide synthase in its entire form has been described. main reasons are the length of these large size enzymes and the flexibility imposed by the linkers between domains, thus making them very difficult to crystallize. numerous questions about domain-domain interactions, spatial arrangement of this complex machinery, substrate specificity and stereochemistry are still unanswered. addressing the structural and functional characterization of ppsc would then help answering these questions and provide valuable information for drug design. to overcome the length-and flexible-dependent problem originating from the presence of multiple domains and linkers, we decided to study domains expressed alone. for this purpose, we used our domain trapping strategy to identify soluble fragments representing a single domain from ppsc [1] . it has the advantage of not relying on the bioinformatically designed domain boundaries and can even sometimes include parts of linkers to obtain more soluble fragments. using this strategy, we were able to identify relatively small and highly soluble fragments representing each domain of ppsc, thus facilitating the downstream structural and functional characterization. more than 20 fragments have been submitted to crystallization trials. among these, 5 gave crystals and allowed us to determine the x-ray structure of ppsc at, er, in addition to the dh domain in complex with a substrate analog for which activity was confirmed in vitro. the computational design of proteins that bind small molecules remains a difficult challenge in protein engineering. the ability to computationally design native-like interactions with high accuracy and efficiency would be an asset towards therapeutic development, enzyme design, and engineering functional proteins. we have developed a systematic approach to designing interfaces. we first identify ligands with naive binding affinity to our protein scaffold, then use rosettaligand to computationally dock the ligand while designing the interface for a tighter interaction. this way, we are taking a 'shot in dim light' for design as opposed to a 'shot in the dark', allowing us to more thoroughly investigate the successful and not-so-successful designs, and improve the computational methods. of 3500 ligands screened, we identified 28 weakly-binding hits in the range of 340 -1110 mm. thus far, rosettaligand has successfully designed one tighter protein-ligand interface, from 312 mm to 21 mm. in progress experiments include designing and experimentally validating more designed interfaces. structural studies of human acidic fibroblast-growth factor (fgf1) mutants with a probable anticancer activity lectins are carbohydrate-binding proteins ubiquitously present in nature. they play a role in biological recognition phenomena involving cells and proteins. the interaction lectin-carbohydrate is highly specific, and can be exploited for the development of nanoparticles containing on their surface lectins specifically directed to carbohydrate residues present only on malignant cells and absent on healthy ones (1) . lectins have been found to possess anticancer properties and they are proposed as therapeutic agents, binding to cancer cell membranes or their receptors, causing cytotoxicity, apoptosis and inhibition of tumor growth. some lectins are able to prevent the proliferation of malignant tumor cells because they recognize the t-antigen (gal b 1-3galnac) found specifically on the surface of tumor cells (2) . the main problem is that their use as a detection agent for the t-antigen in clinical studies is not possible because the immune system can recognize them as foreign molecules and develop an immune response. previous studies with x-ray crystallography made in our laboratory have characterized a lectin found in mushrooms called bel b-trefoil which has antiproliferative activity on tumor cell lines, because it contains three binding sites for the t-antigen. unlike other lectins with this property, bel b-trefoil shows structural homology with a human protein, acidic fibroblast growth factor (fgf1) (3). superposition of their structures suggests that the human protein could be mutated to contain at least one of the binding sites for the t-antigen. such mutations should create in fgf1 the potential capacity of recognizing tumor cells with less immunogenicity than the fungal protein. fgf1 is mitogenic and chemotactic, and mediates cellular functions by binding to transmembrane receptors, which are activated by ligand-induced dimerization requiring heparin as co-receptor. to reach our purpose, the fgf1 cdna was cloned into a bacterial plasmid and then mutated in five different positions to eliminate its mitogenic activity and to engineer in the protein the t-antigen binding capacity. attempts to crystalize the mutants of fgf1 were made using the hanging drop technique with the final aim to carry out their structural characterization by x-ray diffraction analysis of the crystals. the de novo synthesis of proteins in response to the activation of cellular signaling pathways is a crucial element of many high-level biological processes, including the synaptic plasticity underpinning memory formation in the brain. while of fundamental biological importance, there has been a shortage of tools with which to specifically target pools of newly synthesized proteins of interest for study. thus, we have developed timestamp and smash, methods for drug-dependent tagging, or destruction, respectively, of newly synthesized copies of proteins of interest. both methods rely on protein tags that remove themselves by default via an internal hepatitis c virus (hcv) ns3 protease, but which are retained in the presence of cell-permeable small molecule protease inhibitors. the timestamp tag contains split yfp halves and epitope tags which are reconstituted and preserved, respectively, on proteins of interest following drug application, whereas the smash tag contains a strong degron which remains attached to proteins of interest following drug application, resulting in their clearance. one limitation of time-stamp and smash is that they can only be used to independently manipulate one protein of interest at a time. furthermore, the application of timestamp and smash to study endogenous protein pools in mammals has not yet been explored. here, we report on efforts to extend these techniques by reengineering ns3 proteases which can be inhibited by two different drugs orthogonally to one another. by incorporating different drug resistance mutations into two ns3 protease variants, we engineered ns3 protease domains that are inhibitable either by asunaprevir only, or by telaprevir only. we found that these tags permit simultaneous and independent control over the newly synthesized pools of two proteins of interest within the same population of cells. we also report the development of transgenic knock-in mouse strains incorporating timestamp and smash tags, which allow the interrogation of newly synthesized pools of specific endogenous synaptic proteins in the context of their endogenous regulatory elements, and without relying on overexpression. infectious diseases are often diagnosed by the presence of specific antibodies that are produced in response to the invading pathogen. one example are antibodies that are present in patient blood after infection with the dengue virus serotype 1 and that are directed against an epitope on the virus' nonstructural protein 1 (ns-1). traditional antibody diagnosis relies on time-consuming multi-step assays that require sophisticated equipment in a laboratory environment. a promising alternative are protein switches that are based on bioluminescence resonance energy transfer (bret). these switches comprise a luciferase (nanoluc) and a green fluorescent protein (mneongreen), which are connected via a semiflexible linker. the linker contains two epitope sequences of ns-1 to which the antibodies bind specifically. if no antibodies are present nanoluc and mneongreen are held in close proximity via two helper domains and bret can occur; thus green light originating from mneongreen is visible. if antibodies are present, they bind to the specific epitopes in the linker of the switch and cause stretching of the linker and therewith break the interaction of the helper domains. as a result, nanoluc and mneongreen are separated in such a way that bret cannot occur anymore; thus only blue light originating from nano-luc remains visible. using this principle, monoclonal anti-ns-1 antibodies were detectable in a controlled buffer system and in spiked plasma samples. furthermore, the developed antibody switch was applied to plasma samples of macaques after a primary infection with dengue virus serotype 1. signal readout was possible using a laboratory-based plate reader as well as the camera of a standard smartphone. we demonstrate that this bret-based protein switch can quickly detect antibodies in solution in a single-step assay format using simple equipment for signal readout, such as a standard smartphone. this simplified antibody detection platform has the potential to be carried out outside of a laboratory, thus in areas with limited laboratory infrastructure and a high number of diverse infectious diseases. proteins expressed from more than two-thirds of the human genome reside within intracellular compartments. of these proteins many are important disease-related targets such as kras and c-myc which cannot be easily addressed by conventional small molecule approaches. some of the weaknesses of small molecules can be addressed by biologic drugs, for example high target specificity and inhibition of protein-protein interactions. the challenge for biologics is how to engineer recombinant proteins to access the intracellular space. one strategy is to use systems evolved by bacteria and viruses to deliver material inside the cells. an example of such pathway is used by pseudomonas exotoxin a (pe). the modularity of pe allows the catalytic domain to be replaced with a biologic payload against desired intracellular target. an additional benefit of pe-based delivery is a possibility of targeting the drugs only to relevant cells in the body by modifying the cell-targeting domain of the pe. the aim of this project is to deliver functional payloads against k-ras and c-myc into the cell using a pseudomonas exotoxin a translocation domain. we used phage and ribosome display to select antibody mimetics that bind k-ras and c-myc. here, we present their activity in biochemical assays and the initial results on generation of pe-based constructs. (1) . hemagglutinin is synthesized as ha0 molecule assembled as noncovalently bound homotrimers on the viral surface. this precursor protein is cleaved by trypsin-like proteases to yield two subunits ha1 and ha2 linked by a single disulphide bond (2) . ha0 is also post-translationally modified by n-glycosylation (3). it is well established that the virus hemagglutinin is the main antigen, inducing the neutralizing antibodies. in the attempt towards developing influenza vaccine production (the egg-based manufacturing lasts several months) that would be faster and safer the utilization of recombinant antigen alone is currently being observed. recently we demonstrated that yeast produced influenza h5 protein although cleaved into two subunits induced strong immunological response in mice (4) . in this report, we describe the biochemical and immunological characterization of the h5 antigen, based on hydrolytic domain of the h5n1 gene, with deletion of multibasic cleavage site and expressed in yeast system. the ha encoding gene from h5n1 virus with deletion of 18 nucleotides was cloned into ppiczac vector. rha fusion protein with his6-tag was secreted into the culture medium and was purified to homogeneity in one step using ni-nta agarose. the efficiency of the antigen purification was 200 mg/l. glycosylation sites of rha were determined using lc-ms-ms/ms. analysis of the n-linked glycans revealed that the rha is glycosylated at the same sites as the native ha in the vaccine strain. next we investigated if the hemagglutinin with deletion of the cleavage site oligomerize into higher molecular forms. to determine the oligomeric forms of the recombinant antigen various approaches were applied e.g. native-page, size exclusion chromatography or dynamic light scattering. as a final experiment to measure the size of oligomers in a protein sample a combined technology sec-mals was conducted, using multi angle light scattering (mals) as a detector. the immunological activity of rha was tested in chicken and mice, where antigen elicited high immune response. the data presented here demonstrate that new influenza antigen produced in p. pastoris is highly immunogenic and might be consider as a candidate for subunit vaccine. structural motifs capture redundant patterns that frequently occur in proteins. motifs associated with contiguous fragments of structure (i.e., secondary structural motifs) are well studied and have been successfully used to capture "rules" describing sequence-structure relationships in protein design and structure prediction. we have extended this concept to motifs that capture tertiary information-(i.e., tertiary structural motifs or terms. we have discovered that a relatively small alphabet of terms describes the known structural universe (all secondary, tertiary and quaternary information in the pdb) at sub-angstrom resolution. this alphabet of universal motifs reveals the remarkable degeneracy of the protein structure space, with just a few hundred terms sufficient to accurately capture half of the known structural universe. we have begun to demonstrate the considerable promise this structural alphabet has for applications such as protein design, structure prediction, and docking. we have developed a novel protein design framework that selects amino acid sequences, given a desired structure, using solely information from the universal terms. we show that given a native backbone, this framework recovers the native sequences to a level on par with state-of-the-art atomistic protein design methods, indicating that the motifs capture the salient structural rules governing native proteins. further, predicted sequence distributions agree closely with observed evolutionary variation. given the apparently high degeneracy among even complex features of protein structure, methods based on mining the pdb for tertiary information should provide ample opportunities for advancement in problems of computational structural biology. sortase-mediated synthesis of protein-dna conjugates for sensitive biosensing bedabrata saha 1 , marieke op de beeck 1 , remco arts 1 , maarten merkx 1 1 in recent years, semisynthetic protein-dna conjugates have emerged as attractive biomacromolecules for different applications in bio-nanotechnology, biosensing, diagnostics and therapeutics. in protein-dna conjugates, synthetic oligonucleotides allow the construction of desired molecular architecture with high specificity, while maintaining the original functionality of the protein molecules for desired application. however, the synthesis of site-specific and stoichiometric protein-dna conjugates can be challenging. due to the diversity in composition and physico-chemical properties of the proteins, few generic strategies are available for conjugation of protein molecules to a dna scaffold. a common approach is to use thiol-based covalent conjugation, but the introduction of additional cysteines can lead to the formation of intermolecular disulfides or interfere with the formation of native disulfide bonds. as an alternative, here we have developed a site-directed protein-dna conjugation strategy based on sortase mediated trans-peptidation reaction. the sortase recognizes a 'sorting motif' (i.e. lpxtg, x any amino acid), which is recombinantly introduced by site-directed mutagenesis at the cterminal end of the protein molecule. the sortase cleaves the t-g peptide bond and catalyzed the formation of a new amide bond between the lpxt peptide and the n-terminal amine of any molecule bearing an n-terminal oligoglycine motif. for this purpose, a triglycine motif was introduced at the 5'end of single-stranded dna (ssdna). on-column synthesis of triglycine modified ssdna, protected on a controlled pore glass beads, simplified the purification process and enhanced the yield of triglycinemodified ssdna (> 90%). we used this conjugation strategy in several biosensing applications. for example, we used the method to conjugate ssdna linkers at the c-termini of a range of single-chain antibody fragments (scfv) and applied these constructs to allow oriented display of capture molecules on biosensor surfaces. ssdna-scfv were using an excess of triglycine modified ssdna, we achieved 55% conversion scfv-ssdna conjugate, which can be further purified by in two step purification process consisting of ni-nta affinity column and ion-exchange chromatography. we also extended this sortase-based conjugation strategy to develop a bioluminescence based assay for sensitive target oligonucleotide detection. in this regard, the 5' and 3' end triglycine-modified ssdna molecules were successfully conjugated with a bret protein pairs, nanoluc luciferase and mneongreen fluorescent protein. the introduction of a c-terminal sortase-his5 tag and and n-terminal strep-tag allowed efficient purification of theseprotein-ssdna conjugates from excess oligonucleotides and unreacted protein. mass spectrometry based proteomics to identify the protein differences in human breast milk from breast cancer patients and controls devika channaveerappa 1 , roshanak aslebagh 1 , kathleen f. arcaro 2 , costel c. darie 1 1 breast cancer is the second leading cause of cancer death in women. about 12% women in the us develop breast cancer. death rates due to breast cancer have been declined over the years due to advancements in mammography and treatment. although, mammography helps in the early detection of breast cancer, it has few limitations. dense breast tissue makes mammogram less accurate. breast milk can be assessed to evaluate the risk of one getting breast cancer by comparing the proteomes of breast milk from healthy and breast cancer suffering individual. this study makes use of mass spectrometry based proteomics to identify the differences between the control and cancerous samples which would further help in identifying potential biomarkers for breast cancer. firstly, sds-page was used to separate the proteins from the whole milk sample. the gel bands for each sample was then excised and cut into small pieces. the gel pieces were washed and trypsin digested in order to extract the peptides. peptide mixtures in the solution were cleaned using c18 zip-tipp and then analyzed by liquid chromatography-tandem mass spectrometry (lc-ms/ms). 60 minutes and 120 minutes gradient were used for lc-ms/ms analysis. raw data obtained were converted to pkl files using proteinlynx global served (plgs version 2.4). raw data were then submitted to mascot database search for protein identification. the mascot results were then exported as .dat files and further analyzed using scaffold version 4.1 software. three breast cancer milk samples were investigated against healthy control milk samples. in the sds-page gel, after coomassie staining, the protein patterns did show minor differences. after lc-ms/ms analysis, the proteins identified by mascot database search were imported into the scaffold software and compared for the relative ratio between the proteins from the milk sampled from control donors and the donors with breast cancer. there were significant differences identified in the proteomes of the two sets of samples. some of the proteins were upregulated in the breast cancer samples and some were down regulated when compared with the controls. additional investigation of more breast milk samples is ongoing. this study focuses on identifying biomarkers directly in the milk of donors with breast cancer. leukolike vectors: leukocyte-inspired nanoparticles claudia corbo 1,2 , alessandro parodi 1,2 , roberto palomba 1,2 , roberto molinaro 1 , michael evangelopoulos 1 , francesco salvatore 2,3 , ennio tasciotti 1 1 the houston methodist research institute, 2 fondazione irccs sdn, 3 nanomedicine aims to improve drug efficiency by enhancing targeting and biocompatibility, and reducing side effects. multiple surface modifications have been proposed to provide nanocarriers with these features, based on complex synthesis processes and very often inefficient in contemporary providing biological tolerance and targeting properties [1] . bio-inspired approaches based on surface coatings developed from the purified cell membrane of immune cells represents a new paradigm shift for the development of carrier enable of prolong circulation and proper tumoritropic capabilities. we showed that nanoporous silicon (nps) particles coated with leukocyte cellular membranes -leukolike vectors (llvs) -possess cell-like properties [2] . llvs can escape macrophage uptake, delay sequestration by the reticulo-endothelial system, target tumor inflamed vasculature and accumulate within the cancer parenchyma [2] . llvs were fully characterized for their shape, size, surface charge and coating through dynamic light scattering and scanning electron microscopy. in addition we characterized the content and function of the leukocyte's proteins transferred onto the llvs coating through high-throughput proteomic analysis and the results revealed the presence and the correct orientation of several important markers of leukocytes: cd45, cd47 and mhc-i were identified as key players in determining llvs biocompatibility, while leukocyte associated function-1 (lfa-1) and mac-1 contributed to the llvs targeting ability and bioactivity towards inflamed endothelium [3] . recent investigation showed that the coating induced the formation of a singular protein corona (i.e. the protein adsorption layer) on the surface of the nanoparticles compared to negative control following in vivo injection. in addition, the proteolipid coating favored active extravasation of the llvs in the tumor vasculature by molecular mechanisms similar to those used by tumor infiltrating leukocytes. this work shows that is possible to transfer biologically active leukocyte membrane proteins onto synthetic nanoparticles, thus creating biomimetic carriers retaining cell-like functions that are not affected by the protein corona effect that occurs in vivo. the targeting of the inflamed endothelium can be applied to a broad range of diseases and the approach used to formulate the system could open new avenues for the fabrication of the next generation of personalized treatments by using as cell membrane source the immune cells of patients. references: [1] alessandro parodi, claudia corbo, armando cevenini, roberto molinaro, roberto palomba, laura pandolfi, marco agostini, francesco salvatore, ennio tasciotti. enabling cytoplasmic delivery and organelle targeting by surface modification of nanocarriers. nanomedicine uk. accepted. steroid hormone receptors are intracellular receptors that initiate signal transduction in response to steroid hormones, including oestrogen and androgens. generally, the binding of the steroid to the nuclear receptor induces the protein to form a dimer and relocate onto the chromatin, although the order of these events may vary. the location of receptor binding on the chromatin is defined by specific hormone response elements (hre). once located, the receptor promotes gene activation by the recruitment of other co-factors. it is this process that makes the complex of receptor protein and co-factors play a pivotal role in the regulation and activation of genes. the failure to regulate this process correctly is a key step in the development of several endocrine-driven cancers. for example: oestrogen receptor positive (er1) breast cancer is one of the most common forms of cancer and accounts for 70% of all breast cancer cases. in er1 tumours, the oestrogen receptor (er) drives the tumour growth and cell proliferation. understanding the interactions of the er with other proteins, either directly or indirectly, can provide vital insight to the regulation of the system that drives this cancer. the progesterone receptor (pr) has also been implicated in breast cancer, and the androgen receptor (ar) is a known driver in the majority of prostate cancers. to meet the challenges of elucidating these systems, we have developed methods to purify and analyse cross-linked regulatory complexes bound to dna by mass spectrometry (chip-ms). this allows for the enrichment of proteins involved in gene regulation. chip-ms, combined with tandem mass tags (tmt), makes it possible to realise a quantitative method to investigate the dynamic network of interactions between proteins within complexes that undertake the regulation of biological systems. chip-seq is a well-established method for identifying where these protein complexes are bound to the genome. this work focuses on how to combine these technologies with my previous development of cross-linking coupled mass spectrometry techniques (xcms) to provide a strategy for visualising the dynamic organisation of the proteins on the chromatin. global kinetic analysis of caspase protein substrates in cell lysate reveals selective roles and target specificity olivier julien 1 , min zhuang 1 , arun wiita 1 , james wells 1 1 caspases are cysteine proteases that play important roles in development, cell differentiation and cell death. however, the limited number of known caspase substrates hinders our understanding of caspase function. here we performed a non-biased identification and kinetic analysis of caspase-2 and caspase-6 proteolytic substrates in cell lysate, using an enzymatic n-termini enrichment approach followed by mass spectrometry. we identified 235 and 871 potential substrates for the initiator caspase-2 and putative executioner caspase-6, respectively. our results not only confirm known substrates but also identify many more new substrates with the precise location of proteolysis. given the emerging roles of caspases-2 and 26 in inflammation and neurodegeneration, these new substrates may provide molecular insight into the progression of related diseases. the sequence consensus logo of caspase-2 targets was very similar to a classical executioner caspase motif (devd), while caspase-6 revealed a vevd motif. using selected reaction monitoring (srm), we quantified the kinetics of proteolysis of a large subset of these substrates by measuring the appearance of the caspase cleavage product over time. in the end, we measured 50 and 276 kcat/km values for individual substrates cut by caspase-2 and caspase-6, respectively. by comparing these data with our previous analysis of caspase-3, 27, and 28, we found that substrates that are shared between caspases are often cleaved at rates that differ by orders of magnitude. thus, despite having nearly identical primary sequence motifs, the caspases exhibit remarkable substrate specificity that may reflect their specialized roles within the cell. the rockefeller university, 2 new york university school of medicine, 3 johns hopkins university school of medicine line-1 (l1) retrotransposons are catalysts of evolution and disease whose sequences comprise a significant proportion of the human genome. despite tremendous influence on genome composition, l1 rnas only encode two proteins. consequently, l1 particles include a combination of permissive host factors that are essential to their lifecycle as well as repressive factors that constitute defenses against l1's mutagenic activity. we previously characterized host proteins associated with synthetic and natural human l1 retrotransposons, as expressed in cell culture, using a combination of techniques including metabolic labeling and affinity proteomics. to build on these analyses, we have implemented a series of 2d separations and post-purification treatments to produce a multi-dimensional interactomic characterization of affinity isolated l1s. these studies have revealed the presence of at least two populations of putative transposition intermediates that may exhibit distinctive intracellular localizations. we report a comprehensive, quantitative survey of the proteins partitioning within these distinct l1 populations and their associated in vitro activity. our observations provide a basis for the classification of l1 interactors with respect to their physical and functional links, facilitating hypotheses to direct in vivo experimentation. polyubiquitin recognition by continuous ubiquitin binding domains of rad18 probed by modeling, small-angle x-ray scattering and mutagenesis sangho lee 1 , trung thanh thach 1 , namsoo lee 1 , donghyuk shin 1 , seungsu han 1 , gyuhee kim 1 , hongtae kim 1 1 rad18 is a key protein in double-strand break dna damage response (ddr) pathways by recognizing k63-linked polyubiquitylated chromatin proteins through its bipartite ubiquitin binding domains ubz and lrm with extra residues in between. rad18 binds k63-linked polyubiquitin chains as well as k48linked ones and mono-ubiquitin. however, the detailed molecular basis of polyubiquitin recognition by ubz and lrm remains unclear. here, we examined the interaction of rad18(201-240), including ubz and lrm, with linear polyubiquitin chains that are structurally similar to the k63-linked ones. rad18(201-240) binds linear polyubiquitin chains (ub2, ub3, ub4) with similar affinity to a k63-linked one for diubiquitin. ab initio modeling suggests that lrm and the extra residues at the c-terminus of ubz (residues 227-237) likely form a continuous helix, termed 'extended lr motif' (elrm). we obtained a molecular envelope for rad18 ubz-elrm:linear ub2 by small-angle x-ray scattering and derived a structural model for the complex. the rad18:linear ub2 model indicates that elrm enhances the binding of rad18 with linear polyubiquitin by contacting the proximal ubiquitin moiety. consistent with the structural analysis, mutational studies showed that residues in elrm affect binding with linear ub2, not monoubiquitin. in cell data support that elrm is crucial in rad18 localization to dna damage sites. specifically e227 seems to be the most critical in polyubiquitin binding and localization to nuclear foci. finally, we reveal that the ubiquitin-binding domains of rad18 bind linear ub2 more tightly than those of rap80, providing a quantitative basis for blockage of rap80 at dsb sites. taken together, our data demonstrate that rad18(201-240) forms continuous ubiquitin binding domains, comprising ubz and elrm, and provides a structural framework for polyubiquitin recognition by rad18 in the ddr pathway at a molecular level. optimization of a protein extraction method for the proteomic study of pozol cynthia teresa leyva-arguelles 1 , carmen wacher 2 , rosario vera 3 , romina rodr ıguez-sanoja 1 1 instituto de investigaciones biom edicas, unam., 2 facultad de qu ımica, unam., 3 instituto de biotecnolog ıa, unam key words: proteomics, fermentation, pozol pozol is a mexican traditional no alcoholic beverage elaborated by various ethnic groups in the southeastern of mexico. pozol is obtained from the natural fermentation of nixtamal (heat-and alkali-treated maize) dough. the main carbohydrate in maize dough is starch (72-73%), because others such as sucrose, glucose and fructose are mostly lost during nixtamalization; so, the starch remains as the major carbohydrate available for fermentation [1] . a wide variety of microorganisms have already been isolated from the fermentation of pozol; these microorganisms include fungi, yeasts, lactic acid bacteria, and non-lactic acid bacteria [2] . however, only few bacteria are amylolytic in this fermentation and all of them are weakly amylolytic [1] . in an attempt to explain how a very low content of soluble sugars can support a diverse and abundant microbiota, a proteomic approach was designed to understand the fermentation of pozol [3] . nevertheless, the extraction of proteins from pozol remains a limiting step in proteomic analysis mainly due to the complexity of the sample. on the basis of the aforementioned reasons, the aim of this work was to obtain a suitable extraction method of proteins for proteomic analysis. therefore, the fermentation of pozol was continued for 48 h and samples were taken at 0, 9, 24 and 48 h. for each sample, the total sugar content was determined by the dubois et al. method [4] and protein extraction was performed by two methods: a) direct extraction from the dough [3] and b) initial extraction of microorganisms and soluble proteins (this work). comparison between the two protein methods was performed on two-dimensional gels with silver stain. then, gels underwent to image analysis by the image master 2d platinum software. comparing the 2d-gels, more proteins spots were obtained with method b than that with method a, indicating a more efficient protein extraction with method b. although, using method a higher concentration of total proteins was observed, they were mostly maize proteins, that in turn overlap and reduce the efficiently extraction of the microbial low abundant proteins. then, method b allows a better extraction of those low abundant proteins and removes sample components that may interfere with the determination. these results could help us to find the proteins involved in carbohydrate metabolism of the microbiota and finally elucidate the dynamics of pozol fermentation. proteomics has been applied to the enology field for numerous purposes including fermentation control, improvement of fermentation processes, ensuring wine quality, etc. according to rodriguez et al., (2012), the information provided by wine proteomics is not only useful for these intentions, but also offers excellent prospects for innovation and diversification of winemaking processes in the near future. in this context, our group has focused research on the identification of proteins that might be important for yeast survival under typical wine elaboration conditions (standard fermentation, sherry wine biological aging and sparkling wine second fermentation) as well as proteins that configure the content of metabolites which are ultimately responsible for wine quality. by using novel proteomic (offgel fractionator and ltq orbitrap xl ms) and metabolomic techniques (sbse-td-gc-ms) we have identified a high amount of up-regulated proteins involved in processes like oxidative stress response (in biological aging) or protein biosynthesis (in second fermentation) as well as thirty-three proteins directly involved in the metabolism of glycerol, ethanol and seventeen aroma compounds excreted by the yeast under biological aging conditions. further, in order to validate proteome data; null mutants of genes codifying proteins up-regulated in the biological aging condition were constructed. analyses of correlated phenotypes are in progress. this technique and its combination with metabolomics within the enology context will provide enough knowledge to design or choose yeasts or conditions that satisfy wine production and/or wine characteristics such as color/aroma/texture/flavour profile demands of winemakers and consumers. additional binding sites for cytochrome c on its redox membrane partners facilitate its turnover and sliding mechanisms within respiratory supercomplexes blas moreno-beltr an 1 , antonio d ıaz-quintana 1 , katiuska gonz alez-arzola 1 , alejandra guerra-castellano 1 , adri an vel azquez-campoy 0 , miguel a. de la rosa 1 , irene d ıaz-moreno 1 1 ibvf, ciccartuja, universidad de sevilla -csic, 2 bifi -iqfr (csic), universidad de zaragoza, 3 departamento de bioqu ımica y biolog ıa molecular celular, universidad de zaragoza, 4 gliding mechanisms of cytochrome c (cc) molecules have been proposed to shuttle electrons between respiratory complexes iii and iv within plant and mammalian mitochondrial supercomplexes, instead of carrying electrons by random diffusion across the intermembrane bulk phase [1] [2] . in this work, the binding molecular mechanisms of the plant and human cc with mitochondrial complexes iii and iv have been analyzed by nuclear magnetic resonance and isothermal titration calorimetry. our data reveal that both cc-involving adducts possess a 2:1 stoichiometry -that is, two cc molecules per adduct -. the presence of extra binding sites for cc at the surfaces of complexes iii and iv opens new perspectives on the mitochondrial electron transport chain, where membrane respiratory complexes can be either in independent, free diffusional motion or forming macromolecular assemblies. in the latter context, such new binding sites for cc facilitate the turnover and sliding mechanisms of cc molecules within supercomplexes. indeed, the accommodation of several cc molecules between complexes iii and iv in supercomplexes provide a path for cc diffusion from complex iii to iv. such path could have physiological significance in the electron flow, which is controlled in supercomplexes to optimize the use of available substrates [3] [4] [5] . can bio-functionalities be deciphered from protein sequence information using computational approaches? background: the processes of uncovering bio-functionalities such as pharmacological activities, disease processes, physiological and structural properties by means of clinical approaches are irrational. this is because they are resource and time consuming. sometimes, they involve sophisticated and expensive equipments, reagents and animal tissues. contrarily, sequence information-based computerized approaches are rational and have become relevant in assessing bio-functionalities. they include geno2pheno [coreceptor] [1] , position-specific scoring matrix (pssmsi/nsi and pssmcxcr4/ccr5) [2] , and informational spectrum method (ism)-based phylogenetic analysis (istree) [3] . aim: this presentation demonstrates how bio-functionalities could be deciphered from sequence information using computational approaches. method: ism procedure and peptides, vipmfsals and capagfail are engaged. results: protein sequences of the peptides are converted into bio-functionality (affinity). affinity between the two peptides is demonstrated as significant amplitudes at the point of common interaction also referred to as consensus frequency, signifying remarkable affinity. discussions: bio-functionalities of bio-molecules are known to be expressed in one or two genes, which have been found to provide as much biological information as the bio-molecules. this indicates that biological characteristics, represented in these genes and proteins can now be extracted from their sequence information. for example, multi-drug resistances arising from a variety anti-microbial agent from several classes including alkaloids, flavonoids, etc can be retrieved from the sequence information of their encoding genes (mdr1 and mdr11). similarly, translation of hiv infection to aids disease can be extracted from the protein sequence alterations in the hiv gp120. similarly, effectiveness of anti-retroviral agent, maraviroc on the hiv isolate h2bx2 and ndk can be deciphered from the sequence information of their v3 observed at the predicted sequences. these positions are important as they surround the cleavage site in the three-dimensional structure, and are probably less tolerant to change. moreover in previous studies, cys at p1 position has been shown to be the dominant determinant for cleavage efficiency, while cys, pro and glu at p2 position have also been shown to be correlated with increased cleavage efficiency of ns3/4a protease. for adv2 cysteine protease, on the other hand, bsst produces similar significant results for both type 1 (xgx-g) and type 2 (xgg-x) consensus cleavage sites, where p2 and p1' positions have gly with highest percentage in type 1 (xgx-g) while p2 and p1 positions have gly in type 2 (xgg-x). these indicate that the bsst seems to provide a powerful methodology for predicting the substrate specificity for the hcv ns3/4a serine protease and adv2 cysteine protease, which are targets in drug discovery studies. protein plasticity improves protein-protein binding description chiara pallara 1 , juan fern andez-recio 1 1 an accurate description of protein-protein interactions at atomic level is fundamental to understand cellular processes. however the current structural coverage of protein-protein interactions (i.e. available experimental structures plus potential models based on homologous complex structures) is below 4% of the estimated number of possible complexes formed between human proteins.1,2 for these reasons, computational docking methods aim to become a complementary approach not only to solve the structural interactome but also to elucidate the basis of the protein-protein association mechanism. in spite of the advances in protein-protein binding description by docking, dealing with molecular flexibility is a major bottle-neck, as shown by the recent outcomes of the capri (critical assessment of prediction of interactions) experiment.3 this data clearly confirms that the protein dynamics plays a key role in protein-protein association. the use of conformational ensembles generated from unbound protein structures in combination with computational docking simulations might represent a more realistic description of protein-protein association. here, we present the first systematic study about the use of precomputed unbound ensembles in docking, as performed on a set of 124 cases of the protein-protein docking benchmark 3.0.4 the primary aim of our work is to understand the role of the protein conformational heterogeneity in protein-protein recognition. to do this, small conformational ensembles were automatically generated starting from the unbound docking partners, and then an extensive analysis of their binding properties was performed in the context of pydock docking scheme. 5 the results show that considering conformational heterogeneity of interacting proteins can improve docking description in cases that involve intermediate conformational changes in the unbound-to-bound transition. more interestingly, we found that protein plasticity increases chances of finding conformations with better binding energy, not necessarily related to bound geometries. the relevance for future docking methodology development and for understanding protein association mechanism will be discussed. purpose of the research: there is increasing interest in the development of protein scaffolds that can be used to develop affinity reagents that are alternatives to antibodies. the affimer scaffold is based on the cystatin protein fold. the affimer scaffold is biologically inert, biophysically stable and capable of presenting a range of designed or random binding surfaces defined by peptides inserted at 2 different loops. the result is highly specific, high affinity interactions with a wide range of targets including ones that are inaccessible to antibodies. affimers are designed to work in the same way as the very best antibodies, but with a number of key advantages. affimers are quick to develop (typically 7 weeks) without using animals. they contain no disulphide bonds, are expressed easily in e. coli and have no batch to batch variability. affimers are small molecules (108 aa, 12 kda), robust and stable (resistant to ph range, thermally stable and not sensitive to edta). affimers can be a direct replacement for antibodies -no process or workflow change required -and perform identically to antibodies in assays such as elisa, facs, ihc, western blots, affinity purification, microarray and potentially therapeutics. we describe some applications of the technology in regards of affimer development for custom targets on one hand and for the biomarker discovery workflow using affimer microarrays on the other. main results: by screening of our very large (3 x 1010) library against yeast sumo protein we identified affimers with high affinity allowing their use for elisa. moreover, no cross-reactivity was observed when affimers were used on western blots leading to a unique band specific to yeast sumo when compared to human proteins. a library of 25,000 random affimers, expressed in e. coli, was printed on glass microscope slides and challenged with plasma from children (n5104) with sepsis and from healthy children (n524). unsupervised hierarchical clustering based on the 25,000 affimers allowed differentiation between the control and patient samples. 200 affimers were found to differentially bind proteins between the 2 groups with a > 2 fold change. the affimer arrays identified a strong signature of sepsis and roc curve analysis allowed confident prediction of disease (auroc of 0.9). affinity purification and preliminary mass spectrometry analysis identified known biomarkers of sepsis and also potentially novel biomarkers not previously associated with this disease. major conclusions: this work demonstrates the scope of affimer affinity reagents to develop alternative binders to antibodies, where affimers perform identically in most assays without the disadvantages associated with antibodies. moreover, affimers enable a new protein microarray-based biomarker-discovery workflow and we predict that array-based validation of signatures identified using discovery arrays prior to affinity purification and mass spectrometry will offer a cost-and time-effective methodology compared to purely mass spec-driven workflows. tau pathologies, called 'tauopathies', are related to several neurodegenerative diseases including alzheimer disease (ad). in ad, tau protein is observed hyper-phosphorylated and aggregated as paired helical filament (phf). the neuronal tau protein is an intrinsically disordered proteins (idps). nuclear magnetic resonance spectroscopy (nmr) is here used to study the tau protein phosphorylations and protein-protein interactions (ppis). in in vitro assays, tau phosphorylation by rat brain extract is considered as an hyperphosphorylation model that was furthermore pointed out to enable tau aggregation [1] . in a first step, we have identified all the phosphorylation sites of rat brain extract phosphorylated-tau, using the analytical capacity of nmr. we showed that the protein is modified at 20 ser/thr sites. among the kinases that we have characterized so far using tau as substrate, only the extracellular signal-regulated kinase2 (erk2) shows an ability to modify in vitro tau protein on so many sites. we have indeed identified 14 phosphorylated ser/thr-pro motifs out of 18 potential phosphorylation sites in the sequence of full length 441-residue tau. in addition, we showed using transmission electron microscope (tem) a similar in vitro aggregation capacity of erk-phosphorylated tau protein compared to that of rat brain extract phosphorylated-tau. this shows that phosphorylation by the erk kinase generates an hyperphosphorylated tau. given the high efficiency of erk towards tau, we have next looked into the mechanism of tau recognition. erk kinase possesses two well-characterized docking domains: d recruitment sites (drs) and f recruitment sites (frs), which recruit complementary docking sites and increase the specificity and efficiency of the interaction with both its upstream regulators and downstream substrates [3] . as the interaction between tau protein and erk2 kinase is analyzed by nmr spectroscopy, multiple sites of interaction are observed along the tau sequence, similar to drs docking sites, all located in the so-called microtubule binding domain of tau. these sites are short sequences loosely matching the reported consensus for d sites w1-3uxu (w, u, and x refer to positively charged, hydrophobic, or any intervening residues, respectively) [3] , and also the reverse sequence uxuw 1-3.to confirm the mapping of the interaction, two tau recognition sites were produced as recombinant peptides of about 20 amino-acid in fusion with an n-terminal his-tag sumo. interaction assays using 2d [1h, 15n] hsqc spectra of the peptides confirm their binding to erk kinase. the potential of these peptides to inhibit erk activity with tau as substrate is now being investigated. while rigid-body docking has become quite successful for predicting the correct conformations of binary protein complexes, determining whether two given proteins interact remains a difficult problem. successful docking procedures often give equally good scores for pairs of proteins for which there is no evidence of interaction. studies investigating what we define as the 'pre-docking' problem via in silico approaches have only recently become feasible with the help of supercomputers and gridcomputing systems. in a previous work, on a restricted set of protein complexes, we showed how predictions of interacting partners could be greatly improved if the location of the correct binding interface on each protein was known. experimentally identified complexes are found to be much more likely to bring these two interfaces into contact, at the same time as yielding good interaction energies. we present data from a complete cross-docking (cc-d) study of a database of 168 proteins, including the treatment of more than 14,000 potential binary interactions. the performance of the interaction index we developed to predict binding probability compares well with other methods. by studying the interaction of all potential protein pairs within a dataset, cc-d calculations can also help to identify correct protein interaction interfaces. the present large-scale study also reveals the influence of various protein families (enzyme-inhibitor, antibody-antigen, antigen-bound antibody, etc.) on binding specificity, showing, in particular, the distinctive behavior of antigenic interfaces compared to enzymes, inhibitors or antibodies. the performance of our approach is encouraging. although identifying interaction interfaces significantly helps in the identification of interacting proteins, further refinements will be necessary to make in silico cross-docking a viable alternative to high-throughput experimental methods. whole-protein mass spectrometry reveals global changes to histone modification patterns in hypoxia sarah wilkins 1 , kuo-feng hsu 1 , christopher schofield 1 1 chemistry research laboratory, oxford university cells respond to limiting oxygen availability (hypoxia) by altering the gene expression profile. this primarily involves changes at the level of transcription via the activity of hypoxia-responsive transcription factors, although increasing evidence suggests that changes in chromatin structure (i.e. from a condensed 'silent' state to a more open or 'active' state) are required in order for transcription to take place. in particular, post-translational modifications (ptms) to histones have an important regulatory function in gene expression under hypoxic conditions. the n-terminal tails of histone proteins are accessible to a set of enzymes capable of 'writing' and 'erasing' ptms including acetylation, methylation, ubiquitylation, sumoylation and phosphorylation. to date, studies in hypoxia have employed antibody-based methods to investigate changes in histone modifications, and so have focused on individual marks in isolation. the interplay between coexisting ptms is thought to be much more important than the effect of any single mark. therefore, a global view of the histone modification profile is essential to gain a complete understanding of the function of histone ptms and their roles in gene regulation. in this study, we apply whole protein mass spectrometry to investigate hypoxia-induced changes in histone marks. this 'top-down' approach provides insight into combinational modification patterns that are difficult to establish by antibody-based methods or peptide ms analysis. we investigated changes in the global ptm profiles of histones from a range of human cell-lines and tissues under severe hypoxia (<0.1% o2). we find that hypoxia causes a shift in the overall profile towards a more highly modified state, with significant changes in methylation and phosphorylation. marked changes in histone ptms were also observed following treatment of cells with epigenetic inhibitors and commonly used hypoxia mimetics, including several iron chelators currently in clinical trials for the treatment of anaemia. finally, we show that this method can be used to identify the histone variant h2ax, whose phosphorylation at serine 139 is an indicator of double-stranded dna breaks in cancer. overall, these data provide important insights into the epigenetic changes associated with hypoxia in normal and disease contexts. we hope to further develop this method in combination with different labelling strategies to enable quantitative analysis of histonemodifications in cells. mass spectrometry-based protein biomarker discovery in neurodevelopmental disorders interactions. there is currently no biological diagnosis or known cause of asd. slos is characterized by a cholesterol deficiency due to a mutation on the 7dhcr gene. approximately 1/20,000 babies are born with slos. diagnosis is achieved by measuring cholesterol and 7-dehydrocholesterol (7dhc) levels in the blood, however, there is currently no proven treatment for slos. because of this, research is increasing to determine biomarkers for these disorders. here, samples from people with asd (sera and saliva) and slos (saliva), and matched controls were analyzed using a combination of gel electrophoresis (tricine-page, sds-page and blue native page), in gel digestion or insolution digestion and nanoliquid chromatography-tandem mass spectrometry (nanolc-ms/ms) to investigate differences between the proteomes of people with these neurodevelopmental disorders and matched controls. several alterations in protein expression were identified. these differences may lead to potential biomarkers for diagnosis, possible therapeutic targets and an altogether better understanding of the disorders. understanding protein recognition using structural features protein-protein interactions (ppis) play a crucial role in virtually all cell processes. thus, understanding the molecular mechanism of protein recognition is a critical challenge in molecular biology. previous works in this field show that not only the binding region but also the rest of the protein is involved in the interaction, suggesting a funnel-like recognition model as responsible of facilitating the interacting process. further more, we have previously shown that three-dimensional local structural features (groups of protein loops) define characteristic patterns (interaction signatures) that can be used to predict whether two proteins will interact or not. a notable trait of this prediction system is that interaction signatures can be denoted as favouring or disfavouring depending on their role on the promotion of the molecular binding. here, we use such features in order to determine differences between the binding interface and the rest of the protein surface in known ppis. particularly, we study computationally three different groups of protein-protein interfaces: i) native interfaces (the actual binding patches of the interacting pairs), ii) partial interfaces (the docking between a binding patch and a non-interacting patch), and iii) back-to-back interfaces (the docking between non-interacting patches for both of the interacting proteins). our results show that the interaction signatures in partial interfaces are much less favoured than the ones observed in native and back-to-back interfaces. we hypothesise that this phenomenon is related to the dynamics of the molecular association process. back-to-back interfaces preserve the exposure of the real interacting patches (thus, allowing the formation of a native interface), while in a partial interface one interacting patch is sequestered and becomes unavailable to form a native interaction. structural characterization of the cytoplasmic mrna export platform laboratory of cellular and structural biology, the rockefeller university., 2 laboratory of mass spectrometry and gaseous ion chemistry, the rockefeller univ., 3 university of california, san francisco, 4 the new york structural biology center, 5 department of biochemistry, faculty of medicine, university of montreal mrna biogenesis is an intricate process that begins within the nucleus and culminates with the remodeling and nuclear export of the mrnp particles through the nuclear pore complex (npc). defects in this conserved mechanism have been shown to cause serious human diseases. the protein assembly that performs the last steps in mrnp biogenesis and export is located at the cytoplasmic face of the npc and is formed by 14 different proteins, organized into several subcomplexes whose arrangement and molecular architecture are poorly understood. in this study we applied an integrative approach, combining cross-linking and mass spectrometry (cx-ms), electron microscopy and available high-resolution structures, to describe the molecular architecture of the endogenous npc cytoplasmic mrnp export machinery. we generate a hybrid, close-to-atomic structure of the yeast native nup82 complex, the core of the assembly. our map also reveals how the nup82 complex organizes the entire cytoplasmic mrnp export machinery, and how this in turn docks into the architectural core of the npc. mapping of phenotypic profiles into our structures allows us to generate a first functional map of the ensemble. we expect that our map will serve as a framework to understand the molecular mechanisms underlying this key step of mrnp biogenesis. study of candidate proteins to pore associated with p2x7 receptor in different cell types carla oliveira 1 , anael alberto 1 , mônica freitas 2 , luiz alves 1 1 laborat orio de comunicac¸ão celular -fiocruz, 2 centro nacional de ressonância magn etica nuclear -ufrj aim: the p2x7r is a purinergic receptor, which differs from others subtypes due to its structural and pharmacological characteristics. when exposed for extended time or to high concentrations of its agonist (atp), promotes an increase in membrane permeability, allowing the passage of molecules up to 900da. there is a controversy among several authors that leave in doubt if this receptor needs a second protein for the pore formation and which protein could be. we select five pore-forming proteins: trpv1, trpa1, connexins-43 (cx-43), pannexin-1 (panx-1) and vdac. we believe that different mechanisms and proteins could be associated with p2x7r, depending on the cell type and their microenvironment stimuli. in this context, our main goal is identify possible proteins that could be associated with the p2x7r pore in different cells and species. methods and results: we started with rt-pcr technique of cell lines: j774.g8, n2a, u373, u937, hek-293 and primary cells from wistar mouse and swiss mice. we used different primers and pcr cycle for each target at different species. we observed that the p2x7, panx-1 and cx-43 are the most abundant and are present in all cell types except the absence of p2x7 in u373 cells and panx-1 in mice macrophages and u373 cells (n>3). however, trpv1 was seen at n2a and u937 cells and trpa1 in and primary cells from mouse and mice and in j774.g8 cells (n>3). regarding to the vdac, it is present in mouse macrophages, j774.g8 and hek-293 cells (n>3). the further steps, we verified if those proteins could be physically associated with the p2x7r. we coimmunoprecipitated the p2x7r of j774.g8 (with or without atp), mice macrophages, hek-293 and u937 cells. the samples were applied in two separated 12.5% bis/acrylamide gels: one destined to mass spectrometry (ms) and the other to western blot. at this point, we confirmed the presence of p2x7r, and observed several others proteins associated to p2x7r at different cell conditions, mainly when we exposed, j774.g8 cell, to 5 mm atp (n53). at this condition, we found by ms, hsp70, 75, and 90; alpha and b tubulin; myosin va; alpha, b and g actin; malate and lactate dehydrogenase (n51). although u977 and hek-293 had not received atp treatment, we found several proteins associated to p2x7. the next step was to immunoprecipitated those proteins in j774.g8 (treated or not with atp) and use it to verify if p2x7 are physically associated to them. as result we saw the p2x7 associated to panx-1 in j774.g8 cells. conclusion: we conclude that the p2x7r activated by extracellular atp triggers the recruitment of variety different proteins. at this condition, we can suggest that maybe there is a conformational change, regardless of the numerous recruitment structural proteins. in addition, apparently, the pore-forming protein pannexin-1 is associated with p2x7r, and the others pore forming proteins (vdac, cx-43, trpv1, trpa1) seems not be linked to p2x7r at j774. recently, we developed a series of molecular modeling tools for structure-based studies of protein functions and interactions. these tools are publicly available as web servers that are easily operated even by non-specialists: cabs-fold server for protein structure prediction [1] ; cabs-flex server for modeling of protein structure flexibility [2] ; aggrescan3d server for prediction of protein aggregation propensities and rational design of protein solubility [3] ; and cabs-dock server for prediction of peptide binding sites and peptide docking [4] . the web servers are freely available from the laboratory website: http://biocomp.chem.uw.edu.pl/tools sandy on 1 , pinghui feng 2 1 university of southern california, keck school of medicine, 2 developing a technique to detect deamidated proteins and peptides using rig-i sandy on, pinghui feng university of southern california, norris comprehensive cancer center, department of microbiology, and molecular biology, los angeles ca perhaps the most notable type of post-translational modification of proteins and peptides into a higher order structure is deamidation of asparagine and glutamine. deamidation occurs when an amine group is removed, degrading the molecule for purpose of regulating intracellular levels. previous studies have demonstrated that this notable post translational modification has been uncovered over time for use in dna recombinant technology as well as use as a biological clock to facilitate the rapid turnover of biologically important components of the cell. while the effects of this non-enzymatic chemical reaction have been widely studied, the method to uncover modification sites over a large quantity of proteins remains an issue. one of the most common types of deamidation is of asparagine and glutamine residues. at this time, most researchers will depend on mass spectrometric based proteomic techniques for identification of these post-translational sites. the issue is that mass spectral analysis of deamidated proteins and peptides is complication and can lead to misassigned identification attributed by an overlapping of 13c peak of the amidated form with the deamidated monoisotopic peak; these two peaks are only separated by 19.34 mda. while these issues can be mediated by using a mass spectrometer with a high mass measurement accuracy, and high resolving power, it is essential to establish simpler methods for identifying substrates that have undergone deamidation. if deamidation is present, different protein bands will be exhibited in the western blot, which will be compared to a triple mutant rig-i, which resists deamidation, to observe the location of this modification on the protein. with enough testing, i will determine specific sites of digestion and use this information to make conclusions of unknown proteins. i will make results regarding whether the protein has been modified based on the digestion sites. i will use mass spectrophotometry analysis to compare the proteins on a wider scale and double check my results. i have narrowed it down to a couple of different digestion sites that indicate deamidation. though the analysis work can be tedious, it is crucial to ensure the sites we isolate are accurate in order to establish this technique. from my research, we can apply this method for wider scale use such as in clinical settings. in areas of inflammation of parkinson's' patients, we can review specifically the infected cells versus uninfected and isolate the proteins, usually deamidated, responsible often smaller in size and more specific. in addition, research articles have already shown that suppressing modification of certain cells such as bcl-xl playing a major in leading the regulation of cancer cell death by apoptosis. by leading the discovery of a simpler methods to uncovering deamidation in cells, researchers will more easily and quickly be able to scan through various proteins, some of which discovered eventually may play pivotal roles in cancer research. influenza virus (iv) hemagglutinin (ha) is a homotrimeric integral membrane glycoprotein that mediates receptor-binding and membrane fusion. it constitutes the prominent viral surface antigen and a main target for neutralizing antibodies. bacterial, recombinant ha-based vaccines indicate high potential to confer protection against highly pathogenic (hp) avian iv (aiv) h5n1 and arise as alternative for the traditional egg-or cell culture-based manufacturing. relatively short time of bacterial has production can be of great importance in case of a pandemic. escherichia coli produced protein, based on the ha sequence of a/swan/poland/305-135v08/2006(h5n1) hpaiv*, has been successfully expressed in the form of inclusion bodies at institute of biotechnology and antibiotics. refolded and purified antigen was obtained in a soluble form, isolated by reversed phase hplc and identified with peptide mass fingerprinting using matrix-assisted laser desorption/ionization time of flight mass spectrometry (maldi tof/tof ms). the performed research in a great extent allowed to confirm the amino acid sequence of the recombinant ha (rha) assumed based on the cdna and allowed to establish the location of a total of six disulfide bridges. however, during purification and storage of the rha, apart from desired higher order rosette-like structures of the protein, other non-native species resulting from posttranslational modifications, misfolding, aggregation and degradation may occur what results in reduced vaccine potency. here, besides the properly folded monomers, we indicate non-native aggregates induced by disulfide crosslinking. moreover, several free cysteine residues and unexpected intrachain s-s were identified in rha tryptic peptide maps. cys 43 was found most susceptible to formation of disulfide bridges between the distinct chains of rha. the above findings allow to assume that not all rha particles fold to form the native structure. reduced cys residues exhibit tendency to undergo oxidation and unconnew strategies and approaches to understand how antibodies recognize and neutralize snake toxins represent a challenge to improve the antivenoms. the neurotoxic activity of micrurus venom is carried majority by two distinct proteins families, 3ftx and pla2. the conserved structural folding of these toxins can be appreciated as model to generate inhibitors against them. in this regard, monoclonal antibodies (mabs) can be used as tool to find hot spots for inhibit the toxins and represent the first step in order to develop recombinant neutralizing molecules. in this work our goals were analyse a set of monoclonal antibodies against the most toxic components of m. altirostris venom by proteomics approaches. the venom was fractionated; its major toxic proteins identified by in vivo tests based on murine lethal toxicity analyses (approved by the ethical committee for animal experimentation from center of health and science of the federal university of rio de janeiro -no. 01200.001568/2013-87). the toxic components were used to generate a panel of five monoclonal antibodies. elisa and antivenomics results allowed us identify the specificity of all mab and their neutralizing efficacy was measured by in vitro tests. three mabs showed reactivity towards 3ftx and two against pla2. all monoclonal antibodies against 3ftx lack a broad recognition. however, we identified a pair of monoclonal antibodies able to recognize all pla2 molecules of m. altirostris venom and showed a synergism to inhibit the catalytic activity of them. moreover, we challenge monoclonal antibodies against to micrurus venom for inhibit the pla2 activity of naja naja, specie taxonomically out of micrurus cluster. our results showed that pla2 of m. altirostris venom share a pair of conserved antigenic regions and draw attention to use these epitopes to miming antigen to generate antibodies for antivenom production. moreover, face to the cross reactivity and the pla2 activity inhibition capability by mabs towards the naja naja venom, our results highlight the conservation of neutralizing epitopes across the elapidae family. protein-protein interactions are known to play key roles in the most important cellular and biological processes such as signaling, metabolism, and trafficking. one major goal of structural biology is the structural characterization of all protein complexes in human and other organisms. these efforts can be complemented by computational approaches. in this context, computational docking attempts to predict the structure of complexes from their monomeric constituents. the docking problem presents two main challenges: the generation of structural poses or sampling, and the identification of the correct structures with a scoring function (sf). docking methods can be successful if the interacting partners undergo small conformational changes. however, in a general situation, these algorithms generate a large number of incorrect predictions, and therefore the predictive success strongly depends on the accuracy of the sf used to evaluate the docked conformations. a variety of strategies have been developed to score putative protein-protein docked complexes. they are usually based on atomic level potentials, residue level potentials, or a combination of both. in current work, we have evaluated 73 different sf, taken from cchappi server, on the results of 3 different rigid body docking methods, ftdock, zdock, and sdock, using the docking benchmark 4.0 and a docking set built from capri scorers experiment. our results show 9 sf that showed better or similar success rate than the in-built sf. some of these sf increase the docking success rates especially for flexible or weak-binding cases, which are the most challenging for docking. 6 of them are residue level sf robust enough to detected solutions in cases with large conformation change. in particular we found two sf that shows outstanding robustness, one designed for protein modeling and shared among docking methods, and the other is for protein docking which is also the best success rate in the top100 ranking in the capri scorers set. the other 3 atomic level sf display high success rate to find a solution within weak binding proteins. the 2 most successful sf are shared between the docking methods and display high success rate in the hard cases of the benchmark 4.0 and in the capri scorers set. the difference between them in the resolution level at which they work, one being atomistic the other residue-based. we found that they success rate vary according to the docking method chosen, allowing them to explode different properties of the sampling used. this way to characterize a protein complex can help to develop new combined scoring functions in protein docking or a new ranking strategy to enhance the success rate. multi-ptk antibody: a powerful tool to detect a wide variety of protein tyrosine kinases (ptks) isamu kameshita 1 , noriyuki sueyoshi 1 , yasunori sugiyama 1 1 the eukaryotic protein kinases consist of large families of homologous proteins and play pivotal roles in various cellular functions. these enzymes are classified into two major groups; protein serine/threonine kinases and protein tyrosine kinases (ptks). ptks are believed to be involved in various cellular events such as cell cycle, proliferation, differentiation, apoptosis, and cell adhesion in multicellular eukaryotes. as many as 90 ptk genes have been identified in the human genome and many of these ptks are known to be closely correlated with various diseases such as cancer. therefore, it is important to elucidate the expression profiles of the entire ptk family in cells and tissues. to investigate the expression profiles of the cellular ptks, we produced an antibody that detects a wide variety of ptks. for production of the antibody, antigenic peptides corresponding to amino acid sequences of a highly conserved region (subdomain vib) of ptks were synthesized and immunized to balb/c mice. among various antigens, a peptide with 11 amino acids, cyvhrdlraan, efficiently produced a polyclonal antibody with a broad reactivity to ptks. we established a hybridoma cell line producing a monoclonal antibody, yk34, which appeared to cross-react with various ptks. at least 68 ptks could be detected by yk34 antibody, as evidenced by its reactivity with the recombinant src tyrosine kinases whose subdomain vib had been replaced by those of the other ptks. when differentiated hl-60 cells were analyzed by western blotting after two-dimensional electrophoresis with yk34 antibody, we observed significant changes in the immunoreactive spots in hl-60 cell extracts along with the changes in the morphology of the cells. these results suggest that the multi-ptk antibody, yk34, will be a powerful tool for the analysis of a variety of cellular ptks. analysis of the siglec-9 and hvap-1 interactions leonor carvalho 1 , vimal parkash 1 , heli elovaara 2 , sirpa jalkanen 2 , xiang-guo li 4 , tiina salminen 1 1 structural bionformatics laboratory, department of biosciences, 2 medicity research laboratory, 3 department of pharmacology, drug development and therapeutics, 4 sialic acid-binding immunoglobulin (ig)-like lectins (siglec) are type i transmembrane proteins. siglec-9 has an n-terminal v-set domain followed by two c2-set domains in the extracellular region. it contains an immunoreceptor tyrosinebased inhibitory motif (itims) in its cytoplasmic tail and can function as an inhibitory receptor by dampening the tyrosine kinase-driven signaling pathways. these proteins are expressed primarily on leukocyte subsets and, thus, are thought to be involved in regulation of leukocyte functions during inflammatory and immune responses. recently, phage display screening experiments identified siglec-9 as leukocyte surface ligand for human vascular adhesion protein 21 (hvap-1; aoc3 gene product) and their interaction was confirmed by cell adhesion and enzymatic assays (kivi et al., 2009; aalto et al., 2011) . based on our preliminary data, hvap-1 sugar units with sialic acid (sa) might mediate interactions with the v-set domain in siglec-9. furthermore, it is known that the siglec peptides binding to hvap-1 are located in the ce loop of the second c22-set of domain (siglec-9_c22). based on current hypothesis an arginine in siglec-9_c22 interacts with the tpq residue in the active site of hvap-1. the ce loop of siglec-9_c22 has two arginines (r284 and r290) and, therefore, the interacting arginine is unclear. we will now study the interaction mode of hvap-1 and siglec-9 in silico to predict the role of the arginines in the c22 domain and the role of sa-binding using the 3d model of the full-length ectodomain of siglec-9 and the hvap-1 crystal structure. the in silico analysis will be conducted in parallel with experimental site-specific mutational studies and the result will be combined to elucidate the mechanism of hvap-1-siglec-9 interaction. adam middleton 1 , catherine day 1 1 attachment of ubiquitin to substrate proteins regulates almost all cellular processes, including protein degradation and cell division. ubiquitylation involves a cascade of three families of proteins: ubiquitin activating (e1), ubiquitin conjugating (e2) and ubiquitin ligase (e3) enzymes. the 8.5 kda protein can be attached as a monomeric moiety or as a polyubiquitin chain, and the type of modification spells out the 'ubiquitin code' that directs the fate of the substrate. polyubiquitin chains can be formed via eight different linkage types, and the arrangement of chain formation is typically directed by the e2 enzymes. forming a polyubiquitin chain involves binding of two molecules to the e2: the donor (ubd) and acceptor (uba) ubiquitin. ubd is linked to the e2 via a thioester bond between its c-terminal gly and the active site cys of the e2, and when primed for catalysis it interacts with a particular face of the e2. in contrast, coordination of uba by e2s is transient and cannot be easily measured; however, uba binding defines the linkage type of polyubiquitin chains. the e2, ube2k, directs lys48 chain synthesis, which results in modified proteins being degraded by the proteasome. we generated a stable form of the ube2kub conjugate and crystallized it, and showed that both ube2k and its ubiquitin conjugate are monomeric. using molecular docking, we modelled the position of both ubd and uba and investigated the interfaces with site-directed mutagenesis. these experiments led to a molecular model that revealed how ube2k can synthesise lys48-linked ubiquitin chains. this molecular explanation provides a foundation for understanding how other e2s generate lys48-linked polyubiquitin chains. the two chromophorylated linkers of r-phycoerythrin in gracilaria chilensis marta bunster, francisco lobos-gonz alez, jos e aleikar v asquez, carola bruna, jos e mart ınez-oyanedl 1 fac de cs biol., universidad de concepci on the two chromophorylated linkers of r-phycoerythrin in gracilaria chilensis. francisco lobos-gonz alez, jos e aleikar v asquez, carola bruna, jos e mart ınez-oyanedel, marta bunster. departamento de bioqu ımica y biolog ıa molecular, facultad de ciencias biol ogicas, universidad de concepci on. phycoerythrin is a phycobiliprotein present in phycobilisomes in gracilaria chilensis as a complex with chromophorylated linker proteins. our interest is to discover the role of these linkers in the function of phycobilisomes. phycobilisomes(pbp) are auxiliary light harvesting protein complexes in charge of channeling energy towards photosystem ii in alga, cyanobacteria and cryptophyta. this is possible thanks to fluorescent proteins called phycobiliproteins (pbp) and the chromophores (phycobilins, open-chain tetrapyrrols) attached to specific cysteines. phycobiliproteins share a common general structure; they are organized as (alfab) heterodimers which themselves assemble as trimers(alfab)3 or hexamers (alfab)6; this complexes are organized in high order structures to form the core and the rods. besides pbps, pbs have linker proteins in charge of the assembly and stabilization of the complex, and also it has been proposed that they collaborate in the fine tuning of the energy transfer steps between chromophores. these linkers are located within the rods, the rod-core interface, the core and the core-membrane interface. although most linker proteins are colorless, chromophore bearing linkers have been described, which suggest its participation in the energy transfer process. two of them, g 31 and g 33 are associated to r-phycoerythrin in gracilaria chilensis, nevertheless the information available on these linkers in eukaryots is still limited. to understand how these linkers collaborate with the function of the phycobilisome, we need structural information, especially the coordinates of all the chromophores present in the complex; we have sequenced both linkers from the genomic dna, performed sequence analysis and also we have purified the linkers by anion exchange, molecular sieve and hpl chromatography. the characterization was performed by denaturant electrophoresis, absorption and emission spectroscopy and by mass spectrometry. the results show that they have molecular masses as predicted, with a peptide signal for chloroplasts, an internal sequence repeat; residues 67 -170 with residues 179 -273 for g 31 and residues 107-200 with residues 219-315 for g 33, and the presence of conserved cysteine residues putative sites of chromophorylation. the spectroscopy shows that they have different composition of phycobilins and a very short t1/2. a preliminary model for both linkers shows that they belong to aa structural class and that they share a common fold (heat like motifs) frequently involved in protein-protein interactions. dept. of phys., chuo univ., 2 grad. sch. of inform. sci. and eng., tokyo tech, 3 rigid-body docking algorithms are useful for predicting tertiary structures of near-native protein complexes. however, this algorithms generate many protein complex poses including false positives. then, near-native poses are searched in a post-docking process. there are many computational softwares with rigid-body docking algorithms, for example, zdock. we developed a high-performance protein-protein interaction prediction software, megadock, which is basically used on supercomputing environments for a large scale and network level in this work, we then tried to use these docking softwares and the profile method for understanding mechanisms of protein-protein interactions. we focused on some physicochemical properties, electrostatic and hydrophobicity, of a set of protein complex poses generated by a rigid-body docking process. from these poses, we obtained sets of possible interacting amino acid pairs. a set of interaction profiles has some information of docking spaces. from the view of a network prediction, the docking spaces of a set of protein complex poses are one of the properties for discriminating native protein-protein pairs from non-native pairs. in this work, ensemble docking process is performed by megadock ver. 4.0 and zdock ver. 3.0.1. cluster analysis is used with profiles of physicochemical properties. we used a dataset composed of typical 44 monomer-monomer protein pairs and will discuss mainly differences between native and non-native protein pairs. the structural studies of the two thermostable laccases from the white-rot fungi pycnoporus sanguineus marta orlikowska 1 , grzegorz bujacz 1 1 institute of technical biochemistry, lodz university of technology, poland laccases (ec 1.10.3.2, benzenodiol oxygen oxidoreductases) are enzymes that have the ability to catalyze the oxidation a wide spectrum of phenolic compounds with the four-electron reduction of molecular oxygen to water [1] . it has been found that the active site is well conserved in between laccases from different organisms. it contains four copper atoms: one paramagnetic type 1 cooper (t1) that is responsible for their characteristic blue color and where the oxidation of the reducing substrate occurs, one type 2 cooper (t2) and two type 3 coopers (t3) that conform a trinuclear cluster in which molecular oxygen is reduced to two molecules of water [2] . laccases are present in many different species and they have been isolated from plants, fungi, prokaryotes, and arthropods in most cases laccases are monomeric glycoproteins of around 500 amino acids with molecular weights in the range of 60-85 kda. the various functions carried out by those enzymes include the antagonistic ones such as their involvement in lignin biosynthesis (in plants), lignin degradation, pigment production, fruiting body formation, pathogenesis (in fungi) and spore protection against uv light (in bacteria) [1, 3] . the diversified functions of laccases make them an interesting enzyme for study from the point of view of their structure, function and application. laccases of white-rot fungi (wrf) are of special interest because one of its role is to degrade lignin and most of them are extracellular enzymes helping purification procedures [1] . during the last two decades, there has been an increasing interest in the genus pycnoporus for its ability to overproduce high redox potential laccases as the ligninolytic enzymes. we present the crystal structures of two thermostable lacasses produced by strain pycnoporus sanguineus cs43 (laci and lacii). the molecular weights of laci and lacii, determined by sds-electrophoresis, is 68 and 66 kda, respectively [3] . both isoforms shows high amino acids sequence similarity (91%) between them and high thermal stability, at 508c and 608c. they remained active at high concentration of organic solvent (acetonitrile, ethanol or acetone). the unique properties make them promising candidates for industrial applications in wasterwater treatment. laci exerted a higher thermal and ph stability, tolerance against inhibitors and was a more efficient catalyst for abts and dmp (laccases substrate) then lacii [3] . based on the structures we would like to understand the isoforms differences that confers laci a markedly better performance than lacii in ph and thermal stability as well as better resistance to inhibitors. analysis of liver proteome in cystathionine ß-synthase deficient mice using 2d ief/sds-page gel electrophoresis, maldi-tof mass spectrometry, and label-free based relative quantitative proteomics izabela bieli nska 1 , łukasz marczak 1 , hieronim jakubowski 1,2 1 institute of bioorganic chemistry, polish academy of sciences, 2 rutgers university, new jersey medical school homocysteine (hcy) arises from the metabolism of the essential dietary protein amino acid methionine. levels of hcy are regulated by remethylation to met and transsulfuration to cys. cystathionine bsynthase (cbs) catalyzes the conversion of homocysteine to cystathionine (first step of transsulfuration reaction). human cbs deficiency is a recessive inborn error of homocysteine metabolism that casues severe hyperhomocysteinemia (hhcy) and diverse clinical manifestations, including fatty liver disease [1] . although the causes of fatty liver disease in cbs deficiency have been studied the underlying mechanism is not understood. we hypothesize that cbs deficiency induces changes in gene expression that could impair liver homeostasis. to test this hypothesis and gain insight into hepatic functions of cbs we analyzed the liver proteome of cbs -/-and cbs 1/1 mice [2,3] using 2d ief/sds-page gel electrophoresis and maldi-tof mass spectrometry (n514) we identified twelve liver proteins whose expression was significantly altered as a result of the cbs gene inactivation. expression of three proteins was upregulated and of nine down-regulated by the cbs-/-genotype. two up-regulated liver proteins are involved in iron metabolism (ftl and fth). those proteins are associated with oxidation stress and inflammation. third up-regulated liver protein (cbr3) is related to oxidation-reduction process. the downregulated protein are involved in the hydrolysis of n-acylated or n-acetylated amino acids (acy1), regulation of endopeptidase activity (a1at4), cholesterol biosynthetic process (fpps), amino acid degradation (huth), cellular calcium ion homeostasis and l-ascorbic acid biosynthetic process (rgn). using label-free based relative quantitative proteomics (n58) we identified fourteen liver proteins whose expression was significantly altered as a result of the cbs gene inactivation. expression of four proteins was up-regulated and of ten proteins was down-regulated. the down-regulated liver proteins are linked with regulation of bone mineralization and inflammatory response (ahsg) or regulation of mrna splicing (roa2). the up-regulated liver proteins are involved in tricarboxylic acid cycle (suca), oxidation-reduction process (cy250), cholesterol metabolic process, iron ion homeostasis (fech), fatty acid metabolic process (ssdh; eci1) and response to oxidative stress (lonm). our findings suggests that cbs interacts with diverse cellular processes, including lipid metabolism, that are essential for normal liver homeostasis. deregulation of genes involved in lipid metabolism provides a possible explanation for fatty liver disease associated with cbs deficiency. transcription factors play central roles in coordinating developmental processes, as evidenced by the increasing number of transcription factor-related developmental disorders being uncovered by nextgeneration sequencing and genome-wide studies of copy number variation. the action of a transcription factor in regulating gene expression depends on interactions with other transcription factors, coactivators/co-repressors and chromatin modifying and remodeling complexes. transcription factors are commonly regulated by post-translational modifications. however the study of protein-protein interactions and post-translational modifications of transcription factors by common techniques such as coimmunoprecipitation and mass spectrometry is hampered by the difficulty in preserving interactions and modifications through cell lysis. to circumvent this issue, we developed a bioluminescence resonance energy transfer (bret) assay, which allows protein-protein interactions to be observed in live cells. in this assay, a protein of interest is expressed as a fusion with luciferase from renilla reniformis, and its putative interaction partner as a fusion with yellow fluorescent protein (yfp). upon addition of a cell-permeable substrate, the distance-dependent non-radiative transfer of energy from luciferase to yfp is quantified by measurement of light emission at two wavelengths to assess the interaction between the two fusion proteins. to validate the utility of this assay for investigating transcription factor interactions, we confirmed homodimerization of the foxp2 transcription factor, haploinsufficiency of which causes a rare and severe speech and language disorder, as well as interaction of foxp2 with other members of the foxp family. we also confirmed the interaction between foxp2 and multiple candidate interactors identified through yeast two-hybrid assays, including the autism-related transcription factor tbr1, the co-repressors ctbp1 and ctbp2, and post-translational modification enzymes of the pias family. the role of pias enzymes in sumoylation -the covalent modification of proteins with small ubiquitin-like modifier (sumo) proteins -led us to further explore this process, which is notably difficult to investigate because of the dynamic and labile nature of the modification, which is also typically present on only a minor fraction of molecules of a given protein. combining the bret assay with gel-shift techniques we demonstrated that foxp2 is sumoylated. finally, we used the bret assay to examine the effects of etiological foxp2 variants in speech and language disorder on protein-protein interactions and post-translational modification. in summary, the bret assay is a sensitive, reliable and potentially high-throughput technique for exploring protein biology in the context of live cells. we have demonstrated applications of the assay in validating putative protein-protein interactions, assessing posttranslational modifications, and investigating functional effects of protein variants identified in patient cohorts. these investigations have provided novel insights into the function of the foxp2 transcription factor in neurodevelopment and into the etiology of foxp2-related speech and language disorder. the directly interaction between pres1 of human virus b and human heat shock protein 70 (hsp70) deqiang wang 1 , chen ke 1 , jun zhang 2 1 key laboratory of molecular biology on infectious disease, 2 the department of cell biology and genetics the directly interaction between pres1 of human virus b and human heat shock protein 70 (hsp70). hepatitis b virus (hbv) has infected 2 billion people worldwide, and 350 million of them are chronically infected. the chronic virus infection, a major public health problem worldwide, leads to bout two-thirds of hepatocellular carcinoma (hcc). the hbv envelope consists of the large (l), middle (m) and small (s) envelope proteins, which contain pres1-pres2-s, pres2-s, and s domain alone, respectively [2] . the pres1 domain is believed to mediate virus attachment to the high-affinity receptor. yan et al employed a novel technique to propose sodium taurocholate co-transporting polypeptide (ntcp) as the candidate hbv receptor, and consequently, ntcp is a target for a new family of anti-hbv agents [3] . whereas, it remains a query to clarify that ntcp is the only or major hbv receptor in vivo. to illuminate if other host proteins cooperatively participate the hbv infection, we detect the interaction between pres1 and many candidate host proteins. fortunately, we have found that the human heat shocking protein 70 (hsp70) could directly interact with the pres1 domain of the hbv virus protein. both the pull down and the size exclusion chromatography experiments verify that the grp78 have the ability binding to pres1. whereas, whether the interaction between hsp70 and pres1 relates to the hbv infection need further experiments to clarify. 4 the member sponsorship in the vast world of naturally occurring peptides, where more than 7000 peptides are known and approximately 140 peptide therapeutics are currently being evaluated in clinical trials (fosgerau & hoffmann, 2015), the rapid and accurate determination of their physicochemical properties is key in peptide drug discovery. among these properties, hydrophobicity is crucial for understanding molecular recognition and biomolecular aggregation. hence, there is a great interest in determining hydrophobicity scales for amino acid structures. in this work, octanol/water partition (log p) and octanol/water distribution (log dph, fig. 1 ) of n-acetyl-l-amino-acid methyl amides were determined by means of quantum mechanical ief-mst solvation calculations taking into account the intrinsic conformational preferences of each amino acid according to dunbrack's libraries (dunbrack & karplus, 1993; 1994) . the results reveal log d7.4 differences for a-helical and b-sheet conformations in arg, lys, hid, asn, gln, met, cys, leu and ile. furthermore, by decomposing the octanol/water transfer free energy into electrostatic and non-electrostatic components, we estimated that the non-electrostatic cost of transferring the amino acid side chain amounts to 23.9 6 3.0 cal/mol.å2, in agreement with previous estimates reported in the literature. comparison of our scale with other theoretical and experimental hydrophobicity scales yields satisfactory results, leading to correlation coefficients ranging from 0.61 to 0.94. additionally, the mstderived hydrophobicity scale led to significant correlations with the rp-hplc retention factors measured for eight decapeptides (r 5 0.97) and for 195 influenza virus hemagglutinin 13-mer (ac-ypydvp-dyaslrs-amide) peptides (r 5 0.80). finally, the hydrophobicity scale was able to reproduce the experimental log p for 118 random neutral peptides (r 5 0.92) and log d7.4 for '01:15 random charged peptides (r 5 0.95), fig. 2 . future studies will address the application of this methodology to nonproteogenic amino acids, the prediction of peptide hydrophobicity at global and atomic level in peptides, and the scoring of peptide-protein interactions. docking-based tools for discovery of protein-protein modulators docking-based tools for discovery of protein-protein modulators. protein-protein interactions (ppis) play an essential role in many biological processes, including disease conditions. strategies to modulate ppis with small molecules have therefore attracted increasing interest over the last few years. although protein-protein interfaces (ppifs) are considered difficult to target with small molecules given its lack of well defined cavities. successful ppi inhibitors have been reported into transient cavities from previously flat ppifs. recent studies emphasize on hotspots (those residues contribute for most of the energy of binding) as promising targets for the modulation of ppi. pydock algorithm is one of the few computational methods that use energy of solvation to predict protein-protein interfaces and hotspots residues. we present an approach aimed at identifying hotspots and transient pockets from predicted proteinprotein interfaces in order to find potential small molecules capable of modulating ppis. the method uses pydock to identify ppifs and hotspots and molecular dynamics (md) techniques to propose putative transient cavities. we benchmarked the protocol in a small set of protein-protein complexes for which both structural data and ppi inhibitors are known. the method applies to the unbound proteins of the complexes the fast fourier transform algorithm, followed by the energy-based scoring from pydock to calculate the normalized interface propensity (nip) values derived from rigid-body protein docking simulations to predict the ppifs and hotspots residues without any prior structural knowledge of the complex. then we used md to describe the possible fluctuations of the interacting proteins in order to suggest transient pockets that could be useful as targets of small molecules for the modulation of ppis. finally, we evaluated by ligand docking, the validity of predicted hotspots and pockets for in silico drug design. we found that the nip-based method from pydock protein-protein docking identifies hotspots residues that are located within the binding site of known inhibitors of ppis. predicting ppifs from a three dimensional structure is a key task for the modulation of ppis. the use of the nip-based hotspots prediction method improve the identification of transient cavities from md simulation when compared to known binding cavities. this approach can be extremely useful in a realistic scenario of drug discovery targeting ppifs, when there is no information at all about the protein-protein complex structure. protein complexes are the fundamental molecular organizations that assemble multiple proteins to achieve various biological processes. identification of protein complex membership should provide a genotype-phenotype map to elucidate human gene-disease associations. it has been routinely assumed that network clusters with dense connections inside and sparse connections outside would form functional protein complexes. therefore, searching highly modular subgraphs in protein-protein interaction networks was explicitly or implicitly implemented in the algorithms to find protein complexes. however, to our surprise, we found a large portion of complexes with a medium-to-low modularity from the analysis of 719 experimentally confirmed protein complexes. we also discovered that these complexes have cellular functions enriched in highly time-and space-dependent expression, such as signal transduction or subcellular localization. we further developed an algorithm to find such complexes by weighing network connections to capture transient interactions with intrinsically disordered regions. we confirmed that our method improved the identification of biologically relevant members of protein complexes and covered more complexes with a medium-to-low modularity. furthermore, newly discovered subunits in protein complexes could explain more disease-gene associations, indicating its utility to expand current genotype-phenotype map of human diseases. expanding template-based protein-protein complex prediction using ab-initio docking sergio mares-s amano 1 , luis angel rodr ıguez-lumbreras 1 , juan fern andez-recio 1 1 structural characterization of protein-protein interaction (ppi) networks is crucial for understanding the underlying molecular mechanisms whereby life processes and disease arise. however, due to inherent limitations of experimental techniques, such characterization only covers an extremely reduced fraction of the human ppi network (interactome). recent studies have shown that although available structural templates may suffice to model a significant proportion of the interactome, model accuracy and binding specificity remain unsolved problems. consequently, improving the ability to predict ppis structurally will help to provide a better 3d profile of the known interactome, which may ultimately lead to the development of new therapeutic applications. here we show a novel approach that combines templatebased modeling with protein-protein computational docking to the structure-based prediction of ppis. our approach samples different protein-protein structural models derived from docking simulations. models are subsequently ranked using a function that incorporates an energy-based scoring term and a structural template similarity score. the energy-based scoring function includes electrostatics, van de waals and desolvation calculations, whilst the template similarity score accounts for the degree of structural similarity of models against a high-resolution and diverse dataset of structural templates. our approach highly improved the predictive success rate over individual ab-initio docking and templatebased techniques across a large benchmark dataset, including 176 protein-protein complexes. when compared to the performance of the ab-initio docking algorithm, we found that the approach increased consistently the success rate, by approximately 30%, for the top 1, top 5 and top 10 solutions. the success rate improvement was even more notorious when the comparison was performed against the predictions from the traditional template-based docking. though incorporating ab-initio docking expands considerably the scope of the template-based docking method, challenges remain for interacting proteins in which high conformational changes occur upon binding and also the size and diversity of the repertoire of structural templates needs to be increased. is essential for the development of multicellular organisms. in mammalian cells, early events in pcd involve the release of cytochrome c (cc) from mitochondria to the cytoplasm, so letting cc play a key role in assembling the apoptosome and triggering apoptosis. in plants, pcd is part of a general process -the so-called hypersensitive response -in which mitochondrial cc is likewise released into the cytosol but its further role and cytoplasmic partners remain veiled. such a coincidence in cc release made us think of a common link for pcd in such evolutionarily distant species along evolution. to go deeper in understanding the pcd-dependent role of cc, a proteomic approach based on affinity chromatography with cc as bait was run using human and plant cell extracts. upon combining this approach with bimolecular fluorescence complementation (bifc), a total of eight and nine unknown proteins interacting with cc under pcd conditions were identified in human and plant cells, respectively [1, 2] . such novel cc-partners -which are located in the cytoplasm and even in the nucleus -are involved in protein folding, translational regulation, oxidative stress, dna damage, energetic and mrna metabolism [3] . strikingly, some of the novel human cc-partners are closely related to those for plant cc, so indicating that the evolutionarily well-conserved event of cc release from mitochondria could involve a common signalosome consisting of a wide range of common targets [3] . to also understand such a promiscuity of cc from a structural point of view, the cc surface residues involved in complex formation with each one of its counterparts were mapped by using nmr spectroscopy. the resulting data shows that the heme crevice of cc is at the cc-partner interface in most of the complexes, which is in agreement with the vast majority of known redox adducts of cc. in contrast, however, to the high turnover number of the redox cc adducts inside the mitochondria, the complexes formed by cc under pcd conditions lead to the formation of rather stable nucleo-cytoplasmic ensembles. altogether, these findings suggest that extra-mitochondrial cc interacts with nuclear and/or cytoplasmic pro-survival, anti-apoptotic proteins in both humans and plants so as to lead living cells to dye. keywords: cytochrome c, programmed cell death, signalosome. post-translational phosphorylation often modulates the function of proteins. in particular, they affect the role that cytochrome c (cc) plays in cell life and death [1] . cc is phosphorylated in vivo in tyr48 and tyr97 residues [2, 3] , but recently, two new phosphorylation sites have been described at positions 28 and 47 [4] . hence, we aim at understanding the structural and functional changes induced by thr28 and ser47 phosphorylation cc. for this purpose, we designed two phosphomimetic mutants of cc by replacing either thr28 or ser47 by the canonical amino acid aspartic acid (t28d and s47d). as control, two other mutants at the same two positions (t28a and s47a) were analyzed so as to differentiate the effects due to the presence of a negatively charged residue. remarkably, the s47a mutant is significantly less stable than the wild-type species. we found that phosphorylation at position thr28 diminishes the redox potential and oxygen consumption. in addition, t28d mutation affects the ability of cc to bind the distal site pcc1, thereby suggesting that phosphorylation at this position affects the electron carrier capacity of cc. mass spectrometry (ms) is widely used techniques to gain knowledge about biomolecules [1, 2] . it produces a high amount of data which is often presented as a list containing thousands of proteins. that list usually contains few hits interesting for our research. the pocess to select those proteins may include integrating experimental with annotation data. it requires spending some time in both, performing calculus and searching in databases. in this poster we present msbiodata analysis tool, a web service thought to deal with this tedious work. with this tool, researchers can set rules to select the most interesting hits in his lists using both, experimental data and gene ontology [3] annotation. the data can be upload to the web using an excel spreadsheet or a flat files in a mztab format, and rules are easily constructed by means logical sentences. those sentences are composed by one or more terms linked by logic operators (and and or). each term in the logical sentence indicates to our program the conditions 1 that selected hits must meet. once the alysis is finished, the results are delivered by email. msbiodat analysis tool do not requires any programming knowledge to be used and is freely available at: http://msbiodata.innomol.eu keywords bioinformatics/data analysis/proteomics/data mining/ mass spectrometry. beside the rate of protein synthesis, the regulation of protein degradation plays a crucial role in the white muscle protein accumulation and overall fish growth. intracellular proteolysis in salmonid species, such as atlantic salmon, salmo salar l. and rainbow trout, oncorhynchus mykiss walb., was studied to evaluate the basic mechanisms of protein degradation that could possess a potential target to regulate the body mass accumulation in farmed fish. a number of white muscle proteases such as cathepsins b, l, and d, proteasomes, and calcium-dependent proteases (m-and m-calpains), was studied in the juvenile specimens of different size-and age-groups both wild and farmed salmonids. the correlations between the protease activity and expression levels and morphometric characteristics of fish were found. the size-and age-related differences in intracellular protease activity revealed in fish muscles indicate both general role of proteolysis regulation in salmonid growth and the specific role of the individual proteolytic enzymes as well. the data on negative correlation of cathepsin d and calpain activity in muscles and the rate of weight increase in juvenile salmonids were obtained. a revealed positive correlation of cathepsin b activity and morphometric parameters in fish young presumably indicates its primary contribution to non-myofibrillar protein turnover. ubiquitin-proteasome system seems to contribute to background protein turnover as the proteasome activity was not corresponded with growth rate. summarizing the data obtained the autophagy-lysosomal and calpain-related protein degradation pathways were recognized to be directly involved in body growth and muscle protein retention in salmonid fish. the work was carried out using technical facilities of ib karrc ras equipment centre and financially supported by the russian science foundation, grant no. 14-24-00102 "salmonids of the north-west russia: ecological and biochemical mechanisms of early development". solving the proteomic organization of fitness-related genes in uropathogenic escherichia coli in life threatening sepsis. nowadays, complete genomes for almost all major bacterial pathogens are available, helping researchers to identify virulence factors. however we still ignore how these genes are organized at the proteome level and how this association influences bacteria pathogenicity. we integrated available databases on upec e. coli (strain cft073) to investigate the genomic and proteomic organization of genes related to upec fitness in the host. intriguingly, we found that most fitnessrelated genes have orthologs not only in other pathogenic strains but also in non-pathogenic bacteria such as e. coli k-12. these genes are organized in clusters and operons with similar structure. by integrating protein-protein interaction data we observed that genes with high impact on fitness also display a highly clustered organization when compared to other genes. overall, our results show that proteinprotein interaction clusters associated to upec fitness in the host represent a promising target for the design of new antibiotics. elucidating the molecular mechanisms by which the hnh endonuclease gp74 activates the terminases in bacteriophage hk97 (2) . hnh endonucleases are characterized by two highly conserved his residues and an asn residue(3). gp74 is essential for phage head morphogenesis, likely because gp74 enhances the activity of the hk97 terminase enzymes toward the cos site (4) . notably, enhancement of the terminase-mediated cleavage of the phage cos site requires the presence of an intact hnh motif in gp74. mutation of the canonical metal binding his in the hnh motif abrogates gp74 mediated-terminase activity. although phages are widely studied, there is no definitive structural or mechanistic evidence as to how the hnh endonuclease within gp74 functionally interacts with the adjacent terminase enzymes to facilitate phage morphogenesis. previous work on hnhcontaining bacteriophage proteins does not address explicitly how the requirement for divalent metal binding at the hnh endonuclease site induces interaction with the terminase enzymes that are so crucial for phage dna packaging during morphogenesis (4, 5) . in addition, gp74 possesses no sequence similarity to hnh proteins for which the structure has been determined (3), making structural studies of gp74 necessary. toward these ends, we use nuclear magnetic resonance (nmr) spectroscopy to probe metal and terminase binding of gp74 in the wild type state and bearing metal binding mutations. we also report backbone resonance assignment of gp74. our nmr studies have elucidated residues within gp74 required for metal binding and terminase activity. these data are being used to assess the role of specific gp74 residues in phage morphogenesis. together, this work will identify the enigmatic role describing how metal binding in hnh endonucleases is crucial in the replication and morphogenesis of phages. meat production from pigs for human consumption is a resource heavy process, indeed every part of the animal that is not used constitutes a protein food-chain loss, which is neither economically nor environmentally viable. the goal of this project is to better harness slaughterhouse waste such as the keratin rich pig bristles and nails through microbial conversion. instead of using identified single microorganisms, it is the goal to define microbial consortia where microorganisms synergistically show the ability of efficient keratin degradation/conversion. candidate consortia have been obtained by selecting for microorganisms growing on enriched media that contains milled pig bristles as sole carbon and nitrogen source. by using mass spectrometry and various biochemical analyses to investigate keratinolytic enzymes, methods will be established for identifying and characterizing suitable consortia. protein families likely to be involved are keratinases, which are specialized proteases including serine, cysteine and metallo proteases, as well as systems capable of reducing or otherwise breaking disulfide bonds which are highly abundant in hair and nails. furthermore, interactions and symbiosis of microorganisms in a consortium will be investigated at the meta-proteomics level. the project will lead to development of biotechnological degradation of keratin rich fibers, and provide new insights into functional dynamics and efficacy of microbial consortia. a comprehensive protein domain analysis to map cancer-type-specific somatic mutations interpretation of the genome-wide association studies (gwas) of cancer patients to find cancer-typespecific biomarker is challenging due to the mutational heterogeneity of cancer types. network approaches to find cancer-type-specific variants and biological pathways are increasing since genes tend to act together to display phenotypic or disease outcomes. phenotype similarity has proven to reflect the relationship of functionally related genes. we applied phenotype similarities between various diseases for expanding molecular connections of cancer-type-specific variants to discover cancer-type-specific modules. specifically, cancer-type-specific variants of 7 cancer types from the cancer genome atlas (tcga) were analyzed to find phenotype-inferred relationships among the variants. we find that cancer variants that cause the similar disease phenotypes tend to be linked as a cluster of biological pathways or functions. moreover, cancer-type-specific modules could explain the underlying pathogenicity of specific symptoms which manifest in particular cancer types. cancer-type-specific modules and pathways found from phenotype similarity/dissimilarity based on cancer symptoms improved the discrimination performance to sort cancer-type-specific variants to accurately predict patient groups. our method will be further developed to find genetic biomarkers for the diagnosis or prognosis of specific cancer types pk-009 engineering a stable, symmetric membrane protein scaffold amanda duran 1 , jens meiler 1 1 computational protein engineering has the potential to contribute to various fields including drug design, protein therapeutics, and materials science. protein-ligand interface design and the construction of large, stable proteins rely on stable scaffolds. symmetry is a great tool for protein stability both in protein engineering and nature. several membrane protein structures exhibit pseudo-symmetry and are proposed to be the result of gene duplication, fusion and diversification events originating from a monomeric gene. aquaporins (aqp) are a class of membrane proteins that exhibits a two-fold inverted pseudo-symmetry. the escherichia coli aqp glycerol facilitator protein (glpf) was originally computationally engineered to be perfectly symmetric in sequence and presumably in structure. the symmetric gene was assembled, cloned, and expressed. however, after facing many challenges experimentally, the computational study has been expanded to 13 aqps of known structure for a more extensive symmetric backbone search. mammoth structural alignment was used to align the structures to their inverted counterparts. cutpoints were calculated based on a-carbon distance. finally, the rosetta protein modeling software suite was used to refine and energetically minimize the symmetric backbones. from over 1500 generated symmetric backbones, 20 candidates were chosen for experimental verification. these studies are ongoing.currently, the symmetric backbone models have scored to be more stable than the wild-type proteins. experimental verification of these symmetric backbones will provide valuable information for the current state of membrane protein modeling and design using computational methods. intrinsically disordered proteins drive heritable transformations of biological traits daniel jarosz 1 , james byers 1 , sohini chakrabortee 2 , sandra jones 3 , amelia chang 2 , david garcia 1 1 stanford university, 2 whitehead institute for biomedical research, 3 rockefeller university the transmission of information from one generation to the next generally occurs via nucleic acids. the only known protein-based molecular memories are prions, which drive heritable biological traits based upon self-templating changes in protein conformation. these protein-based genetic elements have previously been identified systematically, but at least three do not share the sequence biases or structural characteristics that have informed such studies. here we employed a comprehensive library of yeast proteins to examine the breadth of protein-based inheritance. transient overexpression of more than forty proteins created new traits that were heritable and beneficial. some shared properties of known prions, but most employed distinct genetic and biochemical mechanisms to act as elements of inheritance. traits with these characteristics were common in wild yeast strains and could also be elicited using orthologous mammalian proteins. the inducing proteins were strikingly enriched in intrinsically disordered sequences that have been widely conserved across evolution. intrinsically disordered proteins are associated with human disease and with dosage sensitivity in yeast, flies and worms. our results suggest another widespread role for such intrinsically disordered sequences: induction of heritable epigenetic switches that transform phenotypic landscapes and drive adaptation to stressful environments. prediction of binding affinity in protein complexes: contacts do matters almost all critical functions in cells rely on specific protein-protein interactions. understanding these is therefore crucial in the investigation of biological systems. despite all past efforts, we still lack a thorough understanding of the energetics of association of proteins. here, we introduce a new and simple approach to predict binding affinity based on functional and structural features of the biological system, namely the network of interfacial contacts. we assess its performance against a protein-protein binding affinity benchmark and show that both experimental methods used for affinity measurements and conformational changes have a strong impact on prediction accuracy. using a subset of complexes with reliable experimental binding affinities and combining our contacts-and contact types-based model with recent observations on the role of the non-interacting surface in protein-protein interactions, we reach a high prediction accuracy for such a diverse dataset outperforming all other tested methods. free radical oxidation -a new method for obtaining stable protein coatings on magnetic nanoparticles magnetically targeted nanosystems (mtnss) are now considered to be applicable in different areas of biology and medicine such as hyperthermia, magnetic resonance imaging, immunoassay, cell and molecular separation, a smart delivery of drugs to target cells. proteins are promising materials for creation of coatings on magnetic nanoparticles (mnps) due to their biocompatibility, an ability to protect magnetic cores from influence of biological liquids and prevent agglomeration of mtnss in dispersion, their possible functional activity as therapeutic products and biovectors. the creation of stable protein coatings with retention of native properties of molecules is still an important biomedical problem because of disadvantages of the commonly used methods such as formation of a polydisperse ensemble of particles, nonselective linking of proteins leading to cross-linking of macromolecules in solution, and desorption of coatings. a novel method in obtaining stable single-layer coatings assembled from protein molecules on the surface of magnetite nanoparticles has been developed. it is based on protein liability to free radical modification, leading to the formation of intermolecular covalent cross links. free radicals are locally generated on the surface of nanoparticles via the fenton reaction thereby proteins adsorbed on the surface are subjected to the cross-linking. o-phenylenediamine was used for detection of free radical generation initiated by nanoparticles. the proteins drastically differing in their structure and properties, namely, serum albumin, thrombin and immunoglobulin g were selected for creating the protein coatings. the properties of the obtained coatings and their stability have been studied with the help of dynamic light scattering (dls), uv/vis spectrophotometry, antibody-antigen test and the method of spectral-fluorescent probes. albumin molecules in mnps coatings have been shown to retain their capability of binding with a dye and be conformationally stable. the dye 3,3'-di-(g-sulfopropyl)25,5'diphenyl-9-ethiloxacarbocyanine-betaine interacting with albumin with a growth of fluorescence and with partial cis-trans conversion of the dye has been used. it has been proven that coatings composed of protein macromolecules are 1) stable, 2) formed around individual nanoparticles and 3) have several nanometers in thickness. the free radical linking of thrombin and immunoglobulin g on the surface of nanoparticles has been shown to almost completely keep native properties of the protein molecules. the free radical linking method reveals new possibilities for design of single-layer multiprotein polyfunctional coatings on the surfaces of all the nano-, micro-and macroobjects containing metals of variable valence (for example, fe, cu, cr). the spectral-fluorescent investigation was supported by the russian foundation for basic research, project nos. 13-03-00863 and 14-03-31196mol_a. regulation of neuronal snares by accessory proteins shrutee jakhanwal 1 , reinhard jahn 1 1 regulation of neuronal snares by accessory proteins 1shrutee jakhanwal and 1reinhard jahn 1department of neurobiology, max planck institute of biophysical chemistry, fassberg, goettingen, germany-37075. synaptic vesicle exocytosis lies at the heart of the process of neurotransmitter release. and, the family of proteins that is central to the process of synaptic vesicle exocytosis is the family of snare proteins. there are three kind of neuronal snare proteins namely syntaxin, snap25 and synaptobrevin. these three snare proteins interact through their snare-motifs to form a highly stable four-helix bundle, which in turn, pulls two membranes together to mediate fusion. years of work in this field have established that the four-helix bundle is critical for the membrane fusion to occur. however, the process of regulation of snare-mediated fusion remains very poorly understood. the major regulatory proteins involved in the process are munc 18, munc 13, synaptotagmin and complexin. the major aim of my project is to obtain a closer look at the regulation process of snare-mediated fusion by focusing on the interaction between the snare proteins and the regulatory proteins. to achieve this objective, i express and purify the different proteins involved in the process of snare-mediated fusion and thereafter subject them to appropriate biochemical characterization. in order to assess the role of the purified proteins in the process of fusion, i reconstitute them into liposomes and perform in-vitro lipidmixing assays. these assays are based on f orster resonance energy transfer (fret). based on the discretion of assessing the protein-protein or protein-lipid interactions, either the proteins or the lipids can be fluorescently labeled. also, the lipid compositions can be varied in order to assess the effect of lipid on the function of the respective protein. fluorescence-based anisotropy measurements can also provide information about the degree of freedom of a protein, indirectly providing information about the kinetics of a reaction. employing these techniques, i observe that munc 18-1 leads to displacement of syntaxin from a complex of syntaxin and snap25. also, a complex of syntaxin and munc 18 is resistant to the action of the aaa-atpase, nsf and its co-factor asnap, implicating this complex as a strong candidate for acting as the starting point for the process of neurotransmitter release. munc 18 also appears to enhance lipid-mixing by interacting with the snare-complex. further investigations on the same lines can provide very useful insights into the process and can help us unravel the secrets that underlie the beauty of the exquisitely regulated process of neurotransmitter release. binding of thymidine nucleotides to a viral thymidine monophosphate kinase aldo a. 3 centro de investigaci on en alimentaci on y desarrollo theme: biochemistry there is great interest in the evolution and activities of fish trypsins, since they appear to have evolved into different families. the cdna for trypsin iii from the monterey sardine (sardinops sagax caerula) was obtained and its deduced amino acid sequence matched its identity with a purified protease from the fish by mass spectrometry analysis. molecular modeling of sardine trypsin iii compared to other homologs showed a typical trypsin fold with all the cognate components for catalysis, and specific amino acid distribution that are possible factors that explain the cold adaptation. from phylogenetic analysis, sardine trypsin iii belongs to the novel y family, which is proposed to have evolved for cold adaptation. the obtained recombinant trypsin iii showed a low catalytic efficiency, but it remained active at cold temperatures, similar to other cold-adapted trypsins. the cold-adaptation of sardine trypsin iii opens a wide range of biotechnological applications for this protease and is also interesting from the serine protease structure-function relationship point of view. fungicidal mechanism of scolopendin 2, a cationic antimicrobial peptide from centipede heejeong lee 1 , dong gun lee 1 drastically (from 6.5 x 10-3 to 2 x 10-3 colonies) upon deletion of this 130 residues domain from the full length trai. we are investigating the structure and function of this very c-terminal end of trai using nmr spectroscopy. for the backbone assignment we used slice-selectively homonuclear broadband decoupled spectra along with standard experiments. three-bond scalar coupling constants were obtained through real-time j-upscaling experiments. with the backbone assignments, we have the first hand evidence which shows that his domain is for the most part intrinsically disordered, but contains short a-helical regions. structural development, interaction studies to find the binding partner and transition of disorder to order orientation of this domain will be further investigated in this project. here we investigated a model system where mab aggregation is induced by increasing the ionic strength (nacl) at low ph. the aggregation depends both on protein and sodium chloride concentration. with nanoparticle tracking analysis (nta) and micro flow imaging (mfi) the aggregation formation was further characterized. aggregation can be partially reverted by lowering the ionic strength as determined by soluble monomer concentration measurement using se-hplc: parts of insoluble aggregates could be solubilized as soluble aggregates, dimers or even monomers. a quasi equilibrium is formed in between the subtypes. the whole aggregation process was examined by ftir and cd-spectroscopy to identify structural changes of the mab. screen of protective additives: the effect of osmolyte additives on aggregation kinetics and final aggregate concentration is investigated, revealing protective effects in both cases. in a screen with more than 200 compounds not only the aggregation propensity was studied but also structural changes. the aggregation index (quantity for colloidal stability) and the melting point (quantity for conformational stability) measured by differential scanning fluorimetry were determined. the used mtp format screen has potential for buffer optimization and formulation development. structural biology and protein dynamics tetraspanin cd81 has a broad range of cellular functions, such as integrin association forming tetraspanin-enriched domains, synapse formation between b and t cells, cell adhesion, motility, invasion and signalling. furthermore, cd81 is one of the four receptors involved in the cell entry of hepatitis c virus (hcv) and therefore infection onset, one of the major causes for chronic liver disease resulting in cirrhosis and hepatocarcinoma. human cd81 large-extracellular-loop (hcd81lel) is composed of a "stalk" and a "head" subdomain; with the latter interacting with hcv-e2 glycoprotein. we present four novel hcd81lel crystal forms. analysis of the fourteen independent observed hcd81lel high-resolution x-ray structures suggests that the dynamism of the hcd81lel head-subdomain is an inherent molecular property, an observation supported also by molecular dynamics (md) studies. we classify the conformations in three distinct clusters (closed, intermediate and open) , which are seen both in the crystal structures and in the molecular dynamics simulations. the md simulations also show that conformational variability is modulated by ph changes, with distinct probability for each cluster at acidic and neutral ph. furthermore, in silico docking of the recent e2core structure with three of the major types of hcd81lel head-subdomain clusters highlights hydrophobic interactions as the major forces in the e2core: hcd81lel recognition mechanism. we propose that the flexibility of the hcd81lel is exploited by hcv at different stages of cell entry from virus attachment to internalization and fusion with the endosomal membrane. our results provide important insights on the basic mechanism governing hcv binding to hcd81, and can help structure-based drug design of entryinhibitors of hcv. allophycocyanin of gracilaria chilensis: from gene to function jorge dagnino-leone 1 , jos e martinez-oyanedel 1 , marta bunster-balocchi 1 1 universidad de concepci on theme: structure-function relationship of proteins the phycobilisomes (pbs) are auxiliary photosynthetic complexes that allow cyanobacteria and red algae to enhance the energy uptake in the range of 490-680 nm. in gracilaria chilensis, an eukaryotic red algae, pbs is composed of phycoerythrin (pe), phycocyanin (pc) and allophycocyanin (apc); these proteins possess chromophores which capture energy and then transfers it to photosytems. pbps are oligomers of a ab heterodimer; it oligomerizes into a trimer (ab)3, this trimer has discoidal shape and it is associated in hexamers (ab)6, several of this hexamers forms cylinder-like structures. pbs has 2 components: antennas and core. the antennas are composed of pe and pc, whose function is to capture energy between 490-570 and 590-625 nm respectively and transfer it to the core. the core is formed by apc, which can absorb energy in the 620-650 nm range. apc emission allows transferring energy to the photosystems with high efficiency. pbs is also composed by linker proteins which allow the correct assembly of pbs and possibly regulate the energy transfer. the main goal in our group is to build an atomic model of the gracilaria chilensis phycobilisome. we have solved the crystal structure of pe and pc and created an antenna model. at present we are working in apc and the chromophorilated linker proteins. the objective of the present work is to create a model of the core of gracilaria chilensis; to achieve these we have used molecular biology, biochemistry and bioinformatics techniques. we designed oligonucleotides primers for the four allophycocyanin subunits genes and for the globular domain of the apce linker. these primers were used in pcr experiments to obtain the genes sequences. the sequences were translated to a aminoacid sequences and used to build a 3d model for apc subunits and trimers using the software modeller. on the other hand we purified and analyzed the spectroscopic properties of apc from gracilaria chilensis using absorption and fluorescence spectroscopy. we also determined apc oligomerization state using gel filtration. molecular docking using the cluspro server was performed to obtain a hexamer and apc cylinder models. based on electron micrographs obtained by our lab a tri-cylindric core model was built. all the models were submitted to a molecular dynamics using gromacs software. finally we determine possible energy transfer pathways in the core model applying the extended forster equation, spectroscopic data from literature and the transition dipole moments of each of the chromophores present in the core. as conclusion of this work we built the first atomic model of gracilaria chilensis phycobilisome core and propose energy transfers pathways inside the core in the context of a phycobilisome. novel practical strategies to access artificial metalloenzymes marco filice 1 , jose miguel palomo 1 1 departamento de biocat alisis, instituto de cat alisis, csic protein chemistry and engineering since the first report, the design of artificial metalloenzymes has rapidly been converted into an important topic in biological and inorganic chemistry due to their potential applications in synthetic chemistry, nanoscience and biotechnology. the combination of a catalytically active organometallic moiety with a macromolecular host has permitted the creation of biohybrids, a new kind of heterogeneous catalytic entities combining the attractive features of both homogeneous and enzymatic systems. presenting our most recent achievements in this research area, here we describe two novel powerful and promising approaches focusing the practical synthesis and large scale production of heterogeneous artificial metalloenzymes showing chimeric activity. the first strategy is based on the in situ synthesis of noble metal nanoparticles and their supramolecular assembly with a microbial lipase from candida antarctica (fraction b) finally creating an ultra-active organometallic-enzyme heterogeneous nanobiohybrid. in the second approach, combining different protein engineering protocols (molecular biology, orienting immobilization, solid-phase bioorganic modification and bioinformatic tools), an orthogonal solid-phase strategy creating novel unnatural catalytic sites was designed and optimized. the application of such a strategy onto the structure of the lipase from geobacillus thermocatelunatus permitted the generation of a heterogeneous artificial metallolipase with chimeric activity. as proof-of-concept, the combinatorial library of generated artificial metalloenzymes obtained by both strategies was successfully assessed in a set of different synthetic reactions (selective c-c bond formation as suzuki, heck or diels-alder reactions) and also combining both activities (metallic and enzymatic) in cascade processes such as dynamic kinetic resolution of amines or production of arylamines. the obtained results were excellent in all cases. extending this strategy to other enzymes, proteins and catalytic metals, we envisage the creation of a combinatorial library of programmable artificial enzymes useful for a wide set of applications (i.e. fine organic and medicinal chemistry, bioremediation or biomedicine). proteomic examination of the yeast nuclear pore complex dynamics protein turnover and exchange nuclear pore complexes (npcs) are proteinaceous assemblies situated in nuclear envelopes of eukaryotic cells. the main function of the npc is the selective transport of macromolecules. npcs also partake in other functions, such as nuclear organization and gene regulation. the core scaffold of the npc is thought to be a stable structure, while the peripheral components exchange at various rates. however, these phenomena have not been elucidated in detail. the recent findings that yeast daughter cells get a higher proportion of the old npcs and the core scaffold hardly turns over raise the possibility that the exchange of the peripheral nucleoporins can be a repair mechanism. yeast provides a useful organism for the interrogation of nucleoporin exchange, as it performs closed mitosis; hence the only mixing of npc constituents is due to exchange. we have developed a panel of genetic tools providing for conditional induction and repression of nucleoporins. by combining these switches with stable isotope metabolic labeling and affinity capture, cross linking coupled to mass spectrometry, we are able to distinguish between pre-existing and newly synthesized proteins and quantify their relative amounts in the npc. our preliminary findings are in agreement with results obtained in other organisms: the core scaffold of the npc (inner ring, outer ring) appears to be stable, however does exchange slowly over time, while peripheral components exchange faster. by looking at the exchange rates of yeast nucleoporins we hope to gain insight into the npc biology of actively dividing eukaryotic cells. active site clustering identifies functional families of the peroxiredoxin superfamily angela harper 1 , janelle leuthaeuser 2 , patricia babbitt 2 , jacquelyn fetrow 3 1 department of physics, wake forest university, 2 department of molecular genetics and genomics,-wake forest university, 3 departments of physics and computer science, wake forest university bioinformatics understanding the relationships between proteins is vital to increasing our knowledge of the protein universe. while there are large databases of sequence information, the massive data influx over the past decade has prevented adequate classification of proteins at the molecular function level. however, it has been previously suggested that a protein's active site information may correlate with these known molecular functional differences; thus, active site profiling was developed to use residues around the active site of a protein to relate proteins. subsequently the deacon active site profiler (dasp) was developed to create these active site profiles and search them in a database, such as gen-bank, in order to find proteins with similar active site environments. by using dasp to computationally cluster proteins based on the similarity of their active site profiles, the peroxiredoxin (prx) superfamily was analyzed through active site similarity methods. the residues from the active site of each prx structure were extracted and clustered, and these profiles were iteratively searched in genbank through a multi-level iterative sequence searching technique (misst). the prx superfamily has been studied by experts, allowing the results of these searches to be compared to a well-annotated group of proteins. while previous sequence based evolutionary methods have been unable to identify functional differences between some subgroups of the prxs, notably the ahpc-prx1 and prx6 subgroups, misst discretely separates these subgroups. classifying prx proteins into functionally relevant groups using computational active site similarity methods lays the foundation for an automated process for identifying protein functional groups beyond the prx superfamily. synthesis and conformational studies of glycoprotein n homolog of bovine herpesvirus 1 (bhv-1) by using cd, nmr and molecular modelling it serves as a chaperone for viral glycoprotein m and, in its gm-unbound form, acts as an inhibitor constraining the transporter associated with antigen processing (tap). the ul49.5/gm complex formation is required for the maturation and proper trafficking of both viral proteins. in the absence of gm, ul49.5 blocks transport of antigenic peptides by tap and their mhc i-restricted presentation. the molecular mechanism of ul49.5 activity still remains elusive. in order to investigate the structural requirements for biological function ul49.5 study was conducted using cd, nmr and molecular dynamics methods. the data obtained with the use of high purity synthetic peptides encompassing ul49.5 confirmed the presence of an alpha-helix structure, formed preferentially in the presence of dodecylphosphocholine (dpc) micelles as a membrane-like environment. in order to determine the three-dimensional structure of ul49.5 protein in the present work its nmr solution structure in the presence of membrane-like environment was performed. the nmr data were used as a set of restraints for a simulated annealing protocol that generated 3dstructures of the colin johnson 1 , sara codding 1 1 membrane proteins resealing of tears in the sarcolemma of myofibers is a necessary step in the repair of muscle tissue. defects in this repair process are responsible for muscular dystrophy and cardiomyopathy. the repair pathway is triggered by the influx of calcium through lesions in the membrane, which result in membrane fusion and patching of the wound. recently dysferlin has been identified as a calcium binding protein essential for sarcolemma repair, as well as other snare mediated exocytotic events including cytokine and acid sphingomyelinase secretion. in this presentation we demonstrate a direct interaction between dysferlin and the snare proteins syntaxin 4 and snap-23. in addition, fret and in vitro reconstituted lipid mixing assays indicate that dysferlin accelerates snare heterodimer formation and snare mediated lipid mixing in a calcium sensitive manner. our results suggest a model whereby dysferlin acts as a calcium sensing snare effector for exocytosis and membrane fusion. exploring the therapeutic potential of a peptide derived from a poxviral immune evasion protein: nmr determination of the solution structure of viper and its inactive mutant toll-like receptors (tlrs) have a role in viral detection leading to cytokine and ifn induction, and as such they are targeted by viruses for immune evasion. the poxviral protein a46 has been identified to inhibit tlr signaling by interacting with tir domain-containing proteins of the receptor complex to collectively inhibit all tlr adaptor proteins that positively regulate transcription-factor activation (1). one 11 aa peptide (kysf-klilaey) termed viper (viral inhibitory peptide of tlr4) was reported to retain the inhibitory properties of full length a46 against tlr4 signaling. a 9r homopolymer delivery sequence at the c-terminus provided delivery of the peptide into cells. structural comparisons are presented between 9r-viper, which is active in preventing tlr4-dependent cytokine induction in cell culture, and a mutant that exhibited loss of function (9r-viper l6a,e10a), through solution nmr spectroscopy. we find that despite a relatively minor sequence difference, the loss of hydrophobicity as well as negative electrostatic interactions result in subtle but potentially significant differences in the region of the peptide proposed to interface with tlr4. reference: wake forest university, 2 wake forest university, 3 university of california san francisco protein function prediction the elucidation of protein molecular function lags far behind the rate of highthroughput sequencing technology; thus, it is essential to develop accurate and efficient computational methods to define functional relationships. protein clustering based on sequence similarity has emerged as a simple, high-throughput method for defining protein relationships, but sequence-based techniques often inaccurately define molecular function details. active site profiling (asp) was previously developed to identify and compare molecular details of protein functional sites. protein similarity networks were created using both active site similarity and sequence similarity for four manually curated superfamilies, and results demonstrate that asp-based clustering identifies detailed functional relationships more accurately than sequence-based clustering. building on this, two iterative pipelines were developed using active site profiling and profile-based searches to cluster protein superfamilies into functional groups. first, the two level iterative clustering process (tulip) utilizes active site profiling and iterative pdb searches to divisively cluster protein structures into groups that share functional site features. across eight superfamilies, tulip clusters exhibit high correlation with expert functional annotations. subsequently, the multi-level iterative sequence searching technique (misst) utilizes iterative profile-based genbank searches to identify protein sequences that belong in each tulip group. the results indicate that these asp-based methods accurately and efficiently identify functionally relevant groups through a process that can be applied systematically and on a large-scale. moreover, the approach can be applied more quickly than detailed manual curation, suggesting its value in guiding annotation efforts. dept. biochemistry and molecular biology. university of valencia, 2 lab of peptide and protein chemistry. centro de investigaci on pr ıncipe felipe membrane proteins changes in the equilibrium between pro-survival and pro-apoptotic members of the b-cell lymphoma-2 (bcl-2) protein family at the mitochondrial outer membrane (mom) induce structural changes that committed cells to apoptosis. bcl-2 homology-3 (bh3)-only proteins participate in this process activating pro-apoptotic effectors and promoting permeabilization of the mom. the membrane association of bh3-only proteins is a controversial issue due to the lack of a canonical carboxyl-terminal (c-terminal) transmembrane (tm) domain. we used an in vitro transcription/translation system to study the insertion capacity of these hydrophobic c-terminal regions of the bh3-members bik, bim, noxa, puma and bmf into microsomal membranes, and an escherichia coli complementation assay to validate our results in bacterial cells. furthermore, we have fused these hydrophobic regions to gfp to investigate the subcellular sorting. these results will allow further refinement in the elaboration of the bcl-2 protein-protein and protein-membrane interactome network. alexis peña 1 , flaviyan jerome irudayanathan 1 , shikha nangia 1 1 syracuse university, dept. of biomedical and chemical engineering computational modeling, biostatistics, biomedical and chemical engineering tight junctions (tj) are vital intracellular barriers that are responsible for regulating paracellular transport. claudins, a family of abstract small transmembrane proteins with approximately 27 members, are an integral part of the tj strands. tight junctions provide molecular-level protection and prevent infection and toxins from entering the body; in the same sense tjs allow nutrients and vital solutes to pass through. claudins are associated with various diseases including metastatic cancer as well as an entry point for many viruses. despite their importance and abundance in all cell membranes and their ubiquitous nature, the exact 3-d structure of claudins has remained elusive to traditional x-ray crystallographic and nmr studies. in this investigation, a computational approach was used to determine the claudin structure of claudin 1-10. homology modeling, molecular dynamic simulations, and reverse mapping were employed to predict the protein structures with relative accuracy. understanding structure of claudin proteins and its interaction at the molecular level can lead to effective drug delivery technology. determination of optimal conditions for an isothermal titration calorimetry essay to obtain kinetic parameters of trypsin i from pyloric caeca of monterey sardine (sardinops sagax caerulea) idania emedith quintero reyes 1 , francisco javier castillo y añez 1 , enrique fernando vel azquez contreras 1 , roc ıo sugich miranda 1 , david octavio corona mart ınez 1 , aldo alejandro arvizu flores 1 , ivet cervantes dom ınguez 1 1 protein kinetics determination of optimal conditions for an isothermal titration calorimetry essay to obtain kinetic parameters of trypsin i from pyloric caeca of monterey sardine (sardinops sagax caerulea) trypsin is the most studied alkaline protease and it s very common to found isoforms from this protein as the case for monterey sardine (sardinops sagax caerulea); as it shows an expression of trypsin i and trypsin iii according to the cdna characterization. trypsin i was determine to be a cold adapted enzyme as it shows a higher catalytic efficiency (kcat/km) than the mesophilic counterparts. the kinetic parameters were obtained by spectrophotometric essays, which are not fallible for all the enzymes because native, recombinant or mutant enzyme activity could be below the detection limit of the assay, opaque or turbid solutions interfere with spectrophotometric detection, etc. alternative tools as the isothermal titration calorimetry (itc) can measure enzyme kinetics using thermal power generated by the enzymatic conversion of substrate to product; were the rate of reaction is directly proportional to thermal power. the objective of this study was to stablish the optimum conditions to obtain kinetic parameters of trypsin i from pyloric caeca of monterey sardine using itc. to reach the objective trypsin i was purified from viscera of monterey sardine using molecular exclusion and affinity chromatography obtaining a yield of 1.1 mg/ml. at 208c kcat and km of tryipsin i form monterey sardine were 14.6 s-1 and 1.4 mm respectively. at 158c were 13.6 s-1 and 4 mm (kcat and km) and at 48c kcat was 0.454 s-1 and km 0.52 mm. the kinetic parameters obtained by spectrophotometric assay at 258c were kcat and km 436 s-1 and 1.8 mm respectively. at 208c the kcat was 409.7 s-1 and km 1.8 mm and at 158c kcat 288 s-1 and km 3mm. comparing the values obtained for kcat with the spectrophotometric essay were higher 29 fold than those obtained by itc and the values in km were similar by both methods. even though the differences in kcat, we can reassert the psychrophilic behavior of trypsin i as the catalytic efficiency is higher by both methodologies. in the understanding that the kinetic behavior of enzymes is important to not only understanding biochemical pathways and catalytic mechanisms but is again a fruitful area for drug discovery and development; so the itc provides a universal approach to determining the kinetic behavior of enzymes and can yield in a single experiment a complete set of kinetic parameters for an enzyme-catalyzed reaction that can be applied for the different alkaline proteases from pyloric caeca of monterey sardine (sardinops sagax caerulea). mysterious world of stress-responding sigma factors in bacillus subtilis olga ramaniuk 1 1 protein-dna interaction bacterial transcription is mediated by the rna polymerase holoenzyme containing sigma factors -essential proteins for the initial step of transcription that recognize and bind to promoter dna. the primary sigma factor is essential in exponential phase of growth while alternative sigma factors are active during transcription under stress conditions. this project has three main aims. the first aim is to explore the binding properties of b. subtilis alternative sigma factors; specifically, whether sigma factors lacking the autoinhibitory domain 1.1 can bind to promoter dna in the absence of rnap. the second aim explores whether rnap associated with alternative sigma factors is regulated by the concentration of the initiation nucleoside triphosphate. the third aim is to define the regulon of sigma i. in order to achieve our aims, 7 out of 17 alternative sigma factors were successfully purified using affinity chromatography and ion exchange chromatography. we set up in vitro transcription system with selected sigma factors and initiated experiments with sigma i regulon determination. results named above and our future findings will help to better understand gene expression regulation on the level of transcription initiation. this work was supported by grant no. p305-12-g034 from the czech science foundation. assessing the costs and benefits of protein aggregation protein aggregation and cell fitness protein aggregation has been associated with numerous diseases but also with important cellular functions such as epigenetic inheritance. here we present a population genetics approach to infer the costs and benefits of protein aggregation on cell fitness. this information is crucial to understand how cellular systems tolerate the formation of protein deposits and which factors modulate this event. using our experimental system, we measured different protein aggregation effects (deleterious, neutral or beneficial) within the same genomic background. single cell analyses, within the same population, showed stochastic variability in the aggregate's size and in its effect on cell fitness. our data indicates that, in certain conditions, protein aggregation can enhance population variability and survival expectancy. overall, these results suggest that the presence and formation of protein aggregates could be almost harmless whereas the associated gain and loss of function are critical for the cell. revealing the key role of negatively charged residues of heme sensor proteins involved in geobacter sulfurreducens' signal transduction pathways marta a. silva 1 , telma c. santos 1 , teresa catarino 2 , carlos a. salgueiro 1 1 ucibio-requimte, departamento de qu ımica, fct-unl., 2 instituto de tecnologia qu ımica e biol ogica, unl signal transduction proteins bacterial chemotaxis systems sense and regulate the microbe mobility in response to environmental conditions. such mechanisms constitute a striking example of cell motility to gain advantages for cell survival and permit the bacteria to fill important niches in a diversity of anaerobic environments [1] . geobacter sulfurreducens (gs) is an anaerobic bacterium with a considerable respiratory versatility whose genome encodes for an unusual family of methyl-accepting chemotaxis proteins (mcp), each containing at least one heme c-binding motif [2] . these sensor proteins, gsu0582 and gsu0935, are involved in signal transduction pathways mediated by chemotaxis-like systems [3] . the thermodynamic and kinetic characterization of the sensors gsu0582 and gsu0935 by visible spectroscopy and stopped-flow techniques, at several ph and ionic strength values revealed that sensor gsu0935 midpoint reduction potentials are lower than those of gsu0582 at all ph and ionic strength values and the same were observed for the reduction rate constants [4] . the origin of the different functional properties of these closely related sensor domains are rationalized in the structural terms showing that gsu0935 has two extra negatively charged residues in the vicinity of the heme group, which have no counterpart in gsu0582: glu89 and asp57. residue asp57 is less exposed compared to glu89 and it was suggested that its carboxylic group might have a role in the modulation of the heme reduction potential of gsu0935. to investigate this, both residues were replaced by a positively charged amino acid (lysine) and by a neutral one (asparagine or glutamine). for the mutants with enough expression, a functional characterization was carry out, using several spectroscopic techniques, including uv-visible and cd, together with kinetics and potentiometric measurements. significant changes on the reduction potential values are observed when a negative charge is replaced by a positive one at position 57 or 89. therefore, the decrease of the reduction potential in asp57 and glu89 mutants reinforces the hypothesis that the higher reduction potential observed for heme sensor domain gsu0582 is related with the less negative electrostatic surface around the heme. this work provides, for the first time, evidence for the co-existence of two similar methyl-accepting chemotaxis proteins functioning in different working potential ranges. these proteins are responsible to allow geobacter sulfurreducens triggering an adequate cellular response in different anoxic subsurface environments. 1 national autonomous university of mexico, faculty of medicine, 2 national autonomous university of mexico, faculty of chemistry, 3 national autonomous university of mexico, institute of chemistry molecular evolution the glycolytic enzyme triosephosphate isomerase (tim) is an oligomeric (b/alpha)8 barrel that catalyses the interconversion of d-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate in a diffusion-limited reaction. although each subunit has its own active site, naturally occurring monomeric tims have not been reported; in fact, monomer association is very tight. tim topology is well conserved among the three domains of life. nevertheless, their folding mechanism and inhibition properties vary across species. comparative studies of proteins have proved to be very useful in understanding the relationship between sequence and physicochemical properties, however, they lack the capacity to give a more integrative and evolutive correlation. in order to elucidate how the catalytic properties, the oligomerization state and the stability of extant tims arose, in this work we examined the molecular history of eukaryotic tim through ancestral protein reconstruction methods (maximum likelihood) and the subsequent physicochemical characterization of the resurrected enzymes. we first characterized in detail the protein corresponding to the last common ancestor of animals and fungi (tim63). the cd and fluorescence spectra of tim63 are similar to those of extant tims. secondary structure is lost in a cooperative transition with tm 5 68.78c. the enzyme loses activity upon dilution suggesting that only the dimer is active. dilution experiments followed by isothermal titration calorimetry indicate that dissociation enthalpy is small; moreover the heat capacity change observed is three times higher than the one predicted for a rigid body dissociation process, suggesting partial unfolding of the monomers. when compared with extant tims, the catalytic efficiency of tim63 is reduced 10-fold, whereas binding of pgh, a transition-state analogue, shows a similar thermodynamic signature. these data indicate that although monomer association may have been less tight in ancestral tims, catalysis has been always linked to oligomerization. analysis of the crystal structure of tim63, obtained at 1.9 å resolution, suggests that the lack of four salt bridges observed in the interface of extant tims is responsible for the low dimer stability. in order to test this hypothesis we also studied the stability of four younger reconstructed ancestors that acquired the salt bridges in two different phylogenetic lineages. we found a correlation between the appearance of stabilizing interactions in the interface, dimer stability and catalysis; suggesting that these salt bridges are partially responsible for extant dimer stability and shed light on the dimeric nature of extant tims. receptor protein-tyrosine phosphatases: dimerization, receptor kinase interaction and allosteric modulation elizabeth dembicer 1 , damien thevenin 1 1 department of chemistry, lehigh university theme: receptor tyrosine kinase and receptor protein phosphatase signaling many cell-signaling events are regulated through reversible tyrosine phosphorylation of proteins, which is controlled by the counterbalanced actions of two key enzyme families: protein tyrosine kinases and protein tyrosine phosphatases. interestingly, both families include transmembrane receptor-like enzymes, namely the receptor tyrosine kinases (rtks) and the receptor-like ptps (rptps). while the regulation and actions of many rtks are well characterized, the mechanisms controlling the enzymatic activity of rptps and how they interact with their substrates remain to be fully explained. thus, understanding how these receptors function and interact will give fundamental insights into how tyrosine phosphorylation is finely tuned in cells, and how it can be modulated. increasing evidence indicates that rptps, like rtks, are regulated by homodimerization. however, it appears that homodimerization inhibits the activity of most rptps. even though the transmembrane (tm) and the juxtamembrane domains have been proposed to be involved in this process, there is no clear structure-based proposal for the role of these regions. moreover, several rptps have been identified as candidate regulators of rtks. in particular, the receptor-type tyrosine-protein phosphatase eta (ptprj; also known as dep1 or cd148) is capable of attenuating egfr tyrosine phosphorylation. physical interactions of egfr with ptprj at the cell surface have been documented, but the basis for these interactions is unknown. here, using a dominant-negative transcriptional activator-based assay (dn-aratm), and mutagenesis analysis, we show that: (1) ptprj has a strong tendency to homodimerize, (2) ptprj heterodimerizes with egfr through tm-tm interactions, (3) these interactions are mediated by specific residues, and can be modulated by the delivery of peptide binders. this work represents the first structure-function study of rptp-rtk interaction, and may not only result in significant progress towards a better understanding of the basic biology of rptps in cancer cells, but also offer new possibilities for targeting protein tyrosine phosphatases for therapeutic modulation of egfr in oncology. inhibiting egfr dimerization and signaling through targeted delivery of juxtamembrane domain peptide mimics using phlip anastasia thevenin 1 , kelly burns 1 , janessa guerre-chaley 1 , damien thevenin 1 1 regulating receptor tyrosine kinase signaling the elevated phosphorylation of key regulatory tyrosines on oncogenic signaling proteins that result from aberrant protein tyrosine kinases activity plays well-abstract established roles in promoting tumorigenesis and in the high frequency with which resistance arises to existing therapeutic treatment. for instance, this is the case for the epidermal growth factor receptor (egfr). thus, there is a clear need for novel specific targeting methods to inhibit the activity of receptor protein tyrosine kinases, such as egfr, in cancer. egfr becomes activated upon ligand binding to the extracellular domain, leading to receptor dimerization. the juxtamembrane (jm) domain of egfr is critical for intrinsic tyrosine kinase activity and receptor dimerization by stabilizing the active conformation of egrr through the formation of a antiparallel helical dimer. therefore, peptides mimicking the jm domain -if specifically delivered to cancer cells -have the potential to prevent egfr dimerization, receptor activation, downstream signaling, and thus to attenuate aberrant egfr activity in cancer cells. here, phlip (ph low insertion peptide), a peptide that can selectively target cancer cells and tumors based solely on their extracellular acidity, is used to selectively translocate the jm domain of egfr in cancer cells to prevent egfr dimerization. at ph above 7, phlip is soluble and unstructured, however, when exposed to lower ph such as observed in tumors, phlip inserts as a transmembrane (tm) alphahelix, allowing the direct translocation of cargo molecules into the cytoplasm. using the dominant negative arac-based transcriptional reported assay (dn-aratm), which assesses jm and tm domain interactions in cells membranes of e. coli, we show that phlip-jm is able to disrupt egfr dimer by 50%. current work is focused on testing the ability of such phlip-jm peptide conjugate to perturb egfr homodimerization and decrease downstream signaling through soluble kinases, such as akt and erk, in cancer cells. the thumb subdomain of yeast mitochondrial rna polymerase is involved in processivity, transcript fidelity and mitochondrial transcription factor binding gilberto velazquez 1 , luis brieba 2 , rui sousa 3 1 universidad de guadalajara, 2 langebio cinvestav, 3 university of texas healthsscience center at san antonio dna protein interaction abstract single subunit rna polymerases have evolved two mechanisms to synthesize long transcripts without falling off a dna template: binding of nascent rna and interactions with an rna:dna hybrid. mitochondrial rna polymerases share a common ancestor with t-odd bacteriophage single subunit rna polymerases. herein we characterized the role of the thumb subdomain of the yeast mtrna polymerase gene (rpo41) in complex stability, processivity, and fidelity. we found that deletion and point mutants of the thumb subdomain of yeast mtrna polymerase increase the synthesis of abortive transcripts and the probability that the polymerase will disengage from the template during the formation of the late initial transcription and elongation complexes. mutations in the thumb subdomain increase the amount of slippage products from a homopolymeric template and, unexpectedly, thumb subdomain deletions decrease the binding affinity for mitochondrial transcription factor (mtf1). the latter suggests that the thumb subdomain is part of an extended bindingsurface area involved in binding mtf1. design principles of membrane protein structures vladimir yarov-yarovoy 1 , diane nguyen 1 1 membrane protein structure membrane proteins play key role in cellular signaling and ion transport. statistical analysis of expanding database of high-resolution membrane protein structures in protein data bank (pdb) provides useful information about membrane protein structure and function. we used rosettamembrane software (yarov-yarovoy v et al (2006) proteins) to analyze 300 unique alpha helical membrane protein structures in pdb and derive knowledge based energy function for membrane protein structure prediction, membrane protein-protein docking, and membrane protein design. the rosettamembrane residue environment energy term is based on amino acid propensities in hydrophobic, interface, and water layers of the membrane and depends on the residue burial state -from being completely buried within a protein environment to being completely exposed either to the lipid or water environments. residue buried state is determined from the number of residue neighbors within 6 and 10 å spheres. the rosettamembrane residue-residue interaction term is based on the propensities of amino acid pairs to be in close proximity to each other within hydrophobic, interface, and water layers. results of our statistical analysis reveal fine details of favorable and unfavorable environments for all amino acids types in all membrane layers and residue burial states. we find that large hydrophobic amino acids are favorable facing the hydrophobic core of the lipid bilayer. small amino acids are favorable facing the protein core within the hydrophobic layer of the membrane. aromatic or positively charged amino acids and favorable facing the lipid head groups. residue-residue interactions are often favored between polar and charged amino acids and also between some of small and large hydrophobic amino acids inside of the protein core within the hydrophobic layer of the membrane. these data will be useful for rational design of novel membrane protein structures and functions. coordinated gripping of substrate by subunits of a aaa1 proteolytic machine ohad yosefson 1 , andrew nager 1 , tania baker 1 , robert sauer 1 1 protein quality control' or 'protein degradation' hexameric aaa1 protein-remodeling machines use conserved loops that line the axial pore to apply force to substrates during the mechanical processes of protein unfolding and translocation. an open question in the aaa1 field is whether pore loops from different subunits of the hexameric ring grip the substrate coordinately (all six subunits involved), independently (one subunit at a time involved), or partially coordinated (two or three subunits at a time). to answer this question, we studied covalently linked hexamers of the e. coli clpx unfoldase bearing different numbers and configurations of wild-type and mutant pore loops and challenged these variants with protein substrates with a broad range of stabilities. we find that successful unfolding of increasingly resistant substrates requires the coordinated action of a greater number of wild-type pore loops. our results support a mechanism in which a power stroke initiated in one subunit of the clpx hexamer results in the simultaneous movement of all six pore loops, which coordinately grip and apply force to the substrate. structure and function of the toc159 m-domain, and its role in targeting the preprotein receptor to the chloroplast outer envelope membrane matthew smith 1 , shiu-cheung lung 2 , prem nichani 1 , nicholas grimberg 1 , j. kyle weston 1 , shane szalai 1 , simon chuong 2 1 deartment of biology, wilfrid laurier university, 2 department of biology, university of waterloo chloroplast biogenesis and function rely on the import of thousands of nucleus-encoded preproteins from the cytosol. preprotein import is supported by the toc and tic (translocon at the outer and inner envelope membranes of chloroplasts) complexes, which work cooperatively to translocate preproteins across the double-membrane envelope to the chloroplast interior. toc159 is one of the preprotein receptors of the toc complex, is also encoded in the nucleus and post-translationally targeted to the chloroplast, and is comprised of 3 distinct domains: 1) the intrinsically disordered n-terminal acidic (a-) domain; 2) the central gtpase (g-) domain; and 3) the c-terminal membrane (m-) domain that anchors the protein to the chloroplast outer membrane (com) through an unknown mechanism. the m-domain has no known homologues and does not contain a predicted trans-membrane domain, but does contain intrinsic chloroplast targeting information at the extreme c-terminus. the m-domain also contains a predicted b-helix motif, which may be important for anchoring the protein to the com. we are interested in characterizing the structure of the m-domain and determining the nature of its association with the com, as part of our larger goal of understanding the role toc159 plays in protein import into chloroplasts. we are also interested in defining the precise nature of the targeting information contained within the extreme c-terminus of toc159, elucidating the targeting pathway that is used, and whether other com proteins use this pathway. we will present our most recent data on the structure, function and targeting of the toc159 m-domain. structural investigation of nlpc/p60 protein acquired by trichomonas vaginalis through a lateral gene transfer event jully pinheiro 1,2 , augusto simoes-barbosa 1 , david goldstone 2 1 microbiology, school of biological sciences, university of auckland, 2 structural biology, school of biological sciences, university of auckland trichomonas vaginalis is an extracellular flagellated protozoan parasite that causes the most common non-viral sexually transmitted disease, with approximately 200 million cases worldwide annually. nevertheless, the biochemical processes behind t. vaginalis infection and its interaction with the vaginal microbiota are still not well defined. in 2007 the draft genome sequence of trichomonas vaginalis strain g3 was described, identifying 60,000 protein-coding genes. of these, nine genes encode nlpc/p60-like members. this superfamily is widely represented in the different kingdoms of life and has diverse enzymatic functions, such as amidases, endopeptidases and acetyltransferases. previous studies have shown that members of this superfamily hydrolyze specific peptide linkages in bacterial cell walls affecting germination, vegetative growth, sporulation and division or cell lysis/invasion. as a typical eukaryote, the protozoan parasite t. vaginalis does not have a cell wall itself. previous studies suggest that the t. vaginalis nlpc/p60 genes were acquired via lateral gene transfer from bacteria and must have an important function, possibly controlling the vaginal microbiota and aiding parasite invasion and infection. to investigate the function of the nlpc/p60 family of proteins in t. vaginalis we have expressed, purified and crystallized a member tvag_119910 and report its three-dimensional structure, determined at 1.5 å resolution, by x-ray diffraction. the structure of the protein reveals a typical papain-like fold resembling peptidoglycan hydrolases from the nlpc/p60 family with a conserved cysteine and histidine; forming the catalytic residues. the protein contains two bacterial sh3 domains at the n-terminus. this domain acts as a general binding domain and is likely to aid the interaction of the nlpc/p60 domain with substrate components. combined with biochemical and enzymatic characterization, the structure of this nlpc/p60 protein will help to elucidate the molecular origin of its hydrolase activity and to decipher their putative role in the parasite infection. novel dna polymerases from red sea brine-pools: new potential polymerases for pcr application masateru takahashi 1 , etsuko kimura 1 , mohamed salem 1 , ulrich stingl 1 , samir hamdan 1 1 protein biotechnology the polymerase chain reaction (pcr) is a key tool in medical and biological research. the most common pcr reaction relies on the thermal cycling method that consists of repeated cycles of heating and cooling steps for dna melting and extension by the dna polymerase, respectively. the introduction of new dna polymerases to the market is a major area of development that tremendously helped in improving the performance and quality of pcr. nonetheless, pcr still requires optimization of salt and metal ion concentrations leaving a room in the market for introducing new dna polymerases that are robuster in their salt and metal ion concentration dependence. in this study, we will present the characterization of a novel archaeal dna polymerase from the red sea brine-pool (termed br3) and demonstrate how its enzymatic activity reflects on every aspects of the environment of the brine-pool -high tolerance to concentrations and types of salts and metal ions including utilization of zn21 ions in its active site. these results suggest that the brine-pool microorganisms are likely to contain novel chemical pathways to deal with its exterior harsh conditions. we will further show the mechanism of br3 polymerase how it was adjusted to be active in harsh condition. structural basis for the identification of the n-terminal domain of coronavirus nucleocapsid protein as an antiviral target ming-hon hou 1 , shing-yen lin 1 , chia-ling liu 1 , yu-ming chang 2 , jincun zhao 3 , stanley perlman 3 1 institute of genomics and bioinformatics, national chung hsing university., 2 institute of biological chemistry, academia sinica., 3 department of microbiology, the university of iowa drug discovery coronaviruses (covs) cause numerous diseases, including middle east respiratory syndrome and severe acute respiratory syndrome, generating significant health-related and economic consequences. covs encode the nucleocapsid (n) protein, a major structural protein that plays multiple roles in the virus replication cycle and forms a ribonucleoprotein complex with the viral rna through the n protein's nterminal domain (n-ntd). using human cov-oc43 (hcov-oc43) as a model for cov, we present the 3d structure of hcov-oc43 n-ntd complexed with ribonucleoside 5'-monophosphates to identify a distinct ribonucleotide-binding pocket. by targeting this pocket, we identified and developed a new coronavirus n protein inhibitor, n-(6-oxo-5,6-dihydrophenanthridin-2-yl)(n,n-dimethylamino)acetamide hydrochloride (pj34), using virtual screening; this inhibitor reduced the n protein's rna-binding affinity and hindered viral replication. we also determined the crystal structure of the n-ntd-pj34 complex. on the basis of these findings, we propose guidelines for developing new n protein-based antiviral agents that target covs. thermal and structural stability of ß-glucosidases gh1 maira artischeff frutuoso 1 1 departamento de bioqu ımica do instituto de qu ımica da universidade de são paulo enzymology we compared the stability of thermophilic b-glucosidases gh1 to mesophilic ones in the presence of denaturants as urea and high temperature by following the transitions between the native and unfolded states by tryptophan fluorescence, enzymatic activity and differential scanning fluorimetry (dsf). the bacterial b-glucosidases (bgla) and (bglb) of the mesophile paenibacillus polimyxa and bglucosidase (bglthm) of the thermophile thermotoga maritima were expressed as recombinant proteins in novablue (de3) and purified by affinity chromatography (ni-nta resin). these recombinant enzymes have very similar folding type structure (b/a)8 barrel, as shown in crystal structures and exhibited a characteristic peak between 330 and 340 nm in the tryptophan fluorescence spectra, indicating that those proteins are folded. circular dichroism analysis in the far-uv region (190 nm to 240 nm) also showed typical spectra of folded proteins with secondary structure composition of 47% of a-helix and 13% of b-sheets for bgla, 61% of a-helix and 2.5% of b-sheets for bglb and 30% of a-helix and 20% of b-sheets for bglthm. the average degree of accessibility to the exposed tryptophan residues in the native enzyme to increasing concentrations of the acrylamide suppressor (stern-volmer constant -ksv) is greater to bgla (9.49), but similar to bglb (3.17) and bglthm (3.84). the thermal stability determined by dsf was higher for bglb (tm 43.8 c) than for bgla (tm 35. 2 c) . the bglthm was stable at 478c and remained stable for up to 4 h at 808c. in addition the thermal inactivation kinetics at 478c evaluated by the relative remaining activity showed that bgla denaturation (kinactivation of 1.9 s-1) is faster than bglb (kinactivation of 31.3 s-1). on the other site, bglthm inactivation at 958c was a two-step process, which exhibited an initial fast step (kinactivation of 2.9 s 21) followed by a slow step (kinactivation of 0.2 s-1). the chemical denaturation by urea followed using tryptophan fluorescence showed a transition pl-039 covalent structure of single-stranded fibrinogen and fibrin oligomers cross-linked by fxiiia. the influence of free radical oxidation anna bychkova 1 , vera leonova 1 , alexander shchegolikhin 1 , marina biryukova 1 , elizaveta kostanova 1 , mark rosenfeld 1 1 n. m. emanuel institute of biochemical physics, russian academy of sciences protein structure and function native fibrinogen is a key blood plasma protein whose main function is to maintain hemostasis by virtue of producing the cross-linked fibrin clots under the effect of thrombin and fibrin-stabilizing factor (fxiiia). fxiiia-mediated isopeptide g-g bonds are known to be produced between g polypeptide chains of adjacent fibrinogen or fibrin molecules. but there are apparently conflicting ideas regarding the orientation of g-g bonds. in this study several peculiarities of self-assembly of fibrin(ogen) and induced oxidation of the proteins have been studied with the aid of elastic and dynamic light scattering, uv-, ftir-and raman spectroscopy methods. in the presence of fxiiia both the non-oxidized and oxidized fibrinogen molecules has been shown to bind to each other in the "endto-end" fashion to form the flexible covalently cross-linked fibrinogen homopolymers. to identify the orientation of g-g bonds in fibrin protofibrils a novel approach based on self-assembly of soluble cross-linked fibrin protofibrils and their dissociation in the urea solution of moderate concentrations has been applied. the results of elastic and dynamic light scattering coupled with analytical ultracentrifugation indicated the protofibrils to exhibit an ability to dissociate under increasing urea concentration to yield single-stranded structures entirely brought about by g-g bonds. the results of this study provide an evidence to support the model of the longitudinal g-g bonds that form between the g chains end-to-end within the same strand of a protofibril. since fibrinogen is known to be sensitive to ros the mechanisms of fibrinogen and fibrin self-assembly under induced oxidation have been investigated. in both cases the polypeptide chains of the oxidized fibrin(ogen) proved to be involved in the enzymatic cross-linking more readily than those of unaffected molecules. the enhancing role of the d:d interaction under oxidation could be considered as an compensatory mechanism in the assembly of fibrin when the d:e interaction is impaired. the experimental data on fibrinogen and fibrin oxidation acquired in the present study, being combined with our earlier findings, make it reasonable to suppose that the spatial structure of fibrinogen could be evolutionarily adapted to some ros actions detrimental to the protein function. the study was supported by the rfbr, research projects 14-04-31897mol_a and 15-04-08188a. structural and thermodynamic analysis of co-stimulation receptor cd28 phosphopeptide interactions with grb2, gads, and pi3-kinese sh2 domains in addition to the signaling produced by the binding of antigen-major histocompatibility complex to tcell receptors, co-stimulatory signals from other receptor-ligand interactions are required for full activation of t-cells. the cd28 receptor on the t-cell surface has been well characterized, and the binding of ligand to cd28 is critical for producing co-stimulatory signals. cd28 has no enzymatic activity and its cytoplasmic region consists of 41 amino acids that contain the sequence ymnm, in which the tyrosine residue is phosphorylated by kinase. the phosphorylated sequence, pymnm, is recognized by src homology 2 (sh2) adaptor proteins, such as growth factor receptor binding protein 2 (grb2), grb2related adaptor downstream (gads), and the phosphatidylinositol 3-kinase (pi3-kinase) regulatory subunit, p85. the consensus sequence for the binding of grb2 sh2 and gads sh2 is pyxnx, and that of p85 n-terminus sh2 (nsh2) and c-terminus sh2 (csh2) is pyxxm. we reported the high-resolution crystal structure of grb2 sh2 in complex with the cd28 phosphopeptide [higo et al., plos one 8, e74482, 2013] , and recently determined those of gads sh2, p85 nsh2, and p85 csh2. these data along with the results of binding thermodynamics analyzed using isothermal titration calorimetry, helped to elucidate the molecular recognition mechanisms of cd28 by adaptor proteins. the sh2 proteins were overexpressed in escherichia coli, and were purified using affinity and gel-filtration chromatography. the cd28 phosphopeptides, 8-residue (octp) and 12-residue (ddcp02), were synthesized using the solidphase supported technique, and were purified using reversed-phase chromatography. the crystals were obtained by the hanging-drop vapor diffusion method. x-ray diffraction data were collected at synchrotron radiation facilities, and the structures were determined by the molecular replacement method. the models of grb2 sh2, gads sh2, p85 nsh2, and p85 csh2 in complex with octp were refined at 1.35, 1.2, 1.0, and 1.1 å resolutions, respectively. the crystal structures showed that the phosphotyrosine phosphate moiety directly interacted with the side-chain of arginine in sh2, which is common in all complex structures. in the grb2 sh2 and gads sh2 complexes, the side-chain of asparagine at the py12 position forms a pair of hydrogen bonds with the main-chain amide and carbonyl groups of lysine in sh2. alternatively, in the p85 nsh2 and csh2 complexes, the side-chain of methionine at the py13 position is located in hydrophobic pockets of nsh2 and csh2, in which the hydrophobic interactions of csh2 would be stronger than those of nsh2. this idea is supported by the observed binding thermodynamics. the binding affinity of csh2 to ddcp02, because of a favorable enthalpy change, is about 10-fold higher than that of nsh2. the binding affinity of grb2 sh2 to ddcp02 is similar to that of gads sh2 to ddcp02, and is about 10-fold lower than that of nsh2 to ddcp02. these results indicate that the contribution of hydrophobic interactions of nsh2 and csh2 at the py13 position are stronger than those of hydrogen bonds of grb2 sh2 and gads sh2 at the py12 position. novel kinetochore protein complex from silkworm holocentric chromosomes takahiro kusakabe 1 , hiroaki mon 1 , jaeman lee 1 1 the kinetochore, which consists of centromere dna and a multilayered protein complex, plays important roles in chromosome organization and segregation. interactions between chromosomes and spindle microtubules allow chromosomes to congress to the middle of the cell, and to segregate the sister chromatids into daughter cells in mitosis, which is followed cytokinesis. in contrast to monocentric chromosomes, in which the centromere is normally present at a single region on each chromosome, the holocentric chromosomes have centromeric activity along the entire length of the chromosome. it has been known that the silkworm, bombyx mori, has holocentric chromosomes since 1970s, none of silkworm kinetochore proteins, however, have been identified so far. here we report the identification of a novel set of genes for outer kinetochore proteins in silkworm by using bioinformatics and rna interference-based screening. under the hypothesis that depletion of essential kinetochore genes causes cell cycle arrest in mitosis, we performed rnai in the silkworm cell line, bmn4-sid1, targeting a set of candidate genes. knockdown of five genes caused significant cell cycle arrest at the g2/m phase. we also found that these five proteins make a complex, and that all of them are localized along the chromosome arms, indicating that the silkworm kinetochore extends along the chromosome. inactivation of bine aldehyde dehydrogenase from spinach by its physiological substrate bine aldehyde to contend with osmotic stress caused by drought, salinity, or low temperatures some plants synthesize the osmoprotectant glycine bine (gb) from bine aldehyde (bal). the last step-the irreversible nad1dependent oxidation of bal-is catalyzed by aldh10 enzymes that exhibit bine aldehyde dehydrogenase (badh) activity. we here report that the spinacia oleracea badh (sobadh) is reversibly inactivated by bal in the absence of nad1 in a time-and concentration-dependent mode to approximately 50% of the original activity. inactivation kinetics are consistent with a partial reversible, two-steps mechanism that involves the formation of an active non-covalent enzyme•bal complex before formation the inactive enzyme-bal complex. crystallographic evidence indicates that in the enzyme previously inactivated by bal the aldehyde forms a thiohemiacetal with the nonessential cys450 (sobadh numbering) located at the aldehyde-entrance tunnel, thus totally blocking the access to the catalytic cysteine. accordingly, bal does not inactivate the c450s sobadh mutant. two crystal structures of the inactivating enzyme-bal complex showed that the trimethylammonium group of bal is inside the active-site aromatic box, as in the productive way of binding. this explains why the inactivation of the a441i mutant-where the binding of the trimethylammonium group is hindered-requires non-physiologically high bal concentrations, while the a441c mutant-where the binding is allowed-is inactivated similarly to the wildtype enzyme. cys-450 is conserved in most plant aldh10 enzymes of known sequence, and in all of them with proven or predicted badh activity. inactivation by bal appears therefore to be a common feature of plants badhs. this short-term regulation may be of great physiological importance since the irreversibility of the badh-catalyzed reaction would unbalance the nad1/nadh ratio if the aldehyde concentrations are high, the nad1 concentrations low and the reaction is not slowed down. plants badhs are prone to this situation since they work under osmotic stress conditions, when high bal concentrations are required for the synthesis of high levels of the osmoprotectant gb. the partial nature of the decamer possesses a donut shaped structure with 10 calcium ions on the surface available for interactions with carbohydrate molecules. binding specificity was evaluated for 20 carbohydrates using differential scanning fluorimetry (dsf) that showed bjcul interacts with galactose and lactose but less with glucose and sacarose. surprisingly, high levels of thermostabilization of bjcul was achieved with the antibiotic aminoglycosides geneticin (g418) and gentamicin in a calcium concentration dependent manner, but not kanamycin. intriguingly, while lactose and galactose inhibited erythrocyte agglutination by bjcul, g418 and gentamicin did not affect hemagglutination implying a second site of binding. dsf analysis also suggested the presence of a second binding site for the antibiotics and crystallization of the complexes are in progress in order to understand fully this new binding mechanism of c-type lectin with antibiotics. ab initio modelling of structurally uncharacterised antimicrobial peptides mara kozic 1 1 institute of integrative biology, university of liverpool ab initio modelling of structurally uncharacterised antimicrobial peptides mara kozic 1* 1 institute of integrative biology, biosciences building, university of liverpool, crown street, liverpool l69 7zb, united kingdom * mara.kozic@liverpool.ac.uk antimicrobial resistance within a wide range of infectious agents is a severe and growing public health threat. antimicrobial peptides (amps) are among the leading alternatives to current antibiotics, exhibiting broad spectrum activity. an understanding of the structure of a protein can lead us to a much improved picture of its molecular function. furthermore, an improved understanding of structure-function relationships facilitates protein design efforts to enhance their activity. currently, the 3d structures of many known amps are unknown. to improve our understanding of the amp structural universe we have carried out large scale ab initio 3d modelling of structurally uncharacterised amps. such ab initio modelling is facilitated by the typical small size of amps as well as their tendency to contain disulphide bonds, these providing valuable additional information to simulations. preliminary results reveal unexpected similarities between the predicted folds of the modelled sequences and structures of well-characterised amps. for example, lacticin q was revealed to contain a helical bundle fold that bears a striking resemblance to enterocin 7a. we also found a remarkable similarity between the predicted structure of silkworm 001 peptide and b-hairpin amps such as tachyplesin i. our results improve the understanding of the structure-function relationship of amps. surface aggregation-propensity as a constraint on globular proteins evolution susanna navarro 1 , marta diaz 2 , pablo gallego 2 , david reverter 2 , salvador ventura 1 1 institut de biotecnologia i biomedicina and departament de bioquimica i biologia, 2 institut de biotecnologia i biomedicina, universitat aut onoma de barcelona in living cells, functional protein-protein interactions compete with a much larger number of nonfunctional interactions. theoretical studies suggest that the three-dimensional structures of present proteins have evolved under selective pressure to avoid the presence of aggregation-prone patches at the surface that may drive the establishment of anomalous protein contacts. however, no experimental evidence for this hypothesis exists so far. the a-spectrin sh3 domain (spc-sh3) has been used as a protein model to decipher the sequential aggregation determinants of proteins. here we use it to address the structural determinants of protein aggregation and their link to protein evolution. to this aim we exploit aggrescan3d (a3d), a novel algorithm developed by our group, which takes into account both protein structure and experimental data to project aggregation propensities on protein surfaces. we used a3d to design a series of spc-sh3 variants with progressively stronger aggregationprone surfaces and characterized their thermodynamic, structural and functional properties. our data support evolution acting to constraint the aggregation propensities of globular protein surfaces in order to decrease their potential cytotoxicity and the protein quality control machinery acting to buffer this negative selective pressure. utilizing 3d structure for the annotation of structural motifs in the conserved domain database narmada thanki-cunningham 1 , noreen gonzales 1 , gabriele marchler 1 , myra derbyshire 1 , james song 1 , roxanne yamashita 1 , christina zheng 1 , stephen bryant 1 , aron marchler-bauer 1 , farideh chitsaz1 1 1 conserved domain database, structure group cbb/ncbi/nlm/nih the conserved domain database (cdd) is a protein classification and annotation resource comprised of multiple sequence alignments representing ancient conserved domains. cdd protein domain models are curated by ncbi and use 3d protein structure explicitly to define domain extent and the location of conserved core structures, and to provide accurate alignments between diverse family members via structure superposition. cdd also imports external collections such as pfam and tigrfam. recently, a novel class of annotation labeled as "structural motifs" has been introduced to supplement current capabilities. these annotations define compositionally-biased and/or short repetitive regions in proteins, which are difficult to model as functional domains conserved in molecular evolution. structural motifs include transmembrane regions, coiled coils, and short repeats with variable copy numbers. for many types of short tandem repeats, a few position-specific score matrices (pssms) suffice to annotate more than 90% of the known instances of that structural motif. unfortunately, a lack of sequence similarity within coiled-coil regions prohibits the development of only a few generic models; therefore, models for coiled-coil regions in the context of specific families have been developed using the spiricoil database as a reference. increased coverage of coiled-coil regions in cdd, specific site annotations of these structural motifs as well as their representation on the webpages will be discussed. specific in vivo ultrasound imaging of e-selectin expression in tumors using a microbubble contrast agent covalently attached to the peptide ligand iellqar, known to bind to e-selectin [2] . however, it was observed that this probe has a limitation in the imaging of cardiovascular diseases where higher shear stresses prevent microbubbles from remaining attached to the target. therefore, peptides with higher eselectin affinity are needed to design probes capable of imaging these diseases. in this context, automated docking and molecular dynamics methodologies were combined and applied to different e-selectin binding peptides. these studies predicted the energetically more favorable binding mode as well as the key interactions between the peptide ligands and the e-selectin receptor. some of these peptides were prepared by solid-phase peptide synthesis and their interactions with e-selectin analyzed by surface plasmon resonance technique. the results showed that these peptides have different affinities for e-selectin. these data were correlated with the computational studies and evaluated to obtain crucial information of the key recognition elements needed for higher e-selectin affinity. these recent results will be presented. burkholderia pseudomallei is the causative agent of melioidosis, a serious invasive disease of animals and humans in tropical and subtropical areas. sedoheptulose-7-phosphate isomerase from b. pseudomallei (bpgmha) is the antibiotics adjuvant target for melioidosis. in general, bpgmha converts dsedoheptulose-7-phosphate to d-glycero-a-d-manno-heptopyranose-7-phosphate (m7p). this is the first step of the biosynthesis pathway of ndp-heptose responsible for a pleiotropic phenotype. therefore, this biosynthesis pathway is the target for searching novel antibiotics increasing the membrane permeability of gram-negative pathogens or adjuvants synergistically working with known antibiotics. the crystal of this enzyme has been solved at 1.9 å resolution. there is an active site pocket where a putative metal binding site is located. to find out inhibitors of bpgmha, in-silico virtual screening with zinc, a free database of commercially-available compounds, has been performed. tens of thousands of chemical compounds were docked into the active site of bpgmha. a number of putative bpgmha binding compounds better than m7p were found using surflex-dock included in the sybyl software package. characteristics of these compounds were surveyed and classified to identify common binding properties with bpgmha. mapping the structure of laminin using cross-linking and mass spectrometry gad armony 1 , toot moran 1 , yishai levin 2 , deborah fass 1 1 weizmann institute of science, department of structural biology, 2 weizmann institute of science, israel center for personalized medicine laminin, a 800 kda heterotrimer, is a major element in the extracellular matrix (ecm). within the ecm, laminin contributes to the adhesion and migration of cells, both in health and disease. the laminin trimer was observed by rotary shadowing electron microscopy to be cross shaped: the three short arms of the cross are formed by the amino-terminal halves of the three subunits, whereas the long arm of the cross holds the three chains together in a long coiled coil. the narrow and flexible arms of the laminin cross complicate studying its structure to high resolution by crystallography or electron microscopy single particle reconstruction. to advance our understanding of this remarkable quaternary structural assembly, we have used cross-linking and mass spectrometry to analyze the organization of the laminin trimer. this technique was validated by known crystal structures of isolated laminin domains. in all cases the crystal structure distances agree with the cross-linker length. the identified cross-links were particularly helpful in assigning the register and the subunit order of the long coiled coil due to the high content of cross-linkable residues in this region. using known x-ray crystal structures, homology modeling, and distance restraints provided by two cross-linker chemistries, a clearer picture of the laminin quaternary structure is obtained. non-sequential protein structure alignment program mican and its applications shintaro minami 1 , george chikenji 2 , motonori ota 1 1 dept. of info. sci., nagoya univ., 2 dept. of comp. schi. & eng., nagoya univ. in some proteins, secondary structure elements are arranged spatially in the same manner, but they are connected in the alternative ways. analysis on such non-sequential structural similarity in proteins is important because it provides a deeper understanding of the structural geometry of protein. this can be also observed even in the homologous proteins, indicating the non-sequential structural similarity is significant in the protein evolution. however, the non-sequential structural similarity in proteins is less investigated. we developed a novel non-sequential structural alignment program mican, which can handle multiple chains, inverse direction of chains, c$�lpha$models, alternative alignments, and non-sequential alignments. we performed comprehensive non-sequential structural comparison among homologous proteins in the same scop superfamily by using the mican program. based on the result, we found that approximately 8% of superfamilies include at least one protein pairs showing non-sequential structural similarity. 85% nonsequential structurally similar pairs are aligned in a simple way, e.g. circular permutation, $�$strand flip/ swap, but 15% are complicated. interestingly, most of such complicated non-sequential similarities can be explicable by combination of 2-4 simple non-sequential relationships. this result indicates that accumulation of simple structural changes in the course of protein evolution produces completely different fold homologs. as early as 1919, ritter surmised that the cell's molecules cooperate to form a "special apparatus and an organised laboratory". 1 despite supporting evidence from srere, mcconkey and others, efforts to understand molecular organisation in vivo are still in their infancy. however, important aspects of the cell interior have already been revealed. for example, weak molecular interactions structure the cytoplasm into time-evolving, functional zones. 2 weak interactions are difficult to capture and can preclude protein detection in cells by many biophysical techniques, including nmr spectroscopy. 3, 4 we explored the effects of cell-like milieus on the cytochrome c (cyt c)-flavodoxin (fld) interaction. these oppositely charged proteins interact weakly with a number of cognate partners. neither cyt c4 nor fld is detectable by nmr in escherichia coli confirming their "sticky" nature ( figure 1a) . the cyt c-fld interaction was assessed in buffer, 8% polyacrylamide gels and in solutions containing 100 g/l of macromolecular crowders ( figure 1b) . 1h, 15n hsqc nmr revealed that the interaction was transient in buffer, proceeding via the known binding site for both proteins. substantial line broadening was effected in crowded and confined solutions suggesting that the cyt c-fld complex is stabilised under native-like conditions. the stabilising effect of macromolecular crowders was also observed by native gel electrophoresis and crystallization. these findings coincide with spitzer and poolman's model for cytoplasmic structuring, emphasising the role of charge-charge interactions and crowding in the formation of macromolecular "clusters". 5 the implications for cytoplasmic structuring will be discussed alongside related investigations of cationic protein interactions in e. coli extracts. 3, 4 detergent:protein ratio. the transmembrane b-barrel of bama is folded in either micelles, bicelles or nanodiscs, however an n-terminally attached single potra5 domain is flexibly unfolded, due to the absence of stabilizing contacts with other protein domains. measurements of backbone dynamics show distinct time scales of dynamic behavior for bama b-barrel and parts of its extracellular loop l6, revealing high local flexibility within the the lid loop. this work presents the first high-resolution 2d solution nmr spectra of the bama barrel and establishes improved biochemical preparation schemes, which will serve as a platform for structural and functional studies of bama and its role within the bam complex. protein arginine methylation is a widespread and important posttranslational modification in eukaryotic cells, shown to be involved in the activation or repression of transcription, modification of the splicing machinery, signaling, and dna repair. mammalian protein arginine methyltransferases include a family of nine sequence-related enzymes that transfer one or two methyl groups onto the terminal guanidino groups on arginine residues, producing monomethylarginine only (mma, type iii), symmetric dimethylarginine (sdma) and mma (type ii), or asymmetric dimethylarginine (adma) and mma (type i). while prmt1, 2, 3, 4, 6, and 8 have been characterized as type i enzymes, and prmt5 as a type ii enzyme, the role and activity types of the two final members of this family of enzymes, prmt7 and prmt9, had been unclear due to conflicting results in the literature, and the substrates for these enzymes had been elusive. both prmt7 and prmt9 are distinct members of the family with two methyltransferase or methyltransferase-like domains and containing acidic residues in otherwise well-conserved substrate double e binding motif, features not seen in the other prmt enzymes. recent work in our laboratory confirmed prmt7 as the only type iii mma-forming enzyme in the group, with a unusual low temperature optimum for activity, and a heretofore not seen preference for a basic stretch of residues in an r-x-r sequence for methylation. mutations of the acidic residues in the substrate-binding motif results in a loss of the specific r-x-r activity and the appearance of a g-r-g specificity typical of many of the other prmts. the physiological substrate of prmt7 has yet to be confirmed, although histone h2b is an effective in vitro substrate. prmt9, on the other hand, had no reported activity, until immunoprecipitation from hela cells showed it pulled down two splicing factors, sf3b2 and sf3b4, in a complex. amino acid analysis showed that prmt9 methylates sf3b2 to produce both mma and sdma, thus making it the second type ii enzyme in mammals. prmt9 knockdown results in modulation of alternative splicing events. this enzyme appears to be relatively specific for the sf3b2 protein; a peptide containing the methylatable arginine residue was not found to be a substrate, and typical substrates of other prmts are not recognized by prmt9. we found that the position of the methylated arginine residue in sf3b2 is important, and the acidic residues in the substrate-binding motif also play an important role in substrate recognition. thus, prmt7 and prmt9 represent unique members of the mammalian prmt family. hydrogen peroxide levels, endogenous hormones (cytokinins, salycilic acid, as well as jasmonic acid and its conjugates), polyphenolics and terpenoids in a model system of a. alba in vitro with inhibition of rootng and stimulation of callusogenesis by means of individual and combined cytokinin and cytokinin/ auxin treatments. results: it was established that inhibition of rooting and stimulation of callusogenesis caused by benzyl adenine (ba) or combinations of ba and indole-3-butiric acid (iba) in vitro were related to elevation of sesquiterpenoids in the essential oils, as well as polyphenolics content, accompanied by a drop of stress hormones, bioactive cytokinins and preservation of oxidative stress and lipid peroxidation levels, as compared with non-treated control. individual treatments with either iba or ba, also increased the sesquiterpenoid content in the essential oil of the plant, in a concentration related manner, this effect being more profound after ba treatment. in addition, ba treated plants exhibited a drop of protein levels of the aerial samples, as well as profound differences of enzymatic activity in the callus tissues, as compared with callus of plants treated with different combinations of ba and iba. conclusion: the results of the present work indicate that alterations of endogenous phytohormonal levels, caused by exogenous plant growth regulators treatment, might be the mediator between primary and secondary metabolism by means of affecting protein levels and activity of key enzymes in vitro. three different additives ((0.1% (v/v); formic acid, acetic acid, ammonium format with formic acid) have been investigated in response to ion intensity of esi-ms for individual hnp 1-4 in saliva. kinetex v r column separation efficiency was evaluated using two different column dimensions (50 x 2.1 mm and 50 x 3 mm.) and two different stationary phases (c18 and c8). kinetex v r column (homogenous porous shell) performance was also compared to new ultra ace v r (encapsulated bonded phase) column. sample optimisation revealed that the spe method removes interference from salivary glycoproteins and consequently yields larger peak area (30-90%) for all hnps. hnps were extracted by spe with a recovery of 80-91%. the meoh: h2o: acetic acid (0.1%) provided enhanced (p>0.05) hnp1-3 ion intensities. the kinetex v r c8 (50 x 3.0 mm, 2.6 mm) column facilitated a better separation efficiency of the four hnps as compared to the ultra core super c18 ace v r (50 x 3.0 mm, 25 mm) column, the kinetex v r c18 (50 x 3.0 mm, 2.6 mm) and the kinetex v r c18 (50 x 3.0 mm, 5 mm) column. the relative levels of the hnps were determined in healthy volunteers before and after a rigorous exercise regime: it is possible that prolonged strenuous exercise will affect oral innate immunity and therefore also the level of salivary defensins. hnp1-3 are traditionally detected in an enzyme-linked immunosorbent assay (elisa) which does not discriminate between the different hnps due to their structural similarities. there has therefore been a need to develop a mass spectrometry method that will discriminate between the defensins. as part of the method validation, the hnp1-3 level was determined by elisa and the data was compared with the lc-ms data. here we present this cross-validation; the data revealed no significance difference between the two methods (r25 0.96) which confirms that the developed lc-ms method is and equal sensitive method for the detection of these potential antimicrobial markers. this method can easily be adopted for similar molecular weight of peptides as hnps and also for any other biological matrix. moonlighting proteins: relevance for biotechnology and biomedicine luis franco serrano 1 , sergio hern andez 1 , alejandra calvo 2 , gabriela ferragut 2 , isaac amela 1 , juan cedano 2 , enrique querol 1 1 institut de biotecnologia i biomedicina. universitat aut onoma de barcelona, 2 laboratorio de inmunolog ıa, universidad de la rep ublica regional norte-salto multitasking or moonlighting is the capability of some proteins to execute two or more biochemical functions. the identification of moonlighting proteins could be useful for researchers in the functional annotation of new genomes. moreover, the interpretation of knockout experiments, in which the result of a gene knocking does not produce the expected results, might be enhanced. the action of a drug can also be facilitated because it might have an off-target or side effect with somewhat hidden phenotypic traits. it would be helpful that bioinformatics could predict this multifunctionality. in the present work, we analyse and describe several approaches that use protein sequences, structures, interactomics and current bioinformatics algorithms and programs to try to overcome this problem. among these approaches there are: a) remote homology searches using psi-blast, b) detection of functional motifs and domains, c) analysis of data obtained of protein-protein interaction databases (ppis), d) matches of the sequence of the query protein to 3d databases (i.e., algorithms like pisite), e) mutation correlation analysis between amino acids using algorithms like mistic. remote homology searches using psi-blast combined with data obtained from interactomics databases (ppis) have the best performance. structural information and mutation correlation analysis can help us to map the functional sites. mutation correlation analysis can only be used in very specific situations because it requires the existence of a multialigned family of protein sequences, but it can suggest how the evolutionary process of second function acquisition took place. we have designed a database of moonlighting proteins, multitaskprotdb (http:// wallace.uab.es/multitask/). from this database we determine the frequencies of canonical and moonlighting coupled functions (being an enzyme and a transcription factor the highest), the percentage of moonlighting proteins involved in human diseases (65% of the human moonlighting proteins in the database) and the percentage of moonlighting proteins acting as a pathogen virulence factor (20% of the moonlighting proteins in the database). correlation between potential human neutrophil antimicrobial peptides (hnp 1-3) and stress hormones in human saliva nadia ashrafi 1 , frank pullen 1 , birthe nielse 1 , cris lapthorn 1 , fernando naclario 2 1 university of greenwich (faculty of engineering and sciene), 2 university of greenwich (centre of sports science and human performance) numerous studies have investigated the effect of exercise on mucosal immunity but the focus has mainly been on salivary immunoglobulins lysozymes and hormones (cortisol, testosterone). this is not surprising given that iga and igg are the predominant immunoglobulins in saliva and there is a relationship between mucosal immunity and upper respiratory illness. it is well known that physical and mental stress provoke the release of cortisol from hypothalamic pituitary adrenal axis, by which stress can modulate various immune responses. in general, cortisol and growth hormones helps to induce the activation of neutrophils. to date, this study represents the first study that investigated the correlation between human neutrophil alpha defensins family against cortisol (stress hormone) and testosterone (growth hormone) in human saliva before and after exercise or training. twelve resistance trained athletes volunteered to participate in the study. participants consumed supplements during exercise and the hnp 1-3, cortisol and testosterone response was investigated pre, post 30 and 60 minutes of the workout. the correlation between salivary antimicrobial peptide (hnp 1-3) and stress hormone (cortisol and testosterone) has been investigated using elisa. cortisol showed no significant (p 5 0.818) difference for (pre to 30 min post) between cho and pl (cho: 483.07 6 912.77 ng/ml; pl5 583.82 6 1134.33 ng/ml) conditions but a strong trend (p 5 0.074) was observed for (pre to 60 min post) post (cho: 1023.19 6 1500.40 ng/ml; pl5 1480.33 6 2214.80 ng/ml) condition. testosterone showed no significant (p 5 0.167; p 5 0.156) difference for (pre to 30 min post) between cho and pl (cho: 23.32 6 44.11 ng/ml; pl5 10.40 6 14.19 ng/ml) and for (pre to 60 min post) post (cho: 26.42 6 19.11 ng/ml; pl5 23.72 6 17.91 ng/ml) condition. hnp 1-3 showed no significant (p 5 0.348) difference for (pre to 30 min post) between cho and pl (cho: 72.26 6 148.82; pl5 125.20 6 70.00) conditions but significant difference (p 5 0.026) was observed for (pre to 60 min post) between cho and pl (cho: 35.18 6 182.69; pl5 228.74 6 151.63) condition. the present findings suggested that there is no correlation between salivary hnp 1-3 and cortisol for (pl: r2 5 0.02 and cho: r2 5 0.01); hnp 1-3 and testosterone (pl: r2 5 0.20 and cho: r2 5 0.10). a worth note from previous study which suggested that using murine skin model (an increase in endogenous glucorticoids (cortisol) by physiological stress reduced mrna levels of antimicrobial peptide (cathelicidin). it is not clear that the correlation between hormones and antimicrobial peptide has been affected by the time interval of the exercise. both cortisol and antimicrobial peptide demonstrated a transient increase after exercise but it is surprising that they are not correlate to each other. one of the hypothesis from the present finding could be cortisol responses slow and it will be interesting to do further research with longer interval. the second hypothesis demands a further investigation to determine the synergism between substances. school of biomolecular and biomedical science, conway institute, ucd., 2 king saud university, sciences, biochemistry department. the crystal structure of a human glucose 6-phosphate dehydrogenase (g6pd) shows that each subunit has two nadp1 sites; in addition to a catalytic site there is a "structural" site which is distant from the catalytic coenzyme site. mutations causing severe deficiency tend to cluster round and close to the dimer interface and the structural nadp1, indicating that the integrity of these areas is important for enzyme stability and therefore for maintenance of activity. in order to understand the molecular basis of g6pd deficiency, and to have a clearer indication about the role of some features of the threedimensional structure, a fuller study of the second, "structural" nadp1 binding site is needed. human g6pd controls the first committed step in the pentose phosphate pathway. it catalyses the oxidation of glucose 6-phosphate to gluconolactone 6-phosphate, generating nadph which is essential, amongst other things, for protection against oxidative stress. the human enzyme can be active in dimer or tetramer forms. human g6pd of "structural" nadp1 per subunit of enzyme. this tightly-bound nadp1 can be reduced by g6p, probably following migration to the catalytic site. the importance of nadp1 for stability is explained by the structural nadp1 site, which is not conserved in prokaryotes. after removing the tightly bound "structural" nadp1 the enzyme is still active but not stable. the effects of different nadp1 fragments on the stability of human recombinant g6pd have been investigated. nadp1 is crucial for the long term stability of human g6pd, and only one of nadp1 analogues which is adenosine diphosphate ribose -2'-phosphate was able to slightly promote the stability of enzyme. . molecular characterization of specific positively selected sites in mammalian visual pigment evolution miguel a. fern andez-sampedro 1 , eva ramon 1 , brandon m. invergo 2 , jaume bertranpetit 2 , pere garriga 1 1 grup de biotecnologia molecular i industrial., 2 visual rhodopsin is a member of the g-protein coupled receptors superfamily. this membrane protein consists of a 11-cis-retinal cromophore bound to a seven transmembrane protein, opsin, by means of a protonated schiff base linkage. it has an important role as a dim light photoreceptor in the retina of the eye. by statistical models, where episodic selection in rhodopsin is tested on one branch of the phylogeny against a background of neutral or purifying selection on the rest of the tree, we have found some significant evidence of specific positively selected sites in early mammalian divergence. we have chosen the three amino acid sites identified with the highest posterior probability of having been targets of positive selection to perform experimental studies, i.e. 13 (positively selected from m to f), 225 (positively selected from r to q) and 346 (positively selected from s to a). we have constructed, expressed, immunopurified and functionally characterized the proposed candidates, f13m, q225r and a346s rhodopsin mutants located at the n-terminus, the transmembrane domain and the c-terminus region of the protein respectively. from the analysis of the molecular features of the f13m mutant, we conclude that position 13 is very important for protein folding and also for proper protein glycosylation, since we only could observe cromophore regeneration after its rescue in the double cysteine (n2c/ d282c) mutant background that stabilizes the n-terminal extracellular domain of the protein. our results also show that mutants q225r and a346s alter the g-protein activation rate, and hydroxylamine susceptibility in the dark-adapted state. in the case of q225r, disrupting critical interactions with the neighbouring y136 of the conserved d/ery motif, critical in gt activation, could cause the lower gt activation ability. the mutant a346s would create a potential additional phosphorylation site in the protein which could affect rhodopsin phosphorylation after photoactivation and, in turn, could affect the binding affinity of arrestin, a regulator of rhodopsin deactivation. this extra phosphorylation site could provide an evolutionary explanation for the enhanced response observed in the case of gt activation. in conclusion, these results highlight the importance of molecular investigations of positive selected sites in rhodopsin evolution and the relevance of structural and functional analysis of these sites in unravelling the molecular basis of visual pigment evolution. natural evolution sheds light on modern drug resistance in protein kinases marc hoemberger 1 , christopher wilson 1 , roman agafonov 1 , dorothee kern 1 1 the anti-cancer drug imatinib exhibits highly specific binding to the human kinase and oncogene abl with a three thousand fold weaker affinity for the structurally and functionally very similar kinase src. it has been shown recently that the major difference in binding of imatinib to abl and src stems from an induced fit after binding of the drug. to further understand the mechanism of imatinib binding to its target we used ancestral sequence reconstruction (asr) and resurrected enzymes along the node from the common ancestor of abl and src up to the extant kinases. we show that imatinib affinity is gained towards the evolution of extant abl while it is lost towards evolving src. the combination of asr and crystallographic data of the ancestors in addition to kinetics data allowed us to identify a subset of residues involved in imatinib specificity sufficient to switch from an intermediate binder to a tight binder. preliminary data shows that a network of hydrogen bonds and packing interactions stabilize the kinked p-loop conformation for tight binders thus allowing for more interactions between the kinase and the drug. strikingly, many of these residues were identified in human cancer patients as "hot spots" for the development of resistance mutations. further investigation into the identified subset of residues in combination with these commonly found imatinib resistance mutations will allow us to understand emerging drug resistances better. an evolutionary view of the cold adapted catalysis of enzymes vy nguyen 1 , christopher wilson 1 , dorothee kern 1 1 the diversity in protein function that we see today arose as a result of life adapting to a cooling earth. how did enzymes, the catalysts of many crucial cellular processes, achieve this cold adaptation? this is a challenging question to answer because ancient sequences of proteins that existed billions of years ago are not available. to address this question we used ancestral sequence reconstruction to create adenylate kinase (adk) enzymes from the divergence of anaerobic and aerobic firmicutes towards modern day thermophilic, mesophilic and psychrophilic organisms. adk is a phosphotransferase that catalyzes the conversion of two adp molecules into atp and amp. we make the following observations. first, all ancestral enzymes are active with optimal catalytic rates linearly corresponding to the temperature of the environments where these proteins would have been found. most strikingly, the catalytic rate of our oldest adk ancestor exhibits a higher enthalpy of activation at low temperatures as compared to the modern thermophilic adk. this suggests a large enthalpic penalty had to be paid for reactions to occur at cold temperatures in an ancestor that existed in a hot environment. second, several high resolution crystal structures of extant proteins that we solved (1.2å -1.6å), show that the oldest ancestors were more rigid than the modern adks due to an intricate salt-bridge network. this work, thus shows for the first time, the molecular and thermodynamic determinants of cold adaptation in an enzyme over a time period that spans billions of years. induced oxidative modification of plasma and cellular fibrin-stabilizing factor anna bychkova 1 , tatiana danilova 1 , alexander shchegolikhin 1 , vera leonova 1 , marina biryukova 1 , elizaveta kostanova 1 , alexey kononikhin 0 , anna bugrova 1 , evgeny nikolaev 0 , mark rosenfeld 1 1 n. m. emanuel institute of biochemical physics, russian academy of sciences, 2 institute for energy problems of chemical physics, russian academy of sciences the main function of plasma fibrin-stabilizing factor pfxiii is to catalyze the formation of the intermolecular covalent cross-links between both gand afibrin polypeptide chains. the crosslinking crucially affects mechanical strength of fibrin and its resistance against fibrinolysis. the precise role of cellular fibrin-stabilizing factor cfxiii remains poorly understood. pfxiii is a heterotetramer (fxiii-a2b2) consisting of two single-stranded catalytic a subunits (fxiii-a2), and two identical single-stranded inhibitory/ carrier b subunits (fxiii-b2). the subunits are held together by weak non-covalent bonds. contrary to plasma fxiii, cfxiii is a dimer (fxiii-a2) devoid of b subunits. as well as many other proteins circulating in the bloodstream, pfxiii is known to be a target for reactive oxygen species (ros) causing processes of protein oxidative modification. since the conversion of pfxiii to the active form of the enzyme (fxiiia) is a multistage process, ozone-induced oxidation of pfxiii has been investigated at different stages of its enzyme activation. the biochemical results point to an inhibition of enzymatic fxiiia activity depending largely on the stage of the pfxiii conversion into fxiiia at which oxidation was carried out. uv-, ftir-and raman spectroscopy demonstrated that chemical transformation of cyclic, nh, sh and s-s groups mainly determines the oxidation of amino acid residues of pfxiii polypeptide chains. conversion of pfxiii to fxiiia proved to increase protein susceptibility to oxidation in the order: pfxiii < pf-xiii activated by thrombin < pfxiii in the presence of calcium ions < fxiiia. with the aid of massspectrometry it has been demonstrated that oxidation leads to decreasing fxiii-a and fxiii-b coverage both in the forms of zymogen and in the presence of calcium ions. a group of amino acid residues involved in oxidation modification of pfxiii is identified in this study. the oxidation of either cfxiii or cfxiiia has revealed an almost complete loss of enzyme activity caused by dramatic changes in the primary and secondary structure of the proteins detected by the ftir data. taking into account these new findings, it seems reasonable to assume that the inhibitory/carrier fxiii-b subunits can serve as scavengers of ros. hypothetically, this mechanism could help to protect the key amino acid residues of the fxiii-a subunits responsible for the enzymatic function of fxiiia. the study was supported by rfbr, research project no. 15-15-04-08188a. mass spectrometry study was supported by the russian scientific foundation grant no. 14-24-00114. performance and quality. making microcalorimetry simple with microcal peaq-itc natalia markova 1 , ronan o'brien 1 , mark arsenault 1 1 microcal, malvern instruments ltd. dynamic interactions involving biomolecules drive and regulate all biological processes. studies of biomolecular interactions are fundamentally important in all areas of life sciences. data provided by isothermal titration calorimetry (itc) enables scientists in academia and industry to directly and quantitatively characterize these interactions in solution. microcal peaq-itc, the latest generation of microcal itc instrumentation, offers a whole range of solutions for addressing current bottlenecks associated with interaction analysis. among the most recognized challenges are the needs to adequately address a broad range of binding affinities and to reliably interpret binding data complicated by the presence of inactive protein fraction or inherent uncertainty in the concentration of a ligand. consistently high performance of microcal peaq-itc enables increased confidence and data resolution when measuring low heats at low or uncertain sample concentrations and complex binding modes. the new microcal peaq-itc analysis software allows for utomated data analysis, minimizing analysis time and user subjectivity in assessing data quality. data quality is determined and advanced fitting performed in a few seconds per experiment allowing for analysis of large data sets of 50 or more experiments in a matter of seconds. glutamine-rich activation domain of transcription factor sp1 -biochemical activity and structure jun kuwahara 1 , chisana uwatoko 1 , emi hibino 2 , katsumi matsuzaki 2 , masaru hoshino 2 1 faculty of pharmaceutical sciences, doshisha women's university, 2 graduate school of pharmaceutical sciences, kyoto university transcription factor sp1 is ubiquitously expressed in a mammalian cell, activates reasonably large subset of mammalian genes, and is involved in the early development of an organism. the protein comprises two glutamine-rich (q-rich) regions (a and b domains) located in its n-terminal half, while three tandem repeats of c2h2 zinc finger motif at its c-terminus binds directly to a gc-rich element (gc box) of dna. in general, q-rich domain is one of the typical motifs found in trans-activation domain of transcription factors together with acidic and proline-rich domains. transcriptional signal of sp1 are transmitted via interaction between q-rich domains of sp1 and different classes of nuclear proteins, such as tata-binding protein (tbp) associated factors (tafs) in components of basic transcription factor complexes (tfii). in addition, self-association of sp1 via q-rich domains is also important for its regulation of transcriptional activity. it has been considered that an sp1! molecule bound to a 'distal' gc-box synergistically interacts with another sp1 molecule at a 'proximal' binding site. although formation of multimers via q-rich domains seems functionally important for sp1, little is known about relevance between biological activity and structural nature of q-rich domains. we analyzed nature of glutaminerich domains of sp1 by biochemical and physicochemical methods. we found that q-rich domains do not have clear secondary structure whereas they can indicate biochemical activity. detailed analysis of nmr spectra indicated interaction between the domains. the q-rich domains of sp1 might be one of the intrinsically disordered proteins (idp). chipping away at the yeast proteome: redesigning an e3 ubiquitin ligase for targeted protein degradation michael hinrichsen 1 , lynne regan 1 1 one of the central goals of synthetic biology is to exploit biological systems in order to produce compounds of therapeutic or industrial value 1. often, these efforts are complicated by the many natural biochemical pathways in cells that can compete for the same small molecule precursors. currently, the most common solution is to simply delete the genes coding for the competing enzymes 2. while such an approach has been successful, it is only applicable to nonessential genes and can produce unintended off-target effects such as decreased cell viability 2. an alternative strategy is to instead target proteins directly for degradation. using this strategy, scientists would first grow cultures of engineered cells to high densities under permissive conditions (i.e. targeted proteins are stably expressed). then, once sufficient cell density has been reached, enzymes of competing pathways would be rapidly degraded, resulting in the rapid production of high concentrations of the compound of interest. we propose to create such a tool by reengineering the c-terminus of hsp70 interacting protein (chip), an e3 ubiquitin ligase. chip recognizes substrate proteins through a short c-terminal peptide tag on target proteins3. we have shown that fusing this tag to non-native substrates is sufficient for ubiquitination in vitro (data not published). cellular assays have also been performed in s. cerevisiae, a model organism commonly used in metabolic engineering applications1. as a number of native yeast proteins possess c-termini similar to that of chip's native substrates (data not published), it was necessary to develop an orthogonal chip-peptide pair. this was achieved by replacing chip's natural tpr ligand-binding domain with a ligand-binding domain engineered previously in the regan lab 4. the altered chip construct has been shown to be active both in vitro and in vivo, and produces an altered growth phenotype when targeted against an enzyme involved in uracil biosynthesis. future work will focus on further kinetic characterization of the engineered enzyme, increasing its activity, and introducing the system into a proof of concept synthetic biology application. advances in modern sequencing techniques have resulted in an explosion of genomic data. correctly classifying this new wealth of information can be daunting not only because of the sheer volume of sequence data, but also because the propagation of erroneous and less-than-ideal names and functional characterizations in the current databases gets in the way of functional classification by mere sequence similarity. we are investigating the extent to which protein domain architecture can be utilized to define groups of proteins with similarities in molecular function, and whether we can derive corresponding functional "labels", starting with some of the most common domain architectures found in bacteria. to this end, we have developed an in-house procedure called sparcle ('specific architecture labeling engine') that lets us track and examine specific or sub-family domain architectures, resulting from annotating protein sequences with domain footprints provided by the conserved domain database (cdd), which includes hierarchical classifications for many common domain families. we will discuss how the proteins are grouped into specific architectures, our successes in assigning functional labels, and the major limitations we have encountered to date. while we will be able to assign functional labels to a large fraction of protein models derived from genome sequences, this effort has the added benefit of pointing out insufficient coverage and resolution of the current protein domain model collections that constitute cdd. we will also discuss alternative procedures that utilize pre-computed domain annotation for clustering protein sequences at a level that is well suited for functional labeling. we hope that this preliminary study will help to identify approaches that facilitate rapid and accurate annotation of genomes with a minimum of manual intervention. pegylated amyloid peptide nanocontainer delivery and release system self-assembly of telechelic peg end-capped with hydrophobic dipeptides collagen stimulating effect of peptide amphiphile c16-kttks on human fibroblasts self-assembly of palmitoyl lipopeptides used in skin care products bioactive films produced from selfassembling peptide amphiphiles as versatile substrates for tuning cell adhesion and tissue architecture in serum-free conditions influence of elastase on alanine-rich peptide hydrogels interaction between a cationic surfactant-like peptide and lipid vesicles and its relationship to antimicrobial activity self-assembled arginine-coated peptide nanosheets in water toll-like receptor agonist lipopeptides self-assemble into distinct nanostructures approved drugs containing thiols as inhibitors of metallo-ß-lactamases: a strategy to combat multidrug-resistant bacteria references leukotriene a4 hydrolase -an envolving target. inflammatory diseases -immunopathology, clinical and pharmacological bases the bifunctional enzyme leukotriene-a, hydrolase is an arginine aminopeptidase of high efficiency and specificity lipoxygenase and leukotriene pathways: biochemistry, biology, and roles in disease a critical role for lta4h in limiting chronic pulmonary this work was supported by the czech science foundation (project p305/11/0708) and czech academy of sciences the tn antigen-structural simplicity and biological complexity bel b-trefoil: a novel lectin with antineoplastic properties in king bolete (boletus edulis) mushrooms acknowledgements: cynthia leyva-arg€ uelles is supported by a personal grant from conacyt, mexico. this work is supported by conacyt grant 131'06:15 and papiit grant in218714 sequence information-based deciphering of biofunctionalities using ism-based techniques has fetched calculation of biological functionalities, designing of biomedical device called computer-aided drug resistance calculator, the understanding of the mechanism of hiv progression to aids [4], and others. they have compared the efficacies of drugs and vaccines, which formed the basis for the innocentive award (id 9933477) for assessing vaccine potency. conclusions: deciphering biological features without engaging reagents, equipments and animal tissues but biological data such as sequence information is one novel, feasible genotypic hiv-coreceptor tropism prediction with geno2pheno [coreceptor]: differences depending on hiv-1 subtype a reliable phenotype predictor for human immunodeficiency virus type 1 subtype c based on envelope v3 sequences available: http:// istree.bioprotection.org signal processing-based bioinformatics methods for characterization and identification of bio-functionalities of proteins an empirical framework for binary interactome mapping estimating the size of the human interactome coming to peace with protein complexes? 5th capri evaluation meeting pj-022 cabs-dock web server for protein-peptide docking with significant conformational changes and without prior knowledge of the binding site while other docking algorithms require pre-defined localization of the binding site, cabs-dock doesn't require such knowledge. given a protein receptor structure and a peptide sequence (and starting from random conformations and positions of the peptide), cabs-dock performs simulation search for the binding site allowing for full flexibility of the peptide and small fluctuations of the receptor backbone cabs-flex: server for fast simulation of protein structure fluctuations cabs-fold: server for the de novo and consensus-based prediction of protein structure cabs-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site mechanism of folding and binding of an intrinsically disordered protein as revealed by ab initio simulations modeling of protein-peptide interactions using the cabs-dock web server for binding site search and flexible docking cabs-fold: server for the de novo and consensus-based prediction of protein structure cabs-flex: server for fast simulation of protein structure fluctuations aggrescan3d (a3d): server for prediction of aggregation properties of protein structures cabs-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site staphylococcal pathogenicity island dna packaging system involving cos-site packaging and phage-encoded hnh endonucleases the etiology of asdis unknown, but it is believed that it involves genetic and environmental components. the purpose of this work is to assess the possible involvement of food contaminants, such as mycotoxins, in the etiology of asd. the hypothesis is that the mycotoxins ingested with the diet could bind to proteins and expose the entire organism,including cns, to the negative effects of xenobiotics, in genetically predisposed patients. in this study some possible protein targets for the mycotoxinswere identified to evaluate if the bond between any protein target and the mycotoxin in exam could play a role in asd. twelve mycotoxins were selected (ochratoxin a, gliotoxin, aflatoxin b1, aflatoxin b2, aflatoxin m1, aflatoxin m2, aflatoxicol, a-zearalanol, b-zeralanol, zearalenone, deoxynivalenol, patulin),which are contaminants of milk and cereals.for each of these molecules,possible protein targets were searched by a reverse docking approach using the idtargetserver[2].from the results given by idtarget, human protein targets expressed in the brain or involved in brain diseaseswere selected. subsequently, a direct docking was made using auto-dock 4.2 [3], in orderto verify the strength of the interaction between selected proteins and each mycotoxin, and to identify the mycotoxins' binding site on each of the selected protein. finally, the bond of some mycotoxins to selected protein targets has been experimentally tested. for each mycotoxin, idtarget returned thousands of possible protein targets,and only those with the best binding energy were selected and evaluated. among them, human protein targets that are expressed in the brain or that are involved in cerebral diseases,have been selected; moreover the protein targets that were not human but that idtargetselected for five or more mycotoxins, were replaced with their human counterparts. at the end of the procedure, nineteen protein targets have been identified for the following direct docking approach. from the docking results, eight proteins have been selected for experimental tests, having a predicted binding energy lower than 27 kcal/mol. finally, the interactions between acetylcholinesterase (ache), b-secretase (bace1) and neuroligin-4, x-linked (nlg4x) with aflatoxin b1, aflatoxin b2, gliotoxin, ochratoxin a and deoxynivalenol, were evaluatedusing fluorescence spectroscopy and microscale thermophoresis. these experiments confirmed the presence of an interaction between bace1 and aflatoxin b1 idtarget: a web server for identifying protein targets of small chemical molecules with robust scoring functions and a divide-and-conquer docking approach the calculation of spatial structure and "assembling" of the whole protein from the obtained peptide structures were performed by using molecular dynamics of the protein in the fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (popc) [4]. the obtained structural model may contribute to identification of ul49.5 active sites and elucidation of its mode of action nmr structural studies of membrane proteins acknowledgments: polish national centre for research and development -grant number 178479 pl-017 functional and mechanistic studies of dysferlin, an essential protein in cell membrane repair references moreover the 'm' parameter, which represents the denaturant effect on the protein stability, is 669 cal•mol-1 for bgla and 860 cal•mol-1 for bglb albert einstein college of medicine protein structure modeling, protein-protein interaction computational modeling of ini1/smarcb1 and novel insights into its interaction with hiv-1 integrase savita bhutoria1 epsteain bar virus, nuclear antigen)1. ini1/smarcb1 has no known structural homologues, and its amino-acid sequence yields little insight into its function. a detailed understanding of structure-function relationships is hampered by the lack of structural information for ini1. computational methods that model protein/peptide structures with sufficient accuracy to facilitate functional studies have had notable successes. we carried out combination of sequence analysis ab initio structure modeling and dynamics studies of integrase binding domain of ini1 and found it to be similar to that of phospholipase a2 activating protein, plaa. structural similarity with this distant protein suggests divergent evolution of the two proteins. the modeled structure sheds light on various protein-protein interactions of ini1. by integrating the experimental studies about the binding, we have shown through docking, how a fragment of ini1 binds to the hiv-1 in. molecular docking and experimental studies indicated that two proteins bind tightly through charged/polar residues surrounding a hydrophobic cleft. these studies provide first modeled structure of ini1/smarcb1 or any component of the swi/snf complex, and provide structural basis for in-ini1 interactions. this molecular interpretation of the intermolecular interactions is expected to facilitate design of inhibitors as novel class of anti-hiv-1 therapeutic agents ): e60734. with their catalytic activity towards rna substrates, other biological properties have been reported and evolution studies suggest an ancestral host-defence function in vertebrates. indeed, genetic studies confirmed a rapid molecular evolution within the family, a distinctive trait for host defence proteins exposed to a changing pathogen environment. previous studies from our laboratory characterized the wide spectra antimicrobial activity of two highly cationic human rnases: the eosinophil rnase 3 and the skin derived rnase ribonucleases 6 and 7 have antimicrobial function in the human and murine urinary tract structural determinants of the eosinophil cationic protein antimicrobial activity two human host defense ribonucleases against mycobacteria, the eosinophil cationic protein (rnase 3) and rnase 7 the regulatory mechanism that we are reporting will contribute to prevent both nad1 exhaustion and accumulation of the toxic bal. to the best of our knowledge, this is the first report of a novel reversible covalent modification of an aldh enzyme involving its own substrate anna lewandrowska 1 , aldona jeli nska 1 , agnieszka wi sniewska 1 acknowledgement: this research was supported by the inactivation of the fxr gene reduces aqp2 expression and impairs urine concentrating ability, which leads to a polyuria or urine dilution phenotype. we have previously found that pon1-/-mice exhibit a polyuria phenotype and produce twice as much 24-h urine as their wild type pon11/1 littermates (borowczyk k et al. metabolism and neurotoxicity of homocysteine thiolactone in mice: evidence for a protective role of paraoxonase 1 development and application of novel non-ewald methods for calculating electrostatic interactions in molecular simulations ikuo fukuda 1 , narutoshi kamiya 1 the most time-consuming part of molecular simulation is the calculation of long-range interactions of the particles. in particular, appropriate treatment of the electrostatic interaction is critical, since the simple truncation cannot be used due to the slow decay of the coulombic function. thus, it is highly demanded to calculate the electrostatic interactions with high accuracy and low computational cost. for this purpose we have developed the zero-multipole (zm) summation method [1]. in this method the artificial periodic boundary conditions are not necessary and the fourier part evaluations are not needed, in contrast to the conventional ewald-based methods. instead, a pairwise function that is suitably redefined from the coulombic function is used with a cutoff scheme. the underling physical idea is simple: (a) in a biological system, a particle conformation for which the electrostatic interactions are well cancelled is more stable than other conformations [2]; (b) since such well-cancelled conformations are essentially physical, we should clip a subset of such a conformation out of the conformation within an ad-hoc given cutoff sphere and calculate the interactions only from this subset. this idea is realized by a rigid mathematical consideration that leads to the deformation of the coulombic function. the efficiency of the zm method has been validated in applications to fundamental systems sema 3a) is a protein originally described as an axonal chemorepellent cue involved in many physiological processes ranging from embryonic development to bone homeostasis or immune responses sema3a signal transduction requires the formation of a heteromeric complex with neuropilin-1 (nrp1) and plexina [2]. in addition, sema3a interaction with nrp1 is modulated by the furin protease cleavage at its c-terminal basic domain this c-terminal basic domain has also been suggested to mediate the binding to glycosaminoglycans (gags), an association that locates sema3a to perineuronal nets and enhances its function in restricting neuronal plasticity and inhibiting axonal regeneration in the central nervous system two peptides corresponding to the highly positively charged regions on the domain were shown to bind to immobilized heparin by surface plasmon resonance (spr) and the affinity dramatically increased when the complete domain was assayed. the binding was confirmed by nuclear magnetic resonance (nmr) and circular dichroism (cd) the conserved cysteine within this motif, necessary for the dimerization of sema3a [7], is also critical for the helix formation. in addition, fluorescence spectroscopy studies showed that the n-terminal region also has a contribution in the binding to gags. we acknowledge the financial support from the european union seventh framework programme (fp7/2007-2013) under the project vision semaphorin 3a: a new player in bone remodeling neuropilins lock secreted semaphorins onto plexins in a ternary signaling complex furin processing of semaphorin 3f determines its anti-angiogenic activity by regulating direct binding and competition for neuropilin semaphorin 3a displays a punctate distribution on the surface of neuronal cells and interacts with proteoglycans in the extracellular matrix semaphorin 3a binds to the perineuronal nets via chondroitin sulfate type e motifs in rodent brains mechanistic basis for the potent anti-angiogenic activity of semaphorin 3f. biochemistry collapsin-1 covalently dimerizes, and dimerization is necessary for collapsing activity prior attempts to create functionally relevant groupings of proteins in the crotonase superfamily suggest that this superfamily is difficult to cluster functionally due in part to the functionally diverse nature of the protein superfamily. we have developed two novel procedures to combat this difficulty: tulip (two-level iterative clustering process), a process that utilizes structural information from active sites to cluster protein structures into hypothesized functional groupings, and misst (multi-level iterative sequence searching technique), a process that uses the protein groupings created in tulip as a starting point for iterative genbank searches and further clustering after each search. through these two methods, the total coverage of the crotonase superfamily has increased, and the generated groups contain proteins from subgroups and families that did not have a structural representative. novel hypothesized functional protein groupings have been created, most notably for a large number of proteins that lack annotation data at the subgroup or family level, and for proteins of the enoyl-coa hydratase family fernandes 1 , teresa sorbo 2 , ivan duka 2 , lia christina appold 3 , marianne ilbert 4 , fabian kiessling 3 cnrs, umr 7281, 5 ucibio-requimte, faculdade de ciências e tecnologia e-selectin is a cell-adhesion molecule induced on the surface of endothelial cells in response to cytokines. its upregulation has been reported in many disorders, including inflammatory and cardiovascular diseases, tumor angiogenesis and metastasis [1]. this profile suggests e-selectin as a promising target to develop molecular imaging probes for the detection of these diseases cyt c with 1 equivalent of fld in media mimicking the cytoplasm. these include 8% polyacrylamide gel, 100 g/l bovine serum albumin or polyvinylpyrrolidone 40, and buffer alone for comparison. electrostatic surface representations of the proteins are shown with their in-cell spectrum pl-060 a search for anti-melioidosis drug candidates targeted to d-glycero-d-manno-heptose-1,7-bisphosphate phosphatase from burkholderia pseudomallei bpgmhb converts dglycero-d-manno-heptose-1b,7-bisphosphate to d-glycero-d-manno-heptose-1b-phosphate. this is the third step of the biosynthesis pathway of ndp-heptose responsible for a pleiotropic phenotype. therefore, this biosynthesis pathway is the target for inhibitors increasing the membrane permeability of gram-negative pathogens or adjuvants synergistically working with known antibiotics. to find inhibitors of bpgmhb, we performed homology modeling of bpgmhb and in-silico virtual screening with zinc, a free database of commerciallyavailable compounds. tens of thousands of chemical compounds were docked into the active site of bpgmhb. a number of putative bpgmhb binding compounds better than d-glycero-d-manno-heptose-1b,7-bisphosphate were found using surflex-dock included in the sybyl software package crystal structure of dimeric d-glycero-d-manno-heptose-1,7-bisphosphate phosphatase from burkholderia thailandensis ewha womans university we have solved the crystal structures of d-glycero-d-manno-heptose-1,7-bisphosphate phosphatase from burkholderia thailandensis (btgmhb) catalyzing the removal of the phosphate at the 7 position of d-glycero-d-manno-heptose-1,7-bisphosphate. it belongs to the haloacid dehalogenase (had) superfamily with an a/b rossman fold composed of six parallel b-strands sandwiched between two sets of three a-helices it reveals a conventional rossman-like a-b-a sandwich fold with a novel b-sheet topology. its c-terminus is longer than its closest relatives and forms an additional b-strand whereas the shorter c-terminus is random coils in the relatives. interestingly, its core structure is similar to that of enzyme iib(cellobiose) from e. coli (eciib(cel)) transferring a phosphate moiety. in the active site of the closest eceiib(fruc) homologues, a unique motif cxxgxaht comprising a p-loop like architecture including a histidine residue is found. the conserved cysteine on this loop may be thiolated to act as a nucleophile similar to that of eciib(cel). the conserved histidine residue is presumed to accommodate negatively charged phosphate during enzymatic catalysis leonor morgado 1 , kornelius zeth 1,2,3 , bj€ orn m. burmann 1 , timm maier 1 bama is a b-barrel membrane protein with five periplasmic n-terminal polypeptide transport associated (potra) domains. the bama structure has been determined recently by x-ray crystallography (2,3), however its functional mechanism is not well understood. this mechanism comprises the insertion of substrates from a dynamic, chaperone-bound state into the bacterial outer membrane, and nmr spectroscopy is thus a method of choice for its elucidation we demonstrated that knocked down autophagy by shrna (shatg5, shbecn1, and shatg12) and chloroquine (cq) could enhance high dose of uvb induced cell death in odc overexpressing hela and mcf-7 cells. here, we also observed that knocked down odc in odc overexpressing hela and mcf-7 cells inhibited autophagy and enhanced high dose of uvb radiation. because of atg12 can regulate cell apoptosis and utophagy. site directed mutagenesis was used to mutant the amino acid which can regulate cell apoptosis and autophagy on atg12, respectively in these two odc overexpressing cells. according to the results fish ß-parvalbumin acquires allergenic properties by amyloid assembly using atlantic cod b-parvalbumin (rgad m 1) displaying high ige crossreactivity, we have found that formation of amyloid fibers under simulated gastrointestinal conditions accounts for the resistance to acid and neutral proteases, for the presence of membrane active species at gastrointestinal relevant conditions and for the ige-recognition in allergic patient sera. incorporation of the anti-amyloid compound epigallocathequin gallate prevents rgad m1 fibrillation, facilitates its protease digestion and impairs its recognition by ige. conclusions: rgad m 1 amyloid formation explains its degradation resistance, its facilitated passage across the intestinal epithelial barrier and the epitope architecture as allergen autophagy could degrade the citrullinated and unfolding protein. herein, padi2 could enhance autophagy in jurkat t cells and lead to a degradation of p62 and the accumulation of lc3-ii. autophagy and apoptosis are two critical mechanisms which participate against cellular stress, cell activation, survival and homeostasis. pad2-overexpressed jurkat t cells caused the activation of th17 cells to increase mrna expression of cytokines, such as il-17, il-21, il-22 and tnfa. cytokines provoked caspase expression and led to caspase-mediated cleavage of beclin-1 which was an important factor of apoptotic signaling. knockdown of bcen1 rescued cell survival due to the increase of bcl-xl and the decrease of caspase-3. we suggested that padi2 participated in the activated t cell-induced autonomous death through triggering er stress pathway studies on secondary metabolites production and proteins and enzymes of in vitro cultivated artemisia alba turra and relations with some endogenous phytohormones yuliana raynova 1 , krassimira idakieva 1 , vaclav motyka 2 , petre dobrev 2 , yuliana markovska 3 , milka todorova 1 , antoaneta trendafilova 1 , ljuba evstatieva 4 switzerland aim: artemisia alba turra is an essential oil bearing shrub, characterized with great variability of the essential oil profile of wild grown plants, related to genetic, geographic and environmental factors. it was previously established that inhibition of rooting in vitro caused by cytokinin/auxin treatment affected the essential oil profile of the plant and these changes were also related to bioactive endogenous cytokinin levels in vitro (1, 2) cytokinin and auxin effect on the terpenoid profile of the essential oil and morphological characteristics of shoot cultures of artemisia alba terpenoid profile of artemisia alba is related to endogenous cytokinins in vitro salivary hnp 1-3 are conventionally measured using an enzyme-linked immunosorbent assay (elisa) which does not discriminate between individual hnps due to their structural similarities. considering the biological importance of salivary human neutrophil a-defensin (hnps), there is therefore, a need to develop an analytical method that will discriminate between the defensins. an lc-ms method has been established for the separation and detection of hnp 1-4. the method has been optimised, validated and applied to examine the relative level of hnp 1-4 in participants undertaking a circuit resistance training workout. to date, no studies have systematically investigated the effect of acute (min to hours) and chronic (days to weeks) change in salivary adefensins family before and after exercise by lc-esi-ms systems and models calorimetry showed no difference in dissociation constants at these ph values, while the binding stoichiometry is increased 2.5 fold. furthermore, the binding stoichiometry varied 7 fold among the two alginates corresponding to their difference in average molecular weight and in addition 20 fold higher binding affinity was found with the high as compared to the low molecular weight alginate. in conclusion, the binding stoichiometry of b-lactoglobulin with alginate increases by a factor that correlates to the average molecular weight of the alginate and also a much higher affinity was found for the high molecular weight alginate. acknowledgements: this work is supported by the danish council for the presence of mucins and other high molecular weight glycoproteins in saliva makes the direct analysis of defensins difficult. the lc-ms method was linear for concentrations of hnp-2 between 0.05 and 1 ng/ml (r2 5 0.99) with a lod of 0.05 ng/ml. inter and intra assay precision was 0.94 -15%, respectively. saliva sample were clean up by solid phase extraction (spe) and without-solid phase extraction (wspe) 2.10 mm internal diameter column in relation to the method transfer . during lc-ms optimisation genome-wide docking database (gwidd) provides the most extensive data repository of structures and models of ppi on a genomic scale. currently, we are expanding the gwidd dataset to 800,365 ppi in 1,652 organisms, up from 128,818 ppi in 771 organisms in the previous release. the ppi data were imported from intact and biogrid databases and were subjected to in-house modeling pipeline. gwidd current implementation contains 11,073 experimentally determined complexes, and 12,426 sequence homology and 28,811 structure homology models of complexes. the user-friendly interface offers flexible organism-specific search with advanced functions for a refined search for one or both proteins. the new gwidd version includes also a new interactive visualization screen that allows to view search results in different residue representations with the emphasis on the ppi interface refolding and activation of recombinant trypsin i from sardine fish (sardinops sagax caerulea) amyloid is detectable in human dental plaque and is produced by both clinical and laboratory strains of s. mutans, further supporting a functional role. s. mutans lacking p1 demonstrates residual amyloid forming properties, however, a mutant lacking sortase, the transpeptidase which covalently links p1 and several other proteins to the peptidoglycan cell wall, is defective in cell-associated amyloid-like properties. the objectives of this study were to identify additional amyloid forming proteins of s. mutans and to evaluate the effects of buffering conditions and ph on the ability of the identified proteins to form amyloids. a p1-deficient mutant strain was grown to stationary-phase in defined minimal media, and secreted proteins from spent culture supernatants were fractionated by ion exchange chromatography. partially purified protein fractions were tested for binding of the amyloidophilic dyes congo red (cr) and thioflavin t (tht), and for characteristic birefringent properties following staining with cr and visualization under crossed polarizing filters. proteins from fractions that tested positive for amyloid-like material were separated by sds page, and identified by lc/ms. these included wapa, gbpa, gbpb, smu_2147c and smu_63c. recombinant proteins were expressed in escherichia coli, and purified for confirmation and characterization of individual amyloidogenic properties in vitro. recombinant wapa and smu_63c displayed all the biophysical characteristics of amyloid, including visualization of fibrillar aggregates when viewed by transmission electron microscopy. in contrast, gbpa and smu_2147c produced amorphous aggregates. wapa and smu_63c form amyloid at different ph, smu_63c under acidic conditions and wapa under neutral to basic conditions. this suggests that the prevailing environmental ph may represent different in vivo triggers for amyloid fibrillization of different s like other small gtpases, the activity of rheb is dictated by its guanine nucleotide binding states: it is active in its guanosine 5 0 -triphosphate (gtp) bound form and inactive in the guanosine diphosphate (gdp)-bound form. rheb proteins play critical roles in regulating growth and cell cycle, and this effect is due to its role in regulating the insulin/tor/s6k signaling pathway rheb interacts directly with fkbp38 and prevents its association with mtor in a gtp-dependent manner. moreover, fkbp38 bound to gtp-g-s, a nonhydrolyzable gtp analogon, has a much higher binding affinity for rheb than the gdp-bound form the second study contradicted both studies, since they could not detect any interaction between rheb and fkbp38 [8]. to clarify whether there is an interaction and if it is nucleotide dependent, nmr monitored interaction studies were performed employing a c-terminal truncated construct of human rheb (1-170 5 rhebdct) that cannot be farnesylated and the biochemically defined binding region on fkpb38 (fkbp12-like 5 fkbp38-bd). based on our data rhebdct -gdp does not significantly interact with fkbp38-bp. 15n-fkbp38-bd titrated , we observed a weak interaction between rhebdct bound to a gtpanalogon (gppnhp) and fkbp38-bd. mapping of the observed spectral changes on the structure of rheb-gtp suggests that fkbp38 targets the switch 2 region, loop 109-112 and the neighboring b-sheet region. we further analyzed the backbone dynamics of rhebdct -gdp and -gppnhp using 15n relaxtion data (t1, t2 and heteronuclear noe) .based on these data the phosphorylation loop, the switch regions and the loop around residues 109-112 show increased backbone dynamics that modulated by the nucleotide binding 5 international centre for genetic engineering and biotechnology tdp-43 is an rna processing protein that can form inclusions of debatable nature implicated in neurodegenerative diseases. within the putative aggregation domain, repeats of residues 341-366 can recruit endogenous tdp-43 into aggregates inside cells1. recently, we showed that a coil to b-hairpin transition in a short peptide corresponding to tdp-43 residues 341-357 enables oligomerization2. we have used a broad battery of biophysical experiments, including chromophore and antibody binding, electron microscopy (em), circular dichroism (cd), solution and solid-state nmr, and x-ray to shed light on the nature of these aggregates. based on these findings, structural models for tdp-43(341-357) oligomers have been constructed, refined, verified, and analyzed using computational methods, ranging from docking and molecular dynamics simulations to semiempirical quantum mechanics calculations. interestingly, tdp-43(341-357) b-hairpins assemble into a novel parallel b-turn configuration showing crossb spine, cooperative h-bonding and tight side chain packing3 cellular model of tar dna-binding protein 43(tdp-43) aggregation based on its c-terminal gln/asn-rich region structural characterization of the minimal segment of tdp-43 competent for aggregation structural evidence of amyloid fibril formation in the putative aggregation domain of tdp-43 kyungpook national university scolopendin 2, aglqfpvgrigrllrk, is a 16-mer peptide derived from the centipede scolopendra subspinipes mutilans. to investigate its property against fungal and bacterial pathogens, antimicrobial tests were performed. we observed that this peptide exhibited antimicrobial activity in a salt-dependent manner and showed no hemolysis. the circular dichroism (cd) analysis observed that a-helical structure properties. we determined the mechanism(s) of action using flow cytometry and investigated the release of potassium. the results showed that the microbial membrane in escherichia coli o157 and candida albicans was permeabilized with loss of potassium ions. additionally, the bis-(1,3-dibutylbarbituric acid) trimethine oxonol [dibac4(3)] and 3,3'-dipropylthiacarbocyanine iodide [disc3 (5)] assay showed membrane depolarization. using calcein-encapsulating giant unilamellar vesicles (guvs) and fitc-dextran containing large unilamellar vesicles (luvs), scolopendin 2 disrupted the cell membrane and the damage size is between 4.8 to 5.0 nm against composition of microbial plasma membrane of e. coli and c. albicans. thus, we demonstrated that a cationic antimicrobial peptide, scolopendin 2, possesses broad-spectrum antimicrobial effects that formed pore on the cell membrane. structural and functional investigation of the far c-terminal domain (ctd) of the bifunctional enzyme trai using nmr spectroscopy protein structural biology structural and functional investigation of the far c-terminal domain (ctd) of the bifunctional enzyme trai using nmr spectroscopy b.krishna chaitanya, evelyne schrank and klaus zangger institute of chemistry/organic and bioorganic chemistry university of graz, austria corresponding author email id: krishna.bhattiprolu@uni-graz.at bacterial conjugation is a complex process for the horizontal transfer of single stranded dna from one cell to another. this mechanism also leads, for example, to the spread of antibiotic resistance genes and virulence factors among bacterial species. multi-protein complexes formed at the origin of transfer (orit) region of dna and at the cytoplasmic membrane of the bacterial cell, initiate this process. inside the membrane, the relaxosome identifies the single strand for transfer in a plasmid dna, relaxes and unwinds it, whereas the transferosome is involved in pilus formation (type iv secretion system) and transferring the gene through the cytoplasmic membrane. these events take place in the donor bacterial cell along with several other auxiliary proteins [1] the bifunctional enzyme trai of plasmid r1 plays a crucial role in the relaxosome activity, as it contains both a relaxase and helicase domain. to exert its functions on dna, trai works in close co-ordination with other relaxosome proteins like tray, tram and the integration host factor. trai is a 1756 residual protein and contains 3 major domains: n-terminal relaxase domain, a central helicase domain and a c terminal domain (ctd). the structure of the c-terminal domain until residue 1629 has been solved by crystallography, while the structure and function of the remaining 130 residues remained undetermined [2]. there are saxs models and crystallographic structures for different parts of trai and also for the full length protein. prediction of cleavage specificity in hcv ns3/4a serine protease and adv2 cysteine protease systems by biased sequence search threading gonca ozdemir isik 1 , a.nevra ozer 1 1 department of bioengineering,faculty of engineering,marmara university proteases are enzymes which recognize specific substrate sequences and catalyze the hydrolysis of designated peptide bonds to activate or degrade them. due to the biological importance of proteases, it is particularly important to identify the recognition and binding mechanisms of protease-substrate complex structures in drug development studies. the assessment of substrate specificity in protease systems is crucial, where interpreting the adaptability of substrate residue positions can be useful in understanding how inhibitors might best fit within the substrate binding sites and aid in the design of potent selective inhibitors. substrate specificity is generally determined by the amino acid profile, structural features and distinct molecular interactions. besides experimental methods, computational tools for prediction of natural substrate cleavage sites, such as threading, have emerged as useful alternative approaches to provide valuable insights into complex enzyme-substrate interactions. in this work, the substrate variability and substrate specificity of the hepatitis c virus (hcv) ns3/4a serine protease and the adenovirus 2 (adv2) cysteine protease was investigated by the biased sequence search threading (bsst) methodology. using available crystal structures of the proteases, the template structures for the substrate-bound proteases were created in silico by performing various peptide building and docking procedures followed by energy minimization and molecular dynamics (md) simulations. bsst was performed starting with known binding, nonbinding and some random peptide sequences that were threaded onto the template complex structures, and low energy sequences were searched using lowresolution knowledge-based potentials. then, target sequences of yet unidentified potential substrates were predicted by statistical probability approaches applied on the low energy sequences generated. the results show that the majority of the predicted substrate positions correspond to the natural substrate sequences with conserved amino acid preferences, while some positions exhibit variability. for ns3/4a serine protease cleavage, the significant selection for pro at p2 and cys at p1 positions is zearalenone is a mycotoxin produced by fusarium graminearum and related fusarium species. f. graminearum is a powerful plant pathogen and infects major crop plants around the world. acute toxicity of zearalenone is low, but due to its structural similarity to b-estradiol it has binding affinity to the estrogen receptor, which results in interference with hormonal balance. typical effects seen in animals include symptoms like hyperestrogenism and reproductive disorders (reduced fertility, reduced litter size or swelling of uterus and vulva). to reduce the risk for human and animal health posed by the ingestion of contaminated food or feed different decontamination strategies have been studied, including biotransformation. today many microorganisms are known to degrade zearalenone, but for most of them the degradation pathway and formed metabolites remained unknown, hence it is unknown if this degradation also means detoxification. only for the fungal strains trichosporon mycotoxinivorans and gliocladium roseum zen degradation has been studied in detail and loss of estrogenicity of reaction products has been confirmed. we screened for, and isolated zearalenone degrading bacteria from soil samples. the most promising new bacterial isolate was taxonomically assigned to the species rhodococcus erythropolis and designated pfa d8-1. the zearalenone catabolism pathway of pfa d8-1 was found to be identical as known from g. roseum. the primary reaction product, hydrolysed zearalenone, has so far only been postulated in g. roseum. we prepared hydrolysed zearalenone by preparative hplc and showed loss of estrogenicity in assays with the breast cancer cell line mcf7 and the estrogen reporter yeast strain yzhb817. a genomic library was prepared and screened in zearalenone degradation deficient r. erythropolis pr4. the gene encoding zearalenone hydrolase was found and named zena. the hydrolase was identified as member of the a/b-hydrolase family and named zena. it was cloned, recombinantly expressed in e. coli and purified by 6 x his-tag mediated immobilised metal affinity chromatography. activity of his-tagged and untagged enzyme zena was compared in cleared lysate and zena was purified for enzyme characterisation. the influence of ph and temperature on enzyme activity and stability was evaluated and kinetic parameters were determined. a new biding site for snake venom c-type lectins?maria cristina nonato costa 1 , ricardo augusto pereira de p adua 1 , marco aurelio sartim 1 , suely vilela sampaio 1 1 university of são paulo, fcfrp c-type lectins are proteins that bind different glycan molecules by interactions with a calcium atom present in a carbohydrate recognition domain (crd). many organisms (plants, bacteria, virus and animals) use these proteins in various biological events like lymphocyte adhesion, erythrocyte agglutination and extracellular matrix organization. the c-type lectin fold is plastic and possible for about 1013 different sequences, what promoted its adaptation to diverse functions, similarly to the observed for the immunoglobulin fold (1014-1016 sequences). it is comprised of about 110-130 amino acid residues that folds in two fourstranded b sheets sandwiched by two alpha helices. interestingly, c-type lectins present in snake venoms are possible anti-cancer agents since they are toxic to cancer cells and inhibit the adhesion and proliferation of various cancer cell lines. therefore, we have purified a lactose binding c-type lectin from the venom of bothrops jararacussu (bjcul) to study its structure and binding properties to different sugars. bjcul crystals were obtained by vapor diffusion and the structure solved by x-ray crystallography to 2.9 å resolution. bjcul structure is a decamer formed by a pseudo fivefold axis rotation of a dimer hold by a disulfide bond. each monomer binds a calcium atom and possibly another metal at a second and opposed binding site. key: cord-008777-i2reanan authors: nan title: ecb12: 12th european congess on biotechnology date: 2005-07-19 journal: j biotechnol doi: 10.1016/j.jbiotec.2005.06.005 sha: doc_id: 8777 cord_uid: i2reanan nan in the last 20 years biotechnology has made tremendous progress in its different application fields: red biotechnology, the use of biological methods for medical purposes, is firmly established in the development of new drugs. the use of plant or green biotechnology is under controversial discussion in politics and public. nevertheless, genetically modified herbicide and insect resistant crops are cultivated to a large extent. industrial biotechnology, now often named white biotechnology, seems widely underestimated in the public perception. it includes all industrial processes for the production of chemical products and enzymes, which fully or partly rely on the biological toolbox of nature. white biotechnology processes are carried out in a contained environment, typically in a bioreactor in a dedicated industrial plant. well-known examples are the fermentative productions of antibiotics, amino acids, vitamins and enzymes, products related to medical, food and feed applications. many products like the amino acids glutamic acid, lysine, threonine and tryptophane are exclusively produced using microbes in large scale industrial processes. in other cases, like the water soluble vitamin b2, biotechnological processes successfully replaced chemical productions, due to lower costs and improved ecoefficiency. in contrast to this, most industrial chemicals and polymers are produced by chemical synthesis based on oil and gas. however, there are some examples for bioproducts among industrial chemicals. the solvents acetone and butanol, for instance, were manufactured by fermentation for several decades in the last century. since the 1950s these fermentations have been replaced by more efficient and cheaper chemical synthesis. recently, new pilot and production processes for biopolymers like pha or biomonomers like 1,3-propanediol or lactic acid were announced by different companies. currently, ethanol is by far the largest white biotech product by volume. in brazil, where bioethanol is used as liquid transportation fuel, the annual production is in the range of 15 mio m 3 . bioethanol is of growing importance also in the united states. business consultants predict a tremendous growth of biotechnological products within the chemical industry. high prices for crude oil, dropping prices for renewable resources, and scientific progresses nourish the expectation that industrial biotechnology will replace many bulk chemicals. is this realistic? will we switch from a petrochemical industry to a biobased chemistry within the next years? based on economic considerations it can be stated that this is a long term goal. to achieve this it remains a scientific challenge to make renewable raw materials available for competitive bioproduction of bulk chemicals at low costs. conversion of lignocellulosic material to fermentation sugar may be a solution. also green biotechnology can contribute to the supply of cheap fermentation raw materials. innovative ideas for downstream processing or further chemical conversion of fermentation products are required to enter the chemical value chains. furthermore, the identification of new higher value bioproducts is a chance for short term successes in white biotechnology. enzyme and protein engineering has the potential to create new biomolecules, metabolic engineering can contribute to develop new metabolic pathways, may be even for unnatural compounds. by continuously increasing the efficiency and throughput of dna sequencing we, together with colleagues, have sequenced the human genome and the genomes of all the major model organisms. the challenge now centers on understanding these vast instruction sets. our ability to read these instructions must be enhanced through collection of key additional data sets. one productive path for delineating the functional sequences and inferring their function is comparative sequence. the mouse genome sequence, for example, led to estimates that only 5% of the human genome is functional. sequencing of an extensive set of additional mammalian genomes promises to define these functional sequences with a resolution of less than 10 base pairs. on a different course, we have sequenced the chimpanzee genome to learn what has changed in the evolution of humans. beyond providing for the first time a catalog of the differences between the two genomes, the comparison of the chimpanzee and human genomes reveals the patterns of neutral mutation and regions that deviate from that. the talk will summarize these and related findings. the sequence of additional primate genomes will help delineate what has changed specifically in humans and add power to the analysis. ultimately, capturing human sequence variation and correlating with phenotypic variation will be required to understand function. but learning what these functional elements do requires new sets of experimental data. for this, we have turned to the nematode c. elegans. in this simple system most of the ∼20k genes have been defined and experimentally confirmed. beyond the hundreds of genes with already known mutants, two centers are systematically producing gene knockouts or rnai can be used to inhibit any gene temporarily. sequences of three caenorhabditis species are already available, and two more are underway. expression data has been collected for all the genes under many conditions and time points through development. to enhance the resolution of expression data and to simplify phenotypic analysis of embryonic mutants, we are developing a system that will automatically trace the cell lineage and assign gene expression to precise cells with high temporal resolution. the latest results with the system will be described. in the longer term, this and similar datasets should provide an understanding of how the genome specifies the form and behavior of the worm. uhlen department of biotechnlogy, albanova university center, royal institute of technology (kth), stockholm, sweden here, we present a new protein atlas database (www.proteinatlas.org) showing the expression and localization of human protein in normal and cancer tissues. the atlas is based on the use of antibodies (agaton et al., 2003) to generate high-resolution immunohistochemistry images representing 48 normal tissues and 20 different cancer types (uhlen and ponten, 2005) . each antibody is used to generate more than 500 individual images and each image has been annotated by a pathologist (kampf et al., in press) . the database has been created by the swedish human proteome resource (hpr) and the program has been set-up to allow the exploration of the human proteome with antibody-based proteomics (nilsson et al., in press) . the basic concept is to generate, in a systematic and high-throughput manner (uhlen and ponten, 2005) , specific antibodies to all human proteins, and subsequently used these for functional analysis of the corresponding proteins in a wide range of assay platforms, including (i) a protein atlas for tissue profiles (kampf et al., in press) , (ii) specific probes to evaluate the functional role of individual proteins, and (iii) affinity reagents for purification of the specific proteins and their associated complexes for structural and biochemical analyses. ments, most effective source of variation was perturbation in growth medium, followed by perturbation in growth rate. effect of gene deletion on data variation was found to be less apparent when compared to other perturbations. a significant similarity in variation of metabolome and mrna data was observed, which may be used as the key point for integration of these two sets of data in functional analysis of genes. projection to latent structures (partial least squares, pls) is used for integration of transcriptome and metabolome data. comparison of pca and pls shows that linear model constructed via pls to predict the metabolome data does not make use of all the variation in transcriptome data. thus, pls allows the discrimination between the portion of gene expression change that affects the metabolome profile and the portion that is not directly effective on metabolome. both pca and pls can be used to detect the open reading frames (orfs) which are the main sources of variation in transcriptome data and/or effective on metabolome profile. extracellular metabolomics to accelerate the discovery of key genes involved in fibre degradation silas g. villas-bôas, geoffrey lane, graeme attwood, adrian cookson agresearch limited, grasslands research centre, tennent drive, private bag 11008, palmerston north, new zealand. e-mail: silas.villas-boas@agresearch.co.nz (s.g. villas-bôas) the genome of the hemicellulose-degrading microbe clostridium proteoclasticum is been sequenced and an array of candidate genes with diverse activity relevant to fibre degradation have been identified by automated gene annotation methods. c. proteoclasticum falls within the butyrivibrio-pseudobutyrivibrio assemblage of rumen bacteria which are though to play an important role in the degradation of plant hemicellulose-lignin complexes which limit fibre degradation in the rumen. for new zealand it makes strategic sense to invest in microbial genomics efforts applied to agriculture where the country holds a strong competitive advantage and where ruminants constitute the vast majority of farmed animals. in conjunction with dna sequencing, proteomics and transcriptomics (micro-array analysis) we are using metabolomics as an additional functional genomics tool for gene discovery. we have established a footprinting approach for microbial metabolome analysis focused mainly on metabolic intermediates of polysaccharide degradation to provide quantitative information on end products of fibre-degrading enzymes. a gc-ms method has been developed that is able to resolve complex biological mixtures containing mono-, di, and oligosaccharides, in addition to a series of organic acids. we are currently phenotyping a series of c. proteoclasticum mutants to validate our analytical methodology and we are going to fully characterize the fibrolytic ability of c. proteoclasticum to be compared with other fibre-degrading microbes. we believe that our metabolomics data will complement current proteomic analysis of fibre-degrading enzymes and micro-array analysis of gene expression from a series of mutants by providing direct evidence of the metabolic function of key genes involved in fibre-degradation processes. many gram-negative bacteria utilize cell-to-cell communication systems that rely on diffusible n-acyl homoserine lactone (ahl) signal molecules to monitor the size of the population in a process known as quorum sensing (qs). in human pathogens this form of gene regulation ensures that the cells remain invisible to the immune system of the host until the pathogen has reached a critical population density sufficient to overwhelm host defenses and to establish the infection. the qs regulon of pseudomonas aeruginosa and burkholderia cepacia, two important pathogens for patients suffering from cystic fibrosis, has been studied by proteome analyses. comparative twodimensional gelelectrophoresis of pre-fractionated protein mixtures (extra-, surface-, and intracellular proteins) coupled to mass spectrometry analysis or n-terminal sequencing has been employed to recognize and identify qs-controlled proteins. our findings strongly support the importance of ahl-mediated cell-cell-communication as a global regulatory system and suggest that qs control also operates via post-translational mechanisms. as qs has been proven to be a central regulator for the expression of pathogenic traits and biofilm formation in opportunistic human pathogens it represents a highly attractive target for the development of novel anti-infective compounds. functional genomics technologies (transcriptomics and proteomics) have been exploited to validate the target specificity of natural and synthetic qs inhibitors, thus having a great potential as alternative therapeutics for the treatment of bacterial infections. modeling cell cycle complex formation from high-throughput data sets lars juhl jensen european molecular biology laboratory, meyerhofstrasse 1, 69117 heidelberg, germany. e-mail: jensen@embl. de to analyze the dynamics of protein complexes during the mitotic cell cycle, we integrated data on protein interactions and gene expression. the resulting time-dependent interaction network for the first time places both periodically and constitutively expressed proteins in a temporal cell cycle context, thereby revealing novel components and modules. we discover that most complexes consist of both periodically and constitutively expressed subunits, suggesting that the former control complex activity by a mechanism of just-in-time assembly. consistent with this, we show that additional regulation through targeted degradation and phosphorylation by cdk1 (cdc28p) specifically affects the periodically expressed proteins. alessandra luchini, andrea callegaro, silvio bicciato department of chemical engineering processes, university of padova, padova, italy. e-mail: alessandra.luchini@unipd.it (a. luchini) since transcriptional control is the result of complex networks, analyzing dynamical states of gene expression is of paramount importance to detect the multivariate nature of biological mechanisms. although hundreds of studies fully demonstrated the relevancy of microarrays in describing different physiological conditions, to reconstruct complex interaction pathways it is necessary to analyze the temporal evolution of transcriptional states. however, a robust experimental design for identifying differentially expressed genes over a temporal window would require large amounts of microarrays. unfortunately, replicates for each time point and experimental condition are not always available, because of cost limitations and/or biological samples scarcity. in addition, common data analysis tools, like anova, require replicates and disregard correlation structure among times. we present a method for the identification of differentially expressed genes in un-replicated time-course experiments. the procedure does not assume any model or distribution function, takes into account the correlation of data, and does not require sample replicates at the various time points, other than the presence of an initial time point for all analyzed conditions. the identification of differentially expressed genes as the result of a system perturbation is formally stated as a hypothesis testing problem in which a defined statistic is used to rank transcripts in order of evidence against the null hypothesis. specifically, (i) data are structured so that measurements are correlated in time, within the same biological condition; (ii) the null hypothesis is formulated so that changes in expression levels at different time points are equivalent; (iii) time point t0 represents the system before the perturbation. therefore, modulated genes are detected testing the statistical significance of expression differences between physiological states at each time point, once corrected by the variability at t0, and given an empirical null distribution constructed using permutations. statistical significance is assessed by the q-value. the method has been tested on time-course microarray experiments aimed at studying the temporal changes of gene expression in: (i) skeletal muscle cells treated with a histone deacetylase inhibitor (iezzi et al., 2004) and (ii) immature mouse dendritic cells (dc) exposed to larval and egg stages of s. mansoni (trottein et al., 2004) . differentially expressed genes, identified using the proposed algorithm, have been compared with results obtained from anova model and sam paired test. the biological significance and soundness of selected transcripts was also verified using global functional profiling by means of ontotools. results demonstrate that this novel procedure allows the identification of biologically relevant genes using half of the replicates required by standard model-based approaches. carbon sources. interesting data on the expression profile of the sty and paa genes in pseudomonas sp. y2 have been obtained, and have raised new questions on styrene and paa degradation by this bacterium. the calcium-dependent antibiotic (cda) is a lipopeptide synthesised non-ribosomally and produced by streptomyces coelicolor a3(2). cda contains several non-proteinogenic amino acid residues. hydroxyphenylglycine (4-hpg) is one of the unusual amino acids in the structure of the cda and vancomycin groups of antibiotics. for the members of the vancomycin group of antibiotics, the 4-hpg residue plays crucial roles in the structure and function of the final glycopeptide antibiotic. to reveal the putative biosynthetic pathway of this amino acid in cda, a standard "double crossover replacement strategy" was used to delete 4-hydroxymandelic acid synthase (4-hmas, encoded by hpd) from s. coelicolor mt1110 and 2377, using the delivery plasmid pzmh3. there was no cda production in the disrupted strains. plates containing a gradient of hydroxymandelic acid were used to restore cda production in both s. coelicolor mt1110 hpd and 2377 hpd. exogenous supply of 4-hydroxyl phenylglyoxylate and 4-hydroxyphenylglycine reestablished cda production by the hpd mutant. feeding analogs of these precursors to the mutant resulted in the directed biosynthesis of novel lipopeptides with modified arylglycine residues (mutasynthesis). a cxcl2 tandem repeat promoter polymorphism is associated with susceptibility to severe sepsis in the spanish population n. maca-meyer 1 , c. flores 1 , l. pérez-méndez 1 , r. sangüesa 2 , e. espinosa 2 , j. villar 1 : 1 research institute, hospital universitario n.s. de candelaria, s/c tenerife 38010, spain; 2 department of anesthesiology, hospital universitario n. s. de candelaria, s/c tenerife 38010, spain. e-mail: nmacame@ull.es (n. maca-meyer) sepsis describes a complex clinical syndrome resulting from a systemic inflammatory response to bacteria, and remains an important cause of mortality in the intensive care unit. cxcl2 chemokine (or mip-2) exhibits a pivotal role in the immune response, and several functional studies in animal models of sepsis have catalogued cxcl2 as a candidate gene for the development of sepsis. we have performed a case-control association study of cxcl2 gene variants and susceptibility to severe sepsis in 179 hospitalised patients and 364 healthy individuals. after the examination of linkage disequilibrium in the region, we analysed whether two promoter polymorphisms (snp rs3806792 and a newly described polymorphic short tandem repeat d4s3454) were associated with the syndrome. we found a significant association of common variants at d4s3454 with the development of severe sepsis (heterozygote carriers or 2.82; 95% ci 1.10-7.24, and homozygote carriers or 3.65; 95% ci 1.41-9.43; mantel-haenszel χ 2 test for linear trend p = 0.0006). the risks remained significant even after a genomic control adjustment, based on 20 additional genotyped polymorphisms not linked to the candidate gene. these preliminary results suggest that cxcl2 gene variants may contribute to the development of severe sepsis. kasper møller 1 , ana paula oliveira 1 , jens nielsen 2 , mark johnston 1 : 1 center for microbial biotechnology, biocentrum, technical university of denmark, denmark; 2 department of genetics, school of medicine, washington university, st. louis, usa glucose is the preferred carbon and energy source for most cells. in saccharomyces cerevisiae, a complex regulatory network ensures that s. cerevisiae ferments glucose to ethanol even in the presence of oxygen. to obtain a better understanding of this crabtree effect and the logic of the glucose signalling network in s. cerevisiae, we are analyzing glucose sensing and signalling in the related species saccharomyces kluyveri, which exhibits much less of a crabtree effect (it prefers not to ferment glucose when oxygen is available). we show that there are only two major glucose transporters in s. kluyveri, and that these are regulated in response to the availability of glucose via a glucose sensor and a signalling pathway similar to the glucose induction (rgt2/snf3-rgt1) pathway in s. cerevisiae. we have used dna-microarrays for s. kluyveri to find targets of the s. kluyveri glucose induction pathway, as well as to evaluate the global response to a change in environment from growth on ethanol to growth on glucose. this study identifies a number of differences in the regulation of glucose uptake and global responses to glucose between s. kluyveri and s. cerevisiae, which may contribute to their different glucose metabolism. detection and analysis of microrna using lna probes nana jacobsen, christian lomholt, peter mouritzen, peter stein nielsen, mikkel noerholm exiqon a/s, bygstubben 9, dk-2950 vedbaek, denmark. e-mail: mouritzen@exiqon.com (p. mouritzen) micrornas are a class of short endogenous rnas that act as post-transcriptional modulators of gene expression. growing evidence suggest that micrornas exhibit a wide variety of regulatory functions and exert significant effects on cell growth, development, and differentiation. recent studies have shown that human microrna genes are frequently located in cancer associated genomic regions and perturbed microrna expression patterns have been observed in many malignant tumors. we have exploited the significantly improved hybridization properties of lna oligonucleotides against rna targets to design lna-modified dna probes for detection of different micrornas in animal and plants by northern blot analysis, microarray hybridization and in situ hybridization. we will describe the results obtained from detection and analysis of different micrornas in c. elegans, zebrafish, mouse, and plants. in addition, we will describe a novel lna-based method for expression profiling of mature micrornas by quantitative rt-pcr. expression profile of the sty and paa genes in pseudomonas sp. y2 by means of dna microarrays david bartolomé-martín 1 , david juck 2 , m a teresa del peso-santos 1 , charles w. greer 2 , julián perera 1 : 1 departamento de bioquímica y biología molecular i, facultad de ciencias biológicas, universidad complutense de madrid, 28040 madrid, spain; 2 environmental microbiology group, biotechnology research institute, national research council canada, montréal, que., canada h4p 2r2. e-mail: perera@bio.ucm.es (j. perera) dna microarrays are a new and powerful tool to study gene expression in very diverse systems. environmental biotechnology and biodegradation are some of the fields of research where this technology may be very promising. pseudomonas sp. y2 is a bacterium able to grow in minimal medium plus either styrene (sty) or phenylacetic acid (paa) as the sole carbon and energy sources. this bacterium is the only organism where the genes that code for both the upper (sty genes) and the lower (paa genes) catabolic pathways for the styrene degradation have been described till now. it is unique in having two active copies of the genes encoding the lower pathway (paa1 and paa2 gene clusters). we have designed a dna microarray with the sty and paa genes in order to analyse their expression in the wild type pseudomonas sp. y2, in p. sp. y2 t2 (a paa2 deletion mutant) and in p. sp. y2 c1 (a crc gene mutant). this analysis has been performed on bacterial cultures grown in media with different carbon sources. interesting data on the expression profile of the sty and paa genes in pseudomonas sp. y2 have been obtained, and have raised new questions on styrene and paa degradation by this bacterium. dynamics in induced repression of phosphomannose isomerase pmi40 gene of saccharomyces cerevisiae anssi törmä 1,2 , juha-pekka pitkänen 1,2 , laura huopaniemi 2 , risto renkonen 2 : 1 medicel ltd., haartmaninkatu 8, 00290 helsinki, finland; 2 rational drug design program, department of bacteriology and immunology, haartman institute and biomedicum, university of helsinki, p.o. box 63, 00014 helsinki, finland. e-mail: juhapekka.pitkanen@medicel.com (j.-p. pitkänen) gdp-mannose is the precursor of cell wall biosynthesis in s. cerevisiae. to understand the system level role of gdp-mannose, we studied a conditional knock-out strain of the key enzyme in its synthesis; pmi40. the experimental procedure allowed us to study the order of mechanisms the cells launch in order to adjust to a sudden malfunction in the metabolic machinery. we collected 100 samples from continuous cultivations over 80 h and measured genome-wide gene expression levels, 10 enzyme activities, and concentrations of 30 intracellular metabolites. for sampling we have built a sample robot, which automatically takes and preserves the samples. in order to carry out this magnitude of experimentations and generated data, we have constructed a proprietary software platform to handle all the phases from project management in wet-lab to workflow and pathway management in in silico. after normalization and clustering, significantly changed genes and metabolites were searched for enrichment in biological processes and molecular complexes. further, gene expression levels, metabolite concentrations, and enzyme activities were searched against each other for causality over time. overall, we focused on thorough analysis of our own data and known database data in order to reward our efforts with knowledge. at the transcriptome level, repression of pmi40 led to two major types of activation profiles, one peaking at the time when pmi40p activity and gdp-mannose were depleted and the other later during recovery from the perturbation. the primary response was most enriched with genes known to play roles in mating and filamentous growth and associated with the transcription factors ste12p, tec1p, dig1p, and mcm1p, whereas the secondary response consisted of genes involved in carbon metabolism and associated with the general stress response regulators msn2p and msn4p. skn7p, a high-level transcription factor was associated with both the primary and the secondary response, consistent with its suggested role of coordinating environmental responses and developmental processes. transcriptome of pig ovarian cells: discriminant genes involved in follicular development bonnet a., le cao k.a., low-so g., san cristobal m., tosser-klopp g., hatey f. laboratoire de génétique cellulaire, centre inra de toulouse, castanet-tolosan 31326, france in order to identify genes and gene networks involved in pig ovarian follicular development, we built subtractive suppressive hybridization libraries (ssh) from granulosa cells of healthy follicles (small, medium or large). the rna isolated from these cells was used to hybridize cdna nylon micro-arrays. data analysis using a gaussian linear mixed model showed that 83% of the variability is due to the genes. two hundred fifty one regulated genes (from the 956 expressed) were identified and clustered into three groups according to the follicle size. moreover, we found previously identified genes such as aromatase, igfbp2 which supported the validity of our experimental model. ramdom forest analysis put forward the most discriminant 11 genes between the three follicle classes. this study put forward gene sets such as those involved in cell modeling, regulation of transcription, apoptosis during follicle growth. the next step will be to describe more precisely the spatio-temporal expression patterns at the mrna levels of the genes identified by these experiments. microalgae constitute a significant source of valuable natural products, e.g. sulfated polysaccharides, polyunsaturated fatty acids, and phycobiliproteins that find applications in wide range of industries, including food, pharmaceutical, agricultural and cosmetics. however, genomic and molecular genetic studies of microalgae lag far behind those of higher plants. in order to accelerate red microalgal genomic studies by taking advantage of current genomics and post-genomic technologies, we have generated expressed sequence tag (est) databases of two red microalgae porphyridium sp. and dixoniella grisea grown under various physiological conditions. to date we have sequenced 7210 and 6231 ests of porphyridium sp. and d. grisea, respectively. the sequence assembly resulted into, ca. 2000 non-redundant unigenes for each microalga, only 40% of which were identified by similarity to sequences in the public databases. porphyridium sp. and d. grisea unigenes were compared with the whole-genome predicted proteomes of three microalgae and those of representative eukaryotic and prokaryotic organisms. both microalgae have highest similarity to the red microalga cyanidioschyzon merolae. the order of sequence similarity to other organisms examined was arabidopsis thaliana, oryza sativa, chlamydomonas reinhardtii, thalassiosira pseudonana (diatom), saccharomyces cerevisiae, caenorhabditis elegans, archaea and cyanobacteria. although red microalgae are considered as phylogenetic bridge between prokaryotes and eukaryotes, our data show that the red microalgae have strong similarity to eukaryotes and only distant similarity to prokaryotes. gene expression profiles were studied by analyzing cdna and subtraction libraries constructed from algae grown under various physiological conditions. we observed that top three most abundant ests in the stationary phase of porphyridium sp. were adp ribosylation factor like-1, flavohemoglobin and adp ribosylation factor-1. in addition, we have identified several genes which were specific to nitrate-and sulfate starvation. the sarco(endo)plasmic reticulum ca 2+ -atpase (serca, "the calcium pump"), is responsible for pumping the ca 2+ released into the cytoplasm during muscle contraction back into the sarcoplasmic reticulum store while proton are pumped the opposite way as counter-cations. these transport processes go against the concentration gradients and are therefore energy consuming. the energy is derived from atp hydrolysis via formation and break-down of a phospho-enzyme intermediate. over the last year a number of new crystal structures have been published which have added to our understanding of how this task is accomplished, which provides an impressive insight to the mechanism of a molecular pump. rhomboids are a family of intramembrane serine proteases that are widely conserved throughout evolution. among diverse functions discovered so far, rhomboids participate in intercellular signalling, parasite invasion, membrane dynamics and bacterial quorum sensing, making them potentially valuable therapeutic targets. the identification of physiological substrates and of selective inhibitors will be key towards their evaluation as drug targets. we have developed an in vitro cleavage assay to monitor rhomboid activity in the detergent solubilised state, enabling the first isolation of a highly pure rhomboid with catalytic activity. analysis of purified mutant proteins suggests that rhomboids use a serine protease catalytic dyad instead of the previously proposed triad, and gives insights into subsidiary functions like ligand binding and water supply. this work was supported principally by embo and the mrc. structure and target-specificity of thioredoxin h kenji maeda 1 , anette henriksen 2 , per hägglund 1 , christine finnie 1 , birte svensson 1 : 1 biochemistry and nutrition group, biocentrum-dtu, technical university of denmark, dk-2800 kgs. lyngby, denmark; 2 biostructure group, carlsberg laboratory, dk-2500 valby, denmark. e-mail: kenji@biocentrum.dtu.dk (k. maeda) thioredoxins are ubiquitous small proteins with protein disulphide reductase activity. thioredoxins can alter the structures and the activities of various target proteins by reducing their disulphide bonds. seeds of several plants are abundant in cytosolic thioredoxins referred as h-type. in barley, two thioredoxin h isoforms, hvtrxh1 and hvtrxh2 that share 51% sequence identity but differ in temporal and spatial distributions were previously identified and characterised. in the present study, the relationship between structures and targetspecificities of h-type thioredoxins are analysed. the 3d-structures of hvtrxh1 and hvtrxh2 are determined by x-ray crystallography as the first crystal structures of thioredoxin h. comparison of the structures shows that the majority of solvent exposed residues near the active sites are conserved between the two isoforms. this is in agreement with previously observed similarity in target-specificity of the two isoforms. thioredoxins from organisms distantly related to barley, such as e. coli, have highly similar folds but different surface charge distributions compared to barley thioredoxins. a comparison of the target-specificities of hvtrxh1, hvtrxh2, e. coli thioredoxin and several thioredoxin mutants will be attempted to reveal the structural features that influence specificity of barley thioredoxin h isoforms. enbrel is a dimeric fusion protein consisting of the extracellular ligand binding portion of the human 75 kda (p75) tumor necrosis factor receptor (tnfr) linked to the fc portion of human igg1. the cho-expressed molecule contains both n-and o-linked oligosaccharides with a total carbohydrate content of 20% by mass. the o-linked oligosaccharides were released by hydrazinolysis and their structure determined by exoglycosidase sequencing and maldi-tof mass spectrometry. to locate precisely the o-linked sites, the glycosylation heterogeneity of tnfr:fc was simplified by treatment with n-acetyl neuraminidase and n-glycanase. the remaining molecule, which only carries core o-linked glycan structures, was cleaved by trypsin and analyzed by lc-ms. precise localization of o-glycosylation sites was determined based on the concept of a specific modification of the o-glycosylated serine into 2aminopropenoic acid and o-glycosylated threonine into 2-amino-2butenoic acid. the deficit in mass resulting from this transformation was the marker used to localize the modified residues on the peptides by tandem mass spectrometry sequencing (ms-ms). ms-ms spectrum of enbrel glycopeptides were interpreted based on the presence of 2-aminopropenoic acid and 2-amino-2-butenoic acid, resulting in a complete map of o-linked glycans precisely located at 10 different sites. anu mursula, beatrix fahnert, sari krapu, eija-riitta hämäläinen, ritva isomäki, peter neubauer bioprocess engineering laboratory and biocenter oulu, university of oulu, oulu, finland. e-mail: anu.mursula@oulu.fi (a. mursula) wnt proteins form a highly conserved family of secreted glycoproteins important in cell-cell signaling events during embryogenesis and adult tissue maintenance. impairments within this complex signaling pathway can lead for example to developmental defects in embryos, degenerative diseases and cancer. respectively, wnt proteins can be used as tools in basic research concerning wnt function, developmental biology, screening for interacting compounds, and for medical applications (e.g. therapeutics, stem cells). hence, recombinant wnts provide a valuable basis for these purposes. however, production of recombinant wnt proteins is challenging, because they contain multiple disulfide bonds making the folding very difficult. a process for production of murine wnt-1 in e. coli has been developed in our laboratory. the knowledge obtained from this research has also been applied to the expression of other wnts, namely wnt-4 and wnt-6, and can be used to approach other cysteine-rich proteins as well. since the expression level of wnt proteins is rather low so far, tools for monitoring and optimizing the production process have been established. by means of this sandwich hybridization method the level of target (wnt) mrna can be measured. the technique has already been applied to analyzing wnt-1 mrna levels. probes also for wnt-4 and wnt-6 have been generated. thus, transcription of wnt genes in all kind of cells (e.g. tissue, recombinant hosts) in general as well as kinetics of transcription can be studied using these tools. in growth factor signaling, stimulation of cell-surface receptors first triggers activation of the receptor itself and then of a large number of intracellular effector molecules. the stimulus is integrated with a host of other cellular processes, leading to cytoskeletal changes, activating transcriptional programs in the nucleus and ultimately resulting in cell proliferation, differentiation or motility. classical signaling pathways and networks depict potential protein-protein interactions only in a static form. in the cell, these interactions are dynamic and occur in an ordered fashion. here, we apply a mass spectrometric method that converts temporal changes to differences in peptide isotopic abundance in order to study the global dynamics of signaling events. briefly, three cell populations are metabolically labeled with either normal arginine or a 13 c substituted form, or a 13 c 15 n variant (stable isotope labeling by amino acids in cell culture, silac). each population was then stimulated with egf for a different time period and tyrosine phosphorylated proteins were affinity purified with anti-phosphotyrosine antibodies. the proteins from the precipitated complexes were quantitatively analyzed and identified using lc-ms/ms. arginine containing peptides occurred in three forms, directly indicating protein activation at the corre-sponding time point. combination of two experiments sharing one common time point of activation then generated five-point dynamic profiles. from the 202 proteins quantified, we identified 81 signaling proteins, including virtually all known egfr substrates and 31 novel effectors, and the time course of their activation upon egf stimulation. discriminating proteins involved in the signaling network from unspecific binders was straightforward as these presented an activation profile. we have now further extended this study by directly measuring in vivo phosphorylation sites in response to growth factor stimulation and monitoring the time evolution of the phosphorylation events. finally, we determined and quantitatively compared the global egf and pdgf tyrosine phosphoproteomes in human mesenchymal stem cells and revealed a control point in their differentiation into bone-forming cells. such global activation profiles provide a novel perspective in cell signaling and will be crucial to model the highly dynamic signaling networks in a systems biology approach. klaus schneider 1 , dave g smith 2 , steven skaper 2 , alastair d. reith 1 : 1 discovery research, glaxosmithkline, coldharbour road, harlow, essex, uk; 2 neurology & gastrointestinal cedd, glaxo-smithkline, coldharbour road, harlow, essex, uk over the last 15 years, progress in signal transduction research has revealed an astonishing degree of complexity in cell signalling which is manifested in positive and negative regulations and feedback loops within signalling pathways and by cross-talks between pathways, all of which are highly cell-type dependent. it has become evident that protein phosphorylation by protein kinases plays a major role in this complex regulation of cell signalling (hunter, 2000) . due to the importance of signal transduction in disease processes, many protein kinases may constitute key targets for disease intervention. yet, the lack of a full understanding of the regulation, the activation and, importantly, of downstream substrates of particular protein kinases requires often more detailed studies before initiation of resource-intensive efforts to find disease-modifying molecules. technologies for the study of protein kinase signalling include 32 p labelling, mutational and knock-out studies and more recently rna interference. these tools are complemented by approaches that are based on proteomic technologies developed over the course of the last 10 years. in this presentation, the scope of proteomics technologies to contribute to an understanding of kinase signalling will be discussed. an overview of available technologies will be given and results will be presented from a proteomic study of glycogen-synthase kinase 3 (gsk3) signalling (coghlan et al., 2000) . novel findings will be presented on a study of gsk3 inhibition in a primary neuronal cell line by differential 2d gel electrophoresis, which resulted in the identification of more than 40 proteins that were significantly regulated. proteome analysis is typically done by nanospray lc/ms in order to achieve higher sensitivity and thus a greater number of protein identifications. however, nano-scale lc systems can be more challenging to use and maintain. to obtain the best chromatographic performance, connections must be made carefully to minimize band broadening. improved chromatographic performance can enhance the mass spectrometric results by tandem ms as a greater number of peptides can be detected. a microfluidic chip-based system has been developed (yin et al., 2004) that minimizes the number of connections and the delay volumes. this work evaluates the performance of this device against the traditional nanospray approach. a yeast extract sample was separated by sds-page and bands were excised from the gel for further analysis. after in-gel digestion, the sample was analyzed by both traditional nanospray and the microfluidicbased chip device. after protein database searching, the identified proteins and the protein sequence coverage's were compared for the two approaches. the microfluidic device was demonstrated to be equivalent or better compared to the traditional approach. yin, h., killeen, k., brennen, r., et al., 2004. anal. chem. 77, 527-533 . plant cytochromes p450 (p450s) play key roles in the biosynthesis of most bioactive compounds with agronomic and therapeutic applications. a collection of about 120 plant p450s was expressed in yeast. the cdnas were isolated from the model plant with a sequenced genome arabidopsis thaliana, some others from wheat, helianthus tuberosus or vicia sativa. they were expressed under the control of a galactose-inducible promoter in an engineered strain of saccharomyces cerevisiae in which the gene of the native p450 reductase was replaced with the gene of a p450 reductase from a. thaliana under the control of the same galactose-inducible promoter, in order to provide an optimal context for plant p450 expression and activity (pompon et al., 1996) . an original procedure was designed for the high-throughput functional screening of this enzyme collection. it is based on the detection of oxygen consumed during the catalytic reaction by a fluorochrome embedded in the bottom of the microwell plates. this method was validated using several recombinant p450s of known activity. it also allows for a very efficient screening for enzyme inhibitors. the advantages and limits of the method will be discussed. this work was carried out with the support of génoplante programme (no 2001004) . reference pompon, d., louerat, b., bronine, a., urban, p., 1996. methods enzymol 272, 51-64. 3 folding of a bacterial membrane protein studied by protein engineering daniel e.otzen, pankaj sehgal, peter a. christensen department of life sciences, aalborg university, sohngaardsholmsvej 49, dk -9000 aalborg. e-mail: dao@bio.aau.dk (d.e. otzen) we have carried out a kinetic analysis of the folding of the 4-helix transmembrane protein dsbb in a mixed micelle system consisting of varying molar ratios of sodium dodecyl sulfate and dodecyl maltoside. this analysis incorporates both folding and unfolding rates, making it possible to determine both the stability of the native state and the process by which the protein folds. the analysis also takes into account the composition of the mixed micelles, which is different from the bulk detergent composition. refolding and unfolding are consistent with a three-state folding scheme involving the sdsdenatured state, the native state and an unfolding intermediate that accumulates only under unfolding conditions at high mole fractions of sds. the temperature-dependence of the folding reaction displays an unusual decrease in heat capacity accompanying unfolding, which probably reflects the amphiphilic environment of the membrane protein. destabilization of dsbb by different short-chain alcohols correlates very well with the alcohols' respective hydrophobicities. data from a series of ala-scanning mutants tentatively identify a nucleus for folding, which is relatively diffuse and involves all four helices. we are currently complementing this work with studies of the association of peptides corresponding to individual transmembrane segments of dsbb. the reca protein of e. coli plays a crucial role in homologous recombination and dna repair. the recombination process takes place in a filamentous complex, in which the protein monomers are arranged in a helical manner around a single-stranded dna (ss-dna). in the presence of atp the filament can accommodate a second, double-stranded dna (ds-dna) and the strand exchange reaction can occur. the three-dimensional structure of reca itself and its complex with adp have been determined by x-ray crystallography. the active nucleoprotein filament, however, has only been studied at low resolution. both electron microscopy (em) and small-angle neutron scattering (sans) indicate significant differences between the structures of the active nucleoprotein filament and the compressed, inactive filament of only reca. we have presented a structural model of the reca protein in its active filament with ss-dna, using data obtained by linear dichroism (ld) polarized-light spectroscopy, based on a technique, we call "site-specific linear dichroism", which allows the orientation of a set of amino acids to be determined from ld data by systematic modification of the protein. here, we show that ld data of the nucleoprotein filament with ds-dna is over all similar to the data of the complex with ss-dna, indicating that the orientation as well as internal structure of reca in the active filament is not significantly altered when the bound dna is changed from single-stranded to double-stranded. this result supports the idea that the strand exchange reaction occurs without large conformational change of the reca protein. the choline-binding modules: a powerful biotechnological tool jesús m. sanz instituto de biología molecular y celular, universidad miguel hernández, elche, spain choline-binding modules (chbms) are present in some virulence factors of streptococcus pneumoniae (pneumococcus). the most extensively studied chbm is c-lyta, the carboxy-terminal domain of the pneumococcal cell-wall amidase lyta. the three-dimensional structure of choline-ligated c-lyta is built up from six loop-hairpin structures ("choline binding repeats", chbrs) forming a left-handed ␤-solenoid with four choline binding sites. although the structure of the ligand-free form is not yet known, our folding studies suggest that it is more loosely packed, with a partially unfolded amino-terminal region and a stable carboxy-terminal moiety that is extremely resistant to chemical denaturation (maestro and sanz, 2005) . the affinity of c-lyta for choline and other structural analogues allows its use as an efficient affinity tag for overexpression, immobilization and single-step purification of proteins of biomedical interest (c-lytag fusion protein purification system). this system presents many advantages when compared to current commercial methods, namely simplicity, compatibility with buffers and robustness. the availability of multiple supports that specifically bind chbms (such as multiwell plates) has recently allowed the development of a new procedure for the immobilization of c-lyta-containing hybrid proteins that may be used in proteomics, diagnostics and peptide display. in this communication, we present our last results about the stability, folding and engineering of c-lyta, together with a compendium of the current biotechnological potential of this protein, and highlight the productive link between basic molecular studies and their application. many of the modern approaches for studying disease compare steady state functions, such as repair, growth, and regulated gene expression within the various biological compartments organised by specialized function, be it mitochondria or blood vessels. the assignment of protein identities, which are linked to key biological mechanisms, which are associated with disease processes and disease progressions are an important area of this work (marko-varga and fehniger, 2004) . today, the technology available for studying proteome expression and resolving exact protein and peptide identities in complex mixtures of biological samples allows global protein expression within cells, fluids, and tissue to be approached with confidence. this confidence is due in part to reproducible repetitive sampling and analysis technologies including robotics data acquisition and high level mass spectrometry including both laser-desorbtion and electro spray ionisation. the precision in defining differences between normal and diseased steady states is aided by the creation of compiled reference and master data sets and by new methods for multiplexing the analysis of samples in groups. the establishment of key representative reference proteome systems representing the dynamic changes in protein expression during disease will be vital to the interpretation of changes observed in specific samplings of disease states and specific cells obtained from these samples. the creation of reference databases of proteins linked to disease pathways will play an important role in furthering our understanding of the "proteome of disease". examples will be given where protein expression patterns have been generated from compartments within tissue sections. marko-varga, g., fehniger, t.e., 2004. j. proteome res. 3, 167178. 2 adaptation of the saccharomyces cerevisiae proteome to nutrient limitations studied by metabolic stable isotope labeling and mass spectrometry albert j.r. heck netherlands proteomics centre and utrecht university, the netherlands. e-mail: heck@npc.genomics.nl. url: www.netherlandsproteomicscentre.nl one of the major aims of proteomics is to provide quantitative data on differential protein expression levels. recently, mass spectrometry-based methods have been introduced that can provide quantitative data on differential protein expression, mostly using stable isotope labeling (goshe and smith, 2003) . we opted for metabolic labeling as this provides efficient means to quantify differential protein expression, and has the advantage that all proteins are labeled universally (romijn et al., 2003) . in their natural habitat microorganisms encounter non-optimal growth conditions and often growth is limited by one nutrient. microorganisms need to respond rapidly to changes in the environment in order to survive. in the present study, we investigate the proteome response of chemostat cultivated wildtype saccharomyces cerevisiae to two different nutrient limitations, namely carbon and nitrogen limitation. yeast was metabolically labeled in well-controlled chemostat cultures. 14 n and 15 n labeled proteins were separated using 1d gel electrophoresis followed by rp-lc-esi-ms on a lc-q. relative quantification was performed by using relex software (maccoss et al., 2003) . we quantified 759 proteins, using on average 8 peptide peak pairs per protein. this analysis revealed that 419 proteins showed a significant increase/decrease in expression level. the functional annotation of these proteins revealed that the yeast cells change expression levels of enzymes involved in metabolism of the growth-limiting compound. the protein expression ratios were compared with corresponding transcript levels. moreover, we compared the accuracy of quantifica-profiles mainly reflected differences in cellular origins in addition to different functional roles. mass spectrometric analysis identified 82 proteins pertaining to several functional classes, i.e. acute phase proteins, antioxidant proteins and proteins involved in protein synthesis/maturation/degradation, cytoskeletal (re)organization and in lipid metabolism. several proteins not previously implicated in nerve regeneration were identified, e.g. translationally-controlled tumor protein, annexin a9/31, vitamin d-binding protein, ␣-crystallin b, ␣-synuclein, dimethylargininases and reticulocalbin. real-time pcr analysis of selected genes showed which were expressed in the nerve versus the dorsal root ganglion neurons. in conclusion, this study highlights the complexity and temporal aspect of the molecular process underlying nerve regeneration and points to the importance of glial and inflammatory determinants. yeasts plasma membrane macromolecular components involved in stress resistance paola branduardi, paola paganoni, danilo porro dipartimento di biotecnologie e bioscienze, università degli studi di milano-bicocca, piazza della scienza, 2-20126 milano, italy. e-mail: paola.branduardi@unimib.it (p. branduardi) the plasma membrane is a universal structure of living cells constituting an essential barrier dividing and defining the intracellular from the extracellular environment. it is consequently easy to deduce the crucial role played by said structure for any cell of any living organism, and especially for unicellular organisms, since all the information deriving from the external environment as well as many of the consequent cellular responses have to pass through this barrier. unicellular organisms, thanks to easy manipulation and cultivation techniques, can represent a very useful model for studying the plasma membrane function and response. in addition microorganisms, and among them yeasts, can be considered advantageous cell factories for recombinant productions. in this contest, the implementation of any process of production has to take into account, among others, the response and the tolerance of the host to the external environment. from these considerations derives the interest of our group to analyse the main macromolecular components of yeasts plasma membranes (proteins, lipoproteins and lipids), isolated from cells grown under different stress conditions, with particular attention to acidic environments. here, we present our recent data about separation and identification (by sequencing analyses) of lipoproteins isolated from the conventional yeast saccharomyces cerevisiae as well as from the non-conventional and acid tolerant yeast zygosaccharomyces bailii cell cultures grown in different conditions. in parallel, the protein fraction is under evaluation through a differential 2d proteomic approach and consequent analyses. effect of fungal polysaccharides on the expression of pancreatic proteins in streptozotocin-induced diabetic rats sang woo kim 1 , hye jin hwang 1 , kwang bon koo 2 , jang won choi 2 , jong won yun 1* : 1 department of biotechnology; 2 department of bioindustry, daegu university, kyungsan, in an attempt to search novel biomarkers for monitoring diabetes prognosis, we examined the influence of the hypoglycemic fungal extracellular polysaccharides (eps) on the differential expression of pancreatic proteins in streptozotocin-induced diabetic rats. the results of diabetic study revealed that orally administrated eps exhibited excellent hypoglycemic effect, lowering the average plasma glucose level of the diabetic rats to 52.3%. pancreatic proteome were analyzed by 2-de system, which separated more than 2000 individual spots. the 2-de analysis demonstrated that thirty-four proteins from a total of about 500 matched spots were differentially expressed, of which 26 spots were identified as the proteins whose expression has previously been associated with diabetes. twenty-two overexpressed and twelve underexpressed proteins were significant (p < 0.05) between the healthy and diabetic rats, and the altered proteins were restored (p < 0.05) upon eps treatment. it was first found that carbonyl reductase (18.6-fold, p < 0.001) and mawdbp (31.4-fold, p < 0.01) were surprisingly upregulated upon diabetes induction, and then those two protein concentrations were completely restored by eps treatment. moreover, we obtained eight unidentified proteins that have not been reported to be related with diabetes mellitus. these results evidenced the effect of fungal eps on searching potential markers for diagnosis and therapeutic manipulation of diabetes mellitus. although molecular basis of protein modulation after eps administration in diabetic rats was not verified in this study, the results of the proteomic analysis provide impetus for further studies of mining the biomarkers for diabetic therapy. the model established in our experiment is expected to mimic human diabetic status, which will help us to interpret the roles of biomarkers in diabetic state. the use of polyol-responsive monoclonal antibodies in immunoaffinity chromatography and as a probe for unfolding of wild-type and altered (t103i) amidase from pseudomonas aeruginosa s. martins 1 , j. andrade 1 , a. karmali 1 , a.i. custódio 2 , m.l. since immunoaffinity chromatography is a powerful protein purification technique of interest in proteomics, monoclonal antibodies (mabs) against mutant (t103i) amidase from p. aeruginosa were raised by hybridoma technology. in order to identify mabs that bind t103i amidase tightly but release under gentle conditions, hybridoma clones secreting polyol-responsive mabs (pr-mabs) were previously screened. nearly 10% of elisa assay-positive hybridoma produced clones secreting pr-mabs with potential application as ligands for immunoaffinity chromatography. to select the optimal conditions for amidase elution, an elisa-elution assay was carried out, with two of these clones (f6g7; e2a6). the dissociation of ag-ab complex required 10% of propylene glycol and either 0.25 m (nh 4 ) 2 so 4 or 0.25 m nacl. the binding of purified mab of igm class (e2a6) to wild-type and mutant amidases was investigate by direct elisa, which revealed that it recognised specifically a common epitope on both amidases. conformational changes on antigen molecule were studied. mab e2a6 showed a higher affinity for heat denatured forms than for native forms as revealed by affinity constants suggesting that the mab recognizes a cryptic epitope. the effect of mab e2a6 on amidase activity was also investigated. the binding of mab to wild-type and mutant amidases exhibited an inhibition and activation of 60% as a function of time, respectively. this pr-mab is useful as a probe to detect conformational changes in native and denatured amidases as well as a ligand in immunoaffinity chromatography, which is of great interest in protein purification and proteomics. fragility and solubility of non-classical inclusion bodieš s. peternel 1 , a. ristič 1,2 , v. gaberc-porekar 1 , v. menart 1,2 : 1 national institute of chemistry, ljubljana, si-1000; 2 lek pharmaceuticals d.d., ljubljana, si-1000. e-mail: spela.peternel@ki.si (š. peternel) human granulocyte colony stimulating factor (g-csf) is a pharmaceutically important cytokine. when overexpressed in escherichia coli, it is usually accumulated in the form of inclusion bodies (ibs) . when produced at 42 • c classical insoluble ibs are formed while at 25 • c non-classical ibs containing a high amount of correctly folded g-csf are formed. as higher fragility and solubility of non-classical ibs were noticed, we decided to check whether bacterial cell disruption method has any influence on their mechanical stability and solubility. enzymatic lysis, sonication and homogenization, methods often used for disruption of bacterial cells during the isolation of ibs were compared. lysozyme treatment of bacterial cells appears to be mild enough disruption method not influencing the integrity of ibs. homogenization of bacterial cells at high pressure (100.000-120.000 kpa) shows no impact on classical ibs while some loss of target protein from the non-classical ibs is observed. sonication seems to be most harmful as even at rather low sonication altitudes, noticeable disassembling and solubilization of non-classical ibs occurs while no effect on classical ibs is perceived. our studies show that non-classical ibs are much more fragile and soluble than classical ones. therefore, one should extremely carefully choose the method for cell disruption to avoid undesirable loss of the target protein. the danish tick ixodes ricinus parasitize three different hosts both mammals and birds during the 3-year life cycle. the aim of this study was to identify the last blood host being the host, which the nymph had parasitized before molting to the adult instar. the reason for the study was to reveal the origin of the host contributing the most to the life cycle of the tick and thereby the maintenance of tick-borne diseases in denmark. the most common tick-borne diseases are lyme borreliosis and tick-borne encephalitis (tbe) causing illness in both animals and humans. we analyzed adult ticks, which were collected from known hosts. the analysis was performed at different heat stable proteins, which could be detected during the off host period by elisa. we found that heat stable proteins could be used as identification markers for host recognition. mushroom polysaccharides alter the expression of diabetesassociated proteins in the liver of streptozotocin-induced diabetic rats hye-jin hwang 1 , sang-woo kim 1 , kwang-bon koo 2 , jang-won choi 2 , jong-won yun 1* : 1 department of biotechnology, daegu university, kyungsan, kyungbuk 712-714, korea; 2 department of bioindustry, daegu university, kyungsan, kyungbuk 712-714, korea. e-mail: jwyun@daegu.ac.kr (j.-w. yun) in the present study, we investigated the influence of the hypoglycemic fungal extracellular polysaccharides (eps) on the differential expression of liver proteins in streptozotocin (stz)-induced diabetic rats. the results of diabetic study revealed that orally administrated eps exhibited an excellent hypoglycemic effect, lowering the average plasma glucose level in eps-fed rats to 52.3%. in the next step, we analyzed the differential expression patterns of rat liver proteins from each group, to discover potent candidates for diabetesassociated proteins. a total of 69 proteins of the 2-de gel were expressed differentially between diabetic and healthy rats. among them, 34 proteins were upregulated and 35 proteins were downregulated upon diabetes induction. many of these changes were in accordance with observations in previously published studies. surprisingly, the altered levels of most proteins in diabetic group were fully or partially restored to those of non-diabetic control group by eps treatment. moreover, we obtained 13 unidentified proteins that have not been reported to be related with diabetes mellitus. although molecular basis of protein modulation after eps administration in diabetic rats was not verified in this study, the results of the proteomic analysis provide impetus for further studies of mining the biomarkers for diabetic therapy. potential is still limited by the differences observed in the structure of plant and mammalian n-glycans. indeed, theses differences and particularly the presence of ␤1,2-xylose and ␣1,3-fucose glycoepitopes are responsible for the immunogenicity of plant n-glycans. in order to reduce the structural differences between plant and mammalian n-glycans, current strategies are to knock out plant-specific glycosyltransferases or to humanize plant n-glycans by expression of mammalian glycosyltransferases in plants. in the present study, we have expressed a human ␤1,4-galactosyltransferase in alfalfa. in order to further increase the efficiency of the human ␤1,4-galactosyltransferase in the plant golgi apparatus, we have exchanged the endogenous targeting signal of this human glycosyltransferase for the ones from plant glycosyltransferases recently characterized in our laboratory. we will illustrate this approach of targeted expression with the results obtained by fusion of the catalytic domain of human ␤1,4-galactosyltransferase with the n-terminal sequence of a plant glycosyltransferase that targets the fusion to the very early compartments of the golgi apparatus. the efficiency of natural versus targeted expression of human ␤1,4galactosyltransferase in alfalfa will be compared in term of n-glycan humanization. altogether, our results clearly illustrate that we are now on the way to get perfect copy of mammalian glycoproteins in alfalfa plants. construction of recb-recd gene fusion and analysis of fusion enzyme activities oytun portakal 1 , gerald r. smith 2 , pakize dogan 1 : 1 department of biochemistry, hacettepe university medical school, 06100, ankara, turkey; 2 divisions of basic sciences, fred hutchinson cancer research center, seattle, wa 98109-1024, usa. e-mail: oytun@hacettepe.edu.tr (o. portakal) protein folding is a fundamental process to gain protein function. in an oligomeric protein, the interaction between polypeptides affects the folding process and assembly to the holoenzyme. recbcd is a heterotrimeric and multifunctional enzyme that plays an essential role for major pathway of homologous recombination in e. coli. it is composed of recb, recc and recd gene products. recd is the fast motor unit of the recbcd enzyme. recd also plays a role for high affinity dsdna binding, nuclease activity and chidependent regulation. this study was designed to test the hypothesis that recd polypeptide regulates the essential reca loading activity. the approaching of the study was to fuse recd gene to subsequent recb gene and to observe the changes in enzyme activity and structure. for these purpose two genetic fusion mutations, two-nucleotide deletion and three-codon substitution were created at the overlap sites (ta) of recb and recd genes. fusion mutations were constructed by phage-mediated recombination system, which is called recombineering. this technology requires red function but not host reca protein function. here, we showed the recbd fusion polypeptides in crude extracts. genetic characterization tests were revealed that both fusion enzymes are recombination proficient and have wild-type phenotype. biochemical assays demonstrated that recbdc fusion heterotrimers have dsdna exonuclease, unwinding and chi cutting activities. they were also resistant to dna damaging agents. western blot analysis also detected a wild type length recd polypeptide together with recbd fusion polypeptides and a decreased heterotrimer compared to wild type. our findings suggest that recb-recd genetic fusions may affect recd assembling to the heterotrimer, but not affect it's native folding. sandwich immunoassay-a simple strategy for enhancement of the sensitivity and the specificity in prostate specific antigen detection based on surface plasmon resonance cuong cao, sang jun sim department of chemical engineering sungkyunkwan university, 300 chunchun-dong, jangan-gu suwon, prostate cancer is a deadly disease in men. prostate specific antigen (psa) has been proved to be the most reliable and specific biomarker in preoperative diagnosis, monitoring and followup of patients with prostate cancer. in this study, a biochip based on surface plasmon resonance was fabricated to detect psa at concentrations ranging from 1 to 1000 ng/ml. to reduce nonspecific binding, the chemical surface of sensor was constructed by using various ethyleneglycol mixtures of different molar ratios of hs(ch 2 ) 11 (och 2 ch 2 ) 6 cooh and hs(ch 2 ) 11 (och 2 ch 2 ) 3 oh. we also biotinylated the sams surface to enhance the orientation of protein immobilization. by using this surface, spr-based psa detection gave a positive ru value at the fist response in the whole range of psa concentrations. however, this ru value could get better and more reliable by simply applying a secondary interactant, the psa polyclonal antibody, in sandwich immunoassay. the results shown this approach could satisfy our purpose without modify the secondary interactant, which has usually been done by the other report. expression of epitopic domains of human coagulation factor viii in escherichia coli amir amiri yekta 1,2 , naser amirizadeh 3 , alireza zomorodipour 1* , fariba ataei 1 : 1 department of mol genet. national institute for genet eng & biotechnol tehran-iran p.o. box: 14155-634, tehran, iran; 2 islamic azad university of jahrom, jahrom, iran; 3 department of hematol, faculty of med, tarbiat modarres university, tehran, iran. e-mails: amir amiriyekta@yahoo.com (a.a. yekta), * zomorodi@nrcgeb.ac.ir (a. zomorodipour) human factor viii (hfviii) plays major role in the intrinsic pathway of blood coagulation and is used to treat individuals with hemophilia a for bleeding episodes. many researches have been focused on the molecular aspects of this protein. in this regard, epitopes of hfviii as well as their corresponding antibodies have many important applications. bacterially produced fviii-epitopes are capable to neutralize the alloantibodies that inhibit hfviii activity. the purpose of present study was to over-express two epitope-containing fragments of fviii in e. coli under t7 promoter (novagen). two dna fragments from light-and heavy-chains of hfviii (942bp-c1c2 and 1644bp-a1a2, respectively) were subcloned in the expression vector. the use of his 6 -tagged tail was also considered for detection and purification purposes. in each of the examined clones, a protein of expected size was detectable. in the c1c2-expressing clone the specificity of the over-expressed protein was confirmed by its reaction with the rabbit serum directed against native hfviii as well as anti-his-tag antibody. in the heavy chain-related-expressing clone, the expression level was low, but it was detectable by immunoblotting experiments. manipulations of the growth as well as induction may be required. the over-expression of the other epitopes reported in the heavy chain may be achievable by the expression of (a) sub-fragment(s) of this region. the over-expressed his-tagged c1c2-related protein was appeared to be trapped in the cell as non-soluble inclusion bodies. therefore, after homogenizing of the induced recombinant cells, the nonsoluble fraction was dissolved in a solution of denaturant (guanidine hydrochloride) and subjected for the purification, using a ni-nta resin (qiagen) followed by protein measurement. accordingly, an expression level of 5 mg/l (of culture) of the purified c1c2-related peptide was obtained. the recombinant hfviii c1c2-derived peptide has provided useful mean for further experimental and medical applications. isotachophoresis has almost exclusively been applied for contracting and stacking samples ions before zone electrophoretic separation of proteins. this study attempts to apply microfluidic isotachophoresis (itp) as a high resolution analytical method for proteins. beta-lactoglobulin and other milk proteins with slightly different pi were labelled with fluorescent red 646 and analysed by the micralyne tk system using microfluidic glass chips, either with simple cross (sc) or double cross (tt) injection or designed 2d-itp-cze chips with double tt injection and sc for transfer to the second dimension cze channel, efficiently non-covalently coated with 0.6% w/v epoxy-polydimethylacrylamide to lower electroendoosmosis. capillary zone electrophoresis (cze) in borate or phosphate buffer was reproducibly perform for more than 75 consecutive runs using upchurch tm reservoirs glued to the wells to enable larger buffer volumes and greater run-to-run stability. finally, isotachophoretic anionic separation of the proteins were done using phosphate (ph 8.1) or chloride (ph 8.5) as leading ion and -amino-caproic acid (ph 8.9) as terminating ion. the effect of narrow cut ampholytes as spacers needs further investigations. the perspective aim is to combine the migrating itp separated zones with second dimension capillary zone electrophoresis as a new microfluidic proteomic 2danalysis. development of strategies for heterologous expression of glucose dehydrogenase from the halophilic archaeon halobacterium sp. nrc-1 juan carlos cruz-jiménez 1 , lorenzo saliceti-piazza 1 , rafael montalvo 2 : 1 chemical engineering, university of puerto rico-mayagüez campus, mayagüez 00680, puerto rico; 2 biology, university of puerto rico-mayagüez campus, mayagüez 00680, puerto rico. e-mail: juancruzj@hotmail.com (j.c. cruz-jiménez) halophilic archaea are excellent model organisms and valuable for biotechnology applications; they are easy to culture in the lab, genetically tractable, and exhibit a variety of interesting and useful characteristics. most halophilic archaea require 1.5 m nacl to sustain growth and structural integrity. among the 2.630 genes in halobacterium, we are studying the gene encoding a glucose dehydrogenase, gene id is 446, located between the 345205 and 346272 bases (halobacterium sp. nrc-1 genome project). this extremozyme is bioengineerable, and its use as a model for studying biocatalysis in aqueous/organic and nonaqueous media has not been explored to date. the utilization of enzymes in organic solvents has several potential advantages over aqueous systems. a major benefit is the increased solubility of many substrates, resulting in higher concentrations of reactant and products, hence reducing and purification costs and simplifying recovery protocols. cells were grown aerobically during seven days at 37 • c in a complex medium, harvested by centrifugation and their genomic dna extracted. for cloning of the gene, primers were designed based on the sequence recently published by the halobacterium sp. nrc-1 genome project. the forward (5 -ccgcatgcgcc cacagtccc-3 ) and reverse (5 -ccggcctctagaacggcctgg-3 ) primers were designed to incorporate restriction sites for sph i and xba i, respectively (in bold). we are pcr amplifying the genomic dna and developing methods for the heterologous expression using the mesophilic escherichia coli, as well as purifying the enzyme. the purification procedure will be carried out using high resolution methods based on the protein's halophilicity. bioinformatics methods will be used to facilitate conforming of protein function and for comparison with a native enzyme. quantitative measurements by mass spectrometry of hundreds of proteins simultaneously using the new proteinchip systemseries 4000 p. iversen, e. fernvik ciphergen biosystems inc., symbion research park, fruebjergvej 3, dk-2100 copenhagen, denmark. e-mail: piversen@ciphergen.com (p. iversen) most mass spectrometry methods used in proteomics allow for the identification of multiple proteins in a limited number of complex samples, but lack the ability to assess the quantity of the proteins and their modifications. however, mounting evidence shows specific cleavage of well-known proteins as being strong candidates for specific biomarkers, and in order to discover these biomarkers one has to be able to monitor the quantity and mass of hundreds of proteins from hundreds of complex samples reproducibly. the new series 4000 instrument in connection with proteinchip arrays ® from ciphergen biosystems enables this. the new 4000 series instrument is optimized for sensitivity, reproducibility and quantitation. new ion optics allows the use of higher acceleration voltages thus increasing the sensitivity, but without lowering the resolution. a new method of blanking the detector in connection with a non-linear gain of the detector also increases the sensitivity to the effect that igg can be detected down to 0.2 fmol. furthermore, the unique design of the instrument permits the detection of proteins with great variation in both mass and concentration and thus making it ideal for proteomics studies. a unique feature of the 4000 series instrument is the possibility to normalize the output by controlling the laser and detector so that results can be read with equal precision on different instruments, which is not often possible in mass spectrometry where individual instruments yield different results. this feature is vital in the validation of research results beyond individual laboratories. the coupling of liquid chromatography with mass spectrometry is now firmly established as a routine method for the identification of proteins that have been subjected to enzymatic digestion. in an on-line lc-ms experiment, the column eluent is coupled to the electrospray source via an emitter and any tryptic peptides present in the mixture are mass analyses as they elute from the hplc column. should there be any co-eluting species in the eluent, these will be separated in the mass analyser by their mass-to-charge ratio. it has become increasingly clear that relative quantification of protein expression changes is important in modern biology and medicine. several current approaches have been developed that utilise stable isotope labelling of samples in combination with separation and subsequent analysis by mass spectrometry. however, we have recently described an lc-ms strategy where quantification is achieved via normalisation of the ms datasets and comparison of the peptide intensities (of the observed tryptic peptides) across samples is performed. in this case, it is desirable to perform replicate injections and hence reduce statistical errors. this approach places a requirement upon good chromatography, especially in terms of retention time reproducibility. in addition exact mass measurement of the eluting ions is required as well as the ability to generate reproducible and reliable peak intensity, or area, calculations for the eluting tryptic peptides. the ability to measure the mass to charge ratios of ions accurately, across injections and across samples, increases confidence that the same ions have been matched from each sample injection. in this presentation our current strategy for the relative quantification of proteins will be discussed using, as examples, complex protein mixtures from salmonella enterica, eschericia coli and human serum. proteomic analysis for the production of rhctla4ig in transgenic rice cell cultures using dige ji-suk cho, song-jae lee, inha university, difference in gel electrophoresis (dige) technology using fluorescent dyes is a novel method, which simplifies the process of detecting and matching proteins between multiple gels by allowing the separation of up to three separate protein samples in the same gel. it provides accurate quantitative and reproducible differential expression values for proteins in several samples. recombinant human cytotoxic t lymphocyte-associated antigen 4-immunoglobulin (rhctla4ig) was produced in the transgenic rice suspension cell cultures using ␣-amylase promoter system. this system is efficient for the production of recombinant proteins, as it secretes target proteins into culture medium under sugar-depleting condition. in this study, the intracellular proteins expressed at both growth and induction stages of culture were separated and analyzed using 2-d dige. each sample from different conditions and internal standard were labeled with n-hydroxy succinimidyl ester-derivatives of cy2, cy3 and cy5 dyes and run within a single dige gel. using decyder tm software, 2218 spots were detected with two-fold thresholds with 95% confidence and it was found that 60 proteins underwent significant change during the production of rhctla4ig with normalization method improving data distribution. a pooled sample mixture for the picking gel was prepared with deep purple staining and analyzed with mass spectrometry. in addition, the intracellular rhctla4ig spots were identified with western blot analysis using goat anti-human igg (fc) antibody after dige gel was transferred to pvdf membrane. study of substrate specificity of rnr-exoribonucelases using hybrid proteins ana barbas, mónica amblar, cecília m. arraiano instituto de tecnologia química e biológica, ean, 2784-505 oeiras, portugal. e-mail: ab@itqb.unl.pt (a. barbas) the ribonucleases are essential enzymes responsible for the regulation of gene expression and have shown to be important for biotechnology purposes. for instance, commercial mutants deficient in ribonucleases have been quite relevant for the over-production of recombinant proteins. escherichia coli rnase ii is a processive 3 -5 exoribonuclease prototype of the rnr family that has homologues widespread in the majority of the sequenced genomes. by sequence alignment it has been proposed for the rnr type proteins the existence of three different domains: an n-terminal cold shock nucleotide binding domain (csd), a rnb catalytic domain, and a c-terminal s1 nucleotide binding domain. we have constructed several rnase ii deletion mutants to enable the characterization of each domain. these studies have allowed us to determine that both csd and s1 are involved in the binding of the enzyme to the rna substrate, being the s1 domain the most important. in rna-binding proteins it has been shown that the s1 domain's conformation is highly conserved. however, it is not known whether the substrate specificity is s1-dependent. in order to characterize the s1 domain and verify if it is directly related to substrate specificity, we have constructed rnase ii hybrid proteins in which the s1 domain was substituted by the s1 of two other exoribonucleases, rnase r (rnii-rnr) and pnpase (rnii-pnp). preliminary results have demonstrated that both quimeric proteins are capable of binding and degrading various rna substrates. in addition, studies are currently being carried out to verify the possibility that s1 domain of pnp in the hybrid protein might be involved in multimerization and/or interaction with other proteins. the murine monoclonal antibody igg1, anti-digoxin was produced in a rolling bottle fermentor. purification was performed on a protein g column. cd spectra were recorded on a jasco-810 spectropolarimeter. protein concentrations of 20-50 g/ml and path length of 1 cm were used for measurements in a far uv region. all measurements were performed in a cell holder thermostand with an accuracy of ±0.2 at 25 • c. at this temperature the predominance of ␤-strands is indicated. large conformational changes occur at 78 • c. at this temperature the spectra tense to irregular ␤-strands and unordered structures. these evidences confirming temperaturedependent conformational changes of protein and also high thermal stability of mentioned monoclonal antibody. generation of monoclonal antibodies for the assessment of protein purification by recombinant ribosomal coupling janni kristensen, kim kusk mortensen, hans peter sørensen 1 laboratory of biodesign, department of molecular biology, aarhus university, gustav wieds vej 10 c, dk-8000 aarhus c, denmark. e-mail: hans.peter.sorensen@teknologisk.dk (h.p. sørensen) we recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of escherichia coli. recombinant proteins were covalently coupled to the e. coli ribosome by fusing them to ribosomal protein 23 (rpl23) followed by expression in an rpl23 deficient strain of e. coli. this allowed for the isolation of ribsomes with covalently coupled target proteins which could be efficiently purified by centrifugation after in vitro proteolysis at a specific site incorporated between rpl23 and the target protein. to assess the efficiency of separation of target protein from ribosomes, by site specific proteolysis, we required monoclonal antibodies directed against rpl23 and gfp. we therefore purified rpl23-gfp-his, rpl23-his and gfp from e. coli recombinants using affinity, ion-exchange and hydrophobic interaction chromatography. these proteins could be purified with yields of 150, 150 and 1500 g per gram cellular wet weight, respectively. however, rpl23-gfp-his could only be expressed in a soluble form and subsequently purified, when cells were cultivated at reduced temperatures. the purified rpl23-gfp-his fusion protein was used to immunize balb/c mice and the hybridoma cell lines resulting from in vitro cell fusion were screened by elisa using rpl23-his and gfp to select for monoclonal antibodies specific for each protein. this resulted in 20 antibodies directed against rpl23 and 3 antibodies directed against gfp. antibodies were screened for isotypes and their efficiency in western immunoblots. the most efficient antibody against rpl23 and gfp were purified by protein g sepharose affinity chromatography. the purified antibodies were used to evaluate the separation of ribosomes from gfp, streptavidin, murine interleukin-6, a phagedisplay antibody and yeast elongation factor 1a by centrifugation, when ribosomes with covalently coupled target protein were cleaved at specific proteolytic cleavage sites. proteomic analysis for the production of rhctla4ig in transgenic rice cell cultures using dige ji-suk cho, song-jae lee, inha university, difference in gel electrophoresis (dige) technology using fluorescent dyes is a novel method, which simplifies the process of detecting and matching proteins between multiple gels by allowing the separation of up to three separate protein samples in the same gel. it provides accurate quantitative and reproducible differential expression values for proteins in several samples. recombinant human cytotoxic t lymphocyte-associated antigen 4-immunoglobulin (rhctla4ig) was produced in the transgenic rice suspension cell cultures using ␣-amylase promoter system. this system is efficient for the production of recombinant proteins, as it secretes target proteins into culture medium under sugar-depleting condition. in this study, the intracellular proteins expressed at both growth and induction stages of culture were separated and analyzed using 2-d dige. each sample from different conditions and internal standard were labeled with n-hydroxy succinimidyl ester-derivatives of cy2, cy3 and cy5 dyes and run within a single dige gel. using decydertm software, 2218 spots were detected with two-fold thresholds with 95% confidence and it was found that 60 proteins underwent significant change during the production of rhctla4ig with normalization method improving data distribution. a pooled sample mixture for the picking gel was prepared with deep purple staining and analyzed with mass spectrometry. in addition, the intracellular rhctla4ig spots were identified with western blot analysis using goat anti-human igg (fc) antibody after dige gel was transferred to pvdf membrane. similarity searches and multiple alignment of s 1 and s 2 protein of sars-cov for modeling 3d structure and its evolution (origin) mohammad soltany rezaee rad, iman tavassoly, negar mottaghi, banafsheh rezaee. e-mail: mohammad.soltany@gmail.com (m.s.r. rad) aims: the exact origin of the cause of severe acute syndrome (sars) is still an open question. nowadays 8 recombinant origins for this virus have been found. s 1 and s 2 subunit of spike protein of this virus are the most important proteins responsible for severe acute respiratory syndrome. in fact they are glycoproteins of this virus exist on its surface. they are responsible for mediating fusion of viral and cellular membrane. the classification and modeling 3d structure of this virus can help us to suggest new ideas about its charististics and function, which may lead to new therapeutic and preventing modalities. methods: we used nucleotide sequence of s 1 and s 2 subunit of s (spike) protein for multiple alignments. we have done multiple alignments with different bioinformatics software (clusterx, entrez) for comparing the sequence with the other viruses and, we used weblab view software for modeling and identifying 3d structure of these proteins. findings: the similarity searches on nucleotide sequence of this protein with the 30 single strands rna (ssrna) shows the virus belong to a known classification named coronaviridae. these 3d structures show the responsibility of s protein in this syndrome. another findings based on these alignments is an important similarity between these subunits and genome of hiv-1 showing they have familiar mechanism in pathogenesis. discussion: multiple alignments are powerful tool in classification of new recombinational virus and emerging infection. 3d structure model of this virus is an important guide to understand the mechanism of this virus. the shape of glycoprotein that modeled with bioinformatics software can help us in understanding mechanism of binding this virus to human cells. this fact can be used in designing drug and vaccine to cure and prevent the sars. blocking these origins and sites leads to inhibiting the virus attachment. also the similarity between this virus and hiv-1 shows us that both of them have similar proteins that cause pathogenesis of these viruses. the simulated moving bed (smb) technology is a continuous countercurrent chromatographic separation technique that has been applied successfully in the last years to a number of significant problems. an smb consists of a series of fixed bed chromatographic columns connected in a loop, and outperforms column chromatography in terms of productivity and solvent consumption. the use of smb instead of batch processes for bioseparations, i.e. separations involving large and rather complex molecules with multiple 3d configurations depending on parameters such as ph, temperature, etc., is becoming of greater and greater interest. examples of these are therapeutic proteins, antibodies and plasmid dna among others. for all chromatographic processes in this field, one of the most crucial issues is the cleaning of the chromatographic media with a special solvent system, an operation usually referred to as cleaning in place (cip). in single column chromatography this is easily done off-line, but this is not compatible with standard smb operation. in order to overcome this limitation, the standard smb configuration has been modified by adding a dedicated section plus an additional section for the re-equilibration of the freshly cleaned column with the working solvent before it is re-inserted into the smb loop. in such a way, cip according to gmp criteria can be incorporated into the smb unit and operation, which is then called cip-smb. this new smb configuration is also related to the three fraction separation unit called 3f-smb that has been recently introduced and applied to the separation of nucleosides. in this work we apply cip-smb using a size exclusion stationary phase to the separation of plasmid dna from the filtered cell lysate solution. plasmid purification has become a key issue in the last years as a result of the advances in gene therapy, whereas traditional laboratory methods are not always suitable for therapeutic purposes. we report about separation performances, which are then discussed in the light of smb design criteria and compared to column chromatography performance. computer guided optimization of adsorptive bioseparation processes bernt nilsson department of chemical engineering, lund university, 221 00 lund, sweden. e-mail: bernt.nilsson@chemeng.lth.se separation processes like chromatography can be highly nonlinear and the behavior can sometimes be hard to predict. optimization of preparative chromatography is often done experimentally, which is both time consuming and expensive. a model-based approach to optimization is therefore an attractive and challenging way to overcome some drawbacks in the traditional working procedure in biotechnical industry. efficient model-based optimization for industrial needs requires three parts; models, methods and tools. model-based methodology requires a set of chromatography column model structures, which can capture the phenomenon of interest. for instance they have to capture column load variations, elution profile changes, operation condition disturbances, column configurations and stationary phase properties. to derive a reliable model for optimization it has to be calibrated and validated to experimental data, which requires an efficient calibration procedure. different calibration procedures are discussed and compared. after validation the model can be used in the design of a separation step. to do a robust design a set of requirements have to be fulfilled. the column size and operation conditions are used to optimize the performance of the step, which requires a constraint nonlinear optimization method. the choice of objective function for optimization and corresponding constraints are not obvious and the resulting operation conditions are often not robust. therefore there have to be additional methods available for analysis of the performance, like sensitivity and robustness analysis. optimization of the purification of antibodies is discussed and exemplified. the work with mathematical models and numerical methods has to be supported by a set of computer tools of different kinds in order to solve industrial problems effective. there is a need for different kinds of tools; customized tool to solve specific problems by a non skilled user and general toolbox for the expert. an example of a toolbox is presented. tina tarmann, alois jungbauer department of biotechnology, university of natural resources and applied life sciences, vienna, austria plasmids and viruses are the contemporary vehicles for genetherapy and genetic vaccination. extremely promising results have been reported from in-vitro, in-vivo and clinical studies. currently a lot of these compounds are manufactured with a technology which has been directly transferred from laboratory to pilot scale without further engineering. membrane based separations, chromatographic separations and precipitation have been employed for separation of plasmids and viruses. chromatographic separation have been designed with aim of protein separation. thus such processes suffer from either mass transfer limitations or low capacity. monoliths without intraskeleton mesopores and chromatography particles with giga pores are excellently suited for adsorption and separation of plasmids and viruses. low mass transfer resistance and high capacity compared to conventional beaded materials can be observed. adsorption kinetics were derived from infinite and finite bath methods and isotherms were constructed. these data also suggest that a conformational change of the plasmids takes place upon adsorption. discussion of the mass transfer properties and an example of scale up of a chromatographic separation process using these novel materials will be shown and discussed in respect to already existing processes. the recent developments in molecular therapies such as non-viral gene therapy and dna vaccination have fostered the development of efficient plasmid dna (pdna) purification processes. the separation of supercoiled (sc) and open circular (oc) isoforms is one of the key steps in the large scale purification of pdna vectors intended for a therapeutic use. although escherichia coli produces mainly the more compact sc pdna isoform, oc, linear and denatured pdna isoforms are usually present and are likely to be less efficient in transferring gene expression. for this reason, regulatory agencies specify that more than 90% of pdna in a therapeutic product is in the sc isoform. in this work histidine-base recognition is explored as a mean to separate pdna isoforms. the agarose gel used here combines the mild hydrophobic characteristics of an epoxy spacer arm with a pseudo-affinity histidine ligand. chromatographic profiles were obtained by injection of native plasmid (sc + oc) samples in the histidine-agarose support showing an efficient and baseline separation of both isoforms. the high resolution obtained with this support indicates that the method is potentially applicable to the separation of pdna at preparative and analytical scale. affinity ligand development with a novel encoded bead screening technology ib johannsen, versamatrix a/s, gamle carlsberg vej 10, dk-2500 valby, denmark. e-mail: www.versamatrix.com the presentation describes a new invention for fast development of affinity ligands, where up to 20,000 ligands can be screened onbead and identified in a few hours. combinatorial synthesis by the split and mix procedure is a powerful technique for generating vast numbers of diverse chemical compounds on polymer beads with relatively little effort. traditionally, the technique is hampered by the laborious spectroscopic and chemical analysis, needed to determine the exact structures of the ligand on selected beads. in this way, 6-12 month analysis time could easily be spent just to analyze a tiny fraction of the library. in the versaffin tm technology each bead is encoded, individually tracked, and identified during the synthesis and screening. this decreases the whole ligand development time from months to weeks and increases the amount of information significantly. the bead code further enables evaluation of the ligandprotein binding under varying binding and elution conditions. the instrument for reading the encoded beads and for quantifying the amount of bound protein is presented. the encoded beads we use are based on functional cross-linked polyethylenglycol (peg), which is compatible with water as well as most organic solvents. thus, the combinatorial synthesis can be carried out in organic solvents and the resulting compounds can be evaluated, still bound to the parent beads, under aqueous conditions. a further advantage of using peg based beads for on-bead screening is the fact that peg is biologically inert and therefore does not interfere in a bioassay. in the biopharmaceutical industry, pressure is mounting to shorten development times and thereby time to market, e.g. in the field of monoclonal antibodies, generic processes have been established which allow for more rapid development from gene to production of pre-clinical and proof of concept/phase i material. for non-mab products from various expression systems, productspecific approaches still prevail. however, for most product types similar issues like clearance of process-and product-related impurities, overall purity and yield, or manufacturing issues have to be dealt with in downstream process development. integrated and timely approaches based on process science and developed orthogonal analytical tools are often hampered by tight time frames and limited resources available. on the other hand, thorough understanding and analytical characterization of product characteristics but also of (process-related) impurities are pre-requisites for fully exploiting separation power and for achieving final purities way above 95% in a robust and cost-effective manner. from primary separation to polishing steps, we have made several attempts to improve the efficiencies of process steps themselves but also of ways to develop them. the strategies applied comprise implementation of innovative processing tools, rational streamlining and optimization of a sequence of unit operations, tech transfer and scale up considerations. also in this context, the applicability of scale down and ultra scale down models for process development and optimization purposes, their potential for speeding up and their limitations will be discussed. chromatographic monoliths are rather new chromatographic stationary phases. they consist of a single piece of a highly porous material. the pores are interconnected forming a network of channels. since the transport mechanism is predominantly based on convection, mass transfer between mobile and stationary phase is significantly enhanced resulting in short separation times. because of that they seem to be an ideal support for separation and purification of extremely large molecules like proteins, dna or even viruses. in this talk, various features of the monoliths like high porosity, fast mass transfer, surface accessibility and dynamic binding capacity will be described. effect of each feature on the separation and purification efficiency will be discussed in terms of molecular size and properties. while chromatographic monoliths are already widely accepted in microchip fluid devices, capillary columns as well as analytical columns, very few reports about preparative monolithic columns can be found. reasons for lack of preparative chromatographic columns will be elucidated and preparation strategy for construction of several liter volume methacrylate based monoliths will be presented. finally, several examples of biomolecule purification like large proteins, plasmid and genomic dna and viruses on cim convective interaction media ® monolithic columns will be given. further, their application as bioreactors and supports for solid state synthesis will be demonstrated. hubbuch institute of biotechnology, forschungszentrum juelich, 52425 juelich, germany. e-mail: m.schroeder@fz-juelich. de (m. schroeder) the intraparticle diffusion coefficient is an important parameter for modeling of chromatographic separation processes. a new method based on dynamic measurements of intraparticle concentration profiles of proteins in process chromatographic media with a confocal laser scanning microscope is presented. the diffusion coefficient is determined by fitting experimental data to a spherical diffusion model. excellent agreement of experimental data with simulation results is obtained. the diffusion coefficient is measured for seven proteins in sepharose 6 ff, spanning molecular weights from 14.3 to 160 kda. in addition, multicomponent diffusion processes for combination of differently sized proteins are analyzed and the influence of adsorbed proteins on the diffusion coefficient is measured in sp or q sepharose ff. taken together the presented method allows measuring the diffusion coefficient of proteins in process chromatographic media in a packed column. use of automated docking for predicting chromatographic behavior of proteins in hydrophobic interaction chromatography andrea mahn, m. elena. lienqueo contreras university of chile, santiago, chile in the present work, we have extended and automated the methodology proposed by mahn et al., 2005 for predicting protein behavior in hydrophobic interaction chromatography (hic). this methodology is based on the good correlation level between the average surface hydrophobicity of the interfacial zone (local hydrophobicity lh) and protein retention time in hic, for only three different ribonucleases. for determining the lh it is necessary to select the most probable protein-ligand conformation. in this work, we have determined the most probable conformation, of more than 12 proteins, using (i) first, the module insight ii affinity by accelrys for providing automated docking (grid method) of ligands (phenyl) to the proteins (100 conformations); (ii) then, the different probable docked protein-ligand conformations were automatically scored using the module insight ii ludi by accelrys; (iii) after that, each conformation was clustered and each cluster was scored by using the average score of each cluster (iv) finally, the most probable conformation was selected using a function based on the number of cluster components, and the average score value. then, when the most probable conformation was selected, the local hydrophobicity (lh) was calculated using the graphical representation and analysis of structural properties (grasp) program. the results have shown an acceptable correlation level (r > 0.90) between lh and the experimental dimensionless retention time (drt). in view of these results, we consider that this methodology could be used to adequately represent the chromatographic behavior in hic for all kinds of proteins (with a heterogeneous and homogeneous surface hydrophobicity distribution) and without a large number of tedious experiments, but only using computational simulation and adequate score criterion. potato tuber proteins are nutritious and show potential as functional ingredient in food systems. however, the present bulk processing technology can only recover byproduct protein for animal feed use. an expanded bed adsorption (eba) process for isolating functional food-grade protein from crude potato starch effluent was previously developed. moderate capture efficiency (20-25%) of the total crude protein was most likely caused by diffusion limitations and aggregated protein, inaccessible for adsorption. we employed the same adsorption ligand attached to agarose-tungsten carbide beads to create stable beds of 2.0-2.7× expansion using flow rates at 400-750 cm h −1 . a pilot scale eba process was run in a commercial processing plant over a three month campaign of starch production from potatoes of mixed variety. fresh crude effluent (150-300 l/cycle) was applied to a column (20 cm × 2 m) containing 20 l of resin. protein capture by eba was reliable in operation, producing a refined protein material, which after dewatering and gentle drying, showed improved functionality over heat-coagulated protein produced at the same plant. overall productivity increased. however, finding a robust operating window of predictable productivity is challenging since the potato fruit water is complex and deteriorates easily. from breakthrough curves, it is observed that the major bulk protein, patatin, displays non-langmuir adsorption behavior. this may indicate a range of interactions for different species of the same protein. chlorogenic acid (ca), the main polyphenolic substance in potato tuber, causes enzymatic browning and undesirable flavor changes, but polyphenols can also react with protein. assessing the effects of interacting cell components therefore applies to the bioprocessing of plant material. at present the acceptance of biochip technology for on site use, e.g. diagnosis or environmental control is hindered by rather expensive and complex instrumental systems. there is a need to provide reliable and cost-effective systems that can be operated with minimal training. the construction of electronic biochip microarrays using semiconductor technology enables the construction of compact systems with high integration at acceptable production costs. the key feature of the fully electrical biochip technology are micro arrays made in advanced si-technology and carrying several array positions with interdigitated nanometer gold electrodes on its surface. the chips are fabricated by standard silicon fabrication methods allow-ing high volume production and to minimise the cost per chip. the advantage of fully electronic microarrays is the intrinsic high spatial resolution and direct signal coupling of the biosensing element and the transducer. the function of fully electronic biochips is also based on the electrochemical transduction and quantification of the formation of affinity complexes on the chip surface. a portable device for field applications and point of care diagnosis have been designed and manufactured. the amperometric device enables the recognition of biomolecular interactions by measuring the redox recycling products of elisa enzyme labels. the highly sensitive signal transduction is achieved with a 16-channel interdigitated ultramicroelectrode array. one major advantage of fully electronic microarrays is the direct signal coupling of the biosensing element and the resulting robustness and opportunity for miniaturisation. those electrical biochip arrays have been adapted for the detection of all types of affinity complexes, such as for dna, rna, proteins and haptens. self assembling of capture oligonucleotides via thiol-gold coupling have been used to construct the array chip nucleic acid interface. thus e.g. pathogenic micro organisms have been identified and quantified via their genomic dna or ribosomal rna respectively. another application based on immobilized antibodies is shown to sense extreme low concentration of bioagent toxins. for processing the assay formats and the electrical read out of the detection of affinity complexes a modular fully automated measurement system has been developed. it is manufactured in industrial lines and available at market. dynamics and self-assembly of organic molecules on surfaces revealed by high-resolution, fast-scanning stm flemming besenbacher interdisciplinary nanoscience center (inano), university of aarhus, dk-8000 aarhus c, denmark. e-mail: fbe@inano.dk in the interdisciplinary area of nanoscience and nanotechnology, the adsorption and self-assembly of organic molecules on singlecrystal surfaces have attracted much attention lately due to the potential applications in fields ranging from molecular electronics to biocompatible interfaces. the supramolecular structures formed upon deposition of molecular species on solid surfaces depend on the molecular architecture and the distribution of functional groups on one hand, which determines the thermodynamically stable molecular arrangement, and on the other hand, on kinetic factors like thermal diffusion, spontaneous rotations and conformational dynamics. i will show how the unique aspect of our aarhus stm and the time-resolved, high-resolution stm imaging can be used to obtain important new insight into the dynamics, and can provide very important new information on the atomic-scale realm and on the dynamics of molecular nanostructures. the time-resolved stm data are visualized in the form of stm movies (see www.inano.dk/spm) which can subsequently be analyzed in order to extract quantitative information on the activation energy, the prefactors and the adsorbate-promoted diffusion. i will specifically discuss: (i) the self-assembly of guanine quartets on au(1 1 1) and the influence of cooperative hydrogen bonds, and (ii) the molecular recognition in binary mixtures of dna bases. g molecules are found to self-assemble into a hydrogen-bonded network of g-quartets, whose structure corresponds perfectly with the quartet structure of telomeric dna determined by x-ray crystallography. the strong preference of g molecules to form quartets can be explained by a cooperative effect that strengthens the hydrogen bonds within the g-quartet network over the hydrogen bonds in isolated dimers. by means of a combination of stm experiments and dft calculations we compare the 2d molecular networks formed on deposition of the binary mixtures g-c (purine-pyrimidine pair of complementary bases) and a-c (purine-pyrimidine pair of non-complementary bases). we find that the non-complementary bases segregate into islands of pure a and a network of pure c, whereas the complementary bases g and c form a network that cannot be separated by annealing up to the desorption temperature for c. high-resolution stm images allow us to identify the structures for the enhanced thermal stability as structures that contain g-c bonds, possibly with the same structure as the watson-crick pairs in dna molecules. kühnle, r., et al., 2002 . nature 415, 891. otero, r., et al., 2004 . angewandte chemie int. ed. 43, 2092 . otero, r., et al., 2004 . nat. mater. 3, 779. otero, r., et al., 2005 . angewandte chemie int. ed. 44, 2. rosei, f., et al., 2002 . science 296, 328. schunack, m., et al., 2002 . biomedical and pharmaceutical companies are using an increasing number of carbohydrate polymers in the formulation of drugs. one such polymer with highly attractive features is chitosan that can be produced from crustacean shells. chitosan is non-toxic, biocompatible and biodegradable. chitosan can be formulated as nanoparticles or membranes and have enhanced several bioprocesses. among the well-documented features are enhanced drug uptake by tight junction relaxation and enhanced in vivo uptake and protection of nucleic acid formulations. chitosan research has increased throughout this decade. research programs are addressing the potential of chitosan applications but preparations with variable molecular size and charge are not easily available. specifically companies working with the development of new drugs and enhancement of drug functionality are in need of formulation technology that provides well characterized biocompatible material. in the chitosan innovation consortium the danish companies, coloplast, novozymes biopolymers, pipeline biotech, and zgene, together with the research center inano (aarhus university) and bioneer a/s have developed a series of chitosan preparations suitable for research of biopharmaceutical applications (www.chitosan.dk). the ability to obtain functional formulations is currently being tested in both in vitro and in vivo experiments. the consortium participants have established a platform from which chitosan processing, characterization and formulation technology can be extracted. by providing specified chitosan preparations the polymer feature can be adjusted to fit specialized biopharmaceutical applications. high throughput bioprocessing govind rao center for advanced sensor technology, umbc, baltimore, md 21250, usa the post genome era holds a great deal of promise. an enormous number of new proteins await study. these will require sophisticated culture techniques, as cells will have to be grown under large numbers of environmental conditions to elucidate expression triggers. unfortunately, unlike molecular biology, bioreactor technology is little changed since its inception. the primary reason has been a lack of sensor technology that can be readily employed to monitor the cellular environment. we will take a look at the current status of the technology and report on promising optical sensor technology that permits low-cost high throughput cell culture and fermentation. noninvasive sensors that monitor ph, po 2 and pco 2 and high sensitivity solutions for glucose and glutamine measurements will be presented. in addition, the mixing characteristics that determine bioreactor performance will be examined at the small scale and their relevance to the large scale will be demonstrated. on-line monitoring and fed-batch operation in shake flask and micro titre plate cultures jochen büchs 1 , frank kensy 1 , markus jeude 1 , tibor anderlei 2 , barbara dittrich 3 , doris klee 3 : 1 biochemical engineering, rwth aachen university, aachen, germany; 2 ac biotec, jülich, germany; 3 textile chemistry and macromolecular chemistry, rwth aachen university, aachen, germany although methods of molecular biology has led to rational design of micro-organisms to suit our requirements, screening of large numbers of strains and media is still one of the most important tasks in biotechnology. batch operation of shaken bioreactors and absence of on-line monitoring is still the general state of the art for that purpose. it is also a very common practice in screening projects that only the final product titre is measured at the end of the culture for the evaluation of the "best performers". in the recent years several approaches were introduced to follow microbial cultures also in shaken bioreactors like shake flasks or micro titre plates (mtp's). it became obvious that the cultures can behave quite unexpected and most relevant and essential information is lost, if only the final product titre at the end of the cultures is utilised for evaluation. as a result, the screening may be directed to an unknown and non-intended direction or may even fail. this is demonstrated with some examples in this contribution. new methods and techniques are introduced to measure the oxygen and carbon dioxide transfer rate and the respiratory quotient in shake flasks and the optical density, nadh fluorescence, ph and do 2 in mtp's. if the desired product can be fused to a fluorescence protein, like gfp or yfp, also the product formation can be monitored on-line in mtp's. it is of utmost importance that the operating conditions of the applied shaking bioreactors are suitable and shaking motion is not stopped during the measurement. otherwise, e.g. power input, mixing and oxygen supply is interrupted and the micro-organisms will ongoingly rearrange their metabolism to cope with these disturbances of their environmental conditions. another problem of screening is the commonly applied batch operation mode. a lot of microbial systems display an overflow metabolism, substrate or osmotic inhibition or are characterised by a catabolite repressed product formation. in all these cases, batch operation is not the preferred operation mode and, therefore, these cultures are run in fed-batch in larger scales. in particular, it is nearly impossible to screen systems, which are catabolite repressed by the carbon source, in batch mode in defined mineral media. after initial growth has led to a nearly complete consumption of the carbon source, the product formation is derepressed. but then no more carbon source is available to continue with production. it is quite questionable whether in batch operation mode suitable strains can be selected for later fed-batch operation or not. this consideration has resulted in the development of a new technique which allows to run the screening in fed-batch operation mode. this technique is applicable in shake flasks as well as in mtp's. dramatic increases in product titre between 4-and 400-folds were observed under these conditions compared to conventional batch screenings. mikkel nordkvist, john villadsen center for microbial biotechnology, technical university of denmark, dk-2800 lyngby. e-mail: mnq@biocentrum.dtu.dk (m. nordkvist) efficient mixing and mass transfer are highly important in the chemical industry and in the fermentation industry. poor mixing can result in low yield and variable product quality in a number of cultivation processes, and mass transfer can easily become the limiting step in aerobic cultivations, especially at high cell density. we have tested a new tank reactor system, where liquid is withdrawn from the bottom of a tank, rapidly circulated, and injected back into the bulk liquid through the nozzles of rotary jet heads. liquid feed as well as gas is added in the recirculation loop and thereby distributed via the rotary jet heads. solid feed in powder form can also be added in the loop with advantage, and heat is efficiently removed in a plate-type heat exchanger, which is part of the loop. the system has a very simple design with no internal baffles or heat exchange area, and between batches the rotary jet heads are used for cleaning in place. a number of applications ranging from dispersion of liquid and powder to mass transfer will be presented. mass transfer applications include baker's yeast cultivation and oxidation of lactose to lactobionic acid by a carbohydrate oxidase. fast and accurate analytical information that can be used to rapidly evaluate the interactions between biological systems and bioprocess operations is essential for optimization of biological production processes. we have researched and developed a multiplexed microbioreactor system for the parallel operation of multiple microbial fermentations. the microbioreactors have working volumes from 5 to 150 l, and are instrumented for real-time monitoring of dissolved oxygen, ph and optical density. the growth profiles obtained with escherichia coli compare favorably to results obtained from conventional 500 ml batch bioreactors. we also demonstrate the use of our microbioreactors coupled to dna microarray analy-sis, as a tool for accelerated discovery and elucidation of metabolic pathways and gene expression profiles. the multiplexed system represents a significant step towards high-throughput data acquisition and has the potential to replace current instrumented bioreactors, which are bulky, expensive to run, and require many mechanical manipulations. design of a laboratory scale bioreactor to study solid-state tobacco fermentation m. di giacomo, l. nappi, d. silvestro, m. paolino, d. parente r&d biology department, british american tobacco italia, naples 80126, italy italian toscano cigar production is based on the fermentation of dark fire-cured tobacco. the process starts with the rise of leaf moisture to levels of water activities assuring development of the wild phylloplane microflora in the absence of free water. the intense growth of microorganisms modifies leaf characteristics (ph rise from acidic to alkaline condition) contributing to define the toscano typical smoke profile. tobacco fermentation takes place in great bulks of 500 kg which cause considerable amount of heat evolution as a function of the metabolic activities of the microorganisms. this heat accumulates and temperature can rise to as high as 70 • c. a laboratory cylindrical packed-bed bioreactor was designed to work under isothermal conditions. the reactor was ideal to ferment small quantities of tobacco (200 g) and was made up of a column aerated from the bottom with humidified air and placed in a thermoregulated room. experiments were conducted with constant temperature and air flow. moreover, bioenrichment experiments were conducted in the presence of different microbial starter cultures. fermentation courses were monitored by measuring microbial counts and chemical/physical modification of the substrate. with this laboratory scale system we obtained different kinds of information on the role and dynamics of the microorganisms involved in the fermentation process and on the influence of different environmental conditions. for the future, the design of an adiabatic device for tobacco fermentation is planned. on-line liquid chromatography as a process analytical technology for monitoring and control of biotech processes rick e. cooley 1,2 : 1 process analytics center of excellence, dionex corporation, sunnyvale, ca, usa; 2 eli lilly and company, indianapolis, in, usa (retired) biotech processes, used to produce an active pharmaceutical ingredient (api), generally differ from small molecule api manufacturing processes in that the starting materials tend to be more variable, more complex, and the product of interest in lower concentration due to the fact that they originate from a biological rather than a chemical process. this complexity and low starting concentration has generated a high level of interest in developing technologies that can be utilized to increase yield and reduce variability in the initial bioreactor phase, as well as, downstream isolation and purification operations. the use of process analytics, or on-line analytical measurements, is a technology approach that can contribute to increased process understanding and control. this presentation will provide examples of how various analytical measurement technologies have been utilized to monitor typical bioprocess unit operations leading to increased automation and control. examples of the use on-line liquid chromatography to monitor bioreactors, process scale chromatography columns, and enzymatic reactions will be presented in more detail. application of advanced monitoring strategies for recombinant protein production karl bayer department of biotechnology, university of natural resources and applied life sciences, vienna a-1190, austria high yield in combination with the required quality are the key objectives of large-scale production of recombinant proteins using different host/vector systems. however, in the past the efficient production of recombinant proteins was frequently limited due to inadequate exploitation of the cell factory and deficiencies in process design. to achieve optimal exploitation of microbial cell factories the key requirement is to enhance the monitoring capabilities to improve the insight into the host metabolism dynamics and to cope with limited understanding of the interaction of recombinant protein synthesis with host cell metabolism. since each protein exerts an individual influence on the host/vector system, the selection of appropriate analytical methods is even more important. in order to overcome these problems high throughput technology platforms, such as dna microarrays for transcriptome and differential 2-d electrophoresis (dige) for proteome analysis provide extensive data to screen for significant analytes and provide an appropriate basis for in silico modelling and reverse engineering of regulatory networks. taking advantage from such an iterative process experimental design will be improved and further aid to increase the performance of modelling. in addition, transcriptome data are used to screen fast stress responsive promoters to set up gfp based reporter gene fusions for in-situ monitoring of the metabolic load due to recombinant gene expression. moreover chemometric methods are frequently applied to model complex data from easily obtainable on-line data sets to overcome the limited monitoring capabilities due to the high complexity and nonlinearity of biological reactions and reaction networks. this strategy has been successfully applied to model bdm (bacterial dry matter), pcn (plasmid copy number) and the amount of recombinant protein using data sets acquired from off-gas analysis (o 2 consumption, co 2 evolution), alkaline consumption rate, in-situ capacitance and multi-wavelength fluorescence measurements. at-line monitoring of bioprocess-relevant marker genes using an electric dna-chip britta jürgen 1 , daniel pioch 1 , le thi hoi 1 , jörg albers 2 , rainer hintsche 2 , stefan evers 3 , karl-heinz-maurer 2 , michael hecker 4 , thomas schweder 1 : 1 institut für pharmazie, ernst-moritz-arndt-universität, 17487 greifswald, germany; 2 ebiochipsystems, 25524 itzehoe, germany; 3 vtb-enzymtechnologie, henkel kgaa, 40191 düsseldorf, germany; 4 institut für mikrobiologie, ernst-moritz-arndt-universität, 17487 greifswald, germany. e-mail: britta.juergen@uni-greifswald.de (b. jürgen) the gram-positive bacteria bacillus licheniformis and bacillus subtilis represent important industrial hosts for the production of enzymes (e.g., proteases and amylases) or antibiotics (e.g., bacitracin). both organisms are attractive for this purpose because of their apathogenity and their classification as gras organisms (generally regarded as save). moreover, their easy cultivation and their high natural capacity to secrete proteins into the growth medium qualify them for the industrial overproduction of homologous or heterologous proteins (simonen and palva, 1993) . for the control of the physiological state and the productivity of the production cells efficient analysis tools are of great interest. a prerequisite for the evaluation of the physiological state of cells during industrial fermentation processes is the analysis of so-called process-relevant marker genes, the expression of which indicates unfavorable growth and production conditions (schweder and hecker, 2004) . by means of proteome and transcriptome analyses we have identified critical process-relevant genes of b. licheniformis and b. subtilis cells under different nutrient limitation conditions and during industrialclose bioprocesses. dna-chips with probes for such process-relevant marker genes could be valuable diagnostic tools for the monitoring of the cellular physiology during microbial bioprocesses. in order to provide reliable tools for the monitoring of the cell physiology during microbial bioprocesses, we have developed a fast mrna analytical approach, which allows an at-line monitoring of the transcriptional activity of selected marker genes during bioprocesses. this approach is based on an easy, fast and reliable rna isolation procedure and the measurement of specific mrnas by means of an electric dna-chip barken et al., 2004 a robust bioprocess is crucial to ensure the consistent process performance and provide the high quality of product for drug manufacturing in biopharmaceutical industries. existing methodologies for bioprocess design do not involve establishing mechanisms to achieve the desirable robust bioprocesses and have low capacity in handling uncertainty in the product manufacturing. also, the solutions are often obtained step wise and do not account for interactions between the steps. despite its importance, the robustness of a bioprocess has not been properly defined and studies carried out in statistic sense are often retrospective. in addition, the computational cost is expensive due to using a line search algorithm for finding an optimal operating solution. finally, the existing methodologies are difficult to apply to the whole bioprocess in biopharmaceutical industries. this paper attempts to define rigorously a measure for process robustness and presents a new methodology for evaluating the robustness of bioprocess operations and their performance. the methodology is based on the concept of 'windows of operation' which shows the whole range of possible operating regions. the methodology also establishes a lower bound for the largest variation of a design variable to ensure the performance. these bounds are achieved by min-max optimization techniques. a direct search algorithm has been developed and its computational cost is much lower than the line search algorithm. results include visualization of robust operating regions and a set of indices which compare the performance of different operating strategies. the capabilities and efficiency of this methodology are illustrated by applying it to the centrifuge selection for the clarification of high solids density cell broths. the research work will impact considerably upon robust bioprocess operation. when grown on methanol, pichia pastoris is able to synthesize proteins to high titres as well as secreting and glycosylation, thereby making this organism a very interesting host for the production of recombinant drugs at large scale. the methanol residual concentration has been reported to strongly influence the specific productivity, the optimum concentration being around 3 g/l. a suitable monitoring and control technique is therefore necessary to study and improve the productivity of p. pastoris fermentations. the current research aims at showing how a mid-infrared spectrometer (atr-ftir) can be calibrated in-situ in order to monitor and control p. pastoris fermentations. this method is simple and fast, and eliminates the need of both standards preparation and off-line calibration. it is based on the observation that during fed-batch processes, only substrate and biomass concentrations vary significantly. the method therefore consists in adding a known amount of methanol at the beginning of the process, just after inoculation, and subsequently calibrating the instrument. financial support for the swiss national science foundation is gratefully acknowledged. implantable biomaterials are subjected to inflammatory responses mediated by adherent phagocytes such as monocytes and macrophages. these cellular responses and behavior have been shown to be dependent on the type of protein that adsorbs to the surface. surface modification is necessary to control and prevent protein adsorption, and thus modulate the inflammatory responses. hydrophilic surfaces that adsorb minimal amounts of protein are considered useful for minimizing the inflammatory reactions to biomaterials. in this study we have used two routes to modify polyethylene terephthalate (pet) films: (1) a wet-chemical method for attachment of linear polyethylene glycol chains (mpeg); and (2) gas-phase plasma polymerisation of diethylene glycol vinyl ether (degve) to generate peg-like surfaces. the surface chemistry was assessed by x-ray photoelctron spectroscopy (xps), fourier transform infrared spectroscopy (ftir) and time-flight secondary ion mass spectrometry (tof-sims). the two pegylated surfaces were compared for their ability to minimise both fibrinogen adsorption and the adhesion and activation of macrophage-like human leukocytes. adsorbed fibrinogen has been shown to be one of the key proteins in stimulating inflammatory responses to biomaterials. adsorption was investigated quantitatively using 125 i-radiolabeled human fibrinogen. in addition, the conformation of the adsorbed protein was tested using an antifibrinogen monoclonal antibody in an enzyme-linked immunosorbent assay. the results showed that pegylated surfaces adsorbed up to 90% less fibrinogen, and that unfolding of adsorbed fibrinogen was more pronounced on the linear mpeg layers than on the peg-like plasma polymer surfaces. adhesion of in-vitro differentiated macrophage-like u937 cells was reduced on both the peg-like plasma polymer surfaces and the linear mpeg layers compared to the unmodified pet surface, but cells adhering to the peg-like plasma polymer surfaces secreted less tumor necrosis factor-␣ (tnf-␣) than cells adhering to the linear mpeg layers. thus, the linear mpeg surface is relatively efficient at reducing adhesion of macrophage-like cells, but those cells that do attach are in a more activated and proinflammatory state. analysis of ceramides from biological samples m. budvytiene 1 , j. liesiene 1 , b. niemeyer 2 , n. ceramides in human skin play an important role in the regulation of cell growth, differentiation and apoptosis. moreover, they are involved in the numerous signaling pathways. the growing interest in the investigations of ceramides physiological functions requires efficient separation methods of ceramides from biological resources and sensitive analytical methods. in this work some sensitive and selective methods, involving thin layer chromatography (tlc), high performance liquid chromatography (hplc), mass spectrometry (ms) and nuclear magnetic resonance spectroscopy (nmr) were employed for the separation and characterization of ceramides from human foreskin. epidermal lipids were extracted from human foreskin for 24 h at room temperature using three solvent mixtures (chloroform/ethanol/water 1:2:0.5, v/v/v); (chloroform/ethanol 1:1, v/v), and (chloroform/ethanol 2:1, v/v). ceramides retention char-acteristics in tlc and hplc were compared with the retention of commercial standards. the best separation was obtained using normal phase column packed with hilic silica 3 m. the elution was performed using mixture of chloroform and ethanol 50/50 (v/v) as an eluent. two commercial standards n-stearoyl-sphingosine cer(ns), r f = 0.51, and n-palmitoyl-sphingosine cer(np), r f = 0.50, were selectively separated with hplc system in above mentioned conditions. the retention time of cer(np) and cer(ns) was 4.31 and 5.19 min, respectively. the same lipids were detected by hplc in the human foreskin extracts. the structure of the lipids from collected fractions was confirmed by means mass spectrometry and nmr. the physiological functions of the separated ceramides are investigated. production recombinant human thymosin-␣ 1 overexpressed as intein fusion protein in e. coli roman s. esipov, vasily n. stepanenko, anatoly i. miroshnikov, shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, ul. miklukho-maklaya 16/10, moscow, 117997 russia. e-mail: esipov@ibch.ru (roman s. esipov) medicines based on polypeptides consisting of 30 and more amino acid residues are widely spread in pharmaceutical market at the present time. practically all polypeptide medicines known are prepared by general chemical synthesis that caused high cost of their production. that's why biotechnological way of polypeptide medicines preparation using recombinant gene expression in bacteria seems to be promising. during our work we design hybrid construction allowing solving a problem of specific cleavage of target polypeptide from the hybrid protein using system of protein splicing from new england biolabs. in case of thymosin-␣ 1 production system intein mediated purification with affinity chitin (impac system)-binding tag has been used, where the modified sce vma from s. cerevisiae has been applied as intein. in contrast to other systems, impact allows the preparation of target protein without using of serine protease and other factors that may cleave the hybrid protein. in the presence of thiol reagents, such as dithiothreitol, mercaptoethanol, or cystein the hybrid protein can be site-specifically cleaved to give intein, the target protein and small fragment of nextein. using of this system does not allow to obtain the target protein with ser, cys or thr on n-terminal of protein, because in those cases target protein and n-extein ligation product will be formed. ser is n-terminal amino-acid in thymosin-␣ 1 . it was recently found that some metal ions essentially affect the splicing. we tried to use zncl 2 and we have found that, in case of intein-thymosin-␣ 1 , the maximal yield of target polypeptide and the minimal yield of splicing products are observed in the absence of dithithreitol and in the presence of 0.5-1 mm zinc chloride in buffers on all stages of thymosin ␣ 1 isolation. the structure of recombinant thymosin-␣ 1 of human was confirmed by the determination of n-and c-terminal amino-acid sequences and by maldi tof mass-spectrometry. we present a microbioreactor with thermoelectric cooling to inactivate cellular metabolism by cell culture freezing. small-scale cultivation methods have gained increased importance and their development has been supported by advances in bioprocess monitoring methods. yet efficient sampling methods for off-line analysis remain important where in-situ real-time measurements are difficult, for example for intracellular metabolite concentration or enzyme activity measurements, and for all methods which are invasive by nature, such as protein purification. freeze-stop measurements of metabolite levels are ideal, because they are inert, i.e. do not require the addition of a chemical. in large systems, the chilling time is often the limiting factor, and alternative methods for cell metabolism inactivation are required, such as the spraying of the cell suspension in 60% methanol at a temperature of −40 • c, or the use of boiling buffered ethanol. due to the small thermal mass of microsystems, shorter chilling times can be expected. sample cooling to 4 • c in a microbioreactor has been presented previously. in this contribution, we investigate sample freezing to completely inactivate the cell metabolism from microbioreactor working volumes of approximately 100 l. the use of multi-parameter flow cytometry for characterisation and monitoring of insect cell-baculovirus fermentations in a mechanically-agitated bioreactor bojan isailovic 1 , alvin w. nienow 1 , ian w. taylor 2 , ryan hicks 2 , christopher j. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf-21 cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf21 infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp486 coding for am-cyan coral protein, which emits natural green fluorescence. the optimization of a fermentation process requires the organism to be cultivated under desirable conditions, which depends on how well the fermentation process is controlled. inadequate mixing and mass transfer are responsible for the heterogeneous environment at large scale in terms of nutrient concentration and ph profile, resulting in lower product yields. these have been associated with, inadequate control of ph and the production of acetate or formate in response to over-feeding of glucose and oxygen deficiency. rapid analyses of substrates and indicator metabolites in a fermentation process is critical for optimal control. this can be achieved in real time with nir spectroscopy. in this study, nir-spectroscopy has been applied to monitor the concentrations of glucose, acetate, formate, ammonium hydroxide and biomass in the cultivation of e. coli (w3110). a comparison of partial least square models built using water standards, synthetic medium standards and fermentation samples has been made. the control of the brewing process is important for improving product quality, and lowering costs. infrared spectroscopy is a technique that can be used at-line and on-line to rapidly measure component concentrations of unprocessed whole broth samples in real time. in this study, both mid -infrared (mir) and near-infrared (nir) spectroscopy have been used and compared for the monitoring of ethanol, flavour components, wort sugars, biomass and specific gravity during the brewing. partial least-squares regression (pls) was used to model relationships between component concentrations and spectra. the performance of these models was evaluated in terms of the standard error of prediction (sep), number of pls factors and the correlation coefficient (r). calibration models were constructed using spectra acquired for multi-component mixtures, intended to simulate brewing fermentation conditions, and actual brewing fermentation samples. chemometric results indicated that nir is a powerful tool for accurately measuring sugar, ethanol and biomass concentrations. when choosing an expression host for production of a specific recombinant protein, one can essentially select from a multiplicity of different systems. while e. coli bacterium is usually the starting point for any cloning and expression effort, there is no universal expression host that would work optimally for all proteins. practical issues to consider include e.g. need for post-translational modifications and protease activity of the host. we have produced recombinant hiv-1 nef in different host cell systems: e. coli, p. pastoris yeast and stable drosophila s2 insect cells. using strain/cell line development, production and purification data from practical experiments, we were able to conduct a techno-economical comparison of the different host cell systems. the annual production goal was set at 100 mg of high-purity nef. this was supposed to be produced campaign-wise in 1-2 batches using laboratory-scale bioreactors and other equipment. in this study, it was shown that although the production costs of the different systems were in the same range, the production in e. coli was most inexpensive, and the s2 cell system was the most expensive. regardless of the selected host system, the labour costs incurred the most expenses. when comparing different stages of the work (strain/cell line development, bioreactor production and down-stream processing), the strain development, the most man-hours demanding stage, involved approximately half of the costs of every production system. although e. coli was the most inexpensive host system for producing nef, it has some definite disadvantages: e.g. the production of endotoxins and the disability to perform post-translational modifications. if these disadvantages are of importance, the production must be done using more expensive system. modelling and control of industrial fermentation j.k. rasmussen, s.b. jørgensen capec, department of chemical engineering, technical university of denmark, dk-2800 lyngby, denmark. e-mail: jkr@kt.dtu.dk (j.k. rasmussen) fed-batch processes play a very important role in chemical and biochemical industry. fermentations in biochemical industry are most often carried out as fed-batch processes. present control schemes do not utilize the full potential of the production facilities and may fail to achieve uniform product quality and optimal productivity. the introduction of model based control strategies is considered difficult because suitable models are not readily available. first principle engineering models can be used but the usually limited knowledge of the regulatory network in the micro-organism makes model development very time consuming. another strategy is to use a purely data-driven approach where only limited prior knowledge of the process is required. a framework for generation of such blackbox models is used in this project. this framework is called "grid of linear models" (golm), it uses a large number of linear models which each describes the behaviour of the process within a certain time interval. the combination of these models results in a model which covers the entire time span of the fermentation and approximates the nonlinear time varying behaviour. a procedure for deriving golm models from operational data has been developed and because they consist of a large set of linear models it makes them suitable for model predictive control implementation with iterative learning capabilities from batch to batch. iterative learning model predictive control (ilmpc) based on a golm model is being implemented on a fermentor at novozymes a/s. the results will be evaluated in terms of the controller's capability to ensure uniform product quality and reject both intra and inter batch process disturbances. the model based approach renders optimization of the process recipe possible by using the ilmpc capability. this opportunity provides a great potential for increase of productivity and reduction of cost. j.m. viader-salvadó, j.a. fuentes-garibay, l.j. galán-wong, m. guerrero-olazarán institute of biotechnology, biological science school, autonomous university of nuevo león, san nicolás de los garza, n.l., méxico, the production of recombinant trypsin and trypsinogen has been reported as difficult due to a probably toxicity on host or its instability. in an effort to attain high-level production of shrimp trypsin for aquaculture applications, we have evaluated shrimp trypsinogen production by recombinant pichia pastoris strains in 5-l bioreactors. a p. pastoris gs115 mut+ containing in its genome the litopenaeus vannamei trypsinogen cdna fused in frame to saccharomyces cerevisiae ␣-factor secretion signal, previously constructed in our laboratory, was used. four three-step fermentations (glycerol batch, glycerol and methanol fed-batch) were carried out. the glycerol batch step (2 l of basal salts medium, 50 g/l glycerol, 8.8 ml biotin 0.02%, 8.8 ml ptm1, 250 l 289 antifoam, ph 5 adjusted to with 28% nh 4 oh) was carried out until glycerol was completely exhausted (21 h). the glycerol fed-batch step was carried out feeding with 50% glycerol (12 ml/l biotin 0.02% and ptm1) at 0.8 ml/min by 9 h and 45 min of posterior starvation. the methanol fed-batch step was carried out feeding with 100% methanol (12 ml/l biotin 0.02% and ptm1) by 133 h using a methanol concentration on/off feedback control to maintain constant the methanol concentration in the culture medium to 1 g/l. in all the fermentations the air flow rate and the agitation were set at 5 l/min and 800-1000 rpm, respectively. with the four fermentation assays, the influence of the ph and temperature in the production phase to the recombinant shrimp trypsinogen production were evaluated. in the four fermentations, at the end of the second step a biomass of 250 g/l wet weight were obtained (od600 230). the methanol demand in the four fermentations surprisingly was not increasing rather initially it was 0.37 ml/min, after 32 h decreased 2.5 times for 29 h, increased to 0.49 ml/min for 23 h and afterwards it was decreased manually to a constant value of 0.3 ml/min for that the dissolved oxygen will not decrease to values less than 20% (last 48 h). the total protein amount in the culture medium supernatant increased during the production step until values of 1.6 g/l (assay at ph 6), 6.5 times more than the worst assay (ph 3) recombinant shrimp trypsinogen production was confirmed by sds-page (about 500 mg/l) and trypsin enzymatic activity was detected using bapna as substrate after trypsinogen activation with shrimp hepatopancreas extract. large conformational change on giant dna induced by ascorbic acid: a nobel scheme on its antioxidative activity yuko yoshikawa, emi sakai, yoshiko oda department of food and nutrition, nagoya bunri college, nagoya 451-0077, japan ascorbic acid is often regarded as an antioxidant in vivo, where it protects against dna damage by scavenging reactive oxygen species. in the present, we will show another potent scenario on the protective effect of ascorbic acid through a significant structural change of giant dna. recently, we examined the effect of ascorbic acid on the higher order structure of dna through single molecular observation with fluorescence microscopy, and found that ascorbic acid generates a pearling structure in giant dna molecules, where elongated and compact parts coexist along a molecular chain. the results of observations with atomic force microscopy indicate that the compact parts assume a loosely packed conformation. as the extension, here we study the protective effect against double-strand breaks by reactive oxygen at different concentrations of ascorbic acid, in relation to the change of the higher order structure of giant dna. we have performed a real time observation on the doublestrand breaks on individual dna molecules by use of fluorescence microscopy. we have found that the double-strand break is markedly protected when ascorbic acid exists over millimolar concentrations. it is found that such a protective effect of ascorbic acid corresponds well to the above mentioned change on the higher order structure of dna. it has been reported that human circulating immune cells, such as neutrophils, monocytes and lymphocytes, accumulate ascorbic acid in millimolar concentrations. therefore, it is expected that the ascorbic acid concentration that induces the large conformational change on dna may be of physiological significance. , y., et al., 2004 . febs lett. 566, 39-42. yoshikawa, y., et al., 2003 . plant proteins are increasingly being used as an alternative to proteins from animal sources and substantially contribute to the human diet in several developing countries. there are many process both industrial and food based which employ protein hydrolysis and hydrolytic products have a wide variety of applications from industrial fermentation media to food additives. traditionally, proteins are hydrolysed by chemical means. acid hydrolysates of protein are used to produce food ingredients and flavour compounds. however, hydrolysis by chemical reagents produce potentially hazardous byproducts and these non-selective hydrolysis products cannot easily be defined. the use of enzymes allows for selective hydrolysis of protein and thus produces a potentially safer and more defined material. the present investigation describes the effects of substrate, enzyme and hydrolysate concentration on the hydrolysis of corn gluten. the corn gluten was hydrolysed by a commercial protease preparation neutrase. the protein hydrolysis reactions were carried out in 0.2 l of aqueous solutions at the temperature of 50 • c and ph 7 and were monitored by using ph-stat method. the degree of hydrolysis (%) and soluble protein concentration depending on time were investigated by using 1, 2, 4, 6, 8 and 10% (w/v) substrate concentrations; and 0.25, 0.4, 0.5, 0.6 and 0.75, 1% (v/v) enzyme concentrations; and 25, 50, 75 and 100% (v/v) this paper describes medium optimization for urease production by aspergillus niger ptcc 5011 by one-factor-at-a-time and orthogonal array design methods. the one-factor-at-a-time method was used to study the effects of carbon and nitrogen sources on urease production. among various carbon and nitrogen sources used, sucrose and yeast extract were the most suitable for urease production, respectively. subsequently, the concentration of sucrose, yeast extract and mineral sources were optimized using the orthogonal array method in two stages. the effects of nutritional components for urease production by a. niger ptcc 5011 in the first and second stages were in order of urea > niso4 > sucrose >kh 2 po 4 > k 2 hpo 4 > cacl 2 > yeast extract > mgso 4 and yeast extract > sucrose > k 2 hpo 4 > kh 2 po 4 > urea > cacl 2 > niso 4, respectively. the optimal concentrations of nutritional components for improved urease production were determined as 20g/l sucrose, 0.85 g/l urea, 3.4 g/l yeast extract, 0.03g/l niso 4 ·6h 2 o, 0.5 g/l mgso 4 ·7h 2 o, 0.04 g/l cacl 2 , 0.35 g/l kh 2 po 4 , and 0.35 g/l k 2 hpo 4 . these results showed that urea, niso 4, yeast extract and sucrose had significant effect on urease production by a. niger ptcc 5011. tween 80 and mgso 4 had negligible effect on urease production by this strain. the subsequent confirmation experiments determined the validity of the models. maximum urease activity in optimized media by onefactor-at-a-time and orthogonal array methods were about 1.14 and 2.74 times greater than with the basal medium, respectively. carbon sources create fingerprint fermentation characteristics pınar ç alık 2 , güzide ç alık 1 , tunçer h.özdamar 1 : 1 bre lab, department of chemical engineering, ankara university, 06100 ankara, turkey; 2 ib lab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: calik@eng.ankara.edu.tr (g. ç alık) this work reports on a systematic investigation of the interactions between the single-carbon sources, i.e. glucose and citric acid, and complex-medium components, i.e., carbon and nitrogen sources, in enzyme fermentation processes, i.e. serine alkaline protease (sap; ec 3.4.21.62),) and ␤-lactamase (ec 3.5.2.6), with the oxygentransfer and ph conditions to demonstrate the influences of carbon sources that create the fingerprint fermentation characteristics, moreover, their influences on the product and by-product formations and the intracellular reaction rates. the influence of the medium composition i.e. citric acid-, glucose-, molasses-and soybean-based media together with the oxygen transfer (ot)-and ph-conditions applied, on the product and by-product distributions and ot characteristics were investigated in batch bioreactors. for sap, in general, under uncontrolled-ph operations the variation of the medium ph in sap fermentation process has a tendency to increase in the sap production phase; however, depending on the carbon source used, its behaviour changes in the early stages of the fermentation as the consequences of the directed functioning of the intracellular bioreaction network. the loci of the dissolved oxygen (do) curves also strongly depend on the carbon source(s) utilised, in addition to the applied ot conditions. the complex media profiles are significantly different compared to the defined media as the ph and do profiles are interrelated owing to the bioreactor operation conditions affecting the metabolic reaction network. the highest volumetric oxygen uptake rates were obtained with soybean-based medium that was ca. three-fold higher than the values reported in citrate-based and glucose based media, and ca. 1.5-2-fold higher than the values reported in molasses-based medium. the significant changes, moreover, the drastic change observed with the use of soybean-based complex medium are due to the compositions of the fermentation media used, and its influence on the intracellular bioreaction network. thus, we conclude that the change in medium composition based on the carbon source changes the fermentation characteristics under the designed bioreactor operation conditions that appear as the fingerprints of the bioprocess. effects of oxygen transfer on ␣-amylase production by b. amyloliquefaciens nurhan güngör, güzide ç alık bre lab, department of chemical engineering, ankara university, 06100 ankara, turkey ␣-amylase (e.c. 3.2.1.1) a commercially important enzyme used in food, textile, detergent, brewing, paper, and animal feed industries; hydrolyses ␣-1,4 glucosydic bonds in amylose, amylopectin, and related polysaccharides. optimum medium composition and influence of bioreactor operation parameters on ␣-amylase production with high yield and selectivity were determined together with the metabolic flux distributions using b. amyloliquefaciens (nrrl b-14396) which is found to be a good producer of the enzyme. systematic investigation of oxygen transfer in relation with the metabolic fluxes for ␣-amylase is not available in the literature. shake-flask experiments were conducted in 0.5 dm 3 airfiltered bioreactors in orbital shakers with agitation and heating controls (b. braun, certomat-bs1). laboratory-scale bioreactors were composed of agitation, heating, foam, dissolved-oxygen and ph-controlled 1.0 and 3.5 dm 3 systems (b. braun, biostat q; and chemap). after separation of the cells with a sorval rc28s ultracentrifuge, ␣-amylase activity was measured by the dns method (bernfeld, 1955) . amino acid concentrations were determined with a hplc (waters), protein and organic acid concentrations were measured with a hpce (waters, quanta 4000e) (ç alık et al., 1998) . oxygen transfer characteristics in the bioreactor systems were calculated using the dynamic method (rainer, 1990) . in the mass flux balance-based analyses, a pseudo-steady state approximation for the intracellular metabolites and the accumulation rates of the extracellular metabolites measured throughout the fermentations in considerations of the biochemical feature of the system were used to acquire the flux distributions. in laboratory scale, the effects of different c sources i.e., glucose, fructose, maltose, lactose and soluble starch; n sources i.e., (nh 4 ) 2 hpo 4 , (nh 4 ) 2 so 4, and nh 4 cl and/or their concentrations; and the operation parameters, ph and temperature on cell growth, substrate utilization, ␣-amylase and byproduct concentrations, and ␣-amylase activity were investigated. in pilot scale, the fermentation and oxygen transfer characteristics of the bioreaction system together with the metabolic fluxes were determined. oxygen transfer regulates benzaldehyde lyase production in e. coli pınar ç alık iblab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: pcalik@metu.edu.tr the effects of oxygen transfer rate on benzaldehyde lyase (bal) production by puc18::bal carrying recombinant e. coli on a defined medium with 8.0 kg/m 3 glucose were investigated in order to finetune the bioreactor performance, in v = 3 dm 3 batch bioreactors at five different conditions with the parameters at, i.e. q 0 /v r = 0.5 vvm and n = 250, 375, 500 and 750 min −1 and; q 0 /v r = 0.7 vvm and n = 750 min −1 . the concentrations of the product and by-products amino acids and organic acids were determined in addition to bal activities. medium oxygen transfer rate conditions and uncontrolled ph operation at ph 0 7.25 are optimum for maximum bal activity, i.e. 860 u/cm 3 at 12 h, and productivity and selectivity. on the bases of the data, response of the intracellular bioreaction network of r-e. coli to oxygen transfer conditions were analysed using a mass-flux balance based stoichiometric model that contains 102 metabolites and 133 reaction fluxes. the results reveal that metabolic reactions are intimately coupled with the oxygen transfer conditions. oxygen transfer rate showed diverse effects on the product formation by influencing metabolic pathways and changing metabolic fluxes. metabolic flux analysis was helpful to describe the interactions between the cell and the bioreactor by predicting the changes in the fluxes and the rate controlling step(s) in the metabolic pathways. therefore, knowing the distribution of the metabolic fluxes during the growth, and bal and by-product formations provide new information for understanding physiological characteristics of the r-e.coli, and reveals important features of the regulation of the bioprocess and opens new avenues to successful application of metabolic engineering. the detection and diagnosis of pathogenic bacteria causing many diseases to the human body is an area of important research to public welfare. food is the most important energy source to humans, but it can give rise to disease caused by pathogenic bacteria not performing adequate detection tests. oligonucleotide-based microarrays are becoming increasingly useful for the analysis of expression profiles and polymorphisms among interested genes. here, we checked the possibility of development of oligonucleotide-based microarrays for detection and diagnosis of foodborne pathogenic bacteria. the oligonucleotide chip technology was applied to one control strain and seven foodborne pathogenic bacteria strains. it was designed repeated spots of eight hyperspecific and two highly conserved (control) capture probes from 16s rdna sequences. in order to validate experimental quality and to certificate specificities among specific spots at a glance by 2d and 3d views, quantitative visualization tool was developed. using the proposed oligonucleotide chip, we could classify and diagnose species and even subtypes of some pathogens. induction of in vitro neuro-muscular junctions using neuroblastoma and fibroblast cell lines for facilitating receptor-binding studies with botulinum toxin arindam chaudhury, bal ram singh department of chemistry and biochemistry, university of massachusetts dartmouth, 285 old westport road, north dartmouth, ma 02747-2300, usa. e-mail: g achaudhury@umassd.edu (a. chaudhury) botulinum toxin (bont), the most potent biological toxin known, is responsible for botulism, a fatal paralytic disease of the neuromuscular transmission. it blocks the release of acetylcholine at the neurotransmitter junction of the synapse. the objective of the current study was to induce in vitro neuro-muscular junctions through co-culturing of nerve and precursor-muscle cell lines. presently no known primary cultures or cell lines are available for nerve-muscle co-culture, thus validating the current work. j2-3t3 fibroblast cell line was first adapted to grow in media conducive for growth of sh-sy5y neuroblastoma cell line. the two cell lines were then splitted and co-cultured and observed for junction formations. light and fluorescent microscopic studies revealed en plaque (twitch-type) and en grappe (bulbous nerve endings) nerve-muscle junctions. growth rate of j2-3t3 cells decreased substantially when the media was initially changed. structurally they were more spindle-shaped than the normal reticular shapes of j2-3t3 cells, when grown in a media tailor-made for them. the formation of nerve-muscle junctions were confirmed using markers specific for each cell type. future work is focusing on receptor identification for the botulinum toxin in the established in vitro neuro-muscular junctions and also the transcellular translocation of the toxin. fourier-transform infrared (ftir) spectrometers have recently enjoyed widespread popularity in bioprocess monitoring applications due to their non-invasiveness and in-situ sterilizability. their online applicability creates an interesting opportunity for process control and optimization. however, the precision and accuracy of the predicted analyte concentration values directly depend on the quality of the measured signal and the robustness of the calibration model. instability and time drift in the measured spectra are currently one of the main obstacles in ftir monitoring. the intensity of the detected signal is influenced both by random noise and structural drifts and offsets. as a result, it is often necessary to scale the measured spectrum with respect to a constant reference spectrum, a technique similar to the internal standard approach used in analytical assays, such as hplc. applying this technique has lead to a noticeable decrease in the standard error of prediction in the monitoring of an anaerobic s43 fermentation of the saccharomyces cerevisiae yeast. in order to test the robustness of the calibration model and to increase its resistance to signal instability, random spikes of known amounts of analytes were introduced into the measured medium. this approach can be used to fine-tune the calibration model on-line and is currently one of the aspects investigated in this laboratory. the effect of the stringent response induction on l-valine biosynthesis by corynebacterium glutamicum ilze denina 1,2 , longina paegle 2 , liga zala 1,2 , maija ruklisha 2 : 1 faculty of biology, university of latvia, kronvalda blvd. 4, riga lv-1586, latvia; 2 institute of microbiology and biotechnology, university of latvia, kronvalda blvd. 4, riga lv-1586, latvia. e-mail: ilzede@hotmail.com (i. denina) the present study was focused on methods of the stringent response induction and on investigation of its effect on valine overproduction by isoleucine auxotrophs of corynebacterium glutamicum. the intracellular level of guanosine 5 -diphosphate 3diphosphate (ppgpp) increased and bacterial growth rate (µ) decreased during the short-term experiments when the exponentially growing cells were exposed to isoleucine limited conditions. the induction of the cellular stringent response resulted in a drastic increase in the activity of acetoxydroxy acid synthase (ahas), also by a significant increase in valine production. in contrast, an increase in ahas activity and valine synthesis by c. glutamicum was not achieved when bacterial growth was down-regulated in a ppgpp-independent manner. these results demonstrated that induction of the ppgpp-mediated stringent response might be significant in order to increase valine overproduction by c. glutamicum. infections with human cytomegalovirus (hcmv) continue to be an important health problem in certain patient populations, such as newborns, graft recipients of solid organs, or bone marrow and aids patients. in these groups, hcmv is a major cause of morbidity and mortality. the complex biology of hcmv necessarily begins with an initial interaction between the envelope of the infectious virus and the host cell. glycoprotein b (gb) is the major antigen, on the envelope of hcmv, for the induction of neutralizing antibodies. the region between aa 552 and 635 of hcmv gb (termed antigenic domain 1, ad-1) has been identified as the immunodominant target for the humoral immune response following natural infection. screening methods for detection of neutralizing antibodies have not been used because they are costly and labor intensive and thus far are not feasible for use on a large scale. for the development of reliable and inexpensive serodiagnostic tests the ad-1 of hcmv glycoprotein gp58, which are known to bind neutralizing antibodies, was expressed in prokaryotic systems. in this work, one prokaryotically expressed fusion protein which codifies ad-1 with ␤-galactosidase was used. the influence of different process conditions, on the production of the fusion protein containing the ad-1 as well as sugars addition to the fermentation medium was investigated. in order to analyze the expression of fusion protein, the ␤-galactosidase activity was followed throughout the fermentation. lysis process was also optimized and some final confirmation tests about protein antigenicity were performed. polyenzymic systems for the preparation of drugs based on modified nucleosides d. chuvikovsky, r. esipov, t. muravyova, a. miroshnikov shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, ul. miklukho-maklaya 16/10, moscow 117997, russia. e-mail: esipov@ibch.ru (r. esipov) considerable progress in the preparation of nucleoside analogues was achieved by combination of chemical and biochemical transformations. enzyme-catalyzed chemical transformation is now widely recognized as practical alternative to traditional organic synthesis in pharmaceutical and chemical industries. pentofuranosyltransfer reaction catalyzed by nucleoside phosphorylases was successfully employed for the synthesis of a variety of base-and sugar-modified nucleosides. enzymes involved in the metabolism of ribose phosphate and deoxyribose phosphate, such as ribokinase and phosphopentomutase, were used for the preparation of sugar-modified nucleosides. nucleosides phosphorylases (thymidine phosphorylase (tp), uridine phosphorylase (up) and purine nucleoside phosphorylase (pnp)), ribokinase and phosphopentomutase from e. coli have been cloned and overexpressed in e. coli. fast and efficient methods for the purification of nucleosides phosphorylases have been developed. the amount of purified protein was about 140 mg/l of cell culture, corresponding to 6300, 16,800 and 4200 units, respectively, of the up, tp and pnp. synthesis of medicinal drugs ribavirin (1-(␤-d-ribofuranosyl)-1,2,4-triazole-3-carboxamide), cladribine (2-chloroadenine-9-␤-d-2 -deoxyribofuranoside) and fludarabine (2-fluoroadenine-9-␤-darabinofuranoside) with the use of recombinant enzymes were studied. several important factors affecting the modified nucleosides production (ph, temperature, enzyme concentration, donor/acceptor ratio) were investigated and optimized. under optimum conditions ribavirin, cladribine and fludarabine produced in the reaction mixture in yields of 84, 85 and 80%, referred to 1,2,4-triazole-3-carboxamide, 2-chloroadenosine and 2-fluoroadenosine, respectively. aggregation and adsorption of fibroin molecules in aqueous solution won hur school of biotechnology and bioengineering, kangwon national university, chunchon 200-701, korea. e-mail: wonhur@kangwon.ac.kr fibroin, the structural protein from bombyx mori, is composed of heavy chain (generally called 'fibroin') and light chain polypeptides of about 370 and 25 kda, respectively. this study investigated the aggregation of fibroin and the adsorption between fibroin and surfaces. the variations of particle size and zeta potential were investigated by electrophoretic light scattering spectrophotometer (els). the adsorption of fibroin on surface was investigated in a continuous flow system by biacore applied surface plasmon resonance(spr) technique. the particle size and zeta potential range of aqueous fibroin were 140 nm, ±20 mv, respectively. iso-electric point(ph iep )of fibroin was ph 4.29. the amount of fibroin adsorbed on a gold surface was less than 0.1 g/ml even in the presence of high concentration of fibroin. the modification of gold surface was accomplished by applying chemicals known to form self-assembled monolayer, those are carrying nh 3 + , coo − , benzene ring and peptide that similar structure with fibroin. the adsorbed amount of fibroin on the self-assembly monolayers (sams) increased in the following order: nh 3 + > benzene ring > coo − > peptide surface. the deposition of fibroin in aqueous solution on non-waven fabric was affected by nacl and high temperature. ph influences metabolite profiling of ␤-lactamse producing b. licheniformis nazari̇leri 1 , pınar ç alık 1 , ali şengül 2 : 1 ib lab, department of chemical engineering, metu, 06531 ankara, turkey; 2 gülhane sch med, dept immunol, 06018 ankara, turkey. e-mail: e115715@metu.edu.tr (n.i̇leri) ph conditions in the bioreactor affect product and by-product formations by influencing metabolic pathways and changing metabolic fluxes, based on its influence on, i.e. dna transcription, protein synthesis, transport mechanism, atp generation and cellular energetics. whereupon, some fermentation processes favours uncontrolled-ph conditions while others favours controlled-ph conditions. on the bases of the interactions between the cell and the bioreactor through a process, carried out at either uncontrolled-or controlled-ph conditions, intracellular ph can be widely different and variable during the fermentation process. consequently, if one aims towards a quantitative understanding of the cell metabolism, one has to take into account the time variations of the intracellular ph and its effects on the in-vivo kinetics of the metabolic steps involved. moreover, since the presence of dormant or dead cells in the cultivation medium have negative effect on the synthesis of the production of desired product; the physiological state of the culture has great importance. in this context, the effects of ph on the regulation of intracellular ph, transport mechanism, and metabolic activity of b. licheniformis during production of ␤-lactamase (ec 3.5.2.6), an industrial enzyme catalyzing the hydrolysis of beta-lactam ring in beta-lactam antibiotics, was investigated. in addition, the physiological state of the organism and its effect on the production were observed. the optimal controlled-ph operation was found to be ph 6.75 with 54 u/cm 3 enzyme activity. the intracellular and extracellular na + , k + ion concentrations increased significantly throughout the process with increasing ph. on the other hand, the intracellular nh 4 + ion concentration was relatively constant. isolation and characterization of angiotensin-i converting enzyme inhibitory peptides by use of anti-peptide antibody fida hasan 1 , megumi kitagawa 2 , yoichi kumada 1 , naoya hashimoto 1 , masami shiiba 2 , shigeo katoh 1 , masaaki terashima 2 : 1 graduate school of science and technology, kobe university, nada, kobe 657-8501, japan; 2 department of human science, kobe college, okadayama, nishinomiya 662-8505, japan inhibitory peptides against angiotensin-i converting enzyme can be promising bio-functional peptides as natural alternatives for the non-peptide ace inhibitory drugs. these peptides are inactive within sequences of parent proteins and can be released during enzymatic digestion or food processing. immunointeraction is very effective for the purification of proteins and peptides with high purity. in this study, ace inhibitory peptides from hydrolysate of bonito meat were isolated by an anti-peptide antibody column and hplc, and kinetics of production of these ace inhibitory peptides was studies. an anti-peptide antibody against an ace inhibitory peptide, which was found by kohama et al. from tuna was obtained by immunization of the antigen peptide pc-iace (kkpthikwgd). water extract of bonito meat was digested at 37 • c in a modified gastric juice, 1.76 mg/ml nacl containing 312 g/ml pepsin (ph 2). peptides in hydrolysates were purified by use of an affinity column coupled with the antipeptide antibody and separated by hplc equipped with a reverse phase column (cosmosil 5c18-ms-ii, 4.6 cm × 150 cm). amino acid sequences and ic 50 values of the potent ace inhibitory peptides were determined. sds-page and western blotting experiments clarified that bonito protein contained peptides having similar sequence to the antigen peptide. a fraction of retention time 18-28 min in hplc purification samples showed high inhibitory activity, and several peptides in this fraction were separated. after 48 h digestion, two major inhibitory peptides, herdpthikwgd and pthikwgd, were found to be relatively stable in the gastric juice. kluyveromyces marxianus physiology on several levels of carbon, nitrogen sources and oxygenation during inulinase production silva-santisteban yépez, o. bernardo, francisco maugeri department of food eng./unicamp, 13081-970, campinas, sp-brazil. email: maugeri@fea.unicamp.br (f. maugeri) inulinase produced by yeasts is an interesting alternative compared with the one produced by filamentous molds, as culture conditions can be better controlled. during the assays, it was observed that inulinase production levels varied with nutritional conditions in batch culture. kluyveromyces marxianus atcc 16045 culture is described by two main phases, the first one being the growth phase, where substrate consumption and basal inulinase production were performed, and the second one being the phase where some metabolites are uptaken and high inulinase production is observed. the metabolic fluxes analyses were used to describe the cell physiology in the first phase, in a variety of conditions of sucrose and ammonium sulfate concentration and aeration condition. the metabolic network included the main metabolic pathways such as glycolisis, pentose phosphate pathway, krebs cycle, oxidative phosphorylation and biomass biosynthesis. the physiology in this phase was correlated with high inulinase production in the second phase. it was also noticed that inulinase production diminished when sucrose was in high concentration, leading, additionally, to ethanol production. in these terms, it was unveiled a kind of crabtree effect performed by this strain. forward extraction of l-aspartic acid from fermentation broths by reverse micelles and backward extraction by gas hydrate methodö. aydogan 1 , e. bayraktar 1 ,ü. mehmetoglu 1 , m. parlaktuna 2 , t. mehmetoglu 2 : 1 department of chemical engineering, faculty of engineering, ankara university, tandogan, ankara 06100, turkey; 2 department of petroleum and natural gas engineering, middle east technical university, ankara 06531, turkey. e-mail: mehmet@eng.ankara.edu.tr (ü. mehmetoglu) recently gas hydrate method has been applied as a technique for backward extraction of amino acids from reverse micelle systems. in this study, backward extraction of l-aspartic acid was investigated by gas hydrate method. at the first stage, production of l-aspartic acid was carried out using 50 ml of 0.5 m ammonium fumarate (ph 9.5) as substrate at 37 circc in an orbital shaker at 150 rpm for 4 h. e. coli (atcc 11303) was used as biocatalyst. at the end of reaction excess fumaric acid was extracted in reverse micelle phase. then forward extraction of l-aspartic acid was carried out with injection method in reverse micelle phase. for back extraction, co 2 is used to form gas hydrates crystalline structure. back extraction experiments were carried out between 35-37 bar g pressure and at 2 circc. at the end of the back extraction l-aspartic acid was obtained in crystalline form. the results indicate that recovery of l-aspartic acid from reverse micelles by forming gas hydrate can be achieved with a yield of 55.3%. consequently, gas hydrate method can be used as a new technique for backward extraction of amino acids from reverse micelles. aerobic and anaerobic cultivations of aspergillus niger on different nitrogen sources susan meijer, gianni panagiotou, lisbeth olsson and jens nielsen center of microbial biotechnology, biocentrum-dtu, technical university of denmark, dk-2800 lyngby, denmark in this study, we aim at creating a succinic acid producing strain of a. niger. a. niger is known to be a strictly aerobic organism, meaning it is not able to use the reductive part of the tca cycle to produce succinate. during aerobic growth a. niger uses oxygen as electron acceptor in the respiratory chain, thereby reoxidizing the produced nadh to nad + . however, under anaerobic conditions other compounds than oxygen, such as no 3 − are required as electron acceptor (denitrification). this process consists of no 3 − reduction to nh 4 + coupled to substrate-level phosphorylation that supports growth under anaerobic conditions. in the present study, our aim was to investigate the effect of different nitrogen sources on the physiology of a. niger during growth under aerobic and anaerobic conditions. aerobic growth experiments on three different nitrogen sources; ammonium, nitrate and nitrite, showed that ammonium and nitrate could be consumed by the filamentous fungus. nitrite on the other hand could not facilitate growth, indicating the absence of a nitrite uptake system. however, under anaerobic conditions notable growth was only observed on nitrate. these data support the hypothesis of the existence of an alternative electron acceptor that might facilitate anaerobic growth of a. niger. among the therapeutic proteins derived from mammalian cells, recombinant antibodies received a great deal of attention as a prominent product through biotech pipelines toward the marketplace. they now occupy about 25% of the estimated medicines in clinical development and many more antibodies which lead the value of the market going forward are reported. there are various environmental factors affecting rcho cell cultures such as medium components, temperature, ph, and byproducts (ammonia, lactate, and, etc.) . because most of mammalian cells are very sensitive to their environmental change, appropriate control of environmental parameters is a very important consideration to enhance cell growth and production of target proteins. balanced addition of limiting medium components plays an essential role on improvement of cell density and product concentration. temperature and ph are easily adjustable process parameters, being reported to influence cell growth and recombinant protein production. ammonia and lactate are well-known byproducts which have an inhibitory effect on cell growth when their concentrations exceed a specific level. in this work, effects of various environmental factors including temperature, ph, amino acids, vitamins, hormones, and metabolic byproducts on cell growth and recombinant antibody production were investigated in the cultivation of recombinant chinese hamster ovary cells. the most suitable condition of each environmental condition was proposed for enhancement of the cell growth and the productivity of recombinant antibody. the present study was carried out in order to assess the protective effects of calycosin-7-o-␤-d-glucopyranoside isolated from astragali radix (ar) on hyaluronidase (haase) and the recombinant human interleukin-1␤ (il-1␤) induced matrix degradation in human articular cartilage and chondrocytes. we isolated the active component from the n-butanol soluble fraction of ar as haase inhibitor and structurally identified as calycosin-7-o-␤-d-glucopyranoside by lc-ms, ir, 1 h nmr, and 13 c nmr analyses. the protective effect of arbu on the matrix gene expression of immortalized chondrocyte cell line c-28/i2 treated with haase was investigated using a reverse transcription polymerase chain reaction (rt-pcr). its effect on haase and il-1␤-induced matrix degradation in human articular cartilage was determined by using a staining method and calculating the amount of degraded glycosaminoglycan (gag) from the cultured media. pretreatment with calycosin-7-o-␤-d-glucopyranoside effectively protected against matrix degradation of the human chondrocytes and articular cartilage. therefore, it would appear that calycosin-7-o-␤-d-glucopyranoside from ar is a potential natural ant-inflammatory or anti-osteoarthritis agent and can be effectively used to protect against proteoglycan (pg) degradation. catechol-o-methyltransferase (comt) is an enzyme that catalyses a variety of endogenous and exogenous catechol substrates by transferring a methyl group from s-adenosylmethionine (sam) to either the meta-or the para-hydroxyl group of the catechol ring. the enzyme has a physiological role in the metabolism of the catechol estrogens, inactivation of the catecholamine neurotransmitters such as dopamine and epinephrine and detoxification of a variety of xenobiotic catechols. comt activity has been identified in various tissues; however with the developments in molecular biology and gene technology, the production and purification of large amounts of recombinant comt is a good option for biochemical, pharmacological and structural studies. in this work, cultures of recombinant e. coli harbouring a model plasmid were grown in a 500 ml shake-flask containing 125 ml of complex medium. the influence of medium composition and induction time on comt production, recovery and clarification by sonication, ammonium sulphate precipitation and purification by hydrophobic interaction chromatography onto a butyl-sepharose column will be presented and discussed. bioactive bacterial exopolysaccharides corinne sinquin 1 , karim senni 2 , jacqueline ratiskol 1 , farida guéniche 2 , jean guézennec 1 , gaston godeau 2 , sylvia colliec-jouault 1 : 1 ifremer, 44311 nantes cedex 3, france; 2 ea2496 université rené descartes, 92120 montrouge, france. e-mail: corinne.sinquin@ifremer.fr (c. sinquin) interest in mass culture of microorganisms from the marine environment has increased considerably, representing an innovative approach to the biotechnological use of under-exploited resources. marine bacteria associated with deep-sea hydrothermal conditions have demonstrated their ability to produce in an aerobic carbohydrate-based medium, unusual extracellular polymers (guezennec, 2002; colliec-jouault et al., 2004) . these exopolysaccharides (eps) present original structural features that can be modified to design innovative bioactive compounds and improve their specificity. with the aim of promoting biological activities, chemical modifications (depolymerization and substitution reactions) of one eps produced by vibrio diabolicus have been undertaken (raguenes et al., 1997) . the structure of the native eps has been described (rougeaux et al., 1999) the potential of the eps derivatives as therapeutical agents will be presented. physiological responses of e. coli to glucose and oxygen shifts in fed-batch fermentations jaakko soini 1 , christina saarimaa 1 , arne matzen 2 , peter neubauer 1 : 1 bioprocess engineering laboratory, department of process and environmental engineering, university of oulu, oulu fi-90014, finland; 2 sanofi-aventis, germany. e-mail: jaakko.soini@oulu.fi (j. soini) in high-cell density fermentations e. coli cells are often subjects of transient changes in microenvironment around them. these changes can be, for example, medium component gradients or differences in oxygen availability. we have studied the physiological response of e. coli w3110 cells to simultaneous oxygen limitation and overfeeding of glucose. the aim is to obtain more information of physiological changes for better understanding of the bottlenecks in such processes. the response of the cells for glucose and oxygen shifts was studied by analyzing key metabolites and proteins and mrna transcript levels. the transcript levels were measured using a sandwich hybridization technique (rautio et al., 2003) proteomic analysis was carried out by 2d-electrophoresis and the metabolite analysis by hplc. the main focus of this study is to monitor the expression patterns of marker genes involved in mixed acid fermentation, glycolytic pathway and tricarbonic acid cycle. rautio et al., 2003 . sandwich hybridisation assay for quantitative detection of yeast rnas in crude cell lysates. microb. cell fact. influence of ner on genetic instability of the (ctg/cag) tracts in bacterial chromosome sylwia szwarocka 1 , paweł parniewski 2 : 1 department of microbiology and immunology, university of łódź, 90-237 łódź, banacha 12/16, poland; 2 centre for medical biology, polish academy of sciences, 93-232 łódź, lodowa 106, poland many human hereditary neurological diseases, including fragile x syndrome, myotonic dystrophy and friedreich's ataxia, are associated with expansions of triplet repeat sequences (trs) (cgg/ccg, ctg/cag and gaa/ttc) in or near specific genes. mechanisms that mediate the expansions and deletions of trs include dna replication, repair and recombination. many investigations suggest that the structural properties of the trs play a consequential role in their genetic instabilities. nucleotide excision repair (ner) is the major cellular system in both prokaryotes and eukaryotes and recognises damages due to distortion of the dna helix. involvement of ner in the hairpin loop repair that can form within ctg tracts has been reported. the participation of this repair systems in the trs instability was investigated in e. coli only on multicopy plasmids. the results showed that deficiency of some ner functions dramatically affects the stability of long (ctg/cag) inserts. in this work we present a chromosomal model to study the instability of the trs in e. coli. we introduced the (ctg/cag)n tracts into the chromosome of e. coli and used strains with some deficiency of the ner and investigated genetic stability of these tracts after multiple recultivations. in general, our results show that the (ctg/cag)n repeats are much more stable in the chromosome than in plasmids. these data may suggest that instability of trs in plasmids is associated with interaction between repetitive tracts on different plasmid molecules inside the cell. however, mutations of ner genes may increase (uvra and uvrb mutants) or decrease (uvrc and uvrd mutants) stability of the trs in the e. coli chromosome. this study was partially funded by the kbn grant 2 p05a 01927. performance analyses of a multi-stage integrated fermentation process for lactic acid production hsun-tung lin, feng-sheng wang department of chemical engineering, national chung cheng university, chia-yi 621-02, taiwan. e-mail: chmfsw@ccu.edu.tw (f.s. wang) in this work, we considered a multi-stage integrated continuous fermentation process for producing lactic acid. each stage consists of a mixing tank, a fermenter, a cell recycle unit and an extractor. the generalized kinetic model is first applied to formulate the integrated process. we have compared the overall productivity and conversion of the integrated process with those of two simplified processes. from the design equations, we obtain that three processes have the identical overall conversion. however, the proposed process has the greatest overall productivity. the specific kinetic model for lactic production (youssef et al., 2000) was applied to the integrated process in order to find the maximum overall productivity. two optimization problems are respectively considered to determine the optimal stages, operating conditions and design variables. the first problem supposes that the integrated process has the equal working volume ratio for each fermenter. such a process requires four stages to yield the maximum overall productivity and the nearly complete overall conversion. however, if the working volume ratio for each stage is considered as the decision variables in the second optimization problem, three stages is enough to achieve the identical overall productivity. youssef, c.b., guillou, v., olmos-dichara, a., 2000. contr. eng. pract. 8, 1297-1307. modelling of the binding of ligands to macromolecules jørgen m. mollerup department of chemical engineering, building 229, dtu, 2800 lyngby, denmark a variety of factors that govern the properties of proteins are utilized in the development of chromatographic processes for the recovery of biological products including the binding and release of protons, the non-covalent association with non-polar groups (often hydrophobic interactions), the association of small ions (ion exchange) and the highly specific antigen-antibody interaction (affinity interactions). the fulcrum point in the understanding and modelling a chromatographic separation is the adsorption isotherm that determines the peak shape at preparative load. to enable an efficient chromatographic process development strategy it is necessary to conduct theoretical and experimental investigations of the adsorptive behaviour of proteins. thermodynamically consistent models for ion exchange chromatography and hydrophobic interaction chromatography have been developed and can be utilised in the simulation of a chromatographic separation. besides, measurements on hic media can be utilised to determine the cohn salting-out coefficient. the lig-and binding process can frequently be coupled to associated structure changes in the protein, the ligand or both. this gives rise to nonlinear adsorptive behaviour known as cooperativity which cannot be modelled using conventional models which displays convex behaviour. examples of cooperative behaviour are the reversible binding of oxygen and carbon monoxide to haemoglobins and the binding of nad + to yeast glyceraldehydes 3-phosphate dehydrogenase. in the paper we discuss the modelling of reversible binding of mobile as well as immobilised ligands to macromolecules and compare modelling to experiment. comparative analysis of the temperature policy for processes with a deactivating native enzyme i. grubecki, m. wojcik department of chemical and biochemical engineering, university of technology and agriculture, 85-326 bydgoszcz, ul. seminaryjna 3, poland a comparative analysis of the temperature policy for an enzymatic reaction with michaelis-menten kinetics in a batch reactor has been carried out. both isothermal and optimal temperature policies for processes with deactivating native enzyme have been considered. in the model, the thermal deactivation was described by a first-order reaction, and the arrhenius-type dependence between rate parameters and temperature was assumed. as an indicator for a direct comparison between the isothermal and optimal temperature policies the quotient of conversions under identical initial and final condition was used. a method was presented to calculate this indicator, which is based on the analytical and numerical solutions. this method can be of great importance for the industrial practice. application of changeable temperature policy could result in significant increase in conversion when ratio of activation energy for deactivation and activation energy for reaction is high. studies on the impact of mixing during brewing using near and mid-infrared spectroscopy georgina mcleod 1 , alvin w. nienow 1 , graham poulter 2 , reg wilson 3 , henri tapp 3 , christopher j. the control of the brewing process is important for improving product quality, and lowering costs. infrared spectroscopy is a technique that can be used at-line and on-line to rapidly measure component concentrations of unprocessed whole broth samples in real time. in this study, both mid -infrared (mir) and near-infrared (nir) spectroscopy have been used and compared for the monitoring of ethanol, flavour components, wort sugars, biomass and specific gravity during the brewing. partial least-squares regression (pls) was used to model relationships between component concentrations and spectra. the performance of these models was evaluated in terms of the standard error of prediction (sep), number of pls factors and the correlation coefficient (r). calibration models were constructed using spectra acquired for multi-component mixtures, intended to simulate brewing fermentation conditions, and actual brewing fermentation samples. chemometric results indicated that nir is a powerful tool for accurately measuring sugar, ethanol and biomass concentrations. the optimization of a fermentation process requires the organism to be cultivated under desirable conditions, which depends on how well the fermentation process is controlled. inadequate mixing and mass transfer are responsible for the heterogeneous environment at large scale in terms of nutrient concentration and ph profile, resulting in lower product yields. these have been associated with, inadequate control of ph and the production of acetate or formate in response to over-feeding of glucose and oxygen deficiency. rapid analyses of substrates and indicator metabolites in a fermentation process is critical for optimal control. this can be achieved in real time with nir spectroscopy. in this study, nir-spectroscopy has been applied to monitor the concentrations of glucose, acetate, formate, ammonium hydroxide and biomass in the cultivation of e. coli (w3110). a comparison of partial least square models built using water standards, synthetic medium standards and fermentation samples has been made. template refolding utilizing biospecific interactions shigeo katoh 1 , yoichi kumada 1 , nanae maeshima 1 , daisuke nohara 2 : 1 graduate school of science and technologykobe university, kobe 657-8501, japan; 2 department of biomolecular sciencegifu university, gifu 501-1193, japan. e-mail: katoh@kobe-u.ac.jp (s. katoh) recombinant proteins over-expressed in e. coli are often accumulated as insoluble particles called inclusion bodies. proteins in inclusion bodies must be solubilized by a denaturing agent, such as urea and guanidine hydrochloride, and refolded to recover their native structures having biological activities. in bioprocesses it is important to obtain high refolding efficiencies and high throughputs at high protein concentrations. in refolding operation, a denatured protein solution is usually added batch-wise into a large volume of a refolding buffer in order to start refolding by reducing the concentration of a denaturant and to prevent aggregate formation of renaturing molecules. thus, a large volume of a stirred tank is required, and the concentration of protein after renaturation becomes low. biointeractions between a pair of biomolecules, such as enzyme-inhibitor, antigen-antibody and hormone-receptor, are highly specific and have been used for detection and separation of biomolecules. these interactions may be used as templates for refolding of target molecules, which can be captured with the templates and are prevented from aggregate formation and, in the case of proteases, from autoproteolysis. the specific interaction might promote refolding of the target molecules. these might improve the refolding efficiency. the biointeractions between antigen-antibody and enzyme-inhibitor were used for efficient refolding in packed columns, in which template ligands (antibody, inhibitor) were coupled on gel support. denatured solutions of target molecules (carbonic anhydrase and s. griseus trypsin) were mixed with refolding buffer and supplied to the affinity column coupled with the template ligands for refolding. with refolding in the column, higher refolding efficiencies were obtained than those by the batch dilution method with relatively low concentrations of denaturants. by increasing the adsorption capacity of the column, throughput of refolding can be increased without decrease in the refolding efficiency. the use of multi-parameter flow cytometry for characterisation and monitoring of insect cell-baculovirus fermentations in a mechanically-agitated bioreactor bojan isailovic 1 , alvin w. nienow 1 , ian w. taylor 2 , ryan hicks 2 , christopher j. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf-21 cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf21 infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp486 coding for am-cyan coral protein, which emits natural green fluorescence. carbon sources create fingerprint fermentation characteristics pınar ç alık 2 , güzide ç alık 1 , tunçer h.özdamar 1 1 bre lab, department of chemical engineering, ankara university, 06100 ankara, turkey; 2 ib lab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: calik@eng.ankara.edu.tr (g. ç alık) this work reports on a systematic investigation of the interactions between the single-carbon sources, i.e., glucose and citric acid, and complex-medium components, i.e., carbon and nitrogen sources, in enzyme fermentation processes, i.e., serine alkaline protease (sap; ec 3.4.21.62),) and ␤-lactamase (ec 3.5.2.6), with the oxygentransfer and ph conditions to demonstrate the influences of carbon sources that create the fingerprint fermentation characteristics, moreover, their influences on the product and by-product formations and the intracellular reaction rates. the influence of the medium composition i.e. citric acid-, glucose-, molasses-and soybean-based media together with the oxygen transfer (ot)-and ph-conditions applied, on the product and by-product distributions and ot characteristics were investigated in batch bioreactors. for sap, in general, under uncontrolled-ph operations the variation of the medium ph in sap fermentation process has a tendency to increase in the sap production phase; however, depending on the carbon source used, its behaviour changes in the early stages of the fermentation as the consequences of the directed functioning of the intracellular bioreaction network. the loci of the dissolved oxygen (do) curves also strongly depend on the carbon source(s) utilised, in addition to the applied ot conditions. the complex media profiles are significantly different compared to the defined media as the ph and do profiles are interrelated owing to the bioreactor operation conditions affecting the metabolic reaction network. the highest volumetric oxygen uptake rates were obtained with soybean-based medium that was ca. three-fold higher than the values reported in citrate-based and glucose based media, and ca. 1.5-2-fold higher than the values reported in molasses-based medium. the significant changes, moreover, the drastic change observed with the use of soybean-based complex medium are due to the compositions of the fermentation media used, and its influence on the intracellular bioreaction network. thus, we conclude that the change in medium composition based on the carbon source changes the fermentation characteristics under the designed bioreactor operation conditions that appear as the fingerprints of the bioprocess. kinetic resolution of racemic benzoin with different lyophilized microorganisms ç . babaarslan 1 ,ü. mehmetoglu 1 , a.s. demir 2 : 1 ankara university, faculty of engineering, department of chemical engineering, 06100, ankara, turkey; 2 middle east technical university, department of chemistry, 06531 ankara, turkey. e-mail: barslan@eng.ankara.edu.tr (ç . babaarslan) the biocatalytic resolution of racemates is valuable tool for enantioselective synthesis and proved to be a convenient method for obtaining optically enriched compounds from their racemic form. in this work, enantiomerically pure benzoin which is one of the 2-hydroxy ketones was synthesized by kinetic resolution of racemic benzoin using different lyophilized microorganisms as lipase sources. the effect of lyophilized microorganism type, solvent type and acyl donor type on enantioselectivity were studied. in kinetic resolution experiments, lyophilized microorganism was resuspended in the media containing solvent, racemic benzoin and acyl donor at 30 • c and 150 rpm on orbital shaker. the reaction was followed by tlc during the experiment and the enantiomeric ratio of benzoin was determined by hplc analysis using chiral cell ob column. the best enantioselectivity value was obtained with lyophilized rhizopus orayzae cbs 112-07 as ee s = 16% and ee p = 30% (conversion = 35%) using thf as solvent and vinyl acetate as acyl donor. otimizing the fermentation broth for tanase production by a new isolated strain paecilomyces variotii vania battestin, gláucia pastore, gabriela macedo department of food science, unicamp, p.o. box 6121, campinas, cep 13083-862, são paulo, brazil tannase is an inducible enzyme that catalyses the breakdown of ester linkages in hydrolysable tannins, resulting in gallic acid and glucose. the fermentation broth can use by-products as wheat bran, rice or oats, adding tannic acid. the use of by products or residues rich in carbon source for fermentation purposes an alternative to solve pollution problems that can be caused by an incorrect environmental disposal. in the present study we have optimized the production of an extracellular tannase by a new isolated paecilomyces variotii using response surface methodology. the first step was identify the variables having a significant effect on enzyme production. the variables evaluated were temperature, residues ratio (coffe: wheat bran), concentration of tannic acid, salt solution during 3, 5 and 7 days of fermentation time. results showed that temperature, residues ratio (coffe: wheat bran) and tannic acid had significant effects on tannase production. commercial wheat bran (cwb) and coffe rusk residues (cr) were used as solid substrate. for fermentation the mediun was composed by, cwb:cr were mixed with distilled water and transferred into 250 ml capacity erlenmeyers flasks and autoclaved at 120 • c for 20 min. the medium was then inoculated with spores (5.0 × 10 7 ) and the flaks were incubated at 32 • c. tannase was assayed according to the methodology of mondal et al. (2001) . acording to the statist analyses, the optimum conditions to produce tannase was the range of temperature (29-34 • c); tannic acid (8.5-14%); residues % (coffe: wheat bran) (50:50) and 5 days fermentation time. the enzyme production increased 8.6 times more enzyme production than that was obtained before this optimization. how to cope with fda's pat-initiative with respect to fermentation process monitoring and control marco jenzsch 1 , andreas luebbert 1 , rimvydas simutis 2 : 1 institute of bioengineering, martin-luther-university halle-wittenberg, halle (saale), germany; 2 process control department, kaunas university of technology, kaunas, lithuania with its pat initiative, fda forces drug manufacturers to increase their activities in innovative manufacturing techniques, and, more than previously, to focus on quality assurance. the agency particularly places emphasis on making use of modern process supervision and control techniques such as up-to-date process analytics, multivariate data acquisition and analysis tools in order to improve process monitoring and control. in this contribution we show by means of practical examples how this guidance can be applied to cultivations of genetically modified microorganisms. a comparison of different multivariate state estimation techniques will be presented and compared with more knowledge-based techniques such as the extended kalman filter. the comparison was made for the model system gfp expressed from e. coli bacteria (bl21/de3/gfp) for which more than 40 full data sets are available. all these techniques have already been used during real protein formation at productionscale fermenters, with the same success. hence, the requirements expressed in the pat initiative can immediately be put into practice. feedback control of the recombinant protein production processes based on such estimations is show for several cultivation systems. simple parameter adaptive controllers are compared with model supported controllers, for instance, generic model controllers and model predictive controllers. the results clearly show that we have at hand a rather extended arsenal of feedback control procedures that can be used successfully to tightly control the processes even along set-point profiles of physiological variables such as the specific growth rate (µ). again, fda's suggestion with respect to "control in the engineering sense" can be applied immediately to reduce batch-to-batch variances and thus to increase process quality. extending life by alternative respiration? alexander kern, franz hartner, anton glieder institute of biotechnology, graz university of technology, a-8010 graz, austria. e-mail: a.kern@tugraz.at (a. kern) alternative oxidase transfers electrons directly from the ubiquinol pool in mitochondria to oxygen, allowing cell respiration in presence of complexs iii and iv inhibitors like antimycin a or cyanide. electron transfer by alternative oxidase is not coupled with proton transfer across the mitochondrial membrane, thereby uncoupling the supply of small metabolic intermediates by the central metabolic pathway from energy production in the cell. alternative oxidase is present in mitochondria of plants, many fungi and a few, mostly crabtree-negative yeasts, but not in p. angusta (hansenula polymorpha) and s. cerevisiae. alternative oxidase has multiple functions in different organisms. it is involved in stress answers, in programmed cell death, maintenance of the cellular redox balance, and also citric acid accumulation in a. niger. we isolated the alternative oxidase gene from the methylotrophic yeast p. pastoris in order to study its effects on the cellular energy content, respiratory activity, its protective role against oxidative stress. our results indicate the importance of an exact regulation of the alternative oxidase due to its impact on many cellular functions. new types of energy efficient fermenters with better mass transport, mixing and cooling properties than the current crop of rushton turbine derived tank bioreactors are likely to be required in the future. such fermenters will be needed in order to meet the increasing pressure on costs for low price commodity type products such as single cell protein or food and technical grade enzymes, and to meet the demands of the new wave of white biotech, in which bio-produced chemicals must be made at prices competitive with those of the traditional chemical industry. with this in mind, a prototype pilot scale (500 l) u-loop fermenter has recently been commissioned at biocentrum-dtu. in this fermenter, liquid circulation is driven by a propeller pump through a vertical u-shaped pipe, which is connected at the top with a de-gassing tank. we present here a study of liquid mixing and dispersion in the prototype u-loop fermenter. sub-sequently we show that the results can be described with the tanks in series model. mixing was characterised using pulses of nacl tracer, which were detected with a conductivity probe in various parts of the fermenter. bodenstein numbers (bo) were determined for flow rates corresponding to a linear fluid velocity of 1.05 m/s in the 'legs' of the reactor and showed that the majority of the mixing occurred in the top degassing part (bo = 29) rather than in the u-loop section (bo = 422). it was also observed that the time for mixing to 90% homogeneity after tracer pulse addition was a function of the number of cycles through the reactor (3-3.5) within the range of flow velocities (u) studied (u = 0.41 m/s to u = 1.74 m/s). the mixing time to 90% homogeneity was between 44.6 s (at u = 1.74 m/s) and 181 s (at u = 0.41 m/s). today many biotechnological processes are operated at suboptimal conditions and according to best practice. however, the current industrial development is towards analyzing more parameters and in particular there is a large interest in analysis of biological/biochemical variables. the quality of products and also the possibility to optimize production in submerged cultivations would be greatly enhanced if more on-line/real-time information were at hand. the present investigation was undertaken with the aim of evaluating the potential in using multi-wavelength fluorescence for monitoring and control of filamentous fungi fed-batch cultivations. a recombinant a. oryzae expressing a heterologous lipase was applied as model system. spectra of multi-wavelength fluorescence were collected every five minutes with the bioview ® system (delta, denmark) and both explorative and predictive models, correlating the fluorescence data with the important biological parameters cell mass and lipase activity, were built. the models will be presented, furthermore, advantages and disadvantages of multiwavelength fluorescence for monitoring of cultivation processes will be discussed. moving from r&d to pharmaceutical development is a costly process. it is therefore of paramount importance to design a manufacturing process that combines robust and well-documented technological platforms. therapeutic recombinant proteins designed for human administration should be as close to the authentic product as possible. here, the use of a scalable process and an economically sound affinity tag can be a relevant choice. the tagzyme tm system has been designed to allow for the precise removal of amino terminal affinity tags. the system is based on the use of recombinant aminopeptidases including dipeptidyl peptidase i (dapase tm ). dapase tm is currently produced under cgmp providing a suitable strategy for its use in pharmaceutical production. the tagzyme tm system is superior to other methods since: (1) it is based on exopeptidases, precluding, e.g., unspecific protein cleavage reported when using so-called site-specific endoproteases. (2) it has been tested for production of more than 200 recombinant proteins. (3) it is easily scalable from lab scale to kg of processed protein. (4) it allows the use of his-tags for commercial production without patent infringment, due to our ipr position. (5) the commercial use of tagzyme tm does not require any licensing, only purchase of the enzyme(s). (6) the use of aminopeptidases for pharmaceutical production has been extensively documented for approved drugs. (7) a number of therapeutics is currently being developed using tagzyme tm . (8) unizyme can assist in the optimization of the dsp to enable further cost reduction in the process. these aspects will be discussed and illustrated in the presented poster. website sphingolipids are biologically active molecules involved in the regulation of a large quantity of biological responses. they function in cell proliferation, survival and death (apoptosis) as messengers. dysregulation of apoptosis has significance in numerous pathological conditions including cancer. several anticancer agents act by increasing tumor cell ceramide (a kind of sphingolipid) content. so, a novel approach to cancer therapy would be the pharmacological manipulation of sphingolipid metabolism. in this study, sphingolipid metabolism in baker's yeast s. cerevisiae is used as a model system as many of its sphingolipid related genes and proteins have been characterized. gepasi-biochemical kinetics simulator was used for metabolic control analysis (mca) of the above-specified system. the concentration control coefficients (ccc), flux control coefficients (fcc) and elasticity coefficients were calculated, and their significance in identification of anticancer drug targets is determined. elementary flux modes were also identified and metabolic pathway analysis (mpa) was performed. quantitatively, control effective flux (cef) values were used for potential drug target identification. the results from mca and mpa indicate almost the same potential drug targets: serine palmitoyl transferase, ceramide synthase and ceramidase. drugs against these targets are in preclinical and clinical development. for the identification of new potential drug targets, the cccs, fccs, cefs and elasticity coefficients were examined with an objective function of maximizing the cell ceramide concentrations. it was found that manipulation of inositol-1-phosphate synthase and phosphoinositide kinase activities have considerable effects on ceramide concentrations. if a drug targeting the two enzymes at the same time is designed, it might give a better outcome in terms of cancer therapy. in recent years, there is a growing interest in utilization of airlift reactors (alrs) to biotechnological processes. nevertheless, their industrial application still remains limited because of a lack of reliable studies on transfer phenomena and mixing enabling a suggestion of suitable scale-up procedure. the way to more widely utilization of alrs to biological processes lies in experimental research (on a model medium as well as on a real fermentation medium) followed by mathematical modelling and scaling-up of the processes. this paper deals with a modelling of a glucose-gluconic acid fermentation by a. niger in an internal loop airlift reactor. knowledge of the stoichiometric relationship in the key reaction provides a good opportunity for estimation of substrate and product concentration. the model is based on material balance equations and has been adjusted to experimental data obtained from three internal loop airlift reactors (10.5, 40 and 200 l) . in the model, the alr is divided into ideal stirred tanks in series. in each zone (tank) of the alr the material balance is calculated in two phases (the gas and the liquid phase). this work was supported by the slovak scientific grand agency, grant number vega 1/0066/03 alkaline phosphatase (ap, e.c. 3.1.3.1) is a thermolabile enzyme which is indigenous to all dairy products. it has an inactivation temperature slightly above the value that is required to destroy the most resistant pathogenic microorganism likely to be found in milk. due to that feature, this enzyme is used as an indicator of proper pasteurization. the effect of temperature treatment on the activity of ap was investigated in raw cow's and goat's milk. the stability of alkaline phosphatase in raw milk was compared with the stability of this enzyme in a 0.1 m potassium phosphate buffer with ph 6.6. the ph value of the buffer was approximately the same as that of raw milk. the inactivation curves were measured in the temperature range from 54 to 69 • c. ap in cow's milk was completely inactivated at 69 • c during 60 s but approximately 30% of activity remained at 54 • c after 100 min of treatment. the time required for a complete inactivation of the enzyme in the raw cow's milk was reduced from 90 to 1 min as the temperature increased by 11 • c. heat treatment of goat's milk caused the decrease of activity of the enzyme in the same temperature range as in the case of cow's milk. the increase of temperature from 58 to 68 • c reduced the inactivation time from 35 min to 40 s. the study of thermal stability of the alkaline phosphatase in the buffer solution showed that the time required for inactivation of enzyme was significantly shorter than in milk. milk thus had a protective effect on the activity of alkaline phosphatase. the experi-mental curves were fitted simultaneously using kinetic models where the initial heating period was considered. this work was supported by a grant of 6th framework program of eu, project foodpro, no. sme-2003-1-508374. during the process of separation, purification and concentration of monoclonal antibodies (mabs) at industrial scale, the chromatographic unit operations have an important role. three different protein-binding modes are employed: ion-exchange, hydrophobic and affinity binding. two adsorbent properties are of uppermost importance: a high selectivity and adsorption capacity. in the case of ion-exchange/hydrophobic chromatography, the binding of charged proteins can be affected by ph and ionic strength. in this work, the adsorption capacity of eight commercially available adsorbents designed for separation of mabs (mabselect, rprotein a sepharose ff, poros 50a, prosep-va, fractogel emd se hicap (m), sp sepharose ff, mep hypercel, s ceramic hyperd f) was measured as a function of ph. as a model mab and contaminant proteins, human immunoglobulin (igg), human serum albumin (hsa) and horse skeletal muscle myoglobin (myo) were used. the resin properties were investigated within the range of ph 4-8. the experiments were conducted in a batch-mode, individually for each model protein. the results showed that ion-exchange and hydrophobic resins provided the best selectivity for igg at ph 6. the selectivity of affinity adsorbents was essentially unaffected by ph, however, the highest capacity for igg was at ph 7. another investigated aspect was the dynamics of protein binding. the solution of individual protein in contact with tested adsorbent was circulated through an uv spectrophotometer, what enabled the measurement of time-dependent decrease of protein concentration. the results indicated that affinity adsorbents with a rigid matrix needed approximately four times shorter time to reach the adsorption equilibrium with igg in comparison with a gel. the gels, however, provided higher adsorption capacity. at ion-exchange resins, the time necessary to adsorb 99% of total amount of igg was about 0.5-2 h. the affinity adsorbents were highly selective and therefore they adsorb very small amount of tested contaminant proteins (hsa, myo). the adsorption capacity was saturated by 50% in less than 25 min in all cases of dynamic adsorption measurements. this work was supported by a grant of 6th framework program of eu, project aims, no. nmp3-ct-2004-500160. microtechnology has for several years been applied within chemical reaction engineering. the advantages of microtechnology are that it makes it possible to develop light weight and compact systems, and the systems enable large surface-to-volume ratio, which results in low mass-transfer distances. in addition, parameters like pressure, temperature, residence time, and flow rate are more easily controlled. the use of microtechnology is also beginning to find its ways into the field of biotechnology. what we are aiming at is the development of a microreactor that can be applied as a production tool in industry as an alternative to conventional enzymatic reactors. our strategy is to use a small plate of a suitable material with microchannels fabricated into its surface, and the approach is to covalently couple enzymes into the microchannels. substrate can then be pumped through the channels and the enzymatic conversion will take place within the channels. as model enzyme in the development of the microreactor we are applying celb, a thermostable ␤-glycosidase from pyrococcus furiosus. kinetic resolution of racemic benzoin with different lyophilized microorganisms ç . babaarslan 1 ,ü. mehmetoglu 1 , a.s. demir 2 : 1 ankara university, faculty of engineering, department of chemical engineering, 06100, ankara, turkey; 2 middle east technical university, department of chemistry, 06531, ankara, turkey. email: barslan@eng.ankara.edu.tr (ç . babaarslan) the biocatalytic resolution of racemates is valuable tool for enantioselective synthesis and proved to be a convenient method for obtaining optically enriched compounds from their racemic form. in this work, enantiomerically pure benzoin which is one of the 2-hydroxy ketones was synthesized by kinetic resolution of racemic benzoin using different lyophilized microorganisms as lipase sources. the effect of lyophilized microorganism type, solvent type and acyl donor type on enantioselectivity were studied. in kinetic resolution experiments, lyophilized microorganism was resuspended in the media containing solvent, racemic benzoin and acyl donor at 30 • c and 150 rpm on orbital shaker. the reaction was followed by tlc during the experiment and the enantiomeric ratio of benzoin was determined by hplc analysis using chiral cell ob column. the best enantioselectivity value was obtained with lyophilized rhizopus orayzae cbs 112-07 as ee s = 16% and ee p = 30% (conversion = 35%) using thf as solvent and vinyl acetate as acyl donor. chromatography is one of the most important unit operations at separation, purification and concentration of monoclonal antibodies (mabs) at industrial scale. since these proteins belong to the group of immunoglobulins, their molecular weight (about 150,000 g/mol) or hydrodynamic radius (10.7 nm), respectively, is relatively large. the adsorbents used in ion-exchange/affinity chromatography of these biomolecules should thus provide a high pore accessibility coupled with a high value of specific surface area in order to ensure a sufficient ligand density and a high binding capacity. in this study, the pore accessibility of eight commercially available adsorbents designed for separation of mabs (mabselect, rprotein a sepharose ff, poros 50a, prosep -va, fractogel emd se hicap (m), sp sepharose ff, mep hypercel, s ceramic hyperd f) was investigated via size exclusion of standard-sized molecules (glucose, sucrose and dextrans with molar weight range 1200-40 × 10 6 g/mol) at nonbinding conditions. the experiments were conducted in a batch and column-mode. the batch experiments provided absolute partition coefficients, which were calculated from a mass balance and represent the fraction of pore water accessible to a solute. it was found that several adsorbents contained a small fraction of very small pores (less than 1 nm), whereas some adsorbents contained a significant fraction of pores larger than 100 nm. the column measurements provided relative partition coefficients, which were calculated from the retention volumes of solutes and represented the relative accessibility of pores, scaled between the accessibility of the smallest and largest solute used. when the absolute partition coefficients were recalculated into the relative form, it was found that the coefficients obtained by both methods correlated very well. the relative partition coefficients of solutes with the hydrodynamic radius of 10 nm (corresponding to mabs) was about 0.2-0.4 at ion-exchange and hydrophobic adsorbents and 0.6-0.8 at affinity adsorbents. the relation between hydrodynamic radius of the solutes and their partition coefficients was successfully described with the giddings random plane model. this work was supported by a grant of 6th framework program of eu, project aims, no. nmp3-ct-2004-500160. the transfer of laboratory results to a larger scale is often a critical step in process development to industrial application. the objective of this study was to scale-up the bioreactor for the fructosyltransferase production based on the results obtained in a 3 dm 3 stirred bioreactor. the investigations made in this bioreactor provided a clear picture of the effect of medium composition on the obtained fructosyltransferase (ftase) activity but the influence of mixing intensity was unequivocal. the increase of agitation rate had a positive effect up to a certain level where both fructosyltransferase and biomass production increased. the final optimal yield factor of ftase per dry cell mass obtained in the laboratory bioreactor was 10,300 u g −1 . we studied the effect of oxygen transfer on the process of ftase production at a larger scale, in 12 and 100 dm 3 mechanically stirred bioreactors and in air-lift bioreactors, 20 and 60 dm 3 whilst the medium composition was kept constant. the yield factors of ftase were comparable in both mechanically stirred bioreactors and they were about 7500 u g −1 . this decrease compared to that in the laboratory bioreactor could be explained by a slower cell growth. this fact was also confirmed by that glucose was not depleted till the end of fermentation and free fructose concentration was also lower. the yield factor of ftase was 7400 u g −1 in the 60 dm 3 air-lift reactor and 6100 u g −1 in the 20 dm 3 air-lift bioreactor. the lower yield of ftase in 20 dm 3 bioreactor was caused by a better biomass growth. this work was supported by slovak scientific grand agency, grant numbers vega 1/0066/03 and 1/0065/03 and by science and technology assistance agency, grant number apvt-20-025704. the poster gives an overview of the objectives and achieved results of an interdisciplinary project on direct product isolation from crude feedstocks using magnetic micro adsorbents in combination with suitable magnet technology. the project was funded by the deutsche bundesstiftung umwelt and was running between august 2002 and november 2004. in the course of the project several milestones could be met, which can be looked at as critical key points on a route towards an industrial realization of the process. among these milestones are: (i) the production of magnetic micro adsorbents with high capacity and selectivity in batches up to 50-100 g; (ii) the proof that the micro adsorbents can be reused many times; (iii) generation of a variety of recombinant tagged, active enzymes; and (iv) the design, assembly and operation of a fully automated pilot plant capable of generating approx. 5 g/h (≈95% purity) protein. the process was also simulated by help of the software tool superpro designer and simple mass balance and sorption equilibrium approaches were used to derive rules for estimating optimum process parameters and productivities. finally an environmental performance evaluation was conducted externally by the german dechema. in this study the effect of fed-batch cysteine addition to a culture of a high-gsh-accumulating yeast strain on the metabolism of glutathione was investigated. it is known that cysteine is the rate limiting amino acid in the biosynthesis of gsh. the influence of the consumption rate of cysteine on glutathione metabolism and growth of s. cerevisiae mt-32 was determined. the results show that for rates of consumption below a critical value the microorganism growth is similar to a culture without feed of cysteine, but glutathione production is increased two-fold. on the other hand, if cysteine consumption rate is above the critical value the changes of cell metabolism implies ethanol accumulation in the extracellular media which diminishes biomass synthesis. the maximum specific glutathione production in this case is maintained at two-fold; however, gamma-glutamylcysteine accumulation is increased. cysteine present in culture media directs cell metabolism to a greater synthesis of ammonia and amino acids. hydrophobic interaction chromatography (hic) exploits the hydrophobic properties of protein surfaces for separation and purification by performing interactions with chromatographic sorbents of hydrophobic nature. in contrast to reversed phase chromatography this methodology is less detrimental to the protein and is therefore more commonly used in industrial scale as well as in bench scale when the conformational integrity of the protein is important. hydrophobic interactions are promoted by salt and thus proteins are retained in presence of a cosmotropic salt. when proteins are injected on hic columns with increasing salt concentrations under isocratic conditions only, a fraction of the applied amount is eluted. the higher the salt concentration the lower is the amount eluted protein. the rest can be desorbed with a buffer of low salt concentration or water. it has been proposed that the stronger retained protein fraction has partially changed the conformation upon adsorption. this has been also corroborated by physicochemical measurements. the retention data of five different model proteins and 10 different stationary phases were evaluated. partial unfolding of proteins upon adsorption on surfaces of hic-media were assumed and a model describing the adsorption of native and partial unfolded fraction was developed. furthermore we hypothesize that the surface acts as catalyst for partial unfolding, since the fraction of partial unfolded protein is increasing with length of the alkyl chain. stationary phases for bioseparation of glycoproteins j. aniulyte 1 , j. liesiene 1 , b. niemeyer 2 : 1 department of chemical technology, kaunas university of technology (ktu), radvilenu pl. 19, 50254 kaunas, lithuania; 2 institute of thermodynamic, helmut-schmidt-university/university of the federal armed forces hamburg, holstenhofweg 85, hamburg, germany d-22043 nowadays glycomics raise new challenges for affinity chromatography related with an abundance of glycoconjugates in living organisms and with scaling-up of the preparative processes. economics, efficiency and practicality dictate the search of novel chromatographic biospecific adsorbents that could contribute to enhancing the productivity of the affinity separation process. the purpose of the work was to prepare cellulose-and silica-based biospecific adsorbents with immobilized lectins and to evaluate them for the affinity chromatography of glycoproteins. cellulose-based matrix granocel and silica coated with hydrophilic polymers were used as a support. the effect of support characteristics, such as pore size, chemistry of active groups and their density on the support' surface on lectin immobilization and on the efficiency of adsorbents obtained were evaluated.three different methods were used for the activation of the support: oxidation with sodium periodate, modification with pentaethylenhexamine (spacer arm of 18 atoms) and carbonyldiimidazole activation.cona and wga two lectins of different molecular weight and shape were selected to notice differences resulting from the size and diffusion behaviour. chromatographic performances of the adsorbents were studied applying two different glycoproteins (god and fetuin) carrying specific terminal glycomoieties of mannose (god), and n-acetylglucosamine (fetuin) for specific interaction with cona and wga, respectively. the adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to 7.4 mg per ml support and a high recovery (up to 93%). it was shown that spacer arm affected ligand coupling kinetic as well as the chromatographic behavior of the adsorbents obtained. the adsorption isotherms of god onto cona adsorbents reveal an adsorption behavior with high and low affinity binding sites. the dissociation constant k d of the ligand-sorbate complex is approximately 1 × 10 −6 m, and 0.4 × 10 −5 m, respectively. it was supposed that the second step is related to the sorption of solvated god onto already adsorbed god forming sorbate dimers. cell disruption and chromatography are key unit operations in the downstream processing of an intracellular product. the cost involved in the extraction and purification of intracellular products can be reduced by selective release of proteins and reduction in the number of steps involved in the purification. the extent of disruption can be varied to provide a selective release, limiting the release of the contaminant proteins. the particle size distribution of the cell debris in the resulting suspension depends on the extent of disruption. expanded bed adsorption chromatography allows for the direct capture of the proteins from an unclarified suspension. this technique allows for the integration of solid-liquid separation, concentration and preliminary purification in one unit operation. a perfectly stable expanded bed can be obtained by choosing the appropriate flow conditions and a suitable adsorbent. the difference in the density between the adsorbent and the cell debris in the suspension, permits the cell debris to flow through the column without blocking, whilst the protein molecules in the suspension are adsorbed onto the adsorbent. after sample application, the bed is washed with buffer and the proteins eluted from the column in the packed bed mode. the presence of the cell debris in the feedstock influences the expansion of the bed and the adsorption of protein molecules. the physical properties of the suspension obtained after cell disruption depends on the extent of disruption. the particle size distribution of the cell debris, the viscosity and the release of soluble proteins and other intracellular components are influenced by the extent of disruption. the influence of the extent of disruption of e. coli on the expansion of the bed and the adsorption of ß-galactosidase is presented in the current study. e.coli cells were disrupted at different operating pressure using a high pressure homogenizer. the resulting crude homogenate is subjected to expanded bed adsorption chromatography using streamline deae as adsorbent. the disrupted suspension was characterised in terms of viscosity, density, particle size distribution of the cell debris and the extent of protein and ß-galactosidase released. the interaction between cell debris and adsorbent was quantified as the cell transmission index (ratio of the amount of cells present in the sample before and after passing through the bed). the expansion of the bed at a constant settled bed height and flow rate was measured. the influence of the cell debris on the extent of adsorption of ß-galactosidase has been quantified in terms of dynamic binding capacity (dbc) at 10% of the inlet concentration. the dbc of ß-galactosidase that was released by disruption at 7500 psi (5%, w/v, w/w, 1 pass) was found to be 100 u/ml of adsorbent while dbc of samples disrupted at 2500 psi (5% w/v, w/w, 1 pass) was 67 u/ml of adsorbent. the extent of disruption of e. coli over a wide range and its effect on the expansion and adsorption will be presented. study of dna binding during expanded bed adsorption and factors affecting adsorbent aggregation ayyoob arpanaei 1 , niels mathiasen 1 , timothy hobley 1 , owen rt thomas 1,2 : 1 center for microbial biotechnology, building 223, biocentrum-dtu, technical university of denmark, 2800, lyngby, denmark; 2 department of chemical engineering, university of birmingham, edgbaston, b15 2tt, uk. e-mail: aa@biocentrum.dtu.dk (a. arpanaei) the adsorption of sonicated calf thymus dna (as a model dna molecule) to biosepra q hyper z adsorbents was evaluated in batch and expanded bed modes. stability of the expanded bed during feedstock loading was also studied. two batches of prototype q hyper z (batch 1 and 2) were examined, which had ionic capacities measured to be 122 and 147 mmol cl − /ml support respectively. in all adsorption experiments a 50 mm tris-hcl ph 8 buffer was used. maximum static binding capacities of adsorbent batches 1 and 2 were determined to be 20.9 and 23.8 mg dna/ml particle, respectively. dynamic binding capacity at 10% breakthrough (dbc 10% ) was measured in a 1-cm diameter eba column containing 6.7±0.5 cm settled bed with a feed of 20 g/ml dna. dbc 10% of the adsorbents were 7.4 and 12.7 mg dna/ml support for batches 1 and 2, respectively in buffer containing no salt. however, the maximum dbc 10% for batch 1 (10.1 mg dna/ml support) and 2 (18.9 mg dna/ml support) were obtained in buffers containing 0.25 and 0.35 m nacl, respectively. further increases in salt concentration led to a decrease in dbc 10% for both adsorbent batches. the bed compression during loading that was observed in experiments at high conductivities (achieved by adding salt) was less than that seen with low conductivity (2 ms/cm) solutions. aggregation of adsorbent particles and channeling of flow were not observed in the presence of salt concentrations more than 0.1 m. the effect of different concentrations of dna during loading in the presence of 0.15 m nacl was studied. it was found that increasing dna concentrations in the feed from 20 to 40 g/ml, 60 to 80 g/ml resulted in a decrease of dbc 10% by 16, 24 and 30%, respectively. the bed compressed slower during loading of feedstock with low dna concentrations compared to that for higher concentrations. the expanded bed showed a partly reversible compression behavior during feedstock loading. this is attributed to the electrostatic interaction between dna adsorbed on the particles surface and rearrangements of dna strands as the number of free ligands on the adsorbent surfaces decrease during loading. large-scale production of plasmid dna for gene therapy and dna vaccination applications has become necessary as a result of the increasing number of approved protocols using non-viral vectors for gene delivery. a major challenge of large-scale plasmid production is to establish a robust cgmp manufacturing capable of producing hundreds of milligrams or grams of a pharmaceutical grade product. alcohol and salt precipitation are operations largely used in the early steps of plasmid downstream processes. however, there are few systematic studies on the influence of these precipitation agents in the final plasmid recovery and purity. in this work, alcohol and salt precipitation steps used in a plasmid purification process developed by our group have been optimized aiming at large-scale production. the optimization of alcohol precipitation indicated that almost 100% of the pdna precipitated when 0.6 vol. of isopropanol were used. the studies also indicated that the precipitation profile was strongly influenced by pdna initial concentration. finally, the final plasmid recovery and purity after a sequential alcohol and salt precipitation were strongly dependent on the concentrations of these precipitation agents. thus, a commitment between high recovery and purity level should be made during the development of the downstream processes. comparison of novel and conventional processes for protein refolding and initial purification h. ferré 1,2 , u. jørgensen 1 , l. scale down of downstream processing unit operations is convenient for assessing process alternatives, particularly if feedstock is scarce. in this study it was imperative to use the smallest possible scale for comparison of a new system for continuous protein refolding and direct expanded bed adsorption (eba) capture with a traditional process composed of discrete operations of batch renaturation, centrifugation, microfiltration and packed bed chromatography (pbc). minimisation of the scale was restricted by the eba step: the smallest practical scale being a 1cm diameter column with 5-6 cm of settled bed, expanded two fold. in order to permit a fair comparison a similar column diameter and adsorbent volume was used in the packed bed process. in both alternatives, chelating media charged with cu 2+ was used and a feedstock of denatured hat-tagged human beta-2 microglobulin (hat-h␤2m). following batch refolding and clarification, the performance of the packed column was severely hampered due to fouling of the top adapter. reducing the protein loaded to the packed bed to 50% of dbc working lead to a recovery of 68.5% at a purity of 87% and 4.6-fold concentration. the eba-based process performed unimpeded and productivity was calculated to be 8% higher than for that employing a packed bed. however, due to the severe scale restrictions placed on the eba process, which limited optimisation, significant productivity improvements of eba over packed bed are expected at larger scale. high gradient magnetic filtration has the potential for rapid processing of large volumes of crude bioprocess liquors when magnetic adsorbents are employed. the binding of a protein to a superparamagnetic solid support provides a unique selective 'handle'. typically the focus is placed on using the magnetic handle for direct capture of a protein from a fermentation broth. however, magnetic adsorbents may provide solutions to a range of downstream processing problems and in this presentation we illustrate this with a number of case studies. using whey as a model system, we show that the extent of the tryptic hydrolysis (ca. 0.2 mg/ml added enzyme) of proteins could be controlled by adding benzamidine-linked magnetic adsorbents after a given period (4-15 min), followed by removal of the loaded adsorbents using a magnetic filter. hydrolysis was stopped effectively and approx. 50% of the added trypsin could be recovered. a coupled process was devised for the refolding and purification of inclusion body proteins. solubilised (in 8 m urea) inclusion bodies of recombinant histidine affinity tagged human beta 2 microglobulin (hat-h-beta2m), were refolded by dilution in a pipe reactor (14 s), then captured directly on cu(ii) charged magnetic immobilised metal affinity adsorbents in a second pipe reactor (10 s residence time). loaded adsorbents were retained in a magnetic filter, then washed and the protein eluted. a generic framework for the prediction of scale-up when using compressible chromatographic packings r. tran 1 , j. joseph 1 , a. sinclair 2 , y. zhou 1 , n. packed bed chromatography is the pre-eminent technique in the downstream purification of many biological products. the aspect ratio of a packed bed has a significant effect on the column pressure drop by virtue of wall support which is reduced at low aspect ratios. this can result in unexpectedly high pressures during manufacturing caused by the compression of the matrix via drag forces due to fluid flow through the bed. the need for an accurate model to predict flow conditions at increasing scale is essential for the scaling-up of chromatographic processes and for avoiding bed compression during operation so that maximum throughput can be achieved. several studies have generated correlations which allow for the prediction of column pressure drops but have either been mathematically complex, which makes their practical use unfeasible, or they have used highly specific empirical constants and hence require a large amount of experiments to be performed before they can be used. in this study, we have established relationships to link the critical velocity of operation, to bed height (l), column diameter (d c ), feed viscosity (µ) and also to the matrix rigidity through the level of agarose cross-linking (a%). the correlation is straight forward to use and involves very few system-specific constants thus significantly reducing the need for any preceding laboratory-scale experimentation. this paper describes the series of experiments that were performed to establish the correlation, using a range of cross-linked agarose matrices (2-10%), at various aspect ratios, fluid flow rates and varying viscosities (0.9-1.85 mpas). a mathematical model was developed where parameter estimation for multi-variables was achieved by least squares optimisation. the model can be used to predict the extent of compression in industrial chromatography applications and will be useful in the development of chromatographic operations and for column sizing. institute of process engineering, swiss federal institute of technology, zurich. e-mail: makart@ipe.mavt.ethz.ch (s. makart) simulated moving bed (smb) technology receives increasing attention in biotechnology and in the biopharmaceutical industry as it enables an increase in productivity per unit mass of stationary phase, reduced solvent consumption, and fast and reliable scale-up. combining continuous chromatographic separation unit and reactor should enable the production of biopharmaceuticals and fine chemicals with high purity and yield at the same time. due to the increasing demand of enantiopure intermediates in the pharmaceutical industry, biocatalytic processes gain more and more importance because of their excellent enantioselectivity. yet the application of biocatalytic carbon-carbon bond formation on process scale is often hampered by an unfavourable equilibrium position and difficult downstream processing due to substrate/product mixtures. coupling a continuous separation unit to such a process would improve the feasibility by driving the reaction to completion and thus increasing the overall yield. we will discuss the design of such an integrated biocatalytic/smb process, taking the formation of l-allo-threonine from glycine and acetaldehyde, catalysed by the glycine-dependent aldolase glya from e. coli, as a model reaction. the enzyme exhibits absolute stereoselctivity at the c-alpha atom, whereas selectivity is less strict at c-beta. in situ product removal, by the integration of an smb unit, would aid to maintain a high diastereomeric excess as it shortens the residence time of the products in the reactor, in addition to shifting the reaction to the product side. the in-line coupling of the chromatographic unit to the enzyme reactor requires the use of the same solvents for reaction and separation, so the choice is limited to aqueous solutions close to physiological ph, limiting in turn the possible stationary phase materials. in a screening of different cation exchangers, amberlite cg-120 ii gave promising results: threonine is more retained than glycine, acetaldehyde is poorly and the cofactor plp is not retained. adsorption isotherms were determined by the retention time method and a smb under process conditions was simulated. by improving the packing of the column, i.e. achieving a more even particle size distribution, we tried to further increase the efficiency of the separation step. the application of enzymes for the synthesis of optically active substances is nowadays of growing importance in the pharmaceutical industry. this requires a proper cultivation of the microorganism as well as a posterior isolation process yielding a constant catalyst quality at high purity. goal of this project is the development of an integrated process for the production and isolation of a lipase from trichosporon beigilie and its posterior application for the enantioselective synthesis of pharmaceutical products. the cultivation of the microorganism is optimised in a laboratory and pilot-scale fermenter in a fed-batch mode. parameters like media composition, temperature, ph and aeration rate are set up. taking advantage of the localisation of the enzyme (covalently attached to the cell membrane) the first step of product isolation consists of a continuous cell disruption. optimal results are achieved with the continuous bead mill disruption process (68% enzyme release with a specific activity of 0.8 u/mg of protein). the non disrupted cells are recycled as inoculums for a new cultivation, increasing the yield of the overall process (by 5-times in the pilot-scale fermenter). in order to isolate the product two different process sequences are considered. the first one consists of an extraction (peg and phosphate buffer) coupled to an ion-exchange chromatography (q-sepharose ff). the second one applies a precipitation step with ammonia sulphate followed by a hydrophobic interaction chromatography (sepharose-hic) provid-ing a lipase yield of 72% (8-times higher than the one provided by combining extraction-chromatography). an ultrafiltration process is used in order to concentrate the lipase and its final properties (molecular weight, isoelectric point, activity, stability and kinetic data) are studied using p-nitrophenylacetate as model substrate. the relevance of the obtained product for its application in the pharmaceutical industry is proven by transforming (r,s)-naproxen-methylester into (s)-naproxen acid with an enantiomeric excess of >99% (after 24 h). biotensides (sugar fatty acid esters, sfaes) find nowadays a wide range of applications in pharmaceutical, personal care and food industry because of their biocompatibility, biodegradability and special surfactant properties. goal of this project is the development and optimisation of an integrated process for the enzymatic synthesis of sfaes from renewable sources to be used in cosmetic formulations. the following figure shows the scheme of the overall process. commercial and also new screened lipases are applied in the reaction between sugar and fatty acid. the mixture grade of the initial reaction system is increased by ultrasounds taking into account the influence on the catalyst characteristics and also the necessity of an organic solvent as adjuvant. the reaction takes place in an enzymatic membrane reactor (emr) equipped with an ultrafiltration membrane which retains the catalyst. the separation of the by-product (water) from the rest of the components can be achieved by means of a pervaporation unit which coupling to the emr allows the semi-batch process. in order to separate the esters from the fatty acid a stepwise elution chromatography method is developed using silica as adsorbent and ethyl acetate and methanol as eluents. with this system 91% of the dimer is isolated with purity (hplc) of 93%. the application of a dialysis membrane technique allows the separation of 80% of the fatty acid by building ester micelles changing the polarity of the organic solvent used as eluent. solubility and crystallisation properties of recombinant bacillus halmapalus ␣-amylase cornelius faber centre for microbial biotechnology, biocentrum dtu, building 223, 2800 lyngby, denmark a comprehensive knowledge of solubility properties is a prerequisite for the efficient design and operation of bulk enzyme recovery processes, however, complete phase diagrams are only available for very few proteins, in particular lysozyme of high purity. here, we present the results of detailed solubility studies in aqueous solutions of an industrially relevant ␣-amylase of technical grade. experiments were conducted in small scale batch mode (working volume of 1 ml). the influence of selected cations and anions from the hofmeister series on the stability of the ␣-amylase was examined. the hofmeister series for anions was followed in the correct order at all salt concentrations studied, i.e. from 0 to 1 m, whereas the series was reversed for monovalent cations at concentrations up to 0.5 m, with the exception of lithium. to further investigate why the position for lithium was different to the hofmeister series established for lysozyme, the zeta potential of protein solutions at low concentrations of selected salts was determined. the results of these measurements indicate a pronounced effect of lithium on the zeta potential, as compared to other salts. in particular, the ph of zero zeta potential (i.e. the pi) was shifted approximately 0.5 ph units towards alkaline conditions in the presence of lithium, whereas the pi stayed almost constant for sodium and potassium. since the solubility exhibits a minimum at ph-values at or near the protein's pi, shifts in ph caused by salt addition are important to identify and quantify to avoid uncontrolled phase separation. the measurement of the zeta potential of proteins in solution holds significant promise as an attractive tool for understanding and controlling processes that are operated close to the solubility limit and which are often plagued by uncontrolled precipitation or crystallisation and thus rely on carefully chosen operating conditions. polyphenolic interactions with potato proteins during industrial expanded bed adsorption processing sissel løkra 1 , knut olav straetkvern 1 , bjørg egelandsdal 2 , gerd vegarud 2 : 1 department of natural science & technology, hedmark university college, n-2317 hamar, norway; 2 norwegian university of life sciences, 1432 as, norway. e-mail: sissel.lokra@lnb.hihm.no (s. løkra) in plant extracts it has been shown that polyphenols have a tendency to react with proteins, either by covalent or non-covalent interactions. these reactions can induce changes in the surface properties of the proteins, and, e.g. cause proteins to be insoluble and precipitate at ph-values below their isoelectric point. potato proteins have a high nutritional quality and show interesting functional properties in food systems. moreover, chlorogenic acid (ca) and caffeic acid constitute about 90% of the total polyphenol content of potato tuber. we have experienced expanded bed adsorption (eba) chromatography to be a method well suited for recovering industrial proteins from potato starch effluent. the process separates proteins from polyphenolic pigments, fiber and minerals. during the adsorption step, patatin, the major potato tuber protein shows complex binding kinetics demonstrated by breakthrough curves. in addition to diffusion limitations in the eba resin, changes in protein structure and surface properties probably are likely to affect this adsorption behavior. reactions between ca and patatin might result in a range of interactions for different species of the same protein. this project therefore aims to assess the interactions between ca, patatin and other major tuber protein fractions and how these changes affect the protein capture in eba. changes in size and charge are screened in 2-d electrophoresis and analyzed further. samples of different protein fractions are taken from breakthrough curves and dynamic binding capacity experiments in model systems with real feedstock. sandwich hybridisation assay for analysis of brewery contaminants s. huhtamella 1 , m. leinonen 1 , t. nieminen 1 , a. breitenstein 2 , p. neubauer 1 : 1 bioprocess engineering laboratory, university of oulu, finland; 2 scanbec gmbh, halle, germany. email: peter.neubauer@oulu.fi (p. neubauer) here we describe the development of a sensitive, cultivationindependent analytical method for the analysis of brewery contaminants which can be performed within three hours in crude sample extracts. the method is based on 16s rrna detection by a paramagnetic bead based sandwich hybridization assay (sha) with two oligonucleotide probes designed to either detect the species or a group of contaminants. the signals were read out either by a fluorimeter (rautio et al., 2003; leskelä et al., 2005) or potentiometrically with an electric biochip instrument (ebiochip systems) . this assay is advantageous over rt-pcr becasue it only detects viable cells and the method can be directly applied to crude cell extracts without prior purification. we describe the principle of designing and evaluating a series of groupspecific lactobacillus probes and the optimisation towards effective cell lysis and high assay sensitivity. the applicability of the sha was evaluated with real brewery samples and the results were compared to routine tests. in all steps of the evaluation the reliability and usability of the method was prioritised. the optimised method combined with a 24 h pre-enrichment period gave reliable results, had a detection limits of about 10 4 -10 5 cells per assay and was easily applicable in a brewery environment. biodesulfurization is one of the possibilities studied by the researchers to attain the maximum sulfur levels imposed for a near future by governments (european directive, 2003) . rhodococcus erythropolis igts8 is a natural and strictly aerobic microorganism able to remove the sulfur atom from dibenzothiophene (dbt) in a selective way (4s route (oldfield et al., 1997)), obtaining 2hydroxibifenyl (hbp) and sulfate. growth is carried out using the experimental procedure performed in previous works dealing with the inoculum built up, media composition and operational conditions (del olmo et al., 2005a (del olmo et al., , 2005b . this work is focused to determine the oxygen uptake rate during the production of the biocatalyst. four experiments were carried out at a biostat b fermentor (braun biotech.) using as only variable the constant stirrer speed used: 150, 250, 400 and 550 rpm. oxygen uptake rate have been determined by means of two methods: dynamic technique at different times during growth for few seconds to ovoid influences and from oxygen profile when the term dealing with oxygen transfer rate is known (predicted by the model proposed in a previous work (garcía-ochoa and gómez, 2004)). our values obtained from the techniques used present the same tendency in all the runs carried out: our values from dynamic technique is always lower than our values obtained from the oxygen profile. these values are modeled and the difference observed is explained due to the cellular economy principle: during the seconds employed in the dynamic technique determinations microorganism do not produce 4s route enzymes. it was studied different methods to recover a p. salmonis antigenic protein from recombinant e. coli cells. this protein has shown be highly effective in vivo vaccine. it has the ability to stimulate salmon immune system protecting them against of aggressive disease salmonid rickettsial syndrome resolving by this way a great problem of salmonid aquaculture. biomass obtained from iptg induced e. coli bl21 (de3) codon plus culture was used for soluble and insoluble antigenic protein recovery. it was evaluated recuperation by glass bead mill, freezing and thawing, osmotic shock and lisozyme/edta treatments, all of them applied in single or combined way. biomass was measured by dry weight of cells, soluble protein concentration was quantified according to bradford method, and antigenic protein was identified by sds-page and western-blot analysis. cells treated with lisozyme/osmotic shock and then glass bead mill the soluble protein was a 62.9% of the dry weight cell mass whereas using lisozyme/edta and glass bead mill as a single treatment only a 24.4 and 22.6% were obtained respectively. the freezing and thawing disruption treatment released less than 5% of soluble protein, as well as the osmotic shock procedure too. the sds-page and west-ernblot analysis revealed that the antigenic protein must be purified from the insoluble cell fraction when physical or mechanical disruption methods were employed and from the soluble cell fraction when chemical or enzymatic treatments were used. we propose investigate in further studies the inclusion bodies formation to design an efficient purification procedure for the target protein. the iso-peroxidase pox2 from garlic bulb allium sativum that represented the major peroxidase activity was purified to homogeneity. the enzyme is monomeric and has a molecular mass of 36 kda, and a pi around 9. the optimum temperature ranged between 25 and 40 • c, while optimum ph was around 5. pox2 appeared remarkably thermostable since it retained 50% of its activity at 50 • c for at least 6 h. in addition, the enzyme was stable at a ph range from 3, 5 to 11. kinetic constants were calculated, the apparent k m values were 500 and 150 m for gaïacol and h 2 o 2 , respectively. the high thermostability of pox2 may represent clear advantages in a number of processes including immobilizing peroxidase and use it as a biosensor to detect oxidant component as h 2 o 2 and other peroxides. immobilization of pox2 was achieved by binding covalently the enzyme to a sepharose matrix (bead and membrane va epoxy). the immobilized peroxidase showed great stability at heat and storage than the soluble enzyme. the native enzyme retained 55% of its activity at 60 • c for 10 mn while the immobilized pox2 retained full activity for 35 mn at the same temperature. in other side, the free enzyme retained full activity for at least one month and a half during storage at 25 • c, and lost 50 m of its activity after 2 months. the immobilized form of pox2 retained complete activity for 2 months at the same temperature. the immobilized enzyme was used to detect h 2 o 2 in some food components such as milk and fruit juices. in a second study, same experiments were performed in order to detect the smaller quantity of added h 2 o 2 to the farming milk. purpose: a new research field has been created to begin to address protein function at level of regulation of enzyme activity. this new area has been given the name chemical proteomics, or activity-based proteomics (abps), and makes use of small molecules that can covalently attach to catalytic residues in an enzyme active site. the selectivity of the chemically reactive group allows specific proteins or protein subset to be tagged, purified and analyzed. methods: this molecule (abps) has three subsets: tag, linker, and warhead. warhead is a nucleophile and attach to active site. linker is a polypeptide that makes a simple connection between warhead and tag. tag is fluorcent or radioactive material that facilitates the detection of drugs. findings: several diseases such as cancer, rheumatoid arthritis and osteoporosis are associated with elevated levels of protease activity. serine hydrolyses abps have been used to profile enzyme activity in a diverse range of cancer cell lines. in studies comparing metastatic and non-metastatic human breast cancer models, it was shown that the former exhibited a higher activity of a ␥-glutathione-s-transferase, an enzyme that has not previously been associated with breast cancer. discussion: additionally, abps can be used to develop robust screens for small molecule inhibitors of a specific enzyme target within a large family of related enzymes. this method of inhibitor screening allows compounds to be assayed for both potency and selectivity against a set of related in complex biological samples. this technique is able to identify novel enzymatic proteins and drugs and has the potential to accelerate the discovery of new drug target. a cyclodextrin glycosyltransferase (cgtase) from a new isolated strain from bacillus clausii e16, was purified through q-sepharose, gel filtration chromatography and deae-sephadex a-50. the mw of the pure enzyme was 75 kda with sds-page. the enzyme displayed optimum ph value and ph stability at ph 6.0 and in range of 6.0-11.0, respectively. the optimum temperature and thermostability were at 55 • c and up to 60 • c by 1 h, respectively. the k m and v max were 2.85 mg/ml and 80.0 mol/min mg and 0.83 mg/ml 13.45 mol/min mg using maltodextrin and soluble starch, respectively. the isoeletric point was 4.8 and the n-terminal region of the pure enzyme was sequencing by maldi-tof-ms. the ratio of ␣-, ␤and ␥-cd was 0.29:1.00:0.79 and 0:1:0 with maltodextrin and soluble starch at 2.5%. application of magnetic separation technology for recovery of immobilised lipases nadja schultz 1 , anke neumann 1 , george metreveli 3 , matthias franzreb 2 , christoph syldatk 1 1 chair of technical biology, university of karlsruhe, engler bunte ring 1, d-76131 karlsruhe; 2 forschungszentrum karlsruhe, institute of technical chemistry, water-and geotechnology; 3 inst. für wasserchemie, engler bunte ring 1,76131 ka. e-mail: nadja.schultz@ciw.uni-karlsruhe.de (n. schultz). url: www.fzk.de/itc-wgt (m. franzreb) first results on the development of the magnetic separation technology for the recovery of immobilised lipase from a 2-phase-system, which should be suitable for a large scale use in future, known as high gradient magnetic separation (hgms) are presented. the application of immobilised lipases makes the reuse of the enzyme in a process possible and is therefore interesting for industrial applications. in this study immobilised lipase is used in a 2-phase-system. here the new approach to recycle and reuse the lipase, immobilised on magnetic particles, from a 2-phase-system with the help of the new high gradient magnetic separator (hgms) is examined in 30 ml scale. as model enzyme for the immobilisation on magnetic microparticles (polyvinyl alcohol (pva), 1-2 m) the commercially available (novonordisc) lipase a (cala) from candida antarctica was used. necessary for screening of immobilisation methods and characterisation of the immobilised lipase (candida antarctica) was the development of a robust, simple and rapid chromophoric activity assay. therefore the pnpp-lipase assay was optimised for direct application on immobilised lipases (in preparation schultz et al., 2005) . further more a ph-stat-assay for measuring the activity of free and immobilised cala in a 2-phase-system of tributyrate and buffer was optimized for this system. another important basis for the realisation of the recovery of immobilised lipases was to optimise the immobilization technique of the lipase. furthermore approaches for the explication of generally empirical based immobilization techniques on insoluble support were made. hereby we successfully applied the zeta potential measurement on the immobilization behaviour of the lipase cala. for to determine the operating temperature for the biomagnetic separation procedure we studied stability analysis of free and immobilised lipase cala at different temperatures (37, 25, 4, −20 • c) and at different ph values (ph 6, 7 and 8). the optimal temperature and ph value for the free and immobilised lipase was determined. presently and constructively on the so far developed methods and techniques we intensively work on the demonstration and feasibility of the recovery of immobilized lipase from a 2-phase-system. challenging approaches and first results on the recovery of immobilized lipase from a 2-phase-system in 30 ml scale were shown already. nowadays glycomics raise new challenges for affinity chromatography related with an abundance of glycoconjugates in living organisms and with scaling-up of the preparative processes. economics, efficiency and practicality dictate the search of novel chromatographic biospecific adsorbents that could contribute to enhancing the productivity of the affinity separation process. the purpose of the work was to prepare cellulose-and silica-based biospecific adsorbents with immobilized lectins and to evaluate them for the affinity chromatography of glycoproteins. cellulose-based matrix granocel and silica coated with hydrophilic polymers were used as a support. the effect of support characteristics, such as pore size, chemistry of active groups and their density on the support' surface on lectin immobilization and on the efficiency of adsorbents obtained were evaluated. three different methods were used for the activation of the support: oxidation with sodium periodate, modification with pentaethylenhexamine (spacer arm of 18 atoms) and carbonyldiimidazole activation. cona and wga two lectins of different molecular weight and shape were selected to notice differences resulting from the size and diffusion behaviour. chromatographic performances of the adsorbents were studied applying two different glycoproteins (god and fetuin) carrying specific terminal glycomoieties of mannose (god), and n-acetylglucosamine (fetuin) for specific interaction with cona and wga, respectively. the adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to 7.4 mg per ml support and a high recovery (up to 93%). it was shown that spacer arm affected ligand coupling kinetic as well as the chromatographic behavior of the adsorbents obtained. the adsorption isotherms of god onto cona adsorbents reveal an adsorption behavior with high and low affin-ity binding sites. the dissociation constant k d of the ligand-sorbate complex is approximately 1 × 10 −6 , and 0.4 × 10 −5 m, respectively. it was supposed that the second step is related to the sorption of solvated god onto already adsorbed god forming sorbate dimers. quantitative methods in high throughput screening of aqueous two phase systems matthias bensch, björn selbach, jürgen hubbuch institute of biotechnologie 2, forschungszentrum jülich, germany purification of biopharmaceuticals is one of the most expensive and at the same time least understood steps in bioprocesses. during the process development for protein production, short time to market and the demand for cheap processes dominate today's process development. one way of reducing process costs is to implement integrative processes. aqueous two phase systems (atps) combine the advantages of removing cell debris and simultaneously purifying and concentrating the target protein, however, to the cost of highly complex systems which are difficult to predict and optimize. using high throughput screening techniques in the development of atps processes thus seems to be an ideal candidate for achieving both a reduced development time and an economical process without the need for preliminarily well characterized systems. in this study, we use the robotic system tecan freedom evo tm as an automation platform for the evaluation of aqueous two phase systems. central to this workstation are the integrated hardware as liquid handler, gripper, reader and centrifuge. we have created high throughput methods for rapid parameter estimations. as a first step, pipetting and mixing had to be calibrated for the use of highly viscous polymer solutions which are common in atps. the focus of the current work lies on the integration of the automatic preparation and analysis of two phase systems in microtiter plate scale. the robotic platform can now automatically create aqueous two phase systems and measure characteristic values such as binodal curves, protein concentrations and protein distributions between the two phases. the major bottleneck of hts processes, namely the rapid analysis of impure systems, is tackled by using automated elisa tools. depending on the intended use, the high number of measured partition coefficients and yields can be used for modelling or rapid process design. today, most optimisations of chromatography separations are based on experimental work and rule of thumb. the pat initiative has opened up for a model-based approach for downstream processing of pharmaceutical substances. this work uses a nonlinear chromatography model to optimize an ion exchange separation step. the general rate model with langmuir mpm kinetics described the behaviour of the components in the column. the optimal operating points using both productivity and yield as objective functions were found. the optimizations were run with both igg and bsa as target proteins respectively to compare their optimal operating points. the requirement on the optimal operating point was a purity of at least 99%. this requirement was added to the optimization problem as a nonlinear inequality constraint. flow rate, loading volume, start salt concentration in elution, elution gradient and cut points were used as decision variables in the optimization. the more retained component, bsa, was much easier to separate from igg with a gradient elution than igg from bsa while still retaining a high productivity and yield. the higher load volume at the optimal operating point, with bsa as target protein, causes a displacement of igg and thereby improving the separation. a high productivity at the yield optimum was still possible with bsa as target protein. both a lower productivity and yield was obtained with igg as target protein. optimisation and robustness analysis of a hydrophobic interaction chromatography step niklas jakobsson, marcus degerman, bernt nilsson department of chemical engineering, lund university, p.o. box 124, se-221 00 lund, sweden process development, optimisation and robustness analysis for chromatography separations are often entirely based on experimental work and generic knowledge. the present study proposes a method of gaining process knowledge and assisting in the robustness analysis and optimisation of a hydrophobic interaction chromatography step using a model-based approach. factorial experimental design is common practice in industry today for robustness analysis. the method presented in this study can be used to find the critical parameter variations and serve as a basis for reducing the experimental work. in addition, the calibrated model obtained with this approach is used to find the optimal operating conditions for the chromatography column. the methodology consists of threes consecutive steps. firstly, screening experiments are performed using a factorial design. secondly a kinetic-dispersive model is calibrated using gradient elution and column load experiments. finally the model is used to find optimal operating conditions and a robustness analysis is conducted at the optimal point. the process studied in this work is the separation of polyclonal igg from bsa using hydrophobic interaction chromatography. department of biochemistry and microbiology, ict prague, technicka 3, prague cz-166 28, czech republic the display of novel metal binding sites on the surface of the biosorbent represents potent tool to increase its binding capacity and improve selectivity. in this study, the 15.8 kda transcriptional regulator merr of mercury-inducible mer operon of tn21 exhibiting high affinity and selectivity towards hg 2+ , was displayed on the surface of s. cerevisiae. to achieve this, merr was genetically fused with gene encoding c-terminal domain of ␣-agglutinin which resulted in covalent attachment of the of the fusion protein on the cell wall glucan via glycosylphosphatidylinositol anchor. to evaluate the performance of such modified whole-cell biosorbent with specific regard to hg 2+ , we constructed a new biosensor e. coli strain, which utilizes kanamycine resistance gene as a reporter under the control of mer promoter. it allowed determination of hg 2+ in a range of 5-100 nm by simply monitoring the growth in the hg 2+ /kanamycine-containing media. the effect of genetic engineering of s. cerevisiae surface by merr became significantly pronounced in biosorption experiments with solutions containing 10 m hg 2+ when modified cells accumulated 2.5-fold more hg 2+ than the control strain expressing mere anchoring domain. sensitivity analysis of amino acids in simulated moving bed chromatography ju weon lee, chong ho lee, yoon mo koo center for advanced bioseparation technology, inha university, inchon 402-751, korea. e-mail: ymkoo@inha.ac.kr (y.m. koo) the difficulty of simulated moving bed (smb) design is that the optimization of the operation conditions relies on the determination of accurate adsorption isotherms. most smb chromatograph is carried out under nonlinear conditions, and the nonlinear behavior should be considered properly in the equilibrium isotherms. the other difficulty is the smb operation which has the characteristics of continuous process, all flow rates and switching time of valves should be maintained during the operation of smb. if the disturbances of operating conditions and isotherm parameters are occurred, it affects the zone flow rates and the migration velocity of the solutes, and these effects change the internal profiles of the solutes. therefore, it is the reason of decreasing the purity and the yield of products the objective of this work is to consider the sensitivity of isotherm parameters and operating parameters in smb chromatography process. two amino acids, phenylalanine and tryptophan, separation by smb process is selected as control system. application of ph and po 2 probes during bacillus caldolyticus fermentation: an additional approach in improving a feeding strategy johannes bader 1 , boris neumann 2 , karima schwab 1 , milan popovic 1 , rakesh bajpai 3 : 1 studiengang biotechnologie, fachbereich v, tfh-berlin, seestr., 13347 berlin, germany 2 proteome factory ag, dorotheenstr. 94, 10117 berlin, germany; 3 department of chemical engineering, university of missouri-columbia, w2061 ebe, columbia, mo, usa. e-mail: popovic@tfh-berlin.de (m. popovic) bacillus caldolyticus,a thermophilic microorganism, is a good producer of thermostable liquefying ␣-amylase. during optimisation of initial and feeding media for fed-batch fermentation a two component feeding containing starch and casitone was found advantageous. to approach the optimal feeding rate the method published by akesson et al., 2001 was extended to two component feeding. the key idea, discussed in this presentation, was using the po 2 and ph probing signals to determine if the feeding of one or the other component should be increased or decreased. each of the probes offers information of different areas of feeding condition. to prevent excessive feeding of starch the ph probe is preferable. in case of excessive casitone feeding the po 2 probe responds in very authentic way enabling together with the ph signal reliable and reproducible evaluation of feeding strategy. however a congruent response of po 2 and ph probes means the approaching of the optimum feeding rate for both components. akesson, m., hagander, p., axelsson, j.p., 2001. probing control of fed-batch cultivations: analysis and tuning. contr. eng. pract. 9, 709-723. antibody immobilization by using the plasma polymerized acrylic acid r. jafari, m.tatoulian, f. arefi-khonsari laboratoire de génie des procédés plasmas et traitement de surface, enscp, upmc, 11 rue pierre et marie curie, 75005 paris, france the objective of this work is therefore to produce a surface containing a high density of cooh functions on the polymer beads (ps) for the covalent immobilization of antibodies. we have investigated the plasma polymerization of acrylic acid in a fluidized bed reactor the polystyrene (ps) beads. for such application, there is a strong need to obtain stable plasma polymerized acrylic acid (ppaa) coating, resistant to washing with water. different physico-chemical analyses have been used (water contact angle measurements (wca), xps and sem analysis) to characterize the ppaa coating deposited on ps beads under different experimental conditions. the xps results showed that the pretreatment of surface of the beads before deposition of acrylic acid plays an important role on the stability of ppaa layer. the instability of the coating is partially due the fact that under certain conditions the coatings are soluble in water and secondly due to the bad adhesion of the polymer beads which are hydrophobic to the growing ppaa coatings. xps as well as tof-sims gives evidence of the immobilization of the antibody. xps results as well as static sims allows to detect nitrogen on the surface of the treated beads which proves the presence of the immobilized antibodies. under optimum condition the ppaa coatings provides the possibility to show a nitrogen uptake which varies between 6.5 and 9% of the apparent stoicheiometry of the surface. holst department of medical physiology, the panum institute, university of copenhagen, dk-2200 copenhagen, denmark glp-1 (glucagon-like peptide-1), a peptide of 30 amino acids secreted by endocrine cells in the gut in response to meal ingestion, was discovered during a systematic search for gut factors capable of enhancing insulin secretion. it turned out to be the most efficacious insulin releaser known, and unlike other factors, was shown to retain its insulinotropic activity also in patients with type 2 diabetes. subsequent research has documented that the peptide not only releases insulin from the beta cells, but also enhance all steps of insulin biosynthesis, up-regulates beta cell gene transcription, and has trophic effects on the beta cells. the latter includes both proliferation of existing cells, neogenesis from ductal precursor cells, and inhibition of apoptosis. the peptide also inhibits glucagon secretion, reduces gastric emptying and reduces appetite and food intake. because of these actions, glp-1 administered to patients with type 2 diabetes dramatically lowers blood glucose as well as glycated hemoglobin levels, and reduces body weight. however, natural glp-1 is extremely rapidly metabolized in the body, and the problem has been how to convert the unstable peptide into a clinically useful agent. the two main problems are its susceptibility to enzymatic degradation by ubiquitous dipeptidylpeptide peptidase iv (dpp-iv) and its rapid renal elimination. a related peptide, isolated from the saliva of a lizard, exendin-4, was found to be a full agonist of the glp-1 receptor, to be resistant to dpp-iv and to be cleared more slowly by the kidneys. this peptide was highly effective in clinical studies and has now (30/4) been approved for diabetes treatment by the fda. other approaches include acylation of glp-1 whereby it attaches to albumin in the body and acquires resistance to dpp-iv as well as a slow renal elimination. also this analogue (liraglutide) has favourable clinical effects. fusion proteins of glp-1 and larger, slowly eliminated proteins in the body are currently being evaluated. small molecule, orally available inhibitors of dpp-iv have been demonstrated to protect endogenous glp-1 from degradation and to be efficacious in both experimental and clinical diabetes, and numerous inhibitors are currently in clinical development. the incretin hormones are released from gut endocrine cells upon meal ingestion. they enhance glucose-induced insulin secretion and nay be responsible for up to 70% of postprandial insulin secretion. the incretin hormones are glucagon-like peptide-1 (glp-1) and glucosedependent insulinotropic polypeptide (gip). in patients with type 2 diabetes (2dm) the incretin effect is severely reduced or absent. in 2dm patients the secretion of gip is normal, but its effect on insulin secretion is almost completely lost. glp-1 secretion, on the other hand, may be impaired, but its insulinotropic actions are preserved and it may restore insulin secretion to near normal levels. substitution therapy with glp-1 might therefore be possible. glp-1 is a product of the glucagon gene and its actions include: (1) potentiation of glucose-induced insulin secretion; (2) stimulation of the expression of ␤-cell genes essential for insulin secretion, including the insulin gene; (3) stimulation of ␤-cell proliferation and neogenesis (by enhancing endocrine differentiation of duct cells) and inhibition of ␤-cell apoptosis; (4) inhibition of glucagon secretion; (5) inhibition of gastrointestinal secretion and motility, notably gastric emptying; and (6) inhibition of appetite and food intake. these actions make glp-1 particularly attractive as a therapeutic agent for 2dm. thus, continuous subcutaneous administration of glp-1 for 6 weeks resulted in a 5 mmol/l reduction in mean plasma glucose and a reduction in hgba1c of 1.3%; a weight loss of 2 kg; improved insulin sensitivity; improved ␤-cell function; and the treatment was associated with no significant side effects. unfortunately, glp-1 is rapidly destroyed in the body by the ubiquitous enzyme, dipeptidylpeptidase iv (dpp-iv). clinical strategies therefore include: (1) the development of metabolically stable analogues of glp-1 viz. activators of the glp-1 receptor; and (2) inhibition of dpp-iv. orally active dpp-iv inhibitors have proven successful in experimental diabetes and several companies are now trying to develop clinically suitable inhibitors. so far the clinical experience is limited, but recent clinical studies have provided proof of concept. metabolically stable analogues/activators include the structurally related lizard peptide, exendin-4 or analogues thereof, as well as glp-1 derived molecules that bind to albumin and thereby assume the pharmacokinetics of albumin. these molecules are effective in animal experimental models of type 2 diabetes, and have been employed in clinical studies of up to 52 weeks' duration. on the basis of these studies it can be concluded that a therapy of type 2 diabetes mellitus based on stimulation of glp-1 receptors is likely to be effective and to become a clinical reality within the not too distant future(1-4). recombinant activated coagulation factor vii (rfviia) was developed to treat bleedings in hemophilia patients, who have developed inhibitors against fviii or fix, and has been demonstrated to have an efficacy rate of 80-90% in major surgery as well as in serious bleedings in such patients. to use rfviia as a hemostatic agent in severe hemophilia is a new concept of treatment, not being a substitution therapy, but using a pharmacological dose of exogeneous rfviia to compensate for the lack of fviii or fix. the administration of extra rfviia has been found not only to bind to tissue factor (tf), but also to the negatively charged phospholipids surface of thrombin activated platelets. hemostasis occurs on surfaces being initiated on the tf-expressing cells as a result of exposure of tf, not normally exposed to the circulating blood, following an injury to the vessel wall. tf is a true receptor protein with an intramembraneous part and an intracellular tail. its ligand is fvii/fviia. as soon as tf is being exposed to the blood, it forms complexes with fviia already present in the circulation. these complexes activates fx and provide the initial limited amount of thrombin molecules activating the co-factors, fviii and fv, as well as fxi and platelets. following the thrombin activation of platelets, negatively charged phospholipids are being exposed on the outer surface of the platelets. on this surface most coagulation proteins bind tightly, facilitating the conversion of fx into fxa and the full thrombin burst, necessary for the formation of a tight fibrin hemostatic plug resistant against premature lysis. in hemophilia patients the initiation of hemostasis is essentially normal, but, since they lack fviii or fix, they do not form the fviiia-fixa complex necessary for full thrombin generation on the activated platelet surface. as a consequence the fibrin plug formed in hemophilia is loose, fagile and easily dissolved resulting in continuous bleeding. pharmacological doses of rfviia have been demonstrated to mediate direct binding of rfviia to the negatively charged thrombin activated platelet surface, thereby generating thrombin formation in the absence of fviii/fix. through this mechanism hemostasis is generated in hemophilia patients independent of fviii/fix. furthermore, by generating more thrombin at an increased rate the formation of stable, tight fibrin hemostatic plugs are facilitated. such fibrin plugs are more resistant against premature lysis and help not only to initiate but also to maintain hemostasis. based on its capacity of enhancing thrombin generation locally on the activated platelet surface, rfviia has been used to ensure hemostasis also in other situations than hemophilia, such as platelet defects including thrombocytopenia. recently, rfviia was shown to be hemostatically effective in patients with profuse bleedings as a result of vast trauma and tissue damage. in these patients with a complex hemostasis pattern including a host of changes leading to an impaired hemostatic function, extra rfviia seems to help generate a burst of thrombin resulting in the formation of a stable hemostatic plug more resistant against the ongoing lysis. in patients with intracerebral haemorrhage, one single dose of rfviia recently was found to limit the expansion of the haemorrhage and thereby leading to improved functional outcome. institute for medical microbiology and immunology, panum 18.3.12, blegdamsvej 3, dk-2200 copenhagen n, denmark. email: s.buus@immi.ku.dk complete genomes from several species including many pathogenic microorganisms are rapidly becoming available along with the corresponding "proteomes". even at the peptide level, the diversity of proteome is enormous and easily represents a unique imprint of the originating organism. it is perhaps not surprising that the immune system considers peptides as key targets. recent immunological advances have shown that mhc molecules act as peptide selectors for immune recognition. we have proposed to generate accurate predictions of peptide binding to mhc and used these to identify immunogenic epitopes directly from genomic data. we have developed an iterative data-driven immunobioinformatics approach where data is used to generate predictors, and predictors are used to select new and complementary data for the next iteration. we have demonstrated the superior performance of this approach compared to a random data selection approach. we have also developed an efficient approach to select the most informative mhc molecules to investigate. the resulting, immunobioinformatics resource represents an immediate and powerful application and interpretation of genomic data, and will enable a rational approach to immunotherapy in the future. allergen specific immunotherapy is a causal treatment for igemediated allergic diseases such as hay-fever, and it has relied traditionally on preparations derived from aqueous extracts of various natural allergenic source materials. the cloning and production of an increasing number of allergens through the use of dna technology has not only facilitated the characterisation and analysis of the allergenic proteins, but also provided the opportunity to use these recombinant proteins instead of natural allergen extracts for the diagnosis and therapy of allergic disease. detailed physicochemical, biochemical and immunological characterisation are essential for the comparison of natural and recombinant proteins, and also provide a basis for developing derivatives. chemically modified allergens with attenuated ige-reactivity are currently used for immunotherapy in order to enable high doses to be achieved with a minimized risk of inducing allergic side reactions. dna technology provides the opportunity to develop and produce hypoallergenic allergen variants using strategies including gene mutation. the design of such variants must ensure that t cell reactivity and immunogenic activity are retained in order to preserve therapeutic potential. the recombinant allergens and their derivatives have several advantages over natural allergen extracts. they are relatively easy to produce in consistent pharmaceutical quality; the problems of natural extract standardisation can be avoided completely; the relative concentrations of the individual allergens can be controlled to obtain optimal dosages; nonallergenic proteins are excluded; the possible risks of contamination are avoided. the first clinical trials with grass pollen allergens and birch pollen hypoallergenic variants have yielded very encouraging results. the use of recombinant polyclonal antibodies (pabs) may improve the treatment of disease caused by complex targets such as infectious agents, when compared to monoclonal antibody therapy. symphogen has developed a method for reproducible production of target-specific fully human pab compositions, so-called symphobodies. the antibody genes are first isolated from donors with an immune response against the target and antibodies are screened for specificity. subsequently, the pabs are expressed in mammalian cells using the sympress technology, which is based on site-specific integration. this procedure ensures that each of the expression constructs encoding the antibody genes are stably integrated at the same site in each of the host cells, thereby eliminating genomic position effects and differential growth and production rates. further, the sympress technology comprises the generation of a polyclonal working cell bank (pwcb) which is used as inoculation material for the manufacturing. these cells display sufficient genetic stability to enable a controlled gmp production of recombinant polyclonal antibodies. symphogen's first product, sym001, is a recombinant human polyclonal rhesus d-specific symphobody preparation consisting of 25 different anti-rhesus d antibodies. this product is intended to be used for treatment of idiopathic thrombocytopenia purpura and prevention of hemolytic disease in newborns. recombinant anti-rhesus d symphobodies were produced and shown to be biologically active against rhesus d. the expression technology provided a compositional reproducibility between batches which is sufficient for manufacturing of such a polyclonal product for clinical use. scaledup production for clinical trials is currently ongoing. stem cells play an important role in renewing tissues such as skin and cornea. they are responsible for the continuous generation of the differentiated epithelium. we have characterized stem cells of the skin and cornea in situ and their fate in vitro in human skin reconstructed by tissue engineering using keratin (k)19. in the outer root sheath of the hair follicle, stem cells (label-retaining cells) present in the basal layer of the bulge area express k19 and present a loosely arranged keratin filament network and low levels of k14 protein in their cytoplasm. in addition, another stem cell population (also labelretaining) is present in the first suprabasal layers. these cells exhibit a very dense keratin network and express k17. three-dimensional tissue constructs (dermis and epidermis) obtained by the self-assembly approach of tissue engineering allow the preservation of k19 positive stem cells in the basal layer of the epithelium. in the eye, the stem cells are located in the limbal part but not in central cornea and they express k19. the epithelium of reconstructed cornea is thinner compared to reconstructed skin and more transparent. the characterization of stem cells in reconstructed tissue is essential to evaluate the long-term survival of these tissues in vitro but also after grafting. these human reconstructed tissues are developed for fundamental (physiological, toxicological studies) and clinical applications such as transplantation for the permanent replacement of damaged organs. lg is holder of the canadian research chair (cihr) on stem cells and tissue engineering. alessandra gliozzi physical department, university of genoa, 16146 genoa, italy hollow nanometer-sized capsules can be prepared by means of different techniques. first "nanocapsules" were liposomes, however they are too unstable for many medical or pharmaceutical applications. in contrast, recently developed polyelectrolyte capsules prepared by means of the layer-by-layer technique are much more stable and seem to be a very promising way for coating living cells or tissues in order to prevent or reduce their immune rejection after implantation. several observations on single living cells encapsulated by the alternative adsorption of oppositely charged polyelectrolytes will be presented. the most relevant result is that cell preserve their metabolic activity, are still capable of dividing and performing specific functions. moreover, a technique to immobilize in defined arrays coated cells expressing green fluorescent protein by using a microcontact printing of polyelectrolytes will be presented. finally, tests performed to study the induction of fibrosis and vascularization by nanocapsules implanted in rat kidney and liver will be presented. over the past decade we have developed methods to generate spontaneously and synchronously beating tissue equivalents from neonatal rat heart cells in the culture dish. these tissue equivalents display the key morphological and functional features of intact myocardium and have been termed engineered heart tissue (eht). to generate ehts, heart cells are mixed with freshly neutralized, liquid collagen i, matrigel and growth supplements and grown in a circular casting mold around a central cylinder, which subjects the cells to a continuous mechanical load. this process is enforced by cyclic mechanical stretch. we use eht mainly for two purposes, as a test bed for the effects of pharmacological or genetic manipulations and for cardiac repair. as a cell culture model, ehts compare with standard 2d monolayer cultures of neonatal rat cardiac myocytes and freshly isolated adult cardiac myocytes. advantages of ehts are their functional similarities with intact heart muscles, the ability to easily measure force of contraction under mechanical load, the pos-sibility to transfect cardiac myocytes inside ehts with adenovirus at high efficiency and the reproducibility in large series. a disadvantage is that contractile function as measured at the end of the culture period also integrates influences on tissue development, cell-cellconnections, extracellular matrix production and on non-myocytes. at present we are working on downscaling the eht method to a 96well format for screening purposes. to use ehts for cardiac repair we created multi-looped ehts from five circular ehts large enough to cover the infarct scar 14 days after coronary artery ligation in rats. ehts survived and formed a layer of muscle tissue on top of the infarct scar. ehts restored undelayed anterograde impulse propagation over the scar, prevented further ventricular dilatation, normalized enddiastolic pressure and relaxation, and partly restored contraction of the scar. thus, the study provides evidence that implanting ehts onto infarcted hearts can improve cardiac contractile function after myocardial infarction. the goal of tissue engineering is the development of skin, bones and even organs to restore, maintain and improve tissue function within the body. the current paper focuses on the investigation of invitro growth of osteoblast cells in different types of scaffolds. three of the scaffolds were made of pcl (polycaprolactone) 10% glycerol and 10% hca(hydroxylapetite). two of the scaffolds were made by compression molding, and one was made by fused deposition modeling utilizing the stratasys. the fourth cerabio was a commercially available product totally ceramic. the pores in compression molding were obtained by putting in 75% volume of sugar either 150 and 595 m which was later removed by leaching. the fused deposition scaffold was made by placing the filaments in a predetermined arrangement. the stratasys system was a computer designed model. the scaffolds were seeded with hfob 1.19 human fetal osteoblast cell line with vigorous shaking overnight and incubating at 37 • c and 5% co 2 . observation of the seeded scaffolds was made after 3 days and 9 days of incubation. the seeded cells were stained with bcip/nbp at 37 • c overnight. the cell proliferation in the 595 and 150 m scaffolds appeared approximately the same with a possible advantage of 595 m. the cerabio sample demonstrated the greatest proliferation among the four scaffolds studied and the stratasys sample exhibited a different type of cell adhesion with the cells were clustered in the interstices of the structure. denise freimark, ruth freitag, valérie jérôme chair of process biotechnology, university of bayreuth, d-95448 bayreuth, germany. e-mail: denise.freimark@uni-bayreuth. de (d. freimark) tissue engineering is emerging as an alternative to bone grafts for the regeneration of defects that do not heal spontaneously. ultimately, the development of an optimum carrier and the identification of ideal inductive factors and cells may enable tissue engineering to provide an improvement over bone grafts in the future. bone formation and repair require a complex cascade involving growth factors, cytokines and angiogenesis. at present the complexity of the molecular mechanisms that control gene expression in bone forming cells in embryo as well as in adult is not fully understood. several factors like bone morphogenetic proteins (bmps), transforming growth factor beta (tgf-␤), vascular endothelial growth factor (vegf) and insuline-like growth factor (igf) have been identified and their ability to stimulate bone formation in vitro and in vivo has been investigated. while much is known about these factors per se, less is known about genetic regulation of artificially stimulated osteogenesis. interestingly, some in vitro investigations showed that only optimal growth factors concentrations lead to effective bone formation whereas higher concentrations had deleterious effects which suggest some variation in the activated regulation pathways. therefore, one of our goals is to analyze kinetics, dose-dependence and synergistic effects of growth factors and cytokines on regulation pathways of bone formation. moreover, the optimal vascularization of the scaffold is a major hurdle in the development of engineered bone. it is well known that: (i) vascular invasion precedes bone growth and (ii) osteogenesis takes place in the vicinity of newly formed vessels. thus, inadequate bone vascularization is associated with decreased bone formation. further analysis of the intercommunication between endothelial cells and osteoprogenitors in co-culture systems could provide key information that could be thereafter used to solve this problem. we propose to add some new knowledge to this complicated puzzle. a first step in our investigation is the production of some of the growth factors mentioned above in recombinant form. these factors are expressed in a novel vector (ptriex tm ; novagen) which allows recombinant protein production in prokaryotic or in eukaryotic systems with a single plasmid. afterwards, we analyze potential synergistic effects of these factors as well as kinetic and dose-depend parameters on the genetic regulation of downstream pathways in osteoblasts culture. in parallel, we develop an in vitro system allowing us to investigate the intercommunication between endothelial cells and osteoprogenitors. there has been significant interest in the therapeutic and scientific potential of human embryonic stem (es) cells since they were first isolated in 1998. if human es cells could be differentiated into suitable cell types, stem cells might be used in cell replacement therapies for degenerative diseases such as type i diabetes and parkinson's disease, or to repopulate the heart following myocardial damage. however, there is a significant shortage of high quality human es cell lines and few research groups have experience in the propagation and manipulation of these cells. we are addressing this important issue using the combined expertise of the stem cell biology laboratory and the assisted conception unit at king's college, london. with local ethical approval and under licence from the uk human fertilisation and embryology authority, we have been establishing high quality human es cell lines from a novel source of human embryos. to date, we have derived three human es cell lines and are now focused on the generation of therapeutically important cell populations, including cells that may have clinical application in degenerative and traumatic injury to the brain and spinal cord, heart, retina and other target organs. wallenberg neuroscience center, department of physiological sciences, lund university, bmc a11, s-221 84 lund, sweden cell replacement therapy for parkinson's disease is based on the idea that implanted dopamine neurons may be able to substitute for the lost nigrostriatal neurons. in rodent and primate models of parkinson's disease it has been shown that transplanted dopamine neuroblasts can re-establish a functional innervation and restore dopaminergic neurotransmission in the area of the striatum reached by the outgrowing axons; that the grafted neurons are spontaneously active and release dopamine in an impulse-dependent manner, at both synaptic and non-synaptic sites; and that they can reverse or ameliorate some of the parkinson-like motor impairments induced by damage to the nigrostriatal system. clinical trials in patients with advanced parkinson's disease have shown that dopamine neuroblasts obtained from fetal human mesencephalic tissue can survive and function also in the brains of pd patients, restore striatal dopamine release, and ameliorate impairments in motor behavior. the principal limitation of this approach is the problems associated with the use of tissue derived from aborted human fetuses, and the large numbers of donors needed to obtain good therapeutic effects. until now, transplantation of dopamine neurons has focused primarily on differentiated neuroblasts and young postmitotic neurons, at the stage of neuronal development that is optimal for survival and growth of the grafted cells. however, progenitors taken at earlier stages of development might prove more effective. efforts are now made to expand multipotent neural stem-or progenitor cells in vitro, and control their phenotypic differentiation into a dopaminergic neuronal fate. initial results suggest that in vitro expanded cells can survive and function after transplantation to the striatum in the rat pd model, but the overall yield of surviving dopamine neurons has been very low. with further development, expanded progenitors or dopamine neuron precursors, possibly in combination with cell engineering techniques, may offer new sources of cells for replacement therapy in pd. stem cell therapy has been very much in vogue for several years now. like gene therapy before it, it has raised unrealistic hopes of cures being available imminently. unlike gene therapy, it can cite proof of principle in the well established practice of bone marrow transplantation which is actually a good example of stem cell therapy. however, most of the uses that are now touted as targets for stem cell therapy, envisage the conversion of the stem cells into lineage restricted progenitor cells or more commonly, the final differentiated cell type. such conversions are extremely difficult to initiate and control. the procedures involve the manipulation, differentiation, and expansion of cell cultures in the laboratory, with unknown long term effects on the genetics and physiology of the cells. these issues are compounded when one considers as source material, human embryonic stem cells, where not only the final cell product requires significant scrutiny, but also there are safety issues surrounding the persistence of undifferentiated cells. on top of all these challenges are commercial (for stem cell companies), clinical, and regulatory pressures which will impact heavily on the pace of progress. nonetheless, despite all these hurdles, various academic groups and companies are making significant progress and examples of such developments in diabetes and cardiovascular repair will be given stem cells have the unique ability to perpetuate themselves while continually replenishing tissues throughout the life of an organism. the era of cellular and tissue regeneration for the treatment of disease and the effects of aging has indeed begun. it has been known that mechanical factors play an important role in the regulation of cell physiology. it is therefore reasonable to believe that mechanical factors also play a significant role in the metabolic activity and differentiation of mscs. in this study, we investigated the viscoelasticity of individual bone marrow-derived adult human mesenchymal stem cells (hmscs), and the role of specific cytoskeletal component -f-actin microfilaments on the mechanical properties of individual hmscs. the mechanical properties of hmscs were determined using the micropipette aspiration technique coupled with a viscoelastic solid model of the cell. for the hmscs under control conditions the instantaneous young"s modulus e 0 was found to be 886 ± 289 (pa), the equilibrium young"s modulus e ∞ 372 ± 125 (pa), and the apparent viscosity 2714 ± 1626 (pas). after exposed to 2 m of chemical agent-cytochalasin d that disrupt the f-actin microfilaments, the young's moduli of hmscs decreased by up to 72% and the apparent viscosity increased by 167%. these findings suggest that microfilaments are crucial in providing the viscoelastic properties of the hmscs, and changes in the structure and properties of them may influence the mechanical properties of hmscs significantly. pharmacologic transcription control of desired transgenes is essential for gene-function analysis, drug discovery, biopharmaceutical manufacturing, design of complex artificial regulatory networks, precise and timely reprogramming of key cell characteristics for gene therapy and engineering of preferred cell phenotypes for tissue engineering. capitalizing on our recent advances in the design of small molecule-responsive transcription control modalities we have used conditional molecular interventions for (i) improvement of specific productivity in biopharmaceutical manufacturing, (ii) transdifferentiation of therapeutically relevant cell phenotypes and (iii) design of artificial microtissues. we will also report on a completely new dimension of transgene control as well as engineering of hysteretic and epigenetic gene networks in mammalian cells. chronic diseases are a growing burden for the individual and society alike. one key factor driving this increase is an ageing population. currently, there are 600 million persons aged 60 years or over and this number is predicted to triple by the middle of the 21st century. effective prevention of irreversible damage to major organ systems requires early diagnosis and treatment yielding significant quality of life to the individual and sparing valuable health care resources. even when safe and effective medicines are available, a remaining problem to successful therapy are issues of patient compliance. historically, vaccines have been one of the major advances towards the longevity we enjoy today, with compliance rates close to 100%. hence, vaccines for early treatment of chronic diseases are ideally positioned for long-term therapy, and will take away the burden of self-medication associated with orally active drugs. here we will discuss a new generation of therapeutic vaccines based on virus-like particles (vlps). by displaying target molecules in a highly repetitive manner on vlps, it is possible to break b cell unresponsiveness in experimental animals as well as in humans. using such vaccines in animals, chronic diseases such as hypertension, alzheimer's disease, obesity and rheumatoid arthritis could be treated. furthermore, vaccination against nicotine resulted in high nictotine-specific antibody titers in humans, greatly facilitating smoking cessation in immunized individuals. interdependence of the impact of methanol and oxygen supply on protein production with recombinant pichia pastoris n.k. khatri, f. hoffmann martin-luther-university halle-wittenberg, institute for biotechnology, halle d-06120, germany. e-mail: f.hoffmann@biochemtech.uni-halle.de (f. hoofmann) the methylotrophic yeast pichia pastoris is a potent expression system for secretion of recombinant proteins. methanol as inductor of the foreign gene expression is also a substrate with high oxygen demand, which can lead to sudden oxygen depletion upon induction. thus, supply rates of methanol and oxygen are major process parameter during protein production with recombinant pichia pastoris. limiting dosage of methanol allowed maintenance of oxygen sufficient conditions during production of a single chain antibody fragment, but the product was degraded from the c-terminal end. in contrast, full-length product accumulated with controlled methanol concentrations despite oxygen limitation. the volumetric methanol uptake rate are limited by the oxygen transfer capacity of the reactor. higher methanol concentrations decreased the biomass yield and thereby increased the specific methanol uptake rate. this enabled prolonged production and yielded fivefold higher product concentrations. at the same time, the accumulation of small molecular weight contaminants was reduced. dosage of pure oxygen accelerated methanol uptake, grow and production. switching to dostat mode upon oxygen depletion led to an arrest of product accumulation, in contrast to persistent methanol feeding. the productivity was tenfold higher than without oxygen. combined with high methanol concentrations, however, fast methanol uptake led to toxication of the cells and early stop of production. flow cytometry revealed that perturbation of oxygen metabolism was followed by partial lysis of the culture. recombinant production of therapeutic proteins poses severe challenges due to their complexity (cystines, subunits, size). formation of the correct disulphide bridges is a prerequisite for activity but difficult to achieve in a prokaryotic host. there proteins mainly fold post-translationally as opposed to eukaryotic co-translational folding. thus the recombinant products are often not soluble and/or active. that is why different production parameters (e.g. host, induction conditions, temperature, compartment, proteinaceous fusion partners, co-expression of chaperones, foldases) are applied in order to gain functional recombinant proteins. the impact of all these strategies cannot be predicted and every problem of the production (expression, solubility, activity) might need to be solved for every target protein separately. nevertheless, much effort has been put into this for almost 3 decades, because they are important targets for the pharmaceutical industry. human growth factors influencing cellular proliferation and/or differentiation are one example. this case study gives an overview of strategies tested within the development of two processes leading to an optimised yield of active protein. murine wnt-1 (wnt family) possesses 23 conserved cysteines (most likely all involved in disulphide bridge formation and one in posttranslational modification) and could only be successfully produced in e. coli (fahnert, 2004) recently despite various attempts for many years. the other target protein is human collagen prolyl-4-hydroxylase being a heterotetramer consisting of two ␣-subunits and ␤-subunits each. the ␣-subunit strongly aggregates if produced separately whereas the ␤-subunit is pdi and is suggested to have a chaperone function. therefore a sequential induction strategy was proposed for this protein . the moss physcomitrella patens has been recently recognized as an ideal producer of recombinant proteins with respect to glycosylation. due to the elaborated post-translational capabilities of moss cells, the glycosylation patterns have been manipulated to obtain similar proteins to those found in animal cells. the protein expression using moss in suspension offers important advantages in comparison to other systems e.g. cho cells. the recombinant proteins can be targeted into the mineral medium, simplifying the down stream processing. moreover, there are neither known moss viruses nor plant viruses that are pathogenic for humans. the moss are cultivated axenically in a filamentous stage, the so called protonema. a pilot 30 l tubular photoreactor is used to characterize the response of p. patens to variations on the culture conditions. the phototrophic culture in bioreactors is systematically investigated, where light quantity and quality, stress, concentration of phytohormones, and moss morphology influence the differentiation, growth, and protein expression. a tight control of the moss morphology in suspension, quantified by image analysis, has shown to be advantageous in order to delay the cell differentiation and maintain the carbon dioxide uptake in long bioreactor runs. the introduced perfused culture system by means of cross flow filtration allowed for a continuous product separation and concentration, and feed back of the productive cells. the characterization of this highly controlled culture system is presented and the potential of p. patens as an alternative tool for molecular farming is discussed. (rnai) is an evolutionarily conserved, endogenous mechanism for sequence-specific gene silencing that uses small double-stranded rnas (called short interfering rnas or sirnas) to direct cleavage or prevent translation of homologous mrnas. harnessing rnai for therapy presents an opportunity for potentially treating a wide variety of diseases. the main obstacle is delivering sirnas into the cytosol of target cells in vivo. although we were able to protect mice from autoimmune hepatitis by hydrodynamic tail vein injection of sirnas targeting fas, this delivery method is unlikely to be adaptable for human use. alternate strategies to deliver sirnas in vivo as small molecule drugs using currently available, clinically acceptable injection methods that have shown promise in mouse models will be discussed. these include local delivery to mucosal surfaces and delivery into specific cells via cell surface receptors using an antibody fragment fused to protamine. these sirna complexes silence gene expression in vivo only in cells bearing the targeted receptor. experiments showing efficient, effective and cell-specific delivery in a mouse tumor model will be discussed. in addition, encouraging preliminary data using rnai for a microbicide to prevent sexually transmitted infection will be presented. rna interference (rnai) holds significant progress as a therapeutic approach to siolence disease-causing genes, particularly those that encode "non-druggable" targets. the key hurdle for rnai therapeutics is in vivo delivery. a critical requirement for achieving systemic rnai in vivo is the introduction of "drug-like" properties, such as stability, cellular delivery and tissue biodistribution, into synthetic sirnas. our progress in achieving in vivo silencing of endogenous genes with chemically modified sirnas will be discussed. hiv-1 replication in human t cells can be inhibited by stable expression of a short hairpin rna targeting the viral nef gene (shrna-nef). however, hiv-1 escape variants emerge after prolonged culturing, and all but one escape mutant acquire a mutation in the shrna-nef target sequence. we observed single and multiple nucleotide substitutions, but also partial or complete deletion of the target sequence. these results demonstrate the sequencespecificity of this antiviral approach. we observed an inverse correlation between the level of resistance and the stability of the shrna/target-rna duplex for most of the escape mutants. however, two escape variants did not follow this pattern, including an escape mutant with a single point mutation at position −7 upstream of the target sequence. these mutants provide a much higher level of resistance than expected based on duplex stability, which is obviously not affected in the −7 mutant. we demonstrate that these mutants adopt an alternative rna secondary structure that occludes the target sequence. this results in reduced shrna-nef binding and provides a novel mechanism for rnai-resistance. to avoid viral escape, one should ideally target hiv-1 with multiple effective shrnas against conserved genome sequences. we performed a large scale screening to identify such targets, and we have identified at least nine genome segments that can be targeted effectively with shrnas. these potent antivirals are currently being assembled in a lentiviral vector for gene therapy applications in hiv-infected individuals. furthermore, we will describe approaches to forecast viral escape routes and to effectively block such evolutionary paths with additional rnai measures. in this study we analyzed the effect of antibodies against electronegative ldl on the development of atherosclerotic lesions in low-density receptor-deficient (ldlr −/− ) mice. two groups of (ldlr −/− ) mice (eight females) fed 0.5% cholesterol-enriched chow were used. the first group received a monoclonal antibody against electronegative ldl (100 g) and the second one received pbs (controls). additionally, other two groups (eight males) of (ldlr −/− ) mice were treated with a polyclonal antibody against electronegative ldl (100 g) or pbs (controls). antibodies were administered by intravenous route one week before starting the hypercholesterolemic diet and then every week over an experimental time of 21 days. afterwards, quantification of atherosclerotic plaque area of heart and aortic arch was done by analysis of the slices stained with oil red/hematoxolin/light green with the image propus software. the passive immunization with either monoclonal or polyclonal antibodies against electronegative ldl significantly reduced the atherosclerotic plaque areas in atherosclerosis-prone ldlr −/− mice. in conclusion, antibodies against electronegative ldl administered by intravenous route may play a protective role in atherosclerosis. supported by fundação de amparoà pesquisa do estado de são paulo (fapesp, scholarships to d.m.g., l.s. and a.b. and grants to m.h.k. and d.s.p.a.). the enzyme asparaginase from the procaryote escherichia coli or erwinia crysanthemi is used for the treatment of lymphoblastic leukaemia. the drug causes immunological reactions in despite of the treatment efficiency. asparaginase may also be obtained from saccharomyces cerevisiae and this enzyme could provide an alternative to its bacterial counterparts. in this study, the periplasmic nitrogen regulated asparaginase ii from s. cerevisiae, that is coded by the asp3 gene, was cloned and expressed in the methylotrophic yeast pichia pastoris under the control of the aox1 gene promoter. the recombinant p. pastoris strain was cultured in shake flasks and in a 2 l instrumented bioreactor. in both cases it was observed specific enzyme yields seven fold higher in comparison to that using a nitrogen derepressed strain of s. cerevisiae, reaching 800 u/g dry cell mass. high cell density cultures carried out in the 2 l bioreactor, in which it was attained 107 g dry cell mass/l, resulted in a dramatic improvement in asparaginase fermentation. as such, it was measured enzyme yields of 85,600 u/l and productivities of 1083 u/l h. department of biochemistry and food chemistry, biotechnology, university of turku, tykistokatu 6, biocity 6th floor, 20540 turku, finland. e-mails: lorenzo.galluzzi@utu.fi; deadoc@libero.it; deadoc@aliceposta.it (l. galluzzi) the bacterial luciferase operon from the bacterium photorhabdus luminescens has been used, since its first description, for exceptionally different applications. these ranged from the environmental monitoring to the cell tagging, from the analysis of cellular metabolism to the high-throughput screening of novel compounds. the wild-type luxcdabe operon has been engineered in countless ways (for instance by changing the order of the constituent genes, by optimizing the codon usage and by coupling it to many promoters) and has been expressed in prokaryotic and eukaryotic organisms in order to meet precise research and commercial needs. upon the operon expression light is emitted as the side product of a chemical reaction catalyzed by the luciferase enzyme, an ␣␤ heterodimer encoded in luxa and luxb genes. the reaction involves the oxidation of a long-chain aliphatic aldehyde and reduced flavin mononucleotide (fmnh 2 ) with the liberation of excess free energy in the form of a blue-green light at 490 nm. the luxcde genes code for the polypeptides (transferase, synthetase, and reductase) forming the fatty acid reductase complex that catalyzes the conversion of fatty acids into the long-chain aldehyde required for the luminescent reaction. noteworthy is that for the production of the substrates for the luciferase both atp and nadph are required, while neither is involved in the actual light emitting reaction (wilson and hastings, 1998) . recently, the coupling of the luciferase operon to regulated promoters lead to the construction of genetically modified bacteria able to sense the presence of chemicals and to respond, in a dosespecific manner, with light emission. this approach has been applied to the detection of antibiotics in samples from the food industry as well as to the detection of heavy metal ions in environmental samples (kurittu et al., 2000; bechor et al., 2002) . in addition, it opened the possibility of screening wide libraries of new compounds looking for molecules with pre-determined features, able to induce the bioluminescent response by de-repressing the lux operon transcription when incubated with the appropriate bacterial sensor. the high throughput and low costs are the main advantages of this system, which shows as well a certain degree of specificity (galluzzi and karp, 2003; galluzzi et al., 2004) . nevertheless, since the in vivo bioluminescence relies upon a complex network of biochemical reactions, under certain circumstances the light emission is not a direct consequence of the lux operon transcriptional induction but it more likely originates at a posttranslational stage. as a matter of fact, one can suppose that a change in the light emission will be observed whenever the concentration of one or more substrates for the lux␣␤ reaction occurs. consequently, all the molecules sharing the ability to impair the delicate chemical equilibrium regarding the compounds involved in bioluminescence will be sensed by the bacteria as inducing compounds. this, in turn, will result in a loss of specificity of the assay. we investigated this aspect of the whole-cell assays based upon the bacterial luciferase operon for drug discovery by means of a reporter plasmid in which the luxabcde genes, rearranged and optimized for the after 60 min (black downward arrow) of incubation at 37 • c under vigorous shaking the following concentrations of trimethoprim were added to the growing cells: 25 g/ml (triangles), 50 g/ml (circles) and 250 g/ml (rumbles). water was administered to control cultures (squares). light emission was quantified every 30 min by means of the wallac victor 2 multilabel counter (perkin-elmer, turku, finland) and normalized to the absorbance, measured at 600 nm with the same device. multi-96 white-walled transparent-bottomed plates were used for the assays (nalge nunc, usa). between the measurements the plates were kept at +37 • c under vigorous shaking. filled symbols refers to the absorbance values (left side y-axis); open symbols to the normalized count per seconds (right side y-axis). expression in gram + organisms, are under the control of the qacr regulatory region from staphylococcus aureus . non-pathogenic s. aureus rn4220 cells bearing the pqaclux plasmid (cultivated in lb broth supplemented with 0,5% d-glucose and 10 g/ml chloramphenicol) emit light upon the specific transcriptional induction with quaternary ammonium compounds, widely used as surface disinfectants and in many over-the-counter drugs . however, the incubation of the same cells with an inhibitor of the dihydrofolate reductase enzyme, trimethoprim (sigma-aldrich chemie, steinheim, germany), enhanced in a dosedependant manner the light emission observed upon the induction with the optimal concentration (1 g/ml) of benzalkonium chloride (bc). the extent of this increase in luminescence varied from 5-10% to more than 200%, according to the trimethoprim concentration and to the measurement time. interestingly, when the same plasmid is carried by escherichia coli xl1 cells, the lux operon is expressed constitutively (since the qacr regulatory region is not functional in xl1 cells) and at much higher levels than in induced rn4220 cells. also in this experimental system the incubation with trimethoprim results in a dramatic increase of the luminescent signal from the cultures. the explanation for these observations can be found in the molecular mode of action of trimethoprim. the inhibition of dihydrofolate reductase, indeed, directly leads to the accumulation of its substrates, among which is nadph, deeply entangled in the biochemical network of reactions centred on the light emission from the lux operon. nadph provides the reducing power to restore the reduced flavin mononucleotide pool and it is as well involved in fig. 2 . xl1/pqaclux growing cells were incubated with the following concentration of trimethoprim: 25 g/ml (triangles), 50 g/ml (circles) and 250 g/ml (rumbles). water was administered to control cultures (squares). light emission and absorbance measurements were performed as previously described for rn4220 cells. filled symbols refers to the absorbance values (left side y-axis); open symbols to the normalized count per seconds (right side y-axis). the diverse level of growth observed for rn4220 and xl1 cells can be accounted by the different antimicrobial activity exerted by trimethoprim towards gram− and gram+ cells and by the lower activity of the promoter which in pqaclux plasmid drives the transcription of the selection marker (chloramphenicol acetyl transferase). the production of the long-chain aldehyde, both substrates of the lux␣␤ heterodimer (wilson and hastings, 1998) . in conclusion, here we demonstrate that the use of light emission from the bacterial luciferase as a transcriptional reporter has to be very carefully controlled, since some molecules (here trimethoprim) could mimic to some extent a specific promoter activation by impairing the delicate intracellular biochemical equilibrium. on the reverse side of the coin, fig. 3 . simplified scheme depicting the bacterial folate metabolic pathway. only part of the reactions and compounds are reported. trimethoprim inhibits the nadph-dependant reduction of dihydrofolic acid into tetrahydrofolic acid catalyzed by dihydrofolate reductase. this results in the accumulation of both substrates, which become available for other reactions, and in the depletion of tetrahydrofolate, the major c 1 carrier in the synthesis of purines, thymidine, glycine, methionine, and pantothenate in bacteria. for antimicrobial purposes trimethoprim is often associated with sulfonamides, with which it displays a synergistic activity (since they act on the same pathway but at an earlier reaction) (scholar and pratt, 2000) . however, it has to be noted that the lux reporter system could be used in many different in vivo experimental setups, where the change in the intracellular concentration of nadph, atp or other metabolites would be sensibly detected. we have carried out the construction of five pichia pastoris strains harboring in their respective genome three different growth hormones cdna's (22 and 20 kda human growth hormones and bovine growth hormone), a shrimp (litopenaeus vannamei) trypsinogen cdna and a bacterial (bacillus subtilis) phytase gene. in all the cases, the same kind of expression vector and the same transformation technique were used. each dna was fused in frame to saccharomyces cerevisiae alpha-factor secretion signal to lead the secretion of the foreign protein into the culture medium. the induction of each recombinant strain, all with his + and mut + phenotype, was carried out in shake flaks using methanol buffered minimal medium (bmm) and growth rates on methanol determined. production level of secreted proteins was evaluated by protein analysis by sds-page. furthermore, the expression with three of the constructed strains was performed in a 5-l bioreactor and the level of secreted protein determined in each case. both human growth hormones (hghs) were secreted into the culture medium with a high degree of purity obtaining up to 64% of hghs of total proteins in crude fermentation medium and 3-18 mg/l of hghs. neither bovine growth hormone, shrimp trypsinogen or bacterial phytase were detected in methanol induced cultures of the respective strains, in spite of the same culture conditions were used for all p. pastoris strains. the growth rates on methanol were different between some strains. in fermentor cultures the hghs production were increased and shrimp trypsinogen was produced and secreted into the culture medium after improving the culture conditions. bovine growth hormone was detected only when the culture conditions were modified. the protein production level for each strain was affected by the proprieties of each recombinant protein produced. competitive advantages of the diagnostic method for invasive amoebiasis using preserved antigenic extracts without using enzymatic inhibitors m.s. flores 1,2* , e. tamez we have patented a method to diagnose invasive amoebiasis using a novel assay that preserves antigenicity of extracts with high protease content without using enzymatic inhibitors (ic:mc). the available tests for serologic diagnosis of invasive amoebiasis like elisa and indirect haemaglutination (iha) do not have consistent results in endemic zones. here we show the advantages of the assay and the validation of this diagnostic test for invasive amoebiasis. we demonstrated the reduction of proteolytic activity of ic:mc compared with the proteolytic activity of crude extract and crude extract with enzymatic inhibitors using assays. we displayed the ic:mc sds-page pattern and the western blot (wb) pattern useful for diagnosis. to search the clinical utility of this test we examined the wb obtained with sera from patients with different liver diseases; 90 patients had invasive amoebiasis and 45 patients had other liver diseases. the results were compared with those of iha test. also we have tested the accuracy of wb using sera from people with multiple intestinal parasites, like giardia lamblia, hymenolepis nana, blastocystis hominis, entamoeba coli, etc. the sensibility of the wb using the preserved amoebic antigens was 99%, specificity was 100%, positive predictive value was 100%, negative predictive value was 98% and accuracy was 99%. the wb did not exhibit cross reactions with sera from persons with intestinal parasites. our test was better than the (iha) test commonly used in endemic zones. these results show the improvement of using the preserved amoebic antigens in diagnostic tests. also they prove the diagnostic accuracy of our new wb test. antibacterial and antifungal activity of heracleum sphondylium subsp. artvinense yasemin kaçar 1 , sema tan 2 , aysun ergene 2 , perihan güler 2 , semra mirici 3 , ergin hamzaoglu 2 , ahmet duran 4 , sinem yildirim 2 : 1 mersin university, faculty of science and literature, department of biology, 33800 mersin, turkey; 2 kırıkkale university, faculty of science and literature, department of biology, 71450 yahsihan-kırıkkale, turkey; 3 akdeniz university, faculty of education, department of biology education, 07400 antalya, turkey; 4 selcuk university, faculty of education, department of biology education, 42300 konya, turkey turkey is covered yearly with a huge number of plant species. about 9222 species are condenced on the region that between asia and europe. many plant species have been used in folkloric medicine to treat various ailments. heracleum l (apiaceae) is include over than 70 species on the world. this variety is represent with 17 species in turkey that seven species are endemic. heracleum sphondylium subsp. artvinense is endemic species for turkey. ethanolic and aquous extract of heracleum sphondylium subsp. artvinense were investigated for their antimicrobial activities against six bacterial species (e. feacalis, e. coli, s. aureus, p. aeruginosa, l. monocytogenes, shigella) and two yeast (c. albicans, c. krusei). both ethanolic and aqueous extract of heracleum sphondylium subsp. artvinense showed antimicrobial activity against the gram negative bacterium (shigella) and gave the best activity against c.albicans. to develop artificial vectors allowing nucleic acid to transfect into mammalian cells are crucial for extending gene therapy. synthetic vectors based on lipid molecules are particularly attractive because of their potential safety. however, the low encapsulation efficiency of nucleic acid is one of the problem to be solved. recent advances in dna-lipid complex have improved this drawback, and now some lipid molecules are used as transfection agents. in particular, cationic lipids interact with negatively-charged cell surfaces and nucleic acids. the former interaction results in delivery of the nucleic acids directly across the cell membrane. on the other hand, the latter interaction improves the efficiency of nucleic acids entrapment. in this study, we developed a preparation method of "nanovesicle" containing nucleic acids by using reverse micellar solubilization. since dna interacts spontaneously with cationic amphiphiles, complete extraction of the dna molecules into an organic phase using dimethyl distearyl ammonium bromide (2c18ab), an oil-soluble cationic surfactant, is achieved. the re-encapsulation of the reverse micellar droplets solubilizing dna by water-soluble surfactants facilitates the formation of nanovesicles. a high salt concentration at the re-encapsulation step promotes the production of nanovesicles containing dna molecules. eventually, more than 80% of dna was encapsulated in this nanovesicles under the condition of 6m nacl and 5% (w/v) cetyltridecyl ammonium bromide (ctab). in addition, the nanovesicles prepared at 45 • c was smaller than that prepared at room temperature. the resultant 2c18ab/ctab nanovesicle was ca. 16 nm. asymmetric and chemically-modifiable vesicle surface are effective to design gene delivery system. moreover, such a small dna (or rna) carrier has a potential for novel gene therapy application from skin. the key players in clinically important inflammatory diseases, endothelial cells and leukocytes, communicate through membranebound cell adhesion molecules (cam's). obvious strategies for therapeutic intervention include specific means to affect the expression of cam's on the cells involved. rna-interference (rnai) is a well known means to achieve specific gene-inhibition. for this study, two cam's were chosen, namely vascular cell adhesion molecule-1 (vcam-1) as a target for inhibition, and intercellular adhesion molecule-1 (icam-1) as a non-target reference. we designed short interfering rna (sirna) oligos for vcam-1 in order to selectively inhibit the expression of this cam in cultured human vascular endothelial cells (huvec). real-time rt-pcr showed an 80% down-regulation of vcam-1 mrna while the expression of icam-1 remained unaffected. neither cam was affected by non-specific sirna. in order to further substantiate the potential use of sirna for therapeutic purposes, we have set out to investigate two things: (1) does vcam-1-specific sirna affect the amount of vcam-1 on the surface of huvec? (2) is adhesion between leukocytes and endothelial cells affected by vcam-1-specific sirna? the manufacturing of plasmid dna (pdna) is crucial to obtain a consistent product for gene therapy applications. although flowsheets for pdna production are established on the basis of experience, simulation tools provide a valuable help for evaluating alternatives. a process designed to produce 23 kg pdna/year is analysed with the superpro designer tool. the target pdna is amplified in escherichia coli. after harvest, alkaline lysis is used to disrupt cells and release pdna and impurities. precipitations with isopropanol and ammonium sulphate are performed to concentrate/pre-purify pdna prior to hydrophobic interaction chromatography. experimental data is used as input for simulation. inventory analysis identified water, yeast extract, tryptone, isopropanol and ammonium sulphate as the major raw materials. major raw material costs (50%) are related to fermentation components. economic analysis indicates a unit production cost of $ 375/g pdna. for a selling price of $ 1667/g, the payback time was 1.2 years and the roi was 88.6%. an environmental analysis highlighted the replacement of the isopropanol precipitation for a microfiltration step as a benefit which would: (i) reduce the cost of raw materials (13.8%), (ii) reduce the environmental impact associated with isopropanol (70%) and (iii) reduce costs associated with the treatment/disposal of liquid waste (32.3%). preparation of chitosan microspheres for controlled release of somatotropin s. simsek 1 , j. introduction: proteins and peptides have received extensive interest for their therapeutic applications in clinical applications. in order to achieve high administration efficacy of proteins, polymeric particulate carriers have been developed as an effective way to control the drug release profile and to protect the protein molecules from degradation. somatotropin also known growth hormone is a protein hormone of about 190 amino acids. growth hormone is of considerable interest as a drug used in both humans and animals. chitosan a natural linear biopolyaminosaccharide is obtained by alkaline deacetylation of chitin. properties such as biodegradability, low toxicity and good biocompatibility make it suitable for use in biomedical and pharmaceutical formulations. the aim of this study was to prepare chitosan microspheres containing somatotropin and to investigate these microsphere formulations in-vitro release properties. methods: somatotropin-chitosan microspheres were prepared as follows: chitosan was dissolved in acidic solution (2%) containing polysorbate 80. sodium sulphate solution (20% w/v) containing somatotropin was added into the chitosan solution and mixed 500 rpm for a hour. resulting suspension was centrifuged 15,000 rpm 15 min at 4 • c. the formed microspheres were freeze-dried and sieved. in-vitro release studies were performed in ph 7.4 phosphate buffer and time interval samples were removed and analysed by bradford protein assay method. results: microspheres were obtained by using chitosan. protein encapsulation efficiency was between 95 and 99%. average particle size of microspheres was between 48 and 57 m. during to in-vitro release studies burst effect was observed with chitosan microspheres. for decreasing the burst effect gluteraldehit, betacyclodextrin and poly ethylene oxide were added to formulations. conclusion: according to our results modified chitosan microspheres are promising vehicles for controlled release somatotropine delivery. cancer immunotherapy using hyperthermia with magnetic nanoparticles and dendritic cells k. tanaka, a. ito, t. kobayashi, t. kawamura, s. shimada, k. matsumoto, t. saida, h. honda department of biotechnology, school of engineering, nagoya university, nagoya, aichi 464-8603, japan. e-mail: h041306d@mbox.nagoyau.ac.jp (k. tanaka) our hyperthermia system utilizes magnetic nanoparticle covered with lipid layer including cationic lipid (magnetite cationic liposomes, mcls) as a heating mediator and necrotic cell death is induced by means of locally generating heat. in this process, heat shock proteins (hsps) are strongly induced and released. dendritic cells (dcs) are potent antigen-presenting cells (apcs) that play a pivotal role in regulating immune responses in cancer, which is in carrying antigens to apcs and in the maturation of dcs by acting as a danger signal. in the present study, we investigated the therapeutic effects of dc therapy combined with mcl-induced hyperthermia on b16 melanoma. in an in vitro study, when immature dcs were pulsed with b16 cells heated at 43 • c for 30 min, mhc class i/ii, costimulatory molecules cd80/cd86, and chemokine receptor ccr7 in the dcs were up-regulated, thus resulting in dc maturation. c57bl/6 mice bearing a b16 melanoma nodule were subjected to combination therapy using hyperthermia and dc immunotherapy. mice were divided into four groups: group i (control), group ii (hyperthermia), group iii (dc therapy), group iv (hyperthermia + dc therapy). complete regression of tumors was observed in 60% of mice in group iv, while no tumor regression was seen among mice in the other groups. increased ctl and nk cell activity was observed on in vitro cytotoxicity assay using splenocytes in the cured mice treated with combination therapy, and the cured mice rejected a second challenge of b16 melanoma cells. this study has important implications for the application of mcl-induced hyperthermia plus dc therapy in patients with advanced malignancies as a novel cancer therapy. fermentation of a marine bacterium for the production of cytotoxic compounds vicky webb 1 , els maas 1 , eiichi akaho 2 , hiroto kambara 2 , debbie hulston 1 , anna kilimnik 1 : 1 marine biotechnology, national institute for water and atmospheric research ltd, kilbirnie, wellington new zealand; 2 faculty of pharmaceutical sciences, kobe gakuin university, kobe 651-2180, japan marine bacteria are a potential source of novel compounds for the pharmaceutical industry. new zealand marine bacteria were isolated from a variety of sources and initially bacterial supernatants were screened using a cell based mtt cytotoxic assay. two bacteria were chosen for further fermentations in different media to optimise cytotoxic compound production. the media used were selected for their diverse ingredients. they were either carbon rich, nitrogen rich, starch rich or a basic seawater medium. the fermentations were extracted using ethyl acetate and methanol prior to assaying. the results showed that cytotoxic compound production was enhanced ten fold in the starch rich medium compared to the other media. the levels of cytoxicity appeared to be depended on the cell line used, with the epithelial lung carcinoma cell line a549 being more sensitive to the cytotoxic compounds than the human fibroblast cell line mrc-5. isolation and identification of marine bacteria from deep-sea sediments els maas 1 , cara brosnahan 1 , vicky webb 1 , helen neil 2 , phil sutton 2 : 1 marine biotechnology, national institute for water and atmospheric research ltd., kilbirnie, wellington new zealand; 2 oceanography, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand polystyrene micro plate activated by utilizing a common co-60 gamma source with a crotonic acid. the well surface have been studied in term of optical quality, protein (h-igg) binding capacity and stability. a significantly enhanced total capacity and strength of binding to grafted surfaces was demonstrated as compared to passive adsorption of the proteins to untreated surfaces.the majority routine laboratory tests for the measurement of rheumatoid factor (rf) are semi quantitative and in order to achieve accurate, sensitive and specific rf assay, we are developed enzyme linked immuno sorbent assay (elisa) for human igm-rf kit. stable and reproducible binding of antigen (human-igg) to well of micro titer plate is a prerequisite for rf-elisa kit. the principle of the assay was that the antibodies in serum of patients with rheumatoid arthritis (ra), commonly rheumatoid factor (rf) are directed to the fc part of human igg. in most cases rf belong to the igm class, the well of micro titer plate are coated with antigen (h-igm), and antibodies (h-igg) binding to immobilized antigen is detected by adding enzyme conjugate (anti human-igm-hrp-enzyme) to the wells, substrate was used for color reaction.the performance characteristics of the assay was, the coefficient of variation (c.v) of intra and inter assay was 4.4% and 2.2% respectively, the linearity is ranging from 86 to 112%, and the recovery for three different sera is ranging from 89 to 106%. the data presented in this paper indicated that the activation of polystyrene micro titer plate by the gamma rays could be use for preparation of igm-rf kit and may be others immunoassay techniques. overcoming the nuclease barrier to gene expression during trafficking of plasmid dna vectors a.r. azzoni 1 , a. tavares 2 , g.a. monteiro 1 , d.m.f. prazeres 1 : 1 centro de engenharia biológica e química (cebq), instituto superior técnico, 1049-001 lisboa, portugal; 2 instituto gulbenkian de ciência, rua da quinta grande 6, 2780-156 oeiras, portugal. e-mail: azzoni@ist.utl.pt (a.r. azzoni) inefficient nuclear delivery of plasmid dna (pdna) vectors is thought to be a bottleneck to gene transfer in gene therapy and dna vaccination utilizing non-viral delivery systems. one of the main barriers found by pdna vectors during trafficking to the nucleus is degradation by a population of endo/exo-nucleases. this barrier may be partially circumvented by shielding the pdna from the nucleaserich cell environment with adjuvants or by using nuclease inhibitors. a different approach that has been studied at the cebq is the generation of pdna vectors that are more resistant to nuclease action a priori. in this work, the nuclease barriers to gene expression are being studied aiming at the generation of pdna with improved resistance to nucleases and thus higher transfection efficiency. by engineering the plasmid labile sequences, new plasmid vectors with an improved resistance to physical-chemical and biological degradation are being generated. this was indicated by an extended half-life of the supercoiled isoforms during storage and when the plasmids were exposed to nucleases present at eukaryotic cell lysates and mice plasma. the intracellular trafficking of the new plasmid vectors through the cytosol of mammalian cells was then assessed by fluorescence in situ hybridisation (fish) and the expression of the reporter protein (egfp) was detected by fluorescence microscopy. identification and evaluation of antibacterial phytochemicals of fishbone fern (nephrolepis cordifolia) rikhia chakraborty 1,2 , promod kumar verma 2 : 1 department of cancer biology, lerner research institute, 9500 euclid avenue cleveland, oh 44195, usa, 2 department of biotechnology, guru nanak dev university, amritsar, punjab 143005, india. e-mail: riar5400@rediffmail.com (r. chakraborty) in this study, different aqueous and organic extracts from the fern, nephrolepis cordifolia, were used for screening tests for antibacterial effects. protein and lipid extracts were first tested for antibacterial activity. subsequently, crude extracts of leaves, roots, and stems were prepared in methanol, ethanol, chloroform, hexane, petroleum ether, diethyl ether, and water using optimized standard protocols. each fraction was tested for anti-microbial effect through agar well-diffusion assay, and paper disc method. the antibacterial spectrum against which the fern is active was thus determined. dosagedetermination for optimum activity was also determined for each of the extracts. bacillus and staphylococcus were used as the indicator test-organisms. the results were very encouraging; being effective even at the 54th day, thus showing that the antibacterial properties were not due to any changes in external factors and physiological effects. ethanol, methanol and chloroform extracts from the subaerial portions had strong anti-microbial properties. agar-well diffusion assay and paper-disc diffusion assay done for different solvent fractions were giving comparable results. 2.5 mg was adequate for maximum effect against b. circulans, s. aureus, s. epididermis, and streptococcus sp. 7.5 mg was adequate as effective dosage for kleb-siella pneumoniae. 10 mg was required for mycobacterium bovis, e. coli. pseudomonas was not showing any susceptibility. given the broad spectrum of activity, especially towards the gram-negative bacteria, nephrolepis cordifolia is definitely a promising plant having pharmacological importance. for a full interpretation of the present results further investigations are necessary to elucidate the different physical and chemical parameters of the active principles and also to determine the mechanism of action. the present work highlights n. cordifolia as a plant having a broad spectrum of antimicrobial activity, a phenomenon very rarely observed in the plant kingdom. bacteria (escherichia, salmonella, proteus, staphylococci and bacillus) were isolated from hospital soil using selective enrichment and growth on selective and differential medium, viz. macconkey's agar, clyed medium and baird parkers medium. confirmation and species identification was carried out by biochemical and serological tests. from these isolates, three different pathogens were used to study multiple antibiotic resistances. e. coli bj 83 showed resistance to ampicillin, streptomycin and cefurixime. s. typhi and s. aureus showed resistance to ampicillin and cefuroxime. assay using octadisc using e. coli and s. typhi showed a broader resistance pattern to antibiotics including amoxicillin, clavulanic acid, cephalexin, chloramphenicol, ciprofloxacin, and cotrimoxazole. the r plasmid profile was studied to understand the mechanism of drug resistance. to combat the problem of drug resistance, a strategy of combined antibiotic response of cultures were studied. such a synergistic combination would possibly have the effect of overcoming multiple antibiotic resistances. for e.g. kanamycin resistance strains were inhibited in presence of kanamycin and cefotaxime. further, the bactericidal activities of antimicrobials in honey and garlic were also tested. the mic of honey was observed to be 4%, while that of garlic was between 0.1 and 1%. honey and garlic were also found to inhibit the growth of organisms in the presence of antibiotics. kanamycinresistant e. coli was unable to grow in presence of kanamycin and 0.08% garlic. these traditional agents, long used in ayurvedic system of medicine in india, could be further explored for potent antimicrobial properties. hepatitis b virus (hbv) infection is s global health problem. assays for hbv antigens and antibodies are widely available and standardized. extremely sensitive qualitative pcr kits are also available for detection of hbv in serum. hbv pcr kit may be useful in assessment of occult hepatitis b in hbcab positive alone subjects and carriers. a positive pcr results show presence of virus articles in serum without considering serologic results. but there are differences in efficiency of hbv dna amplification kits. in this study we compare two commercially available hbv pcr kits for evaluation viremia of hbsag positive carriers and hbcab alone positive subjects. material and methods: of the 368 randomly selected subjects serologically examined for hbv, 49 and 43 were positive for hbsag and hbcab alone respectively. both later groups were tested for hbv dna by two commercial kits, hbv pcr detection kit (cinnagen, iran) and hbv pcr test (pazhohesh azma, iran). dna extraction kits recommended by each manufacturer were used for hbv dna extraction. amplicons in both kits were a highly overlapped fragment in 5 conserved sequence of viral genome. results: of the 49 hbsag positive carriers, hbv dna was detected in 37.4 and 65.3% using kit1 and kit2 respectively. only 28.6% were positive in both kits. in hbcab positive subjects (n = 43), 23.3% were positive by kit 2 and all samples were negative when tested by kit 1. there was a significant difference between two kits. sensitivity of kit 1 and kit 2 were 48.6 and 91.4%, respectively. overall, kit 2 increased the detection rate of hbv dna by 88.2%. discussion: our study show there is a significant variation between these two commercial kits especially in hbcab positive subjects that the copy of virus is very low. from these results, it can be concluded that the unstandardized kits have not compatible results, and pcr test interpretation should be done with great care. . the antibacterial activity of the extracts was evaluated based on the inhibition zone using plate diffusion method. most of the extracts were active against both gram positive and gram-negative bacteria, but pseudomonas aerugenosa, bacillus subtilis and sarcina marcescens, were more susceptible to almost all the extracts. finally, further research will be done to elucidate the nature of the active compound and investigate for peptides by using protein gel immobilization bioassay. short interfering rna delivery and gene silencing using polymeric nanocarrier systems k.a. howard 1 , x. liu 1 , d. oupicky 2 , f. besenbacher 1 , j. kjems 1 : 1 inano, university of aarhus, denmark, 2 department of pharmaceutical sciences, wayne state university, detroit, usa the effectiveness of a drug is determined by the ability to migrate through the body and reach target sites in therapeutically relevant levels. nanocarriers for delivery of bioactive agents are being developed at inano to maximise drug payload at target sites. the inclusion of "biological triggers" into the nanocarrier design is used for modulation of cellular nucleic acid trafficking and increased target interaction. chitosan and peptide-based polymers were used to formulate nanocarriers in the size range 30-250 nm containing small interfering rnas (sirnas) for gene silencing applications. page analysis showed the structural integrity of the sirna was maintained during particle formation. in systems composed of bioresponsive polymers, nanocarrier disassembly and sirna release under cellular conditions were shown, using atomic force microscopy. the time course for sirna uptake into nih cells was visualised using confocal microscopy. in addition, sirna localisation within cells could be modulated by the composition of the polymer used. the ability of the nanocarrier system to mediate gene expression was investigated in a cell line stably expressing enhanced green fluorescent protein (egfp). furthermore, the various delivery systems were tested in a mouse model stably expressing the egfp protein using both nasal and intravenous delivery routes. the use of animals as a source of organs and tissues for xenotransplantation can overcome the growing shortage of human organ donors. however, the presence of xenoreactive antibodies in humans directed against swine gal antigen present on the surface of xenograft donor cells leads to the complement activation and immediate xenograft rejection as a consequence of hyperacute immunological reaction. the graft of genetically modified organ of a swine depleted of enzyme ␣1,3-galactosyltransferase that is responsible for gal antigen origin, would be tolerated with simultaneous administration of medicines decreasing other less severe immunological reactions. to prevent hyperacute rejection it is also possible to modify swine genome by human genes controlling enzymatic cascade of complement or modifying the set of donor's cell surface proteins. for this purpose genetic constructs containing inactivated ␣1,3-galactosyltransferase gene, human cd59, cd55 and cd46 genes controlling complement activation and human genes encoding ␣1,2-fucosyltransferase and ␣-galactosidase enzymes modifying cell surface proteins were prepared. these genetic constructs were transfected into the pig foetal fibroblast using lipofection method. after selection, molecular and cytogenetic characteristic of cells with transgene integrated into the host genome were performed. introduction: protease 2a(2a-pro) of coxsackievirus b3 (cvb3) plays major role in viral replication. in case of infection, viral proteins are being synthesized from viral mrna using host biosynthesis machinery. 2a-pro of virus, after being synthesized, exhibit two critical functions, cleavage of viral proteins and breaking eif4g (eukaryotic initiation factor 4g-formerly called p220) which leads to host cell translational system shot-off. the enzyme plays essential role in viral replication and cellular damage. to understand pathogenicity of infection and also developing potent and selective inhibitors against picornavirus infection, it is necessary to prepare pure 2apro enzyme. in this study an expression system with efficient and high yields was obtained. methods: cdna of 2apro was synthesized using in vitro infection of permissive host through reverse transcription process and was cloned in pet22b(+) and since 2a-pro is a toxic product, naturally before induction its expression will act on the host and damage the cells. for this different hosts were checked and finally, blr(de3) plyss which carries an extra-plasmid for lysozyme expression that minimizes unwanted target protein production (leakage) was selected. on the other hand for biological activity assay, polyclonal antibodies against antigenic sites of p220 was prepared by synthesizing small peptides, corresponding to antigenic site of p220 coupling to klh and injecting subcutanously to rabbit. then, the enzyme and its substrate (hela cells lysate that contain p220) were incubated together for different times intervals. results: the recombinant product was confirmed by sds polyacrylamide gel electrophoresis and immunoblot analysis. also p220 cleavage by 2apro was assessed by sds-page and western blot analysis. cleavage of p220 by r2a-pro was prominent after 24 h. so recombinat 2a-pro with good activity was prepared. application of bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system on production of glycoprotein in larvae of silkworm ayano kageshima, tatsuya kato, misun kwon, enoch y. park department of applied biological chemistry, shizuoka university, ohya 836, suruga-ku, shizuoka 422-8529, japan bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system was applied on production of glycoprotein in larvae of silkworm. the bacmid system of autographa californica nuclear polyhedrosis virus (acnpv) has already been established and widely used. since the acnpv does not have a potential to infect silkworm we developed the first practical bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system directly applicable for the protein expression of silkworm. by using this system, the green fluorescence protein and glycoprotein, human ␤1,3-n-acetylglucosaminyltransferase 2 were successfully expressed in silkworm larvae not only by infection of its recombinant virus but also by direct injection of its bacmid dna. three different kinds of signal sequences were tested for the secretion of glycoprotein into hemolymph of silkworm. signal peptides of prophenoloxidase-activating enzyme and bombyxin: insulin-like brain secretory peptide showed the highest secretion ratio, 99% of total expressed ␤1,3-n-acetylglucosaminyltransferase 2 was secreted into hemolymph of silkworm. using bacmid system 50 mu/ml of ␤1,3-n-acetylglucosaminyltransferase 2 was expressed in hemolymph of silkworm, which was 2-three times higher than that of insect cell. silkworm is one of the most attractive hosts for large-scale productions of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. this method provides the rapid protein production in silkworm, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses. we have established an efficient system for foreign gene expression in lily (lilium longiflorum) pollen in a transient mode. pollen was transformed using agrobacterium via vacuum filtration for 20 min. the pollen germinated for 24 h was analyzed to confirm its foreign gene expression in molecular analysis. mouse fed the transgenic pollen culture for 8 weeks showed immune reaction specific for pollen-derived recombinant protein. and the igg level was highly elevated by one time boosting injection afterwards. the lily pollen system may be suggested as a novel type of biofactory for producing edible vaccine with rapidity. anti-apoptosis engineering with the 30kc6 gene obtained from silkworm shin sik choi, won jong rhee, tai hyun park school of chemical and biological eng., seoul national university, seoul 151-744, korea the chinese hamster ovary (cho) cell line producing recombinant human erythropoietin (epo) was manipulated to express the 30kc6 gene, which was originally obtained from a silkworm. the expression of 30kc6 inhibited serum deprivation-induced apoptosis and increased the cell density and epo expression level per unit cell by five-and two-folds, respectively. an increase in these two factors resulted in a 10-fold increase in the volumetric productivity of epo. compared with the controls, the oligosaccharide structures of the epo synthesized by the cells expressing 30kc6 showed greater homogeneity. the terminal sialylation of the glycans of epo were promoted by the expression of 30kc6. the positive effects of 30kc6 expression on the cell viability and productivity were attributable to the stable maintenance of the mitochondrial activity. these results demonstrate that the cho cell line genetically engineered with the 30kc6 gene has a great potential for use in the production of therapeutic proteins. evaluation of fucoidan-chitosan hydrogels on superficial dermal burn healing in rabbit: an in vivo study a.d. sezer 1 , f. hatipoglu 2 , z. ogurtan 3 , a.l. baş 4 , j. introduction: healing of dermal wounds with macromolecular agents such as natural polymers is one of the research areas of the pharmaceutical biotechnology. fucoidan is a sulphated polysaccharide which is commonly obtained from seaweeds. although a great number of studies on the different pharmacological properties of fucoidan are present, there is very limited information on the fucoidan-based system used in dermal burns. the aim of this study was to prepare chitosan hydrogel containing fucoidan and to investigate this hydrogel formulation for treatment of dermal burns on rabbits. methods: fucoidan-chitosan hydrogels were prepared as follows: the polymers were dissolved in acidic solution and sonicated for removing the air-bubbles then the gels were stored at +4 • c for in vivo studies. seven adult male new zealand white rabbits (mean weight, 3.8 ± 0.6 kg) were used for the evaluation of the gels on superficial dermal burns. the back of the rabbits were depilated and sedated. the wounds were made by circular stamp aluminium caps (3.8 cm 2 ) at 80 • c. four wounds were formed for each rabbit; (a) was treated with fucoidan-chitosan gel, (b) was treated with fucoidan solution, (c) was treated with chitosan gel (without fucoidan) and (d) as a negative control group. biopsy samples were taken at 7, 14 and 21st days at the beginning of the study and each wound site was macroscopically observed and evaluated histopathologically. results: oedema was not observed in all groups after 3 days treatment except controls. after 7 days treatment, fibroplasia and scar were observed on wounds treated with fucoidan-chitosan gel and fucoidan solution. the best regenerate dermal papillary formation and the fastest closure of wounds were observed in group a after 14 days treatment. the wound epithel elongation and thickness values were measured; a (5566 and 179 m), b (3586 and, 146 m), c (3666 and, 162 m), d (3533 and, 134 m) at the end of the study. conclusion: re-epithelization and contraction of the wound area which was treated with fucoidan-chitosan hydrogel were faster than the other groups. the fucoidan-chitosan hydrogel formulations can be suitable for the treatment of dermal burns. aim: to analyze the neutralizing -related activity of antibodies against e1 region of hcv, specific polyclonal antibody was raised by immunized rabbits with synthetic peptide that had been derived from e1 region of hcv with the amino acid sequence e1 antibody [ghrmawdmm). materials and methods: hyper-immune hcv e1 antibodies were incubated over night at 4 • c with serum samples from patients positive for hcv rna, with different viral load, ranged 7-11 million copes/ml, then incubated 90 min to hepg2 cells. rt-pcr and flow cytometry were used to study the inhibition binding and entry effect of e1 antibody. direct immunostaining of e1 antibody conjugated with fitic and flow cytometry analysis showed reduced the mean fluorescence intensity in the samples pre-incubated with e1 antibody compared with samples without e1. of 18 positive serum samples, 13 (72%) samples showed completely inhibition of infectivity as detected by rt-pcr. conclusion: in house produced e1 antibody, blocks binding and entry of hcv virion infection to target cells suggesting the involvement of this epitope in virus binding and entry. isolation of these antibodies that block virus binding and entry will be useful in providing potential therapeutic reagents and for vaccine development. tumor necrosis factor beta (tnf␤) is a pleiotropic cytokine mediating its activity through the same receptors as the structurally related tnf␣. shortening of the n-terminal part has been reported to enhance its cytotoxic activity, with the explanation that removal of the flexible n-terminus reduces steric interferences in the receptor binding process. for studies of relationship between the n-terminal protein structure and physicochemical properties, three different forms mettnf␤, his7tnf␤ and n19tnf␤ were expressed in e. coli. high solubility of n19tnf␤ was expected, however, both analogs his7tnf␤ and n19tnf␤ were predominantly obtained in the form of inclusion bodies (ibs). on the other hand, mettnf␤ was equally distributed between the soluble and insoluble fraction. most probably, the composition of the n-terminal part, such as the exposure of hydrophobic residues in the case of n19tnf␤, or a mildly hydrophobic stretch of seven histidines appended to the natural hydrophobic n-terminus in the case of his7tnf␤, lead to incorrect folding and force aggregate formation. solubilization of ibs was performed under native and denaturing conditions using various concentrations of nls, urea and gndhcl. mettnf␤ ibs were easily dissolved with 0.2% nls, while his7tnf␤ and n19tnf␤ demanded denaturing conditions. structural differences in the nterminal part of various tnf␤ proteins were also reflected in protein refolding characteristics and chromatographic behavior. dilution, ultrafiltration and dialysis were used for refolding, and imac was chosen as the main chromatographic step. our results suggest that not only the length of the n-terminal part of tnf␤ but also the composition and exposure of certain amino acid residues affect its physicochemical properties. enzymatic modification of sphingomyelin long zhang, lars hellgren, xuebing xu biocentrum-dtu. e-mail: lz@biocentrum.dtu.dk (l. zhang) due to its major role in maintaining the water-retaining properties of the epidermis, ceramide is of great commercial potential in cosmetic and pharmaceuticals such as hair and skin care products. currently, chemical synthesis of ceramide is a costly process, and developments of alternative cost-efficient, high yield production methods are of great interest. in the present study, the potential of producing ceramide through enzymatic hydrolysis of sphingomyelin (sm) have been studied. sm is a ubiquitous membrane-lipid and rich in dairy products or by-products. in present study, we have optimized the production of ceramide from sm using phospholipase c from clostridium perfringens. water and enzyme amount had the biggest influence on sm hydrolysis in the system. botulinum neurotoxins constitute a family of bacterial toxins for botulism syndrome in human. the toxins bind with high affinity to nerve cells where they cause a complete inhibition and release of neurotransmitters and thereby produce flaccid paralysis. in this work we have reported isolation of the binding domain of type e neurotoxin by pcr and expressed in a proper expression vector. the output of this investigation can be used as a tool to study the mechanism of binding of holotoxins and also can be useful to study the antibody production against botulism syndrome. the synaptobrevin (vamp2) a protein which play a key role in the fusion and exocytosis of the vesicle of mammalian nerves terminals this protein is substrate different serotypes of botulinum neurotoxins. light chain of clostridium botulinum type b, d, f and g cleave end of neurons. due to intraction of clostridium botulinum neurotoxin with vamp2, this protein can be used as one of the toolsin the detection of poisings cause by this bacteria in the clinical laboratory using the enzyme with proof reading activity the above gene amplified by pcr technique for the production of the recombinant protein, the prokaryotic expression vectors (pet system) was used. a recombinant protein developed on poly acrylamide gel analyzed by western blotting and eliza. most children with down syndrome (ds) are born to younger mothers (<35 years). recent reports linking down syndrome (ds) to maternal polymorphisms at the methylenetetrahydrofolate reductase (mthfr) gene locus have generated great interest among investigators in the field. in this study, forty mothers with their affected outcomes and 100 control mothers were included. all mothers were subjected to complete medical and nutritional history with special emphasis on folate intake through food or oral supplementation. estimation of blood homocysteine level was done. also we examined the two polymorphisms in genes encoding the folate metabolizing enzyme methylenetetrahydrofolate reductase (mthfr), namely, 677c > t and 1298a > c. folic acid intake from food and from vitamin supplements was significantly low (below the recommended daily allowance) in the group of mothers with ds children compared to control mothers (p < 0.01) using student t-test. blood homocysteine was normal in both control and ds mothers. the prevalence of the two polymorphisms, namely, 677c > t and 1298a > c in mothers of ds children (case mothers) (n = 40) was compared with controls (n = 100). frequencies of mthfr genotypes (cc, ct, and tt) at position 677 demonstrated no difference between the case and control groups. genotype frequencies of mthfr at position 1298a (aa, ac, and cc) were different among the case and control mothers. we here report the first study on a possible relation between ds with mthfr 1298a > c genotypes in egypt. our results showed that mthfr 1298a > c polymorphism is a remarkable genetic entity among egyptian females with d.s. children. sufficient folate intake and supplementation is an important preventive strategy in overcoming the risk of nondisjunction. homologous recombination (hr) is the mechanism that permits the creation of genetically engineered strains through gene targeting. in order to further develop gene targeting techniques, notably for higher eukaryotes such as filamentous fungi, it is of crucial importance to fully understand the molecular mechanisms behind mitotic hr. in saccharomyces cerevisiae, a dna double strand break (dsb) is an essential intermediate in meiotic hr. however, as hr occurs at a low rate in mitotic cells, it has been difficult to determine the nature of the event(s) that triggers it. rad52 is a key protein involved in hr and is evolutionarily conserved from yeast to human. to shed light on the molecular events in hr, we have generated a large collection of defined rad52 mutant strains in the yeast s.cerevisiae. a screen of these mutants led to the identification of strains that fail to repair dna dsbs, yet are proficient for homologous recombination. this result strongly suggests that dsbs may not be the major cause of spontaneous mitotic hr and gives new perspectives in respect to novel potential gene targeting substrates. we have analyzed these separation of function mutants in a variety of new assays to obtain a more detailed understanding of their controversial phenotype. our latest results will be presented. the outcome of interferone plus ribavirine treatment of hepatitis c virus (hcv) genotype 4 is unfortunately poor. development of alternative therapy for this genotype is of a paramount importance. inhibition of hcv gene expression in vitro by the use of antisense phosphorothioate oligodeoxynucleotides (s-odn) against internal ribosomal entry site (ires) elements were associated with favorable results. to assess s-odn activity, previous studies utilized viral subgenomic or full cdna fragments linked to reporter genes and transfected into adhered cells or in a cell free system. in the present study we utilized hepg2 cells infected with native hcv rna of genotype 4. the culture system presented herein was shown to support hcv replication on the following bases (1) consistent detection of both plus and minus rna strands for 4 weeks in cells and in fresh culture supernatent, (2) ability of supernatent to infect naive hepg2 cells (3) consistent expression of core and e1 envelope proteins in infected cells throughout the 4 week culture. s-odns against aug translation start site (s-odn-1, nt 326-348) of the viral polyprotein precursor and stem loop iiid within the ires region (s-odn2, nt 264-282) were added to infected cells. intracellular viral replication was monitored by nested rt-pcr of plus and minus strands. the results of these experiments demonstrated that intracellular replication of hcv genotype 4 was completely arrested after 48 h in culture using either s-odn molecule (with more efficacy of s-odn1 than s-odn2) at concentrations as low as 1 m. the inhibitory effect of s-odn appeared to be specific to hcv replication since equal levels of human glyceralehyde 3-phosphate dehydrogenase (gapdh) gene expression were noted pre and post supplementation of s-odns. in conclusion, the present study provides evidence antisense phosphorothioate oligonucleotides have potent inhibitory effect on genomic replication of hcv genotype 4, the most common type in egypt. a sensitive and specific pcr-elisa was developed to detect shigella dysentery in food. the assay was based on the incorporation of degoxigenin-labeled dutp and a biotin-labeled primer specific for shiga toxin genes during pcr amplification. the labeled pcr product were bound to streptoavidin-coated wells of a microtiter plat and detected by an elisa. the elisa detecting system was able to increase the sensitivity of the pcr assay by up to 100-fold, compared with a conventional gel electrophoresis. the detection limit of the pcr-elisa was 0.1-10 cfu dependent upon shigella dysentery serotypes and genotypes of shigatoxin. the entire procedure took about 4 h. isolation, cloning, expression and purification of snap-25 as a substrate of botulinum neurotoxin type a and e m.l. mossavi 1 , f. ebrahimi 1 , j. amani 1* , h. basiry 1 , z. ahmadi 2 : 1 department of biology, faculty of science, imam hussein university, tehran, iran; 2 department of biology, faculty of medicine, bageatallah university, tehran, iran. e-mail: kpjamani@ihu.ac.ir (j. amani) clostridial neurotoxin inhibit neurotransmitter relase by selective and specific intracellular proteolysis of synaptosomal associated protein of 25 kda (snap-25); synaptobrevin/vamp-2 and syntaxin. snap-25 is one of the components that form docking complex in synaptic ends. this protein is substrate for botulinum neurotoxins type a and e. each of these toxin serotypes specifically cleave snap-25 in particular position and there by block docking and synaptic vesicle membrane fusion and finally prevent neurotransmitter exocytosis and transition of neurotic signals. in order to use the protein as a substrate for detection of different type of clostridium neurotoxin in vitro test, the protein was produced by recombinant technique. the dna encoding snap-25 was isolated from rat brain by pcr using the two primer the amplified fragment colonel into expression vector pet32a.the expression protein was purified by affinity chromatography. confirm by different method the his tag fusion protein was digested with entrokinase. the present work deals with the preparation of pure alpha-1antitrypsin (aat) protein from healthy subjects, which can be used in preparing its corresponding monospecific antibody in albino rabbits. this antibody was found very useful in the immuno-diagnosis of pulmonary emphysema. this study has been also concerned with the biochemical changes associated with aat deficiency in pulmonary diseases. to fulfill this work, a group of healthy blood donors was selected for separation of pure aat antigen from blood. the pure aat was used for the preparation of anti-aat, the purity and potency of antibody was checked by titration methods. the biochemical changes were studied in thirty subjects clinically divided into three groups including, control, heavy cigarette smokers with pulmonary emphysema, and non-smoking subjects with pulmonary emphysema. the activity of elastase and hydroxyproline (hp) level as a marker of elastin and collagen breakdown were assayed in bronchoalveolar lavage (bal) fluid. the activity of aat and to inhibit proteases as represented by tryptic inhibitory capacity (tic) was evaluated. serum ceruloplasmin, transferrin and iga level as well as thiobarbituric acid reactive substances (tbars) were estimated in all groups. our data revealed that aat and its tic showed very highly significant decreased levels in all patients with emphysema as compared to control, while the elastase activity and hp level in bal fluid were significantly increased in these patients. serum tbars was significantly increased in such patients associated with increasing level of both ceruloplasmin and transferrin. while, serum iga was significantly increased. furthermore, the biochemical changes were markedly changed in smokers with emphysema than non-smoking subjects. in conclusion, the preparation of anti-aat on the local level is very important where less expensive and less time consuming and can be useful in immuno-diagnosis and prognosis of pulmonary diseases. a new method combined with boosting and projective adaptive resonance theory for analysis of gene expression data from cancer patients hiro takahashi, yasuyuki murase, hiroyuki honda department of biotechnology, school of engineering, nagoya university, nagoya 464-8603, japan. e-mail: h041305d@mbox.nagoyau.ac.jp (h. takahashi) an optimal and individualized treatment protocol based on accurate diagnosis is urgently required for the adequate treatment of patients. for this purpose, it is important to develop that a sophisticated algorithm that can manage large amount of data, such as gene expression data from dna microarray, for optimal and individualized diagnosis. in our previous study, we developed the projective adaptive resonance theory (part) as a gene filtering method and boosted fuzzy classifier with sweep operator (bfcs) as a modeling method. in the present study, we applied the combination of part and bfcs (part-bfcs method) to microarray data of brain tumor (central nervous system tumor) obtained from the website. the method enabled the selection of 14 important genes related to the prognosis of the tumor, i.e., sensitivity for combined therapy with surgery, radiotherapy, and chemotherapy mainly with vincristine, cisplatin, and cytoxan. the constructed model showed about 20% higher accuracy than that of the conventional method. genomic signal analysis of hiv variability based on rt gene p.d. cristea, rodica tuduce d. otelea biomedical engineering center, university "politehnica" of bucharest, romania. e-mail: pcristea@dsp.pub.ro (p.d. cristea) dna sequences genotyped from 60 clade f hiv-1 isolates from romanian patients in the laboratory of the national institute of infectious diseases "matei bals", bucharest, romania, have been studied. the symbolic sequences have been converted into digital genomic signals by using a complex quadrantal representation of the nucleotides described earlier. the cumulated phase and unwrapped phase of a complex genomic signal reflect the statistical distribution of bases and base-pairs, respectively. independent component analysis of the genomic signals has been used to characterize the variability of the f subtype hiv strains isolated in romania. the sequenced segment is of (about) 1302 base pairs, approximately aligning with the standard sequence of hiv-1 (accession nc001802 in genbank) over the interval 1799-2430 bp. this segment, which is currently used for the standard identification and assessment of hiv-1 strains, comprises the protease (pr) gene and two thirds of the reverse transcriptase (rt) gene. only results referring to the analysis of the rt gene region are presented here and used for extracting features of virion isolates and for establishing phylogenetic trees of the studied strains. the analyzed rt encoding segment has the length 1005 bp and is located in the second interval (298-1302 bp) of the analyzed dna segment, respectively along the 2096-3100 bp region of nc001802. taking into account the mutations identified in these sequences, the samples were classified in three groups from the point of view of their resistance to current antiretroviral compounds: sensitive, resistant and multiresistant. the paper presents results for the isolates in which mutations leading to multiple drug resistance have been identified. over expression of secb protein in e. coli enhances the periplasmic expression of human growth hormone m. ghafari 1,2 , a. zomorodipour 2 : 1 islamic azad university of jahrom, tehran, iran; 2 national institute for genet eng and biotechnol., tehran, iran. email: maryamghafari2001@yahoo.com (m. ghafari) among several proteins involved in the secretion pathway of proteins in e. coli, secb plays a key-important role in solubilization of preproteins before processing. in order to increase processing of a human growth hormone precursor (pelb::hgh) which appeared to have problem in processing efficiency, as a possible solution a regulated co-expression of a secb gene was considered. in this regard, we designed an arabinose-regulated secb expressing plasmid compatible with an iptg/lactose-regulated pelb::hgh expressing plasmid. for the construction of the secb expressing plasmid the origin of replication and antibiotic resistant gene (amp) of a pbad vector was replaced by a p15a-ori and a kanamycin resistant gene, respectively. the expression and processing of pelb::hgh preprotein in the two-plasmid containing bacteria in a secb over-expression state was compared to that of the pelb::hgh expression in normal bacteria. although a decline in total expression level of hgh during the overexpression of secb was observable, probably due to presence of two different expressing plasmids, but both the processing efficiency of pelb::hgh and the transport of mature protein into the periplasmic space was enhanced during prolonged arabinose induction. current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts. natural allergen extracts contain a mixture of allergenic and non-allergenic components that are difficult to standardize. recombinant allergens can improve diagnosis and de-sensitization against single component. major cat allergen is a heterodimer composed of disulfide linked 7.8 and 10.1 kda polypeptide chains. both chains of feldi protein were obtained in e.coli system. purification of recombinant proteins was performed in denaturing conditions using immobilized metal affinity chromatography specific for proteins with histidine tag. from 1000 ml culture approximately 13 mg protein for feldi chain 1 and 43 mg of feldi chain 2 were obtained. after purification histidine tag was removed by hydrolysis with thrombin. immunological activity of feldi against serum of patients allergic to cat was narrowed to subgroup of patients allergic to feldi protein by surface plasmon resonance. immunological activity of each chain and renatured heterodimer was also tested using immunoprecipitation techniques against serum of population group. subdoligranulum variabile -a novel member of the human gut micro flora with a high prevalence kim holmstrøm, trine møller bioneer a/s, hørsholm, dk-2970, denmark in 2003 we isolated and cultured for the first time a bacterium from a human fecal sample representing a hitherto unknown member of the clostridium leptum rrna supra generic cluster. the c. leptum rrna supra generic cluster represents one of the 3 major phylogenetic lineages within the human gut microbiota, and is characterized by having only a small proportion of its members actually identified by cultivation compared to the estimated numbers of bacteria contained in this group from culture-independent gut flora analyses. s. variabile is an obligate anaerobe gram negative bacterium with a characteristic pleiomorphic coccoid-droplet-like cellular morphology. its closest previously cultivated relative based on a 16s rdna phylogenetic analysis is faecalibacterium prausnitzii, a rod-shaped and therefore easily distinguishable gram negative bacterium present in high numbers in the human fecal micro flora. based on 16s rdna sequence we designed a s. variabile specific oligonucleotide probe for use in fish analysis to estimate the prevalence of this "new" bacterium in fecal samples collected from healthy human beings. interestingly, we observed a high proportion of s. variabile present in all tested samples, and in some instances we observed a higher prevalence than the more well-known group of bifidobacteria equally estimated by fish analysis. documentation of these results will be presented. several species of sea cucumbers, long an incumbent of traditional medicines were selected as the source of animal based antibiotic compounds. swabs of the inner surface and coelomic fluid (inner fluid) samples from sea cucumber (holothuria atra jaeger) were taken. thirty strains of bacteria were isolated. these strains were grown in different antibiotic production media. nine human pathogenic bacterial species were used as test agents and they are, k. pneumoniae, mrsa, m. luteus, s. thyphimurium, s. epidermitis, s. saprophyticus, b. subtillis, p. aeruginosa and s. pyogenes; only four bacterial strains showed mild antibiotic activity against s. pyogenes and s. thyphimurium. similar testing on two other species, h. scabra and s. variegatus will be carried out. different media, especially antibiotic production enrichment media will also be used. characterization will be done upon obtaining an antibiotic compound, which shows moderate to high activity against at least one of the nine human pathogens used. for plant based medicines, three rhizomes, "cekor," "jerangau" and "bonglai" were analyzed. solvent extraction using ethanol, methanol and acetone was carried out, at a concentration of 20-50 mg per ml solvent. the filtrates were used for the antibiotic testing stage. all the three plant species showed moderate antibiotic activity against m. luteus, s. epidermitis, s. pyogenes and s. saprophyticus. interestingly, the antibiotic activity increased when combinations of the herbal extracts were used. cell-based therapy continues to be a promising avenue for the treatment of duchenne muscular dystrophy, an x-linked skeletal muscle-wasting disease. recently, we have demonstrated that freshly isolated myogenic progenitors contained within the adult skeletal muscle side population (sp) can engraft into dystrophic fibers of non-irradiated mdx 5cv mice after intravenous transplantation. engraftment rates, however, have not been therapeutically significant, achieving at most 1% of skeletal muscle myofibers expressing protein from donor-derived nuclei. to improve the engraftment of transplanted myogenic progenitors, an intra-arterial delivery method was adapted from a previous procedure. cultured, lentivirus transduced skeletal muscle sp cells were transplanted into the femoral artery of non-injured mdx 5cv mice. based on the expression of microdystrophin and gfp transgenes in host muscle, sections of the recipient muscles exhibited 5% to 8% of skeletal muscle fibers expressing donor-derived transgenes. further, donor muscle sp cells, which did not express any myogenic markers prior to transplant, express the satellite cell transcription factor pax7 and the musclespecific intermediate filament desmin after extravasation into host muscle. the expression of these muscle-specific markers indicates that progenitors within the side population can differentiate along a myogenic lineage after intra-arterial transplantation and extravasation into host muscle. given that femoral artery catheterization is a common, safe clinical procedure and that the transplantation of cultured adult muscle progenitor cells has proven to be safe in mice, our data may represent a step towards the improvement of cell-based therapies for dmd and other myogenic disorders. metabolic engineering design of an extracellular hgh synthesis system birgül şentürk 1 , pınar ç alık 2 , güzide ç alık 1 , tunçer h.özdamar 1 : 1 bre lab, department of chemical engng, ankara university, 06100 ankara, turkey; 2 iblab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: ozdamar@eng.ankara.edu.tr (t.h.özdamar) metabolic engineering design of an extracellular human growth hormone (hgh) synthesis system is based on cloning the dna encoding human therapeutic protein together with the signal dna sequence of an extracellular enzyme gene into a host-vector system. in this context, extracellular protease (subc) signal dna sequence, i.e. pre-signal dna sequence, was fused into the frame in front of hgh mature dna sequence, by the use of four primers designed using pcr-based gene splicing by overlap extension method. b. licheniformis chromosomal dna and plasmid carrying hgh cdna were used as templates in pcrs, respectively, for the amplification of the subc signal dna sequence and hgh mature peptide sequence. for the fusion of two target genes, i.e. mature peptide sequence of hgh and, signal dna sequences were amplified separately by pcrs. the primers used at the ends to be joined were designed as complementary to one another by including nucleotides at their 5 ends that are complementary to 3 portion of the other primer. these products were mixed in the next pcr reaction, where one strand from each fragment contains the overlap sequence at 3 end. extension of this overlap by dna polymerase has yielded the recombinant hybrid-gene; and hybrid-gene serve as template for the continuation of reactions for the increase of the concentration in the microreactors. the hybrid gene fragment was first cloned into puc19, and then sub-cloned to pmk4 e.coli-bacillus shuttle plasmid. thus, a new expression vector with high stability and high copy-number was obtained and transferred into host b. subtilis 168 (spo − ). the metabolic flux distributions calculated by the mass balance based stoichiometric model based on the proposed metabolic reaction network for r-b.subtilis were determined by using time profiles of the substrate, dry-cell, hgh, amino acids and organic acids concentrations as the constraints. on the basis of the intracellular bioreaction rates and the interactions with the bioreactor operation parameters, an in-depth insight will be provided for further metabolic engineering design for the extracellular hgh production in r-b.subtilis. medicines based on polypeptides consisting of 30 and more amino acid residues are widely spread in pharmaceutical market at the present time. practically all polypeptide medicines known are prepared by general chemical synthesis that caused high cost of their production. that's why biotechnological way of polypeptide medicines preparation using recombinant gene expression in bacteria seems to be promising. during our work we design hybrid construction allowing solving a problem of specific cleavage of target polypeptide from the hybrid protein using system of protein splicing from new england biolabs. in case of thymosin ␣ 1 production impac system (intein mediated purification with affinity chitin-binding tag) has been used, where the modified sce vma from s. cerevisiae has been applied as intein. in contrast to other systems, impact allows the preparation of target protein without using of serine protease and other factors that may cleave the hybrid protein. in the presence of thiol reagents, such as dithiothreitol, mercaptoethanol, or cystein the hybrid protein can be site-specifically cleaved to give intein, the target protein and small fragment of nextein. using of this system does not allow to obtain the target protein with ser, cys or thr on n-terminal of protein, because in those cases target protein and n-extein ligation product will be formed. ser is n-terminal amino-acid in thymosin ␣ 1 . it was recently found that some metal ions essentially affect the splicing. we tried to use zncl 2 and we have found that, in case of intein-thymosin ␣ 1 , the maximal yield of target polypeptide and the minimal yield of splicing products are observed in the absence of dithithreitol and in the presence of 0.5-1 mm zinc chloride in buffers on all stages of thymosin ␣ 1 isolation. the structure of recombinant thymosin ␣ 1 of human was confirmed by the determination of n-and c-terminal amino-acid sequences and by maldi tof mass-spectrometry. mature embryos of five t. aestivum and five t. durum cultivars formed embryogenic callus on two different media. embryos were removed from surface sterilised seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of murashige skoog and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-d) or 1 mg/l naphtalenacetic acid (naa). the developed calli and regenerated plants were maintained on 2,4-d or naa free ms medium. wheat plants can be regenerated via two different systems. there were significant differences in percentage of callus induction and regeneration capacity on the different initiation medium. among the t. aestivum cultivars, yakar had the highest regeneration capacity in both induction medium. in t. durum cultivars, kiziltan gave the highest regeneration capacity in ms + 2,4 d medium and yilmaz gave the highest regeneration capacity in ms + naa medium. a strong genotypic effect on the culture responses was found for both induction medium. tumor necrosis factor beta (tnf␤) is a pleiotropic cytokine mediating its activity through the same receptors as the structurally related tnf␣. shortening of the n-terminal part has been reported to enhance its cytotoxic activity, with the explanation that removal of the flexible n-terminus reduces steric interferences in the receptor binding process. for studies of relationship between the n-terminal protein structure and physicochemical properties, three different forms mettnf␤, his7tnf␤ and n19tnf␤ were expressed in e. coli. high solubility of n19tnf␤ was expected, however, both analogs his7tnf␤ and n19tnf␤ were predominantly obtained in the form of inclusion bodies (ibs). on the other hand, mettnf␤ was equally distributed between the soluble and insoluble fraction. most probably, the composition of the n-terminal part, such as the exposure of hydrophobic residues in the case of n19tnf␤, or a mildly hydrophobic stretch of seven histidines appended to the natural hydrophobic n-terminus in the case of his7tnf␤, lead to incorrect folding and force aggregate formation. solubilization of ibs was performed under native and denaturing conditions using various concentrations of nls, urea and gndhcl. mettnf␤ ibs were easily dissolved with 0.2% nls, while his7tnf␤ and n19tnf␤ demanded denaturing conditions. structural differences in the nterminal part of various tnf␤ proteins were also reflected in protein refolding characteristics and chromatographic behavior. dilution, ultrafiltration and dialysis were used for refolding, and imac was chosen as the main chromatographic step. our results suggest that not only the length of the n-terminal part of tnf␤ but also the composition and exposure of certain amino acid residues affect its physicochemical properties. high level expression of recombinant growth hormones in e.coli faces common problems such as protein aggregation and inclusion body formation. discussions are raised whether it is more beneficial to obtain soluble protein but to loose expression rates. here we describe an experiment based on the hypothesis that slower expression should result in at least partially soluble recombinant protein. experiments were performed on bovine, chicken and mink growth hormones. expression rate was controlled externally by adjusting cultivation temperature, media, inducer amount, and both induction and cultivation times. another approach to the problem was performed through genetic manipulation. we changed strong t7 promoter to e. coli promoter consensus sequence thus reducing expression rate. recombinant growth hormone was still found to form aggregates, even when expressed at extremely low levelsseveral (2-8) percent of total intracellular protein. we developed optimization scheme of insoluble protein production and showed that expression rate minimization is not influencing recombinant growth hormone solubility in vivo thus suggesting an idea of sequence specific aggregation. to optimize the recombinant protein production in small-scale shake-flask system and high cell density fermentation, new tools have been developed at our laboratory which helps to get knowledge about the physiological state of the cell culture. these tools include (i) a quantitative monitoring system for cellular mrnas based on a sandwich hybridization technique, and (ii) a wireless online monitoring tool (senbit), applicable for standard sensors such as ph, po 2 and temperature for the continuous data collection from shake flasks. the senbit system is a new tool supplying valuable information for the optimisation of the expression of recombinant genes in shake flasks and allowing conclusions towards the reproducibility of shake flask cultures. the presentation will focus on use of the sensitive sandwich hybridization technology for the quantitative analysis of process relevant marker genes in different kind of microbial cell cultures with a focus on the production of recombinant proteins. samples from shake flask cultures and high cell density fed-batch fermentations of the yeast pichia patoris, have been analysed. additionally the mrna analysis was combined with the application of the senbit wireless system to study the production of a recombinant protein in shake-flask cultures of p. pastoris. aside from p. pastoris, mrna sandwich hybridization also was used to monitor product expression in fed-batch fermentation of e. coli for the production of a protein with two subunits by sequential induction. quantitative rna analysis as a tool for optimization of tetrameric collagen prolyl 4-hydroxylase production in e. coli a. neubauer 1 , m. bollok 2 , j. myllyharju 1 , p. collagen prolyl 4-hydroxylase (p4h) involved in the biosynthesis of collagens is an ␣ 2 ␤ 2 tetramer. recombinant expression of p4h in e. coli was described recently . the construct for cytoplasmic expression contains the genes of both subunits in one plasmid under control of different promoters. the ␣ subunit forms inactive aggregates, when expressed separately. in mammalian cells the ␤ subunit is available in large excess and keeps the ␣ subunit in a soluble active form. to mimic this in the bacterial system, we induced both genes sequentially. after induction of the ␤ subunit with iptg, expression of the ␣ subunit was initiated with anhydrotetracycline. here we use the analysis of the product mrnas with a bead based sandwich hybridisation assay (sha) (rautio et al., 2003) for optimization of the fermentation procedure. a high p4h activity was obtained if a high mrna level of the ␣ subunit could be maintained over a longer time. the obtained results illustrate the importance of the second induction for a high level expression of the p4h tetramer. the cells need to be in a "healthy state" with low metabolic load to react efficiently to the second induction. the data illustrate the optimization of a fermentation process by monitoring mrna levels which is of general interest for optimization of products which are difficult to detect. cell-based therapy continues to be a promising avenue for the treatment of duchenne muscular dystrophy, an x-linked skeletal muscle-wasting disease. recently, we have demonstrated that freshly isolated myogenic progenitors contained within the adult skeletal muscle side population (sp) can engraft into dystrophic fibers of non-irradiated mdx 5cv mice after intravenous transplantation. engraftment rates, however, have not been therapeutically significant, achieving at most 1% of skeletal muscle myofibers expressing protein from donor-derived nuclei. to improve the engraftment of transplanted myogenic progenitors, an intra-arterial delivery method was adapted from a previous procedure. cultured, lentivirus transduced skeletal muscle sp cells were transplanted into the femoral artery of non-injured mdx 5cv mice. based on the expression of microdystrophin and gfp transgenes in host muscle, sections of the recipient muscles exhibited 5-8% of skeletal muscle fibers expressing donor-derived transgenes. further, donor muscle sp cells, which did not express any myogenic markers prior to transplant, express the satellite cell transcription factor pax7 and the muscle-specific intermediate filament desmin after extravasation into host muscle. the expression of these muscle-specific markers indicates that progenitors within the side population can differentiate along a myogenic lineage after intra-arterial transplantation and extravasation into host muscle. given that femoral artery catheterization is a common, safe clinical procedure and that the transplantation of cultured adult muscle progenitor cells has proven to be safe in mice, our data may represent a step towards the improvement of cell-based therapies for dmd and other myogenic disorders. collagen and its derived product gelatin are attractive mammalian proteins to be used as model for the production of complex heterologous proteins in plants. the availability of a recombinant product will provide a safer, more homogeneous product than the current animal-derived material. the aim of the project is to investigate the feasibility of a production system for the accumulation of recombinant collagen for conversion to gelatin using barley. the 5 -end of the cocksfoot mottle virus (cfmv; genus sobemovirus) genomic rna sequence, called cfmv -element, has been shown to enhance recombinant protein synthesis in barley (wo 01/55298, mäkinen et al., 1995) . the -element will be used to study whether accumulation levels of complex mammalian proteins can be further increased, using collagen as a model that will serve as basis for exploring the expression of other complex proteins. this system can be study the production of barley-derived recombinant collagen for conversion to gelatin. classical swine fever virus (csfv) is an animal pestivirus which can be used as a surrogate model to elucidate the role of envelope glycoproteins of closely related human hepatitis c virus (hcv). the necessity to use the surrogate models for hcv is due to the fact that this virus cannot be grown in vitro cultures. csfv genome codes for three major antigenic glycoproteins which are located in the same cluster of genes; they are designated as e2 and e0 (e rns ) and e1. glycoproteins form heterodimeric and homodimeric complexes on the external part of viral particles. it is generally accepted that envelope glycoproteins play a major role in the initial stages of viral infection both for csfv and hcv. formation of complexes is needed to effectively infect host cells. we have investigated the formation of glycoprotein dimers by immunoperoxidase monolayer assay and by immunoblotting (western blotting). immunoblotting is a very useful technique in these studies because the complexes are formed via cysteine-cysteine disulphide bonds and they are retained during sds-page under non-reducing conditions. by modifying the glycoprotein genes and by arresting n-glycosylation of e2 and e0 we have investigated which factors influence the formation of complexes. it has been found that some glycosylation inhibitors which act at the early stages of glycan chain processing influence, not only glycosylation, but also the stability of e2 protein, effectively inhibiting the formation of glycoprotein complexes and the yield of the virus. these inhibitors are potential agents for arresting the multiplication and spread of csfv, and its relative -human hcv. recombinant proteins have been produced in a variety of heterologous protein expression systems. eukaryotic unicellular algae have distinct advantages, e.g. it can synthesize complex protein that requires post-translational modification. furthermore, microalgae can be grown in confined environment and thus prevents leakage of genes to the environment. our group has developed a platform technology for the production of recombinant proteins in red microalga porphyridium sp. we have constructed algal transformation plasmid vectors containing a camv 35s promoter and polya signal site. a streptoalloteichus hindustanus bleomycin-resistant gene was used as the selective marker. we have expressed ovalbumin and hepatitis-b surface antigen (hbsag) as model proteins. transformation was carried out by agitating algal cells and vector dna with glass bead. transgenic lines were selected by growing algal cells on agar plate containing 6 g/ml zeocin. positive transgenic lines were selected by screening the colonies by pcr and confirmed by dna sequencing. expression of ovalbumin and hbsag protein was examined by western blot analysis. ovalbumin was found to be expressed inside the algal cells while small hbsag was secreted into the medium due to presence of signal peptide. these findings indicate that red microalgae are capable of producing heterologous proteins. life-material exhibition as ethical interpretation chang shih-lung biotechnology industry study center, taiwan institute of economic research, taipei 106, taiwan. email: schang@tier.org.tw in this thesis we aim to explore the medical-related life-material exhibitions within ntu hospital-the humanity building, and taipei mackay hospital-the historical showroom as abecedarian clues to understand the burgeoning phenomenon-medical museums in taiwan. following these clues, we treat the whole context of medical museums, which transform values through situational construction, as the background to interpret the ethical implications of group val-ues that are transformed in the medical profession. in this thesis, we see medical museums, as the social prescription transforming medical profession, format the ritual context of situation ethics with the cultural construction of life-material. multi-disciplinary interactions and visiting itinerary can be transferred to the exploring horizon of research approach through description and interpretation. among them, we observe that humanistic elements have become essential equipment (or mat'eriel) of medical profession. though humanistic equipment (or mat'eriel) has its bottleneck in the museum situation, it can also unblock new possibilities for museum exhibitions, ethical practice or life ethics. as part of a wide research program aimed at developing new antitumoral agents, we present herein a series of stereoisomeric derivatives of fused tetrahydrofuranes (fthf) substituted with diverse protecting groups either at the primary or secondary hydroxyl groups. unprotected derivatives were also synthesised to investigate the influence of substituents on the in vitro activity of fthf. data on the synthesis, chemosensitivity and apoptosis induction by this series of fthf will be correlated to substitution pattern, stereochemistry and protecting groups, as an aid to the rational design of novel antitumour drugs. establishment of in vitro test systems for pulmonary edema resorption by peptide drug candidates dominik geiger, aswin mangerich, rudolf lucas, klaus p. schäfer, inge mühldorfer 1 department of biotechnology, altana pharma ag, konstanz, germany; 2 imc university of applied sciences, krems, austria tnf-␣ was found to up-regulate the rate of lung liquid clearance (llc) in several animal models by the activity of its lectin-like tip domain. this activity can be mimicked by a circular peptide designated tip. tip was shown to induce llc and consequently pulmonary edema resorption in different animal models. in order to study the mechanism of action of tip, we established two in vitro test systems for edema resorption with the human lung epithelial cell line calu-3: (1) the ability of calu-3 cells for spontaneous dome formation within confluent monolayers was utilized for quantitative examination of tip's activity on active transepithelial fluid transport ("dome assay"). (2) the effects of tip on bioelectrical properties of polarized cell monolayers were studied by using the transepithelial electrical resistance (teer) technology ("teer assay"). dome assay experiments confirmed that dome formation is a sodium dependent process and that tip is able to increase this process. teer assay experiments proofed that tip acts in a polarized and dose dependent manner. in conclusion, there is strong evidence that the dome and the teer assays are suitable systems for in vitro activity testing of tip and other anti-edema peptide drug candidates and are useful for studies on their mechanism of action. increasing safety concerns in gene therapy result in more stringent regulatory requirements. those cover the complete process chain of cell banking, fermentation, and purification. a wide range of applications for pdna requires gram to kilogram amounts for clinical trials and market supply. economic, productive and robust processes are a prerequisite for low cost of goods (cogs). therefore manufacturer of biopharmaceuticals need to address these considerations by developing new production processes meeting the new standards. boehringer ingelheim austria developed a novel pdna production process suitable for large-scale cgmp production. the process is based on e. coli fermentation. the process contains no components from animal origin. the optimized fermentation process yields up to 1 g pdna/l fermentation volume. for isolation of pdna from e. coli alkaline lysis in glass bottles or stirred tanks is commonly used. cell wall structure is destroyed by a combination of alkaline ph and detergents. in our process alkaline lysis is operated in a closed, continuous system directly connected to clarification without using enzymes. we developed a scalable process for pdna purification based on 3 different chromatographic principles. the capture step is carried out by hydrophobic interaction chromatography followed by anion exchange chromatography as intermediate step. final polishing is carried out by conventional size exclusion chromatography in a group separation mode providing also buffer exchange and desalting for the final formulation. the process results in a pdna drug substance of highest quality containing a low level of impurities (genomic dna, rna, proteins endotoxines) suitable for therapeutic applications. depending on the conditions during fermentation and the used host homogeneities of greater than 95% or even 98% are possible, while a high over all yield can be achieved (∼50%). during the development monolithic chromatography supports (cim ® ) were compared with conventional resins and evaluated as potential alternatives. the complete process is monitored by a set of analysis covering cell banking to final purification. new sensitive methods based on hpce, hpiex and fluorometric measurement were developed. whereas many natural amino acids are currently produced by very cost efficient biological processes, the manufacture of methionine is still performed by traditional chemical synthesis. this was mainly due to the poor performances of the currently available producing strains that inhibited the development and commercialization of a biological process to l-methionine. metabolic explorer has recently reinvestigated and developed an efficient biological process that employs an engineered microorganism and utilizes a renewable starting material (corn sugar) as its feedstock and converts glucose into l-methionine. we will describe (a) the general scheme for the engineering of the host organisms and (b) the general approach to maximize carbon and reducing equivalent throughput to l-methionine. to highlight this effort, we will present the global approach developed to improve the process combining metabolic flux analysis, traditional protein biochemistry, molecular biology and fermentation optimisation. saccharomyces cerevisiae is an established 'work horse' of the fermentation industry and modern biotechnology has led to a spectacular expansion of the range of products that can be produced by this yeast. however, for the large-scale sustainable production of chemicals, it is equally important that the range of carbohydrate feedstocks be expanded. especially relevant in this respect is the ability to consume the pentose sugars d-xylose and l-arabinose, which make up a substantial part of plant carbohydrates. wild-type s. cerevisiae strains cannot metabolise d-xylose, but are capable of slowly metabolising its keto-isomer, d-xylulose. therefore, efficient conversion of d-xylose into d-xylulose has long been a key issue in yeast metabolic engineering. non-saccharomyces yeasts that is capable of growing on d-xylose use two enzymes, xylose reductase and xylitol dehydrogenase for this purpose. while both enzymes have been successfully expressed in s. cerevisiae, this is not always compatible with efficient product formation. for example, in the case of ethanol production, the different cofactor specificities of these two oxidoreductases cause massive byproduct formation. theoretically, introduction of a xylose isomerase, which catalyses the interconversion of xylose and xylulose, might circumvent these problems. however, it is notoriously difficult to express bacterial and archaeal xylose isomerases in s. cerevisiae and, until recently, activities of heterologous xylose isomerases expressed in s. cerevisiae were vanishingly low, at least under physiological conditions. a breakthrough was reached when, in 2003, a xylose isomerase gene from the anaerobic fungus piromyces was expressed in s. cerevisiae. while this led to high activities of xylose isomerase, these were not enough to enable fast growth or product (ethanol) formation. in this presentation, we will discuss how a combination of metabolic and evolutionary engineering led to fast and efficient xylose utilization by engineered saccharomyces cerevisiae strains under aerobic as well as anaerobic conditions. furthermore, we will illustrate how evolutionary approaches can be applied to facilitate the utilization of mixed substrates. hyaluronic acid (ha) is a natural and linear polymer composed of ␤-1,3-n-acetyl glucosamine and ␤-1,4-glucuronic acid repeating disaccharide units with a molecular weight (mw) up to 6 mda. it is a major constituent of the extracellular matrices and the synovial fluid. in the last decades, various fields of application including cosmetics, ophthalmology, rheumatology, tissue engineering and drug delivery have been explored, owing to the many important biological functions of ha and its unique physico-chemical properties. however, for some specific applications, the relatively high mw of ha is a limiting factor and the availability of low mw fractions would be highly desired. for food applications, low mw ha has been shown to penetrate the gastrointestinal barrier, thereby increasing the ha bioavailability. moreover, low mw ha fractions are able to re-establish the ha content in the skin and can thus be used in cosmetics as anti-aging and anti-wrinkle agents. finally, low mw ha has shown to prevent oxygen free radical damage in granu-lation tissue during wound healing. a range of methods has been described for the depolymerization of ha to low mw fractions. these techniques involve heat treatment, ultrasonication, uv/gamma irradiation, chemical and enzymatic degradation. we present results on a degradation process of ha originating from bacillus subtilis fermentation into well-defined low mw fractions. the process developed in lab scale is safe, well-controlled and produce low mw ha fractions with narrow polydispersity. moreover, the process is readily up-scalable. the low mw ha fractions are being evaluated in various cosmetic applications. metabolic pathway manipulation for improving the properties and productivity of microorganisms is becoming an established concept. metabolic engineering can be defined as the directed improvement of product formation or cellular properties through the modification of specific biochemical reactions or introduction of new ones with the use of recombinant dna technology. a detailed analysis of the physiological means of the different pathways is needed to be able to introduce modifications aimed to the production of not only important metabolites, but also to understand the fundamentals of cell biology. aimed to produce single compounds, metabolic engineering necessarily includes the modification of the cellular pathway(s) as well as the redirection of the energy toward the production itself. the existing metabolic engineering applications are the culmination of more than two decades of global experience developing processes for the production of fine chemicals, vitamins, nutraceuticals and animal nutritional aids such as amino acids. based on the relative low complexity, the first biotechnological applications have been developed from microorganisms. our laboratory has been engaged in this field since different years. yeasts like saccharomyces cerevisiae, kluyveromyces lactis and zygosaccharomyces bailii have been developed for the production of fine chemicals like lactic and ascorbic acids from d-glucose. in this contribution, we will present the last data obtained. since 40 years amino acid production is in the focus of industrial microbiology. l-glutamate and l-lysine are produced with corynebacterium glutamicum, while escherichia coli is used for l-threonine production. the worldwide market of threonine drastically increases: in 1996 the amount of threonine produced worldwide was 15,000 t and raised to 30,000 t in 2000 and 45,000 t in 2004. concerning predictions of experts the demand of threonine will rise with a two-digit rate of economic growth within the next few years. meanwhile the prices declined. these conditions enforce very efficient production processes. beside the strain development and an optimised downstream procedure the fermentation process is an important target for productivity improvement. strain development is dependent on detailed knowledge of the production strains. with innovative methods we are able to get a close look inside the cells under different culture conditions. these methods have been called 'omics' in recent literature. knowledge about genome, transcriptome, proteome, phosphoproteome, metabolome and fluxome leads to new ideas for strain improvements. data generated by these methods must be based on clearly defined culture conditions. therefore, highly parallel fermentations have to be performed to generate biological parallel samples. cutting-edge technical equipment is the basic requirement for experiments like this. other requirements are optimised sampling for different analysis, technical parallels of analytical steps and a detailed statistical analysis of data. these procedures guarantee distinction between real data and data noise. integration of all these data to a holistic model of the cell is the challenge for the future. by combination of a new strain, process and downstream improvements the plant productivity was increased drastically. we ended up in an optimized, fast and high yield process to scope challenges worldwide market comes up with. rieping, m., hermann, t. (2002); fermentation process for the preparation of l-threonine; wo/0218543. ) are potentially quite useful biocatalysts, as they allow for the regioselective and stereoselective hydroxylation of activated as well as non-activated carbon atoms. in addition, the large number of members of the p450 superfamily exhibits a wide diversity of specificities from which a useful biocatalyst may be selected. from a technical point of view, however, they have significant drawbacks. thus, they usually cannot be produced in large quantities nor recovered or stored without a severe loss in activity. their catalytic activity is mostly quite low, and their operational stability leaves much to be desired. most p450 enzymes require a complex protein/phospholipid machinery for activity, and the final electron donor in the reaction cascade, usually nadph, does require extensive recycling to arrive at a commercially satisfactory process. recently, the use of bacterial cytochromes as hydroxylation biocatalysts has received considerable attention. some of them are natural fusion proteins, which contain the heme and the reductase domains on a single polypeptide chain. they are catalytically much more active compared to, e.g. cytochromes occurring in human tissue. we thus have set out to further improve the technical applicability of these enzymes, and have centered our activities around several bacterial cytochromes. it proved very useful to apply rational mutagenesis and directed evolution to these enzymes, leading to a surprising compatibility of mutant enzymes with a wide variety of substrates. mechanisms for the limited stability of the enzymes were explored, leading to hybrid enzymes with enhanced stability, and the cofactor problem was alleviated using auxiliary enzymes or mediator-based technologies. as a result, a bioreactor based on microbial cytochromes was built and operated for several days. baeyer-villiger monooxygenases represent useful biocatalytic tools as they can catalyze reactions, which are difficult to achieve using chemical means. however, so far only a limited number of these monooxygenases were available in recombinant form kamerbeek et al. (2003) . using a recently described protein sequence motif fraaije et al. (2002) and the available genome sequence information, we were able to identify and overexpress a number of novel bacterial bvmos. one of the overexpressed bvmos was found to be relatively stable as it originates from thermobifida fusca, which grows at ∼60 • c. the enzyme was shown to be active on a broad range of substrates, preferring aromatic ketones fraaije et al. (2005) . the best substrate discovered so far is phenylacetone, hence its name: phenylacetone monooxygenase. we have solved the crystal structure of phenylacetone monooxygenase, which represents the first structure of a bvmo malito et al. (2004) . the crystal structure provides insight into the complex mechanism of catalysis mediated by fadcontaining bvmos. by site-directed mutagenesis we have probed the role of several active-site residues. a crucial role is played by an arginine residue. as phenylacetone monooxygenase shares significant sequence identity (>40%) with all known nadph-dependent bvmos, many of the observed structural features seem to be conserved within this class of atypical monooxygenases. by homology modeling using the phenylacetone monooxygenase structure, catalytic properties of other baeyer-villiger monooxygenases can be explained or predicted. screening for fungal baeyer-villiger monooxygenases l. butinar 1 , j. friedrich 1 , v. alphand 2 : 1 laboratory of biotechnology, national institute of chemistry, ljubljana si-1001, slovenia; 2 groupe biocatalyse et chimie fine cnrs fre2712, université de la méditerranée, marseille, france the asymmetric form of the baeyer-villiger (bv) oxidation (transformation of ketones into lactones) is an important challenge for organic chemistry since the obtained lactones are valuable building blocks for synthesis of countless biologically active products. to date, enzymatic or microbial bv oxidations appears as more successful than their chemical counter-parts. (ten brink et al.) whereas most active bv monooxygenases are produced by bacteria (among which the well-studied enzyme of acinetobacter calcoaceticus), only a few fungal strains expressing bvmo were described (alphand et al., carnell and willetts) . in order to increase the number of available biocatalysts which perform such an asymmetric biotransformations, a screening of fungi belonging to major groups of zygo-, ascoand basidiomycetes was conducted using bicycloheptenone as testsubstrate. surprisingly, a large number of the tested fungi were able to transform the substrate into one to four different lactone isomers. the yields, the enantio-and regio-selectivity of the reaction depended on the fungal strain. alphand, v., furstoss, r., 2000. j. mol. catal. b 9, 209-17. carnell, a., willetts, a.,1992 . biotechnol. lett. 14, 17-21. ten brink, g.j., et al., 2004 . pyranose oxidase (p2ox) is a periplasmic enzyme that widely occurs in basidiomycetes. it catalyses the c-2 oxidation of several aldopyranoses to the respective 2-keto derivatives, transferring electrons to molecular oxygen to yield h 2 o 2 . p2ox is of interest for carbohydrate conversions, as its reaction products (2-keto sugars) can be attractive intermediates in the production of food ingredients. we cloned the gene encoding p2ox, and subsequently amplified a cdna clone by rt-pcr. the cdna was inserted into a bacterial expression vector and successfully expressed in e. coli. properties of the heterologous protein were compared to those of the native enzyme showing that they are essentially identical. both the native as well as the recombinant enzyme were used in biotransformations of sugars. recently, the 3d-structure of this tetrameric enzyme was elucidated. based on structural information, several enzyme variants containing point mutations were constructed and further characterised. two of these variants (e542k and e542r) displayed improvements in stability and certain kinetic properties thus making them attractive for biocatalytic applications. lactones are important compounds for the fragrance and flavour industry. right now the production of lactones is dependant on the import of crude materials from tropical countries. in this project, we want to tackle the manufacture of lactones via a biocatalytic route using p450 monooxygenases. cytochrome p450 monooxygenases catalyse the oxyfunctionalization of non-activated c-atoms. cyp102a1 from bacillus megaterium, cyp102a2 and cyp102a3 from bacillus subtilis are soluble fusion proteins comprising p450 monooxygenase and fad/fmn reductase domains in one polypeptide chain. all three enzymes are highly homologous fatty acid hydroxylases. especially, cyp102a1 also known as p450 bm-3 is well characterized and shows high activity compared to other p450 monooxygenases. the aim of the work is to change selectivity but conserve the high activity that is typical for those enzymes. using methods of structure modelling, rational protein design and directed evolution new mutants of these enzymes with changed regioselectivity are obtained. products of conversion with monooxygenases are intermediates in the production of lactones. the interface of biology and materials science has led to new materials with unique structural and functional properties, and new process technologies with the ability to produce, from "bottoms up", a wide range of biomimetic structures. these materials and their designs have broad application as catalysts, sensors, and devices for use in synthesis, cell and tissue engineering, bioanalysis and screening, and nanoelectronics. we have focused on the generation of sugar-based nanostructures, complete with tailored selectivities and biocatalytic activities at the molecular and nanoscales. these include biocatalytically-generated carbohydrate derivatives that selfassemble with high precision to give novel architectures with functional and responsive properties. izumoring: a strategy for total production of rare sugars ken izumori rare sugar research center, kagawa university, kagawa 761-0795, japan. e-mail: izumori@ag.kagawa-u.ac.jp we found a new enzyme, d-tagatose 3-epimerase (dte), that epimerize all ketohexoses at c-3 position. this epimerase catalyze not only between d-tagatose and d-sorbose, but also d-fructose = dpsicose, l-sorbose = l-tagatose, and l-psicose = l-fructose. this new enzyme offered us a useful key tool to connect all ketohexoses using hexitols as intermediates. the figure shows that all eight ketohexoses can be connected with dte and polyol dehydrogenases (pdh) in a ring. using this ring, we can easily find the pathway to transfer d-fructose to d-tagatose via d-psicose using dte and pdh. various aldose isomerases transform ketohexoses to the corresponding aldohexoses. so, we can connect all 16 aldohexoses with 8 ketohexoses using the enzyme. finally, all hexoses, 8 ketohexoses, 10 hexitols and 16 aldohexoses are connected using enzyme reactions in a ring structure (not shown). this kind of strategy is effective also on transformation of tetroses and pentoses. now, we can produce all monosaccharides; tetroses, pentoses and hexoses by enzyme reac-tions. the bioproduction strategy of all rare sugars (monosaccharides that are rare in nature) is illustrated using ring form structures named as izumoring. we have already succeeded to produce d-psicose in large scale and are now in the progress of mass production of various rare sugars from natural and cheap sugars using izumoring. bioprocess development for chiral intermediates christian wandrey institute of biotechnology, forschungszentrum jülich gmbh, jülich d-52425, germany chiral alcohols, diols, amino alcohols and chiral acids (e.g. hydroxy acids and amino acids) play an important role in pharma and agro synthesis. in the past such chiral intermediates were obtained by racemic resolution via chiral reductions using prochiral precursors. here the problem of cofactor regeneration arises. this problem could be solved by enzyme-coupled or substrate-coupled cofactor regeneration using formate or isopropanol as reducing agent. alternatively, whole cell bioreductions were developed where glucose is used as the reducing agent. in recent years "designer microorganisms" were developed in which oxidoreductases (e.g. alcohol dehydrogenases) were over-expressed in escherichia coli. in such cases, cofactor regeneration was achieved intracellularly with isopropanol as the reducing agent or by coexpression of a formate dehydrogenase, so that once again format could be used for reduction. another route to obtain chiral intermediates is a fermentative approach using classical pathways (like the aromatic amino acid pathway). here, the pathway is interrupted after the intracellular production of chorismate. new chiral intermediates can be obtained by over expression of additional genes, which catalyzed the production of chorismate derivatives leading to cyclohexadiene-transdiols and the corresponding amino cyclitols. the last example can be regarded as an example of industrial biotechnology where glucose is used as starting material (white biotechnology). here bioprocess development is carried out in an integrated approach, in which molecular biochemical engi-neering cares for the optimal intracellular metabolic network and the classical biochemical engineering cares for the optimal environment of the cell in a fermenter. examples will be given which reach (in cooperation with industrial partners) up to kilogram scale. biotransformations are usually involved in just one or very few separate reactions in organic syntheses. the development of a cell-free "system of biotransformations" (sbt), in which a set of enzymes acts in a coordinated fashion in a one-pot synthesis, lead to increased catalytic complexity, selectivity and yield, as well as facilitated operation at reduced costs. the example chosen to prove the usefulness of the sbt-approach is the production of dihydroxyacetone phosphate (dhap). dhap, a c3-compound from glycolysis, is an important precursor for asymmetric c-c-bond formation. so far, the production of dhap is difficult and expensive. for the construction of the dhap-producing sbt, e. coli's glycolysis is isolated from the metabolism to an as large as possible extent by the construction of a multi-ko-mutant. a culture is grown in an appropriate medium, homogenized in the production buffer, and used as the catalytic system. high production yields can be achieved since the production pathway is almost completely isolated from the metabolic network. the employed dhap-producing sbt provides not only a path from glucose to the product, but also an integrated atp-regeneration and nad-recycling system. in first experiments with a tpi-ko-mutant, a dhap-production yield on glucose of 32% could be achieved, without optimizing the system. the system remained active for more than 24 h. up to now, atp cannot be applied in catalytic concentrations, but has to be present in equimolar amounts to glucose. the production yield could be increased by 10% through the addition of phosphate ions as substrate to the reaction, enabling the system to utilize atp more efficiently. these experiments indicate that the sbt-approach is viable and a large potential remains to improve the dhap-producing sbt. for some years novozymes have manufactured a pectate lyase for scouring of textile as an ecological alternative to the traditional harsh chemical treatment, and recently, we at novozymes discovered additional applications for pectate lyases. however, to be commercially attractive more robust pectate lyses had to be developed. in this paper, we will demonstrate how we for two different pectate lyases have improved their stability significantly. as the two enzymes are quite similar in sequence and structure, it was a new discovery for us to find that different concepts of protein engineering had to be used in our attempt to stabilize each individual pectate lyase. the stability of one enzyme was improved by substitutions in the internal of the structure whereas the stability of the other pectate lyase was primarily improved by changing surface residues. starting with knowledge from structural analysis, we have applied rational based protein engineering resulting in few selected variants. also random based protein engineering combined with screening of hundreds of thousands variants was used. in conclusion: the project team showed that by synergistic use of the two approaches, we were able to move faster towards a solution and eventually we succeeded finding new stabilized pectate lyase variants, applicable for new business areas. the importance energy independence as a national goal equals or exceeds that of the moon landing in 1990. the development of a new industry to produce fuel ethanol from woody biomass would increase national security, improve employment and the environment, and provide substantial relief from the debt of imported petroleum. costs associated with the rapid development of this new industry (∼$1.4 billion per year) could be paid by re-assigning 1 cent per gallon from existing federal gasoline taxes, a small price to pay for future energy independence. the corn-to-ethanol industry continues to make a remarkable contribution to our liquid fuel needs through expansion. today, one row of every six rows of corn is converted into fuel ethanol in the u.s. however, this expansion will be limited to 3-4% of total automotive fuel by the economics of corn costs and production. corn can do more! corn stover is the single most abundant agricultural residue in the us and can be used as a feedstock to produce 60-80 gallons of ethanol per dry ton. further expansion with other biomass feedstocks such as agricultural and municipal residues (lignocellulose, woody biomass) could produce over 100 billion gallons of fuel ethanol annually according at a recent joint report by the usda and doe (april, 2005) . current technology has been demonstrated at pilot scale for the production of fermentable sugars from hemicellulose by dilute acid hydrolysis and for the hydrolysis cellulose using fungal cellulose enzymes. biocatalysts such as recombinant escherichia coli have been developed and demonstrated for the efficient conversion of all sugar constituents of biomass to ethanol. a national goal for the full-scale deployment of current technology to produce biomass-based fuel ethanol will allow the us to reduce imported petroleum by 50%. together with increased efficiencies of hybrid vehicles, energy independence could be achieved within 10-20 years. similar gains could be realized by many nations around the world to provide new manufacturing and employment, redistributing wealth and ensuring a cleaner, healthier environment. bioethanol production using thermophilic bacteria marie just mikkelsen, birgitte k. ahring emab, biocentrum-dtu, 2800 lyngby, denmark the industry of bioethanol production is facing the challenge of redirecting the process from fermentation of relatively easily convertible but expensive starchy materials, to complex but inexpensive lignocellulosic biomass. on lignocellulosic hydrolysates, gram-positive thermophilic bacteria have unique advantages over the conventional ethanol production strains. the primary advantages are their natural broad substrate specificities, and in some strains, a high tolerance to lignocellulosic hydrolysates. moreover, ethanol fermentation at high temperatures also has the advantages of high productivities and substrate conversions, low risk of contamination and facilitated product recovery. some thermophilic bacteria naturally produce primarily ethanol from most sugar monomers present in lignocellulosics, but modifications are still necessary to increase ethanol yields. the release of useable sugars from lignocellulose biomass for industrial fuel-ethanol fermentation is often facilitated by a weak acid hydrolysis step. as a consequence, inhibitors such as furfural and 5-hydroxymethylfurfural (hmf) are formed as degradation products of xylose and glucose, respectively. moreover, the fermentative end-product of ethanol is also inhibitory. these, and other inhibitors present an environment, which elicits the expression of stress-related genes in saccharomyces cerevisiae. recently, 65 s. cerevisiae genes have been identified as important in furfural stress tolerance. when furfural is present, yeast with these genes disrupted grows poorly compared to wild-type yeast. a sub-class of these genes suggests that yeast grown under furfural-induced stress may rely upon similar pathways as cells grown under various other stresses, including oxidative, heat, and sorbate. to investigate this link further, we analyzed stress-induced phenotypes such as ros activity, dna damage, and membrane damage in wild-type and mutant yeast exposed to furfural or hmf stress. moreover, we investigated whether overexpression of this sub-class of genes would provide protection from furfural-induced stress and oxidative damage. micro-organism to be used in fermentation of lignocellulose hydrolyzates should preferably have three characters: (a) high ethanol tolerance, (b) resistance to inhibitors found in the hydrolyzate, and (c) a broad substrate utilization range, since the hydrolyzate contains several sugars. in addition to the possibility of controlling the level of potential inhibitors, fed-batch fermentations also permit the parallel uptake of several different monomeric sugars. two strains of saccharomyces cerevisiae, cbs 8066 (a commonly used laboratory strain) and tmb 3000 (a strain isolated from a spent sulfite liquor fermentation plant), were characterized in batch and fed-batch fermentation of a dilute-acid hydrolyzate from spruce. the strains had different abilities to ferment spruce hydrolyzate. the study suggests that the furan reduction capacity of a yeast strain is a key factor for its performance in fermentation of lignocellulosic hydrolyzate. polyketides constitute a structurally highly diverse group of natural products that possess broad ranges of pharmacological properties and represent a major source for novel cancer therapeutics. however, these compounds may be sub-optimal in regard of activity, selectivity, availability and unwanted side effects. in addition, the sustainable production of these valuable metabolites can be a challenge. studying the molecular basis of the biosynthetic pathways may set the basis for improving the production and for rationally engineering derivatives with altered bioactivity profiles, e.g. through targeted knockouts, mutasynthesis ziehl et al. (2005) , and swapping of pathway genes. our results in elucidating and manipulating the biosynthesis of selected antitumoral polyketide metabolites from bacteria (aureothin, chartreusin) and fungi (cytochalasines, rhizoxin) are presented. analyses at the genetic and biochemical levels provided new insights into several unusual biosynthetic features, e.g. non-linear polyketide assembly for the nitroaryl-substituted polyketide aureothin he and hertweck (2003, 2005) , an oxidative rearrangement cascade in the chartreusin pathway xu et al. (2005) , and a fungal iterative pks-nrps hybrid synthase schuemann and hertweck (2005) involved in cytochalasin biosynthesis. the most surprising result was obtained from elaborating the biogenesis of the antimitotic agent rhizoxin from rhizopus sp., which allowed for a significant improvement in large-scale production partida-martinez and hertweck (2005). he, j., hertweck, c., chem. biol. 2003 , 10, 1225 -1232 chem. bio. chem. 2005, 6, glycopeptides such as vancomycin and teicoplanin are the drugs of last resort for the treatment of severe infections caused by antibiotic resistant gram-positive bacteria. glycopeptides inhibit the peptidoglycan biosynthesis by binding as dimers to the d-ala-d-ala termini of the cell wall precursors. amycolatopsis balhimycina synthesizes the vancomycin-type glycopeptide balhimycin, whose structure and biological properties greatly resemble vancomycin and which only differs by its glycosylation pattern. using a "reverse genetics" approach we have identified the 66-kb gene cluster encoding the biosynthesis of balhimycin. by a combination of genetics, biochemistry and analytical organic chemistry, we were able to elucidate the biosynthetic pathway and to assign functions to almost all genes of the cluster. the biosynthesis starts with the pathway-specific provision of the non-proteinogenic amino acids ␤-hydroxytyrosine (␤-ht), hydroxyphenylglycine (hpg) and dihydroxyphenylglycine (dpg) which form together with (n-methyl)-leucine and asparagine the heptapeptide backbone of balhimycin. dpg is synthesized via a polyketide synthase mechanism (pksiii) similar to that known from plant chalcon/stilben synthases (pfeifer et al., 2001) . for the ␤-ht synthesis three genes are essential which form an operon (puk et al., 2004) : bpsd, an nrps binds a tyrosine molecule, which is then hydroxylated by the p450 monooxygenase oxyd. the perhydrolase bhp is required for the release of ␤-ht. subsequently bhaa, a nadh/fad-dependent halogenase catalyzes the chlorination of ␤-ht to form chloro-␤-hydroxytyrosine (puk et al., 2002) , which is needed to stabilize the dimerization. the amino acids are linked by non-ribosomal peptide synthetases (recktenwald et al., 2002) , and the aromatic side chains are interconnected by p450 monooxygenases; a series of reactions which lead to the first antibiotically active intermediate. inactivation of the oxygenase genes revealed the order of the cyclization steps (bischoff et al., 2001) : the oxygenases act in a stepwise fashion in the sequence oxyb, oxya and oxyc. the resulting cross-linked heptapeptide is then finally modified by methylation and glycosylation. the biosynthesis is regulated by the strr-type regulator bbr, which was shown to bind in front of different operons of the balhimycin gene cluster. this ensures the coordinated expression of the biosynthetic genes. the non-producing mutants, defective in the supply of the non-proteinogenic amino acids, were used as recipients in cloning experiments as well as in approaches of precursor-directed biosynthesis by feeding chemically synthesized alternative precursors. thus, novel balhimycin derivatives were generated (weist et al., 2002 (weist et al., , 2004 . bischoff et al., 2001 . angew. chem. int. ed. 40, 4688-4691. pfeifer et al., 2001 . j. biol. chem. 276, 38370-38377. puk et al., 2004 . j. bacteriol. 186, 6093-6100. puk et al., 2002 . chem. biol. 9, 225-235. recktenwald et al., 2002 . microbiology 148, 1105 -1118 . weist et al., 2002 . angew. chem. int. ed. 41, 3383-3385. weist et al., 2004 spectroscopy guided discovery of novel bioactive microbial natural products thomas ostenfeld larsen, michael edberg hansen cmb biocentrum-dtu, technical university of denmark, 2800 lyngby, denmark the task of finding novel bioactive natural products is usually bioassay driven. often a certain type of compound (e.g. polyketide, alkaloid) turns out to be active in an assay. when having generated a promising hit in a bioassay the normal procedure in the drug discovery process usually is to produce a large number of structurally analogous compounds either by traditional chemical synthesis or by combinatorial chemistry in order to study structure activity relation-ships and to find even more active lead compounds. alternatively to chemical synthesis of analogues nature can be explored for structurally similar compounds by uv-spectroscopy guided screening. this work will present a new method for the systematic and automated computer assisted search of full uv spectra in large number of datafiles for both dereplication of known and discovery of new natural products based on the use of the new mathematical algorithm x-hitting. exploring the substrate spectrum of the antibiotic producing bacteria saccharopolyspora erythraea p. krabben, p. oliveira, f. baganz, j. ward department of biochemical engineering, university college london, london wc1e 7je, uk knowledge of substrate utilisation capabilities play an important role in the development of genome scale metabolic models (borodina et al., 2005) and refinement of first generation annotations. furthermore, knowledge of the product formation during catabolism of different substrates provides valuable information about the distribution of metabolic fluxes and thereby forms a basis for rational strain improvement. we present here, data on the substrate utilisation capabilities and the corresponding product formation of s. erythraea. this analysis will help in improving the production of erythromycin and provide clues to the activation of the cryptic secondary metabolic pathways present in the s. erythraea genome. reference borodina, i., et al., (2005) . genome-scale analysis of streptomyces coelicolor a3 (2) modeling of growth and product formation on complex media containing multiple substitutable substrates is a challenge. complex media offers the organism multiple choices of carbon and nitrogen substrates including free amino acids, peptides, soluble and insoluble proteins in addition to the defined sources such as glucose and ammonium sulfate. we present a structured model that accounts for growth and product formation kinetics of rifamycin b fermentation in a multi-substrate complex medium. the model considers the organism to be an optimal strategist with a mechanism to regulate the uptake of the substrate combinations. further, we assume that the uptake of a substrate depends on the level of a key enzyme, which may be inducible. the model also considers control parameters as fraction of flux through a given metabolic branch. the control parameters are obtained using a simple multi-variable constrained optimization. the model parameters were rigorously estimated via a specifically designed experimental plan. the model correctly predicts the experimentally observed growth and product formation kinetics and the regulated simultaneous uptake of the substitutable substrates under different fermentation conditions. the model and the model parameters provide useful insights into the growth and product formation strategy of this industrially important process. this presentation will describe the experimental results, the model development and the relevant model parameters for a. mediterranei s699. the recent surge in oil price and the increasing concern on our environment have generated much interest in the production of chemicals from renewable resources. succinic acid, also called as amber acid, is a four-carbon dicarboxylic acid, which can be used as a precursor of numerous products including biodegradable polymers, green solvents, pharmaceuticals, and bulk and fine chemicals. a new capnophilic bacterium named mannheimia succiniciproducens mbel55e was isolated from the rumen of korean cow. this bacterium can produce large amounts of succinic acid along with some other metabolites such as lactic, formic and acetic acids. we have completely sequenced the genome of m. succiniciproducens and charaterized its genome content in the context of metabolic pathways. we then constructed the genome scale in silico metabolic network for metabolic flux analyses, and carried out metabolic flux analysis under varying environmental conditions. based on the in silico analyses results, we selected several target genes to be manipulated for enhanced succinic acid production. detailed results of metabolic engineering based on genome-scale information will be reported. we have been developing tools for inverse metabolic engineering in order to identify gene targets that improve the phenotype of industrial strains and cells for medical applications. to this end, we create genomic fragment libraries from a source organism and use it to transform the host organism. cells are properly selected in environments that favor the phenotype of interest and genes enriched in these cells are sequenced and used in follow up transformations of cells with specific genetic backgrounds. this overall strategy is complemented with additional tools for modulating gene over-expression, gene deletion, and high throughput clone isolation. we will demonstrate applications of this strategy to the identification of gene targets for improved xylose assimilation in recombinant saccharomyces cerevisiae and improved lycopene production in escherichia coli. based on assumed reaction network structures, nadph availability has been proposed to be a key constraint in ␤-lactam production by penicillium chrysogenum. in this study, nadph metabolism was investigated in glucose-limited chemostat cultures of an industrial p. chrysogenum strain. enzyme assays confirmed the nadpspecificity of the dehydrogenases of the pentose-phosphate pathway and the presence of nadp-dependent isocitrate dehydrogenase. pyruvate decarboxylase/ nadp-linked acetaldehyde dehydrogenase and nadp-linked glyceraldehyde-3-phosphate dehydrogenase were not detected. although the nadph requirement of penicillin-gproducing chemostat cultures was calculated to be 1.5-1.7-fold higher than that of non-producing cultures, activities of the major nadph-providing enzymes were the same. isolated mitochondria showed high rates of antimycin a-sensitive respiration of nadph, thus indicating the presence of a mitochondrial nadph dehydrogenase that oxidizes cytosolic nadph. the presence of this enzyme in p. chrysogenum has important implications for stoichiometric modelling of central carbon metabolism and ␤-lactam production and may provide an interesting target for metabolic engineering. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf-21 cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf21 infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp486 coding for am-cyan coral protein, which emits natural green fluorescence. complete elucidation of the genetic control of a metabolic flux requires the availability of fine-grained expression levels of the gene(s) of interest. we developed a collection of promoters of varying strength for tuning gene expression in the yeast s. cerevisiae. engineered promoters were obtained through random mutagenesis of the constitutive tef1 promoter. eleven mutated promoters were selected by fluorescence-activated cell sorting (facs) spanning gradually increasing activities between about 8 and 120% compared to the native tef1 promoter. data were also confirmed at the level of mrna via rt-pcr. by introducing selectable markers in front of the different tef1 promoter mutations, we provided plasmid collections, which can be directly used to amplify promoter replacement cassettes for genomic integration of the fine-grained promoter collection in front of any yeast gene. l-arabinose, widely distributed in plant kingdom, is a component of plant cell wall. l-arabinose does not abundantly exist in free state in plants, but usually in corn hull, sugar beet pulp, gum arabic, mesquite gum, as the polysaccharide such as arabinoxylan and arabinogalactan. to produce arabinose from agricultural wastes, we screened arabinogalactan degradable strain from compost. thereafter, putative arabinase gene from this strain was cloned (b1029 ts2-8). as a result of spectrometric assay using -nitrophenyl ␣l arabinofuranoside, recombinant showed 3-4-fold higher activity than wild type e. coli strain. after enzymatic reaction with corn fiber, b1029 ts2-8 produced 2.15 g/l of l-arabinose, which was detected on hplc. however, the enzyme activity was very low. so, we are transferring the gene into expression vector system. further characterization study and enzyme engineering to enhance the activity toward corn fiber will be presented in poster. there are only a limited number of hypersaline areas all over the world, which include several locations in turkey such as van lake located in eastern region of turkey. isolation and identification of halophilic and hyperhalophilic microorganisms from such locations is essential for the determination of biodiversity in turkey. high-level production of extremozymes from these microorganisms has also many economical advantages due to their stability at extreme reaction conditions. proteolytic enzymes are the most important group of enzymes produced commercially. of these, proteases produced by alkalophilic microorganisms are investigated not only in scientific areas such as protein chemistry and protein engineering but also find wide application in food, pharmaceutical, leather and detergent industries. in this study, 24 microorganisms isolated from van lake were screened for the presence of extracellular alkaline protease activity. the optimum screening temperature and ph were determined as 37 • c and ph 9.5, respectively. one of the isolates that could grow at 0-20% salinity reached highest levels of extracellular alkaline protease activity. this best producer, which was identified as the halotolerant bacillus pumilus, was found to produce alkaline protease both in the presence and absence of nacl. to improve enzyme production yields, culture conditions such as medium composition, growth ph and temperature were optimized. the effect of different carbon sources, organic and inorganic nitrogen sources on the production of alkaline protease was studied. whereas a mixture of inorganic and organic nitrogen sources induced high protease production, use of only an organic nitrogen source supported poor enzyme production. halotolerant bacillus pumilus produced maximum alkaline protease activity when maltose, yeast extract and sodium nitrate were used as carbon source, organic and inorganic nitrogen sources, respectively. this project was supported by tubitak through project tbag 2321-103t069. in the market of biochemical products a very important role is played by heterologous proteins production, and despite recent advances in mammalian cells exploitation, yeasts can still present advantages as host systems. among them, the spoilage yeasts belonging to the zygosaccharomyces genus have become, due to some peculiar properties, significantly attractive. in particular, z. bailii is characterized by acid resistance, osmotolerance to high sugar and ethanol concentration combined with high biomass yield. despite still little is known about its genetics and cellular biology, our group is working on its development and exploitation for recombinant productions with an integrated approach coupling physiological study with the creation of molecular tools for heterologous proteins production. we previously described and did a patent application regarding the first techniques necessary to transform this yeast and to express and secrete different proteins derived from different sources. here we present and discuss the last advances in optimization of heterol-ogous protein expression in particular, on one side we present a reproducible strategy for target gene deletion, leading to the first z. bailii auxotrophic mutant, and on the other we show the improvement of gene dosage and plasmid stability by building a set of multicopy expression vectors based on the sequences of the z. bailii 2 -like endogenous plasmid psb2 and an integrative plasmid. all the known ␥-butyrolactone autoregulator receptors are highly conserved in the dna binding motif present in their n-terminal portions and have been proposed to play roles as transcriptional regulators in antibiotic production and/or morphological differentiation. previously, kim et al. reported that the cloned scar in streptomyces clavuligerus has several characteristics of the autoregulator receptors in the genus streptomyces. in this study, to clarify the in vivo function of scar, a scar-disrupted strain was constructed by means of homologous recombination after introducing a scar-disruption construct via transconjugation from e. coli. no difference in morphology was found between the wild-type strain and the scar disruptant. however, the scar disruptant showed a 1.8-fold higher production of clavulanic acid than the wild-type strain. the phenotype was restored to the original wild-type phenotype by complementation with intact scar. therefore, the autoregulator receptor, scar, acts as a negative regulator of biosynthesis of clavulanic acid but plays no role in cytodifferentiation of s. clavuligerus. lactate dehydrogenase catalyses the production of lactate from pyruvate. it is the first target for many researches on lactic acid producer microorganisms like rhizopus oryzae. in the present study based on the known sequences of r. oryzae ldha and ldhb genes skory (2000), they were cloned and expressed in a citric acid producer fungus aspergillus niger. the aspergillus niger strains expressing ldha or ldhb gene resulted in increased production of lactate in aspergillus niger. among 50 transformants tested 4 ldha and 5 ldhb expressing strains were found to have higher lactate dehydrogenase activity compared to wild type in the conditions tested. the highest specific activity obtained with ldha transformants was only 2.5 times of the wild type while this was 10 times for one of the transformants expressing ldhb. in addition to increased lactate production citric acid production was also increased. however, gluconic acid production ceased in the ldha or ldhb expressing a. niger strains. the production of lactic acid in a. niger transformants and lactate dehydrogenase a and lactate dehydrogenase b enzymes are being investigated in the chosen strains. selection of n source suitable for production of rhodococcus sp. biomass for the purposes of microbial transformation of 5␣h-epoxypregnanolone (5␣h) and 5 -epoxy-pregnenolone ( 5 ) into their 9␣-hydroxy-derivatives was carried out. three dehydrated and three non-dehydrated n sources were tested. the transformation reaction was carried out in phosphate buffered medium containing 1 g l −1 of the steroid substrate and 0.1 g l −1 cells. the steroids were determined by hplc. the transformation resulted in formation of three derivatives appearing in the reaction medium in the sequence: 4 -3-keto-; 9␣-hydroxy-and 9␣,20␤-hydroxy-epoxy-pregnenolone. a strong influence of the n source on the hydroxylating activity of the biomass was observed. triptose (difco) gave a cell depot actively hydroxylating 5␣h without any significant accumulation of the 4 -3-keto-derivative. the most effective accumulation of hydroxylated derivatives of 5 was observed with biomass grown on freshly prepared meat extract while the commercial products triptose (difco), meat extract (difco) and lactalbumin (flika) gave valuable information about the dynamics of the transformation process. biobleaching of kraft cellulose pulp by poliporus versicolor aysun ergene 1 , nazif kolankaya 2 : 1 kırıkkale university, faculty of science and literature, department of biology, yahsihan, kırıkkale, turkey; 2 hacettepe university, faculty of science, department of biology, beytepe, ankara, turkey the suitability of culture supernatant from poliporus versicolor for use in the biobleaching of kraft cellulose pulp was investigated. p. versicolor was found to grow on mycological broth (1% soytone, 4% d-glucose and 0.5% cellulose pulp). maximal extracellular ligninase production was detected after7 day (7 nkat). the optimum biobleaching conditions are 30 • c and ph: 4.8, with 10 days. in this condition p. versicolor decreased the kapa number from 38.55 to 19.42 and increased brigthness from 28 to 32.7 in 10 day treatment. xylanase production, purification and characterization from a soil isolate, bacillus m-13 ayşegül ersayin 1 , aytaç kocabaş 1 , b. zümrüt ogel 2 , ufuk bakir 3 : 1 biotechnology department, middle east technical university, ankara, turkey; 2 food engineering department, middle east technical university, ankara, turkey; 3 chemical engineering department, middle east technical university, ankara, turkey, 06531. e-mail: ubakir@metu.edu.tr (u. bakir) xylan is a major component of the plant cell wall, representing up to 35% of the dry weight. xylan molecule is a complex polymer consisting of a ␤-d-1,4-linked xylanopyranoside backbone substituted with acetyl, arabinosyl and glucuronosyl side chains. hydrolysis of the xylan backbone is mainly catalysed by endo-␤-1,4-xylanases (ec 3.2.1.8). many bacterial and fungal species are able to utilize xylan as a carbon source. interest in the enzymology of xylan hyrdolysis has increased because use of xylanases in bioconversion of agricultural wastes to valuable products like single cell protein, xylo-oligosaccharides and fuel, in bio-bleaching processes, food and animal feed industries. in this study, xylanolytic nature of a soil isolate bacillus spp., bacillus m-13, has been shown. bacillus m-13 produced multiple xylanases when grown on a liquid medium containing agricultural wastes as the sole carbon source. various agricultural wastes including corn-cobs and cotton waste, with and without pretreatments were used to maximize enzyme production. the major xylanase having molecular weight of 20 kda upon sds-page and a pi of 9.1 upon ief was partially purified by liquid chromatographic techniques 150-fold with 40% recovery, including gel filtration, ion exchange and hydrophobic interaction chromatography. enzymes are important constituents in the laundry detergents due to their contribution to shortening washing times, reduction of energy and water consumption by lowering washing temperatures, provision of environmentally friendlier wash-water effluents and fabric care. however, they can loose a significant part of their activity in the chemically-hostile detergent matrix over a time period of several weeks. therefore, improving the storage stability of enzyme granulates is the main challenge in the development of a new product. the complexity of the detergent matrix implies the presence of a complicated mechanism involved in the inactivation of the enzymes. a combination of factors, such as oxidation by h 2 o 2 released by the bleaching agents, humidity, high temperature, autolysis of enzymes, high local ph in a granule, oxygen, and other detergent components, plays a role in the activity loss. an experimental investigation on the inactivation of the solid-state enzyme during storage has been initiated. the release rate of h 2 o 2 from the bleaching agent, sodium percarbonate, was determined using a simple and accurate method for measuring the gas phase h 2 o 2 concentration. the deactivation kinetics of pure enzyme was determined as a function of gas phase h 2 o 2 concentration and humidity. the preliminary results indicate that humidity plays a significant role in the inactivation mechanism of the detergent enzyme due to a possible increase in the mobility of the enzyme molecule and the surface area exposed to destructive agents. the effect of main granulate ingredients on the stability of the enzyme was investigated and the extent of the protection of each component was estimated. the study is important for the revealing of the phenomena occurring in the detergent matrix during storage. understanding the inactivation mechanism provides a valuable tool for the development of more effective protective coatings and stabilizers. the use of biosurfactants in cosmetic industry has attracted great attention of biotechnological researchers because they consist of two inexpensive, renewable and easily accessible starting agricultural materials: sugar and oil/fat. carbohydrate based products are non-toxic, biocompatible and biodegradable. in addition, the enzymatic processes present many advantages in comparison with the chemical methods, which employ high temperatures in the presense of alkalin catalysts, high-energy consuption and low selectivity of products. sugar esters present vast application, such as for antibiotics, biomaterials, surfactants, cosmetics and so on. we investigated the synthesis of sugar vinyl esters, using protease-catalysed transesterefication method applying protease from bacillus subtilis. sucrose 0.125 m and vinyl ester 0.5 m has been mixed in dimethylformamide at 160 rpm of agitation. at first, we have studied the effects of protease from bacillus subtilis concentrations (5, 10, 20 and 40 mg/ml) as catalyst. afterwards, the influence of the temperature (30 and 50 • c). after that, the influence of the molar ratios (1:1; 1:2; 1:4 m/m) between vinyl laurate (ch3(ch2)10cooch ch2) and sucrose. subsequently, we investigated the effects of water amount, using 0, 5, 10 and 20% of water in dmf. the conversion ratios of sucroseto-sucrose esters were determined decreasing sucrose measurement with hplc. the results showed that the best conditions to produce high activity on the enzymatic reaction was by using 40mg/ml of protease from bacillus subtilis at 30 • c, molar ratio of 1:4 (vinyl laurate:sucrose) and adding 10% of water in dmf. finally, we succeeded in the characterization of vinyl sugar ester, which was produced after 25 hours of reaction by 13 c nmr. the results confirmed the c1substituted sugar mono-ester (1 -o-vinyl lauroyl sucrose). effects of oxygen transfer on ␣-amylase production by b. amyloliquefaciens nurhan güngör, güzide ç alık bre lab, department of chemical engng, ankara university, 06100 ankara, turkey ␣-amylase (e.c. 3.2.1.1) a commercially important enzyme used in food, textile, detergent, brewing, paper, and animal feed industries; hydrolyses ␣-1,4 glucosydic bonds in amylose, amylopectin, and related polysaccharides. optimum medium composition and influence of bioreactor operation parameters on ␣-amylase production with high yield and selectivity were determined together with the metabolic flux distributions using b. amyloliquefaciens (nrrl b-14396), which is found to be a good producer of the enzyme. systematic investigation of oxygen transfer in relation with the metabolic fluxes for ␣-amylase is not available in the literature. shake-flask experiments were conducted in 0.5 dm 3 air-filtered bioreactors in orbital shakers with agitation and heating controls (b. braun, certomat-bs1). laboratory-scale bioreactors were composed of agitation, heating, foam, dissolved-oxygen and ph-controlled 1.0 and 3.5 dm 3 systems (b. braun, biostat q; and chemap). after separation of the cells with a sorval rc28s ultracentrifuge, ␣-amylase activity was measured by the dns method bernfeld (1955) . amino acid concentrations were determined with a hplc (waters), pro-tein and organic acid concentrations were measured with a hpce (waters, quanta 4000e) ç alik et al. (1998) . oxygen transfer characteristics in the bioreactor systems were calculated using the dynamic method rainer (1990) . in the mass flux balance-based analyses, a pseudo-steady state approximation for the intracellular metabolites and the accumulation rates of the extracellular metabolites measured throughout the fermentations in considerations of the biochemical feature of the system were used to acquire the flux distributions. in laboratory scale, the effects of different c sources, i.e. glucose, fructose, maltose, lactose and soluble starch; n sources, i.e. (nh 4 ) 2 hpo 4 , (nh 4 ) 2 so 4, and nh 4 cl and/or their concentrations; and the operation parameters, ph and temperature on cell growth, substrate utilization, ␣-amylase and by-product concentrations, and ␣-amylase activity were investigated. in pilot scale, the fermentation and oxygen transfer characteristics of the bioreaction system together with the metabolic fluxes were determined. bernfeld, p., 1955 . methods in enzymol. 1:149-159. ç alık, p., ç alık, g.,özdamar, t.h., 1998 . enzyme microb. technol. 23, 451-461. rainer, b.w., 1990 . geldanamycin is a benzoquinone ansamycin produced as a secondary metabolite by the actinomycete, streptomyces hygroscopicus var. geldanus, in submerged culture. it is a broad-spectrum antibiotic and exhibits an anti-tumour activity through its interaction with the heat shock protein 90 family of chaperone proteins. the optimal recovery of geldanamycin from fermentation broths is the focus of the presented work. the application of adsorbent resins was assessed and the viability of developing a solid phase extraction process for geldanamycin was determined. it was found that recovery of geldanamycin from fermentation broth was possible using adsorbent resins and the use of resins facilitated the recovery of a product stream of high purity. the composition of the fermentation broth had an impact on the performance of the resins and it was found that assessing performance on the basis of experimentally derived data was more apt than studying the kinetics of adsorption alone. adsorptive processes are, by their nature, difficult to optimise and this was found to be the case when optimising the recovery of geldanamycin from partially clarified fermentation broth. considerable effort was required to optimise geldanamycin adsorption, via examining the effect of environmental conditions and process system configuration, and geldanamycin desorption, via examining the effect of environmental conditions and investigating selective elution patterns. it is well known that halophilic eubacteria synthesize compatible solutes in order to face the high ionic strength environment in which they proliferate. these biomolecules are gaining more and more importance as biotechnological tools in a wide array of applications, and the recently developed novel bioprocesses enabled large-scale production of these compounds and therefore commercial distribution. however, there is still interest in the optimization of the production process of sole hydroxyectoine that was demonstrated to have a superior stabilization capacity. in this research project, we optimized growth conditions of marinococcus m52 to obtain high yield of hydroxyectoine. their production proved faster in the batch experiments at a higher oxygen supply, even if the stationary phase was comparable in all cases. the mf experiments showed a final biomass which was 5-fold that obtained in the corresponding batch process. in addition the monitoring of compatible solutes production showed that in the last 24h experiment hydroxyectoine accounted for 80-90% of the total content, accumulating up to 13-15% of the cell dry weight. studies for improving downstream process for ectoine and hydroxyectoine recovery showed that short permeabilization cycles in water are effective in a temperature range between 45 • c and 55 • c using a ratio 1:4/biomass:water. moreover, we evaluated the ability of ectoine to stabilize lactic acid bacteria during freeze-drying and to protect human cells from heat stress. in particular, the compatible solutes were added to the medium of confluent keratinocytes before subjecting the cells to heat stress, or lps insult. rt-pcr and western blot analysis demonstrated the hsp70b' gene over-expression in heat stressed human keratinocytes treated with ectoine. finally, we demonstrated that even at low concentration (50 mm) these compatible solutes are able to diminish cell death in lactic acid bacteria due to lyophilization procedure. among all existing alternative energy sources, biomass-derived bioethanol is especially advantageous since it is clean, sustainable and potentially inexpensive. the actual production of bioethanol is divided into a pre-treatment step, an enzymatic hydrolysis step and a fermentation step. while fermentation has been practiced by humans for centuries, our knowledge of enzymatic hydrolysis is still limited. nevertheless, it is well accepted that hydrolysis is a synergism among three classes of enzymes, ␤-glucosidase, endoglucanase and cellobiohydrolase. furthermore, a complete and efficient hydrolysis is only achieved when the enzymes are in the correct proportion. the common enzyme proportions have so far been based on the natural enzyme abundance as produced by the microorganism or mainly been determined by a trial-and-error approach. in this study, however, we used metabolic control analysis (mca) as a modelling tool to gain fundamental knowledge about enzymatic hydrolysis and to design an optimal enzyme mixture. using gepasi, a free software, the degree of control of each reaction step or each enzyme towards the overall hydrolysis can be calculated. our hypothesis is that the amount of each enzyme used for hydrolysis should be proportional to the degree of control of the enzyme. with mca, a significant amount of time, labour and reagents can be saved on developing hydrolysis enzyme mixture. furthermore, this study should demonstrate the usefulness of mca on understanding enzyme-catalyzed reactions outside the cell. process optimization for fed-batch fermentation of bacillus thuringiensis subsp. israelensis arindam chaudhury, gopinathan c department of biotechnology, university of calicut, calicut, kerala 673635 india. e-mail: g achaudhury@umassd.edu (a. chaudhury) bacillus thuringiensis (bt) is a desirable biopesticide because of its low cost and lack of toxicity. the use of bt in developing countries is limited due to process complications and economic non-feasibility of the fermentation process. in the present study, we have shown how regional production, using inexpensive alternatives for carbon and protein sources, can effectively reduce the cost of mass production of bt. while using alternative media supplements, the biomass production, nor the larvicidal activity was hampered. in addition, the positive effects of sparged aeration and the indispensable role of yeast extract were also proved. this work provides the first experimental proof of delineating the sporulation process and delta-endotoxin production. the role of various buffering agents and additives in increasing biomass production and early sporulation were also investigated. for the production of coenzyme q 10 (coq 10 ), an electron carrier in the respiration chain with antioxidant activity. with decrease of dissolved oxygen level from 20 to 5%, the intracellular coq 10 content increased about 4-fold, yielding 2 mg per g-dry cell weight at 5% dissolved oxygen level. azide significantly increased the intracellular coq 10 content, with the highest value of 5.3 mg per g dry cell weight in the presence of 0.45 mm of sodium azide. however, dnp (up to 200 m) and h 2 o 2 (up to 10 m) did not affect the intracellular coq 10 content, indicating proton gradient release and oxidative stress do not affect the synthesis of coq 10 . these results show that restricted electron flux by limited oxygen supply and the addition of azide increases the intracellular coq 10 content. fourier transform infrared spectroscopy (ft-ir), combined with in situ heat sterilizable attenuated total reflection (atr) probes, constitutes a promising and versatile technique for on-line monitoring of bioprocesses. the ft-ir enables rapid determinations of the medium composition without the requirement of sample withdrawal and preparation. in this work the concentration levels of the substrates glycerol and methanol were monitored on-line in pichia pastoris cultivation. partial least squares (pls) models were used for obtaining the concentration readings. the glycerol concentration measurement proved to be very reliable and reproducible in the glycerol batch phase. however, the on-line information regarding the glycerol concentration was not utilized for any process control purposes. on the other hand, the availability of on-line information about the methanol concentration proved to be crucial for the successful implementation of the cultivations. the temperature strategy in the methanol fed-batch phase utilized temperatures as low as 10 • c. in order to keep the metabolic activity at a reasonable level the culture was therefore pushed towards the maximal substrate consumption rate, rather than being a conventional substrate limited fed-batch. as a consequence methanol accumulation occurred on occasions. without on-line information about the concentration this accumulation, if sustained, would have resulted in a poisoning of the culture, either from methanol itself, or perhaps more importantly from formaldehyde. therefore, it can be concluded that the ft-ir/atr instrument was very useful in this application. jørgensen department of chemical engineering, denmark technical university, building 229, dk-2800 lyngby, denamrk. e-mail: fpd@kt.dtu.dk (f.p. davidescu) modeling biochemical reaction network in microorganisms still represents a challenge due to the very large number of enzyme catalyzed biochemical reactions, to the very complex system and to the many feed-forward and feedback regulation mechanisms. the presented approach to model such a system is based on the stochastic grey-box modeling framework proposed by kristensen et al. (2003) . this methodology consists of parameters estimation based on a prediction error method followed by different statistical tests for parameter significance and for model (in-) validity. the methodology furthermore allows estimation of unknown functional relations, e.g. kinetic rates. a set of experimental data zangirolami (1998) obtained during continuous cultures of a high enzyme producing aspergillus oryzae strain. the oxygen concentration was decreased stepwise and the substrate concentration was modified from one experiment to other. a model proposed by agger et al. (1998) is investigated on these data. the primary interest is to develop a physiologically feasible model, also at the low oxygen concentrations often found in industrial practice. microbially produced secondary metabolites such as antibiotics have tremendous economic importance. streptomyces spp. have long been identified as sources of antibiotics and chemotherapeutic compounds, synthesising over 4000 bioactive compounds. geldanamycin is a novel chemotherapeutic agent produced by streptomyces hygroscopicus var. geldanus in submerged fermentation. initial studies have focused on optimisation of media design through understanding and controlling metabolic routes of biosynthesis within the cell. geldanamycin is a by-product of the shikimate or aromatic amino acid biosynthesis pathway. stimulation of this pathway and concomitant production of geldanamycin is achievable through amino acid control. increasing concentrations of primary carbon source greatly influence biomass generation and product formation, as does the inclusion of cations such as magnesium and calcium to the fermentation media. optimisation of production media through balancing minerals, nitrogen, and carbon sources has significantly improved antibiotic yields in shake flask cultures and the development process will be extended into pilot scale through the use of bioreactors. microbiology and biotechnology research group, school of life sciences, napier university, edinburgh, eh10 5dt, scotland. email: m.el-mansi@napier.ac.uk (m. el-mansi) synopsis: during growth of corynebacterium glutamicum on glucose or other glycolytic intermediates, pep carboxylase fulfils an anaplerotic function as it replenishes intermediary metabolism with biosynthetic precursors that are essential for growth and glutamate production. under these conditions, pep carboxylase plays a central role and this in turn is characterised by a high flux control coefficient thus rendering this enzyme an ideal target for metabolic interventions. further analysis in silico revealed that any increases in the concentration of the enzyme was accompanied by increases in flux through the enzyme itself as well as glutamate formation, presumably as a consequence of sustaining a high intracellular level of ␣-ketoglutarate; the immediate precursor for glutamate biosynthesis. a combined approach to enhance periplasmic expression of human growth hormone in escherichia coli, using a modified signal peptide from alpha amylase gene of bacillus licheniformis s.k. falsafi 1,2 , a. zomorodipour 2 : 1 islamic azad university of jahrom, iran; 2 department of mol genet. national inst for genet eng & biotechnol., tehran, iran. e-mail: soheil falsafi@yahoo.com (s.k. falsafi) the alpha amylase gene signal peptide, originated from a strain of bacillus licheniformis, was shown to be able to transport its native protein, when expressed in e. coli the competence of the fusion protein being processed and translocated through the inner membrane is highly dependent on the amino acid sequences in the signal peptide. therefore, in order to increase the expression efficiency of bla signal peptide, we reconstructed the bla signal peptide coding fragment with the following modifications. two rare codons of arg 6 (cgg) and arg 10 (cga) and codons for leu 15 (tta) and pro 23 (cct), in the signal peptide were substituted with their corresponding e. coli major codons. two other changes, including phe 20 (ttc) → leu 20 (ctg) and ala 28 (gcg) → met 28 (atg), were also introduced to increase the processing efficiency. the hgh-expressing plasmid equipped with the modified bla (blaf2) was subjected for further expression analysis in a t7-based expression system. the results obtained from the protein patterns of the induced bacteria indicates in high expression level of hgh preprotein (hgh::blaf2) followed by efficient transfer of the mature hgh to e. coli periplasm. (ip6) has been demonstrated to have a wide range of health benefits such as prevention and therapy of various cancers, amelioration of heart disease, and prevention of renal stone formation as well as complications from diabetes. on the hand, lower phosphorylated forms of inositol, especially inositol trisphosphate (ip3) and inositol tetrakisphosphate (ip4) are important signal transduction molecules within the cells both in plants and the animal kingdom. it has been hypothesized that at least the anticancer function of ip6 is mediated via these lower inositol phosphates. the diversity and practical unavailability of the individual myo-inositol phosphates preclude their investigation. phytases, which catalyze the sequential hydrolysis of phytate, render production of defined myoinositol phosphates in pure form and sufficient quantities. different phytases may result in different positional isomers of myo-inositol phosphates and therefore different biochemical properties. phytases differing in ph optima, substrate specificity, and specificity of hydrolysis have been identified in plants and microorganisms. in this paper the dephosphorylation pathway of the novel phyfauia1 was compared to other bacterial phytate degrading enzymes. preliminary results have shown that phyfauia1 converted ip6 into ip5 (myoinositol 1,2,3,5,6 pentakisphosphates) and another isomer, which is yet to be elucidated. characterization of the novel ␤-peptidyl aminopeptidase (bapa) from sphingomonas sp. 3-2w4 that cleaves synthetic ␤peptides birgit geueke, hans-peter e. kohler environmental microbiology, eawag, 8600 duebendorf, switzerland. e-mail: birgit.geueke@eawag.ch (b. geueke) non-natural peptides, which are capable of evoking a specific biological response, are currently receiving much attention. oligomers of ␤-amino acids (␤-peptides) are representing a group of pharmaceutically interesting peptides because of their very high stability towards enzymatic degradation and their ability to mimic the structure of naturally occurring biologically active peptides. the pharmaceutical potential on the one hand and the high stability on the other hand aroused interest for studies on the environmental fate and the degradation behaviour of this class of compounds. a novel bacterial strain (sphingomonas sp. 3-2w4) that was capable of degrading short ␤-peptides was isolated from an enrichment culture. the ␤peptide degrading enzyme was purified and its gene sequence was determined (bapa). the gene encodes a ␤-peptidyl aminopeptidase (bapa) of 402 amino acids that is synthesized as preprotein with a signal sequence of 29 amino acids. it belongs to the n-terminal nucleophile (ntn) hydrolase superfamily and is the first peptidase that is capable of cleaving amide bonds in ␤-peptides composed of synthetic ␤-amino acids. the biochemical properties of recombinant bapa were investigated regarding its substrate specificity and possible application in the synthesis of ␤-peptides. to produce efficient strains of agaricus bitorquis (quel.) saccardo, which are resistant to high temperatures p. guler, a. ergene, s. tan kirikkale university, faculty of science and literature, department of biology, yahsihan-kirikkale in this study, the culture mushroom agaricus bitorquis (quel.) sacc. the growth of the mycelium and the fructifications under high temperature is examined. the spores taken from the mushrooms that were collected from nature were grouped as a, b, c, d, e. the spores were inoculated into malt extract agar and incubated at 30 • c and primer mycelium was produced. the mycelium discus taken from primer mycelium in 8 mm diameter were inoculated into the center of malt extract agar and incubated at 30, 32, 34, 36 and 38 • c separately. during the incubation period the growth of the mycelium were measured. during the growing period the radial growth speed of the mycelium were taken as criteria. the best mycelium growth for all groups was seen at 30 • c. at 36 • c the e group mycelium and at 38 • c other group's mycelium did not grow. these temperatures were determined as thermal lethal point for the groups. from all the mycelium produced from all temperatures spawn was prepared and with the results taken from these, spawn calendar is prepared. in this research, the spawn was inoculated to compost with mixing system and separately put in culture rooms, temperatures as 30 and 32 • c. at this level the culture mushroom production techniques were used. the harvested mushrooms were inspected morphologically. at this morphological inspections the cap width, cap tissue thickness, stalk thickness and stalk long ness was taken as criteria. in the study the best growth was seen at d group mushrooms and this group mushrooms tyrosinase's activities were measured and graphics were made. introduction: viral contamination of biological products; cause many problems in viral diagnostic laboratories, blood transfusion organizations, and biological producers. bovine viral diarrhea virus (bvdv), from the pestivirus genus, is the most common viral contamination in (fetal) bovine serums (fbs). also, bvdv used as a module, for study hepatitis c virus inactivation due to its similarity in structure and genome. pulsed uv lights (puvls) have this potential to inactivate known and unknown or reemerging viruses as well as prions. two puvl with the wavelengths of 355 and 266 nm, were produced by q-switched nd 3+ :yag laser in its third and forth harmonic, respectively. the energy of each pulse for 355 nm was 12.7 mj/cm 2 and for 266 nm was 35.2 mj/cm 2 . bvdv were produced and titrated in mdbk cell line. mdbk and fbs were already checked for non-cytopathic or cytopathic pestiviruses, using related ag-elisa kit. bvdv suspended in solution with the dilution of 1:2 before exposure. the quartz tube with the minimum uv-absorption in compare with air, used as a container for exposed solutions. calculation of the virus titer, 10 4.3 tcid 50 /ml, was done based on the reed and muench method. bvdv suspended in pbs was exposed into the 3.52-352 j/cm 2 of puvls with the wavelength of 355 nm and also, was exposed into the 1.27-92.25 j/cm 2 of puvls with the wavelength of 266 nm. furthermore, bvdv suspended in fbs was exposed into the 88, 176, 352 and 704 j/cm 2 of puvls with the wavelength of 355 nm and also, was exposed into the 6. 35, 25.4, 63.5, 127 and 190 .5 j/cm 2 of puvls with the wavelength of 266 nm. results: the minimum dose for inactivation of bvdv suspended in pbs with the 355 and 266 nm wavelengths of puvls, were 352 and 92.25 j/cm 2 , respectively. also, the minimum dose for inactivation of bvdv suspended in fbs with the 355 and 266 nm wavelengths of puvls, were 704 and 127 j/cm 2 , respectively. to evaluate the fbs quality to support cell culture, treated fbs with the dose of 190.5 j/cm 2 of 266 nm puvls was used to grow vero cell line in 12 successive passages. the viability of cells in two study groups was identical. the statistical evaluation of two treated groups showed no significant difference, in 12 passages. conclusion: because inexpensive equipment can be used to produce puvls capable of handling different volumes of biologics with operational ease, this viral inactivation technique is cost effective for relevant industries. the procedure has the potential to be combined synergically with other inactivation method. puvls offer a new, nonadditive and chemically safe alternative for the treatment of fbs to inactivate adventitious viruses and to preserve the biological activity necessary for the propagation of cell culture. characterization and gene cloning of the g-resorcylic acid decarboxylase for application to selective production of g-resorcylic acid y. iwasaki 1 , y. ishii 2 , k. kino 1 , k. kirimura 1 : 1 dept. appl. chem., sch. sci. eng., waseda univ., tokyo, japan; 2 bme, asmew, waseda univ., tokyo, japan. e-mail: iwasaki@moegi.waseda.jp (y. iwasaki) for selective production of ␥-resorcylic acid (␥-ra, 2,6-dihydroxy-benzoic acid) from resorcinol (re, 1,3dihydroxybenzene) under mild conditions, we screened various microorganisms and found the reversible ␥-ra decarboxylase (rdc) as a novel enzyme applicable to carboxylation of re to form ␥-ra, in a bacterial strain rhizobium radiobacter wu-0108 1) . rdc catalyzed the decarboxylation of ␥-ra, and regio-selective carboxylation of re to form ␥-ra, without formation of ␣-ra and ␤-ra. the molecular weight of rdc was estimated to be 130 kda by gel-filtration, and that of the subunit was determined to be 34 kda by sds-page, suggesting that rdc is a homotetrameric structure. the gene encoding rdc was sequenced, and a site-directed mutagenesis study revealed that the two histidine residues at positions of 164 and 218 in rdc are essential for the catalytic activity of rdc. through the reactions using e. coli cells highly expressing rdc, 6.7 mm ␥-ra was produced from 15 mm re at 30 • c for 16 h, with a yield of 45%. ishii, y. et al., 2004. biochem. biophys. res. commun., 324, 611-620. laccase biosynthesis in stirred fermenters teresa jamroz, stanislaw ledakowicz, barbara sencio department of bioprocess engineering, technical university, lodz pl 90-924, poland industrial applicability of enzymes is closely related to development of efficient methods of their production. currently, significant interest in lignolytic enzymes, including laccase, has been observed. laccase is an enzyme applied in various industrial branches and environmental processes. broad laccase applicability induces researchers to develop urgently efficient methods for its commercial production. laccase (ec.1.10.3.2. p-diphenol oxidase) is produced by cap mushrooms from the class basidomycetes. this is the so-called white rot fungi which in natural conditions appears on both living and dead wood. as shown in the practice of biotechnological processes, high-efficient strains have low resistance to destructive factors in bioreactors. hence, to preserve a proper morphology and physiological state of an organism, strictly determined culture conditions must be obeyed. this is very important in the case of basidomycetes for which submerged culture in the liquid phase is not a natural habitat. results of studies on laccase production from cerrena unicolor family are discussed. cultivation of active biomass was carried out in stirred tank and rotating disc bioreactors of different volume (b. braun of working volume 12 dm 3 ; fas-01 of working volume 3.5 dm 3 ). experiments in both fermenters were made at impeller revolutions 200 and 300 min −1 , on a modified lindberg substrate. significant differences in the rate and yield of laccase production were reported. an almost three times higher values of laccase activity were obtained in the b. braun fermenter, at rotational speed 300 min −1 . to retain suspended cells in bioreactor a filtration process can be used. the biomass is concentrated by withdrawing cell-free culture broth. if the desired product is dissolved in the broth (extracellular production), the procedure enables the continuous harvest in the cellfree permeate. an application test of filtration system for suspended biomass of aspergillus niger in submerged single stage continuous culture was presented in this report. the system is easy to construct and there is a possibility of its sterile exchange during cultivation. the culture medium contained the following substances (g/dm 3 ): white sugar, 150.0; nh 4 no 3 , 1.5; mgso 4 ·7h 2 o, 0.2; kh 2 po 4 , 0.2; feso 4 ·7h 2 o, 0.05. fermentations were carried out in the lab bioreactor biomer 10. the bioreactor was a standard cstr (continuous stirred tank bioreactor) with working volume of 5 dm 3 . high citric acid concentration in culture medium (p = 108.9 g/dm 3 ), high yield of citric acid (y p/s = 72.6%) and high efficiency coefficient (k ef = 71.1) were observed in single stage continuous culture with biomass retention. oxygen transfer regulates benzaldehyde lyase production in e. coli pınar ç alık, iblab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: pcalik@metu.edu.tr the effects of oxygen transfer rate on benzaldehyde lyase (bal) production by puc18::bal carrying recombinant escherichia coli on a defined medium with 8.0 kg/m 3 glucose were investigated in order to fine-tune the bioreactor performance, in v = 3 dm 3 batch bioreactors at five different conditions with the parameters at, i.e. q o /v r = 0.5 vvm and n = 250, 375, 500, 750 min −1 and; q o /v r = 0.7 vvm and n = 750 min −1 . the concentrations of the product and by-products amino acids and organic acids were determined in addition to bal activities. medium oxygen transfer rate conditions and uncontrolled ph operation at ph o = 7.25 are optimum for maximum bal activity, i.e. 860 u/cm 3 at 12 h, and productivity and selectivity. on the bases of the data, response of the intracellular bioreaction network of r-e. coli to oxygen transfer conditions were analysed using a mass-flux balance based stoichiometric model that contains 102 metabolites and 133 reaction fluxes. the results reveal that metabolic reactions are intimately coupled with the oxygen transfer conditions. oxygen transfer rate showed diverse effects on the product formation by influencing metabolic pathways and changing metabolic fluxes. metabolic flux analysis was helpful to describe the interactions between the cell and the bioreactor by predicting the changes in the fluxes and the rate controlling step(s) in the metabolic pathways. therefore, knowing the distribution of the metabolic fluxes during the growth, and bal and by-product formations provide new information for understanding physiological characteristics of the r-e. coli, and reveals important features of the regulation of the bioprocess and opens new avenues to successful application of metabolic engineering. saprophytic mycobacterium strains belong to the best known microorganisms which have been applied to the pharmaceutical industry for the production of steroid drugs. the mycobacterial cell wall is the permeation barrier to chemical compounds, including lipophiles. using isoniazid (inh), the inhibitor of the mycolic acids biosynthesis, we were able to demonstrate increased ad production and susceptibility to antimicrobial agents. the process of sterol transformation and products accumulation was monitored using gas chromatography. isoniazid was shown to intensify ␤-sitosterol side-chain degradation by mycobacterium sp., and accumulation of 4-androstene-3,17-dione (ad) and 1,4-androstadien-3,17-dione (add), which are the starting materials in the biotechnology of medically important steroids. to confirm these results, the sensitivity of the bacteria to antimycobacterial drugs was performed. the minimum inhibitory concentration mic 50 of rifampicin and erythromycin decreased markedly in the presence of inh. this work was supported by grant nr 3p04c 06923 of the committee for scientific research. for the purpose of high-throughput screening and to reduce experiments with animals in pharma biotechnology biosensor systems gain importance. the principle of a biosensor is the combination of cultured cells and a sensorchip device, which allows the monitoring of cellular activity. in contrast to traditional analytics with a biosensor you can measure on-line cellular activity change caused by an effector as well as the restored activity after privation of the effector (re-native activity). cmos technology can be used for the realisation of various biological sensorchips such as adhesion sensorchips, metabolical sensorchips and electrophysiological sensorchips. the standard cmos technology allows a high reproducibility of the chips, the integration of electronic components on the chip, which reduces the amount of external devices and the combination of different sensors on one chip. in cooperation with the semiconductor company micronas and the biotech company bionas we have realised different types of cmos sensorchips to measure adhesion of a cellular monolayer with interdigitated electrodes (ides), metabolical activity via acidification with ion-sensitive field effect transistors (isfet) and sponteneous neuronal network activity with passive palladium electrodes. microbial agents have been applied to the different stages of pulp and paper processing. the work presented describes a study on the effect of applying ligninolytic enzymes, such as a laccase plus mediator system, on a variety of different types of pine and eucalyptus pulps and subsequently subjecting these to different ageing processes. industrial pulps were obtained from different portuguese pulp and paper companies. the pulps used were (1) unbleached pine pulp from portucel tejo; (2) unbleached eucalyptus pulp from portucel setúbal; (3) bleached eucalyptus pulp from portucel setúbal; and (4) pulp made from recycled paper from renova s.a. several types of handsheets were produced with 2 different grammage namely, 60 and 180 g/m 2 . the prepared handsheets were subject to an aging sequence in three different chambers: ultraviolet radiation (wavelength of 280 nm), temperature (19 • c) and moisture (70%); and thick saline fog at a concentration of 1% and temperature of 35 • c. in order to evaluate the effect of moisture cycles and temperature, two aging sequences were used for each type of handsheet. in the first, the moisture varied (60, 80 and 100%), while the temperature was held constant (25 • c); in the second the temperature varied (60, 70 and 80 • c) and the moisture was held constant (50%). following the aging phase, the handsheets were subject to several chemical (viscosity and index kappa) and physico-mechanical (colour, tensile breaking strength, stretch and the bursting strength) tests in order to characterize the effect of the aging conditions. results will be presented describing the effect of application of the laccase-mediator system on the optical and mechanical properties of the prepared handsheets. fundação para a ciência e a tecnologia, project pocti/ agr/47309/02. aspergillus niger is a filamentous fungi widely used in industry. its growth as freely dispersed hyphae leads to an increase in the medium viscosity and to problem of mass transfer, especially oxygen transfer. oxygen acts both as final electron acceptor in the mitochondrial chain and as nutrient for the biosynthesis of unsaturated fatty acids and sterols. thereby, a lack of oxygen affects the nadh/nad ratio, the atp production, the growth and has a strong influence on the physiology of the microrganism. in the present study, the metabolic changes of a. niger in response to a lack of oxygen was investigated using oxygen limited chemostats combined with nitrogen pulse. under these conditions, the main consequence of a sudden decrease of oxygen availability is an increase in the mannitol production. this work showed that the mannitol biosynthesis, involving the enzyme mannitol-1-p dehydrogenase, helps the reoxidation of nadh when the final electron transport acceptor, oxygen, is limiting. investigation of the lipase activity of the bacteria isolated from olive mill wastewater sevgi ertugrul 1 , nur koçberber 1 , gönül dönmez 1 , serpil takaç 2 1 department of biology faculty of science ankara university 06100 beşevler ankara turkey; 2 department of chemical engineering faculty of engineering ankara university 06100 tandogan, ankara, turkey the bacteria that could grow on media containing olive mill wastewater (omw) were isolated and their lipase production capacities were investigated. the strain possesing the highest lipase activity among 17 strains grown on tributryin agar medium was identified as bacillus sp. the effect of ph on the lipase activity of the strain was investigated in tributryin medium and ph 6 was found to be the optimal. the liquid medium composition was improved by adding different carbon sources and fatty acids into tributryin medium -omitted tributryin -to increase the enzyme activity. the cultivations were performed at 30 • c and ph 6. lipase activity of the bacillus sp. was measured spectrophotometrically through the hydrolysis of p-nitrophenol palmitate. among the media containing different compositions of tricapryn, trimyristin, tributyrin, triacetin, tween 80, glycerol-trioleate, glycerol-trioctanoate, glycerol-tridodecanoate, omw, glucose, and whey; the medium consisted of 20% whey + 1% glycerol-trioleate was found to give the highest lipase activity. cultivation of bacillus sp. in the optimum medium at ph = 6 and 30 • c for 64 h was resulted in the extracellular and intracelluar lipase activities of 15 and 168u/ml, respectively. this study was supported by ankara university biotechnology institute (project no: 2004-151 and 2005-164) . under different abiotic stresses, cell growth and metabolic activity are highly influenced in all types of living organisms medium osmolality is usually one of those factors affecting different types of biological systems in different ways. however, even in the same organ of higher eukaryotes the degree of osmoregulation mechanism is highly variable in different types of cells. therefore, studying the effect of osmotic stress on mammalian cell is very important subject for particular cell line. the effect of hyperosmotic pressure on the kinetics of cell growth of and metabolic activity of mesenchymal stem cells (mscs) and two industrially important cell lines, hybridoma cells and human embryonic kidney cell (hek) were investigated in batch cultures at different osmotic pressures in the range from 325 to 500 mosm kg −1 . in case of mscs cells, the maximal specific growth rate [] of 0.029 [h −1 ] associated with the highest specific glucose consumption rate [-q gluc ] of 0.1129 × 10 −6 [mol cells −1 h −1 ] was obtained in medium of 375 mosm kg −1 . in case of hybridoma cells, osmotic pressure showed not only influence on the kinetics of cell growth and metabolism but also on the monoclonal antibody production. the maximal mab production was obtained in case of cells cultivated under osmotic pressure of 375 mosm kg −1 . further increase in osmotic pressure resulted in significant reduction in growth rate as well as mab production. on the other hand, hek cells were more sensitive to osmotic pressure in industrially used serum free medium and the addition of serum decreased the inhibitory effect of high osmotic pressure on the cells. gustavo g. fonseca 1,2 , andreas k. gombert 2 , elmar heinzle 1 , christoph wittmann 1 : 1 biochemical engineering, saarland university, saarbrücken, germany; 2 chemical engineering, são paulo university, brazil kluyveromyces marxianus cbs 6556 is a potentially interesting yeast strain characterized by a high capacity of conversion of substrate into biomass. however, this yeast has been only marginally studied so far. therefore, we performed a metabolic characterization in batch and chemostat cultures at dilution rates of 0.10, 0.25 and 0.5 h −1 . the specific rate of o 2 consumption (qo 2 ) increased with dilution rate from 2.87 to 11.09 mmol (g dw) −1 h −1 . the respiratory coefficient remained almost stable around 1.0 for all metabolic states investigated. even at the dilution rate of 0.5 h −1 , which is close to the maximum growth rate of the strain of 0.56 h −1 , no significant overflow metabolism was observed. the concentration of extracellular metabolites increased with the dilution rate, but remained below 6% of the carbon consumed as glucose. all carbon balances closed near 100% underlining the consistency of the data. in contrast to s. cerevisiae the respiratory capacity of k. marxianus cbs 6556 is not strongly influenced by the dilution rate in aerobic chemostat or batch cultures, indicating its high potential for biomass-directed applications. a thermostable l-arabinose isomerase for enzymatic production of d-tagatose o. hansen, f. jørgensen, p. stougaard department of enzyme technology, bioneer a/s, kogle allé 2, dk-2970 hørsholm, denmark. e-mail: och@bioneer.dk (o. hansen) d-tagatose, an isomer of d-fructose, is a low-calorie bulk sweetener with a sweetness equivalent to sucrose. d-tagatose has obtained gras approval for use as a food ingredient, and is currently produced by chemical isomerization of d-galactose, which may readily be obtained by hydrolysis of lactose. structurally, d-galactose is closely related to l-arabinose, and it has previously been shown that some variants of l-arabinose isomerase (araa) may catalyze the conversion of d-galactose to d-tagatose, in addition to the metabolic conversion of l-arabinose to l-ribulose. we have screened a number of bacterial araa enzymes for their ability to catalyze the isomerization of d-galactose to d-tagatose. the best enzyme was found in the thermophilic bacterium thermoanaerobacter mathranii (dsm11426). the araa gene of t. mathranii was cloned, sequenced and expressed in e. coli. amino acid sequence comparisons of the t. mathranii sequence and other known araa sequences showed a relatively low sequence identity of about 30%, indicating a distant phylogenetic relationship to the other members of the l-arabinose isomerase group. the t. mathranii enzyme was thermostable with optimal activity at 65 • c and it required manganese ions. unlike other araa variants, the t. mathranii enzyme showed k m values in the same order of magnitude for l-arabinose and d-galactose, suggesting that this enzyme is a versatile isomerase capable of isomerizing structurally related aldoses. the enzyme was immobilized by chemical cross-linking of a crude e. coli cell homogenate, and the immobilized enzyme efficiently converted d-galactose into d-tagatose. currently, we are developing an enzymatic method for industrial production of d-tagatose using the immobilized enzyme. the agricultural production can be negatively affected by different pest insects (pi) and the use of chemical insecticides (chi) has been the traditional method for controlling pi during decades. nevertheless, there are various ecological implications due to the extensive application of chi. a viable alternative for the use of chi in certain agro-systems, is the use of entomopathogenic nematodes (epn) of the genera steinernema and heterorhabditis that are natural pathogens for different pi; besides, the presence of a symbiotic bacterium is necessary for an effective entomopathogenic activity can take place. the nematode/bacterium complex does not represent a risk for the environment. different authors propose that the best alternative for the massive production of epn is the submerged culture within bioreactors; nevertheless, more research is required to have really robust processes. particularly, information regarding the actual hydrodynamics during epn production and its relation with the epn productivities are scarce, among other aspects. the present study deals with the hydrodynamic characterisation during the production of the epn steinernema carpocapsae and its symbiont bacterium xenorhabdus nematophilus, in submerged monoxenic culture in an internal-loop air-lift bioreactor (v l = 4.5 l) using two culture media: one of them containing whey and the other one, agave juice, aguamiel, (agave spp.). process viscosity of the culture broth was determined along the time, exhibiting a maximum value of 20 mpa s. moreover, it was determined that the hydrodynamic conditions were always located within the laminar region (re < 500). at the experimental conditions tested, it can be inferred that the epn productivity are more sensitive to changes in the culture medium composition than on the prevailing hydrodynamic conditions during the fermentations. bacillus thuringiensis is a gram-positive bacterium used as a biological pest control agent. moreover, it is able to produce, several biologically active molecules such as bacteriocins and hydrolytic enzymes among which chitinases that play double roles, fungicide and improving the insecticidal effect of b. thuringiensis deltaendotoxins. a newly isolated b. thuringiensis subsp. kurstaki strain bupm4, was shown to produce a novel bacteriocin named bacthuricin f4. the highest bacteriocin activity was found in the growth medium and evidenced in the late exponential growth phase. upon purification of bacthuricin f4, the specific activity was increased 100-fold. this bacteriocin was heat-stable up to 70 • c and resisted up to ph 3.0. its molecular mass, determined by mass spectrometry was 3160.05 da. direct n-terminal sequencing of bacthuricin f4 revealed the following sequence: dwtxwsxl. the latter was unique in the databases. bacthuricin f4 was active against bacillus species while it had little or no effect on gram-negative bacteria. the bacteriocin produced by the b. thuringiensis strain bupm4 respond to both criteria of thermostability and stability to low phs. thus, it could be used as a source of bacteriocin active against related species of bacillus harmful for agricultural products and as food preservative. the other example of antimicrobial compound produced by b. thuringiensis is a chitinase. we describe the selection of b. thuringiensis high chitinase-producing strain bupm255, and the characterization and the heterologous expression of a novel chitinase encoding gene. the cloning and sequencing of the corresponding gene named chi255 showed an open reading frame of 2031 bp, encoding a 676 amino acid residue protein. both nucleotide and amino acid sequences similarity analyses revealed that the chi255 is a new chitinase gene, presenting several differences from the published chi genes of b. thuringiensis. the identification of chitin hydrolysis products resulting from the activity, exhibited by chi255 through heterologous expression in e. coli revealed that this enzyme is a chitobiosidase. the addition of the sequence of chi255 to the few sequenced b. thuringiensis chi genes might contribute to a better investigation of the chitinase "structure-function" relation. cloning and characterization of s-adenosyl-l-methionine synthetase from pichia ciferrii dscc 7-25 kwon-hye ko 1 , gee-sun yoon 1 , gi-sub choi 1 , joo-won suh 2 , yeon-woo ryu s-adenosyl-l-methionine(sam) has an important role for dna methylation and cell signaling. sam was synthesized from methionine and atp by sam synthetase and play an pivotal function in the primary and secondary metabolism of cells. recent studies have revealed in the effect of sam in case of morphological differentiation in both eukaryotes and prokaryotes. the p. ciferrii produces large quantities of sphingoid base. tetraacetylphytosphingosine(taps), which is a precursor of sphingolipid, could be used for the production of pharmaceuticals and cosmetics. we isolated sam gene from p. ciferrii and cloned it into expression vector for e. coli and p. pastoris, respectively. an 1.2 kb sam-s gene fragment was isolated by low-strigency pcr using degenerated primer. by the analysed primary sequence deduced from dna sequence, this gene included conserved domains similar with other well-known sam synthetase. first of all, sam synthetase gene cloned pgem-t vector and subcloned into histidine tagging system to purify the expressed protein using metal chelating resin. typical characteristic analysis of this enzyme is underway. metabolic networks offer a large variety of different synthesis pathways starting from cheap substrates and leading to interesting high-value compounds, i.e. metabolites. in case an interesting pathway can be disconnected from the remaining metabolic network, the perforated cell or the crude extract could be used for a one-pot multi-step synthesis of the desired compound. pathway isolation, achieved by deletion of genes encoding gene products enabling side reactions, interferes with the viability of the organism, which is a requisite for the production of the system of biotransformation (sbt). in this work, a rational systems biology-derived approach is presented for the design of a sbt. it is illustrated for a sbt allowing for production of dihydroxyacetone phosphate. the design procedure comprises three steps: (i) a production pathway is identified in the metabolic network of e. coli. the e. coli pathway is complemented by two additional enzymes in order to obtain a fully energy and redox balanced production pathway. (ii) an optimal combination of gene knockouts is designed and a suitable growth medium composition is identified, both by a model-based approach: flux balance analysis of a genome-scale metabolic network is used to predict enzyme expression within the wild-type organism on different media, while a mixed-integer optimisation is employed to identify viable mutants as this approach strongly depends on the quality of the fba prediction, available regulatory information on the usage of metabolic pathways and thermodynamic constraints were taken into account. (iii) thermodynamic analysis of the obtained, partially branched sbt reaction cascade revealed the extent of loss in yield by the remaining side reactions. in summary, this systems biology-driven approach potentially enables the substitution of a elaborative multi-step synthesis process by a one-pot enzyme reaction cascade. institute of industrial biotechnology, inha university, incheon 402-751, korea. e-mail: leecg@inha.ac.kr (c.-g. lee) algal biotechnology is drawing increasing interest due to its potential as a source of valuable pharmaceuticals, pigments, carbohydrates, and other fine chemicals. currently, its application is being extended to the areas of wastewater treatment and agriculture. however, lack of suitable photobioreactors (pbrs) makes the cost of algally-derived compounds higher than those derived by chemical synthesis and thus has prevented widespread use of algal cultures. the culture of algae prior to the late 1940s was apparently restricted to laboratory scale operations. experiments on outdoor algal mass production began in the late 1940s with nearly concurrent development of experimental culture facilities in germany and the united states. for the next two decades, outdoor mass culture of algae was undoubtedly the hottest topic in the algal biotechnology area. recent developments of high-density pbrs enable the production of valuable biologically active compounds by algal mass cultures. however, light is almost always the limiting factor in high-density photobioreactors. key factors for successful photobioreactors will be discussed and various photobioreactors will be analyzed and compared for their advantages and disadvantages. the new techniques, such as pigment redcution and application of flashing light and lumostatic operation, will be discussed for possible solutions to overcome the light limitation in high-density microalgal cultures. a quick and reliable method for screening fungal transformants for specific genetic modifications is essential for many molecular applications, for example when one tries to develop a transformation system for a new fungal host. southern analysis is laborious and time consuming. several colony hybridisation methods have been developed for the analysis of a large number of transformants. unfortunately these methods suffer from different disadvantages such as non-specific binding, a limited usability to screen for specific integration and the fact that these procedures always take a few days (van zeijl et al., 1998) . recently, methods for pcr-based analysis of fungal transformants have been described. most of these methods require high quality dna. many methods for dna extraction from fungi have been described in the past few years. these methods often are tedious, time consuming, costly or limited to a small number of samples each time. most of the available protocols include the growth of mycelium in a liquid culture, followed by freeze-drying or maceration in liquid nitrogen and grinding of the frozen material to break the cell walls (cassago et al., 2002) . lately a few methods have been described to isolate dna from fungi suitable exclusively for pcr and appropriate for the simultaneous treatment of a large number of samples. some methods also describe the direct use of mycelium (cooke et al., 1997) in the pcr-reaction mixture. we compared the applicability of a few rapid dna extraction methods for myrothecium gramineum and tested the resulting dna samples on there suitability for pcr-applications. myrothecium gramineum is a filamentous ascomycete used in ongoing research as a new cloning and expression host. five methods were tested. in four of these methods dna was extracted from mycelium (goodwin et al., 1993 and aljanabi et al., 1997) or spores (ferreira et al., 1997 and xu et al., 1995) prior to pcr. a fifth assay used mycelium straight in the pcr-reaction mixture. only this last method seemed useful for myrothecium to isolate dna suitable for pcr. fragments up to 2000bp were amplified. cheese whey is a liquid effluent from cheese-making processes. there is an increased interest in the economic utilization of whey produced by the dairy industries, because the whey is a pollutant, due mainly to its lactose content. the goal of this work was to find the most suitable values of some fermentation parameters for lactic acid production from whey by a lactic acid bacterium, lactobacillus helveticus (atcc 15009). the effects of lactose content, temperature, ph and the supplementation with yeast extract were investigated using surface response methodology. a composite central design was used with three center points, making a total of 27 operational conditions. the region of maximum production is outlined by the following intervals: temperature around 40 • c; lactose concentration between 70 and 85 g/l; concentration of yeast extract between 20 and 25 g/l; ph between 7 and 7.5. fermentation studies on a continuous fermentative process coupled to a vacuum flash evaporator were carried out in lab scale equipment. the phases of this work consisted in an assembly and instrumentation of the prototype and elaboration of a supervisory system coded in labview 6.1, which allows the data acquisition and control through personal computers. the experiments in continuous fermentation used saccharomyces cerevisiae and sugar cane molasses as substrate. the analytical follow up was done through analysis of total reducing sugars, ethanol, glycerol, dry mass and viable cells. the system worked for months uninterruptedly, producing an alcoholic solution at the condenser with 50 • gl. the fermentation operated with concentrations of ethanol at 5 • gl, which is a weakly inhibitory value for the yeast of the process, even when fed with concentrated cane molasses, containing up to 330 g/l of sugar. the result meets the initial goal, which was to operate the system with low level of ethanol and to guarantee high productivity, even in high concentrations of sugar in the feeding. the results showed that system productivity was superior to that of the conventional continuous process. lactic acid (la) is a versatile chemical, used as an acidulant, flavor and preservative in the food, pharmaceutical, leather and textile industries, and for production of biodegradable poly lactic acid (pla). l(+)lactic acid is the only optical isomer for use in pharmaceutical and food industries because human body is only adapted to assimilate this form. in this research, lactic acid production was improved on 20 l fermentor. in our experience, among six strains of lactobacillus were examined for the production of l(+) lactic acid, lactobacillus casei ssp. casei atcc 39392 was selected as a highest l(+) lactic acid producer. optimized medium used for lactic acid production contained (per l) 80 g glucose, 50 g whey powder and 20 g corn steep powder. for a homofermentative process, ph 6.0 was found to be optimal. in order to avoid product inhibition, the produced lactic acid was neutralized using calcium hydroxide. maximum production and productivity of lactic acid in batch system, were 81 g and 1.35 g/lh, but in fed batch system, after 3 feeds of glucose, production and productivity increased up to 360 g and 4 g/lh. saleh a. mohamed molecular biology dept., national research centre, cairo, egypt an extracellular polygalacturonase (pgii) from trichoderma harzianum was purified to homogeneity by two chromatography steps using deae-sepharose and sephacryl s-200. the molecular weight of t. harzianum pgii was 31,000 da by gel filtration and sds-page. pgii had isoelectric point of 4.5 and optimum ph of 5.0. pgii was very stable at the ph 5.0. the extent of hydrolysis of different pectins by enzyme was decreased with increasing of degree of esterification (de). pgii had very low activity toward nonpectic polysaccharides. the apparent km value and kcat value for hydrolyzing polygalacturonic acid (pga) were 3.4 mg/ml and 592 s −1 , respectively. pgii was found to have temperature optimum at 40 • c and was approximately stable up to 30 • c for 60 min of incubation. all the examined metal cations showed inhibitory effects on the enzyme activity. 1, 10 phenanthroline, tween 20, tween 80, triton x-100 and sds had no effect on the enzyme activity. the rate of enzyme catalyzed reduction of viscosity of solutions of pga or pectin was higher three times than the rate of release of reducing sugars indicating that the enzyme had an endo-action. the storage stability of the enzyme in liquid and powder forms was studied, where the activity of the powder form was stable up to one year. these properties of t. harzianum pgii with appreciable activity would be potentially novel source of enzyme for food processing. tarek m. mohamed, biochemistry division, chemistry department, tanta university, tanta, egypt preparation of peroxidase from horseradish, which could be used for commercial applications such as diagnostic kits, was occurred through a simple reproducible method consisting of extraction, ammonium sulphate precipitation, filtration through non-binding protein filter and lyophilization. the purification method was developed allow the preparation of 33 mg of enzyme from 1 kg of horseradish roots. the one mg of enzyme contains 900 units of peroxidase. this value is similar to that produced by sigma (50-1000 unit mg −1 powder). the final preparation is salt free reddish brown powder with free ammonia content less than 0.01 g −1 units. the rz value (a400/a280) of the enzyme, which is a good criterion of purity and heme content, was 2.6. the lyophilized enzyme was stable at −20 • c for at least one year. the liquid form of the enzyme in presence of 0.1% sodium azide was stable up to 25 days at 4 • c, while it lost most of activity at room temperature in the same period. the properties of horseradish peroxidase including km, optimal ph and temperature, activation energy, thermal stability and effect of different compounds were studied. the applicability of this enzyme in determination of serum glucose was performed. the analysis of glucose in human sera gave results using the kit containing the prepared peroxidase similar to those obtained with a commercial glucose kit. lactobionic acid production using lactose oxidase: from laboratory to 600 l scale mikkel nordkvist 1 , per munk nielsen 2 , peter budtz 3 , john villadsen 1 : 1 center for microbial biotechnology, technical university of denmark, dk-2800 lyngby, denmark; 2 novozymes a/s, dk-2880 bagsvaerd, denmark; 3 chr. hansen a/s, dk-2970 hørsholm, denmark. e-mail: mnq@biocentrum.dtu.dk (m. nordkvist) currently, lactobionic acid is mainly a high-price specialty product used, e.g. in solutions for organ stabilization. however, lactobionic acid can also be used as a biodegradable cobuilder in detergents, and it has several applications in food technology. with lower production costs it has the potential to become a bulk chemical. the kinetics for the oxidation of lactose to lactobionic acid by a new carbohydrate oxidase was studied in a 1 l bio-reactor with control of ph, temperature, and dissolved oxygen. the byproduct hydrogen peroxide has a negative influence on the lactose oxidase enzyme, and hence additional experiments were made with addition of catalase to remove hydrogen peroxide, thereby also providing extra oxygen. on the basis of the experiments in 1 l scale, experiments were performed in a 600 l reactor equipped with a new system for dispersion of air to supply the necessary oxygen for the oxidation. the aeration system in the large scale reactor was able to supply oxygen sufficiently fast to give the same production rate, at low values of the air flow rate and the energy input, as was obtained in the high-performance laboratory reactor. the non-characterized gene previously proposed as d-tagatose 3-epimerase from agrobacterium tumefaciens was cloned and expressed in escherichia coli. the expressed enzyme was purified by affinity chromatography on histrap hp, desalting chromatography on hiprep 16/60, and gel filtration chromatography on sephacryl s-300 hr with a final specific activity of 8.89 u/mg. using maldi-tof-ms, the native protein was estimated to have a molecular mass of 32,600 da and a monomeric structure. the purified enzyme exhibited maximal activity at 50 • c and ph 7.5 without the addition of metal ions and at 60 • c and ph 7.0 with 1.0 mm mn 2+ . among various metal ions, mn 2+ was the most effective divalent cation for d-fructose epimerization activity. the addition of mn 2+ significantly increased the thermal stability and the epimerization activity with other ketoses such as d-psicose, d-tagatose, d-ribulose, d-sorbose, and d-xylulose. the activity, substrate affinity, maximum velocity, and catalytic efficiency (k cat /k m ) of the enzyme for d-psicose were higher than those for d-tagatose, which suggests that the enzyme is not d-tagatose 3-epimerase but d-psicose 3-epimerase. the equilibrium ratio between d-psicose and d-fructose was 37:63 at 60 • c with 1.0 mm mn 2+ . when the enzyme was used at 14 u/ml, dpsicose was produced at 211 g/l from 700 g/l d-fructose containing 1 mm mn 2+ after 120 min, corresponding to a conversion yield of 30.2%. the role of ammonium ions in glucosamine formation during the citric acid fermentation process by aspergillus niger m. papagianni 1 , f. wayman 2 , m. mattey 2 : 1 department of hygiene and technology of food of animal origin, school of veterinary medicine, university of thessaloniki, thessaloniki 54006, greece; 2 department of bioscience, university of strathclyde, glasgow, g1 1xw, uk. e-mail: mp2000@vet.auth.gr (m. papagianni) stoichiometric modeling of the citric acid fermentation process by aspergillus niger, in 12-l stirred tank reactor, indicates that nh 4 + ions combine with a c-containing metabolite inside the cell to form a nitrogen compound which is then excreted by the mycelium. the close correlation between calculated and experimental profiles indicates that this metabolic process is rapid and takes place before the c-structure of the glucose has been greatly altered by glycolysis or the pentose phosphate pathway. hplc analysis identified glucosamine as the product of this relationship. a clear effect of medium concentration of nh 4 + on glucosamine formation was observed when fermentations carried out with optimal and sub-optimal ammonium concentrations. (nh 4 ) 2 so 4 addition when medium nitrogen was depleted, enhanced the formation of new cells from the tips of fragmented hyphae and led to glucosamine accumulation in amounts depending to pulse concentration. the fungus reacts in excess ammonium by converting it to glucosamine, to be utilized later when a regeneration process takes place with fragmentation of vacuolated hyphae and subsequent regrowth, depending always on the culturing conditions. however, depending on carbon and ammonium concentration in medium, glucosamine can be secreted in concentrations as high as 50 g/l. about 1000 microorganisms originated from traditional korean food origin were screened for efficient palatinose production. an isolate designated fmb1 was exceptionally efficient in sucrose-palatinose conversion activity. conversion of sucrose into palatinose by fmb1 was much faster than a reference strain of erwinia rhapontici. fmb1 is a gram negative, facultatively anaerobic, motile, noncapsulate, and straight rod-shaped bacterium producing acid from glucose. based on api and 16s rdna analyses, fmb1 was determined to be enterobacter sp. the maximum conversion of 10% sucrose to palatinose and trehalulose by enterobacter sp. fmb1 was achieved within 6 h. the preliminary dna sequencing result of the gene corresponding to sucrose isomerase of enterobacter sp. fmb1 revealed that it showed 87% similarity to that of klebsiella sp. (??). within the scope of an r&d project developed in collaboration with leather tanning portuguese industrial partners a screening of new proteases to be used in the industrial process was performed. a bacillus subtilis strain isolated from alkaline spent purge liquor was shown to be a promising protease producer. microorganism growth was studied for optimisation of temperature, agitation, ph and medium composition either for biomass or proteases production. optimal growth temperature is different for maximum biomass growth (40 • c) and optimal proteolytic activity (43 • c) yielding biomass specific growth rate of 1.6 and 1.4 h −1 , respectively. the achieved proteolytic activities were 5.7 and 7.2 u/ml of protease, respectively. optimised medium composition (7 g/l beef extract, 4 g/l yeast extract, 5 g/l peptone and 0.4 g/l cacl 2 ) yielded a specific growth rate of 1.5 h −1 and 13.9 ku/l of protease, in shake flask experiments. bioreactor experiments (from 1 to 16 l) with the selected medium were performed at 43 • c in order to test aeration rate (1 and 2 vvm), stirring (300-700 rpm) and ph (uncontrolled, controlled at 7 and 8). best protease activity was 64 u/ml in 2 l bioreactor without ph control at 500 rpm and 2 vvm. the proteolytic extract was characterized and compared to commercial bates. results indicate that these proteases can be employed in the purge phase of the leather tanning process in industry. the gram positive bacterium bacillus megaterium is known for its capacity to produce exoenzymes including amylases, proteinases, and penicillin amidase at industrial scale. here, we describe the development of various vectors for the production and export of recombinant heterologous proteins employing b. megaterium signal peptides. the target gene can be cloned directly adjacent to the signal peptide coding sequence (bart et al., 2005) . this arrangement allows for a correct n-terminal sequence of the mature protein after processing by the signal peptidase sipm. using this newly developed protein production and export system lactobacillus reuteri 121 levansucrase (van hijum et al., 2001) was secreted in significant amounts (∼4 mg/l) into the growth medium. fusion of the recombinant levansucrase to affinity tags allowed one-step purification of the recombinant protein from the growth medium. however, fused peptide tags resulted in a decreased secretion of the fusion protein. 1.4 mg his 6 -tagged levan-sucrase were purified per litre of culture. the system was further enhanced via coexpression of a gene for the signal peptidase sipm (malten et al., 2005a) and deletion of the gene for the extracellular protease nprm. developed new tools allow for various strategies of integrated high level production, export and purification of heterologous proteins in b. megaterium. methods for high-throughput screening of secreted enzymes are under development. the determined sequence of the b. megaterium genome, studies using high-cell density cultivations (malten et al., 2005b) and proteome data from batch fermentations implicate new targets for directed genetic optimization of b. megaterium production and secretion strains. novel strong and inducible promoters are currently under investigation. toru matsui 1 , naoya shinzato 1 , hisashi saeki 2 , hitoshi matsuda 2 : 1 center of molecular biosciences, university of the ryukyus, okinawa 903-0213, japan; 2 japan energy co., japan. e-mail: tmatsui@comb.u-ryukyu.ac.jp (t. matsui) optically active epoxides are considered as the potential intermediate for chiral drugs synthesis. although s-styrene oxide (so) have been extensively investigated using styrene monooxygenase from pseudomonas sp., microbial production of r-so with high enantiomeric excess was hardly examined. in this study, r-so producing bacteria from styrene was screened using various alkene assimilating bacteria. r-so with the highest ee (ca. 100%ee) was obtained using ethene utilizing bacteria, identified as mycobacteirum sp., while produced relatively lower at around 70%ee when using propene utilizing bacteria. the alkene monooxygenase gene homologue sequence amplified from the genomic dna revealed a significant similarity to that of etnabc. these bacteria also showed stereoselective degradation of racemic so, suggesting that the produced so might be further stereoselectively degraded to increase the ee. the ethene utilizing bacteira produced not only r-so but also s-epichrolhydrin at high ee.when using arylchloride as the substrate. this research was supported by nagase science and technology foundation. the secretion efficiency of the escherichia coli sec pathway is dependent on the growth phase but not on protein size f.j.m. mergulhão, g.a. monteiro centro de engenharia biológica e química, instituto superior técnico, av. rovisco pais, 1049-001 lisbon, portugal. e-mail: filipem@alfa.ist.utl.pt (f. mergulhão) the secretion efficiency of the escherichia coli sec pathway was evaluated through the expression of green fluorescent protein and human proinsulin fusion proteins. translocation to the periplasm is dependent on the growth phase of the bacterial culture and the highest secretion efficiency is attained in mid-exponential phase. secretion performance is independent of protein size (17-42 kda) and even when the amino acid composition of the secreted proteins is very similar, the amino acid distribution within the protein can affect translocation. in silico prediction analysis suggests that proteins that are prone to form ␣-helix structures are more efficiently translocated. culture medium composition plays an important role on secretion performance with the highest secretion results being obtained in minimal medium. streptokinase is a common fibrinolytic drug. that is used in thrombolytic therapy for long time. to compare with another thermbolytic drugs like tpa, it has lot of advantages. in this present research dna was extracted from s. equisimilis h46a for the first time in iran. streptokinase gene was amplified by using two forward primers and one reverse primer. a common restriction enzyme, bamh-i, was used for cloning. both ends of the pcr products (full length: 1323 bp and mature section: 1245 bp) and the restriction site on mcs of pqe-30 vector were digested. in this study, pqe-30 expression vector was used with high level expression ability for production of recombinant fusion streptokinase with simplifying the purification by employing affinity-metal chromatography method. in addition, the cloning results were controlled by double digestion and sequencing. takesono oxidation of short-chain iso-alkanes was studied with propanegrown resting mycelia of scedsporium sp. a-4. isobutane was oxidized to tert-butanol, but not to isobutanol. isobutanol was used for growth, but both isobutene and tert-butanol were not used for growth. isopentane was oxidized to 3-methyl-1-butanol, 2-methyl-2-butanol, and 3-methyl-2-butanol but not to 2-methyl-1-butanol. 2-methylpentane was oxidized to 4-methyl-1-pentanol, 2-methyl-2pentanol, and 4-methyl-2-pentanol but not to 2-methyl-1-pentanol or 2-methyl-3-pentanol. 3-methylpentane was not oxidized. oxidation of branched alcohols was also studied. application of nadph-dependent 2.5-diketo-gluconic acid reductase for production of l-ascorbic acid claudia pacher 1,2 : 1 division of food biotechnology, department of food sciences and technology, boku, university of natural resources and applied life sciences, muthgasse 18, a-1190 vienna, austria; 2 research centre applied biocatalysis, petersgasse 14, a-8010 graz, austria. e-mail: claudia.pacher@boku.ac.at ascorbic acid is an organic acid with various applications in the food and pharmaceutical industries. at present, the majority of commercially manufactured vitamin c is synthesized via the reichstein process, which is highly energy-consuming, involves considerable quantities of organic solvents and gives an overall yield of about 50%. therefore, during the past decades a lot of research was done to develop biotechnological alternatives for the synthesis of reichstein intermediates by enzymatic or fermentative means, which show some advantages regarding costs and environmental friendliness. one of the fermentation routes runs via 2.5-diketo-d-gluconic acid (2.5-kdg), produced by pectobacter (erwinia) cypripedii. this compound has been reduced by a nadph-dependent 2.5-diketogluconic acid reductase (dkr) from corynebacterium glutamicum to the key intermediate 2-keto-l-gulonic acid (2-klg) before chemical rearrangement leads to the final product. for economical reasons we wanted to express dkr heterologously. based on our long term experience with coenzyme regeneration we wanted also to perform the reaction in homogeneous solution. the spent coenzyme of nadph-dependent dkr has been regenerated by a second isolated nadp-dependent enzyme like glucose dehydrogenase. we describe here the recombinant production, purification and characterization of dkr and the results of enzymatic 2-klg formation by using the recombinant enzyme in a homogeneous system with conjugated coenzyme regeneration. grp78 residing in endoplasmic reticulum (er) functions as a molecular chaperon by associating transiently with incipient proteins as they traverse the er and aiding in their folding and transport. furthermore, the protein can also be induced under various stress condition such as glucose starvation, inhibition of protein glycosylation by tunicamycin, blockage of vesicular trafficking by brefeldin a and er-calcium-atpase pump inhibition by thapsigargin. thus, substances that directly down and up-regulate grp78 transcription are expected to be useful for treatment of cancer and alzheimer's disease, respectively. in the course of our screening program to obtain substances, which regulate grp78 expression, we first constructed an assay system monitored by the expression of a reporter gene. hela cells, which are transformed with luciferase gene under the control of grp78 promoter designated as hela 78c6 cells, respond sensitively to luciferase grp78 induction by er stress such as treatment of tunicamycin. by using this screening system, we isolated pyrisulfoxin as an up-regulator of grp78, and valinomycin, citreoviridin and alternariol as down-regulators. detailed studies on other biological activities were now under way. pdh is an enzyme that was described only several years ago in a number of ecologically related litter-decomposing fungi (agaricales, gasteromycetales). it catalyzes the c-3 and/or c-2 oxidation of several aldopyranoses to the respective keto sugar derivates. pdh shows a very broad substrate range, oxidizing almost all major sugar components of wood polysaccharides, and is implicated to play a role in lignocellulose degradation. agaricus bisporus, the white button mushroom, is an economically significant agricultural crop. the cultivation, which is done by solid-substrate fermentation on straw-and hay-based composted substrate, is sometimes seen as one of only few economically feasible methods for bioconversion of lignocellulosic agricultural waste material. deeper insight in the physiological role of pdh may provide help for mushroom growers to increase yield, improve quality or make new sources of raw materials utilizable. we amplified a fragment of the pdh gene with degenerated primers derived from internal peptide sequences. the screening of a genomic library led to the isolation of the pdh gene. subsequently we amplified a cdna clone by rt-pcr and investigated the transcriptional regulation by different carbon sources on a defined minimal medium. optimization of monoclonal antibody production processes with simulation and scheduling tools demetri petrides, charles siletti intelligen inc., scotch plains, nj 07076, usa. e-mail: dpetrides@intelligen.com (d. petrides) this presentation will review the state of the art in batch process simulation and scheduling tools and their applications in the design and debottlenecking of integrated biopharmaceutical processes. a systematic methodology will be presented for identifying and eliminating size, time, and throughput bottlenecks that limit production in single and multi-product facilities. the methodology will be illustrated with an industrial case study dealing with the optimization of a multi-product facility that produces therapeutic monoclonal antibodies (mabs). mab processes are characterized by a long bio-reaction time (e.g., 1.5-2 weeks for fed-batch operation and 1-2 months for perfusion operation). the cycle time for processing a lot in the recovery and purification train typically takes 3-4 days. consequently, one way of increasing throughput is by installing extra bioreactors that operate in staggered mode and utilize the same recovery train. the result is that multiple batches may be at different stages of completion at any given time. since cleaning equipment (e.g., cip skids) and buffer preparation and holding tanks are shared by multiple steps across many batches, this type of operation leads to time/scheduling bottlenecks that constrain the cycle time and the throughput of a process. the problem becomes more challenging in the context of multi-product facilities and when constraints imposed by the limited availability of resources are considered. our methodology and its computer implementation will illustrate how to systematically identify and eliminate such bottlenecks. the industrial case study will provide a real world example of the methodology. application of two stage continuous cultures of aspergillus niger for citric acid biosynthesis jerzy j. pietkiewicz, malgorzata janczar, wladyslaw lesniak food biotechnology department, university of economics, wroclaw 53-345, poland. e-mail: jerzy.pietkiewicz@ae.wroc.pl (j.j. pietkiewicz) the aim of the work was application test of submerged two stage single stream continuous cultures of aspergillus niger for citric acid production from sucrose. studies were carried out in lab fermenters with working volume of 5 dm 3 . the bioreactors were standard cstrs. in two stage continuous cultures (tscc) high mycelium growth and high citric acid production were observed in the first bioreactor. there was almost four times lower growth of biomass rate and about three times lower citric acid production rate in the second bioreactor. studies on influence of dilution rate in race from 0.0098 to 0.0230 dm 3 /(dm 3 h) on course and efficiency of tscc showed, that the highest citric acid yield (y p/s = 86.3%), high volumetric rate of its production (r pc = 0.958 g/(dm 3 h)) and the highest biosynthesis efficiency coefficient (k ef = 82.7) were obtained with dilution rate d = 0.0148 dm 3 /(dm 3 h). there was also high citric acid concentration (p = 129.5 g/dm 3 ) and low residual sugar concentration (s k = 6.9 g/dm 3 ) in the medium flowing out the second bioreactor in those cultures. the beta-galactosidases in commercial use are of different origins and yeast and fungal lactases present the greatest interest. the yeast lactases present neutral optima ph and are suitable for the hydrolysis of lactose in milk. in this work, the aim was to study the influence of aeration in the production of beta-galactosidase in batch fermentations with kluyveromyces marxianus atcc 46537. the medium composition for culture was as follows (in g/l): lactose pa 50, yeast extract 5, (nh 4 ) 2 so 4 4 and kh 2 po 4 2. the fermentations was carried out at 30 • c, ph 5.5, at 200 rpm starting with an initial cellular concentration of 1 × 10 7 cels/ml, with different aeration rates. the cells were disruped with chloroform 2% (v/v) as solvent. the enzymatic activity was determined as initial rate of lactose hydrolysis at defined conditions. the studies have revealed the importance of aeration on kluyveromyces marxianus in the growth and beta-galactosidase synthesis. the enzymatic activity of fermented medium with 0.5 vvm was 50% higher than one without aeration. furthermore, the cellular growth was faster in the aerobic fermentation than in the anaerobic one. the aeration has taken an important place in the enzymatic synthesis and in the cellular growth, however the results have shown that the aeration rate increase of 0.5-1.5 vvm has not implied a increase in the cellular growth neither in the enzymatic reached activity. the lactose presents in the milk has a solubility of only 20% at 30 • c, and a high percentage of the world population presents intolerance to this sugar, due to the low or absence of the activity of the lactase enzyme in the organism. to minimize such problems, the most viable alternative for nourishing dairy products is the enzymatic hydrolysis of milk, although it is an expensive process due to the high cost of the beta-galactosidase enzyme. an alternative that has been greatly studied is the immobilization of this enzyme, originated from many different sources. there are several immobilization procedures for this enzyme, however, a procedure considered ideal was not obtained yet. the objective of this work was to study the immobilization process of beta-galactosidase from aspegillus oryzae in sodium alginate with commercial gelatin. in the immobilization process was studied the glutaraldehyde influence for 1, 3 and 5%, in the presence of commercial gelatin at the concentration of 2% at the immobilization medium. the activities of the immobilized enzymes were obtained in a stirred micro-reactor, at the temperature 30 • c, ph 4.5 with a 50 gl −1 lactose solution in acetate buffer. the experimental results showed that the immobilized biocatalyst that presented the larger initial activity was the one obtained at the immobiliza-tion medium that contained 5% of glutaraldehyde. after 20 daily determinations of enzymatic activities, a fall of 30, 40 and 24% was verified in the enzymatic activities for the immobilized biocatalysts using glutaraldehyde at 1, 3 and 5%, respectively, however, in all cases, the enzymatic activity reached the half of their initial activity after 10 determinations. hydrolysis of sucrose by immobilized beta-fructofuranosidase in silica eloízio júlio ribeiro, ubirajara coutinho filho faculdade de engenharia química, universidade federal de uberlândia, uberlândia 38400-902, brazil. e-mail: ejribeiro@ufu.br (e.j. invertase, known as beta-fructofuranosidase (ec 3.2.1.26), plays a catalytic role in the conversion of sucrose into glucose and fructose. it is largely used in the food industry to prevent the crystallization of sucrose in sugar mixtures and can be used in enzyme reactors for hydrolysis of sucrose. the objective of this work was to study the kinetic of sucrose hydrolysis by immobilized betafructofuranosidase in a continuous recirculation reactor, evaluated the enzyme stability and determine the effective half-life of immobilized enzyme. invertase was covalently immobilized on sillanized controlled pore silica. nonlinear fitting were used to determine the kinetic parameters for substrate and product inhibition observed in the enzymatic hydrolysis of sucrose. the kinetics studies of immobilized invertase were carried out in a continuous recirculating reactor. the half-time of enzyme inactivation (t 1/2 ) was calculated from the initial rates of the remaining enzyme activity. the model of inhibition by substrate and product adequately represented the enzymatic hydrolysis. the fructose effect was competitive inhibition (k f = 3.1022.10 −4 mol/ml) and the glucose effect was noncompetitive inhibition (k g = 2.2521.10 −4 mol/ml). the effective diffusivity of sucrose into the support was shown to be the same as for sucrose in dilute solution (0.75 × 10 −5 cm 2 /s at 40 • c). the half-time of enzyme inactivation (t 1/2 ) was 1656 h. controlled pore silica showed to be an excellent immobilizing support. the immobilized invertase was very stable at temperatures lower than 50 • c. the intrinsic parameters (k i , k f , k g and v m ) were shown to be similar to the apparent values. the low permeability of mycobacterial cell wall envelopes is a result of the unique composition and organization of the cell wall lipids. the permeability of mycobacterial cell wall can be changed by means of partial disintegration of its compounds. the aim of present work was to characterize the changes in the cell wall skeleton (cws) and non covalently bound free lipids under the influence of isoniazid, the inhibitor of mycolic acids biosynthesis. fatty acid (fames) and mycolic acid methyl esters (mames) obtained from all tested preparations were analyzed by gc/ms analysis. the analysis of free lipids and cws revealed distinct changes in the composition of the frac-tions obtained from the cells exposed to action of the isoniazid. the changes in the quantity of fatty acids in the inh-treated cells indicates that inh interferes with the synthesis of lipidic compounds of the mycobacterial cell wall also. the decreased amount of covalently bound mycolic acids in the cws is responsible for the enhanced penetration of hydrophobic compounds through the cell wall. this work was supported by grant nr 3p04c 06923 of the committee for scientific research. barbara sencio, teresa jamroz, stanislaw ledakowicz department of bioprocess engineering, technical university, lodz, poland the enzyme laccase (ec.1.10.3.2. p-diphenol oxidase) is a subject of research in many centres dealing with improvement of bioprocesses with the use of different white rot fungi species. most strains that produce this enzyme in vitro require inductors initiating its biosynthesis. when cerrena unicolor was applied, it was found that the strain produced laccase very efficiently without additional toxic compounds. to specify optimum conditions for laccase production in a submerged culture, research was undertaken to obtain the most efficient inoculum c. unicolor. the goal of this research was to determine the effect of form and incubation time of inoculum on enzymatic activity of the laccase producing strain. the experimental inoculum was the mycelium prepared on a solid and liquid substrate. basing on results obtained, it was found that the laccase yield was the highest in the cultures where the mycelium was grown on a solid substrate. maximum activity of the c. unicolor strain was achieved on the 16th day of culture, and the amount of laccase produced was higher by, ca. 30% as compared to the mycelium obtained from the liquid substrate. results of these experiments were used to continue studies on the impact of inoculum age. experiments were carried out using an inoculum incubated for 1-3 weeks at the temperature 30 • c in a certomat bs1 shaker at 110 rpm. the best results in the c. unicolor strain culture were achieved using a 7-day-old inoculum. effect of alcohol treatment on hydrolytic activity of candida rugosa lipase serpil takaç, a. ezgiünlü department of chemical engineering, institute of biotechnology, ankara university, 06100 tandogan, ankara, turkey candida rugosa lipase (crl) was treated with 20, 40, and 60% concentrations of methanol (m), ethanol (e), 2-propanol (2p) and 1-butanol (1b) to investigate the changes in its hydrolytic activity toward p-nitrophenylacetate. the treatment included the following steps at +4 • c: (i) stirring crl with phosphate buffer for 24 h; (ii) treating the solutions with alcohols; (iii) stirring treated-crl for 24 h; (iv) centrifugation at 10,000 rpm for 30 min; (iv) dialysis the supernatant against bidistilled water for 39 h. the activity of crls was followed for 120 h at 37 • c in the presence and absence of isooctane. the enzyme activity was measured spectrophotometrically and the protein concentration was measured by lowry's method. it was found that the recovered protein did not change considerably with the type of alcohol; however, decreased with alcohol concentration. in the presence of isooctane, specific activities of the untreated and treated-crls increased compared with those obtained in the absence of isooctane. 1b-crls and e-crls showed higher activities than m-crls and 2p-crls whereas untreated-crl exhibited higher activity than m-crls, e-crls and 2p-crls. the highest and the lowest activities were obtained with 20% 1b-crl and 60% 2p-crl, respectively. the changes occur in the structure of crl after treatments were investigated by electrophoretic analysis. this study was supported by ankara university biotechnology institute (project no: 89) . different genera, species and strains of microorganisms were found to posses different cryoresistance. optimal ways for cryopreservation of microorganisms-producers of antibiotics, microorganisms, used in food industry, agriculture and veterinary have been developed. it was demonstrated, that non-lethal damages could occur in cryopreserved microorganisms after their returning to physiological culture conditions, which were manifested in streptomyces' hypha fragmentation, that of cyanobacteria's, streptococci's chains as a result there was an increase in a number of colony-forming units, a reversible inhibition of microorganisms' proliferative activity in bacillus thuringiensis and lactic streptococci, stimulation of the enzyme processes and antibiotic production. non-lethal damages are repaired during microorganism culturing in the first passage. the cause of non-lethal damages is a reversible inhibition of biosyntheses of protein and nucleic acids respiratory activity. the repairing of non-lethal damages is accompanied by the production of stressproteins, different from heat shock proteins. effect of ph in the 2-propanol treatment of candida rugosa lipase on its enantioselectivity in the hydrolysis of racemic naproxen methyl ester serpil takaç, a. ezgiünlü department of chemical engineering, ankara university, 06100 tandogan, ankara, turkey candida rugosa lipase (crl) was treated with 2-propanol (2p) at the ph values of 1.5, 4, 6, 7.5, 9 and 12 to investigate the changes in its enantioselectivity in the hydrolysis of racemic naproxen methyl ester. the treatment included the following steps at +4 • c: (i) stirring crl with different buffer solutions to maintain the desired ph values for 24 h; (ii) treating the solutions with 40% 2p; (iii) stirring treated-crls for 24 h; (iv) centrifugation at 10,000 rpm for 30 min; (iv) dialysis the supernatant against bidistilled water for 39 h. hydrolyses of racemic naproxen methyl ester to form s-naproxen were performed in shaking flasks at 200 rpm and 37 • c for 192 h in isooctane-phosphate buffer solution biphasic system using treated-crls with the activity of 7.75 u. the concentrations of the enantiomers of naproxen methyl ester and naproxen were determined with hplc. it was found that the treatment ph has an important role on the enantioselectivity and conversion. the highest enantiomeric excess for the substrate, for the product, enantiomeric ratio, and con-version were obtained with crl treated at ph 1.5 as 39, 98, 181 and 29%, respectively. these values were followed with 2p treated crl at ph 12 as 35, 98, 121 and 27%. however, lower enantiomeric excesses, conversions and enantiomeric ratios were obtained at the treatment ph values between 1.5 and 12. the effects of fatty acids, nitrogen (as nh 4 no 3 ), phosphorus (as kh 2 po 4 ), ph value, manganese (mn 2+ ), iron (fe 2+ ) and methanol concentration on growth and production of oxalic acid from post refining fatty acids by a mutant of aspergillus niger xp in submerged fermentation experiments was studied. of the a. niger strains screened, a. niger xp was identified as the best oxalate producer on lipids. the influence of the ph on oxalic acid formation shows that the maximum production rate and higher concentration of product are observed at the ph ranging from 4 to 5. with a medium containing 50 g fatty acids/l, the production reached a maximum of 68 g oxalic acid/l after 7 days. the addition of 1.5% (w/v) methanol to seed culture increased the product yield and concentration of oxalic acid but decreased the amount of an undesired by-product (citric acid). under this condition, the maximum oxalate productivity (14-18 g/l days) was maintained for 2-4 days of fermentation. other results of the experiments show that supplementation of the production medium with manganese and iron enhances oxalate production. fatty acids proved to be a very good substrate for oxalic acid production by a. niger xp giving excellent yields and productivity at low ph. the results are very promising as they may lead to cheap alternative processes for oxalic acid production from renewable lipid resources. department of food engineering, middle east technical university, ankara 06531, turkey. e-mail: banuy@metu.edu.tr (b. yalcindag) laccase (e.c. 1.10.3.2, p-benzenediol:oxygen oxidoreductase), which is an enzyme belonging to the multi-copper oxidase family, catalyzes the oxidation of a broad variety of polyphenols with a preference for p-isomers, which are converted to p-quinones. fungi generally contain several laccases which have been found to be involved in delignification, melanin synthesis and pathogenesis. laccase has also important potential application areas especially in food and chemical industries. after aspergillus fumigatus genome data were released, research on functional analysis of laccases has been initiated in our laboratory. laccase genes of aspergillus nidulans, ya and tila, and laccase and multi-copper oxidase genes of aspergillus fumigatus, abr2 and abr1, were used to analyze a. fumigatus genome for laccases. this sequence analysis resulted in 4 probable laccase genes, one of which was the previously cloned abr2 gene. in this study, one of these genes (aflac1) was further characterized. after sequence alignment and characterization studies, aflac1 was predicted to have 2128 bp having six introns, which makes the protein 606 amino acid long, and the predicted protein sequence showed 63% homology with the dihydrogeodin oxidase of aspergillus terreus and 38% homology to the laccase 2 of botryotinia fuckeliana. aflac1 gene is found within an uncharacterized gene cluster containing genes with homology to glutathione-s-transferase, polyketide synthase, o-methyl transferase, and others. the information obtained from sequence analysis was employed in designing pcr-primers to amplify the aflac1 gene, followed by cloning onto pan52-1 and pan52-4 vectors for heterelogous expression in aspergillus sojae. in addition, by the use of rt-pcr, aflac1 cdna will be cloned and expressed in escherichia coli. furthermore, gene silencing studies will be performed to enlighten the function of aflac1 and associated gene cluster. stability of growth rate of photosynthetic cells is an important factor in designing of effective photobioreactors especially in long term operations. in our experiments, in order to keep operational parameters almost constant, a semi-continuous culture method was developed. in this method, a part of culture broth containing grown cells was repeatedly replaced by fresh medium at a predetermined time interval. the replacement of broth with fresh media could keep the cell concentration, volume of broth and distribution of light intensity constant at initial values throughout the cultivation. it was shown that in one side illumination with a halogen lamp, if the ratio of the light intensity at the front side of a flat plate photobioreactor to that at the rear side was kept lower than 4, the growth rates was sustained in constant levels. however, at higher ratios the growth was followed by rapid decrease after 5-6 h. supplemental illumination with a fluorescent lamp from the rear side of the flat plate photobioreactor could sustain almost stable growth rate. beside of the illumination conditions, increased ferrous ion concentrations in medium could keep the stability of growth rate even in unstable illumination conditions, while consumed ferrous ion was slight. glutathione (gsh) plays a pivotal role in protecting cells from by-products generated by oxidative metabolism. these characteristics make this active tripeptide an important drug for the treatment of liver diseases and is of interest in the food additive industry, therapeutics and sport nutrition. in the first part of the research, a screening was carried out among 48 yeast strains, to find out those able to accumulate higher gsh intracellular levels. two saccharomyces cerevisiae strains proved to be the best gsh producers (1.3%dw), in every samples the presence of s-adenosyl-methionine in traces (0.2% dw) was also evidenced. s-adenosyl-methionine (sam) plays a role in the immune system, maintains cell membranes, participates in detoxification reactions and in the manufacture of brain chemicals and cartilage. the second part of the research was aimed at increasing, in a post fermentative procedure, gsh levels present inside the cells at the end of the growth phase. moreover, time course of sam intracellular levels, to be related with accumulated gsh, was also monitored. cells were then suspended in an appropriate solution containing mineral salts, glucose and the aminoacids, precursors of the two studied molecules. according to this procedure, gsh intracellular levels reached 4.6% dw after 48 h incubation. moreover, gsh levels can be related to sam production (up to 1.3% dw). the presence, in several samples, of intermediate metabolites, such as cystathionine and omocysteine, proved the establishement of an intracellular equilibrium between gsh and sam; this behaviour represents a promising starting point for the set-up of a microbial process for the simultaneous production of the two studied molecules. three acetate mutants of y. lipolytica yeasts, which varied in colony morphology (rough and smooth), were employed for continuous citric acid production from glucose and fructose syrup in a membrane reactor with cell recycle. the strains were compared for their product yields, specific acid production rates and ratios of citric acid to isocitric acid. experiments shoved that glucose syrup was a better substrate for citric acid production by y. lipolytica. citric acid concentration in the effluent ranged from 80 to 120 g/l, depending on the yeast strain used. all y. lipolytica strains produced very low amounts of isocitric acid. its concentration did not exceed 3 g/l. based on the results of these experiments, smooth strain awg-7 was found to be the most suitable for citrate production both from glucose and fructose syrup during long time continuous processes (500 h). in the steady state, the highest citrate productivity (1.3 g/lh) was obtained with this strain, when the feed medium contained 200 g/l of glucose and dilution rate (d) was d = 0.013 1/h. supplementation of the feed medium with bacto-pepton improved the productivity, citric acid yield and stability of the continuous process in the cell recycle fermentation system. for textile dyeing with natural dyes, indigo has an almost unique position as the most blue natural dye. due to the importance of indigo, considerable research has been conducted to replace the chemical synthesis of the dye by an application of biotechnological methods. therefore, we investigated several characteristics of natural indigo derived from polygonum tinctorium and its dyeing properties using silk fabrics, such as washing, perspiration, and light fastnesses. this work was financially supported by program for cultivating graduate students in regional strategic industry from korea industrial technology foundation. glycine oxidase is the product of the yjbr gene of bacillus subtilis that was predicted by sequence homology to be a flavoprotein similar to sarcosine oxidase. glycine oxidase catalyzes the oxidative deamination of various primary and secondary amino acids (e.g. sarcosine, n-ethylglycine, and glycine) and d-amino acids to form the corresponding ␣-keto acids and hydrogen peroxide. previous investigations reported on the cloning and production of the glycine oxidase gene in escherichia coli was up to 1 u/g cell. the present works has improved the expression of the recombinant his-tagged glycine oxidase by 15-fold by using pet28a and rosetta cells under the optimal iptg, temperature and time of induction. the protein obtained represented 30% of total soluble proteins in crude extract. the enzyme was purified to near homogeneity using imac with a 95% recovery and with and specific activity of 1.21 u/mg protein. the enzyme was active towards glycine, sarcosine and different d-amino acids, having in general, a basic ph optimum. the kinetic parameters were also studied, showing a km range from 0.3 to 300 mm. the enzyme was immobilized, and used to obtain pyruvic acid (␣-keto acid) from d-alanine with a good yield. the enzymatic synthesis of lipophilic derivatives of various natural antioxidants including flavonoid glycosides, as well as derivatives of cinnamic acid, was performed using various immobilized lipases in ionic liquids such as 1-butyl-3-methylimidazolium tetrafluoroborate (bmim-bf 4 ) and 1-butyl-3-methylimidazolium hexafluorophosphate (bmim-pf 6 ). the influence of various reaction parameters on the catalytic behavior and the selectivity of lipases was pointed out. a response surface methodology was applied for the optimization of the yield and the productivity of the biocatalytic process. the antioxidant activity of the biocatalytically prepared lipophilic derivatives of natural antioxidants, as expressed on cu 2+ -induced oxidation of low-density lipoprotein (ldl) and total serum, was investigated. process strategy for reduction of proteolysis in pichia pastoris fermentations jan weegar 1 , john dahlbacka 1 , noora sirén 2 , niklas von weymarn 2 , kaj fagervik 1 : 1 faculty of chemical engineering,åbo akademi university, finland; 2 laboratory of bioprocess engineering, helsinki university of technology, finland. e-mail: jan.weegar@abo.fi (j. weegar) the yeast pichia pastoris is a popular host organism for production of recombinant proteins. it is, however, common that the products are degraded by proteases towards the end of the fermentation, resulting in productivity and purity decreases. proteolysis of recombinant proteins in p. pastoris fermentations is affected by the temperature and ph of the growth medium. in this study, it was shown that decreasing the temperature from 30 to 10 • c during the induction phase effectively prohibited proteolysis. on the other hand, the temperature decrease resulted in a reduced maximal methanol consumption rate, which subsequently resulted in a culture highly sensitive to residual methanol. the temperature was slowly decreased according to a predetermined trajectory. as the temperature reached values below 12 • c, the methanol concentration had to be closely monitored and the substrate feed rate adjusted in order to prohibit methanol poisoning as well as to maintain the culture as a substrate limited fed-batch. measurement of protease concentrations revealed that proteases were present at 10 • c, but at this temperature the proteolysis rate was evidently effectively reduced. the recombinant protein produced could almost totally be recovered (i.e. high purity) with this process strategy compared to a constant high temperature culture (30 • c) where severe breakdown of the product was observed. with the growing of an ecological conscience in the public opinion, more and more industrial processes are analyzed for a possible ecologically beneficial alternative. for the production of ascorbic acid, which is nowadays done by the reichstein process, a lot of research was done to develop biotechnological alternatives for the synthesis of reichstein intermediates by enzymatic means, which show some advantages regarding costs and environmentalfriendliness. besides of two-stage fermentation, our approach is to design a tailor-made organism that produces 2-keto-l-gulonic acid, which is the direct precursor of ascorbic acid from glucose or gluconic acid and which can easily be converted to the final product by conventional methods. erwinia (pectobacter) cypripedii, which is a natural producer of 2,5-diketo-d-gluconic acid, was selected as a suitable host for a 2,5-diketo-d-gluconate reductase from corynebacterium glutamicum. to increase the yield of the desired compound we investigate two 2-keto-reductases in the host organism that diminish the yield of 2-keto-l-gulonate by reducing the compound to l-idonic acid, or by metabolisation of intermediates. these two enzymes were investigated with vari-ous biochemical and molecular biological methods, which will be presented. overproduction of bioinsecticides by heat and salt stress and control of dissolved oxygen in cheap media of bacillus thuringiensis nabil zouari, dhouha ghribi, samir jaoua laboratoire des biopesticides, centre of biotechnology of sfax, tunisia, bp:k, 3038 sfax, tunisia bioinsecticides based on preparations of spores and insecticidal crystal-proteins (icps) produced by the bacterium bacillus thuringiensis (bt) proved to be a high tool for fighting some agricultural pests and vectors of diseases. however, the use of bt preparations as commercial insecticides would be prohibitively expensive because it is not easy to reach cheap overproduction of icps during large-scale fermentation. here, we report possibilities to improve delta-endotoxins production as a consequence of responses of bt strains to low levels of heat and salt stress. each stressor results differently in the improvement of delta-endotoxins production, but both were shown to be most efficient at the beginning or the midexponential phase of the cultures which become resistant at the stationary or the sporulation steps. heat stress caused increase of 84% of synthesis yields of the sporulating cells, in contrast, salt caused increase of 25% of spores counts, corresponding to 28% toxins production improvement. combined effects of both stressors lead to toxins production improvement of 66%, yield improvement of 40%. we focused on the overcome of carbon repression catabolite, closely related to oxidative metabolism, by an adequate control of dissolved oxygen in the cheap media we formulated for bt insecticides production. we showed that an equilibrium between the high density of vegetative cells and their ability to synthesize toxins during their sporulation was necessary to take into account. 40% increase of icps production was reached into 3 l fermenter combination of mutagenesis, heat and salt stress and oxidative metabolism control allowed more than 100% improvement of delta-endotoxins. these results are of great importance in practical point of view, since high bioinsecticides concentrations could be produced without decrease of the yields of their production. mechanism and function of the intramembrane-cleaving protease rhomboid marius lemberg 1 , javier menendez 2 , christopher koth 2 , matthew freeman 1 : 1 mrc laboratory of molecular biology, cambridge, uk; 2 ontario center for structural proteomics, toronto, canada rhomboids are a family of intramembrane serine proteases that are widely conserved throughout evolution. among diverse functions discovered so far, rhomboids participate in intercellular signalling, parasite invasion, membrane dynamics and bacterial quorum sensing, making them potentially valuable therapeutic targets. the identification of physiological substrates and of selective inhibitors will be key towards their evaluation as drug targets. we have developed an in vitro cleavage assay to monitor rhomboid activity in the detergent solubilised state, enabling the first isolation of a highly pure rhomboid with catalytic activity. analysis of purified mutant proteins suggests that rhomboids use a serine protease catalytic dyad instead of the previously proposed triad, and gives insights into subsidiary functions like ligand binding and water supply. oligosaccharides and 3-keto-glycosides. availability of the enzyme is, however, hampered by the very slow growth and low production rates of the fungus. cloning of the encoding gene and production of the protein by heterologous expression are therefore a prerequisite not only for any biotechnological application, but further scientific investigations as well. on the basis of peptide sequences degenerated primers were designed, and the resulting pcr fragment was used as a probe to isolate the gene from a genomic library. two very similar genes encoding previously uncharacterized proteins were also found, and flanking regions were amplified using rage-pcr. furthermore cdna clones of all genes were isolated by rt-pcr. for textile dyeing with natural dyes, indigo has an almost unique position as the most blue natural dye. due to the importance of indigo, considerable research has been conducted to replace the chemical synthesis of the dye by an application of biotechnological methods. therefore, we investigated several characteristics of natural indigo derived from polygonum tinctorium and its dyeing properties using silk fabrics, such as washing, perspiration, and light fastnesses. this work was financially supported by program for cultivating graduate students in regional strategic industry from korea industrial technology foundation. the behavior of phytate degrading enzymes isolated from malaysian zea mays root in rice bran media anis shobirin meor hussin 1 , abd-elaziem farouk 1 , hamzah mohd salleh 1 , ralf greiner 2 : 1 biomolecular engineering research group, department of biotechnology engineering, kulliyyah of engineering, international islamic university malaysia, jalan gombak, 53100 kuala lumpur, malaysia; 2 centre for molecular biology federal research centre for nutrition and foods, haid-und-neu-straße 9, d-76131 karlsruhe, germany phytate degrading enzymes catalyze the step-wise release of phosphate from phytate, the principle storage form of phosphorus in plant seeds and pollen. they are widespread in nature, occurring in plants and microorganisms, as well as in some animal tissues. phytate-degrading enzymes have been studied intensively in recent years because of the great interest in such enzymes for phytate degradation and their application for animal feed and human health. from over isolate 140 screened isolates of phytate degrading enzymes, three isolates from the root malaysian maize plantation have shown phytate degrading activity. the production of phytate degrading enzyme was studied using different concentrations [%, w/v] of rice bran media during different stages of cultivation. the dephosphorylation of phosphate from rice bran phytate has shown regulatory effect on the secretion of bacterial phytases. in this conference, we will present data for the characterization of the enzymes. in this paper we present the properties of a phytase purified from a bacterium isolated from malaysian wastewater, which might find application as an animal feed supplement. the phytase described herein is a periplasmic enzyme. the phytase was purified about 180fold to apparent homogeneity using ion-exchange chromatography and gel-filtration with a recovery of 10% referred to the phytatedegrading activity in the crude extract. the enzyme exhibits an activity of about 1106 u mg −1 . gel filtration of the native enzyme on a calibrated sephacryl s-200 column gave a molecular mass of the phytase of 42,000 ± 1500 da with elution position being measured by determination of enzyme activity. lower molecular mass species or higher molecular mass aggregates were not observed. the phytase appeared homogeneous by polyacrylamide gel electrophoresis under non-denaturing conditions at ph 8.3 and 4.8 and gave a single protein band upon sds gel electrophoresis after coomassie staining of the gels. these results indicate that the phytase could be regarded as homogeneous. the estimated molecular mass after sds-page indicated that the phytase having a molecular mass of 41,500 ± 2500 da. consequently, this enzyme is a monomeric protein. the purified enzyme had a single ph optimum at ph 4.5 and was virtually inactive above ph 7.0. at ph 3.0, 40% and at ph 2.5, 20% of the activity at optimal ph was observed. the effect on enzyme stability was studied in the ph range 1.0-9.0 at 4 • c. within 14 days the phytase did not lose any activity in the ph range from 3.0 to 8.0, but at ph values below 2.0 a rapid decline in activity was observed. at ph 1.5, 72% and at ph 9.0, 65% of the initial activity was lost during 24 h. in the range of temperatures studied, 10-80 • c, the optimum temperature for the enzyme was found to be 65 • c. the apparent activation energy was estimated at ph 4.5 from the slope of log v max versus 1/t. the data showed excellent linearity from 15 to 65 • c. the arrhenius activation energy for the hydrolysis of phytate was calculated to be 37.5 kj/mol. in order to check thermal stability, the purified enzyme was incubated at different temperatures, cooled to 4 • c and assayed using the standard phytase assay. the enzyme lost no activity in 10 min at temperatures up to 65 • c. when exposed for 10 min at 70 • c, it retained 50% and at 80 • c 12% of the initial activity. in order to determine the substrate selectivity of the purified phytase, several phosphorylated compounds in addition to phytate, were used for k m and v max estimation by detecting the release of the phosphate ion during hydrolysis using formation of a soluble phospho-molybdate complex in an acidic water-acetone mixture. only phytate was identified as a substrate. the kinetic parameters for the hydrolysis of phytate were determined to be k m = 0.15 mmol l −1 and k cat = 1164 s −1 at ph 4.5 and 37 • c. like other bacterial phytatedegrading enzymes, the purified enzyme showed substrate inhibition. the activity of the purified enzyme was inhibited at substrate concentrations >7 mm. the study of the effect of metal ions on enzyme activity showed that none of them had an activating effect when used at a concentration between 10 −4 and 10 −3 m. mg 2+ , ca 2+ , mn 2+ , co 2+ , ag + , hg 2+ , and cu 2+ had little or no effect on enzyme activity, while zn 2+ , fe 2+ , and fe 3+ showed strong inhibitory effects. the reduced phytate-degrading activity in the presence of fe 2+ and fe 3+ is attributed to a lower phytate concentration in the enzyme assay because of the appearance of a fe-phytate precipitate. when compounds which tend to chelate metal ions, such as o-phenanthroline, edta, oxalate, citrate or tartrate, were tested for their effect on enzyme activity, it was noted that none of them was inhibitory at a concentration from 10 −4 to 10 −3 m. fluoride, a known inhibitor of different phytate-degrading enzymes from bacteria and the hydrolysis product phosphate as well as its structural analogs molybdate, wolframate and vanadate were found to be strong inhibitors of the purified enzyme. flouride inhibited the hydrolysis of phytate with a k i value of 112 mol l −1 . several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (amps) in recombinant bacterial expression systems. in the present work, we investigated the use of the baculoviral polyhedrin (polh) protein as a novel fusion partner for production of a model amp (halocidin 18 subunit; hal18) in escherichia coli. the recombinant hal18 amp could then be hydroxylamine cleaved from the fusion protein and easily recovered by simple dialysis and centrifugation. this was facilitated by the fact that polh was soluble in the alkaline cleavage reaction but became insoluble during dialysis at a neutral ph. importantly, recombinant and synthetic hal18 peptides showed nearly identical antimicrobial activities against e. coli and staphylococcus aureus, which were used as representative gram-negative and -positive bacteria, respectively. these results demonstrated that baculoviral polh can provide an efficient and facile platform for production or functional study of target amps. extensive industrial and food additive applications of succinate have attracted much effort towards finding an environment-friendly alternative to the petrochemical production processes. it is very attractive to engineer s. cerevisiae for succinate production because of its generally regarded as safe (gras) status, ease of genetic manipulation and fermentation. we approached this metabolic engineering problem with a two-step methodology combining modern computational as well as molecular biology tools. in the first step we identified potential metabolic engineering targets leading to high succinate yield and productivity, with the aid of genome scale metabolic model and a bi-level optimization framework using flux balance analysis and quadratic programming. in the next step, various deletion mutants are being constructed and characterized for physiology and succinate production. results from these experiments then will be used to improve the predictions in computational models. so far, we have constructed a saccharomyces cerevisiae mutant deleted in sdh3, which encodes a major subunit of sdh-complex converting succinate to fumarate in mitochondria. the physiology of sdh3 mutant has been characterized in aerobic and anaerobic batch cultivations and in glucose limited chemostat at dilution rate as low as 0.027 h −1 . in aerobic batch fermentations, the mutant showed reduced maximum specific growth rate as compared to the wild-type, and it was incapable of growing on ethanol as sole carbon source, as predicted from the model. interestingly, the mutant showed much higher specific growth rate in anaerobic conditions, close to the wildtype strain. moreover, the chemostat cultivations indicate that the critical dilution rate of the mutant is below 0.027 h −1 . this opens further opportunities to investigate interesting behavior of the mutant and the underlying regulatory processes to improve our understanding of yeast mitochondrial metabolism. department of chemical engineering, yıldız technical university, davutpaşa campus, 34210 esenler/istanbul, turkey. e-mail: dkilic@yildiz.edu.tr (d.k. apar) lactose is the dominant carbohydrate in milks which are, in turn, the only significant natural sources of lactose. a large number of people do not digest lactose properly due to a lack or inactivity of the intestinal beta-galactosidase and they suffer from intestinal dysfunctions -gas, abdominal pain and diarrhea -if their diet contains lactose. moreover, lactose is a sugar with a high bod, low sweetness, low solubility, when compared to the products of its hydrolysis (glucose and galactose) and being a hygroscopic sugar has a strong tendency to adsorb flavours and odours. the hydrolysis of this sugar is very attractive towards the improvement of processes for the production of ice cream and other refrigerated dairy products and it could be very interesting for the development of additives for animal and human alimentation. the enzymatic hydrolysis of lactose is carried out by beta-galactosidases, enzymes that are widely distributed in nature, appearing in micro-organisms, plants and animal tissues. the present investigation describes the effects of the sonication process parameters on enzymatic hydrolysis of milk lactose and enzyme stability. bandelin sonopuls sonicator was used for the lactose hydrolysis experiments. ␤-galactosidase enzyme used is produced from kluyveromyces marxianus. the reactions were carried out in 250 ml of milk. the process variables for the sonicator are duty cycle, acoustic power and enzyme concentration. the amount of residual lactose concentration (g/l) and residual enzyme activity (%) against time were investigated versus process variables. beside of this; the mathematical models depending on the operating conditions were also derived by using the experimental data of lactose concentration and enzyme activity. kinözbek department of chemical engineering, yıldız technical university, davutpaşa campus, 34210 esenler/istanbul, turkey. e-mail: dkilic@yildiz.edu.tr (d.k. apar) over the last decade, the use of plant protein hydrolysates alternative to animal protein hydrolysates in human nutrition has broadly expanded. protein hydrolysates are often used in different nutritional formulations, such as supplementation of drinks to enhance their nutritional and functional properties, or special medical diets. there are many processes which employ protein hydrolysis and hydrolytic products. among these processes, the use of enzymes allows selective hydrolysis of protein and produces a potentially safer and more defined material. in the present study, the effect of the temperature, ph and viscosity on the hydrolysis of corn gluten was investigated using a stirred batch reactor system. the corn gluten was hydrolysed by using neutrase enzyme, a bacterial protease produced by a selected strain of bacillus amyloliquefacien. the reactions were carried out in 0.2 l of aqueous solutions containing 1% (w/v) corn gluten and 0.4% (v/v) enzyme. the degree of hydrolysis (%) and soluble protein concentration depending on the time were investigated at the temperatures 40, 45, 50, 55, and 60 • c; and at the ph values 6.5, 7, 7.5, and 8. to investigate the effect of viscosity, the various amounts of glycerol was added to the reaction solutions to produce viscosities in the range of 1.415-13.43 cp. the degree of hydrolysis (dh) was computed by using ph-stat method. for the soluble protein determination in the hydrolysates samples, the folin-lowry method (1951) was used. polyhydroxyalkanoates (pha), one of the most promising bioplastics for the partial replacement of synthetic polymers like polypropylene, are polyesters produced by bacteria as intracellular storage reserves of carbon and energy. the industrial production of pha is achieved by pure cultures in its natural state or using genetically engineered organisms. the main obstacle to the replacement of synthetic plastics by biopolymers is their great cost difference. research on the field of biopolymers synthesis using mixed cultures and waste organic carbon sources as substrates prove to decrease substantially the production costs of pha. the optimization of pha production under aerobic feeding conditions (adf) was achieved recently in our group, obtaining the highest value of pha content stored by mixed cultures (79.2% of cell dry weight). in this work only a homopolymer of polyhydroxybutyrate (phb) was obtained and since it is a highly crystalline and brittle material its application field is limited. the mechanical and thermal properties of pha can be varied to a great extend by adjusting the monomer composition. the incorporation of different monomeric units, other than hb, in the polymer chain, originates copolymers with improved mechanical properties. optimization of pha production from propionate by a mixed culture was studied varying the carbon and ammonia concentrations. propionate only, acetate alone or a mixture of acetate and propionate were tested. copolymers of hydroxybutyrate and hydroxyvalerate, p(hb/hv), with different compositions were obtained. consequently polymer properties could be manipulated by feeding the selected volatile fatty acid composition. the pharmaceutically important plant species of glycyrrhiza sp. (called licorice) is an important commercial product used as a natural sweetener, anti-inflammatory, anti-cancer and anti-diabetic agents. agrobacterium rhizogenes transformation system was used for the hairy root cultures of licorice g. uralensis. after inoculation of aseptic stem segments the ability of hairy root formation was scored for a period of 6 weeks. mean transformation frequency ranged from 27% (for 8196 up to 31% (for 15834). some transformed genotypes showed significant differences in roots weight, flavonoids and glycyrrhizin (gl) production. the cotransformation rate of licorice intact explants cultivars with lba 9402 tl-dna and the 35s gus gene showed an average of more than 35%. these obtained root cultures were additionly elicited with extracts of biotic elicitor acremonium sp. (endomycorhizal fungus), and were used as an in vitro system to metabolites production. the transformed and elicited hairy roots of g.uralensis were obtained by infection of a. rhizogenes 8196 have produced gl at an yield of 4.5% dry weight on the period of culture as a 30 days. according to tentative analyses the hairy roots cultures of glycyrrhiza species produced flavonoids (liquiritigenin and liquiritigen). more high levels (3.42 g/l) of the total flavonoids production have been identificated on the strains which transformed by lba 9402. this study involved any difference among elicitor treatments and incubation periods for the optimal meabolites production. clearly, the selection of an effective agrobacterium strain for the production of transformed root cultures is highly dependent on the plant species, and must be determined empirically. production, purification and characterization of scytalidium thermophilum phenol oxidases didem sutay 1 , ufuk bakir 1 , zumrut b. ogel 2 : 1 chemical engineering department, middle east technical university, inonu bulvari, 06531 ankara, turkey; 2 food engineering department, middle east technical university, inonu bulvari, 06531 ankara, turkey. e-mail: ubakir@metu.edu.tr (u. bakir) phenol oxidases (pos) are a group of enzymes which are responsible for oxidation of various phenolic compounds in the presence of molecular oxygen. there are different types of pos present in nature and three major groups of these enzymes are laccases (e.c. 1.10.3.2, p-benzenediol: oxygen oxidoreductase), catechol oxidases (e.c. 1.10.3.1, o-diphenol oxidoreductase) and tyrosinases (e.c. 1.14.18.1, monophenol monooxygenase). another group of enzymes, peroxidases (e.c. 1.11.1.7), can also be considered as a member of po family. pos have very wide substrate range and final oxidation products of these substrates are quinones, which are highly reactive molecules and polymerize into brown, red or black waterinsoluble compounds. pos are very common in nature, they can be found in almost all plants, animals and microorganisms. pigmentation and protection from the environment are main functions of these enzymes. pos have different applications in food, pharmaceutical, textile industries and waste-water treatment systems. the objective of this study was po production by the thermophilic fungus, scytalidium thermophilum, purification and characterization of the enzyme. for this purpose, enzyme production was performed either in a shaker-incubator or a temperature, ph and dissolved oxygen controlled 2 l bioreactor (probiotem) to optimize enzyme production medium composition and bioreactor parameters. as the carbon and nitrogen sources, 4% glucose and 0.4% yeast extract were determined as the optimal concentrations, respectively. copper, gallic acid and tannic acid were determined to increase enzyme production. purification was performed by using membrane and chromatographic techniques. hydrophobic, ion exchange and gel filtration columns were used by using a fplc system;äkta prime (amersham biosciences). especially the phenyl sepharose tm high performance column appeared to be very efficient for po purification from scytalidium thermophilum. purified po have been characterized by electrophoretic techniques and kinetic studies. isolation of lipolytic microorganisms from subtropical soils. cloning, purification and characterization of a novel esterase from strain pseudomonas sp. cr-611 núria prim, cristian ruiz, cristina bofill, f.i. javier pastor, pilar diaz department microbiology, university of barcelona. av. diagonal 645, microorganisms or their enzymes are used in a wide range of biotechnological activities such as polymer hydrolysis, synthesis of added-value compounds, sample decontamination, etc. thus, there is an increasing interest for isolating new enzymes and new enzymeproducing organisms for their use in industrial conversions (cherry and fidantsef, 2003) . among these enzymes, lipases, esterases, cellulases, xylanases and pectinases play an important role in many biotechnological processes like those related to pulp and paper processing. three samples of subtropical forest soil from puerto iguazú (argentina) were used for the isolation of autoctonous microorganisms growing in an organic matter-rich environment. a total of 724 pure cultures of bacteria and fungi were obtained and their hydrolytic activities on polysaccharide and lipidic substrates were assayed using olive oil, tributyrin, cholesterol esters, xylan, cellulose and pectin as substrates. among the isolates analysed, 449 were active on one or more of the substrates evaluated, and 43 of them degraded all substrates. nearly half of the strains displayed lipolytic activity, whereas the number of strains active on xylan, cellulose, pectin and cholesterol esters, was much lower. the 76 strains bearing the highest hydrolytic activities were selected and stored for further characterization (ruiz et al., 2005) . among them, strain cr-611, one of the most active isolates on tributyrin, was selected for identification and characterization of its lipolytic system. lipolytic strain cr-611 was identified by morphological, physiological and phylogenetic tests, as a pseudomonas sp., closely related to p. fluorescens. sds-page and zymogram analysis (diaz et al., 1999) of cell extracts and supernatants from the strain revealed a complex lipolytic system consisting of at least two lipolytic enzymes. sequence alignment and clustering of previously described pseudomonas lipases and esterases allowed the design of different sets of primers for the isolation of the lipase/esterase coding genes. a gene coding for an esterase with homology to family vi bacterial lipases (arpigny and jaeger, 1999) was isolated and cloned in escherichia coli. the cloned enzyme was further purified and characterized, showing preference for short fatty acid esters and displaying a typical michaelis-menten kinetics, with no interfacial activation. the substrate profile, together with the kinetic behaviour and sequence similarity of the cloned enzyme to family vi bacterial esterases, allowed to identify this enzyme as an esterase and was named esta6. maximum activity was achieved on muf-butyrate at 55 • c and ph 8.5, suggesting that it could be of interest for biotechnological purposes. microbial xylitol production from agricultural wastes has recently attracted much attention from industries because it has potentials to realize the cheaper production of xylitol with low environmental impact (tada et al., 2004) . in order to realize the effective xylitol production by a xylose utilizing yeast, the oxygen supply is a key for maximizing xylitol yield over consumed xylose (y xl ) because the intracellular xylitol metabolism is strongly influenced by the amount of available oxygen. in the present work, we tried to apply a metabolic reaction model in order to determine the optimal oxygen transfer rate (otr) in a fermentor for maximizing xylitol yield. corn cob hydrolysates containing 25 g-xylose/l was employed as medium for xylitol production by computer-controlled batch cultures using candida magnoliae (ferm p-16522, aist). a metabolic reaction model considering main xylitol metabolisms including glycolysis, pentose-phosphate pathway, tca cycle and cell synthesis was developed. the model allows to estimate various intracellular metabolic flux distributions including a xylitol production rate. the oxygen uptake rate to maximize the ratio of xylitol production rate over xylose consumption rate corresponds to the otr condition to maximize a xylitol yield over xylose consumed. based on the metabolic reaction model, the otr was optimized by a linear programming, the optimal otr and the maximum xylitol yield were estimated as 0.5 mmol o 2 /l h and 0.81 g-xylitol/g-xylose, respectively. the experimental verification using the optimal otr demonstrated that the xylitol yield was greatly improved to 0.75 g-xylitol/g-xylose from 0.6 g-xylitol/g-xylose in our previous study. expression of a bacterial sugar phosphate transporter in s. cerevisiae to release l-glycerol 3-phosphate accumulated by metabolic engineering almut popp, huyen thi thanh nguyen, ulf stahl, elke nevoigt department of microbiology and genetics, university of technology, 13353 berlin, germany. e-mail: a.popp@lb.tu-berlin. de (a. popp) in contrast to glycerol, its phosphorylated precursor l-glycerol-3phosphate (l-g3p) is retained by the plasma membrane. therefore, engineered yeast strains accumulate l-g3p in the cytosol resulting in low overall yield of the desired product and laborious downstream processing. a suitable sugar phosphate transporter in the yeast plasma membrane would overcome these limitations. the glycerol-3-phosphate transporter (glpt) of e.coli is an antiporter and naturally mediates the uptake of l-g3p in exchange with inorganic phosphate. we assume that this transporter would also mediate the excretion of accumulated l-g3p into a phosphate-rich medium if it was present in the plasma membrane of engineered yeast. despite many inconsistencies in codon usage, we were able to express the bacterial glpt gene in yeast. expression was monitored by a c-myc tag added to the c-or n-terminal hydrophilic tail, respectively. the quantity of the construct with n-terminal tag clearly exceeds the quantity of the construct with c-terminal tag. both gene products are located in the endoplasmic reticulum, as shown by immunofluorescence microscopy. obviously, yeast's transmembrane protein sorting machinery does not recognise it as a substrate for the secretory pathway. metabolic flux analysis of c-and p-limited shikimic acid producing e. coli gaspard lequeux 1 , louise johansson 2 , jo maertens 1 , peter vanrolleghem 1 , gunnar lidén 2 : 1 biomath, ghent university, coupure links 653, 9000 gent, belgium; 2 department of chemical engineering, lund university, p.o. box 124, 22100 lund, sweden. e-mail: gaspard.lequeux@biomath.ugent.be (g. lequeux) metabolic flux analysis (mfa) was applied to decipher why plimited e. coli fermentations are more optimal for shikimic acid production in comparison with glucose-limited fermentations. as mfa allows obtaining insight in the intracellular flux distribution over different pathways by only measuring net production and consumption rates of metabolites, under the condition that parallell pathways are removed. to this end a detailed metabolic network model was created and checked for consistency, dead-ends, and parallell pathways. several fermentations were performed at different dilution rates (ranging from 0.05 to 0.3 h −1 ) and different limitations (phosphate and glucose). the e. coli strain used was w3110 with genetic modifications in the aromatic amino acid pathway to enhance shikimic acid production. for each dilution rate, a metabolic model was solved. this way, the evolution of each flux can be followed with respect to the dilution rate. the p-limited cultures showed a better yield which can be explained by the diminished excretion of dehydro-shikimic acid (as is known from literature) and a reduction of the hydrolysis of atp. takasumi hattori, kuniki kino, kohtaro kirimura department of applied chemistry, school of science and engineering, waseda university, tokyo, japan. e-mail: takasumi@suou.waseda.jp (t. hattori) alternative oxidase is a terminal oxidase in respiration chain, which is a branched chain of cytochrome pathway, and inhibited by salicylhydroxamic acid (sham), but not by cyanide. the citric acid-producing fungus aspergillus niger wu-2223l has a cyanide-insensitive and sham-sensitive respiration catalyzed by the alternative oxidase (kirimure et al., 1999) and did not produce citric acid when cultivated with sham (kirimure et al., 2000) . in this study, the transcript levels of alternative oxidase gene (aox1) (kirimure et al., 1999) and activities of alternative oxidase under the conditions of citric acid production were examined during 9 days-cultivation. the amount of aox1 mrna was determined by northern blot analysis, and the specific activity of alternative oxidase as that of duroquinol oxidase. the transcript level and the activity were highest at 2 days at log-phase, decreased during 2 and 4 days, and thereafter maintained at low levels. however, the transcript and alternative oxidase activity was constitutively detected during whole the cultivation periods under the conditions of citric acid production. the sequence analysis of aox1 chromosomal dna revealed the presence of potential binding site of cyclic amp responsive element (cre), stress responsive element (stre) and heat shock factor (hsf) in its upstream region. these results indicated that the expression of alternative oxidase was regulated in the transcription level and alternative oxidase contributes as the main respiration chain during citric acid production. kirimura, k., et al., 1999 . curr. genet. 34, 472-477. kirimura, k., et al., 2000 . biosci. biotechnol. biochem. 64, 2034 -2039 . trichoderma strains are considered to be among the most useful fungi in industrial enzyme production, agriculture and bioremediation. metabolic versatility displayed by these fungi makes it a very amenable source of new gene products. functional analysis of candidate genes goes by two complementary ways: gene overexpression and loss-of-fuction mutants generation. a few expression systems are available mainly to direct constitutive gene expression in catabolite repression conditions. following a genomic approach, we have recently cloned some gene promoters with high expression in glucose in trichoderma harzianum cect2413. a main goal in a gene functional analysis is the generation of knock-out mutants. up to date, there is no reference about successful gene disruptions in t. harzianum mainly due to a very low homolog recombination frequency. rna-mediated gene silencing has been shown as an efficient tool to diminish or totally abolish specific gene expression. especially those strategies based on the use of hairpin constructs allow rapid and easy generation of strains with reduced levels of mrna from genes of interest. we have used the t. harzianum cect2413 tss1 promoter to direct the expression of a hairpin dna construct that induced the appearance of small-interfering rnas (sirnas) and the silencing of the uida reporter gene in a previous uida overexpressing strain. reduced levels of mrna and gus activity correlated with the presence of sirnas. this is the first report on rna-mediated gene silencing in trichoderma and constitutes a useful and promising tool for functional genomic studies in fungal systems. analysis of the metabolic response of escherichia coli to quantitative modulations of the glucose-6-phosphate dehydrogenase based on 13 c-labelling experiments cécile nicolas, fabien létisse, stéphane massou, philippe soucaille, jean-charles portais. e-mail: fabien.letisse@insa-toulouse.fr (f. létisse) microorganisms have an efficient capacity for adapting their metabolism in response to genetic or environmental changes, and understanding metabolic robustness has become an emergent issue. part of the robustness originates from the network organization of metabolic systems, where the interplay between all available biochemical reactions provides alternative mechanisms for compensating the perturbations. recently, 13 c-metabolic flux analysis ( 13 c-mfa) has been applied to escherichia coli knock-out mutants lacking key enzymes to determine the phenotypic effects of structural changes in the metabolic network, providing further evidences for compensatory phenomena. the aim of our on-going work is to understand how the central metabolism in e. coli responds to quantitative alterations at a specific key-point of the metabolic network. the glucose-6-phosphate dehydrogenase (g6pdh), a key enzyme in the central metabolism for which the effects of deleting the gene (zwf) has been already described (zhao et al., 2004) , was chosen as the target. to this aim we have generated a set of expression mutants, i.e. mutants having each a fixed level of expression of the zwf gene. four different levels of expression, leading respectively to g6pdh activity 2; 2.9; 5.7 and 14 times higher than in the wt strain, have been obtained. for each mutant, transcriptomics analysis will be carried out and compared to both the zwf-and wt strains to detect changes in the network structure, and the distribution of fluxes will be measured using 13 c-mfa. the flux maps obtained for the various strains will be compared to evaluate the quantitative response of the central metabolic network to imposed and increased g6pdh activity. metabolic control analysis will be applied to provide insights onto the sensitivity of the measurable metabolic fluxes to g6pdh activity. combination of transcriptomics and fluxomics approaches will provide information on the nature and extent of the compensatory mechanisms. because the activity of a single enzyme is tuned at different levels in knock-out and expression mutants, this investigation provides a situation that mimics gene-level regulation of metabolism. , j., et al., 2004, metab. eng., 6, 164 . with the depletion of the world's petroleum supply, there has been an increasing worldwide interest in ethanol as an alternative, nonpetroleum source of energy. this fact caused increased interest in the new ethanol technology fermentation process research. as reported before, bacteria zymomonas mobilis possesses more advantages than saccharomyces cerevisiae, microorganism used for ethanol production in industrial scale. for that reason, we have focused on the fermentation studies of this facultative bacterium in free and immobilized form. the immobilization of the cells into polyvinylalcohol (pva) hydrogel lens-shaped capsules lentikats, improved the batch fermentation process efficiency nine times. starch, the substrate considered as one of the best of renewable energy source is considered as fuel ethanol feedstock. due to z. mobilis disability of maltose, maltotriose and dextrin utilization, the starch has to be converted into glucose monomers. this pre-fermentation step can overcharged whole ethanol production process. this ineffective part of the process was resolved with immobilization of glucoamylase into lentikats. the system with immobilized enzyme and cell was stabile in continuous mode for 60 days without any significant change in the system efficiency. the combination of cell and enzyme immobilization can significantly improve the efficiency and the cost of ethanol production in industrial scale. fermentation of an inhibitory dilute acid-hydrolysate from spruce using a fed-batch procedure combined with cell-reuse andreas rudolf, gunnar lidén department of chemical engineering, box 124, lund university, se-221 00 lund, sweden. e-mail: andreas.rudolf@chemeng.lth.se (a. rudolf) a well-controlled addition of hydrolysate to the fermentation has proved very efficient in reducing yeast inhibition due to compounds formed during lignocellulose hydrolysis. furthermore, using high cell mass concentrations has been another way of avoiding the negative impact of the inhibitory compounds. if possible, the yeast should therefore be re-used in the process. in the present work a dilute-acid hydrolysate from spruce was fermented using a fed-batch procedure with reutilization of yeast. the fermentation procedure worked satisfactorily, with more than 98% of fermentable sugars consumed in each of the four consecutive fed-batch fermentations performed. the ethanol yields on fermentable sugars reached 0.45 g/g. there was continued cell growth in the repeated fed-batch experiments, with an average cell yield on fermentable sugars of 0.06 g/g. in contrast, only about 20% of the fermentable sugars were consumed within 24 h, when the fermentation of the hydrolysate was run in a batch process. the work shows the potential to re-use the yeast in a suitably designed process. metabolic engineering is defined by bailey in his seminal 1991 paper as "the improvement of cellular activities by manipulation of enzymatic, transport, and regulatory functions of the cell with the use of recombinant dna technology". the manipulation of these functions ultimately results in the manipulation of metabolism, which is the purpose of many biotechnological processes. metabolic engineering has sought its methods and tools in mathematics and the physical sciences, and later in information technology (it) leading to the proliferation of bioinformatics. in this work we propose a novel approach to metabolic engineering that regards it as a business process reengineering (bpr) endeavour. hammer and champy in their celebrated 1993 book define bpr as "the fundamental rethinking and radical redesign of business processes to achieve dramatic improvements in critical contemporary measures of performance, such as cost, quality, service and speed". our thesis is that metabolic engineering with its goal of reengineering the metabolism of the microorganism, is equivalent to business process reengineering (bpr) in business and management. indeed this is essentially what metabolic engineering does to the cell through the use of recombinant dna technology, which can be viewed as a radical redesign of the metabolic process. after all, it causes changes that cannot be attained otherwise, and whose purpose is to achieve dramatic improvements in cellular activities such as the increase in production of some metabolites by orders of magnitude. the cost incurred by the process, which is metabolism in this case, can be, for example, energy requirements or change to a cheaper substrate. in this study we elucidate this parallelism between the two with emphasis on modelling of metabolism and how the concepts of business process modelling can be applied to it. the purpose is not to produce a model, but rather to introduce the modelling methodology and demonstrate its utilisation and benefits and outline its limitations and challenges. we believe that the novelty of our work lies in applying a new paradigm in approaching metabolic engineering that has not been considered previously. thermophilic ethanol production from wheat straw hydrolysate in continuous culture tania i. georgieva, birgitte k. ahring biocentrum-dtu, technical university of denmark, building 227, dk-2800 lyngby, denmark. e-mail: tig@biocentrum.dtu.dk (t. georgieva) ethanol production from lignocellulosic biomass has attracted widespread attention as an unlimited low cost renewable source of energy to transportation fuels due to increasing petroleum use and air pollution towards greenhouse gases. wheat straw available as agricultural residue has been considered as a potential lignocellulosic substrate for industrial bioethanol production. a major technical obstacle to commercialize bioethanol production form lignocellulose (such as wheat straw) is associated with a lack of microorganism able to rapidly and efficiently ferment both hexose and pentose sugars into ethanol and to tolerate the inhibitors present in undetoxified hydrolysates. currently used industrial mesophilic microorganisms (saccharomyces cerevisiae and zymomonas mobilis) are excellent ethanol producer from glucose, however, they are not able to ferment other sugars such as xylose, which is the second most abundant sugar in lignocellulose. thermophilic anaerobic bacteria have been considered for ethanol production from lignocellulosic biomass as an alternative to mesophilic ethanol producing strains, predominantly because of their abilities naturally to ferment the whole diversity of sugars found in lignocellulosic biomass. an increase attention to thermophilies for ethanol production have also arise from broad spectrum of advantages regarding industrial scale ethanol fermentation such as high growth and metabolic rates, low oxygen solubility, reduced risk of reactor contamination, and cost savings via mixing, cooling and facilitated product recovery. in addition, simultaneous co-fermentation of glucose and xylose in a single operation unit could substantially reduced the ethanol production cost. research has been attempted to study the potential of using a thermophilic anaerobic bacterium for continuous ethanol fermentation of lignocellulosic biomass, with particular emphasis on effectiveness of our strain to ferment undetoxified wet oxidized wheat straw hydrolysate with respect to sugar (glucose and xylose) conversion and ethanol yield. the experiment was carried out in a lab-scale reactor operated at 70 • c with wheat straw hydrolysate as a substrate in concentrations from 20 to 80 wt.% equivalent to total sugar mixture of 11-38 g/l. wheat straw hydrolysate (woh) [200 g/l wheat straw, 92.4% dry matter (dm)] was prepared using wet oxidation pretreatment process followed by enzymatic saccharification with commercial enzymes mixture of celluclast1.5, and novozym188 (novozymes, denmark). both xylose and glucose sugars were simultaneously converted to ethanol. the sugar utilization was higher than 90%, and high ethanol yields were achieved. reactor shows good long-term performance (124 days) in terms of operation stability and reactor contamination. maltotriose is the second most abundant fermentable sugar in wort and due to incomplete fermentation, residual maltotriose in beer may cause problems in the brewing industry. to study genes that might improve utilization of maltotriose we used a library with dna from brewer's strains and a laboratory strain and identified a new transporter encoded by mtt1. mtt1 gave lager strain a15 the ability to grow on yp/2% maltotriose in the presence of 3 mg/l of the respiratory inhibitor antimycin a. this transporter gene shares 74% similarity with mph2 and mph3, 62% similarity with agt1 and 91% similarity with mal61 and mal31. moreover, mtt1 shares even higher similarity (98%) with the s. pastorianus mty1 gene (m. salema-oom, unpublished, ncbi accession number aj491328). purified radiolabeled maltotriose and radiolabeled maltose were used to study sugar uptake of lager strains a15 and ws34/70, and of a15 containing mtt1 or mtt1alt, a more efficient, altered version of this gene lacking the 66 basepairs from the 3 end and containing 57 base-pairs of vector sequences. these transport studies show that mtt1 and, especially, mtt1alt encode maltose transporters with relatively high activity towards maltotriose compared to maltose. this study is part of a multi-disciplinary project, funded by the european union (contract no. qlk1-ct-2001-01066) focusing on the development of high-gravity resistant brewer's yeast strains. metabolic engineering of l-phenylalanine pathway in bacillus subtilis yasemin demirci 1 , pınar ç alık 2 , güzide ç alık 1 , tunçer h.özdamar 1 : 1 bre laboratory, department of chemical engineering, ankara university, 06100 ankara, turkey; 2 iblab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: ozdamar@eng.ankara.edu.tr (t.h.özdamar) metabolic engineering design of a recombinant l-phenylalanine (phe) production system is based on coordinated overexpression of the flux-controlling genes in the aromatic-amino acid pathway. based on the insights gained by the work carried out in our laboratories (özçelik et al., 2004) , aroh for the reaction r96 at the branch-point chorismate that connects the preceding reactions of the aromatic group amino acid pathway to the proceeding reactions towards phe, was predicted as the first-, and aroa for dahp synthase (r89) predicted as second-metabolic engineering sites. aroa gene was cloned next to aroh, by the use of four primers designed using pcr-based gene splicing by overlap extension method. the genes were amplified separately by pcrs. the primers used at the ends to be joined were designed as complementary to one another by including nucleotides at their 5 ends that are complementary to the 3 portion of the other primer. these products were mixed in the next pcr reaction, where one strand from each fragment contains the overlap sequence at 3 end. extension of this overlap by dna polymerase has yielded the recombinant two-gene product; and the two-gene product serve as template for the continuation of reactions for the increase of the concentration in the micro-reactors. the two-gene fragment was first cloned into puc19, and then sub-cloned to pmk4 e. coli -bacillus shuttle plasmid. the new expression vector with high stability and high copy-number was obtained and transferred into host b. subtilis. the metabolic flux distributions calculated by the mass balance based stoichiometric model based on the metabolic reaction network for r-b.subtilis were determined by using time profiles of the substrate, dry-cell, phe and other amino acids, and organic acids concentrations as the constraints. on the bases of calculated intracellular fluxes of recombinant b. subtilis carrying pmk4::aroa::aroh, an in-depth analyses of the metabolic engineering design will be presented. ozçelik,i̇., ç alık, p., ç alık, g.,özdamar,t.h., 2004. metabolic engineering of aromatic group amino acid pathway in bacillus subtilis for l-phenylalanine production. chem. eng. sci. 59 (22-23), 5019-5026. the 100% respiratory-deficient nuclear petite amylolytic saccharomyces cerevisiae npb-g strain capable of excreting a hybrid protein possessing both ␣-amylase and glucoamylase enzyme activities was generated and its employment for direct fermentation of starch into ethanol was investigated under both shake flask and controlled bioreactor cultivation conditions. when compared with a standard host strain, higher ethanol concentrations and yields were achieved with the nuclear petite strain under both cultivation conditions. further improvement in ethanol production was achieved by the use of an initial glucose supplement. comparison of the ethanol fermentation performances of the respiratory-deficient npb-g and the parental respiratory-sufficient wtpb-g strain showed an increase of, ca. 48% in both ethanol production yields and ethanol productivities with the respiratory-deficient strain. response surface methodology (rsm) was used as a statistical tool to optimize the initial yeast extract and starch contents of the medium, which resulted in a substantial increase in the stability of the expression plasmid in both strains with concomitant improvement in their amylolytic potentials. high ethanol yields on substrate values of the bioreactor cultures, that were very close to the theoretical yield, indicated that the amylolytic respiratory-deficient strain developed in this study was very effective in the direct fermentation of starch into ethanol. establishing a biotechnology educational framework to support a knowledge-based economy in puerto rico rosa buxeda, lorenzo saliceti-piazza industrial biotechnology program, university of puerto rico, mayagüez campus, mayaguez 00680, puerto rico. e-mail: rbuxeda@uprm.edu (r. buxeda) industrial biotechnology has been identified as a major thrust area of economic development within the past five years for the island of puerto rico. the portfolio of biotechnology manufacturing investments in the island has passed the two billion dollar mark. world known companies like amgen, abbott and eli-lilly lead these investments, which have catalyzed a strong technology transfer to the island. a strong collaboration between industry and academia was needed to provide a well-trained workforce for these company startups. as a result, the university of puerto rico, mayagüez campus (upr-m) developed four initiatives which are part of the educational pipeline in biotechnology. these are: (i) an industrial biotechnology program, a 5-year bs degree which contains a novel curriculum with courses from science and engineering with undergraduate research and industrial internships as part of the degree requirements; (ii) an industrial biotechnology learning center, which provides customized biotechnology and bioprocessing training modules, including lectures and hands-on experiences to train and develop the workforce needed in the biotechnology manufacturing plants; (iii) a biotechnology summer camp, which addresses the high school student population and its main purpose is to educate and advise on the different career paths that can be followed in the field of biotechnology; and (iv) a biotechnology center for research and training in bioprocessing, that will address the development of corporatesponsored research projects to strengthen links between industry and academia in order to build up a knowledge-based economy. our paper will describe each initiative in detail as well as its outcomes and impact on puerto rico's knowledge-based economy goals. isolation of acid phosphatase from sweet potato and immobilization using different adsorbent d. omay, y. güvenilir, n. deveci istanbul technical university, department of chemical engineering, maslak 34769, istanbul, phosphatase enzymes occur in a wide range of plant and animal tissues. they catalyze the hydrolysis of phosphate bonds in organic phosphates, between the phosphate group and rest of the molecule. immobilization of enzymes and biological compounds is currently gaining importance due to its wide variety of applications in the food and pharmaceutical industries and also its biomedical applications. it was reported that enzymes can be activated by complexation with polysaccharides such as chitin or chitosan. the aim of this experimental study was determined as partial purification and isolation of acid phosphatase enzyme and its immobilization. the purification was realized by applying centrifugation, ammonium sulfate precipitation and dialysis respectively. the specific activity of the supernatant was 0.1 u/mg and after 80% saturation this value increased 0.64 u/mg. furthermore, acid phosphatase was investigated using different adsorbent (chitin, chitosan, synthetic zeolite and raw zeolite) and evaluated the storage stability and re-usability of the immobilized acid phosphatase. it was estimated that, acid phosphatase activity was shielded the ratio of 94, 96, 99, and 92% by using raw zeolite, synthetic zeolite, chitin and chitosan respectively under 12 h operation condition. øyvind m. jakobsen 1,2 , michael c. flickinger 3 , svein valla 1 , trond e. ellingsen 2 , trygve brautaset 1 : 1 department of biotechnology, norwegian university of science and technology, norway; 2 sintef applied chemistry, sintef, norway; 3 biotechnol. institute, department of biochemistry, molecular biology and biophysics, university of minnesota, usa aerobic methylotrophs can utilize one-carbon (c 1 ) compounds such as methane and methanol as a sole c-source for growth and energy. the majority of research on these organisms has focused on their biochemical novelity and commercial viability. for the industrial production of bulk products such as the amino acids lysine and glutamate raw material costs and abundance are important, and c 1 sources are thus attractive compared to sugars. bacillus methanolicus can secrete up to 55 g/l of glutamate upon methanol growth at 50 • c (thermotolerant and methylotroph) and mutants producing 35-40 g/l of l-lysine have been selected. we study the genetics for conversion of methanol into biosynthesis of glutamate and lysine, and in the present report we unravel the regulation of genes impor-tant for the consumption and tolerance level for c 1 compounds by b. methanolicus. b. methanolicus has a methanol dehydrogenase gene (mdh) for oxidation of methanol into formaldehyde and a ribulose monophosphate (rump) pathway for assimilation of formaldehyde. we recently discovered that mdh and five rump genes are carried by natural plasmid pbm19 in this bacterium and this represented the first documentation of plasmid-dependent methylotrophy in any microorganism. we here use real-time pcr to analyse the regulation of plasmid-and chromosomally located rump genes, in cells upon methylotrophic and non-methylotrophic growth. high methanol concentrations in the growth medium is cell toxic and the mechanisms for this sensitivity of b. methanolicus is poorly understood. our results indicate that plasmid pbm19 plays a fundamental role for this trait as well and the impact of our results on the biotechnological applications of this bacterium is discusssed. department, national research centre, dokki, cairo, egypt adding a proteolytic enzyme extraction from jack fruit (artocarpus integrifolis) in combination of fermentation process in low fat yogurts manufacture was tried to improve yogurt flavour and rheological properties. experimental yogurts milk contained control, 3.9 (t1), 7.8 (t2) and 11.7 (t3) units/ml milk from crude extracts of plant proteinase. the ph of the product treated with crude proteinase was lower than the control. however, the rate of acidity development during storage slightly increased with increasing the addition of crude proteinase level and progress of storage period of yogurt. the proteolytic activity of all yogurts gradually increased until the end of storage period (15 days). yogurts made from milk treated with crude proteinase preparations were less firm compared with control at all storage periods, where t3 showed more less firm after 15 days of storage being 20.17 g/100 g. generally, increasing units of plant proteinase preparations decreased the firmness. on the other hand, yogurt made from milk pretreated with plant proteinase had higher syneresis, and apparent viscosity than the untreated product. the greatest viscosity was found in t2 and t3 of 433 and 479 mpa s respectively, compared with control of 299 mpa s at 15 days storage. the results indicated that there is an inverse relationship between the amount of units of crude proteinase preparations and susceptibility of yogurt to syneresis. the t2 gained the highest scores (85 points) followed by the control (81.5 points) after 15 days of storage, while yogurt of t3 showed a low scoring being 75. from the foregoing results, it is recommend to use jack fruit (artocarpus integrifolis) as a source of plant proteinases and utilize it to develop a high quality yogurt at a level of 7.8 units of plant proteinases/ml milk. the penicillium chrysogenum oat1 gene encoding a class iii omega-aminotransferase has been cloned and characterized. this enzyme that converts lysine into 2-aminoadipic semialdehyde is important in providing 2-aminoadipic acid, a precursor of penicillin and other ␤-lactam antibiotics. the enzyme is related to ornithine-5-aminotransferases and to lysine-6-aminotransferases encoded by the lat gene located in the bacterial cephamycin gene clusters. expression of oat1 is induced by lysine, ornithine and arginine and repressed by ammonium ions. area-binding consensus sequences and an 8-bp direct repeat associated with arginine induction in emericella (aspergillus nidulans) have been found in the oat1 promoter region. deletion of the oat1 gene resulted in the loss of omegaaminotransferase activity. the deletion mutants were unable to grow on ornithine or arginine as sole nitrogen sources and showed a reduced growth on lysine. complementation of the deleted mutant with the oat1 gene restored growth on ornithine, arginine and lysine to the levels of the parental strain and omega-aminotransferase activity. the strong expression of oat1 gene after induction by the basic amino acids may provide additional 2-aminoadipic acid for the formation of the 2-aminoadipyl-cysteinyl-valine tripeptide for ␤-lactam biosynthesis. morphological characterisation of two high producing strains of penicillium chrysogenum carrying a disruption in the nadph-dependent glutamate dehydrogenase k. rueksomtawin, j. thykaer, h. noorman, j. nielsen center for microbial biotechnology, biocentrum-dtu, technical university of denmark, dk-2800 lyngby, denmark. e-mail: kr@biocentrum.dtu.dk (k. rueksomtawin) metabolic engineering has proven to be useful in optimisation of ␤-lactam production, e.g. constructing superior strains with multiple copies of the ␤-lactam gene cluster. it is however, of equal importance to gain insight into other aspects of the metabolism to establish a general overview in order to apply metabolic engineering for further improvement of the production strains. in that context, the redox metabolism is essential, as it functions as a tightly controlled connection between the different parts of the metabolism. in order to investigate this role of the redox metabolism in more detail, the gdha-gene, encoding the nadph-dependent glutamate dehydrogenase, was disrupted in two industrial strains of penicillium chrysogenum. during physiological characterisation of the two strains it became apparent that considerable changes in the morphology had occurred due to the genetic alteration. since the morphology is an important parameter in process optimisation, an examination of the morphology of the two strains was undertaken. in this work, the morphological differences between the gdha-disrupted strains and the reference strains were comprehensively investigated both during growth on solid media and submerged growth in a flow-through growth chamber. with the advance development of computerized image analysis techniques, the key morphological properties of the individual hyphal elements were quantified. in comparison to the reference strains, the disruption of the gdha gene resulted in a morphological change from short hyperbranched hyphal elements to long elongated hyphal elements with less branches. in the parallel studies with aspergillus nidulans and its corresponding gdha-deleted strain, no difference in the morphology was observed. polyketides (pk) represent one of the largest groups of natural products and are found in fungi, bacteria and plants. since many useful polyketides either originate from sources that are difficult or even impossible to cultivate or are produced in inadequate amounts, we are interested in expressing polyketide synthases (pkss) in heterologous hosts. saccharomyces cerevisiae, aspergillus niger and aspergillus nidulans were chosen as initial hosts, because the techniques necessary to cultivate and manipulate these strains genetically are well established. 6-methylsalicylic acid synthase (6-msas) was chosen as a model pks. replicative plasmids carrying the genes encoding 6-msas from penicillium patulum and phosphopantetheinyl transferase (pptase), respectively, were transformed into s. cerevisiae. in addition, an integrative vector was designed and the gene encoding 6-msas was integrated in the yeast chromosome. batch cultivations on galactose minimal media were performed. the results are presented and in particular the effect of expression mode and type of pptase (bacterial versus fungal) is discussed. furthermore, the progress of the work on expressing 6-msas in a. niger and a. nidulans using an integrative vector system is presented and discussed. the valorisation of functionalized chemicals from biomass resources compared to the conventional fossil production route ben brehmer, wageningen ur agrotechnology & food sciences, workgroup: valorisation of plant production chains, p.o. box 17, 6700 aa wageningen, the netherlands. e-mail: ben.brehmer@wur.nl at some undisclosed point in the foreseeable future, cheap fossil fuels resources will become depleted and the industry will be forced to pursue more difficult reserves with increasingly high extraction costs. most alternatives available and under research do not consider price as the main motivation for replacement, but focus solely on sustainability and environmental benefits. sustainability is an interesting word as there are enough fossil resources scattered around the world to be sustainable in quantity but not sustainable in price. seeing that fossil fuels are derived from prehistoric biomass it is not at all presumptuous to assume that every application and product can be replaced by the biomass of today. in fact, many highly specialised pharmaceutical chemicals already have a biomass origin. yet, not all of the uses of fossil fuels need to be replaced by a comparable carbon based source, such as biomass. energy and transportation in particular do not necessary need to rely on carbon cleavage, whereas practically all of the petrochemicals contain a carbon backbone. the main stipulation in substituting fossil-based chemicals with bio-based chemicals is availability and cost. it is proposed that already today, by utilising existing, recently developed and developing technology, it is economically advantageous for many chemicals to derive from biomass, in particular the functionalized chemicals. the only way to validate this conjecture is to develop a complete comparative life cycle analysis. as opposed to a traditional lca, the "multicriterion" developed here will revolve around energy flows and process efficiency in terms of exergy. the aim is to assess the optimum route with the best production options along the whole production chain while determining any possible limiting factors. using this tool, a systematic production matrix relating several logical source crops and a few key chemicals of varying derivative levels can be created and compared to the conventional fossil routes. combined with economic considerations and some unambiguous environmental factors, the investigation will provide all the information relevant to the industry. the goal is to create an objective and reliable simulation system ratifying the economic and environmental feasibility of exploiting biobased chemicals today and indicate the steps necessary for further improvement. biosynthesis of multi-enzymatic preparation from aspergillus niger ibt-90 useful in textile fabric treatment rita pyc 1 , jadwiga sojka-ledakowicz 2 , tadeusz antczak 1 , joanna lichawska 2 : 1 institute of technical biochemistry, the technical university of lodz, lodz 90-924, poland; 2 textile research institute, lodz 92-103, poland among many methods of producing enzymatic preparations, i.e. by liquid surface fermentation -lsf, submerged fermentation -smf or solid state fermentation -ssf, this last is most advantageous. cultivation in solid state means fermentation on a matrix formed by industrial and agricultural wastes. most often filamentous fungi -due to the lack of available water in the foundation -are the efficient, competitive microorganisms applied in solid state bio-conversion. the aim of research works carried out at the institute of technical biochemistry of the technical university of lodz and at textile research institute, lodz was defining optimal conditions for biosynthesis and testing the possibility of applying multi-enzymatic preparation from aspergillus niger ibt-90 in the treatment of woven fabrics made of natural cellulose fibres. as the result of biosynthesis optimization process, malt sprouts, wheat barn and beet pulp were selected as the best media to obtain enzymes of high pectinolytic activity maintaining at the same time high activity of cellulolytic enzymes and xylanase. the highest obtained activities reached: 2000 0 pm for total pectinolytic activity, 7 j/ml for endoglucanase and 527 j/ml for endoxylanase and they were 2.4-3.0 times higher than those achieved before optimizing process. performed research works demonstrated that the optimum activity of applied enzymatic system is obtained in the range of ph 4.6-4.8. woven fabrics made of flax and cotton fibre blends, subjected to bio-pre-treatment, were evaluated with reference to their sorption properties. comparative evaluation of liquid sorption by woven fabrics allowed to notice efficient enhancement of fibres' sorption capabilities after pre-treatment using enzymes system from aspergillus niger ibt-90. this offers the possibility of substitution of alkali scouring of linen-cotton woven fabrics before their bleaching by bio-treatment. the phylum actinobacter includes many bacteria of industrial importance both for accumulation of primary and secondary metabolites. both primary and secondary metabolites are dependent on precursors and cofactors that are provided by the central carbon metabolism of microorganisms. there are alternative pathways for catabolism of carbon either via the embden meyerhof parnas (emp) and pentose phosphate (pp) pathway or through the entner doudoroff (ed) pathway. the emp pathway is energetically more favorable and has therefore been presumed to be the dominating route for carbon metabolism in bacteria producing secondary metabolites. however, primary metabolism is poorly studied for most actinobacter species as focus traditionally has been on secondary metabolism. with the aim to gain more knowledge about the diversity of central carbon metabolism within the phylum actinobacter, 17 different strains were collected from various sources and strain collections. the strains were grown in minimal medium with supplement of standard vitamin solution and [1-13 c] glucose as carbon source. the 13 c-labeling patterns of proteinogenic amino acids were determined by gc-ms analysis. through this method, the fluxes in the central carbon metabolism during balanced growth were estimated and pathways for carbon metabolism were determined. in particular the labeling patterns of alanine and valine were of interest as they are derived from pyruvate and therefore can be used to distinguish between whether glucose is metabolized through the ed pathway or the emp pathway. nobacter, amycolatopsis balhimycina produces the glycopeptide balhimycin. the balhimycin aglycone is identical to the aglycon of vancomycin, which is a commercial glycopeptide. as a. balhimycina appears to be accessible to genetic modifications and the biosynthetic cluster responsible for balhimycin production is published, this genetically well-characterised academic strain can serve as a platform strain for production of vancomycin analogues derived through combinatorial biosynthesis. the understanding of the physiology of this microorganism is essential for the efficient accumulation of potentially commercial secondary metabolites. the bacterium is capable of growth in a fully defined minimal medium, with the production of balhimycin. the strain was grown at either nitrogen or phosphate limitation and balhimycin accumulation was followed at these conditions. flux analysis of balhimycin production by amycolatopsis balhimycina based on 13 c labelling experiments was performed. the zygomycetes blakeslea trispora and phycomyces blakesleeanus accumulate beta-carotene, ubiquinone (coenzyme q), various different sterols, and other terpenoids, all of them produced via the mevalonate pathway. these fungi are used or could be used for the industrial production of these terpenoids, edible oil, chitosan, and various organic acids. by measuring the specific radioactivity of terpenoids made from radioactive mevalonate, leucine or acetate in the presence of excess glucose in wild types and mutant strains we have concluded that these fungi have separate subcellular compartments for the production of carotene, sterols and triacylglycerols. the terpenoid moiety of ubiquinone is synthesized in the same compartment as ergosterol. these compartments contain separate pools of all their common metabolites, beginning from acetyl-coa. mevalonate carbon atoms do not find their way back to general metabolism, i.e., these fungi lack the "shunt" pathway. the compartments are regulated independently. the very large variations in carotene content caused by many environmental and genetic changes are not accompanied by variations in the ubiquinone content. the ubiquinone content increases when the cultures grow on leucine or acetate as carbon sources and is not affected by illumination. phycomyces, but not blakeslea, increases the production of ubiquinone in presence of oligomycin. lincomycin, produced by streptomyces lincolnensis, is important, clinically used antibiotic. its gene cluster consists of 27 putative open reading frames with biosynthetic or regulatory functions (lmb genes) and three resistance genes (lmra, lmrb, lmrc). the organization of transcription units was determined. the analysis of the lincomycin biosynthetic gene transcripts in various cultivation stages revealed the genes with putative regulatory functions which are transcribed in early stages of cultivation. previous analysis of biosynthetic pathways of lincomycin and functionally different anthramycin antibiotics (anthramycin, sibiromycin, tomaymycin, mazetharmycin and porothramycin) indicates that the genetic information on the lincomycin and anthramycin biosynthesis should share common elements (genes), both biosynthetic and regulatory. hybridization experiments demonstrated presence of several analogues of lmb genes involved in the biosynthesis of anthramycin produced by streptomyces refuineus and porothramycin produced by streptomyces albus. effect of various calcium salts on the erythromycin production by saccharopolyspora erythraea m. rostamza 1 , a. noohi 1 , j. hamedi 2* : 1 department of biology, faculty of science, science and research branch, islamic azad university, tehran, iran; 2 microbial biotechnology lab., department of biology, faculty of science, university of tehran, tehran, iran. e-mail: jhamedi@khayam.ut.ac.ir (j. hamedi) calcium carbonate has the positive effect on the erythromycin, however, because of its low water solubility, caused to clogging the spargers of fermenters and fouling the microfilters. in this research, various soluble calcium salts was added to the fermentation medium and their effect the growth of saccharopolyspora erythraea and erythromycin production was studied in the complex medium consisted soy bean meal, dextrin and starch as major ingredients. the fermentation conditions were 220 rpm, 8 days at 30 • c. the results obtained showed that there is no significant difference between erythromycin concentrations in the medium containing calcium lactate and calcium carbonate. however, erythromycin concentrations in the other calcium salts containing media were less than to calcium carbonate containing medium. optimum concentration of calcium lactate for erythromycin production was 10 g/l. lincosamides and its derivatives are clinically important antibiotics. comparison of gene cluster coding for lincomycin biosynthesis and newly identified cluster of genes for celesticetin biosynthesis revealed new information on functions of several genes common for both biosynthetic pathways. the celesticetin gene cluster was identified by screening the cosmid library of streptomyces caelestis with heterologous probes based on lincomycin biosynthetic genes involved in a part of biosynthesis shared by both antibiotics. sequence analysis of the cluster revealed 24 putative orfs, out of which 18 are lincomycin biosynthetic genes analogues, four are specific for celesticetin biosynthesis and one codes for resistance. the gene cluster is bounded with transposase genes on both sides. in order to clarify function of three putative regulatory genes of lincomycin biosynthesis, the insertional inactivation with the pcr targeting system in streptomyces lincolnensis was done and resulted in differently reduced production of lincomycin. larsen cmb biocentrum-dtu, technical university of denmark, 2800 kgs. lyngby, denmark we present a new algorithm called "x-hitting" for automatic identification of novel bioactive compounds based on full spectroscopic characters of highly complex mixtures of natural product. one of the most dramatic advances in recent drug discovery has been the increase in screening capacity throughput and data handling. therefore, analysis has become the bottleneck in the drug discovery process. the algorithm presented here is investigated and demonstrated on identifying potentially new bioactive compounds. in addition method is shown to have a high performance for automatic identification of known structures. these tasks are referred to as new-hitting and cross-hitting, respectively. finally, the receiver operating characteristics (roc) is introduced to the research field as an important tool for evaluating the performance of the "compound predictor". through examples it is shown, that known cross-hits are identified with high proficiency, and that the new-hitting works on finding new targets represented by analogues and structurally new compounds. a gene encoding a ␥-butyrolactone autoregulator receptor that have a common activity as dna-binding transcriptional repressors, controlling secondary metabolism and/or morphological differentiation in streptomyces was cloned from a natamycin producer, streptomyces natalensis, and its function was evaluated by in vivo. pcr using the primers designed from two highly conserved regions of streptomyces autoregulator receptors gave a 102-bp band, the sequence of which revealed high similarity to the expected region of a receptor gene. by genomic southern hybridization with 102-bp insert as a probe, a 687-bp intact receptor gene (sngr) was obtained from s. natalensis. in vivo to clarify the function of sngr, a sngrdisrupted strain was constructed, and a phenotype was compared with the wild-type strain. the sngr disruptant started natamycin production 6 h earlier and showed a 4.6-fold-higher production of natamycin than the wild-type strain. in addition, sporulation was earlier and 10-fold abundance. the phenotype indicates that the autoregulator receptor protein of s. natalensis acts as a primary negative regulator of the biosynthesis of natamycin and is related to regulation of sporulation. exploring the biocatalytic potential of the novel thermostable baeyer-villiger monooxygenase: phenylacetone monooxygenase daniel e. torres pazmiño 1 , gonzalo de gonzalo 2 , gianluca ottolina 2 , giacomo carrea 2 , dick b. janssen 2 , marco w. fraaije 1 , 1 biochemical laboratory, groningen biomolecular sciences and biotechnology institute, university of groningen, nijenbaeyer-villiger monooxygenases (bvmos) represent useful biocatalytic tools as they can catalyze reactions which are difficult to achieve using chemical means. however, only a limited number of these atypical monooxygenases are available in recombinant form. using a recently described protein sequence motif, a putative bvmo was identified in the genome of the thermophilic actinomycete thermobifida fusca. the nadph-dependent and fad-containing monooxygenase is active with a wide range of aromatic and aliphatic ketones and sulfides. genetic and kinetic data suggest that phenylacetone is the physiological substrate of the enzyme. previously, it was reported that this bvmo exhibits only a moderate enantioselectivity with (r,s)-␣-methylphenylacetone. this poster we will show an overview of the biocatalytic potential of the enzyme as explored so far. interestingly the enzyme has been found to perform highly enantioselective oxidations with a range of ketones and sulfides. this again indicates that this novel thermostable oxidative biocatalyst can be a useful tool for the synthesis of chiral building blocks. the enzyme s-adenosylhomocysteine hydrolase (adohcyase) catalyzes hydrolysis of s-adenosylhomocysteine (adohcy), an inhibitor of transmethylation reactions, into adenosine (ado) and homocysteine (hcy). the catalysed reaction is reversible, and the equilibrium is strongly displaced in direction of the synthesis of adohcy when reaction occurs in vitro. nevertheless, its biotechnological application resides in the synthesis of antivirals. for it, we selected between different producing microorganisms of the enzyme, the gram positive bacterium corynebacterium glutamicum. after designing the specific oligonucleotides, the gene was expressed in escherichia coli using the expression system pet28a+ with iptg. the electrophoretic analysis under denaturing conditions, shows a clear induction and over-expression of a protein with a mw of 49 kda. on the other hand, the immobilization of this recombinant enzyme in a solid support allows to use it as a catalyst for the synthesis of adohcy. the enzyme was purified by imac thanks to the presence of n-terminal 6 × his tag end, and immobilized in eupergit c for the optimization of the production of adohcy, a product of high value. sequencing, cloning, expression, purification and characterization of a novel cytochrome p450 monooxygenase from rhodococcus rubber luo liu, rolf d. schmid, vlada b. urlacher institute of technical biochemistry, stuttgart university, allmandring 31, 70569 stuttgart, germany. e-mail: itbvur@itb.unistuttgart.de (l. liu) the cytochrome p450 monooxygenases are heme-containing proteins, which catalyze a wide range of oxidative reactions (werck-reichhart and feyereisen, 2000) . a monooxygenation activity was observed for the strain rhodococcus ruber dsm 44319. a p450-like gene fragment was amplified by pcr using degenerated primers. for identification of regions that flank this p450-like dna fragment, the method "directional genome walking using pcr" was applied (mishra et al., 2002) . the full size gene encoding a cytochrome p450 enzyme was amplified by pcr from genomic dna and cloned into the vector pet28a(+) for heterologous expression in escherichia coli bl21(de3) cells. the enzyme was purified using metal affinity chromatography. the primary protein structure suggests, that this enzyme is a natural self-sufficient fusion protein consisting of a ferredoxin, a reductase and a p450 monooxygenase. the reductase activity was determined using an exogenous electron acceptor cytochrome c. the reductase domain of this p450 monooxygenase showed a strong preference for nadph over nadh. the substrate spetrum was investigated. in the presence of nadph the p450 enzyme shows hydroxylation activity towards 7-ethoxycoumarin, naphthalene, indene, acenaphthene, toluene and fluorene. mishra, r.n., singla-pareek, s.l., nair, s., sopory, s.k., reddy, m.k., 2002 . directional genome walking using pcr. biotechniques 33, 830-834. werck-reichhart, d., feyereisen, r., 2000. cytochromes p450: a success story. genome biol. 1 (6), 3003.1-3003.9. selective production of monoglyceride consisted of conjugated linoleic acid by penicillium lipase yomi watanabe 1,2 , yoshie yamauchi-sato 3 , toshihiro nagao 1 , satoshi negishi 3 , tadamasa terai 4 , takashi kobayashi 1 , rolf d. schmid 2 , yuji shimada 1 : 1 osaka municipal technical research institute, osaka, japan; 2 stuttgart university, stuttgart, germany; 3 the nisshin oillio group, ltd, yokosuka, japan; 4 osaka institute of technology, osaka, japan conjugated linoleic acid (cla) is a group of c18 fatty acid (fa) containing two conjugated double bonds. it is expected to prevent cancer, adipositas, atherosclerosis etc, and is commertially available in the primary form of free fa, containing almost equal amounts of 9cis,11trans-and 10trans,12cis-cla. it is therefore desired to be converted to a palatable form. for this purpose, we have previously proposed two enzymatic ways to convert cla to monoglyceride, an emulsifier, with penicillium camembertii lipase; (1) sequencial esterification-glycerolysis, (2) esterification at low tem-perature. these methods, however, are time-and energy-consuming. esterification of cla with glycerol under ambient pressure by the lipase produces equal amounts of mono-and diglycerides. in contrast, it was newly found that the reaction under reduced pressure supressed the formation of diglycerides and achieved to produce 90% monoglyceride at 95% esterification. improving the thermal stability of cellobiohydrolases i (cel7a) from t. reesei by site directed evolution frits goedegebuur 1 , lydia dankmeyer 1 , peter gualfetti 2 , brad kelemen 2 , edmundo larenas 2 , paulien neefe 1 , pauline teunissen 1 , colin mitchinson 2 : 1 genencor international bv, archimedesweg 30, 2333cn leiden, the netherlands; 2 genencor international inc., 925 page mill road, palo alto, ca 94304, usa genencor international has been working to produce improved enzyme products for economic conversion of ligno-cellulosic biomass to fermentable sugars. most of this work was performed under a subcontract with the u.s. department of energy for cellulose cost reduction for biomass conversion. cellulolytic biomass conversion is performed in nature by a complex mixture of enzymes. cellobiohydrolases play a key role and all effective cellulase mixtures contain a large excess of cellobiohydrolases over endoglucanases, suggesting that it is the exoglucanase activity that is limiting. the fundamental dependence of reaction rate on temperature predicts that large increases in performance, and decreased enzyme cost, would be achieved if the enzymatic conversion could be operated at elevated temperatures. industrial strains of trichoderma reesei produce cellulases at very high levels and low cost. however, t. reesei cbh1 (hypocrea jecorina cel7a) does not have sufficient stability to survive and perform at high temperatures. this poster shows the thermal stability improvement in t. reesei cbh1 by site directed evolution. sites with increased thermal stability properties were combined and evolved in high temperature stable cbhi variants. the evaluation of lipases as biocatalysts for organic chemistry can be carried out, at laboratory scale, by using soluble enzymes for biotransformations in aqueous media. however, the industrial exploitation of such an enormous potential should require a suitable protocol for immobilization of lipases. the binding of lipases on suitable pre-existing supports should greatly improve the performance of industrial reactors allowing us a continuous use or re-use of such interesting biocatalysts. in addition, lipases, like most enzymes, are not perfect chemical catalysts. lipases may be unstable and they may not have the optimal activity nor the optimal enantio or regioselectivities. in this way, immobilization of lipases, together with its relevance for the performance of each different industrial reactor, could be also used as a tool to improve and optimize some of these parameters. that is, immobilization of lipases, far from an already solved problem, constitutes an exciting field of research in the promising area of industrial bio-organic chemistry. in this work we would like to present useful immobilization methods of several lipases. the lipase immobilised were used for enzymatic hydrolysis of peptidomimetics of structure a. type of immobilzation used can changed the enantioselectivity of biocatalyst prepared. the absolute configuration of products b and c obtained in enzymatic reactions were assigned by chemical correlation. two-component flavin-dependent monooxygenases form an interesting class of flavoenzymes. they consist of two separate proteins; a monooxygenase component, which catalyses an oxygenation reaction in the presence of reduced flavin, and a flavin reducing component, which reduces flavin (fad or fmn) using nad(p)h as an electron donor. a well-known example of this class of monooxygenases is styrene monooxygenase (otto et al., 2004) . due to the ability to form enantiopure epoxides, which are relevant building blocks for the pharmaceutical industry, styrene monooxygenases form a valuable class of enzymes for biocatalysis. while screening a metagenomic library for oxidative enzymes, an indigo-producing clone was found. sequencing the particular clone revealed an inserted fragment of environmental dna encoding a two-component monooxygenase ( many investigations over the recent years have been directed to the production of natural aroma compounds. through biotransformation and bioconversion, aroma compounds considered as "natural" can be produced starting from monoterpenes, generating high value products as rose oxide. rose oxide is found in small amounts in some essential oils such as bulgarian rose oil and geranium oil. (−)-rose oxide is an impacting flavor compound and has a small threshold: 0.5 ppb. application of agro-industrial residues in bioprocess on one hand provides alternative substrates, and on the other hand helps solving pollution problems, that might be caused by the disposal of this residue in nature. the liquid cassava waste, is originated by the pressing of cassava roots. it is considered as a "harmful" pollutant waste due to its high organic charge and presence of cyanide. on the other hand, can be considered rich in nutrients that can be used in other applications. it was found that sporulated surface cultures of penicillium sp. were able to convert citronellol into cis-and trans-rose oxides. other bioproducts were 3,7-dimethyl-5-octen-1,7diol and 3,7-dimethyl-6,7-epoxy-1-octanol. no chemical oxidation or auto-oxidation products were detected in liquid control broths. the experiments were conducted at 30 • c and 160 rpm. when the medium was cassava, the production of rose oxide, 3,7-dimethyl-5octen-1,7-diol and 3,7-dimethyl-6,7-epoxy-1-octanol were insignificant reaching trace amounts. but when the mycelium developed in cassava medium and than transferred to mineral medium (citronellol as c-source) the concentrations of rose oxide increased dramatically, reaching 70 mg/l for the cis-isomer and 30 mg/l for trans-isomer. a mechanistic mathematical model of enzymatic degradation of avicel and phosphoric acid swollen cellulose (pasc) has been proposed. the model is based on the degree of polymerization (dp) of starting substrate, and follows its decline with time, to the final end product -glucose. three enzyme classes, namely, endoglucanase (eg), cellobiohydrolase (cbh) and ␤-glucosidase (bg) are all individually incorporated in the model. the model is, additionally, taking into account cooperative action of the involved enzymes, as well as effects of enzyme inhibition by end-products, cellobiose and glucose. to be able to describe the complex process of enzymatic hydrolysis with a set of differential equations certain assumptions needed to be introduced. those assumptions represent the simplification of an up-to-date knowledge of both substrate and enzyme structure, but also enzyme mode of action. for example, one of the often asked questions is: "what is happening with shorter cellooligosacharides (dp 7 and up) laying on the surface of cellulose chain? are they being adsorbed to the core cellulose chain or partly solubilized to a hydrolysis broth?" to give answers to these questions and confirm mathematical modeling real enzyme hydrolysis data are needed. in this work, four well characterized, highly purified mono-component enzymes from humicola insolens (two eg and two cbh) and one bg from aspergillus niger were used to hydrolyze avicel and pasc. by careful choice of catalyst, some enzyme specific characteristics like presence or absence of cellulose binding domain will also be incorporated into the model. hydrolysis experiments were initially performed by distinct mono-component enzymes, to confirm the basic characteristics of each of the enzyme classes. soluble hydrolysis products (dp 1-6) were analyzed by hplc and detection of non-soluble, higher-dp polysaccharides was performed by technique of polysaccharide analysis using carbohydrate gel electrophoresis. optimisation of halogenase enzyme activity k. muffler 1 , m. retzlaff 2 , k.-h. van pée 3 , r. ulber 1 : 1 technische universität kaiserslautern, germany; 2 technische universität münchen, germany; 3 technische universität dresden, germany. e-mail: muffler@rhrk.uni-kl.de (k. muffler) halogenases provide the opportunity of a regioselective and stereospecific halogenation of organic subtrates in contrast to the class of haloperoxidases. these enzymes allow a gentle synthesis of halogenated organic molecules and are capable to halogenate in specific positions (hammer et al., 1997; keller et al., 2000) , whereas traditional organic synthesis often failures or mainly leads to byproducts (hasegawa et al., 1999) , e.g. halogenation of tryptophan in other positions than position 3. our current research is focussed on tryptophan-5-halogenases, because the 5-br/cl-tryptophan could be applied as a pharmacologically attractive precursor of serotonin. in our work we describe the optimization of an enzyme assay respectively the enzyme activity. for this purpose we use a genetic algorithm. the responsible gene of the fadh 2 dependent enzyme was cloned from streptomyces sp. origin into a pseudomonas fluorescens strain. however, the optimization procedure was done with the purified his-tagged tryptophan-5-halogenase, which was easily obtained from the crude extract of the lysed cells by application of immobilized metal affinity chromatography. the application of algorithms allows an optimization of the multidimensional search problem leading to a global optimum in the search space in contrast to the traditional used one-factor-at-a-time method. latter often failures, because this method does not reflect possible influences the parameters can have on each other. effect of organic solvent type on the enantioselectivity of candida rugosa lipase in the hydrolysis of racemic naproxen methyl ester in biphasic reaction system serpil takaç, deniz mutlu department of chemical engineering, institute of biotechnology, ankara university, 06100 tandogan, ankara, turkey hydrolysis of racemic naproxen methyl ester to produce s-naproxen was carried out in the biphasic system using isooctane, cyclohexane, hexane and toluene with candida rugosa lipase after stirring in the phosphate buffer (ph 7.5, 0.02 m) for 2 h at +4 • c and centrifuging. the hydrolyses were carried out in shaking flasks for 120 h at 200 rpm and 37 • c with the initial substrate concentration of 0.034 m. the concentrations of the enantiomers of racemic naproxen methyl ester in organic solvents and those of naproxen in buffer solution were determined with hplc. it was found that the enantiomeric excess for substrate (ee s ), enantiomeric ratio (e) and conversion (x) decreased in the following order: isooctane > cyclohexane > hexane > toluene. the enantiomeric excess for product (ee p ) was found to be the same for isooctane, cyclohexane and hexane where the lowest ee p was obtained in toluene. the highest ee s , ee p , e and x values achieved in isooctane at the residence time of 120 h were 91, 95, 130, and 49%, respectively. this study was supported by ankara university biotechnology institute (project no: 89). fusarium fujikuroi (gibberella fujikuroi mating group c) produces multiple secondary metabolites such as gibberellins and bikaverin. gibberellins are terpenoid hormones that induce growth and regulate various stages of development in plants. they have numerous applications in agriculture industry. bikaverins are polyketides that have toxicity against different organisms because they inhibit respiration. regulation of polyketide and gibberellin synthesis by nitrogen has been intensively studied in fusarium but little is known about their regulation by carbon source. our main interest is to understand the regulation of biosynthesis of these compounds in f. fujikuroi imi 58289. to investigate this regulation the organism was grown in high nitrogen medium under submerged conditions and then transferred to nitrogen-free media having various concentrations of different carbon sources. the gibberellin production was not affected significantly. on the other hand bikaverin was synthesized enormously when sucrose was used as the only carbon source. high production in sucrose required a minimal amount of the sugar, but did not change appreciably above this threshold along a wide range of concentration. the bikaverin synthesis was repressed when glucose coexisted with sucrose in the medium. the effect of the c source on the expression of key genes, cps/ks (copalyl diphosphate synthase/kaurene synthase) for gibberellin and pks4 (polyketide synthase) for bikaverin biosynthesis is currently under investigation. ment of dormancy and loss of viability in seeds with the passage of time, it lacks any systematic propagation from seeds and is typically propagated through rhizomes. this restricts large scale cultivation of this plant. in vitro propagation of plants is an effective means for rapid multiplication of species, in which conventional methods have limitations. in the pesent study we have analysed the role of various growth promoters and the effects of dark and light incubation on germination of n. alba seeds. the results indicate that in vitro propagation of n. alba from seeds can be applied as an efficient method of multiplication. the study was funded by the state planning commisson (dpt) turkey and the university of ankara vide projects no. 120640. this research was performed for developing of biological treatment process of odor gas such as mek, h 2 s, and toluene, which is generated from the food waste recycling process. to establish the operational conditions of odor gas removal by small-scale biofiltration equipment, it was continuously operated by using toluene as a treating odor object. when the odor treating microorganisms were adhered to fibril form biofilter, high removal efficiency over 93% was obtained by biofilm formation. at 400 ppm of inlet odor gas concentration and 10 s of retention time, the removal efficiency was 76 and 93% in first stage reactor and second stage reactor, respectively. however, the removal efficiency remained over 97% at the operational conditions above 15 s of retention time. post soviet countries are going through the transition stage and are extremely sensitive to new technology, economics or social changes and globalization processes. that is why decision-making and system of regulation of the use of gmo's are very sensitive to number of factors. three levels of factors, the most influential to decision-making: global, regional and local; are identified at the research paper. global level depends on external policy of leaders of gmo regulations. usa and eu have the biggest influence on transition countries, though their positions completely differ. as usa was leader in inventing gmos, it is lobbing newly created biotechnological industry. in eu and other european countries lobbing of biotechnological industry was not as strong as in usa. thus, their national law is stricter. world trade organization and international agreements are also part of global level. regional level. geographical position of country is also very important because every regulation system depends a lot on regulation that is implemented in neighboring countries. countries do not exist in vacuum; they are linked territorially, politically, economically and socially with neighboring states. regulation systems of the transition countries in the eastern europe can be divided into three types: those who have no system of regulation of gmo (belarus, romania, hungary and ukraine); who approved some variety that are treated as safe to the market (poland, moldavia and georgia) and who approved all of the gmos (bulgaria, croatia and russia [before 2004]). but even if a country declares not to use gmo it is rather difficult to control import of such products, because of the lack of the testing laboratories, corruption of the state employees, agreements on intellectual property, and institutional country problems. in spite of global and regional tendency of gmos related regulation the most important part is the local level, namely the national regulation system. depending on national level we are choosing priorities at higher levels. as an example of post soviet countries ukraine is taken, as ukraine is one of the largest countries and one of the biggest exporters of agricultural products in europe. research includes the analysis of attitude to gm product, their potential risks and benefits of three categories that influence decision-making the most: farmers, gm experts and non-governmental organizations. recombinant microorganism development for extracellular benzaldehyde lyase production hande kaya 1 , pınar ç alık 1 , tunçer h. ozdamar 2 : 1 iblab, department of chemical engineering, metu, 06531 ankara, turkey; 2 bre laboratory, department of chemical engng, ankara university, 06100 ankara, turkey. e-mail: e119497@metu.edu.tr (h. kaya) in this study, the extracellular production of the benzaldehyde lyase (bal, ec 4.1.2.38) that catalyses the synthesis the enantiopure 2-hydroxy ketones for drug syntheses, by bacillus sp., was aimed. for this purpose, the signal dna sequence of an extracellular bacillus enzyme, i.e., serine alkaline protease, was fused in front of the bal gene (accession number ax349268) from pseudomonas fluorescens biovar i, using pcr-based gene splicing by overlap extension (soe) method. b. licheniformis (dsm 1969) chromosomal dna was used as sap gene (accession number x03341) template for the synthesis of sap signal sequence. bal gene was amplified by using the plasmid carrying bal gene, puc18::bal. thereafter, the signal peptide of sap with its own promoter was fused in front of the bal gene by soe method. the hybrid gene first cloned into puc19 plasmid, thereafter sub-cloned into pbr373, pmk4 and phv1431 shuttle vectors. the escherichia coli-bacillus plasmids carrying the hybrid gene pre(subc)-bal was transferred into bacillus subtilis npr− apr−, and bacillus licheniformis. the influence of the host bacillus species on bal production on a defined medium with glucose was investigated in bioreactor systems. for each of the recombinant (r-) bacillus species, effects of initial glucose concentration on cell growth and bal production were investigated; and, physiological differences and similarities between the wild-type and r-bacillus species are discussed. thereafter, the benzaldehyde lyase production capacities of recombinant e. coli and b. subtilis are compared in terms of cell concentration and bal volumetric and specific activities. for the comparison bal gene was cloned into prseta vector which is under the control of strong t7 promoter and expressed in e. coli bl21 (de3) plyss strain. the variations in by-product distributions with each recombinant organism and yields are also discussed. phosphoketolases (ec 4.1.2.9) are thiamine diphosphate (thdp)-dependent enzymes, that play a crucial role in the pentose phosphate pathway (ppp) of heterofermentative and facultative homofermentative lactic acid bacteria, and of the d-fructose 6phosphate shunt of bifidobacteria. reports affirm that cellulomonas flavigena can use the ppp when is cultured under anaerobic conditions. a genomic library of c. flavigena constructed in -zap express vector was screened. four positive clones were isolated, in vivo excised and the resulting pbk-cmv phagemids, each containing a 4.0 kb insert, were characterized by restriction analysis and dna sequencing. the open reading frame (orf) of the dxylulose 5-phosphate/d-fructose 6-phosphate phosphoketolase gene, xpkl, was located from nucleotide 54 to 2466. the xpkl orf encoded a 804 amino acids-residue polypeptide (xpkl) with a calculated molecular mass of 89,000 da, a value coincident with that estimated by comparative sds-page (about 90,000 da). a putative ribosome binding site (gggagc) is present 11-5 nucleotide upstream of the translational start of the xpkl polypeptide. the c. flavigena xpkl polypeptide sequence was 66% identical to dxylulose 5-phosphate/d-fructose 6-phosphate phosphoketolase from bifidobacterium sp., bifidobacterium gallinarum, gloeobacter violaceus and bifidobacterium adolescentis. this analysis also revealed highly conserved regions. lactococcus lactis is a main diacetyl-producing bacteria by citrate metabolism in dairy products. the transport of citrate in these bacteria is dependent on citrate permease that is encoded by citp gene. previous studies of the citqrp operon in escherichia coli mutants showed that citp message is considerably stabilized in rnase iii mutant. so, in the context of the citrate metabolism research, the characterization of the lactococcal rnase iii enzyme is very important for the dairy industry. rnase iii is an endoribonuclease which has an important role in rrna processing and control of gene expression. with the aim of studying lactococcal rnase iii we have cloned the rnc gene from l. lactis ssp lactis il1403 in the broad host range pls1rgfp vector. this plasmid includes the gfp gene, encoding the green fluorescent protein (gfp), cloned under the control of the pm promoter, that is inducible by maltose. maltose induction of the lactococcal rnc expression showed a 5-fold increase of rnc transcription from this plasmid. activity assays for lactococcal rnase iii were standardized using crude extracts and a substrate specific for b. subtillis rnase iii. the results showed that this substrate was specifically cleaved by lactococcal rnase iii and its activity induced by maltose. lac-rnc was cloned in pet15 vector and the corresponding six-histidine-tagged rnase iii protein was overproduced in e. coli bl21 (de3) strain by iptg induction. the protein was purified by affinity chromatography using hplc system and was shown to be active by in vitro activity assays using the lac-rnase iii specific substrate mentioned above. we have also cloned lactococcal rnc gene and studied its expression in an e. coli rnc deletion mutant ( rnc). complementation assays performed in e. coli demonstrate that the lactococcal rnase iii (lac-rnase iii) is able to process rrnas and to regulate the levels of polynucleotide phosphorylase (pnpase). these results demonstrate that the lactococcal enzyme is able to substitute the ec-rnase iii not only in the rrna processing, but also in the processing of mrnas. the amount of lactococcal rnc transcript in an e. coli rnc strain was 3.3-fold higher than in the wild type strain, suggesting that the e. coli rnase iii triggers the degradation of the heterologous rnc mrnas. the results obtained have shown that lac-rnase iii is an interesting enzyme for biotechnological purposes. objectives: the pharmaceutical and food industry has an increasing demand for selectively glycolized active agents. in our application isomaltose can be synthesized by immobilized dextransucrase, which transfers a glycosyl residue from sucrose (substrate) to glucose (acceptor). as the reaction proceeds, isomaltose can act as an acceptor and is converted into undesired follow-up products called isomalto-oligosaccharides, imos. we investigate on two approaches to avoid imo formation, selective adsorption of isomaltose (ergezinger et al., 2005) and the co-entrapment of dextranase adsorbate, which breaks imos down to isomaltose. results and conclusions: the first part of our research concerns the adsorption of dextranase on bentonite, which complies with langmuir model. at complete saturation (0.8 g g −1 ) our immobilisate exhibits an activity of 16,000 u g −1 . a kinetic analysis does not reveal significant differences between the adsorbed and free form of enzyme (k m,bentonit : 14.1 ± 0.7 m versus k m,free : 13.0 ± 0.7 m). thus, bentonite displays a high binding capacity paired with favorable kinetic properties. beyond that we investigate the activity of dextransucrase in co-immobilisates, which is reduced during coimmobilization due to interactions with the adsorbate. among various co-immobilisates, the one containing dextranase bound to preblotted bentonite imparts the highest activity (40% as compared to control: immob. dextransucrase). the molar yield coefficient of coimmobilisates y isomaltose/sucrose surpasses coefficient of control by 13%. further on we will characterize mass transfer of dextranase substrate into alginate matrix as well as bentonite-dextransucrase interactions. and kefir. a second approach is to use yeast as a production organism to produce natural folates for fortification. here we investigate and discuss the folate content in skq2n, a diploid strain of saccharomyces cerevisiae, when cultured in different media and at different stages of growth. the aim is to gain a basal knowledge of the folate production profile, forms of folate produced and degree of leakage to the surrounding medium, in relation to the culturing medium and physiological state of the cells. danisco innovation, danisco a/s, langebrogade 1, po box 17, dk 1001 copenhagen k, denmark we at danisco a/s (copenhagen, denmark) have revealed a new starch degrading pathway by the discovering several new enzymes and metabolites in fungi and algae. these new enzymes include glucan lyases, dehydratases and tautomerases, which proved to be useful in biocatalysis. these new metabolites proved to be useful as both antioxidants and antimicrobials for food and non-food applications. this pathway is named as anhydrofructose pathway of starch and glycogen degradation. this technology is referred to as the anhydrofructose technology. diet is evolving from nourishing populations via providing essential nutrients to improving health of individuals through nutrition. modern nutritional research focuses on health promotion and disease prevention, on protection against toxicity and stress, and on performance improvement. as a consequence of these ambitious objectives, the disciplines "nutrigenetics" and "nutrigenomics" have evolved. nutrigenetics asks the question how individual genetic disposition, manifesting as single-nucleotide polymorphisms (snps), copy-number polymorphisms (cnps) and epigenetic phenomena, affects susceptibility to diet. nutrigenomics addresses the inverse relationship, i.e. how diet influences gene transcription, protein expression and metabolism. the mid-term objective of nutrigenomics is integrating genomics (gene analysis), transcriptomics (gene expression analysis), proteomics (global protein analysis) and metabolomics (metabolite profiling) to define a "healthy" phenotype. the long-term deliverable of nutrigenomics is personalised nutrition for maintenance of individual health and prevention of disease. the major challenges for -omics in nutrition and health still lie ahead of us, some of which apply to -omic disciplines in general while others are specific for -omic discovery in the food context: (i) the integration of gene-and protein expression profiles with metabolic fingerprints is still in its infancy as we need to understand how to (a) select relevant sub-sets of information to be merged, and (b) resolve the issue of the different time-scales, at which transcripts, proteins and metabolites appear and act; (ii) the definition of health and comfort is less of a clear-cut case than the one of disease; (iii) -omics in nutrition must be particularly sensitive: it has to reveal rather many subtle than a few abundant signals to detect early deviations from normality; (iv) in the food context, health cannot be uncoupled from pleasure, that is, food preference and nutritional status are interconnected. transcriptomics serves to put proteomic and metabolomic markers into a larger biological perspective and is suitable for a first "round of discovery" in regulatory networks. metabolomics, the comprehensive analysis of metabolites, is an excellent diagnostic tool for consumer classification. the great asset of this platform is the quantitative, non-invasive analysis of easily accessible human body fluids like urine, blood and saliva. this feature also holds true to some extent for proteomics, with the constraint that proteomics is more complex in terms of absolute number, chemical properties and dynamic range of compounds present. proteomics in the context of nutrition and health has the potential to (a) deliver biomarkers for health and comfort, (b) reveal early indicators for disease disposition, (c) assist in differentiating dietary responders from non-responders, and, last but not least, (d) discover bioactive, beneficial food components. independent of the context of application, proteomics represents the only platform that delivers not only markers for disposition or condition but also targets of intervention: the only way to intervene in a biological condition and to modulate its outcome is interfering with the proteins involved. it is evident that not only comprehensive analyses with one discovery platform (lateral integration of information) are required but also vertical integration between different -omic levels are indispensable for a deeper understanding of disposition, health, environment and diet (desiere, 2004) . a major "vertical integration issue", to date unresolved, is given by different timescales of transcript production, protein expression and metabolite generation (nicholson et al., 2004) . the transcript machinery usually responds fast to an external stimulus (seconds to minutes), the proteins may be expressed within minutes to hours (and have a halflife from minutes to even months) and metabolites vary significantly during the day and depend on latest dietary input. this means that data, which seem to correlate qualitatively (e.g. reflecting the same pathway), may not necessarily be related time-wise. rather, they may represent different responses at different time points and, possibly, to different stimuli. comprehensive -omic analyses is an essential building block of "systems biology", which can be defined as follows (clish et al., 2004) : systems biology is the comprehensive analysis of the dynamic functioning of a biological system (cell, organ, organism or even ecosystem) at gene, protein and metabolite (or higher organizational) level, achieved by comparison of two defined biological states of this system, typically before and after perturbation. while a comprehensive list of components (genes, proteins, metabolites) of a given biological system is a pre-requisite for this kind of research, the main reasoning for the "system view" is that only information on the interactions between the components gives clues to function of the entire network. a systems biology approach has recently demonstrated the power of proteomics to dissect immunity and inflammation. toll-like receptor recognition and signalling was elucidated and showed, how bacterial "barcodes" are read and interpreted in order to trigger an adapted immune response (aderem and smith, 2004) . in order to address some of the challenging objectives of -omics-driven nutritional research, we have addressed (a) the effect of early antibiotic administration on the maturation of intestinal tissues, (b) protein discovery in human milk, (c) the effects of polyunsaturated fatty acids on gene expression and lipid profile in the liver, and (d) biomarkers for intestinal stress. (a) antibiotics and gut maturation: the effects of early administration of antibiotics on intestinal maturation were assessed at the gene expression level in a rat model. (b) human milk: rapid enrichment and iterative, consolidated identification of immunologically relevant milk proteins was achieved through the employment of restricted-access media and a tailored proteomic strategy (labéta et al., 2000; lebouder et al., 2003; panchaud et al., 2005) . (c) fatty acids and liver transcriptome/lipidome: epidemiological studies have correlated higher intakes of poly-unsaturated fatty acids (pufas) with lower incidence of chronic metabolic disease. the molecular mechanisms regulated by pufa consumption were examined assaying the liver transcriptome and lipid metabolome of mice fed a control and a pufa-enriched diet (mutch et al., 2005) . (d) gut stress markers: as a first step, we catalogued protein expression along the jejunum, ileum and colon of the rat intestine and found gut segment-specific proteins (marvin-guy et al., 2005) . the innovative combination of a neonatal separation model with proteomic analysis allowed us to study, whether early life psychological stress may impact the adult gut neuromuscular protein expression and the approach revealed specific protein biomarkers. omics for engineering lactic acid bacteria willem m. de vos wageningen center for food sciences, wageningen university, the netherlands. e-mail: willem.devos@wur.nl. url: http://www.wcfs.nl/ lactic acid bacteria (lab) are high at-rich gram-positive bacteria that have a well-established record in industrial food fermentations where they contribute to conservation, flavour and texture. in addition, several lab are used as food-grade hosts for the production of enzymes, peptides or metabolites. finally, lab are exploited in functional foods that contribute to the health and well-being of the consumer. a variety of metabolic engineering approaches have allowed for the improvement of many attributes of lab. these approaches have been facilitated by the possibility of uncoupling of growth and metabolite production in lab, the wealth of genetic tools that allow modulation of gene expression in a dynamic range, and the determination of several complete lab genomes (de vos et al., 2005) . we have developed lactobacillus plantarum as a paradigm for lab engineering by experimental and modelling approaches, the application of functional and comparative genomics, and the implementation of other post-genomics avenues (kleerebezem et al., 2003; smid et al., 2005; de vos et al., 2004) . examples of optimizing the production of vitamins and other cofactors, the impact of these engineering approaches on the global transcription and metabolite profiles, and determining the l. plantarum activity in the human host will be discussed. solanum tuberosum (potato) is the fourth major crop worldwide and used for food, feed and biotechnological applications. to fully realize the biosynthetic potential for production of starch, protein and metabolites, we conducted an extensive quantitative profiling of the expressed genes of mature potato tuber. a total of 58,322 sage (serial analysis of gene expression) tags of 19 nt representing 22,235 different tags were analyzed. the 695 tags seen 10 or more times were assigned a tentative function by comparison to homologous genes. contrary to the transcript profile of rice seedlings (gibbings et al., 2003) the storage organ of potato is not dominated by transcripts encoding storage proteins. transcripts for four types of protease inhibitors, a metallothionein and a lipoxygenase were more prominent than patatin isoforms. the lactic acid bacterium lactococcus lactis is used extensively in the production of fermented milk products. during cheese production the bacterium experiences many changes in its immediate environment, as a result of its own reactions. the most severe change is the accumulation of lactic acid, which changes the ph of the medium until growth is totally inhibited. we have focused upon a survey of these dairy related stress responses, as a means of constructing more robust strains. when l. lactis starter cultures are produced in rich media, they will experience an initial period with purine limitation after being added to the milk substrate, a stress condition that in several studies have been found to induce cross resistance towards a number of other stresses. we have analyzed both general purine nucleotide (atp and gtp) and specific gtp limitation in chemi-cally defined medium, using both proteomics and transcriptomics. the differential expression analyses were performed with a custom designed dna microarray of pcr amplified probes. the two stress conditions resulted in very different stress responses, both at the transcriptomic and proteomics level. from a new study on the temporal expression pattern of l. lactis during growth in milk, we present preliminary data showing differential expression of genes and proteins of the purine stress stimulon as well as other stress stimulons. cell physiology of the yeast saccharomyces cerevisiae glucose repression mutants ∆snf1, ∆snf4 and ∆snf1∆snf4 was studied in batch and glucose limited chemostat cultivations. detailed physiological studies were performed on cells grown in batch using glucose, galactose, or glucose-galactose mixture as a carbon source. during growth on glucose-galactose mixtures it was shown that after glucose was consumed, galactose consumption remained repressed for about 15 h in ∆snf1 or ∆snf4 mutants, and for more then 40 h in ∆snf1∆snf4 mutant, whereas it only lasted 6 h in wild-type cells. the global transcriptional response in the glucose repression mutants was studied using chemostat cultures. s. cerevisiae wild type and the mutants were grown in glucose limited aerobic chemostats at a dilution rate of 0.1 h −1 . biological triplicates were performed for each strain. to identify transcriptional responses of the glucose repression mutants, statistical tests, clustering method and a model-driven analysis method were used. the global transcription data analysis experiments showed that genes involved in hexose transport, carbohydrates metabolism, respiration, and signal transduction were differently expressed in ∆snf1 and ∆snf4 mutants comparing to wild type cells. combination of gene expression data and gene-scale metabolic model indicated changes in the metabolic sub-networks among studied glucose repression mutants. genomics technologies have recently been introduced into food and nutrition science for identifying targets of molecular actions of nutrients as well as non-nutrient components of foods. changes in the transcriptome, proteome and metabolome have been determined for assessing the molecular actions of zinc as an essential micronutrient and of flavonoids in processes such as colon carcinogenesis and atherosclerosis. zinc is essential for the structural and functional integrity of cells and plays a pivotal role in the control of gene expression. zinc deficiency effects in human cells and an animal model (rats) were analyzed by microarrays and showed that a low intracellular zinc concentration caused major alterations in the steady-state mrna levels of several hundred target genes-dependent on the tissues studied including liver, brain, muscle, intestine and kidney. proteome analysis from the same samples by 2d-page followed by peptide mass fingerprinting via maldi-tof-ms identified similarly a large set of proteins with altered expression level but allowed a common theme of action to be identified. although pleiotropic in first view, the obtained pattern of zinc-affected genes/proteins may represent a reference for defining the zinc regulon in mammalian cells. flavonoids occurring in large number in plant species are considered protective agents in a variety of processes including inflammation and cancer development. we have studied the effects of around 80 selected flavonoids in a screening program and identified for compounds such as flavon, genistein or quercetin by genomics technologies their putative mode of action in colon cancer models and endothelial cells. as part of a collaborative effort employing human endothelial cells and blood mononuclear cells from a human intervention trial with soy isoflavones (genistein/daidzein) the effects of the flavonoids on the stress-response to oxidized ldl and homocystein was analyzed. a set of markers of anti-inflammatory and anti-apoptotic activity was identified for genistein and daidzein and cell biological studies confirmed that both compounds prevented programmed cells death in stressed endothelial cells. in the food processing industry, unwanted presence of extremely heat-resistant bacterial endospores creates major problems due to their capability to survive classical thermal treatments and their ability to subsequently germinate and form actively growing vegetative cells. screening of spoilage isolates using genomic typing techniques to visualise putative genome-based biomarkers allowed us to classify strains according to the degree of thermal resistance of their spores. in addition, we showed that sporulation in the presence of ingredients rich in calcium ions promotes thermal resistance of developing spores and correlates with the expression of specific (marker) genes (see oomes and brul, 2004) . finally, the molecular program that forms the basis of spore germination has been assessed using genome-wide expression analysis. noticeably genes involved in dna-repair were transiently expressed in germinating wild-type spores. also, surprisingly, it was found that spores contain significant levels of ribosomal and messenger rnas. degradation of these rna molecules upon spore thermal injury was found to be characteristic for their thermal resistance and predictive for their subsequent outgrowth behaviour. this finding is currently being patented. the information on spore presence, predictions of their thermal resistance and process survival chances, is used to structure a process management system to facilitate optimal food quality assurance and allow for real time analysis in case of the need for quality control. the information on spore presence, predictions of their thermal resistance and process survival chances is now being integrated. this is used to formulate the conditions for a process management system with state of the art food production quality assurance, which allows for real time analysis in case of the need for quality control. the interplay of the pectinase spectrum of aspergillus niger as revealed by dna microarray studies elena martens, jac benen, johan van den berg, peter schaap fungal genomics group, laboratory of microbiology, wageningen university, dreijenlaan 2, 6703 ha wageningen, the netherlands. e-mail: elena.martens@wur.nl (e. martens) the saprobic fungus aspergillus niger is an efficient producer of extracellular enzymes many of which show carbohydrate modifying activities. these enzymes have gras status and therefore are widely used in the food and feed industry. after determination of the genomic sequence of a. niger by dsm, it was estimated that only a fraction of the potential of secreted enzymes is currently characterised. database mining using the proprietary genome sequence has resulted in the identification of more then 80 genes encoding enzymes involved in the depolymerisation of the back bone and the site chains of the complex polysaccharide pectin. additional enzymatic activities required to remove methyl and acetyl esters, present in pectin were also observed. by using dna microarrays we have sought to gain insight into the complex regulation of all the genes involved in pectin degradation. a. niger was cultivated on sugar beet pectin and on the monomeric constituents of pectin, viz. galacturonic acid, rhamnose and xylose. subsequently the corresponding transcriptomes were analysed. we will report on our findings concerning the regulation of the expression of the genes involved in the degradation of pectin and its main constituent-galacturonic acid and the consequences for the interplay of the encoded (novel) enzymes. since reactive oxygen species (ros) are formed in all living organisms a wide range of antioxidative enzyme systems are present to keep the system in balance. when an animal is slaughtered the cellular anti-oxidative capability is reduced, resulting in an accumulation of ros followed by an increased oxidation of dna, lipids and proteins. generally lipid oxidation is a well-known problem, causing increased rancidity during prolonged storage, of especially fatty fish. the implications of protein oxidation are, however, more unclear also in respect to quality decay of fish. protein oxidation causes a wide variety of amino acids modifications, where of the most studied is carbonylation of proline, argenine, lysine or threonine. these carbonyl groups can be labelled with 2,4-dinitro-phenylhydrazine. combining two-dimensional gel electrophoresis with immunoblotting enables the detection of carbonyl groups for each single protein. the results presented here, reveal that both during frozen storage and tainting of rainbow trout protein oxidation/carbonylation increases, furthermore there is an increase in oxidation/carbonylation for distinct proteins. anne-marie neeteson european forum of farm animal breeders, benedendorpsweg 98, 6862 wl oosterbeek, the netherlands society is concerned about food, animal welfare, food safety, new technologies, scientists and industry. these elements are all present in genomics for farm animals. therefore, it is important to build awareness in scientists and industry, start a dialogue with stakeholders and society, and to be transparent in a pro-active way. this paper will address the issues at stake for scientists and industry, when it comes to genomics and animal health. it will combine the results of imperical, ethical and sociological efforts in three eu funded projects. (c) the proper use of genomics in relation to infectious diseases in production animals, and the role of the scientist in the development in new technologies in this field, are being addressed in european animal disease genomics network of excellence (ead-gene, http://www.eadgene.org/). some observations are that: (1) genomics does not concern changing the gene. however, acceptability of any discovery dealing with living beings and edible products, is not obvious just like that! animals have a symbolic and emotional load. (2) genes are still related to eugenics, in the mind of people. genes as such cause reluctance, but it is seen as positive if the use of medication can be reduced, and if animals will be better resistant to disease. (3) consumers are in favour of consumer education, compulsory labelling and the imposition of minimum standards. the inclination to pay more for foods produced according to desired standards relates closely to income level. (4) animal welfare is the major issue citizens mention as a concern. the focus of breeding organisations on productivity should be counterbalanced by serious attention to the animal's needs in order to avoid unnecessary negative impact on the welfare of the animals. (5) when technical specialists and lay people communicate, they tend to use different languages: they use the same words, but with rather different interpretations. so transparency of breeding practices and clear definitions of terminology will be essential for effective communication among all stakeholders. (6) food safety and human health are the major concern for most people, when it comes to making a choice. during the latest decades research within the field of animal genomics has in general been following the same strategies as those used within the field of human genomics, although with much less resources. the porcine genome has been characterized intensively through the development of linkage maps, comparative maps and physical maps. until a few years ago it had not been anticipated that it would be possible to embark on whole genome sequencing of animals genomes. however, because of technological developments and much lower costs for sequencing, several animal genomes have now been assembled/are on the way to being assembled. the initial step towards sequencing the porcine genome was taken by the sino-danish pig genome project. the efforts within this project have now generated approximately 3.84 million genomic shotgun sequences and 700.000 expressed sequence tags (ests). the shotgun sequences have been included in a three-species alignment to make an initial evolutionary analysis. the results show that pig is much closer to human than mouse is. the ests represent 5 -end sequences from a total of 98 non-normalized cdna libraries. based on assembly and annotation of the ests the structure of the porcine transcriptome has been analysed. the relevance of assembling the porcine genomic sequence is justified both from the perspective of sustainable animal breeding and from the fact that the porcine model is an important research platform because of the anatomical, physiological, biochemical and metabolically similarities with man. examples of functional genomic studies both aimed at sustainable animal breeding and aimed at exploiting the pig as a model for medical studies will be discussed. genomics refers to global, systematic and high throughput approaches that allow collecting large amount of data and thus offer new possibilities for analysis and understanding biological processes. we will present some new knowledge related to reproduction in farm animals resulting from three different strategies. (1) functional analysis of gene and protein expression: the transcriptome and the proteome analysis allowed to identify new genes and proteins whose expression is associated with processes of ovarian follicular growth and atresia as well as oocyte maturation in bovine and porcine species, maturation of spermatozoa in the different compartments of epididymis. farm animals produce food as cost effectively as possible, however this may have negative side effects for their health and welfare. trade off processes between production on one hand and reproduction and health on the other hand play a crucial role. the principles of selective breeding for the best of naturally occurring variation has proven to be able to balance an increased level of production and quality of life for the animal. every year, the economic value of the genetic gain achieved by the breeders and carried over to the producers is 1.5% of the economic value of eu farm animal production. consequently, a conservative estimate of the gain from animal breeding is, every year d 1.2 billion in europe. recent developments, such as the sequencing of the genomes of the human, chicken and cow, together with high throughput laboratory techniques, means that there are new opportunities to enhance quality of life. the goal of this paper is to give an overview of the options offered by genomics for enhanced quality of life with focus on identifying relevant gene variants and technologies for large scale tracking and tracing. selective breeding for the best of naturally occurring variation remains the same as in traditional systems, but by pinpointing the relevant gene variants along genomics it is possible to identify directly the animals best selected for high production without comprising health and welfare. the combination of full genome sequences, software tools, study of functional physiological processes cost-effective high-throughput snp genotyping and comparative mapping have the (proven) potential to identify relevant gene variants, e.g. pork color, boar taint, general disease resistance. functional mutations have direct option of application in breeding programs. unfortunately this is not the case for genetic markers due to cost of genotyping and inconsistent phenotypic effects. new technologies for snp genotyping are cost effective and enable large scale genotyping (1.000 of animals/day). a selection of the best technology and strategic use of these opportunities enable tracking and tracing. the application of this technology offers new opportunities for quality of life, both for animal and humans. background: studies have shown that prebiotic and probiotic consumption alters the gastrointestinal flora, modulates the immune system, inhibits genotoxicity and has a protective effect on colon carcinogenesis. however, the effect of synbiotic consumption on these parameters in subjects at risk of colon cancer has not until now been investigated. aim: to determine if a synbiotic (prebiotic and probiotics together) modulates cancer risk biomarkers in human subjects at risk of colon cancer. methods: a 12-week randomised, double blind, placebo controlled, ethically approved trial of a food supplement containing lactobacillus gg, bifidobacterium bb-12 and raftilose synergy 1 (prebiotic) was performed in 37 colon cancer subjects who had undergone 'curative resection'. faecal and blood samples were obtained before (t1, week 0) midway through (t2, 6 weeks) and following intervention (t3, 12 weeks). rectal biopsies were obtained at t1 and t3. the effect of synbiotic consumption on the faecal flora was assessed using standard plate count techniques. genotoxic damage was measured in single cells derived from biopsies using the comet assay. fw was prepared by diluting faeces 1:1 in dmem, ultracentrifugation and sterile filtration. the genotoxic (comet assay) and cytotoxic potential (almar blue assay) of fw was determined. peripheral blood mononuclear cells were isolated from blood and cytokine production in vitro assayed by elisa. natural killer cell cytotoxic activity and the phagocytic and respiratory burst activity of monocytes and granulocytes in whole blood were determined by flow cytometry. results: in the synbiotic group faecal numbers of bifidobacteria significantly increased (p < 0.001) and lactobacilli increased although not significantly (p = 0.0674) while coliforms decreased (p < 0.05). enterococci, clostridium perfringens and bacteroides were unaffected. in the placebo group bifidobacteria decreased (p < 0.001), the other bacterial groups were unaffected. in biopsies genotoxic damage was increased in the placebo group at t3 versus t1 (p = 0.0301) but was unchanged in the synbiotic group. the genotoxic and cytotoxic potential of fw was unaltered. synbiotic consumption significantly increased (p < 0.05), ifn-␥ production by pbmcs but il-2, il-10, il-12, and tnf-␣ production was unaffected. natural killer cell, phagocytic and respiratory burst activities were unaltered. conclusion: synbiotic consumption did not have a strong immunomodulatory effect on the systemic immune system in this study, nor did it influence the genotoxic and cytotoxic potential of fw. however, synbiotic consumption altered the composition of the gut flora to a more beneficial composition as well as protecting against genotoxic damage in vivo, suggesting a protective effect of synbiotics against colon carcinogenesis. emmaårsköld, malin svensson, halfdan grage, peter rådström, ed w.j. van niel applied microbiology, lund institute of technology, lund university, p.o. box 124, sweden lactobacillus reuteri is used today in a variety of dairy products as a probiotic bacterium. several lactic acid bacteria have the ability to produce different kinds of exopolysaccharides (eps), which have the potential to be used as an alternative biothickener. however, the yield of eps is too low to be profitable in the food industry. to optimise the environmental conditions for eps formation a l. reuteri strain was chosen for a factorial design study. the factors used in this experiment were temperature (30-43 • c), ph (4.5-6.5) and sucrose concentration (50-150 g/l); for each factor three different values were chosen. the strain was grown in batch mode using a semi-defined medium at constant ph and sucrose as the carbon source. the results obtained with 27 fermentations revealed that the highest eps formation was found at 37 • c, ph 4.5 and 100 g/l of sucrose. sucrose did not further affect the eps formation above a concentration of 100 g/l. temperature and ph were significant for the eps formation, but only temperature was significant for growth. a central composite design study was chosen for further optimization of the ph and sucrose concentration for maximum eps formation. also the gene expression of the sucrase enzyme responsible for eps formation was investigated using qrt-pcr. the data were used to develop a model for growth and eps formation. dendritic cells (dc) play a pivotal immune regulatory role in the th1, th2 and treg cell balance. dc are present in the gut mucosa and may thus be target for modulation by gut microbes. here, we screened a large panel of human gut-derived lactobacillus and bifidobacterium spp. for dc polarizing capacity: bone marrow-derived murine dc were exposed to lethally irradiated bacteria and cytokine and dc surface markers were analyzed. substantial differences were found among strains in their capacity to induce proinflammatory cytokines, while the differences for anti-inflammatory cytokines were less pronounced. bifidobacteria were weak il-12, il-23 and tnf-␣inducers, while both strong and weak cytokine-inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12, il-23 and tnf-␣ production induced by otherwise strong cytokine-inducing strains, while il-10 production remained unaffected. those lactobacilli with greatest capacity to induce il-12 were also most effective in up-regulating surface markers. surface marker up-regulation was however reduced in the presence of weak il-12-inducing strains. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. cell surface-associated glycolytic enzymes from lactobacillus plantarum 299v mediate adhesion to human epithelial cells and extracellular matrix proteins s.m. madsen, j. glenting, a. vrang, p. ravn, h.k. riemann, h. israelsen, m.r. nørrelykke, a.m. hansen, m. antonsson, s. ahrné, h.c. beck bioneer a/s, hørsholm dk-2970, denmark among the main selection criteria of lactic acid bacteria for probiotic use, the ability to adhere to intestinal epithelial cells, mucus, or extracellular matrix proteins is considered important. using a proteom based approach we identified a group of novel surface proteins that are non-covalently bound to the cell wall of the probi-otic bacterium lactobacillus plantarum 299v. surface proteins were extracted and analysed by gel electrophoreses followed by mass spectrometry analysis. the surface proteins included glycolytic enzymes like, e.g glyceraldehyde 3-phosphate dehydrogenase, which usually is a typical intracellular enzyme. a collection of lactobacillus species was screened and the phenomenon of surface-associated glycolytic enzymes was found in many of the analyzed species. this is to our knowledge the first example of surface-associated glycolytic enzymes in probiotic bacteria. however, in pathogenic bacteria these enzymes are well known and their surface localization is involved in adhesion to human epithelial cells and invasion. we suspect that the presence of these enzymes on the surface of probiotic bacteria could prevent adhesion of pathogenic bacteria and possibly also involve other probiotic activities such as immune modulation. binding studies showed that the surface-associated glycolytic enzymes of lb. plantarum 229v were able to bind to caco-2 intestinal cells and extracellular matrix proteins like fibronectin. scientific and industrial interest on exopolysaccharides (eps) synthesized by microorganisms and on their chemico-physical properties has been quickly growing in the last years. many strains of lactobacilli produce eps allowing them to adhere to human mucosae and therefore to have probiotic effects such as the stimulation of immune response and even antitumoral activity of these molecules has been claimed. futhermore prebiotic actions of eps beneficially affect the human host health improving the properties of indigenous microflora. in this research we have studied two novel interesting lactobacilli strains: lactobacillus plantarum dsmz 12028 and a particular human isolated strain of lactobacillus crispatus that have probiotic potentialities and are good eps and l(+)-lactic acid producers. the aim of this work has been to characterize these strains and their metabolites (eps, organic acid and bacteriocins), to state them as probiotics and to study their adhering ability on human cells. we have studied the physiology of l. plantarum and l. crispatus in shaking flasks as well as their optimal fermentation conditions to obtain high cell density cultures suitable for use as starters in the food industry and eventually for probiotic preparations. fermentation experiments have been performed in a bioreactor equipped with microfiltration (mf) modules using a semidefined medium and various carbohydrates in different culture conditions (aeration, temperature). the kinetic of eps production has been followed and according to results both strains have shown a growth-related production ranging from 200 to 400 mg/l. in vitro studies concerning the ability of these strains to adhere to human mucosae are in progress as well as the structural characterization of the exopolisaccharides. previous studies have shown that compression coating improves the storage stability of freeze-dried lactobacillus acidophilus, although this stability is related to the degree of cell injury, which in turn is related to the compression pressure used. compression coating has also been found to improve the survival of freeze dried l. acidophilus during exposure to simulated gastric fluid (sgf). the aim of the present work is to create a compression coated l. acidophilus formulation, with targeted release at the terminal ileum and beginning of the colon in the human gastrointestinal tract. dissolution studies were performed using a phosphate buffer with a ph of 2 and 6.8, to simulate gastric fluid and intestinal fluid (sif), respectively. cell viability was monitored using multi-parameter flow cytometry (mpfc), together with traditional cfu/ml counts. mpfc was used to identify live, dead and stressed cell populations, using the fluorescent stains propidium iodide (pi), 3,3 -dihexylocarbocyanine iodide (dioc 6 (3)) and to-pro-3. results show that an enteric coating material, eudragit l100-55, is both suitable for compression coating, and enhancing the survival of cells when exposed to sif. pectin usp 100 has also been shown to promote targeted release of the cells. the opium poppy papaver somniferum contains more than 80 tetrahydrobenzylisoquinoline-derived alkaloids. it is the source of the narcotic analgesics codeine and morphine, which accumulate in specialized internal secretory cells called laticifers. in the aerial parts of the plant, the laticifer cells are anastomosed, forming an articulated network. laticifers are found associated with the vascular bundle in all plant parts. the morphinan alkaloids morphine, codeine and thebaine are found both in roots and in aerial plant parts and specifically accumulate in vesicles within laticifers. the benzo[c]phenanthridine alkaloids sanguinarine and 10-hydroxysanguinarine are found in root tissue. the syntheses of sanguinarine and of the tetrahydrobenzylisoquinoline latex alkaloid laudanine are completely understood at the enzyme level. nearly all enzymes of morphine biosynthesis have also been described. in more recent years, cdnas encoding enzymes of alkaloid biosynthesis in p. somniferum have been isolated and characterized. the cell-specific localization of several of the enzymes of morphine, sanguinarine and laudanine biosynthesis has also been described. with knowledge of many of the gene of alkaloid formation and their sites of expression, the metabolic engineering of p. somniferum for tailored alkaloid profiles is now being undertaken. an agrobacterium tumefaciens-based transformation and a regeneration protocol have recently been developed specifically for narcotic tasmanian cultivars. the various cdnas encoding genes of alkaloid biosynthesis in p. somniferum are being systematically reintroduced into the plant to achieve engineered plants with altered alkaloid profiles. the first results have now been obtained with sense and antisense genes stably expressed in a regenerated tasmanian cultivar. the ultimate goal of exploiting the genes of alkaloid biosynthesis is to produce transgenic medicinal plants of specific alkaloid content that would facilitate commercial production and improve our understanding of the factors that regulate biosynthesis as well as provide experimental systems with which to investigate the ecological role of alkaloids in planta. integrated transcript and metabolite profiling for gene discovery in plant natural product pathways richard a. dixon, lahoucine achnine, bettina deavours, mohammed farag, marina naoumkina, lloyd w. sumner plant biology division, samuel roberts noble foundation, ardmore, ok 73401, usa the rich diversity of chemical structures found in the plant kingdom arises in large part from a limited number of basic chemical scaffolds (e.g. terpene, polyketide) that are modified by a limited number of chemical substitution types (hydroxylation, glycosylation, acylation, prenylation, o-methylation, etc.) . much of the diversity is brought about by the substrate-and/or regio-specificities of the substitution enzymes. in contrast to the large collections of gene sequence and transcript level data available on-line, little detailed information exists on the plant (secondary) metabolome. promiscuity of substrate specificity in vitro may complicate attempts to assign functions to genes of secondary metabolism accessible to researchers through various cdna library collections. using the isoflavonoid and triterpene pathways in medicago species as examples, we describe how integrated metabolite and transcript profiling approaches can aid functional genomics, help explain metabolic regulation, and provide tools for assessing the impacts of genetic modifications in plant secondary metabolism. focused and non-targeted approaches were used to assess the impact associated with introduction of new high flux pathways in arabidopsis thaliana by genetic engineering. transgenic a. thaliana plants expressing the entire biosynthetic pathway for the tyrosine derived cyanogenic glucoside dhurrin as accomplished by insertion of cyp79a1, cyp71e1, and ugt85b1 from sorghum bicolor accumulated 4% dry-weight dhurrin with marginal inadvertent effects on plant morphology, free amino acid pools, transcriptome and metabolome. in a similar manner, plants expressing only cyp79a1 accumulated 3% dry-weight of the novel tyrosine derived glucosinolate, p-hydroxybenzylglucosinolate with no morphological pleitropic effects. in contrast, insertion of cyp79a1 plus cyp71e1 resulted in stunted plants, transcriptome alterations, accumulation of numerous new glucosides derived from detoxification of intermediates in the dhurrin pathway, and in loss of the brassicaceae specific uv protec-tants sinapoyl glucose and sinapoyl malate as well as kaempferol glucosides. the accumulation of new glucosides in the plants expressing cyp79a1 and cyp71e1, was not accompanied by induction of glycosyltransferases, demonstrating that plants are constantly prepared to detoxify novel xenobiotics. the pleiotrophic effects observed in plants expressing sorghum cyp79a1 and cyp71e1 were complemented by retransformation with s. bicolor ugt85b1. accordingly, insertion of high flux pathways directing synthesis and intracellular storage of high amounts of natural products is achievable in transgenic plants with marginal inadvertent effects. arabidopsis thaliana-distinct function in gene transcription dynamics? jeppe madura larsen, brian stougaard vad, søren mølgaard, kell andersen, mads n. davidsen, klaus d. grasser department of life sciences, aalborg university, 9000 aalborg, denmark in recent years it has been shown that introns to some extent can regulate expression in the eukaryotic cell. insertion of introns in the 5 utr in arabidopsis genes has shown to increase gene expression at both rna and protein level. in order to investigate if 5 utr introns have distinct characteristics, we analyse these in the well annotated arabidopsis thaliana genome published by the arabidopsis genome initiative. 12,898 loci annotated with full-length cdnas were analysed and 1989 loci (15.4%) containing 5 utr introns were isolated. we studied if the genes containing these introns showed different patterns in alternative splicing and gene function (gene ontology classification) compared to the remaining genes not containing this intron type. 1802 of the isolated loci (90.6%) contained only one 5 utr intron and these were used for further analysis, where it was investigated if the 5 utr introns had a characteristic size distribution. genes containing transcripts with 5 utr introns were more subjected to alternative splicing (9.63% versus 2.39%) and had a tendency to be more involved in cell regulatory functions compared to genes without this intron type. it was also found, that 5 utr introns was characteristically size-distributed. we identified thee predominant sizes of approximate 110, 270 and 390 bp compared to only one for orf introns. this suggests widespread multiple splicing events in 5 utr introns. the results presented here suggest that 5 utr introns have distinct characteristics and function in gene transcription dynamics. cytochrome p450 monooxygenases appear to be involved in the biosynthetic pathways of a large variety of primary and secondary metabolites in microbial, animal and plant cells. in particular, cytochrome p450 scc catalyzes the conversion of cholesterol into pregnenolone-the precursor of all steroid hormones in mammalian steroidogenic tissues. cytochromes p450 are also involved in the biosynthesis of different plant steroid derivates that play important role in regulation of plant growth and development. therein, investigation of possible influence of cytochrome p450 scc expression on plant regulatory system is of a great interest. this report devoted to the investigation of transgenic tobacco plants, which have been generated by the transformation with recombinant plasmid pgbp450f constitutively expressing cyp11a1 cdna of the bovine cytochrome p450 scc . the transgenic state of the plants was confirmed by southern blot analysis. transgenic plants are phenotypically different from the control ones. in particular, they obtained a substantially higher growth rate and are larger than wild type plants. we have demonstrated that incubation of fragments of the transgenic plants leaves in [ 14 c]-labeled cholesterol containing medium results in formation of the radioactively labeled product with chromatographic mobility corresponding to pregnenolone. the presence of this metabolite in the steroid fraction of lipid extracts obtained from the transgenic plants leaves was confirmed by gas chromatography mass spectrometry (gc-ms) method. the data obtained indicate that cytochrome p450 scc synthesized in transgenic plants displays its specific catalytic activity. biotechnological production of glucose isomerase enzyme with streptomyces olivochromogenes for production of fructose syrup from hydrol m. hashemiravan, a. sadat barikani department of food science and technology, azad university (pishva, varamin unit), institute of food science and agriculture, tehran, iran the use of glucose isomerase for isomerization and production of fructose syrup was performed by selected industrial strain of streptomyces olivochromogenes ptcc 1457. growth of microorganism and production of enzyme in different culture media was studied, and effects of different parameters such as phosphate and aeration was evaluated. the growth of microorganism at 28 • c, caused a production of high amount of enzyme. the production of enzyme was considered in two culture media (a and b). medium (a) was selected for the higher production amount of enzyme. the highest amount of enzyme production was seen in medium a, which was 36.4 giu/ml, after 80 h. the use of baffles in culture flasks, increased the amount of enzyme production, four times more. the production of enzyme was increased, 1.25 times more, in phosphate deficient medium. the cells containing enzyme (intra cellular glucose isomerase) was separated by centrifuge, and extraction and release of enzyme was performed by ultra sonication, that is a physical-mechanical method. this method released about 89.9% of total intracellular enzyme. the best length of time for sonication was found to be 4 min. experiments showed that optimum ph and temperature of the enzyme were 7.5-8 and 80 • c, respectively. the highest activity of the enzyme was observed at ph 5.8 for up 120 min. at the time of isomerization reaction the existence of magnesium ions showed to be necessary and omission of this ion cause a decrease of enzyme activity in isomerization process, but this effect was not necessary for the enzyme activity results showed that treatment of glucose syrup at temperatures of 40, 60 and 80 • c, by the enzyme, caused 48%, 50% and 52% of isomerization, respectively. efficiency increase of high acetic acid production with the use of acetobactereace iranian native strains mutation m. hashemiravan 1 , a. alirezasadat barikani 2 : 1 department of food science and technology, azad university (pishva, varamin unit) institute of food science and agriculture, iran; 2 young iranian researchrer's club, tehran, iran the first step in the present research is the isolation of acetobactereace native strains from fruits (such as grape or apple) and fresh vinegar. this separation has been done with the use of effective isolation methods and optimized mediums. ten strains were isolated effectively, the bio chemicals test were performed, each of them were detected, classified and nominated with a special code. two methods have been used for mutation: (a) mutation with the ultraviolet radiation; (b) mutation with nitrous acid. in the first method the microbial cells were treated with us radiation for different periods of 15, 30, 45 and 60 s, and the effect of the uv mutation was assessed. as a result, the period of 45 s was determined as the optimum mutation periods. in the second method, the microbial cells were first washed with the acetate buffer 0.2 m with the ph of 4.5, then 10 ml nitrous acid 0.07 m was added and was mixed for 2-10 min. finally the sampling was done in the periods of 2, 4, 6, 8 and 10 min and was transferred to a plate containing the medium of ethanol-phenol red-agar. the two methods have been compared with each other. each method has its own advantages and disadvantages. the mutation pathway in method (b) is more stable and conducted, while mutation with the uv radiation method, change the position of thiamine-cytosine bases absolutely randomly. finally, the best mutant site was inoculumed with the medium containing alcohol 16%, acetozyme gz 0.6 g/l, acetozyme d 1 g/l, acetic acid 1.5% and 1 l double distilled water. the acetic acid was produced in the rate of 16 g/100 ml. also the mutant strains were detected with scanning electron microscope (sem) and interesting photographs were taken from mutant cell. the performed experiments were planned with the use of taguchi statistical method (qualitek 4). this research has been performed in the irost as the thesis of ph.d. in food biotechnology industry. isotachophoresis has almost exclusively been applied for contracting and stacking samples ions before zone electrophoretic separation of proteins. this study attempts to apply microfluidic isotachophoresis (itp) as a high resolution analytical method for proteins. beta-lactoglobulin and other milk proteins with slightly different pi were labelled with fluorescent red 646 and analysed by the micralyne tk system using microfluidic glass chips, either with simple cross (sc) or double cross (tt) injection or designed 2d-itp-cze chips with double tt injection and sc for transfer to the second dimension cze channel, efficiently non-covalently coated with 0.6% (w/v) epoxy-polydimethylacrylamide to lower electroendoosmosis. capillary zone electrophoresis (cze) in borate or phosphate buffer was reproducibly perform for more than 75 consecutive runs using upchurch tm reservoirs glued to the wells to enable larger buffer volumes and greater run-to-run stability. finally, isotachophoretic anionic separation of the proteins were done using phosphate (ph 8.1) or chloride (ph 8.5) as leading ion and -amino-caproic acid (ph 8.9) as terminating ion. the effect of narrow cut ampholytes as spacers needs further investigations. the perspective aim is to combine the migrating itp separated zones with second dimension capillary zone electrophoresis as a new microfluidic proteomic 2danalysis. genotypical differences affecting the response of pisum sativum to differing boron/iron applications e.e. hakki, u. zeynep, m. hamurcu, a. tamkoc, m.b. babaoglu, s. gezgin department of field crops, faculty of agriculture, selcuk university, kampus, konya 42079, turkey. e-mail: eehakki@selcuk.edu.tr (e.e. hakki) boron and iron are among the microelements required for the proper development of the vegetative and generative tissues of plants. though iron is present in high amounts in almost all soil types, its bioavailability to crops is extremely reduced, hence most of the plants face an iron defficiency problem and while on one side crop productions effected, on the other hand the nutrition problems come up to human through contagious nutritional chains. boron is also among the most problematic micronutrients of the major crop plantation areas of turkey. both defficiency and toxicity problems exist in a total of about 50% of the central anatolian soil where pea is among the legumes cultivated. application of varying levels of boron and iron combinations in greenhouse and the analysis of plant aquisition via icp-aes as well as determination of the effects of the element combinations both in morphological and molecular levels are the aim of our studies. the genetic bases of the response differences of plant genotypes to b and/or fe, were investigated through the applications of molecular marker techniques. considerable growth rate and stem size differences were detected within the parents (wild-type versus cultivar) and the f9 plants. the presence of efficient genotypes to high micronutrient levels are expected to help us increase the cultivation of the crop in problematic areas as well as in exploring the molecular bases of the microelement uptake mechanisms. increase in sulfite production by accelerating sulfate uptake in brewing yeast t. fujimura, y. kodama, y. nakao, n. nakamura, w. miki institute for advanced technology, suntory ltd., mishima-gun, osaka 618-8503, japan sulfite plays a role as an antioxidant, which stabilizes beer flavor. therefore, it is important to control the sulfite concentration during fermentation. sulfite is produced as an intermediate in the sulfate assimilation pathway in yeast. we have already reported that over-expression of a lager yeast-specific ssu1 gene, encoding a sulfite efflux pump, leads to increase of sulfite production (fujimura et al., 2003) . in the present work, we have clarified that there are two types of sul2 genes (scsul2 and non-scsul2) each encoding a high affinity sulfate permease in lager brewing yeast. eighty percent and 86% identity are found by comparing the dna sequences and the deduced amino acid sequences, respectively. a comparative functional analysis of the two genes has been performed aimed at achieving further increases in sulfite production by accelerating sulfate uptake. over-expression of scsul2 and non-scsul2 has been achieved by transformation of lager brewing yeast, saccharomyces pastorianus. experiments have been done with and without expression of non-scssu1. the resultant transformants have been evaluated by fermentation tests in wort. over-expression of either scsul2 or non-scsul2 failed to show significant effect on sulfite formation. a combination of over-expression of non-scsul2 and non-scssu1 resulted in two-fold higher sulfite production compared with overexpression of only non-scssu1 and four-fold higher compared with the parental strain. these results suggest that the non-sc-gene types significantly contribute to sulfite production in lager brewing yeast. , t., et al., 2003. functional phytases (myo-inositol hexakisphosphate phosphohydrolase) catalyse the release of phosphate from phytate (myo-inositol hexakisphosphate), the predominant form of phosphorus in cereal grains, oilseeds and legumes. possible applications of phytases have been suggested in animal nutrition to increase mineral bioavaliability and to decrease phosphate pollution in area of intensive life stock management and in human health. zea mays is one of the cereals that contain high amount of phytate as the major phosphate storage compound. over 200 bacteria were isolated and screened for phytases from the halosphere, rhizosphere and endophyte of malaysian maize plantation. the phytase activity of the isolates was screened by a modification of the ammonium molybdate method. the highest extracellular phytase activity was detected from bacteria that isolated from the endophyte of the maize root. in this paper, results for 24 isolates chosen for media, temperature and ph optimization will be presented. production of plant proteinase from jack fruit seeds (artocarpus integrifolis) and its influence on rheological and sensory characteristics of low fat yogurt el-sayed el-tanboly dairy sciences department, national research centre, dokki, cairo, egypt adding a proteolytic enzyme extraction from jack fruit (artocarpus integrifolis) in combination of fermentation process in low fat yogurts manufacture was tried to improve yogurt flavour and rheological properties. experimental yogurts milk contained control, 3.9 (t1), 7.8 (t2) and 11.7 (t3) units/ml milk from crude extracts of plant proteinase. the ph of the product treated with crude proteinase was lower than the control. however, the rate of acidity development during storage slightly increased with increasing the addition of crude proteinase level and progress of storage period of yogurt. the proteolytic activity of all yogurts gradually increased until the end of storage period (15 days). yogurts made from milk treated with crude proteinase preparations were less firm compared with control at all storage periods, where t3 showed more less firm after 15 days of storage being 20.17 g/100 g. generally, increasing units of plant proteinase preparations decreased the firmness. on the other hand, yogurt made from milk pretreated with plant proteinase had higher syneresis, and apparent viscosity than the untreated product. the greatest viscosity was found in t2 and t3 of 433 and 479 mpa s, respectively, compared with control of 299 mpa s at 15 days storage. the results indicated that there is an inverse relationship between the amount of units of crude proteinase preparations and susceptibility of yogurt to syneresis. the t2 gained the highest scores (85 points) followed by the control (81.5 points) after 15 days of storage, while yogurt of t3 showed a low scoring being 75. from the foregoing results, it is recommend to use jack fruit (artocarpus integrifolis) as a source of plant proteinases and utilize it to develop a high quality yogurt at a level of 7.8 units of plant proteinases/ml milk. an effective process for the chemical-biotechnological utilization of distilled white lees was studied. a first treatment with hydrochloric acid allowed the solubilisation of tartaric acid. the influence of temperature, amount of hcl and reaction time were considered through an experimental design. under the optima conditions 77 g/l from white distilled lees and 45.6 g/l from red distilled lees were recovered. the tartaric acid was precipitated as calcium tartrate so that it can be isolated from the rest of the raw material compounds. the solid residue was used as an economic nutrient for lactic acid production by lactobacillus pentosus using trimming wastes as substrate. the lactic acid concentrations and volumetric productivities achieved were similar to those obtained using distilled lees without tartaric acid recovery as nutrient. toasted wine was traditionally produced in galicia, northwest of spain. nowadays this technique is being recovered. grapes after harvesting are air dried in order to concentrate sugars, acids and flavor compounds. raisings are pressed to obtain a must with high sugars concentrations. two different grape wines were prepared concentrating the sugars up to 37 and 57 brix, respectively. in order to get a better knowledge of the problems involved, synthetic media simulating the grape musts were prepared. theses musts were used to optimize the initial sugar concentration, the amount of nutrients required, the optimum temperature to carry out the fermentation and the influence of the type and amount of yeast. under the best conditions some fermentations with grape must were carried out to produce wines with intense aroma and flavor notes and high residual sugar concentrations. in this studies, bacillus sp. e1 strain was isolated from koreanstyle fermented soybean paste and it was producing the biological response modifier (brm). the brm activated the b cell selectively. it was identified the bacillus licheniformis e1. the brm was purified by ion-exchange chromatography and gel filtration. chemical properties of brm: molecular weight of brm was estimated to be about 1,594,000 da. sugar content of brm was 33.0% (w/w) and glucosamine (35.1 mol%) was the high level. protein content of brm was 4.3% (w/w) and serine (17.2 mol%) was the high level. infra-red absorption spectrum was showed the characterization of glycoprotein. biological properties of brm: the brm which isolated from fermented soybean paste was similar to that of bacillus licheniformis e1 by immuno-fluorescence assay. we confirmed that the brm was capsular substance of b. licheniformis e1. potato nitrogen concentrate (pnc) is a highly viscous liquid with high complex nitrogen content produced from the protein-fraction in potato starch extraction. the concentrated extract is rich in minerals and ␣-amino nitrogen. although ␣-amylase nowadays is mainly produced exploiting bacillus production systems there is still considerable demand for fungal ␣-amylase from aspergillus oryzae origin. the aim of the experiments to be reported here was to investigate, if pnc can replace commonly used complex nitrogen sources in the production of fungal ␣-amylase. the following data have been measured in pnc pretreated by diluting to 1/2 and clarifying by centrifugation. total-n: 8.4 g n/l; ␣-amino-n: 2.8% (w/v) (as glycine); soluble protein (bradford): 51.2 mg/l (as bsa); total carbohydrates: 110.0 g/l; reducing sugars: 5.6 g/l; dry weight: 57.3% (w/w). in the following experiments nitrogen sources were replaced on the basis of their ␣-amino nitrogen content. the carbon source for all experiments was maize starch. the formation of ␣-amylase by a. oryzae atcc 1011 in shake flasks -using pnc (centrifuged or not), yeast extract, malt extract, casein hydrolysate or meat extract -was compared to "standard" cultivation with corn steep liquor. the experiments showed only small differences in ␣-amylase titers using complex nitrogen sources. no remarkable differences were observed in the resulting biomass. in general no differences in enzyme productivity and biomass formation could be seen after 50 h of incubation. especially the bench top bioreactor experiments indicated an optimal fermentation time of about 100 h. cultivations of a. oryzae atcc 1011 were carried out in bench top bioreactors. comparing cultivations in a medium with pnc as the sole complex nitrogen source to one containing csl as such no significant differences both in the formation and amount of ␣-amylase and the fungal growth were observed. thus pnc might be able to replace complex nitrogen sources such as csl or even the more expensive yeast extract and casein hydrolysate in fungal amylase production systems. 1.19) is involved in the metabolism of inositol and catalyzes the conversion of d-glucuronic acid to l-gulonic acid with nadph as a cosubstrate posterior to the oxidation of inositol to glucuronic acid by the enzyme inositol oxygenase. although the yeast sporobolomyces oryzicola (nakase and suzuki, 1986) is not able to grow on inositol as the sole carbon source, intracellular glucuronate reductase can be found in cells grown in a medium containing d-glucuronic acid. the enzyme could be a useful tool in the design of a specific quantitative assay for glucuronic acid, e.g. in so called energy drinks. the organism was grown in media containing either glucose and glucuronic acid or only glucuronic acid and difco yeast nitrogen base. whereas growth on both media was similar in shake flask culture, hardly any growth in either medium was observed in bench top bioreactors. the influence of dissolved oxygen tension was investigated and the relevant data will be shown. the formation of intracellular glucuronate reductase activity by sp. oryzicola is inducible by media containing glucuronic acid. no activity is found in cells grown in a medium containing only glucose as the carbon source. besides the activity against d-glucuronic acid, activities against 5-ketogluconate and -at very low levelsagainst galacturonic acid and the lactone of glucuronic acid were detected. the enzyme activity is stable up to 35 • c. the ph has relatively low influence on the activity against glucuronate, whereas the reduction rate of 5-ketogluconic acid is optimal at ph 7.0-7.5 with significantly lower values at ph 6.0 and 8.0, respectively. data on the kinetics of the conversion of both glucuronate and 5-ketogluconate will be shown. nakase, t., suzuki, m., 1986. j. gen. appl. microbiol. 32, 149-155 . the multiple nutritional and functional impacts of food fermentation on human health have been widely accepted (reddy and pierson, 1994; hugenholtz et al., 2002) . however, the related role of the involved microorganisms to the nutritional effect from the fermented food is still not well defined and the mechanisms involved are still largely unknown. the present study was to investigate iron bioavailability in carrot juice fermented by two selected lab strains, l. pentosus fsc1 and ln. mesenteroides fsc2. after digestion by gi enzymes, the juice was supplied to fully differentiated caco-2 cells to study iron uptake and transepithelial transport by caco-2 cells from the digested juice. our data revealed strain specified changes in iron bioavailability in carrot juice fermented by these two strains. after in vitro digestion with pepsin and pancreatic-bile enzymes, the best yield of soluble iron was from ln. mesenteroides fsc2 fermented juice. surprisingly, the l. pentosus fsc1 fermented juice yielded about five times higher uptake iron as compared to fresh juice, while ln. mesenteroides fsc2 fermented juice was not significantly different from the fresh juice. interestingly, the transepithelial transferred iron across the cell line was however better from ln. mesenteroides fsc2 fermented juice than from l. pentosus fsc1 fermented juice. to summarise, our study showed that level of soluble iron after in vitro digestion does not necessary indicate iron absorption, especially in the case of lab fermented food. data on improved iron uptake from l. pentosus fsc1 fermented juice indicated exiting of promoter(s) for iron absorption in such juice that is not related to the production of organic acids and lowering ph effect. peng zhang, herve vanderschuren, martin stupak, wilhelm gruissem institute of plant sciences, 8092 zurich, the tropical root crop cassava (manihot esculenta crantz) is a major source of food for approximately 1 billion people worldwide. in sub-saharan africa, more than 200 million people rely on cassava as their major source of dietary energy. in many parts of africa and latin america, cassava leaves are a vegetable source for daily uptake. cassava is grown mostly by poor farmers under marginal environmental conditions and in areas where few other crops can sustain competitive yields. the crop is therefore fundamental for subsistence farming and food security, but it is also very susceptible to stresses common in the areas and conditions where it grows. in many parts of africa, reliable cassava production is strongly impacted by infections with the african cassava mosaic geminiviruses (cmgs), a rapidly spreading disease that causes large yield losses. in the coastal areas of east africa, cassava production now is threatened by another devastating disease, cassava brown streak disease (cbsd). cassava plants are also frequently attacked by many pests, such as cassava hornworm and stemborers. several reports also indicate that greater leaf longevity, especially under drought conditions, could be important for increasing yields and/or the stability of production in cassava, as well as improve the access to an important nutrient source. conventional breeding efforts have attempted to address the constraint to cassava production, but with limited success. the new tools of biotechnology can change this situation by offering new approaches to the challenges of cassava. these new technologies have the potential to make cassava much more productive, a better source of nutrients, and profitable to grow, hence, greatly contributing on the sustainable development of tropical agriculture. recently we have developed biotech cassava with value-add traits, including resistance to cassava mosaic virus, prolonged leaf life and insect resistance. new strategies are also explored to increase protein content of cassava storage roots. we are currently undertaking pilot studies with two teams of leading scientists and experts for projects to test acmv-resistant transgenic cassava lines in africa and lines with extended leaf retention at ciat, colombia under field conditions. this development of substantially equivalent improved transgenic cassava lines is part of a larger study to analyze the need, effectiveness and biosafety of biotech cassava for agricultural production. the goal of the pilot studies will be the development and coordination of a broader project that produces important and novel scientific results, valuable information on the need and impact of biotechnology at the subsistence farming level, and a sound scientific basis for the development of guidelines for biosafety assessments and release of transgenic organisms into the environment and agricultural production in africa and latin american countries. this study was conducted to reveal the effects of different pretreatments on obtaining haploid plants by using the anther culture in pepper capsicum annum l. cultivars demre sivrisi and sirena. buds were collected at uninucleate microspore stage. anthers collected from buds were cultured in ms medium containing different hormones and hormone concentrations. experiment results revealed that when sirena anthers were pre-treated cold at +4 • c for 24 h and kept in darkness at 25 • c for a period of 1 week gave good results. in the case of demre sivrisi anthers were pre-treated cold at +4 • c for 48 h and kept in darkness at 25 • c for a period of 1 week gave good results. on the other hand no cold pretreatment to anthers resulted with low embryo formation. similar results were also observed on the anthers kept at 35 • c for 1 week as callus was produced in some petri dishes but no regeneration was observed. as a conclusion, since no cold pretreatment to anthers resulted with low embryo formation it is possible to say that cold pretreatment should be applied to anthers in pepper another culture studies. the yeast d. hansenii ufv-170 was tested in this work in batch experiments in synthetic media at constant initial substrate concentration (100 g l −1 ) under variable oxygenation conditions. to get additional information on its fermentative metabolism, a stoichiometric network was proposed on the basis of the general knowledge available in the literature on xylose metabolism in pentose-fermenting yeasts and the specificities of xr and xdh activities in d. hansenii and checked through a bioenergetic study performed using the experimental data of product and substrate concentrations. it can be stressed that under strongly oxygen-limited conditions xylitol production was negligible, whereas under semi-aerobic conditions maximum xylitol production (p max = 76.6 g l −1 ) and yield (y p/s = 0.73 g g −1 ) were obtained. a progressive decrease in these parameters was observed under fully aerobic conditions, suggesting that xylitol-producing yeasts require limited oxygen conditions, which is species-dependent. the proposed model, which utilizes the experimental specific rates of substrate consumption and product formations, allows estimating the main bioenergetic parameters. besides, it proved to be an effective tool to investigate different metabolic situations and showed how they can influence the flux distribution of the carbon source and the bioenergetics of this biosystem. the effect of disinfectants on fungi anne svendsen, pernille skouboe bioneer a/s, hørsholm dk-2970, denmark prevention of mould spoilage of foods can only be carried out successfully, if the species, which are actually spoiling the food product, are known. a very limited number of fungal species has been associated with the spoilage of each food category. proper disinfection of production facilities is very important to avoid mould spoilage. resistance of moulds to disinfectant treatments are known and different species have shown different response to the same disinfectant. to obtain proper disinfection it is important to know the resistance of the spoilage fungi against different disinfectants. in this study the effect of disinfectants on the spoilage fungi of cheese, rye bread, liver paté and fruit juice was investigated. in collaboration with five food companies the dominating species responsible for spoilage of each food product were isolated and used for testing. commercial disinfectants and disinfectants "under development" were tested. tests were performed in suspension and on surfaces, the methods used were modified after en 1650 and en 13697. considerable variability in fungicidal effect among the species was observed. some disinfectants were ineffective at low temperature. some disinfectants showed different effect in suspension and on surfaces, resulting in an effective kill in suspension and almost no effect on surface. the identification of effective disinfectants in the food industry includes: (1) testing against the specific spoiling species of the food product, (2) testing on surface, not only in suspension, (3) test parameters adapted to the food manufacturing plant. intensified research efforts in recent years confirm the major importance of the microbial flora in the gastro-intestinal tract for human health. ingestion of prebiotic oligosaccharides increases the number of the desirable bacteria like bifidobacteria and lactobacilli in the colon. we are looking at beta-galactosidases from lacto-bacillus spp. for the production of galacto-oligosaccharides (gos) because we speculate that the enzymes of probiotics will form gos with high prebiotic potential. in this present study, purified betagalactosidases of selected lactobacillus strains were used for the production of gos from lactose. different enzyme reactor set-ups, both discontinuous and continuous, were tested and compared. temperatures up to 37 • c and ph values between 6 and 6.5 were required for satisfactory enzyme stability during the process. enzyme source, substrate concentration and the level of substrate conversion were found to be critical process parameters for gos yields and composition. yields of up to 40% (w/w) of total sugars were achieved when the initial lactose concentration was 200 g/l. capillary electrophoresis (ce) and hplc with pulsed amperometric detection were the analytical tools for investigating the influence of reactor type, enzyme source and conversion level on gos composition. the prebiotics market is increasing rapidly and is expected to more than double until 2010 to about 180 million d world-wide. therefore, the development of enzymatic processes on an industrial scale is a high priority goal of our research. starter addition does not always succeed in improving standardisation and quality of the complex sensory properties of traditional fermented foods. in many cases the added strains do not grow as well as the environmental strains present in the production plant. here, a method of geometric simplification (by dichotomy) of a complex ecosystem found on a raw milk livarot (82 strains) was tested on cheese curd. by a limited number of cultures, successively, 40 out of 82, 20 out of 40 and 10 out of 20 strains were selected on the basis of two criteria (i) respect of the taxonomic proportion, (ii) generation by the daughter ecosystems of an odour close to the one of the mother ecosystem. finally a sub-ecosystem of 10 strains gave an odour similar to the one of the more complex mixture. the use of molecular methods (pcr-sscp) permitted to follow the main species growing. mother and daughter ecosystems were characterized by sensory analysis and gc-ms. probably because of an important redundancy of the strain functions, the method was very efficient. this method may permit to improve a lot the set up of mixture of strains and species used in fermented food industry. effect of the dilution rate on the exopolysaccharide production by bifidobacterium longum atcc 15707 c. shene, m. rubilar, s. bravo universidad de la frontera, chemical engineering, av. francisco salazar 01145, casilla 54-d temuco, chile exopolysaccharides (eps) producing lactic acid bacteria are used in dairy industry (cheese and yogurt) due to the rheological properties that these compounds confer to the products. preliminary results also suggest the use of eps as health-promoting (anti-tumor and immunostimulatory actions) ingredients. bifidobacteria are grampositive bacteria natural inhabitants of the gut of warm-blooded animals and man. a number of investigations have shown that bifidobacteria promote host health mainly because of the reduction proliferation of some pathogenic bacteria through acid synthesis. in this work results obtained in the experiments carried out to test the capability of b. longum atcc 15707 to synthesize eps are presented. continuous culture fermentations were carried out at dilution rates between 0.04 and 0.44 h −1 . composition of the culture media was that of the mrs broth. biomass concentration presents higher values (2.9-3.2 g l −1 ) at dilution rates between 0.1 and 0.2 h −1 . biomass growing at these rates is difficult to pellet and adheres to the fermentor walls behavior that was not observed at other growth conditions. eps from cultures grown at these rates were preparated and fractionated. authors wish to thank the chilean conicyt for the economical assistance given through the project fondecyt 1050602. high pressure-low temperature (hplt) inactivation processes were performed on bacillus subtilis vegetative cells at various conditions. at atmospheric pressure, lowering the temperature to as low as −45 • c was found to have minor anti-microbial effects. upon application of high pressure various phase transitions occurred in the microbial suspensions under study. after pressure treatment at 150-450 mpa, cells were plated under optimal conditions to assess cell viability. treatments at 250-450 mpa and −25 • c were the most effective in inactivation. in these cases, ice i-iii solid-solid phase transition was observed. in addition, we hypothesised that intracellular thawing (solid-liquid phase transition) had already occurred while the extracellular surrounding was undergoing solid-solid phase transition. this double effect is suggested to be key in mediating the observed large drop in viability. we speculate that more cells survived after treatment at −45 • c compared to the same treatment at −25 • c because both the extra-and intracellular surrounding remained fully frozen. at −45 • c a solid-solid phase transition was observed when pressure was higher than 350 mpa. a metastable state of ice i was observed at 250 mpa treatment. results from the current study will be presented (see also shen et al., ifset in press) . the data call for a mechanistic evaluation of the effects of hplt as an anti-microbial treatment. such data are currently being gathered and will be used in defining optimal hplt process conditions for the food industry. the influence of saccharomyces cerevisiae, kloeckera apiculata and candida pulcherrima mixed cultures on the selected alcohols formation during model fermentation pawel satora, tadeusz tuszynski department of fermentation technology and technical microbiology, food technology faculty, agricultural university, cracow, poland. e-mail: psatora@ar.krakow.pl (p. satora) for the study five yeast species were chosen, isolated from successive stages of plum fruits spontaneous fermentation: from the beginning (candida pulcherrima, kloeckera apiculata, saccharomyces cerevisiae w4), middle (s. cerevisiae w54) and final fermentation (s. cerevisiae k1). to characterize the potential influence of yeast mixed cultures on the selected alcohols formation, wick-erham synthetic medium (10% glucose) was fermented by mixed cultures of two and three yeast species. after distillation, ethanol, propanol, isobutanol, isoamyl alcohols, hexanol and 2-phenylethanol were determined using gas chromatography. findings were compared with the results obtained after monoculture fermentations. the use of mixed cultures resulted in increasing of glucose utilization rate, ethanol and fusel alcohols formation (except propanol) and decreasing of methanol synthesis. the samples fermented using two yeast species characterized higher (about 10%) amount of volatile compounds in relation to monocultures. it takes note of especially high level of ethanol (av. 44.3 g/dm 3 ), methanol (16.7 mg/dm 3 ) and isoamyl alcohols (47.6 mg/dm 3 ). the positive feature of triple cultures using was limitation of methanol and fusel alcohols synthesis that was accompanied by relatively high ethyl alcohol production (av. 41.5 g/dm 3 ). the consumption of sugar syrup becomes increasingly significant in industrial processes due to economic advantages and the easy of use. the production of sucrose syrup using enzymatic hydrolysis represents the safest alternative, once the reaction does not produce any toxic or undesirable substance. this work consists on the production of sugar syrup by immobilized inulinase from kluyveromyces marxianus, with two alternatives process: (a) syrup enriched with fructooligosaccharides or solely with glucose and fructose. the process is comprised by the following stages: production and purification of the enzyme in optimized conditions, immobilization of the enzyme in solid support and the conversion of sucrose in a fixed bed bioreactor with the immobilized enzyme. the final composition of the product can be a mixture of glucose, fructose, sucrose and fructooligosaccharides or a mixture of fructose and glucose, according to the operational conditions. the bioreactor can be operated continually for approximately 5 months with the same biocatalyst. the product from this process is ideal for applications in the food products such as sweet, candies, chocolates, yogurts, etc. besides, the prebiotics properties of the fructooligosaccharides, is a beneficial stimulant of the intestinal flora, which gives to the product a functional property. studies on plant microbial interactions using azotobacter sp. as bio-inoculants towards soil fertility baljeet singh saharan faculty of biotechnology, jcdm college of engineering, sirsa 125055, india. e-mail: baljeet.saharan@gmx.de, baljeet br@yahoo.co.uk (b.s. saharan) high nitrogen fixing, phytohormone producing isolates of azotobacter, azospirillum, acetobacter and pseudomonas were used as inoculants on wheat and cotton with varying doses of nitrogen under field conditions. bio-inoculants were selected on the basis of yield, dry weight and survival rate of bacteria under field conditions. seeds of wheat variety wh 711 were treated with different biofertilizers using nitrogen level of 90, 120 and 150 kg ha −1 and one level of p, i.e. 60 kg ha −1 in field along with control. under field conditions, maximum yield was obtained with azotobacter chroococcum e 12 at 90 (2506 ± 0.04 kg ha −1 ) as well as 120 kg ha −1 (2817 ± 0.07 kg ha −1 ) followed by a. chroococcum ht 57 (2482 ± 0.16 kg ha −1 ) and avk 51 (2474 ± 0.37 kg ha −1 ). whereas, with 120 kg ha −1 highest yield was observed with mac 27 (2833 ± 2.59 kg ha −1 ) followed by e12 (2817 ± 0.91 kg ha −1 ) and avk 51 (2804 ± 0.16 kg ha −1 ). maximum height at 90 kg ha −1 was observed with mac 27 inoculation (71.1 ± 5.24 cm) followed by avk 51 (70.8 ± 4.70 cm) and ht 57 (70.3 ± 1.76 cm). various chosen strains were tested with desi (hd 123) and american cotton (h 1098) under similar pot and field conditions as for wheat in the following season. plant height and yield were determined at the time of harvesting whereas survival rate was monitored at various intervals of time. survival rate of inoculated bacteria was determined after 30, 80, and 135 days. highest survival rate was observed in mac 68 ((3.34 ± 2.56) × 10 6 ), which decreased after 80 and 135 days, respectively. (3.38 ± 1.48) × 10 5 , (1.53 ± 0.92) × 10 5 with mac 68 and (2.97 ± 2.01) × 10 5 and (1.26 ± 3.01) × 10 5 with ht 54, respectively. maximum boll weight was with avk 51 (76.2 ± 2.34 g boll wt. plant −1 ) followed by pseudomonas (71.3 ± 1.77 g), ac 18 (61.5 ± 1.73 g) and ala27 (61.4 ± 2.79 g) boll no. plant −1 was maximum with ala 27 and avk 51 (46 ± 2.59 plant −1 ) followed by pseudomonas (34 ± 0.07 plant −1 ). maximum height and dry matter was obtained with pseudomonas (179.7 ± 1.97 cm) and avk521 (146.7 ± 3.49 cm) with variety hd 123 under field conditions. net saving of 20% nitrogen was observed using a. chroococcum (e 12 and avk 51) bioinoculants for wheat and cotton, respectively. to characterize the antioxidative properties of tempeh-fermented food prepared from vicia faba (l. kontu) with the use of rhizopus oligosporus, the sulfhydryl groups content and surface aromatic hydrophobicity of albumins were investigated. the results obtained for tempeh albumins were compared with raw vicia faba and bovine serum albumin (bsa). these results indicate that tempeh fermentation increased antioxidative activity of albumins. the measurements of antioxidative activity were carried out with the use of 1,1-diphenyl-2-picrylhydrazyl (dpph) and 2,2 -azinobis-(3ethylbenzothiazoline-6-sulfonic acid (abts). the albumins of faba bean-tempeh have possessed much higher activity for scavenging free radicals as measured with the dpph and abts (49.4% and 41.1%) than raw seeds (27.9% and 23.2%) and bsa (5.8% and 13.4%), respectively. it has been also found that tempeh fermentation process increased 2.5 times sulfhydryl groups content (43.2 m/mg of albumins) as compared to raw seeds (17.5 m/mg of albumins). the tempeh albumins have possessed lower surface aromatic hydrophobicity than raw seeds (352.3 fi and 692.1 fi, respectively). orange peel characterization and generation of fermentable sugars solutions for the biotechnological production of food additives b. rivas 1 , j.m. domínguez 1 , p. torre 2 , j.c. parajó 1 : 1 department of chemical engineering, vigo university (campus of ourense), polytechnic building, as lagoas, 32004 ourense, spain; 2 department of chemical and process engineering, genoa university, via opera pia 15, 16145 genoa, italy. e-mail: brivas@uvigo.es (b. rivas) the citrus processing industry generates in mediterranean area around 3 millions tonnes of orange peel as byproduct from the extraction of citrus juices in industrial plants. in order to avoid ecological problems and provide an extra profit, this residue was studied in order to generate a suitable substrate for the fermentation process oriented to the production of food additives. orange peels were characterized and the data collected allowed quantifying a 97% of this waste. soluble sugars (21.2%), cellulose (17.0%) and pectin (42.5%) were identified as more important fractions. this material was submitted to two hydrolysis techniques, prehydrolysis (with diluted sulfuric acid) and autohydrolysis (with water) under different experimental conditions. autohydrolysis was selected as the most appropriate technique for the production of suitable fermentation media. finally, the liquors obtained at 130 • c and liquid:solid ratio of 8 g/g, containing 38.2 g/l of sugars, without additional nutrients, were employed to citric acid production by aspergilus niger cect 2090 (atcc 9142, nrrl 599) . the influence of the addition of calcium carbonate and methanol were studied. under the best conditions an effective conversion of sugars into citric acid was attained, showing the viability of the production of fermentable solutions from this industrial waste. today there is an increasing interest in using high gravity fermentation in brewing. high-gravity fermentation involves production of beer wort of up to 18 • p or even higher and results in beer that has more consistent product quality. the main aim of this study is an increased understanding of how brewer's yeast respond to the various stress factors imposed during high gravity beer fermentation and the consequences these stress factors have on the gene regulation and its consequences on the metabolite levels (both intra-and extracellular). higher attenuation of the wort will be achieved by two different techniques: by the addition of highly fermentable adjuncts such as sucrose or glucose syrups and by mashing with addition of microbial enzymes such as pullulanases and glucoamylases. in the first part of the study model fermentation conditions are established, where the sugar uptake and product formation can be studied in details. characterization of the carbohydrate profile is analyzed by hplc. as flavour changes may occur at higher gravities, it is important to study changes in formation of secondary metabolites, especially esters. transcriptome and metabolome analysis will be used to establish how the stressful conditions prevailing under high gravity fermentations may influence the secondary metabolism in saccharomyces cerevisiae. furthermore, analytical aroma characterization of final beer will be studied by spme and gc-ms. detailed analysis of the effect of different stress factors on the cellular response using dna arrays and metabolite profiling will be carried out. dna arrays will be employed to evaluate if specific metabolic pathways are up-regulated or down-regulated as a consequences of the stress factors. naringin, a bitter compound that occurs in citrus fruit juices, may be converted to a nonbitter form by enzyme hydrolysis. the enzymatic complex naringinase was produced in aspergillus niger cect2088 cultures with naringin as inducer (pérez-mateos et al., 2004) . crude extracts from a. niger and purified naringinase from penicillium decumbens were immobilized into a polymeric matrix of polyvinyl alcohol (pva) hydrogel cryostructured in liquid nitrogen. the operating stability of the pva-naringinase beads was tested using synthetic citric juice (gray and olson, 1981) . immobilized enzymes reduced 40% the naringin content at 20 • c and ph 3.2. furthermore, immobilized preparations from aspergillus and penicillium could be re-used through six cycles (144 h) remaining 70% and 38% catalytic efficiency, respectively. financial support from "ministerio de ciencia y tecnología" and feder (no. agl2003-08006/ali). gray and olson, 1981 . j agric. food chem. 29, 1298 -1301 . pérez-mateos, et al., 2004 (1), 230. otimizing the fermentation broth for tanase production by a new isolated strain paecilomyces variotii vania battestin, gláucia pastore, gabriela macedo department of food science, unicamp, p.o. box 6121, campinas, cep 13083-862 são paulo, brazil tannase is an inducible enzyme that catalyses the breakdown of ester linkages in hydrolysable tannins, resulting in gallic acid and glucose. the fermentation broth can use by-products as wheat bran, rice or oats, adding tannic acid. the use of by products or residues rich in carbon source for fermentation purposes an alternative to solve pollution problems that can be caused by an incorrect environmental disposal. in the present study we have optimized the production of an extracellular tannase by a new isolated paecilomyces variotii using response surface methodology. the first step was to identify the variables having a significant effect on enzyme production. the variables evaluated were temperature, residues ratio (coffe: wheat bran), concentration of tannic acid, salt solution during 3, 5 and 7 days of fermentation time. results showed that temperature, residues ratio (coffe: wheat bran) and tannic acid had significant effects on tannase production. commercial wheat bran (cwb) and coffe rusk residues (cr) were used as solid substrate. for fermentation the medium was composed by, cwb:cr were mixed with distilled water and transferred into 250 ml capacity erlenmeyers flasks and auto-claved at 120 • c for 20 min. the medium was then inoculated with spores (5.0 × 10 7 ) and the flaks were incubated at 32 • c. tannase was assayed according to the methodology of mondal et al. (2001) . according to the statist analyses, the optimum conditions to produce tannase was the range of temperature (29-34 • c); tannic acid (8.5-14%); residues percent (coffe: wheat bran) (50:50) and 5 days fermentation time. the enzyme production increased 8.6 times more enzyme production than that was obtained before this optimization. yeast and lactic acid bacteria are two major microbial groups of the most fermented products. a large variety of fermented foods and beverage are made by the activities of both yeast and lactic acid bacteria, simultaneously or successively. during the spontaneous mixed fermentation of lactic acid bacteria and yeast population, it is extremely difficult to control microbial species due to the complexity of the microorganism involved. therefore, we have compared the antimicrobial activity of chitosan against two lactic strains, lactobacillus plantarum and lb. brevis, and yeast strains, saccharomyces cerevisiae to investigate the possible use of non toxic biopolymer chitosan for selective control in mixed culture. the lactobacilli were more sensitive to the inhibitory activity of chitosan than s. cerevisiae. the results suggest the possible use of low molecularweight-chitosan for the control of food fermentation in which both groups of organisms frequently occur together. the effect of vegetable oils on astaxanthin production of phaffia rhodozyma and xanthophyllomyces dendrorhous csaba vágvölgyi, gyöngyi lukács, miklós takó, árpád csernetics, tamás papp department of microbiology, faculty of sciences, university of szeged, p.o. box 533, astaxanthin (3,3 -dihydroxy-␤,␤-carotene-4,4 -dione) is one of the most important carotenoid product. it is used primarily as food colorant and animal feed additive. their effective antioxidant properties linked to a preventive action on various types of cancer and an enhancement of the immune response could lead to expanded commercial applications. among the natural microbial source available, the closely related red pigmented yeasts phaffia rhodozyma and xanthophyllomyces dendrorhous are of great biotechnological interest. these yeasts have desirable properties as biological sources of pigment, including rapid metabolism and producing high cell densities in fermentor, but the commercial production of astaxanthin is limited by the relatively low content in wild-type strains. the purpose of this study was to determine whether the different vegetable oils had an effect on the carotenoid production in p. rhodozyma. effects of media supplemented with corn germ oil, wheat germ oil, sesame-seed oil, palm oil, pumpkin-seed oil, coconut grease, olive oil (extra virgin), olive oil (sanza), sunflower-seed oil and cottonseed oil were tested. studies were performed on both a phaffia and a xanthophyllomyces strain. yeast was grown in yeast-pepton-glucose liquid medium complemented with the appropriate vegetable oil in different concentrations (0.5-2, v/v, %) . after four days the total carotenoid production was determined spectrophotometrically, and it was referred to dry cell mass. palm oil increased significantly the carotenoid production of the phaffia strain, while a similar effect on the xanthophyllomyces strain could be observed with coconut grease. composition of carotenoid compounds in the strains was determined by thin layer chromatography. lutein is considered a nutraceutic compound that has developed an increasing interest since it is one of the two carotenoids that are located in the macula of the human eye. its consumption is associated with the prevention of age related macular disease (amd). industrially, lutein may be produced using a saponification step of a mixture of lutein diesters that are previously extracted with hexane from natural sources. our proposal is to improve the process by catalyzing the same reaction using microbial lipases during the extraction step with hexane. additionally, the use of supercritical fluids represents an extension of enzymology in non-conventional media with process and environmental advantages. this work was developed using extracts from marigold flower (tagetes erecta) in hexane and supercritical carbon dioxide (sc-co 2 ) where the lutein esters were hydrolyzed by two commercial lipases: lipase b from candida antarctica (novozym 435) and lipase from mucor miehei (lipozyme rm 1m). in particular, we focused our interest in the role of water in the system. interestingly our results show an inverse dependence of the initial reaction rate with respect to the initial water activity (awi) for both lipases, a phenomena that seems to be related to the partition of substrates and products in the solid support)and the hexane phase as a function of water. when sc-co 2 was used as solvent an increase in the consumption rate of lutein diesters occurred, reaching conversions of 70% in 24 h. for hexane, the same conversion was reached after 160 h. this result suggests a significant effect of the media on the reaction that can be related to shifts in the partition of compounds that bring the substrates in closer contact with the enzyme. this work also demonstrates that lutein hydrolysis seems to be another potential application of commercial immobilized lipases in the food/nutraceutical market. the enzyme of interest in this work is ␤-galactosidase from lactobacillus sp. (ec 3.2.1.23). ␤-galactosidases catalyze the hydrolysis and transgalactosylation of ␤-d-galactopyranosides (such as lactose). an attractive biocatalytic application is found in the transgalactosylaction potential of these enzymes which is based on the catalytic mechanism of ␤-galactosidases. the products of transgalactosylation, galacto-oligosaccharides, are non-digestible carbohydrates which meet the criteria of 'prebiotics' and therefore have attracted increasing attention. to produce these 'prebiotic' galactooligosaccharides, an inexpensive and efficient process is desired. immobilization of the enzyme ␤-galactosidase on an insoluble support is an attractive tool to make the process of lactose conversion more economical because the enzyme can be recovered and reused during continuous operation. in this present study, we aimed at immobilizing ␤-galactosidase from lactobacillus sp. by covalent linkages on two solid supports which are commonly used for protein immobilization: chitosan and eupergit c. the protein-binding capacity, the immobilization yield, ph and temperature dependency of activity and stability, and the kinetic parameters of immobilized enzymes were studied. higher activity retention of the immobilized enzymes over a broader ph range and at higher temperatures compared to those of the free enzyme was observed. the immobilized enzymes were evaluated in terms of transgalactosylation activity and stability for a to introduce of foreign genes for the important crop plants such as rice, we need a reproducible efficient procedure for regeneration of the calli through somatic embryogenesis. for this intention, we established the best callus induction medium for tarom mahalli and deilamani cultivars and created the method that the regeneration frequency was reached to 48%. calli were induced from scutellar tissues of mature seeds on ms medium supplemented with three level of 2,4-d (2, 2.5 and 3 mg l −1 ) and n6 medium supplemented with five level of 2,4-d (1.5, 2, 2.5, 3 and 3.5 mg l −1 ). for deilamani cultivar the best medium was n6 with 1.5 mg l −1 2,4-d and for tarom mahalli the same medium with 2 mg l −1 were the best. in subculture media, sucrose was used instead of maltose. for regeneration analysis of plantlets, we used two-factorial experiment in base of crd; one factor was regeneration media with six levels (ms medium supplemented with five amount of kinetin and: naa (mg l −1 ) [(6:2), (4:2), (2:0.5), (3:1), (2:1), respectively] and 0.2 mg l −1 2,4-d and 2 mg l −1 bap). other factor was dehydration process with three levels (without dehydration, dehydration with two layers of filter paper for 30 min [prior to transfer to the regeneration medium], and third factor was factor of 2 with substitution of sucrose with maltose [after 2 weeks; 2 sucrose:1 maltose]). we conclude that maltose due to changing in osmolarity proceeding can elevate the regeneration frequency to 48%. therefore, type of carbon source is critical in callus induction and regeneration. márová ivana, hrdličková jana, kubešová jitka, kočí radka, vidláková tereza faculty of chemistry, brno university of technology, purkyňova 118, 612 00 brno, carotenoids are the most widespread natural pigments with important biological activities and applications mainly in food and feed industry. at present many ways including genetic engineering are developed to reach higher production of naturally formed carotenoids using microbial producers. in this work cloning and expression of crt gene cluster from pectobacterium carotovorum in recipient bacterial strain e. coli dh5␣ as well as in yeast strain s. cerevisiae was tested. plasmid vector phsg298 with inserted crt genes was used for transformation of chemically competent e. coli dh5␣ cells, while in s. cerevisiae shuttle vector paur135 was used. transformants were selected based on resistance to antibiotics, formation of orange-coloured transformant colonies, analysis of recombinant plasmid size and lc/ms analysis of carotenoids produced by recombinant cells. the yield of individual carotenoids (lutein, beta-carotene, lycopene) obtained from various bacterial transformants was several fold higher than in natural producer (lutein: 0.2-1.2 g/g of d.w., beta-carotene: 0.1-1.4 mg/g of d.w.). the highest yield obtained in transformed strain was 63.5 g/g of lutein and 15.4 g/g of beta-carotene. the yield of biomass and carotenoids in. transgenic s. cerevisiae was comparable to some industrial red yeast strains (4.5 mg of total carotenoids + 32 mg ergosterol/l; 36 g/l of biomass). so, transgenic yeasts could be suitable for large scale production of carotenoids and/or enriched biomass, while transgenic bacterial producers are perspective above al for high production of rare carotenoids as lutein or lycopene using transformation by specific genes of crt gene cluster. this work was supported by the project msm 0021630501 of the czech ministry of education, youth and sports. two forms of grape seeds, whole and powdered forms, were heated at four different temperatures-50, 100, 150 and 200 • c. after heating, grape seeds were extracted with 70% ethanol (0.1 g grape seed/10 ml of 70% ethanol), and total phenol contents (tpc), radical scavenging activity (rsa) and reducing power of the extracts were determined. thermal treatment of grape seed increased the antioxidant activity of extracts. the maximum tpc and rsa of whole grape seed extract (wgse) were achieved when the seeds were heat-treated at 150 • c for 40 min, while that of powdered grape seed extract (pgse) were at 100 • c for 10 min, and were greater than that of the non-treated control. according to the gc-ms analysis, several low-molecular-weight phenolic compounds were newly formed in the wgse heated at 150 • c for 40 min. these results indicated that antioxidant activity of gse was affected by heating conditions (temperature and time) and physical conditions of grape seeds at the time of heat treatments. analysis of the unexpected phenotypic consequences associated with plant transformation jonathan latham, allison wilson, ricarda steinbrecher econexus, 6, canon frome court, ledbury hr8 2td, uk. e-mail: jrlatham@gn.apc.org (j. latham) transgenic plants often exhibit unexpected phenotypes. such phenotypes could arise from pleiotropic effects associated with the transgene, or they could arise from other sources. a recent econexus report underlined the potential for the process of plant transformation to result in genetic damage to the transformed plant (genome scrambling-myth or reality? transformation-induced mutations in transgenic crop plants: http://www.econexus.info/). the report showed that mutations arising at the site of transgene insertion are often substantial, frequently resulting in loss or rearrangement of chromosomal dna and insertion of multiple superfluous dna fragments. unintended mutations were also documented at other locations in the plant genome. such transformation-induced mutations could provide an explanation for unexpected phenotypes in transgenic plants. we decided to survey regulatory documents and the scientific literature for instances of unexpected phenotypic consequences arising in transgenic plants. this poster documents the preliminary results of our survey. it is intended to assess the range and frequency of unexpected consequences and to examine whether there is sufficient data available to determine their origin. it is our belief that investigating the origin of these unexpected phenotypes should be a principal aim of biosafety research. biotechnological production of metabolites such as carotenoids could be of high interest because of their antioxidative and antimutagenic activities in human body. production of these metabolites by microbial cells is dependent on cultivation conditions. so, presence of exogenous stress in cultivation environment could stimulate biosynthetic pathways of desired metabolites. two non-conventional yeast strains, rhodotorula glutinis and sporidiobolus salmonicolor, were chosen for study of carotenoid production useful in feed industry. hydrogen peroxide, sodium chloride and/or their combinations were used as exogenous stimulators of carotenoid pathway. presence of exogenous stress led to important overproduction of pigments as well as of supplementary studied substances (ergosterol, glycerol). higher adaptability of yeast cells was observed not only in cultivations with one type of stress. combination of stress factors in cultivation media induced significant increase of pigment formation. moreover, under controlled conditions in laboratory fermentor s. salmonicolor produced about eight-fold amount of ␤-carotene (230 g/g) in medium with 2% nacl and 5 mm h 2 o 2 than in control sample. similar result was observed in r. glutinis cultivated in presence of 2% nacl in inoculation medium only (200 g/g of ␤-carotene). the use of stressed biomass of red yeasts in feed industry could have positive effect not only in animal and fish feeds because of high content of physiologically active substances, but it could influence nutritional value and organoleptic properties of final products for human nutrition. this work was supported by the project msm 0021630501 of czech ministry of education. the culture ph significantly affects mycelial growth and morphology, exopolysaccharide (eps) formation, and their molecular properties during submerged cultures of a medicinal mushroom ganoderma lucidum. when the culture ph shifts from 3 to 6, mycelial growth (12.5 g/l) and eps production (4.7 g/l) were favorable compared with other ph-control strategies. the mycelial morphology was also significantly varied upon culture ph: a feather-like pellets were found when the ph was controlled shifting from 6 to 3 at day 4, which was regarded as undesirable morphological form for eps production. compositional analyses revealed that the ratios and chemical compositions of the eps formed in bottom or top fractions of ethanolic precipitates were significantly different upon culture ph. the molecular characteristics of the eps were further investigated using a size exclusion chromatography/multi-angle laser light scattering (sec/malls) system. plant ␤-n-acetyl-hexosaminidase (hex) (ec 3.2.1.52) is reported to have diverse physiological roles like fruit ripening, degradation of reserved glycoproteins in germinating seed and chitin-elicited lignification. in this paper we report the purification and characterization of hex from korean ginseng roots. after extraction with citrate-phosphate buffer, hex was purified to homogeneity using ion exchanger chromatography, hydrophobic interaction chromatography and gel filtration. its molecular weight was determined using gel filtration and mass spectrometer. enzymatic parameters were studied with 4-methyl-umbelliferyl-n-acetylglucosaminide as substrate. the effect of heat stress and weak organic acids on escherichia coli and a comparison of its recovery by the plate count method and flow cytometry monica s. talsania due to the importance of microbiology for human health, methods have been developed to enumerate viable bacteria. dilution plating is seen to be the 'gold standard' for proof of a cells viability. however, the success of this method relies on post sampling growth, which is limited by our ability to grow cells in the laboratory. additionally, stressed or sub-lethally damaged cells, remain undetected. single cell measurements can provide rapid detailed physiological information, and the assessment of population heterogeneity. this work compared the recovery of stressed e. coli as measured by the number of cfu/ml and by multi-parameter flow cytometric analysis. weak organic acids and high temperature-short time processing (htst) were used to stress the cells both methods commonly used during food preservation. it was shown here that flow cytometry is a powerful tool for the enumeration and detailed analysis of any non-culturable microbial population, which is important because cytotoxic compounds and heat stresses used in food preservation often have a growth inhibiting effect but not necessarily a lethal one. paul g. kovalenko molecular biology & genetics nasu, zabolotnogo str. 150, 031473 kyiv, ukraine the pharmaceutically important plant species of glycyrrhiza sp. (called licorice) is an important commercial product used as a natural sweetener, anti-inflammatory, anti-cancer and anti-diabetic agents. agrobacterium rhizogenes transformation system was used for the hairy root cultures of licorice g. uralensis. after inoculation of aseptic stem segments the ability of hairy root formation was scored for a period of 6 weeks. mean transformation frequency ranged from 27% (for 8196 up to 31% (for 15,834). some transformed genotypes showed significant differences in roots weight, flavonoids and glycyrrhizin (gl) production. the cotransformation rate of licorice intact explants cultivars with lba 9402 tl-dna and the 35s gus gene showed an average of more than 35%. these obtained root cultures were additionally elicited with extracts of biotic elicitor acremonium sp. (endomycorhizal fungus), and were used as an in vitro system to metabolites production. the transformed and elicited hairy roots of g. uralensis were obtained by infection of a. rhizogenes 8196 have produced gl at an yield of 4.5% dry weight on the period of culture as a 30 days. according to tentative analyses the hairy roots cultures of glycyrrhiza species produced flavonoids (liquiritigenin and liquiritigen). more high levels (3.42 g/l) of the total flavonoids production have been identificated on the strains which transformed by lba 9402. this study involved any difference among elicitor treatments and incubation periods for the optimal meabolites production. clearly, the selection of an effective agrobacterium strain for the production of transformed root cultures is highly dependent on the plant species, and must be determined empirically. ayse gul nasircillar akdeniz university, biology, akdeniz univ. faculty of art-science, biological department, 07058 antalya, turkey mature embryos of five t. aestivum and five t. durum cultivars formed embryogenic callus on two different media. embryos were removed from surface sterilised seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of murashige skoog and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-d) or 1 mg/l naphthalenacetic acid (naa). the developed calli and regenerated plants were maintained on 2,4-d or naa free ms medium. wheat plants can be regenerated via two different systems. there were significant differences in percentage of callus induction and regeneration capacity on the different initiation medium. among the t. aestivum cultivars, yakar had the highest regeneration capacity in both induction medium. in t. durum cultivars, kiziltan gave the highest regeneration capacity in ms + 2,4-d medium and yilmaz gave the highest regeneration capacity in ms + naa medium. a strong genotypic effect on the culture responses was found for both induction medium. the glycolytic enzyme triosephosphate isomerase (tpi), which catalyses the interconversion of the triosephosphates dihydroxyacetone phosphate (dhap) and glyceraldehyde-3-phosphate (gap), was studied for its control on glycolysis and mixed acid production in lactococcus lactis il1403. we constructed a number of l. lactis strains in which the tpi activity was modulated from 3% to 217% of the wild-type level. the enzyme was found to be present in high excess with 3% tpi activity supporting 30% of the wildtype glycolytic flux, and with 24% of the wildtype tpi activity the glycolytic flux was essentially unchanged. measurements of the upstream metabolites glucose-6-phosphate (g6p), fructose-1,6bisphosphate (fbp) and dhap were essentially unchanged for tpi activities from 24% to 217%, and only in the strain with 3% tpi activity we observed a significant increase in the intracellular dhap concentration. homolactic product formation was preserved throughout the interval of tpi activity studied, though a small increase in the amount of acetate and formate production was observed in the strain expressing tpi at the lowest level (3% tpi activity). the finding of an increased mixed acid pattern under intracellular conditions with a high dhap concentration is in contrast to earlier data from literature, which indicated that the triosephosphates play an important role in regulation of pyruvate metabolism in l. lactis with a negative effect on the mixed acid flux. we have recently shown that alcohols induce the adhesion of l. monocytogenes at low temperatures, presumably accompanied by enhanced exopolysaccharide (eps) production. however, little is known about the mechanisms involved in the formation of biofilm and eps by l. monocytogenes. in the present project, we show that deletion of selected regulatory and up-regulated genes did not abolish attachment, though the degree of alcohol-induction in some cases was affected. we are applying bioinformatics to search for homologues in l. monocytogenes of known eps genes from various gram positive bacteria. this has revealed candidate genes involved in the synthesis of eps, such as genes encoding glycosyltransferases. moreover, we are at present performing dna microarray analysis for the egde strain grown at 10 • c in the presence of 2.5% isopropanol. this data should, combined with the bioinformatic results, give us a good indication of the genes involved in alcohol-induced surface attachment. repetitive-pcr (rep-pcr) was applied in research on non-starter lactic acid bacteria (nslab) in cheese. we first showed that strains previously differentiated by pulsed field gel electrophoresis (pfge) also could be differentiated by rep-pcr. this was partially due to slight changes in the pcr conditions that allowed reproducible amplification of 7-9 kb bands. more than 20 bands were obtained for most strains. a clear differentiation was also obtained between lactobacillus paracasei, lactobacillus plantarum, lactobacillus curvatus and lactobacillus danicus (a new species found in danish and estonian cheeses and traditional starter cultures). we found that this technique is highly reproducible, e.g. identical profiles in three different pcr-machines, two different dna isolation procedures, and different trained personnel. we applied the developed rep-pcr technique to confirm that survivors after heat treatment, were the actual strains introduced and not due to post-pasteurization contamination. we also showed that when we added a cocktail combination of five strains as protecting cultures to cheese, two to three members of this cocktail was dominating the cheese nslab microflora. in control cheeses without the cocktail in most cases other strains dominated, but in a few cases we were able to show cross-contamination between cheese vats. these data indicate that the rep-pcr will be useful to follow development of adjunct cultures as well as provide a reproducible subspecies (e.g. strain) differentiation. rep-pcr is a much quicker and less labour requiring procedure than pfge, and is apparently a much more reproducible technique than what has been seen for rapd. dynamic modeling of lactococcus lactis metabolism and its dynamic behavior for lactate secretion and regulatory characteristics jinwon lee, ui sub jung, hye won lee department of chemical and biomolecular engineering, sogang university, seoul, south korea, 121-741. e-mail: jinwonlee@sogang.ac.kr (j. lee) dynamic metabolic model for lactococcus lactis has been developed in order to analyze a time-dependent behavior of lactate secretion mechanism and probe its regulatory roles. the model was used to compare and analyze the lactate metabolism through in silico simulation and in vitro experimental measurements most of all pyruvate branch point seems to play a major role in producing lactate, and the results of metabolic control coefficient analysis recommend to increase lactate dehydrogenase activity and to decrease nadh oxidase activity. for obtaining more realistic data, we have added some measured flux data including some intermediate metabolites. by combining the simulation results and experimental measurements, we could establish more reliable and robust systematic lactate secretion model. in addition, an efficient parameter estimation method was used to test the exactness of the reported kinetic parameters. what to choose -the fast or the detailed -strategy to get informative profiles of secondary metabolite produced by fungi in culture. chemo-diversity and lead discovery calls for high throughput techniques, but do we need columns will direct infusion esi-ms (dims) do the job. the latter may give matrix effects and lacks resolution resulting in loss of information, while lc-ms analysis takes time and challenge the data processing. results from nano-esi dims and lc-ms analyses of the same extracts important penicillium species are compared. these results illustrate advantages and problems using these techniques for rapid profiling of fungal secondary metabolites, reviling that matrix effects in dims do not seriously hampers detection of important metabolites while the specificity and certainty, for e.g. de-replication is much higher in lc-ms. phenotypic classification of fungi is essential in food biotechnology ulf thrane center for microbial biotechnology, biocentrum-dtu, søltofts plads 221, technical university of denmark, dk-2800 kgs. lyngby, denmark. e-mail: ut@biocentrum.dtu.dk fungi are of great importance in food and food production. the intended use of fungi as cell factories for production of food ingredients is an upcoming issue in food biotechnology; however, this brings up a possible contamination with mycotoxins as a major issue. a reliable identification of the producer strains is crucial as a correct identification at species level following an updated taxonomy is the key to information on functional characters, e.g. useful metabo-lites and potential mycotoxins, growth conditions, resistance, etc. unfortunately, many mycological reports do not specify the taxonomy used or do not pay sufficient attention to taxonomical systems based on classification by functional characters-in contrast they are using a nucleotide sequence based phylogeny, which conveys little -if anything -about function of the organism. this situation is a major challenge for biotechnologists and mycologists in the years to come and will be highlighted by illustrative examples. the commercial interest in functional foods containing sufficient amounts of living probiotics is paralleled by the increasing scientific attention to the beneficial effect in the digestive tract. a daily intake of viable cells is proposed to ensure probiotic effect on consumer's health. one of the approaches which seems to be feasible to enhance probiotic viability and stability is to improve the fermentation conditions. during batch fermentation the viability of lactobacillus gasseri 5714 decreases after reaching a maximal value apparently indicating cell death. in this work, the apparent loss of viability can be avoided during fed-batch fermentation. a three-fold increase in viability is obtained when nutrient concentration was controlled compared with the viability reached in batch cultures. as a consequence, higher biomass concentration and lower specific lactic acid production were obtained. a mathematical model was developed to simulate and describe the effect of nutrient limitation on growth, viability, glucose consumption and lactic acid production. contribution to the metabolic adaptation to food restriction in rabbits (preliminary results) s. van harten 1 , s. borges 2 , p. cravo 2 , l.a. cardoso 1 : 1 instituto de investigação científica tropical, cvz, lisboa, portugal; 2 instituto de higiene e medicina tropical, lisboa, portugal. e-mail: svharten@gmail.com (s. van harten) in order to understand metabolic differences between two breeds of rabbits (halop ab and oryctolagus cuniculus algirus) during food restriction, the activities and expression of key enzymes and hormones of the rabbit were studied. animals from each breed were divided in two groups (ad libitum and restricted), revealing the results a similar difference in glycemic levels between fed and underfed rabbits, with a restriction of 50% of ad libitum feeding in the wild animals (decrease of 23% lw) and 16% of that ingestion in the halop breed (decrease of 32% lw). the activities of glutamine synthetase and glutaminase show a higher reduction of these enzymes in the wild animals superior to that of the halop breed, compromising, in this way, the ammonium detoxification and the entry of residual carbonated groups of the protein catabolism into the krebs cycle. in the latter animals, a rapid mobilization capacity of triacylglycerols (tga) appears to exist, with a rapid catabolism of fatty acids leading to their oxidation. the wild breeds' results reveal a rise of circulating tga, reflecting difficulties in the lipolysis and mobilization of nefa for oxidation. in these underfed animals, phosphoenolpyruvate and pyruvate suffered a large increase and oxaloacetate a decrease. the halop breed revealed results that indicate a diminution of glycolisis, being glucoses' energy substituted by carbonated chains of lipolysis and protein catabolism. hormone results showed a higher decrease in insulin, t3 and igf-1 in the underfed halop animals. in order to confirm the biochemical results, relative quantification of enzyme expression was studied by real time-pcr. since the introduction of genetically modified (gm) crops in 1996, the area under their cultivation has globally increased from 1.7 million hectares in 1996 to 67.7 in 2003. the number of countries adopting gm crops also rose from one country, the usa, in 1996 to 20 in 2003. despite numerous successes public opinion still questions the ecological, moral, ethical considerations and issues concerning altering the natural state of the organisms. in this study, a survey of food shoppers' knowledge, attitudes and perceptions of gm foods was carried out in food outlets in nairobi. the food outlets were determined by simple random sampling. using systematic sampling, shoppers were interviewed at targeted imported food products. focus group discussions were also conducted with farmers at city markets. the survey reflected views of a systematic sample of 387 shoppers in seven food outlets between november and december of 2003. it revealed knowledge at 20%, with positive attitudes and good perceptions towards gm foods (χ 2 = 42.873, d.f. 9, p < 0.001). seventy nine percent of shoppers were willing to buy and consume gm foods (χ 2 = 61.321, d.f. 2, p < 0.001). cross-tabulation of shopper's position on various issues raised in the survey showed a strong correlation between the respondents' respective knowledge, attitudes, and perceptions (r = 0.84). nineteen percent of food sampled tested positive for gms. poisson statistics were used to calculate the number of sample sequences. the statistical tools were obtained from spss version 11.5. the results of this study will be of great interest in determining the use and adoption of gm crops in kenya. it will also guide the development of national foreign food policy on gm foods. the technology should be embraced as soon as it is acceptable to alleviate, drought, famine and hunger estimated to be affecting 3.3 million kenyans today, mostly children. consumers and gm foods: the case of turkeyözlenözgen 1 , mustafa yildiz 2 : 1 department of family and consumer sciences, school of home economics, university of ankara, ankara 06130, turkey; 2 department of field crops, faculty of agriculture, university of ankara, 06110 ankara, turkey the future development of food biotechnology depends on consumer acceptance. scientists are aware that consumer attitudes will have a crucial impact on the process of the food biotechnology. because, food is one of the central features in human life. consumers' attitudes and trusts in the institutions will determine how gene technology will be used in food sector, in the future. recently, research concerned with consumer aspects of gm foods accelerated. but in turkey, the literature that deals with this subject is very limited and sparse. therefore, this research was carried out on the turkish consumers with the purpose of analyzing the consumers' awareness, assessments about benefits-risks, market place and labelling, and trusts in institutions, towards gm foods. this study was based on interviews with consumers who have recently purchased from major malls, during shopping hours. of the four major malls, voluntary male and female consumers were included in the research if they had main or secondary responsibility for household shopping. the questionnaire form was applied to subjects through face-to-face individual interview. the data were analyzed by using statistical methods according to explanatory variables, including age, gender and educational level. findings indicated that consumers' awareness and views about gm foods were connected to selected demographic characteristics. the results of this study can be important for consumer educators, marketing managers and policy makers. benefit-risk perceptions and moral beliefs of turkish consumers towards transgenic productsözlenözgen 1 , haluk emiroglu 2 , mustafa yildiz 3 , ayşe sezen taş 1 : 1 department of family and consumer sciences, school of home economics, university of ankara, 06130 ankara, turkey; 2 faculty of law, university of bilkent, 06590 ankara, turkey; 3 department of field crops, faculty of agriculture, university of ankara, 06110 ankara, turkey the use of biotechnology in production has generated considerable debate involving the benefits-risks and moral beliefs associated with its use. consumer acceptance of genetically modified product is a critical factor will affect the future of this technology. this study was planned and conducted to determine the relationship between product/process related benefit perceptions, product/process related risk percentions and moral beliefs of consumers towards transgenic products. a total of 400 university educated consumers, consisting of 200 males and 200 females, employed at the ministries selected by random sampling method in ankara, were included into study. interview techniques were used in the gathering the research materials. the interview instrument had been prepared considering previous research and literature. answers given to sentences typed likert were scored, used "varimax analysis technique" for validity. in order to test the reliability of questionnaire were calculated "cronbach alpha" as inner consistency coefficient. the t-test were performed for determining the differences dependent on gender and age variables between product related benefit perceptions, process related benefit perceptions, product related risk perceptions, process related risk perceptions and moral beliefs of consumers. the examination of relationships between product/process related benefit perceptions, product/process related risk perceptions and moral beliefs of consumers was made by correlation analysis technique. it is thought that the results of this study are important both for scientists and social scientists. the application of the dna recombinant technology for food production is generating a great debate in our society with the participation of scientists trying to explain the way of obtaining these new foods and which are their implications; environmentalist groups and anti-biotechnology associations that systematically are against to the application of this technology; legislatives bodies and the public in general, represented by consumers' organizations that expresses their right to be informed. considering that university students will be the future professionals and consumers their opinion on this topic will be decisive in its success or failure, this research is aimed to performed a global and comparative study of the agrobiotechnology perception by students from different areas of knowledge and studies. this study was carried out during the academic years 2000-2004, being analyzed a total of 1516 valid surveys. the designed questionnaire included 30 questions relatives to: evaluation of the own knowledge and interest on the topic; evaluation of the information sources mainly used by university students to obtain nutritional information; the opinion about gm food labelling; risks/benefits perception; purchasing intention and support of biotechnology. results obtained showed that 37% of the university students interviewed have a clear positive perception of biotechnology, mainly the students of the health sciences area. these students understand the scientific terminology and they use the university as the main source of information. they support the development of the biotechnology and they consider that in a future it will report them benefits. other group (11%) has a clear negative perception, they are mainly students from law and art history. they do not understand the scientific terminology, they consider that biotechnology will cause them risks, and as a consequence they don't have intention of buying these foods. the technology of the dna recombinant can be also used to introduce in the plants genome the gene that codes a protein of interest for their use as antigen. the application of agrobiotechnology has allowed the development of a new generation of vaccines that try to reduce or to eliminate the inconveniences of the classic ones. for the new vaccines design, the detailed knowledge of the biology of the pathogen is considered. with this knowledge the genes implied in virulence can be inactivated or modified selectively. the term "edible vaccines" it is usually applies to the use of edible parts of the plants (tubers, fruits, leaves, etc.) genetically modified with the purpose to produce specific components (antigens) of a pathogen (virus, bacteria, etc.) against which is wanted to protect a person or animal. however, oral is not the best vaccination route since the quantity of antigen for an efficient immunization it is usually high, being also needed, the co administration of an adjuvant that stimulates the immune answer. on the other hand, it is also important to highlight that the levels of antigen accumulation in transgenic plants is usually lower than the necessary ones. another problem is the irregular accumulation of the antigen in the different parts of the plants, thus difficult the appropriate control of the doses. tannin is polyphenolic component having some antioxidant properties and exists in many plants and fruits. in pomegranate juice this component causes turbidity and haze. during fruit juice clarification by conventional gelatin method, all poly phenolic substances which are responsible for antioxidant activity are removed and as a result the quality of the product is reduced. in the present study tannase enzyme (tannin acyl hydrolase; ec 3.1.1.20) was used to decompose tannin to gallic acid and glucose and as the result the amount of turbidity of the juice is decreased, however, the antioxidant properties remain unchanged since tannin is not decomposed and not separated in the juice as it occurs in the gelatin method. the amount of gallic acid in pomegranate juice samples before and after addition of tannase was measured using hplc tests and the optimum temperature, the enzyme and juice contact time, ph, and solvent concentration for clarification of pomegranate juice were obtained as 45 • c, ph = 5.5, 2 h and 50 mm citrate buffer, respectively. the potential benefits of enzymatic clarification of pomegranate juice, that is preservation of antioxidant activity and hence increasing the quality of the fruit product, in comparison to that of conventional clarification method by gelatin introduce a new technique in turbidity and haze removed in tannin containing fruit juices. the objectives of this study are to investigate the inhibitory effect of low molecular weight chitosan for its use as biopreservatives of foods. the inhibitory activity of chitosan against escherichia coli and staphylococcus aureus was investigated by determining the effect of chitosan treatment on bacterial growth during culture and its effect on the viability of non-growing cells. the activity of chitosan was decreased considerably for both strains when sucrose was added to ts broth containing chitosan. the addition of ethanol affected little on the inhibition of chitosan against s. aureus. the influence of nacl on the activity of chitosan was similar to the influence of sucrose and ethanol. however, the experiments with non-growing cells showed that the ethanol enhance drastically the antibacterial activity of chitosan on s. aureus. the results presented in this paper demonstrate that its antibacterial activity may be affected considerably by common food additives such as sucrose, ethanol and nacl. in lactococcus lactis the enzymes phosphofructokinase (pfk), pyruvate kinase (pk) and lactate dehydrogenase (ldh) are uniquely encoded in the las operon. we have applied metabolic control analysis to study the role of this organisation. earlier work showed that ldh at wildtype level has zero control on glycolysis and growth rate but high negative control on formate production (c j formate ldh = −1.3). we find that pfk and pk have zero control on glycolysis and growth rate at the wildtype enzyme level but both enzymes exert strong positive control on the glycolytic flux at reduced activities. pk has high positive control on formate (c j formate pk = 0.9 − 1.1) and acetate production (c jacetate pk = 0.8 − 1.0), whereas pfk has no control on these fluxes. decreased expression of the entire las operon resulted in a strong decrease in growth rate and the glycolytic flux. increased las expression resulted in a slight decrease in the glycolytic flux. at the wildtype level the control was close to zero on both glycolysis and the pyruvate branches. the sum of control coefficients for the three enzymes individually was comparable to the control coefficient found for the entire operon at the wildtype level; the strong positive control by pk almost cancels out the negative control by ldh on formate production. the analysis suggests that co-regulation of pfk and pk provides a very efficient way to regulate glycolysis, and co-regulating pk and ldh allows the cells to maintain homolactic fermentation during regulation of glycolysis around wildtype level. bovine chymosin is used extensively in cheese production because of its specificity and low proteolytic activity. we are interested in the caprine chymosin as an alternative because in the canary islands cheese has traditionally been made using goats milk with extract from the abomasum of newborn goats as coagulating factor. we isolated and characterized the prochymosin cdna from the abomasum of milk-fed kid goats. this cdna predicts a polypeptide of 381 amino acid residues, with a signal peptide and a proenzyme region of 16 and 42 amino acids, respectively. the caprine preprochymosin has 99% and 94% identity with the corresponding lamb and calf sequences. the cdna fragment encoding prochymosin was fused in frame to the killer toxin signal sequence in a constitutive vector, and to the ␣-factor signal sequence-flag in an inducible expression vector. kluyveromyces lactis pm3-5c, k. lactis sel1, characterized by a "supersecreting" phenotype, and saccharomyces cerevisiae bj3505 were transformed with the recombinant plasmids. activated culture supernatants of yeast transformants showed milk-clotting activity. the flag-prochymosin fusion was purified from bj3505 culture supernatants by affinity chromatography. after activation at acid ph, proteolytic activity assayed toward casein fractions showed that the recombinant caprine chymosin specifically hydrolyzed -casein. the recombinant caprine enzyme could be an alternative milk coagulant in cheese making. lipid accumulation in schizochytrium g13/2s was studied under batch and continuous culture. different glucose and glutamate source concentrations were supplemented in a defined medium. during batch cultivation, lipid accumulation occurred towards the end of the growth phase but ceased when cell proliferation stopped. under continuous culture, as dilution rate decreased from 0.08 to 0.02 h −1 , both cell dry weight and total fatty acid content (tfa) of the cell increased. with a constant dilution rate of 0.04 h −1 , nitrogen limitation induced lipid synthesis (28% tfa) as described for other lipid-accumulating organisms. however, with carbon-limited conditions, some lipid accumulation was still possible, the tfa being 22%. finally, the batch and continuous culture methods are compared for docosahexaenoic acid (22:6, n − 6) production. the objectives of this study is to investigate the inhibitory effect of low molecular weight chitosan for its use as biopreservatives of foods. the inhibitory activity of chitosan against e. coli and staphylococcus aureus was investigated by determining the effect of chitosan treatment on bacterial growth during culture and its effect on the viability of non-growing cells. the activity of chitosan was decreased considerably for both strains when sucrose was added to ts broth containing chitosan. the addition of ethanol affected little on the inhibition of chitosan against s. aureus. the influence of nacl on the activity of chitosan was similar to the influence of sucrose and ethanol. however, the experiments with non-growing cells showed that the ethanol enhance drastically the antibacterial activity of chitosan on s. aureus. the results presented in this paper demonstrate that its antibacterial activity may be affected considerably by common food additives such as sucrose, ethanol and nacl. nitrite-oxidizing bacteria catalyze an essential step of nitrogen elimination in biological wastewater treatment. recently, novel and yet uncultured nitrite-oxidizing nitrospira-like bacteria were found to be abundant in municipal and industrial wastewater treatment systems where they outcompete nitrobacter, which has long been considered as the organism responsible for nitrite oxidation in bioreactors. despite the importance of nitrospira-like bacteria for wastewater treatment and for nitrogen fluxes in natural ecosystems, little is known about their ecophysiology and interactions with other organisms. cultivation-independent molecular techniques were applied to investigate the diversity, distribution, and physiological and genetic features of nitrospira-like bacteria in nitrifying activated sludge and biofilm. a surprisingly high diversity of these organisms was found to exist in these engineered and in natural habitats. moreover, significant physiological differences could be identified among various phylogenetic sublineages in the genus nitrospira. quantitative co-localization analyses performed by novel image analysis software revealed that these metabolic features are reflected by the spatial organization of nitrifiers living in biofilm and activated sludge flocs. based on an environmental genomics approach the genome of a nitrospira-like bacterium found in activated sludge is being analyzed. results obtained so far point at unexpected physiological capabilities of this organism, and allow us to propose that nitrospira-like bacteria may also play roles in the bioremediation of (per)chlorate and chlorite. the activated sludge process is the most common way to remove organic matter, nitrogen and phosphorus from wastewater by microbiologically means. knowledge about the microorganisms involved is fundamental for optimisation of existing plants and development of new plants and process designs. many of the bacteria believed to be involved in nitrification, denitrification, biological phosphorusremoval, and removal of organic matter in full scale plants are now identified by use of molecular methods. recent developments in experimental approaches have allowed the study of the ecophysiology of these uncultured and potentially important bacteria, thus providing a better understanding of their function in full-scale activated sludge ecosystems. relatively few dominant species in each functional group (e.g. denitrifiers and polyphosphate accumulating organisms) seems to be present. some species appear to be very specialized regarding nutrient requirements while others are more versatile. a new method for mercury remediation from industrial wastewater based on the enzymatic reduction of mercury by live mercury resistant bacteria immobilized on the pumice particles has been developed in gbf, germany, and implemented in the industrial scale (unknown). the experience gained during operation of this instalation led to the idea, that the process of bioremediation may be integrated in one bioreactor with the sorption of mercury from wastewater, by immobilization of the bacteria directly on the activated carbon. for this it was necessary to define several significant parameters of the activated carbon used and the sorption process itself. the paper presents results of the equilibrium and kinetics investigations of the process of mercury sorption from aqueous solutions onto seven different types of activated carbon. the effective diffusion coefficients in the particles were obtained from the transient-state experiments and the sorption isotherms, saturation capacity of the sorbents and its dependence on the temperature and ph were identified. then the hydrodynamic and sorption characteristics of the activated carbon bed in a laboratory-scale fixed-bed bioreactor were investigated in different process conditions (mercury concentration, volumetric flow rate, temperature, ph). the results (effective capacity of the bed, dispersion and diffusion coefficients, mass transfer coefficient) enable implementation of this bioreactor for modified, integrated process of mercury bioremediation from industrial wastewaters. research supported by the grant kbn 4 t09c 013 25. bacterial cr(vi) reductases convert the very mobile toxic cr(vi) to the less toxic and less mobile cr(iii). the ability to reduce cr(vi) was studied on cell extracts of ochrobactrum tritici strain 5bvl1 and microbacterium sp. strain 3a. both microorganisms were isolated from the same sample of chromium-contaminated sludge, taken from a wastewater treatment plant. while in the first case activity was found to be associated with the intracellular soluble extract, in the second case it was a process occurring extracellularly. cr(vi) reduction by the intracellular soluble extracts of strain 5bvl1 required the presence of nadh or nadph as electron-donor, while the extracellular fraction of strain 3a only used nadph. several studies were made on strain 5bvl1 intracellular soluble extracts. a k m of 26.11 m cr(vi) and a v max of 5.75 ± 0.13 nmol cr(vi) min −1 mg −1 protein were estimated from the lineaweaver-burk plot and michaelis-menten non-linear regression. the temperature and the ph optima for cr(vi) reduction were 37.5 • c and 5.0, respectively. hyperthermus butylicus is an anaerobic hyperthermophilic crenarchaeon, isolated from the solfataric sea floor off sáo migel island, azores (zillig et al., 1990) . h. butylicus grows at up to 108 • c (optimally between 95 and 106 • c) at ph 7. it can utilize peptides, polysaccharides, and other substrates, as carbon sources to produce acetate, butyrate, and n-butanol. the capability to produce enzymes (e.g. hydrolases, dna and rna polymerases, etc.) that can tolerate and function at temperatures 20 • c higher than most other thermophilic archaea, renders h. butylicus of particular interest to the biotechnology industry. the complete genome sequence of h. butylicus was determined and it contains 1,667,186 bp on a single circular chromosome. 1695 protein encoding genes were identified which use a high level of uug and gug start codons. many of these were assigned functions on the basis of sequence comparisons. our analyses revealed some unusual metabolic properties in h. butylicus. several sugar transporters were identified, although the set of genes required for glycolysis is incomplete. moreover, genes encoding enzymes converting glucose to trioses are absent and no genes encoding enzymes of the pentose phosphate cycle or the kdpg pathway were detected. the h. butylicus genome encodes many proteases and peptidases although the lon proteases, encoded in all other archaeal genomes, are absent. although it was reported that h. butylicus does not utilize free amino acids in the media, genes for amino acid transporters were identified, and several proteins involved in di-or oligo-peptide transport are encoded. genes encoding signal peptidases are absent. we will summarize gene products of special biotechnological interest. reference zillig et al., 1990. j. bact. 172, 3959-3965. 2 hot genomics: insights in the thermophilic lifestyle of thermus thermophilus from its complete genome holger brüggemann 1,2 , anke henne 1 , gerhard gottschalk 1 : 1 göttingen genomics laboratory, institute of microbiology and genetics, university of göttingen, germany; 2 institut pasteur, unité de génomique des microorganismes pathogènes, paris, france thermus thermophilus is an extremely thermophilic, halotolerant bacterium, which was originally isolated from a natural thermal environment. recently completed genome sequences of two strains, hb27 and hb8, provide a solid foundation for investigating many aspects of thermophilic lifestyle; these range from molecular stability determinants to key elements of organismic physiology. in addition, the species has considerable biotechnological potential; many thermostable proteins isolated from members of the genus thermus are indispensable in research and in industrial applications. the closely related genera thermus and deinoccoocus belong to a distinct branch of bacteria called the deinococcus-thermus group. genome comparison of t. thermophilus and d. radiodurans, a mesophilic organism, which exhibits high resistant to radiation, oxidative stress, and desiccation, is of particular interest for the identification and exploration of thermophilic determinants. a large number of orthologs with a high degree of sequence identity are shared between the two species. this opens the opportunity for comparative studies of conformational and chemical thermostability of proteins, as well as for the identification of specific traits for each organism, explaining their unique physiological properties and their intriguing differences in stress tolerance. although strains hb27 and hb8 share a highly conserved chromosome, striking differences can be found between their megaplasmids, which encode a huge proportion of genes not found in the genome of d. radiodurans. possible contributions made by the megaplasmids to a thermophilic lifestyle will be discussed. microorganisms that can live in high temperatures, extreme ph and high salt concentration are called extromophiles. extromophilic microorganisms have extended our knowledge and understanding of fundamental questions such as the origin of life. the ability to grow in extreme conditions and to produce stable proteins makes extremopliles very attractive for the researchers and also for the industry. extremozymes from extremophiles have a great economic potential in many industrial processes, including agricultural, chemical and pharmaceutical applications. concurrent development of protein engineering will increase the application of enzymes from extremophiles in industry. turkey has vast and various ecologi-cal areas, and so it has a broad microbial diversity. based on the extremophilles which defined in the scope of this project, halophilic microorganisms produced industrially important proteins were isolated from ç amaltı saltern area in izmir, turkey. in this work, growth of isolates at different temperature, salinity and ph values were investigated to determine the effects of various growth conditions. eight isolates grow at ph between 6.50 and 8.50 and two isolates at 6.50-7.50. they grow at temperature between 37 and 55 • c and salt concentration between 3% and 25%. the results of some phenotypic characters showed that they are gram (−) and oxidase,ürease, dnase and nitrate reduction are (−), and catalase (+). they used d(+) glucose, maltose, lactose, sucrose, l(+) arabinose, d(+) mannose, glycerol and four of isolates used d(+) xylose as a carbon source. the isolates resistant to erythromycine, ampicilin sulbactam, cefoxitin, penicillin, bacitracin, novabiocin, amikacin and sensitive to ceftazidine, ciprofloxacin, amoxycillin/clavulanic acid, imipenem, chloromphenicol, ceftazidime/clavulanic acid, aztreonam, cefepime, cefotaxime, cefoperazone amoxicillin. this project was supported by tubitak through project tbag 2321-103t069. the technology of producing renewable energy sources such as ethanol, methane and hydrogen from biomass holds the potential of creating in-house energy resources while lowering the emission of greenhouse gasses as demanded by the kyoto protocol. recently, goals were defined for the european union determining that 5.75% of the transportation fuel has to come from biofuels in year 2010. a large-scale implementation of biofuels into the transportation sector will demand that lignocellulosic biomass, which is found in a surplus throughout the world is used as the raw material for the production process. the presentation will include a comprehensive description of the special bio-refinery concept developed in denmark for production of biofuels and other valuable products from straw. the concept includes several innovative steps such as a pre-treatment method using wet oxidation, on-site production of enzymes and a continuous fermentation process using a genetic modified thermophilic bacterium. by co-producing several biofuels in the plant optimal use of the biomass has been assured and the price of for instance of bioethanol is getting close to conventional oil-based fuels. optimizing each step in the bio-refinery, while having the full integration in mind, will be the way to make an economical viable biofuel production. in the presentation we will present our road map for achieving this goal in the nearest future. replacement of gasoline by liquid fuels produced from renewable sources is a high-priority goal in many countries worldwide. one such fuel, which has been found well suited, is ethanol. it may be produced from various lignocellulosic materials, such as forest and agricultural residues, which are fairly inexpensive. to compete with gasoline the production cost must be substantially lowered. ethanol production from lignocellulose comprises the following main steps: hydrolysis of hemicellulose, hydrolysis of cellulose, fermentation, separation of lignin, recovery and concentration of ethanol and wastewater handling. the enzymatic hydrolysis and fermentation can either be run separately (shf) or combined into a simultaneous saccharification and fermentation (ssf). the latter has been shown to result in higher ethanol yields than shf. some of the most important factors to reduce the cost are: efficient utilisation of the raw material by high ethanol yields, high productivity, high ethanol concentration in the feed to distillation and process integration in order to reduce capital cost and energy demand. in the last years we have performed several studies on the hydrolysis and fermentation of various forest and agricultural residues in a mini-pilot to improve the overall yield of ethanol and to reduce the energy demand and production cost. steam pretreatment, with small addition of acid catalyst, has resulted in sugar yields close to 90% of the theoretical for various types of raw materials, e.g. spruce, salix and corn stover. the ssf has been developed and optimized to give high yield of ethanol. for spruce an ethanol yield of about 80% of theoretical based on the composition of the raw material has so far been obtained using a two-stage steam-pretreatment of so 2 impregnated raw material followed by ssf. improvements of the ssf step, in the form of high dry matter content, recirculation of process streams and adapted yeast have resulted in ethanol concentrations around 45 g/l leading to substantial reduction in energy demand and production cost. these improvements have been assessed by techno-economic evaluation to determine the effect on the ethanol production cost. the process has been further optimised by process integration to further reduce the energy demand. the ethanol production cost was estimated to be around 0.38-0.46 euro/l ethanol assuming a yearly capacity of 200 000 tonnes raw material (dry matter). production of bioethanol from spent grain, a by-product of beer production sho shindo, tadanori tachibana, akita research institute of food and brewing, akita-city, akita 010-1623, japan. e-mail: shindo@arif.pref.akita.jp (s. shindo) the breweries generate one million tons of spent grain every year, and about 20% of the spent grain is recycled in japan. therefore, it is environmentally and economically significant to consider the production of ethyl alcohol as biomass energy using the spent grain from the breweries industry. ethyl alcohol production from spent grain with immobilized yeast cells was investigated. spent grains were liquefied by a steam explosion treatment to obtain liquefied sugar. when 1 kg of wet spent grain was treated under the 30 kg/cm 2 pressure for 1 min using a 5 l steam explosion reactor, 60 g of total sugar was obtained from the liquefied spent grain. furthermore, 1.3% (w/v) of glucose, 0.4% (w/v) of xylose, and 0.1% (w/v) of arabinose were produced when the liquefied spent grain was treated with glucoamylase, cellulase, and hemicellulase enzymes. ethyl alcohol production was carried out by immobilized sacchromyces cereviseae and immobilized yamadazyma stipitis simultaneously from liquefied spent grain. both yeast cells were immobilized on the glass beads carrier. xylose and arabinose were consumed after glucose was consumed completely during ethyl alcohol production. 5.8% (v/v) ethyl alcohol was produced from liquefied spent grain that was adjusted 17% of initial sugar concentration after 2 days. the vegetable oils constitute a resource of renewable potential for the production of fuels, becoming a viable alternative when compared to the diesel from petroleum. among the vegetable oils, the extracted oil of the castor plant seeds is a promising alternative source because it is constituted mainly of the ricinoleic acid (12-hydroxy-9octadecenoic) that represents 90% of the total constitution of the oil approximately. the biodiesel obtained from castor oil can be defined chemically as being a mixture of methyl esters or ethyl esters of carboxylic acids synthesized by transesterification reaction of the existent triglyceride and an alcohol of little chain through the use of alkaline or enzymatic catalysts. in this work, we described the results found in castor oil with different degrees of purity. initially, it was made a rheological characterization followed by structural characterization (rmn 13c, rmn 1h and infrared) and thermal characterization (dtg, dta and dsc) of the crude and refined castor oil. it has been also measured the hydroxyl tenor, acidity index, saponification index and iodine index in different oils. later, these results were used to evaluate possible differences in the quality of the biodiesel (ethyl esters) produced in the enzymatic alcoholysis of the castor oil catalyzed by lipases (novozym 435, liposyme rm im and lipozyme tl im). the degree of substitution of castor oil derivative was performed by titration with 0.1n hcl and confirmed by tlc analysis and the results showed conversion rates about 90%. has been demonstrated to have a wide range of health benefits such as prevention and therapy of various cancers, amelioration of heart disease, and prevention of renal stone formation as well as complications from diabetes. on the hand, lower phosphorylated forms of inositol, especially inositol trisphosphate (ip3) and inositol tetrakisphosphate (ip4) are important signal transduction molecules within the cells both in plants and the animal kingdom. it has been hypothesized that at least the anticancer function of ip6 is mediated via these lower inositol phosphates. the diversity and practical unavailability of the individual myo-inositol phosphates preclude their investigation. phytases, which catalyze the sequential hydrolysis of phytate, render production of defined myoinositol phosphates in pure form and sufficient quantities. different phytases may result in different positional isomers of myo-inositol phosphates and therefore different biochemical properties. phytases differing in ph optima, substrate specificity, and specificity of hydrolysis have been identified in plants and microorganisms. in this paper the dephosphorylation pathway of the novel phyfauia1 was compared to other bacterial phytate degrading enzymes. preliminary results have shown that phyfauia1 converted ip6 into ip5 (myoinositol 1,2,3,5,6-pentakisphosphates) and another isomer, which is yet to be elucidated. in a denitrifying pilot plant reactor, a new obligately anaerobic ammonium oxidation (anammox) process with great potential for nitrogen removal for high strength wastewater was discovered. after transfer of the complex microbial community to a laboratory sbr system, a highly enriched population, dominated by a single anaerobic chemolithoautotrophic bacterium related to the planctomycetes was obtained. the bacterium was purified via percoll centrifugation and characterized as 'candidatus brocadia anammoxidans'. survey of different wastewater treatment plants using anammox specific 16s rrna gene primers and anammox specific oligonucleotide probes revealed the presence of at least four other anammox bacteria, tentatively named 'candidatus kuenenia stuttgartiensis', 'candidatus brocadia fulgida', 'candidatus scalindua wagneri' and 'candidatus scalindua brodae'. a close relative of the two scalindua species, 'candidatus scalindua sorokinii' was found to be responsible for about 50% of the nitrogen conversion in the anoxic zone of the black sea and in the benguela upwelling system along the namibian coast, making anammox an important player in the global nitrogen cycle. electron microscopic studies of all five anammox bacteria showed that several prokaryotic membrane-bounded compartments are present inside the cytoplasm, which are surrounded by unique ladderane lipids. hydroxylamine oxidoreductase, a key anammox enzyme, was present exclusively inside one of these compartments, named the 'anammoxosome'. unique peptides fragments of the purified hao were used to locate the hao gene in genome assembly of 'candidatus kuenenia stuttgartiensis'. the implementation of the anammox process in the treatment of wastewater with high ammonium concentrations was started at the treatment plant in rotterdam, the netherlands, where it is combined with the partial nitrification process sharon. the estimated price for nitrogen removal with partial nitrification and anammox is about 0.75 euro/kg n. gas lift reactors could sustain the highest anammox capacity at 8.9 kg n removed/m 3 reactor per day. an alternative configuration of anammox is the oxygen-limited canon process in which aerobic ammonium-oxidizing bacteria protect anammox bacteria from oxygen and produce the necessary nitrite. maximum nitrogen removal with canon in gas lift reactors was 1.5 kg n/m 3 reactor per day. using several different conditions and parameters, the competition and co-existence of aerobic and anaerobic ammonium-oxidizing bacteria were modeled. in addition to ammonia, urea was also converted after a 2-week adaptation in the canon system. recently it was shown that anammox bacteria can use organic acids as additional energy source. murray moo-young, wa anderson department of chemical engineering, university of waterloo, waterloo, ont., canada n2l 3g1 bioreactors are central to the bioremediation of contaminated environments of water, air or soil. in all three areas of application, bioreactor design is critical to the development of new or improved processes. this overview focuses on the physical limitations of bioreactors caused by biological requirements. the information is based on our own research findings. the need for more applicationsoriented bioremediation research becomes apparent. for technoeconomic reasons, the airlift type has often been the bioreactor of choice for most bioremediations. however, lack of adequate understanding of the quantitative effects of operating conditions on its performance has been an ongoing concern. these effects have been characterized for engineering implementation. to enhance productivity, innovative pretreatment techniques of the polluted sources have also been developed using photocatalytic and chemical oxidation methods. case studies on petrochemical-contaminated water and soil reveal significant enhancement potentials. other studies on microbial biofilters for air bioremediation indicate that the active mass of the biological consortia is not sufficiently understood for rational design. analysis and retrofit design of wastewater treatment facilities using process simulation tools demetri petrides, alexandros koulouris, intelligen, inc., scotch plains, nj 07076, usa. email: dpetrides@intelligen.com (d. petrides) process simulators have been used in the petroleum and chemical industries for over four decades to facilitate the design of new processes and optimize the performance of existing ones. similar benefits can be derived from the use of such tools in the environmental arena, particularly in the field of physical and biological treatment of municipal and industrial wastewater. specifically, process simulators can be used to evaluate and improve options for: (1) more efficient removal of nutrients (e.g., organic nitrogen and phosphorous) that cause eutrofication, (2) estimation and control of volatile organic compound (voc) emissions from open tanks, and (3) more efficient removal and control of hazardous compounds. the potential benefits will be illustrated with cases studies involving both municipal and industrial wastewater facilities. the microbial reduction of metals has showed recent interest as these transformations can play crucial roles in the cycling of both inorganic and organic species in a range of environments and, if harnessed, may offer the basis for a wide range of innovative biotechnological processes. under certain conditions, however, microbial metal reduction can also mobilise toxic metals with potentially calamitous effects on human health. some effluents present heavy metals as soluble compounds, several microorganisms have the capacity to precipitate these metals as insoluble compounds, and this fact allows the collection and separation of these metallic precipitates from contaminated medium. sulfate-reducing bacteria (srb), under anaerobic conditions, oxidize simple organic compounds (such as acetic acid and lactic acid) by utilizing sulfate as an electron acceptor and generate hydrogen sulfide. hydrogen sulfide reacts with heavy metal ions to form insoluble metal sulfides that can be easily separated from a solution. the purpose of this work was study the capacity of desulfovibrio sp. cultures to reduce mixtures of the heavy metals in presence or not of petroleum. for it the experimental design 2 k (k = 5) was carried out. the five studied factors were cr, cu, mn, zn and petroleum. the study was carry out with desulfovibrio sp. batch studies were performed in 50 ml sealed bottles with different concentrations (cr(iii)-10 ppm, cu(ii)-5 ppm, mn(ii)-10 ppm, zn(ii)-15 ppm) of metal sulfate and 2 g l −1 of petroleum. during batch incubation the dissolved concentration of metal studied in supernatant were decreased to undetectable levels for zn (70-100%), however with cu (40-60%), mn (40-70%) and cr (50-80%). the development of continuous process with sulfatereducing bacteria seems to be a suitable alternative to reduce metals in solution from contaminated media such as industrial or mine effluents. after these preliminary results, some experiments in course are focused to study that purpose. reduction of odour emissions from livestock buildings using a bioscrubbing system morten øgendahl, nawaf abu-khalaf, jens jørgen lønsmann iversen department of biochemistry and molecular biology, university of southern denmark, dk-5230 odense m, denmark. e-mail: tvede@bmb.sdu.dk (m. øgendahl) a bioscrubbing system for reducing odour emissions from livestock buildings is presented. the bioscrubbing system consists of two separate units; an absorption column and a water purification module. the absorption column is mounted in the ventilations stacks in the livestock buildings absorbing odorants in the effluent air flow. the odorants are absorbed in a spray of droplets formed by a grid of high pressure nozzles in the inlet of the absorption column. the spray of droplets is extracted from the air flow and pumped to a centrally located water purification module, an inverse three phase fluidised bed bioreactor, where the bio-degradation of the absorbed odorants occurs. the bioreactor features a split sparging system for maximum mixing and aeration. the cleaned water is recirculated to the absorption column. an electronic tongue will quantify key odorants in the bioreactor. the absorption column is designed to be retrofitted into existing livestock building ventilation systems. the water purification module is constructed in standard size units simplifying scaling to match the requirements of individual applications. the total bioreactor volume is increased by increasing the number of standard bioreactors. this work describes a "light off" toxicity bioassay sensor based on whole cell genetically modified bioluminescent bacteria. the biosensor was constructed by mating between the environmentally isolated phenol-degrading acinetobacter sp. strain df4 and the plasmid putk2 that is an inc p␤ plasmid with the bioluminescence genes luxcdabe inserted into a genetic region involved in plasmid replication and transfer. subsequently, the bioreporter designated df4/putk2 and used to investigate phenolics toxicity. among examined phenolics, pentachlorophenol, catechol and nitrophenol recorded the fastest effect on the bioluminescence of bioreporter df4/putk2 over incubation period of 350 min. the effect of various concentrations of phenol and its derivatives either in an individual, duple or triple mixture forms on the bioluminescence response of the constructed bioreporter df4/putk2 were also examined. significant reduction of the bioluminescence was observed whenever a mixture contained pentachlorophenol, catechol and nitrophenol, respectively. to develop a system appropriate to commercialize, the constructed bioreporter df4/putk2 was subjected for immobilization in microtiter plates using several entrapment gels. after a selection of materials was tried, lb/agar was chosen as the most suitable candidate material. characterization of key odour compounds in an air wet scrubber is presented. the key odour compounds represent five chemical groups, i.e. sulphide, alcohol, volatile fatty acids (vfas), phenol and indole. direct aqueous injection (dai) and solid phase extraction (spe) methods were used before injection of key odorants into the gas chromatography-flame ionisation detection (gc-fid). the dai and spe methods were efficient in the identification of odour compounds in the wet scrubber. the spe method had a high recovery and can be more effective in the identification of compounds at low initial concentration. however, dai showed a better linearity and a lower limit of detection (lod) than the spe method. the dai method was the method of choice for characterization, as it is cheaper, easier to handle and highly applicable. at least two odorants, phenol and 1-butanol, were quantified successfully using the dai method. their lod was less than their odour detection limit in the wet scrubber. dai method can be used as a reference measurement method for any further analytical application, e.g. electronic tongue. recent developments in biotechnology enabled the widespread use of microbial enzymes in textile, detergent, food and dairy industries and also in various environmental applications. microorganisms which live at extremes of temperature, ph and salinity, produce extremozymes that offer many exciting opportunities for their use in clean production. in this study, microorganisms were isolated from camaltı saltern area ini̇zmir, turkey. effect of medium salinity on the growth of these microorganisms was determined. seven out of 10 isolates required salt for growth. the salinity ranges at which growth was detected were: 5-25% for two isolates, 6-25% for one isolate, 7-25% for two isolates and 8-25% for one isolate. the isolates were also screened for their capability of producing industrially important enzymes such as amylase, protease, lipase, xylanase and cellulose which are widely used not only in textile, detergent, food and dairy industries but also in various environmental applications. all of the isolates were found to be producers of both amylase and xylanase enzymes at varying salinity array within 5-30% salt concentration range at ph 7.0. extracellular protease activity was detected in the medium of all isolates grown at 5, 10, 15, 20 and 25% salinity at both ph 7.0 (optimum growth ph) and ph 9.0. out of 10 isolates, 9, 10 and 9 were found to produce cellulase enzyme when the salt concentrations were 5, 10 and 15%, respectively. at 20% salt concentration, only one isolate was found to be cellulase enzyme producer. none of the isolates were found to produce lipase enzyme at 5-30% salt concentration range. this project was supported by tubitak through project tbag 2321-103t069. chemical engineering department, middle east technical university, ankara 06531, turkey. e-mail: ubakir@metu.edu.tr (u. bakir) glass and ceramic tiles are very widely used industrial materials. in most cases, periodical cleaning is required to maintain their optical properties such as transparency and visual aspects. because of the ever-growing demand for healthy living, there is a keen interest in materials capable of killing harmful microorganisms. the application of these tiles in care facilities to reduce the spread of infections, in public and residental places to improve hygienic conditions are of general interest. in this study the aim is developing methods to apply thin film coatings on glass tiles to make them anti-bacterial by utilizing photocatalysis and investigating their anti-bacterial properties. semiconductors because of their reasonable band gap energies find great attraction through this purpose. the photocatalytic property of semiconductors are used in this process. oxidising radicals are formed on the coated surfaces and these radicals attacks the organic pollutants and bacteria on contact with the surface. titanium dioxide (tio 2 ) coated surfaces are considered to be very effective against organic and inorganic materials, as well as against bacteria. in the experimental procedure coating solution is prepared by sol-gel technique. after pretreatment of surfaces, the coating solution is applied on the surfaces by dip-coating method. after appropriate thermal treatments, to achieve thin, dense and strong coatings, indicator microorganism is directly applied on the coated surfaces and illuminated under solar simulater light source. finally, the number of surviving microorganisms are determined. in this study, the effects of titanium dioxide (tio 2 ), tin oxide (sno 2 ) solutions and metal doping to these coating solutions on anti-bacterial function were investigated. as a result of this study, the number of escherichia coli that is used as indicator microorganism, on tio 2 and sno 2 coated glasses with respect to the control glass reduced by 80-85% and 40-45%, respectively. doping with metals increased the activity of the coatings, hence the number of surviving microorganisms decreased. activity of a methanogenic ecosystem during the primary contact with a solid support s. michaud, n. bernet, p. buffière, j.p. delgenès inra-lbe, avenue des etangs, f-11100 narbonne, france in this paper, the biological activity during the first initial contact between a methanogenic sludge and a solid support was investigated in batch experiments, at different solid concentrations, using two different granular solid materials and with glucose as the main organic substrate. in all cases, the introduction of a solid material in a methanogenic suspended biomass induced a response of the anaerobic microorganisms, after a lag phase during which biological activity was not detected. this lag phase could be the consequence of a physical stress induced by the first contact between microbial cells and the solid surface. this lag phase was not observed when the biomass used originated from a biofilm reactor, i.e. using a biomass previously exposed to a solid material. a change in the metabolism of organic matter from catabolism and methane production toward production of other compounds could be observed, characterised by a sharp decrease of the methane yield in the anaerobic system. analyses of the gas and liquid phases did not show the production of any new gaseous or soluble compound as the biological end product of this activity. this suggests the production of non-soluble compounds by an anabolic pathway, which could indicate the initiation of biofilm formation. this metabolic activity was shown to be directly correlated to the ratio between the solid surface introduced and the microorganism concentration in the anaerobic culture (m 2 g vs −1 ). from kinetic observations, it could be observed that acetogenic methanogenesis recovered more rapidly than syntrophic propionate and butyrate degradation. evaluating microbial diversity of hydrocarbon degrading bacteria cleantis braithwaite, howard rosser, tawfiq al-ibrahim, hussain, al-bandi research and development center, saudi aramco, dhahran, saudi arabia the analysis of microbial diversity with molecular methods is central to isolating and identifying new and potential biocatalysts resources for research and industry. the ability to degrade hydrocarbon components of petroleum is widespread among bacteria, and is an effective method for remediation of a variety of ecosystems. due to the high carbon content of oil and the low levels of other nutrients essential for microbial growth, treatment of oil with phosphorus and nitrogen is generally required to enhance the growth of hydrocarbon-degrading bacteria and to stimulate oil sludge degradation. in this research study, three types of oily sludges from a gas plant, refinery, and terminal facilities were treated with nutrients. to assess the microbial diversity, both biolog culture method and culture independent polymerase chain reaction (pcr,) denaturing gradient gel electrophoresis (dgge) methods were used. nutrient addition significantly improved oil sludge degradation. we identified and characterized several hydrocarbon degrading bacterial strains that have the ability to convert petroleum. these bacteria included representatives both gram positive and gram-negative genera. there were slight difference in the quantity and type of hydrocarbon degrading bacteria found in the three sites. this is the first molecular analysis of hydrocarbon degrading microbial population in saudi arabian operations. mussel adhesive proteins, including the 20-plus variants of foot protein type 3 (fp-3), have been suggested as potential environmentally friendly adhesives for use in aqueous conditions and in medicine. here we report the novel production of a recombinant mytilus galloprovincialis foot protein type 3 variant a (mgfp-3a) fused with a hexahistidine affinity ligand in escherichia coli, and its ∼99% purification with affinity chromatography. recombinant mgfp-3a showed a superior purification yield and better apparent solubility compared to those of the previously reported recombinant m. galloprovincialis foot protein type 5 (mgfp-5). the adsorption abilities and adhesion forces of purified recombinant mgfp-3a were compared with those of cell-tak (a commercial mussel extract adhesive) and mgfp-5 using qcm analysis and modified afm, respectively. these assays showed that the adhesive ability of recombinant mgfp-3a was comparable to that of cell-tak but lower than that of recombinant mgfp-5. collectively, these results indicate that recombinant mgfp-3a may be useful as a commercial bioadhesive or an adhesive ingredient in medical or underwater environments. cresol, a monomethylated phenol, is an aromatic compound. the environmental protection agency (epa) has determined the carcinogenic potential of cresol. various options are being examined for the degradation of cresol because of their unavoidable large scale production and toxicity. many aromatic hydrocarbons can be used as electron donors aerobically by species of pseudomonas, thus leading to the ring cleavage of these compounds. in the present study, pseudomonas strains were isolated from activated sludge collected from sewage treatment plant. it was repeatedly transferred onto nutrient agar plate to check the purity of the culture. the organism was grown aerobically in an inorganic medium with p-cresol as the solitary carbon source. pseudomonas was confirmed by the expression of green pigment, gram staining and biochemical tests including koh, catalase, nitrate reduction and carbohydrate fermentation reaction. inoculation status was used to determine the rate of degradation of p-cresol. the effect of temperature on p-cresol degradation was studied. moreover, the effect of different concentrations of the aromatic compounds on pseudomonas as well as substrate variability was also documented. phenolic intermediates were estimated colorimetrically using 4-aminoantipyrene, folin-lowry method, uv spectrophotometry and hplc. the results indicated that pseudomonas could degrade up to 300 mg/l of p-cresol within 10 h. pseudomonas sp. exhibited good metabolic versatility and degraded other aromatic compounds including m-cresol and p-hydroxybenzoic acid. we conclude that this strain of pseudomonas has excellent potential for bioaugmenting the degradation of p-cresol-containing waste water treatment units. a considerable amount of waste cooking oil is produced by the restaurant industry worldwide. this poses a significant environmental and economic problem, since high oil and grease concentrations in the sewage system could lead to pipes occlusion and decreased efficiency in water treatment operation plants. therefore, sending these wastes to recycling companies or hazardous waste processors is usually required. yarrowia lipolytica, a well-known lipase producer, requires the presence of lipidic compounds (i.e. vegetable oils) to boost enzyme biosynthesis. in this work, the suitability of waste cooking oil as lipase inducer in submerged cultures of this yeast has been assessed. if successful, this procedure could allow both the degradation of an abundant waste and its valorisation as a raw material for the production of a high added value product. the microorganism was grown in a liquid medium to which various amounts of waste cooking oil were added. biodegradation degrees up to 80% (measured as decrease in cod) were obtained after 3 days of treatment. also, initial glucose concentration in the basal medium seemed to influence the efficiency of the process. on the other hand, addition of waste oil led to a significant increase in lipase production (more than two-fold), compared to oil-free cultures. moreover, chain-length specificity of the produced enzymes was significantly different: high activity towards medium chain length esters was found, which hinted to the occurrence of both lipases and esterases. biodesulfurization: a documental review j. ferrer, simon bolivar university, environmental engineering lab. caracas, venezuela a documental review about larger interest aspects in biodesulfurization technique is showed. especifically, the investigation is related to general framework and the justification of this technique, degradatives pathways elucidated up to now, involved microorganism, important elements in development of bacterial desulfurization and progress areas, and future tendency. in situ bioremediation of a p-nitrophenol contaminated site and assessment of its community structure debarati paul, gunjan pandey, sumeet labana, rakesh k. jain institute of microbial technology, sector 39a, chandigarh 160036, india. e-mail: rkj@imtech.res.in (r.k. jain) biodegradation of p-nitrophenol (pnp), a priority pollutant, was studied as a model system for bioremediation of sites contaminated with nitroaromatic/organic compounds. bioremediation studies were carried out in pnp-spiked soil in small plots under natural field conditions using arthrobacter protophormiae rkj100. role of carrier material was examined by immobilizing the bacteria on corncob powder prior to adding them to soil. these studies demonstrated successful removal of pnp by immobilized cells that were able to deplete pnp completely in 5 days, whereas free cells were able to deplete 75% pnp in the same time period. monitoring the fate of released bacteria revealed fairly stable population of the cells when they were immobilized on corncob powder throughout the period of study. on the other hand, there was a decrease of 2.7 log units in colony forming units of free cells at the end of the study (30 days). bacterial community structure and diversity was also studied for the pesticidecontaminated site wherein the effect of addition of an exogenous strain on the existing soil community structure and on soil functionality was determined using molecular techniques. as revealed by restriction fragment length polymorphism (rflp) studies 45 different phylotypes could be identified on the basis of similar banding patterns. sequencing of representative clones of each phylopyte showed that the community structure of the pesticide-contaminated soil mainly constituted of proteobacteria and actinomycetes. terminal fragment length polymorphism (t-rflp) analysis showed only subtle changes in community structure during the process of bioremediation. bacteriocins encompass an array of structurally different molecules produced by a number of phylogenetically distinct bacterial groups and trigger the killing of the same or closely related species. the recombined escherichia coli strain harboring a bacterocin coding region of xanthomonas campestris pv glycines 8ra was disrupted to obtain cell homogenate. peptidic xanthomonas bacteriocins (pxb) were separated by lowering ph and adding salt. the resulting pxb's were partially purified using ion exchangers, gel filtration. two final active fractions, a and b, were obtained with a yield of 0.005% and 500-1000-fold purification. the activity of pxb was stable at the ph ranging from 7.0 to 10.0. andreja kresal, vanja kokol, vera golob textile department, university of maribor, 2000, slovenia wastewater from textile dyeing industries is characterized by high chemical and biological oxygen demands (cod and bod) and intense color due to the extensive use of synthetic dyes. as dyes of complex aromatic structures are resistant to removal by the typical microbial population and may be toxic to the microorganisms present in the treatment plants, discharge of the wastewater to the treatment plants may lead to its failure. beside, direct discharge of these effluents into municipal wastewater plants and/or environment may cause the formation of toxic carcinogenic and/or unhealthy breakdown products. different chemical and physical methods (adsorption, coagulation-flocculation, oxidation, filtration and electrochemical treatments) for color removal have been proposed, but due capital costs and slow operating speed as well as huge amounts of sludge creation there is still a great need to develop an economic and effective method. the use of lignin degrading white-rot fungi and their enzymes (laccase, lignin peroxidase, manganese peroxidase) has attracted increasing scientific attention due their ability to oxidative degrade a wide range of recalcitrant organic compounds. in the contribution, the decolorization efficacy of different commercial textile reactive dyes (anthraquinone, azo, triphenylmethane) will be investigated after the treatment by laccase from trametes versicolor. in order to examine the reuse of enzymatically decolorized liquors, the ecological suitability and the toxicity of the degradation products after different time of enzyme exposure will be studied. this work was carried out within the scope of research project e! 3100 cawab. influence of heavy metals on growth and extracellular enzyme production of a trichoderma harzianum strain with biocontrol potential l. hatvani 1 , l. kredics 2 , a. szekeres 1 , z. antal 2 , l. manczinger 1 , a. nagy 3 , c. vágvölgyi 1 : 1 department of microbiology, university of szeged, p.o. box 533, h-6701 szeged, hungary; 2 hungarian academy of sciences, university of szeged, microbiological research group, hungary; 3 pilze-nagy ltd. kecskemét, p.o. box 407, hungary. e-mail: kredics@bio.u-szeged.hu (l. kredics) trichoderma species are common soil inhabiting asexual filamentous fungi with teleomorphs belonging to the hypocreales order of the ascomycota division. besides the industrial and clinical importance of the genus, certain strains have been found to cause great losses in mushroom cultivation while other strains are well known to possess high antagonistic activity against several plant pathogenic fungi and therefore used as biocontrol agents. important mechanisms of antagonism include competition and mycoparasitism, which -among others -can be related to the fast growth of trichoderma strains and the production of several extracellular enzymes. the influence of certain, soil-occurring heavy metals on mycelial growth and the secretion of extracellular enzymes involved in competition and mycoparasitism was examined in this study regarding an effective, potential biocontrol isolate of trichoderma harzianum. the metal ions zinc, manganese, copper, iron, lead and mercury were applied at the concentrations of 8, 16, 24, 32, 40, 60 and 80 m, and dry mycelial weight as well as the activities of extracellular ß-glycosidase, cellobiohydrolase, trypsinand chymotrypsin-like protease and n-acetyl-glucosaminidase enzymes were determined. it was found that mercury totally blocked mycelial growth, while other metal ions exerted a much lower influence on growth. the presence of heavy metals did not have a significant effect on the activity of the examined extracellular enzymes with the exception of trypsin-like protease, which showed a four-to six-fold rise in activity in the presence of certain sublethal concentrations of copper. based on these results, our further aim is to develop copper-resistant derivatives by mutagenesis from trichoderma strains with biocontrol potential. since proteases play an important role in mycoparasitism, these strains could be applied within the frames of integrated pest management in combination with copper-containing fungicides, resulting in an enhanced level of crop protection even with reduced amounts of fungicides. this work was supported by grants f037663 of the hungarian scientific research fund and grant omfb-00219/2002 of the hungarian ministry of education. the significance of biocontrol agents (bcas) is that some of them possess good antagonistic abilities against plant pathogenic fungi. a significant number of the most prominent fungi for the purposes of agricultural application belong to the genus trichoderma. in previous studies, in vitro assays on agar plates were reported as the generally used method for the evaluation of antagonistic abilities, as the results of these assays are well transferable to the practical application. the aim of the present study was to develop an accurate, image analysis-based method for the evaluation of the biocontrol characters of bcas. randomly selected trichoderma isolates were tested against fusarium culmorum. in the currently developed method, the areas of the fungal colonies were calculated on petri dishes by measuring the occupied surface of the medium on digital images. the inhibition effect was recorded as the value of biocontrol index (bci), which was calculated from the ratio of the area of the trichoderma colony and the total area occupied by the colonies of trichoderma and the plant pathogen. the proposed method was tested for numerous parameters, and the results revealed that bci proves to be capable for the accurate measurement and scale of the biocontrol abilities of fungal isolates. this work was supported by grants f037663 of the hungarian scientific research fund and grant omfb-00219/2002 of the hungarian ministry of education. the effect of advanced oxidation processes and recirculation on biodegradation of leachates from aerobic landfills liliana krzystek, anna zieleniewska-jastrzębska, stanisław ledakowicz department of bioprocess engineering, technical university of lodz, 90-924 lodz, poland modern landfills are built and operated in a way which allows us to treat them as a special type of bioreactor. simulation of municipal waste biodegradation in lysimeters provides knowledge on basic processes that take place in an aerated landfill. the aim of aeration is to stabilise mainly biodegradable and nitrogen containing components and to reduce methanogenic potential. stabilised leachates from old landfills contain big quantities of refractive carbon compounds that cannot be removed by biological methods. in such case most advantageous is to apply advanced oxidation processes (aops). the objective of this study is an experimental simulation of a landfill aerobic stabilisation and the impact of aops and recirculation of leachate on the reduction of organic load. the performance of the processes was monitored by the reduction in time of basic indices of organic load (bod 5 , cod, toc, vfa, tkn, n-nh 4 + ) and changes in biogas composition. the simulation of aerobic landfill processes was carried out in lysimeters with a fixed bed of household solid waste stabilised during 8 months in anaerobic conditions. leachates taken from the lysimeters were recirculated and subjected to advanced oxidation processes, i.e. ozonation and uv radiation with the addition of h 2 o 2 . experimental studies showed that the aerobic waste stabilisation was a very quick process. during a month the bed was stabilised, reaching a significant reduction of organic load indices. aeration of the lysimeters caused a quick reduction of mainly degradable organic substance (in terms of bod 5 ) and n-nh 4 + and vfa. the reduction of methanogenic potential of the landfill was even faster. the composition of gas at the outlet from the lysimeter changed and after one day already its content was similar to atmospheric air. a more frequent recirculation of leachates enhanced greatly the aerobic biodegradation. it was found that application of advanced oxidation processes (especially ozonation) contributed to a growing reduction of the organic load in the leachates from aerated lysimeters. the application of leachate ozonation resulted in a very high degree of reduction of organic compounds (up to 77%). the objective of the experimental study was to assess the effect of temperature on the extent of aerobic batch biodegradation of potato stillage with a mixed culture of bacteria of the genus bacillus. the experiments were performed at 20, 30, 35, 40, 45, 50, 55, 60, 63 and 65 • c, at ph 7, in five l l working volume stirred tank reactor (str) (biostat ® b, b. braun biotech international). the duration of the process was 120 h. initial cod of the stillage amounted to 51.9 g o 2 /l, the main carbon sources being reducing substances (18.7 g/l), organic acids (determined as their sum) (12.2 g/l) and glycerol (3 g/l). at 65 • c, no cod reduction or biomass increment was found to occur. at the other investigated temperatures, the reduction in cod measured after suspended solids (ss) separation varied from 77.6% (55 • c) to 89.1% (35 • c). without ss separation, cod reduction ranged between 55.6% (20 • c) and 75.1% (35 • c). this indicates that, in terms of the extent of cod reduction, the optimal process temperature was 35 • c and that there was a local optimum at about 63 • c. according to the temperature applied, the content of reducing substances decreased by 84.3-96%, that the organic acids by 91.7-99.6%, and that of glycerol by 91.5-96%. the experiments also produced the following two findings: (1) the rise in temperature brought about a decrease of biomass concentration in the str (measured as ss and bacterial number), and (2) temperature was a factor affecting the demand for ammonia nitrogen (n-nh 4 ), which was the highest at 20 and 60 • c. the high n-nh 4 demand observed both over the higher and lover ranges of the investigated temperature should be attributed to the release of n-nh 4 and to the large amounts of the biomass produced, respectively. the results obtained imply that the extent of potato stillage biodegradation with a mixed bacterial culture was high over a wide range of the investigated temperature. polychlorinated compounds such as tetrachloroethylene (pce) have become serious environmental pollutants. considerable attention has been paid to these organochlorine compounds. this paper describes the molecular analysis of dechlorinating gene in halorespirating bacterium and efficient bioremediation process. an anaerobic bacterium, that dechlorinates pce to tce, was isolated and identified as a species of the genus desulfitobacterium. a novel pce reductive dehalogenase (prda) gene from the desulfitobacterium sp. strain kbc-1 was identified. these prd genes, including membrane anchor protein, were classified as a novel type of pce reductive dehalogenase (approximately 40% homology with the general pce dehalogenase). according to the substrate utility of this strain kbc-1 and phylogenetic analysis of prda, the type of this microorganism may be expected to play the role of a primary degrader of pce in the environment. high efficient bioremediation process so called the restricted aeration system which means microaerobic/aerobic reciprocal bioremediation process was developed. strong modifications take place, as ammonia production with a subsequent rise of the ph value and a rapid heat evolution leading to temperatures of up to 70 • c. little is known about the microbial community in the toscano cigar fermentation and its development as fermentation proceeds. the aim of this study is to investigate the microbial community composition, its dynamic and its influence on the toscano cigar production process. our results show that the fermentation could be divided into three different phases: initially yeasts are the predominant microorganisms while bacterial growth is partially inhibited; the middle phase is characterized by exponential growth of bacteria while yeasts disappear. in the final phase the microorganism population is mostly represented by sporigen microbial species. the occurrence of yeasts in the first phase could be attributed to their ability to grow at low temperature and low ph levels. the bacterial population flourishes after the yeast cells have reached a stationary phase and probably grows on residual nutrients and autolysing yeast cells. yeasts and bacteria involved in the fermentation process were isolated and characterized. the microbial community was investigated by a combination of phenotypic and molecular approaches. the phenotypic characterization was based on both colony and cell morphology. the isolates were then identified by rrna genes sequence analysis. finally, in order to clarify the role of the identified microorganisms in the production process, a preliminary biochemical characterization was carried out. biosensors have undergone rapid development over the last few years; in particular, in environmental field many biosensors using microorganisms and purified enzymes as biological component, were recently studied. benzene is present everywhere with high levels in the cities and sometimes, in petroleum processing plants. it is classified as carcinogenic compound of first class able to cause leukaemia. because the evaluation of benzene requires complex instruments and quite long analysis times, it is required to study alternative systems for benzene detection simple, fast and highly sensitive, such as biosensors. from pseudomonas putida mst, strain previously isolated in our laboratory and able to degrade benzene, we isolated genes encoding for benzene 1,2-dioxygenase and cis-1,2-dihydrodiolbenzene dehydrogenase to use in the development of two different hydrocarbon biosensors based on microorganisms and on purified enzymes. the genes isolated were cloned in pvlt33 and we developed three microbial systems carrying: (1) benzene dioxygenase, (2) dihydrodiol dehydrogenase and (3) benzene dioxygenase-dehydrogenase modified by pcr to obtain enzymes with histidine tag. the cloning was planned to construct recombinant strains able to overproduce the enzymes; the enzymatic activities will be evaluated both using whole cells and purified enzymes. study of operation condition of biofilter using fibril-form matrix for odor gas removal don-hee park, chonnam national university, this research was performed for developing of biological treatment process of odor gas such as mek, h 2 s, and toluene, which is generated from the food waste recycling process. to establish the operational conditions of odor gas removal by small-scale biofiltration equipment, it was continuously operated by using toluene as a treating odor object. when the odor treating microorganisms were adhered to fibril form biofilter, high removal efficiency over 93% was obtained by biofilm formation. at 400 ppm of inlet odor gas concentration and 10 s of retention time, the removal efficiency was 76% and 93% in first stage reactor and second stage reactor, respectively. however, the removal efficiency remained over 97% at the operational conditions above 15 s of retention time. ozonated water is produced using an ozone generator in a container filled with cold water. it is useful for sanitizing the surfaces of various products for which heat or chemical treatment is inappropriate, such as fresh food products. in this study, we investigated the antimicrobial effects of ozonated water and electrolyzed ozonated water against escherichia coli, s. aureus, bacillus subtilis and yeast, saccharomyces cerevisiae for practical use in sanitizing various products. the results demonstrated that the electrolyzed ozone water was effective for the reduction of microbial population at relatively low concentration of ozone. also, the electrolyzed and the ozonated water showed synergistic antimicrobial effects. many xenobiotics can react spontaneously with thiol moieties of glutathione (gsh), forming gsh-conjugates, or via glutathione s-transferases (gst). these enzymes participate in detoxification of potentially harmful compounds from endo or xenobiotic origin. using saccharomyces cerevisiae as experimental model, we observed that cells mutated in the gtt1 or gtt2 genes showed twice as much cadmium absorption than the control strain. we proposed that the formation of the cadmium-glutathione complex is dependent on those transferases, since it was previously demonstrated that the cytoplasmic levels of this complex affect cadmium uptake. the addition of glutathione monoethyl ester (gme), a drug that mimics glutathione (gsh), to gtt1∆ cells restored the levels of metal absorption to those of the control strain. however, with respect to gtt2∆ cells, addition of gme did not alter the capacity of removing cadmium from the medium. taken together, these results suggest that gtt1p and gtt2p play different roles in the mechanism of cadmium detoxification. by analyzing the toxic effects of this metal, we verified that gtt2∆ and gsh1∆ cells showed, respectively higher and lower tolerance to cadmium stress than control cells, suggesting that although gsh plays a relevant role in cell protection, formation of the gsh-cd 2+ conjugate is deleterious to the mechanism of defense. furthermore, analyzing the harmful effects of other xenobiotic, menadione (2-methyl-1,4-napthoquinone), we have also observed that gtt1p and gtt2p isoforms play distinct functions in the process of cell protection as well as in drug remove, since both strains showed lethal phenotypes after direct exposure to 20 mm menadione. however, after adaptive treatments (mild-heat or exposure to a lower menadione concentration), cells acquired tolerance to menadione stress, although the gtt2∆ mutant had still shown a higher sensitivity against drug toxicity. by analyzing the malondialdehyde (mda) produced in response to menadione, we observed that gtt2∆ cells exhibited increased levels of lipid peroxidation, indicating that, during menadione exposure, gsh-conjugates are formed by the same transferase isoform, gtt2p, involved in cadmium stress. financial support: stint (sweden), cnpq and faperj (brazil). polycyclic aromatic hydrocarbons (pahs) are ubiquitous and persistent throughout the environment. they are generally distributed from both natural and industrial sources. many pahs can have a detrimental effect on the flora and fauna of affected habitats through uptake and accumulation in food chains, and in some instances, they induce serious health problems and/or genetic defects in humans. many research efforts have been expended to find a suitable method for remediation of soil and water environments contaminated with pahs. amongst them, the use of ligninolytic fungi is particularly suitable for the development of such processes, since they produce extracellular lignin-degrading enzymes (mnp, lip, laccase, . . .) which degrade a wide range of organic pollutants. coriolopsis rigida has been reported to produce extracellular laccase as the sole ligninolytic enzyme. this makes this fungus particularly suitable for the study of xenobiotics degradation by laccase. the purpose of this research was to obtain high laccase activities by c. rigida in solid state cultures and to determine their ability to degrade anthracene (typical pah). both in vivo and in vitro assays were performed. the former led to 60-80% degradation in 3 days depending on the culture conditions, whereas the latter showed a degradation percentage above 90% in 2 days when low mediator concentration (hbt) was added to the reaction mixture. focus will be given on pressure-driven membrane bioreactors, gastransfer membrane bioreactors and the novel ion exchange membrane bioreactor (iemb). the latter concept has been developed and currently studied by our group. this process, based on integration of donnan dialysis with bioconversion of one or more target pollutants to harmless products, has been modeled and experimentally verified for the removal of various charged inorganic pollutants such as nitrate, perchlorate and bromate by mixed microbial cultures under anoxic conditions. tests of up to 3 months showed a very good operational stability. the essential role of the microbial membraneattached biofilm, which develops naturally in this type of systems, will be also demonstrated and discussed. poly(lactic acid) (pla), which is one of biodegradable plastics, is depolymerized by hydrolysis and releases soluble monomer or oligomer of lactic acid. many bacteria can use the monomer and oligomer as an energy source or a carbon source. in this study, we applied pla to an electron donor for denitrification process of the previously developed bioreactor, which could remove ammonia from wastewater by simultaneously carrying out two biological processes, aerobic nitrification and anaerobic denitrification. a bench-scale bioreactor was constructed with a gel-plate containing pure-cultured cells of nitrosomonas europaea and paracoccus denitrificans and a pla-plate. the pla-plate was prepared by mixing three kinds of plas with different molecular weight and tricalcium phosphate to keep the constant release of the electron donor for a long term. batch treatment experiment with the bioreactor was repeated with an artificial wastewater containing ammonia for 100 days. the bioreactor could remove nitrogen from the artificial wastewater at nitrogenremoval rate of approximately 4 g n/day per square meter of gel-plate surface during the experiment period without an additional electron donor. the performance was equivalent to that obtained with our bioreactor using ethanol as electron donor for denitrification. the bioreactor using pla dose not need an additional pump for serving an electron donor (e.g., ethanol) and a hollow space for serving. therefore, the concept using solid electron donors like a pla would be effective our bioreactor to compact and simplify, and would be possible to develop a portable or disposable bioreactor. leucosporidium antarcticum as a source of enzymes for biotechnology arkadiusz wojtasik 1,2 , marianna turkiewicz 2 , jaroslaw dziadek 1 , pawel parniewski 1 : 1 centre for medical biology pas, 106 lodowa street, 93-232 lodz, poland; 2 faculty of biotechnology and food sciences technical university of lodz, stefanowskiego 4/10 street, 90-924 lodz, poland. e-mail: awojtasik@cbm.pan.pl (a. wojtasik) leucosporidium antarcticum is a psychrophilic yeast able to growth at low temperature. these microorganisms live in antarctic marine waters and are endemic to that cold environment. furthermore, l. antarcticum is also isolated from the digestive tract of antarctic krill euphausia superba. enzymes isolated from coldadapted microorganisms such as l. antarcticum having a specific activity at low temperatures ranging from 0 to 30 • c are considered for utilization at biotechnological applications such as bioremediation, production of polyunsaturated fatty acids of dietary significance and might be a source of industrially useful enzymatic proteins. the main goal of this study was to construct a cdna library of l. antarcticum. the partial cdna library was obtained and some of the clones were analysed. the sequencing analyses allowed us to find an approximately 450 base pair nucleotide sequence which displayed a very high homology to disulfide bond chaperone belonging to the hsp33 family from psychrobacter sp. high similarity of that heat shock protein was found on an amino acid sequence level and was reaching nearly 85%. the main object of our further research is to clone hsp33 family protein gene and to obtain its expression in a mezophilic host strain. also, further clones will be analysed to find other interesting genes encoding the psychrophilic proteins. this work was partially funded by the kbn grant i29/205/05. out of 9000 plant species found in the flora of turkey, about 3000 are endemic. beautiful flowering (geophytes) bulbous plants form an important part of this rich biodiversity. besides use as ornamental plants, these have great potential in perfume and pharmaceutical industry. genera of fritillaria, ornithogalum, muscari, bellevalia, tulipa, galanthus, sternbergia, crocus, arum and biarum have important and critically endangered species with high export potential that enters into this group. most of these are endangered and their collection from wild and export has been banned to conserve them. large scale production and conservation of these species could also be achieved by in vitro techniques. therefore bulb scale and immature embryo explants of sternbergia candida, s. fischeriana, muscari muscarimi, fritillaria imperialis and f. persica were cultured on different nutrient media supplemented with various concentrations of plant growth regulators using different culture applications. large numbers of bulblets were produced (over 100 bulblets/explants) from single immature embryos on nutrient media in most species tested after 12 months of culture initiation. regenerated bulblets were kept at 5 • c for 5 weeks and then transplanted to soil successfully. to our knowledge the present study is the first report for in vitro bulblet production from immature embryos of geophytes. the procedure described here provides a prolific bulblet production system that may form the basis of bioreactor culture and conservation of endemic and endangered geophytes. the commercial use of organofluorine compounds in industrial, pharmaceutical and pest-control applications has dramatically increased over the past few years, resulting in the introduction of numerous new organic compounds into the environment. organofluorine compounds are chemically very stable and are assumed to be resistant to biological degradation. given the chemical inertness of fluorinated organics, their bioactivity, and their potential for accumulation in the environment, it is important to understand their environmental fate and the mechanisms by which they might be degraded. examples of the biodegradation of fluorinated compounds in literature are scarce, being fluorobenzoic acids the most commonly reported. information on the cleavage of carbon-fluoride bonds in synthetic compounds is limited to fluoroacetate dehydrogenase. in this project we try to obtain more insight in the defluorination mechanisms by investigating the diversity of degradation routes for these compounds in several soil bacteria by making use of modern genetic tools. a gram-positive strain capable of aerobic biodegradation of 4-fluorophenol (4-fp) as the sole source of carbon and energy was isolated by selective enrichment from soil samples collected near an industrial site. batch cultures were set up and substrate consumption, accumulation of intermediates and product formation were monitored. the consortium was able to use 4-fp up to concentrations of 448.4 mg l −1 and was able to utilize a range of other organic compounds. stoichiometric release of fluoride ions was measured in batch cultures suggesting that there is no formation of dead-end products during 4-fp metabolism. biobleaching of kraft cellulose pulp by poliporus versicolor aysun ergene 1 , nazif kolankaya 2 : 1 kırıkkale university, faculty of science and literature, department of biology, yahsihan, kırıkkale, turkey; 2 hacettepe university, faculty of science, department of biology, beytepe, ankara, turkey the suitability of culture supernatant from poliporus versicolor for use in the biobleaching of kraft cellulose pulp was investigated. p. versicolor was found to grow on mycological broth (1% soytone, 4% d-glucose and 0.5% cellulose pulp). maximal extracellular ligninase production was detected after 7 days (7 nkat). the optimum biobleaching conditions are 30 • c and ph 4.8, with 10 days. in this condition p. versicolor decreased the kapa number from 38.55 to 19.42 and increased brightness from 28 to 32.7 in 10-day treatment. boron and iron are among the microelements required for the proper development of the vegetative and generative tissues of plants. though iron is present in high amounts in almost all soil types, its bioavailability to crops is extremely reduced, hence most of the plants face an iron defficiency problem and while on one side crop productions effected, on the other hand the nutrition problems come up to human through contagious nutritional chains. boron is also among the most problematic micronutrients of the major crop plan-tation areas of turkey. both defficiency and toxicity problems exist in a total of about 50% of the central anatolian soil where pea is among the legumes cultivated. application of varying levels of boron and iron combinations in greenhouse and the analysis of plant acquisition via icp-aes as well as determination of the effects of the element combinations both in morphological and molecular levels are the aim of our studies. the genetic bases of the response differences of plant genotypes to b and/or fe, were investigated through the applications of molecular marker techniques. considerable growth rate and stem size differences were detected within the parents (wild-type versus cultivar) and the f9 plants. the presence of efficient genotypes to high micronutrient levels are expected to help us increase the cultivation of the crop in problematic areas as well as in exploring the molecular bases of the microelement uptake mechanisms. butachlor is one of the selective systemic herbicide toxins that are act by inhibition of protein synthesis. this toxin is used exclusively in the rice, barely, cotton and wheat farmlands. butachlor is belonging to chloroacetanilide herbicide group, which are consisting of butachlor, alachlor, acetochlor, metolachlor and poropachlor. in the view of bioenvironmental, butachlor is degraded in the soil by microbial activity. its stability is about 6-10 weeks. it is converted to the water-soluble derivatives in soil or water, with a slow evolution of carbon dioxide. because of butachlor is one of the herbicide toxin, it is inhibitor factor against growth of bacteria and microorganisms. microorganisms can be continuing their activities in the limited concentration of butachlor. therefore treatment of industrial wastewater consist of concentrated butachlor by the biological treatment is impossible and it is necessary chemical or physico-chemical treatment are used. in this research, biological treatment methods are used. in the biological treatment, an activated sludge system with volume of 6.5 l is used. in this method, butachlor with concentration of 5 mg/l are treated. removal percent of butachlor for concentration of 2.5 and 3 mg/l are calculated to 41.20% and 41.67%, respectively. removal percent of cod is also calculated to 86%. olive oil mill wastewater (omw) as the effluent of the concern of olive industry has high organic load. the conventional biological treatments despite of their simplicity and rather suitable performance are ineffective for the omw treatment since phenolics possess antimicrobial activity. in order to carry out a proper treatment on omw, use of microorganism able to degrade the phenolics thus, is necessary. the ability of phanerochaete chrysosporium immobilized purification and downstream process of xylitol obtained biotechnologically from hemicellulosic hydrolyzate of corncobs b. rivas 1 , p. torre 2 , j.m. domínguez 1 , j.c. parajó 1 , a. converti 2 : 1 department of chemical engineering, vigo university (campus of ourense), polytechnic building, as lagoas, 32004 ourense, spain; 2 department of chemical and process engineering, genoa university, via opera pia 15, 16145 genoa, italy. e-mail: brivas@uvigo.es (b. rivas) biotechnological production of xylitol from lignocellulosic materials has been widely studied in the recent years with promising results that confirming the possible industrial application of this technology. xylitol purification from fermented broth is the limiting stage of this process. previous works suggest crystallization procedures in order to recovery xylitol from fermented synthetic solutions. the complexity of fermented hydrolyzate not allows direct crystallization. in this work, corncobs hydrolyzate obtained with autohydrolysis-posthydrolysis techniques, detoxificated with activated charcoal and concentrated was fermented to xylitol by d. hansenii. the fermented broth composition was 64.9% of xylitol (68 g/l), 13.3% of other sugars and 21.8% of other compounds that interferes in the crystallization process (as dry matter of the liquor). the fermented media was submitted to an absorption process with activated charcoal and concentrated until a xylitol concentration of 340 g/l. the liquor was then submitted to a second step of precipitation with ethanol, the best results achieved in this study were obtained with an ethanol/liquor ratio of 4. in these conditions this treatment allows to remove a 56.9% of the impurities. the resulting solution was evaporated and crystallized containing 60% of ethanol and a xylitol concentration of 443 g/l. crystallization was performed at t = 5 • c with slightly agitation. after 36 h were separated xylitol crystals with a recovery yield of 13% and a purity degree of 90%. numerous publications have documented that only a minor number of the indigenous prokaryotic organisms found in complex environments such as the human intestine, biogas reactors, and soil are known, and probably only a fraction of this diversity can be accessed using traditional culturing techniques. some of the reasons for this are the lack of knowledge of specific growth conditions, specific nutrients, and obligate coculture requirements. also growth on a solid surface directly exposed to the atmosphere puts a very strong selective pressure on single cells supposed to develop into visible colonies. therefore, the knowledge of these microorganisms is scarce and generally limited to the 16s rrna genes that have been extracted from different environments and cloned for phylogenetic analyses. an obvious approach to circumvent these problems was the development of techniques based upon micromanipulation for isolation of single cells from complex mixtures. continuous development of modern microscopes in combination with the precision of a servo-powered micromanipulator and the development of the modern microscopic micro injectors used in ivf techniques has further aided the manipulation of single cells. this technique, however, does not solve the problems of the non-culturable cells, and other approaches are needed to gain more information about these organisms. an approach to the non-culturable cells could be genomic analysis of isolated single cells without preceding cultivation. this pcr-based technique is widely used for genetic analysis of human cells, but due to the small amounts of dna present in prokaryotic cells it has so far not been possible to produce identifiable amounts of dna from single cell amplification using conventional polymerases. a promising alternative used for amplification of small amounts of dna is the f29 dna polymerase operating under isothermic conditions. applying random hexamer primers, this polymerase carries out a multiple displacement amplification (mda) of high molecular weight dna template. in this study we demonstrate the successful application of mda for selective amplification of genomic dna from a single prokaryotic cell. the yield was >20 mg of amplified genomic dna corresponding to about a 5 billion-fold amplification from a single cell. the technique was used to approach a large group of non-thermophilic archaea found in agricultural soil. our results show that combining mda with fluorescent in situ hybridization and cell isolation by capillary micromanipulation enables an unprecedented ability to investigate new species without cultivation. also this combination of techniques opens for studies of genetic heterogeneity within populations and processes such as horizontal gene transfer. precipitation of zn 2+ , cu 2+ and pb 2+ at bench scale using biogenic hydrogen sulphide produced from the utilization of volatile fatty acids by sulphate reducing bacteria maria teresa alvarez 1,2 , carla crespo 2 , bo mattiasson 1 : 1 department of biotechnology, center for chemistry and chemical engineering, lund university, p.o. box 124, s-22100 lund, sweden; 2 instituto de investigaciones fármaco bioquímicas, universidad mayor de san andrés, la paz, bolivia biological production of hydrogen sulphide (h 2 s) from sulphate using sulphate reducing bacteria (srb) is popular within environmental biotechnology. srb require absence of oxygen, presence of nutrients required for growth and oxidizable organic substrates (to supply hydrogen atoms for reduction of sulphate). many organic wastes have been used as electron donors for the sulphate-reducers in the treatment of acid mine drainage (amd) including straw, hay, sawdust, peat, spent mushroom compost and whey, however, other wastes such as municipal organic waste can be used. the aim of this work was to study the possibility of using srb for the treatment of amd at bench-scale. this process involved three stages: the volatile fatty acid (vfa) production by hydrolytic bacteria from the degradation of vegetables and fruits, the production of h 2 s through the utilization of the produced vfas by sulphate reducing bacteria and the precipitation of metals by using the biologically produced h 2 s. the substrates used for vfa production consisted of tomato, papaya, apple and banana. the h 2 s produced from the degradation of vfas was utilised for the precipitation of an artificial effluent simulating the heavy metal concentrations of a mine located at bolivian andean region, containing approximately 9 mg/l of zn +2 , 8 mg/l of cu +2 and 4 mg/l of pb +2 . the maximum concentration of hydrogen sulphide obtained was approximately 17 mm. removal efficiencies of 97%, 98% and 100% for zinc, cooper, and lead, respectively, were achieved in the present work. at 30 • c during 14 days. bacterial population was determined by counting in a neubauer chamber with optical microscope. sulfate concentration was measured by turbidity method and metal concentrations in the filtered supernatant were measured by icp-aes. the first part of study consists of determine the maximum concentration of each metal at which d. vulgaris and desulfovibrio sp. grow in similar way than control culture (without metal). both cultures tolerate: cr(iii) 15 ppm, ni(ii) 8.5 ppm, zn(ii) 20 ppm. the maximum precipitation percentages were approximately: 25% (15 ppm cr(iii)), 96% (8.5 ppm ni(ii)) and 99% (for d. vulgaris-10 ppm zn(ii) and desulfovibrio sp.-15 ppm de zn(ii)). time to reach the highest precipitation was minor for mixed culture (desulfovibrio sp.) y all the cases. the next part was focused to study the precipitation percentage when metals are present in combination in the same metal levels (cr(iii)-ni(ii), cr(iii)-zn(ii), ni(ii)-zn(ii) and cr(iii)-ni(ii)-zn(ii)). the combination of metals does not affect significantly the bacterial growth and precipitation percentage of metals. this fact supposes an importance advantage so metals are commonly found together in the environment. future experiments are focused in development of this process in continuous operation mode. biosolubilisation and depolymerisation of coal has potential to produce a clean energy source or high value organic products from low rank coals such as lignite or sub-bituminous coal. these complex soluble phenolic compounds are of value as starting materials for biotransformation to value-added compounds such as antioxidants and flavourants. the bioprocess is carried out at ambient temperature and pressure and is perceived to be environmental benign. in the evaluation of coal solubilisation an important quantity for the assessment of process feasibility is the yield, i.e. the determination of the mass of product obtained per unit mass of coal solubilised. to date, results for coal biosolubilisation reported in the literature are qualitative or at best semi-quantitative, indicating trends with operating variables. the process kinetics has not been determined rigorously because measurement of fungal growth during coal solubilisation is hindered by the presence of the solid coal substrate. knowledge of the profile of biomass growth is required for the rigorous determination of the kinetic parameters necessary for process design and optimisation. in this paper, the use of an indirect method for the estimation of the growth and metabolism of fungal biomass by measuring co 2 evolution and o 2 consumption using an off-gas analyser is reported in the study of fungal coal solubilisation. coal determined rigorously because measurement of fungal growth during coal solubilisation is hindered by the presence of the solid coal substrate. knowledge of the profile of biomass growth is required for the rigorous determination of the kinetic parameters necessary for process design and optimisation. biosolubilisation was carried out in a stirred tank slurry bioreactor with working volume of 1.0 l. complete suspension of the coal particles of 650-800 m mean diameter was achieved at an agitation rate of 560 rpm. growth yield coefficients based on coal and oxygen as well as maintenance coefficients were calculated from growth of the fungus under the same conditions using a non-coal carbon source such as glucose. these data were used to determine the stoichiometric coefficients for biomass growth, enabling the biomass production rate to be quantified in terms of co 2 production rate and o 2 consumption rate. a dna-chip platform for parallel detection of microorganisms related to biofilm in industrial systems and drinking water systems pernille skouboe, dorte lauritsen, kim holmstrøm bioneer a/s, kogle allé 2, dk-2970 hørsholm, denmark. e-mail: psk@bioneer.dk (p. skouboe) an oligonucleotide microarray for simultaneous detection and identification of pathogenic bacteria related to technical water systems as well as drinking water has been developed. the approach is based on the use of a tandem hybridization technique with two ribosomal 16s rdna-pcr products, 1000 bp and 500 bp long, generated from two consensus pcr reactions using conserved ribosomal primers end-labeled with cy3 and cy5, respectively. the tandem hybridization technique implies an internally quality control for discrimination between target and non-target signals. the current prototype of the dna-chip platform includes 20 oligonucleotide probes representing 11 different genera (and subgroups of species), e.g. legionella, mycobacterium, aeromonas, campylobacter, vibrio and enterococcus. the platform has been used for detection and identification of species from pure cultures, and initial experiments with water samples from industrial systems have been performed. the potential as well as the limitations of using a dna-chip based detection format in its present form will be documented. particularly, its potential application as a rapid method for initial screening of environmental or food samples will be addresses. the aim is to reduce and optimize the number of samples required for traditional microbiological identification tests. in the course of a project for the development of a novel kind of a mycotoxin inactivating feed additive, the aim of this study was to isolate and characterize microorganisms with the specific ability to enzymatically break down and detoxify fumonisins, a group of structurally related fungal toxins, with fumonisin b 1 (fb 1 ) being the most abundant and -with respect to toxicology -also the most important representative of this group. these toxins are produced as secondary metabolites by some fusarium species such as fusarium verticillioides and f. proliferatum and are naturally occurring contaminants of cereal grains worldwide. they are found especially in maize and maize based products, and are known to be hazardous to human as well as to animal health. a natural feed additive, based on microorganisms and/or enzymes, should ensure the detoxification of fumonisins during feed uptake and digestion via microbial or enzymatic break down of these compounds, by that protecting the animal from the harmful effects of these mycotoxins. besides an extensive screening of microbial strains derived from strain collections, various different natural habitats were investigated for the presence of fb 1 degrading microbial activity, such as intestinal contents of pigs, soil samples, and naturally fumonisin contaminated maize. while testing of nearly 150 organisms from strain collections did not show positive results, fumonisin transforming activity could be detected in one soil sample and a number of maize samples. trials in order to isolate the respective fumonisin degrading microorganisms resulted in a number of strains, whose fb 1 degrading activity could be proven. the most promising bacterial and yeast strains were further characterized with regard to a general taxonomic description, and to different aspects of their toxin degradation behaviour. approaching a more relevant in vivo situation, fb 1 degradation trials in food-and feed-stuffs were conducted. further on, the applicability of the respective organisms as stabilized lyophilisates was investigated. arsenic is one of the most important global environmental pollutants and the toxicological effects are related to its chemical form and oxidation state. arsenite [as(iii)] is reported to be on average 100 times more toxic than arsenate[as(iv)]. this work shows the ability of one strain of the species ochrobactrum tritici to grow in presence of several metals including arsenite, arsenate, selenite, selenate, tellurite and antimonite. its arsenite mic was determined as 50 mm, whereas for arsenate, this bacterium could resist to concentrations upper than 200 mm. we report the identification of two loci involved in high-level arsenic resistance. sequencing of the first locus identified four complete genes in the following order: arsr, arsd, arsa, arsb. the second locus containing genes for arsenic resistance was also characterized. each sequence has been compared with nucleotide and protein databank (blast programs) and significant homology with known orfs coding for arsenic resistance has been found. it is also possible that the phenomenon of high-level arsenic resistance in o. tritici could evolve other genes or loci. the ability of the ␣-proteobacterium o. tritici to tolerate high levels of arsenic in addition to other oxyanions has considerable potential for detoxification and bioremediation of contaminated environments. this work is based on the mathematical modeling of kinetics of a thermophilic bacteria cultivation system. the cultivation proceeded by way of batch and continuous on the synthetic medium with the main carbon source-lactose. this medium simulated an industrial waste approximately. mixed thermophilic aerobic bacteria popula-tion, applied to the wastewater treatment (sludge v&k bystrice pod hostynem), was used to the inoculation. the cultivation system consisted of the laboratory fermentor biostat b (b. braun biotech) with working volume 2 l and the control unit connected with a computer. it is possible the temperature, ph, aeration, stirring and foaming regulation. physical and chemical cultivation conditions were optimized. the chemical oxygen demand (cod), generally expressive the impurity level, was selected as the main parameter for the cultivations run classification. but into the mathematical model also the kinetics of biomass growth, lactose consumption, production of choice metabolites (acetate, lactate, succinate) and dissolved oxygen concentration was included. the modeling was located to two head distinguishable growth phases of the microorganisms. an optimization and identification of mathematical model parameters was practised in the software language psi/c. the difference between simulated curves and experimental data is not statistically significant on the relevancy level 0.05 (f-test). it took place the cod degradation at 74.0% with the average yield coefficient y chsk/x 3.8 g cod/g biomass in the batch process with the air aeration only. there is a better way to the cod elimination (>90.0%)-the aeration of air enriched by the pure oxygen. in experiments with the continuous system was obtained the 69.0% cod decrease after the steady state stabilization. this work was supported by project msm 0021630501. fluorescence in situ hybridization (fish) of whole cells using oligonucleotide probes was applied to study the influence of low temperature and temperature reduction on the bacterial community of biofilm reactors for the removal of chlorophenols (cps). two packed bed reactors were set up for degradation of a mixture of 2-cp, 4-cp, 2,4-dicp, and 2,4,6-tricp as sole source of carbon and energy at 14 • c (ra) and 24 • c (rb) and were inoculated with bacterial consortia adapted to these respective initial temperatures. the performance of the reactors was studied under different conditions of pollutant loading, aeration rate, and hydraulic retention times over 7 months. total chlorophenol removal capacities of 1240 and 1420 mg l −1 day −1 were achieved in the bioreactors ra and rb, respectively, under a total pollutant load of 1440 mg l −1 day −1 . the population of ␤-proteobacteria was the major bacterial community of the biofilm (35-47%) followed by the ␥-proteobacteria (12-6.5%). two bacteria with the ability to mineralize 50 mg chlorophenols l −1 were isolated from the bioreactors and characterized as ralstonia basilensis and alcaligenes sp., both belonging to ␤-proteobacteria. decreasing the temperature by 10 • c (in two steps of 5 • c each) resulted in an increase in the population of ␥-proteobacteria and a decrease in the population of ␤-proteobacteria in both reactors. application of genus specific probes showed an increase in the pseudomonas population from 25% of the ␥-proteobacteria at 14 • c to 59% at 4 • c. the pollutant removal capacity decreased to 548 and 833 mg l −1 day −1 in ra (4 • c) and rb (14 • c), respectively. the ␣and ␦-proteobacteria, cytophaga-flavobacteria and actinobacteria the survey was carried out in urban and rural areas of two cities (ankara and isparta). this paper is only analysing urban people by excluding villagers. urban sample is consisted of 400 urban consumers and 200 professionals. professionals were selected amongst pharmacists, doctors, agricultural engineering's and industrialists that is thought are affective in the process of developing new technologies and also the development of biotechnology in the society. basic data was gathered by a questionnaire including both structured and open-ended questions besides deep interviewed. workforce development for life sciences-the scottish experience carol booth scottish entreprise, uk the presentation will look at, the background and definition of workforce development for scottish enterprise, examine information available to support scottish enterprises economic intervention and where and when to intervene. conclusions emerging from the evidence base will be used to outline scottish enterprises approach to workforce development and look at which actions might be required to address identified issues. moving on to reasons for integrating workforce development into business development and how the life sciences cluster team at scottish enterprise, stakeholders and partners in scotland have approached their current and future contributions to workforce development for life sciences using a variety of projects. the national institute for bioprocessing research and training (nibrt) in ireland is a proposal that will be a state-of-the-art training, research and pilot plant service facility that brings together institutions with complementary expertise and state-of-the-art research technology, and industry partners. these include university college dublin, trinity college dublin, institute of technology, sligo and dublin city university. nibrt is an innovative collaboration between academic institutions at the forefront of biotechnology, cell biology, engineering and pharmacy and industry. for training, two separate training labs, for upstream and downstream training, in addition to 5 research labs are planted. it will also include a state-of-the-art pilot plant fermentation facility for fermentation optimisation, fermentation scale-up, product separation and purification, regulatory aspects and automation. by aligning with industrial demands, the new institute will tailor its training programmes while remaining on the cutting edge of biotechnology research and technologies. the fermentation facility will offer hands-on training workshops and educational modules for outside researchers and companies. these workshops cover the fundamentals of small-scale fermenta-tion, scale-up considerations, and fermentor design and set-up. the training and educational philosophy underpinning the nibrt will focus on the needs of industry with an emphasis on providing training for accreditation of existing industry staff and prepare technicians and graduates for the technical, business, regulatory and professional aspects of the industry. the strategy is to provide specialised modules in nibrt in support of courses established in the higher education institutions, which will provide the certificates, diplomas and degrees. modules will be offered for all categories of students and will be given credits respected by other third level institutions in ireland. the role of professional graduate degrees in meeting current and future biotechnology industry workforce needs a. stephen dahms san diego state university, usa the presentation will review the status of new graduate training models designed to meet the unique needs of the biotechnology industry as it transitions to commercialization. emphasis will be on professional master's degree programs in biotechnology and their various versions, with a focus on operational and funding strategies and industry acceptance. discussion will also centre upon the creation and operation of industry-validated, specialized and highly targeted professional masters degrees in various refined aspects of the drug development process, including regulatory affairs, biomedical quality systems, clinical affairs, management of drug development, management of reimbursement affairs, bioinformatics, etc. the eurodoctorate in biotechnology, new combined mba/phd combined degrees in the molecular life sciences, the u.s. professional doctorate in chemistry and the proposed u.s. professional doctorate in biotechnology will be also discussed. data will also be presented on the current workforce and the industry's projected needs. genetic studies show that mankind is a rapidly expanded population of closely related individuals with very similar disease sensitivity. bad nutrition and infections dominate among the main health problems in the world. apart from malnutrition, the overeating habits of the developed world are now creating problems in the developing world as well. infectious diseases are also a global problem since new contagious agents like hiv, sars and avian flew do not recognize borders. thus, the global responsibilities of modern health care are obvious. many research scientists from and in developing countries find it nearly impossible to use their talents for the benefit of their own countries. some struggle to develop research and education programmes with poor facilities, some leave science completely, and others migrate to more developed countries. the talents of such people are either being wasted or lost completely to their home countries just at the time they are most needed to combat the great humanitarian challenges of hunger, illness and lack of knowledge. europe must strengthen programmes which allow third world scientists to work to their full potential in their home countries or regions. yang beijing genomics institute, chine academy of sciences, beijing, china europe has all the reasons to be proud of being the cradle of modern science and of its achievements and resources in life sciences. as a model of having solved many of the problems that many other counties are now facing, europe is expected by the whole world to make its further contribution to a better future of mankind and to play a more important role in the international community of life sciences. tropical diseases and public health basilio valladares director of the university institute of tropical diseases and, public health, university of la laguna, 38200 la laguna, tenerife, spain. e-mail: bvallada@ull.es the presentation will look at the main research interests of the university institute of tropical diseases. these are the following: 1. immunology and molecular biology of parasites. we express and purify recombinant proteins from leishmania sp. which have been shown to act as immunomodulators and protect against disease such as l25, hsp70, hsp83. the study of acanthamoeba pathogenic factors has also resulted in the isolation and silencing of extracellular proteins related to their pathogenecity, which has a great potential in the development of novel chemotherapeutics. 2. diagnosis of parasites. the immunological diagnosis of leishmaniasis has been one of the main research interests in our laboratory for several years. as a result, we have identified peptides which could be used to develop kits for the immunological diagnosis of leishmaniasis such as hsp70 c-end, l25 n-end, etc. we have also developed some dna based methods for the identification of acanthamoeba species from biological and environmental sources. 3. water quality. biological parameters. our water research group has the expertise to identify and characterise bacterial, viral and parasitic indicators of faecal contamination in diverse water sources including tap water, rivers, reservoirs, sea, etc. this research area has been developed in collaboration with the local sewage treatment plant and reservoir managing authorities. currently, we are establishing a conjoined project with the center for disease control and prevention (cdc) in atlanta, usa for the identification of water-borne emerging pathogens. 4. development and formulation of chemotherapeutic antiparasitic agents. in this field, we evaluate the leishmanicidal activity both in vivo and in vitro of natural and synthetic drugs and synthetic peptides. in a later stage, the drugs which have shown the highest antiparasitic activity have been subjected to cytotoxicity assays and their molecular targets dissected. some of the drugs tested in the last few years have been submitted to patent due to their outstanding activity. finally, in order to allow the commercialization of these drugs, both in vivo and in vitro assays are being carried out to predict their chemical stability and degradation pathways. this will be followed by the use of liofilization and controlled crystallisation strategies for the development of efficient and safe treatments. 5. human and population genetics. tachykinins and their receptors in different tissues and groups of patients and their association with molecular polymorphisms is another one of our research interests. the knowledge of ligand and receptor sequences and their similarities will allow the rational development of drugs with specific activity against these receptors. furthermore, we are also interested in the interspecific variation along the evolutionary scale of these markers. 6. nitrate assimilation group. research in our group is focused on understanding nitrate assimilation in the yeast hansenula polymorpha. several biotechnological companies use this yeast to produce heterologous proteins (hepatitis b vaccine). genetic manipulation techniques for h. polymorpha are available in our laboratory. head of the department of biotechnology, technological institute of canary islands, pozo izquierdo 35119 santa lucía, las palmas, spain single cell analysis by flow cytometry has proved to be a tool to perform simultaneous and rapid measurements related to cell morphology and physiological state. previous studies showed the possibility of quantifying neutral and polar lipids spectrofluorometrically using a lipid specific fluorescent dye, nile red (nr), however the existence of inter and intraspecific variations in the fluorescent response had not been clearly established. in this work, two strains of marine microalgae: crypthecodinium cohnii and tetraselmis suecica, characterized both by high contents of polyunsaturated long chain fatty acids (dha and epa, respectively) and an hypersaline microalgae: dunaliella salina, characterized by a high ␤-carotene production, were grown under different conditions and collected at different growth phases to be used for in vivo lipid quantification with nr by flow cytometry. our results showed a high correlation between the mean fluorescence signal of nr stained cells and the neutral and polar lipid content measured by gravimetry for each strain. in this respect, these data make feasible the development of a rapid method for lipid quantification in monoalgal cultures. however, differences in the dye uptake related to specificity were detected. in this communication we also assess the possibility of use this cytometric technique to select microalgal strains with high lipid and polyunsaturated long chain fatty acids content from mixed samples. performance of such technique would be a good alternative to the time-consuming traditional screening protocols based on gravimetry and gas chromatography and would optimise the search of new commercial strains of microalgae. claverie-martín head of research unit, biomedical research institute, hospital universitario, de n.s. de candelaria, santa cruz de tenerife, spain our group is involved in the cloning and production of proteins of interest to the food and pharmaceutical industry. we have recently expressed in yeast the cdna that encodes the precursor of caprine chymosin. chymosin is the enzyme responsible for the coagulation of milk in the abomasum of unweaned calves. this enzyme is secreted by gastric mucosa cells as an inactive precursor, known as prochymosin. in the acidic conditions of the lumen, prochymosin is converted into the active form by autocatalytic cleavage of the n-terminal prosequence. chymosin is used extensively in cheese production because it cleaves -casein in a specific manner with low proteolytic activity. several biotechnology companies are producing the bovine recombinant enzyme for commercial use in the process of cheese making. we are interested in the caprine chymosin as an alternative because in the canary islands cheese has traditionally been made using goats milk with extract from the abomasum of newborn goats as coagulating factor. it is well known that the activity of these types of extracts varies depending on the age of the animal and the type of food ingested. these difficulties should be overcome using a recombinant caprine chymosin. the caprine mrna used for the synthesis of the cdna was obtained from the abomasum of milkfed kid goats. the cdna fragment encoding the mature portion of caprine prochymosin was fused in frame to a signal sequence in yeast expression vectors. culture supernatants of yeast cells transformed with the recombinant plasmids showed milk-clotting activity after activation at acid ph. proteolytic activity assayed toward casein fractions indicated that the recombinant caprine chymosin specifically hydrolysed -casein (patent 200402025). the recombinant caprine chymosin could be an alternative milk coagulant in cheese making. work is underway to optimise the expression of the new recombinant prochymosin for further purification and characterization. smart molecules for health victor martín head of research, university institute of bio-organics "antonio gonzález", avda. astrofísico francisco sánchez 2, 38206 la laguna, tenerife, spain the instituto universitario de bio-orgánica "antonio gonzález" (iubo) is a multidisciplinary research centre that belongs to the university of la laguna. the iubo is located at the town of la laguna, inscribed on unesco's world heritage list in 1999, and former capital of the canary island of tenerife. the geographical location in addition to its mild climate has made the canary islands to posses a variety of ecosystems with unique plants and animals. the institute was started up in the 1960s with the need to study the natural products and secondary metabolites produced by those marine and terrestrial organisms, thus providing a new source of bioactive products. the main research lines that are being developed at iubo are summarised in the following paragraphs: anticancer agents from natural sources: several natural products and their semisynthetic derivatives are produced at the iubo in diverse joint projects for the development of new antitumour drugs with novel mechanisms of action. as an example we can mention natural products from the mevalonic, shikimic or polyketide pathways. some products have recently shown in vitro reversion of the resistance in multidrug resistant (mdr) tumour cell lines. genetic engineering: in vitro cultures of the plants atropa baetica, maytenus amazonica and m. macrocarpa are developed in order to manipulate their biosynthetic pathways and induce the production in large scale of the secondary metabolites for diverse applications, including arthritis, rheumatism, and back pain. marine organisms and toxins: dinoflagellates are marine organisms responsible for the red tides and food poisoning episodes. among others, okadaic acid and yessotoxin are the most common toxins present in european shellfish. the isolation of these products is best done from the microorganism cultures, since they are present in very low amounts in the natural sources. at the iubo we develop culture systems to provide us with amounts of toxins large enough to perform biological, metabolic and bioactive studies. insecticide and repellents: natural products are being isolated for their use against plagues, specially those affecting agriculture. these projects are run in collaboration with a number of public institutions and agrochemical companies throughout europe and latin america. fine chemicals and pharmachemicals: our institute possesses large expertise in the field of organic synthesis devoted to the synthesis of medicinal substances, with special focus on asymmetric processes. of particular interest is the development of new methodologies for the total synthesis of biologically active substances like polyether toxins, (un)natural amino acids, sphingosine analogs, alkaloids, etc. with an annual source of 100s of new compounds, the fine chemicals and medicinal chemistry branch at iubo have recently started and anticancer screening program in collaboration with the biomedical research unit at the hospital universitario n.s. de la candelaria. the program is committed to the discovery of novel drugs for application in cancer treatment. the outcome of this project in its first year has been outstanding, leading to the finding of several leads that form the basis for current and future projects. institute of canary islands, biotechnological department, playa de pozo izquierdo s/n, 35119 santa lucía, gran canaria, spain the presentation will look at the main research interests of the biotechnological department of the technological institute of canary islands. these are the following: nutrition and feeding in aquaculture: we conduct studies on digestion, absorption, transport, and utilization of the different nutrients applying also different techniques such as histology, enzymology, genetic or immunology among others. aquaculture feeding is the main important cost in fish farms, being higher that the prize of fries, personnel cost or energetic costs. thus, studies conducted on the improvement of diet formulation and the use of different dietary ingredients is one of our main research lines (such as vegetable oils and meals to be used as alternative to fish meal and oil, or carotenoid sources to improve fish colour). not only the use of the different ingredient is studied, but also the effect of these ingredients on fish health, flesh quality, flesh healthy aspects related with human consumption, are being studied. different formulae are being developed and patents of different diets are being obtained. studies on nutritional requirements are also conducted allowing us to patent different formulae in larvae studies, developing microdiets to substitute the high-cost processes associated with live prey in larval nutrition. development of immunostimulants, anti-stress diets, immune techniques to be applied as bio-indicators of fish health and welfare, as well as use of dietary ingredients derived from bio-reactors are other research lines in our group. genetics: genetic techniques applied to aquaculture are being an important tool. microsatellites are being used to determine genealogy of the fish, allowing to decreases important problems in aquaculture such as fish deformities. genetic techniques such as micro-arrays and gene expression are being applied to obtain indicators of stress and health in fish. these technologies also permit to make different selective breeding programs, increasing the accuracy to estimate genetic parameters and evaluating brood-stock. furthermore, this technology allows to obtain a procedure to increase the productivity and quality of fish hatcheries. new species for aquaculture. development of new culture techniques: the diversification of species cultured is one of the main objectives of european aquaculture, since nowadays only four marine species are commercially produced: gilthead sea bream. european sea bass, turbot and salmon. we have been developed rearing techniques for new species such as red porgy or canarian abalone and also we are conducting studies on different new species, such as different sparids species, octopus or yellowtail. new rearing technology: the election of adequate systems for fish growth for each species and site of production and the localization of more appropriate sites for farms, using gis technology are also of special importance in our research team. besides, new larval rearing techniques such as semi-extensive hatchery (mesocoms) were development and nowadays is being used to increase fry quality (survival, no-deformities, better growth). this technology is being exported to other countries in order to offer new technology easy to manage to be implanted in developing countries. alejandro cañeque project officer, canary islands special zone, ministry of finance, c/leon y castillo 431 -4 a planta, edificio urbis, 35007 las palmas, spain. e-mail: acaneque@zec.org the canary islands special zone is the newest tax instrument within the canary islands economic and fiscal regime (ref). it offers a reduced tax rate of between 1 and 5% of corporate income tax for companies setting up a business. the companies must cover the following minimum requirements: create employment and make a minimum investment. the zec offers other tax advantages such as the exemption from paying transfer tax and stamp duty and the canary islands general indirect tax (igic). this tax scheme particularly fosters the biotechnology and pharmaceutical sectors. references fahnert references bechor the antimicrobial drugs high-level production of human collagen prolyl 4-hydroxylase in sandwich hybridisation assay for quantitative detection of yeast rnas in crude cell lysates microbial processes and products press. biotech. bioeng. 218. van hijum microbial xylitol production from corn cobs using candida magnoliae the role of bacillus methanolicus citrate synthase gene, city, in regulatiing the secretion of glutamate in lysine-secreting mutants plasmid-dependent methylotrophy in thermotolerant bacillus methanolicus 1,5-anhydrod-fructose; a versatile chiral building block: biochemistry and chemistry detailed dissection of a new mechanism for glycoside cleavage: the ␣-1,4-glucan lyase ␣-1,4-glucan lyase, a new class of starch and glycogen degrading enzyme ␣-1,4-glucan lyase, a new starch processing enzyme for production of 1,5-anhydro-d-fructose efficient purification, characterization and partial amino acid sequencing of two ␣-1,4-glucan lyases from fungi ␣-1,4-glucan lyases producing 1,5-anhydro-d-fructose from starch and glycogen have sequence similarity to alpha-glucosidases enzymatic description of the anhydrofructose pathway of glycogen degradation i. identification and purification of anhydrofructose dehydratase, ascopyrone tautomerase and ␣-1,4-glucan lyase in the fungus anthracobia melaloma a systems approach to dissecting immunity and inflammation integrative biological analysis of the apoe*3-leiden transgenic mouse innate recognition of bacteria in human milk is mediated by a milk-derived highly expressed pattern recognition receptor, soluble cd14 soluble forms of toll-like receptor (tlr)2 capable of modulating tlr2 signaling are present in human plasma and breast milk proteomics of the rat gut: analysis of the myenteric plexuslongitudinal muscle preparation an integrative metabolism approach identified stearoyl-coa desaturase as a target for an arachidonate-enriched diet the challenges of modeling mammalian biocomplexity rapid enrichment of bioactive milk proteins and iterative, consolidated protein identification by mudpit technology post-genomics of lactic acid and other food-grade bacteria to discover gut functionality genetics, metabolism and application of lactic acid bacteria. fems microbiol complete genome sequence of lactobacillus plantarum wcfs1 functional ingredient production: application of global and metabolic models metabolic engineering of lactic acid bacteria for the production of nutraceuticals reduction in antinutritional and toxic components in plant foods by fermentation the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no 2001k120240-41). the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no. 2001k120240-40) and the microorganisms given to this work by prof. dr. francesco molinari at university of milano, and prof. dr. leyla aç ikel at gazi university. the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no 2001k120240-40) and the microorganisms given to this work by prof. dr. francesco molinari at university of milano, and prof. dr. leyla aç ikel at gazi university. mrs. lynnette fernandez, johanna mäkeläinen, m.sc. and olli rämö, m.sc. are gratefully acknowledged for their help. this work was supported by the health science council, the academy of finland and the tekes-neobio program. supported by the mec of spain (ppq2002-04361-c04-02) and the canary islands government. jmp thanks icic for a postdoctoral fellowship. frpc thanks cajacanarias for a fpi fellowship. tm thanks the spanish mcyt-fse for a ramón y cajal contract. this work was supported by the korean systems biology research grant from the ministry of science and technology, lg chem chair professorship, ibm sur program and bk21 program. this study was supported by ankara university biotechnology institute (project no: 89). this work was partially supported by mec and feder (bio2004-00439), and fundación séneca carm (00842/pi/01). this work was supported by grants f037663 of the hungarian scientific research fund and grant omfb-00219/2002 of the hungarian ministry of education. keto sugars have long been implicated as attractive intermediates or substrates for further chemical or enzymatic reactions, to generate a number of synthetic sugar derivatives and fine chemicals. the quinone-dependent pyranose dehydrogenase (pdh) purified of the basidiomycete fungus agaricus meleagris catalyzes with high specificity the oxidation of the c-3 of glycosidically bound d-glucose, whereas in contrast it oxidizes simultaneously the c-2 and c-3 of free d-glucose. considering the broad substrate tolerance, pdh provides a new convenient tool for high yield production of 3-ketothis work was partially financed by the scientific and technological research council (cicyt, spain), grant ren2001-3224, by the iii pla de recerca de catalunya (generalitat de catalunya), grant 2001sgr-00143, and by the generalitat de catalunya to the "centre de referència en biotecnologia" (cerba). this work was supported by mega a.s. (czech republic) (www.mega.cz) and following vega grants: 1/2391/05 and 1/2390/05. this work was partially supported by mec and feder (bio2004-00439), and fundación séneca carm (00842/pi/01). financed by a marie curie re-integration grant merg-ct-2004-006378 and consejería de educación y ciencia de la junta de andalucía. this project is part of the collaborative research centre sfb 578 "development of biotechnological processes by integrating genetic and engineering methods", which is supported by the german research foundation dfg. this research was supported in part by grants from the hungarian scientific research fund (otka t37471, f46658 and d48537) and the hungarian-spanish intergovernmental s & t cooperation programme (omfb00103/2005). this research was supported in part by grants from the hungarian scientific research fund (otka t37471, f46658 and d48537) and the hungarian-spanish intergovernmental s & t cooperation programme (omfb00103/2005). this research was supported in part by grants from the hungarian scientific research fund (otka t37471, f46658, d48537) and gvop-3.1.1.-2004-05-0471. the authors thank dr. hamid narjiss (director of morocco inra) for the instruction to identify nadorcott mandarin by molecular markers and helpful discussions regarding this paper. this work was partially funded by the program for scientific cooperation cnrst (morocco) and iccti (portugal), the international foundation for science (ifs) support in stockholm (sweden). this work has been financed by xunta de galicia (pgidt03pxib30103pr). sevgil sadettin, gönül dönmez department of biology, faculty of science, ankara university 06100 beşevler, ankara, turkey this study was supported by ankara university biotechnology institute. demet ç etin 1 , sedat dönmez 2 , gönül dönmez 1 : 1 department of biology, faculty of science, ankara university, 06100 beşevler, ankara, turkey; 2 department of food engineering, faculty of engineering, ankara university, 06110 dışkapı, ankara, turkey sulfate-reducing bacteria (srb) that could grow on modified postgate c medium (pc) containing chromium(vi) were isolated from industrial wastewater and their chromium(vi) reduction capacities were investigated as a function of changes in the initial ph values, chromium, sulfate, nacl concentrations and carbon source. the optimum ph value at 50 mg l −1 initial chromium(vi) concentrations was determined as 8. chromium(vi) reduction by srb was investigated at 22.7-98.4 mg l −1 initial chromium(vi) concentrations. at the end of the experiments, the mixed cultures of srb were found to reduce more than 99% of the initial chromium(vi) levels which ranged from 22.7 to 74.9 mg l −1 within 2-6 days period. the effects of initial 0-9.0 g l −1 concentrations of sulfate and 0-6% (w/v) concentrations of nacl to chromium reduction were showed that, the lowest concentrations of sulfate and nacl, were the best for chromium reduction in the pc media including 50 mg l −1 chromium(vi). when the 25% whey was used as carbon source in the pc medium, 99.6% of the 65.9 mg l −1 initial chromium(vi) concentration was reduced within this study was supported by ankara university biotechnology institute. this study was carried out as a part of the project for analyzing and controlling the mechanism of biodegrading and processing entrusted by the new energy and industrial technology development organization (nedo). this work has been financed by xunta de galicia (pgidt03pxib30103pr). lead ions are considered a high pollutant of different waters. in our previous work were selected seven potential sorbent-strains rhodotorula mucilaginosa 1776, rh. aurantiaca 1195, rhodotorula sp. 4, williopsis californica 248, candida krusei 61t, cryptococcus sp. wt of lead ions. it was determined their stability to high concentration (up to 750 mg/l) of lead ions in medium. the ph changes and yeast physiologies of growth were studied in medium with these heavy metal ions. the influences of environmental factors such as ph of solution, age of microbial culture, biosorbent concentration in suspension, alive or living state of biosorbent, time of contact on sorption were investigated. the levels of maximal sorption ability and biomass affinity to heavy metal ions were established by experimentally received sorption isotherms with mathematical modeling of biosorption process separately for everyone researched yeast strain. the sorption isotherms obtained in these experiments for non-living yeast biomass showed that the maximal sorption capacity was 225 and 195 × 10 −6 mol (g sorbent) −1 for rh. aurantiaca 1195 and s. cerevisiae 1968, respectively. in the case of living biomass, the highthis work has been financed by the spanish ministry of science and technology and european feder (project ctm2004-01539). the authors wish to thank dra. m.j. martínez (cib, csic, madrid, spain) for providing coriolopsis rigida. this work was supported principally by embo and the mrc. fed-batch cultivation of haematococcus pluvialis under illumination with leds for production of astaxanthin abdolmajid lababpour, tomohisa katsuda, shigeo katoh department of molecular science and material engineering, kobe university, kobe, hyogo 657-8501, japan photosynthetic microalga haematococcus pluvialis is a most promising microorganism for production of astaxanthin, which has powerful antioxidant activity and is used both for human being as a food supplement and cosmetic; as well as animal farming such as salmon and poultry. the deficiency of nutrients in batch culture of h. pluvialis decreases the growth of cells and increases the induction of astaxanthin as an induction factor. therefore, it is impossible to reach to high cell concentrations in batch cultures. in previous experiments, the medium replacement increased the cell concentration, while accumulation of astaxanthin was not induced without other factors. in this work, the effects of fed-batch addition of culture medium on the cell growth and astaxanthin production in h. pluvialis cultures were studied. h. pluvialis was cultivated in 50 cm 3 culture medium containing sodium acetate, yeast extract, l-asparagines, mgcl 2 ·6h 2 o, feso 4 ·7h 2 o and cacl 2 ·2h 2 o (ph 6.8). light was supplied by panels of blue or red led lamps. the temperature was kept at 20 • c and the culture was mixed with a magnetic stirrer. in fed-batch cultures of h. pluvialis, the cell concentration and production of astaxanthin increased in comparison with those in batch culture. in addition, the operation in feb-batch manner is easier than medium replacement from industrial viewpoints for production of astaxanthin. simvastatin and similar compounds are wide used as antihypercholesterolemic agents. simvastatin is obtained by c-methylation of side chain of lovastatin. this process is not perfect and some unreacted lovastatin is present in the reaction mixture. simvastatin is separated from lovastatin using fungi clonostachys compactiuscula. using of living microbials has some disadvantages such as high cost, difficult product separation from the reaction mixture. we work on overcome these difficulties by separation and immobilization of enzyme that hydrolyses lovastatin ammonium salt in presence of simvastatin ammonium salt. our results of purification and immobilization lovastatin hydrolase will be presented. investigation of peptide antibiotics produced by trichoderma strains isolated from winter wheat rhizosphere a. szekeres 1 , l. kredics 2 , l. hatvani 1 , z. antal 2 , l. manczinger 1 , a. nagy 3 , c. vágvölgyi 1 : 1 department of microbiology, university of szeged, p.o. box 533, h-6701 szeged, hungry; 2 hungarian academy of sciences, university of szeged, microbiological research group, hungry; 3 pilze-nagy ltd. , kecskemét, p.o. box 407, species of the imperfect filamentous fungal genus trichoderma with teleomorphs belonging to the hypocreales order of the ascomycota division are of great economic importance as sources of enzymes, antibiotics, as plant growth promoters, decomposers of xenobiotics, and as commercial biofungicides. peptaibols and related peptaibiotics (prps) are secondary metabolites constituting a family of fungal peptide antibiotics which is constantly growing since alamethicin was isolated from cultures of trichoderma viride. these compounds are linear, amphipathic polypeptides composed of 5-20 amino acids and usually containing several non-proteinogenic amino acid residues, which are representing characteristic building blocks of the structure. one hundred and twenty trichoderma strains were isolated from roots of winter wheat grown in agricultural fields of southern hungary. the identity of species was examined based on morphological and molecular characters. the presence of prps-producing strains among the isolated trichoderma strains was detected by biological tests and the antibiotics were partially purified using a multistep chromatography procedure involving exclusion chromatography, adsorption chromatography and thin-layer chromatography. about 20% of the isolates proved to be able to produce prps. the antibacterial activity of the compounds was tested against staphylococcus aureus, bacillus subtilis, micrococcus luteus and escherichia coli, while the antifungal effect was recorded against fusarium oxysporum, f. culmorum, rhizoctonia solani and pythium debaryanum. ergezinger, m., bohnet, m., berensmeier, s., buchholz, k., 2005. integrierte enzymatische synthese und adsorption von isomaltose in einem mehrphasenbioreaktionsreaktor. cit 77, 167-171. glucansucrases from family 70 of glycoside-hydrolases are transglucosidases that produce ␣-glucans from sucrose, a very cheap substrate, without any use of nucleotide activated sugars. based on sequence analyses, these enzymes have been classified in two families, the family 70 and the family 13 of glycoside hydrolases. among the natural diversity existing in family 70 in which are found the glucansucrases produced by lactic acid bacteria, three enzymes have been selected for their distinctive specificities: dextransucrase from l. mesenteroides nrrl b-512f (dsr-s), which catalyses almost essentially the synthesis of ␣-1,6-linkages, alternansucrase from l. mesenteroides nrrl b-1355 (asr), which produces alternan polymer formed of ␣-1,6and ␣-1,3-alternated linkages and finally dextransucrase from l. mesenteroides nrrl b-1299 (dsr-e), which is responsible for the synthesis of a branched dextran composed of about 70% of ␣-1,6-linkages in the main chain and 30% of ␣-1,2-branched linkages. for all these enzymes, the natural polymerase activity can be shifted towards oligosaccharide production or gluco-conjugate syntheses by introducing acceptors in the reaction medium. a number of sugar acceptors have been successfully glucosylated with the view of developing new functional food products. acceptor glucosylation yield as well as acceptor reaction product structures were shown to be highly dependant on the enzyme specificity. consequently, using glucansucrases of distinctive specificities and varying the acceptors give access to a large variety of applications. amylosucrase, the sole glucansucrase found in family 13 of glycoside-hydrolases is also of great interest for functional food applications. this enzyme is able to synthesize highly resistant amylose from sucrose. again the reaction conditions can be used to modulate the yield and the size of amylose. the aim of our work is to further develop the applications of these enzymes via rational and combinatorial engineering. the most recent results obtained in this field will be discussed. novel food structure engineering concepts with enzymes johanna buchert vtt biotechnology, espoo, finland food structure is a very important quality attribute in food choice, since it affects not only the sensory perception of texture, but also release of flavour. enzymes offer specific means to engineer food structure by creating cross-links to food biopolymers, i.e. to proteins and/or carbohydrates. enzymatic cross-linking of food biopolymers can be exploited to create novel types of structures to foods without any need of added food ingredients. laccases and peroxidases can be used to crosslink ferulic acid containing carbohydrates, such as sugar beet pectin or arabinoxylan. proteins can be crosslinked by different oxidative or transferase type of enzymes. transglutaminases can crosslink protein via formation of isopeptide bond between glutamine and lysine residues. laccase and peroxidase can oxidize tyrosine residues to corresponding radicals, which in turn can further react with different groups in proteins. tyrosinases, on the other hand, oxidize tyrosine to a quinone, which can further react with aromatic ring, amine and thiol groups present in proteins. the biopolymer networks formed can be further engineered by combining adequate processing to the enzyme treatment. in this work the potential of enzymatic food structure engineering is reviewed. asparaginase-mediated reduction of acrylamide formation in baked, fried, and roasted products hanne vang hendriksen, beate kornbrust, steffen ernst, mary stringer, hans peter heldt-hansen, peter østergaard novozymes a/s, dk-2880 bagsvaerd, denmark. e-mail: hvhe@novozymes.com (h.v. hendriksen) in 2002, it was discovered that acrylamide is formed in several potato and grain-based foods (e.g. chips, french fries, toasted bread, biscuits, cereals) and in coffee, all of which have been prepared at high temperatures. the level of this potential carcinogen in the final food appears to range from 50 to 4000 ppb. later that year, the mechanism of acrylamide formation was unraveled, demonstrating that asparagine and reducing sugars are the precursors for acrylamide. this pointed to several potential enzymatic approaches to remove the root cause of the problem by degrading the precursors in situ. here, we demonstrate that asparaginase treatment leads to a more efficient reduction in acrylamide than alternative enzymatic treatments. asparaginase from aspergillus oryzae is used to reduce acrylamide formation significantly in laboratory models of a range of common food products. examples are french fries, biscuits, crisp bread, and fabricated chips. the sensory qualities appear to be constant. the implications for scaling up the processes for industrial food production are discussed. effect of cultivation conditions on folate content in yeast: exploring the potential of yeast as a bio-enrichment vehicle for folate in foods sofia hjortmo 1 , johan patring 2 , jelena jastrebova 2 , thomas andlid 1 : 1 department of chemical and biological engineering, chalmers university of technology, po box 5401, 402 29 gothenburg, sweden; 2 department of food science, swedish university of agricultural sciences, po box 7051, 750 07 uppsala, sweden. e-mail: sh@fsc.chalmers.se (s. hjortmo) over the past 10 years, the interest in health benefits of the b vitamin folate has increased considerably. a good folate status may hinder progression of several diseases such as neural tube defects and downs syndrome in foetus, as well as cancer, dementia, alzheimer's disease and cardiovascular disease in adults. it is however not easy to reach the recommended intake and new strategies have to be developed to increase the folate status. in this project we explore the use folate producing microorganisms for this purpose. many yeasts have the ability to synthesise folate de novo and can thus serve as a source for humans. folate enrichment in fermented foods could be much improved by using starter cultures better at producing folate compared to traditional strains. this is, e.g. applicable to bread making fb32 over-expression of isoprene biosynthetic enzymes in the ␤-carotene producer zygomycete mucor circinelloides tamás mucor circinelloides has been involved to study the carotene biosynthesis genesis of fungi. this fungus is more amenable to molecular techniques than the others traditionally used in carotenogenic studies (e.g. blakeslea trispora and phycomyces blakesleeanus). moreover, mucor has a great advantage: it is a dimorphic organism. this type of morphology is preferred by the fermentation industry because yeast-like growth allows the submerged culture, when usually higher biomass production can be achieved and cells can be more easily separated from the media. β-carotene is a terpenoid-type chemical compound likewise to sterols, quinones or chlorophylls. the production can be increased by improving the non-carotene specific terpenoid biosynthesis. this can be carried out by the overexpression of the genes responsible for the ratelimiting steps of these pathways. in this study, polyethylene glycol mediated transformations of m. circinelloides protoplasts were performed with autoreplicative expression vectors containing the known terpenoid genes of m. circinelloides (e.g. isoa encoding farnesyl pyrophosphate synthase and carg encoding geranylgeranyl pyrophosphate synthase). carotene production of the transformants and the wild-type strains were analysed by high-performance liquid chromatography (hplc). transformants harbouring plasmids with isoa or carg produce about 1.5 times more carotene than the recipient strain, while carotene production increased about two times in the co-transformants containing both type of plasmids. members of the genus rhizopus are important from biotechnological aspects in consequence of their effective extracellular enzyme, alcohol and organic acid production. moreover, rhizopus strains are used for fermentation of various foods, because they are capable of transforming soybeans into edible products. the high affinity iron permease (ftr1) contains both highly conservative and variable regions applicable for phylogenetic comparisons. the aim of this study was the comparative analysis of this gene of different rhizopus species in order to elaborate a simple and fast method to identify these fungi at a species and subspecies level. conserved regions of candida albicans and rhizopus oryzae ftr1 genes (fu et al., 2004) have been analysed to design degenerate primers for polymerase chain reaction. they were used to amplify the homologous regions from different strains of r. oryzae, r. microsporus, r. stolonifer and r. niveus. isolates of the similarly thermophilic rhizomucor miehei and r. pusillus, as well as a strain of m. rouxii were involved in the study as outgroups. deduced protein sequences were aligned and phylogenetic analysis was performed. surprisingly the r. oryzae isolates formed a group completely different with a significant distance from the r. microsporus isolate. r. niveus is currently not distinguished from r. stolonifer var. stolonifer because of morphological considerations. however, phylogeny of ftr1 gene sequences, in agreement with earlier results based on rapd data (vágvölgyi et al., 2004) , raise the need to handle r. niveus as a separate species. sequences and pcr primers useful for identification of all tested rhizopus strains were elaborated. potential application in a lactose conversion process. the prebiotics market is at high demand therefore the development of the process to produce 'prebiotic' galacto-oligosacharides efficiently and inexpensively is our particular interest. citrus, particularly mandarins and clementines, are among the most economically important fruit crops in morocco. besides morphological traits multiple molecular markers have been used for the caracterisation of citrus germplasm. the main aim of this study was to evaluate the moroccan mandarin germplasm and to identify specific polymorphisms among accessions sharing identical name. eighty mandarin and two sweet orange varieties were analyzed by dna markers. issr markers were amplified using 3 anchored primers and analysed by agarose gel electrophoresis. aflp markers analyses were performed using three primer combinations. the dice coefficient was used to estimate genetic similarities and the upgma algorithm was utilised to generate a phenogram depicting the genetic relationships among the acessions. the selection of 9 primers out of the 31 issr primers primarily assayed, allowed us to maximize the average number of amplified fragments analyzed per reaction (7.3), and the percentage of informative polymorphisms (49%). the three combinations of ecori/msei primers revealed 73 reliable aflp markers, 35 (47%) of witch were polymorphic. the range of fragment sizes varied from 100 to 650 bp. contrasting with the phenotypic diversity for agronomic and fruit quality traits, very low variability at the dna level has found among mandarins, which always showed a high (s > 0.81) coefficient of genetic similarity. the molecular marker analyses allowed the clarification of ambiguous denominations and the establishment of phenological relationships. the mandarin cultivars have been clustered into several different sub groups. this study allowed the identification of one issr marker, distinct and specific for the clementine sidi aissa and some aflp markers specific to maroc late and w. navel. many hybrids used in this study presented high coefficient of the similarity with one of their parents, such as siamelo and king of siam (s = 0.92), fortuna and clementine (s = 0.93), kara and king of siam (s = 0.92). this work was partially funded by the program for scientific cooperation cnrst (morocco) and iccti (portugal), the international foundation for science (ifs) support in stockholm (sweden). the new morocco mandarin variety nadorcott became very important in the international market because of high quality, good size, easy peeling and absence of seeds. also known as afourer or w. murcott, this variety was selected in 1981-1982 at the afourer experimental station, inra, located near to beni mellal city, as an original tree among several 18-year-old murcott honney (c. reticulata × c. sinensis) seedling trees. in order to shed additional light on the genetic origin of this variety, we have carried out isozyme and dna fingerprinting analyses. for better interpretation of the nadorcott molecular profiles, others mandarin cultivars, among which murcott honney, were also analyzed by molecular markers. three enzymatic systems (idh, pgm and pgi) permitted the discrimination between nadorcott and his female parent murcott honney. the molecular patterns displayed by these cultivars point out the sexual origin of nadorcott and discard the previously assumed hypothesis for its origin as a mutation of a nucellar zygote. the issr and rapd markers analyses allowed the identification of kinnow; du japon, vietnam; and swett lime as the genetically most closely related mandarins (s ∼ 0.95) to nadorcott. strong genetic similarity was also found with the clementine group, a possible male parent of nadorcott. the analysis by aflp markers confirmed the hybrid origin of nadorcott and the high genetic relatedness (s = 0.93) of this mandarin to its putative female parent murcott honey, and other cultivars as kinnow (s = 0.94) and clementine (s = 0.93). the possibility of an accurate molecular identification of nadorcott by specific molecular markers is of paramount importance for the protection and management of this original moroccan citrus variety. an effective process for the chemical-biotechnological utilization of distilled white lees was studied. a first treatment with hydrochloric acid allowed the solubilisation of tartaric acid. the influence of temperature, amount of hcl and reaction time were considered through an experimental design. under the optima conditions 77 g/l from white distilled lees and 45.6 g/l from red distilled lees were recovered. the tartaric acid was precipitated as calcium tartrate so that it can be isolated from the rest of the raw material compounds. the solid residue was used as an economic nutrient for lactic acid production by lactobacillus pentosus using trimming wastes as substrate. the lactic acid concentrations and volumetric productivities achieved were similar to those obtained using distilled lees without tartaric acid recovery as nutrient. thermophilic cyanobacterial strains that could grow in the bg 11 media was isolated from hot springs and their reactive dye bioaccumulation was studied under thermophilic conditions in a batch system, in order to determine the optimal conditions required for the highest dye accumulation. in the experiments performed with newly isolated synechocystis sp. and phormidium sp., the optimum ph values at about 25 mg l −1 initial reactive dye concentrations was determined as 8. lipases are extremely versatile enzymes, that catalyze both hydrolysis and synthesis reactions. they have a wide range of industrial applications, among which the manufacture of detergents, pharmaceuticals and fine chemicals are outstanding. the fungus rhizopus oryzae has been reported to synthesize a number of commercially interesting enzymes. in this work, its ability to produce extracellular lipases when grown in solid state culture has been assessed. cultures were carried out in erlenmeyer flasks, using a complex medium and several supports, both synthetic (nylon sponge) and natural (barley bran, ground walnut or peanut). the latter appeared to be more suitable for lipase production. since the best results were initially obtained with lipid-containing supports, barley bran cultures were supplemented with a vegetable oil, in an attempt to optimise lipase production and design an efficient procedure for reusing this agroindustrial waste. surprisingly, this strategy did not improve enzyme synthesis. however, when a surfactant (triton x-100) was added to the basal medium, a dramatic increase in extracellular activity was detected (up to 20-fold). the results agreed with those previously obtained in submerged cultures of r. oryzae, in which addition of olive oil did not increase lipase production, while the presence of triton x-100 had a remarkably beneficial effect. also, enzyme concentration in solid state cultures was up to two-fold that of the submerged ones. est maximal sorption capacity was found for yeasts cryptococcus sp. wt and rh. aurantiaca 1195 . these cultures also demonstrated the high sorption affinity, which makes them especially efficient biosorbents at low concentrations of lead ions. the high efficiency of lead elution was shown with 0.1n edta. isolation and identification of marine bacteria from deep-sea sediments els maas 1 , cara brosnahan 1 , vicky webb 1 , helen neil 2 , phil sutton 2 : 1 marine biotechnology, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand; 2 oceanography, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand marine sediments were obtained using a piston corer with associated trigger core (0.5 m, 0.06 m diameter). cores were collected from depths of 270 to 3911 m, along norfolk ridge and across challenger plateau. sediments ranged from coarse carbonate sands in the north to sandy and silty hemipelagic mud with increasing depth and latitude. all samples sites underlie subtropical surface water masses associated with, and south of, the tasman front. sediment samples were aseptically taken from the triggers cores upon recovery. samples were stored in sterile tubes at 4 • c on board the vessel for 3-17days. the core samples were plated on several different agar types and incubated aerobically for 4 weeks at 16 • c. individual colonies were sub-cultured and purified using standard microbiological techniques. morphological and molecular taxonomy revealed that the bacteria isolated from the sediments were closely related to novosphingomonas, halomonas, stappia, glaciecola, pseudoalteromonas and leeuwenhoekiella. phylogenetic trees constructed using 16s rrna gene sequence data showed that two other isolates were unrelated to known genera. the bacterial isolates are currently being investigated for their biotechnological potential. olive oil mill wastewater (omw) as the effluent of the concern of olive industry has high organic load. the conventional biological treatments despite of their simplicity and rather suitable performance are ineffective for the omw treatment since phenolics possess antimicrobial activity. in order to carry out a proper treatment on omw, use of microorganism able to degrade the phenolics thus, is necessary. the ability of phanerochaete chrysosporium immobilized on loofa was studied. the basal mineral salt solution along with glucose, ammonium sulfate and yeast extract were used to dilute omw properly. the fungus did not grow on the concentrated omw. therefore, omw diluted by 20% was used thought this study. the extent of removal in this biotreatment, of total phenolics (tp) and cod were 90 and 50%, respectively. while the color and aromatocity decreased by 60 and 95%, respectively. the kinetic behavior of the loofa immobilized fungus was found to follow monod equation. the maximum growth rate was 0.045 h −1 while the monod constant based on the consumed tp and cod (mg/l) were 370 and 6900, respectively. the control of water pollution has become of increasing importance in recent years. the release of dyes into the environment constitutes only a small proportion of water pollution, but dyes are visible in small quantities due to their brilliance. many dyes are difficult to decolourise due to their complex structure and synthetic origin. the adsorption of reactive dye remazol brilliant blue r (rbbr) on polyelectrolyte complex (pec) was studied in a batch systems. the adsorption parameters determined were: effect of the different values of ph on the adsorption of dye by pec, and the effect of contact time on the amount of rbbr adsorbed (in mg g −1 ). the data indicates that the adsorption capacity of rbbr by pec is dependent on ph. the maximum adsorption at 50 ppm was 88.52% equal to 11.8 mg of dye/g of polymer. the results show a tendency towards greater adsorption for reactive dyes (ph range of 8-12). the effect of contact time was studied at initial concentration (50 ppm) of dye, the amount of rbbr adsorbed for these pec increased and reached a constant value with the increase in contact time. the increase in the extent of removal of dye after 15 min of contact time is less and hence it is fixed as the optimum contact time. the pec show their capacity to remove rbbr to aqueous solutions by adsorption. flocculation of saccharomyces cerevisiae (diastaticus) ifo1958 was studied. cells of ifo 1958 did not flocculate even in the stationary phase without mg 2+ ("mg 2+ -deficient cells") although they began to flocculate strongly 18h after inoculation in the presence of mg 2+ ("complete cells"). cycloheximide completely inhibited induction of floc-forming ability of "mg 2+ -deficient cells". co-flocculation between "complete cells" and "mg 2+ -deficient cells" was investigated by chemical modification. treatment of "mg 2+ -deficient cells" by proteolytic enzymes did not affect the co-flocculation with "complete cells". photo-oxidation or mercaptoethanol-reduction of "mg 2+ -deficient cells" failed to weaken the co-flocculation with "complete cells" while treatment of "mg 2+ -deficient cells" by periodate brought about a significant loss of the co-flocculation. on the contrary, "complete cells" deflocculated by proteolysis or chemical modification of proteinaceous component failed to co-flocculate with "mg 2+ -deficient cells". these findings suggest that "mg 2+ -deficient cells" are non-flocculent because of lack of proteinaceous component essential for flocculation of cells of ifo 1958. the industrial toscano cigar production starts with the dark firecured kentucky tobacco fermentation process. during this phase the present work is a trial to study the portal serum factors which stimulate the cell proliferation of the schistosomules, aiming to find ways to block or inhibit their effects. our previous studies showed that portal serum of human and hamster (highly susceptible hosts) and a 1-50 kd fraction separated from human portal sera by ultrafiltration stimulate cell proliferation in immature schistosomules (20 days old) in vitro. for further identification of the portal serum factors in the range of 1-50 kd that stimulate cell proliferation, schistosomules were incubated in vitro in medium containing 10% fetal calf serum, 10% portal human serum or 10% peripheral human serum or their fractions separated by native electrophoresis followed by electroelution, incubations were performed in presence of bromodeoxyuridine (brdu) in order to measure differences in cell proliferation. the results showed that human portal sera enhanced cell proliferation of schistosomules compared to the peripheral serum. this stimulatory effect was substantially reproduced by fraction separated from human portal serum with molecular weight 20.8 kd. these results may help in designing a drug or antibody therapy to block the stimulating effect of the portal serum fraction and subsequently to disturb the life cycle of the parasite at early stage of development. center of molecular biosciences, university of the ryukyus, okinawa 903-0213, japan. e-mail: naoya-s@comb.u-ryukyu.ac.jp (n. shinzato)oil strage tank sludge, mainly composed of water and solid hydrocarbons (waxes) needs to be treated when harvesting the stored oil. although the sludge treatment by microbial surfactant or microbial cracking are considered as the feasible method, microbial degradation of the waxes (ca. solid n-alkanes) have been reported in a very limited species such as acinetobacter. in addition, long-chain n-alkanes (so called paraffin waxes) are one of the major components of oil, and their resistant properties to biological attack hold up the recovery of oil-polluted environments. in this report, we have screened n-tetracosane (c24) degrading bacteria from soils in okinawan island, an unique sub-tropical area in japan, to know the bacterial diversity and their degrading mechanism.16srdna phylogenetic analysis of the isolates, totally ca 40, showed they were not only acinetobacter and pseudomonas, but also other proteobacteria (alcaligenes), actinomycetes (gordonia, nocardia, and leifsonia), bacillus, staphylococcus, and unidentified ones. they also grew not only the solid n-alkanes but also iso-alkanes, mid-chain n-alkanes as the sole carbon source. results for the biosurfactant production will also be shown. the properties of 188 environmental enterococci were studied. the strains were isolated mainly from surface and waste waters and several strains from sheep manure were also included. species identification was provided by combination of phenotypic (micronaut system, merlin) and molecular detection methods (automated its-pcr, ddl-pcr). several discrepancies were observed when comparing molecular and biochemical identification. six enterococcal species were overall identified; e. faecium and e. hirae were the most abundant ones, almost 80% of isolates belonged to these two species. the distribution of selected genes conferring virulence to enterococci (cyla, gele and esp) was investigated, the positive signal was obtained mainly for e. faecalis strains. the strains were also characterized for the possession of enterocin genes (enta, entb, entp, ent31, entl50ab) and high frequency of enterocins was observed. biosorption of three different dyes (reactive black 5, cibacron brilliant yellow, cibacron brilliant red) onto immobilized scenedesmus obliquus a microalga was investigated in a batch sys-tem. the immobilized alga exihibited the highest dye uptake capacity at the initial ph value of 2.0 for all dyes. the effect of temperature on equilibrium sorption capacity indicated that maximum was obtained at 25 • c for rb5, cby and cbr biosorption. the freundlich, and langmuir adsorption models were used for the mathematical description of the biosorption equilibrium and isotherm constants were evaluated. biocontrol properties of microbially-treated sugar beet wastes in presence of rock phosphate n. vassilev, i. nikolaeva, m. vassileva department of chemical engineering, faculty of sciences, university of granada, c/fuentenueva s/n, granada-18071, spain. e-mail: nbvass@yahoo.com (n. vassilev) the effect of soil application of sugar beet wastes (sb) treated with aspergillus niger in the presence of rock phosphate (rp) on the control of fusarium wilt of tomato were studied. two treatments and a control were used: inoculation with glomus intraradices (am), further inoculation with a. niger grown on sb + rp medium, and the control (c). application of the am fungus increased plant growth, p and n uptake and reduced disease caused by fusarium oxusporum f. sp. licopersici (fol) as compared to non-mycorrhizal control plants. soil amendment with sb + rp + a. niger resulted in 347% and 467% (versus c) higher plant shoot biomass in plant-soil experiments contaminated or not with fol, respectively. in this case, disease severity and number of fol cfu reached the lowest levels while soil phosphatase and beta-glucosidase activities increased compared to all other treatments. fol negatively affected plant root mycorrhization determined in the am treatment while the difference between the mycorrhization of plants grown in the presence and absence of f. oxysporum in sb + rp + a. niger-amended soil was insignificant (53% versus 59%, respectively). in conclusion, the fermentation mixture containing mineralized organic matter, partially solubilized rp, and a. niger biomass could be efficiently used not only in improving plant growth, nutrient uptake and properties of degraded and polluted soils, as previously reported (vassilev and vassileva, 2003) , but also in environmentally-mild management of fusarium wilt. vassilev, n., vassileva, m., 2003. appl. microbiol. biotechnol. 61, 435-440 . biological treatment processes allow for the effective elimination of charged inorganic micropollutants, e.g. a number of oxyanions, heavy metals, etc. from contaminated drinking water supplies. however, dedicated technologies have to be implemented in order to eliminate the target pollutants without changing the quality of treated water, avoiding its secondary pollution by cells, nutrients and metabolic by-products. some innovative technologies, which combine the use of membranes with the bioconversion of charged micropollutants in order to deal with the secondary water contamination problem, will be presented and critically compared. a special on loofa was studied. the basal mineral salt solution along with glucose, ammonium sulfate and yeast extract were used to dilute omw properly. the fungus did not grow on the concentrated omw. therefore, omw diluted by 20% was used thought this study. the extent of removal in this biotreatment, of total phenolics (tp) and cod were 90 and 50%, respectively. while the color and aromaticity decreased by 60 and 95%, respectively. the kinetic behavior of the loofa immobilized fungus was found to follow monod equation. the maximum growth rate was 0.045 h −1 while the monod constant based on the consumed tp and cod (mg/l) were 370 and 6900, respectively. advanced start-up strategy of an anaerobic three-phase turbulent bed reactor treating winery wastewaters r. cresson, h. carrère, n. bernet, j.p. delgenès laboratoire de biotechnologie de l'environnement, institut national de la recherche agronomique (inra), avenue des etangs, 11100 narbonne, france. e-mail: cresson@ensam.inra.fr (r. cresson)the objective of our study was to compare two start-up strategies for an anaerobic biofilm process, to create an effective biofilm and increase the organic loading rate (olr) as quickly as possible. two methanogenic three-phase biofilm reactors have been started, using the same operational parameters (solid hold-up ratio, gas velocity of 1 mm s −1 ), in order to test two different strategies:• maximal load strategy (reactor a): the olr is increased as long as the global amount of removed cod (biogas production) increased. • maximal removal strategy (reactor b): the olr is increased stepwise as soon as the cod removal rate reaches 80%.both reactors have been operated for 90 days, until a volumetric olr of 20 g cod l −1 j −1 , with more than 90% of carbon removal. the total amount of cod removed and methane produced were higher in reactor b (19.6 and 32.2%, respectively). in both reactors, the short hydraulic retention time (hrt) applied during all the experiment caused a rapid wash-out of planktonic bacteria and an exclusive use of the substrate by the attached micro-organisms, which accelerates the biofilm growth. the lag-phase was reduced to approximately 7 days. the reactor submitted to repetitive disturbance by the maximal removal strategy appeared to be more robust when confronted to perturbation like organic overload or nutritional deficiency. experiments have demonstrated capability and the efficiency of the aggressive strategy for controlling anaerobic bioreactor start-up. sustainability is the generally accepted paradigm for future industrial development. the re-integration of waste products into production processes is a major aspect of environmental sustainability. in this study the use of sugar cane molasses is being investigated for the production of bioplastics by mixed microbial cultures, with the added possibility of parallel biohydrogen production. polyhydroxyalkanoates (phas) are polyesters synthesized by bacteria and accumulated as granules in the cytoplasm. studies conducted by this group have shown that mixed microbial cultures subjected to dynamic feeding conditions may accumulate phas up to 80% cell dry weight, a value close to that obtained for pure cultures. volatile fatty acids are good substrates for the production of phas by mixed cultures. on the other hand, sugar molasses, with a very high sugar content (about 50% dry weight), can produce organic acids by fermentation. the two-stage process being implemented in this study includes a molasses fermentation step, in which the high sugar content of the molasses is converted into volatile fatty acids (vfas), and a pha production step, in which the vfas serve as the precursors to the formation of phas under dynamic feeding conditions. moreover, hydrogen can be produced by anaerobic bacteria from carbohydrate-rich substrates giving organic fermentation end products, h 2 and co 2 . to optimize the production of both high-value products, design of experiments (doe) is being used to elaborate a set of experiments to study the effect of ph, hydraulic retention time and organic loading on both the organic acids distribution (which will serve as precursors for pha production in the second step) and h 2 production in the acidogenic fermentation reactor (a 1 l cstr). preliminary results show that the effluent of the acidogenic reactor fed with 10 g/l total sugars and operated at ph 7 and d = 0.1 h −1 (composed mainly of acetate and propionate) can be successfully fed to a polymer-accumulating mixed culture. under these conditions, the h 2 production yield has been estimated at 3.9 mol h 2 /mol sucrose. vanillin is a flavour compound used in food industry, fragrances and pharmaceutical preparations, which is nowadays mainly produced by chemical synthesis. the increased demand of natural products for the food industry as well as the high cost of natural vanillin extracted from vanilla pods has recently stimulated the research for alternatives to produce this compound by a natural way. the microbial transformation of ferulic acid, a phenolic compounds from lignin degradation, is recognized as being the most interesting alternative to produce natural vanillin. the combined effects of initial ferulic acid concentration (s 0 ) and biomass concentration (x 0 ) on vanillin production by resting cells of escherichia coli strain were investigated using response surface methodology. e. coli jm109/pbb1 a recombinant strain producing key enzymes of ferulate catabolic pathway from p. fluorescens bf13 (feruloyl-coa synthetase and feruloyl-coa hydratase/aldolase) was utilized in this work. a 3 2 full-factorial design was employed for experimental design. the results shown a possible inhibition phenomena at a vanillin concentration of about 0.1 g l −1 leading to the accumulation in the fermentation media of secondary compounds like vanillic acid and vanillin alcohol. removal of dissolved nutrients from wastewater using a microalgae biofilter line christensen, suvina sooknandan, jens jørgen lønsmann iversen department of biochemistry and molecular biology, university of southern denmark, odense, a microalgae biofilter can be used for treatment of wastewater from landbased fish farms in order to remove excess amounts of dissolved nutrients such as nitrate, ammonium and phosphate. a bubble column bioreactor has been developed for cultivation and characterization of microalgae. this type of bioreactor is equipped with a control system that enables online determination of the photosynthetic quotient and optimization of light intensity. furthermore the bioreactor has a dualsparging system simultaneously allowing adequate mixing and high gas-liquid mass transfer coefficients. different species of microalgae have been cultivated in batch and fed batch cultures to characterize growth and ability to take up the different dissolved nutrients. the specific growth rate and substrate uptake rate have been determined to compare and select the algal species most suited for use in a biofilter. additionally the composition of lipid, protein and carbohydrates has been measured to determine the nutritional quality of the algae when used as animal feed. at present, biological nitrogen-removal is mostly carried out through several complicated steps. to simplify the present systems for nitrogen-removal, we have investigated a new nitrogenremoval bioreactor using packed gel envelopes capable of simultaneous nitrification and denitrification. the envelope consists of two plate polymeric gels with a spacer in between. ammonia oxidizer, nitrosomonas europaea and denitrifier, paracoccus denitrificans are co-immobilized in the plate gels. when the envelopes are exposed to wastewater containing ammonia, the immobilized n. europaea oxidizes ammonia to nitrite in the outer aerobic surfaces of envelopes. at the same time, as ethanol solution is injected into the internal anaerobic spaces of envelopes, the immobilized p. denitrificans reduces the nitrite to nitrogen gas using the ethanol solution as an electron donor for denitrification. in this way, the envelopes can remove ammonia from wastewater in a single step. we have already reported advantages of our bioreactor in laboratory-scale experiments. in this study, we show our large-scale bioreactor (water volume 1.8 m 3 ) could treat three kinds of wastewater derived from coal power plants. ammoniacontaining wastewater that occurred regularly in a coal power plant was continuously treated with the bioreactor using thirty envelopes for over a year. the bioreactor could remove more than 90% of total nitrogen at hydraulic retention time (hrt) of 24 h. at hrt of 4 h, the bioreactor accomplished a maximum rate (the transformation of nh 4 + to n 2 ) of 6.0 g n/day m 2 of the envelopes' surface. the performance was equivalent to that obtained in the laboratory-scale experiments. furthermore, our bioreactor showed similar nitrogen-removal performances when it treated nitrate-containing wastewater occurring regularly and condensed ammonia-containing wastewater occurring at irregular intervals in coal power plants. these results show that our bioreactor can treat various wastewater containing nitrogen in coal power plants. thus, our concept is effective to simplify the large-scale systems in coal power plants and the other plants. in order to establish an environmentally friendly process for the treatment of metal containing waste, in a portuguese refinery a process involving sulphur oxidizing acidophilic microbes is being considered. bioleaching of metal containing bottom ash, from fluidised bed incineration of sludge resulting from the refinery water treatment station, was performed using a sulphur oxidising acidophilic culture isolated from an acid pool resulting from the weathering of sulphur piles from the claus plant. this sample served as inoculum for liquid medium cultures with 1% sterile sulphur flowers as source of energy. application of monod kinetics to adapted culture growth of free cells presented a value of µ = 0.124 day −1 . yield of sulphur conversion to sulfate after 17 days was η = 78%. in the presence of bottom ash from the incineration of refinery sludges µ = 0.141 day −1 and the yield of sulphur conversion was η = 67.5%. a η fe = 90% removal of iron is obtained from the treated ash. x-ray fluorescence spectroscopy of the solid residue revealed a total removal of metals namely, v, cu, ni, zn and most of the fe after 15 days of bioleaching. the present of heavy metals in the environment is a serious problem. they are commonly present in effluents from mining and industrial activities. usually, chemical conventional methods are very expensive and they have limitations when heavy metals are in low concentrations. at the moment the interest increases for processes that involve microorganisms as alternative method. some effluents present heavy metal sulphates which are soluble compounds. sulphate-reducing bacteria (srb), under anaerobic conditions, oxidize simple organic compounds (such as acetic acid and lactic acid) by utilizing sulphate as an electron acceptor and generate hydrogen sulphide. hydrogen sulphide reacts with heavy metal ions to form insoluble metal sulphides that can be easily separated from a solution. the purpose of this work was to evaluate the ability of srb to reduce cr(iii), ni(ii) and zn(ii) in artificial contaminated solution. desulfovibrio vulgaris and desulfovibrio sp. strains has been tested in this study. batch cultures was carried out in 50 ml sealed bottles with different concentrations of studied metals (1-20 mg/l), with 10% of inoculum bacterial and adapted to postgate's medium c. a gaseous nitrogen current was employed to purge oxygen and obtain anaerobic conditions. the assays were incubated statically represented very low portion (less than 4-5%) of the total bacterial community at all temperatures tested. schistosomules of schistosoma mansoni (20 days old) were incubated in rpmi 1640 medium containing 10% fetal calf serum, 10% hamster portal venous or 10% hamster peripheral venous serum (highly susceptible host) or 10% rat portal venous or 10% rat peripheral venous serum (poorly susceptible host) in presence of bromodeoxyuridine (brdu) in order to measure differences in cell proliferation. also the rate of cell proliferation of s. mansoni were assessed in vivo in hamster to study the cell proliferation in the natural ontogeny of the organism. the rates of cell proliferation as expressed by brdu labeling indices (blis) were determined as a function of time of incubation by immunohistochemistry using monoclonal antibody to brdu. compared to schistosomules cultured in presence of rpmi plus 10% fetal calf serum, blis were increased by 41% in the presence of hamster portal, but not in peripheral serum. while in case of rat, no significant changes were observed in the blis in both portal and peripheral sera. the experiment was repeated using hamster portal and peripheral sera containing different schistosomal igg antibody titres. the results showed decreased values of blis compared to sera which did not contain the schistosomal antibody(ies). the in vivo results revealed that there was no cell proliferation of s. mansoni schistosomules (6 days old) in the lungs. cell proliferation was detected in schistosomules of 17 days old and the results revealed a significant decrement in the brdu labeling indices (blis) with the increase of the age of schistosomules in vivo. the results indicated that hamster portal venous serum (highly susceptible host) could have stimulating factor(s) for schistosomule cell proliferation which is not found in rat (poorly susceptible host) and the presence of antibody(ies) greatly inhibit the cell proliferation. this could be due to the blocking of some portal serum factors, which stimulate the cell proliferation by the antibody(ies). the release of dyes into the environment constitutes only a small proportion of water pollution, but dyes are visible in small quantities due to their brilliance. many dyes are difficult to decolourise due to their complex structure and synthetic origin. the adsorption of reactive dye remazol brilliant blue r (rbbr) on polyelectrolyte complex (pec) was studied in a batch systems. the adsorption parameters determined were: effect of the different values of ph on the adsorption of dye by pec, and the effect of contact time on the amount of rbbr adsorbed (in mg g −1 ). the data indicates that the adsorption capacity of rbbr by pec is dependent on ph, the maximum adsorption at 50 ppm was 88.52% equal to 11.8 mg dye/g of polymer. the results show a tendency towards greater adsorption for reactive dyes (ph range of 8-12). the effect of contact time was studied at initial concentration (50 ppm) of dye, the amount of rbbr adsorbed for these pec increased and reached a constant value with the increase in contact time. the increase in the extent of removal of dye after 15 min of contact time is less and hence it is fixed as the optimum contact time. the pec show their capacity to remove rbbr to aqueous solutions by adsorption. compared to other european nations, the austrian population shows a low level of knowledge in biosciences and a strong denial of gene technology (1). the austrian non-profit organisation dialog<>gentechnik, a scientific society, organizes various activities to raise awareness for the "hot topics" in the life sciences. according to its principle of independence, all activities are funded publicly. projects on behalf of the austrian authorities and international cooperations demonstrate credibility and trust in dialog<>gentechnik (2). a few examples will be presented. dialogue with the public: on the occasion of the first anniversary that the gmo labelling rules became effective, the action "gene technology on my plate" is performed austrian wide in shopping centres. here, consumers are informed about health and labelling aspects of gm food. two days of open discussions were organized in the context of the austrian genome research program gen-au (3): topics were "gene diagnosis" (2002) and "genome research-what is in it for me?" (2004) . an open lab is currently set up in vienna to offer "hands on" experience in life sciences for everybody. motivating students: in the very successful gen-au summer school (3), high school students spend 3-4 weeks in the lab and work with scientists. the best documentations are awarded. in an innovative project, student groups (age 16-18) work on the topics stem cells and cloning and develop units of an elearning course which will be accessible for all austrian schools in the near future. engaging stakeholders: dialog<>gentechnik manages interdisciplinary wor king groups that develop leaflets, brochures and questionnaires on various aspects of gene diagnosis. four products are currently distributed to the public and to health services. (1) european commission. eurobarometer 55.2, december 2001; (2) www.dialog-gentechnik.at; (3) www.genau.at. the aim of this paper is to compare the attitudes of the urban consumers with professionals towards to new technologies, especially to biotechnology. it was tried to find out in which area, medical, agriculture or industry, people can accept biotechnological developments and in which area not accept. key: cord-022940-atbjwpo5 authors: nan title: poster sessions date: 2016-09-07 journal: febs j doi: 10.1111/febs.13808 sha: doc_id: 22940 cord_uid: atbjwpo5 nan ** each poster has been given a unique number beginning with the letter p; the next part relates to the session in which the poster will be presented. moreover, klf5 is also acting on cellular processes such as cell migration, apoptosis, inflammation, angiogenesis and differentiation. previous studies showed a novel role for klf5 as a regulator of proliferation and differentiation in skeletal muscle stem cells. detecting klf5 at the protein level harbored technical obstacles. commercially available antibodies exhibited low affinity, low specificity and failed to recognize post-translationally modified forms that are directly relevant to the function. thus, these obstacles prevent further functional protein studies such as western blots, protein co-immunoprecipitation and chromatin immunoprecipitation (chip) assays. therefore, we used crispr/cas system to establish a stable cell line which carry v5 epitope tag into the n-terminal of klf5 gene. insertion into the target side of klf5 gene via crispr-cas9 system provided an opportunity to overwhelm the above mentioned obstacles. v5 epitope tag would not interfere with the function of the klf5 and also enable us to recognize endogenous klf5 via anti-v5 antibody in the mouse myoblast cell lines (c2c12). we confirmed the targeted insertion into the exon 1 of the klf5 gene both at the dna and protein levels. the conformational dynamics of structural domains plays an important role in functioning of many proteins. the reca proteins from e. coli are known to be the central catalyst of homologous recombination and repair in bacteria. it forms a helical filament on ssdna capable to bind homologous dsdna and catalysis of the exchange of the complementary strand. significant mobility if its c-terminal domain has been observed experimentally by cryo-electron microscopy. however its potential significance for reca protein activities still remains unclear. in this work we investigated this question by construction of a mutant reca protein with artificial disulfide bridge between central and c-terminal domains. the wild type protein has no disulfide bonds, and therefore its native mobility can be restored in vitro, by addition of b-mercaptoethanol. our data suggest that the s-s bridge decreases both the rate of atp hydrolysis in vitro and the e. coli resistance to uv in vivo. thus, our experimental results indicate that the flexibility of the c-terminal domain significantly affects recombination activity of reca protein in vivo and in vitro. hydroxiurea (hu) is an inhibitor of ribonucleotide reductasethe enzyme that catalyzes the process of free dntps synthesis in living cells. treating cells with hu causes diminishment of the nucleotide pool. as a result, single-stranded dna regions are generated, which leads to s-phase checkpoint activation. the progression of replication forks is blocked and the completion of dna replication is prevented. this results in s-phase cell arrest. nevertheless, our results demonstrate that after prolonged hu treatment, the saccharomyces cerevisiae cells seam to escape the arrest and continue the progression of their cell cycle. we show that when cells re-enter the cell cycle, mrc1, but not ctf4 is detached from chromatin. our data also shows that meanwhile, rad53 checkpoint activity is diminished in order to allow s-phase checkpoint escape and completion of the cell cycle. moreover, cells not only continue the cell cycle, but steadily surmount in the presence of hu. all this data indicates that cells have made the decision to compromise s-phase checkpoint and to adapt to the novel environmental conditions in order to survive. as both mrc1 and ctf4 are known to be responsible for polymerase and helicase harmonization during replicative arrest, our data indicates that mrc1 has a more specific role in the process of adaptation. our data demonstrates that mrc1 is a leading protein to regulate the stability of s-phase checkpoint arrested replication forks. zinc finger domain is the most common dna binding domain in metazoa. almost 100 drosophila proteins with c2h2 zinc fingers also have zinc finger associated domain (zad). several proteins with zad (zw5, pita and zipic) were found to interact with cp190 and act as insulator proteins. for some of the zad-containing proteins (for example, weekle and grauzone) it was shown that their zad domains can form dimers with each other. the ability of these proteins to dimerize appears to be especially important in the light of the model suggesting that dna-binding insulator proteins can support genome looping and organization of chromatin structure via interaction with each other. in this work we aimed to understand the role that zadmediated protein-protein interactions play in maintenance of dna loops, focusing on proteins: zw5, pita, zipic and cg6808. first, we performed co-precipitation and yeast two hybrid assays to confirm dimerization of isolated zads in vitro. we observed that only zads from the same protein can specifically interact with each other (homodimerization) and they are unable to interact with zads from different proteins (heterodimerization). we confirmed homo-but not heterodimerization of zads in vivo with coimmunoprecipitation experiments in s2 cells. furthermore, we found that zad proteins can support longdistance interactions in transgenic constructs in flies. using model system with cg6808 protein, we demonstrated that zad is essential for these interactions. proteins without zad cannot maintain loop formation. finally, analysis of chip-seq experiments for zw5, pita, zipic and cg6808 revealed that binding sites of zad proteins often overlap with regions of inter-chromosomal contacts known from hi-c experiments. we conclude that zad-containing proteins can support longdistance genomic interactions and dimerization of zads is necessary for these interactions. this study was supported by the russian science foundation (project №14-24-00166). over the years a large body of clinical knowledge has accumulated on pharmacological effects of drugs on thyroid function. antipsychotics are administered over long periods in humans; therefore their possible adverse side effects should be taken into consideration. the aim of this study is to evaluate the effects of haloperidol and clozapine on plasma t3 and t4 concentrations in adult male wistar rats. fifty rats aged between 14 and 15 weeks (270 ae 30 g) were divided into five groups (n = 10 in each group), and drugs were administered each day intraperitoneally (ip) for 28 days. the first group was a sham group. the other four groups were considered as low and high treatment doses of the drugs. after a one-week habituation period, animals was administered haloperidol (0.05 mg/kg, n = 10 and 2 mg/kg, n = 10) and clozapine (0.5 mg/kg, n = 10 and 20 mg/kg, n = 10). the rats were anesthetized with ether, and bloods were collected by direct cardiac puncture 24 hours after the last injection. the t3 and t4 plasma concentration levels were analyzed with chemiluminescent immunoassay. statistical analysis was performed with ibm spss v20.0. kruskal-wallis and bonferroni tests were used. t4 plasma concentration levels significantly differ between sham (median=8.25 mg/kg) and haloperidol (2 mg/kg) (median=7. 00 mg/kg), haloperidol (0.05 mg/kg) (me-dian=7.70 mg/kg) and clozapine (20 mg/kg) (median=8.32 mg/ kg), haloperidol (2 mg/kg) (median= 7.00 mg/kg) and clozapine (20 mg/kg) (median= 8.32 mg/kg) groups (p < 0.05). however, no significant differences between the groups regarding to t3 plasma levels were observed. in conclusion, haloperidol and clozapine increased the t4 plasma concentrations, but didn't have any significant effect on t3 plasma concentrations. p-02.09.1-003 isolation of lipase producing strains of bacillus obtained from olive wastewater and screening for substrate specificity in this work, wastewater samples of an olive factory from yusufeli (artvin, turkey) were collected carefully. after a centrifugation period of samples, supernatants were applied to a 0.45 lm filter and incubated on lb agar medium for 24 hours. based on differencies of colony morphologies, 13 isolates were selected and purified for identification. 16s lipase activity assay was carried out by rhodamine b. all of the 13 strains exhibited lipase activity. for determining the substrat specificity of isolates, 5 different substrates were used; 4nitrophenyl-butyrate, 4-nitrophenyl-caprylate, 4-nitrophenyl-laurate, 4-nitrophenyl-myristate, 4-nitrophenyl-palmitate. results were measured spectrophotometrically at 405 nm. all of the strains hydrolyzed 4-nitrophenyl-butirat, while there was no activity with 4-nitrophenyl-palmitate. bacillus sp. l3 was the most efficient strain that hydrolyzed all of the substrates. the gene encoding for lipase of bacillus sp. l3 will be cloned and expressed for more analyses and industrial applications. p-02.09.1-004 some quantitative aspects of hair follicle layers differentiation e. vsevolodov 1,2 , v. golichenkov 3 , a. mussayeva 1,2 , i. latypov 2 1 llc "kazcytogen", almaty, kazakhstan, 2 "institute of general genetics and cytology" sc mes, almaty, kazakhstan, 3 lomonosov's moscow state university, moscow, russia in the course of stable hair growth the differentiation of hair bulb cambium cells to several layers with dissimilar cytochemistry and morphology takes place. this means the activation of different genes in the cells of different layers. depending upon the hair diameter some layers may be absent (medulla in the thin hairs). the hair diameters of the carpet sheep breeds vary widely even within the same square mm of the skin. we compared the different layers thicknesses proportions for the follicles with varying hair diameters. the follicle layers were measured on 155 microphotos of transverse histological sections of the follicles made under the standard magnification. all follicles belonged to the same skin biopsy. the measurements were made at the levels just below the fissure separating the hair and inner root sheath appeared. the empirical regressions of the layers thicknesses and of ratios of different layers against hair diameters were counted. the computer model was made on the basis of these regressions which allowed to obtain the absolute parameters of the layers as well as ratios of these parameters for every chosen hair diameter. using this model we found an essential trend in changing the proportions in relative layers dimensions as we choose the follicles with more and more thick hair. when we change the follicles with 30 mcm hair diameter for those with the hair diameter 100 mcm the ratio of hair medulla diameter to hair diameter increases from 0.07 to 0.76. the ratio of hair diameter to the diameter of inner root sheath increased from 0.70 to 0.84. it means that the thicker is the hair the higher proportion of cells produced by cambium are spent to build innermost layers (medulla layer within the hair or hair within the complexinner root sheath + hair). these data may throw some light on positional information mechanism of layers differentiation. lignin is a heterogeneous polymer that constitutes 30% of woody plant cell walls. microorganisms that degrade lignin are fungi, actinomycetes and to a lesser extent, bacteria. in case of industrial applications, the use of fungi is not feasible due to the structural hindrance caused by fungal filaments, requirement of particular culture conditions such as humidity, aeration which are not compatible with industrial processing environments. bacteria are worthy of being studied for their ligninolytic potential due to their immense environmental adaptability. environmental concerns and increasingly stringent emissions standards have led the pulp and paper industry to devise ways to decrease the level of chlorinated lignin residues in its effluents through both production process changes and improved treatment technologies. bleaching with the enzymes is the most promising because the enzymes may be very efficient, and can be used under industrial conditions. the main objective of this study was to investigate the adequency of klebsiella pnuemonia gst (glutathione-s-transferase) pretreatment for bleaching of calabrian pine kraft pulp. for this purpose the following conditions were investigated: enzyme loadings from 3 to 10 u/g pulp basis and the consistecy of the pulp was between 3 and 10%. enzyme at the desired concentration was added to the pulp and the mixture was incubated at 40°c for 2 hours. after the enzymatic pretreatment to determine the optimum conditions the kappa number of all reactions were analyzed according to tappi standarts. as a result of this study we determine the optimum conditons as 5% pulp consistecy, 8u/g enzyme for pulp treatment. after the enzymatic treatment carried out under optimum conditions we are planning to submit a short bleaching sequence and analyze for physical properties such as viscosity and brightness. owing to this bleacing sequence we are going to able to compare the enzymatic and chemical treatments of pulp in bleaching prosess. p-02.09.1-006 biochemical characterization of lipase from bacillus subtilis strain a10 from olive waste water f. ay sal, m. kac ßagan, s. c ß anakc ßi, a. o. beld€ uz karadeniz technical university, trabzon, turkey lipases (triacylglycerol acyl hydrolases, ec 3.1.1.3) are regarded as mild and environment-friendly biocatalysts for triacylglycerols hydrolysis. in addition to this hydrolytic reaction, they also catalyze reverse reactions of esterification, transesterification, and interesterification in non-aqueous environments. substrate, stereo-, regio-and enantio-specificities, and chiral selectivity are certain unique attributes of lipases that make them industrially attractive. these properties are often exploited in the manufacturing of detergent formulations, synthesis of fine chemicals, useful esters and peptides, food processing, paper manufacturing, degreasing of leather as well as in bioremediation. in this study, lipase from bacillus subtilis strain a10 is partially purified and characterized. bacillus subtilis strain a10 is isolated from olive factory from soke (aydin, turkey) and identified with 16s rrna analysis. lipase activity is screened on petri supplemented with rhodamine b. bacteria was grown in lb medium supplemented with 1% olive oil (vol/vol) for 48 hour at 37°c. after incubation, cells were harvested by centrifugation at 11,000 rpm for 5 minutes, resuspended in 50 mm tris-hcl (ph 8.0) buffer, followed by sonication with sartorius labsonic m to release intracellular proteins. q-sepharose is used as ionexchange column chromatography for lipase purification. effects of temperature on activity and stability were determined spectrophotometrically using p-nitrophenyl laurate as the substrate. effects of ph on activity and stability were also determined. the effects of various metal ions and other reagents on the hydrolytic activity were assayed at 37°c. the enzyme was active and stable in the broad ph range of 5.0-10.0 and temperature range of 24-50°c. bacillus subtilis strain a10 have high lipolytic activity. after cloning this enzyme to an expression vector and detailed characterization, this may suggests its usefulness in industrial applications. p-02.09.1-007 investigation of pin1 as a nuclear factor one binding partner s. saritas, a. e. yilmaz, a. kumbasar department of molecular biology and genetics, istanbul technical university, istanbul, turkey the nuclear factor one (nfi) proteins are important regulators of gene expression in the developing embryo and in adult stem cell niches. this transcription factor family has four members: nfia, nfib, nfic, and nfix. nfi proteins bind a consensus sequence on gene regulatory regions as homo or heterodimers. each member of nfi family has a highly conserved n-terminal dna binding and dimerization domain and a diverse proline rich c-terminal transcriptional activation/repression domain. as knockouts of nfi genes display distinct developmental phenotypes, we hypothesized that specificity of nfi protein function may arise from their interactions with binding partners. a yeast-two hybrid screen identified protein interacting with never in mitosis a1 (pin1) as a potential nfib interactor. pin1 is a ubiquitously expressed protein that specifically recognizes and binds to a phospho-serine or a phospho-threonine followed by a proline (ps/pt-p motif), and catalyzes isomerization of peptidyl-prolyl bonds. interestingly, both n-terminal and c-terminal domains of four nfi isoforms contain several conserved putative ps/pt-p motifs and some of these are reportedly phosphorylated. we looked for nfi pin1 interactions in vitro by gst-pulldown and co-immunoprecipitation assays. while gst-pin1 fusion protein interacts with all of four nfi isoforms, it binds nfib most strongly, nfia and nfic moderately, nfix most weakly. moreover, deletion of the cterminal domain leads to loss of nfi affinity for pin1 implicating this domain in nfi-pin1 interactions. co-immunoprecipitation assays where we co-expressed various epitope tagged nfi and pin1 proteins in hek 293t cells showed that pin1 precipitates nfib, as well as other nfi isoforms and nfib can, in turn, precipitate pin1. we are currently carrying out site-directed mutagenesis on nfib to identify the specific residues that pin1 recognizes. we will further explore if this interaction regulates nfi function during embryonic development. that pre-adapt migrating fish to the life in seawater. among others, smoltification induces intense growth of fish that enter the ocean at a size where risk of predation is significantly reduced. skeletal muscle growth depends on a tightly controlled balance between protein synthesis and degradation. protein synthesis driven by hormone regulation is well studied in smoltified atlantic salmon; while less is known on protein degradation occurring via a number of pathways including cytosolic ubiquitin-proteasome system and calcium dependent calpains. the aim of this study was to compare calpain and proteasome enzymatic activities in the skeletal muscles of s. salar parr, pre-smolts and smolts. calpain and proteasome activities were determined by casein or suc-llvy-amc hydrolysis in the skeletal muscles of s. salar from indera river (kola peninsula, russia). our results demonstrated the significant differences in studied protease activity levels between parr and smolts. calpain and proteasome activities in s. salar smolt muscles showed a significant drop compared with that of parr. the negative correlation between proteases activity levels in the muscle tissue and overall fish growth rate was shown. so, our data indicated life stage specificity in skeletal muscle protein degradation capacity in migrating fish. we suppose that intense muscle growth in s. salar pre-smolts is supported by various mechanisms including accelerated muscle protein accretion through the reduction of protease activities. obtained results enhance our knowledge in the mechanisms of atlantic salmon smoltification. the work was supported by the russian scientific foundation, project no. 14-24-00102. p-02.09. [1] [2] [3] [4] [5] [6] [7] [8] [9] the sociodemographic characteristics of the pregnant women who double and triple prenatal screening test h. d€ ulger, s. yabanci€ un meram medical faculty, n.e.university, konya, turkey double and triple prenatal screening tests which are applicable during first and second trimesters of pregnancy predict existent abnormalities at early stage. the aim of this study is to investigate the relationship between positive results of double and triple tests, further confirmatory tests during prenatal phase, postnatal status of babies and maternal age. in this study, double and triple test results of pregnant women who were admitted to meram faculty of medicine during 2009-2013 period were scanned from archive and test results indicating risk were detected. from these results, those which were above cut-off values for down syndrome, trisomy 18, open spina bifida were determined. a questionnaire was carried out with voluntary participants by reaching to these individuals. positive-negative result ratio of all double and triple test results and sociodemographic features such as age, occupation, presence of consanguineous marriage were investigated. all data from archive and answers from survey questions were assessed statistically. participants of the study were 18 to 46 years old and their average age was 29.89 ae 6.56. 219 ofthem (69.30%) were under 35 years of age whereas 97 of them (30.70%) were above 35 years of age. number of pregnancies were scaling between 1 to 13 with an average of 2.56 ae 1.56. 207 of 316 mothers (65.50%) were not undergone amniocentesis, whereas 6 babies with chromosomal abnormalities were detected among 109 mothers who were undergone amniocentesis. in conclusion, there may be regional, sociological and such that reasons for those who were not undergone amniocentesis despite positive double and triple test results. 6 (5.50%) chromosomal abnormalities were detected among pregnancies with increased risk assessment with positive double and triple results. p-02.09. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] the effects of oil on the growth and development of amphibians l. sutuyeva, t. shalakhmetova, a. ondassynova al-farabi kazakh national university, almaty, kazakhstan currently, the pollution of ecosystems by oil and oil products is increasing everywhere. the oil gets into water and ground during oil production, transportation and accidents. as a result, terrestrial animals and hydrobionts are exposed to oil contamination. thus, populations of animals decline. it can be assumed that the most sensitive to the effects of pollutants are animals in early stages of development. amphibians have established themselves as the most convenient bioindicator species. since lake frog (rana ridibunda) and green toad (bufo viridis) are the bioindicator species in kazakhstan, the study of the effects of oil on their larvae was carried out. we used water-soluble fraction of the oil from zhanazhol field (aktobe region) in our test. the larvae of control group were kept in pure water, and larvae of test groupsin aquariums with 0.05, 0.5 and 1% concentrations of the oil fractions. the concentrations were chosen in accordance with the level of pollution of kazakhstan's water bodies with oil. mortality of larvae, morphometric parameters and morphogenesis were studied. it was found that high mortality of larvae is the most visible reaction when exposed to oil. this indicator rose noticeably depending on the doses (0.05, 0.5% and 1%) in both species with percentages 16%, 51% and 78% in r. ridibunda and 22%, 61% and 83% in b. viridis, respectively, while in the control group it was about 10%. furthermore, delayed larval development was detected. thus, the larvae from the control and 0.05% oil group reached gosner stage (gs) 36, tadpoles from 0.5% and 1% groups were at gs-32 and gs-29, respectively, by the 30th day of life. moreover, behavioral abnormalities (sluggish movements) and decreased sensitivity to mechanical stress (touch) were observed under the influence of high concentrations of oil fractions. thus, oil in low concentrations alters the growth and development of tadpoles of anurans, and causes their increased mortality in high concentrations. p-02.09.1-011 effect of catechin loaded plga nanoparticles on glioma cell line histone h1t is a linker histone which binds to dna and contribute in chromatin condensation as well as regulation of specific genes through spermatogenesis. replacement of this histone h1 subtype and hyperacetylation of histone h4 tail, facilitate the replacement of histones with sperm chromatin condensing proteins of tnps and prms. ethical approval and informed patient consent was gained from 12 infertile men referred to royan institute. testicular biopsies were collected from patients through assisted reproductive techniques (art) procedure. based on pathological results samples were classified into the following three subgroups: obstructive azoospermia (as positive control), complete maturation arrest and sertoli cell only syndrome (negative control). chromatin of tissues evaluated for presence/absence of histone h1t protein in regulatory regions of tnps and prms genes using chip-real time pcr. results showed lower incorporation of h1t protein on regulatory regions of tnps and prms genes in two spermatogenic failure group versus positive control. in this study, it can be concluded that the decreased levels of h1t histone variant in testis tissues and failure in chromatin condensation have significant association with male infertility. p-02.09.1-013 serum dickkopf-1 levels in obese children and adolescents that obesity is detrimental to bone health despite potential positive effects of mechanical loading conferred by increased body mass on bones. the wnt/b-catenin pathway is essential for normal osteogenesis. serum dickkopf-1 (dkk-1) is one of the most important inhibitors of the wnt//b-catenin pathway. the aim of this study was to investigate the serum dkk-1 levels in obese and non-obese children and adolescents. materials and methods: the study included 30 obese children and adolescents (14 males and 16 females) aged from 7 to 17 years and 30 healthy normal-weight controls (13 males and 17 females) aged from 6 to 17 years. serum dkk-1 levels were measured by elisa method using commercially available kit. results: body mass index of the obese children was significantly higher than that of non-obese children (p = 0.000). however, there was no significant difference between dkk-1 levels of the groups. (these results are preliminary and the study is continuing). discussion and conclusion: our result showed that serum dkk-1 levels were not changed in obese children and adolescents. p-02.09.1-014 transcriptional regulation of cdo by nuclear factor one proteins b. kutay, c. lektemur, v. g€ uler, a. kumbasar department of molecular biology and genetics, istanbul technical university, istanbul, turkey nuclear factor one (nfi) transcription factors play important roles in regulation of central nervous system development. three of the four members of nfi family, nfia, nfib, and nfix are expressed in neural progenitors, as well as neurons and glia in the embryo. inactivation of these genes in mice show that they function in development of neocortex and hippocampus in the forebrain, cerebellum, spinal cord and precerebellar nuclei of the hindbrain, regulating neurogenesis, gliogenesis, as well as neuronal migration, axonal outgrowth and guidance. all three neural specific nfis are expressed in precerebellar neuroprogenitors, however, only deletion of nfib leads to a delay in development of precerebellar neurons. investigation of misregulated genes in nfib à/à precerebellar neuroprogenitors identified cell adhesion associated, oncogene regulated (cdo) as a potential downstream target of nfib. interestingly, this gene has been reported to be upregulated in nfia à/à hippocampus as well. cdo, a cell surface glycoprotein of the ig superfamily, has been found to regulate neurogenesis in vivo, is highly expressed in the developing brain and can induce neural differentiation by promoting heterodimerization of basic helix loop helix transcription factors with e proteins. bioinformatic analysis of the 5 kb human cdo promoter region identified five nfi binding sites: one cluster in the first 1 kb region, another in the 3.5 kb upstream region. electrophoretic mobility shift and supershift assays showed that nfib binds to all five sites. furthermore, nfib, along with the other neural nfis, inhibits the proximal cdo promoter driven luciferase activity by up to 85% in hek293t cells. preliminary data indicate that nfis bind to sites in both clusters in human neural stem cells (hnscs) suggesting that these sites are functional in vivo. we are currently investigating this possibility through nfi overexpression and silencing experiments that will examine regulation of cdo in hnscs. differentiation. the aim of this study is to investigate bdnf and drd2/ankk1 gene variants in eos development. in this study, 111 eos patients and 138 healthy controls were used. genomic dna extraction was performed from peripheral blood leukocytes. drd2/ankk1 taq1a (rs1800497) and bdnf val66met (rs6265) polymorphisms were determined by real-time polymerase chain reaction (rt-pcr). positive and negative syndrome scale (panss) was used to determine eos severity. for drd2/ankk1 rs1800497 polymorphism, there was a significant difference in the genotype frequencies between patients and controls for the co-dominant model (p = 0.05, or = 1.723; 95% ci: 0.996-2.98). however, no significant relationship was observed in the genotype frequencies of bdnf val66met polymorphism between eos patients and controls (p = 0.489). these results indicate that, drd2/ankk1 rs1800497 genotypes may affect eos development. however, bdnf val66met polymorphism may not be associated with eos. lack of association of bdnf val66met polymorphism may be due to limited number of patients. our findings need to be confirmed by further studies. various dyes used in the textile industry are discharged in large quantities to the receiving environment in the manufacturing process. this is the beginning of a process that is difficult to compensate for environmental and human health. therefore, contaminated areas should be cleaned. in addition, technologies with high polluting potential should be integrated with biological approach and thereby the impurities consisting of dyes should be reduced. in this experiment; burdirect black meta konz (c.i. direct black 38) was intended to decolorization using laccase. firstly, enzymatic decolorization of the dye was determined using spectrophotometry. the wavelengths of maximum absorption of burdirect black meta konz (c.i. direct black 38) was determined between 200 and 800 nm. then, optimization studies have been done. for optimization studies; dye concentration, laccase activity, ph, buffer concentration, temperature, mediator effect, mediator concentration and time parameters were determined. lastly, in optimal conditions, atr-ftir and gc-ms analyzes of ensuring decolorization of dye were analyzed. decolorization of burdirect black meta konz (c.i. direct black 38) was performed successfully and the absence of any metobolite in the decolorization medium has been provided by atr-ftir and gc-ms analyzes. assessing in terms of application, it can be easily applied by provided the reaction conditions in textile factories. laccase is a tool of decolorization of dyes in environmental friendly process. thus for the development of spermatids into mature sperm able to fertilize the oocyte.one of the causes of male infertility is in fact impaired sperm fertilization capacity due to sperm chromatin abnormalities and aberrant protamine replacement.recent research has focused on protamine biology,including protamine gene and protein structure,mechanisms of protamine expression regulation and involvement of the protamines in male fertility.various studies reported abnormal expressions of protamine (prm) genes in sperm of infertile men.the aim of the study is to investigate the gene expression of prm1, prm2 and their relationship with defective spermatogenesis. materials and methods: this study has been performed on 50 infertile and 3 fertile turkish men.total rna was extracted from the sperm pellet using trizol reagent.after rna extraction and cdna synthesis,real-time quantitative polymerase chain reaction (rt-qpcr) was used to determine the expression of prm1 and prm2. results: distinct levels of spermatozoal prm1 and prm2 mrna were found in infertile patients compared to fertile control groups.we found that the mrna levels of prm1 was reduced in 23 (%47), and the mrna levels of prm2 was reduced in 33 (%64) out of 50 infertile patients.in the current study,we found statistical significant association between the prm1 expression and infertility (p < 0.05).although prm2 gene expression was decreased in most of infertile patients compared to fertile control groups,the differences between the groups were statistically insignificant (p > 0.05). discussion: the results of the study suggested that, the protamine expressions which were associated with spermatogenesis may be important in infertility treatment. further studies are required in a large series of different populations to clarify the role both prm1 and prm2 themselves and their mrna expression on male fertility. the study was conducted to characterize the processes of muscle growth in atlantic salmon (salmo salar l.) of different ages inhabited rivers indera and varzuga (kola peninsula, russia) in summer and autumn. the expression levels of genes myosin heavy chain myhc, myostatin (mstn), and myogenic regulatory factors myf5, myogenin) in white muscle were studied in salmon parr of age groups 0+, 1+, 2+ in june and october. the changes in expression levels of mrfs, myhc and mstn indicating the extent of hyperplasia, hypertrophy, and restriction of muscle growth at different ages of parr were revealed. the pattern of age-related changes differed between seasons. especially, the expression of genes myod, myogenin and myhc peaked in yearling parr (1+) in summer, that indicated the high rate of hyperplastic and hypertrophic muscle growth in yearlings (1+). at the same time, the mstn expression level, the negative regulator of muscle growth, was highest in parr at age 1+. possibly, it is the necessary regulation mechanism to attenuate hyperplasia and hypertrophy and control muscle growth. in autumn, the expression level of myhc and myogenin were higher in salmon of age 0+ and 1+ then in 2+, indicating the higher intensity of hypertrophy in parr at both first ages in comparison to 2+. there was no differences in expression level of myod, myf5 and mstn between age groups in autumn. moreover, the expression levels of genes studied were lower in autumn than in summer. thus, it indicated the decrease of muscle protein synthesis and muscle growth rate in autumn. these findings expand knowledge on age-and season-related features of muscle development in young atlantic salmon in their natural habitat. the study was supported by the grant of the russian science foundation no. 14-24-00102. p-02.09.1-020 lmp2 and lmp7 gene polymorphisms in the southeastern anatolia population of turkey d. mihc ßioglu 1 , f. ozbas gerceker 2 1 sanko university, gaziantep, turkey, 2 gaziantep € universitesi, gaziantep, turkey introduction: the low molecular weight polypeptide 2(lmp2) and low molecular weight polypeptide 7(lmp7) genes are located in the class ii region of the major histocompatability complex (mhc) locus on chromosome 6. these genes encode peptides forming the large components of the proteosome complex which degrades short-lived cytoplasmic proteins. due to the significant role of lmp products antigen presentation, these genes can be accepted as strong candidates of susceptibility factors for different diseases. population genetic studies can also contribute to understanding of the possible role of lmp gene polymorphisms. the aim of this study was to determine the allele and genotype frequencies of the lmp2 and lmp7 gene polymorphisms in southeastern anatolia population and to compare these with the frequencies in other populations previously reported. material and methods: a total of 110 healthy and unrelated individuals participated in this study. polymorphism analyses were done by polymerase chain reaction (pcr)-restriction fragment length polymorphism (rflp) method and allele/ genotype frequencies of lmp2 and lmp7 genes were determined. results: a deviation from the hardy-weinberg equilibrium (v2 = 17.97,p < 0.05) was found for the genotype distribution of lmp2 gene polymorphism, while the lmp7 genotypes found to be distributed (v2 = 0.43,p > 0.05). discussion: available allele frequency data for different populations were used to calculate genetic distances and to construct a neighbor-joining tree. among the included populations, nahuas (mexico) population was found to have the lowest genetic distance from the southeastern anatolia-turkey population. conclusion: it can be concluded that, more studies using different types of genetic markers are needed to clarify the filogenetic relationships of southeastern anatolia population with other populations and also the number of population studies on lmp2 and lmp7 genes should be increased to understand their effects as a genetic marker. p-02.09.1-021 investigation of in vitro antioxidant activity of quercetin loaded plga nanoparticles pharmacological effects. but its usage is restricted because of low aqueous solubility, poor bioavailability, poor permeability and instability in physiological medium. these problems can be overcome with encapsulation of quercetin into nanocarriers such as biodegradable plga based nanoparticles. polymeric nanoparticles which have 1-1000 nm particle size and providing controlled released of biological active agent are prepared by using biodegradable and biocompatible polymers. in this study, encapsulation of quercetin molecules into plga nanoparticles was carried with using the single emulsion (w/o) solvent evaporation method. size measurements of the obtained nanoparticles were performed by zetasizer and their size were found 882. 1; 189.25; 436 .9 nm respectively. the morphological features were examined by sem images. antioxidant activities of q5, q7 ve q10 nanoformulations have been investigated by dpph and no (nitric oxide) methods. it is thought that the nanoparticular formulations that is developed in this study can be useful model for the other antioxidant molecules and will provide a significant contribution to the food and pharmaceutical industry. "this research has been supported by yıldız technical university scientific research projects coordination department. project number: 2014-07-04-gep03". p-02.09. the effect of environmental enrichment on spatial memory and certain nmdars, and 5ht2a expressions in rat pups introduction: the aim of the study was to investigate the effect of environmental enrichment exposed during whole childhood on spatial learning and memory and certain nmdars, and 5ht2a in the hippocampi of pups. materials and methods: four-weeks old, male, weaning rats were randomised into 2 groups as enviromental enrichment (ee, n = 12) and standard cage control (scc,n = 12) groups. eeg housed in an enriched environment and sccg were kept in standard cages for 8 weeks. following the experiment the rats were trained and tested in the morris water maze (mwm) , open field test (oft) and forced swim test (fst) in order to assess the neurobehavioural effects of ee. nr2a, nr2b, 5ht2a protein levels were analyzed by western blotting from hippocampi of rats. results: the positive effect of ee was seen at the learning phase in the mwm as 'latency to locate the hidden platform' between groups thoughout the 4 training days showed that eeg located the hidden platform significantly earlier than sccg on days 2, 3 (p = 0.006, p < 0.0001). also eeg significantly spent lower time in the outer zone of the maze on days 2, 3 which was the sign of low anxiety level (p = 0.011, p = 0.049). the parameters of oft which indicated increased locomotion, exploration and low anxiety were significantly higher in eeg (p < 0.05), in fst comparison of groups showed no difference (p > 0.05). the levels of nr2b and 5 ht2a were significantly increased as compared to sccg as well (p < 0.0001, p = 0.003). discussion & conclusion: these findings showed that exposure to ee throughout the whole childhood causes several neurobehavioural effects like increased exploration and low anxiety. these effects may lead to improvement in speed of learning. increase in the nr2b and 5ht2a concentrations which are the receptors that are related to learning and memory in the hippocampi accompanied these changes which may be basis of the neurobehavioural improvements or may provide contribution to positive neurobehavioural effects. p-02.09.1-023 effects of monosodium glutamate exposure during prepubertal term on several biochemical parameters in rats h. i. b€ uy€ ukbayram, d. kumbul doguc ß, i. ilhan, a. y. ismail s€ uleyman demirel university, isparta, turkey monosodium glutamate, which is commonly used in processed foods as flavor enhancer, is considered 'generally recognised as safe' by fda; however many studies have revealed the negative effects of msg.we aimed to evaluate the effects of msg in childhood on several serum parameters. sixty-six rats, (4 weeks old) were divided into 3 groups as control (cg, n = 22; 11 + 11, male+female) , experiment 1 (msg-low dose, e1g, n = 22; 11 + 11, male+female) and experiment 2 (msg-high dose, e2g, n = 22; 11 + 11) groups. msg was administered at 25 mg/kg/d to e1g, 2.5 g/kg/d to e2g for 6 weeks by oral gavage. the rats were sacrified and blood samples were collected from aorta. the blood samples were centrifuged, the serum samples were separated and glucose, alt, total protein, albumin, creatinine, cholesterole and triglyceride levels were analysed by beckmann au 5800 autoanalyser. level of total protein was significantly increased in e1g and e2g groups when compared to cg (p < 0.05). level of alb€ umine was also increased in both egs but significant difference was seen in e2g as compared to cg. creatinine levels were significantly increased in egs when compared to cg (p < 0.05). although the glucose levels in both egs were increased, the increase in e2g was statistically significant (p < 0.05). the alt levels of in egs were also increased but the significant increase was seen in e2g (p < 0.05). the effect of msg seem to be dose dependent and especially effect on carbonhydrate metabolism. increasing doses caused increase in glucose level, and tendency to glucose intolerance. increasing doses of msg also caused increase in creatinine and urea. another apparent effect of msg was detected on alt activity. in conclusion the negative effect of msg on glucose level, liver and kidney functions depends on daily dose intake. consumption of msg seem to be inevitable it has to be restrained in children otherwise early metabolic problems may be future problems for these children. (700 mg/kg) + tartrazine (750 mg/kg) + brilliant blue fcf (600 mg/kg) + ponceau 4r (70 mg/kg) + azorubine (400 mg/kg) + indigotine (500 mg/kg) + erythrosine (10 mg/kg). artificial food color mixture were administered to g 2 and g 3 and drinking water was applied simultaneously to g 1 by oral gavage per day for 3 weeks. after application all rats were sacrificed, the total oxidant (tos)/antioxidant (tas) capacity were analyzed in rats' brain, liver, kidney homogenate and serum with rel tos-tas diagnostics assay kit.the statistical analysis was carried out by using kruskal wallis test. tas and tos levels in liver homogenate were not found significantly different between all groups (p > 0.05). in serum and kidney and brain homogenate, tas levels were not significantly different between all groups. tos levels in g 3 were higher than g 1 and g 2 in serum and kidney and brain homogenate (p < 0.05). exposure to synthetic food colors may increase oxidative stres in vitale organs such as brain, kidney in female rats. these alterations differ according to organ and dose. parallel with increasing trends on healthy eating habits, consumption of prebiotics and probiotic microorganisms have been popular due to their benefits on human health. functional dairy foods such as probiotic yoghurt and cheese are the most common foods including probiotic microorganisms. due to some considerations such as standardization and quality in bulk production, starter cultures are used in industrialised fermentative food production to start fermentation. starter culture basically refers the microorganisms which induce and maintain fermentation of the fermentative foods and starter cultures including probiotic microorganisms are called as probiotic starter cultures. in this study, probiotic cheese starter cultures as a microbial community were investigated using computational systems biology tools. a metabolic network model of probiotic cheese starter culture was reconstructed using microbial community network modeling approach. literature-based genome-scale metabolic models of commonly used lactic acid bacteria were used for the microbial community metabolic model. the microbial community metabolic model simulated metabolic interactions of the microorganisms in the probiotic starter culture. metabolic flux values computed by the metabolic network model also predicted the metabolic pattern of the glycolysis (conversion of lactose), lipolysis (conversion of fat) and especially amino acid catabolism which are associated cheese flavor metabolism. simulations obtained by metabolic network-based analysis of cheese starter cultures can also be used for other fields like genetic engineering, upstream processing of the functional cheese production. p-03.01.1-002 er quality control protein network in cf to modulate f508del-cftr rescued phase ii xenobiotic metabolizing enzymes convert parent compounds into more hydrophilic metabolite by catalyzing conjugation reactions including glutathione and amino acid conjugation, glucuronidation, sulfation and acetylation. this study was aimed to describe the best cell line model for studying phase ii xenobiotic metabolizing nqo1 and gst-pi enzymes. for this purpose, mrna and protein expression of nqo1 and gst-pi enzymes were analyzed in ht29 and sw620 (colon); hepg2 and huh7 (liver); pnt1a and pc3 (prostate) cell lines by qrt-pcr and western blotting techniques, respectively. protein expression analysis revealed that nqo1 protein was expressed in all cell lines and relative protein expression is highest in the hepg2 (100%) and pnt1a (97%) while huh7 (31.9%) showed relatively low expression of nqo1. in addition, nqo1 mrna expression was relatively high in ht29 (1.68 fold) and pnt1a (1.92 fold) when compared with liver cell line hepg2 (1.00 fold). gst-pi protein expression was found very high in huh7 (100%) while there was no expression in hepg2. gst-pi mrna expression was relatively higher in pnt1a (3.56 fold) and ht29 (2.47 fold) when compared with huh7 (1.00 fold). according to these results, choosing the best cell line as model depends on the purpose of the research. for studying metabolism of a chemical by nqo1 and gst-pi or effect of a chemical on translational regulation of these enzymes, it is better to consider protein expression of the cell lines for choosing best model. however, if the aim is to study effect of a chemical on transcriptional regulation of these enzymes, it is better to choose a cell line that expressing highest mrna of gene of interest. in conclusion, considering both mrna and protein expression levels together, the best model cell lines for studying phase ii xenobiotic metabolizing nqo1 and gst-pi are ht29 and huh7, respectively. p-03.01. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] the studies on the pancreatic cells' surface glycoconjugates profiles in rats fed with high fat with lectin labelling methods by flouresans microscopy y. mater, s. beyhan ozdas gebze technical university, kocaeli, turkey in this study, the backbone of the cellular adhesion-recognition mechanism, located in the cell membrane. the study material selected pancreas tissue, has a privileged structures. the pancreas is one of the main organs to aid in digestion. the pancreas functions as an exocrine gland and role in digestion. in addition, the pancreas also functions as an endocrine gland, secreting several hormones into the blood that control the blood levels of glucose and other nutrients. due to the pancreas have been selected for this unique feature. thus, different types of cells in the same sample will be able to study the structure of the surface glycoconjugates. generally researches about determination of carbohydrates in the cell, glycoproteins or/and glycolipids are cut with enzymes. next step, the oligosaccharide mixture obtained, than establishing the complete structure of oligosaccharides and polysaccharides requires determination of branching positions, the sequence in each branch, the configuration of each monosaccharide unit, and the positions of the glycosidic links. this is a more complex problem than protein and nucleic acid analysis. these processes are indispensable for the understanding of the chemical structure of the sugar. whereas in cells using labeled lectins specific sugars, it is possible to accurately determine. in this study, was used triticum vulgaris (wga) labeled with fluorescein (fitc). thus, the cells located on the cell surface and neu5ac (sialic acid) for wga sugar residues were investigated. according to preliminary results of this study, wga labeled with fitc is specifically binding of these sugars. when this study is completed, the differences of sugar on the surface of different type of cells in the pancreas can be distinguished in micrographs. thus, in the cells of the pancreas, the sugar units involved in adhesion-recognition can be possible to determine specifically. large scale gene networks could be topologically analyzed in order to obtain possible global system-level structure cancer gene co-expression networks can have lower connectivity as compared to normal samples. using colorectal tissue gene expression datasets, we observed that tumor specific networks are less connected than normal networks. functional enrichment analysis suggested that cell cycle genes and methylation-associated cell adhesion genes can specifically play a role in the connectivity loss of carcinoma samples. literature confirmation provided a gene network including significant genes playing roles in the intersections between cell cycle, cell adhesion, and cell skeleton dynamics. this network can provide novel insight to our understanding of the molecular mechanisms of colorectal cancer. p-03.01.1-012 tf-mirna circuits specific to epithelial cancers y. oztemur, a. aydos, b. gur dedeoglu ankara university biotechnology institute, ankara, turkey cancer is the most common cause of death in the world but there are still a lot of uncertainties about the exact mechanism taking roles in regulation of it. cancers can be classified according to cell type; in which they start. carcinomas are the most prevalent types of cancer and start in epithelial tissues. they are also named as epithelial cancers (ecs) and make up about 85 out of every 100 cancers. over the past few years, many studies are concentrated on mirnas, which have emerged as important regulators of gene expression like transcription factors (tfs). tfs are regulators at transcriptional level while mirnas are post-transcriptional regulatory key-elements. otherwise the transcription of mrnas and mirnas are known to be regulated by tfs and tfs are the targets of mirnas. therefore, it is crucial to characterize the relation of tfs, mirnas and their targets by building circuits in diseases such as in ecs. for this study, mirna and mrna expression studies including epithelial tumors and normal samples searched in geo and array express microarray databases. 4 mrna studies and 4 mirna study, which were designed for 4 different ecs (breast, lung, ovary and colorectal) were selected to be analyzed. differentially expressed (de) mrnas and mirnas between epithelial tumors and normal samples were extracted (p ≤ 0.05, 2 fold change). among de genes, transcription factors and mirnas were identified and listed for epithelial tumor vs. normal comparison. circuit analysis resulted with remarkable circuit, which was common for all the types of ecs that includes klf4 transcription factor and hsa-mir-145. in the literature hsa-mir-145 and klf4 are known as important regulators in different types of cancer, which indicated that the motifs involving tfs and mirnas might be useful for understanding the regulation of ecs. as a conclusion finding out new and common circuits may aid us in predicting new or alternative diagnostic and/or prognostic biomarkers for ecs. mesenchymal stem cells (mscs) are multipotent stromal cells that can differentiate into a variety of cell types which are used in cell therapy. although they are the most attractive cell type for cell therapy studies, primary mscs lose their differentiation potential with increasing time in culture and passage so they are of limited use. due to this disadvantage, msc lines are more suitable for in vitro researches owing to their immortality. in this study we compared primary bone marrow-derived msc (bm-msc) with bone marrow derived msc line (rcb2153) in terms of cell characteristics and gene expression profiles to determine the functional differences among mscs types. firstly, mscs were identified by using cd29, cd70, cd90 and cd105 as positive markers and cd34 as a negative marker. gene expression profilling was investigated using affymetrix hg-u133-plus2 arrays. the significant go biological process terms and kegg pathway enrichment analyses of the identified degs were performed using david (p < 0.001, fold change≥2). the analysis showed similar pathway clustering in both cell types. the resulting quantitave transcriptome of 754 genes were identified that differentially expressed in msc line versus primer mscs (538 upregulated and 216 down-regulated). functional classification of changed genes was mainly clustered in cell cycle, cell death and mismatch repair. kegg pathway analysis revealed that the genes were significantly enriched in pathways including "cell cycle, dna replication and focal adhesion" pathways. in conclusion, our results indicate that msc lines can be used instead of primary mscs. these quatitative results provide an important basis to adapt cell lines to more closely resemble physiological conditions as oppossed to animal experimentation. this could help to minimize the use of animals in research. p-03.01. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] association between loss of 18q21, gain of 20q13.33 and progression in sporadic colorectal cancer n. belder 1 , b. savas 2 , m. a. kuzu 2 , i. pak 3 , h. s€ umer c ß elebi 1 , a. ensari 2 , h. € ozdag 1 1 biotechnology institute, ankara university, ankara, turkey, 2 school of medicine, ankara university, ankara, turkey, 3 ankara oncology training and research hospital, ankara, turkey colorectal cancer (crc) is one of the most diagnosed cancer and the third leading cause of cancer deaths throughout the world. identifying of copy number variation (cnv) profiles between early and late stage cancers can be useful to understand the progression and aggressiveness of cancer. the main goal of this study was to construct a comprehensive insight of association between cnv and sporadic crc stages in order to identify novel candidate targets which may contribute to tumor progression. affymetrix 6.0 genechip snp arrays were used for characterization of cnvs in tumor and matched normal formalin-fixed, paraffin-embedded (ffpe) tissues from 5 stage i, 17 stage ii and 25 stage iii samples. paired cnv analyses were performed using partek genomic suite 6.6 and genomic segmentation algorithm was performed using a minimum of 10 markers per segment, a signal-to-noise ratio of 0.3 and the cut-off value for the gain and loss was set of 2 ae 0.3. the adjusted p-value ≤0.05 were considered to be significant. whole genome cnv analysis revealed that amplification of 20q13.33 with 4 genes was found the most frequent (76.5%) in stage ii tumors. the most frequent (36%) amplifications were 13q12.2 and 7p22.2 in stage iii tumors. while deletion of chromosome 18q21.2 in stage iii with a frequency of 36% was found the most frequent loss, deletion of 18q21.1 was seen the most frequent (64.7%) in stage ii tumors. two tumor suppressor genes smad2 and smad4 which are found in these deletion regions were common genes between stage ii and stage iii. our results showed that gain of 20q13.33 might have a significant role in the progression of cancer. loss of 18q21 comprising two tumor suppressor genes is also another important finding. 18q21 loss can be a significant prognostic value in colorectal cancer even though validation of target genes requires additional study and larger sample size. this work was supported by tubi-tak project no:109s477. p-03.01.1-016 meta-analysis based mirna signature discriminates cervical cancer from normal samples a. yucel polat, y. oztemur, a. aydos, b . gur dedeoglu biotechnology institute, ankara university, ankara, turkey gynaecological cancers are common problems in female health. squamous cell carcinoma (scc) is a type of these malignancies. this tumor type is derived from pre-cancerous lesions, which is called cervical intraepithelial neoplasia (cin). cin is classified as cin1, cin2 and cin3 according to their dysplasia grade in the cervical tissues. mirnas are small non-coding rnas that were shown to have important roles in the development and progression of various cancers. the aim of this study is identifying mir-nas, which are playing a part in progression of cervical lesions by a ranking based meta-analysis approach. two mrna and three mirna expression studies, which include normal, cin1, cin3 and scc samples were selected from arrayexpress and gene expression omnibus (geo) databases. three mirna studies were combined with anova dependent ranking based meta-analysis program which was developed in our laboratory to find out a mirna signature that can discriminate cin1, cin3 and scc samples from normal samples. the top five mirnas with the highest ranks in meta-list were selected for further analysis. predicted targets of these mirnas were identified by mirdb target prediction tool. additionally two mrna datasets were selected for mirna-target validation studies. common genes, which were obtained from meta-mirna targets and differentially expressed genes between normal and cin1, cin3 and scc groups from two independent studies, were identified and they were subjected to pathway enrichment analysis. pathway enrichment analysis that was performed with 338 common genes showed that these targets were significantly enriched (p < 0.05) in especially cell proliferation, cell survival and cell cycle pathways, which are the key players of cancer development and progression. the meta-analysis results together with validation analysis of their targets may point out the potential roles of mirnas as biomarkers for the diagnosis and the treatment of cervical cancer. p-03.01. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] the hypoglycaemic and regenerative activity of thymbra spicata in alloxanized-diabetic rats thymbra spicata (labiatae), a carvacrol and thymol containing plant, is one of the medicinal herbs used by diabetic individuals to reduce blood glucose in turkey. we investigated the hypoglycaemic and anti-lipemic effects of the aqueous extract prepared from dried leaves and flowers of this plant in alloxanized-diabetic rat model. rats were divided as: diabetic control (group 1), dia-betic+glibenclamide (group 2), diabetic+plant extract (group 3), untreated control (group 4) and control+plant extract (group 5) groups (n = 6 for each group). serum glucose, lipid levels and body weight changes were mesasured and pancreas and liver histology of the rats were examined. each rat in all groups were administered the plant extract (30 mg), and the reference drug glibenclamide (2 mg/kg) by gastric gavage every day for 8 weeks. in group 1, blood glucose, serum alt, ast, triglyceride, cholesterol and ldl cholesterol levels increased while body weights decreased. in group 2, serum glucose, alt, ast, triglyseride and hba1c levels decreased compared to group 1 while cholesterol and ldl levels were high. in group 3, serum alt, ast, trigliserit, cholesterol, ldl levels decreased significantly but serum glucose and hba1c were higher compared to group 4. body weights increased except group 1 and hdl levels were not altered. histologically degenerative changes observed on pancreas of group 1 were decreased in groups 2 and 3. there was no difference on liver histology of the groups. in conclusion, thymbra spicata showed a protective and regenerative effect on diabetic pancreas. the hypo-lipidemic effect of the plant extract was also more effective than glibenclamide possibly due to the flavonoids, saponins and triterpenoids contents in the extract. its hypoglycaemic and protective activity should be tested for different doses and extract preparations and for longer periods. our study suggests that thymbra spicata is an excellent candidate for future studies on diabetes mellitus. with three different transcriptome data sets from the public gene expression omnibus database: time dependent data of dphop mutant, dargr mutant and wild type strain. the dynamic data spanned both primary and secondary phases of the metabolism. statistical results of transcriptome data were used for reporter metabolite analysis and reporter pathway analysis, which identify the metabolites (or pathways) with a significant coordinated transcriptional change in response to gene deletion perturbation in phosphate and nitrogen metabolisms. further, the production of actinorhodin, a pharmaceutically important compound, was modeled in the two deletion strains by calculating the metabolic fluxes subject to transcriptional level constraints on enzyme-coding genes. the metabolic switch from primary to secondary metabolism was highlighted in terms of the activity of pathways and fluxes as a result of the computational analyses in this work, leading to a better understanding of the role of phosphate and nitrogen metabolisms in increasing production levels. introduction: as a member of legume family licorice (glycyrrhiza glabra l.) has been widely used by human kind for many years as food constituent. especially by folks in rural sites licorice consumed widely. beside food constituent licorice has been used for medical purposes as well. licorice found effective with scientific datas on peptic skin infections, ulcers, inflammation, eczema, alzheimer disease, liver disease, and cancers. it also has been used as natural sweetener and food additive for preparing candies, chewing gum and beverage since ancient times. like all other medicine it has not been free of adverse event or toxicological effects. material and methods: alcoholic extracts of plant obtained by maceration process. for in vitro examination of anti-oxidant profile of licorice dpph free radical scavenging, abts cation radical scavenging and cupric ion reducing antioxidant capacity assay applied. application of extract made by oral route to rats for a week. anti-oxidant profile has been evaluated by myeloperoxidase (mpo), arylesterase (ares), total oxidative stress (tos) and total antioxidant status (tas) of serum levels. determination of toxicological effects alt, ast, ldh and alp values studied. histological investigation applied on liver and kidney tissues. results and discussion: results compared with control and standarts. antioxidant potential of licorice has been observed by in-vitro assays. serum mpo and ares values also compared with in-vitro results and correlation between them has evaluated. toxicological investigations made after evaluation of ast, alt, ldh and alp values. conclusion: in vitro assays has showed that licorice has potential anti-oxidant effect. investigation revealed that a mild toxic effect of licorice by biochemical tests. toxicological profile compared with control group and alt, ast values found slightly decreased and a mild elevation has been seen in ldh and alp values. for further and detailed investigation is needed. p-03.01.1-020 on the applications of a metabolic network model of mesenchymal stem cells h. fouladiha, s. a. marashi, m. a. shokrgozar, m. farokhi mesenchymal stem cells (mscs) have several applications in tissue engineering and regenerative medicine. mscs can be very useful in stem cell therapy, because they can be isolated bone marrow or adipose of an adult. these cells have also been used as gene or protein carriers. therefore, maintaining them in a desire metabolic state has been the subject of several studies. here, we have used a genome scale metabolic network model of bone marrow derived mscs for exploring the metabolism of these cells. then, we try to validate the computational results by experimenal tests. we analyzed metabolic fluxes in order to increase stem cell proliferation using the metabolic model. consequently, the experimental results were in consistency with computational results. in the present work, the applicability of the metabolic model was successfully approved. therefore, this metabolic model can be useful in biomedical researches of stem cells. p-03.01.1-021 qtl analysis for body weight and fatness in bxd recombinant inbred mouse strains a. dogan 1 , c. neuschl 2 , r. alberts 3 , g. a. brockmann 2 1 school of medicine, istanbul kemerburgaz university, istanbul, turkey, 2 department for crop and animal sciences, humboldt-universit€ at zu berlin, berlin, germany, 3 helmholtz-zentrum f€ ur infektionsforschung, braunschweig, germany genetic variation in body weight and composition is under the influence of many genes and have different genetic architectures. in the present study, the genetic factors contributing to body weight and fatness were examined under energy rich feeding conditions. growth traits, lean and fat weight, fat mass gain were analyzed to map qtls in a set of bxd ri strains. genome-wide analyses were revealed several genomic loci that control body weight and associated bodily changes in a sex and age-specific manner. the genetic data provided evidence for significant qtls on chromosome (chr) 4, 5, 14, and 16. most likely candidate genes within or near the regions with the highest significance levels were identified. the genes 1700048f04rik, gbe1, a830060n17, and four genes cenpc1, stap1, uba6, gnrhr for example, are suggested as most likely positional candidates accounting for the qtl effects on chr 5 for fat mass, on chr 16 for fat mass gain and on chr 16 for lean weight, and chr 16 for body weight, respectively. our results showed that body composition and fatness are highly complex that many genetic factors regulating and suggested candidate genes, which may help for studies of human fatness. related to serotonergic and gaba systems in response to hormonal changes. the nutrients involved in neurotransmitter synthesis may be the cause of relationship between diet and premenstrual syndrome. therefore, the aim of this study was to investigate the effect of various nutrients and premenstrual syndrome. this study was conducted to 29 healthy women aged 20-37 years. participants were asked to fill in premenstrual assessment form. dietary intakes (three days in each phases) were recorded during premenstrual, menstrual and postmenstrual phases. energy, protein, amino acids, iron, calcium, and magnesium intakes were estimated. statistical analyses were performed using the spss software. friedman tests were conducted and differences were considered to be statistically significant for p-values lower than 0.05. 60.9% of the participants reported premenstrual symptoms and premenstrual symptoms related nutrient intake were increased in these women. it was determined that energy (p = 0.03) and protein (p = 0.001) intakes were higher in the premenstrual phase. during premenstrual phase; tyrosine (p = 0.002), isoleucine (p = 0.001), leucine (p = 0.001), lysine (p = 0.008), methionine (p = 0.008), cysteine (p = 0.008), tryptophan (p = 0.000), and glutamic acid (p = 0.005) intakes were higher than other phases. likewise, iron intake was higher on premenstrual phase (p = 0.054). on the other hand, intake of other potential premenstrual syndrome related nutrients like fat, cholesterol, calcium, magnesium, and vitamin b6 were not significantly different within the menstrual phases. amino acids including tyrosine, tryptophan, glutamine, and vitamin b6 are involved in neurotransmitter synthesis and might be related to premenstrual symptoms. consequently, elevated intakes of dietary protein and some amino acids during premenstrual phase may be related to premenstrual syndrome symptoms. until now far uv cd spectra of only two potexviruses were published. the papaya mosaic virus (papmv) spectrum, measured by leclerc and co-authors contained no obvious anomalies and was similar to the spectrum of isolated papmv coat protein (cp). but measured 30 years earlier by homer and goodmanfar uv cd spectrum of potato virus x (pvx) itself had anomalous character and differed strongly from the spectrum of isolated pvx cp. in the present work we measured far uv cd spectra for two more members of potexvirusgenus: alternanthera mosaic virus (altmv) and potato aucuba mosaic virus (pamv) and their free cps. the altmv virion and altmv cp spectra were similar to each other and to the spectra of papmv and its cp. the pamv spectrum resembled the pvx spectrum in anomalously low ellipticity of the negative band at 208 nm, but in contrast to pvx, did not have additional peak at 228 nm. homologous modeling showed that cp of the three viruses is very similar in the core structure, and the observed difference may be explained by differences in disordered parts of proteins. possible reasons of potexvirus structural variability are discussed and it is suggested that the intravirus potexvirus cps may assume different conformations in different virions of the same preparation or even along the length of one virus particle. this work was supported by the russian science foundation (grant 14-24-00007). the antimicrobial potential of different phenolics was tested on pectobacterium in search of possible mode of action. in this respect, biofilm formation, exoenzyme activity, gene expression and virulence on its natural host (potato, cabbage, calla lily) were performed. also computational approach to show interaction between phenolic compounds and target protein was carried out using docking tools. the virulence determinants of pectobacterium were significantly impaired, at compound concentrations that did not affect bacterial cell growth. these observations suggested a mechanism which specifically interferes with bacterial virulence. since, these virulence determinants in pectobacterium are controlled by quorum sensing (qs), we focused on the effect of phenolics on the qs system in pectobacteria. the study revealed an inhibiting effect of the tested compounds on the expression level of central qs system and controlled genes, using qrt-pcr. also, there was a prominent reduction in the level of qs signal molecules n-acyl-homoserine lactone (ahl) accumulation. in addition infection capability was also practically blocked, which was completely recovered by application of exogenous-ahl. these results were supported by a potential interaction of plant phenolics with qs targets, as shown by molecular docking tool. collectively, results suggest the potential interference of phenolic compounds with qs central components (expi/expr proteins). moreover, it holds potential for future development of control measures against pectobacterium, and possibly other pathogens with similar mode of virulence. saccharomyces cerevisiae has been a key experimental organism for the study of infectious diseases, including double-stranded rna (dsrna) viruses. the l-a dsrna virus family of s. cerevisiae is widely distributed in nature. several versions of l-a virus are described and new ones continue to be discovered. some s. cerevisiae strains along with l-a dsrna possess smaller dsrnas, called m satellites. these dsrnas encode a sole secretable protein, known as k1, k2, k28 and k-lus toxin. l-a genome encodes the gag major structural protein and gag-pol fusion protein, formed by ribosomal frameshifting. gag-pol has transcriptase and replicase activities are necessary for maintenance of both l-a and m satellite dsrnas. so far, it's not known whether certain l-a virus has evolved to maintain a distinct type of satellite dsrna or this phenomenon lacks inherent specificity. we developed universal strategy to obtain full length l-a and m dsrna genomes from s. cerevisiae. complete viral dsrna genomes can now be cloned, as evidenced by l-a-28 dsrna, analyzed and sequenced directly from any yeast strain by means of enzymatic manipulations on total or fractioned rna content. we have identified previously undescribed l-a variant from different yeast strains specifically associated with certain type of m satellites. moreover, we identified for the first time full 5 0 -utr and 3 0 -utr sequences of m2 satellite. highly conserved sequence regions along with variable fragments were discovered at protein level, revealing clear trend to form clusters among different l-a gag-pol proteins. the obtained data suggest that each l-a virus variant can specifically maintain a distinct type of satellite dsrna. p-04.01.1-004 physic-chemical characterization of plga adjuvants for immunization per os t. chudina, d. kolybo palladin institute of biochemisry of the national academy of sciences of ukraine (nasu), kyiv, ukraine antibodies against diphtheria toxin play the most important role in the immunity against corynebacterium diphtheriae. all current diphtheria vaccines have parenteral route of administration. undoubtedly, oral administration of antigens would be the most patient-friendly way of immunization. however, the efficacy of free antigens oral administration is limited by their degradation in the gastrointestinal tract and poor absorption by m-cells. biodegradable and biocompatible polymers, like poly (d,l lactide-co-glycolide) (plga), are widely used for the design of mucosal immunizing agents. importantly, that the way of particle preparation plays an important role in plga biodegradation and antigen release. the aim of this work was to characterize the main physic-chemical properties of two types of plga particles: with immobilized antigen (plga 1) and with encapsulated antigen (plga 2) . we have prepared two types of plga particles containing egfp-sbb proteins (non-toxic recombinant fragment b of dt fused with egfp). the antigen loading efficiency of particles was determined based on the ratio of protein concentration in solution before and after loading and shown better results for plga 2 particles (plga 1 -72.05%, plga 2 -90.02%). the flow cytometry results demonstrated that 99% of plga1 particles conjugated with egfp-sbb, and only 92.2% of plga 2 particles conjugated with protein.the particle sizes had the slight difference by the results of two different techniques (ntanumber based, the software tracks individual particles; dls -scattering intensity weighted), however demonstrate similar patterns. dls data showed that the mean plga 1 particles size was 203.3 nm and plga 2 -211.6 nm. nta data also showed that mean plga 1 particles size a little smaller than plga 2 (183.8 nm and 192.8 nm respectively) . demonstrated differences in the properties of synthesized particles may have an influence on the immunogenicity of the used for oral immunization antigen. p-04.01.1-005 a suitable system for studying the functionality of a plasmodial protein in mammalian cell lines cho-mt58, a mutant cell line was proved to be an appropriate tool for investigating intracellular function of cct. in this cell line, the endogenous cct activity decreases dramatically at 40°c, blocking membrane synthesis and ultimately leading to apoptosis. we have studied the rescuing potential of pfcct in cho-mt58 cells with the isogenic cho-k1 cells as a control. cells after transient transfection were incubated at 40°c and then analysed by facs using the fluorescence of egfp fused to pfcct. the proportion of cells undergoing apoptosis was determined by propidium-iodide staining. we have demonstrated for the first time that heterologously expressed pfcct is able to complement endogenous cct activity in mammalian cells. thus, a suitable system has been established for functional investigation of structural elements of pfcct. in order to reveal the role of different protein sequences in enzymatic function, we redesigned the structural gene of pfcct obtaining a modular system where different domains are easy to be removed or exchanged. here we designed a series of different truncation and deletion constructs to reveal the role of plasmodium specific sequences. in parallel, heterologous expression experiments of different constructs in the mutant cho-mt58 and the wild type control cell lines are performed to validate the reported model system. p-04.01.1-006 host-pathogen interactions: is there a relationship between tlr 4 polymorphisms and tuberculosis in a group of turkish patients? introduction: tuberculosis (tb) is a global health problem and according to world health organization (who) each year more than 2 million individuals die from tb and each year20,000 cases of tb are notified in turkey. malatya is the third largest city in east anatolian region of turkey and tb incidence rate is higher (28.5/100,000) comparing to the general population of the country. for this reason it is important to determine the factors that lead to tb in this population. disease agent can stay in the latent phase for long periods of time after infecting the individuals. while some infected individuals show the symptoms some others never do and even 90% of these never develop clinical disease. various mechanisms take place during the host response to infectious agents. toll-like receptor (tlr) genes are shown to be candidate genes in these responses. materials and methods: in this study 49 tb patients and 50 healthy controls were included. tlr 4 genotyping for rs4986790, rs4986791 was performed by using a commercial taqman snp genotyping assay kit. data were summarized by count and percent. hardy-weinberg equilibrium was tested by chi-square distribution with 1 df. differences between groups due to allelic and genotypic distributions were analyzed by pearson's exact or fisher's exact tests. in all comparisons significance level was considered to be 0.05. results: the single nucleotide polymorphisms (snps) which were subject of this study haven't been screened in turkish population earlier. no significant association was found between tb and the snps we screened in our group of patients. discussion and conclusion: unlike other populations results we couldn't find a significant association between the disease and the genotypes of our patients. the study should be performed in bigger populations in order to confirm the results. p-04.01.1-007 lytic action of bacteriophages as a tool for the obtaining of images p. boltovets 1 , r. radutny 2 , t. shevchenko 3 1 institute of semiconductor physics nas of ukraine, kyiv, ukraine, 2 scientific and technical center of advanced technologies nas of ukraine, kyiv, ukraine, 3 taras shevchenko national univercity of kyiv, kyiv, ukraine obtaining of images by different types of bacteria now became a very special branch of skill at the interface between science and art. however the authors did not found any scientific article, where bacterial lawn was used as the background and the image was formed by the lytic action of the virus (bacteriophage). whereas the mentioned approach could be used not only with artistic aims but for the practical use. the aim of this work was to demonstrate a possibility to obtain the image on the bacterial lawn by the lytic action of the bacteriophage. the bacterial lawn was obtained by the standard metod using the 1.5% agar with the nutrient medium and the 0.7% agar containing escherichia coli culture. stencils with the preparation of the bacteriophage t4 were applied. samples were incubated during the twenty-four hours at +37°c. after that stencils were removed and the samples were stained by coomassie blue r-250 or fuchsine (with further fixation by the 7% acetic acid). several approaches to obtain the image by the lytic action of the virus were applied. first of all stencils made from printing paper and filter paper were compared. it was demonstrated, that the use of filter paper stensil allows to obtain more accurate and controllable images, than the use of the printing paper stensil. in the next series of the experiment the possibility of the reversed stencil use (where the image is formed not by the lytic zone but by the zone of bacterial growth) was demonstrated. also the possibility of the partial staining of the obtained image was explored. it gives an opportunity to obtain polychrome images using available colorants. summarizing the above it should be noted, that it was the first time when the graphical image was obtained by the lytic action of the virus on bacteria. this approach could be used not only for the artistic aims but as well for the practical use, for example, for the restriction of the action of microorganisms in out-of-theway places. burgdorferi the identification and characterization of possible antigens is essential for the improvement of current laboratory diagnostics for lyme disease and vaccine development. in this study, several recombinant b. burgdorferi outer surface proteins have been obtained and their antigenic properties have been evaluated in an effort to characterize novel immunodominant antigens. because b. afzelii and b. garinii are the most prevalent species in latvian ticks, proteins with conserved domains were included in this study. a panel of serum samples of lyme disease patients with early and disseminate disease stage was used. the controls were matched by age and sex to the patients and represented the same geographic area. the results show that proteins of several b. burgdorferi gene families have properties with respect to their candidacy as a subunit assay for a novel lyme disease immunodiagnostic. especially, the difference in their size in a range on the western blot assay may provide good discrimination between protein bands. however, they have potential for diagnosis if used in combination with other antigens but not as a "stand alone" test. in conclusions, this study showed the existing challenges in serological testing of early lyme disease. the conservation of the sequence of antigen between species of b. burgdorferi complex is essential for the most successful serodiagnostic marker candidate. the presence of homologous proteins in treponema species could lead to the cross reactivity in syphilis patients, and should be carefully evaluated. antimicrobial resistance is one of the greatest challenges in modern medicine. there is a pressing need for better understanding of the specific mechanisms that contribute to resistance to optimize existing therapies. in 2013 in georgia extended-spectrum beta-lactamase (esbl)-producing e. coli strain was isolated from the post-surgical sample obtained from gallbladder of the patients with chronic calculous cholecystitis which belongs to the sequence type 23 (st23) complexes with ctx-m 55 gene. is this strain characterized by other differences on a proteome level? are antibiotics against which the strain is resistant inducing the changes in bacterial proteome? the present work was aimed (i) to study the differences on a proteome level (i) between e. coli 92-1917/13-g and attc e. coli-reference strain and (ii) to compare the proteomes of 1917 strain at two conditions: with and without antibiotics. 1917 strain was grown in the presence of three antibiotics: rocephin (ceftriaxone), fortum (ceftazydym) and claforan (cefotaxime sodium) together. proteomic expression was analyzed using two-dimensional gel electrophoresis and mass spectrometry. significant differences were found for several proteins, including putative abc trnsporter arginine protein 2, cystine-binding periplasmic protein, fkbp-type peptidyl-prolyl cis-trans isomerase, outer membrane protein a, d-galactose binding periplasmic protein and some others. the importance of these differences for anti-microbial resistance will be discussed. p-04.01.1-010 molecular characterization of resistance and virulence features in staphylococcus aureus clinical strains isolated from cutanaeus lesions in patients with drug adverse reactions i. lupu 1 , i. gheorghe 2, 3 , m. popa 2, 3 , a. ion 3 , m. mihai 1 , v. lazar 2, 3 , m. c. chifiriuc 2, 3 1 carol davila" university of medicine and pharmacy, bucharest, romania, 2 research institute of the university of bucharest-icub, bucharest, romania, 3 faculty of biology, university of bucharest, bucharest, romania patients treated with epidermal growth factor inhibitors often experience cutaneous adverse reactions. however, the infectious complications of these toxic effects and the contribution of specific pathogens, such as the community emergent methicillin resistant staphylococcus aureus strains. the present study was aimed to identify the types of sccmec and virulence genes profile in clinical s. aureus isolated from cutaneous lesions of different severity degrees in patients with dermatologic toxic effects. this study was conducted on a total of 42 s. aureus clinical strains isolated in 2016 from acneiform reactions pustulae and periungual lesions in patients with drug cutaneous adverse reactions. multiplex pcr was performed on genomic dna from isolates in order to identify the sccmeccentral elements and the virulence genes: bbp (bone bound sialoprotein), ebps (elastinbinding protein), fnbb, fnba (fibronectin-binding proteins), fib, clfa, clfb (clumping factors a and b), cna (collagen-binding protein), luk-pv (panton-valentine leucocidin), hlg (haemolysin), tst (toxic shock toxin). the mrsa phenotype was genetically confirmed by the presence of meci gene in case of 19.04%, meca in 14.28%, sscmec type ivd element in 11.90%, ccrb2 in 7.17% and sccmec types i, iii, iv in 4.76% of the studied s. aureus strains. regarding the virulence genes encountered in s. aureus strains, the most frequent was clfa (90.47% of the isolates), followed by clfb (88.09%), fib (35.71%), hlg (16.66%) and bbp (14.28%). these results confirm the high prevalence of mec i and sscmec type iv elements, usually encountered in communityacquired mrsa strains, in cutaneous isolates from patients with dermatologic toxic effects. more data on the virulence and genetic background of these local strains are needed to appropriately assess the risk of such infections and avoid the inappropriate administration of beta-lactams. p-04.01.1-011 analysis of toxicogenic properties of staphylococcus aureus strains isolated from cows with subclinical form of mastitis in the central area of russian federation. pore-forming toxins and enterotoxins), which are present in s. aureus strains isolated from clinically healthy cows. staphylococcus strains were isolated from cow's milk. disk diffusion method was used to determine the sensitivity to antibiotics. pcr analysis was used for detection of meca, mecc genes and genes of toxins. investigated strains were resistant to oxacillin (14%), vancomycin (8%) . it was found that all strains, which contain meca and mecc genes, showed resistant to more than 5 antibiotics. it was determined that among the investigated strains 13% contained meca, 13% -mecc, 9% contained both meca and mecc. some strains contained genes of panton-valentine leukocidin (pvl) or alpha-hemolysin and several strains contained both types of genes. enterotoxin a (sea) gene was detected in 26.7% of cases, sed -5%, seg -10%, sei -35%. genes of staphylococcal toxins b, c, e, h were not found. the presence of phenol soluble modulin biosynthesis genes was determined: genes of alpha peptide synthesis were found in 93% of strains, beta peptide toxin genes in 73%, delta toxin gene in 100%. it was determined, that clinically healthy animals are carriers of s. aureus strains that cause mastitis. high level of antibiotic resistance was found in strains containing meca and mecc genes. the major part of the strains carried genes of phenol soluble modulin biosynthesis. the role of phenol soluble modulins as well as of pvl and alpha-hemolyzin in the development of mastitis is not completely clear. we conclude that pore-forming toxins have dominant role in the latent form of mastitis. p-04.01.1-012 impact of lactoferrin on the hydrophobicity and adherence to the inert substratum of staphylococcus aureus strains isolated from patients with cutaneous drug reaction skin healing is a complex biological process that requires the involvement of different cell types and humoral effectors. one of the main factors are aggravating and delaying the healing process is represented by the supra-infection with pathogenic or opportunistic microorganisms that grow in specialized consortia embedded in a self-produced extracellular polymeric matrix, called biofilms, which are extremely resistant to any antimicrobials and host immune response. lactoferrin (lf) is an ironbinding glycoprotein which promotes skin healing by enhancing the initial inflammatory phase, but also by inducing an overabundant immune response. the aim of this study was to investigate the influence of lf, one of the main components of innate, humoral anti-infectious immunity on some microbial features, involved in the first steps of the infectious process, such as hydrophobicity and adherence of staphylococcus aureus strains isolated from maculo-pustular lesions in patients with adverse reactions to epidermal growth factor inhibitors. for hydrophobicity measurement the bacterial suspensions were grown in the presence or absence of lf, and then, the "microbial adherence to hydrocarbons test" (math) was performed. the capacity to develop biofilms on inert substrata and the influence of lf on this feature was spectrophotometrically quantified using an adapted microtiter method, after crystal violet staining. our results showed that lf decreased the hydrophobicity and limited the biofilm development of all 42 s. aureus tested strains, in a dose and time dependent manner. the decreasing effect on the microbial hydrophobicity was accompanied by a lowering effect on the adhesion of microbial strains to the inert substratum. in conclusion these observations indicate that lf exhibits a wound pro-healing effect, by limiting the microbial colonization and biofilm formation and thus, the occurrence of infectious complications of skin lesion. p-04.01. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] host-specificity determinants of bacteriophage vb_ecom_fv3 considered vehicles of s.aureus intoxication in humans throughout the world. the objective of the present study was to assess the presence of enterotoxigenic and methicillin-resistant s. aureus in water buffalo milk and dairy products. a total of 100 water buffalo milk and 100 dairy products (50 water buffalo cream and 50 water buffalo cheese) were collected from different dairy farms, smallholders and local bazaars in samsun, turkey. all samples were analyzed using the standard procedure en iso 6888-1 and isolates were confirmed for the presence of the 16s rrna and nuc gene by polymerase chain reaction. s. aureus was identified in 30 of 100 water buffalo milk (30%), 9 of 50 water buffalo cream (18%), and 17 of 50 water buffalo cheese (34%). a total of 99 isolates were confirmed as s. aureus by pcr. genotypic methicillin resistance was evaluated using pcr for the meca gene. out of 99 isolates, 9 (9%) were found to be methicillin resistant (meca gene positive) by pcr. the enterotoxigenic s. aureus was identified in 12 out of 99 (12%) isolates by the mpcr technique. five isolates produced staphylococcal enterotoxins sea (5/12; 41.6%), two isolates produced sec (2/12; 16 .6%), one isolate produced (1/12; 8 .3%) sed, one isolate produced (1/12; 8.3%) see and three isolates produced sec+sed (3/12; 25%) . none of samples were positive for seb. in conclusion, the presence of enterotoxigenic and methicillin-resistant s. aureus in milk and dairy products is of significant for public health concern and also these enterotoxin genes sea and sed are predominant toxins that can cause staphylococcus intoxication in humans. this study was funded by ondokuz mayıs university, samsun, turkey, scientific research project programs (project no: pyo. vet -1904.12 .004) and this article was part of a phd thesis. p-04.01.1-015 identification and biochemical characterization of an immune modulating protein from helicobacter pylori b. kaplan t€ urk€ oz faculty of engineering, department of food engineering, ege university, izmir, turkey helicobacter pylori is able to achieve persistent infection with minimal immune response. the first line of defence during h.pylori infection is through gastric epithelial cells which present toll like receptors (tlr). a family of bacterial proteins which share homology with the toll/il-1 receptor (tir) domain were identified. the structure of btpa from brucella showed that bacterial tir proteins (btp) mimick human tir domain proteins and act on myd88 signaling pathways to suppress tlr signaling. h.pylori might also produce a similar protein. a putative h. pylori tir protein was found based on sequence homology and the corresponding gene; hp1437; was cloned in fusion with an n terminal cleavable 6his-tag. the recombinant protein, 6his-1437 was purified using nickel affinity chromatography. 1437 was subjected to limited proteolysis and the bands were analyzed by peptide mass fingerprinting (pmf). oligomerization of 1437 was investigated by in vitro pull-down and size-exclusion chromatography. 1437, a 239 amino acid protein, has a predicted c terminal tir domain similar to other btps and sequence alignments verified the presence of tir domain signature regions. recombinant 6his-1437 was produced with a yield of 10 mg/l culture. a structurally stable 25 kda fragment was obtained from limited proteolysis which contained the tir domain as verified by pmf. in vitro pull down assays showed 1437 interacts with itself forming dimers as shown by size-exclusion chromatography. tir domain proteins function by interacting with themselves and other tir domains. our results showed that 1437 also form dimers, supporting that it is a btp. current research is focused on solving the structure of 1437 and investigating its interaction with myd88. 1437 might play a direct role in reduced immune response against h.pylori by binding to myd88 analogous to other btps. further characterization of 1437 will provide the first solid evidence of presence of a tir domain protein in h.pylori. p-04.01. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] lipopolysaccharides with different lipid a acylation status from vibrio cholerae and campylobacter jejuni contribute differently to il6 production by bone marrow-derived macrophages k. korneev 1,2 , e. sviriaeva 1, 2 lipid a is a biologically active part of lipopolysaccharide (lps) from gram-negative bacteria that is responsible for the activation of the innate immunity through interaction with toll-like receptor 4 (tlr4) and subsequent production of proinflammatory cytokines. bacteria frequently transform their lipid a so that its recognition by tlr4 is not sufficient for induction of effective antibacterial immune response. we compared biological activity of various lps from pathogenic bacteria vibrio cholerae and campylobacter jejuni. we purified r-form lps for each strain by hydrophobic chromatography. the biological activity of lps preparations was evaluated by their ability to activate production of proinflammatory cytokine il6 by bone marrow-derived macrophages from c57bl/6 mice, using tlr4-deficient macrophages to control for specificity of tlr4 signaling. lps from e. coli and inactive lps from f. tularensis were used as positive and negative controls. lps from v. cholerae demonstrated biological activity similar to that of lps from e. coli, consistent with the presence of highly acylated lipid a in both strains. however, the former was a slightly weaker activator than the latter, because lipid a from v. cholerae had on average shorter acyl chains. lipid a from c. jejuni had on average longer acyl groups than in e. coli, while degree of acylation was lower, and as a result its lipid a displayed significantly lower biological activity. our study demonstrates importance of functional groups of lipid a in the ability of lps to activate production of il6 by macrophages. in line with our previous reports, we confirmed a direct correlation between biological activity of various lps species with their lipid a acylation status: the biological activity increases with increase in the length and in the number of the acyl chains. excess proinflammatory cytokine production through tlr4 activation can cause sepsis, while inefficient activation may result in the failure to clear bacteria. clostridium perfringens phospholipase c (cpplc) is the most toxic extracellular enzyme produced by this bacterium and it is an essential virulence factor in the pathogenesis of gas gangrene. cpplc may lead to cell lysis at concentrations that causes extensive degradation of plasma membrane phospholipids. however, at sublytic concentrations it induces cytotoxicity without causing evident membrane damage. the results of this work demonstrated that the cytotoxic effect of cpplc requires its internalization and the activation of the mek-erk pathway. cpplc internalizartion occurs through a dynamin-dependent mechanism and in a time progressive process: first, cpplc colocalizes with caveolin both at the plasma membrane and in vesicles, and later it colocalizes with early and late endosomes and lysosomes. the results also showed that cpplc requires endocytosis in order to activate mek-erk, because treatment with the dynamin inhibitor, dynasore, prevents cpplc endocytosis, erk 1/2 activation and cytotoxcity. cholesterol sequestration as well as inhibition of actin polymerization also prevents cpplc internalization and cytotoxocity, involving endocytosis in the signaling events required for cpplc cytotoxic effect. once internalized, cpplc induces reactive oxygen species production through the activation of pkc, mek/erk and nfjb dependent pathways. inhibition of either of these signaling pathways prevents cpplc's cytotoxic effect. in addition, it was demonstrated that nfjb inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of cpplc in mice. these data provide new insights about the mode of action of this bacterial phospholipase c, previously considered to act only locally on cell membrane. understanding the role of these signaling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridial myonecrosis. p-04.01.1-020 apoptosis induced by clostridium perfringens phospholipase c is mediated by reactive oxygen species m. flores-d ıaz 1 , l. monturiol-gross 1 , m. j. pineda padilla 1 , c. araya-castillo 1 , a. alape-gir on 1 bacterial phospholipases are lipolytic esterases surface associated or secreted by a wide variety of bacterial pathogens. clostridium perfringens, the most broadly distributed pathogen in nature, secretes a prototype phospholipase c (plc), also called a-toxin, which plays a key role in the pathogenesis of gas gangrene. this toxin causes death to cultured cells and extensive myonecrosis when injected intramuscularly in experimental animals. the results of the present study showed that c. perfringens plc (3-5 ng/ml) induces morphological and biochemical changes characteristic of apoptosis in cultured cells, as determined by scanning electron microscopy. nuclei condensation and fragmentation were observed by fluorescence microscopy and a typical ladder fragmentation pattern of genomic dna was detected by dna in agarose gels. cell death was prevented by the caspases inhibitors z-devd-fmk and z-vad-fmk. c. perfringens plc induces oxidative stress in cultured cells as determined by fluorescence microscopy and flow cytometry using the membrane permeable probe dcfda. different antioxidants including the gluthation precursor nac, several iron chelators and the free radical scavengers tiron and edaravone prevent cell death induced by c. perfringens plc in cultured cells or in mice challenged intramuscularly with 1.2 lg of that toxin. thus, this work provides compelling evidence that superoxide, hydrogen peroxide, and the hydroxyl radical are involved in the cytotoxic and myotoxic effects of c. perfringens plc. furthermore, the data demonstrated that edaravone, a clinically used hydroxyl radical trap, reduced the myonecrosis and the mortality caused by c. perfringens in a murine model of gas gangrene, induced by intramuscular bacterial injection of 10 8 bacteria. this knowledge provides new insights for the development of novel therapies to reduce tissue damage during clostridial myonecrosis. lectins are ubiquitous proteins able to recognize mono-and oligosaccharides with high specificity and low affinity. lectins do not have any catalytic activity, unlike enzymes, and they are not products of the immune system in contrast to antibodies. lectins play a crucial role in cell interactions on molecular level showing their importance in various physiological and pathophysiological processes as well as both mutualistic and parasitic interactions between microorganism and hosts. photorhabdus luminescens is a gram-negative bacterium from the family enterobacteriaceae. the bacteria have a complex life cycle that involves mutualistic and pathogenic interaction with two different invertebrate hosts. it is highly pathogenic towards insect larvae. in addition, p. luminescens lives in the intestine of infective juveniles of nematode heterorhabditis bacteriophora, together forming an effective entomopathogenic complex. we have identified several soluble lectins produced by p.luminescens. in this study, we focus on 4 proteins from p. luminescens, which show a high sequence homology with each other. a wide range of methods was used for structural and functional studies of photorhabdus lectins, e.g. surface plasmon resonance, isothermal titration calorimetry, analytical ultracentrifugation and x-ray crystallography. all lectins from p.luminescens recognize l-fucose and d-mannose. despite being closely related, they differ in fine binding specificities. to determine their biological function, knock-out mutants of p. luminescens are being prepared to study its interaction with axenic nematodes and insect larvae. breast cancer is the major disease of women in developed countries occuring predominantly after the age of 65. triple negative breast cancer (tnbc) is a typical subtype of epithelial breast cancer which lacks estrogen receptor (er), progesterone receptor (pr) and human epidermal growth factor receptor 2 (her2) all together. although various researches have been focused on characterizing tnbc and enlightening different molecular markers with the aim of improving the overall outcome, currently the sole affective therapy action for tnbc is chemotherapy. thus chemoresistance is the main clinical challange and accounts for 90% of failures in terms of treating the disease. multidrug resistance (mdr) is defined as simultaneous resistance towards the drugs which do or do not demonstrate structural resemblance and have different effects on their molecular targets. p-glycoprotein (p-gp) is a membrane protein coded by abcb1 (mdr-1) gene. p-gp is an atp-dependent pump which pumps a wide range of drugs out of the cells including chemotherapeutic agents such as doxorubicin (dox) and pactilaxel. in the present study, tnbc cell line mda-mb-231 was treated with increasing doses of dox, cell viability was examined with srb assay and development of mdr was investigated through mdr assay and rt-pcr. results demonstrated that cell viabiliy decreased significantly with the treatment of higher doses. mdr was shown to be increased when cells were treated with 50, 200 and 800 nm of the drug respectively along with 15 lm of p-gp inhibitor verapamil. rt-pcr results were obtained to be consistent with mdr assay results and indicated increased mdr-1 gene expression with the treatment of dox. especially after 400 nm of dox treatment, mdr-1 was overexpressed to be 59 fold when compared to control. in conclusion, it was demonstrated that mda-mb-231 cells have shown to display elevated resistance to higher doses of dox. p-05.01.1-005 targeting dna damage response pathway in cancer cells under heat stress and the mechanical effect of ultrasound y. furusawa 1 , t. kondo 2 1 toyama prefectural university, imizu-shi, japan, 2 university of toyama, toyama, japan ultrasound (us) has been widely utilized for diagnosis and therapy in many medical fields. the biophysical modes of us are divided into three classes, thermal, cavitation and non-thermal non-cavitation effects. in clinical use for cancer therapy, the thermal effect was utilized for hyperthermia therapy with focusing us on cancer to rise the temperature from 41°c to 44°c, or further which could induce thermal ablation of cancers. cavitation leads to a variety of mechanical stress such as shear stress, shock wave, high pressure, and chemical stress such as free radical formation, both of which have been inferred to act simultaneously on all biological materials. it has been indicated that us induces cell killing, cell lysis, loss of viability, and loss of clonogenicity. recently, we found that heat stress as well as us without thermal effect induce not only dna single-strand breaks but also dna double-strand breaks, a most cytotoxic region of dna, in chromatin dna detected by both gammah2ax staining and neutral comet assay. in response to the stresses which induce dna damage, the dna damage sensor protein kinase, ataxia telangiectasia mutated (atm), atm and rad3 related (atr), and dna-dependent protein kinase (dna-pk) become activated form to initiate signal transduction pathways activating cell-cycle checkpoints, dna repair, and apoptosis. the molecules consisting of dna damage response pathway were expected as therapeutic targets because defects in the response to dna damage agents can be lethal. this work was designed to explore the possible therapeutic targets of the molecules in dna damage response pathways for future us-aided therapy. finally, several kinases (e.g., checkpoint kinase) on dna damage response pathway seems to be the targets for hyperthermia and us therapy. (ural branch) , ekaterinburg, russia, 2 shemyakin and ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia based on the recently synthesized (s)-(2-aminopurin-6-yl) amino acids (gly, ala, val, phe, pro), we obtained a series of novel modified nucleosides using the transglycosylation reaction. for the first time, it has been demonstrated that the corresponding nucleobases are good substrates for the genetically engineered recombinant e. coli purine nucleoside phosphorylase (conversion to nucleosides reached 90-98%). nucleosides, such as ribosides, 2-deoxyribosides, and arabinosides were obtained in high yields (70-88%). it has been found that yield in the transglycosylation reaction does not depend on the structure of the amino acid fragment. the nucleosides synthesized are considered as potential inhibitors of intracellular adenosine deaminase (ad), the increasing activity of which is observed in hepatitis, cirrhosis, hemochromatosis, obstructive jaundice, prostate and bladder cancer, hemolytic anemia, rheumatic and typhoid fever, gout, and cooley's anemia. cytotoxicity of the synthesized nucleosides was tested in the jurkat (model of human t-lymphoblastic leukemia) and el-4 (model of mice t-lymphoblastic leukemia) cell lines. the compounds studied did not exhibit cytotoxic activity compared to the activity of the known antitumor agent nelarabin. the work was financially supported by the russian science foundation (grant 14-13-01077). p-05.01.1-007 dna binding, dna cleavage, antimicrobial activities, antimutagenic and anticancer studies of a schiff base and its complexes n. yildirim 1 , n. demir 2 , m. yildiz 3 1 health services vocational school, c ß anakkale onsekiz mart university, c ß anakkale, turkey, 2 department of biology, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, 3 department of chemistry, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey schiff bases are considered as favored and the most widely used ligands, due to their metal complexes having variety of applications as antibacterial and anticancer agents. the rational design and synthesis of new schiff bases and their metal complexes have been drawing great interest because of their diverse biological and pharmaceutical activities. so, exploring and designing novel molecules that have biological activities and capable of interacting with nucleic acids has a great significance for disease defence and to discover new dna-targeted anticancer drugs for chemotherapy. in this study, we report the synthesis and characterization of a novel schiff base and its ni(ii) and cu(ii) complexes. the minimal inhibitory concentration (mic) of the compounds was screened in vitro against bacteria and yeast cultures using broth micro dilution test. dna binding and dna cleavage activity of the compounds were investigated by uv-vis spectroscopy and agarose gel electrophoresis. antimutagenic activity of compounds were tested in the absence of microsomal enzymes (s9-). also, cytotoxicity of the compounds against hepg2 cell lines was assayed by the mtt (3-(4,5-dimethylthyazolyl-2)-2,5-diphenyltetrazolium bromide) method. consequently, uv-vis spectroscopy studies indicated that the compounds interact with calf thymus dna (ct-dna) via intercalative binding mode. dna cleavage activity studies showed that the cu(ii) complex can effectively cleave pbr322 plasmid dna. compounds inhibited the base pair mutation with high inhibition rate in the absence of s9. also, schiff base complex had cytotoxic activity towards hepg2 cell line, that it was found to be more potent than the control cisplatin. p-05.01.1-008 single particle electron tomography of rnap elongation complex, stalled at position +24 genome in vivo is constantly exposed to the damaging effects of the environment. single-strand breaks (ssbs) are the most frequently occurring dna lesions. accumulation of unrepaired ssbs can interfere with the cells metabolism and increase genomic instability. in vivo, ssbs are repaired in specific pathway, but, in eukaryotic nuclei, dna is organized in chromatin that could affect the accessibility of lesions to sensor proteins. breaks in a template strand induce arrest of rna polymerase ii (polii) in vitro and in vivo and can be revealed in a transcription-dependent manner. our recent biochemical studies identified two key intermediates formed during transcription through a nucleosome by rnap that are nearly homogeneous, active and stable by biochemical criteria (complexes stalled after entering 24 or 42 bp into the nucleosome; ec+24 or ec+41, respectively). hear we produced two complexes, both stalled in the +24 position, one without break in the dna, and the other with introduced ssb at position +12 of a non-template dna strand. complexes were purified using affinity chromatography and applied to a carbon-coated, glow-discharged em grid. tomographic studies were performed at ae70°in a jeol microscope at 200 kv accelerated voltage. images were recorded using a gatan ccd camera. image analysis was performed using the imod software. the resulting structure of the ec+24 complex with no break in dna consist of two domains, connected by a single dna string. the complex with a break introduced into the dna has a more compact appearance and its two domains were connected by two dna strings, thus forming an intranucleosomal dna loop. our data suggest that ssbs in a non-template strand can induce the formation of stable non-productive transcription intermediate. the inhibitory effect of ssbs onto transcription may suggest a possible mechanism for their recognition in vivo with a transcription-dependent pathway. this work has been supported by the rsf grant #14-24-00031. colorectal cancer (crc) is one of the leading causes of cancerrelated deaths in the developed countries. according to 2014 who report new incidence rate of crc in turkey is 6.5% among other cancer types. owing to difficulty of the low allele frequency variations detection, genetic association profiles of crc have not been entirely identified. low allele frequency variations mlh1 à93g>a (rs1800734) promotor substitution, mlh1 415g>c (rs28930073) exonic substitution, mthfr c677t (rs1801133) and apc 1307 t>a (rs1801155) were investigated in this study. these 4 snps "rs1800734, rs28930073, rs1801133, rs180115" are located on 1p36.3, 5q21, 3p22 respectively. colonoscopic investigations were performed on both cancer and control group. the 4 snps were genotyped using kompetitive allele specific pcr technology in 1014 cases and 805 healthy controls. statistical analysis was carried out with cochran-armitage chi-square test. in this study these 3 of the 4 snps in mlh1, mthfr genes were examined for the first time in turkish sporadic crc cases. statistical analysis showed no significant association within our turkish sporadic crc population. percentage of mlh1 à93aa genotype in group aged ≥ 65 was found to be 5.7% in cancer versus 2% in control group. moreover apc 1307a, mlh1 415c alleles were detected only 1 and 3 allele respectively. previously, apc 1307a allele was determined in 53.3% of a turkish cohort. however in the present study apc 1307a allele was detected on 1 allele only. studies showed mlh1 à93 promoter variation as a risk factor for microsatellite instabile crc but for the current study this data is not available. in spite of literature mthfr c677t and mlh1 415g>c snps were not found to be associated with sporadic crc in turkish population. this research demonstrates that importance of population based studies in multifactorial disease. p-05.01.1-010 excision of damaged bases from transcription intermediates by fpg/nei superfamily dna glycosylases k. makasheva, d. zharkov sb ras institute of chemical biology and fundamental medicine, novosibirsk, russia oxidative lesions are abundant due to constant presence of reactive oxygen species in living cells. repair of oxidative base lesions is initiated by dna glycosylases. for example, bacterial fpg and nei dna glycosylases excise oxidized purines and pyrimidines, respectively, from dna. their human homologs, neil1 and neil2, have been reported to show preference towards oxidized lesions in dna bubbles. from these observations, it had been hypothesized that neil proteins may be involved in the repair of lesions in dna bubbles generated during transcription. however, it is not presently clear how neils would behave on bubbles more closely resembling transcription intermediates (e. g., containing the rna strand), and bacterial homologs fpg and nei had never been investigated with bubble substrates. we have studied excision of either 8-oxoguanine (8-oxog) or 5,6-dihydrouracil (dhu) by e. coli fpg and nei and human neil1 and neil2 from single-strand oligonucleotides, perfect duplexes, bubbles with different number of unpaired bases (6 to 30), d-loops with dna or rna and from complexes with rna polymerase. fpg, neil1 and neil2 efficiently excised dhu located inside a bubble. fpg and neil1 was generally more active than neil2 in excision of 8-oxog from ssdna and bubbles. nei, on the other hand, was active only on dhu located in dsdna (either perfect duplex or dna/dna d-loop). fpg and neil1 also have shown activity in d-loops with rna. the presence of an additional unpaired 5 0 -tail of the third strand of d-loops didn't affect the glycosylases activity. the activity of fpg was observed in pre-assembled transcriptional complexes with e. coli rna polymerase and depended on the position of the lesion in the transcription bubble, possibly reflecting local accessibility of the lesion within the elongation complex. this work was supported by rsf (14-24-00093). nucleotide excision repair (ner) is a multistep process that eliminates a wide range of lesions in dna, including uv photoproducts and base modifications by many carcinogenic and chemotherapeutic agents. one of the advanced approaches to ner process investigation is based on reproducing the repair reaction by mixing protein extracts from mammalian cells with model linear dnas, bearing lesions. long linear dnas (137 bp) containing efficiently recognized and processed by ner system lesions (fluoro-azidobenzoyl photoactive lesion fab-dc, nonnucleoside lesions nflu and nant) in both strands have been synthesized. we have demonstrated that dnas containing closely positioned lesions in the both strands represent difficult-to-repair (fab-dc/nflu(+4), fab-dc/nflu(à3)) or unrepairable (nflu/nflu (+4), nflu/nflu(à3), nant/nflu(+4), nant/nflu(à3)) structures. besides, it has been shown that model dnas bearing 2 bulky lesions in opposite positions (fab-dc/nflu(0), nflu/nflu(0)) represent unrepairable structure as well. the model substrates with increasing distance between lesions in the duplex demonstrated the full recovery of substrate properties in ner process (fab-dc/nflu(+8), fab-dc/nflu(à10), fab-dc/nflu(à21), nflu/nflu (+8), and nant/nflu(+8)), whereas the level of specific excision from nflu/nflu(à10), nflu/nflu(à21) and nant/nflu(à10), nant/nflu(à21) was approximately 50% of the nflu/dg or nant/dg dna respectively. it has been shown that modified dna-duplex (54 bp) with fab-dc has decreased structurally dependent affinity for xpc-hr23b compared to duplexes containing lesions in both strands being analyzed (fab-dc/dg, fab-dc/nflu(+4), fab-dc/nflu (à3), fab-dc/nflu(+8), fab-dc/nflu(à10), fab-dc/nflu(-21)) and increased compared to umdna. the data provide an argument that the ner system of higher eukaryotes recognizes and eliminates injured dna fragments on a multi-criteria basis. it is well known that dna plays crucial role in the biological system because of including all the genetic information for cellular function. therefore, the interaction of molecules with dna has gained interest in the medicinal chemistry to explore new anticancer agent. photodynamic therapy which is alternative cancer treatment method depends on free radicals and singlet oxygen to destroy tumor tissue via necrosis and apoptosis. phthalocyanines (pcs) are used for photodynamic therapy because of their absorption of high wavelength light ability and they have high triplet quantum state yields and long lifetimes in triplet states. also they do not have any toxic effect without light. in this study the novel synthesized 4[2-(2morpholin-4-ylethoxy) ethoxy]phthalonitrile substitued zinc(ii), manganese(ii) and copper(ii) phthalocyanines were used. the potential properties of phthalocyanine compounds for photodynamic therapy were purposed to reveal by the preliminary work. for this aim, the mode of dna binding, photocleavage and topoisomerase i inhibition of these compounds were investigated. 4[2-(2-morpholin-4-ylethoxy) ethoxy]phthalonitrile substitued zinc(ii), manganese(ii) and copper(ii) phthalocyanine compounds have been synthesized. the interaction of novel pcs compounds with calf thymus (ct) dna was investigated by using uv-vis spectroscopy, thermal denaturation studies and viscosity measurements. additionally, dna photocleavage and topoisomerase i inhibition studies were performed to pbr322 dna by using agarose gel electrophoresis. the interaction studies indicated that pcs compounds powerfully bound via an intercalation mechanism with ct-dna. these compounds showed efficiently dna photocleavage under irradiation at 650 nm. the all of pcs inhibited topoisomerase i in a dose-dependent manner. all the experimental studies showed that pc compounds might be used agents for photodynamic therapy. p-05.01.1-014 target search by base excision repair dna glycosylases e. dyatlova, g. mechetin, d. zharkov institute of chemical biology and fundamental medicine, novosibirsk, russia the problem of rapid target search in dna is faced by transcription factors, restriction endonucleases, dna repair enzymes and other sequence-or structure-specific dna-binding proteins. theoretically, the fastest target search in dna can be achieved by combining one-dimensional diffusion along the dna contour (processive search) and three-dimensional diffusion (distributive search). the balance between these search modes depends on many factors affecting dna-protein interactions, such as the presence of mono-and divalent cations, competing proteins, crowding effect, etc. presently, the mechanisms of target search are understood only for a handful of enzymes. we have recently developed an assay to study target search by dna repair enzymes, based on cleavage of oligonucleotide substrate containing two targets. thus, the distance between the targets can be precisely controlled, and any modification can be introduced into dna. subsequently, the probability of correlated cleavage (p cc ) is estimated, reflecting the efficiency of enzyme transfer between the specific sites. in this work, we have investigated five repair enzymes: e. coli endonuclease viii (nei), its human homologs neil1 and neil2, and uracil-dna-glycosylases (ung) from e. coli and vaccinia virus. as expected, p cc of all enzymes depended on the ionic strength of the solution and the presence of mg 2+ . ung from vaccinia virus was the most sensitive to these factors, raising questions about its proficiency as a suggested processivity factor of viral dna polymerase. nei, neil1 and neil2 showed a peak of p cc at low but non-zero ionic strength indicating that nonpolar interactions contribute to binding of these proteins to nonspecific dna. this conclusion was also supported by analyzing amino acid conservation in the catalytic core of nei. introduction of bulky fluorescent group between two specific sites greatly reduced the ability of glycosylases to slide along dna. this work was supported by rsf (14-24-00093). p-05.01.1-015 does causes 1800 mhz magnetic field application kras and p53 mutations in colon?: occurences histopatologically and microbiologically changes in colon determination of kirsten rat sarcoma (kras) and p53 gene mutations in colon. materials and methods: in this study, three groups were prepared as control,sham and electromagnetic field (emf) group.1800 mhz radiofrequency (rf) radiation was produced by using an electromagnetic energy generator.the emf group rats were exposed to electromagnetic field for 12 weeks as 45 minutes per day.at the end of experiments, rats were sacrificed under ethyl ether anesthesia and the rat colons were dissected.fecal speciments were collected.fecal dna (for detection of fusobacterium and bacteroides) and colonic dna (for detection of kras and p53 mutations) were isolated.rt-pcr tchnique was used for detection of bacterias and mutations. results: no any differences was observed histopathologically between control and sham groups.erosions and partial losses were observed at mucosal epitelium in the emf group.the corrupted gland structure, the mucosal edema and the inflammatory cell infiltration were observed.the amout of collagen was increased and fibrosis was detected in emf group.goblet cell number decreased statistically significant when compared to control and sham groups (p < 0.05).the amount of fusobacterium increased significantly in emf group compared to controls.the difference was not detected between groups in the amount of bacteroides.all the samples analysed for kras and tp53 mutations in the colon tissue were found to be wild type.no significant difference was observed between the control group and the emf applied group. discussion and conclusion: in conclusions,for 12 weeks 45 minute/day exposure to 1800 mhz emf caused histopatological damage in rat colon.the amount of fusobacterium is increased.emf exposure did not caused to kras and p53 mutations in colon tissue. p-05.01.1-016 synthesis, antimicrobial activity, genotoxicity, dna binding and dna cleavage studies of new glycine methyl ester derivative schiff base there has been an increasing focus on the binding study of small molecules to dna during the last decades, since dna is an important genetic substance in organisms. therefore, the current growing interest in small molecules that are capable of binding and cleaving dna is related to their utility in the design and development of synthetic restriction enzymes, new drugs, dna agents, and also to their ability to probe the structure of dna itself. in recent years, schiff bases have found increased application in pharmaceutical research, organic synthesis, and bio-processes. schiff bases are considered as favored and the most widely used ligands, due to their metal complexes having variety of applications as antibacterial and anticancer agents. in this study, we report the synthesis and characterization of a novel glycine methyl ester derivative schiff base. the minimal inhibitory concentration (mic) of the compound was screened in vitro against bacteria and yeast cultures using broth micro dilution tests. antimutagenic activity of compound was tested in the absence of metabolic activation. also, dna binding and dna cleavage were investigated of compound by uv-vis spectroscopy and agarose gel electrophoresis respectively. consequently, this compound differs significantly in its activity against tested microorganisms. this difference may be attributed to the fact that the cell wall in gram-positive bacteria is a single layer, whereas the gram-negative bacteria cell wall is a multilayered structure, and the yeast cell wall is quite complex. the compound inhibited the base pair mutation in the absence of s9 with high inhibition rate. uv-vis spectroscopy studies of the interactions between the compound and calf thymus dna (ct-dna) showed that the compound interacts with dna via intercalative binding. to date a large number of the sequences in the human genome (g4 motifs) with the potential to form a spatial structure, gquadruplexes is known. g4 motifs were found in the promoter regions of most of the known oncogenes. recent experimental studies have shown that genome instability directly related to the non-canonical dna structures, including g-quadruplexes. in this work we study the distribution of somatic snvs within the g4 motifs in tumor samples with the aim to identify involvement of the motifs in the process of mutagenesis in pancreatic cancer. using the access kindly provided by the international icgc consortium to the database, we analyzed 68 samples of pancreatic ductal adenocarcinoma and 27 samples of pancreatic endocrine neoplasms. we considered only the promoter regions as the richest with g-quadruplex motifs. we found that quadruplex sequences have the ability to focus somatic snvs. this could be explained by the errors of polymerase during replication through secondary dna structures. furthermore, the snvs occur much more often in loops of g4 motifs than in g blocks, without changing the motive. in addition, t>g(a>c) and t>c(a>g) substitutions occur significantly more likely in loops which in turn stabilize the g-quadruplex structure. the cancer-related mutations tend to increasing the length of g blocks. the conservation of g4 motifs may indicate an important functional significance of g-quadruplex structures in human genome. supported by project no. 16-14-10396 of the russian science foundation. background: multiple myeloma (mm) is a rare, leading to bone destruction and marrow failure, largely incurable malignant disease of plasma cells. anemia (mostly normocytic normochromic) is seen in most patients. mean platelet volume (mpv) is a laboratory marker of platelet function and activity, the most accurate measure of platelet size. the aim of this study was to investigate the mean platelet volume (mpv) values in this disease. materials and methods: whole blood samples were collected from 60 healthy controls and 105 patients with mm. the mean age for controls and patients were 54.5 ae 8.5 and 57.4 ae 8.1 years, respectively. mpv levels were calculated with cancer is a chronic disease in the world which is the second leading cause of death, after cardiovascular diseases. benzimidazoles have been known to act as antiproliferative or anticancer agents in chemotherapeutic drug research area. in this regard we aimed to investigate the cytotoxic and apoptotic properties of novel benzimidazole derivatives bearing pyridyl/pyrimidinyl piperazine moiety against a549 lung adenocarcinoma cells. a549 lung adenocarcinoma cell lines were used in the studies. the cytotoxic activities of the tested compounds were determined by mtt assay. detection of apoptosis was performed using annexin v-fitc apoptosis detection kit bd, pharmingen according to the manufacturer's instruction. all measurements were performed on a facs-calibur cytometer. the ic 50 values of the compounds were determined for a549 cell line. compounds 4, 7 and 12 which were including 4-chlorophenyl, 4-nitrophenyl on pyridine ring; 4-fluorophenyl on pyrimidine moiety, had significant cytotoxic activity with ic 50 values lower than 55.33 ae 23.40 lg/ml. compound 12 showed the highest cytotoxic activity with a ic 50 value of 16.67 ae 2.24 lg/ml, whereas cisplatin ic 50 values were 14.0 ae 2.0 lg/ml lg/ml against a549 cells. cytotoxic activity of compound 4 and 7 with a ic 50 value were 55.3 ae 23.4 and 21.7 ae 8.4 lg/ml, respectively. also, compound 12 showed the highest population of early apoptotic cells (39.4%) of the tested compounds which was 5.88-fold higher than for cisplatin. compound 7 produced a comparable population of apoptotic cells with a percentage of 9.5%, respectively according to cisplatin's percentage of 6.2%. it was determined that synthesized compounds 4, 7 and 12 had considerable anticancer activity against a549 cell lines compared to cisplatin. compound 12 including 4-florophenyl on pyrimidine ring was the most cytotoxic compound against the a549 cell line. our study results demonstrated that compound 7, 12 also induced apopototic pathway on a549 cells. p-05.01. in vitro/in vivo antimitotic activity and structure-activity relationships of new glaziovianin a isoflavone series glaziovianin a (gva), isolated from the leaves of the astelia glazioviana, demonstrated cytotoxicity, disrupting microtubule structure and dynamics of hl-60 cells. the aim of the present work was to devise a concise synthetic route toward gva and its derivatives in order to expand structure-activity relationship studies and to investigate their anti-mitotic effect. a concise six-step protocol for the synthesis of gva and its alkoxyphenyl derivatives 9 starting with readily available plant metabolites from dill and parsley seeds was developed. the sea urchin embryo tests confirmed that gva directly affects tubulin/ microtubule dynamics and structure. the b-ring substitution pattern of gva derivatives exhibited strong effects on activity. according to the assay results, the anti-mitotic activity decreased in the following order: gva > myristicin ≥ 3,4,5-trimethoxyphenyl = 4-methoxyphenyl > dillapiol > 3-methoxyphenyl> 3,4dimethoxyphenyl > 2,3,4,5-tetramethoxyphenyl derivatives. a methylenedioxy moiety was essential for the activity of compounds substituted with four b-ring alkoxy groups. the mts assay of the limited panel of cancer cell lines shows that gva displayed the highest inhibitory activity, with ic50 values ranging from 0.27 (a375 cells) to 2.2 lm (mda-mb-231 cells). compounds, containing 3,4,5-trimethoxy and apiol-derived b-rings, respectively, were less active. other isoflavones did not affect cancer cell growth up to 10 lm. anti-proliferative effects of isoflavones 9 observed in both the sea urchin embryo model and human cancer cell lines correlated well. importantly, none of the synthesized isoflavones 9 demonstrated cytotoxicity in human pbmcs, up to 10 lm. in summary, gva and its analogues were synthesized via a scalable six-step reaction sequence. the gva and its analogues containing 3,4,5-trimethoxy and apiol-derived b-rings were found to be promising anti-mitotic microtubule destabilizing agents with low toxicity against human pbmcs. bag-1 is a multifunctional protein which has interactions with a number of cellular proteins; nuclear hormone receptors, bcl-2, hsp70/hsc70 family, growth hormone receptors, raf-1, ubiquitin machinery and dna to regulate cell survival. for this reason, bag-1 is a critical molecular player in the regulation of cell survival signaling and apoptosis mechanism. elevated expression levels of bag-1 are associated with progression of cancer. in the treatment of breast cancer, silencing tools as a promising combined therapy strategies in the presence of classical chemotherapeutics gain importance to investigate interaction networks of cell death and survival signaling pathways. therefore, we aim to understand potential role of bag-1 silencing in the treatment of breast cancer cells with apoptotic agents; cisplatin or paclitaxel. our results showed that, silencing of bag-1 enhanced cisplatin or paclitaxel-induced apoptosis in mcf-7 cells by down-regulating antiapoptotic and upregulating proapoptotic bcl-2 family proteins, changes on cell cycle, upregulation on subg1 phase, activating caspases and cleavage of parp. in addition, knockdown of antiapoptotic bag-1 has a suppressive role in pi3k and akt signaling pathway in mcf-7 breast cancer cells through inhibition of akt phosphorylation and downregulation on pi3k. investigation targets of akt pathway showed that mtor cell survival pathway also affected through bag-1 silencing. bag-1 silencing inhibited mtor signaling via downregulating both rictor and raptor proteins which are the members of rapamycininsensitive mtorc2 and rapamycin-sensitive mtorc1 complexes, respectively. knockdown strategies of bag-1 is important to enlighten the network interactions of bag-1 and clarify its interaction partners in the cells. therefore utilization of bag-1 targeted strategies might further increase therapeutic efficiency of drugs through inhibiting cell survival machinery in the treatment of metastatic breast cancer. p-05.01.1-024 biological activity evaluation of new 3,5,6trisubstituted triazine derivatives bearing different heterocyclic rings against lung cancer cell lines l. yurttas, g. akalin c ß iftc ßi, h. e. temel, b. demir anadolu university, eskisehir, turkey cancer is one of the major death causing disease worldwide. among the various cell types occurs on different organs, lung cancer is one leading cause of cancer death accounting for approximately 26% of all female and 28% of all male cancer deaths in 2013. the resistance development, cytotoxicity and inadequacy are the main encountered problems by the treatment with existing chemotherapeutic agents. therefore, there is continuous need to discover new active and non-toxic molecules. 1-[4-(5,6-bis(4-substituted phenyl)-1,2,4-triazin-3-yl)piperazin-1-yl]-2-[benzimidazole/benzoxazole/benzothiazole-2-yl)thio]ethanone (1-9) derivatives were synthesized with a four-step synthetic procedure using toluil, anisil and 4-chlorobenzil as starting materials. the anticancer activity of the compounds was evaluated using the methods mtt (3-(4,5-dimethylthiazol-2-yl )-2,5-diphenyltetrazolium bromide), brdu (bromodeoxyuridine) assays and flow cytometric analysis against lung cancer cell lines. the lipoxygenase enzyme inhibition activity of the compounds were also investigated using the method described by baylac and racine. compounds was found to have (inhibition concentration) ic 50 values between 135-500 lg/ml. the early and late apoptotic cell percentage was determined as 6.6 for compound 8 by flow cytometric analysis. the lox inhibition activity was found 48.35 ae 3.08 for compound 1. compound 8 bearing 8-chlorobenzil and benzoxazole moieties was found as the most active compound when we evaluate anticancer potential of all compounds. the lox enzyme inhibition was indicated for the compound 1 including methyl substituent on phenyl rings. the dna synthesis inhibition of the compounds has been still studied at the concentrations ic 50 /2, ic 50 and ic 50 x2. p-05.01.1-025 single amino acid substitutions and deletions modulate the drp-lyase activity of human dna polymerase iota n. miropolskaya, i. petushkov, a. kulbachinskiy, a. makarova institute of molecular genetics, moscow, russia dna polymerase iota (pol ι) is a y-family dna polymerase that possesses an unusual combination of properties. due to the special organization of the active site pol ι has a very low accuracy of dna synthesis but possesses an ability to bypass a variety of dna lesions. in addition to the dna polymerization activity, human pol ι also possesses an intrinsic 5 0 -deoxyribose phosphate (drp)-lyase activity. removal of the drp group is a pivotal step in base excision repair (ber) in vivo. although pol b plays a key role in the drp group cleavage and dna synthesis during ber, pol ι was shown to complement the in vitro single-nucleotide ber deficiency of pol b null cell extracts and was suggested to be involved in ber under oxidative stress. the drp-lyase active site in pol ι is still not known. to address the mechanism of the drp-lyase activity of pol ι we obtained a series of pol ι mutant variants including point mutations of conserved lysine residues and deletions in different locations. we purified human pol ι variants from yeast saccharomyces cerevisiae and tested the effect of mutations on the cleavage of an internal 5 0 -drp group in oligonucleotide dna substrates in the presence or absence for me 2+ ions. the experiments revealed several point amino acids substitutions that significantly affected the drp-lyase activity of pol ι, thus suggesting a possible location of the drp-lyase active site. furthermore, we showed that deletions in the n-terminus of pol ι and metal ions modulate its drp-lyase activity, which may play an important role in the regulation of pol ι activities in vivo. this work was supported by russian foundation for basic research grants 15-04-08398-a and 15-34-70002-mol-a-mos and by the russian academy of sciences presidium program in molecular and cellular biology. rosmarinus officinalis, commonly known as rosemary, is an aromatic plant belongs to lamiaceae family. from past to now, rosemary have been used as a traditional medicine to cure for various illnesses such as diabetes, rheumatism and cancer. recent studies have shown that rosemary is effective for various cancer types. in this study we aimed to investigate the effect of rosemary in glioblastoma cells (gbm) by comparison with etoposide and the effect of rosemary by concurrent application with the etoposide. gbm cells (u87 mg) were seeded into the 24 well plates and cultured with dmem supplemented with 10% fetal bovine serum. rosmarinus officinalis tea was prepared just as traditional usage and filter sterilized. at the second day of the culture rosemary in 1/75 (v/v) dilution ratio was given to first group, 40 lm etoposide was given to second group, 1/75 (v/v) diluted rosemary and 40 lm etoposide together were given to third group. after one day incubation cell viability was measured by neutral red assay. it was observed that rosemary reduced the viability of gbm cells by nearly %38, etoposide reduced the viability by nearly % 49 and rosemary with the etoposide reduced the viability by nearly %50. the results showed that rosemary was able to reduce the viability of gbm cells but hadn't got an increasing or inhibiting potential over the etoposide's cytotoxic effect. from our previous studies we know that rosemary increases the proliferation of mouse embryonic fibroblasts. it is considered that rosemary might have a protection potential from dna damages and when rosemary is used with etoposide during the cancer treatment, it might reduce the side effects on healthy cells. in conclusion rosemary promises hope for developing new cancer treatment strategies and reducing the side effects of chemotherapeutics. for further studies it is aimed to examine the effects of rosemary with other chemotherapeutics and if rosemary has got a protection potential from the genotoxic stress. morpholine moiety has been found to be an excellent pharmacophore in medicinal chemistry and a number of molecules possessing morpholine skeleton are the clinically approved drugs. in this present study, we aimed to investigate the possible underlying apoptotic mechanism for the cytotoxicity of new morpholine dithiocarbamate derivatives bearing 1-(2-aryl-2-oxoethyl)-2-substituted benzimidazole moiety on c6 glioma. c6 glioma cell lines were used in the studies. the cytotoxic activities of the tested compounds were determined by cell proliferation analysis using standard (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (mtt) assay. detection of apoptosis was performed using annexin v-fitc apoptosis detection kit bd, pharmingen according to the manufacturer's instruction. all measurements were performed on a facs-calibur cytometer. the ic50 values of the compounds were determined for c6 cell line. compounds 1, 2, 8, 9, and 10 , which were including hydrogen, 4-methyl, 3-methoxy, 3-chloro and 3-floro substituents on phenyl acetyl moiety, had significant cytotoxic activity with ic 50 values lower than 55 lg/ml. compound 10 showed the highest cytotoxic activity with a ic 50 value of 28 lg/ml, whereas cisplatin ic 50 values were 15 lg/ml against c6 cells. cytotoxic activity of compound 1, 2, 8, 9 and 10 with a ic 50 value were 42, 55, 50 and 30 lg/ml, respectively. compound 2, 9 and 10 showed the highest population of early apoptotic cells as 11.5, 10.1, and 10.3% respectively compared to cisplatin (6.2%). also, compounds caused dna synthesis inhibition depend on their ic50 values by brdu assay. conclusions: it was concluded that synthesized compounds had considerable anticancer activity against c6 cell lines. however, compound 2, 9 and 10 including 4-methyl, 3-chloro and 3-floro substituents were the most active compounds against the c6 cell line. also our study results showed that compound 2, 9, 10 induced apoptosis in c6 glioma cells. rutin is a glycosided flavonoid and known to have antioxidant and anti-inflammatory properties.trail induces the apoptosis of tumor cells and has no significant toxic effect on normal cells. although trail is a promising anticancer agent, trail resistance is a major barrier to effective cancer therapy. this study was conducted to examine the utility of the combined use of rutin and trail in prostate cancer cells. pc-3 and du145 prostate cancer cells were treated with rutin (1-1000um) and/or trail (20 ng/ml), cell viability and migration were examined. cell viability was determined by trypan blue exclusion and mtt assay. cell migration was determined by wound healing assay. furthermore, lactate dehydrogenases (ldh) levels of medium were determined as biochemical markers of cell viability. pc-3 and du-145 prostate cancer cells were treated with rutin for 24 and 48 hours incubation and ic 50 doses for 48 hours incubation were determined 250um and 1000um respectively. treatment with rutin, pc-3 cells is more sensitive than du145 cells. rutin and rutin plus trail inhibit prostate cancer cell growth in a dose-dependent manner. treatment with trail has no effect at inhibiting growth of pc-3 and du145 prostate cancer cells. the combination of rutin and trail elicit a synergistic antitumor effect on pc-3 and du145 prostate cancer cells. there is a significant increased in rutin and rutin+trail treatments group of ldh activities with respect to control and trail group. conclusion: present data show that rutin efficiently enhanced trail effects in prostate cancer cells. combined treatment with rutin and trail is more effective than the individual treatments of trail at inhibiting growth of prostate cancer cells. p-05.01.1-030 determination of antigenotoxic, proliferative and cytotoxic properties of ellagic acid since ancient time, people use plant for traditional treatment. plants or fruits are produced different type of secondary metabolites. particularly phenolic phytochemicals from plants play an important role in the prevention and treatment of radical damage by inactivating the reactive oxygen compounds due to their antioxidant properties. however, the structure and the activities of many herbal products are not fully elucidated yet and there are several studies about the toxicity of herbal antioxidants and their possible risks to human health. ellagic acid, phenolic compounds, is an important substance. ellagic acid is a naturally occurring plant phenol found in numerous fruits, including blackberries, raspberries, strawberries, cranberries, walnuts, pecans, pomegranates and wolfberries. different researchers give some information about the biological activities of ellagic acid. in this study, we aimed to determine the cytotoxic, proliferative and antigenotoxic effects of ellagic acid, which is phenolic compounds found in natural products. cytotoxic effects of ellagic acid on huvec is investigated by lactate dehydrogenase (ldh) and cell proliferation (wst-1) methods; and antigenotoxic effects against ccl 4 on human lymphocytes is investigated by single cell gel electrophoresis (comet) methods. the rusults showed that high concentration (100 and 200 lm) of ellagic acid has cytotoxic and mutagenic effects, but showed antiproliferative effects. on the contrary, low concentrations (4, 8, 12.5 lm) of ellagic acid has anticytotoxic and antimutagenic effects. as a conclusion, low concentrations of ellagic acid might be use treatment of some disease. but high concentrations of ellagic acid constitute a risk factors for people. keywords: cytotoxicity, antiproliferation, wst-1, ldh, rtca-sp the constitutive nuclear factor kappa b (nf-kb) activation is widely found in diverse types of hematologic malignancies such as acute myeloid leukemia (aml) and chronic myeloid leukemia (cml) as well as solid tumors. inhibition of nf-kb signaling via proteasome inhibitors such as bortezomib can induce apoptosis in myeloid leukemia cell lines. however it is not clear whether the cytotoxic effects of bortezomib on myeloid leukemia cell lines is due to direct inhibition of nf-kb or another pathway, such as dna damage. in this study, cml cell line k562 and aml cell line hl-60 were treated with bortezomib (bor) , etoposide (eto) and camptothecin (cpt) alone or in dual combination with these drugs, following by measuring the effects on cell viability, apoptosis and signal pathways. the effect on cell viability was determined using the mtt assay. the data were used in combination index and isobologram analysis. the expression levels of apoptototic genes (bcl2, bax and caspase 3), the related dna damage genes (atm and atr) and the involved genes in nf-kb signaling (rela and p50) were determined by real time rt-pcr. we showed that combinations of bor with topoisomerase inhibitors (cpt and eto) exhibited synergistic cytotoxic effect in k562 cell line but not in hl-60 cell line. the combination treatment increased apoptosis and dna damage response. dnadamage-sensing kinases were detected in k562 and hl-60 cells following treatment with bor as similar as topoisomerase inhibitors. bor increased the mrna levels of atm and atr dramatically, which indicated active dna damage in the myeloid cell lines. furthermore, bor induced apoptotic cell death by decreasing bcl2 and increasing bax and caspase 3 levels. these effects of bor were observed to correlated with increasing the p65 expression levels. this study on the mechanism of action of bor indicates that this compound affects several pathways involved in the control of cell cycle progression, apoptosis and dna damage. p-05.01.1-032 analysis of molecular cytogenetic alterations in gastric and colon carcinoma by array-based comparative genomic hybridization (array cgh) introduction: genomic dna regions are frequently lost or gained during tumor progression. we aimed to evaluate tumor samples of patients with gastric cancer and colorectal carcinoma to show these genetic alterations by array-based comparative genomic hybridization (array cgh) method. materials and methods: dna isolation was performed from the tumor samples obtained from sixteen patients with primary gastric adenocarcinoma and twelve patients with colon adenocarcinoma. then, agarose gel electrophoresis was performed in those dna samples. following electrophoresis of dna, array cgh procedure was performed to four patients with gastric adenocarcinoma and three patients with colon adenocarcinoma who had dna breaks with 100-200 kb. results: after array-cgh study, many common genetic changes in gastric and colon cancer genome were determined. in gastric cancer dna samples, common losses were detected in chromosome 1p34. 1, 1p13.3, 1q22, 3q29, 5p13.2, 6q24.1, 7q11.23, 7q22.1, 8q 12.1, 9p12, 11q13.1, 12 q24.31, 14q32.31, 16q22.1, 17q21.2, 19q13.3, and 20q11 .23, and also common gains were detected in chromosome 3p26.1, 6q27, 8q12.1, 15q11.2 and xq25. in colon cancer dna samples, common losses were detected in chromosome 1p34. 1, 3q29, 7p22.2, 7p11.21, 9q34.11, 12q24.31, 17q21.2, 17q25.1, 19p13.3, 19p13.33, 20q11.21, and 20q11.23 , and also common gains were detected.in chromosome 14q32. 33, xp22.33, xp22.11, xp13.3, xp11.1 and xq25 . both in gastric and colon cancer dna samples, common losses were detected in chromosome 12q24. 31, 17q21.2, and 19p13.3 , and common gains were detected in xq25. discussion and conclusion: we think that these common changes, generally in dna loss areas harboring tumor suppressor genes and dna gain areas harboring oncogenes, may important in gastrointestinal tumorigenesis. the dna of every cell is under a constant attack by various mutagenic factors which damage the dna and can cause cell cycle arrest and even cell death. accumulation of dna damage is the basis for cancer development and one of the reasons for aging of the organisms. in order to preserve the integrity of its dna cells have evolved an impressive array of dna repair pathways, which are precisely coordinated with the progression of the cell cycle. one of the first events at the site of dna damage is poly(adp-ribose) polymerase 1 (parp1) recruitment which is a sensor for single strand breaks in dna. parp1 catalyzes the synthesis of poly(adp-ribose) or par which is needed for the recruitment of many other dna repair proteins by means of par-binding domains. we used high speed confocal spinning-disk microscopy of living cells to obtain precise kinetics of recruitment of par-dependent proteins to the sites of laser induced dna damage. our results show that the investigated par-dependent proteins are recruited to dna damage sites in the matter of seconds, they reach peak intensities for 20 to 30 seconds after damage infliction and start dissociating. the recruitment of the proteins is entirely dependent on par because addition of parp inhibitor abbrogated their recruitment. the use of spinning-disk microscopy of living cells allowed us to obtain the kinetics of recruitment of the studied proteins to the sites of dna damage. the results are consistent with the fact that parp1 and par-dependent proteins are quickly recruited to damage sites and generation of par is essential for other dna repair protein recruitment. the precise kinetic curves may serve as a basis for investigating how they will change or if they will change at all when cells are put in different conditions or treated with various chemical substances affecting dna metabolism and repair. introduction: chronic myeloid leukemia (cml) is a myeloproliferative disease associated with reciprocal translocation between chromosomes 9 and 22. bcr-abl fusion gene which exhibits constitutively active tyrosine kinase activity has a main role in cml. the tyrosine kinase inhibitor imatinib is used as a first line treatment in cml patients, but imatinib resistance leads to failure in therapy. the application of imatinib in combination with other anticancer agents may be a strategy to increase the antileukemic effect of imatinib. in this study, we have investigated the antiproliferative effect two novel agents: a benzamide derivative xt5 and a benzoxazole derivative xt2b in combination with imatinib. these molecules were investigated in imatinib-sensitive (k562s) and imatinib-resistant (k562r) cml cell lines. materials and methods: antiproliferative and apoptotic effects were assessed by mtt assays and flow-cytometry, respectively. we also evaluated the effects of these compounds on the expression of apoptosis-related genes bax, bcl-2, bad, bim, bcl-xl and mcl1 by real-time quantitative pcr. results: treatment of k562 cells with xt5 increased the expression levels of the pro-apoptotic genes bax, bad and bim in both sensitive and resistant cells. however, xt2b was not found to have similar effects on k562r and k562s cells. combined application of xt5 increased cell death in the mtt assay. mtt assay demonstrated that ic50 for xt5 treated cells in k562r with imatinib (ic50 = 3.5) is lower than k562r without imatinib (ic50 = 8.5). discussion and conclusion: our results showed that combining xt5 with imatinib has more antiproliferative and apoptotic effect on a cml cell line. as a result combination of xt5 with imatinib can be an alternative approach to overcome imatinib resistance. introduction: the mmr(mismatche repair) system recognizes base-base mismatches and insertion or deletion loops in doublestranded dna, and it degrades the error-containing region of the newly synthesized strand, allowing the polymerase to correctly resynthesize the second strand according to the template sequence. the human mmr system includes the mlh1 and msh2. alteration in expression or a defect in mlh1 or msh2 can cause resistance to anti-cancer drugs used in chemotherapy. the attempt of the mmr system to detect drug induced dna damage, triggers the activation of apoptosis, a mechanism which may enhance the cytotoxicity of chemotherapy. loss of the mmr system would make the neoplastic cell less able to initiate apoptosis. inability to initiate apoptosis could be a mechanism of resistance to drugs. chronic myeloid leukemia (cml) is a clonal disease originating from aberrations in hematopoietic stem cell. imatinib, a tyrosine kinase inhibitor has significantly improved clinical outcome for cml patients. however, patients develop resistance when the disease progresses to the blast phase (bp) and there are several mechanisms involved in imatinib resistance. in this study we investigated the role of mmr system in imatinib resistance. materials and methods: k562s (sensitive) and k562r (resistance) were grown in rpmi-1640. k562r cells were maintained in rpmi-1640 medium supplemented with 5 lm imatinib rna isolation, cdna synthesis, rt-pcr was performed respectively. results: the results demonstrated that expression of mlh1 in k562r cells is dramatically lower than equal amount of imatinib treated k562s cells, whereas msh2 expression level did not change in both cell lines. conclusion: it can be suggested that alteration and down-regulation of mlh1 genes leads to imatinib resistance. p-05.01.1-037 characterization of interaction between rad51 inhibitor dids and human serum albumin d. velic, s. henry, c. charlier, m. popova, p. weigel, j. masson, i. nabiev, f. fleury cnrs/university of nantes, nantes, france 4 0 -diisothiocyanostilbene-2,2 0 -disulfonic acid (dids) has been largely used during the last 30 years for its inhibitory effect on anion transporters and channels. more recently, ishida and colleagues have described a possible mechanism by which dids inhibits rad51-mediated homologous pairing and strand exchange, key processes in dna repair by homologous recombination. thus, dids could act as a potential revertant of radioand chemo-resistance in cancer cells, which is the major cause of failure during therapeutic protocols. new drugs targeting rad51 protein have since been developed with potential use for medical applications. in this context, we attempted to determine the behaviour of dids towards blood and plasma proteins such as serum albumins. firstly, we analysed the effects of several environmental factors such as solvent polarity, which may affect the stability of the molecule. secondly, we analysed the spectroscopic properties of dids in the presence of human or bovine serum albumin proteins. uv-visible absorption, circular dichroism, fluorescence spectroscopy and isothermal calorimetry were used. here we show for the first time that dids can interact with both serum albumins. we have also determined the characteristics of these interactions. the comparison of several dids derivatives led us to identify the essential chemical moiety of this compound involved in the interaction. moreover, by using site competition approaches we show that the main binding site for this molecule is in subdomain ib of the protein. these findings show that the binding of dids to serum albumin proteins may change the equilibrium between the free and bound dids forms, thereby affecting its bioavailability and efficiency against the rad51 recombinase protein. p-05.01.1-038 mechanism of tap73 beta-mdm2 autoregulation p73 is a transcription factor which is the member of a p53 family. it regulates many cellular processes, such as apoptosis, cell cycle, and senescence. in contrast to p53, p73 is rarely mutated in tumors and elevated p73 expression is observed in many types of cancers including hepatocellular carcinoma, neuroblastoma, and lung. defining regulatory mechanisms which control p73 protein abundance and activity will be crucial for the development of new therapeutic strategies for cancers. mdm2 is known as the key player in regulation stability and activity of p53. in addition, p53 induces mdm2 transcriptional activity, and caspase-2, 3 activations which cleave mdm2 n-terminal at asp 367. cleaved form of mdm2 binds p53 and promotes its stabilization. mdm2 suggested as a candidate to modulate p73 activity and stability too. however, an interaction between p73 and mdm2 has not defined well. in this study, we aimed to analyze the role of mdm2 in p73 stability. to define this relationship, firstly, we overexpressed the tap73beta isoform using trex system in hep3b. tap73 beta and mdm2 protein levels were determined by western blot. to examine whether mdm2 mediate tap73 beta protein degradation by the proteasomes, cells were treated with proteasome inhibitor, mg132 for 4 hours prior to analysis. previous studies showed that p53-induced caspase-2 and caspase-3 activation cleaves mdm2. considering this, we firstly examined caspase-3 activation by western blot in hep3b tap73 beta cells. then we analyzed expression of cleaved mdm2 and tap73 beta levels following caspase inhibitor, z-vad-fmk treatment. as a conclusion, tap73 beta-induced full-length mdm-2 expression. furthermore, tap73 beta enhanced cleavage of mdm2 via increased caspase-3 activation. in addition, inhibition of caspase-3 activation caused a decrease in cleaved-mdm2 levels in parallel with tap73 beta expression repression. our results suggested positive regulation between mdm2-tap73 beta. hepatocellular carcinoma (hcc) is one of the most common type of liver cancer and third leading cause of cancer related deaths in worldwide. discovery of new targets is important in survival of hcc patients. p73 is a transcription factor which is the member of p53 family. it has two promoters; while p1 promoter expresses apoptotic ta isoforms, p2 promoter expresses anti-apoptotic dn isoforms. in addition, alternative splicing in c terminal creates many isoforms of ta and dn p73. it has been shown that both tap73 and dnp73 isoforms are expressed in hcc patient tissue and cell lines. the ratio between tap73 and dnp73 affects the apoptotic response, drug response and prognosis. accordingly, identification of the role of p73 and its targets are important in discovery of new treatment strategies in hcc. to understand the role of p73 isoforms in hcc, firstly we performed mtt assays following dna-damaging drugs and multikinase inhibitor, sorafenib treatment to categorize hcc cell lines as resistant or sensitive. after that, we analyzed the expression levels of tap73 isoforms via western blot in all hcc cell lines. then we overexpressed the tap73beta isoform using trex system in hep3b and snu449 cells. these two clones were analyzed for dna damaging drug response by mtt, cell cycle and apoptosis by flow cytometry, and tumor formation by in vitro and in vivo experiments. in scope of our study; 1. only tap73 alpha isoform is expressed in a few hcc cell lines. 2. there is no correlation between basal expression of p73 isoforms and drug responses in hcc cell lines. 3. there is no change in expression of p73 isoforms after treatment of drugs. 4. we showed that the ectopic expression of tap73beta in hep3b arrested the cell cycle in g1/ s and decreased the colony formation. therefore, the capacity of tumor formation of the cells dramatically decreased in scid mice. as a result, we revealed that tap73 beta play role in tumor formation, cell cycle arrest, dna damage responses in hcc. p-05.01.1-040 biochemical characterization of exonuclease iii-family ap endonuclease point mutants reveals role of conserved amino acid residues in the nir-specific enzymes a. mursalimov, z. koshenov, t. yeleussizov, m. redrejo-rodriguez, a. ishchenko, b. t. matkarimov, m. saparbaev national laboratory astana, astana, kazakhstan oxidative dna damage caused by reactive oxygen species is believed to be a major type of endogenous cellular damage. oxidatively damaged dna bases are substrates for two overlapping repair pathways: dna glycosylase-initiated base excision (ber) and apurinic/apyrimidinic (ap) endonuclease-initiated nucleotide incision repair (nir). in the ber pathway, an ap endonuclease cleaves dna at ap sites and 3 0 -blocking moieties generated by dna glycosylases, whereas in the nir pathway, the same ap endonuclease incises dna 5 0 to a number of oxidized bases. majority of characterized ap endonucleases possess classic ber activities and about half of them are able to catalyze nir activity. at present, the molecular basis of dna substrate specificities of various ap endonucleases remains unclear. here, we examined amino-acid sequence requirement of the nir activity of human major ap endonuclease 1 (ape1). amino acid sequence alignment of various ap endonucleases including e coli exonuclease iii (xth), human ape1 and archaeal mth212 revealed conserved amino acid residues in the nir-specific ap endonucleases ape1, mth212 and exoa that are absent in xth. based on these data, we constructed four ape1 point mutants y128h, n174q, g231s and t268d and examined their dna substrate specificities. results obtained from biochemical characterization of ape1 mutants are discussed in the light of the evolutionary conserved dna repair functions of ap endonucleases and whether these functions can be mutationally separated from. since its discovery some 40 years ago, cisplatin has evolved for its efficacy in one of the most used drugs in treatment of various cancer types. huge effort was invested in understanding the action of cisplatin and development of more potent drugs. they target mainly neighboring purine bases of nuclear dna forming covalent intra-or inter-strand cross-links that affect inhibition of replication and transcription, cell cycle arrest, and attempted repair of the damaged nucleotides. if such damage cannot be removed the cell dies. we have studied the details of the binding site of the short oligonucleotide modified by a platinum compound using complementary solution techniques used in modern structural biology, including raman spectroscopy with dft calculations aided interpretation of the obtained vibrational spectra. moreover, the calculated structure of the dna duplex was verified using saxs (small angle x-ray scattering) curve. in our contribution, we will present an nmr structure of a dna cross-linked with a cisplatin derivative containing a cyclohexane ring. at this atomic level resolution, structural features probably influencing cytostatic effects are described and compared with previously published structures. common structural features of previously determined structures are: a significant roll (25-60°) of the guanine bases involved in the cross-link, bending and unwinding of the double helix at the site of cross-link and orientation towards the major groove. also, the platinum-guanine plane angle varies between 19 and 54°. although the experimental structures were often used as the starting models for molecular dynamics (md) simulations, results of these md still leave many questions unresolved. the results of this research have been acquired within ceitec 2020 (lq1601) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. p-05.01.1-043 ercc2/xpd polymorphisms and colorectal cancer risk: a case control study in a north eastern iranian population j. mehrzad islamic azad university, neyshabur, iran excision repair cross-complimentary group 2 (ercc2) is one of the important dna repair genes.ercc2 codon 751 and 312 polymorphisms has been shown to modulate cancer risk. we therefore assessed the relationship between the ercc2 polymorphisms and the susceptibility to colorectal cancer in a case-control study. there were 105 lung cancer cases and matched healthy controls in this study. information concerning demographic and risk factors was obtained, each person donated 2 ml blood for biomarker testing. ercc2 genotypes were determined by t-arms-pcr method. all of the statistical analyses were performed with spss (v 20.0). there was significant difference between the frequencies of ercc2 polymorphism in cancer cases and controls (p < 0.05). the frequencies of ercc2 751 gln allele were 6.2% in controls and 13.8% in cancer cases. the individuals with lys/gln+gln/gln combined genotype were at an increased risk for lung cancer as compared with those carrying the lys/lys genotype (adjusted or=2.80, 95%=ci 1.21à6.48). the above findings indicate that the genetic polymorphism in the ercc2 codon 751 is associated with the risk of colorectal cancer in an iranian population (neyshabur citizenship). peptide pore blockers are potent tools to study structure and function of potassium voltage-gated channels (kv). kcsa-kv1.x chimeras, in which a ligand-binding site of eukaryotic kv-channel is inserted into bacterial kcsa channel, mimic properly the pore domain of kv-channels. a fluorescence-based approach to study the binding of peptide blockers with kcsa-kv1.1-kcsa-kv1.3 chimeras was developed by us. this approach rested on high-level expression of kcsa-kv1.x chimeras in e.coli inner membrane, binding of fluorescently-labeled toxin at the surface of the spheroplast and analysis of competitive binding of studied ligands by laser scanning confocal microscopy (lscm). here we report on a new analytical system for search and study of kv1.6-channel blockers that combines bl21 (de3) cells expressing kcsa-kv1.6 and rhodamine-labelled agitoxin 2 (rh-agtx2) as a fluorescent probe. by tuning cultivation conditions, the high-level of membrane expression of kcsa-kv1.6 was achieved. it was found that lowering both the growth temperature and the concentration of inducer resulted in significant increase in membrane-embedded kcsa-kv1.6. for system validation, wellknown kv1 channel blockers were studied by the method of competitive binding, and equilibrium dissociation constants were estimated for agtx2, osk1, and kaliotoxin. a new system was applied to study molecular determinants of peptide-kv1.6 channel binding using a number of agtx2 mutants constructed by us, whose affinities to kcsakv1.6 were measured. a new bioengineering fluorescent system is a robust and sensitive assay for assessing the binding activity of kv1.6 channel blockers. it can be used to study interaction interfaces of toxinchannel complexes, to search for novel peptide blockers and to develop new potent and selective kv1.6-blockers for scientific and medical purposes. the work was supported by the grant 14-14-00239 from russian science foundation. asparagus racemosus root extracts (ar) have been exhibited to show a wide range of pharmacological benefits. in this study, liposomes of ar were developed and assessed their physicochemical properties and anti-inflammatory activity in monocytic leukemia cell line (thp-1). liposomes containing ratios of ar to lipid and phosphatidylcholine to cholesterol ratio were synthesized by thin-film hydration (tf), reverse-phase evaporation (rev), and polyol dilution (pd). the in vitro anti-inflammatory activity was assessed in terms of inhibition of tumor necrosis factor alpha (tnf-a) in lipopolysaccharide activated thp-1 by elisa. the size of ar liposomes prepared by tf were larger, whereas those prepared by rev and pd were smaller. ar to lipid ratio was shown to have no influence on particle size, whereas zeta potential enhanced with increasing ar to lipid ratio. ar liposomes with lipid ratio of 1:5 achieved the highest value of entrapment efficiency and were at the highest with polyol dilution method. ar was found to have no toxic effects on thp-1 cells. the anti-inflammatory activities of ar and ar liposomes in terms of tnf-a in thp-1 cells were was exhibited to possess the highest values of around 52% at ar concentration of 1 lg/ml and % tnf-a inhibition tended to decline with the increasing amount of ar. this result may be attributed to the increased amount of liposomal particles being uptaken into the cells as a result of the increasing ar concentrations. it can be suggested that ar liposomes could be an alternative choice of topical/transdermal drug delivery for anti-inflammatory activity. p-mis-002 inhibition of ire1 signaling enzyme increases the expression of tumor suppressor genes and modifies their hypoxic regulation in u87 glioma cells d. tsymbal, o. minchenko palladin institute of biochemistry of the national academy of sciences of ukraine (nasu), kyiv, ukraine gliomas constitute one of the most aggressive groups of malignant neoplasms with poor survival prognosis and scarce therapeutic options. plentiful studies have proven the connection between endoplasmic reticulum stress and malignant growth. we have studied the effect of inhibition of ire1 (inositol requiring enzyme 1), which is a central mediator of endoplasmic reticulum stress and controls cell proliferation and tumor growth, on hypoxic regulation of the expression of different proliferation related genes in u87 glioma cells. it was shown that inhibition of ire1 leads to up-regulation of the expression of krt18, cd24, mest, cenpu, myl9, ing1, ing2, mybl1, and mybl2 genes at the mrna level in u87 glioma cells, with more profound changes for mest, mybl1, and cd24 genes. hypoxia leads to up-regulation of the expression of cd24, ing1, and ing2 genes and to down-regulationof krt18 gene in glioma cells. at the same time, inhibition of ire1 modifies the effect of hypoxia on the expression of all studied genes: suppresses effect of hypoxia on ing1 gene, eliminates hypoxic regulation of krt18, cd24, and ing2 genes in glioma cells. the present study demonstrates that inhibition of ire1 enhances the expression of all studied genes and modifies the hypoxic regulation of these gene expressions in gene specific manner and thus possibly contributes to slower glioma cell proliferation, but several aspects of this regulation remain to be further clarified. amplification and clonig of dna polymerase 1 (pol1) of thermus scotoductus k1 isolated from an armenian goethermal spring a. saghatelyan, h. panosyan, a. trchounian, n. birkeland yerevan state university, yerevan, armenia the most important enzyme ''mined'' from thermophilic microorganisms is dna polymerase, which widely used in molecular biological studies. although dna polymerase produced by thermus aquaticus (taq polymerase) was launched into the market long back, isolation of more processive, reliable and stable dna polymerases from other species is a demand. the purpose of this work was to amplify and clone the pol1 gene of t. scotoductus strain k1 recently isolated from an armenian geothermal spring. the draft genome sequence of strain k1 was deposited under accession number ljjr00000000.1. genomic dna was isolated using genelute bacterial genomic dna kit. primers for the pol1 gene were designed manually. the gene was amplified using pfu polymerase, and amplicons (~2.5 kb) were ligated into the pet-21b(+) vector (novagen) and transformed into chemically competent top10 escherichia coli. inserts were sequenced with t7prom and t7term primers, which showed that the gene sequence was correct and in the right reading frame and could be expressed in mesophilic e.coli. dna polymerases patented form different species of thermus are mostly comparable, suggesting that only limited natural variations in taq-like dna polymerase may be discovered. the pol1 gene from k1 shares 99% and 83% similarity with pol1 of t. scotoductus sa-01 (2.0 kb) and t. aquaticus, respectively. although the difference is not huge at sequence level, possible functional differences (e.g. stability, proofreading activity, resistance to different pcr inhibitors etc.) may occur. therefore, it is important to express and purify dna polymerase from strain k1 for further investigations. peptide ligands of the immunoglobulin g fc region identified by screening phage libraries and site-directed mutagenesis n. kruljec, p. molek, t. bratkovic young researcher, ljubljana, slovenia affinity chromatography based on immunoglobulin (ig)-binding proteins, such as staphylococcal protein a and streptococcal protein g, typically represents the initial step in therapeutic antibody purification process. however, this approach suffers from high cost, poor ligand stability and the requirement for relatively harsh elution conditions that can negatively impact activity and immunogenicity of antibodies. compared to protein ligands, peptides represent an interesting alternative due to higher stability and less expensive production. furthermore, the expected lower affinity for immunoglobulins should allow for elution under milder conditions. the aim of our research was to identify short peptide ligands for the fc region of human iggs. we have screened three commercially available phage display libraries of random cyclic and linear peptides for binding to human fc region in solution using an optimized biopanning approach. five non-homologous linear peptides were shown to specifically interact with the fc portion of immunoglobulins as verified by a set of phage elisa assays. individual phage-displayed peptides were able to recognize specific subclasses of igg. the highest-affinity peptide (12l-19fc), which competed for fc binding with protein a, was subjected to mutagenesis studies. we displayed on phage several variants of 12l-19fc with individual amino acid residues exchanged for alanine as well fragments of the parent peptide of different lengths and evaluated binding to fc with phage elisa to identify the minimal binding motif. binding characteristics of the minimized peptide were further analyzed using spr biosensor. the details will be disclosed at the meeting. diverse effects of ganoderma lucidum in combination with tamoxifen citrate and doxorubicin in mcf-7 breast cancer cells ganoderma lucidum, an edible medicinal fungus, has been known with its anti-metastatic, anti-carcinogenic bioactivities and widely used in asian countries in complementary and alternative medicine. however, there is no information regarding its combined usage with tamoxifen and doxorubicin in breast cancer treatment. we investigated the interactions between ganoderma lucidum and tamoxifen or doxorubicin in mcf-7 human estrogen receptor positive breast cancer cell line. anti-proliferative properties of six extracts were assessed by wst-8 method. the most effective extract in inhibition of mcf-7 cell viability was then evaluated in terms of its anti-metastatic activity by boyden chamber assay. apoptosis and cell cycle assays were performed by flow cytometry. ganoderma lucidum ether extract (g.ether) was the most effective extract on inhibition of cell viability among others with ic (50) values of 100 lg/ml and 12.82 lg/ml at 48 h. and 72 h. respectively. we found that g.ether is capable of inducing apoptosis and changing cell cycle dynamics. however, incubation with g.ether did not affect mcf-7 cell motility significantly. we then assessed the interactions between g.ether and tamoxifen or doxorubicin in mcf-7 cells. the interactions between g.ether and cancer therapeutics were examined by combination index analysis and macsynergy ii software. interestingly, g.ether increased the anti-proliferative effect of tamoxifen although exhibited strong antagonism with doxorubicin in mcf-7 cell line. testing the best matrix/analyte combination for maldi tof mass spectrometric detection of steroid hormones, amino acids, vitamins and carbohydrates in spite of numerous advantages, there are serious drawbacks of the application of matrix assisted laser desorption/ionization time-of-flight mass spectrometry (maldi tof ms) for smallmolecule analyses (below 500 da) and quantification. the main problem is the background interference from commonly used maldi matrix materials. the aim of this work is to evaluate maldi tof mass spectra of physiologically relevant small molecules: steroid hormones, vitamins, amino acids and carbohydrates, acquired with several organic, traditional matrices. small volume, 0.5 ll, of each sample solution (testosterone, progesterone, estradiol, l-cysteine, l-alanine, dl-methionine, glutathione, d-(+)-glucose, d-(+)-maltose, vitamin a, vitamin e) was mixed on the sample plate with the same volume of organic matrix solutions (dhb, thap, chca, 9-aa). for each molecule/matrix pair, we determined quantitative and qualitative parameters of ms analysis. to calculate within day and day-today variation we used excel tools (anova tests). in addition, homogeneity of the sample/matrix distribution on the target was also calculated and expressed as the coefficient of variation of a series of measurements. our results show selectivity of the detection of individual molecules related with the matrix applied. the statistical analysis of certain molecule/matrix pairs gave within and day-to-day variations less than 15%. additionally, homogeneity of the sample/ matrix mixture distribution on the target plate was with some matrices, also less than 15%. some of the used matrices have a great potential for the analysis of small molecules with good analytical parameters, with low variations and high homogeneity of samples on the maldi target plate. these results hold potential for quantification of metabolically-significant small molecules and are very promising for future applications of maldi tof ms analyses. stress causes different expression of mitochondrial biogenesis markers in rat steroid-producing cells of adrenal gland and testes i. starovlah, s. radovic, t. kostic, s. andric faculty of science univeristy of novi sad, novi sad, serbia functional mitochondria of steroid producing cells of adrenal cortex and leydig cells of testes are essential for steroid hormones biosynthesis and regulation. the aim of this study was to determine transcriptional profile of mitochondrial biogenesis markers in adrenal cortex and leydig cells by applying in vivo and in vitro studies. immobilization stress (imo), was performed for 2 hours daily for one (1ximo), two (2ximo) or ten (10ximo) consecutive days. in in vitro studies, primary cultures of purified leydig cells from undisturbed rats were stimulated with stress hormone adrenaline, propranolol (nonselective b-adrs-blocker) and prazosin (the selective a1-adrs antagonist). rq-pcr results showed that the transcription of the main regulator of mitochondrial biogenesis, ppargc1a and ppargc1b, significantly decreased in adrenal cortex of 10ximo rats. oppositely, the significant increase of the same transcript was registered in leydig cells from the same rats. in parallel, transcription of ucp1, the mediator of regulated proton leak, decreased in adrenal cortex, but increased in leydig cells of the same group of rats. incubation of leydig cells with adrenaline, increased transcription of the main markers of mitochondrial biogenesis (ppargc1a, ppargc1b, nrf1 and nrf2a). nonselective b-adrsblocker attenuated this effect. the selective a1-adrs antagonist did not change adrenaline-induced stimulation of ppargc1a, ppargc1b, nrf1 and nrf2a transcription in leydig cells, indicating that the most of the effects are probably mediated by b-adrenergic receptors, not by a1-adrs of leydig cells. in summary, the results suggest that reduction of transcription of mitochondrial biogenesis markers could be a possible mechanism that protects body from excessive glucocorticoid production from adrenal glands in stress conditions, while at the same time stimulation of mitochondrial biogenesis markers transcription in leydig cells could serve as mechanism to preserve testosterone production. p-mis-008 generation of new mitochondria is possible protection mechanism of basal steroidogenesis in leydig cells s. radovic, i. gak, t. kostic, s. andric faculty of science, university of novi sad, novi sad, serbia mitochondria are the most important component of stress response in all cells and for steroid-hormones-producing cells they are the starting point for steroid biosynthesis. here we investigated the parameters of mitochondrial biogenesis in these cells from rats exposed to the psychophysical stress by immobilization (imo). imo stress was applied for 2 hours daily for one (1ximo), two (2ximo) or ten (10ximo) days.hormone levels were measured employing eia, elisa kit or ria. mitochondrial membrane potential (δwm) was measured by tmre fluorescence, mitochondrial mass was detected by quantitative analysis of mitotracker-green fluorescence as well as relative intensity of fluorescence, since number of mitochondria and mitochondrial architecture were defined using transmission electron microscopy. relative gene expression and proteins analyses were performed by rq-pcr and western blot. there was positive correlation between δw m of leydig cells and androgens production of leydig cells. both of them were reduced in all stressed rats but partially recovered in 10ximo group. the mitochondrial mass in leydig cells from 10ximo group was increased. transmission electron microscopy analyses showed that acute and two times repeated stress altered architecture of mitochondrial cristae, while 10ximo increased number of mitochondria and recovered mitochondrial architecture. there was significant increase in the expression of the all markers of mitochondrial biogenesis in leydig cells from 10ximo rats compared with other groups. accordingly, stress-triggered mitochondrial biogenesis represents an adaptive mechanism and does not only correlate with but also is an essential for testosterone production, being both events depend on the same regulators. supporting the evidence that stress, a constant factor in life of humans, induces mitochondrial biogenesis in leydig cells, our results indicate this mechanism probably protects the basal steroid production in stress conditions. targeting survival pathways in leukemic cells through synergism of metformin and thymoquinone u. glamoclija, m. suljagic international university of sarajevo, sarajevo, bosnia and herzegovina generation of resistance to current treatment options is common problem in the therapy of many hematological malignancies. combined therapies utilizing compounds with low toxicity that act synergistically, are proposed to overcome this problem. metformin and thymoquinone (tq) are two molecules which have proven safety profile and represent potential candidates for treatment of hematological malignancies. there are more than 150 clinical trials, at different stages, exploring metformin anticancer activity. metformin activates amp activated protein kinase (ampk) leading to inhibition of the mammalian target of rapamycin (mtor) and induction of apoptosis in different cancers. however, human leukemic cells with increased basal protein kinase b (akt) phosphorylation were shown to be resistant to metformin-induced apoptosis. it was found that activity of metformin can be enhanced by combination with akt and/or nuclear factor 'kappa-lightchain-enhancer' of activated b-cells (nf-jb) inhibitors. tq is phytochemical compound that has shown inhibitory capacity on both of these targets. wst-1 assay was used to evaluate the effects of metformin and tq in dhl4 (b cell lymphoma) and k562 (chronic myelogenous leukemia) cell lines. compusyn software was used in order to calculate the combination index (ci). the ci value indicates whether two drugs have synergistic (ci<1), additive (ci=1) or antagonistic effects (ci>1). we have shown that separately, metformin and tq, exhibit dose dependent inhibition of dhl4 and k562 cells. in combinatorial study with fixed constant ratio and simultaneous drug exposure, in dhl4 and k562 cell lines, ci values were 0.68 and 0.66, respectively. to our knowledge, this is the first report showing synergistic effects of metformin and tq in lymphoma and chronic myelogenous leukemia derived cell lines. these promising data are currently being investigated in order to obtain the insight into their molecular mechanisms. for the last decade many methods of calculating and analysing the physical characteristics of dna has been developed. these methods allow to estimate distributions of free energy, propensity to bend, stress-induced duplex destabilization (sidd), electrostatic potential (ep) etc. and most of them have been used for prediction of genomic regulatory site positions. the main idea of such approach is that proteins recognize genome regulatory sites by these physical and chemical properties, so the physical characteristics are used to predict the location of regulatory sites. most of the characteristics mentioned above describe properties of dna at equilibrium or steady state, but we propose to use characteristics of internal dna dynamics. in this work we used the coarse-grained model of dna, developed recently, to simulate dynamics of the dna open states. with this model we were able to calculate trajectories of the open states moving along the molecule and their dynamical characteristics, such as: open state activation energy, size, half decay time and sound velocity in dna. we use distribution of four dynamical characteristics around transcription start site of experimentally found e.coli promoters taken from regulon db to organise them in stable clusters. clusterization was made with ward method and consensus clustering technique was applied to clusterization results for analysis of its consistency. the same procedure was applied to equilibrium dna characteristics for comparison. distribution of go functions among clusters was also analysed. stable promoter clusters obtained with different physical properties share some similarity. it was not surprise that clusters obtained with dynamical characteristics of dna more similar to sidd clusters then to ep clusters. the data highlights the possible role of dna dynamical properties in transcription initiation and its applicability to promoter identification together with other physical and textual properties of dna. chromium complex with 5-hydroxyflavone acts on metabolic pathways the development of novel therapeutic strategies for obesity treatment are urgently required as obesity is currently the main leading cause in type ii diabetes and insulin resistance. among natural compounds, flavonoids have recently gained interest due to their positive role in maintaining blood glucose levels and insulin secretion. their association with trace elements, wellknown for their capacity in increasing the efficiency of insulin, might potentiate flavonoids biological effects. in this context, the aim of our study was to investigate the in vitro changes in energetic metabolism related genes expression profile in the presence of a chromium complex with 5-hydroxyflavone. dna microarray technology was used for a large scale screening of differentially expressed genes in human adipose stem cells (hascs) after 3 weeks of adipogenic induction in the presence of the chromium complex with 5-hydroxyflavone. moreover, perilipin expression was assessed by flowcytometry. the chromium complex with primuletin negatively regulates the expression of key genes involved in adipogenesis and also modulates the expression of the genes associated with triglyceride synthesis and subsequent fat storage in mature adipocytes. consequently, the chromium complex with 5-hydroxyflavone can be further employed in studies on animal models to investigate the possible improvement of metabolic disorders. deinococcus radiodurans is a highly radioresistant and stress-resistant bacterium. despite extensive studies, the mechanisms of transcription regulation that contribute to the stress-resistance are still poorly understood. d. radiodurans encodes multipe stress-related proteins including three members of the gre-family of transcription factors: grea, gfh1 and gfh2. while grea is a universal bacterial factor that stimulates rna cleavage by rna polymerase (rnap), the functions of lineage-specific gfh proteins remain unknown. we cloned, expressed and purified d. radiodurans rnap and gfh factors and their mutant variants and analyzed their properties using various in vitro transcription approaches. we tested gfh effects on rnap activity in promoter, elongation and termination complexes assembled on natural and synthetic dna templates under different conditions. we found that the gfh factors strongly enhance site-specific pausing and intrinsic transcription termination by d. radiodurans rnap but do not act on active transcription complexes and do not compete with the grea factor. uniquely, the pause-stimulatory activity of gfh is greatly enhanced by manganese ions, which are accumulated in d. radiodurans cells under stress conditions, and is modulated by the secondary rna structure. we revealed functionally important regions in the gfh factors and the rnap active site involved in transcriptional pausing. we propose that gfh factors inhibit rna extension in paused complexes through binding within the secondary rnap channel, coordinating metal ions in the rnap active site and stabilizing an inactive enzyme conformation. this may serve as a sensitive mechanism to regulate transcription under stress conditions and coordinate it with dna repair and replication. our data suggest that gre and gfh proteins target different structural states of the transcription elongation complex and reveal functional diversity of the factors that bind within the secondary channel of rnap. from planktonic to biofilm state of growth, flagella formation is turned off, and the production of fimbriae and extracellular polysaccharides is activated. bola protein is widespread in nature and has been associated with several cellular processes. using high-troughput techniques we showed that bola protein is a new bacterial transcription factor, which regulates the switch between motile and sessile lifestyle. it negatively modulates flagellar biosynthesis and swimming capacity in escherichia coli. moreover, bola overexpression favors biofilm development, involving fimbriae-like adhesins and curli production. our recent results show that bola action in these pathways is related with cdi-gmp a relevant intracellular signaling molecule involved in biofilm formation. we demonstrate that bola contributes to a fine-tuned expression of different diguanylate cyclases and phosphodiesterases and c-di-gmp has a negative influence in the bola mrna transcription. herein we propose that bola is a key player in motile/adhesive transcriptional switch, contributing to a fine-tuned regulation of these important pathways. background: deep venous thrombosis (dvt) is an important health problem worldwide. its pathophysiology is multicausal and involves environmental, genetic and acquired factors. factor v leiden (fvl), prothrombin g20210a (pt g20210a), and methylenetetrahydrofolate reductase (mthfr) gene mutations are to predispose to venous thrombosis. the aim of this study was to compare the frequency of fvl, pt g20210a and mthfr polymorphisms between patients with dvt and healthy controls. methods: this study was conducted at the bozok university hospital. total 220 participants were included in this study, 126 patients with dvt and 94 healthy blood donors. in order to identify fvl, pt g20210a, mthfr c677t and mthfr a1298c, the polymerase chain reaction (pcr) method was utilized combined with the amplification refractory mutation system. results: in 126 patients fvl was present in 54 (42.8%) patients while in controls fvl was present in only 18 (19.1%). frequency of fvl was significantly higher in cases as compared to controls (p < 0.05). pt g20210a mutation was present in 13 patients (10.3%) and in 5 healthy participants (5.3%). mthfr c677t and mthfr a1298c polymorphisms were almost equally distributed among patients and healthy participants. however, the concomitant presence of fvl and double heterozygous polymorphisms of mthfr c677t/a1298c was found in 11 patients (8.7%) and in 2 healthy controls (2.1%), showing significant association with deep venous thrombosis. conclusion: in this study, the frequencies of fvl and pt g20210a polymorphisms were found significantly higher in patients with dvt than those in healthy participants. thus, fvl and pt g20210a polymorphisms have a contributory role on the development of dvt in contrast, mthfr c677t and mthfr a1298c genotypes were not associated with a predisposition to development of dvt. but, a combination of double heterozygous polymorphisms of mthfr c677t/a1298c with fvl may be associated with increased risk of dvt. p-mis-016 self-assembling micellar clusters comprising drugs, nanoparticles and fluorescent compounds for bilogical applications when designing drug carriers, the drug-carrier ratio is an important consideration, because the use of wrong drug-carriers relation can result in toxicity as a consequence of poor metabolism and elimination of the carriers. solubility problem of various substances also plays an important role in many aspects of fundamental science and practical field. specifically, it is an important parameter as well as bioavailability, which determines the required concentration of drug in the body needed to achieve a pharmacological response. among the variety of solubilization methods micellar solubilization is widely used as an alternative to the dissolution of poorly soluble drugs. here, we show a specific approach based on sequential selfassembly of nonionoc detergent micelles (t 9 100, tx114) followed by enacpsulation of various nanoparticles (noble metals, magnet etc.), drugs, fluorescent compounds leads to the formation of stable micellar nano-amd microcomplexes. we propose 3 ways of micellar clusterisation. in the first one micelles are modified by semi-hydrophobic chelator followed by addition of metal ion to make cross-linking. the second way is similar to the first one and suggests application of the metal complex with incresed denticity instead of naked metal ion, and the third one involves micelles clusterisation by semi-hydrophobyc metal complex directly. therefore, one can stabilize micellar network by means of 'interactions on interface': semi-hydrophobyc metal complexes are embedded inside micelle due to hydrophobyc interactions. hydrophobic fluorescent compounds-loaded micellar complexes demonstrates better optical response in aqueous media without crystallization. such obtaining clusters are also very flexible and can be modified by nanoparticles to obtain various nanocomposites, such as fluoromagnetic clusters. this work was supported by russian foundation of basic research grants no. 15-43-03214 r_center_a (2015 no. 15-43-03214 r_center_a ( -2016 no. 15-43-03214 r_center_a ( ) and 15-33-20002 mol_a_ved (2015 no. 15-43-03214 r_center_a ( -2016 . lamellipodia and membrane blebs utilize different signalling pathways to induce directional movement of walker carcinosarcoma wc 256 cells in a physiological electric field clear if those reactions are mediated by similar mechanisms. to establish that, we performed proteomic analysis and subsequent investigation of the role of differential signalling pathways in electrotaxis of cells representing various strategies of movement. cells were exposed to ef in galvanotaxis apparatus and their reaction was recorded. in some experiments cells were pre-incubated with erk1/2 or btk-1 inhibitors. the phosphorylation of erk1/2 and btk-1 was determined by western blot analysis. proteomic analysis was performed by ultimate 3000rs lc nanosystem coupled with a q-exactive mass spectrometer. both blebbing (bc) and lamellipodial (lc) cells show cathodal migration in a physiological ef (1 v/cm). comparative analysis of bc and lc cells proteomes revealed about 150 differential proteins. functional analysis in ingenuity analysis pathway allowed to determine the statistically significant signalling pathways in which these proteins are engaged. among the most distinctively regulated pathways are tec kinase and erk/ mapk signalling activated in lc but not bc. it was found that btk-1 is required for directional movement of lc but not for bc cells. moreover, ef induced stronger and faster btk-1 phosphorylation in lc than bc cells. in contrast erk 1/2 activity was not necessary for electrotaxis of lc cells and ef did not induce erk 1/2 phosphorylation. our results reveal that both lamellipodia and membrane blebs can efficiently drive electrotactic migration of wc256 cells but it is mediated by different signalling pathways. this work was supported by a grant from the national science centre 2012/07/b/nz3/02909, poland. newborn screening for congenital hypothyroidism in turkey: a regional evaluation € o. demirelce 1 , n. y. saral 1 , f. b. aksungar 1,2 , a. coskun 1,2 , m. serteser 1,2 , i. unsal 1,2 1 acibadem labmed, istanbul, turkey, 2 acibadem university, istanbul, turkey congenital hypothroidism (ch) is the most common congenital endocrine disorder and the most important cause of preventable mental retardation. it is important to begin the treatment within 2 weeks before the development of brain damage. tsh based newborn screening programs are shown to be useful for implementing early treatment of ch. in this study, regional results of ch screening program in turkey between 2006 and 2015 were assessed retrospectively. we have evaluated the results of marmara, central anatolia, aegean and mediterranean regions in which our laboratories are located. screening was based on tsh determination in dried blood spot specimens. tsh limits determined to be 10 lu/ml for cut off point and 25lu/ml for clinical decision point. tsh was measured using enzyme immune assay (eia). blood spot tsh data for 65857 newborns during this time period were evaluated. permanent or transient ch was determined according to the results of thyroid function tests. confirmed ch cases were based on local endocrinologists' report and initiation of thyroxine treatment. the frequency of neonatal tsh levels were found to be under the cut off level of 10lu/ml in 63513 (96.44%), between 10 and 25lu/ml in 2287 (3.47%) and above the level of 25lu/ml in 57 (0.09%) babies, respectively. recall rate was 3.5%. ch cases of neonatal tsh levels greater than 25lu/ml were 26. the incidence of ch of this group was 1:2532. there were no significant differences in the number of congenital hypothyroidism between males and females (p > 0.05). the preliminary results of our study indicate that the incidence of ch in our region is higher than the worldwide reports as has been proved by preceding studies. iodine deficiency, dyshormonogenesis, highly consanguineous population, may contribute to the high incidence of ch in turkey. newborn screening of ch must be developed for detecting true cases and tsh cut off point must be reviewed for decreasing redundant recall rate. in silico analysis of the first complete genome sequence of lactobacillus acidipiscis species k. papadimitriou 1 , m. kazou 1 , v. alexandraki 1 , b. pot 2 , e. tsakalidou 1 1 agricultural university of athens, athens, greece, 2 institut pasteur de lille, lille, france introduction: lactic acid bacteria (lab) constitute a significant group of microorganisms for the food industry, as they play a key role in food fermentation and consequently in human health. lactobacillus acidipiscis aca-dc 1533 is a gram-positive, motile, rod-shaped lab isolated from traditional greek kopanisti cheese. here we present the in silico analysis of the first complete genome sequence of l. acidipiscis in order to explore the biology of the species. materials and methods: sequencing of l. acidipiscis genome was performed using the hiseq 2000 and pacbio rsii sequencing platform technologies and the genome assembly was validated against an nhei optical map of the l. acidipiscis genome. protein-coding sequences were predicted by glimmer, rrna genes by rnammer and trna genes by the trnascan-se server. potential genomic islands were detected using the island-viewer software tool, prophage regions by phast and the subsystem-based annotation by rast server. finally, the circular representation of l. acidipiscis genome keyed to the cog groups was constructed by cgview server. results: the sequencing analysis resulted in one continuous genomic scaffold of 2,678,726 bp with a g+c content of 39.75%. the genome contains 2,525 protein-coding genes on the chromosome covering up to 82.09% of the genome sequence, 63 trna and 18 rrna. according to the subsystem-based annotation, 1,562 protein-coding genes were assigned to 310 metabolic subsystems. the most abundant of the subsystems are related to carbohydrates (n = 287, 18.37% of total protein-coding genes) and protein metabolism (n = 173, 11.08% of total protein-coding genes). furthermore, three prophage regions were detected; one intact (43.5kb), one incomplete (14.7 kb) and one questionable (29.3 kb). discussion and conclusion: the whole genome analysis of l. acidipiscis aca-dc 1533 provided interesting information about a not well-studied species. investigation of serum irisin levels of patients with metformin taking new onset type 2 diabetes mellitus increases glucose tolerance and energy expenditure and improves carbohydrate homeostasis. metformin is a biguanidine class antidiabetic drug which inhibits liver gluconeogenesis and decreases insulin resistance and is frequently recommended in treatment of new onset type 2 diabetes mellitus (t2dm). irisin has a role in the regulation of energy metabolism pathways and its level in blood of persons with t2dm has been reported to decrease. regarding this relationship, it was aimed to reveal the effect of metformin on serum irisin levels. 20 patients with impaired oral glucose tolerance test were included to this investigation. they were recommended to take metformin and to change their life style, such as exercise and diet. their blood were taken at the beginning and after 1 month. also, a healthy control group (n = 20) was formed from persons with similar age and sexual distribution as the patient group. irisin levels of their sera were measured by enzyme-linked immunosorbent assay (elisa) method. statistical evaluation of the measurements showed no significant difference (p = 0.780) between the irisin levels of the patients at the beginning and after 1 month treatment. a similar result was found between the control and the treated groups (p = 0.170), while a significant difference (p = 0.002) was observed between the control and untreated patients groups. the results obtained from this study do not show a clear and significant change in the blood irisin levels of the patients with new onset t2dm taking metformin together with life style change. a longer period of treatment and a higher number of patients may be needed for more reliable results. thermodynamics of dna ligands binding at specific sites of telomeric g-quadruplex dna g-quadruplexes are a perspective target for anticancer therapy. for example stabilization of the telomeric g-quadruplex dna formed by single-stranded ends of the chromosomes leads to inhibition of telomerase, which is active in 90% of cancer cells. similarly, small molecules targeted to a specific g-quadruplex would inhibit various cellular processes. stoichiometry and affinity of interaction of these compounds to dna is determined by specific structural motifs within a g-quadruplex. rational design of novel chemical compounds requires an in depth knowledge of interactions between known ligands and g-quadruplex structures. experimental methods that are used for determination of thermodynamic binding parameters, such as isothermal titration calorimetry, differential scanning calorimetry, ultraviolet absorption and circular dichroism spectroscopy provide a collective characteristic for all of the ligand molecules bound to dna, while the information on ligand affinity to individual dna binding sites is lost. we propose a complimentary method for detailed analysis of thermodynamic parameters of ligand binding based on the introduction of fluorescent probes in the structure of g-quadruplex. monitoring fluorescence quenching of the fluorescent labels allows to derive binding constants of the dna-ligand interaction at a specific binding site. temperature dependence of the fluorescence quenching determines the thermodynamics of the dnaligand complex formation. since only a proximal ligand is able to quench the fluorescence, this method allows characterization of the ligand binding to a particular site the g-quadruplex structure. the study was supported by project no. 16-14-10396 of the russian science foundation. the correlation between biochemical and dynamic surface tension parameters of calves blood serum during the animal ontogenesis, as well as by various pathologies or poor diet, the imbalance of protein, mineral, lipid components is observed (the changes in all parameters of biological liquids are accompanied of these metabolism peculiarities). the dynamic surface tension (dst) of serum essentially depends on these factors and (in combination with the biochemical parameters) can provide the valuable information for evaluation of the physiological and biochemical status of the organism (can be used as an express test for animal diagnostics in future). the aim of the work was to study dst and biochemical parameters of calve serum, as well as their correlations, as the main indicators of the animals. ) of calve serum were in the range of the normal values for healthy animals and can be considered as reference data for animal science and practice. the obtained results enable us to establish correlations between the dst and biochemical parameters of calves serum. this work was supported by the russian scientific foundation (grant 14-16-00046). the middle strong correlations of dst values of calves serum with the level of total protein, albumin, billirubin, some enzymes and cholesterol, whereas only weak correlations with the other biochemical parameters (urea, calcium, magnesium, phosphorus, etc.) were found. in the veterinary science and practice such correlations are important for the estimation of the organism physiologicaland biochemical status, for general inspections of cattle before vaccination (immunization) or slaughter, for "quick separation" of healthy and ill animals in the case of infection, etc. role of protein kinase c in the regulation of astrocytic glutamine transporter sn1 in ammonia-exposed mouse cortical astrocytes (bisi; 1 lm). total pkc activity was analyzed by a direct pkc assay and phosphoserine detection by western blot (wb) analysis. protein level of sn1 and sn2, second astrocytic gln transporter belonging to system n, in a membrane fraction was also analyzed. the total uptake and system n-mediated (l-ala and l-leu-inhibitable) gln uptake was tested. treatment of astrocytes with ammonia resulted in a decrease of pkc activity, whereas pma treatment increased pkc activity in ammonia-independent way. bisi treatment reversed fully, and ammonia partially, the pma-induced pkc activity. pma treatment resulted in only a slight decrease in sn1 protein level in both control and ammonia-treated astrocytes, while a decrease of total and system n-mediated gln uptake were noted in control astrocytes, an effect not exacerbated by ammonia. in turn, cotreatment with pma and bisi reversed the decrease of total gln uptake and showed tendency towards increase in system nmediated gln transport. the results suggest that: a) ammonia changes the dominating direction of system n transport from release to uptake, which may be related to decreased phosphorylation or to alterations in relative phosphorylation by different pkc isoforms. this inference remains to be verified in further studies; b) changes in system n transporter function induced by ammonia appear to involve mechanisms other than changes in transporter expression. evidence for human ghrelin ghs-r1a and orexin ox1 heteroreceptor complex formation in a heterologous system ghrelin and orexin are two peptides implicated in the regulation of energy balance and modulation of food-related motivation at the level of the midbrain dopamine reward system. their function in the hypothalamic arcuate nucleus and the ventral tegmental area (vta) has already been described, but the modulation at the level of receptors remains unclear. the action of these peptides is mediated by g-protein-coupled receptors (gpcrs): ghrelin 1a and 1b (ghs-r1 a , ghs-r1 b ) for ghrelin, and orexin 1 and 2 (ox 1 , ox 2 ) for orexin. traditional approaches to know the mechanism of neurotransmission of dopaminergic neurons in the mesolimbic system have focused on targeting neuronal receptors as single entities. from the discovery that gpcrs for neuromodulators may form heteroreceptor complexes, our hypothesis is that ghrelin and orexin receptors may interact and form novel functional units that may specifically participate in the central regulation of food intake and energy balance. as a proof of concept we have investigated the potential of human ghs-r1 a and ox 1 receptors to form heterocomplexes. formation of ghs-r1 a -ox 1 receptor heteromers in transfected hek293t cells was detected by bioluminescence resonance energy transfer (bret) and proximity ligation (pla) assays. furthermore, a negative crosstalk was identified in cells co-expressing both receptors by assessing mitogen-activated protein kinase (mapk) and adenylyl cyclase (camp) pathways, and by a label-free dynamic mass redistribution assay. experiments in sources endogenously expressing ghs-r1 a and ox 1 receptors are needed to know the functional relevance of the heteromer. from the negative crosstalk here identified, it is tempting to speculate that ghs-r1 a -ox 1 receptor heteromers are important players in mediating the response to the combination of different orexigenic signals. lysosomal storage diseases which are related to deficiency of specific lysosomal hydrolases resulted to clinical aspects due to accumulation of substrates in different tissues. since dried blood spot (dbs) is non-invasive, low-cost, easy transportable, acceptable enzyme stability compared to leucocyte and/or fibroblast culture, it's recommended as a first screening test. however the false positive rate with dbs sample is higher compared to other samples. we aimed to investigate any possible effect of leucocyte number on enzyme activity in dried blood samples in a retrospective study. we re-evaluated the lysosomal enzyme activity results in regard to leucocyte number among data within last 1 year. enzyme activities had measured by using fluorometric and lc msms method. we determined the correlations between the lysosomal enzyme activities of alpha glycosidase, glycocerebrosidase, alpha galactosidase, sphingomyelinase, galactocerebrosidase and alpha-l-iduronidase in healthy population (n = 220). while glycocerebrosidase and galactocerebrosidase positively correlated with the number of neutrophils, alpha galactosidase, sphingomyelinase and alpha-l-iduronidase positively correlated with the number of lymphocytes. alpha glycosidase activity showed a correlation both lymphocytes and neutrophils. the patients having the glycocerebrosidase enzyme activity which was lower than 0.6 nmol/ml/hour (which is accepted as the cut off value to recall the patients) existed significantly lower number of leukocyte, lymphocyte and neutrophil compared to those of patients having higher enzyme activity than 0.6. our data indicated that the enzyme activity in dried blood samples including low leucocyte number might be found lower than reference intervals resulting in false positive diagnosis. therefore we suggest that the laboratory scientists should evaluate the number of leucocyte levels while they were interpreting data. using dna-markers for estimation of genetical variability of two kazakh sheep breeds a. mussayeva 1,2 , b. bekmanov 1,2 , a. amirgalieva 2 , k. dosybaev 1,2 , z. orazymbetova 1,2 , r. zhapbasov 2 , a. zhomartov 2 , n. zumadillaev 3 , n. zumadillaev 3 1 llp "kazcytogen", almaty, kazakhstan, 2 "institute of general genetics and cytology" sc mes, almaty, kazakhstan, 3 branch "scientific research institute of sheep" llp "kazakh research institute of animal husbandry and feed", almaty, kazakhstan to compare the frequencies of different microsatellite loci in sheep breeds subpopulations genomic structure of edilbay and kazakh archaromerinos was investigated. different methods for homogeneity testing of two breeds were elaborated. inter simple sequence repeats (issr) pcr analysis of the breeds studied displayed species and breed specific fragments with different frequencies (population frequency more than 0.4) there were found rarely met fragments (frequency lower than 0.4). the combinations of these fragments present the specific issr-spectra which arrange genofond profiles of breeds. using panels of microsatellits (recommended by isag) 2 breeds (5 populations) were characterized. informative value and resolving capacity of the sum of 32 str-loci were estimated. wide polymorphism of alleles length was demonstrated both when the breeds were compared and within the breeds. 10 informative markers were chosen for both two breeds, 7 markers being used for both breeds, while other 3 markers were informative for one of the breed only. when the animals of one breed were compared unique alleles which were met only within one of populations were of much interest. for example the allele 174 of bm1824 was met in birlik population of edilbay breed as often as in 59% of animals while in two other populations there were no this allele. in kumtekey population one can meet 70% animals having particular locus (dyms1), while in the other population (cf ablay) this locus was not met at all. basing on genetical distances obtained using fragment analysis phylogenetic relationships between populations were estimated. so for example edilbay population of cf ajar has the larger distance from two other populations (birlik and bayserke-agro) than each of them from one another. two subpopulations of kazakh arkharomerinos breed (cf kumtekei and cf ablay) also have the genetical difference. how preeclampsia affects oxidant status and antiinflammatory potential of breast milk? preeclampsia is a pregnancy syndrome associated with hypertension, proteinuria and edema, leading to maternal morbidity/mortality and preterm delivery. in this study we aimed to investigate if the breast milk of preeclamptic mothers is effected in oxidative status and anti-inflammatory activity in comparison to the breast milk of mothers with healthy pregnancies. for the aim of the study, hyaluronidase and myeloperoxidase activities (mpo), total oxidant status (tos), total antioxidant status (tas), oxidative stress index (osi) and tbars levels were measured in breast milks of 20 preeclamptic mothers and 20 mothers with healthy pregnancies as control group. when the control group and preeclamptic group were compared, hyaluronidase activity, tas, tos and osi levels showed statistically significant differences in the preeclamptic group. hyaluronidase activity was significantly higher in the preeclamptic mothers' breast milk (734 vs 594 u/ml, p = 0.046). while tos levels were significantly higher in the preeclampsia group (7.48 vs 4.51 lmol/l, p = 0.001), the tas levels were significantly higher in the control breast milks (0.557 vs 0.813 mmol/l, p = 0.011). as expected osi levels (tos/tas ratio) were significantly higher in the preeclampsia group. even though the mean levels were higher in preeclamptic group, the difference in mpo activities and tbars levels did not show statistic significance. oxidant status parameters also suggest that preeclampsia effects in both ways by increasing oxidant status and also decreasing antioxidant capacity shifting the balance to the increased oxidant stress side. as the results showed that the preeclampsia group had higher hyaluronidase activity, this can be interpreted as preeclamptic mothers' milk have higher inflammatory potential as this enzyme enhances inflammation by catalyzing the depolymerization of certain acidic glycosaminoglcans. p-mis-030 investigation of relationship between postprandial lipemia and erythrocyte membrane cholesterol level postprandial lipemia is a metabolic condition related to an increase in plasma triglycerides. remnant-like lipoprotein particles are predominant in postprandial phase and they play an important role in development of atherosclerosis. cholesterol is a prominent component of erythrocyte membranes and regulates the membrane functions such as viscosity and permeability. free cholesterol derived from erythrocytes is thought to participate in the atherosclerotic plaque formation. in the current study, it was aimed to investigate the relationship between postprandial lipemia and erythrocyte membrane cholesterol level in healthy subjects. study group included 87 subjects (39 female and 48 male with age range of 18-55 years). then these individuals were divided into three groups according to the values of area under curve (auc) calculated by using triglyceride levels at the fasting state and at 2nd, 4th and 6th hours after the high fat diet (ottt). lipid and erythrocyte membrane cholesterol (emc) values were compared between groups with low and high ottt response. while tc, tg, ldl-c and emc were significantly higher, hdl-c was significantly lower in high ottt response group than low ottt response group. it was not observed any statistically significant difference when compared emc values between women and men study groups. on the other part, it was seen positive correlation between emc and auc (r = 0.33, p = 0.002), tg (r = 0.26, p = 0.016), tc (r = 0.30, p = 0.005), ldl-c (r = 0.29, p = 0.007) in the total study group. it was concluded that, postprandial lipemia may show atherosclerotic tendency not only with atherogenic lipid profile but also with increasing emc. p-mis-031 eu-openscreen: the european infrastructure for chemical biology b. stechmann, p. gribbon eu-openscreen, fmp leibniz institute for molecular pharmacology, berlin, germany small molecules that can be applied as chemical 'tool' compounds (or 'probes') have become indispensable in basic research for the elucidation of fundamental biological mechanisms. they act directly with the protein-of-interest and often allow for the interrogation of biological processes that cannot be properly studied with traditional genetic or rna interference approaches. eu-openscreen (www.eu-openscreen.eu) is the largest emerging academic chemical biology research infrastructure initiative in europe and will provide access for molecular and cell biologists to screening infrastructure, well-characterized highquality chemical libraries, and facilities for medicinal chemistry services for compound optimization. molecular biologists who have a robust and suitable biological assay and are interested in collaboratively developing chemical tool compounds to validate their targets-of-interest are welcome to work with eu-openscreen. selected assays are screened against a collection of more than 100,000 compounds, incl. confirmatory and counter screening, ic/ec50 determination, sar (structure-activity relationships) and qc of confirmed hit compounds. eu-openscreen will start operations in 2017, but it can already look back on a growing number of transnational activities: joint screening projects, exchange of local compound libraries, development of new design principles for its compound collection; exchange of experimental data through its pilot database etc. steps towards an arthrobacter nicotinovorans based biotechnology for production of 6hidroxy-nicotine as the archetypal agonist of nachr, nicotine stands up as a powerful scaffold for developing new alzheimer disease therapeutic agents in form of nicotine derivatives. in this context, arthrobacter nicotinovorans pao1 and its wide range of nicotinederivatives produced when grown on nicotine have a huge biotechnological potential. indeed, the metabolic intermediate 6-hydroxy-nicotine (6hnic) produced by a. nicotinovorans pao1 was shown to bind to the nachrs, and by modulating their function, to sustain spatial memory formation in a rat model of ad. the current work presents the first attempts to produce and isolate 6hnic by using a genetically engineered a. nicotinovorans strain. the growth and the 6hnic accumulation were compared for two strains: 1. a. nicotinovorans pao1 wild type strain and 2. a genetically engineered a. nicotinovorans pao1 strain (part2ndh) containing the nicotine-dehydrogenase (ndh) genes cloned in the nicotine inducible part2 vector. the growth curves were followed spectrophotometrically. the consumption of nicotine and accumulation of 6hnic were monitored by hplc using a mn nucleodur 100-3 c18ec column and 0.1 m sulfuric acid at a flow rate of 1 ml/minute. the growth curve of the part2ndh strain shows that the bacteria grow slower when compared with the wt. as a result, in the wt strain, the nicotine is quickly depleted from the medium and only low amounts of 6hnic are observed. although the sds-page analysis of the total protein extracts from the part2ndh strain did not show clear signs of ndh overexpression, the enzyme is produced and is active, allowing a 5 fold accumulation of 6hnic in the growth medium. the first attempts to purify ndh from the part2ndh strain using imac were nevertheless unsuccessful. in conclusion, using the part2ndh strain for 6hnic production is feasible. further improvements of the growth condition and strain are envisioned (i.e. knocking the ndh downstream genes; adding inhibitors for the downstream enzymes). studies on the impact of butyrylcholinesterase (bche) on the symptoms and progression of cognitive impairments like alzheimer's disease (ad) or other neurodegenerative disruptions speak in favour of selective bche inhibitors as a new approach in future ad pharmacotherapy. some derivatives of quinine and quinidine, present in the cinchona species bark, have already been identified as selective bche inhibitors with respect to acetylcholinesterase (ache); therefore, further investigation of these compounds might result in promising leads for enhanced anti-ad drugs. we synthesised ten quaternary derivatives of cinchonines and their corresponding pseudo-enantiomeric cinchonidines. quaternization of quinuclidine moiety was carried out with groups diverse in size: methyl and differently meta and para substituted benzyl groups. all of the compounds were prepared in good yields, characterized by standard analytical spectroscopy methods, and were tested for their bche and ache inhibition potency. the inhibition potency of the compounds was defined by the dissociation constants of the enzymeàinhibitor complex (ki). all of the tested compounds reversibly inhibited both human bche and ache. the compounds inhibited bche with ki constants in the range of 0.04-30 lm, and ache in the range 2.5-70 lm. five cinchonidines displayed a 95-510 times higher inhibition selectivity to bche over ache, and four of them were potent bche inhibitors with ki constants up to 100 nm. bche affinity toward the studied compounds depended on the size of the substituent on the nitrogen of the quinuclidinium part of the molecule and on the resonance stabilization of the substituent at the quaternized nitrogen. based on the presented results, cinchonidine cd-(pbr) can be pointed out as a potent and selective bche inhibitor that could be considered for further research in alzheimer disease pharmacotherapy. exposure to nmda (100 lm) for 72 h increased the expression of kir4.1 mrna and decreased that aqp4-and gs mrna. the expression of kir4.1 was decreased by 72 h exposure to glu (2 mm) and tnfa (50 ng/ml). at 8 h incubation, nmda induced a decrease of kir4.1 expression in the presence but not in the absence of calcium in the medium. nmda did not alter the expression of nmda receptor subunits. tnfa increased the expression of the nr1 subunit, and decreased that of nr2b mrna. glu decreased the expression of 3 out of 7 subunits. the study demonstrates, to our knowledge for the first time, that prolonged exposure of astrocytes to nmda alters the expression of mrna coding for critical astrocytic proteins. the dependence of the decrease of kir4.1 mrna expression on extracellular calcium suggests the ionotropic nature of nmda receptor stimulation. the effects of nmda receptor stimulation occurred by a mechanism bypassing changes in subunit composition of the nmda receptor. experiments are under way to establish whether the tnfa-induced changes in the expression of nmda receptor subunits contribute to modulation of nmda receptor stimulation by inflammation. the importance of education in reducing preanalytical errors the preanalytical phase includes the request of test, the preparation of patient, the obtaining of sample from the patient, the transport of the sample to the laboratory, and the pretreatment of sample. the preparation of patient and the obtaining of sample are considered as the most common error sources. in order to reduce preanalytical errors, we aimed to provide training for phlebotomists and to also determine their knowledge level about the preanalytical phase before and after these training. it was given the training related with preanalytic phases to 130 pediatric nurses and 350 adult nurses, other phlebotomists in march. the surveys which are made before and after the training were consisted of 20 questions that are related with demographyic features and preanalytic phases. in order to determine the effects of training to the preanalytic phase, the preanalytic error rates before (in february) and after (in april) traning was calculated with the formula of: (the number of rejected samples/the number of total samples)x100. the average age of participants was 35 ae 9 years. it was not found significant difference between their correct answers rate before the training and the education degree of the participants. the correct answer rate before the training was 59% and after the training it was 92%, which showed an increase of 55%. the preanalytic error rates which were 0.61% in february were decreased to 0.40% in april. in our study, the positive results were obtained through the training aimed to reduce the preanalytical errors. by providing regular training to the phlebotomists and also providing pretraining to the beginners, the updating of their information about preanalytic phase can be achieved. in this way, the loss of labor and economic related to preanalytical errors can be avoided and the accurate results can be obtained in short time. curcumin, the active compound of turmeric (curcuma longa) has antiinflammatory, antioxidative and antitumour effects. unfortunately, curcumin has a poor absorption and low stability. both can be solved by encapsulation of curcumin using a proper technique like electrospray. it was reported that piperin, the active compound of black pepper, enhances the intestinal absorption of curcumin and thus its bioavailability. due to these facts it was aimed in this study to nanoencapsulate turmeric extract in order to enhance its absorption and stability. for that purpose, it was encapsulated with the maize protein zein, chitosane and black pepper extract by varying the voltage and flow rate of electrospray and the concentration of the compounds. the nanocapsules were characterised by measuring their particle size and with help of sem photographs. the particle size of the final nanocapsule formulation was 550 nm and had a sufficient stability over a period of 6 months, visually determined. by encapsulating turmeric extract into double layer nanocapsules with help of black pepper extract, zein and chitosane, the turmeric extract could be protected from degradation, which was observed for the pure turmeric extract in form of clearing its yellow colour. analysis of the human genome reveals that potential g-quadruplex sequences are enriched in promoters of the oncogenes. growing body of evidence suggests that g-quadruplexes (g4) may play putative roles in various biological processes, such as the regulation of gene expression. consequently, targeting the oncogenic g-quadruplexes using small molecules is an alternative strategy for the potential treatment of cancers. porphyrin derivatives are promising class of drug in this respect, being nucleic acids binders and generators of reactive oxygen species under visual light irradiation. interaction between porphyrin derivatives and g4 dna from oncogene promoter region has been studied in vitro. we applied chemical probing, circular dichroism spectroscopy and uv melting techniques in order to map the oxidized bases, monitor structural rearrangements and evaluate stability of the resulting dna structures. specifically, we observed that g4 dna is considerably more susceptible to lightinduced modification than duplex dna; 5 0 -terminal tetrads of the g4 dna are preferably oxidized; structural changes induced by oxidation result in decrease of the thermodynamic stability of the g4 dna. irreversibility of these effects on dna make porphyrin derivatives perspective lead compounds for rational design of ligands targeting human oncogenes. the study was financially supported by project no. 16-14-10396 from the russian science foundation. resistin levels in denervated obese rats n. saglam 1 , t. ahmedi rendi 1 , c. kahraman 2 , a. alver 1 1 department of medical biochemistry, faculty of medicine, karadeniz technical university, trabzon, turkey, 2 school of health, d€ uzce university, d€ uzce, turkey the sympathetic nervous system is an important factor affecting the metabolic and secretory function of the white adipose tissue. resistin is mainly expressed by mononuclear cells, also it is expressed by adipocytes, pancreatic cells, and muscle. resistin induces insulin resistance and glucose intolerance in mice. resistin plasma levels depend on fat depots size and sex. resistin levels decrease in short-term fasting in mice, then it increase refeeding. also, it increase as a response to fed with the high fat diet. in our study we aimed to determination of the effect of high-fat diet and denervation on serum resistin levels in rats. in this study 4 experimental groups were formed each consisted of 8 rats. during 10 weeks, first two groups are fed with high-fat diet and other two groups are fed with standart diet which they purchased from research diets company. at the beginning of the feeding periods, retroperitoneal fat tissiues of animals assigned to the first and the third groups were denervated. second and fourth groups were not denervated. at the end of the 10 week feeding periods, blood collected from rats and blood resistin concentration was determinated by elisa. in denervated and fed with high fat diet groups serum resistin levels higher than control groups (p < 0.05). according to our literature research, there are no studies demonstrating the relationship between resistin and the sympathetic nervous system. also, denervation may lead to increase in serum resistin levels. the amount of resistin is possibly reduced by b -adrenergic activation. in conclusion, it was concluded that there is differences on serum resistin levels depending on diet in bilateral denervation of retroperitoneal fat tissues of rats. stress activated protein kinases regulates the ribosomal frameshift rate in est3 gene, encoding subunit of telomerase s. t€ urkel, s. sarica uludag university, bursa, turkey est3 gene (ever shorter telomere) of s. cerevisiae encodes one of the essential subunits of telomerase enzyme. expression of est3 gene is regulated at the translation level by +1 programmed ribosomal frameshift (prf). it is known that the physiological stresses affect telomere length. in this study, we have investigated the effects of stress activated protein kinases snf1p (ampk) and gcn2p (eif2 kinase) on the prf rate in est3 gene. prf rate of est3 gene was quantified in plasmid based expression system. expression vectors were transformed in to the wild type and mutant yeast strains that deleted for snf1, or gcn2 genes. yeast cells were grown in normal conditions or subjected to acid stress, osmotic stress, or glucose limitations to activate protein kinases gcn2p, and snf1p, respectively. prf rate of est3 gene was measured as 13% in the normal growth conditions in the wild type cells. but, the prf rate of the wild type strain grown in glucose limited conditions decreased more than 10-fold, giving less than 1% prf rate. contrary to glucose limitation, osmotic or acid stress activated frameshift rate by 2-fold in the wild type cells and prf rate increased to 25%. when the prf rate was analyzed in gcn2 and snf1 mutants, frame shift rate of est3 was 4-5% in normal growth conditions. when these mutants were subjected to acidic or osmotic stress, prf rate activated slightly. we have also shown that gcn1p and gcn20p, positive regulator of gcn2p, is also essential for the regulation of prf in est3 in response to stress conditions. it is clear that the basal level expression of est3 is highly dependent on the gcn2p kinase complex. gcn2p is also associates with ribosomes, indicating that gcn2p might have a significant function in connecting the stress signals to biosynthesis of the full length est3 peptide. this regulation might also link the biosynthesis of functional telomerase and telomere replications to cell physiology through protein kinases such as snf1p and gcn2p. inflammation might have a role in erosive esophagitis but not in non-erosive reflux disease the relationship between inflammatory activation mechanisms and acid-peptic injured esophageal tissue is not clear. we evaluated whether there are differences between inflammation and tight junctional proteins such as e-cadherine among subtypes of gastroesophageal reflux disease. the aim of this study was to investigate any possible role of inflammation in pathologic mechanism of reflux disease by determining the inflammatory markers in injured esophageal tissue as well as serum of patients. three groups (erosive-ee, n = 18; nonerosive-nerd, n = 12; healthy controls-hc, n = 13) were evaluated with upper gastrointestinal endoscopy. the esophageal biopsies and blood samples were collected. serum e-cadherine levels, nfkb, chitotirosidase (chit), myeloperoxidase (mpo) activities in serum and homogenized tissues were determined. nkfb levels in tissue was significantly higher in subjects with ee (4.9 ae 2.53 ng/mg.prt) versus hc (2.95 ae 1.51 ng/mg.prt, p = 0.018). mpo tissue activities in ee group were significantly lower (0.07 ae 0.06 u/mg.prt) than hc (0.23 ae 0.22 u/mg.prt, p = 0.025) while mpo serum levels were higher in ee (1.15 ae 1.63 ul) versus hc (0.56 ae 0.69 ul, p = 0.045). tissue chit levels were three fold increased in ee versus hc (p = 0.071). none of these measurements showed any differences in nerd group. nfkb and mpo levels had a negative correlation (r=à0.408, p = 0.005) in tissue. nfkb and ecad levels had a positive correlation in serum (r = 0.642, p < 0.0001). inflammatory process might play a pivotal role in injured mechanism only in erosive esophagitis but not in nerd. noninflammatory mechanisms might be responsible such as hypersensitivity in patients with non-erosive reflux disease. d-dimer (a fibrin degradation product) test is used to aid in the diagnosis of intravascular coagulation. the aim of this study is to investigate the correlation between d-dimer levels and other inflammatory markers including procalcitonin. anonymized data on d-dimer, fibrinogen, hscrp, wbc, neutrophil% (neut%) and procalcitonin levels from 50,107 patients (mean age ae sd, 53.37 ae 20.6) were used for the correlation (excel analyze-it v4.60.4) and linear regression (pasw statistics 18 v18.0) analysis between the measured parameters. there was a significant (p < 0.05) age-dependent increase in d-dimer levels between different age groups. patients with the highest d-dimer levels were also found to have an increased frequency of hscrp levels. d-dimer levels showed a significant correlation with hscrp, wbc and neut%. a model describing the positive association between these parameters were built. the resulting equation is as follows: d-dimer = (hscrp*0.054) + (0.011*age) + (0.006*wbc) + (0.04*neut%)à0.929. correlation analysis between procalcitonin and d-dimer levels gave pearson's correlation coefficient of 0.159. our results suggest that the age-dependent variations should be taken into account while interpreting d-dimer test results. in addition, neut% ratio was found to be the most important parameter for estimating d-dimer levels. our equation can be used when the d-dimer test is not available or for control purposes only. in the field of cancer research great hope lies in finding more powerful and selective way for the direct elimination of cancer cells. this task can be solved by means of nanobiotechnology. recent progress in this field has arisen interest in a carbon nanostructurefullerene c 60 . fullerene exhibits not only unique physico-chemical properties and biological activity but also a significant potential to serve as a nanocarrier for selective drug delivery into cancer cells. the aim of this study is to analyze a unique tool for cancer therapy. the main idea is realized by the non-covalent conjugation of c 60 with the well-known anticancer drug -doxorubicin (dox). two types of conjugate with different c 60 -dox ratio (1:1 and 2:1) were studied. conjugates absorbance and fluorescence, size distribution as well as a mass data were recorded utilizing optical and analytical equipment (microplate reader, zetasizer, lc-ms/ms and maldi-tof). in vitro studies were performed including evaluation of c 60 -dox conjugate effects on human leukemic cells (jurkat, ccrf-cem, thp1 ad molt-16) viability. conjugates accumulation and distribution within cancer cells was monitored using fluorescent microscopy accompanied with fluorescence-activated cell sorting. it was evidently proven that both c 60 -dox conjugates were stable and could be used as reliable candidates for biological application. cellular accumulation and distribution studies showed that conjugation of dox with fullerene promoted its entry into leukemic cells. accumulation of dox in the form of conjugates within cancer cells was intensified compared to the free drug. the results show that conjugated dox is more cytotoxic and the value of its ic50 are lower compared with the free dox. obtained results confirm nanocarrier function of fullerene c 60 and the perspective of its application for optimization of doxorubicin efficiency against leukemic cells. comperative investigation of protective effects of tea and tea-related wastes on reducing potaential of h2o2-induced erythrocytes tea processing waste (tpw) formed during the tea production process in tea factories is up to 50,000 tones/year in turkey. tpw is one of the abundant available phenolic biomass among plantal wastes. in this study, black and green teas and their wastes were used. the aim of the study is to determinate the phenolic content and the radical scavenging activities of the samples, and to measure their effects on hydrogen peroxide-induced erythrocyte damage due to analyzing the reducing potential of erythrocyte involving glutathione reductase (gr), glutathione peroxidase (gpx) activities and reduced glutathione (gsh) content. total polyphenol content of samples was determined as mg catechine per dry mass by using folin-ciocalteau reactive and dpph radical scavenging activity was estimated by cuendet method as equivalent catechine standard. in erythrocyte, gsh level was measured by method of sedlak and lindsay while gr and gpx activities were assayed by the methods of bergmeyer and beutler, respectively. the highest phenolic content was observed in green tea and its wastes (p < 0.01) whereas black fiber waste had the lowest phenolic content. therefore, the highest radical scavenging activity and gsh level were detected in green tea and its wastes (p < 0.01). erythrocyte with the extracts of the teas and their wastes had the similar enzyme activities for both gpx and gr. in sum, the teas and wastes have antioxidant activity but, green tea and its leaf waste hade higher antioxidant activity than other samples. the tea wastes might be evaluated as many of protective health products, particularly in cosmetic fields thus, these by-products no application for any area is expected to become an economical value. fluorouracil (5-fu) is a chemotherapeutic drug classified as an "antimetabolite". it works through irreversible inhibition of thymidylate synthase. chemical derivatization of 5-fu with carbohydrtates is being investigated widely in order to enhance its bioavailability, therapeutic efficiency and to reduce its toxicity. however, water solubility of the newly derived compounds is usually very low. so, in order to obtain a pharmaceutically relevant formulation they need to be formulated appropriately. in this study, we prepared micellar delivery system for the new tetra-o-acetylglycose derivative of 5-fu synthesized via "click reaction", namely f1-[{1 0 -(2″,3″,4″,6″-tetra-o-acetyl-b-dglycopyronosyl)-1 0 h-1 0 ,2 0 ,3 0 -triazole-4 0 -yl}methyl]5-fluorouracil. since the water solubility of this compound is very limited, we tested its solubility in several pharmaceutically relevant solvents by visual estimation after stiring increasing amount of the compound in 1 ml of solvent for 48 h. to estimate the carcinogenic potential of this compound, salmonella/microsome mutagenicity assay (ames test) was performed in four histidine-requiring strains of s. typhimurium, tester strains ta98, ta 1537 (for the detection of frameshift mutations) ta100 and ta 1535 (for detection of base pair substitutions) according to the oecd guideline 471. the drug was solubilized (500 lg/ml) with no precipitation in lutrol ò -f68/ethanol/water (2.25:2.25:5.5 , wt/wt) micelles (7.3 ae 1.0 nm). the results of ames test were negative so the compound neither produced frame shift mutations nor base pair mutations in s. typhimurium strains. the results imply that the new compound can be dissolved in aqueous micellar delivery system in order to be used for further studies, and that it was not mutagenic in the tested s. typhimurium strains. in conclusion, the formulation of the newly synthesized compound is not carcinogenic, and can be evaluated for anticancer activity in vitro and in vivo. integral metabolism parameters of dairy goats during reproductive cycle periods d. solovyeva, e. zarudnaya, s. zaitsev moscow savmb, moscow, russia study of the goat metabolism at different periods of the reproductive cycle allows to correct feeding ration, to increase the age of the productive use of animals and to receive high-quality products. the aim of the work was to determine the metabolic parameters of blood serum of goats, expressed in terms of biochemical parameters and interfacial tensiometry and study their relationship to metabolic processes in the body goats depending on the age and the period of the reproductive cycle. the 90 healthy goats were divided into 5 groups. the dynamic surface tension (dst) parameters were obtained from dependences of a surface tension (r) vs. time (t): at t?0 (r 0 ), at t = 0.02 s (r 1 ), t = 1 s (r 2 ) and t?∞ (r 3 ). this work was supported by the russian scientific foundation (14-16-00046). all animals had 4-5% fat content. the contents of total protein (5.4%), albumin (8.4%) and urea (8.8%) are higher for the lactating animals as compared to the normal goat values. the levels of total cholesterol (18.0%) and creatinine (6.2%) are higher for the lactating animals. in lactating animals have the highest level of, which along with high phosphorus level talks about the intensification of energy processes during lactation. the correlations were found between the biochemical and dst parameters of the goat blood: lipids or cholesterol levels with r 0 (r = à0.40), r 1 (r = à0.72), r 2 (r = à0.89); total protein or albumin levels with r 1 (r = à0.42), r 2 (r = à0.56), r 3 (r = à0.90); aminotransferase activity with r 2 (r = à0.47), r 3 (r = à0.63). the correlations were found between the total protein and albumin levels with k 0 (r = 0.54), k 1 (r = 0.44); glucose levels and r 1 (r = 0.47), r 2 (r = 0.43). thus, the dst and biochemical parameters of goats have strong correlation relationships that are important for biomedical and veterinary applications. the relation of the severity of atherosclerotic disease with oxidative stress in patients with stable coronary artery disease h. sezen harran university, sanliurfa, turkey introduction: because, to the best of our knowledge, the relationship of total oxidant status (tos) and total antioxidant status (tas) with the severity of stable coronary artery disease (cad) has not been investigated in the literature so far, the present study was conducted to address this issue. materials and methods: this study consisted of 349 consecutive patients and controls who underwent coronary angiography. for each patient, the total gensiniscore (gs) was calculated andthose with a gs of >20 were classified as the high gs group (hgg), and those with a gs less than 20 were defined as the low gs group (lgg). the total oxidant status (tos) and total antioxidant status (tas) levels were measured using the erelmethod. the osi, which is an indicator of the oxidative balance, was calculated as the percentage ratio of tos to tas. results: the tas was lower in the hgg than lgg. the tos and osi were higher in the hgg than lgg. the correlation analysis showed that gs was negatively associated with the tas and positively with the tos and the osi. the multivariate analysis showed that age, tos, and hdl-c were independent variables for a high gs. the cut-off level of 10.9 lmol h 2 o 2 equiv./ l for serum tos levels predicted high gs with a sensitivity of 70% and a specificity of 56%. discussion: information on the severity of atherosclerosis is requiredtopredicttheprognosis of an individualpatientandtodetermine the proper treatment modality. the gs system has beenproventodemonstratethe severity of atherosclerotic disease. inthepresentstudy, thepatientswith a high gs had increasedlevels of oxidants. inaddition, tos was an independentindicator of theseverity of atherosclerosis. the optimal cut-offvaluefor tos topredict high-gens score was 10.9 (sensitivity 70% and specificity 56%). conclusions: the results suggest that the severity of atherosclerosis in stable cad is associated with increased oxidative status. evaluation of roemerine as a multidrug resistance pump inhibitor f. g. avci 1 , c. velioglu 1 , e. recber 1 , c. unsal 2 , g. gulsoy 2 , b. sariyar akbulut 1 1 marmara university, istanbul, turkey, 2 istanbul university, istanbul, turkey efflux by multidrug resistance (mdr) pumps is a common defense mechanism used against antimicrobials. by pumping the drugs out, these pumps significantly reduce the efficacy of drugs. one approach to overcome this limitation is offered by the combinatorial therapies where drugs are co-administered with together with pump inhibitors. by simply preventing the efflux of the drug, the presence of inhibitors enhance drug efficacy. (-)-roemerine is an aporphine type alkaloid with significant antibacterial (against bacillus cereus, escherichia coli) and antifungal (against candida strains) activities. interestingly, (-)-roemerine was also found to enhance the cytotoxic response mediated by vinblastine in multidrug-resistant kb-v1 cells. in the same study, this finding was linked to its possible interaction with p-glycoprotein, a eukaryotic mdr pump. taking this finding as the starting point, the current study investigates the potential of roemerine as an inhibitor of the p-glycoprotein homologue pump, bmra, in bacillus subtilis 168. the antimicrobial agent berberine was used as the model agent since its efficacy is reduced by efflux through mdr pumps. to this end, bacillus subtilis 168 cells were subjected to 100 lg/ml berberine, a value well below the mic. this concentration only slightly retarded growth for 2 hours but then cells resumed their regular growth. upon addition of 25 lg/ml (-)-roemerine to the bacillus subtilis 168 cells treated with berberine, growth pattern changed, indicating possible interaction with bmra. further investigation for the change in the expression of bmra was achieved with real time pcr analysis. glucose oxidase is an enzyme that catalyzes the oxidation of glucose to d-glucono-1,5-lactone and hydrogen peroxide. we hypothesized that enzyme would cause a double negative effect on cancer cells, by reducing the presence of glucose in cancer microenvironment and producing reactive oxygen species. to increase enzyme stability and enhance cellular uptake we encapsulated the enzyme with a thin acrylamide layer. the purpose of this work was to optimize the synthesis of these glucose oxidase nanoparticles and investigate their effect on cancer cells. nanoparticles containing single glucose oxidase were synthesized in two steps; first by introducing the vinyl groups onto the surface of enzyme by acyloylation followed by polymerization step with acrylamide monomers. encapsulated enzymes are approximately 150 nm in size and retain most of their activity. after the optimization of nanoparticles, the anticancer potency of these nanoparticles was in vitro tested in mcf-7 breast cancer cell line. according to results, both nanoparticles and free enzyme are capable of inhibiting viability of cancer cells in a similar manner at very low concentrations. currently we are investigating mechanisms involved in this viability inhibition. initial results demonstrated that glucose supplement does not rescue cells from death induced by the activity of glucose oxidase, suggesting an oxidative stress related cause of inhibition. further studies are required to elucidate the exact mechanism. until now there is no determined advantage of glucose oxidase encapsulation against proteolysis. however, encapsulation may induce the accumulation of enzyme in cancer microenvironment. furthermore results suggest that glucose oxidase has a high effect on the viability of mcf-7 breast cancer cells indicating that this enzyme may have a potential use in cancer treatment. studies on the interaction of human phospholipid scramblase 1 with c-terminal domain of topoisomerase iia u. sivagnanam, s. n. gummadi applied and industrial microbiology lab, bhupat and jyoti mehta school of biosciences, indian institute of technology madras, chennai, india human phospholipid scramblase 1 (hplscr1) is a multifunctional protein that plays key roles in several cellular processes including apoptosis, tumorigenesis, anti-viral defense, cell signalling and several protein-protein interactions. it has been shown that hplscr1 interacts with the c-terminal domain of topoisomerase iia (topo iia) and enhances its decatenation activity in vitro. the interacting region in topo iia was identified but till date, no reports exist on the binding region in hplscr1. this study aims to identify the region of hplscr1 that interacts with topo iia. to identify the topo iia interacting sites in hplscr1, nterminal deletion constructs of hplscr1 viz δ25-hplscr1, δ50-hplscr1, δ75-hplscr1, δ100-hplscr1 and δ160-hplscr1 were generated by pcr, cloned, overexpressed and purified to homogeneity using ni 2+ -nta purification. the cterminal domain (ctd) of topo iia was cloned in pgex6p-1 and was expressed as a gst fusion protein. gst pull down assays will be performed with the deletion constructs of hplscr1 and the gst-ctd-topo iia. the binding region in hplscr1 will be confirmed by peptide competition assays. our initial results show that the decatenation activity of topo iia was enhanced when the topo ii was pretreated with hplscr1. δ100-hplscr1 did not show any enhancement of the decatenation activity compared to full length hplscr1. hence, the binding region could be in the 1-100 region of hplscr1. further deletions were done in the 1-100 region of hplscr1 as described earlier. gst-pull down assays and decatenation assays will be performed for the deletion constructs to narrow down the region of hplscr1 that binds to topo iia. we conclude that hplscr1 interacts with and enhances the activity of topo iia and the 1-100 region of hplscr1 is critical for enhancement of decatenation activity. further work is under progress to identify the exact topo iia binding region of hplscr1 and the physiological relevance of this interaction in the cell. a. ugur kurtoglu 1 , v. aslan 2 , e. kurtoglu 2 1 department of biochemistry, antalya education and research hospital, antalya, turkey, 2 department of hematology, antalya education and research hospital, antalya, turkey beta-thalassemia is a common autosomal recessive disorder resulting from over 200 different mutations of the beta-globin genes. our aim was to creat a mutation map of beta thalassemia in province of antalya, turkey. in this study, mutation analysis of a total 150 of beta-thalassemia patients followed up at the thalassemia center of the antalya education and research hospital, antalya, turkey, were included. according to our results, the ivs 1.110 is the most frequent mutation type in our province same as other geographical regions of turkey. the most frequent mutations in heterozygous or homozygous patients are ivs 1.110, ivs 1.6, ivs 2.1 and ivs 1.1. our results indicate the importance of micromaping and epidemiology studies of thalassemia, which will assist in establishing the national prevention and control program in turkey. keywords: beta-thalassemia, beta-globin gene, mutation p-mis-052 investigation of the in vitro effects of some antibiotics on the purified beta-glucosidases from the rat liver n. t€ urkmen 1 , h. kara 2 1 karadeniz technical university medical biochemistry department, trabzon, turkey, 2 balikesir university veterinary faculty biochemistry department, balikesir, turkey beta-glucosidases catalyzes the hydrolysis of the glycosidic bonds to terminal non-reducing residues in b-d-glucosides and oligosaccharides.b-glucosidases are widely distributed in the living world.b-glucosidases which in mammals, primarily found in the liver and kidneys;lysosomal b-glucosidase (gba1),non-lysosomal b-glucosidase (gba2), cytosolic b-glucosidase (gba3),intestinal lactase-phloriz the hydrolase (lph). liver tissues of wistar-albino rats were homogenized with homogenizer in the extraction buffer and crude extract was obtained after centrifugation.ammoniumsulfate precipitation range designated crude extract was purified by sepharose 4b-ltyrosine-1-naphthylamine hydrophobic gel.commercially availabled antibiotics were prepared with substrate buffer.it was investigated inhbition effects of cefuroximesodium, ampicillin-sulbactam, amoxicillin trihydrate/potassium clavulanate, cefazolin sodium, gentamicin sulfate and ceftriaxone disodium antibiotics onto gba2.inhibition types and k i values of related antibiotics were determined with p-npg substrate.lineweaver-burk plot was used for that purpose. rat liver gba2 was purified at 30.2-fold with 43.4% yield.gba2 was illustrated 58 and 110 kda at sds-page.ic 50 value of ampicillin/sulbactam antibiotic for gba2 was found 62.97 mg/ml with competitive type inhibition and other antibiotics didn't inhibit. purfication methods are being used in the literature for the purified b-glucosidase from different sources.purified gba2 was illustrated 58 and 110 kda at sds-page.about molecular weight of bglucosidases is presented different information in the literat€ ure. this has been reported because of acid beta glucosidases are abnormal migration at the acrylamide or agarose gels.it was investigated inhbition effects of various antibiotics onto purified gba2.ampicillin/sulbactam antibiotic inhibited to purfied gba2 at the competitive type.similiar antibiotics studies have been made in the literature for different enzymes. effect of glutamine on insulin resistance and endoplasmic reticulum stress g. aydogdu 1 , p. b. sermikli 1 , a. abbasi taghidizaj 2 , e. yilmaz 2 1 ankara university, institute of science, ankara, turkey, 2 ankara university, biotechnology institute, ankara, turkey obesity and diseases are one of the most important public health problems of the world.excess fat storage in adipocytes leads to the release of increased amounts of non-esterified fatty acids, glycerol, hormones, cytokines, which are factors involved in the development of insulin resistance that cause type 2 diabetes. one of the major differences between obese and lean individuals is the amino acid concentration in the circulation. although there are many studies about the amino acid metabolism associated with insulin resistance in obese individuals, the effect of glutamine metabolism in insulin resistance mechanisms are not well understood yet. glutamine can be used as fuel and its levels in tissues and circulation can regulate cell responsiveness to insulin and cellular metabolism. therefore, glutamine is a potentially important factor that might help us better understand insulin resistance and type 2 diabetes. to determine whether glutamine effect on insulin resistance and endoplasmic reticulum stress, 3t3-l1 cell is treated with different concentration of glutamine and analyzed by western blot for er stress markers. our results indicated that glutamine reduced endoplasmic reticulum stress and related with that attenuated insulin resistance. in case of transport of amino acids, insulin resistance, how it is affected when we have the information about the important tips on energy requirements and metabolism reach insulin resistance and type-2 diabetes treatment is likely to reveal a possible new targets. how does different lead levels affect tsh, ft3, ft4, vitamin b12 and folate? e. ozkan ankara occupational disease hospital, ankara, turkey exposure to heavy metals is increasing with the industrialization of society. one of the most intense exposure to heavy metals is pb on this issue. this study was aimed to determine the relationship between different blood pb levels and serum thyroid hormones (th), vit b12, folate. the cases were 20-65 years old, male individuals who admitted to our hospital between april 2012-march 2016 for periodic inspections because of occupational exposure to pb. the parameters of the cases were retrospectively retrieved. according to their pb levels, exposed workers (n: 9400) were divided into four subgroups; group (g) 1: 0-9.99 lg/dl, g 2: 10-19.99, g 3: 20-39.99, g 4: ≥40 . from these, the number of cases whose th levels were measured (n:3894) given respectively; g 1:3062, g 2:178, g 3:334, g 4:275 cases. also the number of cases whose vit b12 and folate levels were measured (n:2807) given respectively; g 1:2139, g 2:143, g 3:276,g 4:249 cases. levels of pb were determined by icp-ms. th, vit b12, folate were determined by cmia. between the groups formed according to pb levels, there was no significant difference in terms of average t3, tsh and vitamin b12 (p > 0.05). on the other hand there was statistically significant difference between t4 and group 1,3,4 (p < 0.05) but there was no difference between group 2 (p > 0.05). the average folate belongs to the first group was about 10% higher than the other 3 groups, and found that the difference was statistically significant (p < 0.05). there are many publications which have various results between pb levels and t3,t4, tsh. but this study is important to compare the effect of different levels of pb. up to day there was no publication about the relation between different pb levels and vit b12, folate. it was seen that there was no significant clinical relation between different pb levels and thyroid parameters, vit b12. but the low levels of folate in the high pb levels groups shows us that we need further studies about this relationship. fluorescent study of in meso crystallization of membrane proteins with the introduction of membrane protein in meso crystallization 30 years ago by landau and rosenbusch, a new era of membrane protein structural research has emerged (1). since that time this method became associated with a number of major breakthroughs in the field (2) including exceptional successes in structural studies of microbial rhodopsins and g-protein coupled receptors (3) . here we used fluorescence microscopy to study in meso crystallization process of bacteriorhodopsin. several observations provide new insights into the in meso crystallization process. the crystallization starts with formation of microcrystals, followed by growth of a dominating crystal at the expense of smaller ones and formation of a depletion zone around it. these observations demonstrate an ostwald ripening mechanism of the in meso crystal growth. the depletion zone formed around the growing crystal is consistent with the previously proposed analogy relating in meso crystallization with the crystallization in a microgravity convection-free environment. this work is supported by rsf 14-14-00995. (1) landau, e. m.; rosenbusch, j. p. proc. natl. acad. sci. u. s. a. 1996, 93, 14532à14535. (2) caffrey, m. acta crystallogr., sect. f: struct. biol. commun. 2015, 71, 3-18. (3) katritch v., cherezov v., stevens r.c. (2013) . annu rev pharmacol toxicol 53, 531-556. p-mis-058 stamp1 is critical for both ar and mtor signaling in prostate cancer cells x. sheng, y. jin, f. saatcioglu university of oslo, oslo, norway androgen receptor (ar) signaling plays a central role in the initiation and progression of prostate cancer (pca), including when the disease progresses to castration-resistant pca (crpc). the second central signaling pathway in pca, similar to various other cancers, is the pi3k-akt-mtor signaling. importantly, these two oncogenic pathways cross-regulate each other in pca cells by reciprocal feedback, thereby maintaining tumor cell survival even when one is suppressed. we have previously identified that the six transmembrane protein of prostate 1 (stamp1) promotes pca cell proliferation as well as inhibits apoptosis through, at least in part, regulating the erk mapk signaling. human pca cell lines lncap and vcap were used in the study. colony formation, soft-agar growth, prostatosphere formation assays were performed. for in vivo xenograft experiment, the cells were implanted subcutaneously into the flanks of nude mice. here, we show that stamp1 knockdown caused defects in colony formation, anchorage-independent growth and prostatosphere formation in lncap and vcap cells both in vitro, as well as tumor formation and growth in vivo. this may be due to the impaired ar and mtor signaling in these cells upon stamp1 knockdown. interestingly, in the crpc cell line 22rv1, where-stamp1 knockdown did not affect mtor signaling, there was a remarkable repression of tumor take rate and growth. these results clearly indicate that stamp1 is essential for both ar and mtor signaling, and is crucial for pca growth in vitro and in vivo. however, the detailed molecular mechanism requires further investigation. taken together, these data unveil a critical role for stamp1 in coordinating the ar and mtor signaling pathways in pca cells, solidifying the basis for its pro-survival effects in pca, including in advanced disease. quantification of 2d thin layer chromatograms using 2d gel analysis software and gel documentation system o. kaynar, m. ileriturk, d. kaynar, s. kurt ataturk university, erzurum, turkey introduction: thin layer chromatography (tlc) is an important chromatographic technique that is widely used as a cost-effective method for rapid-sensitive analysis of compounds in plants, animals, and humans. however, one dimentional (1d) tlc is not sufficient for the separation of complex compounds. therefore, two-dimentional (2d) tlc was developed. the quantitative evaluation of plates are performed with tlc scanners or documentation systems. however, these systems specific for 1d plates, and cannot be adapted to quantitative evaluations of 2d plates. in this study, the applicability of the gel documentation systems and 2d analysis software for the analysis of 2d tlc plates were examined. material and method: 2d tlc of lipids: 1st dimension: methyl acetate/n-propanol/chloroform/methanol/0.25% kcl (25/ 25/28/10/7 v/v); 2nd dimension: chloroform/methanol/acetic acid/ water (90/40/12/2 v/v); detection: charring. 2d tlc of aminoacids: 1st dimension: 1.5% (v/v) formic acid; 2nd dimension: toluene/glacial acetic acid (10:1 v/v); detection: uv. phospholipid and aminoacid standards, each include 6 different classes were developed by 2d tlc. plates visualized with biorad geldoc xr, and band volumes on plates were calculated with biorad pdquest 2d gel analysis software. for the method validationa) 5 plates containing same 6 lipid classes were developed in the same day, and results were used for the calculation of intra-assay cv;cv% = average of each sample standard deviation/mean of sample 9 100 b) 6 plates containing same 6 lipid classes were developed in 6 different days, and results were used for the calculation of interassay cv; cv% = standard deviation of each sample average/mean of the plates 9 100 results: volume of each phospholipid and aminoacid had less than 10% intra and inter-assay cv. conclusion: gel documentation system with 2d gel analysis software can be used for the quantitative analysis of the 2d tl plates both at uv and visible light. the role of na + k + atpase activity in the vasodilatory effect of n-acetylcysteine introduction: spasm occurred at the stage of and after the preparation of arterial grafts used in coronary artery bypass surgery (cabg) is effective on morbidity and mortality in the first 24 hours of postoperative patients. n-acetyl cysteine (nac) that vasodilatory effect is known,may be considered as a suppressor agent for vasospasm developing during cabg. however, for the prevention of complications that may arise during or after cabg mechanism of these vasodilatory effects should be described. this study was aimed to investigate the role of atpase enzyme on the vasodilatory effect of nac. materials and methods: in this study, 28 adult male wistar albino rats were used. rats were separated into four groups as control rats (g1), 2 mm nac (g2), 5 mm nac (g3) and 10 mm nac (g4). a portion of the thoracic aorta isolated from rats was used for the relaxation response recording, and the other portion was used for measurement of nakatpase activity. isolated smooth muscle rings are suspended in the 20 ml organ bath containing krebs solution for relaxation responses. in all groups, level of smooth muscle contraction were allowed to reach a plateau by adding 60 mm kcl to the organ bath. then, in the first 10 minutes of application relaxation responses which created by adding nac to the medium were recorded and the maximum relaxation responses were measured. nak atpase activity was determined using the mazzanti method. groups means were compared by one-way analysis of variance (anova). the threshold for statistical significance was set at .05. results: the contraction force decreased in all nac dose groups compared to control group and this reduction was statistically significant (p < 0.05). similarly, nak atpase activity is also decreased in a dose dependent manner (p < 0.05). the findings obtained in this study suggest that vasodilator effect of nac formed in thoracic aortic smooth muscle was associated with the activity of the enzyme na k atpase. in the presented study, we isolated and characterized a novel feather-degrading bacterium that shows keratinolytic activity. a bacillus uk69, which was isolated from the soil samples taken from farmland on kahramanmaras sutcu imam university campus, showed high keratinolytic activity when cultured on feather meal medium. the enzyme activity was studied in the ph range of 5.0-12.0. the optimum temperature for keratinase activity was investigated by varying the incubation temperature between 20°c and 80°c. optimum keratinolytic activity was observed at 60°c and ph 10.5. the enzyme was stable at 60°c. the activity was investigated in the presence of some chemicals, including sds, tween 80, dmso, triton x-100, edta, nacl, zncl2, cacl2, glucose. the keratinolytic activity was inhibited by all chemicals tested to some degree. the molecular weight of keratinase was determined by polyacrylamide gel (10%) using standard molecular weight marker and estimated about 37 kda by sds-page. the keratinase isolated from bacillus uk69 could be used in biotechnological processes i.e. feather degradation, wastewater treatment and in industrial processes, such as detergent, food and leather industries. introduction: hemoglobinopathies, including thalassemia, abnormal hemoglobins, constitutes a major group of inherited disorders of hemoglobin synthesis. the reduced or absent of the beta (b) or alpha (a) globin chains of the adult human hemoglobin molecule results beta or alpha thalassemias, leading to imbalanced a-globin/non a-globin chains. the aim of this study was to give a quik desicion with a/b-globin mrna ratio for sequencing of a or b gene, when the anemia is not detectable. materials and methods: mrna and cdna extraction of 25 bthalassemia and 15 a-(including two of 3.7 kb del./hbs) thalassemia subjects and normal controls were accomplished using the high pure rna isolation kit and transcriptor first strand cdna synthesis kit, respectively, following the manufacturer's instructions. we used cdna as a template in the real-time pcr amplification using primers specific for a, b globin genes. amplification was performed in a lightcycler ò 480 instrument. the a/b-globin mrna ratio of each sample was calculated based on the 2 àddct method. results: a/b-globin mrna ratios calculated in a-thalassemia subjects relative to normal control as a result of numbers of defective a-globin genes. the a/b-globin mrna ratio was found higher in b-thalassemia subjects. coinheritance of a-thalassemia in hb s subjects concluded a stabile a/b-globin mrna ratio as per a-thalassemia or b-thalassemia subjects. discussion and conclusion: instability in a/b-globin chains is a significant factor of thalassemia disease severity and can be used before deciding type of gene sequencing when the anemia is not detectable. this study indicates that imbalance in globin gene expression could be demonstrated by measuring a/b-globin mrna ratio, which was conveniently and accurately determined by qrt-pcr and give an information about globin gene function which gene should be correct to investigate an individual for globin gene mutation. p-mis-067 self-assembling peptides mimic supramolecular biochirality r. garifullin 1,2 , m. o. guler 1 1 bilkent university unam, institute of materials science and nanotechnology, ankara, turkey, 2 kazan federal university, institute of fundamental medicine and biology, kazan, russia supramolecular chirality is rooted in asymmetric spatial arrangement of structural elements (e.g. molecules or units with higher hierarchy). self-assembled systems giving rise to this kind of chirality are of great importance because they closely resemble natural biological systems and potentially can lead to new advanced functional materials. in the process of self-assembly, both molecularly chiral and achiral structural units can organize into chiral nanostructures. chiral arrangement of chromophore molecules in space is known to result in emergence of chiroptical properties of a chromophore. organization of pigment-protein complexes into macrodomains in green plants gives rise to biochirality emanating from long-range chiral order of complexes. owing to this order, macrodomains start to absorb circularly polarized light intensively and thus exhibit huge circular dichroism (cd) signal. in our study, a simple approach which was aimed at mimicking the biochirality phenomenon makes use of self-assembling peptide amphiphiles and their interactions with pyrene chromophore. designed peptide amphiphiles are capable of self-assembly into nanofibers with chiral interior, which in principle gives an opportunity to achieve long-range chiral order. two modes of interactioncovalent and noncovalentwere utilized in order to induce supramolecular chirality. covalent interaction mode included direct covalent attachment of pyrene to peptide sequence. upon self-assembly of peptide amphiphile into nanofibers intense circular dichroism phenomenon was observed. noncovalent interaction mode envisioned encapsulation of pyrene molecules in the hydrophobic core of nanofibers of another peptide amphiphile. co-assembly of peptide amphiphile and pyrene molecules led to chiral order and intense cd signal. in addition, it was possible to control the sign of cd signals by using either of peptide isomers, l or d. p-mis-068 pon1 activity in hdl subgroups of obese, overweight and normal weight subjects objective: the aims of this study were isolation of hdl-c subgroups by using precipitation method, determination of pon-1 activity in both total and hdl3 subgroups, and evaluation of performance characteristics of pon-1 activity measurement method in newly diagnosed obese, overweight and normal subjects. material and methods: the study population consists of newly diagnosed 71 obese, 40 overweight and 30 normal subjects. fasting morning blood samples were taken from all study groups. hdl3 subgroup was obtained by heparin-mn-dextran sulphate precipitation method and cholesterol was measured with direct (homegenous) hdl-c method. hdl2-c concentrations were calculated with the subtraction of hdl3-c from total hdl-c. hdl3-c and total pon-1 activity were determined by using eckerson method. non-hdl3 pon-1 activitiy was calculated with subtraction of hdl3 pon-1 activity from total pon-1 activity. results: total hdl-c, hdl2-c and hdl3-c concentrations and the activity of total pon-1 and hdl3 pon-1 were found lower in obesity according to overweight and normal subjects (p < 0.001). negative correlations were found between body mass index and hdl3-c, total pon-1 and hdl3 pon-1 (r = à0.244, p < 0.005; r = à0.247, p < 0.005; r = à0.199, p < 0.05, respectively). conclusion: our findings indicated that hdl-c metabolism and lipoprotein associated antioxidant defense mechanisms were adversely affected with obesity. in conclusion we think that precipitation method using for separating hdl3 subgroup, is simple and cost effective for routine applications in clinical laboratories. besides hdl3-c measurements, pon1 activity, measurement of total and hdl3-c subgroup might be helpful to evaluate the atherosclerotic process in obese subjects. keywords: obesity, body mass index, paraoxonase, hdl subgroup, cholesterol p-mis-069 hepatitis e virus antibody prevelance among persons who work with animals in north cyprus introductions: hepatitis e infection is a major cause of viral hepatitis in many developing countries. the objectives of the present study was to determine the seroprevelance of hev infection in peoples who work with animals in northern cyprus. materials and methods: prevelance of hev infection were determined in study 4 group population: persons without occupational exposure to animals; persons who work with animals; veterinarian and butcher. a total of 400 blood samples were collected. all serum samples were tested elisa using a commercially available kit according to the manufacturer's instructions. ti-test were used for istatistical analyses. p > 0.05 was accepted as significant value. results: in a study of 400 blood donors (334 male, 66 female), the overall prevelance of anti-hev igg antibodies were 3.0%. the blood samples were collected 5 different areas. the prevelance of anti-hev igm antibodies was 0.25% and he was 44 years and acting a butcher during 20 years. the prevelance of anti-hev igg of women were approximately two fold higher than men. no significant difference in anti-hev prevelance was observed between the age of the blood donors. according to the anti-hev igg prevelance, the without occupation expose to animal animal were 1%, the animal husbandry were 7% and the veterians and the butcher were 2% were found. discussion: the prevelance of anti-hev in the north cyprus (3%) was found low such as the prevelance of the turkey (5%). the prevelance of anti-hev igg in animal husbandry were higher that the other groups because of they may be more spend of time and contact with animals. the prevelance of igm results suggested that the possibility of outbreaks may be low in north cyprus. conclusion: this study was the first seroprevelance analysis of north cyprus according to the population number.the further studies could be included the seroprevelance of anti-hev from the animals. most errors in the clinical laboratory occur in the preanalytical phase the aim of this study was to investigate the causes and rates of rejected samples, regarding to certain test groups in our laboratory. this study was designed on the rejected samples between january 2015 and january 2016. clinical chemistry, coagulation, hormone, cardiac markers, total urine evaluation and other (ethanol level, hba1c, hb electrophoresis, neonatal bilirubin, drug level, blood gas, fecal occult blood) test groups were included. the total number of specimen and rejected samples was obtained from the hospital information system retrospectively. types of inappropriateness were evaluated as follows: erroneous coding, clotted specimen, hemolysis, insufficient volume, incorrect patient, incorrect tube and inappropriate specimen. it was determined that 873343 blood samples were sent to our laboratory in one-year period. 0.37% of them were rejected because of preanalytical errors. erroneous coding was found as the most common rejection cause (33%). rejection rates of clotted specimen, hemolysis, insufficient volume, incorrect patient, incorrect tube and inappropriate specimen were found to be 11%, 15%, 19%, 2%, 4% and 16% respectively. in our study, erroneous coding was the most common cause of preanalytical errors. education of medical secretaries is relevant and important as can be seen in the decrease of sample errors and the resulting quality improvement. glycosylated hemoglobin test (hba1c) is important for screening, diagnosing, and monitoring diabetes and prediabetes. however, hba1c levels may dependent on patient ethnicity suggesting that the diagnostic cut-offs should be evaluated for specific populations. therefore, our aim in this study was to evaluate the efficiency of hba1c for predicting diabetes in comparison to oral glucose tolerance test (ogtt) results for turkish population. the study included anonymous lab results (acibadem labmed laboratories in turkey) of 6270 patients (3913 female, 2351 male) aging 40.4 ae 11.6 years (15-86) who had an initial diagnosis of diabetes. glucose and insulin levels during ogtt were measured after the initial administration of 75 g sugar (0hour), 1-hour and 2-hour. these parameters were statistically analyzed in comparison to simultaneous hba1c results. glucose measurements at 1 hour had better distinction power (p < 0.05) between these individual groups than initial and 2-hour glucose measurements. the average hba1c (%) levels for healthy, pre-diabetic and diabetic individuals were 5.4 ae 0.4, 5.7 ae 0.4 and 6.2 ae 0.7, respectively. roc curve analysis showed 23.4% sensitivity and 98.2% specificity for the clinically accepted hba1c cut-off value of 6.5%. hba1c cut-off value of 5.9% had a higher sensitivity of 67.8% and comparable specificity of 85.7%. the highest discrimination power between healthy, pre-diabetic and diabetic individuals was observed at glucose concentration at 1-hour after sugar administration in ogtt test as opposed 2-hours generally used for diagnosis. low sensitivity was observed for the clinically adapted 6.5% cut-off value of hba1c. the cut-off value of 5.9% for hba1c was found to be more sensitive with comparable specificity than the 6.5% cut-off values for diabetes screening in our population. our results suggest that 5.9% for hba1c should be considered for diabetes cutoff value for turkish population. induction of the glutathione-dependent detoxification capacity is involved in the hepatoprotective effect of silymarin against acetaminophen-induced hepatotoxicity y. kim, d. kwon, c. ahn seoul national university, seoul, south korea recent findings in this laboratory showed that silymarin was capable of promoting hepatic glutathione (gsh) synthesis via a modification of the transsulfuration reactions in the liver. to investigate its pharmacological significance, we examined the hepatoprotective effect of silymarin against liver injury induced by acetaminophen (apap). adult male mice were treated with silymarin (200 mg/kg, po) every 12 hours for a total of 3 doses prior to an apap challenge (500 mg/kg, ip). the apap-induced liver injury was assessed by histopathological examination and measurement of changes in plasma enzyme activities, lipid peroxidation and formation of nitrotyrosine protein adducts in the liver. plasma levels of apap and its major metabolites were monitored for 24 hours to estimate the metabolic transformation of apap. also protein and activity of the major cyp subtypes involved in the metabolic activation of apap into a toxic metabolite were determined in liver of the mice treated with silymarin only. silymarin pretreatment attenuated the apap-induced liver injury significantly when determined 24 hours later. plasma concentrations of apap, apap-glucuronide or apap-sulfate in plasma were not changed, but thiol conjugates of apap, such as apap-glutathione, apap-cysteine and apap-n-acetylcysteine, were elevated significantly in the mice pretreated with silymarin. however, silymarin treatment did not affect protein expression of cyp2e1, cyp1a2, or cyp3a11 in the liver. also hepatic microsomal enzyme activities measured using p-nitrophenol, ethoxyresorufin and erythromycin as substrates, were not increased by silymarin, indicating that the elevation of apap-thiol conjugates should be attributed to an augmentation of the gsh conjugation capacity. it is suggested that silymarin may protect the liver against an electrophilic substance-induced toxicity by increasing gsh availability which would enhance the detoxifying capacity of liver cells. prostate cancer (pca) is the second leading cause of death among men in western countries. we have previously found that the six transmembrane protein of prostate 1 (stamp1) promotes pca cell proliferation as well as inhibits apoptosis through, at least in part, regulating the erk/mapk signaling. we also found that stamp1 is highly mobile in pca cells and shuttles between the plasma membrane and the golgi, often found in vesiculotubular structures in the cytosol. using advanced imaging techniques, we have now characterized the trafficking of stamp1 from the plasma membrane to early endosomes in lncap cells, by analysing its dynamic targeting to the three main endocytosis pathways: clathrin-mediated endocytosis, caveolae/lipid rafts, and the arf6-dependent pathway. we found that stamp1 fused to cyan fluorescent protein (cfp-stamp1) is present at the plasma membrane where it accumulates in punctate structures. live cell confocal imaging showed that these puncta were dynamic over time indicating that stamp1 may be constitutively delivered to the plasma membrane and removed from it by endocytosis. co-expression of cfp-stamp1 with various fluorescent protein markers revealed that cfp-stamp1 puncta corresponded to lipid rafts that were labelled with caveolin-1-rfp or antibodies against flotillin. live cell imaging showed that cfp-stamp1 and caveolin-1-rfp disappeared at the same time from the same region of the plasma membrane suggesting that lipid rafts are likely to be responsible for stamp1 internalization. notably, stamp1 was absent from other endocytosis structures such as clathrin-coated pits/vesicles. further work is needed to determine whether stamp1 internalization is required for its function, such as its link to erk signaling, and whether interference with lipid rafts influences stamp1 effects on pca cell proliferation and survival. antithrombin-iii, mpv and plasma total homocysteine levels in behcet's disease introduction: behcet's disease is a multi-systemic and chronic inflammatory vasculitis of unknown etiology characterized by recurrent oral and genital ulcers, uveitis, arthritis, arterial aneurysms, venous thrombosis and skin lesions. platelet indices such as mean platelet volume (mpv) is a standart indicator of platelet function in disease pathophysiology. antithrombin, a glycoprotein synthesized in the liver, is the major plasma inhibitor of thrombin thus modulating blood coagulation. antithrombi-iii (at-iii) is a enzyme even moderate deficiency significantly increases the risk of thrombosis. homocysteine (hcy), that is formed during the metabolism of methionine. several clinical studies have clarified that elevated blood hcy levels are related to atherosclerotic disease. in our study, we investigated ovocystatin is one of the best characterized members of cystatin superfamily of protease inhibitors, and it has been frequently used for pathophysiological studies as the model protein, representative for this superfamily. its application has been supported by high structural similarity to human cystatin c as well as several common biological activities. as regard to biological activity, cystatins, including ovocystatin, are best characterized as inhibitors of cysteine proteases of papain family (c1), such as cathepsins b, h, l and s. these inhibitors participate in intra-and extracellular control of proteolytic events, both in physiological and pathological states. in the recent decade also new activities of cystatins, not assigned to inhibition of papain-like cysteine cathepsins, were found. these activities are associated with an alternative active center for legumain-type proteases in the molecule. here we report a chemical modification of ovocystatin that disables the anti-papain activity of the inhibitor but does not affect its anti-legumain activity. the chemical knockout has been obtained by reaction with 2-hydroxy-5-nitrobenzyl bromide (hnbb) that covalently modifies the trp104 residue in the molecule. the reaction has been monitored by uv-vis and fluorescence spectroscopy. the anti-papain activity of the inhibitor has been measured colorimetrically against bana as a substrate. the anti-legumain activity was assessed fluorometrically using z-ala-ala-asn-amc. the reacted inhibitor exhibited an additional, characteristic for hnbb, band at 410 nm in uv-vis scan. accordingly, an ablation of trp fluorescence was also observed. the molecule fully retained the anti-legumain activity, while only residual antipapain activity (10%) was observed. the modified ovocystatin can be a useful molecular tool for studying the physiological and pathological processes specifically associated with legumain activity. 3 departments of medicine (hematology/oncology) and biochemistry and molecular biology, university of louisville, james graham brown cancer center, louisville, ky, united states 6-phosphofructo-2-kinase/fructose-2,6-bisphospatase (pfkfb) family of enzymes are responsible for the conversion of fructose-6-phosphate (f6p) to fructose-2,6-bisphosphate (f2,6bp) and vice versa, and f2,6bp is an allosteric activator of phosphofructokinase-1 (pfk1), a rate-limiting enzyme of glycolysis. among the four identified pfkfb isozymes (pfkfb1-4), pfkfb2 is the least studied isozyme in human cancers. there exists two different splice variants of pfkfb2, variant-1 and variant-2, coding two different isoforms, isoform a and b, respectively. in this study, we first analyzed the effect of k-ras(g12d)induced oncogenic transformation on pfkfb2 expression in pancreatic duct cells. we found that oncogenic k-ras induction in immortalized pancreatic duct cells (ipde) was associated with decreases in total pfkfb2 mrna and protein expressions (mrna; ipde: 1 ae 0.15; ipde+kras: 0.78 ae 0.24 and protein; ipde: 1 ipde+kras: 0.55). we then, checked individual expressions of splice variants and observed that while pfkfb2 splice variant-1 (p2-v1) expression was reduced by k-ras induction (ipde:100; ipde+kras:81.50), pfkfb2 splice variant-2 (p2-v2) expression was increased (ipde:100; ipde+kras:125.70). then, we checked effects of p2-v1 and p2-v2 on glycolytic phenotype of ipde and ipde+kras cells. over-expression of pfkfb2 variants increased f2,6bp concentration (p2-v1: 1.96; p2-v2: 1.72 fold; compared to empty vec), glucose uptake (p2-v1: 16%; p2-v2: 30%) and glycolysis (p2-v1: 20%; p2-v2: 30%) in ipde+k-ras cells. we next analyzed the subcellular localizations of pfkfb2 isoforms and observed that both pfkfb2 isozymes localize to the nucleus, with more prominent nuclear localization of p2-v1 compared to p2-v2. also, nuclear localization ratio of p2-v2 increases after oncogenic transformation with mutant k-ras. taken together, these results suggest that pfkfb2 may have a role in the glycolytic phenotype of pancreatic cancers characterized with hyperactive k-ras signaling. effects of p38 map kinase inhibitors on mda-mb-231 cell line introduction: p38 mapk phosphorylates serine and/or threonine residues of the target proteins. the activation of p38 mapk leads to cell growth, differentiation, survival or apoptosis. in this study, we tested the effect p38 mapk sb203580 and sb202190 on mda-mb-231 cells to further elucidate the controversial role of p38 mapk on cell proliferation or cell migration. materials and methods: mda-mb-231 cancer line was cultured in rpmi-1640 supplemented with 10% fbs. the cytotoxic and cell migration effects of sb203580 and sb202190 inhibitors were tested by mtt assay and wound assay, respectively. the effects of both inhibitors on proliferation and adhesion of md-mb-231 cells were determined by icelligence system. results: it was found that sb202190 p38 map kinase inhibitor was more effective than sb203580. however, no significant effects of low doses of 1 lm and 5 lm of both inhibitors were seen on cell proliferation as compared to the dmso-treated control cells for up to 96 hours as determined by icelligence system. on the other hand, both sb203580 and sb202190 significantly prevented cell proliferation at a concentration of 50 lm. both sb203580 and sb202190 significantly reduced cell migration in a time-dependent manner at a concentration of 50 lm. then, we tested whether each p38 mapk inhibitors have any effect on cell adhesion during a treatment period of 3 hours using icelligence system. only 50 lm concentration of sb202190 reduced cell adhesion for about 1.5 hour (p < 0.001). conclusion: p38 mapk inhibitors sb203580 and sb202190 differentially affect cell proliferation, survival and migration. acknowledgements: this study is financially supported by dumlupınar university, scientific research project no 2015-85. mutagenicity of a series efficacious benzoxazine derivativesa new approach to evaluate ames test data e. foto 1 , f. zilifdar 1 , s. yilmaz 2 , t. sarac ßbasi 1 , i. yalc ßin 2 , n. diril 1 1 hacettepe university, ankara, turkey, 2 ankara university, ankara, turkey testing safety of drug candidates is as crucial as evaluating their efficacy in early drug development. we previously synthesized a series of 1,4-benzoxazine-3-one derivatives showing significant antimicrobial, in vitro anticancer, topoisomerase i inhibitory activities and studied their several mechanisms of action. in this present study, we have evaluated mutagenic activities of these compounds and their potential metabolites. moreover, we aimed to develop a new statistical algorithm available for structureactivity relationship analysis to identify the regions responsible for the activity. to evaluate mutagenicity of the compounds, ames salmonella/microsome test was used. salmonella typhimurium ta98 and ta100 strains were used to detect for frameshift and basepair substitution mutagens, respectively. additionally, mutagenicity of potential metabolites of them were evaluated by adding metabolic activation system (s9) which was prepared from a pool of male sprague dawley rats. results were evaluated with student's-t test. following regression model estimation analysis, we detected minimum mutagenic doses of all tested compounds for generating a 3d-common features pharmacophore model with hiphop method. according to the results, only bs12, bs13, bs16 and bs17 exhibited strong mutagenic effects on both strains in the presence and absence of s9. additionally bs10, bs7, bs1 and bs15 (in the absence of the s9), bs18, bs4 and bs7 (in the presence of the s9) showed weak mutagenic effects on ta98. hiphop analysis results revealed that mutagenicity was increased in the presence of aromatic desactivating groups which might form hydrogen bonds at the position of r3 and hydrophobic groups at the position of r2 of the benzene ring in the structure of benzoxazine. the new statistical approach developed in this study can be useful for assessing the ames test data available for structure activity relationship analyses. background: recently more than thirty different diseases can screen simultaneously with expanded newborn screening (nbs) programs by tandem ms.expanded nbs with tandem ms is performed routinely at akdeniz university hospital central laboratory since 2008.the aim of this study was to evaluate our nbs results with some second-tier and confirmatory tests. materials and methods: nbs results (n = 1863) were evaluated in dried blood samples which sent to our laboratory for the study between august 2014 and august 2015. electrospray ionisation (esi)triple quadrupole mass spectrometer (shimadzu lc-ms/ms 8030,japan) was used for nbs analysis,acylcarnitine and amino acid profile were screened with mrm (multiple reaction monitoring) spectrum within 2 minutes.second-tier tests were performed as urine organic acid analysis by gas chromatography-mass spectrometry (gc-ms),plasma and urine quantitative amino acid analysis by high pressure liquid chromatography (hplc).pathological nbs results were assessed in three separate groups as amino acid metabolism disorders, fatty acid oxidation defects and organic acidemias. results: metabolic diseases were found in 69 (3.70%) patients by the second-tier tests performed.there were detected amino acid metabolism disorders in 13,organic acidemia in 42,fatty acid oxidation defects in 14 patients. conclusions: the reason of high positive results in our laboratory could explain that our study includes both screening and monitoring of previously diagnosed metabolic patients.nbs is performed in only a few centers in turkey although there were the national screening programs included nbs in many foreign countries.more expanded nbs programmes in our country is required to start treatment of patients before irreversible damage is not occured. although many reports indicate the involvement of calpain in several human pathologies, it is not yet clarified how the protease can recognize the substrates to digest and how can escape to its natural inhibitor calpastatin. answers to these questions have been obtained by identifying specific intracellular localizations of calpain and its substrates and analyzing the interactions of the protease with calpastatin. these studies were carried out using human sknbe neuroblastoma cells. protein-protein interactions and intracellular localization of calpain and the related proteins were determined by immunoprecipitation and isolation of membrane microdomains. we have observed that small amounts of calpain-1 are localized in lipid rafts microdomains together with n-methyl-d-aspartate receptor (nmdar) containing nr1/nr2b subunits. immunoprecipitation experiments have demonstrated that nmdar containing nr1/nr2b subunits, calpain-1, hsp90 and neuronal nitric oxide synthase (nnos) but not calpastatin and calpain-2 are present in specific protein complexes. thus, in this localization calpain activity is regulated by hsp90 that reduces the affinity for ca 2+ of the protease. cell stimulation with nmdar agonists induces calpain activation that specifically cleaves the subunits nr2b of the receptor promoting changes in lipid rafts organization and internalization of nmdar without affecting cell viability. moreover, in these conditions, also nnos is digested and converted in the active form by calpain-1. our data suggest a physiological role of calpain-1 at specific cell sites. the protease inserted in lipid rafts microdomains is in strict contact with its targets and escapes to calpastatin which is not inserted in these structures. following an increase in ca 2+ influx, the activated protease regulated by hsp90, promotes the removal of nmdar from the plasma membranes, decreasing ca 2+ entrance through this receptor-channel and protecting cells from ca 2+ overloading. tissue transglutaminase 2 (tg2) is a multifunctional protein complex that can act as a crosslinking enzyme, gtpase/atpase, protein kinase and protein disulfide isomerase. at the cell surface, tg2 was shown to be involved in adhesion, migration, invasion, growth, epithelial mesenchymal transition and hence implied in the metastatic development of many different tumor types. renal cell cancer (rcc) is one of the most common type of cancer in adult males that generally grows as a single tumor within a kidney. our previous findings indicate that the increased expression of tg2 in rcc results in tumor metastasis with a significant decrease in disease-and cancer-specific survival outcome. herewith, the role of tg2 in cell migration of rcc was investigated in this study by transducing the model rcc mouse cell line renca with a series of tg2 mutant constructs. renca cells were transduced by lentiviral particles encoding wttg2, transaminase-defective tg2-c277s form with low gtpbinding affinity, gtp-binding deficient form tg2-r580a, and transaminase-inactive tg2-w241a. in order to investigate the role of tg2 transamidating and gtpase activity in cell migration, scattering assay was used where 7 colonies for each mutant clone was followed for a time interval of 24 hours. our results showed that non-transduced control and tg2-c277s mutant renca cells demonstrated a similar migration pattern with a 20% of scatter activity. on the other hand, 52% colonies formed by renca cells overexpressing wttg2 and tg2-w241a mutant scattered away from each other. a small insignificant increase in scattering was seen in 28% of the total number of 10 colonies for renca cells overexpressing tg-r580a construct. data from this study supports that gtp-binding activity of tg2 is the drive force in migration driven scattering of renca cells, suggesting that inhibitors targeting the gtp-binding activity of tg2 may serve as a new therapeutic approach in the treatment of rcc. background: in this study, we aimed to investigate the relationship between level of vitamin d with subclinical hypothyroidism and subclinical hyperthyroidism. material and metod: study groups planned as three groups such as euthyroid (n = 11966), subclinical hypothyroid (n = 1040), subclinical hyperthyroid (n = 795). serum tsh, free t4 (ft4) and free t3 (ft3) levels were determined by chemiluminescence immunoassay and serum 25-hydroxy (oh) vitamin d 25 (oh) d level were determined by liquid chromatography-tandem mass spectrometry. euthyroidism was defined as a normal level of tsh (range, 0.4 to 4.2 miu/l), ft4 (range, 0.8 to 2.3 ng/dl) and ft3 (range, 2.3 to 4.2 ng/dl). subclinical hypothyroidism is defined as an elevated serum tsh level associated with normal total or free t4 and t3 levels. subclinical hyperthyroidism is defined as low serum tsh levels associated with normal free t4 and free t3 levels. results: subclinical hyperthyroid group had significantly higher 25 (oh) vitamin d levels compared to the euthyroid and subclinical hypothyroid groups (p < 0.05). 25 (oh) vitamin d levels in subclinical hypothyroid group was not statistically significant when compared with the euthyroid group. food processing wastes provide carbon sources in high amounts for fermentative microorganisms to produce energy. converting carbon-rich biomass into bioethanol through fermentation by microorganisms both provides energy requirement for humankind and also decrease pollutant gases like co 2 , no x and so x (ghorbani et al., 2011) . fermentation processes for bio-ethanol production could be achieved by saccharomyces cerevisiae, zymomonas mobilis, and escherichia coli. bacterial hemoglobin (vitreoscilla hemoglobin, vhb) is the first and best characterized prokaryotic hemoglobin molecule. the function of vhb is supporting the cellular respiration through binding to oxygen at microaerobic environment, transferring it to the terminal respiration oxidases (geckil et al., 2004) and thus improving growth and productivity of the microorganisms. in this study, e.coli strains fbr5, ts3 and ts4 were used as ethanologenic microorganisms. expression of vhb in ts3 is lower than in ts4 strain. for the efficient ethanol production effect of different inoculum sizes, sugar species and sugar concentrations in the growth medium were investigated. vhb expression increased effectively the viability of ts3 strain by up to 1.8x10 9 cfu per ml of fructose (3%, w/v) supplemented lb medium starting with small inoculum for fermentation. this indicates that vgb expression should be at the certain level to maintain sufficient the cell growth for ethanol production. geckil h, gencer s (2004) . production of l-asparaginase in enterobacter aerogenes expressing vitreoscilla hemoglobin for efficient oxygen uptake. applied microbiology and biotechnology 63: 691-697. ghorbani, f., younesi, h., sari, a. e., najafpour, g. (2011) . cane molasses fermentation for continuous ethanol production in an immobilized cells reactor by saccharomyces cerevisiae. ethanol production from dairy industry by product using bacterial hemoglobin t. sar, g. seker, a. g. erman, m. yesilcimen akbas gebze technical university, depertment of molecular biology and genetics, kocaeli, turkey bioethanol production from biomass has a great potential to reduce greenhouse gases emissions. ethanol has several applications in industries (chemical, medical, pharmaceutical, food etc.) in the form of raw material, solvent and fuel. one of the most abundant liquid wastes is cheese whey generated from dairy industries. whey powder is concentrated form of whey and contains lactose and also protein, lipid, minerals and vitamins. vitreoscilla hemoglobin (vhb) is the first bacterial hemoglobin. the main function of this molecule is to improve oxygen transfer to cellular oxidases and thus supporting cellular growth and productivity at low oxygen levels (kallio et al. 1994) . in this work, e. coli strains fbr5, ts3 (low level vhb expressing) and ts4 (high level vhb expressing) were used as ethanol producing microorganisms. fermentation medium containing whey powder supplemented with lb material was inoculated with these strains and incubated for 48 hours at 37°c and 180 rpm in a 1000 ml erlenmayer flask. the ethanol production was improved over 300% by using lower vhb expressing strain. the ethanol levels (v/v, %) were determined as 1.08, 4.99 and 1.51 for fbr5, ts3 and ts4 strains respectively. it is shown that the certain levels of vhb could be useful tool to increase the growth and productivity of ethanol from dairy industry wastes. kallio p.t., kim d.j., tsai p.s. and bailey j.e. (1994) . bioethanol is usually produced from cellulose, hemicellulose and lignin. the lignocellulosic wastes should be hydrolysed into fermentable sugars by using enzymes or dilute acids before microbial fermentation. acidic hydrolysis methodology is cheaper than enzymatic hydrolysis but it can cause production of some inhibitors like aliphatic acids, which affect the growth of microorganisms. vitreoscilla hemoglobin (vhb) is the first described prokaryotic hemoglobin. the recombinant strains carrying vgb gene (e. coli, p. aureginosa) which encodes vhb showed increased bacterial growth, productivity of metabolites compared to untransformant counterparts under low oxygen concentrations [nasr et al., 2001; geckil et al., 2004] . in this study, ethanologic e. coli strains fbr5, its derivative strains ts3 (vgb+) and ts4 (vgb+) were used. ts4 was constructed in such that it could express more vhb than ts3. bioethanol production by these strains in presence of lignocellulosic hydrolysates derived inhibitors was investigated. different acetic acid concentrations (2.5-10 mm) were used as inhibitors from lignocellulose hydrolysate. 10.0 mm acetic acid was used as an inhibitor. the growth of vhb expressing ts3 and ts4 strains was inhibited about 31% after 48 hours fermentation time. strain fbr5 was inhibited as high as 76% by using the same inhibitor including growth medium. it was shown that the expression of vhb could improve growth and productivity in presence of lignocellulosic inhibitors. differentiation of preadipocyte, also called adipogenesis, leads to the phenotype of mature adipocyte. however, excessive adipogenesis is closely linked to the development of obesity. thus, any drug or chemical that can inhibit adipogenesis may have preventive and/or therapeutic potential against obesity and related diseases. azd1208, an inhibitor of the family of pim kinases, is known for anti-cancer activity. here we investigated the effect of azd1208 on adipogenesis in 3t3-l1 preadipocytes. notably, azd1208 treatment led to a concentration-dependent inhibition of both lipid accumulation and triglyceride (tg) synthesis during the differentiation of 3t3-l1 preadipocytes into adipocytes with no cytotoxicity. on mechanistic levels, azd1208 strongly reduced not only the expression levels of ccaat/enhancer-binding protein-a (c/ebp-a), peroxisome proliferator-activated receptor-c (ppar-c), fatty acid synthase (fas), and perilipin a but also the phosphorylation levels of signal transducer and activator of transcription-3 (stat-3) during adipocyte differentiation. furthermore, azd1208 largely decreased leptin, but not adiponectin, mrna expressions during adipocyte differentiation. collectively, these results demonstrate that azd1208 inhibits adipogenesis in 3t3-l1 preadipocytes and the inhibition is largely attributable to the reduced expression and/or phosphorylation levels of c/ebp-a, ppar-c, fas, perilipin a, and stat-3. effect of intrauterin exposure to artificial food colourings on dna damage in rats in many research genotoxic potential of food additives has been investigated. however there are few findings about the effect of artificial food colourings (afc) on dna. in this experimental study, we aimed to analyze whether in utero exposed artificial food colourings would have effect on dna and cause damage.thirteen female rats were included to the study which were equally divided into two groups as control (cg, n = 15) and food colouring (fcg, n = 15) groups. a mixture of nine food colours were given daily to fcg by oral gavage from preconception to birth. no adverse effect level (noael) of artificial food colourings for each colouring was administered to fcg. three months after the birth, 6 offspring from each group were selected randomly as control (cg) and experiment (eg) groups. then they were sacrified under anesthesia. for performing the alkaline comet comet assay leukocytes were seperated from whole blood samples. the alkaline comet assay was performed. the extent of dna damage was assessed from the length of dna migration derived by subtracting the diameter of the nucleus from the total length of the image and graded into 5 categories and these grades were converted into arbitrary unit (au). differences between the means of data were compared by independent samples t test. the results were given as the meanaesd, p values of less than 0.05 were considered as statistically significant. although the extent of dna damage was higher in eg, the comparison of experiment (13.50 ae 1.76) and control (11.66 ae 1.36) groups showed no statistical difference (p = 0.072). relationship between glucocorticoid receptor gene polymorphisms and recurrent depression l. aydogan, i. benli, z. c. ozmen, i. butun gaziosmanpasa university medical faculty, department of biochemistry, tokat, turkey objective: sensitivity to glucocorticoids varies between individuals and these differences have been implicated in the etiology of psychiatric diseases such as depression. recent studies have found relationship between common glucocorticoid receptor (gr) gene (nr3c1) polymorphisms and unipolar or bipolar depression. the nr3c1 gene is a candidate gene affecting depressive disorder risk and management. the aim of the present study was to evaluate the relative distribution of specific polymorphisms of nr3c1 (bcl1 and rs33388) in recurrent depressive disorder (rdd) patients. methods: our study included 100 volunteers with recurrent depressive disorder and 100 healthy individuals without any mental illness. depression was assessed by hamilton and madrs depression scale. nr3c1 gene polymorphisms were detected by real-time pcr, with hybridization probe method. allele and genotype frequencies at two loci (bcli and rs33388) were investigated in rdd patients and controls. results: genotype distribution among rdd patients and the control group for bcl-1 (g/c) were as follows: cc 3% and 5%, gc 27% and 34%, gg 70% and 61%, respectively. there was not a significant difference when the frequency of the allele (p = 0.204) and genotype frequency (p = 0.382) were compared between the patients and the control. genotype distribution in the rs33388region (a/t) of the patients and controls were tt 29% and 25%, ta 49% and 54%, aa 22% and 21%, respectively. allele frequency (p = 0.841) and the genotype frequencies (p = 0.754) were not significantly different among the groups. conclusion: numerous nr3c1 gene polymorphisms were previously reported in association with modification of depressive disorders. the results of our study showed no association between gr genotype and recurrent depressive disorder. nr3c1 polymorphism does not play a role in the development of recurrent depressive disorder. thymoquinone (tq) has been shown to supress the proliferation of various tumor cells, while it is minimally toxic to normal cells. the aim of this study is to investigate the potential therapeutic effects of tq on cell proliferation, apoptosis, invasion, migration, colony formation and wound-healing in sh-sy5y human neuroblastoma cell line. sh-sy5y cell line treated with 5-400 lm tq by solving medium for 24, 48 and 72 h considering a time-and dose-dependent manner. the cytotoxic effect of tq was determined by mtt method. total rna was isolated by trizol reagent. cdna synthesis was performed by using commercial kit. mdm2, p53, p21, akt, pten, cdk4, cyclin d1, caspase-3, -8, -9, -10, bcl-2, bax, parp, bcl-xl, bid, dr4, dr5, puma, noxa, mmp-2, -9, timp-1, -2 and gapdh gene expression profiles were analysed by real-time pcr method. effects of tq in sh-sy5y cells on invasion, colony formation and cell migration were detected by matrigel-chamber, colony formation assay and woundhealing assay, respectively. statistical analysis were performed with rt 2 profiles array data analysis by using student's t test. ic 50 value of tq in sh-sy5y cells was detected as 15 lm at 48 th hours. by rt-pcr results, it was determined that tq caused a decrease in the expression of mdm2, akt, cdk4, cyclin d1, bcl-2 and mmp-2. it is also observed that tq caused a significant increase in the expression of p53, pten, caspase-3, -10, bid, dr4, puma, noxa and timp-1. it was also found that tq in sh-sy5y cells suppressed invasion, migration and colony formation by using matrigel invasion chamber, wound healing and colony formation assay, respectively. in conclusion, we demonstrate that tq significantly effect cell cycle, apoptosis, invasion, migration and colony formation of sh-sy5y cells. tq may be a potential candidate as chemotherapeutic agent for the treatment of neuroblastoma. more studies have to be performed to profile the mechanisms and genome wide effects of tq to prove its therapeutic potential. dna aptamers can achieve a very high affinity to the target due to the potential of developing broad target-binding interface. however, classic strategy selection of aptamer binders is a challenging task requiring multiple rounds of panning and post-selection optimization. we have developed fast and convenient technique for the selection of dna aptamers based on the offrate selection and tandem affinity purification (tap). we constructed and produced in e.coli recombinant chimeric protein, comprising two affinity tags (his6 and gst) separated from each other and from the target protein (anthrax protective antigen domain iv, padiv) by sumo protease recognition polypeptide and synthetic cleavage site for the anthrax lethal factor (lf). the protein bound to aptamer library is first captured by imac resin, cleaved by sumo protease, captured by gst resin and eluted by lf following the lines of the tap method. the gst-captured aptamer-target complexes were subjected to the off-rate selection using soluble padiv as the competitor. multiple selection rounds are cumbersome and can result in carryover. high abundance of moderate affinity aptamers in the resulting pools obtained by classic selection approaches suggests that the procedure to counter-select them at the beginning of panning is needed. reduction of the contact duration between the aptamer library and the target was crucial for efficient selection of high-affinity binders. on the other hand, tap prevents contamination, and bundled with the off-rate selection, allows for clean isolation of high-affinity binders with affinity in the low nanomolar range. the developed technique is applicable for efficient selection of high affinity dna binders to soluble recombinant proteins and their fragments. dna aptamers obtained will be further used for the development of diagnostic and therapeutic tools for the detection and treatment of anthrax. the work was supported by russian science foundation research grant no. 14-15-00630. the role of macab efflux pump in protection of serratia marcescens against antibiotics and oxidative stress the emergence of bacterial multi-drug resistance is a growing problem of public health worldwide. bacterial drug efflux pumps are membrane protein complexes that function to expulse drugs from the cell. they play a crucial role in the rising rates of antibiotic therapy failures. the homolog of macrolide-specific pump macab was identified in opportunistic pathogen serratia marcescens and was used in this study to characterize its role in protection against antimicrobials and other processes beyond the active efflux of antibiotics. here we used method of serial dilutions to determine minimum inhibitory concentration (mic) for s. marcescens sm6 wild type (wt) and its isogenic δmacab mutant strains. we also used h 2 o 2 survival assay to evaluate the ability of wt and the mutant strain to withstand an oxidative stress. finally, we used b-galactosidase assay to evaluate macab promotor activation in the reporter strain and followed macab expression by western blotting analysis using macab-6xhis strain. we show that in contrary to its e. coli homolog, macab pump in s. marcescens is not involved in the protection against macrolides but instead it is required for protection against aminoglycosides. we further show that similar to its salmonella typhimurium homolog, s. marcescens macab is essential for protection of bacteria against h 2 o 2 . transcriptional analyses demonstrate that while low level of macab promotor activity can be detected after 2 hours of growth in lb-broth there is at least 5-fold increase in expression in response to the presence of h 2 o 2 . on the protein level macab can be detected starting from 3 hours of growth in lb-broth and it reaches maximum expression on 16 hour of growth. our data suggest that macab pump in s. marcescens is involved in protection of bacteria against aminoglycoside antibiotics and is crucial for protection against reactive oxygen species. we are currently working on identification of macab substrate with anti-h 2 o 2 properties. antiproliferative and apoptotic effects of noscapine on mcf-7 and mda-mb-231 human breast cancer cell lines approximately 10-17% of breast cancers are negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. these are most aggressive tumor and a clinical problem because of lack of targeted therapies. noscapine is an alkaloid from opium. noscapine is a microtubule-interfering agent. it causes mitotic arrest, induces apoptosis. in this study, we aimed to investigate the effects of noscapine in mcf-7 and mda-mb-231 human breast cancer cell lines. the cytotoxic effects of docetaxel, tamoxifen, and noscapine on the mcf-7 and mda-mb-231 cell lines were analyzed by roche xcelligence system. the cells were cultured in 10% fetal bovine serum containing dulbecco's modified eagle medium at 37°c in a humidified atmosphere containing 5% co 2 . 24 h after seeding, the cells were treated with 4 different doses of docetaxel (12.5 to 100 nm), tamoxifen (12.5 to 100 lm), and noscapine (12.5 to 100 lm). cultured cells were harvested, fixed with 10% formalin, and centrifuged. pellet was blocked, fixed, and embedded in paraffin. paraffin-embedded cells blocks were sectioned at 4 lm thickness and stained with h&e, ki-67, bcl-2, cyclin-d1, and bax. sides were assessed under a light microscope. quantification of the analyzed proteins were evaluated by the percentage of positive cells. all drugs showed cytotoxic effects on both cell lines. all drugs inhibited the proliferation of breast cancer cells, but effects were dependent on time and dose. all drugs were especially more effective on mcf-7 cells. immunohistochemical examinations revealed that tamoxifen was more effective on mcf-7 cells, hovewer docetaxel and noscapine were more effective on mda-mb-231 cells. tamoxifen has more apoptotic and antiproliferative effects on mcf-7 cells. docetaxel and noscapine showed more apoptotic and antiproliferative effects on mda-mb-231 cells. noscapine may be an effective anticancer agent due to antiproliferative and apoptotic effects on breast cancer cells. negative selection of dna aptamers to reduce non-specific binding in solid-phase-based selection procedures carryover by binders specific to the components of the selection system can be a serious issue in hampering the aptamer selection campaign. solution-or "mass"-based techniques still cannot substitute classic phase-separation strategies. one approach to prevent selection of "passenger" phase-specific (plastic, beads) or blocking agent specific aptamer species is their depletion from the initial library pool. our aim was to develop the universal technique for removal of such aptamers exemplified by bsa-and casein-specific binders, while preserving the initial library complexity. the dna aptamer library was subjected to three rounds of depletion using magnetic beads with covalently attached casein and bsa. to ensure high depletion efficiency, beads were pelleted in a 15-ml centrifuge tube by a neodymium magnet through a 10-cm cushion of 20% sucrose, thus preventing weakly bound aptamers from re-populating the library. high complexity of the input library helped to avoid pcr amplification after depletion rounds preventing the library bias introduced by dna amplification. the depletion effciency was confirmed by real-time pcr. resulting oligonucleotide sub-library was analyzed for binding to the targets using solid-phase real-time pcr assay. we have shown that three rounds of panning under the conditions employed provided full depletion of the initial dna pool from nucleic acid structures capable of binding to protein competitors and hampering the process of aptamer selection. we compared selection efficiency of aptamers specific to type a botulinum neurotoxin light chain in depleted vs undepleted library. the yield of the target-specific aptamers was 10-fold higher in the library subjected to the depletion procedure. removal of undesired binders from aptamer libraries appears an important step of solid-phase selex procedure. it can become a useful approach in optimizing solid-phase selex. the work was supported by russian science foundation research grant no. 14-15-00630. epithelial mesenchymal transition (emt) is a critical trans-differentiation program driving cancer metastasis. patients showing signs of emt or presence of distant metastasis have poor prognosis. another well-known feature of decreased cancer-associated survival is the lack of anti-cancer immune responses. thus we hypothesized that the emt and anti-tumor response should be linked via altered secretion of soluble factors by metastatic cells. all cell lines were grown in dmem. emt status of crc cell lines were assessed by investigating canonical markers of emt. cytokine/chemokine expression of crc cells was performed using r&d systems antibody arrays and validated using ccl5 sandwich elisa and rt-pcr. the mechanism of action of zeb1/2 on ccl5 promoter has been studied by luciferace assay and chip. ccl5 coding region was cloned into pcdna3.1 and stably transfected into dld-1 cells. ccl5 deficient ct26 cells were generated using lentivirual shrna transduction. cells overexpressing or knock/down ccl5 were injected orthotopically into mice. t lymphocyte (til) infiltration in respect to ccl5 and sip1 expression was studied using ihc or flow cytometry. emt status catagorised 13 crc cell lines into epithelial, intermediate epithelial, intermediate mesechymal and mesenchymal. cytokine/chemokine antibody arrays showed a significant increase in ccl5 in induced dld-sip1 cells. elisa, multiplex assays and rt-pcr confirmed a significant increase of secreted ccl5 in the induced dld-sip1 cells as well as mesenchymal crc cells as compared to epithelial ones (p = 0.027). promoter studies showed that zeb1/2 bind to ccl5 promoter and and activate ccl5 gene expression. no metastasis was observed for dld-1 cells overexpressing ccl5 but significant alterations of tumour associated lymphocytes were identified in syngeneic orthotopic crc models. our data shows that ccl5 is up-regulated by emt inducing transcription factor sip1, and mesenchymal (metastatic) crc cells secrete significantly more ccl5 compared to epithelial (non-metastatic) ones. ccl5 did not induce emt per se but abundant secretion of ccl5 by metastatic crc cells was a crucial regulator of immune infiltrate in crc. inhibiting ccl5 in metastatic crc may have a therapeutic potential. barley (hordeum vulgare l.) belongs to the grass family, poaceae (gramineae). it is the fourth most important cereal crop after wheat, maize and rice and is among the top ten crop plants in the world. talbina was used to be recommended for the sick and for one who is grieving over a dead person. talbina is made by adding one or two tablespoon of barley flour (must be 100 percent wholegrain barley flour) to one-and-a-half cups of water and placed on low heat for 10-15 minutes (optional: add milk or yoghurt and sweeten with honey). the main objectives of this investigation were determine the a-tocopherol contents and antimutagenicity activity of talbina (hordeum vulgare l.). our results showed that the total tocopherol content was in the range of 0.25 to 1.03 lmol/g fw. talbina extract was shown to have greater antimutagenic activity observed in the 2500 lg/plate concentration s. typhimurium ta98. at all the doses antimutagenic response was significant at (p < 0.01) against both the strains with a percent mutagenicity decrease from 40 to 25 for ta98 followed by ta100 with percent antimutagenicity from 30 to 11. the results of the study concluded that talbina is a better antimutagenic agent than vitamin e and combination of vitamins did not produce any synergistic activity. the compounds containing thiadiazoles have diverse applications as antifungals, anticancer agents, antibacterial, antiinflammatory drugs, antidepressants and carbonic anhydrase inhibitors according to literature. in this study some novel thiadiazole compounds [(1,4,10,13)-tetrathia[4.4] (2,5)-1,3,4-thiadiazolophane; (4,16)dioxo-1,7,13,19)-tetrathia[7.7](2,5)-1,3,4-thiadiazolophane; (4,7,19,22)-tetraoxo(1, 10, 16, 25 )-tetrathia[10.10](2,5)-1,3,4-thiadiazolophane and (4,7,10,22,25,28)-hexaoxo(1, 13, 19 ,31)-tetrathia [13.13] (2,5)-1,3,4-thiadiazolophane] were used to evaluate the cytotoxicity on healthy human lymphocytes and the antibacterial activities. cytotoxicity tests were perfomed using mts assay and the trypan blue test. cells were incubated with the compounds for 72 hours. at the end of the each 24 hour, cell vitality was assessed by measuring the absorbance (490 nm) of each well using a microplate reader for mts assay. in addition, viability percents of the cells were determined after trypan blue test. as a result, the compounds showed cytotoxicity in a dose dependent manner. for the concentrations of 1:1000 of 0.5 mg/ml, the cytotoxic effect was eliminated. also, antioxidant capacity was determined using 2,2-diphenyl-1-picrylhydrazyl (dpph) reagent. moreover, the antibacterial activities of the compounds were analyzed using a microdilution test against e. coli and s.aureus. compounds having various concentrations showed different antibacterial effects against these two bacteria. arabidopsis thaliana ecotypes vary in their ability to utilize organic p substrates insufficient quantity of inorganic phosphorus in soil is an evergrowing problem that affects many fields of agriculture. unlike inorganic phosphates, organic phosphorus compounds are very common in many soil types, but plants are often unable to efficiently utilize them. to better characterize the extent of natural variation in the ability of the model plant arabidopsis thaliana to grow on organic phosphorus compounds, we grew 19 arabidopsis ecotypes on several organic and inorganic sources of phosphorus. plants were grown in liquid or solid media containing naphosphate, phytate and atp as the sole supply of phosphorus or in absence of any phosphorus source. after several weeks of growth, plants were assayed for changes in their morphological and physiological characteristics. phytate was shown to be the least preferred source of phosphorus compared to inorganic phosphate and atp. the rate of biomass accumulation in all ecotypes decreased in the following order from inorganic phosphate to atp to phytate. lateral root formation was markedly reduced in the absence of any phosphorus source or in the presence of phytate. we also showed that phosphomonoesterase activity in intact roots increased when plants were grown on atp and phytate. overall phosphorus content in leaves and roots was similar when plants were grown on atp or inorganic phosphate, but it was markedly reduced on phytate. substantial differences between ecotypes were also observed in root length, p content in ash and phosphomonoesterase activity in intact roots. our analysis of the ability of arabidopsis ecotypes to grow on several different phosphorus sources provides a unique opportunity to investigate the degree of natural variation in this plant's ability to adapt to different nutritional environments. analysis of many important morphological and physiological changes observed in these plants can further extend our understanding of the full range of plant responses to phosphorus availability. laboratory tests are important in terms of confirmation of diagnosis given by clinics and implementation of appropriate treatment protocols for patients. laboratory tests used by the clinics have been increased in parallel with time.there are many reasons for increased use of the test such as increase of elderly population, increase in standard of care, lack of information and shortening of turn around time. unnecessary laboratory testing also constitute one of the reasons for increased use of laboratory tests. in our study we aimed to investigate the unnecessary laboratory testing for fpsa test. fpsa tests which are ordered with total psa tests that values of less than 4 ng/ml or greater than 10 ng/ml were accepted as inappropriate initial testing. 9759 fpsa tests were evaluated as unnecessary laboratory testing. the clinic which ordered the maximum unnecessary laboratory testing with 3315 was urology within all the clinics. although to the restrictions about the ordering of total psa and fpsa tests there were no decrease in the number of unnecessary laboratory testing. unnecessary usage of laboratory testing may cause increase of false positive results, increase in the use of invasive testing, unnecessary drug consumption and increase of healtcare costs. some precautions may be effective in reducing unnecessary tests such as to inform clinicians about the cost of laboratory tests, to increase the clinician education programs and to develop usage of disease specific diagnostic algorithms about test ordering. local clinical validation of blood collection tubes although the tubes with gel and clot activator are widely used due to the advantages, there are ongoing discussions about the effects of the blood collection tube on clinical outcomes in the analysis of biochemical parameters. therefore, we aimed to prove the local clinical validation of the new produced blood collection tubes with low-volume. the blood samples of 40 patients who referred to the hospital phlebotomy unit were collected using holder into the 3 different tubes. first tube was 5 ml glass tube and with no additive, second was 5 ml tube with gel separator, third was 1 ml tube with gel separator. serum was separated and immediadiately analysed for 25 biochemical parameters. the difference between the analyte amounts in the different tubes was evaluated using paired t-test. the clinical significance was evaluated using significant change limit. bias (%) between the other tubes with the reference tube was also evaluated according to the ''allowable total error". when we compared the other test tubes to a glass tube which was assumed reference tube, total protein, albumin, amylase, calcium, triglyceride, cholesterol, hdl-cholesterol, total and direct bilirubin, iron, gamma glutamyl transferase, magnesium, phosphorus results were statistically significant. but the results of all the analytes were within the significant changes limit and the allowable total error was not significant. while a biochemical parameters have analysed, it may be absorbed into the gel and this may caused from factors such as the chemical structure of the gel, analyte itself, the residence time in the gel, storage temperature and volume of the sample e.g. as well as the leaking of gel material to the sample was reported to be another factor for affecting the analysis. despite these factors, we observed that neither gel-clot activator tube with low nor high volume affect the clinical results. the research of the frequency of interference in thyroid function tests interference is defined as the effect of substance in the sample which changes the correct value of laboratory results. the frequency of interference in immune techniques is varied. the frequency of interference depends on population of the study, technique for detecting the reaction and researcher's method. unexpected or inconsistent results with clinical findings should suggest the possibility of interference. in this study it is aimed to investigate the frequency of interference in thyroid function tests (tsh, ft3, ft4) which are the most common requested laboratory tests. thyroid function tests of 47915 patients are analyzed in ankara numune education and research hospital in october 2014-may 2015. five samples which had the incompatible results with clinical findings are re-evaluated just because of the suspicion of interference. the detection of interference included; repetition of test via different immune techniques, serial dilution, polyethylene glycol (peg) precipitation and incubation with heterophilic blocking tubes (hbt). the results of two different immune techniques and before/ after incubation with hbt showed no significant difference. linear curves had observed in serial dilution. after peg precipitation; below 40% of recovery had obtained in one sample, therefore it is interpreted as macro-tsh. the frequency of interference in thyroid function tests for 8-month study period was 0.01%. no information is found about the best test for defining the cross reaction. it is also aforethought that interference should not be excluded by using any single procedure. p-mis-105 development of polyclonal and monoclonal antibodies against fatty acid binding protein 4 (fabp4/ap2) a. abbasi taghidizaj, g. aydogdu, b. p. sermikli, e. yilmaz ankara university, ankara, turkey recombinant proteins and antibodies can be use for therapeutic or diagnostic purposes which produced in many different host organisms. the technique for the production of immortal cell making single antibody, fusing target antibody-forming b lymphocyte precursor with a suitable myeloma cells. the fused hybrid cells (called hybridomas), as a cancer cell will reproduce rapidly and will produce large amounts of the desired antibodies. fatty acid binding protein (fabp4) is a well characterized intracellular lipid transport protein and plays a key role in the intracellular fatty acid transport and adipose tissue metabolism. fabp4 as a adipokine that regulates glucose homeostasis and has various features for metabolic syndrome associated with obesity. in this study, production of monoclonal antibodies against immunogenic fabp4 protein made by recombinant dna technology. recombinant his-fabp4 was expressed in e.coli and purified. balb/c mice used for immunization and serum anti-fabp4 antibodies determined by enzyme-linked immunosorbent assay (elisa). hybridoma cells created by fusion of splenocytes and myeloma partner cells. after selection of antibody producing cell clones, injecting hybridomas into the peritoneal cavity in balb/c mice ascites fluids was obtained. we have selected fifteen hybridoma clones that produced antibodies specific for fabp4, as shown by western blotting and immunocytochemistry. as a result we produced mabs that will be useful for the scientific community working on fatty acid binding proteins and lipid metabolism. in near future, therapeutic approach for this antibody maybe a possibility in metabolic syndrome. thioridazine, an anti-psychotic drug, inhibits migration, invasion and epithelial mesenchymal transition in breast cancer cell lines thioridazine (thz), an antipsychotic drug, exhibits anti-angiogenic effects on breast cancer cell lines. however the mechanistic insight in exerting antiangiogenic effect is not clearly understood. the objective was to investigate the role of thz in epithelialmesenchymal transition (emt) by using cell migration assay, scratch assay, western blot (wb) and immunocytochemistry. thz treatment reduced cell viability on mda-mb-231, mcf-7 and cd44 + /cd24-cells and ic50 values of thz were found to be 9 lm, 16.4 lm and 18 lm respectively, at 24 hours. invasion potency of mcf-7, cd44 + /cd24-and mda-mb-231 cells were determined as 29%, 24%, 16.5% when compared to relevant treatment controls. migration potency of mcf-7, cd44 + /cd24-and mda-mb-231 cells was determined as 28.5%, 63.2%, 61% respectively. among the three cell lines mda-mb-231 cells display enhanced invasive and migration ability when compared to other cell lines. western blotting results demonstrate that thz significantly increases e-cadherin, cytokeratin-18, b-catenin, while inhibiting n-cadherin, vimentin, fibronectin. immunocytochemistry studies revealed decrease in e cadherin and a concomitant increase in vimentin level for all three cell lines upon treatment with thz. moreover thz significantly inhibited the cell migration, invasion and emt in mda-mb-231, mcf-7 and cd44 + /cd24 cell lines by suppressing mesenchymal markers. in conclusion, these data suggest that thz might be a novel anti-proliferative and anti-metastatic agent for treatment of breast cancer. effect of seasonal temperature and humidity on urine density in children environmental heat and humidity are important factors affecting hydration status in childhood. hereby, we aimed to investigate the effects of seasonal climate changes on urine density of children living in mediterranean climate, cyprus. 1700 healthy 0-18 year children's (850 girls, 850 boys) age, sex and urine density results were collected retrospectively for three consecutive years. the correlation of urine density with each seasonal and 12 months' average temperature and humidity has been analysed. the urine density results had a positive correlation with temperature (r = 0.083, p = 0.001) and a negative correlation with humidity (r= à0.072, p = 0.003). mean urine density in spring was higher than that of autumn (p = 0.02) and winter (p = 0.00). mean value of summer was higher than autumn (p = 0.03) and winter (p = 0.00). 0-24 months age group had lower urine density. evaluation of urine density based on gender and puberty revealed no statistically significant difference. seasonal mediterranean climate changes have an impact on urine density in children which may affect hydration status especially in infants < 2 yrs of age. during high temperature seasons ensuring adequate water intake is essential in this age group in mediterranean climate. p-mis-108 implementation related to the use of antibiotics and data sources by community pharmacists in north cyprus as the resistance to antibiotics is gaining importance in today's world;the solution to this problem is possible through a common consciousness of the doctor who prescribes antibiotics,the pharmacist who sells and the patient who consumes antibiotics. irrational use of drugs is an economic and medical problem in many developed and developing countries around the world.the aim of this study is to determine the sales ratio of non-prescription antibiotics in pharmacies which is the biggest category of the antibiotic group sold as well as the indications that lead to its' prescription. eighty-four pharmacies out of 168 pharmacies located in north cyprus were involved in the study with 50%stratified systematic sampling, questionnaires were filled and a consent form was signed by the participating pharmacists. the pharmacists involved in the study stated that non-prescribed antibiotics were demanded from the pharmacists and all except two (97.6%),responded positively to this demand. it has also been identified in the study that 41.5% of the daily sale of antibiotics in the first half of the year 2014 was non-prescribed. the most purchased antibiotics either with or without prescription was found to be the penicillin and its derivatives with 76.2% and upper respiratory tract with 86.9%. when the level of selfawareness of the pharmacists was examined, the rate is found in north cyprus to be (41.5%),compared with the studies conducted in greece,italy,malta and spain 47% and egypt 50.4%that designated the non prescribed antibiotics purchased from the public pharmacies. the rate of sale of non-prescribed antibiotics in north cyprus has been found to be at a higher level compared to the rates in many developed and developing countries. furthermore, the upper respiratory tract infections are amongst the most common viral causes which lead to a high consumption of both prescribed and non-prescribed antibiotics. this study was supported by turkish viral hepatitis prevention society. acrylamide has cytotoxic, antiproliferative and apoptotic effects on human lung adeno carcinoma cell line a549 acrylamide (aa), a widespread substance in many fields, forms in foods during high temperature processing such as baking, roasting, frying. aa is a potent neurotoxic, genotoxic and clastogenic agent being a strong electrophile and forming adduct with biological molecules or potent nucleophiles. up to now, several studies confirmed the toxicity of acrylamide to several organs. on the other hand, aa is reported to have inhibition effects both on proliferation and differentiation of different cancer cells in a time and dose-dependent manner. in addition, natural and synthetic acrylamide derivatives are also used as potent anti-cancer agents. moreover, inhibition concentration (ic50) values of aa against these cancer cells have not been investigated in detail yet. thus, the goal of this study is to investigate the cytotoxicity of aa on a549 cells including with ultrastructural and morphological effects. ic50 value of aa on a549 cells for 24 h was detected with mtt (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h-tetrazolium bromide) colorimetric assay. we evaluated morphological changes under confocal microscopy and ultrastructural changes under transmission electron microscopy (tem). our results demonstrate that aa inhibits the proliferation of a549 cells in dose-dependent manner and ic50 on a549 cells was found to be 4.6 mm for 24 hours. confocal microscopy evaluations showed that aa caused nuclear condensations, fragmentations, cytoskeleton lacerations and membrane blebbing. tem results revealed membrane blebbing, chromatin condensations and cell shrinkage. although aa is a probable carcinogen substance, it drastically inhibited cell viability in dose-dependent manner. from microscopic assessments, aa is suggested to induce apoptosis in a549 cells. in conclusion, the present study confirms the high potential of aa for cytotoxic, antiproliferative and apoptotic activity on a549 cells. however, appropriate aa dose is critical to prevent its possible adverse effects. effect of hemolysis and lipemia on some immunochemical tests in beckman coulter unicell dxi 800 immunoassay analyzer c. yilmaz, s. yildiz, m. senes, v. fidanci, d. y€ ucel ankara training and research hospital, ankara, turkey the aim of the study was to investigate the effects of in vitro hemolysis and lipemia on 25 immunoassays studied by the beckman coulter unicell dxi 800 immunoassay analyzer. we prepared a serum pool without hemolysis, lipemia and icterus. baseline serum pool concentrations of 25 tests were measured by the beckman coulter unicell dxi 800. d _ ifferent serum pools, six for hemolysis and five for lipemia, were spiked with increasing concentrations of hemoglobin (0.6, 1.2, 2.4, 4.5, 6.6 and 8.6 g/l hemoglobin) and intralipid (0.625, 1.25, 2.5, 5 and 10 g/l intralipid). the hemolysate was prepared by osmotic shock method. intralipid (20%, baxter, deerfield, il) was used to mimic the effect of lipemia. the hemolysis (h), lipemia (l) and icterus (i) indices were measured on beckman coulter au 5800. after spiking the pools, the 25 tests were measured again in duplicate on beckman-coulter dxi 800 analyzer. a change of 10% from baseline results was taken as evidence of interference and the interfered tests were also evaluated according to total analytical error based on analytical imprecision and intraindividual biological variation. we observed a positive interference due to hemolysis for folat, vitamin b12, testosterone and by lipemia for cortisol. there was a negative interference of hemolysis for ca 19.9, ca 125, ca 15.3, insulin, pth and e2, and of lipemia for progesterone, ca 19.9, vitamin b12 and pth. we found clinically significant effect (>total analytical error) of hemolysis on folate and insulin, and lipemia on cortisol. investigation of the effect of two different p38mapk inhibitors in rats subjected to isoproterenol-induced acute myocardial injury: an experimental study objective: acute myocardial infarction is a serious acute condition. in the current study, we aimed to investigate the possible effect of two different mitogen-activated protein kinase (p38mapk) inhibitors in rats subjected to isoproterenol (iso)induced myocardial injury. materials and methods: a total of 32 male wistar-albino rats were equally and randomly seperated into four groups as follows: control, iso, iso plus sb203580 andiso plus tak-715. treatment agents were orally administered and myocardial injury was induced by subcutaneous injection of iso. serum cardiac troponin-i (ctni), ischemia modified albumin (ima), heart fatty acid binding protein (hfabp) levels and paraoxonase-1 (pon-1) activity, tissue tos (total oxidant status), tas (total antioxidant status), tt (total thiol), tumor necrosis factor-a (tnf-a) levels, superoxide dismutase (sod) and glutathione peroxidase (gsh-px) activity levels were measured. tissue mrna levels of nf-jb, p38 mapk and nuclear factor erythroid 2-related factor 2 (nrf2) were analyzed. heart tissues were also immunohistochemically and histopathologically evaluated. results: both compounds have led to a decrement in myocardial damage, apoptosis, ctni, ima, hfabp, tos, and tnf-a levels, nf-jb, p38 mapk, phosphorylated c-jun n-terminal protein kinase (pjnk 1/2) expressions. on the other hand, the applied treatment increased sod, gsh-px, tas and tt levels, as well as phosphorylated extracellular signal-regulated kinase (perk 1/2) and nrf2 expressions. conclusion: data established from the current study suggest that administered agents have protective effect against cardiac injury induced by iso, which was more prominent in rats received sb203580 treatment. p38mapk inhibitors may constitute a useful choice as cardioprotective agents due to their antiinflammatory, antioxidant and anti-apoptotic effects. keywords: _ isoproterenol, myocardial infarction, myocardial ischemia, p38 mitogen-activated protein kinases, sb203580, tak-715. silicosis composes the vast majority of occupational lung diseases. silicosis, caused by inhalation of crystalline silica, is a chronic lung disease characterized by parenchymal nodules and pulmonary fibrosis. the susceptibility of patients with silicosis to infection is thought to be due to toxic effects of silica on pulmonary macrophages. ada activity is considered as a nonspecific marker of t cell activation and cellular immunity. this study aimed to compare the serum ada activity in silicosis patients with spirometric values. in this study there were 35 males in each groups which contained patients with silicosis (group 1), individuals having similar symptoms with silicosis from same occupational area (group 2) and healthy subjects (group 3). routine hematological and biochemical parameters were also measured. the serum ada activity and spirometric values (fev1, fev1%, fev1/fvc, fev1/ fvc%, fef25-75 and fef25-75%) were compared. the average age of group1, 2 and 3 are 38.5 ae 10.6, 39.5 ae 2 and 51 ae 10.5 years, respectively. there was a significant difference between group 1 and 3 in terms of the ada level (p < 0.05). there was a negative correlation between ada activity and fev1, fev1%, fev1/fvc, fev1/fvc%, fef25-75 values. elevated serum ada activity has been shown in many diseases with induced cellular immunity. despite initially toxic effects were lead to a little immunological reaction in patients with silicosis, continuation of this immunological response is important in some chronic manifestations of silicosis. the release of chemotactic factors and inflammatory mediators cause the migration of polymorphonuclear leukocytes, t lymphocytes and macrophages. in this study, the ada activity was significantly higher in patients with silicosis than others. increased immunity in patients with silicosis is being considered, increasing ada activity might be help of earlier recognition of these patients and to take better quality of life. atlantic salmon (salmo salar l.) is an important model system in evolutionary and conservation biology that provides fundamental knowledge into population persistence, adaptive response and the effects of anthropogenic change. the role of behavioral and body size variation in environmental adaptation of atlantic salmon is well known, by contrast, the underlying biochemical mechanisms are largely unknown. intracellular proteases, such as cathepsins b and d in lysosomes and calpains and proteasome in cytosol, due to their metabolic and regulatory role may contribute to phenotyping speciation of salmon young. we examined the activity of intracellular proteolytic enzymes in skeletal muscles of atlantic salmon parr from two local habitats of the varzuga river (the main channel and small tributaries) differing in hydrological and feeding parameters. calpain and proteasome activities were determined by casein or suc-llvy-amc hydrolysis in the skeletal muscles of s. salar from varzuga river (kola peninsula, russia). it is known that salmon parr originated from a common hatch became phenotypically divergent during the settle in the biotopes. reliable difference in studied enzyme activities in the salmon parr from two local habitats was found; furthermore, calpain and cathepsin b proteolytic activities were found to negatively correlate with parr body size. muscle proteolytic activity data support an idea on protease contribution to environmentally-driven adaptation and speciation process in fish. the work was supported by the russian scientific foundation, project no. 14-24-00102. the phylogenetic analyses of anthriscus (apiacea) species from turkey based on non-coding "trn" regions of chloroplast genome p. yilmaz sancar 1 , m. tekin 2 , s. civelek 1 1 firat university, elazig, turkey, 2 cumhuriyet university, sivas, turkey anthriscus pers. (apiaceae) species belongs to apiaceae family and is represented by 16 genus on the world and by 8 genus in turkey. anhriscus species are used extensively for treatment various disease such as asthma, alzheimer and show anti-tumoral, anti-microbial, antioxidant features. for determining exact species which treat disease it is necessary sorting species correctly with molecular markers to support morphological features. anthriscus species were defined by examining insufficient quantity of samples in turkey flora. besides, no detailed study was found in our country after flora study. for this reason a revision study was made with the aim of solving some systematical problems in 2013 by tekin. the result of the study provided important contribution to the systematic of the species in turkey. however a molecular study was also required for building the obtained results on a more solid ground. in this study, the aim to reveal systematic and phylogenetic relationship among species of anthriscus in turkey, by using trnl-f region in chloroplast genome. dna was isolated by ctab method and amplified in pcr by using e-f primaries. the obtained data was evaluated by mega 7.0 program and phylogenetic tree was prepared by using maximum likelihood method. according to the phylogenetic tree that we prepared by using the sequence line up of trnl-f section, it was observed that a. cerefolium, a. caucalis and a. tenerrima species completed their speciation and an isolation with other species in terms of speciation was provided. it was also observed that a. kotchi, a. sylvestris subs. sylvestris, a. sylvestris subs. nemarosa and a. lamprocarpa'nın provided hybridization among themselves but they did not complete their speciation. it was determined that a.lamprocarpa var. chelikhii which is one of the two different varieties of a. lamprocarpa is actually a new sub-species. this fact was supported by molecular data obtained from the study we made after morphologic data. introduction: excessive production of androstenedione can becaused by defects of adrenal steroid biosynthesis, tumors of ovarian and adrenal origin, polycystic ovarian syndrome, increased peripheral sensitivity to androgens, and increased peripheral production of androgens. most epidemiologic studies use enzyme-linked immunosorbentassay (elisa) to measure sex steroid hormones because they have acceptable turnaround times and arerelatively inexpensive. mass spectrometry-based methods are currently the most specific quantitative analytical methods for steroid determination. mass spectrometry methods are independent of matrix effects or cross-reactivity. in this study, a new liquid chromatography-tandem mass spectrometry (lc-ms/ms) method was developed. materials and methods: for serum androstenedione measurement, 50 ll of internal standard (d5-11 deoxycortizol) in methanol was added to 250 ll standart or serum and centrifuged at 4.500 rpm for 10 minutes to remove the precipitated proteins. supernatant was transferred to clean tubes and this procedure was performed twice. the supernatant was collected and dried under a nitrogen gas flow at 60 • c and dissolved in mobile phase. 60 ll was injected in to the ultra performance liquid chromatography analytical column for chromatography. elisa study was conducted with drg (lot. no. 50k074) brand kit. results: method comporison between lc-ms/ms and elisa was found slope value 18,412, intercept value à22.87 and r² value 0.1033. the regressione quation was elisa= à2.861782 + 4.905103 lc-ms/ms. discussion and conclusion: method comparison study presented higher results in elisa compared to lc-ms/ms. in our opinion, this might due to the interference in elisa systems. our lc-ms/ms method allows rapid, sensitive and specific determination of androgens in plasma and serum.the specificity of liquid chromatography-tandem mass spectrometry (lc-ms/ ms) offers advantages over immunoassays. heparins play an important role in cell growth, differentiation, migration and invasion. however, the molecular mechanisms of heparin mediated cellular behaviors are not well defined. to determine the effect of heparin on gene expression, we performed a cdna microarray in a hepatocellular carcinoma cell line and found that heparin regulates transcription of genes involved in glucose metabolism. in this study, we showed a new role of heparin in the regulation of thioredoxin interacting protein, which is a major regulator of glucose metabolism, in hepatocellular carcinoma cell lines. we determined the importance of a unique carbohydrate response element located on its promoter for the heparin-induced activation of thioredoxin-interacting protein and the modulatory role of heparin on nuclear accumulation of carbohydrate response element associated proteins. we showed the importance of heparin mediated histone modifications and downregulation of enhancer of zeste 2 polycomb repressive complex 2 expression for heparin mediated overexpression of thioredoxininteracting protein. when we tested biological significance of these data; we observed that cells overexpressing thioredoxininteracting protein are less adhesive and proliferative, however they have a higher migration and invasion ability. interestingly, heparin treatment increased thioredoxin-interacting protein expression in liver of diabetic rats. in conclusion, our results show that heparin activates thioredoxin-interacting protein expression in liver and hepatocellular carcinoma cells and provide the first evidences of regulatory roles of heparin on carbohydrate response element associated factors. this study will contribute future understanding of the effect of heparin on glucose metabolism and glucose independent overexpression of thioredoxin-interacting protein during hepatocarcinogenesis. prolidase activity in chronic obstructive pulmonary disease and asthma t. g€ uc ßl€ u 1 , h. s€ urer 1 , g. bilgin 2 , d. y€ ucel 1 1 ankara training and research hospital, medical biochemistry department, ankara, turkey, 2 ankara training and research hospital, chest diseases department, ankara, turkey chronic obstructive pulmonary disease (copd) is a consequence of an underlying chronic inflammatory disorder of the airways that is usually progressive and causes dysregulation in the metabolism of collagen. and asthma is a disease where there is an accumulation of collagen in the reticular basal membrane of the airway leading to chronic inflammation. prolidase has an important role in the recycling of proline for collagen synthesis and cell growth. we measured and compared prolidase activity in healthy individuals with copd and asthma patients to find out that whether its activity might reflect disturbances of collagen metabolism in the patients. 60 patients with copd, 60 patients with asthma and 20 healthy control subjects with similar age range and sex were included in our study. the patient and control groups do not have any other chronic disease. serum prolidase activity was measured in the patient and control groups. ferritin and alpha-1 antitrypsin concentrations were also compared. there was no significant difference between serum prolidaz activities of asthma and copd patients. serum prolidase activities of both copd and asthma patients were significantly lower than those of the control subjects (p < 0.05). there was no significant difference for ferritine and alpha-1 antityripsin levels between the groups. the prolidase activity is significantly lower in asthma and copd patients comparing with control subjects. the collagen metabolism may be undergone to a change in these patients. hence, there may be an effect on the accumulation of collagen in the reticular basal membrane. the results suggest that collagen turnover are altered by the development of copd and asthma in human lungs, and prolidase activity may reflect disturbances of collagen metabolism in these pulmonary diseases. monitoring serum prolidase activity may be useful in evaluating fibrotic processes and in the chronic inflammatory lung diseases in human. acyclovir molecule in the active site of e. coli purine nucleoside phosphorylase (on the basis of x-ray study) i. kuranova 1,2 , v. timofeev 1,2 , n. zhukhlistova 1 , y. abramchik 3 , t. muravieva 3 , r. esipov 3 1 shubnikov institute of crystallography of fsrc "crystallography and photonics" ras, moscow, russia, 2 national research centre "kurchatov institute", moscow, russia, 3 shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia e. coli purine nucleoside phosphorylase (pnp), which catalyzes the reversible phosphorolysis of purine ribonucleosides, belongs to the family i of hexameric pnps. due to key role in the purine sulvage pathway pnps are attractive targets for drug design against some pathogens. they also used widely in biotechnology for the synthesis of nucleoside analogues as well as for the activation of the prodrugs in anti-cancer gene therapies. the acyclovir (acv), acyclic derivative of guanosine, is antiviral drug for the treatment of some human viral infections. the crystalline complex of e. coli pnp with acyclovir was prepared by co-crystallization using counter diffusion in capillary through the gel layer. the set of x-ray data at 100 k from single crystal grown in space (sp. group p6 1 22) was collected on the spring-8 synchrotron-radiation facility (japan) and the structure was solved at 2.32 a resolution, using the molecular replacement method (pdb id 5i3c). acv molecule was located in the nucleoside binding pocket of the enzyme in two conformations. the phosphate binding site was occupied by so4 ion. the hydrogen bonds network and hydrophobic interactions stabilising acv molecule in the active site as well as the conformational changes upon ligand binding were described. the comparison of e. coli pnp/acyclovir complex and the similar complexes of bacillus subtilis pnp (pdb id 4da7) and human pnp (pdb id 1pwy) allowed to establish the peculiarities of acv binding of in the e. coli enzyme. gonadotropins are glycoprotein hormones that regulate normal growth, sexual development, and reproductive function. these are large, up to 40 kda proteins, which are synthesized and secreted by the gonadotropic cells of the anterior pituitary gland. these hormones may vary in the level of glycosylation depending on the tissue and the metabolism cycles. follicle-stimulating hormone (fsh) and upon binding to fsh receptor, a g-protein coupled receptor (gpcr), regulates the development, growth, pubertal maturation, and reproductive processes of the body. human chorionic gonadotropin (hcg) and luteinizing hormone (lh) act via a shared gpcr (lh receptor) and regulate mechanisms essential for ovulation, early pregnancy and placental function in females as well as spermatogenesis and testosterone production in males. activation of gpcrs by these hormones can be measured by monitoring formation of cellular cyclic adenosine monophosphate (camp). the level on camp was measured using a f€ orster resonance energy transfer (fret)-based biosensor tepacvv (h74) kindly provided by dr, kees jalink. the biosensor was expressed using the developed bacmam gene delivery system (recombinant baculoviruses carrying the transgene under a strong mammalian promoter). kgn cells expressing the fsh receptor and cos7 cells expressing the lh receptor served as study objects. monitoring of specific gpcr activation in living cells, allows detection of only the biologically active agonists, which has real impact in quantification of large hormones. differences in levels of hormone glycosylation may affect their biological function. investigation of this phenomena is planned for near future. detection of biological activity of gonadotropins is of importance for pharmaceutical industry, where today the concentration of recombinant proteins is mostly estimated using immunological assays only. development of a colorimetric aptasensor for the detection of peanut allergen protein ara h 1 in food samples b. bora ege university, izmir, turkey food allergy, especially peanut allergy is a life-threatening problem, and severe reactions against these foods can be observed. since unintnded consumption of non-labeled foods is the most dangerous risk, any residual allergen protein should be tested and labeled by the manufacturers. an aptamer based colorimetric test is a powerful alternative to commercially available rt-pcr and elisa test kits. the main objective of this study is to develop an aptamer based colorimetric test fort he detection of major peanut allergen protein ara h 1. ara h 1 aptamer was used to recognize any residual peanut major allergen protein ara h 1 in food samples. recombinant ara h 1 protein was produced and puirifed to be used as a target. ara h 1 aptamer was used in combination with a blocking sequence, to prevent non-specific binding event, a biotinylated complementary strand to the blocking sequence, and finally strp-hrp interaction in order to facilitate colorimetric reaction. optimal blocking sequence length was optimized and introduced to the 3 0 site of aptamer sequence to construct an aptamer-hairpin structure. liberation of the blocking sequence allows biotinylated complementary strand to bind to the blocking sequence and consequently str-hrp conjugate to achieve color development that is proportional to the target concentration. since, the aptasensor will be used for the detection of ara h 1 in food samples, total protein extraction from chocolate samples was also optimized. in order to lower the detection limit of aptasensor, aptamer coupled magnetic bead based pre-enrichment assay was aslo optimized for the total protein extraction. as a result, a sensitive, fast and reliable aptamer based colorimetric assay was developed for the detection of peanut allergen protein from food samples. moreover, the assay has the advantages like ease of application and low cost which makes the assay a promising and a powerful alternative to commercially available rt-pcr and elisa tests. the association between lipid parameters and waist circumference in female university students in turkey s. ozen, a. cort sanko university, department of nutrition and dietetics, gaziantep, turkey a high waist circumference is associated with an increased risk for type 2 diabetes, dyslipidemia, hypertension, and cvd in patients with a bmi in a range between 25 and 34.9 kg/m 2 . monitoring changes in waist circumference may be helpful, in addition to measuring bmi, since it can provide an estimate of increased abdominal fat even in the absence of a change in bmi. objective of the study was to find an association between plasma lipid profile and anthropometric parameters (waist circumference percentage of body fat and body mass index (bmi)) in abdominal obesity in turkish university students. lipid profile and anthropometric parameters of obesity were studied in a sample of 30 women. students with high bmi (>24) had higher values of low-density lipoprotein (ldl), triglycerides (tg) and cholesterol (c) than students with low bmi (<24) but these differences were not significant. high-density lipoprotein (hdl) levels were non-significantly higher in low bmi (<24) student group. waist circumference, percentage of body fat was higher in high bmi (>24) group than low bmi (<24) group. waist circumference, percentage of body fat was positively correlated with bmi in both samples (bmi (>24) and bmi (<24)). students were grouped depend on their waist circumference. healty individuals who had lower than 80 cm waist circumference had decreased tg levels compared to cardiovascular risk group who had higher waist circumference than 80 cm. this study shows an association between waist circumference, percentage of body fat, body mass index and lipid parameters in young female university students. with regard to the relationship, the screening females for central obesity to prevention of cardiovascular disease are recommended. a new biotechnological product from propolis with low allergen: anti-inflammatory effect propolis is extensively used in food industry due to its special medical properies (antioxidant, antimicrobial, antiseptic, antibacterial, anti-inflammatory and antimutagenic effects). even these positive properties it may cause some allergic reactions in consumers with allergic predispositons. previously, we demonstrated that biotransformation of propolis by some special strains of lactobacillus plantarum (10, 8014, aatc strains) might decrease the allergenic molecules in propolis. in this study, we aimed to investigate the effect of biotransformation of popolis on it's antiinflammatory activities. before biotransformation, propolis samples were treated with different solutions (10% ethanol and polyethylene glycol -peg 40%) and different method (ultrasonic treatment 300 w/25 o c/ 30 minutes) in order to facilitate solvation of solid samples which are very dense and not suitable for fermentation. fermantations were performed at 30 o c/48 hours under constant agitation conditions. the anti-inflammatory activity was determined in-vitro conditions using hyaluronidase's analysis and the xanthine oxidase activity. the highest inhibition (%) of radicals produced by xanthine oxidase was determined in solid samples treated by peg prior to biotransformation and using of l.plantarum 8014 strain during fermentation (88.43%), followed by liquid samples treated by ultrasonic method prior to transformation (86.21%). concernig the results of hyaluronidase activity (%) inhibitions, the best value were determined in the solid sample treated by peg prior to biotransformation and using of l.plantarum 8014 strain during fermentation (93.93%). results indicated that the anti-inflammatory activities of analysed samples are quite high and depending of used extraction methods prior the biotransformation and used specific strain of l.plantarum could be optimized in terms of other required parameters. faceanti-mullerian hormone is not predictive for poor neonatal outcome aim: anti-mullerian hormone (amh) is a growth factor specific to ovaries. it is commonly used to predict ovarian reserve and outcomes of fertility treatments. recently, low levels of amh have been shown to be related to hypertensive diseases of the pregnancy and the risk of preterm labor. the aim of this study was to investigate the diagnostic performance of amh levels of mothers to predict poor neonatal outcome in term pregnancies and the relationship between amh and birthweights of the newborns. materials and methods: 187 patients, having delivery beyond 37 weeks, and who did not have any other medical problems were included in the study. the patients had normal 50 g. oral glucose tolerance test results. they were divided as 3 groups, based on their newborns' birthweight as "2500 g. and 4000 g.". level of amh was determined by elisa method. results: there was not any relation with the amh of the mothers and the poor neonatal outcome of the newborns, in all 3 groups. also no siginificant difference was observed in amh levels of the patients having delivery in early term and late term periods. when the patients of the same group were evaluated; amh levels were irrelevant to age, gravidy, delivery week, body mass index, the weight gain during pregnancy, and poor neonatal outcome. conclusion: amh is not a predictive factor for poor neonatal outcome and it is not a determinant of the weight of the newborn. objectives: the aim of the study was to investigate the effects of differing amounts of hemolysis on serum high sensitvity troponin i (hs-tni), ck-mb mass and myoglobin measurements. materials and methods: we prepared serum pools having troponin i, ck-mb and myoglobulin concentrations at low (5.33 ng/l,1.5 ng/ml and 34.3 ng/ml respectively), normal (39.25 ng/l, 3 ng/ml, 197.9 ng/l respectively) and high (7345 ng/l, 22 ng/ml, 574 g/ml respectively) values. the osmotic shock method was utilized to prepare a hemolysate. hemolysate was added into serum pools increasing concentrations of hemoglobin (0.65, 1.3, 2.5, 5, 7.26 and 9 .42 g/l hemoglobin). troponin i, ck-mb (mass) and myoglobin concentrations were measured in duplicate by beckman coulter access 2 analyzer. the hemolysis indices were measured on beckman coulter au 5800. a change of 10% from baseline results was taken as evidence of interference and the interfered tests were also evaluated according to total analytical error based on analytical imprecision and intraindividual biological variation. results: we found a positive interference due to hemolysis for ck-mb (mass) at low concentrations (1.5 ng/ml), and a negative interference for myoglobin at low concentrations (34.3 ng/ml) and high concentrations (574 ng/ml). conclusions: ck-mb increase and myoglobin decrease in hemolyzed samples with hemoglobin ≥9.42 g/l, but the bias might not be clinically significant (< total analytical error) in samples. a retrospective study to determine a reliable marker for selective screening of pompe disease lysosomal storage diseases (lsd) are rare inherited metabolic disorders caused as consequence of a deficiency in a specific enzyme required for lysosomal function. pompe disease is one of these disorders with deficiency of a-1,4 glycosidase enzyme with an incidence of 1:4,500-1:33,000. as enzyme replacement therapies are available nowadays, early diagnosis is crucial and selective screening is a rational method to reach pompe patients among people who administer to healthcare with lsd suspected symptoms. this study aims to examine the relationship between basic biochemistry parameters and a-1,4-glycosidase activities retrospectively, in order to find a key parameter for selective screening of pompe disease. for this reason a-1,4glycosidase, creatine kinase (ck), creatine kinase-mb (ck-mb) activities calcium, phosphate levels of those who had been suspected to be lsd patients and administered to our laboratory for analysis are examined retrospectively. out of 134 patients's examined,14 of them were diagnosed with pompe disease depending on clinical findings & low a-1,4glycosidase activity. enzyme activities of pompe patients were 0.337 nmol/ml/hour as lsd suspected patients'activities had a mean of 2.78 nmol/ml/hour (p = 0.00).comparison of ck activity was compared results showed significant difference between pompe patients and lsd suspected patients. even though ck activity levels of the lsd suspected patients were much higher (400vs41-171u/l) than reference interval, the levels of the pompe disease patients' were still more than twice of the lsd suspected group (956vs400u/l, p = 0.02). ck-mb, ca, p levels didn't show a significant difference. a strong (-) correlation (p = 0.005 r=à0.240) was observed between a-1,4-glycosidase and ck activities (n:134). selective screening is a rational way to diagnose rare diseases. this study's results show that ck activity can be used as a key parameter to determine patients for selective screening of pompe disease within lsd suspected population. the functional effect of stem cells on the reproductive organs infertitility is considered as a major health problem of recent century. importance of stem cell is increasing so it is searched new features and supposed to be involved in the infertitility treatment where oxidative stress and apoptosis play importany role. we aimed to investigate the beneficial effect of the stem cells related to free radicals and cell death on testis and ovary. biopcy model of wound healing was created in the rat testis and ovary with ppd syringe where stem cells were delivered by injection. rats were divided into four groups including controls, sham, wound healing and wound healing with stem cell. after the creation of the wound, bone marrow-derived mesenchymal stem cells from the tibia of the mature rats and medium were administered to ovaries and testes. following the applications, ovary and testis samples were investigated for oxidative stress and apoptosis by immunohistochemistry. in comparison with the medium and stem cell applications without a medium support, it was meaningfully determined that healing effect in testicles and ovaries were spotted specifically on the seven day. tissues were analysed for these staining by h-score and h-score results were determined using one-way anova test statistically. our results show the positive effects which clinic applications can bring by displaying the great contribution of the stem cell application in the treatment of testicle and ovary damage. these findings suggest that transplantation of the mesenchymal stem cells may help to promote better enviroment for the reproductive organs by the effect on oxidative stress and apoptosis. the further studies of these results in the molecular level can lead the way to solve the problem of infertility, to increase the percentage of success in the ivf and icsi techniques and more importantly to perform a differentiation from a somatic cell to a germ cell. the antimicrobial activity of 2 (5h)-furanone derivative on staphylococcus aureus nosocomial infections caused by methicillin-resistant staphylococcus aureus strains are known to be a reason of many infectious diseases like osteomyelitis, endocarditis, sepsis etc. being organized in biofilms these bacteria become extremely resistant to antimicrobials and host immune system leading to difficulties in treatments. here we report the effect of 2 (5h)-furanone derivative possessing sulfonyl group and l-menthol moiety (f105) on biofilms formed by s. aureus atcc29213 and mrsa cells. while exhibiting relatively high minimal inhibiting concentration -mic (16 mg/l), clear synergy with a number of antibiotics was found in the checkerboard assay. thus, in the presence of 2 mg/l of f105 the mic of kanamycin was decreased 4-fold, and the mics of both erythromycin and ampicillin were lowered 2-fold. at the concentration of 80 mg/l f105 also completely inhibited the biofilm formation by s. aureus; the cell growth was suppressed by two orders of magnitude as judged by differential fluorescent staining with syto9 and propidium iodide. the addition of f105 to preformed 24 h-old biofilms increased the fraction of red-stained (dead) cells of both s. aureus atcc29213 and mrsa strains uniformly throughout the whole profile of the biofilm. the quantitative analysis of clsm microphotographs revealed that f105 at concentration of 80 mg/l led to death of up to 98% of biofilm-embedded cells. this fact suggests that f105 efficiently penetrates into the biofilm matrix and kills the cells without visible damage of biofilm structure. in summary, furanone f105 seems to be a promising compound for drugs design to treat biofilm-embedded s. aureus. this work is supported by the russian science foundation, project №15-14-00046 and the german academic exchange service (№91531398). pneumonia is an inflammatory lung disease which can be associated with inadequacy of host defense system and the proliferation of various pathogenic microorganisms into the lower respiratory tract. community acquired pneumonia (cap) is one of the leading causes of death in elderly. the incidence of pneumonia in people aged 65 and over is 3-5 times more than young adults. creactive protein (crp) is an acute-phase protein of hepatic origin that increases following interleukin-6 secretion by t cells and macrophages. procalcitonin (pct) is a peptide precursor of the hormone calcitonin, the latter being involved with calcium homeostasis. it is composed of 116 amino acids and is produced by parafollicular cells (c cells) of the thyroid and by the neuroendocrine cells of the lung and the intestine. the level of pct rises in a response to a proinflammatory stimulus, especially of bacterial origin. the aim of this study was to compare crp and pct levels in young and elderly patients with pneumonia. recently diagnosed 43 young and 46 elderly patients with pneumonia and their respective aged matched controls (n = 41, n = 42) were enrolled this study. crp and pct levels were by immunoturbidometric and by elisa methods respectively. crp and pct levels for young control and patients and elderly control and patients respectively are 2.32 ae 2.25 mg/l, 0.26 ae 0.13 ng/ml, 20.71 ae 34.61 mg/l, 0.64 ae 1.09 ng/ml, 2.32 ae 2.14 mg/l, 0.27 ae 0.07 ng/ml and 31.41 ae 31.0 mg/l, 0.49 ae 0.83 ng/ml. young patients with pneumonia have significantly higher crp and pct levels than their controls (p < 0.001 and p < 0.028). elderly patients with pneumonia have significantly higher crp levels than their controls (p < 0.001). crp and pct are important markers in the diagnosis of pneumonia. effect of serum albumin concentration on total and ionized calcium z. adiyaman, c. yilmaz, s. a. peker, d. y€ ucel ankara training and research hospital, ankara, turkey objective: the aim of the study is to investigate in vitro effect of albumin concentration on total and ionized calcium concentrations. materials and methods: a serum pool with low albumin (3.10 g/dl) and normal calcium (9.79 mg/dl) concentrations was prepared from leftover sera. from this serum pool, two parts, each of 10 ml were aliquoted. purified albumin, 0.3 g, was added to one of these pools and albumin concentration was determined as 6.1 g/dl. the low and high albumin pools were mixed at different ratios and pools with 3.89, 4.78, and 5.37 g/dl albumin concentrations. total calcium and albumin concentrations of these 5 pools were measured at a beckman-coulter au5800 analyzer and ionized calcium was measured at a radiometer abl 800 blood gas analyzer in triplicate. total and ionized calcium concentrations were evaluated as compared to those of the original pool with an albumin concentration of 3.10 g/dl. results: total calcium concentrations are increased with the increasing albumin concentrations: 1.2%, 1.58%, 2.85%, and 3.59%, respectively. whereas, ionized calcium concentrations were decreased with increasing albumin: 3.0%, 6.1%, 7.4%, and 8.6%, respectively. conclusions: when total allowable error limits based on biological variation were considered, total calcium concentrations are significantly increased at >5 g/dl albumin concentrations. ionized calcium is significantly affected by 0.8 g/dl and over albumin concentrations. a regression equation based on albumin concentration may be useful for corrected ionized calcium concentrations. relationship between lipoprotein (a) and hba1c in patients with type ii diabetes , is a complex lipoprotein consisting of ldl and apolipoprotein(a). lp(a) is a risk factor for coronary artery disease and stroke. the relationship between lp(a) and diabetes mellitus is not clear. in this study, the relationship between lp(a) and glycemic parameters such as hba1c and fasting glucose concentration was investigated. lp(a), hba1c, fasting glucose, triglyceride, total cholesterol, ldl-and hdl-cholesterol concentrations were screened retrospectively from july 2013 to july 2016. there were 1831 patients with these test results at the same time. the patients were grouped according to hba1c values: group i < 5.7% (n = 468), group ii 5.7-6.4% (n = 739), and group iii >6.5% (n = 624). the relationship between these parameters were statistically within each group and all groups. there was not a statistically significant difference between the lp(a) concentrations of group i and group ii. lp(a) concentrations of group i and ii were significantly higher than those of group iii.. _ in total, lp (a) was negatively correlated with hba1c (r = 0.09; p < 0.01), but there was not a significant correlation with fasting blood glucose. _ in groups, there was a significant and negative correlation between lp(a) and fasting glucose in only group i. the negative correlation between lp(a) and glycemic parameters is interesting in patients with diabetes. despite lp(a) is an independent risk factor for cardiovascular diseases, on the contrary to expectations, lp(a) concentrations are decreased in diabetes. effect of blood collection through intravenous lines on hemolysis erroneous results are one of the most important causes of medical errors and may lead to unnecessary investigations or inappropriate interventions. total testing process consists of preanalytical, analytical and postanalytical phases. hemolyzed specimens that one of the most common source of preanalytical errors are frequently observed in laboratory practice and associated with incorrect laboratory results. blood collection through intravenous lines frequently results in hemolysis especially at eds and icus. in this study, we aimed to compare the effect of blood drawing by using bd luer-lock adapters and injector on the hemolysis rates at the ed. 60 patients who has been admitted to the ed were included in this study. all samples were drawn from newly inserted iv lines. the first blood sample was drawn with injector and the second one was drawn with luer-lock adapters to vacuum tubes. after the centrifugation routine chemistry tests and hemolysis indices were analysed on a beckman coulter au680 analyzer for each serum tube. the statistical significance of differences between two tubes was calculated with paired samples t test and statistical significance was accepted as p < 0.05. there were statistically significant differences between the two groups of tubes for the following parameters: ldh, ck, ast, k + , total bilirubin, protein, albumin, alp, calcium and hemolysis index (p < 0.05). the use of luer-lock adapters instead of injector could reduce the hemolysis rate. because of it reduces false results and unnecessary investigations, this approach will be more appropriate and cost-effective in ed. hemolysis and test rejection: are we following a reliable process? introduction: in laboratories, some blood samples are rejected due to hemolysis. we usually cancel only some of the tests that are affected by hemolysis. however, the frequency of the test cancellation may be relative. each test is affected in different degrees of hemolysis; some of them are not even affected at all. in this study, we aim to investigate unnecessary cancellations and explain the relationship between hemolysis and test results according to their kit inserts. materials and methods: we measured hemoglobin levels of 50 hemolyzed serum using drabkin method (abbott). interference studies are conducted using clsi protocol nccls ep7-p is written in kit inserts. target values (100%) and their change due to different degree of hemolysis have been defined. results: hb concentration ranges of hemolyzed sera were found from 44 to 849 mg/dl. according to kit inserts, aspartate aminotransferase (ast) test results deviate 5.7% from the target when the degrees of hb are 62 mg/dl. when the degree of hemoglobin is 125 mg/dl, the test strays about 11.6%. potassium levels increase (108%) at 125 mg/dl hb while this increase reaches to 17.9% at 250 mg/dl hb. sodium, calcium, ck, crea, total bil, lipase are not significantly affected even at 2000 mg/dl. in lactate dehydrogenase (ldh) tests, test reporting is not allowed at any hemolysis level. alt increases 11%, at the 1000 mg/dl hb. ast and potassium results were excluded from patients' reports even though those samples had low hb. some of them were reported despite of excess hemolysis. some tests are even blocked without ever being studied. discussion: prior to the approval of the lab specialist, technicians decide whether to cancel the tests affected by the hemolysis according to the visible hemolysis based on their personal knowledge. conclusion: we should use the hemolysis index, in which standards would be defined via guidelines. this way, all technicians and specialists could know which results are false. the dna-binding hu-proteins are present in all bacteria and belong to the family of nucleoid-associated proteins. these proteins can be considered precursors to eukaryotic histones. gene knockout of hu-proteins partially inhibits the growth of bacteria, their ability to resist various stressing factors and in some cases leads to their death. since the spatial structure of hu-proteins is highly conserved it is possible to create inhibitors that will affect them in a broad spectrum of pathogenic bacteria. in the present work the preparation of the recombinant hu protein from mycoplasma gallisepticum, crystallization of this protein, and x-ray diffraction study of this protein has been reported. the crystallization conditions for studying protein were found by the hanging-drop vapor-diffusion method. found conditions have been adapted to the counther-diffusion method in the capillary. the x-ray diffaction dataset from grown crystals have been collected using synchrotron radiation. 3d-structure of the hu protein from mycoplasma gallisepticum have been determined with 3a resolution. structural features of the investigated protein are described. this work is supported by russian scientific fund (15-14-00063). a novel sensitive disposable indium tin oxide (ito)-based electrochemical immunosensor was developed for simple, rapid and sensitive biomonitoring of sox2. sox2 is a cancer biomarker and used for detecting of small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, skin cancer, prostate cancer, and breast cancer. in this study, indium thin oxide (ito) thin film was used as working electrode. carboxyethylsilanetriol was also used for electrode modifying so as to obtain self-assembled monolayers. the formed self-assembled monolayers were activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (edc)/n-hydroxysuccinimide (nhs) chemistry. edc was used as a heterobifunctional crosslinker. nhs was used in conjunction with the crosslinker edc. anti-sox2 antibody was used as a biorecognition element and it was covalently immobilized onto the ito electrode modified with carboxyethylsilanetriol. immobiliztion steps were characterized by cyclic voltammetry (cv), electrochemical impedance spectroscopy (eis), and scanning electron microscopy (sem). the optimal immobilization conditions for the best sensitivity of the new immunosensor were investigated. under optimal conditions, this immunosensor demonstrated a wide linear range (0.0025-2 pg/ml) with a detection limit as low as 0.02 ng/ml sox2. furthermore, the developed sox2 immunosensor had good storage stability, repeatability and reproducibility. in this work, we successfully fabricated disposable ito thin film based electrodes for sensing the interaction between sox2 antigen and anti-sox2 antibody by electrochemical impedance spectroscopy and cyclic voltammetry. and our developed immunosensor has an acceptable performances for the detection of sox2 antigen, exhibits low detection limit, has selective and reproducible results in immunoreaction analysis. we are thankful for the support from t € ub _ itak (the scientific and technological research council of turkey, project number: 113 z 678). applying multiple linear regression model to determine the relationship between anti mullerian hormone with age, luteinizing hormone, follicle stimulating hormone and estradiol: a data mining study introduction: anti mullerian hormone (amh) has a widely used in our life because it is a good indicator of reproductive age to estimate the time of menopause. the purpose of this retrospective data mining study is the estimate of ovarian reserve by using amh and determines relationship between other indicators which are luteinizing hormone (lh), follicle stimulating hormone (fsh), estradiol and age. materials and methods: 25.294 women members were included this retrospective data mining study who were applying to acıbadem labmed laboratory. multiple regression analysis of age related changes of amh (18-45) and lh, fsh and estradiol were investigated. beckman gen ii elisa kit was used for amh and the technique of electrochemiluminescence and roche elecsys cobas analyzer were used for the measure of other hormones. results: amh shows meaningful correlation between lh, fsh, estradiol and age but also seen there is no correlation between progesterone. after the multiple linear regression analysiz amh= 11.018-(0.220 9 age)à(0.066 9 fsh) + (0.044 9 lh)à(0.004 9 estradiol) is detected and the model's r 2 = 0.627 is also detected. conclusion: nowadays there are lots of methodology were developed the estimate the function of ovary and biological age of ovarian. age, fsh, lh and estradiol show ovarian reserve by indirectly. this study shows the mathematical relationship between amh and the other indicators and results are thought to lead to future developments. antioxidant and anticancer effect of artemisia absinthium extract on colon and endometrium adenocarcinoma cells plants have always been among the common sources of medicines that have many phytochemicals with various bioactivities, including antioxidant and anticancer activities artemisia absinthium (ar) has been used as an antipyretic, antiseptic, anthelmintic, tonic, diuretic, and for the treatment of stomachaches in turkish folk medicine. this study aimed to investigate antioxidant, cytotoxic, genotoxic and apoptotic effect of methanol extracts of ar activities on the human colon (dld-1) and endometrium (ecc-1) adenocarcinoma cell line. total phenolic, flavonoid content, and antioxidant activities were determined using suitable methods (abts, cuprac i.e). cytotoxic effects of ar on cells were determined by mtt and neutral red uptake assays. genotoxicity was evaluated by comet assay and, apoptosis induction were detected by apoptosis elisa and acridine orange staining methods at the half maximal inhibitory concentrations (ic50) levels. it was determined that extract have shown antioxidant activity in all tests and that they could be considered as a source of natural antioxidants. cytotoxic effects were concentration-time dependent. specifically, apoptotic and genotoxic effect increased at 100 and 200 lg/ml concentrations by 48 hours. we found that ar extract had antiproliferative, genotoxic and apoptotic effects on the human cancer cell lines dld-1 and ecc-1. however, further studies at molecular level are required to support our findings and to elucidate chemopreventive and chemotherapeutic effects of ar on colon and endometrium cancers. keywords: artemisia absinthium, antioxidant, anticancer, apoptosis, genotoxicity introduction: colorectal cancer is considered as a major gastrointestinal. this cancer is the second cancer related cause of death after lung cancer in worldwide. we designed a vaccine chimeric including cea and ca19-9 against colorectal cancer (ce-ca). materials and methods: the construct were analyzed by bioinformatics softwares. in this study, the ce-ca gene was optimized using the codon bias of e.coli and synthesized by biomatik company. then construct (ce-ca) was cloned into an expression vector and recombinant constructs transferred to e.coli bl21de bacterium and desired recombinant protein was expressed. recombinant protein was purified using ni-nta affinity chromatography. the content of secondary structures was obtained by circular dichroism (cd) spectrum. then recombinant protein was confirmed using western blot analysis and indirect elisa method. results: sds-page analysis showed that the recombinant protein was highly expressed and purified. western blot analysis confirmed recombinant protein. also cd spectrum confirmed predicted structures by bioinformatics tools. the elisa results showed significantly high affinity toward recombinant ce-ca protein. discussion: based on many studies, cea as potential immunogenic candidate could be considered in vaccine studies. also ca19-9 is a cell-surface antigen that has significant increase of expression in colorectal cancer, thus as marker of colorectal cancer. based in available data, these two antigens, in combination can provide specificity for production of colorectal cancer vaccine. conclusion: these findings suggest that ce-ca as potential immunogenic candidate which could be considered in future vaccine studies and detection of colorectal cancer. flow cytometric cell cycle and apoptosis analyses of some wild animal species a. tas, e. koban bostanlar tubitak, marmara research center (mrc), genetic engineering and biotechnology institute (gebi), animal genetic and reproductive biology laboratory, kocaeli, turkey cell biobanking; more specifically cryopreservation of biological diversity, is promising as a tool to preserve wild animals as well as domestic ones via nuclear transfer. in this study, we investigated the viability and cell cycle characteristics of 5 wild animal species (fallow deer, red deer, wild sheep, wolf, wild goat). auricular tissue samples were maintained in pbs+2%psa. tissues were seeded on 35 mm petri dishes containing dmem/high glucose supplemented with 20% (v/v) fcs and incubated 5%co2 in air at 95% relative humidity and at 37°c. after seeding, the medium was unchanged for 7 days and then it was changed in every 2 days for 25 days at maximum. once the cells were obtained; flow cytometric cell cycle and apoptosis analyses were done. in terms of apoptosis, all the groups showed high viability rates (over 95%) in culture when compared with the negative control (38%). the cell cycle comparisons were made between serum-starved cells and roscovitine treated cells, both for which untreated cells were used as control, which revealed different results for different species. there was no difference found between serum-starved cells and roscovitine treated cells for red deer and wolf. the serum-starved cells resulted in higher g0/g1 phase for fallow deer and wild goat. on the contrary, roscovitine treated cells resulted in higher g0/g1 phase for wild sheep. as a result; the cells obtained from wild animals had high viability and g0/g1 phase rates. therefore, they may serve as a donor cell source for nuclear transfer studies.(grant: tubitak kamag, turkey, 109g016). the interaction of different types of antibiotics with endothelial cells in the presence of nanoparticles the interaction of nanomaterials with cells and lipid bilayers is critical in many applications such as phototherapy, imaging, and drug/gene delivery. the aim of this study was to investigate the interaction of nanoparticles (fe 3 o 4 ) or nanoparticles fused with different antibiotics with cell membranes in order to reveal changes in the membrane organization. endothelial cells were used to determine the effect of different antibiotics (gentamicin, kanamycin, amikacin, penicillin, polymyxin, neomycin, cefotaxime, bacitracin, moxicillin, erythromycin, streptomycin and vancomycin) on the membrane organization. for recording the anisotropy of cell suspensions treated with antibiotics or nanoparticles fused with antibiotics we used 1-4-trimethyl-6-phenyl 1, 3, 5 hexatrien p-toluenesulfonate (tma-dph). we decided to use nanoparticles fused with antibiotics because they contain small amounts of antibiotics which makes them less toxic than simple antibiotics,which is very important in patients with genetic diseases such as cystic fibrosis, that should be treated with antibiotics for a long time. our results showed that at temperatures between 31 and 35°c simple nanoparticles decreased the membrane fluidity. at physiological temperatures (37-39°c) nanoparticles fused with antibiotics (gentamicin, vancomicin, cefotaxim, bacitracin, amoxicillin) increase more the membrane rigidity compare with simple antibiotics or nanoparticles.erythromycin, polymyxin and penicillin increase the membrane rigidity at 37°c, and at 39°c the same effect was obtained in the presence of nanoparticles fused with these antibiotics,suggesting that the nanoparticles are dependent to temperature for penetrating the membrane. in conclusion the membrane fluidity does not depend on antibiotics types, the modification are present in many antibiotics irrespective of class type.the presence of nanoparticles fused with antibiotics is very important for long term treatment. objectives: hypertension is an important cardiovascular risk factor for the development of atrial fibrillation (af). increased atrial electromechanical coupling time interval measured by tissue doppler is accepted as an important factor for prediction of af development in hypertensive patients. monoamine oxidases (maos), are enzymes which catalyze the oxidation of monoamines. 8-isoprostane is considered as an indicator of oxidative stress. mao activity and 8-isoprostane levels were measured in some diseases. however, there are no information on 8-isoprostane levels and mao activity in newly diagnosed patients with stage 1 hypertension has not been observed in a study of literature. aim: this is the first study, we aimed to evaluate the levels of mao and 8-isoprostane in newly diagnosed patients with stage 1 hypertension. the study included 60 newly diagnosed stage 1 hypertensive patients with no other systemic disease. 30 patients were selected as randomized (17 women, 13 men; range of age 46-74 years) and 30 healthy individuals as control (15 women, 15 men; range of age 44-72 years). all the underwent tissue doppler echocardiographic examination. blood samples were taken from patients and controls and, the levels of mao and 8-isoprostane in serum samples were measured by elisa. results: baseline blood pressures, electrocardiographic and echocardiographic findings, and atrial electromechanical coupling were similar in both groups (p > 0.05). compared to the control group, the activity of mao and 8-isoprostane levels were found significantly higher in patients (p < 0.05). conclusion: increased 8-isoprostane level indicate that there is oxidative stress in newly diagnosed patients with stage 1 hypertension. also, increased mao activity may be biochemical biomarkers for the diagnosis of hypertension. keywords: hypertension, monoamine oxidase, 8-isoprostane p-mis-147 determining the indirect reference intervals for complete blood count parameters in bursa, turkey reference intervals (ris) for laboratory test results are defined as the most commonly used diagnostic tool in medicine. therefore, careful determination of ris by the laboratory for use is a very important task. although c28-a3 guideline recommends the direct ris (dris) calculated from healthy subjects, ris can be calculated from laboratory data which are called as indirect ris (iris). the study was carried out at the central laboratory for clinical chemistry, teaching and research (uludag university, bursa, turkey) . the results of the laboratory analyses from 142,591 males, 215,658 females, stored for approximately one year, were used for statistical analysis. data for hospitalized patients and for ambulatory patients from the intensive care unit were eliminated. furthermore, we used evidence based criteria to enrich the health-related values. a modified bhattacharya procedure was used to estimate the iris from hospital patient data. the nested anova was used to evaluate variations among genders and ages. cell dyn analyzer (abbott diagnostics, il, us) was used for the measurements of complete blood count. the obtained iris were also compared the dris determined in our previous ri study and the ris suggested by the manufacturer. we found that the ris of rbc, hb and hct required strong gender partition and calculated the ris of rbc, hb and hct separately. the observed iris for wbc, sub-fractions of wbc and plt in both genders are in good accordance with the dris reported in previous study. age-related changes were noted for rbc, hb, and hct. the calculated iris for rbc, mcv and rdw are different from the ris suggested by the manufacturer. we believe that, using this relatively easy technique, every laboratory can produce its own iris, divided, where possible, according to sex and age and according to local conditions. these ranges can be complementary to dris obtained for reference individuals according to the ifcc recommendations. the principal sigma 70 subunit, involved in transcription of most house-keeping genes in escherichia coli, was also shown to induce rnap pausing during transcription elongation, by interacting with promoter-like motifs in the transcribed dna. such pauses were proposed to play important roles in the regulation of phage and cellular genes. e. coli contains six alternative s subunits but little is known about their ability to induce transcriptional pausing. we expressed and purified alternative s subunits of the sigma 70 family and tested their effects on transcription elongation in vitro on natural and synthetic dna templates containing consensus promoter motifs. the structure of the paused complexes was analyzed by dna footprinting methods. in vivo analysis of transcription was performed using reporter genes placed under the control of corresponding promoters. we demonstrated that the stationary phase sigma 38 subunit induced efficient rnap pausing on both synthetic and natural dna templates containing promoter-like motifs in initially transcribed regions. in contrast, the sigma 32 and sigma 28 subunits did not affect rna elongation. we showed that the sigma 38 -induced pausing depends on sigma contacts with both nontemplate dna strand and rnap core. the pausing results in formation of backtracked transcription elongation complexes which can be reactivated by gre factors that stimulate rna cleavage by rnap. our results for the first time reveal transcriptional pausing induced by an alternative s subunit. analysis of sigma 38 -dependent promoters shows that a substantial fraction of them contains potential pause-inducing motifs suggesting that such pausing may be a widespread phenomenon. we propose that sigma 38 -dependent pauses may play important roles in genetic regulation and modulate the binding of transcription repressors or activators to promoter regions. the crosstalk between streptococcus pneumoniae rnase r, ribosomes and translation c. b arria, s. domingues, c. arraiano instituto de tecnologia qu ımica e biol ogica, lisbon, portugal ribonucleases (rnases) are enzymes that ensure maturation, degradation and quality control of rna thus, contributing to the maintenance of the optimal amount of each transcript in the cells. escherichia coli rnb family of enzymes is present in all domains of life and includes rnase r, rnase ii and the eukaryotic rrp44/dis3, dis3l1 and dis3l2 proteins. in streptococcus pneumoniae only rnase r was identified. rnase r, encoded by the rnr gene, hydrolyzes rnas starting from the 3 0 end. rnase r level is increased in several stress conditions such as heat shock, stationary phase or cold shock, conditions in which most of the proteins translation is blocked. moreover, rnase r is the only exoribonuclease able to degrade highly structured rnas without the help of a helicase which is critical at low temperatures. here, we investigated the role of this enzyme by comparing the wild type strain with an rnr mutant strain. for that purpose we performed northern blots analysis of transcripts involved in translation. also, we investigated rnase r connection to the ribosome and polysome fractions using sucrose gradient polysome separation and western blots. in this study, we highlight the importance of s. pneumoniae rnase r in translation. we show that this enzyme interacts with ribosomes mostly with the 30s subunit at 37°c. moreover, in the absence of this enzyme we have observed a decrease in the amount of the 70s ribosomal subunit, concomitantly with the increase of 30s and 50s subunits. rnase r seems also to modulate the amount of the elongation factors ef-tu and ef-g transcripts. nevertheless, preliminary results further suggest other roles of rnase r in translation. modified nucleotides are present in many rna species in all domains of life. the biosynthetic pathways of such nucleotides are well studied. however, much less is known about the degradation of rnas and the salvage of modified nucleotides, their respective nucleosides or heterocyclic bases. using an e. coli uracil auxotrophic strain, we screened the metagenomic libraries for genes, which would allow the conversion of 2-thiouracil to uracil and thereby lead to the growth on a defined synthetic medium. we show that a novel gene encoding previously uncharacterized domain of unknown function (duf) is responsible for such phenotype. we have purified this recombinant protein and demonstrated that it contains a fe-s cluster. the substitution of cysteines, which have been predicted to bind such clusters, with alanines abolished the growth phenotype. we conclude that this domain is required for conversion of 2-thiouracil into uracil in vivo. this work is supported by the research council of lithuania (lmt, mip-103/2015 modified nucleotides are present in almost all classes of rna. they have great chemical diversity and are critical for rna folding, stability, interaction with cellular proteins and thereby for various cellular processes such as translation, stress response, and signaling pathways. biosynthesis of pyrimidine nucleotides and their modified derivatives in rna is well studied. nonetheless, not much is known about the cellular degradation of these compounds and the enzymes catalyzing such processes. using an e. coli uracil auxotrophic strain, we screened metagenomic libraries for genes encoding isocytosine deaminases. three novel genes were obtained, one of which encodes a protein similar to 8oxoguanine deaminases. the other two encode proteins resembling hydroxydechloroatrazine ethylaminohydrolases. we confirmed that these proteins are functional in vivo, allowing growth of e.coli on minimal medium with isocytosine. we also demonstrated that such purified recombinant enzymes catalyze the conversion of isocytosine, but not cytosine, into uracil in vitro. natural products display special attributes in the treatment and prevention of various human diseases, including cancer. a significant number of organic compounds from plants exhibit anticancer properties as attested by in vitro and in vivo studies. emerging evidence supporting the antineoplastic activity of natural compounds has rendered them promising agents in the fight against cancer. in this study, skin from limnio grape, a red greek grape variety that is indigenous to the greek island of lemnos, was extracted using mixtures of methanol, water and acetone; the apoptosis-inducing properties of these extracts were studied in the human ovarian malignant adenocarcinoma cell lines tov-21g and tov-112d. for this purpose, tov-21g and tov-112d cells were treated with limnio grape skin extracts at a range of concentrations, at 37°c, for 24, 48 and 72 hours. untreated cells incubated for the same time intervals served as controls. cell viability was determined by measuring metabolic activity (colorimetric mtt assay) and observing cell membrane integrity (cell staining with trypan blue). after the determination of the optimal concentration of the extract, total rna was extracted from treated and untreated (control) tov-21g and tov-112d cells. after determination of rna concentration and subsequent first-strand cdna synthesis, mrna expression analysis of apoptosis-related genes was performed with rt-pcr using gene-specific primers. an increasing percentage of non-viable cells was observed by increasing cell exposure time and extract concentration. distinct modulations of the expression of apoptosis-related genes at the mrna level were also observed, mainly concerning bcl2, bclx, bax, bak1 and bcl2l12, along apoptosis induction. in conclusion, the cytotoxic properties of limnio grape skin extracts against ovarian malignant adenocarcinoma cells merit further investigation. the intrinsic apoptotic pathway seems to be the major mechanism of action induced by these plant extracts. almost all eukaryotic mrnas are polyadenylated by a complex machinery that recognizes the poly (a) signal, cleaves the mrna and adds the poly (a) tail. 70% of human genes harbor multiple poly (a) signals. alternative polyadenylation (apa) generates transcript isoforms with different 3 0 utr (untranslated region) lengths due to the use of proximal or distal poly (a) signals. hence, tightly regulated apa has been observed in normal physiological settings as well as in diseases. considering that 3 0 utr shortening cases have been linked to increased protein levels, we hypothesized deregulated apa to be one of the potential cancer related mechanisms. we investigated the 3 0 utr alterations in er(+) breast cancer patients and cell models compared to normal breast tissue, using gene expression data and a probe-based quantification tool, apadetect. based on means of proximal to distal probe sets, slr (short-long ratio) were calculated as an indication apa. significance analysis of microarrays (sam) determined significant genes. the gse numbers of the datasets are gse2034, gse7390 and gse9761. we analyzed two datasets of er(+) breast cancer patient samples (n = 209, n = 135) compared to normal breast tissue (n = 82) using apadetect and sam. a total of 184 3 0 utr shortening and 253 3 0 utr lengthening events were detected in breast cancer samples compared to normal breast tissue. ontology analysis suggested almost all the 3 0 utr shortening genes were proliferation related and were indeed reported to be upregulated in breast cancer. to further investigate the connection between apa and era status, we used data from a cell line model; wild type or era transfected mda-mb-231 cells that are otherwise of triple negative nature. our results suggested that most of the genes are 3 0 utr shortened or lengthened via direct binding of era to dna. our results suggest involvement of apa mechanisms in era action mechanisms. possible link between era regulated transcription and apa remains to be elucidated. contamination of nucleic acids (na) as a result of na extraction protocols may result an inaccurate measurement of dna copy number. agarose gel electrophoresis and spectrophotometric methods are commonly used to check dna purity. however, the resolution of these methods may not be good enough for special applications such as determination of dna copy number and separation of base pairs (bp) that are close in their bp number. in this study, we have developed a new method for separating na's ranging between 75-20000 bp also detecting the impurities in dna solution in 1%, 5% and 10% ratios to the dna of interest. the developed method was validated using the in-house dna fragments of 100, 150 and 200 bp. the dna mixture analyzed using analytical hitachi elite lachrom hplc using the guard and analytical columns tskgel dna-npr, 2.5 lm, 4.6 mm id 9 0.5 cm and tskgel dna-npr, 2.5 lm, 4.6 mm id 9 7.5 cm, respectively. the validation of the analysis was performed by running each sample five times on three different days. the linearity of the detector response was established by plotting a graph to quantity versus area of 200 bp dna. the lod and loq were then measured by calculating the minimum level at which analyte can be readily detected and quantified. the ratios calculated with hplc were compared to the ratios calculated by quant-it kit. recovery values were calculated for each measurement and the uncertainty were calculated for each ratio. the method was found linear for 200 bp in the range of 0.4 ng to 800 ng dna with the regression coefficient of r 2 =0. 9992. lod and loq for the 200 bp dna was found to be 0.39 ng and 1.56 ng, respectively. the recovery values for the 1%, 5% and 10% impurity ratios were found 101.74. 97.41 and 99.47, respectively. the purity of the synthetic dna was determined by hplc and related uncertainty was calculated. the developed method is a simple alternative to electrophoresis and spectrophotometric methods with higher resolution and separation range. physical and chemical factors can disturb the conformation of proteins maturing within the cellular secretory pathway. in response to unfolded proteins the cell activates several stress signaling and adaptive response mechanisms. the aim of our study was to investigate small non-coding rnas as the potential regulators of cellular response to unfolded proteins (upr). for this, we conduct the next generation sequencing of small rna and transcriptome analysis of mrna from jurkat cells exposed to dithiothreitol (dtt), which reduces protein disulfide bounds. analysis of mirnas reveals the differential expression of 104 mirnas. we observe a decrease in the normalized amount of reads aligned to mirna loci in stressed cells. affymetrix analysis with subsequent gsea reveals downregulation of reactome mirna biogenesis pathway (fdr = 0.096). the length distribution of small rnas revealed 32 nt-peak corresponding to trna-derived fragments, amount of which was increased by 2.6-fold under dtt treatment. the trna isotypes that gave rise to almost 76% and 86% of all 32nt rna fragments in stressed and control cells, respectively, include glycine, glutamic acid, aspartic acid and valine. the vast majority of 32nt fragments produced from these trnas are precisely phased 5 0 halves with the characteristic cleavage patterns generated by rnase a angiogenin (ang). observed upregulation of tirna in stressed cells is accompanied with upregulation of ang mrna and down-regulation of angiogenin inhibitor 1 (rnh1). we speculate that translational repression, associated with observed tirna, is an additional mechanism of reducing global protein synthesis in response to dtt-induced stress. collectively, our findings reveal the increase in tirna, the differential regulation of mirna expression together with the global mirna downregulation as the most prominent small rnome reprogramming events and possible fine-tuned levels of post-transcriptional regulation upon dtt-induced cellular stress response. global gene expression changes after spinal cord injury j. k. hyun 1,2,3 , j. kim 1,2 , j. y. hong 1 1 dankook university, cheonan, south korea, 2 institute of tissue regeneration engineering (itren), cheonan, south korea, 3 the neuronal regeneration is hardly achieved spontaneously after spinal cord injury (sci), and the restoration of somatic and autonomic functions after sci is also challenging in the clinical field. the pathophysiology of sci is extremely complex and many in vitro and in vivo studies continued to report opposite results each other in spite of the same treatments, therefore a fundamental analysis such as an extensive assay of global gene expression is required to find a way for spinal cord regeneration. in this study, we aimed to detect the changes of global gene expression after spinal cord contusion in rats according to the time sequence. the spinal cord tissues at contusion site were sequenced after spinal cord contusion in rats using rna-sequencing technology. for time sequence analysis, five time points was determined; 1 hour, 1 day, 1 week, 1 month and 3 months after spinal cord contusion, and sham operated rats at each time point were used as controls. quantitative rt-pcr analysis was also performed to validate expression changes of candidate genes in each category. we found that the pattern of changes in gene expression at acute and subacute stages was quite different from that at chronic stage, especially genes associated with with neurotrophin signaling and apoptosis pathways. most of gene expression levels of inflammatory cell markers were increased and peak during acute stage (1 hour to 1 week) and maintained until chronic stage. some of regeneration-associated genes (rags) including brain derived neurotrophic factor, glial cell derived neurotrophic factor and ciliary neurotrophic factor were increased at 1 hour or 1 day after sci. we concluded that the information of gene expression level according to the time sequence after sci might be useful to determine treatment strategies for spinal cord regeneration especially in chronic stage. p-01.02.2-011 3 0 utr length isoform generation profile in a differentiation model alternative polyadenylation (apa) is the regulated selection of a specific poly(a) signal among other proximal and/or distal signals on the 3 0 utrs (untranslated region) for the endolytic cleavage and addition of a poly(a) tail to form the mature mrna. consequently, position of the poly(a) site determines the length of the 3 0 utr which is known to harbor microrna and rna binding protein sites. such apa isoforms have already been linked to altered protein levels and even functions. therefore we hypothesized apa to be one of the mechanisms to generate isoform diversity in proliferating and differentiated cells to better understand the molecular basis of cancer. we used a combinatorial in silico and in vitro approach to analyze a well known enterocyte differentiation model; caco-2 cells. initially we analyzed gene expression datasets for the proliferative and differentiated caco-2 cells using a probe based apa detection tool. to better understand the significance and to validate these results, we used proliferating and differentiated (day10) caco-2 cells and tested sample apa events by rt-qpcr. 3 0 utr isoforms were identified by using 3 0 race pcr. we identified 43 genes (32% of all apa events) to undergo 3 0 utr shortening in differentiated cells compared to proliferating cells. on the contrary 91 genes (68% of all apa events) went through 3 0 utr lengthening events. several genes have been validated to follow the pattern that was seen in apa detection tool so far. to begin understanding the mechanism behind these observations, we are investigating potential inducers of apa during the complex events of differentiation. our next aim will be to further validate and investigate the consequence of such isoform generation events both in the context of differentiation in colon cancer cells. recognition of phosphorylated threonine-4 of rna polymerase ii c-terminal domain by 3 0end processing apparatus o. jasnovidova, m. krejcikova, k. kubicek, r. stefl central european institute of technology, masaryk university, brno, czech republic rna polymerase ii has evolved an array of heptad repeats with the consensus sequence y1-s2-p3-t4-s5-p6-s7 at the c-terminal domain (ctd) of its largest subunit, rpb1. phosphorylation of serines (s2, s5, and s7) and tyrosine-1 orchestrate the binding of rna processing and transcription factors in the site of transcription. several recent studies showed that also threonine-4 site can be phosphorylated which has a number of functional consequences. to reveal the structural basis for the recognition of threonine-4 phosphorylated ctd, we set out to investigate several proteins factors that were implicated with a high levels of threonine-4 ctd phosphomarks using integrative structural biology. one of them, a factor involved in the 3 0 -end processing and transcription termination, showed a high affinity to the phosphothreonine ctd. using nuclear magnetic resonance spectroscopy (nmr), we determined its structure bound to the ctd phosphorylated at threonine-4 that reveals a direct read-out of the phosphothreonine. altogether, our data provides the first insights into the recognition of this poorly understood ctd mark that plays important role in the ctd code of rna polymerase ii. the results of this research have been acquired within ceitec 2020 (lq1601) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. introduction: the treatment of brain tumor glioblastoma (gbm) is still one of the greatest challenge. anti-inflammatory drug indomethacin (ind) mainly acting through the inhibition of cyclooxygenase (cox) has also anti-cancer activity including brain tumors. the aim was to investigate how ind effects an immortality enzyme telomerases' activity. materials and methods: monolayer and spheroid cultures of t98g human gbm cell line were used to evaluate the effects of ind (100 lm) on cell proliferation, viability, apoptosis, cell cycle, camp levels, the levels of apoptotic and anti-apoptotic proteins, morphology (sem) and ultrastructure (tem) for 72 hours. results were analyzed using the student's t-test. results: ind decreased cell proliferation (p72 < 0.01), cell viability (p72 < 0.000001), cell rate at s phase (p72 < 0.000001) and g2 + m phase (p72 < 0.001), camp levels (p72 < 0.01), the levels of pdgfr-a (p72 < 0.001), mrp-1 (p72 < 0.05), nf-jb (p72 < 0.01) and cox-2 (p72 < 0.01) in comparison to control group. ind mildly increased apoptosis (p72 < 0.001) and caspase-3 levels (p72 < 0.01). interestingly, ind increased htert levels (142%, p24 < 0.001; 154%, p72 < 0.0001). sem evaluation showed that ind led to decreased and shortened microvilli, the lost of cell interactions and the conversion of many cell shapes from spindle to oval. many cell remnants in the intercellular area, intact cell membranes, many dense lipid droplets and few autophagic vacuols in the cytoplasm were observed under tem. discussion and conclusions: the effect of ind on telomerase activity can only be found in 8 publications at pubmed research that they only showed its' inhibitory effect in colon, gastric, head and neck cancers. in contrast to previous studies, it was shown for the first time that ind increased telomerase activity in gbm cells and this increase was independent from cox-2 and other tested factors. p-02.02.2-002 interaction between fibrinogen and insulin-like growth factor binding protein-1 under physiologic conditions and influence of diabetes mellitus type 2 on this interaction n. gligorijevic, o. nedic institute for the application of nuclear energy, university of belgrade, belgrade, serbia fibrinogen is plasma glycoprotein and principle participant in blood coagulation. it interacts with many proteins, including insulin-like growth factor binding proteins (igfbps). one of them, igfbp-1, is controlled by insulin. metabolic changes due to diabetes mellitus (dm) affect igfbp-1. besides glucose regulation, igfbp-1 stimulates wound healing. we have investigated complexes formed between fibrinogen and igfbp-1, their change in dm type 2 (dm2) patients and involvement in fibrin clot. samples from adult healthy persons and dm2 patients were studied: plasma, isolated fibrinogen and fibrin. the amount of igfbp-1/fibrinogen complexes was determined using immunoblotting. immunoprecipitation and lectin affinity chromatography were used to confirm interaction between fibrinogen and igfbp-1. in vitro incubation of fibrinogen with excess glucose or methylgyoxal (mgo) was employed to demonstrate influence of glyco-oxidation on complexes. results have shown that igfbp-1/fibrinogen complexes can be differentiated from igfbp-1 oligomers and igfbp-1/alpha-2macroglobulin complexes. the amount of igfbp-1/fibrinogen complexes was lower in patients with dm2. complexes participated in fibrin clot formation, the amount being significantly lower in patients' samples. the quantity of igfbp-1 monomer in fibrin clot was greater in patients' samples. in vitro experiments revealed that complexes undergo glyco-oxidative modifications leading to their reduced formation, cross-linking and increased acidity (faster electrophoretic movement). isolated fibrinogen from patients with dm2 was additionally able to bind exogenous igfbp-1. since igfbp-1 stimulates wound healing, directly and by delivering igfs, igfbp-1/fibrinogen complexes may be seen as igfbp-1 storage instrument, ready to participate in fibrin formation and to assist in damage repair. reduction of complexes due to glyco-oxidative stress in patients with dm may be part of the mechanism responsible for impaired coagulation process. human interferon gamma (hifnc) is a proinflammatory cytokine involved in the regulation of nearly all phases of immune and inflammatory responses. its abnormal expression is associated with the aetiology of many inflammatory and autoimmune diseases. recently we have been exploring the idea to counteract the over-expression of the endogenous hifnc by competitive inhibition with inactive hifnc mutants. they are designed to have preserved affinity to the hifnc receptor, but to be deprived in their capability to trigger the intracellular signal transduction. to this end a library of mutants was created and two potential hifnc antagonists were selected for further investigations: a single point mutant k88q (q substitution for k in position 88) and a double mutant with additional substitution in the n-terminus. both mutants and the wild type hifnc were expressed in e. coli employing the established by us methodology for large scale production of aggregation-prone proteins in soluble native form. the purified mutants were screened for interferon activity (antiproliferative assay), binding affinity (isothermal titration calorimetry) and ability to compete with the wild type for the hifnc receptor (competition assay on wish cells). the selected mutants demonstrated 100 (single mutant) and 1000 (double mutant) times lower antiproliferative activity than the wild type. measuring the binding thermodynamic parameters, we proved that the receptor binding affinity of both mutants was preserved, which is an indication for their potential to compete with the wild type hifnc for its receptor. finally, the biological assay performed on wish cells showed a distinct dose-dependent competition between the wild type hifnc and the mutants. based on the results presented in this study we conclude that the two hifnc mutants are potential candidates for autoimmune therapy based on selective suppression of the endogenous hifnc activity. mesencephalic astrocyte-derived neurotrophic factor (manf) is an er (endoplasmic reticulum) stress-inducible protein and widely expressed in mammalian tissues. it has been identified as a secretory protein that protects cells against er stress-induced damage. er-stress is one of the main mechanisms that play a role in ischemia/reperfusion (i/r)-induced renal injury. recent studies demonstrated that manf can protect cardiac myocytes and cortical neurons against i/r-induced injury. moreover, it has been suggested that it has a restorative effect in ischemic injury. nevertheless, the function of manf in i/r-induced renal injury is still not known. in the present study, we investigated the function of manf by manipulation its expression level in ischemic acute renal failure model established in proximal tubular kidney cells (hk-2 cells). for this purpose, the cells were transfected with either manf sirna or manf encoding plasmids for silencing or over-expression of manf, respectively. then, the cells were exposed to hypoxia-reperfusion (h/r) induction for indicated times. evaluations of cell viability were determined with wst-1 reagent. the changes in protein levels of h/r-induced stress markers were analyzed byimmunoblotting. the results showed that the overexpression of manf has provided a significant resistance to h/r-induced cell death, whereas silencing of manf has rendered the cells more susceptible to death. it was also determined that the pretreatment of cells with manf conditioned medium caused a decrease in cell death. additionally, oxidative/nitrosative stress (os/ns) and er stress levels were decreased with over-expression of manf and increased by silencing of manf in hk-2 cells. taken together, our study suggests that manf may have a protective role against h/r-induced renal cell injury, possibly through the reducing effects on os/ns and er stress. p-02.02.2-005 his-flag tag as a fusion partner in insect expression systemgain or loss? e. krachmarova 1 , m. tileva 1 , k. maskos 2 , i. ivanov 1 , g. nacheva 1 1 institute of molecular biology "roumen tsanev", sofia, bulgaria, 2 proteros biostructures, martinsried, germany human interferon gamma (hifnc) is a glycoprotein playing major role in the regulation of innate and adaptive immunity. glycosylation is not essential for hifnc activity but is important for its stability, half-life and protease resistance in blood. the commonly used hifnc in therapy and research is produced in e. coli and therefore is not glycosylated. bearing in mind the above mentioned shortcomings of the non-glycosylated hifnc we expressed it in mammalian cells and transgenic mice, however very low yields were achieved. to obtain glycosylated hifnc, here we employed a secretory expression of n-terminal his-flag fusion protein in baculovirus-infected insect high five ò cells. this small hydrophilic tag is designed to not affect the proper folding of the target protein and to facilitate the detection and purification procedures. in parallel the same fusion was expressed in e. coli cells. the fusion proteins were purified to high degree of purity by affinity and size-exclusion chromatography. bioassay carried out on wish cells showed that the antiproliferative activity of both fusion proteins was 500 times lower than that of the native hifnc. this result shows that, in contrast to the generally hold view, the n-terminal his-flag tag interferes with the biological activity of hifnc despite of the protein glycosylation. in order to restore the biological activity we attempted to remove the his-flag tag enzymatically. surprisingly, we found that the fusion protein obtained from insect cells was resistant to enterokinase, independently of the enzyme source and experimental conditions, whereas the protein isolated from e. coli was susceptible and the tag-free protein showed fully restored biological activity. we are prone to explain the enterokinase resistance of the fusion protein from insect cells with either the specific conformation of the glycosylated protein or with the interaction of the carbohydrate residues with the enzymatic activity of the enterokinase. p-02.02.2-006 development of fluorescence assay for highthroughput screening system based on flow cytometry for directed evolution of cellobiose dehydrogenase cellobiose dehydrogenase (cdh) is an enzyme produced by phanerochaete chrysosporium and it has been already successfully cloned in other organisms. one of the most important roles of cdh is removing products of cellulose degradation. cdh is very important for biofuel and biosensor industry. for improvements of enzyme properties we have used directed evolution. the most important step is to develop screening system that reflects properties of interest. screening in microtiter plates (mtp) is expensive, time-consuming and has low throughput with a small number of variants detected (10 3 -10 4 in months). the aim of this work was the development of screening system for mutant libraries of cdh expressed on surface of yeast cells based on fluorescent enzymatic assay and flow cytometry. the screening method should be capable of screening cellobiose dehydrogenase variants mutated for higher activity and higher thermostability by error prone pcr. the fluorescent assay was beta-galactosidase (ec 3.2.1.23) also known as lactase is the enzyme that typically catalyzes hydrolysis of beta-1,4-d-galactosidic linkages in beta-d-galactosides, including disaccharide lactose, with glucose and galactose as end reaction products. this enzyme is able to catalyze synthesis of oligosaccharides, in particular galactooligosaccharides via galactosyl transfer reaction. arthrobacter sulfonivorans beta-galactosidase of unique for prokaryotes extracellular localization may find application in food industry for manufacturing lactose-free dairy products and in pharmacology as bioactive principle of medicines prescribed for patients suffering from lactase deficiency. the study was aimed at cloning of the gene encoding a. sulfonivorans beta-galactosidase, purification and characterization of the enzyme. a novel extracellular beta-galactosidase from a. sulfonivorans was recovered with an overall 207-fold purification, a 7.7% yield and specific activity 16 300 uámg à1 protein. the subunit molecular mass of the enzyme determined by sds-page analysis equalled 125 kda. it was found that the enzyme displays pi 5.35, prefers ortho-nitrophenyl-beta-galactoside as substrate (km 27 mm) and shows maximum activity at 40°c and at ph 7.5-9.5. the beta-galactosidase gene was isolated from the genomic dna library of a. sulfonivorans, sequenced, cloned and deposited in the genbank database under accession number km277894.1. it was established that the gene carries an open reading frame consisting of 3132 bp (1043 amino acids) and encodes beta-galactosidase referred to glycosyl hydrolase family 2 (cazy database). p-02.02.2-009 different splice-forms of tdrd7 protein mutated in cataract's and glaucoma's interacts with s6k1/2 o. skorokhod, v. filonenko department of cellular signalling, institute of molecular biology and genetics nas of ukraine, kyiv, ukraine ribosomal s6 kinases (s6k) are important players in cellular pi3k/mtor signalling network, deregulation of which has been associated with methabolic disorders, inflammation and cancer. previously we had identified a novel binding partner of s6k1 -tdrd7 (trap). tdrd7 is a scaffold protein detected in complexes involved in the regulation of cytoskeleton dynamics, mrna transport, protein translation, non-coding pirnas processing, transposons silensing. it was reported recently that mutations in human tdrd7 result in cataract and glaucoma formation, defined by elevated intraocular pressure (iop) and optic nerve damage. the aim of our study was to confirm s6k-tdrd7 interaplay and study its role in cells. bioinformatical analysis of tdrd7 sequence revealed the presence of potential phosphorylation sites of s6k2. using in vitro kinase assay, we have demonstrated that recombinant s6k2 phosphorylate 3 from 5 fragments of tdrd7. formation of s6k2-tdrd7 complexes in vivo was further confirmed by coimmunoprecipitation using anti-s6k2 and anti-tdrd7 antibodies generated previously in rat brain lysates. this interaction was further confirmed by confocal microscopy, oleksandr had shown that tdrd7 co-localize with s6k2 in hepg2 cells, predominantly in perinuclear region, enreached for one of the tdrd7 isoforms identified previously. moreover, we have detected that c-terminal synthetic peptides of s6k2 with methylated arg interfere with tdrd7 from hepg2 lysates. the physiological characteristics of s6k2-tdrd7 interaction and the role of this complex formation in neuropathology's development need further investigation. many biological function of placenta are performed not just a set of individual proteins, but also different oligomeric structures and complexes. herewith, activities of complexes may considerably differ from activities of individual proteins. therefore, identification and characterization of placental multi-protein complexes is an important step to understanding the placenta function. the aim of the present work was to investigate a composition and biological functions of the very stable high molecular mass multi-protein complexes (spc) from placenta of healthy mother. we isolated spcs (~1000 kda) from the soluble fraction of three human placentas. light scattering measurements and gel filtration showed that the spc is stable in presence salts, acetonitrile and triton x-100 in high concentrations, but efficiently dissociates in the presence of 8 m urea and 50 mm edta. such a stable complex is unlikely to be a random associate of different proteins. it was shown the spc includes a number of proteins with molecular weights of 2 to 180 kda. several protein components of the spc were identified, including serum albumin, transferrin, iggs, annexin a5 and other proteins. serum albumin, transferrin and protein with molecular weight 14,1 kda are the main proteins of the complex. it was shown high the spcs from three placentas possesses dnsase and catalase activities. an addition, investigation of cytotoxic effect on human cancerous cell lines has shown that the spcs reveal high cytotoxicity. antibody-cytochrome b5 fusion protein, characterization and applications for antibody development process antibodies have recently become an essential tool being a part of immunodiagnostics, therapeutics and as a valuable instrument in life science research. an enormous number of options utilizing a various tags were used to create a universal antigen-binding domain, which can be easily detectable, highly soluble and might be produced in high yields with low costs, but no multipurpose solution exists yet. we addressed the question whether a single tag could be found for enhancing solubility of recombinant fab antibody fragment and providing its detection and accurate quantification by rather simple method. a new application for hemeproteincytochrome b 5 as the antibodies fusion partner were proposed. we have constructed of recombinant fab antibody fragment cytochrome b 5 fusion protein. we have shown that cytochrome b 5 enhance expression of fab antibodies fragments in bacterial system, and could be a versatile tool for recombinant proteins folding, redox (oxidation) state studies and for their precise concentration determination in the turbid solutions. fusion fab-b5 protein has a stable red color and characteristic absorbance spectrum with the maximum absorbance at 413 nm in oxidized environment. cytochrome b 5 change its spectrum maximum depending on environmental redox potential and its folded state, so one can track these events in real time spectrophotometrically. binding activities of fab-b 5 fusion protein and hybridoma secreted immunoglobulin were measured by biolayer interferometry and elisa. no significant difference between them was revealed. due to this feature we can distinguish the chimeric protein of interest in complex mixtures and control the process of recombinant proteins expression and purification in real-time. besides, cytochrome b 5 fusion tags multiples recombinant antibody yield (from 2 to 3 times) and doesn't affect antigen-binding properties. the bb125-135 site of fibrin molecule is the site of fibrin protofibrils lateral association l. urvant palladin institute of biochemistry nas of ukraine, kyiv, ukraine previously we showed that fibrin-specific monoclonal antibody i-3c (monab i-3c) inhibited the fibrin protofibrils lateral association. we suggested that the epitope of monab i-3c in bb118-134 of coiled-coil region of fibrin molecule coincides with the site involved in fibrin protofibrils lateral association. the aim of this study was to localize the site of protofibrils lateral association in fibrin molecule using the synthetic peptides bb121-138, bb125-135 and both their scrambled version, and bb109-126 peptide. monab i-3c was isolated from hybridoma culture medium by affinity chromatography on fibrin-sepharose. turbidity analysis was used to study the effect of synthetic peptides on fibrin polymerization. the interaction between peptides and monab i-3c was investigated by spr method using plasmon-6 device. we investigated the effect of synthetic peptides which corresponded to amino acide sequences of fibrin molecule bb109-126, bb121-138, bb125-135, and the scrambled versions of bb121-138 and bb125-135 peptides on a binding to monab i-3c and on the fibrin polymerization process. in spr analysis was showed that bb121-138 and bb125-135 peptides, but not their scrambled version, binds to monab i-3c, immobilized to a chip. turbidity data showed that only bb121-138 and bb125-135 peptides caused the 2-fold decrease of the rate of the lateral association of protofibryls at the concentration 2.2 9 10 à4 m and 2.7 9 10 à4 m, respectively. both of them decreased the final clot turbidity. our data let us to suggest that the bb125-135 site is the site that involved in protofibryls lateral association. it has been recently shown that irisin immunoreactivity was altered in gastrointestinal cancers. as known hematological malignancies was one of the most common malignancies through world, but no study was present how irisin was changed in this type of cancers. therefore, purpose of this was to investigate how immunoreactivity to irisin was altered in hematological malignancies (blood cancers). we used an antibody from phoenix to demonstrate how a 12 kda band after deglycosylation of irisin altered in hematological malignancies. here we first time showed that irisin tissue immunoreactivity from acute lymphoblastic leukemia (all) and acute myelogenous leukemia (aml) patients was increased when compared with unaffected biological tissue parts. from the immune-histochemical (ihc) investigations it is concluded that hematological tissue and blood cells may be another source of irisin and increased with cancer, thus this finding might help to enlighten pathophysiology of hematological malignancies. the value of urine neutrophil gelatinaseassociated lipocalin (ngal) in acute heart failure n. serdarevic clinical centre, sarajevo, bosnia and herzegovina introduction: renal dysfunction is very common in heart failure (hf) and neutrophil gelatinase-associated lipocalin (ngal) is used as an early marker of acute renal tubular injury. recent studies have been reporting that ngal is inhibitor of inactivation of matrix metalloproteinases (mmp-9) which results in enhanced proteolytic activity with prolonged effects on collagen degradation. due to its relation to extracellular matrix degradation in myocardium and infammation, we hypothesized possible increased ngal expression in hf besides it renal dysfunction etiology. patients and methods: in study were included 30 patients hospitalised with signs and symtoms of ahf. urine samples for ngal analysis were collected at admission and analysed by the chemiluminescent microparticle immunoassay (cmia) for the quantitative determination of neutrophil gelatinase-associated lipocalin in human urine (abbott, architect analyzer). refferent range for urine ngal is 0-135 ng/ml. on admission blood samples for bnp (brain natriuretic peptide) analysis were drawn and tested by architect bnp chemiluminescent microparticle immunoassay (cmia), abbott laboratory. results: the mean age of the patients (male= 30, female= 30) was 68.86 years (sd 10.92 years). among them 18 (30%) patients was diagnosed as a hf-pef (hf with preserved ejection fraction) while 42 (70%) as a hf-ref (hf with reduced ejection fraction). mean bnp values was 1616.71 pg/ml (sd 1511.15 pg/ml) and mean lvef was 33.66% (sd 14.19%). mean urine ngal was 60.91 ng/ml (sd 78.72 ng/ml). we found significantly positive, but weak correlation among ngal and bnp only by pearson correlation test (r = 0.139, p = 0.464, wilcoxon signed rank test z = à4.782 p < 0.5). conclusion: bnp levels are elevated in hf with reduced and preserved ejection fraction. urine ngal is not elevated in acute heart failure, but it is slightly positively correlated with serum bnp values. converging evidence implicates the intermediate and medial mesopallium (imm) of the domestic chick forebrain in memory for a visual imprinting stimulus. a number of learning-related changes have been found in plasma membrane and mitochondrial proteins of imm. for broader analysis of these changes we employed two-dimensional gel electrophoresis/mass spectrometry approach and identified differentially expressed proteins in membrane-mitochondrial fraction of the imm across chicks with different estimated levels of imprinting 24 h after training. we further inquired whether the amounts of those proteins in the imm and a control region (posterior pole of the nidopallium, ppn) are correlated with memory for the imprinting stimulus. learning-related increase in the amounts of the following proteins was demonstrated in the left imm, but not in the right imm or left and right ppn: (i) membrane cognin;(ii) a protein resembling the p32 subunit of splicing factor sf2;(iii) voltage dependent anionic channel-1;(iv) dynamin-1; (v) heterogeneous nuclear ribonucleoprotein a2/b1. obtained results indicate that the molecular processes involved in learning and memory of imprinting cover a wide range of cellular activities, including stabilization of protein structures, increased mrna trafficking, synaptic vesicle recycling and specific changes in the mitochondrial proteome. the aim of this work is to study the substrate and inhibitory properties of uridine derivatives in the reactions catalyzed by e.coli up in order to shed some light on the substrate's conformation in the productive complex with the enzyme. we studied the e.coli up-catalyzed phosphorolysis of uridine and its derivatives modified in the heterocyclic base and the sugar moiety. the kinetic constants (km, ki, kcat ) of the phosphorolysis reaction of near 30 uridine derivatives were determined. the combined kinetic (nnna, 35, 2016) and structural data (acta crystallogr., d70, 3310, 2014) provide clear evidence that up binds uridine in the most energetically unfavorable conformation, which, to the best of our knowledge, has no precedents in the enzymes of nucleic acid metabolism. this is possible due to multiple interactions between the substrate and the protein environment (active site residues) mainly through hydrogen bonds. these results are important for understanding the mechanism of action of this class of enzymes. an analysis of the conformations of nucleosides in solution and rotational barriers suggests that the energy difference between the ground state of uridine and uridine complexed with up may be high as 63-69 kj/mol. the binding in a high-energy conformation results in the weakening of the glycosidic bond. the observed conformation of uridine complexed with sulfate (mimetic of phosphate) may be very similar to its conformation in the transient state. until now, foxp1 (forkhead box p1) has been identified as a tumor suppressor in several correlation studies in breast cancer. although, foxp1 is defined as a transcriptional repressor that interacts with other transcription factors in various mechanistic studies, there is no study that explains its repressor functions in breast cancer biology. here we demonstrate the repressor function of foxp1 on nfat (nuclear factor of activated t cells) and the migratory effect of this repression in mda mb 231 breast cancer cells. we performed co-immunoprecipitation experiments for the investigation of protein-protein interaction between two transcription factors. protein-protein interaction on dna was investigated with emsa and transcriptional effects of foxp1 on nfat, lusiferase reporter assay was performed. wound healing assay was used to analyse the effects of overexpression of foxp1 on tumor cell migration. our results showed that foxp1 has protein-protein interaction with nfat on dna and enhances breast cancer cell migration by repressing nfat transcriptional activity and foxp1 shows oncogenic function by regulating breast cancer cell motility. introduction: phosphodiesterase 5 (pde5) is one of phosphodiesterase lead to hydrolyzing cgmp.the cgmp signaling pathway has an important role in proliferation of cells. previous studies showed pde5 was increased in cell lines cancers thus pde5 inhibitors can used as efficacious therapeutic option for treatment of cancers. the current study was to investigation the effect of hydroalcoholic achillea.wilhelmsii extract (hawe) on the pde5 gene expression and cgmp signaling in the mcf-7 er + and mda-mb-468 er à . methods and materials: the ed50 of the hawe on both cell lines were examined by using mtt viability test then the expression of pde5 and cgmp concentration were measured in timedependent manner (in the ed50) by real-time rt-pcr and colorimetric assay respectively. results: treatment with the hawe showed, 25 lg/ml is ed50 for both cell lines and the hawe lead to reduction in pde5 mrna expression and evaluation of intracellular cgmp showed an increase pattern in the time-dependent manner. conclusion: our results showed that the hawe has anti-proliferative property in the mcf-7 and mda-mb-468, cell lines of breast cancer through the cgmp pathway, these data suggested that the hawe can be potential source for the isolation of effective anti-proliferative molecules. keywords: achillea.wilhelmsii, breast cancer, anti-proliferative, phosphodiesterase, cgmp signaling pathway. outer membrane protein g (ompg) is a stable monomeric porin having 14-stranded beta barrel form from e.coli. its exact function is not fully understood; however, it allows the passage of molecules up to 900 da in neutral ph but the pore is closed by going through a conformational change under ph 5.5. as being monomeric and having ph-dependent gating characters, it is suitable for biosensor and targeted drug delivery applications. an attempt on ompg is to create a larger pore while its stability is undisturbed. ompg-16s is obtained by adding 38 amino acids to the primary chain in order to have a 16-stranded beta barrel porin. ompg-16sl is formed by further adding 6 amino acids to loop l6 and by replacing 7 lysines with arginines. ompg-16s and ompg-16sl mutants are investigated by fourier transform infrared spectroscopy (ftir) and compared with ompg-wild type (wt) in terms of ph-dependent conformational changes and thermal stability. each mutant is prepared in na-phosphate buffer pd 5.5/7.5 and infrared spectra are recorded. further, temperature profiling are recorded for the range between 22 to 106°c. results show that both mutants are responsive to ph changes. while turning the ph from acidic to neutral, beta sheet signals shift to lower wavenumbers showing difference in secondary structure, implying the existence of closed and open states. on the other hand, mutant proteins show structural differences compared with the wt protein. porins are known for their remarkable thermal stability. the mutans retain this character by having transition temperature of $ 75°c, although this is less than the wt transition at $ 85°c. in conclusion, two mutants show signs of open and closed states as ompg-wt and even if the mutants are less stable than ompg-wt. this study shows that the attempted alterations in ompg structure are successful in terms of ph-response but it needs improvement in terms of stability when necessary. nad is a key factor in the regulation of mitochondrial metabolism. besides its vital role as redox carrier, nad serves as substrate for protein adp-ribosylation and deacetylation, modifications which modulate enzyme activities in mitochondria. these functions depend on how nad levels are maintained in this organelle. in human cells, mitochondrial nad is segregated from the cytosolic pool and can be synthesized from nmn, which is probably imported into the matrix. here, we tested whether the nudix pyrophosphatase nudt13 participates in the regulation of the mitochondrial nad pool. this enzyme has a predicted nadh pyrophosphatase zinc ribbon domain and a mitochondrial targeting sequence at its n-terminus. however, it has not yet been functionally characterized. we overexpressed nudt13 endowed with a c-terminal flag-epitope in human cells. to evaluate changes in the mitochondrial nad concentration, we used a reporter system which includes the overexpression of the catalytic domain of poly(adpribose) polymerase 1 (parp1) within the organelles (mitoparp). thereby mitochondrial nad is converted into protein-bound poly(adp-ribose) (par). the extent of par formation correlates with the mitochondrial nad availability and is detected by western blotting. our results established that nudt13 is indeed a mitochondrial protein, as it was localized exclusively to these organelles. moreover, when nudt13 was overexpressed along with the mitoparp detector system, a dramatic decrease of par was observed. the obtained results indicate that nudt13 is enzymatically active upon overexpression in the mitochondrial matrix and that it might cleave nad, thereby modulating its organellar level. however, at this point we cannot exclude the possibility of direct par cleavage by nudt13. further characterization of nudt13 will define its substrate specificity and clarify its role in mitochondrial metabolism. the incidence of increase in colorectal cancer (crc) worldwide has become a major health problem. early diagnosis and treatment of crcs are of importance for improving survival. in the present study, it was aimed to investigate chemopreventive effect of rosmarinic acid and evaluate the angiogenesis process in azoxymethane (aom)-induced crc model. male sprague-dawley rats were randomly divided into a control group, aom-induced rat colorectal cancer group (15 mg/kg body weight aom; ip, weekly for four weeks), and rosmarinic acid (5 mg/kg body weight; oral, daily for four weeks)-treated group. in addition to the standart diet of the all groups 15.8% peanut oil was added throughout the experiment. the all rats were sacrificed at the end of 30 weeks. biochemical examinations were performed in rat plasma. histopathological adenocarcinoma rates were observed in 87.5% of aom group. the incidence of adenocarcinoma was showed a reduction in the treatment group. significant increases in plasma tos and mcp-1 levels were found in the aom group compared to controls. these increases were reduced in the treatment groups but no significant. a significant increase was detected in tas levels in the treatment group when compared to the aom group. significant decreases in plasma adiponectin levels were found in the aom and the treatment groups compared to controls. in conclusion, treatment with rosmarinic acid reduced the occurrence of inflammation and was helped to maintain the oxidant-antioxidant balance in the model of aom-induced rat colon cancer. mitochondrial genome, while being strongly reduced in course of evolution, still codes for several proteins. the vast majority of them are components of the respiratory chain complexes. to produce these proteins, the system of mitochondrial translation is presented in the organelles, which is in common close to that in bacteria. translation initiation in bacterial cells is orchestrated by three protein factors called if1, if2 and if3. the orthologs of the two latter proteins are commonly found in mitochondria. however, mitochondrial if3 could not been identified in several groups of organisms, including s.cerevisiae, for a long time. recently we have shown that baker's yeast protein aim23p possesses a function of mtif3. however, the mitochondrial translation has not been stopped in the yeast strain without aim23p which is surprising taking into account the fact that if3 is obligatory for the translation in bacterial systems. instead of blocking of mitochondrial protein synthesis in absence of aim23p, we observed the translational imbalance: the synthesis rate of the complex v subunits was increased while the synthesis rate of the complex iv subunits was repressed. thus, in addition to its general role in translation initiation, aim23p might specifically affect the biosynthesis of individual mitochondrial-encoded protein species. our genetic experiments have revealed that, indeed, aim23p is almost indispensable for cox1p synthesis, and that it affects the translation of cox1 mrna through its 5 0 -utr, like classical mitochondrial translational activators. this is in accordance with our measurements of complex iv activity which is several times less in yeast lacking aim23 gene than in the wild-type. taken together, our results point on the multiple role of aim23p in mitochondrial translation: in addition to its function as mitochondrial if3, it specifically regulates the amount of complex iv subunits and its activity. p-02.02.2-024 the circulating betatrophin and irisin levels in polycystic ovary syndrome patients with and without insulin resistance introduction: polycystic ovary syndrome (pcos) is the most common endocrine/metabolic disease in women around the world, characterized by oligo-or anovulation, polycystic ovary, and/or hyper-androgenism. insulin resistance (ir) and obesity are common findings in patients with pcos. irisin is a recently identified myokine secreted from skeletal muscle in response to physical activity. irisin has been postulated to induce the differentiation of white fat tissue into brown fat tissue. betatrophin is a currently discovered new hormone proposed to stimulate b-cell proliferation. in this study we investigated the levels of irisin and betatrophin in pcos patients. materials and methods: our study group was consisted of 40 patients with pcos and 20 healthy volunteers. patients group was divided into two subgroups according to presence of ir. (pcos+ir and pcos-ir). the oral glucose tolerance test (ogtt) and the homeostatic model assessment (homa-ir) were performed to assess glucose tolerance and insulin sensitivity. irisin and betatrophin levels were measured by elisa method. results: circulating irisin was significantly higher in the pco-s+ir subgroup than the control group (p < 0.022). circulating betatrophin was significantly lower in both patients subgroups than the control group (p < 0.008). there was no negative or positive correlation between irisin and betatrophin levels. discussion: these data suggest that irisin and betatrophin may act a role together in the ir mechanism in pcos patients. butyrylcholinesterase (bche) synthesized in liver has long been associated with hyperlipidemia, type 2 diabetes and obesity. there are also reports on bche knockout mice becoming obese. the exact involvement of how bche interacts with lipids is still not clear. previously we displayed a correlation between leptin, waist circumference, fat mass and bche levels. recently, we have also shown that bche overexpression in hepg2 cells is regulated by alpha linoleic acid. as the next approach on the analysis of lipid metabolism and bche interaction, we considered the capability of bche to hydrolyze lipids. human serum bche was purified by subsequent deae-tris-acryl m and procainamide chromatography. the purified bche was utilized in a modified acid lipase assay with the acid lipase substrate 4-methylumbelliferyl palmitate (4-mu-palmitate). as the second alternative substrate trioleic acid was utilized. the triolein hydrolysis was measured by the nefa kit. verification that bche hydrolysis of these lipid substrates was not due to another esterase was done by iso-ompa inhibition studies. also, lectin binding studies with bche and rca were carried out to rule out non-specific esterase activity. using purified human serum bche and hepatic lipase as control enzyme we found that bche is able to hydrolyze the acid lipase substrate 4-methylumbelliferyl palmitate (4-mu-palmitate). we found that bche hydrolyses this molecule at ph 8 rather better than at ph 7.4. at ph values, purified human bche has a km value that was 10 times bigger than that of human pancreatic lipase. with the bigger molecule the triolein, the difference between the km values of bche and pancreatic lipase was smaller. bche seems to hydrolyze triolein with an efficacy comparable to approximately 25% that of human pancreatic lipase. our results display that another function of bche may be its lipid hydrolyzing activity. p-02.02.2-026 determination of regional reference ranges for erythropoietin with laboratory data mining serum erythropoietin (epo) levels are the main regulator factor of erythrocyte production and increase in response to hypoxia. our region is a location dominated by hypoxic conditions due to the high attitude. in this study we aimed to investigate the mean serum epo levels in the living conditions of our region. two hundred and eighty epo results from our laboratory data whose hemoglobin levels were normal were evaluated in the study. mean serum epo levels were analyzed via chemiluminescence method in beckman coulter dxi 800 auto analyzer. the epo levels of samples was 12.26 ae 9.32 mul/ml (ranged between 3.00 and 48.57) mul/ml. when we performed ae 1 sd for the studies population we determined normal serum epo levels were as 2.94-21.58 mul/ml. the upper limit determined by our results was 17% higher than that of determined by the manufacturer as 18.5 mul/ml and the lower limit determined by our results was 17% higher than that of determined by the manufacturer as 2.59 mul/ml. normal serum epo levels were considerable for our region and the upper and lower limits were higher than those of determined by the manufacturer. more detailed studies considering the physical properties of participants including a higher number participants are necessary. subclinical hypothyroidism is the precursor to hypothyroidism because it has a tendency to transform into hypothyroidism. subclinical hypothyroidism is considered one of the risk factors causing metabolic syndrome. metabolic syndrome can be characterized by plasma level of apelin released from adipocytes. in the present study, we aimed to measure serum apelin level of patients with subclinical hypothyroidism and compare them with serum apelin level from healthy individuals. our study group included 31 patients diagnosed with subclinical hypothyroidism and 23 healthy volunteers. serum samples were obtained from each participant for the measurement of apelin. these were then stored at à20•c until the time of analysis. serum apelin concentrations were determined using an enzymelinked immunosorbent assay. the mean serum apelin levels of subclinical hypothyroidism and control groups were 79 ng/l, control group 60 ng/l respectively. there was no statistically significant difference in terms of the mean apelin levels between the groups (p > 0.05). apelin levels didn't show significant correlation with bmi (p > 0.05). in the present study, no significant difference of serum apelin level was observed between patients with subclinical hypothyroidism and healthy control subjects. however, the apelin levels were higher in the patients with subclinical hypothyroidism than in the control group. the possible relationship between thyroid hormones and apelins is critical to understanding the etiopathogenesis of metabolic disorders. the mitochondrial erv1/mia40 import system does not impact cytosolic fe-s cluster protein maturation and iron regulation erv1 is a sulfhydryl oxidase that partners with the import receptor mia40 to import small cysteine-rich proteins into the mitochondrial intermembrane space. it has also been suggested that erv1 has an additional role in maturation of cytosolic fe-s cluster proteins and regulation of iron homeostasis in s. cerevisiae. however, these studies were performed on one particular erv1 mutant strain (erv1-1) that we discovered has additional defects in glutathione (gsh) metabolism. since gsh is required for iron regulation and cytosolic fe-s cluster assembly, this complicates our understanding of erv1 0 s role in these processes. we discovered that the erv1-1 strain originally tested for fe-s cluster defects was the only strain to exhibit defects in the cytosolic fe-s enzymes. mitochondrial and cytosolic fe-s protein activities in the other erv1 and mia40 mutants tested were similar to the wt control. in addition, while all the erv1 and mia40 mutants tested exhibit temperature-dependent defects in mia40 oxidation, only the erv1-1 strain has significantly reduced gsh levels and more oxidized gsh: gssg redox state. we determined that the cause of gsh depletion in the erv1-1 strain is an additional mutation in the gene encoding the glutathione biosynthesis enzyme (gsh1) that compromises gsh1 protein folding and/or stability. to address whether gsh deficiency in the erv1-1 mutant is the underlying cause for the cytosolic fe-s cluster defects and iron misregulation for this strain, we measured fe-s protein activity, iron-regulated gene expression, and iron accumulation in erv1 and mia40 mutant strains. only the erv1-1 strain exhibited iron misregulation and accumulation of mitochondrial iron, while exogenous gsh rescued these defects. these results demonstrate that the defects in cytosolic fe-s enzymes and iron homeostasis in erv1-1 are due to gsh depletion and neither erv1 nor mia40 play significant roles in cytosolic fe-s cluster assembly and iron homeostasis. human c-peptide is a 31 amino acid polypeptide, which is secreted into blood from b-cells in the pancreas where pro-insulin undergoes a post translational modification and cleaved into insulin and c-peptide. human c-peptide concentrations in blood plasma and urine reflect the level of insulin resistance associated b-cell function and can point out insulin secretory failure. the reference intervals in blood plasma and urine are 0.5-10.0 ng/ml and 40-150 ng/ml respectively. c-peptide measurement in urine and plasma provides a guide for therapy in diabetes. this study describes a method for the development and validation of picaa (peptide impurity corrected amino acid analysis) method for the determination of the purity of the human c-peptide which could be used as a reference material to measure cpeptide concentrations in plasma. two different methods were performed for the picaa; aaa-id-ms/ms for quantification of constituent amino acids following hydrolysis of the material and rp-hplc-esi-tof ms for determination of the peptide related impurities. the result of the aaa id ms/ms method was corrected for the amino acids originating from the impurities. id ms/ms-aaa was performed with zivak ò hplc and zivak ò tandem gold triple quadrupole ms equipped with a phenomenex ez:faast 4l aaa column (250 9 2 mm i.d). the mobile phase was composed of, a: 20 mm ammonium formate (af) in water, b: 10 mm af in acetonitrile (acn). the intact peptide analysis was performed by a hitachi lachrome elite hplc and bruker microtof-q mass spectrometer equipped with a capcell pak mg-ii c18 column (150 9 2 mm i.d., 5 mm particle size). the purity of the synthetic c-peptide was determined by picaa analysis and related uncertainty was calculated. traceability to si was established using the amino acid standards of which the purity was determined by tub _ itak ume using qnmr analysis. picaa is a simpler alternative to the full mass balance approach which requires large quantities of the peptide material. p-02.02.2-034 heat shock proteins: complementary therapies in brain tumors with viscum album e. onay-ucar, s. n. biltekin 1 istanbul university, faculty of science, department of molecular biology and genetics, vezneciler, istanbul, turkey cancer is one of the lethal diseases in the world. different cancer types possess overexpressed hsps levels. viscum album extracts with their anticancer and antioxidant properties are being used in cancer therapies. biochemical composition of this plant is known to vary its features depending on the host trees and time of harvest. in our previous study, it has been found that v.album inhibited hsp27 expression and induced caspase-dependent apoptosis in c6 rat gliomas. the aim of the current study is to find out whether different v.album extracts have different effects on hsps expression level and apoptosis in c6 glioma cell line or not. in this study, three different extracts of v.album were compared for their potential inhibition effects on hsps. the cytotoxic effects of extracts have been determined via mtt test. different experiment groups were set up subjected to heat shock and/or incubated without any heat shock application. overexpression of hsps was induced by heat shock at 42°c for 1 h in c6 cells. expression levels of hsps were determined by western blot analysis. the apoptosis inducing effect was also evaluated via caspase-3 activation in c6 glioma cells. pretreatment of the cells with non-toxic dose (10 lg/ml) of v.album extracts prior to heat shock, reduced significantly the expression levels of hsps. similarly, pretreatment with the extracts prior to heat shock increased apoptosis via caspase-3 activation in c6 glioma cells. these results will be utilized in the determination of the relation between extract composition and stress protein expressions. these results suggest that different extracts of v.album are able to down regulate expression of hsps, and induce apoptosis. this warrants further exploration as a potential resource of bioactive compounds that can be used in cancer therapy. future studies targeting hsps for the development of chemosensitizers may help improve the treatment of cancer in combinational therapy. biological drugs (biologics) are the fastest-growing category of therapeutics among those approved by the agencies for drugs regulation. most biologics are proteins designed for parenteral use. however, proteins are characterized by poor pharmacokinetic and safety profiles. peg-coating (poly-ethylene glycol coating) of biologics provides several benefits, including an increased half-life related to reduced renal clearance, an increased stability to degradation, and a reduced immunogenic/antigenic response. preservation of the three-dimensional structure and activity of the pegylated form is a strict requirement for human use. the recombinant proteins used for this studies (as-sod, superoxide dismutase; mmp12, matrix metalloproteases 12; ansii, l-asparaginase ii) were cloned and then over-expressed in escherichia coli. pegylation reactions were performed using commercial reagents. all the protein samples were purified and analyzed by solution and solid-state nmr (fields from 700 mhz to 950 mhz). we developed new protocols to prepare samples of pegylated proteins, demonstrating that solid-state nmr spectra of exceptionally good quality can be obtained for pegylated proteins in the sedimented state (obtained by either ultracentrifugation or rehydration of freeze-dried samples); surprisingly, sedimentation of pegylated proteins to this end has never been attempted. the spectral quality is comparable toor better thanthat of the corresponding crystalline samples. the excellent quality of the solid-state nmr spectra would make it possible to perform extensive resonance assignment and even a conventional full structure determination of biologics. the proposed method is based on the comparison of a standard twodimensional solid-state nmr spectrum of the sedimented pegylated protein with that of the crystalline state of the native proteinfor which the x-ray structure is available. all eukaryotic creatures hereditarily have natural defense mechanisms and are protected from the infections with this defense mechanism. antimicrobial peptides (amp) contain 10-50 amino acid content, are positively charged with amphipathic feature. the antimicrobial activities of amps are thought to be depended on the microbiocidal effects by binding to the surface of microorganisms and creating pores in their membranes. defensins are both effector and mediator small antimicrobial peptides of the immune system. these peptides in cationic and amphipathic structure have broad spectrum antibacterial, antifungal and antiviral features. defensins regulate the innate and acquired immune systems by suppressing proinflammatory responses during infection. mammals have three structural subfamilies of defensins. these show differences according to the trisulfide arrays in their structure and are classified as a,b, h defensins. human beings have tissue-specific six functional a defensins. human hnp-1 and hnp-4 encoded by defa1, defa3 and defa4 genes are firstly expressed in neutrophils. human hdp5 and hdp6 encoded by defa5 and defa6 are firstly expressed in paneth cells in the intestines and play important role in the defense and homeostasis. human beings have many pseudogenes such as defap and deftp in addition to these functional genes. according to literature data, defensins play an important role in defense against microbial placements on mucosal surfaces. in addition, the antimicrobial spectra of defensins include gram negative and gram positive bacteria, fungi and viruses. in addition to their antimicrobial efficiency, they can accelerate the wound healing due to their mitogenic effects on epithelium cells and fibroblasts. bile salt hydrolase (bsh) enzyme catalyzes the hydrolysis of glycine and/or taurine-conjugated bile salts into amino acid residues and the free bile acids that reduce cholesterol. however, some intestinal bacteria have an excessive deconjugation of tauro-conjugated bile salts and production of secondary bile acid having potential harmful side effects to the host. the catalytic mechanism and substrate preference of such bsh enzyme is not clear. in this study, bsh gene from lactobacillus plantarum gd2 strain was cloned, expressed, characterized in escherichia coli blr(de3) strain, and then val-58 and phe-129 amino acids, supposed to be responsible for substrate preference, were substituted for met-58 and ile-129 amino acids respectively by site directed mutagenesis. the hydrolysis activities and stability of the mutant recombinant bsh (mrbsh) enzymes were examined along with six different bile acids by ninhydrin assay and sds-page respectively. ninhydrin test results indicated that wild-type recombinant bsh (wrbsh) hydrolyzed six major human bile salts with an apparent preference towards glycine-conjugated to tauro-conjugated bile salts. however, the activities of mrbsh/phe129ile enzyme are 29%, 23%, 13%, 14%, 0% and 0% of the activity of wrbsh against to glycocholic acid (gca), glycodeoxycholic acid (gdca), glycochenodeoxycholic acid (gcdca), taurocholic acid (tca), taurodeoxycholic acid (tdca) and taurochenodexycholic acid (tcdca) respectively. the activities of val58met mrbsh enzyme are 79%, 93%, 74%, 43%, 44% and 28% of wrbsh against to gca, gdca, gcdca, tca, tdca and tcdca respectively. our findings support the suggestion that bsh enzymes recognize their substrates predominantly at the amino acid moieties and not at the cholate moieties. however, further pcr-based site-directed mutagenesis and structure-driven computational and theoretical approaches are required for the precise determination of their substrate specificities and the selection of probiotic bacteria. we deposited bacteriorhodopsin in purple membranes under applied electrical field onto ito (indium tin oxide) support. purple membranes film, highly oriented in one direction, was placed between two ito electrodes. we studied dependence of electrical properties of these films on light illumination. we argue that this setup can be used for functional studies of microbial rhodopsins. in opposite to already published results where this system was used as a photocondensor for studying functional properties of bacteriorhodopsin, we studied electric properties of such systems and we found strong light dependence of resistivity of bacteriorhodopsin in purple membranes films. optogenetics is already used in study of neuronal cells cooperation in vitro and in vivo by means of microbial rhodopsinsion pumps and channels incorporated in membranes of neurons changing their electrical potential while receiving a light quantum by laser or led source. best perspectives optogenetics will give after successful transfer to medical applications, such as the treatment of blindness, treatment of disorders like parkinson's disease etc. but to achieve these we need a broad set of tools, optogenetics tools, highly specialized to solve specific problems of neurophysiology. to the creation of such tools our work is dedicated. new optogenetic tools can be made by mutations in existing ones altering their properties (mainly spectral characteristics, selectivity and conductivity) or some promising mutations in conserved residues can be found in existing organisms. a halophilic archaeon halosimplex carlsbadens is a host of protein of our interest. according to the theoretical data based on the alignment with br and the 3d structure model of this novel protein, we suppose this protein functions like the light-driven h+ pump: all the key residues are the same or at worst have the similar properties, except one in the position 96leucine instead of the aspartic acid. a gram-positive bacteria deinococcus-thermus phylum syntheses rhodopsin with substitution of this aspartic acid to alanine. sphingomonas paucimobilis has rhodopsin where aspardic acid in position 96 is changed to serine residue. and one yet uncharacterised guillardia theta rhodopsin even has the same as br motif (d85, t89, and d96) but according to alignment is closer to chr2 even the last one motif is e85, t89, n96. it is expected that all of them will show us new properties. though the further experimental data are essential. the work is supported by rsf 16-15-00242. evaluation of some thymus proteins in patients with crimean congo hemorrhagic fever i. b€ ut€ un 1 , s. sahin 1 , f. duygu 2 1 university of gaziosmanpasa, department of biochemistry, tokat, turkey, 2 oncology education and reasearch center, ankara, turkey crimean congo hemorrhagic fever (cchf) is a tick-borne viral zoonotic disease. it has a high fatal rate (%5-30). tokat is one of the cities having the most reported cchf cases, in turkey. clinical presentation of the disease varies widely among patients. thymic peptides are small molecules synthesized by thymic epithelial cells. they play role in the immune response, as well as anti-inflammatory process. fourty patients referring to the hospital with tick-contact history and/or presenting clinical manifestations consistent withcchf and with positive pcr results for cchf virus in blood samples were included to the study. the wbc and platelet values at application and before the patients were discharged were recorded. the healthy control group consisted of age and gender matched healthy volunteer adults free of any chronic disease. thymosin alpha1 (ta1), thymuline and thymosin beta4 (tb4) were studied by the elisa method in this study. biochemical parameters were also analysed. ast and alt values were significantly higher (p < 0.01) and plt and wbc levels were significantly lower in the cchf group (p < 0.05). levels of tf, ta1 and tb4 were found to be significantly higher in cchf (p < 0.01). there was no mortality during the study period. duration of hospitalization was 6.73 ae 2.74 days. levels of tb4 were significantly correlated with duration of hospitalization (r= à0.351, p = 0.036). alt levels were significantly correlated with tf levels (r = 0.413, p = 0.010). 17 patients received ffp and apheresis for the supportive treatment, while 5 patients received only ffp and 2 patients got only apheresis. 16 patients did not get any of these blood products. there was not a statistically significant differences in thymus peptides among these treatment groups (p > 0.05). we report 40 survived cchf patients with elevated thymic peptides. pathogenesis of cchf has many points to be highlighted. thymic peptides may play role in the clinical situation of the patients with the disease. the effect of methocarbamol on the peroxiadse activity of human erythrocyte hemoglobin hemoglobin is released to blood circulation, after red blood cells lysis. it is carried in circulation by binding to haptoglobin. in normal persons, no free hemoglobin is observed in the blood, because most of hemoglobin is in the form of haptoglobin complex. in some diseases that are accompanied by hemolysis, the amount of released hemoglobin is higher than its complementary haptoglobin. as a result, free hemoglobin appears in the blood, which is a toxic compound for these patients. free hemoglobin has been showed to have peroxidase activity and considered a pseudoenzyme. in this research, the effect of methocarbamol on the peroxidase activity of human hemoglobin was studied. our results showed that the drug inhibited the pseudoenzyme by un-competitive inhibition. both k m and v max decreased by increasing the drug concentration. k i and ic 50 values were determined as 6 and 10 mm, respectively. molecular docking results showed that methocarbamol did not attach to heme group directly. a hydrogen bond connected nh 2 of carbamate group of methocarbamol to the carboxyl group of asp126 side chain. two other hydrogen bonds could be also observed between hydroxyl group of the drug and ser102 and ser133 residues of the pseudoenzyme. p-02.02.2-042 dca reduces viability and down regulates mapk protein activations in human malignant mesothelioma cells and pericardium. microarray analyze results performed in mm patients revealed that one of the most prominent changes is upregulation of many genes involved in glycolysis and the krebs cycle. dichloroacetate (dca) is an inhibitor of pyruvate dehydrogenase kinase (pdk) that enhances the oxidative activity of cells by activating pyruvate dehydrogenase (pdh) in mitochondria. dca has shown as a promising anti-neoplastic agent that re-sensitizes cancer cells to apoptosis. the aim of this study is to elucidate the coupling between pdk inhibition and mm cell proliferation and cell cycle. human malignant mesothelioma (spc212) cell line was used as a model for dca treatments. cell viability was measured by mts assay; mapk protein activations and expressions were assessed by western blotting; cell cycle profile was analyzed by flow cytometry. statistical analysis was performed by utilizing one-way anova test. results showed that dca reduced viability of spc212 cells in a concentration and time dependent manner. protein analysis indicated that mapk pathway was down regulated at concentrations greater than 50 mm. moreover, primary cell cycle analysis has indicated arrestment at g2/m phase in 24 hours. our findings corroborate with recent reports where dca treatments resulted in reduction of viability and g0/g1 and g2/ m arrest in other cell lines. abnormalities in mapk signalling play a critical role in the progression of cancer. here, we showed for the first time that dca decreased mapk activation in 24 h. our results suggest that dca is an anti-prolifertive agents for mm cells in vitro. however, it requres extra analysis with other mesothelial cells. future study will focus on investigating relation between mapk and mitochondrial apoptosis. adrenomedullin (adm) is a vasodilator peptide consisting of 52 amino acids. adm is synthesized in many tissues. and is a biologically active peptide that has various effects including vasodilatation, the regulation of vascular endothelial function and adjusting adipogenesis. hypoxia inducible factor 1 alpha (hif 1a) is a subunit of a heterodimeric transcription factor hypoxia inducible factor 1. it is the master transcriptional regulator of cellular and developmental response to hypoxia. the dysregulation and overexpression of hif1a by either hypoxia or genetic alternations have been heavily implicated in cancer biology as well as a number of other pathophysiologies. in our research, the adm and hif 1-a levels in heart, kidney and lung tissues of rats were investigated in control, hypoxia, control+adm and hypoxia+adm groups. rats in hypoxia groups were provided hypoxic environment containing of 10-12% oxygen and 88-90% nitrogen for 1 week. rats in adm groups were injected intraperitoneally in a dose of 1.25 nmol/kg for four days before the collection of the tissues. the control group was oxygenated with normal air. the control and treatment groups were formed from 7-8 animals and adm, hif 1-a levels were measured in taken tissues with immunoassay method. the aim of this study was to investigate the reaction of the organism when exposed to hypoxic conditions and the effect of adm over hif 1-a level. adm levels and hif 1-a in heart tissue were found decreased in hypoxia group, and adm levels increased in hipoxia+adm group. hif 1-a levels decreased in hypoxi+adm group. adm levels in liver tissue were found decreased in hipoxia and control+adm groups than control group. hif 1-a levels were higher in control+adm group. adm has a role in angiogenic process, and our experiment showed that adm reacts earlier than hif 1-a, and affects its synthesis. organism increases its vascularization as a reaction to hypoxic condition, and adm treatment may provide a rapid adjustment. p-02.02.2-044 covalent conjugation and characterization of immunogenic protein of toxoplasma gondii and polyacryclic acid as vaccine candidate r. c ß akir koc ß yildiz technical university, department of bioengineering, istanbul, turkey toxoplasmosis is a major medical and veterinary disease caused by toxoplasma gondii which infect approximately half of the world's population. this infectious disease especially gains importance in pregnant women and immunodeficient individuals. also t. gondii infection has economic importance. however, there are only one attenuated-live t. gondii vaccine for veterinary uses and no vaccine against t. gondii is available for humans. therefore development of an effective vaccine would be extremely valuable for preventing disease in human and veterinary medicine. subunit vaccines are very attractive vaccine candidates but there is low antigenicity problem when they are used alone. polymers themselves don't stimulate immune response while they used with antigenic structure of various infective agents enhance immune response because when proteins are covalently conjugated with hydrophilic polymers, (1) their circulatory-lives and stability (in different ph and temperature values) enhance (2) binding to proteases and clearance by the reticuloendothelial-system decreases. in this study, immunogenic protein of t.gondii and polyacrylic acid with immune stimulant properties was covalently conjugated and conjugation was demonstrated by size-exclusion chromatography (sec) and fluorescence spectroscopy. it is significant to detect time of death in case of a sudden death for medical and legal concerns. there is no known method that can be used for post mortem time detection. based on this deficiency pmi detection in narrow time frame is a big problem. in this study, we aimed to investigate and determine timedependent expressional changes of apoptotic markers by western blot technique. 14 postmortem skeletal muscle were analyzed 12hour periods in first 36-hour after death. 2nd and 3rd 12-hour periods were statistically significant (p < 0.005). keywords: post mortem interval, time of death, apoptosis. hyaluronidases are excessively found in nature and involved in numerous biological functions. hyaluronidases primarily degrade hyaluronic acid (ha) and have significant role in fertilization during acrosomal reactions. therefore, the measurement of hyaluronidase enzyme activity may provide valuable information about acrosomal function and the fertilizing ability of the sperm. the aim of this study was to investigate the semen hyaluronidase enzyme activity changes among four different sheep breeds (akkaraman, suffolk, merino, and kıvırcık). in this research, ten ram testis tissues from each sheep breed, a total of 40, were cut and collected on ice. ovine testicular hyaluronidase of four different sheep breeds was purified from a crude ammonium sulfate-precipitated fraction of an extract of ram testis. the semen hyaluronidase enzyme activity differences between the sheep breeds were examined by spectrophotometrically monitoring the appearance of ha at 232 nm. analysis of variance test was used to examine the possible mean differences among the four different sheep breeds. the observed mean differences in enzyme units for kıvırcık, suffolk, akkaraman, and merino were as follows 891.60, 817.60, 729.40, and 681.60, respectively. the observed mean differences in absorbance values for kıvırcık, suffolk, akkaraman, and merino were as follows 0.4455, 0.4088, 0.3648, and 0.3408, respectively. the results showed that the observed mean differences in enzyme units and absorbance values among the four different sheep breeds were not statistically significant. despite that, in average kıvırcık had higher values for the activity of each sample and yet it had the smallest values for standard deviation. therefore, in order to achieve higher enzyme activity and more homogenous samples kıvırcık breed should be preferred. what is extra to learn from protein drying measurements? hydrations of soluble proteins are crucial for their functionality. therefore elucidating the details of protein hydration is still of interest in the proteins' action mechanisms. this is the motivation of the present study. in order to study protein hydration, changing concentrations of the well-studied serum albumin protein was measured with the spectroscopic techniques like uv-vis and ft-ir spectroscopy. spectral data is analysed and calculations were performed on the data to extract the relevant changes in the protein. experimental parameters' variation in association with the spectral changes implies the involvement of protein structure and hydrogen bonding in the drying process. the protein's reactions may not be merely a feature of the protein structure in the common sense but it could be related directly to the protein hydration states as well. this is understandable since it is already known that enzymatic proteins lose their functionality when they are dried while this drying may or may not involve dramatic structural changes. on the other hand, here it is claimed that the role of water in gaining the functionality that was lost in the dried state is not just about enhanced diffusion processes and the dynamicity but could be related to the functionality of water in the energy transfer processes as well. investigating the cellular effects of the aldoketo reductases akr1b1 and akr1b10 in hct-116 colon cancer cells b. taskoparan, e. g. seza, m. s. ceyhan, s. banerjee middle east technical university, ankara, turkey aldo-keto reductases (akr) are nad(p)h dependent oxidoreductases are best characterized as glucose reducing agents, and have been implicated in diabetic pathophysiology. increased expression of akr has been associated with tumors of lung, breast, prostate, cervix, ovarian and colon. two members of the akr superfamily that have been associated with different cancer types are akr1b1; aldo-keto reductase family 1, member b1, and akr1b10; aldo-keto reductase family 1, member b10. both are 36-kda cytosolic reductases that are similar in both amino acid sequence identity (71%) and tertiary structure with the (a/b) 8 barrel topology. while hct-116, a colorectal cancer cell line, cells expresses akr1b1 robustly, there is no expression of akr1b10. in this study, we have stably knocked down akr1b1 through shrna technology and overexpressed akr1b10 in hct-116 cells. comparisons were made with a known akr inhibitor sorbinil. with the knock down of akr1b1, we have observed reduced cellular proliferation, enhanced apoptosis, delay in cell cycle progression, reduced expression of mitogenic proteins and a decrease in activation of the inflammatory transcription factor nuclear factor kappa b (nf-kappab). interestingly, although akr1b10 overexpression did not affect cell proliferation, apoptosis or cell cycle, some effect was observed with nf-kb signaling. our data indicate that, although closely related, akr1b1 and akr1b10 have very different contributions towards signaling pathways in colorectal cancer. comparison of different nisin quantification methods and optimization of nisin production by lactococcus lactis z. girgin ersoy, g. demir, m. f. cesur, s. tunca gedik gebze technical university, kocaeli, turkey nisin, which is produced by certain strains of lactococcus lactis, is the only bacteriocin approved by world health organization (who) as a food additive. it prevents the growth of foodborne bacteria which cause food spoilage. nisin research and applications necessitates developing an accurate and reproducible method for its quantification. the agar diffusion bioassay is the most widely used method for quantifying nisin, although it has limitations especially diffusion-related difficulties of the active substance. in the present study, "agar diffusion bioassay", "enumeration of colony forming units", "colorimetric assay" and "flow cytometry" methods were compared with each other to determine antibacterial activity of nisin on micrococcus luteus. moreover, this study also covers the results about the effect of different cultural conditions to optimize nisin production by l. lactis. galactose, lactose and their combination in m17 medium (ph 7) boosted nisin production at 25°c, as the addition of 1.5 lg/ml hemin into the fermentation broth. to our knowledge, this is the first study showing the usage of "flow cytometry" method to determine nisin activity of fermentation broth filtrates. p-02.02.2-051 coronaviral nucleocapsid protein is an antiviral target for drug development institute of genomics and bioinformatics, national chung hsing university, taichung, taiwan between 2003 and 2004, the severe acute respiratory syndrome (sars)-cov caused a worldwide epidemic and had a significant economic impact in the countries affected by the outbreak. recently, the middle east respiratory syndrome human coronavirus (mers-cov) was found in patients with severe acute respiratory tract infections in the middle east and south korea. as is true for all coronavirus infections, there are no efficacious therapies currently available against coronaviral diseases, making the development of anti-coronavirus compounds a priority. the cov genome consists of positive-sense, single-stranded rna approximately 30 kb, and it contains several genes encoding several structural and non-structural proteins that are required for progeny virion production with a conserved order. the n proteins exist in the center of the viral particle and represent a helical structure complex. nucleocapsid protein is most abundant structural protein of covs, binds the viral rna genome to form the virion core, leading to the formation of a ribonucleoprotein (rnp) complex or to a long helical nucleocapsid structure, that is important for maintaining the rna in an ordered conformation for replication and transcription. the cov n protein is also involved in the regulation of cellular processes, such as gene transcription, interferon inhibition, actin reorganization, host cell cycle progression, and apoptosis. two strategies to inhibit oligomeric n protein function have been reported. the first strategy is to discover antiviral agents that target the rna-binding site. the second one is to impair normal n protein function by interfering with monomer-oligomer equilibrium. our recent studies suggest that n proteins in infections caused by coronaviruses will be useful antiviral drug targets because they serve many critical functions during the viral life cycle. post-translational modification of vascular endothelial growth factor (vegf) in colon cancer cells s. tunc ßer, e. solel, s. banerjee middle east technical university, ankara, turkey vascular endothelial growth factor a (vegf-a), commonly referred as vegf, is a potent secreted mitogen crucial for tumor initiation and progression. the gene for vegf is translated into a number of splice isoforms that lead to 121, 165, 189 and 206 amino acid proteins, with different receptor-binding and matrixbinding properties. in the present study, we discuss the functional significance of post-translational modification/processing of vegf 165 isoform in hct-116 colon cancer cells. we also focus on the role of calcium in the post-translational modification of vegf 165 . we show that vegf 165 undergoes n-linked glycosylation in hct-116 cells. perturbation of cellular calcium may affect vegf driven malignant phenotypes. p-02.02.2-053 novel methods for modulating the activity of bcl2 family proteins in apoptosis p. rowell, j. miles, a. wilson, t. edwards apoptosis, also known as programmed cell death, is an essential cellular process, but is implicated in several human diseases, including diabetes and cancer, when it is up-or down-regulated respectively. bcl-2 family proteins are major players in the control of intrinsic, or mitochondrial apoptosis; they respond to intracellular stress signals, function through protein-protein interactions and converge on the mitochondrial outer membrane to cause membrane permeabilisation, release of cytotoxic molecules, and initiation of an apoptotic cascade that leads to cellular demise. our work aims to identify molecules able to bind and modulate the activity of several key players in the bcl-2 family, including the pro-survival members bcl-2, bcl xl and mcl1, and the death promoting family member bax. adhirons, novel non-antibody peptide display scaffolds developed at the university of leeds, have been used to construct a phage display library containing over 10 10 clones, and form a key part of the strategy to identify such molecular modulators. adhirons able to selectively bind individual bcl-2 family members have been identified, in vitro assays carried out to test for modulatory activity, and xray crystallography used to elucidate details of how they interact with their target proteins. more recently, studies have been carried out to identify adhirons able to target multiple bcl-2 family members, with the aim of selectively inhibiting defined groups of proteins in cells. this work provides opportunities to differentiate the activities carried out by different bcl-2 family proteins in apoptosis, enabling us to better understand how their dysregulation contributes to human disease. biophysical and evolutionary study of the structural flexibility of adp-dependent sugar kinases from mesophilic and psychrophilic archaea r. zamora 1 , v. castro-fern andez 1 , c. a. ramirez-sarmiento 1 , e. a. komives 2 , v. guixe 1 1 facultad de ciencias, universidad de chile, santiago, chile, 2 department of chemistry and biochemistry, university of california, san diego, united states of america the capability of extremophiles microorganisms to live at low temperatures is mainly attributed to the high structural flexibility of its enzymes. several sequence and structure features have been associated to a high structural flexibility that enables metabolic processes to occur at low temperatures. during evolution, the general mechanism adopted by these enzymes has been to reduce the free energy of the transition state rather than the michaelis constant, k m . increased structural flexibility and decreased affinity for its substrates in psychrophilic enzymes is compensated by an increase in the catalytic rate, k cat . few psychrophilic enzymes have been reported to performance the optimization of their catalytic efficiency (k cat /k m ) by decreasing k m values. we use the adp-dependent kinase sugar family of archaea as a model, to identify particular structure and sequence features of a psychrophilic enzyme that would make this enzyme more flexible than their thermostable homologues. we characterize the bifunctional psychrophilic enzyme phosphofructokinase/glucokinase from methanococcoides burtonii (mbpfk-gk) and the bifunctional mesophilic enzyme phosphofructokinase/glucokinase from methanococcus maripaludis (mmpfk-gk) by spectroscopic, biophysical and computational techniques. the comparison showed that the absence of two ion pairs is primarily responsible for the increased structural flexibility accounted in the psychrophilic model. this increase in structural flexibility is reflected in the exponential increase in the k m values with temperature. additionally, we reconstruct the sequences of all ancestral enzymes between the current enzymes and their last common ancestor, which was used to trace the occurrence of these electrostatic interactions during evolution in the adp-dependent sugar kinase family. our results suggest that electrostatic interactions are a dominant feature in the transition from psychrophilic to thermophilic environments. fondecyt 1150460. elucidating the domain swapping mechanism of the forkhead domain of human foxp1 fox transcription factors control gene transcription during key processes, such as embryogenesis and immunity, and feature a conserved dna-binding domain known as forkhead. while most forkhead domains are monomeric, solved structures of members from the p subfamily (foxp) show that they can oligomerize by domain swapping (ds), a mechanism where protein segments are exchanged between subunits leading to an intertwined dimer. the biological relevance of ds in foxp has been stressed by disease-causing mutations that impair this process. however, for many proteins ds takes days to occur and requires global protein unfolding. thus, the current mechanism impedes a conciliation of the occurrence of ds of foxp1 in a biological context. here, we elucidate the biological feasibility of this process by biophysically characterizing the ds mechanism of the forkhead domain of foxp1 using size exclusion chromatography (sec), circular dichroism, and hydrogen-deuterium exchange mass spectrometry (hdxms). our results show that ds of foxp1 occurs at micromolar protein concentrations, within hours and is energetically favored. remarkably, dimeric foxp1 follows a threestate n 2 « 2i « 2u folding mechanism, where dimer dissociation into a monomeric intermediate (i) precedes protein unfolding, in contrast to other ds proteins. using sec and hdxms, we show that the i state of foxp1 largely resembles the native state, but has a larger hydrodynamic radius and a higher deuterium uptake in regions that maintain the compact monomer, suggesting that the i state is an 'open' conformation en route of ds. finally, we compared the local flexibility of the dimer and monomer of foxp1, showing that only the hinge region connecting ds segments exhibits different deuteration rates. our results show that ds in foxp1 follows an unusual threestate folding mechanism that proceeds through transient structural changes rather than needing protein unfolding as in most ds proteins. (fondecyt 1130510, 11140601). p-02.02.2-057 the sustained release of growth factor proteins following their implantation in tissue engineering j. jang bone tissue engineering has become a promising approach for bone regeneration. however, insufficient vascularization during bone regeneration, particularly with large bone defects, results in poor and unsustainable bone formation due to central necrosis. therefore, vascularization following implantation in vivo is essential to the successful formation of new bone tissue. we evaluated the release profile of vegf from bgs using a novel fluorescence-based retention assay, which revealed that vegf loaded on bgs can be released in a sustained manner without an initial burst (near zero-order cumulative release) with a controlled release rate of 13.6% per week for up to 7 weeks. in contrast, an elisa-based release assay showed vegf to have an early burst-release profile for the first week. however, the biological activity of vegf released from the bgs was preserved over the 7-week release period, which is consistent with the sustainedrelease profile observed in the fluorescence-based retention assay. we developed a novel fluorescence-based retention assay to evaluate the release of vegf from bgs. this fluorescence-based retention assay, which detects the vegf that remains on bgs, reveals that vegf loaded on bgs can be released in a sustained manner, with a minimal initial burst, for up to 7 weeks. these results indicate that the sustained biological activity of the vegf released from bgs over the full 7-week period promotes bone regeneration, and suggest its potential use for bone tissue engineering. irisin is a recently discovered myokine which regulates energy metabolism and is associated overweight. we aimed to evaluate serum irisin levels in the patients with morbid obesity. a total of sixty patients with morbidly obese and thirty healthy control subjects were included in this study. all participants were measured body weight and height, the lipid profile, and plasma glucose, hba1c, insulin and irisin levels. irisin levels were measured by elisa method. serum irisin levels were significantly lower in morbidly obese patients than healthy controls (p < 0.05). there was no statistically significant difference in terms of age or gender. serum irisin was negatively correlated with bmi, insulin levels, and homa-ir (p = 0.006, p = 0.046, p = 0.048, respectively). our study revealed lower irisin levels in morbidly obese patients with respect to control subjects. the lower irisin levels observed in morbidly obese patients might suggest a loss of browning of subcutaneous adipose tissues. p-02.02.2-059 pka inhibition restores adenosine uptake in renal tubular epithelial cells under high d-glucose conditions w. garrido, j. catal an, s. alarc on, r. san mart ın universidad austral de chile, valdivia, chile introduction: diabetic nephropathy (dn) is the leading a cause of end-stage renal failure whose pathogenesis must to be elucidated. the progression of dn has been associated with elevated levels of adenosine. extracellular adenosine availability is regulated by the activity of the equilibrative nucleoside transporters (ents). due to the ents have putative sites of phosphorylation our objective was to evaluate the role of pka and ck2 kinases on ents activity. methods: adenosine uptake (10 lm adenosine, 60s, 22°c) was assayed in hk2 cells preincubated with 5 mm or 25 mm d-glucose for 24 h and exposed to 10 lm 8-br camp, 10 lmh89 or 100lm tbb for 1 h. plasma membrane and intracellular proteins were fractionated by the biotinylation method and ent1 and ent2 contents were quantified by western blot. results: high d-glucose in hk2 cells inhibited the uptake of adenosine. this effect was reversed using a pka inhibitor (h89) through an increased ent2 uptake activity. we noticed this pka inhibitor did not regulate the plasma membrane localization of ent1 or ent2 under normal d-glucose (5 mm) or high d-glucose conditions (25 mm). also, we saw that tbb (ck2 inhibitor) decreased the activity of ent1 and ent2 under normal glucose conditions, decreasing the localization of ent1 at cell surface, while the membrane localization of ent2 decreases under the effect of tbb and high d-glucose conditions. conclusions: pka inhibition reversed the effect of high d-glucose, increasing the uptake of adenosine mediated by ent2. this could be a new target for the restoration of adenosine levels in dn. relation between serum lipo (a), plasma fibrinogen, red cell distribution width and mean platelet volume in healthy adult men the aim of this study was to investigate the relationship between the serum lipo (a) and plasma fibrinogen, red cell distribution width (rdw) and mean platelet volume (mpv) in healthy adult men. for this purpose, 37 healthy adult men have normal physical examination and laboratory findings and not use any drug were included to the study. serum lipo (a) levels and hematologic parameters (fibrinogen, rdw-sd and mpv) were measured by autoanalyzer and commercial kits. the mean of the age of the persons was 27.2; body mass index was 24.2; serum lipo (a) level was 0.21 and plasma fibrinogen level was 1.61. there was significant positive correlation between the serum lipo (a) and plasma fibrinogen levels (r = 0.246; p = 0.025), significant positive correlation between the serum lipo (a) and rdw-sd (r = 0.267; p = 0.004) and significant negative correlation between lipo (a) and mpv (r= à0.205; p = 0.027). the plasma fibrinogen and the serum lipo (a) levels have been known as the risk factors for cad (coronary artery disease) increase together in healthy adult men. similar findings have been reported in cad patients. it has reported that elevated rdw is associated with intracoronary thrombotic burden and may be associated with the severity and instability of acute myocardial infarction. in addition, mpv is predictor of severe atherosclerosis and may be used for the prediction and identification of cardiac risks in cad patients. our findings show that elevated rdw and decreased mpv may predict to increased risk of cad in the future, in healthy adult men. follicular fluid is rich in peptides, which significantly influence the growing oocyte. due to existence of a link between kisspeptin (metastin) cells and gonadal steroids kisspeptin might manipulate the gonadotropin axis and folliculogenesis. in this context, the study was planned to investigate for the first time that the follicular fluid (ff) and serum concentration of kisspeptin in high and poor responders undergoing in vitro fertilization (ivf)/intracytoplasmic sperm injection (icsi). biological samples were collected from twenty infertile women with polycystic ovary syndrome (pcos) and 20 poor responder participants undergoing controlled ovarian stimulation (cos) with gonadotropin-releasing hormone (gnrh) antagonist protocol for ivf/icsi treatment. kisspeptin concentrations were measured in serum and follicular fluid by using elisa, whereas fsh and lh levels were detected by routine laboratory methods. it was found that kisspeptin levels were significantly lower in serum and follicular fluid of infertile women with pcos. kisspeptin levels were correlated with fsh and lh level in infertile women with pcos. it can be concluded that low level of kisspeptin might inhibit gnrh release that might cause to the inhibition of fsh and lh release and might disrupt folliculogenesis. decline in serum and ff levels of kisspeptin might be possible cause of anovulation and subfertility in pcos subjects. cryptococcus albidus d24 is a newly identified yeast isolates from petroleum area in _ izmir as a lipase producer. the molecular weight of the enzyme is 36.31 kda as found. optimum temperature was 40°c and half-life times were 180, 27, 9 and 0.59 min at 30, 40, 50 and 60°c, respectively. optimum ph value was 8.0. however, it shows significant ph stability at ph values 4.0 and lower. the existence of acetone in the solution as a solvent enhanced lipase activity. cryptococcus albidus d24 lipase was able to catalyze the esterification reaction between fructose and palmitic acid to produce fructose palmitate using acetone as the solvent. due to its stability in organic solvents, we propose that in order to increase the yield of fructose palmitate, we could immobilize d24 lipase. therefore, the effect of immobilization on kinetic parameters of d24 lipase was investigated. different immobilization materials and methods were used to find efficient support materials for d24 lipase immobilization. additionally, fructose palmitate production processes will be optimized with immobilized lipase. introduction: the diagnosis of osteoarthritis (oa) is based on clinical symptoms and radiographic findings. it is known that the pathologic changes at the molecular level in the joint cartilage tissue start before symptoms appear in oa. c1q tumor necrosis factor-related protein 1 (ctrp-1), c1q tumor necrosis factor-related protein 3 (ctrp-3) and kallistatin are related to many different cellular processes including bone and cartilage tissue metabolism. the aim of this study was to investigate any probable association between the serum ctrp-1, ctrp-3 and kallistatin levels and diagnosis and radiologic staging of knee oa patients. materials and methods: this study included 60 patients with knee oa and 30 healthy individuals for control purposes. the patient group was divided into four stages radiologically. ctrp-1, ctrp-3 and kallistatin levels were measured in serum samples of patient and control groups with elisa method, and the differences between the groups were analyzed with statistical methods. results: the levels of serum ctrp-1 in the patient group were significantly higher than in the control group (p = 0.001), serum ctrp-3 and kallistatin levels were not statistically different (in order of p = 0.251, p = 0.160). in the patient group, there was not a significant difference between serum ctrp-1, ctrp-3 and kallistatin levels and radiologic stages (respectively p = 0.811, p = 0.715, p = 0.202). there was a significant positive correlation between the radiologic stage and patient's age, body mass index, western ontario and mcmaster universities arthritis index and visual analogue scale values (respectively p = 0.001, r = 0.510; p = 0.010, r = 0.331; p = 0.001, r = 0.683; p = 0.001, r = 775). discussion and conclusion: serum ctrp-1 levels were detected significantly increased in patients with knee oa, but there was no significant difference in ctrp-3 and kallistatin levels. there was not a significant association between the radiologic stage and levels of ctrp-1, ctrp-3 and kallistatin. enzymes in microorganisms, especially termophilic bacteria are more attractive in biotechnology and molecular biology due to the higher catalytic activity. turkey is rich in geothermal resources and it is important to determine unknown microbial content of geothermal sources. in this study, water and sludge samples were taken from ayder, kizilcahamam and gonen hotsprings. firstly, ph, temperature, salt concentration, gram reaction, mobility, endospore formation, oxidase and catalase tests were carried out as conventional characterization. molecular characterizations of isolates were achieved by fames, rep-pcr and 16s rrna sequencing. finally, test isolates were evaluated according to enzyme production capability by petri dish. as result of conventional tests, isolates were determined as gram positive, mobile-rod shaped, aerobic, oxidase, catalase and endospore positive. the growth range for ph and temperature of the isolates were determined as 5-9 and 50-65°c. in consequence of the salt test, the test isolates were grown at 2-10% nacl. 19 of thermophilic isolates were selected by rep-pcr and according to 16s rrna sequencing analysis test isolates were belonging to bacillus, geobacillus, anoxybacillus and brevibacillus genus at a range of 98-99%. enzyme tests showed that, some of the isolates were able to produce protease (f17, f31, f76, f6, f99 and f19), amylase (f41, f76, f42, f98 and f10), cellulase (f17, f41, f31, f6, f99 and f19), xylanase (f17, f31, f76, f99 and f19) and lipase (f17, f31, f6, f99 and f19). it can be concluded that, geothermal regions are rich in bacillus and related genera. fame analysis was particularly insufficient for diagnosis of thermophilic microorganisms, but rep-pcr was successful in separation of organisms at species and even subspecies levels. most of our bacterial isolates have industrially important enzyme production capacities. it is a pioneer result to use bacteria for industrial applications which need higher temperatures. p-02.02.2-065 warburg effect was investigated by studying various metabolic molecules and assays in mammalian cell lines z. i. kanbagli, b. kiratli, h. cimen yeditepe university, istanbul, turkey majority of the different cancer cells switch their metabolism from oxidative phosphorylation pathway to glycolytic pathway; in order to meet excessive energy requirement, which is also called warburg effect. acetylation is one of the most crucial post-translational modifications playing key roles in metabolic reprograming. in this study, the relationship between acetylation dependent changes in energy metabolism and apoptotic pathways were investigated in pnt1a, du145, hela, hep3b, hek293t, shsy5y. immunoblotting experiments were applied by using antibodies against acetylated-lysine to examine the changes in overall acetylome. candidate proteins displaying elevated acetylation was identified with mass spectrometry based-proteomic analysis. glucose transporter 4 (glut4) was used to detect insulin-stimulated glucose transport, total oxphos rodent antibody cocktail to identify variations in complexes which are responsible for most of the atp production. caspases (casp-3, -9) to unreveal different activation levels of apoptotic pathway among the cell lines. mitochondrial membrane potential was measured by using rho-damine123 by employing confocal microscopy. the expression level of respiratory chain complex iv subunit mtco1 and casp-3 was higher in hek293t compared to other cell lines. casp-9 was upregulated in cancer cell lines, mostly in hep3b. glut-4 levels were downregulated in cancer cell lines in contrast to healthy cell lines. findings imply that these proteins might have significant roles leading to variable metabolic and apoptotic activity of each cell line during energy production. due to the results, mtco1 might be important in adaptation of different cell lines to regulate the overall respiratory chain complex activity. reduction in glut4 level demonstrates insulin desensitization in cancer cell lines, which might lead to metabolic defects in these cells. besides, since p53 has a repressive effect on glut4, it also can lead us to study about p53 levels. the effect of inhibition of pi3k/akt/mtor signaling pathway on receptor tyrosine kinase expression in breast cancer cells g. tunali, a. l. dogan department of basic oncology, hacettepe university cancer institute, ankara, turkey the increased expression and activation of receptor tyrosine kinases (rtk) (egfr, her2, her3) play important roles in breast cancer pathogenesis. her2/her3 interaction is the most potent heterodimer and it causes oncogenic pi3k/akt activation. inhibitors of pi3k/akt pathway (akt inhibitor and pi3k/mtor dual inhibitors) lead to increase in rtk levels and activities while blocking signaling pathway. in this study, the time dependent effect of dual pi3k/akt inhibitor pi-103 on receptor tyrosine kinase expressions' in breast cancer cells was investigated. two breast cancer cell lines, mda-mb-468 cells (which has egfr overexpression and pten deficiency) and skbr-3 cells (which has her2 overexpression) were evaluated for the effect of dual inhibitor. these cells were treated with dual pi3k/akt inhibitor pi-103 for different time periods (1-24 h) . egfr, her2 her3 total rtks expression and pi3k/akt pathway inhibition (p-akt and p-p70s6k expression) were evaluated by western blot. in mda-mb-468 cells, there were significant decrease in p-akt and p-p70s6k proteins' expression during the first 3 h. this inhibition was followed by reactivation of the signaling pathway after 6 h. in skbr-3 cells, p-akt and p-p70s6k proteins' expression were significantly decreased during the first 6 h. the pi3k/ akt signaling pathway in these cells were reactivated after 12 h. basal expression of egfr and her3 in mda-mb-468 cells and basal expression of her2 and her3 in skbr-3 cells were found to be very high. transient inhibition of akt and mtor protein kinase activation in tumor cells followed by reactivation of signaling pathway did not result in a time-dependent difference on egfr, her2 and her3 expression levels. these results suggest that dual pi3k/mtor inhibiton by pi-103 may trigger receptor tyrosine kinase reactivation due to the signal distruption without affecting total protein expression level. site-specific bioorthogonal reactions are one of the significant tools for discovering different aspects of biological systems in live cells. the reactions should be highly stable and rapid in physiological conditions. various chemical tools can be used in bioorthogonal reactions to monitor biological systems, therapeutics, microscopy and diagnostic applications in live cells. synthetic covalent chemistry in the study of biological systems has been used to label biomolecules selectively in their native environment. for example, small synthetic fluorophores can be added to biomolecules without disturbing other molecular biological pathways. aldehydes or ketone-based functional handles can be attached onto protein at specific sites via chemoenzymatic reactions. labeling of carboxy terminus of a-tubulin has been successfully studied in our previous studies by replacement of wild type tyrosine with unnatural amino acid 3-formyltyrosine in the presence of tubulin tyrosine ligase enzyme (ttl)-as its role can suggest whether certain cancer cells might grow more aggressively than others. in this work, we highlight the synthesis and spectroscopic properties of azacoumarin chemical probes to study tubulin-tubulin tyrosine ligase (ttl) system in live cells. significant increase in fluorescence quantum yield or a red shift on absorption and emission maxima is observed when the conjugated product is formed. bioorthogonal fluorescent labeling is such a favorable reaction to perform rapid kinetics, localization and high site-specificity in cell environment. newly synthesized azacoumarin fluorophores should therefore not only be useful for studying ttlbased biological systems, but also would enable broad range of high-yielding and fast diagnostics for future biolabeling applications in biochemistry, cell biology and beyond. binase is an extracellular ribonuclease from bacillus pumilus which shows antiviral and antitumor activities in cell cultures. however, the action of binase on intracellular functions and processes has not yet been identified. here, for the first time we report the whole transcriptome analysis of binase-treated human lung adenocarcinoma epithelial a549 cells. a plasmid-based reverse genetics system and colorimetric cell viability assay were used to identify the binase internalization and binase cytotoxicity towards a549 cell line, respectively. for the whole cell transcriptome analysis a549 cells were treated with 100 lg/ml of binase for 24 h followed by mrna extraction and library preparation. sequencing was performed on solid 5500xl wildfire next-generation sequencer. we found that binase internalized into a549 cells after 2 h of incubation. the binase at 100 lg/ml was absolutely non-cytotoxic towards a549 cell line and was active in the cell culture medium during 48 h incubation. the analysis of rna-seq data showed that among 13 thousands of protein coding transcripts 791 transcripts were up and down regulated by binase, among them 79 transcripts were induced and repressed. binase repressed the production of s100a16 and tnxb which act cancer biomarkers, scn8a and drd4 which play a crucial role in cancer metastasis and responsible for pediatric tumors, respectively. the induction of transmembrane protein transcript abcb11 by binase can help binase to internalize into the cell as abc transporters are often account for transporting drugs across the cellular membrane. binase induced the production of nlrp3, rasgrp1and alpk2 transcripts which can activate apoptosis, cytokine or t cell activation in cancer cells. thus, binase exerts different effects in cancer cells. the rnaseq data obtained will help to understand the mechanism of binase anticancer action. .72) is a specific group of phosphatases capable of hydrolyzing myo-inositol 1,2,3,4,5,6-hexakisphosphate (phytate) with the formation of less phosphorylated inositol derivatives (from mono-to pentaphosphate). three major types of phytases are recognized on the basis of the first phosphate group hydrolyzed by the enzymes: 3-phytase, 4/6-phytase, and 5-phytase. due to the stereospecific way of phosphate release from the phytate molecule by the action of phytases, these enzymes by themselves and their composition may serve as a potential alternative for production of myo-inositol phosphate isomers with therapeutic properties. chemical synthesis of these compounds is inefficient and costly. pantoea sp. strain 3.5.1 showing high phytase activity was isolated from the forest soil sample of the republic of tatarstan, russia. in this study the main objective was the cloning and expression of pantoea sp. 3.5.1 phytase gene in e. coli. first, we amplified the phytase gene (agpp) from the genomic dna of the bacteria using specific primers phmh_dir and phmh_rev. size of phytase gene corresponded to 1729 base pairs. during the optimization of amplification conditions it was found that the optimum temperature for primer annealing was 66°c. this temperature increases the specificity and efficiency of annealing. then the pcr-product of agpp gene was cloned into the pbad myc/ his vector first. on the next step we carried out subcloning of the agpp into a pet28a expression vector. multicopy plasmid pet28a/agpp contained the sequence of the phytase gene of pantoea sp. 3.5.1 under t7 promoter. the corresponding recombinant protein was expressed in e. coli as a fusion with a 6 his-tag and was detected by western blotting. recombinant phytase was purified via affine chromotography on the ni-nta column and displayed high phosphomonoesterase and phytase activities. bag-1 (bcl-2 associated athanogene) is a multifunctional protein that interacts with diverse array of cellular targets and modulates a wide range of cellular processes, including proliferation, cell survival, transcription, apoptosis, metastasis and motility. in human cells bag-1 exists as three major isoforms (bag-1s, bag-1m and bag-1l) derived by alternative translation initiation from a single mrna, which allows interactions with various molecular targets such as hsp70/hsc70 molecular chaperones, components of the ubiquitylation/proteasome machinery, bcl-2, raf-1 kinase, nuclear hormone receptors and dna. our work aims to investigate how altered bag-1 expression levels affect cell survival in mda-mb-231 (er, pr and her2/neu negative) breast cancer cell lines. we first cloned bag-1l gene to a cloning vector, later we transfected mda-mb-231 cells for overexpression of bag-1. we also used bag-1 sirna to silence bag-1 gene. western blot analysis was applied to demonstrate relative expression levels of bag-1, its interacting partners and certain proteins which are important for apoptosis pathway. we performed xtt cell viability assay for bag-1 overexpressed cells to checkbag-1's impact on cell survival, and observed enhanced survival rates on cells compared to that of the untreated cells with bag-1 overexpression. in addition, our study revealed that once bag-1 forms a complex with c-raf/b-raf/hsp70/akt/bcl-2, modulation of cell survival was observed. we believe that once the exact localization and involved molecular mechanisms of bag-1 and its isoforms are found, the role of each bag-1 isoform in cell survival can be understood better. this can further provide routes to study tumor development. the aim of this study is testing the recombinant glp1 encapsulation into a biocompatible material. we also tested if it can be a therapeutic candidate drug for type 2 diabetes. the incretin hormones, which are also named as endogenic peptide hormones have become a more attractive therapy for type 2 diabetes because of different physiological effects. in circulation, glp1 is cleaved by ddp4 in a very short time. if glp1 can be protected from cleaving, the effective time of glp1 would be increased and by this way it can replace the therapy of insulin. chemical synthesis methods of peptides are limited because of low efficiency and high cost. the production of peptides by recombinant e. coli is an alternative way because of effective production, simplicity and low cost. however, the major disadvantage derived from the recombinant e. coli is the frequent formation of inclusion body. for that reason, extra methods are needed for obtaining soluble recombinant peptides. glutathione s-transferase (gst) tag is commonly used as affinity and solubility tag to improve the solubility of recombinant peptides. in this study, we cloned and heterologously produced glp1 using the gst fusion system in e. coli. affinity purification of recombinant protein was achieved by using glutathione immobilized columns. characterization of the gst-tagged glp1 was performed by sds-page. the purity of fusion protein was found to be 65%, as confirmed by glp1 elisa kit. then, the fusion protein was encapsulated in a chitosan coated polygalacturonic acid. the different ph stability and in vitro release tests also in different phs was studied. morganella morganii is an opportunistic pathogen capable of causing a wide range of clinical infections. it is known that microbial metalloproteases play an important role in the development of bacterial infections. thus, investigation of m. morganii metalloproteases has a particular interest. bacteria were grown in lb medium at 37°c. as a bioinformatics tool blast was used. for molecular biological experiments, thermo scientific kits and sibenzyme enzymes were used. pbad/myc-his plasmid was used as an expression vector. bacterial transformation was carried out by heat shock method. bacterial cells were disrupted by sonication. gene expression products were analyzed by western blotting. to analyze the actinolytic activity of bacterial extracts sds-page electrophoresis was used. the putative metalloprotease gene (an cp004345.1) has been found in the genome of annotated strain of m. morganii kt. its amino acid sequence has partial homology (37%) with actin specific metalloproteases grimelysin from s. grimesii and protealysin from s. proteamaculans. using specific primers the gene with 99% homology was identified in the genome of clinical isolate of m. morganii 4. rt-pcr analysis showed that this gene had the maximum expression at 48 h of growth. in addition, the cellular extract of m. morganii 4 had small actinolytic activity. cloning of the gene into e. coli dh5a cells led to the synthesis of the 35 kda protein. it is known that the highest expression of serratia proteases is observed at 48 h of growth, and the molecular weight of the mature proteins is 32 kda. it was shown that metalloprotease gene of m. morganii 4 expressed at the same time of growth. moreover, the recombinant e. coli cells synthesized protein with the similar weight (35 kda) which perhaps is a mature form of the metalloprotease from m. morganii. as a result, in the genome of m. morganii 4 the metalloprotease with similar properties to grimelysin and protealysin proteases was identified. the preliminary characterization of p-ii like protein glnk from lactobacillus brevis z. iskhakova 1 , d. zhuravleva 1 , a. laykov 1 , k. forchhammer 2 , a. kayumov 1 1 kazan federal university, kazan, russia, 2 eberhard-karls university tuebingen, tuebingen, germany the p-ii proteins in bacteria, archaea and plants regulate the activity of a variety of proteins in response to specific metabolic signals which affect their structural state and interaction ability. among various bacteria belonging to lactobacillus only some species have genes encoding pii protein in the genome. here we report the preliminary characterization of pii-like protein lbrglnk from lactobacillus brevis. while the amino acid sequence alignment revealed only 50-70% homology of lbrglnk with other well studied pii proteins, lbrglnk also has the atp binding motive gdgk. trimeric structure of lbrglnk was confirmed in vitro by size exclusion chromatography, suggesting possible similarities of lbrglnk properties with pii proteins. the isothermal titration calorimetry revealed a preferential binding of adp (kd = 50 lm) over atp (kd = 357 lm) suggesting that they compete for binding to lbrglnk. neither 2-oxoglutaric acid nor other nucleotides were interacting with lbrglnk in itc measurements. the mutation gly91ala in the atp binding motive completely abolished the interaction with both adp and atp. the pull down of lbrglnk with l. brevis cell extract allowed identification of chaperonin grol, transketolase and glnr-like transcriptional regulator from merr family as most probable partner proteins for interactions with lbrglnk. this work was supported by the russian foundation for basic research (project no. 15-04-02583a background: hemophilia is a bleeding disorder due to the deficiency in coagulation factors viii (hemophilia a) or ix (hemophilia b). hemophilia patients are essentially treated with intravenous replacement of the missing or dysfunctional factors fviii or fix by recombinant proteins. these therapies often induce the generation of acquired antibodies, and thus, novel approaches are needed. most recent hemophilia strategies target the tissue factor pathway inhibitor (tfpi), which is the major inhibitor of the coagulation cascade, particularly of the extrinsic tenase complex. anti-tfpi agents have been empirically developed such as aptamers, peptides, monoclonal antibodies. we have followed a structure-based strategy, to design a mutated fxa that would show more affinity for tfpi, and thus trap this inhibitor. tfpi exists as two isoforms are folded as multi-kunitz domains related by linkers. the second kunitz type domain of tfpi (tfpi-k2) is known to bind the catalytic site of fxa. methods: the molecular complex of tfpi-k2-fxa was modeled and submitted to molecular dynamics (md), allowing the identification of low-spots interaction. modified fxa with theoretically stronger interaction with tfpi-k2 were predicted using md. the mutants and wild type proteins were expressed in hek cells, and their processing status was checked. they were tested by western blotting, by chromogenic activity using a specific substrate of fxa, by thrombin generation assay in fviii depleted plasma. finally, their binding to a tfpi-k2 peptide array was compared. results: the mutants showed better efficiency to restore thrombin generation in plasmas from hemophiliacs and displayed stronger binding to tfpi-k2 than the wild type fxa. conclusions: the proof of concept of the synergistic approaches of md and mutagenesis was obtained and an efficient tfpi trap was designed. the mutated fxa is a candidate for a new hemophilia therapy. organophosphorus acid anhydride (op) nerve agents are potent inhibitors which disrupt the mechanisms of neural transmission. organophosphorus acid anhydrolase (opaa; e.c.3.1.8.2) is a class of enzyme that potentially acts on phosphorus anhydride bonds, reported intracellularly in diverse organisms, albeit notably the enzyme belongs to alteromonas species are more extensively studied. whereas mass-transfer problem is a major issue in native whole cell biocatalysis, new anchor system derived from the n-terminal domain of ice-nucleation protein from pseudomonas syringe inav (inav-n) was used for the first time to display opaa onto the cell surface. tracing of the recombinant protein and its activity assay showed a successful presentation of opaa and its significant ability for biodegradation of organophosphorus compounds. further studies on bacterial fractions confirmed that opaa is remarkably located on the outer membrane. the specific activity of recombinant bacteria to degrade diisopropylfluorophosphate (dfp) was measured at 260 u/mg of cell wet weight, which almost all was observed in the outer membrane fraction. recombinant cells could also degrade chlorpyrifos (cp) compound in 195.6 u/mg activity. it can be concluded that inav-n anchor is efficient for targeting opaa on the cell surface and can effectively eliminate the masstransfer problem in native whole cell bioconversion system. proper spatial and temporal organization of proteins involved in cell signal transduction is crucial for the specific and efficient information transfer. scaffold proteins coordinate the action of signaling molecules by their physical binding and organization in multiprotein complex assemblies. multiple protein binding is often mediated through intrinsically disordered regions of the scaffold, where the interaction epitopes are defined by linear peptide motifs. using a hub scaffolding protein axin as a paradigm, we have employed peptide microarray technique to identify the binding epitopes for axin interaction partners at high resolution. this enabled us to design axin-derived peptides corresponding to the respective binding epitopes that compete for the interaction in vitro. by transfection of chemically stabilized competitive peptides directly into the cells, we have shown the effect of specific interaction blocking on axin-mediated signaling in vivo. our data demonstrate a proof of concept for a rational design of inhibitors of protein-protein interactions that allow specific intervention with single function of the targeted protein (i.e. recruitment to the axin complex). contrary to the inhibitors that completely disrupt the protein function (e.g. inhibition of a kinase catalytic site), this approach provides a tool for investigating specific action within the axin complex, while the other cellular functions of the targeted protein remain preserved. the results of this research have been acquired within cei-tec 2020 (lq1601) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. de novo design of an artificial biocatalyst using immunoglobulin template became rather routine procedure due to the achievements of molecular biology and crystallography. recently the 'reactibody' approach was developed based on the chemical selection of catalytic repertoires from immunoglobulin library followed by expression of these biocatalysts in eukaryotic system. in this study we structurally characterize the a5 antibody, its kappa and lambda variants, in order to understand the difference on the active site between a5 and a17 which although there are two antibodies sharing very high homology and sequence identity their active residues are located in a different region of the antibody. the structures of the a17 antibody kappa and lambda variants have been already determined, there was no structural information though about the a5 antibody. the structural analysis revealed dramatically different angle in position of nucleophilic residue tyr33 and area of solvent accessible surface. the structural difference of active center reflects on kinetics of the a5 organophosphate modification. both variants of antibody bind with organophosphate throw induce-fit mechanism, but rate of the step of induce fitting is different (k obs are 35 s à1 and 16 s à1 for a5kappa and a5lambda respectively). this observation may hint at novel means of regulation of velocity and specificity of artificial biocatalysts. this study was supported by grant #rfmefi61614x0009. translation elongation factor 1ba (eef1ba) is a component of a heavy form of translation elongation factor 1 (eef1h). it functions as a catalyst of gdp/gtp exchange in translation elongation factor 1a (eef1a) restoring its active conformation appropriate for aminoacylated trna binding. eef1ba forms a tight complex with translation elongation factor 1bc (eef1bc) via the n-terminal domain, while its c-terminal domain executes the catalytic activity. eef1bc has been shown to enhance the attributed to the c-terminal domain catalytic activity of eef1ba. this suggests that the eef1ba n-terminal domain may influence the guanine nucleotide exchange process. to test this hypothesis we prepared a set of n-terminal truncated forms of human eef1ba and checked their activity in the guanine nucleotide exchange assay on both isoforms of eef1a, eef1a1 and eef1a2. we showed that recombinant eef1ba is a non-globular monomeric protein in solution with an elongated shape by analytical ultracentrifugation approach. the truncation of the dispensable for the catalytic activity n-terminal domain of eef1ba resulted in significant acceleration of the rate of guanine nucleotide exchange in eef1a2 comparing to full-length eef1ba. similar effect on the catalytic activity of eef1ba was observed after its interaction with eef1bc. in contrast, the effect of full-length eef1ba and its truncated forms on the rate of guanine nucleotide exchange in eef1a1 was similar but relatively modest compared to eef1a2. this can be explained by higher rate of spontaneous gdp dissociation from eef1a1 comparing to eef1a2 and lower affinity of eef1a1 to eef1ba. thus, we propose that the n-terminal domain of eef1ba via flexible linker region may interfere with eef1a binding to the cterminal catalytic domain that results in a decrease of the overall rate of guanine nucleotide exchange reaction. the formation of a tight complex between eef1bc and eef1ba n-terminal domains abolishes this inhibitory effect. p-02.02.2-079 assessment of quantitative proteomics results in large-scale data-independent with fragmentation spectra reproducibility measure reduces variation and allows to use lowintensity signals organisms with reduced genomes that lack the vast majority of transcriptional or translational regulation systems tend to adapt to changing environment with a variety of subtle changes in protein abundances. as soon as relative changes for most proteins fall below 50%, the power of traditional label-free proteomic analysis rapidly becomes insufficient for robust profiling of hundreds of samples. intoduction of frament-by-fragment and sample-by-sample signal quality assesment in mrm and dia experiments helps to increase accuracy of methods and at the same time reintroduce cases which could have been excluded during bulk quality assessment due to lower signal-to-noise ratio for several fragments. 240 samples of mycoplasma gallisepticum were acquired in data-independent manner on sciex tripletof 5600+ mass-spectrometer (swath acquisition) during the year. the samples were produced from mycoplasma gallisepticum culture cultivated at different temperatures. the signals for each fragment were extracted with vendor-supplied software with the theoretical fragment ions for each peptides instead of spectral library. the results were used for relative protein quantitation in two manners the first conventional method included direct use of peptides with top 3 most intense signals. the second included selection of peptides and ions for quantification for each pair of samples based on the reproducibility of fragmentation patterns after computing the areas of chromatographic peaks for each ion. spectral angle was used as a distance measure for fragmentation patterns for clustering. further, a base set of detected ions was selected for each peptide and a subset for comparison of each pair of runs. the first method resulted in quantitation of 354 proteins across all samples with variation across lc-ms replicates was 21% on average, and the second approach led to quantitation of 515 proteins in total, 390 of them across 75% of samples, all with the variation about 11% on average. p-02.02.2-080 interaction of plasminogen fragments k 1-3 and k 5 with fibrin fragment dd t. yatsenko, v. rybachuk, l. kapustianenko, s. kharchenko, o. yusova, t. grinenko palladin institute of biochemistry of nas of ukraine, kyiv, ukraine introduction: plasminogen interaction with specific binding sites in c-terminal d-domains of fibrin molecule initiates the activation process of proenzyme and subsequent fibrin clot lysis. the sites are exposed under fibrin polymerization. plasminogen kringle domains ensure the proenzyme interactions with fibrin clot. in this study, we investigated the binding of human plasminogen kringle fragments k 1-3 and k 5 with human fibrin fragment dd and their effect on glu-plasminogen interaction with dd. results: kringle-containing fragments k 1-3 and k 5 reduce plasminogen activation by tissue-type activator on fibrin fragment dd. the level of glu-plasminogen binding to dd is decreased by 50-60% in the presence of k 1-3 and k 5. fragments k 1-3 and k 5 have high affinity to fibrin fragment dd (dissociation constant is 0.02 lm for k 1-3 and 0.054 lm for k 5). analysis of k 1-3 and k 5 binding to fibrin fragment dd with reduced disulfide bonds showed the interaction of both plasminogen fragments with c-c-chains of fragment dd. k 1-3 interacts with complex of fragment dd-immobilized k 5 as well as k 5 with complex of fragment dd-immobilized k 1-3. the plasminogen fragments do not displace each other from binding sites located in fibrin fragment dd, but can compete for the interaction. analysis of k 1-3 and k 5 binding to fibrin fragment dd with reduced disulfide bonds showed the interaction of both plasminogen fragments with aand c-chains of fragment dd. conclusions: widely known specific plasminogen-binding site located in aa148-160 region of fibrin molecule is not a single binding sequence of fibrin peripheral domains or plasminogenbinding site is not linear and contains amino acid residues from other polypeptide chains of fibrin d-domains. fibrin fragment dd contains different binding sites for plasminogen kringle fragments k 1-3 and k 5, which can be located close to each other. possible plasminogen kringle-binding sites are located in aand c-chains. p-02.02.2-081 implementation of budded baculovirus particles for characterization of ligand binding to g protein-coupled receptors a. allikalt, a. rinken university of tartu, tartu, estonia g protein-coupled receptors (gpcrs) constitute the largest class of membrane receptors involved in regulation of signal transduction into the cell in response to various extracellular stimuli. for that reason, gpcrs have become important targets for variety of drugs. as these receptors are present in native tissues at very low concentrations, efficient recombinant expression systems are needed to produce sufficient amounts of protein. we have shown that budded baculovirus particles, which display gpcrs on their surfaces can be used as a source of receptors for the investigation of ligand-receptor interactions. this expression system can be used for radioligand binding assay as well as for fluorescence anisotropy-based assay (fa). we have validated the system with budded baculovirus particles produced in spodoptera frugiperda (sf9) cells expressing human dopamine d 1 receptors using [ 3 h]sch-23390 and bodipy-fl-skf-83566 as reporter ligands for corresponding assays. this system has many advantages, for example good signal to noise ratio, homogeneity of the receptor, high expression levels and long-term stability of the receptor preparation. fa method allowed real-time monitoring of reporter ligand binding in the absence and presence of different dopaminergic ligands, giving information about their kinetic properties. association, as well as dissociation of the bodipy-fl-skf-83566 itself were rapid with an apparent half-life of t 1/2 = 38.5 ae 0.3 s for association (2 nm) and t 1/2 = 73.4 ae 3.8 s for dissociation. we determined the pharmacological profiles of different dopaminergic ligands in displacement binding assays with membranes of sf9 cells or budded baculovirus particles. the data were in good agreement for both membrane preparations tested in radioligand binding as well as in fa assay. obtained results indicate that budded baculovirus particles can be proposed as a source of gpcrs for performing fluorescence anisotropy as well as radioligand binding assays. gastrointestinal (gi) cancer includes a variety of cancer types affecting the structures and functions of the gi system, encompassing the gi tract and the accessory organs of digestion, from the esophagus, stomach, biliary system and pancreas to the intestine, rectum and anus. despite the significant advances however, much remains to be learned in the spectrum of gi cancer. several investigators have shown that both gas6 and its receptors, axl, sky, and mer are expressed in various types of cancers. however, the expression level of gas6 in gi cancer remains unclear. the aim of the study was to determine and compare plasma gas6 levels in gi cancer patients. 15 female and 27 male patients were included in the study (n = 42): 21 colorectal, 8 gastric, 4 pancreatic, 3 liver, 2 ampullary, 2 gall bladder and 2 esophageal. from all gi cancer patients, 2 ml venous blood was collected in citrate tubes before surgery. blood samples were centrifuged at 3000 g for 10 min, and plasma samples were carefully removed and stored in à80°c prior to use. the level of plasma gas6 was measured using a commercial developmental elisa kit (r&d systems, minneapolis, mn) which is validated by our laboratory. plasma gas6 levels in cancer patients were determined as follows: 1.84 ae 1.1 ng/ml in colorectal; 1.16 ae 1.1 ng/ml in gastric; 1.63 ae 0.61 ng/ml in pancreatic; 3.91 ae 0.89 ng/ml in liver; 2.15 ae 0.26 ng/ml in ampullary; 1.34 ae 0.46 ng/ml in gall bladder and 2.32 ae 1.7 ng/ml in esophageal cancer. preliminary findings indicate that there is a relation between gi cancers and plasma gas6 levels. taken together, these results suggest that gas6 may be a candidate biomarker for diagnostic use in gi cancer. inhibition of gas6 would be an attractive therapeutic target for slow down the progression of gi cancer. monday 5 september 12:30-14:30 computational biology p-03.03.2-001 computational approaches as an assay for blactam hydrolysis in class a b-lactamases c. tooke university of bristol, bristol, united kingdom b-lactam hydrolysing enzymes, in particular carbapenem-hydrolysing enzymes, are an increasing clinical threat. herein we show that molecular dynamics (md) and combined quantum mechanics/molecular mechanics (qm/mm) approaches are a predictive tool of carbepenemase activity in class a b-lactamases. b-lactam drugs are the most prescribed class of antibiotics worldwide, especially in the treatment of gram-negative pathogens such as klebsiella pneumoniae and escherichia coli. these organisms produce b-lactamases, enzymes which hydrolyse the b-lactam ring, a key resistance mechanism. class a b-lactamases have the ability to hydrolyse carbapenems, termed 'last resort' antibiotics. in particular, the kpc (klebsiella pneumoniae carbapenemase) family are the most clinically important, and recently identified natural kpc variants show increased hydrolytic activity against ceftazidime, a third generation cephalosporin. here we use computational simulations of b-lactam hydrolysis by b-lactamases. in particular, molecular dynamics (md) combined with qm/mm approaches have been used to model the deacylation of the carbapenem meropenem across 8 class a b-lactamases. this method has been extended to model cephalosporin hydrolysis across class a b-lactamases, including kpc variants. these approaches calculated the free energy barriers and correctly distinguished carbapenemases from carbapenem-inhibited enzymes. preliminary results suggest this protocol is also a predictive tool for ceftazidime hydrolysis. further, md simulations of 5 kpc variants (single and double amino acid changes) were analysed to identify structural changes in the active site, highlighting that variants differ in the size of the active site opening, corresponding with experimentally derived kcat values. these computational assays provide a predictive tool of b-lactam hydrolysis and has potential to provide insights into important mechanistic differences both across class a b-lactamases and within the same families. p-03.03.2-002 computational design of a novel polyglutamic dendrimer-based platform as an anticancer therapeutic approach poly (glutamic acid) (pg)-dendrimer as potential nanocarriers for cancer therapies, to specifically deliver tumor associated antigens (taa)mannosamine and melanato target cells and to modulate cancer antigen intracellular trafficking within the cytoplasm to promote an efficient and selective antitumor immunotherapeutic effect. the theoretical structures were obtained using x-plor software. the molecular dynamics simulation of pg-g4-dendrimer and taas was performed using desmond. the electronic properties of the structures were determined by semi-empirical methods using mopac. docking studies of taa to pg-g4-dendrimer to mannose receptor (mr1) were performed using hex 8.0.0 software. taa lumo atoms were conjugated to homo atoms of pg-g4 dendrimer using maestro software. results showed that pg-g4-dendrimer displays 64 carboxylic end groups available for covalent interaction with taas. the homo molecular orbitals of the dendrimer was located on the a-carbon of the carboxylic acid groups from backbone chain and it preferentially interacts with lumo molecular orbitals of amine group from taas. no differences in the gap energy of homo/lumo of all pg-g4-conjugates. taas bind preferentially to a-carbon of cooh of backbone chain instead of cooh from side chain. docking results showed that majority of taa conjugated pg-g4-dendrimer binds to the core of the mr1 receptor. increasing of the number of mannosamine conjugated to pg-g4-dendrimer more close and stable is the conjugated to the receptor. this system shows promising results as a novel functionalized pg-dendrimers for cancer therapy. theileria parva is one of the the economically important protozoan of the theileria genus belong to apicomplexa phylum which include plasmodium spp. and toxoplasma gondii, causative agents of malaria and toxoplasmosis respectively. this parasite is the disease agent of tick-borne east coast fever (ecf) ranks first among the tick-borne diseases of cattle in sub-saharan africa. the disease caused by the parasite affects a large proportion of domestic and wild animals and leads serious economic losses in the world. major problems in dealing with this illness are the high cost of drugs, development of resistance, and absence of effective vaccines. thus, it is important to develop an efficient and affordable antitheilerial agent. for this aim, 1-deoxy-d-xylulose-5 phosphate reductoisomerase (dxr) which subjected to identify novel drug aganist malaria and toxoplasmosis, of theileria parva was selected as potential target for improving novel inhibitors aganist ecf. a computational molecular modeling approach was conducted to determine the 3d structure of tpdxr by phyre2. energy minimisation and root mean square deviation (rmsd) was performed by 3drefine and superpose servers. to ensure the quality of modelling, stereochemistry, energy profile and residue environment of modelled structure were checked by different servers and possible ligand binding pockets were identified by metapocket 2.0 server a reliable 3d model for dxr from t. parva was modeled by using 3au9 as a template. the ca rmsd and the backbone rmsd deviations for the model and the template crystal structure were found to be 0.85 and 0.86 a, respectively. the ramachandran plot for the predicted model by rampage reveals that model shows an acceptable stereochemistry. top three considered possible binding pockets have been identified. these results have important implications for future screens aimed at finding new and safe molecular entities active against tpdxr through docking studies. p-03.03.2-004 molecular binding profile of protoberberine alkoloids on amyloid precursor proteincleaving enzyme 1 (bace1) as a drug candidate for alzheimer's diseases g. yalcin 1 , i. yildiz 2 1 biotechnology institute, ankara university, ankara, turkey, 2 department of pharmaceutical chemistry, faculty of pharmacy, ankara university, ankara, turkey alzheimer's disease (ad) is the most prevalent neurodegenerative disorder that leads to dementia and nowadays over 46 million people live with dementia worldwide. because of the prevalence and economic burden of the disease, drug development studies have picked up speed and scientists especially focused on natural products. ad is basically characterized with tau hyperphosphorylation and accumulation of amyloid b (ab) proteins. ab proteins are generated from sequential cleavages of amyloid precursor protein (app) by b and c secretases, and b-site app cleaving enzyme 1 (bace1) is a b secretase essential for ab production. the alkaloids represent a very extensive group of secondary metabolites, with diverse structures, distribution in nature and important pharmacological activities. protoberberine alkaloids, which belongs to isoquinoline alkaloid class, are widely arranged in many species of the berberidaceae, annonaceae, fumariaceae, papaveraceae, and other plant families. recent searches showed that some of the protoberberine alkaloids such as berberine, palmatine, jatrorrhizine, columbamine, magnoflorine prevents the progress of neurodegenerative disorder. however, the mechanisms of them are not absolutely clear. therefore, we have aimed to elucidate the binding and affect mechanism of these alkaloids on the bace1 open and closed forms in here. for this purpose, molecular docking studies were applied for these natural products to the both forms of bace1 by using autodock vina and it was subjected to explicit solvent simulations by amber molecular dynamic package. our preliminary studies indicate that gly34, thr72, gln73, phe108, tyr198, lys224, thr232, arg235, thr329 residues of binding pocket have affiliations with all of the mentioned alkaloids and the binding of them generates alterations on closed form of bace1. the complexity of animal milk needs to apply numerous approaches and methods for its investigations. an understanding of the processes occurring in the milk can be used, for example, for quality control of the products. fat and protein are main components of milk which have a significant influence at its colloid properties, such as dynamic surface tension (dst). the application of regression-correlation analysis to milk data enables to develop a reliable quantitative model. the aim of our investigation was to perform the regression analysis to establish the relationship between above-mentioned parameters. for this purpose, we used a statistical software packages r version 3.1.2. dst was determined by bpa -1p tensiometer. milk fat (f) and protein (p) contents were measured by analyzer bentley 150. this work was supported by the russian scientific foundation (grant 14-16-00046). obtained formulas characterized the degree of influence of fat and protein contents of a milk sample for each of the dst parameters (r 0 , r 1 , r 2 , r 3 , k 0 , k 1 ): r 0 = 61.1538 + 0.8908 * p à 1.2953 * f r 1 = 63.3297 + 0.5658 * p à 1.4132 * f r 2 = 55.4274 + 0.4585 * p à 1.0143 * f r 3 = 45.6265 + 0.34634 * p + 0.05197 * f k 0 = 6.0986 + 0.1543 * p + 0.1351 * f k 1 = 10.7832 à 0.1470 * p à 1.0302 * f these formulas show that the maximum total effect of fat and protein contents influences at r 0 and r 1 . a significant coefficient (> 1) before the fat is observed in the formula, which describes the value of the tilt of final part of the tensiogram (k 1 ). the resulting regression equations have fundamental importance. with their help it is possible to calculate the dst parameters without their experimental determination, positioning fat and protein contents data. obtained dst parameters promote more complete characterization of the properties of the milk that may be used for dairy products. p-03.03.2-006 molecular studies of scorpion toxin and its mutants interactions with voltage-gated potassium channels the voltage-gated potassium kv1.3 channel is mostly expressed in neurons and immune cells. its blockage has a high therapeutic potential, for example, specific inhibitor shk toxin is undergoing clinical trials on psoriasis. goal of the current study was an interface analysis in complexes of hybrid channel kcsa-kv1.3 with peptide blockers agitoxin and its mutant forms. 3d structure was generated by homology modeling method using complex of mutated kcsa channel with charybdotoxin (pdb-code 2a9h) as a template and equilibrated by molecular dynamic simulation in gromacs software. analysis of hydrophobic and stacking interactions, hydrogen and ionic bonds of the toxin and potassium channels was performed for representative frames with optimal toxin orientations using program platinum and apbs software package. we performed contacts energy characteristics estimation to predict key toxin residues for binding process and possible mutation sites for changing selectivity against kv1.x channels. the results of investigation are in good agreement with the experimental values of binding constants, obtained by competitive binding assays. results of the conducted investigation may find an application in fundamental science and drug design. the research was supported by the russian science foundation grant no. 14-14-00239. simulations were performed using the supercomputing center of lomonosov moscow state university. p-03.03.2-007 homology modeling and molecular docking study of the paraoxonase-1 and its polymorphic variants q/r 192 and m/l 55 for non-statin lipid lowering drugs paraxonase-1 (pon1) enzyme is an hdl associated ester hydrolase exhibiting paraoxonase, arylesterase and lactonase activity, and reduces the formation of atherosclerosis blocking the ldl oxidation and reducing levels of oxidized lipids. in this study, molecular docking approach and molecular dynamics simulation were applied for finding the affinity of non-statin lipid-lowering drugs to pon1 and its polymorphic structures pon1 q/r 192 and m/l 55. fibrates (bezafibrate, ciprofibrate, clofibrate, fenofibrate, gemfibrozil), phytosterols (beta-sitosterol, brassicasterol, campesterol, stigmasterol) and other lipid lowering drugs (ezetimibe, niacin, orlistat, probucol, and sibutramine) was obtained from pubchem database. x-ray crystallographic structure of human pon1 and its polymorphic variants pon1 q/r 192 and m/l 55 was generated via 'modeller', homology modelling software, from human-rabbit hybrid x-ray crystal structure of pon1 (pdb code: 3sre). 10 ns molecular dynamic simulation analysis was performed using gromacs 4.5.5. affinity of lipid lowering drugs to pon1 and its polymorphic variants was predicted by molecular docking approach using autodock 4.2 suite. unlike other lipid lowering drugs that they have negative δg values for affinity, probucol, orlistat and betasterol was calculated by positive δg values (18.7, 12.3 and 1.4 kcal/mol). these values suggest that they may have no affinity to pon1 q/r 192 polymorphic structure. in all drug groups, brassicasterol and stigmasterol to pon1-m/l 55 and sibutramine to pon1 q/r 192 were calculated as the highest affinity. in generally, phytosterols predicted by high affinity to pon1 and m/l 55 polymorphic structures in comparison to other lipid lowering drugs. our study demonstrated that phytosterols predicted as high affinity compounds on pon1 structures may reduce the activity of antioxidant pon1 enzyme. this study need to be supported by in vitro and in vivo detailed studies. prolactin and its cognate receptor, prolactin receptor (prlr), are involved in over 300 distinct functions in mammalians. the mammalian prlr gene consists of 10-13 exons and several 5 0 and 3 0 regulatory sequences. in this study, gaps and annotation errors in the rat prlr gene were corrected by comparing the genomes of mammals and rodents and new putative exons were identified. the rat prlr gene sequences from two different sources (rnor_6.0, nc_005101.4 and rn_celera, ac_000070.1) were used and primary analysis showed that both sequences contain several gaps (varying from 0.35 to 4 kbp), corresponding to about 5.6% (10-11 kbp) of the gene. using the rat known prlr mrna exon sequences, it was found that the rnor_6.0 prlr gene has two exon-10 (one is about 2 kbp long and the other immediately after this). comparisons of mammalian and rodent prlr gene structures showed that the 2 kbp stretch is an assembly artifact. by comparing both gene sequences (and also other available rodent prlr genes), the gaps in the rat prlr gene were reduced from 5.6% to 3.8% (from 11 kbp to 7 kbp). functional annotation of the gene revealed that r. norvegicus prlr gene could have two more additional exons, exon-12 and -13, similar to mus musculus prlr gene. in mammals, prlr mrnas contain non-protein coding exons in the 5 0 utr (exon-1 and -2). in rats, there are 5 exon-1 variants, resulting from alternative promotor usages. studies on the rat and mouse prlr genes revealed that both rodents share 4 common non-protein coding exon-1 variants. in conclusion, it is found that the rnor_6.0 version of the prlr gene has the highest number of unidentified base pairs (corresponding to 5.6% of the gene) and the second exon-10 is the assembly artifact. the rat prlr genes in both databases have several gaps and our corrected version is the best available and characterized form of the rat prlr gene. in silico affinities of some statins to paraoxonase-1 enzyme the structure of the statins (atorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin) was obtained from pub chem database, and x-ray crystal structure of pon1 (pdb id:3sre) from protein data bank. modeller software was used for homology modeling of pon1 and its polymorphic variants that's called as pon1 q/ r 192 and m/l 55. amino acid sequence of human serum pon1 (uniprot: p27169) was used as the modeller template. all molecular dynamics simulations were carried out with gro-macs 4.5.5 software. molecular docking calculations on each of the polymorphic structure of the pon1 was performed with auto dock4.2. suite. for each substrate, y71 residue showed open conformation in pon1 and m/l 55 polymorphic structures while q/r 192 polymorphic structure showed closed conformation. in comparison between structures of pon1 variants, in most cases statins had lower affinity to q/r 192 polymorphic structure than to the other variant. in this study, among statins, atorvastatin showed lowest but simvastatin highest affinities to pon1. by considering that the high affinity drugs can have reducing effect of pon1 activities, it may be more appropriate to use the low affinity statins in hyperlipidemia treatment. however, these findings need to be supported with in vivo and in vitro studies. p-03.03.2-010 self-assembly of lipidoids for sirna uptake and release mechanisms studied by molecular dynamics simulations o. acar 1 , d. alpay 2 , a. r. atilgan 1 , c. atilgan 1 1 sabanci university, istanbul, turkey, 2 northwestern university, evanston, united states small interference rna (sirna) has the ability to bind a specific mrna which provides silencing of selected genes. nanocarriers made out of self-assembled lipidoids encapsulate sirna and deliver them into target cells effectively. in this study, a library of lipidoid structures is constructed and studied by molecular dynamics (md) simulations in different solvents, including sodium acetate, to ferret out their self-assembling mechanisms. the effect of the protonation state of the head group of lipidoids on the final shape of the self-assembled carrier is also studied. we further examine the role of the size of hydrocarbon tails in the packing. we study the final topology and the geometry of the self-assembled lipidoids both in the presence and in the absence of sirna. we find that stable clusters form with as few as 20 chains. for lipidoids having neutral head groups, clusters are in the form of dense bundles, while those with charged head groups form spherical capsids which are depleted of the salt on the inside and having a salt rich phase on the outside. in the self-assembled structure, lipidoids are arranged so as to expose the nitrogen and oxygen atoms to the solvent. while partial capsids with these properties also form at lower lipidoid numbers, 200 chains are necessary to form a fully closed capsid. in the presence of the sirna, the capsid assembles around the nucleotide. the free energy to remove the sirna from the assembly is calculated via repeated steered md calculations utilizing jarzynski's equality relating it to the irreversible work along and ensemble of trajectories. we therefore determine an optimal tail length for the most stable nanostructure, paving the way for designing nanocarriers with high efficacy. milk is one of the most valuable products for humans and attracts a lot of interest of researchers in various fields such as biochemistry, biology, food science and technology. the methods of milk study are quite varied. we chose the combination of the ultrasonic and dynamic surface tension (dst) measurements with the possible correlations among the obtained parameters. the aim of this work is to study the correlation between the parameters of milk, such as a content of fat, protein, lactose, minerals, dry milk solids and dst parameters. for this purpose we used milk analyzer 'klever-1m' and tensiometer 'bpa-1p'. three groups of animals were formed from clinically healthy holstein cows at the age of 4-5 years according to the fat content in the milk sample. group i -5 cows (milk fat content 4.01 ae 0.41%), group ii -6 cows (milk fat content 3.32 ae 0.14%), group iii -11 cows (milk fat content 2.73 ae 0.20%). this work was supported by the russian scientific foundation (grant 14-16-00046). the biochemical parameters of the milk samples of all three groups are in the range of the 'normal' values for healthy holstein cows: protein content varies from 3.0% to 4.2%, lactose and mineral content varies from 4.6% to 0.7%, respectively. the dst parameters (r1, r2 and k0) for the group i have strong positive correlations with the fat content in the studied milk samples. at the same time for the groups ii and iii the fat content in the milk indicates only medium positive and weak positive correlations with the r1, r2 and k0. obtained absolute values of the dst parameters of the milk samples showed some differences between all three groups. thus, the dst parameters are changing in direct proportion to fat content in the milk sample that can be explain by the primarily role of the milk lipids in the formation of the water/fat surfaces (such as fat globules, lipid-protein particles, etc.). p-03.03.2-012 exploration of allosteric paths in caspase molecules using energy dissipation e. n. bingol, o. sercinoglu, p. ozbek sarica marmara university, istanbul, turkey caspases are highly regulated aspartate-specific cysteine proteases that have major roles in programmed cell death; apoptosis. effector caspases are at the terminal step of the pathway, hence they are considered as death switches. with the discovery of the presence of allosteric sites, these molecules attracted the attention of the pharmaceutical studies and became drug targets. as a result of the binding of small molecules to the dimeric interface, active site loops are shifted to an unfavorable position. this is associated with a network between distal allosteric sites and the active site loop. an energy dissipation model was applied in order to analyze this matter in further detail. perturbation of specific residues enable us to determine a possible signaling network in proteins using external energy as an input, while focusing on the dispersion of this energy between residues throughout the structure. molecular dynamics simulations were performed with and without energy perturbation using namd software with charmm27 force field. energy perturbation was applied by increasing the velocity of a chosen residue at the desired time step of the initial md simulation. energy change of each residue was calculated upon the application of perturbation. as a result, residue response times, corresponding to the time of the response of a residue after the perturbation of another chosen residue, are obtained. combining reponse time data with a residue interaction network, it is possible to construct a final network that shows the communication started by perturbation within the molecule. it is shown that perturbation of allosteric sites result in the disruption of the catalytic sites given in literature. our findings support this and also gives a little detail of the possible communication between distal allosteric site and the active site loops. this finding enables the usage of this methodology for similar structures where the exact allosteric mechanism is yet not known. p-03.03.2-013 effect of complex mammalian membrane models with multiple membrane components on ras protein nanoclustering a. farcas 1,2 , l. buimaga-iarinca 2 , c. floare 2 , l. janosi 2 1 faculty of physics, babes-bolyai university, cluj-napoca, romania, 2 national institute for research and development of isotopic and molecular technologies, cluj-napoca, romania ras proteins are essential for the cellular signal transduction that regulates cell proliferation and differentiation and act as binary switches between gdp and gtp forms. a wide range of human tumors are associated with defective ras protein signaling. the production of permanently activated ras proteins is correlated with mutations in ras genes. experiments and computer simulations have shown that membrane-bound ras proteins form nonoverlapping dynamic nanosized subdomains (nanoclusters) in activation state-/isoform-dependent manner. we performed coarse-grained molecular dynamics simulations to investigate the effect of complex mammalian membrane models on formation and evolution of ras nanoclusters. a fundamental part of the plasma membrane is the phospholipids bilayer, which contains phosphatidyl-choline (pc), phosphatidylethanolamine (pe), phosphatidyl-serine (ps), sphingomyelin (sm) and cholesterol (chol). the nature of lipid-lipid and protein-lipid interactions was studied in binary (pc:chol) and quinary mixtures (pc:pe:ps:sm:chol). because the polar lipids are not uniformly distributed between the two leaflets of the membrane, the construction of the plasma membrane with five-component lipid mixtures took into account the asymmetry between the outer and inner mono-layers. the phospholipids chain saturation (combined with the presence of cholesterol) constitute the dominant factor in phase separation and was, therefore, modeled in different lipid tail combinations for various headgroups. using microsecond timescale simulations of membraneembedded ras proteins, we have shown that the nanoclusters are spontaneously forming dynamic structures whose behavioral characteristics is modulated not only by the ras isoform, but also by the complexity of the membrane model. furthermore, we showed that variations in the plasma membrane lipid composition have important implications in the localization of ras protein nanoclusters. optogenetics comprises biological methods to achieve gain or loss of function of well-defined events in specific cells of living tissue by means of targetable control tools that respond to light and deliver the effector function. microbial rhodopsins (mrs) have been established as powerful light-sensitive tools for optogenetics. acting as ion pumps or channels, mrs are used to induce cell (de)polarization to control neuronal activity in a wide range of living organisms. mrs are membrane proteins found in a large clade of organisms, including eukaryotes, bacteria, and archaea. they share a common architecture of 7 transmembrane a-helices and a covalently linked retinal, which is employed to absorb photons for energy conversion or the initiation of cellular signaling. major efforts are put into screening of natural and generating of synthetic mrs with desirable properties for optogenetics, e.g. ion selectivity. however, experimental study of mrs is difficult and resource consuming owing to, among other factors, low expression levels and protein stability. thus, there is a need in developing of computational tools for identification of mrs with desirable properties. we used non-redundant atomic structures of mrs taken from protein data bank to develop a set of numerical descriptors that reflects functional properties of mrs. then, we calculated the descriptors for non-redundant sequences of mrs with known function taken from the uniprot database, resulting in the feature matrix. we applied the support vector machine and the 5fold cross-validation procedure, using the feature matrix as the training set. as a result, we obtained the classifier that discriminates mrs in terms of the ion selectivity, e.g. na + , h + , or cl à pumps, with high precision. finally, we used the derived classifier on a test set of proteins and identified mrs for the further experiment in vivo. rational design of peptides with required stability and functional activity properties becomes a real instrument for the new generation drug development. the reca bacterial protein (and human rad51 homolog) is considered to be the central catalyst of homologous recombination, a mechanism essential for the accurate repair of double-strand dna breaks. dna repair via homologous recombination requires reca nucleoprotein filaments assembly. using seqopt (http://mml.spbstu.ru/seqopt/), a novel method for a-helix sequence optimization we present the successful design of peptide sequences capable to maintain a very stable a-helix structure and to inhibit reca activity. novel a-helical 18 amino acids peptide is constructed based on reca-dna complex structure. we observed in vitro inhibition of reca atp hydrolysis, dna strand exchange reaction and reca filament formation. also, we observed lower e. coli resistance to uv and sos-response suppression in vivo. computational identification of promiscous enzyme activity for the morita-baylis-hillman reaction k. ozturk, s. sayin, n. celebi olcum yeditepe university, istanbul, turkey enzyme promiscuity attracts considerable attention in terms of enzyme evolution, protein engineering and biocatalysis. especially, development of highly efficient novel biocatalysts starting from promiscuous enzymes that have the catalytic machinery to perform desired chemistry is an intense area of research in recent years. in this work, we computationally explored the catalytic promiscuity of natural enzymes for the synthesis of morita-baylis-hillman (mbh) adducts, which display antitumoral activity against human cervical cancer cells, by mining structural protein databases using quantum mechanically optimized theoretical active site models (theozymes). catalytic interactions in the active sites of selected hit proteins with potential mbh activity were evaluated in solvated dynamic environment using molecular dynamics simulations. computational screening of the protein data bank for the quantum mechanically determined optimal arrangement of catalytic functional groups for the target mbh reaction successfully identified an enzyme with experimentally determined promiscuous mbh activity. ras proteins mediate a wide variety of signal transduction pathways that regulate cell growth, proliferation and differentiation. these proteins are small gtpases that act as binary switches between gdp-bound 'off' and gtp-bound 'on' states. oncogenic point mutations of ras are associated with~15% of all cancers and up to 90% in specific tumors and many developmental disorders. both experimental and in silico results showed that the membrane-bound ras proteins form non-overlapping dynamic nanosized subdomains called nanoclusters in an activation state-/ isoform-dependent manner. we performed coarse-grained molecular dynamics simulations in order to investigate the formation and evolution of ras nanoclusters in mammalian model membranes. ras proteins were inserted into the cytoplasmic side of the plasma membrane model (di-c16:0-phosphatidyl-choline: di-18:2-phosphatidyl-choline: cholesterol 5:3:2) where they formed highly dynamic nanoclusters, both in size and in composition. furhermore, we found that the presence of ras protein nanoclusters has a significant impact on the model membrane behavior. properties such as phase behavior, diffusion coefficient, surface tension and lipid tails order parameter are also influenced by the temperature variation of the model membrane. we have investigated dynamics in three different crystal forms of ubiquitin, as well as ubiquitin in solution, with particular emphasis on (i) conformational exchange between b turn type i and ii in the region 51-54 and (ii) rocking dynamics where protein molecules as a whole undergo subtle reorientational motion within the confines of the crystal lattice. experimentally, both motional processes have been probed using relaxation dispersion techniques, including recently developed near-rotary-resonance dipolar relaxation dispersion experiments. thereby it has been determined that rocking motion in one of the crystal forms (pdb id 3n30) occurs on the time scale of tens of microseconds, whereas the conformational exchange has characteristic time constant of ca. 100 ls. using molecular dynamics simulations, we have shown that the similarity of motional time scales is not accidental: bi↔bii exchange and rocking motion appear to be coupled. we have investigated the mechanisms of this coupling and predicted a number of point mutations that are expected to abrogate (or enhance) rocking. the crystals of ubiquitin containing these mutations have been modeled in silico. we have also investigated the interactions (in particular, crystal contacts) that control the balance between bi and bii conformations in different crystal forms. finally, we have used md simulations as a basis for chemical shift calculations and illustrated how relaxation dispersion effects can emerge as a function of bi↔bii exchange in conjunction with the rocking motion. the md simulation study was supported by rsf grant 15-14-20038. serine/threonine kinases are attractive targets in targeted cancer therapy due to their overexpression in several forms of cancer. flavonoids are highly bioactive plant secondary metabolites that are important in human health due to their antioxidant property. quercetin, a natural flavonoid derivative, has been shown to regulate several signal transduction pathways and is in phase i clinical trial as an anticancer drug. this study explored the inhibitory potential of quercetin and its derivatives using in silico methods like molecular docking and molecular dynamics simulations. quercetin and its derivatives were observed to bind to several serine/threonine kinase family proteins with binding energy significantly better than other known inhibitors and commercially available drugs. this study thus sheds light on the atomic level interactions that define the polypharmacological nature of quercetin and its ability to interfere with a number of cancer pathways. introduction: noninvasive prenatal diagnosis (nipd) of the fetal rhd status by rhd genotyping of the maternal plasma was initially applied in alloimmunized pregnant women. fetal rhesus d status detection for management of rhd incompatibility using circulating cell-free fetal dna from maternal plasma or serum is now accepted by many obstetricians in europe as reliable and useful. the aim of the study was to detect fetal rhd specific antibodies in maternal plasma using a nanopolimer based electrochemical biosensor. materials and methods: a three-electrode system, consisting of a gold electrode, an ag/agcl reference electrode and a pt counter electrode, was accommodated in a 10-ml electrochemical cell. anilin and jelatin were used for immobilization of rhd antibody. the polimerization was occured at 319 nm uv light. antibodies of rhd antigen were detected using differential pulse method at between 0.4 and 0.6 v potentials by observing the differentiations in the current values. results: the rhd status of the fetus was predicted in 20 rhdnegative pregnant women (8-36th week of pregnancy). rhd antibody were detected in maternal bood using biosensor in 15 of the fetuses. the results were confirmed with real-time pcr. the fetuses found rhd (+) for exon 5 and 7 of rhd gene by multiplex real-time pcr. discussion and conclusion: biosensors based studies might be useful, because they allow to monitor the molecular interactions in real-time providing qualitative and quantitative information, through kinetics, affinity and concentration analyses. we found that more advantages in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, specific, economic, practical and less time-consuming. fetal rhd detection at low concentrations and in the early week of pregnancy is possible with this method. p-03.03.2-022 investigation of phylogeography of cricotopus sylvestris (diptera: chironomidae) using mitochondrial and nuclear molecular markers the family chironomidae is one of the most widely distributed insect families of diptera, and this family is distributed in all continents and all habitats from the tropics to the arctic in lakes, streams and puddles. in this study, we aimed to determine the dispersal of c. sylvestris using molecular phylogenetic markers not only in turkey but in the world and to reveal from where this species may have entered to turkey in the past. c. sylvestris larvae were collected from 8 lakes across turkey. after total genomic dna extraction from body of larvae, fragments of two mitochondrial genes, cytochrome c oxidase subunit i (coi) and cytochrome b (cytb), and one nuclear gene, carbomyl phosphate synthase domain (cps) of cad, were amplified and sequenced. in addition, several sequences of these three genes of c. sylvestris from different countries of different continents such as south corea, japan, canada, denmark, and sweden were obtained from genbank. all sequences were aligned using mega 6 and bioedit version 7.0.9.0 and were used for phylogenetic analyses. neighbour-joining (nj) tree was created in mega 6 and paup 4.0b10 with 1000 bootstrap replicates. maximum likelihood (ml) analysis was performed in raxmlgui 1.0 using gtrgamma model with 1000 bootstrap replicates. beast v1.8.0 was used for bayesian analysis. our phylogenetic analyses indicated that the japanese, south corean and american c. sylvestris were different from european and turkish members. turkish members of c. sylvestris were closely related to european ones according to our bayesian, nj and ml analyses. in turkish members, c. sylvestris collected from hazar and c ß ıldır lake was more ancient than those from marmara, sapanca, c ß ıldır, aygır, beys ßehir, e girdir and sıhke lakes. in conclusion, our results clearly suggest that several transoceanic dispersal events among the continents may have occurred and that the entrance of turkish c. sylvestris to turkey may have been from southeast and northeast of the country. metagenomics is providing great help to explore world of unculturable microorganisms in the natural samples to enhance our knowledge about microbial diversity. here, we have performed metagenomic analysis of fresh water lake bacterial community using 454 pyrosequencing techniques. we have observed a wide array of bacteria from phylum proteobacteria and family enterobacteriaceae as well as very few viruses from podoviridae, siphoviridae and unclassified phages. we have conducted a metagenomics analysis with the primary focus on the examination of the community of bacteria in a fresh water lake ecosystem. roche gs flx software gave us total 156 253 reads (with an average read length of 795.274 bp). there were 15 226 contigs having > 100 bp sequence length whereas 10 481 contigs with > 500 bp sequence length. for further analysis we have taken contigs with > 500 bp only. further, we have analyzed the microbial community composition using blastn/blastx against nt/nr databases with e-value cutoff of 10 à5 . ≥70% of total contigs were mapped to the reference with ≥60% contig match coverage. the community analysis revealed that domain bacteria is predominantly present (99.8%) in the water sample, followed by eukaryota (0.02%), viruses (0.08%) and other sequences (0.02%). most abundant phyla was proteobacteria (99.8%) and the most dominant family was enterobacteriaceae (89%) followed by xanthomonadaceae (10%), vibrionaceae (0.4%), pasteurellaceae (0.1%), shewanellaceae (0.07%). we performed functional analysis of all 5974 contigs using rapid annotation using subsystems technology (rast) 4 which detected 15 319 coding sequences and 197 rnas in 619 subsystems. among the classified cds from rast showed major cds hits for enzymes involved in the subsystems amino acids and derivatives and the carbohydrate metabolism. the great diversity of microorganisms present in the lake may reflect the human activity in the area. maldi-tof mass spectrometry is a ubiquitous and widespread tool for protein identification. once the protein sequence is unavailable, unambiguous identification cannot be performed, and predictability is limited by the presence of sequenced homologous proteins. we present a statistical approach to predict a number of structural, localization and functional properties of unknown proteins by direct analysis of mass distribution shapes of their post-cleavage fragments obtained from maldi-tof mass spectrometry data. secondary structure of proteins is best predicted by their specific cleavage at the inertial hydropathy group amino acid residues (filmv), with thermolysin (afilmv) being the closest commercially available reagent, leading to distinguishing between proteins with presumably ahelixes or b-sheets with 90% accuracy. cellular localization of proteins is best predicted by their specific cleavage at the external hydropathy group amino acid residues (dehknqr), exemplified by gluc(phosphate)+lysc(dek) cleavage. protein location in the cell membrane and its localization character (monotopic/ transmembrane, single-pass/multi-pass transmembrane) are predictable with~75% accuracy by this single cleavage, with optimal combination of 3-4 cleavages improving the accuracy tõ 80%. functional prediction of proteins is the best among membrane-associated proteins with characteristic structural conformations. we attribute the differences in the mass distribution shapes to the characteristic clustering of amino acids residues with respective hydropathy properties that are involved in the formation of 3d structural conformations of proteins. the suggested approach allows for a non-parametric statistical prediction of uncharacterized proteins from their maldi-tof mass spectrometry data without knowledge or reconstruction of their primary sequence. potential applications include proteomic studies of organisms with unavailable genomic sequences and highly variable proteins analysis. the cancer genome atlas (tcga) represents a comprehensive database of genomic, transcriptomic and epigenetic alterations across more than 20 tumor types. earlier we developed cross-hub tool aimed at multi-way analysis of tcga data in the context of gene expression regulation. in the present work, the software was updated with new features that are described below. crosshub is a python-based application. one of the features of crosshub is the combining tcga rna-seq co-expression analysis to encode chip-seq data in order to reveal most possible transcription factor (tf) targets and coupling mirna-mrna co-expression to several algorithms of mirna target prediction in order to enhance its efficacy. the key feature of the updated crosshub version is the analysis of the associations between expression ratio of tf to its targets and tf mutation status. this allows identification of tfs that are functionally (in)activated with driver mutations in a particular cancer type. the second novel feature of crosshub is the analysis of associations between 'tf-to-targets' expression ratio and tumor characteristics (tnm classification, pathological stage), patient follow-up, etc. in turn, this analysis may result in the identification of 'tf-targets' functional relations that are important for disease progression, tumor invasion, response to chemotherapy. thus, crosshub was supplemented with new features that can be useful for comprehensive tcga data analysis. the updated version of crosshub is freely available at http://sourceforge.net/ projects/crosshub/. this work was financially supported by the russian foundation for basic research (grants 15-04-08731, 16-16-00114 and 15-34-70055) and ras presidium program 'molecular and cellular biology'. p-03.03.2-026 mutations leading to increased rnase production and streptomycin resistance in bacillus pumilus bacillus pumilus strain 3-19 which was derived from soil-isolated b. pumilus 7p using chemical mutagenesis is characterized by resistance to streptomycin (str, up to 500 lg/ml) and ability to produce extracellular enzymes in quantities almost 10-fold higher than the parent strain. these features make the 3-19 strain suitable for industrial production of rnase (binase) which is known for its antitumour and antiviral properties and can be used as an rna-degrading tool in molecular biology. the whole genomes of both mutant and wild-type b. pumilus strains were sequenced recently. to reveal the exact genetic features responsible for rnase overproduction and str resistance we have fulfilled comparative genome analysis of b. pumilus 7p and 3-19 strains. facilities of rast server, edgar platform and additional bioinformatics tools (plasmid finder, prophinder, bl2seq) were used. it is found that both b. pumilus genomes under study contain an intact prophage, while only wild-type strain bears a 6 kb cryptic plasmid. none of the systems is inactivated in mutant strain according to the results of metabolic reconstruction. 3.4% of total cdss differ in 3-19 strain in comparison to 7p one, 36% of them are hypothetical. the altered genes are involved in membrane transport, cell wall composition, chemotaxis, spore formation, carbohydrate metabolism, dna metabolism, translation and transcription regulation. mutation (k56n) leading to str resistance is identified in 30s ribosomal protein s12p. regulatory and coding regions of binase gene have no modifications. candidate genes which can account for binase overproduction are selected. mutation k56n is classical in str resistance and leads to enhancement of decoding accuracy while decreasing elongation speed. rnase overproduction is brought about by non-specialized mechanism since other hydrolases are also overproduced in mutant strain. genes encoding extracellular serine protease, sporulation initiation phosphotransferase f, gnat-family acetyltransferase and cell wall modifying enzymes are reported previously to increase production of degradative enzymes. the action of encoded by them proteins lead to increase of stability and release of secreted proteins to environment and to derepression of their transcription from negative regulators bacillus pumilus strain 7p has been identified on its ability to produce ribonuclease and different extracellular proteases. in order to increase inherent biosynthesis of proteases the 7p strain was screened on culture medium supplemented by streptomycin. derivative b. pumilus strain 3-19 gains the resistance to streptomycin and also shows the increased ribonuclease activity. we used genomes of both these strains to explore streptomycin susceptibility and increased activity of hydrolytic enzymes. whole-genome shotgun sequencing was performed using a combination of pyrosequencing and ion semiconductor sequencing, which provided 23x (7p) and 30x (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) overall genome coverage. assembled genome sequences of 7p and 3-19 strains included 9 and 8 scaffolds > 500 bp with a calculated genome size of 3 577 758 bp and 3 572 739 bp, respectively. the gc content was 42%. both draft genomes have been deposited at genbank (jojx00000000.2 for 7p and jhud00000000.2 for [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] . detailed comparative genomic analyses of strains have been performed. we calculated average nucleotide identity (ani) values between the genomes of our strains and 9 completed b. pumilus genomes deposited in ncbi database. two b. pumilus strains (sh-b9 and safr-032) revealed the max. ani value (95.17% and 94.65%, respectively). b. pumilus sh-b9 strain has been used as a reference for snp calling in strains 7p and 3-19. 3828 snps for the 7p strain and 862 for the 3-19 strain were classified as nonsynonymous variants. 20 radical nucleotide substitutions from the 3-19 genome were not found in 7p genome. among them, the mutation in the 56 codon of rpsl gene (coding 30s ribosomal protein s12) is probably associated with resistance to streptomycin. also, two mutations in rpob and nusa genes (coding rna polymerase and transcription termination factor rho, respectively) may be related to increased enzymes activity. both our strains contain 143 protease-coding genes. twelve of them are encoding extracellular proteases. here we propose an algorithm that can predict an antibodies mutant forms with desired specificity. this algorithm allows to determine the position and type of amino acid residue for mutagenesis. approach is based on a hybrid method of quantum and molecular mechanics (qm/mm) that allows to understand the reaction mechanism and the role of active center amino acids. catalytic antibody a17, that is able to hydrolyze pesticide paraoxon, was selected as a model. however, the hydrolysis efficiency of paraoxon by a17 antibody is only 9 m à1 min à1 , that is insufficient for using this antibody as antidote. the main fundamental goal of our study is to determine the necessary conditions for improving the binding reaction of paraoxon by catalytic antibody a17. the hybrid qm/mm method allows to study the reaction mechanism of interaction of a17 with paraoxon. it was shown that the reaction proceeds via the classical s n 2 mechanism. the key step of the reaction is the proton transfer from the catalytic residue tyr-37 to paraoxon. qm/mm approach determines position for mutagenesis -leu-47 in light chain. for one of the mutant in this position -leu47argwere predicted (i) increased probability of formation of a hydrogen bond between the catalytic moiety and paraoxon compared to the wild type antibody and (ii) smaller value of the diffusion coefficient, which reflects the best positioning of paraoxon in the active center. steady-state kinetic analysis shows that leu47arg exhibits a 70-fold increase in k 2 /k d compared to a17 (90 m à1 min à1 vs. 1.35 m à1 min à1 ). double mutant leu47arg/ser35ala also has improved constants of interaction with paraoxon in comparison with the wild type antibody, however, a single mutant leu47arg still binds paraoxon three times better, that may be due to the fact that the serine in 35 position increase the nucleophilicity of tyr37. thus, our results are in line with our computed predictions. this work was supported by rfmefi60414x0069. due to high prices of meat and meat products, low quality raw materials like offal tissues are commonly used in turkey. in the retrospect of the studies for evaluating and detection of unwanted tissues in the sample is basic histological examination. the light microscopy techniques are very strong method if a researcher qualification is enough. a new researcher-independent method must be developed. therefore, different tissues and organs constitute of unique mrna and protein. our method is based on this event, so the antigenic sites of the tissues can be detected by selected antibodies. the first set of the antibodies are for detecting muscle and adipose, consist on muscle actin and adipose triglyceride lipase. this set is used for calibration on standard meat sample. the second set of the antibodies are detecting of offal tissues, consist on trrap and casein. anti-casein antibody is selected because the mammary gland usage in grinded-meat is very common. immuno-staining started with hier (heat mediated epitope retrieval), then classical ihc method applied to slides with dab-chromogen. after all process completed the slides were photographed by las (leica application suite) on microscope. the capture settings were remained same on both sets. image capture size is 1392x1040 pixels and field of view (fov) is 449 9 335 lm. all the image files were converted to binary for threshold operation. the threshold values of first set and second set were calculated and their ratios were compared. the formula is based on the distribution (dst) of pixel intensity (int) over threshold (thrs) values on all fov (axis: the results are good enough to detect the unwanted micro-structures on 80% raw meat and 20% offal tissue. calculations proofed with imagejò. future application of this method and opencv-based software algorithm is to port the source code to a single board computer (sbc) with a digital microscope connected. monday 5 september 12:30-14:30 mechanisms of pro-inflammatory diseases p-04.02.2-001 the effects of raas inhibition in rate limiting step by aliskiren on testicular torsion injury in rats testis torsion is a urological emergency condition that results in necrosis of the testis if the condition is not treated. unfortunately treatment of testis torsion is not fully understood, therefore clinical and experimental studies are performed continuously. reninangiotensin-aldosterone system (raas) contributed to pathophysiology of several diseases. aliskiren (als) inhibits the renin on the first step of this system. our aim is to investigate the protective effect of aliskiren on unilateral testis damage caused by experimental testis torsion and detorsion. the forty-eight rats were separated into eight groups of six animals: sham, sham+als 200 mg/kg (oral) group, torsion group (tor), torsion/detorsion group (tor/det), tor+als 100 mg/kg (oral) group, tor+als 200 mg/kg (oral) group, tor/det+als 100 mg/kg (oral) group, tor/det+als 200 mg/kg (oral) group. in the tor and tor/det groups, the left testes were rotated 720°clockwise together with the spermatic cord and tunica vaginalis in the scrotal space. the left testes of the rats were subjected to torsion and detorsion during 2 h. after experimental procedures, testicular tissues were examined by histopathologic and molecular analyses. the il-1b and inos mrna expressions were increased in tor and tor/det groups when compared with sham group. both doses of aliskiren administration decreased these expressions in tor/det groups. the stereological results revealed that aliskiren administration promote the numerical density of mature spermatids in tor and tor/det groups. the numerical densities of tor/det+als100 and tor/det+als200 groups were similar and these two groups have significant difference when compared to the tor and tor/det groups. the administration of als may be useful for preventing ischemic damage on unilateral testes injury in rats. this study supported by a tubitak 3001 project, coded 114s048. 3 ) has recently been recognized as a potent immunomodulator which acts through regulation of gene expression involved in immunity response thus affecting various inflammatory and autoimmune diseases. the study was aimed at investigating hepatoprotective role of d 3 in vdr-mediated regulation of pro-inflammatory factors in diabetic liver. materials and methods: type 1 diabetes was induced in male c57bl/j6 mice by i.p. injection of high-dose stz (150 mg/kg b.w.). after 2 weeks of stz-induced diabetes animals were treated with/without d 3 (15 iu/mouse per os) for 8 weeks. blood serum 25ohd 3 was assessed by elisa. rel-a, vpf, inos and vdr expression was measured by qrt-pcr and/or western-blot. results and discussion: diabetes caused two-fold reduction of serum 25ohd 3 level, indicative of d 3 deficiency. significant alterations in d 3 -endocrine system were found as is evident from reduced expression of cyp27a1, cyp2r1, vdbp and vdr at transcriptional and translational levels. these changes were accompanied by a marked increase in mrna and protein levels of inflammation markers rel-a, vpf and inos in hepatic tissue of diabetic mice. diabetes also led to structural lesions in liver tissue. complete restoration of 25ohd 3 content and partial normalization of liver tissue structure were achieved after d 3 treatment. d 3 administration partially normalized expression of cytochromes involved in d 3 metabolism and hepatic pro-inflammatory factors. d 3 treatment prevented overexpression of rel-a and phosphorylated p65/rel-a translocation to hepatocellular nuclei that is most likely mediated through 1,25(oh) 2 d 3 and vdr. conclusion: study confirmed that diabetes-induced liver abnormalities are associated with chronic inflammation that can be linked to impaired d 3 metabolism and deficiency. our findings demonstrate protective vdr-mediated effect of vitamin d 3 against diabetes-induced liver injury. p-04.02.2-003 lavandula stoechas extract increased glucose uptake and protein levels of key signaling molecules in insulin resistant c2c12 muscle cells s. savranoglu 1 , h. ipek 2 , s. arslan 3 , h. delig€ oz 4 , a. r. t€ ufekc ßi 5 , i. demirtas 5 , t. boyunegmez t€ umer 6 1 graduate program of biology, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, 2 graduate program of bioengineering, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, 3 deparment of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, 4 department of chemical engineering, faculty of engineering, pamukkale university, denizli, turkey, 5 department of chemistry, faculty of sciences, c ß ankiri karatekin university, c ß ankiri, turkey, 6 department of molecular biology and genetics, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey introduction: the aim of this is to identify remedial effects of lavandula stoechas, anatolian traditional medicine, against metabolic disorders developed on the ground of insulin resistance. ethyl acetate extract (eae) of l. stoechas was investigated in c2c12 myotubes which were made insulin resistant by free fatty acid (ffa) treatment, for its effects on glucose uptake and as well as on the activation of akt-1 (by pakt/ akt ratio) molecule which plays a central role in insulin signaling through serine (473) phosphorylation. in addition, the protein level of lipoprotein lipase (lpl) enzyme was also evaluated. material and methods: c2c12 cells were made insulin resistant by palmitic acid (ffa) and effects of eae on p-akt (ser473)/akt ratio and lpl level were determined by sds-page/western blot. the effect of eae on glucose uptake in insulin resistant cells were determined by the 2-deoxyglucose uptake assay. results: eae at 25 and 50 lg/ml significantly increased the glucose uptake 120 and 182% compared to insulin resistant control cells. metformin at 2 mm increased this parameter up to 132%. eae increased pakt ser473/akt level 43-37% and lpl expression 50-92% for 25 and 50 lg/ml, in insulin resistant myotubes, respectively (p < 0.05). discussion: eae of l. stoechas improved impaired insulin sensitivity through both enhancing glucose uptake and activation of akt1 molecule through ser473 phosphorylation. in addition, it also considerably increased lpl level which has very important function in lipid metabolism. conclusion: overall, results demonstrated that l. stoechas contain phytochemicals which can be effective for the prevention and also treatment of insulin resistance and associated conditions. our research group is on the way for the identification of these 'bioactive' molecules with bioassay guided fractionation studies. tubitak (projectid: 112t442) supports this study. achievement of complete pain control is very difficult task, which requires a search for new molecular targets during the analgesic substances development. considering the importance of glial cells and their signaling molecules, development of new gliotropic therapeutic methods is a promising direction in pain treatment. polyunsaturated fatty acids, including docosahexaenoic acid demonstrating anti-inflammatory and antioxidant activity are of considerable interest. docosahexaenoic acid (dha, 22:6 n à 3) analgesic activity was studied using a chronic constriction injury (cci) rat model. animals were subcutaneously injected with dha emulsion at a dose of 4.5 mg/kg (125 mm/kg) daily during 2 weeks after surgery. collection of material for subsequent immunohistochemistry investigation was performed on day 28. we clearly demonstrated that the activation of neurokinin neurotransmission and nnos synthesis are coincided with the astroglial activation in the spinal cord dorsal horn (scdh) superficial lamina during neuropathic pain development. however, the detailed mechanisms of interaction between substance p (sp)-, no-ergic systems and astrocytes in the spinal cord remain to be elucidated. systemic administration of dha to cci animals reduced neurogenic pain intensity and duration, leading to an earlier stabilization of paw weight distribution and preventing the development of degenerative changes in denervated limb. this drug treatment reduced the level of the sp-and no-ergic neurotransmission and decreased astrocytosis in the scdh superficial lamina. thus, the ability of dha to affect nociception is a promising and safe alternative to current pharmaceutical therapeutics. immunohistochemistry studies carried out with the russian science foundation financial support (agreement no. 14-50-00034), obtaining dha and all manipulations with animals of the material was funded by rfbr according to the research project no. 16-34-00023 mol_a. p-04.02.2-005 circulating endothelial-derived apoptotic microparticles and aopps are related to highsensitive troponin t in patients with chronic hepatitis c infection the aim of this study was to evaluate non-standard risk factors for cardiovascular events, such as endothelial dysfunction assessed by endothelial-derived microparticles (emps) (cd144 +/ cd31 + ), advanced oxidation protein products (aopps), and low-grade inflammation, that are potentially associated with elevated levels of high-sensitivity troponin t (hs-tnt) and n-terminal pro-brain natriuretic peptide (nt-probnp) in patients with chronic hepatitis c (chc). methods and results: eighty-six chc patients and 60 healthy control subjects were enrolled in the study. circulating levels of hs-tnt, nt-probnp, aopps-albumin (the ratio of aopps to albumin content), emps (cd144 +/ cd31 + ), hs-crp, and tnf-a were assessed. compared with chc patients, the chc patients with diabetes mellitus (dm) had higher levels of emps (cd144 +/ cd31 + ) and aopps-alb, which were associated with elevated hs-tnt levels (≥13.3 pg/ml). nt-probnp positively correlated with tnf-a level in all chc patients and this correlation was stronger in diabetic patients. in multivariate logistic regression analysis, the independent factors associated with the presence of elevated hs-tnt levels were the presence of dm (p < 0.001) as well as high levels of aopps-alb, apoptotic emps (cd144 + /cd31 + /an-v + ), and nt-probnp (p = 0.04, p = 0.03, p = 0.04 respectively). conclusion: the prevalence of elevated hs-tnt were increased significantly in the diabetic patients with chronic hepatitis c. hs-tnt was related to non-standard risk factors for cardiovascular events, and circulating endothelial-derived apoptotic microparticles (cd144 + /cd31 + /an-v + ) level was an independent predictor for elevated hs-tnt levels, potentially indicating some abnormalities in the myocardium. apnea; and healthy individuals; and assessing the connection between the pain and the dimension of the sleep disorder. material and methods: 60 patients who were diagnosed with obstructive sleep apnea and 40 healthy individuals who were similar in terms of age and gender were included in this study. the patients, who were diagnosed with obstructive sleep apnea with the examination and sleep tests, were assessed according to the 1990 american college of rheumatology (acr) criteria in terms of fms. serum d vitamin level was measured by using the ultra performance liquid chromatography method. findings: when the fibromyalgia syndrome and obstructive sleep apnea and pure obstructive sleep apnea patient groups are compared with the control group, the vitamin d level was found to be low at a significant level (p = 0.038, p = 0.001, respectively). no significant difference was found between the vitamin d levels in fibromyalgia syndrome, obstructive sleep apnea and pure obstructive sleep apnea patient groups. a negative correlation was found between the number of the sensitive points and vitamin d levels in fibromyalgia syndrome patients (p = 0.013). results: it has been concluded that the obstructive sleep apnea and fibromyalgia syndrome patients have low vitamin d levels, and this situation must be considered in treatment modalities. on the other hand, the results obtained in the study make us consider that vitamin d metabolism is not influential in the pathogenesis of the fibromyalgia syndrome and obstructive sleep apnea togetherness. p-04.02.2-007 decreased chitotriosidase activity and levels in familial mediterranean fever discussion: familial mediterranean fever is an inflammatory disease. several cytokines and inflammatory mediators are playing role on pathogenesis of the disease. although ıt has been demonstrated that the increased concentrations of cht in patients with fmf. we found lower cht activity and concentrations in patients with fmf. conclusion: serum cht enzyme activity and concentrations may not be considered as a biomarker in fmf patients taking colchicine. new studies are needed to evaluate the changes of the enzyme activity, concentration and the role of cht in patients with colchicines negative patients. chronic hyperglycemic state leads to an increase in subclinical systemic inflammatory response in diabetes mellitus type 2 (dmt2) patients. inflammation-based scores, neutrophil to lymphocyte ratio (nlr), platelet to lymphocyte ratio (plr) and red blood cell distribution width to platelet ratio (rpr) are biomarkers able to quantify systemic inflammation. the aim of the study was to investigate association of the inflammation-based scores with short-and long-term glycemic control markers, and whether they could be used as indicators of glucoregulation in dmt2 patients. the cross-sectional study included 92 dmt2 patients, treated at the primary health care centre zenica from december 2015 to april 2016, distributed into groups according to glycated hemoglobin (hba1c) values: a (n = 59, hba1c ≤7.0%) and b (n = 33, hba1c > 7.0%). complete blood cell count, fasting blood glucose (fbg) and hba1c measurements were determined at the primary health care centre zenica and at the department of laboratory diagnostics, cantonal hospital zenica by standard laboratory methods. all statistical tests were performed using spss 19.0. p values fasting blood glucose and hba1c were significantly higher in the group b compared to the group a (p < 0.0005). there was no significant difference of nlr, plr and rpr between the groups (p = 0.50; p = 0.220; p = 0.525, respectively). significant correlation of inflammation-based scores with fbg and hba1c was found only between plr and hba1c in the group a of dmt2 patients (r = 0.328, p = 0.011). inflammation-based scores could gather meaningful clinical information, either diagnostic or prognostic, on a variety of hyperglycemic, inflammatory, cardiovascular and thrombotic disorders. since there was no statistically significant difference of nlr, plr and rpr between dmt2 patients with good and poor glycemic control, we conclude that these scores could not be used as indicators of glucoregulation in dmt2 patients. chronic inflamation plays a central role in the development and progression of diabetes and in the pathogenesis of its comlications. the neutrophil-lymphocyte ratio (nlr) and platelet-lymphocyte ratio (plr) are indicators of subclinical inflamation. mean platelet volume (mpv) is one of the platelet function indices that reflects the platelet production rate and stimulation. we investigated the association of nlr, plr and mpv with prediabetes and type 2 diabetes mellitus (t2dm) and determine whether or not these are reliable markers for diagnosis. we evalueted 76 people's results who were carried out oral glucose tolerance test (ogtt). acording to 2-h values of plasma glucose in the ogtt; 1. group (normal glucose tolerance: ngt): under 140 mg/dl (n = 42), 2. group (prediabetic: impared glucose tolerance (igt)): ranging from 140 mg/dl to 199 mg/dl (n = 25), 3. group (firstly diagnosed diabetic by ogtt): above 200 mg/dl (n = 9). 4. group is clear diabetic without complication (taking treatment) group (n = 34). we compered nlr, plr, mpv and some biochemical markers between four groups. there are significantly differences between all groups in nlr (p = 0.004) and plr (p = 0.021) values. nlr values are significantly higher in prediabetic (1. it is recognized that a chronic low-grade inflammation and an activation of the immune system are involved in the pathogenesis of insulin resistance and type 2 diabetes mellitus (t2d). this study aimed to analyze the long-term impact of altered metabolism in female t2d patients at the level of mediators of inflammatory response. this study included 65 femalet2d patients and 107 control subjects, which were recruited at the clinical center university of sarajevo and the general hospital tesanj. in this study the effects of glycemic control on markers of the inflammatory response crp, fibrinogen, leukocytes, sedimentation, and cytokine il-6, were analyzed. all subjects included in this study were free of evidence infections, surgery, thyroid disease, polycystic ovarian syndrome, active liver and kidney damage. all biochemical analyses were performed by employing standard ifcc protocols. results from this study demonstrated significant increase of fibrinogen (p = 0.0001), crp (p = 0.001), il-6 (p = 0.013), leukocytes (p = 0.0001) and sedimentation rate (p = 0.008) in female t2d population compared to control subjects. interestingly, a significant correlation was shown between crp and hba1c (p = 0.035), il-6 and glucose (0.032), il-6 and bmi (0.007). in our study, female t2d compared to the healthy population had significantly higher levels of fibrinogen, leukocytes, il6, crp and sedimentation. other studies conducted in female population associated elevated levels of il-6 and crp with t2d independent of other risk factors for diabetes. crp being most robust predictor of diabetes. studies have shown that crp is an important predictor of t2d for the female but not the male population. thus, our data suggest that inflammation play an important role in the pathogenesis in female diabetic population. a more detailed study on a far larger number of subjects should point out fact if they can effectively be used as biomarkers in the primary prevention of t2d in this population. objectives: bone and mineral metabolism disorders hold an important place among the complications after renal transplantation. the purpose of this study was to demonstrate the relationship between vitamin d, calcium, phosphorus metabolism with graft function and to measure 1,25(oh) 2 d 3 levels with lc-ms/ ms in renal transplant recipients. design and methods: this study included 30 renal transplant recipients (10 female, 20 male; mean age: 40.30 ae 12.86) from living related donors were transplanted. blood samples were collected immediately before and after transplantation at month 6. serum creatinine, bun, calcium, phosphorus, alkaline phosphatase, glucose, albumin, pth, 25(oh)d and 1,25(oh) 2 d 3 levels were measured. gfr values were estimated by ckd-epi. plasma 1,25(oh) 2 d 3 levels were determined in a lcms-8040 triple quadrupole tandem mass spectrometer (shimadzu corporation, japan) by mrm. spss 20.0 software was used for statistical analysis. results: although plasma 1,25(oh) 2 d 3 levels significantly increased (p = 0.0001), we did not find any significant differences for serum 25(oh)d levels after transplantation. when posttransplant levels of serum phosphorus, pth, creatinin, bun and alp levels were found to be significantly decreased (p = 0.0001, p = 0.011 for alp), we observed significantly higher calcium and gfr values (p = 0.0001). vitamin d insufficiency was present 13.3%, deficiency 36.7%, severe deficiency 50% before transplantation, insufficiency was also seen 26.7%, deficiency 50%, severe deficiency 23.3% after transplantation at month 6. conclusions: in our study, all patients were found to vitamin d deficiency and insufficiency. determination of vitamin d deficiency and consequently treatment with vitamin d supplements could lead to better graft surveys. free fatty acids (ffa) represent important link between obesity, insulin resistance, type 2 diabetes (t2d), and dyslipidemia. increased adiposity, as approximated by body mass index (bmi), correlates well with increased serum levels of leptin-adipocyte derived hormone implicated in the regulation of adipose mass and alterations in insulin action and secretion. the main objective of the present study was to investigate the potential association of serum ffas with leptin levels in healthy and newly diagnosed type 2 diabetic subjects. this study involved 13 newly diagnosed type 2 diabetics and 13 healthy subjects. all participants in the study were free of evidence of hepatitis, viral infection or active liver and kidney injury. for biochemical analyses of glucose, glycosylated hemoglobin (hba1c), and lipid profile, standard ifcc protocols were used. analysis of free fatty acids (ffas) was done by gas chromatography, while serum leptin levels were determined by the elisa kit. in addition to the expected differences in glucose, hba1c, and bmi, our results also showed significant differences in leptin, myristoleic, palmitic, linolenic, arachidic, and arachidonic acids between t2d and control subjects. in healthy subjects, a significant correlation was demonstrated between glucose and lauric, arachidic, arachidonic acid levels, body weight, and bmi. newly diagnosed diabetics showed significant association between glucose and lauric, myristoleic and linolenic acid levels; with leptin being associated with myristic and palmitoleic acid levels. interestingly, in all participants, significant association was found between glucose and hba1c, glucose and leptin, myristoleic, arachidic, and bmi as well as between leptin, arachidic acid, and bmi. thus, our data point out association of different types of ffas with leptin levels in newly diagnosed type 2 diabetics. however, further studies should be done in larger number of patients to confirm our results. rheumatoid arthritis (ra) and ankylosing spondylitis (as) are chronic inflammatory diseases with distinct clinical manifestations in many ways. the aim of this study is to evaluate the serum levels of molecules which may be used as markers for angiogenesis and vascular leakage in the processes of two clinically different pictures, ra and as. 30 ra patients, 30 as patients and 30 healthy volunteers with mean age of 30-50 were included in the study. serum levels of vegf, angiopoetin-2 and tie-2 were measured by enzymelinked immuno-sorbentassay (elisa) using a commercially available kit. serum nitricoxide levels were evaluated by the griessreaction. serum vegf, ang-2 and no levels were significantly higher in the as group ml, p < 0.001; p < 0.001; p < 0.05). no differences were found between as and ra for tie-2 (p > 0.05). vegf, ang-2, tie-2 and no levels were positively correlated in both as and ra patients (p < 0.001), but no correlation was detected between clinically activation index das-28, basda _ i scores and laboratory measurements such as sedimentation, crp and anti-ccp (p > 0.05). when the diagnostic performance of the parameters were evaluated with the roc analysisonly the performance of the ang-2 in as patients was sufficient (auc (95% cl): 0.718, p < 0.05). elevation of angiogenic factors in the serums of as and ra patients supports the role of angiogenesis in the etiopathogenesis of these diseases. however, lack of relationship between disease activity leads to not to use these factors as a marker for clinical follow-up. only ang-2 measurements may be useful for the differantial diagnosis. the evaluation of ischemia modified albumin as an early biomarker of acute myocardial infarction introduction: acute myocardial infarction (ami), remains a leading cause of morbidity and mortality worldwide. early diagnosis of ami is very important because early treatment may reduce the extent of injury to the myocardium. currently, biomarkers of myocardial necrosis such as myoglobin, ck-mb and troponins are highly sensitive and exhibit good specificity. however, these biomarkers increase after tissue injury, approximately 4-6 h after the cardiac event and detect only the consequences of prolonged ischemia. recently, ischemia modified albumin (ima) has been assessed and found to be very useful for the diagnosis of myocardial ischemia and it is considered as a serum biomarker. the aim of the present study was to evaluate the serum level of ima and determine the relation between patients with ami and control group, in order to verify its potential as a novel marker for early detection of mi. materials and methods: the study was performed with 25 patients and 37 healthy controls. blood samples from all subjects were collected by venipuncture in plain tubes, and immediately centrifuged at 4000 g for 10 min at 4°c. the serum samples were stored at à20°c until analysis. the serum levels of ima were determined using the cusabio biotech human ischemia modified albumin, elisa kit according to manufacturer's instructions. the results are given as international units/milliliter (iu/ml). results: our findings revealed that ima showed no significant difference between the groups. conclusion: our results suggest that ima assay is not a sensitive marker for early detection of ischemic hearth disease and cannot be used alone for the diagnosis of ami. prospective studies are needed to identify ima's potential as a biomarker for ami. p-04.02.2-015 neutrophil-to-lymphocyte ratio and platelet-tolymphocyte ratio in polycystic ovary syndrome polycystic ovary syndrome is a complex and multifactorial disease with metabolic dysfunction and the etiopathogenesis is not well established. emerging data suggest that adiposity and chronic low-grade inflammation are involved in the development of the metabolic dysfunction. neutrophil-to-lymphocyte ratio (nlr) and platelet-to-lymphocyte ratio (plr) have recently been investigated as two new inflammatory markers used in the assessment of systemic inflammation in many diseases. the purpose of the study was to investigate their relation with pcos patients. the study population consisted of 44 patients with polycystic ovary syndrome and 23 healthy women controls. nlr and plr obtained by dividing absolute neutrophil to absolute lymphocyte count and absolute platelet count to absolute lymphocyte count, respectively. the neutrophil count (4.41 ae 1.6 vs. 3.77 ae 1.23, p < 0.05) and platelet count (320.34 ae 55.11 vs. 283.96 ae 52.17, p < 0.05) were higher in patients with pcos compared to the control group. lymphocyte count was 2.08 ae 0.43 in pcos patient and 2.30 ae 0.57 in control group. the nlr and plr of pcos patients were significantly higher compared to those of the controls (2.18 ae 0.92, 1.69 ae 0.57 p < 0.05, 158.31 ae 32.85, 128.59 ae 31.94 p < 0.05, respectively). in this study we found nlr and plr were significantly increased in patients with pcos compared to healty control. nlr and plr were two useful inflammatory markers for assessment of patients with pcos. imbalance in neurotransmission in conjunction with neuroinflammation contribute to neurological dysfunction observed during acute liver failure (alf). own observations indicate that alf in a mouse model is associated with altered expression and/or intracellular distribution of synaptic proteins. since neutralization of tgf-b1 appears to improve the neurological score in alf mice, we examined the possibility that increased levels of tgf-b1, caused by liver damage, may affect the expression of selected synaptic proteins. expression and/or cytoplasmic vs. membrane distribution of a number of functionally critical synaptic proteins in cerebral cortex and blood tgf-b1 were measured in c57bl6 mice with alf induced by single i.p. injection of aom (100 mg/kg of b.w.) and after neutralization of tgf-b1 induced by single i.p. injection of ab-tgf-b1 (1 mg/kg) 2 h before aom injection. in alf mice, blood tgf-b1 was increased, and the expression of presynaptic proteins: synaptophysin and synaptotagmin was increased in the cytosolic (s2) fraction by~45% and 30%, respectively, but was slightly depressed in the membrane (p2) fraction by~20% and~15%. aom induced an increase of postsynaptic proteins: psd-95 and nnos by~40% in p2 fraction. tgf-b1 neutralization resulted in a reduction in the expression of presynaptic proteins by~30% in s2 fraction and~20% in p2 fraction, in control animals and normalized their amount in the cytosolic fraction after aom injection, but was ineffective with regard to psd-95 and nnos. the results indicate that in alf mouse, neutralization of cytokine tgf-b1 normalizes synaptophysin and synaptotagmin expression in the synaptoplasm, without affecting their synaptic membrane content. effect of tgf-b1neutralization appear to be confined to the presynapse. 25% of acute pancreatitis (ap) patients develop severe acute pancreatitis (sap), which is is resulted in multiple organ dysfunction syndrome. an extensive inflammatory response occurs due to inflammatory mediators synthesized and secreted during sap. since preventing the inflammation in sap is important in the prognosis of the disease, new drug candidates having strong antiinflammatory effects will provide a new concept for therapeutic strategies against acute pancreatitis. non-steroidal anti-inflammatory drugs (nsaids) show their effects by inhibiting cyclooxygenases (cox-1 and cox-2) and they play an important role in the pathogenesis of acute pancreatitis. since conventional nsaids inhibit both cox-1 and cox-2, they have serious side effects on gastrointestinal system. therefore, new highly selective cox-2 inhibitors having fewer side effects are needed. in the present study, selective cox-2 inhibitory activities and cytotoxic effects of new series of 2-benzoxazolinone and thiazolo [3,2-b ]-1,2,4-triazole derivatives previously synthesized as specific cox-2 inhibitors with no side effects on gastrointestinal system were investigated. permeability of the compounds was tested by pampa using caco-2 cells. compounds were found highly selective, non-toxic and permeable. ap was induced in rats via retrograde injection of stc into the pancreatic duct system. rats were pre-treated with saline or celecoxib or the new compounds before stc injection and sacrificed 24 h later. the severity of ap was evaluated using biochemical and histopathological analyses. edema, inflammation, hemorrhage and acinar cell necrosis were detected in the pancreatic tissue of sap group. sap was remarkably increased serum lactate dehydrogenase, ast, alt, lipase and amylase activities and serum tnf-a, il-1b, il-2, il-6 and il-8 levels. tissue myeloperoxidse activity was also increased. pretreatment with the novel compounds reserved all these biochemical and histopathological parameters. alopecia areata (aa) is an inflammatory disease which is affects hair follicles, and sometimes nails. it is suggested that cytokinemediated immunity plays an important role in etiopathogenesis of aa. this study was planned to evaluate the serum ykl-40 and tgf-b1 levels of patients with aa. 40 patients with aa and 40 healthy volunteers were recruited into the study. fasting venous blood samples were collected from the participants and serum was obtained by centrifugation. serum ykl-40 and tgf-b1 levels were measured by enzyme linked immunosorbent assay (elisa). serum tgf-b1 levels in the patient group were significantly lower compared to the control group whereas serum ykl-40 levels were significantly higher in patient group. tgf-b1 levels of men and women with aa were found to be significantly lower than that of controls. while serum ykl-40 level of male control group is higher than the male patients, there were no significant differences between women groups. the increased serum ykl-40 levels in aa patients suggests that ykl-40 plays a crucial role in the pathogenesis of aa. arterial immune mediated inflammation participates centrally in all stages of the development of atherosclerosis, from the initial lesion to the end-stage thrombotic complications. although emerging evidence supports augmented cardiovascular morbidity and mortality in cutaneous psoriasis (psc) and psoriatic arthritis (psa) as compared to the general population its underlying mechanism is poorly understood. here we analyzed the inflammatory burden in recent onset of psa patients without traditional cardiovascular risk factors (cvrf) in a transversal study measuring carotid intima media thickness (cimt) (measured with ecodoppler), and proatherogenic inflammatory molecular markers like c-reactive protein (crp), interleukin 6 (il-6), and soluble intercellular adhesion molecule-1 (sicam-1) in comparison with control patients. cimt values are similar in psa (0, 59 ae 0.045*) and psc (0, 62 ae 0.10) patients. however, both of them were significant increase compared with control (0.436 ae 0, 05). regarding inflammatory markers il-6 serum levels in patients with aps was higher than pcs (18 ae 2.9) and healthy controls (16, 3 ae 2.5) but the difference did not achieve statistical significance (*p > 0.05). on other hand mean of sicam-1, value from patients with recent onset of psa is significant higher than controls. psc remain without significant changes compared to control (*p > 0.05). in addition mean value from patients with recent onset of psa is significantly higher than in controls (*p < 0.05) and psc group. overall, preliminary findings suggest for the first time that patients with early psa, without evident traditional cvrf have significant increased values of cimt, sicam-1 crp against the general population control group. this data strongly supports that early cv molecular markers are increased after the first symptoms and signs of this disease even in the absence of traditional cardiovascular risk factors. furthermore, this give new windows for a proper treatment. p-04.02.2-020 protective effect of trail against proinflammatory cytokines on pancreatic beta cells correlated with decrease in dr5 and increase in dcr1 expressions universitesi, antalya, turkey introduction: proinflammatory cytokines are known to have destructive effects on beta cells, which contribute to type 1 diabetes (t1d) development. the combinatory effects of three of these cytokines in particular, namely tnf-alpha (tnf-a), ifngamma (ifn-c), and il-1beta (il-1b), are claimed to render beta cells prone to t cell-mediated destruction. the recently identified anti-inflammatory feature of tnf-related apoptosis-inducing ligand (trail), its possible protective role in this process. in this study, the effects of applications of trail with tnf-a, ifn-ᵞ, and il-1b on beta cell viability and correlation of these effects with trail receptor expression patterns were investigated. methods: glucose-responsive insulin-secreting nit-1 mouse beta cell lines were treated with tnf-a, ifn-ᵞ, il-1b, and soluble trail (strail) individually and in various combinations. cell viabilities were determined at 24 and 48 h by mtt assay. trail ligand and receptor expression profiles on nit-1 cells, and alterations in receptor expression levels following cytokine applications were determined by western blotting analysis. results: trail treatment did not have any cytotoxic effects on nit-1 beta cells at 48 h, while increasing cell viability following il-1/ifn/trail and il-1/tnf/trail combined applications. substantial levels of death receptor 5 (dr5) expression were detected on nit-1 cells before applications, yet it displayed decreased levels at 48 h of trail treatment. lower levels of decoy receptor 1 (dcr1) expression detected prior to treatments increased significantly in contrast. discussion: the fact that trail co-treatment with tnf-a, ifn-ᵞ and il-1b increased cell viability in nit-1 beta cell lines along with reduction in dr5 death receptor expression and an increase in the decoy receptor dcr1 expression, points out to a possible protective effect of trail in insulitis, and strengthens its potential as a putative therapeutic molecule in prevention of beta cell loss. behc ßet's syndrome (bs) is a multisystemic inflammatory disorder with a strong and complex genetic background. being a prevalent disorder both in turkey and also in the ancient trade road 'silk road' countries, bs is an important cause of impairment and disability owing to its chronic and relapsing nature. besides, bs is reported to be an important cause of mortality among the young male patients. while the epidemiology of bs is substantially well documented, currently, the etiology, the molecular mechanisms underlying its pathogenesis, and the classification of the disorder remain to be elucidated. our aim was to disclose the disease mechanisms at molecular level in turkish bs patients by obtaining, comparing, and analysing the transcriptome data of bs patients with age and gender matched healthy controls. for this purpose, by using the affymetrix hg u133 plus 2.0 microarrays, peripheral blood cell mrna profiles of 30 bs patients (b) and 15 matched healthy controls (c) were obtained. following bioinformatics, gene ontology, and pathway analysis, validation experiments of the identified prominent mrnas were performed by qrt-pcr methodology. the comparison of b vs. c yielded differentially expressed gene numbers of 30 and 625 for the chosen fold changes of 2.0 and 1.5 respectively (p ≤ 0.05 for both). during gene ontology and pathway analysis, immune system process, immune system diseases, systemic lupus erythematosus, arthritis, and intestinal immune network for iga production categories/pathways were significantly enriched. clustering analysis revealed a molecular signature which accurately distinguished b and c samples, while the qrt-pcr analysis successfully validated the chosen mrna transcripts. this study documented differential expression of a large number of immune system and immune disease related genes in bs patients. the uncovering of the molecular disease mechanisms of bs will point to novel candidate molecules to be targeted for the treatment of the disorder. obesity is a public health problem in developed countries and worldwide with increasing prevalence through a relationship primarily with atherosclerotic cardiovascular diseases as well as several metabolic disturbances such as increased insulin resistance and diabetes. although several studies identified obesity as an independent risk factor for atherosclerotic cardiovascular diseases, the mechanism underlying the increased cardiovascular risk in obese patients has not been clearly delineated. adma, no, endothelin-1 and homocysteine are an indicator of endothel disfunction that plays an important role in the pathophysiology of atherosclerosis. in our study, obese children and the control group were compared in terms of adma, no, endothelin-1 and homocysteine, we also investigated whether there is a correlation between these parameters. 58 obese and 30 healthy children, participated in the study. when the obese group was compared to the healthy controls, the adma level of the obese group were significantly higher than those of the control group but there was no statistically significant difference in no, endothelin-1 and homocysteine. increased adma level might trigger the pathogenesis of atherosclerosis starting from the childhood years onward. that is why controlling obesity in this age group with diet and other treatment modalities will prevent the mortality and morbidities that will be seen in adult years. inh deficiency leads to the formation of bradykinin causing to dilation of blood vessels. furthermore, the study conducted by shagdarsuren, on the damage done by c1-esterase, demonsrates that the complement system and triglyceride levels are affected. we investigated lipid oxidation and fetuin a levels in patients with c1 _ inh deficiency. materials and methods: 44 people with c1 _ inh and 44 people without any illnesses were taken into the study. fetuin a was studied using an el _ isa kit from raybio (usa). ferrous ion oxidation-xylenol orange test was used to find looh serum levels. sh (free thiol groups) test was studied with regards to ellmans method modified by hu. _ ibm spss 20.0 was used for statistical results. results: in assessments made between the healthy and the illness groups, there was significant differences in the levels of fetuin (p = 0.035), looh (p = 0.000) and sh (p = 0.047). when pearson correlation analysis was performed, we detected a significant positive correlation between fetuin a and looh levels (r: +0.326) discussion and conclusion: in these patients, lipids is secreted from the adipose tissue. in response, anti-atherosclerotic fetuin a levels were risen. patients also possessed increased lipid peroxidation, this increase shows positive correlation with fetuin a levels. in conclusion, we identified that sh with antioxidant properties have increased levels. aim: high fructose corn syrups are found in soft drinks, juice beverages, breakfast cereals, most of the processed foods. it has been shown that high dose of fructose intake may lead to a reduction in the number of hepatocytes, deterioration of liver function, increasing reactive oxygen species and liver steatosis. the aim of this study was to explore whether caffeic acid phenethyl ester (cape) has any potential protective effect on high fructose diet-induced fatty liver model. materials and method: totally fifty rats were divided into five groups. control group, %10 fructose administered group, cape group, %10 fructose + cape administered group and ethanol group. after 6 weeks, liver oxidant and antioxidant status, and blood tnf alpha, il-6, and il-8, tissue nfkb levels were quantified. protein levels were investigated against, nfkb and p-nfkb antibodies and normalized and analyzed against b-actin antibody by western blotting. results: serum tnf-alpha, il-6, il-8 levels were found to be increased in fructose group compared with the control group (p < 0.05). in liver tissue of 10% fructose administered group, mda, protein carbonyls and no levels were higher than control group. however sod activity did not show any difference among the groups. in the fructose administered group, caspase 3 showed liver apoptosis and was considered as positive. acquired data revealed that nfkb protein level was decreased in the presence of cape while increment in nfkb protein level was observed in the fructose administered group compared with control group. in case of pnfkb antibody, increment observed in fructose only and both cape and fructose administered groups, respectively. in cape only administered group, there was a decrement in the level of pnfkb protein. conclusion: depending on further analysis, experimental findings are expected to implicate the role of cape as a protective agent on high fructose diet-induced fatty liver model in relation of inos, nfkb and p-nfkb pathways. the investigating association of hepcidin levels with iron homeostasis and inflammation variables in pregnant women with intrauterine growth restriction a. g. agg€ ul 1 , n. uzun 1 , e. c ß inar tanriverdi 2 , h. € un 1 1 agri ibrahim cecen university, agri, turkey, 2 nenehatun maternity hospital, erzurum, turkey this study was designed to investigate hepcidin levels and their associations with iron homeostasis and inflammation variables in pregnant women with intrauterine growth restriction (iugr). a total of 88 pregnant women were included in this study. pregnant volunteers were divided into two groups (30 healthy pregnant women and 58 pregnant women with iugr). serum hepcidin, total free iron, ferritin, transferrin, transferrin receptor and interleukin-6 (il-6) levels were measured by elisa. also, hemoglobin (hb) and c-reactive protein (crp) levels were determined in serum samples from the healthy pregnant women and the pregnant women with iugr. there were significant differences in hepcidin, ferritin, transferrin receptor, crp and il-6 levels between healthy pregnant women and pregnant women with iugr. hepcidin, ferritin, crp and il-6 levels in pregnant women with iugr were significantly higher than healthy pregnant women (p). the mediators of systemic inflammation in lipopolysaccharide-induced neonatal sepsis rat model sepsis is an excessive inflammatory response that causes shock, multi-organ failure and high mortality. foreign bacterias and lipopolysaccharides lead to stimulation of endothelial cells to produce biologically active mediators such as proinflammatory cytokines and chemokines, cell adhesion molecules, and growth factors. then these mediators could be act on targets, which were involved in the initiation of systemic inflammation in neonatal sepsis. our aim was to indicate a protective role of thalidomide and etanercept, which have anti-tnf-a activity on systemic inflammatory response in lipopolysaccharide (lps)-induced neonatal sepsis rat samples. thirty 7-day-old wistar rats were randomly divided into five groups: a control group that received normal saline, a sepsis group that received lps, thalidomide, etanercept and both thalidomide and etanercept treatment group that were administered with therapeutic agents 6 hrs after lps injection. the rats were sacrificed at 24 hrs after lps or normal saline injection (n = 6). hepatic tissue tnf-a, il-6, icam-1 and pdgf levels were determined by enzyme-linked immuno sorbent assay (elisa) method in all groups. in sepsis group, tissue tnf-a, il-6, icam-1 and pdgf levels were statistically significantly higher than in controls (p < 0.001). at same time, pretreatment with both thalidomide and etanercept were found statistically dramatically decreases the levels of tnf-a, il-6, and pdgf when compared to sepsis group (p < 0.001). there were no significant differences in the icam-1 levels between the all treatment groups and the sepsis group. higher liver tissue tnf-a, il-6, icam-1 and pdgf levels are associated with severe bacterial infection. these proinflammatory cytokines and angiogenic factors may be important in the endothelial dysregulation seen in sepsis. therapeutic agents used in the present study can be help to avoid devastating effects of neonatal sepsis. n-stearoylethanolamine (nse)is saturated minor compound of natural origin that represents the large family of signaling lipids n-acylethanolamines, which belong to endocannabinoid system. considering the crosstalk between obesity-induced inflammatory response and its key role in synaptic dysfunction and neurodegeneration, our current study aimed to investigate the biological effect of nse on brain tissue under high fat diet-induced insulin resistance. previously we found that nse administration to insulin resistant rats caused normalization of liver and pancreas lipid composition followed by the improvement of glucose tolerance and insulin sensitivity (decline in serum insulin level and homa-ir value). moreover, this effect of nse correlated with inhibition of nf-kb translocation into the nucleus of peritoneal macrophages and decreased pool of serum tnfa level in obesity-induced insulin resistant rats. further experiments showed that fat overload triggered significant reduction in the level of main phospholipids (phosphatidylethanolamine, phosphatidylcholine and sphingomyelin), while there were no changes in cholesterol content. nse at a dose of 50 mg/kg during 2 weeks of administration to insulin resistant rats showed a tendency to restore the phospholipid level that was accompanied by increased neural cell survival (91%) compared to rats without treatment (84%). neuroinflammation accompanied by intensive reactive oxygen species (ros) production impairs neurotransmission in a wide range of neurodegenerative pathologies. flow cytometry is used for quantitative analysis of global dna methylation, but fluorescence microscopy is mostly preferred to qualitatively reveal intranuclear localisation of dna methylation and its copattern with other markers. both methods use a similar immunostaining protocol. in this study, we aimed to compare these methods concerning the detection of the global amount of dna methylation. for this, mouse embryonic fibroblasts were cultured either with or without phenol red and then stained for dna methylation or positive controls (histone, betaactin, phosphoakt) by specific antibodies, or nonspecific control antibodies. some cells were incubated with trypan blue before or after the addition of antibodies. fluorescence intensities were measured by the green fluorescence channel (530/30 nm). autofluorescence spectrum of cells was analysed, and fluorescence channel used for dna methylation detection was changed to red (650 nm lp). a poor discrimination between signal and noise was detected due to cellular autofluorescence interfering with specific detection of dna methylation by flow cytometry but not by microscopy. it was also the case for the other markers examined. conventional advances to reduce autofluorescence such using phenol red free culture media or trypan blue quenching were not effective, but using the red channel regarding autofluorescence spectra allows detecting specific staining of dna methylation by flow cytometry. but, green channel did work well for microscopy analysis. findings show that flow cytometry detection of dna methylation requires much attention to quench cellular autofluorescence compared to detection by fluorescence microscopy. one reason could be that flow cytometry detects all cellular content, but manual image-based analysis can exclude cytosolic components. these results suggest the usability of flow cytometry and microscopy as complimentary methods for dna methylation detection, but optimisation to reduce autofluorescence is crucial for flow cytometry. objectives: lung cancers are divided in two main groups as small cell lung cancer (sclc) and non-small cell lung cancer (nsclc) . docetaxel (dtx) and cisplatine are chemotherapeutic that has an anti-tumor activity against various solid tumors. the growing resistance against dtx and cisplatine (cis) still continues to be the biggest obstacle for the treatment success of nsclc patients. deguelin (deg.) is a natural plant derivative and has an encouraging activity against a lot of human cancers. the comparison of the treatment activity of the separate and combined usage of deg., which is a potential chemotherapeutic agent, and dtx, cis which are used in standard treatment, is aimed in this study. material-method: the ic50 doses of dtx, cis and deg. on the a549 and h1299 nsclccell lines were determined via the cell vitality tests in our study. the active concentrations determined were applied to nsclc cell lines as deg., dtx, cis and their combinations. the impacts of the medicine are studied by applying flow cytometric analyzes (apoptosis, cell cycle), glutathione and reducted glutathione, colony formation, migration and angiogenesis analyzes on the treated cells and measuring the oxidative stress index (osi). statistical analyse program, rstudio (v.0.98.501) and the r-script language were used to examine the differences between the agents. the states in which the pvalue was lower than 0.05 were accepted as statistically meaningful. results: we found that deg. has pro-apoptotic, anti-migratory and cytotoxic potential on lung cancer cells. deg. amplified cis and dtx-related anti-cancer efficacy (increased apoptotic cell content and cytotoxicity, reduced migration). also, deguelin pretreatment sensitized the cells dtx-treatment (reduced ic50 values). these effects were remarkable in p53-mutant cells. conclusion: deguelin, solely, has anti-cancer potential on nsclccells. both deguelin pre-treatment and combinantion with standart chemotherapeutics result in enhanced anticancer efficacy. the 83% of the lung cancers are non-small cell lung cancers (nsclc). despite docetaxel (dtx) and cisplatine (cddp) are agents used in the standard treatments of these patients and the recent improvements in the treatments, the response and remission rates observed on the patients are relatively nominal. selenium (se) is an essential diet component and is introduced to have a preventive impact on different levels of cancer. the aim of our study is to investigate the impacts of selenium addition on anticancer feature and tumor prevention before or/and during nsclc standard treatment. the ic50 doses of dtx, cddp and selenium on the a549 and h1299 (p53 mutant) nsclc cell lines were determined via the cell vitality tests in our study. the active concentrations determined and the stipulated available concentrations were applied to cell lines as dtx, cddp, se combinations. the impacts were compared by applying flow cytometric analyzes (apoptosis, cell cycle), glutathione and reducted glutathione, western blot analyzes on the treated cells and measuring the oxidative stress index (osi) and thioredoksin reductase activity. selenium pre-treatments reduced dtx-related ic50 concentrations at lower doses in both nsclc cells. however, cddprelated ic50 concentrations reduced dose-dependent manner. selenium supplementation also altered cell-cycle charactheristics at several concentrations and combination regimens. the remarkably higher osi values were observed after dtx treatment and osi levels were found to be lower in selenium pre-treated nsclc cells. selenium sensitizes nsclc cells to dtx treatment at lower concentrations. however, this effect is obtained dose-dependent fashion for cddp regimen. breast cancer is the most common female malignancy worldwide. human epidermal growth factor receptor 2 (her2) is overexpressed in 30% of breast cancers in association with aggressive phenotypes. the prognosis of metastatic breast cancer remains poor in spite of advances in therapy. as such, her2 has long been studied as a potential target for anticancer drugs. the modulation of intracellular signaling pathways leads to altered cell metabolism that triggers tumorigenesis and adapts cells to cancer cell metabolism. this characteristic hallmark of cancer metabolism is known as warburg effect meaning energy production via enhanced glycolysis. despite of several studies in breast cancer metabolism, little detail exists on the link between her2 overexpression and warburg effect. we have committed examining the nature of aerobic glycolysis in her2 overexpression. in breast cancer cell line mcf7, her2 overexpression (mcf-her2) results in mitochondrial dysfunction with low mitochondrial membrane potential (δᴪm) and ros accumulation. intracellular iron levels are also higher in mcf7-her2 cells than vector control (mcf7-vec). additionally, mcf7-her2 cells show enhanced levels of atp and lactate in association with increase in glucose levels. we have found that complex i activity increases in mcf7-her2 and decreases in knockdown of her2 in hcc1954 cells that is her2 positive breast tumor cell line. based on these results, we conclude that there is a link between her2 overexpression and metabolic indicators of warburg effect. expression and methylation analysis revealed 11 microrna genes deregulated by methylation and new potential target genes of mir-375 and mir-127-5p in breast cancer micrornas (mirnas) and methylation of mirna genes play a great role in epigenetic deregulation in malignant tumors. the aim of our study was to assess the contribution of methylation to expression level alterations of 20 mirna genes and to search for novel potential targets of these mirnas. to analyze alterations in expression we used qpcr technique with 2 references (rnu6, rnu48) and 30 paired (tumor/normal) breast cancer (bc) samples. for methylation analysis a methylation specific pcr and the same set of bc samples were used. significant downregulation was shown for mir-125b-5p, -129-5p, -132-3p, -193a-5p, -34b-3p, -212-3p, -127-5p, and -17-5p (p ≤ 0.05, fisher's exact test) in bc. we observed 10 mirna genes to be hypermethylated and mir-191hypomethylated. hypermethylation for 3 of these mirna genes was shown for the first time: mir-132, -137, and -1258 (41-34% of bc cases). a significant correlation between methylation and expression alterations was revealed for 5 mirnas with downregulation: mir-125b-5p, -129-5p, -132-3p, -193a-5p, and -34b-3p (spearman's correlation coefficient (rs) was in the range à0.71 to à0.81, p ≤ 0.01), and for 3 mirnas with both scene (down-and upregulation) as well: mir-148a-3p, -203a, and -375 (rs = à0.72 to à0.93, p ≤ 0.01). comparative analysis of the data on expression alterations of 20 mirna genes and 6 protein-coding genes, which were predicted as targets by mirwalk 2.0, revealed the negative correlation between expression levels for some potential mirna-mrna interaction pairs. for example, for pairs mir-375/rhoa, mir-375/rassf1(a), mir-127-5p/dapk1 (rs = à038 to à0.46, p ≤ 0.05). thus, both mirnas and methylation affect regulatory networks in bc. novel potential mirna-mrna interaction pairs could be useful in the development of bc therapy approach. this work was financially supported by grant 14-15-00654 from the russian science foundation. the authors thank the n.n. blokhin cancer research center for tissue samples. clear cell renal cell cancer (ccrcc) with metastases has pour prognosis: 5-year survival is about 9%. micrornas (mirnas) and methylation of mirna genes play a great role in epigenetic deregulation in malignant tumors. the aim of our study was to find out mirnas which methylation contributed to ccrcc progression, including metastasis, and to reveal potential target genes of these mirnas. to analyze methylation status, we used a methylation specific pcr as a method and a representative set of 70 paired (tumor/ normal) ccrcc samples. we also used 19 post-mortal renal tissues from individuals without cancer history as additional control. for expression analysis we used qpcr method and 45 paired ccrcc samples. we observed 9 mirna genes (mir-124a-1/-2/-3, -9-1, -9-3, -34b/c, -129-2, -193a, -107) to be hypermethylated, (p ≤ 0.05, fisher's exact test), 3 mirna genes to be hypomethylated and mir-203a with both scene (hyper-and hypomethylation was detected). methylation of 7 mirna genes (mir-124a-2/-3, -34b/c, -129-2, -107, -148a, -203a) correlated with advanced stage and/or tumor size and/or dedifferentiation. hypermethylation of mir-129-2, mir-203a, and mir-107 significantly correlated with metastasis presence (p < 0.05, fisher's exact test). besides, preliminary data revealed the positive correlation between hypermethylation of mir-129-2 and up-regulation of 3p protein-coding genes: rarb(2), rhoa, nkiras1, and chl1, which were predicted as targets by mirwalk 2.0 (spearman's correlation coefficients (r s ) was in the range 0.35-0.53, p ≤ 0.05). in conclusion, novel supposed interactions of mir-129-2 with target genes could be useful as missing chains in signaling pathways. tests for hypermethylation of mir-129-2, mir-203a, and mir-107 could be suggested as markers of metastasis and pour prognosis of ccrcc. because of difficulty in diagnosis and treatment hc is a clinical problem: early symptoms of hc are often non-specific and surgical resection is the only curative treatment for hc. it is well known that epigenetic alterations are linked to cancer development. the purpose of this study was to determine potential mechanisms of epigenetic regulation of genes related to energy metabolism in hc. we have performed bioinformatics analysis of the cancer genome atlas (tcga) project rna-seq data with crosshub software and found a number of genes involved in glycolysis and differentially expressed in cholangiocarcinoma. qpcr analysis revealed significantly decreased expression of pgm1 and eno3 genes in a majority of hc samples which were known as up-regulated in other human cancers according to the literature date. on the basis of tcga methylation dataset (450k illumina microarrays) we supposed that cpg methylation of pgm1 and eno3 promoters may play a role in their inactivation. using bisulfite sequencing study we identified several regions within the gene promoters (pgm1:~750 bp and 330 bp upstream tss; eno3:~750 bp downstream tss) that are frequently methylated in hc samples (up to 60%, 12/20) with down-regulated pgm1 and eno3 expression. thus, we demonstrated frequent and significant pgm1 and eno3 down-regulation associated with hypermethylation of the specific regions within the gene promoters in hc. the pattern of pgm1 and eno3 gene promoter methylation suggests a possibility of ones to be used for the hc diagnosis and development new strategies for therapy. this work was financially supported by grant mк-8047.2016 .4 from the president of the russian federation. the work was performed using the equipment of eimb ras 'genome' center. introduction: the development of stomach cancer is a multifactorial and complex process and includes multiple epigenetic, genetic alterations and dietary/non-dietary factors. iodine as an antioxidant may play a protective role against gastric cancer. the aim of this study was to investigate the changes in iodine level in rat with stomach cancer induced by n-methyl-n 0 -nitro-n-nitrosoguanidine (mnng). materials and methods: a total of 62 sprague dawley rats were randomly divided into six groups. 55 rats were administered with mnng (200 lg/ml) by oral gavage on days 0, 14 and 21 to initiate stomach cancer. during the experiment, 28 rats died and those surviving were sacrificed in the 3rd, 5th, 7th, 10th and 12th months of the experimental period (group i, ii, iii, iv, v, respectively). the control group (group vi) contains 7 rat which are given only food and water for 12 months. the stomach tissue was examined histopathologically. and also, iodine levels in stomach tissue was determined using the foss method. results: a decrease in iodine level was determined in stomach cancer tissue of rats in group i-v compared with normal healthy stomach tissue in group vi. when the control (group vi) iodine level was taken as % baseline, the % iodine levels of all groups were determined as follows 71.78, 52.79, 40.76, 25.33 and 11.96 for groups i-v, respectively. the pathological diagnosis of gastric cancer was adenocarcinoma. discussion and conclusion: the iodine levels of group i were higher than those of group ii (p < 0.01) and of groups iii, iv and v (p < 0.001) and also were lower than in the control group (p < 0.001). iodine deficiency as one of the risk factors of stomach cancer strongly supports the necessity for the application of effective iodine prophylaxis in the areas with iodine deficiency. iodine supplementation might be useful in stomach cancer therapy and therefore, further research is warranted. this study was supported by ataturk university (project number: 2005/147). effect of water extract of turkish propolis on mitochondrial membrane potential in human laryngeal epidermoid carcinoma cell lines propolis is the generic name for the resinous substance collected by honeybees from the buds of various plant sources and it is used by bees to seal holes in their honeycombs, smooth out the internal walls, and protect the entrance of bee hive against intruders. the aim of this study is to investigate what kind of changes the turkish propolis cause on mitochondrial membrane potential (mmp) of human laryngeal epidermoid cell lines (hep-2), by considering its anticancer features. water extract of turkish propolis (wep, 1-3 mg/ml) and ethanolic extract of turkish propolis (eep, 0.075-3 mg/ml) were prepared and incubated with hep-2 cell lines (24, 48, and 72 h). mmp was investigated with a flourometric method by using dioc 6 (3,3 0 -dihexyloxacarbocyanine iodide). the most significant mmp decrease was seen on 72rd hour. both wep and eep extracts at all concentrations decrease mmp according to that of control. the recent studies have shown that propolis extracts have induced apoptotic cell death by decreasing mitochondrial membrane potential in various cancer cells. it was concluded that both wep and eep decreased mitochondrial membrane potentials on hep-2 cell series according to control (0 concentration) depending concentration and time. there are numerous transcription factors involved in the regulation of the inducible gene expression. thus, transcription of proinflammatory genes, steroid hormone receptors, etc. is controlled by the group of factors triggering gene expression which includes nf-kb. another group of factors is involved in the formation of the structure of the chromatin of the inducible genes regulatory regions, providing competence for gene expression. it is expected that this group of factors includes the proteins of nf1 (nuclear factor 1) family. there are few data suggesting that the nf1 factors maintain potentially active state of the chromatin of the hormone-dependent gene promoter regions. these findings initiated studies of the correlation between presence of the nf1 transcription factors on the chromatin of a gene regulatory region and the functional state of the gene in vivo. as a model we chose the rat tryptophan dioxygenase (tdo) gene which is expressed tissue-specifically in the liver under control of glucocorticoid hormones. three constitutive dnase i-hypersensitive regions are identified in the regulatory region of this gene. to conduct the study we used rat liver and kidney. the basic methods were electrophoretic mobility shift assay (emsa), immunoblotting assay and chromatin immunoprecipitation combined with real-time pcr (chip-qpcr). using emsa we found that the proteins of nf1 family interact with the constitutive dnase i-hypersensitive regions in vitro. immunoblotting assay of the protein fraction from rat liver used in emsa experiments showed the presence of the nf1-b1 isoform. chip-qpcr revealed statistically significant differences in the level of the factor nf1 enrichment of the tdo gene regulatory region between the rat liver and kidneys at p < 0.05. these data suggest the involvement of the nf1 proteins in the formation of the chromatin structure of the rat tdo gene promoter region. reciprocal (9;22) translocation and bcr-abl fusion protein that is responsible for developing leukemia are observed in more than 95% of chronic myeloid leukemia (cml) cases. epigallocathecin-3-gallate (egcg) is a green-tea flavonoid and egcg is proposed as a natural anti-cancer agent. histone modifications which contain histone deacetylases (hdac) and histone acetyltransferases (hat) are parts of epigenetic regulations. hdacs play important roles in different human malignancies including leukemia via activation of abnormal signaling pathways. hdac inhibitors have become remarkable therapeutic molecules for malignancies. the aim of this study is to determine the expression changes of leukemia-related hdacs with the treatment of egcg in k-562 cells. the cytotoxic effect of egcg on k-562 cells was determined in time and dose dependent manner by wst-1 analysis. total rna was isolated from k-562 cells. reverse transcription procedure was performed for cdna synthesis and gene expressions were detected by rt-qpcr. the expression level of hdac4, hdac6, hdac11 gene that supports cell proliferation was down-regulated 2.29, 2.56, 3.81 folds in k-562 cells treated with ic50 dose of egcg, according to control, respectively. our current findings suggest that is a polyphenol egcg may be a hopeful agent in treatment of cml by hdac inhibitory effect. chronic lymphocytic leukemia (cll) is a disorder of morphologically mature but immunologically less mature lymphocytes and is manifested by progressive accumulation of these cells in the blood, bone marrow, and lymphatic tissues. carbonic anhydrase (ca) is a metalloenzyme which is widely distributed in the living world, and it is essential for the regulation of acid-base balance. anti-ca antibodies have been reported in many disorders, such as systemic lupus erythematosus, sj€ ogren's syndrome, rheumatoid arthritis, endometriosis, idiopathic chronic pancreatitis, type 1 diabetes and graves' disease. the goal of this study was to investigate carbonic anhydrase i and ii (ca i and ii) autoantibodies in cll. 38 patients with cll and 39 healthy controls were included in the study and ca i and ii autoantibody levels were investigated by elisa. the ca i autoantibody levels of cll group were significantly higher than the healthy group (p = 0.006) while there was no statistical difference between serum ca ii autoantibody levels of the groups (p = 0.301). we found a significant positive correlation between hemoglobin and hemotocrit levels in patients with cll (r = 0.931, p = 0.0001). cut-off value of 0.072 absu for anti-ca i was associated with 92% sensitivity and 41% specificity and a cut-off value of 0.047 absu for anti-ca ii was associated with 48% sensitivity and 85% specificity for predicting cll. the ca i autoantibody levels in patients with cll were found higher compared to control group and the results suggest that ca i autoantibody may be involved in the pathogenesis of cll. genetic and epigenetic aberrations can lead to the activation of oncogenes and inactivation of tumor-suppressor genes (tsgs) followed by the development of malignant tumors. in the present work we evaluated the frequency of alterations of cpg island methylation and dna copy number in 47 paired (tumor/normal) breast cancer (bc) samples using comparative dna hybridization on noti-microarrays and original niman software. the microarrays contained 180 noti-clones associated with 188 chromosome 3 genes. expression alterations were assessed with the use of qpcr technique, ddct method and original atg software. in total, 35 noti-sites with high (15-38% of cases) hypermethylation/deletion (hm/d) frequency were revealed in bc. among genes associated with these sites, there are both known tsgs and tsg-candidates (aldh1l1, vhl, ctdspl, etc.) as well as genes, which involvement in breast oncogenesis was shown for the first time (lrrn1, foxp1, prickle2, etc.). noti-microarray data were verified selectively using bisulfite sequencing for vhl, nkiras1, itga9, lrrc3b, and ctdspl genes. several genes with high hm/d frequency (aldh1l1, ephb1, itga9, and ropn1) were tested for expression alterations using qpcr. frequent (57-90% of cases) and significant (> 2-fold) down-regulation was shown for all of them in bc. the most significant expression loss was observed for aldh1l1 geneon the average 50-fold mrna level decrease in 90% of samples. the involvement of the majority of genes with high hm/d frequency in breast oncogenesis was shown for the first time. these genes are novel tsg-candidates in bc. functional hypermethylation associated with expression loss was shown for aldh1l1, ephb1, itga9, and ropn1 genes thereby strengthening the speculation on tumor suppressor abilities of these genes. methylation and expression analyses of genes, that were revealed by noti-microarrays, were financially supported by grant 14-15-00654 from the russian science foundation. functional hypermethylation of a number of chromosome 3 genes was revealed in colon cancer using noti-microarrays cancer is a disease of genome caused by genetic and epigenetic aberrations. noti-microarrays, that were developed by prof. e.r. zabarovsky, is a unique tool that allows us to simultaneously detect hypermethylation of cpg islands and dna deletionstwo major reasons of inactivation of tumor suppressor genes (tsgs). in the present work, the frequency of chromosome 3 genetic and epigenetic alterations in colon cancer (cc) was evaluated. noti-microarrays, that contained 180 noti-clones associated with 188 chromosome 3 genes, were used for comparative (tumor/normal) hybridization of dna from 24 paired cc samples. data analysis was performed using original niman software. expression alterations were evaluated using qpcr technique and original atg software. in total, 24 noti-sites with 20% and above hypermethylation/ deletion (hm/d) frequency were revealed in cc. among genes associated with these sites, there are several known tsgs and tsg-candidates (for example, vhl, ctdspl, and itga9), but for the majority of genes, involvement in colon oncogenesis was shown for the first time (for example, lrrn1, nbeal2, and ube2e2). the highest hm/d frequency was observed for ankrd28, nkiras1/rpl15, itga9, cmtm6, and gor-asp1/ttc21a genes -38-42%. expression alterations were evaluated for 3 genes with high hm/d frequency (plcl2, prickle2, and ppp2r3a) and significant mrna level decrease (> 2-fold) associated with hypermethylation was shown for all of them in the majority of samples. a number of novel potential tsg-candidates was revealed in cc. functional hypermethylation associated with expression decrease was shown for plcl2, prickle2, and ppp2r3a genes thereby enhancing the suggestion on tumor suppressor function of these genes. this work was financially supported by in many countries, radon is the second leading cause of lung cancer, which accounts from 3% to 14% of cases. it is obvious that the population of all the developed and industrial countries in the world spend most f their time, almost 80%. therefore it is necessary to explore the obtained radiation dose, because of the presence of radon in a room due to the radon emanation from the soil and exhalation from a variety of building materials. the developed countries solve this problem of radon pollution as well as create a special monitoring services. the paper presents some data of 8 genes molecular-genetic analysis from patients with lung cancer who live in almaty located in a foothill area of tectonic faults. the object of research were blood samples obtained from patients diagnosed with lung cancer who are receiving a treatment at the almaty oncology center and living in the city of almaty, where the level of radon activity exceeds the norm approved by the international commission on radiation safety. as a control group relatively people living in the plains, characterized by a lower radon emanation have been considered. to determine mutations in the genes polymerase chain reaction with a subsequent analysis of restriction fragment length polymorphism has been conducted. the pcr products were subjected to hydrolysis by bstni restriction endonucleases haeiii, ras i. disturbances in the genes under consideration to variour types of cancer development. the analysis showed that examinees do not have mutations in the kras gene codons 12-13, which corresponds to a control group consisting of 90 people living in the city of balkhash. on the whole, molecular genetic studies have shown that 44 examined patients do not have mutations in the kras gene. one mutation was been found in the egfr gene. aim: polyps are abnormal growths of tissue that can be found in gastro intestinal system. they are most often found in the colon and rectum. most polyps are noncancerous (benign) however, because of abnormal cell growth, they can eventually become cancerous. the aim of this study is to determine the concentrations of trace element contents in colon and rectum polyp tissues and whether there is any relationship between polyp tissue element levels and the disease. material and method: the present study was conducted on total of 82 individuals including 57 patients and 25 healthy subjects. while receiving normal intestinal tissue from healthy control group; from the patient group both normal tissue and polyp samples were taken during colonoscopy procedure. the concentrations of the elements (al, cr, mn, fe, co, ni, cu, zn, as, se, ag, cd, hg and pb) were determined with induced coupled plasma-mass spectrometer. results: the mean concentrations of cr, mn, ni, se and ag in colorectal polyp tissues of patients were significantly higher than in colorectal tissues of control subjects (p is less than 0.05). on the other hand the mean concentration of cd and pb in colorectal polyp tissues of patients were significantly lower than in control colorectal tissues of control subjects (p is less than 0.05). there was no any significant difference between the groups in terms of concentrations of al, fe, co, zn, as and hg (p is more than 0.05). conclusion: the differences found in some elements between polyps and a control tissues may provide an indication about the role of trace elements in the early stage (polyps) in the colon carcinogenic process and encourages further studies to confirm the involvement of such elements in neoplastic processes. the use of herbal medicines is steadily growing, with approximately 40% of the population use herbs to treat various illnesses in the western world. vitex agnus-castus has been used since ancient times as a remedy. the aim of this study was to investigate the in vitro anticancer activities of vitexagnus-castus oil. for this purpose, the cytotoxicity of vitexagnus-castus oil in sh-sy5y cells was investigated by crystal violet staining. ec50 was found to be 0.5%(w/w) vitexagnus-castus oil for this cell line. this dose was applied to the cell for 24 h, and the cells were harvested for further studies. vitex agnus-castus oil treatment increased bax and p38 mrna levels. on the other hand, bcl-2, bcl2 l1, erk-2, jnk, caspase 3 and 8mrna expression levels were reduced significantly withvitexagnus-castus oil treatment while p53 and pten remained unchanged. these results indicate that another effector caspase such as caspase 6 or 7 may be involved apoptosis process which remains to be elucidated. moreover, mapk pathways, p38 and erk, may be involved in vitexagnus-castus oil induced apoptosis in sh-sy5y cells. these initial observations suggest that this agent might not be useful in treating cancers. further detailed studies should be carried out to elucidate the exact mechanism of vitexagnus-castus oil in neuroblastoma cell lines. melanoma is a skin cancer with a melanocyte origin that can occur in any part of the body that contain melanocytes. while melanoma is less common than other skin cancers, it causes the majority of deaths related to skin cancer. several gene expression databases have shown that interferon regulatory factor 4 (irf4) is upregulated in melanomas, and genome wide association studies linked variation at irf4 locus with skin cancers. irf4 was first identified to have roles in lymphocyte development and function. studies have identified a 'non-oncogene addiction' of malignant cells to irf4 in various hematopoietic cancer types. the aim of this study is to investigate the role of irf4 in melanoma cell lines. lentiviral vectors were used to reduce irf4 levels in melanoma cell lines. a gfp competition assay was performed to study the competitive fitness of melanoma cells with irf4 knockdown (gfp positive cells) over melanoma cells with normal irf4 levels (gfp negative cells). cell cycle profiles were investigated in melanoma cells with irf4 knockdown by propidium iodide staining. migration potential was assessed as well by wound healing assay. our preliminary data showed a decreased competitive fitness for cells with decreased irf4 levels. cell cycle profiling showed increased g0/g1 and decreased g2/m levels in irf4 knockdown cells compared to controls. wound healing assay results showed no difference between controls for cells with reduced irf4 levels. taken together, these results indicate that irf4 knockdown affects the melanoma cell lines' survival and cell cycle profile, suggesting a non-oncogene addiction of melanomas to irf4. these observations are largely similar to previous observations in hematopoietic cancers. unravelling the role of irf4 in melanoma will increase our knowledge about melanoma development and progression and thereby may lead to targeted therapy in melanoma treatment. humans are exposed to various chemicals having beneficial or toxic effects at a time in their daily lives. 7,12-dimethylbenz[a] anthracene (dmba) is a carcinogenic compound produced during the incomplete combustion of carbon-containing compounds. endosulfan is an organochlorine pesticide used against insects on food. morin is an antioxidant, antiinflammatory and chemoprotective flavonoid. this study is aimed to determine the effect of morin in the presence of dmba and endosulfan. for this purpose, 56 adult wistar male rats weighing 170-255 g were randomly selected and divided into eight groups. 25 mg/kg body weight (b.wt.) morin and 5.0 mg/kg b.wt. endosulfan were given to morin and endosulfan treated groups three times in a week. the rats in dmba treated groups were gavaged with 30.0 mg/kg b.wt. dmba three times during the administration period (54 days). cytochrome p4501a (cyp1a) associated 7-ethoxyresorufin o-deethylase (erod) and glutathione s-transferase (gst) activities were measured in rat liver cytosols and microsomes. in addition, liver tissues were evaluated by histopathological analysis. erod activities of control, morin, endosulfan, dmba, morin+endosulfan, morin+dmba, dmba+endosulfan and morin+dmba+endosulfan groups were 71 ae 7, 112 ae 6, 114 ae 8, 126 ae 6, 121 ae 9, 156 ae 12, 151 ae 15 and 185 ae 13 pmol/min/mg protein, respectively. all treatments increased erod activities. gst activities of these groups were 365 ae 14, 430 ae 27, 365 ae 24, 651 ae 57, 460 ae 10, 577 ae 25, 585 ae 23 and 649 ae 33 nmol/min/mg protein, respectively. histopathological studies showed that endosulfan and dmba induced inflammation in the liver tissues and morin reduced their effects. in conclusion, morin treatment increased the metabolism of dmba and endosulfan by inducing cyp1a activity. gst activities of morin+dmba+endosulfan group were not significantly different from those of dmba group. histopathological studies indicated that morin administration reduced the toxic effect of endosulfan and dmba in the liver cells. hepatocellular carcinoma (hcc) is the sixth most common cancer and third most frequent cause of cancer-related death worldwide. molecular mechanisms of hepatocarcinogenesis is still unclear. the impairment of epigenetic mechanisms is implicated in the development of multiple cancers, including hcc. transforming growth factor-beta has been shown to play both tumorsuppressive and tumor promoting roles. transforming growth factor-beta signaling pathway involves activation of smad2 and smad3 by the type i receptor and formation of smad2/3/4 heteromeric complexes that enter the nucleus to regulate transcription. 3-deazaneplanocin a is an inhibitor of the histone methyltransferase ezh2. we aimed to reveal the effect of 3-deazaneplanocin a on transforming growth factor-beta /smad pathway in hepg2 cell line. hepg2, a human liver cancer cell line cultured in dulbecco's minimal essential medium supplemented with 10% fbs. the cells were seeded the day before 3-deazaneplanocin a administration and then the cells were treated with 5 lm 3-deazaneplanocin a for 3 days. expression levels of genes were analyzed by roche lightcyclerò 480. gapdh was used as housekeeping gene. apoptosis assay was performed by the muse annexin v and dead cell assay kit. the unpaired t-test was used to compare variables and p < 0.05 was accepted as statistically significant. 3-deazaneplanocin treatment was significantly reduced transforming growth factor-beta, smads 2-7 in hepg2 cells (p < 0.05). we also found that 3-deazaneplanocin induces apoptosis in treated cell line (p < 0.05). as a result, 3-deazaneplanocin a may take place in treatment of hepatocellular cancer by its inhibitory effect on transforming growth factor-beta /smad pathway and inducing apoptosis in liver cancer cells. brefeldin a (bfa) is a lactone antibiotic first isolated from the fungus eupenicillium brefeldianium. bfa inhibits the transport of secreted proteins from endoplasmic reticulum (er) to golgi apparatus, leading to disruption of golgi function, accumulation of unfolded and not fully incompletely processed proteins in er. bfa also inhibits cell proliferation, phosphorylation and migration of cancer cells. therefore in this study, we investigated the effects of bfa on breast cancer cell proliferation of various phenotypes. in we observed that bfa inhibited the proliferation of all three phenotypes of breast cancer cells, but the effects of bfa were seen at different times and doses. according to time and dose, bfa was observed more effective to mcf-7 compared to other cell lines. physiological, pathological and physical factors. moreover, nlr may represent the two opposing inflammatory and immune pathways that exist together in cancer patients. we aimed to investigate nlr in breast cancer in our population. methods: using data retrieved from the medical records, 66 women diagnosed primary breast cancer met our study inclusion criteria as they had a complete blood count with leukocyte differential performed before any anti-cancer therapy. and 44 women with benign mammary neoplasm/disease, followed up in the outpatient clinics of mammary disease and confirmed with sonographical/histopathological examination, made up our controls. exclusion criteria included laboratory evidence of white blood cells count (wbc) > 10.5 9 10 9 /l. differential leukocyte counts were obtained by bc 6800 (mindray medical international ltd., china), we examined wbc, neutrophil, lymphocyte, platelet counts, and hematocrite, nlr, mean platelet volume values. results: although there is lack of evaluation of tumor-associated neutrophils and lymphocytes, higher nlr median values and lower lymphocyte mean counts (lymphopenia) were shown in women with breast cancer (p < 0.0001). there was a weak negative correlation in breast cancer between nlr values and platelet counts (r s = à0.274; p = 0.026). holmboe] is distributed throughout southern mediterranean europe from spain to the eastern mediterranean on anatolian peninsula of turkey. present study was designed to investigate the in vitro anti-cancer activities of turkish black pine essential oil. the essential oil was extracted by steam-hydrodistillation and its chemical composition analyzed by gc-ms. the major components of the essential oil were a-pinene, b-pinene and trans-b-caryophyllene, respectively. the crystal violet staining method was used to investigate the cytotoxicity of essential oil in sh-sy5y cells. ec50 was found to be 0.75% (w/w) essential oil for sh-sy5y cells. neuroblastoma cells were incubated at 37°c for 24 h. after 24 h, cells were harvested for further studies. bax and p38 mrna levels were significantly elevated in essential oiltreated cells. on the other hand, bcl-2, bcl2 l1, casp-3, casp-8, erk-2 and jnk expression were significantly downregulated. unlike these proteins, p53 and pten mrnas were not changed. in this study, apoptosis was enhanced by turkish black pine essential oil treatment which was activated by the involvement of another effector caspase subfamily, like casp-6 and casp-7. additionally, erk and p38 mapks may be associated with upregulation of the level of bax. based on these results, we suggest that p. nigra subsp. pallasiana essential oil might not be well-suited in cancer treatment. however, further detailed research is necessary to establish the exact role of p. nigra subsp. pallasiana essential oil in sh-sy5y cells. p-05.02.2-026 the protective effect of newly derivatized compound naringenin-oxime and relative to naringenin against cisplatin-induced nephrotoxicity and genotoxicity in rat background: the aim of this study was to evaluate the possible protective effect potentials of newly derivatized compound naringenin-oxime (ng-ox) relative to efficacy of free naringenin (ng) on cisplatin (cis) induced nephrotoxicity and genotoxiticity in rat. methods: totally, fifty six male wistar albino rats were equally divided into eight groups as follows: control; cis treatment (7 mg/kg b.w., i.p.), ng and ng-ox (20 mg/kg b.w., i.p daily for 10 days) alone treatment; cis + ng (20 or 40 mg/kg b.w., i.p daily for 10 days) and cis+ng-ox (20 or 40 mg/kg b.w., i.p daily for 10 days) combination treatment. at the end of the study total antioxidant capacity (tac) levels, total oxidant status (tos), lipid peroxidation (lpo), total thiol, catalase (cat) were studied in homogenate kidney. peripheral lymphocyte cell dna damage was investigated with comet assay results: the results suggest that cis induces oxidative stress resulting in increased tos and lpo reduction thiol, tac and cat in kidney and increased peripheral lymphocyte cell dna damage. the treatment with naringenin and naringenin oxime alone or with cis treatment showed a protective effect against the toxic influence of cp on peroxidation of the membrane lipids and an altering of the total thiol status in the kidney of rats. from our results we conclude that naringenin and naringenin oxime functions as a potent antioxidant and suggest that it can control cp-induced nephrotoxicity and genototoxicity and ng-ox was found more protective than that of ng on cisplatin induced toxicity in rats. keywords: naringenin, naringenin-oxime, antinephrotoxic, antigenotoxic, comet assay. introduction: oxidative damage is considered to play a pivotal role in ageing, several degenerative diseases, and carcinogenesis. lung cancer is the most common type of cancer, resulting in over 1.3 million deaths each year worldwide. accurate and reliable determination of superoxide radicals has been widely investigated using spectrophotometric, electrochemical, amperometric, polarimetric, piezoelectric technologies. among these methods, electrochemical detection is a most promising approach to achieve accurate, separate and rapid superoxide radicals monitoring with using biosensor system. materials and methods: we used a new technic for detecting superoxide radicals in samples. superoxide dismutase (sod) enzyme immobilized on the surface of gold electrode with the help of gelatin, bovin serum albumin (bsa) and glutaraldehyde (ga) crosslinker. for the biosensor preparing benzoquinone selected as a mediator in working buffer and measurements were carried out at à0.7 v. result: for the optimization studies, effect of the bsa, gelatin, glutaraldehyde, ph, buffer concentration on biosensor response. characterization of the biosensor commitment to the work process and answer reproducibility were evaluated. the analytical characteristic of the biosensor were evaluated by measuring the steady state current response to superoxide radical concentrations. the electrochemical response of the enzyme electrode was linearity gradually leveled of at higher concentration. we found that crosslinking of the sod (e.c.1.15.1.1) with glutaraldehyde could be achieved over a wide range of relative mole ratios in 50 mm phosphate buffer at ph 7.5, glutaraldehyde concentration of %2.5. discuss and conclusion: in this study, a new technique for developed sod biosensors has been developed, which features effective combination of sod/gelatin/bsa/ga modified electrode, trapping of sod and glutaraldehyde cross-linking. this technique is reliable and cost effective. the effect of astaxanthin on apoptosis and cell arrest in u87 brain cancer cell line f. s€ og€ utl€ u, b. € ozmen yelken, c ß . kayabasi, a. asik, s. gonca, r. gasimli, s. yilmaz s€ usl€ uer, c ß . biray avci, c. g€ und€ uz department of medical biology, izmir, turkey a brain tumor is a collection, or mass, of abnormal cells in your brain. brain tumors can be cancerous (malignant) or non-cancerous (benign). the brain is one of the least accessible organ that active pharmacological compounds cannot be delivered. the two physiological barriers control and block the entry and exit of endogenous, exogenous compounds. one of these is the bloodbrain barrier and the other is the blood-cerebrospinal fluid barrier. this structures maintain protection of the brain. when there is a cancer case, it can lead to problem. astaxanthin with potent antioxidant properties can cross blood-brain barrier. in our study, we aimed to evaluate the effects of astaxanthin on apoptosis, cell cycle and also migration in brain cancer cell line. in present study, xcelligence real-time cell analyzer was used so as to determine cytotoxic effect of astaxanthin in u87 cell line. changes of apoptosis and cell cycle in u87 cell line exposured to ic 50 dose of astaxanthin (19.5 nm-80 lm) are detected with annexin v-egfp apoptosis detection kit and cycle test plus dna reagent kit with facs, respectively. the result of apoptosis and cell cycle test was analysed in flow cytometry. the group to which active substance was not treated was used as controlled. the wound healing assay performed in order to measure migration ability of u87 cell line to which astaxanthin was treated or not. ic 50 dose of astaxanthin was calculated as 9.20 lm at 48 h by xcelligence rtca sp based on time and dose. astaxanthin decreased the migration ability at rate of 25% in u87 cells treated by ic 50 dose of astaxanthin. astaxanthin had no apoptotic effect on viability in u87 cell line and astaxanthin caused an increase of g2/m phase arrest (1.15 fold) and s phase arrest (1.41 fold). astaxanthin has cytotoxic effects in brain cancer. it determined that astaxantin decreases cell cycle potential at g2/m even a little. the effect of anticancer of astaxanthin should be researched further. interferon regulatory factor 4 (irf4) is a critical transcription factor in development and survival of different cell types including immune cells and melanocytes. furthermore, it has been demonstrated that irf4 expression levels are elevated in several lymphoid cancers, and irf4 is one of the key transcription factors for the survival of these cancers. several genome-wide association studies identified irf4-linked genetic variants to increased melanoma incidence. in addition results from our lab and elsewhere have shown high levels of irf4 expression in melanoma cell lines. furthermore our preliminary results suggest melanoma cells are sensitive to irf4 expression levels. however, there are no published studies about irf4 target genes in melanoma cells. in this study, we are investigating the genome-wide target genes of irf4 in melanoma cell lines via high-throughput sequencing of immunoprecipitated chromatin (chip-seq). we have identified possible irf4 binding regions in loci with known key roles in development of melanocytes from neural-crest cells. one such key factor is mitf, which is the master regulator in melanocyte development and also plays critical roles in melanoma. integrating chip-seq and rna-seq data suggests irf4 as a transcriptional regulator of genes related to progression of melanoma. objectives: aim of this study was to evaluate prognostic importance of selected laboratory parameters (c-reactive protein (crp), gama glutamiltransferaz, ferritin (fer), potassium, chloride, calcium, phosphorus, magnesium, total protein, aspartat aminotransferaz, alanin aminotransferaz (alt), ifn-c, il-6, tnf-a) in non-small cell lung cancer (nsclc). material and methods: 20 patients with nsclc who were treated with chemoradiotherapy (crt) prospectively evaluated. all patients were newly diagnosed tumour. heparinized blood samples were taken from the patients before and after the completion of crt. fer analyzed by chemiluminescence method on beckman coulter dxi 800; ifn-c, il-6, tnf-a were analyzed with elisa kits (boster biological technology) and other biochemical parameters analyzed on abbott architect c16000. post-crt and pre-crt levels compared with survival. results: the lr cox regression analysis revealed that pre-crt ferritin was significantly associated with survival of patients with nsclc (hazard ratio (hr) = 1.007, p = 0.023, 95%ci; 1.001-1.013). it was also demonstrated by lr cox regression analysis, high levels of pre-crt crp was associated with worse outcome of patients (hr = 1.017, 95%ci;1.001-1.032, p = 0.035). after crt, mean alt level was determined as 16.71. there was survival difference in nsclc patients with high post-crt alt (hr = 0.912, 95%ci; 0.841-0.990, p = 0.027). conclusions: there exists a clinically relevant relationship between pre-crt fer concentration and the prognosis of survival in patients with nsclc. elevated fer is the result of inflammation rather than body iron overload. ferritin showed negative correlation with survival so it could be a useful biomarker to indicate bad prognosis of the patients with nsclc. additionally, crp which is easy to detect and feasible for the use in the routine clinical practice should be considered in the prognosis of nsclc patients. keywords: ferritin, nonsmall cell lung cancer, survival, c-reactive protein. epigenetic therapy tries to reverse the aberrations followed to the disruption of the balance of the epigenetic signaling ways through the use of natural and synthetic compounds, active on specific targets, such as dna methyltransferases (dnmts). we previously synthesized some benzoxazole and benzamide derivatives which might have anticancer activities on account of their heterocyclic structure. our studies showed that not only these compounds caused selective cytotoxicity towards cancer cells (hela) with little or no toxicity on normal cells (l929) but also were not genotoxic. in this study, we aimed to test whether these compounds changed global demethylation profile of normal and cancer cells. we used methylation specific comet assay (msc assay) to determine global methylation levels of cells. cells were treated with the tested compounds at ic 50 concentrations for 48 h. slides were prepared as did in alkaline comet assay, then they were incubated with methylation specific restriction enzymes (mspi, hpaii) before electrophoresis. differences in global methylation levels between nontreated control cells and cells treated with compounds were compared by using tail moment data. 5-aza-c, a demethylating agent, was used as reference drug. msc assay results revealed that none of the tested 9 compounds caused hypermethylation on both cell lines. however, global methylation levels decreased statistically (p < 0.05) through both cells treated with c-2 and c-8. only c-3 decreased methylation level on l929 but not on hela. consequently, c-2 and c-8 caused demethylation on hela cells similarly with 5-aza-c at low concentrations. for the reason that dna methylation is regulated mainly dnmt enzymes in the cell, c-2 and c-8 might cause global demethylation in the cell by inhibiting dnmt activity. further studies will be done to support this prediction. overall, macrophages and some subtypes of lymphoid cells are found in tumour stroma. these cells secrete a variety of growth factors, proinflammatory cytokines and chemokines, esp. tnf-a, il-1b and il-6, causing the formation of inflammatory microenvironment around tumour cells. tnf-a and il-1b signaling increases activity of nf-kb pathway. at the same time, il-6, triggers jak-stat signaling pathway, which effector is stat3. nf-kb and stat3 activity facilitates hyperexpression of mir-nas mir-155, mir-181 and mir-21 as well as down-regulates expression of mirnas mir-15/16, mir-199 and let-7. this investigation aims to identify in what way these shifts in mirnaome can lead to epigenome reorganization supporting the cell transformation. mirna targets within gene transcripts were predicted in silico using targetscan software. transcripts of hdac2/4/8/9 and sirt1/5 genes encoding histone deacetylases carry targets for at least one of up-regulated mirnas mir-155, mir-181 or mir-21. also, these mirnas can silence ezh1, mll, mll3, nsd1, setd6/7/8, smyd1, suv39h2 genes encoding histone methyltransferases. mirna mir-21 suppresses gene encoding de novo dna methyltransferase dnmt3b. at the same time, down-regulation of mirna mir-15/16 can allow hyperexpression of gene encoding acetyltransferase elp3. these shifts impair dna and histone methylation, cause the increase of overall level of chromatin acetylation and expression and, therefore, create epigenetic background for reactivation of silent transposons, oncogenes as well as other genes important for cell transformation. immune system can paradoxically facilitate the tumour growth instead of healing. cancer-related inflammation leads to the mir-naome and epigenome shifts contributing to the tumour promotion and progression. lysine acetylation is one of the key mechanisms to regulate chromatin structure and transcriptional activation. acetyl-lysine modifications are recognized by bromodomains, which are small interaction modules found on diverse proteins including histones. among these acetyl-lysine reader proteins is the family of the bet (bromodomain and extra-terminal) proteins which contain tandem bromodomains (bd1 and bd2). the recent discovery of potent and specific inhibitors for the bet family proteins has stimulated intensive research activity in diverse therapeutic areas, especially in oncology, where bet proteins regulate the expression of key oncogenes and anti-apoptootic proteins. several bet inhibitors are currently in clinical trials and reported to exhibit promising clinical activities. however, pleiotropic nature of bet proteins regulating tissue-specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. here, we report the recent progress in the development of bet inhibitors in korea research institute of chemical technology (krict). we have identified the bet inhibitors with a novel scaffold different from the previously reported diazepine and azepine scaffolds and specific for first bromodomains (bd1s). a medicinal chemistry effort is currently made to optimize the pharmacokinetic properties of these lead compounds for further drug development. the experimental data from the biochemical and cell-based assays for these bd1-selective bet inhibitors will be presented. family of small c-terminal domain serine phosphatases (scp), which includes ctdspl, ctdsp1, and ctdsp2, plays a regulatory role in a number of vital processes. in particular, it is shown that ctdspl is capable to activate the retinoblastoma protein (rb) which is well-known tumor suppressor and one of the key cell cycle regulators. although the question on whether ctdsp1 and ctdsp2 dephosphorylate rb is open, high similarity of sequences and three-dimensional structures of phosphatases may indicate the similar function of these enzymes. in the current study expression of scp genes was evaluated by quantitative pcr in 28 non-small cell lung cancer (nsclc) samples. using original crosshub software, that combines an analysis of high-throughput sequencing data of the cancer genome atlas project (tcga) and databases of mrna-mirna interactions (targetscan, mirtarbase, etc), the involvement of mir-183-96-182 microrna cluster in co-regulation of ctdspl/1/2 genes in nsclc was predicted. the significant (5-fold on the average) and simultaneous decrease of mrna levels of ctdspl/1/2 genes was revealed in the majority of nsclc samples (82%, 23/28). such unidirectional expression change and strong positive correlation between phosphatase expression levels (r s = 0.53-0.62, p ≤ 0.01) allowed us to suggest a common mechanism of their inactivation. we evaluated the expression of predicted co-regulators of scp gene expression, mir-183-96-182 family, in examined nsclc samples. as a result, the simultaneously increased levels of all three mir-nas in most nsclc samples (82%, 23/28) and negative correlation with phosphatase gene expression was shown. the results suggest the ability of investigated phosphatases to exhibit tumor-suppressive activity and the involvement of mir-183-96-182 micrornas in the regulation of rb protein activity via inactivation of ctdspl/1/2 in nsclc. cancer is one of the leading causes of death in all around the world. cancer is defined as a disease involving abnormal cell growth with the potential to invade or spread to other parts of the body. tumor markers are substances that are produced either directly by the tumor or as an effect of the tumor on healthy tissue. tumor markers can be used for screening, determining prognosis and monitoring effectiveness of therapy and disease recurrence. the aim of this study is to investigate the frequency of tumor markers orders and the appropriateness of these requests. laboratory information systems data for 2015 were reviewed. for 2015, a total of 35874 patients and 55539 tumor marker requests were included. carbohydrate antigen 19-9, cancer antigen 125, cancer antigen 15-3, prostate specific antigen, alphafetoprotein and carcinoembryonic antigen were measured by chemiluminescence method. according to the data from the year of 2015, both positive tumor markertest resultsratio and the positive patient ratio were 14%. in the patients group with increased marker levels, 48% of the patients had no history of cancer. in the patients group with tumor marker levels in referenceranges, 40% patients with diagnosed cancer history in remission. the ratios of positive tumor markers were 13% forca 19-9, 19% for ca 125, 7.9% for psa, 25%for ca 15-3, 8.4%for afp, and 9.56% for cea. in conclusion; unnecessary test requests increase laboratory work load and health expenses. laboratory and clinical staff collaboration is crucial to increase the appropriate use of tumor markers. dna methylation is an epigenetic modification that is involved in both normal biological and disease states. hypermethylation of promoter regions of tumor suppressor genes have a role in tumor development. therefore, the measurement of promoter methylation of genes can be used for diagnosis and prognosis purposes of cancer. to detect dna methylation alterations in a sample (biopsy, blood, saliva, etc.), sensitive detection systems and optimization of the methods are needed. as a part of a collaboration project between national metrology institute of korea (kriss) and national metrology institute of turkiye (tubitak ume). dna methylation status of apc and gstp1 genes were studied. dna methylation measurements were performed using stepone real-time pcr system and results were analyzed using hrm (high resolution melting) software. the parameters effecting the quantification of dna methylation were found as primers, annealing temperature, pcr cycle number, fluorescence dye and the commercial dna methylation standards used for quantification of dna methylation. since, the accurate measurement of dna methylation is very critical in early diagnosis of cancer and choosing the right therapy, optimization of the method is required. cancer is a disease that includes heterogenic and complex molecular changes. anti-carcinogenic effects of resveratrol, a natural polyphenol, have been proved in a variety of cancer cells. considering the effects of resveratrol, the influence of the signal transduction pathways in the presence or absence of p53 of colon cancer cells is gaining importance. our aim was to investigate the effects of resveratrol in the presence or absence of p53 on cell viability, apoptotic cell death ratio and fold changes of proliferative or anti-proliferative gene expressions, which may have important effects on colon cancer, in hct116 colon carcinoma cells. ic50 doses of resveratrol were determined by wst-1 assay. the apoptotic cell death ratios in treatments of resveratrol were determined by annexin-v-fitc/pi assay for flow cytomety . the changes of ccnd1, fra1, ppard, egfr, birc5, pcna, mcl1, stat3, fos, jun, p27, atf4, trail, puma, gadd45a, rb1, faslg, tnf, socs3 , stat1 gene expressions were evaluated by real time pcr. all data were statistically analyzed by student's t test. our research has revealed that resveratrol (60 lm) causes decrease in cell viability and increase in apoptotic cell death in hct116 p53(+/+) and hct116 p53(à/à) cells significantly (p < 0.05). the fold changes of the gene expressions have shown that resveratrol has significant (p < 0.05) and different effects on the expressions of the genes related with the existence of p53 in hct116 cell lines. therefore we proposed that resveratrol might show proliferative or apoptotic effects related with p53 mutation of colon cancer cells and we predicted that unconscious consumption of resveratrol in colon cancer patient might cause adverse effects. introduction: colorectal cancer (crc) is the third most common cancer worldwide. alterations in methylation profiles of tumor suppressor genes (tsgs) have been recognized as a key mechanism in colorectal cancers. in the current study, we investigated the hypermethylation status of 25 tsgs in colorectal cancer tissues. materials and methods: formalin-fixed paraffin-embedded (ffpe) tissue samples obtained from patients with crc. methylation specific-multiplex ligation dependent probe amplification (ms-mlpa) technique was used to assess the methylation status of 25 tsgs. the findings were evaluated in terms of age, mortality, survival, positive lymph node status, lymphovascular invasion, and perineural invasion. results: hypermethylation-detected patients and hypermethylation-undetected patients were called as group 1 and group 2, respectively. hypermethylation was detected in atm, cdkn2a, and gata5 genes. mortality rate was (12.5%) in group 1 and group 2 (p > 0.05). mean 5-years survival rate in group 1 was 45 ae 3 months and mean 5-years survival in group 2 was 44 ae 3 months (p > 0.05). positive median lymph node count was 10 ae 2 for group 1 and 5 ae 1 for group 2 and the difference was statistically significant (p < 0.05). frequencies of perineural invasion and lymphovascular invasion rate in two groups were 25% (p > 0.05). discussion and conclusion: our findings suggest that tsg hypermethylation found in crc patients may increase the lymph node metastasis. further investigations with larger sample size are required to support our results. boron (b) is known to be important for cell replication and development, but the underlying mechanism remains obscure. recently b has also become important in some specific anticancer processes. some recent reports advise using of some boron compounds for the treatment of specific forms of cancer. for instance, boron-based drugs (bortezomib) are now being developed for use as therapeutic agents with anticancer activities and several other boron-based compounds are in various phases of clinical trials. it has been shown that bortezomib disrupts the regulation of cell cycle and induces apoptosis in both hematologic and solid tumor malignancies except for colon carcinoma. colorectal cancer (crc) is the third most common cancer in men and the second in women, accounting for 10% of all tumour types worldwide. cytotoxic effects of boron compounds on crc cells and changing of its effects related with p53 mutation, which is mutated 50% of cancer cases, have not take part in literature yet. for this purpose; the aim of the study was designed to investigate the effects of borax pentahydrate and disodium pentaborate decahydrate compounds on cell viability, apoptotic cell death ratio and parp protein expressions in p53 (+/+) and p53 (à/à) hct116 colon carcinoma cells lines. the effects of the boron compounds on cell viability were assessed by xtt assay and apoptotic effects and parp protein expression of the compounds were evaluated by flow cytometry and western blot analysis respectively. our results showed that borax pentahydrate (6 mm) and disodium pentaborate decahydrate (12 mm) significantly causes nearly 50% reduction of cell viability at 48 h (p < 0.05). apoptotic cell death ratios and parp expressions revealed that both of the compounds might have a potential for a candidate of anticancer agent. epithelial-mesenchymal transition (emt) is a significant event for metastasis, and could be mediated by several pathways such as pi3k/akt, map kinases and many epigenetic regulators. satb2 is an epigenetic regulator involved in emt and osteoblastic differentiation. since preliminary results indicate that there is a crosstalk between p38 and akt pathways in nsclc cells, we aimed to determine whether this crosstalk has a regulatory effects on emt and satb2 expression in nsclc cells. we used a549 and h1650 cells as a model to evaluate the effects of the crosstalk between p38 and akt on emt of nsclc cells. therefore, cell culture, inhibition of p38 activation via sb203580, transient expression assay for (ca-akt), western blot analysis, sirna transfection for satb2, wound healing and invasion assay were performed in this study. firstly, the expression statues of e-cadherin, satb2, p-p38, p38, p-akt and akt was examined in a549 and h1650 cells by western blot analysis. we observed that e-cadherin and satb2 are downregulated in a549 cells (highly active p38, lowly active akt) compared to h1650 cells (lowly active p38, highly active akt), suggesting that e-cadherin and satb2 are associated with the crosstalk between p38 and akt pathways. our results demonstrated that p38 inhibition in a549 cells leads to decreased pten expression and subsequently increased akt activation. then, we found that p38 inhibition upregulated satb2 expression, and reversed emt in a549 cells. furthermore, alone satb2 knockdown is sufficient to induce emt, and prevented the effects of p38 inhibition on emt. all these results strongly indicate that the crosstalk between p38 and akt pathways might determine satb2 expression and epithelial characters of nsclc cells, and satb2 is a critical epigenetic regulator for emt in nsclc cells. therefore, it is also need to explore how p38 and akt signalling pathways could regulate satb2 expression. this work was supported by tubitak (114s007, 215z283). introduction: lung cancer is a disease characterized by uncontrolled cell growth in the lung tissues. the most common causes of lung cancer are tobacco smoke, radon gas, asbestos, air pollution, and genetic factors. nitric oxide (no) has potential mutagenic and carcinogenic activity and may play important roles in lung cancer. endothelial no, synthesized from l-arginine by endothelial no synthase (enos), inhibits apoptosis and promotes angiogenesis and tumor cell proliferation. the aim of the present study was to examine the possible relationship between enos gene intron 4 vntr and exon 7-g894t (glu298asp) the stressful ecosystems exert strong adaptive pressure and proteins that facilitate these adaptation processes are candidate drug targets. nucleotides are the core of biochemical pathway required for cancer cell growth and replication and genetic changes will lead in oscillation in their pools. although it is questionable whether the warburg effect actually causes cancer, impairing dglucose uptake and metabolism induces oxidative metabolism. lproline (lproline) homeostasis is critical in a constellation of human diseases, in parametabolic linkage between cancer, epigenetics (phang et al. 2013 ) and bioenergetics (pallotta 2013) where degradation and biosynthesis are robustly affected by oncogenes or suppressor genes that can modulate intermediates involved in epigenetic regulation. lproline-fueled mitochondrial metabolism involves the oxidative conversion to l-glutamate by a flavin dependent lproline dehydrogenase/oxidase and a nad +dependent l-d 1 -pyrroline-5-carboxylate dehydrogenase. in saccharomyces cerevisiae an important test tube, put1p and put2p respectively help cells to respond to changes in the nutritional microenvironment by initiating lproline breakdown after mitochondrial uptake (pallotta 2014) . in this preclinical study, low molecular weight compounds were tested for inhibiting lproline mitochondrial transport and put1p/put2p catalytic activities. thus, in seeking for natural bioactive compounds targeting lproline pathway and its substrate channeling (becker's group 2016), we report data using in silico screening and in vitro researches in saccharomyces cerevisiae with genetic background atcc18790 but different phenotypic landscape induced by nutritional stress/ ph changes. cells vitality, dψ measurements, nad(p) + /nad(p) h pool and flavine turnover were determined in spectrofluorimeter microplater reader and via hplc (pallotta et al. 1998 (pallotta et al. , 1999 (pallotta et al. , 2004 pallotta 2011; di martino pallotta 2011) thus in supporting of future cancer therapies with decreasing side effects. evaluation of lymphocyte to monocyte ratio (lmr) in patients with colorectal cancer introduction: inflammation may play an important role in cancer progression and a high neutrophil to lymphocyte ratio (nlr) has been reported to be a poor prognostic indicator in several malignancies. the aim of this retrospective study was to evaluate the prognostic value of nlr, lymphocyte to monocyte ratio (lmr) and platelet to lymphocyte ratio (plr) in patients with colorectal cancer (crc). : 107 patients who were diagnosed with colorectal cancer between january 2010 and january 2016; were evaluated retrospectively. the cutoff value was determined using receiver operating characteristics curve analysis. survival analysis was performed using the kaplan-meier method and log-rank test. the cox proportional hazard model was used to identify the influence of factors related to survival. (tnm stage, tumor differentiation, age, tumour size and lmr) results: receiver operating characteristic curves showed that lmr was superior to plr and nlr as a predictive factor in patients with colorectal cancer. the cutoff value for lmr was 1.82. cancer-specific survival was not significantly different between the high-and low-lmr groups (p = 0.786). age was identified as independent prognostic factor in colorectal cancer (hazard ratio: 3.50; 95% confidence interval: 3.14-351.82; p = 0.004). discussion and conclusion: our preliminary study showed that the lmr was not an independent prognostic factor in crc patients, but additional large sample sized prospective studies will be needed to confirm these findings. the aim of this study is to investigate the effects of luteolin treatment on enzymatic activity of arginase, and ornithine and polyamine levels (putrescine, spermidine spermine) in serum and cancer tissues of ehrlich ascites breast cancer model. balb/c female mice were divided randomly into following groups: healthy control, healthy treatment, cancer control, treatment 1 and treatment 2. 0.2 ml ehrlich ascites tumor cells was inoculated subcutaneously to medial part of left hind leg. healthy treatment and treatment 1 groups, and the treatment group 2 were given 5 mg/kg and 10 mg/kg dose of luteolin, intraperitoneally, for a 12 days period, respectively. luteolin has a hydroxylated flavonoid structure and shows potent antioxidant, anti-inflammatory, and anticarcinogenic properties. luteolin not only leads to cell death in various tumors by suppressing cell survival pathways and stimulating apoptotic pathways, but also sensitize them to cytotoxic therapy. supporting various previous studies, tumor implantation to healthy mice resulted in statistically significant elevation of serum arginase and polyamine levels (p < 0.05) indicating the tumor cells as the main source of this production. furthermore, luteolin treatment abolished this increase in serum arginase and polyamine levels (p < 0.05). tissue measurements of arginase and polyamine levels indicated that luteolin treatment resulted with an increase in these parameters of tumor tissue while the serum levels of them showed a significant decrease. our results revealed that increased tissue arginase and polyamine levels might be related with estrogenic agonistic effect of luteolin on utilized tumor model in this experiment; and decreased serum levels of these parameters while there is a significant increase of them in tissue levels might be a result of a suppression of polyamine efflux from the tumor tissue by inhibitory effect of luteolin on plasma membrane polyamine transporters. hepatocellular carcinoma (hcc) is the third most common cause of cancer-related deaths. around 30-40% of hcc patients are diagnosed at an early stage of the disease. hepatic resection, liver transplantation are common strategies in hcc treatment. even if, most of the patients present advanced-stage tumors and have a restricted survival rate. for the reason, resistance against existing tumor stress conditions have been demonstrated in hcc. hypoxia, hyperglycemia are general stress sources in hcc and result in aggressive cell phenotype, resistance to apoptosis and therapeutic drugs. thioredoxin interacting protein (txnip) regulates cellular responses under stress conditions. over-expression of txnip results activation of oxidative stress and apoptosis. in cancer models txnip is considered as a tumor suppressor gene. however, its role in the development, progression of hcc and mechanisms behind it warrant further investigation. in this study expression levels of txnip were examined in 12 hcc cell lines by rt-pcr and western blotting. txnip expression was significantly high in poorly-differentiated snu-182, snu-387 and snu-423 than the well-differentiated hcc cell lines such as huh-7, hepg2 and plc/prf/5. besides, expression of txnip was examined in 16 non-hcc and 79 hcc tissue samples by immunohistochemical staining. txnip positivity was observed in 40% of well and 80% of poor differantiated hcc tissues. however, no txnip positivity was observed in non-hcc tissues. to investigate whether txnip might be involved in biological responses such as cell proliferation, motility and invasion, we used overexpression and silencing strategies. overexpression of txnip minimally inhibited adhesion and proliferation, whereas boyden-chamber motility and invasion assay showed that invasiveness of cells were increased. our findings suggest that txnip expression is increased in hcc and txnip over-expression is important for invasive phenotype during hepatocarcinogenesis. cardiovascular diseases are the leading cause of morbidity and mortality in the western world. it was shown that ischemic tolerance of the heart can be enhanced not only by ischemic or pharmacological conditioning (pre-and postconditioning), but also by adaptation to chronic hypoxia. different studies have indicated that these cardioprotective phenomena may at least partly share the same signaling pathways. the jak/stat signaling pathway has been demonstrated to participate in the development of cardioprotection by conditiong apparently through the inhibition of gsk-3b. the aim of our present study was to determine whether this pathway also takes part in cardioprotection induced by adaptation to chronic hypoxia. we investigated the effect of inhibitor of jak2 kinase (ag-490) on myocardial infarct size and the jak2/stat3 signalling pathway and other effector molecules that may participate in cardioprotection conferred by adaptation to hypoxia. adult male rats were adapted to intermittent normobaric hypoxia (10% o 2 , 3 weeks, 8 h/day) and part of them recieved ag-490 (3 mg/kg) 15 min before ischemia. control rats were kept under normoxia. infarct size was assessed in isolated perfused hearts. relative expression of the key components of the jak2/stat3 signalling system and other proteins was detected using western blotting. preliminary data indicate that administration of the jak2 inhibitor ag-490 caused a significant increase in infarct size in hypoxic rats. western blot analysis revealed changes in phosphorylation of jak2, stat3 and some other proteins involved in cardioprotection (akt, erk1/2, gsk3b). these results suggest that the jak/stat signaling pathway could participate in the development of a cardioprotective phenotype in rats exposed to chronic hypoxia. however, further research will be needed to clarify in more detail the role of this signalling pathway in the cardioprotective mechanism. p-06.01.2-002 detrimental effect of hypertension on myocardium was reversed by liver x receptor agonist gw3965 hypertension is a cardiovascular disease that causes functional and structural changes in the heart. nuclear liver x receptors (lxrs) are involved in the control of cholesterol and lipid metabolism. however, effect of lxr activation on the hypertensive heart is not well characterized. in this study, the effects of lxr agonist gw3965 on hypertension-induced damage of myocardium were investigated. hypertension was induced by deoxycorticosterone acetate (doca) injection (20 mg/kg, twice a week) following the unilateral nephrectomy in male 8-week-old wistar albino rats for 6 weeks. blood pressure was measured by using tail-cuff method. gw3965 (10 mg/kg/day, i.p.) was administered last 1 week. expression of various markers (grp78, perk, p-perk, ikb-a, nf-kb p65, tnf-a, bax, bcl-2, mmp-2) in the ventricular tissue were examined by western blotting. inflammation and fibrosis were evaluated in histopathological examination. gw3965 treatment reduced systolic blood pressure of hypertensive animals. expressions of endoplasmic reticulum stress markers grp78 and p-perk were increased by hypertension and gw3965 treatment reversed them. hypertension-induced increase in nuclear nf-kb p65 expression and decrease in ikb-a expression were reversed by gw3965 treatment. while bcl-2 expression was lower, bax level was higher than control in the hypertensive animals. in hypertensive group, fibrosis marker mmp-2 expression was augmented and gw3965 treatment reversed this elevation. hypertension-induced increase in interstitial and perivascular collagen deposition and inflammatory cell infiltration in left ventricle were prevented by gw3965 treatment. these results suggest that lxr activation by gw3965 restored the hypertension-induced structural changes of heart in the doca-salt hypertension. methylphenidate (mph) is a psychostimulant prescribed for the treatment of attention deficit hyperactivity disorder (adhd), one of the most common neurobehavioral disorders of childhood and adolescence. in fact, despite the widespread use of mph the full comprehension of its cellular/molecular mechanisms is still elusive, including its effect on blood-brain barrier (bbb). this barrier is a key structure in the central nervous system since it protects the brain and its dysfunction has been described as a critical event in several brain diseases. thus, the aim of the present study was to clarify the effects of mph on the bbb function in both physiological and adhd conditions. for that, we used a rat model of adhd, the spontaneously hypertensive (shr) rats, and wistar kyoto (wky) animals as inbred comparator strain. also, to mimic a clinical dosing schedule for adhd treatment, rats were administered for monday-friday with vehicle or mph (1.5 mg/kg/day or 5 mg/ kg/day, per os) from p28-p55. chronic mph treatment (5 mg/kg/day) promoted cortical bbb permeability in both wky and shr animals; however, more prominent in wky rats. this effect can be explained by the downregulation of claudin-5 and collagen-iv, tight junction and basal lamina protein, respectively. noteworthy, wky animals also showed an increase in the expression of caveolin-1 and in both vascular cell and intercelular adhesion molecules. these bbb alterations led to subsequent infiltration of peripheral immune cells, including cd169 + macrophages. furthermore, hippocampal bbb disruption was only observed in wky rats with 5 mg/kg of mph. here, mph decreased collagen iv expression and upregulated caveolin-1, with no alterations in claudin-5. overall, our results show that chronic exposure to mph can led to brain vascular alterations particularly under physiological conditions. this highlights the importance of an appropriate mph dose regimen for adhd, and also that mph misuse can have a negative effect. regulators of g proteins signaling (rgs) serve several cellular functions varying from tolerance, dependence, neuroprotection, transcription and tumorgenesis. despite their initial role as gtpase activating proteins, evidence suggests that rgs proteins are localized in the nucleus, interact with transcription factors thus regulating transcriptional responses. it was shown that rgs4 directly interacts with and interferes in opioid receptor (or) signaling. rgs4 is mostly expressed in brain and is implicated with brain structural alterations; however, the molecular mechanisms of how rgs4 could be involved in cellular differential functions remains unclear. based on these observations we examined whether rgs4 can regulate transcriptional responses mediated by the stat5b transcription factor. isolated neural stem cells from rgs4 à/à mice were immunostained for the mitosis marker ph3 and the mrna levels of antiapoptotic genes were determined. proliferation assays were performed with brdu staining in neuro2a cells stably expressing rgs4. the functional assays of stat5b transcriptional activation were performed in hek293 expressing either the erythropoietin receptor (epo-r) or the delta opioid receptor (d-or). the present data demonstrate that rgs4 blocks stat5b phosphorylation and transcriptional activation by interfering in stat5b heterodimerization upon epo-r or d-or activation triggered by cytokines or opioids administration. lack of functional rgs4 results in increased mrna levels of stat5b target genes such as the members of the bcl anti-apoptotic family bcl-2, bcl-6 and bcl-xl. this upregulation of stat5b inducible gene transcription results in an increased proliferation rate of neural stem cells. this study demonstrates for the first time a non-canonical function of rgs4 in stat5b mediated transcriptional responses and a novel selective role of rgs4 in transcription. role of the pre-molten globule structure in amyloid fibril formation a. eshaghi department of biology, faculty of science, islamic azad university, mashhad branch, mashhad, iran the major factor that caused extensive research on the protein fibrillation is their crucial roles in important diseases known as the amyloidosis diseases. neurodegenerative diseases, including alzheimer's, parkinson's, diabetes and huntington are the most important types of this disease. understanding the mechanisms of fibril formation and ways of treatment can be useful in reducing this type of disorder. in this project, the fibrillation of carbonic anhydrase protein was investigated as a model. carbonic anhydrase creates two stable intermediated known as pre-molten and molten globule, in different ph solution. this protein at ph between ph 3-4 molten globule structures was formed while the pre-molten form took place under ph 3. in our tests at ph 3.5 when the protein in molten globule structures only the amorphous aggregates were formed. instead, at ph 2.4 in pre-molten globule structure amyloid fibrils formed in the protein. there some reports, indicated the protein from pre-molten globule structure go toward amyloid assembly. even intrinsically unstructured proteins such as alpha-synuclein first took a structure similar to pre-molten globule and then made amyloid fibrils. it seems pre-molten globule structure have the major role in promoting to amyloid fibrils. perhaps drugs that prevent the formation of premolten globule structure have an important role in inhibiting amyloid fibrils. identification of compounds preventing the biochemical changes that underlie the epileptogenesis process and understanding the mechanism of their action is of great importance. we have previously shown that myo-inositol (mi) daily treatment for 28 days prevents certain biochemical changes that are triggered by kainic acid (ka)-induced status epilepticus (se), [1, 2] . however in these studies we have not detected any effects of mi on the first day after se. in the presented study we broadened our research and focused on ka induced other early molecular and morphological changes and influence of mi treatment on these changes. the increase in the amount of voltage-dependent anionic channel-1 (vdac-1), mitochondrial-plasma membrane cofilin and caspase-3 activity was observed in the hippocampus of ka treated rats. administration of mi 4 h later after ka treatment abolishes these changes, whereas under the same time schedule diazepam treatment has no significant influence. the number of neuronal cells in ca1 and ca3 subfields of hippocampus is decreased after ka induced se and mi post-treatment significantly lessens this reduction. no significant changes are observed in the neocortex. obtained results indicate that mi post-treatment after ka induced se could successfully target the biochemical processes involved in apoptosis, reduces cell loss and can be successfully used in the future for translational research. references 1. r. 2010. neuroscience letters, vol. 468, no. 3, pp. 277-281. 2. r. solomonia, et al; 2013. cell. mol. neurobiol. vol. 33, no extracellular deposits of amyloid-b peptide (ab) in brain parenchyma via proteolytic processing of amyloid precursor protein (app) are one of the typical characteristics of alzheimer's disease (ad). these aggregates mainly occur as a result of an increase in ab production or a decrease in its degradation. it was found that the neurotoxicity of ab aggregates is accelerated by acetylcholinesterase (ache). besides, ab-ache complex has a prominent neurodegenerative effect in brain. thus, cholinesterase inhibition and preventing ab production are current treatment strategies for ad. recent studies have shown that methylene blue (metb), a cholinesterase inhibitor with phenothiazine structure, inhibits the formation of amyloid plaques and neurofibrillary tangles. azure b, the major metabolite of metb, has been shown to inhibit ache and bche with ic50 values of 0.486 lm and 1.99 lm, respectively. in the present study, we tested whether azure b, may effectively lower the levels of ab 40/42 . we treated chinese hamster ovary cells, which stably express human wild type app and presenilin-1 (ps70) with 0-15 mm azure b or vehicle for 24 h. to determine the effect of azure b treatment on ab 40/42 levels, we used separate sandwich-based elisas and normalized to total protein levels, determined by bca protein assay. azure b treated ps70 cells were also assessed by propidium iodide in flow cytometry for cellular toxicity. we observed a significant decrease in both extracellular ab 40 and ab 42 levels with a dose range treatment of azure b in ps70 cells. ab 40 levels were reduced by 89.2% in 10 lm and 94.1% in 15 lm azure b-treated cells when compared to control. additionally, ab 42 levels were decreased by 83% in 10 lm and 93.5% in 15 lm azure b-treated cells when compared to control. overall, these preliminary results suggest that azure b may have beneficial effects for the treatment of ad. the effect of green silver nanoparticles (agnps) on the amyloid formation in alphalactalbumin and chaperone action of alphacasein a. ghahghaei, m. dehvari, j. valizadeh formation and deposition of protein fibrillar aggregates in the tissues is associated with several neurodegenerative diseases such as alzheimer's and parkinson's disorders. molecular chaperones are a family of proteins that are believed to have ability for inhibiting protein aggregation. in the present study the effect of different concentrations of green synthesis silver nanoparticles (agnps) from pulicaria undulate l. on the amyloid formation of a-lactalbumin (a-la) and chaperone action of a-casein have been investigated. the effects of the agnps were determined using light scattering absorption, tht binding assay, intrinsic fluorescence assay, ans binding assay, cd spectroscopy and sds-page. light scattering and tht assay results showed that agnps have the ability in preventing aggregation of a-la in a concentration-dependent manner. consistent with these results, sds-page results represented that by increasing the concentration of agnps the adsorption and interaction between agnps and protein have increased. light scattering and tht assay results, also, revealed that the amyloid fibrilation decreased in the presence of both agnps and a s -casein compared to presence of a s -casein alone. fluorescence results, however, show that agnps have no effect on the chaperone ability of a-casein and in fact they perform their protection of protein aggregation action independently. consistent with the above experiments, cd spectroscopy also revealed that agnps have decreased structural changes in reduced a-la in absence and presence of a-casein, both the tertiary and the secondary structure of the proteins. our finding represented that agnps have preventing effects on protein aggregation and have no effect on the chaperone ability of a s -casein. in the main, results of this study show that biosynthesized agnps mediated by >pulicaria undulate l. maybe could be affective as a therapeutic agent for inhibiting aggregation in treatment of amyloidosis disorders. pink1 is a mitochondrial kinase with multiple cellular functions. while loss-of-function mutations of pink1 gene lead to early onset parkinson's disease, its over-expresion is associated with cancer development. parkinson is a multifactorial neurogenerative disease, with a complex aethiology including various cellular stressors. it is now known that genotoxic stress also triggers the release of soluble factors able to induce changes in neighboring cells enhancing the initial lesions, process known as bystander phenomena. althrough the mechanisms are still unclear, recent studies point towards a role for mitochondria in this process. our work investigates pink1 role in intracellular and intercellular stress response, comparatively in various models: fibroblasts (mefs) and neuroblastoma (sh-sy5y) used as a tumoral model or differentiated to a neuronal phenotype. pink1 role in this process was analyzed using genetically engineered pink1 deficient cells exposed to a genotoxic agent, bleomycin. the modified cell lines showed a reduced level of basal atp production. pink1 proved to be involved in cellular vulnerability to stress. despite differences in cellular sensibility between our models, genotoxic treatment of pink1 deficient cells induced consistently higher lesions compared to corresponding wild type variant. pink1 deficient cells showed altered intercellular signaling of stress, impairing bystander phenomena induction, by suppression of signal formation in treated cells, but also by altering the capacity to respond to the signals in neighboring cells. our hypothesis is that pink1 contributes to the management of cellular stress being involved in bystander transmission of detrimental effects through intercellular communication. this is determined mainly by its role in maintaining mitochondrial homeostasy and atp levels, pink1 deficient cells lacking the amount of energy required for rapid dna repair and stress signaling transmission. p-09.02.2-008 intranasal administration of synthetic fragments from receptor for advanced glycation end products prevents memory loss in olfactory bulbectomized mice the receptor for advanced glycation end products (rage) is a member of the immunoglobulin protein superfamily. activation of rage causes brain inflammation, oxidative stress and secretion of beta-amyloid that has been recognized as an essential phase in the development of alzheimer's disease. it is known that the receptor soluble isoform (srage) which lacks the transmembrane and cytosolic domains binds to ligands and prevents negative effects of the receptor activation in in vivo and in vitro experiments. we proposed that potential ligand-binding peptide fragments from srage would demonstrate the same biological activity. we have selected and synthesized 10 peptide fragments from unstructured surface-exposed regions of rage. synthetic peptides were intranasally administrated into olfactory bulbectomized (obe) mice with neurodegeneration of alzheimer's type. we have found that only administration of rage fragment (60-76) effectively prevents the obe murine memory from impairment, leads to decrease of beta-amyloid level and blocks the development of neuronal pathology in the brain of experimental mice. six overlapping fragments of rage (60-76) peptide were synthesized in order to find a site, responding for the therapeutic effect. tests in obe mice carried out with these fragments showed that only the n-terminal part of the molecule is responsible for preventing obe mice memory from impairment. all fragments which do not include n-terminal 60-61 dipeptide have been fully inactive in these experiments. we have proposed that active peptides can interact with beta-amyloid or s100b protein preventing these ligands from binding with rage. this interaction can inhibit the development of neurodegeneration. the aim of this study was to examine effects of social isolation, enriched environment and exercise on learning in rats. the study included 36 female 25 day old wistar rats. the rats were randomly divided into four different groups; control, exercise, social isolation and the enriched environment groups. the social isolation group and the enriched environment group were housed under their specific conditions and the exercise group and the control group were housed in standard conditions during 6 weeks. the rats in the exercise group swam for 6 weeks. after 6 weeks, the rats were evaluated in the morris water maze. brain and blood samples were taken and the hippocampus tissue was dissected. bdnf and ngf levels were measured in these samples. in conlusion, while enriched environment was a positive effect on spatial learning, social isolation was a negative effect on spatial learning and increase thigmotactic behaviors. according to the analysis results ngf and bdnf levels in the hippocampus and plasma did not change with environmental conditions and exercise. time of exposure to social isolation, procedures of the enriched environment, time of exposure to the environment, type and duration of exercise and gender may affect the results. alzheimer's disease (ad) was characterized by dementia that typically begins with subtle recognition failure and poor memory. it slowly becomes more severe and, eventually, incapacitating. the cholinergic system seemed particularly susceptible to synapse loss, especially in cortical regions associated with memory and executive function (1) . recent studies showed that the main cause of the loss of cognitive functions in ad patients was a continuous decline of the cholinergic neurotransmission in cortical and other regions of the human brain (2) . acetylcholinesterase (ache) and butyrylcholinesterase (bche) are hydrolytic enzymes that act on acetylcholine (ach) to terminate its actions in the synaptic cleft by cleaving the neurotransmitter to choline and acetate. both enzymes are present in the brain and detected in neurofibrillary tangles and neuritic plaques. it was suggested that ache predominates in the healthy brain, with bche considered to play aminor role in regulating brain ach levels. however, bche activity progressively increases in patients with ad, while ache activity remains unchanged or declines. both enzymes therefore represent legitimate therapeutic targets for ameliorating the cholinergic deficit considered to be responsible for the declines in cognitive, behavioral, and global functioning characteristics of ad (3). we initiated a study to screen their acetylcholinesterase (ache, ec 3.1.1.7) inhibitory activities, which are the key enzymes taking place in pathogenesis of ad. newly synthesized chiral benzimidazole derivatives with thioure structure showed ic 50 values in the range of 11.55-36.00 nm for ache. this study was financed by turkish research council-tubi-tak (kbag 115z837). p-09.02.2-012 f3-a13 h, novel fingolimod derivative, activates camp-dependent signalling pathway in sk-n-sh cell line g. celik turgut 1 , a. sen 1 , d. doyduk 2 , y. yildirir 2 1 department of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, 2 department of chemistry, faculty of sciences, gazi university, ankara, turkey fty720, a sphingosine 1-phosphate (s1p) receptor modulator, is the first oral disease-modifying therapy to be approved for the treatment of relapsing-remitting multiple sclerosis. in this study, we have synthesized and characterized novel derivative of fty720, namely f3-a13 h, and have determined its underlying camp regulation in sk-n-sh cell lines. for this purpose, we first determined the regulation of the camp response element (cre) activity and camp concentration by f3-a13 h along with fty720 using pgl4.29 luciferase reporter assay and camp immunoassay, respectively. then, we have determined their effect on camp/pka-related gene expression profiles using custom arrays along with fty720 treatment at non-toxic doses. it was found that f3-a13 h significantly activate cre and increase camp concentration in the sk-n-sh cell line, indicating that it activates camp pathway through cell surface receptors as fty720 does. furthermore, f3-a13 h modulates the expression of the pathway related genes that are important in camp signaling pathway. in summary, our data demonstrate that the novel fty720 derivative act as a modulator of camp ultimately by influencing the gene expression via the camp and downstream transcription factor cre pathway. in conclusion, f3-a13 h might contribute future therapies for multiple sclerosis. alzheimer disease (ad) results in memory impairment and accompanied by neuroinflammation, cholinergic deficit and amyloid-beta (ab 1-42 ) accumulation in brain. we found that bacterial lipopolysaccharide (lps) injections or mice immunizations with extracellular a7 nicotinic acetylcholine receptor (a7 1-208 nachr) domain resulted in astrogliosis, decrease of a7 nachr density, accumulation of ab 1-42 in brain and episodic memory impairment. the aim was to reveal main event triggering ad-like symptoms development. c57bl/6 mice were injected with lps, immunized with recombinant a7 1-208 or a7 1-208 endoglycosidase treated to remove carbohydrates. two immunizations with 3 week interval were performed. control mice obtained complete freund's adjuvant injections. mice were tested for memory performance, blood sera were examined for presence and fine specificity of a7 1-208 -specific antibodies and brain preparations were studied for a7 nachr, ab 1-42 and il-6 levels. the original a7 1-208 ('glyc') was more immunogenic than 'deglyc', and their epitopes were recognized with different efficiency. in contrast to lps and 'glyc' a7 1-208 immunization with 'deglyc' a7 1-208 did not stimulate il-6 elevation in brain and had no proinflammatory effect. immunizations with 'glyc' or 'deglyc' a7 resulted in similar a7 nachrs decrease and ab 1-42 accumulation in brain and significant episodic memory decline comparable to those after lps injections. a7 nachr interacts directly with amyloid-beta precursor protein and facilitates its proper processing and metabolism. our data indicate that decrease of a7 nachr density caused by a7 1-208 -specific antibody is critical for ab 1-42 accumulation and episodic memory impairment while pro-inflammatory capacity of a7 1-208 -specific antibody plays secondary role in ad-like symptoms development. in vitro antioxidant and antiacetylcholinesterase activity of achillea millefolium alzheimer diseases (ad) is a neurodegenerative condition without a current effective treatment. increase in reactive oxygen species and lipid peroxidation or decrease in total antioxidant capacity causes oxidative stress-induced tissue damage. it has been suggested that decrease in oxidative stress and inhibition of acetylcholinesterase (ache) activity play a major role in the prevention and slowing of cognitive symptoms of ad. recently, studies have been directed for the discovery of medicinal plants and natural substances that are known to have natural antioxidants. achillea millefolium (a. millefolium) is a traditional herbal medicine that contains natural compounds with antioxidant activity and has been used as a carminative, diuretic, menstrual regulator and wound healer, however the mechanism of its actions are unclear. the aim of our study was to investigate the effects of a. millefolium extracts on free radical production, acetylcholinesterase (ache) activity and lipid peroxidation in vitro. methanol (me) and ethanol (ee) extracts of a. millefolium were prepared to determine (a) in vitro antioxidant capacity (by using 2,2-diphenyl-1-picrylhydrazyl assay, radical scavenging activity, phosphomolibdenum-reducing antioxidant power, ferricreducing antioxidant power, and total phenolic-total flavonoid contents), (b) effects on ache kinetics (by using a colorimetric assay) and (c) effects on sodium nitroprusside-induced lipid peroxidation in mice brain homogenates. me showed a higher antioxidant activity compared with ee in the biochemical assays tested. similarly, me demonstrated significant inhibition of ache activity that was potent than ee. both extracts dose-dependently decreased malondialdehyde content in mice brain homogenates suggesting a strong inhibition of lipid peroxidation. these results showed that a. millefolium has a high antioxidant capacity and antiache activity, indicating a potential use as an adjuvant therapy in ad. b-cells are known to play a key role in multiple sclerosis (ms) progression and autoimmune response. cxcr5 is the main b-cell chemokine receptor that under normal conditions directs their migration to specific areas of secondary lymphoid organs. in ms, areas of demyelinating lesions have been reported to attract bcells due to overexpression of cxcl13, the cxcr5 ligand. we aimed to determine whether snp rs630923 located in the promoter of cxcr5 gene and associated with high risk of multiple sclerosis could have a direct effect on of cxcr5 promoter activity. mef2c binding to dna was assessed using pull-down assay. b-cell stimulation was performed using lps, pma and ionomycin. activities of variants of cxcr5 promoters containing different rs630923 alleles were estimated using luciferase reporter assay. we determined that minor rs630923 allele creates functional mef2c-binding site within one of the regions required for the basal activity of the cxcr5 promoter. cxcr5 promoter containing minor rs630923 variant that is statistically associated with low risk of ms showed significantly decreased activity in stimulated human b-lymphoblastoid cell lines. mef2c has been reported to play an essential role in b-cell survival and b-cell responses. we determined mef2c as the main regulator of rs630923-dependent modulation of cxcr5 promoter activity in b-lymphoblastoid cell lines. this link may be directly related to pathogenic b-cell activities in multiple sclerosis. introduction: parkinson's disease (pd) is the second most common neurodegenerative progressive brain disease with increasing prevalence in aging population. the etiopathogenesis involves many cellular procesess, but is not fully elucidated yet. treatment of pd is based on levodopa and dopamine agonists, but mao-b inhibitors, comt inhibitors, amantadine or anticholinergics may be used as initial monotherapy or as adjuvant therapy. treatment related adverse drug reactions (adrs) are frequent, but cannot be predicted and/or prevented. non-motor adrs, such as nausea, somnolence, hallucinations and hypotension are frequent in dopamine agonist therapy, while dyskinesias along with motor fluctuations are the most common late adrs with levodopa. the aim of our study is to combine clinical data with genetic and epigenetic biomarkers in the algorithm for personalized approach to pd management. materials and methods: we are planning a clinical study to assess the combined impact of selected clinical, genetic and epigenetic factors on the progression of pd, adrs and treatment response. our study will have a retrospective and prospective arm. we will collect peripheral blood samples of pd patients and clinical data. single nucleotide polymorphisms (snps) in the genes involved in dopamine, neurotransmitter and drug metabolism and transport, receptors and signalling pathways will be genotyped. snps within inflammatory, neurodevelopmental, antioxidative defense, synaptic transmission and immune response pathways will also be analysed. in the prospective arm we will isolate the exosomes and check their mirna content at the time of diagnosis and after the treatment initiation. the combined effects of clinical, genetic and epigenetic factors will be analyzed using lasso penalized regression analysis. conclusions: we hope to identify genetic and/or epigenetic biomarkers that may predict progression of pd, adrs and treatment response and may support personalized tratment of pd. most evidence indicates that g protein-coupled receptors form heteromers between them and with other receptors. by allosteric mechanisms, them acquire a multiplicity of unique pharmacological and functional properties. recently, we discovered that dopamine d1 receptors (d1r) and histamine h3 receptors (h3r) form heteromers through which h3r ligands can inhibit d1r function. d1rs also physically interact and modulate ionotropic glutamate nmda receptors (nmdar). in the present work, we investigated if nmdar, d1r and h3r form a heterotrimeric complex in brain. the heteromer expression was studied in slices from both rat and mouse brain cortex by co-immunoprecipitation (co-ip) and proximity ligation assays (pla). the ability of d1r and h3r to interact with nmdar in transfected hek cells was analyzed by bioluminescence resonance energy transfer (bret) with bimolecular fluorescence complementation (bifc) experiments. heteromer properties were studied by analyzing erk1/2 phosphorylation and cell death in cortical slices. endogenous d1r-h3r heteromers were detected in rat and mouse cortical slices, where h3r ligands decreased d1r signaling (erk1/2 pathway) and were also able to block the cell death induced by overstimulation of either d1r or nmdar. by bret experiments in transfected hek cells, we demonstrated that both d1r and h3r form heteromers with nmdar subunit 1a in the presence of subunit 2b. d1r-h3r-nmdar heteromers were detected by bret with bifc. endogenous d1r-h3r-nmdar heteromers were observed in rat and mouse cortex by pla. many systems, including the glutamatergic and dopaminergic, are involved in neurodegeneration. our innovative finding is that d1r, h3r and nmdar form heteromers that may be a point of intervention for cognitive disorders in neurodegeneration. d1r-h3r-nmdar heteromers are expressed in brain cortex and a complex interaction exists between protomers in the heteromer, where h3r ligands act as a 'molecular brake' for d1r and nmdar signaling. studies conducted on obesity and hfd (high fat diet) revealed hypothalamus have crucial roles on development of metabolic diseases. after chronic over nutrition or high fat diet, as a neurodegenerative condition, premise inflammation, neural stress and development of functional impairments are observed. these studies generally focused on changes in neurons, but it's effects on brain vessels are still unknown. in this study, as a neuronal damage infrastructure, changes in hypothalamic vascularity investigated. experiment initiated with 5 weeks old total 40 male wistar rats. in order to acquire obese phenotype, the rats were fed either cafeteria diet as hfd, or normal/chow (standard diet, sd) for 9 months. intravenous glucose tolerance tests performed before sacrification. animals were exposed for 10 s to co2 and then decapitation was performed with guillotine. isolated brains were directly immersed into liquid nitrogen and stored at à80°c. the hypothalamic sections were acquired with the cryostat instrument at different. immunofluorescence was performed on serial sections through the hypothalamus using the antibody smi-71 and cd31. changes in tight junction (tj) proteins (occluding and zone occludin-1) are evaluated via western blot (wb) analysis. the hfd-treated consumed significantly more food than did control animals, when examining average food consumption per day and rats that received the hfd diet weighted significantly more at the end of 9 month diet treatment. there were no differences acquired for glucose tolerance tests. however, after hfd treatment, wb analysis have shown that tj proteins decreased even if hypothalamic micro vessel number increased and smi-71 staining have shown that increased. our primary results have shown that hfd diet can affect hypothalamic vascularity and such changes might initiate neurodegenerations and functional impairments as observed in neuroretinal degeneration in relation to vasculopathy in diabetic patients. defects of mitochondrial trafficking are common problem in many neurodegenerative diseases. its dysregulation can contribute to changes in bioenergetics profile of the cell and can lead to cell death. in our study we investigate distribution of mitochondria and their transport in primary fibroblasts derived from patients with sporadic form of alzheimer's (ad) and parkinson's (pd) diseases. our data revealed that in the most cases the velocity of mitochondrial movement is lower in ad and pd cells in comparison to the control. the most intense differences between ad, pd patients and control group are observed in the case of movement of large mitochondria. owing to the fact that mitochondrial trafficking depends on mitochondrial state, we investigated the 'age' of mitochondria. we observed a diminished mitochondrial turnover in ad and pd fibroblast. evaluation of the mitochondrial distribution within the cell in all 3 groups (ad, pd and control) showed that in the perinuclear area are accumulated 'old' and 'worn out' mitochondria, probably dedicated to remove from the cell. because mitochondrial biogenesis, shape and size depends on fusion/fission proteins we assessed their level within the cell. to summarise, our results revealed alterations in mitochondrial trafficking in fibroblasts derived from patients with alzheimer's and parkinson's diseases in comparison to the healthy control cells. carbonic anhydrases (cas, ec 4.2.1.1) is a zinc metalloenzyme that catalyzes the reversible reactions of co 2 and water. carbonic anhydrases (cas, ec 4.2.1.1) form a family of metalloenzymes that play an important function in various physiological and pathological processes. therefore, many researchers work in this field in order to design and synthesize new drugs. carbonic anhydrase activators are important as much as inhibitors. caas have polar groups to make hydrogen bond in the main body and the activation property of enzyme increaase in this way. caas are have polar groups to make hydrogen bond in the main body and the activation property increaase in this way. furthermore, recent studies suggest that ca activation may provide a novel therapy for alzheimer's disease. in this study ca activators are determined. human carbonic anhydrase isozymes ca i and ca ii are isolated from human blood erythrocyte. hca-i and hca-ii isoenzymes were purified using sepharose-4b-l tyrosine-sulfanilamide affinity colum. finally, hca-i and hca-ii isoenzymes were eluted with appropriate elution buffers. enzyme purity was checked by sds-page. the enzyme activity system contained 0.05 m tris-so 4 ph 7.4, r-nitro phenol in 1 ml total volume. effects of some macrocyclic thiacrown ethers derivates were investigated. enzyme activities were measured at constant substrate and different activator concentrations to find ac 50 value. these compounds are thought to be useful for treating alzheimer's disease. introduction: gender differences in stress models are not studied in detail. we compared different stress conditions on brain bdnf levels, in social isolation (sit) and predator scent tests (pst) in rats. bdnf levels in cortex, hippocampus and amygdala were compared, effects of chronic fluoxetine (flu) treatment were evaluated. methods: 128 rats were used. for sit, animals were kept individually for 1 month and for pst, rats were exposed to dirty cat litter for 10 min at the first day of 1 month stress. flu was given (5 mg/kg, ip) through stress. controls, stress and treated groups were evaluated in elevated plus maze (epm), anxiety scores were calculated. brain bdnf levels were determined in cortex, hippocampus and amygdala by western blot. p < 0.05 were considered significant. results: sit and pst induced anxiety in both male and female rats, females having greater anxiety scores than males (p < 0.05). flu restored anxiety scores in both sexes (p < 0.01) in two settings. male and female rats exhibited reduced cortical bdnf levels in sit (p < 0.001). pst reduced cortical bdnf in females, but increased in males. hippocampal bdnf expression was lowered in sit (p < 0.01) and pst (p < 0.001) in both sexes. female rats had 40% lower bdnf expression than males in amygdala in sit. flu did not restore cortical bdnf in females in both tests, but reduced incresed bdnf levels in males (p < 0.001). flu did not restore reduced brain bdnf in males in hippocampus and amygdala, but restored in hippocampus, in females. discussion: our findings indicate that sex differences must be considered in studies related to mood disorders of animal models, and suggest that bdnf expression in different brain regions are altered differentially in a gender-dependent manner in rats. antianxiety effect of flu is not mediated through increasing bdnf activity in cortex in both genders. increased bdnf in hippocampus and amygdala may reflect its antidepressant effect in female rats, but not in males. perineuronal nets (pnns) are special forms of neural extracellular matrix found around neuron bodies and neurites. hyaluronan and proteoglycan link protein1 (hapln1) is one of the major elements of pnns. hapln1 interacts with tenascins and aggrecan which are other essential pnns components. in most of neurodegenerative disorders caused by neuritogenesis defects, disrupted pnns structure and decreased expression of hapln1 were observed. however, the role of hapln1 in neural differentiation is unknown. the aim of this study is to determine mrna and protein levels of hapln1 during differentiation using pc12 cell line as a neural differentation model, derived from rat pheochromocytoma. after pc12 cells were stimulated to differentiate into neurons by nerve growth factor on days 3, 5 and 7; cells were collected, qrt-pcr and western blot were performed. also, in order to find out whether there is a physical interaction between hapln1 and proteins related to neuritogenesis defects, spinal muscular atrophy (sma) was used as a neurodegenerative disease model. therefore, a detailed hapln1 and survival motor protein 1 (smn1) network analysis were performed in-silico. as a result, we analyzed 3 fold increase in hapln1 mrna level compared to undifferentiated state. on the other hand, a decrease in protein level was detected. this decrease in cellular hapln1 level suggests that, hapln1 is required for formation of pnns structure, thus secreted to extracellular environment at early stage of differentiation. in addition, according to in-silico analysis, an indirect path between hapln1 and smn1 through fibulin2 (fbln2) was detected. fbln2 was also found to be an interaction partner between different matrix molecules such as aggrecan and hapln1 which form a macromolecular meshwork. the results of this study will pave the way for investigating the role of hapln1 and fbln2 in neurodegenerative disease models. also it will help us to understand the mechanism of neuritogenesis defects. determination of properties of bone marrow and tissue-derived mesenchymal stromal/stem cell population in neurofibromatosis type 1 patients neurofibromas, complex tumors deriving from schwann cells and containing fibroblasts, vascular structures and mast cells, are part of the clinical picture in nf1. the risk of malignancy is increased in nf1, wound healing is delayed and keloid formation is frequent. because multiple tissues are involved in malignant and non-malignant manifestations of nf1, we considered the mesenchymal stem/stromal cells (msc) carrying the nf1 mutation might play a role in the microenvironment. mscs affect the biological behaviour of other cells: they alter their proliferation, apoptosis and migration through various secreted growth factors, cytokines, chemokines, or by direct contact. we examined the msc of nf1 patients. methods: the adipogenic and osteogenic differentiation potential of mscs from nf1 and healthy subjects was examined in vitro and by rt-pcr. msc's migration potential was measured in the scracth assay. mscs' interaction with schwann cells and their effect on tumorigenesis was examined in co-culture by apoptosis markers on flow cytometry. results: nf1-mscs' adipogenic and osteogenic differentiation potential was lower than healthy controls as assessed by staining aizerin red s and oil red o and rt-pcr for osteopontin and collagen1. mscs cultured from dermal neurofibroma showed faster closing of the scratch compared to the same patient's normal and caf e au lait skin. on the other hand, mscs obtained from plexiform neurofibroma healed late, while mscs derived from the same patient's caf e au lait skin showed the fastest healing. hepatic encephalopathy with ammonium ions accumulation is accompanied by some disorder in the brain due to toxic material concentration being usually detoxified in the liver. one of the reasons for hyperammoniemia could be some imbalance in brain glutamine metabolisation induced by the key enzymes glutamine transferases (gts), which catalyze the reaction of glutamine transamination resulting in neurotoxic product of a-ketoglutaramate (akgm). akgm is hydrolyzed to a-ketoglutarate and ammonia by x-amidases. in the study, the dynamics of the enzymes activity in the tissues and biological liquids of experimental animals with hepatic dysfunction induced by thioacetamide (taa) was under investigation. white laboratory rats of wistar line (female, weight of 140 g) chronically intoxicated with hepatotrophic toxine of taa. every 2 weeks, some biological samples were collected to assess gt-k and x-amidase activities. x-amidase activity was the highest in the kidney tissue in the control and decreased by 70% in the experimental group. in the experimental hepatic x-amidase activity decreased by 240% compared to those in the control. the average x-amidase activity in the blood serum (0.015 nmols/ mg/min) and in the brain (0.005 nmols/mg/min) differed faintly. maximal gt-k activities were revealed in the kidneys; in the controls, it was about 250% higher than those in the experimental animals. the difference between average enzyme activities in the liver of the control and experimental animals reached 350%. the average gt-k activities in the blood serum and brain of the control and experimental animals were rather similar. the decrease in x-amidase and gt-k activities obtained in the study during hepatic pathology development could testify to imbalance of glutamine metabolism, possibly aimed at declining the level of akgm neurotoxicant under the hepatic dysfunction. acknowledgments: supported by the russian federation ministry of science and education (grant no rfme-fi60414x0116). wilson disease is an autosomal recessive disorder of copper metabolism characterized as neurodegeneration and liver abnormalities. it is caused by defects in the atp7b gene. atp7b is responsible for the sequestration of cu into secretory vesicles, and this function is exhibited by the orthologous ccc2p in the yeast. we aimed to characterize clinically-relevant novel mutations of p.t788i, p.v1036i and p.r1038g-fsx8 in yeast lacking the ccc2 gene. the patients with these mutations have copper storage abnormalities in different parts of their bodies; p.t788i mutation mainly affects the liver and the nervous system, p.v1036i mutation affects the nervous system, and p.r1038g-fsx83 mutation causes damages to the liver. to better understand the effects of these mutations on normal functions of atp7b, we cloned human atp7b gene onto a yeast expression vector and created the same mutations by site directed mutagenesis. then, wild type and mutated forms of atp7b genes were transformed into yeast cells lacking the homologous ccc2 gene for functional comparison. first, we analyzed the expression of atp7b and its variants in yeast cells by a real time pcr approach and western blot to make sure that transformed cells express the plasmids. expression of human wild type atp7b gene in ccc2d mutant yeast restored the growth deficiency and copper transport activity, however, expression of the mutant forms did not restore the copper transport functions and only partially supported the cell growth. our data support that p.t788i, p.v1036i and p.r1038g-fsx83 mutations cause functional deficiency in atp7b functions and suggest that these residues are important for normal atp7b function. in recent years, attempts were made to develop miniaturized potentiometric biosensors which is particularly important to reduce the amount of enzyme and reagents needed. the miniaturization of a biosensor is possible by using an all solid-state polymer membrane ion selective electrode which is cheap, easy to prepare and allow micro-sized construction. the use of all solidstate polymer membrane ion selective electrode as the basic sensing element also has the advantage of providing biosensors that are easy to fabricate, exhibit rapid response and have long life-times. they are also mechanically stable and allow flow-through configuration. genetical and chemical modifications for the alteration of enzyme molecule characteristics are gained considerable importance. enzymes can be modified chemically by using water-soluble polymers or some chemicals. conjugates of natural and synthetic macromolecules with enzymes provides wide usage in medicine and in many fields of biotechnology. in this study, enzyme-polymer conjugates with different molar ratios were synthesized using urease enzyme. in this study micro sized potentiometric urea sensitive biosensor has been developed in which urease-polymer conjugates were immobilized on polymeric membrane ammonium ion selective electrodes whether pvc or derivatized-pvc via glutaraldehyde cross linking reaction. biosensor is not include inner reference electrode and inner reference solution. potentiometric performance of biosensor will be examined with a computer-controlled measurement system designated. the most important features of the obtained micro sized urea biosensor by using enzyme-polymer conjugations were being highly sensitive, having long life-time, easily built at a low cost, and having short response time when compared with conventional potentiometric urea biosensor. also, these biosensors were easily built at micro-construction. this study was supported by grant from the tub _ itak research fund (project number: 114z138). creative drama technique as a new tool to increase enthusiasm and to achieve learning objective for medical students e. y. sozmen 1,2 , e. erem 3 1 faculty of medicine, ege university, izmir, turkey, 2 center for continuing education, ege university, izmir, turkey, 3 recently drama in education techniques have been implemented successfully in education program of primary and secondary high school and positive effects of these techniques on learning ability and attitude of students have been shown. the aim of this study was to organize an education program based on drama in education techniques in a special module of ege university medical faculty and to test any effect of this technique on achievement of learning objectives and student's perspectives on drama. the special module program was on the oxidative stress and antioxidants. the program covered the drama in education sessions (improvisation, role play, game) linked with learning objectives (understanding of free radical generation and free radical reactions in body, evaluation of the effect of free radical reactions in diseases as well as increase the ability usage of scientific information), laboratory work (antioxidant activity determination) and searching a special scientific topic on literature. 12 students (in 3rd year of faculty) who had taken theoretical lecture on this subject a year ago, participated in this special module. the opinions of the students on the program were obtained through a questionnaire form and the increase in knowledge was evaluated via pre/posttest. the mean of pretest point was 2.7/10, that increased to 7.4/10 in the posttest evaluation. 50% of students pointed that they enjoyed participating in drama activities in the pre-questionnaire, this rate was 100% in the final questionnaire. they all remarked that implementation of drama in education was beneficial for their communication skill, helping them to learn more about science and increased their enthusiasm to learn and discuss the scientific information. although the preparation process might take more time and need to hard work for teachers, we concluded that the drama methods as a new tool to increase of participant's interest might be proposed for students in higher education. laboratory-based performance assessment in medical education: an opportunity for connection between scientists and medical students h. tuncel cerrahpasa medical faculty, istanbul university, istanbul, turkey number of medical students who interested in basic medical sciences is declining and medical sciences literacy is falling, it is crucial to develop ways for students and scientists to connect. students need to know that science is an intensely human endeavor, and scientists need mechanisms to bring that truth to the community at large. based on continued interest and experience on the part of faculty, and on student feedback, the development of a more effective and stimulating interactive learning tool was undertaken. an in-depth knowledge of laboratory medicine principles is vital to all practicing physicians. great variation exists in the ways that medical students learn the principles of laboratory medicine. there are a number of programs for electronic media that emphasize laboratory-related skills. some of these are appropriate for medical students in the clinical years. programs that teach skills in common laboratory procedures, such as interpretation of peripheral blood smears and microscopic examination of urine sediment, have been shown to improve student performance. to ensure that important principles are addressed, medical schools should establish goals and objectives specifically related to laboratory medicine and experiment with optimal teaching and assessment methods. we also hope that this study will inspire dialogue among primary care and specialist physicians as to the proper degree of education in this area. ideally, it will encourage scientific studies that address evidence-based possibilities for improving critical laboratory medicine educational outcomes, that is, the training of physicians who optimally use laboratory diagnostics and therapeutics. engaging medical students in scholarly scientific activities and producing clinically competent and research-oriented medical workforces are essential demands, particularly in developing countries. an experimental special study module for medical undergraduate students: learning western blot analysis and detection of b-actin protein expression in tissue and cell culture samples learning, introduce basic principles of laboratory research and to present the results.b-actin is one of six actin isoforms which is mainly expressed in all eukaryotic cells. western blotting is a widely used laboratory technique to determine specific proteins and to evaluate protein expression in tissues and cells. in our study, different concentrations of rat spleen homogenates (25, 50, 75 lg/well) and 50 lg protein/well of human lung and ovary cancer cell lysates were used. the proteins were seperated by 10% sds-page, transferred to pvdf membrane, incubated with specific b-actin antibody and then with hrp-conjugated antibody. protein bands were detected with ecl and densitometric analysis of proteins was quantified by imagej software. differences in protein band intensities were compared using one way anova.a value of p < 0.05 was considered statistically significant. we detected b-actin expression in rat spleen homogenates, human lung and ovary cancer cell lysates, as a 43 kd protein. the protein band intensities were in correlation with protein concentrations. the highest concentration resulted in the highest signal intensity in rat spleen homogenates.b-actin level was higher in ovary cancer cells than in lung cancer cells, although both proteins were loaded equally. the feedback showed that students were very satisfied with this laboratory ssm. they developed their independent study skills, planned a research, worked in a laboratory, learned and performed a technique, western blotting. the hands-on experience is very important for medical undergraduate students for their future scientific career. three-dimensional structure of truncated human kv10. voltage-gated potassium channel kv10.2 belongs to the ether-ago-go family. it has been proved that its mutants are involved in development of neurological diseases and some types of tumor. directed drug design needs knowledge of details of the threedimensional channel structure. the members of the kv10-12 subfamilies are characterized by extremely long n-and c-terminal intracellular tails, which possess a number of structural domains. the n-terminal pas domain in kv10 plays an important role in activation, and is thought to alter the rate of deactivation, possibly by binding at or near the s4-s5 linker at the inner mouth of the pore. here we present an improved 3d structure of the truncated human kv10.2 channel, obtained by single particle em. this channel lacks its cytoplasmic pas domain but it still forms tetrameric particles. earlier we showed that the full length kv10.2 channel is build according to the 'hanging gondola' type, and its cytoplasmic and transmembrane parts are connected by linkers. the cytoplasmic part includes the interconnecting pas and cnbd domains. deletion of the pas domain leads to the conformational change in the cytoplasmic part of the channel. for interpretation of the 3d structures we used homology modeling and molecular dynamics simulation. there are several templates available to the moment including eag domain-cnbhd complex of the mouse eag1 channel, full-length shaker potassium channel kv1.2, c-linker/cnbhd of zelk channels and others. but there are still no templates for many fragments that led to necessity of partial de novo modeling. analysis of molecular trajectory allowed estimating dynamical characteristics of channel, supposing interdomain interactions. results of the conducted investigation have a great interest at both the academic and the industrial levels. the objective of this task program is to enable students to gain scientific attitude and skills for them to be able to deal with the problems they'll confront in future research careers. it's been emphasized in various studies that medical students are keen on conducting scientific research, and it's been stated that they need to be supported in this respect, as they lack the adequate fund of knowledge. this study aims to share information throughout a 5-year performance of the research training program (rtp) conducted at ege university, faculty of medicine. rtp is an educational program applied in addition/parallel to the bachelor's degrees for establishing scientific thought, attitude, proceeding and knowledge of the willing medical students, and enabling them to adopt study skills. the dynamic program produced by the rtp committee in 2011 has been developing each and every year via feedback received from the students. an operating principles, a manual for advisor and an introductory booklet have been laid out. applications are being accepted at the end of first academic year, making announcement to the freshmen in advance. students are being admitted to the program, taking the assessment criterias into consideration. second and third year medical students attend the didactic part of the program during the terms devoted to special study modules. thereafter, they go through the project management phase, and accomplish their projects under supervision of a faculty member until their graduation. 12 first graduates of the program, accomplishing their projects, received their certificates at the graduation ceremony of 2015 graduation. currently, there are 61 students in total from all classes who perpetuate their studies within the program. an inventive training pattern of ege university faculty of medicine, rtp experience is being maintained as a dynamic process and successfully keeps the students advised of conducting scientific researches, cultivating scientific awareness. objectives: objectives selection and verification of blood collection tubes is an important preanalytical issue in clinical laboratories. in this study comparison with the reference glass tube of ayset plastic tubes containing separator gel and assessment for routine clinical chemistry laboratory testing in samples were aimed. materials and methods: thirty-four volunteers were included in the study. samples were taken into two different tubes by two experienced technologists according to the clsi protocol [tube1: z (becton dickinson and company, franklin lakes, nj, usa); tube2: ayset (lot10069, turkey)]. glucose, urea, creatinine, ast, alt, total-cholesterol, triglycerides and high density lipoprotein-cholesterol were analyzed subsequently (olympus au2700) and randomised samples stored at 2-8°c for 24 and 48 h. 0th hour sample was analyzed immediately after collection and accepted as the reference for the comparison of the other samples. a paired t-test and wilcoxon signed rank sum test were used to test the significance of differences between the reference tube and test tubes. results: the difference between the results of reference tube and test tubes for glukose, urea, creatinine, ast, alt, total cholesterol, tg and hdl-cholesterol at 0, 24 and 48 h were statistically no significant (p = 0.09, p = 0.07, p = 0.20, p = 0.16, p = 0.13, p = 0.09, p = 0.05, p = 0.54, p = 0.58, p = 0.46, p = 0.19, p = 0.13, p = 0.66, p = 0.72, p = 0.34, p = 0.11, p = 0.16, p = 0.06, p = 0.08, p = 0.39, p = 0.23, p = 0.63, p = 0.58, p = 0.54, respectively). conclusions: no significant difference was observed between ayset tubes' results and the reference tube's results. e. akin c ß akir d€ uzen laboratuvarlar grubu, istanbul, turkey insulin resistance underlies the development of obesity which is a global health problem. obesity is a main concern of scientists because it's associated with type 2 diabetes, hypertension and some cancers. recently, inflammation centered mechanisms are deeply investigated as well as the effects of anti-inflammatory diets which are highly rich in vitamins, minerals, fibers and healthy oils. these diets are proposed to inhibit or supress the secretion of the inflammatory mediators and also improve the intestinal microflora. the aim in this study is to highlight the increasing trend of publications in regard to insulin resistance and inflammation based obesity along with the effects of antiinflammatory diets used for its treatment in the last decade. we performed a pubmed search with key words of 'obesity and insulin resistance and inflammation' (01/01/2005-15/05/ 2016) (search #1). besides, we performed another search with key words of '(anti-inflammatory diet or dietary supplement) and (obesity or insulin resistance)' (search #2) to highlight the value of anti-inflammatory diets and dietary supplements in combating inflammation based obesity and insulin resistance. search #1 revealed 6763 articles; of these 341 were clinical trials, 18 were observational studies. human studies were 4 552 while animal studies were 2 580. overall, there were 2 229 review articles and 5 meta-analysis in the field. search #2 revealed 4 255 articles of which 796 were clinical trials, 958 were review articles, 46 were meta-analysis. human studies were 2 610 and other animal studies were 1 931. the relationship of metabolic diseases with a low grade inflammatory state has opened a new area of research to understand the consequent causes of inflammation in the human body. the increasing scientific data in the field indicates that antiinflammatory diets may serve as powerful tools to solve the inflammation and the consequent insulin resistance and obesity. medical and biological illustrations for life sciences education: is 'a picture worth a thousand words' in visualizing medicine and science? medical and biological illustrations (mbi) convey complex ideas with just an image and they are powerful intersections of science and art. the clarification of complex pathways via illustrations can be effective means in education as they help the student to visualize the biomolecular world and understand the mechanisms. our aim is to illustrate how a mbi is developed over the example of mechanism of insulin action, via the phosphoinositide (pi) 3 kinase-protein kinase b (akt) pathway. organising one's thoughts and clarifying relationships and then using the optimal complexity level to illustrate the pathway most clearly are the basics of mbi. thus, we made a thorough investigation of insulin mechanism on glucose uptake in skeletal muscle and adipose tissue; a biochemical process that includes insulin receptor (ir), ir substrate, pi3 kinase, pi-dependent kinase 1 and akt. then, we found the 3d structures of molecules via protein databanks and accordingly created drawings and diagrams of each component in both molecular and macrolevels by adobe photoshopò software. graphics tablets and a compatible pc were also used in the production phase. the use of computer hardware/software enables unlimited detail in images and provides the flexibility that classical drawing techniques can not. thus, the final diagram clarifies the underlying molecular mechanisms of a biochemical pathway along with the physiologic actions. recent improvements of computer technology have resulted in the creation, and reproduction of high-quality lower cost medical art. mbi's can be used in education to explain concepts/pathways to students to enhance learning. similarly, mbi's are great tools to show mechanism/procedures to enhance patients' understanding of their medical condition. considering their unquestionable contribution to education, research and patient care, the creation of mbi's should be promoted as a graduate level course and the discipline should be represented at academic level. biochemistry is a compelling field with broad applications in many disciplines like medicine, dentistry, pharmacy and bioengineering. biochemical research increasingly combines ideas from genetics, molecular biology, etc. and collaborates with many disciplines. our objective is to conduct a scientometric analysis of the last decade's postgraduate theses in the field of biochemistry (ptb) in regard to number, collaborations, subject area distribution, etc. to discover the characteristics and trends in turkey. we searched the turkish higher education council's theses database (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) which includes master of science (msc.), doctorate (ph.d.) and specialization (s) theses in all disciplines. an electronic search with the keyword of 'biochemistry' (in the thesis subject area) was conducted, thus it brought all theses either in the biochemistry discipline or theses in other disciplines but have a biochemistry component. we performed data cleaning and further quantitative analyses in excel. we also executed word count analysis on the titles of theses to derive the main subject areas in ptb. of the total of 6374 ptb (2215 s, 2781 msc, 1339 phd theses) 37.6% was in natural sciences while 62.4% was in health sciences. the theses output-growth measured by the compund annual growth rate was 82% over the 10-years. the top 3 clinical disciplines in collaboration with biochemistry were pediatrics, surgery and cardiology, and the top 3 science disciplines were biotechnology, bioengineering and biology. oxidants-stress and antioxidants (1008), endocrine-metabolism (655) and enzymology (608) were the top research areas in ptb, followed by genetics (305) and cancer (252). scientometrics is a powerful tool to understand the direction of science and research. our ptb analysis indicated that prominent areas like stem cell, biosensors, geriatrics are somewhat lagging in turkish biochemistry research while postgraduate education and research in total is growing fast with sound collaborations. the 1st turkish in vitro diagnostic symposium evaluation objective: in vitro diagnostic (ivd) medical laboratory tests and the equipment are closely related the public health, patient safety and the safety of all who utilize these tests. it depends on auditing of the process from the production to the consumption of these materials, that they do not pose a risk to individuals and society. upon these basic requirements; 'turkish in vitro diagnostic symposium: medical laboratory tests' was held in february 2016, organized by the cooperation of turkish biochemical society branch of izmir, and dokuz eylul university health sciences institute. it was intended to shed light on some questions such as, what is the place and importance of ivd in turkey? what are the responsibilities of educational institutions?, what is the role of ministry of health? to put across the conditions of preparing a substructure that may provide achieving success in producing ivd medical devices and in this sector, in our country. material-method: 39 invited speakers attended the symposium, along with the participation of both as lecturers and attendees; ministry of health, turkish standards institution; representatives of manufacturer enterprises; representatives of enterprises manufacturing in turkey; scientists conducting considerable researches on health technology; students and representatives from some of the non-governmental organizations. in addition to the presentations, gathering up the written opinions of the participants, a report was prepared. results: the symposium that lasted for 3 days was realized with 120 participants in total,55 of which from universities;38 representatives of their companies;17 from chamber-institute-public establishments and 10 of which from public hospitals. 94 of the participants were from izmir,26 of them were coming from out of izmir. conclusion: at the end of the symposium,40% of the participants gave feedback. among the feedback selected; 86.2% of the participants evaluated the symposium overall, as successful. 75.6% of them found the symposium successful with regard to its scientific content. their feedback were 55.2% positive in terms of the symposium's contribution to the situation assessment on ivd in turkey, and 44.8% of them stated they would consider participating in the second of the ivd symposium if it is to be organized. perceptions of molecular life science master's students on their scientific and academic competencies and prospective plans for professional development the master's education (me) in molecular life sciences (mls) is aimed at strengthening the knowledge and skills base of the young scientist, preparing him for the competitive academia/industry positions. the rapidly developing pace of science and research forces the master's student (ms) to play a central role in monitoring and guiding his scientific education and professional development (pd). thus, the aim of our study was to examine the perceptions of ms of mls, regarding their scientific and academic competencies. with this data, we planned to analyse if this awareness channels ms to take action and/or matches with their prospective plans. we developed a 15 item online survey with 3 sections (demographic data, current data-contributions of me, competencies and prospective data-action for pd, future plans) and distributed it via e-mail to various postgraduate institutes. at the end of the 10-day period, 138 ms students (in the thesis phase) answered the questionnaire (female: 66.7%, male: 33.3%). the most highly rated activities that contributed to their scientific knowledge and skills gained in the me were laboratory work (73%), visits with their mentors (70%) and theoretical lessons (65%). ms expressed low levels of sufficiency both in theoretical scientific knowledge and laboratory skills (only 43% and 33% sufficiency, respectively). communication skills (80%) and team work (78%) were rated as the highest professional competencies followed by literature search and research planning (both 73%). it was striking that ms perceived themselves as quite insufficient in scientific writing (50%), data analysis (60%) and project writing (61%) while proficiency in english (55%) was the first area they wanted to take action. despite their perceptions of insufficiencies in many areas, a majority (69%) wanted to continue to phd education. these and similar surveys may lead to an increase in selfawareness in ms and the data may contribute to the revision of me. the report of the 1st turkey in vitro diagnostic symposium results the following aspects and suggestions took place in the results report prepared after the symposium: about the national ivd-tc r&d, production, forming quality assurance and innovation strategy and policy the cooperation of the university and the industry is not sufficient most of the industrialists cannot take enough advantage of the support provided by the institutions like tubitak, the ministry of science, technology and industry and the ministry of development the statistics on the ivd-tc in turkey should be carried out as soon as possible national standards should be determined in parallel with the international standards the vat rate of the exported raw materials that would be used in ivd production should be decreased. about the education and training, the job titles and positions the related graduate programs, which would focus on all steps of the whole life cycle related to the ivd-tcs one by one, are not widespread there is no 'postdoc' application in turkey. 'postdoc' staff is needed for insufficient component human resources the lecturers should not be restricted to one discipline only graduate programs on laboratory medicine are needed to be established and spread, in order to train component labor specified on the ivd-tc about research and development there are almost no researches related to product development. this should be associated with the education and training institutions about the research centers currently, there is a real infrastructure on health technology in turkey, but there are difficulties in its instituonalism the insufficient cooperation of the university and the industry does not allow the inventions to turn into products the cooperation supports of r&d, being restricted to tech-nopark and r&d centers, are open fields for improvement p-edu-014 phd training in medical education: career profiles and satisfaction levels of graduates from dokuz eylul university graduate school of health sciences this survey was carried out within the scope of special study modules that is entitled 'phd training in medical sciences' by a group of medical students in deu. the purpose of the survey to investigate the members of health sciences that have successfully completed their phd training in terms of the levels of satisfaction and the status of their career. from this scope we generated two hypothesis: we expected that graduate phd graduates are mostly involved in academy and find their satisfaction levels at moderate level as to phd education. the study was designed as cross-sectional. we reached 166 phd graduates who had graduated from deu graduate school of health sciences between 1991 and 2002 from 12 different departments via e-mail. the survey was included 27 questions, which were prepared in the light of the existing literature. among the 166 phd graduates, 55 (30%) participated in the study. through this survey, perception of phd students on supervisors' scientific and educational abilities, opinions on phd training, productivity of phd training, number of articles published, their position and related satisfaction levels after graduation were investigated. according to the results, more than half of the graduates (%52.7) are well satisficed from the education they had taken. beside this, interestingly we found that %94.5 of graduates prefers staying in the academic positions and %64.8 of them sustains their communication with their supervisors. in conclusion, most of phd graduates were contented with phd training and their career profiles. as a result of this survey, we produced a novel and precise contribution to the literature. in a further study, this survey may extend to other parts of turkey and compile the results in order to get more accurate and inclusive data. phd is an international degree that is reached by conducting an original research after finishing bachelor or master's degree. doctoral degree (phd) can open the door of academic career; on the other hand, a person with a doctoral degree is equipped to carry out important work in research, industry, or public sector. today, gradually increasing number of phd students have brought some problems in phd training. the purpose of this study is to investigate and review activities that have been done by the following international organizations: orpheus: (organization for phd education in biomedicine and health sciences) eua-cde: european university association-council on doctoral education. febs education committee: federation of european biochemical societies these three organizations have done workshops on phd training to pay attention to the following points: *a phd student must take some courses and trainings outside her/his institute, should not be limited to the institute. *the phd training programme should include transferable skills courses. *clinicians, if involved in phd training, should be given free time from their clinic duties. *with regards to potential problems with the supervisor, the institute should provide the student an advisory system. *students should be encouraged to participate in the management of doctoral programmes in the institute. *the students should be given be opportunity to select their own supervisor (thus their thesis area). the phd training has gained quality thanks to these organizations. it is advised that graduate school of health sciences should follow the recommendations and report from these organizations. itsn1 and itsn2 are genes encoded adaptor proteins with multiple isoforms participating in clathrin-mediated endocytosis (cme), mapk signaling and reorganization of actin cytoskeleton. changes in itsns expression can lead to different neurodegenerative disorders and cancers. to date little is known about regulation of itsn genes on posttranscriptional level. the aim of our work was to predict and experimentally confirm target sites for micrornas that could potentially regulate itsns expression. using 8 web servers we analyzed 3 0 utrs of short and long isoforms of human itsn mrnas and found conservative target sites for mir-34, mir-19, mir-129, mir-103/107, mir-194, mir-181 and mir-30 in 3 0 utr of itsn1-s, predicted by 5 servers, mir-203 predicted by 5 servers for itsn1-l, and mir-153, mir-148/152, mir-27, mir-144 and mir-128 predicted by 5-6 servers for itsn2-l. to elucidate potential impact on cme, mapk signaling and actin cytoskeleton regulation by these mirnas we performed enrichment analysis by diana-mirpath server and found that mir-34, mir-19, mir-103/107, mir-181, mir-30 and mir-148 were highly enriched for all analyzed pathways. using regrna 2.0 and scan for motifs services we predicted 12 types of different regulatory elements in 3 0 utr of itsn1 and itsn2: k-box and brd-box, musashi binding element for rbps musashi1 and musashi2, gu-rich element (gre) and au-rich elements (are) that regulate mrna decay. to confirm itsn1-s regulation by micrornas we cloned 3 0 utr of itsn1-s into luciferase reporter vector and transfect hek293 cells by this construction and mir-181a, mir-30a and mir-19a. for mir-181 transfected cells, we found 25-40% decrease of expression of itsn1-s 3 0 utr-bearing construction. for other mirs we did not obtain strong reproducible effect in luciferase assay. these data may confirm mir-181 target site in 3 0 utr of itsn1-s mrna but needed additional research. objective: stroke is an acute neurological disorder that is mostly caused by ischemia in central neural system. 30% of stroked patients lose their life in 1 year, and remaining 1/3 of the living patients continue to their lives as dependent to others. nihss and mrs are two scales which are used in prognosis studies because they can show stroke intensity and after stroke functional recovery. microrna's which have effects on transcription and posttranscription gene regulations are small rna molecules. their roles have been investigated on pathophysiology and treatment of diseases. in this study, it was aimed to detect changes in blood serum levels of mir-146a, -155, -210, 181b, -31, 126, -92a and let-7f of ischemic stroke patients and to investigate role in predicting prognosis methods: 21 patients diagnosed by acute ischemic stroke admitted to neurology service of g€ oztepe hospital and 16 healthy individual were included in the study. after stroke patients' blood samples were taken periodically in 1st day, 1st week, and 3rd month, and at the same time nihss and mrs scores were determined. set 8 mirna blood serum levels were measured by rt pcr results: when compared to the control group, we found that after stroke 1st day peripheric blood levels of mir-146a,-31,-92a and let-7f were significantly low; when 1st week and 1st day serum records were compared there was a significant increase in mir-126 level; and when 1st week and 3rd month records were compared we noted that there was a significant increase in mir-146a,-126 and let-7f levels. from prognosis point of view; after ischemic stroke measurements showed that mir-181b in the 1st day, mir-146a and mir-210 in the 1st week showed positive significant correlation with 3rd month mrs scores (p = 0.002, p = 0.05, p = 0.002, respectively) conclusion: according to the outcomes of this study, after stroke 1st day mir-181b, 1st week mir-146a and 1st week mir-210 levels can be stipulated to use in predicting patients' 3rd month prognosis p-01.03.3-004 inhibitory rna aptamer against lambda ci repressor showed the transcriptional activator activity s. ohuchi, b. suess tu darmstadt, darmstadt, germany because of the variety of functionalities on gene regulation and the easiness of molecular engineering, functional rnas are promising parts for the construction of genetic circuits. artificial affinity rna, or rna aptamer, is one of such genetic parts. in the previous study, an inhibitory rna aptamer against a repressor protein, tetr, was developed as a transcriptional activator [1] . the expression of this aptamer abolishes the repressor activity of tetr, resulting in the elevated gene expression under the control of tetr. because of simplicity of the mechanism, similar transcriptional activators can be generated by using rna aptamers against other repressor proteins. here, we examined the generation of an activator based on an rna aptamer against one of the most frequently applied repressor proteins, lambda phage ci. in vitro selection (selex) was performed targeting a recombinant ci protein employing an rna pool containing 40-nucleotides of a random region. after 6 rounds of selex, the pool rna showed the affinity, as well as the inhibitory activity, against ci in vitro. then, rna aptamers with the transcriptional activator activity were screened from the enriched pool in vivo employing a reporter plasmid on which the expression of a reporter gene, lacz, is repressed by ci. when the variants of the rna pool were transformed to e. coli cells harboring the reporter plasmid, about half of the transformants showed the elevated reporter expression. interestingly, all of these desired rna clones shared the same sequence. quantitative analysis indicated that 35-fold induction of the reporter expression was achieved upon the aptamer expression. our results suggested that diversity of artificial transcriptional activators can be extended by employing rna aptamers against repressor proteins to broaden parts for the construction of genetic circuits. [1] hunsicker, et al. (2009) an rna aptamer that induces transcription. chem. biol., 16: 173-180. p-01.03.3-006 microrna expression signatures between non-atherosclerotic plaque and atherosclerotic plaque in cad with humans, and parallels whole blood rnas and represent a new important class of gene regulators. the present study was designed (i) to investigate the mirna expression profile in human atherosclerotic plaques from peripheral arteries aorta as compared to non-atherosclerotic left internal mamary artery (lima); (ii) to examine the expression levels of mirna in whole with correlation mirnas of aorta tissue. material and methods: thirty-one patients with cad were enrolled in study. lima and aorta tissue samples were obtained during coronary artery bypass surgery. whole blood samples were collected before cabg surgery. each patient with cad was obtained from whole blood, aorta and limas tissues. these tissue samples were immediately soaked in rnalater solution and homogenized using a magnalyser. the rna was extracted using the trizol reagent and the mirneasyò mini-kit. the expression profiles of 738 mirnas were evaluated using highthroughput qrt-pcr. results: we found that mir-497-3p was expressed only in aorta. mir-431-5p and 433 were expressed both aorta and whole blood. 6 mirnas were significantly up-regulated in aorta when compared to lima tissue (fc > 2). 59 mirnas were significantly down-regulated in aorta compared to lima. conclusion: in conclusion, our study suggests that mir-497-3p, mir-431-5p and 433 might be a potential for cardiovascular disease development. also mir-431-5p and 433 might serve as novel non-invasive biomarkers for cad p-01.03.3-007 mir193b regulates cell proliferation and colony formation in pancreatic ductal adenocarcinoma n. gurbuz, e. isildar due to the strong metastatic potential, pancreatic cancer has the highest mortality rate of all major cancers. therefore, the investigators are in urgent need of developing the new alternative therapeutic approaches for the prevention of pancreatic cancer. mirnas, small noncoding rnas, regulates as an inducer or inhibitor in expression of key mediators related molecular mechanisms in cancer promotion. to investigate the effect of mir193b on pancreatic ductal adenocarcinoma cells, we performed the cell viability and clonogenic assays by mts and crystal violet dye, respectively, in panc-1 and miapaca-2 cells transfected with mir193b mimic. our data revealed that mir193b led to decrease the cell viability depending on enhanced mir193b doses, which are 25, 50, 100 and 150 nm, as the ratio of % 23, 60, 82 and 87, respectively, in miapaca-2 cells and as the ratio of % 40, 58, 77 and 89, respectively, in panc-1 cells compared with control condition of each cell. this inhibition mediated by mir193b was also obtained in colony formation both of pancreatic cancer cells. when the induced effect of mir193b on the death of pancreatic cancer cells was compared with gemcitabine, which is currently used as a clinical drug for pancreatic cancer patients, we determined that mir193b was more effective than gemcitabine. based on our findings, it is clearly shown that mir193b serves as a tumor suppressor in pancreatic ductal adenocarcinoma cells. we strongly believed that mir193b gene therapy might be more effective and targeted approach than classical gemcitabine therapy for pancreatic cancer patients. *this work was supported by tubitak (the scientific and technological research council of turkey) grant 114s501. breast cancer is the most common cause of cancer death in women. trastuzumab is a therapeutic agent frequently used against her2+ breast cancers, which has role in approximately 20% of invasive breast cancers. with the discovery of their activity in cancerogenesis, micrornas (mirnas) have become potential candidates to mediate therapeutic actions by targeting genes that are effective in drug response. recent studies have showed that mirnas are induced by targeted therapies. in this study, we aim to find out mirna-mediated mechanism, which is driven by common trastuzumab responsive micro-rnas in her2+ breast cancer. for this purpose, the common trastuzumab responsive mirnas were determined in treated bt474 and skbr3 cells by microarray profiling. two datasets were intersected to find out common mirnas for both cell lines. the overlapping predicted targets of common mirnas were provided by two different mirna-target prediction databases and then a mirna-gene network was built in cytoscape by using networkanalyzer plugin. the most shared target genes were chosen to be analyzed in the ebi-embl gene expression atlas for their expression patterns in breast cancer. 25 common mirnas were found to have overlapped targets in two target prediction algorithms that were used to build the mirna-gene regulatory network. 14 overlapped targets were determined as the most shared genes in the mirna-gene network. expression pattern of each shared gene in the gene expression atlas showed that 12 out of 14 the most shared target genes were strongly dysregulated in several breast cancer types. our results suggest that mirnas might show a common response to the targeted therapies and network analysis can be profitable to have a better explanation of the regulatory mechanisms between drug responsive mirnas and their target genes. revealing the mirna-potential target interactions might provide novel key players that mediate the mechanisms of action in drug treatment. chronic myeloid leukemia (cml) is a clonal disease of primitive pluripotent stem cells that identified with a specific t(9;22) reciprocal translocation that encoding bcr-abl oncoprotein. resveratrol (res) is a natural phytoalexin found in grapes and induces apoptosis, erythroid differentiation and autophagy in leukemic cells. micrornas are small, single strand, non-coding rna molecules that regulate post-transcriptional gene expression via disrupting the stabilization of target transcripts or inhibiting protein translation. in our study we aimed to determine cytotoxic effect of res in k562 human cml cell line and to evaluate the expressions of mirnas that are associated with genetics of leukemia after treatment with res; to investigate target genes of mirnas which show significant expression alterations and molecular mechanisms of res treatment. k562 cells were treated with 100 lm (ic50 dose) res during 72 h and cytotoxicity was evaluated by using wst-1 assay. the rt-qpcr is used for mirna and gene expression analysis. results showed that; res up-regulated tumor suppressor mir-214-3p level 2.87 fold and significantly downregulated hdac1 gene expression (p = 0.003) and upregulated p62/ sqsmt1 gene expression (p = 0.001), according to the control cells.p62/sqstm1 interacts with lc3 and plays role as a critical player in the autophagic degradation of the bcr-abl fusion protein. our findings showed that resveratrol acts as a hdac inhibitor targeting hdac1 and p62/sqsmt1 gene expression level. treatment with hdac inhibitors results apoptosis, cellcycle arrest, cell differentiation, anti-angiogenesis and autophagy. downregulation of hdac1 provides post-translational modification for expression of tumor supressor genes and leads to cell cycle arrest and increases apoptosis. these results could be linked to hdac1 dependent induction of gene associated with autophagy like p62/sqsmt1 and resveratrol could be a therapeutic candidate as a hdac inhibitor for cml treatment. the mirna used in this study are hsa-mir-145, hsa-let-7a, hsa-mir-29b and hsa-mir-155. thereafter, we bought pre-mirnas and their mirnas commercially. we apply them to the a549 cell line in different combination and different concentrations. these mirnas applied solely onto cells or in combination as; four of them, let7 + 145, let7 + 29b, let7 + 155, 145 + 29b, 145 + 155, 145 + let7 + 29b, 155 + let7 + 29b. the cell viability was detected by wst-1 kit in a 96 well plate elisa reader. cells were seeded as 10 000 per well, mirnas incubated with cells for 24 h in an appropriate atmosphere. according to our results some combinations and mirnas didn't alter viability, however 145 + 29b and 145 + 155 combination increased the cell viability dramatically. on the other hand let7 + 29b and 155 only applications decreased the cell viability. the other applications' viability results are not different from the control cells significantly. in this study, we used a549 cell line also called non-small lung cancer (nsclc) cell line and possibly effective mirnas on lung cancer. it is important to exhibit the mirna combinations should be effective on cancer cells' viability. the prospect combinations were determined which is crucial to develop new strategies for cancer treatment. competing endogenous rnas (cernas) act as molecular sponges for the same pool of micrornas through their mirna response elements (mre), titrate mirna levels and thereby regulate gene expression post-transcriptionally. smad interacting protein 1 (sip1), a member of the zeb family is a regulator of epithelial-to-mesenchymal transition (emt) program, which is active during embryogenesis and tumor invasion and metastasis. hence, we investigated the regulation of sip1 by cernas in hepatocellular carcinoma (hcc) cells. among hundreds of sip1 cernas listed at competive endogenous mrna database (cerdb), 14 transcripts (pten, zeb1, ptch1, creb5, acvr2b, enah, robo2, erbb4, e2f3, foxo1, rictor, klf3, ets1, cdk6) sharing at least 9 common mre sites with sip1 were selected and their expression in 9 hcc cell lines were determined by qrt-pcr. ets1 was found to be highly correlated with sip1 in hcc. furthermore, repressing sip1 expression by shrna in hcc cells resulted in decreased expression of ets1, pten and zeb1. our results suggest a possible bidirectional and post-transcriptional regulation of sip1 and its cer-nas in hcc. a meta-analysis for the identification of common microrna signatures in colorectal cancer n. sahar 1,2 , n. belder, h. ozdag 1 biotechnology institute, ankara university, ankara, turkey, 2 comsats institute of information technology, islamabad, pakistan colorectal carcinogenesis (crc) has quite frequent incidence and mortality rates worldwide, despite being studied for decades now. new biomarkers are needed to be identified in addition to the existing ones, due to heterogeneous nature of this disease. the regulatory molecular machinery of a cell, including micrornas (mirnas), contributes to this heterogeneity upon aberrant expression. herein, for a mechanistic understanding of differential gene expression in crc tissue we analyzed mirna expression profiles of 78 crc tumors against 62 normal colorectal mucosa samples, using raw data from e-mtab-752 and e-geod-35834 (affymetrix microarrays), and gse35982 and e-mtab-813 (agilent microarrays) datasets obtained from gene expression omnibus and arrayexpress. raw samples were normalized (different platforms separately) using quartile normalization in brb-array-tools. differential expression of mirnas was identified using cut-off values of p ≤ 0.05, fold change ≥ 1.5 and stringent false discovery rates. mirtarbase and mirwalk2.0 databases were explored to identify validated targets. we found thirty (including mir-21 and mir-183) and thirteen (mir-1, mir-139, mir-375, etc.) mirnas commonly upregulated and downregulated respectively, in both affymetrix and agilent microarray results. predicted targets of these mirnas frequently belong to pathways related to cancer like b-catenin, phosphoinositol-3kinase, and transforming growth factor-b, to name few. moreover, the target genes were significantly enriched in clusters related to cell cycle, cell differentiation and regulation of apoptosis. these promising results will further be compared with differentially expressed gene profiles from a cohort of turkish crc patients. hence the integrated study will refine the panel of diagnostic and prognostic crc markers. hsa-mir-x modulates motility and invasion in triple breast cancer cell line s. noyan 1 , h. g€ urdal 2 , b. g€ ur dedeoglu 1 1 biotechnology institute, ankara university, ankara, turkey, 2 department of pharmacology, ankara university, ankara, turkey breast cancer is a heterogeneous disease and expression levels of certain receptors have demonstrated subtypes which characterize clinically distinct breast tumors. a triple-negative phenotype lacks expression of er, her2 and pr and is known as basallike carcinoma. micrornas are a class of small non-coding rnas that participate in the gene expression in many biological processes. e-cadherin is an important mediator of adhesion in epithelial tissues, and loss of e-cadherin can play a critical role in tumor invasive behavior. another key player of cell integrity pip (3, 4, 5) triphosphate is generated at the leading edge of the cell and leads to cell polarization. pip3 is generated by hydrolysis of pip (4,5) bisphosphate, which is synthesized by pip5k1. any dysregulation in these molecules may support the invasive behavior of the cells. the aim of this study is to find out the role of mirna precursor (hsa-mir-x) in invasion and motility in triple negative breast cancer cells. in this study a triple-negative breast cancer cell line bt-20 was transfected with hsa-mir-x or scrambled control sirna. to check its role in motility and invasion, wound healing and invasion assays were performed respectively. cell invasion was monitored over a period of 24 h by xcelligence real-time cell analyzer using a double-plate and measuring impedance-based signals. additionally emt markers were analyzed by qrt-pcr to explain the molecular mechanisms beneath motility and invasion. we observed that cell motility and cell invasion diminished after transfection of bt-20 cells with mimic for hsa-mir-x. furthermore, qrt-pcr experiments indicated that transfection of hsa-mir-x decreased the expression level of pip5k1c while increasing the e-cadherin expression level. wound healing and invasion assays together with qrt-pcr results support the role of hsa-mir-x in cell motility and invasion. this process might be explained via e-cadherin mediated met or gsk-3-beta related inhibition of invasion. expression level of five micrornas as diagnostic markers in glioblastoma situated in the main brain lobes, but can also be found in other brain regions. while microrna (mirna) as non-coding rnas, play a crucial function in the post-transcriptional regulation of gene expression by mrna degradation or translational repression. in the present study, we aimed to investigate the contribution of gene expression of the five mirnas and to unravel their role in brain tumor cell lines, the mirnas to the risk of gbm tumor. the five gbm cell lines (crl-2365, crl-2366, crl-2948, crl-1690 and htb-15) were evaluated with non-malignant (normal) brain cell line (hcn-2) . determinations of expression level of five mirnas (mir-21, mir-101, mir-138, mir-196, and mir-222) were evaluated by monitoring quantitative rt-pcr (qrt-pcr) technique. the expression levels of four mirnas (mir-101, mir-138, mir-196, and mir-222) were significantly decreased while the expression level of mir-21 was increased in gbm cell lines according to hcn-2 cell line. consequently, these five mirnas can potentially be used as biomarkers for gbm tumor; further studies are mandatory to a better understand and confirm our preliminary findings. background: noncoding rna are known to be crucial molecules with diverse regulatory roles in neural oncology and neurodegenerative disease. the recent study suggested that lncrna anril play role in the development of neuroblastoma and alzheimer disease via binding disease-specific micrornas. material and methods: we used lncrnadisease, hmdd v2.0, mir2disease to predict lncrna-and mir-associated disease in our study. in addition, we utilize tardetscan to search lncrna-mirna interaction. results: disruptions of lncrna anril expression (also named as cdkn2b-as, locus cdkn2a/b (ink4/arf), chromosome 9p21) have been associated with the development of neuroblastoma and alzheimer disease. here, we predicted interactions between noncoding transcripts encoded by locus cdkn2a/b and their molecular partners -microrna. anril can act as decoy while containing sequences that mimic mirna target sites to titer these mirs away from their primary targets thereby act as molecular sponge. using targetscan 7.0, we predicted target sites for hsa-mir-15-p/16-p/195-p/424-p/497-p/6838-p and hsa-mir-125-5p/4319 in anril 3 0 utr. then, we used hmdd v2.0 and mir2disease databases to define if any of these mirs participate in alzheimer disease and neuroblastoma. according to both databases, mir-125 is implicated to alzheimer disease and mir-15 to neuroblastoma. as soon as anril participate in the development of both abovementioned disorders and can have microrna sponge activity, it could potentially positively regulate mir-125 and mir-15 targets by competing with them for micro-rna binding sites thus restoring the expression of target genes. in our further research we plan to experimentally validate predicted microrna sites in anril 3 0 utr. conclusions: we predicted sites for mir-125 and mir-15 in 3 0 utr of anril lncrna that could uncover its possible sponge activity in the development of neuroblastoma and alzheimer disease. aim: matrine excracted from saphora flavescens root and demonsrated that indicates pro-apoptotic and anti-proliferative effect in many types of cancer. acute lymphoblastic leukemia (all) is an acute form of leukemia, or cancer of the white blood cells which characterized by the overproduction and accumulation of lymphoblasts. mirnas play important roles in deregulated cell death mechanisms. we aimed to investigate the effects of critical mirnas during the development of matrine resistance on all cell line ccrf-cem. material-method: ccrf-cem cells were treated with different (0.5-3 mg/ml) concentrations for 72 h and cell viability measurements were carried out with xcelligence device to determine the cytotoxic effects of matrine. mirnas were extracted from treated and untreated ccrf-cem cells using the mirna isolation kit. cdna was synthesised using allin-one first strand cdna synthesis kit. expressions of 44 selected mirnas were analysed with miprofiletm custom mirna qpcr array using the applied biosystem 7500 fast real-time pcr system. results: according to the cytotoxicity assay, it was determined that 'treatment with increasing concentrations of matrine, decreased the viability of the ccrf-cem cell line. expression levels of 44 different mirnas were studied for indicated passages in two replicates. our results showed that hsa-mir-376b-3p (-37,099 fold, p = 0.008), hsamir-106-3p (-16,6795 fold, p = 0.028), hsa-mir-20a-3p (-15,926 fold p = 0.0148), hsa-mir-519a-3p (-11,7398 fold, p = 0.00534), hsa-mir-204-5p (-10,9536 fold, p = 0.0012), hsamir-30b-5p (-9,0631 fold, p = 0.0221), hsa-mir-15b-5p (-8,8971 fold, p = 0.0339), hsamir-106b-5p (-8,8561 fold, p = 0.021), hsa-mir-885-3p (-8,6139 fold, p = 0.00006), hsamir-30a-5p (-8,594 fold, p = 0.009) expression were decreased during the development of matrine resistance. conclusion: these data suggested that especially hsa-mir-376b-3p plays a critical role in the matrine response. ericd (e2f1-regulated inhibitor of cell death) is a newly found lncrna. it is located at 8q24.3. it has two exons and its transcript size is 1745 bp. ericd is regulated by e2f (transcription factor 2) and modulates the cellular response to dna damage. arid3a is a family member of the at rich interaction domain (arid) dna-binding proteins that participate in diverse biological processes like development, cell cycle control, chromatin remodeling and epigenetic regulation. both, ericd and arid3a have just opposite roles in apoptosis in case of dna damage indicating a probability of reciprocal interaction between each other. till now, there is no work related to the interaction between lncrna and arid3a in cancers. herein we try to find a probable interactive role between these in cancers. in this study, 12 different cancer cell lines, 1 osteoblast cell line and 19 different types of normal human tissues rnas were selected for expression analysis of ericd and arid3a. after rna isolation, cdna was converted from their rnas. expression profile analysis of ericd and arid3a in different cancer cell lines and normal tissues was done using imagej program for semiquantitative and 2 (-ddct) method for quantitative rt-pcr. among used cancer cell lines, ericd was highly expressed and arid3a had lower expression in u-2os (osteosarcoma), a-172 (glioblastoma) and a549 (lung cancer). at the same time, ericd expression was lower and arid3a had high expression in hfob 1.19 (osteoblast cell line) and normal tissues like brain and lung . both ericd and arid3a are cell cycle regulated and are commonly regulated by e2f. they have just opposite roles in apoptosis during dna damage. these two genes have a probability of reciprocal interaction between each other in cancer. our results indicate that both ericd and arid3a might have opposite roles in lung cancer, glioblastoma and osteosarcoma. ericd and arid3a might act as cancer promoting and tumor suppressor genes respectively in these cancers. the importance of mirna expressions in infertilty implantation process is controlled with endometrium, factors secreted by the embryos and in accordance with these factors embryo and/or endometrium via receptors on. more than 700 human microrna (mirnas) that are small noncoding rnas were shown to play an important role in intracelluler cycle regulation in both normal and pathological conditions. in this study we aim to identify mirnas and controlling molecules expressions in different time period of endometrium in fertile and infertile cases. the endometrial samples were taken from fertile and infertile patients in proliferation and early secretion periods. the samples are fixed and stained either with hematoxylen-eosin for morphological analysis or with immunohistochemistry for distributions of anti-dicer, anti-drosha, anti-eif2a and anti-eif2c. mir-17-5p, mir-23a, mir-23b, mir-542-3p, mir-21, mir-199a*, mir-705, mir-20a, mir-26a, mir-125b, mir-200a/b/c were analyzed with qrt-pcr. while dicer immunoreactivity was detected weakly only proliferation phase of fertile group, this immunoreactivity were detected strongly in both proliferation and early secretory phases of infertile group. drosha immunoreactivitiy was also weakly detected in the proliferation phase of fertile group, it was moderately detected in both proliferation and early secretory phases of infertile group. eif2a immunoreactivitiy was similar in each groups but there were a few differences between fertile and infertile group. eif2c immunoreactivitiy was negative in all groups. mir-21, mir-199a* and mir-23a were highly expressed in proliferation phase of fertile group, mir-23a and mir-125b were highly expressed in early secretion phase of infertile group. in conclusion, dicer and drosha immunoreactivities and different expression of mirna's were detected in all groups. implantation problems may be reason for different mirna expression which controlling with dicer and drosha in the infertile endometrium in both proliferation and early secretory phases. wheat is an important agricultural crop with an over 615.8 million metric tons harvesting capacity annually. drought and salinity are environmental stress factors that affect yield and quality of wheat, dramatically. there are different defense mechanisms against these stress conditions in plants. altering gene expression profiles by micrornas at post-transcriptional level is one of the most conserved mechanisms among plants. micrornas are an extensive class of noncoding rnas, approximately 22 nucleotide length which regulates the expression of genes by binding to the 3 0 -untranslated regions of specific mrnas. micrornas implicated under salt and drought stress have widely been reported in numerous plant species and wheat genomes in the last years; however, studies on einkorn wheat (triticum monococcum spp. monococcum) are not yet available. the goal of this study is identification of conserved micrornas from einkorn wheat using next generation sequencing technology and bioinformatic analysis. in this study, small rna molecules were extracted from pooled plant samples grown under normal, drought and salinity conditions. sequencing analysis revealed 75 164 unique small rna sequences obtained from 15 139 448 raw reads. after bioinformatic analysis based on comparative genomics approaches, we identified 168 putative mature microrna sequences belonging to 142 distinct microrna families. since chromosomal sequence data is not available for triticum monococcum spp. monococcum, we used available sequences from triticum urartu, a close relative, as template to extract precursor microrna sequences. 111 of precursor sequences showing 100% homology to triticum urartu genome were analyzed for secondary structure prediction using mfold software. data provided in this study is critical to investigate transcriptional regulation of genes involved in stress metabolism in einkorn wheat. the role of vim-as1, a natural antisense transcript, in cancer coding or non-coding transcript. in this regard, vim-as1 is nats located antisense to vimentin gene. in the present study, we aimed to determine tissue and cell line distribution of vim-as1. materials and method: for the tissue expressions analysis, human total rna master panel ii (containing 20 different human tissue samples) was used. total number 14 cancer cell lines and 5 normal cell lines included in the study. for the expression analysis rt-pcr and qpcr methods were used. results: as a result, expression levels of vim-as1 were found to be tissue specific. particularly, while vim-as1 was highly expressed in lung and thyroid gland tissues, its expression was not observed brain, stomach and adrenal gland tissues. also, vim-as1 was also found to be differentially expressed in cancer cell lines. vim-as1 expression was found to be lost in cal29, pc3, and hct116 and highly diminished a549 cancer cells. also, it is found to be highly expressed in bcpap, panc1 and beas2b cells. discussion: results of the current study suggest that vim-as1 may have significant role in the regulation of vim gene in thyroid gland tissues, as it is highly expressed in both thyroid gland tissues and bcpap thyroid cancer cells. moreover, vim-as1 and vim axis can be involved in the formation of lung tumors because vim-as1 was highly expressed in normal lung tissues and beas2b cells and expressed very low levels in a549 lung cancer cells. lung cancer is the leading cause of cancer related deaths in the world and approximately 90% patients with lung cancer ultimately die from metastatic disease. metastasis is the most dangerous step of cancer. in our recently published work showed that akt/nf-kb pathway is continuously active and induces cellular invasion and pten suppresses cellular invasion via inhibition of akt/nf-kb pathway. in this study we aimed to show nf-kb mediated induction of mirna expression can responsible for inducing nsclc invasion. we used chromatin immunoprecipitation (chip) assay kit for detection of tnf-a induced nf-kb mediated mirnas. therefore, h1299 and pc14 cells treated by tnf-a (30 ng/ml) for chip assay. chromatin regions, reading with chip-seq, were analyzed using bioinformatics tools. we also performed additional bioinformatics search to find nf-kb related mirnas which potentially take a role in nsclc invasion. we investigated the effects of mirna which determined at the bioinformatics analysis results on invasion using invasion chamber method. we found 16 mirnas which potentially induced by nf-kb and related with nsclc invasion. our invasion results indicate that mir-548a-3p, mir-548as-3p, mir-8078, mir-1915, mir-6814-3p, mir-548q mimics can induce cellular invasion on h1299, mir-548v, mir-548 h-5p, mir-138-5p, mir-548a-3p, mir-548as-3p, mir-8078 mimics can induce cellular invasion on pc14. we also verified our results by qrt-pcr, because we want to sure that mirnas which can induce invasion, can also transcriptionally regulated by nf-kb or not. we found that mir-548q, mir-548a-3p, mir-584as-3p, mir-1915, mir-8078 in h1299, mir-138-5p, mir-548a-3p, mir-548as-3p, mir-8078 in pc14 can induce cellular invasion by nf-kb. as a conclusion, our investigation indicate that nf-kb can induce nsclc invasion via mir-548a-3p, mir-548as-3p and mir-8078. (this study is supported by tub _ itak grand number 112s636). to understand of the molecular mechanisms of cellular invasion is very important for fight against cancer mechanisms and first step of invasion is emt. cells change phenotypical and genotypical in emt progress. in this study, we focussed on the explore molecular mechanism of tnf-alpha induced cellular invasion of nsclc. we use western blot, qrt pcr and mirna transfect methods for this purpose. we find that tnf-alfa treatment reduce the expression of pten and induce e cadherin expression in a549 cells. when we inhibit nf-kb activity by p65 targeted sirna tnf-alpha can not reduce pten expression means that tnf-alpha inhibits pten expression through on nf-kb. because tnf/nf-kb pathway change the transcriptional level of mir-548as and pten 3 0 untranslated region has recognition site for mir-548as. therefore; we transfected a549 cells by mir-548as. mir-548as transfection leads to inhibition of pten expression. our results indicate that tnf-alpha mediated activation of nf-kb can inhibit pten expression on induction of emt through on mir-548as. (this study is supported by tubitak grand nummber 112s636). introduction: the corpus luteum (cl) is an ephemeral tissue whose regulated secretion of progesterone is essential for maintenance and/or timely termination of pregnancy in rodents. our previous finding that cl of pregnant rats does not possess fas/ fasl system suggests that this tissue may undergo autophagic, but not apoptotic, cell death during regression. we here investigated the presence of autophagic system in cl and its any implications in rodent pregnancy and parturition. materials and methods: lc3 (-i and -ii) expression in cl was estimated by western blot analysis in comparison with progesterone secretion and luteal mass throughout pregnancy. lc3 was also tested by immunocytochemistry. functional implication of autophagy in this tissue was examined by local treatment with bafilomycin a1 (autophagy and v-atpase inhibitor, 6.23 pg/ 0.1 ml/ovary) on day 19 of pregnancy. reproductive, biochemical, and morphological outcomes were evaluated. results: while the expression of cytosol-associated lc3-i was not significantly altered throughout pregnancy, that of autophagosome-associated lc3-ii increased significantly from day 15, showed a peak on day 21, and decreased on day 23 (day of normal parturition). lc3-ii / i ratio had positive correlations with steroidogenic activity and cell size. immunoreactive lc3 was found to be present in the cytosol of steroidogenic cells and showed marked aggregation in cells on day 21. in vivo treatment with bafilomycin a1 resulted in unaltered delivery, but in significant reductions in steroidogenic cell size and neutrophil infiltration compared to vehicle-treated control groups. discussion and conclusion: the ratio of lc3-ii / i in cl was markedly up-regulated in the late phase of pregnancy. manifestation of this autophagy parameter was temporally matched with further structural and functional activation of cl. autophagy may contribute to activation, but not regression, of rodent cl and thus their female reproduction. apoptotic and necrotic effects of low dose bisphenol a in shsy5y neuroblastoma cells b. ayazg€ ok, t. t€ uyl€ u k€ uc ߀ ukkilinc ß faculty of pharmacy, hacettepe university, ankara, turkey bisphenol a (bpa) is a commonly used chemical in industry to make plastics. 'low-dose' term has been expressed for the first time in studies with bpa in 2001. the value of low dose bpa was received as <1lm. in this study, matrix metalloproteinases (mmps) together with their tissue inhibitors (timps), involved in tissue remodeling after i-131 therapy, have been examined in 51 patients (8m/46f) with ptc and 38 (3m/38f) with ptc+ht. peripheral blood samples were collected just before and, subsequently, at 4 days after i-131 administration (3.7 gbq). ptc+ht patients had positive titers of anti-thyroglobulin autoantibodies (tgab). the serum levels of tgab, mmp-2, mmp-9, timp-1 and timp-2 were measured by elisa. there were no significant changes in serum concentrations of mmp-2, timp-2 and mmp-2/timp-2 ratio after i-131 in the two groups. in ptc patients, i-131 administration resulted in an increase with 26% in timp-1 level (p = 0.005) and a reduction with 44% in mmp-9/timp-1 ratio (p = 0.003). in ptc+ht patients it has been observed an increase with 18% in tgab level (p = 0.001), 5% in mmp-9/timp-1 ratio (p = 0.003) and unchanged timp-1 serum concentration. tgab titers were positively correlated with mmp-9/timp-1 ratio (r = 0.51, p < 0.001). our data suggest that radioiodine therapy for ptc patients, but not for ptc+ht, modulates the balance of mmp-9/timp-1 for anti-invasion and anti-migration by augmenting timp-1. in criminal cases, the determination of the time of death of the bodies step in the analysis of events is making a big contribution. today, forensic and molecular methods utilized time of death rather than provide clear information offers potential death time interval. principally, 'time of death' is a term that should be avoided. in forensic science, the postmortem 'interval' is the term to be considered. nowadays, there is no precise molecular method that can be used alone in the determination of pmi. aim of this study is to analyze the usability of ecm components, adamts1,5 and 9 and mmp1,2 and 9 to determine pmi. for this purpose, with iliopsoas muscle tissues provided by ethical board of the ankara institute of forensic medicine, 21 cases have been included in this study, divided into 3 groups according to their pmis: '0-12 h', '12-18 h' and '18-27 h'. from these tissues, western blotting technique is used to analyse the expression of adamts1,5 and 9 and mmp1,2 and 9. it is determined that when pmi increases; adamts-1 and 9 amounts decrease. on the other hand adamts-5 amounts are examined to increased related to the interval., especially on the '12-18 h' dataset. additionally, considering metalloprotease levels, mmp-2 and 9 amounts decrease as pmi increases. unlike mmp-2 and 9; mmp-1 levels increase proportional to the interval, especially on the '18-27 h' dataset. these results are the first data on pmi determination from iliopsoas muscle tissue. in this study, it is suggested that adamts-5 and mmp-1, proteases responsible for ecm degradation, can be used to determine pmi as markers. here we present the first observations of postmortem variation of mmp and adamts activities in skeletal muscle. in recently, popular mmps and adamts s pathways of the relationship between time-dependent changes to investigate the post mortem time interval determination to shed light on the future. the role of functional polymorphisms of matrix metalloproteinases 2 and 9 in spontaneous abortion samples capillaries, which are responsible for maintenance of implantation and placental nutrition, have major effects on mechanisms underlying abortion. matrix metalloproteinases (mmps) function in various cellular pathways. they play a role in patholohical conditions, metastasis; as well as normal physiological functions like tissue and bone regeneration, physiologic function of uterus, ovulation, embryogenesis and embryo implantation. mmp2 and mmp9 (gelatinases) have key roles at organisation of extracellular matrix and affect endometrial implantation. previous studies have shown that mmp2 -1306c>t and -735c>t polymorphisms cause loss of gene function, and mmp9 -1562c>t polymorphism causes a decrease in gene expression, and also gene expression levels are lower in abortion specimens, compared to control specimens. the goal of this study was to investigate the potential effects of functional mmp2 -735c>t and -1306c>t polymorphisms, and mmp9 -1562c>t polymorphism on etiology of abortion. restriction fragment length polymorphism (rflp) method was used to analyze the polymorphisms those evaluated in the study. study group consisted of samples collected from 80 spontaneous abortion specimens, and control group consisted of peripheral blood blood samples collected from 100 healthy subjects. the risk of abortion was 2.2-fold higher in patients with heterozygous -1306c>t polymorphism (p = 0.043). combined genotype analysis showed that the risk of abortion was 3.7-fold higher for patients with normal -735c>t polymorphism, and heterozygous -1306c>t polymorphism (p = 0.021). functional polymorphisms of mmp2 and mmp9 may have a role in etiology of abortion. p-02.07.5-011 changes in the specific extracellular matrix proteins and expression of adamts proteinases and effects of hypoxia in the adriamycin-induced renal fibrosis model renal fibrosis is a common cause of renal dysfunction with chronic kidney diseases. this process is characterized by excessive production of extracellular matrix (ecm) or inhibition of ecm degradation. adamts proteinases, which are widely presented in mammals, have very critical roles in ecm remodeling. we aimed to study the role of adamts proteinases in the pathogenesis of renal fibrosis and the effets of hypoxia by studying adamts expressions in rat kidney after adriamycin (adr) administration. adr was administered intravenously in consequtive two doses (2.5 and 4 mg/kg) to the rats. in addition to control and adr groups, another rats were assigned into three groups as sham, 15 min and 45 min ischemia-reperfusion. after 2 months following the first dose, the expression of adamtss were determined in the renal tissues using western blot analysis. additionally, renal nephropathy and fibrosis were assessed pathological and immuno-histochemical staining methods. in the adr group rats developed renal dysfuntion and tissue damage in favor of renal fibrosis pathologically. it is observed that occurred remarkable changes in the expression of adamtss. it is showed that hypoxia and hypoxia time enhanced tubulointerstitial fibrosis in the rat kidney tissues. expression differences were absorved also in the hypoxia groups, and especially the expression of adamts-1 and -15 genes were showed an increase in various rates. the restricted and different expression pattern of adamts protein profiles in the adr-induced renal fibrosis suggest that adamts family members are related with development and progression of fibrosis. moreover, our findings raise the possibility that the hypoxia promotes renal fibrogenesis. the monitoring of adamts proteinases might contribute to the early diagnosis of renal fibrosis and chronic kidney disease. adams (a disintegrin and metalloproteas) are transmembrane proteins that contain a pro-domain, a metalloprotease, a disintegrin, a cysteine-rich, an epidermal-growth factor like and a transmembrane domain, as well as a c-terminal cytoplasmic tail. they are involved in cell adhesion and they have protease activities. previous studies showed that some adam proteins act in a highly diverse set of biological processes, including fertilization, neurogenesis, myogenesis, embryonic tgf-a release and the inflammatory response. although there are more researches about adam proteins, still the function of all adam proteins remain unclear. we aimed to investigate the potential link of infertilty with adam9, -10, and -17. in this study twenty four patients were included. the patients were classified as normozoospermia (ns; n = 8), oligozoospermia (os; n = 8), azoospermia (as; n = 8) groups. adam9, -10 and -17 protein levels in infertility indviduals were analysed by western blot. adam9 protein level was 1.7 fold lower in the os and as groups than in the ns group. adam10 protein level was 1.36 fold higher in the as group than in the ns group. adam17 protein level was 2 fold lower in the as group accourding to ns group. we observed no change between protein level of adam10 and adam17 of os and ns groups . in conclusion, adam proteins may have a potential role in male infertility. our study is a preliminary and first study on this issue. keywords: adam, infertility. the role of tissue metalloproteinase inhibitors thymus chemokine and thrombospondin-1 on prognosis of crimean-congo hemorrhagic fever s. bakir, m. bakir, s. ersan, a. engin cumhuriyet university, sivas, turkey crimean-congo hemorrhagic fever (cchf) is a disease which is caused by an arbovirus carried by ticks and characterized by the sudden onset of high fever, severe headache, dizziness, back and abdominal pain. cchf pathogenesis is still not resolved today to fully open. therefore, in this study, to determine the level of tck-1, timp-1 and tsp serum samples obtained from cchf patients and the control group is intended to be examined for the pathogenesis and prognosis of the disease. the study sample was created 45 patients with diagnosis of cchf. 45 healthy volunteers were chosen control group matched for gender and similar to in terms of age. tsp, tpc-1 and timp-1 levels of patients and a control group were analyzed using the human elisa kits. serum timp-1 tck-1 and tsp levels in cchf patients were measured significantly higher than the in the control group. cchf pathogenesis of today still have not provided fully open. therefore, it reveals the importance of this work. in our study, presence of high timp-1, tck-1 and tsp levels indicate important direction for pathogenesis and prognosis of cchf disease. p-02.07.5-015 activation of calpain 1 and protein kinase ca promotes a constitutive expression and release of matrix metalloproteinase 9 in peripheral blood mononuclear cells from cystic fibrosis patients matrix metalloproteinase 9 (mmp9) is physiologically involved in remodeling the extracellular matrix components but its abnormal release has been observed in several human pathologies, including cystic fibrosis. we have studied if the alteration in intracellular ca 2+ homeostasis, observed in peripheral blood mononuclear cells (pbmcs), isolated from cystic fibrosis (cf) patients homozygous for deletion of phenylalanine 508 in gene of cystic fibrosis transmembrane conductance regulator (f508del-cftr), could be involved in the abnormal presence of mmp9 in the extracellular fluids of cf patients. pbmcs were isolated from 23 healthy donors and 26 cf patients homozygous for f508del-cftr. mmp9 levels were evaluated following 2 h of cell incubation. our investigations show that all cf pbmcs analyzed constitutively express and release high levels of mmp9; conversely, in pbmcs from healthy donors, expression and secretion of mmp9 are undetectable but both events can be evoked, after 12 h of cell culture, by a possible paracrine stimulation. we have demonstrated that in cf and 12 h-cultured control pbmcs mmp9 secretion is prevented by chelation of intracellular ca 2+ and mediated by the concomitant activation of calpain and protein kinase ca (pkca) and also that mmp9 expression is mediated by the sequential activation of pkc and extracellular signal-regulated protein kinases 1 and 2 (erk1/2). moreover, the rescue of active f508del-cftr reduces the extent of mmp9 secretion, correlating the channel defect to the [ca 2+ ] i dysregulation of cf pbmcs. our results indicate that the high level of intracellular ca 2+ concentration in cf pbmcs, promoting the aberrant activation of both calpain and pkca, induces a constitutive release of mmp9. these data characterize new alterations in mononuclear leukocytes of cf patients that may be of primary importance in the progression of the disease and indicate that pbmcs may contribute to the accumulation of mmp9 in fluids of cf patients. p-02.07.5-016 aebp1/aclp is upregulated in differentiation, injury repair and fibrotic degeneration of skeletal muscle c. € ozdemir 1,2 , u. akpulat 1 , i. onbasilar 3 , c ß . kocaefe 1 1 department of medical biology, faculty of medicine, hacettepe university, ankara, turkey, 2 department of stem cell, institute of health, hacettepe university, ankara, turkey, 3 laboratory animal breeding and research unit, faculty of medicine, hacettepe university, ankara, turkey aebp1/aclp is an ambiguous gene with several attributed functions and cellular events, adipogenic differentiation, cell adhesion, pattern development and fibrosis are the well-understood. aebp1 is the short isoform that acts as a transcriptional repressor by targeting the ap2 promoter and aclp, which is the long isoform that harbors a leader sequence that directs the peptide to the extracellular compartment. the latter is known to be associated with development of the connective tissue, injury repair and fibrosis in certain pathological conditions. aebp1/aclp displays a moderate expression in skeletal muscle where the role is not known. we have investigated the spatial and temporal expression of aebp1/aclp in defined models of skeletal muscle differentiation, injury repair and fibrosis. aebp1/aclp expression is present in steady state dividing myoblasts. this basal expression is upregulated 4 folds upon the induction of differentiation in c2c12 cells. considering that differentiation and post-natal injury repair share several common aspects, we also investigated the expression of aebp1/aclp in acute injury-repair model. in the course of cardiotoxin induced injury, aebp1/aclp expression peaks up to 5 folds in the 6th day of regeneration. this time point concomitantly corresponds to tissue remodelling. since aebp1/aclp is also known to be associated with fibrotic events in chronic pathological conditions, we also have investigated its expression in tenotomy induced skeletal muscle degeneration which mimics endomysial and perimysial fibrosis. aebp1/aclp expression is upregulated up to 10 folds in early time-point samples. these results depict a novel role for aebp1/aclp in extracellular remodeling of the skeletal muscle during injury repair as well as fibrotic degeneration. the source of this expression might come from fibroadipogenic precursers which reside in endomisial area of muscle. our current efforts are focused on presenting of this endomysial expression. the mprbp gene from b. pumilus 3-19 encoding a novel secreted metalloproteinase was identified. based on the primary structure the enzyme has been classified as metzincin metalloproteinase that combines the features of two families: astacin and adamalysin. representatives of the adamalysin family previously have not been described for bacilli. the aim of the work was to elucidate the mechanisms of the gene expression regulation of a new bacillus pumilus 3-19 extracellular metalloendopeptidase. promoter region analysis revealed the presence of potential binding sites for transcription factors spo0a (sporulation) and degu (biodegradation). study of mprbp expression in strain defective in regulatory proteins degs and degu shows that the productivity of metalloproteinase biosynthesis decline in average 60% compared with the strain with a complete degs-degu system. we also studied mprbp expression in strains with a mutation in the gene degu, leading to stabilization of degu~p protein. it is known, that this mutation leads to a multiple increase in the gene expression level, positively regulated by degs-degu system. our data shows a 10-fold increase in metalloproteinase productivity in the recombinant strain. thus, deg-system participates in control of the proteinase synthesis but not only in the regulation of mprbp gene expression. the mprbp expression in the strain deficient in regulatory protein spo0a remained at the level with expression in the strain with the complete spo0a. a similar pattern we observed in the study of mprbp gene expression in strains defective in other spore-specific regulatory proteins (spo0b, spo0f, spo0k, spo0j, sigf, sigh, sigk). these data indicate that mprbp gene expression is free of spo-regulatory proteins. on this basis, we concluded that the expression of metalloproteinase gene is not correlated with the sporulation. p-02.07.5-019 paricalcitol attenuate activity and expression of matrix metalloproteinases in a rat model of renal ischemia-reperfusion injury matrix metalloproteinases (mmps) are endopeptidases involved in the degradation of extracellular matrix. they have been postulated to have a role in the pathogenesis of ischemia-reperfusion injury (iri). in the present study, we investigated the effect of paricalcitol, a synthetic vitamin d analog, on mmps in renal iri. 21 wistar albino rats were divided into three groups: sham operated, ischemia-reperfusion, and paricalcitol-pretreated. iri model was induced by bilateral clamping of renal arteries for 45 min followed by 24 h of reperfusion. the analysis of serum creatinine levels and activities/expressions of mmp-2 and -9 were performed after 24 h of iri. the effects of paricalcitol on activities and expressions of mmp-2 and mmp-9 levels were investigated by gelatin zymography and immunohistochemistry, respectively. the pathological examinations were performed to score tubular damage by light microscopy. creatinine levels increased significantly in the iri group. rats in the paricalcitolpretreated group showed significant decrease in expressions and activities of mmp-2 and mmp-9 during iri. moreover, pathological examinations displayed significantly lower score of tubular damage in paricalcitol-pretreated group. in conclusion, paricalcitol attenuated iri by downregulating the expressions and activities of mmp-2 and -9. p-02.07.5-020 the changes of matrix metalloproteinase 2, 9 activity and hyaluronic acid level in rat's heart and serum under cadmium influence o. fomenko 1 , o. shaulska 1 , y. kot 2 , g. ushakova 3 , a. shevtsova 1 1 dnipropetrovsk medical academy, dnipropetrovsk, ukraine, 2 kharkiv national university, kharkiv, ukraine, 3 dnipropetrovsk national university, dnipropetrovsk, ukraine the changes in the molecular mechanisms of the extracellular matrix degradation under toxic factors are not well known. the main goal of work was the investigation of the mmp2 and mmp9 activity and hyaluronic acid level in the heart and blood serum under cadmium influence at different doses. the 18 wister rats divided to 3 groups were used for the experiment. cdcl 2 x2.5h 2 o in doses 0.1 lg/kg and 1 lg/kg was given to rats intragastrically in drinking water during 36 days. the rats were decapitated under isoflurane anesthesia according to ethical rules; the heart was quickly removed. the relative activity [in arbitrary units (au)] of pro-and active forms of mmp9 and mmp2, total protein (tp) and hyaluronic acid levels were calculated. it was shown that low doses of exogenous cadmium (0.1 lg/ kg) lead to reduced activity of pro-and active forms of mmp9 in myocardium (7.3 ae 0.6 au/mgtp and 7.1 ae 0.6 au/mgtp compare to the 9.67 ae 0.4 au/mgtp and 9.7 ae 0.5 au/mgtp in the control rats accordingly) and in serum (0.95 ae 0.2 au/mgtp and 0.35 ae 0.05 au/mgtp compare to the 1.54 ae 0.05 au/mgtp and 1.49 ae 0.05 au/mgtp in the control rats accordingly), but pro-mmp2 activity in heart was increased (14.5 ae 1.6 au/mgtp compare to the 9.8 ae 0.6 au/mgtp in the control rats); level of ha was decreased in both tissues (0.69 ae 0.16 lg/ml and 3.63 ae 0.3 lg/ml compare to the 1.0 ae 0.13 lg/ml and 3.91 ae 0.3 lg/ml in the control rats accordingly). high doses of cadmium (1 lg/kg) caused a reliable increase of both gelatinase activity in the myocardium: mmp2 increased from 9.65 ae 0.4 au/mgtp to 14.1 ae 0.8 au/mgtp, prommp9to 12.6 ae 1.5 au/mgtp, mmp9to 15.4 ae 1.6 au/mgtp. ha level was increased in serum (4.28 ae 0.1 lg/ml) and decreased in heart (0.49 ae 0.09 lg/ml). the results indicate the dose-dependent and tissue-specific effect of cadmium on mmp-depended protein degradation and level of hyaluronic acid. a disintegrin-like and metalloproteinase domain with thrompospondin-1 repeats (adamts) are a large family of proteoglycanase that show proteolytic activity towards proteoglycans like aggrecan, brevican, neurocan, and versican. interleukin-33 (il-33) is an il-1 cytokine family member that uniquely plays a role as a cytokine and nuclear factor. it is released by necrotic epithelial cells and activated innate immune cells as an alarming danger signal. adamts and il-33 implicated in brain cancer pathogenesis. we aimed to seek the amount of adamts15 in u118 proteolytic enzymes are able to speed up wound healing by removal of the necrotic tissues and fibrin. several investigations have reported that proteases damage also the microbial biofilms formed by opportunistic bacteria including staphylococci on surfaces of chronic and acute dermal wounds. therefore, proteases are seemingly perspective enzymes for biofilm eradication by hydrolysis of both matrix proteins and adhesins, proteins providing cells attachment onto solid surface and other bacteria, as well as by the cleavage of signalling peptides of intercellular communication of gram-positive bacteria. here we report that ficin, a plant protease, efficiently degrades the structural components of biofilm matrix formed by s. aureus and s. epidermidis at concentrations of 10 lg/ml while trypsin and chimotrypsin are used as 1-2 mg/ml solution. the spatial structure of the biofilm was analyzed by atomic force microscopy. after ficin treatment, the biofilm structure became porous, with reduced viscosity. the congo red staining of the treated biofilms confirmed the hydrolysis of the protein component of the matrix. moreover, the biofilm treatment with ficin increased the antimicrobial efficiency of ciprofloxacin against biofilm-embedded cells of s. aureus and s. epidermidis. while 24 h antibiotic treatment did not lead to the increase of dead cells of neither s. aureus nor s. epidermidis embedded into the biofilm matrix, in the presence of ficin the fraction of viable cells decreased significantly. accordingly, soluble ficin appears beneficial for outer wound treatment biofilm eradication and reduces the reinfection risk. the wound-healing activity of ficin requires further investigations. this work was supported by the russian science foundation (project no 15-14-00046). resveratrol (resv) is an antioxidant that belongs to the group of plant compounds, called polyphenols. resv is defined as an antimicrobial substance that is produced by several plants (red grape skin, peanuts and berries) to protect them from rough environments like excessive ultraviolet light, infections and climate changes. as an antioxidant, this polyphenol protects the body against cardio-vascular and cancer diseases. besides, resv has anti-inflammatory, neuro-protective, anti-diabetic and other pharmacological effects. although the positive pleiotropic effects of this polyphenol are well documented, there is a huge need to understand its influence on the biophysical properties of lipid bilayer. in the present work, the interaction of resv with membranes composed of palmitoyl-docosahexaenoyl phosphatidylcholine (pdpc) or palmitoyl-oleoyl phosphatidylcholine (popc), sphingomyelin (sm) and cholesterol (ch) was investigated by means of fluorescence spectroscopy. generalized polarization of the fluorescent probe laurdan (gp) as a function of temperature was used to probe the changes in the fluidity of lipid bilayer induced by different resv concentration. the obtained results showed decreased lipid ordering from 50 to 100 lmol resv and opposite effect from 200 to 500 lmol in pdpc/sm/ch mixtures as compared to the control without resv. the interaction of resv with popc/sm/ch mixtures caused only an increase in the lipid ordering as a function of resv amount. popc and pdpc have the same head group but different fatty acid chains at sn-2. since resv changes the physicochemical properties of lipid bilayer by different ways one might suggest that the interaction of polyphenol with the membrane depends on the level of fatty acid unsaturation. this specific effect of resv on lipid organization could be related to its unique properties to prevent the cell from oxidative stress. neurodegeneration is the umbrella term for the deseases including progressive loss of structure or function up to death of neurons. beta-amyliod peptide is proteolytic fragment of the amyloid protein. the spontaneously formation of selective, voltage-dependent, ion-permeable channels in the lipid bilayers was reported as one of amyliod peptide toxicity mechanisms. the aim of our study was the investigation of the influence of the flavonoids (phloretin, phlorizin, quercetin and genistein) on the membrane activity of amyliod peptides. virtually solvent-free bilayer lipid membranes were prepared from mixtures of phospholipids in 0.1 m kcl (ph 7.4) using monolayer-opposition technique. using spectrofluorimetry we estimated prepared from phospholipids by extrusion the liposomal membrane permeability for calcein. we found that the addition of phloretin into membrane bathing solution led to an signicant increase in the channel forming activity of fragments 25-35 of amyloid peptide, fragment 25-35 of [gly35]-amyloid peptide and 106-126 of human prion protein. addition of other flavonoids did not cause any changes in the steady-state amyloid-induced current. it was found that the effect was caused by electrostatic interaction with the peptide. we found that time course of amyloid induced leakage calcein from liposome's is characterized by two components: the fast one is related to sorption of b-amyloid peptide on the membrane and the slow one is related to the oligomerization of the peptides on the surface of the lipid bilayer. addition of the phloretin simultaneously with b-amyloid peptide to the suspension of liposomes caused significant reduction in time parameters characterizing fast and slow components. from this results we can proposed that phloretin compensates the positive charge of the b-amyloid peptides and leads to the changes in their oligomerization status. the study was supported in part by rfs (14-14-00565) and sp-69.2015.4. ferritin nanocarriers: a focus on a metal-based drug encapsulated inside the protein cavity s. ciambellotti 1 , c. bernacchioni 2 , f. scaletti 3 , p. turano 1 1 cerm (magnetic resonance centre), florence, italy, 2 department of biochemical sciences, university of florence, florence, italy, 3 chemistry department 'ugo schiff', florence, italy ferritin is one of the main player involved in the iron metabolism. the biological role of ferritin is the storage of iron atoms inside the cavity preventing the uncontrolled accumulation of toxic species inside cells. ferritins are polymers constituted by 24 subunits that self-assemble giving rise to an almost spherical nanocage. in vertebrates, ferritins are formed by two distinct subunits, the heavy chain (h), with oxidoreductase activity, and the light chain (l) without catalytic activity. ferritins are promising nanocarriers for the delivery of contrast agents for diagnosis and of drugs for therapeutic purpose. their endogenous origin and the possibility to stabilize and protect the enclosed cargo inside the cavity, make ferritin a biocompatible vehicle. moreover, there are specific receptors on cells that recognize and incorporate ferritin by endocytosis, prospecting a kind of targeted-delivery. following the increasing interest in nanotechnology, we studied the interaction between different isoforms of ferritin with an antimetastatic drug, called nami-a, which is the first ruthenium derived anti-cancer drug to have entered clinical evaluation. this molecule is a metal-based prodrug that can release the metal ion ligands. here, we describe nami-a hydrolysis in the presence of recombinant homopolymers of ferritin followed spectrophotometrically. thanks to characteristic time dependent changes of spectral profile in the uv-visible region, we could detect differences in the hydrolysis process. the formation of a ru-adduct with hferritin was established by uv-visible and circular dichroism spectroscopies, as well as by kinetics measurements that showed inhibition of the ferroxidase activity of h-ferritin. crystallization trials are in progress to identify the binding site of ru by solving the x-ray structure of the complex. finally, we planned to test the cytotoxicity of ferritins pre-incubated with nami-a, using different cancer cell lines. a. cort 1 , t. ozben 2 , a. sansone 3 , s. barata-vallejo 4 , c. chatgilialoglu 5 , c. ferreri 3 1 sanko university, gaziantep, turkey, 2 akdeniz university, antalya, turkey, 3 cnr, bologna, italy, 4 universidad de buenos aires, buenos aires, argentina, 5 national center for scientific research 'demokritos', athens, greece liposomes as biomimetic models of cell membranes were used for examining some novel aspects of drug-metal induced reactivity with unsaturated lipids under oxidative conditions. the chemical basis of cis to trans transformation was ascertained by liposome experiments, using bleomycin-iron complexes in the presence of thiol as a reducing agent that by incubation at 37°c gave rise to the thiyl radical-catalysed double bond isomerisation of membrane phospholipids. the effect of oxygen and reagent concentrations on the reaction outcome was studied. as a chemical biology model for antitumoral strategies, liposomes highlight the role of cell membranes, which are not spectators but important targets of the drug effect, with synergic roles for chemotherapeutic effects. indeed, fatty acid recruitment and membrane formation are attracting a lot of interest in cancer, and in this context the loss of the natural cis geometry and the oxidationinduced lipid remodelling are worthy of deeper studies in antitumoral strategies. furthermore, the interaction between drugs and lipids can be suggestive of novel aspects of chemical reactivity for liposome carriers when circulating in vivo. gpr17 is a g-protein coupled receptor (gpcr), expressed in cells of brain, heart and kidney. it is related to the leukotriene and purinergic subfamilies of the rhodopsin-like class a gpcrs. gpr17 plays controversial role in the brain and spinal cord cells recovery after injuries. it is assumed that gpr17 is one of the cell death regulators immediately following an injury but at later stages it also takes part in tissue regeneration. there are also data implying some role of gpr17 in glucose homeostasis. drugs targeting gpr17 may help with treatments of multiple sclerosis and ischemia. the damage of rat's brain in artificially induced ischemia disease decreased after gpr17 inhibition. in addition, gpr17 takes part in myelin sheath formation, the lack of which is known to be the reason of multiple sclerosis. to better understand functional role of gpr17 and enable design of more efficient ligands we initiated structural studies of this receptor. to improve receptor stability and facilitate crystallization we created a series of chimeric constructs using different fusion partnerssmall soluble proteins inserted into the native amino acid sequence. preliminary experiments were carried out to evaluate the influence of different ligands on the receptor stability and cell surface expression in insect sf9 cells. this work was supported by russian federal target sterols are significant for the structural and dynamical features of cell membranes. among them, cholesterol is the major sterol in mammalian cell membranes whereas stigmasterol is the predominant sterol in plant membranes. stigmasterol varies structurally from cholesterol in having both an ethyl group at carbon 24 and an additional trans double bond between carbons 22 and 23. dimyristoylphosphatidylcholine (dmpc) is a widely studied synthetic lipid, which has a neutral (zwitterionic) pc headgroup and two symmetrical 14-carbon fatty acyl chains. the studies on the interactions of cholesterol and stigmasterol with dmpc membranes at molecular level are very limited. in the present study, a calorimetric comparison of the effects of the animal sterol cholesterol and the plant sterol stigmasterol on zwitterionic dimyristoylphosphatidylcholine (dmpc) multilamellar vesicles (mlvs) was investigated for the first time by using differential scanning calorimetry (dsc). our dsc studies indicate that with the inclusion of increasing cholesterol and stigmasterol concentrations from 5 to 40 mol% into pure dmpc mlvs, the pretransition disappears, the main phase transition shifts to lower temperatures and then disappears at cholesterol and stigmasterol concentration above 25 mol%. the main phase transition enthalpy (dh) is progressively reduced whereas full width at half maximum (dt ½ ) gradually increases with increasing cholesterol and stigmasterol concentrations. moreover, the main phase transition peak consists of overlapping sharp and broad components, indicating the hydrocarbon chain melting of sterol-poor and sterol-rich dmpc domains, respectively. in conclusion, this study shows clearly that both cholesterol and stigmasterol interact effectively with dmpc membranes and cause changes in their structural and functional properties. p-03.03.3-007 trh receptor mobility in the plasma membrane is affected by its interaction with its cognate signaling molecules and by cholesterol depletion r. moravcova, b. melkes, j. novotny department of physiology, faculty of science, charles university in prague, prague, czech republic g protein-coupled receptors (gpcrs) play a fundamental role in transferring information from extracellular environment to the cell interior. some gpcrs are supposed to form signaling complexes with their cognate g proteins and possibly other accessory proteins, which may facilitate the activation of g proteins and thus accelerate signal transmission. here we investigated the role of some components of thyrotropin-releasing hormone (trh) receptor signaling in hek293 cells stably expressing yfp-tagged trh receptor using fluorescence recovery after photobleaching (frap). we observed significant changes in values of the diffusion coefficients if expression of b 2 -arrestin or gb 2 subunit were suppressed by sirna. results of our frap experiments indicated significant difference between control and trh-treated cells as the diffusion coefficient markedly decreased after agonist stimulation. on the other hand, the same decline of the diffusion coefficient value was found after silencing with sirna and subsequent treatment with trh for most of the screened proteins. treatment of cells with 10 à5 m trh led to fast internalization of trh receptor, which was revealed by real-time confocal microscopy. it is known that cholesterol is an essential component of the cell membranes and it exerts modulatory effects on the functioning of various membrane proteins. disruption of plasma membrane integrity by cholesterol depletion using b-cyclodextrin significantly reduced the apparent diffusion coefficient values. interestingly, addition of trh to cells treated with b-cyclodextrin did not further reduced trh receptor mobility. it can be concluded that stimulation with agonist and/or sirna silencing of some components of the trh receptor signaling cascade significantly affects the mobility of trh receptor in the plasma membrane. p-03.03.3-008 l-opioid receptor mobility in the plasma membrane is diversely affected by biased ligands b. melkes, l. hejnova, j. novotny opioid receptors belong to the large family of g protein-coupled receptors (gpcrs), which are currently considered among the most important targets for pharmacological manipulations. during the past few years, a great deal of attention has been devoted to biased agonism. this phenomenon reflects the ability of different ligands to selectively affect the functioning of some gpcrs so they can display marked differences in their efficacies for g protein-or b-arrestin-mediated signaling. here we decided to investigate the effect of different agonists of the l-opioid receptor (l-or) on the lateral mobility of this receptor in the plasma membrane of hek293 cells which were stably transfected with l-or tagged with yellow fluorescent protein (l-or-yfp). it has been found previously that damgo stimulates g-protein-dependent signaling, endomorphine 2 stimulates arrestin-dependent signaling and morphine does not show any significant bias towards these two signaling pathways. in our experiments, we used the fluorescence recovery after photobleaching (frap) method to estimate the diffusion coefficients of l-or-yfp in the resting state and after addition of the above mentioned specific agonists. we observed that addition of damgo increased the value of the diffusion coefficient and addition of endomorphin 2 decreased the value of diffusion coefficient of l-or-yfp. addition of morphine or the l-or antagonist naloxone did not change the value of the diffusion coefficient compared to the resting state. these results indicate that different biased agonists of l-or may differently affect the mobility of this receptor in the plasma membrane. these findings provide new insights into the dynamics of l-or in the plasma membrane and contribute to better understanding of the mechanism of biased agonism at gpcrs, which is of central importance for receptor homeostasis and fine regulation of receptor activity. color tuning and adding potassium selectivity to a light-driven sodium pump k. kovalev 1,2 , v. polovinkin 1,2,3 , v. shevchenko 1,2 , v. gordeliy 1,2,3 1 moscow institute of physics and technology, dolgoprudniy, russia, 2 research centre j€ ulich, j€ ulich, germany, 3 institut de biologie structurale, universit e grenoble alpes, cnrs, and cea, grenoble, france recently a light-driven sodium pump has been discovered, characterized and tested as an inhibitory optogenetic tool. sodium pumping rhodopsin from dokdonia eikasta kr2 has an absorption maximum at 523 nm at ph 7.5 and to create more redshifted variants we analyzed available structures of the kr2 (pdb codes: 4xtl, 4xtn) and did the rational mutagenesis of residues in the retinal proximity region (i.e. m149, g153 and s254). the mutants of kr2 under investigation were: m149a, g153v, m149a/g153v, s254a, s254f, s254g, s254l, s254m, s254n, s254t, s254v, s254y. the protein mutants were expressed in escherichia coli c41 strain, expression was induced by the addition of 1 mm isopropyl b-d-1-thiogalactopyranoside. the cells were then washed trice with unbuffered 100 mm nacl or kcl solution. finally, the ph changes in cell suspensions (od600 = 8.0) were monitored. ph changes upon the addition of 30 lm of protonophore carbonylcyanide m-chlorophenylhydrazone were also measured. the following mutants completely abolished the protein function and were not used for further characterization: s254f, s254l, s254m, s254n, s254v. the remaining mutants have shown sodium pumping activity and s254a mutant has gained an additional potassium pumping activity. all functionally active mutants were purified using ni-affinity chromatography and the absorption spectra were collected for them at ph 7.5 (50 mm na/na-pi, 100 mm nacl). m149a mutant absorption maximum is blue-shifted to 519 nm. g153v and m149a/g153vblue-shift to 470 nm. s254a, a potassium pumping variant,red-shift to 545 nm. s254g, s254yred-shift to 545 nm. s254tno change in absorption maximum position. based on structural analysis of kr2 we discovered another potassium pumping variant and provided the variants with absorption maximum blue-shift up to 53 nm and red-shift up to 22 nm. human endothelin receptors belong to the g-protein coupled receptors (gpcrs) superfamily. this class is pharmacologically important, with more than 40% drugs targeting it. the human endothelin system, which includes endothelin receptors types a and b (etb and eta), plays a highly important role in the blood pressure regulation. endothelium cells produce peptides, known as endothelins 1-4, which activate endothelin receptors and launch cascades of reactions that lead to vasoconstriction or vasodilatation depending on the receptor subtype and the tissue. additionally, endothelin receptors take part in such processes as transmission of nerve impulses, development of neural crest, and regulation of acid-base-salt balance in kidneys. in order to stabilize etb receptor for crystallization trials we introduced a compact soluble protein, apocytochrome b564ril (bril), is the third extracellular loop of the receptor. bril is known to be an effective crystallization driver for gpcrs. the engineered protein was expressed using baculovirus system in sf9 insect cells, purified and subject to variety of pre-crystallization assays. localization of the overexpressed protein in insect cells was visualized via the confocal microscopy. thermal stability of the protein in the presence and absence of ligands was measured by the thermal shift assay. finally, the mobility of the receptor in lipidic cubic phase (lcp) at many different conditions was probed by the lcp-frap (fluorescence recovery after photobleaching) assay. these tests showed that the obtained protein is thermally stable, functionally active and diffuses well in lcp at certain conditions, making it a suitable candidate for proceeding to in meso crystallization trials. this work was supported by the russian science foundation (project no. 16-14-10273). mitochondrial oxidative phosphorylation is the key metabolic pathway for the production of atp. mitochondrial respiratory chain (rc) defects are some of the most commonly diagnosed inborn errors of metabolism with a diverse spectrum of clinical phenotypes. the aim of the study is to evaluate the rc enzyme activities and histopathological findings in muscle biopsies of patients with suspected mitochondrial disease. muscle biopsy samples were collected from 48 pediatric patients. the samples were homogenized in seth buffer using a glass/glass homogenizer. the activities of citrate synthase (cs) and rc enzymes (complex i, ii-iii, and iv) were measured in tissue homogenates by kinetic spectrophotometric assays by schimadzu uv spectrophotometer (uv-1800). non-collagen protein was determined by the modified lowry assay. activities of complex (c) i, ii-iii and iv (cox) were normalized by cs. histopathological investigations included h&e, modified gomori trichrome, periodic acid schiff, oil-red-o, nadh, sdh, cox and atpase stains. deficiency of rc complexes was detected in 39 biopsies (81%). c iv deficiency was most common (60%), followed by c i (40%) and c ii-iii (27%). multiple complex deficiency was present in 40% and isolated deficiency was present in 42% of the biopsies (20% c i, 20% c ii-iii, 60% c iv). cs activity was elevated in 44% of the biopsies. decreased c i/cs, c ii-iii/cs and c iv/cs ratio was observed in 44%, 42% and 52%, respectively. comparing with histological data, biochemical analysis revealed additional findings in 50% of biopsies. complex iv deficiency, either isolated or accompanied by other complex deficiencies, is most common in our cohort. rc analysis may reveal additional findings to histopathological results and careful clinical investigation with correlation of clinical, biochemical and histopathological data is mandatory for the challenging diagnosis of mitochondrial disorders in childhood. investigation of adipocytokines, activity of glut and na + /k + -atpase in rats fed glucose, fructose, starch-based sugars objective: all over the world, shows a significant increase in obesity and diabetes. intake of foods that contain fructose, glucose and starch-based sugar is a potential risk for metabolic syndrome. obesity and diabetes are important effects of high fructose corn syrup (hfcs). we aimed to research, activity of na + /k + -atpase in addition to glucose transporter (glut) 2, resistin, adiponectin and other biochemical markers in rats fed glucose, fructose and starch-based sugars. materials and methods: study was performed on rats and 3 groups were included in the study. rats were fed with chows that were given either normal diet for control group (70% carbohydrate, 20% protein and 10% fat), high fructose (70% carbohydrate (87% fructose and 13% starch), 20% protein and 10% fat), or high sucrose (70% carbohydrate (87% sucrose and 13% starch), 20% protein and 10% fat). rats were fed with chows for 8 weeks. in this process, the weight of the rats were followed. at the end of the experiment, blood is taken in all groups. level of hba1c, glucose, resistin and adiponectin were studied. glut2 and na + /k + -atpase activity were studied in the liver tissue. results: a significant increase in adiponectin levels were determined in rats fed both hfcs and sucrose (p < 0.001). a significant decrease in level of na + /k + -atpase activity were determined in rats fed both hfcs and sucrose (p < 0.001). there was no significant differance level of hba1c, glucose, resistin and glut2 in rats fed sucrose or hfcs (p > 0.05). conclusions: fructose-rich diet has an effect on changes in the atpase activity and is a major risk factor for obesity. keywords: adiponectin, fructose, glucose, high-fructose corn syrup, na + /k + -atpase, resistin. p-03.03.3-014 nucleic acid-biomembrane lipid selfassemblies: from primordial soup to novel genome organization model and cellular nonviral nanotherapies f. k. demirsoy 1 , n. eruygur 2 , e. s€ uleymanoglu 3 1 biotechnology central laboratory, biotechnology institute, ankara university, ankara, turkey, 2 department of pharmacognosy, faculty of pharmacy, cumhuriyet university, sivas, turkey, 3 department of pharmaceutical chemistry, faculty of pharmacy, gazi university, ankara, turkey turkey nucleic acid-cell membrane complexes has attracted research interest as models in gene regulation, cell cycle and division, as biosensors designs, as well as in molecular evolution. zwitterionic phospholipids, complexed with polyribonucleotides by divalent metal cations (mg2+) are considered as genosome vehicles. their formations are studied by spectroscopic, thermodynamic, interfacial and microscopic approaches to build thermodynamic and kinetic models of their structural transitions. dna forms a mg2+-driven ternary complexes with neutral liposomes both at the airwater interfaces and at vesicle surfaces. the described self-assemblies form relevant models for nuclear pore complexes and their further implications in gene expression and functions. such membrane contacts could be considered also in prokaryotic nucleoids important in bacterial segregation, whereas in eukaryotes these complexes can be regarded as focal points for transcription-translationtranslocation processes. the effects of ozone/oxygen mixture on citrate synthase and mitochondrial complex activities of striated muscle tissue of healthy mice we investigated the effects of ozone/oxygen mixture and oxygen on citrate synthase (cs) and succinate dehydrogenase (sdh) activities of striated muscle tissue of healthy mice. thirty-six mice were included the study. firstly muscle samples were taken from all mice's left thigh muscle in under anesthesia (group 0). secondly mice were randomly divided in four groups as: group 1: oxygen was given once at 3 days for 7 days, group 2: ozone/oxygen was given once at 3 days for 7 days, group 3: oxygen was given once at 3 days for 30 days, group 4: ozone/oxygen was given once at 3 days for 30 days. ozone/oxygen mixture and oxygen were given at a dose of 1 mg/ kg groups (1) (2) (3) (4) . after treatment animals were sacrificed, and muscle samples were taken and stored in à80°c for until the analyses. muscle tissues were homogenized in 0.05 m tris-hci and 0.15 m kci. cs and sdh activities were measured with spectrophotometer. cs and sdh activities were expressed as lmol/min/g tissue. cs and sdh activity results were given as mean ae sd. cs activity has been found in group 0 (37.8 ae 11.7), group 1 (46.9 ae 10.8), group 2 (34.7 ae 6.8), group 3 (32.9 ae 7.5) and group 4 (33.9 ae 8.5). sdh activity has been found in group 0 (2.50 ae 1.03), group 1 (2.37 ae 0.77), group 2 (2.52 ae 1.45), group 3 (2.24 ae 1.06) and group 4 (2.59 ae 1.25). there was no statistically significant difference among all groups in terms of cs (p > 0.130) and sdh activities (p > 0.997). there was no difference between all groups in terms of inflammation, muscle fiber size, regeneration or necrosis. vascular structures, connective tissues, lipid and glycogen content of fibers were normal. cytochrome oxidase activity was also normal. ratio of ragged blue fibers of all groups were less than 0.3%, so they were scored as 1. there was no difference among groups for ragged red fiber content. we have not found a significant effect of ozone/oxygen mixture and oxygen on cs and sdh activities of striated muscle tissue of healthy mice. lipidic cubic phases (lcps) consist of bicontinuous lipid bilayers and water channels. lpcs are widely used for membrane proteins crystallization and further determination of their spatial structures by means of x-ray crystallography. this approach was successfully used for studying g-protein coupled receptors structures. usually crystallization initiates by adding the precipitants (buffers with high salt concentrations). here we propose to use photo-switchable analogs of 1-monoolein to change lattice parameter of lpc. we synthetized a number of novel diazo-analogs of 1-monoolein. their structures were confirmed by nmr-spectroscopy and mass-spectrometry. being incorporated in phospholipid vesicles or detergent micelles they subjected to trans-to cis-isomerization under the light exposure at 365 nm. also we characterized the lcp's structures and properties by small-angle x-ray scattering on the rigaku instrument. one of the synthetized compounds, 3-(4-{-[4-(octyloxy) phenyl] diazenyl} phenoxy) propane-1,2-diol (1% mol), was incorporated into the 1-monoolein cubic phase. according to small-angle x-ray scattering data the structure of the monoolein cubic-pn3 m phase with lattice parameter 106.3 angstrem was not affected by insertion of this photo-switchable monoolein's analog. after the light exposure at 365 nm we observed trans-cis-isomerization. in the same time the cubic-pn3 m phase was not changed but the lattice parameter reduced to 98.8 angstrem. this effect on monoolein lpc is similar to the addition of a precipitant to initiate protein crystallization process. the spontaneous return to the initial lattice parameter was completed after 3 days in dark. thus, we demonstrated the possible controlling of the monoolein cubic phase lattice parameters by adding the photo-switchable diazo-derivatives of monoglyceride analogs, which can be used for crystallization of membrane proteins. evaluation of certain protein and phosphoprotein expression levels by using western blot technique in head and neck squamous cell carcinoma a. kalayci yigin 1 , t. cora 2 1 cerrahpasa faculty of medicine, istanbul university, istanbul, turkey, 2 faculty of medicine, selcuk university, konya, turkey introduction: head and neck squamous cell carcinoma (hnscc) is a primer tumor type in head and neck cancers, characterized by aggressiveness, early recurrence and metastasis. while alcohol and smoking play an important role at pathogenesis of disease, deregulation of some signaling pathways, genes and protein levels related to these pathways are considered as important at contribution of development of hnscc. materials and methods: in this study, protein and phosphoprotein expression levels of the frequently phosphorylated sites (egfr, pegfr, igf-ir, pigf-ir, pdgfrb, ppgdfrb, pten, ppten, akt and pakt) were investigated by using a western blot to confirm the expression of mrna in the context of protein levels at normal-tumor tissues of hnscc and sccl-mt1 that is a hnscc tumor cell line and hek-293 that is a normal cell line. results: as a result of western blot analysis egfr, pdgfrb and igf-1r were detected as highly overexpressed cell surface receptors in tumor tissues of hnscc. discussion and conclusion: the findings of this study revealed the overexpression of other cell surface receptors as well as egfr in hnscc. in conclution, potential pathways were identified by determining the cell surface receptors overexpressed in hnscc, these data support each other and may be important in pathogenesis of hnscc. introduction: the investigation of final products of lipid peroxidation is considered as the main mechanism involved in development of pathogenesis, diagnostics and prognosis of various parasitic illnesses. materials and methods: the concentration of lp-ap in the blood was determined in the study group considered of 129 women (65%), and 69 men (35%). results: before antiparasitic treatment, women infected with g. intestinalis showed a statistically significant 1.5 times increase of gpx activity levels; and 1.7 times ada level increase compared to the control group. after the treatment, the cat activity showed a sharp increase, whereas the ada activity decreased by 1.4 times, compared to the average level before the treatment. the results of the blood samples of the infected men with giardiasis, show the statistically significant increase in the level of all the studied parameters of lipid peroxidation, except the total primary production (tpp). the exception was the mda level, remaining significantly increased, in contrast to the control group and to the condition after antiparasitic treatment. in infected men, the level of cat activity was more than 5.8 times higher than that noted in control group. after treatment the levels of ada activity and gpx returned to the values of the control group, while the level of cat activity remained elevated. conclusion: an accumulation of primary and secondary metabolites in the course of giardiasis seems to confirm its involvement in the induction of oxidative-antioxidative homeostasis. antiparasitic treatment in giardiasis leads to normalization of the ap parameters studied in women and men, except the mda content in the blood of men. therefore, additional antioxidant treatment is advised for the antiparasitic therapy of men. in vitro effects of ethanol on rat brain synaptosome and dose-dependent antioxidative role of boric acid ethanol is a psychoactive drug that is very large and used frequently today. it has suppressive effects brain's comminication pathway. depending on its acute or chronic use and the dose, ethanol increase membrane fluidity and it causes oxidative stress. this study deals with the in vitro effects of ethanol toxicity (200 mm) and potential protective effect of different doses of boric acid (ba) (5, 10 and 25 mm) on rat brain synaptosomes. with this aim, five male spraque dawley rats are killed by decapitation under anesthesia. after the frontal cortexes of the rats are taken out, each of them is divided into four pieces. these pieces were used as a sample in five groups (control, ethanol, ethanol+5 mm ba, ethanol+10 mm ba, ethanol+25 mm ba) which include six samples. the synaptosomal fractions are prepared by the homogenization of the frontal cortex pieces and centrifugation for each samples. as markers of ethanol-induced oxidative stress in the synaptosome of the rats, malondialdehyde (mda), nitric oxide (no) and catalase (cat) levels were measured. mda levels in the ethahol group were a quantity increased compared with those in the control group but it unchanged significantly as statistically (p < 0.05). however the mda level in the ethanol+ boric acid (25 mm) group was shown to be significantly decreased compared with that in the ethanol group (p < 0.05). the cat activity of the ethanol group was significantly higher than that in the control group and cat activity of the ba (5 mm,25 mm) groups were close compared with control groups (p < 0.01). no levels in ethanol groups were decreased but unchanged compared with control groups as statistically. neverthless, no levels in ethanol+ boric acid (25 mm) groups were increased (p < 0.05). these results demonstrate that ethanol (200 mm) is capable of triggering damage to rat brain synaptosome and ba could be influential in antioxidant mechanisms against oxidative stress resulting from ethanol exposure. acute myeloid leukemia (aml) is the most common form of acute leukemia in adults and its incidence increases with age. carbonic anhydrases (cas) are zinc-containing enzymes. these enzymes catalyze a very simple physiological reaction, the inter conversion between carbon dioxide and the bicarbonate ion, and are thus involved in crucial physiological processes connected with respiration and transport of co 2 /bicarbonate between metabolizing tissues and lungs, ph and co 2 homeostasis, electrolyte secretion in a variety of tissues/organs, and biosynthetic reactions and many other physiologic or pathologic processes including reproductive tract. investigation of autoantibodies in aml patients have been popular research area in recent years. the aim of the current study was to investigate carbonic anhydrase i and ii (ca i and ii) autoantibodies in the serum of subjects with aml based on the information and considerations of autoimmune relation of acute myeloid leukemia. anti-ca i and ii antibody levels were investigated by enzyme-linked immunosorbent assay method (elisa) in serum samples of thirty patients with aml and thirty healthy peers. anti-ca i and ii antibody titers of aml group were significantly higher compared with the control group (p = 0.000), (p = 0.034), respectively. we found significant positive correlation between anti-ca i antibody and anti-ca ii antibody titers in patients with aml (r = 0.613, p = 0.000). we found significant positive correlation between anti-ca i antibody and anti-ca ii antibody titers in women and the men (r = 0.851, p = 0.000), (r = 0.503, p = 0.080), respectively. at an anti-ca i cut-off point of 0.123 absu, sensitivity was 80% and specificity 100%. at an anti-ca ii cut-off point of 0.097 absu, sensitivity was 60% and specificity 77%. the ca i and ca ii autoantibody levels in patients with aml were found higher compared to control group and the results suggest that ca i and ca ii autoantibodies may be involved in the pathogenesis of aml. aim: behc ßet's disease (bd) is a systemic autoimmune disease. recurrent oral and genital mucosal ulcers, uveitis, and skin lesions are characteristic findings for bd. platelet-lymphocyte ratio reflects a novel marker for romatological diseases. the aim of this study was to investigate the platelet/lymphocyte ratio in behc ßet's disease. methods: whole blood samples were collected from 50 healthy control and 21 patients with behc ßet's disease. the mean age for controls and patients were 40 ae 1.0 and 37 ae 13, respectively (p = 0.639). patients with chronic disease and inflammatory disorders were excluded. thrombocyte and lymphocyte counts were analyzed with abbott cell dyne heamotolgy analyzer. statistical analysis was performed with ibm spss v20. results: platelet counts were higher but not statistically significant in patients with behc ßet's disease compared to control group (289 ae 100 vs. 257 ae 57) (p = 0.088). lymphocyte counts were lower in patients with behc ßet's disease compared to control group (2.56 ae 0.58 vs. 2.69 ae 0.85) (p = 0.513). platelet/lymphocyte ratio was higher but not statistically elevated in patients with behc ßet's disease compared to control group (118 ae 41 vs. 106 ae 49) (p = 0.344) conclusions: platelet/lymphocyte ratio (nlr) and mean platelet volume (mpv) as inflammatory markers recently became popular because of their simplicity, cost effectivity and their advantages to predict clinical prognosis of specific diseases. according to this study's results, platelet/lymphocyte ratio must be analyzed in vast scale patient populations to identify the disease. objectives: systemic lupus erythematosus (sle) is a chronic relapsing autoimmune disease characterized by production of autoantibodies against a series of nuclear antigens and by chronic inflammation. in recent years, neutrophil-lymphocyte ratio (nlr) was determined to be a good indicator of inflammatory status. nlr can be easily calculated from a whole blood count. introduction: neuroblastoma, an embryonal malignancy, is the most common extracranial solid tumor of childhood. untreated neuroblastoma tumors and cell lines are reported to have reduced hla class i expression, rendering them potentially susceptible to natural killer cell killing due to lack of engagement of hla class i-specific inhibitory killer cell immunoglobulin-like receptors (kirs). the aim of this study was to investigate whether kir genes could influence the risk of neuroblastoma and prognosis of the patients. materials and methods: study group consisted of 50 neuroblastoma patients (15 male, 21 female, median age: 36 months) followed at the pediatric oncology clinic of c ß ukurova university medical faculty. control group consisted of 100 healthy children. 14 patients had stage 1, 2, 3 or 4s disease, 36 patients had stage 4 disease. 16 different kir genes were analysed by sequence specific oligonucleotide probe (ssop) analyses. statistical analysis were done using fisher's exact test. results: compared to the control group, neuroblastoma patients had lower expression of activating kir gene, kir2ds3 (p = 0.005), and higher expression of inhibitory kir gene 2dl3 (p = 0.038). additionally kir2ds3 genes were more common in patients with early stages (stage 1, 2, 3 or 4s) (p = 0.023) and kir2dl3 genes were more common in patients with stage 4 (p = 0.044). furthermore, there were no statistically significant differences between the rate of aa and bx genotypes and their centromeric/ telomeric regions of patients and controls. discussion: kir2dl3 gene can have a triggering effect in neuroblastoma pathogenesis; whereas kir2ds3 can have a protective role. investigating nk cell infiltration and kir receptors in neuroblastoma tissue samples will give more insight to the pathogenesis p-04.03.3-008 neuroprotective and immunomodulatory effects of urtica urens s. arslan 1 , g. terzioglu 1 , b. kabalay 1 , a. r. tufekci 2 , a. sen 1 , i. demirtas 2 1 department of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, 2 deparment of chemistry, faculty of arts and sciences, c ß ankiri karatekin university, c ß ankiri, turkey urtica urens (small stinging nettle) has been used for medical purposes in turkey as an alternative therapy. it has been used in the treatment of inflammation that is early, non-specific immune reaction to tissue damage or pathogen invasion, plays an important role in the initiation of neurodegenerative disorders such as multiple sclerosis. however, there are limited studies that investigate anti-inflammatory activity of urtica. therefore, aim of this study is to find out theanti-inflammatory effect of chloroform extract in caco-2 cell line. for this purpose, firstly, chloroform extract of urtica leaves was prepared. chemical composition of extract was determined by lc-ms. the effect of chloroform extract on selected pro-inflammatory and inflammatory proteins such as tumor necrosis factor-a (tnfa), nuclear factor kappa b (nf-jb), c-x-c motif chemokine 10 (cxcl10), and 11 (cxcl11) were determined. whole genome transcriptome analysis was performed by using human ht-12 v4 beadchip. extract treatment caused 35% and 40% increases in protein and mrna levels of nf-jb, respectively. on the other hand, tnf-a protein and mrna levels decreased significantly (24% and 12%, respectively). similarly, cxcl10 and cxcl11 mrna levels decreased 45% and 38%. transcriptome analysis showed that 233 probes were significantly changed (p < 0.05). pathway analysis revealed that the extract altered a group of genes involved in immune response, calcium ion homeostasis and transport, potassium channel complex, g-protein coupled receptor protein signalling pathway, etc. it is well established that calcium is very critical for brain cell death and formation of many brain disease including multiple sclerosis. these observations suggests that urtica maybe used in neurodegenerative diseases. in order to further test this hypothesis experimental autoimmune encephalomyelitis experimentsand activity guided fractionations have been still continuing. this work is supported by tubitak 111t515. p-04.03.3-009 linear low molecular weight a-1,6-glucan from bifidobacterium bifidum bim b-733d is implicated in pathogenesis of celiac disease the bifidobacteria are recognized as human commensals and widely used as probiotics. earlier, we have found (kiseleva et al., benef. microbes, 2013, 4(4) : 375-391) that bifidobacterium bifidum bim b-733d contains low molecular mass (5-7 kda) a-1,6glucans (g anti-tpo and g anti-tg ) that interact selectively with human autoantibodies to thyroid peroxidase (anti-tpo) and thyroglobulin (anti-tg), recognized markers of autoimmune thyroid disease (atd). the aim of the study was isolation and identification of b. bifidum bim b-733d biopolymers (bps) interacting selectively with autoantibodies to tissue transglutaminase (anti-ttg) and antibodies to gliadins (anti-gl), recognized markers of celiac disease (cd). we used affinity chromatography with anti-gl, size exclusion chromatography, 1 h and 13 c nmr spectroscopy, elisa with immobilized bps, tissue transglutaminase (ttg) and gliadins (gl) as positive controls. the bp isolated by affinity chromatography with anti-gl (as more available marker of cd) and size exclusion chromatography was identified by two-dimensional nmr spectroscopy as 5-7 kda linear a-1,6-glucan identical to g anti-tpo and g anti-tg . the functional activity of the bp named g anti-gl , viz., ability to interact selectively with anti-ttg and/or anti-gl was proven by elisa with (i) serum samples of cd patients containing either both anti-ttg and anti-gl without anti-tpo and anti-tg or anti-gl without anti-ttg, anti-tpo and anti-tg vs. serum samples of healthy donors without four types of antibodies and (ii) pure anti-gl vs. pure total igg (without anti-ttg, anti-gl, anti-tpo, anti-tg). since (i) serum samples of cd patients do not contain anti-ttg without anti-gl and (ii) pure anti-gl isolated by affinity chromatography with gliadins (gl) cross reacts with tissue transglutaminase (ttg), additional studies with pure anti-ttg are necessary to find out which of the two antibodies, anti-ttg and anti-gl, bind g anti-gl . in conclusion, we proved that b. bifidum bim b-733d cells contain linear low molecular mass a-1,6-glucan, g anti-gl , that interacts selectively with anti-ttg and/or anti-gl. since g anti-gl is identical to earlier found g anti-tpo and g anti-tg , we hypothesize that the a-1,6-glucan is implicated in pathogenesis of both autoimmune diseases, cd and atd. influences of elevated serum ferritin levels on insulin resistance and non-insulin-dependent diabetes mellitus (niddm) have predicted either because of increased body iron stores or influenced by several inflammatory diseases. low serum 25 hydroxyvitamin d is known to perturb cellular function in many tissues, including the endocrine pancreas, which are involved in obesity and niddm. we planned to investigate the association between 25hydroxyvitamin d with hematologic parameteres and iron status in obesity vs. diabetic patients. study groups consist of control, non-diabetic obese, obese-diabetic and lean-diabetic groups. serum triglycerides, total cholesterol, ldl-c, hdl-c, fasting glucose, hba1c, uric acid, creatinine, ggt,25-hydroxyvitamin d, insulin, crp, esr, total blood count and iron status. apart from the three parameters, there were no significant difference (p > 0.05) between groups. serum ferritin and mchc levels were significantly higher in lean-diabetic patients (p < 0.05). on the other hand, rdw are determined to be significantly lower (p < 0.001) in the non-diabetic obese group. no difference was detected in 25-hydroxyvitamin d levels between the control and the study groups (p > 0.05). non-diabetic obese patients had significantly (p < 0.05) higher levels of tg and lower levels of hdl compared to obese-diabetics. insulin levels were higher in nondiabetic obese and obese-diabetics than lean-diabetics (p < 0.05). this study provides evidence that lean diabetic patients show higher ferritin and mchc levels than obese patients. the increase in serum ferritin and mchc levels is related with altered iron metabolism at cellular level. at late mitosis, the mother cell divides, leaving two daughter cells connected by a thin intercellular bridge (icb). during abscission of the icb, the ingression of the cleavage furrow is formed, and the central spindle microtubules are compacted into the structure known as midbody (mb). the mb is situated within the icb, with the abscission usually occurring at one side of the mb. as a result, only one daughter cell inherits the post-mitotic mb. these mbs can then either accumulate in the cytoplasm or be degraded. recent studies have identified mbs as novel signaling platforms regulating stem cell fate and proliferation. indeed, stem cells as well as cancer cells were shown to accumulate post-mitotic mbs, resulting in reprogramming of the cell fate and conversion to highly-proliferative, stem cell-like phenotypes. it has been proposed that regulated macroautophagy may be playing a key role in mediating pots-mitotic mb degradation. therefore, the experimental approach involved studying the dynamics and function of a protein known as fyco1, which associates with postmitotic mbs and may regulate their degradation. in this study we identified fyco1 as a protein, which associates with post-mitotic mbs and may regulate their degradation. interestingly, fyco1 is also known to be present on autophagosomes, and overexpression of fyco1 can induce the formation of enlarged lc3-containing autophagocytic structures. here we demonstrate that fyco1 knock-down leads to defects in autophagic mb degradation, and that fyco1 functions by targeting endocytic membranes to the autophagic phagophore during early stages of mb degradation. additionally, we showed that fyco1 depletion leads to increased proliferation and cell growth in soft agar. based on all these data, we hypothesize that fyco1 mediates selective mbs degradation via endosome-dependent extension of the phagophore around the post-mitotic mbs, and that mbs may be the regulators of cancer proliferation and progression. p-05.03.3-002 proliferative effect of hypericine on human skin fibroblast cells and identification of the mechanism of action in molecular level to drawbacks associated with efficiency and viral genome integration. in order to improve reprogramming efficiency and compensate for viral transduction, new chemicals have been explored through ipsc research. the aim of this study was to investigate the proliferative effect of hypericine on human skin fibroblast cells (sf) in-vitro, and to identify the mechanism of action in molecular level. the proliferation was measured using the clonogenic and dimethylthiazol diphenyltetrazolium bromide (mtt) assays. real-time quantitative polymerase chain reaction (qrt-pcr) was performed to detect the mrna levels of cyclins (d1 and b1) and cell cycle controller genes (p53 and p21). sf cells were treated with different doses (1 nm-100 lm) of hypericine for 24 h and 48 h. a significant cell proliferation was observed in moderate concentrations (0.1-15 lm; %110-%134), but at high concentrations (25-50 lm) cytotoxic effects emerged in sf cells (ic50 = 23.62 m, r 2 = 0.915). qrt-pcr results revealed that the most proliferative dose of hypericine (15 lm) stimulates cyclin d1. the anti-proliferative activity of hypericine was accompanied by inhibition of cyclin b1 mrna, whereas it induced expression of p53 and p21 genes, and thus apoptosis was observed by dna laddering at the same dose (50 lm). overall results suggested that hypericine can compensate for viral transduction and improve reprogramming efficiency of ipscs by enforcing them in g1 phase. hence we report that hypericine can be a good candidate component for cocktails produced to trigger ipsc proliferation. glioblastoma (gbm) is the deadliest brain tumor. the mean survival time of gbm patients is approximately 12 months, increasing to 14 months after treatment with temozolomide, which is the gold standard chemotherapy. the resistance of gbm to chemotherapy seems to be associated with the blood-brain barrier (bbb) that limits the delivery of chemotherapeutics, and the presence of a population of cells that expresses stem cell-like properties, which are known to be chemo-and radioresistant,5 the glioblastoma stem cells (gscs). the difficulties imposed by these two factors could be reduced by the use of a targeted drugdelivery liposome-based strategy that allows bbb passage and reduces the side effects of chemotherapeutics. the present study evaluated the ability of the f3 peptide-targeted ph-sensitive lipid-based nanoparticle containing doxorubicin (dxr) to target gscs and non-gscs. we evaluated the expression of cell-surface nucleolin by flow cytometry, as well as of stem cell-like markers, in two gbm cell lines. we also determined the ability of gbm cell lines to specifically uptake the f3 peptide-targeted ph-sensitive lipid-based nanoparticles, by flow cytometry, and correlated it with the expression of stem cell-like markers. moreover, to ascertain the impact of intracellular delivery of chemotherapeutic drugs into gbm cell lines, cytotoxicity was further assessed by the mtt assay. our results showed that the f3 peptide-targeted ph-sensitive lipid-based nanoparticles successfully targeted glioblastoma cells and particularly gscs. in addition, the results also provided evidence of the nucleolin overexpression-dependency of this strategy, emphasizing the need to adapt the therapeutic strategy to the individual patient. this study showed that f3-targeted ph-sensitive liposomes may constitute an appropriate strategy to overcome the chemoresistance associated with glioblastoma cells. p-05.03.3-004 leukemic cell plasticiy as a resistance mechanism towards tyrosine kinase inhibitors chronic myelogenous leukemia (cml) is a hematopoietic stem cell disease characterized by the t(9;22)(q34;q11) translocation, which encodes the chimeric tyrosine kinase onco-protein, bcr-abl. the tyrosine kinase inhibitor (tki) imatinib is the first-line treatment for patients with cml. unfortunately drug resistance is one of the main problems observed. while secondary resistance is associated with bcr-abl kinase domain mutations, oncogene amplification and mechanisms interfering with intra-cellular drug concentrations; primary resistance mechanisms haven't been elucidated. we generated high dose imatinib-resistant k562 subclones (k562-ir) by clonal selection to study primary resistance mechanisms in vitro. drug resistance was shown by caspase 3 and annexin v/pi assays. we also showed cellular uptake and function of imatinib with western blot technics. k562-ir cells are not only resistant to imatinib but also to 2nd, 3rd generation tyrosine kinase inhibitors. we demonstrated that k562-ir cells have a highly adherent character, proliferate slowly and are resistant to drug-induced senescence. microarray analysis revealed that k562-ir cells differentially express tissue/organ developement and differentiation genes at high levels. we showed that k562-ir cells forms intact tumor spheroids in 3d cell culture conditions which is a marker of tumor initiating potential. cell surface maker analyses and protein analyses of k562-ir cell population, points towards an epithelial-mesenchymal plastic cell capable of adopting different morphologies. we hypotizied that imatinib and other tyrosine kinase inhibitors may cause the gain of phenotypic plasticity potential in leukemic cells, by interfering with signalling pathways; which in itself may lead to therapy resistance. hypoxia has multiple effects on cancer cells, which are critically involved in tumor progression. hypoxia leads to changes in tumor cell metabolism and can promote cancer cell survival, invasion and metastasis by its critically important role on maintenance of cancer stem cell (csc) phenotype. in this research, human cd133 + cscs isolated from human osteosarcoma cell line saos-2 using macs magnetic separation technique were characterized, and their stemness properties under hypoxic (1% o 2 ) and normoxic (21% o 2 ) conditions were compared in two and three dimensional culture conditions. two different 3d culture techniques (nanofibrous bacterial cellulose scaffolds and scaffold free microtissues) were used to evaluate effects of hypoxia on csc behavior, and the results were compared with the cell behavior in classical 2d culture systems. the morphologies of cells were examined by scanning electron microscopy (sem); rt-pcr and immunocytochemistry staining were used to examine the cancer stem cell phenotype maintenance under hypoxic and normoxic conditions. it is shown that hypoxia supports the expression of stemness markers such as oct3/4, nanog and sox2 compared to normoxic conditions in 3d cultures. although similar effects of hypoxia were observed in 2d cultured cscs, the expression levels of stem cell phenotypeindicative markers were significantly lower on 2d compared to 3d culture systems. this study is seen as an introduction to develop a more relevant 3d hypoxic cancer stem cell based tumor model to study csc behavior and tumor genesis in vitro for testing of novel cancer stem cell therapeutics and to understand signal transduction in cancer stem cells. prostate cancer (pca) is the second most frequent cause of cancer-specific mortality in the world. cancer stem cells (cscs) are a subpopulation of cells that involved in drug resistance, metastasis and recurrence of cancers. the efficacy of natural flavanone apigenin on cell survival, apoptosis and migration of cscs were evaluated. cd44 + cscs were isolated from human pca pc3 cells using a magnetic-activated cell sorting system. pc3 and cscs were treated with different concentrations of apigenin, docetaxel and combinations of the two agents for 48 h. apigenin dose dependently inhibited cscs and pc3 cell viability, and this was accompanied with a significant increase of the cell cycle inhibitors p21 and p27 (kip1). the flavonoid significantly induced apoptosis via an extrinsic caspase-dependent pathway by upregulating the mrna expressions of caspases-8, -3 and tnfa, but failed to regulate the intrinsic pathway as determined by the bax, cytochrome c and apaf-1 in cscs. in contrast to cscs, apigenin induced intrinsic apoptosis pathway as evidenced by the induction of bax, cytochrome c and caspase-3 while caspase-8, tnf-a and bcl-2 levels remained unchanged in pc3 cells. the ability of apigenin to inhibit the proliferation of cscs through apoptosis was confirmed by tali image-based cytometer. the flavanone strongly suppressed the migration rate of cscs compared to untreated cells. significant downregulation of mmp-2 and -9 exhibits the ability of apigenin treatment to suppress invasion. the expressions of pi3k/akt and nf-kb p105/ p50 were significantly decreased after 48 h apigenin treatment. taken together, these data demonstrated that flavonoid apigenin is an invaluable chemopreventive compound that inhibits proliferation, invasion and the stemness properties of cscs. this study was funded by the scientific and technological research council of turkey (tubitak, grant no. 115s356). (pi3k), are frequently found in patients with severe early-onset segmental overgrowth. whilst differences in timing and location of the founder mutation are likely to explain part of the observed disease heterogeneity, it is less clear whether and how quantitative differences in the strength and timing of pi3k activity contribute to phenotypic variability. our aim is to characterise pik3ca mutant-specific signalling as well as to explore the effects of varying the strength and/or temporal pattern of pi3k activation on downstream output specificity in the cell. we are currently employing crispr/cas9mediated gene editing in human induced pluripotent stem cells to generate isogenic disease models of three such activating pik3ca mutations. these cells will be used for signalome profiling by reverse-phase protein arrays (rppa) to compare and contrast mutant-dependent alterations to candidate signalling networks. in parallel, ongoing efforts focus on developing an endogenously expressed optogenetic p110a, allowing precise spatiotemporal control over pi3k signaling to unravel the extent to which pi3kdependent phenotypes are determined by strength of activation and/or dynamic encoding. ultimately, the outcome of this research will yield novel insight into fundamental aspects of pi3k signalling and potentially aid the development of targeted therapies for human diseases of pi3k hyperactivation. e. gov, n. kaya, k. y. arga cancer stem cells (csc) have been proposed to be the cancer initiating cells. because of their highly tumorigenic and drug resistant properties, cscs offer significant potential for developing novel anticancer drugs and therapeutic strategies. in the present study we analysed eight gene expression datasets for breast, ovarian, lung cancer and glioblastoma by comparing gene expression levels between stem cells and tumor cells and integrating them with genome scale biological networks. consequently, mutual molecular signatures (i.e: differential expressed genes, transcription factor, mirna) and biological characteristics were determined via integrative analyses, which might be feasible to uncover the mutual biological mechanism insights behind the cscs. it was identified twenty mutual differential expressed genes in four cancer types; jun and klf6 as transcription factors, egfr and cdk14 as receptors come into prominence as mutual signatures. molecules and pathways that were related to mapk, wnt, p53 signaling and pathways in cancer were the common indicators in csc types. our results provided similarities in gene expression profiles of various cscs and gave clues about the seed of tumorigenesis. this study proposed signatures and pathways that could be considered as effective therapeutic approaches in further experimental and clinical applications to eliminate subpopulation of csc. colorectal cancer (crc) is one of the leading causes of mortality worldwide. metastasis is associated with the presence of circulating tumor cells (ctcs) in the peripheral blood of cancer patients. ctc cut-off values have been shown to predict for poorer overall survival in metastatic breast (≥5), prostate (≥5), and colorectal (≥3) cancer based on assessment of 7.5 ml of blood. in our study, ctcs were detected in blood samples of colorectal cancer patients, using with our modified convenient method for the strategies of ctc enrichment and detection. 7.5 ml peripheral blood samples were firstly collected and peripheral blood mononuclear cells (pbmcs) were isolated from the fresh blood samples by ficoll gradient separation. next, the leukocytes in pbmcs were removed by magnetic microbeads conjugated with cd45 for a negative selection. finally, the retained cells were labeled with anti-epithelial cell adhesion molecule (anti-epcam), cytokeratins (ck8, ck19) and the leukocyte-specific marker as anti-cd45. all samples were analyzed by bd facs aria iii flow cytometry. in total, 10 patients and 7 healthy people were included in this study. the results showed that ctcs were not detected in the blood samples of healhty volunteers, but 3-13 ctcs were detected with ck14, 15, 16, 19-based gating strategy in the blood samples of colorectal cancer patients. it is accepted that the cut off value is 3 ctcs for colorectal cancer and ctc is negative if it is below this value or ctc is considered as a positive, if it is equal to or above this value, which might be an indication for poor prognosis. thus ctc's detection may serve a representative surrogate tumor biomarker for real-time monitoring of disease status and tailoring personalized therapy. cells were grown in culture flasks in a humidified incubator at 37°c with 5% co 2 and were used at the proliferation and confluent stages. cultured cells were exposed to the pemf and prfe. the proliferations of the cells are measured by mtt assay for the effect of emf on the cancer cells. on the other hand the wound healing was investigated by closure of the wound by the cell proliferation with cell morphology using inverted microscope images. the proliferation decreased significantly by the effect of pemf on the semi confluent mcf-7 and mda-mb-231 cells. this effect was observed more prominent on mcf-7. considering prfe therapy this effect is much more pronounced especially for mda-mb-231 comparing with pemf. the phase contrast observations of these results were consistent with mtt analyses. similarly, this effect was seen less for pemf but the proliferation was more suppressed with prfe on the wound models. it was considered that the emf applications could be effective in cancer cells, but this effect has not been studied how it occurs in invasive cancers. in our cell culture study, the appropriate emf applications were found to be effective though the inhibition of proliferation of cancer cells even in invasive cancer but with lower effect. this means that emf applications may support the existing treatment methods of cancer patients and even people who suffer from invasive cancer. metastasis is the one of the most known causes of death in patients diagnosed with cancer. circulating tumor cells (ctcs) are shed from primary tumors and circulating in the bloodstream, and thought to play a key role in metastasis. a hypothesis that ctcs may contribute to metastasis was first introduced in the mid 19th century by thomas ashworth, an austrilian pathologist. in today's research, identification and molecular characterization of ctcs are thought to be a novel target for treatment of cancer and a key factor to understand the metastatic process. existing methods of ctc capture based on the cell search system, flow cytometers, laser scanning cytometers instruments, fiber-optic array scanning technology (fast), isolation by size of epithelial tumor cells (iset), and definition fluorescence scanning microscopy. ctcs are increasingly considered as a 'liquid biopsy' and when liquid biopsy is compared to tumor tissue biopsy, liquid biopsy for ctcs detection can be carried out routinely in patients due to accessibility and ease of blood collection. also, primary tumor sampling may not reflect the actual metastatic conditions, ctcs are thought to be a novel tumor biomarker for real-time monitoring of disease status and tailoring personalized therapy. with futher works, ctcs may be used as liquid biopsies and it might provide better understanding metastatic process, new approaches in cancer diagnostics and treatment. mesenchymal stem cells (mscs) are distributed all over the organism as a source of tissue formation and regeneration. glucose is vital for the proliferation and differentiation of mscs. glucose uptake is mediated by specific glucose transporters of two families, the na-coupled glucose transporters (sglt) and glucose transporter facilitators (glut). the presence and function of glut proteins in human placental amnion derived mscs (hamscs) is unknown. we aimed to investigate the presence of glut1, glut3, glut4 proteins and genes in hamscs isolated from term placentas. mscs were isolated from human term placenta amniotic membrane, the characterization of cells were provided by flow cytometry. mscs were used to assess their chondrogenic, osteogenic and adipogenic differentiation potential. the expression of glut1, glut3 and glut4 proteins was detected in hamscs by immunofluorescence. glut1, glut3, glut4 protein and gene expression in these cells were investigated by western blot and real-time pcr, respectively. flow cytometry analysis results of isolated cells showed that they were positive for cd44, cd90, cd73, cd105 (mesenchymal stem cell markers) and hematopoietic markers cd34, cd11b, cd19, cd44 and hla-dr were negative. the presence of glut1, glut3, glut4 proteins and genes were identified in hamscs. in this study, for the first time in literature, glut1, glut3 and glut4 gene and protein presence was determined in hamscs. therefore, gluts could mediate glucose transport in human amniotic membrane mscs. proliferation and differentiation of mscs in vitro are still not optimized. further studies are required to clarify the complex mechanisms regulating the relationship between glucose and mesenchymal stem cells. disclosure of this relationship may provide a better understanding of glucose-related pathologies such as diabetes. tumors have hierarchically organized heterogeneous cell populations and a small subpopulation of cells, termed cancer stem cells (cscs), is responsible for tumor initiation, maintenance as well as drug resistance. therefore, killing the cscs along with the other cancer cells is gaining an importance. in the present study, it was aimed to evaluate the cytotoxic and apoptotic activity of a novel platin (pt) (ii) complex [pt(hepy) 2 cl 2 ] on mammospheres obtained from mcf-7 human breast cancer line. elevated expression of stemness markers were determined by western blotting. cytotoxicity was assessed using the atp viability assay. effect of the pt (ii) complex on the formation and development of mammospheres was analyzed with sphere formation (sfa) assay. apoptosis was determined via cytofluorimetric analysis (caspase 3/7 activity, annexin-v-fitc and bcl-2 activity) as well as gene expression analysis. cytotoxicity was confirmed with the atp viability assay after the treatment with zvad-fmk (an apoptosis inhibitor) and necrostatin (a necroptosis inhibitor). in addition, alterations in mitochondrial membrane potential were evaluated by jc-1 staining. mammospheres exhibited increased oct-4 and sox2 (stemness markers) expressions compared to parental mcf-7 cells. cytotoxicity by pt (ii) complex was evident in a dose-dependent fashion (1.56-100 lm) . pt (ii) complex significantly prevented mammosphere formation and disrupted mammosphere structure in a dose-dependent manner. pt (ii)-induced apoptosis was determined based on the presence of caspase 3/7 activity, annexin-v-fitc positivity and bcl-2 inactivation. apoptosis was also confirmed with increased tnfrsf10a and hrk gene expressions. in addition to apoptosis, necroptosis was also present as evidenced with increased mlkl expression. mitochondrial membrane was depolarized. in conclusion, the pt (ii) complex seems to be a powerful apoptosis-inducing compound on cancer stem cells, thereby warrants further in vivo experiments. cancer is a disease which arises from destruction of growth and proliferation mechanisms in cells and is the second leading cause of death worldwide [1] . in the development of primary cancers, the head and neck cancer is accounting for approximately 550.000 new cases annually around the world [2] . laryngeal cancer is a type of head and neck cancer in which malignant cells arise from the mucosal tissues of the larynx [3] . cancer might spread from primary tumor by getting into the lymph and blood vessel system and forms secondary tumor. greater than 90% of deaths in cancer patients are attributed to metastasis [4] . circulating tumor cells (ctc's) provide an opportunity to understand the metastatic process of cancer patients. identification and molecular characterization of ctc's in the peripheral blood of cancer patients is a promising research area in the field of biomarker development and novel treatment targeting in today's cancer research [5] . the detection of ctc methods include cell search system, flow cytometry, high-definition fluorescence scanning microscopy, fiber-optic array scanning technology, isolation by size of epithelial tumor cells, and laser scanning cytometers [6] . in our study, 7.5 ml of peripheral blood samples were collected from larynx cancer patients and healthy volunteers and the samples were analyzed by bd facs aria iii flow cytometry via biomarkers epcam, ck19, ck8 for positive selection and cd45 for negative selection [7] . according to the results of our study; ctcs were detected in larynx cancer patients by our newly modified method whereas there was no ctc's detection in the samples of controls. thus, this study may provide us monitoring of the treatment process of larynx cancer and this method might be used as diagnostic, prognostic, and predictive biomarkers in cancer therapy as a liquid biopsy. prostate cancer is the second most common cancer and the fifth leading cause of death from cancer in men 1 . circulating tumor cells (ctcs) present in the peripheral circulation of cancer patients with different solid malignancies including prostate cancer and have a potential as a liquid biopsy to monitor disease progression and response to therapies at cell and molecular level 2 . one of the general methods in ctc detection is flow cytometry 3 . radical prostatectomy is the most frequently applied procedure in the surgical management of localized prostate cancer. in this surgical operation, the surgeon removes the entire prostate gland with the seminal vesicles. a radical prostatectomy procedure can be done using the da vinci robotic system (intuitive surgical, sunnyvale, ca, usa) 4 . robotic surgery has been suggested to have fewer complications, lower risk of infections and shorter recovery period following robotic radical prostatectomy 5,6 . in this study, our aim was to detect ctcs before and after robotic radical prostatectomy in clinical localized prostate cancer patients. the ctc detection study was performed with our modified method in which 7.5 ml of peripheral blood samples were collected from each prostate cancer patient and healthy individual; the samples, using with biomarkers epcam, ck19, ck8 for positive selection and cd45 for negative selection, were analyzed by bd facs aria iii flow cytometry 7 . according to our results, we detected ctcs in the peripheral blood samples of prostate cancer patients before robotic radical prostatectomy. however, following this surgical procedure no ctc or decreased number of ctss was detected. our study might contribute to understand disease progression after robotic radical prostatectomy in clinically localized prostate cancer patients that warrants further research. keywords: circulating tumor cells, prostate cancer, flow cytometry, robotic radical prostatectomy. p-05.03.3-017 determination of effect cytotoxic, apoptotic, caspace-3 activity and mrna expression levels of apoptototic related genes of vulpinic acid on breast cancer cell lines n. kilic ß, s. aras, d. cansaran-duman ankara university, ankara, turkey breast cancer is the most common cancer types in women. several drugs used to treat breast cancer patients are developing resistance to the treatment for this reason success rate falls. therefore the discovery of alternative therapeutic agent and molecular detection of anticarcinogen effect because of treatment for cancer patients may be a source of hope for the contributions. in this study, different concentrations (1.562, 3.125, 6.25, 12.5, 25, 50 , 100 lm) vulpinic acid (va) lichen seconder metabolite was determined to cytotoxic, apoptotic effect and caspase-3 activity in breast cancer cells (mda-mb-231, mcf7, bt-474, sk-br3) and normal cell (mcf12a). in addition to the quantitative real-time pcr (qrt-pcr) using apoptose specific primers (tp-53, bcl-2, bax, birc-5, gapdh, caspase-3, caspase-7, caspase-8, caspase-9) and sybr green dye were performed to determine expression patterns of transcript level in cancer cell lines, using gapdh as a reference gene. the antiproliferative characterization of va effects identification of the gene set at molecular level and we determination role of va on apoptotic pathway. according to our study, va is demonstrated significantly (p < 0.05) effect cytotoxic, apoptotic, caspase-3 activity. beside this, dose dependent expression patterns decreased apoptose spesific genes (except of bcl-2) mrna levels from six to eleven fold change more than untreated va cell lines. va will be used as candidate molecule for effective treatment on breast cancer in the future. glycosylation largely determines the variety and functions of proteins. paucimannose, a mannosidic n-glycoepitope has long been thought to be specific for plants and invertebrates. recently, it has also been detected in mammalsin physiological conditions (stem cells) and in pathophysiological conditions (inflammation and cancer). in glioblastoma cells, paucimannose also seems to play a role in cell proliferation. glioblastoma is the most frequent brain tumor in adults with poor prognosis due to a lack of suitable treatments. we hypothesize that paucimannose could be a promising new biomarker as it is otherwise rarely found in mammals. therefore, paucimannose levels were investigated in different glioblastoma cell lines differing in their proliferation rate and tumorigenicity. the highest paucimannose levels were detected in low proliferating, nontumorigenic cells. furthermore, we found that modulation of paucimannose function by application of a specific antibody regulated cell proliferation and the capability of cells to form colonies in soft agar. these data support a functional role of paucimannosidic epitopes in tumorigenic processes. glioblastoma multiforme (gbm) is the most lethal type of malignant brain tumors. recently, gbm stem cells (gscs) have been studied in great deal and accepted that they have a legitimate role in tumor formation, development, chemo-resistance and recurrence. in this study, it is aimed to investigate new therapeutic targets within apoptosis related molecules to select and eliminate cd133+ gscs effectively. ten primary gbm cells were isolated from gbm tissue samples and they were cultured among with the 4 gbm cell lines (u87, u138, u118 and t98). cd133+ and cd133à cells were seperated by macs method via anti-cd133 (ac133) antibody from cultured cells and cell lines. rna isolation from cd133+ and (à) cells, cdna synthesis was performed. finally, by performing pcr array, mrna expression levels of 88 genes were detected. proper results were collected and analysed statistically. according to the results of pcr array; it has been found that cd133+ cells express approximately 223 fold tnfrsf21 and 20 fold tnfsf7 when they are compared with control cells. tnfsf7 binds to cd27 that is expressed on the surface of tcells. cd27 does not have a death domain, instead it has a cytoplasmic tail which binds to trafs. trafs act as adaptor molecules that are related with jnk and nf-jb signalling pathways. tnfrsf21 (dr6) is a death receptor which are known for transmitting the pro-apoptotic signals from outside to the inside of the cell. it negatively regulates t-cell activation and the release of few cytokines. as a conclusion, tnfsf7 and tnfrsf21 both are found on immune system cells, mostly on t-cells, which may mean that gbm stem cells act as a immune system cells to avoid the elimination by the immune system. to conclude, acting as an immune system cell and promoting survival via tnfsf7 and tnfrsf21, these molecules may be essential markers to target cd133+ gbm stem cells. the effect of docetaxel on p53, sin3a and mdm2 gene expression in mcf-7 breast cell line docetaxel is a cytotoxin effective in treating breast cancer. it stabilizes microtubules and causes catastrophic cell cycle arrest in g2/m. it also initiate signaling through cell death pathways that result in programmed cell death. in this study, it was aimed to investigate apoptotic and cytotoxic effects of docetaxel has on the mcf-7 breast cancer cells line. in this study, mcf-7 breast cell line was applied different doses docetaxel (10 nm, 100 nm, 1 lm, 10 lm, 100 lm) as 24 h and 48 h. mtt analysis was performed to the mcf-7 breast cancer line in control group and groups of docetaxel. afterwards, evaluation of apoptosis by tunel and levels of p53, sin3a and mdm2 gene expression by real-time pcr were determined in an order. it was observed cell variable was significant lower in docetaxel groups compared to control group (p < 0.05) in 24 h as mtt analysis. the lowest cell viabilty was determinated in group applied 100 lm docetaxel. while the lowest positive cell density was determinated in control group, it was observed apoptotic cell density gradually increased with increasing docetaxel concentration in groups treated docetaxel (p < 0.05). the highest p53, si3a and mdm2 expressions were apperared in 100 nm docetaxel group compared to control group. human alpha-fetoprotein (afp) and afp receptor binding domain (afprbd) are able to bind and internalize effectively by wide range of human tumor cells and tissues. as other vector molecules afprbd has insufficient quantity of chemical groups which can be conjugated with drugs or diagnostic agents. conjugation of vector molecules with macromolecular polymer carriers like dendrimers aims to solve this problem. our study describes influence of afprbd-dendrimer-doxorubicin conjugate surface charge on intracellular trafficking routes and toxicity. the amineterminated (g2) and acetyl-terminated (g2 12 ) 2nd generation pamam dendrimers carrying doxorubicin (dox) were used to synthesize conjugates with afprbd. unmodified by afprbd g2 and g2 12 dendrimer derivates labeled with dox were absorbed by the cells at 37°c with different efficiency. g2 12 -dox derivate characterized much slower internalization rate than nonacetylated g2-dox. only g2 12 -dox shown partial colocalization with lysosomal marker lamp2 after 4 h of incubation. internalization of afprbd-g2-dox and afprbd-g2 12 -dox did not show significant difference. at the same time, both conjugates contained afprbd wyкy almost fully associated with lamp2 already after 30 min of incubation. cytotoxicity results revealed that ic50 levels of g2 12 -dox and afprbd-g2 12 -dox coincided and demonstrated a bit higher activity against sensitive to dox skov3 and resistant skvlb cells than afprbd-g2-dox conjugate after 72 h of incubation. at the same time, after 1 h of incubation afprbd-g2-dox and afprbd-g2 12 -dox were much more than g2 12 -dox and g2-dox. we may conclude that there is significant difference in ways of dendrimers internalization by tumor cells depending on nature of surface chemical groups. on the other hand, chemical modification of dendrimer conjugated with does not afprbd influence dramatically on the protein trafficking and resulting cytotoxic effect. russian scientific foundation supported this study (no. 15-15-10013 1.40) , a key enzyme in glycolysis, catalyzes conversion of phosphoenolpyruvate (pep) into pyruvate with regeneration of adenosine triphosphate (atp). the key regulator of the metabolic alterations found in tumor cells is the glycolytic isoenzyme pyruvate kinase type m2 that is generally expressed in all proliferating cells and overexpressed in all tumor cells investigated to date. during carcinogenesis a shift in the pyruvate kinase isoenzyme equipment always takes place, such that the tissue-specific isoenzymes disappear, and m2-pk is expressed. breast carcinoma, the third most common cancer worldwide, accounts for the highest morbidity and mortality. breast cancer tissue analysis confirmed the upregulation of m2-pk in breast cancer, and high m2-pk levels were associated with poor prognosis of breast cancer patients. materials and methods: poly hema (mac) nanoploymers were immobilized by binding covalently with sulphur atoms on the gold electrod's surface. pyruvate immobilization was actualysed with cross linking reagent glutaraldehyde. biosensor was developed by preparing pottasiumferrociyanide, selected as a mediator. results: cyclic voltammograms have been carried out at between~0.4 and 0.6 v potentials vs. ag/agci. m2-pk activty was detected by using differential pulse method at between 0.3 and à0.25 v potentials by observing the differentiations in the current values. in the optimization studies, some parameters such as optimum ph, temperature, concentration of glutaraldehyde and p-hema-mac, were investigated. discussion and conclusion: the method developed for the measurement of the tumor m2-pk activity by using biosensor. we found that more advantageous in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, economic, practical and less time-consuming. piruvat kinase tumor m2-pk activity determination at low concentrations is possible with this method. p-05.03.3-023 tie2/tek: a potential biomarker for targeting glioblastoma stem cells role in angiogenesis, endothelial cell survival, proliferation, migration and adhesion. therefore, tie2/tek could be a potential target for therapeutic strategies directed against glioblastoma stem cells and their microenvironment. in this study, we investigated the gene expression levels of tie2/tek in both cd133+ gscs and cd133à gbm cells. gbm primary cells were freshly isolated from glioblastoma tissue samples and cultured in dmem supplemented with 10% fetal calf serum and 1% penicillin-streptomycin at 37°c in 5% co 2 -humidified incubator. we isolated cd133+ and cd133à cells from 10 gbm primary cells using macs system. following rna isolation from healthy brain tissues, cd133à and cd133+ cells, cdna synthesis was performed. finally, according to microarray protocol, cell surface marker panel array was applied. expression levels were analyzed using the delta delta ct method. statistical analysis was performed using spss software for windows version 13.0. tie2/tek gene expression was determined as 50.07 fold higher in cd133+ gscs than normal brain tissue (p < 0.05). morever it was determined 7.52 fold higher compared to normal brain tissue in cd133à (p < 0.05). according to our results tie2/tek expression was higher in gscs, indicating that tie2/ tek may be a potential marker for targeting cancer stem cells in gbm. this research has been supported by the scientific and technological research council of turkey (no:114s189). adenosine inhibited the breast cancer stemlike cell population through erk1/2 pathway s. m. jafari, m. aghaie cancer stem cells (cscs) are immortal tumor-initiating cells that can self-renew and drive tumorigenesis in various cancers, including breast cancer and others solid cancers. in a study indicated that extracellular atp reduces tumor sphere growth and cancer stem cell population. but at present, there are no reports available in literature on the effect of adenosine on breast cancer stem cells. in this study we evaluated the effect of adenosine inhibition and its mechanism of action in breast cancer stem cells isolated from breast cancer cell lines. our result showed that adenosine significant reduces breast cancer stem cell population. reduction of erk1/2 protein levels was also observed after treatment cancer cells with adenosine. in conclusion, our results indicate that adenosine decreases the breast cancer stem-like cell population through erk1/2 pathway. taxanes are commonly used for the treatment of many cancers as chemotherapeutic drugs that resistance to these agents has become a major clinical obstacle. taxane based chemotherapy drugs such as paclitaxel, docetaxel and cabazitaxel bind microtubules and inhibit to microtubule polymerization appear to stimulate programmed cell death. taxane-resistance to cancer has not been clearly in progression and development of drug resistance. multiple mechanisms are involved in the drug efflux proteins as multidrug resistance protein, differences in amino acid sequences among the b-tubulin isotypes. we investigated taxane resistance with different doses of paclitaxel, docetaxel and cabazitaxel in prostate cancer stem cells. we compared the expression level of apoptotic proteins, and its functional role in resistance mechanisms in cd44 + /cd133 + prostate cancer cell lines. taxane drugs were categorized as concentration-dependent or time-dependent. cabazitaxel caused a time-dependent and dose-dependent reduction in cell viability in all tested cell lines. resistance activity was consistently higher in docetaxel in prostate cancer cells compared with the other drugs. there are many different response of clonogenic formation cd44 + /cd133 + cells with resistance to docetaxel, paclitaxel and cabazitaxel in prostate cancer stem cells. the innate of prostate cancer resistances are important characterization steps and critically limits treatment outcomes therefore novel drugs must be focus on antiresistance and molecular based combinations. mesenchymal stem cells (mscs) are self-renewing cells with ability to differentiate into organized, functional network of cells. mscs isolated from various tissues including adipose tissues, bone marrow, umbilical cord, placenta and pancreas have different differentiation and proliferation potential. good knowledge of the metabolism and proliferation mechanisms of stem cells is required for stem cell therapies. glucose is an important molecule in the culture of stem cells. glucose concentration affects the differentiation and proliferation potential of stem cells. the aim of the study was to investigate the proliferation status by identifying the proliferating cell nuclear antigen (pcna) expression under normoglycemic and hyperglycemic conditions in mscs. mscs were isolated from human term placenta amniotic membrane. characterization of the isolated cells was performed using flow cytometry. chondrogenic, osteogenic and adipogenic differentiation potential of these cells were investigated. characterized cells were cultured in normoglycemic and hyperglycemic conditions for 24 and 48 h and the expression of pcna protein expression in these cells were investigated by western blot. flow cytometry analysis showed that isolated cells were positive with mesenchymal stem cell markers cd44, cd90, cd73, cd105 and negative with hematopoietic markers cd34, cd11b, cd19, cd44 and hla-dr. western blot result of pcna protein expression statistically significantly increased in human amniotic membrane mscs under hyperglycemic conditions for 24 and 48 h culture. the glucose content of stem cell medium is important because glucose is an effective molecule of the proliferation of stem cells. proliferation of mscs in vitro are still not optimized. when the relationship between glucose and stem cells be understood, it will provide a better understanding for the glucose-related pathologies such as diabetes during pregnancy. prostate cancer (pca) is the second most common type of cancer among men in the world. it is revealed that some gene, protein and metabolite sets control the pca, however the whole metabolomics changes are not completely understood yet. pca is common among older men, and this is an important health problem in developed countries. sarcosine is the n-methyl derivative of the glycine amino acid. glycine n-methyl transferase produces sarcosine from glycine. besides, it is metabolized to glycine by sarcosine dehydrogenase. in 2009, high level of sarcosine in urine was associated with pca by sreekumar et al. they identified sarcosine as a pca biomarker that was significantly increased in urine during prostate cancer progression to metastasis. following this study, several studies have been published indicating sarcosine as a pca biomarker. in our study, a preliminary biosensor system was fabricated for determination of sarcosine in urine by using sarcosine oxidase. sarcosine oxidase was immobilized on au electrode surface using gelatin as an immobilization matrix. glutaraldehyde was used as a cross-linking agent to avoid the loss of the enzymegelatin mixture. optimization and characterization studies were carried out. sarcosine concentrations were detected carefully with the developed biosensor system. the fabricated preliminary biosensor is a promising system that can allow lower detection limits after surface modifications. activation of the epithelial-mesenchymal transition (emt) program in tumor cells is associated with invasiveness and stemness. recent studies implicate emt-inducing molecules in reprogramming energy metabolism. the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (pfkfb3) regulates glycolysis by producing fructose 2,6-bisphosphate (f2,6bp). given that pfkfb3 is induced by several established emt-inducers in tumor cells, e.g. hif-1a and ras, we hypothesized that pfkfb3 may be involved in regulation of the emt in tumor cells. silencing of pfkfb3 in pancreatic adenocarcinoma cell lines panc1 and s2vp10 was achieved using specific sirna molecules. mrna and protein expressions of the cdh1 gene (encoding e-cadherin, an established epithelial marker), as well as zeb1 and snai1 genes, by real-time quantitative (q)-pcr and western blot, respectively. immunfluorescence analysis was performed to visualize e-cadherin protein expression on plasma membrane. in order to test the effect of pfkfb3 on the invasive ability of the cells, a matrigel invasion assay was performed. ectopic expression of zeb1 was achived by transfecting cells with a plasmid carrying zeb1 cdna. cells that were depleted of pfkfb3 exhibited markedly increased cdh1 mrna and e-cadherin protein expressions and reduced snai1 and zeb1 mrna expressions. immunfluorescence analysis confirmed the upregulation of the e-cadherin protein on plasma membrane. silencing of pfkfb3 caused approximately 40% reduction in matrigel invasion, compared to non-targeting sirna. inhibition of the matrigel invasion caused by pfkfb3 depletion does not appear to be associated with reduced zeb1 expression, as ectopic expression of zeb1 did not reverse the effect of pfkfb3 silencing on invasion. taken together, these data suggest that pfkfb3 may be required for the maintenance of the mesenchymal phenotype and associated traits in pancreatic adenocarcinoma cell lines. introduction: leukemias are neoplasms that arise from hematopoietic cells initially proliferate in the bone marrow, and then disseminated in the peripheral blood, spleen, lymph nodes and eventually to other tissues. lymphomas occur primarily in the lymph nodes, but can be extended in peripheral blood and bone marrow infiltrate. aim: to determine the values of haematological parameters the control and test groups. to determine the prevalence of types of chronic leukemia in relation to the experimental group. compare haematological parameters in relation to the type of chronic leukemia. materials and methods: a prospective-retrospective study included subjects who were made laboratory hematology in oj clinical chemistry and biochemistry ukcs. blood tests conducted on the hematology analyzer siemens advia 2120 hematology system and abbott cell dyn 3700 and microscopic analysis of the peripheral blood smear. results and discussion: according to the age of respondents test group was established mild form of anemia, a red blood cell count is totaled 3.69 ae 1.13x10 12 , which is signifycantly lower compared to the control group. the average number of leukocytes was significantly higher in subjects studied groups and amounted to 136.91x10 9 , with a maximum value of 580 x10 9 . in the peripheral blood of patients with chronic leucosis has established a significantly higher number of cells compared to the control group (p = 0.001), while the number of monocytes was a significantly smaller. in the group of patients with chronic leukosis largest number had chronic lymphocytic leukemia (70%), and chronic myeloid leukemia had 30% of respondents. conclusion: subjects with cll were statistically older than patients with cml, and as regards the gender structure, men have dominated in cll and cml in women. white bloodline was found that the number of leukocytes in both forms of chronic leukemia high above the reference value. p-05.03.3-032 effect of enzymatic and non-enzymatic isolation methods of endometrial stem cells on their cell proliferative potential and mesenchymal stem cell characteristics human endometrial stem cells (hescs) are responsible for the monthly renewal of the basal layer of the human endometrium by facilitating stromal and vascular regeneration. in this study, hescs were isolated with three different isolation methods including non-enzymatic and enzymatic digestion using trypsin and collagenase type 1. the effect of these three isolation methods on the acquisition of mesenchymal stem cells (msc) and on hesc proliferative potential was evaluated through flow cytometric analysis of cd surface markers and wst-1 tetrazolium salt assay. our findings indicate that hescs isolated with these three methods have statistically similar cell proliferation rate at 24 h time point. however, at 48 h time point, hescs isolated with the non-enzymatic and collagenase type 1 method displayed a higher expansion in cell number when compared to the hescs isolated with trypsin. the late passage of hescs isolated with non-enzymatic and trypsin methods showed the highest proliferation rate in comparison to the hescs obtained via collagenase type 1 isolation method at 24 h, 48 h and 72 h. the three isolation methods for the early passages of hescs had a resemblance in their msc profile with no significant difference. on the other hand, late passage hescs isolated using trypsin non-enzymatic method showed a higher cd31and lower cd44 profile. moreover, late passage of hescs isolated with non-enzymatic method displayed a significant reduction in their cell surface cd90, cd73, and cd105 surface expression levels. only hescs isolated with collagenase type 1 did not present a significant shift in their mesenchymal cd marker profile from early to late passages, taken together results from this study suggest that the longterm maintenance of mesenchymal markers can only be achieved in cell isolation with collagenase type 1, while non-enzymatic method is more suitable to obtain higher msc cell yield for immediate use. hepatocellular carcinoma (hcc) abundantly arises on the viral and/or chemical-induced cirrhosis in liver. cirrhosis is defined as one of the premalignant stage hcc in which microenvironmental changes occurred such as uncontrolled production of collagen type i and activation of hepatocyte growth factor (hgf)/c-met signaling. it has been shown that epcam+/cd133+ subpopulation of cells isolated from hcc tissue can initiate tumor at very low concentration in xenograft model and behaves as hepatic cancer stem cells. however, the molecular mechanisms supporting hepatic stem cell activation are not well understood and knowledge about the role of hgf/c-met pathway in this process is not clear. in this study, we aimed to define effect of collagen type i and hgf induction on the cell behaviours of epcam+/ cd133. epcam+/cd133+ cells were sorted by magnetic separation from huh-7 cells. then proliferation and invasion of cells were analyzed under the hgf induction as well as branching morphogenesis in vitro. after hgf stimulation, phosphorylation level of c-met increased in epcam+/cd133+ subpopulation. moreover, presence of collagen type i enhanced significantly effect of hgf stimulation in the invasion of epcam+/cd133+ cells. we also have showed that hgf stimulation increased branching tubulogenesis capacity of epcam+/cd133+ subpopulation while it did not effect proliferation of cells. these effects of hgf reverted by c-met inhibitor, su11274, in vitro. all these findings showed that hgf and collagen type i regulates aggressive phenotype as microenvironmental changes via induction of invasiveness of epcam+/cd133+ subpopulation of huh-7. in conclusion, we showed that hgf/c-met signaling causes to get more metastatic phenotype based on invasion and tubulogenesis in epcam+/cd133+ hepatic cancer stem cells in hcc and it might be possible to use c-met inhibitors to target hepatic cancer stem cells during hepatocarcinogenesis. endometriosis is defined by the migration of endometrial mesenchymal stem cells (emscs) into the peritoneal cavity or other site of body rather than uterus in a retrograde fashion. its previously known intracellular crosslinking enzyme called tissue transglutaminase (tg2) was shown to play important roles in the extracellular matrix (ecm) modelling, fibrosis, cell adhesion and migration. we have hypothesized that tg2 might be expressed in emscs and take part in the formation of endometriosis. the difference in the proliferation capacity of emsc isolated from endometrial tissue with/without endometriosis was determined using wst-1 assay and tg2 activity and expression levels were analysed by btc assay and rt-pcr. the biosynthesis and activity for mmp-2 and -9 were investigated with zymography and rt-pcr, respectively. although tg2 activity was found to be 50% less in emscs isolated from endometriotic tissue, these cells showed 9 times higher tg2 protein expression than those isolated from the control tissue without endometriosis. emscs from endometriotic tissue have 2.3 times higher tg2 and 10.3 fold higher itgb1 mrna levels when compared to the cells of healthy group. similar results were observed in sdc-4 gene expression with a 2.5fold increase. endometriotic emscs demonstrated an average of 11.5-fold increase in the mmp-2 activity while a onefold increase was evident in mmp-9 activity when compared to the healthy emscs. emscs from patient group possessed a higher proliferative ability in comparison to that of healthy subjects within 96 h. the fact that emscs from the control tissue showed lower tg2 protein levels with a high enzyme activity suggested that tg2 might be important in the development of endometriosis not only by destabilizing ecm but also enhancing the cell migration. in this context, the upregulation of tg2 along with itgb1 and sdc4 was evident in emscs of endometriosis which was possibly associated with the increase in the activity of mmp-2 and -9. recent studies have indicated that pluripotent stem cells and some stromal stem cells such as mesenchymal stem cells (msc) are metabolically different from their differentiated counterparts. in this study, the cellular mechanisms controlling metabolic changes in stem cells was investigated using wharton jelly mesenchymal/stromal stem cells (wj-mssc). wj-msscs were isolated by the explant method and cultured in dmem-f12 with 10% fbs. endothelial differentiation was induced by the addition of vegf, egf, insulin and hydrocortisone for 6 days. neuronal differentiation was achieved by using commercial neuronal differentiation medium (millipore) for 3 days. in parallel experiments, cellular metabolic activity such as lactate production was measured. the msc characterization was performed by flow cytometry using antibodies against cd90, cd105, cd73 and cd44 (bd human msc analysis kit). the differentiation process was followed by measuring the expression of cd31, cd34 for endothelial and gfap, neu and tyrozine hydroxylase proteins for neuronal cells by immunofluorescence. for gene expression, nanog, cd34 and gapdh genes were analyzed by rt-pcr. differentiation stimuli to endothelial or neuronal cells resulted in a significant decrease in msc marker proteins. expression of stem cell markers other than cd73 were decreased to 2-20%. differentiation induced the expression of cd31, cd34 for endothelial and gfap and neu proteins for neuronal cells. in vitro lactate production was decreased following differentiation in both lineages. neuronal differentiation increased glucose consumption by~48% and the extracellular calcium concentration of these cells was significantly lower than synchronous undifferentiated cells. glycolytic activity is decreased during in vitro differentiation of wj-msscs. metabolic reprogramming and glucose uptake of cells may be an early indicator of the differentiation process in wj-msscs, supporting the view on their metabolic plasticity. store-operated ca 2+ entry (soce) activated by depletion of intracellular ca +2 stores has been shown to control intracellular ca 2+ homeostasis in many physiological and pathological events. stromal interactive protein, stim1, as endoplasmic reticulum (er) ca +2 sensor and orai protein as pore-forming subunit of soc channels play crucial roles in the activation of soce channels. stim1 and orai were reported to have pathophysiological roles especially in hepatocellular carcinoma (hcc). anticancer chemotherapy frequently falls back because of these tumor-initiating subpopulations, tentatively called 'cancer stem cells'. the purpose of this study was to investigate the roles of stim1 and orai on soce in differentiation of huh-7 hccs expressing epcam and cd133 surface adhesion molecules (epcam + cd133 + ). epcam + cd133 + subpopulations in huh-7 cells were separated via flow cytometry and transfected with stim1 and orai-1 over-expressing (oe) plasmids. expression levels were confirmed by rt-pcr. changes in intracellular ca 2+ concentration were monitored via dual wavelength spectrofluorimeter in fura 2-loaded cells. in epcam + cd133 + cells, er ca 2+ release increased without any change in soce compared to that of epcam à cd133 à cells. similar results were observed in stim1-oe epcam + cd133 + cells. on the other hand, increase in orai1 has no effect on either parameter. cancer is globally one of the most death causes. recently, huge improvements occurred in the cancer diagnosis and treatment due to advanced technology, however recurrence occurs almost 30-40% of patients and their survival times decreases. in this study, we aimed to investigate of relationship between the cancer stem cells which are strongly associated with chemotherapy and radiotherapy resistance and recurrence with the non-classical mhc i antigens which have immunosuppressive properties. for this purpose, we immunohistochemically evaluated the expression patterns of cd133, cd44, nanog, oct3/4, hla-g and hla-e in the advanced stage colorectal, gastric and breast cancer and also non malign biopsy samples. we detected that the cancer stem cell markers cd133, cd44, nanog and oct3/4 significantly increased in the advanced stage cancer tissues. however, the immunosuppressive hla-g and hla-e expressions increased only in the colorectal and gastric tumor tissue. in addition to the presence of cancer stem cell like cells in the tumor tissues, increased expressions of hla-g and hla-e may indicate an immune evasive adaptation of tumor cells. according to our findings, the hla-g and hla-e may be potential therapy targets to elimination of cancer stem cells of colorectal and gastric cancers. however, more detailed studies are needed to support our findings and also to determinate of clinical values of these markers. endocannabinoids increase sdf-1 release from human mesenchymal stem cells s. k€ ose 1 , f. aerts kaya 1 , d. uc ßkan c ß etinkaya 1 , p. korkusuz 2 1 stem cell research and application center, hacettepe university, ankara, turkey, 2 department of histology and embryology, hacettepe university, ankara, turkey lipid-structured endocannabinoids are endogenous morphine ligands and present widespread receptor-mediated effects at physiological and pathological levels on the nervous system as well as many other systems. these effects are partially realized through mechanisms affecting cell growth, differentiation, apoptosis and migration at the molecular level. the hematopoietic progenitor cells (hpcs) and mesenchymal stem cells (mscs) form a distinct niche in bone marrow where they interact with each other in harmony. the stromal cell-derived factor 1 (sdf-1/cxcl12) is a chemotactic factor in bone marrow and is released from mscs and their receptor cxcr4 is found in hpcs. with these rationale in mind, we asked if hpcs and msc interaction mediates sdf-1 release via endocannabinoidal system. bone marrow mscs obtained from healthy donors and passage 3 mscs were induced with 200 ng/ml lipopolysaccaride (lps) for 4 h. antagonists for cb1 (am281) and cb2 (am630) receptors were added to cultures for 4 days. after incubation with antagonists msc culture supernatants collected and processed with human sdf-1 beta in elisa medium. analyses demonstrated direct decreasing effect of endocannabinoid receptor antagonists on sdf-1 beta release from bone marrow mscs. in conclusion, endocannabinoidal system regulates sdf-1 release on mscs and directly act on hpcs mobilization in bone marrow microenvironment (niche). this may have a clinical implication on therapeutic mobilization strategies for hscs in hematology clinical applications. implantation is invasion of the embryo into the endometrium and occurs in three stages apposition, adhesion and invasion, via the complex cellular and molecular mechanisms. during these stages, both of maternal endometrium and embryo should be appropriate for the implantation which is the beginning of pregnancy. receptivity of uterine consists in the existence of growth factors such as tgfbeta-1, igfr1, vegf. it is indicated that damages of factors relesead from endometrium and blastocyst prevent implantation. recently, stem cells can be obtained from many sources to use for therapeutic purposes and mesenchymal stem cells derived from bone marrow are the most studied. in our study, it was aimed to investigate molecules play a role in blastocyst implantation after bone marrow derived mesenchymal stem cell application into the rat endometriyum. female rats were divided into three groups which were saline (sf, n:7), media (m, n:7), stem cell in media (m+bmsc, n:7). after vaginal smear technique, female rats in estrous cylcle were injected into the uterine and periton 200 ll saline, 200 ll culture media and 1 9 10 6 cell/200 ll culture media. the pregnant female rats on the 7 day were sacrified and uterine samples removed and were stained with heamatoxylin-eosin histochemically and anti-tgfbeta-1, anti-igfr1, anti-vegf and anti-pcna immunohistochemically and obseved under light microscope. h-score results were determined using one-way anova test statistically. it was found that intraperitoneal administration of stem cells with media, was increased tgfbeta-1, igfr1, vegf and pcna parameters when compared with the intrauterine administration of stem cells. in this study, it was revealed that distribution of molecules play role in implantation were changed due to stem cell application. it is supposed that stem cell treatments can be cured the molecules caused infertility. many unconventional biochemical factors remain to be investigated for their potential effects on stem cells. among others, endogenous gasotransmitter h 2 s, generated from l-cysteine and organosulfur-compounds (oscs) metabolisms, plays very important roles in the central nervous, respiratory and cardiovascular system. slow-releasing h 2 s donors are viewed as powerful tools for basic studies and innovative pharmaco-therapeutic agents for cardiovascular and neurodegenerative diseases. exogenous h 2 s administration is able to promptly scavenge ros, activate myocardial k atp channels and increase pro-cell survival signaling, very likely activating erk and phosphatidylinositol 3-kinase (pi3k)/akt pathways. the effects of h 2 s-releasing agents on the growth of stem cells are not yet widely investigated. therefore, stem cell therapy combined with h 2 s may have great clinical relevance in cell-based therapy for regenerative medicine. the effects of slow-releasing h 2 s agents on the in vitro cell growth and differentiation of human lin à sca1 + cardiac progenitor cells (hcpc) were here studied. in particular, the effects of h 2 s-releasing agents, such as na 2 s, gyy4137 and water-soluble gsh-garlic conjugates (gsgaws), on the cell viability and differentiation of hcpc were here investigated by colorimetric assay, immune-fluorescence microscopy and western-blotting analysis. the treatment with slow-releasing h 2 s donors increased the cell proliferation in a concentration dependent manner respect to the control. moreover, the treatment with gsgaws led to an up-regulation of the expression of proteins involved in the cell adhesion and differentiation processes. these preliminary results highlight on the effects of this gasotransmitter on the stem/progenitor cells and on the possibility to develop functional 3d-systems for cardiac tissue repair, that take into account the relevant biological role of h 2 s in the cardiovascular system. p-06.02.5-002 investigation of the protective effect of boric acid and omega-3 fatty acid in model of acute myocardial infarction changes in myocardial rats ischemic heart disease being the most common cause of the mortality and morbidity in worldwide commonly results from the occlusion or narrowing of the coronary arteries by atheromatous plaque and thus is named as coronary artery disease. male sprague dawley rats were used in the present study. rats were divided into 5 groups with 10 rats in each: control, mi, mi+boric acid, mi+omega-3 and mi+boric acid+omega-3 groups. control rats were treated with 2 ml/day saline, boric acid-treated rats received 100 mg/kg/day boric acid and omega-3-treated rats received 800 mg/kg/day for 28 days by oral gavage. for the experimental mi model, 200 mg/kg izoproterenol-hcl (iso) was administered subcutaneously two times with a 24-h interval in the last 2 days of the boric acid and/or omega-3 treatments. twelve hours after the second dose of iso, general anesthesia was induced. under general anesthesia and spontaneous respiration, ecg recordings were obtained by using a computerized data recording and analysis system (mp100, biopar) and d-ii recordings were used in the analysis. compared to the control group, serum ck-mb, bnp and tnf-a levels were higher in mi group (p < 0.001, p < 0.001 and p < 0.01 respectively). in the heart tissue homogenate, biochemically measured calpain activation and mda were increased (p < 0.01 and p < 0.001, respectively) and pon1 levels were decreased (p < 0.05). according to the ecg recordings, st wave and heart rate were found to be decreased (p < 0.001 and p < 0.001, respectively). on the other hand, all above mentioned parameters were found to be improved in rats treated with boric acid and/or omega-3 after induction of mi. moreover, histological analysis including light microscopy and tem revealed a significant histological improvement in rats treated with boric acid and/or omega-3 after induction of mi. results of the present study suggest that omega-3 and/or boric acid treatment significantly decreases the cellular damage in mi. this is study is aimed at measuring the level of serum heart-type fatty acid binding protein (h-fabp) in patients presenting with diabetic ketoacidosis (dka) and diabetic ketosis (dk) and to determine its role in identifying early period cardiac ischemia by comparing this level with the level of a control group at a comparable age this study was planned to be a prospective study and it included 35 patients diagnosed with dka, 20 patients diagnosed with dk and 20 voluntary pediatric and adolescent healthy control subjects. the h-fabp, creatine kinase-mb (ck-mb) and troponin-i levels were studied in patients with dka and dk as well as in the control group at the time of presentation. for dka patients, their h-fabp values were measured once again after acidosis correction and compared with the values they had at the time of presentation there were no differences among groups in terms of sex, age, height and weight. no statistically significant differences were found among groups with respect to troponin-i values (0.06 ae 0.08, 0.07 ae 0.04 0.04 ae 0.04; p = 0.457). no statistically significant differences were found among groups with respect to ck-mb values (1.48 ae 0.91, 1.55 ae 0.9, 2.09 ae 1.37; p = 0.229). the h-fabp values of dka patients at the time of presentation were found to be statistically significantly higher than those of dk patients and control group (1.17 ae 0.79; 0.79 ae 0.5 0.69 ae 0.36; p = 0.006). the h-fabp value of the dka group at the time of presentation was found to be statistically significantly higher than the value at hour 36 after acidosis correction (1.17 ae 0.79; 0.55 ae 0.28; p = 0.0001) the fact that h-fabp levels were found to be high in pediatric patients diagnosed with dka at the time of presentation suggested that myocardial ischemia had been triggered. in diabetic patients, every ketoacidosis attack may lead to cardiac ischemia, thereby accelerating progress to necrosis. in conclusion, we would like to propose h-fabp as a potential marker for indicating myocardial ischemia. p-06.02.5-004 genome-wide analysis of hypoxic stress response in human cardiomyocytes stress in human cardiomyocytes on a genome-wide scale remains poorly understood. this study aimed to identify the gene expression patterns of adaptive response of the human cardiomyocytes (hcm) to hypoxic stress. in vitro experimental models of hypoxia mimicking in-vivo coronary ischemia, are useful tools to identify molecular pathways involved in myocardial ischemia. in the current study, we cultured ac16-hcms in dmem/f12 with 10%fbs. to simulate hypoxia model, cardiomyocytes were plated in hypoxia chamber (1%o 2 , 5%co 2 , 94%n 2 ) for 3, 6, 12, 24 h and the control group was incubated in normal conditions (5%co 2 , 95%o 2 ). cell viability was determined using mttassay. annexin-v assay was used to monitor apoptosis. gene expression profiling was analysed with affymetrix-hg-u133-plus-2 arrays. following bioinformatic and statistical analyses differentially expressed genes (deg) were classified according to gene ontology using david and kegg pathway analysis tools. according to mtt, annexin-v and hif gene expression results, hypoxia time was determined as 3 h. we identified 649 genes (279 down-regulated and 370 up-regulated) (p < 0.001, fold change ≥1.5) were differentially expressed in hypoxic-ac16 vs. ac16. degs were mainly clustered in cell proliferation, regulation of cell death, cell adhesion and response to stress. furthermore, transcriptome analyses revealed that 'metabolic, cytokinecytokine receptor interaction, hif-1 signaling, tgf-beta, cell cycle and apoptosis' pathways were involved in the hypoxic stress response of human cardiomyocytes. this study provides molecular information regarding gene expression reprogramming of human myocardial hypoxia. the pathways identified in this study may pave the road for translational medicine. this study was supported by tub _ itak project number 111s189. autologous ips cells after reprogrammed into endothelial progenitor cells (epcs) may offer several advantages in the treatment of cardiovascular disorders because of their cardiogenic and vasculogenic differentiation potential. to reach that purpose, we differentiated and characterized mouse ips cells into flk1 + , a well-recognized epc marker. further maturation of epc was characterized by the expression of cd31 and cd133 markers. purified ips cells were differentiated into flk1 + cells with the use of differentiation medium on type iv collagen-coated dishes in the absence of lif. we then analyzed flk1 gene expression and protein levels with qrt-pcr, western blot and immunocytochemical methods on days 2.5 to 7.5. flk1 + cells isolated with macs system and then recultured these cells in differentiation medium with vegf to induce epc cells. following induction, cd31 and cd133 gene expression and protein levels were analyzed with genomic and proteomic methods. after isolating these cells and aggregate overnight, we cultured cells in three-dimensional condition in collagen type i and used differentiated medium including vegf and egf. we found that flk1 expressing cell number reached to a peak level (24%) on day 5.5 followed by a progressive decline subsequently. in the second step, cd31 and cd133 positive cells were generated and enriched during day 4 of induction. we showed optimal time for harvesting flk1 + cells is day 5.5 of initial differentiation. following isolation of flk1 + progenitor cells they were further matured into functional epcs by vegf within 4 days of induction. additionally for evaluation of angiogenic potantial differentiated cells, we monitored epcs behavior along vascular formation in 3d culture. our work demonstrates that epcs could be successfully derived from ips cells and these cells have vascular formation and angiogenic potential in 3d culture. epc drived ips cells play important role in the treatment of cardiovascular disease. p-06.02.5-006 electrophysiological, biochemical and genotoxic effects of luna experience on heart tissue in rat model pesticides are widely used for the control of agricultural, industrial and domestic pests. however, the uncontrolled use of pesticides has diverse effects on ecological system and public health. fungicides are one of the pesticide type used to kill fungi or fungal spores. in this study, the effect of different doses of luna experience, a fungicide, on the cardiac electrophysiology and genotoxicity in rats were investigated. among five groups (5 mg, 10 mg, 20 mg, control and positive control for comet assay) treatment groups received by gavage doses of luna experience for 30 days. electrical activity of heart were recorded using electrophysiological recording techniques. tissue activities of paraoxanase (pon) and arylesterase (are) and level of malondialdehyde (mda) were measured using biochemical methods. comet assay was performed on heart tissue. we calculated genetic damage index (gdi) and damaged cell percent (dcp) from comet assay. it was observed that there is a significant decrease in heart rate in all treated groups as compared with control group (p < 0.05). amplitude of p wave and qrs complex did not change (p > 0.05). in all treated groups, statistically significant differences were found for values of pon, are, mda, gdi and dcp when compared to control group (p < 0.05). according to our results, exposure to different doses luna experience have a probable hazard potential for the cardiac system. the macrocyclic cage complexes iron (ii) clathrochelates are of the interest due to their bioactivity; they are able to inhibit t-7 rna polymerase, possess toxicity to leukemia cells hl-60 and suppress amyloid fibril formation. their binding to serum albumins was reported; the extreme binding affinity to albumins is observed for the compounds bearing carboxy groups. upon this interaction, clathrochelates quench protein intrinsic fluorescence and gain optical activity inducing circular dichroism (cd) signal in 350-600 nm region. here we examine the effect of spatial arrangement (isomery of substituents) of clathrochelates on their binding to globular proteins. we study 6 bis-substituted clathrochelates bearing two same or different isomers (ortho-/meta-/para-) of carboxyphenylsulfid groups. their interaction with bovine (bsa) and human serum albumins, b-lactoglobulin and lysozyme are explored by cd and protein fluorescence quenching method. the binding of compounds to albumins evoked the cd bands of the same shape, but their intensities vary up to 45 times depending on substituents isomery. in the presence of b-lactoglobulin, the intensities, shape, and positions of the induced cdbands differ for the compounds with different isomer groups. the cd bands induced by the lysozyme in the case of di-para substituted clathrochelate are shifted relatively to the bands of other isomeric compounds. the pronounced quenching of protein fluorescence by clathrochelates was observed only in the case of bsa, its intensity depends on the geometry of substituents (9-17 times). the different spatial arrangement (isomery) of carboxyphenylsulfid substituents in clathrochelates causes the distinctions in both their cd-signal induced by interaction with proteins and their effect on the protein fluorescence. the geometry of ribbed substituents is important for their binding to biomolecules (particularly proteins) and is suggested to determine the structure of the formed guest-host complex. 3d bioprinting is a new technology that revolutionized the field of tissue engineering and regenerative medicine, allowing reconstruction of living tissue and organs preferably using the patient's own cells. using a 3d printer we can design biological structures by controlling exact deposition of cells, growth factors and extracellular molecules in a spatially-controlled manner. the aim of this study was to evaluate the differentiation of human amniotic fluid stem cells (afsc) into endothelial progenitor cells using a bioinkò hydrogel photopolymerized in a 3d network resembling vascular tissue. characterization of afsc was performed by flow cytometry, followed by sorting of the cd177 + stem cell subpopulation. cd117 + stem cells were stained with cell tracker red cmtpx and then mixed with bioinkò hydrogel. printing was done using a 150 lm diameter needle, under 1 bar pressure, and 150 mm/min speed. the network models with define distance apart were printed and analyzed by fluorescent microscopy. mtt test was used to evaluate the viability of the cd117 + stem cells. our results showed that afsc remained viable as shown by mtt assay. the fluorescent microscopy images confirm the viability biochemical test showing that the cd117 + cells viability is maintained after 7 days of cultured in bioinkò hydrogel. furthermore, histological section of hydrogel showed that cells have a relatively uniform distribution forming network interactions between cells. flow cytometry assay showed that cd117+ cells expressed endothelial markers such as cd31, cd105, cd133, cd144 and vegfr2. in conclusion 3d printers are useful tools for creating three-dimensional scaffolds that mimics the cell microenvironment where different types of cells could proliferate, differentiate and crosslink with each other forming tissue-like structures. this study aims to reveal the biocompatibility, biodistribution and immunomodulatory impact on the production of inflammatory citokines of magnetite (fe3o4) nanoparticles functionalized with natural compounds with proved antimicrobial and immunomodulatory effects. co-precipitation synthesized fe3o4 were functionalized with plant-derived compounds: eucalyptol, carvone, limonene and b-pinene. characterization was done by ir, sem and hr tem, while in vitro biocompatibility was tested using endothelial human cells (fluorescence microscopy and proliferation assay). in vivo biodistribution was tested in a balbc mouse model at 2 and 7 days post-intraperitoneal injection, followed by experimental organ removal. tissue sections obtained from vital organs were stained with hematoxylin-eosin. production of inflammatory cytokines was assessed by elisa. results demonstrated that, at concentrations of 500 lg/ml, all prepared nanosystems have a good biocompatibility in vitro and in vivo, allowing the development of cultured cells and also not affecting any visible behavior and organ morphology of the mice. microscopy evaluation of the organs sections revealed that nanoparticles are not present in vital organs such as brain, heart, kidney and liver, but aggregates were visible in the lungs and spleen. at 7 days post-injection no visible aggregated were found in the lungs, few dark-brown nanoparticles clusters being visible in the red pulp of spleen. elisa results revealed that fe3o4 functionalized with carvone and limonene significantly stimulated the production of il-2, il-10 and il-6, while reducing the production of tnfa. other nanosystems din not impact significantly on the cytokine production. functional fe3o4 nanoparticles are efficient drug delivery shuttles, able to stabilize pharmacological compounds, such as plant-derived bioactives, and their biocompatibility, specific biodistribution and limited immunomodulatory effects recommend their use in pharmacological formulations. p-07.01.3-004 new approach for cell imaging with fluorescent carbon nanoparticles m. dekaliuk, k. pyrshev, o. demchenko palladin institute of biochemistry, kiev, ukraine in the nanotechnology field, much interest was focused on the new carbon nanomaterials for cell imaging. recently discovered inorganic carbon nanoparticles ('c-dots') due to their excellent fluorescence characteristics and biocompatibility have ample opportunities for their use in imaging and functional transformations in living cells. their distinctive features, such as high brightness, small sizes, high biocompatibility, small negative charge on the surface and very easy methods of their preparation present a good alternative to other nanoscale materials. the focus of our research was to determine the possibility of using c-dots as the easily available probes for apoptotic cells detection. the carbon nanoparticles were prepared from alanine, citric acid, urea, etc by hydrothermal treatment at 180 c. the studies were performed with adherent epithelial vero and hela cell lines (atcc). with these tools we demonstrate that both native and apoptotic cells can be easily visualized. the cdots uptake occurs probably by endocytosis, which allows for much larger their number to accumulate in apoptotic cells. using the different methods of sample preparation, they show the ability for labeling various structural compartments of the cell. for living cells there are the intracellular vesicles and lysosomes. in contrast, in fixed cells the nucleus is labeled preferentially. the fact that apoptotic cells accumulate strongly increased amount of cdots can be efficiently used in flow cytometry for characterizing the cell populations regarding the relative amount of apoptotic cells in different experimental conditions. the application of such cheap and easily accessible nanoparticles provides more opportunities to simplify the popular methods of cell labeling and detection. previously, our studies showed the possibility of using these nanoscale fluorophores for super resolution method sofi. a new electrochemical microbial biosensor for the fast detecting of dopamine and epinephrine based on candida tropicalis immobilized in a carbon paste electrode (cpe) modified with single wall carbon nanotube (swcnt) was described in this paper. the immobilized cells were used as a source of polyphenol oxidase (ppo) to develop voltammetric epinephrine and dopamine biosensor. voltammetric determination of phenolic compounds like epinephrine and dopamine is a simple technique available. direct oxidation of phenols can be used, but the oxidation potentials of this compounds are similar and they can not be detected distinctively. another possibility is the use of biosensors based on the polyphenol oxidase (tyrosinase) enzyme that oxidises the phenolic compounds into their corresponding quinones. by this way phenolic compounds that epinephrine and dopamine that used in this study were detected at the different potential. the effect of varying the amounts of swcnt and microorganism on the response to epinephrine was investigated to find the optimum composition of the sensor. the effects of ph and temperature were also examined. increases in biosensor responses were linearly related to dopamine concentrations between 0.1 and 1.0 mm and epinephrine concentrations between 0.01 and 1.0 mm. limits of detection of the biosensor for dopamine and epinephrine were calculated to be 0.021 and 0.0029 mm, respectively. finally, proposed systems were applied to epinephrine and dopamine analysis in pharmaceutical drugs. objective: it has started a long time ago to search for a material that can replace blood. this material does not require special storage conditions, independently of the recipient's blood group and can be applied to all individual. milk, casein derivatives, starch, saline and ringer were used for this aim in the past. the determination of toxic effect of natural hemoglobin (hb) on human, researchers have focused on development modified blood. in this work, the development of an artificial biomaterial alternative of blood for using as preoperative and operative aims was aimed. material and methods: in our study, ultrapure hb molecules are immobilized on triethanolamine coated magnetic nanoparticles using various techniques. prepared nanoparticles were characterized by ft-ir, ctem, xrd and cyclic voltammetry (cv). the cytotoxic effects of artificial blood were tried on mtt cell proliferation. results: the characteristic peaks of hemoglobin were obtained from ft-ir spectra differently from support. particles size is concluded by using debye-scherrer equation as > 80 nm from xrd spectra. sem and ctem images supported xrd result. cv results showed that hb molecule has à0.418 v cathodic potential against ag / agcl standard electrode. significant differences were not observed in the mtt results (p < 0.01). conclusion: the nanoparticles were obtained in accordance with the intended desired method. it is determined that the hemoglobin molecules give the same potential with natural blood even after 3 weeks of immobilization and carrying oxygen as natural blood. there are statistical differences between results of mtt tests due to used concentration. but, it is considered that decantation advantage of the artificial blood minimized cytotoxic effects. proteoglycans are among the most abundant molecules of the inter-cellular structure and they are present in extracellular matrices of connective tissues. these glycosylated proteins contain one or more (gag) chains that are covalently attached to the core protein and their hydrodynamic function is mainly due to the physicochemical characteristics of this gag component which provides hydration and swelling pressure to the tissue. gag levels excreted via urine are used as a marker to monitor different diseases (chronic renal disease, renal fibrosis, glomerular filtration abnormalities, bladder stones, breast and lung cancers, hypertension and diabetes, etc.) besides the well known mucopolysaccharidoses. however, their detection by using chromatographic methods is hard, because of the high polarity of negative charges and different functional groups such as acetyl sulfates that generate microheterogenity. in this study, we developed molecularly imprinted chromatographic hplc columns for specific heparan sulfate (hsa), chondroitin sulfate (cs) and dermatan sulfate (ds) detection in urine. positively charged acrylamide monomers were first polymerized by precipitation polymerization, to produce polymers which will show specific recognition for gag's via electrostatic interactions and hydrogen bond formation. these gag selective polymers were then filled in the steel hplc columns and columns eluents were chemically degraded. degradation products of gag's were examined offline column coupled with tandem mass spectrometry. the results showed that our imprinted columns separated gag's specifically and sensitively. thus, urine gag's can be specifically determined by using a gag specific molecularly imprinted column. in this study internal standart weren't used because the matrix effect was lower than 5% for each urine samples. %cv of ds, cs and hsa was calculated as; supported lipid bilayers (slb) were started to be used for cell culture studies to focus on cell adhesion, cell signaling etc. testing the stability of slbs is essential to utilize them as cell culture platforms. in this study, the stability of phosphatidylcholine (pc) lipid bilayers on glass was investigated under milli-q water, phosphate buffer saline (pbs) and dulbecco's modified eagle medium culture (dme) medium supplemented with/without serum. the stability was also checked by enriching slb with different lipids. pc-liposomes were prepared by hydrating the dried thin lipid film with pbs and then by extruding the suspension through a polycarbonate membrane. a negatively charged phospholipid, phosphatidylserine (ps, 25%); a positively charged phospholipid, dotap (50%) and cholesterol (25%) were also used for liposome preparation. liposomes were fluorescently labelled and series of slb imaging were taken for a week. in all experiments in milli-q water and pbs, the stability was conserved for 7 days. pc bilayers in medium supplemented with serum showed hole formations on the second day and their number and size increased rapidly in time. when the bilayers were prepared in medium without serum, disruption was lowered but not completely removed as a result of other factors in medium. cholesterol providing an increased rigidity to the membrane caused higher stability. positively charged bilayer structures also showed increased stability. this can be explained by decreased mobility of bilayer as a result of electrostatic interaction between positively charged molecules and negatively charged glass surfaces. decreased mobility decreases the interactions within the medium. lastly, negatively charged bilayers did not show high stability. strong repulsive forces between the negatively charged surface and bilayer probably prevented the integrity of the bilayer and increased the deformation. in recent years the use of biopolymers has gained priority in tissue engineering and biotechnology, both as dressing material and for enhancing treatment efficiency. there is a demand for new biopolymers designed with protease inhibitors and antimicrobials. ll-37 is an important antimicrobial peptide in human skin and exhibits a broad spectrum of antimicrobial activity against bacteria, fungi, and viral pathogens. using lignin which is an abundant carbohydrate polymer in nature and a polyacrylic acid, we prepared a polymer film by plastifying caprolactone and polyacyrlic acid. films were actified to immobilize ll-37. the structure was elucidated in terms of its functional groups by fourier transform infrared spectroscopy (ftir), and the morphology of the film was characterized by scanning electron microscopy (sem) before and after the immobilization process. the amount of ll-37 immobilized was determined by elisa method. 99.9% of ll-37 peptide was successfully immobilized onto the films. antimicrobial activity was determined in the film samples by quantitative antimicrobial activity method. according to the results, ll-37 immobilized film samples were effective on test organisms; gram-positive staphylococcus aureus and gram-negative escherichia coli. in bio-compatibility assays, the ability to support tissue cell integration was detected by using 3t3 mouse fibroblasts. samples were examined under transverse microscope, non-immobilized sample showed a huge cellular death, whereas ll-37 immobilized film had identical cellular growth with the control group. this dual functional film with enhanced antibacterial properties and increased tissue cell compatibility may be used to design new materials for various types of biological applications. p-07.01. [3] [4] [5] [6] [7] [8] [9] [10] in vitro modulation of the cross-talk between macrophages and osteoblasts by titania nanotube-modified ti surfaces p. neacsu, a. mazare, p. schmuki, a. cimpean bone remodeling is a dynamic process that maintains a fine balance between bone formation and resorption, and is highly influenced by the inflammatory state of the local microenvironment. therefore, a proper modulation in the cellular interactions and cytokine expression is a promising approach to achieve enhanced bone healing. as the biomaterials surface has a major impact on cellular behavior, the goal of the current study was to investigate the influence of tio 2 nanotube-modified ti surfaces (ti/tio 2 ) on the cross-talk between raw 264.7 macrophages (mf) and mc3t3-e1 preosteoblasts (ob) in mono-and co-culture systems in comparison with flat ti (cpti). raw 264.7 and mc3t3-e1 cells were seeded on the test surfaces and grown in standard culture conditions for various periods of time. for co-culture studies, the cells were cultivated using a transwell system. inflammatory mediators released by raw 264.7 cells were measured using elisa technique, while the ob capacity to produce calcified bone matrix was evaluated by alizarin red staining. in co-cultures, lps-stimulated tnf-a, il-6 and mcp-1 release was significantly increased at 24 h, while after 7 days only il-6 exhibited higher amounts when compared with mf cultures alone. moreover, the secretion of these mediators by cells exposed to ti/tio 2 was diminished, especially in lps evoked conditions. also, alizarin red staining demonstrated the presence of calcium deposits when ob were co-cultured with mf for 24 h and 7 days, whereas the presence of the mf for 4 weeks significantly inhibited mineralization. on ti/tio 2 surface elevated calcified matrix was observed, as compared with cpti. this study reveals that the overall effect of inflammation suppression induced by ti/tio 2 may contribute to the enhanced mineralization. also, chronic inflammation may inhibit or delay the regeneration process. therefore, an adequate modulation of mf and ob interactions is vital for the biomaterials success in stimulating bone regeneration. p-07.01.3-011 synthesis and characterization of the branched magnetic polymer for drug delivery systems t. tarhan 1,2 , b. tural 2 , s. tural 2 1 mardin artuklu university, mardin, turkey, 2 dicle university, diyarbakir, turkey magnetic nanoparticles (mnp) have gained a lot of attention in biomedical and industrial applications due to their biocompatibility, easy of surface modification and magnetic properties. magnetic nanoparticles can be utilized in versatile ways, very similar to those of nanoparticles in general. however, the magnetic properties of these particles add a new dimension where they can be manipulated upon application of an external magnetic field. this property opens up new applications where drugs that are attached to a magnetic particle to be targeted in the body using a magnetic field. often, targeting is achieved by attaching a molecule that recognizes another molecule that is specific to the desired target area. in recent years, the development of the systems in which drug is delivered magnetically to the target is drawing considerable attention since it is a current issue. it is possible to eliminate the most of the problems caused by high doses of chemotherapy by using the magnetic drug delivery systems. therefore, it is important to design delivery systems with high drug loading capacity. it is necessary to increase the number of reactive groups on the surface of nanoparticles in order to increase drug loading capacity. in this study, we synthesized a novel magnetic surface for drug delivery systems. magnetic dextran-nta (md-nta) was synthesized by using magnetic o-carboxymethyl dextran (ocmd) and nana-bis (carboxymethyl) -l-lysine hydrate (nta) in order to increase the number of reactive carboxyl groups on the surface of biocompatible and biodegradable magnetic dextran. magnetic material (md-nta) which was prepared and characterized by the analysis of transmission electron microscopy (tem), scanning electron microscope (sem), vibrating sample magnetometer (vsm), fourier transform infrared spectroscopy (ftir) and x-ray photoelectron spectroscopy (xps). there are three subtypes of the tgf-b protein that has been reported to be involved in tissue repair process; scar tissue formation has been reported on tissues that has been affected by tgf-b1 and 2 due to high collagen synthesis. on the other hand the other isoform tgf-b3, suppresses the dense collagen production caused by tgf-b1 and prevents the scar formation. to be able to use these growth factors local or iv route, new drug transport systems are needed to protect the bioactivity during the treatment and controlled release. for this purpose poly(lactic-co-glycolic) acid polymer which is widely used in controlled release systems was chosen as the matrix material. aim of the project was to design, formulate, prepare and optimize tgf-b3 loaded plga nanoparticular and/or plga polymeric film drug delivery systems and to test their effect on cell proliferation. tgf-b3 loaded nanoparticles was prepared with emulsion-solvent evaporation method; whereas polymeric film systems was prepared with film castingsolvent evaporation method. following the preparation tgf-b3 loaded drug delivery systems was characterized. quantification and in vitro release of the growth factor tgf-b3 was studied with elisa. hepg2 cell line was used on mtt cell proliferation assay for both tgf-b3 loaded nanoparticles and films on a time course study. nanoparticles and films were prepared and loading efficiency of the nanoparticles were found to be 42.42%. particle size, zeta index and polydispersity index for this formulation were determined as 204.9 ae 10.3 nm, 0.0381 mw and 0.380, respectively. thickness of the prepared films were 286 ae 20.16 nm. additionaly prepared nanoparticles and films were found non-toxic. tgf-b3 nanoparticles and films which were prepared in this study are planned to be used as an effective treatment strategy for wound healing after injury. this project was supported by grand 112s541 from the scientific and technological research council of turkey (tubitak). polyvinylpyrrolidone (pvp) is a biodegradable material and natural polymeric biomaterial in such studies. ganoderma lucidum is a natural material containing triterpenes, polysaccharides, adenosine, polypeptides, and amino acids. these constituents have been shown to exhibit anti-cancer properties, enhance and regulate immunity, resist oxidation and ageing, and promote metabolism and cell proliferation. composites of polyvinylpyrrolidone (pvp) have been prepared by solution intercalation method using ganoderma lucidum at different loading amounts. the characterization of pvp/ ganoderma lucidum composites was made by x-ray diffraction (xrd) and scanning electron microscopy (sem); the interactions between ganoderma lucidum and pvp was determined by ftir-atr; the thermal stability was determined by simultaneous tg/dta. hemocompatibility of the prepared composite samples were investigated by a 96-well plate spectrophotometer. in addition, contact angles and antimicrobial activity of biomaterials were also determined. ftir-atr confirms interactions formed between ganoderma lucidum and pvp. xrd and sem results give evidence that ganoderma lucidum was well dispersed and homogenously in the pvp matrix. thermogravimetric analysis indicated that introduction of clay to the polymer network resulted in an increase in thermal stability. the results of in vitro hemocompatibility test were showed that pvp/ ganoderma lucidum composites are used as biomaterial. the development of synthetic materials, textured polymers and metals and their increasing use in medicine make research of biomaterials' hemocompatibility very relevant. composite material is a multi-phase system consisted of matrix material and reinforcing material. matrix material is a continuous phase and reinforcing material is a dispersed phase. the main two bioactive components of ganoderma lucidum can be broadly grouped into triterpenes and polysaccharides. despite triterpenes and polysaccharides being widely known as the major active ingredients at anti-cancer effect. this study describes the synthesis and characterisation of biocomposites of different molecular weight of peg (polyethylene glycol) as matrix with ganoderma lucidum as a filling material at different loading (%1, %2.5, %5 wt). the composites have been prepared by solution intercalation method using ground and sieved ganoderma lucidum at 25 micron scale. the characterization of composites was made by x-ray diffraction (xrd), scanning electron microscopy (sem) and fourier transform infrared attenuated total reflectance (ftir-atr) also in this study the hemocompatibility and antibactarial properties of composite investigated. when xrd and ftir-atr results discussed, all of the composites using the different loading amunt of ganoderma lucidum (%1, %2.5 and %5 wt) were shown a homogen distribution in the matrix (peg). and an interaction have occured between matrix and filling material. the sem photos have confirmed these results. peg and composites have been detected as hemocompatible. these results showed that they can be used as biomaterials. p-07.01.3-016 evaluation of the genotoxic potential of some nanocomposites by comet assay b. yilmaz 1 , s. dogan 1 , s. celikler kasimogullari 2 1 department of molecular biology and genetics, balikesir university, balikesir, turkey, 2 department of biology, uludag university, bursa, turkey due to its similar nature to the bone, nanohydroxyapatite is a biocompatible particle and poly(methyl methacrylate) (pmma) is a polymer that has been used in dentistry and orthopedic applications for years. in this study, genotoxic potential of pmma/nanohydroxyapatite nanocomposite films composed of polymers having different molecular weights and nanohydroxyapatite fillers in different concentrations (1, 2.5 and 5%) were investigated by comet assay which is a kind of gel electrophoresis that can be used to measure dna damage in individual cells. if the dna is damaged we expect broken ends to migrate apart from the head. at the end of the assay performed after incubation with lymphocytes of healthy humans, we measured the dna damage index (ddi) and percentage of damaged cells (pdc). in addition, to prove the morphological properties of the nanocomposites scanning electron microscope was used and an interaction between the matrix and nanoparticles with a homogeneous dispersion was observed. protein adsorption on stimuli-responsive mixed pdmaema/peo polymer brushes a. bratek-skicki 1,2 , c. dupont-gillain 1 1 universit e catholique de louvain, louvain-la-neuve, belgium, 2 j. haber institute of catalysis and surface chemistry, polish academy of sciences, krakow, poland smart polymer brushes are made of macromolecules that are sensitive to stimuli from the external environment, including ph, ionic strength, temperature, etc. when stimuli-responsive polymer brushes are introduced onto material surfaces, their properties can be adjusted by tuning the environmental stimuli. these brushes can find promising applications across many areas of research, including surface science, nanotechnology, and biotechnology. in our work, the adsorption of human serum albumin (hsa, molecular weight of 66.5 kda, isoelectric point ip at ph 4.7) and lysozyme (lys, molecular weight of 14.3 kda, ip~11) was studied on polymer brushes composed of poly(ethylene oxide) (peo) and poly (2-(dimethylamine) ethyl methacrylate) (pdmaema). peo is a protein-repellent polymer and pdmaema is a polyelectrolyte bearing a variable density of positive charges depending on ph. a gold substrate was modified by these thiolated polymers according to the 'grafting to' method. the obtained polymer brushes were characterized by xray photoelectron spectroscopy, static contact angle measurements and atomic force microscopy. polymer brush formation and protein adsorption were monitored by quartz crystal microbalance. surface characterization of the mixed brushes revealed the presence of both polymers at the surface. conformational changes of pdmaema/peo brushes were experimentally evidenced, and the results indicated that the brushes collapse at ph 9.00 (pdmaema is neutral in such conditions) and were swollen at ph 3.5 (pdmaema is positively charged). protein adsorption was performed at different ph values (3.5-9 .0) and salt concentrations (0.001-0.15m). it was shown that pdmaema has a high affinity to hsa at ph above its isoelectric point. however, the adsorption of positively charged lysozyme in a wide range of ph was not observed. these results indicate that pdmaema/peo brushes are promising candidates for selective adsorption from a mixture of proteins. clay-polymer nanocomposites (cpn) developed in recent years as a new type of inorganic-organic hybrid materials that were conceived for medical uses such as tissue engineering or drug delivery [1] , [2] . the understanding of the structure and physico-chemical properties of cpn is a first step in the investigation of biomaterials, but their potential in this respect is determined by their interaction with living tissue components. in this study, pure kaolinite was intercalated with dimethyl sulfoxide (dmso) and then intercalated kaolinite was modified pyridine, 2-amino pyridine and 2,6-diamino pyridine to expand the interlayer basal spacing. modified kaolinite samples as filler and poly(vinyl chloride) (pvc) polymer as matrix were used in the nanocomposite synthesis. nanocomposites of pvc have been prepared by solvent blending method using thf as a solvent. the material characterizations were carried out by xrd, afm, ftir-atr, dta/tg and dsc. the xrd results reveal the formation of intercalation/exfoliation of modified kaolinite in the pvc matrix. ftir and afm results confirm the presence of nanomaterial in kaolinite/pvc nanocomposites. tga data show that the modified kaolinit/pvc nanocomposites have significant enhanced thermal stability. the glass transition temperature (tg) of pvc nanocomposites is higher than that of pure pvc. in addition, the antimicrobial activity of clay-polymer composites were also determined. introduction: polyhydroxyalkanoates (phas) are biocompatible and biodegradable materials obtained from microorganisms. they are produced in the cytoplasm of several bacteria as energy reserve. the physical properties of poly(3-hydroxybutyrate) (phb), which is from the group of phas, make it a competitive source to petrochemical plastics. phb has potential in order to be used in a variety of application fields such as packaging industry, printing materials, agriculture and food industry. furthermore, phb meets expectations for tissue engineering applications, since it is biocompatible, biodegradable, non-toxic and has good mechanical properties. although its many advantages, blending approach could be needed in order to fulfill all expectations of a material. due to its flexibility, polycaprolactone (pcl) is a promising candidate to be blended with phb. the aim of this study is to construct a scaffold by using phb produced by extreme alkaliphilic b. marmarensis gmbe 72 t (dsm 21297) and commercial pcl as components and investigate its properties. materials and methods: electrospinning method was used in order to construct scaffolds from blend polymer solution containing phb from b. marmarensis and commercial pcl. results: nanofiber structures were observed on scanning electron microscope (sem) images and fourier transform infrared resonance (ftir) analyses have shown characteristic peaks for both phb and pcl. discussion and conclusion: phb could be blended with other polymers in order to enrich its properties. in addition, nanofiber structure of electrospun phb-pcl blend makes it a rewarding material as scaffold for several tissue engineering applications. q fever is a zoonotic disease that is encountered widely around the world, the most common acute form of q fever shows the following symptoms; a sudden fever, shivering, lassitude, headache, anorexia. because this disease does not show specific symptoms its diagnosis is possible with laboratory tests. current diagnostic kits lack effectiveness; this is why the main goal of our studies is to come up with a new diagnostic kit that does not have disadvantages that current diagnostic kits show. with this goal, nine mile i strain (rsa 493), s serologic virulent phase i, were obtained from slovak science academy, virology institute for rickettsia reference and research from who co-operation centre. these cells were purified and lipopolysaccharide (lps) isolation from coxiella burnetii was performed. the polymeric carrier, poly (n-vinyl-2-pyrrolidone-co-acrylic acid) [p(vp-co-aa)] was synthesized and characterized. physical complexes of obtained lps and p(vp-co-aa) with varying ratios. ternary complexes of lps-cu 2+ -p(vp-co-aa) were also synthesized with copper metal mediation. structure and interaction of lipopolysaccharide-p(vp-co-aa) complexes were investigated with zeta-sizer device using zeta potential analysis and ftir spectrophotometry according to the ratios of components, reaction environment conditions and chemical structure of the polymer. the best complex ratio according to analysis results will be used in the future studies for obtaining monoclonal antibodies which will be an important step for obtaining more effective and stable diagnostic kits that can be used for q fever. this 2513 in this study; new types of water soluble polymer-biomolecule conjugates were synthesized using covalent bonding techniques between polymers and co-polymers (varying monomers of polyacrylic acid and acrylic acid) with peptides. different compositions of polymers, varying ratios of biomolecule/polymer and different molecular weight of polymer has yielded new types of bioconjugates. conjugation mechanism, composition and structure were investigated with various physicochemical methods (uv, ftir, hplc, gpc, etc.). the peptide used in this study was the antigenic peptide epitope of sheep-goat pox disease (eakssiakhfslwksyadadiknsenk). whether this peptide series was bound to polymers or whether it was bound to polymer-protein carrier; peptide-specific immunogens that were capable of producing antibodies were synthesised. it is thought that using polymer-peptide bioconjugates that contain just peptide will yield a higher peptide-specific immunogenicity compared to traditional adjuvants. in vitro and in vivo investigations of bioconjugates effectivity is planned to be done in the future studies. p-07.01.3-026 bioinert fluorinated ethylene-propylene copolymer modified for keratinocyte adhesion surface properties are crucial when adhesion of a cell to a polymeric material is required for a biomedical application. one of the methods for polymer surface tailoring is argon plasma treatment. this simple and reproducible method alters the surface properties such as roughness and wettability without affecting the bulk properties of the material. for the modification of the bioinert fluorinated ethylene-propylene (fep), related to teflonò, we employed argon plasma treatment with the powers of 3 and 8 w for 20-240 s. the human keratinocytes of the hacat cell line served as a model cell line for biocompatibility testing. we studied adhesion, proliferation and morphology of the cells on modified fep matrices as well as controls (pristine fep and standard polystyrene tissue culture dish) by means of fluorescence microscopy. further morphological details were acquired by scanning electron microscopy. furthermore, fluorescence microscopy with immunochemical labelling was used to determine the size and distribution of focal adhesions in cells grown on the modified matrices. the overall effect of the matrices on metabolic activity of cultured hacat cells was also evaluated using the wst-1 reagent. the ar plasma treatment of fep matrices improved significantly cell adhesion and proliferation and promoted spreading of the hacat cells compared to the pristine fep, on which cells were not able to spread properly. also, increased metabolic activity rates for cells cultured on modified matrices were found in comparison to the pristine fep. altogether, we found that ar plasma treatment improved the surface properties of fep to such extent, that it allows cultivation of adherent cells on its surface. we therefore propose that ar plasma treatment is a useful method for fep surface modification. p-07.01.3-027 graphene oxide enriched biomaterials display potential for tissue engineering applications tissue engineering (te) requires more efficient systems that favor local tissue regeneration with minimum cytotoxicity. materials based on natural compounds ensure biocompatibility and have better effects for regeneration. graphene oxide (go) has been shown to enhance cell adhesion and to improve the rate of cell differentiation. in this context, the aim of this study was to evaluate if the addition of different concentrations (0.25-1 wt.%) of graphene oxide (go) improves the properties of cellulose acetate (ca) materials for biocompatibility and cell differentiation, in the prospective of using these films for te applications. go-containing ca films were tested for cytocompatibility by quantitative and fluorescence microscopy assays, and compared to the ca control. cell cytoskeleton architecture in contact with biomaterials was revealed by confocal microscopy. furthermore, bioconstructs were exposed to in vitro osteogenic and adipogenic induction and monitored for 21 days. histological stainings were performed to validate differentiation. osteogenic and adipogenic markers gene expressions were assessed via qpcr. ca/go 1 wt.% revealed best biocompatibility among all tested scaffolds. adhesion proved to be dependent on the percentage of go in material's composition. cells cultivated on ca/ go 1 wt.% expressed adipogenic and osteogenic markers earlier than cells cultivated on materials with lower go content. differentiation markers displayed an increasing profile of gene expression from 7 to 21 days post induction, with higher levels registered for materials with high go content as compared to films with low go content and to the control. go added to ca materials positively influenced cell survival, proliferation and cell differentiation. ca/go films represent potential candidates for te applications. the design of appropriate scaffolds remains one of the most important challenges for te. current idea is that the cell-scaffold interaction could drive cell differentiation and be linked to gene expression and protein organization. therefore, their quality is essential and should favour cell attachment, growth, migration, in situ vascularization and release of biochemical and physical factors able to address the cell fate. moreover, for an ideal scaffolding material an adequate and interconnected porosity is relevant for facilitating cell spreading and colonization of the inner layers. a combination of optimal mechanical and biochemical properties were here utilized to design a 3d composite hydrogel scaffold (3d-chs) in order to favour cell-scaffold interactions and promote a functional differentiation of human lin à sca1 + cardiac progenitor cells (hcpc). the biocompatible peg-diacrylate (pegda) was used to prepare hybrid protein-pegda hydrogels with embedded albumin-microspheres (ms) as protein component. ms were able to modulate the mechanical and biological behavior of the scaffold acting as air-reservoir, porogen agents and potential carriers of biomolecules. an increase of the hcpc viability in the ms-concentration dependent manner was observed. moreover, the microarchitecture of the 3d scaffold also plays a key role in the stability and functionality of cellularcomposite systems. therefore, pegda-honeycomb structures were fabricated using microstereolithography process and the hcpc viability and adhesion to the microstructures were assessed. 3d-chss were synthesized embedding honeycombstructures into ms-pegda hydrogel and the effects on cell proliferation, cell-cell interactions and cellular alignment were investigated. these results could be of relevant interest for expanding the knowledge on cell-scaffold interaction processes and to promote the development and the application of 3d-chs for tissue regeneration using the emerging bioprinting technologies p-07.01.3-029 gene expressions of mesenchymal stem cells after osteogenic induction on ceraform bone substitute a. kilic s€ uloglu, e. karacaoglu, h. akel, c ß . karaaslan hacettepe university, ankara, turkey ceraformò, is a synthetic calcium phosphate ceramic with the chemical composition of hydroxyapatite 65% and tricalcium phosphate 35%, with 60-85% pore volume and 100-400 lm pore diameter. in this study adipose tissue derived mesenchymal stem cells were differentiated into osteoblast cells and loaded on cer-aformò. in order to improve cell adherence, ceraformò was covered with fibronectin. the cells were cultivated for 28-day period by osteogenic induction medium. days 1, 7, 14, 21 and 28 were selected as specific intervals for incubations. total rna was isolated and cdna was synthesized. differences in the expression of runt-related transcription factor 2 (runx2), bone morphogenetic protein-2 (bmp-2), and osteocalcin (ocn) were determined by qpcr. the peptidylprolyl isomerase a (ppia) gene was used as an internal control. according to the qpcr results, ocn gene expression was observed on the day 14th, continued to increase in day 28. bmp-2 gene expression was increased in 14, 21, 28 day compared to 7 day. on the other hand, runx2 gene expression was increased only on days 21 and 28. these findings pointed out that the osteogenic induction was successfully activated on fn coated bone material. therefore, these results can be used in bone injury treatment and related disorders. p-07.01.3-030 on the in vitro cytotoxicity of graphene oxide nanomaterials v. grumezescu 1 , i. negut 1 , f. sima 1 , e. axente 1 , l. e. sima 2 1 national institute for lasers, plasma and radiation physics, magurele, romania, 2 institute of biochemistry, romanian academy, bucharest, romania during the last decade, graphene and its derivates have proven unique physicochemical properties, several applications being continuously developed. among them, electronic, catalytic, mechanical, optical, and magnetic properties have attracted huge interests. however, the merging of graphene and graphene oxide (go) with biotechnology is still in its infancy, many challenges remaining unexplored. potential applications are related to biosensors, drug delivery or gene therapy and cells imaging. in order to use gos as drug release matrices for cancer cells targeting, it is necessary to ensure that these molecules do not affect normal cells within tissues. it was shown that the cytotoxicity of graphene nanomaterials is highly dependent on surface functionalization. studies suggest that pristine and reduced go with fewer surface functional groups tend to be more toxic than go. in striking contrast, it has been reported that functionalized graphenes, can significantly reduce the cytotoxicity even at relatively high concentrations. in this study, we report on the comparison between go and protein functionalized go when submitted to in vitro cytotoxicity tests. bovine serum albumin (bsa) was used for the noncovalent go surface conjugation. three case-studies were investigated: aqueous nano-colloids consisting of serial dilutions of both go and go-bsa conjugates, dropcasted thin films and laser-assembled thin films on glass substrates. safe concentration windows were identified by live/dead staining and mts assays for different human melanoma cell lines, while melanocytes and human dermal fibroblasts were used as normality controls. the predominant melanoma subtype is represented by cells bearing braf (v600e) activating mutation. with a view to target this specific melanoma subpopulation, we embedded braf inhibitors into go laser-deposited scaffolds and tested their anti-tumoral effect. our results evidence the high potential of these nanomaterials for biomedical applications. osteoporosis is a skeletal disorder associated with low bone mass and increase in bone fragility due to increased osteoclastic activity. binding of rank ligand to its receptor on osteoclast precursor cells results in the osteoclast differentiation. sirna is a dsrna, used to inhibit the translation of the target gene. the aim of the study is to develop an injectable sirna-delivery system targeted to the bone for osteoporosis treatment. pei (polyethyleneimine) and rank complex was loaded into poly(lactic acid-co-glycolic acid) (plga) nanocapsules which are bound to hydroxyapatite (hap)-specific elastin-like protein (elp) for targeting to bone tissue. plga nanocapsules were prepared by w/o/w double emulsion technique. affinity of elp to hap was determined by ftir. elp was coated on the nanocapsules by using the transition temperature of elp. elp on plga nanocapsules were crosslinked by genipin and binding of elp on plga nanocapsules were studied by xps and tem. the optimum ratio of n (pei) to p (sirna) in the complexes to be loaded into plga nanocapsules were studied by etbr staining and zeta potential measurements with varying n/p ratios and finally pei-dnaoligo encapsulation efficiency of the capsules was determined by picogreen reagent. xps results of elp treated plga (elp-plga) nanoparticles indicated the presence of nitrogen atom (11.7%) in the sample which appeared as a fuzzy halo in tem micrographs. n/p ratios up to 5 show negatively charged particles. when n/p was 5, the zeta potential of complex was neutralized which also resulted in larger particles compared to the others. zeta potential moved to positive values when n/p was higher than 5. the migration of polyplexes with different n/p ratios (1-10) was analyzed by gel electrophoresis. dnaoligo complexes show the same patterns of complexation wih that of sirna and thus were used in the encapsulation efficiency studies instead of sirna. the encapsulation efficiency of pei-dnaoligo in plga nanocapsules was 29%. tuesday 6 september 12:30-14:30 aging p-09.03.3-001 novel benzenesulfonamides exhibit low toxicity on zebrafish embryonic development and selectively inhibit carbonic anhydrase ix with nanomolar affinity in xenopus oocytes introduction: the toxic effects of two recently discovered inhibitors (vd12-09 and vd11-4-2) that selectively and with extraordinary strong, picomolar affinity bind to human carbonic anhydrase (ca) ix, an anticancer target, were investigated on zebrafish embryonic development and in xenopus laevis oocytes. zebrafish has emerged as a promising animal model to evaluate the toxicity of the drug candidates. xenopus oocytes do not natively possess any ca activity and thus became a convenient in vivo model system to study the ph effects and the selectivity of synthetic ca inhibitors. materials and methods: morphological changes in zebrafish treated with the compounds were studied by light-field microscopy and histological analysis. ca activity in xenopus oocytes was monitored by measuring ph in the cytosol and at the outer membrane surface and confirmed by mass spectrometry of lysed oocytes. the toxicity studies showed lc50 values to be 13 lm for vd12-09, 120 lm for vd11-4-2 and 9 lm for ethoxzolamide (eza), a non-selective ca inhibitor commonly used in clinic. the zebrafish exposed to lc50 doses of vd12-09 and vd11-4-2 showed fewer phenotypic abnormalities and less morphological changes compared to the zebrafish treated with the corresponding dose of eza. vd11-4-2 exhibited 10-25 nm ic50 for both intracellularly and extracellularly expressed ca ix in xenopus oocytes while exhibiting strong selectivity over ca ii, ca iv and ca xii. discussion: interestingly, the compounds exhibited 10-fold lower toxicity, induced fewer side effects in zebrafish than eza and the amount of vd11-4-2 needed to cause complete inhibition of ca ix enzymatic activity in xenopus oocytes was 30-fold lower than eza. conclusions: vd compounds did not lead to deleterious effects on the zebrafish embryonic development and reached the ic50 of 10 nm for ca ix in xenopus oocytes. the compounds could be further developed as anticancer drugs. cacybp/sip is present in various cells and tissues, both normal and pathological. in normal tissues, e.g. stomach or colon, cacybp/sip is weakly or barely detected whereas in gastric or colon cancer this protein is expressed at a high level. there are also data indicating that the level of cacybp/sip expression correlates with tumor metastatic potency and multidrug resistance. taking into consideration data that suggest association of cacybp/sip with many vital cellular processes, in this work we decided to investigate the possible mechanism involved in regulation of cacybp/sip gene expression, mainly by transcription factors and, on the other hand, the influence of cacybp/sip on the expression of other genes. we have shown that nfat (nuclear factor of activated t cells) influences the cacybp/sip gene expression and that overexpression of cacybp/sip has an effect on the level of ap-1 and on the activity of nfat and ap-1 transcription factors. by analyzing the cacybp/sip gene promoter sequence we also found potential binding sites for transcription factors from the stat family, which are involved in interferon signaling. microarray data indicate that indeed overexpression of cacybp/sip affects levels of the stat proteins as well as of some interferons and interleukins. based on functional analysis we have found many genes the products of which are involved in immune response. to analyze in more detail the influence of an altered level of cacybp/sip on interferon signaling pathways as well as on factors involved in expression of interleukins, including nfkappab, we plan to apply methods such as luciferase assay, real-time pcr or immunocytochemistry. one of the pathological hallmarks of alzheimer's disease (ad) is the neuritic plaques occurred as a result of the extracellular accumulation of aß peptides formed from amyloid precursor protein (app) via the ß-amyloidogenic pathway. aß42 is more prone to aggregation to form plaques and more toxic to neurons than aß40. in addition to change in app metabolism, the decline in levels of neurotransmitter acetylcholine and cholinergic dysfunction are also observed in ad. thus, current strategies for ad treatment focus on compounds with inhibitory effect on cholinesterases as well as preventive effect on aß aggregation. in our earlier studies, toluidine blue o (tbo), a phenothiazine dye, was shown to be a highly effective inhibitor of cholinesterases with k i values in nm range. we also found that intracellular app and aß42 levels are reduced in human neuroblastoma cells after treatment with tbo. additionally, an earlier study revealed that tbo has a selective inhibitory effect on tau aggregation, the other pathological characteristic of ad. the aim of this study was to investigate whether tbo may effectively lower the level of extracellular aß40/42 in an ad-like cellular model. chinese hamster ovary cells that express human wild type app and presenilin 1, namely ps70, were treated with a dose range of tbo (0-15 lm) or vehicle control for 24 h. after treatment, aß40/42 levels in cell culture media were assayed by separate sandwich-based elisas and normalized to total protein levels, determined by bca protein assay. besides, biocompatibility of tbo was evaluated in the ps70 cells using cell viability assay for flow cytometry. strikingly, all dose ranges of tbo inhibited both aß40 and aß42 secreted into the cell culture media. significant reduction for both aß species was evident at 5 lm (p < 0.05), 10 lm (p < 0.001), and 15 lm (p < 0.001) of tbo vs. vehicle control. in conclusion, these results support the idea that tbo may be used as a therapeutic in ad. monitoring the changes of key molecules participating in the osmo-regulatory response of nucleus pulposus intervertebral disc cells during stress-induced senescence e. mavrogonatou, d. kletsas ncsr 'demokritos', athens, greece introduction: intervertebral disc cells are faced with a harsh extracellular milieu characterized by hyperosmotic conditions, nutrient and oxygen deficiency because of the absence of vascularization and oxidative stress due to the accumulation of their metabolism's by-products. we have previously shown that high osmolality is anti-proliferative for disc cells through the activation of the g1 and g2 cell cycle checkpoints by p53 and p38 mapk, respectively. in addition, we have shown the participation of nine solute transporters, with the a1 subunit of na + /k + -atpase being central in this response. here we assessed the changes in the expression of these key osmo-regulatory molecules during in vitro stress-induced senescence. materials and methods: changes in cell cycle progression were assessed using flow cytometry; overall transcriptional alterations were assessed by whole-genome arrays; differences in expression at the mrna and protein level were revealed by quantitative rt-pcr and western blotting, respectively; knocking-down of selected proteins was performed by sirna. results: high osmolality led to the differential expression of > 200 genes, including nine genes encoding transporters. p38 mapk and p53 were demonstrated to differently participate in the regulation of the aforementioned transporters, while knocking-down of three selected transporters had a distinct outcome on the overall cellular response towards hyperosmotic stress. these molecules were found to show differences in their expression in senescent cells. discussion: given that the presence of senescent cells has been demonstrated in the intervertebral disc in vivo and could most probably attributed to the prevailing stressful conditions, here we showed differences in the expression profile of known key molecules for osmo-adaptation during senescence. conclusion: understanding disc cells' physiology is of outmost importance when designing cell-based therapies for disc degenerative disorders. p-09.03.3-005 smad specific e3 ubiquitin protein ligase 2 (smurf2) and its potential effects on inhibitory transmission in aging adams 1, 2, 4, 5 1 interdisciplinary program in neuroscience, bilkent university, ankara, turkey, 2 national nanotechnology research center (unam), bilkent university, ankara, turkey, 3 department of molecular biology and genetics, bilkent university, ankara, turkey, 4 molecular biology and genetics zebrafish facility, bilkent university, ankara, turkey, 5 department of psychology, bilkent university, ankara, turkey smad specific e3 ubiquitin protein ligase 2 (smurf2) is part of the tgf-b signaling pathway associated with cellular proliferation, differentiation, genomic stability and senescence. moreover, smurf2, via its downstream partners, may regulate inhibitory synaptic transmission. our research group previously found that the smurf2 transcript is significantly higher in old zebrafish brains. thus, smurf2 may alter inhibitory synaptic transmission in aged animals. the focus of this study was to examine age-related changes in smurf2 protein levels and related key inhibitory synaptic proteins; gephyrin (gep), a scaffolding protein for gaba receptors, and gaba a , an ionotropic gaba receptor subtype. additionally, the levels of those proteins were studied in a mutant zebrafish line, which lacks acetylcholinesterase (ache) and is suggested to be a delayed aging model. whole brain tissues were isolated from young, middle-aged and old male and female zebrafish brains (ab/wildtype strain), as well as from old male and female ache mutant zebrafish (ache sb55/+ ). animals were maintained and raised in standard conditions. the extracted brain tissue was homogenized in ripa buffer and subjected to western blot analysis to determine differences in the relative protein expression levels. our preliminary data indicated that smurf2 and gep levels remain stable in the aging brain (p = 0.301, p = 0.335), and in the ache mutants gep levels are increased compared to the wildtype controls (p = 0.001). further analysis of the relationships between smurf2 and gaba a levels and brain aging is ongoing. we predicted that alterations in smurf2 levels would parallel changes in key synaptic inhibitory proteins during the aging process, which was the case for the gep levels. while smurf2 may regulate inhibitory synaptic transmission, the exact roles of those synaptic proteins in the context of normal and delayed brain aging are not known well-understood and the subject of continuing study. in recent years, express the hypothesis that aged individuals are vulnerable to infectious and other inflammatory agents and they become more prone to develop majority of severe age pathologies, including cardiovascular and oncology diseases, neurodegenerative diseases, type 2 diabetes mellitus and inflammatory diseases, etc. one of the central components of immune response is the family of toll like receptors (tlr). there are several opinions that single nucleotide polymorphisms (snp) leading to a loss of function of the respective tlrs can be associated with age and increase the risk of age related diseases, especially cardiovascular diseases (cvd). however, many available studies focusing on tlr snps and cvd are with conflicting results. the aim of this study was to assess the potential interaction between genetic variants of tlr2 and tlr4 and ischemic heart disease (ihd) in kazakhstan population over 45 years old. we evaluated 148 patients with ihd and 144 healthy subjects aged 45 years and over (ethnical kazakhs and russians living in republic of kazakhstan). polymorphic loci of the genes tlr2 rs5743708 and tlr4 rs4986790 were genotyped by pcr with subsequent restriction analysis. our results indicated that the genotype and allele frequencies of tlr2 (arg753gln) and tlr4 (asp299gly) were not significantly different between the 2 groups (p ≥ 0.05). statistical analysis didn't elicit any association between studied gene polymorphisms and predisposition to ihd in individuals over 45 years old (p ≥ 0.05). for these polymorphisms, age, fasting blood sugar and serum lipid levels were not also significantly different among different genotypes in the ihd and control groups. in conclusion, the data shows that there is no interaction between tlr2 and tlr4 and ischemic heart disease (ihd) in kazakhstan population over 45 years old. we plan to include other types of polymorphisms in tlr 2 and tlr 4 genes and increase the volume of patient cohort in our future studies. p-09.03.3-007 evaluation of prognosis with total oxidant/ antioxidant status and some oxidative stress parameters in patients with acute ischemic stroke stroke is the third most common cause of death after coronary heart disease and cancer. strokes are classified into two groups according to their pathology: ischemic stroke and hemorrhagic stroke. ischemic strokes make up 87% and hemorrhagic strokes 13% of all strokes. during ischemic stroke, oxidative stress has been shown to play a major role in the occurrence and progression, formed oxidants also affect cell membranes and genetic material such asdna, rna, and various enzymatic events, and they lead to cell damage. some studies have shown oxidant-antioxidant status but have not shown the relationship with prognosis. this study has investigated the relationship between prognosis and total oxidant/antioxidant status and biochemical parameters in patients with acute ischemic stroke 58 patients, with acute ischemic stroke and 37 healthy controls we reenrolled in the study. blood samples were taken within 1st and 7th days, and after 3rd months in the patient group for analysing serum total oxidant status (tos), antioxidant status (tas), catalase, arylesterase, and thiol. prognosis was evaluated with national institutes of health stroke scale (nihss) and-modifiedrankinscale (mrs) scores. there was no significantly difference between groups by means of serum tas, tos and catalase levels. but arylesterase (p: 0.07) and thiol (p: 0.031) levels were significantly higher in first 24 h blood samplingthancontrolgroup. statistically significant negative correlation was observed between the 3rd month values of tos and nihss score (r = 0.410, p = 0.037). but there was no correlation between mrs scores and serum tas, tos, catalase, thiol and arylesterase. similarly, our findings suggested some serum oxidant levels were increased in acute ischemic stroke patients and total oxidant status might be used in evaluation of prognosis but larger studies are needed. p-09.03.3-008 amylin and preptin regulate glucose homeostasis in infertile women with polycystic ovary syndrome and poor responders undergoing ivf/icsi disrupted glucose homeostasis leads not only metabolic disturbance such as polycystic ovary syndrome (pcos), but also influences oocyte growing. this study was designed to evaluate follicular fluid (ff) and serum levels of glucoregulatory hormones, amylin and preptin, in infertile women with pcos and poor responders undergoing ivf/icsi. human follicular and serum were obtained from 20 infertile women with pcos and 20 poor responder participants undergoing controlled ovarian stimulation (cos) with gonadotropin-releasing hormone antagonist protocol for ivf/icsi treatment. ff and serum amylin and preptin levels were measured by elisa. it was found that ff and serum amylin and preptin were lower in infertile women with pcos when compared with poor responder participants. ff amylin and preptin concentrations were lower than that of the serum amylin and preptin concentrations. decreased follicular fluid amylin and preptin levels suggest that amylin and preptin may have a physiological role in follicular maturation via controlling local glucose homeostasis. despite high serum levels of amylin and preptin in pcos their low concentration within the follicle may be main culprit of defective folliculogenesis seen in pcos subjects. similar to insulin resistance in pcos subjects existence of amylin and preptin resistance support the critical role of both peptides in follicular maturation in pcos. keywords: follicular fluid; amylin; preptin; polycystic ovary syndrome; infertility. the transcription initiation on p3 promoter of xp10 bacteriophage in presence of p7 protein, a modulator of rna-polymerase activity a. shadrin g.k. skryabin institute of biochemistry and physiology of microorganisms, ras pushchino, russia many bacteriophages are able to manage the transcription system of their bacterial host for their own needs. for example, bacteriophage xp10, in the early stages of infection of xanthomonas oryzae inhibits transcriptional activity of bacterial rna-polymerase on majority of promoters via p7 protein, except of bacteriophage p3 promoter responsible for expression of the bacteriophage 'middle' class genes. the focus of this work is to study the mechanism of action of p7 protein in the transcription initiation and identification of the role of the individual elements of p3 promoter of xp10 bacteriophage, enabling x. oryzae rna-polymerase escapes inhibition by p7 protein. we have designed a set of promoter probes representing the combination of sequences of p7-resistant p3 promoter and p7sensitive t5n25 promoter. using fret-based assay it was shown that the truncated probes corresponding to promoter dna downstream à26 position, relative to the transcription initiation start site, did not lead to dissociation of the sigma-factor. longer probes, containing à35 promoter element, induce dissociation sigma-factor. the in vitro transcription experiments show that the deletion of region 4, a sigma-factor domain responsible for interaction with à35 promoter element during the transcription initiation, is not critical for inhibition of rna-polymerase by p7 protein. promoter probe with up-element of p3 promoter had affinity to x. oryzae rna-polymerase a several times higher than a probe containing the consensus up-element for e. coli rna-polymerase . summing up the results, it seems like the transcription initiation on p3 promoter of bacteriophage xp10 can escape inhibition by p7 protein through a high affinity interaction between the up-element and c-terminal domains of the alpha subunit of rna-polymerase x. oryzae. p-09.03.3-010 distribution of soluble form of glial fibrillar acidic protein in the different areas of gerbils brain during development and aging y. kovalchuk, g. ushakova oles honchar dniepropetrovsk national university, dniepropetrovsk, ukraine astrocytes are the most abundant cell type within the cns and play an important role in cns homeostasis and function. glial fibrillary acidic protein (gfap) forms the main astrocytic intermediate filament (if). the overall level gfap in different parts of the brain uneven and depends on the number of astrocyte cells. gfap is very sensitive to any kind of neurodegenerative diseases and aging. during aging, a glial reaction is observed in the human brain, as well as in rat and mouse brains. the aim of our study was to investigate the quantitative astrocytes-specific protein gfap in different areas of the gerbils brain at the first stages of postnatal development and aging. for the study 30 gerbils brains were used and divided into 5 groups (n = 6): 1: newborn animals (1 day), 2-4: 30, 90 and 180 days respectively, 5: animals aged 2 years. the animals were decapitated under mild anesthesia (thiopental), with isolated brain three divisions: the cerebellum, thalamus and hippocampus, which are then used to produce cytosolic protein fractions. the level of gfap in the obtained fractions were determined according to the method of competitive elisa. newborn gerbils found no significant content of soluble form of glial fibrillar acidic protein in all investigated parts of the brain, and a sharp increase of amount within 30 days (in cerebellumamounted to 1.12 ae 0.03 lg/100 mg tissue; to 90-180 days increased to 1.67 ae 0.11 lg/100 mg tissue, and began to grow again in older individuals aged 2 years). unlike the cerebellum, the level of sgfap in hippocampus and thalamus reached the maximum at 30 days p.d. (1.5-1.7 lg/ 100 mg tissue), and unchanged for 180 days. these results revealed that the most intensive development of astrocytes in the cerebellum to 90 p.d. of gerbils, and in the thalamus and hippocampus are formed within the first month of life. the plastid-nucleus located protein whiry1 acts as an upstream regulator of leaf senescence binding to the promoter of senescence associated genes (sags) like senescence marker gene hvs40. in order to investigate the impact of whirly1 on drought stress-induced senescence, transgenic barley plants with a knockdown of whirly1 (hvwhy1kd) were grown under untreated and drought stress conditions. the leaf senescence evolution was monitored by physiological parameters and gene expression studies of senescence and drought stress related genes. to reveal the epigenetic indexing at hvs40 at onset of drought-induced senescence in wild type (wt) and hvwhy1kd lines, stress-responsive loading with histone modifications at 6 gene regions of hvs40 (2 regions in the promoter, one region around translation start site and 3 regions located in the gene body) was analysed by chip and quantified by rtq-pcr. in barley, drought treatment caused acceleration of leaf senescence in wildtype (wt) plants, whereas why1kd lines showed a staygreen phenotype. expression of senescence-associated and drought stress responsive genes expression was delayed in hvwhy1kd indicating that whirly1 protein acts as an upstream regulator of drought stress-induced senescence. the chip results showed that drought treatment is causing in wt a significant increase in the levels of h3k9ac all over the analyzed gene regions, correlating with a massive induction of hvs40 expression, while drought stress caused no substantial increase of h3k9ac in why1kd plants. the results suggest that drought induced expression of hvs40 is under epigenetic control, and furthermore that why1 is involved in this epigenetic control level. oncolytic viral therapy is based on the capabilities of selective lysis of tumor cells and is a prospective trend in cancer disease treatment. in vitro experiments showed that plant rhabdoviruses does not have any direct cytotoxic effect upon sarcoma 37 cells, causes induction of apoptosis in these cells and does not pose any threat to somatic cells of warm-blooded animals, which makes it possible to use this virus for therapy of malignant neoplasms. buckwheat burn virus (bbv), the prototypic member of the family rhabdoviridae, contains surface glycoprotein and which is lectin-active. its carbohydrate branch can aid adhesion of lymphocytes to tumor cells. the present study has addressed the effect of bbv on cancer cell viability. all studies were carried out after 1 week of inoculated with erlich cancernome (2 9 10 6 cells/animal, i. p.) in 2 months male balb/c mice treated at once with or without plant extract with bbv (15 mg/kg, i. p.). by fluorescent microscopy and using two due staining by acredine orange and propidium iodide it was found that in the 3rd day of administration of bbv lead to increasing of necrotic and apoptotic cells on 45% and 4% respectively versus to untreated group. at the same time the viability of investigated cells was impaired too and according to flow cytometry analysis using propidium iodide the amount of dead cells was elevated by fivefold (17.7% versus 3.5% in untreated group). also as was shown in previously reports bbv decreased activity of macrophages in the early stages after injection and it may have a positive effect when using this drug in tumor therapy. when using this drug appears to slow down the possibility of a sharp activation of macrophages, and as a consequence of the development of cytotoxic effect will be prolonged. key words: rhabdoviruses, buckwheat burn virus, cancer, cell viability. plants are considered as one of most promising sources for new antimicrobials, based on the evidence of their use in folk medicine to treat various infectious diseases since ancient times. despite relatively small area size, armenia has large diversity of flora with many endemic species. the main goal of this study was the screening of various parts of 28 herbs (widely being used in armenian folk medicine) for their antimicrobial activities in order to select most prospective plants for further comprehensive studies. plant crude extracts were obtained with maceration technique using five solvents: water, methanol, chloroform, acetone and hexane. agar well diffusion assay was used to evaluate antimicrobial properties of plant crude extracts at 500 lg/ml concentration against escherichia coli vkpm-m17, pseudomonas aeruginosa grp3, bacillus subtilis wt-a1, salmonella typhimurium mdc 1754 and staphylococcus aureus mdc 5233, candida albicans wt-174 and candida guilliermondii hp-17. statistical analysis was done using graphpad prism 5.03. crude extracts of all tested plant materials expressed antimicrobial activity against at least one test strain. most of the tested extracts inhibited growth of both gram-negative and gram-positive bacteria. in contrast, only some plant materials exhibited inhibitory activity against yeast strains. according to obtained data sanguisorba officinalis, rumex confertus, hypericum alpestre, lilium armenum and agrimonia eupatoria possessed the highest and broadest antimicrobial activity. moreover, the results showed that acetone was the most effective solvent for solubilizing antimicrobial compounds from plant materials followed by methanol, chloroform, hexane and water. the results demonstrated high antimicrobial activity of medicinal plants used in armenian traditional medicine. five plant species were selected for further comprehensive studies. besides, acetone was proposed as efficient solvent in antimicrobial screening protocols. p-02.08.5-004 effects of aluminum stress on photosystem-i apoprotein a2 gene (psab) transcription level in lichen xanthoria parietina (l.) th. fr. unal € ozakc ßa ege university, izmir, turkey in this study the effects of shortrerm aluminium (al) toxicity on the lichen xanthoria parietina (l.) th.fr. were investigated at physiological and transcriptional level. lichen thalli were treated with alcl 2 in different doses (0.25, 0.5, 1 and 5 mm). lipid peroxidation and chlorophyll integrity were determined by spectrophotometer. expression level of psab gene was also investigated. chlorophyll a content was significantly (p ˂ 0.05) decreased after 48 hours treatment with 1 mm and 5 mm of al, while chlorophyll b content was increased significantly due to treatment with increased concentration of aluminum. also treatment with 0.25 and 0.5 mm al for 24 hours increased the gene expression level of psab by 35.6% and 21.3% respectively. our results indicated that aluminum treatment has decreased the chlorophyll biosynthesis and increased the lipid peroxidation depending on time and concentration. this study also demonstrates that the psi can be readily photo-inhibited by aluminum stress. in conclusion, 5 mm al exposure for 48 hours could damage the electron transport in photosystem i. p-02.08.5-005 nigella sativa reduces paracetamol-induced nephrotoxicity and oxidative stress in rats: biochemical evaluation background: nigella sativa l. (ranunculaceae) (ns) is traditionally used to treat many conditions such as inflammation. this study evaluates the effects of ns seeds ethanol extract in paracetamol-induced acute nephrotoxicity in rats. material method: forty-eight female wistar albino rats were divided into eight groups: i = sham; ii = sham + 1000 mg/kg ns; iii = sham + 140 mg/kg (n-acetyl cysteine) nac; iv = 2 g/ kg paracetamol; v = 2 g/kg paracetamol + 140 mg/kg nac; vi, vii and viii = 2 g/kg paracetamol + 250, 500 and 1000 mg/kg ns, respectively. paracetamol administration (oral) was carried out 1 h after ns and nac administrations (oral), and all animals were sacrificed 24 h later. result: urea and creatinine levels were determined in serum, while glutathione, malondialdehyde levels and superoxide dismutase activity were determined in the kidney tissues. there were significant increases in the serum levels of urea and creatinine in the paracetamol-administered group. serum levels of urea and creatinine were decreased in all groups administered ns with paracetamol. ns administration dramatically restored sod, gsh, and mda levels in the kidneys. conclusion: the results suggest ns has a significant nephroprotective activity on paracetamol-induced nephrotoxicity. it may be suggested that the antiinflammatory and antioxidant effects of ns ethanolic extract originated from different compounds of its black seeds. p-02.08.5-006 the study of problems of preservation of the birches e. shadenova 1,2 , e. zhumabekov 2 , m. sembekov 2 , m. burchaeva 2 1 institute of general genetic and cytology, almaty, 2 laboratory of genetics and reproduction of forest culture, institute of general genetics and cytology, almaty, kazakhstan nature of deciduous trees have a whole range of various medicinal properties. instead of synthetic hormone substitutes, you can use medicinal infusions and decoctions of natural phytohormones are widely used in both folk and professional medicine. one of these plants is birch, its young leaves and buds. however, they also must be used with caution because overdose of these compounds is very dangerous, not only can you not get the desired effect, but also face the opposite of his action. in our research to mass replication of plants (different types of birches (betula ajanensis, yarmolenko, jacguemontii, maximowiczii, ulmifolia, middendorffii, kelleriana, tianschanica)) we use nutritional medium excluding the application of phyto promoters in order to prevent mutation. the object of research serve as the old, the sick, being on the verge of extinction, mature trees as explant meristema. since from the moment of calling experience and most cultivation occurs at nutritional medium without hormones. as a result of molecular analysis we get without virus, genetically identical plants. molecular certification of different types of birches of interest, both in terms of organizing, and in terms of selection and genetic improvement of valuable forms, identification of lines selected from natural populations and clones obtained in vitro. relationship between clones and installed parent form by comparing profiles amplific pcr products using issr-marking. according to the results of carried out works really recovered clones obtained from one source tree, indicating the potential for certification of clones studied forms of birches pcr. a study performed in the framework of the state grant project "conservation of breeding valuable species of birches". p-02.08.5-007 fractionated triterpenoid glycosides from sea cucumber inhibit invasion and metastasis in human cancer cells sea cucumbers are slow-motioned invertebrates. holothuria polii delle chiaje, 1824 is widely distributed sea cucumber in _ izmir coastline (turkey). it secretes saponins i.e. triterpenoid glycosides (ttg) as secondary metabolites. the aim of this study is to evaluate anti-invasive and anti-migrative effects of fractionated ttgs obtained from h. polii on ht-29, t84 and upci-scc-131 cancer cell lines. the semi-purified ttgs was extracted from h. polii collected from coast of _ izmir-dikili. the four different fractions (fraction a-d) were collected by using hplc (high-performance liquid chromatography) and characterizated with maldi-ms/ ms. the fractions obtained from h. polii extract include holothurin a (1243.50 m/z) and 24-dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer (1227.50 m/z). anti-invasive and anti-migrative effects of the fractions on the cancer cell lines were detected with xcelligence rtca dp system. the results showed that fraction a-d inhibited migration and invasion of human cancer cell lines at 6th and 12th hours compared to control group. this study shows that holothurin a, 24dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer could be evaluated as promising anti-cancer agents for human cancers. acknowledgement: the authors acknowledged the scientific and technological research council of turkey (t € ub _ itak) for financial support (114z211). p-02.08.5-008 alternative splicing regulation of sr proteins in response to environmental stress in chinese cabbage serine/arginine-rich protein (sr protein) family, which acts as rna-binding protein, plays a major role in post-transcriptional regulation of pre-mrna, such as alternative splicing (as). these proteins cause pleiotropic effect by regulating as of pre-mrna in a tissue and developmental stage-specific and stress-responsive manner in arabidopsis. here, we identified 31 genes encoding sr proteins in chinese cabbage (brassica rapa chiifu-401) from brassica database and analyzed their phylogenetic relationship. b. rapa has 31 types of sr protein that are classified into common (sr, rsz and sc) and plant specific (scl, rs2z, rs and sr-like) subfamily similar with arabidopsis. interestingly, the as pattern of most sr genes changed at the late stage (14 and 21 days after germination). to verify the correlation between sr genes and environmental stress, we screened the as pattern of sr genes to various abiotic stress using rt-pcr and a microarray analysis. in particular, the expression level and the as pattern of bra015576 and bra018581 were affected significantly by heat stress. these results suggest that the as regulation by sr protein correlates with adaptation mechanism to the environmental stress in chinese cabbage. p-02.08.5-010 characterization of recombinant prolyl oligopeptidase from myxococcus xanthus and potential use in gluten hydrolysis e. k. kocaazorbaz, f. zihnioglu faculty of science, biochemistry department, ege university, izmir, turkey a recombinant prolyl oligopeptidase from myxococcus xanthus was purified with a specific activity of 224 u mg(-1) by using nickel-metal-chelate affinity chromatography and gel permeation chromatography. the recombinant enzyme had a monomeric molecular weight of 70 kda. its isoelectric point, determined by two dimension polyacryl-amide gel electrophoresis, was close to 6.3. the optimum ph and temperature was estimated as 7.5 and 37°c, respectively. the purified enzyme was stable from ph 6.0-8.5 and able to thermal stability up to 37°c. the k(m) and v(max) values were 0.2 mm and 3.42 micromol/ min per mg. the enzyme exhibited hydrolytic activity for suc-gly-ala-pna, suc-gly-pro-pna, z-gly-pro-pna, igf-1, substance p, whereas no activity for h-gly-pro-pna, h-val-ala-pna, h-arg-pro-pna, h-ala-pro-pna, glu-ala-pna, pro-pna, leu-pna. its proteolytic activity was inhibited by activesite inhibitors of serine protease, z-pro-prolinal pmsf, and metal ions, cd 2+ and hg 2+. the potential use of the enzyme was tested by the hydrolysis of the wheat gluten. the resulting gluten hydrolysate were characterized by means of their antioxidant, antibacterial, trypsin inhibition and prolyl oligopeptidase inhibition activities. keywords: serine protease, prolyl oligopeptidase, bioactive peptides, 2,4,6-trinitrobenzene sulfonic acid. proteomic analysis is probably the best approach to analyze seed germination. however, it is difficult to analyze complex samples and there are many obstacles that must be faced in order to achieve a reasonable proteome coverage. for example, the barley (hordeum vulgare) genome was fully sequenced in 2012, but the uniprot database contains less than 1000 reviewed sequences, which is approximately 16-fold less than for arabidopsis thaliana. here, to improve the barley proteome coverage, we employed several fractionation methods including polyethylene glycol precipitation, strong cation exchange chromatography, off-gel separation, sds-page and acetonitrile elution gradient. proteomic analyses were performed using an lc-ms-based analyses and an uhr q-tof mass spectrometer. the candidate peptides were targeted via selected reaction monitoring (srm) and triplestage quadrupole (tsq) mass spectrometer. in total, 4092 proteins were identified, which represents a three-to four-fold increase compared with the standard shotgun analysis of the same sample. out of these, 2240 were only accessible by one of the techniques and, besides, the detection limits were not similar. we hypothesized that an srm-based targeted analysis will allow detection and quantitation of most of these proteins, even without the application of proteome fractionation. we can conclude that all peptides from the library with ms/ms spectra of the total intensity above 10,000 are easily detectable in the total protein extracts. p-02.08.5-012 transcriptome sequencing based identification of alternative oxidase genes in white waterlily, nymphaea alba alternative oxidases (aoxs) are the terminal oxidases in the respiratory electron transport chain of plants. they reduce molecular oxygen to water with low proton translocation across the inner mitochondrial membrane. in plants, aoxs increase local tissue temperature to release volatile compounds thereby attracting pollinator insects and regulation of mitochondrial retrograde signaling pathway. regulation of retrograde signaling pathway is currently under investigation to improve cultivation studies in many plants. water lilies are aquatic ornamental and economically valuable plants classified under nymphaea family. nymphaea alba, white water-lily, has a special focus since its applications in landscaping of parks and gardens, farming as vegetable and medical applications. however, cultivation of n. alba is a challenging process. we hypothesized that by controlling alternative oxidases, success rate can be increased for n. alba cultivation. to identify alternative oxidase encoding genes in n. alba, we performed transcriptome analysis. by using transcriptome analysis data, aox gene sequences, subcellular localization of aox proteins and structural modelling of aox proteins were predicted. in 272934 transcripts, database search with trinotate tool revealed 77 transcripts with aox domains characterized in known alternative oxidases. blast analysis of these 77 sequences with known aox proteins revealed three distinct aox genes (nalba-aox1, nalba-aox2 and nalba-aox4). after subcellular localization analysis of three identified aox proteins by using targetp server tool, nalba-aox1, nalba-aox2 are predicted as mitochondrial while nalba-aox4 is localized in chloroplasts. template based structural modelling results showed that all identifed proteins are statistically similar to known structure models of corresponding aoxs. most environmental contaminants have toxic and mutagenic effects on living organisms as a result of the activation of free radical formation and inhibition of reparation activity. it is becoming relevant to search for protectors of natural origin from the effects of xenobiotics. many biologically active substances (bas) of inartificial origin are found to be antioxidants and can increase the body's resistance to the toxic and mutagenic effects of a wide range of pollutants. the aim of the study was to investigate the antioxidant and antimutagenic properties of bas from medicinal plants limonium gmelinii (plumbaginaceae) and inula britannica (compositae). the antioxidant potential of plant extracts was determined by the activity of superoxide dismutase (sod), catalase, and the content of malonic dialdehyde. mutagenic and anti-mutagenic properties of the extracts were determined in the test by counting chromosomal aberrations in root meristem of barley seeds. barley seeds were treated with an aqueous solution of unsymmetrical dimethyl hydrazine (udmh), which is highly toxic i class hazardous material, well known pro-oxidant. the results showed that udmh enhanced the process of lipid peroxidation and decreased the mitotic activity. treatment of barley seeds with extracts from i. britannica and l. gmelinii and their germination in the presence of stress factors stimulated antioxidant defenses in the primary roots of barley seeds. increase of the activity of sod and catalase, and reduction of peroxidation level of lipids were observed. cytogenetic study showed no mutagenic activity in plant extracts. when effects of plant extracts and udmh were combined there was a significant reduction in the frequency of structural mutations, induced by the toxicant. conclusion about the presence of the antioxidant and antimutagenic activity in the studied plant extracts is made. the work done within the framework of the mes project (no. gr 0115rk00378). p-02.08.5-014 comparative analysis of cytokinin dehydrogenase inhibition and trans-zeatin treatment in arabidopsis seedlings j. nov ak 1 , v. koukalov a 1 , z. medvedov a 1 , c. martin 1 , j. hradilov a 1 , l. sp ıchal 2 , b. brzobohat y 1 1 mendel university in brno, brno, 2 palack y university in olomouc and centre of the region han a, olomouc, czech republic cytokinins are plant hormones regulating many processes during plant life ranging from germination to senescence. manipulation of cytokinin levels and their impact on plant vitality, production and ability to defend against stresses is in great interest of agriculture. in this work we focused on comparison of inhibitor of the cytokinin degradation incyde (2-chloro-6-(3-methoxyphenyl)aminopurine) and exogenous application of trans-zeatin on arabidopsis thaliana seedlings. transcripts of genes regulating cytokinin metabolism were analysed by rt-qpcr analysis. classical cytokinin root essay revealed that incyde effect is comparable to that of trans-zeatin in a similar concentration-dependent manner. besides a negative effect on the primary root length, both substances induce flavonoid accumulation and an increase in the root hairs formation. histochemical staining of transgenic plants expressing glucuronidase (gus) under cytokinin-responsive promoter of arr5 gene revealed increased gus activity in cotyledons following incyde treatment suggesting diverse localization of cytokinin modulation upon trans-zeatin and incyde treatment, respectively. possible molecular differences originating in different cytokinin population and distribution following trans-zeatin or incyde treatments were monitored on the level of gene expression and via an lc-ms proteome analysis in roots and shoots of 14-day-old plantlets. rt-qpcr analysis revealed an alteration in cytokinin metabolism that could explain observed differences on the proteome level between incyde and trans-zeatin treated seedlings. pharmacologically inhibited cytokinin degradation could be very efficient tool for modulation of cytokinin levels. interestingly, the application of incyde and trans-zeatin shows a contrasting spatial and temporal pattern on molecular levels. incyde represents potent growth regulator with interesting properties useful for agriculture. p-02.08.5-015 the expression yield of prokaryotic alphaamylase is significantly magnified by molecular cloning techniques randomly hydrolyzing glycosidic bond alpha-amylase has been traditionally employed in bread and similar industries. in that regard, increasing the overall expression level of the enzyme is a crucial concern in biotechnology. to reach the goal, appropriate alpha-amylase producing species and expression vector were carefully selected. therefore, genome of bacillus subtilis was extracted and amplified by polymerase chain reaction (pcr) using specifically designed primers. subsequently, the extracted gene was inserted in expression vector pht43 and transferred to e. coli as intermediate host followed by bacillus subtilis host replacement. the recombinant vector was expressed in bacillus subtilis and the expression was evaluated by agarose gel electrophoresis. relative purification of the recombinant enzyme was performed by 50 kda filtration to remove impurities. to identify the biochemical characteristics, starch was used as specific substrate to measure enzyme activity and the enzyme was exposed to various ph and temperatures. the extra-cellular expression of alpha-amylase enzyme was successfully elevated by 5 folds in comparison to the native enzyme. the optimum temperature and ph for the enzyme was carefully determined as 70°c and 6, respectively. the enzyme was stable at 50°c, but thermal stability was dramatically decreased at higher temperatures up to 70°c. kinetic parameters were also measured; vmax was 1.998 u/ml min and km was 3.998 mg/ml. it is concluded that the elevated expression extent of recombinant alpha-amylase together with appropriate qualifications could make the clone a good choice for various industrial applications. flax seedlings of cultivars tmp1919, lira and lines g-1071/ 4_o, g-1071/4_k were treated for 4 and 24 hours with 500 lm alcl 3 solution or distilled water (control). twelve small rna libraries were constructed and sequenced using illumina gaiix. to identify known mirnas, obtained sequences were aligned with mirnas from mirbase (http://www.mirbase.org/). fold change value was calculated to identify up-and down-regulated mirnas under al stress. in total, about 40 million raw reads were obtained and 109 conserved mirnas from 26 families were identified. significant expression alterations in flax plants under al treatment were shown for mir319 and mir390. expression level of mir319 was varied in similar way in resistant and susceptible to al genotypes: mir319 was up-regulated after 4 hours of alcl 3 exposure and down-regulated after 24 hours. mir390 expression was increased after 4 hours of alcl 3 exposure and decreased after 24 hours in susceptible to al flax genotypes (lira, g-1071/4_o), while in resistant genotypes (tmp1919, g-1071/4_k) mir390 level was decreased after both 4 and 24 hours of al treatment. in other plant species, mir319 and mir390 were identified as al-responsive. mir319 targets mrna of tcp (teosinte branched/cycloidea/pcf) transcription factors, which control plant growth. mir390 targets mrna of tas3 protein, which regulates lateral root growth via degradation of arfs (auxin response factors). in flax, the involvement of mir319 and mir390 in response to al stress was shown for the first time. moreover, we revealed diverse expression alterations of mir390 in susceptible and resistant to al genotypes. this work was financially supported by grant 16-16-00114 from the russian science foundation. p-02.08.5-017 association genetics of phenylalanine ammonia lyase (pal) and cinnamyl alcohol dehydrogenase (cad) enzymes involved in lignin biosynthesis of european black poplar (populus nigra) b. taskiran, z. kaya middle east technical university, ankara, turkey populus nigra l. are considered as one of the most economically significant forest trees with respect to production of wood, biomass, and other wood-based products. while wood quality and biomass are directly associated with high cellulose content, lignin emerges as an undesirable polymer for both pulp and biofuel manufacturing industries. the aim of the study is by choosing the superior and eliminating the inferior clones to make a contribution to woody feedstock development and to improve wood quality of populus nigra. to estimate association genetics of pal and cad enzymes which have important functions in lignin biosynthesis, the important germplasms of populus nigra has been sampled from 3 year old poplar trees (285 clones x 2 replicates x 2 ramets) which were grown in behic ßbey plantation clone bank in ankara. additionally, five commercially registered clones and six foreign clones were included to the study to make comparison. the average mean values of cellulose, lignin and glucose content were calculated as 21.8 ae 16.29 lg/ml, 23 ae 4.64 lg/ml, and 35 ae 9.71 lg/ml, respectively. even though for pal and cad enzymes, data gathering process have been still resuming, particular clones have been separated from all in terms of pal and cad activities as expected. key words: populus, poplar, lignin, pal, cad, genetic variation, feedstock p-02.08.5-018 proteomic analysis of the molecular mechanisms of the response of plant seeds to pre-sowing treatment by stressors seed treatment with non-ionizing low-level radiation (nr), such as cold plasma (cp) or electromagnetic field (ef), is a modern eco-agricultural technology for stimulation of plant germination and performance. the molecular determinants of seed response to these treatments are not established and no genomic studies of plant seed response to nr have been reported. we studied the effects of pre-sowing seed treatment, using vacuum (7 min), radio-frequency ef (5-15 min) and cp (2-7 min), on germination and growth of non-oilseed helianthus annuus. to gain an insight into the molecular mechanisms underlying effect of nr on sunflower seed germination and dormancy, we estimated changes induced in the balance of plant hormones and differential protein expression. the results of the germination tests and estimation of seedling morphology showed that response develops in time and is stronger when sowing is performed in 7 days in comparison to 3 days after seed treatment. the 2d dige analysis revealed 38 differentially expressed proteoforms in kernels of seeds treated with cp or ef. proteins involved in biological processes of seed maturation, response to stress, response to abscisic acid stimulus, processes of organonitrogen compound metabolism and glucose catabolism were identified. while expression patterns for majority of the proteins were highly specific to cp and ef treated seed kernels, accumulation of several proteoforms of seed storage proteins (ssp), including vincilin-like, miraculin-like protein and albumin-8 were common for both experimental groups. this suggested that response to nr treatment could be at least partially associated to function of ssps in response to oxidative stress that protects proteins required for seed germination and seedling formation. variation of abundance of distinct proteoforms of helianthinin, vicilin-like and 11s globulin-like ssps suggested that post-translational modifications are involved in regulation of the function of ssps. p-02.08.5-019 suppression of lipopolysaccharide-induced inflammatory responses in raw 264.7 macrophages by tuber extract of cyclamen l. turkey is a prominent centre of plant diversity, being the meeting point of three main floristic zones. geophytes which have underground storage organs such as, tubers, bulbs and rhizomes. cyclamen l. is a tuberous geophyte traditionally used by some people for treating whooping cough, headaches or sinusitis, and confirmed to have antioxidant, analgesic and anti-inflammatory properties by several reports. a prolonged inflammatory response is often associated with chronic diseases such as cancer, arthritis and autoimmune disorders. recently, plant based products are used as an alternative and complementary treatment of these diseases. in this respect, the present study was aimed to determine the effects of three cyclamen tuber extracts on lps-induced inflammatory responses of murine raw 264.7 macrophages. firstly, c. cilicium (endemic), c. pseudibericum (endemic) and c. graecum subsp. anatolicum were collected from different localities of turkey. the tubers of plants were air-dried and grounded to fine powder and then extracted with ethanol. cell viability assay was performed to evaluate the nontoxic concentration in cell line by mtt assay. several measurements were performed including tnf-a, no and il-8 concentration assay by elisa after treatment compared to non treated cells to determine the anti-inflammatory activity. also, tnf-a and inos mrna levels were evaluated by quantitative rt-pcr. the cytotoxic activity which is considered safe on raw.264.7 cell were found as 0.5-5 lg/ml. studied cyclamen taxa inhibited tnf-a and il-8 release on lps stimulated-raw.264.7 in a concentration-dependent manner. among the three cyclamen tuber extracts evaluated, the highest nitrite-associated no inhibitory activity was obtained from c. pseudibericum compared to other two cyclamen l. taxa. collectively, these results suggest that cyclamen tuber extracts possess anti-inflammatory properties. p-02.08.5-020 in vitro hypoglycemic activity of ziziphus jujuba recent reports have indicated that continuous treatment with nutritional jujuba (ziziphus jujuba miller) fruit extracts in diabetic rats improved glucose utilization and produced a significant decrease in the blood glucose. in the present study, hypoglycemic activity of z. jujuba was investigated using various in vitro techniques. the hypoglycemic effect of z. jujuba in phosphate buffered saline which grown in balıkesir was studied by measuring glucose adsorption, glucose diffusion and glucose uptake by yeast cells. the glucose content in the solution measured by spectrophotometrically with commercially kits. the adsorption capacity of the z. jujuba was found to be directly proportional to the molar concentration of glucose. the glucose binding capacity of extract increased in higher glucose concentratrations. there was significant differences were observed between the adsorption capacities of z. jujuba and control samples (p < 0.05). the rate of glucose diffusion was directly proportional to the time. diffusion rate was significantly lower in the solution containing z. jujuba compared to control (p < 0.05). the extract demonstrated significant inhibitory effects on movement of glucose into external solution across dialysis membrane compared to control. the rate of glucose transport across cell membrane in yeast cells was observed to be inversely proportional to the molar glucose concentration. z. jujuba inhibited glucose transport across the yeast cells. the results showed that z. jujuba reduced glucose levels at least by three mechanisms. first by increasing glucose adsorption capacity during postprandial hyperglycemia; second by retarding glucose diffusion rate and third, at the cellular level by inhibiting glucose transport across the cell membrane. all of these decreased the absorption of glucose in the intestinal cells and the concentration of postprandial serum glucose. p-02.08.5-021 cucurbitacin b increased the anticancer effect of imatinib mesylate through inhibiton of matrix metalloproteinase-2 expression in colorectal cancer cells f. bakar ankara university, ankara, turkey several natural products have been investigated for their anticancer effects. among these, cucurbitacin b (cub) has been reported as its inhibitory effects on cancer cell proliferation. matrix metalloproteinases (mmps) belong to endopeptidase family and they are received as potential biomarkers for several types of cancer. the aim of this study is to investigate the effect of cub in combination with imatinib mesylate (im) on mmp-2 mrna expression of human sw480 colorectal carcinoma cells. the cytotoxicity analysis was performed via mtt assay. muse cytofluorimetric analysis system was performed to evaluate apoptotic cell population. the mmp-2 mrna expression was determined by quantitative real-time pcr. this study was supported by scientific and technological research council of turkey grant, sbag-114s871. data obtained from the cell culture experiments were expressed as mean ae sd and one-way anova test was applied for multiple comparisons. cub alone significantly inhibited cell growth at 10 lm and higher concentrations. the most potent effect was observed in cub-im combination treatment with 3.51 lm ic 50 value. in cub-im treated group, the apoptotic effect was higher than cub and im treated groups. cub-im induced apoptosis significantly at 10 lm concentration when compared to control and 1 lm (p < 0.05). cub alone showed inhibitory effects on mmp-2 mrna expression at 1 lm and higher doses significantly (p < 0.05). the results showed that the combination treatment of cub with imatinib synergistically inhibited human sw480 cell growth and induced apoptosis by increasing the anti-histone antibodybound nucleosom levels and annexin v binding. although cub could inhibit mmp-2 expression alone at higher treatment doses, it enhanced the inhibitory effect of im on mmp-2 synergistically in a dose dependent manner. in conclusion, this study suggests that cub combined with imatinib mesylate may enhance the effects of chemotherapy in patients with colorectal cancer. plants are most important parts of natural resources that alternatively referred to synthetic drugs for reasons such as being less side effects and lower costs. ziziphus jujuba miller (z. jujuba), a plant used in traditional medicine, is one of the most important ziziphus species belonging to rhamnacea family. the fruit and seeds of this plant are used different purposes such as antiinflammatory, antioxidant, immune-stimulant and wound healer. in this study, we investigated the antibacterial effects of z. jujuba. the aims of this study were to screen the antibacterial activity of z. jujuba. the extract was obtained from z. jujuba fruits pulverized with the aid of ball mill using 50% aqueous-ethanol solution. extracts were screened for antimicrobial activity against six different standard strains of bacteria by determining minimum inhibitory concentration (mic) according to clsi criteria. serial dilutions are made between 64 mg/ml and 0.031 mg/ml concentration range. the lowest concentration of wells that no visible growth has been accepted as mic value. materials in the mic and lower concentrated wells were transferred to 5% sheep blood agar petri dishes for calculation of minimal bactericidal concentration (mbc). the lowest concentration that no colony formation has been accepted as mbc value. jujuba showed the most potent effect on strain of s. aureus atcc 29213 is gram-positive cocci (mic: 2 mg/ml). the mic values of other gram-positive bacteria s. aureus atcc 43300, e. faecalis atcc 51219, l. monocytogenes f 1483 and m. smegmatis cmm 2067 were detected as 8, 16, 8 and 8 mg/ml respectively. mic values of gram-negative bacilli were detected as >64 mg/ml. consequently, z. jujuba was found to be effective on grampositive cocci bacteria (s. aureus atcc 29213, s. aureus atcc 43300 and e. faecalis atcc 51219). the strongest effect was observed on s. aureus atcc 29213 strain. in contrast, extract showed less effect on gram-negative bacilli. p-02.08.5-023 selective cytotoxic effect of morus rubra extract in human lung cancer cells through enhancing apoptosis and cell cycle arrest cancer is a disease that develops as a result of unlimited proliferation of abnormal cells that occurs due to loss of control over the mechanisms of normal growth and differentiation of cells. morus rubra, known as "red mulberry" belongs to family of moraceae. for many years, the fruits of morus species have been used to treat many diseases in traditional medicine. biological effects of morus species is predominantly attributed to its content of polyphenolic compounds. many studies have evaluated the cytotoxic effects of different morus species, but there is no study about cytotoxic effect of m. rubra. in this study, we aimed to evaluate the cytotoxic effect of m. rubra extract in human lung cancer cells (a549) with regard to apoptosis, cell cycle and mitochondrial membrane potential. cytotoxic effect of m. rubra extract on human lung cancer cells was determined using mtt assay. then, mechanisms of cytotoxic activity of m. rubra extract on a549 cells were examined in terms of cell cycle, apoptosis and mitochondrial membrane potential using flow cytometric methods. m. rubra extract exhibited selective toxicity against a549 cells compared to normal foreskin fibroblast cells. we determined that m. rubra extract increased cell cycle arrest at g 1 phase, the level of apoptotic cells and decreased mitochondrial membrane potential in a549 cells. our results showed that m. rubra extract has pro-apoptotic and antiproliferative effect in a549 cells. further studies are also needed to fully mechanisms underlying this effect of m. rubra extract. p-02.08.5-024 dipeptidyl peptidase iv inhibitory activity of arctium tomentosum l. a. zeytunluoglu 1 , f. zihnioglu 2 1 denizli vocational school of technical sciences, pamukkale university, denizli, 2 department of biochemistry, faculty of science, aegean university, izmir, turkey type 2 diabetes mellitus (t2dm) is rapidly growing metabolic syndrome of multiple aetiologies causing hyperglycaemia with insulin resistance at cellular level. a novel approach in the treatment of t2dm is based on preventing of rapid inactivation of the incretin hormone glucagon-like peptide-1 (glp-1) and glucose-dependent insulinotropic polypeptide (gip) by dipeptidyl peptidase-iv enzyme. in this study; dipeptidyl peptidase iv (dpp-iv; ec 3.4.14.5) inhibitory activity of the aqueous and methanolic extracts of arctium tomentosum leaves were successfully tested in vitro conditions. our study revealed that both aqueous and methanolic extracts obtained from test material had a significant dppiv enzyme inhibitory activity in changing ratio. the ic 50 values were also determined by nonlinear regression curve fit using graph pad prism 5.0 with appropriately diluted of lyophilized arctium tomentosum. diprotein-a (ile-pro-ile) was used as reference inhibitor. a. tomentosum aqueous extracts showed ic 50 4.6 lg/ml while the standard (positive control) diprotin a displayed the ic 50 value of 10.1 lg/ml. this study demonstrates that a. tomentosum aqueous extracts could be a good lead for further development as a new antidiabetic agent. p-02.08.5-026 dna recognition determinants of arabidopsis thaliana b3 transcription factors g. sasnauskas, k. kauneckaite, k. lapenas, v. siksnys institute of biotechnology, vilnius university, vilnius, lithuania transcription, one of the most important cellular processes, is regulated by transcription factors (tf), proteins that often directly interact with gene promoter sequences. tf binding to dna is mediated by various dna binding domains. the b3 tfs constitute a large, plant-specific protein family (approx. 10% of all tf proteins in the flowering plants), which is characterized by the presence of one or several small (approx. 110 amino acids) b3 dna binding domains. currently the b3 tfs are divided into four groups (lec2-abi3/val, arf, rav and rem). the preferred recognition sites were identified for representatives of all groups except the rem family. currently, only a single structure of a dna-bound b3 domain (arf1, arf family) is available, thus the mechanism of site-specific dna recognition by the lec2-abi3/val and rav b3 domains remains poorly understood. based on the arf1-dna structure (pdb 4ldx) we have built homology models of dna-bound b3 domains from a. thaliana abi3 (lec2-abi3/val family) and nga1 (rav family) transcription factors, mutated putative dna-interacting amino acid residues and characterized the dna binding ability of the purified mutants using electrophoretic mobility shift assay and a set of radiolabelled dna substrates carrying various variants of the optimal recognition site. we confirm the importance of several positively charged amino acid residues, which are conserved between the abi3/ nga1 b3 domains and structurally related dna-binding domains of bacterial restriction endonucleases ecorii, bfii and ngoavii; furthermore, we identify residues in the 'n-arm' and 'c-arm' loops that may be involved in specific interactions with the dna bases. our results therefore help us refine the homology models of the dna-bound b3 domains and in the future may help us predict the dna binding properties of currently uncharacterized b3 domains. p-02.08.5-027 immunohistochemical analysis of inhibitory effects of origanum hypericifolium oil on dipeptidyl peptidase iv in streptozotocininduced diabetic rats p. ili 1 , a. zeytunluoglu 2 1 denizli health services vocational high school, pamukkale university, denizli, 2 denizli vocational school of technical sciences, pamukkale university, denizli, turkey diabetes mellitus (dm) is a serious metabolic disorder with micro-and macro-vascular complications that result in a significant morbidity and mortality. glp-1 and gip have significant role in pancreatic beta cells and prevention of inactivation of them by dipeptidyl peptidase iv (dpp iv) inhibition is a novel approach to treatment of dm. origanum hypericifolium (lamiaceae) is an endemic turkish plant and its essential oil is mainly composed of monoterpenes including carvacrol and thymol. streptozotocin (stz) is used to induce diabetes in rats. the aim of this study is to investigate the inhibitory effects of o. hypericifolium essential oil on the dpp iv in stz-induced diabetic rats. the animals (female spraque-dawley rats) were assigned to four groups (group 1: control, group 2: stzinduced diabetic, group 3: o. hypericifolium injected, group 4: stz-induced diabetic and o. hypericifolium injected). dm was experimentally induced in groups 2 and 4 by a single intraperitoneal injection of stz at a dose of 45 mg/kg body weight. in groups 3 and 4, rats were intraperitoneally injected with o. hypericifolium oil at a daily dose of 1 ml/kg body weight for 42 consecutive days. at the end of the experimental period, all animals were sacrificed by cervical dislocation under ether anesthesia and liver and kidney tissues of each animal were rapidly excised. tissues were fixed in sainte-marie fixative. after routine histological processes, samples were embedded in paraffin, immunohistochemical staining for dpp iv was performed on sections and then they were photographed. the immunohistochemical reaction intensity differences were observed between the groups. in conclusion, the immunohistochemical distribution of dpp iv in the tissues that the test oil was applied in the diabetic rats may be important for the investigation of the inhibitory effects of oil on the enzyme. moreover, our findings suggest that o. hypericifolium oil may be used for prevention of diabetic diseases. introduction: all eukaryotic cells need microtubules for purposes of nuclear and cell division, organization of intracellular structure, and intracellular transportation, as well as ciliary and flagellar motility. microtubules are made of polymerized a/btubulin subunits. mec17 is important for microtubules, because it encodes the enzyme that adds acetyl groups to lysine 40 (k40) of tubulin. k40 is largely conserved in a-tubulins of many eukaryotes, and acetylation is thought to stabilize microtubule structure. in algae, the effect of acetylation by mec17 on flagellar motility and phototaxis has not been tested previously. materials and methods: in this study, mec17 mutant chlamydomonas reinhardtii cells were compared to wild-type cells to see the effect on flagellar motility and phototaxis. we tested phototaxis, eyespot size and quantity under the microscopy. in addition to this, we fixed cells and examined them by immunofluorescence microscopy using antibodies to tubulin, acetylated tubulin, and photoreceptor. results: we observed that some mec17 mutant cells contain more than one eyespot. we detected no acetylated-tubulin (ac-tub) by immunofluorescence. the cells still phototax and have normal motility discussion and conclusion: interestingly, mec17 cells still have the ability to phototax and they have normal flagellar motility, even though they contain occasional additional eyespots and no ac-tub. chlorella vulgaris as a model system for screening of plant growth modulators p. volynchuk 1 , e. marusich 1 , r. chuprov-netochin 1 , j. neskorodov 2 , y. mishutkina 2 , s. leonov 1 1 life sciences center, moscow institute of physics and technology, dolgoprudny, 2 center "bioengineering", russian academy of sciences, moscow, russia the discovery of new plant growth modulators became extremely important task as an alternative approach to overcome plants resistance to herbicides and pesticides, which leads to harmful action on plants and land rising, environmental and ecological problems. small molecules provide agricultural biotechnology with valuable tools, which help to circumvent the need for genetic engineering and offer unique benefits to modulate plant growth and development. we developed a system to explore molecular modes of action of plant growth modulators using chlorella vulgaris model. our model allows applying high content screening approach in 96well plate format for fast and robust effect assessment of large number of tested modulators. chlorella v. was grown in climate chamber under optimized constant temperature (20°c) and light conditions (16:8 hours/light:dark). modulating effect of tested compounds was estimated by spectrophotometric measurement of microalgae density at the beginning of the experiment (start point-0.1od) and 48 hours later. to validate our system we used known cytokinins and auxins (10 mm) as positive controls of growth stimulation. we showed that in presence of each compound the density of chlorella v. was increased in 7-11 times range, compared with only 2 times increase in control group. eight new chemicals (10 lm), which demonstrated modulation effect on nicotianatabacum l. pollen and arabidopsis thaliana models, were tested on developed chlorella v. model. positive controls showed no stimulating effect at this concentration, while tested compounds were confirmed as hits and increased the density up to 400%. we demonstrated that developed model system, based on chlorella v., is an effective system for primary screening of plant growth modulators. the main advantages of this system are short time of assay, simplicity of performance, possibility of automation and low cost. selected hits can be recommended as perspective candidates for future test on crop field. sunflower is under a big threat of downy mildew which is a fungal disease caused by plasmopara halstedii. the disease can cause up to an 80% yield loss in sunflower production. downy mildew resistance genes (r) denoted as pl has been discovered to date in sunflower. in recent years, single nucleotide polymorphism (snp) markers have become widely used in plant breeding programs. in this study snp markers have been currently developing for pl 6 , pl 8 , pl 13 , pl arg genes by competitive allele specific pcr (kasp) assay which enables bi-allelic scoring of snps and insertions/deletions (indels) at specific loci. in total 66 sequence tagged site (sts) sequences from ncbi were aligned for pl 6 , pl 8 , pl 13 , pl arg genes to identify conserved regions for each gene. based on the conserved regions, specific pcr primers were designed in order to make sequencing of these genes in five crosses and their f 2 progenies. sequence data will be used to design an allele specific primer maches the target snp and amplifies the target region with the common reverse primer provided by kasp genotyping assay. snp markers linked to pl genes which are being developed in this study, have the potential to be used in marker assisted selection (mas) for sunflower breeding programs. p-02.08.5-031 investigation of the antidiabetic effects of hibiscus sabdariffa, teucrium polium and myrtus communis in hepg2 cells line s. altundag 1,2 1 pamukkale university, denizli, 2 istanbul medeniyet university, istanbul, turkey some antidiabetic plants currently are used in alternative treatment of type ii diabetes. hibiscus sabdariffa, myrtus communis and teucirum polium plants are also known for their antidiabetic properties. hibiscus sabdariffa, myrtus communis and teucrium polium; depending on the impact on hepg -2 cells to investigate the possible mechanisms of type ii diabetes with researches on glucolysis and glucogenesis pathways gene expressions (pk m , glut-2,pepck). plants were obtained in dried state from reliable herbalists in denizli and mersin. plants treated with the extractor device. plants obtained aqueous extract was subjected to lyophilization process. human cancer cells have been used throughout the study. the cytotoxicity of the cells was measured by elisa plate reader . total rna was isolated using trızol ò solution was carried out according to the instructions of the manufacturer's (thermo scientific) recommended procedures were performed, but we have to optimize our own laboratory conditions. pk m , glut-2,pepck genes were synthesized by b _ ioneer. during our study the activation of certain genes (pk m , glut-2,pepck) were examined by real time pcr. in our study hibiscus sabdariffa, mrytus communis and teucrium polium plant to extract applied hepg -2 cell line in the gluconeogenesis and glycolysis pathways in involved in some important genes (pepck, pk m , glut-2) analyzed the expression levels . teucrum polium plant extract is applied in hepg -2 cells the glycolysis pathway genes (pk m glut-2) an increased expression also genes of gluconeogenesis pathway (pepck) were not decreased. however hibiscus sabdariffa and the expression of genes involved in glycolysis and gluconeogenesis pathway mrytus communis plants were observed to have a full effect as diabetic or hypoglycemic . in this context, it is considered that the plant teucrum polium on the line hepg -2 cells showed antidiabetic effect. p-02.08.5-032 purification of b-glucosidase from malatya apricot (prunus armeniaca l.) seeds and some of its biochemical properties h. kara 1 , s. sinan 2 , z. ekmekci 2 , y. turan 2 1 university of balikesir faculty of veterinary, balikesir, 2 university of balikesir faculty of arts and sciences, balikesir, turkey introduction: b-glucosidases are one of the key enzymes in carbohydrate metabolism and located in glycosyl hydrolases (ec 3.2.1) family. plant b-glucosidases have biotechnological significance as they are effective on glycosidic bonds of flavor and aroma precursors in plants. b-glucosidases that located in fruit seeds are important because they affect the amygdalin. aim of this study is purification and partially characterization of b-glucosidase from malatya apricot seeds. materials and methods: apricot seeds were homogenized with extraction buffer to prepare of crude extract. the enzyme protein was precipitated with 60% ammonium sulfate then purified by hydrophobic interaction chromatography using sepharose 4b-ltyrosine-1-naptylamine gel. para-nitrophenyl b-d-glucopyranoside (p-npglc) was used as substrate to determine biochemical properties of the enzyme. the optimum ph was determined using buffers between ph 2-12 and thermal optima was determined using 25-85°c temperature range. inhibitory effects was determined with 1 mm substances. results: the enzyme was 11.1-fold purified with yield of 14%. purified b-glucosidase from apricot seed was visualized about 60 kda molecular weight on sds-page. the kinetic parameters were determined against p-npglc substrate as km and vmax values of 2.5 mm and 58.1 eu, respectively. the optimum ph and temperature were determined 5.0 and 55°c respectively. effects of cacl 2 , kcl, nacl, mgcl 2 , k 2 so 4 , na 2 so 4 , cuso 4 , fecl 3 , pb(ii) acetate, agno 3 , zncl 2 and glucose on purified enzyme activity were investigated. kcl, na 2 so 4, pb(ii) acetat and cuso 4 reduced the enzyme activity. discussion and conclusion: in this study, b-glucosidase was purified from malatya apricot seed and some of its biochemical properties were determined. because this enzyme has pharmaceutical importance hydrolyzing amygdalin. the results showed that immobilized almond b-glucosidase was used to break amygdalin and release -cn compound that effective to shrink cancer mass. p-02.08.5-033 a new affinity gel for purifing polyphenol oxidase enzyme a. erg€ un 1,2 , o. arslan 2 1 balikesir university, science and technology application and research center, balikesir, 2 department of medical chemistry, faculty of science, balikesir university, balikesir, turkey polyphenol oxidase (ppo) enzyme, sometimes called as phenol oxidase, catecholase, phenolase, catechol oxidase or tyrosinase, is considered to be an o-dipenol. ppo (ec 1.14.18.1), a multifunctional copper containing metalloenzyme, is widely distributed in nature. ppo exists in many kinds of plants and fungi, such as banana, mushroom, butter lettuce, napoleon grape, potato, coffee, marula fruit, artichoke heads, longan fruit, tobacco, wheat flour. in this study, a novel affinity chromatography gel was synthesized for purifing ppo enzyme. the affinity chromatography gel was synthesized by coupling aniline as a specer arm to cnbr activeted sepharose-4b. then, p-amino benzoic acid was coupled to aniline as a ligand. ppo was purified from musa sapientum var. cavendishii (banana) by using sepharose-4b-aniline p-amino benzoic acid affinity chromatography gel. % 4.49 yield and 33.4 fold purification were achieved. sds-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent mw of 35 kda. the v max and k m of the purified enzyme were determined 42,628 u/ml min and 9.27 mm, respectively. p-02.08.5-036 phenolic content and antioxidant capacity of gamma irradiated olive leaves m. e. diken 1 , b. kocat€ urk 2 , s. dogan 1 , h. tuner 1 1 balikesir university, balikesir, 2 kavram vocational school, istanbul, turkey in this study, dried in diffirent ways (such as microwave, infrared, convection heaters and under normal atmospheric conditions) olive leaves has been used as experimental material. radiation has been applied to dried olive leaves in three diffirent dosages in room temparature. the amount of radiation to be implemented to the samples have 3, 5, 10 kgy/min. in this study, how gamma rays (radiation) effects phenolic components, total phenolic content and antioxidant capacity of dried olive leaves has been determined. the phenolic components, total phenolic content and antioxidant capacity were analysed with hplc, folin ciocalteu and dpph radical scavenging method, respectivelty. gamma rays is well known as a decontamination method for many foodstuffs and plant materials, being an environment friendly and effective technology to resolve technical problems in trade and commercialization. for this reason, nowadays it is utilized as an alternative method of sterilization. gamma rays are of ionizing radiation. when ionizing radiation interacts with matter, generally it causes a break in the molecular bonds and/or breaks the bonds between the molecules. these intermediates have unpaired electron and called free radical. gamma radiation or the radiation-induced free radicals would break or cause damage to the dna molecules of living organisms. gamma irradiation is predict to change phenolic content and antioxidant capacity in living tissues. phenolic compounds are secondary plant metabolites naturally present in fruits and vegetables. in recent years there has been a growing interest in food phenolics because of their potential health benefits mainly due to their antioxidant and free radical scavenging activity. despite the controversy about potential risks posed by genetically modified plants on human health, environment and microorganisms, cultivation area of these crops increases day by day. this increment has revealed concerns especially related to hgt. hgt studies indicated that antibiotic resistance genes in gm plants have a potential to transfer to soil microorganisms. in this study, hgt of widely used genetic elements such as regulatory sequences, from transgenic plants to bacteria was investigated. three soybean feed and four seed examples from turkish feed manufacturers' association were used for genetic analysis based on foreign gene determination. gmo analysis were conducted by camv 35s promoter-specific pcr in genomic dnas. in gm positive samples, genomic dnas sheared into appropriate fragment size by ultrasonication for the purpose of bacterial transformation. presence of 35s promotor region in fragmented dnas was proved by pcr. escherichia coli dh5a strain was transformed by fragmented dna samples according to cacl 2 method. 35s promotor sequence screened by using pcr in bacterial genomic dnas. as a result of gm screening, all feed and three of the seed samples were found to be transgenic. ultrasonication conditions were optimized for shearing dna's to 450-500 bp for bacterial transformation. fragmented dnas confirmed for carrying intact 35s promotor sequence. none of bacterial genomic dnas were found to be 35s-positive. according to the transformation results, absence of 35s promotor sequence in all tested bacterial genomic dnas, proved the dna samples belonging to gm plants can not transfer into compotent e. coli dh5 under laboratory conditions. for verification of this finding, transformation studies will continue with acinetobacter baylyi bd413 strain which is naturally competent soil bacterium for natural transformation. we believe that all of our findings will contribute to constitute transformation system which can be used as model in hgt studies. p-02.08.5-038 preliminary studies on differential methylation in a and d sub-genomes of upland cotton (gossypium hirsutum l.) four species of cotton (gossypium l.) provide raw materials for the textile industry. among the four species, two have diploid genome and another two have tetraploid genome. tetraploid genome consists of a and d sub-genomes. a sub-genome belongs to asian cotton while d-sub genome belongs to american cotton. previous studies revealed that d sub-genome of gossypium species contributes to the superior yield and quality of tetraploid gossypium l. species (atdt). dna cytosine methylation of four regions of dna sequences located on a and d sub-genomes of gossypium hirsutum l. texas marker 1 (tm-1) was investigated using bisulfite sequencing technique. among the regions studied two could not be located on subgenomes due to sequence identity match between a and d subgenomes. on the other hand two dna regions could be located on a and d sub-genomes using the blast searches. some of the dna sequences located on different sub-genomes showed polymorphic nucleotides including c/t and g/a polymorphisms. in silico analysis indicated that some alleles located on different sub-genomes of cotton have c/t and g/a polymorphisms. c/t polymorphisms between the sub-genomes could be thought as unmethylated cytosine using the bisulfite sequencing technique. this indicated that an extra attention needs to be paid in dna total cytosine methylation studies in polyploid species such as cotton using bisulfite sequencing, methylation sensitive amplification polymorphism (msap), whole-genome bisulfite sequencing (wgbs). otherwise, t in c/t polymorphism between the subgenomes could be thought as unmethylated cytosine. based on the two genomic regions we could conclude that a sub-genome may be more methylated than d sub-genome. differential methylation of genes located on different sub-genomes may provide another mechanism responsible for differential gene expression of genes located on different sub-genomes. p-02.08.5-039 cleaved minisatellite locus (cml) markers for fingerprinting of cotton cultivars grown in turkey e. u. gocer, m. karaca akdeniz university, antalya, turkey after their discovery by alec jeffreys in 1984, minisatellites have been used in genetic studies of many organisms. minisatellites, also called variable number tandem repeats (vntrs), are composed of arrays of longer repeats mostly dispersed throughout heterochromatin (centromeres and telomeres). direct amplification of minisatellite regions of dna using a single core primer is a powerful method to amplify minisatellites (damd-pcr). although the damd-pcr technique has been applied to many plant species, the level of polymorphisms in cotton (gossypium l.) is very low due to very narrow turkish cotton genetic base. the objective of this study was to improve the level of polymorphisms by cleaving minisatellite loci by restriction enzyme digestion. genomic dna samples of twenty-one turkish cultivars, pima 3-79, tm-1 and ps-7 were extracted. twenty-one minisatellite primers were screened using the damd-pcr technique. monomorphic amplicons were digested using several restriction enzymes. three to five micro liters of amplified products were digested with various restriction enzymes. digested products of minisatellite loci were separated in 3% high resolution agarose gels. comparison studies of digested and undigested markers revealed that cleaved minisatellite markers showed polymorphisms in those cotton lines that could not be differentiated by microsatellite and minisatellite markers. this approach was called cleaved minisatellite locus markers (cml). the amplification reactions of minisatellites used touch-down (td) cycling conditions. the use of td offered a simple and rapid means of optimizing polymerase chain reaction (pcr), increased specificity, sensitivity, and efficiency without the need for lengthy optimizations of minisatellite primers. the cml markers were obtained at a 55°c annealing temperature, which is a temperature higher than those used in random amplified polymorphic dna (rapd) inter-simple sequence repeat (issr) markers. p-02.08.5-040 association between cytosine methylation and tissue specific expression of microsatellites a. g. ince, m. karaca akdeniz university, antalya, turkey heritable covalent modification of dna, rna or protein without altering their primary sequences is defined epigenetics. because all biological events are influenced by epigenetics, it is one of the most important fields in science. dna methylation is one of the most important epigenetic mechanisms. dna cytosine methylation is a process by which methyl groups are added to cytosine bases of dna. microsatellites, also knows simple sequence repeats are dna sequences consisting of 1-6 nucleotide repeats. there is a large body of information regarding the relationship between microsatellite instability and abnormal gene expression, and between dna methylation and altered gene expression. however, there is limited information on cytosine methylation of microsatellites. in the present study, we investigated whether there is any association between cytosine methylation and tissue specific expression of microsatellites. genomic dna samples of various tissues and developmental stages of pepper line demre sivrisi (capsicum annuum l.) and cotton line tm-1 (gossypium hirsutum l.) were extracted. cdna samples were synthesized using mrna expressed in pepper tissues. cytosine methylation levels were investigated using bisulfite sequencing methods. screening studies of microsatellites revealed that some genes containing microsatellites were differentially expressed in tissues and developmental stages of pepper. microsatellite containing genes that expressed differently among tissues had also showed different methylation levels in cg, chg and chh (where h refers to a, c or t) contexts. methylation level differences between microsatellites were also observed. as far as our knowledge, it is the first report on differential expression of genes containing methylated microsatellites. p-02.08.5-041 pcr-lg: an alternative way to assign the chromosome location of genes/markers in cotton a. aydin, m. karaca akdeniz university, antalya, turkey assignment of genes and dna markers on chromosomes is very important in life sciences, especially for plant breeding and medicine. there are several methods for the assignment of a gene or dna sequence to a specific location on a chromosome. for example, the most widely used technique is the assignment of fluorescently-labeled gene or dna sequences (markers) on chromosomes using the fluorescently-labeled gene (for instance, fish technique). another example is the construction of a genetic map (linkage map) which orders the targeted genes along the dna strand based on recombination frequency. sequencing is the most precise technique in which coding (gene-containing) and noncoding dna region of genes could be located on a chromosome molecule. aneuploid lines could also be used to locate genes in a specific chromosome, but maintenance of these lines is difficult. here we report the use of chromosome substitution lines to indirectly locate genes/markers on chromosomes. we used chromosome substitution lines (csls) that carry a chromosome pair or chromosome arms from gossypium barbadense l. while the rest of chromosomes belong to g. hirsutum l. a total of 10 chls, a homozygous cotton line tm-1 and a double haploid line pima 3-79 were used as plant materials. twenty microsatellite primer pairs were utilized in touch-down polymerase chain reactions. we developed a method, called polymerase chain reaction to locate gene (pcr-lg), to assign genes/markers on chromosome or chromosome arm. with the use of pcr-lg approach any polymorphic genes/markers between tm-1 and pima 3-79 (g. hirsutum and g. barbadense) could be assigned to a chromosome or chromosome arm. results indicated that if 26 csls were used any polymorphic markers (genes) with known primer pairs could be assigned to cotton chromosomes. although we used cotton chromosome substitution lines to validate the proposed technique, it could be applicable all the species that have chromosome substitution lines. p-02.08.5-042 pecularities of genome variability of antarctic hairgrass deschampsia antarctica desv. from the maritime antarctic deschampsia antarctica desv. (poaceae) is the only grass species native to the antarctic region, adapted to harsh environmental conditions. however, reasons for its unique success remain unexplored. stressful environmental factors can influence a plant genome and cause changes in the chromosome number and morphology and increase genetic variation. therefore, the purpose of our research was to explore alterations in the d. antarctica genome both at the chromosomal and molecular levels and to investigate species genome stability using in vitro tissue cultures. plants used for the study were grown in vitro from seeds collected in the argentine islands region during 2008-2014. chromosome number was determined in plant root apical meristems and specimens prepared from tissue cultures. rrna genes localization were determined using the fish technique. moleculargenetic analysis was performed using pcr with polymorphic issr-primers. new forms of chromosome polymorphism, associated with aneuploidy (2n = 13-27), polyploidy (2n = 13-39) and the occurrence of additional b-chromosomes (2n = 26 + 1-3b), were observed. fish analysis also confirmed that genotypes with a different chromosome numbers varied in the number of 5s rdna and 25s rdna sites. assessment of genetic variation demonstrated a low level of diversity: differences between the plants with different chromosome numbers do not exceed the level of within-population variation. cytology analysis of d. antarctica cultured tissues revealed aneuploidy (up to 60.6 %) with predominance cells with diploid and near-diploid chromosome number irrespective of plant's initial karyotype (diploid, mixoploid or polyploid). we assume that discovered intraspecies chromosomal polymorphism is a manifestation of quick genome reaction to harsh antarctic conditions. whereas the results of molecular-genetic analysis and study of cell cultures of this species suggests on the relative stability of d. antarctica genome. p-02.08.5-043 angiotensin converting enzyme inhibitory activity of morchella esculenta (l.) pers a. zeytunluoglu 1 , i. arslan 2 1 biomedical equipment technology, pamukkale university, denizli, turkey, denizli, 2 biomedical engineering, pamukkale university, denizli, denizli, turkey hypertension is a multi-aetiological, chronic pathophysiology that leads to multi-organ dysfunctions like cardiovascular diseases, strokes, and renal complications. natural extracts play an important role in traditional medicines for the treatment of hypertension and are also an essential resource for new drug discovery. mushrooms are use as therapeutics in alternative and complementary medicine as functional food because of contain a large number of biologically active components that offer health benefits and protection against many degenerative diseases. morchella esculenta is one of the most highly priced edible mushrooms worldwide. it contains a wide range of active constituents which include tocopherols, carotenoids, organic acids, polysaccarides and phenolic compounds which exhibit a wide range of medicinal and pharmacological properties including anti-microbial, anti-inflammatory, immunustimulatory, antitumor and antioxidant. in this study; the in vitro angiotensin converting enzyme-i (ace-i) inhibitory activity of m. esculenta peptides were generated by alcalase hydrolysis were studied. the 5 kda < peptides < 10 kda in the ultrafiltration fractions displayed highest ace inhibition (85.9 ae 5.09% at 140 lg/ml). the results indicate that m. esculenta derived peptides may have potential as functional food ingredients in the prevention and management of hypertension. p-02.08.5-046 modulations of antioxidant enzymes, gsts and catalase, by salvia absconditiflora in hepg2 cell line d. irtem kartal 1 , a. altay 2 1 yuzuncuyil university, van, 2 erzincan € university, erzincan, turkey oxidative stress is considered to play a important role in the pathogenesis of aging and several degenerative diseases, such as cancer. in order to cope with an excess of free radicals produced upon oxidative stress, humans have developed several mechanisms for maintain redox homeostasis. these protective mechanisms either scavenge or detoxify ros, block their production, and include enzymatic and nonenzymatic antioxidant defenses. in enzymatic defenses include glutathione s-transferases and catalase enzymes. many epidemiological studies have revealed that there is a strong correlation between consumption of polyphenol-rich foods and the prevention of certain diseases like cancer, cardiovascular diseases and aging. phenolic compounds are abundant in all plants. so, they form an integral part of the human diet. salvia species, commonly known as sage, have been used since ancient times for more than 60 different ailments ranging from aches to epilepsy. there are around 900 species of salvia, 95 of which are represented in turkey including salvia absconditiflora. in this study, s. absconditiflora collected from metu campus (ankara, turkey) is extracted with methanol and water. effects of the water and methanol extracts on the mrna expressions of antioxidant enzymes gstm1 and catalase in hepg2 cells were investigated by q-rt-pcr technique. it was also monitored the effects of the extracts on the enzyme activities of gsts and catalase by spectrophotometrically. water and methanol extracts decreased gsts mrna expression in hepg2 cells for 48 hours and 72 hours incubation and methanol extract decrease catalase mrna expression for only 72 hours incubation. on the other hand, extracts highly increased the gsts and catalase activities in both hour incubation. overall, these results indicate that s. absconditiflora and/or its components have regulatory activities on antioxidant enzymes and they may have a potential as a therapeutic agent in the treatment of cancer. p-02.08.5-047 transcriptomics and proteomics approach to drought stress mechanism in wheat b. cevher keskin tubitak marmara research center genetic engineering & biotech. institute, kocaeli, turkey birsen cevher keskin, yasemin yıldızhan, oktay kulen, bayram y€ uksel, selma onarıcı, _ ismail t€ urkan, as ßkım hediye sekmen, u gur sezerman, bu gra € ozer identification of novel stress-responsive genes and their role in drought response is an important area for the improvement of the crops. drought-related genes were investigated in leaves and roots of three wheat genotypes after different drought stress treatments by rna sequencing (rna seq) technology and de novo assembly was performed before comparative transcriptome analysis. analyzing 311 gigabases of 100 bp paired end illumina reads from a hexaploid wheat poly(a) rna library, we identified common and new differentially expressed transcripts. selected differentially expressed genes were confirmed by qrt-pcr. we also performed root proteome analysis with nano electrospray ionization source coupled to a high-performance liquid chromatography system (nanouplcàesiàqtofàms) to identify drought-related proteins. totally 191 proteins were differentially expressed in root tissues of tolerant and non-tolerant wheat genotypes. responses of antioxidative defense system to drought stress were comparatively studied in the same wheat cultivars. similarities between protein and rna levels help increase our confidence in novel biomarkers, differences may also reveal other post-transcriptional regulatory junctures. all these analyses will allow us to get a better idea about the possible role of these genes in the drought-response mechanism. the drought-related genes that are functionally characterized could be introduced into agronomically important wheat cultivars. this work offers a resource for accelerating drought-related gene discovery and improving this important crop. p-02.08.5-048 isolation and characterization of a hexose converter from olive s. altunok 1 , e. d€ undar 2 1 department of biology, university of balikesir, balikesir, 2 department of molecular biology and genetics, college of arts and sciences, balikesir university, balikesir, turkey introduction: hexose sugars are key components of glycolysis and photosynthesis. the genes regulating their conversion into one another, is therefore, of great importance for the control of carbon metabolism. in this study, we report isolation and characterization of a cdna associated with conversion of hexoses in olive. the cdna putatively named aldolase based on bioinformatic and experimental analyses. material-methods: characterization based on nucleotide and amino acids were conducted using bioinformatic tools such as nucleotide and protein blast, bioedit, primer3, finchtv, clc genomic workbench, expasy, targetp, sosui and web promoter scan. comparison of the genomic and cdna sequence of the gene and detailed bioinformatic analyses including cellular location, hydropathy analysis, amino acid-nucleotide composition and predicted 3d structure were also conducted using the bioinformatics tools mentioned above. temporal expression pattern of the putative aldolase were conducted using real-time pcr experiments. sds and western blot analyses were completed while biochemical analyses are ongoing. oleocanthal is an important secondary metabolite that has been reported to be useful against important human diseases including cancer. the aim of this study was to identify and characterize the key biochemical and genetic components of oleocanthal biosynthesis. to determine the biochemical components of the pathway, multiple olive cultivars along with their spatial and temporal points were determined. the expression levels of multiple candidate genes were also aimed via real-time pcr. nucleotide blast and protein blast (for comparison of the similarity of the candidate genes with that of other organisms) were conducted on ncbi web page. phylogenetic tree construction, amino acid composition analysis, nucleotide composition analysis, hydropathy analysis and translations through expasy were conducted. primer3 was used to design forward and reverse primers to amplify the target genes from different olive tissues at different times. analysis of the first candiate gene with bioedit program revealed that a+t ratio was more than g+c according to the nucleotide composition analysis. according to amino acid composition analysis isoleucine, lysine and leucine were more than other amino acids while kyte&doolittle hyddropathy analysis revealed that the protein was hydrophilic. abundance of hydrophobic amino acids (leucine and isolecine) along with an abundant hydrophilic amino acid (lysine) suggest the existance of hydrophobic pockets in the protein which may mean a membrane bound protein or a sitoplasmic protein with a strong hydrophobic core. the molecular weight of the protein was 56 kda with a pi of 9.21. the protein was found to have a signal peptide. according to the sosui gramn , intracellular localization was found to be in the inner membrane. analysis of other candidate genes contiunes. acknowledgements: this study was supported by tubitak with grant number 110o108. key words: olive, olea europaea l., secologanin, polymorphism, allele diversity p-02.08.5-050 antioxidant potentials of propolis and its bioactive components, and their effects on cyp2e1 gene expression in ht-29 adenocarcinoma cell line a. altay 1 , d. irtem kartal 2 1 erzincan € university, erzincan, 2 y€ uz€ unc€ u yil university, van, turkey propolis is a resinous mixture that is collected by honeybees from plants, and is combined with beeswax and secretions from the bee's salivary glands plus some pollen. it is a rich mixture of polyphenols, flavonoid aglycones, phenolic acids and their esters. it has been used in traditional medicine for thousands of years because of these ingredients. propolis, just like honey, has been the subject of many studies due to its antimicrobial, antifungal, antiviral and hepatoprotective activities. the cytochrome p450 enzymes are involved in phase i xenobiotic and drug metabolism. cyp2e1 is present in high levels in human tumors demonstrated by qrt-pcr and immunohistochemical studies. in this study, propolis collected from datc ßa (mugla, turkey) is extracted with 70% ethanol. phenolic contents of the propolis were measured by lc-ms/ms. cytotoxic effects of the propolis on ht-29 human colon adenocarcinoma cell lines were examined via xtt colorimetric cell proliferation assay. effects of propolis extract and its main bioactive component caffeic acid on the expression of phase i detoxification enzyme cyp2e1 in ht-29 cells were investigated bu using q-rt-pcr technique. ic 50 values for dpph radicals scavenging activities of the extract was calculated as 0.042 mg/ml. tpc and tfc were determined as 168.6 mg gae/g extract and 141.8 mg qe/g extract, respectively. caffeic acid content of the extract was measured as 10.6 lg/g extract. ic 50 values for xtt assay in 48 hours incubation with the extract and caffeic acid were evaluated as 1.16 mg/ ml and 0.149 mg/ml, respectively. propolis extract and its main phenolic content caffeic acid significantly dicreased cyp2e1 mrna expression with 2.4 and 5.3 fold, respectively in ht-29 human colorectal adenocarcinoma cells for 48 hours incubation. overall, these results indicate that propolis and/or its components have regulatory activities on cyp2e1 expression and they may have a potential as a therapeutic agent in the treatment of cancer. p-02.08.5-051 effects of histone h3 lysine 9 inhibition on gene expression profile in tobacco (nicotiana tabacum l.) differentiation is the characteristic of multicellular organism. cellular dedifferentiation underlies topical issues in biology such as reprogramming in stem cell research, regeneration and nuclear cloning, and has common features in plants and animals. the state of dedifferentiation is evidenced by changes in cell morphology, genome organization and the pattern of gene expression as well as by the capability of plant tissues to differentiate into multiple types of cells depending on the type of stimulus applied. chromatin reorganization appears to be a fundamental theme in cellular dedifferentiation and reentry into the cell cycle both in plants and animals. the chemical modifications of histone proteins which are structural units of the nucleosome, generate 'codes' for the recruitment of proteins or protein complexes that affect chromatin structure and gene expression according to 'histone code' hypothesis. methylation of histone h3 at lysine 9 residue by specific methyltransferases suv39h1 in humans and kyp/suvh4 in arabidopsis generates a'code' for the recruitment of hp1 proteins. in this study, to enhance the dedifferentiation efficiency, chaetocin which inhibits suv39h1 has been used at the germination stage of tobacco seeds in in vitro conditions. our results showed that chaetocin induced callus formation from leaf discs of tobacco in the early stage of the inhibitor application. chaetocin can enhance reprogramming of plant cells in seed development treatment as callus induction. it is known that the formation of callus which is the non-differentiated cell community in plants, is a consequence of the changes in the gene expression profile. it has been found that epigenetic modifications play a crucial role in some of these changes. the definition of the genes related to callus formation by the inhibitation of an epigenetic modification -h3k9 methylation-in tobacco might be used to bear on various dedifferentiation-driven cellular processes. induction of tumor cell death by the use of some phytochemicals that consumed through diet, and derived from medicinal plants opens up new horizons for cancer treatment researches. rheum ribes species, which is studied in this research, is one of the commonly used herbs in pharmacological researches. the high content of phenolic compounds in r. ribes extracts were known to be responsible for the high antioxidant and antibacterial activities. our aim in this study is to assess cytotoxic and apoptotic changes by way of implementing methanol extract of the rheum ribes (root) to the mcf-7 breast cancer cell line. cytotoxic effect of rheum ribes extract was evaluated by using the xtt (2,3-bis(2-metoksi-4-nitro-5-sulfofenil)-2h-tetrazolyum) test. in order to determine the dose of ic50, plant extracts were applied as time and dose dependent in the range of 10-500ug. in 72nd hour, the ic50 value is determined as 400ug. to examine the apoptotic effects of the extract, total rnas were isolated from dose group and the control cells firstly, then cdnas were synthesized. expression profile of the target genes(caspase-3, caspase-7, caspase-8, caspase-9, bax, bcl-2, fas) are determined by qpcr. according to the results, when the control group compared with the cells, it was determined that, while 17.33 and 3.05-fold respectively increase in the gene expressions of caspase-3 and caspase-7 of dose group cells, 15.45-fold decrease in the gene expressions of bcl-2. no significant difference was observed in the other genes examined. based on the obtained data, we believe that methanol extract of the rheum ribes induces apoptosis by activating intrinsic pathway. as a result, this plant species can be a new and effective therapeutic candidate for the medical world in search of alternative anti-cancer approaches, and could shed light on the work to be done in this area. p-02.08.5-053 the effect of ferulic acid and 5-fluorouracil combination on apoptosis in pc-3 human prostate cancer cells prostate cancer is quite often seen in industrialized countries and has the second most common death rate due to cancer after lung cancer in men. 5-fluorouracil (5-fu) is a pyrimidine analog and cell cycle-targeting drug by inhibiting dna synthesis. it has been widely used for treatment of several cancers such as gastric, colorectal, and breast cancers. phenolic compounds found in foods are potential antioxidants of harmful oxidative processes related to cancer and also important due to induction of different mechanism such as apoptosis. ferulic acid (fa; 4-hydroxy-3-methoxycinnamic acid), a phenolic compound, is abundant in fruits and vegetables. the purpose of this study was to investigate combination effect of fa and 5-fu on apoptosis in pc-3 human prostate cancer cell line. the effects of 5-fu, fa, and combination of both of them on cell viability were determined by xtt method. total rna isolation was conducted using trizol reagent. expressions of important genes in apoptosis including casp3, casp7, casp8, casp9, bcl2, bax, fas and cycs were investigated in four groups by qpcr. the ic 50 doses of fa and 5-fu were found to be 300 lm and 60 lm for 48 hours in pc-3 cells, respectively. in order to determine combination effect, pc-3 cells were treated with <ic 50 doses (200 lm fa and 40 lm 5-fu). according to qpcr results, a significant increase in the expressions of bax and cycs genes and a decrease in the expression of bcl2 were observed in the fa group, compared with the control group cells. after the treatment with 5-fu, the expression of casp8 was significantly increased compared with the control group. furthermore, combination of fa and 5-fu significantly increased expression of casp7, casp8, fas and cycs genes. in conclusion, comparing with the single treatments, it was observed that combination of fa and 5-fu affected expression of different genes in apoptosis in pc-3 cell line. p-02.08.5-054 combination of ferulic acid and gemcitabine affects expression of some apoptotic genes in pc-3 human prostate cancer cells prostate cancer, the second causing of cancer-related death in men, is an important cancer type due to the late age of onset, slow the progression, high incidence. gemcitabine is an effective agent in several cancer types including non-small cell lung cancer, pancreatic, bladder and breast cancer. it is known that gemcitabine is used single agent or as a part of combination treatment in prostate cancer therapy. nowadays, new treatment methods have been tested for cancer therapy including natural compounds with low toxicity. ferulic acid (fa) one of these agents, an abundant phenolic compound found in various fruit and vegetables. in this study, objective was to investigate combination effect of ferulic acid and gemcitabine on apoptosis in pc-3 human prostate cancer cell line. cell viability was determined by using xtt method after the treatment with gemcitabine, fa and combination of both of them. total rna was isolated with trizol reagent. expressions of casp3, casp7, casp8, casp9, bcl2, bax, fas and cycs genes are important in apoptosis, were evaluated in four groups by qpcr. the ic 50 doses of fa and gemcitabine were found to be 300 lm and 50 lm for 48 hours in pc-3 cells, respectively. for determination of combination effect, pc-3 cells were treated with <ic 50 doses (200 lm fa and 35 lm gemcitabine). when compared with the control group, qpcr results illustrated, a significant increase in the expressions of bax and cycs genes whereas a decrease in the expression of bcl2 gene in the fa treatment group. after the treatment with gemcitabine, the expression of casp3 and fas genes were significantly increased compared with the control group. furthermore, combination of fa and gemcitabine significantly increased expression of casp3, casp7, casp8, fas and cycs genes. in conclusion, it was observed that combination of fa and gemcitabine affected different genes in apoptosis compared with the single treatments in pc-3 human prostate cancer cell line. pistacia lentiscus linn. is widely distributed in mediterranean europe, morocco and turkey. the resin part of its known as mastic gum and plant called as mastic tree. it has been referred to over centuries as having medicinal properties to treat a variety of diseases. the aim of the present study are to evaluate antioxidant, cytotoxic and antimicrobial activities of heptane, chloroform and methanol extracts from p. lentiscus leaves and mastic gum. the antioxidant activities of chloroform and methanol extracts were assayed with various methods, including tpc, dpph free radical and abts radical cation scavenging activity. also, cytotoxicity of extracts was evaluated and screened against human mcf-7, hela, a549, and ht-29 cancer cell lines and nih-3t3 cells. the viability of cells in 100 lg/ml concentration of extracts was evaluated using mtt assay. antimicrobial activity of extracts were investigated against s aureus, s epidermidis, e coli, p aeruginosa, p vulgaris, k pneumoniae, c albicans, c glabrata, c guilliermondii, c tropicalis, c parapsilosis test organims by the agar well diffusion and broth microdilution methods . according to our results, the methanol extract of p. lentiscus leaves showed the highest dpph free radical scavenging activity (ic50 = 0.16 ae 0.02 mg/ml) and abts radical cation scavenging activity (1.33 ae 0.06 mg/ml). this study also exhibited that methanol extract of p. lentiscus leaves had the highest tpc (126.7 ae 5.7 mg gallic acid equivalents/g extract). the hexane extract of mastic gum showed antifungal activity against all of the tested candida species (6-16 mm / <0.25-8 mg/ml). the methanol extracts of p. lentiscus leaves showed the highest antimicrobial activity against all of the tested bacteria except k pneumoniae strain (8-14 mm/>256 mg/ml). on the other hand, p. lentiscus showed no cytotoxicity against cancer and normal cells. the results suggested that p. lentiscus may be natural source of antioxidant and antimicrobial activities. p-02.08.5-056 antibacterial activity of and chemical composition of alcoholic extract of marjoram against some human pathogens m. ahanjan, m. rahbar mazandaran university of medical sciences, sari, iran herbs enjoy a unique value and importance in sustaining healthy communities in terms of disease prevention (1) . in this regard, marjoram is a plant of the mint family which has antibacterial properties on microorganisms (2) . the current study aims to investigate the anti-microbial activity of the alcohol extracts (i.e., methanol or ethanol) of marjoram plants on the bacteria of staphylococcus (atcc: 25923), aureus e. coli (atcc: 25922), and salmonella enterica (atcc: 13076) and p. aeroginosa through utilizing disk diffusion method. also, the minimum inhibitory concentration and the minimum bactericidal concentrationwere measured through tube. the measurement of minimum inhibitory concentration and minimum bactericidal concentration of ethanol and methanol extracts on e.coli were equal with 100 and 120 milligrams per milliliter, subsequently. moreover, the measurement of the minimum inhibitory concentration and of the minimum inhibitory concentration of marjoram ethanol extraction on staphylococcus aurous was reported to be 90 milligrams and 100 milligrams per milliliter, subsequently. in addition, the amount of ethanol and methanol extracts on salmonella enteric and p. aeroginosa was equal with 80 and 90 milligrams per milliliter, subsequently. the results showed that marjoram alcoholic extract enjoy antibacterial properties. also, among the alcoholic extracts, the ethanol extract has demonstrated to be the most effective extract on salmonella enterica and e. coli and p. aeroginosa. p-02.08.5-057 molecular analysis of serine/arginine rich sc35 splicing factor from olive b. bas 1 , e. d€ undar 2 1 depertmant of biology, university of balikesir, balikesir, 2 department of molecular biology and genetics, college of arts and sciences, balikesir university, balikesir, turkey olive (olea europaea l.) is an evergreen fruit tree adapted to mediterranean climate and rich in tannins, essential oils and organic acids. serine/arginine rich splicing factors are essential in seqeunce specific splicing of pre-mrnas. in this study we report molecular characterization of an serine/arginine rich sc35 splicing factor (oesarsc35sf) that was isolated from a cdna library constructed from olive pedicels. nucleotide blast and protein blast (for comparison of the similarity of the candidate genes from other organisms) were conducted on ncbi web page. amino acid composition analysis, nucleotide composition analysis, hydropathy analysis, open reading frame determiantion, through, molecular weight and the isoelectric points calculations were conducted using online expasy software. primer3 was used to design forward and reverse primers to amplify the target gene from different olive tissues at different times. analysis with bioedit program revealed that a+t ratio was more than that of g+c. oesarsc35sf was a protein consisting of 267 amino acids. as expected, amino acid composition analysis revealed that serines and arginines were more than other amino acids. kyte&doolittle hyddropathy analysis revealed that the protein was hydrophilic. the molecular weight of the protein was 30 kda with an isoelectric point (pi) of 11.5. the protein was found to have a signal peptide. according to the predotar analysis results, intracellular localization was found to be in the mitochondrial. the combined results suggest oesarsc35sf might function as splicing factor as its homologs from the other plants. confirming this hypothesis with futher experimental characterization including biochemical function analysis continues. acknowledgements: this study was supported by tub _ itak with grant number 110o108. keywords: olea europaea l., oesarsc35sf, alternative splcing, pre-mrna splicing, bioedit, pedicel specific cdnas. p-02.08.5-058 application of three-phase partitioning for the purification of peroxidase from kiwano (cucumis metuliferus) z. denli 1 , g. arabaci 2 1 kto karatay university faculty of medicine, konya, 2 faculty of arts and sciences, sakarya university, sakarya, turkey peroxidases are enzymes able to catalyze reduction of h 2 o 2 and oxidize various substrates. the kiwano is an oval shaped fruit which has an orange skin with lots of tiny horns. in this study, peroxidase isolated from kiwano is purified with three-phase partitioning (tpp). kiwano fruit was homogenized and obtained crude enzyme extract. the extract was saturated with 50% (w/v) ammonium sulfate ((nh 4 ) 2 so 4 ) and added t-butanol with the ratio of 1:1.5 (v/v). the lower and interfacial layer was collected. the influence of percent saturations of (nh 4 ) 2 so 4 (50, 65, 75, 85, 95%) and tbutanol ratios (1:0.5, 1:1, 1:1.5, 1:2) to the partitioning behaviour of peroxidase were analyzed. after dialyzed, the interfacial and lower phases were measured for peroxidase activity and protein content. the protein pattern of the peroxidases was evaluated by using gel electrophoresis. peroxidase activity recovery and the purification fold of interfacial and lower phases were 117.4, 1.7% and 0.84, 0.03. therefore, other experiments were continued with interfacial phase. at constant t-butanol with the ratio of 1:1.5, the enzyme activity recovery and purification fold of interfacial phase for saturations of (nh 4 ) 2 so 4 (50, 65, 75, 85%) were 40.9, 43.2, 39.2, 135% and 0.37, 0.31, 0.4, 0.91 . the interfacial phase was not dissolved in 95% (nh 4 ) 2 so 4 . at constant 85% (nh 4 ) 2 so 4 , the enzyme activity recovery and purification fold of interfacial phase for t-butanol ratio (1:0.5, 1:1, 1:1.5, 1:2) were 61.6, 48. 1, 127.6, 126.1% and 1.79, 0.6, 1.84, 1.41 . finally, at optimum conditions (% 85 (nh 4 ) 2 so 4 , t-butanol 1:1.5) after dialyzed interfacial phase, the enzyme activity recovery and the purification fold were 138.8% and 4.46. the results of gel electrophoresis showed that the molecular weight of enzyme was between 30-60 kda. the applications of tpp gave the maximum recovery of 138.8% and 4.46-fold purification. as a result, for purfication of peroxidases, tpp is a rapid, simple and economical technique. accumulating the most robust genes and proteins in elite genotypes without any harmful effect on the potential plant yield is an urgent need to enhance productivity under various stressors. among the stressors, drought is a major challenge for agricultural productivity. brachypodium distachyon, with its close relationship to agriculturally and economically important crops, is an important model plant species. although ongoing transcriptomic analyses in brachypodium distachyon available, proteomic analyses are required to obtain an integrated picture of drought response. in the current study, a comprehensive proteome analysis was conducted on brachypodium leaves under increasing levels of drought stress. to screen gradual changes upon drought stress, brachypodium leaves subjected to drought treatment for 4, 8 and 12 days were collected for each treatment day. the cellular responses were investigated through a proteomic approach involving two dimensional difference gel electrophoresis and subsequent combined tandem mass spectrometry. for the validation of transcriptional expression, the genes encoding selected proteins were examined through quantitative real-time pcr. spot detection on cye-dyed gels revealed a total of 497 distinct spots in brachypodium protein repertoire. a total of 13 differentially expressed proteins (deps), with at least 2-fold changes in abundance, were identified by mass spectrometry and classified according to their functions. the biological functions of deps included roles in photosynthesis, protein folding, antioxidant mechanism and metabolic processes, highlighting the significant degree of overlapping between metabolic alterations induced by drought stress. identified proteins in this study and understanding the molecular mechanisms of drought response and defense mechanisms in plants will contribute to the researches on development of drought tolerant crop species. p-02.08.5-060 immunohistochemical and electron microscopy investigation of tmv-based chimeric virus particles carrying conserved influenza antigen in nicotiana benthamiana recently we obtained and partially characterized set of viral vectors based on tobacco mosaic virus (tmv) genome displaying conserved influenza a m2e epitope from matrix m2 protein. purified chimeric virus particles (cvp) conferred protection of mice against lethal homologous and heterologous influenza virus challenge. we revealed significant difference in symptoms of infections caused by tmv-m2e recombinant viruses containing cysteine (cys) to serine (ser) or alanine (ala) substitutions in the human consensus m2e sequence. accumulation level of m2e-ala recombinant coat protein was significantly higher than m2e-cys/ ser (ratio 5:1). tmv-m2e-ser infection, in contrast to ala mutant, suspended growth and development of nicotiana benthamiana. non-inoculated leaves (14 d.p.i.) were fixed with ethanol and histological sections were incubated with mouse serum to m2e and secondary antibodies conjugated with either hrpo or fitc. cvps of all three mutants were detected in epidermal and stomata cells as well as in sieve elements and minor veins. electron microscopy analysis of mesophyll cells revealed typical rigid helical particles. cys/ser mutants mostly accumulated within ground cytoplasm as aggregates of discrete tubules in parallel arrangement, which were not delimited by lipid membranes. we discovered huge amount of cvps in the cytoplasm and lesser amount diffused in the central vacuole. essential part of ala particles was located in the cytoplasm, but mentioned aggregates were not found and only insignificant number of virions was revealed in vacuole. unlike wild-type tmv, none of the mutants was revealed in chloroplasts. diameters of cvps were as follows: sersingle particles in cytoplasm 13 ae 1. cyanidin is a natural anthocyanidin found in a variety of fruits (grapes, blackberry, blueberry, cherry and cranberry etc.) and vegetables (red cabbage, red onion). this polyphenolic compound is a flavonoid with significant antioxidant activity. cyanidin and its glycosides have vasoprotective effects and can interfere with inflammation, carcinogenesis, obesity, and diabetes. one important role of the macrophages is the release of pro-inflammatory mediators, such as nitric oxide, various cytokines, in response to activation signals, including chemical mediators, cytokines, and bacterial lipopolysaccharide (lps). in this study, we investigated the role of cyanidin chloride in inflammation. anti-inflammatory effects of cyanidin chloride were examined in lps-stimulated murine raw 264.7 macrophages. we observed the level of various inflammation markers such as nitric oxide (no), inducible no synthase (inos), cyclooxygenase-2 (cox-2), tumor necrosis factor-a (tnf-a) and interleukin-8 (il-8) under lps treatment with or without cyanidin chloride. cyanidin chloride inhibited not only no production but also the expression of cox-2 and inos, without any cytotoxicity. cyanidin chloride also attenuated pro-inflammatory cytokines and other inflammation-related markers such as il-8 in a dosedependent manner. in conclusion, cyanidin chloride may be beneficial for the prevention and treatment of anti-inflammatory diseases. p-02.08.5-062 the investigation of centranthus longiflorus plant extacts effects on cell proliferation and apoptosis activity in the cell lines of mcf-7 breast cancer h. askin ataturk university, erzurum, turkey introduction: in the u.s., breast cancer is the second most common cancer in women after skin cancer. current treatment of cancer can be done by surgery, chemotherapy, and radiation therapy. in addition, there is widespread use of complementary and alternative medicine in developed countries. plants and plant extracts play a critical role in the research into new anticancerogenic agents. centranthus longiflorus (cl) is used in alternative medicine for sedative and antispasmodic purposes. a plant of turkish origin, centranthus longiflorus used as traditional turkish medicine have remained uninvestigated for familial hypercholesterolemia, diabetes, coronary artery disease and cancer for their in vitro biological activity despite their use for sleep disorders. in this study, growth-inhibiting and pro-apoptotic effects of hexane, ethyl acetate and ethanol extracts of cl in mcf-7 breast cancer cell line were investigated. material and method: aerial parts of cl were collected in erzurum province. hexane, ethyl acetate and ethanol extraction were done by soxhlet extractor. the plant extracts obtained from cl was analyzed using a gc-ms system. dose-and time-dependent cytotoxic and apoptotic effects of cl were evaluated by mtt cell proliferation kit and cell death detection elisa kit, respectively. manufecturer's protocol was followed for analyses. then, apoptotic genes; caspase3, bax and p53 and antiapoptotic genes; bcl-2 and pi3 expression levels were determined by rt pcr. results: according to our results, cytotoxic effect on mcf-7 cell was only observed in 20 and 50 lg/ml doses of cl. however, any of the application doses showed an apoptotic effect on mcf-7 cells. they exhibited a necrotic effect rather than the apoptotic effect. although alterations in expression levels of these genes were determined, this alterations was statistically insignificant. discussion and conclusion: consequently, we can say that cl have a cytotoxic effect on mcf-7 breast cancer cell lines. p-02.08.5-063 reduction of the chloroplast genome and the loss of photosynthetic pathways in the mycoheterotrophic plant monotropa hypopitys, as revealed by genome and transcriptome sequencing e. gruzdev, a. mardanov, a. beletsky, v. kadnikov, e. kochieva, n. ravin, k. skryabin institute of bioengineering, research center of biotechnology of the russian academy of sciences, moscow, russia genomes of parasitic plants represent interesting model systems to study effects of relaxed selective pressure on photosynthetic function. previous genomic studies of nonphotosynthetic plants revealed reduction of their chloroplast genomes, but the corresponding changes in their nuclear genomes are less known. here we present the data on the transcriptome and the chloroplast genome of the non-photosynthetic mycoheterotrophic plant monotropa hypopitys. the chloroplast genomes were sequenced for two specimens of m. hypopitys, collected in different regions of russia. the cpdnas are 34,800 bp (mon-2kalr) and 35,336 bp (mon-1volr) long and rearranged with respect to each other. both genomes contains genes encoding ribosomal proteins, infa, matk, and 4 ribosomal rna genes. 17 and 19 trna genes were predicted in two cpdna. genes encoding nadh dehydrogenase, plastid rna polymerase, all genes related to photosynthetic apparatus, clpp, ycf1, ycf2, accd, and some genes for ribosomal proteins are missing or became pseudogenes. the reduction of gene content is associated with extensive gene order rearrangement and the lack of inverted repeats. overall, the size and gene content of m. hypopitys cpdna indicates that it is close to the end of plastid genome degradation process. in order to get insights into the changes in the nuclear genome associated with the transition to nonphotosynthetic lifestyle, we sequenced and assembled the transcriptome of m. hypopitys. as expected for holoparasites, we did not found transcripts for the nuclear genes encoding the components of photosynthetic machinery, including photosystem i and ii, cytochrome b6f complex, and ribulose bisphosphate carboxylase. contrary to the holoparasitic plant phelipanche aegyptiaca, almost all genes of chlorophyll biosynthesis pathway from protoporphyrin ix were not found in the m. hypopitys transcriptome. this work was supported by the rsf grant 14-24-00175 and rfbr grant 14-14-00749 (mon-1volr cpdna sequencing). introduction: beta-sitosterol is a substance found in plants. chemists call it a plant sterol ester. it is found in fruits, vegetables, nuts, and seeds. it is used to make medicine. beta-sitosterol is used for heart disease and high cholesterol. the federal food and drug administration (fda) allows manufacturers to claim that foods containing plant sterol esters such as beta-sitosterol are for reducing the risk of coronary heart disease (chd). centranthus longiflorus (cl) is used in alternative medicine for sleep disorders. a plant of turkish origin, cl used as folk medicine have remained uninvestigated for familial hypercholesterolemia, coronary artery disease and preventing colon cancer for their in vitro biological activity despite their use for sleep disorders. we investigated of the chemical constituents from dried aerial parts of centranthus longiflorus. material and method: aerial parts of cl were collected in erzurum province. hexane, ethyl acetate and ethanol extraction were done by soxhlet extractor. the plant extracts obtained from the aerial parts of cl was analyzed using a perkin-elmer gc-ms system. results: ten compounds were obtained and identified as butanoic acid, hexadecanoic acid (palmitic acid), 7-methyl-z-tetradecen-1-ol acetate, octadecanoic acid (stearic acid), diisobutyl phthalate, 9-octadecenamide, octacosane, nonacosane, alfa amyrin and beta sitosterol. the latter two were obtained in all extraction (hexane, ethyl acetate and ethanol). discussion and conclusion: all of these compounds are isolated from centranthus longiflorus for the first time. these findings may shed light on the design of new drugs, the cholesterollowering effect. p-02.08.5-065 role of lutein for the high light-induced inhibition of photosystem ii related reactions in thylakoid membranes of arabidopsis thaliana, wt and lut2 k. dobrev, d. stanoeva, m. velichkova, a. v. popova institute of biophysics and biomedical engineering, bulgarian academy of sciences, sofia, bulgaria photosynthetic reactions taking place in thylakoid membranes of higher plants are extremely sensitive towards different environmental stress conditions such as high and low temperature, high light intensity, uv radiation etc. carotenoids are intrinsic component of photosynthetic pigment-protein complexes and are involved in performing multiple important functions. their role of accessory pigments in absorbing sun light, participation in photoprotection via dissipation of excess absorbed light, deactivating of stress-induced reactive oxygen species and structural role are well documented and recognized. the role of lack of lutein in high light-induced alterations in structural organization and functional activity of the main pigment-protein complexes was evaluated using isolated thylakoid membranes of arabidopsis thaliana, wt and mutant lut2, deficient in lutein, subjected to photoinhibitory treatment for different periods of time. alterations in photochemical activity of photosystem i and photosystem ii were determined by a clark-type electrode in the presence of exogenous electron donors and acceptors. activity of oxygenevolving complex and of the grana and stroma situated photosystem ii reaction centers was evaluated by determination of flash oxygen yields and initial oxygen burst under constant light without donors and acceptors. low-temperature (77k) fluorescence was applied for unraveling of light-induced alterations in energy transfer and interaction between the main pigment-protein complexes. maximal quantum efficiency of psii was registered by pulse amplitude modulated fluorescence method. results obtained are discussed in respect to the importance of lutein for the organization and sensitivity of photosynthetic apparatus towards high light intensity treatment. modern agriculture relies heavily on phosphate rock fertilizer to improve phosphorus availability in many soils, but this approach is not sustainable long-term. phytate (myo-inositol hexakisphosphate) is an organic phosphorus compound often present in many soils. however, phytate can not be utilized by most plants, and its accumulation in soil leads to substantial ecological problems. phytases are enzymes that hydrolyze phytate and release inorganic phosphate. many microorganisms such as bacteria and fungi synthesize highly diverse phytases which are suitable for plant biotechnology. generation of transgenic plants expressing phytases of bacterial origin has been proposed as one option to improve plant phosphorus nutrition. in this study, we generated and characterized transgenic arabidopsis thaliana plants expressing a modified phytase gene paphyc from pantoea sp. under strong camv35s promoter. three individual transgenic a. thaliana lines expressing the bacterial phytase gene, as well as negative control plants harboring the camv35s promoter alone were identified. expression of phytase in plants was verified at both transcription and translation levels. phytase-expressing plants grown on media with phytate as the sole source of phosphorus demonstrated better than wild-type growth rates, shoot dry mass, shoot phosphorus content, as well as higher phytase activity in cell-wall extracts. overall, we show that plants expressing bacterial phytase are capable of better growth on phytate as the only source of phosphorus in laboratory conditions. further research investigating the applicability of using bacterial phytase expression to improve plant growth in soil is necessary to evaluate the different routes of solving the phosphorus deficiency problem in agriculture. p-02.08.5-067 the role of elevated temperature in photoinhibition and recovery of photosystem ii in thylakoid membranes from arabidopsis thaliana a. faik, m. gerganova, m. velitchkova institute of biophysics and biomedical engineering, bulgarian academy of sciences, sofia, bulgaria photosynthesis of higher plants is the principle process to transform light energy into biochemical usable energy. in nature, plants are exposed to the environment where light, temperature, uv-b radiation varied and very often their extreme values that are unfavorable for effective performance of photosynthetic reactions. plant are developed various strategies to cope with stress including radical scavenging enzyme system, accumulation of protective compounds, etc. pigment-protein complexes of photosystem i and photosystem ii and their light harvesting antenna, situated within thylakoid membranes, are involved in the primary reactions of photosynthesis -absorption of light, charge separation and electron transport. photosynthetic process is sensitive towards higher than optimal temperatures, the photosystem ii and oxygen evolving complexes being extremely sensitive to elevation of temperature. in present work pam fluorescence was applied to evaluate the effect of long term action of elevated temperature (38/30°c) on the quantum yield of photosystem ii, non-photochemical quenching and rdf, the latter quantifying the photosynthetic process. in addition, the activity of oxygen evolving complex was determined polarographically in the presence of exogenous electron acceptor 1,4 -benzoquinone. sds-page electrophoresis and western blot were applied to determine the damage of d1 -reaction center protein of photosystem ii. alterations of mutual organization within photosystem ii complex and its antenna and of energy interaction between them were followed by analysis of 77k steady state chlorophyll fluorescence spectra. the simultaneous application of high temperature and high light intensity resulted in a well pronounced reduction of non-photochemical quenching that restore to the initial values after recovery for 5 days at optimal conditions. d1 was also restored while quantum efficiency of photosystem ii did not recuperate to initial values. p-02.08.5-068 reorganization of the main pigment-protein complexes in thylakoid membranes from tomato (solanum lycopersicum) during long term exposure to elevated temperature the changes of earth climate resulted in unfavorable environment for plants. depending on the duration of influence of stress factors, the response of plants includes short and long term acclimation. the population, structure and organization of pigmentprotein complexes within thylakoid membranes are dynamic and flexible, thus providing for the acclimation of the photosynthetic apparatus to the changed environment. the main pigment-protein complexes, involved in energy transduction, are photosystem i, photosystem ii and light harvesting complexes. they are separated in grana and stroma regions of thylakoid membranes but it is well established that they can rearrange as a result of alterations of light intensity, temperature increase and decrease in order to balance the perception and utilization of excitation energy. in present work the effect of long term action of elevated temperature on organization and stoichiometry of main pigmentprotein complexes in the thylakoid membranes from tomato plants (solanum lycopersicum cv. m82) was investigated. three weeks old tomato grown at optimal conditions (22/20°c day/ night temperature and light intensity 250 lmol/m 2 /s) plants were exposed for 2 and 6 days to elevated temperature at 38/30°c. by means of blue-native electrophoresis the effect elevated temperature on the populations of psii (dimmer and monomers) and lhcii (monomers and trimers) was estimated and compared with the same parameters for control plants. the ability of plants to recover from this treatment was checked after 5 days under optimal conditions. the changes of content of chlorophylls and carotenoids were determined at every stage of treatment. based on the results obtained it can be concluded that one of the mechanisms for regulating the energy balance and maintenance of efficient photosynthetic process involves a change in the organization and stoichiometry of the photosystem ii and oligomer state of light harvesting complex ii. aim of the study, was to evaluate the anti-tumor effects of silymarin, curcumin and propolis on leptin-induced mcf-7 cells. mcf-7 cells were incubated various concentrations leptin (physiological, obesity and pharmacologically doses; respectively 10, 100 and 1000 ng/ml) . then different doses of silymarin (10, 20, 40, 80 lm), curcumin (10, 20, 40, 60 lm) and propolis (0.063, 0.125, 0.25, 0.5 mg/ml) were added. after 24, 48, 72 and 96 hours incubation periods 3 different area images were taken digital camera. then using dye release reagent we determined the intensity of apoptosis via colorimetric determination by elisa reader. absorbance was directly proportional the number of apoptotic cells (biocolor cell-apo percentageapoptosisassay). also, we examined the effect of these natural products on proliferation rate of leptin-induced mcf-7 cells for 1, 2, 3 and 4 hours (biovision cell proliferation kit) all experiments were carried out 3 different days, at least 3 times. all of three compounds were stimulate the apoptosis at all time points and all different doses of leptin. the differences was statistically significant at the level of p < 0.05 between 24 and 48 hours. it was found that there were not seen much cells at 72 and 96 hours time points. we thought that most of the cells were gone necrosis instead of apoptosis. the best effective doses on apoptosis of propolis was 0.063 mg/ml, silymarin and curcumin were 20 lm. also, we evaluated the effects of on proliferation rate the mcf-7 cells, we found that only propolis was effective of inhibiting proliferation at all doses of leptin induced mcf-7 cells in 1 hour. we hope this study will be a guide for the further studies in anti-cancer agent development field and show that the natural origin substances cause cancer cells apoptosis and provide targeted treatment for cancer therapy. p-02.08.5-070 investigation of some lichen-derived substances' cosmetic potential for skin protection against ultraviolet b due to the depletion of the stratospheric ozone layer and chronic exposure, the occurrence of various skin diseases have been increased in recent decades. thence, people and cosmetic companies have progressively given more importance natural sunscreen products for the protection from harmful sun rays, especially ultraviolet b rays. we, therefore, isolated some lichen-derived substances; 3-hydroxyphysodic acid and protolichesterinic acid from hypogymnia tubulosa and cetraria aculeate, respectively. chemical characterization and identification of the isolated lichen substances were accomplished by using ftir, 1 h-nmr and melting point analyses. the theoretical uv-vis spectra and 3d conformations of the isolated compounds were determined by using the gaussian 03 software with hf theory at the b3lyp/3-21g level. the dark toxicities and ultraviolet b protection capacities of the substances were lighted up as previously described [1, 2] on hacat human keratinocyte cell line by using mtt cell viability and ldh cellular membrane degradation assays. the obtained results from the assays showed that protolichesterinic acid has a more dark toxic activity on keratinocyte cells than 3hydroxyphysodic acid, and the toxic activities were found sufficient as much as 80% at the highest doses of the substances; 400 lm. however, it was observed that the cytotoxicity of the substances were reduced at the rate of approximately 15% by the irradiation. consequently, we think that the substances block the ultraviolet b rays but their cytotoxic feature is an important limitation to their usage in cosmetic industry. references [1] varol, m., t€ urk, a., candan, m., tay, t., koparal, a. t. (2016) photoprotective activity of vulpinic and gyrophoric acids towards ultraviolet b-induced damage in human keratinocytes, phytotheraphy research 30:9-15. [2] varol, m., tay, t., candan, m., t€ urk, a., koparal, a. t. (2015) a great effort and financial supports have been consumed to explore and design novel sun protection factors due to the unhindered increase of malignant and non-malignant skin diseases caused by the chronic exposure and depletion of the stratospheric ozone layer. the testing of naturally produced compounds seems to be the best and inexpensive way to search for potentially photoprotective substances. on the other hand, as photo-resistant species, lichens are still poorly exploited. norstictic acid was, therefore, isolated from the acetone extract of pleurosticta acetabulum. ftir, 1 h-nmr and melting point analyses were performed to identify the chemical features of norstictic acid. gaussian 03 software with hf theory at the b3lyp/3-21g level was also performed to determine the theoretical uv-vis spectrum and 3d conformation of the isolated compound. the dark cytotoxicity and ultraviolet b-protection capacity of norstictic acid were comparatively tested as previously described [1, 2] by using mtt cell viability and ldh cellular membrane degradation assays. as a result of the experiments, it is observed that norstictic acid has a dark-cytotoxicity as less as 25% at the highest dose of the substance; 400 lm. however, ultraviolet b-induced damage on human keratinocytes was blocked by the lower concentrations of norstictic acid as 25, 50 and 100-lm, and 20% of cells were protected according to the control experiments of irradiated cells. consequently, we think that norstictic acid might be employed as a sun protection factor at the low concentrations, and further studies should be performed. years, researchers have focused on the lichen acids because of their biological activities. it is also suggested that lichens can be used as anticancer agents. vulpinic acid, an important lichen seconder metabolite, has antimicrobial activity and strong antimutagenic, anticancer and antioxidant capacity. nanotechnology has the potential to offer solutions to current obstacles in cancer therapies, because of its unique size (1-100 nm) and large surfaceto-volume ratios. so, in this study we aimed to determine the cytotoxic and proliferative effects of vulpinic acid and magnetic nanoparticles loaded with vulpinic acid (fe 3 there are four species of wild rice known around the world. zizania aquatica l., zizania palustris l. and zizania texana hitche are found in north america, whereas zizania latifolia (griseb) turcz) is native to east asia. cwr mainly grows in the areas along the yangzi river and the huai river in china without any cultivation and domestication. cwr was an ancient grain that has been used in chinese herbal medicine to treat a variety of ailments associated with nutrition, including gastrointestinal disorders and diabetes. our previous studies have demonstrated that consuming chinese wild rice can significantly improve blood lipid profiles and ameliorate high-fat/cholesterol diet-induced insulin resistance. however, compared to the well studied common dietary white rice, active composition and the associated proteomic information of chinese wild rice have yet to be investigated. in this study, we compared and analysed the different proteins between chinese wild rice and white rice by proteomics method. our study provides insights and experimental evidence for further exploration of this ancient medical food in disease prevention and therapy. the homology between cwr and n22 is 64%, but significant differences also exist between the two. we gained new insight by analyzing the biological function of the high reliability (credibility score 67 or higher, p < 0.05) peptide mass fingerprint of cwr2-de electrophoresis revealed differences in protein composition between cwr and n22. information obtained from the pmf indicates that glutelin precursor, caffeoyl coenzyme a (coa) omethyltransferase and putative bithoraxoid-like protein can provide gene evidences for its biological function. p-02.08.5-075 mir396 and growth-regulating factors interaction during maize leaf growth under low-temperature stress g. aktug, f. aydinoglu gebze technical university, kocaeli, turkey microrna (mirna) genes are a class of non-coding small rnas about 21 nucleotide-long which are revealed as regulators of plant growth and stress responses. the mirna mir396 targets and regulates growth-regulating factors (grfs) which are plant specific transcription factors family and this regulation machinery is conserved among plant species. plant growth is a result of cell division and expansion which took place as spatial gradient zones throughout maize leaf which are meristem, elongation and mature zones. cells proliferate in meristem, migrate to elongation zone and finally reach to mature zone to get its final size. it has been shown that mir396 affects cell division by regulating grfs and changes leaf size which are determined by cell number and cell size of leaf. this study aims to investigate the role of mir396 and grfs interaction during maize leaf growth under low-temperature stress. maize seedlings were grown under low-night temperature for stress treatment to generate growth retardation and control conditions as well to make comparative analysis. length of the third and fourth leaves of seedlings was measured every day and leaf elongation rate was calculated to observe stress effects on the leaves. growth zones of fourth leaves were harvested during steady-state growth phase for determining expression level of mir396s and their target by q-rt-pcr. we mined 8 mir396 genes sharing sequence similarity and 8 grf targets. the expression analyses of mir396s and grf5 are proceeding for different growth zones. in conclusion, this is the first study investigating the regulation network between mir396s and grf5 in different developmental stages of maize leaf under low-temperature stress. oeigpd cdna was isolated from a cdna library we constructed from olive pedicels. homology searches for nucleotide, amino acids and alternative open reading frames were conducted utilizing blastn, blastp, and blastx, respectively. nucleotide sequences of homologous genes from other plants were aligned using bioedit and the number of snps were detected. the alignment was then used to generate a phylogenetic tree using mega7 program. another alignment with amino acid sequences of the homologues proteins was also generated to construct a phylogenetic tree displaying oeigpd's position among other plants. various aspects of oeigpd including amino acid composition, hydropathy analysis, isoelectric point (pi) and three dimentional structure of the protein were determined using online software at expasy. multiple primer pairs to amplify the full length open reading frame of the gene, to clone the gene into the expressing vector plate51, and to detect expression through real-time pcr were designed using primer3. amino acid composition analysis revealed that oeigpd contained serine, arginine and isoleucine predominantly while hydropathy analysis suggested it was an hydrophilic protein. isoelectric point (pi) of the protein was calculated as 4.97. the molecular weight of the protein was calculated as 11 kda. analyses continue to determine the polymorphism of oeigpd among olive cultivars, and biochemical function of the gene in olive. p-02.08.5-077 cytotoxic effect of fractionated triterpenoid glycosides from holothuria polii in human cancer cells sea cucumbers are the members of class holothuroidea and they have more than 1100 described living species all around the world. sea cucumbers secrete special secondary metabolites from their body walls and they are called triterpene glycosides (tggs). in this study, cytotoxic activity of fractionated ttgs from h. polii on different cancer cell lines were carried out. h. polii delle chiaje, 1824 samples were collected from dikili-_ izmir. the semipurified extracts were fractioned by using hplc. four different fractions (fraction a-d) were obtained. in order to characterize the fractions, maldi-tof/ms was used. the cytotoxic activity of the fraction a-d were tested on ht-29, t84 and upci-scc-131 cell lines by using xcelligence rtca sp system. the cells were treated with three different concentrations of the fractions for 48 hours. the cell index data were compared with the control group. ic50 values of the fractions for three cell lines were calculated. according to the results, the fractions have holothurin a (1243.50 m/z), 24-dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer (1227.50 m/z). the fraction d was the most effective on all cell lines with ic50 value of 10.46 ae 0.18 mg/l, 11.24 ae 0.57 mg/l and 11.13 ae 0.29 mg/l for ht-29, upci-scc-131 and t84, respectively. in conclusion, sea cucumber ttgs are promising agents for colon adenocarcinoma, oral squamous cell carcinoma and colorectal carcinoma (metastatic) treatment. p-02.08.5-078 effect of horse-chestnut (aesculus hippocastanum) seed extract on matrix metalloproteinases during diabetic wound healing impaired wound-healing in diabetics is a major public health problem. the expression and activation of matrix metalloproteinases (mmps) are also impaired in diabetic wounds according to previous studies. their main function is to degrade the various components of the extracellular matrix. also, they participate physiological processes such as inflammation, angiogenesis, tissue remodeling. horse-chestnut seeds (hc) are rich in saponins and flavonoids. it has been shown that hc has antiinflammatory, antioedema, vessel protective, and free radical scavenging properties. the aim of this study is to determine with molecular signs on cutaneous wound healing effects of the ethanol (50%) extract of hc (hce) seed in rats by excision wound model. this study was conducted on diabetic wistar albino rats, which were injected by a single dose (50 mg/kg i.p.) streptozotocin. diabetic treatment rats were applied topically 15% (w/w) ointment with hce and control rats were applied topically simple ointment, once a day during the experimental period. the gene expression levels of mmp-1, mmp-9 by qpcr and levels of nitric oxide (no), hydroxyproline and malondialdehyde in wound tissue investigated at the end of 3rd, 7th, and 14th days. wound closure was also measured. the hydroxyproline and no levels were significantly increased in the hce treated group versus control after the 3rd and 7th days. the malondialdehyde levels were significantly lower in the treatment group. mmp1 gene (associated with collagen processing and reepithelialization) expression levels in hce treated rats were increased in the 7th day while it was reduced in 14th day. mmp 9 gene (associated with inflammation and gelatinase) expression levels in hce treated rats were decreased in 7th, and 14th days compared to the control. these findings indicate that hce accelerated the cutaneous wound healing process in diabetic rats via mmp1 and mmp9 regulation. p-02.08.5-079 isolation and molecular molecular characterization of vps39/vam6 from olive b. celikkaya, e. dundar molecular biology and genetic at balikesir university, balikesir, turkey vps39/vam6 promotes aggregating and fusing of endosomes and lysosomes. it is a component of a protein complex that is found in vacuole membranes. this gene has been studied from various organisms including humans, drosophila, arabidopsis and rice. no studies on olive vps39/vam6, however, have been reported. the aim of this study is to report information of olive vps39/vam6 including expression pattern and biochemical characterization. vps39/vam6 was isolated from a cdna library we constructed from fruited olive leaves in july. to determine the putative name of the cdna, blast analyses were conducted for nucleotide, open reading frame and amino acid sequence comparisons. bioedit program was used to determine the nucleotide and amino acid composition along with its molecular weight and isoelectric point (pi). hydropathy analysis was conducted using kyte and doolittle program. phylogenetic analysis was done using mega7. cellular localization of the product was predicted using sosui gramn. the three dimentional structure of the protein was calculated using i-tasser and compared to previously known structures using cn3d. the blast and bioedit analyses revealed vps39/vam6 had 836 base pairs coding 120 amino acids with a molecular weight of 13.38 kda, and pi of 6.39. the at/gc ratio was very high (1.5) comparing to its homologs from other plants suggesting to expect significant differences of this gene's function from the others. amino acid composition analysis revealed high rates of serine, leusine and isoleucine indicating a hydrophobic property of the protein. the hydrophobic feature was confirmed by kyte and doolittle analysis while the cellular location was revealed to be extracellular. the hydrophobic nature despite extracellular location suggests it is a membrane associated protein which was confirmed by transmembrane domain analysis. as expected no signal peptide was detected. the 3d structure of the protein was similar to its previously reported homologs. p-02.08.5-080 isolation and characterization of the ribosomal l1 protein from olive s. cinarli, e. dundar department of molecular biology and genetics, balikesir university, balikesir, turkey despite as a ribosomal protein, l1 is known as the inhibitor of the cellular aging gene and it has been reported to have roles in apoptosis. the ribosomal l1 protein is larger than the lsu of ribosome and contains 2 domains as 2-layered alpha/beta domain and 3-layered alpha/beta domain. in ribosomes, it functions in translocation and orientation of trnas. although the ribosomal l1 (rl1) gene has been studied in many plants, reports on olive rl1 (oerl1) are very rare. this study presents molecular characterization of rl1 gene from olive. oerl1 was isolated from a cdna library we constructed from unfruited olive leaves in july. homology analyses were conducted using blast programs. nucleotide and amino acid compositions, molecular weight, isoelectric point (pi) and at/gc ratio were determined using bioedit and expasy programs. cellular location of the l1 protein was determined using sosui-gramn program. signal peptide detection, transmembrane domain detection, three dimensional (3d) structure analysis, and phylogenetic analysis were conducted using signalp 4.1, thhmm, i-tasser/cn3d and mega7, respectively. oel1 was found to have an open reading frame of 909 base pairs coding 220 amino acids that constitutes a molecular weight of 24.9 kda and a high pi of 9.87. lysine, leucine and valine had higher rates. the hydrophilic nature suggested by kyte and doolittle analysis despite high rates of leucine and valine suggests an amphipathic nature of the protein that can bind to both hydrophilic and hydrophobic proteins and / or function in both media. a 1.59 at/gc rate is significant comparing to that of its homologs from other plants. sitoplasmic location predicted by sosui-grann is in agreement with the hypothesis suggesting an amphyphatic nature for oerl1. likewise, no signal peptide was detected and it was predicted to have at least one transmembrane domain. further characterization of oerl1 with respect to expression pattern and biochemical function continues. p-02.08.5-081 isolation and characterization of an olive splicing factor 3b subunit 1 m. nurcin, e. dundar department of molecular biology and genetics, balikesir university, balikesir, turkey splicing factor 3b subunit 1 (sf3b1) functions in the regulation of translation and gene expression. sf3b1 forms u2 small nuclear ribonucleoprotein complex (u2 snrnp). splicing factors 3a and 3b binds pre-mrna at the 5 0 site of the intron branching point. this binding joins u2 snrnp to pre-mrna. although sf3b1 from various plants have been widely studied, no studies on olive sf3b1 (oesf3b1) have been reported. this study reports information on various aspects of oesf3b1. oesf3b1 was obtained from a cdna library we constructed from fruited olive leaves in december. it was putatively identified as a splicing factor using blastn, blastp and blastx. to determine wheter oesfb1 was a sitoplasmic protein, sosui gramn was used. tmhmm was used to detect any transmembrane domains while signal peptide analysis was conducted by signalp. i-tasser and cn3d were used to generate the calculated 3d structure and to compare it with experimentally generated models, respectively. nucleotide and amino acid compositions along with the calculated molecular weight and isoelectric point (pi) were analyzed using bioedit and online expasy software. the phylogenetic trees revealed genetic relationship of olive among other plants based on oesfb1. the orf contained 1950 nucleotides coding 254 amino acids that produce a 28.2 kda peptide with a pi of 5.94. alanine, valine and leucine were found at high ratios suggesting a hydrophobicity which was also predicted by kyte and doolittle analysis. the at rich property of oesf3b1 is not unusual comparing to most plant genes. cellular localization of the gene was suggested to be in mitochondria with no signal peptide indicating oesf3b1 could be synthesizing in mitochondria. the predicted 3d structure of oesf3b1 was similar to experimentally produced structures while some hydrophobic pockets were predicted. further characterization of the gene with respect to temporal and spatial expression pattern and biochemical function continues. p-02.08.5-082 kafirin profile of turkish originated sorghum populations r. temizg€ ul, s. yilmaz, m. kaplan, t. akar department of biology, faculty of science, erciyes university, kayseri, turkey sorghum bicolor l. is the fifth important crop in the world with its high photosynthetic activity and resistance to unfavourable conditions as high temperature, drought, salt, and ph changes. sorghum has attracted great interest due to its intensive usage both as human and animal nutrition, and contribution to resistance against many diseases. some proteins of sorghum exerts reducing effect on nutrient digestion through making connetions with other proteins and/or carbohydrates. kafirin proteins have the highest proportion in grain with a range of 77-82%. they are grouped into a (23-25 kda), b (18 kda), c (28 kda) and d (13 kda) subunits depending on molecular weight, solubility and structure. in the current study, kafirin proteins from turkey originated 282 sorghum populations were acquired through sequential extraction; first, non-prolamines were removed through application of 5% naci concentration, and second, kafirins were obtained using tertiary butanol (60%) and reducing agents. sds-page was conducted for seperating and visualising the subunits of kafirins. the a, b, c, and d subunits of populations were respectively estimated as 97, 85, 95, and 60%. of the total proteins, 74% was identified as a, 12% b, 12% c, 1% d, and %1 non-prolamines. non-prolamin group of proteins were visualised as 18 different bands ranging from 15.6 to 230 kda. c and b group of proteins were only viewed when treated with reducing agents as 2-me and dtt suggesting that they are connected with complex cross-links. however, a group of proteins visualized without using these agents due to not having intra molecular disulphide bridges and inter molecular cross-links. non prolamins, except for 47.8, 45.7, 21.7, 20.3 and 15.6 kda, were able visualised in the presence of reducing agents. transcriptomic analysis of the genes encoding analysed proteins needs to be elucidated for better understanding of the genetic diversity and biochemical characteristics of sorghum. p-02.08.5-083 untargeted metabolomic profiling of romanian and uk tomatoes varieties by high performance liquid chromatography coupled with mass spectrometry c. socaciu 1,2 1 university of agricultural sciences and veterinary medicine, cluj-napoca, 2 center for applied biotechnology ccd-biodiatech at proplanta ltd, cluj-napoca, romania tomato flesh is a rich source of many phytochemicals of high nutritional value, including a large variety of carotenoid derivatives with health promoting properties. metabolomics became the most adequate technology for an accurate chemotaxonomic classification and discriminations between different varieties, based on untargeted profiling or targeted, quantitative analysis. different varieties of tomatoes (b-carotene-rich, lycopene-rich, ketocarotenoid-rich) cultivated in romania and uk were comparatively studied using enriched fractions obtained by a preliminary fractionation of the whole pulp homogenate. two methods were applied for carotenoid extraction: a mixture of hexane/ethanol (1) and chloroform/methanol (2) . the dried extracts were dissolved in ethyl acetate and analyzed by uv-vis spectrometry and hplc-esi(+)qtof-ms (bruker gmbh). the base-peak chromatograms were processed by specific biostatistics software (data analysis and profile analysis) and the molecular identification were determined by comparison with the data base lipidomics gateway (www.lipidmaps.org). the content of carotenoids were significantly higher using extraction (2) , ranging from 4.5 to 7.6 mg/100 g. the major carotenoid derivatives, were represented by lycopene, hidroxy-lycopene, all-trans or cis-beta-carotene, echinenone, all-trans retinyl palmitate, but also sterols, phospholipids, di/tri glycerides and ceramides. the romanian varieties were more rich in polar carotenoids and lipids, in general, while the uk tomatoes proved to be enriched in non-polar derivatives, especially esterified carotenoids, keto-carotenoids and glycerides. new molecules were identified, as good discriminatory markers of each tomato variety. acknowledgements. this work was supported by a grant of the romanian national authority for scientific research and innovation, cccdi -uefiscdi, project nr. 16/2015, pncdi3. glycogen is a multi-branched polysaccharide that serves as the main form of glucose storage in the body, where the main reserves are in the liver and muscle. it has been observed that glycogen metabolism is altered in many tumor types, and that glycogen content is inversely correlated with proliferation rate. in addition, it has recently been described that when glycogen accumulation is forced in glioblastoma u87 cells in hypoxia, senescence is induced and tumor growth is inhibited in vivo. our laboratory has various animal models with different parts of the glycogen metabolism pathway affected. most notably, we have two animal models lacking glycogen: muscle glycogen synthase (gys1 ko) and liver glycogen synthase (gys2 ko) knockout animals. we isolated mouse embryonic fibroblasts (mefs) from gys1 ko to perform replicative senescence assays. in addition, we induced hepatocellular carcinomas in gys2 ko animals via n-nitrosodiethylamine (den) injections in order to track tumorigenesis in animals lacking hepatic glycogen. lastly, we performed partial hepatectomies (phx), which involves the resection of two thirds of the liver, on gys2 kos to evaluate the effect of the lack of glycogen on hepatocyte proliferation. interestingly, we have observed that glycogen levels are increased in human and mouse fibroblasts under replicative senescence, and that mefs depleted of glycogen bypass senescence and immortalize faster than wts. we have also demonstrated that senescence pathways are down regulated in mefs lacking glycogen. furthermore, gys2 kos treated with den show higher tumor burden and mortality than controls. we also evaluated the effect of glycogen on hepatocyte proliferation after phx. gys2 ko mice present faster proliferation and liver regeneration rates, when compared to wt counterparts. collectively, our preliminary data suggest that glycogen metabolism plays a crucial role in the regulation of cell cycle in both physiological and pathological states. it is established that pineal is involved in circadian regulation of testosterone secretion from leydig cells. however, the precise routes of this regulatory involvement are still unknown. as cgmp has been also regarded as modulator of steroidogenesis we sought to study the effects of pineal removal on the circadian pattern of cgmp variations and expression of the genes that encode elements of no-cgmp signaling pathway in adult rat leydig cells. the analysis was performed on testicular leydig cells obtained from pinealectomised and shame pinealectomized rats, in six time point during 24 hours. the pinealectomy was confirmed by serum melatonin eia measurement. the androgen levels were measured by ria; cgmp by eia and gene expression was quantified by rq-pcr. all results were analyzed by cosinor method. data revealed circadian transcriptional pattern of nos2, nos3 (genes encoded no producers) and pde5a (gene for cgmp remover) in leydig cells from adult rats. pinealectomy significantly increased expression of nos2 which lost rhythm and increased and delayed amplitude of nos3 expression. further, pinealectomy initiated cyclic transcription of gucy1b3 and noncyclic transcription of gucy1a3 (genes encoded cgmp producers) and increased mesor and amplitude of pde5 transcription. the transcription of prkg1, the main effector in this signaling pathway was not affected with pineal abolition. additionally, pinealectomy did not influence the circadian transcription profile of coxi2 or other investigated genes (coxi1, nrf1, nrf2a, pgc1a) related to mitochondrial function and biogenesis. finally pinealectomy reversed phase of circadian cgmp oscillation in leydig cells, increased amplitude and slightly advanced peak of serum testosterone oscillation. results suggested pineal influence on circadian rhythm of no-cgmp signaling in leydig cells. further studies based on these data are needed to better understand the relationship between pineal and circadian rhythm of testosterone production. influenza is a contagious respiratory infection caused by a variety of influenza viruses. neuraminidase inhibitors is a new class of antiviral drugs that inhibit influenza viruses. the most popular antiviral agents is oseltamivir, having a commercial name of tamiflu, within anti-influenza antivirals. as well as tamiflu is a member of neuraminidase inhibitor group drug. therefore, this study was performed to determine the effect of tamiflu on cultured human peripheral blood lymphocytes. material and methods: for examining the presence of the indirect mutagenic effect of oseltamivir in iver s9 fraction mix was used. cells were treated with 0.5, 1 and 2 lg/ml oseltamivir, the tamiflu capsule ingradient, for 24 or 48 hours in the absence or presence of an exogenous metabolic activation system. the test chemical did not demonstrate any genotoxic effect dose-dependently but it showed a weak cytotoxicity on cells in this study. on the other hand, some concentrations of tamiflu induced sce and also decreased significantly the proliferation index (p ˂ 0.05) in the absence of s9 mix. result: tamiflu did not induce significant increases of ca or micronucleated cells in vitro in cultured peripheral blood lymphocytes under the treatment conditions used but week sce induction was observed. on the other hand, the weak cytotoxic effects observed disappeared in the cultures treated in presence of the s9 mix. discuss and conclusion: tamiflu weakly induced sce at the highest concentration with/without added s9 mix in cultured human peripheral lympocytes. it could be assumed to be a sce inducer. sces can be increased by several agents that attack dna. tamiflu decreased the proliferation index and nuclear division index at some concentrations thus interferring it as being weakly cytotoxic, though this effect disappeared in the presence of s9 mix applications. this finding is important for showing the inefficiency of tamiflu metabolites on the cell cycle. introduction: chronic renal failure as a result of the progression of diabetic nephropathy is the main cause of mortality in patients with type 1 diabetes. chronic hemodialysis is a life-saving therapy for patients with strong renal disorders. the main goal of hemodialysis is toxins removal from the patient. the monitoring of hemodialysis is the best way for biomedical evaluation of correctness and efficiency of this clinical treatment. according to the published data, the markers of development of diabetes complicated with renal failure are increased levels of glucose, urea and creatinine in the patient blood. today colorimetric and spectrometric methods are most commonly used for determination of the above metabolites in biological samples. however, these methods are complex in application, have low selectivity, and require pretreatment of samples. materials and methods: we propose for levels of glucose, urea and creatinine detection the potentiometric multibiosensor based on ph-sensitive field-effect transistors and immobilized enzymes developed in our laboratory. results: we developed a potentiometric multibiosensor and studied its main analytical characteristics. linear dynamic ranges of determination of substrates were following: 0.08 -1 mm of glucose, 0.1-10 mm of urea, and 0.02 -2 mm of creatinine. it was shown that the potentiometric multibiosensor had good reproducibility, and its bioselective elements were working independently from each other, because test of substrates cross-selectivity was negative. discussion and conclusion: very sensitive, fast and selective multibiosensor for simultaneous measurement of three metabolites in a single cycle based on ph-sensitive field-effect transistors and immobilized enzymes is developed. the developed potentiometric multibiosensor was verified by quantitative analysis of glucose, urea and creatinine in blood serum of patients with diabetic nephropathy. p-03.04.4-002 ph-dependent interaction of asymmetrically charged peptides with a protein nanopore over the past two decades, the ability to use natural or artificial nanopores to probe at uni-molecular level the structural and kinetic features of various bio-molecules (peptides, dna, rna) was successfully achieved. the operating principles of the nanopore-based single-molecule technique are simple: the single macromolecule capture, entry and subsequent translocations through a free-standing, voltage-biased nanopore, depend upon the physico-chemical and topological features of the analyte. the concentration, identity volume and charge of the analyte are then deduced from the analysis of the stochastic current blockade events caused by the trafficked analyte across the nanopore. herein, we used the a-hemolysin (a-hl) nanopore and set up an experimental model providing efficient control of a-hl-peptide interactions, in the presence of a ph gradient across the nanopore. for this, we engineered a 36 amino acids long peptide containing a neutral asparagines-containing sequence, flanked by oppositely charged aminoacid patches at the n-(glutamic acids) and c-termini (arginines), whose length was set as to span a single a-hl protein. when the ph of the solution in contact to the a-hl's b-barrel opening is changed from neutral to acidic values, the electrostatic interactions between the protein's mouth and either the n-or c-terminus end of the peptide occurs, and this influences strongly the dynamics of a peptide translocating the nanopore. we further proved that during the same experiment, peptide entry into the nanopore can be set to occur with either n-or c-terminus end head on, by simply changing the sign of the transmembrane potential across the nanopore. nanopores are emerging as a powerful and broadly applicable tool in biophysics, which allows one to study the features of charged macromolecules under confinement. a few noteworthy examples are: determining the electrophoretic mobility, effective charge and diffusion coefficients of charged molecules; exploring the folding and unfolding of peptides and proteins; analyzing biopolymers trafficking, protein transport, dna translocation, rna and dna sensing and sequencing. herein, we employ single molecule analysis techniques using a wild-type ahemolysin (a-hl) protein nanopore to study the capture and translocation behavior of a short cationic peptide (20 amino acids in length) at an extremely low ph value. our experiments revealed that an effective absorbing field is created by the electroosmotic flow, against the electrophoretic force, which enables the peptide capture inside the nanopore. furthermore, our findings show that the trajectory of a single peptide can be experimentally visualized and the main steps determined: the peptide capture, reversible translocation across the pore's vestibule and lumen regions, and the peptide release from the nanopore. also, the kinetic analysis of the main steps observed allowed us to describe the free energy profile of the peptide interactions with the protein nanopore. the presented work provides evidence for the ability of controlling the dynamics of a single-peptide, its capture and passage inside a a-hl nanopore, that underlie the processes naturally occurring in cells, thus proving a powerful approach for probing single molecule biophysics phenomena, in general. changes in the physical conditions of the cancer microenvironment driven by elevated tissue growth and angiogenesis, may introduce exposure of laminar fluid flow, which effect the key factors of cancer, such as progression, immune-escaping and metastasis. conventional experimental models fail to mimic the physical cues on tumor microenvironment. microfluidic culture techniques allow precise control of fluids, simultaneous manipulation and analysis of cultured cancer cells. here, we present a platform that can be used for the investigation of the role of flow mediated mechanical stimuli on cancer cells. microfluidic cell culture platform was fabricated using polymethyl methacrylate and double-sided adhesive films with 25 9 41 mm dimensions. ovary adenocarcinoma cells (efo-27 and onco-dg-1) were used for the optimization of the platform. to understand the fluid and gas distribution patterns, specific modeling was performed. dynamic microfluidic cell culture and static conventional cell culture conditions were compared for the differences of cancer cell phenotype, such as proliferation, viability, epithelial-mesenchymal transition. we confirmed that, the proliferation and viability of cancer cells are increasing under dynamic fluid flow. the proliferation rate of ovary adenocarcinoma cells was correlated with the increase of fluid flow rate. immunocytochemical analysis showed that fluid flow causes decrease in e-cadherin expression, and increase in n-cadherin and vimentin expressions, which indicate mesenchymal phenotype of cancer cells. our results showed that, cancer cells present different characteristics due to fluid flow of tumor microenvironment. to understand the role of physical dynamics by using microfluidic culture techniques, is a key to elucidate the mechanisms underlying disease progression, and may lead to new diagnostics and therapeutic approaches. (this study was funded by turkish scientific and technical research council (tubitak-214s334). high-sensitive detection of low-affinity antibodies by immuno-pcr with supramolecular olygonucleotide-streptavidin complex detection of low affinity antibodies in blood sera and cell surface outwashes is important both in the study of molecules that bind to cellular receptors (circulating tumor cell masking antibody, for example) and medicine (diagnosis of allergy). low affinity igm and ige antibodies can not sometimes be determined by conventional methods. we using supramolecular oligonucleotide-streptavidin complex formed from single-stranded synthetic oligonucleotide (60 n) contains biotin on 5'-and 3'-ends, and sterptavidin in molar ratio 1:1. this complex represents a structure with equivalent electrophoretic mobility of 600 bp dna and preferred "valency" of streptavidin is 2. this universal immuno-pcr approach make it possible to increase a signal by using several oligonucleotides per one antibody. after the method optimization we achieved 100-1000 times highter sensitivity than elisa. to reduce the matrix effect we used 100-500 fold dilutions of sera samples. this approach achieved a significant advantage, because it allows working with small-volume samples (need only 2 mkl of serum sample). antibodies to the disaccharide galb1-3glcnac (le c ) are typical of the natural antibodies. the igm anti-le c antibodies are found in almost healthy people without the epitope specificity variation. we have shown that the concentration of igm anti-le c antibodies was higher (p ≤ 0.0005) for health donor sera (n = 45; 31.3 ae 4.3 pg/ml) compared with sera from patients with breast cancer (n = 38; 17.0 ae 4.1 pg/ml). sensitivity of igm anti-le c antibodies detection was 100 pg/sample (50 mkl) ie 6.7 9 10 7 molecules. thus for the immuno-pcr detection of antibodies the 10 3 -10 4 tumor cells are sufficient. such amount of cells seems to be a realistic one for detection of antibodies masking circulating tumor cells. this study was supported by a grant from russian science foundation (#16-14-00136) and by russian federation president scholarships donated to d. yu. riazantsev (# sp4413.2015.4 bacterial pathogen detection and identification is of crucial importance for disease diagnosis, bacterial contamination surveys and water quality assessment. we propose herein a novel method for bacterial detection based on the interaction of single gram-negative bacterial cells (i.e.: escherichia coli and pseudomonas aeruginosa) with an ahemolysin (a-hl) protein nanopore embedded in a reconstituted lipid bilayer, at neutral ph. as a consequence of an applied voltage, the negatively charged bacteria suspended in saline buffer solution are electrophoretically driven towards the pore opening, inducing reversible blockages in the ionic current through a-hl. experiments were also performed in the presence of an antimicrobial peptide, cma3, as well as in acidic environment. statistical analysis of the frequency and duration of blockage events allowed us to discriminate between the two types of bacteria. the frequency of interactions was higher for escherichia coli with respect to pseudomonas aeruginosa. adsorption of cma3 peptides on the membrane of bacteria increased the frequency of interactions with the pore, contrary to the expected effect induced by lowering the net surface charge of the cells. in experiments performed at ph = 4, the frequency of blockage events was found to be two orders of magnitude higher, with longer interaction life-times. the net negative charge (7 uncompensated aspartate residues) localized at the entrance of the pore contributes an additional electrostatic repulsion interaction between negatively charged bacterial cells and a-hl. thus, adsorption of cationic peptides at the interface will reduce this repulsive interaction. the same effect was recorded at ph = 4, when the aspartate residues are partially protonated, confirming our understanding of the previously observed results. this method could be further developed and integrated with other techniques, making nanopore-based systems a fast and reliable bacterial detection and identification tool. this study was performed to analyze the effects of tunicamycin (tm) and taurohyodeoxycholic acid (tudca) on thle-3 cells. cells were treated with tm to induce endoplasmic reticulum (er) stress and tudca was administered as an er stress inhibitor. cytotoxicity was evaluated at different times of exposure by incubating cells with increasing concentrations of either tudca, tm or both. thle3 cells were cultured in fibronectin, bovine collagen i and bovine serum albumin coated plates. cell lines were grown in begm media supplemented with epidermal growth factor, phosphoethanolamine, fetal bovine serum, 100 u of penicillinstreptomycin and maintained in a humidified incubator at 37°c and a 5% co 2 atmosphere. cell viability was measured using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay kit. cells were grown to confluence in 96well plates and incubated with 1 ll/ml dmso, 1-10 lg/ml tm, 0.1-5 mm tudca, or 10 lg/ml tm + 0.1-1 mm tudca for 18-48 hours. control cells were prepared in plates containing only medium. at the end of the incubation period, mtt was added to each well and incubation was carried out for 4 hours at 37°c. formazan production was expressed as a percentage of the values obtained from control cells. at all hours of incubation neither dmso or 1 mm tudca was cytotoxic. at 24 and 48 hours incubations 5 mm tudca and 10 lg/ml tm + 1 mm tudca were significantly cytotoxic compared to control, dmso and 1 mm tudca groups. treatment of cells with 0.5 mm tudca 8 hours before administrating 10 ug/ml tm significantly decreased the cytotoxic effect of tm. we conclude that tudca may show cytotoxic effects at 1 mm concentration when treated with tm. therefore 0.5 mm of tudca, administered 8 hours before tm treatment should be applied to protect against er stress. acknowledgement: this study was supported by a grant from the scientific and technological research council of turkey (tubitak; 214s223). recent studies reveals that history of preeclampsia is an independent risk factor for cardiac events and stroke. lipoprotein-associated phospholipase a2 (lp-pla2) is a vascular inflammatory marker associated with cardiovascular diseases (cvd). we hypothesize that vascular inflammation (lp-pla2 mass, activity, index) related genetic variations (pla2g7) increase the risk for developing future cardiovascular disease in women with pe. a group of 150 preeclamptic patients and 50 normal pregnant women were recruited from university of istanbul, cerrahpasa medical school, department of gynecology and obstetrics included into the study. the control group was matched for maternal and gestational age at time point of sampling. preeclamptic patients were starified into two groups; early-onset and late-onset according to the 32 gestational weeks. enzyme-linked immunosorbent assay procedure was used to determine the serum lp-pla2 mass level. lp-pla2 activity were determined by kinetic method. plag7 snp genotyping performed by using the sequenom massarray iplex. the rs1805017 tt genotype had a higher lp-pla2 index (p = 0.015) for early onset preeclampsia, cc genotype had a higher lp-pla2 mass and lp-pla2 index for late onset preeclampsia. no difference were found for control. the rs1809381475 gg genotype had higher lp-pla2 mass and index for late onset preeclampsia (p = 0.008, p = 0.012 respectively). stepwise logistic regression analysis performed to identify cardiovascular disease related variables that independently and significantly contributed to the presence of alleles of rs1805017 and rs1809381475 snps in early, late onset preeclampsia and control group. only lp-pla2 mass was independently and significantly associated with both snps in early onset preeclampsia. the association between lp-pla2 mass, index and rs1809381475, rs1805017 snps might be useful genetic markers to adress future cvd risk in patients with preeclampsia. introduction: b-thalassemia is one of the most monogenic autosomal recessive disorder characterized by defective production of the b-chain of hemoglobin. definition of the b-globin genotype is necessary for genetic counselling in the carriers, and for predicting prognosis and management options in the patients with thalassemia. dna-based diagnosis of b-thalassemias routinely relies on polymerase chain reaction (pcr) and gel electrophoresis. the aim of this study is to develop a new procedure, a dna-based piezoelectric biosensor, for the detection of b-thalassemia ivsi-110 mutation, the most common b-thalassemia mutation in turkey. materials and methods: b-globin gene of genomic dna isolated from whole blood, was amplified by pcr. bioactive layer was constituted by binding 2-hidroxymetacrilate metacriloamidoscystein (hema-mac) nanoploymers on the gold electrode's surface. single oligonucleotide probes specific for ivsi-110 mutation of b-thalassemia were attached to the nanopolymer via reactive cross-linker glutaraldehyde. the measurements were executed by piezoelectric resonance frequency which is caused by binding of pcr products in media with single oligonucleotide probe on the electrode surface. the results were confirmed by the conventional molecular method as arms. results: the piezoelectric resonance frequencies obtained by hybridization of the pcr products on bioactive layer were found 216 ae 12, 273 ae 6, and 321 ae 9 hz for the samples of normal b-globin, heterozygote, and homozygote of ivsi-110 mutation, respectively. discuss and conclusion: the developed biosensor serves as a specific result to ivsi-110 mutation. it could accurately discriminate between normal and ivsi-110 mutation samples. because of low costs, fast results, specificity and high detection/information effectiveness as compared with conventional methods, we can be offered this techique as an alternative to conventional molecular methods. the increasing use of nano-sized materials in the last several years has compelled the scientific community to investigate the potential hazards of these unique and useful materials. one of the most widely used nanoparticles is titanium dioxide. the objective of the research is to investigate the alterations in molecular and cellular responses in culture of primary lymphocytes to tio2 nps. human lymphocytes isolated from heparinized blood of healthy individuals were exposed to tio2 nanoparticles. viability, ros generation, the changes in the expression of genes encoding proinflammatory mediators tnf-a, il-1b and il-8 and dna damage were assessed. human lymphocytes were incubated with nanoparticles of different concentrations and viability was determined in 24 and 48 hours after treatment, respectively. cell viability was decreased by a treatment with nanoparticles in both a time-and concentration-dependent manners. the ability of tio2 to induce ros formation in lymphocytes was evaluated using dcf fluorescence as a reporter of oxidant production. the fluorescence intensity of oxidized dcf was increased in cells treated with nps. this means that ros generation occurred in response to the treatment with tio2. to investigate the expression level of mrna related to the inflammation responses in human lymphocytes real-time pcr was performed. the expression of il-1b, il-8 and tnf-a genes were increased by the exposure to nanoparticles of 10, 20 and 40 lg/ml for 24-48 hours. tio2 nanoparticles were shown to induce the dose-dependent fragmentation of dna strands. much evidence of hazardous health effects of nps has been reported. in this study, viability was reduced under the exposure to tio2. oxidative stress was elevated by the treatment with tio2 nps. oxidative stress can also trigger inflammation signals. induced by exposure to nanoparticles they may cause the translocation to the nucleus of transcription factors, which regulate proinflammatory genes, such as tnf-a, il-1b, il-8. background: endothelial cells (ec) represent one of the primary targets of the major pro-inflammatory cytokinetumor necrosis factor (tnf). development of the new approaches for the treatment of acute and chronic inflammatory conditions, including the strategies aimed to tnf neutralization, requires the usage of the adequate cellular models closely resembling the properties of the endothelium. the endothelium-derived ea.hy926 cell line expresses several inflammation and neoangiogenesis markers in response to activation factors however their expression can differ from the patterns demonstrated by primary ec. the aim of the current study was to compare the expression of the known endothelial cellular markers including receptor of vascular endothelial growth factor-2 (vegfr2) and a v b 3 -integrin on 2d and 3d cultures (spheroids) of ea.hy926. methods: the ea.hy926 cell line was used with permission from dr. edgell. the cells were cultivated in the presence of tnf (25 ng/ml) or vegf a (25 ng/ml) for 5 hours. mrna was isolated using rneasy kit from qiagen and reverse-transcribed with revertaid kit (fermentas). rt-pcr was performed with specific primers. expression of vegfr2 and a v b 3 -integrin was visualized by confocal microscopy using specific monoclonal antibodies and previously developed fluorescent hybrid proteins. results: the expression of a v b 3 -integrin and vegfr-2 increased on the 3d culture compared to 2d according to confocal microscopy and rt-pcr. the aforecited methods revealed elevated expression of a v b 3 -integrin in the 3d culture of the ea.hy926 cell line activated with tnf. also increased expression of vegfr2 in the 3d culture activated with vegf a. then by confocal microscopy, we analyzed our fluorescent hybrid proteins that bind a v b 3 -integrin and vegfr2 on the surface of 2d and 3d cultures as well as antibodies with fluorescent label. conclusions: 3d cultures of the ea.hy926 cell line represent a promising model for the inflammation studies. tumor necrosis factor (tnf) is a trimeric cytokine associated with the inflammatory response to tissue injury and found to possess a key role in rheumatoid arthritis pathogenesis. spd 304 is a highly toxic recently discovered tnf inhibitor that promotes trimer dissociation and lead to the inactivation of the protein. according to the traditional anti-tnf treatment of ra, we aim at extracellular inhibition of this pro inflammatory cytokine as an effective therapy. the project plan comprises design, synthesis and validation of candidate inhibitors (measurement of dissociation constant and aqueous solubility). because of the elevated percentage of insoluble compounds a solubility enhancement protocol has been developed. the experimental procedure was the following:: a. drug design. identification of novel drug compounds are based on two approaches: i) structure based drug design using the 3d structure of tnf and ii) design of more potent and less toxic spd304 analogues. b. drug synthesis. a series of spd304 analogues were in house synthesized while novel candidates discovered by in silico approaches were commercially available. the purity of the majority of the compounds exceeded 90%. c. solubility measurement and enhancement. samples were incubated under specific conditions that can enhance aqueous solubility and solubility measurement with a direct uv method pursued. d. measurement of the dissociation constant. a fluorescence binding assay was used in order to evaluate the inhibitory activity of the compounds. from our results it can be concluded that dmso, peg3350 and b-cyclodextrin can be used for solubility enhancement without interfering with fluorescence assay. however peg3350 -in contrast to dmso-is not suitable for isothermic titration calorimetry measurements. dissolution procedure also plays a crucial role in the levels of solubility reached. finally, it has been shown that some of the studied spd304 derivatives have better dissociation constants than spd304. the effect of exercises on serum bmp-6 levels of knee osteoarthritis cytokines. more complicated approaches are expected to focus on molecular proteins as bone morphogenetic proteins (bmps) of the transforming growth factor (tgf)-beta superfamily. bmps associated with many cellular functions, such as proliferation, differentiation, and apoptosis. bmp-6 is significantly important for the endochondral bone formation. inflamation can induced serum bmp levels in oa patients. the aim of this study is to evaluate the clinical findings of oa patients after the isokinetic exercise together with the serum levels of bmp-6 to sustain the molecular approaches for treatments. a total of 36 patients were included in this study. the groups are formed as follows: group 1, oa patients before the exercise; group 2, oa patients after the exercise; group 3, oa patients before the isokinetic exercise; group 4, oa patients after the isokinetic exercise clinical and biochemical findings were evaluated before and after 3 weeks of the exercise programme. self reported severity of pain was measured using the 100 mm visual analog scale (vas), womac scores were calculated and isokinetic knee muscle strength testing was measured using cybex dynamometer that a standardized protocol previously described was applied in a subject-specific range of motion. serum bmp-6 levels of all patients were studied by elisa method. results represented a better vas and womac scores for all exercise groups after treatment. the serum bmp-6 levels were significantly decreased in group 2 compared to group 1 (222.94 ae 44.66; 246.49 ae 38 .15 respectively, p < 0.01) and in group 4 compared to group 3 (246.74 ae 32.26; 256.23 ae 33.11 respectively, p < 0.05). there is not any statistically differences between group 2 and group 4 (p > 0.05). as a conclusion, the decreased serum levels of bmp-6 may be suggested as a biochemical marker for oa patients during exercise programmes. p-04.04.4-006 tnf-a blokade efficiently reduced severe intestinal damage in necrotizing enterocolitis c. tayman, s. aydemir, i. yakut, u. serkant, a. c ß iftc ßi g€ olbasi devlet hospital, ankara, turkey objectives: to ascertain the beneficial effects of infliximab an inhibitor of tumor necrosis factor alpha (tnf-a) on the development of nec in an experimental nec rat model. material and methods: thirty newborn sprague-dawley rats were randomly divided into three groups as nec, nec+ infliximab, and control. nec was induced by enteral formula feeding, exposure to hypoxia-hyperoxia and cold stress. pups in the nec+ infliximab group were administered infliximab at a dose of 10 mg/kg daily by intraperitoneal route from the first day until the end of the study. all pups were sacrificed on the 5th day. proximal colon and ileum were excised for histopathologic, immunohistochemical (tunel and caspase-3), and biochemical evaluation, including, total antioxidant status (tas), total oxidant status (tos), malonaldehyde (mda), and myeloperoxdase (mpo) and tnf-a activities. results: we observed better clinical sickness scores, weight gain, and survival rate in the nec+ infliximab group compared to the nec group (p < .05). histopathological and apoptosis examination (tunel and immunohistochemical evaluation for caspase-3) revealed lower damage in the nec+ infliximab group compared to the damage in the nec group (p < .01). tissue mda, mpo, tnf-a levels, and tos were significantly decreased in the nec+infliximab group, whereas tas was significantly increased in the nec + infliximab group (p < .01). conclusion: tnf-a blockade with infliximab efficiently reduced the intestinal injury and preserve the intestinal tissues from severe intestinal damage by its complex mechanisms on nec. therefore, it may be an alternative option for the treatment of nec.keywords: tnf-a; infliximab; necrotizing enterocolitis; newborn; protection; rat; treatment p-04.04.4-007 short-term diabetes causes cardiovascular inflammation: anti-inflammatory effect of resveratrol introduction: diabetes is a metabolic dysfunction and has been associated with various disorders including inflammation, cardiomyopathy and coronary artery disease. inflammation is a protective mechanism elicited by the host in response to infection, injury, and tissue damage. the aim of this study was to investigate the effect of intraperitoneally resveratrol administration on cardiac and vascular function in diabetic rats. materials and methods: diabetes was induced in sprague-dawley rats by using injection of streptozotocin (55 mg/kg, i.p.). rats were divided into group i: control, ii: control/20 mg/kg resveratrol; iii: diabetic/vehicle; and iv: diabetic/20 mg/kg resveratrol. histopathological examinations with masson's trichrome and verhoeff-van gieson staining were carried out to reveal cardiac and vascular tissue damage and inflammation. in addition to plasma glucose and cardiac & vascular mda levels were measured by standard enzymatic kits while tnf-a, il-1b, il-18 (mbl) were analyzed by elisa kit. results: final body weight decreased in all groups compared to control. in the diabetic rats, plasma glucose and vascular mda levels were enhanced while cardiac mda was unchanged compared to control. vascular tnf-a, il-1b and mbl and cardiac mbl were increased in the diabetic groups compared to control. discussion and conclusion: it has been found that resveratrol has greatly normalized altered parameters. taken together, resveratrol partly improved cardiac and vascular inflammation induced by diabetes. this may be due to the healing activity of resveratrol on pro-inflammatory markers. p-04.04.4-008 cytokine network is critical in growth hormone-induced resistance mechanism against curcumin which modulates jak/stat/ socs pathway in mda-mb-231 and mcf-7 breast cancer cells m. c ß elik, a. c ß oker g€ urkan, e. damla arisan, p. obakan yerlikaya, n. palavan unsal t.c. istanbul k€ ult€ ur university, istanbul, turkey curcumin (diferuloylmethane), a polyphenolic compound that triggers apoptotic cell death in various cancer cells such as prostate, colon, melanoma and breast cancer. a pituitary-derived hormone, growth hormone (gh) play role in elongation and differentiation of ductal epithelia into the breast terminal and buds. in this study, our aim is to determine the role of inflammation in curcumin induced apoptotic cell death via acting on jak/stat/socs pathway in wt and gh+ mda-mb-231 and mcf-7 breast cancer cell lines. according to mtt cell viability assay curcumin triggers cell viability loss in time and dose dependent manner in mda-mb-231 wt and mda-mb-231 gh+ breast cancer cell lines, respectively. selected concentrations of curcumin as 20 lm (for mcf-7) and 25 lm (for mda-mb-231) decreased cell proliferation and induced apoptosis through causing jak2 dephoshorylation, stat1, 3, 5 dimerization and acting on socs proteins expression in each cell lines. in addition, activated jak/stat/socs pathway, via forced gh expression has been suppressed following curcumin treatment for 24 hours. 20 lm curcumin-induced apoptotic cell death via dephosphorylating jak2 at tyr1007/1008 residues and decreased phospho-stat1, 3 level in both breast cancer cell lines. although curcumin dephosphorylated stat5 in both mda-mb-231 and mcf-7 wt cells, no significant effect has been observed in mda-mb-213 gh+ and mcf-7 gh+ cell lines. in consequence, although forced gh expression induced cell proliferation in mcf-7 and mda-mb-231 breast cancer cells, curcumin overcame gh-mediated resistance mechanism via acting on jak/ stat/socs signaling, which is related to pparg-induced inflammation. acknowledgment breast cancer is one of the highest cancer type among women worldwide. various enviromental and genetic factors such as age, gender, family history, metabolic diseases and gene mutations are involved in the breast cancer pathogenesis. growth hormone (gh), a pituitary derived hormone, has essential role on postnatal growth and development. it is also established that signalling route of gh and its receptor (ghr) activity is increased in different cancer types. curcumin, a nutraceutical deriatives from rhizomes of turmeric (curcuma longa), has potential therapeutic activity against cancer cells, including breast cancer. curcumin inhibits proliferation of cancer cells such as prostate, colon, melanoma, cervical and breast cancer via induction of apoptosis and inflammation. stat5, a major downstream target of gh/ghr signalling, is related to survival, proliferation and differentiation. in this study, our aim was to investigate curcumin-induced apoptotic cell death in gh overexpressed mda-mb-453 breast cancer cells via jak-stat/socs signalling and inflammatory response profile. according to mtt cell viability assay, curcumin decreased cell viability in time and dose dependent manner in wt and gh+ mda-mb-453 breast cancer cell lines. we found that 20 lm curcumin-decreased in apoptotic cell death through inactivity at jak2 which led to dimerization of stat1, stat3, stat5. concomitantly, curcumin affected stat regulating socs proteins in mda-mb-453 breast cancer cell line. in addition, we demonstrated that 20 lm curcumin induced pparg expression and altered inflammatory cytokine signalling cascade. consequently, although gh overexpression led to agressive profile in mda-mb-453 breast cancer cells, curcumin overcame this resistance. inflammation is involved in many systemic disturbances, including osteoarthicular or skin diseases, coordinating the signaling network that contributes to tissue injuries. the aim of our study is to reveal pro-inflammatory messengers at the cutaneous barrier (keratinocytes, fibroblasts, endothelial cells), simulating the dermal impact of active principles, especially polyphenols and flavones from vegetal sources: salvia officinalis, asculum hippocastanum and calendula officinalis. we focused on il6 and il8 cytokines as main mediators of inflammation progression, correlated in keratinocytes with il1a as skin irritation indicator and vegf as pro-angiogenic factor, as well as in endothelial cells with icam-1 and vcam-1 adhesion molecules expression. in order to in vitro mimic the inflammatory conditions, we used targeted stimuli for each type of cells: for fibroblasts and endothelial cells -tnfa, a systemic stimulus, single or combined with pma that activates protein kinase c and up regulates nadph oxidase, which lead to superoxide anion production; for keratinocytescontrolled uv-a and uv-b radiation, simulating the solar damages or potential uv interactions with active principles in light exposed skin. the main analysis technique was flow cytometry: beads bases assay for soluble factors and fluorescent antibodies staining. our results prove the different involvement of polyphenols and flavones in the anti-inflammatory mechanisms, depending of the vegetal source: active principles from salvia officinalis induce a strong inhibition of il6 and il8 in tnfa stimulated keratinocytes, fibroblasts and endothelial cells, reduce the icam-1 over-expression but have no effects on irradiated keratinocytes; biocomplexes from asculum hippocastanum inhibit only il8 release in stimulated fibroblasts, but protect keratinocytes from uv-a and uv-b radiation; compounds from calendula officinalis are active on il8 signaling in fibroblasts and counteracts only uv-b inflammation. ischemia and/or reperfusion injury is one of the most common causes of acute renal failure. ischemia-reperfusion associated with thrombolytic therapy, organ transplantation, coronary angioplasty, aortic cross-clamping, or cardiopulmonary bypass results in local and systemic inflammation. within the endothelium, ischemia produces expression of proinflammatory gene products (e.g. cytokines) and bioactive agents (e.g., endothelin), while preventing other "protective" gene products (e.g., thrombomodulin) and bioactive agents (e.g. nitric oxide). therefore, ischemia induces a proinflammatory state that increases tissue vulnerability to further injury on reperfusion. this experimental study was designed to investigate the protective effect of salvia l. extracts on kidneys from i/r injury. salvia lamiaceae have been used for treatment of some illnesses in turkish folk medicine. forty spraque dawley rats were divided into 5 groups (n = 8). right nephrectomy was performed to all groups. group i: control group; group ii: i/r group; group iii: i/r + 50 mg/kg salvia l.group; group iv: i/r + 100 mg/kg salvia l. group; group v: i/r + 50 mg/kg rosmarinic acid. group. salvia l. and rosmarinic acid for 7 days was given single dose as a gavage. 60 minutes ischemia, 60 minutes reperfusion were applied to groups except control. intracardiac blood samples were taken, high sensitive crp (hscrp), tumor necrosis factor-a (tnf-a), interleukin (il)-6 and interleukin ib (il-1b) levels were detected. serum hscrp levels were also determined in our clinical laboratory using routine standard methods. serum tnf-a, il-6 and il-ib levels were evaluated using an enzyme-linked immunosorbent assay technique. mean values were evaluated by statistical analysis. serum hscrp, tnf-a, il-6, and il-1b concentrations were significantly increased after renal i/r as compared to the control group. our treatment group 50 mg/kg salvia l. and 50 mg/kg rosmarinic acid especially 100 mg/kg of salvia l. were found to show a protective effect against renal structure and function. we concluded that salvia l. extracts could be beneficial in the treatment of renal ischemic injury. but 100 mg/kg salvia l. extract were more effective than 50 mg/kg salvia l. extract and used as synthetic 50 mg/kg rosmarinic acid. acne vulgaris is a common chronic inflammatory skin disease of unknown etiology. excess levels of secretory phospholipase a2 (spla2) contributes to inflammatory diseases and studies indicate that lipoprotein lipase (lpl) has differential effects on several inflammatory pathways. the aim of the present study was to assess serum activity of spla2, lpl and evaluate changes in circulating protein levels of angiopoietin-like protein 3 (angptl3), angptl4, cyclooxygenase (cox) and prostaglandin e2 (pge2). serum from 21 control subjects and 31 acne vulgaris patients with moderate and severe disease was evaluated for levels of spla2, cox, pge2, lpl, angptl3 and angptl4. disease activity was determined according to the national health service (nhs) lambeth and southwark clinical commissioning group guidelines for the management of acne. lipid profile, routine biochemical and hormone parameters were assayed by standard kit methods using autoanalyzers (beckman coulter au5800 clinical chemistry and unicel dxi 800 immunoassay systems). serum levels of spla2 and lpl were significantly increased in acne vulgaris patients compared to age and gender matched controls. no significant differences were found for cox, pge2, angptl3 and angptl4 levels between acne vulgaris patients and controls. the results of this study reveal the presence of a proinflammatory state in acne vulgaris as shown by significantly increased serum spla2 activity. increased lpl activity in serum of acne vulgaris can be protective in patients through its anti-dyslipidemic actions. to our best knowledge, this is the first study investigating spla2, lpl, angptl3 and angptl4 levels in acne vulgaris. future studies are aimed to understand the regulation of spla2 and lpl expression in acne vulgaris patients. acknowledgement: this study was supported by a grant from the scientific and technological research council of turkey (tubitak; #115s940). p-04.04.4-013 8-ohdg and hogg1 levels are as an oxidative dna damage markers in acne vulgaris treated with isotretinoin h. ecevit, m. izmirli, b. gogebakan, e. rifaioglu, d. sonmez, b. bulbul sen, t. sen, h. m. okuyan mustafa kemal university, hatay, turkey acne vulgaris is a skin disease that characterized by comedones, papules, pustules, nodules and cysts at face, back and body skin. isotretinoin is one of the treatment agents in acne vulgaris. about 4 weeks after drug treatment, the amount of sebum which is produced by sebaceous gland reduces keratinization disorder and the number of propionibacterium acnes normalizes. however, isotretinoin is known that has a wide range of side effects. in recent studies, isotretinoin treatment has been shown to increase the oxidative stress. 8-hydroxy-2 0 -deoxyguanosine (8-ohdg), an important indicator of oxidative dna damage, hydroxyl ion is bound at the 8th carbon of guanine. this structure is repaired through a base excision repair mechanism and the human 8-oxoguanine dna glycosylase 1 (hogg1) plays a key role in this processes. in this study we aimed to evaluate the dna damage and it's repair in acne vulgaris before and after 6 months of isotretinoin treatment by measuring 8-ohdg and hogg1 levels. the current study includes 43 acne vulgaris patients who are diagnosed in mustafa kemal university, department of dermatology. 8-ohdg and hogg1 levels were measured by enzymelinked immunosorbent assay (elisa) method for before and after 6 months of isotretinoin treatment. the commercial elisa kits (cloud-clone corp; usa and cell biolabs; usa) were used for the assessment of hogg1 and 8-ohdg, respectively.both 8-ohdg (p as a conclusion, isotretinoin increases dna damage and high serum 8-ohdg and hogg1 levels as a result of isotretinoin treatment may effect on the amount of reactive oxygen species. the pineal gland is a circumventricular organ which serves as a major neuroendocrine gland in the brain. its primary function is the production of melatonin which is controlled by signals from the suprachiasmatic nucleus. melatonin codes the length of the night and it is well recognized for its anti-inflammatory effects. lipopolysaccharide (lps) is the essential component in the outer surface membrane of gram-negative bacteria and act as a strong stimulator of natural and innate immunity in all eukaryotic species. furthermore, lps reduces melatonin synthesis and induces the expression of the serine protease inhibitor 3 (spi-3) in the stat3-mediated manner in pinealocytes. however, the precise function of stat3 in the cell signaling in the pineal gland is not yet known. here we investigated the effect of inhibition of stat3 on lps-induced changes in melatonin levels, expression of arylalkylamine n-acetyltransferase (aa-nat) and spi-3 in the pineal gland. experiments were performed in vitro using organotypic and primary cultures prepared from the rat pineal glands. levels of melatonin and spi-3 were determined from tissue homogenate by enzyme-linked immunosorbent assay (elisa). the pinealocytes were used to carry out sirna stat3 transfection. the successful transfection and subsequent decline in stat3 expression levels were proved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page). the changes in synthesis of aa-nat and spi-3 were studied by rt-pcr. in conclusion, lipopolysaccharide can affect the immunomodulators secreted by the pineal gland. the clarification of the effect of inhibition of stat3 on those immunomodulators is important from the clinical point of view because inhibitors of stat3 are nowadays used as tumour suppressors. silica nanoparticles have a great potential for a variety of industrial, diagnostic and therapeutic applications. in this study, we have evaluated the in vitro effects of amorphous silica nanoparticles (7 nm) using human lung mrc-5 fibroblast as model. cells were exposed to 62.5 lg/ml silica nanoparticles for 24, 48 and 72 hours. the cytotoxic and inflammatory response, and matrix metalloproteinase expression were examined. the pro-inflammatory cytokine il-1b, il-6, il-8, tumor necrosis factor (tnf-a), matrix metalloproteinases (mmp-2, mmp-9, mmp-1) and tissue inhibitor of metalloproteinase-1 (timp-1) were analyzed by western blot method. cytotoxicity was evaluated by lactate dehydrogenase (ldh) released into the culture medium by damaged cells. the level of ldh activity was increased after exposure to silica nanoparticles, in a time-dependent manner compared to control. the protein expression of il-1, il-6, il-8 and tnf-a as well as of mmp-1 and timp-1, was up-regulated whereas those of mmp-2, mmp-9 was down-regulated after 48 and 72 hours respectively. in conclusion, our data indicate that amorphous silica nanoparticles generate a cytotoxic and inflammatory response, as well as an imbalance in extracellular matrix due to the differential regulation of mmps and tissue inhibitor of metalloproteinase-1 in mrc-cells after 48 and 72 hours. p-04.04.4-017 association of fto gene variant (rs8050136) with markers of t2dm and obesity in population from bosnia and herzegovina and kosovo fto (fat mass and obesity-associated gene), recently discovered in a genome-wide association study for type 2 diabetes (t2d) encodes a 2-oxoglutarate-dependent nucleic acid demethylase and is mainly expressed in the hypothalamus. this gene may play important role in the management of energy homeostasis, nucleic acid demethylation, and regulation of body fat masse by lipolysis. the aim of this study was to analyze the association of this single nucleotide polymorphisms (snps) with clinical and biochemical parameters of obesity, t2d, prediabetes and at the level of healthy population from bosnia and herzegovina (bh). the study included 638 patients with t2d and prediabetes and 360 healthy controls both sexes, aged from 40 up to 65 years. patients were recruited at the clinical centre university of sarajevo, university hospital of clinical centre in banja luka, general hospital in te sanj and health centre in prizren. genotyping of analyzed polymorphism was performed by rt-pcr method in cooperation with the department of clinical chemistry, faculty of pharmacy, university of ljubljana (ljubljana, slovenia) and university hospital of charles university (hradec kralove, czech republic). our results did not show significant differences in genotype frequencies of the analyzed polymorphisms between patients with t2d, pre-diabetes and healthy population also, results of logistic regression analyses did not show significant association of risk a allele of fto gene polymorphism -rs8050136 with increased risk of t2d (or = 1.084, 95% ci 0.758-1.551, p = 0.659). a allele was significantly associated with higher values of hba1c, insulin, homa ir index, diastolic blood pressure and higher levels of inflammatory markers (fibrinogen and leukocytes). interestingly, a tendency of association of a allele with higher values of obesity markers (bmi, waist and hip circumference) was noted. further studies are needed on a larger population in order to confirm these results. the water extract of capparis ovata (cowe) has been shown to be used as an alternative medicine for the treatment of multiple sclerosis (ms). cowe was further fractionated and studied for additional anti-neuroinflammatory effects in sh-sy5y cells. for this purpose, the dichloromethane sub-fraction of the cowe extract was tested for its anti-inflammatory effects on selected anti-inflammatory genes believed to be important in ms pathophysiology using sh-sy5y cells. cell viability was assessed using lactate dehydrogenase (ldh) activity in the media conditioned by the crystal violet cell staining. in these cells, levels of the tumor necrosis factor-a (tnfa), nuclear factor kappa-lightchain-enhancer of activated b cells (nf-jb1), glial fibrillary acidic protein (gfap), c-x-c motif chemokine 9 and 10 (cxcl9, cxcl10), matrix metalloproteinase 9 (mmp9), chemokine (c-c) motif 5 (ccl5) and tyrosine-protein phosphatase non-receptor type 11 (ptpn11) were determined by quantitative reverse transcriptase-pcr assay (qrt-pcr). we have found out that the dichloromethane sub-fraction of cowe effectively inhibited the expression of all of the genes given above in sh-sy5y cells. thus, phytochemicals present in the dichloromethane sub-fraction of the cowe extract could be beneficial in preventing/treating neurodegenerative diseases in which neuroinflammation is part of the pathophysiology. studies are underway to identify the individual compound(s) in this subextract of the cowe extract contributing to these effects. this work is supported by tubitak 112s187 and pamukkale university paubap 2014fbe051. p-04.04.4-019 apigenin and luteoline were identified as active anti-inflammatory constitutents of lavandula stoeachas by bioassay guided fractionation h. ipek 1 , s. savranoglu 2 , a. r. t€ ufekc ßi 3 , f. g€ ul 3 , i. demirtas 3 , t. boyunegmez t€ umer 4 1 graduate program of bioengineering, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, 2 graduate program of biology, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, 3 department of chemistry, faculty of sciences, c ß ankiri karatekin university, c ß ankiri, 4 department of molecular biology and genetics, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey introduction: lavandula stoechas, in the genus of lavender, has distinct therapeutic uses among anatolian people. rather than worldwide use of its essential oil in aromatherapy, specifically the aqeous portion as decoction has been traditionally used in anatolia against the components of metabolic syndrome, all of which share a state of chronic inflammation as an underlying cause. the anti-inflammatory constiutents of l. stoechas were isolated using a bioassay guided fractionation in lipopolysaccharide (lps) inflammed raw 264.7 macrophages. materials and methods: an aqeous extract was partitioned into ethyl acetate (eae) and n-butanol fractions. the eae, determined as bioactive extract was seperated into 12 subfractions by column chromatography. e6 was identified as active subfraction subjected to sephadex column to get pure compounds which were then applied to nmr, ir, and uv analyses for structure determination. in raw 264.7 cells, the effects of extracts/fractions/subfractions/compounds on lps induced no production was determined by using griess method. the potential inhibitory effects of each compound on lps induced inos expression were determined by qpcr and western blot. results: p-coumaric acid, apigenin and luteoline were found in the e6, and the first two compounds appeared to be primarily responsible for the anti-inflammatory activity. apigenin and luteoline at 50 lm decreased no production 66 and 80%-ic 50: 56 and 26 lm-by inhibiting inos gene expression 84 and 88% as well as protein expression 94 and 99%, respectively (p < 0.05). conclusion: this is the first time that luteoline and apigenin have been found in eae of l. stoechas, and the anti-inflammatory properties of the eae can be attributed, at least in part, to the presence of these two compounds. we are on the way to gain further insight for the action mechanism of these two active principles as anti-inflammatory agent. tubitak (project id:112t442) support this work. the role of tip60 in the inflammation process immun response generates the first line of host defense during inflammation and plays an important role inducing pro-inflammatory response by generating early response against pathogens. il-6 (interleukin 6) is one of the pro-inflamatory cytokines and its expression increases during the infection to activate the jak/ stat pathway. jak/stat pathway is regulated by hamp (hepcidin antimicrobial peptide). our previous study, we reported that hamp gene expression was decreased in liver-specific tip60 conditional knockout mice, so we thought that tip60 may have a direct or indirect role on inflammation mechanism. tip60 (tat interacting protein, 60 kda) is a member of the myst enzyme family of histone acetyltransferases (hats) and plays an important role in multiple function including cellular signaling, dna repair, cell cycle and apoptosis. in this study, the quantitative gene and protein expression of il-6 were investigated by using taqman real time pcr, western blot and immunohistochemistry analysis in control group, lpsinduced inflammation group and liver-specific tip60 conditional knockout group mouse liver. according to our preliminary results, the gene and protein expression of il-6 was increased in lps-induced inflammation group (p < 0.001, p < 0.001) and liver-specific tip60 conditional knockout group mouse liver (p < 0.05, p < 0.01). our initial data suggest that tip60 may be essential for the inflammation process. this work was funded by grants from the scientific and technological research council of turkey (tubi-tak) (grant number: 114z277). although intracellular reactive oxygen species (ros) level is necessary to maintain cellular homeostasis, elevated intracellular ros level with the impact of unfavorable environmental conditions leads to oxidative stress that may cause damage to dna, proteins and lipids. in case of inflammation, organism seeks to provide cellular homeostatis by increasing ros levels via antioxidant molecules and enzymes. therefore, it was thought that there can be a direct or indirect relation between inflammation and oxidative stress. in this study, inflammation was performed by intraperitoneal injection of lipopolysaccharide (lps). the gene expression and activity of antioxidant enzyme including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glutathione s-transferase (gst), glutathione reductase (gr) and glucose 6-phosphate dehydrogenase (g6pd). additionally, any change of reduced glutathione (gsh), oxidized glutathione (gssg), malondialdehid (mda), and hydrogen peroxide (h 2 o 2 ) level are accepted as an indication for the accumulation of ros, the relative levels of them were also studied. to show our inflammation model was performed in mouse kidney with lps treatment or not, the expression of interleukin (il6), which is accepted as a inflammation marker, was investigated by real time pcr. the expression of il6 was significantly increased in lps treated group. while the level of mda and h 2 o 2 was elevated in lps treated group, gssg was decreased. no changes was seen for gsh level. the correlation was observed between enzymatic and molecular levels. while the gen expression and the enzyme activity of sod, cat, gst, gr, and g6pd were decreased, gpx was increased with inflammation. in conclusion, increasing ros level was observed in the inflammation process and, the antioxidant system was affected at the molecular and protein level. this work was funded by grants from the scientific and technological research council of turkey (tubitak) (grant number: 114z277). the aim of our study is to evaluate effect of vitamin d levels on hemogram parameters including neutrophil %, lymphocyte %, neutrophil % / lymphocyte % ratio (nlr) and mean platelet volume (mpv) in behcet's patients. fifty eight patients with diagnosis of behcet that applied to selcuk university faculty of medicine department of dermatology are recruited to the study. clinical and laboratory characteristics of the patients were obtained from hospital automation. t test was used to examine the differences between the parameters. p < 0.05 was taken to be statistically significant. there was a statistically significant difference between vitamin d values and age (p = 0.036) whereas difference was not significant between vitamin d and neutrophil %, lymphocyte %, nlr, mpv values. according to the literature, there are a lot of studies that show the relationship between vitamin d and hemogram parameters. however, contrary to the previous studies, we were unable to find any significant relationship between vitamin d and these hemogram parameters. these results serve the idea that the effects of vitamin d on the hematopoietic system should be further investigated experimentally and clinically. crimean-congo hemorrhagic fever is a tick-borne disease caused by the arbovirus and characterized by a sudden onset of high fever, severe headache, dizziness, back and abdominal pains. the exact pathogenesis of cchf has not been clarified yet. the aim of this study, clinical cases of cchf in cu, se and zn is to examine the relationship between the concentration of trace elements. the study sample consisted of 30 patients which have been diagnosed with cchf. matched for gender, 30 healthy volunteers were similar to the control group according to age. the patients and control groups, serum cu, zn and se levels were analyzed using atomic absorption spectrophotometer. cchf patients in the group, cu zn and se serum levels were significantly lower compared with the control group. in our study, the cofactor of the antioxidant enzyme cu, zn and se elements were lower. this shows us in cchf disease, a decrease in antioxidant enzyme activity, and suggest that they contribute to the immune system's degradation. p-04.04.4-024 inhibitors of mdm2 ubiquitin ligase as prospective modulators of autoimmunity e. bulatov, a. valiullina, r. sayarova, a. rizvanov kazan federal university, kazan, russia ubiquitin-proteasome system is seen as a pool of promising protein targets for therapeutic impact in many human diseases. mdm2 is an e3 ubiquitin ligase widely studied due to its wellknown role in cancerit negatively regulates p53 oncosuppressor that mediates apoptosis in tumour cells. inhibitors of p53/ mdm2 interaction have long been known as potential anticancer therapeutics. however, recent advances in the field suggest that both mdm2 and p53 might be playing a substantial role in autoimmune processes. we used a small molecule p53/mdm2 inhibitor nutlin-3a to test the effect of p53 activation on peripheral blood mononuclear cells (pbmcs) from both healthy volunteers and patients diagnosed with multiple sclerosis. in our study we employed a variety of molecular biology methods, such as immunoblotting, real-time pcr, mts cell proliferation assay, fluorescence flow cytometry and confocal microscopy. we demonstrated that disruption of p53/mdm2 interaction by nutlin-3a alters the p53 levels and also affects the lymphocyte subpopulations within pbmcs. our findings suggest that p53/mdm2 interaction inhibitors can potentially be used as prospective modulators of immune response in autoimmune diseases such as multiple sclerosis, systemic lupus erythematosus and other. the study was funded by rfbr research grant 16-34-60213 mol_a_dk. can ykl-40 be an inflammatory biomarker in vitamin d deficiency? object: the tumor necrosis factor (tnf) was found to be cytotoxic to tumor cells and to induce tumor regression in mice. except for one member, all receptors to the tnf superfamily bind tnf-related ligands and act mostly on inflammatory system. there are currently 19 tnf superfamily ligands. tnf superfamily ligands share several common features. tnf ligands are generally type ii transmembrane proteins whose extracellular domains are be divided by enzyme to create cytokines. the tnf superfamily currently consists of 29 receptors. tnf family receptors are type i or type iii transmembrane proteins that contain multiple extracellular domains. in this study, we investigated to presence, differences and effects of tnf superfamily and receptors genes in human and mice by using bioinformatics techniques. methods: the nucleotide and amino acid sequence of each protein in human and mice was determined using t-blast-n for homologous sequences. homologous sequences of human tnf family genes found an automated procedure by using psi-blast. the secondary structure of and three-dimensional of the protein were analyzed by psipred and ffas server. netcglyc 1.0 and netphos 2.0 program were used for post-translocation modifications. the web apoptosis database was also used for the lists of domains, proteins containing these domains and their associated homologs. results and conclusion: humans tnf ligands have 17 genes encoding proteins that contain a conserved carboxy-terminal domain. this family of proteins is highly conserved between humans and mice. humans contain 29 genes encoding tnffamily receptors. sequence data from the ncbi databases demonstrated the presence of 24 mouse tnf-family receptors with orthologs in humans and one additional receptor found only in mice. the differences and similarities in the tnfs genes in humans and mice will provide information for understanding the utility and limitations of the mouse models of disease and comparing of immunology outcomes. the development of left ventricular remodeling after acute myocardial infarction is a predictor of shock. the genetic influence on cardiac remodeling, and shock in the early period after acute myocardial infarction are unclear. the aim of the present study was to investigate the relationship between angiotensin converting enzyme (ace) gene polymorphism and modified shock index (msi) in the early period in patients with acute anterior myocardial infarction. overall 140 patients with a first acute ami were included in this study. dna was isolated from peripheral leukocytes. the id status was determined by pcr. based on the polymorphisms of the ace gene, they were classified into 2 groups: deletion/deletion (dd) genotype (group 1, n = 57), insertion/deletion (id), insertion/insertion (ii) genotypes (group 2, n = 83). blood pressure and pulse measurements were performed in all patients within 10 minutes admitted to coronary care unit. msi was defined as heart rate (hr) divided by mean arterial pressure (map). echocardiographic examinations were performed in accordance with the recommendations of the american echocardiography committee. one-way analysis of variance (anova) and chi-square analyses were used to compare differences among subjects with different genotypes. the study was approved by the local ethics committee, and each patient gave a written consent. there were no significant differences among clinical parameters of patients. msi was significantly higher in patients who have ace dd genotype than in patients who have ace id / ii genotypes (1.13 ae 0.52 and, 0.85 ae 0.37, p < 0.05). presentation time hypotension or developing hypotension during admission was reported to be an important predictor of intensive care unit admission besides other vital sign measurements. our results suggested that, ace gene i/d polymorphisms d allele may affect modified shock index in patients with a first acute anterior mi. glucocorticoids (gcs) are widely used in medicine, despite their side effects, e.g. osteoporosis. however, precise molecular mechanisms of gc action, especially on bone marrow (bm) cells, remain controversial. given the osteoprotective role of vitamin d 3 , the aim of our study was to examine prednisolone-induced changes in the rank (receptor activator of nuclear factor kappa-b)/rankl (rank ligand)/opg (osteoprotegerin) pathway of rat bm depending on the state of vitamin d endocrine system. female wistar rats received prednisolone (5 mg/kg b.w.) with and without 100 iu of d 3 (for 30 days). the levels of rank, rankl, opg, 1a-hydroxylase (cyp27b1) in bm were determined by western blotting. vitamin d 3 receptor (vdr) and rankl mrnas were measured by quantitative rt-pcr. 25ohd 3 content in the serum was assayed by elisa. rankand vdr-positive bm cells were quantified using flow cytometry and visualized by confocal microscopy. prednisolone induced a marked increase in rankl and rank levels, while opg level was shown to decrease. this reflects disturbances in cytokine-mediated regulation of bm progenitor cell function. data from flow cytometry indicated a significant growth in the number of rank-positive cells (hematopoietic osteoclast precursors) compared to control. these changes were accompanied by a decrease in the levels of vdr and cyp27b1, which is responsible for 1,25(oh) 2 d 3 synthesis, in bm and 25ohd 3 content in serum. co-localization of vdr and rank in mono-and multinuclear bm cells was observed, indicating a close relation between vitamin d 3 and rank/ rankl/opg pathway. vitamin d 3 co-administration prevented prednisolone-induced changes in bm cells through restoration of vitamin d 3 bioavailability and vdr signaling that resulted in a reduction of the osteoclast progenitor pool in bm. thus, prednisolone-induced imbalance in rank/rankl/ opg system components is associated with impairments of vitamin d endocrine system in bm and can be ameliorated by vitamin d 3 treatment. p-08.01.4-002 heat shock pathway in response to different stress factors the heat shock response is an emergency pathway of the cell, which mediates repair and protection from cellular stress and therefore guarantees the survival of the cell. this stress can range from heat or hypoxia to chemicals and heavy metals. it is highly conserved in all eukaryotic cells and plays an important role during atypical conditions. due to its high complexity, the pathway is not yet completely understood. most important, after activation of the pathway, is the refolding of proteins or, in case of severe misfolding, the depletion of proteins to maintain proteostasis. heat shock factor 1 encoded by the hsf1 gene is known as the main switch point in heat shock regulation. after activation it trimerizes and binds to heat shock elements in target gene promoters. one of these promoters is the hspa1a promoter (hsp72 promoter). the promoter was analyzed by dismantling it to its functional parts. especially three elements, the heat shock elements, were in the focus of this work. in first place parts of the promoter were multimerized and combined with different reporters, like luciferase, by cloning. also mutations in the natural promoter were designed by cloning. the focus now is on the heat shock elements, where hsf1 can bind as a trimer. the idea is that these different elements have various effects on different stressors like heat, chemicals (geldanamycine as hsp90 inhibitor, mg132 as proteasome inhibitor) or heavy metals (cadmium, arsenic, zinc) . this was tested on cells transiently transfected with those promoter variants. for promising variants stable cell lines were created. in these stable cell lines further experiments on mrna level can be conducted. in the last months experiments with the crispr/cas9 system were started. furthermore, experiments on transcriptional (qpcr) and translational (dual-luciferase assay) levels were done as well. in the end we hope to get a clear picture on the regulation of the hspa1a promoter by different stress factors. invasive cancer cells form membrane protrusions, invadopodia, that facilitate cell invasion and metastasis. key players invadopodia include the adaptor proteins tks4 and tks5, the actin regulators cortactin, wip and n-wasp, the kinase src and others. in spite that in the last two decades significant advances in our knowledge of the structure and development of invadopodia have been made, detailed mechanisms they are functioning is not yet available. we have identified a series of new tks4 binding partners including adaptor proteins itsn1, itsn2, crk and grb2, kinase src, amph1, bin1, plcg1 and also another member of the tks family -tks5. it may indicate the possible role of tks4 in transport and sorting of cell vesicles. current data are supported by interaction with the proteins of amph1 and bin1, as their main functions are membrane trafficking and remodeling. adaptor proteins crk, grb2 and itsns are important for the actin cytoskeleton rearrangements, endocytosis and signal transduction. moreover, we have identified and characterized new tks4 isoform -tks4-beta. we suggested that an active state of tks4 is regulated via intramolecular interactions between its proline-rich motifs and own sh3-domains. we have shown the interaction between itsns and other prominent component of invadopodia wip. data from immunofluorescent analysis revealed co-localization of itsn1 and wip at the sites of invadopodia formation and in clathrin-coated pits. we have also demonstrated that the key protein itsn1 and wip and n-wasp can form a complex in cells. together, these findings provide insights into the molecular mechanisms of invadopodia formation and identify itsns as scaffold proteins involved in this process. we have shown the interaction between itsns and other verprolin family members cr16 and wire which play an important role in the reorganization of the actin cytoskeleton. we have demonstrated that cr16 and wire interact with sh3domains of itsns in complex with actin. p-08.01.4-004 correlation between proteomic and phenazine profile of pseudomonas sp. phenazines are widely known compounds with huge variativity of biological activities which are produced by pseudomonas sp. and some other bacteria species. the results of our work shows the correlation between the changes of proteomic profile of pseudomonas aeruginosa caused by a mutagenesis and the secondary metabolism of antibiotics (phenazine) profile. different strains of pseudomonas aeruginosa were obtained using mutagenesis, after that bacterial cells were destroyed by ultrasound. protein-containing fractions were isolated using methanol-chloroform method as well as phenazines compounds were extracted from culture media using liquid phase extraction. obtained proteome was analysed by shotgun-proteomics technique. as the result of the liquid phase extraction phenazine compounds were mainly extracted to the organic phase. this phase was evaporated and re-dissolved in 100% methanol. after sample preparation obtained solutions were analyzed by hplc-agilent 1290 with quadrupole tof mass-detector. results of the analysis were compared with the library of known phenazine compounds mass-spectras generated by cfm-id online resource. obtained phenazine profiles were compared with each other and correlation with the changes in proteome was analyzed. received results promote better understanding of mechanisms of phenazine production. this data opens possibilities for targeted changes in the methabolic pathway in order to obtain phenazine compound with required biological activity. insulators are genomic elements which block enhancer-promoter interaction and prevent spreading of heterochromatin. cp190 protein is an integral component of most known drosophila insulators, it interacts directly with ctcf and pita dna-binding insulator proteins using dimeric btb-domain, but function of cp190 within insulators still remains to be elucidated. recently we described an interaction between cp190 btb-domain and cterminal domain of ctcf insulator dna-binding protein, subsequent deletion analysis allowed us to isolate 50aa fragment within ctcf c-terminal domain sufficient for interaction with cp190 btb, but deletions of flanking regions also lead to the loss of interaction with cp190 in vivo. at the same time crosslinking experiments suggest that a dimer of btb interacts with one molecule of ctcf, presuming that it could recognize two peptide fragments within ctcf c-terminal domain. we solved crystal structure of btb-domain from cp190 insulator protein at 1. 3 a resolution. overall structure is similar to other btb-domains. cp190 btb-domain has peptide-binding groove similar to that previously found in bcl6 btb domain. inspection of btb-domain surface revealed several possible binding sites for polypeptide fragments from ctcf protein. based on these observations a set of point mutations within peptide-binding groove of btb-domain has been designed and we tested ctcf-interaction abilities of these mutants using gst pull-down assay and yeast two-hybrid assay. the most significant impact was found with alanine-substitutions of hydrophobic residues whereas substitutions of hydrophilic amino acids were less effective. therefore our results support that cp190 btb-domain recognizes ctcf protein using peptide-binding groove. this study was supported by the russian science foundation (project №14-24-00166). p-08.01.4-006 comparative study of the fatty acid composition of lipids in the raw meat samples obtained from hybrid sheep one of the most important tasks in the animal biology and husbandry is to clarify the role of animal genetic diversity in providing nutrients to the diversity of animal products. the objective of our work was to study the chemical compositions of raw meat samples obtained from domestic (group i -purebred romanov sheep) and hybrid sheep (group ii -f 3 hybrids of romanov sheep with 12.5% of argali blood). the significant changes in fatty acid composition of the lipid fraction from the fat and muscle tissue of the hybrid sheep as compared to the control were found. the content of saturated fatty acids (sfas) in the fat samples of the hybrid animals was by 12.16% lower (55.12 ae 2.30%, p < 0.01), but polyunsaturated (pufas) or monounsaturated fatty acids (mufas) contents were by 0.64% and 9.43% higher (10.10 ae 0.43 and 28.78 ae 1.77 (p < 0.01), respectively) as compared to purebred romanov sheep. the most pronounced changes were found for palmitic acid (decreased from 27.70% to 14.24%) and for oleic, linoleic, arachidonic acids (increased from 20.37%, 5.58%, 0.35% to 29.98%, 6.10%, 0.70%, respectively). the last two acids together with the linolenic acids belong to the so-called essential acids and very important for the animal metabolism. a similar trend was observed on the composition of the lipid fraction of muscle tissue. sfas, pufas and mufas content in muscle tissue of hybrid sheep was 53.38 ae 0.34, 9.39 ae 1.00 and 34.16 ae 0.39%, that was 11.17% lower (p < 0.001), and 4.13% and 4.67% higher (p < 0.01) compared to purebred romanov sheep. these results emphasized the difference of the pufas/sfas ratios in fat and muscle tissues, respectively) and characterized the biological value of the lipid fraction of fat and muscle tissue. the obtained data gave evidence of the positive changes in the fatty acid compositions of the lipid fractions for the hybrid animals as compared to the purebred sheep. supported by the russian scientific foundation, no. 14-36-00039. foodborne illnesses resulting from the consumption of agricultural commodities contaminated with enteric pathogens are an increasing problem around the world. while various possibilities of produce contamination with pathogens exist, the global warming combined with a widespread use of animal manure in agriculture will likely contribute to an increased number of such outbreaks. thus, phages isolated from different agroecosystems may prove to be useful in detection/biocontrol of enterobacteria in produce. during the investigation of the impact of global warming on the diversity and co-evolutionary dynamics between microorganisms and viruses in lithuanian agroecosystems, a novel enterobacteria phage vb_ecos_nbd2 (nbd2) was isolated from agricultural soil using e. coli novablue for phage propagation. nbd2 genomic dna was isolated from cscl-purified phage particles, and was subjected to illumina dna sequencing. nbd2 is a virulent siphovirus that has a low-temperature plating profile (fails to form plaques at a temperature >30°c). the genome of nbd2 is $ 52 kb long, and has a total of 87 probable protein-encoding genes as well as 1 gene for trna ser . the genome analysis revealed that 20 nbd2 orfs encode unique proteins that have no reliable identity to database entries. among the orfs that encode proteins with matches to those in other sequenced genomes, 64 are similar to proteins from phages that infect different members of enterobacteriaceae, while 3 nbd2 orfs are most similar to those from bacteria. based on the similarity to biologically defined proteins, 32 nbd2 orfs were given a putative functional annotation, including 15 genes coding for morphogenesis-related proteins, as well as 14 associated with dna replication, recombination, and repair. phylogenetic analysis revealed that enterobacteria phage nbd2 is distantly related to phages belonging to the subfamily tunavirinae. this research was funded by a grant (no. sit-7/ 2015) from the research council of lithuania. p-08.01.4-009 the antibiotic novobiocin affects the composition of the escherichia coli proteome n. e. arenas 1 , j. williamson 2 , v. schw€ ammle 2 , s. douthwaite 2 1 universidad de cundinamarca, cundinamarca, colombia, 2 university of southern denmark, odense, denmark novobiocin (nov) is an aminocoumarin which competitively inhibits the atp binding site in the gyrase-b subunit of prokaryotic topoisomerase ii. nov remains a therapeutic choice for treating infections with bacterial pathogens that are resistant to more commonly used drugs. the aim of this study is assess the proteomic response of e. coli strain upon nov treatment. minimum inhibitory concentrations of nov were measured by standard assays. three different e. coli strains (as19, as19-rlma::aph and b) were grown aerobically in nutrient rich lb media at 37°c during one hour. the whole cell proteome (five biological replicates in each sample,) was assessed by lc-ms by using tmt labelling protocol. raw files were imported to proteome discoverer (thermo fisher scientific) and searched together with mascot against the uniprot e. coli reference proteome. mics for nov were determined to be >1000-fold higher the wild-type b-strain of e. coli than for the hypersusceptible as19 strains (1 lg/ml). whole genome comparison of the b and as19 strains were characterized by an increase in proteasome components (6 proteins), chaperones (3), error-prone dna polymerase components (4), ribosomal hibernation factors (3), heat shock response (2), electron transport coupled proton transport (8), pentose phosphate pathway (9), flagellar assembly (4), oxidative phosphorylation (10) and tca cycle (8). whereas ribosomal proteins (45), aminoacyl-trna synthetases (7), rnases (6), abc transporters (17), mismatch repair (3) and sec secretion pathway (4) were significantly down-regulated upon nov treatment. the three e. coli strains respond similarly upon nov treatment and their proteomes showed upregulation of heat shock response with changes in the components of translation and transcription, the proteasome and atp biosynthesis. the changes observed can be used to define the processes that are required for antibiotic tolerance and survival of e. coli against aminocoumarin antibiotics. postnatal growth is under control of pituitary derived hormone, growth hormone (gh) that triggers bone, fat tissue growth and development via acting on protein, carbohydrate and fat metabolism. gh functions on postnatal development by jak2/stat5 signaling following gh:gh receptor (ghr) dimerization. isolated growth hormone deficiency (ighd) is a medical condition of insufficient production of growth hormone (gh) that is caused by mutations on gh-n gene in different ethnic origin children. various mutations within gh has been determined in different populations so far, and glutamic acid to glycine (e33g), asparagine to aspartic acid (n47d), threonine to alanin (t-24a) missense mutations, alanine to serine (a13s) substitution, tryptophan to stop codon (w-7x), gaaa insertion in intron 1 of gh-n gene and both intron 1 (+83c) and deletion of 166. amino acid of gh protein phenylalanine (f166del) mutations were detected in turkish ighd children. the potential role of these mutations on cell growth, proliferation, emt via acting on gh signaling pathway has not been observed yet. all these mutations were performed on wild type gh-n gene inserted pc3.1 vector by site-direct mutagenesis and stable cell line of each gh gene mutations were generated by neomycin selection. although w-7x, e33g, f166del, a13s and n47d mutations suppresses gh signaling via acting on either jak2 dephosphorylation or stat5 downregulation, t-24a, gaaa insertion and deletion of +83c mutations have no significant effect on gh signaling. in addition, each mutation lead different growth suppression effect and colony formation potential and intracellular polyamine levels and odc expression profiles were essential role in emt potential of hek293 cell lines. as a result, w-7x, e33g, f166del, a13s and n47d mutations prevented gh signaling and cell growth and differentiation via polyamine metabolism. pulmonary embolism (pe) is a common cardiovascular emergency and affects a large number of patients. acute pe-induced oxidative stress can lead to the accumulation of specific nitroproteins that may play a role in disease progression. the impact of nitration of a single tyrosine residue often has broad implications on the activity of biologically critical proteins, which has become increasingly related to pathological conditions. in this study, we used a proteomic approach to analyze nitrated serum proteins in patients diagnosed with acute pe and healthy controls. nitrotyrosine (no2tyr)-containing proteins were immunoprecipitated from serum with a no2tyr affinity sorbent. precipitated proteins were separated by sds-page and visualized by coomassie blue staining and western blotting with mouse monoclonal anti-no2tyr antibody. among the numerous immunoreactive bands observed in disease patients, the 138 kda protein band was in-gel digested and analyzed by maldi-tof mass spectrometry (ms). mass fingerprint data sets obtained from the peptide fragment ions matched human collagen alpha-1 (iii) chain (co3a1_hu-man) with mascot algorithm analysis giving a score of 65 (p < 0.05). collagen alpha-1(iii) chain is a fibrillar collagen that is found in extensible connective tissues such as skin, lung, and the vascular system. altered metabolism of collagen and its excessive deposition in the matrix of the connective tissue is a hallmark of chronic interstitial lung diseases. collagen can be measured in serum and bronchoalveolar lavage fluid from patients with numerous chronic interstitial lung diseases. given these considerations, future studies are aimed understand the relevance of no2tyr modifications in co3a1 relating to changes in protein structure and function. recent studies have shown that the genes involved in dislipidemia represent potential loci to be associated with diabetes as a disease. recent genome wide association (gwa) studies have associated rs1260326 in gckr gene and rs4846914 in galnt2 gene with parameters of t2d and diabetic dyslipidemia. in this study, the association of these single nucleotide polymorphisms (snps) with t2d and dyslipidemia was tested in the population from bosnia and herzegovina (bh). our study involved 352 patients with t2d and 156 healthy subjects. biochemical and anthropometric parameters were measured in all participants. after dna extraction, sequenom iplex platform was used for the analysis of galnt2 polymorphism (rs4846914), while polymorphism in gckr (rs1260326) gene was analyzed by using real time pcr. our results demonstrated significant association of gckr rs 1260326 variant with waist circumference (p = 0.003) and fasting glucose levels (p = 0.003) in the control group. no such association was demonstrated for rs4846914 galnt2 gene. in the group of diabetic patients, significant association of gckr rs1260326 variant with levels of bilirubin (p = 0.004) and rs4846914 galnt2 variant with hba1c (p = 0.013) and triglyceride levels (p = 0.043) was also demonstrated. our results suggest an association of variations of gckr and galnt2 genes with specific markers of t2d and dyslipidemia. further studies would be needed in order to confirm these genetic effects in other ethnic groups as well. osteoporosis is the most common metabolic bone disorder affecting the normal bone turnover with low bone mineral density (bmd) and risk of fragility fractures. polymorphisms at the sp1 binding site of the collagen type 1 a1 (col1a1) gene is associated with low bmd. we examined the distribution of col1a1 gene polymorphism in 50 young osteoporotic women and in control group in turkish population. patients had low bmd with t score ≤2.5 sd and controls was 25 healthy women (35-57 years). mean age (51.28 ae 5.8) and (46.56 ae 6.15) respectively. the bmd, as g/cm 2 , was measured in the hip and the lumbar spine (l2-l4) with (dexa). dna was isolated from blood. col1a1 gene was analysed with genomica clinical array system. the x 2 test was used to compare allele and genotype frequences between patients and controls. mean of t score in patients was à3.06 ae 0.42. mean bmd (as g/cm 2 ) was 0.708 ae 0.073, and (1.009 ae 0.823) genotype distribution were18(36%) ss, 31 (%62)ss, 1(%2)ss for patients, and 12(48)ss, 9(36)ss, 4(16)ss for control . patients had 33(%66)s allele, 17(34%) s allele, controls had 67(67%)s allele, 33 (33%)s allele. when genotypes and bmd were compared in patients, there was no significant correlation between osteoporosis and genotypes. the allelic distribution was not significant between patients and controls p > 0.05. genotypic distribution in patients were significantly different. patients had a higher frequency of the ss(%62) than controls (ss %36) p < 0.05. this study shows that high prevalences of the ss genotype at the col1a1 locus, in osteoporosis . _ it is possible that the presence of the s allele causes variation col1a1 and col1a2 mrna's producing abnormal collagene protein. since collagen protein is major protein of bone, it is to be expected that a defect in this protein will produce bone fragility. col1a1 gene should be detected early to initiate preventative therapy for bone health. the biological activity of nigella sativa seeds is mainly attributed to its essential oil component which is pre-dominantly (30-48%) thymoquinone (tq). therapeutic effect of tq was exhibited in many diseases including inflammation, cancer, sepsis, atherosclerosis and diabetes. tq has been reported to exhibit antiproliferative effects on cell lines derived from breast, colon, ovary, larynx, lung, myeloblastic leukemia, and osteosarcoma and inhibited hormone refractory prostate cancer. tq induces apoptosis in tumor cells by suppressing nf-jb, akt activation, and extracellular signal-regulated kinase signaling pathways and also inhibits tumor angiogenesis. the aim of this study was to evaluate the anti tumor effects of tq on hepatoma cells. these antitumor assays include cell viability assay, clonogenic assay, scratch assay and molecular expression studies of death related genes. cells were treated with different concentration of tq in hep3b for cell proliferation by mtt and clonogenic assay. in addition, the metastatic character of tq was investigated by scratch assay in hep3b at 3-6 and 24 hours. the effect of tq was also evaluated at mrna level by real-time-pcr. tq was treated on the hep3b cells in three different concentration, namely 75-50 and 37.5 lm. tq showed the cell cytotoxicity in concentration and time dependent manner. the scratch assay revealed no healing in the scratched area due to the decreased cell viability. maximum permissible dose was 50 lm. proapoptotic genes, bax and bad, and autophagy genes, beclin-1 and lc3, were upregulated in hep3b cells after 24 hours treatment in contrast, antiapoptotic gene, bcl-2, expression level was decreased for hep3b cells after 24 hours. p-08.01.4-019 association of irs1 genetic variation with type 2 diabetes and insulin resistance in patients from bosnia and herzegovina insulin receptor substrate-1 (irs1) encodes the irs1 protein, a substrate for the insulin receptor tyrosine kinase and has a critical role in insulin-stimulated signaling pathways. previous studies showed that irs1 single nucleotide polymorphisms (snps) were associated with type 2 diabetes mellitus (t2d). this is the first study performed in a population from bosnia and herzegovina (bh) in which we examined the association of rs7578326 (g>a), rs2943641 (t>c) and rs4675095 (a>t) with t2d risk and related traits. our study involved 437 t2d patients and 252 healthy subjects. biochemical parameters, including but not limited to insulin, homa-ir, hba1c, glucose, and lipoprotein levels, were measured in all participants. genotyping analysis was performed by mass array sequenom iplex platform in cooperation with lund university diabetes centre, malmo, sweden. statistical analysis was done by spss 23. our results demonstrated a significant difference in frequency of rs4675095 (p < 0.001) and rs7578326 (p = 0.021) snps between t2d patients and control subjects. interestingly, here we showed a significant association of irs1 rs4675095 risk t allele with increased insulin levels (p < 0.001) and homa-ir (p < 0.001) in t2d patients. similarly, rs7578326 variant was also associated with the same markers of insulin resistance in diabetic patients, i.e. insulin levels (p = 0.029) and homa-ir (p = 0.025). no such association was demonstrated for rs2943641. however, this irs1 variant was associated with changes in lipoprotein levels, where risk c allele increased vldl (p = 0.006) and decreased hdl levels. our results suggest that irs1 variants are associated with t2d susceptibility in bh population, thus confirming similar findings in other population cohorts. furthermore, the associations of these variants with markers of insulin resistance and dyslipidemic metabolic changes point to their role as potential t2d biomarkers. the adra2a gene encodes alpha-2a adrenergic receptor which mediates adrenergic suppression of insulin. a genetic variant in adra2a was recently associated with defective b-cell function. the objective of this study was to analyze association of two adra2a polymorphisms (rs553668 a>g and rs10885122 g>t) with type 2 diabetes (t2d) and its related traits. in this study we have included 437 t2d patients and 252 healthy subjects from bosnia and herzegovina (bh). biochemical parameters, including but not limited to insulin, homa-ir, hba1c, glucose, and lipoprotein levels, were measured in all participants. genotyping analysis was performed by mass array sequenom iplex platform in cooperation with lund university diabetes centre, malmo, sweden. statistical analysis was performed by ibm spss statistics 23 software. our data showed that frequencies of both, rs10885122 and rs553668, variants were not significantly different between t2d and control subjects. however, rs553668 risk a allele appear to increase insulin levels (p = 0.0002) and homa-ir index (p = 0.00001). furthermore, this variant also seems to affect vldl levels (p = 0.004) and waist circumference (p = 0.02) in diabetic patients. the genotype analysis of rs10885122 variant demonstrated that risk g allele decreased hdl (p = 0.0004) and increased ldl levels (p = 0.014), as well as affected the waist circumference (p = 0.02) in diabetic patients. interestingly, haplotype analysis demonstrated the association of rs553668a / rs10885122g with higher homa-ir index. here we demonstrated that although both, rs10885122 and rs553668, adra2a polymorphisms were not associated with t2d risk in our cohort, they were associated with markers of dyslipidemic perturbations and insulin resistance in diabetic patients. further studies in larger cohorts are needed in order to explore these possible interactions and confirm our findings. smad-interacting protein 1 (sip1), also known as zeb2 is a member of zeb family transcription factors and was shown to regulate epithelial-to-mesenchymal transition, cell cycle, cellular senescence and cancer stemness. bipartite zing finger motifs at amino and carboxyl termini of the sip1 mediate its binding to ebox sequences in the genome. however, there are only limited data about sip1 target genes. by using a home-made anti-sip1 monoclonal antibody, clone 6e5, we conducted chip-seq in three hepatocellular carcinoma cell lines, namely snu398, plc/prf/5 and sk-hep-1 and found receptor tyrosine kinase-like orphan receptor 1 (ror1) as one of the targets. sip1 dnabinding consensus motif cacctg was found at +1 kb, +30 kb and +50 kb from ror1 transcription start site. chip experiments validated sip1 binding to all consensus motifs in the ror1 gene region. interestingly, the strongest enrichment was at +50 kb suggesting that long-range interactions play an important role in the regulation of ror1 by sip1. sip1 knockdown by shrna in high-sip1 expressing snu398 cells resulted in the repression of ror1 expression. ror1 is expressed in embryogenesis and fetal life, and is absent within most of adult normal tissues. however, overexpression of ror1 was observed in many human cancers, from hematological malignancies to solid epithelial tumors. ror1-positive cancer cells have enhanced proliferation, invasion and metastasis capacities, show resistance to apoptotic stimuli and display cancer stem cell characteristics. therefore, sip1 and ror1 act in similar pathophysiological processes. our finding that ror1 is regulated by sip1 at least in hepatocellular carcinoma cells adds another level of complexity to the molecular mechanisms of proliferation, invasion and stemness of cancer cells. hepatocellular carcinoma (hcc) is the most prevalent primary liver cancer and is one of the leading causes of cancer related deaths. smad interacting protein 1 (sip1), a member of the zeb family of emt inducers, is involved in cellular proliferation, senescence, invasion and metastasis in human tumors. however, genes regulated by sip1 in hcc are yet to be identified. we conducted a chip-seq study in high-sip1 expressing hcc cell line snu398 by using a home-made anti-sip1 antibody, clone 6e5. among 509 annotated genes, we selected six1 for further studies because of its increased expression in multiple cancers and its association with poor prognosis. sip1 dna-binding motif cacctg was found at -3 kb from transcription start site of six1 gene. chip qpcr experiment validated sip1 binding to this region with 19,7 fold enrichment. compared to healthy liver, six1 transcripts were upregulated in 8 of 9 hcc cell lines included in this study. knockdown of sip1 by shrna in snu398 cells caused upregulation of six1. immunohistochemistry studies in hcc tissue arrays showed increased expression of six1 in tumors and inverse association with sip1 expression in a tumor grade dependent manner. therefore, our results strongly suggest an inverse correlation of sip1 and six1 in hcc bone mineral density (bmd) and bone turnover are under genetic control and variations in the vitamin d receptor (vdr) are related to bmd. bmd is known to be affected by 25 -hydroxy vitamin d (25 (oh)d) and intact parathyroid hormon (ipth) levels. we aimed to determine correlation blood levels of vitamin d (vitd), ipth, and vdr gene effect in healthy turkish women. the subjects were 25 healthy women in age 35-57 years. the bmd was measured as a t score in the lumbar spine (l2-l4) with dexa. all subjects had normal t score between (-1.0 to 1.4) sd. vitd was measured by lc-20-at shimadzu. ipth was measured by chemiluminescence method, dna was isolated from blood. the fok i (vdrf-foki) and bsmi (vdrb-bsmi) polymorphisms of vdr gene was analysed with genomica clinical array system. the mean vitd level was (21.06 ae 14.54) lg/l, mean plasma ipth level was (57.96 ae 30.49) pg/ml. pearson correlation test showed no relation of vit d with bmd. there was moderately negative correlation between ipth and bmd (r = à0.3062). genotype distribution and allele frequency of subjects were as follows: 21(84%) ff), 1(4%) ff, 3 (12%) ff genotype in vdrf -fok1 gene, 7 (28%) bb, 14 (56%) bb, 4 (16%) bb in vdrb-bsmi gene. allele frequencies were f: 86%, f:14%; b:56%, b:44%. when fok1 and bsmi were combined, 52%(ff-bb) and 20% (ff-bb) were found as the most frequent genotypes. bsmi frequency was in hardy weinberg equilibrium (p > 0.5). but foki was not (p = 0). it was found that vit d, ipth levels and bmd were in normal levels in all carriers of ff genotype and in combined (ff-bb) type carrying healthy women (52%). the association vdr genotype and bmd may be different in various ethnic and geographical groups. therefore it is worthwhile to assess vdr polymorphism among turkish population. these type of distribution studies of vdr in healthy and in osteoporotic women may enlighten to earlier diagnosis and treatment planning. p-08.01.4-026 determination of hb a1c values in beta thalassemia f. g€ uzelg€ ul 1 , g. s. seydel 2 , a. e. yalin 3 , e. s€ onmez 1 , k. aksoy 1 1 c ß ukurova university, adana, 2 nigde university, nigde, 3 mersin university, mersin, turkey introduction: hemoglobinopathies are most commonly seen hereditary blood diseases worldwide. our aim was to compare the hba1c values measured on cation-exchange high performance liquid chromatography (hplc) in beta thalassemia cases. materials and methods: we collected 3 ml of whole blood k 3 edta containing tubes from forty-nine cases. arms, rflp and dna sequence analysis methodologies were carried out for determination of beta thalassemia mutations. hb a1c values were measured using the agilent 1100 hplc system. results: forty-nine diabetic and non-diabetic patients were diagnosed with beta thalassemias: twenty-one ivs1-110/ivs1-110, one ivs1-1/ivs1-1, one ivs1-5/ivs1-5, two ivs1-6/ivs1-6, two ivs2-1/ivs2-1, one fsc5/fsc5, one fsc44/fsc44, two -30/-30, two cd8/cd8, one cd36-37/cd36-37, one cd8-9/cd8-9, two cd82/cd83-g/ cd82/cd83-g, two ivs1-110/ ivs1-6, one ivs1-110/ ivs2-1, one ivs1-110/fsc44, one ivs1-110/cd39, one ivs1-110/cd8, one ivs1-110/cd8-9, one ivs1-110/ivs1-5, one ivs1-6/ ivs2-1, one ivs1-6/ ivs1-25, one ivs2-745/cd8 and one fsc5/cd37. cases were classified as diabetic (6), prediabetic (11) and non-diabetic (32) introduction: members of aurora kinase family aurora a, b and c are conservative kinases of cell cycle which are encoded by genes aura, aurb and aurc respectively. overexpression of aura and aurb was found in human cancers, especially in prostate cancer. moreover, there is the evidence that aurb interacts with one of the major oncogenic kinases -braf. little is known about implication of aurc in cancer, but it was demonstrated, that it can overlap aurb function and shares its location. we studied expression of genes of these kinases in urine of prostate cancer patients aiming to evaluate their involvement in this disease and their potential as tumor markers. materials and methods: 22 urine samples from patients with prostate cancer were gathered after prostate massage before surgical invasion. we used urine samples from 6 healthy men as control. we obtained cells from each urine sample by centrifugation and isolated rna using standard approach with phenol and guanidine thiocyanate. cdna was synthesized and taken to qpcr reactions. data was statistically analysed. results: expression of all studied genes was detected in urine of patients with prostate cancer and of healthy men. expression of aurb and aurc in cancer samples each was higher than expression of aura. the cumulative expression aurb and aurc was higher than expression of aura in 13 samples from 22. we observed positive correlation between expression of aurc and braf (rs = 0.548, p = 0.01). discussion and conclusion: previous investigation showed, that for normal prostate tissue 90% of aurora family expression was presented by aura. we suppose that presence of aurb and aurc cumulative overexpression means presence of cell cycle deviations in prostate tissue of these patients and might be further studied as prognostic marker. in this study we first showed the correlation between aurc and other carcinogenic kinase braf expression, which opens the perspective for investigation of role of aurc in carcinogenesis. bacillus marmarensis sp. nov. is an extreme obligate alkaliphile isolated from mushroom compost near marmara region of turkey. it can survive at extreme ph values up to 12.5. based on its genome sequence, metabolic pathways for 7 proteases, 6 amylases, 2 cellulases, 1 lipase, n-butanol and a biodegradable plastic poly-b-hydroxybutyrate were annotated. in addition to being a potential extracellular hydrolase producer, its ability to survive in the high ph range of 8.0 to12.5 makes it an attractive microorganism for different industrial applications. in the current study, the adaptation strategy of b. marmarensis sp. nov. to alkaline conditions was investigated using proteomic tools. the organism was grown at two different ph values, 10.0 and ph 12.0. for extraction of whole cell proteins, cells were disrupted with mp bio fast prep device. protein extracts were treated with protease inhibitors and a nuclease mix. salts were removed using a cleanup kit. obtained proteins were separated based of their isoelectric points in the first dimension and then based on their molecular weights in the second dimension. proteins maps of cells grown at these two extreme ph values showed significant differences in protein expression for alkaline adaptation. p-08.01.4-029 biochemical and proteomic analyses of normal human astrocytes and glioblastoma exposed to dichloroacetate treatment f. c. atilgan, h. cimen yeditepe university, istanbul, turkey glioblastoma (gbm) is an aggressive malignant tumor composed of astrocytes in brain tissue. gbm cells utilize glycolysis rather than oxidative phosphorylation to support rapid growth rate which is called warburg effect. dichloroacetate (dca) is an antiglycolytic agent that inhibits pyruvate dehydrogenase kinase (pdk) activity and induces apoptosis via normalizing the mitochondrial activity. this study aimed to demonstrate the metabolic alterations between the normal human astrocytes (nha) and gbm cell lines which are exposed to dca, and to identify the differentially expressed proteins by ms-based proteomic analyses. nha cell line, u87mg and u373 as gbm human cell lines were examined through analyzing the alterations in the glycolysis metabolism upon dca treatment by measuring the variations in the pyruvate levels, lactate dehydrogenase a, pdk3. mts was performed to investigate the effect of dca treatment on cell viability. immunoblotting of pgc1-a, oxphos complexes, and mitotracker green staining was employed to reveal the mitochondrial differences between normal and the cancer cells, and upon dca treatment of these cells. proteomic analyses were utilized for the identification of candidate proteins depending on the acetylation status. in this study, compared to nha, the pyruvate and ldha levels were elevated and pdk3 levels in u87mg were reduced by 14%. due to mts results, ≤10 mm dca treatment showed significant decrease in gbm cells compared to nha cells. immunoblotting and mitotracker green staining results showed increase in mitochondrial mass. elevation in the pyruvate and ldha levels and reduction in pdk3 level in u87mg and u373 cells indicates glycolysis dependent metabolic switch in energy metabolism. proteomic analyses demonstrate that most of the differentially expressed proteins comprised of metabolic enzymes. this study provides novel information about metabolic alterations existing between nha and gbm, which can inspire further studies for therapeutic applications. kidney stone is a complex disease resulting from environmental as well as hereditary factors and principally composes of approximately 75% calcium oxalate (caox) crystals, which are formed through a multi-step process. vitamin d receptor (vdr) gene encodes the nuclear hormone receptor for vitamin d3; downstream targets of this gene are chiefly contributed in mineral metabolism though the receptor regulates a variety of other metabolic pathways. calcium sensing receptor casr plays an important role in sustaining mineral ion homeostasis. the aim of this study is to profile the expression level of vdr and calcium sensing receptor (casr) genes and to unravel their role in rat kidney stone induced by ethylene glycol, in order to explain the underlying molecular mechanisms. total rna were extracted from paired sample before and after ethylene glycol treated of 50 rats. the mrna expression level of vdr and casr gene were measured employing quantitative rt-pcr (qrt-pcr). the mrna expression levels of both genes were significantly down-regulated according to before treated. in conclusion, our data suggest reduced mrna expression in vdr and casr genes might be a risk factor for kidney stone formation. further studies are necessary to verify these findings in different ethnic groups. p-08.01.4-031 apj receptor a445c gene polymorphism in turkish patients with coronary artery disease against different models of expected frequencies/counts to understand the evolutionary dynamics of saars in proteins. we obtained from ensembl the assemblies of genomes/proteomes of human and nonhuman primates (chimpanzee, gorilla, and rhesus monkey), rodents (mouse and rat), and birds (chicken and zebrafinch). the expected probabilities for the occurrence of saars based on their nucleotide frequencies in coding regions and amino acid frequencies in individual protein sequences or across the whole proteome were compared with the observed repeat occurrences. we found that with all three methods and in all eight species the correlation between observed and expected repeat counts decreased above a saar length threshold. the percentage of saar proteins for each amino acid also exhibited variability among species when both the repeat length and counts were taken into account. however, clustering based on saar characteristics generally reflected the known phylogenetic relationships between species. our comprehensive bioinformatics analyses reveal that saars show amino acid-specific occurrence patterns with respect to species as well as saar length. tissue proteins play important roles in biological metabolic processes. the qualitative and quantitative analysis of tissue proteins facilitates the understanding of molecular mechanisms that differentiate between physiologic and pathologic states. health and research institutions routinely prepare formalin-fixed paraffinembedded (ffpe) tissue blocks for histopathology. proteomics on ffpe tissue still requires standardization of tissue solubilization processes to overcome variability in protein extraction results. our aim is to compare the proteomic studies of fresh frozen and ffpe rat renal tissues. fresh frozen and ffpe preparations from renal tissues were included in this study. an adult rat was sacrificed and the dissected kidneys were divided two equal section. one immediately frozen in phosphate buffer, and the other tissue specimen not thicker than 5 mm to allow rapid penetration of the fixative put in 10% buffered formalin for 48 hours. the fresh frozen tissue was dissolved and homogenised in the cold phosphate buffer solution containing protease inhibitors. paraffin blocks were performed from formalin fixed tissue specimens. we have extracted the protein from the ffpe tissues using our previously verified method. we have utilized electrophoresis three times to compare protein yield, number, intracellular and intercellular of homogenised samples obtained from ffpe and fresh frozen kidney samples. the number of proteins identified from fresh frozen kidney tissue has generally been shown to be increased compared with ffpe tissue. decrease of the qualitative results in electrophoretic bands was found similar in all replicative studies. ffpe tissues undergo extensive cross linking between protein/ dna/rna molecules during formalin fixation, which creates inter-molecular crosslinks. on the other hand, ffpe tissues represent a valuable resource to carry out retrospective studies aimed to biomarker discovery in kidney cancer as well as other kidney diseases. background: development of atrial fibrillation (af) during the course of chronic primary mitral regurgitation (mr) is common and represents complex molecular mechanisms. however, the gene expression profile of human atrial fibrillation (af) in the setting of chronic primary mr remains uncharacterized. in the current study, we aimed to compare the gene expression profiles of patients with severe degenerative mr in sinus rhythm (sr) and af. methods: left and right atrial tissue samples were obtained from patients with chronic primary severe mr in permanent af (n = 30) and sinus rhythm (n = 30). we performed a novel micro-dissection technique for thin sections of atrial tissue samples and immediately fresh froze intra-operatively. transcriptomes of left and right atrial appendages of degenerative mitral regurgitation patients with sr versus af were compared by microarray analysis on affymetrix hgu-133 plus 2 platform. bioinformatics, data mining and pathway analyses were conducted on partek gs and webgestalt. genome-wide gene expression profiles were compared between af and sr groups among 54.675 transcripts representing 38.500 well-characterized human genes. differentially regulated genes were evaluated according to fold change (fc ≥ 1.5) with a p-value ≤0.05. results: most remarkable pathways altered in af atrial tissues compared to sr group, were extracellular matrix-receptor interaction; mapk, adipocytokine, and calcium signaling; apoptosis and cardiac muscle contraction pathways. conclusions: this is the first human study of comparative transcriptomics in left and right atrial tissues of patients with af versus sr associated with severe degenerative mr. the main findings of this multidisciplinary translational research provide novel candidate targets for the treatment and prevention of af. in order to acquire iron under iron-limiting growth conditions, bacteria employ specific mechanisms such as production and secretion of siderophores. siderophores are low molecular metalchelating compounds that contribute not only to iron scavenging, but also participate in other important processes including oxidative stress response and cell signaling. serratia marcescens, gramnegative bacterium, could be found in various environments, including wastewater, plant rhizosphere and hospital setting where s. marcescens can cause serious life-threatening infections. in this study, we performed a detailed characterization of the siderophores of the clinically important pigment-free s. marcescens strain sr41-8000 and environmental pigment-producing s. marcescens strain sm6. bioinformatic analysis of these genomes by antismash software revealed the presence of several clusters involved in non-ribosomal peptides synthesis (nrps). we found four nrps clusters in genome of s. marcescens sm6. cluster 1 has a low level of identity to enterobactin gene cluster typical for bacteria producing catechol-like siderophores. second cluster has only 4% of identity to xantholipin biosynthetic gene cluster. clusters 3 and 4 of nrps genes of s. marcescens sm6 did not show any homology to known nrps clusters. in contrast, the genome of s. marcescens sr41-8000 contains only one genetic cluster of nrps genes. this cluster does not have similarity to any of the known bacterial nrps genes. thus, genetic analysis of two isolates of s. marcescens allowed us to identify nrps genetic clusters and showed that the repertoire of these genes is different between strains. we hypothesized that the strain isolated from environment has competitive advantage over clinical isolate due to genetic diversity of siderophores. on the other side, clinical isolate has specific genetic cluster of siderophores which may promote s. marcescens growth and adaptation to the extreme niches present in medical facilities. p-08.01.4-037 the first glance on the genome's structure and activity in hibernator edible dormouse hibernation is a unique adaptive way of survival in extreme environmental conditions where mammals decrease their metabolic rate and demonstrate physical inactivity for prolonged periods of time (up to 6-8 months). remarkably, some hibernating animals have a long average lifespan and the ability to avoid muscle atrophy caused by disuse or immobilization. to identify main molecular pathways behind the protective musculoskeletal adaptation and genome structure in hibernator edible dormouse (glis glis), whole-genome analysis of mrna expression in muscles (m. soleus and m. edl) and lumbar spinal cord samples was conducted. three groups of the dormice: 1) active animals 2) hibernated animals and 3) animals immobilized for 2 weeks in laboratory, were examined. rna libraries have been sequenced using hiseq 2500 illumina platform. coupled with genome dna sequencing provided x10 coverage of the estimated genome, we have assembled de novo transcriptome of the dormice. differentially expressed genes in response to immobilization and hibernation were determined. transcriptional program of these phenotypes was similar. pathways enriched by differentially expressed genes were identified. gene expression of the key muscle proteins and muscle atrophy markers was analyzed. muscle-specific e3-ubiquitin ligases murf1 and mafbx revealed no changes in mrna expression. our study represents the first attempt to elucidate changes in transcription profiles of skeletal muscles and spinal cord during hibernation and hypokinesia in edible dormice. in corroboration to the gene expression data, they demonstrated minimal morphological evidence for muscle disuse atrophy during physical inactivity. edible dormice, thus, can be considered as a novel model organisms in investigation of the genetic mechanisms of hibernation and prevention of muscle atrophy. the work is performed according to the russian government program of competitive growth of kfu and supported by rfbr jsps_a no. 14-04-92116. in response to diverse environmental cues bacteria form complex structured communities called biofilms. the metabolic pathways activated by these cues are remarkably different depending on the species studied. however, they all lead to the formation of an extracellular matrix that holds the cells together. non-pathogenic gram-positive spore-forming soil b. subtilis strain is recognized as a model system for the study of biofilms. to discover the pathways regulating biofilm formation in b. subtilis, we studied the natural isolate of b. subtilis strain 168, and constructed the recombinant strains with knocked out genes of following regulatory proteins: abrb (global transcriptional regulator), degu (two-component response regulator of signal transduction system degs-degu), ccpa (regulator of carbon catabolism) and spooa (regulator of sporulation). in the minimal medium broth b. subtilis 168 wild-type strain forms biofilm with its maximum on 48th hour of culture growth. ph-optimum for biofilms formation by the wild-type strain is in the range of 7.4-8.0. the temperature optimum is in the range from 22°c to 45°c. this corresponds to the natural conditions of the b. subtilis habitat in rhizosphere. the level of biofilm formation by regulatory mutant strains with deleted abrb, degu, ccpa, spooa genes is on average 40% lower than by the wild-type strain. this indicates that global regulatory system controlls biofilm formation process. statistically significant differences in the levels of biofilm formation between regulatory mutants haven't been identified. ph and temperature optima of mutant strains are the same as for the wildtype strain -7,4-8 and 22°c -45°c respectively. the crataegus genus which is a member of rosaceae family, has approximately 200 species worldwide and 24 species in turkey. all plant species in this genus have the common name "hawthorn". crataegus microphylla (c. microphylla) c. koch which is characterised by having erect sepals in fruit and smaller leaves in comparison with the other species, is one of the wild edible fruits in turkey. crataegus species have been used as food and also in folk medicine for the treatment of various diseases. for this purpose, the potential biological properties of crataegus microphylla were aimed to reveal by the preliminary work. in this study, prevention of oxidative dna damage using supercoiled pbr322 plasmid dna, acetylcholinesterase, tyrosinase, a-glucosidase inhibition and antioxidant effects: 2,2diphenyl-1-picrylhydrazyl radical scavenging effect, phosphomolibdenum-reducing antioxidant power, ferric-reducing antioxidant power with total phenolic and total flavonoid contents of the c. microphylla leaves, stem barks and fruits that extracted with ethanol, methanol and water were investigated. the experiments of oxidative dna damage studies and antioxidant activities of c. microphylla extracts showed that methanol and ethanol extracts possessed a strong ability to prevent dna damage and significantly antioxidant activities. methanol extracts of stem barks from c. microphylla exhibited the highest acetylcholinesterase and tyrosinase activities (48.86 ae 4.06% and 85.57 ae 2.01%, respectively), at 200 lg/ml. in addition, ethanol extract of leaves from c. microphylla inhibited the a-glucosidase activity significantly when compared to acarbose. this study explained significant antioxidant, enzyme inhibitory, hypoglycemic, and neuroprotective activities of methanolic or ethanolic extracts prepared with stem bark and leaf from c. microphylla and also strong ability to prevent dna damage that corresponded to antioxidant potential of methanol extracts of leaf and stem bark. the yarrowia lipolytica species (yl) is nonconventional yeast widely used for recombinant protein expression due to its system of post-translation protein modification, which is the most similar to that of higher eukaryotes. yl appears the promising producer of recombinant proteins with much more complicated molecules compared to those of prokaryotic producers. however, an important feature for a producer strain of recombinant proteins is the genes, the expression of which undertakes under controlled conditions, and consequently, search of new effective inducible promoters in the yl genome is of great interest. proteome analysis of the yl cells grown at different ph values (4.0, 5.5, 9.0) showed that under alkaline conditions the amount of mitochondrial porine vdac (voltage dependent anion channel), one of the most abundant protein of the mitochondrial outer membrane, increased significantly. vdac is supposed to let reactive oxygen species out of mitochondria protecting the cell against oxidative stress. therefore, the por1 gene expression, encoding vdac should increase in the stress conditions. the promoter of the por1 gene was used to construct some new expression systems based on yl w29. a new genetic construct bearing a reporter bgalactosidase gene under control of the por1 promoter. b-galactosidase activity was assayed in the cells grown in various ph conditions and exposed to exogenous oxidants such as hydrogen peroxide, menadione, and methyl viologen. it was shown, that in h 2 o 2 and methyl viologen treated cells b-galactosidase activity increased 1.5-2.0 -fold reaching its maximum in the cells, grown at ph of 9.0. thus, we demonstrated high inducibility of the por1 promoter, which is essential for effective action of the expression system based on it and potency of application for transformed lines of producers. acknowledgments: supported by the russian foundation for basic research (grant no 16-34-00634 mol_a). aspergillus nidulans is able to detoxify and catabolize the toxic proline analogue, lazetidine-2-carboxylic acid in nature, azc serves as a plant protectant against infections and consumption. we have obtained evidence that azc is not only non-toxic for the model ascomycete aspergillus nidulans, but it can be utilized as a poor nitrogen source. in order to elucidate the molecular mechanism underlying azc detoxification, we have constructed and studied a. nidulans strains deleted in the cognate genes involved in azc detoxification in pseudomonas and saccharomyces cerevisiae. these genes, found by in silico analysis, encode a putative hydrolase, acha, and an azc acetyltransferase, ngn2, respectively. gene deletion was accomplished through double crossover. a cassette containing the~1500 bp 5' and 3' flanking sequences of each gene, with the afpyrg gene as a selection marker, was contructed. crossing the achad and the ngn2d strains isolated the achad ngn2d double mutant strain. rt-pcr was used for gene expression analysis in the wild type strain, area-loss of function and crea-derepressed mutant strains. our results clearly show that azc can be used as a poor nitrogen source by a. nidulans. this utilization requires a) acha, a putative azc hydrolase, and b) a fully active gaba catabolic pathway, as lack of either amdr or gata abolishes azc utilization. most importantly, the double mutant, achad ngn2d, shows azc toxicity, suggesting that ngn2 is a true orthologue of mpr1, able to detoxify azc, a phenotype that can be observed only in the absence of acha. as a final point, ngn2 was shown to be induced by the presence of azc and and to be under nitro the spatial genome organization plays a great role in the maintenance of the nuclear architecture and regulation of all processes occurring in the nucleus. this system is controlled by a set of special proteins having an architectural function. however, the mechanisms of their action remain unknown. among these proteins are, in particular, zad-domain-containing proteins. zinc finger-associated domain (zad) is a ubiquitous motif of c2h2 zinc finger proteins of drosophila. genes that encode zad proteins are specific for and expanded in the genomes of insects. only a few zad-encoding genes have known functions, and the role of zad is being discussed. up to date there was only one known crystal structure of zad-domain from drosophila transcription factor grauzone (grauzad). here, we present for the first time the crystal structure of the zad-domain of serendipity-d transcriptional activator of the egg-polarity gene bicoid. zad-domain was cloned, overexpressed in e. coli, purified and the structure was solved at 3.5 a by mad technique. detailed analysis of the structure proved that the protein exists in dimeric form and revealed unique spatial organization of the protein, different from those for grauzad. this work is supported in part by russian ministry of education and science grant (14.616.21.0066). mycoplasma gallisepticum is a convenient model object for studying the regulation of transcription because it has a reduced genome, lack of cell wall and many metabolic pathways and also easy to culture and non-pathogenic to humans. due to the nature of the genomic organization and the loss of many of the known regulators, the effect of disrupting the function of some proteins may be a useful tool for studying the regulation of transcription. the gene expression study was performed on agilent onecolor microarray with custom design and random-t7 polymerase primer for cdna synthesis. microarray represents 3366 probes for 678 orf including genes and ncrna. in this work, we have investigated the effect of changes in the level of gene expression of m. gallisepticum for two different types of conditions: a genetic knock-out mutants and the cell response to treatment with sub-lethal concentrations of antibiotics. we characterized transcription of m. gallisepticum when the cell responses to dysfunction of proteins with metabolic potential, possible regulators of expression, in violation of permeability of membrane by cccp, inhibition of ribosomal synthesis by tetracycline, dna gyrase by novobiocin and atp synthase by oligomycin. the data obtained allow to characterize the transcriptional response under different conditions and to identify groups of genes that change expression together. major transcriptional changes were observed in the response of cells under cccp treatment due to uncoupling of the proton gradient and further reducing the membrane potential, as well as under novobiocin treatment due to changing the topology of dna. global problem of oil pollution forces scientists to search for a new safe remediation technologies constantly. careful attention is paid to bacteria, some of which possess additional biotechnologically valuable properties, such as utilization of hydrocarbons and production of biofurfactants. in this regard, we carried out proteogenomic characterization of tsukamurella tyrosinosolvens strain ps2, which was isolated from chemical sludge and capable for alkane degradation and biosurfactant production. whole genome of the strain was sequenced on the miseq (illumina) platform, assembled and annotated. proteome on mineral medium with glucose, sucrose and hexadecane as a sole carbon and energy source was studied. shotgun proteomics approach was performed on hybrid chromatography-mass spectrometry machine (maxis impact). alkane oxidation genes (alkane-1-monooxygenase, rubredoxin and rubredoxin-reductase) under genome sequence, as well as two pathways of trehalose synthesis and genes for mycolic acids production were found. emulsification activity of cell-free culture liquid was about four times higher on hexadecane in comparison with sugars. proteomic profile was different at various culture conditions. all glycolysis genes, beginning with glucose-6-phosphate isomerase to pyruvate kinase, were found on the media with sugar. the medium with hexadecane helped to reveal enzymes involved in the beta-oxidation of fatty acids, for example 2,4-dienoyl-coa reductase, 3-ketoacyl-coa thiolase and enzymes of the initial mycolic acid synthesis pathways. thus we have established that the strain t. tyrosinosolvens ps2 utilizes sugar by glycolysis. also, the bacterium is capable for alkane oxidation followed by beta-oxidation of fatty acids. based on the proteogenomic data, we assume that the bacterium is able to synthesize trehalose lipids, namely, trehalose mycolates. obtained results could be useful to create conditions for increased biosurfactants production. gestational diabetes mellitus (gdm) is a glucose intolerance firstly diagnosed during pregnancy. in this study, we aimed to investigate the association between serum adiponectin, resistin levels and insulin resistance in gestational diabetic patients. a total of 80 patients; 40 healthy pregnant women (control group) and 40 pregnant women diagnosed with gdm (gdm group) were included in this study. serum adiponectin, resistin, glucose, insulin, hba1c levels and lipid parameters were measured. insulin resistance index homa-ir values were calculated. in this study, serum glucose, insulin, hba1c levels and homa-ir were significantly higher in gdm group compared to the control group (p = 0.038, p = 0.011, p = 0.001, p = 0.008, respectively). serum adiponectin levels were significantly lower (p < 0.001); whereas serum resistin levels were significantly higher (p = 0.004) in gdm group than in the control group. it can be concluded that resistin contributes to the formation of insulin resistance, adiponectin plays an important role in the regulation of this resistance and they also have effects on gdm pathophysiology. hematological cancers including acute myeloblastic leukemia (aml) and acute lymphoblastic leukemia (all) in terms of incidence and mortality, are the second most important cancer type in turkey. numerous studies show that cancer patients respond differently to treatment thus supporting the idea of personalized therapy need for individuals. renin angiotensin system (ras) have key roles in aml and all progression and it has been shown by many studies suggests that these system's genes might be good biomarkers for aml and all personalized therapy. we aimed to identify ras gene based homogeneous subgroups of acute leukemia and determine the most effective chemotherapoetic agent for each subgroup. after validation and verification of the results, more effective drugs can be recommended for the use in clinics for chemotherapy of aml and all. results of our preliminary studies showed that we are able to identify subgroups of aml and all as well as correlating each existing subgroup with fda approved drugs. considering the long and highly cost process of developing new drugs for cancer treatment makes the present study all the more valuable. in addition, there is a serious need for change in aml and all therapy since there is no highly effective chemotherapy protocol available for their treatment. welcome trust sanger (wts) and cancer cell line encyclopedia (ccle) databases will be used to determine subgroups of aml and all based on ras genes or whole genome expression using standard deviation and hierarchical clustering analysis. the most effective drugs for each subgroup will be identified using pearson's r correlation analysis with drug sensitivity data (ic50, ic50, amax, aare, etc.) available in same databases. further validation tests will be performed by in vitro validation using aml and all cell lines: drug sensitivity profiles will be determined and gene expression will be shown by q-rt-pcr. p-08.02.5-003 functional polymorphisms of ephx2 in a turkish population h. pinarbasi, i. sari cumhuriyet university, sivas, turkey soluble epoxide hydrolase (seh; ec 3.3.3.2) is encoded by ephx2 and catalyses the degradation of endogenous fatty acid epoxides generated by cyp450 epoxygenases. these fatty acid epoxides such as epoxyeicosatrienoic acids (eets) have been shown to posses vasodilator, anti-inflammatory, anti-platelet, anti-hypertensive, anti-apoptotic, anti-thrombotic and natriuretic effects. it has been reported that eet levels are associated with hypertension, stroke and cardiovascular diseases. individual differences in the ephx2 gene that affect the seh activity may alter the circulating levels of eets. k55r and r287q polymorphisms have been known to cause increased and decreased seh activity, respectively. therefore we aimed to determine the genotype frequencies of these two polymorphisms in a turkish population. k55r and r287q polymorphisms were determined by the real time pcr using double-dye hydrolysis probes or pcr-rflp method. the observed genotype frequencies for k55r polymorphism were 80.8% wild type (aa) and 19.2% polymorphic genotype (ag+gg) and for r287q polymorphism 81.4% wild type and 18.6% polymorphic genotype (ga+aa). the genotype distributions for both polymorphisms were in hardy-weinberg equilibrium. pregnancy is one of manifestations for thrombophilia factors, which in its turn leads to various complications of its course. one of the markers of hereditary thrombophilia is mutations in the folate cycle mtr, mtrr and mthfr genes. insufficient intake of folate during pregnancy disrupts the functioning of the genome, leading to miscarriage, violation of embryogenesis and various fetal malformations. however, results of studies on the role of hereditary thrombophilia in the occurrence of complications during pregnancy are rather contradictory. aim of this study was to determine the frequency of alleles and polymorphic variants of folate cycle genes mtr a2756g, mtrr a66g and mthfr c677t in women of kazakh ethnic group with pregnancy complications. we used real-time pcr. blood samples for dna isolation were obtained from 129 pregnant women. the main group consisted of women (n = 90) which had a history of two or more pregnancy complications in the form of pre-eclampsia, eclampsia, missed abortion, miscarriage, and etc. control group consisted of women (n = 39) with two or more normal pregnancy outcomes, and had no complications during pregnancy in history. average age of women in experimental group was 32.0 ae 0.50 years compared with control of the age 33.6 ae 0.33. the analysis of the frequency distribution of alleles of genes in experimental group of women with complications of pregnancy revealed no significant differences relative to the control group. analysis of the distribution of polymorphic variants of folate cycle genes showed significant difference between the study and control groups in the occurrence frequency of heterozygotes for the mutant allele g in the gene mtrr a66g (or = 2.89, ci 95% = 1.25-6.71; v 2 = 6.376, p < 0, 05). no significant differences in alleles between homozygous wild-type and homozygous mutant alleles were observed. this work was funded by the mes kazakhstan (gr 0115rk00287 project number). p-08.02.5-005 a study on the association between rs6918698 polymorphism in connective tissue growth factor gene and pseudoexfoliation syndrome pseudoexfoliation syndrome (pes) is a disorder of the extracellular matrix characterized by the production and progressive accumulation of an abnormal fibrillary material in many ocular tissues. pes prevalence is 11.3% above the age 40 in turkey. since pes is characterized by excessive synthesis of elastic microfibrillar components throughout the body, growth factors can have important roles in the pathophysiology of pes. human connective tissue growth factor (ctgf) is a protein expressed in a variety of tissues, including the anterior chamber of the eye. ctgf coding gene has several genetic polymorphisms. rs6918698 g/c single nucleotide polymorphism (snp) is found at position à945, in promoter region. the presence of a c allele for rs6918698 is critical for transcriptional suppression of the ctgf gene which would reduce ctgf production. aim of this study was to investigate if there is any association between pes and rs6918698 polymorphism of the ctgf gene. study population consisted of 60 patients with pes and 60 controls. blood samples were collected by g€ ulhane military medical academy, department of ophthalmology, ankara, turkey. genotypes were assigned by pcr followed by restriction fragment length polymorphism analysis. genomic dnas were isolated from whole blood samples using manual dna isolation. the frequency of ctgf rs6918698 polymorphic allele g was 0.442 in patients, and 0.450 in controls (0.967, p = 1.000). distribution of genotypes was gg: 31.7%, gc: 48.3% and cc: 20.0% among patients, while gg: 35%, gc: 40.0% and cc: 25.0% (or = 1.162, p = 0.689) in controls. statistical analysis showed that there is no significant relationship between ctgf rs6918698 snp and pes. these are the preliminary findings of a research project which is the first study analyzing the relationship between ctgf rs6918698 snp and pes. this work did not point out a role for ctgf rs6918698 in the risk for pes. a significant relationship might be found when the study population is enlarged. p-08.02.5-006 evaluation of rs11136000 single nucleotide polymorphism of clusterin gene in pseuodoexfoliation syndrome risk pseuodoexfoliation syndrome (pes), an age-related systemic disorder, is characterized by production and accumulation of abnormal fibrillar extracellular material in anterior structures of the eye. clusterin (clu) is a multifunctional glycoprotein produced and secreted by almost all cell types and is found in all body fluids and in accumulated pes material. under cellular stress conditions, clu provides inhibition of stress-induced precipitation and aggregation of misfolded proteins. clu expression level in pes patients is unexpectedly low and this could be due to single nucleotide polymorphisms (snp) on the gene coding for clu. rs11136000 c/t polymorphism has been found to be associated with alzheimer's disease and pathophysiology of alzheimer and pes are similar. this study aimed to determine whether rs11136000 snp of clu gene have a role in the development of pes. study population consisted of 60 patients with pes and 60 controls. blood samples were obtained from g€ ulhane military medical academy, department of ophthalmology, ankara, turkey. genomic dnas were isolated from whole blood of subjects using manual dna isolation. genotypes were assigned by pcr followed by restriction fragment length polymorphism analysis. t allele frequency of pes patients was 0.425 and that of controls was 0.442 (0.934, p = 0.794). the distribution of genotypes was cc: 30.0%, tc: 55.0% and tt: 15.0% among patients while cc: 28.3%, tc: 55.0% and tt: 16.7% (0.922, p = 0.841) in controls. there was no statistically significant difference between pes patients and controls in terms of tt genotype and t allele frequency. these are the preliminary findings of a research project which is the first study analyzing the relationship between clu rs11136000 snp and pes in turkish population. this work did not point out a relation for polymorphic genotype in the risk for pes. however, a relationship between clu rs11136000 polymorphism and pes can be found when we enlarge the study population. the tumor suppressor tp53 is the most frequently mutated gene in head neck squamous cell carcinoma cancer and represents a known transcription factor and tumor suppressor gene that regulates different microrna and target genes. the aim of our work is to construct the transcriptional and post-transcriptional network regulated by tp53 and to evaluate the difference at mrna and protein expression levels of the tp53 target genes in hpv negative head and neck squamous cell carcinoma (hnscc) patients with distinct tp53 mutation states and to elucidate the molecular mechanism that underlie the poor prognosis of tp53 mutation. to show the tp53 mutation landscape and its prognostic relevance for survival, we used cbioportal for cancer genomic analysis. we downloaded mutational profiles of 243 hpv negative hnscc patients. employing different databases we constructed the tp53 regulatory network. and then, to evaluate the effect on mrna, protein and microrna regulated by tp53 we used the mrna and protein expression profiles of patients from tcga. our results show that hotspot, truncating and missense mutations have statistical significance in the univariate analysis. the tp53 regulatory network show the involvement of important target involved in the progression of hnscc and the deregulation of protein expression of an important key epigenetic modifier ezh2 was significantly associated with tp53 mutational state. ezh2 is a member of the polycomb group protein enhancer zeste homolog 2 which is known to be directly repressed by tp53 and indirectly by the activation of mir-200a and mir-200b. we found a significant up-regulation of ezh2 that depend from tp53 mutation. it is important to understand the difference in mrna and protein expression of tp53 regulatory network that could depend from its mutational state. this finding suggest that ezh2 might be a potential therapeutic target for hnscc. p-08.02.5-008 next generation sequencing based approach for monitoring of minimal residual disease in acute lymphoblastic leukemia minimal residual disease (mrd) monitoring is widely used to evaluate efficiency of chemotherapy and to choose a strategy for further treatment in acute lymphoblastic leukemia (all). the most commonly used approaches for mrd detection are based on flow cytometry and qpcr. these methods have several important limitations including insufficient sensitivity, complicated experimental setup and false positivity. the newly developed next generation sequencing (ngs) approaches could overcome the existing limitations in mrd monitoring. here we describe a new mrd monitoring approach based on targeted deep sequencing of malignant rearrangements. first, we identified bcr/tcr rearrangements specific for the leukemic clones in initial bone marrow samples of 10 all patients. for this, we used sanger sequencing of the products of 19 multiplex pcr, performed with bcr/tcr specific primers combined according to the optimal frequency distribution of v/j-genes in healthy donors. second, we analyzed concentration of malignant clone rearrangements, identified at the first step, in dna samples obtained from bone marrow after 36 days of treatment. for this purpose, we performed ngs of 10 libraries for each identified leukemic rearrangement. four libraries were amplicons of bcr or tcr gene rearrangements generated using characteristic v and j segment specific primer combination. six additional libraries were amplicons of the same primer combination from the same dna sample which contained initial leukemic dna spike-in (in concentrations corresponding to 1 per 100, 1000 and 10,000 cells) for a calibration curve generation. using this approach, we analyzed 7 all clone specific rearrangements for three patients and calculated concentration of the leukemic clones by using the calibration curve. for one patient we didn't find any leukemic cells and for two patients we found 1 leukemic cell per 100,000 analyzed cells. znf365 is considered as a transcriptional target for p53 and plays an important role in the homologous recombination, mitosis, centrosome dynamics. as was shown by gwas some snps in znf365 have strong association with the risk of breast cancer (bc). however, it was unclear whether the same snps are associated with risk of bc in kazakhstan. therefore two polymorphisms (rs10995190, rs10761659) of znf365 were investigated in kazakh population in this study. the present case-control study was carried out with participation of 444 kazakh females with bc and 390 cancer-free donors. additionally, subtypes of bc, stratified by estrogen receptor (er+/à), progesterone receptor (pr+/à) and human epidermal growth factor receptor 2 (her2+/à) status were estimated. pearson p-value, odds ratio, 95% confidence interval tests were applied to data analysis. significant differences were found in allele frequency and genotype distribution at rs10995190 locus in znf365 between the patients and control groups (p = 0.013 for allele; p = 0.007 for genotype). moreover, significant association with bc was revealed for rs10995190 after dividing patients according to er+/ à, pr+/à and her2+/à status of the tumor. the g allele was associated with er+ (p = 0.01, or = 1.69, 95%ci:1.11-2.56), pr+ (p = 0.01, or = 1.78, 95%ci:1.13-2.79) and gg genotype with her2-bc carriers (p = 0.02, or = 1.66, 95%ci:1.04-2.63). also, g allele can be considered as a risk factors in er+/ pr+/her2-luminal type of tumor (p = 0.028, or = 1.79, 95% ci:1.06-3.05). our findings correlate with the data of several gwas where the association of the rs10995190 polymorphism with higher mammographic density and the risk of breast cancer have been shown. the obtained results allow us to consider g allele and gg genotype of rs10995190 as a marker of bc risk with predictive value, restricted to er, pr and her2 status of the tumor in the kazakh population. breast cancer (bc) is the most common cancer among women in most of countries. alternative variants of low-penetrance genes such as fgfr2 (rs2981582), tox3 (rs3803662), map3k1 (rs889312), lsp1 (rs381798) are shown to have high frequency in north america, south-east asia, australia, europe populations and a multiplicative effect on the development of bc. in this study was investigated assosiasion between alleles/genotypes combinations of these genes with increase/decrease of bc risk. the case-control study included 625 bc patients and 672 healthy women from kazakh and russian populations. genotyping was performed by pcr-rflp methods. combined effect of allele and genotype variations in four different genes on bc risk was assessed by apsampler algorithm. the fisher exact test, odds ratio (or) with 95% confidence intervals (95% ci) were applied to data analysis. according to obtained results combinations of allele c of tox3 rs3803662 and a of map3k1 rs889312 (p = 0.006, or = 2.2), also allele c of tox3 rs3803662 and c of lsp1 rs381798 (p = 0.02, or = 1.47) associated with increased bc risk in the russian population. consequently, combinations with c allele of tox3 rs3803662 contribute significantly to bc risk with p-value = 0.04, or = 1.86. on the contrary, tt genotype of tox3 rs3803662 with p = 0.04, or = 0.53 and its combination with allele t of lsp1 rs381798 with p = 0.03, or = 0.47 determine a bc risk reduction in russian population. in addition, a risk combination of allele c of lsp1 rs381798 and a of map3k1 rs889312 was found in kazakh population (p = 0.02, or = 1.35). studies have shown that a genetic predisposition to bc can be determined by the cumulative effect of individual alleles and genotypes and possible epistatic interactions of studied genes. obtained combinations of alleles and genotypes can be considered as complex genetic markers of bc and may be used as predictive. cancer that is caused by excessive proliferation of cells and reduced apoptosis is a pathological condition. currently, studies that are comitted with breast cancer are great important early detection and diagnosis of breast cancer. after the discovery of cisplatin as chemoterapy drug, new transition metal based complexes have been developed for treatment of cancer. in this study, anti-cancer activity of azo-azomethide ligand and its mononuclear metal complexes is studied on human cancer cell lines (mcf-7) and mouse fibroblast (l929) cell lines. cells were studied four different concentrations (12,5; 50; 75; 100 lm). xtt (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2h-tetrazolium-5-carboxanilide) protocol was applied after 24 and 48 hours. in our study 10% fetal bovine serum (fbs), 1% l-glutamine, 100 iu/ml penicillin and 10 mg/ml streptomycin in dmem (low glucose) were used. cancer cell lines in dmem medium is produced in 95% humidity and 5% co 2 incubator at 37°c. anti-cancer activity of synthesized complexes were determined on mcf-7 and l929. in the biological activity studies, synthesized compounds showed higher anticancer activity than positive control (5-fu). finally, our new synthesized complexes can be suggested that potent ajan for anti-tumuour for breast cancer drugs. large interindividual differences in response to chemotherapy present an important issue in cancer treatment. malignant mesothelioma (mm) is an aggressive tumor with poor prognosis, treated mostly with gemcitabine/cisplatin (gem/cis) or pemetrexed/cisplatin (pmx/cis) chemotherapy. as both clinical characteristics and genetic variability may affect treatment outcome, our aim was to construct and validate clinical-pharmacogenetic prediction models of treatment outcome in mm for both chemotherapy regimens and to develop an algorithm for genotype-based treatment recommendations. clinical-pharmacogenetic models were built on 71 gem/cistreated and 57 pmx/cis-treated mm patients. pharmacogenetic scores were assigned by rounding the regression coefficients. gem/cis model was validated on 66 independent mm patients. model predicting outcome of gem/cis chemotherapy included crp level, histological type, performance status, rrm1 rs1042927, ercc2 rs13181, ercc1 rs3212986, and xrcc1 rs25487. values ranged between 0 and 3.4; cutoff value of 0.75 had sensitivity of 0.62 and specificity of 0.81. patients with higher score had shorter progression-free and overall survival (p < 0.001). in the validation group, positive predictive value was 0.74 and negative predictive value was 0.56. model predicting outcome of pmx/cis chemotherapy included crp level, mthfd1 rs2236225, and abcc2 rs2273697 with scores ranging between 0 and 3.9. cutoff value of 2.7 had sensitivity of 0.75 and specificity of 0.61. patients with higher score had lower probability of good response and shorter progression-free survival (p < 0.001). clinical-pharmacogenetic models could enable stratification of mm patients based on their probability of response to gem/cis or pmx/cis and improve treatment outcome. this approach could be used for translation of pharmacogenetic testing to clinical practice as it would facilitate the selection of the best treatment option for each patient. p-08.02.5-014 evaluation of anti-diabetic potential of circiliol and circilineol using caco2 cell line diabetes mellitus is a metabolic disorder, and many people are suffering from this disease in the worldwide. oral hypoglycemic agents such as sulfonylureas and biguanides are currently used for the treatment. however, studies searching for a more effective anti-diabetic agents are being carried out continıously. based on that we aim to investigate the potential anti-diabetic effects of circilineol and circiliol isolated from teucrium alyssifolium extract, using in vitro cell culture models. for this purpose, the anti-diabetic actions were investigated by applying model caco2 (colorectal adenocarcinoma) cell line. we determined the level of ag (alpha-glucosidase), sglt1 (sodium-glucose transporter-1) and glut1-5 and glucose transport. neither ag activity nor sglt1 activity was increased with either circiliol or circilineol treatment in caco2 cells compared to positive control. similarly, neither the activity nor the expression level of glut1*-5 was increased in caco2 cell line with either circiliol or circilineol treatment relative to control. in conclusion, these results strongly suggest that circiliol and circilineol do not possess any anti-diabetic potentials.supported by tubitak 114z640 and paubap 2014fbe029 the purpose of this study was to characterize and assess the impact of a novel magnetite (fe3o4) nanosystem functionalized with the natural origin compound eugenol (e) on the pseudomonas aeruginosa virulence, biofilm formation and qs signaling in order to advance research aimed to find alternative and personalized therapeutic approaches for severe infections produced by this opportunistic pathogen. fe3o4 nanoparticles were obtained by a co-precipitation method and functionalized with analytical purity e. functionalized nanoparticles (fe3o4@e) were characterized by ir, sem, tga and hr tem. one laboratory and 9 p. aeruginosa clinical isolates were utilized in the study. growth and biofilm formation were assessed by an adapted microdilution method followed by absorbance reads and viable count analysis in dynamics (6, 12, 24 and 48 hours of treatment). soluble virulence factors production was assessed by enzyme activity evaluation of bacteria grown on specific media. the expression of qs core genes was analyzed by qrt-pcr and a luminescence assay. results demonstrated that the average size of the obtained nanosystem ranges 5-9 nm, particles are relatively homogenous and have a low tendency to form aggregates. subinhibitory concentrations of fe3o4@e limited biofilms formation in a time and strain dependent manner, and significantly inhibited the production of toxin pore forming enzymes (haemolysins and lipases) in most strains. the expression of lasi and lasr genes was three fold downregulated, while the expression of pqsr was upregulated in planktonic cultures suggesting that pqs signaling may be involved in virulence modulation after nanoparticle stimulation. the modulation of bacterial virulence and molecular signaling by functional nanoparticles utilized in subinhibitory amounts offer valuable perspectives to develop personalized antimicrobial approaches based on molecular communication control that clearly modulate pathogenicity and progression of the infectious process. p-08.02.5-016 specimen processing and handling for plasma ammonia measurement b. sarac ßligil 1,2 , s. abus ßo glu 2 , f. aky€ urek 2 , b. € ozt€ urk 2 1 selc ßuk medical school, konya, 2 biochemistry department, selcuk university faculty of medicine, konya, turkey objectives: ammonia requires special processing and handling conditions due to its' unstability. in this study, our aim to investigate a preanalytical factor (delayed analyze) affecting plasma ammonia measurement. design and methods: blood samples were obtained from 20 healthy volunteers. for determining different handling and storage conditions, the following protocols were applied: first protocol (a) transportation on ice and separation (centrifugation at 0°c) within 15 minutes of collection and analyze immediately. second protocol (b) transportation on ice and separation (centrifugation at 4°c) within 30 minutes and analyse refrigerated 2-8°c 2 hours (a1, b1) and 4 hours (a2, b2). all plasma ammonia levels was analyzed enzimatic glutamate dehiydrogenase methods by abbott architect c 8000 clinical chemistry analyzer. results: there were statistically alterations in all protocols compared to first protocol. prolonged centrifugation time for plasma ammonia lead to have higher results (38.6 versus 29.75 lg/dl, p < 0.001). in all protocols including a1, a2, b1, b2 also cause an elevation in plasma ammonia results (35.7, 37.4, 39.5 and 41.2; p = 0.032, p < 0.001, p < 0.001, p < 0.001, respectively). conclusions: ammonia concentration in the blood sample increases over time due to high concentrations in cells as erythrocyte or platelets (three fold). blood samples collected for ammonia determination should be stored on ice, and measured immediately. the wnt/b-catenin signaling pathway has been considered to be a factor in the development and progression of colorectal cancer. many studies have demonstrated that the presence of mutations or polymorphisms in ctnnb1(gene encoding b-catenin; mostly mutations in exon 3) can lead to aberrant activation of wnt/bcatenin signaling at the onset of various types of malignancies, including colorectal cancer. the aim of our study was to assess ctnnb1alteration in the patients with colorectal cancer and compared their tumor and normal tissues. a total of 30 paraffin-embedded colorectal tumor specimens were obtained from department of pathology in cerrahpasa medical faculty. also a total 30 paraffin-embedded normal tissue was used from same cases as a control group. ten-micrometer-thick tissue sections were placed on a glass slide and stained with he. then the tissue sections were dehydrated in graded ethanol solutions and dried without a cover glass. dna was extracted from the tissues with 100 ll of extraction buffer at 55°c over night. the tubes were boiled for 10 min to inactivate the proteinase k. ctnnb1 exon 3 was amplified by pcr. sscp is used to observe any difference between the 2 groups. genomic dna was isolated as described above. 10 ll of each pcr products mixed with 10 ll denaturating buffer and were denatured by heating at 95°c for 10 minutes in, and then were rapidly cooled on ice. the denatured pcr samples are run on 12% acrylamide/ bis gel in 0.59 tbe buffer for 3.5 hours at 200 v at room temperature with water cooling system. the gel was stained with silver staining method. migration and adhesion involve continuous modulation of cell motility, beta-catenin play major roles. beta-catenin gene alterations frequency range between 0 and 16% in colorectal cancer according to the different published studies. in our study no significant differences were found in the ctnnb1 exon 3 between the tumor group and normal groups. p-08.02.5-018 theranostic approach in agressive recurrent meningiomafirst experience in turkey m. o. demirkol 1 , b. uc ßar 2 1 koc ß university, istanbul, 2 american hospital, istanbul, turkey meningiomas arise from the meningothelial cells of the arachnoid membranes of the leptomeninx, which are attached to the inner layer of the dura mater. meningiomas can be classified into three grades (i-iii): grade i meningiomas which are benign, exhibit slow growth; grade ii (atypical) and grade iii (anaplastic) meningiomas, which have a much more aggressive clinical behaviour. meningiomas express non-steroid hormones, including somatostatin. in the brain, somatostatin-a cyclic tetradecapeptide neuropeptide-is believed to act as a neurotransmitter and neuromodulator. somatostatin performs its physiological functions by binding to specific receptors (sstri-sstrv). sstr2 exhibit high affinity for octreotate (tate). tate is a polar, watersoluble peptide that does not penetrate the intact bbb (brain-blood barrier). pet and scintigraphic imagings can only demonstrate somatostatin receptor positive intracranial lesions if the bbb is disrupted. in this aim, dotatate (dota-dphe1, tyr3-octreotate, tate) has been labelled with the positron emitter 68 ga and the beta and gama emitter 177 lu. in this case, we conducted a study to evaluate peptide receptor radionuclide therapy (prrt) planning based on pet/ct imaging of meningioma in the department of nuclear medicine and molecular imaging at the amerikan hospital. [ 68 ga] dota 0 -tyr 3 -oc-pet/ct has been established as the imaging modality of choice for the diagnosis and management of patient with skull-base malign meningioma (rapid progress -to 41 9 48 9 52 mm. from 31 9 37 9 35 mm. in 20 d.-after fifth operation). due to its high sstr2 selectivity, [ 177 lu]-tate showed significantly lower uptake/dose delivered to normal tissues, gatate-pet represents the imaging strategy of choice for an accurate assessment of sstr2 expression levels. although some studies have not shown a clear advantage over pet/ct, there is some evidence that it will have an advantage in selected body sites such as the head and neck, liver, and the pelvis. p-08.02.5-019 cardiovascular diseases can be treated by using 'tetr-odd-vp16' and 'hre' hypoxia inducible systems a. celik 1 , t. kaya 2 , s. cigdem 1 , m. g€ und€ uz 1 1 department of medical genetic, turgut ozal university, ankara, 2 faculty of medicine, turgut ozal university, ankara, turkey ischemia is an insufficient supply of blood to a tissue or organ, usually due to a blocked artery by a blood clot. up to now, the number one cause of death worldwide is caused by ischemia and related conditions such as heart attack or stroke. hif-1a is a transcription activator that functions as a master regulator of oxygen homeostasis. hif-1a protein levels increase under hypoxic conditions as a result of decreased o2-dependent prolyl-hydroxylation, ubiqutination and degradation. we aimed to break up clots in blood vessels and to prevent damage caused by ischemia by using hypoxia inducible systems. we added oxygen dependent degredation domain (odd) of hif1a between and in front of tetr dna binding domain and vp16 transactivation domain, so that tetr-odd-vp16 or odd-tetr-vp16 could activate transcription of tissue plasminogen activator (tpa) controlled by tetracycline response element (tre), in a hif1a independent manner. in addition, we also designed tissue plasminogen activator (tpa) under control of hypoxia response element (hre) of hif1a target genes. western blotting and immunofluorescence assay results showed the expression and nuclear localization of tetr-odd-vp16 and odd-tetr-vp16 constructs under hypoxic conditions, but not normoxic. in addition, using fluorometric reporter systems and tpa enzymatic assay we proved functionality of these constructs under hypoxic conditions. final apporoach to our project is predicting kinetic enzymatic activity of tpa during break up blood clots by using matlab. the results of the present investigation showed, the developed hypoxia responsible systems that can be engineered into endothelial cells to prevent ischemia related cardiovascular diseases. p-08.02.5-020 osteogenic potential assessment of some original scaffolds with magnetic properties new advances in bone tissue engineering demand the development of materials that can not only replace bone, but also regenerate the damaged tissue based on external or even internal stimulus. magnetic materials inside bone scaffolds are known to be a promoting factor for bone healing especially when the therapy is accompanied by application of external magnetic stimulation. based on a recent report, the presence of iron oxide in hydroxyapatite can improve the radio opacity and osteoblast proliferation. in this view, this study focuses on the development of silk fibroin-magnetite biocomposites for potential uses in bone tissue bioenegineering. such novel composites possess good mechanical properties, biocompatibility and biomineralization potential by in vitro tests and could become smart arhiectures, able to stimulate bone regeneration. a new culture model was developed by exposing a 3d cell/ scaffold bioconstruct to a continuous magnetic field during 3 weeks of osteogenic induction. in this view, mc3t3-e1 murine osteoblastes progenitor cells were seeded inside the novel silk fibroin-magnetite biocomposites and subjected to osteogenesys in a magnetic field during 21 days. osteogenic specific markers were evaluated every week in the presence and absence of the field. our results showed that the osteogenic marker's expression started earlier when mc3t3-e1 cells were exposed to the magnetic field. consequently, in our experimental model, the magnetic field had a benefic effect on the osteogenic differentiation process as mc3t3-e1 cells differentiated more efficiently in its presence. these results suggest that the bone healing process could be improved in the presence of a magnetic field. nevertheless, further in vivo studies on animal model should be employed for validation. p-08.02.5-021 impact of physical activity performed on different times of day on cardiac and skeletal muscle damage in trained and untrained male subjects s. algul, m. kara, o. ozcelik y€ uz€ unc€ u yil university, van, turkey introduction: physical activity elevates creatine kinase (ck) and creatine kinase myocardial band (ck-mb) levels which have been considered to be an indirect marker of skeletal and cardiac muscles damage. purpose: impact of physical activity performed on different times of day on serum levels of ck and ck-mb were investigated in trained and untrained male subjects. materials and methods: trained (n = 10, 18.3 ae 0.1 yr, 61.4 ae 2.3 kg) and untrained (n = 10, 18.6 ae 0.1 yr, 63.2 ae 2.4 kg) subjects performed three soccer matches (60 min) in field (30 m versus 50 m) in morning (m), afternoon (a) and at night (n) on separate days. the study protocol was approved by the local ethics committee. venous blood samples were taken at onset and at end of match. serum ck and ck-mb levels are measured using autoanalyser. data are expressed as mean ae s.e., compared by wilcoxon-signed rank and mann-whitney u-tests. p < 0.05 was accepted as statistically significant. results: ck and ck-mb levels increased in three matches in both groups (p < 0.05). importantly, there were significant increases in ck-mb levels in a and n exercises compared to m exercise (p < 0.05) in trained (10.6 ae 1.6 u/l versus 14.2 ae 1.9 u/l, 35% (m) 9.7 ae 1.3 u/l versus 18 ae 2.6 u/l, 66% (a) 9.5 ae 1.1 u/l versus 16.3 ae 2.0 u/l, 103% (n) and also untrained groups (8.6 ae 0.8 u/l versus 11.5 ae 0.8 u/l, 42% (m), 7.3 ae 0.5 u/l versus 11.9 ae 0.9 u/l, 85% (a) 7.9 ae 0.9 u/l versus 13.8 ae 0.9 u/l, 76% (n). discussion: increased metabolic stress or muscle damage during physical exercise elevate serum ck and ck-mb levels. however, higher percentage of increase in ck-mb levels in a and n exercise may reflects additional increases in cardiac muscle stress despite the similar skeletal muscle stress as indicated by ck levels. conclusion: considering the observation of higher percentage increase in ck-mb levels in untrained and also trained subjects, the caution should be taken while performing an exercise in a and n time especially in subjects who has cardiac weakness. p-08.02.5-022 regional assessment of hematological and discrimination indices of complete blood count for beta-thalassemia screening beta-thalassemia is one of the most common genetic abnormality causing health problems worldwide. blood count and film of beta thalassemia trait and iron deficiency anemia have similar features. therefore, several simple screening indices have been developed for differentiating between these diseases. it was to asaimed to assess the hematological parameters and discrimination indices in patients with betathalassemia trait who admitted to our hospital. the parameters of complete blood count (cbc) in 141 subjects (51 males and 90 females) diagnosed by mutational analysis (pcr, gene amplification, dna sequencing) between 2013 and 2015, were retrospectively screened and the thalassemia status of patients was assessed in terms of discrimination indices (eng-land&fraser (ef), green&king (gk), mentzer (m), ricerca (r), shine&lal (s-l), srivastava (s), ehsani and sirdah). the percentages of being above the cut off value were detected by ef 29.72%, gk 30.4%, m 26.35%, r 6.75%, s-l 8.78%, s 35.13%, ehsani 25.67% and sirdah 29.72%. the percentages of falsely negatives for the indices of ricerca and shine&lal were lower than others. morever, when the first three common mutations of our study were considered, 5 out of 23, 7 out of 21 and 4 out of 21 patients were up to the cut off values in terms of e&f, g&k, m, s, ehsani and sirdah indices for ivs-i-110 (g>a), ivsii-1(g>a) and heterozygous codon 8 deletion (-aa), respectively. the molecular diagnosis and prenatal detection for families at risk is important because of the difficulties of treatment in this disease. however, the use of discrimination indices may be valuable for distinguishing of thalassemia trait from iron defiency anemia when the equipment of molecular diagnosis are limited. in our study, ricerca and shine&lal had lower falsely negative results than others. nevertheless, further studies to detect diagnostic perfomance of discriminant indices should be conducted. p-08.02.5-023 novel therapeutic agents in the development of effective drug combinations to treat glioma s. avdieiev 1 , l. gera 2 , r. hodges 2 1 institute of molecular biology and genetics, kyiv, ukraine, 2 university of colorado, aurora, co, united states glial tumors are driven by multiple molecular aberrations that cannot be controlled by a single targeted agent. so, it is possible to expect that the combined multitarget anti-cancer therapy aimed simultaneously at different elements of tumor formation mechanisms will be more effective and will promote the extension of patients' life. to find out which drug combinations will enable the development of therapeutic regimens with improved effectiveness and decreased toxicity, the cytotoxic effects of several bradykinin antagonists (ba) were analyzed for different glioblastoma (gb) cell lines. among all the ba under investigation, bkm-570 appeared to be the most effective, with ic50 values of 4 lm and 3.3 lm in rat glioma c6 and human glioblastoma u251 cell lines, respectively. bkm-570 suppressed erk1/2 and akt1 phosphorylation in u251 cells. temozolomide (tmz), the firstline anti-gliomic drug used in clinics, has only a temporary positive effect and severe side effects in gb patients. we showed that the combination of bkm-570 and tmz led to significant potentiation of tmz cytotoxicity at sub-therapeutic concentrations. recombinant proteins with cytotoxic properties are promising agents for complex therapeutic applications. we revealed that the glioma-associated protein chi3l2 inhibited the viability of u251 cells more effectively than tmz. furthermore, the combination of chi3l2 and bkm-570 resulted in an additive cytotoxic effect. chi3l2-mediated decrease of cell viability was associated with a g1/s transition arrest. chi3l2 provoked the dramatic reduction of prb phosphorylation and a significant decrease of cyclin d1 expression, as well as a substantial increase in p53 level. in addition to the accumulation of p53, we observed the upregulation of cdk inhibitor p21. therefore, g1/s arrest in chi3l2-treated cells could be realized via activation of prb, downregulation of cyclin d, and activation of p53. harmful hereditary mutations in brca1 and brca2 are one of the most important risk factors of breast cancer. the aim of this study is to determine the mutations which are associated with breast cancer in people which is diagnosed breast cancer and/or have breast cancer-diagnosed family members by sanger sequencing and thus provide predictive and prognostic utility. our ongoing study is present the genetic variations in brca1 and brca2 genes in breast cancer-diagnosed 12 patients, that one of them is male, and 1 person yet healthy whose brca2 was sequenced by sanger sequencing. the data were analyzed by using seqscape software 2.6 and detected variations were compared with literature. in brca1, we determined 16 different benign genetic variations and 1 variation with unknown significance and 1 variation which has not in literature. in brca2 gene of 12 patient and 1 healthy person,14 benign variations,2 variations with unknown significance,7 variations which has not in literature and 1 mutation were determined. this mutation is c.3189-3192delgtca and is located in occr. c.9257-74a>c variation in brca2 gene, was determined in only male patient.c.32t>a variation in exon2 of brca2, was observed in only the youngest patient who has no family member with breast cancer and healthy person. while this variation takes place in literature as variation with 'uncertain clinical significance', an in silico program mutation taster speculated as 'disease causing' for this variation. also, almost all of variations with 'unknown significance' literature knowledge were determined in only one and different cases. this situation increases the possibility of being pathogenic of this variations. the our findings until now can contribute to variations with uncertain clinical significance in the literature. also the variations that have not in the literature but we suggest the possible relation with breast cancer as an estimate may be added to literature by expanding the study. p-08.02.5-025 inhibition of the recombinant human butyrylcholinesterase with paraoxon and coumarin analog of soman organophosphorus compounds (op) represent a class of extremely toxic chemicals that are used as warfare agents. uncontrolled utilization of op is highly dangerous due to their high potential to be efficient poisons in terrorist attacks. current therapy of op poisoning include intravenous administration of atropine and acetylcholine reactivators however, it does not completely eliminate brain damage effects. alternative experimental therapy against op poisoning is utilization of bioscavengers that irreversibly react with op and rapidly inactivate them. recombinant human butyrylcholinesterase (rhbche) is one of the most promising candidates as bioscavenger due to its pharmacokinetic characteristics and broad spectrum of op neutralizing activity. here we investigated in vitro inhibition of rhbche with two model oppesticide paraoxon (pox) and coumarin analog of soman (gd c ). both op lead to rapid and irreversible inactivation of rhbche that was monitored using ellman assay and fluorescence measurements. bimolecular inhibition rate constants dramatically differ between pox and gd c that could be explained by steric hindrance in soman analog. the next steps forward creation of catalytic bioscavengers based on rhbche should be done based on mechanisms of op-rhbche interactions. this work was performed in frame of grant rfme-fi60414x0069. non-hodgkin lymphomas (nhls) represent a heterogeneous group of malignancies that arise from the lymphoid system. at the present time exist a lot of drugs for the nhls therapy, but mostly all of them are unsafe and there is no consensus regarding the best treatment protocol. to increase the efficacy and safety of therapeutic b-lymphocyte depletion in lymphomas and leukemia's it would be preferable to induce the death of pathological b cells without affecting normal b cells to prevent side effects. similar to other types of cancer, nhls arise by a multistep accumulation of genetic aberrations that induce a selective growth advantage of the malignant clone. all b-cells of organism have a unique cell surface markerantigen b-cell receptor (bcr). we generate novel approach for personalized non-hodgkin lymphomas therapy based on peptide specific to malignant cells surface receptor. for this purpose we designed new lentiviral peptide library screening technique based on fluorescent reporter cells system. herein 7aa peptide library was used for screening of nhl's malignant receptor agonist. patients' lymph nodes biopsy samples mrna was used as a source of malignant bcr nucleotide sequence. variable domain of the lymphoma bcr was used for chimeric receptor generation, where bcr vh/vl part responsible for agonist recognition and bottom part of receptor was retranslate signal to the reporter gene. in this embodiment of the method, very large numbers of candidate 7aa peptides expressing lentivirus and eukaryotic reporter cells are packaged together in a format where each is capable of replication, thereby forging a direct link between genotype and phenotype. after four rounds of screening we discover peptides specifically interacted with malignant bcr's. selected peptide ligands were fused with chimeric antigen receptor for expression on t-cells. modified tcells selectively eliminate nhl's malignant cells ex vivo. this work was supported by grant rfmefi60714x0061. introduction: pesticides are used to prevent damage of unwanted insects, rodents, plant, moss and other pests. excessive use of pesticides may cause adverse effects in animals and humans. chlorpyrifosethylene (cpe) is an organophosphate pesticide, used in many agricultural products such as figs, cherries, olives worldwide; caused acute poisoning and chronically oxidative stress. rosadamascena mill (rosaceae) is a rose, used for production of rosewater (rw) and rose oil worldwide. rosaceae products are consumed in food and cosmetic industries. materials and methods: in this study, we investigated that cpe and rw effects on kidney tissues of rats. this study was included 32 adult male rats, divided into 4 groups. each group included 8 rats. i.group: control (regular feed), ii.group: cpe (0.3 mg/kg/day), iii.group: rw (100 mg/kg/day) and iv.group: cpe (0.3 mg/kg/day) + rw (100 mg/kg/day). following 15 days, kidneys were taken after sacrificed. analyzes were performed that malondialdehyde (mda), nitric oxide (no) as oxidant; superoxide dismutase (sod), glutathione reductase (gr) as antioxidant parameters. results: as compared with control, mda and no levels in cpe were a significant increase was determined (p conclusion: cpe is shown that significant increase on oxidant parameters, but significant decrease on antioxidant parameters. rw occurs opposite situation. similarly results of cpe, rw + cpe increased oxidant parameters, but decreased antioxidant parameters. these changes are lower than only cpe. these results showed that positive effects both rw and rw + cpe increasing on antioxidant parameters, also decreasing on oxidant parameters. we provide the comparative analysis of the reduced glutathione (gsh), reactive oxygen species (ros), a-tocopherol levels, an intracellular labile zn 2+ pool and esterase activity of red blood cells of patients with diagnosed components of the metabolic syndrome (ms)arterial hypertension and diabetes mellitus type ii (ah + dm + ). as the comparison groups were selected patients with one diagnosed component of msarterial hypertension (ah + dm à ) or without any diagnosed component of ms (ah -dm -). patients of all investigated groups were at the hospital treatment with a diagnosis coronary heart disease (chd) ii degree. human blood was obtained from normal donors and patients with chd ii stage. cytosolic esterase activity was assessed using calcein-am test. ros level was evaluated using cm-h 2 dcf-da. gsh level was estimated using lowry method. an intracellular labile zn 2+ pool was assessed using fluozin-3-am. investigations were performed on the specord m40, hplc system lc-20 prominence (shimadzu) and facscantoii (bd). a significant decrease of the intracellular level of labile zn 2+ in erythrocytes of patients with ah + dm + compare with ah + dm à and ah à dm à was shown. this fact confirms our assumption concerning the important role of zinc homeostasis in the etiopathogenesis of diabetes mellitus type ii. a direct relationship between the intracellular zn 2+ level modification and erythrocytes esterase activity of patients with chd ii degree was observed. moreover, in ah + dm + group of patients this relation was more marked. the unidirectional alteration in the erythrocytes redox state (gsh and a-tocopherol levels reduction, ros formation activation) was revealed at the whole of investigated chd patients groups (ah + dm + , ah + dm à , ah à dm à ). however, the pathological erythrocytes response on in vitro action of the different antioxidants (n-acetylcysteine, ascorbic acid, a-tocopherol, quercetin) had a diverse character that can be a significant test under antioxidant therapy prescription. it is known that long-term social isolation and the disorder of natural circadian rhythm is considered an important stress factors, which cause a variety of metabolic and mental disorders. it should be noted that the impact of the stresses takes up a larger area, according to, review of the action of their mechanism is one of the topical issues of modern science. it is estimated that as a result of stress the metabolic processes change in the organizm. because of this, we've studied the functionality of the antioxidant system in laboratory rat heart muscle cells and blood under psycho-emotional stress. it was found that quantitative changes of nitric oxide (no) was initiated the process of lpo, which caused oxidative stress in the cells and decreased antioxidant enzymes activity, such as catalase,sod, gpx and gr. the results suggested that psycho-emotional stress was accompanied by oxidative stress, causing a reduction in the intensity of energy metabolism in cardiac muscle cells, which was further strengthened by the fact that the activity of the enzymes involved in atp synthesis in mitochondria was reduced. also, we've studied exogenous creatine positive and negative affects on energy metabolism and blood lipid spectrum. based on this, we proposed that psychological stress is one of the factors contributing to the development of various cardiac diseases. the importance of free radical lipid transformations, which differ from the peroxidation processes, was pointed out for the first time in our laboratory studies. we have found that ros can induce free radical destruction processes of sphingolipids with c-c-bond cleavage [lipids, 2011, 46:271-276; lipid insights, 8, 1-9] . in case of acylated sphingolipids, they can undergo decomposition with c-c-bond cleavage upon direct uv irradiation [photochem. photobiol., 2012, 88:899-903] . it was of interest to establish the possibility of photosensitized decomposition reactions of not acylated sphingolipids, which do not absorb an ultraviolet. in this work we studied photosensitized reactions of sphingosines containing a free amino group, and low molecular compounds, which simulate their structure, such as aminoalcohols (serinol). as photosensitizers, the salts of transition metals, hydrogen peroxide, and acetone were used. oxygen was removed by bubbling with argon to reduce the probability of side reactions during photolysis of sphingolipids, such as oxidation processes (including oxygen reactions with alkyl radicals). we have shown that the action of uv-radiation on aminoalcohols and sphingosines in aqueous solutions in the presence of photosensitizers induces their destruction with c-c bond rupture. the main carbonyl product of sphingosines free radical destruction was an unsaturated aldehyde -2-hexadecanal. it was found, that 2-hexadecenal possesses a wide spectrum of biological activity: it promotes reorganization of the cell cytoskeleton and modifies the redox state of the cells [febs journal 282 (suppl. 1), abstracts: mem. biol. s5, lipid signaling & dynamics, p. 235.] . the results of this study can expand the frontier of research regarding free radical lipid damage, which could contribute to a better understanding of the origins of diseases associated with the activation of free radical processes in living organisms. structural basis for the 14-3-3 proteindependent inhibition of apoptosis signalregulating kinase 1 protein kinase ask1 (apoptosis signal-regulating kinase 1) is a member of the mitogen-activated protein kinase kinase kinase (map3k) family that plays a crucial role in immune and stress responses. the activity of ask1 is regulated through homo-oligomerization and interaction with other proteins including the 14-3-3 protein which binds to the phosphorylated motif located at the c-terminus of the kinase domain of ask1 and suppresses its catalytic activity through unknown mechanism. under stress conditions, ask1 is dephosphorylated at ser966 and the 14-3-3 protein dissociates. this dissociation is then one of the factors that lead to the activation of ask1. we performed low-resolution structural analysis of the kinase domain of ask1 (ask1-cd) bound to 14-3-3 using chemical cross-linking, analytical ultracentrifugation and small angle x-ray scattering. the low-resolution structural analysis shows that ask1-cd binds to the 14-3-3 protein in two to two stoichiometry through a small binding interface involving surface of 14-3-3 outside its central channel and several regions from the c-lobe of ask1-cd. the complex is dynamic and conformationally heterogeneous. phosphorus nmr and time-resolved fluorescence measurements, together with low-resolution structural analysis, indicate that binding of ask1-cd to 14-3-3 modulates conformation of ask1's activation segment. these results suggest that the 14-3-3 binding suppresses the catalytic activity of ask1 through direct structural modulation of its activation segment. our study provides new insight into the interaction between the kinase domain of ask1 and 14-3-3 and offers a plausible structural explanation for the 14-3-3 protein-dependent inhibition of ask1 kinase activity. introduction: thymoquinone (2-methyl-5-isopropyl-1,4-benzoquinone, tq) exerts a great antitumor activity against different types of cancer cells. a growing body of evidence indicates that reactive oxygen species (ros) generation followed by modulation of the akt and mapk pathways is a general mechanisms underlying the tq antitumor action. however, the data of tq effects on the mapk pathway are conflicting. to date, the activation or inhibition of the mapk protein family seems to depend on the cell type and on the tq concentration used. in order to elucidate the antitumor potential of tq against gliomas and the underlying molecular mechanism, tq influence on c6 rat glioma cells functioning was studied. results: it has been shown that the cultivation of c6 cells with tq in concentrations of 10 -100 lm during 24 hours strongly inhibits cell proliferation and induces cell death with id 50 of 60 lm. at the same time, tq induces ros generation and intracellular gsh depletion in a dose-dependent manner, that is followed by the mitochondrial potential decrease. interestingly, ros production has only cytoplasmic, but not mitochondrial origin in cells challenged with tq at the concentrations up to 100 lm. two-electron reduction of tq by dt-diaphorase attenuates tq anticancer efficiency whereby inhibition of dt-diaphorase by dicumarol increases tq-induced c6 cell death by 25 %. we analyzed mapk pathways involvement in c6 cells growth inhibition at tq treatment. it has been shown that inhibition of the erk pathway by pd98059 and jnk pathway by sp600125 does not influence on tq-induced effects. on the contrary, the specific phosphoinositide-3-kinase (pi3k) inhibitor (ly294002) abrogates tq-induced growth arrest. conclusion: antitumor effects of thymoquinone on c6 glioma cells is a result of ros generation and intracellular gsh depletion, that is followed by mitochondria disfunction, and growth arrest via pi3k pathway. assessment of oxidative stress and antioxidant defense activity parameters in patients with hiv-infection is of great importance, especially for hiv-positive women of reproductive age planning to have children. data of 53 women of reproductive age with hiv-infection analyzed: patients with hiv-monoinfection (n = 26) and patients with co-infection (hiv and hepatitis b and / or c) (n = 27). as a control we used the data of healthy women (n = 28). serum and hemolysate used as material for the study. lpo-aod products were determined by spectrophotometric and fluorometric methods. average value of initial lpo products -diene conjugates was significantly increased in the group with hiv-co-infected compared to control (1.57 times; p = 0.001) and group with hivmonoinfection (1.4-fold; p = 0.027). the level of secondary products -ketodienes and conjugated trienes increased in patients of both groups compared to control (2.16 times (p = 0.0002) and 2.43-fold (p < 0.0001), respectively). at the same time isolated double bonds and tba-active products content showed no significant changes (p > 0, 05). total antioxidant activity parameter decreased 1.28 fold (p = 0.0273) in the group with hiv-monoinfection compared to control. decrease in activity of the primary antioxidant enzyme -superoxide dismutase (p = 0.0077, compared to the control and p = 0.0035, compared with the group with hiv-monoinfection) and the level of a-tocopherol (1.22-fold to control and 1.25 fold to hiv-monoinfection) was detected in the group with hiv-coinfection. 1.29-fold higher content of retinol in hiv-coinfection group (compared to the control) revealed. in women with hiv-coinfection the oxidative stress was significantly higher than in women with hiv-monoinfection. suggested to include antioxidant supplements in the complex pathogenetic therapy in women with hiv-coinfection (hiv and hepatitis b and / or c), which will contribute to women's ability to bear children. p-09.04.4-008 acute different doses of malathion induce cholinesterase inhibition, glucogenic enzymes and histopathological change in rat liver malathion, which is an organophosphorus compound, is a widely used pesticide all over the world. despite its benefits, malathion has many toxic effects on many tissues including liver. we designed to evaluate the acute different doses of malathion on cholinesterase (che) inhibition, gluconeogenic enzymes and histopathological change of rat liver. for this purpose 4 groups were formed. rats in group 1 served as control group animals which were only given corn oil. group 2, group 3 and group 4 were administered 100, 200, 400 mg/kg of malathion, respectively, dissolved in corn oil by oral gavage. the rats were sacrificed after 24 hours following administration and the livers of rats were removed. the liver che, alanine aminotransferase (alt), aspartate aminotransferase (ast) and lactate dehydrogenase (ldh) were studied using autoanalyzer. histopathological investigation was performed using microscope. the liver che activities of group 2, group 3 and group 4 were inhibition percentage of 53%, 43%, and 51% respectively, comparison of group 1's che activity. the liver alt, ast and ldh increased in group 3 and group 4 compare to group 1 and group 2 (p < 0.013). we also observed that there was vacuolar and hydropic degeneration in liver of group 4. according to our result, acute administrations of malathion result in hepatotoxic effects with increasing doses. background and aims: an imbalance between free radicals and antioxidants is closely linked to the onset of an acute myocardial infarction (ami). the aim of this study was to investigate the antioxidant status and the lipid peroxidation in patients admitted to hospital immediately after ami. methods: the study population comprised 19 patients with ami and 14 healthy subjects. patients that had an acute myocardial infarction (ami) less than 72 hours since onset were selected for this study. antioxidant status was assessed by lactonase activity of paraoxonase (pon1 dhc), trolox equivalent antioxidant capacity (teac) and plasma uric acid level. malondialdehyde was used as marker of lipid peroxidation. results: compare with the control group, the levels of mda and pon1 dhc were significantly higher in group with ami (p < 0.005, respectively p < 0.001). elevated levels of mda (p < 0.005) were found in patients with ami compare with the control subjects. ami patients had also statistically significant reduced levels of teac (p < 0.005) comparative with healthy subjects. no statistically significance was found for plasma uric acid level in subjects from our study. conclusion: a high lipid peroxidation correlated with a decreased teac activity suggest an exacerbated oxidative stress in subjects admitted to hospital immediately after an ami. p-09.04.4-010 dealing with copper toxicity: new insights into copper detoxification in yeast copper (cu), an essential metal, is a double-edged sword, as its essential nature is counterbalanced by the toxic effect that it can exert on cells when not properly controlled. as such, organisms have evolved defence mechanisms against cu toxicity, and in the yeast saccharomyces cerevisiae, the transcription factor ace1 orchestrates several of those mechanisms, by activating cu-detoxification genes. in s. cerevisiae iron (fe) uptake is partially dependent on cu, as the membrane multicopper-oxidase fet3, which is part of the high-affinity iron uptake system, requires cu as a cofactor. aft1, the low iron-sensing transcription factor in yeast, is known to regulate the expression of fet3 gene. however, we found that a strain lacking fet3 is more sensitive to cu surplus conditions than a strain carrying the aft1 gene disrupted. this finding suggests that under such conditions another regulator came into play and controls fet3 gene expression. we next evaluated whether ace1 could be the alternative regulator of fet3 under cu excess. to test this hypothesis we first constructed the double mutant aft1ace1 and assayed its fitness under cu surplus. as expected, the double mutant is much more sensitive to cu than the single aft1 or ace1 mutants. we next monitored the expression of fet3 gene in cells lacking ace1, using yeast-one hybrid and qrt-pcr approaches. our data clearly indicates that fet3 expression is dependent on ace1 when cu is overly abundant. altogether our data unveil a novel mechanism of cu detoxification relying on the activation of fet3 by ace1 in an aft1independent way. experiments to understand the consequences of this regulation in terms of cu detoxification are currently being undertaken. in joint degeneracy, reactive oxygen species manifest their toxicity both through intrinsic reactivity and the inflammatory process activation, leading to cartilage dysfunctions, injuries of matrix proteins and cytokines stimulation. the study is focused on the identification of oxidative balance modulation (enzymes and oxygen free radicals) by a bioactive extract obtained from small sea fish. the cellular support was represented by the chondrocyte line chon-001 derived from human long bones (atcc ò crl-2846 tm ), that assure the reproducibility of a standardised biological system. the oxidative stress was induced through stimulation with il1b, a cytokine-factor that promotes the protein catabolism and also with tnfa, the initiator of pro-inflammatory activation, combined with pma, the activator of protein-kinase c, triggering of oxygen reactive species generating cascades. the antioxidant effect was compared with known antioxidants: vitamin c, x3 fatty acid, n-acetyl -cysteine. the acellular antioxidant/antiradical screening was done using two complementary techniques for total antioxidant status evaluation and completed by measuring cellular catalase (cat) and superoxidedismutase (sod) activity, correlated with intracellular hydrogen-peroxide and superoxide anion monitoring through flow cytometry. the antioxidant properties of the bioactive extract proved in acellular systems are confirmed at cellular level by the involvement of the product in the enzymatic cascade cat -sod, potentiating the catalytic action of the enzymes, and by the decline of both intracellular reactive oxygen species (the hydrogen peroxide decrease with 50%, the superoxide anion is reduced with 31% compared with the stimulated control). the demonstrated antioxidant synergy assures a complete cellular protection induced by the small sea fish extract in human condrocytes. introduction: alzheimer's disease (ad) is a progressive neurodegenerative disorder characterized by memory loss, cognitive impairment. oxidative stress is a contributory factor in ad pathogenesis. glutathione (gsh) is the main antioxidant cellular defence. the ratio of gsh/gssg shows the redox status of gsh, and plays important role in maintaining intracellular redox homeostasis. the current study was carried out to determine oxidative dna damage and ratio of gsh/gssg which plays an important role in protection of target molecules from oxidation in the patients with ad. methods: the study subjects were consisted of patients with ad (n = 67) and age matched control group (n = 42) who were treated and followed in the cerrahpasa medical faculty hospital, department of neurology and department of internal medicine/ geriatrics. dna strand breaks and h 2 o 2 -induced dna damage were determined in lymphocyte dna with comet assay. the gsh and gssg levels in the erythrocyte lysates were measured by using a commercial spectrophotometric kit. the ratio of gsh/gssg were calculated. statistical analysis was performed with spss 22 software package. results: dna strand breaks and h 2 o 2 -induced dna damage were found to be higher (p = 0.000 for all), the ratio of gsh/gssg was found to be lower (p = 0.024) in the ad group than control group. there was no significant difference between male and female for dna strand breaks and h 2 o 2 -induced dna damage in the ad group, but ratio of gsh/gssg were higher in male when compared with female (p = 0.045). no significant difference was found between the men of ad group and men of the control group for gsh/gssg ratio whereas women of the ad have a lower gsh/gssg ration than those in the women of the control group (p = 0.009). conclusion: increased systemic oxidative dna damage and dna susceptibility to oxidation may be resulted from diminished gsh/gssg ratio in ad patients. this finding shows the importance of antioxidant support in ad management. p-09.04.4-013 validation of a liquid chromatography-tandem mass spectrometry method for the measurement of lipid peroxidation product 8iso-prostaglandin f2a in urine m. kant 1 , m. akıs ß 1 , h. _ is ßlekel 1,2 1 department of medical biochemistry, school of medicine, dokuz eylul university, izmir, 2 department of molecular medicine, school of medicine, dokuz eylul university, izmir turkey 8-iso-prostaglandin f 2a (8-iso-pgf 2a ) is a reliable indicator of lipid peroxidation resulting from oxidative lipid damage and is postulated as a gold standard biomarker for the evaluation of oxidative stress. the aim of this study was to validate non-invasive and highly accurate stable isotope dilution-multiple reaction monitoring liquid chromatography-tandem mass spectrometry (sid-mrm lc-ms/ms) method for identification and quantification of 8-iso-pgf 2a . twenty four hour urine samples were collected from healthy volunteers at medical school of dokuz eylul university for analytical performance studies. lc-ms/ms analyses were performed on conventional hplc coupled to a triple quadrupole ion trap mass spectrometer equipped with a turboionspray tm source. analyst software version 1.5 were used for data analyses. mrm transitions used were m/z? 250/164 for 8-iso-pgf 2a and m/z? 255/169 for 8-iso-pgf 2a-d 4 . analytical performance of the method was evaluated by linearity, selectivity, sensitivity, precision and accuracy studies using pure standards and samples extracted from urine of healthy volunteers. the linear calibration range for 8-iso-pgf 2a was determined as 20 ng/ml by using standards ranging from 0.25-50 ng/ml. analytical sensitivity of the method was determined by lod with s/n of 3 and loq with s/n of 10. lod and loq for 8iso-pgf 2a were 2.4 9 10 à2 and 38 9 10 à2 ng/ml, respectively. the assay stability and reproducibility were assessed by the precision and accuracy of intra-and interday measurements (n = 10). the intra-and interday cvs for 8-iso-pgf 2a were 4.5% and 1.2%, respectively. sid-mrm lc-ms/ms method for absolute quantification of 8-iso-pgf 2a was optimized and validated in our laboratory and therefore this highly accurate method can successfully be applied to clinical patient samples. p-09.04.4-015 synergistic anticancer effects of sulforaphane and cisplatin through the induction of apoptosis and autophagy following oxidative stress in malignant mesothelioma cells malignant mesothelioma is characterized by poor responsiveness to current chemotherapeutic drugs, usually owing to high resistance to apoptosis. here, we investigated chemosensitizing effects of phytochemical resveratrol, in combination with cisplatin, on malignant meothelioma cells. cell viability was evaluated using mtt assay. cell apoptosis was detected with dapi staining, caspase 3/7 activity assay and flow cytometry. cell cycle distributions, ros levels and mitochondrial membrane potential were determined using flow cytometry. the expression of cell cycle-, apoptosis-, and autophage-related proteins was measured with western blotting. the combination treatment of cisplatin and resveratrol (cis/res) synergistically induced apoptosis, as evidenced by typical cell morphological changes, the appearance of a sub-g 0 /g 1 peak, an increase in the annexin v(+) cells and the cleavage of caspase-3 and parp. cis/res increased ros production and depolarization of mitochondrial membrane potential with an increase in the bax/bcl-2 ratio. these changes were attenuated by pre-treatment with n-acetylcysteine, suggesting that cis/res induced apoptosis through oxidative mitochondrial damage. compared with msto-211h cells, cis/res was less efficient in killing h-2452 cells. h-2452 cells exhibited an enhanced autophagy to cis/res, as observed by an increase in viable cells exhibiting intense lysotracker red staining and up-regulation of beclin-1 and lc3a. inhibition of autophagy by bafilomycin a1 rendered cells more sensitive to cis/res-induced cytotoxicity and this was associated with induction of apoptosis. these data indicate that the increased resistance of h-2452 cells to cis/res is closely related to the activation of self-defensive autophagy, and provide the rationale for targeting the autophagy regulation in the treatment of malignant meothelioma. stress oxidative induced by chemotherapy with cyclophosphamide (cp) causes vulnerability in sperm and decline of fertility. this study was aimed to evaluate the role of ethyl pyruvate (ep) in the amelioration of fertility and growth of primitive embryo in animals that received cp. 24 adult male mice (6-8 weeks) were randomly divided into 3 groups: control group received normal saline (0. 2 ml/day, ip), cp group received cp (15 mg/kg/week,¬ ip), the cp+ep group received ep (40 mg/kg/day, ip) along with cp, were treated for 35 days. 8 mice from each group were arranged for evaluation of sperm quality and in vitro fertilization (ivf) too. afterward, the separated oocytes from 72 ovulation stimulated mice were conducted to evaluation of ivf and embryo development. the results revealed that cp caused notable decrease in percentage of fertilization in cp group, but administration of ep along with cp caused an increase in the percentage of fertilization in comparison to cp group. the percentage of the two cell zygotes in cp+ep group, and the percentage of blastocysts in control and cp+ep groups were higher than that in cp group (p < 0.05). results showed that the total number of arrested embryos in control and cp+ep groups was decreased in comparison to cp group (p < 0.05). the percentage of arrested embryos type i, ii, and iii in cp+ep group was decreased in comparison to cp group, but that decrease was significant only in types i and ii (p < 0.05). the average motility, viability, nucleus maturity and sperm morphology were decreased significantly in cp group in comparison with control and ep groups, whereas ep caused significant increase of these parameters (p < 0.05). also, the percentage of dna damage was increased significantly in cp group in comparison with control and ep groups (p < 0.05). the results of this study indicated that the ethyl pyruvate was able to suppress free radicals and enhance the ivf and quality of sperm in cp treated animals. malathion, which is a member of organophosphate chemical family, is used to control pests and is a widely used pesticide all over the world. however it is also known to be highly toxic on many tissues including pancreas. to test this we set 4 groups to administer a single dose of malathion dissolved in corn oil via oral gavage at the doses of 100 mg/kg (group 2), 200 mg/kg (group 3) and 400 mg/kg (group 4). only plain corn oil was given to control group (group 1). the rats were sacrificed 24 hours after administration of the chemical and the pancreases of rats were removed. in an attempt to evaluate the dose dependent response, we measured amylase and lipase activities, insulin, malondialdehyde (mda), total oxidant status (tos) levels in rat pancreases. all of the parameters were measured spectrophotometrically. we found that pancreas insulin levels significantly increased in group 4 compare to group 1, besides the insulin levels of group 3 and group 4 were significantly higher than group 2 (p < 0.013). pancreas amylase and lipase activities significantly decreased in group 3 and group 4 compare to group 1 and group 2 (p < 0.013). however, there was no significant change in pancreas mda and tos levels (p > 0.05). according to correlation analysis, when pancreas amylase levels declined, lipase levels were decreased simultaneously and there was a strong positive correlation between them (p < 0.01). in addition, when the comparison was evaluated as a binary, while pancreas amylase and lipase levels diminished, pancreas insulin levels increased and a strong negative correlation between them was found (p < 0.01). according to our result, acute administrations of malathion leads to alterations of insulin, amylase, and lipase levels with a dose dependent manner, while it does not to change oxidant status. the aim of this study is to evaluate the potential toxic effects of mancozeb on the stress biomarkers such as catalase (cat) activity, malondialdehyde (mda) level and protein levels in the brain tissue of zebrafish (danio rerio). mancozeb, is a synthetic fungicide contaminating aquatic environments as a potential toxic pollutants, was investigated in the present study for acute toxicity. zebrafish groups (m-low and m-high) were exposed to different doses of mancozeb (5 mg l-1 and 7.5 mg l-1) for 120 hours except the control group. catalase (cat) activity, malondialdehyde (mda) level and total protein levels were determined by spectrophotometer. the results showed that cat activity and mda levels were decreased in all experiment groups. protein levels were increased in experiment groups when compared to the control group. in conclusion, the changes in the cat activity and mda levels were time and as well as mancozeb dose-dependent. furthermore, the biochemical parameters of mancozeb exposure on zebrafish, showed that mancozeb has significant effect on the zebrafish and/or aquatic organisims. paracetamol (para), which is antipyretic and analgesic, is widely used around the world. paracetamol can be recommended for moderate or mild pains especially in pregnancy as an analgesic. it is known that, paracetamol can cause hepatotoxicity or nephrotoxicity. we were aimed that in this study to show potential hepatoprotective and nephroprotective effect of betaine against long term paracetamol using at therapeutic doses. it has been prepared 3 groups, control, para and para+-betain groups. paracetamol and betaine were administered by gavage to pregnant rats, from first day to the last day of pregnancy (aprox. 21 day). 2 ml physiological saline (%0.9 nacl solutions), 30 mg/kg/day paracetamol, 30 mg/kg/day paracetamol and 800 mg/kg/day betain was given by orally to control, para and para+betain groups respectively. the day of the birth, newborn rats anesthetized by ether and after decapitated. 8 newborn rat's liver and kidney tissues used for biochemical analysis [malondialdehyde (mda), reduced glutathione (gsh), nitric oxide (no) and paraoxonase-arylesterase (pon-are)] and 5 rat's liver and kidney tissues used for histological studies. we showed that, mda and no levels was significantly increased, while pon activities decreased. on the other hand gsh levels and are activities was decreased but these decline wasn't significant in the liver and kidney para group. these biochemical results showed hepatotoxicity and nephrotoxicity in neonates which can be formed in long term maternal paracetamol using at therapeutic doses. also our histological findings was support these biochemical results. we used betaine against potential hepatotoxic and nephrotoxic effect of long term maternal paracetamol using at therapeutic doses for neonates. betaine has antioxidant properties and also used as a methyl donor for transsulfuration reactions in the cell. biochemical and histological examinations showed that betaine protected the tissue injury relatively. p-09.04.4-020 lipid rafts are involved in modulation of ca 2+ responses induced by glutoxim and molixan in macrophages pharmacological analogues of oxidized glutathione (gssg) disulfide-containing drugs glutoxim ò (gssg disodium salt with dmetal nanoaddition, «pharma-vam», st. petersburg) and molixan ò (complex of glutoxim with inosine nucleoside) have found clinical application as a wide range immunomodulators and hemostimulators. however, the cellular and molecular mechanisms of these drugs action are still unclear. previously we showed for the first time that glutoxim and molixan cause biphasic intracellular ca 2+ concentration ([ca 2+ ] i ) increase due to ca 2+ mobilization from thapsigargin-sensitive ca 2+ stores and subsequent store-dependent ca 2+ entry in rat peritoneal macrophages. it is known that key proteins involved in ca 2+ signaling are localized in discrete plasma membrane lipid rafts domains. lipid rafts are cholesterol and sphingolipids enriched microdomains that function as unique signal transduction platforms. thus, the aim of the present work was to elucidate the possible involvement of lipid rafts in glutoxim and molixan effects on [ca 2+ ] i in macrophages. [ca 2+ ] i measurements were performed with fura-2am microfluorimetry. to examine the involvement of lipid rafts in the effect of gssg-based drugs on [ca 2+ ] i we used methyl-bcyclodextrin (mbcd), widely used to remove cholesterol from membranes, thus disrupting the lipid raft domains. we have shown for the first time that macrophage preincubation with 10 mm mbcd for 1 hours before molixan addition causes significant inhibition of both ca 2+ mobilization (on average, by 77.6 ae 9.2%) and subsequent ca 2+ entry (on average, by 82.3 ae 10.5%), induced by molixan. similar results were obtained in experiments with glutoxim. thus, we have demonstrated for the first time that mbcd significantly decreases both phases of glutoxim-or molixan-induced ca 2+ responses in macrophages. the results suggest that intact rafts are required to initiate complex signaling cascade activated by glutoxim or molixan and leading to [ca 2+ ] i increase in macrophages. plant-derived natural substances (phytochemicals) with potent pro-apoptotic activity towards cancer cells in vitro are considered as promising nutraceuticals in anticancer therapy. nevertheless, due to their relatively low bioavailability, administration of high doses of nutraceuticals that are not achievable in vivo seems to exert potentially negligible physiological effects in clinical trials. thus, there is a need for revealing novel cytophysiological effects of low doses of phytochemicals towards cancer cells. in the present study, we have considered thirty one nutraceuticals and selected four phytochemicals acting at low micromolar range (5 to 20 lm) against phenotypically different mcf-7, mda-mb-231 and sk-br-3 breast cancer cells, namely diosmin, sulforaphane, ursolic acid and betulinic acid. nutraceuticals inhibited cell proliferation and caused changes in the cell cycle that was accompanied by elevated levels of p53, p21, p16 and/or p27. apoptosis (annexin v staining, multicaspase and mitopotential assays) was observed exclusively when nutraceuticals were used at the concentration of 20 lm, whereas at the concentration of 5 lm, stress-induced premature senescence was noticed (sa-b-gal activity). nutraceuticals diminished the levels of glut-1 and selected glycolytic enzymes. nutraceuticals promoted oxidative and nitrosative stress as judged by increased production of total reactive oxygen species, total and mitochondrial superoxide, nitric oxide and protein carbonylation. nutraceuticals also stimulated dna single and double strand breaks that was accompanied by atm phosphorylation and to a lesser extent by histone h2ax phosphorylation and 53bp1 foci formation. taken together, several responses to nutraceutical treatment were observed in breast cancer cells that may reflect the heterogeneous nature of cancer cell populations. this study was supported by grant from the national science center, 2013/11/d/nz7/00939. nucleolus is thought to be a stress sensor and oxidative and ribotoxic stimuli may cause the inhibition of rrna synthesis by the inactivation of transcription factor tif-ia/rrn3 that is accompanied by the relocation of nucleolar proteins and p53-based cell cycle arrest and/or apoptosis. as nutraceutical-mediated modulation of nucleolar activity may be considered an attractive anticancer strategy, in the present study, we have investigated the effects of three selected nutraceuticals, namely sulforaphane, ursolic acid and betulinic acid on nucleolus state in three breast cancer cell lines (mcf-7, mda-mb-231 and sk-br-3). we found that low dose nutraceutical treatment resulted in p21-mediated inhibition of cell proliferation, a decrease in rrna synthesis, shifts in lamin a/c and b1 pools, changes in the nucleolar protein levels and their carbonylation, and changes in nucleolus size and number. breast cancer cells differed in erk activity that resulted in different patterns of erk activation/inhibition, phosphorylation status of s6 and autophagy induction upon nutraceutical stimulation. nutraceuticals also affected dna methylation parameters, namely the levels of dnmt1, dnmt3a and dnmt3b that resulted in both global dna hypo-and hypermethylation. taken together, after nutraceutical treatment, nucleolus-centered cellular response was revealed in breast cancer cells of different phenotypic characteristic that may be considered a potential target of anticancer therapy. this study was supported by grant from the national science center, 2013/11/d/nz7/00939. p-09.04.4-023 rate of apoptosis in human macrophages infected with leishmania tropica promastigotes infection of the cells with parasites or exposing cells to heat stress induces a cellular stress. in the present study human macrophages are infected with leishmania tropica promastigotes and exposed to heat stress. the measurement of cytoplasmic histone-associated dna-fragments was carried out using elisa technique. visualization of apoptotic cells was performed by the terminal deoxynucleotidyl transferase dutp nick end labeling staining method (tunel). degree of oxidative stress on cell is evaluated by measuring nitric oxide (no), malondialdehyde (mda), reducte glutathion (gsh) levels and superoxide dismutase (sod) activities. results of the elisa technique showed that infection of macrophages with promastigotes induced apoptosis rate significantly (p < 0.001), heat stress however decreased the rate of apoptosis in infected macrophages remarkably (p < 0.001). high levels of apoptosis rate in infected macrophages and drastic decdrease in apoptosis in heat subjected macrophages infected with promastigotes are confirmed by visualisation of apoptotic cells using tunel method. levels of glutathion (gsh) in infected macrophages decreased significantly (p < 0.05), while malondialdehyde (mda) levels increased notably (p < 0.05). however, no statistical significant alterations were detected in the nitric oxide (no) values and superoxide dismutase (sod) activities. results of the present study indicates that infection of human macrophages with leishmania tropica induces a cellular stress response, characterized by decreased values of gsh and increased levels of mda. increased rate of apoptosis in infected macrophages may be due to the increased cellular stress caused by leishmania tropica amastigotes. decreased rate of apoptosis measured in heat exposed macrophages infected with promastigotes indicates an extention in life span of macrophages. measurements of the parameters characterizing the redox and inflammatory processes in blood are essential for diagnosis and prognosis of type 2 diabetes mellitus (t2dm), but also for monitoring the effectiveness of medical treatments. along with other biochemical effects, hormonal imbalance leads to modified transport function of erythrocytes due to changes in enzyme systems involved in upholding of cellular homeostasis through a rapid degradation of altered proteins, being the second line of defense against the free radicals, which degrade and eliminate the damaged molecules. some of these enzymes are hemoglobin peroxidase (pa) and esterase (ea). the aim of this research study was to identify new parameters with a potential role of biochemical markers in t2dm like hemoglobin peroxidase and esterase activity from erythrocyte. our data showed that pathological processes involved in t2dm imply an increased enzymatic activity of pa (73.85%), which correlates with increased levels of ea (47.9%) and glycated hemoglobin (hba1c) (13.51%), compared with control group. the variables hba1c, pa and ea are correlated: the identified pearson correlation coefficients r = 0.637 and r = 0.573 respectively, have an associated probability of p < 0.001 and p < 0.006 a value that indicates a strong positive correlation between the dependent variables pa and ea and independent variable hba1c. based on these results we concluded that together with glycosylated hemoglobin assay, all the studied parameters can be successfully used as extra test for diabetes associated with oxidative stress and disorders in erythrocyte functions or blood rheology. the radioprotective effects of propolis and caffeic acid phenethyl ester on radiationinduced oxidative/nitrosative stress in brain tissue s. taysi 1 , e. demir 2 , k. cinar 3 1 gaziantep university, school of medicine, gaziantep, 2 haran university, sanliurfa, 3 department of neurosurgery, medical school, gaziantep, turkey head and neck cancer patients treated with radiotherapy suffer severe side effects during and following their treatment. efforts to decrease toxicity of irradiation to normal tissue, organs and cells have led to searching for cytoprotective agent. investigations for effective and non-toxic compounds with radioprotective capability led to increasing interest in antioxidant such as propolis and caffeic acid phenethyl ester (cape). the aim of this study was to evaluate the antioxidant and radioprotective effects of propolis and cape on radiation-induced oxidative/nitrosative stress in the brain tissue. fourty sprague-dawley rats were randomly divided into five groups. group 1 (irradiation (ir) + propolis) received total cranium irradiation and propolis was given orally through an orogastric tube daily. group 2 (ir+cape) received total cranium irradiation plus cape, was dissolved in dimethyl sulfoxide (dmso) just before giving to the rats, intraperitoneally (ip) every day. group 3 (ir) received 5 gy of gamma irradiation as a single dose to total cranium plus 1 ml saline daily. group 4 received daily plain dmso. group 5 received daily plain saline. at the end of the 10 day time period, xanthine oxidase (xo), nitric oxide synthase (nos) activities, nitric oxide (no•) and peroxynitrite (onoo -) levels were significantly higher in ir group compared to all other groups. in conclusion, the results suggest the radioprotective ability of propolis and cape involving prevention of radiation-induced oxidative/ nitrosative damage. p-09.04.4-027 role of leptin and adiponectin in obesityassociated oxidative stress e. becer 1 , a. c ß irakoglu 2 1 near east university, nicosia, cyprus, 2 istanbul university, istanbul, turkey objective: increased oxidative stress is one of the major characteristics of obesity and obesity-related complications. adipokines also induce the production of reactive oxygen species and generating oxidative stress in physiological and pathological conditions of obesity. the aim of this study was to determine the association of leptin and adiponectin levels with body mass index, lipid parameters and oxidative stress biomarkers in obesity. methods: the study included 150 obese and 120 non-obese subjects. plasma leptin and adiponectin levels (ng/ml) were measured using commercially available enzyme-linked immunosorbent assay kits. serum lipid, superoxide dismutase, malondialdehyde and antropometric parameters were measured. results: obese and non-obese subjects did not differ in age, while plasma glucose, total cholesterol, triglycerides, ldl cholesterol and leptin levels were significantly higher and mean hdl cholesterol and adiponectin levels were significantly lower in obese than non-obese subjects. the plasma leptin (p < 0.001) and adiponectin (p = 0.003) levels were significantly correlated with bmi in both obese and non-obese subjects. in obese subjects, leptin levels were significantly correlated with superoxide dismutase (p < 0.01) and malondialdehyde (p < 0.01). strikingly, adiponectin was significantly correlated with superoxide dismutase (p = 0.02) and malondialdehyde (p = 0.0012) levels in obese group. conclusion: our results suggest that leptin and adiponectin levels are associated with defective antioxidant status and increased lipid peroxidation which may have implications in the development of obesity related health problems. clinical trials of biologically active plant substances show a significant preventive effect in cancer, cardiovascular diseases and peptic ulcer disease in the form of both nutritional supplements and therapeutic intervention. anthocyanins contained in dark berries show great antioxidant potential, with the most important including a reduction in oxidative degradation of lipids or tyrosine oxidation by peroxynitrite. our previous studies of the antioxidant properties of extracts from vaccinium corymbosum, aronia melanocarpa and sambucus nigra, however, indicated that their activities largely depend on the method of extraction. while quantitative determination of anthocyanins pointed to a disproportionately larger content of anthocyanins in isolates from lyophylized berries, their scavenging activities against hydroxyl radicals was surprisingly the lowest. inflammatory processes, vascular damage, atherosclerosis and others are caused by oxidativenitrosative stress, so we tested their efficiency to scavenge no degradation products. we found that only purified extracts of lyophilized berries showed the most significant effects against no degradation products, with efficacy of around 50%. an extract from aronia showed greater than 60% efficiency, and a net ethanol extract from all the berries showed a 30% effect. cleaned ethanol extracts showed the lowest effects, while aronia initiated production at a concentration of 25 mg/l. conversely, all acetone extracts consistently initiated no degradation products. these findings are in complete contrast to their determined action against reactive oxygen species. in summary, it follows that a particularly adjusted lyophilized extract of the berries could be responsible for the increased biological activity of no and the observed biological and pharmacological activities of anthocyanins in circulatory disorders. the study was supported by grant vega 1/0782/15 and 1/0018/16. p-09.04.4-032 attenuation of dysfunction, oxidative stress and apoptosis by resveratrol in benzo(a)pyrene exposed ins1-e 832/13 pancreatic beta cell line s. c ß elik, b. baysal faculty of medicine, afyon kocatepe university, afyonkarahisar, turkey diabetes is one of the most important problems in the world. this disease is a very important health problem due to affects many different organs and systems. it has been well established that, environmental pollutants had deleterious effects on glucose metabolism, and caused insulin resistance and type 2 diabetes. with this investigation, it was aimed to investigate the effects of benzo(a)pyrene on pancreatic beta cells and treatment affects of resveratrol. in this study, rat ins-1e beta cell line was used. after reaching the appropriate number of cells culture operations by cell culture, benzo(a)pyrene (20 lm, 24 hours) application have been made after resveratrol (80 lm, 48 hours) application. after incubations oxidative stress, insulin secretion (in cell and in medium), cell proliferation and apoptosis were analysed in cells by biochemical and molecular techniques. b(a)p application resulted in no increase and resveratrol also increased this level of no. resveratrol increased the tas levels decrased by b(a)p, and tos levels were also increased by resveratrol interestingly. osi levels determined with tas and tos levels, has no significant change between groups. gsh levels were decreased by b(a)p while resveratrol increased its' level to control level. mrna expression levels of beta cell functions related genes ins-1, ins-2 and sirt-1 were increased by resveratrol treatment. insulin levels in cell and in medium were increased after resveratrol treatment. mrna expression level of foxo-1 gene was increased while pdx-1 was decreased by resveratrol treatmeant. b(a)p suppressed the mrna expression of p53 gene, but resveratrol increased. the effects of b(a)p on pancreatic beta cells and the protective effects of resveratrol on this cells were investigated in vitro with this research firstly. the results obtained from this research showed that oxidative changes, functional impairment and carcinogenetic effects of b(a) p in pancreatic beta cells could be blocked by resveratrol. the protective effect of vitamin e (alphatocopherol) on ischemia-reperfusion injury in rat liver ischemia-reperfusion (i/r) process is usually used during transplantation and resection of the liver but liver dysfunction and cellular death due to lack of oxygen in tissues, changes in the balance of oxidant/antioxidant in favor of oxidants, and inflammatory response are inevitable during this process. in the present study, it was aimed to investigate whether vitamin e(alpha-tocopherol), an antioxidant agent, has a protective effect against liver ischemia reperfusion injury in rats by using morphometric methods. for this purpose, 21 adult sprague-dawley male rats were divided into 3 groups as; control, ischemia / reperfusion (i/r), and vitamin e+ischemia/reperfusion (evit +i/r). in experimental groups, the total hepatic ischemia was applied for 45 minutes followed by a 24 hour of reperfusion. in the treatment group, 7 days before ischemia 40 mg / kg dose of vitamin e was administered intraperitoneally once a day. after the termination of the reperfusion, the rats were perfused by cardiac way and liver tissues were dissected. following volume and weight calculations, the livers were subjected to the standard histological preparation methods and embedded in paraffin. serial sections at 5 lm thickness were obtained from these blocks, stained with hematoxylineosin, and analyzed with morphometric methods. in light microscopic examinations of the i/r group, irregularity in lobules, dilatation in central veins and sinusoids, extensive areas of necrosis and pycnotic nuclei were seen in hepatocytes. the volume density of sinusoids to liver parenchyma was estimated as 16% in the control group, whereas it was 36% in the i/r group. this value was decreased to 32% in the evit + i/ r group. however, no significant difference was found among the groups in the lobule area calculated by the point counting method. these results show that intraperitoneal vitamin e administration for 7 days prior to ischemia partially inhibits damage caused by ischemia-reperfusion injury in the liver. the leaves, fruit and bark of annona muricata, a member of the annonaceae family, are commonly used in the traditional medicine of tropical and subtropical regions. in recent years, many studies have shown their anti-cancer, anti-convulsant, antiarthritic, anti-parasitic, anti-malarial, and anti-diabetic activities. it should be noted that these characteristics have been described using different types of extracts from different parts of the plant. our studies have focused on the systematic characterization of activities most easily accessible from an aqueous extract of leaves, with hitherto documented antihypertensive and hepatoprotective effects. we found that the extract shows almost 54% efficiency against hydroxyl radicals. with increasing concentrations, the effectiveness weakened, reaching a second peak (40%) at a concentration of 50 mg/l. the scavenging activity against no degradation products maintained a continuously increasing trend with a maximum at a concentration of 100 mg/l. surprisingly, the extract initiated peroxynitrite production in a similar trend, except at 50 mg/l, where it scavenged peroxynitrites with relatively high efficiency, up to 34%. these findings are consistent with the elevated levels of reduced glutathione detected after incubation of liver mitochondria with extract to a maximum concentration of 50 mg/l, with subsequent sharp decline. the activity of glutathione s-transferase was decreased, although not significantly. this indicates a reduction of metabolic processes of compounds, allowing their action over a longer period of time. in a live system, even antihypertensive effects can be observed. however, a significant outflow of gsh to create the gsh adducts of active substances, and particularly s-nitrosoglutathione from increased production of peroxynitrites, can cause liver toxicity. the study was supported by grant vega 1/0782/15 and 1/0018/ 16. the role of polyamine metabolism in curcumin induced apoptosis via reactive oxygene species (ros) generation in growth hormone (gh) overexpressed t47d breast cancer cells r. genc ß, a. coker gurkan, e. d. arisan, p. obakan yerlikaya, n. palavan unsal, n. palavan unsal t.c istanbul k€ ult€ ur € universitesi, istanbul, turkey autocrine growth hormone (gh) signaling triggers cell proliferation, growth, metastasis and drug resistance in cancer cells. polyamines (pas) play an essential role in cell cycle, proliferation, growth and carcinogenesis of various cancer types such as prostate, colon and breast cancer. odc (ornithine decarboxylase) is the key enzyme in the pa biosynthetic pathway that is under control of antizyme (az) and antizyme inhibitor (azi) activity. pa catabolic enzymes polyamine oxidase (pao) and spermine/spermidine acetyle transferase (ssat) by-products triggers reactive oxygene species (ros) generation and apoptotic cell death. curcumin decreased cell viability in dose and time dependent manner in t47d wt and gh+ cells. although forced gh expression induced cell proliferation, 20 lm curcumin inhibited cell invasion. curcumin (20 lm) induced apoptosis by acting on intrinsic and extrinsic pathway in both cell lines. moreover, curcumin supressed odc, azi expression in wt and gh+ t47d cancer cells. although curcumin decreased az expression in t47d wt cancer cell, increased az expression was determined in t47d gh cancer cell. pao and ssat expressions were upregulated in t47d gh+ cells. concomitantly, putrescine levels were increased in t47d gh+ cancer cell compared to wt cells and curcumin depleted spermidine and spermine levels in wt and gh + t47d cells. curcumin induced-apoptotic cell death via ros generation and co-treatment of n-acetyl cysteine (nac) overcame curcumin effect. conclusion, forced gh expression triggers cell proliferation and growth via acting on polyamine metabolism and curcumin-triggered ros generation was prevented by nac treatment in t47d wt and gh+ breast cancer cells. acknowledgment: this study was supported by tubitak 1001 research project (project no: 113z791). the radio-protective effects of propolis and nigella sativa oil on oxidative/nitrosative stress in liver tissue of rats exposed to total head irradiation s. taysi our purpose is to investigate propolis and nigella sativa oil (nso) for their antioxidant effects on the liver tissue of rats exposed to ionizing radiation. a total of 32 sprague-dawley rats were divided into five groups to test the radioprotective effectiveness of of propolis and nso administered by orogastric tube. appropriate control group was also studied. biochemical parameters in liver tissue of rats were determined by spectrophotometer. xanthine oxidase (xo), nitric oxide synthase (nos), superoxide dismutase (sod) activities, nitric oxide (no•) and malondialdehyde (mda) levels were higher in ir group while glutathione-s-transferase (gst), glutathione peroxidase (gsh-px) level in the ir group were lower in this group when compared to the other groups. gst activity in ir plus propolis group was statistically higher than in all other groups. propolis and nso clearly protect liver tissue from radiationinduced oxidative stress. the effects of royal jelly on the antioxidant parameters in the breast tissues of the rats with breast carcinoma treated with paclitaxel or not effects of royal jelly on the breast tissue antioxidant parameters in rats with breast carcinoma treated with paclitaxel or not. 8-12 weeks old female sprague-dawley rats (n = 37) included in current study were divided into 5 groups: control group (n = 8) with healthy rats; breast cancer group (n = 8); breast cancer group (n = 7) treated with 15 mg/kg paclitaxel injection (once a week for 3 weeks); breast cancer group (n = 7) treated with 100 mg/kg royal jelly (by oral gavage for 30 days); and finally breast cancer group (n = 7) treated 100 mg/kg royal jelly in addition to 15 mg/kg paclitaxel injection. rats with breast carcinoma was obtained at 150th days after a single dose injection of 50 mg/kg n-methyl-n-nitrosourea (mnu). all cancer groups were followed by 30 days with treatment of paclitaxel and/or royal jelly. the antioxidant parameters in rat breast tissues, superoxide dismutase (sod) and catalase (cat) activities were determined by spectrophotometric colorimetric methods and glutathione (gsh) by high performance liquid chromatography (hplc). all the antioxidant parameters decreased in breast cancer group without any treatment (p < 0.05). but, statistically non significant increases were observed by paclitaxel and royal jelly treatment (p > 0.05). this study indicated that royal jelly supplementation can not be sufficient to increase the antioxidant parameters in breast cancer. we are going to continue to identify the effects of royal jelly on breast cancer detail. the effects of n-acetylcysteine on microsomal and serum paraoxonase 1 activities at high fat diet induced obese rats obesity is a chronic disease that develops from the interaction between genotype and environmental factors and increase in the accumulation of body fat. it is related with glucose and lipid metabolism disorders, cardiovascular diseases and increased oxidative stress. paraoxanase 1 (pon1) is an enzyme which plays a protective role in oxidative stress, inflammation and liver diseases. it has been suggested that pon1 has protective effects against high fat diet induced obesity and obesity related disorders. n-asetylcysteine (nac) is a potent antioxidant due to its ability to stimulate glutathione synthesis. the aim of this study was to evaluate the microsomal and serum pon1 enzyme activities (paraoxonase, arylesterase and lactonase) at high fat diet induced obese rats in the presence of nac. this study consisted of control, obese and nac-supplemented obese (2 g/l nac) groups. eighteen sprague-dawley rats were randomized into three groups (n = 6). control rats were fed by standart food and obese and nac groups were fed by high fat diet. the microsomal and serum paraoxonase, arylesterase and lactonase activities were determined by colorimetric methods. serum paraoxonase, arylesterase and lactonase activities decreased in obese and nac groups when compared to control groups. on the other hand microsomal paraoxonase, arylesterase and lactonase activities increased in nac group when compared to control and obese groups. however there was no statistically significant difference between the groups. it has been concluded that the microsomal and serum paraoxonase 1 enzyme activities did not change at high fat diet induced obese rats in the presence of n-asetylcysteine. reactive oxygen species, playing an active role in the early and late course of acute pancreatitis, lead to dysfunction of cell membrane and releasing of lysosomal enzymes, and thereby to the injury in pancreatic cells. gallic acid, found in vegetables such as green tea, is an active component which has antioxidant, antiinflammatory, antiviral, anticancer activities. the aim of this study was to investigate the effects of gallic acid in experimental acute necrotizing pancreatitis (anp) model in rats. eighteen male sprague-dawley rats were divided into three groups (6 rats in each group). group 1: sham + saline; group 2: anp induced by intraductal glycodeoxycholic acid and intravenous cerulein; and group 3: anp + gallic acid (100 mg/kg/day, intraperitoneal). at the end of 18th hours, pancreas histopathology was examined. the levels of serum amylase as a diagnostic marker of pancreatitis, interleukin-6 (il-6), total antioxidant status (tas), nitrite + nitrate, total thiols as antioxidant marker and thiobarbituric acid reactive substances (tbars) to measure malondialdehyde (lipid peroxidation product) were determined by spectrophotometric methods. serum amylase, il-6, plasma tbars levels were significantly higher but total thiols levels were lower than sham group in anp group without treatment (p < 0.05). however; tas and nitrite + nitrate levels did not show any significant difference (p > 0.05). on the other hand, while serum amylase, il-6 and tbars levels were lower, total thiols levels higher in gallic acid treatment group than in the untreated anp group, but statistically insignificant (p > 0.05). in conclusion, gallic acid treatment is beneficial but not sufficient to treat the acute necrotizing pancreatitis in rats. p-09.04.4-043 evaluation of oxidant/antioxidant system parameters, il-6 and il-10 levels in amniotic fluid of pregnancies with down sydrome b. _ imge erg€ uder 1 , s. bahsi 1 , t. bahsi 2 , v. topc ßu 2 , a. bakir 2 1 ankara universty faculty of medicinel, ankara, 2 zekai tahir burak research and training, hospital genetic center, ankara, turkey introduction and aim: down sydrome (ds) can be diagnosed at high-risk of down syndrome pregnancies by invasive prenatal testing. in this study we aimed to demonstrate antioxidant/oxidant system markers, il6 and il10 levels in amniotic fluid samples of pregnancies affected by ds. materials and methods: for this purpose we collected amniotic fluid samples from 18 pregnancies affected by down sydrome and 36 normal healthy pregnancies who applied to zekai tahir burak research and training hospital genetic center and were proceeded with amniocenthesis. in the amniotic fluid samples; malondialdehyde (mda), superoxide dismutase (sod), glutathion peroksidase (gsh-px) xhantine oksidase (xo), catalase (cat), adenozine deaminase (ada), nitric oxide (no), nitric oxide senthase (nos) enzymatic activities were evaluated by spectrophotometric methods, il6 and il10 levels are evaluated by elisa. for statistical analysis student's t-test and spearman corralation analusis are used. results: it was found that sod levels are significantly elevated in study group compared to control group (p < 0.05). besides this, in study group, cat and il-6 levels are found singnificantly lower than control group (p < 0.05). we couldn't find any significant difference between two groups in terms of mda, gsh-px, xo, no, nos, ada ve il-10 levels (p > 0.05). discussion and conclusion: there is an important decrease in inflammation compared to normal pregnancie in the amniotic fluid of pregnancies having ds. based on these results, sod enzyme may be used as a marker for prenatal diagnosis of ds. for this purpose these experiments should be tried on larger sample groups. the aim of our work was to compare prooxidant and antioxidant properties of linalool, which is the oxygenated monoterpene compound reported to be a major volatile component of the essential oil of several aromatic species, in hep g2 cells. cytotoxicity of linalool was assayed by celltiter-blue ò cell viability assay. malondialdehyde levels result in membrane damage in hep g2 cells were assayed by using fluorometric method. hep g2 cells were incubated with linalool at 24, 48 and 72 hours. the viability of hep g2 cells decreased in a manner dependent upon concentration and incubation time. the ic 50 values were calculated 81.5 lg/ml (24 hours), 72.7 lg/ml (48 hours) and 64.7 lg/ml (72 hours). but, cell viability of hep g2 cells increased when the cells preincubated with linalool at lower concentrations (˂ic 50 ) against h 2 o 2 cytotoxicity. membrane-damaging effects of linalool were increased with accelerating concentrations. on the other hand, membrane damaging effect of h 2 o 2 was decreased when the cells preincubated with linalool before h 2 o 2 incubation. oxygenated monoterpene linalool had both prooxidant and antioxidant properties showing membrane damaging and protective effects on hep g2 cells depend on concentration. postprandial lipemia is primarily characterized by increasing triglyceride levels after the lipid rich meal. postprandial lipemia may cause oxidative stress by increasing free radical production and increasing oxidative stress could be responsible for the development of many diseases. plasma oxidant-antioxidant status was evaluated in healthy individuals with postprandial hypertriglyceridemia generated by performing oral triglyceride tolerence test (ottt). the study group included 86 subjects (38 female and 48 male). ferric reducing ability of plasma (frap), total thiol and thiobarbituric acid reactive substances (tbars) levels were determined by colorimetric methods at fasting and 2nd, 4th and 6th hours following ottt. the levels of frap and thiol were significantly higher in males than females (p = 0.0001 and 0.042, respectively). thiol levels decreased significantly in both gender at postprandial 2nd, 4th and 6th hours as compared with fasting condition (p = 0.0001). while tbars levels increased at postprandial 2nd hour, that was only significant for male individuals (p = 0.0001). it has been concluded that postprandial lipemia may change oxidant-antioxidant balance in favor of oxidants and gender is an important criteria while assessing the oxidant-antioxidant status in postprandial lipemia p-09.04.4-046 ischemia modified albumin and c-reactive protein levels in prediabetes prediabetes is a state of abnormal glucose homeostasis characterized by the presence of impaired fasting glucose, impaired glucose tolerance, or both. the aim of this study was assess serum ischemia modified albumin (ima) in prediabetes and determine its correlation with other risk factors for chronic complications such as inflammation and hyperglycemia. glucose, insulin, total cholesterol, hdl cholesterol, triglycerides, albumin, c-reactive protein (crp) and ima were measured in 30 patients with prediabetes and 30 controls. prediabetes patients had higher levels of ima and crp in comparison with control subjects but there was no significant difference between groups for ima. significant positive correlation was observed between crp and fasting glucose, insulin. there was no significant correlation between ima levels and the parameters tested. we have shown higher level of crp in prediabetes. these results support the hypothesis that chronic inflammation may be involved in development of hyperglycaemia. p-09.04.4-048 the effects of s _ io 2 nanoparticles of rat uterine smooth muscle specially used in textile field sio 2 nanoparticles on uterus smooth muscle was aimed to be researched. in this study 64 wistar albino female rats were used. rats were separated in 4 groups as control, dose 1 (250 lg/ml), dose 2 (500 lg/ml) and dose 3 (1000 lg/ml). nanoparticle's size was chosen as 20 nm. preparations of four groups were evaluated for biochemical and histological examinations. all isolated uterine smooth muscle stripts except the controls were treated with sio 2 for two hours. in biochemical examinations in order to evaluate oxidative stress level of malondialdehit (mda), activity of superoxide dysmutase (sod) and glutathione peroxidase (gsh-px) were measured. in histological examinations via electron microscope ultrastructure of uterus was examined as well as apoptotic cells detected with immunofluorescent labeling method. intergroups differences were defined by statistical analysis. while mda level increased depending on the dosage, sod level was decreased depending on the dosage. gsh-px rate was decreased for each dosage with respect to control. however, no significant difference is detected between groups. in electron microscopic examination no changes were observed in uterus ultrastructure with compare to control. however, in immunofluorescent labeling it was detected that apoptosis increased in dosage groups with compare to control group. as a result, it was thought that application of sio 2 nanoparticles, in 20 nm size and in 250, 500 and 1000 lg/ml dosages caused of oxidative stress and apoptosis. this results suggested that sio 2 has toxic effects on uterine smooth muscle. uterine myomas are the most common benign pelvic tumors arising from myometrium. they are rarely associated with mortality but responsible for significant morbidity and have adverse effects on quality of life especially in reproductive age women. reactive oxygen species and superoxide dismutases, as well as sex steroids play important roles in the reproductive physiology processes. in addition, oxidative stress and impaired antioxidant defense system have been linked to pathophysiology of various diseases including malignant gynecological disorders. clinical investigations indicate that women with myoma may have increased risk of developing malignant tumors particularly sarcomas. the present study aimed to investigate the possible role of oxidant and antioxidant status in myomas. blood and urine samples of 21 myoma patients were collected. activities of erythrocyte antioxidant enzymes [copper-zinc superoxide dismutase (cuzn-sod), catalase (cat), glutathione peroxidase (gpx1)] and levels of lipid peroxidation biomarkers [plasma malondialdehyde (mda) and urine 8-epi-prostaglandin f 2a (8-epi-pgf2a)] were determined. the results were compared with those of 21 controls. the groups were matched in terms of age, body mass index, smoking habit, coexisting chronic diseases, menopausal status and sex steroid hormone levels. all antioxidant enzyme activities were higher (37% for cuzn-sod, p = 0.003; 52% for cat, p0.05) and the levels of lipid peroxidation biomarkers were lower (%59 for mda, p = 0.011 and 43% for 8-epi-pgf2a, p > 0.05) in myoma patients compared to controls. correlation analyses showed a significant negative correlation between erythrocyte cuznsod activity and plasma mda levels (r = -0.431, p = 0.005). the decreased lipid peroxidation may be the consequence of elevated antioxidant enzyme activities and the data suggests a protective role of activated antioxidants especially cuznsod and cat in patients with myoma. p-09.04.4-050 investigation of ischemia-modified albumin levels in patient with acute limb ischemia introduction: acute limb ischemia commonly occurs as a result of embolus caused by cardiac origin and which may end up with limb loss or even death if left untreated. thrombosis are usually seen where the arteries give branches and tendency to atherosclerosis is more serious at these sites. involvement of several arteries in either embolus or in situ thrombosis limits the adequacy of collateral circulation. restriction of blood flow due to arterial stenosis or occlusion often leads patients to complain of muscle pain on walking. any further reduction in blood flow causes ischemic pain at rest, which affects the foot. early recognition may prevent limb loss or death. ischemia can alter the capacity of the amino terminus of the albumin to bind free metal atoms such as cobalt, copper and nickel. this new, chemically changed albumin is called ischemia modified albumin (ima). ima is a sensitive marker of myocardial ischemia, skeletal muscle ischemia, pulmonary embolism, mesenteric ischemia and stroke. therefore, in this study it was aimed to investigate the ima level in acute limb ischemia. materials and methods: in this study, 24 patients with acute limb ischemia (li group; mean age 62 years) and 34 healthy individuals (control group; mean age 42 years) were included. ima levels were detected in control and li group by elisa (organo teknika, avusturya) using ima el _ isa kit. results: ima values were compared with nonparametric methods mann whitney u test, and significantly decreased ima level was statistically significantly different between li group and control group (p < 0.001). conclusion: there is a significant increase in serum ima in limb ischemia, so that alterations also might be clinically useful in the diagnosis of limb ischemia, but should be supported with further studies. object: polycystic ovary syndrome (pcos) is a multifaceted disorder with a pathogenetic pathway that is not fully understood yet. apart from hormonal derangements, insulin signaling defects and adipose tissue dysfunction, oxidative stress, defined as an imbalance derived from excessive formation of oxidants in the presence of limited antioxidants defenses, has been actively implicated in the etiology of the syndrome. the aim of this study was to determine of serum myeloperoxidase activity (mpo), paraoxonase 1 activity (pon1) and arylesterase activity (are) in patients with pcos. material and methods: the study was carried out on 150 women consisted of 103 patients with pcos and 47 healthy ones as control. serum pon1 activities were measured spectrophotometrically using diethyl-p-nitrophenylphosphate as substrate. phenylacetate was used as substrate for are measurement, and are activity was determined by measuring absorbance of the resulting phenol at 270 nm. molar absorptivity coefficients were used in the calculation of pon and are activities as 1 nmol phenol/ml serum/min. result: the mpo and are activities were significantly lower in the patient groups when compared with the control group (72.09 ae 60.66 -119.74 ae 90.81 u/ml p < 0.001, 47.43 ae 12.21 -83.93 ae 26.38 u/ml p < 0.001, recpectively). the pon1 activities are higher in the patient group (145.55 ae 98.23 u/ml) compared to the control group (153.33 ae 101.90 u/ml) are found, but are not statistically significant. conclusion: lower serum mpo and are activities might contribute to the increased susceptibility for the development of diseases risk in women with pcos. because free oxygen radicals are thought to contribute to the complication of many chronic diseases, the pcos may be related to oxidative stress. subclinical hypothyroidism, defined as an elevated serum thyroid stimulating hormone level associated with serum thyroid hormone concentrations within the reference range. free radicalmediated oxidative stress has been implicated in the pathogenesis of thyroid disorders. the ischemia-modified albumin (ima) has been proposed as a marker of protein oxidative damage, which has been found to reflect hypoxic stress. this study aimed to investigate the influence of subclinical hypothyroidism on serum ima levels. thirty-one subclinical hypothyroidism patients and 27 control subjects were enrolled in the study. albumin, ima were measured and ima/albumin ratio was calculated. to determine the ima levels the measurement method based on albumin cobalt binding assay was used. serum ima levels of patients with subclinical hypothyroidism were 0.58 ae 0.08 absu, ima levels of control subjects were 0.49 ae 0.08 absu. ima levels were significantly higher in patients with subclinical hypothyroidism patients than in control subjects (p < 0.0001). when ima values were normalized for albumin concentrations, the ima/albumin ratio was also significantly elevated in patient group compared to control group (p < 0.01). ima levels are increased in patients with thyroid dysfunction. elevated levels of ima can be a clinically useful marker of protein oxidative damage in subclinical hypothyroidism. p-09.04.4-054 the effects on endothelial dysfunction of quercetin in streptozocin-induced diabetic rats excessively produced in pathologic conditions. ultimately, imbalance between oxidants and antioxidants results with oxidative stress (os). in this study, we investigated some os parameters in standard (20% protein, 70% (7% sucrose) carbohydrate, 10% lipid) and sucrose (20% protein, 70% (35% sucrose) carbohydrate, 10% lipid) diet fed bdnf heterozygous mice liver tissues. the male c57bl6 strain wild type (+/+) and bdnf heterozygous (+/à) mice (5 weeks) were obtained. the animals were fed ad libitum by special standard and sucrose diets. twenty four mice were divided into four groups and each group consist six mice. all mice were fed for 16 weeks. first group involved in c57bl6 wild type mice and fed by standard diet. second group contained c57bl6 bdnf heterozygous mice and fed by standard diet. third group consisted c57bl6 wild type mice and fed by sucrose diet. fourth group involved in c57bl6 heterozygous mice and fed by sucrose diet. in first group, mda levels, sod and cat activities were higher than other groups. in second group, cat activities were lower than other groups. but, we could not find any statistically significant differences between all groups about mda, sod, cat levels in bdnf heterozygous mice liver tissues. in conclusion, standard and sucrose diet feeding may not affect mda, sod and cat levels in bdnf heterozygous mice liver tissues. brain-derived neurotrophic factor (bdnf) is member of neurotrophin family which plays critical roles in the development, differention, survival, maintenance of the central and peripheral nervous systems. bdnf also contributes to food intake and body weight control. bdnf heterozygous mice display increased body weight and mild hyperphagia. expression of bdnf is not limited to the brain, it also express some peripheral tissues like adipose tissue, liver, kidney, skeletal muscle, heart. even though roles of bdnf are well known relatively in central nervous systems, effects of this protein is not clear in peripheral tissues. as mentioned before, it is expressed in organs involved in energy, lipid and glucose homeostasis, including the liver, adipose and muscle tissues, but its role there remains unclear. in this study, we aimed to investigate role of bdnf on liver oxidative stress parameters in heterozygous mice model fed fat diet induced obese mice. in this study, we used c57bl/6 mice inbred strain wild type and bdnf heterozygous (+/à) mice. animals were divided to two groups: wild type (n = 5) and bdnf heterozygote mice (n = 5). the animals were fed ad libitum by high-fat diet during 4 month. weight gain was recorded every 15th days. in liver tissues were measured malondialdehyde (mda), superoxide dismutase (sod) and catalase (cat) by spectrophotometric methods. liver mda levels decreased in obese bdnf heterozygous mice compared to obese wild type group and statistically significant difference between groups. bdnf heterozygous mice cat activities were higher than the other group and this difference was statistically significant. there was no statistically significant difference between the groups in terms of sod activities. it has been concluded that the mda levels and sod enzymes activities changed at high-fat diet induced obese bdnf heterozygous mice compared to wild type mice liver tissues. p-09.04.4-060 disturbances of microelements profile in serum of overweight/obese adult females with acute and persistent pro-inflammatory chlamydia pneumoniae infection p-09.04.4-061 determination of reactive oxygen species induced dna damage using modified cupric reducing antioxidant capacity (cuprac) colorimetric method s. uzunboy, s. demirci c ß ekic ß, r. apak department of chemistry, istanbul university, faculty of engineering, istanbul, turkey reactive oxygen species (ros) term is a common name of a group of species. hydroxyl radical and singlet oxygen can be taken into account as ros samples. ros may be formed as a result of endogenous or exogenous reasons. although ros have some beneficial functions, they should be balanced by antioxidants (aox). excessive amounts of ros can attack biological macromolecules including dna. dna damage is usually related with mutagenic and carcinogenic changes. that's why determination of dna damage is so important and there are a great many studies in literature comprising different techniques. one of the most common of them is the 'comet assay'. but application of the method and interpretation of the results is not easy. investigation of certain dna damage products is also very common. these methods usually need expensive instrumentation such as using tandem mass spectrometry. on the other hand, depicting total dna damage on a certain product may cause misinterpretations. in the presented study, dna was decomposed by hydroxyl radicals produced by fenton method. in the study while dna is not cuprac reactive the oxidation products can react with the cuprac reagent. the effect of aox was also investigated. for this purpose, selected aox compounds were added to the reaction medium. because of their radical scavenging effect, the cuprac absorbance decreased in the presence of aox. in the presence and absence of aox, absorbance differences were calculated. the calibration graphs between final concentration and absorbance differences were drawn for each aox. gallic acid was determined as the most effective one among the tested aox samples. for statistical comparison with the presented study, tbars was used as reference method. direct use of dna as a probe material to determine oxidative damage may be an advantage to understand dna hazard in biological systems. the proposed method can be applied in all laboratories having a spectrometer as a cost-effective and simple procedure. p-09.04.4-062 effects of alpha-1 antagonists on oxidative system of rat heart tissue benign prostate hyperplasia is a progressive process occurring in the stromal and epithelial components of the prostate. alpha-1 receptor blocking agents are used for relaxation of the smooth muscles in the prostatic stroma. our aim was to investigate the effects of alpha-1 antagonists on oxidative system of rat heart tissue. 33 male wistar albino rats were divided into 5 groups randomly. 1) tamsulosin (1 mg/kd/day), 2) terazosin (5 mg/kg/day), 3) doksazosin (25 mg/kg/day), 4) alfuzosin (10 mg/kg/day), 5) control. all drugs were administered every other day single doze via oral. rats were sacrificed after 30 days. heart tissue was taken for biochemical analysis. malondialdehyde (mda), nitric oxide (no), protein carbonyl (pc) levels and superoxide dismutase (sod), glutathione peroxidase (gsh-px) enzyme activities were determined in supernatant samples. there was not an significant difference between terazosin, doxazosin, alfuzosin, tamsulosin groups in means of sod, mda and gsh-px levels. no levels were significantly different between tamsulosin group and the control group (p = 0.004). in addition, tamsulosin group and terazosin group were also significantly different (p = 0.032). according to these results we can say that tamsulosin group had higher no levels than control and terazosin group. tamsulosin's enhancer effect on no levels leads to relaxation of the heart muscle and vascular relaxation, and so fewer side effects than other alpha antagonists. the effect of rat liver tissue radical metabolism and the protective role of hippophae rhamnoides l. on cold and immobilization stress model cold and immobilization stress is a widely used model for study the changes that occur on oxidant-antioxidant balance. hippophae rhamnoides l. (seabuckthorn; sbt) a unique and valuable plant has recently gained worldwide attention, mainly for its medicinal and nutritional potential. this study was aimed to investigate the protective role of sbt which is a natural herbal product with high antioxidant content on oxidative and nitrosative stress induced by cold and immobilization stress in rats. 32 wistar albino rats were divided into 4 groups randomly. control (i.p. physiological saline), sbt (i.p. 200 mg/kg/48 hours sbt), stress (i.p. physiological saline; 6 hours cold + immobilization stress) and sbt + stress (i.p. 200 mg/kg/48 hours sbt; 6 hours cold + immobilization stress) groups were formed. 3nitrotyrosine levels were determined by elisa whereas total antioxidant capacity, total thiol, total glutathione, nitrite-nitrate levels and superoxide dismutase and glutathione peroxidase activities were measured by colorimetric methods. sbt + stress group nitrite-nitrat (p = 0.0001), total glutathione (p = 0.0001) levels and glutathione peroxidase activities (p = 0.0001) were found to be significantly higher whereas superoxide dismutase activity was found to be lower (p = 0.007) when compared to stress group. there was no significant differences between stress group total thiol and total antioxidant capacity levels compared with stress + sbt group. stress + sbt group oxidative and nitrosative stress marker 3-nitrotyrosine level was found to be significantly higher when compared with control group (p = 0.002) whereas there was no significant differences between stress and stress + sbt groups. all this data show that sbt has antioxidant properties on cold and immobilization induced oxidative and nitrosative stress in rat liver tissue. obesity is a major health problem with growing incidence and accompanying complications. its relation with diminished cognitive functions was reported. this study aims to evaluate the effects of obesity induced oxidative stress and metabolic alterations on the cognitive functions of children and adults. 33 children and adolescents with obesity (age: 8-18); and 33 age and gender matched healthy subjects were enrolled. all subjects completed the battery tests of cnsvs via computer. the scores were compared by using commercial software (ibm spss statistics 19). biochemical parameters, malondialdehyde (mda) and protein carbonyl (pc) levels were estimated. mda and pc levels were significantly higher in subjects with obesity (0.78 ae 0.16 lmol/l;198.30 ae 84.45 nmol/ml) than the controls (0.5 ae 0.10 lmol/l; 125.35 ae 43.52 nmol/ml) (<0.001). there was statistically significant difference between study and control groups on all cognitive performance domains. significant correlation was detected between mda, pc levels and the cognition indexes. children with obesity should be evaluated for the cognitive functions, together with the metabolic follow-up. obesity induced oxidative stress may be the reason of the diminished cognition in children as well as the changes in the lipid profile and inflammation, but we need larger study groups to lighten these complex process. p-09.04.4-067 relative contribution of nitric oxide synthase (nos) isoforms to oxidative/nitrosative stress in the cerebral cortex of rat with acute liver failure (alf) acute liver failure (alf) is associated with deregulation of nmda/cgmp/no signaling and oxidative/nitrosative stress in the brain. however, the relative roles of the different nos isoforms and the mechanisms underlying alterations in their activities during alf are not fully clear. here we investigated gene and protein expression of nos isoforms, nos activity, enos uncoupling and total no production in cerebral cortex of rats with thioacetamide (taa)-induced alf. sprague dawley rats (250-280 g) received three i.p. injections of taa (300 mg/kg) at 24 hours intervals. the brain cortex expression nos isoforms (enos/inos/nnos) was measured by real-time pcr and western blot, nos activity was tested by monitoring the conversion of radiolabeled arginine to citrulline. reactive oxygen species (ros) were quantified in the presence of nos substrate l-arginine, using the carboxy-h 2 dcfda probe. no was measured with the griess procedure. the enos expression was decreased, whereas the enos dimmer/monomer ratio and nnos/inos expression were elevated in taa treated rats. while the total nos activity was decreased, the inos activity was elevated and no concentration tended to increase. ros production was elevated by taa. unspecific nos inhibitors l-name and l-nna attenuated ros production in both control and taa rats, but with higher efficiency in the latter case. ca 2+ chelation had almost the same effect as pharmacological nos inhibition suggesting that ca 2+ -independent inos activity is not the main source of ros. incubation with high dose of tetrahydrobiopterin (bh4) with which is critical for enos dimerization and subsequent no production also reduced ros production indicating the enos uncoupling phenomenon in taa cortex. the study points to enos downregulation due to lowered protein expression and uncoupling as a novel mechanism contributing to enhanced superoxide o 2 anion formation, and confirms the role of inos/nnos in enhancing no synthesis in alf-affected brain. introduction: diabetes mellitus (dm) is an endocrine disorder of world which is characterized by altered blood glucose levels and related complications including hepatic injury. myrtus communis l. (mc) is widely used by diabetic patients in the folk medicine of turkey as well as they are used worlwide. it is known that of leaves, oil and fruit of myrtus communis l. (mc) have therapeutic effects on diabetes mellitus (dm). this study was aimed to analyze the possible antidiabetic and hepatoprotective effects of mc berries in streptozotocin (stz) induced diabetic rats. materials and methods: a total of thirty rats composed of six groups as each included five rats were used. 40 mg/kg stz was injected once to animals to induce dm. after stz injection, rats were exposed to three different ethanol extracts of mc berries (250, 500 and 1000 mg/kg) by oral gavage during 14 days. alanine aminotransferase (alt) and aspartate aminotransferase (ast) levels were determined in serum and glutathione (gsh), malondialdehyde (mda) levels and superoxide dismutase (sod) activity were determined in liver tissue. results: mc administration provided significant reducement in the altered serum glucose, ast and alt levels in all diabetic groups. mc extract showed significant antioxidant activity by altering sod activity and gsh level and reducing mda levels in diabetic rats compared to controls (p < 0.05). serum ast and alt levels were reduced by mc administration in all diabetic groups. mc administration provided significance increment in sod activity and gsh level, and significant reduction in mda levels compared to controls (p < 0.05). the maximum hypoglycemic and antioxidant effects were observed at 1000 mg/kg dose of mc. background: human serum paraoxonase 1 (pon1) is a calcium dependent esterase that hydrolyzes organophosphates and also arylesters such as phenyl acetate. pon1 prevents ldl oxidation by hydrolyzing lipid peroxides. pon1 is inhibited by various chelating agents, heavy metal ions, and sulfhydryl reagents. in our study we investigated the effect of calcium on ldl oxidation of purified pon1 q192r isoenzymes. methods: pon1 q192r isoenzymes were partially purified from human serum. both allozymic forms were treated by preincubation with 1 mm edta for 15 minutes. ldl oxidation was induced by copper ions. formation of thiobarbituric acid-reacting substances (tbars) was used as a measure of lipid peroxidation. homocysteine thiolactonase (htlase activity) and arylesterase activities were measured spectrophotometrically by using homocysteine thiolactone and phenylacetate as the substrates. results: addition of 1 mm edta to partially purified hdl-pon1 q192r isoenzymes inhibited 100% of htlase and arylesterase activities. inactivation of pon1 for arylesterase/htlase activity by the addition of edta did not reduce the abilities of both allozymic forms in protecting ldl from oxidation. conclusion: ca +2 -dependent inhibition of pon1 q192r arylesterase/htlase by using the metal chelator edta, did not alter pon1's ability to inhibit ldl oxidation. pon1's ability to protect ldl from oxidation may not require calcium. p-09.04.4-070 evaluation of cholinesterase inhibitory effect, anti-radical and anti-lipid peroxidation activities of mentha pulegium i. hamad 1, 2 1 college of applied medical sciences, aljouf university, aljouf, saudi arabia, 2 faculty of medicine, bahri university, khartoum, sudan introduction: many studies indicated that intake of dietary and medicinal plants is effective in preventing or suppressing many diseases, therefore, there is a growing interest in plant'sbioactive compounds. mentha pulegium, is widely used in gulf countries in herbal teas or as additives in commercial spice mixtures for many foods to offer aroma and flavor. the aim of this study is to investigate the in vitro radical activity, the total phenol and flavonoid content, anti-lipid peroxidation and the cholinesterase inhibitory effects of mentha pulegium methanol extract. methods: the acetylcholinesterase and butyrylcholinesterase inhibitory potentials of extracts, were evaluated by colorimetric assay. the in vitro antioxidant activity was measured by dpph assay, the total phenols content was measured by folin-ciocalteau assay, the flavonoids content by the alcl 3 colorimetric method, and the protective effect of menthe mentha pulegium extracts against lipid peroxidation was evaluated using a liposome oxidation system. results: the methanol extract showed a scavenging activity nearly equivalent to vitamin c which is attributed to its high phenolic and flavonoid contents. the extract possessed protective effect against lipid peroxidation in a dose dependent manner. the methanol extract shows very little anticholinesterase activity as compared to the standard compound, physostigmine. conclusion: results presented here indicate that mentha pulegiumpossess strong antioxidant activity and protective effects and they can therefore be used as a natural additive in food, cosmetic and pharmaceutical industries. type 2 diabetes mellitus is a long term metabolic disorder that is characterized by hyperglycemia and insulin resistance. because of the hyperglycemia and free radicals, diabetes can cause cellular instability. micronuclei is a sensitive indicator of genetic damage and a marker of dna damage. micronuclei is also a morphological marker of chromosomal instability. in this study, we aimed to evaluate the frequencies of micronuclei in papanicolaou stained buccal cells of type 2 diabetic patients. a total of 30 type 2 diabetic patients and 30 healthy individuals were involved into our study. buccal smear samples that belong to these individuals were stained by using papanicolaou method for cytologic examination and the stained slides were evaluated by light microscopy (olympus bx-51). cells with micronuclei in each papanicolaou stained buccal smear sample were counted under light microscopy. the frequency of micronucleated epithelial cells were seen as significantly higher in type 2 diabetic patients than the control group (p < 0.05). one of the boron compounds is sodium perborate tetrahydrate (nabo 3 .4h 2 o), which the most widely used solid peroxygen compounds. it is used in safety bleach formulations, detergents and tooth powders. as known these products are commonly used in daily life. however, the actions on blood antioxidant defenses of sodium tetraborate against reactive oxygen species are not identified yet. it is reported that oxidative stress caused by ros damages. in this study, we searched enzyme activities of superoxide dismutase (sod), catalase (cat), glutathione-s-transferase (gst), glutathione reductase (gr), glutathione peroxidase (gsh-px) and glucose-6-phosphate dehydrogenase (g6pd) also the effect sodium perborate tetra hydrate on activities of these enzymes from human erythrocyte under in vitro conditions. according to our findings sodium perborate tetrahydrate caused significant (p < 0.05) increasing in the cat activity from red blood cell. the other antioxidant enzyme activities (sod, gst, gr, gsh-px and g6pd) did not show any changing by influence of sodium perborate tetrahydrate. metabolism of obese individuals could be exposed to risk of chronic low-grade pro-inflammatory effect and oxidative stress. some inflammatory and oxidative markers have been studied recently. plasma total antioxidant status (tas) and total oxidant status (tos) parameters can be non-invasive markers of diseases such as fatty liver disease, laparoscopic procedures (pneumoperitoneum), accompanying inflammatory condition like urinary tract infection, diabetic neuropathy, chronic hepatitis. the study groups have been comprised of two groups with normal to over-weight children. tas and tos levels were detected and the oxidative stress index (osi) was computed as a marker of the grade of oxidative stress. the over-weight group displayed higher levels of fasting glucose, insulin resistance, the body mass index. also, we know that insulin resistance leads to increased lipolysis and free fatty acid output. higher tos as well as crp is related to the group, also lower tas than other group is shown. crp levels in plasma were positively correlated with insulin and glucose levels. in addition, there was a significant relationship between osi and insulin resistance in the over-weight group. tas and tos are together more accurate sings of oxidative and antioxidative status of people. as well as a raise over weight-related subclinical inflammation and a fall antioxidant capacity is significant even in children. this condition may eventually develop the risk of long-term vascular damage. the effects of hydrogen peroxide pretreatment on antioxidant enzyme activities in calli tissues of two eggplant genotypes under salinity o. yasarkan 1 , e. aky€ uz 1 , g. baysal furtana 2 , s. s. ellialtioglu 3 , r. tipirdamaz 4 1 nezahat g€ okyigit botanic garden, istanbul, 2 department of biology, faculty of sciences, gazi university, ankara, 3 department of horticulture, faculty of agriculture, ankara university, ankara, 4 department of biology, faculty of sciences, hacettepe university, ankara, turkey the effects of hydrogen peroxide (h 2 o 2 ) pre-treatment on catalase (cat) and superoxide dismutase (sod) were investigated and lipid peroxidation measured as malondialdehyde (mda) content of the calli from salt-sensitive (artvin) and salt-tolerant (mardin) eggplant genotypes under salinity stress. the seeds from each genotypes were germinated on ms medium for 4 weeks and hypocotyl tissues from these plantlets were used as explants for calli induction on ms medium including 1 mg/l 2,4-d and 0.1 mg/l kinetin. as for the pre-treatment, the subcultured calli tissues were transplanted on the mediums containing 50 and 100 lm h 2 o 2 for 48 hours and then transplanted on the mediums including 150 mm nacl for 24 hours. antioxidant enzyme analysis and mda measurement was carried out for the control, nacl-only, h 2 o 2 -only and h 2 o 2 pre-treated tissues. pre-treatment with h 2 o 2 decreased the deleterious effects of salt stress on mda contents. in comparison with salt stressed groups, h 2 o 2 pre-treatment with or without nacl reduced mda content especially in artvin. comparing two genotypes, a decrease was observed on sod activity in artvin genotype and an increase in mardin genotype by comparison of salt stressed groups. higher increase on sod activity was observed in 100 lm h 2 o 2 + nacl groups on each genotypes. comparing two genotypes, a decrease was observed on sod activity in artvin genotype and an increase in mardin genotype by comparison of salt stressed groups. higher increase on sod activity was observed in 100 lm h 2 o 2 + nacl groups on each genotypes. the result showed pre-treatment of 100 lm h 2 o 2 induced acclimation of the plants to salinity. in addition, 100 lm h 2 o 2 pre-treatment, as a stress signal, could trigger the activation of antioxidant enzymes in calli and in this way alleviated the oxidative damage in calli growth under salinity. the investigation of effects of ghrelin and cannabinoid cb1 receptor agonist and antagonist on oxidant and antioxidant mechanisms on brain tissues of penicillininduced epileptic rats the aim of this study is to investigate the individual effects of ghrelin and cannabinoid type1 (cb1) receptor agonist acea, the antagonist am-251 and the interaction of these two different systems on oxidant and antioxidant systems in the brain, cerebellum and brain stem tissues of penicillin-induced epileptic rats. in this study 56 male wistar albino rats were used weighing 20-250 g. each group was consisted of 8 rats. study groups: 1: control, 2:penicilin(500 iu), 3:penicillin(500 iu) + ghrelin(1 lg), 4:penicillin(500 iu) + am-251(0.25 lg), 5:penicilin (500 iu) + acea(7.5 lg), 6:penicillin(500 iu) + am-251(0.25 lg) + ghrelin (1 lg), 7:penicillin(500 iu) + acea(7.5 lg) + ghrelin(1 lg). than the levels of mda, gpx and sod are measured in plasma and tissue samples of these rats. penicillin was found to be induced lipid peroxidation in the brain, cerebellum and brain stem tissues in our study. ghrelin and acea, which both have anticonvulsant effects, were shown to be effective in reversing the oxidative damage caused by penicillin and proconvulsant am251 was found to further increase the oxidative stress caused by penicillin in these tissues. ghrelin also was found to suppress the oxidative stress caused by am251 in the cerebellum tissue but it did not contribute to antioxidant effects produced by acea. since the role of oxidative stress in epilepsy has been established, it may be suggested that ghrelin and acea may have anticonvulsant effects via their antioxidant features. the discovery of inhibitors for enzymes that metabolize endogenous ghrelin and cannabinoids through new studies may contribute to the improvement of seizure resistance in epilepsy. accelerated atherosclerosis in patients with ankylosing spondylitis (as) give rise to increased cardiovascular morbidity and mortality. endothelial dysfunction could be the initial process in the development of atherosclerosis. human endothelial cell-specific molecule-1 (endocan) is a novel human endothelial cell-specific molecule. therefore, we assessed serum endocan levels and carotid intimamedia thickness (cimt) as a surrogate marker of atherosclerosis in patients with as. a total of 57 patients with a diagnosis of as according to newyork ctriteria and 50 control subjects were included in our study. serum endocan, interleukin-6 (il-6), tumor necrozis factor-a (tnf-a), c reactive protein (crp) and cimt were measured in all participants. serum endocan, il-6, tnf-a levels were measured with elisa. the other parameters were done by routine biochemical methods. as patients exhibited increased serum endocan levels and cimt compared to matched controls (p < 0.05). whereas, serum il-6, tnf-a were similar between grous. in patient with as, there were no significant differences between active and inactive patients by means of il-6, tnf-a, endocan and cimt. in as group, cimt correlated with disease duration and age (r = 0.597, p = 0.000; r = 0.721, p = 0.000). we could not find any significant correlation between serum endocan levels and parameters studied. our study shows increased cimt in as patients without traditional risk factors such as increased bmi, lipid profile compared to controls. although we found increased circulating endocan levels in patients with as, the other factors could affect increased atherosclerosis in this population because of lack of correlation between endocan and cimt. probable biomarkers could be related to increased cimt in patients with as should be investigated in larger study groups. keywords: ankylosing spondylitis, atherosclerosis, carotid intima media thickness, endocan p-09.04.4-079 investigation of pentose phosphate pathway and oxidative stress in erthrocyte infected babesia ovis a. bildik, t. karagenc ß, p. a. ulutas, n. aysul, h. aksit adnan menderes university, aydin, turkey introduction: babesia infections occur in cattle, sheep, goat, horse, dog, cat pig and rodents. in this study, the effects of babesia ovis living and present in the erythrocytes to glucose metabolism was researched. at the same time, biochemical parameters were also associated with parasitemia. materials and methods: babesia ovis (israel) cell culture was provided from dr. abel martin gonz alez oliva (portugal). culture passaged 48 or 72 hours according to parasitemia state (8-10%). biochemical analyses were performed in erythrocyte culture in which parasitemia between 1% and 16%. cell counts and hemoglobin concentration of erythrocytes culture suspension were measured at cell counter instrument and than it was washed 3 times with physiological saline, erythrocyte suspensions were stored at-80oc analysis. gssg (oxidize glutathione), gsh (reducte glutathione), nadph, glukoz 6 p dehydrogenaz, gshpx (glutathione peroxidase), gshrx (glutathione reductase) were determined by commercial kits. all experiments were done in duplicate, the results were calculated by the number of erythrocytes. results: parasitemia was positively correlated with gsh, nadph and gshrx (p < 0.05). a correlation between other biochemical parameters was not observed. discussion: the pentose phosphate pathway in erythrocytes has an important role such as to provide pentose sugar required for the synthesis of nucleic acid, to reduce glutathione, to produce nadph and to protect from methemoglobin accumulation. in studie sthat naturally infected erythrocytes with babesia parasites, it was seen to be caused to oxidative stress, however gsh results in these investigation were obtained differently . conclusion: according to the results of this study that performed in vitro, it can be suggest that their glutathione metabolism and pentose phosphate pathway of parasites may active.key words: babesiosis, gsh, gssg, nadph, g6pdh, gshpx, gshrx p-09.04.4-080 in vitro protective effect of betaine on peroxidative injury caused by ethanol and aspirin exposure on rat brain synaptosomes i. sogut 1 , g. kanbak 2 1 istanbul bilim university vocational school of health services, istanbul, 2 eskisehir osmangazi university medical school department of biochemistry, eskisehir, turkey aspirin intake of specific daily doses are advised by doctors to postmenapausal women and men above 40 years of age to prevent heart attack and even cancer in recent times. in this study, the aim is to investigate the in vitro cytototoxic effects of different doses of ethanol (50 mm, 100 mm ve 200 mm) alone and together with 100 lg/ml aspirin, and possible protective role of 1 mm betaine on rat brain synaptosomes. 15 male sprague dawley rat forebrains were divided into equal pieces and pooled to form 10 study groups. synaptosomal fractions extracted from pooled rat brains were incubated with different doses of ethanol, aspirin and betaine, and malondialdehyde (mda) levels, an important indicator of cellular damage, were measured. a significant increase (p < 0.05) was observed in mda level of 200 mm ethanol group compared to control group. different doses of ethanol (50 mm, 100 mm ve 200 mm) + aspirin exposure significantly increased (p < 0.001) mda levels compared to controls, whereas betaine administration significantly decreased (p < 0.001) lipid peroxidation caused by ethanol + aspirin treatment. we conclude that ethanol and ethanol + aspirin administration increases lipid peroxidation in rat brain synaptosomes while betaine helps prevent this peroxidative membrane injury.keywords: aspirin, betaine, ethanol, malondialdehyde (mda) p-09.04.4-081 analyses of mitochondrial biogenesis in hepatocellular carcinoma treated with berberine f. aygenli, h. c ß imen yeditepe proteomics and mass spectrometry group (yediprot), genetics and bioengineering, yeditepe university, istanbul, turkey objective: berberine (bbr) has been demonstrated to have anticancer activities against various cancer types, particularly hepatoma. in this project, we aimed to reveal the effect of bbr treatment on mitochondrial biogenesis through sirtuins and hif-1a in hepatocellular carcinoma cell line, hep3b under hypoxia. method: hep3b cells were subjected to normoxia (21% o 2 ) and hypoxia (1% o 2 ) in the presence or absence of bbr treatment. the amount of bbr was optimized via cell viability (mts) assay under normoxia. then, immunoblotting experiments were performed to identify the effect of bbr on hif-1a, pgc-1a, and sirtuins involved in mitochondrial stress. the variation in the oxphos complexes and the level of reactive oxygen species (ros) were also measured to investigate the effect of bbr on mitochondrial energy stress state. results: here, we present that cell viability was significantly decreased at 25 lm. bbr treatment has shown significant reduction in hif-1a and sirt6 which responsible for up-regulation of glycolysis. also, succinate dehydrogenase (cii) and cytochrome c oxidoreductase (ciii) of the oxphos complexes were downregulated without any change in nadh dehydrogenase (ci) or atp synthase (cv). bbr significantly abolished to oxidative stress under hypoxia, which was demonstrated as a reduction in the level of reactive oxygen species by decreasing on sirt3 expression. bbr induces the overexpression of sirt1 and its deacetylated-pgc-1a, which might be an indicator of being a potent protective agent against hypoxia by normalizing mitochondrial function and inducing mitophagy in impaired mitochondria caused by deficiency of glycolysis and oxphos. conclusion: detailed information about the communication between hif-1a and sirtuins and their relation to mitochondrial energy production was provided with the alteration of their activity by bbr treatment. it is highly expected that bbr and its derivatives might become important during the development of supplemental therapies. introduction: reactive oxygen species are involved in a variety of biological phenomena, such as carcinogenesis, inflammation and aging. among the targets of ros, dna appears most important in tumor biology since it is firmly established that cancer is a genetic disease. ros induce several kinds of dna damage, including strand breakage and dna-protein cross-linkage. fruit of zizyphus jujuba, a traditional chinese herb widely consumed in asian countries, has been reported to possess several vital biological activities. this study intends to evaluate their antioxidant activity on glioblastoma cells. materials methods: cell survival was quantified by colorimetric mtt assay. human gliblastoma cells were pretreated with 100 lm h 2 o 2 after 30 minutes 100 lm ziziphus jujuba essential oil was added to the cells for three hours. then, the cell homogenates were taken and glutathione, total oxidant and total antioxidant capacity and nitric oxide levels were estimated using spectrophotometric methods. results: ziziphus jujuba treated cells could prevent intracellular glutathione from being depleted following an exposure of h 2 o 2 . also our data suggest that ziziphus jujuba is effective in preventing h 2 o 2 induced oxidative stress and nitric oxide levels. discussion: some research showed that h 2 o 2 was over produced in the pathological process of acute and chronic neuronal toxicity, the toxicity effect of b-amyloid on the cultured neuron and neuronal cell line was mediated by h 2 o 2 . the traditional medicine recommend several medicinal plants for providing relief from various inflammatory diseases. many research has been reported that the essential oil from seeds of helping to prevent the oxidative stress and neuronal diseases in brain. introduction: toxicity by oxygen radicals has been recommended as a major cause of cancer, heart disease, and aging. oxygen radicals and other oxidants appear to be toxic in large part because they start the chain reaction of lipid peroxidation. most of the analytical techniques for peroxide determination are generally time consuming and not very suitable for routine or on line analysis. we aimed to design a new biosensor for rapid determining of oxidant agent hydrogen peroxide. materials and methods: all reagents were of analytical grade unless stated otherwise, and were purchased from sigma aldrich. firstly, the 2-hidroxymetacrilate metacriloamidoscystein nanoploymers were immobilized by binding covalently with sulphur atoms on the gold electrod's surface. free nh 2 groups of catalase enzyme make schiff bases between nanopolymer's carbonyl groups, then immobilization was actualysed with cross linking reagent glutaraldehyde. we developed a biosensor system preparing ferrociyanide, selected as a mediator, in the buffer solution. results: polyhemamac nanopolymer and catalase complex were immobilized by glutaraldehite to construct a hydrogen peroxide biosensor. the responses of the biosensor are therefore proportional to the oxidation peaks of the complex at +0.019 v potential. the cyclic voltammograms obtained from the experiments showed that, pottasiumferrociyanide mediator complex positively affected the biosensor responses for hydrogen peroxide determination. discussion and conclusion: as a result of this study, the method developed by the catalase enzyme electrode was found to be more advantageous in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, economic, practical and less time-consuming. since biosensor technology provides economical, practical, specific and sensitive results for the determination of hydrogen peroxide, it was improved very efficiently. p-09.04.4-085 impact of amoxicillin, gentamicin and cefazolin sodium antibiotics on antioxidant gene expression and enzymatic activities in mouse liver p. g€ uller 1 , h. budak 2 , m. sisecioglu 2 , m. c ß iftci 3 1 department of chemistry, faculty of science, atat€ urk university, erzurum, 2 department of molecular biology and genetics, faculty of science, atat€ urk university, erzurum, 3 department of chemistry, faculty of arts and sciences, bing€ ol university, bing€ ol, turkey reactive oxygen species (ros) are highly reactive molecules, which are produced by living organisms as a natural byproduct of the normal metabolism and environmental factors. living organisms have the antioxidant defence systems to block harmful effects of ros. the imbalance between oxidants and antioxidants is termed oxidative stress. the antioxidant defence mechanisms are divided into two groups as enzymatic and nonenzymatic defences. enzymatic defence mechanisms consist of enzymes like superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glucose-6-phosphate dehydrogenase (g6pd) and glutathione s-transferase (gst). the present study was designed to determine the effects of gentamicin, amoxicillin and cefazolin sodium antibiotics on the hepatic antioxidant system and to determine any possible correlation between enzymatic and molecular levels. for this reason, effects of these antibiotics on the transcription of the antioxidant system has been investigated by real time pcr, and then the enzyme activity of these enzymes have been measured in whole liver homogenate obtained from control group and the drug administered groups mice. our results demonstrate that administering antibiotics led to crucial inhibition of all antioxidant enzyme activity. while significant transcriptional activation for sod and cat was seen in the gentamicin treated group, the transcription of gst and g6pd was decreased. however transcriptional activation was seen for sod and cat in amoxicillin administered group, the transcription of gst was decreased as compared with the control group. in the cefazolin sodium treated group, while cat and gst transcription were elevated significantly, the expression of sod and g6pd were decreased. in conclusion, gentamicin, amoxicillin and cefazolin sodium affect the hepatic antioxidant system at the molecular and protein level. this work was supported by scientific research project of ataturk university of turkey (grant no: 2013/296). p-09.04.4-086 protective effects of curcumin supplementation on oxidant/antioxidant system changes created by organic phosphorus pesticide poisoning organic phosphorus pesticides (opp), widely used in agriculture or as insecticides in home, cause adverse health effects. chlorpyrifos is one of the most commonly used opp. we aimed to investigate the possible protective effects of curcumin (cur) supplementation, the principal curcuminoid of turmeric, on poisoning symptoms and oxidant/antioxidant system changes caused by chlorpyrifos. adult sprague-dawley rats were used. cur (30, 100 and 300 mg/kg) were administered orally for 5 days. on the sixth day, chlorpyrifos (279 mg/kg, s.c.) was administered. twenty four hours after chlorpyrifos administration, body weights, locomotor activities and body temperatures of rats were measured. following the measurements, rats were decapitated and the blood, brain and liver tissue samples were taken and prepared for the biochemical and histopathological measurements. chlorpyrifos administration increased the malondialdehyde (mda) levels but decreased catalase (cat), superoxide dismutase (sod), glutathione reductase (grx) concentrations and reduced/oxidized glutathione (gsh/gssg) ratio in the blood samples, brain and liver tissues compared with the control group (p < 0.05-0.001). the concentration of advanced oxidation protein products (aopp) were increased only in the brain tissue after chlorpyrifos administration (p < 0.001). cur administration reduced all of these changes (p < 0.05-0.001). similarly, cur at the doses of 300 mg/kg reduced the decreases in body weight, body temperature and locomotor activity with chlorpyrifos (p < 0.001). additionally, the histopathological damage scores induced by chlorpyrifos (p < 0.05-0.01) were decreased by the administration of cur (p < 0.05-0.01). our findings suggest that cur supplementation can ameliorate the poisoning effects of chlorpyrifos via supporting the antioxidant mechanisms and cur could be used for protective purposes against oxidative stress and tissue damage caused by chlorpyrifos. the effect of ogtt applied for screening in pregnancy on adenosine deaminase and xanthine oxidase activity in normal and prediabetic pregnant women z. c. ozmen, k. deveci, i. benli department of biochemistry, gaziosmanpasa university medical faculty, tokat, turkey objective: it was reported that the activities of adenosine deaminase (ada) were different in normal pregnant women and pregnant women with gestational diabetes mellitus (gdm). it was also stated that the activity of xanthine oxidase (xo) was increased in pregnant women with gdm. the objective of this study was to evaluate if glucose have effects on oxidative stress in prediabetic women by affecting ada and ox after 50 g ogtt which was applied in pregnant women for screening. methods: the serum specimens of 39 pregnant women who applied to the outpatient clinic of the obstetrics and gynecology department and had 50 g ogtt, were used in this study. ada and xo activities were analyzed in the serum specimens taken from the normal (n = 20) and prediabetic pregnant women (n = 19) in the 0th and 60th minutes of ogtt. ada and xo activities were measured with the spectrophotometric method and the u/l enzyme activity was calculated. findings: there was no significant difference between the 0th and 60th minutes regarding the ada activities in the normal and prediabetic pregnant woman groups. however, we observed a significant difference between 0th and 60th minutes regarding the xo enzyme activity in normal pregnant women (p = 0.001). in normal pregnant women, the median xo enzyme activity in the 0th minute was 0.29 (0.11-1.33) u/l and it was 0.8 (0.25-2.26) u/l in the 60th minute. nevertheless, there was no correlation between the xo activity and glucose level. as to the prediabetic pregnant women, there was no significant difference between the xo enzyme activities in 0th and 60th minutes. the results of our study showed that the xo activity increased as a response to ogtt in the normal pregnant women compared with the prediabetic pregnant women. this finding made us think that the oxidative stress caused by ogtt did not affect the xo response in prediabetic pregnant women and that there would be some adaptive mechanisms against the chronic exposure to high level glucose. rainbow trout (oncorhynchus mykiss) aquaculture continuously increases in turkey. the objective of the present study is to increase the productivity in fish farming of rainbow trout just via intervention in physical cases without the effects of any chemicals and investigate whether this conditions cause oxidative stress. in this experiment eight tanks were used and 44 rainbow trout larvae were placed in each tank and these tanks were illuminated with light in different wavelengths; natural sunlight, and incandescent long-wave (red light), medium-wave (green light) and shortwave (blue light) led lights. the experiment took 64 days. biochemical changes in rainbow trout exposed to light in different wavelengths (red, green, blue) were analysed via the variations in gr, gst, g6pd, gpx, sod and cat enzyme activities, which are significant for enzymatic antioxidant defence system and in ache activity, which plays an important role on central nervous system. maximum activity change in liver tissue was observed for gst and g6pd enzymes in fish grown under green light and for sod enzyme in fish grown under blue light. in gill tissue, sod and g6pd activities were affected the most, and in brain tissue, these were gst and sod activities. it was observed that the average weight of the fish increased 1.2 times under red and blue lights and 1.3 times under green light. the highest weight increase was observed under green light, however, antioxidant enzyme activities increased in the liver and gills and decreased in the brain tissue under this light condition. in conclusion, it was observed that productivity was 1.2 times under red light when compared with control group and it was determined that the fish grown under red light can tolerate oxidative stress more than other wavelength. p-09.04.4-089 effect of nigella sativa on biliary obstructioninduced oxidative stress and apoptosis in rats human safety concerns, since that these agents may not only cause acute toxicity via inhibition of acetylcholinesterase but they can also induce delayed toxicity in the nervous system. a key interest to the current work is the potential correlation between gene expression and cytoskeletal protein changes in differentiating neural cells exposed to sub-lethal neurite outgrowth inhibitory concentrations of specific ops, which was addressed by analysing the underlying changes in the levels of cytoskeletal gene expression and protein levels. to assess the molecular effects of op exposure, phenyl saligenin phosphate (psp), chlorpyrifos (cpf) and its metabolite chlorpyrifos oxon (cpfo) were applied at the point of induction of differentiation of rat c6 glioma and mouse n2a neuroblastoma cells and incubated for 24 hours. at sub-lethal concentrations (1, 3, 10 lm) all three ops used in this study were able to inhibit the development of neurites with no significant effect on cell viability, as determined by neurite outgrowth and mtt reduction assays. to understand the possible effects of ops on cytoskeletal gene expression, primers for genes encoding glial fibrillary acidic protein (gfap), biii tubulin, growth associated protein 43 (gap43) and neurofilament heavy chain (nefh) were optimized for qpcr analysis and the levels of the corresponding proteins were detected by western blot analysis. exposure to ops caused in most cases a reduction in the levels of cytoskeletal proteins, and the results from qrt-pcr analysis also indicated reductions in the gene expression of gfap in c6 cells, and of nefh and biii tubulin in n2a cells, in a dose dependent manner. thus, the observed changes in protein levels are at least partly due to altered gene expression. curcumin is extracted from a perennial herbaceous plant known as curcuma longa. in recent years, considerable interest has been focused on curcumin due to its use to treat a wide variety of disorders without any side effects. earlier studies have shown that curcumin has anti-apoptotic, anti-inflammatory, antiproliferative, antiangiogenic, anticancer and antiplatelet activities. the goal of the present study was to investigate the effects of curcumin on peroxy radical-induced oxidative changes in human platelets. 15 healthy volunteers were enrolled in the study. none of the study participants were on anticoagulation therapy. citrated venous blood samples were centrifuged at 200 g for 10 minute to obtain platelet-rich plasma (prp). the platelet pellet was washed and suspended with tris-nacl buffer. then, platelets were incubated with h 2 o 2 absence and presence of curcumin (50-500 lg/ ml) for 1 hours at 37°c. to determine the preventive effects of curcumin on the oxidative stress and apoptosis induced by peroxy radicals in human platelets were determined by measuring levels of of lipid peroxidation, total antioxidant capacity, caspase 3, 8 and 9 activities, and mitochondrial membrane potential. additionally, we also studied the effects curcumin on platelet aggregation induced by adp. pre-treatment of platelets with curcumin caused a marked reduction in oxidative stress, activation and apoptotic markers in a dose-dependent manner. on the other hand, pre-treatment of platelets with increasing doses of curcumin resulted in inhibition of platelet aggregation induced by adp. in the light of our findings, we suggest that curcumin may have a therapeutic potential to prevent platelet activation related disorders. people have been using mushrooms in the treatment of diseases as well as food, for centuries. most of the edible and inedible mushroom species were used in important medical studies and their effects were begun to be used in the treatment of diseases. this study focuses on the hepatoprotective effects of tricholoma anatolicum, which is endemic specie in turkey, against oxidative stress based on hydrogen peroxide (h 2 o 2 ) on hepg2 liver cancer cell line. t. anatolicum used in this study was extracted with the help of ultrasonication and fraction methods. then the cytostatic effects of extracts on hepg2 cells were explored and their hepatoprotective effects were determined. moreover, various concentrations of aqueous extract (ehta) of t. anatolicum were determined by hepg2 cells's 24-48-72 hours effect analysis on their cellular morphology, xtt and real-time cell analysis in of xcelligence device. ehta extract's cell pathway (apoptosis and necrosis) effects on hepg2 cells were determined with flow cytometry method with the help of annexin v-apc and 7aad fluorescent dye. finally, the phenolic compounds found in ehta extracts were determined with the help of hplc methods. according to xtt cytotoxicity analysis, the ehta extract values were determined as follows: 24 hours ic 50 > 2000 lg/ml, 72 hours ic 50 . furthermore, according to the real-time cell analysis made with xcelligence, ehta extracts were found to be; 24 hours ic 50 = 241.539 lg/ml, 48 hours ic 50 = 418.135 lg/ml, 72 hours ic 50 = 285.694 lg/ml. increasing concentrations of ehta extracts were determined to direct hepg2 cells to apoptosis. moreover, considering the hplc analysis -according to the reference point of 100 mg in 100 g sample-within ehta extracts, catechins and vanillic acid peaked. the final results revealed that t. anatolicum's effect on hepg2 was cytostatic at low doses, and cytotoxic at high doses. p-09.04.4-093 relationship between serum ceruloplasmin levels and coronary blood flow background: there is growing evidence that oxidative stres plays an important role in the development of the slow coronary flow (scf) phenomenon. ceruloplasmin (cp) is a copper containing metalloenyzme which has antioxidant functionthrough its ferroxidese 1 activity and is associated with cardiovascular diseases. we aimed to investigate the relationship between scf and serum cp level. methods: patients who underwent elective coronary angiography and had no significant epicardial coronary disease were included in the study. patients who had thrombolysis in myocardial infarction frame counts (tfcs) above the normal cutoffs were considered to have scf and those within normal limits were considered to have normal coronary flow (ncf). a total of 90 patients (55 subject as scf and 35 subjects as ncf) were analyzed. 5 ml blood samples were taken from the groups to study ceruloplasmin activity. serum ceruloplasmin levels were determined spectrophotometrically. results: the serum cp levels were statistically lower in scf group than in the ncf group (414 ae 79 versus 469 ae 99 ng/ml, p = 0.009). also there was a significant correlation between serum cp levels and tfcs (r = à0.378, p = 0.013). conclusion: the findings of this study suggests that patients with scf had lower serum cp levels correlated with tfcs. we concluded that reduced serum cp levels might represent a biochemical marker of scf. introduction: sleeve gastrectomy (sg) has been used for the surgical treatment of morbid obesity, as a first step or definitive treatment. alterations of thyroid hormones in gastrointestinal surgery were previously studied. the aim of the present study was to determine the effects of triiodothyronine (t3) supplementation on oxidative stress parameters in anastomotic tissue level. materials and methods: twenty-four male wistar albino rats were divided into control (n 12), and experimental (n 12) groups. rats were underwent a sg, with a hand-sewn suture. experimental group rats received a single dose of t3 (400 mg/100 g) in postoperative day. rats were sacrificed on postoperative day 7. serum thyroid stimulating hormone (tsh), free t3 (ft3), and free thyroxine (ft4) were analysed using elisa. each tissue was homogenized in ice-cold pbs (ph: 7.4) and centrifuged at 2000 rpm for 20 minutes (4°c) to avoid contamination with cellular debris. the supernatants were used to measure total oxidant status (tos), total antioxidant status (tas), nitric oxide (no) and malondialdehyde (mda) levels. all tissue parameters were analysed by spectrophotometric methods. oxidative stress index (osi) values were calculated. results: rats given t3 hormone had not decline in ft3 levels compared with the control groups. a significant decrease in ft4 levels was found in t3 given rats on postoperative day 7. whereas tissue tos levels did not alter by thyroid hormone treatment, tas levels significantly decreased. osi values were not statistically different in tissues. tissue no levels were also similar in both groups. mda levels increased in t3 given rats compared with the control group. discussion and conclusion: this study showed that anastomosis after sleeve gastrectomy is associated with decreased ft4 level. although tos levels and osi values were similar in both groups, t3 supplementation induced lipid peroxidation by increasing tissue mda levels that might deplete tissue antioxidant level. reactive oxygen species (ros) are reactive chemical molecules, which are produced by living organisms as a natural byproduct of the normal metabolism and environmental factors. although intracellular ros level is essential molecules for the signal transduction pathways, elevated intracellular level of ros leads to oxidative stress that causes damage to dna, proteins and lipids. therefore, excessive ros levels have to be eliminated by antioxidant defence systems. tip60 (tat interacting protein, 60 kda) is a histone acetyltransferases (hats) that catalyses multiple functions in metabolism such as dna repair, apoptosis, etc. we thought that if tip60 has a role in signal transduction and apoptosis, it might have direct or indirect relationship with the antioxidant system. the present study was designed to determine the impact of tip60 gene on the hepatic antioxidant enzymes including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), and glutathione reductase (gr) both gene and protein level. for this reason, quantitative gene expression analysis on the antioxidant system has been investigated by real time pcr, quantitative protein expression has been investigated by western blot analysis, and then the activity of these enzymes have been measured in whole liver homogenate collected from control and liver-specific tip60 conditional knockout mice. additionally, since any change of reduced glutathione (gsh), oxidized glutathione (gssg), malondialdehid (mda), and hydrogen peroxide (h 2 o 2 ) level in the cell might be an indication for the accumulation of ros, the relative levels of them were also studied. our data showed that the absence of tip60 affects the antioxidan system both gene and protein level. in conclusion, our initial data suggest that tip60 may be essential for the (ros) homeostasis and redox regulation. curcumin is a major chemical component produced from the rhizome of the plant curcuma longa. lt has been demonstrated that curcumin has an antioxidant, anti-inflammatory, and antiproliferative effects and, protects tissues against ischemia/reperfusion (i/ r) injury. i/r has detrimental effects on transplanted organs including uteri. the major consequence of l/r injury is oxidative stress leading to the generation of ros. uterine transplantation (ut) has been gaining popularity around the worid in the past few years. the aim of our study was to examine the antiapoptotic effects of curcumin on uterine l/r injury. the rats were randomized into three groups of seven rats each, group i consisted of rats that did not receive any treatment, group ll exposed to 0.5 hour of lschemia and 1 hour of reperfusion, group iii of rats that received intraperitonealy curcumin (200 mg/kg) 0.5 hour before the induction of i/r. then, the rat uterine tissue levels of mda, tac, and activities of caspase 3, 8 and 9 were measured. furthermore, the apoptotic index was determined immunohistochemically by the tunel method using light microscopy. biochemical analysis results showed that curcumin decreased the mda and caspase-3 and 9 ieveis, and increased the uterine tissue levels of tac but, caspase 8 activity did not changed by curcumin suggesting that curcumin induces apoptosis via intrinsic apoptotic pathway. on the other hand, an high apoptotic index was observed in i/r group (27.37 ae 11.56%) and decreased after treatment with curcumin (8.69 ae 7.49%). in conclusion, we demonstrated the protective effect of curcumin on apoptosis immediately after reperfusion induction in uteri and we can say that curcumin could improve ir injury and decrease apoptotic index. we propose that curcumin may be a novel approach for improvement of uteri i/r injury. glutathione and the related enzymes belong to the defence system of the tissues against chemical and oxidative stress. these enzymes especially glutathione s-transferase are often overexpressed in tumor cells and are regarded as a contributor to their drug resistance and are thought to play an important role in cancer progression. the purpose of this study is to evaluate the protective effects of chlorophylline as an antioxidant molecule which has inhibitory effects on gst p1-1 on chemically-induced breast cancer model. in our previous work, we had observed that this molecule led to proliferation in breast cancer cells. in this study, n-methyl-n-nitrosourea (mnu) used for inducing carcinogenesis in eighteen, 21-day-old female sprague-dawley rats. chlorophylline and mnu solutions were injected intraperitoneally when the rats were 21, 28, 35 and 42 days old. their weight and tumor diameters were measured throughout the 5 months study period. at the end of the study, all animals were sacrificed and determined both glutathione levels and related enzymes activities (gluathione s transferase, glutathione reductase and glutathione peroxidase) in tumor and tissues such as liver, kidney, heart and spleen were studied and analyzed. as a result, in breast cancer model, glutathione and related enzyme activities were protected by chlorophylline treatment whereas mnu made them decreased compared to the control group. in conclusion, chlorophylline with antioxidant features decreased the toxic effect of mnu by regeneration of glutathione and enhancement of its related enzyme activities. the use of antioxidant molecules, because of proliferative effects and defence-oriented behaviours, should be discussed in cancer therapy. p-09.04.4-100 effect of overexpression of bacillus catalase on lactococcus lactis nisin production z. girgin ersoy, s. tunca gedik gebze technical university, kocaeli, turkey nisin, has been used commercially (e234) in food preservation for approximately 40 years. it's the only bacteriocin which is approved by world health organization as a food additive. the fundamental problem that limits nisin usage in food preservation is low product yield by producer strains. because of high commercial potential of nisin, studies about increasing the production efficiency of nisin is kept in the forefront in recent years. since nisin biosynthesis and bacterial growth are occuring in parallel to each other, conditions that promote growth are also expected to encourage nisin production. it is known that, when supplied with exogenous heme, lactococcus lactis cells can respire under aerobic conditions and produce higher energy which in turn cause higher biomass. however, aerobic conditions also cause oxidative stress since catalase enzyme, which detoxify hydrogen peroxide, is absent in l. lactis. in this study, to complete the missing component of the defence mechanism of l. lactis, catalase (kate) gene of aerobic bacterium bacillus subtilis was overexpressed in facultative anaerobe l. lactis cells. for this, kate gene of b. subtilis was amplified by polymerase chain reaction (pcr). plasmid constructions were established in e. coli by using an e. coli-l. lactis shuttle vector and then the recombinant plasmid was transferred to l. lactis cells by electroporation. the presence of catalase activity in the recombinant strain grown on the solid medium was first detected by dropping hydrogen peroxide directly on the cells, then with enzyme assays. fermentation studies are going on to determine nisin production of the recombinant strain. to the best of our knowledge, this study presents the first preliminary results that shows the effect of overexpression of catalase gene on nisin production. cancer is among the leading causes of morbidity and mortality worldwide. chemotherapy is one of the major cancer treatment strategies. remarkably, natural products have garnered increased attention in the chemotherapy drug discovery field because they are biologically friendly and have high therapeutic effects. humic acid (ha) is a natural product which is forming during decomposition of organic matter in humus. in recent years, there are some resarches on the medical use of humic acid. the present study investigated anticancer effects of ha in several human cancer cell lines. ha was purchased from sigma-aldrich. in this study, we used several human cancer cell lines: the human breast cancer cell line, mcf-7, colon cancer cell line, ht-29, lung adenocarcinoma cell line, a549, and servical cancer cell line, hela. the cells were maintained in dmem medium supplemented with 10% heatinactivated fbs and 1% penicillin/streptomycin. cells were grown in petri dishes in a humidified atmosphere containing at 37•c. five different concentrations (100 ug/ml, 50 ug/ml, 25 ug/ ml, 10 ug/ml, 5 ug/ml) were prepared using a stock solution of ha. the cell proliferation and migration was measured. on the other hand, the apoptotic mechanisms induced by ha in cancer cells were investigated using "apoptosis antibody array kit". the effects of ha on cancer cell lines were evaluated over 72 hours. according to our results, ha induced a decrease in ht-29, a549 and hela cell numbers in a dose-dependent and time-dependent manner. contrary to this, ha induced proliferation of mcf-7 cells in dose dependent manner. ha inhibited cell migration in a dose dependent manner except mcf-7 cell line. it was also determined apoptotic pathways in cancer cells induced by ha. it was concluded that ha has an inhibitory effect on certain some cancers. since the effect of ha on tumor progression is unknown, further studies are needed to clarify the rol of ha on cancer activity. p-09.04.4-102 chronic immobilization stress in rats: fluoxetine and amisulpride protects against chronic immobilization stress-induced biochemical alterations in the present study, the effects of amisulpride and fluoxetine on serum total sialic acid (tsa) and lipid bound sialic acid (lsa) levels was investigated in the rats exposed to chronic immobilization stress. the study was administered using 40 male wistar albino rats weighing 200-250 g. rats were divided into five groups (n = 8/ group). group i comprised the control group, group ii was exposed with saline + immobilization stress (30 minutes daily immobilization stress for 15 days and 0.5 ml saline was administered perorally 30 minutes before immobilization), group iii was exposed amisulpride (10 mg/kg/day) + immobilization stress, group iv was exposed fluoxetine (10 mg/kg/day) + immobilization stress and v. group was exposed amisulpride (10 mg/kg/ day) + fluoxetine (10 mg/kg/day) + immobilization stress. statistical analysis showed that the saline + stress, amisulpride + stress and amisulpride + fluoxetine + stress groups was significantly higher than the control group with regards to tsa levels (p < 0.05). whereas, the fluoxetine group was significantly lower than the group regarding tsa levels (p < 0.05). on the other hand, saline group was significantly higher than the control group with regards to lsa level (p < 0.05). whereas, no significant differences in lsa levels were observed in the amisulpride, fluoxetine and amisulpride + fluoxetine groups, as compared to the control group (p > 0.05). the present study demonstrated beneficial effect of fluoxetine and amisulpride on the concentration levels of lsa and tsa in stress. p-09.04.4-104 protective effect of borax and boric acid on total sialic acid and lipid-bound sialic acid levels against 3-methylcholanthrene and benzo(a)pyrene induced oxidative stress in rats s. ekin 1 , g. oto, f. g€ ok, y. karakus, d. yildiz 1 y€ uz€ unc€ u yil university, van, turkey the present study was performed to investigate total sialic acid (tsa) and lipid bound sialic acid (lsa) levels as possible in vivo chemoprotective effect of borax (bx) and boric acid (ba) again-st3-methylcholanthrene (3-mc) and benzo(a)pyrene (b(a)p) induced oxidative stress in rats. the rats were divided into nine groups of six rats each. group i: control, untreated animals were given % 0.9 nacl, group ii: the b(a)p were administered 25 mg/kg via ip. four times. group iii: the 3-mc-treated animals were administered 25 mg/kg via ip. four times, group iv: ba was given 300 mg/l/day with water. group v: bx was given 300 mg/l/day with water. group vi: b(a)p 25 mg/kg via ip four times + ba 300 mg/l/day dosage with water. group vii: 3-mc 25 mg/kg via ip four times + ba 300 mg/l/day with water. group viii: b(a)p 25 mg/kg via ip four times + bx 300 mg/l/ day dosage with water. group ix: 3-mc 25 mg/kg via ip four times + bx 300 mg/l/day with water. the experimental period was continued for 150 days. statistical analysis showed that the 3-mc + ba group was significantly higher than the control group with regards to tsa and lsa levels p < 0.001, p < 0.001,p p-09.04.4-105 effects of aluminum exposure on trace elements in rat tissues b. ozturk kurt, s. ozdemir department of biophysics, cerrahpasa medical faculty, istanbul university, istanbul, turkey aluminum (al) is the most abundant metal and the third most abundant element in the earth's crust. people are constantly exposed to al which is found in most rocks, soils, waters, air and foods, due to a result of an increase in industrialization and improving technology practices. the study was designed to examine the possible effects of aluminum exposure in different durations on trace elements in rat tissues. twenty-four healthy male wistar rats weighed 180-200 g were randomly divided into three groups: control group (gc) received only drinking water, short-term group (gs) and long-term group (gl). the study groups were orally exposed to 40 mg/kg body weight alcl 3 in drinking water for 8 and 16 weeks, respectively. at the end of the treatment period, rats were sacrificed and the kidney, liver, brain and cerebellum tissues were removed to analyse the levels of al, ar, b, ni, si, cr, cu, fe, mg, mn, se, cu and zn by icp-oes. the statistically significant increase were determined in cerebellum al, cu, as, b and cr levels in gl according to the gc. while as levels were statistically increased, ni levels were decreased in gl in the kidney and liver. while cu, mg and cr levels were higher, se and b levels were lower in the gs than gc in the brain. there were no significant difference in si and mn levels. as a result of our study, it may be concluded that al accumulation may lead to changes in tissue trace element levels. tacal, o., 266 tacal, ö., 321 take, g., 412 takic, m.m., 417 taldykbayev, z., 416 talim, b., 292 tamashevski, a., 382 tamer, f., 144 tamer, l., 245, 279, 395 taneva, s., 209 taniyan, g., 275, 306 tanrisev, m., 288 tanriverdi, e.c., 245 tanriverdi, k., 279 tarhan, m., 161 tarhan, t., 315 tartar, s., 387 tas, a., 204, 293 taskin, a., 406 taskin, t., 339 taskiran, e., 271 taskiran, b., 328 taskoparan, b., 221 taspinar, r., 301, 302 tastan, ö., 271 tatli, ö., 340 tauraite, d., 206 tay, t., 344 tayman, c., 354 taysi, s., 389, 392 teker, h.t., 270 tekes, s., 361 tekin neijman, s., 410 tekin, m.h., 188, 297 tekin, g., 191, 256 tekin, m., 196 tekin, n., 155 tekin, g., 256 telci, d., 187, 307 telefoncu, a., 151 temel, h.e., 158, 159, 160 temelie, m., 267 temizgül, r., 347 temlyakova, e., 168 teplova, v., 372 terashima, r., 284 tercan avci, s., 197 terekhov, s., 381 tereshenko, o., 304 terzi gulel, g., 149 terzi, e., 300, 302 terzi, e., 301 terzioglu, g., 296 terzioglu, o., 272 testoni, g., 348 tetik vardarli, a., 282 tevdoradze, t., 148 tevzadze, l., 148 tezcan, ö., 308 tezcanli kaymaz, b., 282 thielens, n., 228 thomaidou, d., 265 thomas, a., 228 thornton, j.d., 216 ticea, a.c., 168 tikhonova, a., 287 tileva, m., 209, 210 timofeev, v., 198, 202 timofeev, v., 202 timofeeva, e., 383 tipirdamaz, r., 401 tiryaki, m., 300, 302 tok, m., 140 tokay, e., 365 toker, a., 360, 402 tokgun, o., 284 toksoy öner, e., 138 tokyol, ç., 390 toman, r., 317 tomasi, a., 410 tombul, n., 333, 337 tooke, c., 231 topaloglu, h., 292 topbas, m., 200 topcu, b., 247 topcu, c., 292 topcu, g., 144, 358, 377 topçu, v., 393 topcu, c., 417 topçuoglu, c., 193, 416 topel, h., 197, 264 toprak, b., 223 toprak, m.s., 351 torac, e., 384 tosner, z., 383 tosun, m., 308, 354 toy, h., 143 toymentseva, a., 239 tozkoparan, b., 246 trabulus, d.c., 256 trantirek, l., 229 trantirkova, s., 229 trchounian, a., 165, 324 trizna, e., 200, 289 troshagina, d., 371 tro t, m., 269 truncaite, l., 149, 363 tsakalidou, e., 170 tsarkov, d., 233 tsarkova, m., 233, 235 tsigara, e.g., 206 tsigara, m.g., 206 tsverava, l., 266 tsvetkova, e., 253 tsyba, l., 362 tsyganov, d., 158 tsymbal, d., 165 tüfekçi, a.r., 241, 358 tufekci, a.r., 296 tufenkci, h., 139 tuglu, m.i., 300, 309 tuglu, i., 200 tuli, a., 142, 182, 237, 403 tuli, a., 352 tulubas, f., 247 tunali, g., 226 tunca gedik, s., 143, 408 tunca gedik, s., 221 tunçdemir, m., 396 tuncel, h., 378 tuncel, h., 273 tunçer, s., 222 tuncer, e., 161, 261 tuncer, z.s., 395 tuncer, b., 133 tuncer, e., 273 tuncez akyurek, f., 185 tunçez akyürek, f., 359 tuner, h., 333 tüney kizilkaya, i., 325 tural, b., 315 tural, s., 315 turan, i., 330 turan, m., 161, 261 turan, v., 173 turan, y., 333 turan, c., 411 turano, p., 217, 290 türel, s., 329, 341 turhan, t., 193, 242 turhan, y., 316, 317 turk, s., 373 turkan, a., 219 türkcan kayhan, c., 135 turkekul, k., 299 türkel, s., 176 turkeri, o.n., 245 turkeri, n., 141 turkkan, b., 257 turkmen, s., 401 türkmen, n., 180 turkon, h., 223 tutar, y., 161 tüten, a., 351 tutkun, e., 196, 275 tüylü küçükkilinç, t., 285 tuz, m.u., 321 twardovska, m., 335 tyapkina, o., 371 tzartos, s., 268 photoprotective activity of vulpinic and gyrophoric acids towards ultraviolet b-induced damage in human keratinocytes evaluation of the sunscreen lichen substances usnic acid and atranorin pleiotropic anticancer activity of selected nutraceuticals against mcf-7 bucharest, 3 national institute for marine research and development "grigore-antipa uk in some adult and elderly populations the acute and/or persistent infection with the common intracellular respiratory pathogen chlamydia pneumoniae (chl) may be associated with increased risk of developing obesity or cardiovascular disorders. thus, 19 microelements modifying oxidative stress status were determined by icp-ms/ms in the hno 3 diluted serum samples collected from chl-positive adult females (n = 39) living in urbanized area in poland. chl infection was confirmed by igg+ antibody elisa and real-time pcr assay. all females were classified under their body-mass index values to the normal-weight (nw), over-weight (ow) and obese group (ob) although there are many drugs currently used in the treatment of peptic ulcer, such a drug providing radical treatment without side effects is not available. since oxidative stress is involved in peptic ulceration, this study was designed to investigate antiulcerogenic and antioxidant effects of hippophae rhamnoides l. ether extract on indomethacine-induced stomach ulcer in rats. materials and methods: thirty-five sprague dawley male rats (weights ranging 180-220 g) were randomly divided into 7 groups, as each composed of 5 rats. after hippophae rhamnoides l. leaf ether extracts of 100 mg/kg, 250 mg/kg and 500 mg/kg doses and 20 mg/kg doses of famotidin orally administered, 25 mg/kg doses of indomethacine were orally applied to rats in order to make ulcer. on the sixth hour of indomethacin administration all rats were sacrificed using thiopental (50 mg/kg). the stomachs were removed, and ulcer areas were evaluated macroscopically. superoxide dismutase activity (sod), glutathione (gsh) and malondialdehyde (mda) levels in stomach tissues of rats were determined by elisa method with respective kits conclusions: we can conclude that the ether extract of hippophae rhamnoides l. leaves reduces free radical formation and has antiulcerogenic effects on stomach tissue control group; 2 hours torsion/4 hours detorsion group (t/d); all other groups were saturated for four days egcg, cape and egcg+cape (10lml/kg). sections were taken from bouine's-fixed and paraffin-embedded testicular tissue blocks and stained with h&e. immunohistochemistry was applied for the detection of pi3k, akt and mtorc. intensities were evaluated as mild (1), moderate (2) or strong (3). serum 8ohdg, plasma mda levels were analyzed using elisa method. results were analyzed by anova statistical test. testis samples in control group exhibited normal histological morphology. disorganization and separation of seminiferous tubule cells and accompanying interstitial edema and vessel dilation were while mda level decreased significantly in cape+egcg group, 8ohdg level showed significant increase in cape group. in conclusion, cape and egcg exerted protective effects on tt. effects may be achieved through pi3k/ akt/mtorc pathway involved in cell proliferation, angiogenesis, apoptosis. prophylactic use of egcg prior to tt surgery improved testicular morphology, therefore could prevent destructive effects of tt lmol/l), c18 (0.58 ae 0.278 lmol/l)), respectively. conclusions: this study is important for konya region, mitochondrial fatty acid b-oxidation disorders studies subject areas. this study is the first study to assess acylcarnitine levels of patients living in our region. we believe that our results will be useful for future studies. key words: acylcarnitine, mass spectrometry, dried blood spot p-09.04.4-137 binding of fas and cu(ii) ions to hsa changes its cys34 thiol group antioxidant capacity and carbonylation pattern with methylglyoxal binding of cu(ii) ion (0.1 mol/mol hsa) led to increase of k' value if fish oil extract was present, but for other fas k' value decreased. the content of free hsacys34-sh decreased for 10% after cu(ii) ion binding, and during 24 hours incubation at 37°c, it was further decreased for 10% (stearic acid, mixfas) and 20% (myristic, fish oil extract, oleic acid). carbonylation of fa-hsa-cu(ii) complexes with mg (20 mol/ mol hsa), lead to decrease in cys34-sh content depending on fa present: 30%-35% for myristic and stearic acid, 40% for oleic acid and mixfas and 40% for fish oil extract. carbonylation of fa-hsa-cu complexes could contribute to further enhancement of oxidative and carbonyl stress in diabetes as well as other diseases 151 pirto ek p-05.03.3-007 anti-proliferative and inducing apoptosis of the hydro alcholic achelia. wilhelmsii extract on human breast adenocarcinoma cell lines mcf-7 and mda-mb-468 background: vitamin d deficiency is associated with several conditions and/or diseases like inflammation, atherosclerosis, cardiovascular disease and mortality. several studies showed that lower vitamin d levels were associated with high serum levels of inflammatory biomarkers. ykl-40 is a glycoprotein, secreted by macrophages, neutrophils and different cell types. it is also associated with inflammation and pathological tissue remodeling. in this study, we aimed to evaluate relationship between the vitamin d deficiency and ykl-40 levels. methods: our study group includes 45 subjects with vitamin d deficiency (group 1) and 40 age and sex-matched healthy subjects with normal serum levels of vitamin d (group 2). plasma 25 (oh) vitamin d levels were measured with liquid chromatography-tandem mass spectrometry (lc-ms/ms) method. plasma ykl-40 analysis was performed by elisa. serum hs-crp levels were measured with nephelometric method. results: plasma vitamin d levels below 20 ng/ml were accepted as vitamin d deficiency. although we could not find any significant differences by means of serum hs-crp levels between groups (p > 0.05), plasma ykl-40 levels were significantly higher in group 1 than group2 (p < 0.05). conclusions: in literature, vitamin d deficiency is associated with inflammation. in our study, we found similar hs-crp levels between groups and higher ykl-40 levels in group 1. vitamin d deficiency may be related to increased ykl-40 levels in terms of causing chronic inflammation.keywords: vitamin d deficiency, ykl-40, inflammation. evaluation and comparison of tnf-family ligands and receptors genes in mice and humans by bioinformatics techniques stance called plaque builds up inside the coronary arteries. apelin is a novel endogenous peptide with inotropic and vasodilatory properties and is the ligand for the angiotensin receptor-like 1 (apj) receptor. we aimed to determine genotype and allele frequencies of apj receptor a445c gene polymorphism in turkish patients with cad and healthy controls by rflp-pcr. this study was performed on 159 unrelated cad patients and 62 healthy controls. we obtained aa, ac and cc genotype frequencies in cad patients as 41.5%, 49.1% and 9.4%, respectively. in the control group, frequencies of genotypes were found as 35.5% for aa, 48.4% for ac and 16.1% for cc. we did not observe difference in apj receptor a445c polymorphism between cad patients and healthy controls (v 2 = 2.178; df = 2; p = 0.336). the a allele was encountered in 66% (210) of the cad and 59.7% (74) of the controls. the c allele was seen in 34% (108) of the cad and 40.3% (50) of the controls. allele frequencies were not significantly different between groups (v 2 = 1.57; df = 1; p = 0.225). the frequencies of apj receptor a445c genotype were not significantly different between control and patients. individuals with cc genotypes had significantly higher weight, systolic and diastolic blood pressure levels and systolic blood pressure than other genotypes, p ≤ 0.05. in addition, hdl-c level was found decreased, but this reduction was not statistically significant. contrarily, the low levels of weight, sbp, dbp and tc were statistically significant in the subjects with aa genotype in cad. in conclusion, cc genotype carriers may have more risk than other genotypes in the development of hypertension in cad. we suggest that this polymorphism may not be a marker of cad, but it may cause useful in function of the apelin/apj system and may be a genetic predisposing factor for diagnostic processes and can be helpfull in finding new treatment strategies. p-08.01.4-032 comparative genomics/proteomics analyses of single amino acid repeat containing proteins across different vertebrate taxa a. g. keskus, o. konu department of molecular biology and genetics, bilkent university, ankara, turkeyconsecutive runs of single amino acids lead to overrepresentation of certain physicochemical properties in protein sequences. researchers also demonstrated a link between single amino acid repeat (saar) containing proteins and neurodegenerative diseases as well as biological functions. moreover, saar frequencies were shown to vary across species based on selected orthologous proteomes and/or proteins. hence, analysis of whole proteomes across multiple vertebrate taxa may provide additional species-and sequence-specific trends for saars. in addition, there is a need for testing the observed saar occurrencesthe aim of this study is to evaluate the effect of quercetin (q) on liver injury secondary to cerulein induced-acute pancreatitis (ap). for this reason, rats were randomly divided into four groups (8 rats for each group) control group received physiological saline, four time and dimethyl sulfoxide, two time, at 1 hours intervals, intraperitoneally (i.p.). cerulein group received cerulein (50 lg/ kg-rat weight, in physiological saline), four times, and dimethyl sulfoxide (1%), two times, at 1 hour intervals, i.p. quercetin pretreatment (q+cer) group received quercetin (100 mg/kg-rat weight, in dimethyl sulfoxide) one time, one hour before cerulein treatment and physiological saline, one time, six hour after cerulein treatment. quercetin post-treatment (cer+q) group received dimethyl sulfoxide, one time, one hour before cerulein treatment and quercetin, one time, six hour after cerulein treatment. cerulein treatment increased significantly vascular congestion in hepatic cells. quercetin treatment also decreased significantly vascular congestion. the liver mda and carbonyl levels in cerulein group were significantly higher than the control group (p < 0.01, p < 0.001, respectively). the mda and carbonyl levels in q+cer group decreased significantly compared to the cer group (p < 0.001). the mda, carbonyl, mpo levels in cer+q group were significantly lower than the cer group (p < 0.001). the gssg/gsh ratio of q+cer and cer+q groups were significantly lower than the cer group (p < 0.05, p < 0.001, respectively). the sod activity in cer group was significantly lower than the control group, but the sod activity in q+cer and cer+q groups was significantly higher than the cer group (p < 0.05).this study shows that quercetin treatment was reduced the severity of liver injury secondary to cerulein induced-ap as reflected by changes in the parameters of hepatic oxidant and antioxidant. p-09.04.4-029 identification of water extract of propolis components by using different columns in gas chromatography-mass spectrometry propolis is a natural material obtained by honey bees from various plants. protective effect of propolis against damages of free radicals is due to different compounds within propolis. the aim of this study is to identify qualitatively and quantitatively the chemical composition of water extract of propolis (wep) provided by erzurum region using rtx-1 and rtx-5 ms column of gas chromatography-mass spectrometry (gc-ms) and to compare with two columns.in this study, wep of 25 mg/ml was prepared, cleared by membrane filter of 0.45 lm and freezed at à80°c. then, it was lyophilized up to dry form and derivatized to apply for gaseous form. 7 mg of dry extract was reacted with 350 ll pyridin + 700 ll bis-trimethylsilyl trifluoroacetamide (bstfa) mixture including 1% trimethylchlorosilane (tmcs) and incubated for 30 minutes at 100°c. all analyses were performed with shimadzu gcms-qp2010 ultra by using a flame ionisation detector (fid). rtx-1 and rtx-5 ms capillary columns and helium for carrier gas at a flow time of 1 ml/minute were used. injection was applied on split mode at 250°c. derivatized propolis sample was injected as 2 ll, initial column temperature was adjusted as 60°c, then increased to 240°c with increments of 3°c. total analysis time was determined as 62 minutes. relative percent amount of separated compounds was calculated from total ion chromatogram with computerised integrator. all components were defined by using nist and wiley libraries.peaks obtained from rtx-1 column were much more than those of rtx-5 ms. on the other hand, analyses performing with both two columns have similar carbohydrate, aromatic acid and other acid contents.consequently chemical constituents of wep were determined qualitatively and quantitatively with gc-ms. it was concluded that rtx-1 column among both columns differentiating for polarities may produce more compounds in the propolis analysis. introduction: aquatic environment can be mostly contaminated by mixtures of metals. biochemical parameters have gain importance to characterize the effects of metals on aquatic organisms. glutathione s-transferase (gst) and its substrate glutathione (gsh) are important parameters of antioxidant defense system of fish metabolism due to their vital role in xenobiotic conjugation. objective: the goal of the current study to evaluate the changes in gst and gsh levels in response to cd, zn and cd+zn effects after 14 and 28 exposure days. materials and methods: fish were obtained from cukurova university fish culturing pools (turkey). fish were exposed to 1.0 lg/ml of cd (cdcl 2 .h 2 o) and zn (zncl 2 ), and their mixture, for 0, 14 and 28 days. at the end of the exposure period, liver tissues were dissected and homogenized in a phosphate buffer (ph 7.4). homogenates were centrifuged at 10,000 g (30 min, +4°c). supernatants were stored at à80°c until the analysis. one-way anova was used to compare data (mean ae se) followed by duncan's test (p < 0.05). results: gst and gsh changes were recorded as decreases after all metal exposures at day 14. although 28 day exposure was found as less effective, combined effects caused significant decreases in gst and gsh levels. also longer exposure durations were appeared to be more effective in that situation. conclusions: significant decreases in gst and gsh levels could be occurred due to increased reactive oxygen species caused by metals particularly their combined effects. metal type, their single and combined effects and also exposure duration should be also taken into account when considering the antioxidant system response. gst and gsh might be considered as sensitive biomarker in toxicity assessment of metal mixtures.financial support: this study was supported by a grant from c ß ukurova university (turkey).background/aims: the activation of lectin-like oxidized low density lipoprotein receptor-1 (lox-1) on endothelial cells leads to intracellular oxidative stress and inflammation and a feed-forward cycle of injury in diabetes, since both oxidized low density lipoprotein (oxldls) and glucose increase lox-1 expression. quercetin (qr) is part of a subclass of flavonoids called flavonols. polyphenolic compounds affect the development of atherosclerosis not only through antioxidant properties but also by modulation of serum lipids, thereby influencing the immune and inflammatory processes associated with the development of atherogenic diseases. we investigated the effects of dietary qr on endothelial dysfunction mediated by oxidized low density lipoprotein (oxldl)/lectin-like oxidized low density lipoprotein receptor-1 (lox-1) in animal model of type 2 diabetes mellitus. methods: we compared 4 groups of male adult wistar albino rats: a control group, an untreated diabetic group, diabetic groups treated with qr, and qr group. diabetes was induced by a single injection of stz (50 mg/kg). animals were kept in standard condition. in 30 day after inducing diabetic, serum was collected for biochemical parameters. glucose, lipid profiles, microalbuminuria, oxldl and lox-1 levels were determined. results: serum triglyceride, ldl, vldl levels in diabetic control group (without treatment) was significantly higher than control group (normoglycemic untreated group). supplementation with quercetin decreased serum total cholesterol and increased hdl-cholesterol compared with the control group. serum oxldl and lox-1 levels in diabetic control group (without treatment) were significantly higher than control group (normoglycemic untreated group). conclusions: consumption of quercetin reduced oxldl and lox-1 levels. thus, quercetin could be effective in improving hyperglycemia, dyslipidemia, and endothelial damage in type 2 diabetes. p-09.04.4-055 investigation of some oxidative stress parameters in bdnf heterozygous mice liver tissue a. bodur 1 , i. ince 1 , i. abidin 2 , a. alver 1 objectives: the aim of this study was to evaluate the possible protective effects of nigella sativa (ns) against cholestatic oxidative stress and liver damage in the common bile duct ligated rats. methods: a total of 24 male wistar albino rats were divided into three groups:sham control, bile duct ligation (bdl) and bdl+received ns; each group contain 8 animals. the rats in ns treated groups were given ns (0.2 ml/kg) once a day orally for 4 weeks starting 3 days prior to bdl operation. results: the changes demonstrating the bile duct proliferation and fibrosis in expanded portal tracts include the extension of proliferated bile ducts into lobules, mononuclear cells, and neutrophil infiltration into the widened portal areas were observed in bdl group. treatment of bdl with ns attenuated alterations in liver histology. the alpha smooth muscle actin (a-sma), transforming growth factor beta (tgf-b1) and the activity of tunel in the bdl were observed to be reduced with the ns treatment. bdl significantly increased the tissue hydroxyproline (hp) content, malondialdehyde (mda) levels, and decreased the antioxidant enzyme (superoxide dismutase (sod) and glutathione peroxidase (gpx)) activities. ns treatment significantly decreased the elevated tissue hp content and mda levels and prevented the inhibition of sod and gpx enzymes in the tissues. conclusion: the data indicate that ns attenuates bdl-induced cholestatic liver injury, bile duct proliferation, fibrosis, apoptosis and oxidative stress. the hepatoprotective effect of ns is associated with antioxidative potential. organophosphorous compounds (ops) are used widely as pesticides for agricultural purposes, as oil additives or as flame retardants. however, due to their widespread use, there are major introduction: in this study, the antiulcerogenic effect of a water extract (cawe) obtained from a spices sample, cinnamomum aromaticum, was investigated using indomethacin-induced ulcer models in rats. materials and methods: experimental groups consisted of six rats. antiulcerogenic activities of 50, 100, 200 and 400 mg/kg body wt. doses of the cawe were determined by comparing the negative (treated only with indomethacin) and positive (famotidine) control groups. results and discussion: although all doses of the cawe showed significant antiulcerogenic activity as compared to negative control groups, the highest activity was observed with 400 mg/kg body wt. doses (45%). the cawe showed similarly antioxidant activity when compared with trolox and ascorbic acids used as positive antioxidants. in addition, the activities of catalase (cat) and myeloperoxidase (mpo) enzymes were determined in the stomach tissues of rats and compared with those of the negative and positive control groups to expose the effects of these enzymes on antiulcerogenic activity. the enzymatic activities of cat and mpo and lipid peroxidation (lpo) level in indomethacin-administrated tissues were increased significantly by indomethacin in comparison to control groups. these enzymes and lpo level were decreased, however, by the cawe. in contrast to lpo level, cat and mpo activities, glutathione (gsh) level was decreased by indomethacin and increased by all doses of cawe and famotidine. the present results indicate that the cawe has a protective effect in indomethacin-induced ulcers, which can be attributed to its antioxidant potential. introduction: thiol groups (-sh) are important anti-oxidants and essential molecules protecting organism against the harmful effects of oxidative stress. the aim of our study is to evalute thiol-disulphide homeostasis with a novel and automated method in patients with prostate cancer (pc) before and after radical prostatectomy (rp). material and methods: 18 patients with prostate cancer and 17 healty control subjects were enrolled into the study. plasma samples were collected from patients before rp and 6 months after the rp operation. thiol-disulphide homeostasis was determined with a recently developed novel method. prostate specific antigen, albumin, total thiol, native thiol, disulphide and total antioxidant status (tas) were evaulated and compared between the groups. results: native thiol levels were 419.8 ae 54.87 lmol/l in the control group, 350.7 ae 46.35 lmol/l in the patients before rp, and 364.3 ae 46.88 lmol/l in patients after rp. native thiol, total thiol and tas levels were significantly higher in the control group than the patients before rp (p values < 0.001). native thiol, total thiol and tas levels were higher 6 months after rp compared to before rp in patients, but these changes were not significant statictically (p values 0.3, 0.3 and 0.09 respectively). discussion and conclusion: our study demonstrated that antioxidant defense mechanism was weakened as indicated by the decreased thiol levels in the patients with pc. increased oxidative stress in prostate cancer patients may cause metabolic disturbance and have a role in the pathogenesis of prostate cancer. p-09.04.4-108 is there any relation between 50 g oral glucose challenge test and serum total oxidant-antioxidant status in pregnant woman? the purpose of this study was to test the hypothesis that any degree of antepartum screening for gestational diabetes mellitus with oral 50 g glucose challenge test (gct) should be associated with oxidant-antioxidant status.in this prospectif study, oral glucose challenge test was applied to 25 pregnant women aged 25-40 years and at 24-28 weeks of gestation. plasma glucose concentrations were measured initial, 1 hour and in addition to test 2 hours after ingestion of 50 g glucose. at the same time serum insulin, cortisol, total antioxidant status (tas), total oxidant status (tos) levels were measured and the oxidative stress index (osi) was calculated.ten pregnant women (forty percent) had a positive glucose challenge test (gct). a positive moderate relation with initial and 1 hour serum total antioxidant status (tas) levels (r = 0.68) and the oxidative stress index (osi) (r = 0.67) was found. there was a positive weak correlation with initial and 2 hours total oxidant status (tos) levels (r = 0.22) but statistically significance difference was not found (p > 0.05).in this study after ingestion of 50 g glucose serum total antioxidant status (tas), serum oxidant status levels (tos) and serum oxidative stress index (osi) levels were higher than the initial levels.the results of this study suggest that antepartum screening for gestational diabetes mellitus with 50 g oral glucose challenge test (gct) weakly associated with oxidant-antioxidant status and to confirm this results the longer follow-up studies with more participants are necessary.wheat (triticum ssp.), cultivated for centuries in the middle-east, central asia, europe, and north-africa, is one leading staple crops around the world, and its marginally grown ancestor einkorn (triticum monococcum ssp. monococcum), possesses rich gene resources for wheat improvement and have bioactive compounds reducing and preventing chronic diseases such as diabetes, cancer, alzheimer, and cardio vascular diseases, beside their nutritional properties. however, as more attention has been given to wheat cultivars with strong gluten, protein content, starch composition, and resistance to biotic and abiotic stresses in bread wheat and yellow-colored pasta product in durum wheat health compounds such as fibers, phytochemicals, and bioactives have been underestimated so far. the aim of this study was, then, to examine the total phenolics and flavonoids, quantify their phenolic acids, a-tocopherol by high performance liquid chromatography (hplc), and their 2,2-diphenyl-1-picrylhydrazyl (dpph) scavenging activity of bread (triticum aestivum l.), durum (triticum turgidum ssp. durum desf.) wheat cultivars and einkorn (triticum monococcum ssp. monococcum) wheat populations collected from different provinces (bolu and kastamonu) of turkey. ferulic acid (148.67-764.04-lg/g), p-coumaric (5.06-54.09-lg/g), and total phenolic content (ranged 2.06-8.11-lmol gae/g) of einkorn populations were significantly higher than bread and durum wheat cultivars. results suggested the possibility of production of einkorn wheat populations, and hopefully cultivars rich in particular health beneficial component(s) may provide benefit to the consumers. in addition, higher phenolic content of einkorn may offer novel wheat genetic resources for the improvement of new wheat cultivars and the development of wheat-based functional foods. oxidative damage due to ischemia and acute kidney injury (aki) after coronary artery bypass graft (cabg) surgery are the leading complication during this process. in the kidney, ischemia/ reperfusion injury contributes to aki that is a clinical syndrome with rapid kidney dysfunction and high mortality rates. some animal and clinical studies have demonstrated an increase in serum and urinary neutrophil gelatinase-associated lipocalin (ngal) expression after renal ischemic injury.in this study, our aim was to investigate the relationship betwwen ngal and oxidative stress parameters due to ischemia caused by total perfusion time (tpt) in patients who have undergone on-pump cabg. materials and methods: the study was conducted in 30 patients who received on-pump cabg at university of istanbul, cerrahpasa medical faculty, department of cardiovascular surgery. blood samples were collected prior to surgery and after 2 hours following the termination of cardiac pulmonary bypass (cpb) . following centrifugation, serum samples were separeted and stored at -80 c until analysis. serum ngal, ima (ischemia modified alb€ umin), pco (protein carbonyl), nt (nitrotyrosine), lpd (lipid peroxide) levels were determined by elisa procedure. results and discussion: serum ngal, pco, nt levels in after 2 hours following cpb were significantly higher than the before surgery (p < 0.001, p < 0.001, p serum ngal levels in after 2 hours following cpb was found to have positive correlation with ima, pco, nt, and lpo levels (r = 0.59, p < 0.01; r = 0.77, p < 0.001; r = 0.68, p < 0.001; r = 0.77, p < 0.001 respectively). ngal levels were positively correlated with total perfusion time after cbp (r = 0.37, p < 0.05).the results of our study show that, increased ngal levels 2 hours after cpb were positively correlated with oxidative stress parameters and total perfusion time. investigation of the effects of thymoquinone against indomethacine induced gastric damage in rats introduction: incidence and high cost of acute stomach mucosa damages make this issue a very interesting issue for study. for this reason, it is aimed to investigate the effects of different dosage of thymoquinone (tq) against indomethacine induced gastric damage in rats. material and method: in our study, six groups of 36 wistrar male rats were used. groups are named as healthy, ind control, famotidine (40 mg/kg) and three different doses of tq (0.5, 1 and 2 mg/kg). while any treatment or drug administration will done on healthy group, model was generated to other groups by giving fam or tq with tap water via oral gavage. 5 min later, 25 mg/kg ind administrated to each rat. animals were sacrified about 6 hour later and stomach samples of each groups were collected for macroscopic study and gsh levels measurements. results: lower doses of tq is more effective and all tq groups exhibit reduced ulcer region with respect to the ind control group. gsh level of ind control group is lower than healthy group. the gsh level of tq, especially in lower doses, and fam groups statistically exhibit an increase in gsh level. conclusion: it is observed that ind induced gastric damage cause ulcer and increase in free radical. it is determined that lower doses of tq (0.5, 1 and 2 mg/kg) is also exhibit a protective effect on ind induced model. it is tought that quinone in tq structure have a strong redox feature and this feature clean up the free radicals caused by ind, it reduces the oxidative stress and protect the stomach from ulcer. anzer honey is the most famous honey in turkey with many endemic species flowers. the anzer plateau is located rize province of eastern black sea region. in this study, antioxidant and anti-hyaluronidase and anti-urease activities were investigated of the plateau bee pollen. the antioxidant capacity was determined by total phenolic content (tpc), total flavonoids contents (tfc), ferric reducing antioxidant power (frap) and 2,2diphenyl-1-picrylhydrazyl (dpph) radical scavenging activity. atherosclerosis is the leading cause of mortality worldwide, and as a chronic inflammatory disease, caused by a complex interplay between inflammatory and oxidative events.quercetin, a plant derived flavonoid and a well-known antioxidant, has shown great promises with regards to its protective effects against oxidative stress.however due to its physicochemical properties, the optimum pharmacokinetic behavior is a challenging issue.herein, we aimed to fabricate quercetin loaded solid lipid nanoparticle (quer-sln) to improve the bioavailability and therapeutics efficiency. furthermore the in-vitro capacity of quer-sln for ameliorating tnf-a induced oxidative stress in human endothelial vein cell (huvec) was evaluated. quer-slns were prepared by simple hot homogenizing method and characterized by means of drug loading (dl), encapsulation efficiency (ee), cytotoxicity, size, zeta potential and morphology. antioxidant activity of plain quercetin and quer-slns were then investigated using intracellular reactive oxygen species (ros) detection method (dcfh-da assay) by facs flowcytometryin conclusion, the results here showed superior control of oxidative stress by quercetin nanosystem as compared to plain quercetin. precirol based slns as a biocompatible/biodegradable lipid, may provide a novel drug delivery system for quercetin with improved beneficiary impact in atherosclerosis. objective: zinc is known as an antioxidant essential trace element. we aimed to evaluate the dose-dependent effects of zinc on the oxidant-antioxidant system in liver, kidney and brain tissues of rats and the histological alterations in the absence of oxidative stress (os). material and methods: thirty-nine female weighing about 150 gr wistar albino rats were divided into four experimental groups as ad libitum (al) diet (control), al diet + 3 mg/kg zn sulfate (low dose; group 1), al diet + 12 mg/kg zn sulfate (middle dose; group 2) and al diet + 25 mg/kg zn sulfate (high dose; group 3). zn sulfate solutions were administered 0.2 ml/day orally for 13 days and in day 14 rats were sacrificed and tissues were excised for detecting malondialdehyde (mda), advanced oxidation protein products (aopp), superoxide dismutase (sod), glutathione peroxidase (gsh-px), glutathione reductase (gr), and glutathione-s-transferase (gst) activities. histological evaluation was also performed to confirm the effects of zinc. results: in liver tissues aopp levels decreased in all groups receiving zinc as compared to the control group. liver mda levels were increased in group 1 and 3; sod and gsh-px levels were both increased while gst levels were decreased in all groups compared to control. gr levels were increased only in group 2. in kidney; aopp level was decreased only in group 1 and sod level was only decreased in group 2 as compared to control while gr levels were increased in all doses of zinc. in brain; aopp, gsh-px and gr levels were decreased in all groups receiving zinc as compared to control group. sod activity in brain tissues was increased by the administration of middle dose of zinc (group 2). gst level was decreased in only group 1 conclusions: the biochemical and histological findings of this study suggest that zinc has various effects on liver, kidney and brain tissues in the absence of os.key words: zinc, liver, kidney, brain introduction: this study aimed to investigate the effect of diabetes mellitus (dm) on oxidative stress and antioxidant capacity in humour aqueous (ha) and venous serum using total antioxidant capacity (tac) and total oxidative stress (tos) levels in serum and ha in cataract patients. materials and methods: in this study patients were divided into two groups. group 1 was composed of patients with type 2 dm and cataract and group 2 was composed of patients with cataracts who are not accompanied by dm and cataract patients who are not accompanied by systemic diseases. each group consisted of 20 patients, totally 40 patients were included in the study. the ha which was collected from the eyes at the beginning of the cataract surgery and venous blood serum collected from the same patients were analyzed. in both groups, ha and serum tac and tos levels were measured with elisa. results: : serum tac levels in the dm group were significantly lower than in the control group (p < 0.05). tos serum levels in dm group was statistically higher than the control group (p < 0.05). differences between tac and tos levels were not statistically significant when compared the two groups' ha results (p > 0.05). group 1, divided into two subgroups according to their hba1c levels, there was no statistically significant difference between the subgroups when hba1c levels were compared with the relationship between serum and ha's tac and tos levels (p > 0.05). there was not an association between the gender, age and the levels of tac-tos in both groups (p > 0.05). discussion and conclusion: presences of dm is the only risk factor for increase of oxidative stress and decrease of anti-oxidant capacity in patients without a systemic complication of dm and diabetic retinopathy. in our study, diabetic patients without retinopathy showed similar ha tos and tac levels to healthy individuals, this finding indicates that blood-aqueous barrier is protected in these patients. the effect of ferulic acid against testicular ischemia/reperfusion injury in rats u. sac ßik 1 , g. erbil 1 , z. c ß avdar 2 , c. ural 2 1 department of histology and embriyology, school of medicine, dokuz eyl€ ul university, izmir, 2 department of molecular medicine, health science of institute, dokuz eyl€ ul university, izmir, turkeytestis torsion is one of the urologic emergencies occurring frequently in neonatal and adolescent period. testis is sensitive to ischemia/reperfusion (i/r) injury and, therefore, ischemia and consecutive reperfusion cause an enhanced formation of reactive oxygen species (ros) that result in testicular cell damage and apoptosis. ferulic acid, known as an antioxsidant, is a phenolic acid found in seeds and leaves of the plants. we aimed to investigate potential protective effect of ferulic acid against testis i/r injury.thirty five wistar rats were randomly divided into 5 groups; control, ethyl alcohol, ischemia, i/r, i/r-ferulic acid groups. animals were exposed to 2 hours of ischemia followed by 2 hours of reperfusion. ferulic acid was administered (100 mg/ kg) before reperfusion intravenously. testicular cell damage was examined by h-e staining and pas. tunnel, active caspase-3, inos and mpo were evaluated by immunostaining. malondialdehyde (mda), glutathione (gsh) levels, glutathione peroxidase (gpx) and superoxide dismutase (sod) activities were assessed by biochemical methods.histological evaluation showed that ferulic acid pretreatment reduced significantly testicular cell damage and decreased tun-nel, caspase 3 positive cells; inos and also mpo expression. in addition, ferulic acid administration decreased significantly the mda levels increased by i/r. morever, ferulic acid increased significantly the sod activity levels, which was decreased by i/r. there were no statistically significant differences in the levels of gsh and gpx activity in all groups.the present results suggest that ferulic acid is a potentially beneficial agent in protecting testicular i/r. background: in heart failure (hf), angiotensin antagonists (aa), beta-blockers (bb), spironolactone, diuretics and acetylsalicylic acid are often used. top 3 pharmaceutical groups reduce mortality. on the increased oxidative stress (os) in patients with hf, it is known to have beneficial effects of certain groups of drugs. however, the net effect of these drugs in os is unknown. the aim of this study was to investigate the effects of drugs used in hf on os. materials and methods: 133 patients were included in the study. all of the patients had systolic heart failure and all of them were under treatment. drugs used by the patients were recorded. the levels of total antioxidant status (tos), total oxidant status (tas), the enzymatic activity of ceruloplasmin, paraoxonase-1 and arylestherase were measured according to erel's method. serum total thiol levels were measured with sh modified hu method and the lipid hydroperoxide levels were measured with the ferrous ion oxidation xylenol orange assay. the percentage tos / tas was determined as osi. results: in patients treated with acetylsalicylic acid (asa), spironolactone, beta blocker and furosemide, there were increased tos, decreased tas and osi (p < 0.05). in patients treated with angiotensin blockers, increased tas and looh, and decreased sh were found (p < 0.05). in patients treated with nitrates and ccb, tos and osi were found decreased. correlation analysis showed that increased tas correlated with the use of angiotensin blockers, asa, furosemide and beta blocker positively; and with the tos and osi level correlated with the use of spironolactone, asa spironolactone and furosemide (p < 0.05). conclusion: current medical agents that are being used in hf are effective in reducing os in hf patients. one of the effective mechanisms to reduce the mortality of some of these drugs may decrease os.key words: heart failure, drug use, oxidative stress p-09.04.4-119 across adjacent ring formed titanium phthalocyanine-mediated photodynamic therapy alters and degrades filamentous actin cytoskeleton and internal membranes photodynamic therapy (pdt) is widely accepted as a promising and minimally invasive treatment strategy due to its applicability on a wide range of cancer diseases. this clinically approved treatment method relies on the dramatic production of singlet oxygen and reactive oxygen species (ros) in target tissue to evoke apoptotic cell death [1] . we, therefore, focused on the intracellular ros accumulation, internal membrane degradation, filamentous actin cytoskeleton alteration and nucleus morphology changes induced by pdt-mediated across adjacent ring formed titanium phthalocyanine which was previously synthesized bis(ethane-1,1p-phenol-2,2-p-phenoxy) phthalocyaninatotitanium (iv).characterization of the synthesized metallophthalocyanine was accomplished by using uv-vis, ir, 1 h-nmr and maldi-tofmass spectroscopies. the dark and pdt-mediated activities of bare and phosphonolipids (max. 5%) charged titanium phthalocyanine (0.625, 1.25, 2.5, 5 and 10 lm) were determined on a549 human lung carcinoma and hacat human keratinocyte cell lines by using intracellular ros assay, dioc(6), tritc-phalloidin and dapi staining protocols. waltmann pdt 1200 l was used as the non-toxic light source at 100 j/cm 2 fluence and 150 mw/ cm 2 fluence rate. the experiments showed that pdt-mediated titanium phthalocyanine leads to significant and concentration dependent reactive oxygen species accumulation. moreover, internal membrane degradation, apoptotic bodies on nucleus and filamentous actin cytoskeleton alteration were observed. consequently, the activity mechanism of pdt-mediated titanium phthalocyanine seems to be in a tight relationship with ros accumulation-mediated internal membrane degradation, filamentous actin cytoskeleton alteration and apoptotic pathways activation. introduction: retinal vein occlusion (rvo) is a common retinal vascular disorder that can affect visual acuity and cause blindness in elder population. sulphur containing aminoacids such as cysteine (cys), cysteinylglycine, glutathione, homocysteine and c-glutamylcysteine are reported to be associated with the pathogenesis of rvo. thiols are organosulfur compounds that are formed of a carbon-bonded sulfhydryl group. sulphur containing aminoacids slightly contribute the composition of plasma thiol pool. thiols can undergo oxidation reaction via oxidants and form disulphide bonds our purpose is to research the relationship between a novel oxidative stress marker serum dynamic thioldisulphide homeostasis and retinal vein occlusion. materials and methods: 22 rvo patients and 22 controls were included in the study. native thiol, total thiol, disulphide levels are measured in the serum samples of rvo and control group by using an automated method described by erel et al. also disulphide/native thiol and disulphide/total thiol ratios were calculated. results: there were no significant difference between the rvo and control group in native thiol, total thiol, disulphide disulphide/native thiol and disulphide/total thiol ratios.(p > 0.05 for all) conclusion: our study is the first report evaluating the dynamic thiol-disulphide homeostasis in rvo patients by a newly developed method by erel et al. further large sample sized studies investigating the levels of sulphur containing aminoacids may additionally be planned to verify this study. purpose: the purpose of this study was to evaluate markers of systemic oxidative stress and antioxidant capacity in subjects with severity of osas. methods: a total of 106 osa patients were included in the study (18 controls, 14 with mild, 14 with moderate, and 60 with severe osa). patients were grouped according to apnea-hypopnea index (ahi) as mild, moderate and severe osa. patients with ahi<5 served as control group. known risk factors for oxidative stress, such as age, sex, obesity, smoking, hypelipidemia, and hypertension, were investigated as possible confounding factors. plasma arylesterase, total oxidative stress (tos), total antioxidant capacity (tac), total thiol, catalase (cat) levels were measured for all patients. results: the mean age was 52.49 ae 12.9 years and 40.6% (43/ 106) of the study population was female. plasma arylesterase, tos, tac, total thiol, and cat plasma values were not different between mild, moderate, severe osa groups and controls (p > 0.05). catalase levels were significantly lower in women patients with severe osa compared to healthy women controls (p < 0.05). there was a negative correlation between ahi and serum total thiol levels (r = à0.289, p < 0.05) in severe osa groups. conclusionthe present prospective study provides evidence that osa might be associated with decreased antioxidant burden possibly via catalase way. results: the sera 8-ohdg, mda and il-6 were significantly higher in diabetic group than control group (p < 0.05). although there was a notable positive correlation between mda and 8-ohdg, there was no a relationship between 8-ohdg or mda with il-6. discussion: in agreement with previous studies our data illustrated that high levels of oxidative stress is associated with increased production of oxidized lipids and nucleobases in diabetic patients compared to control group. also enhanced proinflammatory cytokine, il-6, induced inflammmation in these patients.conclusion: oxidative stress and inflammation play pivotal roles in the development of diabetes and can cause major complications in dm. so we suggest that early detection of these measurable indicatores can help to diagnosis the severity or presence of some complication in diabet. the effect of quercetin on erythrocyte glucose-6-phosphate dehydrogenase enzyme activity in ethanol treated rats in this study, we aimed to evaluate the effects of ethanol on erythrocyte (g-6-p-d) enzyme activity and the effects of quercetin on erythrocyte g-6-p-d activity in the recovery of the effects of ethanol.rats were randomly divided into four groups. the control group (n = 6) received physiological saline. the quercetin group (n = 7) received quercetin (120 mg/kg/ day) via i.g. route the alcohol group (n = 7) received ethanol (80% v/v, 1 ml/day) via i.g. route. the alcohol + quercetin group (n = 5) received 1 ml of ethanol (80% v/v) 2 hours after quercetin treatment (120 mg/ kg/day). experimental procedures were peformed for 30 days. erythrocyte g-6-p-d activity was found to be higher in the quercetin group than those in the alcohol group (p < 0.001). in the alcohol group, the erythrocyte g-6-p-d activity was found to be significantly decreased than those in the control group (p < 0.001). statistically significant differences were observed in erythrocyte g-6-p-d activity between the alcohol group and the alcohol + quercetin group (p < 0.001).as a conclusion, our results demonstrate that ethanol decreased erythrocyte g6pd activity and quercetin was found to be beneficial in the prevention of toxic effect raised by ethanol.key words: erythrocyte, ethanol, g6pd, oxidative stress, quercetin p-09.04.4-126 effects of the sulphasalazine to the cerebral hypoxia reperfusion injury in rat background: cerebral ischemia/ reperfusion (i/r) injury is still a difficult process to treat and rehabilitate today. this study was designed to investigate beneficial effects of sulfasalazine in cerebral i/r injury in rat. methods: except control group (n = 5), 20 wistar albino rats were divided into four groups for acute and chronic stage investigation of i/r injury, and temporary aneurysm clips were attempted to both internal carotid arteries for duration of 30 minutes. four hours later, except control, sham-a, sham-c groups, 40 mg/kg once a day sulfasalazine was administered to animals, orally. animals were sacrified and then necrotic neuronal cells of hippocampal ca1, ca2, and ca3 region, and cortical necrotic neurons, perivascular edema, pyknotic neuronal cells, irregularities of intercellular organization (iio) were counted and scaled histopathologically. tissue il-1b, il-6, malonyldialdehyde (mda), myeloperoxidation (mpo), no, and tnf-a levels were measured by using elisa, too. results: sulfasalazine could reduce perivascular edema, iio, cortical and hippocampal neuronal cell death in both stages. it could decreased mda in acute stage, but not reduce il-1b, il-6, mpo, no, and tnfa levels. it could increased il-1b levels in chronic stage but not affect to il-6, mpo, mda, no, tnf-a levels.conclusion: sulfasalazine could improve histopathological architecture of hypoxic tissue in both stages of i/r injury. it could inhibit lipid peroxidation cascades in acute stage but not affect to tissue mpo, no, il-6, and tnf-a levels in any stage in rat. these results suggested that therapeutic mechanisms of sulfasalazine should be investigated by using more specific laboratory methods in future studies.key words: antiinflamatory, cerebral hypoxia reperfusion injury, sulfasalazine, stroke. camel and horse milk xanthine oxidase (xo) was found to catalyze the reduction of nitrate and nitrite to nitric oxide (no) under aerobic condition. to date, mammalian nitrate reductase (nar) and nitrite reductase (nir) have not been identified. no, a gas, is found to control a seemingly, limitless range of functions in animals.one assay was used to determine nar and nir activities of milk xo: (1) nitrite formation from nitrate by nar, and (2) nitrite utilization by nir. nitrite concentrations were determined by using sulfanylamide and n-(1-naphtyl)-ethylenediamine, which form red color measured at 485 nm.these activities of the milk xo require nadh as a physiological electron donor. high xo, nar and nir activities are detected only after heat treatment (80°c, 5 min) of the fresh milk in the presence of molybdate. in both camel and horse milk nar activity of xo was almost two times higher than its nir activity. it is well known that xo can be reversibly converted from the dehydrogenase form to the oxidase through the oxidation of sulfhydryl groups. cysteine and, to a lesser extent, glutathione increased nir activity of milk xo but not its nar activity. the mechanism of this increase of nir activity remains unclear and is currently under study. substitution of tungsten for molybdenum under above conditions gave no detectable nar and nir activity of milk xo. the molybdenum site-directed inhibitor, tungsten inhibited in a dose-dependent manner. therefore, nitrate and nitrite are clear to interact with mo center of xo.camel and horse milk are traditional drinks in central asia and kazakhstan. therefore, it is very important that xo provide a mechanism for generation of no in camel and horse milk where nitric oxide synthase, no producing enzyme, does not exist. p-09.04.4-128 the influence of phytomedicine on metabolic processes of white rats undergone to ionized radiation the study of peroxide lipids oxidation (plo) process is used as one of stability parameters of organism's changes and as a key mechanism for understanding of adaptation reactions and of pathogenesis of different diseases. it's determined by high biological activity of products which are formed in the plo reactions, in this relation lipids with high contents of fat acids play important role. to investigate the influence of phytomedicine eminium regelii on the metabolic processes (peroxide lipids oxidation) of white rats' organism in conditions of ionized radiation.the animals were exposed to ionizing radiation (gamma-radiation 60 co) on the radiotherapeutic equipment teragam in a dose of 6 gy and received phytomedicine eminium regelii in a dose of 2.5 mg/kg orally within 14 days following the ionizing radiation exposure. gamma-rays caused the increase of lipid peroxidation (lpo) primary (dc) and secondary products' (mda) concentrations in spleen, liver, thymus and adrenal glands.treatment by phytomedicine resulted in contents of dc decreased in 3 times in spleen, in 7 times in thymus, in 5 times in adrenal glands, in liver in 4 times, in lymph nodes of small intestine it in 3 times. mda decreased in liver up to 6 and 3 times, in spleen in 3.2 times, in thymus in 9 times, in liver in 6 times, in adrenal glands in 12 time, no changes in lymph nodes of small intestine.the effect of phytomedicine treatment of organisms exposed to sublethal dozes of gamma-radiation results in the lpo primary and secondary products concentrations decrease in spleen, liver, thymus and adrenal glands. p-09.04.4-131 evaluation of serum levels of ischemia modified albumin (ima) in bipolar disorder patients k. € unal 1 , c. topc ßuoglu 2 , m. cingi 3 1 clinic of biochemistry, ankara polatli duatepe public hospital, ankara, 2 clinic of biochemistry, ankara numune training and research hospital, ankara, 3 clinic of psychiatry, ankara numune training and research hospital, ankara, turkey introduction: bipolar disorder is one of the most debilitating psychiatric disorders characterized by disruptive episodes of mania/hypomania and depression. considering the complex role of biological and environmental factors in the etiology of affective disorders; recent studies have focused on oxidative stress, which may damage nerve cell components and take part in pathophysiology. aim of our study is to contribute these data about oxidative stress in bipolar disorder, by detecting ischemia modified albumin (ima) levels of bipolar disorder patients in remission and also by comparing these results with healthy controls. methods: study population consisted of 35 patients meeting the diagnostic and statistical manual of mental disorders, fifth edition (dsm-5) criteria for bipolar disorder i. 36 healthy subjects were included as control group (hc). serum ischemia modified albumin (ima) levels of all participants were determined. results: statistical analysis on serum ischemia modified albumin (ima) levels did not show any significant difference between bipolar disorder patients in remission and healthy controls.conclusion: studies on oxidative stress in bipolar disorder have reached controversial results up till now. in this study, no statistically significant difference was detected between oxidative parameters of bipolar disorder patients in remission and healthy controls. in order to evaluate oxidative stress in bipolar disorder comprehensively, further studies are needed. keywords: bipolar disorder, ischemia modified albumin (ima), oxidative stress p-09.04.4-133 xanthine oxidase and adenosine deaminase activity in patients with familial mediterranean fever (fmf) objective: fmf is an autosomal recessive dissease which is characterized by recurrent fever and inflammation of serous membranes. in this study we measured serum adenosine deaminase (ada) and xanthine oxidase (xo) levels in fmf cases. method: serum ada levels were measured with a sensitive colorimetric method described by giusti and xo levels were analysed by the method of worthington in 30 fmf patients and 30 healthy controls. results: there was a significant difference in xo and ada levels between controls and cases. ada and xo levels were higher in patents with fmf. humic acid (ha) is a natural product which is forming during decomposition of organic matter in humus. in recent years, there are some resarches on the medical use of ha. the present study was undertaken in order to evaluate the anticancer properties of the ha using a prostate cancer and osteosarcome cell lines pc-3, sjsa1 as an in vitro model system.ha was purchased from sigma-aldrich. the cells were maintained in dmem medium supplemented with 10% heat-inactivated fbs and 1% penicillin/streptomycin. cells were grown in petri dishes in a humidified atmosphere containing at 37•c. five different concentrations (100 ug/ml, 50 ug/ml, 25 ug/ml, 10 ug/ ml, 5 ug/ml) were prepared using a stock solution of ha. we measured cell proliferation and migration to understand of progression effects of ha in pc-3 and sjsa1 cell lines in vitro.according to our results, ha treatment caused cytotoxicity and induced cell death in vitro in pc-3 cells with an ic50 value of 67.9 lg/ml. contrary to this, ha induced proliferation of sjsa1 cells in dose dependent manner. ha demonstrated the highest proliferatif activity against sjsa1 cells with an ic50 value of 100 > lg/ml. on the other hand, cell migration was reduced in pc-3 cell line and interestingly, migration was accelerated in sjsa1 cell line.our study may provide new insights into the regulatory effect of ha in cancer, but further studies are needed to clarify the role of ha in cancer pathogenesis. the febs journal 283 (suppl. 1) (2016) key: cord-257167-rz4r5sj7 authors: nan title: abstracts for the 29th annual meeting of the japan neuroscience society (neuroscience2006) date: 2006-12-31 journal: neuroscience research doi: 10.1016/j.neures.2006.04.004 sha: doc_id: 257167 cord_uid: rz4r5sj7 nan the brain basis of conscious experience is one of the great unsolved mysteries of science. how can a material object became aware of the world around it and of its very own awareness? many scholars think this question is unanswerable. new approaches to this age-old mind-body problem have recently been encouraged by the development of pet and mri and the powerful tools of modern neuroscience. we are now witnessing the explosive growth of a new field, called cognitive neuroscience which focuses on behavior and uses classical reaction-time paradigms together with new computer-based technology. a parallel approach is to cross-correlate formal aspects of conscious experience as the brain spontaneously pursues its regular trajectory through the objectively defined states of waking nrem and rem sleep. at the level of the brain it is possible to record pet and mri and by using an animal model to analyse cellular and molecular mechanisms. the advantage of this approach, which i call the conscious state paradigm, is that it, is quantitative, integrative, and holistic (in the rigorous sense of that word). the brain basis of activation (a) input-output gaining (i) and modulation (m) can now be described in relation to the changes in consciousness associated with them. the data can be used to create a three-dimensional model (aim) which describes the brain-mind trajectory in its state space. neural circuits in many parts of the developing nervous system exhibit highly rhythmic episodes of electrical activity. such activity has generally been considered to fine tune connections that are initially made by axons responding to a complex array of molecular guidance cues. however, chick and mouse spinal cords exhibit activity at early stages as motoneurons are still migrating and beginning to extend their axons. modest alterations in the frequency of such episodes produced changes in the expression of several guidance/recognition molecules and caused motor axon pathfinding errors. interestingly the type of pathfinding decision, the binary dorsal-ventral choice or the subsequent pool specific fasciculation and projection of axons to specific muscles, was differentially affected by decreasing or increasing the frequency respectively. thus, activity generated by developing circuits interacts at early stages with the molecular signaling pathways that govern the formation of precise circuits and any drugs that alter this activity could adversely affect circuit formation. supported by nih grant ns19640. sl2-1-1 frontal cortex and higher-order motor control: preferential use of multiple premotor and prefrontal areas dependent on behavioral context jun tanji brain science research center, tamagawa university, machida, tokyo, japan in the cerebral cortex of primates, there exist a number of motor areas rostral to the primary motor area and caudal to the prefrontal cortex. although each area has been defined based on anatomical and physiological criteria, functional roles played by each of them have been the matter of much debate. in fact, in a recent trend of research reports, it is popular to stress commonalities rather than specificities in the use of multiple cortical areas in motor control. for instance, the involvement of the primary motor cortex in cognitive aspects of motor behavior has been an eye-catching subject of recent reports. the involvement of the prefrontal cortex in movement planning has also been inferred. up to a certain extent, it is true that the use of different areas have some factors in common. however, it is a mistake to ignore profound differences in the use of each area, depending on specific aspects of motor behavior. in this lecture, i will describe such differences that could be clarified only when neural activities are examined properly, by studies designed to reveal individual aspects of functional significance of each area. sl3-1-1 neuronal functions and molecular motor, kinesin superfamily proteins, kifs: from transport of synaptic proteins and mrnas, to brain wiring, neuronal survival and higher brain function nobutaka hirokawa department of cell biology and anatomy, graduate school of medicine, university of tokyo, japan the intracellular transports are fundamental for neuronal morphogenesis, functioning and survival. to elucidate this mechanism we have identified and characterized kinesin superfamily proteins, kifs, using molecular cell biology, molecular genetics, biophysics, and structural biology. kifs play essential roles on neuronal function and survival by transporting synaptic vesicle precursors (kif1a/kif1bbeta), nmda type(kif17) and ampa type(kif5s) glutamate receptors and mrnas(kif5s) such as camkii ␤ mrna and arc mrna with a large rna-transporting protein complex containing at least 42 rna related proteins such as hn rnp-u, staufen, and fmrps. kif2a is fundamental for correct brain wiring by suppressing elongation of axon collaterals through depolymerizing microtubules in growth cones. kif3 suppresses tumorigenesis by transporting ncadherin/beta catenin containing vesicles from golgi to plasma membrane in the neuroepithelium. kif4 controls the activity-dependent survival of postmitotic neurons by regulating parp-1 activity in brain development. thus, kifs play significant roles not only on various neuronal functions but also on brain wiring, development and higher brain functions such as memory and learning. tsukahara award 2-1 what we can learn from the functional recovery after brain injury tadashi isa national institute for physiological sciences, okazaki, japan it is believed that when a part of a neuronal system is damaged, some of the lost functions can be taken over by residual systems through training. such concept is considered as the basis of neurorehabilitation, however, the mechanism of the "take-over" is not well understood. in this talk, i will present our recent progress in two lines of studies using non-human primate models related to this issue. the first topic is on the recovery of dexterous finger movements after lesion of the lateral corticospinal tract at the cervical spinal cord. after the virtually complete lesion, relatively independent finger movements can be recovered in 1-3 months. we have found that bilateral primary motor and ventral premotor cortices are involved at various stages of the recovery process by combining the pet imaging and reversible local inactivation technique. in the second, i will talk about the visuo-motor processing in monkeys with unilateral lesion of the primary visual cortex (v1). after complete ablation of v1, the monkeys recover performance of visually guided saccades toward the blind field in 2 months. saccades to the blind field have low sensitivity and less accurate. however, the monkeys can perform surprisingly cognitively demanding tasks in the blind field. i will discuss on our hypothesis on the bottom-up and top-down control of learning during recovery. takuji iwasato riken brain science center (bsi), wako-shi, saitama, japan in the rodent primary somatosensory (barrel) cortex, the configuration of the whiskers on the face is topographically represented as "barrels", discrete modules of layer iv neurons and thalamocortical afferent (tca) terminals. while barrel formation is an important model of the establishment of patterned topographic connections between the sensory periphery and the brain, the molecular mechanisms underlying this process are poorly understood. we developed and applied mouse reverse genetic technologies to examine these molecular mechanisms. we have focused on the nmda-type glutamate receptor (nmdar) and calcium-stimulated adenylyl cyclases, as nmdar-and camp-cascades are central to various types of neuronal plasticity, both in adulthood and during development. series of global and region-specific knockout mice have revealed the roles of these molecules in the patterning of barrel cortex and differentiated the specific mechanisms at presynaptic tca terminals compared to those at postsynaptic cortical neurons. research funds: presto (jst), kakenhi 17023055, kakenhi 15029261 sy1-1-01-2 distinct roles of two 7-pass transmembrane cadherins in neurite growth control tadashi uemura 1,6 , yasuyuki shima 1,6 , keisuke sehara 2 , manabu nakayama 3 , shinya kawaguchi 4 , mikio hoshino 5 , yoichi nabeshima 5 , tomoo hirano 4,6 1 graduate school of biostudies, kyoto university, kyoto, japan; 2 school of science, japan; 3 kazusa dna research center at chiba, japan; 4 graduate school of science, japan; 5 graduate school of medicine, japan; 6 crest, jst, japan drosophila flamingo (fmi) and mammalian celsr1-3, which are 7pass transmembrane cadherins, have been considered to mediate the regulation of neuron contact-dependent neurite growth. we show that mammalian 7-pass transmembrane cadherins celsr2 and celsr3 are activated by their homophilic interactions and regulate neurite growth in a distinct manner. both gene-silencing and co-culture assay showed that celsr2 enhanced neurite growth whereas celsr3 suppressed it. our result suggested that celsr2 had a stronger activity as g-protein coupled receptor than celsr3 did, most likely due to a difference of a single amino acid residue in the transmembrane domain, and this functional difference resulted in distinct effects in neurite growth regulation. thus, neuron-neuron interactions modulate neurite growth differentially through this couple of 7-pass transmembrane cadherins. masatoshi takeichi riken center for developmental biology, kobe, japan for synapse formation, axons need to recognize their specific partners, and subsequently stabilize their contacts. while a number of cell surface molecules should be involved in such processes, cadherin adhesion molecules play a role. when cadherin activities are blocked, synaptic contacts become destabilized in cultured neurons. this is also the case in vivo; e.g., in the neural retina whose cadherin activities are impaired without perturbing their overall architecture, a certain class of synaptic contacts does not normally form. another series of our study demonstrate that the cadherins cooperate with nectins, a subfamily of ig-domain molecules, for establishment of axon-dendritic contacts: in early hippocampal pyramidal neurons, nectin-1 is preferentially localized in axons; and nectin-3, in both axons and dendrites. we present evidence that the heterophilic binding between axonal nectin-1 and dendritic nectin-3 is important for facilitating the axon-dendritic attachment; and cadherins seem to be required to stabilize the nectin-initiated contacts. thus, multiple classes of adhesion molecules work together to ensure the correct linking between axons and dendrites. hitoshi sakano department of biophysics and biochemistry, university of tokyo, tokyo, japan we have studied how the olfactory sensory neurons (osns) expressing the same odorant receptor (or) gene converge their axons to a specific set of glomeruli in the olfactory bulb (ob). retrogradestaining of osn axons indicated that the dorsal/ventral (d/v) arrangement of glomeruli in the ob is correlated with the expression areas of corresponding ors along the dorsomedial/ventrolateral axis in the oe. in contrast, the anterior/posterior (a/p) arrangement of glomeruli appears to be independent of the epithelial locations of osns and more dependent on the expressed ors. it was found that g proteinmediated camp signals regulate the positioning of glomeruli along the a/p axis in the ob. we also found that multiple sets of cell adhesion molecules, e.g., ephrin-as and eph-as, are expressed in a complementary manner, whose transcription levels are uniquely correlated with the expressing or species. we propose that differential levels of repulsive/adhesive interactions of axon termini may regulate the sorting of like-axons during the process of osn projection. research funds: crest, jst, kakenhi 14104026 sy1-1-01-5 lamina-restricted guidance of hippocampal mossy fibers hajime fujisawa 1 , fumikazu suto 2 1 nagoya university, nagoya, japan; 2 institute of genetics, mishima, japan axons from different sources terminate at particular dendritic segments of target neurons in a laminal fashion. one important issue to be addressed is how individual axons are instructed to invade and arborize in particular laminae. projection of hippocampal mossy fibers is one of good experimental models to analyze molecular mechanisms that govern lamina-restricted termination of axons, because the fibers project to the proximal dendritic segment of ca3 pyramidal cells. we here report the following three mechanisms that provide lamina-restricted projection of mossy fibers. first, a neural repellent sema6a is expressed in ca3 pyramidal cells and principally suppresses invasion of mossy fibers to ca3. second, the repulsive signal of sema6a is mediated by plexin-a4 expressing in mossy fibers. third, the repulsive activities of sema6a are attenuated by plexin-a2 in the proximal dendritic segments of ca3 pyramidal cells, resulting in the segments permissive for mossy fibers to invade. over the course of development, children become increasingly able to control their thoughts and actions (i.e., cognitive control). the term cognitive control is an umbrella term for a set of putative control processes. these control processes may reach adult levels at different rates, depending on the rate of functional development of the specific brain structures involved. the structure most closely associated with cognitive control is prefrontal cortex (pfc). pfc is composed of what are believed to be functionally distinct subregions, including ventrolateral, dorsolateral, rostrolateral, and medial pfc. i will discuss the control processes associated with each of these regions, and how the functionality of these regions differs between school-aged children, adolescents, and young adults. three fmri studies will be presented, focusing on (1) working memory maintenance and manipulation, (2) rule representation and task-switching, and (3) relational reasoning. based on these data, i will discuss some general points about neurodevelopment changes in cognitive control, and outline the approach that our laboratory has taken in our developmental cognitive neuroscientific research. sy1-1-09-5 towards manipulative neuroscience based on non-invasive brain decoding in atr computational neuroscience laboratories, we proposed several computational models such as cerebellar internal models, mosaic, modular and hierarchical reinforcement-learning model. some of these models can quantitatively reproduce subject behaviors given sensory inputs and reward and action sequences which subjects received and generated. these computational models possess putative information representation such as error signals for internal models, action stimulus dependent reward prediction, and they can be used as explanatory variables in neuroimaging and neurophysiology experiments. we named this approach as computationalmodel-based neuroimaging, as well as computational-model-based neurophysiology. this new approach is very attractive and appealing since this is probably the only method with which we can explore neural representations remote from either sensory or motor interfaces. but, sometimes limitation of mere temporal correlation between the theory and data became so apparent, and we started to develop a new paradigm 'manipulative neuroscience' where physical causality is guaranteed. research funds: nict, karc sy1-1-09-6 neural mechanisms in williams syndrome-insights from neuroimaging andreas meyer-lindenberg nimh, bethesda, md, usa williams syndrome (ws), a rare disorder caused by hemizygous microdeletion of −25 genes on chromosome 7q11.23, has long intrigued neuroscientists with its unique profile of striking behavioural abnormalities, such as hypersociability, combined with a differential impact on cognitive functions, with some types of abilities only mildly affected while others are severely impaired. ws, thus, raises fundamental questions about the neural mechanisms of social behaviour, the modularity of mind, and brain plasticity in development, and provides a privileged setting to understand genetic influences on complex brain function in a bottom-uph way. recent months have seen dramatic advances in uncovering the functional and structural neural substrates of ws and a beginning understanding of how these are related to dissociable genetic contributions characterized both in special participant populations and animal models. we will review neuroimaging work indicating abnormal function and structure in subsystems of visual processing, long term memory, and emotional regulation and social cognition, and discuss advances in relating them to the underlying molecular biology of this unique syndrome. daisuke yamamoto tohoku university graduate school of life sciences, japan the fruitless locus of drosophila was originally recognized by its mutants, the males of which preferentially court males rather than females. the fruitless primary transcript is subject to sexually dimorphic splicing mediated by transformer, and encodes a group of proteins that are putative transcription factors of the btb-zn finger protein family. the male-specific fruitless protein is expressed in small groups of cns neurons of males, but not of females. fruitless masculinizes these neurons thereby establishing the neural substrates for male-typical behavior. by experiments that label individually the neurons that express fruitless, we have identified a subset of brain interneurons that display marked sexual dimorphism in their number and projection pattern. fruitless supports the development of those neurons with male-specific dendritic fields, which are programmed to die during development in females as a result of the absence of fruitless. thus, the fruitless protein expression can produce a male-specific neural circuit likely used for heterosexual courtship by preventing cell death in identifiable neurons. hitoshi okamoto riken brain science institute, wako, saitama, japan the emotional behavior depends on the evolutionarily most conserved neural circuits. especially the fear behaviors involve the basal telencephalic nuclei such as the amygdala and the nucleus accumbens. thanks to the progress in understanding of the telencephalic development among different species, we can determine the correspondence of the parts between the teleost and mammalian telencephalons. with these in mind, we initiated the characterization of the emotional neural circuits in the zebrafish brain which are amenable to various modern technology. we already reported the asymmetric axonal projection from the left and right habenulae which act as the relay station to conduct the emotional information from the telencephalon to the monoaminergic neurons in the midbrain and the hindbrain. to investigate whether such asymmetric neural circuits cause the laterality for emotional behaviors, we are now in the process of establishing the paradigms for combining the behavioral assay with genetic manipulations to control the activity of the emotional neural circuit in zebrafish. research funds: kakenhi (17023501) sy1-1-17-3 a molecular biological approach for songbirds to study learned vocal communication kazuhiro wada, erich jarvis duke university, usa songbirds possess one of the most accessible neural systems for the study of brain mechanisms of behavior, particularly that for learned vocal communication. however, neuroethological studies in songbirds have been limited by the lack of high-throughput molecular resources and gene manipulation tools. to overcome this limitation, we generated a resource of full-length cdnas for gene expression analyses and functional gene manipulation in songbirds. we constructed total 21 full-length cdna libraries from brains in different behavioral and developmental conditions. with these cdnas, we created a novel database and 18 k songbird cdna array. we used the arrays to reveal a set of 33 genes regulated by singing behavior. their molecular functions spanned most cellular and molecular categories, including signal transduction, structural, and synaptically released molecules. with the full-length cdnas, we were able to express proteins of singing-regulated genes in targeted brain area, using a lentiviral system. this resource now opens to more thoroughly study molecular neuroethological mechanisms of behavior. research funds: uehara memorial fellowship to k.w. and nih grant to e.j. -1-17-4 stepping pattern learning using mice: histochemical identification of activated neuronal circuits takashi kitsukawa graduate school of frontier biosciences, osaka university, osaka, japan identifying brain areas and neuronal circuits activated in a behavior is a critical step in understanding how the brain works in that behavior. also, identifying neuronal types involved in a behavior is a key step toward connecting behavioral approaches with molecular and genetical approaches. an efficient method of clarifying neuronal types activated by behavior is histochemical identification of neuronal types combined with c-fos staining. i would like to introduce our work as an example of using this method. in order to understand the neural processing involved in sequential motor skill learning we built a wheel running system in which a mouse learns sequential stepping patterns. we double-stained brain sections from mice which performed this task with c-fos and a neuronal marker such as enkephalin, substance p or nitric oxide synthase, each of which denotes a particular neuronal type. our results indicate that particular types of stiriatal neurons are activated during this learning, suggesting that cortico-striatal circuits are involved. synaptic plasticity that is dependent on precise timing of spikes between pre-and postsynaptic neurons plays important role in development and plasticity of brain functions. such spike-timingdependent plasticity (stdp) has attracted wide attentions because of its high computational power and physiologically plausible induction. we previously demonstrated that long-term potentiation was closely associated with structural plasticity of dendritic spines. however, how stdp is associated with structural changes has not been elucidated. we here report that paired two-photon uncaging of a caged-glutamate compound at a single spine and postsynaptic spike of whole-cell clamped neuron rapidly induced long-lasting bidirectional structural plasticity of spines in hippocampal ca1 pyramidal neurons. our results indicate that stdp is intimately associated with bidirectional structural plasticity at the level of single spines. research funds: kakenhi 17680033 sy1-2-02-4 role of camkii as a structural protein that stabilizes actin cytoskeleton in dendritic spines kenichi okamoto, radhakrishnan narayanan, yasunori hayashi massachusetts institute of technology, usa actin serves as a major cytoskeleton which maintains spine structure and exists in a equilibrium between f-actin and g-actin. tetanic stimulation causes a persistent shift of actin equilibrium towards f-actin which enlarges dendritic spines. but the mechanisms which maintain these changes remain elusive. we propose that camkii ␤ acts as an actin stabilizing molecule to maintain spine structure. camkii ␤ is not only an abundant f-actin binding protein, it can also make oligomers. we found that camkii ␤ oligomer crosslinks f-actin and stabilizes actin depolymerization kinetics. in spines, camkii ␤ oligomer slows down actin dynamics and camkii ␤ is enriched in spines by actin polymerization. the suppression of endogenous camkii ␤ alters spine shape to filopodia-like structures. these experiments suggest that camkii ␤ plays a role as a major stabilizer of the actin cytoskeleton to maintain spine structure. we also found that camkii ␤ detaches from f-actin in an activity dependent manner. we will discuss how camkii ␤ maintains actin equilibrium in activity dependent dendritic plasticity. ryohei yasuda duke university medical center, usa the small gtpase protein ras plays central roles in calcium signaling important for many forms of synaptic plasticity and regulation of neuronal excitability. using 2-photon fluorescence lifetime imaging microscopy in combination with a fret-based ras activity sensor, we visualized the activity of ras signaling with high spatiotemporal resolution. our studies indicate that calcium entry due to action potentials causes ras to activate in a supra-linear manner (yasuda et al., 2006) . furthermore, in response to single spine stimulation using 2-photon glutamate uncaging, ras activation initially occurs at the stimulated spine, subsequently spreading into its parent dendrites and nearby spines. these results suggest that nonlinear filtering by ras regulators as well as the spatial spreading of ras and ras regulators shape spatiotemporal patterns of ras signaling. hiroshi shibasaki takeda general hospital, kyoto, japan involuntary movements are unintended, generalized or focal, movements of abnormal nature, and include tremor, myoclonus, dystonia, chorea/ballism, athetosis and dyskinesia. myoclonus is characterized by abrupt, shock-like movements caused by brief muscle contraction (positive myoclonus) or abrupt cessation of on-going muscle contraction (negative myoclonus), or their combination. depending on the estimated origin, it is classified into cortical, brain stem, and spinal myoclonus. cortical myoclonus is short in duration (50 ms). by back averaging eeg or meg time-locked to spontaneous myoclonus, a cortical activity is demonstrated in the corresponding area of the contralateral primary motor cortex immediately preceding the myoclonus (by 20 ms for hand). it is mediated by fact-conducting corticospinal pathway. cortical myoclonus is often stimulus-sensitive (cortical reflex myoclonus), showing extremely enhanced cortical responses to somatosensory or visual stimulus, and enhanced longloop transcortical reflexes. these findings, together with transcranial magnetic stimulation, suggest increased excitability of sensorimotor cortex in cortical myoclonus. mark hallett ninds, bethesda, md, usa there have been many recent advances in the understanding of the physiology of focal dystonia. three main avenues of research have shown abnormalities in cortical inhibition, sensory processing including sensorimotor integration, and plasticity. this lecture will emphasize the abnormal inhibition. abnormal inhibition appears to be the most direct cause of unwanted muscle contractions that make up both the involuntary spasms and the overflow movements also seen in this condition. a loss of inhibition is seen in spinal and brainstem reflexes, but these changes are likely secondary to cortical abnormalities. cortical inhibition is also diminished as demonstrated most clearly with transcranial magnetic stimulation. gaba content may be decreased as shown with magnetic resonance spectroscopy. a particular type of defective inhibition is surround inhibition, the inhibition that normally operates to sharpen fine skilled movements. studies are now in progress to determine the synaptic mechanisms of surround inhibition and how this becomes abnormal in dystonia. understanding about inhibition in dystonia has led to some new treatments including some non-invasive cortical stimulation methods. research funds: nih intramural program sy1-2-10-3 basal ganglia-cortical systems reinforcing tonic motor activity in health and disease peter brown sobell department, institute of neurology, uk the synchronisation of neuronal activity in the beta frequency (∼20 hz) band has been noted in healthy primates, including humans, at both striatal, putamenal and cortical levels. it is most obvious in the motor cortex during tonic motor activity and is suppressed by voluntary movement. in this talk i will develop the idea that beta band synchronisation in the basal ganglia-cortical loop promotes tonic/postural contraction at the expense of new movements. thus, spontaneous phasic increases in beta activity in healthy subjects can be shown to be associated with a slowing of voluntary movements and a reinforcement of transcortical stretch reflexes. beta synchrony is also greatly exaggerated in untreated parkinson disease, where it may bias against new movement and contribute to bradykinesia and rigidity. excessive dopaminergic stimulation, either during treatment for parkinson disease, or in conditions such as dystonia, may overly suppress beta activity in bg-cortical loops leading to excessive movement. recordings of local field potentials in the basal ganglia of patients with movement disorders will be described that support this schema. research funds: mrc sy1-2-10-4 coding of reward value of actions and valuebased action selection in the basal ganglia a damage of the nigrostriate dopamine system results in severe impairments of voluntary movements as well as involuntary behavioral states like rigidity, akinesia and tremor as typically observed in parkinson disease. recent studies revealed that long-term potentiation of corticostriatal synaptic transmission occurs dopaminedependent manner, and that neuronal firing related to external stimuli and body movements are modulated by whether the stimuli and movements are associated with reward or not. we recorded striatal neurons of monkeys who chose between left-and right-handle-turns based on the estimated reward probabilities of the actions. during a delay period before the choices, activity of more than one-third of striatal projection neurons was selective to the values of one of the two actions. during handle-turns, another subset of neurons was activated. these results suggest representation of action values in the striatum, which can guide action selection in the basal ganglia circuit. roles of the basal ganglia circuit in voluntary and involuntary action selection will be discussed. in vivo reporter gene imaging is expected to be a powerful tool in gene and cell therapy monitoring. we designed a new pet reporter gene system with f-18 fluoroestradiol (fes) and human estrogen receptor ligand binding domain (herl), which would work in various tissues with little physiological effect. we have been evaluating its potential in gene therapy monitoring constructing a plasmid co-expressing thymidine phosphorylase (htp), a factor works for revascularization, as therapeutic gene and herl. cos7 cells transfected with the plasmid expressed the both proteins and, when the plasmid was in vivo electroporated into mouse calf muscle, the electroporated muscle accumulated significantly higher amount of fes that the control side. this system was successfully applied to es cell transplantation monitoring also. inducible herl expression system was stably transfected into mouse es cells and viable es cells could be detected in vivo using fes. these data support the prospect that our in vivo reporter gene system would be useful in gene/cell transplantation therapy monitoring. tetsuya suhara molecular imaging center, national institute of radiological sciences, chiba, japan the molecular imaging using positron emission tomography enables to visualize various brain molecules with radio labeled ligand. neurons and glias express various receptors and transporters and those can be a specific target of the imaging. the functions of those molecules can be examined in various types of pharmacologically or genetically modified animal models. amyloid precursor protein transgenic mice provide the target of in vivo imaging of amyloid protein and glial reaction. because pronounced neuronal death is frequently heralded by microgliosis, in vivo analysis of glial activation in a quantitative manner could be a powerful means for assessing neuroglial degeneration. on the other hand, clinical finding of molecular imaging can also provide important cues for the basic research targets. since there is no ideal animal model for psychiatric disorders, the abnormal dopamine d1 receptor found in clinical research indicates a possible therapeutic target of negative symptoms of schizophrenia. the bidirectional interaction between basic research and clinical research using the molecular imaging technique can expand our knowledge in brain disease. sumiko mochida department of physiology, tokyo medical university, tokyo, japan in presynaptic terminals, packages of neurotransmitters, synaptic vesicles (svs), are localized at specific sites in different stages by regulation of proteins complexes. the recent outstanding studies have revealed molecular mechanisms of presynaptic structure and function. for svs, new proteins were found and the anatomy of vesicle structure was clarified. readiness for transmitter release, svs are docked and primed at the cytomatrix at the active zone where proteins complex formation is regulated by phoshorylation. new kinase sad-1 found at the sv and active zone or pka phosphorylates specific proteins. sv exocytosis is triggered by conformational changes in the fusion proteins complex when ca 2+ sensing protein was activated. synchronies of sv fusion are maintained by a ca 2+ sensing protein, synaptotagmin i. after transmitter release svs are recycled: surprisingly, recycled svs are shared between synaptic boutons regulated by cytoskeletal and motor proteins. these new findings suggest fine mechanisms in presynatic terminals that regulates transmitter release. shigeo takamori 21st century coe program, department of neuorology and neurological science, tokyo medical and dental university, tokyo, japan synaptic vesicles are storage organelles for neurotransmitters that recycle in the presynaptic terminals. to achieve their functions, i.e. neurotransmitter uptake and membrane fusion, they have to be equipped with specified proteins that play essential roles for each process. since decades ago, we have witnessed major advances in our knowledge about the molecular constituents on synaptic vesicles and we've functionally characterized many key players on membrane fusion machinery such as snare proteins and the rab gtpases and on neurotransmitter uptake such as vesicular transporters and vacuolar h + -atpase. however, a detailed picture of a vesicle membrane with all of its constituents is not yet available. in the present study, we have applied a combination of biochemical and biophysical approaches on purified synaptic vesicles from rat brains in order to arrive at a comprehensive and quantitative description of synaptic vesicles. in particular, with a newly developed counting method for synaptic vesicles in solution, we estimated the copy number of each molecule in a single synaptic vesicle. sy1-3-11-3 sad: a novel kinase implicated in phosphoproteome at the presynaptic active zone toshihisa ohtsuka department of clinical and molecular pathology, faculty of medicine/graduate school of medicine, university of toyama, toyama, japan sad is a serine/threonine kianse, which has been shown to regulate various neuronal functions during development, including clustering synaptic vesicles, maturation of synapses, and axon/dendrite polarization: these have recently been revealed by genetic studies in c. elegans and mice. to test if sad is also involved in synaptic functions at mature neurons such as neurotransmitter release, we have recently isolated and characterized human orthologues of sad. interestingly, sad localizes both on synaptic vesicles and at the presynaptic active zones. moreover, sad, together with prominent active zone proteins cast and bassoon, is tightly associated with the active zone cytomatrix. in accord with its unique localization at the nerve terminals, sad appears to be involved in a late step of neurotransmitter release, via direct phosphorylation of another active zone protein rim1. thus, these results suggest that sad regulates not only neural polarization during development but also neurotransmitter release at mature synapses. toshiaki sakisaka 1 , takeshi baba 1 , sumiko mochida 2 , yoshimi takai 1 1 department of molecular biology and biochemistry, osaka university graduate school of medicine, osaka, japan; 2 depatment of physiology, tokyo medical university, tokyo, japan two types of factors are involved in ca 2+ -dependent neurotransmitter release: the snare system and its regulators. the regulators of the snare system include many factors, such as the rab3 system, munc-13, munc-18, doc-2, and tomosyn. we have previously reported that tomosyn, a syntaxin-1-binding protein, works as a molecular clamp that controls free syntaxin-1 availability for the formation of the snare complex and thereby regulates synaptic vesicle exocytosis. here we show that pka-catalyzed phosphorylation of tomosyn decreases its binding to syntaxin-1, resulting in enhanced the formation of the snare complex. conversely, rock phosphorylates syntaxin-1, which increases the affinity of syntaxin-1 for tomosyn and forms a stable complex with tomosyn, resulting in inhibition of the formation of the snare complex. thus, tomosyn is likely to be an upstream regulator of the snare system, whose activity is regulated via well known signal transduction pathways. tei-ichi nishiki department of physiology, okayama university, okayama, japan a synaptic vesicle membrane protein, synaptotagmin i is thought to be a ca 2+ sensor for neurotransmitter release. however, the physiological contributions of its ca 2+ -binding domains (c 2 a and c 2 b) are still unclear. we have studied the roles of aspartate (asp) residues in the c 2 b ca 2+ -binding sites. in synaptotagmin i deficient neurons, although synchronous release was abolished as previously reported (cell 79, p. 717) , asynchronous release was significantly increased. this defect was completely rescued by expressing wild-type synaptotagmin i, indicating the crucial roles of synaptotagmin i for triggering synchronous release and suppressing asynchronous release. synaptotagmin i with mutations in the second or third asp inhibited synchronous release, but still could suppress asynchronous release. thus, we conclude that synaptotagmin i maintains the synchrony of transmitter release in two ways. ca 2+ binding to the c 2 b is essential for synchronizing release. suppressing of asynchronous release seems not to require ca 2+ binding to the c 2 b because mutation in the second asp inhibits ca 2+ binding, yet still allows the protein to suppress asynchronous release. yukiko goda, kevin j. darcy, kevin staras, lucy m. collinson mrc cell biology unit and lmcb, university college london, uk it has been assumed that vesicle replenishment at central synapses operates autonomously at individual presynaptic terminals. we tested the classical model of a compartmentalized synaptic vesicle cycle using fluorescent styryl dyes in combination with methods of fluorescence recovery after photobleaching and correlative light and electron microscopy in cultured hippocampal neurons. we found that endocytosed, recycling synaptic vesicles travel along axons and incorporate into non-native synapses by an actin-dependent mechanism. these newly-incorporated vesicles underwent exocytosis upon stimulation, demonstrating that they form part of the functional recycling pool at their host synapses. our findings indicate that synaptic vesicle recycling is not confined to individual presynaptic terminals but rather a substantial proportion of synaptic vesicles are shared constitutively between synapses. research funds: mrc, nih and narsad hiroshi kunugi department of mental disorder research, national institute of neuroscience, ncnp, japan neurotrophins such as brain-derived neurotrophic factor (bdnf) and neurotrophin-3 (nt-3) have been implicated in the phathogenesis of several neuropsychiatric diseases including schizophrenia, mood disorders, and neurodegenerative diseases. in the past decade, we have systematically screened bdnf, nt-3 and its low-affinity receptor p75 genes for polymorphisms and their possible association with neuropsychiatric diseases. as a result, three polymorphisms of the bdnf gene (c270t in the 5 noncoding region, bdnf-linked polymorphic region, and val66met), three polymorphisms of the nt-3 gene (g-3004a, microsatellite in intron 1, and gly-63glu) and a missene polymorphism of the p75 gene (ser205leu) have been found to be associated with susceptibility to schizophrenia, bipolar disorder, depressive disorder, or alzheimer's disease, although some contradictive negative results have also been reported. here i summarize these findings, review the relevant literature, and discuss future directions of the promising role of the genetic variations of neurotrophins and p75 in neuropsychiatric diseases. recently, a single nucleotide polymorphism (val66met) in the bdnf gene resulting in a prodomain substitution at position 66 from a valine (val) to methionine (met) has been shown to lead in humans to altered hippocampal size and function, and susceptibility to neuropsychiatric disorders. we have recently determined in vitro that bdnf met aberrantly engages a specific vps10 protein, sortilin, that is part of a highly specialized sorting machinery that regulate bdnf trafficking to secretory pathways. in order to determine whether these trafficking defects are responsible for impaired hippocampal functioning, we have developed a transgenic knock-in mice containing the genetic variant bdnf (bdnf met/met ). we have determined that there is a regulated secretion defect for bdnf met , as well as altered hippocampal structure and function in these bdnf met mice, in a manner similar to that reported in humans with this variant bdnf. thus, this bdnf met/met mouse may provide an in vivo model system to inform human studies focused on associations of this variant bdnf with clinical disorders. research funds: nih grant# ns052819 sy1-3-19-6 processing of bdnf and brain disorders masami kojima 1,2 1 aist, osaka, japan; 2 sorst, jst, saitama, japan the fact that pro-and mature neurotrophins elicit opposite effects through p75 neurotrophin receptor (p75ntr) and trks, respectively suggests that proteolytic cleavage of proneurotrophins is an important mechanism that controls the direction of neurotrophin actions. here we examined the effects of two rare single nucleotide polymorphisms (snps); 372 (t/g) and 378 (t/g) of the human bdnf gene, causing amino acid substitution (r125m and r127l) near the cleavage site. western blot analysis and two-side elisa demonstrated that these snps prevented the cleavage, resulting in secretion of probdnf, but not mature bdnf. these snps did not affect intracellular distribution or mode of secretion of the protein. application of the uncleavable probdnf (probdnfml) elicited apoptosis of cerebellar granule neurons, but inhibited dendritic growth of basal forebrain cholinergic neurons. together, these results reveal structural determinants for the cleavage of probdnf, and demonstrate distinct functions of probdnf for different populations of neurons. we have now analyzed the brain functions of the mice expressing this form of bdnf. sy1-4-04-1 pleiotropic effects of gdnf in regulation of enteric nervous system development hideki enomoto laboratory for neuronal differentiation and regeneration, riken center for developmental biology, kobe, japan formation of the enteric nervous system (ens) is governed by multiple extracellular signals at a given time during development. inactivation of the gene encoding gdnf, ret or gfr␣1 leads to nearly complete absence of enteric neurons during early development. although this finding establishes gdnf as an essential extracellular signal acting at the initial stage in ens development, little is known about whether and how enccs continue to depend on gdnf later in development. we have generated mice in which function of gfr␣1, the high affinity receptor for gdnf, is conditionally inactivated in a time-specific fashion. we will show how gdnf signal influences cell migration, differentiation, proliferation and survival of developing enteric neurons, and discuss the biological significance of the findings in development and regeneration of the nervous system in general. bone marrow stromal cells (mscs) including the primitive pluriopotent mesenchymal stem cells and the multipotent adult progenitor cells, are attractive targets for cell and gene therapy for the range of central nervous system disorders. we present using replicationincompetent hsv-1 vector that msc population can be efficiently engineered to secrete a series of various cytokines in the large quantities and in long term in vivo to be able to treat the ischemic stroke of the brain potentially. three kinds of gene-transferred mscs, hgf, il2ss+fgf-2, and vegf were prepared and directly transplanted into the lesioned brain of rat transient middle cerebral artery occlusion model. each growth factor gene-transferred mscs achieved the remarkable amelioration of neurological symptoms and apparent decreasing of infarct volume comparison with native research funds: kakenhi (14657350) sy1-4-04-4 hgf gene therapy for the treatment of spinal cord injury masaya nakamura 1 , akio iwanami 1 , kazuya kitamura 1,2 , yoshiaki toyama 1 , hideyuki okano 2 1 department of ophthalmology surgery, keio university, tokyo, japan; 2 department of physiology, keio university, japan hepatocyte growth factor (hgf) has recently been reported to exhibit neurotrophic activity and to play a role in angiogenesis. in this study, we demonstrated the validity of hgf for treatment of spinal cord injury (sci) in adult rats. first, we analyzed temporal expression of hgf and c-met in the injured spinal cord. hgf-mrna expression was relatively low in the acute phase of sci compared with c-met mrna expression. hypothesizing that hgf is insufficient immediately after sci, we induced sci at th10 level in adult rat 3 days after injecting herpes vector-mediated gene transfer of hgf (hgf group) or lacz (control) into spinal cord. motor function was evaluated by bbb score. 1 or 6 weeks after injury, histological analyses were performed. there were significant decreases in apoptotic cell number and significant enhancements of angiogenesis and gap43+fibers in hgf group compared to the control group. animals of the hgf group showed better functional recovery than the controls. these findings suggest that hgf could have therapeutic effects for sci. hiroshi funakoshi, toshikazu nakamura division of molecular regenerative medicine, osaka university graduate school of medicine, osaka, japan hgf was initially identified and molecularly cloned as a mitogen for primary hepatocytes (nakamura et al., 1989) . recently, hgf is found to be a novel neurotrophic factor for various types of neurons, such as hippocampal, cerebral cortex, cerebral granular, motor, and sensory neurons. mutant c-met/hgf receptor knock-in mouse reveals that hgf decreases neuronal survival and axonal elongation of several types neurons, including motor, sympathetic and cerebral granular neurons, during development (maina et al., 1999) . therefore, it is possible to speculate that hgf could play an important role in the retardation or regeneration of neurons in neurodegenerative diseases. here we show the examples of beneficial effects of hgf on model animals of different neural and neurodegenerative diseases, using several delivery methods for hgf including gene therapy approach. we also present the possible application of hgf in modifying the neurogenesis for the disease. references maina et al., 1999 . nature neuroscience. nakamura et al., 1989 . nature. hiroyuki kato center for clinical medicine and research, international university of health and welfare, nasushiobara, japan we examined stroke patients using fmri at acute/subacute and chronic stages, and visualized areas of brain activation during paretic hand movements. normal hand movement activated the contralateral primary sensorimotor cortex, supplementary motor areas, and ipsilateral cerebellum. at the acute/subacute stages, we observed reductions of these activations and/or addition of activation in ipsilateral cortex or contralateral adjacent cortex during paretic hand movements. at the chronic stages, recovery of activation and/or persistent addition of activation were observed. thus, motor functional recovery was accompanied by restoration of brain activation and/or appearance of additional activation within the motor network of the brain. the findings suggest that cortical motor reorganization as well as recovery from reversible injury plays a role in the restoration of motor function. interestingly, the time period during which reorganization occurred was limited to first 1-2 months after stroke, suggesting the presence of a critical period. in the cat, illert et al. (1977) first demonstrated that disynaptic pyramidal excitation in forelimb motoneurons can be mediated via propriospinal neurons located in the c3-c4 segments. in contrast, recently it has been shown that polysynaptic pyramidal epsps are only rarely observed in forelimb motoneurons of macaque monkeys and humans. we reexamined the indirect corticomotoneuronal inputs in the primates, and obtained the following evidence for the pathway. (1) in the macaque, recordings from forelimb motoneurons showed polysynaptic pyramidal epsps after blockade of glycinergic inhibition by strychnine. moreover, we recently identified c3-c4 propriospinal neurons, which receive pyramidal inputs and project to forelimb motor nuclei. (2) in human arm motor units, magnetic stimulation of the motor cortex produced multiple peaks at short latency in the post-stimulus time histogram, whose total duration was longer than the corresponding value of a finger muscle. stimulation of the pyramidal tract in the medulla could also produce multiple peaks, though in a lower frequency. functions of the pathway both in physiological and pathological conditions will be discussed. bisphenol-a (bpa) has been extensively evaluated for toxicity in a variety of tests as the most common environmental endocrine disruptors. we previously reported that prenatal and neonatal exposure to bpa potentiated central dopaminergic neurotransmission, resulting in supersensitivity to psychostimulant-induced pharmacological actions. many recent findings have supported the idea that astrocytes, which are a subpopulation of glial cells, play a critical role in neuronal transmission in the central nervous system. we found that in vitro treatment with bpa caused the activation of astrocytes, as detected by a stellate morphology and an increase in levels of gfap. a low concentration of bpa significantly enhanced the ca 2+ responses to dopamine in both neurons and astrocytes. these findings provide evidence that bpa induces dopaminergic changes in neurons and astrocytes. this phenomenon may, at least in part, contribute to the enhancement by bpa of the development of psychological dependence on drugs of abuse. mami yamasaki, yonehiro kanemura the department of neurosurgery, clinical research institute, osaka national hospital, national hospital organization, osaka, japan l1cam(l1) is a member of the immunoglobulin superfamily of cell adhesion molecules. x-linked hydrocephalus, masa syndrome and certain forms of x-linked spastic paraplegia are now known to be due to mutations in the gene for l1. therefore, these syndromes have been reclassified as l1 syndrome. we performed a nation-wide l1gene analysis and identified li gene mutations in 35 families with l1 syndrome. all the patients showed developmental delay in various degree. we discussed genotype and phenotype correlations, a striking correlation between the mutation class and the severity of symptoms and molecular basis of severity of developmental delay. the loss of extracellular domain functions like l1-mediated cell adhesion and cell migration is considered to be responsible for molecular genesis of ventricular dilatation and disturbance of the functions of cytoplasmic domain would cause symptoms related axon growth in l1 syndrome. research funds: kakenhi (16390424), (16689025) sy1-5-05-4 rett syndrome and developing brain yoshiko nomura segawa neurological clinic for children, japan rett syndrome (rtt) is a neurodevelopmental disorder with mental retardation, autistic feature, and stereotyped hand movements. hypofunction of the brainstem monoaminergic neurons is suggested. pathology showed no degeneration. methyl-cpg-binding protein 2 gene (mecp2) located at xq28 is the causative gene. types of mutation at different functional domains are correlated to clinical severities. x-inactivation also influences phenotypic variability. mecp2 was thought as a global transcriptional repressor, but finding of bdnf as a target gene suggest its role in the neuronal activity-dependent gene regulation. genetic heterogeneities have been suggested and the mutation of cyclin dependent kinase-like 5 gene (cdkl5) manifest as atypical rtt. the mutations of mecp2 are found in other clinical conditions, such as x-linked mental retardation, angelman syndrome, autism, and severe neonatal encephalopathy. thus, the evaluation of rtt gives the clue to study the clinical, pathophysiological, biological and molecular correlation of not only rtt but also other neurodevelopmental disorders. in our previous studies, we have proposed that ros and/or ros-mediated signal play(s) an essential role in 6-ohda-induced, caspase-dependent apoptosis. in contrast, mpp+-mediated death is not blocked by caspase inhibition and is accompanied by an increase in intracellular free calcium. subsequently, we have demonstrated that mpp+ induces release of cytochrome c but not activation of caspase and proposed that depletion of atp and/or calcium-activated calpain-mediated degradation of procaspase-9 are responsible for the absence of subsequent activation of caspases. furthermore, we have identified that degradation of several important proteins by activated calpain and proteasome system is linked to mpp+-mediated dopaminergic neuronal death. as such, we have found that one of onconeural proteins seems to play a role as a potential survival factor, degradation of which is involved in mpp+-induced cell death. taken together, we reason that distinct set of proteases activation is involved in experimental models of pd. therefore, novel strategies interfering activation of these proteases may contribute to prevention of dopaminergic neuronal death. satoshi ogawa department of neuroanatimy, kanazawa university of medical school, ishikawa, japan we discuss the role of er-stress in neuronal cell death in snpc by introducing two models. upregulation of pael-receptor in the substantia nigra pars (snpc) of mice induces endoplasmic reticulum (er) stress leading to a decrease in tyrosine hydroxylase and death of dopaminergic neurons. the role of er stress in dopaminergic neuronal vulnerability was highlighted by their enhanced death in mice deficient in the ubiquitin-protein ligase parkin and the er chaperone orp150, suggesting parkin dysfunction result in er-stress mediated neuronal cell death. conversely, transgenic rats overexpressing megsin (tg meg), a newly identified serine protease inhibitor (serpin), demonstrated intraneuronal periodic-acid schiff (pas) positive inclusions, which distributed throughout the deeper layers of cerebral cortex, hippocampal ca3, and substantia nigrta. enhanced er stress was observed in dopamine neurons in snpc, accompanied with loss of neuronal viability and motor coordination. in both subregions, pas-positive inclusions were also positive with megsin. these data suggest that enhanced er stress causes selective vulnerability in a set of neuronal populations. noradrenaline (na) transmission modulates synaptic excitability and plasticity through distinct receptor subtypes. accumulating evidence has suggested that the central na system modulates consolidation and reconsolidation of long-term emotional memory. here we show that the na system is particularly important for retrieval of reconsolidated emotional memory. the mutation of the gene encoding tyrosine hydroxylase causes a deficit in conditioned taste memory after its reactivation. this memory deficit is restored by pharmacological stimulation of na activity before the test and is also restored by intra-amygdala na stimulation through ␣1or ␤-adrenergic receptors. moreover, intra-amygdala na stimulation in the wild type animals increases their susceptibility to recall reconsolidated memory. our findings indicate that the amygdalar na system, primarily through ␣1and ␤-adrenergic receptors, acts to improve the retrieval of reconsolidated memory trace. shigeru morinobu, shigeto yamamoto, shigeto yamawaki departmnet of psychiatry and neurosciences, hiroshima university, hiroshima, japan as psychophysiological reactivity on exposure to cues resembling an aspect of the trauma is the major symptom in ptsd, it is hypothesized that impaired extinction may be involved in ptsd. rats subjected to single prolonged stress (sps) exhibit the enhanced negative feedback of the hpa axis, exaggerated startle response, and analgesia. thus, sps is a good model of ptsd. we examined whether extinction of fear memory was impaired in sps rats, using the contextual fear conditioning. sps rats exhibited the significant longer freezing during re-exposure to the context 2-4 days after the conditioning. furthermore, repeated administration of d-cycloserine markedly inhibited the development of enhanced freezing in sps rats. we measured the levels of nmda receptor subunits (nr1, nr2a, 2b, 2c), glycine transporter 1, and eaac1, by real-time pcr. no significant changes were found in the hippocampus. based on these findings, it is speculated that the increase in other types of glutamate transporters or nmda receptor modification may play a role in impaired extinction in sps rats. ichiro masai masai initiative research unit, riken, wako, japan in human, there are hereditary retinal diseases such as retinitis pigmentosa. to understand these molecular mechanisms, we performed a large-scale mutagenesis using zebrafish as an animal model. here we report two zebrafish mutants, twilight (tli) and eclipse (els), both of which show no normal erg and okr response. in the tli mutant, photoreceptors initially differentiate but degenerate later. electron-microscopic analyses revealed that photoreceptive membranes are severely disorganized in the tli mutants, suggesting that tli is required for the formation of photoreceptive membranes. in the els mutant, photoreceptors seem normal in morphology, suggesting that phototransduction is compromised. we found that the els gene encodes cgmp phosphodiesterase 6 ␣ -subunit (pde6c), a component of cone-type pde. since genetic mutations of pde6c have not been reported in human, the els mutant provides a good model for studying roles of cone-pde6 in visual functions. shinichi nakagawa 1 , masatoshi takeichi 2,3 , fumi kubo 1,3 1 nakagawa initiative research unit, riken, wako, japan; 2 riken cdb, kobe, japan; 3 department of biostudies, kyoto university, kyoto, japan the marginal region of the optic vesicle contains retinal stem cells that remain undifferentiated and proliferate for a much longer period compared to other progenitor cells in the central retina. we have previously shown that wnt2b, a signaling molecule expressed in a region neighboring the stem cell area, functions as a putative stem cell factor that endows undifferentiated retinal cells with the characteristics of the stem cells. interestingly, wnt2b inhibits cellular differentiation in the absence of notch activity, a well-known signaling receptor that inhibits neuronal differentiation. wnt2b antagonizes proneural gene functions independent of the notch signaling pathway, presumably through unidentified transcriptional repressors. we have isolated several candidate genes that are upregulated upon an activation of the wnt signaling pathway, and some of them are expressed in the stem cell containing region. physiological roles of those genes will be discussed. research funds: kakenhi (16570184) sy1-6-06-3 identification of cell lineage of retinal progenitor cells by cell surface markers sumiko watanabe, hideto koso, shinya satoh department of molecular and developmental biology, university of tokyo, tokyo, japan i would like to discuss about early cellular developmental stages of retina, which we identified by examination of the expression pattern of cell surface markers. we found c-kit and ssea-1 to be spatiotemporal markers of distinct populations of retinal progenitor cells, and these cells dramatically changed their expression profiles of c-kit and ssea-1 during development. c-kit-positive cells expressed various immature retina specific genes; and later onset of rhodopsin expression and stronger proliferation activities were observed. c-kit/ssea-1 double-positive cells showed stronger proliferation activities than ckit single-positive ones. although the number of ssea-1-positive cells was augmented by beta-catenin signal, c-kit-positive cells were positively regulated by notch signaling, suggesting that c-kit and ssea-1 have intrinsically distinct characters. prolonged expression of c-kit by a retrovirus resulted in promotion of proliferation and the appearance of nestin-positive cells in response to scf, suggesting a role for c-kit in retinal development. the retinal photoreceptor cells play a primal and central role in the phototransduction system. they are susceptible to deterioration in human retinal diseases, which lead to severe visual impairment. we have been demonstrated that transcription factors, otx2 and crx play critical roles in retinal photoreceptor development. while otx2 is a key molecule for retinal photoreceptor cell fate determination, crx is essential for the terminal differentiation and maturation of photoreceptors. meanwhile, the photoreceptor cell is a highly polarized neuron and also has epithelial characteristics such as adherens junctions. our investigation of a role of apkc, which has been proposed to play a critical role in the establishment of epithelial and neuronal polarity, in differentiating photoreceptors has shown that apkc is required for the formation of outer & inner segments and ribbon synapse. in addition, we also found that photoreceptor polarity formation has important roles in proper retinal lamination. we would like to present our recent analysis of photoreceptor development. research funds: kakenhi (16790070, 16027257, 17024061) raj ladher riken center for developmental biology, kobe, japan the inner ear translates mechanical energy into neural signals that the appropriate centers of the brain can decode into balance or sound information. the inner ear forms from bilateral thickened discs of ectoderm located on either side of the hindbrain, early during development. induction of the inner ear is mediated by localized signals emanating from the paraxial mesoderm. in the chick, the inner ear is induced by localized fgf19 found in the mesoderm. we find that although fgf19 can induce the inner ear, it is unable to support differentiation of the inner ear. differentiation, that is the development of the chick inner ear hair cells, is triggered by another family member fgf3 and is actually inhibited by fgf19. for full functionality, the inner ear needs to be integrated into the larger auditory complex, made up of the middle ear, the external ear and the auditory centers in the hindbrain. these components develop from diverse origins but are intimately linked during development. we have been trying to understand how integration occurs and present one model by which this could occur. research funds: center support grant, mext leading projects grant sy1-6-06-6 how is olfactory receptor-dependent axonal wiring conducted? shou serizawa, kazunari miyamichi, haruki takeuchi, yuya yamagishi, tokiko tsubokawa, hitoshi sakano department of biophysics and biochemistry, crest jst, university of tokyo, tokyo, japan in the olfactory system, termini of primary axon segregate depending on the type of olfactory receptor (or) expressed, forming the olfactory sensory map. to study how the or-dependent axonal wiring is conducted, we analyzed the gene expression profile in the olfactory epithelium of the transgenic mouse in which the majority of olfactory sensory neurons (osns) express a particular or gene. we found that the expression of the immunoglobulin superfamily gene kirrel2, encoding homophillic adhesion molecule, is down-regulated in the transgenic mouse compared to the wild type control. the expression level of kirrel2 in each osn is found to be correlated with the type of or species expressed in the osn. moreover, kirrel2 promoted fasciculation of osn axon termini in the mosaic gain-of-function experiment. here, we propose that the information of which type of or is expressed in the osn is converted to the expression level of kirrel2 which determines the adhesiveness of axon termini, contributing to or-dependent segregation of osn axons. in spite of its morphological similarity to the other species in the melanogaster species subgroup, drosophila sechellia has evolved distinctive physiological and behavioral characters adapting to its host plant morinda citrifolia, known as the tahitian noni fruit. the ripe fruit of m. citriforia contains hexianoic acid and octanoic acid, the main components of the odor from the fruit. d. sechellia is attracted to these two fatty acids, while the other species are repelled by them. using inter-species hybrid between d. melanogaster deficiency mutants and d. sechellia, odorant binding protein 57e was identified as the gene responsible for this behavioral difference among the species. obp57e forms a gene cluster with obp57d, and these two genes are expressed in the same cells associated with the chemosensory organ. the history of dynamic obp57d/e-cluster evolution was revealed by comparison of the genomic sequences of the obp57d/e region obtained from 30 species phylogenetically located between d. melanogaster and d. pseudoobscula. sy1-6-14-4 an approach of dissociating complex traits into fine genetic elements using consomic strains of mouse aki takahashi 1,2 , akinori nishi 1,2 , toshihiko shiroishi 1,2 , tsuyoshi koide 1,2 1 sokendai, kanagawa, japan; 2 national institute of genetics, shizuoka, japan much of the genetic variation that underlies most behavioral traits is complex and is regulated by loci that have quantitative effect on the phenotype. we have previously shown that laboratory strain c57bl/6 (b6) and wild-derived strain msm/ms have great differences in many behavioral traits. consomic strains were established by natural mating between b6 and msm, and those strains have the same genetic background as b6 except for one chromosome from msm. by examining bunch of consomic strains on many behavioral trait, such as spontaneous activity, anxiety-like behavior, pain sensitivity, and social behavior, we were able to map which chromosome have a locus or loci affecting those phenotype. one strain b6-17msm, which have msm chromosome 17, showed increased fear responses and riskassessment behavior, and thus it is thought that there is a locus/loci related to the emotionality. to identify the gene in the loci, we have made congenic strains, and successfully narrowed the locus down in the telomeric region. research funds: kakenhi (16.12064) sy1-6-14-5 cloning of the major quantitative trait locus underlying capsaicin resistance in mice capsaicin is the main compound of hot chili peppers, and induces sensations of heat and pain. however, sensitivity to capsaicin differs among individuals. a genetic approach using a mouse model reveals some quantitative trait loci for this sensitivity. capsaicin resistance linked on chromosome 2 (capsq1) is the major locus affecting reduced taste sensation in kjr mice. here we show that intracellular recycling of capsaicin receptor (trpv1) was impaired in kjr neurons in contrast to that of c57bl/6j mice. by searching the candidate genes, eh domain-containing four (ehd4), a trp-binding scaffold protein encoding gene was found. ehd4 binds to c-terminal of trpv1. three mutations were found in ehd4 of kjr, which remarkably diminished the binding, leading to changes in the intracellular distribution of trpv1. this study is the first genetic dissection associated with capsaicin/heat resistance in a nature strain and shows a novel binding protein to trpvs. sy1-6-14-6 comprehensive behavioral analysis of genetically-engineered mice tsuyoshi miyakawa hmro, kyoto university, kyoto, japan one of the major challenges in the life sciences of the post-genome era is to elucidate the functions of the genes at the level of individual animals. final output level of the functions of the genes expressed in the brain is behavior, indicating a need for systematic investigations of the behavioral significance of the genes. in our laboratory, we use a "comprehensive behavioral test battery" for genetically-engineered mice to reveal causal relationships between genes and behaviors. the battery covers broad areas of behaviors, from simple reflexes to highly cognitive functions. so far, we have assessed more than 45 different strains of mutant mice with the battery. surprisingly, more than 90% of the strains showed at least one significant behavioral phenotype, suggesting that a large part of the genes expressed in the brain may have some functions. representative results for a few strains of mutant mice and the meta-analytic results of the combined data will be presented. also, a potential impact of our approach to "large-scale neuroscience" will be discussed. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird hiroshi takashima department of neurology and geriatrics, kagoshima university, kagoshima, japan inherited neuropathies are clinically and genetically heterogeneous. at least 28 genes and 12 loci have been associated with charcot-marie-tooth disease (cmt) and related inherited neuropathies. most causes of inherited neuropathy have been discovered by positional cloning technique and in the past two years, the pace of cmt gene discovery has accelerated. these recently discovered cmt causing genes/proteins include those which, although showing unpredictable correlations with the peripheral nervous system, are definitely important for the peripheral nerve. their discovery should pave the way for dramatic progress in the understanding of peripheral nerve biology. on the other hand, genotype-phenotype correlations of these genes are also important in order to understand the pathomechanisms of inherited neuropathy since, based on mutation studies, a large number of genes associated with both the demyelinating and axonal forms of cmt have been identified. to clarify the specific features and molecular mechanisms, we reviewed recent progress in cmt research, especially cmt4f caused by prx, and scan1 caused by tdp1. sy1-6-22-2 gangliosides are important for the maintenance of the nodes of ranvier nobuhiro yuki, keiichiro susuki department of neurology, dokkyo university school of medicine, tochigi, japan gangliosides are abundant in vertebrate nervous system, but the function has yet to be elucidated. some patients with guillain-barre syndrome have autoantibodies to gangliosides such as gm1, who show failure of peripheral motor nerve conduction. sensitization of rabbits with gm1 can produce the disease model. in ventral roots from the paralyzed rabbits, igg and complements deposited on the nodes of ranvier, and sodium channel clusters were disrupted. in ganglioside-deficient mice with disrupted gm2/gd2 synthase gene, motor nerve conduction velocities were reduced in the sciatic nerves. some myelin loops failed to contact the paranodal axolemma, and potassium channels were aberrantly localized at the paranodes. the abnormality became prominent with age. these findings using different models showed that gangliosides are important for the maintenance of the node of ranvier and saltatory conduction along the myelinated nerve fibers. hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan neuropathic pain caused following nerve injury is one of important issues in neuroscience as well as clinics, since its pain pathway is apparently distinct from that in healthy humans and naive experimental animals. this is clearly evidenced by the finding that the tactile information is converted to noxious one in allodynia characterized in neuropathic pain. in our recent paper (nature medicine, 2004) , we firstly demonstrated that lysophosphatidic acid (lpa) and its receptor (lpa1) activation initiate the neuropathic pain. in this and following studies we proposed that the demyelination of nociceptive fibers reorganizes the nociceptive spinal inputs through sprouting and electrical synapses (ephapses). i will discuss four issues, lpa-induced demyelination of dorsal root fibers using in vivo and ex vivo culture models, the signal transduction of underlying lpa-mediated downregulation of myelin proteins, evidence for sprouting and ephapses following demyelination and the origin of injury-specific lpa production in terms of demyelination and allodynia. research funds: kakenhi (17109015) toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan axons of adult central nervous system are capable of only a limited amount of regrowth after injury, and an unfavorable environment plays major roles in the lack of regeneration. some of the axon growth inhibitory effects are associated with myelin. three myelin-derived proteins have been identified to inhibit neurite outgrowth in vitro. these proteins induce activation of rho in some neruons. inhibition of rho or rho-kinase promotes axon regeneration in vivo. these findings establish rho and rho-kinase as key players in inhibiting regeneration of the central nervous system. i will review recent findings regarding the signaling mechanism of axon growth inhibitors. our experiments suggest that several new candidate proteins may be axon growth inhibitors. these proteins activate not only rho/rhokinase but also other signals to inhibit neurite outgrowth from some neurons in vitro. these findings suggest that agents that block the multiple signals elicited by these axon growth inhibitors may provide efficient tools that produce functional regeneration following injuries to the central nervous system. sha mi biogen idec, usa lingo-1 is a cns-specific protein expressed in both neurons and oligodendrocytes. in neurons, lingo-1 mediates the inhibition of axonal growth as a component of the ngr1/p75/lingo-1 and ngr1/troy/lingo-1 signaling complex. inhibition of endogenous lingo-1 by soluble lingo-1 or dominant negative lingo-1 can reverse the inhibition of neurite outgrowth by myelin components. soluble lingo-1 treatment significantly improves functional recovery of spinal cord injured rats as determined by bbb scores. soluble lingo-1 treatment promotes axonal regeneration and reduced axon dieback in the corticospinal tract, rubrospinal tract, and optic nerve. in oligodendrocytes, lingo-1 mediates the inhibition of differentiation and myelination. loss of lingo-1 function using dominant negative lingo-1, lingo-1 rnai, or soluble lingo-1 or lingo-1 knockout increased oligodendrocyte differentiation and myelination, whereas over-expression of lingo-1 led to inhibition of oligodendrocyte differentiation and myelination, in vitro and in vivo. the discovery of a significant role for lingo-1 in neurons and oligodendrocyte biology are an invaluable step for understanding cns axon regeneration and myelination. alex reyes new york university, usa neurons in the auditory cortex exhibit a wide range of firing patterns. to elucidate the cellular properties and circuitry that give rise to these responses, a 2d sheet of excitatory and inhibitory neurons were reconstructed in vitro using an iteratively-constructed network (icn) modified to contain both feedback and feedforward circuits. a disc of neurons was stimulated and the resultant firing pattern and spread was documented. simultaneous whole-cell recordings were performed from pyramidal and interneurons in a slice preparation of the mouse auditory cortex. a computer simulated the activities of thalamic neurons and calculated the net synaptic conductance that would be generated by their firing. this waveform was converted to current, injected into the recorded neurons via a dynamic clamp circuit, and the resultant firing documented. using the icn method, we reproduced the firing of a realistic network of excitatory and inhibitory neurons. we replicated many of the responses recorded in vivo. morever, the firing patterns of neurons depend substantially on their distance from the stimulus center and on the identity of the local interneurons. research funds: nih dc005787-01a1 sy1-7-15-2 neuronal avalanches reveal neuronal wirings of layer 2/3 cell assemblies jun-nosuke teramae, tomoki fukai brain science institute, riken, japan how cortical neurons process information crucially depends on how their local circuits are organized. spontaneous synchronous neuronal activity propagating through neocortical slices displays highly diverse, yet repeatable, activity patterns called 'neuronal avalanches'. they obey power-law distributions of the event sizes and lifetimes, presumably reflecting the structure of local cortical networks. however, the explicit network structure underlying the power-law statistics remains unclear. here, we present a neuronal network model of pyramidal and inhibitory neurons that enables stable propagation of avalanche-like spiking activity. we demonstrate a neuronal wiring rule that governs the formation of mutually overlapping cell assemblies during the development of this network. the resultant network comprises a mixture of feedforward chains and recurrent circuits, in which neuronal avalanches are stable if the former structure is predominant. we investigate how the resultant power laws depend on the details of the cell-assembly formation as well as on the inhibitory feedback. research funds: kakenhi (17700318) sy1-7-15-3 spike-timing dependent and homeostatic plasticity from an optimality viewpoint taro toyoizumi 1 , jean-pascal pfister 2 , kazuyuki aihara 1,3 , wulfram gerstner 2 1 department of complexity science and engineering, university of tokyo, japan; 2 school of computer and communication science & bmi, epfl, japan; 3 aihara complexity modelling project, erato, jst, japan maximization of information transmission by a spiking neuron model predicts changes of synaptic connections that depend on timing of pre-and postsynaptic spikes as well as on the postsynaptic membrane potential. under the assumption of poisson firing statistics, the synaptic update rule exhibits all the features of the bienenstock-cooper-munro rule, in particular regimes of synaptic potentiation and depression separated by a sliding threshold. the learning rule is found by maximizing the mutual information between presynaptic and postsynaptic spike trains under the constraint that the postsynaptic firing rate stays close to some target firing rate. an interpretation of the synaptic update rule in terms of homeostatic synaptic processes and spike-timing dependent plasticity is discussed. research funds: grant-in-aid for jsps fellows 03j11691 and sci. res. on priority areas 17022012 from mext of japan, and swiss natl. sci. found. 200020-108097/1 sy1-7-15-4 timing computations in the auditory brain stem john rinzel center for neural science and courant institute of mathematical sciences, new york university, usa sound localization involves precise temporal processing by neurons in the auditory brain stem. the first neurons in the auditory pathway to receive input from both ears can distinguish interaural time differences (itds) in the sub-millisecond range. these cells in the mammalian medial superior olive have specialized biophysical features: two dendrites, each receiving input from only one side; very short membrane time constant; specialized ionic channel properties, including a low-voltage activated k+ current, i-klt. this i-klt contributes to phasic firing (one spike in response to a step of current), precise phase-locking, and extremely timing-sensitive coincidence detection. we will describe the temporal feature-selecting properties of mso cells based on biophysical (hh-like) modeling, in vitro electrophysiology and application of concepts from dynamical systems theory and coding theory. neuronal information is often inferred by counting spike numbers over tens to hundreds of milliseconds. however, if relative spike timings at the scale of milliseconds would carry information, neuronal circuits could have large information capacity. in response to various visual inputs, the retina fires spike bursts separated by hundreds of milliseconds of silent periods. onsets and spike numbers of these bursts are highly reproducible. we asked if spike patterns, i.e., combinations of interspike intervals within single bursts, carry information. using the retinas of salamanders and mice, we found that bursts have various spike patterns, which are unique to the preceding inputs. differences in spike patterns at the scale of milliseconds encode differences in the input as long as 200-300 ms. when single bursts contain three or more spikes, the multiple interspike intervals combinatorially encode multiple features of the input. this suggests the spike patters are not determined sorely by slowly modulating instantaneous firing rates. we propose that the retina encodes multiple features in hundreds of milliseconds of input into burst spike patterns at the scale of milliseconds. accumulating evidence reveals that the generalized seizure activity can produce regenerative, in addition to degenerative, structural changes in the hippocampus, including the enhancement of progenitor cell division of dentate granule cells. although the regulatory mechanisms underlying such neurogenesis are unknown, we hypothesized that newly generated granule cells may contribute to the reorganization of the hippocampal formation in the early course of seizures, constituting a possible substrate for epileptogenicity. to address this issue, we examined the division of dentate granule cell progenitors in rats after kainic acid administration, or perforant path kindling. the results indicate that initial limbic seizures trigger the enhancement of dentate progenitor cell division, but progenitor cells may become unreactive to prolonged generalized seizures. the degenerative process is not necessary for triggering the upregulation. it is also suggested that newly generated granule cells may play a role in the network reorganization that occurs during epileptogenesis. the molecular basis underlying such neurogenesis will be discussed. keiichi itoi 1 , ikue otaki 1 , saya suzuki 1 , yasunobu yasoshima 2 , kazuto kobayashi 2 1 laboratory of informational biology, graduate school of informational science, tohoku university, sendai, japan; 2 institute of biomedical science, fukushima medical university, japan in order to examine functional roles of the noradrenergic (na) neurons in the locus coeruleus (lc) we developed a novel method to ablate specifically the na neurons in the lc, and examined the behavioral and stress responses using the animal model. a transgenic mouse line was used in which human interleukin-2 receptor ␣ subunit (hil-2r␣) was expressed under the control of dopamine ␤-hydroxylase gene promoter. anti-hil-2r␣ antibody fused to pseudomonas exotoxin was microinjected into bilateral lc of a transgenic mouse stereotaxically to destroy specifically the na neurons. as behavioral paradigms, elevated plus maze and open field test were used. plasma adrenocorticotropin levels were measured following lipopolysaccharide injection intraperitoneally, as an immune stress. thus, the effect of lc ablation how it affects the behavioral and stress responses will be elucidated. -8-16-5 integrated circuits controlling the stress response james p. herman department of psychiatry, university of cincinnati, oh, usa the hypothalamo-pituitary-adrenocortical (hpa) axis is a primary stress-response system in all vertebrates. the end-product of hpa activation, glucocorticoids, serve the general function of redirecting bodily resources to meet a real or perceived challenge. however, prolonged glucocorticoid secretion has deleterious effects on metabolism, immune function and behavior, making control of hpa activity a priority for the organism. this control is exerted in large part by limbic structures in the brain. our studies indicate that the amygdala, hippocampus and prefrontal cortex play major roles hpa axis regulation. the amygdala is primarily stress excitatory, whereas the hippocampus has an inhibitory influence on hpa activity. the role of the prefrontal cortex is considerably more complex; its prelimbic region is primarily stress inhibitory, whereas the infralimbic region may participate in stress activation. all of these regions exert their influence via subcortical relays to hypothalamic paraventricular nucleus (pvn) neurons controlling the hpa response, allowing convergence of information from multiple limbic sources prior to the pvn. sy1-8-24-1 molecular mechanism for the inverse incidence of parkinson's disease and cancer: synuclein as stimulator of tumour differentiation makoto hashimoto department of chemistry and metabolism, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative disease and cancer are major age-associated disorders. however, the pathogenesis of these diseases may be in sharp contrast, since the former is featured by cell death, whereas, the latter is associated with immortalization. in parkinson's disease (pd) research, smoking, the risk factor for a variety of cancers, had been known to reduce the risk of pd. furthermore, epidemiological studies described that the incidence of cancer was reduced in pd patients. recent study provides evidences of the inverse relationship of pd and some cancers at the molecular level. for example, loss of neuroprotection of dj-1 is causative for familial pd, while increased expression of this molecule stimulates oncogenesis. in this context, we show that proteasomal inhibition by ␣-synuclein, which has been thought as one major pathogenic mechanism for pd, may induce differentiation of cancer cells. thus, unifying approach on the basis of the opposite pathogenic mechanism to neurodegenerative disease and cancer might uncover unexpected findings in both fields. kiyomitsu oyanagi department of neuropathology, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative diseases and malignant tumors develop symptoms usually at middle or old-age in humans. however, it is well known that critical periods of some malignancies are in fetal period, which are (1) leukemia in patients exposed with atomic bomb during the iind world war, and (2) brain tumors in rats with ethylnitrosourea administration. as to neurodegenerative diseases, (3) many genetic/familial diseases show clinical symptoms at the middle or old age. (4) epidemiological study revealed that emigrants from guam to the main land of usa show relatively high incidence of amyotrophic lateral sclerosis, and the critical period of exposure to some environmental noxiousness was considered to be childhood/adolescence. (5) relating to parkinson disease, low magnesium intake over generations induced selective degeneration of the dopaminergic neurons in the substantia nigra in rats [oyanagi et al., in press] . these findings indicate that not only certain malignant tumors but also some sporadic neurodegenerative diseases may be induced originally by the insults in embryonic stage/childhood. to understand the role of synuclein, the major component of pathological inclusions, we examine the expression of synuclein in the embryonic mouse cerebral cortex. we found that a-synuclein and b-synuclein were predominantly detected in the subplate neurons, which are known to enter programmed cell death at a postnatal stage. in another line of inquiry, we are interested in a zinc finger protein containing poz domain, rp58, which functions as a sequence specific transcriptional repressor and involved in cortical layer formation. when the rp58 gene is disrupted, apoptosis is enhanced, and a-synuclein, but not b-synclein, is upregulated in the mutant cortex, suggesting that a-synuclein is involved in the cell death. interestingly, in the mutant cortex the expression of s-phase marker, pcna increased, suggesting that rp58 mutant mice are useful to analyze the relation among neurodegeration, synuclein and cell cycle. minoru saitoe, junjiro horiuchi, daisuke yamazaki tokyo metropolitan institute for neuroscience, tokyo, japan age-related memory impairment (ami) is a striking feature of ageassociated neuronal dysfunction. to identify gene mutations that affect ami, we screened ∼100 drosophila lines and found that heterozygous mutants for the pka catalytic subunit (dc0/+) exhibit robust suppression of ami without affecting memory at young ages. this result suggests a causal relationship between pka and ami. of particular interest, igf/pi3k/akt signaling, which results in decreased gsk3 activity, has also been shown to ameliorate ami. both pka and gsk3 phosphorylate the microtubule-associated protein tau, causing tau aggregation and neurodegeneration. while igf signaling suppresses activity of gsk3 at young ages, declining igf levels during aging may increase gsk3 activity in aged animals. in support of this idea, we found suppression of ami in flies fed gsk3 inhibitors. we hypothesize that similar to the mechanisms occurring in neurodegenerative diseases, tau phosphorylation by pka and gsk3 causes neuronal dysfunction during normal aging. research funds: kakenhi sy1-8-24-5 molecular mechanism of cancer progression by gamma-synuclein koji okamoto radiobiology division, national cancer center research institute, tokyo, japan synucleins, a family of small proteins consisting of three known members, are implicated in both neurodegenerative disorder and tumorigenesis. ␣synuclein is involved in the formation of pathologically insoluble deposits characteristic of neurodegenerative diseases such as alzheimer disease and parkinson disease, whereas overexpression of ␥synuclein is associated with progression of breast and ovarian cancer. however, the normal cellular function of synucleins remains largely unknown. in order to get an insight into biological function of synucleins, we focus on cancer progression induced by ␥synuclein. we introduced ␥synuclein into breast cancer cells in order to recapitulate malignant transformation of breast cancer. using such cells, the attempt to elucidate the biochemical function of ␥synuclein is underway. the impact of synuclein over-expression, especially on known tumor suppressor pathways such as the p53 pathway, will be discussed. research funds: kakenhi ryuichi sakai growth factor division, national cancer center research institute, 5-1-1 tsukiji, chuo-ku, tokyo 104-0045, japan numbers of growth factors and their membrane receptors which possess tyrosine kinase activity are involved in proliferation and differentiation of the neural system. shc family docking molecules conduct signals directly downstream of various growth factor receptors as substrates and binding partners of these tyrosine kinases. in the neural systems, two unique shc family molecules, shcb and shcc, are found to be specifically expressed and analysis of mice lacking these proteins revealed that they have redundant functions during mammalian neural development as mediators of ngf/trka signaling. it was recently found that tyrosine phosphorylation of shcc is frequently detected in majority of neuroblastoma cell lines. we showed that hyperphosphorylated shcc detected in some of neuroblastoma cell lines is associated with constitutively activated anaplastic lymphoma kinase (alk) caused by the gene amplification. identification of binding partners of shcc and expression of mutant shcc in several cancer cell lines revealed novel roles of shcc as a regulator of differentiation and proliferation of neuroblastic tumors. research funds: kakenhi sy1-8-24-7 identification of estrogen receptor target genes and role of their gene products in cancer and nervous system satoshi inoue 1,2 1 department of geriatric medicine, university of tokyo hospital, tokyo, japan; 2 research center for genomic medicine, saitama medical school, saitama, japan estrogen has crucial roles in the cancer growth and in the neural function. here, we have isolated and characterized novel estrogenresponsive genes to clarify the molecular mechanism of the estrogen action in target cells using genomic binding-site cloning (gbsc) method. one of the first identified genes is the estrogen-responsive ring finger protein (efp). efp expression was observed in uterus, mammary gland and certain regions of the brain where er is also expressed and positively regulated by estrogen. we revealed that efp targets proteolysis of 14-3-3 sigma, a negative cell cycle regulator that causes g2 arrest and that efp is an essential oncogenic factor in breast cancer growth. on the other hand, another gene identified by gbsc is nr2d, an nmda receptor. this gene was regulated by estrogen in the hypothalamus, together with er, pr and efp. these estrogen responsive genes could mediate roles of estrogen action in specific organs, utilizing differential mechanisms as well as sharing common mechanisms. keiji tanaka 1 , hossein esteky 2 , kiani roozbeh 2 , tadashi sugihara 1 , gang wang 3 1 riken brain science institute, wako, saitama, japan; 2 institute for studies in theoretical physics and mathematics, tehran, iran; 3 kagoshima university, kagoshima, japan individual cells in the monkey inferotemporal cortex, which is the final unimodal stage along the ventral visual pathway, respond to moderately complex features, but not to objects nor to object categories. then, questions arise where and how view-general objects and object categories are represented. a possibility is the representation by a population of inferotemporal cells. to examine it, we recorded responses of 670 inferotemporal cells to 1100 object images in a fixation task. we also conducted psychophysical experiments with monkeys to determine conditions for view-invariant object recognition. the results suggest that a population of inferotemporal cells represent object categories and their relational structure, and that the representation is common to nearby views of objects with up to 60 • rotation. research funds: kakenhi 17022047 alexander thiele 1 , gene stoner 2 , louise s. delicato 1 , mark roberts 1 1 university of newcastle upon tyne, uk; 2 the salk institute, japan a variety of different roles of synchronized activity for sensation and perception have been proposed, ranging from object binding, through attentional enhancement, to mechanisms of learning. we have employed different paradigms to investigate the role of neural synchrony in visual perception and attentional selection in the awake macaque monkey. using two different tasks and stimulus conditions, well suited to probe the role of feature binding in the motion domain, we found no support for the idea that neuronal synchrony in macaque area mt underlies the binding of an object's component features. recent reports have focused on the role of synchrony in the mediation of attention. we will discuss the role of synchronized activity in primate v1 while attentional load was varied, and how it could be mediated by cholinergic mechanisms. research funds: hfsp, wellcome trust, bbsrc sy2-1-01-3 context-dependent changes in noise correlation in mt william newsome, marlene r. cohen stanford university and howard hughes medical institute, usa changes in the correlated firing of a pair of neurons may provide a metric of changes in functional circuitry within the nervous system during ongoing behavior. we studied dynamic changes in functional circuitry by analyzing the noise correlations of simultaneously recorded mt neurons in two behavioral contexts: one that promotes cooperative interactions between the two neurons and another that promotes competitive interactions. we created cooperative or competitive contexts by changing the axis of motion of the discrimination task from trial to trial. we found that identical visual stimuli indeed give rise to differences in noise correlation in the two behavioral contexts. specifically, noise correlations were higher in the cooperative than in the competitive context. this result suggests that mt neurons receive inputs of central origin whose strength changes with the task structure. the changes in correlation appear to reflect differences in how mt neurons are pooled for the purpose of perceptual discrimination, and may derive from higher-level cognitive processes such as feature-based attention. research funds: howard hughes medical institute sy2-1-01-4 effects of task demands on color processing in area te of the monkey hidehiko komatsu 1,2 , kowa koida 1 1 national institute for physiological sciences, okazaki, japan; 2 sokendai, okazaki, japan color vision has two different functions, namely, categorization and discrimination, and the same color stimulus can be processed according to these two functions depending on task demands. lesion studies suggested that inferior temporal (it) cortex of the monkey plays a key role in color vision, and we have recently found that color selective neurons are concentrated in a small region in area te of it cortex. to study how the color selective responses in this region are affected by the task demands, we trained monkeys a color categorization task and a color discrimination task using the same set of color stimuli, and analyzed how the responses are affected. we found response magnitudes of many neurons changed between two tasks while the color tuning is well reserved. in several extreme cases, large gain change almost completely eliminated the responses in one task. these results suggest that color signals are gated by the top-down signal representing task demands in area te and color channels specific to different tasks are formed at this level of the visual cortex. yoichi sugita neuroscience research institute, aist, tsukuba, japan early visual experience is indispensable to shape the maturation of cortical circuits during development1. monocular deprivation in infancy, for instance, leads to an irreversible reduction of visually driven activity in the visual cortex through the deprived eye and a loss of binocular depth perception2-4. here, i show exposure only to monochromatic illumination in infancy results in the disruption of color perception. infant monkeys were reared for nearly a year in a room where the illumination came from only monochromatic lights. after extensive training, they were able to perform color matching. but, their judgment of color similarity was quite different from that of normal animals. furthermore, they had deficits in color constancy; they could not compensate for the changes in wavelength composition. these results indicate that early visual experience is also indispensable for color perception. research funds: crest sy2-2-02-1 dendritic growth, spinogenesis and synaptogenesis in response to neurosteroids in the developing purkinje cell kazuyoshi tsutsui 1 , hirotaka sakamoto 2 , katsunori sasahara 1 , hanako shikimi 1 , kazuyoshi ukena 1 , mitsuhiro kawata 2 1 laboratory of brain science, faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima, japan; 2 department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan new findings over the past decade have established that the brain synthesizes steroids de novo from cholesterol. such steroids synthesized de novo in the brain are called neurosteroids. recently we have identified the purkinje cell as a major site for neurosteroid formation in the brain. this is the first demonstration of de novo neuronal neurosteroidogenesis in the brain. in mammals, the purkinje cell actively synthesizes progesterone and estradiol de novo from cholesterol during neonatal life, when cerebellar cortical formation occurs. subsequently, our recent studies on mammals using the purkinje cell have demonstrated organizing actions of neurosteroids. both progesterone and estradiol promote dendritic growth, spinogenesis and synaptogenesis via each cognate nuclear receptor in purkinje cells. research funds: kakenhi (15207007 and 16086206 to kt) sy2-2-02-2 roles of estrogen receptors in the regulation of socio-sexual and emotional behaviors-studies with knockout mice and rnai sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan the gonadal steroid estrogen plays a major role in the regulation not only of female reproductive behavior but also an array of social and emotional behaviors in both sexes, by acting through intracellular estrogen receptors (ers), ligand dependent transcription factors. a series of studies using single and double knockout mice for er-␣ and/or er-␤ genes have revealed that activation of er-␣ and er-␤ differentially regulate a number of behaviors as well as neuroendocrine functions. our studies have suggested a unique role of activation of er-␤ in the hypothalamic and limbic brain areas, dorsal raphe nuclei and locus coeruleus in the regulation of socio-sexual and emotional behaviors. in this talk, our findings from behavioral studies using er-␣ and er-␤ knockout mice along with possible brain mechanisms underlying the behavioral effects will be first overviewed. our most recent studies on brain site-specific manipulation of er gene expression with the use of small interference rna combined with adeno-associated virus will then be presented. research funds: kakenhi (17330151, 17051001) sy2-2-02-3 sex steroid receptor function in sexual behavior shigeaki kato 1,2 , takashi sato 1 , takahiro matsumoto 1,2 1 imcb, university of tokyo, tokyo, japan; 2 erato, jst, saitama, japan androgen actions are believed to mediate nuclear androgen receptor (ar)-mediated gene regulations. ar is a member of nuclear receptor, and acts as a hormone-induced transcription factor to control of target genes through chromatin remodeling/histone modification. we generated the floxed ar mice to avoid testicular feminization mutant (tfm) abnormalities with infertility, and then crossed with female ar(−/+) heterozygoutes expressing cre to generate ar(−/−) female mice. the ar(−/y) ko males grew healthy with typical features of tfm abnormalities, and genital organs were atrophic with a marked decrease in the serum testosterone level, but with normal estrogen level (kawano et al., 2003) . no sexual behaviors and reduced aggressive behaviors were seen in ar(−/y) male mice (sato et al., 2004) . female ar ko mice were normal in sexual behavior but exhibited premature ovarian phenotype (shiina et al., 2006) . together with these results, the ar function will be discussed in terms of ar function as a transcription factor. references kawano, h., et al., 2003 . pnas usa 100, 9416. sato, t., et al., 2004 . pnas, usa 101, 1673 . shiina, h., et al., 2006 research funds: probrain sy2-2-02-4 annexin 1: a mediator of cell-cell communication in the neuroendocrine system julia buckingham 1 , helen christian 2 , john morris 2 1 imperial college london, uk; 2 department of human anatomy and genetics, university of oxford, uk annexin 1 (anxa1) plays an important part in mediating the regulatory effects of glucocorticoids (gcs) on neuroendocrine function, particularly within the hpa axis. it is expressed by folliculostellate (fs) cells in the pituitary gland and by ependymal cells and activated glia in the hypothalamus but not by classical secretory cells. gcs act on cells expressing anxa1 to cause the translocation of the protein to the plasma membrane at points with particular accumulation at points where the cells make contact with endocrine cells. this process is effected via a non-genomic mechanism and is dependent upon phosphorylation, lipidation and a transport protein, possibly abca1. the released protein then acts, via cell surface receptors on the endocrine cells to suppress stimulus-evoked peptide release. the nature of the anxa1 receptor is unclear but, increasing data suggest that members of the formal peptide receptor family may be important in this regard. katsuhiko nishimori 1 , yuki takayanagi 2 , masahide yoshida 1 , yoshiyuki kasahara 1 , masaki kawamata 1 1 graduate school of agricultural science, tohoku university, sendai, japan; 2 department of physiology, jichi medical university, minamikawachi-machi, japan we examined the behaviors of mice lacking oxtr gene and discovered that oxtr null females displayed impaired nurturing behavior, and their pups showed defect in ultrasonic vocalization, instead, increased locomotor activity by social isolation test. those are implying impaired mother-infant relationship. oxtr null males also showed more aggressive and having social amnesia as well as the phenotype of oxt null mice. in addition, oxtr null mice failed to maintain their body temperature after acute cold exposure. their rectal temperature rapidly dropped in comparison of that of wildtype animals at 5 • c ambient temperature. our studies demonstrate that oxtr plays a critical role in regulating several aspects of social behavior and the other physiological function, and may have important implications for developmental psychiatric disorders, such as autism. research funds: grant-in-aid for scientific research (b) (14360046) sy2-2-08-1 cortical mechanisms mediating visuomotor control of primate grasp roger n. lemon ucl institute of neurology, uk primates demonstrate an exquisite ability to precisely shape their hand when grasping an object. a network of parietal and frontal motor areas is thought to play a key role in this behaviour. our work shows that: hand shape can be unambiguously determined from emg activity of hand and digit muscles. information about grasp is represented by neuronal populations in the ventral premotor cortex (area f5); f5 activity shows graspspecific discharge soon after an object becomes visible, well in advance of activity in primary motor cortex (m1). local field potential activity in f5 and m1 is also tuned to grasp, and there is strong beta coherence between f5 and m1, indicating reciprocal transmission of information. this is also seen in synaptic responses of m1 neurones to stimulation of f5 (and vice-versa). single pulse stimulation in f5 strongly modulates corticospinal outputs from m1 through corticocortical pathways between these two areas. paired-pulse tms can probe the excitability of these pathways in humans. facilitation of meps is both object and muscle specific and indicates that activity in these pathways is selectively enhanced during object grasp. research funds: wellcome trust, bbsrc sy2-2-08-2 where tactile signals are ordered in time shigeru kitazawa 1,2 1 department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; 2 crest, japan science and technology agency, saitama, japan how does the brain order successive events? it is generally accepted that the brain can resolve the order of two stimuli that are separated in time by 30 ms. this applies to temporal order judgment of two tactile stimuli, delivered one to each hand, as long as the arms are uncrossed. however, crossing the arms caused misreporting (that is, inverting) of the temporal order. the reversal was not due to simple confusion of hands, because correct judgment was recovered at longer intervals (e.g., 1.5 s). when the stimuli were delivered to the tips of sticks held in each hand, the judgment was altered by crossing the sticks without changing the spatial locations of the hands. we recently found that temporal order judgments of tactile stimuli are strongly affected by visual distractors and/or eye movements. the results suggest that tactile stimuli are ordered in time only after they are referred to relevant locations in space, where multiple modalities of sensory signals converge. results from functional imaging support this idea. sy2-2-08-3 decision making and underlying neural mechanisms-auditory-visual ambiguity solving and preference shinsuke shimojo 1,2 1 biology/cns, california institute of technology, pasadena, ca, usa; 2 jst.erato shimojo implicit brain function project, atsugi, japan we explore mechanisms underlying crossmodal ambiguity solving (passive decision), and preference (active decision). we've employed the illusory flash effect, where a single flash appears to be doubled when accompanied by two sounds. 122-channel meg was employed, while the observer reported number of flashes. partial directed coherence was applied to see if there was a causal influence by the auditory on the visual cortices. the results indicate a strong causal influence in a-v direction in alpha (8) (9) (10) (11) (12) and ranges only in the illusion-reported trials, while stimulus parameters were identical. no such difference was found in v-a direction. for preference, the observer's gaze shifted towards the to-be-chosen stimulus (face) before conscious decision. our fmri study shows activity specific to preference task in the ventral amygdala and the ventromedial prefrontal. while such results enable the same causality analysis, it also raises a question as to what determines active/passive nature of decision. research funds: jst.erato, hfsp sy2-2-08-4 why look there? insights from spatial neglect and the medial frontal cortex masud husain ucl institute of neurology, uk why do we look where we do? studies in humans show that when we look at a scene, our initial fixation patterns can be predicted to a high degree of accuracy. our eyes go to the most salient locations where local feature contrast is greatest. these findings have led to the concept of a salience map which directs attention and gaze bottomup. in humans, damage to the right posterior parietal cortex often leads to dramatic neglect of the left side of space. recent research has begun to unravel the components of this syndrome, demonstrating several underlying mechanisms. these include a disturbance of the salience representation, a failure to keep track of spatial locations across saccades and difficulty in sustaining attention over time. gaze is directed not only bottom-up by but also top-down by voluntary mechanisms. our recent investigations of human medial frontal regions reveal important roles for the supplementary eye field and the pre-supplementary motor areas in the control of competing eye movement plans and deciding where to look. parietal and medial frontal gaze regions appear to play different, complementary roles in controlling why we look where we do. research funds: wellcome trust (061140) sy2-2-08-5 recognizing self actions through externalized eyes atsushi iriki 1,2 1 symbolic cognitive development, riken brain science institute, saitama, japan; 2 cognitive neurobiology, tokyo medical and dental university, tokyo, japan we can recognize ourselves and our own actions through the mirror or video images. thus, human can use such apparatus as externalized eyes (sensory tools), while non-human animals can normally use tools as extension of their effectors (motor tools) at most. human fmri studies revealed that the right temporo-parietal junction region and the mesial superior frontal gyrus are involved in perceiving and manipulating the representation of the self actions under different third person perspectives. japanese monkeys could be trained to use a hand-held video camera as a manipulable extension of their eyes only when their own vision was gradually transferred to the distant cues via motor-tools to extend their body images. the emergence of novel cortico-cortical projections between temporo-parietal junction and the intra-parietal cortex was described in monkeys that were trained to use motor tools, therefore, integrate the tool in their own body image. thus, presence of a self-objectification mechanism is suggested for acquisition of sensory tools as externalized eyes to recognize self actions. yoshiyuki kubota division of cerebral circuitry, nips, okazaki, japan gabaergic nonpyramidal cells in the neocortex are composed of several different subtypes. we found that most of gabaergic cell types, including fs basket and somatostatin martinotti cells, that innervate dendritic spines in addition to the somata and dendritic shafts. most postsynaptic spines also received an asymmetrical input, called double innervated (di) spines. to better characterize the other asymmetrical input on the di spines, excitatory presynaptic terminals were stained by immunohistochemistry for two types of vesicular glutamate transporters (vgluts): vglut1, existing mostly in cortical pyramidal cells, and vglut2, found in thalamocortical fibers. gabaergic inputs were rarely observed in spines innervated by vglut1-expressing terminals (n = 289), but were found in-10% of spines innervated also by vglut2-expressing terminals (n = 444). symmetrical synapses on di spines were positive for gaba, as shown by postembedding immunohistochemistry. these results indicated that some thalamocortical inputs are likely selectively inhibited at the spine level by gabaergic synapses from cortical nonpyramidal cells. research funds: kakenhi sy2-3-03-2 gabaergic recruitment of excitation by cortical axo-axonic cells gabor tamas, csaba varga, gabor molnar, szabolcs olah, pal barzo, janos szabadics university of szeged, hungary the axon has the lowest threshold for action potential generation and axons in the cerebral cortex receive input only at the axon initial segment exclusively from axo-axonic cells (aacs), which use the dominant inhibitory neurotransmitter, gamma-aminobutyric acid (gaba). thus, aacs are considered as strategically placed inhibitory neurons controlling cortical information flow. we applied multiple patch clamp recordings in slices of rat and human neocortex and found that single spikes in aacs can trigger action potentials in pyramidal cells and initiate stereotyped series of multiple synaptic events in the cortical network. the excitatory action of aacs is based on a depolarized reversal potential for axonal relative to perisomatic gabaergic inputs as determined in paired perforated patch recordings. powerful axo-axonic depolarization from the resting membrane potential is supported by a ∼44-fold decrease in the potassium-chloride co-transporter 2 (kcc2) expression from somatic to axon initial segment membranes detected by quantitative immunogold labeling. in my talk i will describe the integrative and plasticity properties of thin basal dendrites of cortical pyramidal neurons. these dendrites receive the majority of the cells' synaptic inputs, so determining their integrative and plasticity properties is of prime importance. previous studies have most often reported global linear or sublinear summation in these dendrites. using confocal imaging and dual-site focal synaptic stimulation of identified thin dendrites in rat neocortical pyramidal neurons we show that thin dendrites provide a layer of independent computational "subunits" that sigmoidally modulate their inputs prior to global summation. next i will describe the plasticity rules used by these fine basal dendrites putting a special emphasis on the role of nmda-spike in local synaptic plasticity processes. yumiko yoshimura department of visual neuroscience, research institute environmental medicine, nagoya university, nagoya, japan neocortical circuits contain fine-scale networks of excitatory neurons interconnected precisely. we previously showed that layer 2/3 pyramidal cells in visual cortex share common excitatory inputs from layer 4 and from within layer 2/3, when they are directly connected. here, we tested whether inhibitory cells are incorporated into the fine-scale specificity of excitatory connections. we recorded photostimulation-evoked synaptic currents from pairs of layer 2/3 cells, consisting of one inhibitory cell and one pyramidal cell in rat visual cortex slices, and measured the extent of common inputs to the pairs based on cross-correlation analysis. fast spiking inhibitory cells shared extensive common excitatory inputs with neighboring pyramids only when the pairs of cells were reciprocally connected. adapting inhibitory cells shared little or no common input with neighboring pyramids, regardless of their direct connectivity. therefore, fine-scale specificity depends on the type of inhibitory cell and on the direct connectivity between neighboring pyramidal-inhibitory cell pairs. research funds: kakenhi (17023026,17500208) sy2-3-03-5 local circuitry of cortical inhibitory neurons edward callaway, takuma mori, xiangmin xu the salk institute, usa we used laser scanning photostimulation to map local input to inhibitory neurons in layer 1 of rat visual cortex and layer 2/3 of mouse barrel cortex. mouse studies used transgenic animals with gfp expressed in subsets of inhibitory neurons. in layer 1, axondescending cells receive excitatory input predominantly from layer 2/3 while neurogliaform cells receive stronger input from deeper layers. layer 2/3 neurons also receive inputs that vary systematically by cell type. two subtypes of martinotti cells, distinguished by calretinin (cr) expression, also differ in morphology and intrinsic physiology. cr+ martinotti cells receive excitatory input predominantly from layer 2/3, while the cr− martinotti cells also receive strong excitation from layer 4. irregular-spiking basket cells also receive strong excitatory input from layers 2/3 and 4, but they often have a gap at the top of layer 4, with little or no input. fast-spiking basket cells and pyramidal cells in mouse barrel cortex receive input indistinguishable from cells in rat visual cortex, with strong input from layers 2/3 and 4, and only weak input from deeper layers. research funds: nih sy2-3-03-6 physiological genomics of cortical microcircuits sacha b. nelson brandeis university, czech republic cortical microcircuits are comprised of highly diverse neuronal cell types that differ in their morphology, synaptic connectivity and intrinsic electrophysiology. presumably, these phenotypic differences are orchestrated and maintained by unique transcriptional programs. in order to begin to reveal those programs we have recently developed methods for measuring genome-wide gene expression from small numbers (30-50) of fluorescently labeled, hand-sorted neurons. subsets of pyramidal neurons and gabaergic interneurons were labeled genetically with gfp or anatomically with fluorescent microspheres. labeled neurons were characterized electrophysiologically and sorted for expression analysis. the resulting expression profiles revealed highly diverse expression of molecules involved in cell-cell signaling and cell-cell adhesion, as well as transcription factors. based on this diversity of expression we constructed a taxonomic tree using an unsupervised clustering algorithm, that correctly reflects known relationships between cortical cell types. research funds: r03 ey015273 , mcknight neuroscience of brain disorders award sy2-3-09-1 axon guidance mediated by localized ca 2+ signals in the growth cone hiroyuki kamiguchi laboratory for neuronal growth mechanisms, riken brain science institute, wako, japan axonal growth cones migrate along the correct paths, not only directed by guidance cues but also contacted by local environment via cell adhesion molecules (cams). many guidance cues attract or repel the growth cone via asymmetric ca 2+ signals. its turning direction depends on the occurrence of ca 2+ -induced ca 2+ release (cicr) through the ryanodine receptor type 3 (ryr3). the activity of ryr3 is controlled by cams via camp and pka. in this way, axon-guiding and cam-derived signals are integrated by ryr3, that serves as a key regulator of axon guidance. attractive ca 2+ signals facilitate intracellular membrane transport to the leading front and subsequent vamp2-mediated exocytosis on the side with elevated ca 2+ . in contrast, repulsive ca 2+ signals do not trigger such membrane dynamics. growth cone attraction, but not repulsion, is prevented by inhibition of vamp2-mediated exocytosis. the results indicate that growth cone attraction is driven by asymmetric membrane dynamics and that growth cone repulsion is driven by different mechanisms, not simply by changing the left/right polarity of the same molecular machinery. sy2-3-09-2 molecular mechanisms for signaling through plexin-a1 hitoshi kikutani, atsushi kumanogoh, toshihiko toyofuku research institute for microbial diseases, osaka university, suita, japan sema3a acts as a guidance cue for axons through a receptor complex comprising neuropilin-1 as the ligand-binding subunit and plexin-a1 as the signal-transducing subunit. the ferm domain-containing gef, farp2, associates directly with plexin-a1. sema3a induces the dissociation of farp2 from plexin-a1, resulting in activation of farp2's rac gef activity, rnd1 recruitment to plexin-a1 and down regulation of r-ras. simultaneously, the ferm domain of farp2 sequesters pipki␥661 from talin, thereby inhibiting its kinase activity. these activities are necessary for sema3a-mediated repulsion of outgrowing axons. plexin-a1 also functions as a ligand binding receptor of a transmembrane semaphorin, sema6d and contributes to cardiac morphogenesis. sema6d exerts a migration-inhibitory activity on cells from the ventricle and a migration-promoting activity on those from the conotruncal segment. plexin-a1 forms a receptor complex with off-track in the ventricle or with vegf receptor type 2 in the conotruncal segment, which are responsible for the effects of sema6d on the respective regions. research funds: crest sy2-3-09-3 axonal transport elicited by axon guidance molecules yoshio goshima department of molecular pharmacology & neurobiology, yokohama city university graduate school of medicine, yokohama, japan for the wiring of individual neurons together into an orderly network, control by axon guidance molecules of navigation to their targets is a critical event, and molecular components destined for specific subcellular domains of neuron must be targeted correctly. we previously reported that semaphorins3a (sema3a), a repulsive axon guidance cue, increases the rate of bi-directional axonal transport in dorsal root ganglia (drg) . to address if the individual molecules rides on the sema3a-facilitated cargo, we used time-lapse imaging of several egfp-fusion proteins expressed in drg. sema3a stimulated the transport of neuropilin-1::egfp, plexin-a4::egfp, and fyn::egfp, which are the components of sema3a signaling, but not neuropilin-2::egfp. interestingly, sema3a accelerated the anterograde transport of trka::egfp. these facts suggest that sema3a selectively facilitates the transport of its own signaling components and that sema3a may modulate ngf-sensitivity of neurites by accelerating the transport of trka. kozo kaibuchi department of cell pharmacology, nagoya university graduate school of medicine, japan neurons are one of the most highly polarized cells, comprised of two structurally and functionally distinct parts, axon and dendrites. however, it remains largely unknown how neuronal polarity is established. we previously showed that collapsin response mediator protein-2 (crmp-2) is enriched in growing axon, and play a crucial role in axon specification. crmp-2 interacts with tubulin dimers to promote microtubule-assembly, and binds to sra-1, an effector of rac1 to regulate wave-dependent reorganization of actin filaments. crmp-2 links kinesin-1 to tubulin dimmers and sra-1, and participates in the kinesin-1-dependent transport of tubulin dimmers and the sra-1/wave complex to growing axons. we also found that the par-6/par-3 complex and the ras/pi3-kinase/akt/gsk-3b pathway are involved in neuronal polarization. akt appears to phosphorylate gsk-3b and inactivates its kinase activity downstream of ras/pi3-kinase, thereby increasing non-phosphorylated active crmp-2 which promotes axon growth. this time, i summarize and discuss functions of these polarity molecules in regulation of neuronal polarity. research funds: grant-in-aid for creative scientific research sy2-3-09-5 regulation of actin dynamics during neurite initiation and axon guidance frank gertler, adam kwiatkowski, doug rubinson, erik dent, leslie mebane mit, usa nervous system development requires extensive cell migration and elaboration of neurites that become axons and dendrites. axons are guided to their targets by motile growth cones. both whole cell and growth cone migration involve dynamic remodeling of the actin and microtubule cytoskeleton in response to environmental cues. the ena/vasp protein family regulates cell migration and axon guidance. ena/vasp proteins modulate actin remodeling and promote the formation of long, sparsely branched actin networks, such as those found in filopodia. results of recent work on phenotypes arising in mice lacking all three ena/vasp proteins (mena, vasp, evl) will be presented. such animals exhibit a "cobblestone cortex" in which groups of neurons migrate beyond the pial membrane. the mutants also contain little if any cortical axonal fiber tracts. analysis of primary cells from the mutants indicates ena/vasp function is required for neurite initiation. mutant neurons express differentiation markers but form few, if any filopodia and exhibit alterations in actin-microtubule interactions. kimitaka anami department of psychiatry, national center hospital for mental, nervous and muscular disorders, ncnp, tokyo, japan recent years, studies using eeg and fmri in simultaneous manner has become flourished. this is because the feasibility that any eeg events is, in principle, able to be mapped on the mri tomographic view has attracted many researchers. applications of this methodology are to basic eeg researches including event-related potentials and background activities, and as clinical aspect, localization of epileptic foci. and applications of this methodology is not matured yet but still developing. in this presentation, we will introduce our study using this method to epilepsy and to other eeg events. masaya misaki 1,2 , takashi abe 1,2,3 , shigeyuki kan 1,4 , satoru miyauchi 1,5 1 national institute of information and communications technology, kobe, japan; 2 japan society for the promotion of science, tokyo, japan; 3 graduate school of biosphere sciences, hiroshima university, higashi-hiroshim, japan; 4 kyushu institute of technology, kitakyushu, japan; 5 crest, japan science and technology agency, tokyo, japan recording fmri and an eeg simultaneously is effective for studying spontaneous brain activities. we used this method to examine the relationship between an eeg rhythm and a bold signal. some studies have hypothesized that the hemodynamic response for a change in power of certain eeg frequency bands, such as alpha waves, is canonical in shape. however, the appropriate response shape for a change in the rhythmic eeg has not yet been determined. we measured the eeg and fmri simultaneously while subjects were in a resting or sleeping state. we applied nonlinear regression analysis using an artificial neural network to detect correlations between the changes in rhythmic eeg waves and fmri signals without a priori hypothesis of the response shape. research funds: crest of jst and grant-in-aid for jsps fellows norihiro sadato, hiroshi toyoda department of cerebral research, national institute for physiological sciences, okazaki, japan previous studies have demonstrated a nonlinear relationship between blood oxygenation level dependent (bold) response and stimulus parameters. however the origin of this nonlinearity still remains unclear. to investigate the origin for the nonlinearity of bold response, we underwent simultaneous measurement of fmri and near infrared spectroscopy (nirs) . temporal dynamics of the responses in oxy-, deoxy-and total hemoglobin concentrations as well as bold signal were simultaneously measured during a visual stimulation with various durations. to quantify the degree of the nonlinearity of responses, we introduced a model using an impulse response function modified with additional nonlinearity scaling. this model was applied to the nirs measures as well as bold responses. the nonlinearity of the response in oxygen extraction fraction (oef) was also estimated from nirs measures. the non-linearity of bold was almost identical to oef across the wide range of nonlinearity of the neuronal responses. and hence we conclude that the non-linearity of bold responses to the neural activity is mainly due to the nonlinear response of oef. the bold-fmri signal is ambiguous regarding the underlying neurophysiology. in our work we attempt a) to better understand the neurophysiological basis of fmri and b) to improve on the information obtained by functional brain imaging by adding additional information, e.g. obtained by electrophysiological measurements. in one series of experiments, we combined transcranial magnetic stimulation with near-infrared imaging in order to clarify how changes in deoxy-hb concentration (the inverse of bold) is related to neuronal inhibition. in another series of experiments, we combine eeg with fmri in order to identify bold correlates of neuronal background rhythms such as alpha rhythm, mu rhythm, etc. in a third series of experiments, we combine fmri with the measurement of high-frequency oscillations in eeg. the latter is an expression of evoked spike burst in the somatosensory cortex, i.e. this kind of measurements adds the information about action potentials to fmri haruhiko akiyama tokyo institute of psychiatry, japan activation of microglia is a part of host defense mechanisms in the brain. microglia remove invading microorganisms as well as cell debris that contains hazardous substances such as lysosomal proteases. brain is particularly vulnerable to the immune and inflammatory attacks and, therefore, has multiple mechanisms that regulate the immune and inflammatory responses more strictly than other organs. nevertheless, many neurodegenerative lesions are associated with activated microglia and low-grade, but sustained, inflammation. neuroinflammation is a term that refers to such conditions. in alzheimer's disease (ad), microglia play a central role for phagocytic removal of amyloid beta-protein (abeta) from the brain. the process is enhanced by complement activation. however, these cellular and humoral responses to abeta may be toxic to neurons in ad. neuroinflammation could be a double-edged sword in the brain. in patients with neurodegenerative diseases, complication of systemic inflammatory diseases, depletion of some neurotransmitters such as catecholamines, and the presence of brain lesions may adjunctly upregulate neuroinflammation, which further accelerates neuronal degeneration. makoto sawada department of brain life science, riem, nagoya university, japan microglia, macrophage-like cells in the cns, are multi-functional cells; they play an important role in removal of dead cells or their remnants by phagocytosis in the cns degeneration as well as are one of important cells in the cns cytokine network. they are thought to be originated from mesoderm, and to be similar cells to other tissue-resident macrophages. activated microglia have been shown to remove potentially deleterious debris and promote tissue repair by secreting neurotrophic factors at the neuronal injury sites, however, they can release potentially cytotoxic substances in vitro, and at least so-called fully activated form of microglia which are observed at the injury site in aids dementia is completely neurotoxic. these suggest that some factor(s) may contribute to change microglial phenotype from protective to toxic, but the detail is not clear. recently we generated hiv-derived nef protein tranduced microglia. they are found to increase both the potential to produce o2-and mpo-like peroxidase activity resulting in the neurotoxicity. therefore, the target protein(s) of nef might to be involved in the control of microglial neurotoxicity. there is abundant evidence that extracellular atp have an important role in pain signaling. the focus of attention now is on the possibility that atp receptor of microglia might be involved in neuropathic pain. neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. this type of pain state is generally resistant to currently available treatments. we recently reported that the expression of p2x4 receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly p2x4 receptors produce a reduction of the neuropathic pain behaviour (2003. nature 424, 778-783) . more recently, we have reported that brain-derived neurotrophic factor (bdnf) released from microglia by the stimulation of p2x4 causes the depolarizing shift in reversal potential of anion in li neurons of rats with nerve injury (2005 ( . nature 438, 1017 ( -1021 . understanding the key roles of these atp receptors may lead to new strategies for the management of neuropathic pain. research funds: kakenhi (15209051) sy2-5-05-4 pet imaging of microglia using peripheral benzodiazepine ligands richard b. banati university of sydney, australia brain disease often results in significant changes in the functional state of microglia, the brain's resident tissue macrophages. the response is thought to be an important step in the pathophysiology of traumatic, inflammatory, neoplastic and degenerative brain disease. part of the structural and functional plasticity of microglia is the de novo expression of the 18 kda transposor protein or "peripheral benzodiazepine binding site" (pbbs). the pbbs is bound by the isoquinoline pk11195, which labeled with carbon-11 can be used for positron emission tomography (pet). using 11c-(r)-pk11195 pet in inflammatory and neurodegenerative brain disease as well as receptor autoradiography, we have shown that distributed regional pbbs up-regulation correlates with clinical deficit and mirrors the histologically described activation of microglia in the penumbra of focal lesions, but importantly also in the distant, anterograde and retrograde projection areas of the lesioned neural pathway. sy2-5-10-1 application of simulation of light propagation in tissue to nirs imaging of brain function eiji okada department of electronics and electrical engineering, keio university, japan in nirs imaging, the functional image is obtained from the variation in intensity of detected light caused by concentration change in haemoglobin in cortical tissue. the serious problem of nirs imaging is ambiguity in light propagation in the head caused by the scattering of tissue. this poses results in poor spatial resolution and contrast in the image. the development of simulation model to calculate light propagation in the head to deduce the path length in the brain and the spatial sensitivity profile is very important to improve the nirs imaging. in this study, simulation of light propagation in the head model for nirs imaging is briefly reviewed. the heterogeneity of the tissues in the head, especially low-scattering cerebrospinal fluid (csf), has a strong effect on the light propagation in the brain. the sensitivity to concentration change in haemoglobin in the cortical tissue is improved by the effect of the csf. the simulation of nirs imaging indicates that the intensity and size of activated region in the functional image depend on the relative position of activated region to fibre pairs. yoko hoshi integrated neuroscience research team, tokyo institute of psychiatry, tokyo, japan quantification of near-infrared spectroscopy (nirs) data has been a central issue in the nirs field. over the past 25 years, many approaches to quantification have been tried, and in the case that hb concentration changes are global within the tissue, the quantitative accuracy of time-resolved spectroscopy (trs) and phase-resolved spectroscopy (prs) has been established. when the changes are localized, however, as with functional brain activation, the difficulty of quantification has not yet been fully overcome because elimination of the effects of hemodynamic changes in the extracerebral tissue is also challenging. the temporal profile of detected light intensity measured by trs carries information about depth-dependent attenuation, because light that penetrated into deeper positions in the head is detected later. thus, several time-domain methods to determine absorption changes with depth resolution have been proposed. diffuse optical tomography (dot) is also a potential technique for quantitative detection of focal changes in cerebral hemodynamics. in this symposium, i will outline these approaches. sy2-5-10-3 brain functional imaging in cerebral ischemic disorders: comparison of nirs and fmri kaoru sakatani department of neurological surgery, nihon university school of medicine, tokyo, japan we compared the evoked cerebral blood oxygenation (cbo) responses measured by nirs and activation maps of bold-fmri in stroke patients with mild and severe (misery perfusion) cerebral ischemia (ci). in the age-matched controls, deoxyhemoglobin concentrations decreased with increases in oxyhemoglobin and total hemoglobin in the primary sensorimotor cortex (psmc) during contralateral motor tasks. the psmc on the non-lesion side exhibits normal cbo response pattern. on the lesion side, the mild ci did not affect the cbo response pattern, but the severe ci caused an increase of deoxyhemoglobin during the task associated with increases of oxyhemoglobin and total hemoglobin. the mild ci caused only slight reduction of the activation volumes of bold imaging on the lesion side; however, the severe ci, caused markedly reduction of the activation volumes on the lesion side. misery perfusion caused marked reductions of activation volumes of bold imaging associated with an increase of deoxyhemoglobin during activation. bold-fmri should be performed, giving consideration to the baseline circulatory conditions. masato fukuda, toru uehara, masahiko mikuni department of psychiatry and human behavior, gunma university graduate school of medicine, gunma, japan near-infrared spectroscopy (nirs) has been increasingly employed in psychiatry researches such as personality (2005 . neuropsychobiology 52, 45), aging (2004 . neuroimage 22, 1715 , and psychiatric disorders ("progress in schizophrenia research", nova science publishers, 2006) . frontal lobe reactivity was investigated using multichannel nirs machines in unipolar depression, bipolar depression, and schizophrenia (2004. biol. psychiatry 55, 501; 2006. neuroimage 29, 172) by monitoring changes of oxy-hemoglobin concentration ([oxy-hb]) every 0.1s during a verbal fluency task. the unipolar depression was characterized by smaller [oxy-hb] increase, the bipolar depression by comparable but delayed [oxy-hb] increase, and the schizophrenia by reduced [oxy-hb] increase during the task period followed by [oxy-hb] re-increase during the post-task period, suggesting reduced, preserved but delayed, and inefficient frontal lobe reactivity, respectively. collaborators: itsuro ida, akihiko oshima, makoto ito, tomohiro suto, masaki kameyama, yutaka yamagishi, toshimasa sato, masashi suda sy2-5-10-5 clinical application of nirs to neurorehabilitation optical imaging using near-infrared spectroscopy (nirs) is suitable for assessing cortical activation during human gait because of its flexibility and portability. in healthy subjects, walking induced increase of oxygenated hemoglobin levels that centered in the medial sensorimotor cortex and supplementary motor area. in hemiparetic patients with stroke, cortical activation was characterized by asymmetrical activation in the sensorimotor cortex and recruitment of the premotor and prefrontal cortex. a longitudinal study revealed that locomotor recovery was associated with improvement of asymmetrical activation in the sensorimotor cortex as well as enhanced activation in the premotor cortex. sensorimotor stimulation by facilitation technique induced enhanced activation in the motor related areas, particularly in the premotor cortex. partial body weight support and speed-dependent exercise decreased sensorimotor activation, suggesting relative shift of locomotor control to the hierarchically lower structures including the spinal cord. thus nirs may contribute to establishing brain-based strategies for neurorehabilitation. research funds: funds for comprehensive research of aging and health sy2-5-10-6 measurement of language related brain activities during recovery from aphasia eiju watanabe 1 , yumiko muroi 2 , chizuru nakajima 2 1 department of neurosurgery, jichi medical university, tochigi, japan; 2 department of neurosurgery, tokyo metropolitan police hospital, tokyo mechanism which supports the recovery of language after aphasia is not well understood. it has long been discussed that language related areas including the regions surrounding the language area and compatible cortex on non-dominant side could be candidates which support the recovery. several studies suggest the compensation by these areas using fmri and pet. we used optical topography (ot) to know the participation of these areas during the recovery from the aphasia. we measured 17 aphasics who showed recovery from the aphasia after apoplexy. word generation task was used. in seven cases ot was measured more that twice. seven cases showed the activity on the non-dominant frontal lobe. they all showed activities on the dominant frontal lobe in the follow-up measurements along with the deactivation of non-dominant side. these findings showed dynamic participation of non-dominant frontal lobe during the recovery phase suggesting that the rehabilitation protocol should be changed according to the area activated in each phase. tamami fukushi research institute of science and technology for society (ristex), japan science and technology agency (jst), tokyo, japan recent development of neuroscience has provided remarkable scientific discoveries, as well many newer philosophical, ethical, legal and social issues. for example, the consequences of the progress of non-invasive neuroimaging technologies, such as functional magnetic resonance imaging (fmri) and near infrared spectroscopy (nirs) show the possibility to read the mind of others, which may lead the criminal and commercial applications. brain-machine interface (bmi) technology and pharmacological manipulation of the human brain can cause the unpredictable enhancement of human ability. in advance to the expansion of "applied neuroscience", neuroscientists should consider "what the ethical problem is in the current neuroscience" and "how we learn and interact with the ethics". in this symposium, the panels will talk about the history of neuroethics then provide the ethical aspects of basic research. we will also discuss the future perspective of the neuroethics in japan and world in terms of sharing the problems across neuroscientists, ethicists, mass media, and public. judy illes stanford university, usa akin to genetic testing in the 1990s, the translation of neuroimaging capabilities from the laboratory to the clinical setting has raised ethical questions about how new diagnostic and predictive information will be managed in the absence of effective treatments for certain diseases, about the timing of technology transfer and handling of technology that falls in the regulatory gray zone between research and clinical use, and what impact increasing opportunities for selfreferral to health care will have on patient-physician relationships, medicine, and society overall. potential off-label uses of advanced neuroimaging outside the health care setting -in law, education, employment and even for national security -are already being tested and debated. we will discuss how these issues converge in 21st century neuroethics, the presence of neuroethics in the international domain, and the critical role of ethics in neuroscience in the future. sy2-6-06-3 neuroethics from primate's eyes naotaka fujii bsi, laboratory for symbolic cognitive development, japan neurophysiologists working on monkeys have been trying to understanding how their brains are working. the aim of the studies was not merely revealing functions of primate's brain but also trying to extrapolate the findings in primates onto human brain functions. this was true but not really true due to technical limitations which prevented us from expanding the findings in primates directly to the human brain function. however, recent advancement of technology has changed the world. findings in primates can be directly applied to human studies with or without researcher's intention. technologies invented in primate physiology are now readily transferred into human without much discussion. brain machine interface is one example. now, monkey's brains are forcing us to think about social impact of our research from ethical view, which we have not discussed before. as an experienced primate neurophysiologist but with little ethical training, i will discuss 'what is ethically correct primate research' and 'how our scientific contribution has to be made' from very practical and ground level of neuroethics. sy2-6-06-4 neuroethics of nurturing the brain takao k. hensch critical period mechanisms group, riken brain science institute, japan at no time in life is the brain so easily shaped by experience than in infancy and in early childhood. it is during these critical periods that neural circuits acquire language and other basic brain functions. unraveling mechanisms that limit such dramatic plasticity to early life would pave the way for novel paradigms or therapeutic agents for rehabilitation, recovery from injury or improved learning in adulthood. conversely, a deeper insight into early postnatal brain development will provide valuable inspiration for effective brain-based education methods for our children-perhaps the greatest potential contribution of neuroscience to society. this raises urgent and important ethical questions for our consideration: is there an "ideal" brain we should be nurturing? to what extent can/should drugs be used not merely to correct developmental disorders but also to enhance performance? how do we determine what is good or bad for the maturing brain? research funds: riken bsi sy2-6-06-5 neuroethics beyond laboratories and across cultures daofen chen national institute of neurological disorders and stroke, usa recent progress in systems and cognitive neuroscience poses new ethical challenges to both investigators and to the funding agencies that support scientific investigations. potential uses of many of these recent advances go beyond their intended medical applications. a growing array of neurotechnologies capable of monitoring or even intervening in human cognition makes it imperative to consider the social, ethical, and legal implications of these scientific advances. while it once might had been possible to conduct research with naive ignorance of its societal implications, this is no longer the case. moreover, we need to be cognizant that modern brain science is profoundly influenced by the distinct cultural and social values held by different sectors of the world population. we will discuss what can be done from the perspective of funding agencies to facilitate intercultural dialogue, foster mutual understanding, and develop a framework and strategies to address emerging neuroethical issues and prioritize our future efforts in neuroscience research. sy2-6-06-6 bridging neuroscience and public: neuroethics in cultural contexts osamu sakura 1,2 1 interfaculty initiative in information studies, university of tokyo, tokyo, japan; 2 research institute of science and technology for society (ristex), jst, japan to bridge between neurosciences and public society-it should be one of the important aims of neuroethics. for this purpose we need to draw the outline of neuroscience within the cultural context. the method and the result of natural sciences are universal, of course, but its social consequences are highly variable among cultures. although the systematic comparative researches are open to the future, we should discuss how the neurosciences could create the healthy relationship between the public society, especially focusing on the method for public participation and on the previous successful cases. mitsuru kawamura 1,2 , akira midorikawa 3 , yoshiki kaneoke 4 , shinichi koyama 1 , masato taira 5 , argye hillis 6 1 showa university school of medicine, japan; 2 crest, jst, saitama, japan; 3 national institute of neuroscience, ncnp, tokyo, japan; 4 national institute for physiological sciences, okazaki, japan; 5 nihon university, tokyo, japan; 6 johns hopkins university, baltimore, usa this symposium aims to provide an opportunity to talk between clinical neuropsychologists and neuroscientists. focusing on the visual system, we will discuss up-to-date studies from neuropsychological and neuroscientific viewpoints. the topics include motion perception in brain-damaged patients, neuroimaging of motion perception, surface and depth perception in brain-damaged patients, and neuroimaging of surface and depth perception, and neuropsychological and neuroimaging studies of visual attention. we will discuss consistency and inconsistency of our findings, and discuss what to do in order to produce synergy between clinical neuropsychology and neuroscience. research funds: kakenhi (17022035), crest sy2-6-11-2 impairment of surface perception in patients with ventral occipital damages shinichi koyama 1 , mitsuru kawamura 1 , akira midorikawa 2 , yoshiki kaneoke 3 , masato taira 4 , argye hillis 5 1 showa university, tokyo, japan; 2 national institute of neuroscience, ncnp, tokyo, japan; 3 national institute for physiological sciences, okazaki, japan; 4 nihon university, tokyo, japan; 5 johns hopkins university, baltimore, usa we examined the perception of faces and objects in two patients with ventral occipital damages, using psychophysical techniques. patient 1 was a 61-year-old woman with bilateral damage in the fusiform and parahippocampal gyri. although she could recognize pictures of famous people, she frequently failed to decide the races of unfamiliar faces and occasionally failed to see the hollow-face illusion (gregory 1970) . in addition, her performance in object identification task became poorer when the objects were presented in inverted (negative) pictures. patient 2 was a 63-year-old men with bilateral damage in the lingual and fusiform gyri. his recognition of faces and objects became poorer when they were presented in inverted pictures. based on the above results, the role of the ventral visual cortex in the perception of faces and objects will be discussed. research funds: grant-in-aid for jsps fellows sy2-6-11-3 how do pictorial cues influence 3d information processing in the parietal association cortex? masato taira 1,2 1 arish, nihon university, tokyo, japan; 2 department of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan pictorial cues are one of the most influenced cues for 3d perception. basically, it is thought that the parietal association cortex processes 3d visual information by binocular disparity cues and the temporal association cortex processes that by pictorial cues in the concept of two visual information processing systems in the brain. however, recent studies have suggested that there are many crosstalk of 3d information between these association areas. our recent studies have shown that a group of neurons in the parietal cortex (area cip) is involved in perception of 3d surface orientation and used both disparity and pictorial cues. functional mri data in human also suggest that pictorial cues, such as attached and cast shadow, are processed in the parietal cortex in 3d perception. furthermore, human psychophysical data gives us some insights of how the pictorial cues influence 3d information processing in the parietal association cortex. research funds: kakenhi 17022038 sy2-6-11-4 case report of a patient with posterior cortial atrophy who relatively preserved perception of moving objects akira midorikawa 1 1 national center of neuroscience, national center of neurology and psychiatry, tokyo, japan it has been presented that severe type of bálint syndrome behaves like a blind person; however, it also reported that there are some cases who behave like a blind person but could walk without collision. up to today several cases have been reported, but the underlying mechanism of the phenomenon has not been mentioned. in this report, i will talk about a patient with bálint syndrome due to degenerative disease known as posterior cortical atrophy (pca), who could not only walk around without collision but also play catch very well, nevertheless having blind like behavior. the crucial visual information underlying these phenomenons was assumed to be motion parallax induced by not only objects movement but also self movement. in addition, discrepancy between the patients who could walk and not walk will be discussed. research funds: crest, japan science and technology agency sy2-6-11-5 neural mechanism underlying visual perception of motion as revealed by non-invasive human study yoshiki kaneoke department of integrative physiology, national institute for physiological sciences magnetoencephalography (meg) measures the neural activity representing the synchronized inputs to the millions of pyramidal neurons in the localized cerebral cortex. thus, it will show us another aspect of the neural activity related to the specific brain function that would not be revealed by the recording of single neuronal activity. our meg studies have revealed several important findings in the human visual motion detection system. first, the response properties for the apparent motions indicate the importance of the human mt/v5+ for the perception and the existence of the parallel processing for the motion and light blinking. second, the existence of the spatiotemporal filtering mechanism for the perception of motion speed is shown by the various motion stimuli. third, we present the evidence that the spatial integration of the speed without direction information occurs in our visual system. based on the results, scalar fields theory for the spatial integration of motion is proposed to explain various complex motion perception. research funds: kakenhi (15500221) sy2-6-11-6 neural correlates of visual attention argye hillis 1 , mitsuru kawamura 2 , akira midorikawa 3 , yoshiki kaneoke 4 , shinichi koyama 2 , masato taira 5 1 johns hopkins university, usa; 2 showa university, tokyo, japan; 3 national institute of neuroscience, ncnp, tokyo, japan; 4 national institute for physiological sciences, okazaki, japan; 5 nihon university, tokyo, japan in this paper i report a series of studies of unilateral spatial neglect (usn) in acute stroke, demonstrating a frequent double dissociation between stimulus-centered neglect and viewer-centered neglect, and showing that these types of neglect can be modality-specific. other data reveal that different types of usn are observed when there is hypoperfusion of temporal cortex versus parietal cortex. still other data provide evidence that severity of neglect depends on the volume of hypoperfused tissue in acute stroke, and that reperfusion results in early recovery of neglect. finally, i will review new evidence that right usn is common after left cortical infarcts or hypoperfusion in acute stroke, but the distribution of types of usn is very different from the distribution of types of usn after right hemisphere stroke. takeo kubota, takae hirasawa, kaoru nagai department of epigenetic medicine, university of yamanashi faculty of medicine, chuo, yamanashi, japan how are brains controlled molecularly? this is one of fundamental questions in neuroscience. several lines of evidences have suggested that genes are more strictly controlled in the brain than in other organs. epigenetics is one of such systems to control expression of the genes not based on dna sequence, but based on 'beyond the dna sequence' (chromatin modifications and small rnas). the failure of this system is known to result in neurodevelopmental diseases, such as an autistic rett syndrome. it has recently been demonstrated that the epigenetic system is affected by an environmental stress after birth, and that the system is associated with neural differentiation and cell fate determination and human brain diversity. these findings imply that epigenetics is an important research field to understand mechanisms of neural development and mental diseases. topics from update epigenetic researches in neuroscience will be discussed in this symposium. as one of epigenetic disorders, we introduce studies of rett syndrome (rtt) and its model mouse. rtt is a neurodevelopmental disorder, characterized by mental retardation and peculiar behavior. mutations of the mecp2 gene, encoding methyl-cpg binding protein 2, cause rtt. mecp2 acts as a transcriptional silencer. abnormal expression of some genes due to mecp2 dysfunction is thought to be the first step of rtt pathophysiology. to understand how mecp2 mutation makes rtt, we have two approaches that are morphological investigation of brain and discovery of mecp2-downstream genes. using mecp2-null mice (mecp2 −/y ), we revealed small number of dendritic spines and premature postsynaptic density at presymptomatic period. premature synaptogenesis may be the initial neuronal changes of rtt. we also found that mecp2 directly regulates expression of insulin-like growth factor binding protein 3 (igfbp3) gene in human and mouse brains. pathological features of mecp2 −/y have the similarity of igfbp3 transgenic mice, which show reduction of early postnatal brain growth. over-expression of igfbp3 due to lack of mecp2 may lead to delayed brain maturation. growing evidence suggests that alterations in the epigenetic status such as dna methylation and histone modifications in brain are involved in the behavioral response to environmental factors and the pathogenesis of psychiatric diseases. however, in contrast to mrna profiling, there are few established methods for profiling the genomewide epigenetic status to date. we developed a method for profiling the genome-wide dna methylation pattern using tiling arrays, and focused our analysis on human brain samples derived from psychiatric patients and control subjects. in this symposium, general picture of the genes that are heavily methylated or non-methylated in human brain, and the relationship between dna methylation and mrna expression patterns will be presented. understanding what produces neuronal diversification has been a longstanding challenge for neuroscientists. the recent finding that long interspersed nucleotide elements-1 or l1 (line-1) retroelements are active in somatic neuronal progenitor cells provided an additional mechanism for neuronal diversification. together with their mutated relatives, retroelements sequences constitute 45% of the mammalian genome with l1 elements alone representing 20%. the fact that l1 can retrotranspose in a defined window of neuronal differentiation, changing the genetic information in single neurons in an arbitrary fashion, allows the brain to develop in distinctly different ways. this characteristic of variety and flexibility may contribute to the uniqueness of an individual brain. however, the extent of the impact of l1 on the neuronal genome is unknown. the characterization of somatic neuronal diversification will not only be relevant for the understanding of brain complexity and neuronal organization in mammals but may also shed light on the differences in cognitive abilities, personality traits and many psychiatric conditions observed in humans. sy2-7-07-6 notch-induced acquisition of astrocyte differentiation potential of neural stem cells kinichi nakashima 1 , jun kohyma 1 , tetsuya taga 2 , masakazu namihira 1 1 grad. sch. biol. sci., naist, ikoma, japan; 2 inst. mol. embryol. genet., kumamoto univ., kumamoto, japan neurons and astrocytes are generated from common neural stem/precursor cells, however, neurogenesis precedes astrocytogenesis during brain development. we have previously reported that gfap-positive astrocyte differentiation is dependent on the transcriptional activity of stat3. a cpg dinucleotide in the stat3 recognition sequence within the gfap gene promoter is highly methylated at midgestation which becomes demethylated as the brain develops, enabling stat3 to induce gfap expression. thus, it is conceivable that the epigenetic modification such as dna methylation of cell type-specific gene promoter controls the switch from neurogenesis to astrocytogenesis in the developing telencephalon. here we report that neurons, which are generated earlier than astrocytes can potentiate neural precursors at midgenstation to differentiate into astrocyte through the activation of notch-signaling. the activated notch-signaling accelerates demethylation of stat3 binding element in the gfap gene promoter. sy2-8-12-1 neurogenesis and stem cell supply as therapeutic approach to overcome ischemic stroke masayasu matsumoto department of clinical neuroscience and therapeutics, hiroshima university graduate school of biomedical sciences, japan in order to overcome the brain damage caused by ischemic stroke, several strategies have been so far applied. in the present symposium, i will address the following points to be considered prior to clinical application of neurogenesis and/or stem cell supply to repair the damaged brain function. (1) which type of brain infarction will be a future target of this therapeutic approach? (2) which phase of brain infarction (i.e., acute or chronic phase) will be selected as a future timing of therapeutic intervention? (3) which will be the best way to be applied in the clinical settings, neurogenesis control, stem cell supply or both? the present research status and future directions will be presented and fully discussed. research funds: kakenhi sy2-8-12-2 gene therapy for cerebral ischemia setsuro ibayashi, hiroaki ooboshi department of medicine and clinical science, graduate school of medical sciences, kyushu university, fukuoka, japan cerebrovascular disease is the leading cause of the disabled people in japan and western countries. gene transfer technique may be applicable to the treatment of serious types of cerebrovascular disease. cerebral blood vessels have been targeted by gene transfer with intravascular or perivascular approaches. several experimental studies have revealed potential usefulness of gene therapy for prevention of vasospasm after subarachnoid hemorrhage. as for cerebral infarction, studies using various brain ischemia models have shown effectiveness of gene transfer in reduction of infarct size and functional recovery. our recent studies of post-ischemic gene transfer have provided promising results in attenuation of ischemic damages by inhibiting apoptosis, inflammation and vascular permeability. approaches to cerebral ischemia using gene transfer for angiogenesis and neurogenesis appear to be novel and promising strategies. thus, gene therapy has a potential for the future therapy against cerebral ischemia. isao date department of neurological surgery, okayama university, okayama, japan cerebral ischemia is one of the neurological disorders that cell transplantation is expected to be applied. in this presentation, the author will summarize our recent basic research and clinical application reported in the literature. it is now possible to make several types of neurotrophic factor secreting cell line by genetic manipulation. in order to prevent immunological reaction and tumor formation, we have been using encapsulated cell grafting technique. we transplanted several types of neurotrophic factor secreting cell line into the middle cerebral artery occlusion model and could confirm the histological and behavioral efficacy. we have also been using adultderived neural stem cells as donor cells because they have merits to make autografitng possible. as donor tissue, neural protection can be expected similar to fetus-derived neural stem cells. the effect of neural protection increases when neurotrophic factor secreting genes such as gdnf were inserted into neural stem cells. cell transplantation is considered a new therapeutic approach for cerebral ischemia and clinical application is expected. we now know that (1) motor function may recover after minor injury to the primary motor cortex, (2) this recovery is, at least in part, associated with reorganization of cortical motor representation, (3) the molecular mechanism for synaptic plasticity and axonal regrowth is being elucidated, and (4) recent clinical experience revealed that the motor function in patients with spinal cord injury is improved after transplantation of her/his own olfactory mucosa. furthermore, recent neuroimaging techniques can display the cortical functions as we as the specific fiber connections in individual brain. virtually any part of the brain can be approached with the accuracy of millimeters by the current image-guided neurosurgery. those theoretical and technical backgrounds suggest we might be ready for the reconstruction of brain function. nobuyuki nukina laboratory for structural neuropathology, riken brain science institute, japan a major hallmark of the polyglutamine (pq) diseases is the formation of pq inclusions. recently, misfolding has come to be considered one of the primary factors for pq protein aggregation, although, the nature of misfolding is not yet well known. the protein misfolding induced by pq expansion was investigated with our molecular model system using mutant myoglobin which is inserted different size of pq. expanded polyglutamine stretches form intramolecular and intermolecular beta sheets and amyloid fibrils. the surface of the mutant myoglobin with expanded pq was partially unfolded and destabilized. we also investigated the early phase of fibrillization by small-angle x-ray scattering and electron microscopic studies, revealing that the expansion of pq to 50 repeats induced the formation of quasi-aggregate in the earliest stage of the protein fibrillization. this structure could be closely involved in recruitment of various functional proteins into aggregates, leading to the cellular dysfunction that causes pq diseases. furthermore using cellular model system we also studied the aggregates interacting proteins (aips) by analyzing the purified polyglutamine inclusions and the lists of aip including chaperones, proteasome subunits, ubiquitin interacting proteins and others suggest the pathological role of aips in the disease cascades. sy3-1-01-3 neuronal dysfunctions in dentatorubralpallidoluysian atrophy (drpla) shoji tsuji 1 , toshiya sato 2 , mitsunori yamada 3 1 department of neurology, the university of tokyo, tokyo, japan; 2 center for bioresource-based researches, japan; 3 department of pathology, brain research institute, niigata university, niigata, japan to investigate molecular mechanisms of neurodegeneration in drpla, a polyglutamine disease caused by expansions of cag repeats of drpla gene, we have established transgenic mice harboring a single copy of the full-length human mutant drpla gene with 129 cag repeats. the q129 mice exhibited neurological phenotypes similar to juvenile type of drpla characterized by ataxia, myoclonus and epilepsy. electrophysiological studies disclosed age-dependent abnormalities in the globus pallidus and cerebellum. neuropathological studies revealed progressive brain atrophy without obvious neuronal loss and an age-dependent increase in neuronal intranuclear accumulation of mutant proteins with the regional distribution vulnerable to drpla. expression profiling analyses revealed down-regulated genes including camp responsive genes. these results suggest that "neuronal dysfunction", but not the "neuronal cell death", is the essential mechanism of neurodegeneration in drpla. huntington's disease (hd) is caused by an expansion of a cag repeat encoding polyglutamine in the huntingtin protein and involves progressive motor, cognitive and psychiatric symptoms. using a transgenic mouse model of hd, we have shown that environmental factors can dramatically modify the disease process and delay the onset and progression of motor and cognitive symptoms. further, we have attempted to correlate these behavioural findings with changes in gene expression, neuronal morphology, neurogenesis, and cortical plasticity, in an attempt to elucidate cellular and molecular mediators in hd, and understand how gene-environment interactions can modulate these pathogenic pathways. our findings indicate that the modulatory effects of environmental manipulations are mediated by amelioration of specific molecular and cellular deficits, and provide experimental paradigms for the identification of novel therapeutic targets for hd and related brain disorders. sy3-1-06-1 control of neural organization in the developing cerebral cortex yasuto tanabe mitsubishi kagaku institute of life sciences, tokyo, japan in the developing cerebral cortex, the generation of neurons with distinct identities and patterns of connectivity is controlled by a hierarchical series of cellular interactions that culminate in the laminar organization of distinct cortical areas. over the past three years we have begun to examine cerebral cortical development by focusing on three distinct major neuronal subtypes, namely, cajal-retzius cells, cortical projection neurons, and cortical interneurons. the analyses of these distinct neuronal subtypes allowed us to identify several candidate molecules and cellular interactions that might contribute to the laminar and areal organization of the cerebral cortex. in the first part of my talk, i would like to deal with the issue of ontogeny of cajal-retzius cells, and present the way cajal-retzius cells are generated, migrate and finally distribute in the developing cerebral cortex. then, i would like focus my talk on the issue of the way the acquisition of radial migration and axonal trajectory patterns of distinct cortical projection neurons is controlled during the development of the cerebral cortex. research funds: kakenhi 17390086, 17023061 sy3-1-06-2 mechanisms of the regulation of neuronal migration and corticogenesis kazunori nakajima 1,2 1 dept. of anat., keio univ. sch. of med., tokyo, japan; 2 inst. of dna med., jikei univ. sch. of med., tokyo, japan mammalian cerebral cortex has a six-layered structure where the neurons are aligned depending on their birth-date. to determine whether the migration from the ventricular zone (vz) to beneath the marginal zone (mz) is essential for neuronal segregation into layers, we investigated whether migrating neurons have different cell aggregation properties in vitro depending on their birth-dates, even before they arrive beneath the mz. we analyzed vz cells and cells from the intermediate zone (imz) mainly composed of migrating cells, and found that the cells had acquired a birth-date-dependent preferential segregation mechanism in a reelin-independent manner. these findings suggest that cortical neurons acquire a birth-date-dependent segregation property (or fate) before their somas reach the mz. in silico experiments of the reaggregation culture supported that this mechanism might indeed contribute to the layer formation in the developing cerebral cortex in concert with other mechanisms such as reelin signaling. kenji shimamura division of morphogenesis, institute of molecular embryology and genetics, kumamoto university, japan neurons of the thalamus originate in restricted regions of the proliferative zone of the diencephalic compartment before settling in their final locations in the nuclei. to investigate cellular and molecular mechanisms underlying nucleus formation, we analyzed the sequence and pattern of expression of specific markers that distinguish the subsets of neuronal precursors during development of the thalamus. we found that a morphogen-like activity of sonic hedgehog (shh) precisely defines positions of neurons with distinct properties, and that some gabaergic interneurons migrate from their birth place to distant nuclei in a highly organized manner. we also provide evidence that shh produced by the zona limitans intrathalamica (zli), which abuts the prethalamus and thalamus, is likely to be a cue for this directed migration. our results suggest that local production of prespecified neurons coupled with distinct migration properties and local guidance cues such as compartment boundaries could be principle elements for the nucleus formation. layers and nuclei are important functional units in the vertebrate cns. neurons in these structures have common physiological and anatomical features. despite their importance, mechanisms for nucleogenesis are poorly understood. we focused on the lower rhombic lip (lrl)-derived precerebellar neurons, and utilized exo utero electroporation with an enhanced yellow fluorescent protein (eyfp) gene, to study the process of nucleogenesis. after the unilateral transfer of eyfp to the lrl of embryonic day 12.5 mice, eyfp-labelled neurons migrate tangentially from the lrl in two distinct streams, one toward the ventral metencephalon and the other toward the ventral myelencephalon. the former formed the pontine grey nucleus and reticulotegmental nucleus and the latter the external cuneate nucleus and lateral reticular nucleus. before forming the clusters, the labelled neurons begin to migrate toward the ventricle along the radial fibres, and aggregate as they detach from the fibres. perturbation experiments such as introduction of dominant negative constructs and sirna suggested involvement of several molecules in the migration of these neurons. the brains of fetal alcohol syndrome patients exhibit impaired neuronal migration, but little is known about the mechanisms underlying this abnormality. here we show that ca 2+ signaling and cyclic nucleotide signaling are the central targets of alcohol action in neuronal cell migration. an acute administration of ethanol reduced the frequency of transient ca 2+ elevations in migrating neurons and cgmp levels, and increased camp levels. experimental manipulations of these second messenger pathways, through stimulating ca 2+ and cgmp signaling or inhibiting camp signaling, completely reversed the action of ethanol on neuronal migration in vitro as well as in vivo. each second-messenger has multiple but distinct downstream targets, including camkii, calcineurin, pp1, rho gtpase, mapk and pi 3 k. these results demonstrate that the aberrant migration of immature neurons in the fetal brain caused by maternal alcohol consumption may be corrected by controlling the activity of these second-messenger pathways. sy3-2-02-1 membranes, water and diffusion denis j. le bihan shfj/cea, france among 1905 einstein papers is one which unexpectedly gave birth to a powerful method to explore the brain. molecular diffusion was explained by einstein on the basis of the thermal random translational motion of molecules. in the mid 1980s it was shown that water diffusion in the brain could be imaged using mri. a dramatic application of diffusion mri has been brain ischemia, following the discovery that water diffusion drops immediately after the onset of an ischemic event, when brain cells undergo swelling through cytotoxic edema. also, water diffusion is anisotropic in white matter, because axon membranes limit molecular movement perpendicularly to the fibers. this feature can be exploited to map out the orientation in space of the white matter tracks and image brain connections. more recently, it was discovered that diffusion mri could detect transient swelling of activated cortical cells. this represents a significant breakthrough, allowing non invasive access to a fast and direct physiological marker of brain activation. this approach will bridge the gap between invasive optical imaging techniques and current functional neuroimaging approaches in humans, which are based on indirect and remote blood flow changes. sy3-2-02-2 diffusion tensor fiber tractography using a 3tesla mr system yukio miki department of diagnostic imaging and nuclear medicine, kyoto university, kyoto, japan diffusion tensor imaging (dti) is an mr imaging technique that is sensitive to orientation of mobility in water molecules. dti reveals two specific characteristics: diffusion anisotropy; and directional distribution of water diffusivity. white matter shows high diffusion anisotropy, because diffusion is faster in parallel to fiber direction than in other directions. dti of the brain can be reconstructed to display 3d macroscopic fiber tract architecture, in a process known as fiber tractography. with recent advances in actively shielded 3-t magnets and parallel imaging techniques, high-field mr imaging has become practical in clinical settings. we have demonstrated that depiction of most fiber tracts was improved on 3-t tractography compared to 1.5 t. we have also established an integration of tractography and intraoperative subcortical motor-evoked potential, and demonstrated that diffusion tensor tractography of the corticospinal tract using 3-t mr was able to provide interactive information on fiber tracts, depicting the course of eloquent fiber tracts during an operation. to test whether mr tractography is reproducible and reliable, we used this technique to assess acute tiny infarcts located in the supratentorial brain. we analyzed the data of 14 patients who presented to our institute with sensorimotor symptoms. there was an excellent correlation between the location of the infarct as assessed by tractography and clinical symptoms. next, we applied the technique to patients with evolving symptoms after admission to hospital. we specifically assessed the change in the tract-infarct relationship over time. the data showed that, in most cases when there was symptomatic progression, the distance between the tract and the infarct border depicted on dwi diminished. finally, we studied whether the use of tractography could help predict a patient's prognosis. to simplify the analysis, we specifically focused on patients with lenticulostriate artery (lsa) infarcts. we analyzed the correlation between the extent of cst involvement within the infarcts and the severity of motor deficits. the data indicated that the tractographic technique could be useful to predict a patient's outcome. sy3-2-02-4 anatomical and functional tractography: a combined approach with diffusion tractography and corticocortical evoked potential riki matsumoto department of neurology, kyoto university graduate school of medicine, japan recent advances in diffusion-weighted imaging have raised the possibility of in vivo investigations of brain circuitry in humans. the probabilistic tractography provides estimates of the likelihood of a pathway between two brain regions without tensor estimation and thus could trace the fiber pathways beyond regions of low diffusion anisotrophy into the grey matter. however, the results depend merely on anisotropic movement of water molecules and need validation. for presurgical evaluation of epilepsy patients, we developed an in vivo tracking method, cortico-cortical evoked potential, to electrically track the cortico-cortical connections by stimulating a part of the brain through epicortical electrodes and recording the cortical evoked potentials that emanate from a distant region of the cortex via projections. combined with preoperative diffusion analysis, this invasive evaluation provides a unique opportunity to study the cortico-cortical connectivity both functionally and anatomically. results of the combined approach will be presented. parkinson disease (pd) is the second commonest neurodegenerative disorder after alzheimer disease characterized by tremor, rigidity, bradykinesia, and postural instability. pathologically, the most outstanding change is the neurodegeneration of the nigral dopaminergic neurons. although familial forms of pd can be encountered up to 15% of the patients, the remaining cases are sporadic. it has been postulated that nigral neurodegeneration in pd is induced by the interaction of genetic risk factors and environmental factors. epidemiological studies revealed numbers of environmental factors that are positively correlated with increased risk of pd; such factors include pesticide, herbicides, rural living, well water drinking, metals such as manganese and iron, fuel oil, industrial chemicals, and hydrocarbon solvents. in addition, certain employments were reported to be associated with increased risk of pd; these include steel/alloy industry, wood/pulp plant, farming, carpentry, cleaning, orchard, mining, and welding. these studies suggest importance of environmental factors in the pathogenesis of pd. recent progress in these areas will be discussed. masami ishido national institute for environmental studies, tsukuba, japan there are getting much public concerns about children health since environmental factors such as industrial chemicals cause deficit in developing brains. it has been suggested that they may be incident of attention deficit hyperactivity disorder or autism. epidemiologic studies also suggested that parkinson's disease was found in the peoples who were exposed to pesticides in their childhood. thus, we examined the effects of industrial chemicals, called endocrine disruptors, on rat neurodevelopment. oral administration of an endocrine disruptor (12-60 mg/kg) into male wistar rats (from 5 days to 3 weeks of age) significantly caused hyperactivity at 4-5 weeks old. immunohistochemical analyses of the brain tissues at 8 weeks of age revealed a large reduction of immunoreactivity for tyrosine hydroxylase, but not for glutamic acid decarboxylase, both of which are localized in the substantia nigra, suggesting the specific degeneration by the chemical of dopaminergic neurons. tunel-positive cells were seen in the substantia nigra. thus, environmental insults in early life may be of particular etiologic importance. sy3-2-07-3 non-thermal effects of mobile phones upon the rat brain leif g. salford, b. persson, j. eberhardt, g. grafstrom, l. malmgren, a. brun dept of neurosurgery and the rausing laboratory, lund university, sweden we have shown that rf electromagnetic fields can cause significant leakage of albumin through the bbb of exposed rats as compared to non-exposed animals. one remarkable observation is that sar values around 1 mw/kg give rise to a more pronounced albumin leakage than higher sar values -all at non-thermal levels. if the reversed situation were at hand, we feel that the risk of cellular telephones, base-stations and other rf emitting sources could be managed by reduction of their emitted energy. the sar value of around 1 mw/kg is exists at a distance of more than one meter away from the mobile phone antenna and at a distance of about 150 -200 meters from a base station. another remarkable observation in our studies is the fact that a significant (p < 0.002) neuronal damage is seen in rat brains 50 days after a 2 hour exposure to gsm at sar values 200, 20 and 2 mw/kg. we have followed up this observation in a study where 96 animals were sacrificed 14 and 28 days respectively after an exposure for 2 hours to gsm mobile phone electromagnetic fields at sar values 200, 20, 2, 0.2 and 0 (controls) mw/kg. significant neuronal damage is seen after 28 days and albumin leakage after 14. our findings may support the hypothesis that albumin leakage into the brain is the cause for the neuronal damage observed after 28 and 50 days. sy3-2-07-4 the mitochondrial toxin 3-nitropropionic acid: an environmental toxin to study striatal degeneration in huntington disease emmanuel brouillet neuronal death laboratory, ura cea-cnrs 2210, france huntington disease is a neurodegenerative disorder caused by a mutation in the gene encoding huntingtin. the mechanisms underlying the preferential degeneration of the striatum, the most striking neuropathological change in huntington disease, are unknown. the behavioral and anatomical similarities found between huntington disease and animal models of striatal degeneration using the environmental toxin 3-nitropropionic acid (3np) support the hypothesis that mitochondrial defects could play a role in huntington disease. we will discuss the mechanisms of 3np toxicity and show that 3np and mutated huntingtin have certain mechanisms of toxicity in common. in particular, we show that mutated huntingtin can alter the expression of mitochondrial complex ii, the respiratory chain enzyme specifically inhibited by 3np. in summary, the 3np story is a good example showing how the study of environmental toxins can greatly help to elucidate the complex mechanisms underlying chronic neurodegenerative disorders. we recently demonstrated that rats received intrecisternal injection of 6-ohda or environmental chemicals, such as bisphenol a, nonylphenol, p-octylphenol, diethylhexylphthalate or dibutylphthalate, at 5 days of age showed behavioral hyperactivity at 4-5 weeks of age. immunohistochemical studies revealed a deficit in the development of dopamine (da) neurons. adult rats received these chemicals showed degeneration in nigro-striatal da neurons similarly to parkinson's disease. in this study, we investigated the mechanism of 6-ohda-induced neurotoxicity, using pc12 cells as an in vitro model system. we observed the generation of reactive oxygen species (ros) and p-quinone via auto-oxidation of 6-ohda. we also characterized the oxidation of cellular proteins by 6-ohda and the protective effect of antioxidants such as catalase, glutathione, and n-acetylcysteine with different manner. we will discuss about apoptotic cell death pathway including cytochrome c release and caspase activation induced by 6-ohda, ros and p-quinone. sy3-2-07-6 environmental factors in the pathogenesis of alzheimer's disease joanna l. jankowsky california institute of technology, usa epidemiological studies indicate that environmental factors significantly influence the risk of developing alzheimer's dementia. foremost among those factors are education, occupation, and leisure activities. although not universal, most studies have found that individuals with greater education, more challenging occupation, or active leisure hobbies show relative protection against dementia. animal models for alzheimer's disease have recently been used to explore the mechanism of this effect. transgenic mice designed to recapitulate alzheimer's amyloid pathology are protected from functional decline by enriched housing designed to provide cognitive stimulation. both enriched housing and exercise modify the level of amyloid-beta in the brains of transgenic mice, demonstrating that environmental factors can significantly influence brain biochemistry. intriguingly, traditional environmental enrichment can improve cognitive behavior while paradoxically elevating amyloid-beta levels in transgenic mice, suggesting that environmental stimulation may alter amyloid metabolism and cognitive function by competing mechanisms. the efficacy of synaptic inhibition depends on the number of gaba a receptors expressed on the neuron surfaces. in the present study, we have elucidated the role of prip (plc-related inactive protein) in trafficking of the receptors by analyzing prip knockout (ko) mice; the sensitivity to diazepam was reduced as assessed by biochemical, electrophysiological and behavioral analyses of ko mice, suggesting the dysfunction of the ␥2 subunit-containing receptors. we then examined the mechanisms by which prip molecule regulates cellsurface expression of ␥2 subunit-containing receptors. disruption of the direct interaction between prip and the ␤ subunit of receptors by prip-binding peptide inhibited cell-surface expression of ␥2 subunit-containing receptors in gh3 and hek293 cells. constitutive internalization of the receptors was also modified by the peptide. collectively, prip molecules are involved in trafficking of ␥2 subunit containing gaba a receptors to/from cell-surface membrane. research funds: kakenhi (16109010) sy3-3-03-2 involvement of bdnf in the induction of ltp at visual cortical inhibitory synapses yukio komatsu dept. visual neurosci., res. inst. environ. med., nagoya univ., nagoya, japan high-frequency stimulation (hfs) induces long-term potentiation (ltp) at inhibitory synapses of layer 5 pyramidal cells in rat visual cortex. this ltp requires a postsynaptic ca 2+ rise for induction and spike firing of presynaptic cells for maintenance, although the necessary frequency is low, suggesting that ltp is expressed presynaptically and some information must be sent backwards from the post-to presynaptic cells during induction. in this study, we investigated whether bdnf could act as such retrograde messengers. ltp did not occur when hfs was applied in the presence of k252a at 200 nm, inhibiting trk receptor tyrosine kinases selectively at that dose. hfs induced ltp when k252a application was started soon after hfs or when k252a was loaded into postsynaptic cells. ltp did not occur in the presence of trkb-igg or anti-bdnf antibodies. in cells loaded with bapta, the addition of bdnf to the medium enabled hfs to induce ltp without affecting basal synaptic transmission. these results suggest that bdnf released from postsynaptic cells activates presynaptic trkb, enabling the induction of ltp. research funds: kakenhi (17300101) sy3-3-03-3 autocrine mglur1 activation in cerebellar purkinje cells regulates gaba-mediated synaptic inhibition trevor smart, ian c. duguid university college london, uk in the cerebellum, retrograde signalling is important for the induction of short-and long-term changes to synaptic inhibition at interneuron-purkinje cell (in-pc) synapses. endocannabinoids, via cb1 receptors, mediate a short-term decrease in synaptic efficacy, while glutamate, via presynaptic nmda receptors, induces a sustained increase in gaba release. we now report that dendritically released glutamate also acts as an autocrine messenger, activating mglur1 on pcs to enhance synaptic inhibition via the release of endocannabinoids. this process was triggered by repetitive pc stimulation and blocked by uncoupling the mglur1-gq/11 transduction pathway as well as being initiated by direct mglur1 activation during pc depolarisation. glutamate uptake by excitatory amino acid transporters controlled the extent of autocrine mglur1 activation, whilst basal glutamate levels were unable to enhance endocannabinoid release. our study suggests that autocrine mglur1 activation provides a powerful homeostatic mechanism to dynamically regulate inhibitory synaptic transmission. sy3-3-03-4 regulatory mechanism of inhibitory synaptic transmission in the cerebellum shin-ya kawaguchi 1,2 , tomoo hirano 1,2 1 dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; 2 crest, jst, kawaguchi, japan at the gabaergic synapses between inhibitory interneurons and a purkinje neuron in the cerebellum, postsynaptic depolarization induces long-term potentiation of transmission efficacy mediated by gaba a receptors (rebound potentiation: rp). the signaling cascades regulating the induction of rp has been clarified. the balance of activities of protein kinases and phosphatases determines whether rp is induced or not. here we show another molecular mechanism involved in the rp induction. using both electrophysiological experiments and computational kinetic simulation of biochemical reactions, we demonstrate how the long-term potentiation of gaba a receptormediated responses is brought about. rp induction was impaired by inhibition of ca 2+ -activated protease calpain or by disturbance of association of gaba a receptor ␥2 subunit with gabarap (gaba a receptor associated protein). binding of gabarap to microtubule was also involved in the regulation of rp. our results suggest that structural alteration of gabarap caused by calpain activity is critical for establishment of rp. sy3-3-08-1 contribution of hebb's "organization of behavior" to the development of brain science masataka watanabe tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan more than 50 years have passed since the publication of "organization of behavior". this book has been one of the most influential books in neuroscience. around the time of the 50th anniversary of this book, special issues and articles concerned with this book appeared in several journals. his idea, which is a general framework for relating behavior to synaptic organization through the dynamics of neural networks, has stimulated variety of neuroscience researches in relation to, for example, environmental effects on development, naturenurture interaction, memory consolidation and sensory deprivation. however, he also made some mistakes, for example he advocated frontal lobotomy. in this symposium, i will briefly review how influential this book has been on basic and practical neurosciences, and will re-consider the importance and limitation of studying mental processes, such as emotion, memory and thought by exploring brain mechanisms, in reference to the idea of cell assembly, phase sequence and hebb synapse. sy3-3-08-2 detection of cell assembly in neuroscience experiments and brain-machine interfaces yoshio sakurai 1,2 , susumu takahashi 2 1 department of psychology, kyoto university, kyoto, japan; 2 crest, japan science & technology agency, japan the reality of cell-assembly coding in the working brain depends on how we could detect specific properties of cell assembly from multi-neuronal activities in behaving animals. first in the present paper, we show experimental results indicating some of the properties, i.e., functional overlapping of individual neurons and connection dynamics among multiple neurons, that depend on tasks and events being processed and on the distance among the neurons. second, we demonstrate a newly developed method, brain-machine interface (bmi), to test the reality of cell assembly as neural information and the plastic formation of cell assemblies during learning processes. we introduce our recent bmi system with independent component analysis (ica) and specific multi-electrodes and show some neuronal and behavioral data obtained by the bmi system. hebb postulated that coincident activities of pre-and postsynaptic neurons trigger input-specific plasticity. how relevant is it in protein synthesis-dependent late-phase plasticity (lp)? synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific lp. using live-imaging techniques, we measured entry of vesl-1s-egfp into dendritic spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as synaptic tag in many criteria. ve trapping conforms to the hebb's rule in a sense that it required both presynaptic activity and postsynaptic no-pkg pathway, but their coincident time window was far wider (∼h) than that of early-phase plasticity, suggesting an involvement of persistently synchronized rather than transiently coincident activity. no spreading from the activated synapses may persistently prime the postsynaptic tag components at the surrounding synapses, during which brief inputs to these synapses will establish associative and heterosynaptic tags. thus, tagging one synapse would lead surrounding synapses to multiple metaplastic states. tomoki fukai laboratory for neural circuit theory, riken brain science institute, saitama, japan in the cell-assembly hypothesis, cortical neurons are considered to form functional subnetworks depending on a particular demand of information processing. such cell assemblies may be organized through synchronous firing of the constituent neurons and synapses modifiable by hebbian learning. in this talk, i will overview recent in vivo and in vitro experimental findings that provide new evidence for synchronous or precisely timed neuronal activity. i propose a hardwired structure of local cortical networks, "entangled synfire chains", on the basis of the experimental observations of cortical activity. in this model, multiple cell assemblies can be defined by the pattern of neuronal wiring. however, the same experimental findings can lead us to a different type of cortical network models. in this type of models, cortical networks may self-organize to develop a critical dynamical state, which may be useful for realizing a hypothetical "liquid-state machine". i will discuss the characteristic properties of both types of models and the possible implications in cortical computations. research funds: kakenhi (17022036) sy3-3-08-5 impact of hebbian hypothesis on neuroscience keisuke toyama shimadzu institute of basic technology, seikacho, hikaridai, kyoto 619-0237, japan hebb has seeded two major concepts in the modern neuroscience, i.e., cell assembly hypothesis for perception, and hebbian synapses to construct that cell assembly. the former concept stimulated extensive searches for the response selectivity extending from the primary visual cortex to the inferotemporal cortex and even to the hippocampal cortex, while the later concept triggered neuroscience studies of the learning and memory in the developing and adult brains. recently, these concepts refreshed the impact with new dressing of 'dynamics'. cell assembly that was originally assumed to be static, became dynamic and opened a new possibility for the neural computation, combined with dynamic hebbian synapses conceptualized as the spike-timing dependent plasticity (stdp). i would like to discuss about speaker's talks in this context. sy3-4-04-1 tonic gaba a receptor mediated conductances: properties, functions and plasticity alexey semyanov riken brain science institute (bsi), japan communications mediated by non-synaptic receptors are important for information processing in the brain. high affinity extrasynaptic gaba a receptors mediate a persistent "tonic conductance" which reflects their activation by ambient concentrations of gaba. this phenomenon is found in different brain regions, shows cell-type specific differences in magnitude and pharmacology, and changes during brain development. our findings have revealed functional significance of gaba a receptors mediated tonic conductance in the hippocampus. we have shown that it modulates rate-coded information processing by individual neurons, and acts in a cell-type specific manner to regulate the excitability of the local neuronal circuit. the magnitude of the conductance is regulated by efficiency of gaba uptake and membrane potential. gaba a receptor mediated tonic conductance undergoes adaptive plasticity. it is up-regulated in hippocampal pyramidal cells in a model of pilocarpine status epileptics in rats. in mice lacking gad65 the amount of the tonic inhibition is reduced in ca1 hippocampal interneurons, while unchanged in pyramidal cells. sy3-4-04-2 modulatory effects of peri-interneuronal glial cells on neuronal activities in hippocampal ca1 region yoshihiko yamazaki 1 , yasukazu hozumi 2 , kenya kaneko 1 , satoshi fujii 1 , hiroshi kato 1 1 dept. of neurophysiol., yamagata univ. sch. of med., yamagata, japan; 2 dept. of anat. & cell biol., yamagata univ. sch. of med., yamagata, japan glial cells, in addition to their supportive roles in the nervous system, make up a functional unit with neurons and have been suggested to play novel roles in neuronal activities. we focused on interneuron/peri-interneuronal glial cell (pg) pairs in the hippocampal ca1 region and performed dual whole-cell recordings to investigate the modulatory effect of glial cells on neuronal activities. direct depolarization of pg suppressed the excitatory postsynaptic currents in an adjacent interneuron. this suppression was inhibited by adenosine a1 receptor antagonist. moreover, pg activation modulated the firing pattern of the interneuron. since interneurons in the hippocampus are mainly inhibitory and the terminals of a single interneuron make a large number of synapses on a group of pyramidal cells, direct inhibitory regulation via pg would have marked effects on the information processing of neurons in the ca1 region. research funds: kakenhi (15082201) sy3-4-04-3 carbachol-induced beta oscillations in rat hippocampal slices kiyohisa natsume, jun arai graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan rat hippocampus has the cholinergic input from medial septum and diagonal band in vivo. the input involves the generation of hippocampal rhythm, theta, gamma rhythm. to mimic the system, we applied carbachol, a cholinergic agent, to rat hippocampal slices. carbachol can induce beta oscillation as well while carbachol can induce theta, and gamma oscillations in the slices. in the present paper, we introduce the beta oscillations. the application of 30 m carbachol induces beta oscillations which occur intermittently with the interval of 20-30 s. during the intervals, gamma oscillations are induced. the mean frequency of the oscillations is 16.9 ± 0.9 hz (mean ± s.e.m.). the oscillations are induced via muscarinic m1, 3, 4 receptors. the frequencies of them are significantly decreased by the application of bicuculline, a gaba a antagonist. they are sensitive to bicuculline, while theta oscillations are not. it is indicated that the character of beta oscillations are different from those of theta oscillations. the neocortex and the hippocampus are connected by way of the entorhinal cortex and the subiculum. to examine ongoing network interactions among these distinct cortices during neocortical slow oscillations (1-3 hz), we recorded intracellular potentials in single neocortical, entorhinal, subicular, and hippocampal neurons, together with hippocampal field and multi-unit activities in adult anesthetized rats. we have found that (1) most entorhinal and subicular neurons displayed bimodal active (up) and quiet (down) states of membrane potential, in synchrony with neocortical slow oscillations, (2) no bimodal up-down transition was present in hippocampal neurons. hippocampal granule cells were directly driven by entorhinal up-state activity, while ca3 and ca1 neurons discharged during both up and down states, (3) gamma and fast (ripple) oscillations were observed in hippocampal ca1 area irrespective of up-down transition. these observations suggest that entorhinal and subicular regions are "neocortex-like" and hippocampal networks can generate self-organized activity independent of neocortical slow oscillations. the cholinergic neurons in the mesopontine reticular formation (mprf) seem to control sleep-wake cycle and hippocampal activity, because stimulation of the mprf elicits rem sleep and hippocampal theta wave. in this study, we recorded neuronal activity in the mprf and pontine and hippocampal eeg during rem sleep and investigated time-relationship between them. our results are summarized as follows: (1) most of the mprf neurons were active during rem sleep; (2) the mprf activity increased over ten seconds before transition from nrem to rem sleep, i.e. from non-theta to theta period; (3) the theta wave was instantaneously accelerated concomitant with activation of the mprf neurons. these results suggest that cholinergic neuron in the mprf is important in generation and maintenance of rem sleep and theta wave. because hippocampal theta waves are involved in memory consolidation during rem sleep, our findings might help to clarify this mechanism. research funds: kakenhi (17605001) sy3-5-09-1 clock mechanisms of the scn involving in the entrainment to the morning and evening light sato honma, natsuko inagaki, nobuko tokumaru, ken-ichi honma dept. physiology, hokkaido univ. grad. sch. med., sapporo, japan the circadian clock, by entraining to the light-dark cycle of different day length, controls seasonality in biological functions. the mechanism is currently explained by morning and evening oscillators which change their coupling intensity depending on the day-length. by using clock gene expression as a marker of clock functions, we examined the localization and molecular bases of the two oscillators. rats and mice were housed under light-dark (ld) 18:6 h and ld 6:18 h. clock gene expression patterns in the entire scn were examined by in situ hybridization on the first day of constant darkness. the phase relations of per1 and per2 rhythms suggest that light-on resets per1 rhythms in both light conditions, while per2 rhythm also relates to the light-off. in cultured scn of transgenic mice expressing luciferase under the control of per1 promoter, we observed two bioluminescent peaks a day only in the anterior scn from the mice kept in ld 18:6. the finding suggests that two distinct oscillators, which respond to the day-length, reside in the anterior scn. the suprachiasmatic nucleus (scn) is the center of the mammalian circadian clock. tissue transplantation of the scn restores the behavioral circadian rhythm in scn-lesioned mice in spite of the impaired neural connection with the host brain. we have investigated whether grafted scn regulates the circadian oscillator in peripheral organs using the scn-transplanted mice that have a limited time information transmission paths. as a result, the grafted scn restored not only circadian behavior rhythm but also the circadian rhythms of peripheral organs. many of clock genes showed dynamic oscillations with identical phase relationship as shown in intact animals, however, per1 and per2 showed low amplitude of oscillation. the findings suggest that diffusible signal molecules released from the transplanted scn entrain the circadian clock in peripheral organs and that they differentially modulate the expression of clock genes. sy3-5-09-3 genome-wide analysis of adrenal-dependent and independent circadian regulation of mouse hepatic genes norio ishida clock cell biology group, national institute of advanced industrial science and technology (aist), ibaraki, japan recent progress in genome-wide expression analysis has identified hundreds of circadian regulated genes in the suprachiasmatic nucleus as well as in peripheral tissues of mammals. adrenal gland is important for circadian regulation for mammalian peripheral clocks. to identify circadian expressed genes regulated by adrenal glands pathways, we performed dna microarray analysis using hepatic rna from adrenalectomized (adx) and sham-operated mice. we identified 169 genes that fluctuated between day and night in the livers, 100 lost circadian rhythmicity in adx mice. these included the genes for key enzymes of liver metabolic functions such as glucokinase, hmg-coa reductase, and glucose-6-phosphatase. the present study showed that the circadian expression of mouse liver genes is governed by core components of the circadian clock such as clock, and the other 100 genes depend on adrenal glands pathway such as glucocorticoids. hitoshi okamura kobe university graduate school of medicine, japan light is a powerful synchronizer of the circadian rhythms, and bright light therapy is known to improve metabolic and hormonal status of circadian rhythm sleep disorders, although its mechanism is poorly understood. in the present study, we revealed that light induces gene expression in the adrenal gland via the suprachiasmatic nucleus (scn)-sympathetic nervous system. moreover, this gene expression accompanies the surge of plasma and brain corticosterone levels without accompanying activation of the hypothalamoadenohypophysial axis. the abolishment after scn-lesioning, and the day-night difference of light-induced adrenal gene expression and corticosterone release, clearly indicate that this phenomenon is closely linked to the circadian clock. the surge of plasma corticosterone after light exposure indicates that environmental light signals are instantly converted to glucocorticoid signals in the blood and csf. the light-induced clock-dependent secretion of glucocorticoids adjusts cellular metabolisms to the new light-on environment. sy3-5-09-5 neuronal and hormonal control of peripheral clock function through suprachiasmatic nucleus shigenobu shibata, naomi hayasaka, takashi kudo, tsuyoshi yaita department of pharmacology, school of science and engineering, waseda university, tokyo, japan the clock genes are expressed not only in the suprachiasmatic nucleus (scn) of the hypothalamus where the master clock exists, but also in other brain regions and various peripheral tissues. in the liver and lung, clock genes are abundantly expressed and show clear circadian rhythm. although oscillation of clock genes in the liver and lung is controlled under the circadian clock mechanism in the scn, we do not know the resetting signals on peripheral clock function. communication between the scn and peripheral tissues occurs through various systems involving the sympathetic, nicotinic and glucocorticoide functions. this symposium mainly describes both anatomical and physiological experiments to reveal the sympathetic and glucocorticoid control over peripheral clock function. sy3-6-10-1 a to z of gene transfer with adenoviral vector-application to neuronal birth date-specific gene transfer using replication-defective adenoviral vectors, we successfully performed 'pulse gene transfer' into progenitor cells in a neuronal birth date-specific manner. when adenoviral vectors were injected into the midbrain ventricle of mouse embryos between embryonic days (e)10.5 to e14.5, the adenoviral vectors introduced a foreign gene into a specific cohort of birth date-related progenitor cells. this technique allows us to distinguish a cohort of birth date-related progenitor cells from other progenitor cells with different birth dates and to introduce a foreign gene into specific subsets of neurons by performing adenoviral injection at specific times. this adenovirus-meditated gene transfer technique will enable us to examine the properties of each subset of progenitor cells that share the same neuronal birth date. i will explain directions how to use an adenoviral vector and application of an adenoviral vector in my talk. research funds: crest sy3-6-10-2 live imaging in the specific neuronal cells by the combination of transgenic mice and viral vectors kaori kashiwagi, naoaki saito lab. mol. pharmacol. biosig. res. ctr, kobe univ, kobe, japan live imaging analysis has revealed that each protein kinase c (pkc) subtype shows spatio-temporally distinct targeting in response to various stimuli. we demonstrated that the trans-synaptic stimulation induced translocation of ␥pkc-gfp in cerebellar slices from bitransgenic mice (nse-tta/tetop-␥pkc-gfp) which express ␥pkc-gfp in time and region-specific manner. this translocation was not restricted, but propagated from the distal to the proximal dendrites close to the soma of purkinje cells. in order to gain further insight in to the molecular mechanisms of pkc translocation, we introduced viral vectors to primary cultured purkinje cells. the propagative ␥pkc-gfp translocation was also observed in cultured purkinje cells derived from nse-tta mice. the molecular mechanisms of pkc translocation in purkinje cells were analyzed by live imaging with various kinds of viral vectors. the combination of tg mice and viral vectors is useful to understand the physiological role of pkc in the specific neuronal cells. research funds: kakenhi (17024040) sy3-6-10-3 visualization and manipulation of the signaling systems in the cns using sindbis viral vectors sho kakizawa dept. of pharmacol., grad. sch. of med., the university of tokyo, tokyo, japan virus vectors can efficiently deliver genes to neurons and other cells in the nervous systems in vitro and in vivo. because many viral vectors are in common use, it is important to select the best viral vector for each specific application, and a number of factors must be considered when making a decision. sindbis virus is an enveloped plus-strand rna virus belonging to the alphavirus genus of the togaviridae family. the sindbis viral vector is characterized by its effective, rapid, high-level and preferential transduction of neurons. these facts indicate that the vector is a powerful tool for the robust expression of target genes in the specific population of neurons. in this symposium, we will introduce our recent topics on the synaptic functions in the cerebellar systems revealed by visualization and manipulation of signaling molecules, such as nitric oxide and inositol 1,4,5-trisphosphate, in the cerebellar purkinje cells. research funds: grant-in-aid for scientific research on priority areas-molecular brain science-from the ministry of education, culture, sports, science and technology of japan sy3-6-10-4 rescue of phenotypes of null-mutant mice by virus vector-mediated gene transfer kazuhisa kohda, wataru kakegawa, kyoichi emi, michisuke yuzaki dept. of physiol., keio univ. sch. of med., tokyo, japan even after the completion of genome project in major species, functions of many molecules remain uncharacterized. a transgene-based rescue approach is one of the powerful methods to decipher the mechanisms of actions of an orphan receptor; however, it is quite labor intensive and time consuming. here, we have developed a virus vector-based rescue approach and applied to investigate the mechanisms of action of the orphan glutamate receptor ␦2 (glur␦2) in the cerebellum. by introducing a sindbis virus carrying a wild-type glur␦2 into glur␦2-null cerebellum in vivo, we could rescue abnormal phenotypes, such as impaired long-term depression at parallel fiber-purkinje cell synapses. by examining whether a mutant glur␦2 lacking a specific domain could similarly rescue the phenotypes, we could evaluate functional importance of the domain. alternatively, by introducing a partial sequence of the gene of interest into wild-type brain and examining its dominant-negative effect, we will be able to identify the region of the gene product that is functionally important. research funds: kakenhi 16650071 sy3-6-10-5 gene transfer into in vivo cerebellar purkinje cells by hiv-derived lentiviral vectors hirokazu hirai advanced science research center, kanazawa university, ishikawa, japan cerebellar purkinje cells are key elements regulating motor coordination and motor learning. gene transfer into purkinje cells is an effective approach for the study of cerebellar function and treatment against cerebellar disorders. although adenoviral vectors or sindbis vectors are frequently used for gene delivery into neurons, the former has extremely low affinity for purkinje cells, while the latter causes substantial damage to the infected cells. to achieve effective gene transfer into purkinje cells, we used human immunodeficiency virus (hiv)-derived lentiviral vectors. purkinje cells were efficiently transduced without significant influence on the cell viability and synaptic functions. gene expression was also detected, though less efficiently, in other cortical cells, whereas no transduced cells were observed outside of the cerebellar cortex. these results suggest that hiv-derived lentiviral vectors are useful for the study of gene function in purkinje cells as well as for application as a gene therapy tool for the treatment of diseases that affect purkinje cells. research funds: jst/presto, kakenhi (17300100) sy3-7-11-1 physiological basis for stereotaxic surgery in basal ganglia atsushi nambu division of system neurophysiology, national institute for physiological sciences, and school of life science, the graduate university for advanced studies, okazaki, japan stereotaxic surgery in movement disorders such as parkinson's disease dramatically improves the symptoms of such diseases. i assume the physiological basis for the treatment is to disrupt abnormally increased information flow through the basal ganglia. i will discuss the pathophysiology of basal ganglia disorders and the effect of stereotaxic surgery in light of the three major pathways in the cortico-basal ganglia loop, i.e., hyperdirect (cortico-stn-gpi), direct (cortico-striato-gpi) and indirect (cortico-striato-gpe-stn-gpi) pathways, that dynamically control the activity of the thalamus and cortex to perform correct motor programs in correct timing. research funds: kakenhi (17022042) sy3-7-11-2 deep brain stimulation of subthalamic nucleus on parkinson's disease fusako yokochi department of neurology, tokyo metropolitan neurological hospital, tokyo, japan parkinson's disease is a progressive and degenerative disease caused by dopamine deficiency attributed to the degeneration of neurons in the substansia nigra. consequently, various symptoms appear, such as cardinal symptoms, those in the advanced stage and those as the side effects of long-term levodopa therapy. many antiparkinsonian drugs have been developed, but all of the symptoms are not improved by these drugs. stereotaxic surgery was started for treating severe tremor and rigidity in the mid-20th century. stimulation by chronically implanted electrodes in the brain, that is, deep brain stimulation (dbs), has recently been applied and has been shown to have marked effects on the symptoms resisted to conventional treatments. in particular, dbs of the subthalamic nucleus (stn) is an effective treatment for the symptoms. much basic research on the function of stn has been reported, but stn function is still unclear. clinical outcomes including the side effects of stn dbs, the neural activities recorded from stn and the localization of clinical effects are reported in this paper. sy3-7-11-3 firing patterns of basal ganglia neurons and effects of deep brain stimulation in parkinson's disease takao hashimoto center for neurological diseases, aizawa hospital, matsumoto, japan high-frequency electrical stimulation of the subthalamic nucleus (stn), internal segment of the globus pallidus (gpi) and thalamus can improve motor signs in patients with parkinson's disease, however, its mechanism of action remains unclear. a leading hypothesis regarding the development of movement disorders of basal ganglia origin suggests that hyperkinetic and hypokinetic disorders occur as a result of changes in the mean discharge rate in the gpi and substantia nigra, which in turn suppress thalamocortical output. on the other hand, altered firing patterns in the basal ganglia have been reported in mptp-induced parkinsonian animals: increases in bursting activity and periodic oscillatory activity in the gpi and stn, and synchronization of gpi, or gpi and striatal neurons. synchronous oscillation in the basal ganglia may break down independent processing in the motor circuit and disrupt signal processing at the cortical level. kaoru takakusaki department of physiology, asahikawa medical college, asahikawa, japan locomotion is composed of volitional and automatic processes. particular attention is required to perform volitional processes such as avoiding obstacles and accurate limb placements during locomotion. however, we are largely unaware of the automatic control processes of rhythmic limb movements, muscle tone and postural reflexes that accompany locomotion. because each process is seriously impaired in parkinsonian patients, the basal ganglia must play a crucial role in integrating the volitional and automatic processes of locomotion. the basal ganglia contribute to the planning and execution of voluntary movements via basal ganglia thalamocortical loops. on the other hand, recent our findings suggest the importance of the direct basal ganglia outflow to the brainstem where fundamental neuronal networks for controlling postural muscle tone and locomotion are located. in this presentation we discuss the role of the basal ganglia in the integration of volitional and automatic movements during locomotion, which has been less understood aspect of gait control. research funds: grant-in-aid for scientific research (c) and priority area (area no.454) sy3-7-11-5 characteristics of neuronal activity within the globus pallidus interna (gpi) in patients with dystonia yoichi katayama 1,2 1 department of neurological surgery, nihon university school of medicine, tokyo, japan; 2 division of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan dystonia represents disordered muscular tonicity of the trunk and/or extremities, and is often dramatically controlled by chronic gpistimulation in humans, indicating that dystonia is attributable to certain abnormal activity of gpi neurons. little is yet known, however, regarding characteristics of neuronal activity within the gpi underlying dystonia. we analyzed activity of gpi-neurons in patients with dystonia or parkinson's disease, which were recorded during surgery for chronic gpi-stimulation. as compared to gpi neurons in patients with parkinson's disease, gpi neurons in patients with dystonia were distinctive with following three characteristics; firing rate (49.4 ± 31.7 hz, n = 27) was low, firing pattern was often composed of irregular pauses and bursts, and many were responsive to body movement with wide receptive fields. these findings suggest that dystonia may be related to unstable movement-related sensory processing within the gpi. it has been considered that dystonia, which is generally characterized by postural abnormalities and involuntary movements including torsion, is caused by dysfunction of the basal ganglia. the purpose of the present work is to clarify the neural mechanisms underlying the onset of dystonia by analyzing the pathophysiology of a model for torsion dystonia, wriggle mouse sagami (wms). the genomic mutation of wms occurs in the gene encoding plasma membrane ca 2+ -atpase isoform 2 that is located on the 6th chromosome. recent immunohistochemical and electrophysiological investigations on wms have shown that (1) d2-type dopamine receptors are downregulated presynaptically in the striatum, and (2) a large number of purkinje cells in the cerebellum express tyrosine hydroxylase (the synthesizing enzyme for dopamine) and their excitability is greatly reduced. in this symposium, the possible correlation between these data and dystonic phenotypes will be discussed. kei-ichiro maeda reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan gnrh has been well established to regulate reproduction through two modes of secretion: surge and pulse. the surge mode is female-specific and induces lh surge and then ovulation through positive feedback action of estrogen. the pulse mode tonically activates gonads in both sexes with being negatively regulated by gonadal steroids. environmental cues, such as photoperiod or nutrition, are considered to affect reproductive activity through altering pulse mode of gnrh release. pulse mode seems much more robust than surge, because estrogen can induce a surge under a certain condition when the pulse is suppressed. the following four papers aim to unveil the physiological mechanism underlying two modes of gnrh secretion in various experimental models. sy3-8-05-2 metastin: a neuropeptide playing a central role in the regulation of ovulatory cycle hiroko tsukamura, kei-ichiro maeda graduate school of bioagricultural sciences, nagoya university, japan estrous cyclicity is regulated by a sequence of neuroendocrine events consisting of hypothalamus-pituitary-gonadal axis. gonadotropinreleasing hormone (gnrh)/luteinizing hormone (lh) surge is induced by positive feedback action of estrogen secreted by mature ovarian follicles. the central mechanism of positive feedback action of estrogen on gnrh/lh secretion, however, is not fully understood yet. metastin was first isolated as a natural ligand for a g-proteincoupled receptor, gpr54 (2001. nature 411, 613) . recent studies reported that a genetic alteration leading to homozygous loss of function of gpr54 impairs pubertal development in mice and human. we have first demonstrated that endogenous metastin plays a physiological role in inducing ovulation through stimulating gnrh/lh surges to control estrous cyclicity in the female rat (2005. endocrinology 146, 4431) . the present paper focuses on the role of metastin in regulating gnrh/lh surge based on our recent study in rats and discusses possible mechanism underlying positive feedback action of estrogen. suzanne moenter, catherine christian university of virginia, usa a surge in gonadotropin-releasing hormone (gnrh) secretion is the cns signal that triggers the luteinizing hormone (lh) surge, which causes ovulation. the gnrh surge depends on a switch in estradiol (e) feedback from negative to positive and in rodents on time of day, occurring in the pm. treating ovariectomized (ovx) mice with a constant release e capsule (ovx+e) elicits daily pm lh surges; there is no diurnal change in ovx controls. likewise, extracellular recordings of firing activity of gfp-identified gnrh neurons showed no diurnal changes in cells from ovx mice. in contrast, e increased firing in the pm compared to am. gabaergic neurons form a major input to gnrh neurons, and activation of the gabaa receptor can be excitatory in these cells. whole-cell patch-clamp recordings of synaptic activation of gabaa receptors on gnrh neurons revealed e-dependent decreases in transmission during the am (negative feedback) and increases in transmission near surge onset that persisted for some populations of afferents thru the surge peak. together these data indicate one mechanism by which e induces the gnrh surge is by altering gaba transmission to gnrh neurons. yoshitaka oka grad. sch. sci., univ. of tokyo, tokyo, japan the gonadotropin-releasing hormone (gnrh) peptidergic neuronal systems consist of hypothalamic neuroendocrine and extrahypothalamic neuromodulatory gnrh neurons. here, i introduce our recent studies on the physiological properties of neuroendocrine preoptic (poa) gnrh neurons in comparison with the neuromodulatory terminal nerve (tn) gnrh neurons. to study the both types of gnrh neurons, we use a fish model system in which we can easily identify both of them in intact brain preparations in vitro, which is a great advantage over most other vertebrates. the poa-gnrh neurons had quite different basic electrophysiological membrane properties from those of tn-gnrh neurons and showed alternating active and silent phases of firing activities, in contrast to the regular pacemaker activities of tn-gnrh neurons. now that we have various experimental approaches (electrochemical measurement of gnrh release, ca 2+ imaging after single-cell electroporation, single-cell rt-pcr, double patch clamp recording, etc.) in hand, simultaneous multidisciplinary approaches should be possible to study the physiology of gnrh neurons. research funds: kakenhi 15370032 sy3-8-05-5 metabolic regulation of puberty onset using the prepubertal rat model helen i'anson biology department and neuroscience, washington and lee university, usa we hypothesize that glucose availability determines the timing of puberty onset in rats. abdominal fat stores are low in dieted, prepubertal female rats with delayed puberty, suggesting that such rats may be particularly sensitive to dietary fuel changes. such rats are fed a single daily meal and demonstrate decreased blood glucose levels between meals. we hypothesized that such daily hypoglycemic bouts delay onset of puberty during dieting. when glucose supplement was given to prepubertal dieted rats, and they exhibited first estrus with similar timing to previously dieted re-fed rats. conversely, when dieted rats were re-fed ad libitum and simultaneously glucose-deprived, first estrus was delayed. blood glucose levels during glucose-supplementation which induced first estrus, and during glucoprivation and re-feeding which delayed first estrus, were similarly elevated, suggesting that central, rather than peripheral, glucose levels are monitored in the prepubertal animal. in summary, central glucose availability may be an important signal timing puberty onset. research funds: jeffress memorial trust and washington and lee university sy3-8-12-1 molecular mechanisms of thyroid hormone action in developing brain toshiharu iwasaki, noriyuki koibuchi department of integrative physiology, gunma university graduate school of medicine, maebashi, japan thyroid hormone (th) plays an important role in the developing brain. the action is mainly exerted by controlling gene expression through binding to its specific receptor, th receptors (trs). trs are ligand-regulated transcription factors that are expressed in many organs, including brain. trs bind to target dna sequences known as th-response element (tre). coactivators and corepressors are involved in the tr-mediated gene regulation through histone modification. we characterized the coactivator and the corepressor that were expressed in embryo brain. although precise mechanism of the th action for brain development has not been fully clarified, these cofactors may be involved in these actions. th affects brain development only during a limited period, which is referred as the critical period of th action. recently, it has been speculated that environmental chemicals may cause the abnormal brain development. possible mechanism of action of such chemicals on tr system will be discussed. research funds: kakenhi 17510039, 17390060 sy3-8-12-2 thyroid hormone and organic anion transporters in brain hiroyuki kusuhara, yuichi sugiyama graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan organic anion transporting polypeptides (oatps/slco) currently consist of 15 isoforms in human and rodents. they are initially identified as part of detoxification system in the body, and involved in the tissue uptake of xenobiotics, especially amphipathic organic anions. some members accept a variety of structurally unrelated compounds as substrate. oatp1c1 is characterized by its unique substrate specificity, highly selective for thyroid hormones, particularly for t4 and reverse t3, but the transport activity of t3 is quite low. it is predominantly expressed in the brain capillaries and choroid plexus, acting as barrier of central nervous system, where oatp1a4, the transport activities of t4 and t3 are similar, is also expressed. the uptake of t4 and t3 by the brain determined using in situ brain perfusion technique was saturable and inhibited by oatps substrates and inhibitors. these two transporters may play a role in regulation of brain levels of t4 and t3. research funds: the advanced and innovational research program in life sciences from the ministry of education, culture, science and technology sy3-8-12-3 alterations of gene expression profiles in the developing brain by chemicals disrupting thyroid hormonedependent signals takayuki negishi 1 , masaki takahashi 1 , yasuhiro yoshikawa 2 , tomoko tashiro 1 1 department of chemistry and biological science, aoyama gakuin university, kanagawa, japan; 2 department of biomedical science, the university of tokyo, tokyo, japan there is increasing concern about the possibility that environmental chemicals such as polychlorinated biphenyl (pcb) and its hydroxylated metabolites interfere with normal brain development through acting as thyroid hormone disrupting agents. in this presentation, based on comprehensive dna microarray analysis, we demonstrate alterations in gene expression in the brain of neonatal rats perinatally exposed to 4-hydroxy-2,2 ,3 ,4 ,5pentachlorobiphenyl (anti-thyroid hormone-like), as well as in primary cultured rat hippocampal neurons exposed to 4-hydroxy-2,2 ,3,4 ,5,5 ,6-heptachlorobiphenyl (thyroid hormone-like). among genes whose mrna expression levels were affected by these compounds, a number of genes essential for the establishment of synaptic networks were detected, suggesting that long-lasting effects on higher brain functions may result from exposure of the developing brain to these compounds. hydroxylated metabolites of pcbs (oh-pcbs) have chemical structures similar to thyroid hormones (ths). we reported that low doses of two types of oh-pcb inhibited th-dependent extension of purkinje cell dendrites (2005. dev. brain res. 154, 259) . koibuchi et al. (2004) clarified that they interfere with th-dependent gene expressions in reporter gene assays. further, takasuga et al. (2004) detected some pcb and oh-pcb congeners in human csf, of which 4oh-cb187 is the highest concentration. to determine its effects on developing neurons, we examined 4oh-cb187 in rodent cerebellar culture cells. interestingly, 4oh-cb187 promoted dendritic development of purkinje cells in the absence of th and increased significantly their survival numbers. these results indicate that oh-pcb congeners may disrupt normal brain development by different mechanisms depending on their chemical structures. we have reported that rat pups exposed to an antithyroid agent, propylthiouraci l (ptu), via maternal milk exhibit hyperactivity, impairment in spatial learning and social interaction, and audiogenic hypersensitivity extending to audiogenic seizures, thus this ptu rat can be a possible candidate of animal model for autism. in ptu rats, the delay in migration of the extragranular cells of cerebellum, and in innervation from inferior olive nuclei to purkinje cells were shown. these neurons transiently expressed serotonin-ir, therefore we treated ssri or 5-htp to examine the relevance of serotoninergic function. the treatment of 5-htp but not ssri recovered the delay of cell migration. these effects of serotonin manipulation in ptu rats on the behavioral impairment and the development of cns will be discussed. it is hoped that embryonic stem (es) cells will be used in transplantation therapy for neurological diseases. however, because grafts of neural stem cells derived from es cells may contain residual undifferentiated cells, there would be a risk for teratomas. to reduce this risk, we applied to es cells herpes simplex virus thymidine kinase (hsv-tk) gene and ganciclovir (gcv) treatment. stable mouse and cynomolgus monkey es cell lines expressing hsv-tk were obtained. gcv sensitivity was higher in undifferentiated es cells than in es cell-derived neural stem cells. es cell-derived neurons were resistant to gcv treatment. nude mice with transplants of undifferentiated es cells expressing hsv-tk formed teratomas, but the tumor growth was suppressed after the gcv treatment. suicide gene delivery might increase the safety of the use of es cells in cell replacement therapy. enzymatic degradation of chondroitin sulfate is known to promote axonal regeneration in the central nervous system. the physiological role of chondroitin sulfate up-regulated after injury was examined in the nigrostriatal dopaminergic system which was unilaterally transected and treated with chondroitinase abc. in transected mice, dopaminergic axons did not extend across the lesion. chondroitin sulfate was up-regulated around the lesion and a fibrotic scar containing type iv collagen deposits were developed in the lesion center. in chondroitinase abc-treated mice, numerous dopaminergic axons were regenerated across the lesion. in these animals, chondroitin sulfate immunoreactivity was remarkably decreased and the formation of a fibrotic scar was unexpectedly prevented. these results support our previous supposition that chondroitin sulfate does not act as an obstacle to regenerating axons, but involved in the repair process of the brain injury including the formation of the fibrotic scar (kawano et al., 2005) . reference kawano et al., 2005. j. neurosci. res. 80, 191-202. research funds: kakenhi 16500234 os2a-8-03 neurotransmitters that maintain and suppress the tonic firing of the serotonergic neurons in the dorsal raphe during sleep waking cycles yoshimasa koyama 1 , kazumi takahashi 2 , yukihiko kayama 2 1 cluster of science and technology, fukushima university, fukushima, japan; 2 department of physiology, fukushima medical university, fukushima, japan the present experiment was done to examine, under unanesthetized natural sleep-waking condition, which neural systems were involved to regulate the firing of the serotonergic (5ht) neurons in the dorsal raphe (dr) during sleep waking cycles. using head restrained, unanesthetized rats, single neuronal activity was recorded and each drug was applied iontophoretically or by pressure close to the recording neurons. spontaneous firing of the 5ht neurons in dr were excited by glutamate and orexin a or b. they were inhibited by noradrenaline. an ␣1 receptor agonist (phenylephrine or methoxamine) increased the firing rate during sws or ps, but had no effect when applied during w. in ps-off type dr neurons, cessation of firing during ps was recovered by bicuculline, however in the dr neurons that did not stop firing during ps, bicuculline had almost no effect. os2a-8-04 correlation between regional grey matter volume and proficiency increase in second language: a vbm study arihito nauchi 1,2 , kazuyoshi hirano 2,3 , yukimasa muraishi 2,3 , kuniyoshi l. sakai 1,2 1 department of basic science, university of tokyo, tokyo, japan; 2 crest, jst, japan; 3 secondary education school, university of tokyo, japan although neuroimaging studies have contributed to clarify the brain function, the neural basis of individual variation in cognitive abilities such as language still remains unknown. in the present study using voxel-based morphometry (vbm), a whole-brain unbiased technique for detecting regional differences in mr images, we examined the relationship between the proficiency increase in second language (l2) and grey matter volume among students aged 13-17, who received special classroom training in the use of english verbs. in specific regions including the left lateral premotor cortex and the left inferior frontal gyrus (the grammar center), we found a positive correlation between the regional grey matter volume and improved performance for the grammaticality of english sentences. these results suggest an anatomical basis for the language faculty, such that the capacity of a specific region is related to proficiency increases in l2. os2a-8-05 grammar center activation in honorification judgment of japanese sentences kanako momo 1,2 , kuniyoshi l. sakai 1,2 1 department of basic science, university of tokyo, tokyo, japan; 2 crest, jst, japan one linguistic theory proposes that japanese honorification is a syntactic feature, because syntactic agreement is required between subject/object and honorific forms. to investigate whether such syntactic processing is actually realized in the brain, we examined cortical activation using fmri under several types of normal/anomalous judgment for japanese sentences including honorification. when activation in a honorification task was contrasted with that in a spelling task, we observed significant increase in the left including grammar center (ifg) as well as the bilateral cerebellum for all tested participants. moreover, the lower performance group showed greater activation in the left f3op/f3t and the bilateral cerebellum. these results suggest that syntactic process is required for japanese honorification and that activation in these regions shows modulation according to performance level, even in native language. research funds: crest, jst os2a-8-06 top-down modulation for melody-related activity in the right auditory areas: an meg study takuya yasui 1,2,3 , kimitaka kaga 2 , kuniyoshi l. sakai 1,3 1 dept. of basic science, univ. of tokyo, tokyo, japan; 2 dept. of otolaryngology, univ. of tokyo school of medicine, japan; 3 crest, jst, japan we previously reported right-hemisphere dominance for melody error-induced fields (m140) (neurosci. res. 52, s61). in a subsequent study, we confirmed that m140 was independent from mismatch negativity usually induced by oddballs. in the present study, we examined whether m140 was induced by deviation from a memorized melody. we used four pairs of unfamiliar songs, each pair consisting of an original song and a modified song in which the third note deviated from that of the original. subjects learned these songs and judged whether there were one or two deviations in notes. there was no significant difference in dipole amplitudes between m140 elicited by the original songs and that by the modified ones. however, while m140 without the deviation showed no significant effect of lateralization, m140 with the deviation resulted in significant enhancement in the right hemisphere. these results suggest the existence of memory-induced, i.e., top-down modulation for melody-related activity in the right auditory areas. research funds: crest, jst os2a-8-07 cortical plasticity in adulthood for learning phonics rules for english orthography and phonology makiko muto 1,2 , kuniyoshi l. sakai 1,2 1 department of basic science, university of tokyo, tokyo, japan; 2 crest, jst, tokyo, japan although matching english orthography with correct pronunciation is difficult for second language learners, learning phonics rules may rapidly improve their performance. in the present fmri study, we tested an english matching task during the course of phonics training in 16 sessions, in which infrequent words were visually shown, while matched/unmatched speech sounds were simultaneously presented. comparing the first half of the sessions with the latter half, the left posterior inferior temporal gyrus (the letter center) and a part of the left lateral premotor cortex (the grammar center) showed activation decreases, when the performance was significantly improved. these results suggest that the plasticity of functional systems involving these critical regions is essential for establishing phonics rules and for forming a new link between orthography and phonology. research funds: crest, jst os2a-8-08 hierarchical syntactic processing in the left frontal region: an meg study kazuki iijima 1,2 , naoki fukui 3 , kuniyoshi l. sakai 1,2 1 dept. of basic science, univ. of tokyo, komaba, japan; 2 crest, jst; 3 dept. of linguistics, sophia univ., yotsuya, japan previous erp studies have shown word-related activation based on semantic association or context. however, it remains unclear how syntactic information of preceding words is integrated into the ongoing sentence processing. in the present meg study, we measured brain activity during each of four tasks: a syntactic task, a semantic task, a memory task, and an evaluation task. sentence stimuli consisted of one noun phrase and one verb, where the noun phrase had either an objective or nominative case particle. the first peak of the activity for a verb presentation was observed at the left frontal region as early as 130 ms after the onset. in the objective-case condition, this activity was enhanced only for the syntactic task, while in the nominative-case condition no such task-selectivity was observed. these results are consistent with the current linguistic theory (the minimalist program), which holds that a noun phrase with an objective case particle is directly merged with a verb, to form a new hierarchical level. research funds: crest, jst os2a-8-09 individual difference of brain activity in medial prefrontal cortex and superior temporal sulcus during social cognition koji jimura, seiki konishi, tomoki asari, junichi chikazoe, yasushi miyashita dept. physiol. univ. tokyo sch. med., japan previous neuroimaging studies have reported brain activity in the medial prefrontal cortex (mpfc) and the superior temporal sulcus (sts) during performance of theory of mind tasks. the present fmri study explored individual difference of the mpfc and sts activity by employing false belief paradigms. the task consists of two sessions, study and test. during the study session, subject studied a brief story in which two characters have false beliefs. then, the subject answered questions about the false belief and the fact that constitutes the false belief during the test session. consistent with previous studies, significant activity was observed in the mpfc and the sts during representing the false belief. the individual differences of the mpfc and the sts activity were correlated with psychodiagnostic indices that represent controlled and automatic idealization, respectively. these results suggest that the two indices represent distinct neural mechanisms participating in social cognition. research funds: grant-in-aid from mext (14002005), jsps research fellowship (1611149) os2a-8-10 brain activity of happy facial recognition in mother-daughter relationship jun shinozaki 1 , nobukatsu sawamoto 1 , toshiya murai 2 , takashi hanakawa 1 , hidenao fukuyama 1 1 hbrc, kyoto univ. grad. sch. of med. kyoto, japan; 2 neuropsychiatry, kyoto univ. grad. sch. of med. kyoto, japan relationship between parents and children is special, and affective facial recognition between them should evoke specific neural activity not shared by other personal relationships. eleven healthy females participated in this fmri experiment. the subjects saw happy and neutral faces of their own mothers, and newly learned other subjects' mothers during the scan. when happy face recognition was compared with neutral face recognition, the mother-daughter combination induced greater activity than the non-familial combination in the following areas; the lateral prefrontal cortex, anterior cingulate cortex, middle temporal cortex, striatum, and anterior insula. it has been shown that the lateral prefrontal and anterior cingulate cortices are associated with familial facial recognition, whereas the middle temporal cortex is related to happy facial recognition. the activity in the striatum and anterior insula might be related to positive affection and empathy, respectively. os2a-8-11 anatomical connections among functionally identified brain regions for sentence processing yukari yamamoto 1,2 , atsushi maki 1,2 , kuniyoshi l. sakai 2,3 1 advanced research laboratory, hitachi, ltd., tokyo, japan; 2 crest, japan science and technology agency, saitama, japan; 3 dept. of basic science, univ. of tokyo, tokyo, japan we have functionally identified the left dorsal inferior frontal gyrus (ifg), the left lateral premotor cortex, and the triangular/orbital part of the left ifg (f3t/f3o) as regions associated with sentence and discourse level processing. in the present study, we examined whether there are direct anatomical connections among these regions by using diffusion tensor tractography. f3t and f3o of the left ifg were chosen as seed areas for fiber tracking. fiber bundles that went through two spherical regions were extracted from the tracking data. the central coordinates of these regions were (−54, 27, 21) , (−39, 3, 42) , and (−51, 27, −6) in the standard brain, which are associated with syntactic processing (the first and second coordinates) or sentence comprehension (the third coordinate). direct connections among these regions were consistently observed among the subjects. this result suggests a critical network among multiple regions that are associated with sentence processing. os2a-8-12 effect of the incongruity controlled by semantic distance on visually evoked magnetic fields nobuyoshi harada 1 , sunao iwaki 1 , mitsuo tonoike 2 1 aist, osaka, japan; 2 chiba university, chiba, japan visual incongruities of heads changed on animal pictures, which were controlled by the semantic distance of the word of the animal, were investigated on visually evoked magnetic fields. the semantic distance was decided by the numbers of links of the semantic network of the taxonomic layer in a japanese thesaurus. the words for mammalians were grouped into five semantic categories in the thesaurus. the heads of the animals were changed with those from another semantic category (deviant, d = 4), and with those from an inner semantic category (middle, d = 2), while others were not changed (normal, d = 0). peak amplitudes of waveforms of the root mean square values on the components of 170 ms (f (2/38) = 4.92, p = 0.013) and 220 ms (f (2/38) = 5.23, p = 0.0098) were significantly decreased with increments of the semantic distance in left occipital sensors. the gradient of the decreasing line of the amplitudes of the 170 and 220 ms components indicated the capability of extracting the structure of a typical prototype of the form of the animal. we call this capability, the structural sensitivity for prototype (ssp). ryohei yasuda duke university medical center, usa calcium signaling in dendritic spines is important for many forms of synaptic plasticity. however, the quantitative mechanisms of how calcium elevations are translated into spatial and temporal patterns of biochemical reactions leading to modifications of synaptic strength are unclear. identifying and following the spatiotemporal activation of molecules necessary for synaptic plasticity is crucial for a better understanding of this complex process. to visualize the activity of signaling pathways in neurons deep in brain tissues, we have combined fluorescence lifetime measurements and two-photon microscopy. this technique allowed us to measure spatiotemporal aspects of the activity of signaling proteins including ras gtpase proteins in response to physiologically relevant stimuli with single spine resolution. research funds: burroughs wellcome fund, dana foundation os2p-2-02 mechanisms of p2y purinoceptor-mediated long-term enhancement of inhibitory transmission examined by multiple-probability fluctuation analysis at cerebellar gabaergic synapses yumie ono 1 , xiaoming zhu 1 , takashi tominaga 2 , fumihito saitow 3 , shiro konishi 1,2 1 waseda-olympus bioscience research institute, singapore, singapore; 2 department of neurophysiology, tokushima bunri university, kagawa, japan; 3 department of pharmacology, nippon medical school, tokyo, japan postsynaptic p2y receptor activation by atp enhances ipscs at cerebellar interneuron-purkinje cell (pc) synapses. to investigate the underlying mechanisms, we here employed the non-stationary fluctuation analysis to estimate the number (n) and single channel conductance (i) of gaba a receptors in pcs using whole-cell recordings of evoked ipscs in pcs of rat cerebellar slices before and 5-15 min after application of atp. the atp-induced enhancement of the ipsc amplitude was associated with a significant increase in the single channel conductance, but not the number, of gaba a receptors in pcs: i and n after atp treatment were 160 ± 9.9% and 94 ± 6.1% of the controls, respectively. pretreatment with the protein kinase a inhibitor h-89, but not the calmodulin kinase ii inhibitor kn-62, completely abolished the atp-induced ipsc enhancement. os2p-2-03 activin induces long-lasting nmda receptor activation via scaffolding pdz protein arip1 isao inoue, akira kurisaki, hiromu sugino institute for enzyme research, tokushima university, tokushima, japan calcium entry into the postsynaptic neuron through nmda type glutamate receptors (nmdars) triggers the induction of long-term potentiation (ltp). the ca 2+ permeability of nmdar is regulated by phosphorylation of its tyrosine residues. we report here that activin, a member of the transforming growth factor-b (tgf-b) superfamily, and one of proteins synthesised after ltp, promotes phosphorylation of nmdars and increases the ca 2+ influx through those receptors in primary cultured rat hippocampal neurons. this signal transduction occurs in a functional complex of activin receptors, nmdars, and src family tyrosine kinase, fyn formed on a multimer of postsynaptic scaffolding pdz protein, activin receptor interacting protein 1 (arip1). activin-induced nmdar activation persists more than 24 h, which is complimentary to the transient activation of nmdars by brain derived neurotropic factor (bdnf). our results show that activin is a long-lasting potentiator involved in synaptic plasticity regulatory mechanisms. os2p-2-04 roles of cam kinase i in the hippocampal longterm potentiation kohji fukunaga, takashi komori, shigeki moriguchi department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan cam kinase i (camki) family members are highly expressed in the adult rat hippocampus and camki-alpha is predominantly localized in the cytosol. camki activation requires phosphorylation of thr177 by camkk as an upstream kinase. we here documented a marked increase in camki-alpha-thr177 phosphorylation following ltp induction in rat hippocampal ca1 region. like camkii activation following ltp (fukunaga et al., 1993) , the increased camki-thr177 phosphorylation remained elevated at least for 60 min after ltp induction. the increased camki-thr177 phosphorylation was closely associated with prolonged increases in phosphorylation of creb and myosin light chains in the ca1 region. this is in contrast with transient increases in camkiv and erk phosphorylation. treatment with camkk inhibitor, sto-609 significantly inhibited both creb and mlc phosphorylation with concomitant reduction of ltp in the ca1 region. taken together, camki likely mediates the late phase of creb phosphorylation and an increased mlc phosphorylation in the hippocampal ltp. to investigate how the excitatory postsynaptic inputs of the proximal dendrite effect the information processing of synaptic inputs at the distal dendrite, stimulation was applied to induce bap and epsp at the alveus and the proximal dendrite, respectively. the resulting coincidence of magnitude of bap and epsp at the distal dendrite was enhanced when the bap was delivered at a timing (5 ms) to induce ltp. furthermore, the magnitude of bap at the distal dendrite was attenuated by the input from the proximal dendrite at a timing (20 ms) to induce ltd. these results suggest that the magnitude of bap delivered to the distal dendrite can be amplified or attenuated depending on the relative timing between proximal input and bap. this may be due to an effect on the coding process at the distal dendrite and could support the basis for a novel learning rule in the brain. research funds: kakenhi (17021037) os2p-2-06 mouse brains deficient in neuronal pdgf receptor-␤ develop normally but are vulnerable to injury yoko ishii, takeshi oya, lianshun zheng, masakiyo sasahara department of pathology, faculty of medicine, university of toyama, japan the platelet-derived growth factors (pdgfs) and pdgf receptors (pdgfrs) are widely expressed in the mammalian cns. here, we developed novel mutant mice in which pdgfr-␤ subunit gene was genetically deleted in the neurons of cns to elucidate the role of pdgfr-␤. our mutant mice reached adulthood without apparent anatomical defects. the cerebral damage after cryogenic injury was severely exacerbated in the mutants compared with the controls. furthermore, this exacerbated lesion formation was suggested to be, at least partly, due to the enhanced excitotoxicity after injury, because nmda-induced lesion formation was also extensively enhanced in the cerebral cortex of the mutants without altered nmda receptor expression. this is the first known report to address the postnatal function of pdgfr-␤ expressed in cns neurons, using genetically engineered mutant. it was clearly demonstrated that pdgfr-␤ expressed in neuron protects cns neurons from cryogenic injury and nmda-induced excitotoxicity. early postnatal days (especially the first three weeks in the rat) are the critical period for newborn hippocampal granule cells (gcs) to dynamically migrate from the dentate hilus and form the gc layer. to investigate the mechanism that regulates newborn gc migration, we developed a new slice coculture system. the hilar parts of entorhino-hippocampal slices prepared from postnatal six-day-old (p6) rats that had received a single brdu injection at p5 were substituted with the corresponding region of entorhino-hippocampal slices from p6 rats. after five days in vitro, newborn gcs, detected by brdu and prox1, migrated out of the hilar graft and reached the host gc layer. chronic application of picrotoxin, a gaba a receptor antagonist, facilitated the migration of newborn gcs into the gc layer. these results indicate that gaba a receptors regulate the migration of newborn gcs in early postnatal days. os2p-3-02 cdk5 is required for neuroblast migration in the adult mouse brain yuki hirota 1,2,6 , toshio ohshima 3 , takuji iwasato 4 , ashok b. kulkarni 5 , hideyuki okano 2,6 , kazunobu sawamoto 1,2,6 1 bridgestone lab. keio univ., tokyo, japan; 2 dept. physiol., keio univ., tokyo, japan; 3 dev. neurobiol. riken, tokyo, japan; 4 behavioral gen. riken, tokyo, japan; 5 nih, bethesda, usa; 6 sorst, jst, saitama, japan neuroblasts generated in the subventricular zone (svz) of the lateral ventricles migrate into the olfactory bulb (ob) through the pathway called rostral migratory stream (rms). molecular mechanisms regulating the directional long-distance migration remain largely unknown. here we studied adult function of cyclin-dependent kinase 5 (cdk5) that has been revealed to play a role in neuronal migration in the embryonic brain. crossing the floxed-cdk5 mice to emx1cre mice resulted in decreased size of ob and abnormal distribution of neuroblasts. svz explants from these mice cultured in matrigel showed decreased migration distance. leading process of neuroblasts infected with cre-encoding retrovirus were found in random orientations and frequently failed to migrate out of the svz compared to control cells. these results indicate that cdk5 has a cell autonomous function in neuroblast migration in the adult brain. os2p-3-03 colocaliztion of neuron markers and glial markers in gabaergic neuron progenitors as revealed by singlecell microarray analysis shigeyuki esumi 1 , wu sheng-xi 2 , yuchio yanagawa 3,4 , kunihiko obata 5 , nobuaki tamamaki 1 1 kumamoto univ., kumamoto, japan; 2 fourth military medical univ., xi'an, people's republic of china; 3 gunma univ., maebashi, japan; 4 sokendai, hayama, japan; 5 riken, wako, japan gabaergic neurons and oligodendroglia share many characters in the murine forebrain. both of the cell types has been reported to originate in the medial ganglionic eminence and migrate to the neocortex. in addition, it is reported that they share several glial markers, such as ng2, plp, and cnp at their prematured stages. in order to investigate its nature, we have established a single-cell microarray analysis method. single gfp-positive gabaergic neuron progenitors were corrected from the subventricular zone of the gad67-gfp knock-in mouse neocortex at e18-p0 by dissociation and picking. complemental dna from the single cells was amplified by universal pcr amplification and converted into biotin-labeled crna using t7 rna polymerase. after these procedures, crna sufficient for a microarray analysis was obtained. as the result we found, mbp and s100-␤ expression in the gabaergic neuron progenitors. os2p-3-04 role of ␤-catenin signaling in regulating proliferation of transit-amplifying cells in the adult mouse subventricular zone kazuhide adachi 1,2,3 , masanori sakaguchi 3 , toru yamashita 2,3,6 , yuko fujita 3 , yukiko gotoh 4 , arturo alvarez-buylla 5 , takeshi kawase 1 , hideyuki okano 3 , kazunobu sawamoto 2,3 1 neurosurgery, keio univ. sch. med., tokyo, japan; 2 bridgestone lab. dev. regenerative neurobiol., keio univ. sch. med., tokyo, japan; 3 physiol., keio univ. sch. med., tokyo, japan; 4 inst. mol. cell biosciences, univ. tokyo, tokyo, japan; 5 neurosurgery, ucsf, san francisco, usa; 6 neurol, okayama univ, med, dentistry and pharmaceutical sci, okayama, japan the subventricular zone (svz) continuously produces olfactory bulb neurons in the adult rodent brain. neural stem cells generate migratory neuroblasts via highly proliferative transit-amplifying cells in this region. here, we studied the role of ␤-catenin signaling in the adult mouse svz. ␤-catenin accumulated in the nucleus of only the transitamplifying cells in the svz. activated ␤-catenin signaling promoted the proliferation of transit-amplifying cells, resulting in an increased number of new neurons in the olfactory bulbs. these results suggest that ␤-catenin signaling plays a role in the proliferation of transitamplifying cells in the adult mouse svz. the ciliary marginal zone (cmz) is a region between the neural retina and ciliary epithelium, and contains retinal progenitor cells that give rise to neuron and glia. wnt2b is expressed in cmz, and has been shown to control the differentiation of the retinal progenitor cells. we have isolated a novel bmp antagonist, chick tsukushi (c-tsk), which belongs to the small leucine-rich proteoglycan family. in the eye, the expression of c-tsk is observed in the cmz which is similar with that of wnt2b. to examine the molecular interactions between c-tsk and wnt2b, we co-electroporated them into the optic vesicle at stage 9-10 chick embryo and observed the proliferation of the retinal progenitor cells. we found that c-tsk inhibited the wnt2b activity that sustains prolonged proliferation of retinal progenitor cells. our result suggests that c-tsk controls the proliferation of retinal progenitor cells interacting with wnt2b. to reveal the role of epigenetic gene regulation in neuronal differentiation, we studied subcellular distributions of histone deacetylase (hdac) 9 in developing cortical neurons. an expression vector of gfp-tagged hdac9 was transfected to dissociated cortical cell cultures as well as cortical neurons in vivo. hdac9 was primarily localized in nuclear until 1 week in vitro, but was translocated to cytoplasm in the later stages. such translocation was found in a similar time course after birth in vivo. to examine a possibility that neural activity is involved in the translocation, firing activity of cultured neurons was examined using multi-electrode dishes. as a result, spontaneous firing activity was prominent in the late stages when cytoplasmic translocation occurred. however, ttx addition to the culture medium produced the inverse translocation. these results suggest that activity-dependent intracellular localization of hdac9 contributes to neuronal differentiation in cortical development. research funds: kakenhi (16700286) dept. of physiology, fujita health univ. sch. of med., japan we described electrical synapses in alpha retinal ganglion cells (␣-gcs). precise temporal synchronization of spikes is generated from ␣-gcs (hidaka et al., 2004) . the fraction of open channels in gap junctions were evaluated with techniques of dual patch-clamp, connexin immunocytochemistry, and high-voltage electron microscopy. junction conductance (maximum 2.45 ns) was measured. in high-voltage electron microscopy (hitachi1250m, nips, 2005 , gap junctions (average size 0.86 m long) were present in contacts. in confocal laser-scanning imaging, connexin36 localization at contacts counted gap junctions (seven sites in a pair on average). assuming that the density of connexons would be 5180/m 2 and a single channel conductance is 15 ps, the conductance of each junction would be 45 ns. the presence of seven junctions between a pair will lead to estimate a total junction of 315 ns. the measured conductance could allow to estimate a fraction of open channels as 0.8%. the open fraction is small, when we consider whether electronic transmission acts to synchronize the spikes in the intercellular network. the visual system separates different types of information into parallel, anatomically distinct processing streams. despite their significance for visual processing, the molecular mechanism underlying the physiological stream formation is largely unknown, partially because these physiological streams have not been reported in mice. to identify molecular correlates of functionally distinct streams, we fabricated a custom cdna microarray of higher mammal ferrets. we successfully identified molecules whose unique distribution and developmental profiles define the lgn itself, its constituent layers, or identify cells comprising one of the physiological streams in the lgn. using these molecules as temporal and spatial markers, we investigated mechanisms of the physiological stream formation in the ferret lgn. research funds: kakenhi (17023014), coe research akira muto, herwig baier department of physiology, university of california san francisco (ucsf), usa the visual system operates over a broad range of luminances. this is accomplished by adjustment of photosensitivity, called light adaptation. to study the molecular mechanisms of light adaptation, we screened for zebrafish mutants that showed compromised optokinetic responses (reflexive eye movements to large field motion) after an abrupt dark-to-light transition. in this experimental paradigm, wildtype fish larvae recover their full optokinetic response within about two minutes after being brought back to light. in a screen of almost 2000 genomes, we identified five mutants all of which showed substantially delayed recovery of the okr. positional cloning of one of the loci revealed a mutation in the dna-binding domain of glucocorticoid receptor (gr). gr is known for its role in the stress response, but its function in the visual system is unexplored. we propose that gr is regulating genes essential for light adaptation in the retina. os2p-4-04 multisite recording of the signal propagation pattern in the visual cortex makoto osanai, yusuke takeno, satoshi tanaka, tetsuya yagi graduate school of engineering, osaka university, suita, japan recently, the visual prosthesis systems with implanted stimulus electrode in the visual cortex are developed. but the signal propagation pattern induced by electrical stimuli in the visual cortex is not fully investigated. therefore, we studied the signal propagation pattern induced by electrical stimuli in the mouse visual cortex slice, using a 60 channel multielectrode array and a calcium imaging system. in the electrophysiological study, the responses conducted vertically against the layer of the cortex with layer 4 stimuli and propagated horizontally in the layer 2/3. in the calcium imaging study, the area of the higher calcium concentration region spread vertically with layer 4 stimuli. signal propagation was restricted within several tens m around the stimulus electrode by ap5 + cnqx administration and was completely blocked by ttx administration. administration of bicuculline increased the area of the signal propagation in a dose-dependent manner. we concluded that these restricted patterns of the signal propagation in the visual cortex were due to the inhibitory system. os2p-4-05 presence of two phases in the sensitive period of orientation plasticity shigeru tanaka 1 , toshiki tani 1 , kazunori o'hashi 1,2 , jerome ribot 1 1 brain science institute, riken, saitama, japan; 2 graduate school of life science and systems engineering, kyushu institute of technology, kita-kyushu, japan recently we have revealed that orientation-restricted visual experience induces drastic reorganization of orientation maps in the cat visual cortex. in this study, we examined the effect of release from single orientation exposure on once reorganized orientation maps during the sensitive period using intrinsic signal optical imaging. when kittens were returned to the normal visual environment by removing the goggles after 2 weeks of goggle rearing starting around the age of 3 weeks, the over-representation of the exposed orientation was preserved. on the contrary, when the goggle rearing started around the age of 5 weeks and then the animals were returned to the normal visual environment, orientation maps rapidly changed to represent orientations equally. these findings indicate that the sensitive period of orientation plasticity consists of two phases: orientation map reorganization is irreversible in an early phase and reversible in a late phase. os2p-4-06 residual visuomotor processing in the animal model of blindsight: comparison with normal, near-threshold vision masatoshi yoshida 1,2,3 , tadashi isa 1,2,3 1 dept. dev. physiol., nat'l inst. physiol. sci., okazaki, japan; 2 sch. life sci., grad. univ. adv. stud., hayama, japan; 3 crest, jst, kawaguchi, japan in two macaque monkeys with unilateral v1 lesion performing a visually guided saccade task, saccadic parameters were compared between the saccades to the affected hemifield and those to the intact hemifield. the luminance contrast of the target presented in the intact hemifield was reduced so that the detectability was comparable to that in the affected hemifield (80-90% correct). in the saccades to the affected hemifield, the curvature of the trajectories was smaller and the deviation of the saccadic end points from the target was larger than those to the intact hemifield. these results suggest that without geniculo-striate pathway, online compensation for the variation of the initial saccadic command is not fully functional, thus leading to inaccurate saccades. we propose that the residual visuomotor processing of monkeys with v1 lesion is unlike normal, near-threshold vision. research funds: kakenhi 13854029, kakenhi 16700343 and crest, jst os2p-4-07 comparison of the angle representation in macaque visual areas v1 and v2 minami ito 1,2 , hidehiko komatsu 1,2 1 national institute for physiological sciences, okazaki, japan; 2 the graduate university for advanced studies, hayama, japan previously, we have reported that fairly large number of area v2 neurons has angle selectivity. here, we studied the angle selectivity of area v1, which is the major source of inputs to area v2. we conducted single-unit recordings from the superficial layer of area v1, while animals performed a fixation task. for comparison, we used a similar stimulus set. the stimuli were much larger than the size of the classical receptive fields. area v1 neurons responded mainly to sharp angles (30 • ), straight lines (180 • ) or right corners (90 • ), but not to intermediate angles (60 • or 120 • angle width). this contrasted with area v2, where neurons showed a variety of the optimal angle width including intermediate angles. we also observed several v1 neurons showed fine orientation tuning to short line segments, while weak or no responses were induced by a set of large angle stimuli. we suggest that area v1 neurons largely contribute to representing line components (lines and line-ends) and to sending such information to area v2. os2p-4-08 firing rates and dynamic spatiotemporal patterns of ganglion cells both contribute to retinal information processing xin jin, ying-ying zhang, xue liu, hai-qing gong, pei-ji liang department of biomedical engineering, shanghai jiao tong university, shanghai, china population activities of retinal ganglion cells (rgcs) were recorded using a multi-electrode recording system. single unit analysis showed that firing rate of individual neuron was strongly dependent on the luminance intensity of stimulation. however, population activity of ganglion cells usually showed particular spatiotemporal pattern, in response to a specific velocity of the moving bar. differing from single direction-selective ganglion cell (dsgc), which responds to its preferred direction of movement by firing at its maximal rate, population activity of non-direction-selective ganglion cells may encode the motion information in a temporally ranked manner, independent to their individual firing rates. these results suggest that an efficient and economical coding mechanism may be employed by the retina, where the firing rate of individual neurons and spatiotemporal pattern of population neuronal responses could act in parallel to encode different aspects of visual information. yasuto tanaka, satoru miyauchi, masaya misaki brain information group, nict, kobe, japan visual long-range interaction was reported to be limited in space. here, we show the evidence of long-range interaction extending to an order magnitude larger using the right-left symmetrical configuration. two horizontally collinear gabor signals, one defined as probe and the other cue, were presented at the left and right side of the visual field at mirror symmetrical regions. detection threshold of gs probe reduced with cue-probe separations up to 10 • . the facilitation was highly tuned to the symmetrical locus. furthermore, the facilitation was substantially longer at upper visual field than the lower visual field. the reduction was specific to orientation, phase, and horizontal direction, the results indicate long-range mirror symmetrical interaction across vertical meridian, suggesting symmetrical neuronal communication between early visual cortices. the anisotropy between left-right hemifield (symmetry) and upper-lower hemifield (upper-field advantage) signifies hemifield inhomogenity in human vision. os2p-4-10 integrity of visuospatial attention in a split brain patient noudoost behrad, seyed reza afraz, maryam vaziri, hossein esteky school of cognitive sciences (scs), iran transfer of visual information between hemispheres is severely impaired following transection of posterior part of the corpus callosum. we investigated whether attentive visual object tracking across vertical meridian of the visual field is possible for a posterior callosotomized patient (md). we asked md to track one bouncing ball among four identical distracters while fixating at the center of the screen. target crossed the vertical midline in half of the trials. her performance in crossed conditions was significantly above chance level. also, we asked her to make decision about horizontal alignment of two balls presented simultaneously in one of three conditions: both in right or left hemifield, or each in one hemifield. in this alignment task md was able to compare location of the two bilaterally presented stimuli well above chance level. our data suggest that inter-hemispheric transfer of position information required for spatial attention is preserved without posterior corpus callosum. pei sun, justin l. gardner, mauro costagli, kenichi ueno, r. allen waggoner, keiji tanaka, kang cheng laboratory for cognitive brain mapping, riken brain science institute, wako-shi, japan although the preference for stimulus orientations in human visual cortex has been inferred indirectly in a few studies using fmri, tuning to particular stimulus orientations has not been directly demonstrated using this technique. in an effort towards revealing orientation selectivity and its spatial arrangement in human v1, we have conducted an fmri study with a novel stimulation paradigm and a differential mapping method. we found that responses of the majority of activated voxels were modulated by the grating orientation and individual voxels were sharply tuned to particular orientations. our results provide the first demonstration that orientation selectivity in humans can be directly studied using fmri. os2p-4-12 probing the spatial scale of classifier performance with high spatial resolution fmri justin l. gardner 1,2 , pei sun 2 , keiji tanaka 2 , david j. heeger 1 , kang cheng 2 1 department of psychology and center for neural science, new york university, usa; 2 laboratory for cognitive brain mapping, riken brain science institute, japan recently, classifier analysis with conventional resolution fmri has been used to decode the orientation of a grating stimulus from the fmri responses of early visual cortex. it has been proposed that classifier analysis exploits small but robust orientation biases in voxels that are created by local inhomogeneities in the columnar organization. we have examined this proposal by using classifier analysis to decode stimulus orientation using high spatial resolution fmri (0.75 mm × 0.75 mm × 3 mm voxels) in human v1. we found that many voxels that are weighted heavily in the classifier analysis and carry similar orientation biases closely follow draining veins that are visible on t2*-weighted venograms. we suggest that large draining veins with orientation specific responses, rather than local inhomogeneities in orientation maps, may provide a basis for classifier performance using large voxels. research funds: nrsa fellowship from the nih (1f32ey016260-01) os2p-4-13 relationship between horizontal connections and functional structure in macaque anterior inferotemporal cortex (area te) hisashi tanigawa, kathleen s. rockland, manabu tanifuji riken brain science institute, wako, japan we have studied the relationship between horizontal connections and functional structure in te using a combination of optical imaging, unit recording, and anatomical tracing. intrinsic signal imaging was performed in exposed te, under anesthesia, during presentations of visual object stimuli. this resulted in multiple optical spots evoked by each stimulus. in some animals, subsequently, unit recording was carried out at multiple sites within the imaged region. then, an anterograde tracer was injected into one of the spots. both optical imaging and unit recording revealed regions with stimulus preference similar to that at the injection site. however, these regions and the injection site were not always connected by horizontal axons. some regions sharing a preference to particular stimuli were connected, even though they showed different preferences to the other stimuli. these results suggest that horizontal axons can connect regions with different stimulus preferences in te, in contrast to like-to-like connectivity, as understood in early visual cortices. we recorded single cell responses from the inferotemporal cortex of a fixating monkey while visual stimuli with various durations (18-350 ms; isi = 1 s) were presented. presentation of visual stimuli at all of the tested durations resulted in prolonged responses. the brief presentations evoked multiple phasic responses while the long presentations evoked sustained activities. there was a significant difference in average firing rate of late phase (350-550 ms) of response to optimal stimulus across presentation durations. but no such differences were found for the first phase (70-270 ms). in addition, the optimal stimulus evoked significantly different response magnitudes in the first and second phase particularly in the short presentation durations. but the suboptimal stimulus (∼50% of max response) evoked similar response magnitudes in the first and second phase. these results suggest that stimulus selectivity of inferotemporal cells depends on the stimulus presentation duration and the time window that is used to measure the firing rate. os2p-4-15 the perceptual learning effect in myopes by the lateral masking procedure keiko mizobe 1 , kazuto terai 2 , osamu hieda 3 , shigeru kinoshita 2 1 dept. of ophthal., kyoto second red cross hospital, kyoto, japan; 2 dept. of ophthal., kyoto pref. univ. of med., kyoto, japan; 3 baptist eye clinic hospital, kyoto, japan purpose: the study of the visual cortex revealed the lateral masking collinear configuration modulated the neuronal responses and psychophysical studies also showed perceptual learning improved the visual detection. we asked whether perceptual learning could improve the myopic blurred vision, using the new instrument of the lateral masking technology, neurovision. method: nine low myopes were studied. non-corrected digital visual acuities (va) ranged from 0.4 to 1.2. the logmar average was 0.31. eight sessions of neurovision treatment were performed to each individual. the estimation was done by comparison of va before and after the treatment. results: four eyes showed more than one octave improvement of va. the logmar average of the four was 0.03, improved from 0.50. the residual five eyes showed less or no improvement. the change of logmar average was from 0.15 to 0.08. conclusion: some myopes showed the perceptual learning effects by new treatment, using the lateral masking technology. os2p-5-01 the hindbrain neuroepithelial cells exclude the migrating facial motor neurons by expression of planar cell polarity (pcp) genes hironori wada 1 , hideomi tanaka 1,2 , satomi nakayama 1 , miki iwasaki 1,2 , hitoshi okamoto 1,2 1 laboratory for developmental gene regulation, bsi, riken, japan; 2 crest, jst, japan many neurons migrate tangentially through one cell layer at a specific depth within the brain. in the developing zebrafish hindbrain, the facial (nvii) motor neurons originate in rhombomere (r) 4 and migrate tangentially to r6 near the pial surface of the hindbrain. in this study, we demonstrate that expression of the planar cell polarity (pcp) genes celsr2 and frizzled3a in neuroepithelial cells maintain the nvii motor neurons near the pial surface during their caudal migration in the zebrafish hindbrain. mosaic analyses show that expression of the frizzled3a gene in the surrounding neuroepithelial cells prevented the entry of the nvii motor neurons in the neuroepithelial layer. the demonstration of a role for neuroepithelial cells in excluding differentiated neurons from the neuroepithelial layer may provide new insights into the general mechanisms underlying formation of the layered structures in the mammalian brain, such as in the cerebral cortex. os2p-5-02 disrupted-in-schizophrenia 1 (disc1) regulates the transport of the nudel/lis1 complex to axons via direct interaction with kinesin-1 shinichiro taya, kozo kaibuchi department of cell pharmacology, nagoya university, nagoya, japan disc1 is a candidate gene for susceptibility to schizophrenia. in a scottish family, the chromosome translocation interrupts the coding sequence of disc1. disc1 is reported to interact with nudel, which forms a complex with lis1. although the functional significance of this complex in axon growth and neuronal development has been reported, the transport mechanism of the complex into axons and the functions of disc1 remain largely unknown. here we report that disc1 interacted with kinesin-1, a motor protein of anterograde axonal transport. kinesin-1 interacted with the nudel/lis1 complex through disc1, and these molecules accumulated at the distal part of axons. the knockdown of disc1 by rnai of disc1 induced the delocalization of nudel and lis1 from the axons and reduced axonal growth. the knockdown of kinesin-1 induced the delocalization of disc1 from the axons. taken together, these results indicate that disc1 links kinesin-1 to the nudel/lis1 complex and regulates its transport as a cargo receptor for axon elongation. research funds: mext os2p-5-03 role of a novel collapsin response mediator protein-2 interacting molecule, synaptotagmin-like protein in hippocampal neuron nariko arimura, saeko kawabata, atsushi hattori, kozo kaibuchi department of cell pharmacology, graduate school of medicine, nagoya university, nagoya, japan during the development, neurons recognize the extracellular signals and extend the axons to proper directions. certain kinds of receptors are transported from the nerve cell body to the axon terminal, and participate in the recognition of extracellular environments. however, the mechanism of controlled recruitment of receptors remains unsolved. here, we report that synaptotagmin-like protein 1 (slp1) can mediate the vesicle transport. slp1 is known to associate with rab27. we found that slp1 associates with collapsin response mediator protein-2 (crmp-2), which is a key regulator of axon formation. slp1 could form the trimeric complex with rab27b and crmp-2, and also associate with kinesin-1 through crmp-2. slp1 is accumulated on microtubules at the axonal growth cones, and is co-localized with a receptor of growth factor. these findings suggest that slp1 functions as a mediator of recruitment of certain receptor depending on crmp-2 and kinesin-1. os2p-5-04 absolute quantification of mdr1a, mrp1, mrp4 and bcrp proteins at the mouse brain blood barrier by lc-ms/ms junichi kamiie 1,2 , yuki katsukura 1,2 , sumio ohtsuki 1,2 , xiao-kun cai 1,2 , tetsuya terasaki 1,2 1 graduate school of pharmaceutical sciences, tohoku university, sendai, japan; 2 sorst, jst, japan the abc transporter proteins are thought to limit permeability across the blood-brain barrier as the efflux transporters. however, contribution of each transporter to the bbb function is not clarified. the purpose of this study was to clarify the protein amounts of mdr1a, mrp1, mrp4, and bcrp in the brain capillaries of mouse by newly developed membrane protein quantification method using lc-ms/ms. by this method, the standard curve showed linearity between 10 and 1000 fmol, and amino acid sequence of the detected fragment was confirmed by ms/ms spectrum. in the brain capillaries, the protein amounts of mdr1a, mrp4, bcrp were 3.9, 2.7 and 4.8 fmol/g, respectively, while it of mrp1 was under detection limit of standard curve. this quantitative profile suggests that mrp4 and bcrp function as the efflux transporter at mouse blood-brain barrier as well as mdr1a. os2p-5-05 dominant expression of claudin-5 in highly purified brain capillary endothelial cells sumio ohtsuki 1,2 , hirofumi yamaguchi 1 , saori sato 1 , tmoko asashima 1,2 , tetsuya terasaki 1,2 1 graduate school of pharmaceutical sciences, tohoku university, sendai, japan; 2 sorst, jst, japan claudins are major constituents of tight junctions (tjs). the purpose of this study was to clarify the expression levels of each claudin subtype in brain capillary endothelial cells (bcecs), which form the blood-brain barrier. mouse bcecs were highly purified using endothelial surface antigen (pecam-1) and magnetic cell sorting. mrna expression of caludin-1-23 was measured by real-time rt-pcr. claudin-5 showed the highest mrna expression in the purified mouse bcecs. mrna levels of claudin-1 and -12 were 0.0027% and 0.20% of that of claudin-5. claudin-5 mrna was concentrated in the purified bcecs, while claudin-1 and -12 mrna in the purified bcecs were lower than that in the whole brain. rat claudin-5 mrna was also concentrated in rat brain capillary fraction, but claudin-12 mrna did not. these results suggest that claudin-5 is a dominant tjs protein in bcecs, and expression of claudin-1 and -12, which was reported as tj protein in bcecs, are not restricted in bcecs. os2p-5-06 effects of hydrogen peroxide towards gap junction communication in astrocytes and permeability of blood brain barrier f. ahmad 1 , a. pauzi m. yusof 1 , p.d. mourad 2 , m. bainbridge 3 , s. ab ghani 1 1 universiti sains malaysia; 2 university of washington, seattle, usa; 3 brody school of medicine, east carolina university, nc, usa h 2 o 2 is the main peroxides produced in mammalian cells that consume o 2 . the main source of h 2 o 2 in the brain, produced in large amount, was from the superoxide dismutase catalyzed reaction in mitochondria. therefore, we look into the effects of h 2 o 2 towards the gap junction communication in astrocytes and permeability of blood brain barrier. in this study, by using a h 2 o 2 microsensor, we investigated the level of h 2 o 2 in the brain that altered the permeability of bbb. the microsensor was implanted in the rat's brain and operated amperometrically. we measured h 2 o 2 level from the current generated by the electron transfer at the electrode. we observed a change in permeability when external h 2 o 2 was injected into the brain. fatality occurs when the injected h 2 o 2 exceeds 250 m. these finding showed that the altered paracellular permeability in the presence of h 2 o 2 is caused by a series of events that happen one after another. research funds: short term grants 304pkimia633134 and 304pfar-masi670008 os2p-6-01 somato-ovarian sympathetic reflex discharges in anesthetized rats sae uchida, fusako kagitani, harumi hotta dept. auton. nerv. syst., tokyo metropol. inst. gerontol., tokyo, japan ovarian sympathetic efferent reflex discharges caused by single electrical shock stimulation of spinal (t9-11) afferent nerves or limb (tibial) afferent nerves were studied in urethane anesthetized rats. in central nervous system (cns) intact rats, stimulation of the t9-11 spinal afferent nerve produced early and late a-reflex discharges, and a late c-reflex discharge. after spinalization at the third thoracic level, stimulation of the same spinal afferent nerve produced an a-reflex with the same latency as the early a-reflex in cns-intact rats and an early c-reflex discharge with the similar latency as the late a-reflex in cns-intact rats. on the other hand, stimulation of the tibial afferent nerve produced late a-reflex and c-reflex discharges in cns intact rats, those were not observed after spinalization. it was concluded that ovarian sympathetic aand c-reflex discharges evoked by stimulation of a segmental spinal afferent nerve in cns-intact rats are of spinal and supraspinal origin, and those evoked by tibial nerve stimulation are of supraspinal origin. os2p-6-02 responses of renal sympathetic nerve activity and sodium excretion to 3 days sodium loading in rats misa yoshimoto, nozomi iinuma, rie itokawa, eri hayashi, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan in the present study, a month recording of renal sympathetic nerve activity (rsna) in freely moving rats was made to explore the long-term regulation of rsna and sodium excretion. wistar male rats were instrumented chronically with electrodes for the measurements of rsna and electrocardiogram. after the 7 days recovery period, rsna, heart rate and sodium balance were measured over three weeks. animals were allowed to drink four different concentration of sodium chloride solutions (0, 50, 154, 308 meq./l nacl) over 3 days. the sodium loading with 308 meq./l nacl suppressed rsna significantly and then it gradually recovered while either 0 meq./l nacl or 154 meq./l nacl loading had no effects on rsna. sodium excretion changed significantly in proportion to the each sodium loading levels. these results indicated that the changes in rsna were not always correlated with the changes in sodium excretion in rats. os2p-6-03 cross correlation analysis of respiratoryrelated optical imaging signals yoshitaka oku 1 , haruko masumiya 1 , yasumasa okada 2 1 dept. physiol., hyogo col. med., nishinomiya, japan; 2 dept. med. keio univ. tsukigase rehab. ctr. shizuoka, japan we aimed to establish an objective method to identify the distribution of respiratory-related regions and the timing when these regions are activated relative to the inspiratory activity from optical imaging signals. optical signals were recorded from the ventral medullary surface of neonatal rats in vitro using a voltage-sensitive dye. cross correlation between integrated c4 ventral root (c4vr) activity and each pixel was calculated after cycle-triggered averaging and detrending. the maximum of cross correlation coefficients and the lag at which the cross correlation became maximal (lagmax) were displayed as 3d pseudocolor maps. in all preparations, two respiratory-related regions were consistently identified: (1) a continuous column extending from the para-facial region to the pre-bötzinger complex, and (2) a region corresponding to the ventral horn. pixels where lagmax were negative (meaning that the activity preceded the c4vr activity) tended to be distributed in the para-facial region, and this tendency was more evident when superfusate ph was lowered. os2p-6-04 slow afterhyperpolarization determines the firing pattern of action potentials in rat gnrh neurons masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan gonadotropin-releasing hormone (gnrh) neurons play a pivotal role in the hypothalamo-pituitary-gonadal axis. gnrh neurons must be able to continuously fire in response to depolarizing stimuli. for this type of firing, gnrh neurons may have a certain intrinsic property. to address this issue, we investigated the ca 2+ -activated voltage-independent k + currents underlying afterhyperpolarization. dispersed gnrh neurons from adult gnrh-egfp transgenic rats were cultured overnight and used for an electrophysiological experiment with perforated patch clamp configuration. the gnrh neurons showed a slow afterhyperpolarization current (i sahp ). in contrast to previous reports, the i sahp observed in rat gnrh neurons was potently blocked by an sk channel blocker apamin. in current clamp condition, gnrh neurons evoked a train of action potentials to depolarizing current pulse. apamin increased the susceptibility to spike failure. the results indicate that rat gnrh neurons exhibit an apaminsensitive i sahp , which regulates the firing pattern. research funds: kakenhi 16590180, 16086210 os2p-6-05 the effect of music to sex hormones of elderly person hajime fukui 1 , kumiko toyoshima 2 , kiyoto kuda 1 , katsuhiko iguchi 3 1 nara univ. of edu., nara, japan; 2 grad. school of human sciences, osaka univ. japan; 3 nara city medical clinic, japan it has been known that testosterone or estrogen protects nervous system and regulates cell death in a brain. also, it is pointed out that the decline of t and est accelerates depression. therefore the treatment such as hormone replacement therapy (hrt) has been tried to cure depression and alzheimer's disease. however, it has been pointed out that hrt has serious side effects. on the other hand, there are reports that music influences on a steroid hormone. in addition, it is known that music has certain therapeutic gain toward ad and dementia. in this study, from a point of view of the prevention of ad and dementia, we examined the effect of music to sex hormones of normal elderly person. four males and 36 females participated music session and t and est were evaluated. as a result, in female, in the high hormone group, the values decreased after the session, and in the low hormone group, the values increased. from above, there might be possibility that through a steroid hormone music participates in protection and improvement of function on brain. tuberculous meningitis (tbm) is the most common form of chronic infection of the central nervous system. despite the magnitude of the problem, the general diagnostic outlook is discouraging. this study identifies a specific protein marker in csf, which will be useful in early diagnosis of tbm. we have demonstrated the presence of a 30-kda protein band in csf of 100% (n = 5) of confirmed and 90% (n = 138) of suspected tbm patients out of 153 tbm patients. the 30-kda protein band was analyzed by lc-ms/ms analysis. in the present study we have identified two mycobacterial proteins rv3804c (ag85a) and rv1886c (ag 85b) and one host derived protein as the components of the tbm specific 30-kda protein. involvement of mitochondrial extrinsic and intrinsic apoptotic pathways in dopaminergic neurodegeneration was tested in rotenone-and mpp + -induced rat models of parkinsonǐs disease (pd). hplc-ec, patch clamp, fluorimetry, immunoblot and rt-pcr were used for measuring neurotransmitters/free radicals, membrane currents, caspases activities, levels of proteins and mrna of mitochondria-linked signaling in brain. we report here a retrograde mode of neuronal death via mitochondrial intrinsic pathway in mpp + -, but an extrinsic mode of cell death in rotenone-induced model. drug screening in these models (l-deprenyl as positive control) indicated that quercetin, coenzyme q 10 , vitamin d 3 and melatonin act via interfering the signaling events in neurons. loss of complex-i and -iv activities and changes in some of the protein subunits in pd postmortem brains were confirmed in pd and control cybrids. results from the present study provide evidences for a direct involvement of mitochondria and are suggestive of existence of both intrinsic and extrinsic apoptotic pathways in dopaminergic neuronal death. os2p-7-02 involvement of thioredoxin on the neuroprotective effect of (−)-deprenyl tsugunobu andoh 1 , boon chock 2 , dennis l. murphy 3 , chuang c. chiueh 4 1 dept. applied pharmacol., univ. toayama, toyama, japan; 2 lab. bioch., nhlbi, nih, md, usa; 3 lab. clin. sci., nimh, nih, md, usa; 4 cent, brain diseases and aging, taipei med. univ., taipei, taiwan the present study investigated whether the induction of thioredoxin (trx) involves in the cytoprotective mechanisms of (−)-deprenyl which is known as the inhibitor of mao-b. after confirming (−)-deprenyl protects against mpp + -induced cytotoxicity in human sh-sy5y cells, we observed further that (−)-deprenyl induced trx for protection against oxidative injury caused by mpp+. the induction of trx was blocked by pka inhibitor through a pka-sensitive phosphoactivation of map kinase erk1/2 and the transcription factor c-myc. (−)-deprenyl-induced trx and associated neuroprotection were concomitantly blocked by the antisense against trx mrna in human sh-sy5y cells. consistently, trx increased the expression of mnsod and bcl-2 supporting cell survival. in conclusion, (−)-deprenyl augments the gene induction of trx leading to elevated expression of antioxidative mnsod and antiapoptotic bcl-2 proteins for protecting against mpp + -induced neurotoxicity. os2p-7-03 pgd 2 induces neuronal apoptosis via 15d-12,14 -pgj 2 tatsurou yagami 1 , noboru okamura 2 , toshiyuki sakaeda 2 1 facul. health care sci., himeji dokkyo univ., himeji, japan; 2 kobe univ. grad. sch. med., japan prostaglandin d 2 (pgd 2 ) is abundant in the brain, but its neuropathologic role has been unclear. here, we found that pgd 2 induced neuronal apoptosis in rat cortical cultures. however, a pgd 2 receptor blocker did not suppress neurotoxicity of pgd 2 . little pgd 2 receptor was detected, suggesting an involvement of pgd 2 metabolites in the apoptosis. among pgd 2 metabolites, 15-deoxy-12,14 -prostaglandin j 2 (15d-12,14 -pgj 2 ) caused neuronal apoptosis most potently and rapidly. although 15d-12,14 -pgj 2 is an endogenous ligand for peroxysome proliferator-activated receptor ␥ (ppar␥), ppar␥ activators did not kill neurons, suggesting that 15d-12,14 -pgj 2 induces apoptosis independently of ppar␥ activation. we found specific binding sites of [ 3 h]15d-12,14 -pgj 2 (jbs) in plasma membranes. there was a close correlation between the neurotoxicity of various eicosanoids and their affinity for jbs. in conclusion, we demonstrated that pgd 2 induced apoptosis via 15d-12,14 -pgj 2 in rat cortical neurons, and suggested that jbs in the plasma membrane was involved in the 15d-12,14 -pgj 2 -induced apoptosis. yoshiki iwamoto, daisuke umetsu, shigeru ozaki, naohito terui department of physiology, university of tsukuba, tsukuba, japan stability of a driver's head is crucial for clear vision and consistent, smooth operation of a vehicle. we reported last year that bilateral sternocleidomastoid muscles (scm) of drivers showed a symmetrical increase in activity during forward acceleration of a vehicle. in the present study, we analyzed the relationship between scm activity and vehicle acceleration. emgs of the right and left scm of drivers were recorded during rapid forward acceleration. the time course of the rectified, smoothed emgs did not match that of vehicle acceleration. for a given acceleration, emg was larger when acceleration was increasing than when it was decreasing. we compared emgs and a linear sum of acceleration and its time derivative, jerk. with optimal weights for the two variables and a proper time lag, the linear sum reproduced the emg profile. the optimal weight and lag varied across subjects and vehicles. we suggest that the jerk-related muscle activity may be necessary to quickly restore proper head position after sudden acceleration. grasping is a highly developed movement in primate including human. in contrast to the well-known involvement of cerebral cortex, role of spinal neurons in controlling this behavior has never been examined. here, we show the first direct evidence suggesting the significant contribution of spinal neurons. we trained japanese monkeys to perform the precision grip task, pinching the two springloaded levers with their index finger and thumb, and recorded neural activities through an oval recording chamber implanted over the cervical spinal cord (c6 to t1). majority of the recorded neurons showed movement-related modulation of firing rate, and the modulation sometimes started before movement onset. spike-triggered averaging of muscle activities revealed some neurons had post-spike effects to hand muscles, suggesting that spinal neurons were capable to generate and modulate muscle force during precision grip. we suggest that primate spinal neurons have a significant role in preparation and execution of grasping movement. research funds: kakenhi 17022043 os2p-7-06 compartmentalization of the cerebellar nuclei: aldolase c expression and the olivonuclear projection pattern izumi sugihara, yoshikazu shinoda dept. systems neurophysiol., tokyo med. & dental univ., tokyo, japan the cerebellar cortex is compartmentalized into more than 20 longitudinal stripes by the aldolase c (=zebrin) expression pattern, which is tightly correlated with the topographic olivocortical projection. however, no equivalent compartmentalization has been known in the cerebellar nuclei. we mapped aldolase c labeling of terminals of purkinje cell axons and anterograde labeling of collaterals of olivocerebellar axons in the rat cerebellar nuclei. the cerebellar nuclei were divided into the caudoventral aldolase c-positive and rostrodorsal negative parts, indicating purkinje cells in the positive and negative stripes in the cortex project to the caudoventral and rostrodorsal parts in the nuclei, respectively. olivonuclear projections showed clear topography within these parts, which was completely congruent with the olivocortical topography. these results clarified the compartmentalization of the cerebellar nuclei and supported that the aldolase c expression is tightly related with the functional organization of the cerebellum. we examined a context dependency of neuronal activity of the pedunculopontine tegmental nucleus (pptn) in monkeys during visually guided saccade tasks. about half of movement-related activities occurred for only the saccades to the saccade target in the task, but they did not occur for the saccades outside the task. on the other hand, for the other half of neurons, movement-related activities occurred for every saccade regardless of the task condition. for visual responses, some neurons responded either the initial fixation point or saccade target, and others responded equally to both stimuli. we further analyzed mutual relationship among modulation timing, preferred direction, effect of reward expectation and this context dependency of the activities, and discussed the visuo-motor processing of pptn. in the reinforcement learning theory, the midbrain dopamine (da) neurons send reward prediction error signal to the striatum. the cholinergic pedunculopontine tegmental nucleus (pptn) is one of the strongest excitatory input sources to da neurons. we hypothesized that pptn may play an important role for relaying necessary components of reward prediction error signals to da neurons. during recording of pptn neurons, we utilized reward predictable visually guided saccade tasks where a shape of fixation point indicated a reward volume. for more than half of the neurons, which showed cue related responses, the cue responses were dependent on association of cue feature and reward size. from another population, we recorded reward related activity. in conclusion, pptn neurons may relay both reward and reward prediction signals, sufficient for computation of reward prediction error. research funds: kakenhi (17022027) os2p-7-09 timing activity in supplementary eye field during a saccadic eye movement task shogo ohmae 1 , xiaofeng lu 1,2 , yusuke uchida 1 , toshimitsu takahashi 1,2 , shigeru kitazawa 1,2 1 dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; 2 crest, jst, tokyo, japan to act properly in our daily life, the ability to detect and predict timing of events is always required. how do we deal with timing in the brain? to address this question, we trained two japanese monkeys to perform a visually guided saccadic eye movement task in which the monkeys made saccades to each of 16 targets following a gosignal given at a random timing between 500 and 800 ms after the appearance of the target. we recorded neuronal activity from the supplementary eye field (sef) during the task. we found a group of cells that showed activity related to the length of the delay period from target-on to the go-signal. these cells were classified into two types: (1) those that showed buildup activity during the delay period until the go-signal, and (2) those that displayed changed activity after the go-signal in relation to the length of the delay period. the results suggest that sef is involved in timing the onset of the go-signal during the saccadic eye movement task. in reaching, a spatial visuomotor transformation should occur in our brain. we can make the transformation not only when the relationship between visual and motor coordinates is default, but also when a gain for the relationship is changed, for example, in a microsurgery. we trained monkeys to make reaching movements when visuospatially identical targets were presented on a computer display by aligning a cursor that indicated their hand position, while the gain was systematically changed. we recorded and analyzed movement-related neuronal activity in the ventral premotor cortex (pmv) and the primary motor cortex (mi) during reaction time. it was revealed that a majority of the mi neurons and a part of the pmv neurons showed activity changes depending on executed movement direction, amplitude, and velocity, whereas a number of the pmv neurons exhibited activity consistent to the visual location of the targets, but not to motor parameters such as amplitude and velocity. the results indicate that the pmv contributes to gain control of reaching during visuomotor transformation. local oscillatory changes in the human sensorimotor cortex induced by simple motor tasks were investigated using supragyral and intrasulcal surface electrodes which was temporarily implanted for the treatment of intractable deafferentation pain. time frequency spectrogram and coherence between electrodes revealed that, before and after several hundred milliseconds of the motor execution, the coherence in the premotor cortex increased cooperatively between neighboring electrodes but that the coherence in the intrasulcal primary sensorimotor cortex decreased exclusively. this result reflects that the premotor cortex plays a role in motor planning with diffuse network while the primary motor cortex plays a role in selective motor execution with local motor output unit. the human sensorimotor processing may be hierarchical and similar to an artificial neural computer. we have shown that the trigeminal oral nucleus (vor) neurons with the receptive field in the intraoral structures project bilaterally to either the jaw-closing (jc) or jaw-opening (jo) motor nucleus in the cat. it is known that neurons in the somatosensory cortex project to the trigeminal sensory nuclei in the rat. thus, we conducted this study to reveal whether there are vor neurons that receive cortical projections and project to the jc or jo nucleus in the rat. we injected a retrograde tracer, fluorogold (fg), in the vor, and found many retrogradely labeled neurons in the contralateral rostral primary somatosensory cortex (si). thus, we injected an anterograde tracer, biotinylated dextranamine (bda), in the rostral si, and also fg in the jc or jo nucleus in the same animals. we found a considerable number of fg-labeled vor neurons made contact with bda-labeled axon terminals. these results suggest that si neurons control jawreflexes through vor neurons. tsunehiko kohashi, yoichi oda grad. sch. science, nagoya univ., nagoya, japan the mauthner (m) cells, paired large reticulospinal neurons in teleost hindbrain, are known to initiate fast escape from sudden aversive stimuli. to investigate how the fast escape is established during early developmental stages, we examined motor performance of the escape in zebrafish embryos or larvae, and the contribution of mcell activity on the behavior. the rostral portion of the zebrafish, 30-200 h post fertilization (hpf), was embedded in agar and the tail flip in response to water pulse applied to the head was examined. thirty hpf embryos, in which m-cell has already received trigeminal nerve innervation and is still extending its axon in the spinal cord, showed tail flips contralateral to the stimulated side with longer latency (>9 ms) than larvae (>80 hpf, 3 ms). m-cell activity monitored with confocal ca 2+ imaging during the tail flip (>80 hpf) tightly correlated with the initiation of fast escape, whereas delayed escapes without m-cell firing appeared in some cases (<25%) after 100 hpf. thus, the development of the escape behavior coincided with that of m-cell circuit. junctophilins (jps) expressed in the er/sr interacts with plasma membrane thereby constructing junctional membrane complexes (jmc). we here report that lacking neural jps subtypes exhibit an irregular hindlimb reflex and impaired memory. to define neural mechanism of memory deficit in jp-dko mice, we performed whole-cell patch clamp recording of hippocampal neurons. in wild mice, an obvious afterhyperpolarization (ahp) was observed and its ahp was totally blocked by apamin. by contrast, ahp was absent in the jp-dko mice and was insensitive to apamin treatment. the er ca 2+ release through ryanodine receptors, triggered by glutamate receptor-mediated ca 2+ influx, is essential for the activation of sk channels toward ahp generation in the hippocampal neurons. therefore, jp-mediated jmc formation likely plays an essential role in neural excitability underlying neural plasticity and memory. os2p-8-02 distribution of voltage-gated calcium channel ␣ 2 ␦-4 mrna in mouse central nervous system takeshi houtani, satoru sakuma, masahiko kase, tetsuo sugimoto department of anatomy and brain science, kansai medical university, moriguchi, osaka 570-8506, japan the ␣ 2 ␦ subunits are the auxiliary subunit of voltage-gated calcium channels and modulate the biophysical properties of the pore-forming ␣ 1 subunits. these auxiliary subunits are composed of four genetically different molecules, ␣ 2 ␦-1 to ␣ 2 ␦-4. the distributions of ␣ 2 ␦-1, -2, -3 mrna have been intensively investigated in the rat central nervous system by in situ hybridization, but that of ␣ 2 ␦-4 remains to be determined. we cloned ␣ 2 ␦-4 cdna fragment from mouse brain by rt-pcr and examined the distribution of ␣ 2 ␦-4 mrna-expressing cells in the mouse central nervous system by in situ hybridization using digoxigenin-labeled crna probe. while the ␣ 2 ␦-4 mrna was found to be broadly expressed, some neuronal types or sites such as piriform cortex, hippocampal pyramidal cells, paraventricular hypothalamic nucleus, facial nucleus and motor neurons of the ventral horn had intense mrna expression. our results suggest that ␣ 2 ␦-4 subunit may play an important role in learning and memory, neuroendocrine secretion and somatic motor control. the mushroom bodies of insect brains are essential in associative olfactory learning. here we show that the drosophila larval mushroom body calyx, the dendritic region, is organized in about 34 glomeruli, which we have mapped. individual glomeruli receive specific innervation from second order olfactory neurons. by contrast, they contain dendrites from hundreds of mushroom body neurons (kenyon cells), which show low specificity for individual glomeruli. glomeruli therefore potentially transmit specific sensory inputs to a large fraction of kenyon cells. quantitative analysis of dendritic termini of single larval-born kenyon cells suggests that they arborize in about 6 glomeruli in an apparently random manner. this pattern of connectivity is consistent with a model in which kenyon cell dendrites process olfactory input by a combinatorial mechanism that allows the discrimination of a large number of odors. withdrawn os2p-8-05 hypothalamic defense reaction involves purkinje cells in the flocculus folium p via orexin and gaba in anesthetized rabbits, electric stimulation in the hypothalamic defense area either excited or inhibited "simple spike" discharges in purkinje cells located in folium p of the flocculus. iontophoretic application of an orexin antagonist (sb334867) depressed the excitation, while bicuculline depressed the inhibition. h 1 or h 2 histamine antagonist had no effect. labeling orexin fibers by immunocytochemistry showed that they were most numerous in folium p as compared with other folia of the flocculus. stimulation of the hypothalamic defense area produced little field potentials in the folium p unlike those evoked by mossy fibers. these observations suggest that the excitation and inhibition are mediated by orexin-containing fibers, which contact purkinje cells directly and also indirectly via other gabaergic neurons. os3a-5-01 activity-dependent development of corticispinal synapse in mouse slice co-culture takae ohno, masaki sakurai dept. physiol., teikyo univ. sch. med., tokyo, japan we showed nmda-dependent synapse elimination of corticospinal (cs) tract in vitro in rat. in order to use the genetically modified mice to study the underlying molecular mechanisms of this developmental plasticity, we studied development of cs synapses in c57 bl/6 mice. by recording field epsp (fepsp) along 80 m interval lattice in the spinal gray matter in response to the stimulation of deep cortical layer, we evaluated spatial distribution of synapse formation quantitatively. fepsps were recorded diffusely throughout the spinal gray matter at 7-8 div, then the amplitudes of fepsps in the ventral side began to decrease at 9-10 div, and dominated in the dorsal area at 14 div. cs axon terminals labeled anterogradely with biocytin distributed diffusely throughout the spinal gray matter at 7-9 div but the axons terminals in the ventral area were eliminated until 14 div. this synapse elimination from the ventral side was blocked by apv application from 6 div, indicating that this process is also nmda-dependent. in slice coculture study, we showed that corticospinal (cs) axons grow rapidly and reach the ventral spinal gray until 7 div. the number of those ventral axons is reduced before 14 div. to study the behavior of the cs axons at the single axonal level, we transfected a small number of cortical neurons with eyfp expression vector pcag-eyfp by way of electroporation to visualize them and took the time-lapse images of their axons under the confocal microscope equipped with an on-stage co2 incubator. some axons showed rapid growth, reaching the ventral most part of the spinal gray matter already at 6 div. some axons had collaterals at the dorsal part and retracted the ventral branch while extending the dorsal branch during 7-11 div. some ventral axons showed a fragmented tip during retraction, which was indicative of axonal pruning. these observations provide direct evidence that there are early cs axons that once reach the ventral spinal gray and then retract to stay dorsally. we identified click-iii/camki␥ as a novel brain-enriched isoform of the camk-i family that was lipid-anchored by multiple lipid modifications, prenylation and palmitoylation, resulting in enrichment of click-iii into lipid rafts fractions. in situ hybridization revealed the abundant presence of click-iii transcript throughout the central nervous system in mouse embryos. to test the role of click-iii during early neuritogenesis, a shrna vector specific for click-iii was delivered into dissociated cortical culture. we found that knock-down of click-iii resulted in significant decrease in the number and total length of dendrites. results from introduction of click-iii into a click-iii-null context confirmed this finding. surprisingly, lipid modifications of click-iii seemed to contribute to fully elicit such an effect. we thus uncovered a novel signaling mechanism by which lipid raft insertion and local activation of a camk can be efficiently coupled to actin cytoskeletal signaling during dendritogenesis. os3a-5-06 transcription factor control of dendrite arbor ultrastructure adrian moore, reiko amikura, shiho nakao, andrew liu, emi kinameri riken brain science institute, japan the different functions of neurons in a complex nervous system are reflected in a large diversity of dendrite arbor morphologies. the drosophila larva dendritic arbourization (da) neurons consist of four classes (i-iv) with increasing levels of arbor complexity. these diverse arbor shapes develop due to class specific mechanisms of dendrite branching and outgrowth. here we show that these class specific differences in dendrite arbor morphology are controlled by a combinatorial code of transcription factors. we have developed a system to label individual dendrite arbors then subsequently identify them in electron microscopic sections. using this method we illustrate that the dendrites of class i neurons, with a simple arbor, contain a high density parallel array of microtubules; on the other hand class iv neurons, with a complex arbor, contain a low density meshwork of microtubules. we are presently investigating how these differences in ultrastructure are controlled by the transcription factors making up the combinatorial code. os3a-5-07 segmental and hox related cues are involved in the establishment of the somatotopy yasunori murakami igbmc, strasbourg, france in the rodent, trigeminal sensory inputs are topographically relayed, and mapped in the somatosensory cortex. little is known about the mechanism underlying the development of the somatotopic organization. by fate mapping of specific rhombomeres (r), we found that principal sensory (prv) neurons derived from r3 receive predominantly inputs from the maxillary branch of the trigeminal nerve and uniquely contribute to the whisker map. by conditional inactivation, we found that early expression of hoxa2 in r2 is required for pathfinding and positioning of trigeminal nerve afferents. at later stages, hoxa2 expression in prv neurons provides instructive cues for topographic arborization of maxillary axons. moreover, while prv neurons appeared normally specified, loss of hoxa2 function resulted in selective loss of eph4 expression, and altered axonal projections from prv to the ventral posterior medial (vpm) nucleus of the thalamus, and absence of a postnatal whisker map at any level of the neuraxis. thus, hoxa2 dependent cues are required to determine the territory for whisker representation in r3 and the assembly of a somatosensory circuit. os3a-5-08 the second wave of corticospinal innervation after synapse elimination of the first wave tsutomu kamiyama, masaki sakurai dept. physiol, teikyo univ. sch. med., tokyo, japan in the previous study we showed that the rat corticospinal (cs) terminals and synapses were widely distributed at p7 and those in the venrtolateral (vl) area were eliminated from p8 to p10 and that the number of terminals in the dorsomedial (dm) and vl area began to increase again from p12 and further increased thereafter. in the present study we further studied the subsequent developmental time course of cs terminal distribution. cs axons were anterogradely labeled by injection of biotin dextrane (bda) into the sensorimotor cortex. the number of the terminals began to increase from p12, reaching peak around the third postnatal week. labeling of single or a few by microinjection axons revealed that at p14 some additional cs axon branches appeared within the dorsal column of the target spinal segment and further ramified after entering the gray matter. however, the number of axons did not increase in the brainstem and the upper cervical cord. these suggest that the second wave of innervation is explained mainly by branching of cs axons just before and after entering the spinal gray matter. os3a-5-09 proteomics of the growth cone: i. protein profiling of the growth cone the growth cone is a motile tip formed at the developing neuronal processes, and functions for the accurate determination of the axon pathway and the synaptogenesis. in higher organisms, however, the molecular basis of the growth cone is poorly understood for the present, since the information on the protein localization there is insufficient to explain the growth cone functions. proteomics is a powerful strategy for identifying the protein composition in a given cell or a subcellular compartment, and the application of this method to the growth cone should help us solve the above question. we obtained the whole growth cone (gcp) obtained from neonatal rat forebrain and the membrane subfraction of the gcp (gcm), and then those fractions were analyzed using proteomics. we have identified several hundreds of the distinct proteins of these fractions. here, we show the profiling of gcp and gcm, and will discuss the overview of these protein profiles in relation to the growth cone functions. axonal branching is thought to be regulated by not only genetically specified molecules but also neuronal activity. however, the interplay between these two mechanisms remains largely unknown. to study this issue, we analyzed the role of electrical activity in layer-specific thalamocortical (tc) axon branching by using organotypic cocultures. during the second week in vitro, yellow fluorescent protein-labeled tc axons formed branches primarily in the target layer. spontaneous firing was found to increase when branches were formed abundantly. pharmacological blockade of synaptic transmission diminished layer-specific branching considerably. moreover, time-lapse imaging showed that branching was generated dynamically by elimination as well as addition in the target layer and that blockade of synaptic activity reduced this remodeling. these findings suggest that synaptic activity modifies layer-specific tc axon branching by regulating the remodeling process with molecular cues expressed in the target layer. research funds: kakenhi (17023030), kakenhi (15300107) os3a-6-01 the application of navigation-guided repetitive transcranial magnetic stimulation for intractable deafferentation pain naoki tani, yoichi saitoh, haruhiko k., satoru oshino, masayuki hirata, amami katoh, toshiki yoshimine 1 department of physiology, university of osaka, osaka, japan repetitive transcranial magnetic stimulation (rtms) has been applied to control intractable deafferentation pain (dp). but nobody has investigated which cortical area is the most effective target for pain relief. therefore, we stimulated m1, s1, sma, premotor accurately with a navigation-guided rtms and compared their effects of pain relief. at the same time, rtms (1, 5, 10 hz, 500 stimulations) was compared in 20 dp patients. the pain relief was evaluated with visual analogue scale. high frequency (5, 10 hz) rtms of m1 was the only effective stimulation for treating intractable pain in 10 of 20 patients (50%). the pain relief continued for 3 h significantly. we would like to discuss the mechanism of pain relief with high frequency rtms of m1. os3a-6-02 involvement of atp on nociceptive modulation in rat model of masseter muscle pain yasuo sugiura, noriyuki ozaki, masamichi shinoda department of functional anatomy and neuroscience, nagoya university graduate school of medicine, nagoya, japan we determined the role of p2x 3 r on pressure pain and mechanical hyperalgesia in a newly developed rat model of pain in masseter muscle (mm) . the pain in the mm was assessed by the pressure pain threshold (ppt) defined as the amount of pressure required to induce head flinching. the mm injection of ␣,␤-meatp (p2x 1,3,2/3 rspecific agonist) significantly enhanced the behavioral response to the pressure. this enhanced response was completely blocked by the co-application of ␣,␤-meatp with ppads (p2x 1,2,3,5,1/5,4/5 r-specific antagonist). excessive muscular contraction of mm produced by the electrical stimulation significantly decreased the ppt indicating mechanical hyperalgesia of the mm. administration of ppads to the exerted mm produced a complete recovery of decreased ppt. p2x 3 rpositive neurons innervating the exerted mm increased in trigeminal ganglia. our results suggest that p2x 3 r plays an important role in pressure pain, and mechanical hyperalgesia caused by excessive muscular contraction of mm. the present study was undertaken to investigate the change in the activation of the nociceptive neuronal circuit under a neuropathic pain-like state. here we found sciatic nerve ligation (snl) produced a marked increase in the number of c-fos-positive cells in the periaqueductal gray (pag). using the fluoro-gold (fg) microinjection into the pag, numerous fg-labeled cells were detected in the hypothalamus. in the arcuate nucleus (arc) of the hypothalamus, the immunoreactivity (ir) for an excitatory neuronal maker, fosb was increased, whereas the ␤-endorphin (␤-ep)-ir was decreased 7 days after snl. furthermore, the subpopulations of ␤-ep-positive cells were co-labeled with fosb in the arc. the present data suggest that the hypothalamus can be received by snl-induced concomitant nociceptive signals, leading to continuous activation of neurons projecting to the pag. this phenomenon, in turn, indirectly controls pain transmission in the dorsal horn through the descending antinociceptive pathway. os3a-6-04 the cantor-like patterns in rat hippocampal ca1 pyramidal neurons tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca1. this prediction includes an attractor dynamics expected in the associative network, which was proposed by many authors, since marr's theory of simple memory in the hippocampus. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to find cantor-like patterns experimentally from the ca1 pyramidal neurons. temporally associated and non-associated electrical stimulations were delivered to schaffer collaterals, and membrane potentials were recorded by patch-clamp recording method. in our results, cantor-like patterns were observed in hippocampal ca1 pyramidal neurons. young songbirds shape their songs using memorized tutor songs and auditory-vocal feedback. we prevented zebra finches from hearing their own vocalizations by exposure to loud noise after 35 days of age, before which they had been reared with song tutors from birth. when the noise stopped at 102-200 days of age, the birds sang unstable and noisy song syllables that did not resemble the tutor syllables. the similarity to the tutor syllables steadily increased until the time of song crystallization (30 days later). these findings show that the memory of tutor syllables still exists well beyond the normal age of song crystallization (d90 of age) and that zebra finches can develop songs using the memory well after the normal period of song development. the temporal order of syllables resembled the tutor model only in birds released from the noise before 80 days of age. thus, different schedules and processes may govern the learning of syllable phonology and syntax. in addition to well-characterized areas, a novel adult neurogenic region; the temporal germinal layer (tgl) was identified in rats (takemura, 2005) . a tracer study revealed that there is an interconnection between the dorsal part of the tgl and the lateral nucleus of the amygdala, suggesting a functional implementation of tgl neurogenesis in amygdala-dependent emotional memory processing. to investigate this possibility, we performed a tgl region-specific low-dose irradiation, which can selectively kill proliferating cells and hence can reduce neurogenesis, using a gamma knife. the tgl-irradiated rats expressed a significantly increased tone-related long-term fear memory, indicating a functional significance of the tgl neurogenesis for aversive memory reduction. we (tsukada and pan, 2005) systematically examine the functional difference between spatio-temporal learning rule (stlr) proposed by tsukada (1996) and hebbian learning rules in a single-layered neural network, computing their ability to differentiate spatiotemporal sequence. in this paper, we tested physiologically the cooperative plasticity without a postsynaptic spike in the ca1 hippocampal network. tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca1. they also predicted chaotically transitory dynamic behavior called chaotic itinerancy in the hippocampal ca3. this prediction includes an attractor dynamics expected in the associative network, which was proposed by marr and others. the time series of events, which could be output from ca3, may be encoded in ca1 in an efficient way. the proposed cantor coding is effective, because the topology of time series is naturally measured on the cantor set since each element of cantor set represents a single time series. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to make more realistic model of ca1, using 2-compartment model of neuron, and we found the cantor coding of information of time series in the model ca1. it is known that neurons can propagate action potentials with high temporal precision. however, it is unclear how precisely closely neighbouring neurons synchronize and whether they can code information. here we show that sub-millisecond synchronization can code information as well as the discharge rate modulation. we found that closely neighbouring pyramidal neurons in the ca1 region of the hippocampus synchronize with sub-millisecond precision. the optimal frequency bands for transmitting these synchronizations matched the beta, gamma and fast-ripple oscillations. moreover, we found that the synchronizations were commonly coupled with rate modulations in relation to both internal (retention and comparison) and external (stimulus and motor) events. the synchronization often occurred in relation to stimulus inputs even when rate modulation was clearly absent. therefore, our results suggest that sub-millisecond synchronization plays an important role in propagating information in the hippocampus. the alterations of cerebral motor function by chronic ischemia are poorly understood, since no motor symptoms are noticeable in most of the cases. we evaluated spatial distribution and intensity of eventrelated desynchronization of beta band (beta-erd) evoked in motor area using synthetic aperture magnetometry in 15 patients with chronic ischemia due to diverse vascular occlusive diseases (n = 12) and moyamoya disease (n = 3). contrary to the normal motor activation, ipsilateral beta-erd was dominant during grasping task of affected hand in 8 patients. this abnormal activation was obscured by self-paced finger tapping requiring more selective hand motor programming. and it was more frequently observed in the atherosclerotic hypoperfusion (with white matter change) than in other pathogenesis. ipsilateral beta-erd may be a new indicator of subclinical functional alteration in motor cortices caused by chronic ischemia. os3a-7-02 hypothermia protects against cerebral ischemia by suppressing ␦pkc activation takayoshi shimohata 1,2 , heng zhao 2 , gary steinberg 2 1 department of neurology, brain research institute, niigata university, niigata, japan; 2 department of neurosurgery, stanford university, stanford, usa hypothermia protects the brain from ischemia, but the underlying mechanisms of this effect are not fully elucidated. ␦pkc is reported to induce apoptosis upon activation. its activity is modulated by phosphorylation, translocation and proteolytic cleavage. we investigated effects of hypothermia on ␦pkc activation using a rat permanent distal mca occlusion model. mild hypothermia (30 • c) reduced infarct size by 84%. western blots indicated that ␦pkc cleavage increased markedly in ischemic core but moderately in penumbra after stroke, which is suppressed by hypothermia (p < 0.05). p-␦pkc (t505) dephosphorylated after stroke; this effect is blocked by hypothermia. full-length and cleaved form ␦pkc as well as p-␦pkc (s643) translocate from the cytoplasm to the mitochondria and nucleus, which is suppressed by hypothermia. ␦pkc activator suppressed the protective effect of hypothermia. taken together, hypothermia blocks ␦pkc activation after focal ischemia. this effect might contribute to hypothermic neuroprotection. calcium responses in situ following ischemia remain unclear. we sought to determine, in rats, the calcium changes following transient forebrain ischemia. in anesthetized adult rats, 4-vessle occlusion was induced. fluo-3/am was microinjected, and the fiber-coupled confocal microscope [imaging fiber bundle coupled to the microlensattached nipkow-disk scanner (csu-21, yokogawa, japan) equipped with 10× objective lens] was inserted into the brain. 4-vessle occlusion induced comparable ischemia in both hippocampus and frontal cortex. fluorescence intensity of fluo-3 increased up to 115%, and persistently increased up to 130% during 20-min reperfusion, indicating the long-lasting ca 2+ increase in the ca1 region. in contrast, in the frontal cortex, 10-min ischemia increased fluorescence intensity during ischemia but not reperfusion. in the ca1 region but not in the frontal cortex, transient forebrain ischemia induces long-lasting increase in ca 2+ in situ. research funds: kakenhi #16390407, #16047212 os3a-7-04 reevalution of classical view on resident microglia: neutrophils may play more critical roles than resident microglia at acute phase of ischemic and traumatic brain insults hiroaki matsumoto 1 , h. watanabe 1 , y. kumon 1 , t. ohnishi 1 , chi ii 2 , y. imai 2 , j. tanaka 2 1 dept. neurosurgery, ehime university, japan; 2 dept. molecular and cellular physiology, ehime university, japan resident quiescent microglia (mg) are thought to respond quickly to a variety of pathologic events in the brain, by proliferating and producing a number of bioactive substances including proinflammatory cytokines and nitric oxide (no). in the present study, however, we found that the majority of resident mg died through apoptosis within 36 h after the onset of ischemic and traumatic brain insults. we further noticed that traditional mg markers isolectin b4 and cd11b recognized with ox42 antibody histochemically stained neutrophils, which were identified by neutrophil-specific elastase, rather than iba1+ mg or macrophages. accumulation of neutrophils was observed at the very early phase of the insults, while they expressed proinflammatory cytokines and inducible no synthase. iba1+ amoeboid-shaped mg started to accumulate 3 days after the insults. the data prompted us to reevaluate the roles and the fate of resident mg in the brain. os3a-7-05 insulin regulates the hepatic clearance of amyloid ␤ peptide tetsuya terasaki 1,2 , chihiro tamaki 1 , sumio ohtsuki 1,2 1 graduate school of pharmaceutical sciences, tohoku university, sendai, japan; 2 sorst, jst, japan the liver is the major organ that eliminates amyloid ␤-peptide (a␤) from the circulation, and we have revealed that low-density lipoprotein receptor-related protein 1 (lrp-1) is a molecule responsible for the hepatic clearance. since epidemiologic investigations suggest the high incidence of alzheimer's disease in diabetes mellitus, the purpose of this study was to clarify the effect of insulin on the hepatic clearance of a␤ . insulin infusion into the rat portal vein increased lrp-1 expression in plasma membrane fraction of liver, but did not affect the expression in whole lysate. insulin treatment also increased the hepatic uptake of a␤(1-40), which reached 1.6-fold greater uptake than non-treated control after 10 min treatment. increase of the hepatic uptake of a␤(1-40) by insulin was concentration dependent (ec 50 = 230 pm), and was completely suppressed by rap (2 m), an lrp inhibitor. these results suggest that insulin induces translocation of lrp-1 to the plasma membrane of hepatocytes, leading to increase of a␤ hepatic clearance from the circulation. research funds: sorst, jst os3a-7-06 mr images of intra-arterially administered microglia surrounding ␤-amyloid deposit in the rat brain the therapeutic use of microglial cells has recently received some attention for the treatment of alzheimer disease (ad), but few noninvasive techniques exist for monitoring cells. here we present a magnetic resonance imaging (mri) technology to track micrgolia cells injected intra-arterially in a rat model of ad. we labeled microglia expressing gfp with resovist using the hvj-e vector. we administered labeled microglia into the carotid artery of the rats. mri revealed clear signal changes attributable to resovist-containing microglia in a␤-injected areas. this study demonstrates the usefulness of mri for non-invasive monitoring of exogenous microglia, and suggests a promising future for microglia as therapeutic tools for ad. extravasation of protease-activated receptor (par) activators, such as thrombin, into brain parenchyma can occur after blood-brain barrier breakdown in a number of cns disorders, which causes pathophysiological changes in neurons and glial cells. to elucidate the mechanism of thrombin-induced activation of astroglial cells, we used 1321n1 astrocytomas that show a characteristic retraction of bipolar protrusions after activation of pars with thrombin. the thrombin-induced morphological change of 1321n1 cells was inhibited by an inhibitor of ip 3 receptors, 2-aminoethoxydiphenyl borate (2-apb) or an endoplasmic reticulum ca 2+ -atpase inhibitor, cyclopiazonic acid (cpa). in parallel, thrombin-induced mobilization of ca 2+ was inhibited by 2-apb and cpa. moreover, removal of external ca 2+ accelerated the reversal of thrombin effects. these results suggest that refilling of ca 2+ store by ca 2+ entry play an important role in the cytoskeletal dynamics of astroglial cells. to clarify the occurrence range of neurofibrillary tangles (nft), we reexamined an autopsied alzheimer patient with the onset at age 38 and a 25-year-clinical course. the brain showed severe atrophy (630 g). microscopic examination disclosed that all telencephalic neocortices had nft of more than 100 and sp of more than 10. all limbic cortices and nuclei had nft of more than 100 and sp of more than 10. although there was no sp, various numbers of nft were observed in the following structures: claustrum 85, caudate 9, globus pallidus 6, hypothalamus 22, meynert's nucleus 4, thalamus 74, substantia nigra 6, central gray 12, locus ceruleus 6, purkinje cells 0, posterior root ganglion 0, adrenal medulla 3. this study revealed that there exist nft-rich neurons and free neurons. the latter includes purkinje cells and posterior root ganglion cells. considering the pathogenesis of nft, it must be valuable to clarify qualitative/quntitative differences between nft-rich neurons and free neurons. os3a-7-09 transcriptional regulation of androgen receptor in aging mouse brain androgen receptor (ar) mediates action of androgen, which is involved in memory, behavior and other brain functions that deteriorate with advancing age. in aging mice brain, ar mrna expression was measured by rt-pcr, ar promoter methylation by southern hybridization, and proteins binding to promoter by emsa. ar mrna level was significantly higher in male than female, and it was downregulated by testosterone, but upregulated by estradiol in adult mice. female mice exhibited higher methylation of ar promoter than males. methylation was increased by testosterone, but decreased by estradiol. furthermore, dnasei accessibility to ar promoter was reduced in males, increased by gonadectomy but reduced by sex steroids in adult male. incubation of brain nuclear extract with 32plabeled ar promoter yielded three specific complexes. the intensity of these complexes varied with age and sex. these findings show that ar mrna expression and promoter methylation are inversely regulated by sex steroids in the adult mice cerebral cortex. such regulation of ar expression might influence androgen action and consequently brain function during aging. reliability of synaptic transmission depends on the efficiency of transmitter removal from the synaptic cleft, as well as on the release machinery and the postsynaptic response mechanism. it has been shown in various synapses that postsynaptic and glial excitatory amino acid transporters (eaats) contribute to glutamate removal. however, the role of presynaptic eaats remains unclear. using mouse retinal slices, we examined the contribution of eaats at the rod to rod bipolar cell (rbc) synapse. the kinetics of the rbc current evoked by electrical stimulation of rods was slowed by pharmacological blockade of eaats. recordings of the evoked rbc currents from eaat subtype-deficient mice and the eaat-coupled anion current revealed that functional eaats are localized to rod terminals but not to postsynaptic or glial cells. model simulations suggest that rod eaats are densely packed near the release site, and that rods are equipped with an almost self-sufficient glutamate recollecting system. trpv4 is a thermosensitive trp channel, and activated by body temperature. we found functional-trpv4 was expressed in soma, dendrites and synapses in the neurons. since trpv4 was firstly cloned as an osmotically activated channel, we hypothesized trpv4 might be involved in volume regulation of the spines. therefore, we quantified the spine volume changes by glutamate stimulation, and confirmed trpv4 expression related to the volume increase of spines. next, we compared the resting membrane potential (rmp) between wild type and trpv4-deficient neurons at 37 • c, and found rmp in wild type was more depolarized by approximately 5 mv than rmp in trpv4-deficient neurons. we also performed current-injection experiments in both neurons, and found that trpv4-deficient neurons required much bigger currents to get their firing. thus, we conclude that trpv4 is involved in regulation of both neural activity and spine motility in hippocampus. os3p-2-04 a system for rapid uncaging in defined patterns and its application hiroshi kojima department of intelligent information systems, tamagawa university, tokyo, japan neurons integrate many sysnaptic signals at dendrite. understanding these information processes is a central topics in experimental and computational neuroscience. the use of focused laser beam for uncaging can provide fine spatial resolution to analysis of neural function. however, most experiments were carried out either at spatial locations or in a very simple scanning patterns. we developed a system for performing uncaging in arbitrary pattern in order to emulate realistic neural activity. our system is capable of patterned photorelease of caged neurotransmitters at 100 locations per 200 ms with submicron resolution. ultraviolet laser light is steered by galvano-mirrors and projected onto the surface of preparations for uncaging the caged chemicals. simultaneously, imaging of neurons are obtained by 2-photon microscopy and electrophysiological experiments can be done. we briefly report the present system for rapid uncaging and its application to neurophysiological research. os3p-2-05 d1-like receptors selectively block p/q-type calcium channels to glutamate release onto cholinergic neurons in the rat basal forebrain a number of molecules have been identified in the sensory ganglia including those involved in the signal transmission to the brain. their functions, however, remain largely unknown. we tried to develop a method enabling to inhibit gene expression in the sensory ganglia in vivo by rnai and to evaluate its effect on the synaptic transmission in the brain slices. for this purpose, we selected the nodose ganglion (ng), in which the neurons sending glutamatergic projections to the nucleus tractus solitarii in the brainstem, are located. in anesthetized young wistar rats, synthetic sirna against the genes coding adenosine a 1 receptors (adora1) was introduced to the ng by electroporation. one to five days after sirna delivery, the expression level of adora1 in the ng decreased by >90% of that in the non-treated ng, being not accompanied by a change in mrna level for a 2a receptors. this technique might be promising in analyzing the function of specific molecules involved in transmitter release regulation at the brain synapses. nmda-receptors are specific constituents of glutamatergic system in brain responsible for molecular mechanisms of recognition and learning. activation of neurons by nmda results in intracellular generation of reactive oxygen species (ros) and reorganization of cell metabolism. exposure of rodent and human lymphocytes with nmda results in ros increase within the cells which is suppressed by nmda antagonists. moreover we have demonstrated by rt-pcr technique and by using anti-nmda-antibodies the expression of nmdareceptors on lymphocyte membranes. in addition, we shown that nmda receptor dependent signal from lymphocyte membrane is transformed into specific intracellular reactions controlling caspase-3 activity and interferon-␥ synthesis. in the presentation, properties of nmda-receptors and their functional role in immunnocompetent system are discussed. small molecule g-protein arf1 in combination with phospholipase d (pld) is essential for intracellular trafficking of the proteins from endoplasmic reticulum to golgi apparatus. however, it is recently reported that it also regulate ionic channel activity at the cytoplasmic membrane. to examine possible involvement of arf and subsequent pld in regulation of receptor-induced responses in neurons, we recorded k + -current response to dopamine (da) in the ganglion cells of aplysia under conventional two-electrode voltage clamp. intracellular application of arf1 blockers such as brefeldin a, exo1, and arf1 n-terminal peptide, markedly suppressed the da-induced response. furthermore, intracellular application of ␣-synuclein, a specific blocker of pld, significantly depressed the k + -current response to da. these results suggest that arf1 and subsequent pld may regulate the k + -current response induced by da. os3p-3-05 p250gap, a brain-enriched rhogap, is involved in the nmdar-mediated signaling takanobu nakazawa 1 , toshihiko kuriu 2 , ayako m. watabe 3 , toshiya manabe 3 , shigeo okabe 2 , tadashi yamamoto 1 1 div. of oncology, inst. med. sci., univ. of tokyo, tokyo, japan; 2 dept. of cell biol., tokyo medical and dental univ., tokyo, japan; 3 div. of neuronal network, inst. med. sci., univ. of tokyo, tokyo, japan nmdar regulates structural plasticity by modulating actin organization within spines. however, the signaling pathways that link nmdar activity to the postsynaptic actin cytoskeleton are poorly understood. we identified a brain-enriched rhogap, p250gap, which interacts with the nr2b subunit of nmdar. within neurons, p250gap was highly concentrated in the postsynaptic density and co-localized with nr2b and an actin-binding protein, cortactin. p250gap promoted gtp hydrolysis of cdc42 and rhoa in vitro and in vivo. nmdar stimulation led to de-phosphorylation and redistribution of p250gap. when over-expressed in dissociated neuron, p250gap suppressed the activities of rho gtpases, which resulted in spine elongation. taken together, the results suggest that p250gap is likely to be involved in nmdar activity-dependent actin re-organization in spines. os3p-3-06 non-static method to directly quantify the transfer of firing correlation from one neural population to another: fokker-planck method hideyuki cateau riken brain science institute, saitama, japan firings of only a few neurons are too weak to be transmitted safely, to activate other neurons to fire, or to contract muscles. therefore, we implicitly assume that brain function is exerted by macroscopic population of neurons. to characterize how a macroscopic neural population behave, the simulation method provide an indirect approach. many single neuron simulation runs need to be performed first before extracting macroscopic features by statistically averaging. unlike this method, the fokker-planck (fp) method directly evaluates the macroscopic features, thereby giving a clearer insight into function achievable with neuronal population. despite the lasting interests in firing correlation in coding and conveying information, theoretical studies on it have been largely confined to complicated simulation studies. here, we provide a first non-static fp analysis to directly calculate how correlation and population rates are transferred from one population to another and elaborate a dynamical interplay between these macroscopic quantities at work in time. os3p-4-01 spatial frequency tuning of disparity-selective neurons in macaque v4 hironori kumano 1 , seiji tanabe 2 , ichiro fujita 2 1 grad. sch. of engineering science, osaka univ., osaka, japan; 2 grad. sch. of frontier biosciences, osaka univ., osaka, japan to examine whether convergence across spatial frequency channels contribute to stereoscopic processing, we recorded single neuron activity from area v4 of awake, fixating monkeys. for each neuron tested, we first measured the spatial frequency tuning with sinusoidal gratings or two-dimensional (2-d) filtered noise images, and then examined the disparity tuning with both correlated and anticorrelated dynamic random-dot stereograms (rdss). neurons with broader spatial frequency tuning had more attenuated disparity tuning for anti-correlated rdss. in a subset of v4 neurons, we analyzed responses to various combinations of binocular disparity and spatial frequency by using 2-d filtered noise stereograms. the disparity tuning of most v4 neurons was consistent across a range of spatial frequencies to which they were sensitive. we suggest that v4 neurons pool disparity signals across spatial frequency channels to create an unambiguous representation of stereoscopic depth. os3p-4-02 predicting the monkey's behavioral choice in a stereoacuity task from neuronal responses in area v4 hiroshi shiozaki, seiji tanabe, ichiro fujita lab. cognitive neurosci., grad. sch. frontier biosciences, osaka univ., japan many neurons in visual area v4 of macaque monkeys are selective for binocular disparity. most disparity-selective neurons in v4 are sensitive to small changes in disparity near zero, suggesting that they might contribute to stereoacuity. however, the role of these neurons in stereoscopic depth discrimination has not been directly addressed. we recorded single unit activity from v4 while a monkey was engaged in a fine stereoscopic depth discrimination or stereoacuity task. the monkey was trained to report by saccadic eye movement whether the center region of a random-dot stereogram was nearer or farther than its immediate surround. trial-to-trial fluctuation of visual responses of v4 neurons was correlated with the monkey's subsequent behavioral choice. given the cell's disparity preference, an ideal observer can predict the monkey's upcoming behavioral response from the visual response of v4 neurons. the results suggest that v4 neurons are involved in mediating stereoacuity. os3p-4-03 the role of disparity energy and binocular matching processes in stereopsis takahiro doi, seiji tanabe, ichiro fujita lab. cognitive neurosci., osaka univ., japan the early visual system computes disparity energy of stereo images. some of the next stages retain this information, while other stages perform further computation to solve the stereo correspondence problem. we addressed how the energy and correspondence computations underlie stereopsis. we asked human subjects to discriminate depth of random-dot stereograms with various amounts of disparity. at each disparity level, we manipulated the proportion of dots with the same luminance contrast between the two eyes by reversing the contrast of some dots in one eye. at small disparities, the proportion of correct choices increased monotonically from chance to perfect as the proportion of the same-contrast dots was increased. at large disparities, the subjects perceived reversed depth when contrastreversed dots dominated, and the proportion of correct choices reached only chance level when the two types of dots were balanced. the results suggest that the correspondence and energy computations underlie fine and coarse stereopsis, respectively. we introduce a novel receptive field (rf) analysis, lsrc, which can reveal various aspects of visual receptive fields that were undetectable previously in a single measurement. the visual stimuli are standard wide-field 2-d ternary dynamic random noise, generally refreshed every 26-40 ms. unlike the conventional reverse correlation which computes a spike-triggered average (sta) of the stimuli themselves, lsrc computes the sta of the spectra of localized regions of the stimuli. both simulations and recordings from cat v1/v2 neurons demonstrate that lsrc is capable of revealing details of complex cell rfs, cross-orientation suppression, variations of orientation tuning within rfs that might lead to shape selectivites. since the stimuli can cover a wide visual field area, and few assumptions are made regarding specific shapes or features in stimuli, lsrc is highly suitable for multi-neuron, multi-area studies spanning retina, v1, and especially areas beyond. research funds: mext(13041033), jsps(13308048), coe21 os3p-4-06 analysis of center-surround organization of v1 neurons as a high-order receptive field hiroki tanaka, izumi ohzawa graduate school of frontier biosciences, osaka, japan responses of area 17 (v1) neurons are influenced by stimuli not only in their classical receptive field (rf) center, but also in its surround. such a center-surround organization may be considered as a unified higher-order rf. we have sought to obtain detailed structures of such a rf by harmonic analyses of responses to drifting contrast-modulated sinusoidal gratings that cover both the center and surround regions. of 52 cells analyzed, 80% showed spatial frequency tuning curves that were well fitted with gaussian. by taking the inverse fourier transform of these curves, spatial center-surround rf was obtained as gabor functions with spatial phases between ±90 degrees. highly asymmetric structures were observed for cells with strong surround suppression. estimated sizes of center and surround were well correlated with those from size tuning curves. moreover, there was no space-time tilt in the center-surround rf. the results suggest that neurons with surround suppression are capable of coding various spatial forms of higher-order features (figure-ground borders), but are insensitive to motion of such stimuli. os3p-4-07 spatial organization of receptive fields of complex cells in the early visual cortex kota sasaki 1 , izumi ohzawa 1,2 1 grad. school of eng. sci., osaka univ., japan; 2 grad. school of frontier biosci., osaka univ., japan little is known about the quantitative internal structure of the receptive fields (rf) of complex cells, although this is crucial for understanding how a complex cell acquires its function by collecting inputs from neurons in the preceding stage. therefore, we have analyzed the relationship between the spatial 2nd-order interaction kernels and the rf envelopes of complex cells. extracellular single unit recordings were performed in anesthetized and paralyzed adult cats. threevalued (i.e. gray, dark, and bright) dynamic white noise stimulus with 51 × 51 dots was presented over an area 2 to 4 times larger than the rf of a complex cell. for each dot location, a 2nd-order kernel and its envelope (by hilbert transform) were calculated. the rf envelope of the neuron was determined by summing the envelopes of 2nd-order kernels at all locations. 2nd-order kernels had roughly comparable extent as the rf, and contained 3.3 subregions on average (n = 53). among 35 complex cells, whose rf envelopes were elongated, 16 cells exhibited the horizontal elongation. research funds: mext(13041033), jsps(13308048), coe21 os3p-4-08 orientation tuning of neuron in cat lateral geniculate nucleus tomoyuki naito 1 , osamu sadakane 1 , masahiro okamoto 2 , hironobu osaki 3 , hiromichi sato 1 1 grad. sch. med., osaka univ., osaka, japan; 2 grad. sch. front. biosci., osaka univ., osaka, japan; 3 med. sch., osaka univ., osaka, japan we examined the orientation selectivity of lgn neurons of anesthetized cats and found that although about 19% lgn neurons showed significantly orientation-biased response to the grating with optimal size and spatial frequency (sf), and that 97% of lgn neurons exhibited significant orientation selectivity to gratings with diameter larger than its classical receptive field (crf) and sf higher than the optimal for crf response. two stimulus-size tuning curves measured for responses to stimulation with the optimally-or null-orientated grating exhibited profile similar to each other under the optimal sf condition. however, high sf grating caused stronger surround suppression for response to the orthogonally oriented stimulus than that to the optimally orientated stimulus. our results suggested that elliptic crf center produces orientation-biased response of lgn neurons. furthermore, surround suppression of lgn neurons tuned to particular stimulus orientations enhances orientation selectivity of lgn neurons. os3p-4-09 temporal dynamics of suppressive receptive field surround in cat v1 satoshi shimegi, hiroyuki kida, ayako ishikawa, hiroshi sakamoto, hiromichi sato graduate school of medicine, osaka university, toyonaka, japan in the primary visual cortex (v1), a neuronal response to stimulation of the classical receptive field (crf) is suppressively modulated by the stimulus presented at the receptive field surround (srf). using stationary flashes (500 ms) of sinusoidal grating with optimal parameters and varying radii as stimuli, we examined the temporal dynamics of the surround suppression in v1 cells of anesthetized cats. stimulus slightly larger than the crf caused suppression in early response (<100 ms) but not in middle (100-200 ms) and late responses (200-500 ms). as stimulus size was further enlarged, the middle and late responses were remarkably suppressed while the early response was only moderately or weakly suppressed. radius of surround suppressive field progressively expanded in temporal sequence from 2.5 deg (early response) to 5 deg (middle response) and 7.5 deg (late response). thus, modulation of early response seems to reflect whether stimulus is larger than crf size or not, and late response to reflect how wide area is stimulated. research funds: kakenhi (15500259) os3p-4-10 spatial-frequency dependent surround suppression in cat v1 ayako ishikawa 1 , satoshi shimegi 2 , hiroyuki kida 3 , hiromichi sato 2 1 grad. sch. front. biosci., osaka univ., osaka, japan; 2 grad. sch. med., osaka univ., japan; 3 grad. sch. eng. sci., osaka univ., japan we examined the temporal dynamics of the surround suppression of visual response in terms of spatial-frequency (sf) tuning of neurons in cat v1. we used a stationary flash (duration, 500 ms) of a circular sinusoidal grating patch with optimal orientation and sf as crf stimulus, and that of an annulus (50 ms) with optimal orientation but varying sf as srf stimulus. first, we stimulated crf and srf simultaneously (stimulus-onset-asynchrony (soa) = 0) and analyzed time course of surround suppression. sf tuning of the surround suppression changed along time course of response, and effective sf of surround suppression shifted from the sf lower than that optimal for crf response (c-sf) to that near c-sf. next, changing soa, we examined surround suppression on different temporal phases of crf response. soa-dependency of surround suppression changed according to the temporal phase of response. these results suggest that multiple mechanisms with different sf-and temporal characteristics are involved in the surround suppression. os3p-4-11 contrast-dependency of spatial summation property in cat v1 and lgn masahiro okamoto 1 , tomoyuki naito 2 , osamu sadakane 2 , hiromichi sato 2 1 grad. sch. front. biosci., osaka univ., japan; 2 grad. sch. med., osaka univ., toyonaka, japan we examined contrast-dependent change in a receptive field (rf) size and strength of surround suppression of neurons in the primary visual cortex (v1) and the lateral geniculate nucleus (lgn) of anesthetized cats. rf structure was modeled by spatial interactions of excitatory and inhibitory gaussians. both in v1 and lgn, ratio of gaussians (rog) model captured size-tuning curves of responses better than difference of gaussians (dog) model. under the high contrast stimulus condition, the peak of size tuning curve shrank by 0.76 and 0.68 times in v1 and lgn, respectively. in lgn, surround suppression was strengthened under high contrast stimulus condition, but in v1, the strength of surround suppression did not affected by stimulus contrast on average. we conclude that 1) rog model describes the surround suppression better than dog model both in v1 and lgn, 2) under high contrast stimulus condition, there is a reduction of rf size with a shrinking of excitatory gaussian, which is confirmed with rog model. hiroyuki kida 1 , satoshi shimegi 2 , ayako ishikawa 3 , hiroshi sakamoto 3 , hiromichi sato 2 1 grad. sch. eng. sci., osaka univ., japan; 2 grad. sch. med. sci., osaka univ., japan; 3 grad. sch. front. biosci., osaka univ., japan in the primary visual cortex (v1), neuronal responses to stimulation of the classical receptive field (crf) were suppressed by the presence of stimuli at surround receptive field (srf). we examined whether the suppression varied according to spatial configuration of srf stimuli in v1 neurons of anesthetized cat. the crf stimulus was a circular patch of sinusoidal grating with optimal stimulus parameter. srf was divided into 8 flanks (45 • step), and stationary stimulated with an annulus, oppositely-faced flanks (2-fk) or a flank (1-fk) stimulus. the durations of stimulus presentation were 500 ms for crf and 50 ms for srf stimulation. localized srf stimulation with either 2-fk or 1-fk exerted significant suppression on crf responses. according to the analysis of spatiotemporal change in srf effects, there was no particularly suppressive srf area for 1-fk stimulation throughout the crf response. however, 2-fk stimulation of end position to crf had strong and long-lasting suppression on responses during 80-200 ms after onset. os3p-4-13 temporal-frequency dependency of receptive field size and surround suppression in lgn and v1 osamu sadakane 1 , tomoyuki naito 1 , hironobu osaki 2 , masahiro okamoto 3 , hiromichi sato 1 1 grad. sch. med., osaka univ., japan; 2 med. sch., osaka univ., japan; 3 grad. sch. front. biosci., osaka univ., osaka, japan spatial summation property of neurons in the primary visual cortex (v1) varies depending on stimulus parameters (e.g., stimulus contrast). in this study, we examined how temporal frequency (tf) of grating stimulus affects size-tuning properties of cat v1 neurons. our results showed that, when the tf was higher than the optimal, the strength of surround suppression became weak and receptive field size became larger, suggesting that v1 neurons change their spatial property according to tf in such a way that neurons integrate wide visual field for fast moving stimulus, whereas localized field for slow stimulus. we also tested the effect of changing stimulus size on tf tuning curve. consistent with above-mentioned results, large grating made the peak and the high cut-off of tf-tuning curve higher than those for small grating. in the lateral geniculate nucleus (lgn), we obtained basically similar results to those of v1 neurons, suggesting that the subcortical tf tuning property contributes to that in v1. ryo sasaki, takanori uka department of physiology (i), juntendo university, tokyo, japan a few studies have shown that basic tuning functions in early visual cortex change during visual perceptual learning (schoups et al. 2001; yang and maunsell 2004) . the change in neuronal sensitivity in these studies, however, is small compared to the improvement in behavioral sensitivity. here we hypothesized that the read out of information from sensitive neurons was modified by learning. to test this hypothesis, we investigated whether learning modifies neuronal sensitivity or read out of middle temporal (mt) neurons during learning of a depth discrimination task. two monkeys were trained to report the depth of moving dots (near or far), and we recorded from isolated mt neurons during the course of training. the monkeys showed improvement in discrimination thresholds across 70 daily sessions. in contrast, the sensitivity of mt neurons did not change, whereas the correlation between neuronal activity and the monkey's behavioral choice increased during the course of training. these results suggest that plasticity due to perceptual learning occurs within the neural pathway following area mt. we developed an in vivo method to localize the fine tip of a glassinsulated tungsten microelectrode for chronic recording using 4.7 t mri. the scan conditions were first optimized by imaging a microelectrode that was sunk into copper sulfate solution. the microelectrode tip was precisely localized up to a resolution of 50 m under particular geometrical scan condition. we then examined the applicability of the method in vivo under this optimized scan condition in the temporal cortices of three monkeys. the microelectrode was penetrated into the dorsal or ventral bank of the superior temporal sulcus and the tip was localized by the high-resolution mri. the accuracy of this method was validated by comparing the localized positions of the microelectrode tips with the corresponding electrolytic lesion marks in histological sections. a transient signal change in diffusion-weighted image of the brain has been detected in human visual cortex. the time course of this signal was ahead of the bold signal and characterized by a steep onset. diffusion-mri thus represents a new exciting mechanism for fmri. in order to increase its efficiency we aimed at defining a diffusion response function (drf) as a counterpart of the hemodynamic response function (hrf). an volume of interest was defined using spm with a boxcar function. gamma-variate functions were used to model the steep onset. the parameters of the drf were estimated by fitting the time-course with the drf convolved with a boxcar. although the magnitude of the signal change (around 1%) was smaller than that of bold (>2%), the temporal profile showed a constant precedence of the diffusion signal by 2.4s. os3p-5-03 new insights on normal and pathological brain function from tomographic analysis of magnetoencephalographic signals laboratory for human brain dynamics, brain science institute (bsi), riken, wako-shi, japan tomographic analysis of magnetoencephalography (meg) data combines exceptional temporal resolution with accurate localization, at least for places a few centimeters away from the center of the head [moradi, et al., neuroimage; ioannides et al., cerebral cortex] . this unique capability of probing brain function across the entire cortex and deep brain structures from milliseconds to minutes in the same experiment has already provided new insights about normal [ioannides et al., cerebral cortex;ioannides et al., neuroimage] and pathological [ioannides et al., j. neurosc.] brain function. novel ways of analyzing meg data provide direct measures of regional brain activity over much longer timescales. these new methods are used in ongoing studies to probe the nature of global brain activity in different states of awareness (e.g. different stages of sleep) and explore the relationship between estimates of electrophysiological activity derived from meg with hemodynamic measures of brain activity. os3p-5-04 spatial registration of stand-alone fnirs data to mni space ippeita dan, archana singh, masako okamoto national food research institute, japan the registration of functional brain data to the common brain space offers great advantages for inter-modal data integration and sharing. however, this is difficult to achieve in functional near-infrared spectroscopy (fnirs) because fnirs data is primary obtained from the head surface and lacks structural information of the measured brain. therefore, we present a method for probabilistic registration of fnirs data to the standard montreal neurological institute (mni) template through international 10-20 system without using the subject's magnetic resonance image (mri). the standard deviation in probabilistic registration thus performed for given head surface points is approximately within 1 cm. this means that if the spatial registration error is within an acceptable tolerance limit, it is possible to perform multisubject fnirs analysis to make inference at the population level and to provide information on positional variability in the population, even when subjects' mris are not available. stochastic perturbation in scale is a basic property of biological systems and generates scale-independent structuration and functional dynamics in spatial and temporal patterns, which can be characterized by fractal dimensionality. it allows a user-independent evaluation and does not rely on subjective evaluation in image assessment. we have used a box-counting algorithm in scale-space segmented images to determine the mass fractal dimension of ventricles in different neurological disorders. three groups of subjects [alzheimer disease (ad), obstructive hydrocephalus (oh) and normal controls] were examined. mass fractal dimension is high for ad (1.33), approaching unity (∼1.02) for oh, and in between for control (1.14). statistical analysis was performed and significant differences were observed for these groups (p < 0.01). the observations are accounted by a flow dynamics heterogeneity model. the implications are that stochastic structuration and fractal dimension may be useful to track temporal progression of disease and assess therapeutic management. thrombin, a serine protease essential for blood coagulation, also plays an important role in injury associated with intracerebral hemorrhage. in this study, we revealed that mitogen-activated protein kinase (mapk) pathways contribute to thrombin-induced brain injury in two experimental models. firstly, we employed organotypic cortico-striatal slice cultures. application of thrombin to slice cultures resulted in cortical neuronal injury and striatal shrinkage. the cortical neuronal injury was ameliorated by inhibition of extracellular-signal regulated kinase (erk) but not p38 mapk, while the striatal shrinkage was prevented by both of them. secondly, thrombin was injected into rat striatum. thrombin-induced brain injury determined by immunoreactivity of neuronal marker was reduced by inhibition of erk and p38 mapk. these results suggest that mapk pathways play important roles in thrombin-induced brain injury and they should be therapeutic targets against neurodegeneration associated with blood-brain barrier destruction. positron emission tomography was used to study brain activations during motor imagery of standing and during performance of standing posture in parkinson's disease (pd). eight pd patients performed mental and motor tasks: (1) resting, (2) staring at a standing human object, (3) thinking of standing, (4) standing with eyes open, (5) standing with eyes closed. regional cbf data analyzed by spm2 were compared with normal counterparts. the cerebellar vermis was more activated during imagination of standing in the pd group than in healthy group. as seen in healthy subjects, standing also activated the primary sensorimotor foot area and cerebellar vermis in pd patients, but the between-group comparison generated greater activations in the vermis and prevuneus in pd. the cerebellar vermis engages in postural balance both in mind and reality, and the precuneus may play a more important role in postural control in pd. os3p-6-03 potentiation of nmda receptor-mediated current by metabolic failures through glycine release facilitation in the hypoglossal motoneurons of the rat yu kono 1,2 , eiji shigetomi 2 , kiyoharu inoue 1 , fusao kato 2 1 dept. neurol., jikei univ., sch. med., tokyo, japan; 2 lab. neurophysiol., jikei univ., sch. med., tokyo, japan to elucidate the mechanism underlying the selective vulnerability of motoneurons (mns) to metabolic failures (mfs), we compared the membrane current responses of mns and non-mns to mfs. experiments were performed on neurons in the hypoglossal nucleus (xii) and dorsal motor nucleus of the vagus nerve (dmx) in the young rat brainstem in the presence of ttx. mfs were induced by nacn or oxygen deprivation. in xii neurons, mfs induced large persistent inward currents accompanied by marked increase in strychnine-sensitive synaptic inputs, indicating facilitation of glycine release onto xii neurons. furthermore, nmda receptor-mediated current evoked by exogenous nmda was increased by nacn. in dmx neurons, mfs evoked outward currents without affecting synaptic inputs. these pre-and postsynaptic responses to mfs in mns might play a role in their selective vulnerability in various neurodegenerative diseases including the amyotrophic lateral sclerosis. os3p-6-04 effects of mdma on serotonergic neurons in rat organotypic mesencephalic slice culture including the raphe nuclei yuichi suzuki, megumi higuchi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharmaceu. sci., kyoto univ., kyoto, japan 3,4-methylenedioxymethamphetamine (mdma) is a recreational drug of abused which has been shown to increase serotonin (5-ht) release and cause degeneration of 5-htergic nerve terminals via 5-ht transporter, although the mechanisms are unclear. in this study, we developed rat organotypic mesencephalic slice culture including the 5-htergic raphe nuclei, and examined the effects of mdma and methamphetamine (meth) on 5-ht release and 5-htergic neurotoxicity. immunohistochemical studies for tryptophan hydroxylase revealed abundant 5-htergic neurons around the raphe nuclei. treatment with a 5-htergic neurotoxin 5,7-dihydroxytryptamine dramatically reduced the tissue contents of 5-ht and its metabolite, which was blocked by a selective 5-ht reuptake inhibitor. mdma and meth (0.1-1000 m) increased 5-ht release, and reduced the tissue contents of 5-ht and its metabolite at higher doses. the mesencephalic slice culture including the 5-htergic raphe nuclei may be useful to examine the mechanisms underlying 5-htergic neurotoxic effect of mdma in vitro. os3p-6-05 studies on drug dependence (rept. 421): involvement of platelet-derived growth factor (pdgf) receptor in the morphine-induced rewarding effect masami suzuki, minoru narita, michiko narita, tomoko takeuchi, yasuyuki nagumo, keiichi niikura, tsutomu suzuki dept. of toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan the present study was undertaken to investigate the involvement of platelet-derived growth factor (pdgf) receptor in the morphineinduced rewarding effect in rodents. extensive coexpression of tyrosine hydroxylase with pdgf receptor was apparently observed in the rat ventral tegmental area (vta). the levels of dopamine and its major metabolites in the nucleus accumbens (n.acc.) were markedly increased by the microinjection of pdgf into the rat vta. the morphine-induced rewarding effect was suppressed by intra-vta microinjection of pdgf receptor fc chimera. the increased level of dialysate dopamine produced by morphine in the rat n.acc. was significantly decreased by intra-vta injection of pdgf receptor fc chimera. these findings suggest that the stimulation of -opioid receptors in the vta by morphine leads to the activation of pdgf receptor, which may be directly responsible for the morphine-induced rewarding effect in rodents. os3p-6-06 prostaglandin d 2 is a strong mediator of neuroinflammation in genetic demyelinating mouse model prostaglandin (pg) d 2 , an inflammatory mediator, mainly produced by hematopoietic pgd synthase (hpgds). microglial activation and gliosis are commonly observed during the neuroinflammation. in twitcher (galct wi/twi ), a genetic demyelinating mouse model, we found that hpgds expression was upregulated in activated microglia accompanied by the dp1 receptor induction in hypertrophic astrocytes. using primary culture of glial cells, we demonstrated that activated microglia produced large amount of pgd 2 by hpgds and that astrocytes expressed both dp1 and dp2 receptors and were activated by pgd 2 . we found that gliosis and demyelination were well suppressed in hpgds-or dp1-null twitcher and twitcher treated with an hpgds-inhibitor. these results suggest that pgd 2 is a key molecule of neuroinflammation involved in the demyelination. research funds: 09670806, 12558078 os3p-7-01 on a sodium channel distribution enabling high frequency signal processing go ashida 1,2 , kousuke abe 2 , kazuo funabiki 1 1 grad. sch. medicine, kyoto univ., kyoto, japan; 2 grad. sch. informatics, kyoto univ., kyoto, japan some auditory neurons, such as the owl's nucleus laminaris (nl) cells, can sense very high frequency signals (up to 8 khz). from the theoretical point of view, it seems exceptionally difficult to handle these high frequency signals because the membrane time constant is far longer. first, we discuss a biophysical mechanism of shifting the membrane time constant by connecting the large cell body (soma) with the small node of ranvier. next, we discuss the effect of sodium channel distribution on the impedance function of the membrane. sodium conductance in the soma amplifies low frequency signal components below 1khz, while that in the node does up to 10 khz. last, as a typical example, we discuss the capability of high frequency signal processing in the owl's nl neuron. some biological evidences indicate that sodium channels in the nl neuron are distributed mainly in the nodes but less in the soma. by using an nl neuron model, we show that a neuron with low somatic sodium conductance and high nodal sodium conductance can achieve fine sensitivity to high frequency signals. interaural time difference (itd) is calulated using axonal delay lines and coincidence detector neurons (nucleus laminaris:nl). however, little is known about the cellular mechanisms of coincidence detection. here, we report the results of in vivo intracellular recordings from the barn owl's nl. we used coaxial glass electrodes in which one (microelectrode) was inserted into a patch-electrode type capillary. the inner sharp electrode was protected by the outer one during penetration of the cerebellum. we isolated 140 nl cells from 16 owls and achieved intracellular recordings in 38 of them, as judged by a sudden dc potential drop and the resting membrane potential (mean rp = 58 ± 17 mv). nl neurons produced small spikes and oscillatory potentials whose waveform closely resembled the superposition of the tones delivered to the two ears (sound analogue psps:sap). the amplitude of saps varied as a function of itd. spike rates changed in linear proportion to the amplitude of sap. we evaluated sound localization ability of vision impaired and sighted persons by using a 'two-sound sources discrimination test' in a semianechoic darkroom. in total, 13 vision impaired (7 blind and 6 low vision) and 15 sighted persons participated. the stimuli were pure tone pulses. for each trial, the same single sound pulse was emitted consecutively from a pair of speakers with the same angle either left or right from the midline of the subject. localization ability was assessed whether the subjects are able to discriminate two sound sources or not in each trial. the discriminability of the blind subjects slightly exceeded that of sighted subjects but the difference was not significant. the discriminability of the low vision subjects, on the other hand, was significantly lower than that of blind or sighted. it was suggested that a peculiar 'object perception' of blind persons is not able to measure by means of 'two-sound sources discrimination test.' os3p-8-01 autocrine bmp signaling in astroglia sensitizes the glial scarring masahisa yamada 1 , runa araya 1 , naoto kitamura 1 , yuji mishinsa 2 1 yamada unit, riken bsi, saitama, japan; 2 nihs, nieh, nc, usa bone morphogenetic proteins (bmps) affect growth of glial cells however, contribution of bmps during glial scar formation is unknown. to study the role of bmp signaling in vivo, we disrupted bmpr1a, one of the type i receptors for bmps, in a telencephalic neuronal stem cell-specific manner. we found that aberrant architecture of microvessels that led to a failure in maintaining the blood-brainbarrier in the mutant mice. although mutant mice showed inflammation around the cortical microvessels, proliferation of hypertrophic reactive-astrocytes in the mutant mice was attenuated. disruption of astroglial bmpr1a expression by cre-adenovirus recapitulates the same phenomena. bmps were upregulated in reactive astrocytes in after brain injury. knocking down of bmpr1a by small interfering rna in primary astrocytic culture negatively affected their astrocytic growth injured by scratch, which reinforced the importance of autocrine bmp signaling in astrocytes. this result opens up the understanding of novel mechanisms underlying the autocrine bmp signaling on glial scarring after cns injury. the present study was undertaken to evaluate the functional role of the glial cells in the induction of stress. here, we found that aging mice promoted anxiety-like behaviors as characterized by both the light-dark and elevated plus-maze tests, and they exhibit an increase in astrocytes in the cingulate cortex. a robust increase in gfap-positive astrocytes was noted in the cingulate cortex of nerve-ligated mice that exhibited the anxiety-like behavior. in contrast, iba1-positive microglial cells were dramatically increased as compared to that in control mice and some of them were co-localized with brdu-like immunoreactivity in the hippocampus of mice exposed to chronic psychological stress. our results indicate that the increase in astrocyte or microglia in the cingulate cortex or hippocampus may lead to emotional disorders including aggravated anxiety under aging, chronic pain-like state or exposure to chronic psychological stress. withdrawn os3p-8-04 fucosylation prevents overshooting of the migration by the vagus motor neuron precursors shigeharu kinoshita 1,2 , hideomi tanaka 1,2 , sachiko tsuruoka 3 , hironori wada 1,2 , hitoshi okamoto 1,2 1 riken bsi, wako, japan; 2 jst crest, kawaguchi, japan; 3 riken rrc, wako, japan the vagus motor nuclei are important as the autonomic center for the maintenance of homeostasis. aberrant positioning of nuclei is implicated in the etiology of the sudden infant death syndrome (sids). therefore, control of precursor cell migration into the right position may be crucially important. the zebrafish embryo has two vagus motor nuclei, the dorso-laterally and medially located nuclei (dmx and mmx). the dmx precursors are born near the floor plate, migrate dorso-laterally and then are accumulated at the defined position. in the towhead mutant embryos, ectopic neurons are distributed between bilateral dmx where precursors aberrantly migrate in dorsal direction and fail to stop at the right position. positional cloning and mrna rescue analysis identified towhead as a gdp-mannose 4,6 dehydratase (gmds), a key enzyme for de novo synthesis of a gdp-fucose. as a result, the mutant embryos showed exclusive reduction of fucosylated glycans. our findings represent that fucosylation is responsible for maturation of these neurons. in development of the drosophila visual center, photoreceptor cells extend their axons (r axons) to the lamina ganglion layer and trigger proliferation and differentiation of synaptic partners (lamina neurons) by delivering the inductive signal, hedgehog (hh). this mechanism helps to establish an orderly arrangement of connections between the r axons and lamina neurons, termed a retinotopic map because it results in positioning the lamina neurons in close vicinity to the corresponding r axons. it is found that the bhlh-pas transcription factor single-minded (sim) is induced by hh in the lamina neurons and is required for the association of lamina neurons with r axons. in sim mutant brains, lamina neurons undergo the first step of differentiation but fail to associate with r axons. as a result, lamina neurons are set aside from r axons. the data reveal a novel mechanism for regulation of the interaction between axons and neuronal cell bodies that establishes precise neuronal networks. research funds: kakenhi (17024013) os3p-8-06 initial molecular steps in synaptogenesis in vivo: trans-synaptic interaction of cell adhesion molecule is involved in postsynaptic assembly of psd95-homolog dlg hiroshi kohsaka, etsuko takasu, akinao nose department of physics, university of tokyo, tokyo, japan trans-synaptic interaction via cell adhesion molecules (cam) is essential in constructing synapse structures. although this notion has been supported by various studies in vitro, evidence in vivo has been lacking. here we used live-imaging and genetic analysis to show that a drosophila cam fasciculin2 (fas2) mediates early interaction between pre-and postsynaptic cells in synaptogenesis in vivo. by visualizing gfp-tagged fas2 genetically expressed on a muscle, we found fas2 accumulated at postsynaptic site just after the contact between growth cones and its target muscle. genetic and deletion analysis implied that trans-synaptic interaction with presynaptic fas2 is crucial for the postsynaptic localization of fas2. in addition, postsynaptic localization of a scaffolding protein dlg, psd95-homolog, and glutamate receptors was impaired in fas2 mutants. these results provide the first in vivo evidence that trans-synaptic cell adhesion molecule has a role in inducing the assembly of synapses. gaudilliere brice harvard medical school, usa postsynaptic differentiation of dendrites is an essential step in synapse formation. we report here a requirement for the transcription factor myocyte enhancer factor 2a (mef2a) in the morphogenesis of postsynaptic granule neuron dendritic claws in the cerebellar cortex. a transcriptional repressor form of mef2a that is sumoylated at lys403 promoted dendritic claw differentiation. activity-dependent calcium signaling induced a calcineurin-mediated dephosphorylation of mef2a at ser408 and thereby promoted a switch from sumoylation to acetylation at lys403, leading to inhibition of dendritic claw differentiation. our findings define a mechanism underlying postsynaptic differentiation that may modulate activity-dependent synapse development and plasticity in the brain. research funds: ns4102, ag11085 ps1a-a002 characterization of mrna species that are associated with postsynaptic density fraction by gene chip microarray analysis we previously reported the partial identification by random sequencing of mrna species that are associated with the postsynaptic density (psd) fraction (tian et al., 1999) . we report here further characterization by gene chip analysis of the psd fraction-associated mrnas, which were prepared in the presence of rnase inhibitor. we confirmed that a large number of mrna species are associated with the psd fraction and found that mrnas encoding various postsynaptic proteins were highly concentrated in the psd fraction. we identified some mrna species that were highly concentrated in the psd fraction. we also constructed a cdna library using the psd fraction-associated mrnas as templates, and identified 1152 randomly selected clones by sequencing. our data suggested that the psd fraction-associated mrnas are a very useful resource, in which as yet uncharacterized genes are concentrated. tian et al., 1999. mol. brain res., 72, 147-157. research funds: kakenhi (17500252) ps1a-a003 the distribution of snap-25 protein is regulated in isofom-specific manner makoto itakura, saori yamamori, kouta takano, masami takahashi department of biochemistry, kitasato university school of medicine, sagamihara, japan two isoforms of snap-25 derived from exon 5 splicing are expressed in brain. we generated two specific antibodies for snap-25a and b, and studied the distribution in rodent brain. there was a sticking difference in expression of snap-25a and b during early postnatal period. snap-25b was low at the birth and increased remarkably thereafter. by the contrast, snap-25a increased transiently and attained a maximum level around seven day after birth. furthermore, there seemed to be a difference in their distributions in plasma membrane, since a substantial amount of snap-25b but not snap-25a was recovered in raft-enriched fractions of triton x-100-treated lp1 membrane after sucrose density gradient centrifugation. immunohistochemistry demonstrated that snap-25b was widely distributed throughout brain, whereas, snap-25a was restricted to some particular regions of brain. these results indicate that expression and distribution of snap-25 protein are regulated differently in isoformspecific manners, and snap-25a and snap-25b play different functional roles in brain. ps1a-a004 erc(elks/rab6ip2/cast) regulates syaptic short-term plasticity by recruiting bmunc13-2 to the active zone camkii in the postsynaptic sites is localized as a psd-anchored or a cytoplasmic form. camkii in the two sites is interchangeable by its translocation. translocation and targeting of this kinase to appropriate subcellular compartments are crucial for its physiological function. we have previously suggested that postsynaptic camkii is also localized in lipid raft microdomain (2001. mol. brain res. 89, 20-28) . in this report, we proved the lipid raft localization of camkii by detergent-treatment and successive sucrose floatation assay of spm or cos7 cells expressing camkii, and by cholesterol depletion from membrane using mbcd. we also investigated the mechanism and properties of camkii targeting to lipid raft. camkii targeted to lipid raft microdomain possibly through protein-protein interaction. our data suggest that lipid raft microdomain is a major site of camkii distribution, as well as postsynaptic density and cytoplasmic region, at the postsynaptic site. glial glutamate transporters, glast and glt-1, are co-localized in processes of bergmann glia wrapping excitatory synapses on purkinje cells (pcs). although glast is expressed six-fold more abundantly than glt-1, the decay kinetics of climbing fiber (cf)mediated excitatory postsynaptic currents (cf-epscs) in pcs in glast(-/-) mice are not significantly different from those in wildtype mice. here we attempted to clarify the roles of glial glutamate transporters in cf-pc synapses using glast(-/-) and glt-1(-/-) mice, and a novel antagonist of glial glutamate transporters, (2s,3s)-3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate. our results indicate that glial glutamate transporters can retain the fast decay kinetics of cf-epscs in the normal range if a small proportion (approximately 20%) of functional transporters, glast and/or glt-1, is preserved. glutamate is well known as an essential neurotransmitter in nervous system. how glutamate-mediated synaptic transmission is controlled in neural circuit of live animal, however, remains to be poorly understood. we found that the loss-of-function mutations in vglut (vesicular glutamate transporter) encoded by eat-4 gene led to abnormal sensory behaviors including thermotaxis in c. elegans. thermotaxis defect of eat-4 mutant was caused by malfunction of both thermosensory neuron afd and its downstream interneuron ria, suggesting that thermal signals from afd or ria to their downstream neurons are transmitted by glutamate through eat-4 vglut. a mutation in avr-14 glutamate receptor also led to abnormal thermotaxis. we are trying to investigate whether avr-14 functions in the downstream neurons of afd or ria, and to identify other glutamate receptors involved in thermotaxis. through the analysis of thermotaxis neural circuit, we are hoping to reveal the mechanisms of glutamate-mediated synaptic transmission at neural circuit level. ps1a-a008 biochemical characterisation of the vesicular glutamate transporter 1 stephan schenck, shigeo takamori department of neurology and neurological science, 21st century coe program, tokyo medical & dental university, tokyo, japan vesicular glutamate transporters (vgluts) load synaptic vesicles with glutamate, the major excitatory transmitter in the brain, thus making these transporters of outstanding importance for the function of the central nervous system. the three known isoforms of these secondary active transporters have been characterised in terms of tissue distribution, developmental expression patterns and some pharmacological features. while the third isoform constitutes only a minor fraction, vglut1 and vglut2 are abundantly expressed in the brain with a complementary distribution pattern and divergences in the expression profile during ontogeny. so far, no clear difference in the function of vglut1 and vglut2 has been found. to further characterise the specific properties of the transporters we make use of the vglut1-ko mouse which gives us the opportunity to investigate a brain devoid of vglutl. we focus on synaptic vesicle fractions from ko-mice to study the vglut-associated transport biochemically. in recent years, three isoforms of vesicular glutamate transporters (vgluts) have been molecularly identified in mammals. histological investigations have revealed that the distribution of three vglut isoforms in the cns is largely complementary with limited overlap, suggesting that differential expression of vglut isoforms may contribute to functional diversity in glutamatergic synapses. however, functional differences among the isoforms remained poorly understood. to get insights into their isoform-specific property, we searched for interacting protein(s) to the c-terminus of vglut1 by yeast two-hybrid screening and found endophilin a1. as expected for the interacting molecule to vglut1, endophilin a1 was typically localized to vglut1-positive synaptic terminals in cultured hippocampal neurons. we are currently investigating physiological significance underlying their direct interaction and co-localization. the aim of this study is to investigate the molecular basis for lactate utilization. hippocampal neuronal culture was continuously superfused with glucose or lactate solution and spontaneous excitatory postsynaptic currents (sepscs) were recorded from a voltage-clamped pyramidal neuron. in lactate solution, amplitude of epscs was decreased in 60∼90 min, followed by spontaneously recovered after120 min, while epsc in glucose medium remained unchanged. application of apv+ni in lactate medium, spontaneous recovery was not observed. in neuron cultures, incorporation of 14c-lactate was gradually increased, which was suppressed by applications of inhibitors for calcium calcium channels or protein kinase c. in glial cell cultures, incorpotation of lactate was initially maintained. increased expression of monocarboxylate transporter (mct) 2 was demonstrated in the lactate medium. results suggested that increased mct2 expression of neurons may lead to utilization of lactate to sustain synaptic function via calcium-dependent manner. yumei wu 1 , kazuhito tomizawa 1 , shuang liang 2 , iori ohmori 1 , teiichi nishiki 1 , kohji takei 2 , hideki matsui 1 1 dept. of physiol., okayama univ., okayama, japan; 2 dept. of neurosci., okayama univ., okayama, japan synaptic vesicle endocytosis is regulated by phosphorylation of endocytotic proteins, such as amphiphysin (amph) i and dynamin i. here, we show a novel type of regulation of vesicle endocytosis by proteolysis. in mouse hippocampal slices, amph i was found to be cleaved by a ca 2+ -activated protease, calpain during prolonged depolarization or stimulus trains. the calpain-cleaved n-terminal amph i fragment lost its ability to bind dynamin and inhibited transferrin uptake as overexpressed in cos-7 cells, indicating that the calpain cleavage of amph i inhibits endocytosis. amph i in hippocampus was also cleaved by calpain in vivo after kainate seizure. although the second administration of kainate caused less severe seizure activity than the first one, this relieved second seizure was not observed in pre-treatment with a calpain inhibitor, allm during the first seizure. thus, the proteolytic activity of calpain could protect neurons from excitotoxicity by inhibiting vesicle recycling. synaptic vesicles (svs) are effectively recycled by endocytosis for continuous synaptic transmission. previously, we have suggested that a high level of synaptic transmission is maintained by recycling of svs through two types of endocytosis operating coordinately (27th this meeting). in the present study, we labeled endocytosed svs at nerve terminals of drosophila with fluorescence dyes, fm1-43 and fm4-64, and also measured quantatively exocytosis and endocytosis of svs, using these dyes. egfp-labeled cacophony ca2+ channels and anti hrp stained the active zone and non-active zone at synapse, respectively. imaging analysis revealed that two distinct types of endocytosis of svs occurred at the active zone and the non-active zone of motor nerve terminals. we have previously shown that baclofen, a gaba b receptor agonist, inhibits exocytosis in synapses of mouse hippocampal neurons. syntaxin 1a is also known to modulate exocytosis. to characterize the molecular mechanisms involved, the inhibitory effects of baclofen in neurons transfected with antisense oligonucleotide to syntaxin 1a were investigated by patch-clamp recording and counting the number of release sites. transfected neurons showed higher frequency of miniature epscs and stronger inhibition by baclofen than controls, but no change in number of sites. increased exocytosis is thus induced by increases in transmitter release per site, rather than by more sites due to neurite sprouting. these results suggest that gaba b receptor shares part of the mechanism involved in modulation of exocytosis with syntaxin 1a in mouse hippocampal neurons. we have previously shown a transient localization of tubulin (tub) during synaptic vesicle (sv) cycling in drosophila nerve terminals. the tub localization is detected during sv recycling, while microtuble (mt)-loop is observed throughout sv cycle. in this study, we characterized the two distinct tub localizations and showed their relation with sv pool formation. axonal mts and mt-loops abounded in acetylated (acetyl) tub. the transient localization was either polymerized or depolymerized, and organized by non-acetyl tub. taxol decreased the non-acetyl tub localization but not mt-loops, and inhibited exo/endo cycling pool (ecp) formation. in boutons containing mt-loops, ecp formation was also inhibited. acetyl mt-loops tend to be stable whereas presynaptic non-acetyl tubs are either free dimers or dynamic mts. these results suggest that presynaptic dynamic tub, especially non-acetyl tub, controls ecp formation. presynaptic tub dynamics may regulate functional presynaptic plasticity by controlling sv pools. research funds: grant-in-aid for jsps fellows the mechanism by which pregnenolone sulfate (pregs) enhances synaptic transmission was studied at the rat calyx of held. pregs increased the amplitude of evoked epscs, without affecting that of spontaneous miniature epscs, indicating that the site of its action is presynaptic. pregs facilitated presynaptic voltage-gated ca 2+ channel (vgcc) currents via accelerating their activation kinetics, but had no effect on k + currents, resting conductance, or action potential waveforms. in simultaneous pre-and postsynaptic recordings pregs did not change the relationship between presynaptic ca 2+ influx and epscs, suggesting that exocytotic machinery downstream of ca 2+ influx was not involved in the pregs effect. neither bapta nor gtp␥s loaded into presynaptic terminals blocked the effect of pregs. we conclude that pregs enhances transmitter release via facilitating vgccs by a novel mechanism, which is independent of intracellular ca 2+ or g-proteins. ps1a-b017 spine targeting of endocannabinoid synthesizing enzyme, diacylglycerol lipase-␣ in the cerebellum and hippocampus endocannabinoids are neuromodulator that is released from postsynaptic neurons, acts retrogradely on presynaptic cb1 cannabinoid receptor, and induce suppression of transmitter release. to understand the retrograde signaling mechanisms, we investigated subcellular localization of a major endocannabinoid biosynthetic enzyme, diacylglycerol lipase-␣ (dagl␣), in the mouse brain. in the cerebellum, dagl␣ was predominantly expressed in somatodendritic membrane of purkinje cells, and highly concentrated at the base of spine neck. however, dagl␣ was excluded from the main body of spine neck and head. in hippocampal pyramidal cells, dagl␣ was selective to spines, but widely distributed within spines. these results indicate that dagl␣ is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two cell types. synprint site of voltage-gated ca 2+ channels interacts with synaptotagmin. however, its physiological role is not entirely clear. here we report that ap-2 subunit can directly bind with synprint site. this interaction was ca 2+ -dependent, being weaker at concentrations higher than 200 nm. in contrast, the interaction of synaptotagmin with synprint was optimal at 100 m ca 2+ , being weaker at lower or higher concentrations. the binding domain of synprint for ap-2 and synaptotagmin was indistinguishable, and these proteins competed with each other for the synprint site. to assess physiological role of these interactions, we made a peptide containing synprint site, and loaded it directly into the nerve terminal at the calyx of held. this peptide blocked endocytosis measured with capacitance, and gradually diminished exocytosis upon repetitive presynaptic activations. we conclude that ca2+ channel synprint site makes ca 2+ -dependent interactions with ap-2 and synaptotagmin thereby contributing to vesicular endocytosis. ps1a-b019 acl-4, an evolutionarily conserved acyltransferase like gene is required for normal synaptic transmission in c. elegans naoko hara, takao inoue, yasukazu takanezawa, hiroyuki arai department of health chemistry, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan it is generally accepted that various phospholipid molecular species are formed by phospholipids acyltransferase reactions. however, the physiological significance and the molecular mechanism of the remodeling are largely unknown. to address these questions, we focused on evolutionarily conserved acyltransferase like genes in c.elegans acl-1˜14, and generated their deletion mutants. the mutants of acl-4 gene, which is predominantly expressed in neurons and muscles, showed no apparent phenotype. however, the mutants exhibited severe movement abnormalities in fat-3 mutant background in which long chain polyunsaturated fatty acids are depleted. pharmacological analysis revealed that these mutants showed presynaptic defects in synaptic transmission. these abnormalities were rescued by neuron specific acl-4 expression, suggesting that certain phospholipid species produced by acl-4 are involved in maintaining normal synaptic transmission and motility of c.elegans. daisaku yokomaku 1 , hussam jourdi 1 , akiyoshi kakita 2 , tadasato nagano 1 , hitoshi takahashi 3 , nobuyuki takei 1 , hiroyuki nawa 1 1 dept. mol. neurobiol., brain res. inst., niigata univ., japan; 2 brain resource center, brain res. inst., niigata univ., japan; 3 dept. pathology, brain res. inst., niigata univ., japan scaffolding proteins containing pdz domains interact with synaptic receptors and cytoskeletal components and are therefore implicated in synaptic development and plasticity. little is known, however, about what regulates the expression of the pdz proteins and how the levels of these proteins influence synaptic development. here, we show that ligands for epidermal growth factor (egf) receptors (erbb1) decrease a particular set of pdz proteins and negatively influence synaptic formation or maturation. in neocortical cultures, egf decreased the expression of grip1 and sap97. moreover, egf treatment resulted in a decrease in the frequency of pan-pdzimmunoreactive aggregates on dendritic processes. these findings revealed a novel negative effects of erbb1 receptor ligands that attenuates the expression of the pdz proteins and inhibits postsynaptic maturation in developing neocortex. takatoshi iijima 1 , eriko miura 2 , keiko matsuda 1 , tetsuro kondo 1,3 , masahiko watanabe 2 , michisuke yuzaki 1 1 dept. physiol., sch. med., keio univ., tokyo, japan; 2 dept. anatomy, hokkaido univ., sch. med., sapporo, japan; 3 mol. neurophysiol., aist, tsukuba, japan cbln1 is a member of the c1q and tumor necrosis factor families predominantly produced in cerebellar granule cells. recently, we have shown that cbln1 is secreted as a glycoprotein and plays crucial roles in synaptic plasticity and synaptic integrity of purkinje cells. although other members of the cbln family, cbln2-4, are known to be expressed in the brain, their precise expression patterns and biochemical properties remained unclear. here, we show that each cbln member is expressed in various regions of developing and mature brains. all cbln family members could form both homomeric and heteromeric complexes each other in heterologous cells. like cbln1, cbln2 and cbln4 were secreted as glycoproteins, whereas cbln3 was retained in the endoplasmic reticulum. these results suggest that each cbln member is potentially involved in synapse development and plasticity in various brain regions. s-scam is a synaptic membrane-associated protein with pdz domains, a guanylate kinase domain and ww domains. it interacts with various synaptic components including nmda receptor subunits, psd-95 and neuroligin. as we previously reported, s-scam is recruited to excitatory synapses by ␤-catenin. s-scam forms a ternary complex with neuroligin and psd-95. more importantly, s-scam is involved in synaptic accumulation of neuroligin and subsequently affects the localization of psd-95 at excitatory synapses. in the course of these studies, we observed signals detected by anti-s-scam antibody at inhibitory synapses. we have here examined whether s-scam is indeed localized at inhibitory synapses in hippocampal neurons. we have raised questions which molecules s-scam interacts with at inhibitory synapses and which role s-scam plays in the assembly of inhibitory synapses. eriko fujita, yuko tanabe, takashi momoi division of differentiation and development, department of inherited metabolic disorder, national institute of neuroscience, ncnp, oawahigashi, tokyo, japan igsf4/ra175 (ra175), which is a member of immunoglobulin superfamily having pdz binding domain at c-terminals, has ca2+independent homophilic trans-cell adhesion activity. ra175 participates in synaptic junction and epithelial junctions in various tissues including testis. homozygous null (ra175-/-) male is infertile and shows the defective elongating spermatids and fails to mature further. ra175 interacted with par-3 being involved in the polarity of epithelial cells via pdz binding domain at c-terminals. par-3 was colocalized in the cell adherent region of p19 embryonal teratocarcinoma cells during ra-induced differentiation into epithelial-like cells and mainly localized in the spermatid of ra175+/+ testis, whereas it was undetectable in the spermatid of the ra175−/− testis. ra175 and jam-c were localized around the head portion of spermatid and ra175 deficiency provided the abnormal polarization of the jam-c, which is necessary for the differentiation of round to elongated spermatid. jam-c inhibited the interaction between ra175 and par-3. research funds: 12324122 izumi kawabata, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan coordinated development of excitatory and inhibitory synapses is critical for both stability and temporal fidelity of neuron network in the hippocampus. however, there have been few analyses on postsynaptic molecular assembly in interneurons during development. to address this question, we examined dynamic properties of psd-95 clusters in cultured hippocampal interneurons. higher density of dendritic psd-95 clusters was observed in interneurons at 11 div. at 18 div, this difference was less prominent, mainly due to >3-fold increase of psds in excitatory neurons. psd-95-gfp imaging revealed lower rate of cluster appearance/disappearance in interneurons at 11 div. the higher rate of cluster turnover in excitatory neurons, together with their higher rate of net cluster increase, may explain the delayed boost of cluster density. photobleaching of psd-95-gfp revealed similar kinetics in two neuron types, suggesting additional determinants of cluster dynamics apart from the steady-state assembly rate. possible involvement of other postsynaptic molecules in interneuron psd dynamics is now being investigated. ps1a-c025 two-photon imaging of immature dendritic protrusions and astroglial processes in hippocampal slice cultures hideko nishida, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan several lines of evidences indicate roles of astroglia in synaptogenesis, possibly mediated by either cell adhesion or diffusible factors. however, structural evidences supporting this claim are virtually lacking, mainly due to technical limitations in simultaneous imaging of neuronal and astroglial structures. here we visualized astroglia and pyramidal neurons in hippocampal slice cultures by combining adenovirus-mediated, cre-dependent expression of gfp with electroporation of rhodamine-dextran. two-photon time-lapse imaging of immature dendritic protrusions and astroglial processes in 3-7div slice cultures revealed longer lifetime of dendritic protrusions having experienced astroglial contacts than those without contacts. dendritic protrusions with astroglial contacts also showed higher tendency to form spines. furthermore, expression of mutant rac1 in astroglial cells induced significantly longer, non-spiny protrusions than control. these findings suggest an involvement of direct astroglia-filopodia contacts in subsequent maturation of dendritic protrusions. taiko imura, fusao kato lab. neurophysiol., jikei univ. sch. med., tokyo, japan application of p2x receptor agonists to the neurons in the nucleus of the solitary tract (nts) results in glutamate release facilitation (kato & shigetomi, 2001; shigetomi & kato, 2003) . recently accumulated evidence indicates that astrocytes affect the neuronal excitability by releasing gliotransmitters such as atp. this study was performed to determine whether such astrocyte-neuron interaction takes place in the nts. first, we analyzed the spatial localization of these cells by immunohistochemistry. a large number of gfap-positive cells with processes in the close apposition to the neun-positive neurons were found. second, we analyzed the effect on synaptic activity of localized application of atp using laser-based photolysis of caged atp in brainstem slices. uncaging of atp at neuronal dendrites (2 s, 4-micrometer diameter) resulted in an immediate rise in mepsc frequency, in a manner sensitive to p2x receptor antagonists. these results provide supports for the possible interaction between astrocytes and neuronal presynaptic terminals. research funds: kakenhi (17300123) ps1a-c027 ealy synapsin i accumulation in a granule cell axon at the filopodial attachment site of developing rodent purkinje cell dendrites in vitro isao nagata, junko kimura-kuroda department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan synapse formation between the parallel fibers (pf) and dendrites of purkinje cells (pc) occurs at an early stage in the developing cerebellar cortex of the neonatal rodent. however, the precise spatio-temporal pf-pc interaction has not been elucidated. we have found that growth of pc dendrites was initiated by the attachment of axonal neurite bundles of granule cells orienting at right angles in several types of 2d-and 3d-cerebellar cultures. here, we investigated the expression of a synaptic vesicle marker, synapsin i, in granule cell axons by multiple immunofluorescence labelings in these cultures. synapsin i was first expressed at the filopodial attachment site of a pc dendrite as a cluster of faint punctate deposits in a long axon, then they appeared to gather into a slender and finally into a small round deposit. thus, the filopodial attachment of the juvenile pc dendrites to the axons of granule cells may induce rapid formation of presynaptic terminals via local clustering of synaptic vesicles. ps1a-c028 integrative spike dynamics of rat ca1 neurons: an in situ multineuronal imaging study takuya sasaki, rie kimura, norio matsuki, yuji ikegaya department of pharmacology, university of tokyo, tokyo, japan the brain operates through a coordinated interplay of numerous neurons. our new technique with large-scale optical recordings reveals the diversity of synaptic integration in hundreds of neurons. in hippocampal slices bolus-loaded with calcium fluorophores, we stimulated the schaffer collaterals and monitored the bulk presynaptic activity from the stratum radiatum and individual postsynaptic spikes from the ca1 stratum pyramidale. single neurons responded to varying synaptic inputs with unreliable spikes, but at the population level, the networks output a linear sum of synaptic inputs. the network activity varied from trial to trial, even though given constant stimuli. this variation emerged through time-varying recruitment of different neuron subsets, which were shaped by correlated background noise. our imaging approach enables linking single-cell behaviors to their communal dynamics, and we discovered that, even in a relatively simple ca1 circuit, neurons could collectively be engaged in complex information processing. it is assumed based on previous in vitro experiments by other researchers that mglur6 connects with syntenin at the dendrites and mglur7 with pick1 at the axon terminal in on cone bipolar cells. to prove this possibility, we investigated wild-type mouse retinas immunohistochemically and confirmed their co-localized immunopositive labels at the respective places. next, we examined which scaffold protein would connect with mglur7 that was known to be ectopicly expressed in the dendrites of mglur6-deficient on cone bipolar cells. we observed no pick1 but only syntenin at the mglur6-deficient dendrites, and also the syntenin immunopositivity was co-localized with mglur7 immunopositivity. these findings suggest that mglur7 connects with syntenin in place of mglur6 that was knockout from the on cone bipolar dendrites. noriko trpv family is identified as thermosensitive, ca 2+ -permeable channels. trpv1, expressed in sensory neurons, is activated by noxious heat above 42 • c, whereas trpv 4, expressed in keratinocytes, is sensitive to moderate temperatures (>34 • c). here we examined the role of trpv1 and 4 in regulation of body temperature (bt) by using infrared laser as a heat stimulus. in wild type mouse, though the laser irradiation which caused the increase in skin temperature up to 55 • c did not induce the change in bt, desensitization of trpv1 with capsaicin resulted in the increase in bt. on the other hand, in trpv4-knockout mouse, moderate thermal radiation (>43 • c) caused the increase in the bt. the processing of noxious and moderate thermal radiation stimuli may depend on the trpv1 and 4 respectively. research funds: kakenhi (17657052) ps1a-c032 generation and biochemical analysis of a glur␣2 knockout mouse hirotsugu azechi 1 , manabu abe 1 , rie natsume 1,2 , kenji sakimura 1,2 1 department of cellular neurobiology, brain research institute, niigata university, niigata, japan; 2 sorst/jst, saitama, japan glur␣2(glur2) is a key subunit of ampa receptors, since it is a critical determinant of their calcium permeability. to clarify the molecular function of glur␣2, we generated a conditional glur␣2 knockout mouse using the cre/loxp recombination system. we first established a "floxed" mutant line gra2f using c57bl/6(b6) es cell line renka. the homozygous floxed mutants showed no significant abnormalities, thus our gra2f was used as a target of glur␣2 line. by crossing gra2f and tlcn-cre that expressed cre in germ line cells, glur␣2 null ko mice were produced, but most of them died within 3 days after birth. to overcome the lethality, the glur␣2 mutation was transferred onto b6/129 or b6/cd-1 genetic background. subcellular fractionation and quantitative immunoblot showed changes in the amount of ampa receptor subunits. these results indicated a significant role of glur␣2 in the distribution of functional ampa receptors in vivo. ps1a-c033 gisp: a novel brain specific protein that binds to the gaba b1 subunit and promotes its surface expression here we report the identification and characterisation of a novel brain specific 130 kda protein, gaba b r interacting scaffolding protein (gisp), that interacts directly with the gaba b 1 subunit via a coiledcoil domain. gisp coimmunoprecipitates with gaba b 1 and gaba b 2 from rat brain. in cultured hippocampal neurons gisp displays a punctate dendritic distribution and colocalises with gaba b receptors. when co-expressed with gaba b rs gisp increases the amount of gaba b 1 protein and also promotes gaba b 1 surface expression in the heterologous cells. furthermore, gisp increases surface expression of gaba b 1/gaba b 2 complexes. these results suggest that gisp is involved in the forward trafficking and stabilisation of gaba b rs. thus gisp is an novel gaba b 1-binding protein potentially involved in the cell surface and/or synaptic targeting of the gaba b rs. three distinct isoforms of vesicular glutamate transporters (vglut1-3) have been cloned and shown to exhibit differential distribution patterns in the brain. recent work shows the presence of vgluts in synaptic-like microvesicles (slmvs) of endocrine cells. mammalian pineal melatonin-secreting cells, pinealocytes, contain numerous slmvs which likely accumulate glutamate to inhibit melatonin synthesis. vglut1 and vglut2 seem to participate in this glutamate accumulation. in the present study, we found that vglut3 mrna is also expressed in the adult rat pinealocytes. vglut3 immunoreactivity (ir) was distributed throughout the pineal gland, and was co-localized with vglut1-ir or vglut2-ir in many, but not all, processes of pinealocyte. these data indicate that there are some subpopulations of slmvs which differ in the kind of vglut isoforms contained and/or in their combinations, suggesting vglut isoform-dependent sorting of slmvs to pinealocyte processes. kenzi saito 1,2,3 , kenji nakamura 4 , toshikazu kakizaki 1,3 , satoe ebihara 5 , masakazu uematsu 6 , shigeo takamori 7 , minesuke yokoyama 4 , shiro konishi 4 , masayoshi mishina 3,8 , jun-ichi miyazaki 9 , kunihiko obata 10 , yuchio yanagawa 1,3 1 gunma univ., maebashi, japan; 2 sokendai, hayama, japan; 3 sorst, kawaguchi, japan; 4 mitsubishi kagaku inst. life sci., machida, japan; 5 kumamoto univ., kumamoto, japan; 6 toyohashi univ. tech., toyohashi, japan; 7 tokyo med. den. univ., tokyo, japan; 8 univ. tokyo, tokyo, japan; 9 osaka univ., suita, japan; 10 riken, wako, japan the vesicular gaba transporter (vgat) loads gaba from neuronal cytoplasm into synaptic vesicles and is selectively expressed in inhibitory neurons containing gaba and/or glycine. to assess the functional role of vgat in development, we have disrupted the gene encoding vgat using cre-loxp system. western-blot analysis showed that vgat protein was absent in the homozygous embryos, indicating that the mutation had generated in a null allele. vgat knockout mice died around birth. all vgat knockout mice displayed cleft palate and omphalocele. our results suggest that vgat plays essential roles in both palate formation and ventral body wall development. research funds: kakenhi (17052002) ps1a-c036 postnatal changes in the colocalization of vglut1 and vglut2 immunoreactivities at single axon terminals of the mouse neocortex kouichi nakamura 1,2 , akiya watakabe 3 , hiroyuki hioki 1 , fumino fujiyama 1 , yasuyo tanaka 1 , tetsuo yamamori 3 , takeshi kaneko 1,2 1 dept. morphol brain sci., grad. sch. med., kyoto univ., japan; 2 crest, jst, japan; 3 div. brain biol., nat. inst. basic biol., okazaki, japan vesicular glutamate transporter (vglut)1 and vglut2 accumulate transmitter glutamate into synaptic vesicles. the vgluts show a complementary expression pattern in the brain, but colocalize at single axon terminals in some synapses. here we quantitatively evaluated postnatal changes in the colocalization of vgluts at single axon terminals of the developing mouse neocortex by using a pixel-based correlation coefficient (cc) as an index of the colocalization. the cc was calculated from pixel values for vglut1 and vglut2 in each pixel of confocal micrographs of double immunofluorescence-labeled brain sections. in the barrels, the cc showed a prominent increase transiently around p7. the cc was higher in area s1 than areas m1 and area v1 throughout postnatal development. our results indicate that the colocalization of vgluts in the neocortex is regulated in an age-, area-and layer-specific manner. gaba b receptors mediate slow and prolonged synaptic inhibition in the brain, and are members of the g protein-coupled receptors. here we have investigated the role of 5 amp-activated protein kinase (ampk), as an endogenous regulator of gaba b receptor function. site-specific mutagenesis identified multiple phosphorylation sites for ampk within the cytoplasmic tails of both gaba b r1 and r2. the activation of ampk regulated stability of gaba b receptors coupling with k + channels. together highlights a novel role for ampk in regulating the functional properties of gaba b receptors, by direct phosphorylation. given the role of ampk as a sensor of cellular stress this potential mechanism may be relevant in regulating the efficacy of synaptic inhibition under anoxic conditions and during periods of high synaptic activity. takao hirai, hiroaki nishio department of molecular pharmacology, faculty of pharmacy and pharmaceutical sciences, fukuyama university, hiroshima, japan serotonin (5-hydroxytryptamine, 5-ht) is a central neurotransmitter that is widely implicated in the regulation of mood and cognition, and is a peripheral signaling molecule that affects hemostasis, immune function, intestinal physiology, and other systems. there is increasing evidence for contribution of neuronal system to regulation of bone metabolism. this study was thus aimed at elucidation of possible functional expression of serotonergic system in mouse osteoblasts. rt-pcr analysis revealed constitutive expression of mrna for several 5-ht receptor subtypes, 5-ht transporter (5-htt) and vesicular monoamine transporter 1 (vmat1) in primary cultured mouse osteoblasts and mc3t3-e1 osteoblastic cells. sustained exposure to fluoxetine, a selective 5-ht reuptake inhibitor, significantly prevented increase in alkaline phosphatase activities and mineralization in mc3t3-e1. these results suggest that serotonergic system may be functionally expressed to regulate mechanisms underlying cellular differentiation and maturation in mouse osteoblasts. junko motohashi department of physiology, keio university school of medicine, tokyo, japan hotfoot mice are spontaneous mutants with ataxic phenotype. most hotfoot alleles identified so far have deletions of one or more exons coding for portions of the n-terminal domain of the ␦2 glutamate receptor (glur␦2). however, because only genomic dna was available for most hotfoot mutants, it was unclear whether truncated forms of glur␦2 were actually translated and involved in the ataxic phenotype. here, we report that a newly identified hotfoot mutant, ho15j, was caused by a new type of intragenic deletion of the grid2 gene, which was indeed translated as glur␦2 lacking 52-amino acids in the n-terminus. mutant glur␦2 proteins were retained in the soma of purkinje cells and degraded. as a result, ho15j mice exhibited a severe motor discoordination on rotarod tests. furthermore, these mice exhibited sustained innervation of purkinje cells by multiple climbing fibers, and impaired long term depression, which is thought to underlie motor learning. these results indicate the importance of the n-terminal domain in glur␦2 signaling and cerebellar functions. research funds: kakenhi (16200024) ps1a-d040 role of the dry motif in melanin-concentrating hormone receptor 1 in signaling yumiko saito 1 , yoshimi aizaki 1 , mituse nakano 2 , kei maruyama 1 1 dept. pharamacol., saitama med. sch., saitama, japan; 2 international university of health and welfare, tochigi, japan considerable attention has been focused on the functional importance of the highly conserved dry triplet in class a g protein-coupled receptors (gpcr). here we investigated the role of asp140, arg141 and tyr142 in the dry of rat melanin-concentrating hormone receptor 1 (mch1r). in transfected cells, mutation of asp (d/a) resulted in nonfunctional receptor despite of showing moderate level of cell surface expression and an apparent affinity to mch. d/a mutation occurred with no increase in basal signaling pathway, suggesting no indication for constitutive activity. y/a mutation also yielded a loss of function phenotype that is similar to d/a mutation. mutation of the arg (r/a) showed higher ec50 value in signaling with a decrease in mch binding, while the level of cell surface expression exhibited only moderate decrease. these data suggest that a function for dry motif different from that widely accepted for class a gpcrs in regulating mch1r-mediated signal pathway. in this study we confirmed functional heteromultimerization between a 1 r and p2y 1 r electrophysiologically using xenopus oocyte expression system. when a 1 r and p2y 1 r were coexpressed, application of non-hydrolyzable atp analogue induced g i/o response, showing formation of functional heteromultimers with a unique phenotype. it was also observed that the heteromultimers can activate g q/11 pathway by atp analogue and also g i/o pathway by adenosine analogue, maintaining the features of the original subunits. ps1a-d043 dual signaling via metabotropic glutamate receptor1␣ is regulated by a cytoskeletal protein 4.1g michihiro tateyama 1,2 , yoshihiro kubo 1,2 1 department of biophysics and neurology, nips, aichi, japan; 2 sorst, jst, saitama, japan the g protein-coupled metabotropic glutamate receptor 1␣ (mglur1) is known to functionally couple to different types of g proteins. recently we have reported that the signaling pathways through mglur1 are differentially regulated by different types of ligands, glutamate and gd 3+ . on the other hand, several cytoskeletal proteins have been reported to interact with the c-terminal cytoplasmic tail of mglur1. these proteins, such as homer and 4.1g, are also known to change the membrane expression of and modulate the function of mglur1. here we investigated whether or not these cytoskeletal proteins regulate the multi path signaling of mglur1. interestingly, the functional couplings of mglur1 to gq and gs pathways were altered by co-expression of 4.1g, but not by homer. deletion of the c-terminal tail abolished the effect of 4.1g, indicating that the interaction of 4.1g with the c-terminal tail of mglur1 regulates the multi path signaling. ps1a-d044 modulation of the eaac1-mediated glutamate uptake by the addicsin mutant mitsushi j. ikemoto 1,2 , saori akiduki 1,2 1 age dimension research center, aist, ibaraki, japan; 2 graduate school of science, toho university, chiba, japan addicsin is a murine homologue of rat glutamate-transporterassociated protein 3-18 (gtrap3-18), an inhibitory modulator of neural glutamate-transporter excitatory amino acid carrier 1 (eaac1). it contains two potential pkc phosphorylation motifs at positions 18-20 and 138-140. however, its physiological function remains almost unknown. to clarify a significance of these pkc phosphorylation motifs, we investigated eaac1-mediated glutamate transport activity in c6bu-1 cells provided with a mifepristone-inducible expression of addicsin (wt), its mutants mutated at serine 18 into alanine (s18a) or at serine 138 into alanine (s138a). as compared with wt, s18a had no inhibitory effect on glutamate transport activity under exposure to 100 nm pma, and had increased glutamate transport activity under normal condition. by contrast, s138a had the same glutamate transport activity as that of wt. thus, the eaac1-mediated glutamate transport activity may be regulated by a pkc-dependent phosphorylation at serine 18 in addicsin. kaori akashi 1 , manabu abe 1 , toshikazu kakizaki 1 , rie natsume 1,2 , kenji sakimura 1,2 1 department of cellular neurobiology, brain research institute, niigata university, japan; 2 sorst-jst, saitama, japan kainate type glutamate receptors are composed of various combinations of glur␤1-3(glur5-7) and glur␥1-2(ka1-2) subunits. although their physiological functions and subunit compositions have been inferred from various studies, they are still not clear. to clarify the functions and subunit dynamics of kainate receptors, we generated glur␤2ko mice from c57bl/6 es cell line. the glur␤2ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, the amounts of glur␥1 and glur␥2 proteins were significantly decreased in the crude fraction of ca3 region of glur␤2ko. furthermore, subcellular localizations of both subunits were also changed in glur␤2ko. these results suggested that native kainate receptors might function as heteromeric channels (glur␤/␥) and the glur␤2 subunit might determine subcellular localization of the glur␥ subunits, similar to the roles of nmda receptor glur subunits determining stability and distribution of the glur subunits. ps1a-d046 sema4d/plexin-b1 activates gsk-3␤ via r-ras gap activity, inducing growth cone collapse yuri ito, izumi oinuma, hironori katoh, manabu negishi laboratory of molecular neurobiology, graduate school of biostudies, kyoto university, kyoto, japan plexins are receptors for repulsive axonal guidance molecules semaphorins. we have recently reported that semaphorin 4d (sema4d) receptor plexin-b1 induces growth cone collapse by functioning as an r-ras gap. here we characterized the downstream signaling of plexin-b1-mediated r-ras gap activity, leading to growth cone collapse. sema4d suppressed the endogenous r-ras activity in hippocampal neurons, in parallel with dephosphorylation of akt and activation of gsk-3␤. ectopic expression of the constitutively active mutant of akt, myr-akt, or treatment with gsk-3␤ antagonist suppressed the sema4d-induced growth cone collapse. the r-ras gap activity was necessary for plexin-b1-induced dephosphorylation of akt and gsk-3␤. plexin-a1 also induced dephosphorylation of akt and gsk-3␤ through its r-ras gap activity. thus, we conclude that plexin-b1 dephosphorylates akt and gsk-3␤ through r-rasgap activity, inducing growth cone collapse. to find proteins having relations in receptor trafficking, we searched human genome database and selected hepatocyte odd protein shuttling (hops) as a candidate gene. hops had three transmembrane domains, and expressed abundantly on a brain tissue. hops was detected in membranous regions from subcellular fractionation and immunohistochemistry. hops was recruited to membranous structures when overexpressed in cos7 cells. when expressed in hippocampal cultures, hops enhanced the amplitude of mepsc. from antibody feeding assay, we discovered that hops enhanced the recycling of glur2. hops was co-immunoprecipitated with grip1 (glutamate receptor interacting protein 1) when they were co-transfected to hek cells. thus, it was suggested that hops had roles in synaptic transmission enhancement by stabilization of surface glur2 via grip1 binding. serine must be taken up into neurons for their survival, because neurons lack serine biosynthetic enzyme. we have recently identified a serine transporter asc-1. a neural amino acid transporter snat2/ata2 also transports serine. we investigated their roles as serine transporters by comparing the localization of these serine transporters in the rat brain. the asc-1 immunoreactivity (asc-1-ir) was detected in dendrites and somata of pyramidal neurons. the snat2-ir was widely detected in neurons, whose intracellular localization was similar to that of asc-1-ir. deferent from asc-1-ir, snat2-ir was also located in astrocytes and ependymal cells, especially around capillary blood vessels and ventricles. these results suggest the significant contribution of asc-1 and snat2 to the neuronal uptake of l-serine. snat2 might also accumulate l-serine in astrocytes from the extracellular spaces including blood and csf. ps1a-d049 neuronal glutamate transporter eaat4 controls climbing fiber-mediated presynaptic inhibition of gabaergic transmission at cerebellar interneuron-purkinje cell synapses shin'ichiro satake 1 , si-young song 2 , shiro konishi 3 , keiji imoto 1 1 natl. inst. physiol. sci. (nips), okazaki, japan; 2 mitsubishi kagaku inst life sci, tokyo, japan; 3 tokushima bunri univ., sanuki, japan through extrasynaptic diffusion and activation of presynaptic ampa receptors in bc terminals. we here examined possible roles of glutamate transporters in this cf action. the eaat4/glt-1 blocker threo-3-methylglutamate, but not the glt-1 blocker dihydrokainate, augmented the cf-induced inhibition. cf stimulation obviously inhibited gabaergic transmission onto pcs in the lobule iii, where eaat4 expression was low, whereas the cf-induced inhibition was minimal in the lobule x, where eaat4 was abundant. the results suggest that eaat4 plays a major role in regulating the concentration of cf transmitters, possibly glutamate, in the route of its extrasynaptic diffusion, and determining the degree of cf-induced inhibition of gaba release from bcs depending on the regional difference of eaat4 expression in postsynaptic pcs. chitoshi takayama 1 , yoshiro inoue 1 1 department of molecular neuroanatomy, hokkaido university school of medicine, sapporo, japan gaba mediates inhibitory transmission in the adult central nervous system (cns). in contrast, gaba induces depolarization in the immature cns. this developmental shift from depolarization to hyperpolarization may be caused by decreasing of the intracellular chloride ion concentration regulated by two chloride ion co-transporters, na-k-2cl co-transporter 1 (nkcc1) and k-cl co-transporter 2 (kcc2). in this study, we focused on kcc2, which lowers the intracellular chloride ion concentration, and examined the developmental localization of the kcc2 with special reference to the neuronal development in the cerebellum. kcc2 was negative in the proliferating and migrating neurons. post-migratory neurons, which formed synapses, expressed the kcc2. the kcc2-protein was localized at the membrane of dendrites and cell bodies, whereas growth cones, axons and terminals were negative. these results suggested that formation of synapses might induce kcc2-expression and localization, and gabaergic transmission might shift from excitation to inhibition after synapse formation. akinori nakajima 1 , hisashi mori 1 1 molecular neuroscience, university of toyama, toyama, japan the actions of many neurotransmitters are mediated by the members of a superfamily of receptors coupled to heterotrimeric guanine nucleotide binding proteins (g-proteins). the dopamine receptors are classified into two categories, d1-like and d2-like according to their pharmacological properties. the d1-like receptors consist of d1 and d5 receptor, and are coupled to the adenylyl cyclase activating g proteins (gs). in the present study, we have generated a series of d1 receptor mutants and examined the effect on the gs coupled receptor signaling. we found that the expression of the third intracellular loop (3i loop) domain of d1r fused with egfp effectively reduce camp production mediated by d1 and d5 receptors. interestingly, we also identified that the 3i loop domain of d1r interfere with gs coupled beta2 adrenergic receptor signaling. these results suggest that the third intracellular loop of the d1 receptor is a primary determinant in its coupling to gs signaling. the activation of phosphatidylinositol-linked d1-like dopamine receptor profoundly suppresses the exaitatory transmission in the developing hippocampus yoshinobu noriyama 1 , yoichi ogawa 2 , hiroki yoshino 1 , masayuki yamashita 2 , toshifumi kishimto 1 1 dept. psychiatr.; 2 dept. physiol i, nara med. univ., kashihara, japan we studied the effect of dopamine (da) on gabaergic and glutamatergic transmission in neonatal rat hippocampus from the early period of synapse formation by whole-cell patch-clamp recordings from ca1 pyramidal cells. da (100 m) profoundly decreased gaba a receptor-mediated postsynaptic currents to 32% in the first postnatal week, when gaba provides excitatory drive. da also decreased ampa receptor-mediated excitatory post synaptic currents to 29% in the second postnatal week, when glutamate responses first appear. the da-induced inhibition declined after these periods. the receptor subtype involved in the da-induced inhibition was phosphatidylinositol (pi)-linked d1-like receptor, since skf 83959, a selective agonist for pi-linked d1-like receptor, clearly mimicked the action of da. these results suggest that the activation of pi-linked d1-like receptor profoundly suppresses the excitatory transmission during the early period of synapse formation in the developing hippocampus. ayuka ina 1 , jinko konno 1 , sachine yoshida 1 , hideki ohmomo 1 , hitoshi kawano 2 , fumihiro shutoh 1 , haruo nogami 1 , setsuji hisano 1 1 graduate sch., comprehensive human sci, univ. tsukuba, tsukuba, japan; 2 tokyo metro inst neurosci, tokyo, japan supporting critical neurobiological roles of glutamate in mouse corticogenesis, we recently reported that cortical cells express vglut1 or -2 mrna at early fetal ages. to know roles of fetal vglut in cortical development, we studied expressions of vglut proteins in mouse fetuses by immunohistochemistry. on embryonic day 13 (e13), vglut1 immunoreactivity (ir) was first detected in the marginal zone (mz), subplate (sp) and intermediate zone (imz). on e15, vglut1-ir was seen as puncta close to l1-ir thalamocortical fiber tracts in the sp and also localized to fiber tracts expressing l1or tag1-ir in the imz, whereas vglut2-ir was first observed in the sp and upper imz where l1-ir existed. these results show that vglut1ir corticofugal fibers appose to elongating vglut2-ir thalamocortical fibers, suggesting that vglut may play a crucial role in glutamatemediated axon guidance to determine thalamic innervation patterns in the developing cortex. ps1a-d054 developmental changes in the mechanism underlying activity-dependent swelling of the hippocampal ca1 regions michie kon 1 , yoichi avil 2 , hiroshi tsubokawa 1,2 1 dept. of information engineering, tohoku univ., sendai, japan; 2 grad. schl. of information sciences, tohoku univ. to investigate the mechanisms underlying swelling of brain cells in association with neuronal activity, we analyzed interactions between changes in cell volume and synaptic activities in mouse hippocampal slices. swelling of several areas within the ca1 region were detected as increases in transmittance of near infrared light (irt). field epsps (feps) were recorded simultaneously from the stratum radiatum of ca1 region. in adult mice, repetitive stimulation of afferent fibers induced transient increases in irt at both somatic and dendritic regions in a frequency-dependent manner, which was temporally associated with feps. application of the bicuculline, a gaba-a receptor antagonist, reduced these optical signals. however, in mice under 15 days old, the optical signals did not follow by high-frequency stimulation of inputs, and were not affected by an application of bicuculline. these results suggested that gaba-dependency in the mechanisms of cell volume regulation developmentally changes in the hippocampal ca1 region. in the present study, the effects of bilateral injections of glutamatergic agents into the hippocampal ca1 region on morphine-induced conditioned place preference (cpp) were investigated in rats. subcutaneous administration of different doses of morphine (2.5-10 mg/kg) produced a dose-dependent cpp. using a 3-day schedule of conditioning, it was found that intra-ca1 administration of nmda receptor antagonist, mk-801 (2 and 4 g/rat) significantly attenuated the morphine (7.5 mg/kg)-induced cpp. moreover, nmda receptor agonist, nmda (0.1, 0.2 and 1 g/rat) significantly potentiated the morphine (2.5 mg/kg)-induced cpp. these results suggest that the development of morphine-induced cpp may be related to nmda and mk-801 receptors in that the glutamatergic system can modulate opiate reward. takahiro sonomura 1 , kouichi nakamura 2,3 , hiroyuki hioki 2 , masanori uemura 1 , takeshi kaneko 2,3 1 dept. anatomy for oral sciences, grad. sch. med. and dent., kagoshima univ., kagoshima, japan; 2 dept. morphological brain sciense, grad. sch. med., kyoto univ., kyoto, japan; 3 crest, jst the majority of neostriatal neurons are medium-sized projection neurons with spiny dendrites and have so far been classified into three groups: striatonigral neurons producing ppd, and striatopallidal neurons producing ppe, and striatoinnominatal neurons producing pptb. these projection neurons are regulated in part by dopaminergic input from the substantia nigra pars compacta. it has been assumed that d1 receptor are expressed in striatonigral neurons and d2 receptor are expressed in striatopallidal neurons. in recent years, molecular cloning work has shown that there are at least five dopamine receptor genes (d1, d2, d3, d4, d5). in this study, the double-labeling method combining in situ hybridization and immunocytochemistry revealed how these five dopamine receptor subtypes are distributed among three projection neuron groups. the cerebellar tissue is good model system for the analysis of neuronal development, since dynamic neuronal development such as migration and axonal and dendritic outgrowth after birth. in the present study, we examined the localization of chondroitn sulfate proteoglycans (cspgs) by cspg-specific antibodies and lectins. cspgs are mainly observed at molecular layer in developing cerebellum (p10-15) but they scarcely seen at external granular layer. electron microscopic observation demonstrated that phosphacan, one of cspgs, is localized at axonal membrane of parallel fibers. moreover, phosphacan inhibited adhesion and axonal extension of cerebellar granular neurons, while it promoted axonal fasciculation of their aggregated cultures. thus, cspgs, inhibitory molecules for axonal extension, are participated in axonal guidance cue in developing cerebellum. the vasopressin neurons are well knwon to show structural plasticity during chronic physiological stimulation such as salt loading. in the present study, salt loading significantly diminished the levels of chondroitin sulfate proteoglycans (cspgs) in vasopressin neurons. this downregulation is possibly due to proteolysis by tpa, since (1) tpa immunoreactivity was observed at neurosecretory granules of vasopressin dendrites and terminals, (2) salt loading increased protein and mrna levels of tpa in the somata and dendrites in the supraoptic nucleus but reduced protein levels of it in the terminals of the neurohypophysis, (3) depolarizing agent released tpa from isolated neurosecretosomes, (4) tpa knockout mice revealed lower ability of osmotic homeostasis and vasopressin release. thus, it is probable that tpa is participated in regulating structural plasticity of vasopressin neurons by degrading cspgs. chondroitin sulfate (cs) proteoglycans are essential for neuronal morphogenesis, including neural migration, survival and neurite formation in the developing brain. cs chains are modified by various sulfotransferases generating diverse sulfation patterns, which are assumed to be involved in the selective binding to various proteins such as growth factors. in this study, we analyzed the expression patterns of several cs sulfotransferases in the developing mouse cerebrum. using in situ hybridization analysis, it was revealed that cs sulfotransferase mrnas (u2st, galnac4-6st, d4st) were expressed in various types of cells, especially in the ventricular zone, and the cortical plate neurons just below the marginal zone. immunohistochemical analysis with anti-cs antibodies revealed that cs were highly expressed in the ventricular zone and the marginal zone. these results suggest that the cs structural domains generated by these cs sulfotransferases are involved in the regulation of the proliferation of neural progenitor cells and neuronal migration. nobuaki maeda 1 , maki ishii 1 , isao nagata 2 , yumiko shimazaki 1 1 dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; 2 dept. of brain structure, tokyo metro. inst. for neurosci. chondroitin sulfate (cs) is a long polysaccharide with enormous heterogeneity that binds with various proteins in a structure-dependent manner. previously, we revealed that cs is involved in the morphogenesis of the purkinje cell dendrites. in this study, we analyzed the expression of cs in the postnatally developing cerebellum using monoclonal antibodies that recognize specific structural motifs in cs. among the epitopes recognized by these antibodies, the expression of mo-225 epitopes, glca(2s)␤1-3galnac(6s) (d unit)containing structures, remarkably increased during development. detailed immunohistochemical analysis indicated that d unit-rich cs was deposited between purkinje cell surface and the processes of bergmann glia. furthermore, it was found that pleiotrophin bound to d unit-rich cs on phosphacan distributed around purkinje cells. these observations suggest that d-type structure in cs is important for the signaling of pleiotrophin, which play roles in purkinje cell-bergmann glia interaction. nobuna fukazawa 1 , mineko kengaku 2 , nobuaki maeda 1 1 dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; 2 lab. for neuronal cell polarity, riken bsi, wako, japan ptp is a receptor-type protein tyrosine phosphatase, which is synthesized as a chondroitin sulfate proteoglycan that pleiotrophin-ptp signaling regulates the morphogenesis of purkinje cell (pc) dendrites. we previously revealed that ptp associated with delta/notchlike egf-related receptor (dner), which mediates the pc-bergmann glia (bg) interaction and regulates morphological differentiation of these cells. here, we found that ptp was expressed by both pcs and bgs and the expression by pc occurred at relatively late developmental stage. ptp showed patchy distribution in the dendritic shafts of pcs, which partially overlapped with the localization of dner. furthermore, we revealed that multiple tyrosine residues in the cytoplasmic domain of dner were phosphorylated and that these tyrosine phosphorylated residues were efficiently dephosphorylated by the ptp catalytic domain. these results suggested that ptp participate in the pc-bg interaction by regulating tyrosine phosphorylation level of dner. masahiko tanaka 1 , tohru marunouchi 1 1 division of cell biology, institute for comprehensive medical science, fujita health university, toyoake, aichi, japan cerebellar purkinje cells have the most elaborate dendritic trees among the neurons in the cns. to investigate the cellular and molecular mechanisms of dendrite development of purkinje cells, we cocultured purkinje cells on a coverslip with other cerebellar cells such as granule cells and astrocytes on the cell culture insert of 3 m pore size. when purkinje cells were co-cultured with granule cells, dendrite development of purkinje cells was promoted in comparison with that in control conditions. this co-culture effect was abolished by addition of a glutamate antagonist in the cultures. in contrast, dendrite development of purkinje cells was inhibited when purkinje cells were co-cultured with astrocytes. we propose that (i) glutamate secreted by granule cells and diffused through the porous membrane of the cell culture insert promotes the dendrite development of purkinje cells and (ii) astrocytes inhibit the effect of glutamate through their glutamate transporting activity. heparan sulfate (hs) proteoglycans regulate neural development through the interaction with cell surface proteins and extracellular matrix molecules. an extracellular endosulfatase, sulffp1, has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp1 gene, and examined the motor control. homozygotes appeared to be normal, showing no sign of ataxia. performances of the rotarod and beam-walking tests were normal compared with the control mice. both short-term and long-term adaptations in the optokinetic response were normal, while the gains in optokinetic response and vestibulocular reflex were significantly reduced. heparan sulfate (hs) proteoglycans regulate a number of developmental signaling through interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific sulfation patterns in hs, but the mechanism generating such modifications has not been fully elucidated. here we show that a new class of hs endosulfatases plays an important role in brain development. the mice deficient in either sulffp1 or sulffp2 appeared to be normal, while most of the double knockout mice died soon after birth. mutant brains had higher content of 6-o-sulfated disaccharide units in hs, suggesting a role of sulffps in heparan sulfate remodeling in vivo. the double mutant brains were smaller than the controls and showed some axon guidance defects. these data demonstrate that specific hs modification generated by sulffps is important for normal brain development. recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. here, we examined roles of 5-hydroxytryptoamine (5-ht), noradrenaline (na) and dopamine (da) in the formation of dendrites and synapses by dissociation culture. embryonic day 16 or 18 rat cerebral cortex was cultured in the presence of 5-ht, na or da. after 4 days, we analyzed dendrite formation using anti-map2 antibody. after 14-21 days, we analyzed synaptogenesis with anti-psd-95, anti-synaptophysin, and anti-map2 antibodies. the addition of 5-ht (100-10000 nm), na (100-1000 nm) or da (10-1000 nm) increased dendritic length of pyramidal neurons. 5-ht (100-1000 nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that dendritic outgrowth may be promoted by 5-ht 1a receptor, ␣ 2a receptor and d 2 receptor, while inhibited by 5-ht 2a and ␤receptors. in addition, synaptogenesis was promoted by 5-ht 2a and 5-ht 2c receptors, whereas inhibited by 5-ht 1a receptor. tatsuya mori, tomoe wada, takahiro suzuki, naoyuki inagaki department of cell biology, nara institute of science and technology, nara, japan most neurons have polarized shape consisting of a single long axon and multiple dendrites. several proteins have been implicated in the establishment of neuronal polarity; however, the mechanism for neuronal polarization is not well understood. in this study, with proteomic approach, we identified a novel protein, singar, as one of the proteins which are up-regulated during neuronal polarization of rat cultured hippocampal neuron. singar was expressed specifically in brain and developmentally up-regulated during neuronal polarization in vitro and in vivo. in 293t cell, singar associated with p85 and p110, the subunits of pi3kinase which is considered as one of the key molecules in neuronal polarization. moreover, inhibition of singar by rna interference induced the formation of multiple axon-like neurites. these data suggest that singar ensures the formation and maintenance of neuronal polarity by suppressing the formation of surplus axons. ps1a-e068 lrfn2, a neuronal leucine-rich repeatcontaining transmembrane protain can interact with psd-95 naoko morimura, takashi inoue, kei-ichi katayama, jun aruga laboratory for comparative neurogenesis, riken bsi, saitama, japan in a variety of organisms, proteins with leucine-rich repeat domain (lrr) function significantly in neural development. lrfn, a neuronal lrr transmembrane family, was expressed in the brain specifically. expression of lrfn2 was low in embryonic brain, and increased dramatically after birth. in the rat dissociated hippocampal neurons, lrfn2 protein was detected predominantly at mature dendrites, where it was accumulated at spines and colocalized with psd-95, a postsynaptic scaffold protein. we examined the physical interaction between lrfn2 and psd-95 by immunocrecipitation and pull-down assay, since lrfn2 contains class i pdz domain-binding motif at its c-terminal tail. we revealed that lrfn2 associated with psd-95/nmda receptor complex in the brain extracts and lrfn2 directly bound to psd-95 via its pdz domain-binding motif. in this study, we suggest that lrfn2 may play an important role in the regulation of synaptic functions. tsuya taneda, shingo miyata, hiroaki okuda, masaya tohyama department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan protein arginine methylation is a common post-translational modification catalyzed by a family of protein arginine n-methyltransferases (prmt1-8). among the prmt proteins, the prmt3 has some characteristic motifs in the n-terminal tract which follows its active methyltransferase site. although little attention has been paid to protein methylation in the nervous system. first of all, we have examined the distribution of the prmt3 in the rat brain. the prmt3 was expressed in the cell bodies and dendrites in the hippocampal neurons. further, the ontogenetic analysis revealed the prmt3 expression increased from the perinatal stages to the adulthood. these findings suggest that the prmt3 relates to the neural function in the young and adult brain. furthermore in order to study the role of the prmt3 in the brain, we tried to identify novel interacting proteins with the prmt3 in rat hippocampal neurons using tandem affinity purification assay coupled with mass spectrometry. ps1a-e070 proteomics of the growth cone: ii. the systematic immunostaining analysis of the growth cone proteins identified by the proteomic research motohiro nozumi 1,2 , michihiro igarashi 1,2 1 div mol cell biol, grad. sch. med dent sci; 2 trans-diciplinary res program, niigata univ., niigata, japan proteomics is a powerful method to understand the molecular composition of a given cell or a compartment of the cell. in the accompanying paper, we applied this method to the growth cone from the rat forebrain, and we identified more than several hundred proteins there. although the proteins have been determined using the powerful methods, the localization of each protein in the neuron should be confirmed; thus, we checked the immunostaining in the cultured rat cortical neurons. currently, we have already performed the immunocytochemistry concerning more than 150 identified proteins including cytoskeletal components, signaling molecules, receptors, and cell adhesion molecules. by quantitative analyzing the fluorescent intensity using the digital imaging, we classified the growth cone proteins into several groups. we have found more than twenty proteins specifically localized in the growth cone by this analysis. research funds: kakenhi 17023019; project-promoting grant from niigata univ ps1a-e071 netrin-1 is involved in the sensory axonal projection toward the spinal cord as a repulsive guidance cue tomoyuki masuda 1 , keisuke watanabe 2 , kazuhiro ikenaka 2 , katsuhiko ono 2 , hiroyuki yaginuma 1 1 dept. anat., fukushima med univ. sch. of med., fukushima, japan; 2 div neurobiol bioinfo, nat inst physiol sci, aichi, japan in higher vertebrate embryos, the ventral spinal cord exerts chemorepulsion for dorsal root ganglion (drg) axons to orient them toward their targets. netrin-1 is known to be a chemorepellent for a subset of axons, the role of netrin-1 for ventral spinal cord-derived repulsion is, however, unknown. by employing culture assays, we report here the involvement of netrin-1 in this repulsion. in the mouse embryo at e11, netrin-1 is expressed in the floor plate and the dermamyotome, and the netrin-1 receptor unc5c is expressed in drg neurons. we show that hek-cell aggregates secreting netrin-1 repelled chick e5 drg axons. moreover, using function-blocking antibody against netrin-1, we revealed the fact that netrin-1 plays an important role in ventral spinal cord-derived repulsion. together, these findings suggest that the ventral spinal cord repels drg axons by secreting netrin-1 to shape the initial trajectories of drg axons. research funds: grants-in-aid on priority area (c) (mecst 17590166 to t.m.) hitoshi maeda, masaki sakurai department of physiology, teikyo university school of medicine, tokyo, japan in the previous reports, we showed that in the early development, corticospinal synapses (cs) were formed widely in the spinal gray matter but those in the ventral side were eliminated later in an activity dependent manner. however, the property of postsynaptic cells to cs input is poorly understood. in the present study, we investigated the electrophysiological and morphological properties of the neurons that receive cs synapses in the acute spinal cord slices of neonatal rat. the postsynaptic neurons that were confirmed by the stimulation of the posterior funiculus, where the cs tract is located in rodents, were whole cell patch clamped and labeled by neurobiotin tm . responsive neurons are widely distributed in the p6 neonates, but the ventral neurons became unresponsive after p9. the majority of the ventral neurons are of multipolar type with large somata showing "repetitive" or "phasic" firing patterns; on the other hand, most of dorsal neurons have smaller somata and multipolar branches with "single" or "phasic" patterns. keisuke watanabe 1 , hirohide takebayashi 1,2 , kazuhiro ikenaka 1 , katsuhiko ono 1 1 div. neurobiol. bioinfo., natl. inst. physiol. sci., okazaki, japan; 2 dev stem cell biol. program, ucsf, usa netrin-1 is a long-range diffusible factor that exerts chemoattractive or chemorepulsive effects on developing axons growing to or away from the neural midline. however, it is not known whether netrin-1 also exerts chemoattractive effect on ventral-ward migrating dorsal interneurons in the developing spinal cord. to test this hypothesis, we examined dorsal interneuron migration in netrin-1 −/− background, using olig3-lacz knockin allele, which marks most of ventral-ward migrating dorsal interneurons. in the embryonic spinal cord of olig3 +/lacz ;netrin-1 −/− mice, ventral migration of olig3 cells was significantly impaired. furthermore, a netrin receptor, dcc was expressed in olig3-positive cells. these results suggest that netrin-1 exerts chemoattractive effects on ventral-ward migrating dorsal interneurons in vivo. netrin-g1 and netrin-g2 are vertebrate-specific membrane-anchored members of the unc-6/netrin family that have no affinity to classic netrin receptors and their function is unknown. here we show that netrin-g1 and netrin-g2 proteins are selectively distributed on axons of distinct pathways, and each interacts with a specific receptor on target dendrites. netrin-g1 and netrin-g2 differentially bind to lrrcontaining proteins, ngl-1 and a related molecule nag14, in vitro. ngl-1 and nag14 in the mouse brain are concentrated in distinct dendritic segments, corresponding to lamina-specific termination of axons expressing netrin-g1 and netrin-g2, respectively. furthermore, in netrin-g1 and netrin-g2 deficient mice, in which axonal pathfinding is normal, there is selective mislocation of individual receptors within dendrites. together, these results suggest that axonal netrin-g proteins transneuronally regulate the localization of distinct receptors on dendrites, and thereby determine the properties of subdendritic segments. jinhong huang 1 , ryuichi sakai 2 , teiichi furuichi 1 1 lab. molecular neurogenesis, brain science institute of riken, saitama, japan; 2 division of cell growth factor, national cancer center of japan, tokyo, japan cas is a tyrosine-phosphorylated docking protein that is indispensable for the regulation of actin cytoskeletal organization and cell migration in fibroblasts. the neuronal function of cas, however, is poorly understood. here we report that cas is dominantly enriched in the brain, especially the cerebellum, of postnatal mice. during cerebellar development, cas is highly tyrosine phosphorylated and is concentrated in the neurites and growth cones of granule cells. cas coimmunoprecipitates with src family protein tyrosine kinases, crk, and cell adhesion molecules. the axon extension of granule cells is inhibited by either rna interference knockdown of cas or overexpression of the cas mutant lacking the crk binding motifs. these results demonstrate that cas acts as a key scaffold to link the proteins associated with tyrosine phosphorylation signaling pathways to the granule cell axon elongation. research funds: huang was a postdoctoral fellowship recipient of jsps in in vitro cerebellum-pons-medulla block preparations isolated from neonatal rats on p4-p8, stimulation of parallel fibers produces excitation of purkinje cells lasting for 600-800 ms. this unusually prolonged response is observed in the lateral region of the cerebellum (paraflocculus/flocculus), where purkinje cells develop primary dendrites on p5-p7. since -agatoxin iva and -conotoxin mviic abolished the prolonged response, we suggest the involvement of p/q type ca channels. immunohistochemical labeling revealed that p/q type ca channels emerged in paraflocculus/flocculus and uvula/nodulus lobules on p4 and that they then locate in purkinje cells, in cell body on p5 and in primary dendrites on p6-p7. the parallel development of p/q type channels, primary dendrites, and the occurrence of prolonged parallel fiber-purkinje cell transmission suggests their causal relationships. naoya ichikawa, yasuo kitagawa, tatsuhiko kadowaki graduate school of bioagricultural sciences, nagoya university, aichi, japan we have recently identified a novel gene, mahya, which is specifically conserved between hymenoptera and deuterostome. mahya encodes a secretory protein with a follistatin-like domain, two immunoglobulin domains, and a c-terminal novel domain. mouse mahya genes (mmahya-1 and mmahya-2) are expressed in the olfactory bulb, hippocampus, and cerebellum of the adult brain. we have found that mmahya-1 protein is specifically synthesized in the pre-migratory granule cells and localized at the molecular layer of the postnatal cerebellum. these results suggest that mmahya-1 is involved in either the migration of granule cells or the dendritic maturation of purkinje cells. we will further report the analysis of the functions of mmahya-1 for the early cerebellum development. toshitaka morishima 1 , erina fukushi 1 , kazuto kobayashi 2 , naohiro hozumi 1 , sachiko yoshida 1 1 toyohashi university of technology, toyohashi, japan; 2 honda electronics co. ltd., toyohashi, japan in cerebellar development, granule cells migrate with elongation their axon, called parallel fibers, and form neuronal circuit in molecular layer. although density and thickness of parallel fibers are important information for cerebellar development, few were simple and useful methods. we have proposed a new method for two-dimensional acoustic impedance imaging for developing cerebellar slices. an acoustic impedance microscopy was obtained by mechanically scanning the transducer and the reflection intensity was interpreted into local acoustic impedance of no treated acute slices with no invasion. the developing parallel fibers were clearly observed as the contrast in acoustic impedance, whereas they were cloudy in immature egl from neonatal rat. the reflection from molecular layer enlarged and floated to deep layer, so that its spatial pattern was changed during cerebellar development. this imaging method is believed to be a powerful tool for observation of neuronal development, as neither fixation nor staining is required. tatsuro yamamoto 1 , hideyuki dekimoto 1 , tomiyoshi setsu 1 , masahiko watanabe 2 , mikio hoshino 3 , yo-ichi nabeshima 3 , toshio terashima 1 1 dept. of anat., kobe univ. grad. sch. of med., kobe, japan; 2 dept. of anat., hokkaido univ. grad. sch. of med., sapporo, japan; 3 dept. of pathol and tumor biol, grad. sch. of med., kyoto univ., kyoto, japan a mutant mouse, cerebelles (cbll), lacks the entire cerebellar cortex but survives into the adult. the responsible gene for this mutation is ptf1a, whose expression is lost in this mutant. in the present study, we examined cerebellar afferent and efferent systems of this mutant mouse by neural tracing methods with a combination of immunohistochemistry. the injection of fluoro-gold (fg) into the cbll thalamus resulted in retrograde labeling of neurons in the contralateral cerebellar nuclei. these fg-labeled neurons were glutaminase-positive. after the injection of bda into the cbll lumbar cord, spinocerebellar terminals projecting to the deep cerebellar nuclei were anterogradely labeled in spite of absence of the cerebellar cortex. these findings suggest that afferent and efferent systems of the cerebellar nuclei of the cbll are preserved in spite of absence of the cerebellar cortex. kumiko ishida 1 , tomoko nishiyama 1 , hitoshi tatsumi 1 , masahiro sokabe 1,2 1 department of physiology, nagoya university graduate school of medicine, nagoya, japan; 2 icorp, cell mechanosensing project, japan science and technology corporation sprouting and synaptic reorganization of the mossy fiber (mf) are commonly found in the hippocampus of temporal lobe epilepsy patients. as the muscarinic agonist, pilocarpine, can induce similar morphological changes, hippocampal slices treated with this drug have been widely used as a model of epilepsy. we found that pilocarpine induced a transient retraction and subsequent elongation of the neurites of granule cells in the slice cultures; the retraction was peaked approximately 12 h and the elongation started at approximately 24 h after the drug application. tetrodotoxin strongly inhibited both the retraction and elongation, while the bdnf sequestering protein, trkb/fc, retarded only the elongation. this result suggests that na + channel dependent neuronal excitation and following activitydependent bdnf releases are essential in the biphasic morphological changes induced by pilocarpine in hippocampal slices. rieko muramatsu, yuji ikegaya, maki k. yamada, norio matsuki, ryuta koyama laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, the university of tokyo hippocampal granule cells extend their axons, i.e. the mossy fibers (mfs), from the dentate gyrus (dg) to the area ca3. once this oneway projection is disrupted, the mfs retrogradely innervate granule cell dendrites and make excitatory synapses that induce epileptic neural activities in the dg. to clarify the mechanism that regulates normal, anterograde mf projections, we used a co-culture system of hippocampal slices. when a dg slice from a gfp(+) rat was juxtaposed to the ca3 region of a host hippocampal slice from a wild type rat, the gfp(+) mfs ran through the host ca3 toward the host dg but failed to invade it even after ten days in vitro. thus the dg seemed to serve as a barrier that blocks retrograde projections of mfs. however, the mfs extended into the dg when forskolin, an activator of adenylate cyclase, was chronically applied. these results suggest that the dg has a mechanism supporting anterograde mf projections to ca3, which is regulated by the levels of adenylate cyclase activation. calcitonin gene-related peptide (cgrp) is a 37 amino acid neuropeptide that is widely distributed in central and peripheral nervous systems. cgrp is expressed from early developmental stage in rat brain, suggesting that cgrp may be involved in not only neurotransmission but also neural development. but roles of cgrp in neuronal development of cerebral cortex and hippocampus remain unclear. in the present study, we made dissociation culture of cerebral cortex and hippocampus of embryonic day (e) 16 or e18 rat. dendritic outgrowth of pyramidal neurons was analyzed after 4 days using anti-map2 antibody. synapse formation was analyzed after 2-3 weeks, using anti-psd-95 and anti-synaptophysin antibodies. in the presence of cgrp (10-1000 nm), both dendritic length and synaptic density were increased. however, the number of dendritic branching was not affected. these results suggest that cgrp promotes dendritic outgrowth and synapse formation. chisako kanamaru, kazunori suda, kouji senzaki, takashi shiga university of tsukuba, graduate school of comprehensive human sciences, tsukuba, japan recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. in this study, we examined roles of 5hydroxytryptoamine (5-ht) and noradrenaline (na) in the formation of dendrites and synapses using dissociation culture of rat hippocampus. embryonic day 18 rat hippocampus was cultured in the presence of 5-ht or na. after 5 days, we analyzed formation of dendrites using anti-map2 antibody. after 21 days, we analyzed formation of synapses using anti-psd-95, anti-synaptophysin, and anti-map2 antibodies. the addition of 5-ht (500 nm) or na (500 nm) increased dendritic length and number of branches of pyramidal neurons, whereas decreased number of primary dendrites 5-ht (100-1000 nm) and na (10-1000 nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that ␣ 2a receptor promotes dendritic outgrowth, while ␤ receptor suppress dendritic outgrowth and branching. in addition, 5-ht 2a receptor and ␣ 2a receptor promote synapse formation. kenji amano down syndrome cell adhesion molecule (dscam) knock-out (ko) mouse died within 24 h after the birth. to investigate possible etiology of the neonatal death, we examined the respiratory activity using whole body plethysmography and the c4 inspiratory activity using brainstem-spinal cord preparation. the respiratory activity of dscam-ko mice using plethysmography was irregular frequency and small amplitude accompanied with apnea. furthermore, c4 inspiratory activity also showed irregular frequency and narrow duration of the bursting. we then analyzed spatio-temporal pattern of the respiratory neuronal activity using combination of the voltage-sensitive dye (di-2 anepeq) and the imaging system (micam02). in dscam-ko mice, the optical signal which precedes c4 inspiratory activity was depressed. these results suggest that pre-inspiratory neuronal network, which determines respiratory rhythm, does not develop normally in dscam-ko mice and causes lethal respiratory dysfunction. ps1a-f088 hippocampal cells cultured on 3d collagen substrate secrete a dense extracellular matrix, supporting neuritic outgrowth shantanu sur, thomas launey, masao ito brain sc. inst., riken, japan the brain extracellular matrix (ecm) influences neuronal migration and morphogenesis. we explored how hippocampal cells modify their extracellular environment when seeded onto collagen gel, a major component of the ecm. after 2 weeks in vitro, neurons formed a dense layer, >0.1 mm below the gel surface, with neurite outgrowth toward the surface, within the top gel layer (tgl). initially, we thought that hippocampal cells were penetrating the gel, following partial degradation of the collagen matrix. however, (1) collagenasespecific inhibitor did not affect cell depth, (2) limiting gliosis by antimitotics reduced the thickness of the tgl by 40%, (3), neither glial nor neuronal cell body were found in the tgl by gfap/map2 detection, (4) neurite outgrowth was observed only within this tgl, but not toward the bottom of the gel. to see whether the tgl is the remains of the initial collagen substrate, we embedded fluorescent beads in the collagen gel before cell seeding. the tgl was completely devoid of beads after 2 weeks, suggesting that the tgl is newly formed by ecm material, largely secreted by glial cells. emi kumamaru, tadahiro numakawa, yuki yagasaki, hiroshi kunugi disorder research, national institute of neuroscience, ncnp, tokyo, japan the level of glucocorticoid is regulated through hpa axis, and glucocorticoid itself has a negative feedback effect on hpa axis. however, under the intense stress, the glucocorticoid level is increased, and the high level of it is suggested to induce neuronal damage and to cause the mood disorder. on the other hand, it is possible that the reduction of neuronal function mediated by bdnf is partly related to the cause of the disorder. therefore, in the present study, we investigated the effect of glucocorticoid (dexamethasone, dex) on synaptic maturation and function enhanced by bdnf in early developing hippocampal neurons. we found that bdnf increased the expression of synaptic proteins including glutamate receptor and presynaptic protein, however, pretreatment with dex significantly inhibited the up-regulation of these proteins by bdnf. further, increase in release of glutamate and in intracellular ca2+ by bdnf was suppressed after dex pretreatment, suggesting that dex inhibits the maturation of synaptic function mediated by bdnf. takashi ueyama 1 , kazuto kujira 1 , tetsuya kawabe 2 , takao ito 1 , yoshihiro tsuruo 1 1 department of cell biology and anatomy, wakayama medical university, wakayama, japan; 2 department of cardiovascular medicine, wakayama medical university, wakayama, japan in this study, we investigated the effect of castration on the emotional stress response in the brain by comparing the c-fos expression in response to immobilization stress (imo) between castrated rats (cast) and sham-operated rats (sham). increased c-fos immunoreactive cells in response to imo were observed in septum, thalamus, hypothalamus, midbrain, pons and medulla oblongata in accordance with previous findings. in cast compared with sham, the numbers of c-fos-ir cells were significantly lower in the medial parvocellular part of paraventricular hypothalamic nucleus, while they were significantly higher in the supraoptic nucleus and medial amygdaloid nucleus. these data suggest that neuronal activity in these areas is influenced by systemic androgen level. this may underlie the pathophysiology of partial androgen deficiency in aged men (padam). research funds: grant-in-aid for scientific research (c) (17590600) ps1a-f091 metabolic and glucagon response of a genetically heat-tolerant rat to ambient heat and cold fujiya furuyama 1 , hitoo nishino 1 , takehiro yahata 2 1 nagoya city university graduate school of medical sciences, nagoya, japan; 2 nayoro city college, nayoro, japan the inbred fok rat was developed by us using heat selection and inbreeding for generations. fok rats avoided serious multisystem disorders caused by heat stroke and by extreme dehydration. saliva spreads widely over the whole ventral body surface in fok rats. however, no strain difference was not found in vitro in the salivation rate, suggesting exsisting of a negative feedback loop between the central thermoregulation system and evaporation system. on the other hand, body temperature of the fok rats did not decreased in a extream cold environment as those in control rat strain. thermogenesis induced by cold in fok rats was larger than those in control rat strains. the larger increase in thermogenesis was partly attributable to glucagon-induced thermogenesis in brown adipose tissue. blood levels of triglryceride was lower, but polyunsaturated fatty acids were higher in fok rats than those in control rat strains. these changes can be considered to be results of genetically acquired heat-tolerance. oxidative stress is involved in the degeneration of nigrostriatal dopaminergic system in parkinson s disease (pd). vitamin e is a potent antioxidant, and its retention and secretion are regulated by alpha-tocopherol transfer protein (ttp) in brain. dysfunction of ttp has been shown to result in systemic deficiency of vitamin e in human and mice. in the present study, we using the ttp knockout mice, investigated the effect of vitamin e deficiency in pd development by generating mptp mouse model of pd. we confirmed that vitamin e depleted in the brain of ttp knockout mice completely. while the mptp treatment decreased striatal dopamine in the all three ttp genotypic groups, there were no significant differences among them. our results suggest that vitamin e does not play a major protective role in mptp-induced nigrostriatal dopaminergic neurodegeneration in the brain. priyanka dikshit 1 , anand goswami 1 , nobuyuki nukina 2 , nihar ranjan jana 1 1 national brain research centre, india; 2 laboratory for structural neuropathology, riken brain science institute, 2-1 hirosawa, wakoshi, saitama 351-0198, japan a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins, often associated with various chaperones and proteasome components. but, how the polyglutamine proteins are ubiquitinated and degraded by the proteasome is not known. here, we demonstrate that the expanded polyglutamine proteins that are misfolded, become ubiquitinated. secondly, we identified chip ubiquitin ligase that is able to target polyglutamine expanded huntingtin and ataxin-3 for the misfolding-dependent ubiquitination and degradation by the proteasome. the over expression of chip reduces the aggregate formation and cell death mediated by expanded polyglutamine proteins and the suppressive effect is more prominent when chip is over expressed along with hsc70. finally, we show that the expression of chip is increased in the expanded polyglutamine protein expressing cells. hypothalamic-pituitary-adrenal axis is central to the regulation of stress response. for the comprehensive detection of genes responsive to stress, we identified and catalogued the entire partial complementary dna sequences (expressed sequence tags (ests)) from rat hypothalamus. we have identified the total of 11,092 ests (5,442 non-redundant sequences). 2858 of them matched known genes of rodents in the genbank databases, but 2584 remained unknown. now we classified a full set of hypothalamic ests on the basis of their functional domains. complete profile of them will be presented in the meeting. these ests will also be applied to a cdna microarray for stress experiments. the present study will provide a refined genomic resource for molecular studies of animal models of stress-related disorder. research funds: grants-in-aid from the ministry of health, labor and welfare shinya yanagita, seiichiro amemiya, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, japan our previous study suggests that acute running is one stressor activating corticotropin-releasing hormone (crh) neurons in the hypothalamic paraventricular nucleus (pvn). many studies have reported that several weeks of voluntary running improved stress tolerance during non-exercise stress. it is, thus, possible that housing in cages attached running wheel can alter activation of stress-related neurons during acute running. in this study, we examine the effects of 0, 2, or 4 weeks prior wheel running (i.e. housing in the cages attached running wheel) on activation of stress-related neurons, such as pvn, central nucleus of amygdala (cea), locus coeruleus, dorsal raphe, ventral tegmental area (vta), and prefrontal cortex during acute running using immunohistological methods in rats. prior wheel running altered activation of various stress-related neurons during acute running, especially markedly decreased activation of cea, and increased that of vta. these results suggest that prior wheel running influences stress-related neuronal activity during acute running. ps1a-f096 transforming growth factor-␤ in the brain regulates fat metabolism during exercise kazuo inoue, toma ishikawa, wataru mizunoya, tetsuro shibakusa, tohru fushiki division of food science and biotechnology, graduate school of agriculture, kyoto university, kyoto, japan we have previously reported that the concentration of transforming growth factor-␤ (tgf-␤) increases in the cerebrospinal fluid of rats during exercise and that an increase in fat oxidation was observed following intracisternal administration of tgf-␤. these results led us to postulate that tgf-␤ in the brain regulates the enhancement of fatty acid oxidation during exercise. to test this hypothesis, we carried out respiratory gas analysis during exercise while inhibiting the effect of tgf-␤ in the brain using intracisternal administration of anti-tgf-␤ antibody or sb-431542, an inhibitor of the type 1 tgf-␤ receptor (t␤r1). we found that each reagent blocked the increase in fatty acid oxidation. these results suggest that brain tgf-␤ has a role in enhancing fatty acid oxidation in peripheral tissues during endurance exercise, and this regulation is executed at partly via the t␤r1 signal transduction system. yoshii takanobu it has been demonstrated that vasopressin (avp) might play a role in anxiety-related behavior. we hypothesized that traumatic stress changes avp activity and avp contribute to the symptom of ptsd. we carried out in situ hybridization (ish) for avp mrna expression and avp immunohistochemistry (ihc) with an experimental paradigm of single prolonged stress (sps) as ptsd model. sd male rats were exposed to sps (2 h restraint; 20 min forced-swimming; ether anesthesia) then they were put in untouchable situation for 7 days. avp mrna expression significantly decreased in the son. ihc showed no significant change in avp-ir, but after additive stress (forced swimming 15 min), avp-ir in the son was significantly diminished. we considered that the stress decrease avp synthesis, but has little effect to the storage of avp. mumeko tsuda, takaaki ozawa, aosa fukushi, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan neonatal maternal separation (ms) is known to affect anxiety and fear responses in adult whereas its effect on socio-sexual behaviors is not fully understood. in the present study, we examined the effect of ms on an array of emotional and socio-sexual behaviors in both sexes of c57bl/6j mice. pups were separated from mothers daily (3 h) on postnatal days 1 through 14. starting at 13 weeks of age they were tested for (1) emotionality and anxiety levels in open field (oft), light-dark transition (ldt), and elevated plus maze tests; (2) responses to social stimuli in social investigation (sit) and social preference tests; and (3) socio-sexual behaviors in aggressive and sexual behavior tests. overall, there was no apparent effect of ms on behaviors measured in the oft and ldt except for higher levels of exploration in the ms group compared to the non-stressed (ns) group in both sexes. during the sit, social investigation time and general activity in ms females were much lower than those in ns females suggesting ms females may be more fearful to social stimuli. in the present study, we investigated the effect of environmental stress applied during perinatal period on spatial learning activity of mouse evaluated by morris water maze test. mice were exposed to the noise of 90 db (so), or were forced to swim (sw). these manipulations were performed for 15 min once a day at 2 weeks after birth (from postnatal days 14 to 18) or 3 weeks after birth (from postnatal day 21 to 25). normal mice were left undisturbed (no). the spatial learning activity was tested at the age of 6 weeks. it was found that the spatial learning activity of both so and sw mice manipulated 2 weeks after birth was impaired as compared to no mice. so mice manipulated 3 weeks after birth exhibited the same learning behavior as no mice, while that of sw mice manipulated 3 weeks after birth was impaired. present results indicated that the effect of the environmental stress on the learning activity of the adolescent mice might be dependent on the period of the stress manipulation. kin-ya kubo 1 , yukiko yamada 2 , mitsuo iinuma 2 , yasuo tamura 2 , fumihiko iwaku 1 , kazuko watanabe 3 , minoru onozuka 4 1 dept. oral anat., asahi univ. sch. dent., japan; 2 dept. ped. dent., asahi univ. sch. dent.; 3 dept. physiol., gifu univ. sch. med., japan; 4 dept. physiol. and neurosci., kanagawa dent. coll., japan recent studies have suggested that occlusal disharmony is related to temporomandibular arthorosis and braxism, which may come from a hypothalamic-pituitary-adrenal (hpa) axis. in addition, aged mice with masticatory dysfunction show deficits in spatial memory, being due to various pathological changes in the hippocampus, suggesting the link between malocclusion induced by abnormal occlusion and hippocampal pathology. in this study, to prove this hypothesis, we examined the effect of this malocclusion on plasma corticosterone levels, the numbers of hippocampal neurons and spatial performance in water maze in samp8 mice. this treatment age-dependently advanced a decline in spatial memory, an increase in plasma corticosterone levels, and a decrease in neuron density in the hippocampal ca3 region. the results suggest that abnormal occlusion may progress hippocampal neuron loss via stress, thereby leading to senile deficits in memory. yurie nakamoto, go mugishima, mitsuko sato, masako miwa, mitsunobu yoshii division of psychobiology, tokyo institute of psychiatry, tokyo, japan it has been shown that pbr are increased after acute stress and decreased under chronic stressful conditions. in our previous studies, expression of pbr was significantly correlated with trait anxiety in normal human subjects, which might reflect polymorphism of the pbr gene. in addition, males appeared to have higher pbr densities than females in their prime lives. the present study was designed to analyze these sexual differences in rats. blood samples were obtained from adult male and female slc wistar rats immediately after acute random electrical footshock and also from these animals after chronic social isolation (for 12 weeks after weaning). in naïve, male rats expressed higher densities of platelet pbr than females. chronic social isolation caused a marked increase in platelet pbr in male rats compared to female. the results indicate that pbr responses to environmentally induced stress are much less in female, probably under the influence of estrogen. kanako tambara 1 , yayoi kitamura 1 , junichi tanaka 2 , yukio hattori 1 , yasushi hayashi 1 1 department of human nutrition, notre dame seishin university, okayama, japan; 2 department of curriculum, teaching and memory, naruto university of education, tokushima, japan we investigated the effects of exogenous putrescine on stressinduced hyperthermia (sih) in male c57bl/6j mice after systemic injection of putrescine to clarify the role of brain putrescine in stressful conditions. in addition, we examined the effects of spermidine, spermine, and the anxiolytic diazepam on sih. the rectal temperature of singly housed mice was measured twice at a 10-min interval, to measure the basal temperature (t 1 ) and stress-enhanced temperature (t 2 ), respectively. the difference ( t = t 2 − t 1 ) gives the sih. in control mice, t was approximately 1 • c. pretreatment with diazepam caused dose-dependent inhibition of the sih. similarly, putrescine reduced t, although it caused a dose-dependent decrease in t 1 . furthermore, spermidine and spermine also lowered t and t 1 at doses lower than that of putrescine. these results suggest that endogenous brain putrescine and other polyamines have an anxiolytic-like effect in stressful conditions. eriko iguchi, yasuhiro tanaka, toshiyuki matsuoka, shuh narumiya department of pharmacology, kyoto university, kyoto, japan prostaglandins (pgs) are synthesized in many organs including the brain. of their synthesis, the rate limiting step depends on cyclooxygenase (cox), which has two subtypes, cox-1 and cox-2. it has been known that, under some stressful conditions, cox-2 is induced in some neurons and increases pgs production. but the roles of the increased pgs under stress are not fully elucidated. in this study, we restrained mice in small tubes individually for 6 h and subjected them to the elevated plus maze task 24 h later. these mice showed more anxiety. immunohistochemistry showed significant induction of cox-2 by restraint in some parts of the brain, such as cerebral cortices and amygdala. next, we examined the effect of indomethacin on this stress-induced anxiety. indomethacin is expected to reduce pgs production. mice treated with indomethacin stayed on open arms longer than control mice. these data suggest that pgs synthesized during stress may have anxiety-increasing effect. ps1a-g104 imaging brain and immune association accompanying cognitive appraisal of acute stressor to investigate association between brain and immune systems accompanying cognitive appraisal of an acute stressor, we recorded 15o-water positron emission tomography, cardiovascular, neuroendocrine, and immune indices, when 11 male subjects conducted a mental arithmetic task in a high controllability (hc) condition and a low controllability (lc) condition. activation in the orbitofrontal (ofc) and medial prefrontal (mpfc) cortices was observed in the lc compared to the hc. furthermore, significant correlations between brain activation and hr, hrv, bp, and nk cells were found commonly in the ofc in the lc, but not in the hc. thus, the ofc is a pivotal region for top-down regulation over immune activity accompanying cognitive appraisal on a stressor. wei zhang 1 , takesi sakurai 2 , yasuitirou fukuda 3 , tomoyuki kuwaki 1,3 1 dept. molec. integ. physiol., chiba univ., japan; 2 dept. pharmacol., univ. tsukuba, japan; 3 dept. autonom. physiol., chiba univ., japan we have previously proposed that orexin plays as a master switch to elicit multiple efferent pathways of the defense response. it is still open question, however, how information of stressor activates the orexinergic neurons. in this study, we examined possible afferent nuclei to activate orexinergic neurons. in urethane-anesthetized mice, a gaba-a receptor antagonist, bicuculline, was microinjected into the amygdala or the bed nucleus of stria terminalis (bnst), of which electrical stimulation induced simultaneous increases in blood pressure, heart rate, and respiration. bicuculline dose-dependently induced cardiorespiratory excitation in both orexin neuron-ablated and wild-type mice. however, dose-response curve was rightward shifted in the former. we conclude that the amygdala and bnst constitute one of the afferent pathways to the orexinergic neurons that involved in the defense response against stressor. in this study, developmental changes of anxiety behavior as well as myelin formation were investigated in male balb/c mice. the early-weaned mice had lower number of entries to the open arms of elevated plus maze at the age of 3-8 weeks, indicating persistent higher anxiety. high performance thin layer chromatography analysis was conducted for amygdaloid galactosyl ceramide, which is a typical lipid of myelin. the early-weaned mice had higher levels of galactosyl ceramide at the age of 5 weeks, and an electron microscopic study suggested increased number of myelinated axon and reduced diameter of myelinated axon in the basolateral amyglaloid nucleus. these results suggest that the early weaning induces precocious myelin formation in the amygdale between 3 and 5 weeks of age, which would be related to higher anxiety state in the early-weaned mice. research funds: sasakawa sci. res. grant takefumi kikusui, yuji mori veterinary ethology, university of tokyo, tokyo, japan we previously reported that early-weaned mice developed persistent increase in anxiety as well as aggression. in this study, developmental changes of brain derived neurotrophic factor (bdnf) protein levels were investigated in early-weaned icr mice. the early-weaned male and female mice had lower number of entries to the open arms of elevated plus maze at the age of 3 weeks, and this change was persistently observed in males. concurrently, the early-weaned males showed decrease of bdnf in the prefrontal cortex between 3 and 8 weeks of age, and in the hippocampus at the age of 3 weeks. however, there was no difference of bdnf expression in females. in addition, the early-weaned males, but not females, showed reduced brdu immunoreactivity in the dentate gyrus. these results suggest that the deprivation of mother-infant interaction during the late lactating period augments the anxiety in the adulthood by decreasing the level of bdnf in the pre-limbic system, and that these stress responses are sexually dimorphic, i.e., male is more vulnerable to early weaning stress. research funds: kakenhi #14760187 ps1a-g108 a systematic analysis of genetic factors associated with behavioral diversity between msm and c57bl/6 koide tsuyoshi 1,2 , aki takahashi 1,2 , toshihiko shiroishi 2,3 , akinori nishi 1 1 mgrl, national institute of genetics, mishima, japan; 2 sokendai, hayama, japan; 3 mammalian genetics lab, nig, mishima, japan in the previous study conducting a multi-phenotype behavioral tests, we observed a great difference of the behavioral phenotype between mouse strains, msm and c57bl/6. in order to elucidate a genetic factors underlying the behavioral difference, we analyzed a series of consomic strains which are made by replacing one of the chromosomes with that of msm strain. the behavioral data clearly indicated involvement of multiple genetic factors for each behavioral phenotype. one of the consomic strains, b6-6cmsm, which carries chromosome 6 of msm, showed extreme behavioral differences from c57bl/6. the strain showed lower activity in home cage and novel cage, and showed decreased number of transition in the light dark box test. by conducting analyses of composite interval mapping and a series of sub-consomic strains, we successfully identified genetic loci for the behavioral phenotype. tomoko soga 1 , yu kajiyama 2 , shigenobu shibata 2 , hiroshi kunugi 1 1 department of mental disorder research, national institute of neuroscience, center of neurology and psychiatry, tokyo, japan; 2 department of electrical engineering and bioscience, waseda university the hypothalamus-pituitary-adrenal (hpa) axis plays an important role in the pathophysiology of depression. alterations of brain derived neuronal factors (bdnf) have been implicated in depression. we examined the effects of synthetic glucocorticoid (dexamethasone; dex) on emotional behavior and gene expression of hpa-related molecules and bdnf in mice. dex treatment for 4 days after birth showed a significant decrease in locomotor activity and a significant rise in the time of immobility during forced swimming test. dex treatment to mature mice resulted in significant decrease in the number of entries into the open arm during elevated plus maze test. there was no change in gene expression of hpa-related molecules in dex-treated group. bdnf gene expression decreased significantly in dex-treated group, which showed behavioral abnormalities. our results lend further support for the involvement of glucocorticoid and bdnf in depression-related behavior. sachiko chikahisa, hiroyoshi sei, atsuko sano, kazuyoshi kitaoka, yusuke morita department of integrative physiology, the university of tokushima graduate school, tokushima, japan music is known to be able to elicit emotional changes including anxiolytic effect. the gonadal steroid hormone estrogen (e2) has been associated with anxiety levels. in this study, we examine whether the effect of music on anxiety is related with ovarian steroid in female mice. behavioral paradigms measuring anxiety (open field, elevated plus maze, dark-light transition and marble burying test) were tested in gonadally intact (sham-operated) and ovariectomized (ovx) female mice treated with placebo (ovx + placebo) or chronic estradiol (ovx + e2) replacement. in three behavioral tests except for open field, sham-operated mice exposed to music showed less anxiety than those exposed to white-noise and silence, while ovx + placebo mice did not show these effects at all. ovx + e2 mice showed the anxiolytic effect of music only in the marble burying test. these results suggest that exposure to music reduce anxiety levels, and ovarian steroids may be, at least partially, involved in the anxiolytic effects of music observed in female mice. tatsuhiro yasuda free, tokyo, japan strength and periodicity of periodical air pressure ascent around ones' ears induced by others' respiration may impact upon ones' awaken level, i.e. cognition. the air vibration acts upon tympanic membrane and then cochlear receptor stereocilia transforms it to neural signals which are sent via cochlear nucleus to inspiration nucleus in the medulla, and inspiration is induced. simultaneously afferent signals generated by external intercostals contraction are forward to medulla, thalamus and cortical areas. the stimuli with larger strength and periodicity compared to ones body size yields to auditory startle reflex. continuation of this may induce hyper ventilation or tension. if the input may be lasting with smaller strength and periodicity, insufficient diaphragm activity after hypoxia and gasping fade-out may induce afferent signal shortage that shrinks various cortical neural activity. lasting this situation may fall into depression. suitable timing of inspiration inducing may keep good mood, strong motivation and effective cognition. body system is suggested to own inherent observer that detects alerting or safe state so called homunculus. kenichi sasaguri 1 , takero in general, it has been proposed that the mandibular retrusive position resulted from either malocclusion or inadequate occlusal reconstruction is one of the causes of indefinite complaint. we determined whether the malocclusion model influences brain activities by using fmri study. the results indicated that in some of volunteers, significantly bold signals in the hypothalamus and the amygdala, being associated with emotion and/or stress increased during clenching. it is, therefore, suggested that malocclusion influences the whole body through emotional system, thereby causing the indefinite complains. ps1a-g113 synaptic organization between the amygdaloid axon terminals and the parvicellular reticular formationprojecting neurons in the retrorubral field of the rat toshiko tsumori, yi qin, shigefumi yokota, tatsuro oka, yukihiko yasui dept. anat. & morphol. neurosci., shimane univ. sch. med., izumo, japan the retrorubral field (rrf) is known as one of the areas containing numerous dopaminergic neurons in the midbrain. in the present study, we showed that the axon terminals from the central amygdaloid nucleus (ace) made synaptic contacts with non-dopaminergic rrf neurons sending their axons to the parvicellular reticular formation (rfp), where many premotor neurons projecting to the orofacial motor nuclei have been well known to exist. the ace axon terminals, which usually contain small pleomorphic vesicles and occasionally contain both small pleomorphic vesicles and large dense-cored vesicles, formed symmetrical synapses with cell bodies and dendrites of the rfp-projecting rrf neurons. moreover, most of these axon terminals showed glutamic acid decarboxylase immunoreactivity. the present study suggests that the ace exerts inhibitory influences upon the non-dopaminergic rfp-projecting rrf neurons to control orofacial movements closely related to emotional behavior. research funds: kakenhi (15500238) ps1a-g114 involvement of nr2b tyrosine-phosphorylation in emotional responses mediated at the amygdala mina delawary 1 , takanobu nakazawa 1 , yuji kiyama 2 , toshiya manabe 2 , tadashi yamamoto 1 1 div. of oncology, ims, univ. of tokyo, tokyo, japan; 2 div. of neuronal network, ims, univ. of tokyo, tokyo, japan nr2b is tyrosine-phosphorylated, with tyr-1472 being its major phosphorylation site. to investigate the role of tyr-1472 phosphorylation, we generated mice with a tyr1472phe knock-in mutation (yf/yf mice). in the elevated plus-maze test, time spent in open arm was reduced in yf/yf mice as compared to that in wild-type mice. similar phenotype was seen in the corticotropin-releasing factor (crf) overexpressing mice. this phenotype of yf/yf mice was canceled by the administration of crf receptor antagonist. as expected, in yf/yf mice, the expression level of crf in the amygdala was increased compared with that in wild-type mice. in the slice of amygdala from wild-type mice, nmda application induced de-phosphorylation of tyr-1472 and up-regulation of crf mrna level. given that crf is important in emotional responses, these data strongly argue that phosphorylation of nr2b is involved in the control of emotional responses by regulating crf content. ps1a-g115 increase in anxiety in transgenic mice overexpressing camkii in forebrain previous studies have shown that ␣calcium/calmodulin dependent protein kinase ii (␣camkii) plays important roles in aggressive and fear response in mice. to understand roles of alpha camkii in emotional behaviors, we have generated transgenic mice overexpressing ␣camkii in forebrain. because these mutant mice showed increase in anxiety in open field and elevated zero maze tests, we here examined effects of administration of selective serotonin reuptake inhibitor (ssri) on anxiety-related behavior of these mutant mice. treatment with ssri suppressed anxiety-related behavior of camkii mutant mice, suggesting that camkii mutant mouse is a mouse model of anxiety disorder. to investigate the mechanisms for increase in anxiety led by overexpression of camkii, we next compared the expression profiles between wild and mutant mice using dna micro array. these mutant mice showed abnormal changes in expression levels of genes related to ca 2+ signal transduction in hippocampus. yumiko ikeda, katsunori kobayashi, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan environment is known to influence behavior of animals. however, cellular and synaptic mechanisms underlying behavioral changes by environment remain largely unknown. we examined effects of changes in environment on locomotor activity and mossy fiber (mf) synaptic transmission in hippocampal slices. in mice housed in enriched condition for 5 weeks, locomotor activity and longinterval (200, 1000 and 5000 ms) paired-pulse facilitation (ppf) at mf synapses were reduced. in contrast, in mice housed in isolated condition for 5 weeks, there was no detectable change in either the total ambulation distance or the magnitude of ppf. we compared properties of the mf synaptic transmission with the locomotor activity in individual mice used in all experiments and found that the magnitude of synaptic potentiation induced by dopamine was negatively correlated with the ambulation distance. our results suggest that the modification of the hippocampal mossy fiber synaptic transmission could be involved in the environmental regulation of locomotor activity. yilong cui 1,3 , yosky kataoka 1,2 , yasuhisa tamura 3 , yasuyoshi watanabe 2,3 , hisao yamada 1 1 department of anatomy and cell science, kansai medical university, osaka, japan; 2 department of physiology, osaka city university graduate school of medicine, osaka, japan; 3 molecular imaging research program, riken frontier research system, saitama, japan during long-term intracranial self-stimulation (icss; electrical stimulations to the hemi-lateral medial forebrain bundle of rats by their lever pressing behavior at 50-60 times/min), inhibition periods (less than 10 times/min) were often observed 2 h after start of icss. we have been demonstrated that the inhibition was not induced by thermal effect on the neural function or by muscular fatigue. furthermore, the inhibition period was significantly decreased by pre-treatment with ns-398, a selective cox-2 inhibitor. these observations indicate that the arachidonic acid cascade is involved in inhibition of long-term icss and would be in weariness or fatigue sensation. male bluegill, lepomis macrochirus, is known to display alternative reproductive tactics. "parental" males defend nests and provide parental care, and "satellites" or "sneakers" are non-nesting, attempting to achieve parasitic fertilizations via sperm competition. in teleost and other non-mammals, arginine vasotocin (avt), the homologue of mammalian avp, is known as an important hypothalamic peptide involved in the alteration of reproductive behavior. behavioral evaluation and immunohistochemical study in preoptic area (poa) were conducted in parental and satellite bluegills to clear the role of avt in teleost reproductive tactics. parentals displayed more aggressive and courtship behavior than satellites and satellite males had significantly more cells than parentals, while the size of avt cells showed no difference between the male morphs. these results suggested that hypothalamic avt might play some part in the central control of reproductive behavior in teleost. ps1a-g119 impulsive choice in domestic chicks: context dependence and dissociation between delay and handling cost toshiya matsushima 1 , naoya aoki 2 , andras csillag 3 1 biology, hokkaido univ., sapporo, japan; 2 agriculture, nagoya univ., nagoya, japan; 3 anatomy, semmelweis univ., budapest, hungary choice between small/immediate reward and large/delayed reward has been widely used as a behavioral measure of impulsiveness. to study how ecological factors shaped underlying neural processes, we examined week-old chicks in four different tasks with identical economical consequences. in task 1, chicks chose between small reward (one pellet) delivered immediately and large reward (six pellets) after a delay up to 3 s. in task 2, chicks chose between small reward located at 20 cm and large reward at −140 cm, where cues signaled the distance of invisible food. task 3 was similar to the task 2, except that cues signaled the food quantity. in task 4, total handling time differed due to lowered food accessibility, while the delay was kept identical. lesion experiments revealed that ventral striatum was specifically involved in choices based on anticipated proximity (but not quantity), whereas arcopallium (association cortex analogue) in choices based on anticipated handling cost. research funds: kakenhi (15370033, 17021018) ps1a-g120 analysis of the brain regions associated with the dance language of the honeybees taketoshi kiya, takekazu kunieda, takeo kubo dep. biol. sci., univ. tokyo, tokyo, japan social animals have highly developed communicative abilities. the worker honeybees (apis mellifera l.) can transmit location of food sources by the dance language. in spite of the simple structure of the honeybee brain and the stereotyped dance behavior, its neural mechanisms remain totally unknown. previously, we found active brain regions in the dancing workers (dancers) by using a novel immediate early gene, kakusei, as a marker for neural activities and found its prominent expression in the small-type kenyon cells (skcs) of the mushroom bodies. here, we report that kakusei was similarly expressed in the skcs of the foraging workers (foragers), which do not always show the dance behavior. in contrast, the skcspreferential kakusei expression was not observed in the brains of the orienting workers, which were flying to learn the hive location. these results imply that the activities of the skcs in the dancer brain are neither due to dance presentation itself nor sensory inputs during foraging, but complex information processing accompanying the foraging behavior. c. elegans wild type animals are usually attracted to nacl, but show avoidance behaviors after being conditioned with nacl and starvation (food−/nacl+). this behavioral plasticity is not induced under the food−/nacl− or food+/nacl+ conditions. we isolated learning-defective mutants including pe401, which had a missense mutation in the casy-1 gene. several casy-1 deletion mutants also showed learning defects. casy-1 has an extensive similarity to human calsyntenin/alcadein, which is a single-pass transmembrane protein with cadherin-like repeats localized to the postsynaptic membrane of cns synapses. alcadein forms a stable tripartite complex with app and x11l/mint2. however, after dissociation of x11l, alcadein is susceptible to cleavage by protease(s). we found that casy-1 was expressed mainly in neurons and functioned at the adult stage. we are now investigating the localization pattern of the gfp-tagged protein, and whether casy-1 can also be proteolytically cleaved. ps1a-g122 insulin-like signaling is required for association between temperature and feeding state in c. elegans eiji kodama 1 , atsushi kuhara 1 , akiko mohri 1,2 , kotaro kimura 1,3 , masatoshi okumura 1 , masahiro tomioka 4 , yuichi iino 4 , ikue mori 1,5 1 div. of biol. sci., nagoya univ., japan; 2 present address: univ. of texas, health sci. cent., usa; 3 present address: natl. inst. of genet., japan; 4 mol. genet. res. lab., univ. of tokyo, japan; 5 inst. for advanced res., nagoya univ., japan c. elegans can associate cultivation temperature with feeding state. mutations in ins-1 encoding insulin homologue caused defective associative learning, mutations in daf-2 and age-1 encoding the homologues of insulin receptor and pi 3-kinase, respectively, suppressed the defect of ins-1, and the mutation in daf-16 encoding forkhead transcriptional factor caused the learning defect. this suggests that ins-1 antagonizes daf-2 insulin-like signaling for associative learning. interestingly, age-1 animals associate their cultivation temperature with feeding-state quicker than wild type. this defect was rescued by expressing age-1 in some head interneurons. in addition, the activity of these interneurons were down-regulated by starvation through ins-1. we suggest that insulin-like signaling modulates the neuronal activity of interneurons essential for associative learning. ps1a-g123 analysis of ttx-8: novel thermotaxis gene conserved among various organisms akiko miyara, akane ohta, yoshifumi okochi, masatoshi okumura, ikue mori laboratory of molecular neurobiology and institute for advanced research, nagoya university, nagoya, japan c. elegans can memorize the food condition in relation to the cultivation temperature and migrate to the cultivation temperature when looking for the food. this response to temperature is called thermotaxis. several neurons and genes required for thermotaxis have been identified, but molecular mechanism of thermotaxis is still poorly understood. the ttx-8(nj21) and ttx-8(nj34) mutants are obviously defective in thermotaxis and partially defective in chemotaxis. we revealed that ttx-8 encodes novel protein and is expressed in many neurons and functions in several neurons responsible for the thermotaxis behavior. the predicted protein structure of ttx-8 is similar to ric-3, identified in c. elegans at first and conserved among several species (halevi et al., 2002 (halevi et al., , 2003 . ric-3 is thought to be required for the maturation of acetylcoline receptor (halevi et al., 2002) , so ttx-8 may play a similar role such as folding, assembly, transmission or anchoring of some kind of membrane protein. ps1a-g124 analysis of aho-3 mutant that cannot associate cultivation temperature with feeding state in c. elegans nana nishio 1 , akiko mohri 1,2 , eiji kodama 1 , atsushi kuhara 1 , mizuho koike 1 , kotaro kimura 1,3 , ikue mori 1,4 1 div. of biol. sci., nagoya univ., japan; 2 present address: univ. of texas, health sci. cent., usa; 3 present address: natl. inst. of genet., japan; 4 inst. for advanced res., nagoya univ., japan the nematode c. elegans can associate cultivation temperature with feeding state: well-fed animals migrate to and starved animals avoid from the cultivation temperature on a temperature gradient. to identify genes required for this associative learning, we screened mutants that are defective in starvation-induced cultivation temperature avoidance. we isolated aho-3(nj15) mutants that were normal in thermotactic migration after cultivated well-fed state and normal in response to food in locomotion assay (sawin et al., 2000) , indicating that they are normal in temperature and food recognition and may be defective in the associative learning. aho-3 gene encoded a predicted hydrolase and the molecular properties have not been characterized yet, although aho-3 is a highly conserved protein throughout yeast to human. currently, we are trying to dissect the molecular and cellular analysis of aho-3 gene further. we have demonstrated aversive conditioning in lymnaea using 100 mm sucrose presentation as the appetitive stimulus (cs) and mechanical tactile stimulation to the head as the noxious stimulus (ucs). we measured the feeding response before and after pairing with the aversive stimulus to determine whether learning alters the innate preference for sucrose. we also measured the neuronal activity of b3, located in the buccal ganglion. an associative memory, lasting 24 h, was produced with 20 pairings of cs and ucs. the learning was characterized by a shift in the response to the ucs from a whole body withdrawal response to the cessation of feeding behavior. b3 neuron responded with repetitive impulse discharge regularly as fictive feeding patterns to a sucrose application in naive animals, on the other hand cs application failed to generate regular impulse activity rather it resulted in generation of epsps in the conditioned animal. this can interpret that the conditioning decreased the excitability of b3 neuron activity thus to decrease the fictive feeding behavior. yasutaka nomura 1 , dai hatakeyama 1 , tetsuro horikoshi 1 , etsuro ito 2 , manabu sakakibara 1 1 lab. neurobiol. engr, sch. high-tech, tokai univ., numazu, japan; 2 cris, hokkaido univ., sapporo, japan calexcitin, low molecular weight gtp-binding protein is found to be phosphorylated in the visuo-vestibular conditioned hermissenda at the type b photoreceptor. we found positively stained neurons to anti-calexcitin antibody (gift from dr. kuzirian) at the cerebral and pedal ganglion in the circumesophageal nervous system of conditioned lymnaea with two different ways. one was the same conditioning paradigm as hermissenda and the other was taste aversion conditioning. both of these conditioning response is the whole-body withdrawal. no positive neuron was found in naïve animal. neurons in cb cluster and pea cluster showed both positivity to calexcitin and serotonin. this suggested the functional role in conditioning. ken honjo, katsuo furukubo-tokunaga graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan the fruit fly drosophila melanogaster has been utilized as a successful model to study underlying mechanisms of learning and memory. we have established a novel larval olfactory paradigm and found that appetitive and aversive memories are considerably different in their stability whereas both are localized to the mushroom bodies (mbs). we found that larval memory induced by sucrose lasts six times longer than that induced by quinine although the initial learning performances are comparable. by expressing shi ts1 in larval mbs, we demonstrate that disruption of neural output from mbs abolishes both appetitive and aversive memory indicating that both memories are stored before the mb output synapses. moreover, we show that disruption of either creb or amnesiac functions abolishes appetitive but not aversive memory. thus these data suggest that appetitive and aversive reinforcements stimulate different intracellular and/or intercellular signaling pathways generating distinct memory components in mbs. motomi matsuno 1 , minoru saitoe 1,2 , tim tully 3 1 tokyo metropolitan institute for neuroscience, tokyo, japan; 2 department of biology, tokyo metropolitan university, japan; 3 cold spring harbor laboratory, usa we identified ruslan as a novel memory mutant, and found that it encodes a cell adhesion molecule, klingon (klg). klg belongs to the immunoglobulin superfamily and was originally identified as an essential gene for the development of photoreceptor neurons. we report here that klg is necessary for long-term memory as well as early-phase memory. we show that klg expression is dependent on neural activity and functions as a downstream of both the transcription factor, creb and the cell surface receptor notch, both of which are well known to function in ltm formation. transgenic expression of klg improves memory of a klg mutant. since klg protein localizes along the surface between neuropil and neuropil glia, we propose that klg mediates an interaction between neurons and glia that is required for memory formation. we have investigated the ability of context-dependent olfactory learning in the cockroach, periplaneta americana. we trained one group of cockroaches to associate peppermint odor (conditioned stimulus, cs, p) with sucrose solution (appetitive unconditioned stimulus, us+), and vanilla odor (cs, v) with saline solution (aversive us, us−) under illumination (l), and to associate p with us− and v with us+ in the dark (d). another group received training with opposite stimulus setup (l: v+/p−, d: v−/p+). before training, cockroaches preferred v over p. 1 day after training, the former group significantly preferred p over v under illumination but preferred v over p in the dark, and the latter group displayed the invert odor preference. result of the control experiment excluded the possibilities that conditioning hours of the day or its order was used as cues to disambiguate the meaning of css. thus cockroaches are capable of disambiguating the meaning of cs odors according to the visual context. hidehiro watanabe, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan a century had passed since pavlov reported classical conditioning of salivation in dogs. however, the cellular mechanisms underlying this conditioning remain obscure. in insects, salivation is regulated by salivary neurons of the subesophageal ganglion which innervate the salivary grand. here, we established antennal classical conditioning of salivation and that of activities of salivary neurons in cockroaches, periplaneta americana. in insects, antennae are elaborate sense organ that processes many sensory modalities including odor and taste. we found that responses of salivary neurons to an odor was increased after repetitive pairing of the odor with sucrose or saline solution presented to an antenna, but those to an odor paired with water or tactile stimulus presented to an antenna did not changed. the level of salivation to sucrose-associated odor was significantly greater than that to non-associated odor. these results are the first to suggest the classical conditioning of salivation in non-mammalian species. these results are useful to study neural mechanisms underlying classical conditioning of salivation. research funds: kakenhi 17005050 ke zhang 1 , jian z. guo 1 , ai k. guo 1,2 1 institute of neuroscience, chinese academy of sciences, china; 2 institute of biophysics, chinese academy of sciences, beijing, china the cooperation of dopamine system and other brain cortices is essential for decision-making in mammal. drosophila can make clearout choice in visual flight simulator when facing conflicting visual cues based on the saliency of the cues previously trained to follow. here we show this behaviour is impaired when the transmission of dopaminergic neurons or mushroom bodies (mb), was genetically silenced by gal4/uas-shi ts1 system, suggesting that this behaviour is mediated by dopaminergic system acting through mb, a structure shown to be densely innervated by dopaminergic fibers. however, the dopaminergic and mb synaptic activities were required only during the early choice period (<4 min), but not for the sustenance of the chosen flight path. thus the dopaminergic system and mb are specifically devoted to the cognitive function exemplified by the flyǐs choice behaviour and further studies of the circuit in drosophila may help to understand the neural basis of higher cognitive functions. sae unoki, yukihisa matsumoto, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan in mammals, the dopaminergic reward system plays ubiquitous roles in reward learning. previous studies in insects suggested that octopamine (oa) and dopamine (da) mediate various kinds of reward and punishment signals in olfactory learning. however, whether such roles can be generalized to learning of sensory signals other than odors remained unknown. we pharmacologically studied the roles of oa and da in appetitive and aversive forms of visual pattern learning in crickets. crickets injected with oa receptor antagonists exhibited no significant levels of appetitive visual learning, but aversive one was unaffected. the opposite influences were observed by injection of da receptor antagonists. our finding that oa and da participate in reward and punishment conditioning in visual learning, together with results of previous studies in olfactory learning, suggests ubiquitous roles of the octopaminergic reward system and dopaminergic punishment system in insect learning. this suggests conserved roles of aminergic reinforcing systems among different phyla. aiko watanabe, neal a. hessler laboratory for vocal behavior mechanisms, riken brain science institute, saitama, japan in adult songbirds, neural turnover occurs in hvc, a forebrain motor control nucleus. cells labeled by bromodeoxyuridine (brdu), a cell birth marker, appear in the ventricular zone, migrate into hvc, and some of them mature into projection neuron. to assess the role of neurogenesis in adult song plasticity, we deafened adult bengalese finches, whose songs are disorganized and become plastic within the first month after deafening, and then stabilize. deafened birds had more brdu-labeled cells in hvc than control birds within the first month. more tunel-stained apoptotic cells also tended to be seen in hvc of deafened birds. however, number of the brdu-labeled cells decreased 2 months after deafening, when the songs had stabilized. most of the brdu-labeled cells in hvc of deafened birds were immunoreactive for a neuron-specific marker, hu. additionally, amount of singing in deafened birds, which may affect amount of neurogenesis, did not significantly differ from that in control birds. these results suggest that the amount of neurogenesis is related to adult song plasticity. yasko tobari 1,2 , kazuo okanoya 1,2,3 1 lab. for biolinguistics, riken-bsi, wako, japan; 2 grad. sch. of sci. and tech., chiba university, chiba, japan; 3 presto, jst. kawaguchi, japan a set of brain nuclei controls song production in songbirds. among these nuclei, the robust nucleus of arcopallium (ra) is the telencephalic site of direct projections onto vocal motor neurons and respiratory premotor neurons. the projections of ra to the mudulla included the tracheosyrigeal part of the hypoglossal nucleus (xiits), which innervates the syrinx, the birds , vocal organ, and respiratoryrelated nucleus, retroambigualis (ram) were present in bengalese finches. in this study, we have focused our attention on the descending projections of ra, with a view to the presence of contralateral projections to xiits and ram, using in vivo tract-tracing technique. the results indicated that ipsilateral and contralateral projections of ra to respiratory-vocal nuclei in the brainstem were defined in adult male bengalese finches. birdsong is composed of various song elements that have typical frequency modulation. each element is aligned in own sequential rule. especially in bengalese finches, the sequential rule obeys finite state grammar. it has been focused what neural mechanism enables such a complex sequential rule. in order to learn and maintain their own song, they have auditory neural representation of their own song in the forebrain area hvc. we collectively recorded the activities of hvc neurons driven by all possible element pair stimuli. the results show that most of neurons in hvc respond not only the sequence included in their own song but also the sequence not included. each neuron has typical response distribution toward the whole element sequence. in addition, the distribution property is different among neurons in same individual. taken together, information of the entire song element sequence would be stored in the neural ensemble of these neurons as a population coding. hironobu sakaguchi department of physiology and biological information, dokkyo university, school of medicine, japan avian vocal learning provides a good model for human speech learning. young male songbirds learn to imitate their tutor's song during a specific time in development, which is referred to as a sensitive period. many behavioral studies have shown that vocal learning is affected by a song template and social factors. if a young bird is raised without a tutor's song template (father) and/or social contacts with other birds, including its mother and siblings, it produces an abnormal isolated song, meaning that isolation delays the sensitive period for song learning. here, we investigated for the delayed song learning of socially isolated zebra finches from new tutors. consequently, isolated birds, exposed to new tutors from day 120, developed the zebra finch-typical song (song syntax), similar to song acquisition in young birds during the sensitive period of song learning. however, they were not able to imitate the syllable phonology from new tutors. the differences between two aspects of song organization suggest that the schedules and processes of the learning of phonology may be different from those of song syntax. ps1a-h137 facilitatory effects of oxytocin on synaptic plasticity in the olfactory bulb and olfactory learning in young rats fumino okutani, jing-ji zhang, guang-zhe huang, hideto kaba department of integrative physiology, kochi medical school, nankoku, japan oxytocin (ot) within the olfactory bulb (ob) has been reported to be important for the induction of maternal behavior and recognition of offspring. the activity of mitral cells, olfactory relay neurons in the ob is inhibited by granule cells via reciprocal dendrodendritic synapses. electrophysiological studies have revealed that ot modulates mitral cell activity by acting on mitral and granule cells. in a classical conditioning paradigm, young rats show aversion to the odor that has been paired with foot shock. our studies have shown that plasticity in the ob is critical for this olfactory learning. pups that received ot infusion into the ob in the presence of citral odor developed an aversion to the odor without shock, suggesting that ot infusion has a facilitatory effect on olfactory learning. using ob slices, long-term potentiation (ltp) was induced in field epsps recorded in the granule cell layer. ot administration also facilitated ltp. these results demonstrate that ot is involved in olfactory learning in young rats. research funds: kakenhi 17023034 ps1a-h138 the gaba a receptors in the ventral pallidum are involved in the retrieval of conditioned taste aversion in rats tadashi inui, tsuyoshi shimura, takashi yamamoto div. behav. physiol., dept. behav. sci., grad. sch. human sci., osaka univ., japan we examined the effects of microinjections of gaba a receptors antagonist bicuculline into the ventral pallidum (vp) on the retrieval of conditioned taste aversion (cta). in experiment 1, rats received a pairing of saccharin or quinine hydrochloride (cs) with an i.p. injection of 0.15 m lithium chloride (us). after this conditioning, vehicle or bicuculline was bilaterally infused into the vp just before the re-exposure to the cs. the microinjections of bicuculline significantly increased the intake of saccharin cs, but not quinine hydrochloride. in experiment 2, rats were presented with saccharin as cs via an intraoral cannula. the microinjections of bicuculline significantly increased ingestive responses and decreased aversive responses. these results suggest that the gaba a receptors in the vp play an important role in the expression of ingestive and/or aversive responses to saccharin cs during the retrieval of cta so that the microinjection of bicuculline might increase the intake of saccharin cs. research funds: kakenhi (17730431) ps1a-h139 transient blockade, but not genetic deficiency, of c-fos gene expression impairs long-term memory in taste aversion learning roles of c-fos gene expression and its protein product, fos, in conditioned taste aversion (cta) learning were examined using the antisense oligodeoxynucleotide (odn) method in rats and in mice carrying c-fos gene deficiency. infusion of antisense odn (as-odn) directed against c-fos mrna into the parabrachial nucleus (pbn), but not into the amygdala or insular cortex (ic), impaired the acquisition, while infusion of randomized and inverted control odns had no effect. suppression of fos synthesis in the amygdala or ic impaired the retention. retrieval of an acquired cta was not impaired by as-odn infusion into the pbn or amygdala. in contrast, mice carrying c-fos gene deficiency showed normal acquisition and retention. the present results suggest that the fos-mediated signals in the pbn, amygdala or, ic plays key roles in the acquisition and/or consolidation, but not the retrieval, of long-term cta memory. ps1a-h140 gaba receptors in the deep cerebellar nuclei are essential for mouse eyeblink conditioning classical eyeblink conditioning is a useful experimental system to analyze the neuronal substrate underlying learning and memory. the knowledge on the mouse eyeblink conditioning is far less compared with rabbit's. we examined the role of the deep cerebellar nuclei (dcn) during delay eyeblink conditioning in c57bl/6 mice by using gaba a receptors agonist and antagonist. in the acquisition tests, in which muscimol (msc) or picrotoxin (ptx) was injected from beginning of training, acsf-injected control mice learned this task, but both msc-and ptx-injected mice showed a significant impairment in acquisition of conditioned response (cr). in the retention tests, in which the drug was injected after acquisition of training, cr % in acsf-injected mice were kept over 80%, while those in the mscand ptx-injected mice decreased to 30%. these results revealed that gabaa receptors in the dcn play important roles in acquisition and retention of mouse eyeblink conditioning. various forms of synaptic plasticity are found in cerebellar circuits, but their significance in motor leaning remains unknown. in the cerebellum, delphilin is expressed selectively in purkinje cells (pcs) and localized exclusively at parallel fiber (pf) synapses, where it interacts with glutamate receptor ␦2 that is essential for long-term depression (ltd) and motor learning. here, we showed that ablation of delphilin proteins facilitated ltd induction at pf-pc synapses and enhanced optokinetic response adaptation without affecting histology. this finding suggests that threshold regulation of ltd at pf-pc synapses is a limiting step for motor learning efficiency. ps1a-h143 post-training cerebellar cortical activities are necessary for transfer of memory trace of motor learning from cortex to nuclei soichi nagao 1,2 , takehito okamoto 1 , fumihiro shutoh 1,3 1 lab for motor learning control, riken bsi, saitama, japan; 2 sorst, jst, saitama, japan; 3 dept. anat., grad. univ. tsukuba, ibaraki, japan one-hour optokinetic training induces short-term adaptation of horizontal optokinetic response (hokr) gains in mice. succession of 1 h daily training for 1 week induces long-term adaptation. we recently reported that the memory trace of adaptation of hokr is initially acquired within the cerebellar flocculus through long-term depression (ltd), and later transferred to the vestibular nuclei for consolidation. in order to reveal the neural mechanisms underlying the memory transfer, we reversibly inactivated the neural activities of flocculus bilaterally by local application of muscimol immediately after the end of daily training. mice treated with muscimol showed depressed long-term adaptations, while the short-term adaptations were intact, suggesting that the neural activities of cerebellar cortex in a certain period after training are necessary for the transfer of memory trace from flocculus to vestibular nuclei. research funds: kakenhi (16500204) ps1a-h144 modification of gene expression in the cerebellar cortical neurons related with long-term motor learning yuji t. katagiri 1,2 , takehito okamoto 1 , shin-ichi nishimura 1 , fumihiro shutoh 1,3 , soichi nagao 1,4 1 lab. for motor learning control, riken bsi, saitama, japan; 2 univ of the air, chiba, japan; 3 dept. of anatomy, human comprehensive science, grad. univ. tsukba, japan; 4 sorst, jst, japan we recently reported that the cerebellar ltd plays a crucial role for both acquisition and consolidation of memory trace of long-term motor learning using the adaptation paradigm of mouse horizontal optokinetic eye movements (shutoh et al., 2006) . in order to listup the molecules involved in the motor learning, we sampled total rna from the cerebellar flocculus of short-and long-term adapted mice, and quantified amounts of gene expression by the microarray methods. we found that the number of genes modulated by longterm motor learning much exceeded that modulated by short-term motor learning, and the number of down-regulated genes were larger than that of up-regulated genes. we furthermore examined the gene expression of purkinje cells by the laser micro-dissection and quantitative rt-pcr methods. ps1a-h145 influence of spatial cues on hippocampal neuronal activity in spatial navigation tasks in mice hippocampal neurons were recorded while mice performing spatial tasks of searching for unpredictable and predictable rewards. the influence of spatial cues, including distal and proximal cues, on the response of hippocampal cells that exhibited place-related activity was examined. place cells predominantly shifted their fields accordingly by changes of visual and auditory distal cues, and fewer cells shifted their fields by changes of proximal cues. these results provide evidence that hippocampal neurons of mice can use flexibly information of spatial cues to represent the environment, and this ability is important for spatial learning. son ho 1,2 , t kobayashi 1,2 , e hori 1,2 , k umeno 1,2 , t ono 1 , h nishijo 1,2 1 system emotional science, univ. of toyama, toyama, japan; 2 crest, tokyo, japan we investigated a role of the hippocampal formation (hf) in encoding of a moving object in an open field. rats acquired icss rewards if they moved freely. then, a remote-controlled car was placed inside the open field. the rats could receive icss if it chased and approached the car. of a total of 133 place cells recorded, activity of 40 was significantly modulated by the car speed and/or distance between the car and rat; 21, 12 and 7 cells displayed distance-dependent, car speeddependent, and distance and car speed-dependent firing, respectively. furthermore, six cells, which did not show the place field in reference to rat position, but showed the place fields in reference to car position. in a control experiment, the same car was introduced, but the rats could receive icss rewards without relation to relative distance between the rat and car. so far, 16 place cells were recorded in this experiment. of these, six and three place cells displayed distancedependent and car speed-dependent firing, respectively. the results suggest that hf encodes not only spatial information of own location, but also that of other moving object in an environment. hisae gemba, kazuko nakao, ryuiti matsuzaki, yusaku amaya department of physiology, kansai medical university, moriguchi, japan cortical field potentials were recorded by electrodes implanted on the surface and at a 2.0-3.0 mm depth in the cortex of monkeys in the process of learning somatosensory-initiated hand movements and then analyzed. it was found that an s-n, d-p potential, at about 40 ms latency from stimulus, in the caudal bank of the left arcuate sulcus (homolog of broca's area) was related to recognition learning (association of stimulus with movement), and that an s-n, d-p potential in the motor and somatosensory cortices, and areas 5 and 7, contralateral to the operating hand, was related to skill learning (making movements quicker and more appropriate). in visuo-initiated hand movements, the left prefrontal cortex was related to recognition learning; the motor and somatosensory cortices, and area 5 to skill learning, as previously reported. this indicates that motor programming for somatosensory-initiated and visuo-initiated hand movement differs. computational studies of hippocampal function generally assume that ca1 performs a match-mismatch comparison of memory retrieval with sensory input. here we investigated this comparator model using an ensemble recording during task behaviors in the rat. we employed directional memory-guided alternation and visual cue discrimination tasks for the same animal. after training, the animals tended to predict a next direction according to the alternation paradigm even in the visual cue discrimination task. during this task, we found that some ca1 neurons showed specific bursts when a predicted event did not occur or along the trajectories of their corrective movements from a wrong cite to a correct cued one. these data suggest that ca1 plays an important role in the mismatch detecting and correcting process of behavior. n-methyl-d-aspartate (nmda) receptor has high permeability to ca 2+ but is blocked by mg 2+ in a voltage-dependent manner. this property is a molecular basis of nmda receptor-dependent long-term potentiation, which is thought to play a central role in learning and memory. we have generated the genetically engineered mice in which mutated nmda receptors defecting in mg 2+ binding ability are expressed specifically in the granule cells of the dentate gyrus, the entry point to the hippocampal trisynaptic circuit. the mutant mice showed a variety of behavioral abnormalities including hyperactivity, impaired prepulse inhibition. to elucidate the effect of mutation on the information processing in the hippocampus, we recorded the place-related activity from hippocampal ca1 cells, the output stage of hippocampal circuit. the link between the behavioral anomaly and the hippocampal activity is discussed. mikako sakurai, ko zushida, masayuki sekiguchi, keiji wada department of neurodegenerative diseases, national institute of neuroscience, ncnp, tokyo, japan uch-l1 is a component of the ubiquitin system. uch-l1 is expressed at high levels in the hippocampal neurons. however, the functional role of uch-l1 in synaptic plasticity and behavior is not understood. we examined behavior and synaptic plasticity in gad mouse which is an autosomal recessive spontaneous mutant carrying an intragenic deletion in the gene encoding uchl1. gad mice have significantly impaired performance in the open field and one-trial passive avoidance tests. theta burst stimulation (tbs) of shaffer collateral in hippocampal slices from gad mice elicited decremental long-term potentiation (ltp) in the area ca1. in contrast, non-decremental ltp was induced in control wild-type mice. the maintenance of tbsinduced ltp in the wild-type mice was impaired by actinomycin d, an inhibitor of transcription, whereas tbs-induced ltp in gad mice was insensitive to actinomycin d. these results suggest that uch-l1 is a molecule participating in the synaptic plasticity elicited by tbs and the memory function. ps1a-i151 learning stages in rat operant reversal task and cross-correlation between hippocampal and prefrontal local field potential powers yoshinori izaki, tatsuo akema department of physiology, st. marianna university school of medicine, japan to investigate whether the relationship between hippocampus (hip) and prefrontal cortex (pfc) spontaneous local field potentials changes with leaning stages, we analyzed cross-correlation (cc) of these local field potential powers during operant reversal training sessions. rats were trained with initial discrimination task until a stable discriminative performance was achieved (learning stage 0). then the rats received the reversal training. learning stages examined were as following: the first training session (stage i), leaning stage for s+ (stage ii) and for s− (stage iii). different changes of the cc in some frequency-band powers with learning stages were observed. the cc in higher gamma-band (64-120 hz) was strong at stage 0 and changed with leaning stages. particularly, the cc decreased to almost zero at stage ii. these results suggest that functional connection between hip and pfc is reflected in this frequency-band and changes with learning stages. ps1a-i152 longitudinal fiber systems in the dentate gyrus of the rat norio ishizuka, yoshitomo umitsu department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan longitudinal fiber systems in the dentate gyrus of the rat were investigated by anterograde labeling method of pha-l and retrograde labeling method with fluorescent dyes. the flattened hippocampal formation allowed sections to be cut perpendicular to the full septotemporal axis of the dentate gyrus. injection of pha-l into the hilar region elucidated that two longitudinal fiber systems existed in the dentate gyrus. the first fiber system gives rise to projections to the superficial portion of the dentate molecular layer, and the longitudinal axonal trajectory of this system ceased within the range of about 1.5 mm from the injection level. in the second fiber system, axonal terminations began to appear at the level of 1 mm apart from the injection level and were distributed in further full septotemporal extent of the dentate molecular layer. the axonal arborizations of the second system were found in the deepest portion of the dentate molecular layer immediately above the granular cell layer. in the experiment of fluorescent dye injection, several kinds of cells in the hilus were retrogradely labeled. ryoichi moki, ryang kim, hisahiro umeeda, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan recent studies have shown that when conditioned fear memory is retrieved, fear memory becomes labile and requires gene expressiondependent reconsolidation for the re-storage. in addition, previous our study using conditional creb mutant mice indicated that creb is required for reconsolidation of conditioned fear memory. we also observed protein synthesis-dependent reconsolidation of spatial memory using morris water maze. in this study, to understand the mechanisms of reconsolidation of spatial memory, we examined a role of creb in reconsolidation of spatial memory. using conditional creb mutant mice that enable to induce the inhibition of creb activity in a tamoxifen-dependent manner, we found that inhibition of creb activity leads to disruption of spatial memory after the retrieval. this result indicates that creb is required for reconsolidation of spatial memory. shunsuke hasegawa, hirosi hosoda, satoshi kida department of bioscience, tokyo university of agriculture bhlh-pas transcription factor bmal1 ubiquitously expresses in brain. bmal1 functions by forming a heterodimer with either clock or npas2, which has been known to play important roles in control of circadian rhythm and memory formation, respectively. to understand roles of bmal1 in forebrain function, we generated conditional mutant mice that enable to induce the inhibition of bmal1 function in a forebrain. using a dominant negative mutant of bmal1 (bmal1 r91a) that forms a heterodimer with clock but loses the binding activity with e-box (hosoda et al., 2004), we generated transgenic mice expressing this mutant under the control of tetracycline-dependent promoter. these mutant mice were crossed to transgenic lines expressing tetracycline-dependent transcription factors (tta) under the control of alpha camkii promoter. we observed the expression of bmal1 r91a in several double transgenic lines in a tta-dependent manner. behavioral analyses showed that these mutant mice showed an impairment of memory formation, indicating crucial roles of bmal1 in learning and memory. stress sometimes causes memory deficits. and chewing has been shown to reduce stress. however, the chewing-related mechanism in stress-induced memory deficits is unclear. we thus examined the effects of chewing on spatial memory using morris water maze and fos induction in the hippocampus and amygdala in stressed mice. when mice were exposed to restraint stress, reduction in learning ability and density of fos-positive cells in the dg and the bla was seen, but not in the mice chewing a thin wooden bar during stress exposure. the results suggest involvement of the amygdaloidmechanism by which chewing may prevent the stress-induced impairment of hippocampus-dependent memory. seiichiro amemiya, shinya yanagita, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, tokyo, japan we examined the effect of background noise (bgn) on spatial learning and its neuronal activity related to arousal and stress using maze task and c-fos immunostaining. rats performed maze task under different intensity of bgn (0, 70, or 100 db; intermittent white noise). 70 db bgn induced significant decreases in number of error and time to goal in maze compared with 0 and 100 db. although bgn increased fos positive acetylcholinergic neurons (fos-chat) in mesopontine tegmentum (mt) regardless of the intensity, fos-chat in basal forebrain (bf) increased intensity-dependently. in locus coeruleus (lc) and cortex, fos positive cell increased intensitydependently. furthermore, 100 db bgn remarkably enhanced fos expression in stress-related nuclei, such as paraventricular nucleus and central nucleus of the amygdala. these results suggest that bgn improve spatial performance by enhancing arousal following activation of cholinergic neurons in mt and bf, and lc neurons. however, higher bgn intensity could evoke over-arousal and stress responses, thereby prevent the maze task. siriporn chattipakorn 1,2 , anucha pongpanparadorn 2 , wasana pratchayasakul 2 , anchalee pongchaidacha 2 , nipon chattipakorn 2 1 fac. dent. cmu, chiang mai, thailand; 2 cert, cmu, chiang mai, thailand current pharmacotherapy of ad is the use of ache inhibitors. previous in vitro study showed that tde inhibited ache activity. this is the first study investigating the effects of tde on cortical ache activity and neuronal activity in in vivo. we used fos immunohistochemistry to determine the neuronal activity and the colorimetric method to investigate cortical ache activity following the single injection of various tde doses. mean fos-positive neurons in cortex were 101 ± 14, 124 ± 19 and 108 ± 22 in the groups administered 250, 500 and 1000 mg/kg tde, respectively. cortical fos-positive neurons in all three tde-treated groups were greater than those in the control group. percent inhibition of cortical ache activity was 17.4 ± 6.3, 22.7 ± 6.9 and 16.6 ± 5.0 for 250, 500 and 1000 mg/kg tde, respectively. these ache inhibitory effects were significantly different from the control. these findings suggest that tde could be beneficial as a possible novel therapeutic agent for ad. we showed the effects of a 2-h tde administration in animals on the inhibition of cortical ache activity and the enhancement of cortical neuronal activity. ache activity in circulation following a 2-h tde administration was not different compared to the control. this study investigated that the effects of tde on circulating ache activity (cache) in animal models was time-dependent. we used the colorimetric method to investigate cache activity in rats following the single administration of tde at various doses at different time courses (10, 60 and 120 min). percentage inhibition of cache activity following a single tde injection at doses 500 and 1000 mg/kg significantly decreased at 10 and 60 min after tde injection, but not at 120 min. cache inhibitory effects among two doses of tde administrated groups at various time courses were not significantly different. these findings suggest that tde may be a short-acting ache inhibitor. donepezil, galanthamine and tacrine are acetylcholinesterase (ache) inhibitors used for treatment of alzheimer's disease. we examined the neuroprotective mechanisms of ache inhibitors against apoptotic glutamate neurotoxicity using cortical neurons. we show that they protect neurons through mechanisms other than ache inhibition. the protective effects are mediated through nicotinic receptors (nachrs). donepezil and galanthamine protect neurons through ␣4and ␣7-nachr and kinases involved in pi3k-akt pathway, and increase the levels of phosphorylated akt and bcl-2. these results suggest that these ache inhibitors express their neuroprotective effects against glutamate neurotoxicity through nachrs and that donepezil and galanthamine protect neurons through pi3k-akt pathway via ␣4and ␣7-nachrs. ps1a-i160 arachidonic acid preserves hippocampal neuron membrane fluidity in senescent rats yasuto kashiyae 1 , yoshiyuki ishikura 2 , shigeaki fujikawa 2 , yoshinobu kiso 2 , manabu sakakibara 1 1 lab. neurobiol. engr., tokai univ., numazu, japan; 2 inst. health care sci. suntory, shimamoto, japan previous studies indicate that long-term dietary supplementation with arachidonic acid (aa) in 20-month-old rats (oa) effectively restores performance in a memory task and induction of long-term potentiation in the hippocampus to the level of young control animals (yc). this study examined fluorescent recovery after photobleaching (frap) in yc, old control (oc), and oa neurons in hippocampal slice preparations. three measures: mobile fraction (mf), diffusion constant (d), and time constant (τ), were estimated among yc, oc, and oa. each of these parameters was significantly different between oc and yc, suggesting that membrane fluidity is lower in oc than in yc. in contrast, d and τ were almost comparable in oa and yc, indicating that hippocampal neuronal membranes supplemented with aa were more fluid than those in oc, whereas the fraction of available molecules remained smaller than in yc. long-term administration of aa to senescent rats might help to preserve membrane fluidity and maintain hippocampal plasticity. ps1a-i161 thimet oligopeptidase co-exists in gfap-and cd11b-positive glia in rat pc/rsc treated with mk-801 takeshi kato 1 , mohammad arif 1 , michiyuki yamada 2 , toshiyuki chikuma 3 , md. mahiuddin ahmed 4 1 lab. natural info. sci., grad. sch. integr. sci., yokohama city univ., yokohama, japan; 2 grad. sch. integr. sci., yokohama city univ., yokohama, japan; 3 dept. hygien. chem., showa pharmaceut. univ., machida, japan; 4 dept. r&d, bioelectro. anal. sci., japan thimet oligopeptidase (ep24.15) hydrolyzes not only neuropeptides but also the peptides generated by proteasomes. in the present immunohistochem study we found that mk-801 activated gfapand cd11b-positive glia cells in rat posterior cingulate/retrosplenial cortex (pc/rsc) 3 day after the treatment. mk-801 also increased ep24.15 and prolyl oligopeptidase. immunohistochem data showed that ep24.15 co-localized with gfap and cd11b positive glial cells. since mk-801 causes schizophrenia-like psychosis and produces neurotoxicity in adult rodent brain, we further examined the pretreated effect of neuroleptics. clozapine co-administration suppressed the increased ep24.15 in the pc/rsc. these data suggest that ep24.15 in the astroglia and microglia cells of rodent brain might in part control positive and/or negative schizophrenia symptoms. ps1a-i162 effect of age and sex steroids on the expression of alzheimer's disease presenilin (ps) 1 and 2 in the mouse brain soumi ghosh, m.k. thakur banaras hindu university, india alzheimerǐs disease is a neurodegenerative disorder characterized by the impairment of cognition and memory. these functions are improved by supplementation of sex steroids. the genes causing lateonset of ad, presenilin (ps) 1 and 2, code for highly homologous integral membrane proteins. the proteolytic fragments of these proteins are main biological components. we have analysed the effect of age, sex and gonadal hormone supplementation on ps expression at protein level by western blotting. ps1 shows a significant decrease with aging in both males and females. however, there is no significant variation in expression of ps1 and ps2 with sex. gonadectomy also lowers the level of presenilin proteins in old age. ps2 protein shows increase in expression with gonadal hormone treatment in both ages, but estrogen supplementation to old mice lowers ps1 level. these modulatory effects of age, sex and gonadal hormones on ps proteins may explain the therapeutic interventions of hormone replacement therapy. research funds: ministry of science and technology, india ps1a-i163 effects of the monomeric, oligomeric and fibrillar beta-amyloid peptides on the proliferation and differentiation of adult neural stem cells from svz dept. of pharmacol., seoul natl. univ., south korea the subventricular zone is the largest neurogenic area of the adult brain. in this region, neural stem cells (nsc) serve to produce newly generated neurons and glia cells throughout adulthood. however, the common neurogenesis of nsc cannot replace neuronal loss in alzheimerǐs disease (ad) induced by amyloid deposits composed mainly of amyloid␤proteins. in vitro, we examined the effects of various form of a␤peptide on the proliferation and differentiation of nsc from svz of 10-week-old adult mice. in this study, a␤42 peptide was prepared three forms of aggregating stage, monomeric, oligomeric and fibrillar a␤42 peptide. we found that treatment of nsc with oligomeric form of a␤42 peptides remarkably increased the number of neurospheres during proliferation and neurons during differentiation in-vitro. we also found that these neurogenesis was accompanied by morphological change of neuron. the number of secondary and tertiary neurites increased at submicromolar concentrations of oligomeric a␤42 peptide without shrinkage of axonal length. in alzheimer's disease (ad) brain, the formation of senile plaque with accumulated microglia is observed. although the role of microglia in ad is not clarified, their involvement in a␤ clearance is noted. high mobility group box protein-1 (hmgb1) is a non-histone chromosomal protein. here, hmgb1 was associated with senile plaques and protein level was increased in ad brain. diffuse hmgb1 immunoreactivity was observed around dying neurons in the kainic acid-and a␤1-42 (a␤42)-injected rat hippocampi. hmgb1 was not co-localized with a␤ in transgenic mice which show massive a␤ production without neuronal loss. furthermore, co-injection of hmgb1 delayed the clearance of a␤ and accelerated neurodegeneration in a␤42-injected rats. these results suggest that hmgb1 released from dying neurons may inhibit microglial a␤ clearance and enhance the neurotoxicity of a␤. perineuronal nets consisting of chondroitin sulfate proteoglycan (cspg) and hyaluronic acid (ha) are associated with distinct populations in mammalian brain. in the present study, we observed perineuronal net-like structure by rat cortical neurons in dissociated culture using wisteria floribunda lectin, ha binding proteins, and cspgspecific antibodies. this perineuronal net-like structure was observed often at parvalbumin-positive neurons, indicating gabaergic ones. it is well known that perineuornal nets-containing neurons are survive against alzheimer disease in human. to elucidate significance of perineuronal nets in alzheimer disease, we applied beta-amyloid peptide into cultured cortical neurons. perineuronal nets-containing neurons were resistant against beta-amyloid peptide, while negative neurons were often dead. these results indicate that perineuronal nets are participated in protecting neurons from cytotoxic substances such as beta-amyloid. ps1a-i166 x11-like protein regulates metabolism of app in the mouse brain yoshitake sano 1,2 , tadashi nakaya 2 , shigeyoshi itohara 1 , toshiharu suzuki 2 1 riken bsi, saitama, japan; 2 hokkaido university, neuroscience, sapporo, japan abnormal metabolism of amyloid beta precursor protein (app) results in the accumulation of beta amyloid (a␤) in the brain, and contributes to the pathogenesis of alzheimer's disease. app has a functional sequence in its cytoplasmic domain, the yenpty motif, which is involved in trafficking, internalization, and metabolism of app. x11-like protein (x11l) was identified as a molecule that interacts with the motif and regulates app metabolism in cultured cells (1999 . j. biol. chem. 274, 2243 2003. j. biol. chem. 278, 49448) . there is no evidence, however, that endogenous x11l suppresses app metabolism and a␤ generation in vivo. to examine the physiologic role of x11l in app metabolism in the brain, we generated x11l null mutant mice. the mutant mice developed normally without gross anatomic brain abnormalities. there were increased amounts of cterminal fractions cleaved at the ␤-site and a␤, but the amount of total app was unaltered in the mutant mouse brain. these results suggest that x11l suppresses the production of a␤ by inhibiting ␤secretase-induced proteolysis of app. it is still unknown how human's central nervous system (cns) controls its body system to keep the body balanced. this study aims to analyze the characteristics of spectral response of body sway in eyes open and in eyes closed during static upright stance based on a pid control model. in this model, body sway in medial-lateral direction is considered, and the body is simply modelled as a multi-link inverted pendulum system. spectral response analysis showed the gain varied with input frequency and time lag. peaks of the gain were intensively influenced by controller's parameters (kp, kd and ki). parameters identification showed that kd is decreased in eye-closed. by simulation, the spectral responses of the pid model quite agreed with the experimental data. the results proved that the spectral characteristics of body sway is determined by the dynamics of body system and its controller's parameters, suggest the balance-keeping control in cns can be modelled as a pid controller. nuclear dysfunction is a critical element of the pathology of polyglutamine (polyq) diseases. proteome analysis of soluble nuclear proteins in the nuclear matrix of neurons expressing normal or mutant huntingtin or ataxin-1 protein by 2d-electrophoresis and tof-mass delineate that mutant at1 and htt proteins similarly reduce transcriptional factor x1 and x2. immunoprecipitation and pull-down assays support interaction between polyq and factor x1 and x2. immunohistochemistry of hela cells and primary neurons reveal sequestration of factor x1 and x2 into inclusion bodies and reduction of them in the nuclear matrix. compensatory expression of factor x1 and x2 ameliorates poly-q pathology in htt-/at1-expressing neurons and transgenic drosophila. these results suggest that factor x1 and x2 are critical regulators of polyglutamine disease pathology and could be a target for developing therapeutics. ps1a-j170 ba1-42 was reduced in rat brains fed with coconut juice n. radenahmad, p. subhadhirasakul psu, thailand young coconut juice (ycj), cocos nucifera (arecaceae), believed to contain phytoestrogen and other sex hormone-like substances, was investigated for its possible beneficial effects on halting dementia in ovariectomized (ovx) rats, a model system for the postmenopausal condition. sixty ovx rats were divided into six groups, 10 rats/group (g). group 1 received e2 at 2.5 g/kg per day; groups 2 and 3 received ycj at 20 ml, and 100 ml/kg day, respectively, once everyday. group 4 received ycj 100 ml/kg plus e2 at 2.5 g/kg day twice a week, all for 5 weeks. the other two were ovx and sham-operated controls. using a chemiluminescent immunoassay, circulating e2 in group 3 was insignificantly different from the control groups. after rats were sacrificed, brains were removed, fixed and paraffin embedded for ihc staining. using anti-␤-amyloid 1-42 antibody, this alzheimer pathology was found in cytoplasm and dendrites, but not in nuclei or axons, of pyramidal cells both in hippocampus and in layer 3 and layer 5 of cerebral cortex. it was found that amyloid deposition in frontal, temporal and hippocampus of rat brains in group 3 was lesser than ovx and control groups. amyloid deposition was correlated with e2 serum at r = −0.4. ps1a-j171 correlation between semantic memory and regional gray matter volume of anterior aspect of right temporal lobe in normal elderly subjects. a voxel-based morphometry yasuyuki taki 1 , shigeo kinomura 1 , kazunori sato 1 , shinya uchida 1 , ryoi goto 1 , kentaro inoue 1 , ichiro tsuji 2 , hiroyuki arai 2 , ryuta kawashima 3 , hiroshi fukuda 1 1 department of radiology and nuclear medicine, institute of development, aging and cancer, tohoku university, sendai, japan; 2 tohoku univ. grad. school of med., sendai, japan; 3 niche, tohoku univ., sendai, japan the purpose of this study was to determine whether there is a correlation between semantic memory and regional gray matter volume in community-dwelling normal elderly people by voxel-based morphometry. we collected brain magnetic resonance images of 111 community-dwelling normal elderly subjects. we performed multiple regression analysis of raw score in the wais-r information subtest, gender, and regional gray matter volume. the volumes of the right superior and middle temporal gyri showed significant positive correlations with raw score in the information subtest. our study indicated that normal elderly individuals show a significant correlation between regional gray matter volume and semantic memory. research funds: (h13-kenko-008), (h13-choju-007, h13-choju-023) ps1a-j172 effects of fluoxetine on the cognition of patients with mild cognitive impairments arash mowla, azadeh pani shiraz university of medical sciences, iran recent researches suggest a role for monoaminergic hypofunction in age related cognitive decline. in several studies selective serotonin reuptake inhibitors demonstrated neurogenesis in hippocampus. we studied the effects of fluoxetine on cognition of patients with mild cognitive impairment (mci). fifty-two non-depressed patients with mci were randomly assigned to take fluoxetine or placebo. the patients were administered mini-mental status examination (mmse) and wechsler memory scale iii (wmsiii) pre intervention. twenty-six patients completed the 8 weeks trial. treatment response was defined as a final mmse and wms-iii scores. the patients in the fluoxetine group showed improvement in mmse and immediate and delayed logical memory scores of wms-iii. the placebo group had not significant changes in the cognitive measurements. fluoxetine enhanced memory and cognition in the patients. this was consistent with pervious studies that emphasized on the role of fluoxetine in improving memory and promoting neurogenrsis in the hypocampus. however, this conclusion should be tempered by the small sample size. lisa l. cook 1 , d.g. goodenowe 1 , y. yamazaki 1 , j. flax 2 1 phenomenome discoveries inc., saskatoon, canada; 2 precisionmed inc., san diego, ca, usa dementia affects about 10% of the population over the age of 65 and can result from various neuropathological conditions. currently, there is no way to differentiate specific forms of dementia (alzheimer's disease (ad), vascular dementia, etc.) prior to autopsy. pdi has discovered an 8-metabolite biomarker panel within the serum of patients with ad, non-ad dementia and healthy non-demented controls that can simultaneously differentiate the type of dementia and identify cognitive impairment using a non-targeted metabolomics technology based a fourier transform ion cyclotron resonance mass spectrometry (fticr-ms). the accurate measurement of the metabolite mass is sufficient to elucidate its molecular formula, thereby leading to metabolite identification, explication of biological significance and efficient biomarker validation. the 8-metabolite biomarker panel could provide a non-invasive method to aid in the diagnosis of specific subtypes of dementia. the development of a high throughput assay for these markers will also be presented. neurons become photosensitive by genetically introducing one of green algae-derived protein, channelrhodopsin-2 (chr2). in this study, we quantitatively investigated the rapidness of the light-gated current of chr2 expressed in pc12 cells using blue led light. the light-gated current consists of two components, inactivating and noninactivating. the magnitude of inactivating component was almost linearly related to the light intensity. the non-inactivating component showed the tendency to saturate at high illumination. we also found that the activation rate is about 10-fold faster than the inactivating rate, but both are linearly dependent on the light intensity. since the photoactivated current was very rapid in both onset and offset, the neuronal firings were phase-locked to short light pulses in an acute slice of hippocampus. it is suggested that the genetic expression of chr2 is one of the most ideal photostimulation methods of a genetically identified neuron with defined activity patterns in intact nervous system. yujiro hattori 1 , shigeki ohta 1 , kenji hamada 2 , naofumi yamada-okabe 2 , yonehiro kanemura 3 , hideyuki okano 4 , yutaka kawakami 5 , masahiro toda 1,6 1 neuroimmnology research group, keio univ., tokyo, japan; 2 chugai co. ltd., kanagawa, japan; 3 inst. cli. res., onh, japan; 4 physiology, keio univ., tokyo, japan; 5 cellular signaling, institute for advanced medical research, keio univ., tokyo, japan; 6 neurosurgery, keio univ., tokyo, japan to identify neuron specific genes, we performed two gene profiling techniques, dna microarray and est analysis. in this study, we focused on genes expressed specifically in the normal brain tissues but not in glioma tissues and identified the human kiaa1110 gene which was a homologue of rat synarfgef (po). rt-pcr analysis revealed that the human kiaa1110 homologue was expressed only in adult brain tissue. western blot and immunocytochemical analyses showed the kiaa1110 protein was expressed in adult brain tissues and differentiated neuronal cells but not in fetal brain tissues nor neural stem/progenitor cells. in conclusion, we identified an adult neural-specific gene using the combined gene profiling method and our results suggest the usefulness of this method to identify tissue specific genes. ritsuko the objective of this study was to find the proteins related to sexual differentiation and to elucidate its molecular mechanism. methods: developing hypothalamic and cortical cells from fetuses on embryonic day 15 were dissociated. after the cells were treated with 100 nm estradiol-17beta (e2) or ethanol for 8 days, proteins were extracted and labeled with cydyes. two-dimensional difference gel electrophoresis (2d dige) was then performed. the differential protein spots were analyzed by software analysis, subject to in-gel digestion, and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof-ms). results: more than 4000 spots were detected from 2d dige. compared with ethanol treatment, e2 increased the expression of 501 spots in the hypothalamic cells and 169 spots in the cortical cells (p < 0.2, difference >1.5). proteomics analysis showed different effects of e2 for hypothalamic and cortical cells. in order to relate cellular brain structure to function, it is necessary to manipulate neural circuits at the level of individual cell types. genetic methods for neuronal inactivation combined with cell-typespecific promoters will achieve this goal. here, we have developed a genetic method for quickly and reversibly inactivating in vivo mammalian neurons using allatostatin receptor (alstr), which causes neuronal hyperpolarization when treated with peptide ligand allatostatin (al). in rat barrel cortex neurons expressing alstr, al reversibly inactivated neuronal activity evoked by electrical stimulation of the whisker pad. both inactivation and recovery were seen within several minutes. we also confirmed the effectiveness of the al/alstr system in ferret visual cortex, lateral geniculate nucleus (lgn), and monkey lgn. therefore, the al/alstr system will be a powerful tool to investigate neuronal circuits and function. prospective purification of neural stem cells (nsc) through the specific cell surface marker is crucial for functional recovery of the damaged brain. in the last meeting, we showed that living nsc were enriched from mouse whole brain as positive cells for erythro-phytohemagglutinin (e-pha), which binds to complex type asparagine-linked oligosaccharide (n-glycans). in this study, by using facs system, we found that high selective affinity of e-pha binding to the brain cells; e-pha negative cells were neurons, mid-positive cells were nsc, and highly positive cells were endothelial cells, respectively. ligand blot analysis revealed the existence of the e-pha binding proteins different from the known selective nsc markers in e14 days brain homogenate, suggesting that n-glycosylated proteins could be distinctive markers for nsc. masahiro waza, hiroaki adachi, masahisa katsuno, makoto minamiyama, fumiaki tanaka, manabu doyu, gen sobue department of neurology, nagoya university, nagoya, japan the pathogenic gene product of spinal and bulbar muscular atrophy (sbma) is polyglutamine (polyq)-expanded androgen receptor (ar), which belongs to hsp90 client protein family. 17-allylamino-17-demethoxygeldanamycin (17-aag) is a new derivative of geldanamycin that shares its important biological activities but shows less toxicity. 17-aag is now in phase ii as a potential anti-cancer agent because of its ability to selectively degrade several cancer-related client proteins. we examined the efficacy and safety of 17-aag in a mouse model of sbma. administration of 17-aag significantly ameliorated polyq-mediated motor neuron degeneration by preferential proteasome degradation of mutant ar. the ability of 17-aag to preferentially degrade mutant protein would be directly applicable to sbma and other neurodegenerative diseases. modulation of hsp90 function by 17-aag has emerged as a candidate of molecular targeted therapy for neurodegenerative diseases. the avian embryo has long been a popular and an excellent model for studying vertebrate development because of its classical manipulative advantages. in the present study, we tried to develop regulated gene transfer by using the tet regulatory system in the chick. the reverse tetracycline-controlled transactivator was expressed under the control of the motor neuron (mn) specific hb9 promoter. tetracycline responsive elements were used for inducible gfp expression. after these constructs were introduced into neural tube by in ovo electroporation, gfp expression was induced in spinal mns in the presence of doxycycline. approximately 5-20% of mn express gfp very intensely whereas the remaining mns never express gfp, suggesting that, although the transgene is induced in limited numbers of mns, once activated, cells express a large amount of the protein product of the experimental gene. thus, this strategy can be applicable for a variety of experiments that require specially and temporally regulated gene expression in the chicken embryo. ps1a-k181 selective collection of catecholaminergic (ca) neurons in the brain and its application to gene expression analyses hiroaki nakamura 1 , yoshiyuki ishii 1 , kazuto kobayashi 2 , yasufumi sato 3 , keiichi itoi 1 1 lab. info. biol., grad. sch. info. sci., tohoku univ., sendai, japan; 2 dept. mol. genet., fukushima med. univ., japan; 3 inst. develop., aging, cancer, tohoku univ., japan ca neurons are involved in a wide spectrum of physiological functions in the brain. most ca neurons are localized in the brainstem and hypothalamic regions and typically make clusters of cells, among which the noradrenergic (a1, a2 and a6) and dopaminergic (a9, a10 and a12) neurons predominate. in order to explore functional roles of these neurons, we collected ca neurons selectively using tyrosine-hydroxylase (th)-green fluorescent protein (gfp) transgenic mice in which gfp was expressed under the control of th gene promoter. in fetal mice, most gfp-positive neurons expressed th-immunoreactivity in limited brain regions including the locus coeruleus (lc). therefore, lc-containing region was dissected under fluorescent microscopy, and neurons were dispersed by treating with trypsin, then gfp-positive cells were sorted out by flow-cytometry (facs). rna was extracted from the gfp-positive (th) neurons, reverse-transcribed, and analyzed by pcr. atg4b has been shown to play an important role in the processing of lc3, a mammalian homologue of yeast atg8, but the tissue distribution of atg4b remains unknown. to understand the role of atg4b in rat tissue cells, we prepared an antibody to atg4b and pc12 cells in which atg4b expression was knocked down by rnai. in rnaitreated pc12 cells where atg4b expression was 10% of that in the wild-type pc12 cells, the expression of cytosolic lc3-i was similar to that in wild-type cells. the knockdown cell lysates, however, suppressed cleavage of prolc3 to lc3-i. moreover, the expression of atg4b mrna was high in the cerebellum and olfactory bulb, while its protein was evenly distributed in the brain. immunostaining for atg4b was intense in neurons, especially in the cerebellum. these results suggest that atg4b plays a major role in the processing of lc3, while autophagy is deeply associated with the metabolism in neurons, especially in the cerebellum. ps1a-k183 spatial and time-dependent transneuronal propagation of swine coronavirus (hemagglutinating encephalomyelitis virus, hev) in the rat central nervous system after its hind footpad inoculation transneuronal propagation of hev 67n strain into the central nervous system was examined after its subcutaneous inoculation (10 5 pfu) in the rat hind footpad. on day 3 post-inoculation (p.i.), antigenpositive neurons were detected in cell groups of the ipsilateral spinal cord of lumber segments. on day 4 p.i., they increased in number, and higher-order transneuronally infected neurons were observed in restricted brain areas that project to the spinal cord. on day 5 p.i., the viral infection became more extensive and complex, and neurological signs appeared from this period. in this model the 4th day would be critical for the analysis of the long-distance connections. hev can be used as a novel tracing probe, being equivalent to other reported virus probes. hiroyuki hioki 1 , hiroshi kameda 1 , hisashi nakamura 1 , taro okunomiya 1 , koji ohira 1 , kouichi nakamura 1,2 , takahiro furuta 1 , takeshi kaneko 1,2 1 dept. of morphol. brain sci., grad. sch. of med., kyoto univ., kyoto, japan; 2 crest, japan vesicular stomatitis virus g-protein (vsv-g) pseudotyped lentiviral vectors are useful vectors for gene transfer into the central nervous system. vsv-g achieves a broad transduction spectrum and lentiviral vectors provide an efficient vehicle to integrate transgenes into dividing and non-dividing cells. thus, vsv-g pseudotyped lentiviral vectors with ubiquitous promoters, such as human cytomegalovirus (hcmv) promoter, infect and express transgenes in neuronal and glial cells. the purpose in this study is to explore neuron-specific promoters and to quantitatively examine their characteristics. at first, we used five kinds of well-known neuron-specific promoters; hsyn1, rta1, mcamkii, rnse and hpdgf promoters. then, we developed new hybrid promoters by a combination sequence of hcmv enhancer and neuron-specific promoters listed above. all of the new hybrid promoters dramatically improved expression of reporter gene (gfp), but the specificity deteriorated in the rat striatum, thalamus and neocortex. although green fluorescent protein (gfp) is a useful tool to label living neurons, neuronal processes are not completely labeled with gfp. in the present study, we tried to develop dendritic membrane-targeted gfp using non-prolilferative lentivirus vector with human synapsin i promoter. palmitoylation site of gap43 n-terminal and myristoylation site of fyn n-terminal were first tested for membrane targeting of gfp. myristoylated gfp (myrgfp) was efficiently localized at the plasma membrane of infected neurons, but not palmitoylated gfp. since myrgfp was located at both dendritic and axonal membranes, we further added the putative dendrite-targeting or basolateral targeting signals, such as c-terminals of telencephalin (tlc), fc ␥ ii ␤ receptor (fcr), polymeric immunoglobulin receptor (pigr), and low density lipoprotein receptor (ldlr), to c-terminal of myrgfp, and compared their efficiency on dendrite targeting. recently, we developed recombinant rabies virus vectors which were expected to act as a potential neurotracing tool. the vectors infected neurons specifically from axon terminals and were transported to the downstream neurons trans-synaptically. by using two different recombinant vectors, each of which expresses reporter protein of different kind, we attempted double labeling of a neuron. it was expected that we could detect and visualize the divergence or convergence of a neurocircuit. in the study, we could demonstrate the efficiency of double labeling in vivo. in the present study, we examined the potential of this technique particularly in terms of the quantitative detection of double labeled neurons in complicated neurocircuit. the experiments were performed in the hippocampus and the neighboring cortices of rats. we could show that this technique is also useful for the quantitative analysis of neurons which forms projections to different region of the brain. shuchen lee, lihao ge institute of neurobiology, institute of brain science, fudan university, shanghai, china glycine receptors on bullfrog retinal cone photoreceptors were characterized by immunocytochemical and whole-cell patch clamp techniques. cone terminals were both gly␣1 and gly␤ immunoreactive. in freshly dissociated cones, an inward current could be induced while glycine was focally applied to the terminal. the glycine-induced current was strychnine-sensitive and reversed in polarity at a membrane potential, close to the equilibrium potential of chloride ions. these results suggest that glycine, which may be released by glycinergic inplexiform cells, could modulate functions of cone photoreceptors. ␦-catenin has 10 armadillo motifs and a carboxyl terminal type i pdz ligand. in neurons, ␦-catenin is enriched in the postsynaptic density, where it serves as a link between the adherens junction and the post-synaptic protein complex including the nmda and ampa receptors. electrophysiological recordings from ca1 hippocampal neurons overexpressing ␦-catenin demonstrated that ␦catenin increased the ampa receptor-mediated epsc but had no significant effect on the nmda receptor-medicated epsc. the effect of ␦-catenin on the ampar-epsc was medicated by its pdz ligand. in cos7 cells, co-transfection of ␦-catenin/grip showed that ␦-catenin regulated the membrane localization of grip through its pdz ligand. co-transfection of ␦-catenin/grip/gulr2 increased the surface expression of glur2 in cos7 cells compared with grip/glur2 or ␦-catenin/glur2 transfection. this study points to ␦-catenin as a regulator of glur2 receptor trafficking. inseon song, kunihiko obata, alexey semyanov bsi, riken, japan gaba a receptor mediated tonic conductance is a major component of membrane conductance which determines the way how neuron integrates incoming synaptic signals as well as input-output characteristics of the cell. we measured density of picrotoxin (gaba a receptor antagonist) sensitive holding current (which reflects gaba a receptor mediated conductance) in interneurons of hippocampal ca1 area in wild type (wt) and gad65 knockout (ko) mice. this parameter was twice lower in gad65ko mice (wt: 2.0 ± 0.5 pa/pf, n = 7; gad65ko: 0.9 ± 0.3 pa/pf, n = 8; p = 0.038). the total membrane conductance was similar in both types of animals suggesting adaptive compensation. application of gaba (5 m) increased tonic current in both type of mice by the same amount. no significant difference in amplitude or frequency of spontaneous ipscs was detected, although their decay time was shorter in gad65ko animals (wt: 12.4 ± 0.7 ms, n = 14; gad65ko: 10.8 ± 0.6 ms, n = 12; p = 0.047). the changes in inhibition which we have found may explain previously reported behavioral abnormalities in gad65ko. research funds: bsi, riken hiroki mutoh, thomas knopfel lab. for neuronal circuit dynamics, bsi, riken, wako, japan olfactory glomeruli constitute the first stage of central odor processing. yet, their role in integration of odor information is only partially understood. we previously discovered that 5 hz olfactory nerve (on) stimulation induces long-term depression (ltd) in young (p5 to p8) mice. the present experiments were designed to understand in more detail the molecular mechanisms underlying on ltd. bath application of dhpg, a selective group i mglur agonist, induced on ltd and occluded subsequent 5 hz stimulation-induced ltd. on ltd was not induced by activation of group ii or iii mglur agonists. the dhpg-induced on ltd was mediated by mglur1 but not by mglur5 because it was antagonized by the mglur1 antagonist ly367385 but not by the mglur5 antagonist mpep. expression of dhpg-induced on ltd was accompanied by an increase in paired-pulse ratio suggesting that on ltd is caused by a decrease of release probability. we propose that mglur1 is expressed at the on. on ltd may be important for establishment and maintenance of odor maps in the olfactory bulb but may also involve in the regulation of the sensitivity for specific odorants. ps1p-a004 involvement of dopamine system in long-term potentiation of thalamo-prefrontal cortex pathway masatoshi takita 1 , michiko ohtomi 2 1 cognition and action group, national institute of advanced industrial science and technology (aist), ibaraki, japan; 2 department of biomolecular science, faculty of science, toho university, chiba, japan a mesocortical dopaminergic (da) input to prefrontal cortex (pfc) with the d1 receptor is necessary for long-term potentiation (ltp) to occur at hippocampal-pfc synapses, which is involved by working memory (wm) in rats. here the da system was investigated in another wm-involved pathway from mediodorsal nucleus of the thalamus (md) to pfc. preliminarily, local perfusion of the d1 antagonist sch23390 into pfc by using a microdialysis method impaired md-pfc ltp but the d2 antagonist sulpiride did not. extracellular da levels in the pfc robustly increased after the tetanus of md (by 110-120%). as a result both excitatory synaptic inputs to the pfc involved the wm-related da profile, implying da system enables a contrast-emphasis for cooperative crosstalk among several neuroplasticities in the pfc to selectively store intersectional information of multiple brain areas. neuronal activity is necessary for postnatal maturation of synaptic connections only grossly laid out in the neonatal brain. in sensory cortices, synaptic maturation involves strengthening of sensory-evoked responses and development of receptive field (rf) maps with defined rf size and shape. evoked activity is thought to shape synaptic maturation in sensory cortices by mechanisms of competitive hebbian plasticity. dendritic excitability, mediated by voltage-gated na + channels, is required for active backpropagation of axosomatic action potentials (aps) and initiation of dendritic spikes; backpropagating aps and dendritic spikes enable forms of synaptic hebbian plasticity, such as spike-timing dependent plasticity (stdp). here we examined the role of dendritic excitability in synaptic maturation of layer 2/3 pyramidal neurons in the rat somatosensory barrel cortex. in the present study we compared ltp induction in neocortex of captreated and normal rats in present of gaba antagonist, picrotoxin (ptx). the result of present experiment showed that ptx plays an important facilitatory role in the induction of ltp in both normal and cap-treated group. in cap-treated group, in present of ptx, the ltp responses significantly were higher than normal group. we conclude that the enhancement of ltp by ptx can be explained by product of competition between excitatory and inhibitory pathways or synapses. these results suggest that gabaergic system has an important role in synaptic plasticity. also, these results indicated that gabaergic inhibition has been increased in cap-treated group. tohru kurotani, komatsu yukio department of visual neuroscience, research institute of environmental medicine, nagoya university, nagoya 464-8601, japan we showed in previous study that somatic inhibitory synapses of neocortical layer 5 pyramidal neurons undergo long-lasting depression and potentiation depending on the intrinsic firing pattern of the cell that mimics slow wave sleep (sws) and arousal states. in the present study, using a minimal stimulation method, we recorded somatic ipscs from layer 5 pyramidal cells in visual cortical slices prepared from rats at sws like state under urethane anesthesia and in those prepared from rats at arousal state. the average amplitude of somatic ipscs recorded in slices from the former group was significantly larger than that recorded in slices from the latter group. the mean rise time, decay time constant of ipscs and the mean input resistance of the cells were not significantly different between these two groups. the present results further confirmed that the somatic inhibition in neocortical layer 5 pyramidal neurons is bidirectionally modified in accordance with behavioral state. corticothalamic fibers (ct), originated from cerebral layer 6 pyramidal cells, make excitatory synapses with both thalamic relay neurons and reticular neurons. since these pyramidal cells abundantly express kainate receptors (kars) mrna, we studied the effect of kainate on the presynaptic function of the two ct synapses in mouse thalamic vb nucleus. bath application of kainate (200 nm) depressed ct-epscs and increased the paired pulse ratio in relay neurons. in contrast, kainate at the same concentration facilitated ct-epscs and decreased the paired pulse ratio in reticular neurons. these results suggested that kars differentially regulated release at the two ct synapses. furthermore, high frequency stimulation of ct depressed relay cell synapses but facilitated reticular cell synapses. blocking endogenous kars abolished these effects. because reticular cells are the main source of inhibitory input to relay neurons, we suggested that endogenous kars presynaptically regulate the balance of excitatory and inhibitory inputs to thalamic relay neurons. to examine the involvement of ntr1 in the regulatory mechanisms for ltp in the amygdala, we utilized ntr1-knockout (ko) mice. we performed whole-cell patch-clamp recordings from the pyramidal neurons in the basolateral amygdala (bla), where da-nt neurons project. we found that the bla-ltp, induced by la stimulation, was significantly greater in ntr1-ko mice than in wild-type mice. the bla-ltp in ntr1-ko mice was attenuated by sulpiride, a d2 receptor antagonist. these results suggest that d2-ntr1 interaction regulates the extent of ltp in the mouse la-bla synapses. ps1p-a010 facilitation of axonal plasticity in recovery from traumatic brain injury and the role of tnf␣ in mouse model recent studies suggest axonal plasticity as possible mechanism of recovery from brain injury. apart from that, tnf␣, an inflammatory cytokine, has also been suggested to serve neuroprotective roles. the present study evaluated motor function recovery after controlled cortical impact (cci) brain injury, and also the facilitation of plasticity by biotin dextran amine (bda) axonal tracing in tnf␣ko mice and wild type (wt) mice. mice were subjected to left sided cci or served as sham controls, and were evaluated by composite neuroscore and rotarod over 28-day period. bda was injected in right cerebral cortex to observe new axonal connections. so far, we observed recovery of motor function in wt mice, whereas tnf␣ko mice showed continuous functional deficit. we also observed greater number of new axonal connections in wt mice. our results suggest that tnf␣ is necessary for functional recovery after brain injury, and axonal plasticity may be the mechanism involved. disuse of synaptic activity causes homeostatic adaptation presynaptically and/or postsynaptically. here we show that in hippocampal autaptic cultured neurons tetrodotoxin-induced chronic inactivity increases the fraction of high vesicular release probability pool with the entire readily releasable vesicle pool size remained intact. kinetics of short-term plasticity and unchanged apparent ca 2+ sensitivity indicate that ttx-induced presynaptic modification is unlikely due to an increase in the fusion rate crucial for the ca 2+ at the final fusion step. in addition, analysis of neurons genetically lacked the synaptic vesicle protein synaptotagmin-1, and timing-dependent rescues using two different viruses provide a novel conception, namely, vesicle machinery requires prolonged period so that the fast burst vesicle pool orchestrates presynaptic homeostasis system underlying "vesicle mobilization". ps1p-a012 inhibitory modulation of the hippocampal ca3 transmission and plasticity by glucagon-like peptide-2 jun-ichiro oka, takashi iwai lab. pharmacol., fac. pharm. sci., tokyo univ. sci., japan glucagon-like peptode-2 (glp-2) is a proglucagon-derived peptidehormone in the intestine and brain. we reported that glp-2 (i.c.v.) improved the concussive brain injury-or scopolamine-induced amnesia in mice. however, the mechanisms of glp-2 effects on hippocampal neurons are unclear. in this study, we investigated the effects of glp-2 on the synaptic function of neurons in the acute hippocampal slices. hippocampal slices (400 m) were prepared from 7 to 35 days wistar rats of both sexes. patch-clamp recordings were made from pyramidal cells of the ca3 in the whole-cell mode using glass microelectrodes (resistance: 4-8 m ). in extracellular recordings, field excitatory postsynaptic potentials (fepsp) were evoked with a bipolar tungsten electrode, placed in the mossy fibers. glp-2 (10 nm-1 m) inhibited spontaneous excitatory postsynaptic current. glp-2 (10 nm) did not affect fepsp amplitude or the paired-pulse ratio, but attenuated the long-term potentiation. these results suggest that glp-2 may play an inhibitory role in the dg-ca3 transmission. ps1p-b013 quantitative imaging of exo-endocytosis at mossy fiber presynaptic terminals of hippocampus by genetically expressed fluorescent probe takuya hkima, rikita araki, toru ishizuka, hiromu yawo dept. of dev. biol. and neurosci., tohoku univ. grad. sch. of life sci., japan both presynaptic and postsynaptic mechanisms are proposed for the synaptic plasticity. however, the presynaptic mechanisms have been analyzed indirectly on the postsynaptic responses. it has been difficult to quantify the exocytosis at the presynaptic terminals, particularly those in vivo or in acute slices. to measure exocytosis directly, we applied the synaptophluorin (sph) method to the individual presynaptic terminals in hippocampal slices of a mouse genetically expressing a conjugate protein of vamp-2 and superecliptic phluorin selectively in the mossy fiber terminals. the sph fluorescence at individual mossy fiber terminal was increased by nerve stimulation and was followed by its reduction which is blocked by bafilomycin a1, a vesicular h+-atpase inhibitor. therefore, the rising phase of sph fluorescence corresponds to exocytosis whereas the decreasing phase to endocytosis and subsequent re-acidification of vesicles. this method would enable us to evaluate the presynaptic contribution to synaptic plasticity. jyoti parkash, gurcharan kaur gndu amritsar, india we have earlier reported that gnrh nerve terminals in the me continue to express high levels of polysialylated form of neural cell adhesion molecule (psa-ncam) in a cyclic fashion and psa-ncam covers both the gnrh axon surfaces and the associated glial cells in the proestrous phase rats indicating that psa plays important role in the neurosecretory activity in hypothalamus. to further establish the functional significance of psa-ncam molecule, we have studied the expression of psa-ncam on gnrh axon terminals and glial cells in the proestrous phase of cycling rats as well as gaba and pbz treated proestrous rats by using dual immunohistofluorescent staining. both gnrh and psa-ncam immunostaining was much higher in proestrous phase rats, whereas, gaba and pbz treatments significantly reduced their expression. the expression of pst has been studied within gnrh cell bodies as well as at their terminals by combining in situ hybridization with immunohistofluorescent in poa and me-arc regions of cycling female rats as well as in gaba and pbz treated proestrous rats. cortical plasticity has important roles in the development of neural circuits in sensory cortices. however, the roles and mechanisms for various types of ltp and ltd are not clear. we investigated supragranular ltp and two types of supragranular ltd in the slices obtained from the rat auditory cortex, and compared their properties. frontal cortical slices were prepared from male wister rats. supragranular field potentials elicited by the stimulation applied to layer vi were recorded. ltp was induced by tetanic stimulation (ts, 100 hz for 1 s) applied to layer vi. ltd was induced by low-frequency stimulation (lfs, 1 hz for 900 s) applied to layer vi. ltd was also induced by ts applied to supragranular layers near the recording site. lfs-induced ltd and ts-induced ltd were completely abolished in the presence of 50 m apv, 3 m bicuculline, but not 500 m mcpg. lfs-induced ltd and ts-induced ltd occluded each other, suggesting that that both types of ltd share cellular and molecular mechanisms. kazuyoshi kawa department of neurophysiology, tohoku university, graduate school of medicine, sendai, japan using slice-patch techniques, synaptic transmission in neurons of the area postrema (ap) of the rat was studied. when 20 mm kcl was applied from a "y tube" to ap neurons (whole-cell clamped at −10 mv), massive inhibitory postsynaptic currents (ipscs) were induced. most of the evoked ipscs were blocked by bicuculline confirming gabaergic identity. when nicotine (5-100 m) or capsaicin (0.1-1 m) was applied to ap neurons, robust appearance of ipscs with gabaergic identity was induced. after blocking action potential generation in the slice with tetrodotoxin (1 m), nicotine and capsaicin could still induce gabaergic ipscs. interestingly, responses to capsaicin of the synaptic facilitation showed marked desensitization even after 5 min of rigorous washout. it is concluded that nicotinic receptors, as well as capsaicin receptors (presumably, trpv1), are expressed at gabaergic presynaptic terminals in area postrema neurons and play a distinctive role in controlling autonomic neural functions. research funds: grant from the smoking research foundation (japan) takako morimoto-tanifuji 1 , akira komatu 2 , akinao nose 1 1 dept. phys., univ. tokyo, tokyo, japan; 2 dept. physiol., sch. med., tokyo women's med. univ., tokyo, japan the molecular mechanisms that target neurotransmitter receptors to the postsynaptic membrane and keep them clustered remain unknown. we investigated how the localization of glutamate receptors (glurs) is regulated in neuromuscular junctions (nmjs) of drosophila 3rd instar larvae. there are mainly two classes of glurs, containing either gluriia or iib. gluriia has a sequence predicted as ca 2+ -permeable site. when camkii was inhibited by the expression of inhibitory peptide, ala, the content of gluriia in synapses was dramatically increased and the mean amplitude of extrajunctional potential (ejp) was enhanced. the expression of constitutively active form of camkii (t287d) resulted in decreased gluriia content and enhanced gluriib content. although miniature ejp amplitude was reduced, ejp amplitude was normal in t287d expressing larvae, suggesting the existence of some homeostatic mechanisms. taken together, camkii regulates the localization of glurs in a subunitspecific manner and modulates synaptic function in nmjs. ) . notably, neuronal dnrs from dnr1* flies did not show mg 2+ blockade, and dnr1* flies displayed significant impairment in transcription-dependent long-term memory (ltm) but not in transcription-independent acquisition and short-term memory. we identified salient increases in genes involved in l-ltp formation, e.g. homer, and activin, as well as the increase in genes involved in ltm, e.g. staufen, upon ltm formation. however, such increases were absent in dnr* flies. transcription for ltm is mediated, at least, by transcription factors such as creb, adf-1, and notch. we examined how mg 2+ blockade of dnr links to these transcription factors. research funds: kakenhi ps1p-b020 response properties of wind-sensitive giant interneurons in the 4th-instar nymphs of the cricket tetsuya matsuura 1 , masamichi kanou 2 1 dept. of welfare eng., iwate univ., morioka, japan; 2 dept. of biology, ehime univ., matsuyama, japan the response properties of four wind-sensitive giant interneurons (gis) 8-1, 9-1, 9-2 and 9-3 in the 4th-instar nymphs of the cricket gryllus bimaculatus were investigated. air current was presented to the animal from 12 different directions in the horizontal plane. the intensity-response curves showed that the response magnitudes of gi 8-1 increased with stimulus velocity up to 300 mm/s regardless of the stimulus direction. the response magnitudes of gi 9-1 reached a plateau at a stimulus velocity of 30 mm/s in most stimulus directions. the response magnitudes of gis 9-2 and 9-3 increased with stimulus velocity up to 300 mm/s regardless of the stimulus direction. the directional sensitivity curves revealed that the preferential directions of the gis in nymphs were the ipsilateral-side in gi 8-1, the ipsilateralfront and contralateral-rear in gi 9-1, the ipsilateral-rear in gi 9-2 and the ipsilateral-front in gi 9-3, designated with respect to the side of the ventral nerve cord containing the axons, which were basically the same with those of adults. yasuyuki ishikawa, sadao shiosaka division of structural cellular biology, nara institute of science and technology, nara, japan long-term potentiation (ltp) is an enhancement of synaptic strength that may contribute to information storage in the mammalian brain. ltp expression can be regulated by previous synaptic activity, a process known as "metaplasticity." we report a novel form of cellwide metaplasticity in hippocampal area ca1. serine protease, neuropsin, is involved in the regulation of synaptic plasticity. neuropsin increased the stability of ltp induced later at the same inputs via l-type vdcc. moreover, neuropsin-deficient mice impaired l-ltp induction by l-tbs. our findings have revealed the effects of neuropsin on the conversion of e-ltp to l-ltp. ptp, a form of presynaptic short-term plasticity, is mediated by a transient increase in transmitter release probability caused by tetanic stimulation. although it has been known that ptp is induced by the elevation of presynaptic ca 2+ , the molecular mechanism of ptp is poorly understood. in order to elucidate the specific role of presynaptic trkb receptors in ptp, we analyzed ptp using hippocampal slices from conditionally gene-targeted mice in which the knockout of the trkb gene is restricted to presynaptic sites in the ca1 region. we found that ptp induced by the 100-hz tetanus was reduced in mutant mice, and that ptp in control mice was partially reduced by an n-type ca 2+ channel blocker, while ptp in mutant mice was unaltered by the blocker. thus, these data suggest that the n-type ca 2+ channel-dependent component of ptp requires trkb receptor activation. research funds: jsps and mext of japan kiyoshi ohnuma 1 , kunihiko kaneko 1,2 , makoto asashima 1,2 1 grad. sch. of arts & sci., univ. of tokyo, tokyo, japan; 2 jst, tokyo, japan measuring fluctuations or population distributions of a system can be used to understand the dynamics of the system. we have used this approach to study intercellular interaction between neuronal cells. here we show that the shape of the population distribution of intracellular ca 2+ concentration ([ca 2+ ] i ) may change because of nonsynaptic communication. we loaded pc12 cells with a ca 2+ indicator, indo-1 am, and the [ca 2+ ] i of more than 10,000 cells was measured using flowcytometry. the [ca 2+ ] i distribution of unstimulated single cells had a long right tail, suggesting that [ca 2+ ] i is usually low but sometimes becomes high. on the other hand, the distributions of cell clumps and depolarized single cells were bell shaped, suggesting that many ca 2+ -related mechanisms such as channels and pumps were activated by nonsynaptic communication or by depolarization to change the shape into a normal distribution according to the central limit theorem. our results suggest that measuring the distributions is useful in researching intercellular interaction. na x is a sodium channel involved in sensing the sodium level of the body fluid. our recent studies showed that na x is specifically localized to perineuronal lamellate processes of specialized glial cells in the circumventricular organs, the cns organs involved in the sodium reception. however, molecular and cellular mechanisms underlying the sodium reception of the glial cells has not been elucidated. to address this issue, we developed a functional expression system of the channel protein in cultured glial cells, and found that na x enhances glucose uptake and lactate release in an extracellular sodium-dependent manner. these results suggest that na x alters the state of energy metabolism of the glial cells by sensing a physiological increase of the sodium level. the state of inexcitable glial cells thus play a key role for the control of excitable neural cells in the circumventricular organs. we have isolated spinesin/tmprss5 from human and mouse cns. in mouse cns, four isoforms (types 1-4) were expressed. subcellular localization analysis revealed that type 4 (full length) spinesin was predominantly localized to the er, golgi apparatus and plasma membrane, whereas type1 variant was localized to the cytoplasm. furthermore, we performed expression analysis of m-spinesin in some cell lines. nsc34 and nb2a derived from neuronal cell express only type 4, whereas os3 and kt-5 derived from astrocyte express both type 4 and type 1. interestingly, it was observed that the level of spinesin mrna was increased by a dibutyryl cyclic amp treatment only in os3 and kt-5. we analyzed promoter region of m-spinesin gene, and identified that 5 -flanking region from −224 to −188 bp was essential for m-spinesin gene expression. however, this region did not involve camp-dependent regulation of m-spinesin expression. these results indicate that cell-specific expression and regulation of spinesin gene may play multifunctional roles in cns. it has recently been elucidated that l-serine (l-ser) is one of the glia-derived neurotrophic factors in the brain and its biosynthetic enzyme 3-phosphoglycerate dehydrogenase (phgdh), which is the first committed enzyme of l-ser biosynthesis in the phosphorylation pathway, is selectively expressed in glial cells, but not in neurons. since l-ser seems to be important in retinal functions as well, we investigated in the present study the cellular distribution of phgdh in the mouse retina. phgdh immunoreactivity was detected in müller cell soma in internal nuclear layer, being close to internal plexiform layer. immunopositive profiles were cellular processes surrounding rod spherules and retinal neurons in internal nuclear layer through nerve fiber layer. it was suggested that müller cells contribute in l-ser synthesis and its transportation to neurons in the retina. astrocytes frequently show spontaneous intracellular ca 2+ signals, such as intra-and intercellular ca 2+ waves; however, their physiological roles remain elusive. the overexpression of an ip 3 -hydrolyzing enzyme, ip 3 5-phosphatase, suppressed the spontaneous ca 2+ signals in rat hippocampal astrocytes in culture without noticeable effects on their viability. hippocampal neurons were cultured on a monolayer of astrocytes, and their neurite outgrowth was analyzed. the total neurite length and the number of proximal dendrites and branches decreased significantly when neurons were cultured on the monolayer of ca 2+ -signal-deficient astrocytes. moreover, time-lapse imaging revealed that the extension speed of growing neurites was markedly reduced on ca 2+ -signal-deficient astrocytes. these results indicate that spontaneous ca 2+ signals in astrocytes are essential for glial cells to promote neurite outgrowth. katsuyasu sakurai 1 , noriko osumi 1,2 1 tohoku univ. sch. med., japan; 2 crest, jst, japan astrocytes are the most numerous cells in mammalian brain tissues, although factors regulating their structure and function are still poorly understood. we have previously reported that pax6 transcription factor is expressed in gfap positive cells in the rat hippocampus. in the present study, we first investigated the expression patterns of pax6 in postnatal mouse brain and found that pax6 was expressed in almost all astrocytes in the cerebral cortex. to address the role of pax6 in the astrocytes, we examined the morphology of the astrocytes in the wild type (wt) and pax6 heterozygote mutant (sey/+) mice at 8 weeks. confocal imaging revealed that arborization and extension of the astrocytes were poor in sey/+ mice as compared with the wt. in primary culture, the astrocytes isolated from sey/sey cortex showed no morphological difference. however, 6 and 24 h after dibutyryl-camp treatment, the majority of the wt astrocytes had undergone the conversion from a polygonal to stellate shape, while sey/sey astrocytes rarely showed this response. these results suggest that pax6 regulates the morphology of astrocytes, thereby being involved in astroglial functions. we raised mouse monoclonal antibody (mab) dim21 to study its distribution and function in cell membrane and found not only its preferential reaction with ptdglc on tlc, but also its labeling in rodent cns (yamazaki et al., 2006) . we previously reported a unique expression of dim21 ag in developing mouse brain, especially in cell membranes of embryonic radial glia (kinoshita et al., 2005) . we show here that mab dim21 also recognizes adult neural stem cells and glial cells at postnatal period. dim21, brdu and gfap co-expressed in cells of mouse neurogenic subventricular zone. we discuss a possibility that the dim21 ag may be expressed in the radial glia/astrocyte lineage cells. the bone morphogenetic protein (bmp) receptors are thought to have a role in neural patterning of early neuronal development. the bmp receptor is widely expressed throughout the central nervous system (cns) including cerebellum. however, the physiological roles of bmp signaling in mature brain remains obscure. to understand bmp function in cns, we generated a transgenic mouse line that conditionally overexpresses bmp signaling through the type i receptor alk2 (alternatively known as avcri) in a purkinje cell-specific manner using a cre-loxp system. we bred this mouse line with the cre transgenic mouse line of which expression was driven by l7 promoter. tissue specificity of cre recombination was monitored by a bicistronically expressed egfp following a constitutively active alk2 cdna. increased bmp signaling was confirmed by ectopic phosphorylation of smad1/5/8 (p-smads) in purkinje cells. we will discuss functional changes of the purkinje cells which receive excess amount of bmp signaling through alk2. lipopolysaccharide component of the cell wall of certain bacteria is pyrogenic whose administration to spinal cord injured animals was found to inhibit glial scar formation. glial scar being considered as an impediment for axonal growth, it had been proposed in 1950s and 1960s that sub-febrile doses of pyrogen could be considered for spinal cord injury repair research. we tested this ignored hypothesis in paraplegic bonnet monkeys and found that such sub-febrile doses of bacterial pyrogen derived from salmonella typhi was indeed effective in preventing the glial scar formation in short-term and at least prolong the formation of such scar in long term. therefore, pyrogen therapy may be considered as an adjunct to other strategies such as transplantation approaches to treat spinal cord injury. kavita seth, r.k. chaturvedi, s. shukla, a.k. agrawal dev. tox. div., industrial toxicology reserch center, lucknow, india crosstalk between neurons and glial cells (astrocyte and microglia) in neurodegenerative conditions such as parkinson's disease has gained attention of more than supportive interaction. here contribution of glial cells in 6-ohda induced degeneration of dopaminergic neurons was investigated. glial cultures showed significant loss in cell viability after 48 h (34 and 19%) and 72 h (56 and 33%) exposure to 10 −4 and 10 −5 m 6-ohda respectively. it was accompanied by morphological changes and induction of gfap, s-100 and ox42. 6-ohda (10 −6 m, 72 h) was found to cause a significant impairment in 3 h glutamic acid uptake (31%) and gsh levels (27%). further neurons (in coculture with 6-ohda pre exposed glial cells) on exposure to 6-ohda (10 −6 m), showed loss of th expression and significant neuronal cell death (34%). the results of the present study suggest that 6-ohda may impair glial cell functioning, which eventually affect neuronal fate making them more vulnerable toward toxic insults. nestin is an embryonic intermediate filament component, which is transiently expressed by the immediate precursors to neurons and glia during brain development. we studied nestin distribution in the olfactory system after injection of diethyldithiocarbamate in adult rats to cause reversible lesion of the olfactory epithelium (oe). the oe presented a near-complete destruction at 1 day after injection, then started to repair at 3 days and returned to the normal levels at 6 weeks. nestin was expressed in olfactory ensheathing cells (oecs) of the olfactory mucosa at 3∼7 days, but not in those of the olfactory bulb (ob). simultaneously strong expression of nestin was detected in certain population of astrocytes in glomeruli. the reversion of astrocytes in glomeruli to immature phenotype may reflect their involvement in reinnervation of glomeruli. (ng2) is currently considered a marker of multipotent progenitor cells in the brain. in the present study, most iba1+ cells accumulated in stab wounds and ischemic lesions were found to express ng2, of which molecular weight of its core protein was higher by 10 kda than that of ng2 expressed in contralateral brain region. this was due to the lack of shedding of ng2 in the brain lesions. we found that iba1+ cells accumulated in stab wounds and ischemic core lesion, most of which were ng2+/pdgfra+. furthermore, some of these cells expressed gfap, nestin, cd163 and von willebrand factor. ng2+ mg isolated from stab wounds often formed cell aggregates bearing alkaline phosphatase activity turned into cells with neuroectodermal phenotypes in serumfree culture medium. these variety of antigens expressed by ng2+ mg in brain lesions may be related to their multipotentiality to regenerate damaged brain tissue. saroj sharma, l.k. singh, b. ray, t.s. roy 1 all india institute of medical sciences, india oculomotor nerve (on) supplies most of the extra-and intraocular muscles. it shows changes with normal ageing, metabolic and degenerative diseases. though there are various studies on the on, no definitive data regarding the morphometry and the fine structure is available. so, in the present study, neural and the connective tissue organization of the extradural part of the on from 20 cadavers were studied. light microscopy revealed multi-fascicular nerve with myelinated fibers of various calibers. small sized myelinated fibers were noted at the junction of the central and the paracentral zone of most of the nerves. using unbiased stereology techniques the size of myelinated fiber axonal areas showed a multi-modal distribution and presented range from <1 to 40 m 2 . most of the fibers were myelinated and counts produced a mean of 16,891 (12,000-21,500). ultrastructurally, difference in the compactness of arrangement of connective tissue was observed with advancing age. the cell junctions of the perineurial cells and the endoneurial capillaries were observed. myelin thickness ranged from 0.29 to 3.16 m (from fetal age to 60 years age). during the development of the drosophila visual system, retinal axons project to the optic lobe through the optic stalk. the optic stalk is composed of glial cells and adopts tube-like structure. fak is a non-receptor protein tyrosine kinase involved in many aspects of cell behavior including cell migration through the regulation of actin or microtubule dynamics. in drosophila fak (dfak) mutant animals, the optic stalk was abnormally broadened and retinal axons were defasciculated. cdgapr encodes one of gaps that regulate rho-family gtpases. putative cdgapr mutants showed dfak-like phenotype. since dfak and cdgapr interacted genetically, they are likely to act in the same signaling pathway to regulate cytoskeletal rearrangement via rho-gtpases. tissue specific rescue experiment showed that dfak autonomously acts in the glial cells. our results suggest that dfak and cdgapr regulate glial cell rearrangement to establish precise tubelike structure of the optic stalk and organized retinal axon projection. astrocytes are thought to be active participants in synaptic plasticity in the developing nervous system. spontaneous gabaergic postsynaptic activity is reported to be decreased in small neurons of the caudal nts at the end of the first postnatal week. to investigate whether astrocytes might be involved in this phenomenon, we examined developmental expression of gfap, an astrocytic marker. gfap began to be immunohistochemically expressed in the caudal nts at p5-10. costaining with calbindin, a marker for a certain type of small neurons, showed that gfap positive processes were thereafter closely apposed to soma of small calbindin neurons. electron microscopy showed that some astrocytic processes were interposed between orphan gabaergic varicosities and soma of small neurons at the specific developmental stages. these findings indicate that astrocytes may participate actively in regulating the postnatal differentiation of local neural network of the caudal nts. hitoshi ozawa, naoyuki yamamoto, nobuhiko sawai, hao-gang xue department of anatomy and neurobiology, nippon medical school, tokyo, japan it is well known that the hypothalamo-pituitary-adrenal (hpa) axis is an important system for responding and mediating the stress. in addition, hippocampus is also an important area for the stress response. in the hippocampus, the expression of glucocorticoid receptor (gr) has been reported in the ca1, ca2 pyramidal neurons and the dentate gyrus neurons. on the other hand, while astroglia around the hippocampus also expresses gr, the morphological and functional changes under different corticosteroid condition have not been well elucidated. in the present study, we investigated morphological changes of astroglia around pyramidal neurons. under the lack of corticosteroids, astroglia showed well developed morphology with the spread fibrous processes, however the changes recovered to the control level with corticosterone replacement. these suggested that the astroglia were directly regulated by glucocorticoids as associated with the changes of hippocampal neurons. ps1p-d042 impact of s100b on hippocampal spontaneous activities in anesthetized and epileptic conditions seiichi sakatani 1 , akiko seto-ohshima 2 , shigeyoshi itohara 2 , hajime hirase 1 1 neuronal circuit mechanisms research group, japan; 2 lab. for behavioral genetics, bsi, riken, wako, japan s100b is a calcium binding protein mainly expressed in astrocytes and has a role in synaptic plasticity and learning. in order to assess the physiological roles of s100b, we have recorded hippocampal spontaneous activities from urethane anesthetized s100b ko and wt mice. typical eeg patterns including theta (3-8 hz) and sharp wave associated fast ripple (120-180 hz) oscillations were observed in both populations and these patterns were indistinguishable between the wt and ko. when epileptic activity was induced by kainic acid (i.p.), a difference appeared in ca1 radiatum, where ictal event was characterized by hyper-synchronous gamma band (30-80 hz) activity. while both populations developed ictal event within 40 min, mean power during the development was significantly smaller in ko mice. our results suggest that deficiency of s100b does not have a profound impact on neural activity in normal conditions. however, when neural activity was raised, activation of s100b related pathways could potentially be activated. yoshiko takagishi 1 , erina okabe 2 , xiaoyang sun 1 , sen-ichi oda 2 , yoshiharu murata 1 1 riem, nagoya univ, nagoya, japan; 2 grad sch bio-agricult sci, nagoya univ, nagoya, japan shambling (shm) is a spontaneous mouse mutation that causes neurological and motor deficits, characterized by ataxia and the hind limb paralysis. we have recently identified the shm gene that encodes caspr, which constitutes paranodal junction of myelinated nerves. to determine whether the mutation alters the node of ranvier, we performed morphological analysis of myelinated nerves in shambling mice. by electron microscopy, we found that paranodal loops were disorganized and septate-like transverse bands were absent in mutant mice. immunohistochemistry revealed that caspr was diffusely located at the paranodal region, though the staining was extremely weak in mutant sciatic nerves. contactin, a component of the paranodal junction, was distributed similar pattern to that of capsr. further, k + channels were mislocalized to the paranode, while na + channels were normally restricted to the node. these findings suggest that the mutation disrupts the paranodal structure and may disturb salutatory conduction of myelinated nerves in shambling mice. ps1p-d044 regulation of hippocampal neurocircuit activity by glutamate transporter glt-1 noriko koganezawa, shinsuke muraoka, ken-ichiro tsutsui, toshio iijima div. systems neurosci. tohoku univ. grad. school of life science, japan glial cells are now recognized as an essential functional element in synapses. in order to investigate their function, we focused on the activity of glt-1, the glutamate transporter which is expressed in the astrocytes of hippocampus, in the rat brain slice preparations. response to an electrical stimulation of the schaffer collaterals was recorded using the optical imaging technique. by combining the application of the glutamate receptor blockers (nbqx, ap5) and the glt-1 blocker (dhk) with the signal subtraction, we could visualize the activity of glt-1 as a slow, tonic rise of the optic signal following electrical stimulation. then we evaluated the function of glt-1 by applying its blocker dhk. an obvious reduction of neural activity was observed in the hippocampal neurocircuit after application of dhk. furthermore, the blocking of glt-1 function in the ca3 region was elicited by much lower concentration of dhk than that in the ca1 region. ps1p-d045 monoclonal antibody rip specifically recognizes 2 , 3 -cyclic nucleotide 3 -phosphodiesterase in oligodendrocytes the antigen recognized with monoclonal antibody (mab)-rip has been used as marker for oligodendrocytes and myelin sheaths. however, the rip-antigen has been unknown yet. to identify the rip-antigen, we performed immunopurification with mab-rip using the differentiated cg-4 cells lysate. maldi-qit/tof ms n analyses revealed that one of molecules was 2 ,3 -cyclic nucleotide 3phosphodiesterase (cnp). immunocytochemical and immunohistochemical studies showed that rip-antigen colocalized with cnp in rat cerebellum, cultured rat oligodendrocytes and cg-4 cells. moreover, the same localization was also observed in rat cnp1 transfected hek293t cells. overall we first demonstrated that the antigen labeled with mab-rip is cnp in oligodendrocytes. the expression of bdnf gene is regulated by four promoters (pi-piv), and is under activity-dependent control. until now, it has been established that bdnf pi is activated by ca 2+ signal via cre. on the other hand, neuron-restrictive silencer cis-element (nrse), located in bdnf pii, represses bdnf gene expression through binding nrsf and recruiting hdac in non-neural cells. here, we found that nrse repressed the activity of bdnf pi in neuron. using rt-pcr and chip assay, the bdnf exon i expression and the histone acetylation of bdnf pi were increased by the administration of ca 2+ signals or hdac inhibitor. in addition, nrsf bound to bdnf pii in neurons but was detached by ca 2+ signals. these results suggest that bdnf pi activity is regulated by creb and nrsf through an alteration of chromatin structure. since creb and nrsf are playing an important role in neuronal differentiation, it is considered that the bdnf pi is deeply involved in the regulation of neurogenesis. singo suzuki 1,2 , hisatsugu koshimizu 1 , megumi kashihara 1 , tomoko hara 1 , masami kojima 1,2 1 research institute for cell engineering; 2 sorst, jst, japan brain-derived neurotrophic factor (bdnf) plays a crucial role in synapse development, especially, in the central nervous system (cns) . although this concept is now accepted extensively, the underlying molecular mechanisms are poorly understood. here we show that 3-day treatment with bdnf leads to a significant increase in cholesterol content in primary neuron. this change was in its dose-dependent manner and blocked by co-application of a cholesterol synthesis inhibitors. to understand the molecular relationship between cholesterol content and synapse development, we estimated the amount of cholesterol and sv proteins in lipid raft fractions prepared from cultured cortical neurons. the results indicated that bdnf treatment increased the amount of cholesterol and sv proteins in lipid rafts, but not in non-rafts fraction. these data suggested a possibility that bdnf regulated synapse development by increasing the amount of cholesterol and sv proteins in synaptic rafts. (p38) is known to be expressed in the cells of the central nervous system, and supposed to be involved in the control of cell proliferation, differentiation and survival. in this study, we found that p38 expressed in the neural progenitor cells at embryonic 14 days (e14) mice brain. to ascertain the function of p38 on these cells, we treated the cultured e14 cells with the selective chemical inhibitor for p38 (sb203580) for 7 days, and determined the number of neural progenitor cells. the inhibitor specifically enhanced the number of neural progenitors compared to the control cells. this result suggests the involvement of p38 in the proliferation and/or survival of neural progenitor cells in developing mouse brain. hemragul sabit 1 , takashi yamazaki 1,2 , takeshi oya 1 , yoko ishii 1 , masakiyo sasahara 1 1 pathology ii, university of toyama, toyama, japan; 2 oral and maxillofacial surgery, university of toyama, toyama, japan purpose: we had reported the increase of pdgf-b and active src in rat peripheral nerve regeneration. here examined activation of pdgf receptors (pdgfrs) and signals in the peripheral nerve regeneration. method and result: crushed sciatic nerve was removed on 3 to 28 days after injury, and activation of pdgfrs, mapks, akt and p38 were examined by phosphoprotein purification kit (qiagen) and western blot. expression of pdgfrs increased from 3 to 21 days after injury. p-tyr was highly detected from 3 to 21 days after injury, and activation of pdgfrs also increased during this period. activation of erk and jnk increased up to 7 days after injury and then gradually decreased. activation of akt and p38 continuously increased from 3 to 28 day after injury. conclusion: pdgfrs and their signals were activated in rat peripheral nerve regeneration. autocrine signal of pdgf may contribute to the regenerative processes, such as proliferation and differentiation of schwann cells and axonal extension. cbln1 is a cerebellum-specific protein structurally related to the c1q and tumor necrosis factor families. recently, we have shown that cbln1 is secreted from cerebellar granule cells (gc) and controls synaptic structure and plasticity of gc-purkinje cell (pc) synapses. however, because cbln1 was previously shown to serve as a precursor of a pc-specific peptide cerebellin, it remains unclear whether cbln1 needs to be processed before it trans-synaptically activates signaling pathways in pc. here, we show that purified recombinant cbln1 proteins, which formed a hexamer, preferentially bound to spines on pc dendrites. furthermore, cbln1 mutants that did not form a hexamer lost the binding affinity to pc spines. although cerebellin peptide may also contribute to different aspects of signaling, these results indicate that cbln1 released from gc directly bind to postsynaptic pc as a hexamer and activates signaling pathways in pc. activity-dependent gene expression in neurons contributes to expressing a variety of neuronal functions including a long-lasting neuronal plasticity. recently, we found that specific kinds of mrna can be stabilized in an activity-dependent manner. to elucidate the mechanisms for activity-dependent mrna stabilization, we have focused on bdnf, which is a member of neurotrophin family and plays an important role in exerting neuronal functions. we constructed firefly luciferase gene fused to 3 -untranslated region (utr) of bdnf mrna to investigate the effect of the 3 utr on the calcium signal-mediated mrna stabilization. in cultured neurons, we found that the degradation of firefly luciferase-bdnf3 utr mrna induced by the treatment with actinomycin d was prevented by calcium signals evoked via l-type voltage-dependent calcium channels (l-vdccs) and nmda receptors. we are now investigating to identify the cis-regulatory elements involved in the calcium signal-mediated stabilization of bdnf mrna using a series of mutant bdnf3 utr. recently, it has been established that bdnf and pacap regulate the expression of a group of genes which encode proteins involved in expressing neuronal functions. in this study, we found that the treatment of cultured rat cortical neurons with bdnf or pacap acutely induced the mrna expression of the activityregulated cytoskeleton-associated protein (arc) and homer1a, whose products are necessary for the synaptic plasticity. bdnf induced arc mrna expression through the activation of trkb-erk/mapk pathway, whereas pacap induced it partly through the activation of nmda-receptors. using affymetrix genechips, we are now investigating a comprehensive profile of gene expression controlled by bdnf or pacap in cultured rat cortical neurons. ps1p-e056 bmp4 expression in the adult rat brain bone morphogenetic protein-4 (bmp4) is a member of the transforming growth factor ␤ (tgf-␤) superfamily and plays important roles in multiple biological event. although bmp4 expression has been well described in the early development of central nervous system (cns), little information is available for its expression in the adult cns. we, thus, investigated bmp4 expression in the adult rat cns using immunohistochemistry. bmp4 is intensely expressed in most neurons and their dendrites. in addition, intense bmp4 expression was also observed in the neuropil of the gray matters where high plasticity is reported, such as the molecular layer of the cerebellum and the superficial layer of the superior colliculus. furthermore, we found that astrocytes also express bmp4 protein. these data indicate that bmp4 is more widely expressed throughout the adult cns than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that bmp4 plays pivotal roles also in the adult brain. ps1p-e057 hgf as a target-derived trophic factor for rat nigro-striatal dopaminergic (da) system during post natal development wakana ooya, hiroshi funakoshi, toshikazu nakamura div. molecular regenerative medicine, osaka univ. grad. sch. med., osaka, japan hgf is a novel neurotrophic factor in vitro on da neurons. however, little is known about expression and biological activities of hgf in nigral-striatal system in vivo. here we show that hgf is a targetderived trophic factor for rat da system. real-time rt-pcr revealed that c-met mrna was expressed in substantia nigra (sn) and striatum (str), while hgf mrna was expressed in str but not in sn in programmed cell death period. hgf, c-met, phospho-c-met, th, da transporter immunostaining revealed the presence of concentration gradient of hgf from sn to str and c-met was phosphorylated in da nerve end during early postnatal development. phospho-c-metpositive da neurons decreased at later developmental stage, while it became prominent in oligodendrocytic leanage. hgf application into str increased da neuronal number and neurites and modified oligodendrocyte maturation, while opposite effects were observed by the application of blocking antibody for hgf. therefore, hgf may be a critical trophic factor for nigro-striatal da system development. the neuroprotective effects of g-csf were reported in neurological disease models. in the present study, we examined whether g-csf can protect dopaminergic neurons from mptp-induced cell death in a pd. the mice were intraperitoneal injected with mptp for five consecutive days, g-csf is intraperitoneal administered two days and one day before first mptp injection, and 30 min before each mptp injection. in our results, g-csf significantly prevented mptpinduced loss of th-positive neurons, and increased bcl-2 protein, decreased bax protein expression. these findings suggest that g-csf has therapeutic potentiality to protect mptp-induced cell death through increasing the level of bcl-2 expression, decreasing the level of bax expression in c57bl/6 mice. kazue takahata has been shown to increase the expression of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, and the activity of superoxide dismutase 1. here, we evaluated the effects of (−)-bpap on the phosphorylation of mitogen-activated protein kinase (mapk) and akt in slice cultures as well as in an in vivo model. (−)-bpap significantly increased phosphorylation levels of mapk, but not those of akt. (−)-bpap attenuated the decrease in nigrostriatal tyrosine hydroxylase immunoreactivity of 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine-treated mice. (−)-bpap may exert antiparkinsonian activity through neuroprotective effects on dopaminergic cells in addition to catecholaminergic enhancement in parkinsonian substantia nigra. shyuichi maeda 1 , yoko tohyama 1 , shinichi kohsaka 2 , tadashi kurihara 1 , kazuyuki nakajima 1,2 1 soka university, hachioji, tokyo, japan; 2 dept. of neurochem., national institute of neuroscience, kodaira, tokyo, japan astrocytes (ast) are a cell type that supports cns not only nutritionally but also neurotrophically, by supplying neurotrophic factors (ntf) required in the neuronal survival, maturation and protection. however, the ability of ast to produce/secrete ntf has not been accurately known. thus, in the present study, we investigate the capacity of ast to produce/secrete various ntf in vitro. ast were prepared from the mother culture of neonatal rat brain. the ntf in the conditioned medium (cm) were detected by immunoblotting. the analysis of neurotrophins in the cm revealed that ast produce/secrete ngf, bdnf and nt-3, and promote the production of them by stimulation with lipopolysaccharide (lps). furthermore, tgf␤2 among tgf␤ family was detected in the cm of ast, and the production was enhanced by stimulation with lps. these profiles of ast were different from those of microglia, suggesting the differential regulation of ntf by glial cells. ritsuko katoh-semba 1 , chiaki nakagawa 1 , masako tsuzuki 1 , motoko matsuda 1 , satoshi ichisaka 2 , yoshio hata 3 1 inst. dev. res., aichi human ser. ctr., kasugai, japan; 2 div. neurobiol., tottori univ., sch. med., yonago, japan; 3 div. integrative biosci., tottori univ. grad. sch. med., yonago, japan the sleep-awake rhythm is formed during the early period after birth. the rhythm is very important for the functional development of brain. when the rhythm formation is disturbed, autism-like behaviors are often observed. brain-derived neurotrophic factor (bdnf) is known to be one of the factors forming circadian rhythms. we have found increases in levels of bdnf in the entorhinal cortex as well as the visual cortex from adult male rats 12 h after beginning an 8-h phase advance of the light-dark cycle. here we planned to reveal the effects of the phase advance on neurotrophins in juvenile rats. we first examined circadian changes in the concentrations of bdnf and neurotrophin-3 (nt-3) in selected brain regions from 14-day-old male rats and compared to those from adults. the changes in levels of bdnf and nt-3 were observed in the neocortex and hippocampus. objective: this study was aimed to investigate the possible beneficial effects of granulocyte colony stimulating factor (g-csf) compared to methylprednisolone (mp) in experimental spinal cord injury (sci). materials and methods: (in vivo) adult female sprague-dawley rats had moderate sci (200 kdyne, ih injury device) at t8/9 and were assigned to three groups; a (placebo), b (mp treated; 30 mg/kg i.v. immediately after injury), c (g-csf treated; 15 g/kg i.v. for 5 days after injury). animals were assessed with the bbb locomotor rating scale for 6 w post injury and then killed for assessment of tissue sparing around the lesion. result: the behavioural recovery rates of the group c was as good as group b and significantly better than that of group a. morphological assessment showed better tissue sparing in group b and c compared to group a. these results suggest that g-csf is a possible neuroprotective agent in sci. research funds: kakenhi (16390427) ps1p-e064 glutamate signals enhance the expression of rnase-l in primary cultured cortical neurons of mice chie sugiyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan mitochondrial dysfunction results from a decline in the mitochondrial rna (mtrna) transcripts and mitochondrial enzyme activity, as well as from mitochondrial dna (mtdna) damage. to evaluate involvement of mtdna expression in glutamate-induced neuronal death, in this study, we examined the effects of glutamate exposure on mtrna level in cultured cortical neurons of mice. cultured neurons (12 div) exposed to glutamate for 15 min at 100 m. glutamate exposure led to a decrease in mrna of nd1 and nd6, which are subunits of nadhubiquinone oxidoreductase, before cell death. since mtrnas level is regulated at least in part by rna degradation mediated by rnase-l, we next examined the effect of glutamate on expression of rnase-l. rt-pcr analysis revealed that glutamate was effective in increasing the level of rnase-l mrna at least 2-12 h after treatment. the increase in the expression of rnase-l was abolished by the nmda receptor antagonist mk-801. these results suggest that the activation of nmda receptor by glutamate reduces mtrna level probably through enhanced expression of rnase-l in cultured cortical neurons. yuka gotoh, kiyokazu ogita dept. pharmacol, setsunan univ, osaka, japan expression of dj-1 is enhanced by oxidative stresses. although exact functional significance of dj-1 has still unknown, it is thus proposed that dj-1 is protective against neural damage under oxidative stresses. in this study, we tested expression of dj-1 in the hippocampus damaged by trimethyltin (tmt) treatment in mice. tmt was systemically injected into mice to cause neural damage in the dentate gyrus selectively. immunohistochemical analysis indicated that dj-1 was markedly increased in the molecular layer of the dentate gyrus on days 4 and 14 post tmt injection. on day 14 post tmt injection, enhanced expression of dj-1 was observed in the stratum lucidum of the ca3. in glutathione-depleted mice, tmt was more effective in enhancing expression of dj-1, compared with that in untreated mice. furthermore, double staining of dj-1 and gfap demonstrated that most of cells highly immunoreactive to dj-1 were co-localized with gfap in the dentate gyrus of tmt-treated animals, but not of untreated animals. these results suggest that dj-1 is enhanced in the dentate astrocytes activated by tmt treatment. kei higuchi, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan the systemic administration of trimethyltin (tmt) is known to induce granule cell death in the dentate gyrus of mice. we have previously shown that an injection of tmt (2.8 mg/kg, i.p.) led to significantly reduction of granule cells in the dentate gyrus 2 days later, with visually apparent recovery of the granule cell layer 14 days afterward. in this study, we examined the effects of glucocorticoids on tmt-induced damage in the dentate granule neurons of mice. tmtinduced neuronal cell damage was assessed by the immunohistochemical analysis using an antibody against single-stranded dna. the systemic injection of dexamethasone (0.5-5 mg/kg) led to a significant reduction in neuronal damage induced by tmt in the dentate gyrus. the neuronal damage induced by tmt at the dose of 2.0 mg/kg was enhanced by adrenalectomy. dexamethasone was effective in completely preventing this neuronal damage in adrenalectomized animals. taken together, these results suggest that glucocorticoid released from adrenal cortex may be capable of protection against tmt-induced dentate granule cell death in mice. masami ishido national institute for environmental studies, tsukuba, japan melatonin, a secretory product of the pineal gland, has antitumor activities and is involved in the regulation of circadian, seasonal rhythms and in inducing osteoblast differentiation. furthermore, melatonin is reported to be a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. in this chapter, antioxidant nature of melatonin was demonstrated to prevent the cultured neural cells from apoptosis induced by endocrine disrupting chemicals, maneb. neurotoxicity of maneb (1 g/ml) on the pc12 cells was elicited through apoptotic cell death. activation of caspase-3/7 was associated with this process. a fluorescence rationing technique using mitochondrial dye revealed that maneb altered mitochondrial membrane potential of the neural cells. however, melatonin (1 nm) could largely prevent the neural cells from the neural toxicant by inhibition of both caspase-3/7 activation and disruption of the mitochondrial transmembrane potential. thus, melatonin could be a powerful free radical scavenger against manebcaused mitochondrial dysfunction in pc12 cells. ps1p-e068 kinesin superfamily protein 4 (kif4) regulates activity-dependent neuronal survival by suppressing parp-1 enzymatic activity ryosuke midorikawa, yosuke takei, nobutaka hirokawa department of cell biology and anatomy, university of tokyo, tokyo, japan in brain development, apoptosis is a physiological process that controls the final numbers of neurons. here we report that the activitydependent prevention of apoptosis in juvenile neurons is regulated by kinesin superfamily protein 4 (kif4), a microtubule-based molecular motor. the c-terminal domain of kif4 is a module that suppresses the activity of poly (adp-ribose) polymerase-1 (parp-1), a nuclear enzyme known to maintain cell homeostasis by repairing dna and serving as a transcriptional regulator. when neurons are stimulated by membrane depolarization, calcium signaling mediated by camkii induces dissociation of kif4 from parp-1, resulting in upregulation of parp-1 activity, which supports neuron survival. after dissociation from parp-1, kif4 enters into the cytoplasm from the nucleus, and moves to the distal part of neurites in a microtubule-dependent manner. we suggested that kif4 controls the activity-dependent survival of postmitotic neurons by regulating parp-1 activity in brain development. research funds: 1684304 ps1p-e069 expression of hsp105, apg-1, and apg-2 in the hippocampal neural cells by trimethyltin masanari orita, kiyokazu ogita dept. pharmacol, setsunan univ, osaka., japan we tested changes in expression of high-molecular-weight heat shock proteins (hsps) in the hippocampal dentate gyrus in vivo and in the cultured cortical neurons in vitro after trimethyltin (tmt) treatment, which caused neuronal damage in the dentate gyrus and cultured hippocampal neurons. tmt (2.8 mg/kg) was systemically injected into mice, and then an immunohistchemical analysis was performed to identify cells immunoreactive to antibodies against hsps, neun, and gfap in coronal sections of hippocampus. tmt was effective in enhancing the expression of hsp105, apg-1, and apg-2 in the granule cell layer of the dentate gyrus, but not in ca1-ca3 pyramidal cell layer, 16 h to 2 days later. double staining of neun and these hsps revealed that these hsps expressed by tmt almost co-localized with neun in granule cells of the dentate gyrus. whereas hsp105 highly expressed in survival neurons in the culture, apg-1 and apg-2 highly expressed in damaged neurons with nuclear condensation. taken together, the high-molecular-weight hsps may be involved in neuronal survival and damage caused by tmt. brain irradiation is often performed in patients with brain tumors. however, little has been known about radiosensitivity of neurons, especially in the developmental stages. in this study, we investigated the effect of irradiation on immature neurons with that on mature neurons. primary neuronal cultures were prepared from fetal rat hippocampi at embryonic day 18. thirty gray of x-irradiations were performed on the cultured cells at 7 or 21 days in vitro (div). then the cells were fixed at 24 h after the irradiation with dapi. at 7-div, irradiation significantly increased the number of nuclear pyknosis of neurons. in contrast, radiation did not induce any nuclear pyknosis of neurons at 21-div. this indicates that the radiosensitivity of 7-div immature neurons is higher than that of 21-div mature neurons. glutamate receptors are believed to be involved in various neurological disorders via its excitotoxicity. ataxic mice lurcher (lc) are caused by a mutation in the ␦2 glutamate receptor (glur␦2), which shows constitutive channel activities in purkinje cells and leads to the cell death. thus, lc is the first example of neurodegeneration caused by chronic excitotoxicty. interestingly, glur␦2 is also suggested to regulate autophagy via its association with beclin. however, it is unclear how excitation caused by constitutive channel activities is related to the autophagic pathway and cell death. here, using heterologous cells in vitro, we show that continuous influx of na + , but not ca 2+ , was necessary and sufficient to induce autophagic cell death. in addition, we found that intracellular atp levels and subsequent activation of map kinase are involved in this process. junko taniguchi a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins that are ubiquitinated and often associated with various transcription factors, chaperones and proteasome components. but, how the expanded polyglutamine proteins or their aggregates elicit a complex pathogenic responses in the neuronal cells are not fully understood. here, we demonstrate that the expression of expanded polyglutamine proteins down-regulates the nf-kb-dependent transcriptional activity. expression of expanded polyglutamine proteins increases the stability and the levels of ikb-a and its phosphorylated form. we have also found that various nf-kb subunits and ikb-a aberrantly interacts with the expanded polyglutamine proteins and associates with their aggregates. finally, we have shown several nf-kb-dependent genes are down-regulated in the expanded polyglutamine protein expressing cells. molecular mechanisms for selective neuronal death in polyglutamine diseases remain to be clarified. by microarray analysis, we compared gene expression profiles in cerebellar granular cells under expression of normal and mutant ataxin-1 and found a novel gene down-regulated in response to mutant ataxin-1 in cerebellar granular neurons. we named the novel gene maxcell (mutant ataxin-affected gene in the cerebellum). nothern blot shows that maxcell mrna in human brain is expressed in cerebellum and cerebral cortex. immunohistochemistry with anti-maxcell antibody shows cytoplasmic stains of neurons but not glial cells in mouse brain. confocal microscopy shows that maxell-egfp is colocalized with ribosomal protein s6 as a ribosomal marker. we are analyzing the function of maxcell protein that might relate to sca1 molecular pathology. ps1p-f080 permeability transition in mitochondria isolated from cold perfused brain and spinal cord-a detailed comparison of calcium sensitivity the purpose of this study was to compare the sensitivity of isolated brain and spinal cord mitochondria to ca 2+ -induced permeability transition (mpt). the spinal cord is more traumatized than the brain during the extraction because it takes more time. in order to minimize confounding factors, we induced severe hypothermia in animals prior to removal of tissue and isolation of mitochondria. sensitivity to ca 2+ -induced mpt was evaluated in brain and spinal mitochondria in energized and de-energized model of swelling with or without mpt inhibitor, cyclosporin a (csa). the present findings imply that the general features of mpt are similar in brain and spinal cord mitochondria and that mpt may be an important pharmacological target in disorders affecting the spinal cord. the role of cyclophosphamide monohydrate (cp), which is known as an immunosuppression drug, in the central nervous system (cns) has not been elucidated. in the present study, we found that treatment with cp prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, cp exposure induced the activation of both the map kinase (mapk) and pi3 kinase (pi3k) pathways. interestingly the up-regulation of bcl-2, a survival promoting molecule was observed after cp treatment. these observations suggest that cp protects cns neurons from neuronal damage through intracellular signaling pathways. the research of cell death mechanisms has rapidly progressed. however, cell death is an inherently difficult process to measure. to investigate the roles of cell death in vivo, we introduced scat3 probe. scat3 is an indicator protein for caspase-3 activation that uses fret between two types of fluorescent protein, ecfp and venus, linked by a peptide containing the caspase-3 cleavage sequence. using this probe, we could monitor the activation of caspase-3 at the singleneuron level in culture. we will discuss about neural cell death through the detection of caspase activity. keiichi seko, koichi kawada, chie sugiyama, masanori yoneyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan trimethyltin chloride (tmt) is a kind of organotin derivates that are known to induce neuronal damage in human and rodent. in this study, we examined tmt-induced neuronal death in mouse primary cultured cortical neurons in vitro and the frontal cortex in vivo of mice. in vivo analysis using mice revealed that injection of tmt (2.8 mg/kg, i.p.) led to an increase in single-stranded dna-positive cells, as well as in dnase ii-positive cells, in the frontal cortex 2 days later. in cortical neurons, tmt exposure for 48 h led to a marked decrease in the cell viability, as well as to an increase in nuclear condensation and ldh released. tmt exposure was effective in activating dnase ii in the nucleus. in addition, caspases 3 and 8, but not caspase 9, were significantly activated by tmt treatment. cytochrome c release was not affected by tmt treatment. the caspase inhibitor zvad-fmk completely prevented tmt-induced neuronal death. these results suggest that tmt-induced neuronal death is involved in caspases and dnase ii activated by mitochondria-independent pathway in cortical neurons. we investigated the pattern of hippocampal damage and the levels of brain polyamines after systemic injections of trimethyltin (tmt) chloride (2.5 mg/kg, i.p.) in 3-(3w) and 7-week-old (7w) icr mice. in addition, we measured the brain tin level following tmt injection. tmt induced marked, localized cell death in granule neurons of the dentate gyrus in 3w mice. by contrast, slight, diffuse neuronal damage was found in the ca1 and ca3 subfields and dentate gyrus of 7w mice. the hippocampal putrescine level was elevated markedly in 3w mice on tmt administration, whereas a minor putrescine increase was detected in 7w mice. there was no difference in the brain tin level between these two age groups. these results revealed the age-dependent vulnerability of mice hippocampal neurons to tmt administration, and suggest that massive activation of polyamine metabolism is associated with tmt-induced neurodegeneration. withdrawn ps1p-f086 establishment of memory guided actions of taking food with tweezers in monkeys naoki hirai 1 , toshinori hongo 2 , kimisato naito 2 , shigeto sasaki 2 1 department of physiology, kyorin university, school of medicine, tokyo, japan; 2 tokyo metropolitan institute for neuroscience, tokyo, japan monkeys learned a task of taking food with tweezers (twz) under the visual guidance. the task consisted of sequential actions of looking at twz, grasping it by hand simultaneously shifting gaze to food, bringing twz to food, and picking it up with twz. brief interruption of vision for 0.3-1.0 s during any actions by a liquid crystal shutter disrupted the ongoing actions, indicating that each action needed visual information as guidance. this contrasted with the task of taking directly with hand, which was done without vision. with repeated practice, they developed a mode of using more memory and somatosensory cue as guidance. they directed their gaze to invisible food in advance, and when vision of 0.1 s became available, they grasped twz, brought the twz to memorized location of food and grasped the food without vision. these results show that they acquired food taking actions using twz based on memory and somatosensory cues, the latter allowing monkeys to use twz as an extension of the hand. masahiko nishimura, yoshihiko yoshii university of ryukyus, okinawa, japan we have experienced that the patients with arm impairments by brain disorder were difficult to manipulate the tools with the paralyzed arm, and healthy arm. lt. parietal lobe and ifg are commonly recognized tools-semantic neuro-system. however, nobody knows a neural network contributed to suitability for a purpose of toolsmanipulation. we examined an fmri to evaluate the brain activation of tools-manipulation in 17 volunteers. experiment was performed by three tasks, control task is a forearm rotation, task1 is simulation of tools-manipulation, task2 is execution of tools-manipulation. we found different brain regions by this experiment. task2 to investigate the role of synchronous firing in the prefrontal cortex (pfc), we performed cross-corelational analysis of the pfc neurons, while monkeys performed a path-planning task, which required multiple steps of actions to reach goals. first, we analyzed synchrony among pfc neurons during the execution period in comparison with that during the preparatory period. we found that neuronal synchrony was enhanced transiently for each step of movement during the execution period. next, we examined relationship between neuronal synchrony and task-related activities. we found that the relationship between neuronal synchrony and response selectivity of pfc neurons was more distinct during the preparatory period than during the execution period. we would discuss dynamical roles in neuronal synchrony in planning multiple steps of actions. the functional significance of primate medial prefrontal cortex in the selection of action has been unclear. we studied neuronal activity in this region while monkeys were performing a variant of conflict solving task in which visual cues instructed them to push either the left or right. the location of the cue was either compatible (congruent) or incompatible (incongruent) with the target's location. we found a focus of reaching-related neurons in the medial prefrontal cortex rostral to the pre-sma. the activity of neurons in this newly identified area was dependent on conflict. intracortical microstimulation in this area did not evoke eye movements, distinguishing this area from the sef. we found that the local field potential in this area, but not in other areas, differed when congruent and incongruent trials were intermixed, and when only the congruent trials were presented repeatedly, suggesting the involvement of this area in the selection of actions is dependent on the task demand. masaki maruyama, peter fenwick, andreas a. ioannides laboratory for human brain dynamics, riken-brain science institute, saitama, japan we used infrared corneal reflection, sampling at 1 khz, to record simultaneously and independently the 12 • horizontal saccades of each eye for 12 subjects. two paradigms were used, in go-only sessions saccade direction with the cue to move, and in go/no go sessions, saccade execution, direction and move. mutual information (mi) analysis showed the two eyes were most consistently yoked for position than for velocity, but both provided adequate signals. mi showed coupling between the start and end of saccades and the importance of velocity signals in their ballistic nature. surprisingly leftward movement latency was longer to peak-velocity and showed more complex mi interactions. comparing go-only to go/no go saccades, significant differences were longer onset latencies and a higher eye velocity before the end of saccades. a recent meg study using this protocol, found just before and during the saccade the additional go/no go difficulty led to more interaction between left and right brainstem and cerebellum. these could be related to eye velocity changes with the higher cognitive loading. wriggle mouse sagami (wms) has been presented as a mouse model for dystonia, as it is characterized by postural and motor impairments, such as sever tremor, sustained muscle contractions of the limbs, and wriggling of the neck and trunk without coordination. by extracellular unit recordings under awake conditions, we analyzed neuronal activity in the basal ganglia and the cerebellum of this mutant mouse. in the basal ganglia (globus pallidus, entopeduncular nucleus, substantia nigra pars reticulata), neither rates nor patterns of spike discharges were significantly different as compared to normal mice. on the other hand, the discharge rate of cerebellar purkinje cells in wms was markedly decreased. these results suggest that the decreased activity of purkinje cells may be responsible for movement disorders in wms. hiromi hirata division of biological science, nagoya university, nagoya, japan wild-type zebrafish respond to mechanosensory stimulation with multiple fast alternating trunk contractions at 1 day, whereas bandoneon (beo) mutants contract trunk muscles on both sides simultaneously. muscle voltage recordings confirmed that muscles on both sides of the trunk in beo are likely to receive simultaneous synaptic input from the cns. recordings from motor neurons revealed that glycinergic synaptic transmission was missing in beo mutants. furthermore, immunostaining with glyr antibody failed to show clusters in beo neurons. these data suggest that clustering defect of glyrs at synapse causes the impairment of glycinergic transmission and abnormal behavior in beo. indeed, mutations in the glycine receptor beta subunit were identified in beo. this is the first direct demonstration that glyr␤ is essential for physiologically relevant clustering of glyrs in vivo. since glycine receptor mutations in humans lead to hyperekplexia, a motor disorder characterized by startle responses, zebrafish bandoneon mutant should be a useful animal model for this condition. medium-sized spiny projection neurons in the striatum receive inputs from gabaergic and cholinergic interneurons as well as from extrinsic sources, including the cerebral cortex. in the present study, the effect of gabaergic modulation on striatal projection neuron activity was investigated by infusion of the gaba a receptor blocker gabazine in the vicinity of the recorded neurons in monkeys who performed a memory-guided reaching task. the gabazine infusion enhanced the activity of striatal projection neurons in response to both cortical stimulation and task events, while the neuronal activity specific to the task events was decreased. these results suggest that local gabaergic input may play an important role in fine tuning of striatal projection neuron activity. research funds: kakenhi (17022042) ps1p-f094 dependence of synchrony in the subthalamic network on temporal characteristics of afferent inputs katsunori kitano, fumito kosuga department of human and computer intelligence, ritsumeikan university, japan the subthalamic nucleus (stn) and the external segment of global pallidus (gpe) constitute the indirect pathway of the basal ganglia and highly modulate the basal ganglia functions. the evidence that the emergence of synchronized oscillatory activity in the network of the two nuclei is relevant to movement disorders such as parkinson's disease shows temporal structures of the neuronal firings play an important role for the functions. among possible underlying mechanisms for the abnormal activity, the characteristic membrane properties of stn neurons is likely to be one of the most crucial origins. to clarify the detailed mechanism, we focus on and investigate the dynamical properties of the neuron theoretically and numerically with the model neuron. we apply the phase reduction method to the dynamics of the neuron to analyze the stability of synchronous activity. in particular, how the stability depends on temporal characteristics of afferent inputs to the neurons as well as the intrinsic membrane properties are investigated. during phasic voluntary movement, electromagnetic oscillatory activities of ∼20 hz around the central sulcus show decrement and increment (erd/ers, respectively), that are assumed to reflect the cortical activation and inhibitory/recovery process respectively. we investigated the correlation between personality and these oscillatory changes. from 35 healthy subjects, high and low scorers (n = 12 each) of novelty seeking dimension on the psychometry were selected. magnetic fields were recorded while they performed selfpaced movements of their right index fingers, and frequency analysis was carried through the beta band (18-24 hz). high ns group showed less amount of erd in the left hemisphere, smaller magnitude, larger latency of ers in the right hemisphere and smaller amount of baseline activity in both hemispheres than low ns group. it was suggested that individuals with high ns trait may have less inhibition after the movement and higher readiness during resting state. despite the emerging methodology of combined fmri and tms, the quantitative relationship between tms intensity and bold signals is poorly understood. eight healthy subjects were scanned on a 3-t scanner, with an mri-compatible figure-of-eight tms coil attached for eliciting right hand movement. bold measurement was performed with the stepping stone sequence (tr = 2.7 s) with online monitoring of meps. the intensity of tms pulses was varied from 30% to 95% at a 5% step (frequency at ∼0.15 hz). bold signal changes were assessed in the primary motor cortex. a sharp increase in bold signals was observed above 80% stimulation. bold signals were weakly but significantly correlated with tms intensity adjusted by the resting motor threshold (r = 0.46, p = 0.001). this finding gives a theoretical background for the application of fmri with tms to cognitive brain regions. ps1p-f097 shift of activation areas induced by hand movement during recovery from post-stroke hemiparesis: an nirs study kotaro takeda 1,2 , yukihiro gomi 1 , itsuki imai 3 , nobuaki shimoda 1,2 , hiroyuki kato 1,2 1 international university of health and welfare, ohtawara, japan; 2 crest, jst, kawaguchi, japan; 3 nasu neurosurgical center, nasushiobara, japan we investigated the cerebral hemoglobin (hb) changes in hemiparetic stroke patients under voluntary hand grasping task from acute to chronic phases by using near infrared spectroscopy (nirs). fortyfour channels (22 channels on each side) were placed on the scalp overlying both sensorimotor cortices, and the cerebral hb changes were observed during four to six cycles of 15 s task and 30 s resting periods while sitting on a chair. the amounts of oxy-hb change were significantly increased in the bilateral sensorimotor areas during hemiparetic hand grasping at the acute phase, though the significant increase was mainly observed in the contralateral sensorimotor area during hemiparetic hand grasping at the chronic phase and during normal hand grasping at all phases. this result suggests that the functional recovery from post-stroke hemiparesis may be attributed to neuronal reorganization of sensorimotor areas via recruiting ipsilateral cortex. research funds: crest, jst todd pataky 1,2 , rieko osu 2 , hiroshi imamizu 2 , mitsuo kawato 1,2 1 computational brain project, icorp, jst, japan; 2 atr cns laboratories, japan movement direction encoding in primate single cortical cells has been widely documented. this study was designed to test whether this directional tuning is observable at the voxel level in human fmri. three subjects performed 1 hz isometric force pulses to seven targets separated by 30 • in the shoulder/elbow flexion-extension plane. while in mri, online force feedback was provided by a 2d strain gauge. twenty repetitions of each condition were performed in 12 s blocks (tr = 2 s). all subjects showed broad activation over the contralateral motor area, and from functional rois an average of 661 voxel time series were extracted. many voxels exhibited continuous cosine-like tuning with movement direction. decoding using linear svm revealed that while correct classification rate was only 31.3% (chance: 14.3%), errors were distributed normally about the target such that 80.4% (chance: 59.2%) of the data was classified correctly to within 60 • . these data demonstrate that non-invasive neuroimaging is sufficiently sensitive to study the problem of coordinate system representation. the behavioral thermoregulation is important for living in various temperature environments. however, the neural mechanism of behavioral thermoregulation is poorly understood. in this study, we aimed to establish a new model to analyze the neural mechanism of behavioral thermoregulation using zebrafish. we investigated whether zebrafish perform behavioral thermoregulation against heating. when water temperature was changed from 28 • c, fish showed repetition of short time swimming in the range 32.5-40 • c. the frequency of the heat induced escape behavior was increased with temperature dependant. these results suggest that the heat induced escape behavior is a part of behavioral thermoregulation. the heat induced escape behavior was observed stably in 3-5 days past fertilization, indicating that the neural mechanism which control behavioral thermoregulation is matured in 3 days. in conclusion, we established an effective new model to analyze behavioral thermoregulation. ps1p-f100 to learn with one limb or two: limited transfer between unimanual and bimanual skills recent studies on neural activity in primary motor cortex of nonhuman primates suggest that unimanual and bimanual movements are controlled by partially overlapping neural processes. here we demonstrate that unimanual and bimanual motor learning also reflect a partially overlapping process. first, motor adaptations to reach with a novel force field applied to a limb could not be fully transferred to the same limb across unimanual to bimanual conditions, and vice versa. second, learning acquired during unimanual reaching could not be fully eliminated by repeated bimanual reaching with no loads, and vice versa. rather, some learning remained intact (but invisible) until the original context was again performed. lastly, two conflicting force fields can be learned simultaneously if they are separately associated with unimanual and bimanual reaching. these results support the view of partially overlapping neuronal processes and illustrate the intimate relationship between neural control and motor learning. research funds: jsps and nserc rieko osu 1 , ken-ich morishige 2 , jun nakanishi 1,3 , hiroyuki miyamoto 2 , mitsuo kawato 1 1 atr computational neuroscience labs, kyoto, japan; 2 kyushu institute of technology, japan; 3 jst-icorp, japan human can execute multiple motor tasks by using the same limbs, which makes human different from industrial robots. recently the optimal feedback control hypothesis has given a significant impact on the motor control community because it produces an optimal behavior for a given task by avoiding offline computation of optimal desired trajectory that would result in suboptimal behavior in the presence of noise. it, however, requires considerable amount of resources and learning to realize multiple tasks on nonlinear system. on the other hand, a desired trajectory enables the brain to share resources with multiple tasks and save learning time by dividing a difficult problem into easier sub-problems of plan and execution. considering the modularity of the brain and viability for nonlinear system, the hierarchical implementation is a better solution for global optimality as a versatile creature. here, we experimentally demonstrate that the hand variance modulation during multiple via-point tasks supports the existence of a desired trajectory. the purpose of this study is to computationally predict arm-reaching movements and posture controls from neuronal activity of premotor (pm) and primary motor area (mi). the activity was collected with single-unit recording method during the animal performing a visually guided arm-reaching task. electromyograms (emgs) and kinematics were also measured. we reconstructed the emgs from the neuronal data using a linear regression model, and then we estimated the kinematics from the reconstructed emgs with an artificial neural network model and proportional derivative controller. as a result, these serial processes allowed us to accurately predict the kinematics during both moving and maintaining her posture from the activity. the advantage of our bmi system is to estimate not only the kinematics but also the muscle tension from the neuronal activity. we have recently reported essential role of the tongue in breastfeeding in the hypoglossal (xii) nerve-injured newborn rats. of particular interest were the findings that the rates of the amounts of milk intake in the unilateral xii nerve-injured p1 pups of the surviving cases increased greatly between p4 (30% of the control value) and p7 (52%), suggesting adaptive tongue movement during development. this study was undertaken to reveal underlying basic mechanisms for such adaptation focusing on neural plasticity allowing effective suckling. after resection of the ipsilateral xii nerve on p1, dii, a postmortem neuronal tracer, was applied to the contralateral uninjured xii nerve of p4 and p7 pups. dii-labeled neuronal fibers were successfully traced within the tongue and were found to extend over the xii nerve-injured side with gradual increase from p4 to p7. we show evidence for functional neural plasticity that allows effective suckling in the xii nerve-injured newborns with suckling disturbance. previously we reported that decorticated rats showed abnormal righting movements in the air when dropped from the supine position, while the air righting reflex (arr) could be evoked purposefully (180 • turn around the body axis) in decerebrated ones. thus, the basal ganglia might send interference signals to the arr center via the midbrain tegmentum. to clarify its functional roles in arr control, we examined arr movements in rats with the midbrain lesioned. wistar, male rats were prepared; after the posterior cerebral cortex was removed by sucking, the superior colliculus and surrounding structures were ablated in various degrees. arr movements were examined post-operative 1, 4, and 7 days. in rats with the superior colliculus lesioned extensively on both sides, arr onset were delayed and body turn around the longitudinal axis was weakened, so that either insufficient or no rotation occurred in the air. furthermore, coordination between the body and tail rotations was lost in many cases. the ablated region may relay cortical signals that give a top priority to the arr center. ps1p-g110 role of plateau potentials in feeding system of aplysia kurodai aiko kinugawa 1 , tatsumi nagahama 2 1 dept. of life sci., grad. sch. of sci. & technol., kobe univ., kobe, japan; 2 fac. phar. sci., toho univ., funabashi, japan rhythmic motor activities seen in the animal behaviors can be generated by specific neural circuits termed the central pattern generator (cpg). in the feeding system of aplysia kurodai, the le neuron we identified produces the long-lasting plateau potentials and may be a cpg element. during the feeding-like responses duration of the depolarization of the follower neurons was shortened by hyperpolarization of the le. in this study we found that the le plateau potentials had refractory periods and they were turned to activation periods by application of large depolarizing currents. and various depolarizing pulses tended to produce the stable plateau potentials with almost constant depolarizing size and duration, suggesting that the le can supply the constant long-lasting depolarizing outputs to the follower neurons even when it receives various length and intensity of excitatory inputs from the presynaptic neurons. the le may be an important cpg element to determine the size and duration of the basic depolarization of many buccal neurons. withdrawn ps1p-g112 neural organizations for vocal control in a social rodent, the deguneural organizations for vocal control in a social rodent, the degu naoko tokimoto 1 , sayaka hihara 1 , kazuo okanoya 2 , atsushi iriki 1 1 lab for symbolic cognitive development, bsi, riken, japan; 2 lab for biolinguisutics, bsi, riken, japan vocalizations of most animals are innate, the region for the direct control of such sound is known to be localized in the pag. on the other hand, a few animals with the cortico-medullary projection path can learn a new sound. in this research, we investigated about vocal control in pag of social rodent, the degu (octodon degu). it is known that degus have fifteen kinds of vocal repertoires, and that their courtship song has a complex structure. we verified the hypothesis by electrical stimulation of the pag that the neural mechanism of degus that enables complex vocalization differs from that of guinea pigs with simple vocalization. guinea pig is near relation with degu. as a result, in guinea pigs, each sound is controlled in different area of the pag. in degus, however, multiple sounds are controlled by the same area, and the different sound was occasionally evoked by the different kind of stimulation. the sound with the time-series specific to the spontaneous vocalization of degu was not emitted. the effects of a heat-and steam-generating sheet (hsg sheet) on autonomic nerve activity and bowel movement were examined in women suffering irregular defecation, the hsg sheet was applied to the lumbar or abdominal regions, causing the temperature between the sheet and skin to increase to about 38.5 • c. application of the hsg sheet to either the lumbar or abdominal region significantly increased the rate of miosis in the pupillary light reflex. as for changes in r-r, the hf increased after application, suggesting that the parasympathetic nerve system had become dominant. bowel movement assessed by electrogastrography increased in amplitude. based on the above findings, we concluded that the application of an hsg sheet to the lumbar or abdominal region may lead to dominant parasympathetic nerve activity and improve gastrointestinal motility. ps1p-g115 prostaglandin e 2 -induced thermogenesis involves a gaba-receptive mechanism in the preoptic area toshimasa osaka national institute of health and nutrition, 1-23-1 toyama, shinjuku,162-8636, japan unilateral microinjection of pge 2 into the region around the rostroventral wall of the third ventricle (av3v) elicited thermogenic, tachycardic, vasoconstrictive, and hyperthermic responses simultaneously in urethane-chloralose anesthetized rats. the magnitude of these responses increased dose-dependently in a range of 20-1000 pg, except for the vasoconstrictive response. next, the effects of pretreatment with a gaba a receptor antagonist, bicuculline methiodide (0.5 mm, 100 nl), microinjected into the preoptic area (poa) ipsilateral or contralateral to the pge 2 injection site was examined. this treatment alone had no effect on the o 2 consumption rate and temperatures of colon and skin but elicited a bradycardic response. however, all pge 2 -induced responses were blocked 10 min after the pretreatment with bicuculline, and recovered at ∼70 min. pretreatment with vehicle saline had no effect on the pge 2 -induced responses. these results suggest that the gaba-receptive mechanism in the poa is required for the pge 2 -induced thermogenesis. tetsufumi ito 1 , hiroyuki hioki 2 , kouichi nakamura 2 , takeshi kaneko 2 , yoshiaki nojyo 1 1 dept. anat., univ. of fukui, fukui, japan; 2 dept. of morphological brain sci., kyoto univ., kyoto, japan although gaba-immunoreactive (ir) fibers in the rat superior cervical ganglion (scg) were thought to originate in small cells located in the cervical sympathetic trunk (cst), almost all gaba-ir axon terminals showed markers for sympathetic preganglionic neurons (spns) in our recent report. in this study, we performed series of experiments to confirm the origin of gaba-containing fibers. gad67-ir fibers were not found in dorsal roots (drs), but in ventral roots (vrs), stellate ganglion, cst, and scg. gad67-positive somata were not found in dr ganglia and cst, but in intermediolateral (iml) nucleus of thoracic spinal cord (tsc). after intraperitoneal injection of fluorogold (fg), to label the entire spns, some fg-ir neurons were also positive for gad67. we injected sindbis virus, an anterograde tracer, in iml, and some labeled terminals in scg showed gad67-ir. after cutting t1-t4 vrs and drs, almost all gad67-ir fibers were abolished in scg. these results indicate that gaba-containing fibers in scg originate from spns in iml of tsc. masato nagahama 1 , ning ma 1 , reiji semba 2 , satoru naruse 3 1 dept. of anat. ii, mie univ. school of med., tsu, japan; 2 inst. for developmental research, kasugai, japan; 3 dept. of int. med., nagoya univ. graduate school of med., nagoya, japan aquaporin 1 (aqp1) is first found as a water-transporting protein and has been demonstrated in various organs and tissues. in the present study, we have demonstrated the presence of aqp1 immunoreactivity in a particular neuronal subtype in the enteric nervous system (ens) of the rat ileum. aqp1-immunoreactive (ir) neurons simultaneously expressed a neuronal marker huc/d. moderate numbers of aqp1-ir neuronal somata were found in the myenteric plexus, and a very few were found in the submucosal plexus. aqp1-ir neurons can be classified as dogiel type i cells, which have several short processes and a single long process. many aqp1-ir fibers were found both in the myenteric and submucosal plexi. many aqp1-ir varicose fibers were closely associated with neuronal somata in the ganglia, whereas other aqp1-ir fibers penetrated into the muscle layers. these results suggest that aqp1-ir neurons probably play a significant role within the ens to control gut functions. research funds: kakenhi (13137204) ps1p-g118 abdominal expiratory nerve activity in the decerebrate neonatal rat center for medical sciences, ibaraki prefectural university of health sciences, ibaraki, japan the abdominal expiratory activity was recorded from the iliohypogastric nerve in the decerebrate, vagotomized, paralyzed, ventilated neonatal rat at postnatal days 1-4. the increase in the volume and frequency setting of the artificial ventilator (fio2 = 50%, fico2 = 0%) failed to make the rat apnea. under this condition, the phrenic nerve showed unstable rhythmic inspiratory bursts, and the tail pinch increased the respiratory frequency. although the iliohypogastric nerve showed expiratory discharges, their amplitudes and shapes were not consistent. when fico2 was increased, the cycle period was prolonged and the abdominal expiratory activity was enhanced. in many rats, the iliohypogastric nerve showed biphasic discharges that consisted from the pre-and post-inspiratory discharges. the preinspiratory discharge has larger amplitude and shorter duration than the post-inspiratory discharge. since the post-inspiratory discharge was usually small or indistinguishable in the adult rat, the present results suggest that the pattern of abdominal expiratory activity will change during the postnatal development. we investigated the role of gabaergic neurons in the rostral ventrolateral medulla (rvm) in central respiratory control. we used gad67-gfp knock-in mice in which we could identify gabaergic (i.e., gfp-positive) neurons in a living condition. we recorded gabaergic neuron activities (n = 40) in medullary transverse slices. about 20% of gabaergic neurons were inspiratory, and all of the remaining neurons were non-respiratory. about 50% of gabaergic neurons recorded in the superficial rvm were co 2 inhibitory, and all of the remaining neurons were co 2 insensitive. we suggest that gabaergic inhibition in the rvm respiratory neuron network is mediated mainly by inspiratory neurons. gabaergic neurons are also involved in central chemosensitivity. we investigated two groups of people with a different initial level of an emotional tension before intellectual loading (il). first group had the initially increased emotional level, stressed group (sg), and the second group had not, calm group (cg). reaction time (rt) of simple visual sensorymotor reaction and asymmetry of skin potential level (spla) in two facial zones: forehead and nasal were measured. from research groups, subgroups with 0 and 10 mv spla were extracted. thus the distinction in the greater rt gain after il in subgroups with 10 mv forehead spla, in comparison to subgroups 0 mv forehead spla. such law was common for both for sg and for cg. in two subgroups of 0 and 5 mv nasal zone spla in sg the greater rt gain after il was in 0 mv nasal spla subgroup, and for cg in 5 mv subgroup. it was shown, that individual features of il performance are related to spl lateralization. but the low of the relationships of spl asymmetry in nasal zones depends on the level of emotional tension of investigated groups. mitsuko kanamaru, ikuo homma department of physiology, showa university school of medicine, tokyo, japan we have reported that serotonin (5ht) in the dorsomedial medulla oblongata in mice increases tidal volume and minute ventilation via 5ht2 receptors. peripheral administration of p-chlorophenylalanine (pcpa) reduces the whole brain 5ht level. the present study examined whether peripheral pcpa pretreatment affects hypercapnic ventilatory responses in mice. adult male mice (c57bl/6n) were pretreated with pcpa (0.1 g/10 ml/kg body weight) or saline intraperitoneally for consecutive 4 days. on the next day, each mouse was placed into a double chamber plethysmograph to obtain respiratory flow curves. one hundred percent o 2 inhalation was changed to stepwise 5, 7 and 9% co 2 in o 2 inhalation every 5 min. hypercapniainduced increases in tidal volume and minute ventilation during 9% co 2 inhalation were reduced by pcpa pretreatment; these results suggest that 5ht may increase tidal volume in hypercapnic ventilation. saori nishijima, kimio sugaya, minoru miyazato division of urology, department of organ-oriented medicine, faculty of medicine, university of the ryukyus, okinawa, japan we investigated the effect of gosha-jinki-gan on bladder activity and the autonomic nervous system in rats. forty-two female rats were divided into a control diet group and a 1.08% gosha-jinki-gan diet group. after 4 weeks, continuous cystometry with physiological saline or 0.1% acetic acid solution and biochemical analysis were done. the amplitude of bladder contraction with physiological saline was lower in the gosha-jinki-gan diet group than in the control diet group, and plasma dopamine and serotonin levels were also lower in the gosha-jinki-gan diet group. when cystometry was done with 0.1% acetic acid, the interval between bladder contractions was shortened in the both groups. however, the interval and duration of bladder contractions were longer in the gosha-jinki-gan diet group than in the control diet group. therefore, it is suggested that gosha-jinki-gan inhibits bladder activity by maintaining the balance of the sympathetic nervous system and the parasympathetic nervous system at a low level. satoko suzuki, shinya yanagita, seiichiro amemiya, ichiro kita graduate school of science, tokyo metropolitan university, japan we examined the effects of negative air ions (nai) on physiological responses and neuronal activity with c-fos immunohistochemistry. in addition, we investigated the effect of vagotomy to reveal afferent pathways of nai stimulation. we analyzed neuronal activity of the paraventricular nucleus of hypothalamus (pvn), the locus ceruleus (lc), the nucleus ambiggus (na), and the nucleus of solitary tract (nts). nai significantly decreased blood pressure, heart rate, and respiratory rate, and increased hf component which is an index of parasympathetic nervous activity. nai decreased c-fos expression in the pvn and lc, and enhanced in na significantly. after vagotomy, the physiological responses and changes of c-fos expression in pvn, lc, and na was disappeared. furthermore, increase of c-fos expression in nts induced by nai was also disappeared. these results suggest that effect of nai on sympathetic and parasympathetic nervous activity was induced by reducing the activity of the pvn and lc, whereas enhancing the na activity, and that these effects of nai was caused through vagus nerve. yusuf o. cakmak, umit suleyman sehirli school of medicine, university of marmara, turkey previous assessments of the autonomic nerve supply of testis from vagus and brainstem nuclei were conflicting in the literature. we challenged this consensus by using neuronal tracer fluorogold in rats. fluorogold dye solutions was injected unilaterally under the capsule of rat testis. rats were sacrificed by transcardiac perfusion-fixation in the fifth and seventh days after injection. brainstem of the control group, subdiaphragmatic vagotomy group and main group rats were dissected. in the main group the fluoroscent-labelling were dense in area postrema. dorsal vagal nucleus, nucleus of solitary tract and nucleus ambiguous were also labelled. these preliminary data provide an evidence of testicular innervation by vagus nerve. taking into account that brainstem structures could be labelled from the testis, it can be assumed that the areas detected might be involved in the neural control of testicular functions. the results of this study cautioned that innervation of the testis may not be fully explained by innervation from pelvic and paraaortic ganglia. research funds: scientific researches comittee of medical school of marmara university ps1p-g127 differential control of renal and lumbar sympathetic nerve activity during freezing behaviour in conscious rats yoshimi tahara, misa yoshimoto, keiko nagata, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan the present study was designed to examine sympathetic and hemodynamic responses to loud noise exposure, which induced freezing behaviour, in chronically instrumented rats. wistar male rats were instrumented with electrodes for measurements of renal (rsna) and lumbar (lsna) sympathetic nerve activity and catheters for measurements of systemic arterial and central venous pressure. rats were exposed to 90 db white-noise for 10 min. 90 db noise exposure resulted in an immediate and significant increase in rsna while lsna did not change significantly during the exposure in sham-operated (so) rats. there was a significant difference in the response between rsna and lsna during the 90 db noise exposure in so rats. sinoaortic denervation attenuated the magnitude of the increase in rsna while it had no influence on the changes in lsna observed in so rats. these data suggest that arterial baroreceptor significantly contribute to the differential control of rsna and lsna during freezing behaviour in conscious rats. here, we first demonstrated that in both the kolliker-fuse nucleus (kf) and the rostral ventral respiratory group (rvrg) region, phrenic nucleus (phn)-projecting neurons were embedded in the plexus of axons originating from the ventrolateral subnucleus of the nucleus of the solitary tract (vlnst) and that the vlnst axon terminals made synaptic contacts with somata and dendrites of the phnprojecting neurons, using a combined anterograde and retrograde tracing technique. secondly, we indicated that some of the vlnst neurons innervate both the kf and the rvrg by way of axon collaterals, using the double-labeling method. using retrograde tracing combined with in situ hybridization for mrna encoding glutamic acid decarboxylase 67 (gad67), we finally showed that most of the kf/rvrg-projecting vlnst neurons expressed gad67 mrna. these results suggest that vlnst neurons may exert inhibitory influences upon the phn-projecting kf/rvrg neurons for inspiratory control. we have examined whether the neurons of the dmv have direct synaptic contacts on the myenteric ganglia using wga-hrp. the myenteric ganglia of the stomach were composed of four types of neurons. the average numbers of axosomatic terminals per profile were 2.0 on the small neurons, 3.1 on the medium-sized neurons, 1.2 on the large neurons, and 4.2 on the elongated neuron. most of the terminals contained round vesicles and formed asymmetric synaptic contacts on the small, medium-sized and large neurons. about 80% of the axosomatic terminals on the elongated neurons contained pleomorphic vesicles and formed asymmetric synaptic contacts. when wga-hrp was injected into the dmv, many anterogradely labeled terminals were found around the myenteric neurons. the labeled terminals were large (3.16 m), and contacted exclusively the somata. most of them contained round vesicles and formed asymmetric synaptic contacts. serial ultrathin sections revealed that almost all neurons in a ganglion received projections from the dmv. ps1p-h131 neuronal mechanisms of respiratory rhythm modulation induced by external k + concentration change in the newborn rat brainstem-spinal cord preparation hiroshi onimaru, ikuo homma dept. physiol., showa univ. school of med., tokyo, japan it has been suggested that two distinct rhythm generators (pfrg-pre-i and pre-bötzinger insp) for respiration in the medulla possess different sensitivity to various neuromodulators. we hypothesize that the dominancy of these rhythm generators to determine basic respiratory rhythm depends on the back ground stimulation level. to verify this hypothesis, we studied neuronal mechanisms of respiratory rhythm modulation induced by external [k + ] change. we recorded membrane potential of pre-i neurons, c4 nerve and facial nerve activities. addition of 4 mm k + to the standard superfusate decreased burst rate of c4 activity. addition of 6 or 8 mm k + caused initial inhibition of c4 burst and subsequent high frequency c4 burst. the facial nerve burst was depressed. pre-i neuron was depolarized strongly by application of high k + , and the burst activity was disturbed and action potentials were inactivated. results suggest that pfrg-pre-i or pre-bötzinger insp rhythm generator is dominant in low or high back ground stimulation level, respectively. research funds: kakenhi (16500208) ps1p-h132 regulation of synaptic transmission in the reticular formation of medulla oblongata by substance p we have examined the response of neurons in the reticular formation near the nucleus ambiguous (na) to the administration of substance p (sp). whole-cell recording was applied to the postsynaptic neurons in coronal slice preparations of medulla oblongata isolated from infant rat. bath application of sp (1 m) increased or decreased the frequency of spontaneous activities. several neurons were clamped at −60 mv and recorded epscs evoked by electrical stimulation to dorsoventral adjacent area from recording neurons. in several neurons, evoked inward epscs were augmented by sp perfusion. i-v curve suggested that voltage dependent current was both augmented and not changed by sp. our previous studies have shown that administration of neurokinin 1 receptor (nk1r) antagonist near the na inhibited gastric and respiratory movement in anesthetized rat. these results indicated that sp affect to both post and presynaptic nk1r and regulate the transmittance efficiency to generate the output signal of certain autonomic reactions. ps1p-h133 effects of local warming in the back or abdominal region by means of a heat-and steam-generating sheets on physiological response in the low temperature room to investigate effects of local warming of the body on physiological functions as well as subjective feeling, eegs, ecg, respirometer, bis (bispectrum) index, blood pressure (bp), and local skin temperature of the body were monitored while a steaming heat pack was put on the lower lumber or abdominal region of the subjects for 1 h in the cold room. in the control experiment without the heat pack, lf/hf of hr variability (lf/hf-hr) and lf of bp variability (lf-bp) increased, while hf of hr variability (hf-hr) and skin temperature decreased, suggesting elevation of sympathetic nervous activity. in the warming experiment with the heat pack, an increase in lf/hf-hr and/or lf-bp was suppressed and hf-hr increased. we will discuss these autonomic data in relation to subjective unpleasant or pleasant feeling, eeg and bis data. junichi arai, yasuhisa endo, ryouichi yoshimura, huan wang kyoto institute of technology, japan in the ventromedial hypothalamic-lesioned animals, the abnormal cell proliferation in liver and pancreas are thought to be due to the vagus hyperactivity and/or the sympathetic repression. we conducted the co-culture system of several cell lines and demonstrated that the proliferation of hepatocytes and min-6 cells (a cell line of pancreatic b cells) were stimulated by the administration of carbachol, when they were co-cultured with cell lines of endothelial cells or smooth muscle cells. these effects were also found in the filter-insert coculture system, but never seen in the culture using single cell line. we discuss the possible mechanism of their intracellular signal transduction. research funds: kakenhi to study the correlation between the trans-cranial oxy-and deoxyhemoglobin (hb) dynamics and sbp, we measured hb dynamics (f-nirs ® , omm-3000, shimadzu corp. japan) over the frontal area and sbp (finapres ® , bp monitor, ohmeda 2300, usa) at the right middle finger from 12 volunteers (22.8 ± 2.1 years). mild thermal stimuli (17 ± 1 • or 40 ± 1 • ) were administered every 2 min alternatively to the left hand. some area showed positive correlation between the oxy-hb and sbp, the other showed negative correlation between them. hb dynamics over the frontal area have any correlation to sbp to some extent. so, trans-cranial nirs should be discussed carefully for neural activation. we thank shimadzu corp. for the use of omm-3000. we examined the effects of color environments on cognitive function in 12 healthy subjects and 12 patients after traumatic head injury using p300 components and loreta analyses. the examination was performed in color environments of red, green, or black using visual oddball tasks with photographs of a crying baby face as the target stimuli. the p300 latency in the red environment was significantly shorter in controls than in patients. the p300 amplitude in the red environment was significantly larger in controls than in patients. loreta analysis demonstrated that the neurological activities in the occipital lobes, left tonsillar nucleus, anterior cingulated gyrus, and brodmann area 10 in the red environment were significantly higher in controls than in patients. hironori nakatani, cees van leeuwen riken brain science institute, saitama, japan some figures, such as rubin's vase/face and the necker cube, have two or more distinct interpretations and are, therefore, called 'ambiguous'. when an ambiguous figure is presented continuously for a period of time, we experience spontaneous switching between the alternative interpretations. as this occurs without any changes in the figures themselves, perceptual switching phenomena are eminently suitable to study how perceptual processes are influenced by the intrinsic dynamics of neural activity. we analyzed eye-movement and eeg during perceptual switching in the necker cube. blink probability showed a peak about 800 ms before the button press responses. we found that only blinks that appeared around the peak time led to a characteristic spatiotemporal pattern of eeg. our results indicate that some, but not all, blinks play an active role in perceptual switching processes. ps1p-h139 neural basis of social cognition investigated by functional near infrared spectroscopy and electroencephalograms recorded from the whole brain tsuneyuki kobayashi 1,2 , mikinobu takeuchi 1,2 , takahiro omote 3 , naoyuki yosimura 3 , etsuro hori 1,2 , kazuo sasaki 3 , taketoshi ono 2,4 , hisao nishijo 1,2 1 system emotional science, univ. toyama, toyama, japan; 2 crest, japan science and technology agency, japan; 3 bio-information engineering, univ. toyama, toyama, japan; 4 molcul. & integ. emotional neurosci., univ. toyama, toyama, japan neural basis of social cognition was investigated by functional near infrared spectroscopy (fnirs) and electroencephalograms (eegs). a head cap for recording fnirs and eegs was set on heads of subjects. the probes of the fnirs imaging systems (103 channels) and/or electrodes of the eeg system were attached on the heads of the subjects. the subjects were required to perform social cognition tasks to discriminate (1) human facial stimuli with different gaze directions and (2) simple animation videos representing social interaction. whole brain hemodynamic images were superimposed on the 3d reconstructed mri images of the brains. now we are analyzing hemodynamic images and eeg data related to social cognition, and the results indicated some heterogeneity of the cortex in social cognition. hiroshige takeichi 1 , sachiko koyama 2 , ayumu matani 3 , andrzej cichocki 1 1 riken, wako, japan; 2 hokkaido university, sapporo, japan; 3 university of tokyo, kashiwa, japan to evaluate the level of spoken sentence comprehension objectively and quickly, electroencephalograms (eeg) were recorded from five japanese adults, while they were listening to fifty-second spoken sentences. natural japanese (native) and spanish (foreign) sentences were modulated in amplitude by an eleventh-order m-sequence at 40 hz, and played twice: forward and backward. evoked responses to the modulation were analyzed as follows: (1) circular cross correlation functions were calculated between the eeg data and the m-sequence for each subject. (2) the functions were averaged across subjects. (3) independent component analysis (ica) was applied to the averaged functions and independent eeg components were estimated for each stimulus for each subject. (4) phase-locked component responses to the modulation were inspected. as a result, two components showed differential responses to the comprehensible forward japanese and the other incomprehensible stimuli. research funds: jst and kakenhi (17022004) perceptual rivalry, such as ambiguous figure perception and binocular rivalry, reflects the flexibility of our brain, because it produces fluctuating perception though an unchanging stimulus. in this study, we carried on meg recordings of healthy subjects while they reported perceptual alternation of bistable apparent motion. we investigated power and phase synchronization analyses of meg signals and compared the spatiotemporal patterns during spontaneous perceptual alternation (rivalry condition) with the externally-triggered alternation (replay condition) to extract the inherent dynamics of perceptual alternation. as results, we detected transient anterior-posterior synchronizations in advance of subjects' reports of perceptual alternation in the rivalry condition. these results suggest that these synchronized activities are involved in a higher-order process inducing spontaneous alternations in perceptual rivalry. ps1p-h142 the reflection of category perception of sound in the auditory evoked n1m magnetic responses to periodic complex sounds with equivalent acoustic parameters except for 2 different fundamental frequencies (f0) and 12 different spectral envelopes of vocal, instrumental and linear shapes were recorded to clarify the cortical representation of timbre categorization. responses to vocal and instrumental (nonlinear) sounds were localized significantly anterior to linear sound responses. n1m source strength for nonlinear sounds was significantly larger than that for linear sounds. n1m peak latency only for vocal sounds was not affected by f0. these results suggest that perceptual categorization was reflected in n1m source strength and location (linear or nonlinear), and in n1m latency (vocal or nonvocal). sunao iwaki 1 , hiroko kou 1 , kouichi sutani 1 , mitsuo tonoike 2 1 national institute of advanced industrial science and technology, osaka, japan; 2 chiba univ., chiba, japan interactions between neural activities detected at multiple brain regions involved in the visual target detection processing were assessed using meg and the causal modeling. meg signals were measured during subjects performing a visual infrequent target detection task. distributed source model was used to infer the dynamic neural activities at the multiple regions and the structural equation modeling (sem) was then used to compare two possible causal models underlying the generation of major event-related components, namely p3, related to the target detection. we used akaike information criteria (aic) and goodness-of-fit index (gfi) as measures of the goodness of the models. the results of the comparison of two possible sem models, whose major difference was on the contribution of the activities in the parieto-temporal region to the generation of p3 components, suggested the involvement of frontal and anterior cingulate cortex in the early p3 component (p3a) and the contribution of the parietal and temporal regions to the later component (p3b in our study, we investigate whether or not bilinguals use distinct neural substrates to recognize words in their first and second languages (l1 and l2, respectively). we compared the brain activity of 11 chinese learners of japanese as l2 with that of 28 japanese natives studied in our previous study. we obtained written informed consent from each subjects. in data analysis, we used spm2. while natives showed specifically greater activation in the left middle temporal gyrus than learners, learners showed specifically greater activation in the bilateral parieto-occipital and left occipito-temporal junction than natives. these results indicate that there are distinct neural substrates for word recognition of l1 and l2. neural activations for lexical processes were measured using noun, vowel, and pseudo-character decision tasks with magnetoencephalography (meg) and functional magnetic resonance imaging (fmri) on ten right-handed subjects, and their time courses were analyzed with an fmri-constrained meg-multi-dipole method. the average activations rose at latencies around 100 ms in the occipital gyrus or cuneus (og/cuneus) and ventral occipito-temporal areas (vot), and at latencies around 200 ms in the posterior superior temporal and inferior parietal areas (pst/ipl), anterior temporal area (at), and posterior inferior frontal gyrus (pifg). the differences in activation between tasks are considered to reflect visual-form process in the og/cuneus and r.vot, phonological process in the l.pst/ipl and l.pifg, and semantic process in the l.at. the decay of activation for these areas was found to be well fitted to exponential functions with time constants around 500 ms. the effectiveness of a habituation/dishabituation paradigm for determining the cerebral dominance for language was examined using a 1.5 t fmri. healthy right-handed adult volunteers with prior written informed consent were instructed to listen to analysis-synthesized words. after habituated to a single word presented repeatedly, the subject was presented with contrastive words which comprised comparison and habituation words in a pseudo-random order. the two blocks were repeated alternately for 6 times. comparison words were phonemic or intonational derivative of the habituation word, and presented in respective sessions. the results showed that the left auditory cortex responded more to the phonemic contrast, and the right to the intonational contrast, which is in line with other paradigms/techniques for determining cerebral dominance, while the present paradigm demands little effort on the subject. the issue that whether meaning of kanji words is accessed from orthography, or from both orthography and phonology representations is still debated. the present fmri study investigated brain areas underlying the use of orthography and/or phonology in kanji reading by engaging subjects in semantic categorization task with homophone and orthographic similarity effects. fifteen native japanese volunteers participated. stimuli were pairs of definitions and their target words, including correct words and foils. the subjects were asked to decide the correct target words of definitions. the results showed that homophone versus non-homophone foils increased activation of the left fusiform and middle frontal gyri. orthographically similar versus dissimilar foils increased activation of the left middle and inferior frontal gyri. these findings reflected the roles of both orthography and phonology in kanji reading. moreover, homophone versus non-homophone minus orthographically similar versus dissimilar foils revealed activation of the left fusiform gyrus. this might suggest the role of this area in character-to-sound conversion of kanji words. chieko takamiya 1 , mie matsui 2,3 , tsuneyuki kobayashi 2,4 , hisao nishijo 2,4 , michio suzuki 2,5 , yasuhiro kawasaki 2,5 , masayoshi kurachi 2,5 , jun nakazawa 6 , kyo noguchi 7 , hikaru seto 7 1 neuropsychiatry, univ. toyama, toyama, japan; 2 crest, japan science and technology corporation, japan; 3 psychology, univ. toyama, toyama, japan; 4 system emotional science, univ. toyama, toyama, japan; 5 neuropsychiatry, univ. toyama, toyama, japan; 6 developmental psychology, univ. chiba, chiba, japan; 7 radiology, univ. toyama, toyama, japan an individual has a theory of mind (tom) if he imputes mental states to himself and others. this ability is necessary for our well-rounded social communication. we used functional magnetic resonance imaging (fmri) in ten healthy subjects to study the neural mechanisms underlying tom. we adopted the picture sequencing tasks which demanded inferring mental states to self and others as tom task. as a result, there were significant brain activations in the medial frontal cortex and middle frontal gyrus. these activations coverged with a part of results in previous neuro-imaging studies on tom and social cognitive functions. objective: the purpose of this study was to investigate the neural bases of evaluation of ambiguous facial expression using whole brain functional magnetic resonance imaging (fmri). methods: participants underwent fmri scanning during which they performed a task evaluating facial expression of human (happy or sad). the task consisted of three conditions: ambiguous, middle, and high intensity of facial expression. pictures were chosen from atr facial expression image database. results: subtraction between ambiguous and other conditions revealed the activation of anterior cingulate cortex and prefrontal cortex in evaluation of ambiguous expression. the present results suggest that these area may be involved in evaluation of ambiguously expressed emotions. motoaki sugiura 1 , atsushi sekiguchi 2 , keisuke wakusawa 2,3 , yuko sassa 2,4 , hyeonjeong jeong 2,5 , kaoru horie 5,6 , shigeru sato 5,6 , ryuta kawashima 2,6 1 miyagi university of education, sendai, japan; 2 niche, tohoku univ., japan; 3 dep. pediatrics, tohoku univ. school of medicine, japan; 4 ristex, jst, japan; 5 gsics, tohoku univ., japan; 6 lbcrc, tohoku univ., japan using an fmri, we examined the cortical mechanisms for risk perception during observation of risky tool usage. normal subjects were presented with a picture of a naturalistic situation involving two actors, in which risks related to a tool and the direction of action were modulated in a two-factorial design. after the fmri, each subject self-evaluated the degree of risk in each picture. main effects of object-and direction-related risks were observed in the left ventromedial prefrontal cortex, and dorsolateral parieto-frontal network, respectively, suggesting that the object-and direction-related risk signals are separately processed in these networks. significant positive correlation between self-evaluated risk and cortical activation was observed in the anterior part of the left superior frontal sulcus, suggesting an involvement of this region in phenomenal risk-perception. in this fmri study, we identified cortical areas where activation during experience of risky situation is correlated with the harm avoidance (ha) scores, subscale of temperament and character inventory (tci). forty-six healthy subjects performed a rule speculation task in risky, normal, and safe situations in fmri. each situation was arranged for subjects to gain 10, 50, 100 points or lose 100, 50, 10 points, respectively. cortical activation induced by experience of risky situation was estimated. a significant positive correlation with the ha scores, was observed in activated areas in the right anterior insula in risky versus safe comparison. the results suggested that activation in this region predicts the individual difference in behavioral response to risky situation. this finding indicates that the right insula underlies individual difference in response to risky situation. ps1p-h152 brain activation related to the evaluation of absolute and relative value of outcome juri fujiwara 1 , masato taira 2,3 , toshio iijima 1 , ken-ichiro tsutsui 1 1 div. sys. neurosci., tohoku univ. grad. sch. life sci., sendai, japan; 2 arish, nihon university, tokyo, japan; 3 appl. sys. neurosci., nihon univ. grad. sch. med. sci., tokyo, japan one way to evaluate the behavioral outcome is in terms of absolute gain or loss (absolute value), but the evaluation can also be achieved by comparing the outcome with the possible outcomes of unchosen options (relative value). here we attempted to disentangle the brain processes involved in the absolute and relative value evaluation by using event-related fmri. subjects were instructed to compete with a computer to maximize the income in a task, in which they had to choose one option out of two, each of which were associated with either 0 yen or a gain or loss of 200, 400, or 800 yen. in each trial, a choice period was followed by a serial presentation of the outcomes of the chosen and unchosen options. we analyzed the brain activity during the presentation of each outcome. the activation changes related to the evaluation of absolute and relative value were observed mainly in the basal ganglia and in the cerebral cortex, respectively. ps1p-i153 neural activation during experience-based reasoning chisato suzuki 1,2 , takashi tsukiura 1 , hiroko mochizuki-kawai 1 , yayoi shigemune 1,2 , toshio iijima 2 1 neurosci. res. inst., aist, japan; 2 div. systems neurosci., tohoku univ., japan the aim of this study is to investigate neural activations when reasoning future events based on experienced events. before fmri, subjects encoded two kinds of four-scene comics; the complete version with four scenes and incomplete one without the last scene. after encoding, subjects performed three tasks during fmri. in the first task, subjects chose a last scene associated with the first scene encoded in the incomplete version (memory-based reasoning: mr), whereas in the second task, subjects recognized a last scene encoded in the complete version (memory: m). in the third task, subjects chose a last scene appropriate to the first scene in the new comics (reasoning: r). activations specific to mr was found in a relatively anterior part of the left pfc and right pfc. the common activations between mr and m were identified in the right mtl, whereas a relatively posterior part of the left pfc was activated commonly between mr and r. the findings suggest that the network including bilateral pfc and right mtl may contribute to the experience-based reasoning of future events. to assess neural responses to reciprocal mindreading in socially strained human relationships, we performed an fmri study in 16 healthy subjects who participated in the chicken game. statistical parametric mapping showed that the counterpart effect (human versus computer) activated the anterior paracingulate cortex (pcc) and the posterior superior temporal sulcus (sts). when we analyzed the data to evaluate whether the subjects made aggressive or reconciliatory choices, the posterior sts showed that the counterpart had a reliable effect regardless of risky or safe decisions. in contrast, a significant opponent x selection interaction was revealed in the anterior pcc. it could be inferred that the posterior sts and the anterior pcc play differential roles in mentalizing; the former serves as a general mechanism for mentalizing, while the latter is exclusively involved in socially risky decisions. creativity is the ability to generate new and original ideas. the most of studies of creativity used linguistic tasks which involve multiple aspects oflinguistic information processing in addition to creativity. we used new artistic creativity task such as designing new tools, in which we could quantitatively evaluated the creativity by the originality (os: originality score) of the products. using fmri, we observed bold signal change during designing task in art students (trained) and non-art students (untrained). we observed clear difference between two groups; in the trained highly creative group, the os is correlated with the interhemispheric difference of neural activities of the prefrontal cortex with right hemisphere dominance. in the untrained group we saw no such correlation. thus, our result supports the notion that both right prefrontal dominance and the increase of interhemispheric cooperativity could be the source of the artistic creativity. ps1p-i156 the difference of brain activity elicited by different styles of art hiromi yamamura 1 , yasuyuki kowatari 1,2 , shigeru yamane 2 , miyuki yamamoto 1,2 1 comprehensive human sciences, university of tsukuba, tsukuba, japan; 2 system brain science division, aist, tsukuba, japan artworks are categorized according to time and place where they were produced (cultural effects). surrealistic art is one of those categories and it gives uneasy impression to our mind. we investigated brain activity during viewing pictures of different art styles using functional magnetic resonance imaging (fmri). works of several artists who are well-known as representatives of renaissance, impressionism and surrealism were used as stimuli and results were analyzed by spm2. while renaissance arts or impressionism arts elicited a similar activation pattern in the occipital and inferior temporal areas, surrealisms showed deactivation in parietal with the activation in the right dorsal prefrontal cortex (ba9, ba46). these results suggest that a particular style of artwork may have commonly activated brain regions. research funds: coe(j-3) ps1p-i157 effects of chewing on the activity of the prefrontal cortex in working memory processing: an fmri study in general, it has been proposed that chewing produces holding or enhancing effect on attention. furthermore, recent studies have shown that chewing causes activation of various brain regions, including prefrontal cortex. we therefore examined the influence of chewing on brain activities using fmri. the subjects used were 20-30 aged healthy adults, being conducted continuously to two-back task with intermittent gum-chewing. gum without odors and taste component was used to remove effects other than chewing. the results indicated that chewing tended to increase the bold signals in the prefrontal area including the dorsolateral prefrontal cortex during two-back task. this suggests the possibility that chewing may accelerate the process of working memory. research funds: kakenhi 17,6577 ps1p-i158 the tip-of-the-tongue with an emotional reaction caused by recall of celebrities' names hirohito m. kondo 1 , michio nomura 2 , jun kawaguchi 3 1 ntt commun. sci. labs., ntt corp., atsugi, japan; 2 dept. psychol., tokai women's univ., kakamigahara, japan; 3 dept. psychol., grad. sch. environ. studies, nagoya univ., nagoya, japan the tip-of-the-tongue (tot) phenomenon is a mental state where you cannot recall something though you have every confidence that you know it. the tot state generates emotional reactions, but it is not clear what neural mechanisms are involved in the awareness of frustration. participants were instructed to recall the full names of celebrities when their faces were presented. event-related fmri analysis demonstrated that the anterior cingulate cortex (acc), anterior insular cortex (aic), inferior frontal cortex, intraparietal sulcus, and fusiform gyrus were activated during the tot state with frustration. activity of the acc and right aic was positively correlated with the degree of frustration in unsuccessful retrieval. roi analysis indicated that the acc and right aic were sensitive to retrieval demands and awareness of frustration, respectively. we suggest that the cinguloinsular circuit regulates the self-monitoring processes during the tot state. noriko kudo 1,2,3 , yulri nonaka 1 , katsumi mizuno 4 , kazuo okanoya 1,5 1 riken, bsi, biolinguistics, saitama, japan; 2 chiba university, chiba, japan; 3 jsps, japan; 4 department of pediatrics, showa university, tokyo, japan; 5 presto, jst, japan segmentation of speech stream is a prerequisite for language acquisition. language learners use the transitional probability between vocal tokens to segment continuous auditory stream into distinctive words. we consider that the ability for statistical learning is not specific to language, but more general cognitive competence. and we ask whether this ability could be considered as innate. in this study, we measured erps for 18 neonates within 3 days, in order to examine whether neonates can learn transitional probabilities and statistically segment words. four three-tonal-words were presented in random order without intervals during recording of the eeg. as a result, only the first tone of each word evoked a significant positive component in the frontal area. since this potential is not evident during the first session, this is likely to be due to statistical learning. these results suggest that the ability to distinguish words based on statistical information is innately prepared in humans. using near-infrared spectroscopy (nirs), changes in concentration of oxygenated hemoglobin (oxy-hb) in the prefrontal cortex were evaluated while eleven human subjects performed the paintings appreciation task. in this task, subjects were required to appreciate abstract and representational paintings that appear one after another on a computer monitor. subjects were then required to judge the degrees of interest, beauty, and desirability immediately after the appreciation. it was shown that the peak of averaged oxy-hb change was higher while subjects appreciated abstract paintings. average differentiation for each oxy-hb change revealed that the changes while the appreciation of representational paintings were more accelerated than that while the appreciation of representational paintings. these results suggest the different cortical activity dependent on appreciation of abstract and representational paintings. we used meg to investigate the spatiotemporal cortical activities during mental calculations and their modulation by arithmetic complexity. eleven healthy subjects have participated in the study. three conditions were considered: easy: add three (3) to a two-digits number without carry-over; difficult: stimuli were the same as easy, but with carry-over; nocalc: add zero to the two digits number. probe stimuli were presented 1 s after the presentation of task stimuli (a pair of two-digit and one-digit number), and the subjects were required to respond by lifting the right index or middle finger. root-mean-square values for 12 different meg sensor groups covering entire cortical area were calculated to evaluate local signal power in each condition. increased neural activities in the bilateral frontal/prefrontal and the parietal regions during both calculation conditions were observed in the latencies around 700-900 ms. the activities in the bilateral prefrontal and the left parietal areas in the same latencies were found to be complexity-dependent, i.e., increased activities in these regions were observed in difficult condition compared to easy condition. we investigated an effect of auditory feedback on self-produced speech in children with and without autism by measuring the lombard effect. ten children with autism (9:4-14:11) and 18 agematched typically developing children (9:5-15:2) were instructed to name 50 pictures of objects aloud in control and masking conditions. in masking, weighted-white noise was continuously delivered through a headset. the subjects' speech responses were recorded from a microphone. in typically developed children, the enhancement (masking/control) in masking was significantly greater (duration = 1.2 ± 0.2, loudness = 1.4 ± 0.3) than in the children with autism (duration = 1.1 ± 0.2, loudness = 1.2 ± 0.3) (p < 0.001). the present findings suggest that deficits in speech audio feedback in autistic children and this could be one of the reasons for their delay in speech development. since the mechanism underlying the effect of low power laser irradiation on the soft tissue is still unknown, we examined whether it can influence the muscle contraction as well as its fatigue in the frog (xenopus laevis) gastrocnemius or not. muscle tension continuously induced by a supramaximal stimulus to the sciatic nerve at 0.5/s chronologically attenuated and showed a simple fatigue curve. direct irradiation of laser (532 nm, 180 mw) to the muscle surface (28.3 mm 2 ) significantly delayed its attenuation (p < 0.05). when the rest period was set between stimulating sessions and the laser irradiation was applied during the rest period, averaged muscle tension during stimulating period for 2 min decreased according to the session sequence. however, comparing with no or cooling application during the rest periods, such laser irradiation case significantly delayed the muscle fatigue (p < 0.05). it is suggested that laser irradiation has a potential to more activate atp synthesis during as well as after muscle contraction. ps1p-i165 nedl1, a novel e3 ubiquitin ligase for dishevelled-1, targets mutant superoxide dismutase-1 and interacts with p53 yuanyuan li 1,2,3 , kou miyazaki 1 , toshinori ozaki 1 , akira nakagawara 1 1 division of biochemistry, chiba cancer center research institute, chiba, japan; 2 production technology development center, the furukawa electric co., ltd., ichihara, japan; 3 hisamitsu pharmaceutical co., ltd., tokyo, japan we have cloned a novel hect-type e3 ubiquitin ligase gene termed nedl1. previous study has shown that nedl1 is exclusively expressed in neuronal tissues and its expression level is high in favorable neuroblastomas and undetectable in unfavorable ones. dishevelled-1, a regulatory molecule in the wnt signaling pathway, was identified as the physiological target of nedl1 for uniquitination and proteasome-mediated degradation. on the other hand, nedl1 bound and ubiquitinated mutant (but not wild-type) sod1 in a mutant sod1 type-dependent manner, which is proportionally related with the fals severity. in the present study, we show that nedl1 physically bound p53, and induced apoptosis in a p53-dpendent manner. taken together, our results suggest that nedl1 may play a critical role in neuronal cell death occurring in fals through interacting with mutant sod1 and p53. spinal and bulbar muscular atrophy (sbma) is an inherited motor neuron disease caused by the expansion of polyglutamine tract within the androgen receptor (ar). chip (carboxyl terminus of hsc70interacting protein), u-box type e3 ubiquitin ligase, has been shown to interact with hsp90 or hsp70 and ubiquitylates unfolded proteins trapped by molecular chaperones and degrade them. we demonstrated in a neuronal cell model that transient over-expression of chip reduced the monomeric mutant ar more than the wild-type, suggesting that the mutant ar is more sensitive to chip than is the wild-type. we also demonstrated high expression of chip ameliorated motor impairments in the sbma transgenic mouse model. these findings suggest that chip over-expression ameliorates sbma phenotypes in mice by reducing nuclear-localized mutant ar, which probably due to enhanced mutant ar degradation. we performed an electrophysiological study demonstrating inhibition of spontaneous muscle action potentials within a co-culture of rat muscle and spinal cord by exposure to patients with guillain-barré syndrome (gbs) serum, as well as purified igg, from selected patients with gbs. using a whole-cell recording technique, we then investigated the effects of serum and purified igg from patients with gbs on voltage-dependent calcium currents (vdcc) in ngf-differentiated pc12 cells and cerebellar purkinje cells. serum from selected patients with gbs and purified igg from some serum of patients with gbs inhibited ca 2+ current in both cells. these results suggest that muscle weakness in some patients with gbs might be induced by changes in p/q-type calcium channel function within motor nerve terminals. the aim of the present study was to explore the possible role of cox-2 inhibitor, rofecoxib in pentylenetetrazol (ptz, 40 mg/kg, i.p.)induced kindling. rofecoxib was administered orally daily 45 min before either ptz or vehicle. seizure severity was measured according to a prevalidated scoring scale. biochemical estimations were performed on the 16th day of ptz treatment. chronic treatment with rofecoxib (2.0 and 5.0 mg/kg, p.o.) for 15 days showed significant decrease in ptz-induced kindling score. chronic treatment with ptz significantly increased lipid peroxidation, nitrite levels (no levels), and myeloperoxidase levels and decreased the reduced glutathione (gsh) levels in brain homogenate, which was reversed with rofecoxib treatment. research funds: university supportted study ashish dhir, shrinivas kulkarni uips, panjab university, chandigarh, india the objective of the present study was to elucidate the effect of cyclooxygenase inhibitors on pentylenetetrazol (ptz)-induced (80 mg/kg) convulsions in mice with possible mechanism of action. various cox-inhibitors were administered 45 min prior to the ptz administration. onset, duration of clonic convulsions and percentage mortality/recovery were recorded. pretreatment with cox-inhibitors aspirin (10 and 20 mg/kg, p.o.), naproxen (7 and 14 mg/kg, p.o.), nimesulide (1-5 mg/kg, p.o.) or rofecoxib (1-4 mg/kg, p.o.) dose dependently showed protection against ptz-induced convulsions. rofecoxib (1 mg/kg) or nimesulide (1 mg/kg) also enhanced the subprotective effect of diazepam or muscimol showing gabaergic modulation of cox-2 inhibitors. cox-2 inhibitors also antagonized the effect of flumazenil (4 mg/kg) against ptz-induced convulsions further confirming the gabaergic mechanism. ps1p-j171 cell proliferation after domoic acid-induced neuronal damage in adult rats domoic acid (da) is structurally related to kainic acid, which is a rigid analogue of the putative neurotransmitter l-glutamate that causes neuronal excitation. da-induced convulsions affects limbic structures such as hippocampus and entorhinal cortex. in this study we examined the neuronal damage after intraperitoneal da administration and cell proliferation in the adult rat brain. the most extensive neuronal cell damage was observed in ca3 subfield as evaluated by he staining, while tunel positive cells were mainly observed in the granular cells of cerebellum and dentate gyrus (dg) of the hippocampus. to elucidate the relations between damage and cell proliferation, we examined bromodeoxyuridine (brdu) labeled cells. brdu labeled cells were detected in dg and the granular cells of cerebellum. the cell proliferation was not associated with damage. ps1p-j172 a-type potassium channel truncation mutation in temporal lobe epilepsy the role of voltage dependent calcium channels on the pentylenetetrazol (ptz) kindling induced learning deficits was investigated in rats. in this study animals were divided into three groups. in the test group verapamile were injected in the hippocampus (4 mg/4 min). after 20 min kindling was established in rats with ptz. the control animals were the same age and undergone the same treatment in term of acsf injections and post-kindling waiting time as the kindled animals. and in sham group the animals received saline. one month after induction of kindling spatial learning and memory was tested by morris water maze. results showed that intra-hippocampal injection of verapamil significantly decreased spatial learning, suggesting that only working memory impaired but reference memory remain intact. the results with this study suggest that intera-hippcampal injection of verapamil significantly impaired spatial learning in rats. we showed that 8-oxoguanine (8-oxog) in mitochondrial (mt) dna and cellular rna increased significantly in the ca3 subregion of the mouse hippocampus after kainate administration. laser scanning confocal microscopy revealed that 8-oxog accumulated greatly in mtdna of the ca3 microglia. wild-type and mth1-null mice, the latter lacking an ability to hydrolyze 8-oxo-dgtp and 8-oxo-gtp to the monophosphates to avoid their misincorporation into dna or rna, exhibited similar degree of the ca3 neuron loss after kainate administration, however, levels of 8-oxog accumulated in mtdna and cellular rna in the ca3 microglia were significantly increased in mth1null mice in comparison to wild-type mice. we thus demonstrated that mth1 efficiently suppresses the accumulation of 8-oxog in both cellular dna and rna in the hippocampus, especially in microglia, caused by excitotoxicity. ps1p-j176 transcription factor nrf2 regulates brain response to kainate-induced excitotoxicity yukihiko dan 1 , kosuke kajitani 1 , noriko yutsudo 1 , ken itoh 2 , masayuki yamamoto 2 , yusaku nakabeppu 1 1 kyushu univ., med. inst. bioreg., div. neurofunc. genomics, japan; 2 univ. tsukuba, grad. sch. comp. hum. sci., japan nf-e2 related factor 2 (nrf2) is the key transcription factor that serves to transmit the inducer signal to an antioxidant response element (are), a cis-acting element required for gene expression of a battery of proteins acting on anti-oxidative stress and detoxification of electrophiles. since loss of nrf2 has been reported to increase neuronal death under increased oxidative stress, nrf2 seems to play a role for neuroprotection. administration of kainite, a potent agonist of an excitatory neurotransmitter glutamate, to rodents produces epileptiform seizures followed by a delayed loss of pyramidal cells in the ca3 subregion of hippocampus. to unveil the functional significance of nrf2 in the brain, we compared seizure responses between wild-type and nrf2-null mice after systemic kainate administration. we found that nrf2-null mice exhibited an increased susceptibility to the kainate-induced seizure, and their loss of the pyramidal cells and gene expression profiles are now under investigation. ps1p-j177 synaptic plasticity and 4-aminopyridineinduced epileptic discharges in rat hippocampal slices makoto otani, tetsuo furukawa, kiyohisa natsume department of brain science and engineering, kyushu institute of technology, kitakyushu, japan four-aminopyridine (4-ap) at the concentration below 0.1 mm suppresses k d channel and induces the epileptic discharges in rat hippocampal slices. in the present study, the involvement of the activation of nmda receptor on the ictogenesis of the 4-ap induced discharges in ca3 region was studied. ten m 4-ap induced the epileptic discharges with the frequency of 0.33 ± 0.04 hz (mean ± s.e.m.; n = 3) and the amplitude of 1.23 ± 0.86 mv. when ap-5, an nmda receptor blocker, was applied to the pre-established epileptic discharges, the frequency and the amplitude of the discharges did not change significantly. on the other hand, when ap-5 was applied from the ictogenesis period of the discharges, the discharges did not appear. these results suggest that the nmda receptor-dependent synaptic plasticity involves in the ictogenesis of 4-ap-induced epileptic discharges. chronic exposure of cultured astrocytes to morphine is reported to induce differentiation of the cells. using primary astrocyte cultures, we observed that under thyroid hormone (th) deficient conditions, morphine significantly decreased cell viability. further studies showed that the loss of cell viability was due to apoptosis of the cells. the effect is attenuated by th supplementation to the culture medium. the observed effect of morphine appears to be mediated through the opioid receptor since the opioid antagonist, naloxone, inhibited the decline in cell viability. 7ni, a specific inhibitor of nnos, completely blocked loss of cell viability suggesting that morphine induced intracellular no production, leads to cell death. studies suggested that no acts through a cgmp independent pathway. the involvement of no induced cgmp independent pathway in morphineinduced apoptosis during th deficiency has been investigated. collectively, the present study demonstrates that morphine mediated cytotoxicity of astrocyte is critically influence by the level of thyroid hormone in cultured medium. ps2a-a001 influence of conductance-input signal and prior activation history on spike generation in rat somatosensory cortical neurons takashi tateno 1 , hugh p.c. robinson 2 1 engineering science, osaka university, osaka, japan; 2 university of cambridge, uk in the cortex, a profusion of electrophysiological cell types, which form specific synaptic connections, is becoming apparent. a quantitative understanding of the dynamics of different cell types when responding to complex, natural inputs, is an important prerequisite for understanding the cortical network. neurons compute by transforming excitatory and inhibitory synaptic conductance inputs into a spike train output. we have examined the properties of synaptic conductance inputs which are most effective in evoking spikes, by injecting broad-band excitatory and inhibitory conductance inputs, and using spike-triggered reverse correlation and wiener-kernel estimation to calculate the average conductance input trajectory (acit) preceding spikes. the time course of the acit provides a general description of a neuron's response to dynamic conductance stimuli. our analysis showed that the acit reflects both previous stimulus history and previous discharge history, and that the relative influences of these two factors depend on the cell type. amyotrophic lateral sclerosis (als) is a rapidly progressive neuromuscular disease caused by the destruction of motor neurons. our study has investigated the effects of als-csf on voltage-gated calcium p/q-type channel (␣1a) expression in pre synaptic terminals of rat spinal motor neurons. csf from als and non-als (neurological patients) was injected into the 3-day-old rat pup spinal subarachnoid space at the rate of 1 l/2.5 min. the rats were sacrificed 48 h after csf injection and spinal cord sections were processed for immunocytochemistry with p/q-type channel ␣1a antibody and also for cytochrome oxidase labeling. als-csf significantly increased p/qtype channel expression compared to csf from non als patients. als-csf significantly decreased cytochrome oxidase activity in the rat spinal motor neurons, which may be a sign of degeneration. it is probable that, toxic factors present in the als patients csf might induce the expression of p/q-type channel observed in pre synaptic terminals synapsing on the spinal motor neurons. ps2a-a003 on the membrane potential profile of ca1 pyramidal cells recorded with voltage sensitive dye imaging in rat hippocampal slices takashi tominaga 1,2 , yoko tominaga 1 1 dept. neurophysiol., kagawa sch. pharmaceutical sci., tokushima-bunri univ., kagawa, japan; 2 lab. for dynamics of emergent intelligence, riken bsi, hirosawa 2-1, wako, saitama, japan integration of membrane potential response in a single neuron is a basis of neuronal calculation. we have been aiming to visualize this with voltage sensitive dye (vsd). hippocampal slices, with its unique laminar structure, allow us to assign optical signals to particular membrane fractions. but, it has not been clear whether the profile of optical signal could be a measure of membrane potential profile. to solve this, we visualized rather steady membrane potential change caused by perfusion of high potassium medium. a steep peak in optical signal was seen along stratum pyramidale. an application of ttx diminished this peak, and made the optical signal profile flat along the cell. thus, we concluded that the specificity of the vsd is small. with "neuron", by assuming a population nature to the optical signal, the membrane potential profile in a response to stimulation was successfully simulated. ps2a-a004 overexpression of inwardly rectifying k + channel 2.1 in hippocampal slice culture masayoshi okada, hiroko matsuda department of physiology, kansai medical university, japan the expressions of mrnas for the inwardly rectifying k + channel (kir) 2.1 have been reported in mammalian central nervous system, but regulation of expression or its role in synaptic transmission remains unknown. in our rat hippocampal slice cultures, the endogenous kir current was hardly detected with whole cell recordings in the ca1 pyramidal neurons. then, egfp and kir2.1 expressing virus vectors were constructed, and infected to the neurons in the slices. the vectors succeed to express the kir current, and the translocation of the fusion protein to the plasma membrane was also observed. furthermore, the overexpression significantly reduced the raise in whole-cell membrane potential evoked by depolarizing current injection, suggesting that kir plays a role of noise-filter for synaptic input in central neurons. takeshi otsuka, mieko morishima, yasuo kawaguchi div. cerebral circuitry & structure, nips, okazaki, japan layer 5 pyramidal cells are heterogeneous in morphological and physiological properties, and project to multiple subcortical areas. although recent studies have addressed anatomical features of pyramidal cells identified projection regions, little is known about intrinsic membrane properties of these subtypes. here, we obtained whole cell recordings from rat frontal layer 5 pyramidal cells that project to the striatum (ccs) or pontine nucleus (cpn), identified by injection of fluorescent retrograde tracer to these regions. firing properties of pyramidal cells had similarity depending on the projection regions. ccs cells showed strong adaptation of successive spike intervals in response to the depolarizing current injection. however, cpn cells exhibited very little spike frequency adaptation during current injection. we also examined synaptic inputs from layer 2/3 neurons to these subtypes by single cell stimulation, and detected excitatory inputs in both subtypes. our results suggest that physiological properties of layer 5 pyramidal cells are correlated with their subcortical target. this study aimed to clarify expressional changes in types 1 and 2 of ryanodine receptors (ryr1 and ryr2) in the cerebellum of a ca 2+ channel ␣ 1a subunit mutant, rolling mouse nagoya. semi-quantitative rt-pcr revealed altered mrna signal levels of ryr1 but not ryr2 in the rmn cerebellum: a less ryr1 mrna signals than in the control cerebellum. well consistent with the semi-quantitative rt-pcr results, ryr1 immunostaining in soma and primary dendrites of purkinje cells was less intense in rmn than in control mice. in contrast, ryr2 immunostaining was detected in cerebellar glomeruli but the staining intensity was not different between rmn and controls. the present study suggests that somatodendritic ryr1 expression in purkinje cells was decreased in the cerebellum of rmn. this may suffer ryr1-mediated ca 2+ release, contributing altered ca 2+ homeostasis in the rmn purkinje cells. ps2a-a007 dopamine-based modulation of lateral amygdala neuron excitability: a possible involvement of potassium current ryo yamamoto, yoshifumi ueta, noubuo kato integrative brain sci. med., kyoto univ., kyoto, japan the amygdala and dopaminergic innervation thereonto are considered to cooperatively regulate emotional states and behaviors. in the present slice experiments, we investigated the effects of dopamine (da) on lateral amygdala (la) neurons by whole cell recordings. application of da depolarized la neurons, reduced the action potential threshold, and induced slow afterdepolarization (sadp). this sadp was induced voltage dependently, and lasted for more than 5 s. d1 receptor agonists induced the same sadp. previous reports have repeatedly suggested that sadp is triggered by calcium influx. consistently, calcium channel blockers or chelating intracellular calcium inhibited the present da-induced sadp. a membrane conductance decreased at the peak of sadp current (i sadp ). also, i sadp was suppressed by including cesium in the pipette solution. these results suggest that the present da-induced modulation of la neuron excitability may depend on a potassium current that can be masked by calcium influx. toru aonishi 1,2 , hiroyoshi miyakawa 3 , masashi inoue 3 , masato okada 2,4 1 tokyo institute of technology, japan; 2 brain science institute, riken, japan; 3 tokyo university of pharmacy and life science, japan; 4 the university of tokyo, japan it has been reported that amplification of ap paired with epsp boosts the induction of ltp. there are two alternative hypotheses of such amplification mechanisms; one is activation of the na channel and other is inactivation of the a-type k channel. which is essential? in this talk, by mathematical analyses and the neuron simulator, we demonstrate that the balance of inward and outward currents, which can be controlled by down/up-regulation of the a-type k channel induces a divergence of the membrane input resistance, i.e. a singularity, and such super-sensitivity is the fundamental mechanism for boosting amplification of ap paired with epsp. the balance of na and a currents is essential for controlling dendritic integration manners. we also show that the down-regulation of the a-type k channel, which modifies the ratio between the inward and outward currents, leads to a drastic change from amplifying ap mode to shunting epsp mode. miharu komai 1 , maya yamazaki 1,2 , mika tsujita 1 , manabu abe 1 , rie natsume 1,2 , kenji sakimura 1,2 1 department of cellular neurobiology, brain research institute, niigata university, niigata, japan; 2 sorst/jst, saitama, japan we previously reported that stargazin family (␥2, ␥3, ␥4, and ␥8) not only promoted ampa receptor surface expression but also modulated receptor activity and channel property (yamazaki et al., 2004) . therefore, we assumed these family proteins were auxiliary subunits of ampa receptors. to prove this hypothesis, we generated ␥4 subunit knockout (ko) mice using the cre/loxp recombination system and analyzed their phenotypes. the ␥4 subunit ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, on western blot analysis, protein expression levels of ampa receptor subunits were reduced in ko mice compared with those in wild-type at postnatal day 11, while the reduction was not so significant in adult brain. these results suggested that ␥4 might regulate dynamics of ampa receptor subunits during early development. in the cns, neural damages, such as hypoxia, ischemia and degenerating diseases, are often accompanied by disturbances in the ph environments. ambient ph plays as a significant signal for neural functions. microglia (brain phagocytes) express abundant voltagegated proton (hv) channels which have extremely high selectivity for h + and potent h + efflux ability. exposure to na-lactate (ph 6.8) induced cell acidosis and activation of the hv channels. the channel activation was characterized by increased conductance, facilitation of activation kinetics, prolongation of deactivation kinetics and a shift of the activation voltages to negative potentials. consequently, the hv channel could open more easily over a wide range of the membrane potential during lactic acidosis, and may contribute to a quick relief of the cell acidosis. mari sasaki, masahiro takagi, yasushi okamura okazaki institute for integrative bioscience, aichi, japan here we report a novel four transmembrane protein similar to the voltage sensor domain (vsd) of the voltage-gated channels that exhibits activities of a voltage-gated proton channel. voltage-gated proton channel currents have classically been described in snail neurons and recently in mammalian blood cells. however, the molecular basis underlying this channel has been elusive. here we identify a novel cdna clone named as mouse voltage-sensor domain only protein (mvsop1). cells overexpressing this protein showed depolarization-induced outward currents accompanied by tail currents during repolarization, which reversed at equilibrium potentials for protons. imaging analysis demonstrated that phin recovers rapidly after an acid load in mvsop1-transfected cells. mvsop1induced currents exhibited two key features of native voltage-gated proton channels: ph-dependent gating and zn 2+ sensitivity. neutralization of a positive charge in the s4-like segment caused shift of the voltage-conductance relationship, suggesting that it plays important role in gating. oscillatory extracellular electric fields have been observed in mammalian brains. the electric fields modulate neuronal excitability and synaptic events. to investigate the effect of the oscillatory electric fields on the ca1 pyramidal neuron, we applied sinusoidal electric fields to the rat hippocampal slice and recorded voltage responses with a voltage sensitive dye (rh414). application of sinusoidal electric fields induced transmembrane voltage oscillations in all the layers of the ca1 region. in the pyramidal layer, the amplitudes of the responses to the 1-hz field were the largest. the amplitudes were decreased monotonically when the frequency of the fields became higher. however, in the stratum radiatum, the amplitudes of the responses to the 3-10-hz fields were larger than those to the other frequencies. the frequency preference in the dendritic region may be an underlying mechanism for the synchronization of the membrane potentials among large population of neurons within the theta frequency range. acid sensing ion channels 2 (asic2) have proposed to constitute mechanoreceptors and nociceptors. we examined the localization and characterization of asic2-expressing cells in rat central nervous system (e17-p14) using immunohistochemical techniques. asic2positive fiber first appeared in brain stem and spinal cord at e17-18 stage. asic2-expresseing cells appeared in white matter of brain stem and spinal cord at e20 stage. in early postnatal stages asic2expressing cells appeared in corpus callosum, cerebellar medulla and dorsal horn of spinal cord at p4 stage. these cells were identified as an oligodendroglia by oligodendrocyte specific antibody and immunoelectron microscopy. these results are suggested the hypothesis that the function of asic2 mediate the myelin formation in the developmental stages of central and peripheral nervous system. masato shino, seiji ozawa, yasuhiko saito department of neurophysiology, gunma university graduate school of medicine, maebashi, gunma, japan nucleus prepositus hypoglossi (nph) is involved in horizontal eye movement. previously, we found nph neurons exhibiting a characteristic firing pattern in response to depolarizing current pulses (fil neurons). fil neurons exhibited a spike train with a long first interspike interval (1st isi) that is attributed to a large, slow hyperpolarization (ahp) after the first spike. in this study, we investigated ionic conductances underlying the long 1st isi by whole-cell recordings in rat slices. application of 100 m apamin, an sk-type ca 2+ -activated k + (kca) channel blocker, shortened the 1st isi and decreased the amplitude of the slow ahp. the shortening of the 1st isi was observed when membrane potentials were depolarized. moreover, application of 3 m mibefradil, a t-type ca 2+ channel blocker, shortened the 1st isi. these suggest that the firing pattern of fil neurons arises from activation of sk-type kca channels induced by ca 2+ influx through t-type ca 2+ channels. research funds: kakenhi (c) (17500270) jafar vatanparast 1,2 , mahyar janahmadi 1 , houri sepehri 2 , ali haeri-rohani 2 , ali reza asgari 1 1 neuroscience research center, shaheed beheshti medical sciences university, tehran, iran; 2 dept. of biology, university of tehran, tehran, iran the roles of the ionic channels and muscarinic receptors in paraoxon (px) induced burst firing in snail neurons were studied using current clamp method. px (0.6 m, within 5 min) increased the frequency of spikes and shortened ahp. slow waves of depolarization with superimposed bursts were recorded within 20 min. atropine blocked the depolarization shift but not the other effects of px. px was able to reversibly decrease the duration of calcium spikes elicited in a na + free ringer. this effect observed in the presence of atropine and was along with a decrease in the duration of ca 2+ spike ahp and an increase in the spike frequency. the px blockade of ca + channels may decrease the activation of ca 2+ dependent k + channels that underlies ahp. blockade of these channels possibly makes the neurons susceptible for burst induction, while activation of metabotropic muscarinic receptor by px underlies the depolarization shift with associated bursts. dendritic membrane properties are reported to be non-uniformly distributed in a single neuron and the non-uniformity could be important for synaptic integration. however their distribution is still unclear. we estimated distribution of membrane resistance by fitting a compartment model to voltage imaging data of membrane response in hippocampal ca1 slices to perturbation, such as propagating epsp induced by synaptic inputs and biphasic response to extracellular electric field. by numerical simulations, we found that these imaging data were consistently reproduced if we assume a step function as distribution of membrane resistance. this implies that a steep decrease of membrane resistance exists in distal dendrite of hippocampal ca1 pyramidal neuron. it is known that cooling-induced desensitization of cold receptors, however, its intracellular mechanism has remained unresolved. in this study, we analyzed molecular mechanism of desensitization of cold/menthol receptors (trpm8). repeated menthol application induced trpm8 desensitization. this desensitization was depended on extracellular ca 2+ , indicating that involvement with ca 2+ -dependent kinase. pkc activator (pma) desensitized trpm8 and go6976 (pkc inhibitor) abolished pma-induced trpm8 desensitization. pma similarly desensitized wild type trpm8 and mutant trpm8, in which serine or threonine residues in some putative pkc phosphorylation sites were replaced by alanine. pma treatment did not induce internalization of trpm8. as the basis of cooling-induced desensitization of cold receptors, we conclude that cooling-activated trpm8 causes pkc to desensitize trpm8 itself. yosuke sawada 1 , hiroshi hosokawa 1 , kiyoshi matsumura 2 , shigeo kobayashi 1 1 dept. of int. sci. and tech., grad. sch. of info., kyoto univ., kyoto, japan; 2 dept. of info. sci. and tech., osaka institute of technology, osaka, japan cooling below 17 • c evokes cold pain sensation. however, the molecular basis of the cold pain sensation is still unknown. trpa1 is activated by pungent compounds stimuli. if trpa1 responded to cooling to noxious cold range, it could be candidate for evoking cold pain sensation. however, whether trpa1 is activated by cooling or not is still controversial. here, we investigated that trpa1-expressing hek293 cells responded to noxious cold stimuli. whole-cell recording demonstrated that cooling below threshold evoked inward current. threshold temperature was 17.5 ± 2.7 • c. in inside-out singlechannel recording, cooling activated trpa1 directly. single channel conductance was 74.1 ± 18.8 ps. single channel currents showed inword rectification. in conclusion, trpa1 is the cooling activated cation channel. yoshiki matsuda, foong yen ang, jinsun yoon, noriko ebisu, satoshi takahashi, shinichi kogure dept. bioengin., soka university, tokyo, japan hyperplarization-activated and cyclic-nucleotide-gated nonselective cation channels (hcn1-4) have been demonstrated in the cns. since they contribute to various physiological functions including neuronal pacemaking activity, setting of resting membrane potential and generation of paroxysmal discharge, we examined their expressions as well as functions in the pns using the frog (xenopus laevis) sciatic nerves. western blot analyses for hcn1-4 demonstrated that samples from the nerve and the heart showed an hcn2 band whereas those from the dorsal part of skin and the gastrocnemius did not, and that immunoreactivities for hcn1, hcn3 and hcn4 could not be found in those samples. when an hcn channel blocker, zd7288 was applied on the stimulus portion of sciatic nerve and the nerve was elicited at 0.5/s by a duration of 10 ms pulse with supramaximal intensity, the generation of anode-break-excitation rather than cathode-makeexcitation was significantly blocked (p < 0.01). it is suggested that hcn2 channels exist in the pns and they contribute to the burst or recurrent discharges. ifenprodil, a clinically used cerebral vasodilator, interacts with several receptors, such as ␣ 1 adrenergic, n-methyl-d-aspartate, serotonin and receptors. however, the molecular mechanisms underlying the various effects of ifenprodil remain to be clarified. here, we show that ifenprodil inhibited g protein-activated inwardly rectifying k + (girk; kir3) channels, which play an important role in the inhibitory regulation of neuronal excitability in most brain regions and the heart rate, expressed in xenopus oocytes. in contrast, kir1.1 and kir2.1 channels in other kir channel subfamilies were insensitive to ifenprodil. the girk currents induced by -opioid receptors or ethanol were also attenuated in the presence of ifenprodil. the inhibitory effects of ifenprodil were not observed when ifenprodil was applied intracellularly. our results suggest that inhibition of girk channels by ifenprodil, at submicromolar concentrations or more, may contribute to some of its therapeutic effects and adverse side effects. ps2a-b022 proliferation of rat c6 glioma cells is controlled by the concentration-sensitive na + channel (na c ) shigeru yoshida, hiroyuki yamaguchi, takashi takeuchi, hokuto tanaka, yoshiyuki morimoto, teruki hagiwara department of life science, kinki university, higashi-osaka, japan the role of na + as a regulator of cell growth was studied using the tumor cell line (c6), which has a large quantity of concentrationsensitive na + channels (na c ; c = concentration). cell proliferation was suppressed when [na + ] o was raised from control (140 mm) to 190 or 240 mm. an increase in [na + ] i was revealed by an image processor in c6 cells loaded with a na + indicator (sbfi), under high [na + ] o conditions. [na + ] i elevation was augmented by ouabain or by bumetanide (na + /k + /cl − cotransporter blocker), while it was decreased when na c expression was inhibited by rnai techniques. the real-time pcr method revealed that the expression level of the immediate early gene egr-1, which is involved in cell growth, was concomitantly reduced. it is to be noted that similar alterations in cell growth, egr-1 level and [na + ] i were induced by a na + ionophore (monensin) without raising [na + ] o . these data indicate that na + enters through na c upon [na + ] o increase, and [na + ] i elevation itself is responsible for these phenomena. hiroshi kuba, takahiro ishii, harunori ohmori dept. physiol., univ. kyoto, kyoto, japan na + channels are concentrated in the axon to generate action potentials. however, little is known about how distribution of na + channels contributes to the activity and function of single neurons. in avian nucleus laminaris (nl), neurons act as coincidence detectors for sound source localization, and are tuned to both characteristic frequency (cf) and interaural time difference (itd) of sounds. we show here in the chick that nl neurons have distinct distribution of na + channels along the axon and optimize the itd sensitivity depending on their cf. neurons of high and middle cf (higher than 1 khz) had small action potentials, and had no na + currents in the somatic membrane, but clustered only in the axon at some distance from the soma (20-50 m). while, neurons of low cf generated large overshooting spikes, and na + channels were clustered closer to the soma (5 m) in the axon. thus, nl neurons had a spike generator on the axon, at a greater distance from the soma with the increase of cf. by computer simulation, these unique distributions of na + channels were found essential to enhance the coincidence detection. research funds: kakenhi (17021021) il-sung jang 1 , in-sun choi 1 , eun-ju park 1 , jin-wha cho 1 , man-gee lee 2 , byung-ju choi 1 1 kyungpook national university, school of dentistry, south korea; 2 kyungpook national university, school of medicine, south korea bisphenol a (bpa), an endocrine disrupter, is contained in cans, polycarbonate bottles and some dental sealants. here we report the effect of bpa on gaba a receptors using a conventional whole-cell patch clamp technique from acutely isolated rat ca3 pyramidal neurons. bpa itself elicited a postsynaptic current, which is highly sensitive to bicuculline, in a dose-dependent manner. bpa increased postsynaptic currents induced by gaba at lower concentrations (<10 m), but decreased those induced by gaba at higher concentrations (>100 m). in addition, bpa decreased both the current amplitude and decay time constant of gabaergic mipscs. finally, mechanisms underlying bpa-induced modulation of gaba a receptors will be discussed. we recently generated nav1.1-deficient mice and showed that these mutant mice developed epileptic seizures and died prematurely. we have now used these nav1.1-deficient mice as negative controls to examine nav1.1 distribution in the mouse brain using rna in situ hybridization histochemistry and immunohistochemistry. at low magnification, nav1.1 expression was higher in the thalamus, superior colliculus, inferior colliculus, pons, medulla and cerebellar nuclei relative to other brain regions. contrary to previous studies indicating a somato-dendritic nav1.1 distribution, in the present study, higher magnification analysis revealed that nav1.1 is predominantly distributed to axons in some brain parts. this apparent discrepancy may reflect the lack of specificity of anti-nav1.1 antibodies used in these previous studies, none of which utilized nav1.1-deficient mice. based on our findings, we propose that nav1.1 might be involved in propagating action potential to presynapses. keiji miura 1,2 , masato okada 2,3,4 , shun-ichi amari 4 1 department of physics, kyoto university, kyoto, japan; 2 "intelligent cooperation and control", presto, jst, japan; 3 department of complexity science and engineering, university of tokyo, chiba, japan; 4 brain science institute, riken, saitama, japan we considered a gamma distribution of inter-spike intervals as a statistical model for neuronal spike generation. a gamma distribution is a natural extension of poisson process and it can generate spike trains with various irregularities. the model parameters consist of a time-dependent firing rate and a time-independent spiking irregularity. because the environment changes over time, the firing rate varies for each interspike interval. we used a novel method of information geometry to estimate the spiking irregularity whatever the functional form of the firing rate is. our estimator is simple and easily applicable to experimental data. the estimator is useful for characterizing spiking irregularity which varies among neuron types. it may be possible to classify neurons into functional groups according to their spiking irregularities. research funds: grant-in-aid for scientific research (nos. 14084212 and 16500093) mitsuyo watanabe, yuko ishimaru, taketo nakadai, tomoyuki kanamatsu graduate school of bioengineering, soka university, tokyo, japan we examined the effect of colchicine, inhibitor of axonal flow, on cerebral amino acid metabolism in the rat. the rats were injected with [1-13 c] glucose intravenously (1 g/kg) 24 or 48 h after the intraventricular injection of colchicine (75 g/10 l) and the amino acid fractions were extracted from the brains at 10, 20 or 40 min after the glucose injection. the amount of [1-13 c] glucose in the cerebra was increased, however, the 13 c incorporation into glutamine, glutamate, gaba and aspartate from [1-13 c] glucose were decreased. only glutamine concentration in all amino acids was increased in the cerebra of the colchicine group, compared to those values in the control group. the microdialysis analysis showed that the amount of gln in the dialysate was increased by three times in the colchicine group compared with the control group. these data may suggest that the glycolysis of glucose is decreased and that the influx of glutamine from blood to brain occurs with neuronal dysfunction induced by colchicine. these results indicate that a ␤ alters the bhlh gene expression in neural stem cells toward cell death. ken kojima, akiko nishida, shinji takebayashi, jyuichi ito department of otolaryngology-head and neck surgery, graduate school of medicine, kyoto university, japan basic helix-loop-helix (bhlh) transcription factors play crucial roles in development of the central and peripheral nervous systems. to visualize expression of hes1 or hes5 gene, phes1-and phes5-egfp transgenic (tg) mice were generated (ohtsuka et al., 2006) . in each transgenic mouse, a promoter of hes1 or hes5 gene drives enhanced green fluorescent protein (egfp) gene. in the inner ear, it is suggested that hes1 or hes5 regulate cell division and differentiation of sensory and supporting cell progenitors via notch signaling pathway. by use of immunohistochemical technique, we examined distribution of gfp expressing cells in the inner ear of the transgenic mice from embryonic day 10 (e10) to postnatal day 60 (p60). in the phes1-egfp tg mouse inner ear, gfp immunoreactive (gfp-ir) cells were detected from e10 to p60. in the phes5-egfp tg mouse inner ear, gfp-ir cells were observed from e16.5 to p15. gfp-ir cells in phes1-gfp tg mouse are candidates of sensory cell progenitors in mature mammalian inner ear. ohtsuka et al., 2006. mol. cell neurosci. ps2a-c033 expression of zfh-5 in the developing mouse brain: mrna, antisense rna and protein expression yuriko komine 1 , kenji nakamura 2 , motoya katsuki 1 , tetsuo yamamori 1 1 national institute for basic biology, okazaki, japan; 2 mitsubishi kagaku institute of life science, machida, japan zfh-5 is a transcription factor containing three homeodomains and 18 zn fingers and expressed in differentiating neurons. we have reported that the level of zfh-5 mrna is negatively regulated by antisense transcripts of the zfh-5 gene. in several types of neurons, including pyramidal cells in the hippocampus and granule cells in the cerebellum, the zfh-5 antisense rna is expressed prior to the mrna; as the level of the antisense rna gradually decreases, zfh-5 mrna starts to be expressed. recently, we have raised an antibody against mouse zfh-5 and examined the expression profile of the zfh-5 protein. in the most regions of the brain, the protein expression pattern consisted with that of mrna. however, in the several types neurons mentioned above, zfh-5 protein was not detected even when the zfh-5 mrna was already expressed. this observation together with other data suggested that the zfh-5 protein level is regulated by several mechanisms including suppression by the antisense rna and translational control. takashi inoue 1 , maya ota 1 , katsuhiko mikoshiba 2 , jun aruga 1 1 laboratory for comparative neurogenesis, riken bsi, saitama, japan; 2 laboratory for developmental neurobiology, riken bsi, saitama, japan zic family zinc-finger proteins play various roles in animal development. in mice, five zic genes (zic1-5) have been reported. despite their partially overlapping expression profiles, mouse mutants for each zic gene show distinct phenotypes, suggesting the functional redundancy of zic proteins. it is expected that the common and specific roles of mouse zic proteins can be clarified by studying compound mutant mice. in the present study, we characterized zic1/zic3 compound mutant mice. mice carrying homozygous zic1 mutant allele together with zic3 null allele showed defects in midline structures, including abnormalities in forebrain and thalamus. especially, the compound mutants showed severe anatomical abnormalities in the dorsal and ventral telencephalon and olfactory system, which are not obvious in either zic1-or zic3-single mutant. these observations indicate that zic1, in cooperation with zic3, have an essential role in controlling proliferation and differentiation of the neuronal projenitors in the medial telencephalon. chiaki maruyama, haruo okado department of molecular physiology, tokyo metropolitan institute for neuroscience, japan rp58, a novel zinc finger protein containing a poz domain, functions as a sequence specific transcriptional repressor. rp58 gene disrupted mice show severe abnormalities in brain cortical layer formation, suggesting that rp58 has a crucial role in cerebral development. to understand the role of this protein in brain development, we examined rp58 gene expression in mouse embryo and adult brain by in situ hybridization. as a result, we found that rp58 transcripts are first detected at embryonic day 10 in the neuroepithelium of the spinal cord and telencephalic vesicle. in the day 12-13 embryos, rp58 transcripts are predominantly observed in the preplate region but not in outside the nervous system. at e15, rp58 transcripts were detected throughout the neocortex and hippocampus, but not in the thalamus and striatum. in the cortex, the transcripts were detected primarily in cortical neurons, but not in the marginal zones and ventricular zone. in adult mice, rp58 is expressed in neocortical and hippocampal neurons and granule cells in the cerebellar cortex toshiki kameyama, fumio matsushita, yuzo kadokawa, tohru marunouchi division of cell biology, fujita health university, toyoake, japan neural zinc finger (nzf) proteins are transcription factors with dnabinding domains of c2hc-type zinc finger motifs. using p19 cells, we demonstrated that nzfs were expressed transiently during neuronal differentiation, and forced expression of nzf cdnas resulted in neuronal differentiation. these results suggest that nzf family have a function regulate neuronal differentiation. to elucidate in vivo functions of nzf family in detail, we generate knockout mice of nzf-2 and nzf-3 respectively. nzf-2 null mice are born alive, but die within 10 min after birth with cyanosis. on the other hand, nzf-3 null mice are viable, fertile and appear normal. these mice look normal morphologically. then we generate double knockout mice of nzf-2 and nzf-3 by intercrossing. double knockout mice have a forelimb posture abnormalities like arthrogryposis multiplex congenita. and we find out that the spinal nerves projecting forelimb and trunk are decrease dramatically in the double knockout mice embryo. , 2006) . in the present study, to examine the role of runx3 in the development of drg in more detail, we examined the development of drg neurons in runx3-deficient mice from the early embryonic stages to birth, using various markers for subpopulation of drg neurons. in newborn runx3−/− mice, parvalbumin-positive drg neurons were greatly reduced in number, whereas calretinin-positive neurons were slightly decreased. similar decreases were observed in embryonic days 14.5 and 15.5. shin hisahara 1,2 , susumu chiba 2 , hiroyuki matsumoto 2 , yoshiyuki horio 1 1 department of pharmacology, sapporo medical university, sapporo, japan; 2 department of neurology, sapporo medical university, sapporo, japan in mammalian cns, the function of histone deacetylase sirt1 is still unclear. recent studies indicated that sirt1 interacts with nuclear receptor co-repressor (n-cor) and n-cor represses intracellular domain of notch-icd activation of the hes1 promoter. we performed overexpression of sirt1 and n-cor in neurosphere by nucleofection, then induced differentiation. we found remarkable promotion for neural differentiation by overexpression of sirt1 and n-cor in the sirt1 with n-cor. sirt1 and n-cor suppressed hes1 transcription by notch-icd in the luciferase assay. hes1 transcription was suppressed in overexpression of sirt1 and n-cor, suggesting that interaction between sirt1 and n-cor represses hes1 transcription. consistent with this, chip assays revealed that not only n-cor but also sirt1 bind to the promoter of hes1 gene. taken together, these results indicate that sirt1 and n-cor accelerate neural differentiation of the undifferentiated cells via binding hes1 promoter site and repressing hes1 transcription. yasushi maruyama 1 , mitsuhiko kurusu 2 , masataka okabe 3 , katsuo furukubo-tokunaga 1 1 grad. school life and envir. sci., univ. tsukuba, japan; 2 natl. inst. genetics, mishima, japan; 3 inst. dna medicine, jikei univ. school of medicine, japan during brain development, a large number of neurons are generated by proliferation of neural stem cells. with a characteristic proliferation mode that persists through development, the neuroblasts of drosophila mushroom bodies (mb) provide an attractive model system to study mechanisms of neural stem cell proliferation. here we show that tailless (tll), a member of the orphan nuclear receptor super family, has a crucial function in maintaining cell cycle progression of the mb neuroblasts. mosaic analysis demonstrates that cell autonomous activity of tll is crucial for maintenance of the mb neuroblast cell cycles. moreover, gain-of-function analyses confirm instructive functions of tll in maintaining neuroblast activity. we propose that tll plays pivotal roles in proliferation of the mb neuroblasts and suggest a conserved mechanism of neural stem cell control with the tll/tlx homologs in both drosophila and vertebrate brains. kouji senzaki, masaaki yoshikawa, shigeru ozaki, takashi shiga graduate school of comprehensive human science, university of tsukuba, ibaraki, japan runx family transcription factor is an important component of tgf-␤ and bmp signaling. we reported previously that runx3 mrna is expressed in the dorsal root ganglion (drg) from the early developmental stages, and that runx3 regulates axonal projection of trkcexpressing proprioceptive drg neurons (inoue et al., 2002) . furthermore, we announced previously that runx3 mrna is expressed in cranial ganglia of v, vii, viii, ix and x in mouse developmental stages. the expression was restricted to subset of neurons in each ganglion. to examine the influence of runx3 on the differentiation of trigeminal ganglion neurons, we investigated the expression of neurotrophin receptors, calcium binding proteins and neuropeptides in trigeminal ganglia of runx3 knockout mice using immunohisitochemical staining. we found the decrease of trkc-expressing neurons in trigeminal ganglia of neonatal runx3 knockout mice, however, we observe little change in the proportions of nuen-expressing neurons. kouko tatsumi 1 , hirohide takebayashi 2 , takayuki manabe 1 , kazuhiro ikenaka 2 , akio wanaka 1 1 dept. anatomy, nara med. univ., kashihara, nara, japan; 2 division of neurobiology and bioinformatics, nips, nins, okazaki, aichi, japan our previous study with double labeling of brdu and cell lineage markers suggested that a number of astrocytes were differentiated from resident oligodendrocyte progenitor (opcs)-like cells in the injured adult brain. and we found out that these opcs expressed ng2proteoglycan and olig2 at early phase after injury. to directly trace the lineages of these opcs, we employed double transgenic mice that express tamoxifen-sensitive creer under the control of the olig2 promoter together with rosa-egfp reporter. the gfp positive cells were detected around the injured region, and the almost all of these cells co-expressed gfap at late phase after injury. furthermore, we confirmed that the morphological characteristics of these cells were those of the astrocyte by immunoelectron microscopy. our results clarified that dormant opcs in vivo differentiate into astrocytes in adult injured brain, and suggested that these cells participate in glia scar formation after brain injury. olig2 is a bhlh transcription factor, essential for oligodendrocytes (ols) and motoneurons differentiation in the spinal cord. however, differentiation of olig2 lineage cells in the forebrain is largely unknown. here we examined fates of olig2 expressing cells in the fetal forebrain by tamoxifen (tm)-inducible cre-loxp system. olig2-creer knockin mice were mated with reporter mice, and tm was injected at embryonic day (e) 12.5 or 14.5, when most of olig2+ cells are observed in the basal forebrain. the olig2+ cells at e12.5 gave rise to more neuron than glia that included both ols and astrocytes. majority of neuronal olig2 lineage cells differentiated into gabaergic neurons, and a lesser number, into cholinergic neurons. the olig2+ cells at e14.5 generated more glial cells than neurons. these results indicate that olig2 lineage cells generate three major types of neural cells with a stage dependent manner, and may have multiple functional roles on neural differentiation in the fetal forebrain. mana igarashi 1,2 , masato yano 1,2 , satoru hayashi 1,2 , hirotaka j. okano 1,2 , hideyuki okano 1,2 1 dept. physiol., keio univ. sch. med, tokyo, japan; 2 sorst jst, japan the mammalian neuronal hu rna binding protein family is homolog of drosophila elav protein which is essential for differentiation and maintenance of the nervous system. in mammals, neuronal hu expresses in both early postmitotic and mature neurons and has ability to induce neuronal differentiation by binding to the utrs of specific target mrnas. to understand the molecular mechanism of hu induced neuronal differentiation, we purified hub associated complexes. among them, nf90 family, a double strand rna binding protein which is one of hu associated proteins, is known to bind to utrs of p21, p27 and tau mrna known as hu targets. we generated rabbit polyclonal antibodies against nf90 and nf45, binding partner of nf90, respectively. in mouse embryonic brains, we found that nf45/90 expressed highly in postmitotic neurons where neuronal hu proteins are highly distributed. moreover, we found that hu and nf45/90 formed mrnp complexes in mouse brain extracts. we will discuss the role of hu binding partners in neuronal differentiation through post-transcriptional regulation. sachiko the pallium is specified as a homologous field in the vertebrate telencephalon. however, little is known about how species-specific pallial structures are generated during embryogenesis. to address this issue, we compared several neuronal subtypes and their migration patterns in the developing pallium of the mouse and quail. cell tracing analysis revealed that neurons born at the dorsal pallium tangentially migrated in the developing quail telencephalon, as in the mammalian cortex. next we investigated distribution of later-born neurons in the quail telencephalon using laminar specific genes (er81 and brn2) in the cerebral cortex. in situ hybridization and immunohisitochemical studies indicated that these neuronal markers were expressed in discrete regions of the developing quail telencephalon. our data suggest that early stages of cortex/pallium development are comparable between the mammalian and avian embryos, whereas neuronal specification in later stages is regulated by distinct mechanisms in each species. research funds: kakenhi (16027202) ps2a-d047 protein expression in hippocampal cells dissociated and re-cultured from organotypic slice cultures we established a re-cultivation technique of hippocampal cells dissociated from long-term cultured organotypic slices. protein phenotype of the cells was analyzed using immunocytochemical technique. antinestin immunoreactivity was observed in cells with short processes 2 days in the re-cultivation. the anti-nestin immunoreactivity was progressively declined, whereas number of cells expressing anti-␤iii tubulin immunoreactivity increased through the re-cultivation for 1-4 weeks. presence of neurons, astrocytes and oligodenderocytes was examined using anti-␤iii tubulin, anti-glial acidic fibrillary protein and rip antibody, respectively. apart from the cells expressing one of the markers, the cells marked with multiple sets of antibodies were observed. these results suggest that protein expression was changed backward in normal differentiation course in hippocampal cells once matured in organotypic slices. we have shown that perineuronal ng2 + cells are major populations of proliferating cells in the cerebral cortex of rats. in the adult cortex, ng2 is known as a marker for oligodendrocyte progenitor cells (opcs) that retain ability to proliferate and differentiate into new oligodendrocytes. however, it is still unclear whether all ng2 + cells in the neocortex are the opcs. we investigated about subtypes of ng2 + cells found in the perineuronal regions of the cerebral cortex using cell markers. two subtypes of perineuronal ng2 + cells could be distinguished by the subcellular localization of gst-protein. one is nuclear type, the other is cytoplasmic type. only the nuclear gst-+ cells have the proliferative activity. these data suggest that the nuclear gst-+ /ng2 + cells in the perineuronal territory are progenitor cells engaging in reproduction of cortical cells. muguruma keiko, su hong-lin, matsuo-takasaki mami, watanabe kiichi, sasai yoshiki neurogenesis and organogenesis group, riken center for developmental biology, kobe, japan in this study, we report in vitro generation of math1 + cerebellar granule cell precursors and purkinje cells from es cells by using soluble patterning signals. when neural progenitors induced from es cells in a serum-free suspension culture are subsequently treated with bmp4 and wnt3a, a significant proportion of these neural cells become math1 + . the induced math1 + cells mitotically active and express markers characteristic of granule cells precursors (pax6, zic1, and zipro 1). after purification by facs and coculture with postnatal cerebellar neurons, es cell-derived math1 + cells exhibit typical features of neurons of the external granule cells layer, including extensive motility and a t-shaped morphology. interestingly, differentiation of l7 + /calbindin-d28k + neurons (characteristic of purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by fgf8 rather than by wnt3a. this is the first report of in vitro recapitulation of cerebellar neurons by using the es cell system. sachiyo misumi, kim hye-jung, hideki hida, hitoo nishino department of neurophysiology and brain science, nagoya city university graduate school of medical science, nagoya, japan regulation of the cell cycle plays an important role in cell proliferation, differentiation, and apoptosis. we have shown that pretreatment with cell cycle blocker increase the number of neurons from neural stem or progenitor cells (npcs) without influencing apoptosis after differentiation. in this study, we investigate the molecular mechanism of neuronal differentiation by cell cycle arrest. in rt-pcr, the expression of p21 cip1 , p27 kip1 and p57 kip2 mrnas were elevated during differentiation to neuron from npcs. especially, prolonged enhancement of p27 kip1 mrna was shown. transfection of p27 kip1 into npcs induced activation of neurod promoter and increase of number of ␤tublin iii-positive cells. treatment with deferoxamine to npcs from e12.5 rat midbrain and hb1.f3 cell line did not activate erk and akt phosphorylation during the treatment. date suggest that prolonged p27 kip1 elevation is related to enhanced production of neuron from npcs, and that cell cycle regulation in g1/s phase did not activate mapk and pi3-k signaling. yuichi tanaka 1 , yusuke tozuka 1 , dai muramatsu 1 , kin-ichi nakashima 2 , tatsuhiro hisatsune 1 1 departement of integrated biosciences, graduate school of frontier sciences, university of tokyo, kashiwa, japan; 2 graduate school of biological sciences, nara institute of science and technology, ikoma, japan we previously reported no definite evidence for in vivo neurogenesis in adult neocortex. however, we also confirmed dividing cells in this area. in this study, we analyzed the characteristics of adult cortical nestin+ cells. in vivo, they belonged to ng2+ and olig2+ cells, showed slowly proliferating ability compared to those in adult dentate gyrus. for in vitro analysis, we precisely isolated progenitor cells by percoll gradient procedure. they differentiated into tuj-1+ or map-2+ neuronal cells by adding retinoic acid or bdnf. more than 90% of newborn neurons expressed gabaergic neuronal markers, gaba, gad or calretinin. we also purified nestin-gfp+ cells from nestin-gfp transgenic mice using the facs system, and confirmed their neuronal potential. moreover, integration of a neural bhlh transcription factor neurod1 significantly promoted this neurogenesis. we demonstrated neurogenic potential of adult cortical nestin+ cells. mie gangi 1 , michiko imanishi 2 , teiko kuroda 2 , masao tachibana 1 , masahiko takada 2 1 department of psychology, graduate school of humanities & sociology, university of tokyo, tokyo, japan; 2 tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan a kv3 subfamily of voltage-gated k + channels is thought to play an important role in high-frequency repetitive firings. it is unknown which subtype of kv3 channels is expressed in the frog retina where ␥-range oscillatory spikes are evoked presynaptically by light stimulation. we found immunohistochemically that kv3.1b and kv3.3 were expressed both in the mouse and frog retinas. however, a laminar pattern with two bands in the inner plexiform layer was displayed by kv3.3 in the frog retina and by kv3.1b in the mouse retina. it has been shown that mammalian cholinergic amacrine cells express kv3.1b. thus, the differential expression of kv3 channels may reflect their functional diversity between the frog and mouse retinas. hiroshi jouhou 1,2 , kazunori yamamoto 1 , masayuki hara 1 , akinori homma 1 , akimichi kaneko 3 , masahiro yamada 1 1 tokyo metropolitan univ., hino, tokyo, japan; 2 astellas pharma. inc., osaka, japan; 3 sch. rehabili., seijoh univ., aichi, japan in order to interpret the formation of receptive field surrounds in the retinal neurons, hirasawa and kaneko (2003) proposed a phmediated mechanism to substitute for the gaba-mediated feedback hypothesis from horizontal cells (hcs) to cone photoreceptors. to verify the idea that the depolarized hcs release protons we measured, by a fluorescent ratio imaging technique, the ph of the immediate external surface (ph o ) of hcs isolated from carp or goldfish retina. when hcs stained by 5-hexadecanoylaminofluorescein, a phsensitive lipophilic dye, were depolarized by application of kainate or by high extracellular k + , ph o acidified. the amount of ph o acidification was monotonically dependent on the amount of depolarization, as much as 0.21 ± 0.05 ph unit by 100 mm k + . acidification of pho was suppressed by 0.4 m bafilomycin a1, a specific inhibitor of v-atpase, suggesting that the hc depolarization enhanced an outward proton movement by the outward electrogenic h + pump. ps2a-e055 analysis of spread of activity in the local circuit of superior colliculus by using multi-channel field potential recording system penphimon phongphanphanee, katsuyuki kaneda, tadashi isa national institute for physiological sciences, japan to study how the visual signal is processed in the local circuit of superior colliculus (sc) from the superficial layers (ssc) to the deeper layers (dsc), we analyzed the propagation of excitation following the electrical stimulation of the ssc by using a planar 64-channel field potential recording system in slice preparations obtained from 16 to 24 days old mice. stimulation at ssc induced negative field potential with short latency and short duration (100-200 ms) at the recording site in ssc adjacent to the stimulating site. after application of bath containing 10 m bicuculline, the same stimulus induced a large negative field response with long duration (200-400 ms) that spreads laterally in ssc and ventrally to dsc. these responses disappeared after application of 50 m apv, when only short latency response remained. the results suggest that when gaba a receptormediated inhibition is reduced, visual signal in the ssc propagates to the dsc as large response with long duration and nmda receptors contribute to propagation of the response. osamu hosoya 1 , ken tsutsui 2 , kimiko tsutsui 1 1 dept. of neurobiol. and neuroanat., okayama univ. grad. sch. of med., dent., and pharm. sci., japan; 2 dept. of genomics and proteomics, okayama univ. grad. sch. of med., dent., and pharm. sci., japan amphiphysin ir (amph ir) is alternatively spliced variants of amphiphysin i which is specifically expressed in retina. amph ir is composed of conserved domains including the n-terminal bar, the central clathrin/ap-2 binding, and the c-terminal sh3 domains and the variant specific two novel insertions (a and b). insert a may be a determinant for the retina-specific expression. insert b has no significant homology to known proteins and two shorter transcripts with 3 -truncations in the insert were also expressed. recently, we found that a human retinal pigment epithelia cell line, arpe-19, also expresses amph ir. arpe-19 thus can be a useful tool for investigating the cellular function of amph ir in retina. immunofluorescence analyses with arpe-19 cells revealed that amph ir occasionally colocalized with mitochondria, raising the possibility that amph ir may participate in structural or functional organization of mitochondria. further characterization of the variant is under investigation. hironori takamura 1 , satoshi ichisaka 2 , chihiro hayashi 2 , hirotoshi maki 2 , yoshio hata 1 1 div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; 2 div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan monocular deprivation (md) induces significant plasticity in the primary visual cortex (v1) during critical period. it was reported that inhibition of extracellular signal-regulated kinase (erk) activity in the visual cortex suppressed the ocular dominance plasticity. if erk is involved in the mechanism of this plasticity, visual deprivation would change the activity of erk in v1 and such change might be induced only in the critical period. to test this possibility, we examined effects of md on the amount of phosphorylated (activated) erk (perk) in the rat visual cortex. by md, we found a significant decrease in the amount of perk in v1 receiving deprived eye inputs in both young and adult rats. as to the subcellular localization of erk, we found a significant increase of the nuclear perk only after md in young rats. these results suggest that erk signaling might be regulated by different mechanism between young and adult rats. research funds: kakenhi (176959) ps2a-e058 rapid pre-synaptic weakening by experiencedependent competition in mouse visual cortex nobuko mataga, yoko mizuguchi, takao hensch neuronal circuit development, riken brain science institute, saitama, japan in the binocular zone (bz) of mouse visual cortex, critical period (cp) plasticity is accompanied by a transient loss of spines on pyramidal cell dendrites. to explore a correspondingly rapid and local pre-synaptic refinement by sensory deprivation, excitatory intracortical or thalamocortical axon terminals were visualized in the bz by vesicular glutamate transporters (vglut)1 and vglut2, respectively. a complementary distribution of vglut1 and vglut2 was established by postnatal day (p)18 and both signal intensities increased further by p29-30 (peak cp). the immunoreactivity for vglut2 decreased around layer iv after brief monocular deprivation (4dmd) during the cp. interestingly, both signals in all layers were lower in the bz contralateral to an eye injected with ttx than in the ipsilateral bz, consistent with the stronger functional plasticity and rapid dendritic refinement as compared to 4dmd. these results suggest that rapid and local weakening of excitatory inputs corresponds to dendritic spine pruning during experience-dependent competition. reiko meguro, masao norita department of sensory and integrative medicine, niigata university, graduate school of medical and dental sciences, niigata, japan we investigated how the geniculate and the extra-geniculate visual systems reorganize by monocular deprivation at birth. using anterograde/retrograde tracer, biotinylated dextran amine (bda), we made a small injection into the dorsal lateral geniculate nucleus (dlgn) or the lateral posterior nucleus (lp) of the degenerated side of the monocular deprived rat. the geniculate projection terminated mainly in layer iv of area 17, with a small projection to layer vi of areas 17 and 18a. cells in layer vi of area 17 projected to dlgn. in addition, cells in layer v of area 17 projected to dlgn, which is not observed in normal rats. in area 18a, cells in layers v and vi projected to dlgn. the projection from lp terminated mainly in layer iv of 18a. cells in layers v and vi of area 18a projected to lp. smaller number of cells in layer v of area 17 also projected to lp. these findings suggest that major parts of visual system developed normally, but some developed cross talk between geniculate and extra-geniculate systems. ps2a-e060 activity dependent plasticity of feedback projection from the primary visual cortex to the dorsal lateral geniculate nucleus miho yoshida 1 , takemasa satoh 2 , yoshio hata 1 1 div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; 2 div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan the projection from the lateral geniculate nucleus (lgn) to the primary visual cortex (v1) shows significant morphological plasticity responding to visual experiences in early life. such experiencedependent plasticity enables the geniculocortical projection to form functionally specific connections. it is not clear whether similar plasticity operates in other neural connections in the visual system. therefore, we tried to investigate the plasticity of feedback projection from v1 to the lgn. we focused on the density of type 1 metabotropic glutamate receptor ␣ (mglur1␣) in the lgn which locate postsynaptically at synapses of feedback projection. immunohistochemical signal for mglur1␣ in the lgn decreased after elimination of v1, showing that this signal reflects density of functional feedback synapses. to explore the activity dependent plasticity, we examined the effect of cortical activity blockade on the mglur1␣ signal in the lgn of young rats. yu morishima 1 , hiroshi sakamoto 2 , takahumi akasaki 1 , yoshio hata 1 1 div. integrative biosci., tottori univ. grad. sch. med. sci., japan; 2 div. neurobiol., sch. life sci., fac. med., tottori univ., japan monocular deprivation (md) during postnatal development reduces cortical response to the deprived eye and input axons serving the deprived eye retract. however, when md is combined with continuous inactivation of the visual cortex by muscimol, cortical neurons lose their response to the open eye and the open eye axons retract. to clarify mechanisms underlying the two forms of ocular dominance (od) plasticity in different direction, we examined other characteristics of them, (1) how rapidly the reverse od shift proceeds and (2) whether the shift is induced only in young animals. we infused the cortex with muscimol in 4-week-old kittens and in adults. the reverse od shift was observed after 6 days md, but not significant after 3 days md. in adults, od distribution remained unchanged. morphological change of individual input axons was also examined after 6 days md. the reverse od shift might reflect a mechanism of developmental plasticity that has a slower time course than the normal od shift. ps2a-e062 experience-dependent plasticity in the absence of ampa receptor subunits in mouse visual cortex youichi iwai 1 , nafiseh atapour 1 , john renger 1 , john roder 2 , peter seeburg 3 , takao hensch 1 1 neuronal circuit dev., riken, bsi, wako, japan; 2 mount sinai hospital, toronto, canada; 3 max planck inst. med. res., heidelberg, germany two ampa receptor subunits (glur1, 2) play prominent roles in hippocampal models of homosynaptic plasticity (ltp/ltd). brief monocular deprivation (1d md) rapidly alters both the phosphorylation state and surface expression of glur1 in visual cortex. here, we addressed whether these coincidental events are essential for subsequent ocular dominance (od) plasticity. mice lacking glur1 (ko) displayed little ltd in visual cortical slices, while baseline transmission was normal. they also exhibited normal visual receptive field properties in vivo and shifted responsiveness toward the open eye after 4d md during a typical critical period. the rate of plasticity appeared somewhat slowed, as 20d md eventually led to full od shifts. in glur2 ko mice, even 4d md robustly activated od plasticity. thus, experience-dependent modification of ampa receptors is not essential for plasticity in vivo, although glur1 may contribute to the very earliest stages. shigeyoshi higo, nobuaki tamamaki department of morphological neural science, kumamoto university, kumamoto, japan virus-assisted transduction with reporter genes is a useful technique to investigate morphology of neurons in the central nervous system. however, the mechanisms to induce reporter expression in vivo often depend on gene-manipulated mice. since mice are not the best experimental animal for the study of mental disorder, we developed an adenovirus in which gfp expression is driven by dlx2 promotor and dlx5/6 enhancer. this virus labels gabaergic neurons and oligodendrocyte in the wild-type mouse neocortex and allows us to trace gfp-labeled axons of gabaergic neurons in serial brain sections. we used this virus to investigate gabaergic neurons with long projecting axon branches beyond a functional area. the virus was injected into the stratum oriens of the mouse hippocampal field ca1 and revealed a nonpyramidal neuron projecting to ca3 and fimbria. further we shall introduce this virus to the cat brain and investigate axon branches of gabaergic projection neurons in the neocortex. akiko yamashita 1 , takao oishi 2 , motoharu hayashi 2 1 div. appl. system neurosci., nihon univ. grad. sch. med. sci., tokyo, japan; 2 dept. cell. and mol. biol., primate res. inst., kyoto univ., inuyama, japan gabaergic cells in the cerebral cortex are divided into subgroups: parvalbumin (pv)-, somatostatin (som)-, calretinin (cr)-, and calbindin-containing types. to clarify inhibitory system in primates, we determined coexistence of these molecules and proportions of these subtypes within gabaergic cells in the various cortical areas. pv, som or cr did not coexist with each other in primates as observed in rodents. more than 60% of gabaergic cells contained pv; showing that pv cells are more abundant in primates than in rodents. proportion of som cells in gabaergic cells was smaller in the primary visual area (5.3%) than in other areas, such as the prefrontal (10.8%), primary motor (9.8%), somatosensory (10.0%) and secondary visual areas (8.5%), indicating cortical differentiation in gabaergic system of the primate cerebrum. our recent retrograde labeling studies in mice and cats showed that the neocortical areas are connected not only by excitatory neurons but also by gabaergic projection neurons. in order to address the importance of the gabaergic projection neurons in the neocortical information processing, we need to know the branching pattern and postsynaptic elements of the gabaergic projection axons. since more than 80% of the gabaergic projection neurons showed npy immunoreactivity, we used npy-cre transgenic mouse that express cre in npy neurons and adenovirus that encodes gfp in the downstream of floxed stop to label the gabeergic projection axons. after injection of the adenovirus into deep layers of the npy-cre mouse neocortex and immunoperoxidase staining of gfp in the brain section, we could reveal gabaergic neurons in a golgi-like image with their axons. also this method seemed to allow us to label gabaergic projection neurons retrogradely. koji ikezoe 1 , guy n. elston 2,3 , tomofumi oga 1 , hiroshi tamura 1,3 , ichiro fujita 1,3 1 osaka univ., japan; 2 univ. queensland, australia; 3 crest, jst, japan layer iii pyramidal cells in adult monkeys exhibit systematic differences in their dendritic morphology among cortical areas. basal dendrites of cells in visual association cortex such as inferior temporal area te spread more extensively and are more branched than those in the primary visual cortex (v1). pyramidal cells in prefrontal cortex, such as area 12, have even more dendritic branches than those in area te. here, we investigated whether a similar regional difference in the dendritic morphology was present in infant monkeys. we stained individual layer iii pyramidal cells in v1 (n = 52), area te (n = 46), and area 12 (n = 43) of a 3-week old monkey (macaca fascicularis) using intracellular dye-injection techniques in lightly fixed tissues. the number of branches and the tangential extent of dendrites was greatest in area 12, followed by area te, and v1. thus, considerable heterogeneity in pyramidal cell structure already exists 3-weeks after birth. hiroaki matsushita 1 , mahito ohkuma 1 , masami watanabe 2 , ei-ichi miyachi 1 1 department of physiology, fujita health university, aichi, japan; 2 department of perinatology, institute developmental research, aichi, japan acetylcholine (ach) receptors are believed to be expressed in developmental and regenerative process of retinal neurons. we performed the patch-clamp recording and fura-2 based calcium imaging in cat retinal ganglion cells (rgcs). under whole cell clamp conditions, transient sodium currents and action potentials were observed in all of normal or axonal regenerated rgcs. however, these currents and spikes were not observed in the 50% of axotomized rgcs. bath application of 100 m carbachol, an ach receptor agonist, rose [ca 2+ ] i in 22% of normal rgcs. although the 85% of rgcs responded to carbachol at 3 days after axotomy, no responsive rgcs appeared during 7-15 days. ach responsiveness recovered in axonal regenerated rgcs (17%). since pycnotic cells were observed few days after axotomy, ach may modulate neurotrophic effect in survived rgcs. these results suggest that ach is an important marker for neuronal degeneration and regeneration in cat rgcs. research funds: kakenhi (17500217 to em, 16591780 to mw) kenichiro miura, masakatsu taki, hiromitsu tabata, kenji kawano dept. integrative brain sci., grad. schl. of med., kyoto univ., kyoto, japan the initiation of smooth pursuit eye movements is facilitated by the bottom-up attention to the target (hashimoto et al., 2004) . to study the effects of the attention on the processing of second-order motion stimuli, we recorded smooth pursuits in three humans with a dualpurkinje-image eye tracker. the pursuit target, presented on a crt monitor (75 hz), was a gaussian patch of texture displayed on a neutral gray background. the gaussian envelope moved at 30 deg/s, while the texture consisting of black and white random-noise pixel blocks remained stationary (drift-balanced stimulus). the number of the frames displaying the target before the motion onset was selected to manipulate the attention to the target, either eight frames (107 ms) or only one frame (13 ms). the initial tracking responses were larger when the target became visible eight frames before the motion onset. the result suggests that the second motion processing underlying the smooth pursuit initiation is facilitated by the attention to the target. ps2a-e069 motion picture effects on eye movements and blood flow in the frontal area atsuhiko iijima 1,3 , tohru kiryu 2 , kazuhiko ukai 3 , takeshiko bando 1 1 div. integrative physiol., grad. sch. med., niigata univ., niigata, japan; 2 div. inform. sci., grad. sch. & tech., niigata univ., niigata, japan; 3 dept. appl. phys., sch. sci. & tech., waseda univ., tokyo, japan motion pictures taken by rider's view of motocross bike elicited horizontal eye movements coherent to the motion vectors in some subjects, and not coherent in the other subjects, while those taken by passenger's view of roller coaster evoked similar eye movements in all of the subjects. 20 subjects watched the two-dimensional motion pictures in random order. eye movements were measured by a binocular video oculography (newopto), and head movements were measured by a magnetic motion sensor (polhemus). blood flow in the frontal area was simultaneously monitored with a near infrared spectroscopy (hamamatsu). the patterns of eye movements and the blood flow variation during movie presentation changed in relation to motion components of the movie. possible mechanisms of the differences will be discussed. kiyoto matsuura 1 , kenichiro miura 1 , masakatsu taki 1 , hiromitsu tabata 1 , naoko inaba 1 , kenji kawano 1 , frederick a. miles 2 1 grad. schl. med., kyoto univ., kyoto, japan; 2 lab. sensorimotor res., nei, nih, bethesda, md, usa human ofrs show winner-take-all behavior when elicited by moving grating patterns composed of two sinusoids (sheliga et al., sfn 2005) . we recorded the ofrs to the motion of vertical grating patterns composed of two sinusoids of spatial frequency 3f and 5f, which created a repeating pattern with beat frequency, f, in two monkeys. motion consisted of successive steps (100 hz), each one-fourth of the wavelength of the beat, so that with each step the two components shifted one-fourth of their wavelengths and had opposite directions, the 5f forwards and the 3f backwards. the contrast of the 5f was fixed at 4, 8, or 16%, while the contrast of the 3f was varied from one-fourth to four times the contrast of the 5f. when the contrast of the 3f (5f) was less than about half that of the 5f (3f), the 5f (3f) dominated initial ofr: winner-take-all. thus, the motion processing underlying the ofr in monkeys, like that in humans, includes nonlinear interactions. masazumi katayama, takahiro fujita department of human and artificial intelligence systems, faculty of engineering, university of fukui, fukuki, japan when executing prehension movement to grasp an object such as a tool, we plans the hand shape and grasping position to grasp a target object. while, goodale proposes the hypothesis that the roles of two visual streams (dorsal and ventral streams) are "vision for action" and "vision for perception", respectively. from the above points of view, we investigated independence of visual estimation and motor execution for grasping position of a target object. in this experiment, grasping positions were measured under the following four conditions: visual estimation without grasping, grasping without lift-up movement, grasping and lift-up movement and visual estimation without grasping. as a result, we found that grasping positions of visual estimation are significantly different from grasping positions of motor execution in the second and third conditions (p < 0.05). we concluded that grasping positions of visual estimation and motor execution are independent and these results support the goodale's hypothesis. ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst. niigata univ., niigata, japan cortical sensory areas are divided into modality-specific domains such as the visual and auditory cortices, in which sensory neurons are driven by modality-specific inputs. recently, wallace et al. found that multimodal neurons clustered in deep layers are present near the borders between sensory cortices. multimodal properties of these neurons may be explained by three types of inputs: overlapped projections from the thalamus, projections from multi-modal sites, or overlapped horizontal projections from the modality-specific sensory cortices. in this study, we tested the third possibility. we prepared the mouse cortical slices including the visual and auditory cortices. the horizontal activity propagation elicited by local electrical stimulation were visualized using flavoprotein fluorescence imaging. these results indicate that cortical areas between the visual and auditory cortices receive horizontal projections originated in the visual and auditory cortices, suggesting that multimodal horizontal connections are important for the multimodal properties of sensory neurons. jumpei naito 1 , yaoxing chen 2 , yukiko tanada 1 1 dept. animal sci., teikyo univ. sci. & tech., uenohara, japan; 2 china agricul. univ., beijing, china twenty white-leghorn chicks (p0-8) were perfused with 1% paraformaldehyde through the heart under deep anesthesia of nembutal (32 mg/kg bw). two to three small crystals of dii were implanted into the optic tectum, thalamus, or hypothalamus under a dissecting microscope. a total of 225 rgcs were classified into six groups according to the somal area and dendritic field (naito and chen, 2004). group ic projected dominantly to the tectum. group is and iiis showed high hypothalamic-and thalamic-dominance, respectively. group iic was non-specific in the central projections. group ivc was tectal-dominant. patterns of the dendritic stratification were counted to 32 in 121 tec-rgcs, 28 in 77 tha-rgcs, and 16 in 38 hyo-rgcs. of these stratification patterns, many patterns were common among tec-, tha-, and hyp-rgcs. in contrast, the rgcs that showed a same dendritic pattern were consisted of a single rgc in most of the non-common rgcs, and their dendrites extended mainly to the superficial inner plexiform layer (sublayers 1-3). yasuro atoji, shouichiro saito laboratory of veterinary anatomy, gifu university, gifu, japan the present study was examined afferent and efferent fiber connections of the intermediate part of the caudal nidopallium (nci) in the pigeon by a tract-tracing method. in the present study we define nci an area which is located lateral to the field l complex and ventral to ncl. following a ctb injection into nci, a large number of neurons was labeled in nci, the mesopallium, and intermediate arcopallium (ai) and in the thalamic posterior dorsointermediate and posterior dorsolateral nuclei. contralateral ai contained a small number of labeled neurons. a few labeled neurons were found in lst. few labeled cells were found in ncl, field l, piriform cortex, or hippocampal formation. following a bda injection into nci, a large number of labeled fibers extended in nci, mesopallium, and ai. lst contained a small number of labeled fibers. few labeled fibers were located in ncv and limbic regions. the diencephalon contained very few labeled fibers. in summary, nci has strong reciprocal connections within nci itself and with the mesopallium and ai, and little connections with the limbic system. hidenori horie 1 , kenji yuda 3 , eiichi okawa 4 , katsuyoshi maruyama 4 , hiroshi uozato 5 , hiroko horie 1 , satomi nakajima 1 , kenkichi tanioka 6 , yuji ohkawa 6 , tomoki matsubara 6 , wolfram tetzlaff 7 1 advanced res. centr. biological sci., waseda univ., tokyo, japan; 2 technomaster co. ltd., yokohama, japan; 3 kikuna yuda eye clinic, yokohama, japan; 4 healthcare business co., matsushita electric ind. co. ltd., yokohama, japan; 5 dept. ophthalmol. & visual sci., kitasato univ., kanagawa, japan; 6 nhk sci. technical res. labo., tokyo, japan; 7 icord, univ. british columbia, vancouver, canada we describe here a highly effective method to improve visual acuity of children with myopia and adult with presbyopia by repeatedly offering a visual object at variable distances while keeping the apparent retinal projection size of the object constant using a novel electronic device. in our experiments on human subjects, we used an lcd screen that was rhythmically moved between 25 and 70 cm toward and away in a high speed (top speed: 1000 mm/s) from the subjects. the device significantly improved visual performances in over 60% of the school-aged children with myopia and 80% of adults with presbyopia. hiroyuki miyamoto 1 , toral s. surti 2 , takao k hensch 1 1 laboratory for neuronal circuit development, riken brain science institute, wako, japan; 2 san francisco, usa competitive plasticity of binocular response following monocular deprivation (md) is prominent in the primary visual cortex (v1) during an early critical period. recently, md has been shown to enhance head-tracking behavior induced by slow rotation of grating stimuli in adult mice and is critically dependent upon the integrity of v1. here, we addressed to what extent these two types of plasticity induced by the same md share common mechanisms. adult mice lacking a gaba-synthetic enzyme (gad65 ko), which do not exhibit ocular dominance (od) plasticity by brief md during the critical period, showed normal optomotor acuity and enhancement with 4 day md. od shifts did not correlate with optomotor enhancement in these mice. finally, early md spanning the entire critical period had no effect on optomotor acuity through the deprived-eye. these observations support the view that adult perceptual learning and classical od plasticity are independent. junya hirokawa 1 , miquel bosch 2 , shuzo sakata 3 , yoshio sakurai 4 , tetsuo yamamori 1 1 division of brain biology, national institute for basic biology, okazaki, japan; 2 mit, ma, usa; 3 rutgers university, nj, usa; 4 department of psychology, kyoto university, japan the brain is able to integrate information from different sensory sources to enhance behavioral responses. to identify the neuronal populations responsible for multisensory enhancement in rats, we have mapped the activation of neurons during an audiovisual integration paradigm (sakata, et al., 2004) by the expression of c-fos. a pronounced c-fos upregulation was found in superior colliculus and in layer iv and deep layer of latero-medial secondary visual area (v2lm). local injection of gaba agonist muscimol into this region selectively suppressed the behavioral enhancement related to multisensory integration, while no suppression was found by the injection into primary auditory and visual areas. these results suggest a key role of v2lm in integration of auditory and visual information to facilitate the behavioral reaction for bimodal stimuli. takashi shinozaki 1 , youichi miyawaki 2 , tsunehiro takeda 1 1 department of complexity science and engineering, university of tokyo, chiba, japan; 2 mathematical neuroscience, riken bsi, saitama, japan drifting grating patterns with different motion directions independently presented to the two eyes induce two sets of perceptual rivalries: interocular rivalry (left or right eye's image) and motiontype rivalry (pattern or component motion). we studied this double rivalry process based on psychophysical and magnetoencephalography (meg) measurements. pattern-motion percept exclusively arose and persisted for a long duration whereas component-motion percept was soon followed by percept of either of left or right eye's single motion direction. reaction time (rt) measurement showed that the pattern-motion was perceived faster than left or right eye's motion direction. we then compared meg signals among those perceptual conditions and found a meg response of interocular rivalry in the latency range expected from the result of rt measurement. these results suggest that the double rivalry process has a hierarchical structure in which motion-type rivalry is resolved before interocular rivalry. visual stimuli evoke several brain potentials with relatively precise time courses. the role of these brain potentials in visual object categorization is not clear. in this study we recorded event related brain potentials (erp) while subjects participated in a face/non-face categorization task. gray face and non-face natural object images were presented briefly (10 ms) followed by a noise mask with pseudo randomly selected stimulus onset asynchrony (soa = 0-990 ms). subjects reported presentation of face or non-face images by pushing one of the two assigned keys. we found that the face category discrimination performance significantly declined only in short soa (10 and 20 ms) with a larger impact of masking on non-face discrimination. in erp, the peak amplitude and latency of p1, n170 and area under curve of a late positive potential expanding from 300 to 500 ms were correlated with the subjects behavioral performance. the effect of backward masking on early erp components may be due to altering sensory processing of visual stimuli while the effect on late erp potential could be related to its impact on decision making processes. yasushi naruse 1 , ayumu matani 1,2 , tomoe hayakawa 2,3 , norio fujimaki 2 1 university of tokyo, kashiwa, japan; 2 nict, kobe, japan; 3 teikyo university, tokyo, japan to study the process of alpha rhythm resetting, we investigated the relationship between visual evoked potential and the seamlessness: how much the phase angle of prestimulus alpha rhythm and the backward-extrapolated phase angle from poststimulus alpha ringing synchronize. alpha ringing is an evoked potential in alpha frequency band around 500 ms in latency. eight clinically normal adult volunteers participated in the experiment, in which the subjects passively viewed a series of 1000 flash stimuli with their eyelids closed throughout the experiment. eeg was simultaneously recorded during the experiment. we classified the trials into four subsets owing to the seamlessness, and then averaged the trials in each subset. the result showed that the larger the amount of the alpha rhythm resetting is, the larger the p100 amplitude becomes. this suggested that a factor of the variability of the p100 amplitude is the amount of the prestimulus alpha rhythm resetting. research funds: a grant-in-aid from the ministry of education, culture, sports, science and technology (no. 16300083) hitoshi sasaki 1 , takuya ishida 1 , masayoshi todorokihara 2 , junichiro miyachi 1 , tahei kitamura 3 , ryozo aoki 1 1 dept. physiol. & biosignal., osaka univ. grad. sch. med., suita, japan; 2 dept. phys. & elec., osaka pref. univ. grad. sch. eng., sakai, japan; 3 dept. elec. eng. & elec., col. industri. tech., amagasaki, japan recently it has been shown that noise can improve detection of sensory stimuli in several modalities. here we investigated whether visual contrast detection sensitivity can be improved by adding a certain amount of noise. contrast detection thresholds of a light changing brightness periodically were measured with or without overlapping noise in five normal participants. the contrast detection threshold, measured by using the psychophysical method (up-anddown method), decreased at around the threshold level of the noise intensity. these findings are consistent with our previous findings obtained by using another psychophysical method and confirm that noise can improve signal detection in human visual perception. narumi katsuyama 1 , nobuo usui 1 , izuru nose 1,2 , masato taira 1,3 1 department of applied system neuroscience, nihon university school of medical science, tokyo; 2 faculty of human science, bunkyo university, saitama, japan; 3 arish, nihon university, tokyo, japan when an object is moving, perception of its 3d trajectory in depth can be strongly influenced by the trajectory of its cast shadow. for example, a ball moving in a diagonal trajectory can appear to rise in a frontal plane when the shadow moves along the horizontal trajectory (rising configuration) or to roll in depth when the shadow follows the same trajectory as the ball, while the trajectory of ball is identical. using fmri, we found that several visual areas, including human mt and the posterior sts and the posterior parietal cortex, are activated in the comparison between rising configuration and ball only condition. additional correlation analysis by modifying the slope of the shadow' s trajectory also showed activation in the posterior part of sts and the posterior parietal cortex, including precuneus. these results suggest that cortical areas in the temporal and parietal cortex might be involved in the processing of apparent motion of ball induced by the moving cast shadow. ps2a-f083 local area network in the gerbil's auditory cortex: reversible focal inactivation with infrared laser irradiation akira yamamoto, hiroshi riquimaroux gratuate school of engineering, doshisha university, scnrl, japan this study investigated local area networks in the primary auditory cortex (a1) and the anterior auditory field (aaf) by blocking neural activities with the near-infrared laser irradiation (wave length = 830 nm). in previous in vivo studies, the laser irradiation could focally inactivate neural activities in a few minutes after the irradiation started, while the activities recovered in a few minutes after its cessation. by using this technique, the present study examined corticocotical relationships in the auditory cortex of the mongolian gerbils (meriones unguiculatus). cf (constant frequency) and fm (frequency modulated) tones were presented to anesthetized animals, and neural responses were extracellularly recorded contralaterally to the ear of stimulation. when irradiated aaf area and recorded neural responses from ai, the irradiation changed phasic responses into tonic responses, and vice versa. these results indicate that there are functional connections within ai or aaf, and between ai and aaf. takashi doi, hiroshi riquimaroux department of knowledge and engineering and computer sciences, doshisha university, kyoto, japan in a previous behavioral study, ablation of right auditory cortex (ac) made the discrimination between ascending and descending frequency modulated (fm) tones by mongolian gerbil (meriones unguiculatus) difficult (wetzel et al., 1998) . this result indicates that some neurons in gerbil's right ac represent the directions of fm sweeps. actually, we could find direction-dependent neurons and these neurons were mainly in anterior auditory field (aaf). in aaf, bfs are gradually shifted along the rostrocaudal direction, and the same bfs are arranged in dorsoventral direction (thomas et al., 1993) . moreover, aaf has dense synaptic connections within the area (budinger et al., 2000) . we made network models based on this structure of aaf and could gain similar responses to the actual responses of directiondependent neurons. this result suggests that aaf in gerbil's ac has good structure to process fm tones. research funds: a grant to rcast at doshisha univ. from mext, innovative cluster creation project by mext it has been demonstrated that the auditory space, namely the direction of a sound source, is represented topographically in the mammalian superior colliculus (sc). however, it is unclear as to how this auditory space map of the mammalian sc is formed in the auditory pathway. the present study investigated the topographical representation of auditory space in the external nucleus of the inferior colliculus (icx) of anesthetized gerbils. the icx is the major auditory nucleus that has projections to the sc. the stimuli were 50-ms noise bursts whose azimuths varied on the horizontal plane in the virtual acoustic space. single-unit responses were recorded from the icx. the majority of units exhibited some degree of spatial selectivity and preference for the azimuth contralateral to the recorded side. for supra-threshold stimulus only, there were topographical gradients of preferred azimuths in the icx. however, the spatial tuning width and preferred azimuth of the units depended markedly on stimulus level. the results indicate that in mammals, the formation of a rigorous auditory space map is incomplete at the icx level. manabu toyoshima 1 , yasuo takeda 2 , yasushi shimoda 1 , kazutada watanabe 1 1 department of bioengineering, nagaoka university of technology, nagaoka, japan; 2 department of clinical pharmacy and pharmacology, kagoshima university, kagoshima, japan nb-2 that we isolated and identified is a neural cell recognition molecule belonging to contactin subgroup. we reported previously that nb-2 expression is prominent in the auditory system. nb-2 knockout mice exhibit impaired neural function in the auditory system. these findings indicate that nb-2 is indispensable for the function of auditory system. here we report the detailed analysis of the nb-2 localization using anti-nb-2 monoclonal antibody that we produced recently. immunohistochemical analysis of the rat brain showed that nb-2 was detected not only in all brain regions of the auditory pathway, but also in substantia nigra (sn), caudate putamen (cpu) and fibers projecting from sn to cpu. the nb-2 immunopositive cells in sn are restricted to gabaergic neurons. since gabaergic neurons play essential roles in the development and function of the auditory system, it is highly likely that nb-2 regulates the development and/or function of gabaergic neuron in the auditory pathway. reiko nagashima, kiyokazu ogita department of pharmacology, setsunan university faculty of pharmaceutical sciences, osaka, japan sensorineural hearing loss can be caused by a variety of insults, including acoustic trauma. there is compelling evidence that reactive oxygen species (ros) are formed in the cochlea during acoustic stimulation. glutathione (gsh) protects against the hearing loss through scavenging ros generated by noise. in this study, we investigated the changes in expression of gamma-glutamylcysteine synthetase (gcs) gene, which is the rate-limiting enzyme in de novo gsh synthesis, in the cochlea following acoustic stimulation. nuclear extracts were prepared from the cochlea at various time points after acoustic stimulation (4 khz octave band, 125 db, 5 h), and then subjected to electrophoretic mobility shift assay to determine activator protein-1 (ap-1) dna binding. ap-1 binding was increased 2-12 h after the exposure. rt-pcr and immunostaining revealed that noise exposure was effective in elevating the expression of gcs in the cochlea 2 h later. taken together, ap-1 may participate in the expression of gcs gene in the cochlea after acoustic stimulation. masaharu kudoh, ryuichi hishida, katsuei shibuki 1 department of neurophysiology, brain research institute, niigata university, niigata, japan multiple formants compose vowels. we have previously reported that bilateral lesions including in the auditory cortex (ac) of rats impaired discrimination learning between synthesized vowel-like sounds with multiple formants, while discrimination between stimuli of a single formant or pure tones was not significantly impaired. in the present study, we determined the responsible auditory fields, which were required for the discrimination leaning between vowel-like sounds. water-deprived rats were trained to discriminate between two sounds including four different formants. licking a spout during presentation of one sound was rewarded with water while the other was not. surprisingly, local lesions in the primary ac or the ventral association cortex had no clear effect on the discrimination learning. in contrast, the dorsal association areas impaired the discrimination learning. these findings indicate that the dorsal auditory association cortex plays a critical role in discrimination learning of vowel-like sounds with multiple formants. hiroaki tsukano, yamato kubota, manavu tohmi, masaharu kudoh, katsuei shibuki department of neurophysiol, brain research institute, niigata university, niigata, japan we used transcranial flavoprotein fluorescence imaging for visualizing cortical responses to missing fundamentals in mice. c57bl/6 mice were anesthetized with urethane. the skull on the auditory cortex was exposed and covered with liquid paraffin to keep the skull transparent. cortical images of green fluorescence in blue light were recorded by a cooled ccd camera. responses in the auditory cortex elicited by sound stimuli (5-26 khz for 500 ms) exhibited mirrorsymmetrical tonotopic maps in the primary auditory cortex (ai) and anterior auditory field. the activity patterns in ai elicited by 5 khz were different from those elicited by 20 or 25 khz. however, the areas activated by 5 khz were also activated by the mixture of 20 plus 25 khz but not by that of 19 plus 26 khz, suggesting that cortical responses to missing fundamentals in ai were visualized using flavoprotein fluorescence imaging. hiroko kosaki national priting bureau, tokyo, japan we constructed a functional scheme of macaque auditory by distribution of calcium binding protein, parvalbumin (pv). auditory cortex is consisted of one core (primary cortex), and five surrounding rings, which correspond with secondary, tertiary, quaric, and quintic cortices. parvabumin showed a graduation, that is, inner core is most pv-rich, and outer rings showed the decrease of pv concentration. comparing with pv staining in visual cortex, these six-levels suggested similar hierarchic and reciprocal structure, which are proposed by deyoe and vanessen by analysis of feed-forward and feedback connections. akihisa kimura, tomohiro donishi, keiichiro okamoto, yasuhiko tamai department of physiology, wakayama medical university, wakayama, japan tonotopically comparable subfields of the primary and non-primary auditory areas in the rat cortex have similar topographies in the projection to the medial geniculate body but reverse topographies in the projection to the thalamic reticular nucleus (trn). in the present study, we determined how cortical and thalamic afferents intersect in the trn with regard to tonotopic organization. in light of the fact that a subset of auditory cells in the trn responds to visual or somatosensory stimulus, we also explored the potential sources of cortical and thalamic afferents that would set up polymodal sensory interaction in the trn. small injections of biocytin into the trn, which were made with guidance of electrophysiological recording of auditory response, resulted in retrograde labeling. retrogradely labeled cortical and thalamic cells exhibited distinctive patterns of distribution depending on the injection sites. the results indicate anatomical nodes in the auditory trn that would implement selective relay of auditory and/or polymodal sensory inputs. ps2a-f092 functional connections between the core and belt fields of the guinea-pig auditory cortex observed by optical recording and partial cortical inhibition using muscimol junsei horikawa, daisuke uchiyama, tatsunori matsui, shunji sugimoto department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan guinea pigs were anesthetized with ketamine and responses to puretones in the auditory cortex stained with a voltage-sensitive dye rh795 were recorded with a photodiode array. after determining the core (ai and dc) and belt fields, ai or dc was inhibited by putting a muscimol-containing agar piece on each field. the inhibition of ai resulted in reduction of responses in the belt fields by 55-90%, whereas the inhibition of dc resulted in reduction only by 20-50%. the reduction by ai inhibition was larger in the anterior and ventral belt fields and that by dc inhibition was larger in the posterior belt fields than in the other fields. further inhibition of dc after the ai inhibition or vice versa resulted in suppression of the responses in all the fields. these results suggest that the responses of the belt fields are elicited mostly via connections from the core to the belt fields and the belt fields receive differential connections from ai and dc. masataka nishimura, hiroyuki kaizo, wen-jie song department of electrical, electronic, and information engineering, graduate school of engineering, osaka university, suita, japan the auditory cortex of many mammals has a core area and surrounding belt regions. in guinea pigs, the primary auditory area, the secondary auditory area, and many belt regions have been reported. however, the activity of the belt regions has not been fully examined. using a high-resolution optical imaging system, we examined cortical responses to tone stimulations in anesthetized guinea pigs. the auditory cortex of six guinea pigs was exposed to the ventral end and stained with the voltage-sensitive dye rh-795. a novel field in the ventro-anterior region was identified based on its isolated responses to a pure tone stimulation and the relatively long latency of the responses. the field was located ventro-caudal to the primary auditory area, and was close to the ventral edge of the auditory cortex. we thus named the field as ventro-caudal field (vc). smooth frequency gradient was observed in vc in rostro-caudal direction, with the frequency axis in opposite direction to that of the primary auditory area. yoko kato 1,2 , kazuo okanoya 1 1 laboratory for biolinguistics, riken bsi, wako, saitama; 2 graduate school of humanities, university of chiba, chiba, japan bengalese finches sing complex courtship songs. to sing complex sequences, they require auditory feedback during singing. song nucleus nif has a projection from primary auditory area field l and then it projects to sensory/motor nucleus hvc. moreover, bilateral lesion of nif cause song deterioration on complex sequences (hosino and okanoya, 2000) . we recorded auditory responses by multiunit activity from nif and field l. auditory responses of nif showed selectivity to bird's own song (bos) than its reversed song (rev). comparing selectivity of nif and field l, nif showed stronger selectivity than field l. however, nif did not show sequence dependent selectivity. these results suggest that nif relays auditory information and enhances bos selectivity. however, we did not observe a direct evidence that nif related to generation of complex sequences. we started to record responses extracellularly from ac neurons of guinea pigs. in general, animals show a stereotyped pattern of behaviors; they have a quiet, almost-motionless period, usually for tens of min. during this period, animals do not appear to sleep but be sensitive to the environmental disturbance. thereafter they usually fall asleep with their eyes closed. during this presleeping period, the best frequency tone was repeatedly presented 100-500 times at a fixed interval, through a speaker at 50-70 db spl. responses to such repetitive tones are apparently irregular, with the occurrence of spikes in most trials but no spikes in some trials. however, if all the trials are accumulated, there was global phase alternation every a few to tens of seconds. one phase constitutes relatively high rates of spike occurrence, while the other very low rates of spike occurrence. we suppose that, unlike a machine, the brain has a unique mechanism that automatically turns on and off the cortical processing of the redundant sensory stimulus. masashi sakai, sohei chimoto, ling qin, yu sato department of physiology, university of yamanashi, japan a periodic click train produces a continuum of several perceptual qualities: (i) at low repetition rate (<10 hz), the individual clicks are clearly heard as discrete events so that the entire train produces "rhythm" percept, (ii) at high repetition rate (>100 hz), the entire train is heard as a single continuous event leading to a strong "pitch" percept, and (iii) in the transition range, the periodicity can still be detected as "roughness". we physiologically explored how those perceptual qualities are represented in the primary auditory cortex in awake cats. we found that distinct population of cells conducted two coding modes: (i) representing low-rate stimuli through stimuluslocking activity (i.e., temporal code) and high-rate stimuli as only onset responses or (ii) exhibiting sustained responses with generating larger amount of discharges at higher repetition rate (i.e., rate code). in addition, pure-tone stimuli elicited onset responses or sustained responses in each of these cell populations, respectively. we will discuss functional consequences and spike evocation mechanisms of each population. atsuhito toyomaki 1,2,3,4,5,6 1 hokkaido university, sapporo, japan; 2 hokkaido university, sapporo, japan; 3 sakushin gakuin university, utsunomiya, japan; 4 kobe shoin women's university, kobe, japan; 5 riken, wako, japan; 6 sakushin gakuin university, utsunomiya, japan gaps in a continuous sound play important roles for perception of voiceless consonants (i.e./k/,/p/,/t/) and japanese special mora (sokuon). we recorded auditory evoked responses to short gaps and tones from children (8-12 years old, n = 8) and adults (19-23 years old, n = 12). there were six gap conditions with durations of 8, 16, 32, 64, 128 and 256 ms embedded in a continuous tone and six tone conditions with the same durations. the frequency of all the tone was 500 hz. the responses elicited by the onset of gaps differed between the children and the adults: the responses in children were significantly larger and more sustained than those in adults for all the durations. in contrast, an n1 and p2 complex followed the onset of all the tones in all the subjects. thus development time course of neural process is conceivably different between gaps and tones. ps2a-f098 an fmri study on pitch control of voice using transformed auditory feedback method akira toyomrua 1 , tamaki miyamoto 2 , atsushi terao 2 , sachiko koyama 2 , takashi omori 2 , harumitsu murohashi 2 , shinya kuriki 2 1 jst, saitama, japan; 2 hokkaido university, sapporo, japan auditory feedback plays an important role in natural speech production. in the present study, we conducted an fmri experiment while subjects performed a transformed auditory feedback (taf) task to delineate the neural mechanism for control of pitch. the subjects were required to vocalize a and to hold the pitch of a feedback voice constant. in taf condition, the pitch was altered suddenly two or three times, whereas in non-taf condition the pitch was not modulated. under the taf condition, auditory feedback control is selectively expected to work more strongly than the non-taf condition. thus, a comparison between these conditions could neatly extract brain regions involved in auditory feedback control of pitch. as a result, right supramarginal gyrus, right frontal lobe (ba9), right anterior insula, left premotor area and right superior temporal gyrus showed greater activation (12 subjects, p < 0.05 corrected). this result suggests that auditory feedback of pitch is mainly controlled by the right hemisphere. sachiko koyama-takeichi 1 , yuko toyosawa 2 , fumiya takeuchi 3 , michinao matsui 4 , shinya kuriki 1 1 research institute for elecronic science, hokkaido university, sappro, japan; 2 jst, saitama, japan; 3 hokkaido university, school of medicine, japan; 4 kobe shoin institute for linguistic science, kobe, japan sounds with relatively long duration elicit a sustained component (slow field, sf). in the present study, we recorded cortical magnetic responses elicited by vowels and examined whether sf differs between native and non-native vowels (n = 11). four synthesized vowels were used as stimuli (stimulus duration 600 ms). two of the vowels (a, o) are native for japanese and the other vowels (ae, schwa) are not. two inter-stimulus intervals were used (1200/4800 ms). for the native vowels, an early sf (250-300 ms) was larger for the long than for the short interval session in both hemispheres. for the non-native vowels, the early sf was larger for the long than the short interval session only in the right hemisphere. neither an effect of interval nor hemisphere was significant for a late part of sf (400-600 ms) regardless of stimulus types. research funds: japan science and technology agent (brain sciences and education), kakenhi (16500166) ps2a-f100 spatio-temporal representation of frequencymodulated sounds in the auditory cortex revealed by optical imaging shunji sugimoto 1,2 , junsei horikawa 1 1 department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan; 2 riken brain science institute, wako, japan optical imaging (voltage-sensitive dye, rh 795) showed spatiotemporal response patterns for frequency-modulated (fm) sounds in the multiple fields of the guinea pig auditory cortex. an fm sound evoked a strong onset response spreading widely over the cortex, which was followed by a later response moving across the iso-frequency contours in the core fields. the location of the later response was corresponding roughly to the instantaneous frequency input of each fm sweep. on the other hand, a pure tone evoked a wide-spreading onset response followed by strong and long-lasting inhibitory effects. the later response to an fm sound appeared clearly when the frequency of the fm sweep was modulated over a wider range of frequencies, while it was diminished when the sound frequency was less modulated. these results imply that the cortical representation of such a later response contributes to a detection of frequency modulations in sounds. yamato kubota, kuniyuki takahashi, ryuichi hishida, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan mitochondrial flavoprotein fluorescence is intimately coupled with energy metabolism. if the flavoprotein fluorescence is photobleached, energy metabolisms and neural activities can be inactivated. we applied this photo-inactivation technique to demonstrate auditory signal transmission from the anterior auditory field (aaf) to the primary auditory cortex (ai). cortical responses in aaf and ai after sound stimuli (5-20 khz) were visualized using transcranial flavoprotein fluorescence imaging in mice anesthetized with urethane. after determination of tonotopic maps, the auditory cortex was irradiated with strong blue light derived from a xenon lamp for 40 min, while the surface either aaf or ai was covered with a piece of carbon paper for preventing photo-inactivation. although photoinactivation of ai had almost no effect on the responses in aaf, photo-inactivation of aaf significantly reduced the responses in ai. these results suggest the presence of auditory signal transmission from aaf to ai. kousuke abe 1 , go ashida 1,2 , kazuo funabiki 2 1 graduate school of informatics, kyoto university, kyoto, japan; 2 hmro, faculty of medicine, kyoto university, kyoto, japan sound signals are translated to dispersed sporadic firing of the auditory nerves, and are converged to the third auditory station called the nucleus laminaris (nl) in birds. in vivo intracellular recording from owl's nl cells revealed that sound waveforms are observed in the postsynaptic membrane potentials (sound analogue potential; sap). we simulated synaptic inputs to the owl's nl neurons by recruiting the convergence of phase-locked excitatory inputs. several parameters such as the degree of phase-locking, the number of convergence and the time course of a unitary synaptic input affected the amplitude of sap, the amplitude of dc depolarization and the spectral features of synaptic noise in a complex manner. biophysical mechanisms for recreating sound waveforms by synaptic potentials will be discussed. takashi nihashi 1 , shigenori takebayashi 2 , masahiko bundo 2 , masazumi fujii 3 , toshihiko wakabayashi 3 , jun yoshida 3 , hiroyuki fujisawa 1 , kazunori ando 1 , kazumasa hayasaka 1 1 department of radiology, national hospital for geriatric medicine, obu, japan; 2 department of neurosurgery, national hospital for geriatric medicine, obu, japan; 3 department of neurosurgery, nagoya university school of medicine, nagoya, japan we identify si, using fmri in a routine scan for the patient who need a surgical approach. the activation of the brain with a tumor is complicated. we considered the pattern of the response. twelve patients were participated in this study. using 1.5 tesla mr imager, tactile stimulation was applied to bilateral palm, respectively. the statistical threshold was set for individual. contralateral activation on si was found in 11 out of 12 patients in the affected hemisphere. when region is near central sulcus, the multiple sites were activated. on the other hand, when the tumor is from central sulcus, the activation is simple: contralateral si. this method is useful to decide si in affected hemisphere in a short time. however, there are great inter-individual differences due to the locations of the tumor. takayuki iwano, shinya yamamoto national institute of advanced industrial science and technology (aist), neuroscience research institute, tsukuba, japan to examine how body surface with low spatial resolution is represented in the brain, we conducted a tactile identification task on toes. subjects (n = 8) lay on their backs with their eyes closed, and one of their toes was touched with a toothpick. the subjects were required to identify the toe by verbal response. the subjects responded correctly when the great or fifth toe was touched (cf. fein, 1987) . surprisingly, subjects tended to misidentify the second toe as the third (47.2%), and the third toe as the fourth (37.2%), while the reverse misidentification rarely occurred (third as second, 4.5%; fourth as third, 7.1%). this unidirectionality suggests that misidentification arises not only from large overlapping receptive fields associated with the toes, but from some additional factors such as a lack of experience with visuotactile integration, which could be used to reshape the toe receptive fields. ps2a-g105 effects of saccades on subjective temporal order of somatosensory signals toshimitsu takahashi 1,2 , shunjiro moizumi 1 , ayami okuzumi 1 , humine saito 1 , shigeru kitazawa 1,2 1 department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; 2 crest, jst, saitama, japan morrone et al. (2005) recently reported that subjective temporal order of two successive visual stimuli was reversed when the stimuli were delivered just prior to the onset of a saccade. in this study, we examined whether saccades affect temporal order judgments of tactile stimuli. right-handed subjects were required to make a visually guided rightward saccade (24 • ), and to judge the order of successive tactile stimuli that were delivered one to each hand at various timing relative to the onset of the saccade. with a stimulation interval of 100 ms, subjects generally judged the order correctly as long as the stimuli were delivered after the saccade. however, they often misreported (i.e., inverted) the order when the stimuli were delivered just prior to the onset of the saccade (within 300 ms). the results show that the reversal effect of saccades is multimodal and further suggest that multimodal brain areas are involved in ordering sensory events in time. ps2a-g106 function-directed organization of the postcentral somatosensory cortex representing oral structures takashi toda 1,3 , miki taoka 2,3 1 department neuroscience oral physiology, osaka university graduate school of dental sciences, suita, japan; 2 secondrary cognitives neurobiology, tokyo medical & dental university, tokyo, japan; 3 department physiology, toho university school of medicine, tokyo, japan the representation of oral structures in areas 3b and 1 of four conscious macaque monkeys was studied by recording single-neuron activities. a total of 115 electrode penetrations were made in areas 3b and 1. in 44 penetrations, pairs of adjacent neurons along the track had receptive fields (rfs) on continuous oral portions with or without overlapping, or otherwise on the same portion. in the remaining 71 penetrations, however, 14% of adjacent pairs (311/2153) had rfs on discrete but functionally-related sets of oral portions, e.g., the lip and tongue tip, the cheek mucosa and lateral margin of the tongue, the corresponding portions of the upper and lower lips, the corresponding portions of the palate and tongue, etc. we speculated that such an organization in areas 3b and 1 might be responsible for forming composite rfs of area 2 neurons. those composite rfs often covered discrete but functionally-related oral portions as reported earlier. research funds: kakenhi (12771111, 14771029) miki taoka 1 , michio tanaka 1 , hisayuki ojima 1 , atsushi iriki 2 1 secondrary cognitives neurobiology, tokyo medical and dental university, tokyo, japan; 2 laboratory of symbolic cognitive development, riken brain science institute, wako, japan we previously reported neuronal projections from the hand region of the second somatosensroy cortex (sii) to higher motor cortices (vetral premotor cortex etc.) suggesting that sii may be related to motor control of the hand movement. in the present study, we investigated the activities of sii hand neurons during voluntary movements. we recorded 168 neurons from two animals that were active when animals took small pieces of food by hands and put them into the mouth. among them (44% contra-, 55% bi-and 1% ipsilateral hand movements), we could determine receptive fields for only 43 neurons (25%). most of activities (138 neurons) were related to a certain phase of movements such as reaching, pinching a food piece, and putting it into the mouth. we found neurons showing phasic activities just before/after a certain phase, for example, just before pinching the object, or just after putting it into the mouth. those results suggest that sii hand neurons code the start or end of a certain act of hand. takahiro furuta 1,2 , kouichi nakamura 1 , takeshi kaneko 1 1 department of morphological brain science, graduate school of medicine, kyoto university, kyoto, japan; 2 crulrg, laval university, canada we investigated response properties of whisker-responsive neurons in the nucleus interpolaris (spvi) combining juxtacellular recording and a piezo-stimurator. the spvi is one of the first relay stations in the vibrissal system. rostral part of the spvi sends axons to the posterior thalamic nuclear group, whereas the caudal part of the spvi projects to the ventral lateral part of the ventral posterior medial nucleus. in the rostral part of the spvi, the vast majority of recorded neurons were multi-whisker responsive neurons, which are considered as projection neurons. in the caudal part of the spvi, about a half of neurons were mono-whisker-responsive neurons, which are thought as local circuit neurons. almost all neurons had angular tunings to upward deflection of whiskers in the rostral part of the spvi, while neurons in the caudal part of the spvi exhibited various angular tunings to all directions. these results indicate that the spvi could be divided into two sectors by response properties. seiji komagata 1,2 , shanlin chen 2 , hiroki kitaura 1 , masaharu kudoh 1 , minoru shibata 2 , katsuei shibuki 1 1 department of neurophysiology, brain research institute, niigata university; 2 department plastic surgery, school of medicine, niigata university, japan we visualized neural responses in the mouse somatosensory cortex using transcranial flavoprotein fluorescence imaging. mice were anaesthetized with urethane (1.7 g/kg, i.p.), and somatosensory responses were elicited by vibratory brush stimulation (50 hz for 1 s) applied to the left plantar forepaw. changes in green fluorescence in blue light were observed in the right somatosensory cortex. immediately after cutting the left median and ulnar nerves, the somatosensory responses were almost completely abolished. however, the responses appeared again within a few hours after the partial denervation, and almost complete recovery was observed a few weeks after the partial denervation. the recovered responses were eliminated by cutting the remaining radial and muscle cutaneous nerves. the rapid recovery of the responses observed in the present study may explain the mechanisms for allodynia and cortical plasticity in the somatotopic maps. shin-ya nakamura, takaaki narumi, ken-ichiro tsutsui, toshio iijima division system neuroscience, tohoku university graduate school of life science, sendai, japan in the rat somatosensory pathway, information received with a whisker is conveyed to the barrel cortex via trigeminal and thalamic nucleus mainly by two parallel pathways, the lemniscal and paralemniscal. the former includes the nucleus of trigeminal prv and thalamic vpm, which are known to contain neurons selective to the direction of whisker stimulation, and the latter includes trigeminal spvi and thalamic pom. in this study, we examined the specific involvement of the lemniscal pathway to the discriminative perception of whisker stimulus direction. rats were trained to perform a single-whisker directional discrimination task, and the task performance was evaluated before and after the selective electrolytic lesion or muscimol inactivation of each trigeminal and thalamic nucleus. the lesion or inactivation of prv or vpm significantly impaired the task performance, whereas those of spvi or pom did not. this result suggests the specific involvement of the lemniscal pathway in the single-whisker directional discrimination. kumiko yokouchi 1 , nanae fukushima 1 , tetsuhiro fukuyama 2 , akira kakegawa 1 , tetsuji moriizumi 1 1 department of anatomy, shinshu university school of medicine, matsumoto, japan; 2 department of pediatrics, shinshu university school of medicine, matsumoto, japan to know sensory cues for suckling behavior, rat pups of the suckling period received unilateral or bilateral resection of the infraorbital (io), mental (m) or lingual (l) nerves responsible for sensation of the upper and lower lips, and the tongue. for comparison, unilateral or bilateral removal of the olfactory bulb was done in these pups. the control, unilaterally io or m nerve-injured, and unilaterally bulbectomized pups showed successful suckling by their access to the mother's nipple, oral contact to it and long-lasting sucking. the bilaterally bulbectomized pups could not have access to the nipple. the bilaterally io nerve-injured pups could have access to the nipple with no oral contact, while the bilaterally m nerve-injured pups showed successful suckling. suckling behavior of the bilaterally l nerve-injured pups was characterized by frequent oral contacts for a very short duration, resulting in ineffective milk-intake. the results show fundamental roles of olfaction and sensation of the upper lip and the tongue in suckling. takahiro kawashima 1 , takeshi kawano 1 , hidekuni takao 1,2,4 , kazuaki sawada 1,2,4 , hidekazu kaneko 3,4 , makoto ishida 1,2,4 1 department electrical & electronic engineering, toyohashi university of technology, aichi, japan; 2 issrc, toyohashi university of technology, aichi, japan; 3 aist, hsbe, ibaraki, japan; 4 jst, crest, japan a si microprobe array (probe: au-coated recording site at the tip, 2 m in diameter, 60 m in length; array: 40-m pitch) to record neuronal activities has been developed by using selective si probe growth technique. to examine electrical properties of the array, single motor unit action potentials evoked by the electrical stimulation of the sciatic nerve of a rat were recorded in the left tibialis anterior muscle. signal-to-noise ratio of observed signals decreased with probe impedance, suggesting that lower impedance is better for recording small action potentials. however, lower impedance makes more difficult to record local signals, because signals observed at probes with low impedance were highly correlated (r = 0.91). to record local signals, it is necessary to decrease the area of the recording site of each probe at the expense of an increase in the impedance. research funds: kakenhi (17206038), the 21st century coe program "intelligent human sensing" ps2a-g113 a microelectrode positioning system for longterm extracellular recording of multiple neuronal activities hidekazu kaneko 1 , hiroshi tamura 2 , shinya s. suzuki 1 , takahiro kawashima 3 1 aist, hsbe, ibaraki, japan; 2 laboratory of cognitive neuroscience, graduate school of frontal bioscience, osaka university, osaka, japan; 3 department electrical & electronic engineering, toyohashi university of technology, aichi, japan a novel microelectrode-positioning system was devised that tracks a target neuron by countermoving a microelectrode against uncontrollable movements of brain tissue. the system automatically adjusted the position of a seven-core microelectrode such that the amplitude of each spike of a target neuron at the tip was the same as that at a lateral recording site (differential mode). the differential mode was compared with a conventional (peak-search) mode in which the spike amplitude of a target neuron at the tip was continually maximized. the differential mode was more stable to forced electrode movements and more sensitive to small displacements than the peak-search mode. furthermore, the differential mode enabled stable recording of not only spikes of a target neuron but also those of non-target neurons for over 2 h. seiji matsuda 1 , takehiro terashita 1 , tetsuya shimokawa 1 , kyoujy miyawaki 1 , yuji miguchi 1 , takuya doihara 1 , jie chen 1 , shuang-yan gao 1 , chun-yu li 1 , min wang 1 , zhong wang 1 , bing xue 1 , naoto kobayashi 1,2 , kazuhiro shigemoto 3 1 department anatomy, ehime university of medicine, ehime, japan; 2 medical education c, ehime university of medicine, ehime, japan; 3 department of hygiene, ehime university of medicine, ehime, japan this study shows the phylogenetic development of cajal's initial glomeruli (ig) and dogiel's pericellular nests (pcns) in the dorsal root ganglion of the healthy adult frog, chick, rat, and rabbit. the three-dimensional architecture of the neurons was observed in ganglia by scanning electron microscopy, after removal of the connective tissue. the proportion of neurons having ig or pcns increased with increasing phylogenetic complexity in the species examined here. in the chicks, the stem processes were longer and sometimes tortuous. typical ig were observed not in frogs or chicks, but in rats and rabbits. dogiel's pcns also have been observed in the drg of rats and rabbits. the nerve fibers in the pcns were less than 1.2 m in diameter and had some varicosities. some pcns contain tyrosine hydroxylase-positive nerve fibers and varicosities. masayo okumura, eiji kondo matsumoto dental university, institute for oral science, shiojiri, japan we established a rat nerve injury model using axotomy of the inferior alveolar nerve (ian), and investigated its effect on gene expression in the trigeminal ganglion. microarray analysis three days after surgery showed the up-regulation of some genes which are regulated by transcription factor stat3, whereas other genes known to be regulated by stat3 were not detected. stat3 is expressed in many tissues and plays various roles. however, there have been few reports about the role of stat3 in the peripheral nervous system, despite its welldocumented activation in the central nervous system after injury or stress. the aim of this study is to elucidate the role of stat3 in gene expression in the trigeminal ganglion after ian injury. at various time points, we analyzed and investigated changes of gene expression which are known to be influenced by stat3 and stat3phosphorylation, which indicates transcriptional activity, as well as cell types in which the genes and stat3 are expressed. these results should help us understand injury-induced change mechanisms of the peripheral nerve. hirofumi hashimoto 1 , susumu hyodo 2 , makoto kawasaki 1 , minori shibata 1 , takeshi saito 1 , hiroaki fujihara 1 , takashi higuchi 3 , yoshio takei 2 , yoichi ueta 1 1 department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; 2 laboratory of physiology, department of marine bioscience, ocean research institute, university of tokyo, japan; 3 department of integrative physiology, university of fukui, japan adrenomedullin 2 (am2) (identical to intermedin) belongs to the super family of am. centrally administered am and am2 activated oxytocin (oxt)-secreting neurons and increased plasma oxt level in rats. in the present study, we examined the effects of central administration of am2 on oxt-secreting neurons and sympathetic outflow in comparison with that of am in conscious rats. effects of central administration of am2 was stronger than those of am and the effects of am2 on oxt secreting neurons could not be blocked completely by pretreatment with cgrp or/and am receptor antagonists. these data suggested that am2 would have unknown receptor except cgrp and am receptor. arata oh-nishi 1 , makoto saji 1 , taku uchida 1 , sen-ichi furudate 2 , nobuyuki suzuki 1 1 division of brain science, kitasato university, graduate school of medical science, kanagawa, japan; 2 division of reproduction and fetal development, kitasato university, graduate school of medical science, kanagawa, japan the mechanism whereby neonatal hypothyroidism impairs cognitive function has not been well studied. in this respect, nmda receptors are thought to be crucially involved in cognitive and memory function. we have examined the effect of neonatal hypothyroidism and hyperthyroidism on the nmda receptor function, using rats treated with methylmercaptoimidazole (mmi), which specifically blocks the biosynthesis of thyroid hormone and mmi-treated rats injected with thyroxine, respectively. dose-response curves indicated that the sensitivity to nmda of the nmda receptors was significantly reduced in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. concomitant with this observation, western blot analysis showed that the nmda receptor subunit nr1 expression significantly decreased in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. our lab demonstrated that estradiol is endogenously synthesized within hippocampal neurons in the adult male rat (pnas, 2004) . here we report that the density and morphology of spines of pyramidal neurons in ca1 region are rapidly altered by treatments with nm estradiol and bisphenol a (xenoestrogen). hippocampal slices are incubated with estradiol or bisphenol a for 2 h, and then neurons were injected iontophoretically with lucifer yellow. three-dimensional imaging of neurons is performed by confocal laser microscopy, and the analysis of individual spines is performed by neurolucida software. the results showed that in ca1, both estradiol and bisphenol a induce a significant increase in the total spine density, especially the density of thin spine. synaptic plasticity of hippocampal neurons is demonstrated to be rapidly modulated by estrogen and xenoestrogen. we investigated the effects of stress on enhanced green fluorescent protein (egfp) expression in the arginine vasopressin (avp)-egfp transgenic (tg) rats. after bilateral adrenalectomy and intraperitoneal administration of lipopolysaccharide egfp fluorescences were increased in the parvocellular division of the paraventricular nucleus and the external layer of the median eminence. this tg rat is a convenient tool to study dynamic changes of avp expression in the hypothalamus under stressful condition. chitose orikasa, yasuhiko kondo, yasuo sakuma department of physiology, nippon medical school, tokyo, japan we report here a sex difference expression of somatostain mrna within the sexually dimorphic nucleus of the preoptic area (sdn-poa), the volume of which depends on gonadal hormones during the ontogeny. in infant rats aged day 8-15, the volume of somatostain mrna-positive region within the poa was significantly larger in males than in females and overlapped the sdn-poa in both sexes. the sdn-poa visualized by nissl staining in adjacent sections agreed precisely with the extent of somatostain mrna-positive cellular distribution. orchidectomy of males neonates and estrogen treatment of female pups reverse brain phenotypes when examines on day 15. the staining of somatostain mrna in individual neurons was diminished when examined on day 35 or 70, albeit that the sex difference of the volume of somatostain mrna-positive region persisted thoughtout the observed period. somatostatin may play a role in the establishment of the sdn-poa, which lacks classic nuclear receptor for estrogen. ps2a-g121 short chain sugar acid, 2-buten-4-olide, activates oxytocin-secreting neurons in the hypothalamus of rats makoto kawasaki 1 , tatsushi onaka 2 , hirofumi hashimoto 1 , hiroaki fujihara 1 , yoichi ueta 1 1 department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; 2 department of physiology, jichi medical school, tochigi, japan 2-buten-4-olide (2-b4o), an endogenous sugar acid, which may be involved in the regulation of feeding. we examined the effects of 2-b4o on the hypothalamo-neurohypophyseal system in rats. the plasma oxytocin (oxt) levels were significantly increased at 15-60 min after intraperitoneal (i.p.) administration of 2-b4o (100 mg/kg), whereas plasma arginine vasopressin (avp) levels did not change. dual immunostaining revealed that fos-like immunoreactivity (li) was predominantly observed in oxt-secreting neurons in the paraventricular and the supraoptic nuclei 120 min after i.p. administration of 2-b4o. in addition, many fos-li neurons were also observed in the nucleus of the tractus solitarius (nts) after i.p. administration of 2-b4o. these results suggest that peripherally administered high dose of 2-b4o activates oxt-secreting neurons in the hypothalamus through the activation of the nts neurons. ps2a-g122 estrogen receptor ␣ gene promoter activity is a marker for the sexually dimorphic nucleus of the preoptic area tomohiro hamada, yasuo sakuma department of physiology, nippon medical school, tokyo, japan the volume of the sexually dimorphic nucleus in the preoptic area (sdn-poa) is two to four times larger in male rat than in female, however function of this nucleus has not well known. in contrast, estrogen causes the sexually dimorphism by acting in perinatal periods. recently, transgenic rats expressing enhanced green fluorescent protein (egfp) under the control of an estrogen receptor (er) ␣ promoter were generated to tag er␣-positive neurons in the brain. in the present study, we examined gfp expression could be used a marker for the sdn-poa. gfp labeled cells were distributed in the core of sdn-poa of male and female transgenic rats and in the majority of these cells included er␣, immunohistochemically. both area and number of gfp expressed cells in the sdn-poa were larger in male than in female, however, female gfp cells in the sdn-poa showed concentrated distribution than male. these results suggest that gfp labeled cells in sdn-poa could be useful marker to make clear the function of the sdn-poa. recent studies on gonadal steroids imply that testosterone and estradiol are involved in learning and memory with modification of excitatory synapses in the hippocampus. although previous in vivo studies have demonstrated that these steroids increase the number of dendritic spines in neurons, it is still unclear whether each steroid has a direct effect on the modulation of the spatio-temporal patterns of dendritic morphogenesis. in the present study, we investigated steroid-induced morphological changes using cultured hippocampal neurons derived from neonatal or embryonic mice. the neurons were transfected with venus-actin. time-lapse images were taken by laser scanning confocal microscope during steroid treatment. testosterone but not estradiol increased the number of spines/filopodia of the dendrites within 4 h. these results obtained from in vitro studies suggested that testosterone affects dendritic morphogenesis of hippocampal neurons in short term. tetsuya kimoto 1,2 , shinpei higo 1,2 , yasushi hojo 1,2 , kouhei nakajima 1,2 , hironori nakanishi 1,2 , hirotaka ishii 1,2 , suguru kawato 1,2 1 department of biophysics & life science, university of tokyo, tokyo, japan; 2 crest, jst, japan hippocampus is one of the main target of sex steroids (androgen and estrogen) and stress steroids (corticosteroids). neuronal signal transmission in the hippocampus is modulated acutely by these steroids, and we recently demonstrated that the hippocampus of the adult male rat contained enzymes required for the synthesis of these steroids. however, the full diagram of hippocampal neurosteroid synthesis has not been obtained yet. in the present study, we therefore investigated the synthesis of sex steroids and corticosteroids in the hippocampus of adult male rats, by monitoring the metabolism of tritiated steroids with hplc system. ps2a-g125 gaba depolarizes gnrh neurons isolated from adult gnrh-egfp transgenic rats chengzhu yin, nobuyuki tanaka, masakatsu kato, yasuo sakuma nippon medical school, department of physiology, tokyo, japan gnrh neurons are essential in the reproductive neuroendocrine system. in regulation of gnrh neurons, gaba may be one of the major players, especially in relation to gnrh/lh surge. we, therefore, performed a cell-physiological analysis of gaba action on rat gnrh neurons. cells were dispersed from adult gnrh-egfp transgenic rats and cultured overnight. gnrh neurons, were applied to the perforated patch-clamp configuration with gramicidin d. gaba evoked cl − conductance, which was almost completely blocked by either picrotoxin or biccuculin. the reversal potential of the response was ranged from −40 to -10 mv in identified gnrh neurons in both sexes. there was no difference in the reversal potential among the stages of estrous cycle. in unidentified neurons, however, the reversal potential was more negative than -50 mv and most of them were ∼−80 mv. in conclusion, gnrh neurons isolated from adult rats express gabaa receptor and its reversal potential is more positive than the resting potential. although the neural activation in the subfornical organ (sfo) by angiotensin ii (angii) is widely regarded for the increments of angii-induced water intake and vasopressin release, galanin (gal) have been reported to inhibit them. therefore, gal may inhibit neural activity of angii-sensitive sfo neurons. rt-pcr analysis demonstrated existences of all mrnas of gal receptor subtypes, galr1, galr2 and galr3, in the sfo. in extracellular recording on sfo slice preparation, gal dose-dependently led to inhibition of neural activity. all gal sensitive neurons showed excitatory response by angii. galr1 selective agonist m617 induced inhibitory responses, as well as gal. in patch-clamp recordings, gal induced outward current in some neurons. these results suggest that gal inhibits neural activity in sfo neurons through, at least partially, outward current following activation of galr1. hiroaki fujihara 1 , tomoki fujio 1 , david murphy 2 , yoichi ueta 1 1 department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; 2 molecular neuroendocrinology research group, the henry wellcome laboratories for integrative neuroscience and endocrinology, university of bristol, bristol, uk we have generated transgenic (tg) rats expressing an arginine vasopressin (avp)-enhanced green fluorescent protein (egfp) fusion gene. in this study, we investigated the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight after drinking 2% saline for 5 days in 3, 6, 12 and 24 months old tg rats. in 3 and 6 months, there were no difference between tg rats and tg(−) rats about the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight under normal condition and salt loading. in aged tg rats (12 and 24 months old), there were no obvious changes in water balance. these results suggest that the expression of avp-egfp transgene does not disturb body fluid homeostasis in tg rats. ps2a-h128 prolactin-releasing peptide is a potent mediator of stress response in the brain through the hypothalamic paraventricular nucleus takashi mera 1 , hiroaki fujihara 2 , hirofumi hashimoto 2 , makoto kawasaki 2 , tatsushi onaka 3 , takakazu oka 1 , sadatoshi tsuji 1 , yoichi ueta 2 1 department of neurology (division of psychosomatic medicine), school of medicine, university of occupational and environmental health, japan; 2 department of physiology, school of medicine, university of occupational and environmental health, japan; 3 department of physiology, jichi medical school we examined the effects of restraint stress (rts), nociceptive stimulus and acute inflammatory stress on the prolactin-releasing peptide (prrp) gene expression in the hypothalamus and brainstem. moreover, we examined the effects of pretreatment with an anti-prrp antibody on nociceptive stimulus-induced c-fos gene expression in the hypothalamic paraventricular nucleus (pvn). rts, nociceptive stimulus and acute inflammatory stress upregulated the prrp gene expression in the brainstem. pretreatment with anti-prrp antibody significantly attenuated nociceptive stimulus-induced c-fos gene expression in the pvn. these results suggested that prrp is a potent and important mediator of stress response in the brain through the hypothalamic pvn. taieb bousejin 1 , afsaneh eliassi 1 , nasser naghdi 2 , ali ghanbari 1 1 ghsemi; 2 pastor institute, tehran, iran the purpose of this study was to consider the role of the ventromedial hypothalamus (vmh) d1 receptors on histamine-induced gastric acid secretion (gas). the animals were anasthetized and guide cannulas were implanted unilaterally above (0.5 mm) vmh. animals were anasthetized and two polyethylene tubes were introduced into the stomach through esophagus and pylorododenal junction. iv infusion of histamine in sham grup induced marked increase in gas with a peak response that started from 30 min up to the end of experiments (90 min). at the peak acid response, the vmh microinjection skf38393 (0.1,1) significantly reduced the amount of gas (p < 0.001). there was no any effect by microinjected sch23390 (0.1) into the vmh. injecting skf38393 into the vmh, 5 min after sch23390, had no effect on gas in compare with control. but, the acid suppressant effect of skf38393 was completely removed by peripheral injection of sch23390 (p < 0.01). our results show that the vmh d1 dopamine receptors have regulatory mechanisms of gas by interaction with h2 receptors through an inhibitory neural pathways. zhilin song, celia d. sladek department of physiology, uchsc, aurora, co, usa although prior studies demonstrated expression of p 2x purinoceptors in supraoptic neurons (son) and indicated their importance in atp stimulated vasopressin release, in studies monitoring the effect of atp on intracellular ca ++ ([ca ++ ] i ), we have obtained evidence that p 2y purinoceptors (p 2y r) are important in the response to atp. atp stimulated [ca ++ ] i increase was maintained in ca ++ -free medium and reduced by pretreatment with thapsigargin to deplete [ca ++ ] i stores. p 2y r agonists increased [ca ++ ] i in son, with p 2y1 r agonist being the most effective. the possibility that p 2y1 r mediates atp induced [ca ++ ] i increase in son was further evaluated using a p 2y1 r antagonist, mrs2179. atp stimulated increase in [ca ++ ] i was greatly attenuated by mrs2179 (100 m) in 2 mm ca ++ medium. in ca ++ -free medium, there was no significant response to atp in the presence of mrs2179. furthermore, combined treatment with mrs2179 and ppads (10 m, a p 2x r antagonist) also abolished the [ca ++ ] i response to atp. these results demonstrated that p 2y1 r mediates a large portion of the [ca ++ ] i response to atp challenge in son. ps2a-h131 analysis of the ontogenic expression of enzymes for brain neurosteroids in the male rat hippocampus hirotaka ishii 1,2 , yasuhiro sonoki 1,2 , aizo furukawa 2,3 , yasushi hojo 2 , tetsuya kimoto 1,2 , suguru kawato 1,2 1 department of biophysics and life science, university of tokyo, tokyo, japan; 2 crest, jst, japan; 3 kurihama national hospital, japan brain neurosteroids are steroids synthesized endogenously in the brain. our recent studies have demonstrated that the adult male rat hippocampus is equipped with a complete machinery for the synthesis of androgen and estrogen. to define the physiological role of brain neurosteroids in the hippocampal development and function, detailed information about the expression profiles of enzymes for brain neurosteroids in the hippocampus is essential. this study have comprehensively investigated the temporal patterns of enzymes for brain neurosteroids in the male rat hippocampus from postnatal day 1(pd1) to the adult stage using rt-pcr/southern blotting. enzymes required for the synthesis of estradiol from cholesterol were expressed form pd1 to pd14 with a higher level than in the adulthood. these results indicate that the rat hippocampus synthesizes estradiol more vigorously during the postnatal stage than in the adulthood, which may play an important role in the hippocampal development and function. hideo mukai 1,2 , gen murakami 1 , shirou kominami 3 , john h morrison 4 , william g.m janssen 4 , tetsuya kimoto 1,2 , suguru kawato 1,2 1 department of biophysics and life sciences, graduate school of arts and sciences, the university of tokyo, meguro, tokyo 153-8902, japan; 2 crest project, jst, japan; 3 faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima 739, japan; 4 kastor neurobiology of aging laboratories, fishberg research center for neurobiology, usa estrogens elicit rapid non-genomic effects on the synaptic transmission, and spinogenesis in the hippocampus. however, the existence of estrogen receptor alpha (er␣) still remains elusive. with highly purified antibody rc-19, mass spectrometric analysis identified er␣ in the hippocampus and immunohistochemistry showed er␣ localization in principal neurons of ca1, ca3, and granule cells in dentate gyrus. further, western blot revealed that er␣ is contained in psd fraction, confirming the observation with immunoelectron microscopy. these results imply that the synaptic er␣ mediates the effects of estrogen in hippocampal neurons. ken takumi gnrh neuron is the key modulator of reproductive systems, directly regulating the synthesis and secretion of gonadotropins from anterior pituitary gland. gnrh neurons have been reported to be contacted by various neuronal systems, suggesting that the biosynthesis and release of gnrh is controlled by a complex of excitatory and inhibitory inputs. however, anatomical studies which quantified the direct input on gnrh neuron are few. in this study, we quantitatively analysed glutamatergic and gabaergic input onto gnrh neurons of the rhesus monkey by immunofluorescence method and confocal laser scanning microscopy; the close appositions between gnrh neuron and axon terminals immunoreactive for either vgluts or vgat were counted and the densities of the appositions on the dendrites and soma were calculated. sabine gouraud 1 , song t. yao 1 , jing qiu 1 , julian fr paton 2 , david murphy 1 1 university of bristol, hw-line, uk; 2 department of physiology, bristol heart institute, university of bristol, united kingdom the neuropeptide hormone vasopressin (vp) is produced in the magnocellular neurons of the hypothalamic supraoptic (son) and paraventricular (pvn) nuclei and stored in the posterior pituitary (pp). dehydration evokes an increased expression of the vp gene in magnocellular neurons and a massive release of vp from the pp in the circulation to promote the water conservation at the kidney level. in parallel, a functional remodelling of the hypothalamo-neurohypophyseal system (hns) is observed but poorly understood. we investigated this activity dependent plasticity of the hns using proteomic (2d fluorescence difference gel electrophoresis (dige)) combined with maldi mass spectrometry approaches to identify proteins that change in abundance in the son and the pp from 3 days dehydrated rats. a truncated form of prosaas, a granin-like neuroendocrine peptide precursor known as a potent inhibitor of the prohormone convertase 1, has been found decreased in the pp and increased in the son. ichiro nishimura, masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan function of gonadotropin-releasing hormone (gnrh) neurons is regulated by gonadal steroid estrogen. however, the precise mechanism of estrogen action upon these cells has not been clarified. we investigated a direct action of estrogen on the regulation of potassium current in gnrh neuronal cell line gt1-7. delayed rectifier potassium current (i k ) and large-conductance calcium-activated potassium (bk) current were recorded by patch clamp configuration in gt1-7 cells cultured in dmem supplemented with 10% fbs for 3 days. bk current was increased by addition of 17␤-estradiol (e2) in culture medium in a physiological concentration range. this action of e2 was blocked by ici-182,780, a potent estrogen receptor (er) antagonist. we further examined whether e2 acted through er ␣ or er ␤ by using selective agonists ppt and dpn, respectively. the dpn augmented the bk currents similar to the effect of e2 but ppt had no effect. e2 had no effect on the i k . these results indicate that e2 increases the bk current by activating er␤ without affecting the i k . research funds: kakenhi 16590180, 16086210 ps2a-h136 myelin protein zero is one of the components of the detergent-resistant membrane microdomain fraction derived from rat pituitary katsutoshi taguchi 1 , haruko kumanogoh 2 , shun nakamura 2 , seiji miyata 3 , shohei maekawa 1 1 department of biosystems science, kobe-university, kobe, japan; 2 division of biochemistry and cellular biology, national institute of neuroscience, ncnp, tokyo, japan; 3 department of applied biology, kyoto institute of technology, kyoto, japan a membrane microdomain enriched in cholesterol and glycosphingolipids was found to contain many signal transducing and cell adhesion molecules. here, we studied the components of the membrane microdomain fraction derived from rat pituitary, and found specific enrichment of several proteins in this fraction. one of them, 25 kda protein, was identified as myelin protein zero (p0) from mass analysis and this result was confirmed by western blotting that a specific antibody to 25 kda band reacted to an authentic p0 prepared from rat sciatic nerve myelin. p0 is a type i transmembrane glycoprotein and a member of the immunoglobulin superfamily. the expression of p0 has been believed to be restricted to the peripheral myelin in mammals. our result, however, indicates that p0 expresses more widely and participates in cell communications. mari ogiue-ikeda, norio takata, suguru kawato department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan 17␤-estradiol (e2) has a rapid effect on synaptic transmission. recently, we found that hippocampal neurons synthesize e2 (hojo et al., 2004) , and express estrogen receptor ␣ (er␣) at synapses. endocrine disrupters are representative estrogenic industrial compounds. while their disrupting effects on reproductive organs are well documented, their effects in the central nervous system are almost unknown. in this study, we investigated the effects of e2 and endocrine disrupters (des, bpa, np, op and tbt) on nmda-induced ltd in the rat hippocampal ca1, ca3 and dg with a custom made multi-electrode measuring system (med64). ltd was enhanced by e2 dose-dependently in ca1, ca3 and dg. des, bpa, np, op and tbt had similar or different effects on ltd dose-dependently. our results suggest that estradiol and endocrine disrupters rapidly modulate synaptic plasticity in the hippocampus and that the action of endocrine disrupters can be quantitatively analyzed by measuring the modulation of ltd of the hippocampal neurons. we examined the effects of chronic salt loading on the hypothalamic expressions of the green fluorescent protein (gfp), arginine vasopressin (avp) and oxytocin (oxt) genes and body fluid balance in avp-enhanced (e) gfp transgenic rats. chronic salt loading caused marked increase of the egfp fluorescence in the hypothalamoneurohypophyseal system in transgenic rats. there were no differences of the avp and oxt gene expressions in the hypothalamus, plasma avp and oxt levels and water balance between nontransgenic and transgenic rats under normal condition and after salt loading. humoral responses to chronic salt loading were maintained in avp-egfp transgenic rats. takeshi saito 1 , takushi x. watanabe 2 , tomoko urabe 2 , hirofumi hashimoto 1 , hiroaki fujihara 1 , yukio hirata 3 , yoichi ueta 1 1 department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; 2 peptide institute, inc., osaka, japan; 3 department of clinical and molecular endocrinology, tokyo medical and dental university, tokyo, japan salusin-␤ was newly discovered as a bioactive endogenous peptide. low concentration of salusin-␤ stimulates the secretion of arginine vasopressin (avp) from perifused rat hypophysis. salusin-␤ coexists with avp, but not oxytocin, in the rat magnocellular supraoptic (son) and paraventricular nuclei (pvn). to further investigate the physiological role of salusin-␤ in body fluid homeostasis, we examined the effects of salt loding for 5 days on salusin-␤-like immunoreactivity (li) in the son and pvn of rats by immunohistochemistry. the marked increase of salusin-␤-li in the son and pvn were observed in the salt loaded rats. the result suggests that salusin-␤ may play a role of body fluid balance by regulating avp release. naoyuki yamamoto 1 , hao-gang xue 1 , yuji ishikawa 2 , yoshitaka oka 3 , hitoshi ozawa 1 1 department of anatomy and neurobiology, nippon medical school, tokyo, japan; 2 national institute of radiological sciences, chiba, japan; 3 department of biological sciences, graduate school of science, the university of tokyo, tokyo, japan the terminal nerve gnrh (gonadotropin-releasing hormone) system, an extrahypothalamic peptidergic system, is thought to modulate neural circuitries involved in the control of motivational status for certain behaviors in teleosts. the major afferent source to the gnrh neurons is a midbrain nucleus, the nucleus tegmento-terminalis in percomorph teleosts, while a comparable nucleus appears to be missing in cyprinids teleosts. here, we examined the presence of such an afferent pathway in medaka oryzias latipes. injections of a dii crystal into the cluster of gnrh neurons resulted in labeled cells in the midbrain tegmentum, and injections to the midbrain tegmentum resulted in labeled terminals close to the gnrh neurons. these results suggest that the afferent pathway to the gnrh neurons is a character shared by "advanced" teleosts like medaka and percomorphs. takeshi yamazaki 1 , eiji munetsuna 1 , asuka kamogawa 1 , suguru kawato 2 , shiro kominami 1 1 graduate school of integrated arts science, hiroshima university of higashihiroshima, japan; 2 graduate school of arts and science, tokyou university of tokyo, japan tributyltin, tbt, an endocrine disruptor, induced increases in estradiol content in rat hippocampal slice culture. to analyze molecular mechanism of stimulation of estrogen synthesis, we determined mrna contents of estrogen biosynthetic enzymes and activity of p450arom in the hippocampus. method: the cultured hippocampus slices from 10 days male rat were treated with 100-1000 nm of tbt for 48 h. after the treatment, total rna was extracted and the levels of mrna of estrogen synthetic enzymes were quantified by real-time rt-pcr. activity of p450arom was determined by quantification of [3h]estradiol from [3h]testosterone. result: forty-eight hours treatment of hippocampal slices with 100 nm tbt induced increases in mrna contents of p450arom, and with 1000 nm tbt induced that of 3␤-hsd. estradiol content was increased by the treatment with 100 nm tbt, but not affected by 1000 nm tbt. tbt may modulate estradiol synthesis by alteration of expression of p450arom. the medial preoptic area (mpoa) is an important neural site for regulation and maintenance of sleep. studies have indicated that gabaergic neurons and terminals at the mpoa are active during sleep. present study was carried out to elucidate the contribution of gaba-a receptor at the mpoa in sleep-wakefulness (sw) in male wistar rats. the sw was assessed by chronically implanted electrodes for eeg, eog, and emg. a bilateral guide cannula was also implanted for drug injection into the mpoa. after recovery, three baseline sleep recordings were taken for 4 h on different days. bicuculline methoiodide (gaba-a receptor antagonist) at a dose of 30, 60, 90 and 120 ng in 200 nl was injected bilaterally into the mpoa in different groups of rats and their sw was studied for subsequent 4 h. the 30 ng dose of bicuculline methoiodide had minimum effect whereas 60 and 90 ng produced arousal. maximal wakefulness was observed at dose of 90 ng with no further increase in wakefulness at higher dose of 120 ng. the results suggest the involvement of gaba-a receptors at the mpoa in sw. yoshiaki isobe 1 , hiroyuki tsuda 2 1 department of neuro-physiology and brain science, nagoya city university, graduate school of medical sciences, nagoya, japan; 2 department of molecular toxicology, nagoya city university graduate school of medical sciences, japan locomotor activity in rodents shows free-running circadian rhythms even under the constant light. constant light exerts a promoting effect on hepatic carcinogenesis. after the partial hepatectomy, hepatic cell proliferation is regulated by circadian rhythm information (via wee1). to know the relation of proliferating factor (cell cycle) with circadian rhythmicity, locomotor activity against a diethylnitrosamine (den), widely used to initiate the hepatic neoplastic foci, is analyzed in preliminary. den was injected (i.p., 200 mg/kg) on rats during the free-running condition under the constant dim light (dd) and constant light (ll). the effects of den were gentle under the dd. however, under the ll, phase delay accompanying the elongation of circadian period () was observed. decrement of an amount of activity in 24 h after the den administration was obvious under the ll compared with that under the dd. this study was designed to investigate the central regulating system of hypothermia during maintenance phase of hibernation. although intracerebroventricular (icv) injection of naloxone (non-selective opioid receptor antagonist) and naloxonazine, (1 antagonist) were effective, naltrindole (␦ antagonist) and nor-bni ( antagonist) did not interrupt the hibernation. the increment of c-fos expression was observed in arcuate nucleus (arc) at 1 h after from hibernation onset compared with before hibernation. in addition, a localized ␤-endorphin-like immunoreactivity (␤-end ir) was observed in neuronal perikarya in arc at 1 h after from hibernation onset. although ␤-end ir in arc got weak, the ␤-end ir of nerve fibers in preoptic nucleus (pon) got strong with progression of hibernation. these results suggest that the ␤-endorphin was transported to pon from arc by axonal flow and then played an important role in maintenance of hypothermia via 1-opioid receptors in hibernation. wei-min qu, zhi-li huang, naomi eguchi, yoshihiro urade, osamu hayaishi department of molecular and behavioural biology, osaka bioscience institute, osaka, japan prostaglandin (pg) d 2 is a potent somnogenic substance, and isomerized from pgh 2 through the action of pgd synthase (pgds). pgds has two distinct types, the lipocalin-type pgds (l-pgds) and hematopoietic pgds (h-pgds). selenium compounds have been reported to decrease sleep by inhibiting pgds in rats. to clarify what type of pgds inhibition is involved in sleep reduction by selenium or whether selenium intoxication decreases sleep, we intraperitoneally injected secl 4 into l-pgds and h-pgds knockout (ko), and their wild-type (wt) mice. in wt mice, secl 4 decreased rapid eye movement (rem) and non-rem sleep for 5 h after injection and, concomitantly, increased wakefulness. similar results were observed in h-pgds ko mice. in contrast, l-pgds ko mice did not exhibit any significant changes in sleep-wake profiles after secl 4 administrations. these findings indicate that pgd 2 plays an essential role in the maintenance of the sleep state under physiological conditions, and l-pgds is a key enzyme for the production of pgd 2 involved in sleep-wake regulation. under baseline conditions, h 1 r ko mice showed essentially identical sleep-wakefulness cycles to those of wild-type (wt) mice but with fewer incidents of brief awakening (<16 s epoch), prolonged duration of non-rapid eye movement (nrem) sleep episodes, a decrease in the number of state transitions between nrem sleep and wakefulness, and a shorter latency for initiating nrem sleep after an intraperitoneal injection of saline. the h 1 r antagonist pyrilamine mimicked these effects in wt mice. these results indicate that h 1 r is involved in the regulation of behavioral state transitions from nrem sleep to wakefulness (huang et al., 2006) . ps2a-h147 dissociation of responsibility in firing activity to dim light between the optic nerve and the suprachiasmatic nucleus neuron of mice koichi fujimura, ai fukushima, takahiro nakamura, toshihiro jogamoto, kazuyuki shinohara division of neurobiology & behaviour, nagasaki university, graduate school of biomedical science, nagasaki, japan the involvement of light response in the optic nerve to the firing activity of suprachiasmatic nucleus (scn) neuron was investigated by extracellular single unit recordings from the optic chiasma and the scn in mice. recordings were carried out during the early night in a light:dark cycle, and the illuminations were applied to a contralateral retina with a high-power led (λ = 500 nm). the scn neurons responded to the light in intensities above 10 11 photons/cm 2 /s and were activated maximally at around 10 15 photons/cm 2 /s, they were about 1.6 log units less sensitive than optic fibers with high sensitivity. a sustained illumination in the intensity range between suprathreshold for the optic fibers and subthreshold for the scn neuron did not suppress the subsequent light response in the scn neurons, except in a few neurons. these results suggest that the most of the light responsive scn neurons are driven by any inputs independent of the high sensitive optic fibers. masayuki ikeda, tomoyoshi kojiya department of biology, faculty of science, toyama university, japan the hypothalamic suprachiasmatic nucleus (scn) has a pivotal role in the mammalian circadian clock. scn neurons generate circadian rhythms in action potential firings and neurotransmitter releases, and the core oscillation is thought to be driven by clock gene transcription-translation feedback loops. we have found robust circadian rhythms in the cytoplasmic concentration of ca 2+ in scn neurons. since cytosolic ca 2+ regulates diverse cellular systems, we have hypothesized that the cytosolic ca 2+ rhythms may mediate the cellular output from the clock gene oscillations. here, to address the clock gene functions on the ca 2+ rhythms, mouse bmal1 and its dominant negative sequence (mbmf1r5) are transfected into the organotypic culture of scn with a yellow cameleon ca 2+ sensor by the gene gun. the results demonstrated that over-expression of bmal1 or mbmf1r5 significantly inhibited the circadian ca 2+ rhythms and thus we concluded that the native bmal1 rhythm is essential for cellular output processes of the murine clock system. ps2a-h149 the activation of ␣2 adrenergic receptor increases the frequency of carbachol-induced ␤ oscillation in rat hippocampal slices masafumi nakano, jun arai, kiyohisa natsume kyushu institute of technology, kyushu, japan recently it is found that locus ceruleus (lc) activation suppresses ␤ rhythm in hippocampus in vivo. noradrenergic fibers derived from lc project to hippocampus. carbachol, a cholinergic agent, can induce ␤ oscillation in rat hippocampal slices like ␤ rhythm in vivo. in the present study, the effect of epinephrine on the generation of carbachol-induced ␤ oscillation in ca3 region of rat hippocampal slices. carbachol (30 m) induced ␤ oscillation with the frequency and the amplitude of 14.8 ± 0.1 hz, and 0.7 ± 0.1 mv, respectively (mean ± s.e.m.; n = 3). epinephrine (50 m) significantly increased the frequency of 16.2 ± 0.1 hz (**p < 0.01), not change the amplitude. clonidine (50 m), an ␣2 receptor agonist, alone significantly increased the frequency at the concentration of 50 m (*p < 0.05). yohimbine, an ␣2 receptor antagonist, suppressed the oscillation. these results suggest that the application of adrenaline will increase the frequency of hippocampal ␤ rhythm via ␣2 receptor. attractor dynamics of recurrent neural network are believed to play an important role in information processing in the brain. we recorded transient activities of two neuron groups by two tetrodes apart 0.4 mm from each other in the hippocampal ca3 region in vitro and applied micro-iontophoresis of glutamic-acid near the tetrodes to activate the neurons selectively. it was found that number of spikes during twosite (pairing) stimuli is fewer than the total number of spikes during the single-site stimuli, suggesting synaptic interaction in the network. peri-stimulus time histogram (psth) of ensemble as well as individual neuronal activity in response to the single-site stimuli applied far from the recording site composed of transient (latency 100-300 ms), oscillatory (2-4 hz) and sustained responses. following the pairing stimuli, the psth showed change in transient response properties (8/9 slices). these results suggest the pairing stimuli would change attractor dynamics of the neural network in the ca3 region. ryozo aoki 1 , hiroshi wake 2 , hitoshi sasaki 1 , kiyokazu agata 3 1 dept. physiol. & biosignal. osaka univ. grad. sch. med., suita, japan; 2 dept. elec. eng. & elec. col. industri. tech., amagasaki, japan; 3 dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan by insertion of a stainless-steel monopolar electrode to the head of planarian, continuous waveform of electrical potential could be first observed in microvolts. the frequency spectrum showed an almost monotonously decreasing distribution likely as 1/f, ranging from 10 − 1 to 10 + 2 hz. during the eeg recordings the planarian was kept still by cooling in several degrees. when it was cooled down to lower temperatures the amplitude of eeg was suppressed, and by warming again restored with spikes provably due to motions. this eeg active state continued beyond 40 min after the electrode insertion but the amplitude gradually decreased, and became natural noise at the time up to 60 min. by observing the sample it turns out the sticked head was degraded. strong photo stimulation suppressed this eeg signals and recovered after over 30 min. however little response to light pulse stimuli was observed on the eeg spectrum. mariko uchida 1 , hiroki sato 1,2 , naoki tanaka 2 , atsushi maki 1,2 1 japan science and technology agency, crest, saitama, japan; 2 advanced research laboratory, hitachi, ltd., saitama, japan previous studies about electroencephalography (eeg) described that alpha-wave power (the frequency band from 8 to 12 hz) decreases and the sleep spindle power (from 12 to 16 hz) increases in falling asleep. the purpose of this study is to analyze the crosscorrelation between the eeg power changes (eegpc) of each band and the cortical hemoglobin concentration changes (hbcc) during sleep. we measured optical topography (ot) and eeg simultaneously. the hbcc was measured at eighty-eight positions covering whole head of subject by ot probes. five females and eight males participated in this measurement. the results showed the high correlation between eegpc and hbcc at the location of dorsolateral prefrontal area, both in the period of (i) dominance of alpha-wave and (ii) dominance of sleep spindle. the time lag from eegpc to hbcc was from 1 to 8 s in (i), and from 8 to 15 s in (ii). we examine these differences between (i) and (ii) in detail. carnitine deficiency disturbs fatty acid oxidation under the fasting condition (fc). we show herein that nocturnal locomotor activity (la) was reduced under fc and recovered to normal by carnitine injection in jvs −/− mice, a model of systemic carnitine deficiency. as judged from eeg/emg profiles, jvs +/+ mice showed prolonged wakefulness under fc, but jvs −/− mice revealed disruption of the prolonged wakefulness with a high frequency of non-rem sleep. as the orexinergic arousal system plays an important role in la, we determined orexin neuronal activity in the fasted mice. fasted jvs −/− mice had fewer c-fos + orexin neurons in their lateral hypothalamus and a reduced orexin-a content in their csf, suggesting that the fasted jvs −/− mice exhibited reduced la and fragmented of wakefulness due to suppressed orexin neuronal activity. juhyon kim 1 , kazuki nakajima 1 , yutaka oomura 2 , kazuo sasaki 1 1 div. of bio-information eng., univ. of toyama, toyama, japan; 2 dept. of integrat. physiol., kyushu univ., fukuoka, japan novel peptide, orexin, identified in the lateral hypothalamus (lh) participates in the regulation of sleep-wakefulness. orexin-containing neurons in the lh project to the pedunculopontine tegmental nucleus (ppt). the ppt is one of brain sites which control sleepwakefulness. thus, we examined effects of orexin on ppt neurons electrophysiologically using brain slice preparations in rats. applications of orexin depolarized the membrane potential of ppt neurons dose-dependently, and the depolarization was associated with the increase in membrane resistance. when extracellular k + concentration was increased, the magnitude of the depolarization significantly decreased. when extracellular na + was replaced by n-methyl-dglucamine, the magnitude of the depolarization also decreased significantly. these results suggest that the ionic mechanism for orexininduced depolarization includes k + channel, non-selective cation channel and/or na + /ca 2+ exchanger, and that orexin participates in the regulation of sleep-wakefulness via the excitatory effect on ppt neurons. ben-shiang deng 1 , wei zhang 1 , akira nakamura 2 , masashi yanagisawa 3 , yasuichiro fukuda 2 , tomoyuki kuwaki 1,2 1 dept. molec. integ. physiol., chiba univ., japan; 2 dept. autonom. physiol., chiba univ., japan; 3 dept. molec. genet., univ. texas, usa we examined whether the respiratory chemoreceptor reflex in prepro-orexin knockout mice (ko) was blunted or not, and if so, whether supplementation of orexin restored the abnormality. we also studied whether pharmacological blockade of orexin in the wildtype mice (wt) resulted in a similar abnormality. ventilation was recorded by whole body plethysmography before and after intracerebroventricular injection of orexin-a, -b, sb-334867 (an orexin receptor antagonist), or vehicle. data were examined for only awake periods because sleeping distorts the chemoreflex. hypercapnic ventilatory responses but not hypoxic responses were attenuated in ko. similar abnormality was reproduced in wt treated with sb-334867. icv injection of orexin partially restored the hypercapnic chemoreflex in ko. our findings suggest that orexin plays a crucial role for co2-sensitivity at least during waking periods. research funds: kakenhi 16590162,17590183 junko hara 1 , taizo matsuki 2 , katsutoshi goto 1 , masashi yanagisawa 3 , takeshi sakurai 1,2 1 department of pharmacology, basic medical science (coe), university of tsukuba, ibaraki, japan; 2 yanagisawa orphan receptor project, erato, jst, tokyo, japan; 3 howard hughes medical institute and department of molecular genetics, university of texas, dallas, texas, usa when the production of inflammatory cytokines is stimulated by acute inflammatory, the nonrem-sleep amount of animals increases. this is possibly due to changes in the biological activity of the tnfalpha system. besides their important function in sleep regulation during acute immune response, cytokines also seem to be involved in physiological sleep regulation. orexins (hypocretins) are recently identified neuropeptides that are derived from a common precursor peptide. recent studies suggest that specific degeneration of orexincontaining neurons occurs in brains of human narcolepsy patients, suggesting critical roles of these neurons in the regulation of vigilance states. here, we examined the effects of inflammatory cytokines on the activity of orexin neurons, by means of patch-clamp recording. these effects might also possibly be involved in the pathophysiology of narcolepsy. ps2a-i157 prenatal exposure to bisphenol a enhances avoidance response to predator odor and impairs sexual differentiation of olfactory response of medial amygdala neurons tetsuya fujimoto 1 , kazuhiko kubo 2 , shuji aou 1 1 dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; 2 dept. otorhinolaryngol., chidoribashi hospital, fukuoka, japan prenatal exposure to bisphenol a (bpa) impairs the sexual differentiation of exploratory behavior and enhances depressive behavior (fujimoto et al. 2006) . in this study, the effects of bpa on general motor activity and avoidance response to predator odor and olfactory responses in medial amygdala neurons were examined. the smell of fox predominantly suppressed locomotor activity and enhanced avoidance response by bpa. in the electrophysiological study, male medial amygdala neurons showed selective excitatory responses to predator odors. this type of neurons did not respond to plant odors. in contrast female amygdala neurons did not show such selectivity. the sex difference in this neuronal response pattern was attenuated by bpa exposure. these findings suggest that bpa impairs sexual differentiation of medial amygdala neurons which affect emotional responses to the olfactory cues of predators. research funds: grants-in-aid for scientific research (no. 16209006, s.a.) shuji aou 1 , tetsuya fujimoto 1 , yumi ichihara 1 , kimiya narikiyo 1 , toru ishidao 2 , hajime hori 2 , yukiko fueta 2 1 dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; 2 dept. environm. manage., sch. health sci., univ. occup. environm. health, kitakyushu, japan 1-bromopropane (1-bp), an ozone-depleting substance replacement, has neurotoxicity and exhibited reproductive toxicity in adult animals. in this study, we investigated the effects of prenatal exposure to 1-bp on sexual differentiation of reproductive and non-reproductive behaviors. pregnant rats were exposed to 700 ppm of 1-bp during prenatal period. the open-field test, lashley iii maze test and sexual behavior were evaluated at adult age. 1-bp significantly reduced the locomotor activity and the number of entries into the center area in female rats but not in males in the open-field test. in sexual behavior, the number of ear wiggles, an index of proceptive behavior, was decreased and the rejection score was increased in female rats. these results suggest that 1-bp is the potential candidate of endocrine disruptors which affect brain development. (16651027) ps2a-i159 changes in hippocampal excitability of rats prenatally exposed to 1-bromopropane yukiko fueta 1 , toru ishidao 1 , susumu ueno 2 , yasuhiro yoshida 3 , hajime hori 1 1 department of environmental management, school of health sciences; 2 department of pharmacology; 3 department of immunology, school of medicine, university of occupational and environmental health, kitakyushu, japan inhalation exposure to 1-bromopropane (1-bp), a substitute for ozone depleting compounds, alters the function of gabaergic system in the hippocampus of adult male rats. but the neurotoxcitiy induced by prenatal exposure has not been well investigated. in this study pregnant rats were exposed to 1-bp (700 ppm) during gestational day 1-20 (6 h/day), and the hippocampal excitability in pregnant rats and their offspring was examined. basic excitability was enhanced and disinhibition was observed in the hippocampus of pregnant rats. offspring, however, exhibited an enhancement of averaged s/r curve of ps in the ca1 at the pnd 12-15. conversely, s/r curves of fepsp as well as ps in the ca1 were inhibited at the age of 6-8 weeks. our results suggest that 1-bp causes hyperexcitability in pregnant rats, and disrupts basic excitability in the ca1 of the offspring during development. research funds: grant-in-aid for exploratory research (16651027) ps2a-i160 effects of endocrine disrupting chemical bisphenol a on the development of mouse cerebral cortex keiko nakamura 1,2 , kyoko itoh 1 , takeshi yaoi 1 , tohru sugimoto 2 , shinji fushiki 1 1 dept. pathol. appl. neurobiol., kyoto pref. univ. med, kyoto, japan; 2 dept. pediatr., kyoto pref. univ. med, kyoto, japan bisphenol a (bpa), a widely distributed xenoestrogen, has been shown to disrupt thyroid hormone function. we have thus studied whether prenatal exposure to low-doses of bpa affects morphology and the expression of thyroid hormone-dependent genes in murine fetal neocortex. pregnant mice were injected subcutaneously 20 g/kg of bpa daily from embryonic day 0 (e0). control animals were injected vehicle alone. for evaluating cell proliferation, neuronal differentiation and migration, bromodeoxyuridine (brdu) was given to pregnant mice and processed for immunohistochemistry. the total rna was extracted from embryonic telencephalons at different embryonic period. brdu-labeled cells were decreased in the ventricular zone at e14.5 and e16.5, whereas those cells increased in the cortical plate at e14.5, as compared with control mice. some of the genes associated with neurogenesis and thyroid hormone function were upregulated in bpa-treated group. research funds: jsps grant 15390334 keiko ikemoto 1 , teruko uwano 2 , hisao nishijo 2 , taketoshi ono 2 , masayuki ito 3 , ikuko nagatsu 4 , katsuji nishi 5 , shin-ichi niwa 1 1 dept. neuropsychiat., fukushima med. univ. sch. med.; 2 toyama med. pharm. univ.; 3 faculty med., mie univ., mie, japan; 4 fujita health univ. sch. med., toyoake, japan; 5 dept. leg med., shiga univ. med. sci., japan we examined the effect of maternal repeated cold stress (rcs) on development of catecholamine neurons of offsprings using by tyrosine hydroxylase (th) immunohistochemistry. rcs was loaded to pregnant rats between day 9 and 19 after fertilization. pups were perfused at postnatal day 8. in the frontal cortex, the number of largesized (more than 7 m in diameter) th-immunoreactive (-ir) varicosities was significantly smaller in prenatally rcs rats than controls. in the locus coeruleus of prenatally rcs rats, th immunoreactivity was less than that of controls. in the medullary c1/a1 catecholaminergic field, the size of th-ir neurons was smaller and the quantity of thir fibers were less in prenatally rcs rats, although there were not significant differences. it was suggested that prenatal rcs impaired development of catecholaminergic neurons, especially noradrenergic neurons of neonates. ps2a-i162 developmental exposure to pentachlorophenol affects thyroid hormone responsive gene in the brain but not stress response maiko kawaguchi 1,2,3 , kaori morohoshi 3,4 , rie yanagisawa 3 , erina saita 5 , gen watanabe 6,7 , masatoshi morita 3 , kazuyoshi taya 6,7 , hirohisa takano 3,4 , toshiyuki himi 1,2 , hideki imai 3,8 1 dept. toxicol and pharmacol., facul. pharmacy, musashino univ., tokyo, japan; 2 res. inst. pharmaceut. sci., musashino univ., tokyo, japan; 3 nation. inst. for environ. stud., ibaraki, japan; 4 grad. sch. environ. sci., univ. tsukuba, ibaraki, japan; 5 wildlife rescue veterinarian associ., tokyo, japan; 6 facul. agriculture, tokyo univ. agriculture & technol., tokyo, japan; 7 the united grad. sch. veterinary sci., gifu univ., gifu, japan; 8 div. environ. health sci., dep. social med., facul. med., miyazaki univ., miyazaki, japan antiseptic pentachlorophenol (pcp) treatment to rats affects thyroid hormone (th) system, which is essential for normal development of central nervous system. in this study, we show the exposure to pcp during gestation and lactation suppressed plasma th level, and induces gene expression of neurogranin and th receptor ␤, which play a role in neural formation. the present data suggest that pcp may affect central nervous system development, though stress response was not affected by pcp exposure. ps2a-i163 the effect of psychological stress during pregnancy on the open-filed behavior, the forced swim test, the fos expression in the brain, and the level of plasma corticosterone in offspring rat hiroshi abe, noriko hidaka, kei odagiri, yuko watanabe, yasushi ishida dept. of psychiatry, miyazaki med. coll., univ. of miyazaki, japan one group (psy) was born from the dams which observed, during their pregnancy, that another rat was exposed to the foot-shock stress in a communication box. the other group (c) was born from the dams not exposed to such stress. psy, comparing to c, showed decreased activities in the open-field test and prolonged immobility time in the forced swim test. on the other hand, there were no significant differences between the number of fos immunopositive cells in various regions of the brain in two groups before and after the foot-shock. however, plasma corticosterone was elevated in psy compared with c. these results suggest that the prenatal psychological stress might enhance reactivity to novel environment and depressive behavior induced by forced swim, and chronically elevated level of corticosterone might be involved in this neurobiological substrate. akane nakasato, yasushi nakatani, yoshinari seki, hideho arita department of physiology, toho university school of medicine, tokyo, japan to evaluate roles of da and serotonergic (5-ht) systems in stressinduced anxiety, we measured brain da and 5-ht levels before, during and after a forced swimming test (fst) in autistic model of the rat. the model rat was made by exposing a pregnant rat to valproic acid (vpa). our previous study demonstrated that the autistic model exhibited abnormality of 5-ht system and behavioral impairments related to autism. in the present experiment, we gave a prolonged fst for 60 min in the model rat, which frequently experienced to be drowned after the immobility time during fst. brain da and 5-ht levels were measured from samples collected from the prefrontal cortex (pfc). we found a gradual and steady increase in pfc da level during fst, although 5-ht level showed only transient augmentation. behavioral alteration after fst was characterized by an increased appetite during light phase (sleep) of circadian cycle. we suggest that the feeding abnormality may be caused by the stress-induced anxiety mediated by mesocortical da system. shigeo masaki, eiko aoki, satoshi yonezawa, atsuo nakayama dept. embryology, inst. developmental res., kasugai, aichi, japan neuroligin (nl1-4) is a family of neuronal cell-surface proteins to be involved in intercellular junctional formation and signalling. recently, several studies have implicated nl3 and nl4 in autistic disorders. nls have a relative identical structure (∼70%); nl1 and nl2 localize in the glutamatergic excitatory, and inhibitory synapses, respectively, while nl3 seems express in the olfactory glia, but nl4 distribution is unknown. here we have generated antibody against human nl4, and explored its distribution in the post mortem human tissues. in the central and peripheral nervous system, nl4 was expressed exclusively in the neurons, and was especially abundant in particular subsets of neurons, including neurons producing nonapeptides. nl4 was observed in paraneurons and some endocrine cells outside the cns. these results suggested that nl4 is important for neuroendocrine function. nl4 cdna was transfected to in neuroblastoma. formed spine-like structures on the cells expressing nl4 were rough and thicker than those of nl1 or nl3 transformants. it suggested the unique activity of nl4 for synapse formation. motopsin is a serine protease secreted from pyramidal neurons of cerebral cortex and hippocampus. recently, the truncation of motopsin gene has been reported to cause non-syndromic mental retardation. however, the underlying mechanisms are yet to be elucidated. we report here that the knockout (ko) mice deficient in the expression of motopsin exhibit morphological abnormality. golgi-cox staining revealed that spine density on both apical and basal dendrites of hippocampal ca1 pyramidal neurons in the ko mice significantly decreased than in the wild type mice. similarly, spine density tended to decrease at cingulate cortex of the ko mice than wild type. our results suggest that motopsin affects dendritic spine formation and/or stabilization. mental retardation is a frequent disorder affecting 1-3% of the population. recently the truncation of motopsin/neurotrypsin gene has been identified in algerian family in which four out of eight children affected by a severe impairment of cognitive functions with an iq below 50. here we report that knockout (ko) mice lacking motopsin gene mildly impaired water maze performance and social behavior. the ko mice significantly delayed the latency to the platform area on a probe test of hidden version of morris water maze although they showed the similar performance to wild-type mice during training session. in a social memory test, the ko mice showed significant elongation of sniffing time to an intruder, despite of normal performance of social memory. our results suggest that the ko mice provide insights into the molecular mechanisms important for development of cognitive functions. natsue yoshimura 1 , daisuke horiuchi 1 , tomoyuki miyashita 2 , minoru saitoe 2 , hitoshi okazawa 1 1 department of neuropathology, medical research institute, tokyo medical and dental university, tokyo, japan; 2 tokyo metropolitan institute for neuroscience, tokyo, japan polyglutamine tract binding protein-1 (pqbp-1) was originally isolated as one of the candidates for polyglutamine disease related protein. recently, several groups has reported about pqbp-1 disease that pqbp-1 mutant causes x-linked mental retardation (xlmr). to investigate the function of pqbp-1 in xlmr pathology, we produced two kinds of flies, human pqbp-1 overexpression flies (hpqbp1 flies) and drosophila pqbp-1 knock-down flies (dpqi flies), and examined olfactory learning and memory to analyze their memory consolidation process from short-term memory (stm) to long-term memory (ltm). the hpqbp1 flies showed memory impairment in ltm. in current study, we analyze memory abilities of the dpqi flies to observe detailed function of pqbp-1 in memory formation. seiji hayashizaki, masahiko takada tokyo metropolitan institute for neuroscience, usa when two alternatives are available in instrumental behavior, animalǐs behavior is biased toward responding on one lever with which each behavioral response results in delayed large reward delivery, and against responding on the other lever with which each response results in immediate but small reward delivery. this has been used as an index of impulsive behavior and is known to be susceptible to lesions of brain structures such as the basolateral amygdala (bla) and the nucleus accumbens (na). it has been shown that the bla and na are involved in maintaining reward seeking behavior with a secondary reward when a secondary reinforcer is available. thus, a question arises as to how the behavioral response on the delayed lever is maintained through functions exerted by these structures when no secondary reinforcer is available. to this end, we implanted cannulae bilaterally and electrodes into the bla and na to identify neuronal substances and activities involved in the mediation of 'putative secondary reward' without secondary reinforcer. xue-zhi sun 1 , sentaro takahashi 1 , yoshihisa kubota 1 , rui zhang 2 , chun cui 2 , yoshihiro fukui 2 1 natl. inst. radiol. sci., chiba, japan; 2 sch. med. tokushima univ., tokushima, japan heavy ion irradiation has the feature to administer a large radiation dose in the vicinity of the endpoint in the beam range, and its irradiation system and biophysical characteristics are different from ordinary irradiation instruments like x-or gamma-rays. using this special feature, heavy ion irradiation has been applied for cancer treatment. the safety and efficacy of heavy ion irradiator have been demonstrated to a great extent. for instance, brain tumors treated by heavy-ion beams became smaller or disappearance. however, fundamental research related to such clinical phenotypes and their underlying mechanisms are little known. in order to clarify characteristic effects of heavy ion irradiation on the brain, we developed an experimental system for irradiating a restricted region of the rat brain using heavy ion beams. the characteristics of the heavy ion beams, histological, behavioral and elemental changes were studied in the rat following heavy ion irradiation. yukio imamura department of psychiatry, university of ottawa, on, canada nmdars contain two nr1 subunits paired with two nr2 subunits. nr1 and nr2 (a-d) subunits harbor the glycine and glutamate binding sites, respectively. nmdars are localized in both synaptic and extra-synaptic areas, but they are found at higher density within the synapse. after the peak of synaptogenesis, the nr1/nr2a complex, characterized by rapid offset kinetics, dominates at the synapse, while the nr1/nr2b complex, characterized by slow kinetics, predominates in the extra-synaptic area. the activation of extrasynaptic nmdars by glutamate escaping from the synaptic cleft during episodes of high synaptic activity suggests that they may have a different role. using whole-cell voltage-clamp recordings from ca1 pyramidal neurons from mice (at 12 weeks of age), we found that following induction of ischemia, ifenprodil, a selective nmdar-nr2b antagonist, reduced the inward current of the isolated nmdar at extra-synaptic site while it had less effect at the synaptic nmdar. the molecular mechanisms involved are currently under investigation and these new data will be also presented at the meeting. in the present study, we observed expression and changes of mineralocorticoid receptor (mr) and glucocorticoid receptor (gr) in the gerbil hippocampal ca1 region after ischemia. in blood, corticosterone levels increased biphasically at 30 min and 12 h after ischemia, and thereafter its levels decreased. in the sham group, mr and gr immunoreactivities were weakly detected in the ca1 region. by 3 days after ischemia, mr and gr were not significantly altered in the ca1 region. from 4 days after ischemia, mr and gr immunoreactivities were detected in astrocytes and microglia in the ca1 region, and at 7 days after ischemia. the specific distribution of corticosteroid receptors in glia may be associated with the differences of mr and gr functions against ischemic damage. the present study was investigated the effects of early treadmill training after cerebral infarction in rats. we determined whether treadmill exercise changes cellular expression of caspase-3 and midkine in the mca area. stroke was induced by a 90-min mca occlusion using an intraluminal filament. rats were exercised for 20 min each every day on a treadmill. brain damage in ischemic rats was evaluated by infarct volume. exercised and non-exercised rat brains were processed for immunocytochemistry to quantify the areas of caspase-and mkimmunoreactive calls. no significant differences in infarct volume were found between rats trained with treadmill and non-exercised controls. cellular expressions of mk were significantly increased in striatum (glia) of the exercised rats. treadmill exercise was shown to suppress the decrease in caspase-3 expression in the penumbra. the present study showed the exercise after cerebral infarction might have important implication for post-ischemic recovery. ps2a-j174 reversed astrocytic glutamate transporter glt-1 crucial to the ca 2+ paradox-like insult-induced neuronal death in neuron/astrocyte co-cultures tatsuro kosugi, koichi kawahara, takeshi yamada, motoki tanaka lab. of cellular cybernetics, graduate school of information science and technology, hokkaido univ., sapporo, japan "ca 2+ paradox" is the phenomenon whereby the intracellular concentration of ca 2+ paradoxically increases during reperfusion with normal ca 2+ -containing media after brief exposure to a low ca 2+ solution. the present study aims to characterize the ca 2+ paradoxinduced cell injury in neuron/astrocyte co-cultures. prior exposure of the cultures to a low ca 2+ solution for 60 min significantly injured only neurons after reperfusion with a normal ca 2+ medium for 24 h, but astrocytes remained intact. after the onset of reperfusion, the intracellular concentration of na + in astrocytes increased significantly during the reperfusion episode, resulting in a reversal of the operation of the astrocytic glt-1. the present findings suggested that ca 2+ paradox-induced accumulation of na + in astrocytes was involved in the reperfusion-induced excitotoxic neuronal injury resulting from the reversed operation of astrocytic glt-1 during the reperfusion episode. common genetic mutation in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (cadasil) has been associated with missense mutations of notch3 concerning cysteine residues within the extracellular amino-terminal region. we report new mutations of two japanese cadasil families, which did not directly involve a cysteine residue. exons of the notch3 were amplified by pcr and subsequently analysed for dhplc and direct sequence. the first patient carried the missense mutation c577t, which results in pro167ser. the second patient carried the missense mutation c302g, which results in arg75pro. new mutations had not changed the number of cysteine residues, but coding the extracellular amino-terminal region of the notch3 receptor which may involve an alteration in the ligand binding or putative dimerisation properties. ps2a-j177 mci -186, a radical scavenger, protected cortical neurons from cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol 3kinase madinyet niyaz 1 , tadahiro numakawa 2 , yoshinori matsuki 1 , emi kumamaru 2 , yuki yagazaki 2 , harumi kitazawa 1 , hiroshi kunugi 2 , motoshige kudo 1 1 pathology department of tokyo medical university, tokyo, japan; 2 department of mental disorder research, national institute of neuroscience, ncnp, tokyo, japan the role of mci -186, a radical scavenger, in the central nervous system (cns) has not been fully elucidated. in the present study, we found that treatment with mci -186 prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, we found that mci -186 exposure induced the activation of both the map kinase (mapk) and pi3 kinase (pi3k) pathways and that the mci -186-dependent survival effect was blocked by the inhibitors, u0126 (an mapk pathway inhibitor) or ly294002 (a pi3k pathway inhibitor). these results suggested that mci -186 exerts a protective effect on cns neurons via enhancing survival-signaling pathways in addition to a role such as a radical scavenger. osamu tokumaru 1 , noriko yoshimura 1 , tetsuro sakamoto 1 , takaaki kitano 2 , naoko nisimaru 1 , isao yokoi 1 1 dept. physiol., sch. med., oita univ., japan; 2 med. edu. ctr., sch. med., oita. univ., japan protective effects of ethyl pyruvate (ep) on energy metabolism of rat brain exposed to ischemia were investigated by 31 p-nuclear magnetic resonance (25 • c). brain slices were incubated in standard artificial cerebrospinal fluid (acsf) with 2 mm ep (ep-1), acsf replaced by acsf with 2 mm ep after ischemia (ep-2), or acsf only (control). the brain slices were exposed to ischemia by stopping the perfusion for 1 h. high-energy phosphate, creatine phosphate (pcr) and ␥-atp, levels were measured. decrease in pcr level was not different among the three groups when exposed to ischemia. but increase in pcr level after the reperfusion was significantly larger in ep-1 than in control (p < 0.01). these results indicate that ep is effective in the reperfusion period and is more protective when administered before ischemic exposure. the importance of timing of administration of ep in clinical use was suggested. research funds: grant-in-aid for scientific research (c) #17591639 from mext to t.k. hideaki tamai 1 , kuniko shimazaki 2 , norimasa seo 1 1 department of anesthesiology and critical care medicine, jichi medical university, graduate school, tochigi, japan; 2 department of physiology, jichi medical university, tochigi, japan we investigated the effects of acupuncture on cell proliferation in the dentate gyrus (dg) and the lateral ventricle (lv) of adult rats. in this study, acupuncture was performed at the acupoints neiguan (pc6), yintang (ex-hn3) and sanyinjiao (sp6), which have been used for the enhancement of conscious and functional recovery in stroke patients. eight weeks old male wistar rats were used in the experiment. through 5-bromo-2,-deoxyuridine (brdu) immunohistochemistry, a significant increase in cell proliferation in the dg of the acupunctured group was observed. however, the cell proliferation in the lv was not affected with the acupoints pc6, ex-hn3 and sp6. the present findings indicate that the sensitivity on cell proliferation in the dg by acupuncture stimulation is higher than in the lv. yukio ago 1 , keiko takahashi 1 , shigeo nakamura 1 , akemichi baba 2 , toshio matsuda 1 1 laboratory of medicinal pharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan; 2 laboratory of molecular neuropharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan this study examined the effect of isolation rearing on anxiety-related behavior of mice in the staircase test, an animal model of anxiety. the staircase test consisted of placing an experimentally naive mouse in an enclosed staircase with five steps. in group-reared mice, an anxiolytic diazepam increased the number of steps climbed to the top step of the staircase, but did not affect the frequency of rearing behavior. the anxiogenic drug ␤-cca increased the number of rearing, but did not affect the number of steps climbed. on the other hand, methamphetamine increased the number of steps climbed to the second step. in these circumstances, isolation-reared mice showed an increase in the numbers of steps climbed to the top step and rearing in the staircase. these findings suggest that isolation rearing increases in exploratory and anxiety-like behaviors in mice. tomonori fujiwara 1 , tatsuya mishima 1 , takefumi kofuji 2 , kimio akagawa 1 1 department of cell physiology, kyorin university school of medicine, mitaka, tokyo, japan; 2 radio isotope laboratory, kyorin university school of medincine, mitaka, tokyo, japan hpc-1/syntaxin 1a is believed to regulate the exocytosis of synaptic vesicles. in order to examine the neurophysiological function in vivo, we have produced hpc-1/syntaxin1a knock-out mice. surprisingly, the null mutant mice revealed normal development and basal synaptic transmission in cultured hippocampal neurons appeared to be normal. however, in conditioned fear memory test, consolidation of the memory was impaired in homozygous mutant mice but not in heterozygote. however, once memory consolidation was acquired, the extinction process was disturbed in homozygote. we further examined latent inhibition of cued fear memory (li) to access behavioral property. interestingly, li was suppressed both in heterozygous and homozygous mutant mice unlike the case of conventional conditioned fear memory test. implication of these behavioral abnormalities in hpc-1/syntaxin1a knock-out mouse will be discussed. research funds: kakenhi (15700292) ps2a-k182 effects of local administration of the gaba agonists into the hippocampus ca1 area on active avoidance learning and serotonergic systems in the administration area in rats satoko hatakenaka 1 , hiroko miyakubo 1 , junichi tanaka 1 , yasushi hayashi 2 , yukio hattori 2 , masahiko nomura 3 1 department of curriculum, teaching and memory, naruto university of education, tokushima, japan; 2 department of human nutrition, notre dame seishin university, okayama, japan; 3 department of physiology, saitama medical school, saitama, japan in fischer 344 male rats, bilateral injections of the ␥-aminobutyric acid (gaba) a agonist muscimol into the ca1 area slightly decreased the avoidance rate in an active avoidance task. similar injections of the gaba b agonist baclofen enhanced the avoidance rate. there are significant differences between the muscimol-and baclofen-treated groups in the avoidance rate, implying that gaba a and gaba b receptors have the opposite action on the performance of avoidance learning. perfusion with muscimol through the microdialysis probe decreased the serotonin metabolite 5-hydroxytryptamine (5-hiaa) concentration in the ca1 area, whereas baclofen perfusion had no effect, suggesting that the gabaergic system may exert to inhibit the serotonin release in the ca1 area through gaba a receptors. sawako arai, taku nagai, kenji takahashi, hiroyuki kamei, kazuhiro takuma, kiyofumi yamada lab. neuropsychopharmacol, kanazawa univ., kanazawa, japan we performed immunohistochemical c-fos mapping after a prepulse inhibition (ppi) test of the startle reflex in mice. startle stimulus increased the number of c-fos-positive cells in the somatosensory cortex, nucleus accumbens shell and the caudal pontine reticular nucleus (pnc), while prepulse trials without startle stimulus increased c-fos expression in the lateral globus pallidus (lgp). in mice subjected to startle stimulus with prepulses, most of the startle stimulus-induced c-fos expression was diminished but c-fos expression remained in the lgp. prepulse-induced c-fos expression in the lgp was colocalized with gad-67. fluoro-gold infusion into the pnc and the pedunculopontine tegmental nucleus (pptg) retrogradely labeled neurons in the pptg and lgp, respectively. microinjections of phaclofen, but not picrotoxin, into the pptg impaired ppi of the startle reflex. these results suggest that gabaergic neurons in the lgp which project to the pptg play a crucial role through the activation of gaba b receptors in the ppi of the startle reflex. shiho kitaoka 1 , sho koyasu 1 , akinori nishi 2 , tomoyuki furuyashiki 1 , toshiyuki matsuoka 1 , shuh narumiya 1 1 department of pharmacology, university of kyoto, kyoto, japan; 2 department of physiology, university of kurume, kurume, japan prostaglandins e2 (pge 2 ) exert their actions in various organs through specific receptor, ep1 to 4. the previous study suggests that ep1 modulates da system. to investigate the roles of ep1 in da system, we examined ep1ko mice with behavioral sensitization induced by cocaine. the administration of cocaine elevated da concentration in the nucleus accumbens up to ∼300% in both wild-type and ep1ko mice. however, increase of locomotor activity in ep1ko mice was significantly lower than that in wild-type mice. because locomotor activity is closely related to dopamine d1 receptor (d1r) signaling, we tested the density of d1r and d1r signaling with phosphorylation of darpp-32. there were no differences in d1r binding. d1r signaling was significantly attenuated in the striatal slices from ep1ko mice. the effect of d1r agonist on locomotor activity was also attenuated in ep1ko mice. these results indicate that pge 2 has enhancing effects on locomotor activity via ep1 by potentiating the d1r signaling. central serotonin (5-ht) function has been implicated in impulsivity. the present study examined rats with 5-ht depletion by parachloroamphetamine (pca) in simple and reversal go/no-go visual discrimination tasks, and analyzed the relationships between learning performance and focal concentrations of 5-ht and its metabolites (5-hiaa) in the brain. for both tasks, significant negative correlations between learning performance and 5-ht and 5-hiaa concentrations were observed in the medial prefrontal cortex and nucleus accumbens. in contrast, for reversal task only, significant correlations between learning performance and 5-ht and 5-hiaa concentrations were observed in the orbitofrontal cortex and amygdala. these data suggest the regional difference of 5-ht roles on selective indices of impulsivity. yuki sato 1,2,3 , tatsushi onaka 2 , norimasa seo 1 , eiji kobayashi 3 1 dept. anesthesiol., jichi med. univ., tochigi, japan; 2 dept. physiol., jichi med. univ., tochigi, japan; 3 div. organ replacement research, center for mol. med., jichi med. univ., tochigi, japan cyclosporine is widely used for preventing allograft rejection. however, in a considerable number of transplant recipients, cyclosporine causes neuropsychological side effects such as confusion, depression, and anxiety. cyclosporine inhibits calcineurin activity and forebrain-specific calcineurin knockout mice exhibit deficits in social behaviour. it is thus possible that cyclosporine causes psychological side effects via disturbing social interactions. here, we examined effects of cyclosporine upon anxiety and social behaviour in mice. calcineurin did not significantly change percent entries into open arms and time spent on open arms in the elevated plus maze test. on the other hand, in the social interaction test in home cage, cyclosporine increased the number of particles in home cage, an index of social activity. all these data suggest that impaired social interaction is a cause of psychological side effects of cyclosporine. to investigate the distribution of functionally activated vestibularrelated brainstem neurons during postnatal development, ombined immuno-/hybridization histochemistry of c-fos expression was performed in sprague-dawley rats (p1-21; adult). conscious animals were subjected to rotational or translational stimulus which activates hair cells of the horizontal semicircular canals or utricle, respectively. neuronal activation within brainstem nuclei was defined by the expression of c-fos. labyrinthectomized controls and normal stationary controls showed only a few sporadically scattered fos-expressing neurons. with rotational stimulation that comprised cycles of constant angular acceleration and deceleration, fos-labeled neurons were observed by p4 in the vestibular nucleus and downstream relay stations of vestibular pathways, such as the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc, ioa, ioc, iok). a later maturation time was evidenced for the utricular system. fos-labeled neurons were only identifiable in the vestibular nucleus by p7; in the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc and io␤) by p11. within the vestibular nucleus of p7-9 rats, neurons activated by canal or utricular inputs were intermingled throughout its rostro-caudal length. in p21 and adult rats, neurons activated by canal or utricular inputs were intermingled in localized regions of the medial and spinal vestibular nuclei. however, neurons in the rostral half of spinal vestibular nucleus were activated only by utricular inputs. taken together, we have demonstrated that canal-and otolithrelated brainstem neurons that encode rotational and translational movements in the horizontal plane are histologically segregated and exhibit different developmental time frame. to determine whether perineuronal nets (pn) within the vestibular nuclei contribute to plasticity of central connectivity, we studied the presentation of pn within the vestibular nuclei during development (rats, p1 to adult) and after unilateral labyrinthectomy (ul) in the adult. histochemistry with the lectin wisteria floribunda agglutinin was used to map pn about neun-immunopositive neurons within the vestibular nuclei. in normal postnatal rats, pn was detectable by p3 in the vestibular nucleus as fuzziness about neuronal cell bodies. from p9 onwards, the fuzzy pn progressively consolidated into a network organization. the fuzziness was no longer observable after p12. during postnatal development, the number of neurons showing pn increased with age, reaching the adult level by p21. with ul, the pn network on the lesioned side remained compact until 5 days post-lesion when the fuzziness reminiscent of that in early postnatal rats became evident. by 11 days after ul, the pn of some neurons resumed the network pattern as was observed in normal adult rats. this phenomenon was found in the pn of the remaining neurons by 14 days after ul. the pn on the labyrinth-intact side showed the compact network of uninjured age-matched rats. taken together, our findings indicate pn changes that suggest possible correlation with vestibular nuclear neuronal function both during postnatal development of normal rats as well as in adult rats following destruction of the ipsilateral inner ear. minori ueda, takayuki suzuki, hiroyoshi miyakawa laboratory of cellular neurobiology, tokyo university of pharmacy and life science, tokyo, japan dynamics of transmitters in the synaptic cleft depends on many processes such as transmitter release, uptake and diffusion. to better understand these processes, we analyzed ampar-and nmdarmediated epscs and synaptically induced transporter currents (stcs) elicited with high-frequency stimuli. recordings were made from pyramidal cells and astrocytes in the ca1 region of rat hippocampal slices, 100 hz/20 pulse tetanic stimulations were delivered to schaffer-collaterals, and the evoked currents during the course of tetanic stimulation were isolated. the decay time course of the last isolated stc during the tetanic stimulation was not significantly different from that of the first. while the amplitude of the ampar-mediated epscs showed significant decay in the presence of cyclothiazide, there was no marked decay of the amplitude of the nmdar-mediated epscs. these findings imply that synaptic fatigue and saturation of glutamate transporters do not take place during the course of high-frequency stimulation at 100 hz. ikuko yao 1 , hiroshi takagi 1 , hiroshi ageta 1 , tomoaki kahyo 1 , ken hatanaka 1 , kaoru inokuchi 1 , mitsutoshi setou 1,2 1 mitsubishi kagaku institute of life sciences, tokyo, japan; 2 university of tokyo, tokyo, japan; 3 okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan we identified and characterized a novel ubiquitin ligase named scrapper. scrapper is an f-box protein which has leucine rich repeat and c-terminal membrane localization sequence, highly expressed in neurons throughout the brain. to investigate the physiological role of scrapper in the neuron, we recorded mepscs from the neuron over-expressed the egfp-tagged full-length scrapper construct or truncated form of scrapper constructs. they exhibited a strong suppression or enhancement in the frequency of mepscs while showing a non-significant change in mepsc amplitude, rise, and decay time compared with neurons expressing egfp. the passive membrane properties of neurons such as membrane resistance (rm), series resistance (rs), and membrane capacitance (cm) were not statistically different from those of control. these data suggests a presynaptic effect of scrapper protein. ps2p-a003 presynaptic membrane potential-dependent regulatory mechanism of transmitter release tetsuya hori, tomoyuki takahashi department of neurophysiology, university of tokyo graduate school of medicine, tokyo, japan in simultaneous pre-and postsynaptic recordings at the calyx of held, we addressed the mechanism underlying presynaptic membrane potential-dependent changes of transmitter release. a weak sustained depolarization (e.g., 10 mv, 1 s) of calyceal nerve terminal potentiated epscs despite that it diminished presynaptic action potential (a.p.) amplitude. as we further depolarized the terminal epscs became eventually depressed concomitantly with a marked reduction in the a.p. amplitude. when presynaptic ca 2+ currents (i pca ), induced by an a.p.-waveform command pulse, were used to evoke epscs, a weak sustained depolarization enhanced i pca and epscs in parallel. this epsc facilitation was robust at the calyx of held both in rats and mice, but was almost absent in p/q-type ca 2+ channel knockout mice. we conclude that the p/q-type specific ca 2+ channel facilitation plays an essential role in the facilitation of transmitter release following presynaptic depolarization. hiroshi takagi 1 , koji ikegami 1 , ken hatanaka 1,2,3 , yoko fujiwara-tsukamoto 1 , mineo matsumoto 1 , ikuko yao 1 , mitsutoshi setou 1,2,4 1 mitsubishi kagaku institute of life sciences, japan; 2 presto, japan; 3 school of pharmaceutical sciences, the university of tokyo, japan; 4 okazaki institute for integrative bioscience, japan a variety of post-translational modifications to the exposed cterminal tails of tubulin, such as detyrosination/tyrosination, polyglycylation and polyglutamylation would play a crucial role in the neuron. however, evidence for the implication of these modifications in regulating the translocation of channels and receptors is currently unavailable. of the modifications, polyglutamylation is highly abundant in the mammalian brain, thus, this modification might account for the translocation of channels and receptors in the mammalian brain. in the rosa22(−/−) mouse, which shows a gross loss of polyglutamylated ␣-tubulin, transient a-type currents were largely suppressed in hippocampal pyramidal neurons in vitro. we provide herein, using rosa22 mice, the evidence for the implication of ␣tubulin polyglutamylation in the regulation mediated a-type k current. satoshi kawasaki 1 , shingo kimura 1 , reiko fujita 2 , shuji watanabe 1 , kazuhiko sasaki 1 1 dept. of physiol., sch. of med., iwate medical univ., morioka, japan; 2 dept. of chem., sch. of lib. arts & sci., iwate medical univ., morioka, japan application of dopamine (da) induces a slow na + -current response in the identified neurons of aplysia ganglia under voltage clamp. this type of response is produced by the activation of trimeric g-protein sensitive to cholera toxin (ctx) as previously reported. the na +current response to da was gradually and irreversibly depressed after intracellular injection of clostridium difficile toxin b, which is known to inactivate all types of rho family g-proteins. intracellular application of clostridium botulinum exoenzyme c3, a specific toxin to rhoa-c, also depressed the da-induced response irreversibly. furthermore, the da-induced current response was significantly depressed by gap domain of p50rhogap applied intracellulary. in contrast, gef domain of rhogef dbs had a tendency to increase the response. these results suggest that the da-induced na + -current response may be regulated by the activation of rho family g-protein. the ␦2 glutamate receptor (␦2r) plays a crucial role in cerebellar functions. although ␦2r has a putative channel pore domain, and ␦2r displayed ca 2+ -permeable channel activities in lurcher mutant mice, it has been unclear whether wild-type ␦2r functions as a channel. here we introduced a ␦2r transgene, which had a mutation (gln618arg) in the putative channel pore conserved in ca 2+ -permeable glutamate receptors, into ␦2 −/− mice. surprisingly, a mutant ␦2r transgene, as well as a wild-type transgene, rescued all abnormal phenotypes of ␦2 −/− mice, such as ataxia and loss of long-term depression. these results indicate that ca 2+ influx through ␦2r is not required for its function in the cerebellum in vivo, and that wild-type ␦2r may not function as a ca 2+ -permeable ion channel. research funds: kakenhi (17700316) and takeda science foundation ps2p-a007 distribution of tarp -8 on hippocampal neurons and its key role in synaptic and extrasynaptic expression for ampa receptors masahiro fukaya 1 , mika tsujita 2 , maya yamazaki 2 , etsuko kushiya 2 , manabu abe 2 , kaori akashi 2 , masanobu kano 3 , haruyuki kamiya 4 , kenji sakimura 2 , masahiko watanabe 1 1 department of anatomy, hokkaido university school of medicine, sapporo, japan; 2 department of cellular neurobiology, brain research institute, niigata, japan; 3 department of cellular neuroscience, graduate school of medical science, osaka university, suita, japan; 4 department of molecular anatomy, hokkaido university school of medicine, sapporo, japan the -8 is one of four transmembrane ampar regulatory proteins (tarps). pre-and post-embeding immunogold visualized -8 on excitatory synaptic and extrasynaptic membrane. in -8-ko mice, ampars were reduced in hippocampal homogenates (46% of control) and psd fraction (35%). immunogold labeling also exhibited reduction of extrasynpatic (47%) and synaptic (35%) ampars in ca1 pyramidal cells. the reduction of extrasynaptic receptors was particularly severe on dendrites (36%) and spines (38%). ampar-mediated responses were reduced at ca1 synapses (52%). therefore, -8 is the major auxiliary subunit of hippocampal ampars. etsuko tarusawa 1 , yugo fukazawa 1 , elek molnar 2 , masahiko watanabe 3 , ryuichi shigemoto 1,4 1 div. cerebral structure, nips, okazaki, japan; 2 mrc, univ. of bristol, bristol, uk; 3 hokkaido univ., sapporo, japan; 4 sorst, jst, kawaguchi, japan relay cells in the dorsal lateral geniculate nucleus receive two types of glutamatergic inputs; retinogeniculate (rg) and corticogeniculate (cg) synapses. it has been shown that the synaptic transmission at both rg and cg synapses is mediated via ampa and nmda receptors. however, how ampa and nmda receptors are expressed in these two types of synapses have not been elucidated. we examined the expression pattern of ampa and nmda receptors in rg and cg synapses using sds-digested freeze-fracture replica labeling (sds-frl). the sds-frl revealed that synaptic size of individual rg synapses was significantly smaller than that of cg synapses. rg synapses expressed 1.7 to 3 times higher density of ampa receptors than cg synapses. on the other hand, cg synapses expressed 1.6 to 3 times more nmda receptors than rg synapses. these results indicate differential effects on the relay cell by the retino-and cortico-geniculate inputs through ampa and nmda receptors. katsuyuki kaneda 1,2,3 , hitoshi kita 1 1 dept. of anat. & neurobiol., univ. of tennessee, memphis, tn, usa; 2 japan society for promotion of science, tokyo, japan; 3 dept. of developmental physiology, nips, okazaki, japan to investigate the properties of synaptically induced slow responses in globus pallidus (gp) neurons, whole-cell recordings were performed using rat brain slice preparations. repetitive stimulation of the gp and internal capsule induced mixed fast epsps/ipsps followed by a slow ipsp (sipsp), and a long-lasting slow depolarization (sdepo). bath application of nbqx, cpp, and gabazine blocked the mixed epsps/ipsps. the gaba b receptor antagonist cgp55845 abolished the sipsp. an mglur1 antagonist, but not an mglur5 antagonist, partially blocked the sdepo. in addition, cgp55845 enlarged the amplitude of fast ipscs, but not of epscs, that were evoked during the repetitive stimulation, suggesting an involvement of presynaptic gaba b receptors in gaba release. these results indicate that synaptically released gaba and glutamate can evoke gaba b receptor-and mglur1-mediated responses in the gp. contribution of these responses to the control of gp activity will be discussed. research funds: nih and the jsps ps2p-a010 essential contribution of glutamate to gaba depolarization involved in hippocampal seizure-like activity yoko tsukamoto 1 , yoshikazu isomura 1,2 , michiko imanishi 1 , tomoki fukai 2 , masahiko takada 1 1 system neurosci., tokyo met. inst. neurosci., tokyo, japan; 2 neural circuit theory, riken bsi, saitama, japan we have previously shown that neuronal synchronization is achieved by excitatory gabaergic and glutamatergic inputs during a hippocampal seizure-like afterdischarge. however, it still remains unclear how the gaba response is converted from inhibitory to excitatory in the process of afterdischarge induction. here we traced the time-course of amplitude and reversal potential of gabaergic transmission in pyramidal cells and interneurons entraining the afterdischarge, and examined influence of glutamate on the conversion of gaba response. the gaba reversal potential in pyramidal cells rose to spike-threshold levels for >20 s after the induction. gaba amediated cl-influx lasted for 1.5 s, and then glutamate enhanced the conversion effectively in a gaba a -independent manner, which was dependent on an extracellular k increase. coapplication of gaba and glutamate caused a similar oscillatory activity. the results show gaba and glutamate may cooperatively induce as well as maintain seizure-like activity. michiko nakamura 1 , yuko sekino 1,2 , toshiya manabe 1,2 1 division of neuronal network, department of basic medical sciences, institute of medical science, university of tokyo, tokyo, japan; 2 crest, jst, japan profound activity-dependent facilitation of synaptic transmission at hippocampal mossy fiber synapses is a unique and functionally important property. in the present study, we found that this synaptic strengthening was partially mediated by presynaptic gaba a receptor activation during the developmental period (p < 30), using electrophysiological methods and optical imaging. in immature animals (p10), fiber volley amplitudes were activity-dependently increased during short-train stimulation of mossy fibers. this fiber volley facilitation was significantly decreased by either inhibition of gaba a receptors or suppression of gaba release from interneurons. these results suggest that gaba released from inhibitory interneurons and gaba a receptors on mossy fibers contribute to activity-dependent facilitation of the excitatory synaptic transmission during development. takuya nishimaki, il-sung jang, jyunichi nabekura dep. dev. physiol., nips, sokendai, okazaki, crest, jst, japan lateral superior olive (lso) is the first auditory center. during the early postnatal period, the inhibitory synaptic inputs to lso neurons from medial nucleus of the trapezoid body (mntb) change from predominantly gabaergic to glycinergic. we focused on metabotropic gaba b receptor (gaba b r) as the key molecule of difference between gaba and glycine. in immature lso neurons postsynaptic gaba b r could activate k + channels, but this effect ceased by the third postnatal week. baclofen, a gaba b r agonist, reduced ipsc amplitude at mntb-lso synapses in neonate (<p5) with a significant change in the paired-pulse ratio. the presynaptic effect of baclofen was gradually decreased with development (p2-18). in situ hybridization and immunohistochemistry experiment revealed gaba b r expression in mntb neurons was also gradually decreased. the property of mntb-lso synapses in gaba b r knock out mouse remained immature at p14-16 compared with control mouse. thus, gaba b receptor seems to play an important role in development of synaptic property. pyramidal cells in the hippocampal ca1 area receive gabaergic input from interneurons, which make synapses on the soma, dendrites and the axon initial segment (ais). here, we used the sdsdigested freeze-fracture replica labeling method to visualize the cell surface distribution of the ␣1, ␣2, and ␤2/3 subunits quantitatively on distinct subcellular compartments of pyramidal cells. labelings for these subunits were accumulated over clusters of intramembrane particles (imp) on the protoplasmic face (p-face) of the plasma membrane, indicating that many imp clusters of a certain size represent gabaergic synapses. the synaptic labelling densities for gaba-a receptor subunits were not significantly different on the tested subcellular compartments. double labelling experiments showed that the majority of synapses contains ␣1, ␣2, and ␤2/3 subunits. in the cerebellar cortex, the effects of ␣-adrenoceptor activation on inhibitory synaptic transmissions have not yet been fully understood. therefore, we investigated the effects of the ␣ 1 -or ␣ 2 -adrenoceptor agonist on firing rates of presynaptic interneurons (ins). presynaptic cell-attached recordings showed that spontaneous activity of gabaergic ins was enhanced by the ␣ 1 -adrenoceptor agonist phenylephrine, while reduced by the ␣ 2 -adrenoceptor agonist clonidine. immunohistochemical studies showed that ␣ 1 -adrenoceptors were expressed in the molecular layer and the purkinje cell (pc) layer, while ␣ 2 -adrenoceptors were observed in cell bodies of pcs and ins. these results suggest that noradrenaline enhances inhibitory neurotransmitter release via ␣ 1 -adrenoceptors, which were expressed in presynaptic terminals and somatodendritic domains, whereas suppresses the excitability of ins via ␣ 2 -adrenoceptors, which were located in the presynaptic in soma. thus, cerebellar ␣-adrenoceptors play roles in a presynaptic dual modulation of gabaergic inputs from ins to pcs. research funds: kakenhi (16700344) ps2p-b015 involvement of basal pkc and erk 1/2 activities in constitutiveinternalization of ampa receptors in cerebellar purkinje cells tetsuya tatsukawa 1 , takahiko chimura 2 , azumi matsumoto 2 , kazuhiko yamaguchi 2 1 lab. for motor learning control, riken, bsi, japan; 2 lab. for memory & learning, japan ampa receptor (ampar) internalization provides a mechanism for cerebellar ltd, which is the underlying basis of motor learning and motor coordination. however, the relationship between cerebellar ltd and constitutive ampar internalization in parallel fiber (pf)-purkinje cell (pc) synapses remains unclarified. in the present study, we demonstrated that tetanus toxin (tetx, a blocker of exocytosis mediated by vamp) infusion into pcs caused the rundown of pf-epsc amplitude through the constitutive ampar internalization, and then addressed question whether intracellular signals involved in cerebellar ltd induction modify the constitutive ampar internalization at pf-pc synapses using electrophysiological and immunocytochemical techniques. our results suggest that the regulation of actin polymerization is involved in the surface expression of ampars and the surface expressing ampars are constitutively internalized depending on both basal pkc and mek-erk1/2 activities at pf-pc synapses. taisuke miyazaki 1 , kohichi tanaka 2 , masahiko watanabe 1 1 department of anatomy, school of medicine, hokkaido university, sapporo, japan; 2 department of molecular neuroscience, tokyo medical and dental university, tokyo, japan glutamate released to the synaptic cleft has to be removed by glutamate transporter. in the cerebellum, glutamate transporter glast is richly expressed in bergmann glial cells (bg) that enwrap synapses, dendrites and somata of purkinje cells (pcs). in this study, anatomical analysis was applied to glast-deficient mice. in the mutant mice, lamellate processes from bg fibers became atrophic, resulting in incomplete sealing of synaptic cleft. furthermore, the distribution of climbing fiber (cf) terminals was decreased proximally. by anterogradely labeling, multiple cf innervation was demonstrated to be formed by ectopic innervation to and from nearby proximal dendrites, particularly at their crossing point. these results suggest that glast is essential for complete ensheathment of pc synapses and for the establishment of cf mono-innervation by suppressing local ectopic innervation between nearby pc dendrites. miwako yamasaki 1,2 , kouichi hashimoto 2 , taisuke miyazaki 1 , masahiko watanabe 1 , masanobu kano 2 1 dept. of anatomy, grad. sch. of med., hokkaido univ., sapporo, japan; 2 dept. of cellular neuroscience, grad. sch. of med., osaka univ., suita, japan the ␥ isoform of protein kinase c (pkc␥) is highly expressed in cerebellar purkinje cells (pcs), and its deficiency impairs the late phase of climbing fiber (cf) synapse elimination. in the present study, we analyzed multiple cf innervation in pkc␥-deficient mouse by immunohistochemistry combined with anterograde tracer labeling of cfs. in general, cf innervation regressed proximally in the pkc␥-deficient mouse. in some mutant pcs, a considerable part of the proximal dendrites lost association with cf terminals. in the majority of multiply innervated pcs, single main cf innervated broad region of proximal dendrites, whereas surplus cfs innervated the somata and basal portion of the proximal dendrites. moreover, immunoelectron microscopy revealed synaptic contact of cf terminals to somatic spines. these results suggest that pkc␥ is crucial for strengthening innervation by a main cf and elimination of surplus cfs from proximal somatodendritc compartments of pcs. keiji imoto 1,2 , takashi kodama 1,2 , ryuichi shigemoto 2,3,4 , yugo fukazawa 2,3 , yasuo mori 5 1 division of neural signaling, nips, okazaki, japan; 2 school of life science, graduate university for advanced studies (sokendai), okazaki, japan; 3 division of cerebral structure, nips, okazaki, japan; 4 sorst, japan science and technology agency, kawaguchi, japan; 5 department of synthetic chemistry and biological chemistry, kyoto university, kyoto, japan a murine ataxic mutant, rocker, has a point mutation in the ca v 2.1 (p/q-type) ␣ subunit gene and shows the mildest phenotype among the series of ca v 2.1 mutants. although functional defects of the mutant channel were relatively mild, we found that synaptic transmission in parallel fiber-purkinje cell synapses was considerably impaired. we demonstrated that this impairment was mainly attributable to postsynaptic changes, which included reduction of the number of ampa receptors in psd, whereas the presynaptic function remained normal. we also found the dendrites of purkinje cells showed poor arborization in rocker mice. these lines of evidence indicate that ca v 2.1 exerts its strong influence on postsynaptic maturation and dendritic structure. bai-chuang shyu, chia-ming lee, wei-chih chang institute of biomedical sciences, academia sinica, taipei, taiwan roc the aim of the present study was to investigate synaptic transmission and organization of thalamus evoked activities in the anterior cingulate cortex (acc) using a novel mouse brain slice preparation protocol. the mono-and poly synaptic responses in the acc were excited by direct thalamic activation. the synaptic transmission involves both ampa and nmda glutamate receptors. several thalamus-evoked responses were identified: the early negative component (n1) emerged in layer vi near cingulum. subsequently a positive component (p) appeared in layer vi. the second negative component (n2) became apparent in layers ii/iii and v. then the activities spread downward to layer vi and developed as n3 component in a medial-to-lateral direction. we confirmed that functional thalamocingular activities were preserved in our newly developed brain slice preparation. the thalamus-evoked responses were activated and progressed along a lateral-medial-lateral trajectory loop in the acc and this process was modulated extensively by glutamatergic synapses. ps2p-b020 target-dependent extracellular ca-dependence of the short-term plasticity of the solitary complex synapses in the rat lab. neurophysiol., dept. neurosci., jikei univ. sch. med., tokyo, japan the discharge frequency-dependence of the transmission efficiency at the synapses from the vagal primary afferents to the second-order neurons in the solitary complex (sc) forms the primary filter of the visceral information. such frequency filtering of the synaptic transmissions from the solitary tract (ts) to neurons in the sc depends on their short-term plasticity as evidenced by strong correlation between frequency-dependence and the paired-pulse ratio (ppr). here we analyzed the mechanism underlying distinct pprs among distinct types of synapses. in synapses from ts to the neurons in nucleus of the solitary tract, which show prominent low-pass filtering, ppr significantly increased from 0.4 ± 0.2 to 1.1 ± 0.2 (n = 6; mean ± s.d.) by reducing [ca 2+ ] o from 2 to 0.5 mm, whereas that in low-pass synapses from ts to the neurons in dorsal motor nucleus of the vagus was affected only slightly. these results suggest that the synaptic frequency filtering in the sc is under distinct control depending on the function of the postsynaptic target systems. ps2p-b021 response of spinal monosynaptic reflex to high frequency stimulation in newborn rat the susceptibility of synaptic transmission to the stimulation frequency is a characteristic feature of immature animals, and has been attributed to the incompleteness of transmitter release mechanisms. in an isolated spinal cord preparation of newborn rat, monosynaptic reflexes (msrs) evoked by dorsal root stimulation, were mediated by both nmda and non-nmda glutamate receptors. msrs were constant in amplitude at 1/15 s, and were depressed completely by cnqx. when stimulus rate was increased to 1/s, msr amplitudes were greatly reduced initially, and recovered later. this recovery of msr was eliminated by apv. non-nmda component of msrs was eliminated completely at 1/s, but appeared in constant amplitudes in the presence of cyclothiazide. from these results, non-nmda glutamate receptor mediated almost most of msrs at 1/15 s, but not at 1/s due to desensitization. in contrast, nmda glutamate receptor does not mediate msrs, but activated at 1/sec, in place of non-nmda receptor. in conclusion, incompleteness of transmitter release mechanisms could not be solely attributed to the feature of immature synaptic transmission. kenichi ono 1 , keisuke shiba 2 , ken nakazawa 3 , ichiro shimoyama 4 1 department of otolaryngology, chiba university, chiba, japan; 2 department of otolaryngology, kimitsu central hospital, chiba, japan; 3 department of integrative neurophysiology, chiba university, chiba, japan; 4 section for human neurophysiology, research center for frontier medical engineering, chiba university, chiba, japan we determined synaptic source of the respiratory-related activity of laryngeal motoneurons (lms) using spike-triggered averaging of lm membrane potentials triggered by spikes of medullary respiratory neurons in decerebrate, paralyzed cats. in inspiratory (i) lms, monosynaptic epsps were evoked by spikes of i neurons with augmenting firing patterns, and monosynaptic ipsps were evoked by spikes of expiratory (e) neurons with decrementing firing patterns and of i neurons with decrementing firing patterns. in elms, monosynaptic ipsps were evoked by spikes of i neurons with decrementing firing patterns and of e neurons with augmenting firing patterns. we conclude that various synaptic inputs from respiratory neurons contribute to shaping the respiratory-related trajectory of lm membrane potentials. ps2p-b023 in-vivo gene transfer of exogenous protein to the primary afferent neurons chiaki yamada 1,2 , eiji shigetomi 1,3 , fusao kato 1 1 lab. neurophysiol., jikei univ. sch. med., tokyo, japan; 2 dept. basic biol. sci., kyoritsu univ. of pharm., tokyo, japan; 3 jsps, japan in order to understand the mechanism how sensory information is transmitted to the brain, it is indispensable to identify the functional roles of already-identified molecules related to synaptic transmission between the afferent fibers and the central second-order neurons. here we developed a method for efficient in-vivo gene transfer into the neurons in the sensory ganglia and evaluated expression of their gene products. in the anesthetized young wistar rats, we injected pcaggs-egfp plasmid vector (through courtesy of drs. j. miyazaki and k. nakajima) into the nodose ganglion (ng) at the neck and transferred it by electroporation. two days after transfection, the ng was extirpated and fixed to observe egfp signals. a large portion of somata in the ng as well as the centrally and peripherally projecting afferent fibers expressed high levels of egfp with few damaged cells. this technique might enable to analyze the function of specific molecules in the transmitter release from the sensory afferent termini in the brain. research funds: kakenhi (17650116, 176865) ps2p-b024 laminar sources of synaptic input to layer 1 neurons in rat visual cortex takuma mori, edward m. callaway salk institute, usa layer 1 of the mammalian neocortex consists of interneurons, including late-spiking neurogliaform cells and non-late-spiking axondescending cells. very little is known about connectivity of these cells with cells in other cortical layers. we examined the laminar sources of local excitatory and inhibitory functional synaptic inputs onto individual layer 1 neurons of rat visual cortex, using scanning laser photostimulation. we find that axon descending cells get excitatory synaptic inputs primarily from layer 2/3. neurogliaform cells receive more excitatory inputs from layers 4 and 5. the dominant inhibitory synaptic inputs onto both types of cells originate from layer 2/3. these neurons also get inhibitory inputs from layers 1 and 4-6. the presence of convergent excitatory and inhibitory synaptic inputs from multiple cortical layers suggests that the computations in layer 1 are highly integrative. takako nishi 1 , tsukasa gotow 2 , shiro nakagawa 2 1 ins. nat. sci., senshu univ., kawasaki, japan; 2 dept. neurol., kagoshima univ. grad. sch. med.-den. sci., kagoshima, japan four extra-ocular photoreceptors, the photoresponsive neurons, a-p-1, es-1, ip-1 and ip-2 which respond directly to light, exist in the abdominal ganglion of the mollusc onchidium. a-p-1/es-1 of these neurons respond to light with a depolarizing receptor potential, whereas light hyperpolarizes the other ip-1/ip-2. the present study was conducted to examine the functional significance of the above ip-1/ip-2, having a peak sensitivity at 510 nm light. the body wall of the animal transmitted enough 510 nm to hyperpolarize ip-1/ip-2 in the ganglion covered by the body wall. ip-1/ip-2 were coupled with weak electrical synapses and involved in a spontaneous beating or bursting spike activity. ip-1/ip-2 were not coupled to a-p-1/es-1 one another. finally, ip-1/ip-2, the first order photosensory cells were also the second order interneurons or motoneurons relaying various sensory inputs and innervating the pneumostome and viscera. ps2p-c026 cellular mechanism of interaural level difference calculation in the auditory brainstem of the chicken tatsuo sato, iwao fukui, harunori ohmori department of physiology, faculty of medicine, kyoto university interaural level difference (ild) and timing difference (itd) are the major two cues to localize the sound source, and are processed separately. although the details of itd processing are well investigated, not much is known about ild. in the barn owl, it is proposed that ild is calculated in lldp (posterior part of the dorsal lateral lemniscal nucleus). we confirmed already that in vivo lldp calculates ild in the chicken as well. in this work using the patch clamp recording with the brainstem slice preparation, we found that lldp neurons fire tonically under the regulation of two k + channels activated at low and high voltage, and receive both excitatory and inhibitory inputs representing the contralateral and the ipsilateral sound intensity, respectively. we are intending to clarify the cellular mechanism of ild calculation as the interaction of both excitatory and inhibitory synaptic inputs. to understand the functional organization of the cns, it is essential to know how sensory information is processed within the cns. we have been approaching this topic by following the ontogenetic patterning of neural circuit formation related to the cranial and spinal sensory inputs using optical imaging. in this study, we surveyed developmental organization of neural networks related to the olfactory nerve in the embryonic chick forebrain. stimulation applied to the olfactory nerve elicited epsp-related optical signals in the olfactory bulb from the 7-day old embryonic stage. the epsp was mediated by glutamate, and nmda-and non-nmda-receptor components were identified. in more developed stages, in addition to the responses in the olfactory bulb, another response area was discriminated within the cerebrum, which seemed to correspond to the higher-ordered nucleus of the olfactory pathway. the results suggest that the olfactory pathway is functionally generated at early stages of development when neural networks related to other visceral and general somatic sensory inputs are also in the process of developing. yoko momose-sato, katsushige sato dept. physiol., tokyo med. dent. univ. sch. med., tokyo, japan in an accompanying study, using a voltage-sensitive dye recording technique, we examined the developmental organization of the olfactory pathway in the embryonic chick forebrain, and showed that functional synaptic transmission in the olfactory bulb was expressed at around e7. it is known that odor stimuli elicit oscillatory events in the olfactory bulb in various species. we found that oscillatory activity was also generated in the chick olfactory bulb during embryogenesis. at early stages of development (e7-e8), postsynaptic responserelated optical signals evoked by olfactory nerve stimulation exhibited a simple monophasic waveform that lasted a few seconds. as development proceeded, the pattern of the optical signal became complicated, and oscillatory activity was observed in a later phase of the postsynaptic response. the oscillation was restricted to the olfactory bulb, and this spatial pattern was different from that of the propagating wave activity termed the depolarization wave. we examined spatio-temporal patterns of the oscillatory activity in different stages, and studied its developmental dynamics. akiyoshi shimada 1 , nyberg tobias 1 , nahoko kasai 1 , yuriko furukawa 2 , keiichi torimitsu 1,2 , kunihiko obata 3 , yuchio yanagawa 2,4 , tadaharu tsumoto 2,3 1 ntt basic research laboratories, ntt corporation, kanagawa, japan; 2 sorst, jst, saitama, japan; 3 neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; 4 department of genetic and behavioral neuroscience, graduate school of medicine, gunma university, gunma, japan in the immature stage of cerebral cortex, gaba a receptor activation depolarizes rather than hyperpolarizes the membrane potential of neurons. cortical neurons of glutamic acid decarboxylase 67-green fluorescence protein (gad67-gfp) knock-in mice at postnatal day 0-1 were cultured on a microelectrode array. spontaneous firing of cortical neurons appeared within the first 5 days in vitro (div). when a gaba a receptor antagonist, bicuculline, was added, four types of modulation of firing pattern were observed until at least 14 div: increase, decrease, disappearance and no change. these results and an additional analysis suggest that a portion of the spontaneous activity emerged through an activation of gaba a receptors. ps2p-c030 pathway specific signal modulation by purinergic receptors in the parallel processing system makoto kaneda 1 , toshiyuki ishii 2,4 , yasuhide shigematsu 3 , toshihiko hosoya 2 , yukio shimoda 3 1 dept. physiol., keio univ. sch. med., tokyo, japan; 2 bsi, riken, wako, japan; 3 mri, tokyo womens' medical univ., tokyo, japan; 4 dept. biomolecular science, univ. toho, funabashi, japan the retina divides visual information into on-and off signals, and processes them with independent subsets of neurons. so far no difference between these pathways has been found for neurotransmitter functions. we have previously found that p2x2-purinoceptors are selectively present on the off-type, but not on the on-type cholinergic amacrine cells of the mouse retina. we therefore asked if atp has different functions in the two pathways. as expected, only the off-type, but not the on-type, cholinergic amacrine cells responded to atp. this response was mediated by the p2x2-purinoceptors. furthermore, a p2x-purinergic receptor antagonist activated the tonic phase firing of the off ganglion cells, but not of the on cells. thus atp has off-pathway specific modulatory functions mediated by p2x-purinergic receptors. bhupesh mehta, gulnaz begum, preeti g. joshi department of biophysics, national institute of mental health and neuro science, bangalore, india cultured hippocampal neurons in network exhibit synchronized oscillations of cytosolic ca 2+ . these oscillations are an inherent property of various cell types and are essential for the maintenance and development of neuronal plasticity, and for normal brain functioning. the synchronized ca 2+ oscillations are primarily controlled by glutamate receptor activation, but may be modulated by a variety of other factors. these spontaneous oscillations are inhibited by activation of purinergic receptors. we observed a dose dependent inhibition of the oscillatory activity by purinergic receptor agonists atp and utp, but an enhanced oscillatory pattern was observed when the action of 2mesatp was observed. the inhibition of 2mesatp response by p2y1 receptor specific antagonist mrs 2179 indicates that different subtypes of purinergic receptors play different role in the regulation of synchronized ca 2+ oscillations. data will be presented on the p2 receptor mediated regulation of synchronized ca 2+ oscillations in cultured hippocampal neurons. mahomi kuroiwa, akinori nishi department of pharmacology, kurume university school of medicine, kurume, japan presence of additional d1-like dopamine receptors that selectively couple to gq/phospholipase c (plc) has been proposed. in recent studies, skf83959 was shown to selectively stimulate plc-linked d1-like dopamine receptors. darpp-32 is a phosphoprotein that regulates the efficacy of d1 receptor signaling. activation of d1 receptors that couple to golf/pka signaling leads to darpp-32 phosphorylation at thr34. we investigated the effect of skf83959 on darpp-32 phosphorylation at thr34 by using mouse neostriatal slices. treatment of neostriatal slices with skf83959 increased thr34 phosphorylation. the effect of skf83959 was enhanced by a plc inhibitor, u73122. in contrast, the effect of skf83959 was partially blocked by a d1 receptor antagonist, sch23390, and the sch23390-insensitive increase in thr34 phosphorylation was completely abolished by an adenosine a 2a receptor antagonist, zm241385. thus, analysis of darpp-32 phosphorylation revealed that skf83959 activates at least three signaling cascades in the neostriatum: (1) plc, (2) d1 receptor/golf/pka, (3) adenosine a 2a receptors. ps2p-c033 thiamylal may more effectively reduce neuronal excitability in cerebral cortex than that in hippocampus naoshi fujiwara division of medical technology, niigata university school of health sciences, japan effects of thiamylal (a barbital derivative anesthetic) on excitation propagation in the cerebral cortex and hippocampus were analyzed using membrane potential imaging. cortical and hippocampal slices of the c57bl6 mouse were dyed with a voltage-sensitive dye rh414. in cortical slices, electrical single-pulse stimulation to the layer v evoked transient depolarization in the vicinity of stimulated site, and then this excitatory response propagated to the layers ii-iii and widely expanded in these layers. the excitation propagation in the layers ii-iii was significantly inhibited by thiamylal (200 mol/l). in hippocampal slices, excitation propagation elicited by the same stimulation to the ca1 stratum radiatum remained without significant changes in the presence of thiamylal at the same dose. on the other hand, an ampa/kainate receptor antagonist cnqx (20 mol/l) inhibited excitation propagation in both cortical and hippocampal slices. the results suggest that thiamylal more effectively reduce neuronal excitation in cortex than that in hippocampus. research funds: grant-in-aid from jsps no.15390471 ps2p-c034 brain-derived neurotrophic factor regulating ampa receptor translocation to postsynaptic density through ip3r and trpc calcium signaling hiroko nakata, shun nakamura national institute of neuroscience, tokyo, japan the change in the number of postsynaptic ampa-type glutamatergic receptors (ampars) by neuronal activity is implicated in the excitatory synaptic plasticity. here, we showed that bdnf induced ampar translocation to postsynaptic site of the dissociated cortical pyramidal neurons. using calcium imaging, we located the spines that were activated by bdnf. with thus identified spines, we analyzed the ampar subunit glur1 localization to postsynaptic density (psd) by immunostaining. we found that bdnf increased the ratio of surface glur1 at psd within 20 min. bdnf selectively translocated ampar, but not nmdar. the essential signaling was ca 2+ transients evoked by the activation of trkb/plc␥ pathway. both the intracellular ca 2+ rise, that is, ca 2+ release from ip3r and ca 2+ influx through storeoperated cation channel trpc contributed to the increase of the surface expression of glur1 at psd. these results suggest an important role of bdnf in the regulation of ampar trafficking by spatial and temporal ca 2+ signaling regulation. research funds: health sciences research grant of nano-1 hiroyuki konishi 1 , kazuhiko namikawa 1,2 , keiji shikata 3 , yuji kobatake 1 , taro tachibana 3 , hiroshi kiyama 1 1 dept. anat. & neurobiol., osaka city univ., grad. sch. med., osaka, japan; 2 dept. anat., asahikawa med. col., hokkaido, japan; 3 dept. bioeng., osaka city univ., grad. sch. eng., osaka, japan activation of akt-mediated signaling pathways is crucial for injured neurons to survive and regenerate. in this study, we attempted to identify novel substrates for akt by using an antibody, which recognized phosphorylated consensus motif by akt. using pc12 cells, which overexpressed constitutively active akt, we screened protein spots that exhibited increased immunostaining by the antibody, and consequently identified the neuronal intermediate filament protein, peripherin. using some mutant forms of the recombinant proteins, the phosphorylation site was determined in vitro. we then generated an antibody against phosphorylated peripherin, and demonstrated that peripherin was phosphorylated not only in akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. these results suggest that peripherin is a novel substrate for akt in vivo and that its phosphorylation may play a role in motor nerve regeneration. the truncated trkb receptor, t1, is involved in the control of cell morphology via the regulation of rho proteins via rho gdi1. however, it is unclear whether t1 regulates the downstream of rho signaling and the actin cytoskeleton. in this study, we investigated this question using c6 rat glioma cells, which express t1 endogenously. rho gdi1 was dissociated from t1 in a bdnf-dependent manner, which also causes decreases in the activities of rho-signaling molecules such as rhoa, rock, pak and erk. moreover, bdnf resulted in the disappearance of stress fibers in the cells treated with lpa, an activator of rhoa, and in morphological changes in cells. furthermore, a competitive assay with cfp fusion proteins of t1-specific sequences reduced the effects of bdnf. these results suggest that t1 regulates the rho signaling pathways and the actin cytoskeleton. anjana kalita eukaryotic gene expression laboratory, national institute of immunology, new delhi, india jak and stat are activated in response to many cytokines and growth factors. this pathway has been primarily studied in non-neuronal origin cells. it has been shown that stat activated cellular transformation, occurs through transcription regulation of specific genes like c-myc, cyclin d1, bcl2. the present study was undertaken to identify target genes of jak/stat pathway in response to tnf-alpha & igf-1 in neuronal origin cells. we also looked at the mechanism of activation of these target genes. we saw that jak/stat pathway was involved in upregulation of cyclind1 in response to igf-1 both at protein & rna levels but no change was observed in c-myc, bcl2 levels. interestingly, mapk-erk, which is also known to activate cd1 in other systems, had no effect on the cd1 level in neuronal cells. on the other hand, pi3kinase not only activated jak/stat pathway but also affected on cyclind1 level. thus it appears that jak/stat is activated by pi3k, which in turn upregulates cyclind1 level. pi3k also activated erk without changing cd1 level, thus we show that jak/stat pathway is important for survival of neurons, activated by pi3k. cd1 is a one of the major downstream targets of jak/stat. hiroko inoue 1 , haruhisa kawasaki 2 , ritsuko fujii 2 , tohru yoshioka 2 , kazunori sango 3 , toshihiko kadoya 4 , hidenori horie 2 1 sch. of sci. and eng., waseda univ., tokyo, japan; 2 advanced res. center for biol. sci., waseda univ., tokyo, japan; 3 tokyo metropolitan inst. for neurosci., tokyo, japan; 4 pharmaceut. res. lab. kirin brewery co., ltd., gumma, japan oxidized galectin-1 (gal-1/ox) stimulates macrophages to promote axonal regeneration in peripheral nerves after nerve injury. but, the mechanism how gal-1/ox stimulates macrophages remains to be clarified. in the present study with rt-pcr and western blotting, we searched for the molecules contributing to the mechanism. rt-pcr analysis with cultured rat peritoneal macrophages revealed that the application of gal-1/ox enhanced mrna expression of nerve regeneration-related factors such as il-6, il-1 ␤. however, those of gdnf, ngf, bdnf, nt-3, nt-4, igf-i, and igf-ii scarcely detected in control condition were not changed by the application of gal-1/ox. since il -6, il-1, and lif can promote axonal regeneration in the in vitro axonal regeneration model, the up-regulation of these cytokines by the treatment with gal-1/ox may enhance nerve regeneration after nerve injury. kennichi katou, taku iwamoto, satoshi kida dep. of bioscience, tokyo univ. of agriculture, japan ␣camkii has been known to play essential roles in synaptic plasticity. in response to increase in ca 2+ concentration, ␣camkii is activated by the interaction with ca 2+ /cam. prolonged increase in ca 2+ level leads to further activation of ␣camkii through autophosphorylation at t286. to understand the molecular dynamics of ␣camkii activation in vivo, we have tried to visualize and monitor the ␣camkii activity in various types of cells using fret. in hela cells, increase in ca 2+ level induces continuous interaction between ␣camkii and cam and conformational change of ␣camkii, indicating that the conformational change of ␣camkii mainly reflects the interaction with cam. in sh-sy5y cells, the increase in ca 2+ level induces only transient interaction between ␣camkii and cam but the conformational change of ␣camkii is maintained following the termination of the interaction with cam, suggesting that conformational change of ␣camkii is induced by the interaction with cam and then maintained by autophosphorylation. thus, mechanisms for regulation of ␣camkii activation is cell-type dependent. akifumi kamata, yusuke takeuchi, kohji fukunaga dept. pharmacol., grad. sch. pharmaceu. sci., tohoku univ., sendai, japan ca 2+ /calmodulin-dependent protein kinase ii (camkii) is highly expressed in brain including dopaminergic neurons, however, the role of camkii in the dopaminergic neurons remains to be unclear. camkii consist of four isoforms, ␣, ␤, ␥ and ␦, and differential isoforms are implicated in the different neural functions. we have examined the isoforms of camkii expressed in rat substantia nigra (sn). rt-pcr and immunoblot analyses revealed that the ␥ and ␦ isoform mrnas including ␦3 isoform, a nuclear isoform of camkii, were predominantly expressed in sn. the immunohistochemical study also confirmed the preferential localization of the ␥ and ␦ isoforms in the sn dopaminergic neurons, and immunoreactivity against anti-camkii␦1-4 antibody was detected in both nucleus and cytoplasm. furthermore, we defined expression of bdnf mrnas having the exon ii and iv in sn. taken together with our previous observation, the camkii␦3 isoform was involved in the expression of bdnf in sn dopaminergic neurons. ps2p-d041 the role of ca 2+ /calmodulin-dependent protein kinase ii in neuronal functions revealed by inactivated camkii␣ knock-in mouse yoko yamagata 1,2 , hiroyuki sakagami 3 , keiji imoto 1,2 , kunihiko obata 4 , yuchio yanagawa 5 1 okazaki, japan; 2 sokendai, okazaki, japan; 3 tohoku univ., sendai, japan; 4 riken, wako, japan; 5 gunma univ., maebashi, japan ca 2+ /calmodulin-dependent protein kinase ii (camkii) is one of the major protein kinase in the central nervous system and proposed to play important roles in various neuronal functions. we engineered knock-in mice with the inactivated ␣ subunit of camkii by replacing k42 with r42 to study functional significance of camkii activity in intact animals. as expected, camkii activity was selectively reduced, while camkii subunit protein levels were comparable to those of wild type controls in homozygous mutants. in situ hybridization revealed normal expression patterns of camkii subunit mrnas, including dendritic localization of camkii␣ mrna. further analyses of these mice will be presented in the meeting. research funds: a grant-in-aid for scientific research from japan society for the promotion of science (kakenhi , #17500218) ps2p-d042 characterization of ca 2+ -dependent membrane binding proteins of rat brain shohei maekawa 1 , katsutoshi taguchi 1 , haruko kumanogoh 2 , shun nakamura 2 1 division of biosystems science, grad. sch. of sci/tech, kobe-u kobe 657-8501, japan; 2 division of biochemistry and cellular biology, national inst. neurosci. kodaira, tokyo 187-0031, japan ca 2+ -dependent membrane binding proteins were purified through a ca 2+ -dependent membrane binding and egta-induced membrane dissociation protocol from a crude triton solubilized membrane fraction. molecular characterization of these ca 2+ -dependent membrane binding proteins through lc-ms/ms identified annexin vii, annexin xi, and copin isoforms, in addition to annexin vi and neurocalcin␣. since the membrane fraction contains phosphatidylethanolamine (pe), cholesterol, and phosphatidylcholine (pc), the membrane binding activity of annexin vi, a major protein in this fraction, was studied using artificial liposomes. annexin vi showed a pe-dependent, but not cholesterol-dependent liposome binding activity. immunostaining of annexin vi in cultured neurons showed a punctuate staining of neuronal cell membrane and this spots increased during maturation. these results suggest the participation of annexin vi in the membrane cycling at the presynaptic region. ps2p-d043 generation of dominant negative mutants of transcription factor crest takuzou simo, kenichi kato, hiroshi hosoda, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan transcription factor calcium-responsive transactivator (crest) has been shown to be required for dendritic growth of neuron. previous studies have shown that crest contains two functional domains; multifunctional domain (mfd; aa251∼322) required for transactivation, nuclear localization and homodimerization and c-terminal transactivation domain (ctd; aa238∼402). to further understand the function of crest on brain function, we tried to generate the dominant negative mutant of crest to use mouse genetics study. indeed, we generated fusion proteins of gal4-dbd with mutants of crest; crest c ( 238∼402) lacking c-terminal transactivation domain, mfd and mfd-nls that is a fusion protein of mfd with nuclear localization signal (nls). from the experiments using gal4 one hybrid system, we found that mfd-nls shows the strong dominant negative effects on transcriptional activation by crest. ps2p-d044 characterization of iq-arfgef, a novel exchange factor for arf6, in mouse brain hiroyuki sakagami, hisatake kondo department of cell biology, tohoku university graduate school of medicine, sendai, japan adp-ribosylation factor 6 (arf6) regulates cytoskeletal dynamics and membrane trafficking that are critical processes for synaptic plasticity. to understand its neuronal functions, we have focused on guanine nucleotide exchange factors (gef) for arf6. in this study, we determined the entire sequence of a partially identified cdna (mkiaa0522) encoding a novel sec 7 domain. the deduced protein contained coiled coil, iq, sec7, plekstrin homology domains. interestingly, it contained a type i pdz domain-binding motif at its cterminal tail. arf pull-down assay demonstrated its ability to active arf6 more selectively than arf1. thus, we named this protein as iq-arfgef. in situ hybridization analysis demonstrated the preferential expression of iq-arfgef in the forebrain and cerebellar granule cells. furthermore, iq-arfgef mrna was localized not only in the hippocampal neuronal layers but also in their dendritic fields. our present findings suggested that iq-arfgef may play important roles in dendrites through activation of arf6 and interaction with various pdz proteins. eriko miura 1 , masahiro fukaya 1 , tokiharu sato 2 , kazushi sugihara 3 , masahide asano 3 , katsuji yoshioka 2 , masahiko watanabe 1 1 dept. anat., hokkaido univ. sch. med., sapporo, japan; 2 dept. mol. cell. biol., cancer res. inst., kanazawa univ., kanazawa, japan; 3 adv. sci. res. center, kanazawa univ., kanazawa, japan the c-jun n-terminal kinase (jnk) is one of the major mitogenactivated protein kinases (mapks), and jsap1 (or jip3) is a jnkassociated scaffold that controls the specificity and efficiency of jnk signaling cascades. here we studied its expression and distribution in mouse brains. jsap1 mrna was expressed in developing and adult brains, showing spatial patterns similar to jnk1∼3 mrnas. in embryos, jsap1 immunolabeling was intense for progenitor cells in the ventricular zone and external granular layer, and for neurons and glial cells differentiating in the mantle zone. in adults, jsap1 was distributed in spines, dendrites, perikarya, and axons of various neurons, and often associated with ser and cell membrane. this wide spatiotemporal expression suggests its fundamental role in mediating external stimuli to jnk mapk pathway and other intracellular machineries. yasukazu hozumi, kaoru goto department of anatomy and cell biology, yamagata university school of medicine, japan diacylglycerol kinase (dgk) is involved in intracellular signal transduction as a regulator of a second messenger, diacylglycerol, and consists of several isozymes. to address the functional implications of dgk isozymes in the brain, we have raised specific antibodies against the isozymes. by immunoblot analysis, we found that the immunoreactive bands for dgk␤, -␥ and -were detected in the light membrane/microsome enriched fraction of rat brains, suggesting that these isozymes localize to the internal membrane system. immunohistochemical examination on rat brain revealed that dgk␤ was detected as dot-like structure in the cell membrane, and dgk␥ as round-shaped structure in the juxtanuclear region of neurons. dgk was detected in the perinuclear region and in the dendrites of purkinje cells. these data show that dgk isozymes localize differentially to the internal membrane system in neurons, suggesting that dgk isozymes play unique roles in distinct internal membrane system. kotaro hattori 1 , shigeo uchino 2 , tomoko isosaka 1,3 , shinichi kohsaka 2 , takeshi yagi 3 , shigeki yuasa 1 1 dept. ultrastractural res., nat. inst. neurosci., ncnp, kodaira, japan; 2 dep. neurochem., nat. inst. neurosci., ncnp, kodaira, japan; 3 kokoro biology group, fbs, osaka univ., suita, japan recently, we have found that fyn-mediated tyrosine-phosphorylation of nmda-r subunits is required for haloperidol-induced catalepsy. haloperidol induced catalepsy in the control mice, but such response was significantly reduced in fyn-deficient mice. fyn activation and enhanced tyrosine-phosphorylation of the nmda-r nr2b subunit were induced after haloperidol injection to the control mice, but both responses were significantly reduced in fyn-deficient mice. both nr2b phosphorylation and the nmda-induced calcium responses increased after dopamine d 2 -r blockade in cultured striatal neurons of the control, but not in fyn-deficient neurons. accordingly, d 2 -r antagonist activates striatal fyn and the subsequent tyrosine phosphorylation of nr2b alters striatal neuronal activity, thereby inducing catalepsy. we also report further proteomics analysis on this molecular pathway. hiroaki okuda, shingo miyata, tsuya taneda, atsushi yamaguchi, shinsuke matsuzaki, natsuko kumamoto, masaya tohyama department of anatomy & neuroscience, graduate school of medicine, osaka university, osaka, japan the schizophrenia is a well-known debilitating psychiatric disorder. recent studies focused on genetic contributions to schizophrenia and have revealed several susceptibility genes, including dysbindin (dtnbp1: dystrobrevin binding protein 1) at 6p22.3. however it is difficult to explain the relationship between the onset of the schizophrenia and only single genetic factor. since, there is an important another factor, an environmental factor, for the schizophrenia. although little attention has been paid to the importance of both factors for the schizophrenia in the nervous system. therefore, to investigate the mechanism of the onset of the schizophrenia, firstly we performed a yeast two-hybrid screening, using the n-terminal region of dys-bindin as a bait. as a result, we detected several transcriptional factors. thus, we further examine that these interactions regulate the expression level of particular genes in the neurons. ryota adachi 1 , naoko oda 2 , ryuzo shingai 1,2 1 21st coe program, iwate university, morioka, japan; 2 department of welfare engineering, faculty of engineering, iwate university, morioka, japan the response to environmental stimuli is important to live for organisms. the soil nematode caenorhabditis elegans responds well to chemical and thermal cues known as chemotaxis and thermotaxis behavior. c. elegans is attracted to some ions, amino acids and alcohol that are byproducts of bacteria, and migrates to the temperature of cultivation with food on a temperature gradient. the sensory neurons detected these stimuli are in the head region. the neural network and genes required in each neuron are well known. however, it is under investigation about the integration of chemical and thermal stimuli. to understand the mechanism of this integration, we investigated the chemotaxis behavior under each temperature using the worms cultivated under each temperature. wild type worms cultivated at 10 • c showed decreased chemotaxis response to water-soluble chemicals and alcohol. alteration of assay temperature did not affect response to cl − but that to na + . to discuss more detail, we used thermotaxis abnormal mutants for chemotaxis assay. takashi takekawa 1 , masaki nomura 2,3 , toshio aoyagi 2 , tomoki fukai 1 1 riken brain science institute, saitama, japan; 2 graduate school of informatics, kyoto university, kyoto, japan; 3 crest, japan science and technology agency, kawaguchi, japan the neuron model proposed by izhikevich can exhibit a variety of firing patterns of cortical neurons and gives an efficient mean to conduct large-scale network simulations. however, simplified models may lose essential properties of real neurons, and hence their properties must be compared with those of more realistic models. in fact, our previous studies have shown that two neuron models with almost identical membrane potential profiles sometimes have quite different phase responses and synchronization properties. here, we study the phase responses of the izhikevich model showing fast rhythmic bursting and demonstrate that it exhibits synchronization properties similar to those shown by complex conductance based models. furthermore, its synchronization properties are consistent with those of realistic models in other firing patterns. therefore, we may conclude that the model with tuned parameters is suitable for simulating the dynamic behavior of large-scale networks. honghai cui, hirotaka sakamoto, mitsuhiro kawata department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan the amygdala plays a critical role in stress responses, especially for modulation of anxiety and fear. in the present study, we investigated the effects on the neuronal morphology for severe stress in the amygdala by using single prolonged stress (sps) paradigm as ptsd (post-traumatic stress disorder) model. rats were perfused 7 days after sps, and intracellular injections of lucifer yellow were carried out in neurons of central (cea) and basolateral amygdala (bla). at the cellular level, we observed a significant increase of dendritic arborization in bla pyramidal neurons but no effect on the cea neurons. these results suggest that sps-induced structural plasticity of the bla provides a cellular substrate for affective disorders that are triggered in ptsd. takahiko kawasaki 1,2 , tatsumi hirata 1 1 division of brain function, national institute of genetics, sok-endai, mishima, japan; 2 prest, kawaguchi, japan in macrosmatic mammals such as mice, olfactory information is detected by two distinct sensory organs, the olfactory epithelium and the vomeronasal organ, and their signals are integrated into the main olfactory bulb (mob) and the accessory olfactory bulb (aob), respectively. the secondary projections from the mob and aob first run parallel in the lateral olfactory tract and eventually diverge into the discrete targets in the telencephalon. although recent studies have revealed general principles in the primary projections, it has been largely unknown how the secondary projections from the olfactory bulbs are constructed. therefore, we investigated the secondary olfactory projections in mutant mouse lines for several axon guidance molecules and their receptors using a specific dye labeling technique, especially focusing on the distinctive pathways of mob and aob neurons. ps2p-d053 an essential role for click-iii/camki␥ in regulation of neurite extension natsumi ishihara, sayaka takemoto-kimura, hiroyuki okuno, haruhiko bito dept. of neurochem., univ. of tokyo, tokyo, japan click-iii/camki␥, a membrane-anchored camk, is a novel member of the camki family that acts downstream of camkk. in order to uncover the biological function of this kinase, we first examined its subcellular distribution and found that tagged click-iii was concentrated at the tips of filopodia-like processes in cultured hippocampal neurons, in close vicinity of actin cytoskeleton. phenotypic studies in pc-12 cells expressing various mutants of camkk and click-iii revealed that depolarization-induced activation of camkk-click-iii signaling is sufficient to robustly induce neuritogenesis. interestingly, click-iii localization in membrane fractions was regulated via sequential lipidification in an activity-dependent manner. furthermore, lipidification mutants of click-iii were impaired in their ability to facilitate activity-dependent neuritogenesis. our results thus indicate a novel role of click-iii in activity-dependent neurite formation and extension. takuro maruyama 1 , masahiro matsuura 2 , nobuhiko yamamoto 1 1 grad. sch. of frontier biosci., osaka univ., suita japan; 2 riso kagaku corp., tsukuba japan during development, thalamocortical (tc) axons invade into the cortex and stop growing in layer 4. to study tc axonal targeting, we examined the effect of the membrane proteins that are expressed in the developing cortex. the dorsal thalamus dissected from embryonic rat brain was dissociated and plated on the dishes that were coated with preclustered fc-tagged extracellular domains of several candidate molecules. after four days in vitro, axonal growth was enhanced on the substrata with low concentrations of ephrin-a5, sema7a or kit ligand, while a growth-inhibitory effect was found in higher concentrations. a tendency for tc axons to prefer or avoid the region containing these proteins was observed in a new culture method, in which purified proteins were printed on filter membranes in a real laminar size. these results suggest that ephrin-a5, sema7a and kit ligand may contribute to regulating tc axonal growth in the developing cortex. research funds: kakenhi 17023030, kakenhi 15300107 ps2p-e055 expression pattern of the guidance molecules and their receptors during gnrh neuron migration from the nose to the ventral forebrain shizuko murakami 1 , katsuhiko ono 2 1 dept. anat., juntendo univ. sch. of med., tokyo, japan; 2 div. of neurobiol. and bioinfo., nat. ins. for physiol. sci., okazaki, japan using in situ hybridization, we examined the expression pattern of attractive and repulsive guidance molecules and their receptors in the migration pathway of gnrh neurons of chick embryos. netrin-1 expression was seen in the ventral hypothalamus, whereas was absent in the nasal region and the rostral forebrain. a few gnrh neurons expressed netrin-1 receptor neogenin. slit-1 was expressed in the forebrain, but its receptor robo-1 was not expressed in gnrh neurons. semaphorin (sema) 3a expression was detected in the olfactory-forebrain region. within the forebrain, sema3a was absent in the restricted region of the medial forebrain where gnrh neurons migrated in association with a subset of olfactory fibers. at the end of this pathway, sema3a expression was observed in the caudal forebrain, and then gnrh neurons changed their course ventrally. many gnrh neurons expressed sema3a receptor neuropilin-1, suggesting that sema3a may act as a repellent during the migration of gnrh neurons. kazuto fujishima 1,2 , qing-hua jin 1 , junko kurisu 1 , tomoo hirano 2 , mineko kengaku 1 1 riken, bsi, lab. for neural cell polarity, saitama, japan; 2 department of biophysics, graduate school of science, kyoto university, kyoto, japan during development, cortical pyramidal neurons elaborate their dendrites in correct pattern and orientation in response to various environmental factors such as neurotrophins or guidance molecules. dendrite morphology is also regulated by cell-cell contacts with neighboring cells. recent studies have implicated notch signal as a candidate for such a contact-dependent regulator of dendrite patterning in postmitotic cortical neurons. we previously showed that delta/notch-like egf-related receptor (dner), neuron-specific transmembrane protein, acts as a notch ligand and mediates neuron-glia interaction during cerebellar development (eiraku et al., 2005) . dner is expressed in the dendrite of developing cortical pyramidal neurons. here, we analyzed the role of dner in the development of pyramidal neuron dendrites. we quantitatively compared the dendritic morphology of cortical pyramidal neurons in wild-type and dner knock out mice. reelin signal controls neuronal migration in the developing brain. because the binding of reelin to apoer2/vldlr induces dab1 phosphorylation, dab1 is a critical component of reelin signal. recent studies suggested that reelin signal is involved not only in neuronal migration but in other aspects of neural development. we investigated a possible function of the reelin signal in the filopodia and neurite formation. administration of reelin protein increased number of filopodia in a lesser extent than bdnf treatment. pp2 suppressed effects of reelin and bdnf on filopodia formation, suggesting that both signal pathways involve src kinase function. in spite of these similarities of reelin and bdnf functions in the filopodia formation, bdnf did not phosphorylate dab1. reelin treatment of the neurons derived from dab1 deficient mice did not induce significant increase in the number of filopodia. these results suggest that the filopodia and neurite formation involves the reelin-dab1 signal pathways. hidenobu mizuno 1,2 , tomoo hirano 1,2 , yoshiaki tagawa 1,2 1 dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan; 2 crest, jst, kawaguchi, japan the left and right hemispheres are interconnected via the corpus callosum. in mouse visual cortex, callosal axons from one hemisphere make dense projections to a restricted region at the area 17/18 border of the other hemisphere, where they terminate in layers 2/3 and 5. we used in utero electroporation-mediated gene transfer of gfp to assess distribution of callosal projections during development. gfp-labeled callosal axons arrived at the 17/18 border and started to invade the cortical plate around p5. they generated exuberant axonal arborizations by p13 then underwent remodeling to form the adult pattern by p15. neonatal enucleation did not affect the development, suggesting no apparent role of retinal activity. exogenous expression of a potassium channel kir2.1 in callosal projection neurons, a manipulation known to reduce neuronal firing, attenuated axonal arborizations in layers 2/3. area-specific targeting to the 17/18 border was not affected. results suggest that certain stages of callosal axon development require neuronal activity in cortex. ps2p-e060 identification of a novel tetraspan membrane protein, nplp, that is up-regulated after the facial nerve axotomy chihiro akazawa, yoh sasaki, masato hoshi, yasuko nakamura, shinichi kohsaka department of neurochemistry, national institute of neuroscience, ncnp, japan facial nerve axotomy of adult rats, with its extensive ability to regenerate, provides us of a useful model in which to study the molecular mechanisms of nerve regeneration. to identify genes up-regulated during regeneration processes, we constructed a subtractive library enriched for cdnas expressed in the injured facial nucleus. we identified a novel putative tetraspan protein designated as neuronal pmp22like protein (nplp) that has a distant homology to pmp22/claudin family proteins. the mrna expression of nplp was detected on the cell bodies of neurons meanwhile mrna of pmp22 resided on the cell bodies of schwann cells. the nplp mrna level significantly increased in motor neurons of axotomized facial nerve, hypoglossal nerve or sciatic nerve. nplp has a large extracellular domain that may interact with other membrane molecules. overexpression of nplp showed the microspike formation in pc12 cells. our results suggest that nplp plays an important role in the regeneration of injured motor neurons. shinsuke shibata 1 , shin-ichi sakakibara 2 , hirotaka j okano 1 , hideyuki okano 1,3 1 dept. physiol., keio univ., sch. med., japan; 2 dept. histol. neurobiol., dokkyo med. univ., japan ; 3 crest-jst, japan musashi (msi) is an evolutionarily conserved rna-binding protein. drosophila-msi regulates asymmetric cell division in neural development. in mammalian cns we identified two members, msi1 and msi2, which are co-expressed in neural precursor cells including neural stem cells, suggesting msi proteins regulate the immaturity of cns stem cells. to reveal the roles of msi family in vivo, we generated msi1 and msi2 deficient mice. msi1−/− mice frequently died of obstructive hydrocephalus with aberrant cell proliferation of cerebral aqueduct. msi2−/− mice survive to adult show poor weight gain, low spontaneous movement and temperature insensitivity, with the malformation of pns. msi1−/− msi2−/− mice die in a few hours after birth with severe defects in the brain, including malformation of hippocampal dentate gyrus. in vitro and in vivo analysis show msi family proteins control the expression of several downstream target molecules post-transcriptionally, and play important roles cooperatively in mammalian cns and pns development. youhei koshimizu 1 , satoshi yamagoe 2 , kazuo suzuki 2 , michiko ohtomi 1 1 department of biomolecular science, graduate school of science, toho university, chiba, japan; 2 department of bioactive molecules, national institute of infections diseases, tokyo, japan we have previously suggested that lect2 (leukocyte cell-derived chemotaxin 2) is expressed in neurons and may play a role in the regulation of both dendritic extension and morphology of astrocytes in the mouse brain. however, the mechanisms of these regulations have not yet been clarified. in this study, we carried out multi-immunostaining against lect2 and tau or map2 in neuronal cell cultures and in tissues of adult mouse brain to examine the functional sites of lect2 in neurons. at the early stages in cultured neurons, lect2 was remarkably localized in the vicinity of membrane of soma, and was detected around root and end of neurites. in tissues of adult mouse brain, lect2 was localized to soma, axons and dendrites in neurons. these results suggest the possibility that lect2 influences the extension of dendrites and axons from the early stage of neuritic development. furthermore, it is thought that lect2 may regulate the dendritic and axonal morphology in mature neurons. mitsuru noda 1,2 , takahiro moriya 1 , hideyuki terazono 1 , suguru kudo 5 , masahiro yamaguchi 3 , kenji yasuda 4 , izumi nagata 2 , kazuyuki shinohara 1 1 div. neurobiol. & behav., nagasaki univ. grad. sch. biomed. sci., nagasaki, japan; 2 dpt. neurosurg. radiol. nagasaki univ. nagasaki, japan; 3 dpt. life sci., grad. sch. arts & sci., univ. tokyo, tokyo, japan; 4 dpt. physiol., grad. sch. med., univ. tokyo, tokyo, japan; 5 special division for human life technology, national institute of advanced industrial science and technology (aist), japan neural stem cells (nscs) are defined as self-renewing, multipotent progenitor cells that give rise to neurons, astrocytes and oligodendrocytes. using single-cell-based on-chip cell-cultivation system, we analyzed the process of neural differentiation and examined the effects of bdnf. the individual nscs from e12.5 nestin-promoter gfp transgenic mice were placed into 32 pairs of agar microchambers connected by microchannels and were differentiated in the presence or absence of bdnf. we observed that a part of nscs exhibited neuron-like morphology and extended some neurites. the application of bdnf increased the rate of neurite outgrowth. thus we addressed the process of neural differentiation in a single-cell-based level and demonstrated the effect of bdnf. ayumi kyuka, ryoichi yoshimura, yasuhisa endo department of applied biology, kyoto institute of technology, kyoto, japan in order to investigate the relationship between neurite outgrowth and target cells, ng108 cells were co-cultured with vascular smooth muscle cells (sm-3). sm-3 cells promoted the neurite outgrowth, but repelled the contact of their growth cones. the dna microarray analysis indicated that neuropilin-1 mrna was specifically up regulated in ng108 cells. but the western blotting analysis did not show the significant increase of neuropilin-1. sema3a, a candidate of ligands for neuropilin-1, was found in ng108 cells rather than in sm-3 cells. the immunocytochemical analysis revealed that neuropilin-1 was increased in the surface of ng108 cells co-cultured with sm-3 cells or cultured with the conditioned medium of sm-3 cells. these results suggest that neuropilin-1 may be transferred from cytoplasmic pool to surface of ng108 cells by some soluble factors released by sm-3 cells. we will discuss about kinetics of neuropilin and chemorepellents. yoshikuni edagawa 1 , j. nakanishi 2 , h. hamada 1 , y. yokomachi 1 , k. yamaguchi 3 , n. takeda 1 1 inst. biomed. eng., waseda univ., tokyo, japan; 2 riken, japan; 3 kanagawa univ., japan we show a novel method for controlling neurite outgrowth with a photochemical micropatterning technology, which is based on the photoreactive molecule switching its character by irradiation of uv light. this molecule formed a self-assembled monolayer on a glass coverslip, which was used for cell-culture surface. for the neurite outgrowth, pc12 cell was recruited as a model of neuron. by irradiating uv light at small spots on the cell-culture surface, single cells were specifically attached to the corresponding spots with intermediated ecm. to draw narrow paths for neurite outgrowth, new regions were irradiated in adjacent to the cell bodies attached on the spots. ngf stimulated each pc12 cell to extend a neurite along the narrow path: the elongation required both photo-activated path and ngf. the successive irradiation in front of developing neurite achieved bending or branching of the neurite. these results indicate that the timing and direction of neurite outgrowth can be controlled with this functional culture system. tetsuhiro kakimoto, hironori katoh, manabu negishi lab. of molecular neurobiology, grad. sch. of biostudies, kyoto univ., kyoto, japan n-wasp is important for actin polymerization and is strongly expressed in brain. toca-1 was shown to be required for cdc42 to activate n-wasp in vitro, although its physiological role is unknown. here we studied neural function of toca-1. toca-1 is strongly expressed in developing brain. knockdown of toca-1 in pc12 cells significantly enhances neurite elongation. consistently, overexpression of toca-1 suppresses neurite elongation through its amino terminus including the f-bar/efc domain, which induces plasma membrane invagination. in addition, knockdown of n-wasp also enhances neurite elongation in pc12 cells, in clear contrast to the previous report using dominant negative mutants. on the other hand, knockdown of toca-1 in rat hippocampal neurons enhances axon branching a little but not axon elongation, while knockdown of n-wasp enhances both axon elongation and branching. these results suggest that a vesicle trafficking regulating protein toca-1 regulates different aspects of neuronal morphology from n-wasp. research funds: kakenhi (17079003) ps2p-e067 pragmin, a novel effector of rnd2 gtpase, stimulates rhoa activity and regulates neurite outgrowth hironori katoh, hiroko tanaka, manabu negishi kyoto university, japan the rho family small gtpase rnd2 is specifically expressed in brain. although rnd1 and rnd3 display an antagonistic action for rhoa, signaling pathways of rnd2 are not well known. here we have performed a yeast two-hybrid screen using rnd2 as bait and identified a novel rnd2-effector protein, pragmin (pragma of rnd2). pragmin is predominantly expressed in neurons, including cortical and hippocampal neurons. in in vitro and in vivo binding assays, pragmin specifically bound to rnd2 among the rho family gtpases in a gtp-dependent manner. rnd2-bound pragmin significantly stimulated rhoa activity and induced cell contraction through rhoa-and rho-kinase-dependent pathway in hela cells. in pc12 cells, expression of pragmin inhibited the ngf-induced neurite outgrowth in response to rnd2, and knockdown of pragmin by a pragmin-specific small interfering rna enhanced the neurite elongation. therefore, these results suggest that rnd2 controls neurite outgrowth by regulating rhoa activity through pragmin. ps2p-e068 regulation of growth cone motility by collapsin response mediator protein-2 (crmp-2) masumi iketani 1 , yanai shigeki 1 , fumio nakamura 1 , yoshio goshima 1,2 , kohtaro takei 1,2 1 dept. of mol. pharmacol. & neurobiol., yokohama city univ. grad. sch. of med., yokohama, japan; 2 crest, japan sci. & tech. agency, kawaguchi, japan nerve growth cone located at a distal tip of a neurite is known to be the site governing axonal growth and guidance. collapsin response mediator protein-2 (crmp-2) is enriched in the distal part of axon and participated in axonal formation and growth as well as growth cone collapse by semaphorin signaling. the local functions of crmp-2 within growth cones, however, remains unclear. chromophoreassisted laser inactivation (cali) that can inactivate selectively a target protein with high spatial and temporal resolutions. acute localized loss of function of crmp-2 in the entire growth cone region resulted in temporal growth arrest of neurite in chick dorsal ganglion neurons. asymmetric inactivation of crmp-2 in a half region of growth cone caused growth cones to turn temporally toward the inactivated region. these findings suggest that a spatial distribution of crmp-2 activity within the growth cone can regulate growth cone motility and direct neurite outgrowth. research funds: crest, jst ps2p-e069 eph receptors are negatively controlled by protein tyrosine phosphatase receptor type o takafumi shintani 1,2 , masaru ihara 1,2 , hiraki sakuta 1,2 , hiroo takahashi 1,2 , ikuko watakabe 1 , masaharu noda 1,2 1 division of molecular neurobiology, national institute for basic biology, okazaki, japan; 2 crest, jst, kawaguchi, japan eph receptors are involved in numerous events such as cell movements, axonal guidance, the formation of synapses, and the maintenance of synaptic plasticity. eph receptors are activated by autophosphorylation of tyrosine residues upon the binding of their ligands, ephrins, however, the protein tyrosine phosphatases (ptps) responsible for the negative regulation of eph receptors have not been elucidated. here, we identified protein tyrosine phosphatase receptor type o (ptpro) as a specific ptp that efficiently dephosphorylates both epha and ephb receptors as substrates. using the retinotectal projection system, we show that ptpro controls the sensitivity of retinal axons to ephrins, and thereby plays a crucial role in the establishment of topographic projections. ps2p-e070 neogenin acts as a displacement factor that releases rho from rho-gdi katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan repulsive gudiance molecule (rgma) is a potent inhibitor of axon regeneration in the adult central nervous system. rgma inhibits mammalian cns neurite outgrowth by a mechanism dependent on activation of the rho/rho-kinase pathway. here we show that direct interaction of the rho gdp dissociation inhibitor (rho-gdi) with neogenin which is a receptor of rgma initiates the activation of rhoa, and this interaction between neogenin and rho-gdi is strengthened by rgma. we also found that neogenin facilitates the release of prenylated rhoa from rho-gdi. thus, understanding of the molecular mechanisms of rgma may be useful for developing methods to overcome the inhibitory signals. nobuyuki fukushima, masumi nakashima, ryou tokunaga, ryutaro moriyama department of life science, kinki university, higashiosaka, japan lysophosphatidic acid is an extracellular signaling lipid that regulates neurite morphology in neuronal cells through g protein-coupled lpa receptors, including lpa 1 and lpa 2 . here we examine how lpa 3 , third lpa receptor subtype, is involved in neuronal development by using pheochromocytoma 12 (pc12) cells. pc12 cells expressed low levels of endogenous lpa 3 mrna. when pc12 cells were infected with retrovirus expressing lpa 3 and treated with nerve growth factor under serum-free conditions for 3 days, neurite formation was observed in 55% of total infected cells, which was similar to that in control virus-infected pc12 cells. however, the mean length of neurites was increased in pc12 cells expressing lpa3, compared with that in control pc12 cells. interestingly, multiple neurite sprouts were frequently observed in lpa-treated lpa 3 -expressing cells. indeed, the numbers of neurites and neurite branching points per cell were increased in these cells. these results indicated that lpa 3 was involved in neuritogenesis and branch formation. masao sakurai 1 , tomoko aoki 1 , kyoko ishikawa 1 , shingo yoshikawa 2 , john b. thomas 2 , chihiro hama 1 1 cdb, riken, hyogo, japan; 2 salk institute, usa in drosophila, odor information is represented as a combination of activated glomeruli in the antennal lobe (al). each glomerulus is formed by specific synaptic connections between olfactory receptor neurons (orns) and projection neurons (pns). in an attempt to address the question of how these glomeruli are stereotypically organized during development, we identified wnt5 as a regulatory factor for glomerular patterning. in the wnt5 mutant, several glomeruli were shifted to ectopic positions, and this phenotype was partially rescued by wnt5 expression in orns. previous studies in embryos have shown that wnt5 repels the axons expressing derailed (drl). in the als of drl mutants, however, some glomeruli were located at incorrect positions, with the pattern different from that in the wnt5 mutant. this and other genetic data suggest that drl is an antagonist for wnt5 signaling. we propose that wnt5 signal transmitted from orns to pns provide one of mechanisms for glomerular patterning in drosophila. ps2p-e073 a homeodomain transcription factor, homothorax, controls precise dendritic and axonal targeting of the antennal lobe projection neurons in the drosophila brain mai ando, yoko totani, katsuo furukubo-tokunaga grad. sch. life envir. sci., univ. tsukuba, japan the precise neuronal connectivity in the nervous system depends on specific axonal and dendritic targeting of individual neurons. temporal and spatial regulation of gene expression by transcription factors is though to play seminal functions in determining wiring specificity. we are interested in the identification of the genes that control specification and connectivity of antennal lobe projection neurons, which convey olfactory information from the primary olfactory center to the higher order structures such as mushroom bodies and lateral horns. here, we show that homothorax (hth), a homeodomain protein, is expressed in most of the projection neurons. marcm mosaic analyses showed profound targeting defects in both dendritic and axonal projection patterns. currently, we are looking for genes that are either downstream or work cooperatively with hth. based on these data, we will discuss the functional roles of hth in the specification of antennal lobe projection neurons. ps2p-e074 differential microarray analysis of mushroom body transcripts using chemical ablation ayako ino 1 , masatomo kobayasi 1 , taiki nakajima 1 , lydia michaut 2 , indrayani ghangrekar 1 , kuniaki takahashi 3 , ryu ueda 3 , kaoru saigo 4 , walter j. gehring 2 , katsuo furukubo-tokunaga 1 1 grad. sch. life envir. sci., univ. tsukuba, japan; 2 biozentrum, univ. of basel, switzerland; 3 genet. strains res. ctr., natl. inst. genet.; 4 dept. biophys. biochem. grad. sch. sci., univ. tokyo, japan mushroom bodies (mbs) are the centers for olfactory associative learning in the drosophila brain. we have surveyed mb transcripts using a drosophila whole-genome microarray and identified 1,465 genes that exhibited reduced expression levels with pharmacological mb ablation. among the identified genes, we have further selected 102 genes that exhibited a most consistent reduction of expression levels in the ablated brains. in situ hybridization analyses confirmed preferential mb expression patterns for the majority of the identified genes. moreover, we also demonstrate that rnai of many of the identified genes causes mild to strong mb defects. these results provide novel and genome-wide insights into the repertoire of the genes that control differentiation and function of the mb neurons in brain development and plasticity. mikiko inaki 1 , yoshie suzuki 1 , hiroyuki aburatani 2 , akinao nose 1 1 dept. phys., univ. tokyo, tokyo, japan; 2 rcast, univ. tokyo, tokyo, japan in drosophila neuromuscular junction, the axons of individual motoneurons precisely recognize and project to specific muscle cells. to understand the molecular logic of target specificity, we tried to identify systematically target recognition molecules expressed on individual muscles. we focused on two neighboring muscles, 12 and 13, which are innervated by distinct motoneurons, and searched for genes that are differentially expressed between them by using wholegenome microarrays. by comparing gene expression data from the two muscles, we identified ∼170 genes with more than two-fold difference in expression level. for 26 candidate genes out of 33 (78%), the differential expression was confirmed by in situ hybridization and/or quantitative pcr. we focused our functional analyses on those encoding transmembrane or secreted proteins, with confirmed differential expression pattern. ectopic expression and/or knockdown of some of them altered the target specificity of muscles 12 or 13, implicating them in neuronal target recognition. makoto aoki, hiroshi segawa, mayumi naito, hitoshi okamoto laboratory for developmental gene regulation, brain science institute, riken, saitama, japan the sensory neurons have two axons, the central and peripheral axon. previously, we have demonstrated that peripheral axons of sensory neurons are completely abolished in the zebrafish embryos overexpressing the lim domains of islet-2. to find the molecules which regulate the formation of peripheral axons of sensory neurons in the islet-2 signaling cascade, we have created cdna library from trigeminal sensory neurons of zebrafish larvae. most of clones were known genes which are implicated in signal transduction, transcription and cytoskeltal formation, but several clones show no similarity with any known genes. most of them show cns or sensory neuron-specific expression patterns and the expression level of these genes were affected by the overexpression of the lim domains of islet-2. loss of function and gain of function studies of these clones, whose function are unknown, showed that these clones might be involved in the development of peripheral axons or the formation of neural circuitry. hiroshi yamamoto, kiyokazu agata development of biophysics, graduate school of science, kyoto university, kyoto, japan highly conserved netrins are known as bi-functional guidance molecules, attracting some axons and repelling others. they act through receptors of the deleted in colorecal cancer (dcc) and unc5 receptor families. freshwater planarians can proliferate by fission and decapitation, the anterior piece regenerates a tail, and the posterior piece regenerates a head. the planarian central nerve system (cns) can be used as a model for studying neural regeneration and understanding how we can rebuild nervous system after injury. we have identified eight netrin related genes from planarians, dugesiajaponica, including four different netrin homologues and three unc5 homologuesand one dcc homologue. we analyzed expression patterns of eight genes in both intact and regenerating planarians, and then conducted rnai experiments to investigating their functions in the process of regeneration of visual neurons. here we will summarize these results and speculate their molecular actions during eye regeneration in planarians. ps2p-e078 analysis of the significance and mechanism of neurite elimination using c. elegans as a model yu hayashi 1 , takaaki hirotsu 2 , eriko kage 1 , hideaki takeuchi 1 , hirofumi kunitomo 3 , yuichi iino 3 , takeo kubo 1 1 dept. biol. sci., grad. sch. sci., univ. tokyo, tokyo, japan; 2 dept. biol., fac. sci., kyushu univ. grad. sch., japan; 3 mol. genet. res. lab., univ. tokyo, japan during brain development, a large amount of neural connections once formed undergo elimination, the mechanism and physiological significance of which are poorly understood. we previously showed that elimination of neurites occurs in the nematode c. elegans and that a novel transcription factor mbr-1 is involved in this process. in the present study, we attempted to clarify the significance of this phenomenon. the mbr-1 mutant is defective in odor adaptation, and we therefore hypothesized that neurite elimination is critical for maturation of the olfactory circuit. by executing cell-specific rescue experiments, we suggest that mbr-1 functions in a set of interneurons involved in olfactory processing. we are now examining whether neurite elimination occurs in these neurons using gfp reporters. we expect that our research provides a useful model for genetic dissection of neurite elimination. taro asakura 1 , ken-ichi ogura 1 , yoshio goshima 1,2 1 dept. of mol. pharmacol. and neurobiol., yokohama city univ., grad. sch. of med., yokohama, japan; 2 crest, japan sci. and tech. agency, kawaguchi, japan netrin is an evolutionary conserved axon guidance molecule. in c. elegans, netrin/unc-6 is secreted from ventral cells, and attracts some axons ventrally and repels dorsally. one of the neurons guided by netrin/unc-6 is the hsn neuron. the hsn neuron of c. elegans has interesting axon guidance. netrin/unc-6 is required for the first ventral growth of the hsn neurons. however, what molecules participate in the axon guidance is largely unknown. in this study, we found that the vulval precursor cells strongly expressed netrin/unc-6 during the axonal guidance of the hsn neurons. silencing of netrin/unc-6 only at the vulval precursor cells resulted in abnormal axon guidance of hsn. in addition, expression of netrin/unc-6 only at the vulval precursor cells in unc-6 null mutants partially restored the hsn guidance defects. these results suggest that netrin/unc-6 expressed in vulval precursor cells plays essential roles on axon guidance of the hsn neuron. tomomi sato, takanori hamaoka, hidenori aizawa, hitoshi okamoto brain science institute, riken, saitama, japan the optic tectum is a visual center in vertebrates. it receives topographically ordered visual input from the retina in the superficial layers and then sends motor outputs from the deeper layers to the premotor reticulospinal system in the hindbrain. although it is well known how the retinal axons project to the tectum, it has not yet been well understood how the tectal axons project to the hindbrain. here, we established a stable transgenic line tg(brn3a-hsp70:gfp) in zebrafish to visualize the retinotectal and the tectobulbar projections. to explore the question, we developed an efficient single-neuron labeling system in combination with cre/loxp and gal4/uas systems. using the system, we found that the tectum projected to each hindbrain segment with a two-dimensionally ordered pattern. in addition, we showed that ephrinb2a was involved in the patterned projection from the posterior tectum to the hindbrain segment rhombomere 2. these results suggest a neuroanatomical and a molecular basis for the motor map in the tectum. research funds: grant-in-aid for young scientists (b) 16700296 masahiko taniguchi 1 , yoshinori mikami 2 , tomoyuki masuda 3 , tomoyuki yoshida 2 , naoto matsuda 2 , masayoshi mishina 2 , takao shimizu 4 1 department of biochemistry, cancer research institute, sapporo medical university, sapporo, japan; 2 department of molecular neurobiology and pharmacology, graduate school of medicine, university of tokyo, japan; 3 department of anatomy, fukushima medical university, japan; 4 dapartment of biochemistry and molecular biology, faculty of medicine, university of tokyo, japan semaphorin gene family contains a large number of secreted or transmembrane proteins, and some of them function as the repulsive and attractive cues of axon guidance during development. here we report the identification of zebrafish semaphorin 6d (sema6d). the orf of sema6d is 3054 bp (1018 aa). zebrafish sema6d protein showed a 70.8% identity with mouse sema6d protein. to clarify the expression pattern of sema6d, in situ hybridization was performed. in the embryos, sema6d is expressed in the lens and hindbrain. we also found that sema6d has the repulsive activity. these results indicate that sema6d repels specific types of the axons and functions in the nervous system development. research funds: kakenhi on priority areas from the mext (17023013) mayumi yamada 1 , shohei maekawa 2 , seiji miyata 1 1 department of applied biology, kyoto institute of technology, kyoto, japan; 2 division of bioscience, graduate school of science and technology, kobe university, kobe, japan obcam belongs to the immunoglobulin superfamily cell adhesion molecule (cam) and was synapse-specific cam in cortical and hippocampal neurons in vivo. however, it is uncertain how obcam is involved in synaptic functions. in this study, we showed that obcam was highly concentrated on spines of cultured mature neurons. the inhibition of obcam function with its specific antibody resulted in a decrease in the number of presynaptic terminals and postsynaptic spines. the suppression of obcam mrna expression with its specific antisense oligonucleotide also resulted in impairment of synapse formation. in contrast, overexpression of obcam augmented the formation of synapses. moreover, obcam was internalized via a raft-dependent pathway and the internalization was promoted by increased neuronal activity. these results suggest that obcam, a spine-specific cam, is a critical modulator of synaptogenesis and its surface expression is dynamically regulated in response to neuronal activity. ps2p-f083 semaphorin 4d inhibits ␤1 integrin activity through the r-rasgap activity of plexin-b1 izumi oinuma, hironori katoh, manabu negishi lab. mol. neurobiol., grad. sch. biost., kyoto univ., kyoto, japan plexins are cell surface receptors for semaphorins and regulate cell migration in many cell types. we have recently reported that the semaphorin 4d (sema4d) receptor, plexin-b1 functions as a gap for r-ras, a member of ras family gtpases implicated in regulation of integrin activity and cell migration. here we characterized the role of r-ras downstream of sema4d/plexin-b1 in cell migration. activation of plexin-b1 by sema4d suppressed the ecm-dependent r-ras activation, r-ras-mediated pi3-k activation, and ␤1 integrin activation through its r-ras gap domain, leading to inhibition of cell migration. in addition, inactivation of r-ras by overexpression of the r-ras-specific gap or knockdown of r-ras by rna interference was sufficient for suppressing ␤1 integrin activation and cell migration in response to the ecm stimulation. thus, we conclude that r-ras activity is critical for ecm-mediated ␤1 integrin activation and cell migration, and that inactivation of r-ras by sema4d/plexin-b1-mediated r-ras gap activity control cell migration by modulating the activity of ␤1 integrins. shun hamada 1 , emi fukuda 2,3 , shota katori 2,3 , takeshi yagi 2,3 1 dept. nutrition health sci., fukuoka women's univ., fukuoka, japan; 2 grad. sch. frontier biosci., osaka univ., osaka japan, 3 crest, jst, japan cnr/protocadherin (pcdh)␣ is a subfamily of the clustered pcdhs that comprise >50 cadherin-related molecules and are encoded by three gene clusters (␣, ␤ and ␥). the molecular features and synaptic localization of the clustered pcdhs have raised the possibility that they are synaptic recognition molecules. we have demonstrated that overexpressed pcdh␣ family proteins alone in several cell lines are rarely transported into the plasma membrane. furthermore, we found that a stretch of about fifty amino acids located at the c-terminus of pcdh␣s interfered the trafficking to the cell surface. in the present study, we compared the transport properties of a series of the cytoplasmic region truncation mutants and found that truncation mutants lacking 34 or more c-terminal residues were detectable at the cellular surface suggesting a role for lysine-rich motif in the c-terminus of pcdh␣s in the intracellular retention. mdga1 is a novel cell surface glycoprotein similar to ig-containing cell adhesion molecules (igcams) with functions in migration and process outgrowth. mdga1 is expressed by layer 2/3 neurons throughout the neocortex at p7 mice, but is absent in adults. between e15.5 and late p0, stages that span the generation and radial migration of layer 2/3 neurons, mdga is expressed in patterns consistent with its expression by migrating layer 2/3 neurons, suggesting a role for mdga1 in controlling their migration and settling in the superficial cortical plate. we performed loss-of-function studies using rna interference (rnai) with in utero electroporation into the lateral ventricle at e15.5 to transfect progenitors of superficial layer neurons. we found that an rnai suppressing mdga1 protein blocks proper migration of superficial layer neurons to the superficial cortical layer. we conclude that mdga1 acts cell autonomously to control the migration of superficial layer cortical neurons. in various pathological conditions, activated microglia mediate immune responses to injured cns neurons. however, it is not clear whether and how activated microglia affect neurons via direct contacts. this study aimed at examining whether direct contacts between microglia and hippocampal neurons increase following cns injury and whether telencephalin (tlcn), a dendrite specific adhesion molecule, which potentially binds to immune cells, mediates the direct contact. hippocampal neurons were damaged by local injection of excitotoxin, kainic acid (ka). compared to control animals, ka-injected mice showed higher density of contacts between activated microglia and dendrites of ca1 pyramidal neurons. contacts with longer interface appeared in ka-injected mice. these results suggest the importance of direct contacts for the immune response of microglia to injured neurons. similar contact formation was also observed in tlcn-deficient mice, indicating that the direct contacts are mediated by other molecules than tlcn. kilon is belonging to immunoglobulin superfamily of cell adhesion molecules and contains three igg-like domains. western analysis revealed that the expression levels of kilon is low at early neuronal culture and increased with progress of culture days. immunocytochemical observation showed that kilon was localized at elongating axon and growth cones but not at dendrites on 7 days in vitro (div), while kilon was observed at synapses, mainly at presynaptic terminals on 14 div. similar tendency was observed in kilon immunohistochemistry of brain sections in vivo. kilon was observed at axonal fibers of the cerebral cortex on postnatal day 2, but it was seen at synapses in adult brains. these results suggest that kilon is axonal cell adhesion molecule to control axonal guidance and/or extension. ps2p-f088 analysis of mice that show abnormal expression of neuroglycan c, a central nervous system-specific transmembrane proteoglycan sachiko aono 1 , yoshiyuki kuroda 1 , fumiko matsui 1 , yoshihito tokita 1 , keiko nakanishi 1 , michiru ida 1 , masahito ikawa 2 , masaru okabe 2 , katsuhiko ono 3 , atsuhiko oohira 1 1 institute for developmental research, aichi human service ctr., kasugai, japan; 2 research institute for microbial diseases, osaka university, suita, japan; 3 national institute for physiological sciences, okazaki, japan neuroglycan c (ngc) is a membrane-spanning chondroitin sulfate proteoglycan that is exclusively expressed in the central nervous system. to study the role of ngc in the brain, we produced two strains of ngc-mutant mouse by gene-targeting; a mouse strain with no ngcexpression and a strain with low expression (knockdown mice). both mice were viable and fertile. they did not show obvious abnormalities in gross brain anatomy. to examine their behavioral phenotype precisely, the ngc-knockdown mice were subjected to several kinds of behavioral tests sequentially. they displayed obvious abnormalities in morris water maze and passive avoidance tests, suggesting that ngc is involved in learning and memory. we are now carrying out the same experiments using the ngc-knockout mice. research funds: kakenhi (17500246) ps2p-f089 phosphorylation of extracellular signal-regulated kinase in aged rats with acute face inflammation koichi iwata 1 , tatsuhisa watanabe 2 , ikuko suzuki 1 , junichi kitagawa 1 , akiko ogawa 3 , kenro kanda 4 , kazunao kuramoto 5 1 dept. of physiol., sch. of dent., nihon univ., tokyo, japan; 2 dept. of oral and maxillofacial surgery, sch. of dent., nihon univ., tokyo, japan; 3 dept. of oral diagnosis, sch. of dent, nihon univ., tokyo, japan; 4 shinjuku vocational school of acupuncture, moxibustion and judo therapy, tokyo, japan; 5 division of research animal center, tokyo metropolitan institute of gerontology, tokyo, japan the capsaicin-induced perk expression was studied in the aged rats (30-34 months) following noxious face stimulation. a large number of perk-li cells were expressed in the superficial laminae of the trigminal spinal nucleus in adult and aged rats following subcutaneous capsainsin injection into the whisker pad region. the larger number of perk-li cells was expressed in adult rats than aged rats following intravenous administration of naloxone before capsaicin treatment. the present results suggest that the descending modulation system was impaired in the aged rats, resulting in the abnormal pain sensation advancing age. hirokazu katsura 1 , koichi obata 1 , masafumi sakagami 2 , koichi noguchi 1 1 department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; 2 department of otorhinolaryngology, hyogo college of medicine, hyogo, japan recent studies demonstrated that the activation of extracellular signal-regulated protein kinase (erk) 1/2 and p38 mitogen-activated protein kinase (mapk) in dorsal root ganglion (drg) neurons contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk5 (also known as big mapk 1 or bmk1) is activated in the drg and participate in pain-related behaviors in the complete freund's adjuvant (cfa) model. peripheral inflammation induced an increase in the phosphorylation of erk5, mainly in tyrosine kinase a-containing small-to-medium-diameter drg neurons at days 1 and 3 after cfa injection. furthermore, time course of phosphorylated-erk5 level in the drg matched the emergence of cfa-induced pain hypersensitivity. our data suggest that activation of erk5 in drg neurons may contribute to the development of inflammatory pain. ps2p-f091 activation of erk5 in drg neurons contributes to acute pain toshiyuki mizushima 1,2 , koichi obata 1 , takashi mashimo 2 , koichi noguchi 1 1 department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; 2 department of anesthesiology, osaka univ. recently, we have reported that phosphorylation of extracellular signal-regulated protein kinase (erk) 1/2 and p38 mitogen-activated protein kinase (mapk) occurred in primary sensory neurons in response to natural noxious stimulation of the peripheral tissue, i.e., activity-dependent activation of erk and p38 in dorsal root ganglion (drg) neurons. however, there has been no study examining erk5 (also known as big mapk 1 or bmk1) activation in drg neurons after noxious stimulation of normal tissue. here, we report intensity-dependent erk5 phosphorylation in drg neurons by painful stimulation. noxious stimulation induced phosphorylated-erk5 in small-to-medium diameter sensory neurons with a peak at 2 min after stimulation. furthermore, we found a stimulus intensitydependent increase in the number of activated neurons. our data suggest that activation of erk5 in drg neurons may contribute to acute pain induced by noxious stimulation. koichi obata, koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, japan there is compelling evidence indicating that the activation of extracellular signal-regulated protein kinase (erk) 1/2 and p38 mitogenactivated protein kinase (mapk) in the dorsal root ganglion (drg) and spinal cord contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk5 (also known as big mapk 1 or bmk1) is activated in the drg and spinal cord and participate in pain-related behaviors in the l5 spinal nerve ligation (snl) model. l5 snl induced an increase in the phosphorylation of erk5 not only in the injured l5 drg, but also in the spared l4 drg at day 7 after surgery. furthermore, l5 snl induced a striking increase in erk5 phosphorylation in glial cells in the ipsilateral dorsal horn. our data suggest that activation of erk5 in the drg and spinal cord may contribute to the development of neuropathic pain. atsushi sakai 1 , minoru asada 1 , naoki seno 2 , hidenori suzuki 1 1 department of pharmacology, nippon medical school, tokyo, japan; 2 pharmaceutical research center, kyowa hakko kogyo co., shizuoka, japan glial cell line-derived neurotrophic factor (gdnf) has been known to alleviate the neuropathic pain. however, the mechanisms of gdnfinduced analgesia remain almost unclear. gdnf binds to gfr␣-1, which forms receptor complex and signals intracellularly through ret. recently, neural cell adhesion molecule (ncam) has been found to be an alternative signal-transducing receptor for gdnf. here, we report that ncam is involved in gdnf-induced analgesia in a rat model of the neuropathic pain. ncam mrna expression was decreased in the ipsilateral dorsal horn of the spinal cord after the nerve injury, but gdnf treatment returned its expression to the normal level. treatment with ncam antisense oligodeoxynucleotide blocked the analgesic effect of gdnf without affecting ret phosphorylation. these results suggest that activation of ncam signaling may provide a new strategy to relieve intractable chronic pain. ps2p-f094 interleukin-1␤ enhanced the excitability of trigeminal root ganglion neurons via activation of satellite glia following inflammation mamoru takeda 1 , jun kadoi 1 , msanori nasu 2 , masayuki takahashi 1 , shigeji matsumoto 1 1 dep. physiol and 2 res. cent. for odont. nippon dent. univ., japan the present study was investigated whether activation of satellite glial cells modulates the excitability of trigeminal root ganglion (trg) neuronal activity via the il-1␤ paracrine mechanism following inflammation. two days after cfa into the whisker pad area, the mean number of trg neurons that were encircled by glial fibrillary acidic protein and il-1␤-immunoreative satellite cells were significantly increased compared with those in the control. fg labeling was used to identify the trg neurons innervating the site of inflammation. in the fg-labeled small trg neurons, the occurrence of il-1␤ induced depolarization in inflamed rats was larger than that in control rats. il-1␤ application significantly increased the firing rate evoked by depolarizing pulses in the inflamed neurons compared with the control neurons. these results suggest that activation of trg satellite glial cells modulates the excitability of trg neuronal activity via the il-1␤ paracrine mechanism following peripheral inflammation. junichi kitagawa 1 , mamoru takeda 2 , jun kadoi 2 , yoshiyuki tsuboi 1 , shigeji matsumoto 2 , koichi iwata 1 1 dept. of physiol., sch. of dent., nihon univ., tokyo, japan; 2 dept. of physiol, sch. of dent. at tokyo, japan, nippon dental univ., tokyo, japan the present study was designed to elucidate an involvement of the primary afferent neurons in the trigeminal neuropathic pain using the rats model with chronic constriction nerve injury of the infraorbital nerve (ion-cci). the mechanical escape threshold was significantly lower in ion-cci rats at day 3 after ion treatment and the threshold decrement was lasting more than day 14. single unit activities of ion were recorded from the ion-cci rats. the firing frequency was significantly higher in a␦ fibers in ion-cci rats as compared with naive at day 3-14 after ion-cci. whole cell patch clamp recording was performed from the middle trg neurons. ik and ia currents were significantly smaller and ih current was larger in ion-cci rats than that of naive rats. the present results suggest that ik, ia and ih currents are involved in abnormal firing of trg neurons in the rats with ion-cci, resulting in neuropathic pain in trigeminal region following peripheral nerve injury. hisako urai, munehiro uda, katsuya kami graduate schools of sport and exercise science, osaka university of health and sport sciences, osaka, japan mechanical hyperalgesia of skeletal muscles has been known to occur following intense eccentric contraction such as downhill running (dhr). the present study examined the number of c-fos-positive neurons in spinal dorsal horn to determine peak of mechanical hyperalgesia following dhr in rats. furthermore, we investigated whether glial cells are activated in dorsal horn with excitation of secondary afferent neurons (san). rats performed an intermittent bout of dhr for 90 min. at 6, 12, 24, 48 and 120 h post-dhr, the rats were applied a weight on the right triceps surae muscle. immunohistochemical staining for c-fos and gfap on spinal cords was performed by freefloating abc method. the number of c-fos-positive neurons detected in superficial dorsal horn were increased at 6 h, peaked at 12 h and then decreased. intense gfap immunoreactivities were also detected at 6 and 12 h post-dhr. these results suggest that dhr generates mechanical hyperalgesia by increasing responsiveness of san, and moreover astrocytes may regulate excitability of san. katsuya kami, hisako urai, munehiro uda department of health science, osaka university of health and sport sciences, osaka, japan a production of inflammatory cytokines is increased in injured skeletal muscles. the present study examined relationship between production of inflammatory cytokines in skeletal muscles and fospositive neurons in spinal dorsal horn following downhill running in rats. the rats performed the downhill treadmill running for 90 min at 25 m/min. after the running, rats were applied the weight on the gastrocnemius muscles for 30 min, and then spinal cord and soleus muscles were removed from the rats. productions of il-1beta, il-6 and tnf-alpha in soleus muscles and expression of fos protein in dorsal horn were examined using immunohistochemical approach. at 6 h post-running, number of fos-positive neurons was increased, peaked at 12 h and then decreased to control level at 24 h post-running. vigorous inflammatory reactions with necrotic myofibers in soleus muscles were observed at 2 days post-running. these results indicated that increased numbers of fos-positive neurons in dorsal horn are induced prior to vigorous inflammation of skeletal muscles. shinichi sugiyo 1 , yusuke sakai 2 , aya masawaki 3 , takashi shimoda 2 , masayuki moritani 1 , motohide takemura 1 1 dept. oral anatomy and neurobiology, osaka university grad. sch. of dentistry, osaka, japan; 2 dept. of fixed prosthodontics, osaka university grad. sch. of dentistry, osaka, japan; 3 dept. of dental anesthesiology, osaka university grad. sch. of dentistry, osaka, japan diabetes mellitus is among the most common causes of painful peripheral neuropathy, worldwide. we examined if there exist the diabetic rat (dm)-specific difference in nociceptive behavioral and c-fos immunoreactivity (ir) by formalin test. injection of formalin into the upper lip 2 weeks before streptozotocin injection induced biphasic specific pain related behavior (prb) for 90 min. first phase was greater in dm than in the control rat (ctrl). in dm, second phase was much greater than ctrl. c-fos ir in the trigeminal caudal nucleus was also greater in dm than in ctrl. these results indicate that dm induced greater prb and c-fos expression following formalin injection into the rat upper lip. yasuko kozaki, satoshi hurune, fukushi kambe, hisao seo, kazue mizumura res. inst. environ. med., nagoya university, nagoya, japan we have reported that prostaglandin ep3 receptor (ep3r) activation attenuates the desensitization of bradykinin (bk)-induced increase of intracellular calcium ([ca 2+ ] i ) in a ptx-sensitive manner in cho cells expressing canine ep3r and mouse bk b2 receptor (b2r). in this study, we examined the involvement of protein kinase a (pka) in the desensitization of the bk response. when bk (1 nm) was applied twice with a 6-min interval to the cells expressing b2r, the second [ca 2+ ] i increase by bk was markedly attenuated. however, the pretreatment with a specific inhibitor of pka, h-89 (1 m) restored the second response. to further confirm camp increase by bk, the expression of a camp responsive reporter gene was examined. bk (10 pm) treatment for 1 h significantly increased the reporter gene expression. it is likely that bk increases the level of intracellular camp, and thus activates pka, resulting in the desensitization of the bk response. these results suggest that the desensitization of bkinduced increase in [ca 2+ ] i was at least in part mediated by pka. ps2p-f100 contribution of peripheral 5ht2a or 5ht3 receptors to fos expression in the trigeminal spinal nucleus (vsp) produced by the masseter muscle injury of rats keiichiro okamoto, akihisa kimura, tomohiro donishi, yasuhiko tamai dept. physiology, wakayama med univ., japan we have recently reported that orofacial nocifensive behavior evoked by the masseter muscle (mm) injury is attenuated by blocking peripheral 5ht2a or 5ht3/r in male rats with tmj inflammation. here we tested if these two 5ht/r subtypes contribute to fos responses in vsp after mm injury. formalin injection into mm produced fos-like immunoreactivity (li) in several areas of vsp and c2. fos-li was distributed mainly in the ventrolateral trigeminal subnucleus interpolaris/caudalis transition (vl-vi/vc) and vc/c2 transition regions. the number of fos-li induced by mm injury was increased in these areas in cfa-evoked tmj-inflamed rats for 7 days compared to naive rats. we tested if local 5ht2a or 5ht3/r antagonist affects fos expression in both groups. the number of fos-li in the vc/c2 but not vl-vi/vc region was reduced when drugs were injected locally prior to formalin injection in tmj-inflamed rats. these data suggest that peripheral 5ht2a and 5ht3/rs play critical roles in mediating mm nociception during tmj inflammation. keiko abe, hidemasa furue, kohei kga, go kato, toshiharu yasaka, akihiro tamae, toshihiko katafuchi, megumu yoshimura department of integrative physiology, graduate school of medical sciences, kyushu university, japan we examined the postsynaptic effects of 5-ht on substantia gelatinosa (sg) neurons in slice preparations of rat spinal cord and their relationship to the morphological features. in ∼60% of sg neurons examined, 5-ht induced an outward current. the outward current was mimicked and suppressed by a 5-ht 1a agonist and 5-ht 1a antagonist, respectively. in ∼10% of sg neurons, 5-ht evoked an inward current which was mimicked by a 5-ht 3 agonist. the outward current was observed mostly in excitatory neurons such as vertical cell, while the inward current was induced in an inhibitory neuron, islet cell. these findings suggest that 5-ht inhibits excitatory neurons and excites inhibitory neurons in the sg through activation of 5-ht 1a and 5-ht 3 receptors, respectively. the reciprocal postsynaptic actions of 5-ht on sg neurons in addition to presynaptic inhibitory effects on primary afferents might play an important role in descending control of nociceptive transmission by 5-ht. we examined effects of levobupivacaine, ropivacaine, bupivacaine and r-bupivacaine on epscs in substantia gelatinosa (sg) neurons of the spinal dorsal horn evoked by dorsal root stimulation, and on action potentials in dorsal root ganglion neurons generated by the dorsal root stimulation. in sg neurons, levobupivacaine reversibly suppressed the amplitude of monosynaptic a␦ and c fiber-evoked epscs. however, a␤ fiber-evoked epscs were slightly inhibited in amplitude. on the other hand, bupivacaine equally suppressed those three fiber-evoked epscs. in drg neurons, ic 50 of bupivacaine and r-bupivacaine were almost equal on a␤, a␦ and c neurons. however, ic 50 of levobupivacaine and ropivacaine on a␦ and c neurons were lower than that on a␤ neurons. the present results suggest that pure s (−) enantiomers especially levobupivacaine effectively inhibits noxious transmission to the spinal dorsal horn by the blockade of ap conduction through a␦ and c fibers. ps2p-g103 nitric oxide-dependent long-term potentiation revealed by real time imaging of nitric oxide production and neuronal excitation in spinal dorsal horn hiroshi ikeda, kei kusudo, kazuyuki murase dept. human & artificial intelligence systems, univ. fukui, fukui, japan no plays an important role in the induction of long-term potentiation (ltp) in spinal dorsal horn, which is believed to underlie hyperalgesia and allodynia. in this study, to elucidate the relationship of no to ltp, we measured the spatiotemporal distribution of no signal with the no-sensitive dye, and neuronal excitation with the voltagesensitive dye, in rat spinal cord slices. in superficial dorsal horn, neuronal excitation evoked by dorsal root stimulation was potentiated for more than 2 h after low-frequency conditioning stimulation (lfs). in the same slices that exhibited ltp, no was produced and distributed in the superficial dorsal horn during lfs. ltp and production of no were inhibited in the presence of no synthase inhibitors and an inhibitor of heme oxygenase, the synthetic enzyme for carbon monoxide (co). research funds: kakenhi to hi (17700306) and km (16500262) and grants from novartis foundation and promotion of science and sumitomo foundation to hi tao liu, tsugumi fujita, akiko koga, masafumi kosugi, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the effect of a pla 2 activator melittin on inhibitory transmission in the substantia gelatinosa (sg; lamina ii of rexed), we applied the blind whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. in about 90% of neurons examined, melittin (1 m) superfused for 3 min gradually increased the frequency and amplitude of spontaneous glycinergic inhibitory postsynaptic currents which were recorded at 0 mv in the presence of bicuculline. this action was visible about 2 min after the beginning of its superfusion and subsided within 8 min after washout. these melittin actions were reduced in extent by a pla 2 inhibitor 4-bromophenacryl bromide, while being unaffected by tetrodotoxin, and also by inhibitors of cyclooxygenase (cox) and lipooxygenase (lox). it is concluded that pla 2 activation pre-and postsynaptically enhances glycinergic transmission in sg neurons, possibly not through metabolites of cox and lox; this action would contribute to a modulation of nociceptive transmission. research funds: kakenhi (17500275) ps2p-g105 presynaptic p2y 1 receptor-mediated enhancement of inhibitory synaptic transmission in the rat spinal dorsal horn terumasa nakatsuka, shugo koga, tsugumi fujita, tao liu, masafumi kosugi, eiichi kumamoto department of physiology, faculty of medicine, saga university, saga, japan using whole-cell patch-clamp recordings, we examined whether the activation of p2y receptors can modulate synaptic transmission in dorsal horn (dh) neurons of adult rat spinal cord slices. bath applied 2-methylthio adp (2mesadp, 30 m), a p2y receptor agonist, did not change excitatory transmission, but clearly increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (ipscs) in about 20% of dh neurons recorded. miniature ipsc in the presence of ttx was increased in frequency by 2mesadp with no change in the amplitude. the 2mesadp-induced increase in miniature ipsc frequency was attenuated in extent by mrs2179 (30 m), a selective p2y 1 receptor antagonist. these results indicate that the activation of presynaptic p2y 1 receptors enhances inhibitory but not excitatory synaptic transmission in a subpopulation of dh neurons. thus, spinal p2y 1 receptors can be involved in an inhibitory effect on pain transmission. research funds: kakenhi (17700370), the japanese health sciences foundation (kh21006) ps2p-g106 presynaptic enhancement by proteinaseactivated receptor-1 agonist peptide of glutamatergic excitatory transmission in rat substantia gelatinosa neurons tsugumi fujita, terumasa nakatsuka, akiko koga, tao liu, masafumi kosugi, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan we have previously reported that proteinase-activated receptor (par)-1 but not par-2 agonist (each 1 m) enhances glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons. the present study examined a detail of the par-1 mediated enhancement by applying the whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. par-1 agonist (sfllrn, 1 m) reversibly increased the frequency of spontaneous epsc without a change in the amplitude and also in holding current at -70 mv. this facilitatory action was resistant to tetrodotoxin, and was not seen in the presence of par-1 antagonist (yfllrnp, 1 m). these results indicate that the activation of par-1s existing in nerve terminals in the sg results in an increase in the spontaneous release of l-glutamate from there. it is suggested that par-1 activation in glutamatergic neuron terminals in the sg may be involved in the modulation of nociceptive transmission from the periphery. research funds: kakenhi (16700340) ps2p-g107 effect of tramadol metabolite m1 on glutamatergic excitatory transmission in rat spinal dorsal horn neurons akiko koga, tsugumi fujita, tao liu, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the antinociceptive effect of tramadol, we examined the effect of m1, which is one of its metabolites, at 1 mm on glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons of an adult rat spinal cord slice by using the whole-cell patchclamp technique. bath-applied m1 reduced the frequency but not amplitude of spontaneous excitatory postsynaptic currents (epscs) at −70 mv. this action was not seen in the presence of a -opioid receptor antagonist ctap (1 m). m1 also reduced the peak amplitudes of epscs which were monosynaptically evoked at −70 mv by stimulating primary-afferent a␦-and/or c-fibers in a spinal cord slice with an attached dorsal root. we conclude that m1 inhibits the quantal release of l-glutamate from nerve terminals in the sg through the activation of -opioid receptors; this action is not distinct in extent between primary-afferent a␦-fiber and c-fiber transmission. this effect of m1 would give a cellular basis for the antinociceptive effect of systemically-administered tramadol. narihito iwashita, natsu koyama department of physiology, shiga university of medical science, otsu, japan in our previous study, subcutaneous injection of glutamate into the human forearm evoked pain and produced skin temperature increase around the injection site. these results suggest peripheral glutamate receptors contribute to nociceptive signaling and neurogenic inflammation. in order to further investigate which subtype of glutamate receptors is involved in neurogenic inflammation, effect of nmda receptor antagonist mk-801 or non-nmda receptor antagonist cnqx was evaluated in hindpaws of pentobarbital-anesthetized rats. attenuation of skin temperature increase induced by simultaneous mk-801 injection with glutamate was larger than that of skin temperature increase induced by simultaneous cnqx injection with glutamate at the same concentration. on the other hand, inhibition of paw edema formation by cnqx was stronger than by mk-801. these data demonstrate that peripheral nmda receptor predominantly contributes to vasodilatation, while peripheral ampa/ka receptor predominantly contributes to increase of vascular permeability in glutamate-induced neurogenic inflammation. ps2p-g109 studies on pain control system (rept. 57): changes in phosphorylation of nr2b-contained nmda receptor in the spinal cord obtained from rats with painful neuropathy following chronic ethanol consumption kan miyoshi, minoru narita, michiko narita, tsutomu suzuki dept. toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan chronic ethanol consumption produces a painful peripheral neuropathy. mechanical hyperalgesia was clearly observed during ethanol consumption and even after ethanol withdrawal in rats, and it lasted for 14 weeks. under these conditions, the immunoreactivities of phosphorylated-ser-1303 nr2b (p-ser-1303 nr2b) subunit and phosphorylated-conventional protein kinase c (p-cpkc) were significantly increased in the spinal cord following chronic ethanol consumption, whereas p-tyr-1472 nr2b subunit immunoreactivity was not changed in this region. the hyperalgesia induced by chronic ethanol consumption was significantly attenuated by repeated i.p. injection of ifenprodil, a selective nr2b-containing nmda receptor antagonist. these findings provide evidence for a substantial role of the phosphorylation of cpkc-dependent nr2b-contained nmda receptor in the development or/and maintenance of ethanoldependent neuropathic pain-like state in rats. ps2p-g110 prolonged depression of nociceptive response in the prefrontal cortex with high frequency stimulation of the amygdala yumi izawa, yoriko kawakami dept. physiol. tokyo women's medical university, tokyo, japan high frequency stimuli (hfs, 100 hz, 20 a, 30 s) delivered to the basolateral nucleus of the amygdala (bl) induced prolonged depression of the nociceptive specific response in the prefrontal cortex (pfc). we examined the receptor mechanism underlying this depression of pfc neuron activity. extracellular neural activities, induced by nociceptive stimulation applied peripherally, were recorded in the rat pfc. inhibitory effects of hfs delivered to the bl on nociceptive responses were blocked by specific antagonists of a metabotropic glutamate receptor (mglur) or nmda receptor microinjected locally into the pfc. dopamine depletion, produced by 6-ohda injected into the substantia nigra, also reduced the inhibitory effects of hfs. the mglur and dopamine receptor mediated prolonged depressions of nociceptive responses were induced by hfs of the amygdala. our results suggest that emotional condition modulates pain sensation. ps2p-g111 the nav1.3 sodium channel pathologically reconfigures the thalamic pain amplifier-generator after spinal cord injury bryan c. hains, stephen g. waxman yale university school of medicine, usa spinal cord injury (sci) induces pain-related phenomena associated with the aberrant expression of nav1.3, a rapidly repriming voltage-gated sodium channel. in this study we hypothesized that, following sci, neurons in the thalamus undergo similar electrophysiological changes linked to nav1.3. four weeks post-sci, nav1.3 protein was upregulated within thalamic neurons, where unit recordings revealed increased spontaneous discharge, afterdischarge, hyperresponsiveness to innocuous and noxious peripheral stimuli, expansion of peripheral rfs, and bursting. these properties persisted after interruption of ascending spinal barrage. lumbar intrathecal administration of specific antisense oligodeoxynucleotides against nav1.3 caused a significant reduction in nav1.3 expression and reversed electrophysiological alterations. these results show, for the first time, a change in sodium channel expression within neurons in the thalamus after injury to the spinal cord, and suggest that these changes contribute to altered processing of somatosensory information after sci. tomoki fukuda 1 , hiroyuki ichikawa 2 , ryuji terayama 2 , tomosada sugimoto 2 1 department of oral maxillofacial rehabilitation, okayama university, okayama, japan; 2 department of oral function and anatomy, okayama university, okayama, japan ib4-sap is a neurotoxin designed for targeting primary nociceptors with ib4 binding sites on the cell surface. however, the exact cell spectrum that is affected by the toxin has not been thoroughly investigated. we, therefore, unilaterally injected ib4-sap (2.5 l of 0.067% solution for each ganglion) directly into the 4th and 5th lumbar (l4 and 5) dorsal root ganglia (drgs). three weeks later, the rats were killed and drg sections were stained for ib4-binding. after counterstain, the cell body size of neurons were measured. ib4-sap reduced the total number of drg neurons in l4 and 5 ganglia combined by 21% (11652 ± 2213 on untreated side versus 9208 ± 1609 on treated side). small neurons (<1200 m 2 ) were reduced by 28% whereas large ones (≥1200 m 2 ) were not affected. ib4-binding neurons were mostly small (≥96%) and were reduced by 46%. the number of small neurons, that were not stained for ib4-binding, increased by 35% (1994 ± 286 versus 2700 ± 600). schuichi koizumi 1 , kaoru nasu-tada 1 , makoto tsuda 2 , emiko kunifusa 1,2 , kazuhide inoue 2 1 div. pharmacol., natl. inst. hlth. sci., tokyo, japan; 2 dept. mol. system pharmacol., grad. sch. pharmaceut. sci., kyushu univ., fukuoka, japan although microglial p2x4 receptor, a key molecule for the mechanical allodynia, is increased after peripheral nerve injury, the molecular mechanisms underlying its upregulation remain unknown. here, we describe the influence of fibronectin on p2x4 receptor expression in microglia. microglia that were cultured on fibronectin-coated dishes showed a marked increase in p2x4 receptor expression. western blot examination of the spinal cord from rat with spinal nerve injury indicated that fibronectin was upregulated on the ipsilateral side. interestingly, intrathecal injection of atp-stimulated microglia revealed that microglia cultured on fibronectin-coated dishes was more effective in the induction of allodynia than microglia cultured on control dishes. taken together, our results suggest that spinal fibronectin is elevated after the peripheral nerve injury and it may be involved in the upregulation of the p2x4 receptor in microglia, leading to neuropathic pain. research funds: mf16, kakenhi (170230570) ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. of biomed. sci., nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root (dr), which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. 2004). however it remained whether lpa has direct actions on myelinated schwann cells (sc). in the present study we examined the direct effects of lpa on dr fibers in ex vivo culture system. scanning electron microscopy (sem) study revealed that lpa caused a swelling and disruption of myelinated fibers at 24 h. in transmission em analysis, the addition of lpa caused a disruption of myelin sheath of a␦-and a␤-fibers. on the other hand, it was found that c-fibers were separated to each other by scs in naive fibers. following the addition of lpa, c-fibers showed direct contacts and some of them were uncovered. all these effects were also observed either with or without dr ganglion. thus, it is suggested that lpa has direct actions on myelinated and unmyelinated scs to cause demyelination of a-fibers and to uncover c-fibers. research funds: kakenhi 17109015 ps2p-g115 lysophosphatidic acid (lpa) down-regulates myelin associated proteins in cultured dorsal root fibers norikazu kiguchi, ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. biomed. sci. nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root, which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. 2004). these treatments also caused a decrease in myelin protein and their gene expression levels. here we report the biochemical evidence underlying this demyelination in cultured fibers. the addition of lpa at 1 mm decreased the protein levels of myelin basic protein (mbp) at 24 h. this action was significantly inhibited by botulinum neurotoxin/c3 (bont/c3). on the other hand, lpa also caused a decrease in gene expression of various myelin proteins, such as mbp, pmp22, mag, p0 in cultured fibers. the maximal decrease was observed all at as early as 3 h after the addition of lpa. bont/c3 and y27632 abolished the lpainduced down-regulation of mbp gene. all these findings suggest that the down-regulation of gene expression of myelin proteins is through rhoa-rock pathway underlying lpa-induced demyelination. neuropathic pain arise from peripheral never injury. the purpose of this study was to explore behavioral characteristics and investigate the involvement of nmda receptors and opioid receptors in the behavioural responses following spared nerve injury (sni). the hind paw withdrawal threshold to cold-and mechano allodynia and heatyperalgesia were tested at 0 and 3, 7, 14, 21, 28 days after operation. pre-emptive co-administration of mk-801 and morphine were tested. sni produces mechanical and cold allodynia and heat hyperalgesia. co-injection of morphine and mk-801 decreased cold-and mechanoallodynia, but had slightly effect on heat-hyperalgesia. the present data demonstrate that the sni procedure result in severe changes in behavioral responses in whether hyperalgesia or allodynia. coadministration of both drugs seems to be more effective to alleviate induced neuropathic pain. satoshi deyama 1,2 , naomi akiyama 1 , mikie hirata 1 , takayuki nakagawa 2 , shuji kaneko 2 , masabumi minami 1 1 dept. of pharmacol., grad. sch. of pharm. sci., hokkaido univ., sapporo, japan; 2 dept. of mol. pharmacol., grad. sch. of pharm. sci., kyoto univ., kyoto, japan the bed nucleus of the stria terminalis (bst) is involved in the regulation of negative affective states such as anxiety and fear. in this study, we examined the role of the noradrenergic (na) transmission within the bst in the negative affective component of pain in rats. we found that excitotoxic lesion of the bst attenuated intraperitoneal acetic acid-or intraplantar formalin-induced conditioned place aversion (cpa) without reducing nociceptive behaviors. we showed that na release within the bst was significantly elevated by these noxious stimuli. intra-bst injection of a ␤-adrenoceptor antagonist timolol significantly suppressed these noxious stimuli-induced cpa without affecting nociceptive behaviors. these results suggest that visceral and somatic noxious stimuli-induced na release within the bst contributes to the negative affective, but not sensory, component of pain. noriyuki ozaki, mariko kawai, yasuo sugiura department of functional anatomy and neuroscience, nagoya university, graduate school of medicine, nagoya, japan neonatal maternal separation induces visceral hyperalgesia in colon. this study compares the effects of maternal separation on response sensitivity to gastric and colorectal distension in long-evans rats. maternal separation was performed for 3 h per day between postnatal day 1 and 14. visceral sensitivities were assessed in stomach and colon at 10 weeks of age by visceromotor responses induced by either gastric or colorectal distension. somatic pain sensitivities were also assessed by von frey filaments and radiant heat. in contrast to the response to colorectal distension, maternal separation induced decreased response to gastric distension, especially in male rats. no difference was found between control and separated rats in somatic pain sensitivities. these results indicate that maternal separation differentially modulates visceral pain sensation in stomach and colon. research funds: grant-in-aid for scientific research 16500216 ps2p-g119 change by aging in muscular mechanical hyperalgesia after lenghtening contraction k. mizumura, t. taguchi, t. matsuda, t. nasu res. inst. environ. med., nagoya univ., nagoya, japan our previous experiments have shown that the mechanical threshold of the edl muscle underwent lengthening contraction (lec) lowered 1 to 3 days after exercise in rats (8 w old). c-fos expression in the superficial dorsal horn increased in l4 spinal segment when the edl muscle was compressed 2 days after exercise. from these results we have concluded that the muscle became hyperalgesic after lec. in the present experiment, we examined whether this hyperalgesia after lec changes along aging. male sd rats 8, 80 (81-84) and 130 (131-133) w old were used. the basal mechanical threshold (randall-selitto method) of edl muscle tended to be higher in 80 w old rats, but not significant. after lec, the threshold started to decrease 1 day after lec in all three age groups. it returned to the pre-lec level 4 days after lec in 8 and 80 w old rats only. recovery of 130 w old rats delayed up to 7 days after lec. increased c-fos expression in the superficial dorsal horn was observed in l4 as well as in l5 in 130 w old rats. these results suggest that hyperalgesia occurs in larger areas and lasts longer in aged animals. tong liu, hong p. wei, chun y. yuan, ai k. guo institute of neuroscience, chinese academy of sciences, china drosophila can display complex courtship behavior. male-male courtship behavior shown in some fly mutants, but here we report for the first time that the male-male courtship behavior can be induced by disturbance of dopamine level. to up-regulate dopamine level, uas-th/th-gal4 males were used, which showed high level of dopamine and performed active male-male courtship behavior. this behavior was attenuated by decreasing dopamine level either through drug breeding or genetic method. the increased courtship behavior in uas-th/th-gal4 males is specific to male partners, because the males courted females normally. to down-regulate dopamine level, pale ts , th temperature sensitive mutant was used. when raised at restrictive temperature, pale ts showed obvious attraction to wild type males. our study shows that the high level or low level of dopamine can induce male-male courtship behavior in active or passive manner. athushi yokoyama 1 , masaharu akita 2 1 kanagawa life-science res., japan; 2 kamkura, kanagawa, japan we have developed the screening system for drug and chemical compounds of food by the used of ratwhole embryo culture. the advantages of whole embryo culture are to examine the direct effects of l-calnitin (lcal) on embryo and also to find the non-teratogenic agent (d-calnitin:dcal). as the testing agent, lcal was examined in this study using the rat embryo cultured from day 11 to 13 of gestation. in treated embryos of lcal, the embryonic heart beat, the crown-rump length, the embryo weight and the total embryonic somites were not decreased. on the other hand, the malformation (the defects of neural tube) and the short size of head length were observed in the embryos cultured with lcal. in treated embryos of d-calnitin (dcal), there parameter was not decreased. the observed malformation of lcal was not observed in the embryos cultured with dcal. these results might be due to the differences between lcal and dcal in the embryo toxicity. yoshihisa uenoyama, kenji takase, junya hirata, hiroko tsukamura, kei-ichiro maeda laboratory of reproductive science, nagoya university, nagoya, japan the mechanisms underlying the pubertal increase in gonadotropinreleasing hormone (gnrh) secretion are poorly understood. recently, metastin was found to stimulate gnrh secretion and mutations of its receptor are associated with lack of puberty. effect of immunoneutralization of endogenous metastin in the brain on the onset of puberty was examined to clarify the physiological significance of metastin in timing the puberty. when wistar-imamichi strain female rats received an infusion of anti-metastin antibody into the third ventricle during days 25-39 of age, they did not show the first estrus before 52 days of age with mean age of 54.7 ± 2.0 day. in contrast, most of normal mouse igg-treated controls showed the first estrus by 43 days of age with mean age of 42.8 ± 2.2 day. the age of vaginal opening was also delayed in the anti-metastin-treated rats. thus, the present study demonstrates that the puberty onset was delayed by immunoneutralizing central metastin. central metastin may be involved in timing the onset of puberty in female rats. kenji takase, yoshihisa uenoyama, shunji yamada, hiroko tsukamura, kei-ichiro maeda lab. of reproductive science, nagoya university, aichi, japan metastin has been considered to be involved in triggering pulsatile gonadotropin-releasing hormone (gnrh)/luteinizimg hormone (lh) secretion to time the onset of puberty. the present study aimed to determine expression of metastin, a novel kiss-1 gene product, in the rat brain during peripubertal period. wistar-imamichi strain female rat shows vaginal opening on around days 29 of age (d29), and first estrus on around d34. brain tissues were obtained on d21, 26, 31, 36 and 41. kiss-1 mrna expression in the arcuate nucleus-median eminence region (arc-me) and anteroventral periventricular nucleus (avpv) increased significantly from d21 to 26 and was kept at a high level thereafter. gpr54 mrna expression in the medial preoptic area increased significantly from d21 to 31. metastin-immunoreactive cells were not found on d21 but were apparent in the arc-me on d26 onward. these results indicate that metastin expression increases in the arc-me and avpv before vaginal opening, suggesting that metastin triggers the onset of gnrh/lh secretion in female rats. toshiyuki saito 1 , sei-etsu fujiwara 1 , kenjiro konno 2 , takashi yamaguchi 3 , tetsu nemoto 4 , etsuko kasuya 1 , ryosuke sakumoto 1 1 anim. neurophysiol. lab., natl. inst. agrobiol. sci., tsukuba, japan; 2 inst. exp. anim. res., fac. med., gunma univ., maebashi, japan; 3 grad. sch. sci. & eng., yamagata univ., yonezawa, japan; 4 sch. health sci., fac. med., kanazawa univ., kanazawa, japan in the present study, we recorded and examined local field potentials (lfps) in the hippocampus of piglets performing an operant task by a radio-telemetry system. under halothane anesthesia, a pair of tungsten electrodes was implanted into the hippocampus and fixed on the surface of the skull with a transmitter using dental cement. after recovery from surgical procedures, the piglets were moved to a training cage. in the lfps, spike-shaped waves were frequently found just before the piglets pushed a switch with their noses. these waves may represent some of the hippocampal neural activities associated with switch manipulation for getting a food reward. yasuo osawa department of bioscience, tokyo university of agriculture, tokyo, japan memory extinction is an inhibitory learning rather than forgetting or erase of conditioned memory. from the view of treatment of phobia and post traumatic stress disease (ptsd) caused by fear memory, it is important to find the drugs to facilitate extinction of fear memory. importantly, previous studies using pavlovian fear conditioning have shown that d-cycloserine, a nmda receptor agonist, facilitates memory extinction. in this study, to examine whether d-cycloserine is applicable for the treatment with another type of fear memory, we investigated effects of d-cycloserine on extinction of aversive memory in mice. indeed, we performed conditioned taste aversion (cta) task, where the ingestion of a novel taste is paired with transient sickness. our results indicated the injection of d-cycloserine before but not after the re-exposure to cs facilitates extinction of cta. ps2p-g126 hippocampal neural responses during a conditional delayed stimulus-response task in awake mice nobuhide kitabayashi 1 , teruko uwano 1,3 , anh tran 2,3 , eturou hori 1,3 , taketoshi ono 2,3 , hisao nishijo 1 1 system emotional science, univ. of toyama, japan; 2 integrative neurosci, molecular and integrative emotional neurosci., univ. of toyama, japan; 3 crest, japan to investigate a hippocampal (hf) involvement in the representation of temporal sequence in mice, neural responses were recorded during performance of a conditional delayed stimulus-response association task. a trial was initiated by one of two different conditioned tones. after a 2 s delay, two serial reinforcements with an intervening delay was presented; aversive air puff-delay-tube protrusion to evoke licking sucrose solution and the opposite order of the same reinforcements. of 85 hf neurons, 26 responded to the tones, the reinforcements, and during the delay. some neurons responded to a presentation of a sensory stimulus, and other responded differentially during the delay depending on the reinforcement sequence. the results suggest a crucial role of the hf in representation of serial events in episodic memory in mice as well as in rats and primates. further studies will be conducted using genetic modified-mice to clarify the neural substrate in episodic memory. naoko inoue 1 , atsu aiba 2 , kaoru inokuchi 1 1 mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; 2 grad. sch. med., kobe univ., kobe, japan vesl-1s/homer-1a and vesl-1l/homer-1c are splicing isoforms encoded by the vesl-1 gene. vesl proteins bind and regulate mglur1/5, ip3 receptor, ryanodine receptor, and trp channel at the postsynapse. the expression of vesl-1s is upregulated by tetanic stimulation that elicits l-ltp. vesl-1s is thought to play a critical role in the conversion from short-term to long-term memory (ltm). in this study we generated vesl-1s gene-targeting mice (ko) and examined whether vesl-1s plays a role in the ltm formation. analysis with the contextual fear conditioning revealed a defective in consolidation process, reconsolidation process, and remote memory formation in ko. ko further showed an enhanced freezing decrement within a test session, indicating faster within-session extinction. in contrast, consolidation process of the extinction was normal in ko. these results demonstrate that the vesl-1s protein plays critical roles in various processes of the ltm formation. we now examine the signaling pathways important for ltm formation that are altered in ko. hiroshi ageta 1 , r. migishima 1 , s. kida 2 , k. inokuchi 1,3 1 mitsubishi kagaku inst. life sci (mitils), japan, 2 tokyo univ. agricul., japan; 3 crest, jst, japan memory process consists of at least four distinct phases, acquisition, consolidation, maintenance, and retrieval. activin ␤a mrna increases following l-ltp induction in the hippocampus, suggesting that activin plays a role in the memory formation. here, we generated activin and follistatin (antagonizes activin function) transgenic mice in which the transgene expression was tightly regulated by dox in a forebrain-specific manner (tet off system). transgene expression was turned off or on within 3 d by (+/−) dox. contextual fear conditioning with these mice revealed that activin function is required during maintenance phase of fear memory for one week retention. furthermore, activin plays a role in the re-consolidation process. thus, fear memory that was once acquired and consolidated tightly could be "erased" by inhibiting the activin function during maintenance phase. these mice are useful for the study of ptsd. ps2p-h129 sex differences in the effects of chronic estrogen treatment on fear conditioning in c57bl/6j mice takaaki ozawa, mumeko tsuda, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan it has been suggested that estrogen may play a role in the regulation of learning and cognitive functions. although most of previous studies have focused on elucidating facilitatory effects of estrogen on learning in females, estrogen is also known to affect various behaviors in males. in the present study, we investigated the effects of different doses of estrogen on fear conditioning (fc) learning in both sexes of mice. gonadectomized c57bl/6j mice were implanted with a silastic capsule containing 0, 5, 10 or 20 g of estradiol benzoate. since it is possible that estrogen may indirectly modify learning by affecting general activity, emotionality and anxiety levels, we tested the mice in open-field and light dark transition paradigms prior to fc. mice were then conditioned for fear responses (freezing) to tone stimulus and tested for both contextual and cued fc responses. we found that estrogen facilitated both types of fc learning in females, whereas it inhibited them in males especially at a higher dose, with a small effect on emotional behaviors. ps2p-h130 analysis of brain regions activated during memory consolidation in passive avoidance task zhang yue department of bioscience, tokyo university of agriculture, tokyo, japan short-term memory (stm) is labile. to generate long-term memory (ltm), stm is stabilized through a process known as memory consolidation. importantly, previous studies have shown that memory consolidation requires the function of transcription factor creb whose activation induces c-fos expression. in this study, we tried to understand molecular mechanisms of consolidation of passive avoidance memory that has been known to be amygdala and hipocampusdependent. indeed, we investigated brain regions that are activated following the learning by analyzing the expression level of c-fos using immunocytochemistry. consistent with previous study, we observed increase in c-fos expression in amygdala and hippocampus. more interestingly, we also found this increase in prefrontal cortex, indicating that prefrontal cortex plays critical roles in memory consolidation in light-dark passive avoidance task. hiroshi nomura, norio matsuki laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan we have demonstrated the effect of ethanol on reactivated fear memory for the first time, using contextual fear conditioning. rats were conditioned with mild footshock, reexposed to the training context, immediately injected with ethanol or saline, and finally tested 48 h after reexposure. ethanol-treated groups expressed longer freezing and the effect lasted for 2 weeks. reactivation was necessary for the effect. the injection of ethanol itself did not induce a fearful response. as memory retrieval triggers memory extinction and reconsolidation, we investigated whether extinction process is involved in this ethanol effect. increasing retrieval time did not enhance freezing by ethanol, suggesting that ethanol had no effect on memory extinction. post-reactivation injections of anisomycin revealed that retrieval triggered reconsolidation. moreover, picrotoxin inhibited the memory enhancement by ethanol. these studies demonstrate that ethanol enhances reactivated contextual fear memories via activation of gaba a receptors. ps2p-h132 analyses of brain regions activated in reconsolidation and extinction phases of contextual fear memory nori mamiya, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan the retrieval of conditioned fear memory by conditioned stimulus (cs) initiates two processes; reconsolidation or extinction. we previously found that the change in memory stability after retrieval (reconsolidation) associates with memory extinction. to understand the regulatory mechanisms of memory stability after the retrieval at the anatomical level, we here investigated the brain regions that are activated in reconsolidation and extinction phases. we measured the levels of phospho-creb inducing changes in neural plasticity following the re-exposure to cs. short re-exposure to cs inducing reconsolidation increased in phospho-creb in amygdala and hippocampus. in contrast, longer re-exposure inducing extinction increased in phospho-creb in amygdala and prefrontal cortex. these results indicate that distinct brain areas are activated in response to short or long re-exposure to cs and suggests that amygdala plays crucial roles in the interaction between reconsolidation and extinction. ps2p-h133 analysis of molecular mechanism for the destabilization of retrieved contextual fear memory akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan reconsolidation acts to stabilize, whereas extinction tends to weaken the expression of the original memory. to understand the mechanisms for the regulation of memory stability after the retrieval, we have investigated the relationship between reconsolidation and extinction using contextual fear conditioning. we previously found that memory extinction is associated with regulation of fear memory stability, indicating the interaction between memory reconsolidation and extinction phases. in this study, we compared molecular signatures of reconoslidation and extinction using mice. pharmacological experiments using antagonists for cannabinoid receptor 1 (cb1) and l-type voltage-gated calcium channels (lvgccs) indicated that both cb1 and lvgccs are required for memory extinction but not consolidation and reconsolidation. more interestingly, blockade of either cb1 or lvgccs function prevents the disruption of the original memory by protein synthesis inhibition. these results suggest that cb1 and lvgccs are required for not only memory extinction but also the destabilization of reactivated memory. hotaka fukushima, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan previous our studies using contextual fear conditioning revealed three distinct time-dependent phases following memory retrieval: stable, reconsolidation, extinction phases. to understand the nature of memory processing following retrieval, we examined the effects of reexposure on memory reconsolidation and extinction using light-dark passive avoidance task. this task is thought to allow us to discriminate between reconsolidation and extinction phases at the time point when mice enter dark box from light box. brief re-exposure to light box did not affect the stability of fear memory (stable phase). further extending re-exposure to light box triggered the requirement of protein synthesis for re-storage of fear memory (reconsolidation phase). in contrast, entry from light into dark box initiated extinction of fear memory (extinction phase). additionally, using pharmacological blockade of cb1 and lvgccs, we also found that cb1 is required for only memory extinction but that lvgccs are required for memory extinction and reconsolidation. wakoto matsuda 1 , takahiro furuta 1 , kouichi nakamura 1,2 , takeshi kaneko 1,2 ps2p-h136 difference in organization of corticostriatal and thalamostriatal synapses between patch and matrix compartments of rat neostriatum fumino fujiyama 1 , tomo unzai 1 , kouichi nakamura 1,3 , sakashi nomura 2 , takeshi kaneko 1,3 1 department of morphological brain science, kyoto university, kyoto, japan; 2 department of physical therapy, kyoto university, kyoto, japan; 3 crest, japan the striatum, which has patch/matrix compartments, receives glutamatergic inputs from cortex and thalamus. in the present study, the differences in synaptology of these inputs between both compartments were examined. axon terminals positive for vesicular glutamate transporter (vglut)2, thalamostriatal inputs, were less dense in patch region, whereas vglut1-positive corticostriatal inputs were evenly distributed. quantitative analysis revealed 84% of vglut2positive synapses in patch region were formed with spines, whereas 70% in matrix region were made with dendritic shafts. in contrast, the targets of vglut1-positive inputs were mainly spines in both regions. moreover, vglut2-positive axospinous synapses in patch region were larger than vglut1-positive ones. the present observation suggests that thalamostriatal connection is more plastic in patch region. research funds: kakenhi (16500217, 17022024,16200025, 17022020 (0131),17650100) ps2p-h137 single cell tracing of thalamostriatal projection neurons with reference to patch and matrix compartments of rat striatum tomo unzai 1 , fumino fujiyama 1 , takeshi kaneko 1,2 1 department of morphological brain science, university of kyoto, kyoto, japan; 2 crest, japan the striatum consists of patch and matrix compartments, and receives glutamatergic inputs mainly from the cerebral cortex and thalamus. thalamic intralaminar nuclei are known to project exclusively to matrix compartment. on the other hand, it has not been clarified which thalamic nuclei project to patch compartment. in the present study, we combined single cell tracing with immunohistochemistry for mu opioid receptor, which is specifically expressed by patch neurons, to reveal the distribution of thalamostriatal axon terminals in relation to striatal compartments. recombinant sindbis virus expressing membrane-targeted green fluorescent protein (palgfp) was injected into the rat thalamus. a single neuron in the thalamic paraventricular nucleus extensively projected to the striatum and preferentially to patch compartment compared with matrix compartment. the axons were also distributed in the thalamic reticular nucleus, accumbens nucleus, amygdala, and cerebral cortex. research funds: kakenhi (16500217, 17022024, 16200025, 17022020(0131) , 17650100) ps2p-h138 lesion of the nucleus accumbens dopamine system shortens the lever pressing interresponse time and delays the response initiation in mice yuji tsutsui 1 , kayo nishizawa 1 , nobuyuki kai 2 , kazuto kobayashi 2,3 1 dept. of psychology, fukushima univ., japan; 2 dept. mol. genet., fukushima medi. univ., japan; 3 crest, jst, kawaguchi, japan dopamine transmission is thought to be important for rodents to perform operant behaviors such as lever pressing. the lever pressing experiment was conducted to examine the effects of 6-ohda injections into the nucleus accumbens (acb) in c57bl/6j mice. all mice were trained to press the lever for a food pellet using a fixed ratio 5 (fr5) schedule. the mice were injected with ascorbate vehicle or 6-ohda into the acb, and then tested post-surgically using the fr5 schedule again. the 6-ohda-injected mice showed the acceleration of response speed, which was revealed by the shortening of interresponse time between each of the five lever pressings, and the suppression of the initiation of the response to the next step. this suppression of initiation was revealed by the increase of time from the last presentation of food to the next initiation. these results suggest that the acb dopamine system is important for the initiation and control of the operant behaviors in rodents. hideshi shibata laboratory of veterinary anatomy, tokyo university of agriculture and technology, fuchu, tokyo, japan retrosplenial area 29 is one of the important structures for spatial memory and behavior in the rat. to understand more fully the functional roles played by area 29, it is essential to clarify the neural circuitry subserving these functions. in the present study, we analyzed the organization of frontal cortical projections to area 29 in the rat, using retrograde transport of cholera toxin b subunit (ctb). ctb injections into area 29d retrogradely labeled cells in the orbital cortex and the caudal parts of the anterior cingulate and primary and secondary motor cortices. ctb injections into area 29c labeled cells in similar cortical regions, except for the orbital cortex. ctb injections involving areas 29a and b labeled cells in the caudal part of the anterior cingulate cortex. the results show that the orbital, anterior cingulate, and primary and secondary motor cortices have a different pattern of projections to each subdivision of area 29, suggesting different functional roles played by each subdivision of area 29 in spatial memory and behavior. eiichi jodo 1 , yoshiaki suzuki 2 , tadahiro katayama 1 , ken-yo hoshino 2 , yukihiko kayama 1 1 dep. of physiol., fukushima med. univ., fukushima, japan; 2 dep. of neuropsy., fukushima med. univ., japan it has been shown previously that the dopaminergic neurons in the ventral tegmental area (vta) selectively respond to a stimulus repeatedly paired with reward stimuli in a classical conditioning paradigm. since the vta receives dense projection from the medial prefrontal cortex (mpfc), such response selectivity of vta neurons may in part be produced by inputs from the mpfc. however, few studies have compared the firing pattern between these two regions. our present experiment was designed to make such a comparison in freely moving rats. two different tones were sequentially presented, one of which (target, 30%) was paired with intracranial simulation of the reward area. the unit activity was recorded from the mpfc and/or the vta. pfc and vta neurons exhibited phasic excitation with the peak latency of about 0.1 s to both tones, while only the target tone induced sustained activation of firing activity lasting until presentation of the reward. masato inoue, akichika mikami primate research institute, kyoto university, inuyama, aichi, japan to investigate the neuronal mechanism in the ventrolateral prefrontal cortex (vlpfc) and inferotemporal cortex (it) for holding information for object and their order of presentation, we examined single neuronal activities in the vlpfc and it while monkeys were performing a serial probe reproduction task. in the task, two sequentially presented objects were memorized and then a target object was selected from memorized objects based on a color stimulus. in 19% out of 438 vlpfc neurons, the delay-period activity showed objectselectivity and order-selectivity. in only 6% out of 254 it neurons, the delay-period activity showed object-selectivity and order-selectivity. the starting time of the order-selective activity was earlier in the vlpfc. these results suggest that the vlpfc plays a role in holding information for object and their order of presentation and the it receives information for object and their order of presentation from the vlpfc. masao yukie, yasutaka oosawa department of behavioral physiology, tokyo metropolitan institute for neuroscience, fuchu, japan relational memory theory (eichenbaum et al., 1994) has been proposed from evidence that the hippocampal damage in rats impairs learning of transverse patterning task (a+ versus b−; b+ versus c−; c+ versus a−). very recent monkey study (alvarado et al., 2005) demonstrated that lesion of the hippocampus produced a significant impairment in that task and supported such a theory. in our study, however, ischemic damage of the hippocampus has not impaired learning of such a transverse patterning task (yukie et al., 2005) . in the present study, we examined effects of lesion of the monkey perirhinal cortex on transverse patterning task using two sets of 2d visual stimuli presented in a wgta. our three monkeys with perirhinal lesions failed to attain a learning criterion within a training limit of 75 sessions in phase 3, although they learned easily the four problems in phases 1 and 2. our results suggest that the perirhinal cortex, but not the hippocampus, is important for learning of transverse patterning task, that is, for formation of relational memory. yasuko sugase-miyamoto 1 , noriyuki higo 1 , munetaka shidara 2 1 neuroscience research inst., aist, tsukuba, japan; 2 grad. sch. of tsukuba univ., tsukuba, japan a recent dopamine d2 receptor study using antisense cdna showed that d2 receptor in rhinal cortex is crucial for learning associations between visual stimuli and reward schedules. neuronal responses in the perirhinal cortex differentiate the visual cues only when the cues are associated with the schedule states, while those in area te are related to physical attributes of the cue independently of the schedule states. to investigate the cellular substrate for d2mediated associative learning, we examined monkey temporal lobe immunohistochemically with a d2 receptor antipeptide antiserum. d2 receptor immunoreactivity was observed in the pyramidal cells in layers ii-vi of the rhinal cortex and area te. the signal was mainly observed in cell bodies, and also in both apical and basal dendrites for some cells. the signal in layers v-vi was stronger in area 36 of the perirhinal cortex than in area teav. the differential localization between area 36 and te suggests the differential roles of the two areas in associative learning process. by using axonal transport of fast blue, diamidino yellow and tritiated amino acids, we determined the afferent and efferent connections of the retrosplenial cortex (rsp) in the macaque monkey. the rsp receives heavy projections from the subiculum, presubiculum and the caudal entorhinal areas (ec-ecl), and projects back to the presubiculum and the ec-ecl. the supracallosal portion of the rsp has connections primarily with the caudal half of the subiculum and presubiculum, as well as the lateral zone of the ec-ecl. the caudoventral portion of the rsp is, in contrast, mainly connected with the rostral half of the subiculum and presubiculum as well as the medial zone of the ec-ecl. the two portions of the rsp, thus, have access to different portions of the medial temporal lobe. these results indicate that there are two distinct neural systems in the retrosplenial-medial temporal network. hideko nakano 1 , natsuko yoshida 2 , kiyohisa natsume 3 1 kyushu kyoritsu university, fukuoka, japan; 2 kyushu institute of technology, fukuoka, japan; 3 kyushu institute of technology, fukuoka, japan eeg activity was examined in english rhythm acquisition of japanese students who learn english as a foreign language (efl). we measured theta, alpha and beta rhythms of five subjects while they were reading aloud the materials and listening to the audio-recording, using eight electrodes attached to their skulls. the result shows that the increase of theta power at f3 and f4 was the highest and suggests that the theta rhythm at f3 and f4 may have a relationship to the process of english rhythm acquisition. moreover we found the highest increase in theta power when the subjects began to orally reproduce every line of the rhythm materials. this finding was observed in three right handlers except a left handler and a right handler who had just returned after 6-month english study experience in australia. these results suggest that the change of theta power at frontal areas may be more closely related to the japanese efl learners' english rhythm acquisition. research funds: kakenhi (1565081) ps2p-h146 neural correlates of music retrieval: an eventrelated fmri study using sparse temporal sampling takamitsu watanabe 1 , sho yagishita 1 , hideyuki kikyo 1,2 1 department of physiology, the university of tokyo school of medicine, tokyo, japan; 2 department of molecular neuroimaging, national institute of radiological sciences, chiba, japan we investigated neural correlates of music memory using eventrelated functional magnetic resonance imaging and sparse temporal sampling technique with originally composed musical materials. written informed consent was obtained from all the subjects in accordance with the declaration of helsinki, and the experimental procedure was approved by the institutional review board of the university of tokyo school of medicine. a 1.5 t scanner system was used (te = 50 ms; tr = 14 s; acquisition time = 3.0 s). we demonstrated that the right hippocampus, bilateral lateral temporal cortices, left prefrontal cortex and left precuneus are involved in music retrieval. in addition, performance-based analysis suggested that the right hippocampus is associated with the accuracy of music memory. in this fmri study, we determined the neural correlates of the intellectual excitement. sentences describing facts in natural and human science were visually presented, and subjects judged whether they know the fact or not. after the fmri, each subject self-evaluates subjective "intellectual excitement" of each sentence. positive correlation with the self-evaluated intellectual excitement for known facts and novel facts were analyzed. significant correlation between cortical activation and self-evaluated intellectual excitement for novel facts was observed in the left and the right parahippocampal gyrus and for known facts was in the left orbital part of inferior frontal gyrus. it suggests the cortical areas related to self-evaluated intellectual excitement are different between getting of novel knowledge and recognition of existing knowledge. hyeonjeong jeong 1 , motoaki sugiura 3 , yuko sassa 2 , keisuke wakusawa 2,4 , kaoru horie 1,5 , shigeru sato 1,5 , ryuta kawashima 2,5 1 gsics, tohoku university, sendai, japan; 2 niche, tohoku university, japan; 3 miyagi university of education, sendai, japan; 4 department of pediatrics, school of medicine, tohoku university, japan; 5 the lbc research center, tohoku university, japan a foreign language word is learned and retrieved either in daily situations (situation) or written text (text), and memory transfer is required when the learning and retrieval modes are different. in this experiment, normal japanese subjects learned korean words in the situation and text modes in video clips. during a subsequent fmri session, subjects were presented with the learned words in different movie clips; half of the learned words was presented in the same mode as in the learning session (match), and the rest was presented in a different mode (mismatch). comparison of the mismatch with match condition revealed significant activation in the orbital part of the left inferior frontal gyrus. the results suggest that this area plays a role in the memory transfer of foreign language words when the learning and retrieval modes are different. georgina e. cruz 1 , christie l. sahley 2 , kenneth j. muller 1 1 physio. & biophys., univ. of miami, miami, fl, usa; 2 biol. sci., purdue univ., west lafayette, in, usa in some animals much is known at the level of single synapses about mechanisms underlying behavioral sensitization, but in no system is the involvement of interactions at the network level well understood. the s-cell network of the medicinal leech is a chain of electrically coupled interneurons spanning the nerve cord with distributed sensory input and motor output and is crucial for sensitization of reflex shortening. its firing increases with sensitization although few additional s-cells initiate impulses during the reflex. we tested the hypothesis that the initial burst of impulses from the s-cell in the stimulated segment suppresses initiations in adjacent segments. hyperpolarizing the central s-cell to reduce its firing during skin stimulation markedly increased the number of initiations in adjacent s-cells, which corroborated the limited expansion of initiation sites seen in the behaving animal. a computational model of s-cell refractoriness further supported the idea of interaction among s-cells during sensitization. research funds: nih, u.s.a. ps2p-h150 sensory/motor modules regulating the development of peer social relationship mamiko koshiba 1,2 , shun nakamura 2 1 jst, crest, japan; 2 ncnp social intelligence is indispensable for animal's survival and could have evolved to language capability. further, as a recent problem in japan, 'fewer children' supposedly causes the more tight interaction of child-parent, reciprocally the less between siblings or friends. in order to study the genetic and epigenetic development of peer social relationship after birth, we controlled peer interaction through limiting a particular sensory/motor modality as social deprivation and examined the effect on the active attachment behavior of domestic chick to conspecific mates. the chick has a merit of being precocial and unique in higher animal with no need of parent-care. comparing to the chicks reared as a group, the isolated chicks didn't develop their active attachment to peers. meanwhile, the behavior study with the chicks deprived not sensory/motor function itself, but only social interaction in auditory, visual, olfactory or tactile system, suggested that vocal communication at least must play a key role for the development. dna-chip study along the different social context brought candidates of social genes. shogo sakata 1 , minoru hattori 2 1 department of behavioral sciences, hiroshima university, higashi-hiroshima, japan; 2 graduate school of biosphere science, hiroshima university, higashi-hiroshima, japan peak interval (pi) 30-s procedure is a very good method to investigate for timing. six male wistar rats were trained for five days a week in pi 30-s procedure over 30 days. the 3-s bin of lever press responses on probe trials showed a clear peak point. the temporal distributions had the peak time of regression curve fitting with the gaussian function. the peak time corresponded to near the 30-s with reinforcement durations. then nicotine was administrated to the rat by intraperitoneal injection before daily pi 30-s session. results showed that the peak time in the nicotine administration was slightly leftward shift compared to the saline injection. however the pattern of temporal distribution of responses was not changed by the nicotine treatment as well as control condition. it suggests that the nicotine administration affects on the time perception that was reflected by the peak durations of responses. ps2p-i152 the effect of random practice schedule on arbitrary stimulus-response association learning satoshi tanaka 1,2 , ritz oshio 1,3 , norihiro sadato 1,4 , manabu honda 5,6 1 nips, okazaki, japan; 2 jsps, tokyo, japan; 3 nagoya univ., nagoya, japan; 4 ristex, jst, tokyo, japan; 5 sorst, jst, tokyo, japan; 6 ncnp, tokyo, japan previous studies suggest that randomly ordered practice facilitates retention and transfer of motor skills compared to blocked or regularly ordered practices. it remains unclear, however, whether the advantageous effects of random practice can be expended to cognitive skill learning in humans. we examined the simultaneous learning of multiple arbitrary stimulus-response (s-r) associations under three different practice schedules: blocked, random and regularly ordered. behavioral data indicate that subjects performing the random practice showed better performance of the retention and transfer of learning compared to those performing the blocked or regularly ordered practice. the present result indicates that random practice schedule is effective also for s-r association learning, which are considered as a bridge between motor control and cognitive control. ps2p-i153 sports rats show increased level of bdnf in the cerebellum, possibly learning and memorizing well masaki morishima, sayuri hara, yutaka nakaya dept. nutrition and metabolism, univ. of tokushima, japan previously, we reported that the activation of hippocampal norepinephrine neurotransmission following a decrease in monoamine oxidase a was observed in sports, a novel hyper-running rat on wheel. this study assessed whether sports show increased bdnf levels and better learning and memory. compared to control, both protein and mrna levels of bdnf in cerebellum were significantly elevated in sports even without wheel running, and slightly increased in hippocampus. in the cerebellum of sports, trkb/pi3k pathway was activated, whereas mapk pathway was activated in the hippocampus. locomotor activity assessed by the open field test showed that the sports were significantly more active in center coat than control. in the passive avoidance test, sports did not enter a dark area at next time indicating that sports showed better passive avoidance learning. these results suggest that bdnf signaling of sports were activated from trkb to mapk and pi3k in the hippocampus and cerebellum, respectively, and that these signaling pathways might play an important role in learning and memory. research funds: kakenhi (17500429) ps2p-i154 selective manipulation of working memory through d1 and d2 receptors: computer simulation shoji tanaka, hiroki yata dept. of electrical & electronics eng., tokyo, japan though a number of experimental results suggest that working memory processes are controlled by the dopaminergic system, its mechanism is still unclear. to elucidate the mechanism, we have constructed a model of the prefrontal cortical neural circuit for working memory. the neurons in the model are leaky integrate-and-fire model with ampa, nmda, gaba, and leak conductances and have dopamine d1 and d2 receptors. the computer simulation with this model shows that d1 receptor activation mainly affect working memory activity itself, while d2 receptor activation affect the termination of working memory, being consistent with the experimental result. the simulation also mimics the hyper-and hypo-dopaminergic states. under such conditions, like schizophrenia, simulated pharmacological treatments using agonists and antagonists of d1 and d2 receptors indicate efficacies of some these treatments for the restoration of working memory. in conclusion, this kind of simulation shows how dopamine controls working memory by using the synergism of the actions of dopamine, glutamate, and gaba. kozo sugioka, tomiyoshi setsu, tatsuro yamamoto, toshio terashima div. anat. & dev. neurobiol., dept. neurosci., kobe univ. grad. sch. med., kobe, japan we examined activity and habituation in rats with experimentallyinduced abnormal morphogenesis of the hippocampus. pregnant rat (jcl:wistar) was injected with saline or 25 mg/kg mam on the 15th day of gestation. the activity of male and female offspring was measured for each 1 h light and dark period, and the habituation to the visual stimulation was observed by measuring the activity with every 1 min interval for 1 h under 5 min dark/light alternative schedule during weaning and adult periods. activity was measured using infra-red sensor in a home-cage placed in the experimental room. the mam-treated rat showed hyperactivity for dark-period during both weaning and adult periods, and showed retarded habituation during weaning period. sex difference of behavioral alteration was evident during adult period in both groups. these behavioral disorders were discussed in relation to the mam-treated rat showing abnormal hippocampus (disruption of the ca1 pyramidal layer and ectopic neuron mass). ps2p-i156 long-lasting tagging of functionally activated neurons in the mouse brain naoki matsuo, leon reijmers, mark mayford the scripps research institute, la jolla, ca, usa immediate-early genes (iegs) have been widely used as activity markers for mapping neurons involved in specific animal behaviors including learning and memory. however, conventional ieg approaches that use immunohistochemistry or in situ hybridization allow to detect neurons only shortly after their activation and does not enable genetic manipulations. here we have developed transgenic mice that allow selective and long-lasting tagging of neurons that were activated in a given brain region at a given time point. the mice consist of two components; c-fos promoter driven tetracycline-controlled transactivator (tta) and teto promoter regulated feedback loop. when strong neuronal activity occurs in the absence of tetracycline analogs such as doxycycline (dox), c-fos promoter driven tta initiates the teto-linked expression of mutant tta (tta*) that is not inhibited by dox. this teto-linked gene expression is then maintained indefinitely by feedback activation via the tta* even in the presence of dox. using this system, we have examined the expression of bicistronic teto promoter driven tau-lacz and egfp-glur1. hamid gholamipour 1 , shirin babri 2 , khameneh saied 3 1 department of physiology, university of tabriz, tabriz, iran; 2 university of tabriz, iran; 3 university of tabriz, iran diabetes mellitus is one of the most prevalent diseases in the world. because hippocampus is an important area for memory formation, the present study is scheduled to investigate the effect of insulin injection in ca1region of hippocampus on memory formation. fifty male rats were divided into five groups. (1) control (2) sham operation (3) test (4) diabetic/saline (5) diabetic/insulin. groups 4 and 5 were made diabetic by treatment with stz (50 mg/kg, i.p.). in all but the control group, two canula were stereotaxically implanted in ca1 region of hippocampus. learning was tested and compared between groups through passive avoidance test. results showed that in the test group the latency increased as compared to control and sham groups (p < 0.05). compared to sham group diabetic/insulin group showed increased latency (p < 0.05) but no significant difference was found between diabetic/saline and diabetic/insulin groups. in conclusion, according to the results obtained in this study, insulin facilitates memory in intact rats but not in diabetic sex differences in hippocampus-dependent memory formation are well documented, but the mechanisms are poorly understood. the ca 2+ /calmodulin (cam) kinase cascade regulates gene transcription in the hippocampus, which is required for long-term memory (ltm) formation. we hypothesized that sex differences in transcriptional regulation may account for the sexual dimorphisms in memory formation. we tested this idea by studying the role of cam kinase kinases (camkks). using mouse molecular genetics we found that camkk␤ is required for spatial, but not contextual ltm. consistent with the impaired spatial memory formation, camkk␤ null mutants lacked spatial training-induced creb activation and had impaired late ltp. in contrast to camkk␤, camkk␣ is required for contextual, but not for spatial ltm. furthermore, female camkk mutants had normal spatial and contextual ltm. thus, we show that there are malespecific mechanisms to regulate gene transcription that may explain sex differences in hippocampus-dependent memory formation. akshay anand, sudesh prabhakar, monika bhatia, c.p. das department of neurology, post graduate institute of medical education and research, chandigarh, india background: parkinson's disease has a prevalence rate of 19 per 100,000 in india. we studied the park 4 polymorphism in north indian population and parkin expression in early parkinson's disease (n = 30) and sporadic parkinson's disease (n = 30). methods: pcr, sscp, rflp and direct sequencing analysis were used to screen mutations. results: our results revealed homozygous exonic mutations in exon-1, 3 and 6 in early pd and exon-1 and 12 in sporadic pd, heterozygous mutations in exon 4 and 9 in five early pd and one sporadic pd patient. frequency of s/n polymorphism was significantly high suggesting that exchange of serine to asparagine at position 167 of protein affects the secondary structure or hydrophobicity of the protein resulting in pathogenicity. our facs analysis of these samples indicates reduced parkin expression correlating with severity of mutations. conclusions: we conclude that high frequency of parkin 4 mutations in pd population in india affect parkin expression resulting in pd. wanida tripanichkul 1 , kittisak sripanichkulchai 2 , david finkelstein 3 1 faculty of medicine, srinakharinwirot university, thailand; 2 faculty of medicine, khon kaen university, thailand; 3 university of melbourne, australia emerging data suggests beneficial effect of estrogen for parkinson's disease (pd), yet the exact mechanisms implicated remain obscured. activated glia observed in mptp mouse model and in pd may participate in the cascade of deleterious events that ultimately leads to dopaminergic nigral neuronal death. estrogen can modify glial expression of inflammatory mediator, such as cytokines and chemokines implicated in neurodegeneration. to determine whether estrogen-elicited neuroprotection in pd is mediated through glia, adult male c57bl/6 mice were pretreated with 17beta-estradiol (e2), injected with mptp on the day 6 and brains were collected on day 11. e2 pretreatment decreased nigral neuronal loss and diminished striatal fibers deficit induced by mptp. the neuroprotective effect of e2 was coincident with an attenuation of a glial response within the snpc and striatum. these findings propose that e2 neuroprotection in mptp mouse model may mediate through reactive glia inhibition. ps2p-i163 effect of angiotensin-converting enzyme inhibitor perindopril in mptp-treated mice; immunohistochemistry and in vivo electron spin resonance (esr) study rumiko kurosaki 1 , fumihiko yoshino 2 , masaichi chang-il lee 2 1 dept. of food sci. and nutrition, showa women's univ., tokyo, japan; 2 clin. care and med. div. of pharmaco., kanagawa dental college, japan we investigated the effects of perindopril on the dopaminergic system and the oxidative stress in mice after mptp treatment. administration of perindopril showed dose-dependent neuroprotective effects against striatal dopamine and its metabolites depletion after mptp treatment. we have reported that th, gfap, pv, nnos and cu, zn sod positive cells in the substantia nigra was changed after mptp treatment in our immunohistochemical study. the administration of perindopril significantly attenuated mptp induced changes of these immunopositive nigral cells. we could measure increased oxidative stress in the brain of mptp and perindopril treatment mice using by in vivo esr technique. our results provide further evidence that the ace inhibitor perindopril may offer a novel therapeutic strategy for parkinsonǐs disease. research funds: kakenhi (17700577) ps2p-i164 recruitment of calbindin into substantia nigra dopamine neurons suppresses the onset of parkinsonian motor signs shigehiro miyachi 1 , kaori sawada 1 , haruo okado 1 , atsushi nambu 2 , masahiko takada 1 1 tokyo met. inst. neurosci., fuchu, tokyo, japan; 2 natl. inst. for physiological sci., okazaki, japan there is a consensus that dopaminergic neurons in the substantia nigra that express calbindin, a calcium-binding protein, are selectively invulnerable to parkinsonian insults. based on this notion, an attempt was made to test the hypothesis that parkinsonism may be suppressed by recruitment of calbindin into a subpopulation of nigral dopamine neurons that does not normally contain calbindin. an adenoviral vector expressing calbindin was injected unilaterally into the striatum of macaque monkeys, to let calbindin express in the dopaminergic neurons via retrograde transport. two to three weeks later, the parkinsonism-inducing drug mptp was systemically administered several times. parkinsonian motor signs, such as muscular rigidity and flexed posture, appeared only on the side ipsilateral to the calbindin recruitment when cumulative doses of mptp exceeded threshold for their bilateral onset. toru yasuda, hideki mochizuki, yoshikuni mizuno department of neurology, juntendo university school of medicine, tokyo, japan using the serotype-2 raav vectors, we have recently reported the protective effect of parkin on the ␣-synuclein (␣s)-induced nigral dopaminergic neurodegeneration in a rat model. here we investigated the neuronal specificity of ␣s toxicity and the effect of parkin co-expression in a primate model. another serotype (type-1) of raav (raav1) carrying αs cdna (raav1-␣s), and a cocktail of raav1-␣s and raav1 carrying parkin cdna were unilaterally injected into the striatum of macaque monkeys, resulting in protein expression in striatonigral gabaergic and nigrostriatal dopaminergic neurons. the injection of raav1-␣s alone caused a decrease of th-immunoreactivity in the striatum, while there was no effect on gabaergic neurons. in the presence of overexpressed parkin, ␣s seemed to be less accumulated and/or phosphorylated at ser129 residue in gabaergic neurons. these suggest that the ␣s toxicity is not expressed in non-dopaminergic neurons but the ␣s-ablating effect of parkin is exerted in all neurons introduced in primates. tomokazu oshima, yohsuke narabayashi narabayashi memorial laboratory of neurology, neurological clinic, tokyo, japan rigidity in aged parkinsonians is often intractable against surgeries. we investigated how their rigidity scored by updrs was related with ␤-band local field potentials (␤-waves) of the surgical targets in thalamic ventrolateral nucleus (vl) and posteroventral pallidal internal segment (pvp). forty patients aged 70-80s gave informed consent for thalamotomy and/or pallidotomy. we divided the patients into groups with rigidity of 0.5-1 (i), 1.5-2 (ii), 2.5-3 (iii), and 3.5-4 (iv). the ␤waves were rated with total periods (%) of 13-27 hz wavelets in 3-s sample records. rigidity was re-scored after the surgeries. the vl ␤-waves were rated 65-70% in groups i-iii with a slightly increasing tendency for increasing rigidity, but declined to about 55% in group iv. the pvp ␤-waves were 60-80%, but with a decreasing tendency for increasing rigidity. the surgeries alleviated rigidity in all the groups, but were least effective in group iv with least vl and pvp ␤-waves. the results suggest that the pathology of aged parkinsonian rigidity develops beyond the pallido-thalamic pathway. yoshihisa tachibana 1,2 , hirokazu iwamuro 1,3 , masahiko takada 4 , atsushi nambu 1,2 1 div. syst. neurophysiol., natl. inst. physiol. sci., okazaki, japan; 2 sokendai; 3 dept. neurosurg., univ. tokyo, tokyo, japan; 4 dept. syst. neurosci., tokyo met. inst. neurosci., fuchu, tokyo, japan to approach a new therapy for parkinson's disease, extracellular unit recordings combined with microinjections of glutamate-related drugs were performed in the external and internal segments of the globus pallidus (gpe/gpi) of mptp-treated parkinsonian monkeys (macaca cyclopus). compared with the normal state, spontaneous oscillatory discharges were so often observed in the gpe/gpi and the subthalamic nucleus (stn) of the parkinsonian monkeys. microinjections of ionotropic glutamate receptor antagonists into the vicinity of recorded gpe/gpi neurons reduced their abnormal oscillations. these results suggest that glutamatergic excitatory input from the stn contributes to the oscillatory activity of gpe/gpi neurons, and that intrapallidal injections of ionotropic glutamate receptor antagonists may ameliorate some of parkinsonian symptoms. one of the pathological features of parkinsonǐs disease (pd) is loss of dopaminergic neurons in the substantia nigra pars comapacta (snpc). and it has been known that ␣-synuclein is involved in the neuronal loss. during the dopaminergic neuronal loss, activated microglia were centered in snpc. we hypothesize that ␣-synuclein may play a role in microglial activation to migrate to the pathological regions and to perform the neuronal cytotoxicity. we demonstrated that ␣-synuclein induced the cd44 expression on microglia and also enhanced the mt1-mmp expression to shed off cd44 at the cell surface and degrade surrounding ecm to open the migratory way. a53t mutant ␣-synuclein showed greater level of cd44 shedding and cell migration. extracellular treated ␣-synuclein also increased cd44 and mt1-mmp expressions dose-dependently. among the multiple signaling pathways, erk pathway was involved in ␣-synuclein induced cell migration. these induced cell migration were also confirmed in human pd patients. research funds: national creative research initiative grant (2004 grant ( -2006 ps2p-j169 serotonergic fibers are involved in the conversion of l-dopa to dopamine in the striatum and the substantia nigra pars reticulata of parkinsonian model rats ryohachi arai 1 , hiromasa yamada 1 , yoshinari aimi 1 , ikuko nagatsu 2 1 department of anatomy, shiga university of medical science, otsu, japan; 2 fujita health university school of medicine, japan dopaminergic neurons in the substantia nigra pars compacta (snc) project their axons to the striatum (st) and their dendrites to the substantia nigra pars reticulata (snr). dopamine released from these axons and dendrites is important in the regulation of motor activity. in parkinson's disease, dopaminergic neurons in the snc degenerate. l-dopa is the most effective drug for this disease. we hypothesize that, in parkinson's disease, a part of administered l-dopa is converted to dopamine in serotonergic fibers of the st and snr. here we produced parkinsonian model rats by the unilateral injection of 6hydroxydopamine into the snc, and found that serotonergic fibers in the st and snr were immunohistochemically positive for dopamine after l-dopa administration in the rats. therefore, it is possible that serotonergic neurons may be involved in the therapeutic effects of l-dopa for parkinson's disease. in mptp-induced pd monkey, reactive microglia are observed around neurons in nigra several years after mptp treatment and may be related to the progression of pd. to evaluate if reactive microglia in striatum and/or nigra of mptp-induced pd mice are present for a long time after mptp administration, like pd monkey. iba 1-and tb4distribution in microglia were immunohistochemically investigated at 0 h and 7 days after twice mptp-treatments (one treatment comprised of 4 intraperitoneal injections of 20 mg/kg mptp at 2 h interval) to c57bl/6 and balb/c at 6 months (mo) interval. the recognizable change of iba 1-and tb4-distibution in microglia of both mice strains was observed even 6 mo after the first treatment. the twice mptp treatments tended to aggravate the symptoms in both mice strains, compared with once treatment. these results suggest that reactive microglia are present for a long time after the treatment by mptp and must play a role in the chronic progression of pd. ps2p-j171 activated microglia affect the nigro-striatal dopamine neurons differently in neonatal and aged mice treated with mptp hirohide sawada 1 , ryohei hishida 2 , yoko hirata 3 , kenji ono 4 , hiromi suzuki 4 , shin-ichi muramatsu 2 , imaharu nakano 2 , kunihiro tsuchida 1 , toshiharu nagatsu 1,4 , makoto sawada 4 1 school of medicine, fujita health university, aichi, japan; 2 division of neurology, jichi medical university, japan; 3 department of biomolecular science, gifu university, japan; 4 research institute of environmental medicine, nagoya university, japan microglia play an important role in inflammatory process of parkinson's disease. we examined the effects between neonatal and aged microglia activated with lps on the nigro-striatal dopamine (da) neurons in mice treated with mptp. by mptp administration to neonatal mice, the number of da neurons in the substantia nigra was significantly decreased, whereas that in mice treated with lps and mptp was recovered. on the contrary, the number of da neurons of the 60 week-old mice treated with mptp was significantly decreased with lps treatment. these results suggest that activated microglia in neonatal mice have neurotrophic potential, in contrast to the neurotoxic effect in aged mice. hyposmia is one of the most characteristic symptoms of parkinson's disease (pd). it may occur even before the motor symptoms start. in the olfactory bulb (ob), dopaminergic cells were present at glomerular layer. furthermore, it has been reported that ob contains neural stem cells. thus, ob has attracted attention because of its unique regenerative potential. in the present study, we established isolation of neurosphere forming cells (nsfcs) derived from adult mice ob, and examined proliferation potential in ob after dopaminergic neuronal loss induced by mptp, a selective toxin for dopaminergic neurons, utilized frequently as pd model. the number of neurospheres derived from adult ob was not decreased with mptp administration, rather significantly increased. we also evaluated nsfcs differentiation into neural subtypes. the isolation of neural stem cells has helped to establish the cellular basis of neurogenesis and the exciting potential for transplant-mediated treatment of degenerative cns disease like pd. ps2p-j173 phosphorylation of erp57 in adult rat brain with neonatal 6-ohda treatment qinghua li, yasuyoshi watanabe department of physiology, osaka city university graduate school of medicine, osaka, japan dopaminergic neuron degeneration occurs in sporadic parkinson's disease (pd), but the mechanism of sporadic pd is not clarified. we prepared neonatal dopamine depleted rats, by i.c.v. injection of 6-ohda at 3(p3) and 6 days after birth, to investigate the mechanism of dopaminergic neuron degeneration. at p56, tyrosine hydroxylase (th) immunostaining cells were significantly reduced in the substantia nigra, and th immunostaining fibers were significantly reduced in the striatum, thus this model mimics the selective dopaminergic neuron degeneraion in sporadic pd. by two-dimensional electrophoresis we found that a certain protein was phosphorylated in the 6-ohda lesioned rats at p56, and it was identified as disulfide-isomerase a3 precursor (erp57) a kind of molecular chaperone of the endoplasmic reticulum (er) by maldi-tof ms. the result suggests that the phosphorylation of erp57 may have the key function to induce dopaminergic neuron degeneration and somehow relates to the pathogenesis of sporadic pd. katsunori nishi department of neurology, tokyo metropolitan institute for neuroscience, tokyo, japan regrowth of survived dopaminergic (da) neurons after the administration of psi, a potent proteasome inhibitor, was examined in vitro. dissociated cell co-culture was prepared from embryonic rat mesencephalon and striatum. psi (20 or 25 nm, 48 h) was applied to cultures at 7 days in vitro and succeeding changes of da neurons were investigated up to 35 days. more than 95% of da neurons reduced in number after the administration of psi, and a few truncated da neurons, devoid of neurites, being observed. non-da neurons were less severely affected at these concentrations of psi. regrowth of da neurites was observed approximately 2 weeks after the administration of psi and continued during the observation period. in most of the regrowing da neurons, one of the processes extended far longer than the rest, suggesting that severely injured neurons retain the capacity to reextend axons. regrowth was less remarkable in mesencephalic culture lacking striatum indicating that target cells are necessary for this effect. in conclusion, psi-damaged da neuron has strong regrowth potential in vitro. ps2p-j175 specific expression of proapoptotic factor pag608 on motor neurons in spinal cords of l-dopatreated parkinsonian models ikuko miyazaki, masako shimizu, francisco j. diaz-corrales, maria f. esraba-alba, masato asanuma dept. of brain sci., okayama univ. grad. sch. of med., dent. and pharmaceut. sci., japan we previously identified a proapoptotic gene, p53-activated gene 608 (pag608), as a dopa-induced gene in the striatum of l-dopa-treated parkinsonian models, which increased p53 expression to promote apoptosis by its nuclear translocation. last year, we also reported specific induction of pag608 in the internal capsule of l-dopatreated and constitutive expression in the smi-32-immunopositive motor neurons in the pontine nucleus and motor nuclei of trigeminal nerve and facial nerve. in the present study, we examined distribution of pag608 in the spinal cords of l-dopa-injected parkinsonian rats by immunohistochemistry. l-dopa treatment showed inducing tendency of pag608 expression on the motor neurons in the anterior corn and lateral corticospinal tract of spinal cords. the expression of pag608 in the motor nuclei of cranial nerves and its induction in the spinal cords suggests its possible involvement in motor dysfunction such as dyskinesia. ps2p-j176 an approach to the generation of ar-jp mouse model: crossbreeding of pael-r transgenic mice with parkin knockout mice hua-qin wang 1,2 , yuzuru imai 2 , haruhisa inoue 1,2 , ayane kataoka 2 , sachiko iita 2 , nobuyuki nukina 2 , ryosuke takahashi 1,2 1 neurology, university of kyoto, kyoto, japan; 2 bsi, riken, saitama, japan since loss of parkin e3 activity appears to be causal of ar-jp, accumulation of potentially toxic parkin substrates should result in degeneration of da neurons. however, parkin knockout mice show no different da neuronal loss even at old ages, presumably due to relative short lifespan of mice. pael-r is one of the best characterized parkin substrates. we generated pael-r transgenic mice and crossbred it with parkin knockout mice. pael-r transgenic mice showed modest alterations in dopamine metabolism and behavioral deficits without displaying obvious dopaminergic neuronal loss at the age of one year. however, when pael-r transgenic mice were crossbred with parkin knockout mice, the da neuronal loss was induced in a pael-r gene dosage-dependant manner. these results strongly support that pael-r accumulation substantially contributes to dopaminergic neurodegeneration in ar-jp. parkinson's disease, a common motor disorder, is caused by a degeneration of dopaminergic neurons in the substantia nigra. after dopamine denervation, an over-activity of glutamatergic pathways has been found and that is implicated in the neuropathology of parkinson's disease. previous study (lai et al., 2004) have found that application of an antisense oligodeoxynucleotide specific for nr1 have successfully knockdown the expression of nr1 gene expression in the striatum of 6-hydroxydopamine-lesioned rats. in the present study, modulation of gene expression of nr1 was re-addressed using a small interfering rna (sirna) specific for nr1. in pc12 cells, reductions of nr1 proteins after a single application of nr1 sirna were found by western blot experiments. and after one single application of nr1 sirna in the striatum of the lesioned rats, a significant reduction in apomorphine-induced rotation was found. slight reductions in the levels of nr1 immunofluorescence were found in the striatum after the sirna treatments. lai et al., 2004. neurochem. int. 45, 11-22. research funds: faculty research grant, frg/04-05/ii-27, hong kong baptist university ps2p-j178 homocysteine and parkinson's disease: effects of acute intranigral administration on dopaminergic system g. chandra, k.p. mohanakumar indian institute of chemical biology, kolkata, india homocysteine (hcy) is implicated in a number of geriatric multisystem disorders and patients with hyperhomocysteinemia exhibit profound neuropsychological abnormalities. parkinsonǐs disease (pd) patients receiving long-term l-dopa therapy are reported to have elevated plasma hcy levels. we studied whether hcy is neurotoxic to the nigrostriatal dopamine (da)-ergic system in sd rats. animals infused unilaterally in substantia nigra pers compacta (snpc) with hcy (0.25-1 mol in 1 l) showed dose dependent loss of da and its metabolites, in the ipsilateral striatum on 19th day. animals with 1 mol hcy exhibited significant motor disabilities and spontaneous and da-ergic drug-induced turning behaviors. in these animals a clear loss of neurons was visible in snpc, which were shown to be daergic by tyrosine hydroxylase immunoreactivity. intra-raphe infusion of hcy did not alter the neurotransmitter levels in the serotonergic perikarya or terminals. these results indicate the toxic potential of hcy to the da-ergic system and suggest that chronic l-dopa therapy in pd patients may further deteriorate the disease. ps2p-j179 ubiquitin proteasome system was impaired by the aggregate formation of mutant ␥pkc found in sca14 takahiro seki 1 , takayuki shimahara 1 , naoko adachi 2 , naoaki saito 2 , norio sakai 1 1 dept. mol. pharmacol. neurosci., grad. sch. biomed. sci., hiroshima univ., hiroshima, japan; 2 lab. mol. pharmacol., biosig. res. ctr., kobe univ., kobe, japan we have previously demonstrated that several mutant protein kinase c gamma (␥pkc), found in several families of spinocerebellar ataxia type 14 (sca14), are susceptible to cytoplasmic aggregation and cause cell death in cho cells, indicating that this property is involved in the etiology of sca14. however, the relationship between the aggregate formation of mutant ␥pkc and cell death remains unclear. accumulating evidences indicate that the impairment of the ubiquitin proteasome system (ups) is related to the pathogenesis of many neurodegenerative disorders. therefore, we examined whether the aggregate formation of mutant ␥pkc affects ups function. the immunoreactivities for ubiquitin and proteasome were intensely accumulated in the aggregates of mutant ␥pkc. decreased proteasome activities were also observed in cells having aggregated mutant ␥pkc. these results indicate that the aggregation of mutant ␥pkc exert cytotoxic effect via the impairment of ups. it is well known that oxidant stress is involved in many pathologic conditions including brain ischemia and neurodegenerative diseases. recently, however, another type of stress, endoplasmic reticulum (er) stress has also been reported to be associated with such diseases. er stress is characterized by accumulation of unfolded proteins in the er that is caused by inhibition of protein modification, disturbance of ca 2+ homeostasis or oxygen deprivation. we recently reported that targeting disruption of herp, a novel er stress-related gene, caused f9 cells vulnerable to er stress. using these cells, we developed a screening system for molecules that suppress er stress. approximately 300 compounds have been screened, and we found some molecules that protect human neuroblastoma cells against er stress and oxidative stress. we speculate that this system could provide novel therapeutic targets to the er stress and oxidative stressrelated diseases. toshiyuki araki 1 , yo sasaki 2 1 department of peripheral nervous system research, national institute of neuroscience, ncnp, tokyo, japan; 2 washington university school of medicine, st. louis, missouri, usa axonal degeneration which is observed in a variety of neuropathological conditions or physical damage to axons is a self-destructive program that is independent from programmed cell death. we previously reported that increased nicotinamide adenine dinucleotide (nad) production by the overexpression of nicotinamide mononucleotide adenylyltransferase1 (nmnat1) or exogenously applied nad can protect neurites from degeneration caused by mechanical or neurotoxic injury of neuronal cells. the mammalian nad biosynthesis is mediated by at least 6 different kinds of enzymes and each enzyme converts different substrate to nad or its precursors. here we investigated whether overexpression of these enzymes or exogenous application of nad precursors protects neurites from degeneration through increased supply of nad. cocaine is considered to affect spine morphology and the composition of postsynaptic density (psd) of medium spiny neurons in nucleus accumbens (nac). we examined the accumulation of several proteins altered by cocaine challenge after withdrawal of repeated cocaine administration in psd fraction of rat nac at different time points. total psd protein yield was decreased at 10 min, but next increased at 2 h and returned to basal at 6 h after cocaine challenge. actin showed a similar pattern but was maintained at high level at 6 h. both psd-95 and glur1 were increased between 2 h and 6 h like actin. by contrast, some proteins such as drebrin were decreased after the peak at 2 h. interestingly, the 20 s proteasome subunit demonstrated a dramatic upregulation at 2 h. these data suggest that the composition of psd proteins is regulated by proteasome activity as well as actin cycling. it is possible that some proteins may be removed from psd by proteasome following transient requirement for organizing psd in the nac of chronic cocaine-administrated animals. ps2p-k184 effects of mdma and 5-meo-dipt on serotonin transporter and dopamine transporter yosuke yamauchi, takaya izumi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharm. sci., kyoto univ., kyoto, japan by two electrode voltage-clamp recordings from xenopus oocytes heterologously expressing serotonin transporter (sert) or dopamine transporter (dat), the effects of two addictive agents, 3,4methylenedioxymethamphetamine (mdma) and 5-methoxy-n,ndiisopropyltryptamine (5-meo-dipt), on sert and dat were examined. as previously reported, mdma (0.3-10 m) dose-dependently induced transport-associated, inward current response in the sertexpressing cells. interestingly, mdma-induced current response was also observed in dat-expressing cells. on the other hand, 5-meo-dipt (0.3-300 nm) evoked an outward current response in sertexpressing cells similarly to that of selective 5-ht reuptake inhibitors. no current response was observed when 5-meo-dipt was applied to dat-expressing cells. these results suggest that mdma is transported not only by sert but also by dat, and that 5-meo-dipt suppresses the spontaneous transport activity of sert. junichi kitanaka 1 , nobue kitanaka 1 , tomohiro tatsuta 1,2 , yoshio morita 2 , motohiko takemura 1 1 department of pharmacology, hyogo college of medicine, nishinomiya, japan; 2 department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan we examined the effects of pretreatment with clorgyline on morphine-induced behavioral changes and antinociception. a single administration of morphine (30 mg/kg, i.p.) to male icr mice induced a hyperlocomotion. the anova analysis revealed statistical significance of a morphine effect (hyperlocomotion) and of a clorgyline pretreatment x morphine interaction effect (inhibition), but not of an effect of clorgyline pretreatment. clorgyline pretreatment itself did not affect the spontaneous locomotion. clorgyline at a dose of 0.1 mg/kg but not other doses tested significantly potentiated morphine-induced antinociception evaluated by tail flick but not hot plate test. clorgyline at the doses of 1 and 10 mg/kg significantly inhibited dopamine and serotonin metabolism. these results suggest that clorgyline showed its inhibitory effect on morphine-induced hyperlocomotion, but not antinociception, through mao inhibition. recent studies in our laboratory have shown that methamphetamine (meth)-induced hyperlocomotion and behavioral sensitization in mice were inhibited by clorgyline, an irreversible monoamine oxidase inhibitor. in this presentation, the effect of clorgyline pretreatment on meth reward was assessed by conditioned place preference (cpp) paradigm, using an apparatus developed with supermex ® sensors. although intact male icr mice showed a significant cpp for meth (0.5 mg/kg, i.p.), pretreatment with subchronic clorgyline (0.1-10 mg/kg, s.c.) did not affect the magnitude of cpp. pretreatment with clorgyline significantly decreased apparent dopamine and serotonin turnovers in the striatum in a dose-dependent manner. these results indicated that clorgyline pretreatment did not influence meth reward in mice. of lobeline pretreatment on methamphetamine-induced stereotypy and monoamine metabolism in mice motohiko takemura 1 , nobue kitanaka 1 , tomohiro tatsuta 1,2 , yoshio morita 2 , junichi kitanaka 1 1 department of pharmacology, hyogo college of medicine, nishinomiya, japan; 2 department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan the effects of lobeline, an alkaloid constituent of indian tobacco, on methamphetamine (meth)-induced stereotypy and monoamine metabolism were investigated in male icr mice. pretreatment with lobeline (3.0-30 mg/kg, i.p.) 15 min prior to drug challenge significantly decreased an intensity of stereotypies and increased its latency to onset in a dose-dependent manner. in saline challenge groups, doses of lobeline examined did not affect the spontaneous locomotion nor induce any stereotyped behaviors. the range of lobeline doses examined except 30 mg/kg did not affect apparent monoamine turnovers in the brain regions including striatum 20 min after drug challenge. these results suggested that the inhibitory effect of lobeline (3.0-10 mg/kg) on meth-induced stereotypy did not attribute to the change in the brain monoamine metabolism. kazuto sakoori, niall murphy riken bsi, wako-shi, japan previously we showed that endogenous nociceptin suppresses drug reward. here, we examined the effect of blockade of nop receptors on methamphetamine (meth) induced behavioral sensitization in order to understand the role of endogenous nociceptin in the chronic response to addictive drugs. first, nop receptor ko and wt mice were treated with 1 mg/kg meth and locomotor activity measured daily for 14 days. wt mice showed gradually increasing sensitivity to meth with repeated treatment of meth, whereas nop receptor knockout mice did not. next, 5 nmol ufp-101 (a nop receptor antagonist) and 1 mg/kg meth were co-administrated to mice and locomotor activity measured daily for eight days. ufp-101 strongly suppressed locomotor activity. thus, it was unclear if ufp-101 suppressed behavioral sensitization to meth during chronic drug treatment. however, when challenged with meth after four or more days without treatment, ufp-101 co-administrated mice showed a lower locomotor response. these results suggest that endogenous nociceptin facilitates the plastic changes induced by chronic treatment with addictive drugs. the influence of olanzapine (a d2dopamine receptor antagonist) on the morphine-induced conditioned place preference (cpp) in male and female mice was investigated in the present study. subcutaneous (s.c.) injection of morphine (1-10 mg/kg, three drug sessions) induced place preference both in male and female mice. intraperitoneal (i.p.) administration of olanzapine (0.5-5 mg/kg) induced place aversion (cpa) in female mice but not in male mice. administration of olanzapine (1, 2.5 and 5 mg/kg, i.p.) reduced both the acquisition and expression of morphine-induced cpp in male and female mice. however, olanzapine (5 mg/kg, i.p.) caused more than 80% mortality in female but not male mice. the effects of olanzapine were reversed by l-arginine (20 mg/kg, i.p.) pre-administration. in conclusion, it seems that olanzapine reduced morphine effects in part via a nitric oxide (no) mechanism. feed-forward associative learning (ffal) theory of cerebellar motor learning proposed by the author presumes that higher motor centers have place-coding systems and the same systems are shared by the cerebellum. when a new motor learning proceeds with respect to a certain movement, previous learning results of the movement will turn out to be modified or erased. ffal theory presumes that transferred memory from the cerebellar cortex to nucleus will serve as the maintenance of the previous learning. from this line, many aspects of saccadic adaptation are successfully demonstrated by computer simulation based on the theory. another theoretical issue is the credit assignment problem of motor error. a motor error is generally an integrated result of maladjusted multiple learning elements, and is to be decomposed to each element credit. this problem naively leads to an idea of a dual redundant system for movement, one for execution and the other for error decomposition. ffal theory naturally and simply resolves the credit assignment problem and demonstrates a computer simulation of motor learning of multi joint movement system, using the place-coding hypothesis. ps2p-d191 regulation of camp responsive element binding protein to stress in rat amygdala and hippocampal formation the department of anatomy and histology, shanghai medical school, fudan university, china amygdala (am) and hippocampal formation (hf) are important structures relating with emotional learning and memory. transcription factor, camp-responsive element binding protein (creb) in am and hf plays important roles in memory modulating processes. creb is a nuclear protein and is wldely accepted as prototypical stimulusinducible transcription factor. creb is activated in response to a vast array of physiological stimuli and then becames phosphorylated creb (pcreb). neurophysiological and neuropharmacological studies said that creb may regulate gene transcription and protein synthesis to maintain the long term and sustaining changing of synaptic efficiency during the long-term process of synaptic plasticity. but we cannot tell exactly via what kind of neurons in am and hf creb regulate these processes. we used the animal model, forced swimming (fs) as emotional stimuli and the experiment methods such as, immunocytochemistry, western-blotting with anti-pcreb antibody. the distributing profiles and changing rules of pcreb immunoreactive nuclei in amygdala and hippocampal formation of both control and experiment groups were investigated. the neuronal types of pcreb immunoreactive nuclei were analyzed by double-labelling immunocytochemistry with anti-pcreb, anti-glu and anti-pv antibodies. the results were: (1) the number of pcreb immunoreactive neuclei and total amount of pcreb in the subnuclei of rat amygdala, dentate gyrus (dg) and cornu ammonis 3(ca3) were increased after fs. the rule of this kind of changing was of region-and time-specific. (2) pcreb immunoreactive neuclei were expressed in glutamate immunoreactive neurons and were devoid in interneurons. these results suggested that pcreb in limbic system regulated the fs process and the regulation was finished via exciting neurons, glutamate neurons. hideto takahashi 1,2 , tomoaki shirao 1 1 dept of neurobiol & behav.; 2 ercgsm, gunma univ. grad. sch. of med., maebashi, japan dendritic spines are developmentally-regulated and activitydependent polymorphic structures based on actin cytoskeleton. drebrin is a spine-rich actin-binding protein regulating spine morphogenesis during development. here we find that chronic blockade of ampa receptors (ampar) inhibits synaptic drebrin clustering during development of hippocampal neurons, but not that of nmdar. further, the analysis of fluorescence recovery after photobleaching for egfp-drebrin a reveals that only 22.7 ± 3.0% of drebrin in the spine is stable, with a turnover time of 5.8 ± 0.4 min. blockade of ampar by 20 m cnqx reduces the population of stable drebrin (8.9 ± 3.6%), and has no effect on a turnover time. on the other hand, blockade of nmdar by 100 m ap5 has no effect on the population of stable drebrin, whereas shortens a turnover time (4.0 ± 0.3 min). these data suggest that ampar activities increase the binding capacity of drebrin in spines, and therefore promote drebrin clustering at spine synapses. instead, nmdar activities regulate spine-shaft shuttling of drebrin. itsuko nihonmatsu 1 , yoshito saitoh 1 , kaoru inokuchi 1,2 1 mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; 2 crest, jst, tokyo, japan dendritic protein synthesis requires dendritic localization of mrnas in neurons. however, ultrastructural localization of these mrnas have not been well described. here we employed in situ electronmicroscopic technique to examine the precise localization of ␣camkii mrna in dendrites. ␣camkii mrna was located at the specific sites of dendritic shafts of pyramidal neurons, close to the spines, rather than in a diffused manner. we observed an increase in the ␣camkii mrna signals at the synaptic layer undergone l-ltp in the hippocampal dentate gyrus in unanesthetized freely moving rats. the increase was transient and returned to the basal level at 1 h. the alteration in the ␣ camkii mrna localization in dendrites may reflect a functional change in the translational apparatus along with synaptic plasticity. reiko okubo-suzuki 1,2 , daisuke okada 1 , kaoru inokuchi 1,2,3 1 mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; 2 yokohama natl. univ. environment information sci., kanagawa, japan; 3 crest, jst, japan late-phase long-term potentiation (l-ltp) depends on de novo protein synthesis. synaptopodin (synpo), an f-actin-associated protein, increases in the activated synapses following l-ltp induction. spine volume and f-actin content in the spines also increase during l-ltp. to reveal the roles synpo plays in the regulation of spine volume and f-actin content, we examined synpo-egfp (se) localization and spine volume in the hippocampal neurons using time-lapse confocal imaging techniques. se-overexpression did not alter spine volume, but the amount of se in spines positively correlated with the spine volume. pharmacological activation of the nmda receptors increased both spine volume and synpo content in spines. furthermore, experiments with ptk2 cells indicated that synpo stabilizes f-actin. these results suggest that synpo synthesized in soma and transported into the activated spines following l-ltp induction stabilizes spine f-actin that may lead to the maintenance of increased spine volume. mineo matsumoto 1 , mitsutoshi setou 1,2 , kaoru inokuchi 1 1 laboratory for molecular gerontology, mitils, japan; 2 laboratory for nano-structure physiology, nips, japan subcellular localization of rna is an efficient way to localize proteins to a specific region of a cell. a requirement for dendritic rna localization and subsequent local translation has been demonstrated in several forms of experience-dependent synaptic plasticity. in spite of several attempts to identify these rnas, the population of rna species present in dendrites as a whole has not been well described. here we show the results of microarray analyses with rnas isolated from rna granule or synaptosome fractions prepared from the rat brain. these analyses revealed the complex nature of the dendritic rna population, which included rnas that were not expected to be in the dendrites. neural activity caused by an electroconvulsive shock triggered a redistribution of the dendritic transcriptome towards the synaptosome, a translationally active region. our results suggest that the redistribution of dendritic rnas is one of the mechanisms regulating local translation in response to synaptic inputs. ps3a-a005 an activity that traps vesl-1s protein into spines serves as synaptic tag synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific late-phase plasticity, but it has not yet been substantiated. original idea of synaptic tagging is supposed to regulate protein entry into synaptic region including spines. using live-imaging techniques, we measured entry of vesl-1s-egfp into spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as the synaptic tag in many criteria. ve trapping required synergistic activation of postsynaptic no-pkg pathway and an activity abolished by ttx at 1 m, but not 50 nm. because 50 nm ttx is supposed to suppress na channels only postsynaptically, we concluded that ve trapping is a hebbian-like process that requires both pre-and postsynaptic activities. however, their coincidence time window was far wider (hrs) than that of early-phase plasticity, suggesting a requirement of persistently synchronized, rather than transiently coincident, activities, and a possibility of metaplastic states for late-phase plasticity. ps3a-a006 acute effects of dehydroepiandrosterone sulfate (dheas) on the synaptic transmission and plasticity in rat hippocampal slices yuxia xu 1 , ling chen 2 , masahiro sokabe 1,3,4 1 dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; 2 dept. physiol., nanjing med. univ., nanjing, china; 3 sorst cell mechanosening, jst, nagoya, japan; 4 dept. mol. physiol., nips, okazaki, japan the neurosteroid dehydroepiandrosterone sulfate (dheas) is known to improve memory and learning in mammals. recently we report that chronic administration of dheas facilitates the induction of ltp in the rat hippocampus. to elucidate the underlying synaptic mechanism of the dheas effects, we examined in this study the acute effects of dheas on the synaptic transmission and plasticity at the ca1 region in rat hippocampal slices. an application of 0.1 dheas for 10 min to the slice augmented instantly the epsp, which was terminated within 30 min. however, even 1 h after the drug application, a subthreshold tetanus could induce ltp without alteration of ppf. this facilitating effect of dheas on ltp induction was blocked by a coapplication of a nmda receptor antagonist with dheas for 10 min, suggesting that the dheas effect involves a sustained modulation of the postsynaptic signaling mediated by nmda receptor. xiaoniu dai 1 , ling chen 2 , masahiro sokabe 1,3,4 1 dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; 2 dept. physiol., nanjing med. univ., nanjing, china; 3 sorst cell mechanosensing, jst, nagoya, japan; 4 dept. mol. physiol., nips, okazaki, japan to know whether 17␤-estradiol (e2) can protect ca1 neurons from functional deficit due to ischemia, adult male wistar rats were subjected four-vessel occlusion (4vo) for 10 min, and the effect of e2 against this ischemic injury was examined. the electrophysiological properties of ca3-ca1 synapses were examined by a real-time optical recording method 7 days after ischemia. the ischemic brain showed a decreased synaptic transmission and an impairment of ltp induction but no alteration in paired-pulse facilitation. administration of e2 (1 mg/kg) 3 h before 4vo was able to protect ca1 neurons from these ischemic synaptic dysfunctions. the estrogen receptor-␣ selective agonist ppt (2 mg/kg) produced a similar protective effect, but the estrogen receptor-␤ agonist dpn (8 mg/kg) did not. above results suggest that e2 can protect neurons not only from cell death but also from functional damages caused by cerebral ischemia. ps3a-a008 non-genomic rapid effects of estradiol on hippocampal synapses: multi-electrode dish analysis kohei nakajima 1 , mari ogiue-ikeda 1 , yuki oishi 2 , suguru kawato 1,2 1 department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo at komaba, tokyo, japan; 2 department of physics, university of tokyo, tokyo, japan estradiol has a non-genomic, rapid effect on synaptic transmission, which is manifested within seconds to minutes. recently, hippocampal neurons were shown to synthesize estradiol de novo, and to express estrogen receptor ␣ (er␣) at synapses. although these results imply that estradiol rapidly modulates synaptic plasticity through synaptic er␣, there are few electrophysiological evidence about it. here we investigated effects of estradiol on ltd by using wild type, er␣ hetero and er␤ hetero mouse hippocampal slices with a multi-electrode dish (med, panasonic). med enabled us to measure epsps in ca1, ca3, and dentate gyrus simultaneously. hippocampal slices were perfused with estradiol before nmda-induced ltd. we found that estradiol enhanced ltd both in wild type and er␤ hetero mouse, but not in er␣ hetero mouse. our data suggested non-genomic rapid action of estradiol through synaptic er␣. withdrawn ps3a-a010 morphological changes of dendritic spines mediated by glucocorticoid receptor (gr) in rat hippocampus yoshimasa komatsuzaki 1 , gen murakami 2,3 , tetsuya kimoto 2,3 , suguru kawato 2,3 1 college of humanities and sciences, nihon university, tokyo, japan; 2 department of biophysics and life sciences, university of tokyo, tokyo, japan; 3 crest, jst, japan modulation of hippocampal synaptic plasticity by glucocorticoids has been attracting much attention, due to its importance in stress responses. dendritic spines are essential for memory storage processes. here we investigated the effect of dexamethasone (dex), a specific agonist of glucocorticoid receptor (gr), on density and morphology of dendritic spines in adult male rat hippocampus by imaging of lucifer yellow-injected spines in slices. the application of 100 nm dex induced rapid modulation of the density and morphology of dendritic spines in ca1 pyramidal neurons within 1 h. the total spine density increased from 0.88 spines/m to 1.36 spines/m. dex significantly increased the density of thin and mushroom type spines, however only a slight increase was observed for stubby and filopodium type spines. because the presence of 10 m cycloheximide, an inhibitor of protein synthesis, did not suppress the dex effect, these responses are probably non-genomic. hideki tamura 1 , yuji ikegaya 2 , sadao shiosaka 1 1 division of structural cell biology, naist, nara, japan; 2 laboratory of chemical pharmacology, university of tokyo, tokyo, japan the capacity of activity-dependent synaptic modification is essential in processing and storing information, yet little is known about how synaptic plasticity alters the input-output (i-o) conversion efficiency at the synapses. in the adult mouse hippocampus in vivo, we carefully compared the i-o relationship, in terms of presynaptic activity levels versus postsynaptic potentials, before and after the induction of synaptic plasticity and found that synaptic plasticity led synapses to respond more robustly to inputs, that is, synaptic gain was increased as a function of synaptic activity with an expansive, power-law nonlinearity, i.e., conforming to the so-called gamma curve. in extreme cases, long-term potentiation (ltp) and depression (ltd) coexist in the same synaptic pathway with ltp dominating over ltd at higher levels of presynaptic activity. these findings predict a novel function of synaptic plasticity, i.e., a contrast-enhancing filtering of neural information through a gamma correction-like process. research funds: 21st century coe research ps3a-a012 actin organizations within single dendritic spines in ca1 pyramidal neurons studies with two-photon photoactivation naoki honkura, masanori matsuzaki, haruo kasai center for disease biology and integrative medicine, faculty of medicine, the university of tokyo, japan the major cytoskeleton of dendritic spines is filamentous actin (factin). we have here investigated sub-spine actin organizations using two-photon photoactivation of pa-gfp fused with ␤-actin in rat ca1 pyramidal neurons. we found segregated and discontinuous organizations of two pools of f-actin, dynamic and stable pools, which turned over with time constants of 1.2 min and 17 min, respectively. fractions of the stable f-actin pool were greater in larger spines, therefore, the entire f-actin pool was more stable in larger spines. we succeeded in visualizing a retrograde flow of f-actin in the dynamic pool from the apex to the base of spine, and found that both the speeds (0.2-1.2 um/min) and lengths (0.2-0.7 um) of the f-actin flow were greater in spines with larger head volumes. moreover, spine heads rapidly shrank when actin polymerization was blocked by latrunculin a, suggesting that the rate of actin polymerization in each spine actively and continuously determines the volume of spine head via the length of f-actin. tomoharu nakamori 1 , katsushige sato 2 , kohichi tanaka 1 , hiroko hamazaki 1 1 mol. neurosci., tmdu, tokyo, japan; 2 physiology, tmdu, tokyo, japan the visual wulst (vw) in the thalamofugal pathway in chicks is known to have a critical role in the visual learning. to understand the function of the vw in the learning process of imprinting, we investigated the neuronal activity of vw region in chick brain. the slice stained with a voltage-sensitive dye was prepared for a multiple-site optical recording. when chicks were reared in quasi-dark condition, the extent and amplitude of response induced by electrical stimulation were different between at 1 or 4 days post-hatching (p1 or p4), and at p7. this corresponds to behavioral data showing that chicks have high ability of visual learning in imprinting behavior until p4, but they lose this ability at p7. in addition, the light-exposed chick showed larger optical response than the dark-rearing one. the optical response in the vw was partly inhibited by the glutamate-and gaba-receptor antagonists. these results suggest that the glutamatergic as well as gabaergic neurons are active in the area including vw and that the neuronal activity of vw affects the learning ability for imprinting. withdrawn ps3a-b015 effect of estrogen on hippocampus in male and female mice takanori sugawara 1 , shinji hayashi 1 , victoria luine 2 1 graduated school of integrated sience, yokohama city university, yokohama, japan; 2 department of psychology, hunter college, city university of new york, new york, usa we examined structural difference in the hippocampal neurons with golgi stain among the male, the female and the female treated with estrogen neonatally. the mice were gonadectomized and received 17 ␤-estradiol (e2) or oil-vehicle injections at adult before golgi impregnation. spine densities 10 m of apical dendrites of the pyramidal neurons in the hippocampus ca1 region were calculated with categorization into three shapes, i.e., mushroom type with large head, thin type and filopodia-like type. as a result, only in the female not estrogen treated neonatally, the mushroom type and total spine densities were increased but the thin type spine density was decreased by e2 treatment in adult. the present results indicate that estrogen given at adult induces an enlargement of spine to mushroom type and generates new spines only in the female mice not treated with estrogen neonatally. thus, dendritic spine formation seems sexually dimorphic and depends on the sex steroid environment during the neonatal period. jun-ichi goto 1,2 , takafumi inoue 2,3 , akinori kuruma 1 , katsuhiko mikoshiba 1,2,3 1 lab. developmental neurobiology, brain science inst., riken, saitama, japan; 2 div. molecular neurobiology, inst. medical science, univ. tokyo, tokyo, japan; 3 calcium oscillation project, icorp-sorst, jst, tokyo, japan changes in synaptic efficacy at the parallel fiber (pf)-purkinje cell (pc) synapse are postulated to be a cellular basis for motor learning. although long-term efficacy changes lasting more than an hour at this synapse, i.e., long-term potentiation and depression, have been extensively studied, relatively short lasting synaptic efficacy changes, namely short-term potentiation (stp) lasting for tens of minutes, have not been discussed to date. here we report that this synapse shows an apparent stp reliably by a periodic burst pattern of homo synaptic stimulation. this stp is presynaptically expressed, since it accompanies with a reduced paired-pulse facilitation and is resistant to postsynaptic ca 2+ reduction by bapta injection or in p/q-type ca channel knockout cerebella. this novel type of synaptic plasticity at the pf-pc synapse would be a clue for understanding the presynaptic mechanisms of plasticity at this synapse. aya ishida, wataru kakegawa, michisuke yuzaki department of physiology, keio university, tokyo, japan mitogen-activated protein kinase (mapk) cascade is thought to be essential for the synaptic plasticity and learning. in the hippocampus, three different mapk subfamilies, including extracellular signalregulated kinase (erk), p38 mapk and c-jun nh2-terminal protein kinase (jnk), have been shown to selectively regulate different forms of synaptic plasticity -long-term potentiation (ltp), longterm depression (ltd), and depotentiation after ltp, respectively. although erk was previously shown to play a role in cerebellar ltd in cultured purkinje cells, the role of mapks has not been systemically studied. here, we examined the effect of specific inhibitors of three different mapks on ltd by patch-clamp recordings from cerebellar slices. we found that u0126, a specific inhibitor for erk activation, significantly inhibited ltd induction, whereas sb203580 and sp600125, antagonists for p38 mapk and jnk, respectively, had no effect. therefore, unlike hippocampal ltd, cerebellar ltd was dependent on erk, suggesting involvement of different intracellular downstream pathways. ps3a-b018 regulation of ampa receptor trafficking by aaa atpases in cerebellar purkinje cells: are nsf and vcp playing complementary or antagonistic roles? thomas launey 1 , chou-chi li 2 , yumiko motoyama 1 , junko yamaoka 1 , masao ito 1 1 riken brain sci. inst., japan; 2 national cancer institute, nih, ma, usa the number of postsynaptic ampa receptors (ampar) is regulated by interactions with multiple protein complexes, throughout its synthesis, maturation, transport, synaptic insertion and degradation. aaa atpases influence several of these stages, the most extensively studied being nsf's contribution to ampar trafficking. in cerebellar purkinje cell (pc), we show that valosin containing protein (vcp), an atpase with high homology to nsf, is bound to ampa receptors in pc's dendritic compartment. following glur2 co-ip from molecular layer, vcp was detected by ms/ms and by monoclonal anti-vcp. pull-down assay showed a direct interaction between vcp and glur2 c-term domain, requiring vcp n-term domain and both the nsf and pdz binding domains of glur2. glur2 phospho-ser880 promotes vcp complex dissociation, suggesting a relation with synaptic plasticity. further, pep2m-related peptides, thought to interfere specifically with nsf-regulated ampar trafficking, also blocked the glur2-vcp interaction. yuichi kitagawa 1,2 , shin-ya kawaguchi 1,2 , tomoo hirano 1,2 1 dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; 2 crest, jst, kawaguchi, japan at inhibitory synapses on a cerebellar purkinje neuron (pn), postsynaptic depolarization induces long-lasting potentiation of the gaba a receptor (gaba a r) responsiveness (rebound potentiation: rp). previous studies have clarified the molecular mechanism regulating rp induction. whether rp is induced or not is determined by the balance of activities of protein kinases (camkii and pka) and phosphatases (pp-1 and calcineurin). to understand the complex behavior of biochemical reactions systematically, a kinetic simulation model to analyze the behaviors of signaling network was developed. computer simulation reproduced the bistable states of gaba a r phospholyration according to stimulation patterns, which apparently corresponded to whether rp was induced or not. we further studied the systematic property of the molecular network, and obtained several experimental predictions. these possibilities were evaluated by experiments such as immunocytochemistry using cultured pns. ps3a-b020 long-term depression of synaptic transmission in a songbird motor nucleus essential for song learning yuki haruta, yachun huang, neal hessler vocal behavior mechanisms riken brain science institute, japan in order to fully understand the neural basis of song learning, it is critical to characterize forms of synaptic plasticity that could be involved in this process. we previously reported that, in synapses of the song motor nucleus ra, participation of postsynaptic nmda receptor nr2b subunits and presynaptic transmitter release both decrease from young birds to adults. here, we tested whether synaptic function could be modified in a similar way by acute stimulation. after pairing slight postsynaptic depolarization with presynaptic stimulation, ltd was reliably induced at both hvc and lman inputs in juvenile birds from 35 to 42 days old. this depression required activation of postsynaptic nmda receptors, and was expressed by decreased transmitter release, which required activation of cannabinoid receptors. no ltd could be induced in normal birds over 60 days old, when song learning is nearly complete, but ltd remained possible in birds over 60 days old who had been isolated from song tutors, and thus retained the capacity for learning. ps3a-b021 involvement of ca 2+ -permeable ampar in the repetitive-ltp induced synaptic enhancement (rise) yukiko ueno, keiko tominaga-yoshino, akihiko ogura graduate school of frontier biosciences, university of osaka, osaka, japan we showed previously that 3 exposures to glu of cultured rat hippocampal slices at 24 h intervals produced a long-lasting enhancement in synaptic strength accompanied by synaptogenesis (rise). we examined here whether the conversion of ampar subunits occurred during the development of rise. immunochemical staining for ampar subunits, glur1 and glur2, showed that the number of glur1-positive puncta increased transiently after the repeated glu exposures, whereas the number of glur2-positive puncta increased gradually and persistently. jstx (a ca 2+ -permeable ampar blocker) suppressed fepsp amplitude recorded at ca3-ca1 synapses by 20-40% in the period corresponding to the transient increase of glur1-positive puncta. this transient increase should represent the delivery of ca 2+ -permeable (glur2-lacking/glur1-including) ampar to synaptic sites. furthermore, jstx application at that period blocked the rise production. these results suggest that the transient delivery of ca 2+ -permeable ampar to synaptic sites is involved in the rise production. yoshihiro egashira, tsunehiro tanaka, yuji kamikubo, yo shinoda, keiko tominaga-yoshino, akihiko ogura osaka univ. grad. sch. frontier biosciences, toyonaka 560-0043, japan long-lasting synaptic plasticity, the cellular basis of long-term memory, is assumed to be associated with protein synthesis. using cultured rat hippocampal slices, we previously found that a long-lasting synaptic enhancement coupled with an increase in the number of synaptic structures was established after 3 inductions of ltp, not after its single induction. this synaptic enhancement required protein synthesis for its establishment. we recently found an apparently mirror-image phenomenon; 3 inductions of ltd led to a long-lasting synaptic decrement coupled with a decrease in the numbers of synaptic structures. to know whether this synaptic decrement also requires protein synthesis, we induced ltd 3 times (24 h intervals) by applications of dhpg (a type i mglur agonist), during or after which anisomycin (a protein translation blocker) was applied. we found that anisomycin did not block the induction of ltd but blocked the establishment of the long-lasting synaptic decrement. haruo mizutani, tetsuya hori, tomoyuki takahashi department of neurophysiology, graduate school of medicine university of tokyo, tokyo, japan bath-application of 5-ht (10 m) attenuated the amplitude of evoked epscs and facilitated paired-pulse ratio without affecting the miniature epsc amplitude, suggesting that its site of action is presynaptic. the 5-ht 1b receptor agonist cp93129 mimicked the presynaptic inhibitory effect of 5-ht. 5-ht 1b receptor antagonist nas-181 reversed the 5-ht inhibitory effect, indicating that the 5-ht induced inhibitory effect occurs by mediating 5-ht 1b receptors. the presynaptic inhibitory effect of 5-ht became weaker as animals matured. in whole-cell recordings from calyceal presynaptic terminals, 5-ht attenuated voltage-dependent calcium currents, but had no effect on potassium currents. this 5-ht effect was characterized with a marked desensitization, but sustained under the fast calcium chelating agents, bapta. these results suggest that 5-ht, upon activating 5-ht 1b receptors, inhibits presynaptic calcium channels thereby inhibiting transmitter release and induces receptor desensitization by calcium influx at the immature calyceal synapse. takako ohno-shosaku 1 , masato ano 1 , yuki hashimotodani 2 , tadasato nagano 3 , masanobu kano 3 1 dept. impair. study, grad. sch. med. sci., kanazawa univ., kanazawa, japan; 2 dept. neurophysiol., grad. sch. med., osaka univ., osaka, japan; 3 dept. cell. neurosci., grad. sch. med., osaka univ., osaka, japan retrograde endocannabinoid signal contributes to activitydependent modulation of synaptic transmissions in various brain regions. endocannabinoid release is triggered by depolarizationinduced elevation of intracellular calcium level or activation of gq-coupled receptors. here we report that nmda receptors can also contribute to generation of endocannabinoid signal. inhibitory postsynaptic currents (ipscs) were recorded in cultured hippocampal neurons prepared from newborn rats. application of nmda induced a transient suppression of cannabinoid-sensitive ipscs but not cannabinoid-insensitive ipscs. the nmda-induced suppression of ipsc was blocked by a cannabinoid receptor antagonist. these results indicate that activation of nmda receptors induces the endocannabinoid release, and suppresses the inhibitory synaptic transmission through activation of presynaptic cannabinoid receptors. the most caudal region of the rat spinal cord, the conus medullaris has a simple anatomical feature, which lacks ventral as well as dorsal root fibers and somatic motor neurons in the ventral horn. a small number of neurons distribute around the central canal, and some of them are nitric oxide synthase (nos) positive. a dense distribution of nerve fibers immunoreactive to cgrp, sp, and npy was found in dorsal part of the conus medullaris similarly to that of other spinal cord levels. in addition, enk-, 5-ht-, and th-immunoreactive varicose fibers were richly distributed throughout the sectional plane. to analyze this unique structure may provide valuable information on the basic neural cytoarchitecture and fiber connections of the spinal cord, particularly for the intraspinal circuitry. for this purpose, we made an electron microscopic study using nadph-diaphorase histochemistry combined with immunohistochemistry for neuronal markers. adenosine has been known to be a neuro-modulator in the nervous systems and four types of adenosine receptor are identified (a1, a2a, a2b and a3). adenosine a1 and a3 receptors have been reported to inhibit high-threshold ca channel currents in neurons. to investigate the interaction between adenosine a1 and a3 receptors in rat striatum neurons in culture, l-type ca channel currents were recorded by whole-cell clamp method before and after administration of a1 agonist (cpa) and a3 agonist (2-cl-ib-meca). ca currents were decreased after administration of low concentration of cpa and 2-cl-ib-meca as reported previously. although ca currents were decreased by 2-cl-ib-meca in the presence of cpa, ca currents applied with cpa were not decreased on cells in the presence of 2-cl-ib-meca. at administration of cpa and 2-cl-ib-meca on cells simultaneously, ca currents were not decreased. these results suggested that adenosine a3 receptor may inhibit adenosine a1 receptor throughout a intracellular pathway in neurons. ps3a-c028 influence of extracellular gaba and taurine to gaba a receptor-mediated actions in radially migrating cortical plate cells with identified by in utero electroporation t. furukawa 1 , j. yamada 2 , k. inoue 1 , y. yanagawa 3 , a. fukuda 1, 2 1 dept. physiol., hamamatsu univ. sch. med., japan; 2 dept. biol. info. process, grad. sch. elec. sci. & tech., shizuoka univ., hamamatsu, japan; 3 dept. developmental and integrative neurosci., gunma univ. sch. med., gunma, japan it is well known that role of gaba a -r mediated actions is important for early cns development. the radially migrating cells may affected by the actions. gaba content in the brain of gad67-gfp knock-in mouse decrease compared with the wild type mice. therefore, we investigate the influence of the circumferential gaba concentration to radially migrating cells. furthermore, as it was known that gaba a -r is affected by taurine, the influence of taurine to radially migrating cells was also investigated. there was no significant difference in distribution of radially migrating cells that was labeled by means of electroporation. evoked gaba a -r mediated currents of labeled cells had dose-dependent manner and had no differences among genotypes. therefore, we have examined the influence of circumferential taurine to gaba a -r mediate actions. takashi hayakawa 1 , hiroyuki hioki 1 , kouichi nakamura 1,2 , hisashi nakamura 1 , takeshi kaneko 1,2 1 dept. morphol. brain sci., grad. sch. med., kyoto univ., kyoto, japan; 2 crest, jst, kawaguchi, japan we previously reported that almost all vesicular glutamate transporter 3(vglut3)-immunoreactive (ir) cells were also gabair in neocortex and choline acetyl transferase (chat)-ir in caudate-putamen in rat. although, in dorsal and median raphe nuclei, many vglut3-positive cells showed immunoreactivity for 5-hydroxytryptamine (5ht), a significant proportion (12.3%) of vglut3-postive cells was 5ht-negative. in this study, triple immunofluorescence staining was performed for vglut3, 5ht and one of the following proteins: neuronal nuclear antigen (neun), glial fibrillary acidic protein (gfap), glutamic acid decarboxylase (gad67) and tyrosine hydroxylase (th). our results showed that all of the vglut3-positive/5ht-negative cells were immunoreactive for neun but not for gfap. furthermore, we found that these vglut3positive/5ht-negative neurons didn't show any immunoreactivities for gad67 nor th, and thus it is indicated that there is a group of exclusively glutamatergic vglut3-positive neurons in these nuclei. research funds: kakenhi 16200025, 17022020, 17650100 ps3a-c030 cortico-striatal and fast-spiking cell activity in the rat frontal cortex during cortical oscillations in vivo: modulation by serotonin m victoria puig 1 , mika ushimaru 1 , yoshiyuki kubota 1 , akiya watakabe 2 , tetsuo yamamori 2 , yuchio yanagawa 3 , yasuo kawaguchi 3 1 div. cerebral circuitry, nips, okazaki, japan; 2 div. brain biology, nibb, okazaki, japan; 3 dept. genetic and behavioral neurosci., gunma univ. graduate school of med., japan we studied how cortico-striatal (cs) and fast-spiking (fs) cells are modulated by slow-wave-sleep (sws) oscillations and by serotonin (5-ht). cs and fs cells were recorded simultaneously with the electrocorticogram in the secondary motor area of anesthetized rats that expressed a gfp in gabaergic interneurons. fs displayed a highsuccess excitation to striatal stimulation, suggesting a control of cs over fs. during sws, both cs and fs fired during the up-states though with different patterns. the stimulation of the dorsal raphe promoted longer up-states. moreover, 60% of the cs were inhibited by 5-ht through 5-ht 1a r and 6% were excited through 5-ht 2a r. however, 44% of the fs cells were inhibited and 28% excited. these results show that cs cells are more inhibited by 5-ht than fs. the expression of 5-htr was confirmed by in situ hybridization. research funds: jsps pe04061 and 15300110 ryohei tomioka, kathleen rockland laboratory for cortical organization and systematics, riken brain science institute, saitama, japan in small mammals, gabaergic neurons have been shown to contribute to ipsi-and contralateral cortical projections. here, we report in monkey as well that some gabaergic neurons send long-distance projections. identification was partly based on golgi-like labeling of the dendritic tree, achieved by injecting adenovirus as a retrograde tracer in areas v4, teo, or tep. aspiny or sparsely spinous nonpyramidal neurons were clearly visualized in the white matter or, less frequently, in cortical gray matter, in mainly layer 3 but also in layer 5 and/or 6. in each of the 3 cases, about 50-100 gabaergiclike neurons were scored, with a preferential location anterior to the injection sites. in addition to their characteristic dendritic morphology, the neurons were identified as positive for gabaergic neuronal markers; namely, gad67, somatostatin, or nos. thus, we conclude that gabaergic projection neurons are phylogenetically conserved; but more work is needed to determine (1) their other features, (2) possible species variability, (3) their functional significance. supported by riken bsi. withdrawn ps3a-c033 regional, cell type, and layer-specific differences in cholinergic modulation of neocortical neurons allan gulledge 1,2 , susanna b. park 2 , greg j. stuart 2 , yasuo kawaguchi 1 1 national institute for physiological sciences, japan; 2 div. neurosci., jcsmr, australian national university, canberra, australia we examined cholinergic modulation of pyramidal and nonpyramidal neurons in 3 neocortical areas (prefrontal, somatosensory, and visual cortex). transient ach exposure (100 m) inhibited layer 5 pyramidal neurons in all areas via activation of an sk-type potassium conductance. pyramidal neurons in layers 2/3 were generally less responsive to ach, but ach inhibited layer 3 cells in visual cortex. prefrontal layer 5 pyramidal neurons were more responsive to ach than were layer 5 cells in other areas of cortex. fast spiking (fs) nonpyramidal neurons were completely non-responsive to ach, even at very high concentrations (5 mm). on the contrary, ach generated fast, nicotinic receptor-mediated responses in 37% of non-fs interneurons (24 of 65 cells). laminar or regional differences in ach responses were not observed in nonpyramidal neurons. these data suggest that ach may act to inhibit the output of cortical projection neurons while preserving information processing in superficial neurons. toshikazu kakizaki 1,2 , kenzi saito 1,3 , yuchio yanagawa 1,2 1 department of genetic and behavioral neuroscience, gunma university graduate school of medicine, maebashi, japan; 2 sorst, jst, kawaguchi, japan; 3 sokendai, hayama, japan a major inhibitory neurotransmitter gaba is synthesized by glutamate decarboxylase (gad), and is accumulated into synaptic vesicles by vesicular gaba transporter (vgat). another inhibitory neurotransmitter glycine could be transported into synaptic vesicles by vgat, and be co-released with gaba. several molecules related to gabaergic or glycinergic neurotransmission are expressed in nonneural tissues, suggesting that gabaergic and glycinergic systems exert their activities outside the cns. vgat-deficient mice die in the perinatal period, and display omphalocele, defect in ventral body wall closure, suggesting that gaba and/or glycine are involved in body wall formation. to further investigate whether gaba is essential for the ventral body wall formation or not, we have been examining how the body wall developed in the gad67-deficient mouse fetus. ps3a-c035 gaba mediated glutamate release from developing cerebellar cortex and ca sensitivity sachiko yoshida, miyuki ohshita, masakazu uematsu, shoichiro hirano, shinya tanaka, naohiro hozumi toyohashi university of technology, toyohashi, japan gaba (␥-amino butyric acid) and glutamate are known to play important roles as modulators in the survival and development of cerebellar neurons. during cerebellar development, gaba-mediated responses, gaba excitations, become depolarized inducing an increase in intracellular calcium concentrations, and are thought to have important trophic effects. many observations of gaba excitations using cultured cells have been reported, whereas few using acute slices. we recently reported the spatial nature of glutamate and gaba releases from acute slice with an enzyme-linked assay system and ccd imaging technology. in the present study, we evolved this measurement system to allow observations of spontaneous or gaba-mediated glutamate release from developing postnatal acute cerebellar slices. glutamate was released spontaneously, but gaba-mediated glutamate release appeared from postnatal 4 to 6 day in egl. its release, especially from premigratory zone, was inhibited by ni 2+ , but cd 2+ couldn't. we suggest that gaba excitation induces granule cell migration. ps3a-c036 gabaergic fiber in the rat trigeminal motor nucleus reorganized following masseter nerve transection hiroyuki hayashi 1 , hiroaki wake 2 , junichi nabekura 2 , osamu takahashi 1 1 department of histology, kanagawa dental college, yokosuka, japan; 2 national institute of physiological science, okazaki, japan it has been reported that gabaergic nerve terminals are seen in the trigeminal motor nucleus (vm) of the rat, and that there are primary afferent inputs from the muscle spindle of masticatory muscles to the vm cell bodies. we recently found that the number of these gabaergic fibers projecting to vm is markedly reduced in postnatal development. in this study, to elucidate the possibility that the re-arrangement of gabaergic circuits could be reproduced after neuronal injury, we examined the effect of axonal injury of the masseter axon on the gabaergic circuits in the vm. two to eight weeks after unilateral surgical transection of the masseter nerve of rats, gabalike immunoreactive (gaba-ir) varicosities were examined using immunofruorescence technique. the significant increase in number of gaba-ir varicosities were seen after eight weeks of the operation. this result suggest that gabaergic inputs may play one of important role for reorganization of afferent inputs in the vm. akiko arata 1 , kunihiko obata 2 , jonathan davies 3 , mark bellingham 3 , peter g. noakes 3 1 lab. for memory & learning, riken-bsi, wako, japan; 2 obata res. unit, riken-bsi, wako, japan; 3 sch. biomed. sci., univ. queensland, queensland, 4072, australia during embryonic development, approximately half of the motoneurons (mns) undergo programmed cell death. this process depends also on glycinergic and/or gabaergic synaptic activity, as suggested by increased mn number in gephyrin-deficient mice (banks et al., 2005) . we investigated the involvement of gaba alone in the mn death using gad67-deficient mice, in which cerebral gaba is reduced to less than 10% of the wild-type. mn numbers at embryonic day (e) 18 were counted by the method of banks et al. brainstemupper spinal cord blocks were prepared from e18 embryos and subjected to electrical recording from the c4 and c8 ventral roots and also gaba measurement. in gad67-deficient embryos, increase in number of brachial mns (139%) and decrease in both spontaneous discharges in the c4, c8 roots and gaba content (less than 20%) were observed, compared with those of the wild-type littermates. gaba might control cell death in developing network. abolghasem esmaeili, joe lynch, pankaj sah queensland brain institute, the university of queensland, australia the amygdala has key role in processing emotional information. distribution of gaba a receptor subunits is crucial for understanding physiology and pharmacology properties of these receptors in the amygdala. we examined the pharmacology of gaba a receptors by expressing different subunit combinations in hek293 cells and comparing the pharmacology with specific gabaergic inputs in the amygdala. dmcm blocked the actions of gaba at expressed ␣1␤1␥2 and ␣2␤1␥2 combinations (75% reduction) but had no effect at ␣1␤1␥1 or ␣2␤1␥1. in slice recordings dmcm blocked ipscs by 70% in the lateral amygdala and had variable effects in the central amygdala. diazepam and zolpidem enhanced ipscs in the lateral whereas the response in the central amygdala was either reduction or enhancement. real time pcr and western blotting revealed differences in the distribution of gaba a receptor subunits between the lateral and central amygdala. we conclude that in the lateral amygdala all inputs have ␥2 subunits whereas in the central amygdala some inputs contain ␥1 while others contain ␥2 subunits. masayuki kobayashi department of pharmacology, nihon university school of dentistry, tokyo, japan noradrenergic agonists have different effects on the excitatory neural transmission according to their subtypes in rat cerebral cortex. the present study aimed to explore what kind of second messengers and the precise site of synaptic membrane, pre-or postsynaptic, is involved in these noradrenergic modulation. the suppressive effect by activation of ␣ 1 -adrenoceptors was mediated by protein kinase c, and excitatory effect by activation of ␤-adrenoceptors was mediated by camp/protein kinase a cascade. phenylephrine suppressed inward currents evoked by puff application of glutamate, and it decreased mepsc amplitude and increased mipsc frequency. isoproterenol increased mepsc frequency and decreased mipsc amplitude. gaba-induced postsynaptic currents were suppressed by isoproterenol. these results suggest that phenylephrine may decrease postsynaptic currents through glutamate receptors and increase the release probability of gaba from presynaptic terminals. on the other hand, isoproterenol may facilitate glutamate release and suppress gaba a receptor-mediated postsynaptic currents. ps3a-d040 hydrogen sulfide modulates synaptic transmission in rat hippocampal neurons mamiko tsugane 1 , takashi iwai 2 , yasuo nagai 1 , junichiro oka 2 , hideo kimura 1 1 dept. mol. genetics, nat'l. inst. neurosci., ncnp, tokyo, japan; 2 lab. pharmacol., fac. pharm. sci., tokyo univ. sci., chiba, japan hydrogen sulfide (h 2 s), which is a well-known toxic gas and facilitates the induction of hippocampal long-term potentiation, has been proposed as a neuromodulator in the brain. the aim of this study is to understand the mechanism of regulation on synaptic transmission by h 2 s. we examined the effect of h 2 s on spontaneous excitatory postsynaptic currents (sepsc) as well as paired-pulse facilitations using both whole-cell and field potential recordings from rat hippocampal slices. sodium sulfide (na 2 s), a donor of h 2 s, reduced the amplitude of field excitatory postsynaptic potentials and increased the ratio of paired-pulse facilitation. the frequency and the amplitude of sepsc were initially reduced by na 2 s then gradually increased, while the inward currents elicited by glutamate were not significantly suppressed by na 2 s. these observations suggest that h 2 s may modulate glutamatergic synaptic transmission by suppressing the release of a transmitter. several studies show that activation of locus coeruleus (lc) play an important role in the symptoms of opiate withdrawal. in this study the effects of lc inactivation on self-administration of morphine and on morphine withdrawal syndrome in rats has been investigated. male rats were anaesthetized and implanted with silastic catheters inserted in to the right jugular vein. after 5 days animals were fitted and the external end of the catheter was connected with a syringedriven pump, then were placed in the self-administration apparatus. lc was inactivated by (1 l) lidocaein (2%) 5 min before training. animals were allowed to self administer morphine (1 mg/kg per inf.) ten consecutive daily 2-h session. during all morphine self administration session lever pressing was measured. our results show that: (1) lc inactivation produced a significant decrease in the initiation of morphine self administration during all session. after the last test session morphine withdrawal symptom signs (mws) precipitated by naloxone were measured. (2) most of mws were decreased by lc inactivation in comparison with morphine group. these results suggest that extracellular atp plays a dual role in astrocytic ca 2+ wave propagation with activation of distinct purinergic receptors in the hippocampus of the rats. the electrophysiological analysis of the rescue effect of 17␤ estradiol from glucocorticoid activity yuki oishi 1 , suguru kawato 2 1 department of physics, graduate school of science, university of tokyo, tokyo, japan; 2 graduate school of arts and sciences, university of tokyo, tokyo, japan it is well known that stress reduces several activity of brain. especially, hippocampus is the largest target of stress. these phenomena are caused by glucocorticoids which are synthesized at adrenal when suffering stress. on the other hand, 17␤ estradiol is one of the neuro protective factors and rescues neural death caused by several neurotoxins, such as ␤-amyloid, glutamate, glucocorticoids. in this study, we focused attention on the acute effects of steroid hormones and researched the effects of glucocorticoids and estradiol on rat hippocampal long term potentiation (ltp), which is the index of learning and memory. the results was that corticosterone (glucocorticoid of rat) acutely reduced ltp via glucocorticoid receptor. 17␤ estradiol rescued this reduction via estrogen receptor ␣ and ␤. so we found that 17␤ estradiol affected not only neuro protection but synaptic protection from stress-induced suppression of synaptic transmission acutely. ps3a-d044 the hypothalamic neuropeptide y neuron system of rats after long-term, high-dose dexamethasone treatment jinko konno, ayuka ina, sachine yoshida, hideki ohmomo, fumihiro shutoh, setsuji hisano lab. neuroendocrinol., graduate sch. comprehensive human sci., univ. tsukuba, ibaraki, japan effects of dexamethasone (dex) on hypothalamic neuropeptide y (npy) expression were evaluated with semi-quantitative in situ hybridization and immunohistochemistry. adult male wistar rats received an injection of dex (0.2 mg/100 g b.w., sc) or sesame oil (vehicle control) everyday for 9-10 days. the two and intact rats (intact control) were decapitated, and the hypothalamus was dissected out, fixed and cut into paraffin sections. npy-immunoreactive axonal varicosities in the external zone of the median eminence were apparently more frequent in the dex-treated rat than in controls. npy hybridization signals in the arcuate nucleus were significantly higher in the treated-rat than in controls. no difference was found between both control animals. these results indicate stimulatory effects of dex on hypothalamic npy production and suggest enhanced npy influences on pituitary function. akiko shingo, idumi yamashita, shozo kito lab. of neuroscience, hyogo university, hyogo, japan we examined estrogen-like actions of isoflavones in the cerebral cortex and hippocampus on the basis of our previous data that estradiol induces igf-1 mrna expression, upregulates estrogen receptors and facilitates ere binding in these brain areas. materials are ovxed and non-ovxed rats. each group of rats were divided into the following groups. a: rats fed with phytoestrogen-free control diet, b: rats fed with diet with soy bean-derived estrogen and c: rats fed with control diet combined with chronic intraperitoneal injections of minimum dose of ␤-estradiol. after feeding, rats were sacrificed to remove the cerebral cortex and hippocampus. expressions of mrnas of igf-1, estrogen receptors ␣ and ␤, and ere binding were analysed. as the results, it was revealed that isoflavones induced increased expression of mrnas of igf-1 and estrogen receptors in both ovxed and non-ovxed rats. difference between estrogen receptor ␣ and ␤ in responses to isoflavones were analysed. isoflavones feeding increased ere binding as much as chronic injections of estrogen did in the ovxed rats. research funds: kampo science foundation, japan ps3a-d046 mechanism of central metabolic control by tgf-beta in the rat brain: using the rat with depletion of hypothalamic noradrenaline teppei fujikawa, kazuo inoue, tohru fushiki division of food science and biotechnology, graduate school of agriculture, university of kyoto, kyoto, japan we have previously reported that activated transforming growth factor-beta (tgf-beta) increase in the rat brain during exercise. intracranial administration of tgf-beta induced an increase in fat oxidation, free fatty acid and keton body in the blood. these results suggest that activated tgf-beta in the rat brain participates in metabolic control of peripheral tissue by cns. it is, however, not known how tgf-beta increases in specifically fat oxidation. many investigations suggest that hypothalamus is essential for central metabolic control. in addition, some reports suggest that noradrenergic system in the hypothalamus may play important role for fat oxidation. in this study we measured concentration of extracellular noradrenaline (na) in the hypothalamus by using microdialysis after injection of tgf-beta. then, we measured respiratory exchange ratio and serum samples, after administration of tgf-beta in the rat with depletion of hypothalamic na by injection of 6-hydroxydopamine. ps3a-d047 the effect of brain-derived neurotrophic factor (bdnf) on neuropeptide y (npy) neurons in the mouse corpus callosum: an examination using organotypic brain slice culture ryoichi yoshimura, kazuto ito, yasuhisa endo department of applied biology, faculty of textile science, kyoto institute of technology, japan the morphology of neuropeptide y (npy) neurons existing in the corpus callosum (cc) and the effects of brain-derived neurotrophic factor (bdnf) on the npy neurons were examined by using organotypic slice culture system. bdnf treatment significantly increased the number of the npy-immunopositive cell bodies and fibers in cc assessed with immunocytochemistry. electron microscopy demonstrated that the npy immunoreactivities were mainly localized in the regions associated with accumulating synaptic or cored vesicles in cc nerve fibers. the sectional area of npy-positive fibers was larger in the bdnf-treated culture than in the control culture. the number of nerve fibers adjacent to the npy-positive fibers was also larger in the bdnf-treated culture than the control. these results suggest that npy may play a key role in the neuronal regeneration, and bdnf takes part in the development of npy neuron fibers as well as the increase of the number of npy neurons in cc. reiji semba 1 , kimi watanabe 1 , munekazu komada 2 1 institute for developmental research, aichi human service center, aichi, japan; 2 graduate school of medicine, kyoto university, kyoto, japan d-serine is hypothesized to be a glia-derived neurotransmitter activating the nmda receptor because d-serine was reported to be formed and localized exclusively in astrocytes. however, we reported strong immunoreactivity of d-serine in some axons. to reveal which cells are producing d-serine in the brain, an in situ hybridization study of serine racemase, the enzyme producing d-serine from l-serine, was performed. using antibodies against neun, a neuronal marker, gfap, an astrocyte marker, and cnpase, an oligodendrocyte marker, type of the cells containing the mrna was examined. coincidentally with our immunohistochemical study of d-serine, strong signals for serine racemase mrna were found in some neurons while weak signals were found in astrocytes. present results suggest that d-serine will be a neurotransmitter activating the nmda receptors produced in a specific type of neurons. takatoshi hikida 1,2 , asif k mustafa 2 , kenji hashimoto 3 , kumiko fujii 2,4 , kazuhisa maeda 2,5 , hiroshi ujike 6 , richard l. huganir 2 , solomon h. snyder 2 , akira sawa 2 1 dept. of systems biology, obi, suita, japan; 2 depts of neurosci. & psychiat, johns hopkins univ. med., baltimore, maryland, usa; 3 chiba univ. forensic mental health, chiba, japan; 4 dept. of psychiat, shiga univ. med. sci., shiga, japan; 5 div. of neuropsychiat, tottori univ., yonago, japan; 6 dept. of neuropsychiat, okayama univ., okayama, japan accumulating evidence from both genetic and clinical studies suggests a critical role of d-serine in schizophrenia (sz). we identified and characterized pick1 as a protein interactor of the d-serine synthesizing enzyme, serine racemase (sr). d-serine levels in the hippocampus and frontal cortex of pick1 knockout mice were significantly lower than those of their wildtype littermates at age of p7, but not in adults, suggesting regulation of pick1 on sr at developing stage. in case-control association study, we observed an association of the pick1 gene with sz, which is more prominent in disorganized sz. our findings suggest that pick1 contributes to sr activity, d-serine production, and nmda neurotransmission in the pathophysiology of sz. ps3a-d050 epileptiform activity is inhibited by taurine which can activate glycine and gaba a receptors in immature rat hippocampus akihito okabe 1,2 , werner kilb 2 , ileana l. hanganu 2 , taizhe qian 2 , daiichiro nakahara 3 , atsuo fukuda 1 , heiko j. luhmann 2 1 dept. of physiol., hamamatsu, japan; 2 inst. of physiol., mainz, germany; 3 dept. of psychol., hamamatsu, japan many studies indicate that the underlying mechanism of epileptic seizures differ between children and adults. the depolarizing gabaergic responses in immature neurons may contribute to higher epilepsy susceptibility. to investigate whether taurine, a neurotransmitter found in high concentrations in the immature cns, modulates epileptiform activity in immature hippocampus, we performed field-potential recordings in neonatal rat hippocampal ca3 region of an intact preparation. 5 mm taurine blocked epileptiform activity induced by mg 2+ free acsf and 20 m 4-ap. this taurine effect was prevented by the glycinergic antagonist strychnine and the gaba a antagonist gabazine. inhibition of taurine uptake by ges also suppressed epileptiform activity in strychnine and gabazine sensitive manner. these results suggest that taurine mediates an inhibition in immature hippocampus via glycine and gaba a receptors that suppresses epileptiform activity. ps3a-d051 responses of pge 2 in undifferentiated and differentiated ng108-15 cells kayoko matsushima 1 , takashi imanishi 1 , akinori kawaguchi 1 , tetsuyuki wada 1 , shigeru yoshida 2 , seiji ichida 1 1 school of pharm. sci., kinki univ., osaka, japan; 2 school of pharm. sci., kinki univ., osaka, japan; 3 school of pharm. sci., kinki univ., osaka, japan; 4 school of pharm. sci., kinki univ., osaka, japan; 5 school of sci. & eng., kinki univ., osaka, japan; 6 school of pharm. sci., kinki univ., osaka, japan our previous findings showed that 5-ht-and bk-induced [ca 2+ ] i increases were enlarged in differentiated ng108-15 cells. for the next stage, we investigated the effect of pge 2 , an inflammatory mediator for 5-ht and bk, on the cells. ng108-15 cells were loaded with fura-2/am, and the change in [ca 2+ ] i was monitored by an image processor. the results showed: (1) pge 2 -induced response was decreased when ng108-15 cells were differentiated by bt 2 camp, (2) 10 −5 m ah6809 and sc19220 irreversibly inhibited pge 2 -induced response by about 50% and 26%, respectively, while 10 −5 m ah23848 and sulprostone had no effect, and (3) pge 2 -induced response was abolished under ca 2+free conditions in about 70% of both ng108-15 cells. these results indicate that the response to pge 2 , via ep1 and ep2 receptors, significantly decreased during differentiation. mitsumasa murano, fumihito saitow, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan the most of cerebellar outputs are generated as a result of synaptic interaction in the deep cerebellar nuclei (dcn) and by the electrical membrane properties of dcn neurons themselves. this study aimed at examining mechanisms underlying the serotonergic modulations of both the gabaergic transmission at the purkinje-to-nuclear cell synapses and the membrane properties of dcn neurons using cerebellar slices prepared from 11-to 21-day-old rats. bath application of serotonin (5-ht) decreased the amplitude of stimulation-evoked ipscs in dcn neurons in a dose-dependent manner. furthermore, slow inward currents ware observed in dcn neurons during 5-ht application. under the current-clamp recording, 5-ht markedly depolarized and increased action potential discharges of dcn neurons. taken together, these results suggest that 5-ht facilitates the voluntary activity in dcn neurons by both pre-and post-synaptic mechanisms. ps3a-d053 searching for endogenous ligands of trace amine receptors in mammals (1) akira komatsu 1 , airi yamaguchi 2 , noriko makikusa 2 , osamu koizumi 2 1 dept. physiol., tokyo women's med. univ., sch. med., tokyo, japan; 2 neurosci. lab., fukuoka women's univ. fukuoka, japan trace amine receptors were discovered in mammals, but their endogenous ligands have not yet been found. to search for them, we developed a new method to make antibodies against monoamines for immunohistochemistry (ihc). monoamines, phenylethylamine (pea), tyramine (ta) and histamine (ha), were conjugated to a hemocyanine, klh, using an imidoester cross-linker, dimethyl suberimidate (dms). rabbits were immunized by the conjugated macromolecule. the obtained antibodies were assayed by elisa and competitive elisa technique to check their antibody titer and specificity respectively. the antibodies recognized specifically the monoamine-dms part within the complex. for ihc, the rat brain was perfused by 2% dms, post-fixed by 4% formaldehyde and then frozen-sectioned. the antibody against ha revealed the immunoreactive neurons in the hypothalamus, showing that this method is effective to demonstrate the presence and localization of monoamines. the antibodies against pea and ta failed to reveal immunoreactive neurons in the rat brain. ps3a-d054 effects of mg 2+ on neural activity of cultured cortical neurons of the rat and mouse yuriko furukawa 1,2 , nahoko kasai 1 , akiyoshi shimada 1 , keiichi torimitsu 1,2 , kunihiko obata 3 , yuchio yanagawa 2,4 , tadaharu tsumoto 2,3 1 ntt basic research laboratories, kanagawa, japan; 2 sorst/jst, saitama, japan; 3 neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; 4 dept. of genetic and behavioral neurosci., grad. sch. of med., gunma university, gunma, japan it is well known that mg 2+ plays an important role not only in energy metabolism, but also in neural information processing. however, the mechanism of such a role in cns is not well understood. previously we reported that neural activity and the intracellular ca 2+ concentration are largely affected by mg 2+ removal in cultured cortical neurons of the rat. transient glutamate release was also detected. in the present study, we investigated effects of the mg 2+ removal on neural activity in cultured cortical and hippocampal neurons. in particular, we measured the intra-and extracellular mg 2+ concentration and their actions on neural activity using a mg 2+ indicator, kmg-20-am together with fluo4-am. we observed different effects of the mg 2+ removal on gabaergic and non-gabaergic neurons by using gad67-gfp knock-in mice. research funds: jst/sorst ps3a-e055 transient zinc-positive terminations in the developing rat somatosensory cortical system noritaka ichinohe, daniel potapov, kathleen s. rockland lab. for cortical organization and systematics, bsi, riken, usa synaptic zinc (zn) is a neuromodulator used by a subset of nonthalamic glutamatergic connections, and associated with both experiencedependent and developmental plasticity. during development, transiently high levels of synaptic zn occur in both sensory and nonsensory cortical areas. by injecting the retrograde tracer sodium selenite into barrel cortex, we demonstrated a transient subset of zn + thalamocortical neurons from p7-p13. zn + cortical neurons were also labeled, intrinsic and extrinsic, from p5. unlike in the adult, these were in layer 5, instead of layers 2, 3, and 6. at p9, neurons occurred in layers 2, 3, and 5 and, in some areas, layer 4. at p15, zn + neurons first appeared in layer 6; and at p22, there is the adult lamination. as whisking and exploratory behavior commences in the second postnatal week, these transient zn + terminations may play a role in experience-dependent adjustments in cortical circuitry. research funds: bsi, riken and kakenhi no. 17024064 ps3a-e056 systematic comparison of the structure of the serotonin immunoreactive neurons between insect species masaaki iwano 1,3 , ryohei kanzaki 2 , kei ito 1,3 1 center for bioinform., imcb, univ. of tokyo, tokyo, japan; 2 dept. of mechano-inform., grad. sch. of inform, sci. and tech., univ. of tokyo, tokyo, japan; 3 bird, jst, saitama, japan in the vertebrate central nervous system, the distribution of the serotonin immunoreactive neurons (sirns) is known to be preserved remarkably during evolution. systematic comparison of the invertebrate sirns has not been performed, on the other hand. in the current study we analyzed the morphology of the sirns in the brains of holoand hemi-metabolous insects including flies, bees, moths, beetles, crickets, dragonflies and cicadas. in spite of the large variation in the size and cell numbers of the brain, the number and distribution of the sirns were highly consistent between species. for example, we observed either one or two pairs of bilateral sirns with similar morphology that connect specific subregions of the lateral accessory lobe, a candidate pattern generator of the zigzag locomotion of the insect. variation was greater in the antennal lobe, the insect primary olfactory center, where sirns project either ipsil-or contra-laterally depending on the species. maki kagohashi 1,2 , taizo nakazato 2 , shigeru kitazawa 2 1 neurol, juntendo univ., tokyo, japan; 2 physiol, juntendo univ., tokyo, japan in vivo voltammetry has been used for measuring neurotransmitter releases in the brain of behaving rats (e.g. nakazato, 2005) . however, task freedom was restricted by cables connecting the head and the measurement system. to overcome the difficulty we developed a wireless voltammetry system and examined its sensitivity in vitro (kagohashi et al., jns2005). the system consisted of a wireless transmitter with a potentiostat and a signal receiver. in the present study, we reduced the size and weight and measured dopamine (da) currents in vivo with the wireless system mounted on the back of the rat. a single-step voltage pulse (100 to 250 mv for da; 300 to 450 mv for 5ht) was applied at 4 hz through a carbon electrode that was chronically implanted in the striatum. after administration of l-dopa, da currents showed a gradual increase in good agreement with the data measured with conventional systems. the present wireless system would be applicable to measurement of neurotransmitters in various situations (e.g. social interaction). research funds: scientific research on priority areas (mobiligence) hiroyuki yamazaki, tomoaki shirao department of neurobiology and behavior, gunma university graduate school of medicine, maebashi, japan dendritic spines are multiple functional units that receive most of excitatory inputs in central nervous system. in the purpose of finding a novel molecule that is involved in regulation of dendritic spines, we have done a screening of a novel drebrin binding protein. yeast twohybrid system was conducted with drebrin as bait, and a novel drebrin binding protein was isolated. in neurons, this protein was localized primarily in nucleus and dendritic spines. hence, we named it spikar for its unique intracellular localization in spine and karyoplasm. we studied the role of spikar in spine formation. hippocampal neurons were transfected with shrna expression vector for spikar at several developmental stages. in early stage, spikar knock down (kd) did not affect the density of dendritic protrusions that were mostly filopodia. in contrast, spikar kd reduced spine density at the stage of synapse formation. these results suggest that spikar plays a role in the formation of dendritic spines, without affecting the filopodia formation. ps3a-e059 time-lapse analysis of the translocation of drebrin-actin complex from dendritic spines to dendritic shafts by glutamate stimulation toshiyuki mizui 1,4 , yuko sekino 2,3 , tomoaki sirao 1 1 dept. of neurobiol. & behav., gunma univ. grad. sch. of med., maebashi, japan; 2 div. of neural network, inst. med. sci. univ. of tokyo, tokyo; 3 crest, jst, kawaguchi, japan; 4 jsps, japan we have shown that nmda receptor activation induced translocation of drebrin, with retaining its binding to f-actin, from dendritic spines to their parent dendrites. in the present study, we analyzed the time course of gfp-tagged drebrin a (gfp-da) dynamics after glutamate receptor activation. we prepared primary hippocampal cultured neurons, transfected them with gfp-drebrin a expression vector using microinjection methods at 14 days in vitro (div), and analyzed the dynamic localization of gfp-da at 16 div. glutamate stimulation started gfp-da translocating within 10 s and completed in 2 min. after washout of glutamate, gfp-da gradually re-accumulated in the spine, and the fluorescence intensity of gfp-da is fully recovered in 10 min. these data suggest that translocation mechanism of drebrin from spines to shafts is different from that from shafts to spines. research funds: grant-in-aid for jsps fellows ps3a-e060 distribution of the srf co-activator mal in developing mouse brain mitsuru ishikawa 1 , jun shiota 1 , hiroyuki tsutsumishita 1 , hiroyuki sakagami 2 , masaaki tsuda 1 , akiko tabuchi 1 1 dept. biol. chem., fac. pharm. sci., univ. toyama, toyama, japan; 2 dept. cell biol., tohoku univ., grad. sch. medicine, sendai, japan the srf co-activator mal (megakaryocytic acute leukemia) plays an important role in controlling srf-dependent gene, whose expression is regulated by rearrangement of actin cytoskeleton. recent studies with conditional deletion of srf gene demonstrated that srf was required for inducing genes such as egr-1, c-fos,␤-actin but also for neuronal migration and plasticity. in this study, we investigated the expression of mal in developing mouse brain and the role of mal for dendritic morphology. the in situ hybridization analysis revealed that mal mrna was highly and developmentally expressed in hippocampus and broadly expressed in cortex, olfactory bulb. staining of mal displayed cytoplasmic localization at cell bodies and apical dendrites. furthermore, dominant negative mal mutants and rnai led to a reduction of dendritic number, as well as a decrease of srf transcription. these findings indicate that mal is involved in the formation or the stability of dendrites. research funds: kakenhi (17790055) to a.t. shoko shimizu 1 , shinsuke matsuzaki 1 , tsuyoshi hattori 1 , ko miyoshi 2 , masaya tohyama 1 1 department of anatomy and neuroscience, graduate school of medicine, osaka university, japan; 2 department of brain science, graduate school of medicine and dentistry, okayama university, japan disrupted-in-schizophrenia 1 (disc1) was identified as a novel gene disrupted by a (1;11) (q42.1;q14.3) translocation segregating with schizophrenia and affective disorders in a scottish family. kendrin was identified as a protein which interacts with disc1 at centrosome and residues 446-533 of disc1 (kendrin-binding region: kbr) were essential for the interaction with kendrin. in this study, we show that c-terminal of disc1 downstream of kbr is indispensable structure for kbr to interact with kendrin and also essential for disc1 to target to the centrosome. furthermore, we have shown that inhibition of the disc1-kendrin interaction perturbs the tubulin network formation. these results suggest that the c-terminal region of the disc1 is important to the disc1-kendrin interaction and that a truncated form of disc1 lacking the c-terminal downstream of the translocation breakpoint might affect the microtubule organization. tatsuro kumada, yasuhiko nakanishi, atsushi fukuda department of physiology, hamamatsu university school of medicine migratory cells exhibit dynamic morphological changes in the cell soma and process in both normal developmental program and tumor growth. the morphological changes in the cells are correlated with the rate of cell migration and ion transfer such as ca 2+ or cl − . although the highly invasive migration of glioblastoma in the brain is known to be influenced by a variety of ion channels, there were a little evidence about the relationships among the morphological changes and ion homeostasis. to clarify it, we have developed a glioma cell culture system for the simultaneous observation of the cell movement and ca 2+ and cl − imaging. we found that the relatively low density a172 glioma cells actively moved on the substrate. the movement has the correlation with intracellular ca 2+ oscillation in the cells. the relationship between cell movement and intracellular ion levels is further studied. ps3a-e063 involvement of ca 2+ influx in the unpolarized non-vesicular release of fgf-1 hayato matsunaga, hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan little is known of molecular basis mechanisms for the er-golgiindependent or non-vesicular release of fgf-1 lacking a conventional signal peptide sequence. we found that fgf-1 is co-released with s100a13, a ca 2+ binding protein from cultured rat astrocytes upon the serum-deprivation stress. here, we report that fgf-1 is co-released with s100a13 from the axon and dendrites in cultured rat hippocampal neurons upon depolarization stimulation, but serum-deprivation stress leads to release, which is seen in neurites as well as in soma. the interaction between fgf-1 and s100a13 required ca 2+ . the overexpression of s100a13 88-98 mutant lacking an ability of interaction with fgf-1 inhibited the their release, suggesting that s100a13 is a cargo molecule. the release of fgf-1 upon either stimulation was abolished by voltage-dependent n-type ca 2+ channel blocker. these findings suggest that ca 2+ influx may be involved in the unpolarized non-vesicular release of fgf-1. in neuron, intracellular calcium involves a large number of physiological phenomena, including cell migration, differentiation, and neurite outgrowth. pc12 cell is a useful model of neural differentiation and neurite outgrowth, and recently we demonstrated that 5-ht has an effect on neurite outgrowth via the increase in intracellular calcium concentration ([ca 2+ ] i ) in pc12 cells. however, it is unclear how [ca 2+ ] i regulates neurite outgrowth via actin cytoskeleton. in this study, we investigated effects of [ca 2+ ] i on actin dynamics in pc12 cells transfected with yfp-actin. filopodial growth speed and actin retrograde flow were increased by treatment with calcium ionophore, a23187. treatment with calcineurin inhibitors decreased the filopodial growth speed, while treatment with camk inhibitor did not. these effects could contribute to 5-ht induced enhancement of neurite elongation. the actin cytoskeleton is a complex protein network that not only provides cellular structure but is fundamental for cellular dynamics. on stimulation of pc12 cells by ngf, proteins that directly interact with f-actin such as actinin rapidly translocate to the f-actin-rich cytoskeleton. clp36 is a pdz-lim protein which was originally identified as an actinin-interacting protein in skeletal muscles. here, we show that clp36 is endogenously expressed in pc12 cells and plays an important role in actin dynamics during ngf-induced neurite outgrowth. immunofluorescent studies showed that clp36 is accumulated in irregular cell surface and membrane extrusion soon after ngf-stimulation, where colocalized with actin filaments. we next performed rnai experiments to explore the role of clp36 in actin dynamics in growth cones and found that knockdown of clp36 expression lead to the suppression of ngf-mediated neurite outgrowth. in addition, we revealed using clp36 deletion mutants that both of pdz and lim domains are necessary for the proper function of clp36. ps3a-e066 screening of genes expressed preferentially in migrating gabaergic neurons of developing cerebral cortex toshiya kimura 1 , tsuyoshi kobayashi 1 , yuchio yanagawa 2 , kunihiko obata 3 , fujio murakami 1,4 1 grad. sch. of frontier biosci., osaka univ., osaka, japan; 2 grad. sch. of medicine, gunma univ., maebashi, japan; 3 bsi, riken, wako, japan; 4 sorst, jst, japan neuronal migration plays a critical role in constructing brain architecture organization. however, molecular mechanisms underlying this process still remain elusive. in an attempt to identify molecules that regulate the motility of migrating neurons, we focused on migrating cortical interneurons, and performed subtractive hybridization, differential screening and in situ hybridization. subtraction was done between the embryonic and postnatal interneurons, because they robustly migrate prenatally but not postnatally. among the 2208 clones tested, two genes, neuronatin and seizure related gene 6 (sez-6) attracted our attention. they were expressed in the subventricular zone of the embryonic cortex, implicating that these molecules are expressed in interneuron subpopulations. postnatally, mrna signals were hardly detectable. these results raise the possibility that they are expressed preferentially in subpopulations migrating cortical interneurons. yan zhu 1,2 , tomoko matsumoto 1,2 , sakae mikami 3 , takashi nagasawa 3 , fujio murakami 1,2 1 grad. sch. of frontier biosci., osaka univ., japan; 2 sorst, jst, japan; 3 inst for frontier med. sci., kyoto univ., japan long distance neuronal migration takes place typically along the tangential plane of the developing neural tube. the migratory behaviour and the underlying molecular mechanisms of tangential migration are poorly understood. we address these issues using the hindbrain precerebellar system as model system. precerebellar neurons, born dorsally in the lower rhombic lip, migrate in close association with the pial membrane (except inferior olive neurons) ventrally or rostroventrally. we therefore studied the role of pia-secreted chemokine sdf-1 and its receptor cxcr4 in the precerebellar migration. we show that cxcr4 is expressed in the migrating precerebellar neurons, and its expression is down-regulated towards the end of migration. in cxcr4 and sdf-1 knock out mice, migrating precerebellar neurons are less confined to the pial surface. more strikingly, the rostrally-directed migration of pontine precerebellar neurons is severely disrupted, leading to a caudalized ectopic pontine-like cluster. ps3a-e068 involvement of an immunoglobulin superfamily molecule, neph2/mkirre in the migration of precerebellar neurons kazuhiko nishida 1,2 , kazuhide nakayama 1 , saori yoshimura 1,2 , fujio murakami 1,2 1 grad. sch. of frontier biosci., osaka univ., osaka, japan; 2 sorst, jst, saitama, japan neural cell migration plays a crucial role in central nervous system development. in this study, we analyze the involvement of neph family transmembrane proteins of the immunoglobulin superfamily in the migration of precerebellar neurons (pcns). postmitotic pcns derived from the rhombic lip in the hindbrain first migrate tangentially along the pial surface, followed by radial migration to settle at their final positions (kawauchi, d., taniguchi, h., watanabe, h., saito, t., and murakami, f., development, in press ). in situ hybridization analysis showed that among neph family members including neph1, neph2/mkirre, and neph3, only neph2/mkirre was strongly expressed in pcns. expression of neph2/mkirre was detected from e13.5 when pcns migrate tangentially. the expression level became weaker at p1, when pcns stop the radial migration, raising the possibility that neph2/mkirre might be involved in the migration of pcns. we are currently analyzing the function of neph2/mkirre in the migration of pcns. hiroki umeshima 1,2 , toshio ohshima 3 , tomoo hirano 2 , mineko kengaku 1 1 lab. for neural cell polarity, riken bsi, wako, japan; 2 department of biophysics, kyoto university, kyoto, japan; 3 lab. for developmental neurobiology, riken, bsi, wako, japan during lamination of the cerebellar cortex, granule cells exit their final mitiosis at the external granular layer and migrate to the internal granular layer. we analyzed the molecular mechanisms regulating migration of granule cells. using an in vivo electroporation system followed by time-lapse confocal microscopy of a slice culture, we found a dominant negative form of cdk5 (cdk5-dn) disrupted the morphology of granule cells during radial migration. recently, centrosome positioning is thought to be one of the important factors for neuronal migration. double-labeling of the centrosome and the whole-cell images by transfecting centrin2-gfp and rfp enabled us to record dynamic movement of the centrosome during radial migration. we found that the motion kinetics of the centrosome was disrupted by cdk5-dn. based on these results, we will discuss the role of centrosome during neuronal migration. keisuke ito 1,2 , takahiko kawasaki 1,2 , tatsumi hirata 1,2 1 division of brain function, national institute of genetics, mishima, shizuoka, japan; 2 department of genetics, school of bioscience, sokendai newly generated neurons migrate through proper pathways toward their own targets, where they are integrated into specific neuronal circuits. we have analyzed a unique tangential migratory stream of early-generated cortical neurons designated as lot cells, and performed pharmacological perturbations to characterize the intracellular mechanism of the migration. among various drugs, we found that a protein kinase inhibitor, k252a has the most interesting effect on the lot cell migration. during the normal migration, leading processes and cell bodies of lot cells move forward in a coordinated manner, but k252a blocks the migratory movement of cell bodies without inhibiting the extension of leading processes. we also found that k252a has a similar effect on cerebellar granule cells. these phenomena are quite intriguing because the drug seemed to switch the neurons from "whole cell migration" to "neurite extension" mode. we are now analyzing possible targets of k252a, aiming for dissection of these phenomena. the conserved ser/thr kinase unc51 functions with unc-76 to regulate axonal transport in drosophila hiroaki mochizuki 1 , hirofumi toda 1,3 , emiko suzuki 2 , joseph gindhart 4 , toshifumi tomoda 3 , katsuo furukubo-tokunaga 1 1 grad. school life and envir. sci., univ. tsukuba, tsukuba, japan; 2 gene net. lab., natl. inst. genet. mishima; 3 beckman res. inst., city of hope, ca, usa; 4 dep. biol., univ. richmond, va, usa neural network develops through regulated guidance of axons and interconnection among them. despite intensive researches in the past years, genetic mechanisms of axonal development still remain unclear. we have identified the drosophila homolog of unc51, which encodes a ser/thr kinase and is required for axonal formation in c. elegans and mouse. we found that unc51 is essential for neural development in drosophila. loss of function of drosophila unc51 results in reduced locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants. we also found that unc51 genetically interacts with unc-76, an evolutionarily conserved cytoplasmic protein that binds to kinesin heavy chain. in unc51 mutants, unc-76 was separated from synaptotagmin vesicles. these results suggest that unc51 coordinates kinesin-cargo interaction via unc-76 to regulate dynamic axonal transport. ps3a-e072 change in microtubule polarity during the conversion of dendrites into axons kensuke hayashi 1 , daisuke takahashi 2 1 life science inst. sophia university, tokyo, japan; 2 waseda university, tokyo, japan axons and dendrites of neurons differ in the polarity of their microtubules. the mechanism for the difference, however, is not well understood. we found previously that dendrites convert into axons in cultured neurons isolated from rat cerebral cortex. in this study, we examined whether microtubule polarity changes during the conversion. in dendrites of neurons before culture, microtubule polarity was nonuniform. after 24 h of culture, we found that most of microtubules in the original dendrites had their plus ends oriented distal. this indicates that microtubules with their minus-ends distal disappeared during the culture. microtubule movement along actin filaments is a candidate for this mechanism among several types of microtubule movement reported in neuronal processes so far. however, the change of microtubule polarity within dendrites was observed even in the presence of actin polymerization inhibitors. our results suggest a rearrangement of microtubules by a yet-unreported movement in neuronal processes. research funds: kakenhi (16500193) and kakenhi on priority areas (16027207) ps3a-e073 generation and analysis of region-specific rac1-deficient mice hidetoshi kassai 1 , masahiro fukaya 2 , eriko miura 2 , mizuho sakahara 1 , masahiko watanabe 1,2 , atsu aiba 1 1 div. cell biol., kobe univ. grad. sch. med., kobe, japan; 2 div. physiol. sci., hokkaido univ. grad. sch. med., japan rac1 is a member of the rho family of small gtpases, and assumed to be involved in regulation of neuronal development through actin cytoskeletal reorganization. nevertheless, physiological role of rac1 in the cns is poorly understood because of the embryonic lethality of rac1 knockout mice. in this study, we generated and analyzed region-specific rac1-deficient mice (emx1-rac1 ko mice) by the cre-loxp system, in which a promoter for emx1 homeobox gene induces expression of cre recombinase exclusively in the dorsal telencephalon, including cerebral cortex, hippocampus and olfactory bulb. emx1-rac1 ko mice showed partially abnormal layering of cerebral cortex, indicating impaired migration of neuronal cells during cortical development. furthermore, emx1-rac1 ko mice lacked corpus callosum and anterior commissure, both of which connect the left and right cerebral hemispheres. these results suggest that rac1 regulates neuronal cell migration and axonal growth in cerebral cortex. previously we reported overexpression of map1b containing nterminal 126 amino acids promoted neuronal death. to reveal the mechanism of map1b n-terminal induced neuronal death, we searched for the proteins that interact with n-terminal of map1b by two-hybrid system. alpha-tubulin was found to interact with map1b n-terminal and their in vitro interaction was proved with pull-down assay. the interaction of tubulin and map1b n-terminal has not yet been reported. beta-tubulin was also found to interact with map1b n-terminal. when ␤ tubulin was divided in 2 fragment at between amino acid 213 and 214, there was no interaction between ␤ tubulin fragments and map1b n-terminal. interaction needs the continuous region over aa 213 and 214. there were much proportion of round formed cos7 cells in n-terminal containing map1b transfected cells than in n-terminal lacking map1b transfected cells. there might be some interference in interaction between map1b and tubulin in cells express map1b containing n-terminal. otone endo 1,2 , masaaki mizuno 3 , yasukazu kajita 2 , jun yoshida 2 1 department of neurosurgery, ja kainan hospital, aichi, japan; 2 department of neurosurgery, nagoya university, nagoya, japan; 3 department of molecular neurosurgery, nagoya university, nagoya, japan primate es cells have rather different character from rodent ones, but it is inevitable to elucidate mechanism for stable culture, purification and induction into object-oriented differentiation, because human es cells might show wide similarity to cynomolgus ones. we refined the way of large scale culture maintaining totipotency without contacting feeder cells indispensable for primate es cells. our super selective induction method for dopaminergic neurons is also refined, and induced neurons transplanted in vivo which survive without forming tumor such as teratoma for long period, are evaluated not only immunohistologically but eletrophysiologically and ethologically suggesting its enough stability, activity, ability to make neural network system and potentiality to improve clinical symptom of parkinsonism. differentiation of other types of neurons and development of fully functional neural network must be established. shigeki ohta 1 , masae yaguchi 1 , yumi matsuzaki 2 , yoshiaki toyama 3 , yutaka kawakami 4 , hideyuki okano 2 , masahiro toda 1,5 1 neuroimmunology research group, keio univ., tokyo, japan; 2 physiology, keio univ., tokyo, japan; 3 orthopaedic surgery, keio univ., tokyo, japan; 4 institute for advanced medical research, keio univ., tokyo, japan; 5 neurosurgery, keio univ., tokyo, japan we have shown that mouse dendritic cells (dcs) have the ability to induce the proliferation and survival of neural stem cells/progenitor cells (nspcs) in vitro. implantation of dcs into injured mouse spinal cord could improve the motor function through activation of endogenous nspcs in vivo. in this study, to identify an effective dc subtype for the treatment of spinal cord injury (sci), we analyzed the effects of different mouse dc subtypes on the proliferation of nspcs in vitro. among mouse splenic cd11c + dcs, cd8␣ + dcs increased the number of neurospheres most effectively in vitro. furthermore, a significant functional recovery after mouse sci was induced by implantation of cd8␣ + dcs compared to cd11c + dcs. these results suggest that cd8␣ + dcs can be an effective subtype of mouse dcs for the treatment of sci. ps3a-e077 von hippel-lindau protein regulate the neurogenesis in skin-derived precursor cells atsuhiko kubo 1 , hiroshi kanno 1 , takaakira yokoyama 1 , shuichi nakano 2 , naoki sugimoto 2 , nahoko kobayashi 3 , tetsuhiko yoshida 3 , isao yamamoto 1 1 dept. of neurosurgery, yokohama city university graduate school of medicine, yokohama, japan; 2 fiber and dept. of chemistry, konan university, kobe, japan; 3 toagosei co., ltd. corporate research laboratory, nagoya, japan skin-derived precursors (skps), multipotent somatic stem cells, are preferred cell source for autologus cns cell replacement therapy. they are proliferated by the mitogens of egf and bfgf. to investigate the effects of von hippel-lidau (vhl) protein in the neural cell fate commitment, skps were inoculated with hsv vector expressing vhl protein. skps showed promotion of neurogenesis and inhibition of gliogenesis. to detect the intrinsic factors that control lineage commitment, vhl peptides fused with the protein transduction domain (ptd) were synthesized. the ptd-vhl peptides showed rapid cell internalization in nearly 100%, and peptide with the elongin c binding site (residues 157-171) showed a high ability of inducing neuronal differentiation by interacting with jak/stat pathway. these findings are important in its application to the cns cell grafting. ps3a-e078 the effect of pueraria mirifica on erk1/2 and s-100 following sciatic nerve injury in rats pornpen chaiworakul, supin chompoopong department of anatomy, mahidol university, bangkok, thailand to investigate the effects of pueraria mirifica (pm) compared with genistein (g) and estrogen (e2) on the expression of erk1/2 and s-100 following sciatic nerve crush and transection in rats. protein levels of perk1/2 and s-100 in distal segments of nerve at day 7 were determined by western blot analysis. it was demonstrated that pm and g treatments, similar to e2, caused a significant decrease in the expression of perk1/2 levels in both nerve crush and transection injuries. however, transected nerves showed high and sustained levels of erk1/2 phosphorylation. following treatments, levels of s-100 were significantly decreased both in crushed and transected nerves with respect to control group at p < 0.05. this estrogenic effect was blocked by ici182,780. because of their structural similarity to e2, pm may have therapeutic potential in nerve injuries which as previously reported to enhance sfi following sciatic nerve crush in rats after day 7. this study suggested that pm as well as g could enhance nerve regeneration like e2 by interfering with the injury-induced erk signaling pathway. yasuhiro kato 1 , takafumi suzuki 2 , kunihiko mabuchi 2 1 department of advanced interdisciplinary studies, graduate school of engineering, the university of tokyo, japan; 2 department of information physics and computing, graduate school of information science and technology, the university of tokyo, japan mems technologies have been established to fabricate a multichannel neural probe for interfacing with the nervous system. there is, however, no suitable probe for long-term neural recording and stimulation. one main reason is the death of brain tissues damaged by the probe insertion and implantation. thus, a new skeleton-like multichannel flexible neural probe coated with hybrid biodegradable polymer was fabricated. the skeleton-like probe was designed to minimize the volume of the flexible probe and buffer injurious micromotion between the probe and the tissues in a post-implantation. the probe was coated with mixed polyethylene glycol and microspheres with nerve growth factor (ngf) to improve the stiffness for the probe insertion, and deliver ngf for an optimal period to promote regrowth of damaged neural tissues around the probe. damage-induced neuronal endopeptidase (dine) is a newly identified nerve regeneration-associated molecule. it encodes neuronspecific membrane-spanning metalloprotease and belongs to nep/ece family which degrades/processes neuropeptides. although the precise mechanism of dine including substrate is still unclear, dine seems to play a protective role in damaged neurons. the most marked property of dine is a striking response to various kinds of nerve injury in both central nervous system and peripheral nervous system. to clarify the transcriptional regulation of dine after nerve injury, we analyzed 5 untranslated region of dine gene. previously, we found that lif treatment and ngf deprivation additively increased dine mrna. in this study, promoter analysis showed that dine promoter activity was cooperatively up-regulated by atf-3 and stat3, which were induced after nerve injury and activated at the downstream of lif treatment and ngf deprivation. this combination of transcription factors may be pivotal to promote gene expression, which is responsible for nerve regeneration. tomohiro miyashita, takekazu kubo, masashi fujitani, katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan wnt proteins are known as those concerning with formation of central nervous system. we tested whether they play a role in inhibition of axon regeneration after spinal cord injury. cerebral granule neurons from p6-10 wistar rats were cultured. wnt proteins were added into the culture medium. twenty-four hours after culture, neurite length of each neuron was measured. immunohistochemistry was done employing anti-wnts antibody and anti-ryk (wnt receptor) antibody. anti-ryk antibody was injected continuously for two weeks into the subarachnoid space of contused rat spinal cord. locomotor behaviour was evaluated up to six weeks after injury. immunohistochemistry showed that several wnt proteins and ryk were upregulated after spinal cord injury. wnt proteins inhibited neurite outgrowth of cultured cerebral granule neurons. and this effect was abolished by y27632, a rho-kinase inhibitor, and anti-ryk antibody. suppression of wnt proteins may promote axon regeneration and improve locomotor behaviour after spinal cord injury. akihito takeda, richard goris, kengo funakoshi department of neuroanatomy, yokohama city university graduate school of medicine, yokohama, japan in contrast to mammals, spontaneous nerve regeneration after lesion of the spinal cord occurs in fishes. we examined tissue remodeling and axon regeneration after spinal hemisection in the goldfish. in the lesioned spinal cord, neurogenesis reached the maximum level 7 days after the hemisection. glial cells positive for glial fibrillary acid protein (gfap) temporarily increased at the lesion site one day after. many gfap positive cells expressed somatostatin. serotonin (5ht) positive cells increased in number progressively from 1 day to 6 weeks after. six weeks after, the regenerated axons with glial fibers invaded fibrotic scar centered about the lesion site, and 5ht cells surrounded the axons and glia. thus, 5ht may promote these neural elements to invade the fibrotic scar. six weeks after the hemisection, projections from locomotion center in midbrain to spinal motoneurons were restored, and swimming ability was also recovered. these results suggest that the goldfish have ability to reestablish correct projections after the spinal injury. masao koda 1 , yukio someya 2 , ryo kadota 2 , chikato mannoji 2 , tomohiro miyashita 2 , atsushi murata 3 , masashi yamazaki 2 1 department of orthopaedic surgery, togane hospital, 2 department of ortopaedic surgery, graduate school of medicine, chiba university, japan; 3 division of rehabilitation medicine, chiba university hospital, japan objective: anoikis is a type of apoptosis due to the detatchment from the extracellular matrix. preparation of graft cells for cell therapy includes dissociation of cultured cells, which may cause anoikis. here we tested the effect of bdnf for anoikis of schwann cell. methods: (in vitro) schwann cells were cultured from sciatic nerves of neonatal rats. schwann cells were transferred to suspension culture. bdnf was added into the culture medium. cell death was detected 24 h after suspension culture. (in vivo) schwann cells were transplanted with or without bdnf treatment into contused rat spinal cord. immunohistochemistry was performed to detect survival of grafted cells. the olfactory bulb and its caudal extension are unique forebrain regions with the residence of neural stem cells and the ability of persistent neurogenesis. however, evidence for active functional involvement of neural stem cells is still very limited. this study was undertaken to know whether or not newly generated neurons are integrated in olfactory neuronal circuits in the neonatally bulbectomized rats that had been proved to show olfactory discriminative abilities. for this purpose, retroviral vector, a very useful tool to trace neural stem cells, was applied to the anterior part of the subventricular zone of the rats of which olfactory bulbs had been unilaterally ablated at the neonatal stage. we will show cell dynamics of newly generated neurons in the neonatally bulbectomized olfactory nervous system, with special reference to their neuronal circuits. koichi kawada, masanori yonayama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka the subventricular zone (svz) contains undifferentiated cells, which proliferate and generate the olfactory bulb (ob) interneurons. throughout life, these cells leave the svz and migrate to the ob via the rostal migratory stream, where they differentiate. we have shown that trimethyltin (tmt) causes neuronal damage in the hippocampal dentate gyrus. in this study, we examined neuronal degeneration and regeneration in the ob after tmt treatment in mice. ddy mice were given tmt (2.8 mg/kg) to prepare slices for an immunohistochemical analysis using antibodies against single-stranded dna (ssdna), 5-bromo-2 -deoxyuridine-5 -monophosphate (brdu), neuronal nuclei (neun) and nestin. positive cells immunoreactive to ssdna markedly increased in the ob on days 1 after tmt treatment. positive cells immunoreactive to brdu markedly increased in the ob on days 2 after tmt treatment. double staining of brdu and neun in the ob revealed that almost brdu was not incorporated into mature neurons on day 2 after the treatment. these results suggest possible enhancement of neurogenesis in the ob following tmt treatment. ps3a-f087 early migration of human umbilical cord blood neural stem cells transplanted into rat brain miroslaw janowski 1 , hanna kozlowska 1 , marcin jurga 1 , aleksandra habich 1 , elzbieta wanacka 1 , barbara lukomska, krystyna domanska-janik department of neurorepair, medical research center, warsaw, poland many neurological disorders result from progressive cell loss or rapid cell damage. as stem cell technology appeared there is an arising hope for cell replacement therapy and definitive cure. recently, in our laboratory human umbilical cord blood neural stem cell line (hucb nsc) was established. the aim of the study was to analyze the migratory potential of hucb nsc transplanted into intact rat brain. hucb nsc transfected with gfp gene was stereotactically transplanted (tx) into intact brain of csa immunosuppressed adult wistar rats. cell detection was performed 24 h, 48 h, 72 h and 7 days after transplantation using abs anti gfp, hla class i and numa. analysis of rat brains revealed viable gfp positive hucb nsc cells migrating from tx site and dispersed through the host brain tissue 1, 2, 3 and 7 days after grafting. immunohistochemical studies confirmed that these cells were of human origin: hla class i or numa. in future we plan to study their lesion directed migratory potential. heparan sulfate proteoglycans (hspgs) are considered to play roles in cns development, such as axonal guidance. however, little is known about the function of hspgs during nerve regeneration. in this study, we examined the expression of ext2, one of the enzymes for heparan sulfate biosynthesis, after hypoglossal nerve injury. the upregulation of ext2 mrna was detected using in situ hybridization in injured hypoglossal motoneurons, and heparan sulfate glycosaminoglycan was also upregulated in the injured hypoglossal nucleus. we also examined the expression of mrna for hspg core protein. the mrnas for glypican-1 and syndecan-1 were upregulated in injured motoneurons. these results indicate that the synthesis of hspg is upregulated in injured motoneuron and hspg might be involved in nerve regeneration. masami watanabe 1 , hiroe sagawa 2 , masahiro ichikawa 3 , yoshihito tokita 1 1 dept. perinatol., int. dev. res., kasugai, japan; 2 dept. ophthalmol., nagoya univ. sch. med., nagoya, japan; 3 dept. neurosurg., nagoya univ. sch. med., nagoya, japan we examined whether rho/rock inhibitor, y39983, can make injured rgc axons regenerate into the crushed optic nerve (opn) of cats. methods: culture; retinal pieces were cultured in dmem for 14d. after fixation, the neurites were stained with anti-tuj1 antibody to obtain number and length of tuj1 neurites. crush; after an intravitreal injection of drug, the left opn was crushed with thread. on day 12, wga-hrp was injected into the vitreous. sections of opn were reacted for hrp with tmb reaction. masanori yoneyama, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan in this study, we evaluated the effects of glutathione depletion on proliferative activity in neural progenitor cells of 15-days-old embryonic mice. neural progenitor cells were prepared from the hippocampus of 15-days-old embryonic mice by culturing in dmem/f12 medium for 9 days in vitro (div). marked round spheres were formed from cells adhered to each other under the culture conditions in the presence of bfgf and egf, and then subsequently proliferated to form large neurospheres in proportion to the duration of cultivation. to evaluate the effects of glutathione depletion on proliferation in the neural progenitor cells, buthionine sulfoximine (bso) were exposed into cultured neural progenitor cells for a period of 0-9 div. treatment with bso resulted in a marked reduction in endogenous glutathione in the cells. mtt assay revealed that the deletion of glutathione led to a marked decrease in surviving neurospheres cultured for 3-9 div. these results suggest that glutathione would positively regulate proliferative activity and/or survival in neural progenitor cells of murine hippocampus. michio hashimoto, eisuke kawakita, masanori katakura, osamu shido dept. of environ. physiol., sch. of med., shimane univ., japan docosahexaenoic acid (dha), one of the main lipids in brain, plays crucial roles in the development and function of brain neurons. we examined the effect of dha on neuronal differentiation of neural stem cells (nscs) in vitro and in vivo. nscs obtained from rat embryos were propagated as neurospheres and cultured with or without dha for 7 days. dha increased the number of tuj1(+) neurons compared with the control, and the newborn neurons in the dha group were morphologically more mature than in the control. dha decreased the incorporation ratio of brdu, the mitotic division marker, during the first 24 h period. thus, dha promotes the differentiation of nscs into neurons by promoting cell cycle exit. furthermore, dietary administration of dha significantly increased the number of brdu(+)/neun(+) newborn neurons in the granule cell layer of the dentate gyrus in adult rats. these results demonstrate that dha effectively promotes neurogenesis both in vitro and in vivo, suggesting that it has the new property of modulating hippocampal function regulated by neurogenesis. research funds: kakenhi (17590205) ps3a-f092 interaction among cues for visual depth motion perception tomokazu shimizu, akitoshi hanazawa kyushu institute of technology, fukuoka, japan when an object surface approaches or leaves us, we perceive visual depth motion. cues for this motion are change in binocular disparity, change in spatial frequency and optical flow. we investigated interactions among these cues by using visual stimuli in which the cues provides opposite depth motion direction. for the stimulus without optical flow component, spatial frequency was changed continuously by presenting uncorrelated random dot patterns filtered by different band-pass filters. when binocular disparity and spatial frequency was oppositely changed, subjects perceived depth motion corresponding to the change in binocular disparity or special frequency. for the stimulus with optical flow component, a random dot pattern filtered by a band-pass filter was expanded or contracted. when binocular disparity and the other two cues were oppositely changed, subjects perceived depth motion corresponding to the change in the other two cues. when depth motion was perceived from binocular disparity, stimulus image was perceived as changing its size. when from the other cues, depth perception from binocular disparity was suppressed. research funds: coe-j19 kazuyuki takahashi, akitoshi hanazawa kyushu institute of technology, japan in phenomena such as biological motion and structure from motion, a global structure is perceived by an integration of local motion signals. to clarify the fundamental mechanism of this motion integration process, we psychophysically examined the influence of directional motion coherency on motion grouping. moving dots were presented in three apertures that were aligned horizontally. before presenting these stimuli, subjects were instructed to detect a dot moving in a direction among noise dots presented in the central aperture. the dots moving in the same as or different from the instructed direction were presented in the side apertures. the performance of the subjects was the best when the dots in the side apertures moved in the same direction as the instructed one. the performance kept high when the directional difference was up to ± 10 • and declined as the difference increased. the high performance would be due to the grouping of the dots presented in the central and side apertures that have the same or similar motion direction. the underlying motion grouping mechanism was suggested to integrate motion signals that have a certain directional variation. ps3a-f094 effect of spatial context on structure-frommotion perception koshi makino, akitoshi hanazawa kyushu institute of technology, japan when viewing an orthographic projection of dots on the surface of a rotating cylinder, one perceives a transparent rotating 3d cylinder. this phenomenon is called structure-from-motion (sfm). the direction of the rotation is ambiguous. we investigated the influence of spatial context on the perceived direction of the rotation. three spatially separated stimuli were horizontally aligned. subjects reported in which direction the central random-dot sfm cylinder rotated. they perceived the same direction of rotation as the side stimuli when the side stimuli were corotating cylinders whose direction of rotation was disambiguated by binocular disparity. this effect was strong when the stimuli consisted of a small number of dots, and was attenuated as the number of dots increased. the perception was also influenced by translational motion stimuli that had front and back planes comprising oppositely moving random-dots whose depth was specified by binocular disparity. these results suggest that the neural mechanism determining the rotation direction of bistable sfm is strongly influenced by the 3d structure of surrounding stimuli defined by binocular disparity. ps3a-f095 rotational motion aftereffect in positive direction for 3-dimensional random-dot pattern masako ono, akitoshi hanazawa kyushu institute of technology, kitakyushu, japan when viewing a unidirectionally moving pattern followed by a stationary pattern, we will see the stationary pattern moving in the direction opposite to the preceding movement. this phenomenon is well known as motion aftereffect (mae). this mae can be perceived for 3-dimensional motion such as rotating cylinders. we found that adaptation to the rotation of a stereoscopic random-dot cylinder generate mae like phenomenon in the same positive direction as the rotation of the adaptation stimulus (positive mae). this positive mae was strong when cylindrical random-dot was used as a stationary test stimulus. this aftereffect could not be perceived for uniformly distributed non-cylindrical random-dot. although ordinary mae declined in a few seconds, this positive mae remained for a few minutes. this is a new phenomenon that is different from known 3dimensional mae. this finding suggests that the visual system has a mechanism that detect 3-dimensional rotation direction specifically, and this mechanism has a property that gives a bias to the perception of stereoscopic rotation direction in an adapted direction. research funds: coe-j19 takanori uka, ryo sasaki department of physiology 1, juntendo university school of medicine, tokyo, japan crowding refers to a subjectǐs difficulty in identifying a target in the presence of distracters. as a first attempt towards identifying the neural mechanism of crowding, we investigated perceptual crowding using a random-dot kinematogram. human subjects were required to report the direction of moving dots within a center patch (3 deg) of a center/surround display presented 10 degrees to the left of fixation, and to ignore the dots in the surround. motion coherence of the dots in the center patch, as well as surround size varied randomly across trials. motion coherence of the surround was always 0 percent. for each of 11 subjects, we calculated direction discrimination thresholds (at 82% correct) at each surround size. consistent with crowding, thresholds increased when surround size was 4.5 and 6 degrees, compared to those with no surround. surprisingly, however, thresholds decreased when surround size was 9 and 12 degrees, relative to 6 degrees. our results show that the spatial resolution of motion direction discrimination improves when the area we have to ignore exceeds a defined size. ps3a-f097 generation of receptive fields in higher visual areas based on v1 columnar structure: a model study yoshitaka toyoda, yoshiyuki shimizu, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan neurons in higher cortical areas of the visual pathway, such as v2 and v4, respond to stimuli with complex shapes. how do these neurons integrate signals from v1? in particular, does the well-known columnar organization of v1 play a role in determining the shape selectivity of higher-order neurons? to explore these questions, we devised a feed-forward hierarchical model. in our model, higherorder neurons sum the activities of v1 neurons linearly according to a neural receptive field (nrf), a weighting function defined over the cortical surface. the manner a nrf sums over multiple columns determines its shape selectivity. since there is no physiological data regarding possible forms for nrf, we have tested simple functional prototypes, gaussians and gabor functions. reponses of these model neurons are examined using non-cartesian gratings and other stimuli, and compared to published physiological data. about 15% of model neurons exhibit responses similar to those of v2 and v4 neurons. odd-symmetric gabor nrfs tend to generate more of these neurons. taihei ninomiya, takahisa m. sanada, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan when images with different spatial frequencies (sfs) are projected onto the two retinae, a 3-d surface slant is perceived (blakemore, 1980) . the relationship between the binocular receptive fields (brfs) and sf tuning properties indicate that the early cortical neurons can signal slant-in-depth (sanada and ohzawa 2006) . however, their measurements of brf were conducted in the spatial domain, and the sf tunings were tested monocularly. in this study, interactions are examined directly in the sf domain between grating stimuli presented to the left and right eyes. frequency-domain brfs were measured by a reverse correlation technique. both binocular and monocular sf profiles were obtained by this method. we predicted binocular sf (bsf) maps from monocular sf profiles, and compared the prediction and the actual bsf maps to assess the binocular interactions. with this method, neural response properties which previous studies couldn't access were revealed. research funds: mext(13041033), jsps(13308048), coe21 ps3a-f099 consistency of simple cell receptive fields: space and spatial frequency domain measurements yuka tabuchi 1 , kota sasaki 2 , izumi ohzawa 1,2 1 grad. school of frontier biosci., osaka univ., japan; 2 grad. school of eng. sci., osaka univ., japan frequency-domain subspace reverse correlation and 2-d spacedomain dynamic dense noise have become increasingly popular for mapping receptive fields (rf) of early visual cortical neurons. however, it is not known whether results from these methods are mutually consistent. to examine this issue, we compared an rf in the space domain measured by 2-d noise stimuli and an rf reconstructed from the response in the spatial frequency (sf) domain measured by flash grating stimuli of various orientation (or), sf and spatial phase presented in rapid succession. we fitted these two rfs by gabor functions, and examined the consistency of their parameters. all parameters including sf, or, spatial phase, and size of the rf agreed well when an expansive nonlinearity is considered for each cell. the optimal sf obtained in the space domain increased over time to the same extent as that obtained in the sf domain. therefore, responses of a simple cell can be encapsulated in a concise framework of a linear filter followed by expansive nonlinearity. research funds: mext(13041033), jsps(13308048), coe21 ps3a-f100 firing statistics and stimulus selectivity of inferior temporal cortical neurons in the monkey shunta tate 1,2 , hiroshi tamura 1,3 , ichiro fujita 1,3 1 graduate school of frontier biosciences, osaka university, osaka, japan; 2 jsps, japan; 3 crest, jst, japan inferior temporal (it) cortical cells are selective for visual shape, and vary in their spontaneous firing pattern among them. cluster analysis indicated that it cells were classified into five groups based on inter-spike interval (isi) histograms of their spontaneous firing. the first two groups showed a single peak at a long or a short isi in isi histograms. the other three had multiple peaks, whose positions and relative heights varied among the groups. principal component analysis and other analyses of visual responses showed that the five groups differed in their stimulus selectivity for a predetermined set of visual stimuli. stimulus selectivity was sharper in the single-peak groups than in the multiple-peak groups. one of the single-peak groups was modulated by natural images more strongly than the other groups. the results suggest that cells with different firing patterns carry different aspects of visual information, and may perform different functions in the coding of visual object images. supported by jsps and crest. research funds: kakenhi 16-07757 ps3a-f101 spatial-frequency dependency of receptive field size and surround suppression in lgn and v1 hironobu osaki 1 , tomoyuki naito 2 , osamu sadakane 2 , masahiro okamoto 3 , hiromichi sato 2,3 1 med. sch., osaka univ.; 2 grad. sch. med., osaka univ.; 3 grad. sch. front. biosci., osaka univ., osaka, japan in the primary visual cortex (v1), neurons change their responses depending on stimulus parameters such as orientation, size, spatial frequency (sf). we investigated how sf of stimulus affects on stimulus-size tuning property of responses of neurons in v1 (n = 93) and lateral geniculate nucleus (lgn) (n = 63) in anesthetized cats. first, we found that v1 neurons exhibited shifts of their sf tuning from high to low according to a change in stimulus size from small to large. second, we measured stimulus-area summation curve of responses and found that a higher sf stimulus caused a reduction of the receptive field (rf) size and an increase of the surround suppression. similar results were obtained for lgn neurons implying that the relationship between sf and area summation properties observed in v1 has its origin in lgn. these results suggest that the sf tuning of rf surround is broader than that of rf center and this center-surround mechanism reduces redundancy in visual information processing. hiroyuki nakamura 1 , akichika mikami 2 , kazuo itoh 1 1 department of morphological neuroscience, gifu university graduate school of medicine, gifu, japan; 2 department of behavioral and brain sciences, section of neurophysiology, primate research institute, kyoto university, inuyama, japan an extrastriate visual area v3a is considered to be involved in the dorsal stream visual areas, however, its connections are not understood. to demonstrate the cortico-cortical connections of v3a, we injected a bi-directional tracer biotinylated dextran amine into the v3a. our results indicated that the v3a has connections with the occipital, parietal and temporal cortices. the v3a may thus be involved in the visual information processing of both the dorsal and the ventral stream visual areas. in addition to these connections, we found that v3a has commissural connections with the v3, the v3a, the parieto-occipital area, the dorsal parietal area, and the ventral intraparietal area, and receives commissural projections from the dorsal and ventral aspect of secondary visual area v2. these commissural connections may convey ipsilateral visual information near the vertical meridian representations. ps3a-g103 activity of neurons in the isthmo-optic nucleus and its relationship with head movements hiroshi ohno, hiroyuki uchiyama department of information and computer science, faculty of engineering, kagoshima university, kagoshima, japan retinopetal neurons in the isthmo-optic nucleus (ion) send their axons to the contralateral retina in birds. the centrifugal visual projection is thought to be involved in attentional modulation of retinal output. we recorded activity of neurons in the ion in awake, headunrestrained japanese quails using an implanted electrode assembly. head movements were videotaped with a high-speed video camera (100 fps), and were also monitored with a 2d or 3d accelerometer. we found two distinct types of activity pattern: phasic and tonic. the majority of neurons in the ion discharge in a phasic manner. phasic and tonic cells are also different one from another in relation to head movements. phasic cells show phasic elevation of activity 10-40 ms after end of head movements, while tonic cells show tonic suppression during head movements. we will discuss the activity profiles of neurons in the ion in terms of their possible role in visually guided behaviors. ps3a-g104 timing of face specificity in fusiform gyrus responses to stimuli in different parts of the visual field yuka okazaki 1,2 , arman abrahamyan 3 , catherine stevens 3 , andreas a. ioannides 1,2 1 brain science institute, riken, saitama, japan; 2 graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan; 3 school of psychology, university of western sydney, sydney, australia neuroimaging techniques have demonstrated the preferential responses to faces in the fusiform gyrus (fug). event related potential (erp) and magnetoencephalography (meg) studies have shown that such the responses specificity to faces occurs approximately 170 ms (n170) after stimulus onset by comparing with the other objects. in the present study, we examined whether these and earlier fug activities, which have been already identified by our team (within 100 ms), were selective for face. we achieved this by analyzing meg data elicited by static human faces, hands and shoes stimuli placed in fovea and four quadrants. we found robust statistically significant activities for faces in fug about 100 ms after stimulus onset which depended on the stimulus location in the visual field. narihisa matsumoto 1 , shoutaro akaho 1 , kenji fujikumi 2 , yasuko sugase-miyamoto 1 , masato okada 3 1 aist, ibaraki, japan; 2 ism, tokyo, japan; 3 university of tokyo, chiba, japan to understand the temporal aspects of information encoded at a population level in the inferior-temporal (it) cortex, we applied a cluster analysis method to the responses of 45 neurons. each response was recorded while one of the visual stimuli that consisted of geometric shapes and faces of humans and monkeys was presented. population activity vectors of 45 neurons for 38 visual stimuli were clustered by a mixture of gaussian model. we estimated the number of clusters by using variational bayes algorithm. we assumed that the probability of the number of clusters depended on the one at one time step before. in the early period, the population vectors formed three clusters corresponding to global categories (human versus monkey versus shape). in the subsequent period, each cluster expanded to form sub-clusters corresponding to detailed categories. moreover, the number of clusters changed smoothly over time. these results suggest that the responses of it neurons represent different levels of categorical signals separated along the time axis. ps3a-g106 relationship between color and shape selectivity in area teo of the monkey masaharu yasuda 1,2 , hidehiko komatsu 1,2 1 national institute for physiological science, okazaki, japan; 2 sokendai, okazkaki, japan visual objects typically consist of multiple features such as color, shape, texture etc. it is reported that neurons selective for these object features exist in the inferior temporal (it) cortex of the monkey and some of them are selective for more than one of these features. however, little is known about the relationship between the selectivity for different features. last year, we have reported that there exist many neurons in the posterior part of it cortex (area teo) that are selective for both color and shape. to study the relationship between the color selectivity and shape selectivity, we tested the responses of each neuron using all combinations of the sets of colors and shapes, and conducted svd (singular value decomposition) analysis. we found that some teo neurons exhibited selectivities for color and shape that were independent (separable) each other, whereas in some other neurons they were not independent (nonseparable). these results suggest a possibility that color and shape informations interact at cellular level in this area. ps3a-g107 neural correlates of stimulus shape detection in monkey inferior temporal cortex taijiro doi lab. cogn., neurosci., osaka univ., japan we searched for a neural "correlate" of conscious perception of shape by recording neuronal activities from inferior temporal (it) cortex while a monkey performed a 3-choice shape detection task. the monkey was required to judge whether or not a sample stimulus was presented immediately after a forward masking stimulus. when there was, the monkey was required to select the stimulus identical to the sample from three targets, two shapes and one small dot. trial-totrial variation of firing rates of many it neurons correlated with the monkey's seen versus not-seen choices. the mean choice probability (cp) of it neurons was 0.58, a value significantly larger than the chance level. neurons with stronger visual responses exhibited larger cps. we also searched for temporal firing patterns within the spike train from a single neuron or across 2-3 simultaneously recorded neurons, but failed to find any temporal structure related to the monkey's behavioral choice. the results indicate a link between the firing rates of it neurons with conscious perception of stimulus shape. research funds: mext grant (17022025) ps3a-g108 behavioral visual performance of the zebrafish mutant, eclipse yuko nishiwaki 1 , atsuko komori 1 , tomonori manabe 2 , toshihiko hosoya 2 , hiroshi sagara 3 , emiko suzuki 3 , hitoshi okamoto 2 , ichiro masai 1 1 masai initiative research unit, riken, wako, japan; 2 riken bsi, wako, japan; 3 ims, university of tokyo, minato-ku, japan eclipse was identified as a visual zebrafish mutant that does not show both electroretinogram and optokinetic response. in the last meeting, we reported that the els gene encodes the ␣ subunit of cgmp phosphodiesterase (pde6c), which functions in phototransduction in cone photoreceptors. since genetic mutations of pde6c have not been reported in human patients of hereditary eye diseases, the els mutant is a good model for studying physiological roles of pde6c. here we investigated whether the structural integrity of photoreceptors and visual sensitivity are affected in the els mutants. our electron-microscopic analyses revealed that photoreceptors do not undergo degeneration and are maintained in the els mutant until 9 day-post-fertilization. however, we found that visual response to the contrast is slightly affected in larvae heterozygous for the els mutation. these data suggest that the level of pde6c activity is important for the sensitivity of vision. ps3a-g109 localisation of two markers of oxidative phosphorylation in the ageing human retina: an immunohistochemical study tapas nag, shashi wadhwa aiims, india the enzymes of oxidative phosphorylation are known to be affected by reactive oxygen species, which cause mutations in them, leading to reduced energy production. we examined the distribution of two markers of oxidative phosphorylation (nadh-ubiquinol oxidoreductase and cytochrome c oxidase) in the human retina at different ages. eyeballs of donors (age: 50-94 years) were fixed in paraformaldehyde, frozen retinal sections from macular to midperipheral regions cut and immunolabelled for nadh-ubiquinol oxidoreductase (complex i) and cytochrome c oxidase (complex iv; molecular probe, usa). complex i-immunoreactivity (ir) was moderately present in photoreceptors, outer plexiform layer and few ganglion cells from 50 to 80 years of age, and showed a decline and lack of ir in older retinas (85-94 years). complex iv-ir was intensely present in most ganglion cells, outer plexiform layer and photoreceptors from 50 to 91 years of age, and absent at 94 years of age. thus, complex i and iv-ir decline with age, with the former showing an earlier reduction in its ir. the data signify a reduced mitochondrial activity in the retina with ageing. research funds: aiims ps3a-g110 temporal characteristics of neural activity related to target detection during visual search tomoe hayakawa 1 , norio fujimaki 1 , toshihide imaruoka 2 1 nict, kobe, japan; 2 kit, kanazawa, japan meg and fmri experiments were conducted during the orientation singleton search task, and moment magnitudes of dipoles were estimated with an fmri-constrained meg-multi-dipole method to obtain differences between target-present and -absent conditions in each brain region for the whole time course. activity around the cas consisted of a prominent and a subsequent smaller but still obvious peak (117, 215 ms); the first peak showed no difference between conditions while the second peak was significantly larger in the target-present. activity around the pfug had a prominent peak and subsequent small activity (125, 237 ms), whereas the target's presence or not had no influence on either activity. the activity of the right intraparietal sulcus (ips) was significantly larger than that for the left ips at latencies around 196 ms irrespective of the target's presence or not. the results demonstrate that neural activities of multiple regions had different temporal characteristics and the later activity around the cas was related to the target segregation from its surroundings. kaoru amano 1,2 , derek arnold 3 , alan johnston 4 , tsunehiro takeda 1 1 univ. tokyo, chiba, japan; 2 ntt cs lab., kanagawa, japan; 3 univ. sydney, sydney, australia; 4 ucl, london, uk when a moving border defined by small luminance changes (or by color changes) is shown in close proximity to moving borders defined by large changes in luminance, the low contrast border can appear to jitter at a characteristic frequency -a phenomenon we refer to as misc (arnold & johnston, 2003) . in order to reveal the neurophysiological substrates of this illusion, brain activities measured using magnetoenceohalography (meg) were compared with the perceived rate of illusory jitter measured psychophysically. the result showed that the perceived rate was around 10 hz and matched with the alpha frequency of meg. as 10 hz meg responses were enhanced in the presence of illusory jitter relative to the presence of isoluminant motion and physical 10 hz jitter, we believe that the activity is related to illusory jitter generation rather than to jitter perception or to isoluminant motion per se. these results support our hypothesis that misc is generated within cortex by the dynamic characteristics of a cortical feedback circuit rather than by any physical stimulus properties. ps3a-g112 the internal structure and the visual neuron projection patterns of the ventrolateral protocerebrum (vlpr) in the drosophila central brain kazunori shinomiya 1,2 , kei ito 1,2,3 1 center for bioinform., imcb, univ. of tokyo, tokyo, japan; 2 dept. comput. biol., grad. sch. frontier sci., univ. of tokyo, kashiwa, japan; 3 bird, jst visual information processing in the insect brain has so far been analyzed mainly within the optic lobe. many visual pathways are known to project from the optic lobe to a central brain area called the ventrolateral protocerebrum (vlpr). the vlpr is therefore expected to be one of the major higher-order visual centers. the neural circuits in this area, however, remain essentially unknown. our study is to reveal the detailed internal structure of the vlpr, for the first time, using the drosophila brain as a model system. we have identified 12 discrete glomerulus-like structures (gls) in the vlpr, among which at least five are innervated by the visual projection neurons from the optic lobe. we analyzed the detailed internal structure of these gls by visualizing single cells in each visual pathway using the combination of the gal4 enhancer-trap and the flp-out systems, and revealed the directionality of each pathway by specifically labeling the pre-and post-synaptic terminals. ps3a-g113 dynamic reorganization of orientation maps in a late phase of the sensitive period kazunori o'hashi 1,2 , toshiki tani 2 , shigeru tanaka 1,2 1 graduate school of life science & systems engineering, kyushu institute of technology, japan; 2 laboratory for visual neurocomputing, brain science institute, riken, japan we have found that there are two phases in the sensitive period of orientation plasticity: an early irreversible phase and a late reversible phase. in this study, we attempted to elucidate how orientation maps are reorganized in the late reversible phase, performing intrinsic signal optical imaging several times from the same kittens. we observed the over-representation of the exposed orientation even one day after the onset of goggle rearing around the age of 5 weeks. we also found that when the goggles were removed after 1 or 2 weeks of goggle rearing, drastically reorganized orientation maps returned to regular orientation maps that had been established before goggle rearing. these results suggest that once established orientation maps in an early phase serve as template maps to which later rapidly reorganized orientation maps are restored by the release of single orientation exposure. manavu tohmi, seij komagata, yamato kubota, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan fourier analysis of intrinsic signals produced by periodic visual stimuli has been applied for constructing retinotopic maps (kalatsky and stryker, 2003) . in the present study, we used fourier analysis of flavoprotein fluorescence signals for constructing retinotopic maps in the mouse visual cortex. periodic bar stimuli that moved across the visual fields produced periodic fluorescence signals in the visual cortex of anesthetized mice. the fourier components of the signals locked with the periodic stimuli were calculated in each pixel regarding the magnitude and phase. retinotopic maps were constructed based on these components. vascular artifacts could be removed when the stimulus frequency was higher than 0.3 hz, since fluorescence signals but not vascular responses could follow up to these frequencies. combination of flavoprotein fluorescence imaging and fourier analysis is a powerful tool for investigating high-resolution retinotopic maps with short acquisition time in the mouse visual cortex. yoshitake kohei, manavu tohmi, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst, niigata univ., nigata, japan we have reported that ocular dominance plasticity induced by monocular deprivation can be visualized in mice using transcranial flavoprotein fluorescence imaging. another condition for producing ocular dominance plasticity is strabismus, which causes an increase in the proportion of monocular cells in the visual cortex. however, this possibility has not been tested in mice, mainly because surgical operations for producing large and stable shifts in eye position are difficult in mice. in the present study, we designed a new prism goggle for mice. this goggle was attached on the skull of mice during the critical period. the neural responses in the visual cortex of these mice were investigated using transcranial flavoprotein fluorescence imaging. preliminary experiments suggested that the responses in the monocular zone of the visual cortex were not affected in the strabismic mice. however, binocular interaction, which was additive in the binocular zone of normal mice, turned to be more repulsive in the strabismic mice. ps3a-g116 retinotopy-based morphing of brain activity hiroshi ban, hiroki yamamoto, jun saiki graduate school of human & environmental studies, kyoto university, kyoto, japan the topographic visual field map is a fundamental property of the primate early visual cortex. we propose a new method to represent and sample topographic activities in the space of visual field by extending our previous study (maeda et al., 2003. neurosci. res.) . the procedure was as follows. first, eccentricity and visual angle representations were measured for each subject using standard phase-encoding stimuli. second, individual cortical surfaces were reconstructed. third, the transformation between the position in the visual field and that on the cortical surface was established. finally, by using this transformation, brain activities were sampled and then displayed as an image spanning visual field dimensions, each pixel of which represents the activity of neurons representing a given position in the visual field. this retinotopy-based morphing is useful to analyze brain activity related to spatial and form vision and is more reasonable to integrate individual data than normalizing methods based on stereotaxic coordinates and anatomical structures. masahiro yamada 1 , yasuhiro enami 1 , hiroshi jouhou 2 , takehiko saito 3 , kaj djupsund 4 1 tokyo metropol. univ., hino, tokyo; 2 astellas pharma. inc., osaka, japan; 3 suny upstate med. univ., center for vision and ophthal., ny, usa; 4 univ. kuopio, dept. neurobiol., kuopio, finland on-off type amacrine cells are intensely connected with each other by gap junctions (gjs), forming a syncytium with a wide receptive field. we studied effects of external ph (ph 0 ) on the control of cell functions. photoresponses of the cells were recorded intracellularly. slits of light stimuli simplified the estimation of the current flow in the cellular network into a one-dimensional problem. by lowering ph 0 only 0.2 units from the baseline of 7.60, we found a remarkable reduction of the conduction velocity by 20-30%, an increase of the length constant and a hyperpolarisation of the resting potential. based on our theoretical model, combined with measurements of conduction velocity and length constants of the receptive field, we could estimate both gj and plasmamembrane conductances of the cell. thus, we suggest that protons could contribute to the reduction of conductances, especially at the plasmamembrane but also at gjs. ps3a-g118 analysis of the band-pass filtering of the retinal rod by the ionic current model it is known that the rod network behaves like a band-pass filter. it was found that the time to peak of the response was shorter in rods further away from a slit of light. the band-pass filtering behavior has been attributed to an inductance element, i h , or i k(ca) . however, biophysical mechanism underlying the band-pass filter is not fully understood. to analyze the functional roles of ionic currents in the band-pass properties of rods, a model of the rod network was developed. the model incorporates much of the known parameters in rods, i.e., the phototransduction cascade, ionic currents (i ca , i kv , i k(ca) , i h , i cl(ca) ), calcium system and gap junctions between rods. in simulation, the band-pass properties of the rod was analyzed. it was found that single rod itself behaves as a band-pass filter. the mechanism underlying the band-pass filter was examined by changing model parameters. the result suggests that i k(ca) , i cl(ca) and i h are responsible for the bandpass filtering. research funds: kakenhi (17500195) ps3a-g119 stimulus selectivity and correlated spontaneous activity of distant neurons in monkey inferior temporal cortex go uchida, mitsuhiro fukuda, manabu tanifuji bsi, riken, wako, japan in inferior temporal (it) cortices of anesthetized macaque monkeys, we have previously shown that spontaneous spike activities (sas) of 30% (17 of 57) of neuron pairs (inter-neuronal distance >500 m) are significantly correlated. in the present study, to investigate how the correlated sas relate to functional structure in it cortex, we measured stimulus selectivity for each neuron of the 57 pairs and explored similarity of stimulus selectivity by calculating correlation coefficients of responses to 28 visual stimuli. this analysis revealed that the pairs with correlated sas tended to show more similar selectivity than the pairs lacking correlated sas. in addition, model analysis showed that in 71% (12/17) of the pairs the correlation of sas reflect synchronous transition between two activity states: periods with high and low mean firing rates. these results suggest that a network underlying the synchronous state transition provides circuitry that functionally connects distant it neurons showing similar stimulus selectivity. toshiyuki ishii 1,2 , toshihiko hosoya 1 1 bsi, riken, japan; 2 dept. biomolecular science, toho univ., japan understanding the significance of single spikes can be of critical importance in the analysis of neuronal information coding. it is often assumed that the firing rate is the sole carrier of information. however, if fine temporal patterns of spikes would carry information, the system could have large encoding efficiency. the vertebrate retinal ganglion cells fire burst spikes, separated by hundreds of milliseconds of silent periods. here we show that temporal patterns of spikes within these bursts carry visual information. when three or more spikes are fired, the multiple interspike intervals encode the input in a cooperative, non-redundant manner. this suggests that the spike patterns are not sorely determined by slowly modulating instantaneous firing rates. we also found that millisecond-scale structures in the spike patterns encode light intensity waveforms over 200 ms. we propose that the retina compresses hundreds of milliseconds of light sequences into spike patterns at the scale of milliseconds. kazuhiro shimonomura, takayuki kushima, tetsuya yagi osaka university, osaka, japan purpose of this study is to design a neuromorphic hardware model that emulates fundamental architecture and function in the primary visual cortex (v1). we have constructed a binocular vision system consisting of two silicon retinas and simple cell chips and fpga circuits. the silicon retina has a concentric center-surround laplacian-gaussian-like receptive field. the output image of the silicon retina is transferred to the simple cell chips. the simple cell chip aggregates analog pixel outputs of the silicon retina to generate an orientationselective response similar to the simple cell response in v1. this architecture mimics the feed-forward model proposed by hubel and wiesel, and computes physically a two-dimensional gabor-like receptive field. the fpga circuits compute complex cell responses based on the disparity energy model. the system can emulate the neural image of the binocular complex cells responding to natural scene in real-time and is useful to verify computational models of v1 neurons. masayoshi tsuruoka 1 , masako maeda 1 , bunsho hayashi 1 , ikuko nagasawa 2 , tomio inoue 1 1 dept. physiol. showa univ. sch. dent. tokyo, japan; 2 dept. anestesiol. showa, univ. sch. dent. tokyo, japan the present study investigated the involvement of ventral root looping afferent fibers in visceromotor function. under halothane anesthesia, the t13-l2 dorsal roots were cut bilaterally to eliminate thoracolumbar influences. an electromyogram (emg) of the external abdominal oblique muscle evoked by colorectal distention was measured. colorectal distention (80 mmhg) was produced by inflating a balloon inside the descending colon and rectum. emg activity evoked by colorectal distention significantly increased when the colon was inflamed with mustard oil (5%, 1 ml). the increased emg activity significantly reduced following bilateral l6-s3 ventral rhizotomies. a baseline emg did not significantly alter when the l6-s3 ventral roots were cut bilaterally prior to inflammation. following the development of inflammation, there was less of an increase in emg activities. these results suggest that looping afferent fibers in the ventral root are involved in visceromotor function during colon inflammation. ps3a-g123 hypnotic modulation of the cerebral processing of human visceral sensation using positron emission tomography using positron emission tomography (pet), we examined cerebral processing to visceral perception during neutral, hyperalgesic or analgesic suggestion with standard hypnosis. activation within right dorsolateral prefrontal cortex (dlpfc) and right inferior parietal cortex (ba40) was significantly greater (p < 0.001, uncorrected) during rectal distention with analgesic suggestion than with neutral suggestion. on the other hand, activation within right medial frontal cortex (mpfc) was significantly greater (p < 0.001, uncorrected) during rectal distention with hyperalgesic suggestion than with neutral suggestion. this is the first evidence with pet for a modulation of cerebral processing during visceral stimulation by hypnotic suggestion. these results suggest a role of dlpfc and mpfc in the cognitive control of the interoception. the participation of bladder receptors sensitive to cold temperature has been proposed in overactive bladder for decades. bladder cooling reflex (bcr) which consists of immediate sense of urgency and detrusor contraction in response to ice water infusion may be a neuropathic cause of detrusor overactivity (do). recently, urothelial cells display a number of properties similar to sensory neurons and have many sensors including gene for transient receptor potential (trp). we detected cold sensitive receptor trpm8 in the urothelial cell by immunofluorescence in an animal model for boo. intravesical administration of trpm8 agonist (l-menthol: 0.06-6 mm) in freely moving rats, increased the micturition pressure (mp) in either normal (n = 7) or boo rat (n = 8). the micturition interval (mi) did not change in normal rat, but decreased in boo that have do. the results suggest that bcr is enhanced in boo by increasing trpm8 on the urothelium cell of the urinary bladder. ps3a-g125 caudate projection from the vagal responsive site in the thalamic parafascicular nucleus in monkeys shin-ichi ito 1 , a.d. craig 2 1 dept. physiol, shimane univ. sch. med., izumo, japan; 2 atkinson res. lab., barrow neurol inst, phoenix, usa we investigated efferent projections to the forebrain, from the vagal afferent activation focus in the thalamic lateral parafascicular nucleus (pf) (ito & craig, j neurophysiol 2005) . evoked potentials were mapped in the right thalamus from stimulation of the left cervical vagus nerve, and fluorescent dextrans were iontophoretically injected at the response focus. the injection sites were all located in the ventrolateral part of caudal pf, lateral to the habenulointerpeduncular tract, medial to the basal ventromedial nucleus, and ventromedial to the centre median. labeled terminals were found in the caudate nucleus (cd) in all cases. terminal patches extended longitudinally in the head of cd, concentrated in its ventral aspect. dense terminal patches also occurred throughout the tail of cd. these results suggest that visceral information modulates the portion of the striatum that has been implicated in cognitive function, and they implicate the caudate nucleus in the control of heart rate and respiration. research funds: nih grant ns40413 ps3a-g126 ascending general visceral sensory pathways to the telencephalon via the medial inferior lobe in a percomorph teleost, tilapia masami yoshimoto, naoyuki yamamoto, chun-ying yang, hironobu ito, hitoshi ozawa department of anatomy and neurobiology, nippon medical school, tokyo, japan general visceral sense is relayed to the telencephalon via thalamic and hypothalamic centers in mammals and birds. in teleosts, an ascending connection that corresponds to the thalamo-telencephalic pathway is present. however, it remained unclear whether or not a hypothalamo-telencephalic pathway exists in teleosts. the medial inferior lobe (mil), which corresponds to part of the hypothalamus of other vertebrates, is known to receive general visceral sensory inputs from the rhombencephalon in a percomorph teleost tilapia. hence, telencephalic connections of the mil were studied in this study. tracer injection experiments into the mil revealed that this hypothalamic zone projects to the preoptic area, the ventral telencephalon (i.e., vs, vd, and vv), and the dorsal telencephalon (i.e., dm, rdc, and dl). these findings suggest that the mil corresponds to hypothalamic relay zones in mammals (e.g. ventromedial hypothalamic nucleus). tatsushi onaka, yuki takayanagi department of physiology, jichi medical university, tochigi, japan administration of prolactin releasing peptide (prrp) decreases food intake. we have previously shown that an icv injection of anti-prrp antibodies increases food intake. neurones producing prrp are activated after peripheral administration of cholecystokinin octapeptide, a satiety factor. it is thus possible that prrp may mediate satiety signals in the brain. here we examined effects of anti-prrp antibodies upon total amounts of food intake and meal patterns. an icv injection of anti-prrp antibodies increased the total amounts of food intake and amounts of food intake during a meal but did not significantly change meal frequency. these data suggest that prrp may play an important role in the short-term control of food intake and are consistent with a hypothesis that prrp is a satiety signal within the brain. research funds: grant-in-aid for scientific research (c) ps3a-h128 fasting induced long-chain fatty acid receptor gpr120 expression in the anterior pituitary of mouse ryutaro moriyama, shingo imoto, shinya shano, nobuyuki fukushima department of life science, kinki university, higashiosaka, japan g-protein-coupled receptor 120 (gpr120) is known as a receptor for unsaturated long-chain fatty acids. the present study investigated the effect of 48 h fasting on gpr120 expression in several regions of male mouse by real-time quantitative pcr, in situ hybridization and immunohistochemical method. gpr120 mrna expression was highly observed in the anterior pituitary, lung, colon, rectum, skeletal muscle, adipose tissue and testis in normal fed animals. 48 h fasting induced gpr120 mrna expression increase in the anterior pituitary, lung and rectum. in the anterior pituitary, gpr120-like immunoreactive cells were only observed in fasting animals. these results suggest that long-chin fatty acid regulates endocrine function in the anterior pituitary via gpr120 at least fasting period. ps3a-h129 ketone body sensing cells in the lower brain stem to regulate food intake and reproductive functions kinuyo iwata, mika kinoshita, hiroaki sato, hiroko tsukamura, keiichiro maeda laboratory of reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan ketone bodies are used for energy in the brain under malnutrition, such as prolonged fasting. we have previously revealed that 3hydroxybutylate (3hb), one of ketone bodies, sensed by the ependymocytes lining the fourth ventricular walls (4v) in the rat brain to regulate reproductive functions and feeding behavior. the present study was aims to determine if the ependymocytes located on the wall of 4v respond to the change in 3hb. change in the intracellular calcium concentration ([ca 2+ ] i ) in vitro was measured in dispersed ependymocytes taken from the 4v in rats. the present results showed that the [ca 2+ ] i increased in response to 3hb, but the increase was blocked by ␣-cyano-4-hydroxycinnamic acid, which is a monocarboxylate transporter 1 (mct1) inhibitor. immunohistochemistry showed that mct1-immunoreactivities were located on the 4v ependymocytes. these results indicate that the ependymocytes may sense 3hb through a mct1-dependent mechanism. research funds: kakenhi 14360177 ps3a-h130 comparison of hypothalamic histamine release by leptin in normal mice and high fat diet-induced obese mice tomoko ishizuka, kouta hatano, atsushi yamatodani dept. med. sci. and technol, grad. sch. allied hlth sci., fac med., osaka univ., osaka, japan leptin is a satiety factor which is produced by the white adipose tissue. peripheral administration of leptin decreases body weight and food intake acting on the hypothalamus. circulating concentration of leptin is in proportion to body fat mass, however, in obese humans, elevated concentrations of endogenous leptin cannot prevent the accumulation of the adipose tissue. we previously reported that leptin decreases food intake via the activation of the histaminergic system. in the present study, the effect of leptin on hypothalamic histamine release was compared in normal and high fat diet-induced obese (dio) mice. leptin (1.3 mg/kg, ip) reduced food intake in normal mice but not in dio mice, suggesting that dio mice have resistance for exogenous leptin like obese humans. the same dose of leptin increased hypothalamic histamine release in normal mice, while it had no effect in dio mice. these results suggest that the lack of the activation of the histaminergic system partly contributes to obesity in leptin-resistant dio mice. tomoya kitayama, yuri onitsuka, katsuya morita, toshihiro dohi department of dental pharmacology, hiroshima university, hiroshima, japan parkinson disease (pd) is neurodegenerative disorder of the substantia nigra accompanied by depletion of dopamine levels. symptoms of pd include disorder of aspiration and mastication, and dysphagia. in this study, rats injected with 6-hydroxydopamine (6-ohda) resulted in an extension of feeding time and a marked increase in the amount of feed powder on cage floor after clump feeding at 4 weeks after 6-ohda without affect on number of neuron in solitary tract. these rats were transplanted with neural progenitor cells at 0 mm; anteroposterior, +3 mm; lateral and −5 and −7 mm; dorsoventral from bregma at 2 weeks after 6-ohda injection. the treatment shortened feeding time and decreased the leavings on the cage floor, as well as achieving decrease of neuronal death in substantia nigra. however, neural progenitor cells were not detected in substantia nigra. these results suggest that transplantation of neural progenitor cells may better 6-ohda-induced eating disorders via protection of neurons. research funds: grant-in-aid for young scientists b 17791323 most tools used by nonhuman animals are extension of their effectors (motor-tools), while humans can use a kind of tools as substitute for their sensory organs (sensory-tools). to understand biological bases of using such tools, we trained japanese monkeys to use a tool as an extension of the eyes, and analyzed its learning processes to proceed as follows: (1) retrieving the food with a rake (a motortool), (2) retrieving the hidden food with a mirror-attached rake, (3) using the reflected image of the food on a mirror separated from the rake, placed stationally beyond hidden food, (4) moving a mirror hung along the rail by hand to find the food, (5) using a rake with a small camera mounted inside, with which the monkeys searched for the food using the live video image captured by the camera on the monitor. finally, they could use a hand-held camera (a sensory-tool) as a manipulable extension of their eyes. thus, acquisition of using the externalized eyes can be achieved by gradual transfer of their own vision to the distant visual cues via motor-tools to extend their body image. kaori sawada 1,2 , shigehiro miyachi 2 , michiko imanishi 2 , masato taira 1 , masahiko takada 2 1 div. applied system neurosci., nihon univ. sch. med., tokyo, japan; 2 dept. system neurosci., tokyo metropol. inst. neurosci., tokyo, japan to investigate the outflow of information from the temporal lobe to the prefrontal cortex, we injected rabies virus into three prefrontal regions: medial area 9 (9m), dorsal area 46 (46d), and ventral area 46 (46v). the retrograde transsynaptic labeling was examined in the temporal lobe cortex 3 days after prefrontal injections when the second-order neurons were labeled. the labeled neurons were observed in the lateral and medial aspects of the temporal lobe. in the lateral temporal lobe, neuronal labeling from 9m, 46d, and 46v was arranged topographically in and around the superior temporal sulcus. the labeing in the medial temporal cortex was also topographically arranged, such that 9m, 46v, and 46d receive multisynaptic projections from the entorhinal cortex, area 36, and both, respectively. these results suggest that there are parallel streams of information flow from the temporal lobe to the prefrontal cortex. research funds: crest, japan science and technology agency ps3a-h137 new neural activities of reward anticipation and task errors h. ogawa 1 , h. ifuku 2 , t. nakamura 3 , s. hirata 4 1 kumamoto kinoh hosp, kumamoto, japan; 2 fac educ, kumamoto univ., kumamoto, japan; 3 nat kikuchi hosp, kumamoto, japan; 4 dept. psych, kumamoto univ. hosp, kumamoto, japan neural activities at reward phase were recorded from the primary (pgc: areas g, 3 & 1-2) and higher-order (hgc: prco & ofc) gustatory cortices of a monkey engaged in a taste discrimination go/nogo task. a lever had to be pressed after led onset when nacl was delivered, but not to water delivery. reward was given ca 2 s after led offset at correct trials. relations between cues and responses were reversed. of 169 reward-related neurons found, 88.7% showed on type responses and the rest usual expectation responses. three types of on responses were noticed; c-type (n = 96) only at correct trial, i-type (n = 5) at around possible reward onset only at incorrect trials, and c-i type (n = 49) at both. two classes of the c-i type were found; class i increased discharges at correct trials but decreased them at incorrect, but class ii increased them at both. all 3 types were found in both cortices, but most class i were found in pgc and most class ii in hgc. i-type and class ii c-i type may represent error signals and reward anticipation. hiroaki ishida, masahiko inase, akira murata department of physiology, school of medicine, kinki university, japan in the macaque monkey, the ventral intraparietal area (area vip) integrated visual-tactile information in the body centered reference frame. the receptive fields of these neurons mapped on the same body parts in each sensory modality, so this area contributes to own body representation often referred to as body image. recent psychological studies implied that shared body representation of self and other might be required in the brain for social interaction. this means other¸s body image is mapped on own body image in the same neuron. in our experiments, we studied visual-tactile receptive field of the bimodal neuron in vip, then recorded activity during observing the experimenter being touched. some of neurons that had receptive fields anchored on the monkey¸s body showed visual response while the experimenter was being touched on corresponding body parts. the results suggested that bimodal neurons in vip may be related to matching mechanism between own body image and others, then we discussed that this area may contribute to the human social ability such as imitation. daichi hirai 1 , takayuki hosokawa 2 , masato inoue 1 , akichika mikami 1 1 section of brain sciences, primate research institute, kyoto university, inuyama, japan; 2 department of psychology, tokyo metropolitan institute for neuroscience, tokyo, japan amygdala is involved in stimulus-reinforcement association learning, and have neural responses related to prediction of rewarding and aversive outcomes. however, it remains unclear whether representation of reinforcement value in the amygdala depends on other available outcomes in a given trial block. to elucidate how rewarding and aversive infomation are coded in the amygdala, we recorded single neuronal activity in monkey amygdala during delayed color matching task. we compared the neural responses to cue that rewarding outcome in two different stimulus-outcome conditions; one included electrical stimulus as aversive outcome, and the other included only rewarding outcomes. we found amygdala neurons to code the relative preference of available outcomes in a given trial block. ps3a-h140 neuronal correlates of expectation-evaluation based on previous and ongoing contextual memories in the monkey prefrontal cortex kyoko matsuda, toshiyuki sawaguchi lab. cogn neurobiol, hokkaido univ. grad. sch. med., sapporo, japan to expect future events based on the ongoing context and to evaluate it are important for flexible control of goal-directed behavior. to examine a possible involvement of the lateral prefrontal cortex (lpfc) in such functions, we recorded neuronal activity from the lpfc of monkeys that performed an oculomotor task. in this task, the target of a saccade was indicated by combinations of successively presented two cues; symmetrically allocated two objects (cue1), and centrally allocated one of the objects presented in cue1 (cue2). the frequency of which object was presented as cue2, i.e., task context, was manipulated across blocks. we focused on cue2 period and found that a subset of neurons showed object preference depending on current task context (cc type) or previous task context (pc type). cc type and pc type activities may be neuronal correlates of expectation-evaluation based on current and previous contexts, respectively. thus, neuronal processes for expectation-evaluation based on previous and ongoing "contextual memories" may progress in the lpfc. ps3a-h141 anterior insular cortex neurons in monkey are activated when reward might be delivered, such as occurs in gambling takashi mizuhiki 1 , barry j. richmond 3 , munetaka shidara 1,2 1 grad. sch. of tsukuba univ., ibaraki, japan; 2 neurosci. ri., aist, tsukuba, japan; 3 lab. neuropsychol., nimh, bethesda, usa the human insular cortex has attracted interest because it is activated during risk-taking or decision-making tasks in fmri studies. to identify related neuronal signals, we recorded single insular neurons while two monkeys worked in a reward schedule task in 2 conditions: (1) a cue is picked at random so it is uncertain whether a correctly performed trial will be rewarded [uncertain condition], (2) a cue indicates whether the current trial will be rewarded or not [certain condition]. in the uncertain condition 84/120 neurons responded in all trials. in the certain condition 50/84 neurons responded in the rewarded trials only. 48 of these 50 showed significant differences in firing rate between in the first trials after reward and other trials. these insular neuron responses seem related to reward expectancy and recent reward delivery. these neuronal responses might underlie the activation identified in imaging studies during gambling and decision-making tasks. research funds: kakenhi (priority areas17022052), aist masamichi sakagami 1,3 , kosuke sawa 2 , xiaochuan pan 1,3 1 bsrc, tamagawa university, tokyo, japan; 2 senshu university, kanagawa, japan; 3 presto, jst, japan reward prediction behavior based on integration of associative information was investigated. monkeys were trained to perform a sequential association task with symmetric reward by symbolic delayed matching-to-sample procedure. at first, they learned two sequences of stimuli: a1-b1-c1 and a2-b2-c2. after monkeys could acquire the sequences, new pairs of stimuli (i.e., d1 and d2, e1 and e2, etc) were introduced to associated with b1 or b2 (d1-b1, d2-b2, etc). the asymmetric reward rule was instructed by pairing c (c1 or c2) with the reward. after this instruction, reward predictive behavior was tested by using trained sequences and new stimuli. monkeys could show reward predictive behavior for not only a1 and a2, which were associated with c1 and c2 in trained sequences, but also new pairs of stimuli, which were not directly associated either with c or reward. these results suggested that monkeys could use reward predicting information by integration of association among trained sequences, c-reward association, and new stimuli. research funds: kakenhi (17022037), hsfp, presto, jst ps3a-h143 reward predicting activity of prefrontal neuron based on group of stimuli xiaochuan pan 1,2 , kosuke sawa 1,3 , masamichi sakagami 1,2 1 bsrc, research institute, tamagawa university, japan; 2 presto, jst, japan; 3 department of psychology, senshu university, japan ability to anticipate a reward based on grouped events is important for guiding appropriate behavior. the main purpose of this study is to examine the pfc neuronal mechanism involved in predicting reward using learned associations among groups of stimuli. monkeys performed a sequential association task with symmetric reward. at first, they learned two sequences of stimuli: a1-b1-c1 and a2-b2-c2. the asymmetric reward rule was instructed by pairing c (c1 or c2) with the reward block by block. monkeys were also trained with two different orders of stimuli (b-c-a and c-a-b). out of 202 neurons from the lateral pfc, 31% showed reward-related activity in the first cue period. and one third of them (sr type) predicted reward only when a preferred stimulus was presented as a first cue. interestingly, the preference was not based on visual properties of stimulus, but on stimulus-group. the results suggest that about 10% of lateral prefrontal neurons predict reward based on stimulus-groups that were formed through the associative learning. attention evoked by novel stimuli is important for behavioral adaptation to new environment. however, it remains unknown whether the novelty is processed in a specific region of the prefrontal cortex. we trained two monkeys on a pavlovian conditioning task interleaved with an instrumental conditioning task and recorded cell activity from the lateral and medial prefrontal cortex (lpfc and mpfc). in a block of the pavlovian task (pv block), a visual stimulus (cs) was paired with a liquid reward and the trial repeated 3 times. in a following block of the instrumental task, the monkey searched a correct action to obtain the cs as positive feedback. the cs was alternated every 4 pv blocks. in many lpfc cells, responses to the cs were enhanced immediately after the change of cs, while such enhancement was less popular in mpfc. this result suggests that lpfc more contributes to coding of stimulus-novelty than does mpfc. when an outcome of action is uncertain, a top-down attention is directed to the coming outcome. to clarify the neural mechanisms, we trained two monkeys on a task with secondary reinforcers and recorded single cell activity of the medial and lateral prefrontal cortex (mpfc and lpfc). in a pavlovian block (pv block), a visual stimulus was paired with a liquid reward. in a following instrumental block (inst block), the monkey searched a correct action based on the visual feedback. the same visual stimulus as the one presented in the preceding pv block followed a correct action, whereas another visual stimulus followed a wrong action. when the monkey made more than 3 consecutive correct trials, a new pv block started. both mpfc and lpfc cells gradually increased their firing toward the visual feedback when the outcome was uncertain, while the onset of the activity was significantly earlier in mpfc than in lpfc. these results suggest that the top-down attention first occurs in mpfc and propagates to lpfc in individual trials. ps3a-h146 neuronal activity in the presupplementary motor area during a bimanual sequential motor task toshi nakajima 1 , hajime mushiake 1 , jun tanji 2 1 department of physiology, tohoku university school of medicine, sendai, japan; 2 brain science research center, tamagawa university, machida, japan to investigate the involvement of the pre-supplementary motor area (pre-sma) in organizing bimanual sequential movements, we recorded neuronal activity while a monkey was performing a motor task consisting of pronation or supination of either arm, with an intervening delay. in this report, we focus on neuronal activity during a period when the monkey was preparing to start the 2-sequence movements in a memorized order. we made regression analysis of neuronal activity in this period. we found that neuronal activity in the pre-sma rarely reflected muscle activity. instead, we found neuronal activity representing forthcoming actions such as supination, regardless of the arm to be used. we also found neuronal activity that reflected the second movement in a preparatory period before the execution of the first movement. we would demonstrate typical examples of pre-sma neurons and discuss their functional implications. ps3a-h147 neuronal activity in the putamen and cm thalamus during response bias and its complementary process yukiko hori, takafumi minamimoto, minoru kimura dept. of physiol., kyoto prefect univ. med., japan we showed previously that cm thalamus participates specifically in complementary process to response bias (minamimoto et al. 2005) . to study the roles of the putamen and cm in response bias and it complementary processes, we recorded activity of cm and putamen projection neurons from two macaque monkeys performing asymmetrically rewarded go-nogo button press task. instruction of go or nogo activated cm neurons (n = 73) preferentially when the instruction was associated with small reward. the instructions activated 3 groups of putamen neurons preferring small reward-(n = 19), large reward-action (n = 3) and both types of action (n = 26). onset latencies of these putamen neurons and rts in large-reward-go trials were shorter than those in small-reward-go trials by 30-50 and 100-150 ms, respectively. putamen neuron activation lead that of cm neurons by 50-70 ms. these results suggested that the putamen plays a major roles in both response bias and its complementary process while cm participates in the complementary process in concert with the putamen. research funds: kakenhi (17022032) ps3a-h148 encoding expected total rewards and their errors through a series of action choices by dopamine neurons naoyuki matsumoto, kazuki enomoto, minoru kimura dept. physiol. kyoto pref univ. med., japan to examine how dopamine (da) neurons represent reward expectation and its error through a series of action choices, we recorded activity of da neurons in two japanese monkeys making trial-anderror and repetition choices to find a correct, rewarding target among three alternatives. there are trials of first (t1), second (t2) and third (t3) choices with reward probabilities of about 30, 50 and 80%, respectively. monkeys got reward after they hit a correct target, and got one more time by choosing the same target in the next trial (r1, 95%). most da neurons (66/85) responded to the start cue of each trial and reinforcer beep after the choices. magnitude of the start cue responses progressively increased from t1 to t2 and to t3 trials, then decreased in r1 trial. in another task with two repetition trials (r1 and r2, 96%), magnitude of start-cue responses decreased gradually from t3 to r1 and to r2 trials. thus, the start cue responses may reflect expected total rewards through a series of action choices for a goal, while reinforcer beep responses may reflect their errors. research funds: kakenhi (17022032) ps3a-h149 striatal neuron activity during decisions and action selections for probabilistic, scheduled rewards hiroshi yamada 1,2 , hitoshi inokawa 1 , minoru kimura 1 1 dept. of physiol. kyoto prefect univ. med., kyoto, japan; 2 jsps, japan to study roles of the striatum in decision and selection of actions for probabilistic, multiple rewards, we recorded 146 striatal projection neurons from a monkey. after depressing a start button, the monkey chose 1 of 3 target buttons with correct rates at 1st, 2nd, 3rd and repetition trials of 33, 50, 89 and 96%, respectively. correct choices were followed by reward water. neuronal firing rates at starting each trial were related either to expected reward probability or to schedule states to obtain reward twice (11/55) rather than to upcoming choice of target (2/55). during the target choice, another subset of neurons showed firings selective to choosing particular target (29/57) rather than to expected reward probability (12/57). after the target choices, another group of neurons fired related to expected reward (41/56) rather than to chosen action (19/56). our results suggested that striatal neurons encode expected reward probability, schedule states to obtain multiple rewards and choice of actions during decision and action choices for a goal. research funds: kakenhi (17022032), jsps fellows ps3a-h150 encoding of reinforcement after rewardbased action selection by tonically active neurons in the striatum hitoshi inokawa, hiroshi yamada, minoru kimura department of physiology, kyoto pref. univ. of med., japan to study the signals encoded by tonically active neurons (tans) in the striatum, presumed cholinergic interneurons, reward-based decision and action selection, activity of 169 tans was recorded from the putamen and caudate nucleus of a japanese monkey. after depressing a start button, the monkey chose 1 of 3 target buttons at average correct rates of 33 (first), 50 (second), 89 (third) and 96% (repetition choices). correct and incorrect choices were followed by high-tone beep, reward water and low-tone beep, respectively. about a half of tans (80/169) responded differentially to the high and low tone beep respectively. number of responsive tans and magnitudes of the responses to high-tone beep was highest at the first choices, then, decreased gradually at second, third and repetition choices. these results suggested that the tans may encode reinforcement after reward-based action choices which is modified by reward expectation errors and motivation. research funds: kakenhi (17022032) ps3a-i151 representation of value of action, action and its outcome in sub-populations of striate neurons y. ueda 1 , k. samejima 2 , k. doya 3 , m. kimura 1 1 dept. physiol., kyoto pref. univ. med.; 2 brain sci. res. center, tamagawa univ.; 3 irp, oist to know the mechanisms of reward-based action selection in the basal ganglia, we recorded activity of 236 striatal projection neurons of two macaque monkeys performing a free choice task with probabilistic reward. after a 1 s delay, monkeys chose between left-and right-handle turn, followed by water reward at probability of 10, 50 or 90%. a linear regression of neuronal discharge rates showed: 39 neurons encoded reward values of either action during delay period before go signal, with most (64%) of them not having the action value signal in other task epochs. another subset of neurons encoded action signal selectively during action selection after go signal (n = 33), while other 37 neurons encoded presence or absence of reward at reinforcer epoch after the action selection. neurons encoding action values were in more anterior part of putamen than the neurons encoding actions. these findings suggested that sub-populations of striate neurons process action values and selection of actions during rewardbased decision and action selection. research funds: kakenhi (17022032) ps3a-i152 delay period activity of the monkey striatum in duration discrimination task atsushi chiba, ken-ichi oshio, masahiko inase dept. physiol., kinki univ. sch. med., osaka sayama, japan neuronal activity was recorded from the striatum of a monkey during a duration discrimination task. two visual cues (a blue or red square) were presented consecutively followed by delay periods, and the subject then chose the cue presented for the longer duration. durations of both cues, order of cue duration (long-short or short-long), and order of cue color (blue-red or red-blue) were randomized on a trial-by-trial basis. striatal neurons phasically responded during the first cue (c1), first delay (d1), second cue (c2), second delay (d2), and response periods. activity during the d1 and d2 periods was analyzed in this study. firing rates during the d1 period linearly depended on c1 durations. on the other hand, d2 period activity depended on trial types (ls and sl), but not on the variety of c2 durations in each trial type. our results suggest that striatal neurons encode, in the delay periods, not only temporal information with monotonic dependence on cue durations to prepare a comparison to a forthcoming cue duration, but also encode discrimination results between two cue durations. research funds: kakenhi (17021039) ps3a-i153 neuronal activities in the anterior inferior temporal cortex of monkeys during an asymmetrical pair association task based on facial identity satoshi eifuku 1 , ryoi tamura 1 , teruko uwano 1 , taketoshi ono 2 1 dept. integrative neurosci., univ. toyama, toyama, japan; 2 dept. molecular integrative emotional neuroscience, univ. toyama, toyama, japan to elucidate neuronal basis of face memory, neuronal activities in the area teav of monkeys were recorded during a pair association paradigm that involves recognition of facial identity (i-apa task). in the i-apa task, monkeys were required to memorize 4 paired associates of patterns and facial identity. each association has a particular direction, either the 'face to pattern' direction in which a cue stimulus which is a face is associated with a test stimulus which is a pattern, or the 'pattern to face' direction in which a cue stimulus which is a pattern is associated with a test stimulus which is a face. during the i-apa task, neuronal responses to a particular paired associate were identified. many of these neurons showed asymmetrical activities during the delay periods which were dominant in the 'face to pattern' trials. this asymmetrical delay activity are indicative of the crucial role of the teav area in face memory. research funds: kakenhi (16500260 17021016) ps3a-i154 reflexive social attention elicited by biological motion in monkeys and humans yoshiya mori 1 , mikio inagaki 1 , wu lisa 2 , taijiro doi 1 , eishi hirasaki 1 , hiroo kumakura 1 , ichiro fujita 1 1 osaka univ., japan; 2 massachusetts institute of technology, usa determining where another individual is attending and preparing for his/her upcoming action is crucial for members of a social group. here we report that the walking direction of another individual elicits a reflexive shift of visuospatial attention in monkeys and humans. we examined how the reaction time to peripheral visual targets was affected by a prior, brief presentation of a walking biological motion (bm) stimulus. during the task, subjects responded to a target point after the disappearance of the bm stimulus and fixation point. the walking direction of the bm stimulus was not predictive of the target direction, and was irrelevant for performing the task. we found that the reaction times in congruent trials, where the walking direction of the bm stimulus and the direction of the target appearance were the same, were significantly shorter than those of incongruent trials. we believe the attention mechanisms driven by bm may be part of the intentionality inference system. research funds: grants from 17022025 and takeda science foundation ps3a-i155 response properties of posterior parietal neurons during a multidimensional visual search task tadashi ogawa, hidehiko komatsu natl. inst. physiol. sci., aichi, japan the posterior parietal cortex (ppc) is thought to be one of crucial areas to direct spatial attention toward the target in visual search. visual sensory information (e.g. stimulus features) might be integrated in ppc to form a saliency map that controls spatial attention. to examine this hypothesis, we recorded the neural activity from the lateral intraparietal (lip) and 7a areas of monkeys performing a multidimensional visual search task. the monkeys had to make a saccade to either shape or color singletons in a stimulus array depending on the instructed search dimension. ppc neurons increased their activity when the receptive field stimulus became the target. some neurons showed target enhancement depending on the stimulus condition (singleton type and stimulus features), whereas others exhibited it irrespective of the stimulus condition. the mixed existence of these two distinct types of activities suggests that ppc is one of critical stages that integrate feature-dependent signals to produce featureindependent signals identifying the target location toward which spatial attention should be directed. monkeys utilize visual information in social communication. to elucidate visual function to categorize sexes, (1) performance of visually guided sex discrimination task and (2) neuronal activity during the task in orbitofrontal cortex (obf), the region could be related to sex recognition and vision processes, were investigated. monkeys were trained to discriminate the sex of a monkey shown in a picture that was presented on the display. the monkeys pressed the right bar for pictures of males and the left for females to get water reward. as a result, the monkeys were able to discriminate the sexes of monkeys shown in pictures. extracellular recordings of neurons in obf during the task showed that some cells responded to the pictures in a sexspecific manner. the present results suggest that visual information alone sufficiently contribute to discriminate sex in monkeys. obf could be involved in visual categorization of sex. research funds: kakenhi (a) (16209006) (sa) and coe program in kit from the mext ps3a-i157 activities of bursting neurons during color discrimination task in the monkey prefrontal cortex naoki ishikawa 1 , satoshi katai 2 , masanori saruwatari 1 , masato inoue 1 , akichika mikami 1 1 section of brain sciences, primate research institute, kyoto university, inuyama, japan; 2 third department of internal medicine, shinshu university, school of medicine, matsumoto, japan the neurons in the prefrontal cortex of monkeys are involved in the behavioral control of saccadic eye movements. on the other hand, cerebral cortex consists of different types of neurons. in this study, we trained macaque monkeys to perform a delayed matching to sample task with saccadic eye movement. and we classified neurons whether they had burst episode or not, and then classified bursting neurons into fast spiking (fs), fast rhythmic bursting (frb), and intrinsic bursting (ib) neurons (katai et al. neuro 2004) . most of bursting neurons activated during the target presentation or during the saccade period were selective to the target location or saccade direction. these results suggest that the bursting neurons have the significant role in the target selection and decision-making of the eye movement toward the specific direction. atsushi matsumoto 1 , tetsuya iidaka 2 1 department of psychology, nagoya university, nagoya, japan; 2 department of psychiatry, nagoya university, nagoya, japan several studies indicated that gamma band activity (gba: 30-80 hz) reflects the process to form mental representation of objects or information. we investigated whether the gba is observed during subliminal visual word processing as well as supraliminal word processing. gba were observed both in masked and unmasked condition. at the 400-600 ms time window, gba was significantly higher in the word condition compared to the nonword condition in the unmasked condition. similarly, in the masked condition, gba of the word condition was significantly higher than that of the nonword condition at that time window. these results indicate that the unconscious lexical processing was reflected in the gba at that time window. furthermore, at the 600-700 ms time window, gba induced by word was significantly higher than that induced by nonword. this effect was not observed in the masked condition. in addition we found the significant semantic priming effect, indicating that the information of briefly presented words was processed unconsciously. wakayo yamashita, junichi hayashi, tomoki murakami, gang wang department of bioengineering, kagoshima university, kagoshima, japan the purpose of this study was to investigate the dependency of view association learning on the separation of the views. each stimulus set included 16 images (4 objects × 4 views). 4 novel objects were generated by deforming a prototype in four directions. for 30 deg-interval object sets, 4 views were obtained by rotating each object with the interval of 30 deg, 60 deg-interval set and 90 deg-interval set were with 60 deg and 90 deg interval respectively. task performances were evaluated while the subjects performed an object matching task, in which the subjects had to recognize one object from others regardless of the viewpoint. the performance across 60 deg separated views was significantly higher in the trials with 30 deg-interval sets than those with 60 deg-interval sets. similarly, the difference was also found in the performances across 90 deg separated views between those with 30 deg-interval sets and 90 deg-interval sets. the results suggest that the exposure of interpolated views significantly improved the association learning of the views. ps3a-i160 brain regional activity during attention task ( the kana pick-out test, treated as inspecting higher brain function, has been proposed to be suitable for screening dementia, which is widely used among public health nurses in japan. however, few fmri studies while demonstrating the test have reported. we therefore assessed the effect of brain regional activity with computerized kana pick-out test projected on the screen with clicking a mouse button to pick kana out under fmri running. executing the test resulted in significant increases in bold signals in right prefrontal area, bilateral hippocampus and broca's area. the results indicate the existence of the attention pathway from and/or to prefrontal area as association mechanisms for execution of kana pick-out test, suggesting that this test is useful in screening dementia. ps3a-i161 obsessive compulsive symptoms in middle school students and its association with tic disorder, body dysmorphic disorder and trichotilomnia in shiraz, iran, 2005 ashkan mowla, arash mowla shiraz university of medical sciences, iran aim: the aim of this study is to evaluate ocd symptoms, tic disorder, body dismorphic disorder (bdd) and trichotilomnia (ttm) among middle school students of shiraz, iran. methods: 1682 middle school students were selected in a cluster random sampling from the four educational regions of shiraz, iran.persion standardized moci was used to assess obsessional symptoms. for evaluating bdd, tic disorder and ttm symptoms, a semi-structured interview was done according to dsm-iv-tr criteria. results: students with more obsessional symptoms were more girls and demonstrated more positive family history.they were more likely to be from lower socioeconomic class and with lower school average. they also showed more association with body dysmorphic disorder and tic disorder. conclusion: girls especially those from lower socioeconomic class demonstrated more obsessional symptoms. this study, like pervious ones, confirmed bdd symptoms and tics to be more in individuals with ocd symptoms. it was seen that ocd symptoms would affect school performance. ps3a-i162 sirna-induced nr1 knockdown causes hypofunction of nmda-r and cognitive deficit m. saji 1,2 , t. utida 2 , a. ohnishi 2 , k. noda 1 , m. ogata 1 , h. akita 1 , n. suzuki 1,2 1 physiol, health sci. sch. kitasato univ., sagamihara, japan; 2 brain sci., graduate sch. kitasato univ., sagamihara, japan blockade of nmda-r by antagonists causes psychomimetic effects, suggesting involvement of nmda-r dysfunction in mental disorders like schizophrenia. however, the relationship between mental disorders and molecular abnormality has not been cleared. to identify the role of nmda-r in brain function, we performed sirnainduced knockdown of nmda-nr1 using hvj-envelope vectors. we confirmed that marked down-regulation (50%) of nr1 expression occurred only in the hippocampus among various brain regions 4-8 days after intra-ventricular injection of sirna-vector complex. in the hippocampal slice from rats with the nr1 knockdown, the nr1 down-regulation prevented depressive effects of nmda on fepsps, while the treatment did not affect ltp or ltd. in rats with the nr1 knockdown, the nr1 down-regulation caused disruption of prepulse inhibition, while the same treatment did not affect locomotor activity. these results suggest that hypofunction of hippocampal nmda-r by sirna-treatment causes a deficit of cognition. ken hatanaka 1,2 , hiroshi ageta 2 , ikuko yao 2 , kaoru inokuchi 2 , yutaka kirino 1 , mitsutoshi setou 2,3 1 graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan; 2 mitsubishi kagaku institute for life sciences, tokyo, japan; 3 okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan schizophrenia is a severe psychiatric disorder that characterized by psychotic symptoms in particular delusions and hallucinations, reduced interest and drive, altered emotional reactivity and disorganized behavior. to know the molecular mechanisms of the disease, we screened altered gene expression on the brain of schizophrenic patients by using microarray analysis, and found that the expression of ubl3 mrna was significantly decreased in the enthorinal cortex, whose size is known to be reduced in some schizophrenic patients. ubl3 is a highly conserved protein, which has a ubiquitin-like domain (ubl domain) and caax motif which is a membrane localization signal. we found that ubl3 mrna was expressed in the hippocampus, and purkingie cells of the cerebellum. the putative molecular function of ubl3 wil be discussed. research funds: grant-in-aid for young scientists (b), presto ps3a-i164 decreased interneurons in the pax6 mutant mouse limbic system hasumi haba 1 , tadashi nomura 1 , yoshinobu hara 1,2 , noriko osumi 1,2 1 div. dev. neurosci., ctaar, tohoku univ. sch. med., sendai, japan; 2 crest, jst, japan core features of schizophrenia are impairments in certain cognitive functions such as working memory, in which a number of brain regions in the corticolimbic system are involved. recent studies have revealed abnormality in distribution of interneurons in these regions. we have previously found that pax6 heterozygous mutant rats show behavioral abnormalities including impairment in fearconditioned memory and sensorimotor gating. in the present study, we thus analyzed distribution of interneurons in several regions of pax6 heterozygous mutant mouse (sey/+) brain. we focused on three subpopulations of interneurons: parvalbumin (pv)-, calretinin-, and somatostatin-posive interneurons. immunohistochemical studies indicated marked decrease in pv-positive interneurons in two brain regions of sey/+ mice, i.e., the olfactory bulb and the amygdala. reduced number of pv-positive interneurons was observed in the sey/+ amygdala at 8 weeks, but not at 4 weeks. our results suggest that age-dependent decrease of pv-positive interneurons might underlie behavioral abnormalities in sey/+ mice. schizophrenia is a complex genetic disorder, characterized by multiple susceptibility genes. dysbindin (dtnbp1) is a susceptibility gene for schizophrenia. genetic evidence for the association between the disorder and the dysbindin gene has repeatedly been reported in various populations world wide. recently, decreased expression levels of dysbindin mrna and protein have been reported in postmortem brain in patients with schizophrenia. thus, we performed behavioral analysis in sandy mouse, which has a deletion in dysbindin gene and expresses no protein. sandy mouse showed decreased locomotor activity and time in the center in the open field test. and an acute treatment of atypical antipsychotic, olanzapine (0.2 mg/kg, i.p.), improved the decrease in time in the center. moreover, subtle behavioral abnormality was observed in elevated plus maze test and social interaction test in sandy mouse. our results suggest that dysbindin might be involved in anxiety-related behavior in novel environment. research funds: 16790712, 17025055 ps3a-i166 gene expression analysis of dysbindin mrna in peripheral blood in schizophrenia sachie chiba 1,2 , satoko hattori 1 , hiroaki hori 1 , tetsuo nakabayashi 3 , hiroshi kunugi 1 , ryota hashimoto 1 1 department of mental disorder research, national institute of neuroscience; 2 tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan; 3 musashi hospital, ncnp, kodaira, japan although many efforts have been spent to discover a biological marker of schizophrenia, no biological marker has been established. as genetic evidence suggested that dysbindin (dtnbp1) is a susceptibility gene for schizophrenia, we measured dysbindin mrna expression level in peripheral blood samples of 38 patients with schizophrenia and 38 age-sex matched healthy controls by a quantitative real time rt-pcr method. we quantified the expression levels of two major dysbindin transcripts among several known splicing variants. no significant difference in the expression levels of examined dysbindin transcripts was observed between control and schizophrenia. further examination measuring other dysbindin transcripts should be warranted to find a biological marker for schizophrenia. research funds: 16790712, 17025055 ps3a-i167 genetic variation in dysbindin influences memory and general cognitive ability ryota hashimoto 1 , hiroko noguchi 1 , hiroaki hori 1 , tetsuo nakabayashi 2 , satoko hattori 1 , sachie chiba 1 , seiichi harada 2 , osamu saito 2 , hiroshi kunugi 1 1 department of mental disorder research, national institute of neuroscience, national center of neurology and psychiatry, kodaira, japan; 2 musashi hospital, ncnp, kodaira, japan; 3 tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan dysbindin (dtnbp1) is a susceptibility gene for schizophrenia, a neuropsychiatric disorder characterized by cognitive dysfunction. we examined the possible association between genetic variants in the dysbindin gene and memory and iq in 165 healthy volunteers and 72 patients with schizophrenia. individuals who did not carry a protective haplotype had lower performance in several memory domains wms-r, although this haplotype did not affect iq measured by wais-r. a risk independent polymorphism for schizophrenia influences both memory and iq in the opposite direction. these data suggest that dysbindin gene may have impact on the cognitive function such as memory and iq and that memory might be an intermediate phenotype of dysbindin on risk for schizophrenia. research funds: 16790712, 17025055 ps3a-i168 detection of 18f-dopa signal in brainstem monoaminergic nuclei in schizophrenia yuri kitamura 1 , nicola bright 3 , toshio yanagida 1 , masatoshi takeda 2 , paul grasby 3 1 department of physiology, osaka university, japan; 2 department of psychiatry, osaka university, japan; 3 cyclotron unit, imperial college, hammersmith hospital, uk we used 18 f-dopa pet to investigate presynaptic dopamine dysfunction in schizophrenic patients. the object of this study was to test that a schizophrenic cohort would show elevated aadc activity in the substantia nigra, midbrain raphe and locus coeruleus compared to normal controls. all subjects and 18 f-dopa scans were obtained from a database of scans published in mcgowan et al. 2004 , archives general psychiatry. the 14 schizophrenic patients all met dsm-iv criteria on medication and 10 healthy volunteers were compared. we attempted to improve the quality of the 18 f-dopa signal by implementing a fbf-realignment movement correction method. significant increases in 18 f-dopa uptake were found in the striatum, substantia nigra and raphe nuclei of schizophrenic patients (p > 0.02). our result suggests that an elevated presynaptic dopamine function is present in dopaminergic neurons that innervate striatal areas associated with enhanced dopamine activity in schizophrenia. in this study, we analyzed the p300 component of the visual eventrelated potential in 14 patients with schizophrenia and 14 healthy controls, and also performed loreta analysis. the ethics committee of kurume university approved this study. the p300 amplitude for the crying face was significantly smaller in patients than in controls. in controls, the p300 amplitude was significantly larger for the crying face than for the laughing face, while in patients, there was no significant difference in the p300 amplitude between the 2 faces. loreta analysis demonstrated that there were significant differences in the activity in brodmann area 10 between the 2 faces in controls, while in patients, there was no significant activity difference between the 2 faces. stimulation with crying face induced higher activities in the 10 and right 21 areas in controls than in the patients. these results indicated that the cognitive function was influenced by affective stimulus. ps3a-j170 inappropriate input produces schizophrenialike working memory deficits in a simulated neural circuit kensuke nomura 1 , shoji tanaka 2 , koki yamashita 2 , motoichiro kato 1 , haruo kashima 1 1 department of neuropsychiatry, school of medicine, keio university; 2 department of electrical and electronics engineering, sophia university a number of studies indicate that the prefrontal cortex (pfc) is intrinsically linked to working memory (wm) and that dopamine critically modulates wm activity. according to the hypothesis proposed by goldman-rakic and her colleagues, we constructed an electrophysiological circuit model for wm which represents eight directions. the computer simulation with this model shows that the working memory activity is dampened by cue-irrelevant inputs and greater noise inputs lose the directional selectivity of the representation. a lot of studies suggested that increase of noise was related to schizophrenia, especially in wm disturbance. our study indicates that noise inputs cause wm impairment in patients with schizophrenia and that working memory performance is not always positively correlated with the neuronal activity of the pfc. ps3a-j171 pericentrin is localized to the base of neuronal primary cilia in the developing cerebral cortex ko miyoshi, ikuko miyazaki, masato asanuma department of brain science, okayama university, okayama, japan we previously identified pericentrin, a mammalian centrosomal protein, as a binding partner of the product of disc1, a candidate gene for schizophrenia. in this study, we analyzed in vivo expression of pericentrin in the mouse embryo. in the developing cerebral cortex, pericentrin mrna was highly expressed in migrating cells of the intermediate zone, though proliferating neuroepithelial cells and mature neurons revealed a low expression level of pericentrin. the pericentrin protein was shown to be localized to the base of primary cilia in the pre-plate of the developing cerebral cortex, in agreement with a recent study demonstrating the involvement of pericentrin in primary cilia formation. specific subtypes of receptors such as 5-ht6 are known to be localized to the plasma membrane of neuronal primary cilia in certain regions of the brain, and then our results raise the possibility that pericentrin dysfunction may result in perturbed chemosensory function of neuronal primary cilia and increased vulnerability to psychiatric disorders. dysregulation of gr has been thought to play an important role in the pathophysiology of mood disorders. two isoforms of human gr-alpha and -beta arise from alternative splicing of the pre-mrna primary transcripts. previously, we evaluated these two isoforms mrna level in the peripheral white blood cells of the patients with mood disorders. we found that the reduced gr-alpha mrna level in the patients with both bipolar and major depressive disorders, while gr-beta mrna level was not altered. these results suggest that dysregulation of alternative splicing play an important role in the pathophysiology of mood disorders. to test this, we evaluated mrna level of alternative splicing-related sr protein family, which regulate alternative splicing in several genes including gr, in the peripheral white blood cells of the patients with mood disorders. we did not find any differences in 7 of the 10 sr protein mrnas level in the patients compared to healthy controls and now, we are examining other sr family mrnas level. ps3a-j173 alteration of neocortical long-term depression following electroconvulsive shock yoshifumi ueta 1 , ryo yamamoto 1 , shigeki sugiura 2 , kaoru inokuchi 3 , nobuo kato 1 1 dept. integrat. brain sci., grad. sch. med., kyoto univ.; 2 nara med. univ.; 3 mitsubishi kagaku inst. life sci. electroconvulsive therapy is useful in treating drug-resistant depressive disorders, though its mechanism remains unclear. there have been a few reports that studied effects of electroconvulsive shock (ecs) on long-term potentiation. however, its effects on long-term depression (ltd) have not been investigated to date. the present experiments examined roles of ecs in inducing ltd at a variety of corticocortical synapses in rat cortex slices by using whole-cell patch clamp. following ecs, ltd magnitude at layer ii/iii-to-vi pyramidal cell synapses was significantly reduced in comparison to no-ecs subjects. as described in recent microarray studies, homer1a/vesl-1s was identified as one of the most up-regulated molecules after ecs. we therefore injected homer 1a protein by diffusion from patch pipettes. homer 1a injection, as well as with ecs treatments, reduced ltd magnitude only at layer ii/iii-to-vi pyramidal cell synapses, implicating that homer 1a may be a biological mediator of ecs effects. masanori kasai 1 , nozomi miyagi 1 , norio kawashiro 2 , daisuke torizuka 2 1 dept. of chem. & biosci., faculty of sci., kagoshima univ., kagoshima, japan; 2 sanko shokuhin co., ltd., tokyo, japan it is well known that zinc is an essential mineral necessary for a multitude of body functions, including acuity of taste. to know a change of serum level in adjuvant-induced inflammation, we measured a zinc level in serum from male lewis rats received a suspension of complete freund's adjuvant (1.0 mg), injected intradermally into the tail. body weight, food intake and water intake were also measured. all rats showed signs of systemic inflammation (weight loss, hind paw swelling, nodules around eyes and penis) after the 11th day. the rats were sacrificed to measure the serum mineral contents (zn, na, cl, p, ca, k, mg) on the 2nd, 7th, 14th, 21st, 28th and 35th days. the serum zinc level was decreased on all of the measurement and the average of serum zinc (75.1 ± 12.1 g/dl, n = 7) on the 35the day was significantly lower than that in intact rats (139.4 ± 11.9 g/dl, n = 7). this decrease of zinc was correlated with weight loss but not hind paw swelling. other minerals did not show any significant changes throughout the measurement period. ps3a-j175 molecular cloning of a novel candidate for ethanol-responsive genes, yy1ap-related protein (yarp), in rat brain in order to elucidate the molecular mechanisms of etoh action on the cns, we investigated changes in gene expression in the adult rat brain after chronic etoh treatment. by means of cdna subtraction, we identified a candidate for etoh-responsive genes in the hippocampus. cdna cloning and sequence analysis revealed that this gene encodes a novel homolog of yy1ap (yy1-associated protein) and is well conserved in rats and humans. homology search for functional domains predicted that the yarp polypeptide contains nlss', a dnabinding motif, and a chromatin decondensation domain, as well as yy1-binding and transactivation domains previously demonstrated in yy1ap. in the brain, neurons such as hippocampal pyramidal cells were stained by in situ hybridization, and co-expression of yarp and yy1 genes was demonstrated in the same neurons. analogous to yy1ap as a co-activator of transcription factor yy1, it is postulated that yarp can regulate cerebral gene expression in response to etoh treatment. ps3a-j176 excitotoxic degeneration of hypothalamic orexin neurons: involvement of nr2b-containing nmda receptors and rescue by gaba a receptor stimulation hiroshi katsuki, shinsuke kurosu, toshiaki kume, akinori akaike department of pharmacology, graduate school of pharmaceutical sciences, kyoto university, kyoto, japan selective degeneration of orexin neurons, a pathological hallmark of narcolepy, is in part reproduced in hypothalamic slice cultures by application of quinolinic acid (qa), an endogenous nmda receptor agonist. we report here that nr2b-selective nmda antagonists ifenprodil (3 and 10 m) and ro25-6981 (0.1 and 1 m) markedly inhibited degeneration of orexin neurons induced by 24 h application of nmda (45 m) or qa (1.5 mm). we also show that stimulation of gaba a receptors by muscimol (10 and 30 m) or isoguvacine (30 and 100 m) potently inhibited qa cytotoxicity. in addition, the protective effect of gaba (100 m) plus a gaba uptake blocker nipecotic acid (1 mm) was abolished by a gaba a antagonist picrotoxin (100 m). norepinephrine and serotonin did not provide a neuroprotective effect. thus, gabaergic inhibition may be decisive on survival of orexin neurons under excitotoxic stimuli mediated by nr2b-containing nmda receptors. yoshika kurokawa, shinji tsukahara, hidekazu fujimaki national institute for environmental studies, tsukuba, japan to evaluate neurotoxicological influence of volatile organic chemicals (vocs), such as toluene, on hippocampal function, we attempted to develop an in vivo optical imaging technique for the hippocampus of mice with or without receiving voc inhalation. we dissected out the cerebral cortex in mice anesthetized with pentobarbital in order to prepare an optical window for monitoring the dorsal surface of the hippocampus, and stained the hippocampus with voltage-sensitive dye (rh795). we then monitored optical signals responding to electrical single-pulse stimulation to the parahippocampal region or hippocampal formation with a time resolution of 1 ms. we also examined optical signals in the hippocampus during toluene inhalation. as a result, neural excitation of the superficial layer was observed in the hippocampal formation after electrical stimulation. on the other hand, acute perinasal exposure of toluene gas did not alter any signal pattern in the hippocampal formation. we will discuss the usefulness of this technique for examination of the neurotoxicological influence of vocs. ps3a-j178 a simple method for fabricating electrodes array for multichannel neural recording -investigation of the alignment of the array and the measurement system-noriyuki taniguchi 1 , osamu fukayama 2 , takashi sato 2 , takafumi suzuki 2 , kunihiko mabuchi 1,2 1 dept. biomed. eng., univ. tokyo, tokyo, japan; 2 dept. info. physi. comp., univ. tokyo, tokyo, japan various types of electrodes have been developed for use as brain-machine interface (bmi) to record signals from neurons. electrode arrays can be purchased from vendors. however, economic considerations and the adjustment of the array alignment for experimental design still make it worthwhile to develop fabrication methods inhouse. thus we developed a low-cost multichannel microwire array electrodes for recording from the cerebral cortex of conscious rats. the electrodes were able to align for the experimental paradigms. the effectiveness of the arrangement of the array as a bmi device was investigated. the electrodes were implanted in the primary motor cortex of wistar rats. we used a wheel-formed rat exercising kit to measure the walking speed of a rat. the neural signal of the rat and the rotating speed of the wheels were simultaneously recorded. and we evaluated the estimation of the walking speed by multiple electrodes with different alignments. ps3a-j179 on-chip electrophysiological measurement of artificially constructed single-cell based neuronal networks ikurou suzuki 1 , yasuhiko jimbo 2 , kenji yasuda 1 1 department of life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan; 2 department of precision engineering, graduate school of engineering, university of tokyo, tokyo, japan we have developed a single-cell-based on-chip 8 um-diameter multielectrode arrays with an agarose microchambers (amc) for topographical control of the network patterns of living neurons. this system enables flexible and precise control of the cell positions and the pattern of connections through photo-thermal etching. and sampling rates of measurement are 100 khz in 64ch electrodes simultaneously. using this system, we formed a single-cell-based neural network pattern of rat hippocampal cells within the amc array and controlled the growth direction of axon/dendrite selectively using photo-thermal etching methods during cultivation, and recorded the spontaneous firings and evoked responses. moreover, we identified propagation along patterned neural network and found the effects of tetanic stimulation within this neural network. in the meeting we will present the results in detail and will discuss the potential of our method. yuichi yamashita 1 , tetsu okumura 1 , kazuo okanoya 2 , jun tani 1 1 lab. for behavior & dynamic cognition, riken-bsi, japan; 2 lab. for biolinguistics, riken-bsi, japan how the brain generates and learns temporal sequences is a fundamental issue in neuroscience. the production of birdsongs, a process which involves complex learned sequences, provides researchers with a good biological model to study this phenomenon. bengalese finches (bf) learn highly complex songs that have grammatical structure. the underlying neural mechanisms that allow the birds to learn these songs are however not fully understood. to address this issue, we developed a neural network model of bf's songs that might explain how different regions of the brain work together. to test the model, we also conducted empirical experiments on the brains of bf. the model shows that complex grammatical songs can be replicated by simple interactions between deterministic dynamics of a recurrent neural network and random noise. moreover, comparison between the model and the empirical data on real birds shows similar trends. this work is a part of an integrated research project combining model simulations and empirical study. please see also the empirical component of this project as reported by okumura. ps3a-k181 local administrations of muscimol into the nif alter song grammar of the bengalese finches (bf) tetsu okumura 1 , yuichi yamashita 1 , kazuo okanoya 2 , jun tani 1 1 behav & dynamic cognition, riken-bsi, saitama, japan; 2 biolinguistics, riken-bsi, japan songs of passerines are learned behavior which used by males to attract females. their songs consist of several song notes, and these notes are produced in a fixed temporal order. among the passerines, however, bfs sing complex song which follows finite state syntax. the song control system of bf consists of a set of discrete nuclei including the hvc and nif. previous study showed that nif lesioned bfs sung simpler songs, with less phrases to phrases branching. therefore, nif-hvc connection may play important role in generating song grammar. in this study, we perfused nif with muscimol via microdialysis probes as a perturbation on nif-hvc system. following a local perfusion, song grammar was modified. some of chunks in their grammar were disappeared and introductorily notesǐ duration was elongated. nif is also known as one of auditory relay nucleus to hvc. part of the effects is possibly caused by disruption of auditory feedback. we also developed a neural network model of nif-hvc system. please refer yamashitaǐs poster for details of this model. the reason for the emergence of reward expectancy neurons suggested by a model using reinforcement learning and an artificial neural network katsunari shibata 1 , shinya ishii 1 , munetaka shidara 2 1 dept. of e&e engineering, oita univ., oita, japan; 2 grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan in the experiment of multi-trial schedule task to obtain a reward, reward expectancy neurons, which respond only in the non-reward trials prior to the reward trial, have been observed in the anterior cingulate cortex of monkeys. it is difficult to explain directly by reinforcement learning why they do not respond in the reward trial. here, we interprets that such neurons emerge as an intermediate representation to generate appropriate value and actions in reinforcement learning by simulation analysis using a model that consists of an artificial recurrent neural network trained by reinforcement learning. the simulation result suggests that the reward expectancy neurons emerge to realize smooth temporal increase of the state value by complementing the neurons that respond only in the reward trial. [1] s. ishii, et al., "a model to explain the emergence of reward expectancy neurons using reinforcement learning and neural network", neurocomputing, 2006 behavior is adjusted by outcomes of actions. to examine the neural mechanisms of the behavioral adjustment, we recorded single cell activity of the medial prefrontal cortex (mpfc) of two monkeys performing a behavioral adjustment task. the monkey searched a correct action (left or right lever press) on the basis of the two kinds of visual feedback, one (cs+) paired with a liquid reward and the other (cs−) that did not appear in a preceding pavlovian conditioning. cs+ followed a correct action and cs− followed a wrong action. when the monkey made more than 3 consecutive correct trials, a new block of pavlovian conditioning started. we calculated the prediction errors provided by cs+ and cs− on the basis of a reinforcement learning model of action selection. we found that the neuronal activity corresponds to the prediction error of value of the selected action. this result suggests that mpfc contributes to behavioral adjustment by providing prediction errors of action values. makoto miyazaki 1 , shinya yamamoto 2 , sunao uchida 3 , shigeru kitazawa 4,5 1 faculty of hum sci., waseda univ., tokorozawa, japan; 2 neurosci. res. inst, aist, tsukuba, japan; 3 faculty of sport sci., waseda univ., tokorozawa, japan; 4 dept. of neurophysiol, juntendo univ. grad. sch. med., tokyo, japan; 5 crest, jst, saitama, japan our judgment of temporal order of two sensory signals is not always fixed but subject to changes due to prior experiences, such as repeated exposure to a constant stimulus sequence. to date, such perceptual changes occurred so that signals in the order of the most frequent sequence are judged as simultaneous. in this study, we examined temporal order judgment of two tactile stimuli, delivered one to each hand, using stimulation intervals sampled from biased gaussian distributions (mean = ±80 ms, s.d. = 80 ms). previous studies predict that the point of simultaneity would be shifted toward the peak of the gaussian, i.e. toward the most frequent interval. however, the point of simultaneity was shifted away from the peak by about 50 ms. our results disagree with the previous studies, but conforms to a contrasting prediction from a bayesian integration theory. research funds: kakenhi (15200031) ps3a-k185 single measurement of oxy-and deoxyhemoglobin for a functional near infra-red spectroscopy ichiro shimoyama 1 , fumiko sato 2 , ken nakazawa 3 , kenichi ono 3 1 chiba university, japan; 2 field of home economics, faculty of education, chiba univ., japan; 3 department of integrative neurophysiology, graduate school of med. chiba univ., japan to study single dynamics for oxy-and deoxy-hemoglobin to a single task, we measured near infra-red spectroscopy (omm-3000, shi-madzu) over the frontal area (45 channels) for 7 volunteers (19-23 y). thirty tasks were presented visually every 30 s, the subjects were asked to think about the question immediately following the sentences and asked not to think moreover if the question was difficult (e.g., how to cook curried rice? or how to fold paper into a turtle? etc). a comprehension-test was done just after the record. easy/difficult serial tasks were selected, and the oxy-and deoxy-hemoglobin differences between 2 tasks were calculated to obtain correlation coefficients between the oxy-and deoxy-hemoglobin. grand averaged correlation coefficient was −0.4+/−0.45 between the dynamics of the oxy-and deoxy-hemoglobin. the correlation should be considered in discussing neural activation for nirs. we thank shimadzu corp. for providing the nir station. kazuya ishibashi 1,2 , kosuke hamaguchi 3 , masato okada 1,2,3 1 department of complexity science and engineering, graduate school of frontier sciences, university of tokyo, kashiwa, japan; 2 jst, japan; 3 riken bsi, wako, japan a synfire chain is one of the networks which generate stable synchronous pulse packets. although the networks with a single stable synfire state is intensively analyzed by using several neuron models, the networks with several stable synfire states have not yet been investigated so thoroughly. by using leaky integrate-and-fire neuron model we construct a layered associative feedforward network embedded with several memory patterns. we analyse the network dynamics with the fokker-planck equation. first, we analyze the activity of the network when we activated one memory pattern of the first layer. we show that the layered associative network has stable synfire state. second, we investigate the activity when we activated 2 different memory patterns. then we observe several characteristic phenomena, which are not observed in the conventional homogenous synfire chain. we will report the details of those phenomena. research funds: kakenhi (14084212) and (16500093) ps3a-k187 auditory erps can be identified as corresponding stimuli by classifier with naive bayes method akitoshi ogawa 1,2 , sachiko koyama 1,2 , takashi omori 3 , takashi morotomi 4 1 research institue for electronic science, hokkaido university, sapporo, japan; 2 japan science and technology agency, saitama, japan; 3 graduate school of information science and technology, hokkaido university, sapporo, japan; 4 sakushin gakuin university, utsunomiya, japan in an attempt to reversely estimate the input stimulus from measured erps, we developed computational classifier using naive bayes method. correct classification rates could be index values of the erp characteristic. in this study, we applied the classifier to identify auditory erps (n = 13). the erps were elicited by tones (500 hz) with different durations (8, 16, 32, 64, 128, 256 ms) and gaps (8, 16, 32, 64, 128 , 256 ms) embedded in a continuous pure tone (500 hz). to confirm the generality of the method, we used leave-one-out cross validation. erps of each subject were identified by the classifier which was constructed from the others' erps. as a result, the correct rates for 32 and 64 ms were high both for the tones (32 ms, 38%; 64 ms, 61%) and the gaps (32 ms, 46%; 64 ms, 46%). ps3a-k188 determination of channel parameters for construction of a neural model of caenorhabditis elegans kazumi sakaa, akane andoh, taro ogurusu laboratory of bioscience, faculty of engineering, iwate university, iwate, morioka, japan caenorhabditis elegans (c. elegans) is one of the most suitable model animal for investigation of the relationship between the connection and the function of the neural network because its connection was revealed with the electronmicroscopy. on the other hand, it has been difficult to build a precise model neuron because the neuronal electrophysiological data of c. elegans has not been sufficient. we have been developing a precise neural model by extracting parameters required for model of voltage dependent channels from the electrophysiological data by the genetic algorithm with a neural simulator genesis and parallel genesis. using these simulation softwares, not only the optimum parameter set was determined for each channel but also the ratio of the conductances of several channles were determined. we report validity of obtained parameters and the possibility of the existence of unknown channel. supported by grant from jsps. ps3a-k189 theoretical consideration about nmda current change and its effect on synaptic plasticity shigeru kubota, tatsuo kitajima department of bio-system engineering, yamagata university, yonezawa, japan it is well known that nmdar plays an important role in learning and memory. several experiments have shown that the property of nmdar epsc can change within a few weeks after birth, leading to the shortening of its decay time course. since the calcium current through nmdar is involved in ltp and ltd induction, it is possible that such change can work as the modulation of the plasticity rule or higher-order plasticity. here we show by the biophysical compartmental model that the alteration of nmdar property can modulate the calcium influx into the spine, which finally switches plasticity rule. we also show that this type of plasticity switch can promote synaptic competition and separate postnatal synapses rapidly into two groups of either strong or week ones. our results suggest that changing nmdar time course is very useful for the developing animals in order to promote fast and stable formation of the polysynaptic circuit. manish kumar jain department of psychiatry, r.d. gardi medical college, india introduction: i want to inform you regarding the some of challenges coming across my practice with the person with the psychiatric disorder in social rehabilitation like education and training, work and employment, family, groups, social, sexual, environmental and regional, coordination with the other health group and care giver, insurance problems, medical, physical, occipital vocational, languages problems mostly how to give oppurtinies with in the society and many more to be come in future. method: i keep the records with me since i join the medical college and my during practice but this is really challenging to calm down for question with their relatives and care givers. results: it is always to see the experience of the other people including self help groups in this regards and most challenging with near by perfect action and required more interaction with the rehabilitation groups because some are social problems in psychiatric disorder. conclusions: there is big challenge in the for social rehabilitation for the persons with psychiatric disorder as multifactor involvement s are there in this groups with early intervention and long term rehabilitation so that we can produced many working induals with in the society among the person with psychiatric disorder the more interaction among the society and care giver working in this field as well as neuroseiencents working in this field so that we will able to achieve almost complete social rehabilitation as till today we are not able to achieve social rehabilitation up to 50% till now. hepatic encephalopathy (he) refers to acute neuropsychiatric changes accompanying fulminant hepatic failure (fhf). in the present study we investigated changes in lipid composition of membranes isolated from cerebral cortex of rats treated with thioacetamide (taa), a hepatotoxin which induces fhf and thereon he. estimation of phospholipid fatty acid content in cerebral cortex membranes from taa treated rats revealed a decrease in monounsaturated fatty acid namely oleic acid and the poly unsaturated fatty acids ␥-linolenic acid, decosa hexanoic acid and arachidonic acid compared to controls. assesment of membrane fluidity with pyrene, 1,6-diphenyl-1,3,5-hexatriene, and 1-[4 (trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene revealed a decrease in annular membrane fluidity while the global fluidity was unaffected. the level of thiobarbituric acid reactive species-marker for lipid peroxidation also increased in membranes from taa treated rats indicating prevalence of oxidative stress. results from the present study demonstrate gross alterations in cerebral cortical membrane fatty acid composition and fluidity during taa induced he and their possible implications in the pathogenesis of this condition are also discussed. nagatoki kinoshita, shigenobu yonemura cellular morphogenesis, cdb, riken, kobe, japan rho-gtpases are well known as regulators of cytoskeletal reorganization and many cellular morphogenetic movements. however, little is known about their distributions and their physiological functions in vertebrates. immunohistology of chick embryos revealed apical accumulation of rho, rac and cdc42 in neural plate cells, especially in bending hinge points. after neural tube closure, the apical accumulation decreased. coordinately, activities of rho-gtpases and myosin ii in neural plate cells were higher during neurulation than after neural tube closure. inhibitions of actin filament formation, myosin ii-mediated contraction or rho-associated kinase activity affected neural tube formation. inhibition of rho activity induced the disruption of its apical accumulation and the defects of neural tube formation. these results suggest that rho-gtpases in an active form accumulate in the apical surface of neural plate cells and play important roles in neurulation. furthermore, we are screening regulators and effecters of rho-gtpases transiently expressed in neural plate cells during neurulation. setsuko sahara, dennis dm o'leary mnl-o, the salk institute, usa gradients of morphogens are postulated to establish the initial patterning of the mammalian forebrain, but little is known about their downstream targets and the mechanisms of patterning. here we report mouse buttonhead homogoues, the sp gene family, as candidates of downstream of those morphogens: sp5 expression correlates with wnts/bmps in the cortical hem, sp8 with fgfs in the cop, and sp9 with shh in the ventral midline and mge. by using in utero electroporation, we show that sp8 regulates anterior-posterior patterning of the cortex into areas by controlling distinct fgfs that having opposing effects. sp8 and fgf8 exhibit reciprocal induction, indicating that sp8 is a positive feedback regulator of fgf8. surprisingly, though, ectopic expression of both sp8 and its dominant active form shift cortical areas in the opposite manner to fgf8, suggesting that sp8 activates additional targets that overcome fgf8 function. our results indicate that fgf10 is an additional target of sp8, showing effect on patterning similar to sp8. these findings indicate that sp8 balance the proper cortical arealization through fgf8 and fgf10. research funds: nihr37ns31558 ps3p-c003 fyn-fak signal transduction is involved in the radial migration of late-generated neocortical neurons eiko nakahira 1 , kotaro hattori 1 , takeshi yagi 2 , shigeki yuasa 1 1 dept. ultrastructural res., nat. inst. neurosci., ncnp, tokyo, japan; 2 kokoro biology group, fbs, osaka univ., suita, japan fyn tyrosine kinase posphorylates focal adhesion kinase (fak) that is involved in cell migration. taking into account the defective formation of neocortical layers ii-iii in fyn-deficient mice, fyn-fak signal transduction might be involved in the control of the migration of neocortical neurons. accordingly, we analyzed the neuronal migration in the mutant neocortex and compared the phenotypes to the changes induced by fak gene-knock down by foreign gene transfer by means of in utero electroporation. late-generated neocortical neurons exhibited defective radial migration in the mutant and this defect was rescued by the transfer of fyn-expression vector to the neocortical primordium. fyn and fak were colocalized in the migratory neurons, and fak sirna transfer into neocortical primordium induced migration defect similar to that in fyn deficiency. these findings strongly suggest that the coordination of fyn and fak is essential for the radial migration of late-generated neocortical neurons. noriyo ishibashi 1 , kazuko keino-masu 1 , tatsuyuki ohto 1 , satoshi kunita 2 , satoru takahashi 2 , masayuki masu 1 1 dept. of mol. neurobiol., grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan; 2 laboratory animal resource center, univ. of tsukuba, tsukuba, japan heparan sulfate (hs) proteoglycans regulate developmental patterning through the interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific hs structures generated by sulfation and epimerization. a recently identified extracellular sulfatase, sulffp1, has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp1 gene, and examined the brain development. a previous study showed that the brain-specific disruption of the ext1 gene, which encode a hs synthesizing enzyme, led to severe brain defects including hypoplasia of the cerebral cortex and cerebellum. in this study, we thus examined the morphological changes of the cerebellum in the neonatal and adult sulffp1-deficient mice. heparan sulfate (hs) proteoglycans play a crucial role in mediating important signaling by wnt, hedgehog and fgf. recently, novel sulfatases, sulffp1/sulfatase-1 and sulffp2/sulfatase-2, which have hs 6-o-endosulfatase activity have been isolated. since these sulffps are detected in the extracellular space, sulffps are thought to regulate cell surface signaling through hs remodeling. in order to examine the function of sulffp genes in zebrafish, we isolated zebrafish sulffp1 and sulffp2. here we report the isolation and the characterization of the third homologue, sulffp3. sulffp3 has about 56% and 71% overall amino acid homology with sulffp1 and sulffp2, respectively. at 24 h postfertilization, sulffp3 is expressed in the ventral region of spinal cord, whereas sulffp1 is expressed only in the floor plate and sulffp2 is expressed in the lateral floor plate and ventral regions of spinal cord. detailed expression patterns of sulffp3 will be presented. masahiko ajiro, kenichi arai, mika maeda-sato, masuo obinata, wataru shoji dept. of cell biology, idac tohoku univ., japan collapsin response mediator proteins (crmps) are cytosolic proteins involved in neuronal differentiation and axonal guidance. a member of this family, crmp2 was shown to mediate the repulsive effect of sema3a on axons. crmps appear to play more complex roles in axonal differentiation, elongation and branching during development. since less is known about their in vivo function, we studied their roles during development using transparent zebrafish embryos. at early axogenesis stage, zebrafish crmps are expressed in specific patterns. in trigeminal sensory ganglia, crmp2, 3, 4, and 5 are highly expressed. knocking down of these gene results in disorganization of the ganglia, separating into several clusters. however, their axonal patterns including direction, extension, and branching appears normal. same defects were observed in the knockdown of neuropilins, receptor component for class 3 semaphorins. these results suggest that crmps may functionin keeping trigeminal neurons as a ganglia by mediating semaphorin-neuropilin signals. ps3p-c007 developmental origin of diencephalic sensory relay nucley in teleosts y. ishikawa 1 , n. yamamoto 2 , m. yoshimoto 2 , t. yasuda 1 , k. maruyama 1 , t. kage 1 , h. takeda 3 , h. ito 2 1 nat. inst. rad. sci., chiba, japan; 2 nippon med. sch., japan; 3 tokyo univ., japan we propose a novel interpretation of the embryonic origin of cells of diencephalic sensory relay nuclei in teleosts, based on our studies in the medaka embryonic brain. it has been proposed that the relay system in teleosts is unique among vertebrates. teleost relay nuclei, the preglomerular complex (pg), have been assumed to originate from the basal plate (posterior tuberculum, pt) of the diencephalon, whereas relay nuclei in mammals are derived from the alar plate. our results show, however, that many pax6-or dlx2-positive cells migrate laterally and ventrocaudally from the diencephalic alar plate to the basal plate during development. massive clusters of the migrated alar cells become localize in the mantle layer lateral to the pt neuroepithelium, from which the pg appear to differentiate. we therefore consider most neurons in the pg are be of alar, not basal origin. thus, the teleost pg can be regarded as migrated alar nuclei. the organization of the diencephalic sensory relay system may have been conserved across vertebrates. hideyuki dekimoto, yoshihiro oomiya, satoshi kikkawa, toshio terashima, yu katsuyama department anatomy and developental neurobiology, kobe university graduate school of medicine laminaiton is one of features unique to the brain of vertebrates. to understand the evolution of layer formation in the vertebrate brains, we are studying genes which exhibit layer-specific expression. since one of ets family transcription factors, er81 is expressed specifically in the layer v of the mouse neocortex, we selected this gene for the purpose of our study. here we cloned zebrafish er81 homologue (zfer81), and found that the amino acid seuqence of the putative protein is highly conserved throughout the entire length. expression of zfer81 was observed in multiple sites of developing brain. the expression disappears sequentially in some sites, whereas it persisted in other sites until adult stage. er81 expressing sites in the brain was basically conserved between mouse and zebrafish, whereas expression pattern in each site (i.e. telencephalon, tectum) was different. based on these observations, evolution of the gene expression in the brain lamination will be discussed. hiroyuki koizumi, teruyuki tanaka, joseph g. gleeson university of california, san diego, usa doublecortin (dcx), encoding a microtubule-associated protein, is critical for neuronal migration, as mutations result in x-linked lissencephaly in hemizygous males and subcortical band heterotopia in heterozygous females, whereas in mouse, rnai-mediated knockdown but not germline knockout shows abnormal positioning of cortical neurons. dclk (doublecortin-like kinase) is one of the homologous genes of dcx, encodes for protein with an n-terminus that is 70% identical to dcx, but also additional c-terminal protein kinase domain. here, we report that the dclk functions in a partially redundant pathway with dcx in the formation of axonal projections across the midline and migration of cortical neurons in mouse. dosagedependent genetic effects were observed in both interhemispheric connectivity and migration of cortically and subcortically derived neurons. rnai-mediated knockdown of either gene results in similar migration defects. these results indicate the dcx microtubuleassociated protein family is required for proper neuronal migration and axonal wiring. hiraki sakuta 1,2 , hiroo takahashi 1,2 , takafumi shintani 1,2 , kazuma etani 1 , masaharu noda 1,2 1 div. of mol. neurobiol., nibb, okazaki, japan; 2 crest, jst, japan in the developing chick retina, the expression of bmp4 is relieved by that of bmp2 at around e5 with a change from a dorsal high to dorsotemporal high pattern, complementary to that of ventroptin, a bmp antagonist. we previously demonstrated that misexpression of ventroptin altered the retinotectal projection along both the dv and ap axes. here, we show that topographic molecules along the dv axis, together with ephrina2, are expressed in a double-gradient fashion from e6 on like ventroptin and bmp2. when bmp2 expression is manipulated by using the gene-specific knockdown and the reagent-inducible gene expression techniques, the expression patterns of these double-gradient molecules are all changed. moreover, in the bmp2 knockdown and ephrina2-misexpressing embryos, the retinotectal projection is altered along the two axes. the expressional switching from bmp4 to bmp2 thus appears to play a key role in retinal patterning and consequently in topographic retinotectal projection, by changing the direction of the dv axis toward the posterior side during retinal development. noriyuki morita, teiichi furuichi lab. for molecular neurogenesis, riken-bsi, wako, japan the mammalian cerebellum is anteroposteriorly and mediolaterally compartmentalized at the level of neuroanatomy and also at the level of gene expression. to elucidate the molecular mechanisms underlying the establishment and the maintenance of functional cerebellar compartment, genes responsible for mouse cereballar development transcriptome were examined for patterned expression in cerebellum by whole-mount in situ hybridization. not a few known and novel genes were found to be expressed in parasagittal band pattern in the embryonic mouse cerebellum, which could be categorized as "early-onset-genes". parasagittally expressed genes were classified in comparison with the band pattern of en2, wnt7b and pcp2/l7 gene expression in declival vermal lobule, to investigate the correlation between spatial expression profiles and transcriptional regulatory elements. our accumulating data suggest that not only patterning genes like engrailed and wnts, also genes related in later events in neural development such as synaptogenesis are expressed as earlyonset-genes. yasufumi tanaka, tomiyoshi setsu, hideyuki dekimoto, yu katsuyama, toshio terashima kobe university graduate school of medicine, japan the nissl staining of the brains of the adult reeler and normal mice showed that the size of the pontine nuclei (pn) was reduced in the reeler compared with the normal counterpart. the injections of dii and 4di-10asp into the left and right hemicerebellum, respectively, resulted in that only a few pn neurons were doubly labeled in the control, but in the reeler most of pn neurons were doubly labeled. the placements of solutions of dii and 4di-10asp into the left and right cerebellar peduncles of paraformaldehyde-fixed brains resulted in that dii-labeled or 4di-10asp-labeled pontocerebellar fibers made a fascicular formation in the cerebellum of the normal mouse, but such a fascicular formation was not recognized in the reeler and labeled terminals of mossy fibers were randomly arranged along the course of the pontocerebellar projection. reelin mrna and reelin were both expressed in the pn of the normal mice. these data elucidate that the reelin may play a key role in fasciculuation and collateral formation of pontocerebellar projections in addition to cell positioning or migration of pn neurons. kudoh suguru 1,2 , takahisa taguchi 1 1 aist, ikeda, japan; 2 presto, jst the spatiotemporal patterns of spontaneous action potential were analyzed, using the multi-site recording system for extracellular potentials of neurons and the living neuronal network cultured on a 2-dimensional electrode array. the map of functional connections between neurons revealed that each culture contained some hublike neurons and the distribution of the number of functionalconnections approximated a power-law distribution. we confirmed that the spatiotemporal pattern of spontaneous action potentials became more complex pattern along with developmental stage, and the constant pattern of stimulation promote this developmental change. in addition, the spatiotemporal pattern and the functional connections between neurons were drastically re-organized by real-time feedback stimulation. these results strongly suggest that the network structure of the cultured hippocampal neurons is neither stable nor random, but is functionally dynamic and is suitable for certain types of information processing. research funds: presto, jst ps3p-d014 laterality of the human cerebral hemisphere taiko kitamura, jinzo yamada department of anatomy, tokyo medical university, tokyo, japan it has been reported that some functional predominance is located in the right or left hemisphere of the human brain. especially, the speech center and the center related to thought and emotion are located in the left and in the right hemisphere, respectively. in this study, the laterality between the right and the left human hemisphere was investigated macro-anatomically. we measured the weight, the medial-lateral width (m-l), the anterior-posterior lenght (a-p), and the width of the medial surface in the right and the left human hemisphere using in anatomical practice for medical students. the weight of each hemisphere was roughly equal. the m-l was wider in the right side than the left side. the a-p was longer and the width of the medial surface was larger in the left side than in the right side. because of the longer a-p and the larger width of the medial surface in the left hemisphere, it appeared that the left hemisphere overspreads the medial-dorsal marginal surface of the right hemisphere by the naked eye. such overspreading suspects that the left hemisphere develops earlier and faster than the right hemisphere. ps3p-d015 synchrony-induced transition behaviors organized under spike-timing dependent plasticity for retrieving the memorized patterns takaaki aoki 1 , toshio aoyagi 2 1 department of physics, kyoto university, kyoto, japan; 2 graduate school of informatics, kyoto university, kyoto, japan temporally correlated spikes, such as spike synchrony, have been observed in relation to behaviors or cognitions. however, it is unclear how the neurons read out the incoming spike synchronization in the dynamical behavior of network. in this modeling study, considering a network of excitatory and inhibitory neurons organized under spiketiming dependent plasticity, we present a type of network model in which incoming spike synchrony causes a transition between learned activity patterns in the order they were experienced in the learning process. furthermore, using appropriate training patterns, this network exhibits a context-dependent transition, in which the network switches to multiple patterns from a single pattern depending on the temporal structure of neuronal activity at the onset of incoming spike synchrony. this ability of the network may provide one of mechanisms by which a neuronal system can be trained to carry out tasks in a context-dependent manner. shozo kito, maiko kitagawa, akiko shingo lab. of neurosci., hyogo univ., kakogawa japan in our previous studies, we showed that a part of nicotine's beneficial effects on hippocampal and cortical neurons were due to increased igf-1 mrna expressions. nevertheless, the situation may be somewhat different as far as nicotine's effects on the neuronal progenitor cell, which is still on the way of differentiation are concerned. to clarify this problem, nicotine was intraperitoneally injected into 4 weekold wistar strain rats in several doses followed by successive injections of brdu for the next 4 days. then rats were sacrificed and vertical sections of the hippocampus formation were offered for double immunohistochemical staining of brdu/psa-ncam, brdu/neun or brdu/gfap. as the results, numbers of both brdu(+)/psa-ncam(+) cells and brdu(+)/neun(+) cells were much decreased nicotine-dose dependently. on the other hand, as much as 1 mg/kg was needed for nicotine to exert its effect on the number of brdu(+)/gfap(+) cells. these results reveal that nicotine inhibits neurogenesis and plasticity in the hippocampus of adult rats. ps3p-d017 the establishment of the organotypic slice culture of postnatal rat forebrain involving egfp-labeled neural progenitors kaoru sato 1 , james e. goldman 2 1 division of pharmacology, national institute of health sciences, tokyo, japan; 2 department of pathology, columbia university, new york, usa after injecting egfp-encoding retrovirus into p0 rat svz, sagital sections of forebrain were made at p3 and cultured for 6 days. the migration pattern of the egfp-labeled neural progenitors in the cultured slices is almost same as that at the corresponding age. the expression patterns of the glial differentiation-markers were also in accordance with those at the corresponding age. when slices were cultured with anti-␣3 integrin antibody, the migration of the neural progenitors inside svz was significantly enhanced along the rostrocaudal extent. these results suggest that the organotypic slice culture of postnatal rat forebrain is an efficient experimental system for pharmacological studies about migration and differentiation of neural progenitors. radial glia is involved in the contact guidance of neuronal migration and also the neuronal and astroglial precursors. to make clearer the role of radial glia, we developed a method for the selective ablation of a subset of radial glia. it has been reported that tenascin-c (tn-c) is one of the markers for radial glia. accordingly, diphtheria toxin (dt)gene and enhanced green fluorescence protein (egfp)-gene both driven by tn-c gene promoter were co-transferred into the ventricular zone cells of the mouse neocortical primordium by means of in utero electroporation. the numbers of egfp-labeled cells in that tn-c gene promoter and subsequently dt gene are activated selectively decreased by this approach. using this method, the examination of radial glial morphology and neuronal migration following selective ablation is in progress. takayuki manabe, kouko tatsumi, eri makinodan, manabu makinodan, takahira yamauchi department of 2nd anatomy, nara medical university, kashihara, nara, japan it has been well documented that neurogenesis persists at the subventricular zone and the subgranular layer of the dentate gyrus in the adult mammalian brain. in the adult mice, we demonstrated that cells around a cryo-injured cortical lesion had a proliferative activity (labeled with brdu in vivo) and formed neurosphere-like aggregates in the sphere-forming culture condition. significantly lager number of spheres was observed in the culture from the injured hemisphere, which excluded the neurogenic regions (i.e. the svz and hippocampus), than those cultured from the control (contralateral and intact) hemisphere. furthermore, the sphere-forming cells differentiated to neuronal-and glial-marker positive cells in vitro. these results suggest that the cells forming sphere-like aggregates in vitro may function as a kind of progenitor cells in the injured brain. if this is a case, it would be tempting to transplant these sphere-forming cells to cure brain injury or disease. further characterization of the cells is underway. ps3p-d020 localization of neurotrophin receptors trka in pc12 cells: 3d reconstruction analysis of membrane proteins tomoki nishida 1 , hiroshi jinnai 2 , tatuo arii 3 , akio takaoka 4 , ryoichi yoshimura 1 , yasuhisa endo 1 1 department of applied biology, kyoto institute of technology, kyoto, japan; 2 department of polymer science and engineering, kyoto institute of technology, kyoto, japan; 3 national institute for physiological sciences, myodaiji, okazaki, japan; 4 osaka university, mihogaoka, ibaraki, osaka, japan it was previously reported that trka (ngf receptor) was associated with caveolae, small invaginations on the cell membrane, but its subcellular localization is not clarified in detail. we performed immunocytochemistry of trka and caveolin-1 in pc12 cells, analyzed by high-voltage electron microscopy, and reconstructed 3d structure of their subcellular distribution by imod. our results indicated that localization of caveolin-1, known as an integral membrane protein of caveolae, was never found in the invagination structure in pc12 cells, but trka and caveolin-1 immunoreactivities were mainly found as a mesh-like structure in the cytoplasmic matrix. kensuke shiomi, kazuko keino-masu, masayuki masu department of molecular neurobiology, graduate school of comprehensive human sciences, university of tsukuba, tsukuba, japan the wnt signaling plays important roles in cell growth, differentiation, polarity formation, and neural development. previously we identified ccd1, a third-type of the dix domain-possessing protein, as a positive regulator of the wnt/␤-catenin pathway. ccd1 mrna was mainly detected in the neural crest derivatives and differentiated neurons in mouse embryos, suggesting the importance of ccd1 in the wnt-mediated neuronal development. there are three subtypes of mouse ccd1 gene products, ccd1a, ccd1b and ccd1c, which are generated by different promotor usage. mouse ccd1a as well as zebrafish ccd2a has a calponin homology domain which can mediate the interaction with the actin cytoskelton. we found that in the ccdtransfected hela cells, only the type a ccd proteins co-localized with the actin filament. in order to examine the function of the type a ccd proteins, we are now doing overexpression and functional blocking experiments using zebrafish embryos and cell culture. research funds: kakenhi (15770137, 17300098) ps3p-d022 analysis of a role of r-spondin2 on proliferation of the cortical neuroepithelium yumiko hatanaka 1 , masahiro yamaguchi 2 , fujio murakami 3 , masayuki masu 1 1 grad. school of comprehensive human sci., univ. of tsukuba, japan; 2 grad. school of med., univ. of tokyo; 3 grad. school of frontier biosci., osaka univ r-spondin2 (rspo2) is a secreted activator of wnt/␤-catenin signaling (kazanskaya et al. 2004) . rspo2 is expressed in the developing medial cerebral wall and transgenic mice expressing rspo2 in the entire neuroepithelium show enlarged lateral ventricle with a slight increase of brain size (hatanaka et al. 2005) . since wnt3a has a role for expansion of caudomedial cortical progenitor cells (lee et al. 2000) , these findings lead us to the idea that rspo2 may synergistically promote proliferation of cortical neuroepithlial cells together with wnt3a. to clarify their role on proliferation of cortical neuroepithelial cells, we first introduced a ␤-catenin/tcf reporter gene into these cells of embryonic day 11.5 mouse. an application of wnt3a on these cells increased level of the reporter expression, and an addition of rspo2 further increased its level. we are now monitoring incorporation of brdu in neuroepithelial cells to know whether wnt3a and rspo2 directly promote their proliferation. tae sun kim, hideki hida, tomoko narita, sachiyo misumi, hitoo nisino department of neurophysiology & brain science, nagoya city university graduate school medical sciences, nagoya, japan to investigate whether physiological low oxygen during development and cytokines expressed in the dopamine (da)-depleted striatum increase the number of da neurons from es-derived neural progenitor cells (npcs), npcs were treated with cytokine cocktail (il-1␤, il-11, lif, gdnf) or lowered o2 (3.5%), followed by tyrosine hydroxylase (th) immunostaining. low oxygen increased total number of th (+) cells (1.86-fold) as compared to normal o2. cytokine cocktail significantly increased th (+) cells (2.11-fold) compared to nontreated control. treatment of lif and il-1␤ to npcs exhibited major contribution in the effect of cytokine cocktail. data suggest that physiologically relevant low oxygen in development and cytokines and trophic factors that were enhanced in da-depleted striatum cause in the increase of daergic neurons from es-derived npcs. ps3p-d024 structural basis for reelin signaling: determination of receptor-binding site and its three-dimensional structure norihisa yasui 1 , terukazu nogi 1 , mitsuharu hattori 2 , kenji iwasaki 1,3 , junichi takagi 1 1 research center for structural and functional proteomics, inst for protein res., osaka univ., suita, japan; 2 dept. of biomed. sci., grad. sch. of pharm. sci., nagoya city univ., nagoya, japan; 3 core research for evolution and technology (crest) a large secreted glycoprotein reelin acts on target neurons through its receptors (apoer2 and vldlr), resulting in tyrosine phosphorylation of dab1. in the present study, we have carried out structural and functional studies on the reelin signaling. first, we determined the structure of a single reelin repeat by x-ray crystallography. it had a horseshoe-like globular structure with some similarities to carbohydrate binding modules from many enzymes. moreover, electron micrographic 3d reconstruction of four-domain reelin fragment (i.e. r3-6) revealed an elongated rod-like structure. next we determined minimum active unit within reelin. a fragment containing both the fifth and sixth reelin repeats (r5-6) was capable of binding to the receptor (apoer2), and was also able to induce tyrosine phospholylation of dab1 in primary neuronal culture. ps3p-d025 effects of astrocyte-derived factor and cell-cell communication on uni-directional differentiation from mouse embryonic stem cells into neural cells embryonic stem (es) cells uni-directly differentiate into neurons via neuroectoderm and neural stem cells by neural stem sphere (nss) method. cultured with astrocyte-derived factor, colonies of es cells give rise to nsss. we analyzed structure and gene expression of cell spheres formed under various culture conditions, in order to elucidate mechanisms of the uni-directional differentiation into neurons. quantitative real-time rt-pcr analysis demonstrated that the neuronal differentiation did not occur in the cell spheres. these results suggest that astrocyte-derived factor and cell-cell communication are necessary for the differentiation. we have previously established es cell differentiation system, by which we can derive neurospheres containing neural stem/progenitor cells (ns/pcs) with the identity of early caudal neural tube. taking advantage of this culture system, we have recently found conditioned medium of a stromal cell line (cmsc) has the activity to support the formation of neurospheres. this activity was more prominent when cultured at low cell density than when cultured at high cell density, suggesting that it supports the survival of ns/pcs. moreover, rt-pcr analysis of regional identities of the cmsc treated neurospheres revealed elevated expression of pax3 and pax7 compared with those of untreated neurospheres, indicating that cmsc promotes dorsalization of ns/pcs or selective proliferation of dorsal ns/pcs. elucidation of underlying mechanisms may provide important tools to derive early ns/pcs which can generate variety of projection neurons and be applicable to regenerative medicine. research funds: sorst jst ps3p-d027 neudesin, a secreted factor, promotes neural cell proliferation and neuronal differentiation in mouse neural precursor cells neudesin expressed in adult mouse brain encodes a secreted signal with neurotrophic activity in neurons (j neurosci res 79:287, 2005) . most neurotrophic factors are involved in neural cell proliferation and/or differentiation. however, the role of neudesin in neural development remains to be elucidated. neudesin mrna was expressed in the neural precursor cells before the appearance of neurons. therefore, roles of neudesin in neural development were examined using the neural precursor cells. neudesin significantly promoted neuronal differentiation. in addition, neudesin transiently promoted neural cell proliferation early in the developmental process. the differentiation was mediated though activation of the pka and pi-3k pathways. in contrast, the proliferation was mediated through the mapk and pka pathways. the expression profile and activity indicate that neudesin plays unique roles in neural development. ps3p-d028 fabp7 is required for maintenance of neural stem/progenitor cells in the postnatal hippocampus motoko maekawa 1 , miho matsumata 2,3 , yuji owada 2 , shigeki yuasa 1 , noriko osumi 2,3 1 natl. inst. of neurosci., ncnp, tokyo, japan; 2 tohoku univ. sch. of med., sendai, japan; 3 crest, jst pax6 transcription factor is a key player for brain patterning and embryonic neurogenesis, and also expressed in the postnatal brain. we have previously shown that pax6 is necessary for keeping neural stem/progenitor cells in the hippocampus. in this study we have focused on a fatty-acid binding protein fabp7, a downstream of pax6, regulating maintenance of embryonic neural stem/progenitor cells (arai et al., 2005) . fabp7 was expressed in neural stem/progenitor cells in the hippocampal dentate gyrus (dg). 56% of fabp7-expressing cells co-expressed gfap (a marker for early progenitors), and 33% of them co-expressed psa-ncam (a marker for late progenitors). fabp7 expression was also overlapped with pax6, and expression of fabp7 was down-regulated in the dg of pax6 deficient rats and mice. finally, brdu-labeling analysis revealed decreased cell proliferation in the dg of fabp7 knockout mice. taking all together, it is concluded that fabp7 is required for maintenance of neural stem/progenitor cells in dg. ps3p-d029 involvement of the psa-ncam expressing cells in early development of the vascular system of the forebrain momoko miyakawa, tatsunori seki department of anatomy, juntendo university school of medicine, tokyo, japan early development of the vascular system of the forebrain were studied in the chick embryo. staining of vascular endothelial cells by fitctomato lectin and immunohistochemical staining of the surrounding cells were performed on the same cryostat sections of embryos of embryonic day 4-7. sections were examined under a confocal laser scanning microscope. capillaries were found in the lateral pallium and seemed to grow from psa-ncam-positive outer zone to negative inner zone of the pallium. psa-ncam is thought to be expressed in the immature neurons. the rims of capillaries were immunoreactive with psa-ncam in both zones. immunoreaction of doublecortin (neuronal marker) and punctate immunostaining of laminin also were observed on rims of capillaries. by immuno-electron-microscopy it appeared that the endothelium were covered with very thin processes of cells of which outer surface was immunoreactive with psa-ncam. psa-ncam expressing cells may be involved in the development of the vascular system of the forebrain by supporting or guiding the growing capillaries. masaharu kotani 1,6 , shiki okamoto 2 , masato imada 3 , kouichi itoh 4 , atsushi irie 5 , hitoshi sakuraba 6 , hideo kubo 7 1 department of molecular biologu, ohu univ., koriyama, japan; 2 dept. deve. physiol., natl. inst. physiol. sci., okazaki, japan; 3 dept. anatomy, nihon univ. shl. med., tokyo, japan; 4 dept. mol. pharma., univ. tokushima bunri, sanuki, japan; 5 dept. biochem. cell res., tokyo metro. inst. med. sci., tokyo, japan; 6 department of clin. genet, tokyo metro. inst. med. sci., tokyo, japan; 7 dept. med. biol, tokyo metro. inst. med. sci., tokyo, japan as randam-2 shows the highest expression level with the proliferating stage of neural stem cells (nscs), it is thought that the isolation of nscs based on the expression level of randam-2 is possible. in the present, we show that the isolated randam-2 high+ cells enrich nscs. the randam-2 high+ cells had the characteristics as the highly self-renewal capability and potential for multilineage differentiation into neural cells. in contrast, almost all of the randam-2 low+/− cells exhibited not only the extremely low self-renewability but the differentiation capability restricted to neurons. the results demonstrate that randam-2 is a usefule marker for the isolation of nscs by facs. yasuharu takamori 1 , yasuhisa tamura 1,3 , yosky kataoka 1,2 , yilong cui 1,3 , hisao yamada 1 1 department of anatomy and cell science, kansai medical university, osaka, japan; 2 department of physiology, osaka city university graduate school of medicine, osaka, japan; 3 morecular imaging reserch program, riken frs, saitama, japan lamins are major structural proteins of nuclear envelope. three lamin subtypes, a/c, b1 and b2 are mainly present in mammalian somatic cells. to investigate the pattern of lamin expression during neuronal differentiation, we immunohistochemically analyzed the existence of lamins in two neurogenic regions of rat brain; subgranular zone of dentate gyrus and subventricular zone, with confocal microscopy. gfap-positive primary progenitor cells possess lamin a/c (++), b1 (++), b2 (++), psa-ncam-positive subsequent progenitor cells possess lamin a/c (−), b1 (+++), b2 (+), and mature neurons possess lamin a/c (++), b1 (+), b2 (+++), in both neurogenic regions. these observation showed that the composition of lamin subtypes was distinct in particular differentiation stages during adult neurogenesis. yusuke tozuka 1 , yuichi tanaka 1 , tatsuhiro hisatsune 1 1 department of integrated biosciences, university of tokyo, chiba, japan recent work has shown that nestin + neural progenitor cells exist in the adult brain, and suggested that neural activity itself could act directly on these progenitor cells. it has been unclear, however, how do adult progenitor cells sense activity signals from surrounding neural circuit. in the hippocampus where new neurons are continuously produced throughout life, nestin + adult progenitor cells received gabaergic inputs. the gabaergic activity depolarized these progenitor cells, and then promoted their neuronal differentiations. although neuronal production does not readily occur in the adult neocortex, nestin + neural progenitor cells exist in this area too. interestingly, these progenitor cells also received excitatory gabaergic inputs. this gabaergic inputs inhibited their cell proliferations. from these results, we here propose that adult progenitor cells are a direct target of gabaergic neuronal networks, and that this networkto-progenitor cell interaction influences progenitors development by regulating their cell proliferations and/or neuronal differentiations. ps3p-e033 new migration pattern in the postnatal neurogenesis of the dentate gyrus takashi namba 1,2 , hideo namiki 2 , tatsunori seki 1 1 dept. of anat, juntendo univ. sch. of med., tokyo, japan; 2 integrative biosci. and biomed. eng, sch. of sci. and eng, waseda univ., tokyo, japan in the hippocampus, granule cells continue to be generated from embryonic to adult stages. the early postnatal neurogenesis is a transitional state between the embryonic and adult neurogenesis. previously, we have suggested that the postnatal hilus contains astrocytic neural progenitors that divide and differentiate into neuroblasts, and that finally the neuroblasts settle in the granule cell layer (gcl). however, the questions remain how astrocytic progenitors divide and differentiate into neurons, and how the neuroblasts migrate to the gcl. to observe them, we developed a time-lapse imaging system. retrovirus-gfp was injected into the rat hippocampus at p5. three days after the injection, the hippocampal slices were prepared for the time-lapse imaging. the present data show that neuroblasts migrate from the hilus to the gcl, changing the direction of their movement. this is inconsistent with the previous report suggesting simple radial migration (rickmann, et al., 1987) . the dividing pattern is currently under investigation. akiya watakabe 1 , noritaka ichinohe 2 , sonoko ohsawa 1 , tsutomu hashikawa 3 , kathleen s. rockland 2 , tetsuo yamamori 1 1 div. of brain biol, nibb, okazaki, japan; 2 lab. for cortical organization and systematics, bsi, riken, wako, japan, 3 lab. for neural architecture, bsi, riken, wako, japan by using gene expression profiles, we have tried to classify layer 6 neurons in several areas of monkey neocortex. we previously reported that nurr1, ctgf and sema3e mrnas are specifically expressed in subsets of layer 6 neurons. we further show here that cholecystokinin (cck) mrna is expressed in a subset of excitatory neurons in layer 6. by double ish, layer 6 neurons in monkeys are roughly divisible into cck(+) and sema3e(+) subgroups. each subgroup was further subdivided by other markers. tracer experiments showed that cck and sema3e mrna expression correlate well with corticocortical and corticothalamic connectivity, respectively, but the correlation was only partial. from this, we infer that subtypes defined by gene expression may not directly correspond to classical neuronal types. the implication of our findings will be discussed in terms of constancy of laminar structure across areas and species. research funds: kakenhi 17024055 ps3p-e035 rbp-j regulates the cortical laminar formation kenji tanigaki 1 , kazue muraki 1 , norio yamamoto 2 , tasuku honjo 2 1 shiga medical center, research institute, shiga, japan; 2 department of medical chemistry, kyoto university, kyoto, japan precise patterns of cell cycle exit and migration of neural progenitors are crucial for the formation of cortical layer structure. to examine involvement of notch-rbp-j signaling in the cortex laminar formation, we deleted rbp-j from neural progenitors in anatomically restricted areas by in vivo electroporation of cre-expressing plasmids. such studies revealed that rbp-j deficiency caused transformation of glutamatergic pyramidal neurons in layer ii/iii to layer iv neurons with concomitant loss of astrocytes. the loss of rbp-j accelerated neuronal differentiation and changed their laminar fates. in addition, time-lapse studies indicated the migration defect of rbp-j-deficient neurons. the results showed that notch-rbp-j signaling regulates migration of differentiated neurons as well as the timing of the cell cycle exit of neuronal progenitors to determine the laminar and cellular fates of neural progenitors. ps3p-e036 search for the genes that define mammalian cortical progenitor cells using single-cell gene expression profiles ayano kawaguchi 1 , tomoko ikawa 1 , yuya kasukawa 2 , hironori ueda 2,3 , kazuki kurimoto 4 , michinori saitou 4 , fumio matsuzaki 1,5 1 lab. for asymmetric cell division, cdb, riken, kobe, japan; 2 functional genomics subunit, cdb, riken, kobe, japan; 3 lab. for systems biology, cdb, riken, kobe, japan; 4 lab. for mammalian germ cell biology, cdb, riken, kobe, japan; 5 crest, jst, japan in the mammalian brain, cellular heterogeneity of the progenitor cells has largely hindered the molecular analysis of neuronal diversity. to overcome this problem, we randomly picked individual vz/svz cells of mouse embryos, and constructed cdnas from each of them by global pcr amplification method. we could classify these "single cell derived cdnas" into several groups retrospectively based on the expression of marker genes, including cell cycle related genes, transcription factors, and regional marker genes. 30 samples that showed typical marker gene expression pattern of the groups were applied for genechip analysis. the obtained data were confirmed by quantitative pcr and in situ hybridization. by this strategy, we identified nine genes that were specifically expressed in the svz progenitor cells. research funds: kakenhi (15700264) ryosuke tatsuno 1 , tomoaki sai 2,3 , masahiro otsu 2 , kuniko akama 1 , takashi nakayama 4 , tosifusa toda 5 1 grad. sch. of sci. and tech., chiba univ., chiba, japan; 2 lab. regener neurosci., tokyo metropol. univ. fac. health sci., tokyo, japan; 3 dept. orthop. surg., jikei univ. sch. med., tokyo. japan; 4 dept. biochem., yokohama city univ. sch. med., yokohama, japan; 5 proteomics collab. res., tokyo metropol. inst. of gerontol., tokyo, japan embryonic stem (es) cells possess pluripotency and self-renewal. however, the proteomic analysis of neural stem cells and neurons differentiated in vitro from es cells has not so proceeded yet. we investigated the expression levels of proteins during in vitro differentiation of mouse es cells into neurons via neural stem cells by neural stem sphere (nss) method, using 2-d gel electrophoresis and maldi-tof ms. we identified vimentin, creatine kinase, atp synthase beta subunit, and some proteins with no annotation in murine brain the database, which were up-regulated in neural stem cells, and down-regulated in es cells and neurons. these results suggest that the neural stem cells have characteristic protein expression profile. ps3p-e038 identification of se90, a novel gene expressed in the nural progenitor cells shin-ichi sakakibara, kazuhiko nakadate, shiichi ueda department of histology and neurobiology, dokkyo university school of medicine, tochigi, japan identification of the genes regulating neural progenitor or neural stem cell functions is critical to understand the mechanisms of the adult neurogenesis and neurodegenerative disease. we compared the gene expression profile of proliferating neural stem cell cultures with those of differentiated cells. a subtractive library was constructed by using the suppression subtractive hybridization and the differential screening was performed. among two thousand of the differentially expressed subtracted clones, we identified 150 genes that significantly upregulated in neural stem cell culture. these included several novel genes, in addition to the known genes involving in the cell cycle and signal transduction. in situ hybridization and the developmental northern analysis demonstrated that these mrnas were enriched in the germinal neuroepithelium, embryonic ventricular zone and the postnatal subventricular zone surrounding the lateral ventricles. we further analyzed the expression pattern of the novel gene se90 in developing and matured cns. teiichi furuichi 1 , akira sto 1,2 , yukiko sekine 1 , noriyuki morita 1 , tetsushi sadakata 1 , satoshi shoji 1 , jin-hong huang 1 , toshio kojima 2 1 laboratory for molecular neurogenesis, riken brain science institute, japan; 2 comparative systems biology team, riken genome sciences center, yokohama 230-0045, japan mouse cerebellum develops through a series of cytogenetic and morphogenetic events that are genetically coded within the first three weeks of life. we have extensively investigated the spatio-temporal gene expression profiles during the postnatal development of mouse cerebellum by differential display, rt-pcr, genechip, cdna microarray, and in situ hybridization. we have informatively systematized all the profiles in an online neuroinformatics database cdt-db (http://www.cdtdb.brain.riken.jp) with various search functions. we have demonstrated that the postnatal development of mouse cerebellum is genetically programmed by thousands of genes that exhibit differential expression patterns in time and space. further studies on a scale that includes the underlying expression of all genes and more detailed studies on their transcriptional regulation will shed light on the genetic basis for cerebellar development. miwako ozaki 1 , makoto mizuno 2 , kazuhisa sakai 4 , yoshimoto kiyohara 4 , kazuhiko yamaguchi 3 , tsutomu hashikawa 4 , hiroyuki nawa 2 1 institute of biomedical engineering, waseda university, tokyo, japan; 2 department of molecular neurobiology, brain research institute, niigata university, niigata, japan; 3 laboratory for memory and learning, bsi, riken, saitama, japan; 4 laboratory for neural architecture, bsi, riken, saitama, japan neuregulin (nrg), a neurotrophic factor, involved in the development, differentiation and repair of the nervous system, regulates the activation of ion channels and neurotransmitter receptors. in order to examine the molecular mechanism on the relationships between network, synapse formations and higher orders functions, we prepared ig-nrg1 knock out mice (nrg1 type i and iv were disrupted). the mutant mice showed motor disco-ordination and abnormality of synaptic structure in related areas in cerebellar nuclei and cortex. in addition, the number of vesicles in presynaptic neurons decreased in their synapses. the study on cerebellum that is very clear in the network input information would give some suggestions to the relationship between synaptic functions and behaviors. ps3p-e041 psd-95 protein expression in rat oromaxillofacial motoneurons during postnatal development kohji ishihama 1,2 , satoshi wakisaka 1 , shiho honma 1 , akira ito 1,2 , kei azuma 1,2 , mikihiko kogo 1 1 department of oral anatomy and developmental biology, osaka university graduate school of dentistry, osaka, japan; 2 first department of oral and maxillofacial surgery, osaka university graduate school of dentistry, osaka, japan postsynaptic density (psd), which is composed of diverse proteins, involved in synaptic structure, neurotransmission and signal transduction. psd-95 implicates in formation and maturation of excitatory synapses. psd-95 regulates the localization of the nmda receptor by means of binding with nr2. rhythmical oro-maxillofacial activities, such as suckling and chewing, are generated in the brainstem, and we showed that nmda receptors played critical role for the rhythm and pattern generation and signal transmission around the trigeminal motor nucleus during prenatal and early postnatal development. here we examined the temporal distributions of psd-95 protein using with immunohistochemical study, in developing rat brainstem from suckling to mature chewing stage. there was early emergence of psd-95 expression in the interneurons located at medial of the trigeminal motor nucleus. masami miura, masao masuda, toshihiko aosaki neural circuits dynamics research group, tokyo metropolitan institute of gerontology, japan the striatum, an input stage of the basal ganglia, contributes to habit formation as well as motor functions. recent studies suggest that striatal interneurons play an important role in processing of cortical input. we investigated the synaptic connections between interneurons using paired whole-cell recordings and immunohistochemical techniques. we found that fast-spiking (fs) interneurons sent gabaergic inhibitory input to cholinergic interneurons, which were gaba a receptor-mediated and suppressed by gaba b receptor agonist skf97541. in turn, cholinergic interneurons sent cholinergic excitatory input to fs interneurons. because the excitatory postsypnatic potentials (psps) were blocked by hexamethonium and dihydro-␤-erythroidine, the psps were nicotinic acetylcholine receptor-mediated. these results suggest that gabaergic interneurons and cholinergic interneurons mutually influence their excitability and might modulate the activity of striatal local circuits. ps3p-e043 ocular following responses (ofrs) to a brief background motin are modulated in relation to preparation for upcoming pursuit hiromitsu tabata, kenichiro miura, kenji kawano dept. integ brain sci., grad. schl of med., kyoto univ., kyoto, japan recently, our group reported that the ocular responses to a brief perturbation of a small target during fixation increased when subjects (humans, monkeys) were preparing for upcoming smooth pursuit eye movements (spems) rather than preparing for saccades or stationary fixation. here, we report that the increase in ocular responses based on the anticipation of spems was also observed in monkeys when a large-field visual stimulus (background) was moved briefly prior to pursuit. the result indicates that the visual region where the gain of the visuomotor transmission increased is not limited to a small region near the target but spreads to a larger field. in other words, the anticipation of upcoming spems could affect the generation of ofrs. furthermore, directionally biased ocular responses to the brief background motion were observed when the animals repeatedly performed spems toward one direction, implying that the prediction of the upcoming spem direction might cause the directional asymmetry of the visuomotor transmission gain. ps3p-e044 comprehensive characterization of motor neurons related with locomotory central pattern generator in the earthworm by imaging toshinobu shimoi 1 , kenji mizutani 2 , hiroto ogawa 3 , kohji hotta 1 , kotaro oka 1 1 ctr. for biosci. and info, keio univ., yokohama, japan; 2 neuro, karolinska inst, stockholm, sweden; 3 bio, saitama med. sch., saitama, japan in this study, we comprehensively identified and characterized motor neurons concerning with locomotory central pattern generator (cpg) in the earthworm by calcium imaging as multiple recording. the candidates of motor neurons were stained with dextran conjugated calcium indicators using retrograde labeling from projection nerves. we obtained the responses of up to 50 cell bodies of motor neurons and sensory neurons on the ventral surface of the segmental ganglion (25% or less for all neurons on the ventral surface). we analyzed the activity patterns of the candidates of motor neurons using pattern matching method comparing between calcium responses or between calcium responses and locomotory motor pattern. as a result, we detected motor neurons as pairs of neurons having strong synchrony to each other neuron or to motor pattern. these results were great progress to identify motor neurons related with locomotory cpg in the earthworm. ps3p-e045 three dimensional (3d) pursuit eye movement signals in cerebellar dorsal vermis takuya nitta, teppei akao, sergei kurkin, kikuro fukushima department of physiology, hokkaido university school of medicine, sapporo, japan for pursuit of a target moving in 3d space, signals for frontal and vergence-pursuit must be synthesized. studies in our laboratory have demonstrated that 3d pursuit signals are generated in the frontal eye fields, and also present in cerebellar floccular region. however, the majority of floccular purkinje (p-) cells discharged after onset of vergence-pursuit. cerebellar dorsal vermis is another cerebellar area for frontal pursuit. to examine whether 3d pursuit signals are present in this area, we examined simple-spike discharge of vermal pursuit p-cells in 3 monkeys. of a total of 77 p-cells that were examined during both frontal and vergence-pursuit, 46% discharged for both, 40% only for vergence, and 14% only for frontal pursuit. these results indicate that most of vermal pursuit p-cells discharged for vergence and that about half of them had 3d pursuit signals. majority (70%) of these p-cells discharged before onset of vergence eye movements with the typical lead time of 50 ms, suggesting their involvement in the initiation of vergence-pursuit. research funds: kakenhi (17022001) ps3p-e046 information processing in fef-rnrtp pathway for smooth pursuit seiji ono, michael j. mustari division of sensory-motor systems, yerkes national primate research center, emory university, atlanta ga, usa the frontal eye field (fef) cortex is known to play a role in smooth pursuit (sp). this role is supported by fef projections to the rostral nucleus reticularis tegmenti pontis (rnrtp) which projects heavily to the vermis. using multiple linear-regression modeling, we have shown that sp neurons in rnrtp were biased towards eye acceleration. however, the functional characteristics of sp related fef neurons that project to rnrtp have never been described. therefore, we used micro-electrical stimulation to deliver single pulses in rnrtp to antidromically activate fef neurons. the majority of sp related fef neurons that we identified as projecting to rnrtp were most sensitive to eye acceleration and much less sensitive to eye velocity. the neurons in fef-rnrtp pathway carry signals that could play a primary role in sp initiation. our antidromic studies may help address a fundamental question regarding whether basilar pontine nuclei integrate signals from multiple cortical areas or mostly relay signals with little transformation to cerebellum. research funds: nih grants ey13308, rr00165 aya takemura 1 , yumi murata 1,3 , kenji kawano 1,2 1 neurosci. res. insti, aist, tsukuba, japan; 2 dept. integ brain sci., grad. sch. med., kyoto univ., japan; 3 grad. sch. compreh hum sci., univ. tsukuba, japan previous studies in monkeys suggest that the medial superior temporal (mst) area is involved in visual motion processing. to understand the role of the mst in optokinetic nystagmus (okn) and afternystagmus (okan), we examined the effects of bilateral chemical lesions in the mst in two monkeys. when each monkey was injected with ibotenic acid (15 mg/ml, 36-37 l total), the initial rapid rise in okn was reduced. consequently, it took longer for the eye velocity to reach a steady state (i.e., an eye velocity close to the stimulus velocity). by contrast, the steady state okn was not affected and the okan persisted. the initial amplitude and falling time constant of the okan increased. the results suggest that the mst is part of the direct pathway for the initial rapid rise in the okn, but is not involved in the velocity storage mechanism for the steady state okn and okan. smooth pursuit is performed by coordination of eye and head movements. we have reported that the majority of fef pursuit neurons in monkeys with their head free to rotate about a vertical axis were modulated not only during eye-and gaze-pursuit but also head-pursuit to a moving reward feeder while the monkeys fixated an earth-stationary spot without gaze movement. to examine the origin of head-pursuit modulation, we moved the reward feeder in a ramp trajectory at 20 • /s with random intervals. the majority of pursuit neurons discharged before the onset of head movements with the mean lead time of 58 ms. discharge modulation during head-pursuit and passive whole body rotation was not correlated in most neurons. these results suggest that proprioceptive neck inputs or vestibular inputs are not the main origin of head-pursuit modulation. rather, our results suggest that the main origin reflects pursuit commands. ps3p-e049 the local feedback loop of the saccadic system: an analysis of the eye movements induced by pdb stimulation rikako kato department of developmental physiology, national institute for physiological sciences, okazaki, japan saccadic amplitude are controlled by a comparator that calculates dynamic motor error. some models place the comparator in the superior colliculus while others assign this role to the reticular formation. to decide between the two hypotheses one would need to stimulate pathways in between their putative comparators. we stimulated collicular axons descending in the pdb. our data demonstrate that electrical stimulation of the pdb evokes saccades and they always terminate before the end of the stimulus train. the characteristics of evoked saccades are comparable to those spontaneously generated by the cat. our data clearly demonstrate that the feedback path of the local loop of the saccadic system closes downstream of the superior colliculus. katsuo fujiwara 1 , kenji kunita 2 , kaoru maeda 1 , takeo kiyota 1 1 department of human movement and health, graduate school of medical science, kanazawa university, kanazawa, japan; 2 institute for health and sport sciences, osaka city university, osaka, japan we investigated changes in visual evoked potential (vep) during postural adaptation process while subjects maintaining standing posture on an oscillation floor with periodic vision shut. the subjects were 17 undergraduate students. a shutter goggle was used as a vep stimulator which was opened periodically for 30 ms with 800-ms intervals. the oscillation trial (0.5-hz frequency and 2.5-cm amplitude) (65-75 s) was repeated 5 times. postural steadiness was evaluated by mean fluctuation speed of the center of foot pressure. the mean speed decreased as trial was repeated, and reached a plateau before the 5th trial. a significant correlation was shown between 5th-1st trial differences in mean speed and vep amplitude (r = 0.79). this indicates that the role of visual information is different among subjects with various adaptation processes of postural control. ps3p-e051 primary motor cortex contributes to generating manual following response toshitaka kimura 1 , naoki saijo 1 , hiroaki gomi 1,2 1 ntt cs labs, kanagawa, japan; 2 erato shimojo implicit brain function proj, jst, saitama, japan a large-field visual motion during arm movements induces a shortlatency, involuntary arm response called as manual following response (mfr). the mfr exhibits similar features to the ocular following response (ofr) elicited by the similar visual stimulus, with respect to the stimulus-response directional characteristics and the spatiotemporal frequency tuning property. this suggests that computational mechanism is shared for both responses. however, the neural basis of the mfr motor command generation remains unclear, while ofr is known to be generated subcortically. here we show, by using transcranial magnetic (tms) and electrical (tes) stimulation over the primary motor cortex (m1), that (1) an emg response evoked by tms was facilitated during mfr, while that by tes was not, and (2) intracortical inhibition within m1 assessed by paired-pulses tms was reduced during mfr. these results suggest that mfr is generated through activity of interneuronal networks within m1. such cortical mechanisms for mfr generation are distinct from the subcortical processes for ofr generation. naoki saijo 1 , hiroaki gomi 1,2 1 ntt cs labs., kanagawa, japan; 2 erato shimojo implicit brain function proj, jst, saitama, japan when a visual target is suddenly shifted during a reaching movement, we can quickly adjust the arm movement. however, the computational mechanism to generate quick adjustment is still unclear. here we investigated this mechanism from the viewpoint of visuomotor coordinate transformation. we observed the hand responses to the target shifts in 8 radial directions applied during reaching. the data show that the direction of the initial phase (150-200 ms) of hand response acceleration was slightly biased from the corresponding target shift direction, whereas the direction of the late phase (250-300 ms) was little biased. additionally, when we use a target shift having less-motion energy, the response latency greatly increased and the directional bias significantly decreased. these results suggest that the on-line reaching adjustment would be generated by two different mechanisms: a reflexive controller which is induced by visual motion with short latency and generates spatially inaccurate response, and voluntary controller which generates spatially accurate response with long latency. ps3p-e053 spatial relationship between gaze and reaching-target modulates manual following response naotoshi abekawa 1 , hiroaki gomi 1,2 1 ntt cs labs., kanagawa, japan; 2 erato shimojo implicit brain function proj, jst, saitama, japan to explore the functional mechanism of the manual following response (mfr) induced by a large-field visual motion during arm movement, we examine its modulation caused by the spatial relationships between gaze, target, and background. on a large vertical screen placed in front of the subject, full field checker pattern, two markers (upper and lower), and a gray mask around one of the markers, were displayed. in the first condition, subjects kept watching the upper marker, and pointed the upper (congruent) or lower (incongruent) marker instructed before every reaching. the checker pattern suddenly moved either rightward or leftward brief after reaching start. in the second condition, subjects did the same task with watching the lower marker. in both conditions, the mfr amplitude was significantly grater in the congruent condition than in the incongruent condition, whereas the mask location did not significantly affect the mfr amplitude. this suggests that the spatial relationship between gaze and target is important in modulating mfr. misako komatsu, eizo miyashita dept. compu. intelligence & systems sci., tokyo tech., yokohama, japan when a subject performed pointing to a remembered target under eyes fixated, we have reported that endpoints tended to sift closer to the fixation point. moreover, we have noted that the greater the distance between a target and the fixation point, the larger the errors. the result was consistent even when the position of the fixation point was changed. the above tendency was considered to occur in eye-or gaze-centered coordinates. it is open question, however, if the brain correctly compensates the difference of the relative position of eyes to the head? to answer this question, we investigated the dependency of the endpoint errors on the positions of a monitor and the fixation point. the subjects, sitting in front of the monitor, were asked to point a remembered target as accurately as possible using a computer mouse. all the results were consistent with the previous ones regardless of the position of monitor or the fixation point. these results suggest either the eye-position doesn't affect how we recognize the target position, or the brain correctly compensates the eye-position with a fixed head position. ps3p-f055 influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot tetsuro muraoka 1 , takashi obu 2 , kazuyuki kanosue 3 1 asmew, waseda university, saitama, japan; 2 graduate school of human sciences, waseda university, saitama, japan; 3 faculty of sports sciences, waseda university, saitama, japan the aim of this study was to investigate the influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot. the subjects (n = 6) were supine, and their hand was prone. they performed cyclical flexion-extension coordinations of the hand and foot in the iso-(iso) or opposite-(oppo) directions, and those with an elastic load against wrist flexion (el-iso and el-oppo) at 1.5, 1.75, and 2.0 hz. over 99% success rate was observed in all tasks except oppo (78-97%). the in-phase muscle activity of wrist and foot muscles was obserbed in all tasks except oppo. it was suggested that the in-phase muscle activity might be an important factor in a coordinated movement of ipsilateral hand and foot. research funds: the special coordination funds for promoting science and technology, mext, japan ps3p-f056 simultaneous muscle activity stabilizes the coordinated movement of ipsilateral hand and foot takashi obu 1 , tetsuro muraoka 3 , kazuyuki kanosue 1,2,3 1 faculty of human sciences, waseda university, saitama, japan; 2 faculty of sport sciences, waseda university, saitama, japan; 3 asmew, waseda university, saitama, japan in human, voluntary opposite-directional movement (antiphase) of ipsilateral hand and foot is more difficult than iso-directional movement (inphase). the purpose of the present study was to investigate the influence of the coupling of muscle activity on these movements. eight normal subjects lay in supine position with hand prone and their foot was forcedly moved by a dynamometer cyclically at 1, 1.33, and 1.66 hz. they were asked to perform 4 tasks, concentric/eccentric contraction of ankle dorsiflexors with in-phase/antiphase wrist extension/flexion. all tasks were performed successfully. muscle activity of hand flexors was observed in concentric-antiphase and eccentric-inphase tasks, indicating simultaneous muscle activity of hand and foot. it may be suggested that simultaneous muscle activity would make the movement easier regardless of the direction of movement. ps3p-f057 activities of erector spinae muscles during jaw clenching in man kayoko yasunaga 1,2 , tadachika yabushita 1 , kazuo toda 2 , kunimichi soma 1 1 orthodontic science, tokyo med. & dent. univ., tokyo, japan; 2 div. integrative sensory physiology, nagasaki univ., nagasaki, japan recent studies focused the functional relationships between the masticatory and the posture system. the hypothesis of our present study is an existence of functional connections between the masticatory system and the spinal muscles which maintain the posture. therefore, we investigated the effect of the maximum jaw clenching on the spinal muscle activities. bipolar needle electrodes were inserted into erector spinae muscles to record the motor unit activities when the sitting subjects relaxed and performed maximal jaw clenching. as a result, the instantaneous frequencies of the spinal muscles decreased with clenching, compared with relaxed jaw position. our results suggested that there were some relationship between spinal muscle activities and jaw clenching. the effects of bipedal walking on the central nervous systems-influence of bipedal walking on the spinal reflex-naomi wada 1 , sachiko motoyama 1 , futoshi mori 1 , shigemi mori 2 1 department of veterinary physiology, yamaguchi university, yamaguchi, japan; 2 national institute for physiological science, okazaki, japan the one of the biggest questions in the vertebrate evolution is how human got the highly developed brain. many investigators suggest that upright posture and bipedal walking caused remarkable development of brain and produced the human being. the purpose of our experiments is to show the influences of bipedal habits on central nervous systems. we have established the bipedal walking model using rats (rbm) by amputation of forelimbs and training of upright posture and bipedal walking. after training of upright posture and bipedal walking for 12-20 weeks, rats got abilities of the stable upright posture and bipedal walking with symmetrical hindlimb movements between left and right side. in the present experiments, we studied about the effects of bipedal habits on the lumbar spinal reflex. the results of out experiments showed that bipedal habits inhibit the spinal reflex pathways. ps3p-f059 neuronal activity in primary motor cortex during quadrupedal locomotion of the japanese monkey katsumi nakajima 1 , futoshi mori 2 , akira murata 1 , masahiko inase 1 1 dept. of physiol., kinki univ. schl. of med., osakasayama, japan; 2 dept. of vet. physiol., facult. of agr., yamaguchi univ., yamaguchi, japan to elucidate cortical mechanisms related to the control of primate locomotion, we recorded neuronal activity in m1 of the monkey walking quadrupedally on the treadmill. tungsten microelectrodes were inserted into m1 hindlimb region using a custom-made micromanipulator. we found that all neurons recorded in m1 modulated their discharge phasically time-locked to the step cycle or increased their discharge frequency tonically during simple locomotion. the neuron exhibiting phasic modulation peaked once or twice per step. the peak activity occurred at widely different times during the step cycle in different recorded neurons. as the treadmill speed increased, most of recorded neurons increased their discharge frequency. all these results suggest that m1 output in monkeys directly and/or indirectly acts on spinal circuitries generating a basic pattern of rhythmic activity during simple locomotion in a manner different from that in subprimates. research funds: kakenhi (16500271) ps3p-f060 activity of putaminal neurons receiving inputs from motor cortical areas in behaving monkeys sayuki takara 1,2 , nobuhiko hatanaka 1,2 , masahiko takada 3 , atsushi nambu 1,2 1 school of life science, the graduate university for advanced studies, japan; 2 division of system neurophysiology, national institute for physiological sciences, japan; 3 tokyo metropolitan institute for neuroscience, japan the putaminal (put) neurons receive motor cortical inputs and change their activity in relation to movements. to investigate how these inputs contribute to put neuron activity in behaving monkeys, extracellular unit activity was recorded from identified put neurons during the performance of a memory-guided reaching task. based on orthodromic spikes evoked by cortical stimulation, individual put neurons were defined in terms of whether they receive input from the primary motor cortex (mi), the supplementary motor area (sma), or both. the results showed that mi-recipient neuron activity was responsive to the movement, while sma-recipient neuron activity was responsive to the cue stimuli and/or the delay period. the activity of neurons receiving convergent inputs was related to both the movement and the delay period. we previously reported that electrical stimulation of cerebrofugal fibers induced short latency facilitation and succeeding suppression on phrenic activities, while train pulse stimulation of caudal raphe nuclei (raphe magnus, rm, and raphe pallidus, rp) induced suppression or facilitation on respiratory neural activities in cats and rats. in this study, in order to analyze the cerebral and raphe projections to the respiratory neuron network, we examined the effects of stimulation of cerebrofugal fibers and caudal raphe nuclei on activities of ventral respiratory group neurons (vrgs) in the medulla and upper cervical inspiratory neurons (ucins). animals were anesthetized, immobilized and artificially ventilated. stimulation of cerebral peduncle (cp) induced short latency facilitation and succeeding suppression on activities of ucins. stimulation of rm or cp evoked inhibitory postsynaptic potentials in the caudal vrgs. these results suggest that rm and cerebral cortex directly inhibit main respiratory output neurons in vrg. ken muramatsu 1 , sei-ichi sasaki 2 , yuichiro cho 1 , kenji sato 1 1 anatomy and physiological science, tokyo medical and dental university, tokyo, japan; 2 department of physiology, ibaraki prefectural university of health sciences, ibaraki, japan distribution of average diameters of external anal sphincter (eas) motoneurons and peripheral motor fibers were examined in cats. to identify eas motoneurons, horseradish peroxidase was applied to the central cut end of the anal branches of the pudendal nerve. eas motoneurons were found in the onuf's nucleus of s1 and s2 spinal levels. to examine size of peripheral motor fibers, ganglionectomy was performed onl7 -s3 spinal segments which contain afferent fibers of eas muscles. after 3 weeks survival period, anal branches of the pudendal nerve was examined. histograms of the distribution of average diameters of cell body and motor fiber shows unimodal distri bution. also, distribution of muscle spindles of eas muscle were examined by serially sectioning the distal colon and staining with mayer's haemotoxylin and eosin. no muscle spindles were found. these results suggest that eas muscle is controlled without gamma loop. mariko miura, yoshiki iwamoto, kaoru yoshida neurophysiol., univ. tsukuba, tsukuba, japan saccade accuracy is ensured by an adaptation mechanism. the speed and magnitude of adaptation vary greatly across experiments even for the same subject. one factor that might cause this variability is adaptation history. the present study aims to clarify whether preceding adaptation influences subsequent adaptation over several days. gain decrease adaptation was induced in a monkey by stepping the target backward during saccades. adaptation experiments were repeated for 4 consecutive days. we compared adaptation in day1 and that in day4. the gain decrease for the first 700 saccades in day 4 (0.140 ± 0.036) was larger than that in day 1 (0.080 ± 0.021) (p = 0.026, n = 5, paired-t test). the rate of adaptation in day 4 (1.739 ± 0.393 × 10 −4 /sac) was higher than that in day 1 (1.120 ± 0.240 × 10 −4 /sac) (p = 0.011). the overall gain change (1800 saccades) in day 4 (0.219 ± 0.030) was larger than that in day 1 (0.112 ± 0.022) (p = 0.002). thus, both the speed and magnitude of adaptation were increased by preceding adaptation. the present study suggests that the memory of saccadic adaptation is retained for days and facilitates following adaptation. research funds: kakenhi (16300129) ps3p-f064 asymmetry of the anticipatory convergence eye movement haruo toda, takehiko bando div. integr. physiol., grad. sch. med. sci., niigata univ., niigata, japan typically, convergence eye movement is known as symmetric adduction of the both eyes. but asymmetrical convergence also found in the natural condition. these asymmetrical convergence may reflect asymmetries of central control of convergence eye movement. the lateral suprasylvian (ls) areas are extrastriate cortices which receive visual information from v1. the ls has contralateral dominant receptive fields and convergence eye movements evoked from the long latency regions were asymmetrical. cats (n = 7) were trained to start convergence by an alarm signal (buzzer sound or combination of buzz and blinking of led), preceding target movement by 4 s. after training, ocular convergence was elicited by the alarm signal before target movement (predictive open-loop convergence) in 60% of trials. in three cats, we used training with obliquely approaching target. after training, asymmetrical anticipatory eye movements were observed. based on these findings, related ls neuronal activities and results from lesion study, we will discuss the role of ls in asymmetry of anticipatory and visually-evoked convergence eye movement. yusuke uchida 1 , xiaofeng lu 1,2 , shogo ohmae 1 , toshimitsu takahashi 1,2 , shigeru kitazawa 1,2 1 dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; 2 crest, jst, tokyo, japan we examined reward related neural activity in the supplementary eye field (sef). for this purpose, two monkeys were rewarded after each visually guided saccade from a central fixation point to one of 16 targets that were arranged in a radial pattern. a target appeared while the monkeys were fixating on the central point, and the monkeys made a saccade to the target when the fixation point disappeared and held on the target until the target turned off. reward was delivered during or after target-hold period. we found that many sef cells became active during the period of reward delivery (r-cell). more than half of r-cells showed enhancement of the neural discharge in the specific target directions but not other directions in which the same amount of reward was given (rd-cell). interestingly, most of rd-cells displayed activity with the clear directional tuning. these results demonstrate reward dependent activity specific to spatial direction in the sef, and further suggest that sef cells provide reinforcement mechanism. research funds: kakenhi 17022033 ps3p-f066 frontal pursuit area is involved in the retinalslip dependent adaptation of monkey post-saccadic pursuit eye velocity hiromasa kitazawa 1 , soichi nagao 1,2 1 lab. for motor learning control, riken bsi, saitama, japan; 2 sorst, jst, saitama, japan smooth pursuit is under learning control by several brain areas including cerebrum and cerebellum. smooth pursuit velocity is modifiable by repetition of target velocity for a brief period at its onsets. role of cerebellar vermis and hemisphere in the adaptive control of smooth pursuit is suggested by lesion experiments, but the role of frontal pursuit area (fpa) is not known. to reveal possible involvement of fpa in the adaptation of smooth pursuit, we identifying fpa by unit recording and microstimulation, and reversibly inactivated it by local injection of muscimol. we found that inactivation of fpa not only reduced of the velocities of pursuit in the ipsi-and contra-versive directions to the inactivated fpa, but also appreciably depressed its adaptation, suggesting that fpa is involved in the adaptation of smooth pursuit. shinji matsutani department of functional morphology, kitasato university school of nursing, kanagawa, japan distribution of terminals on individual centrifugal axons in the main olfactory bulb was studied using an anterograde tracer to elucidate function of the centrifugal system. the tracer was injected into olfactory cortical areas, and individual labeled axons were traced from serial sections. as already reported in the last meeting, the centrifugal axons had multiple terminals with discrete locations. distribution of these terminals was examined in reconstructed maps in which localization of the terminals was projected onto a sagittal plain. in most axons, the terminals were clustered to form a patch that was stretched in a rostrocaudal direction. it was also common that patches belonging to the same axon were found in distant locations and in both sides of the single bulb. while most of the terminals were seen in the granule cell layer, those located in the glomerular layer and in the external plexiform layer were found following injections into the anterior olfactory nucleus. the centrifugal fibers may couple the activity of discrete and distant subsets of bulbar neurons. ps3p-f068 projection targets of the drosophila taste receptor neurons in the primary gustatory center of the brain takaaki miyazaki 1,2 , kei ito 1,2,3 1 dept. of comput. biol., grad. sch. of frontier sci., univ. of tokyo, japan; 2 center for bioinform., imcb, univ. of tokyo, japan; 3 bird, jst, japan in order to figure out the way of information processing linking gustatory stimulus and taste-associated behavior, systematic knowledge about the underlying neural networks is required. drosophila melanogaster is an attractive model organism for this task, thanks to its relatively simple brain structure and a wide variety of molecular and genetic tools available. gustatory sensory neurons in the labellum of the mouth project their axons via the labial nerve to the suboesophageal ganglion (sog) of the brain. to understand the entire neural circuits of these first-order neurons in the primary gustatory center, we searched for the gal4 enhancer-trap strains that visualize specific neural fibers in the sog and the labial nerve. screening 4,000 strains, we identified about 180 candidate lines. the projection targets of the labeled neurons were classified into seven areas. the terminals of the already identified sensory neurons appear to fall into specific subsets of these areas. research funds: bird, jst ps3p-f069 immunoreactivity and voltage-gated channels of mouse taste bud cells kennji kimura 1 , yoshitaka ohtubo 1 , takashi kumazawa 2 , kiyonori yoshii 1 1 graduate school of life science and systems engineering, kyushu institute of technology, kitakyushu, japan; 2 department of applied chemistry, saitama institute of technology, fukaya, japan mammalian taste buds comprise four heterogeneous cell types, type i to iv, and their collaboration seems to generate taste sensation. we investigated the electrophysiological properties of these cell types except type iv with taste buds preserved in mouse lingual epithelia. type i cells elicited smaller ttx-sensitive, tea-sensitive, and teainsensitive currents in magnitude than other cell types. type ii cells elicited a smaller tea-sensitive current and a larger tea-insensitive current than type iii cells. these results suggest that type ii and iii cells elicit action potentials with different ionic mechanisms, and that the difference results from the functional differences of these cell types. research funds: kakenhi (16300094) and the 21st coe program (center #19) granted by mext of japan ps3p-f070 inositol monophosphatase maintains synapse localization and regulates behavior in the mature nervous system of c. elegans yoshinori tanizawa 1 , atsushi kuhara 1 , hitoshi inada 1 , eiji kodama 1 , takafumi mizuno 1 , ikue mori 1,2 1 lab. of mol. neurobiol., nagoya univ., japan; 2 institute for advanced research, nagoya univ., japan inositol monophosphatase (impase) is suggested to be relevant to bipolar disorder. although lithium is believed to exert therapeutic effect by inhibiting impase in patients, the mechanism underlying lithium therapy is largely unknown. here we show that the loss of impase causes defects in behavior and localization of synapses in c. elegans. mutations in ttx-7 gene encoding impase exhibit defective thermotaxis behavior, which is attributable to the loss of impase activity in the most essential integrative interneuron ria in the nervous system. the ttx-7 mutations also cause mislocalization of synaptic proteins in ria. both behavioral and synaptic defects in ttx-7 mutants were rescued by expression of impase at adult stage and inositol application, and were mimicked by lithium application in wild type animals. these results suggest that impase is required in the mature nervous system for maintaining synapses of the central interneurons in order for animals to behave properly. research funds: kakenhi ps3p-f071 postnatal alterations in expression of vesicular glutamate transporters in the main olfactory bulb (ob) of rats h ohmomo, f shutoh, a. ina, s. yoshida, h. nogami, s. hisano lab. neuroendocr., graduate sch., univ. tsukuba, tsukuba, japan olfactory information is conveyed to the brain by transmission from primary olfactory neurons to mitral or tufted cells. however, little is known about development of these ob glutamatergic neurons in early postnatal life. vesicular glutamate transporters (vglut) have been used as the best histological markers to identify glutamatergic neurons. we here studied expressions of two vglut isoforms (vglut1 and -2) during rat ob development from postnatal day 1 (p1) to p10 by in situ hybridization and immunohistochemistry. at p1 vglut1 immunoreactivity (ir) was detected in all layers except the olfactory nerve layer, and thereafter its localization expanded and intensity increased. vglut1 mrna signals were detectable in the mitral cell layer from p1 to p10. in contrast, vglut2 ir was prominent in the glomerulus at all days examined, and only at p1 and p3 in mitral cells. despite mitral vglut2 ir disappeared at p10, the mrna signals were still detectable. these results suggest that glutametergic neurons in the rat ob continue to develop even after birth. ps3p-f072 v1r genes multiplied in amphibian and expressed in the main olfactory system atsuko date-ito 1,2 , masumi ichikawa 3 , yuji mori 2 , kimiko hagino-yamagishi 1 1 tokyo metrop. inst. med. sci., tokyo, japan; 2 the univ. of tokyo, tokyo, japan, 3 tokyo metrop. inst. neurosci., tokyo, japan in rodent, v1r gene family is expressed specifically in the vomeronasal organ (vno) and is thought to be responsible for pheromone reception. however, teleost fishes lacking for the vno have a single v1r gene, which is expressed in the olfactory epithelium (oe). to examine when the v1rs function as pheromone receptors in the course of evolution, we analyzed the amphibian xenopus tropicalis genome, and identified 23 v1r sequences. these v1rs were not expressed in the vno, but most of them were expressed in the oe of the middle cavity, which is considered for reception of water-soluble odorants. from these results, we speculate that the amphibian v1rs get a chance to receive diverse odorants such as pheromones by gene multiplication and sequence diversification. our results raise the possibility that pheromonal information is transmitted via the main olfactory system. ps3p-f073 analyses of ligand binding sites and snps on sweet taste receptor system in human noriatsu shigemura, a.a. islam, yuki nakamura, shinya shirosaki, yuzo ninomiya sect. oral neurosci., grad. sch. dent science, kyushu univ., japan recent studies have shown that t1r2/t1r3 heterodimer plays a role as a sweet taste receptor. but, mice lacking t1r3 showed diminished but not abolished behavioral and nerve responses to sugars, suggesting t1r3-independent sweetener binding site also exist in mice. in this study, to predict binding sites on t1r2/t1r3 and/or other sweet receptor in human, we measured sensitivity thresholds to various sweet compounds and examined the qualitative similarities. we also used gymnemic acid and ␥-cyclodextrin, which selectively inhibits sweet responses and reduces the inhibitory action of it. the ten sweet compounds were classified into five groups [(1) sucrose, glcose, fructose, (2) saccharin, aspartame, acesulfame-k, glycine, (3) d-phenylalanine, (4) d-tryptophan, (5) l-proline]. in sequencing analysis, four and two snps with amino acid substitution were revealed in t1r2 and t1r3, respectively. these results suggest that there may be at least five binding sites in human sweet receptor system. the individual differences in sweet sensitivities may be due to these snps. keiko yasumatsu 1 , sachiko saito 2 , yuko murata 3 , ding ming 4 , tatsu kobayakawa 5 , robert f. margolskee 4 , yuzo ninomiya 1 1 sect. oral neurosci., grad. sch. dent. sci., kyushu univ., fukuoka, japan; 2 saito sachiko taste and smell research institution, ibaraki, japan; 3 national res. institute of fisheries sci., kanagawa, japan; 4 dept. of physiol. & biophys., mount sinai sch. med., new york, usa; 5 national institute of advanced industrial science and technology, ibaraka, japan the effect of unsaturated fatty acids on taste responses was examined by measuring perceived taste intensity in human, behavioral short-term lick responses and electrophysiological taste responses recorded from the chorda tympani and glossopharyngeal nerves in mice. the results showed that dha and other polyunsaturated fatty acids inhibit responses to bitter taste compounds without affecting other taste stimuli. we also found fatty-acid inhibition on bitter responses in an in vitro g-protein activation assay using bovine taste membrane, but lack of the bitter taste inhibition in ggustducin ko mice. these results suggest that fatty acids specifically inhibit responses to bitter stimuli by suppression of activation of t2r receptors which coupled with ggustducin. ps3p-f075 newborn infant body odor attenuates their mother's postpartum moods shota nishitani 1 , mayumi kokuryo 1 , tsunetake miyamura 2 , kazuyuki shinohara 1 1 div. neurobiol. & behav., nagasaki university, japan; 2 obstet. & gynecol. of miyamura hospital, japan mothers are attracted to the body odor of newborn infants, but little is known about its reason. in the present study, we examined whether the body odor of newborn infants exert effects on moods in postpartum mothers. the body odors of newborn infants were collected from their undershirts. postpartum mothers were exposed to odors of a part of the undershirt with control odors, their own infant body odors or other infant body odors. we used the poms to assess the effects of infant body odors on postpartum moods. this study was approved by the ethics committee of nagasaki university. the infant body odors significantly increased hedonics and friendliness scores, and significantly decreased anxiety, depression and fatigue scores, whether infant odors may be originated from their own infants or other infants. these results suggest that body odors of newborn infants attract their mothers because they have calming effects on postpartum mothers. research funds: japan science and technology agency (jst), research institute of science and technology for society (ristex) ps3p-f076 human prefrontal activity in taste encoding: an fnirs study masako okamoto 1 , mari matsunami 2 , haruka dan 1 , tomoko kohata 2 , kaoru kohyama 1 , ippeita dan 1 1 national food research institute, tsukuba, japan; 2 nippon suisan kaisha, ltd., japan taste remains one of the least-explored human senses. using multichannel functional near-infrared spectroscopy (fnirs), we examined the lateral prefrontal cortex (lpfc) of healthy volunteers (n = 10) while they tasted and encoded the quaternary taste mixtures. the contrast between the cortical activation under encoding conditions and that under control conditions without memory requirement revealed activation in the bilateral ventro-lpfc and the right posterior portion of the lpfc. the activation pattern, which was in line with those that have been associated with intentional encoding of non-verbal materials of other senses, supported an amodal role of lpfc in intentional encoding, at least at a macro structural level. this study also demonstrates that, by using fnirs, lpfc functions on taste can be examined with experimental paradigms comparable to those used for other senses. recently, we performed simultaneous respiration and electroencephalographic recordings during odor stimulation. we sought to identify changes in respiratory pattern, inspiratory phase-locked alpha oscillation (i-␣) and location of dipoles estimated from the potentials. electroencephalographic dipole tracing identified the location of dipoles from the i-␣ in the limbic area and the cortex; the entorhinal cortex, hippocampus, amygdala, premotor area and orbitofrontal cortex. in this study, we compared the respiratory pattern during odor stimulations, i-␣, dipole localizations without habituation with those with habituation of odors. onset of inspiration was used as a trigger for averaging, and potentials were averaged before and after the habituation period. habituation of odor caused to return to the normal respiratory pattern, decrease of amplitudes of ␣, and entorhinal cortex, hippocampus, amygdala were less active. akio tsuboi, takaaki miyazaki, takeshi imai dept. of biophys. & biochem., univ. of tokyo, tokyo, japan vertebrate odorant receptor (or) genes are divided phylogenetically into two distinct classes, the fish-like class i and the terrestrialspecific class ii. in the present study, we systematically analyzed mouse class i or genes (42 subfamilies) to elucidate the expression profiles in the olfactory epithelium (oe) and the projection sites of their olfactory sensory neurons (osns) in the olfactory bulb (ob). in situ hybridization (ish) revealed that most class i or genes (36 subfamilies) were expressed in the dorso-medial zone (zone 1) of the oe. furthermore, there appeared to be no significant differences in the distributions of osns expressing class i genes within zone 1. these results indicate that there is a clear boundary between zone 1 and non-zone 1 areas in the oe. some class i ors are known to possess ligand specificity for aliphatic acids, aldehydes and alcohols. our ish analysis has revealed that osns expressing the class i ors in zone 1 tend to converge their axons on a cluster of glomeruli in an antero-dorsal domain that is assumed to be involved in responses to the aliphatic compounds on the ob. research funds: kakenhi (16500196) ps3p-g079 taste response characteristics of putative interneurons in the rat gustatory cortex tatsuko yokota, kunihiro eguchi, katsunari hiraba department of physiology, school of dentistry, aichi-gakuin university, nagoya, japan previous studies have indicated that the extracellular spike waveforms and discharge rate properties of cortical neurons differed between pyramidal cells and interneurons, the latter tending to have narrower spike-widths and higher discharge rates. taste-sensitive neurons in the rat gustatory cortex were classified according to (1) best-taste profiles and (2) spike-widths which were found to form a bimodal distribution (narrow and broad). narrow-spike neurons had a significantly larger response to nacl than broad-spike neurons, but no differences were found to other tastants. the proportion of narrow-spike neurons in the n-best neurons was higher than that in the h or nh-best neurons. these results indicate that putative interneurons may play an important role in the coding of salt taste information. research funds: kakenhi (11671860) of japan to t.y. yuki sato, nobuhiko miyasaka, yoshihiro yoshihara laboratory for neurobiology of synapse, riken bsi, wako, japan in the fish olfactory system, individual olfactory sensory neurons (osns) are thought to express only one or at most a few different odorant receptors (ors) from the large or family consisting of ∼100 members. here, we investigated the mechanisms underlying or gene choice by using transgenic zebrafish that carried a modified bac containing a zebrafish or gene cluster. replacement of the or coding regions in the bac transgene with reporter genes allowed the reporters to be expressed in a small population of osns in the transgenic fish. in situ hybridization analysis using or-specific probes revealed that or genes expressed in reporter-positive cells were mostly restricted within the same or subfamilies to which the replaced ors belonged. additionally, the reporter-expressing osns projected their axons to a topographically fixed cluster of glomeruli in the olfactory bulb. these findings suggest the hierarchical regulation of or gene choice, whereby an individual osn may express one or gene from a limited subpopulation that is chosen from the entire repertoire in advance. research funds: kakenhi (16300105) ps3p-g081 identification of perisomatic-targeting granule cells in the mouse olfactory bulb hiromi naritsuka 1 , kazuhisa sakai 2 , tsutomu hashikawa 2 , kensaku mori 1 , masahiro yamaguchi 1 1 dep. physiol. grad. sch. med., univ. of tokyo, tokyo, japan; 2 laboratory for neural architecture, bsi, riken, saitama, japan in the olfactory bulb (ob), odor information is processed by the local circuit that includes inhibitory interneurons. granule cells (gcs) are major interneurons in the ob, but their diversity is not well understood. in the ob of adult transgenic mice expressing gfp under the control of nestin gene regulatory regions, we observed gcs with strong gfp expression (referred to as type s cells). their dendrites branched and formed spines within the granule cell layer, internal plexiform layer and mitral cell layer but did not reach the external plexiform layer, where typical gcs make synapses with dendrites of mitral and tufted cells. type s cells had huge protrusions at their dendritic ends, which formed contact with mitral cell somata. electron microscopic analysis revealed the existence of reciprocal synapses between type s cell protrusions and mitral cell somata. characteristic morphology of perisomatic-targeting gcs indicates that they have functions distinct from typical gcs in the ob. keiko moriya-ito, kentaroh endoh, yuuki ishimatsu, masumi ichikawa department of neuroscience basic technology, tokyo metropolitan institute for neuroscience, fuchu, tokyo, japan a coculture system of accessory olfactory bulb (aob) neurons and vomeronasal neurons was established for studying the functional roles of aob neurons in pheromonal signal processing. in this study, the effect of vomeronasal neurons on the development of aob neurons was examined in a coculture system. the densities of dendritic spines were lower in the coculture than in single culture. the ratio of the density of synaptophysin-immunopositive spine/total spine density was larger in the coculture than in the single culture. the volume of spine head was larger in the coculture than in single culture. by electron microscopic observation, the synapses on dendritic shafts were decreased and the synapses on dendritic spines were increased in the coculture. the synapses between aob neurons and vomeronasal neurons were recognized in the coculture. these observations suggest that synapse formation of aob neurons is modified by synaptic contact with vomeronasal neurons. ps3p-g083 nacl induced responses of mouse fungiform taste cells: existence of amiloride sensitive and insensitive taste cells ryusuke yoshida, tadahiro ohkuri, keiko yasumatsu, noriatsu shigemura, yuzo ninomiya sect. of oral neurosci., grad. sch. of dental sci., kyushu univ., fukuoka, japan previous electrophysiological studies showed that the chorda tympani nerve contains two types of nacl-responsive fibers, amiloride sensitive (n-type) and insensitive (e-or h-type) fibers, suggesting the existence of amiloride sensitive and insensitive taste receptor cells in fungiform papillae. in this study, we examined nacl responses of mouse fungiform taste cells in isolated taste bud and amiloride sensitivity of them. some taste cells respond to apical restricted nacl stimulation with increase in firing frequency and their responses were concentration dependent. amiloride mixed with apical nacl solution inhibited nacl responses in some taste cells [amiloride sensitive (as) cells] but not in others [amiloride insensitive (ai) cells]. ai cells responded to other electrolytes such as kcl and hcl. these results suggest the existence of at least two types of nacl sensitive cells, as and ai cells. n-or e-type fiber may selectively innervate as or ai cells respectively. research funds: kakenhi (15209061), kakenhi (17791325) ps3p-g084 integration of olfactory and oral sensory input in the rat insular cortex hideki kashiwadani, kensaku mori department of physiology, university of tokyo, tokyo, japan axonal connections between olfactory cortex and insular cortex suggest that insular cortex integrates olfactory information and information originated from the oral cavity (taste, tactile, temperature). however cellular mechanisms underlying the integration of multimodality are poorly understood yet. in this study, we examined single-unit spike responses of insular cortical neurons to odor stimulation and intraoral water stimulation in urethane-anesthetized rat. we found that more than 25% of recorded neurons in the insular cortex responded to odors. about half of the odor-responsive neurons were activated by intraoral water stimulation, indicating the convergence of olfactory and oral sensory information onto individual neurons in the insular cortex. when odor stimulation and intraoral water stimulation were simultaneously applied, some neurons showed spike responses larger than the responses evoked by each stimulus. the integration of olfactory and oral sensory information in the insular cortex might contribute to form the flavor sensation. research funds: kakenhi (17023012) ps3p-g085 odor combination selectivity of the rat piriform cortex neurons ikue yoshida, kensaku mori dept. physiol. grad. sch. med., univ. of tokyo, tokyo, japan olfactory cortex is thought to integrate signals from different odorant receptors to form the olfactory image of objects. however, the manner of integration at the level of individual cortical neurons is not well understood yet. using single-unit recording method, we examined the response selectivity of individual neurons in a dorsocaudal part of the anterior piriform cortex (apc) to 8 classes of odorous compounds, each class being present in odors from many different vegetables and fruits. individual neurons typically responded to more than 2 classes of odorants. each neuron was uniquely tuned to a specific combination of odorant classes, and different neurons typically showed different odor combination selectivity. single-unit responses to odor mixtures showed mixture facilitation and mixture suppression. these results suggest that individual neurons in the apc can be characterized by the odor combination selectivity and that the apc neurons may integrate signals from different odorant classes. research funds: kakenhi (13gs0007) ps3p-g086 odor-driven activity in the anterior piriform cortex of an in vitro isolated whole brain with the olfactory epithelium takahiro ishikawa 1 , takaaki sato 2 , akira shimizu 1 , ken-ichiro tsutsui 1 , toshio iijima 1 1 div. of systems neuroscience, grad. sch. of life sciences, univ. of tohoku, sendai, japan; 2 res. inst. for cell engineering, aist, amagasaki, japan to examine the neural mechanisms underlying odor-induced response in the anterior piriform cortex (apc), we analyzed odorinduced local field potential (lfp) and multiunit activity in an in vitro preparation, isolated guinea-pig whole brain with the olfactory epithelium. in apc, odor-induced lfps consisted of a phasic initial component followed by a fast oscillatory activity in the beta range (20 hz). by comparison a result of current source-density analysis with unit activity data, we confirmed that the initial component of odor-induced response has a characteristic temporal pattern, generated by a relatively weak direct afferent input, followed by an intracortical associative response, which was associated with a phasic inhibition. the beta oscillation might be generated by the repetition of these network activities. these electrophysiological data were consistent with the results of previous studies that used slice or anesthetized in vivo preparations. ps3p-g087 chemotaxis of c. elegans to concentration gradient of an attractant superimposed on a uniformly distributed attractant lin lin, hiroyuki oikawa, miyako sasaki, tokumitsu wakabayashi, ryuzo shingai department of welfare engineering, iwate university, morioka, japan to investigate the informational interaction between pathways from different sensory inputs to the behavior in the nervous system of c. elegans, chemotaxis toward the concentration gradient of an attractant spotted on a uniformly distributed another attractant was investigated. lysine and chloride ions are water soluble chemoattractants. when 3 m lysine was spotted on ammonium chloride background, 0.003-0.01 m and 0.05 m background did not influence lysine chemotaxis, while 0.03 m background augmented and 0.07-0.1 m background suppressed the chemotaxis. in contrast, when 0.1 m ammonium chloride was spotted on the lysine background, the background did not alter or suppressed the chemotaxis. interaction between informational pathways from different sensory inputs could be seen also in the presentation of an odorant spotted on chemoattractant background, and vice versa. ps3p-g088 glutamate receptors are regulated by the ras-mapk pathway in neural circuit-dependent odor adaptation in c. elegans takaaki hirotsu 1,2,3 , takeshi ishihara 1 , eisuke nishida 3 , yuichi iino 2 1 dept. biol., fac. sci., kyushu univ., japan; 2 mol. genet. res. lab., univ. of tokyo, japan; 3 grad. sch. biostudies., kyoto univ., japan c. elegans shows a decrease in chemotaxis to odorants after exposure to the odorant for 5 min. this plasticity, called early adaptation, requires aiy interneurons, which receive synaptic inputs from olfactory neurons, indicating that early adaptation depends on neural circuit. the ras-mapk pathway is activated by odorant exposure in aiy and plays essential roles for early adaptation. the function of glr-1, a non-nmda type glutamate receptor, in aiy is also important for early adaptation. glr-1 appears to localize at postsynaptic sites in aiy. this localization was changed by odorant exposure in early adaptation. mutation of the ras-mapk pathway impaired localization of glr-1. in vitro kinase analyses revealed the possibility that mapk directly phosphorylates glr-1. these results suggest that the ras-mapk pathway controls odor adaptation by directly regulating glr-1 localization in aiy neurons. kohei ueno 1 , yoshiaki kidokoro 2 1 dept. behav. sci., grad. sch. med., gunma univ., maebashi, japan; 2 inst. mol. cel. reg., gunma univ., maebashi, japan sodium chloride (nacl) is the major substance that induces nacl taste. in rodents, some strains prefer nacl solutions (∼1%), but others do not or even avoid them. although it is reported that the difference is based on the genetic background, the molecular information involved in the difference is not known. in the 26th ns annual meeting, we have shown that nacl preference in several wild-type strains of drosophila melanogaster is variable and p-element insertion in a single gene suppressed nacl preference. here, we carried out the sequencing analysis and found eight single-nucleotide polymorphisms (snps) in the gene. moreover, we found that one of the snps was correlated with nacl preference among wild-type strains. we generated transgenic flies and rescued the low preference phenotype of p-element insertion strain using the gal4/uas system. finally, we examined the expression pattern of the gene and found the gene is expressed in taste organs. taken together, we suggest that the gene is a novel nacl receptor gene. ps3p-g090 spatial and temporal organization of odor representation by moth antennal lobe output neurons shigehiro namiki 1,2 1 graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan; 2 department of mechano-informatics, graduate school of information science and technology, university of tokyo, tokyo, japan the antennal lobe (al) is the first relay station for olfactory information in the insect brain and is the anatomical equivalent of the mammalian olfactory bulb. both systems have common structures called glomeruli, functional units of olfactory processing. odor-evoked spatial and temporal patterns by an array of glomeruli are both important in olfactory coding. but the details of olfactory coding mechanisms are still unclear. we confirmed that projection neurons (pns, al output cells) innervating the same glomerulus had similar olfactory responses in the silkmoth. by pooling data from many pns that innervate identified glomeruli i reconstructed odor representations. i found that olfactory information is encoded by distributed spatiotemporal activity of a pn population and that there are no clear correlation between the similarity of slow temporal patterns of pns and spatial distances of innervating glomeruli. research funds: brain ps3p-g091 medial nucleus amygdala neurons have morphologically and electrophysiologically heterogeneous properties makoto yokosuka 1 , yoshinori sahara 2 , shinichiro horie 2 , masumi ichikawa 3 , shun nakamura 2 1 st. marianna univ. schl. med., kawasaki, japan; 2 ntl. inst. neurosci., ncnp, tokyo, japan; 3 tokyo metropol. inst. neurosci., tokyo, japan we characterize the electrophysiological and morphological properties of the medial nucleus amygdala (mea) neurons using whole-cell recordings in mice slice preparations. most mea neurons showed either tonic-bursting or adapting burst of action potentials to deporalizing currents. biocytin labeling showed that mea neurons possessed bipolar to multipolar cell bodies and dendritic fields covering projection areas from the accessory olfactory bulb. norepinephrine increased the frequency of spontaneous ipscs in some neurons, while serotonin increased spontaneous epscs in others. morphologically and physiologically heterogeneous mea neurons seem likely to produce multiplex outputs of many instinct behaviors. hideyuki matsumoto, kensaku mori department of physiology, graduate school of medicine, university of tokyo, tokyo, japan olfactory sensation sometimes lasts even after odorant stimulation has ceased. neuronal mechanisms for the olfactory afterimage are not well understood yet. single unit recordings from mitral/tufted cells in the mouse olfactory bulb (ob) showed that some neurons continued to discharge for more than 10 s even after the cessation of odorant stimulation. the induction of the sustained spike discharge depended on the intensity of odorant stimulation, and showed an allor-none behavior. spike discharges during the sustained discharge mode phase-locked to the respiration cycle and the phase-locking pattern during the sustained discharge mode differed from that during odor stimulation. these results suggest that neuronal mechanism in the ob may be responsible for the induction of the post-stimulus sustained discharges. the respiratory-phase-locked sustained discharges were recorded from juxta-glomerular cells. this implies that neuronal interactions within the glomeruli are involved in the induction of the sustained spike activity of mitral/tufted cells. ps3p-g093 synaptic transmission shows state-dependent change in the urethane-anesthetized rat olfactory bulb yusuke tsuno, hideki kashiwadani, kensaku mori department of physiology, graduate school of medicine, the university of tokyo, tokyo, japan olfactory cortex (oc) shows a state-dependent sensory gating that is controlled under the modulatory inputs from the basal forebrain and brainstem. since the olfactory bulb (ob) receives the modulatory inputs heavily, neuronal activity in the ob might change in a state-dependent manner. in the present study, we demonstrate a clear state-dependent change in the magnitude of the transmission of granule-to-mitral dendrodendritic inhibitory synapses and olfactory cortex-to-granule excitatory synapses. transmission of granule-tomitral synapses and olfactory cortex-to-granule synapses was facilitated during slow-wave state and suppressed during fast-wave state. in addition, we observed synchronous slow oscillations (about 1 hz) in the granule cell layer of the ob, layer iii of the oc, and the occipital cortex. thus the ob shows state-dependent synaptic modulation and presumably receives top-down periodic signals from the cortex. research funds: kakenhi (17023012) ps3p-g094 rem sleep deprivation decreases na-k atpase phosphorylation gitanjali das, birendra n. mallick school of life sciences, jawaharlal nehru university, new delhi, india it has been hypothesized that "one of the functions of rem sleep is to maintain brain excitability" rem sleep deprivation increases noradrenaline in the brain that increases the na-k atpase activity causing increased brain excitability. however, the molecular mechanism of such increased na-k atpase activity was unknown; although it was known that dephosphorylated state is the active form of na-k atpase. rats were rem sleep deprived by flower-pot method; large platform and recovery from lost rem sleep were carried out as controls. at the end of experiment, brains were quickly removed by cervical dislocation and synaptosomes prepared, which were used for western blotting against phosphoserine and phosphothreonine antibodies as well as for na-k atpase activity. after rem sleep deprivation the activity increased, while the level of phosphorylated form of na-k atpase decreased in the same sample. this confirms our hypothesis that rem sleep deprivation induced increased activity is due to dephosphorylation of na-k atpase. research funds: icmr (govt. of india) and upoe (govt of india) takeshi fujii 1,2 , ken yoshikawa 2 , yuki takatori 1 , koichiro kawashima 2 1 dept. of pharmacol., fac. of pharmaceut sci., doshisha women's coll., japan; 2 dept. of pharmacol., kyoritsu univ. of pharmacy, japan stimulation of muscarinic (machr) and nicotinic (nachr) receptors with respective agonists induces ca 2+ signals in t cells. in the present study, using rna interference approach, we investigated roles of machr and nachr subtypes in ca 2+ signals in ccrf-cem (cem) cells, a human t cell line, as a model of t cells. cem cells express m 1 , m 3 , m 4 and m 5 machr subtypes, and ␣3, ␣5, ␣6, ␣7, ␣9, ␣10 and ␤4 nachr subunits. transfection of anti-m 3 , anti-m 5 and anti-␣7 small interfering rna (sirna) significantly down-regulated respective mrna expression, while no changes were observed in gene expression of other machr subtypes or nachr subunits. ca 2+ signals evoked by oxotremorine-m, a non-selective machr agonist, were reduced by anti-m 3 or anti-m 5 sirna. ca 2+ signals evoked by nicotine were reduced by anti-␣7 sirna. these findings indicate that m 3 , m 5 machr and ␣7 nachr subtypes play major roles in ca 2+ signals to acetylcholine in t cells, and suggest that these receptors are involved in regulation of immune function. research funds: kakenhi (16590060) ps3p-g101 is "seronegative" mg explained by autoantibodies to musk? kazuhiro shigemoto 1 , sachiho kubo 2 , seiji matsuda 3 , naoki maruyama 2 1 dept. of preventive medicine, ehime univ. schl. of med., ehime, japan; 2 dept. of mol. path., tokyo metro inst. for gerontology, tokyo, japan; 3 dept. of integrated basic medical science, ehime univ. schl. of med., ehime, japan muscle-specific kinase (musk) is critical for the synaptic clustering of nicotinic acetylcholine receptors (achr). musk is activated by agrin, which is released from motoneurons, and induces achr clustering at the postsynaptic membrane. although autoantibodies against the ectodomain of musk have been found in a proportion of patients with generalized myasthenia gravis (mg), it is unclear whether musk autoantibodies are the causative agent of generalized mg. in the present study, rabbits immunized with musk ectodomain protein manifested mg-like muscle weakness with a reduction of achr clustering at the nmj. the autoantibodies activated musk and blocked achr clustering induced by agrin or by mediators that do not activate musk. thus, musk autoantibodies rigorously inhibit achr clustering mediated by multiple pathways, an outcome that broadens our general comprehension of the pathogenesis of mg. (shigemoto et al., j. clinical investigation, 2006) research funds: kakenhi (16590831) ps3p-g102 dynamic changes in the thalamo-cortical system associated with thalamic neurodegeneration shin-ichi kyuhou, hisae gemba department of physiology, kansai medical university, japan in purkinje cell degeneration (pcd) mice, degenerating thalamic neurons were found morphologically in the particular thalamic nuclei including the ventral medial geniculate nucleus around postnatal day 50. electrophysiologically, auditory evoked potentials in the primary auditory cortex began to decrease gradually in amplitude from postnatal day 45. analysis of spontaneous cortical field potentials by fast fourier transform, revealed that high frequency oscillation (hfo) of around 25 hz appeared prominently in the auditory cortex. local injection of kynurenic acid, a glutamate receptor blocker, into the thalamus suppressed the hfo in the auditory cortex, indicating that the thalamus is involved in the generation of the hfo. the real time polymerase chain reaction analysis demonstrated the upregulation of the mrna of nmda receptors in the auditory cortex. these results suggested dynamic changes occurred in the thalamo-cortical system after thalamic neurodegeneration in pcd mice. research funds: grant c1 from kansai medical university ps3p-h103 unusually folded sod1 species sequester specific motor molecules and inhibit the axonal transport of their cargos minako tateno 1 , yumiko simazaki 1 , fuminori saitoh 1 , ryosuke takahashi 2 , toshiyuki araki 1 1 national institute of neuroscience (ncnp), tokyo, japan; 2 dept. of neurology, kyoto university, kyoto, japan misfolding of mutant sod1 protein is thought to be responsible for the selective loss of motoneurons in sod1-related familial amyotrophic lateral sclerosis (als), although the molecular mechanisms underlying the toxicity of such unusually folded sod1 species are not yet clarified. since we have detected accumulation of unusual sod1 species in motoneuronal axons from g93a sod1-tg mice, we fractionated the ventral white matter of spinal cords to isolate the unusual sod1 species. immunoprecipitation analyses revealed specific interaction of unusual sod1 species with certain kinds of motor molecules. moreover, the axonal transport of cargos mediated by those molecules was found to be significantly reduced in symptomatic mutant sod1-tg compared with wt sod1-tg mice. these data strongly suggest that the toxic property of unusual sod1 proteins is partially ascribable to the transport inhibition of specific cargos. research funds: grant-in-aid for scientific research c (17590907) ps3p-h104 relationship between the amount of the cathepsin d expression and the symptomatic manifestation of neuronal ceroid-lipofuscinosis in a mouse model masahiro shibata, masato koike, yasuo uchiyama department of cell biology and neuroscience, osaka university graduate school of medicine, japan mice deficient in cathepsin d (cd), a representative lysosomal aspartic proteinase, have been shown to be an excellent model of neuronal ceroid-lipofuscinosis (ncl). here we report that the phenotype of mice in which cd is partially expressed is decided depending on the amount of the protein expression of cd. the proteolytic activity and protein expression of cd in the mutant mice were approximately 30% of those in the wild-type mice, while the growth of the mice appeared intact until postnatal day 24. the mice started to show ncl symptoms on p25, and their life span was prolonged for one to three days, compared to that of the cd-null mice. the protein expression of cd in the heterozygous mice was approximately half of that in the wildtype mice and the mice showed no pathological finding. these results indicate that a threshold of the cd expression required for the manifestation of ncl symptoms in the mice may be present in the range from 30% to 50% of that in the wild-type mice. research funds: kakenhi (10343253) ps3p-h105 neuronal toxicity of expanded polyglutamine depends on intracellular distribution among cells with similar expression levels mamoru satoh, atsuyoshi shimada, noriko kawamura, yoichi chiba, yuko saitoh, hiromi keino, masanori hosokawa dept. pathol., inst. develop. res., aichi human service center, aichi, japan we previously reported that expanded polyglutamine (polyq) tracts induced cellular toxicity of neuro2a cells in the form of massive cytoplasmic aggregates but not of intranuclear inclusion. however, we did not rule out the possibility that such toxicity depends on the level of intracellular expression of polyq. in this report, we compared the toxicity of polyq among cells expressing polyq tracts with a variety of intracellular distribution but at similar expression levels. damages were most remarkable in cells with cytoplasmic massive aggregate in terms of shrunken cellular and nuclear sizes. cells with cytoplasmic homogeneous distribution, cytoplasmic punctate distribution and intranuclear inclusion of polyq tracts were relatively spared. these data suggest that the severity of cell damages depends on the type of intracellular distribution of polyq tracts in cells expressing polyq tracts at similar level. ayumi takamura 1 , katsumi higaki 1 , junichiro matsuda 2 , yoshiyuki suzuki 3 , eiji nanba 1 1 division of functional genomics, research center for bioscience and technology, tottori university, tottori, japan; 2 national institute of biomedical innovation, osaka, japan; 3 clinical research center, international university of health and welfare, tochigi, japan g m1 -gangliodisosis is an autosomal recessive lipid storage neurodegenerative disorder. due to a deficiency of lysosomal ␤-galactosidase, excessive lysosomal accumulation of gm1 is observed in patients and animal model brains. however pathogenesisi of this disease is still unclear. since gm1 is known to be a major sialoglycolipid constituent of plasma membrane (pm) in neuron, we examined the analysis of brain of mouse model. cerebellar granule cells from this mouse showed gm1 accumulation of lysosome and pm and the membrane fluidity was also reduced. gm1-bound phosphorylated trka was markedly decreased in cultured neuron and brain tissues. subsequent plc␥, known as a downstream signal of trka, was also impaired. these results suggest that dysfunction of neurotrophin signaling may cause the onset of neurodegeneration in g m1 -gangliosidosis. katsuya inoue 1,2 , katsuaki endo 1 , takamitsu fujikawa 1 , seijyun fukuda 2 , tatsuo nakamura 2 1 department of physical therapy, university of aino, osaka, japan; 2 institute for frontier medical science, kyoto university, kyoto, japan regeneration of spinal cord injury is an important thema in rehabilitation science as well as basic one. the experiment was designed to reveal the process after spinal cord injury by asphyxia. to establish the animal model of spinal cord injury produced by asphyxia, we used adult cats with aorta occulusion under deep pentabarbital anesthesia. twenty minutes after occulusion electrical reflex activity of spinal cord disappeared. after 40 min occulusion, irreversible functional changes were observed, long term depression of reflex activities and disorders of motorsensory function. we also traced time course of electrical and functional changes after 40 min occulusion. ps3p-h108 development of a rodent behavioral model to study the direct interactions of reward and learning adam weitemier, niall p. murphy riken brain science institute, japan cognitive and reward processes often occur simultaneously, and perhaps interdependently. learning is a necessary condition in many experimental models aimed at assessing the rewarding value of a given stimulus. conversely, reward is often used as an experimental tool to engage mnemonic processes in studies aimed at investigating learning and memory. recent studies have demonstrated shared neurobiology between memory and reward. a direct behavioral interaction between reward and memory has never been studied. cognitive impairments observed in psychiatric conditions of dysregulated reward, such as drug abuse and depression, make this issue important, particularly in light of ongoing efforts to investigate higher brain functions. we are developing a rodent behavioral model with which to directly assess the influence of reward processes on learning and memory. we will introduce our recent progress with this new model, including two variations of the procedure designed to study the influence of reward on memory acquisition and memory recall. tetsuya ando 1 , yuya kawanaka 1 , minoru saito 2 , hiroaki mochizuki 1 , ken honjo 1 , hirofumi toda 1,3 , toshifumi tomoda 3 , akira sawa 4 , katsuo furukubo-tokunaga 1 1 grad. school of life & envir. sci., univ. tsukuba, japan; 2 molecular physiol., tokyo metropolitan inst. neurosci. tokyo, japan; 3 beckman res. inst., city of hope. california, usa; 4 dept. of psych. & neurosci. johns hopkins univ. school of medicine. baltimore, usa the disrupted-in-schizophrenia-1 (disc1) gene, originally identified at the breakpoint of a chromosome (1;11) (q42.1; q14.3) translocation in a scottish schizophrenia pedigree, is a promising candidate gene for schizophrenia and affective disorder. however, cellular and molecular mechanisms underlying cognitive impairments are yet to be elucidated. to address disc1 functions in vivo, we expressed disc1 in drosophila and examined developmental and behavioral phenotypes. overexpression of disc1 resulted in marked suppression of olfactory associative learning in flies whereas it caused no symptoms of neural degeneration even in aged animals. we anticipate that the drosophila system will serve as a novel model system amenable to a variety of genetic manipulations for the study of schizophrenia. ps3p-h110 effect of hypothermia on discrepancy between memory learning ability and anatomical brain damages in rats with neonatal hypoxic ischemic encephalopathy yuji miyatake 1 , ayumi kamo 1 , kenji minato 1 , hitoshi haruna 1 , hiritsugu fukuda 2 , yuji murata 2 , takayoshi hosono 1 1 department of bomedical engineering, osaka electro-communication university, japan; 2 graduate school of medicine, osaka university, japan we investigated the effect of brain hypothermia on neonatal hypoxic ischemic encephalopathy (hie) in hie-model rats using olton t-maze and anatomy. the common carotid artery of 22 of 25 7-day-old rats was ligated and cut under anesthesia. after the operation the rats were put in a box containing 8% oxygen at 40 • c for 15 min. after the insult, 11 of the 22 rats were put in a box at 35 • c for 12 h (hypothermia, h-group). the other 11 rats were returned to their mother without hypothermia (normothermia, n-group). sham operations were performed on three rats (s-group). eight weeks after the operation, their learning and memory ability was assessed by olton t-maze, and no statistical difference was observed in either the working or reference memory in the three groups although the anatomical brain size in the n-group was significantly smaller than in the h-group and s-group. withdrawn ps3p-h112 tau hyperphosphorylation in ts1cje, a partial trisomy 16 mouse model for down syndrome ebrahim abdul 1 , a. shimohata 1 , w. yu 1 , m. yamaguchi 1 , m. murayama 3 , d. chui 3 , t. akagi 2 , t. takeuchi 1 , k. amano 1 , h.s. karthik 1 , t. hashikawa 2 , h. sago 4 , c.j. epstein 5 , a. takashima 3 , k. yamakawa 1 1 research scientist; 2 lab. for neural arch.; 3 lab. for alzheimers disease; 4 div. of fetal med. ncchd; 5 ucsf, usa although down syndrome (ds) or trisomy 21 is the most common genetic cause of mental retardation, its neuropathology remains unclear. ts1cje, a ds mouse model partially trisomic for chromosome 16, shows learning and behavioral abnormalities mimicking ds mental retardation. the trisomic segment, corresponding to parts of human chromosome 21q22, has about 97 genes. importantly, sod1 and app, which may contribute to the ds phenotype, are excluded from the ts1cje trisomic segment. here we report that ts1cje brains show hyperphosphorylation of tau in the absence of nft formation, as well as increased gsk3␤ and jnk/sapk activities without alterations in a␤pp metabolism. our results suggest that genes on the trisomic ts1cje segment other than app and sod1 can cause hyperphosphorylation of tau, which in turn may be critical in the pathogenesis of ds mental retardation. research funds: kakenhi number: 17790737 ps3p-h113 increased oxidative stress and mitochondrial dysfunction in ts1cje, a down syndrome mouse model atsushi shimohata 1 , ebrahim a. s. 1 , m. yamaguchi 1 , w. yu 1 , h. sago 2 , c.j. epstein 3 , k. yamakawa 1 1 lab. for neurogenetics, riken-bsi, japan; 2 div. of fetal med. ncchd, japan; 3 dept. pediatrics, ucsf, usa down's syndrome (ds), caused by chromosome 21(hsa21) trisomy, is the most common genetic cause of mental retardation and affects every major organ in the body. ts1cje is one of a number of segmentally trisomic ds mouse models, and is triplicated for a region of mouse chromosome 16 extending from sod1 to znf295, containing 97 genes syntenic with hsa21. since these mice show learning and behavioral abnormalities mimicking ds mental retardation, ts1cjespecific trisomic segment genes may be involved in the ds phenotype. in the present study, we observed increased levels of reactive oxygen species (ros), mitochondrial function impairment in primary cultured astrocytes and hippocampal neurons, and increased cabonylated proteins in ts1cje brains. collectively, our results implicate dosage imbalanced genes other than sod1 and app in both ros generation and mitochondrial dysfunction, which in turn possibly contribute to the ts1cje ds mental retardation-like phenotype. ps3p-h114 polyinosinic-polycytidylic acid injection in early pregnancy causes the hypomyelination in the hippocampus, but not in the cortex manabu makinodan 1,2 , kouko tatsumi 2 , takayuki manabe 2 , takahira yamauchi 1,2 , eri makinodan 2 , juro shimoda 1 , toshifumi kishimoto 1 , akio wanaka 2 1 department of psychiatry, nara medical university, kashihara, japan; 2 department of 2nd anatomy, nara medical university, kashihara, japan polyinosinic-polycytidylic acid (poly i:c) elicits maternal immune response similar to anti-viral ones. recent studies demonstrated that poly i:c injection into pregnant mice resulted in behavioral changes including deficits in prepulse inhibition in the offspring, rendering this system an animal model of schizophrenia. in the present study, we observed such behavioral abnormalities reproducibly in the experimental group born from poly i:c-injected mice, but not in the control group born from pbs-injected mice. they showed decreased myelination in the hippocampus at juvenile period with unaltered number of oligodendrocytes. on the other hand, myelination in the cerebral cortex did not significantly differ between the experimental and control mice. the hypomyelinaton in the hippocampus at the juvenile period may be a possible cause for the behavioral changes in later periods. joanna doumanis, ritsuko kazama, adrian moore, nobuyuki nukina riken brain science institute, japan the fruitfly drosophila melanogaster is well established as a model system in the study of human neurodegenerative diseases. to model the polyglutamine expansion disease, huntington disease (hd), we have established stable, inducible cell lines expressing n-terminal truncated huntingtin fused to egfp with an expanded (62q) polyglutamine repeat in a drosophila larval central nervous system-derived cell line. induction of expression results in the formation of protein aggregates, characteristic of hd. utilising rnai, we have carried out a high-throughput screen for modifiers of aggregate formation in these cells. 7200 genes, encompassing around 50% of the drosophila genome, were screened, resulting in the identification of 370 candidates that either suppress or enhance aggregation. most candidates identified have mammalian orthologues, validating the use of drosophila to screen for genes relevant to human disease. we established in vivo models of hd by expressing polyq-egfp in the drosophila nervous system and are further characterising selected candidates in our model. the rodent model of harmaline-induced tremor has been used as an animal model of essential tremor. the present study investigated effects of harmaline on olivocerebellar systems of mice and rats. systemic administration of harmaline produced generalized tremors in both types of rodents. immunohistochemical studies revealed significant degeneration of purkinje cells that was associated with activated microgliosis in the cerebellar cortex, following administration of harmaline in rats but not in mice. however, in mice but not rats, microgliosis was induced following administration of harmaline in the inferior olivary nucleus (ion). numbers of neurons in the mouse ion did not decrease, suggesting the possibility that microgliosis in ion might not be a simple neurotoxic effect. presumably, differences in sensitivity of purkinje cells between rats and mice may be related to differences in functional alterations in their respective olivocerebellar systems induced by harmaline. recognition of these species-specific differences is an important consideration for experimental analysis of the rodent model of tremors. ps3p-h117 analysis of ␣-synuclein expression in young mouse model of multiple system atrophy kimiko nakayama, yasuyo suzuki, ikuru yazawa laboratory of research resources, national institute for longevity sciences, aichi, japan multiple system atrophy (msa) is a sporadic neurodegenerative disease that affects oligodendrocytes and neurons in human central nervous system. glial cytoplasmic inclusions (gcis) are diagnostics of msa. gcis are shown to be abnormal accumulation of filamentous ␣-synuclein. yazawa et al. (2005) generated a transgenic (tg) mice overexpressing human wild-type ␣-synuclein in oligodendrocytes under the control of the 2 , 3 ,-cyclic nucleotide 3 -phosphodiesterase (cnp) promoter. tg mouse study demonstrated that formation of gci-like ␣-synuclein inclusions in the oligodendrocyte leads directly to neuronal degeneration, as shown by motor impairment and novel accumulation of mouse ␣-synuclein in neuron. to elucidate the mechanisms of neurodegeneration in tg mice, we prepared primary cultures of neurons and glial cells from tg mice. the cells are examined the effects of ␣-synuclein accumulation. ps3p-h118 dysregulation of sodium channel ␤4 subunit by expanded polyglutamine in huntington disease transgenic mice fumitaka oyama, haruko miyazaki, kazumasa okamura, yoko machida, kurosawa masaru, takashi sakurai, nobuyuki nukina laboratory for structural neuropathology, riken bsi, wako-shi, japan sodium channel ␤4 (␤4) is a very recently identified auxiliary subunit of the voltage gated-sodium channels. we have identified ␤4 as an est that was significantly downregulated in the striatum of hd model mice and found that reduction in ␤4 started at a presymptomatic stage of the hd model mice. in contrast, spinal cord neurons, which generate only negligible levels of expanded polyq aggregates, maintained normal levels of ␤4 expression even at the symptomatic stage. expanded polyq with nls expression suppressed the promoter activity of ␤4 gene in pc12 cells. forskolin, an activator of the camp/pka pathway, did not affect b4 promoter activity, indicating that ␤4 is not camp-responsive gene. these findings strongly suggest that sodium channel ␤4 subunit is a novel molecule, which is an upstream non-camp-responsive gene in hd pathogenesis. ps3p-h119 repeat length-and age-dependent changes in behavioral phenotypes of drpla transgenic mice harboring a single copy of a full-length human drpla gene kazushi suzuki 1 , yuji takahashi 1 , jun goto 1 , mutsuo oyake 2 , toshiya sato 3 , shoji tsuji 1 1 department of neurology, the university of tokyo, tokyo, japan; 2 department of neurology, brain research institute, niigata university, niigata, japan; 3 center for bioresource-based research, brain research institute, niigata university, niigata, japan we carried out detailed analyses of the behavioral phenotypes of drpla transgenic mice carrying an expanded cag repeat of 76 (q76), 96 (q96), 113 (q113), or 129 (q129). in the accelerating rotarod (9w), the latencies of q76, q96, q113 and q129 were 113%, 81%, 34% and 24%, respectively. in the open field, moving distances of q96, q113, and q129 were decreased to 98%, 50%, and 20%, respectively, while that of q76 was increased to 134%. home cage activity was decreased depending on the repeat length. the q113 mice, however, showed increased ratios of the activity during the light time to that during the total day at 8 weeks (115%) and 24 weeks (208%), suggesting that drpla mice display not only impaired motor coordination, but also changes in emotional behavior, and disrupted night and day activity patterns. ps3p-h120 the mice lacking schnurri-2 show multiple behavioral abnormalities related to psychiatric disorders keizo takao 1 , nobuyuki yamasaki 1 , keiko toyama 1 , tsuyoshi takagi 2 , shunsuke ishii 2 , tsuyoshi miyakawa 1 1 hmro, kyoto university graduate school of medicine, kyoto, japan; 2 riken, tsukuba, japan schnurri-2 (shn-2) is a zinc finger transcription factor, a mouse homologue of human hiv-ep2, that binds to nuclear factor kappa b-binding site in the hiv long terminal repeat. shn-2 is known to play important roles in the mammalian immune systems. however, the role of shn-2 in the central nervous system (cns) is still unknown. to investigate the functional significance of shn-2 in mammalian brain, we analyzed the shn-2 knockout (ko) mice using a comprehensive behavioral test battery. shn-2 ko mice were dramatically hyperactive under novel environment and in their home cage. they also showed increased acoustic startle response and impaired prepulse inhibition, indicating their impairment in sensorimotor gating. anxiety-like behavior and depression-like behavior were also significantly reduced in shn-2 mice. our results demonstrate a critical role of shn-2 in cns and suggest that shn-2 ko mice may serve as an animal model of psychiatric disorders. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird ps3p-h121 comprehensive brain-behavior phenotyping of camkii␣ heterozygous knockout mice nobuyuki yamasaki, koichi tanda, keiko toyama, yasuyuki fukui, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan ca 2+ /calmodulin-dependent protein kinase ii (camkii) is a ubiquitous serine/threonine protein kinase that is abundant in brain as a major constituent of the postsynaptic density and critically involved in synaptic plasticity, learning and memory. several behavioral abnormalities of camkii␣ mutant mice were reported, but systematic assessments of behaviors of camkii␣ mutant mice have not been conducted. to analyze the behavioral effects of camkii␣ deficiency, we subjected camkii␣ heterozygous knockout mice to a comprehensive behavioral test battery. the mutant mice showed hyperactivity, decreased anxiety, decreased depression-related behavior, increased offensiveness, selective spatial working memory deficit, and dramatic periodic change of locomotor activity in home cage. to identify the mechanism underlying these behavioral abnormalities, gene expression analysis was conducted. the potential involvement of camkii␣ in pathogenesis/pathophysiology of psychiatric disorders will be discussed. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird ps3p-h122 effects of various factors on the results of a comprehensive behavioral test battery for genetically engineered mice: a factor analytic study hiroshi ougino, nobuyuki yamasaki, koichi tanda, keiko toyama, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan we have been using a behavioral test battery to reveal unknown phenotypes of genetically engineered mice. for the adequate experimental design and interpretation of data, it is essential to know experimental variables which may potentially influence results, and various kinds of factors which underlie many indices measured in the tests. in this study, we investigated the effects of background strains (c57bl/6j, c57bl/6n, c57bl/6c, 129svev, balb/c), body weight, age at test, and start time of test on the results of each test, by analyzing data of more than 1200 mice (, including wild type and mutant mice from 25 strains of genetically engineered mice), which had been tested in our laboratory. also, we conducted factor analyses of a large set of data to examine the relationship between behavioral indices. the potential implications of our findings for the improvement of the behavioral test battery will be discussed. calcium-and calmodulin-dependent protein kinase iv (camkiv) is a protein kinase that activates the transcription factor, camp responseelement binding protein (creb). camkiv has been hypothesized to play a significant role in synaptic plasticity and in learning and memory. however, functions of camkiv in a variety of behaviors, e.g., motor function, nociception, fear, anxiety, depression, learning and so on, have not yet been fully elucidated. to gain more insight into behavioral significance of camkiv, we subjected camkiv−/− mice to a battery of behavioral tests. camkiv−/− mice did not display any deficit in spatial reference memory and working memory tests, but had mild performance deficit in fear conditioning tests. these results indicated selective and specific involvement of camkiv in regulating emotional behavior. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird ps3p-h124 comprehensive behaivoral analysis of ryanodine receptor type3 knockout mouse suzuko ohsako 1 , koichi tanda 1,2 , nobuyuki yamasaki 1 , keiko toyama 1 , hiroshi takeshima 3 , tsuyoshi miyakawa 1 1 kyoto university graduate school of medicine, kyoto, japan; 2 dep. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan; 3 dep. of biochem. and mol biol., tohoku univ. graduate school of medicine, miyagi, japan ca 2+ signaling is essential for the regulation of neuronal processes including synaptic transmission and transmitter release. ryanodine receptors (ryrs) are family of intracellular calcium channels and mediate calcium-induced calcium release from the endoplasmic reticulum. ryr3 is highly expressed in the hippocampus, caudate putamen, and thalamus. to investigate the behavioral effects of ryr3 deficiency, we subjected ryr3 knockoout mice to a battery of behavioral tests. ryr3 knockout mice exhibited hyperactivity and abnormal behavior in social interaction test, while they did not show any deficit in motor function, depression, attention, and working memory tests. these results suggest a role of ryr3 in regulating general locomotor activity and social behavior. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird ps3p-h125 comprehensive behavioral analysis of neuronal nitric oxide synthase knockout mouse keiko toyama 1 , koichi tanda 1,2 , nobuyuki yamasaki 1 , tsuyoshi miyakawa 1 1 hmro, kyoto university graduate school of medicine, kyoto, japan; 2 dept. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan nitric oxide (no) plays several important roles in the brain, including in regulation of synaptic signaling and plasticity. no is synthesized from the amino acid l-arginine by the enzyme nitric oxide synthase (nos). in neurons, no is produced by neuronal nitric oxide synthase (nnos), representing one of three nos isoforms expressed in most tissues. to elucidate function of nnos/no in a variety of behaviors, e.g., activity, motor function, nociception, attention, anxiety, depression, social interaction, learning and so on, we subjected nnos knockout mice to a battery of behavioral tests. nnos knockout mice exhibited increased locomotor activity and decreased depressionrelated behavior. furthermore, they displayed increased social contacts in novel environment and homecage. these results indicate that nnos/no is involved in regulation of their behaviors. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird ps3p-h126 primate model of attention-deficit/hyperactivity-disorders (adhd) shintaro funahashi 1 , keiko shimizu 2 1 grad. sch. human and environmental std, kyoto univ., kyoto, japan; 2 primate res. inst., kyoto univ., inuyama, japan adhd is one of the prevalent childhood psychiatric disorders. children with adhd show hyperactive behavior and attention problems, suggesting prefrontal (pfc) contribution to adhd. adhd is also known as dopamine (da) related dysfunctions, because methylphenidate is the most effective drug for the treatment of adhd. pfc is the cortical area where the strongest da innervation is observed. injection of da-related drugs to pfc produces behavioral deficits in cognitive tasks. these suggest that da-related dysfunction in pfc could be a candidate of biological causes of adhd. to prove this notion, we injected 6-ohda into bilateral pfc to destroy da innervation in infant monkeys and examined whether these monkeys exhibited hyperactivity. 6-ohda injected monkeys showed significant increase of spontaneous activity in test cages. oral administration of methylphenidate reduced spontaneous activity in 6-ohda injected monkeys. these results suggest that monkeys injected 6-ohda into pfc are good candidates of the primate model of adhd. research funds: kakenhi (17021022) ps3p-i127 training-induced recovery of precision grip after primary motor cortex damage in the adult monkey yumi murata 1,2,3 , noriyuki higo 1,3 , takao oishi 1,3,4 , akiko yamashita 5 , keiji matsuda 1 , motoharu hayashi 4 1 neurosci. res. inst, aist, tsukuba, japan; 2 grad. sch. compreh. hum. sci., univ. of tsukuba, tsukuba, japan; 3 crest, jst, kawaguchi, japan; 4 dept. cell mol. biol., primate res. inst., kyoto univ., inuyama, japan; 5 div. applied sys. neurosci., nihon univ. sch. med., tokyo, japan in the present study, we compared the motor recovery between monkeys that received daily training and that did not receive any training after lesion of the primary motor cortex (mi), in order to investigate the effects of postlesion training on motor recovery. we derived a hand representation map in mi, and ibotenic acid was then injected to destroy the digit region, which resulted in hand paralysis. after one or two months of postlesion training, skilled use of the affected hand including a precision grip was recovered. untrained monkeys also became able to grasp objects with their affected hand, but they couldn't use a precision grip. this suggests that recovery of precision grip requires postlesion training. research funds: a grant-in-aid for scientific research on priority areas from mext (17021055) mouse mutants with behavioral abnormality are indispensable tools to elucidate molecular pathways underlying behavior. in order to develop numbers of novel behavioral mutants, we have been carrying out dominant behavioral screening in potential mouse mutants that was randomly induced point mutations by a chemical mutagen enu (n-ethyl-n-nitrosourea). we screened about 2,500 g1 animals (dba/2j × enu-treated c57bl/6j) for home-cage activity, open-field activity, and passive avoidance response, and obtained 13 lines of dominant behavioral mutants. by linkage analysis, the causative genes were mapped in 5 of 13 mutant lines. hyperactivity was predominant phenotype, and 7 of 13 mutants showed hyperactivity in home-cage and/or open-field. we will report the recent results of initial characterization and the progress of fine mapping in these enuinduced mutants. ps3p-i129 ubiquitin signal in neurons of cathepsin ddeficient mouse brains with special reference to the autophagic process masato koike, masahiro shibata, yasuo uchiyama dept. of cell biol. and neurosci., osaka univ. grad. sch. of med., suita, japan we have shown that autophagy contributes to the accumulation of vacuolar structures in neurons obtained from cd−/− and cb−/−cl−/− mice, murine models for neuronal ceroid lipofuscinoses (ncls) (koike et al., 2005) . until recently, it remains unknown what signaling is essential for autophagosome formation. interestingly, in the conditional atg7-knock-out mice where autophagy is absent specifically in the liver, numerous ubiquitinated aggregates are detected in the cytosol of hepatocytes (komatsu et al., 2005) , suggesting that protein ubiquitination may serve as a signal to the autophagic process. we therefore examined the immunohisto/cytochemical localization of ubiquitin and lc3, and found that in our ncl model mice, positive signals for ubiquitin and lc3 were co-localized on the membranes of granular structures in the neuronal perikarya. these results suggest that protein ubiquitination may be involved in signaling for autophagosome formation in ncls. research funds: grant-in-aid for young scientists (b)(17790141) and creative scientific research (16gs0315) ps3p-i130 activation of medial prefrontal cortex neurons by systemic phencyclidine is primarily mediated via ampa/kainate glutamate receptors tadahiro katayama 1 , eiichi jodo 1 , yoshiaki suzuki 2 , ken-yo hoshino 2 , yukihiko kayama 1 1 dept. of physiology, fukushima medical university, fukushima, japan; 2 dept. of neuropsychiatry, fukushima medical university, fukushima, japan it has been shown that tonic activation of the medial prefrontal cortex (mpfc) plays a pivotal role in development of behavioral abnormalities induced by systemic phencyclidine (pcp). however, receptors mediating such activation are not clearly specified, though several studies indicate the increase of extracellular acetylcholine, dopamine, and glutamate in the mpfc. here, we examined effects of local application of those antagonists on increased firing activity of mpfc neurons by systemic pcp in anesthetized rats. after tonic activation of mpfc neurons by pcp had been established, cnqx, sch23390, mecamylamine or scopolamine was locally applied with iontophoresis or gas pressure on the recorded neuron. cnqx reduced pcp-induced augmentation of firing activity to the baseline level, while others gave little changes. these results suggest that pcpinduced activation of mpfc neurons be mediated primarily via ampa/kainate receptors. ps3p-i131 increased depressiveness and decreased sensitivity to antidepressants in calcium/calmodulin-dependent protein kinase iv (camkiv)-knockout mice jiro kasahara 1 , hiroyuki sakagami 2 , hisatake kondoh 2 , kohji fukunaga 1 1 department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan; 2 department of histology, graduate school of medicine, tohoku university, sendai, japan calcium/calmodulin-dependent protein kinase iv (camkiv) is expressed abundantly in the nuclei of neurons and thought to regulate ca-dependent gene expressions mediated by the transcriptional factors such as creb. recently, we found that chronic treatments of the rats with antidepressants increased camkiv activity and creb phosphorylation in the prefrontal cortex, suggesting the importance of camkiv in the effects of antidepressants. this result led us to perform the behavioral assessments of depressiveness and the sensitivity to antidepressants in camkiv-knockout mice by some experimental paradigms. from the experiments, the increased depressiveness and decreased sensitivity to antidepressants were observed in the mice, suggesting the importance of camkiv for the regulation of depressiveness and the effects of antidepressants. ps3p-i132 severity of audiogenic seizures is influenced by multiple factors in vlgr1-mutated mice hideshi yagi 1,2 , makoto sato 1,2 1 division of cell biology and neuroscience, department of morphological and functional sciences, faculty of medical sciences, university of fukui, fukui, japan; 2 research and education program for life science, university of fukui, fukui, japan epilepsy is a highly prevailed disorder and reports are accumulating that demonstrate that single gene mutation causes such disorders. we made vlgr1-mutated mice and found that they showed high susceptibility to audiogenic seizure, one of the reflex seizures provoked by loud noise. to evaluate whether the genetic backgrounds influence on phenotype of the audiogenic seizure in our mice, we made c57bl/6 backcrossed vlgr1-mutated mice and 129/svs4 backcrossed vlgr1-mutated mice. these two backcrossed lines showed different susceptible periods and severity of audiogenic seizure from the original line. furthermore, phenotype of audiogenic seizure was altered by restraining mice from free moving while being exposed to loud noise. these observations suggest that genetic factors and environmental factors may modify the phenotype of seizures and our vlgr1-mutated mice are good model of reflex epilepsies that are evoked by multifactors. ps3p-i133 reduction in the density of parvalbumin-positive cells in the medial frontal cortex of rats behaviorally sensitized to methamphetamine tomoko kadota 1 , ken kadota 1,2 1 department of bioenvironmental medicine, university of chiba, chiba, japan; 2 chiba institute of psychiatry, chiba, japan our previous study demonstrated that the development of behavioral sensitization of rats to methamphetamine (map) corresponded in time with the progress of neurotoxic changes induced in the medial prefrontal cortex (mfc). the present study further examined morpholological changes of rats that were administered a daily dose of 5 mg/kg of map i.p. for 12 days (d 1 d 12) and then withdrawn from the drug for 7 28 days (wd 7 wd 28). the regimen reduced the densities of parvalbumin positive cells (pac); these were probably gabaergic cells and distributed in the strata covering layers ii, iii and v in the anterior cingulate cortex (cg 1) and mfc. the decrease in the density of pac was first observed in cg 1 and then in mfc. the reduction began on d 6 and advanced to higher levels on d 12 and subsequently wd 7. these findings suggest that the behavioral sensitization regimen leads to the deterioration of inhibitory processes in the neural circuits in cg 1 and mfc, particularly in layers ii and iii. ps3p-i134 up-regulation of ␤ 2 -adrenergic receptor immunoreactivity in astrocytes in the spinal cord after dorsal rhizotomy teruyoshi kondo, yoshihiro ishibashi, kei-ichiro nakamura department of anatomy, division of microscopic and developmental anatomy, kurume university school of medicine, kurume, japan stimulation of ␤ 2 -adrenergic receptor (␤ 2 -ar) induces astroglial proliferation and activation after brain injury, but little is known concerning the potential role of adrenergic receptors in the spinal cord. present study demonstrated that rhizotomy induced a marked and prolonged up-regulation of ␤ 2 -ar-immunoreactivity (ir) in the regions of the dorsal root entry zone and dorsal funiculus containing the central processes of the injured primary sensory neurons. ␤ 2 -arimmunoreactive cells coexpressed gfap-ir and were positive for nestin which is characteristic of reactive astrocytes. a population of ␤ 2 -ar-immunoreactive cells were labeled with ki-67, a marker of cell proliferation, indicating some of them went into cell mitotic state. interestingly, a major population of ␤ 2 -ar-immunoreactive cells also exhibited fgf-2-ir. these findings suggest that ␤ 2 -ar may play important roles in astrocytic activation and neuroprotection associated with induction of synthesis of growth factor such as fgf-2. ps3p-i135 effects of lateral fluid percussion injury (fpi) on the optical signals in dentate gyrus of the rat brain slice preparations shin yamashita 1 , norihiro muraoka 2 , hiroshi hasuo 1 , takashi akasu 1 , minoru shigemori 2 1 dept. of physiology, kurume univ. sch. of med., kurume, japan; 2 dept. of neurosurgery, kurume univ. sch. of med., kurume, japan we investigated the effects of experimental traumatic brain injury on the neuronal function in dentate gyrus (dg) using optical recording techniques with voltage-sensitive dye (rh482). horizontal hippocampal slices were obtained from the control and the fpi rats (one week after the single moderate impact). electrical stimulation of perforant path (pp) produced the optical signal spread in the molecular layer of dg. temporal change in the optical signal, obtained from an area on the propagation pathway, had two peaks (fast and slow peaks). increment of stimulus intensity (10-50 v) increased the amplitude of both fast and slow peaks. the intensity for producing the maximal response was 30-40 v. the amplitude of slow peak in fpi group was about 50% larger than that in control group, while the amplitudes of fast peak were not different in the two groups. these data suggest that the excitatory pp synapse onto granule cells of dg is facilitated after fpi. ps3p-i136 comparative study of neural activities in mouse hippocampal slices by flavoprotein autofluorescence and ca 2+ imaging chikafusa bessho, yasuharu mitsushima, ryo matsumoto department of physics, kyoto sangyo university, kyoto, japan recently k. shibuki et al. have succeeded in flavoprotein autofluorescence imaging of neural activities in the rat brain. we examined neural activities in mouse brain (hippocampal) slices by the modified method and ca 2+ imaging. the slices (300 m) were prepared from the block in an ice cold acsf medium using microslicer and incubated for 1 h in the oxygenated medium at room temperature. a slice was placed on a recording chamber perfused with the medium at a flow rate of 1 ml/min. green autofluorescence (>520 nm) of the slices illuminated by blue light (460-490 nm) was observed by an inverted microscope. images of the autofluorescence were recorded using a calcium imaging system. ca 2+ imaging was also performed in the slices. slices were incubated in acsf medium containing 10 m of fluo3/4 am for 1 h at 37 • c. the ca 2+ image was recorded with an excitation wavelength of 460-490 nmand an emission wave length of >520 nm. the autofluorescence and ca 2+ responses wereobserved in slices perfused with l-glutamate (10 mm). takuya hayashi 1 , hiroshi sato 1 , shinichi abe 2 , takashi hanakawa 2 , hiroshi watabe 1 , hidenao fukuyama 2 , babak aldekani 3 , hidehiro iida 1 1 department of investigative radiology, national cardiovascular center research institute, osaka, japan; 2 human brain research center, kyoto university, kyoto, japan; 3 nathan kline institute for psychiatric research, ny, usa we show connectivity pattern between cortex and striatum in macaque and human by using the non-invasive method of diffusionweighted magnetic resonance imaging (dwi). in macaque, the dwibased striatal connectivity of brodmann's area 9 corresponded to that revealed by the tracer (mncl 2 ) tractography. the dwi-based connectivity pattern also isolated a part of the ventral striatum corresponding to the histochemically-specific 'shell' region in both human and macaque. in addition, we confirmed the species-homology in intra-striatal topography of cortical connection by quantitatively analyzing the connectivity; however, we found that human striatum was more intensively connected to prefrontal cortex and less connected to extra-frontal cortices. these results suggest that human striatum has a dominant and specific role in processing prefrontal information. research funds: h17-kokoro-025 ps3p-i138 optical analysis of synaptic transmission by a fluorescent glutamate probe shigeyuki namiki, hirokazu sakamoto, sho iinuma, kenzo hirose department of cell physiology, nagoya university graduate school of medicine, nagoya, japan glutamate is an essential excitatory neurotransmitter in the central nervous systems. for optical analysis of glutamatergic synaptic transmission, we have developed a fluorescent glutamate probe called eos. by imaging with eos, we successfully detected the synaptically released glutamate following axon firings in cultured hippocampal neurons; the spatial distribution of the glutamate release was non-uniforml along dendrites. we also succeeded in monitoring the phorbol ester-induced potentiation of the glutamate release. furthermore, we found spontaneous and stochastic glutamate release which was confined to small regions. neither application of tetrodotoxin nor removal of extracellular calcium blocked the release. high concentrations of sucrose increased the frequency of the release. these features are reminiscent of those of miniature epsc in electrophysiological recordings and thus suggest that the spontaneous release is quantal vesicular release. in conclusion, our probe directly visualizes the presynaptic release. shingo miyata 1,2 , yasutake mori 1,2 , tsuya taneda 1,2 , hiroaki okuda 1,2 , masaya tohyama 1,2 1 department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan; 2 21st coe program, tokyo, japan local protein synthesis in neuronal dendrites is one of the mechanisms that may mediate a rapid and synapse-specific mobilization of proteins from the resident mrnas. a great deal of effort has been made in analyzing the dynamic state of protein synthesis in the living cells, chiefly by quantifying protein level. however, the protein level cannot mirror the spatio-temporal alteration of translation, because it cannot be affected only by protein synthesis but also by other factors like degradation. therefore, it is problematic to visualize the dynamic state of translation by the present methods. to solve the problem, we applied fret (fluorescence resonance energy transfer) technique to in situ detection of the assembly and disassembly cycle among a pair of translation initiation factors (eifs), thereby showing that bdnf and ephrin could potentiate local protein synthesis in the dendrites of hippocampal neurons. ps3p-i140 a model selection of glm applied to fmri data using aic jobu watanabe 1 , fumikazu miwakeichi 2 , andreas galka 3,4 , ryuta kawashima 4 , tohru ozaki 3 , sunao uchida 1,5 1 institute for biomedical engineering, consolidated research institute for advanced science and medical care, waseda university, japan; 2 department of medical system engineering, faculty of engineering, chiba university, chiba, japan; 3 institute for statistical mathematics, tokyo, japan; 4 institute of experimental and applied physics, university of kiel, keil, germany; 5 new industry creation hatchery center, tohoku university, sendai, japan; 6 faculty of sport sciences, waseda university, tokorozawa, japan in the general linear model (glm) that is widely used in analyses of functional neuroimaging data, several combinations of explanatory variables are possible. the akaike information criterion was applied as a basis of comparison and selection among several glms that analyze block-designed functional magnetic resonance imaging (fmri) data. the glms with/without a resting condition, head motion covariates, time derivatives and dispersion of hemodynamic response function were compared. we demonstrate that a combination of these explanatory variables can effectively improve the model and that aic is a useful tool for model selection in fmri studies. ryuzo shingai, katsunori hoshi, tokumitsu wakabayashi department of welfare engineering, iwateuniversity, morioka, japan to investigate the relationship between the behavior and function of the nervous system of caenorhabditis elegans, quantitative analysis of behavior that indirectly represents the internal states of the worm is necessary. we devised an automated analysis system of c. elegans locomotion. the system is well suited for detecting four locomotion states: forward or backward movement, curl and rest. the system was applied to a phenotype that when a worm is transferred from a seeded plate to a bacteria-free plate, the worm shows frequent backing and short duration of forward movement for 20-30 min and then a gradual increase in the duration of forward movement. accuracy of the state identification for wild type and several mutants was sufficiently high, indicating the system is robust in studies of locomotion. ps3p-j148 flavoprotein fluorescence responses elicited by thalamic stimulation in slices obtained from the mouse barrel cortex daiki kamatani, ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst., niigata univ., niigata 951-8585, japan we have reported that whisker trimming induced activity-dependent changes in the barrel cortex of rat cortical slices using flavoprotein fluorescence imaging. however, contribution of thalamo-cortical afferents in this plasticity was not clear, since specific stimulation of thalamo-cortical afferents was not possible in the coronal cortical slices obtained from rats. in the present study, we used the mouse cortical slices that kept thalamocortical connections to the barrel cortex intact. the cortical activities in layer iv were observed as fluorescence responses after thalamic stimulation. the magnitude of the fluorescence responses was increased as the amplitude of cortical field potentials was increased. these cortical responses were suppressed by antagonists of glutamate receptors such as cnqx and apv, and almost completely abolished in the presence of cnqx plus apv. in preliminary experiments, we confirmed that whisker trimming induced activity dependent changes in the barrel cortex of mice. ps3p-j149 effects of implicit emotional processes on encoding-related activations of episodic memory: an eventrelated fmri study yayoi shigemune 1,2 , takashi tsukiura 1 , hiroko mochizuki-kawai 1 , chisato suzuki 1,2 , toshio iijima 2 1 neurosci. res. inst., aist, tsukuba, japan; 2 div. systems neurosci., tohoku univ. grad. sch. life sci., sendai, japan in this study, we investigated the effects of implicit emotional processes on encoding-related activations of episodic memory using fmri. nineteen healthy right-handed male participated in this study. we prepared emotional pictures with three kinds of emotional valence (negative: nega, neutral: neu and positive: posi) and line drawings for encoding. in the fmri scanning, subjects memorized line drawings, which were presented after the emotional pictures. after the scanning, subjects were presented with the names of line drawings, and were required to judge whether or not line drawings with the names were learned. we found significant activations of the right anterior cingulate gyrus specifically in the nega condition, the right lingual gyrus in the neu condition and the right amygdala and pulvinar in the posi condition. these results suggest that encodingrelated activations of episodic memory may be modulated by the implicit primer with emotional valence. ps3p-j150 different neural correlates of stimulus-actiondependent and stimulus-dependent reward predictions revealed by fmri masahiko haruno 1 , kenji kansaku 2 , yu aramaki 2 , mitsuo kawato 1 1 atr cns, kyoto, japan; 2 institute of physiology, okozoki, japan efficient decision making requires multiple reward predictions in switching different contexts and learning. we conducted a human fmri experiment (n = 14) to examine stimulus-action-dependent and stimulus-dependent reward predictions. in condition a, each of two fractal figures specifies a monetary reward associated with a button push (left or right). if the button is pressed correctly, 50 or 10 yen is provided with a probability of 0.8, but only with 0.2 if pressed wrongly. the key difference in condition b is that a fractal determines the reward but not the action. subjects had learned the two conditions fully before scanning. at the fractal onset, the putamen, lateral ventral and medial dorsal prefrontal cortices showed stronger activity correlated with the predicted reward (p < 0.01) in a, while it was more prominent in the caudate, dorsolateral prefrontal cortex and cerebellum in b. the striatum also showed a similar difference correlated with the reward prediction error at reward feedback, suggesting the different neural substrates for different reward predictions. research funds: nict ps3p-j151 brain networks for communicative speech production: feeling inference and speech content production yuko sassa 1,2 , motoaki sugiura 3 , hyeonjeong jeong 1,4 , keisuke wakusawa 1,5 , kaoru horie 6 , shigeru sato 6 , ryuta kawashima 1 1 niche, tohoku university, sendai, japan; 2 ristex, jst, tokyo, japan; 3 miyagi university of education, sendai, japan; 4 gsics, tohoku university, sendai, japan; 5 department of pediatrics, tohoku university, sendai, japan; 6 the lbc research center, tohoku university, japan communicative speech production often accompany inference of the targetperson's feeling. in this fmri study, we segregated the brain networks forthe feeling inference and speech content production processes incommunicative speech production. during presentation of a picture showingan actor's utterance in a balloon, normal subjects covertly talked to theactor (speech), inferred feeling (feeling), or described the action (des). greater activation in the contrasts speech-feeling was observed in themedial prefrontal cortex, and that in the contrast feeling-des wasobserved in the right superior temporal sulcus extending to the temporalpole. the results suggest that these two regions play roles in the speechcontent production and feeling inference, respectively. research funds: the 21st coe program ps3p-j152 the construction of a brain-computer interface using the brain activity measured by near infrared spectroscopy takafumi miyoshi 1 , yasuhisa fujibayashi 2 , yoshiharu yonekura 2 , tatsuya asai 2 1 department of human and intelligence systems, university of fukui, fukui, japan; 2 biomedical imaging research center, university of fukui, fukui, japan people with severe motor disabilities can increase the quality of life if they can communicate with the external world. a brain-computer interface using brain activity is one of the ways to provide such communication without depending on muscular controls. brain activity was measured non-invasively by multi-channel near infrared spectroscopy (nirs) during various motor tasks from healthy subjects. these spatial brain activities were fed to neural networks, and pattern learning was carried out by matching the tasks and the brain activities. we propose that nirs signals may be used to construct a brain-computer interface. ps3p-j153 imaging of brain activity by near infrared spectroscopy in response to various sounds tatsuya asai 1 , kuniyoshi shinya 1 , tetsuo araki 2 , masahiro kusakabe 2 , yasuhisa fujibayashi 3 , yoshiharu yonekura 3 1 department of nuclear power and energy safety engineering, university of fukui, fukui, japan; 2 department of human and intelligence systems, university of fukui, fukui, japan; 3 biomedical imaging research center, university of fukui, fukui, japan brain activity can be monitored non-invasively by near infrared spectroscopy (nirs). in the present study, we measured changes in cerebral hemoglobin concentrations during a listening task using multi-channel nirs from healthy right-handed subjects, and hemispheric dominance for various sounds including verbal sounds was assessed. we have found asymmetrical brain activity when subjects listened to sounds with their left or right ear. these results suggest that hemispheric sound dominance may exist in addition to language dominance in healthy humans. kazuo kitamura 1,2 , winfried denk 3 , michael hausser 2 1 department of cellular neuroscience, graduate school of medicine, osaka university, osaka, japan; 2 university college london, london, uk; 3 max-plank institute, heidelberg, germany we describe a new approach for making targeted patch-clamp recordings from single neurons in vivo visualized using two-photon microscopy. the method involves using a patch electrode to perfuse the extracellular space surrounding the neuron of interest with a fluorescent dye, thus allowing the neuron to be visualized as a negative image and identified on the basis of its somatodendritic structure. the same electrode can then be placed on the neuron under visual control to allow gigaseal formation. we demonstrate the reliability and versatility of the method using recordings from principal neurons and interneurons in mouse and rat barrel cortex and cerebellum. we also show that the method can be used for in vivo juxtacellular labelling in identified cell types. this approach thus offers the prospect of targeted recording and labelling of single neurons in the intact native mammalian brain without the need to pre-label neuronal populations. research funds: wellcome trust, gatsby foundation, jsps and uehara foundation ps3p-k161 analysis on viability of gabaergic neurons in cerebral cortical slices of adult mice yasuyo tanaka 1 , yasuhiro tanaka 1 , takeshi kaneko 1,2 1 dept. of morphological brain science, kyoto univ., kyoto, japan; 2 crest, jst, kawaguchi, japan whole cell clamp recording and intracellular staining in adult brain slices are technically difficult because of their low viability. we analyzed the effect of slice cutting and incubation conditions on viability of cortical gabaergic neurons, using gad67-gfp knock-in mice. we considered gfp positive cells as having survived. we observed more gfp-positive cells in the slices when nacl in cutting solution was replaced with n-methyl-d-glucamine (nmdg) chloride, choline chloride or sucrose. however, the viability was lower after 3 h incubation in nmdg-based solution than in nacl-based solution. cutting at 0 • c did not reduce the number of gfp-positive cells, but decreased gfp fluorescence in single neurons as compared with cutting at 20 • c. the viability after 3 h incubation was better kept at 20 • c than at 0 or 37 • c. we thus recommend that slices be cut at 20 • c in na-free solution, and incubated at 20 • c in nacl-based solution. we thank dr yanagawa for his generous gift of knock-in mice. research funds: kakenhi (16200025,17022020,17650100) ps3p-k162 contribution of reduced and oxidized glutathione to signals detected by magnetic resonance spectroscopy as indicators of local brain redox state takumi satoh 1 , yoshichika yoshioka 2 1 faculty of engineering, iwate university, morioka, japan; 2 iwate medical university, takizawa, japan we evaluated gsh signals by the mega-press (a frequencyselective refocusing technique) signals assessed by magnetic resonance spectroscopy (mrs). gsh gave a single positive signal (2.95 ppm) by mega-press. in contrast, gssg gave a multiplet of reversed signals (3.03, 3.23, and 3.34 ppm). a phantom solution mimicking the normal condition (gsh:gssg = 100:1) gave a single positive peak. gsh was prominent and gssg signals were minimal. thus, the signals originated from gsh, not from gssg. in the phantom solution (creatine: gsh: aspartate: gaba = 7:3:1:1), the creatine signal overshadowed the other signals. through mega-press, a single peak of gsh stood out over other signals. in vivo, the brains of healthy volunteers gave similar signals as the in vitro phantom solution, indicating that the signal originated from gsh. the estimated concentration of gsh in the human brain was 1.9 mm. in conclusion, mega-press allowed us to assess gsh levels in vivo non-invasionally. hiroshi kadota, hirofumi sekiguchi, yasoichi nakajima, yutaka kohno, makoto miyazaki department of sensory and communicative disorders, research institute, national rehabilitation center for persons with disabilities, tokorozawa, japan we investigated the brain regions related to the inhibition of habitual responses by using functional mri. we used the rock-paper-scissors game as an example of a familiar habitual behavior. it is considered that making positive attempts to lose when presented with the gesture of a rock, paper, or scissors is associated with the inhibition of habitual responses. in this study, the subjects were randomly assigned to one of the following two groups: the "win group" and the "lose group." a comparison between these groups showed that the lose group displayed activation of multiple cortical areas in the brain. with regard to the prefrontal cortex, the comparison revealed a higher activation in the left middle frontal gyrus (brodmann area 9) and the right superior and middle frontal gyri (brodmann area 10) in the lose group. these findings suggest that these regions play a role in the inhibition of habitual responses. ps3p-k164 cortical commissural connection in macaque and human callosum using diffusion mri rishu piao 1 , takuya hayashi 1 , hiroshi sato 1 , shinichi abe 1,2 , takashi hanakawa 2 , hidenao fukuyama 2 , hidehiro iida 1 1 national cardiovascular center, osaka, japan; 2 human brain research center, kyoto university, kyoto, japan we investigated the cortical commissural connection in human and macaque using the non-invasive diffusion-weighted magnetic resonance imaging (dwi). we used the probabilistic algorithm to track connection paths between a pair of the left and right homologous in 9 subcortical areas. in macaque, the classification of callosum based on the highest interhemispheric connections paralleled with the results of tracer studies. however, the territory corresponding to the interfrontal connectivity extended more posteriorly than suggested in the tracer studies. the human interhemispheric connectivity showed similar topography in callosum as in the current macaque study, except that the connectivity territory of the frontal areas extended more posteriorly than in macaque. this study revealed that the commissural connectivity of the two species has a common intra-callosal topography. ps3p-k165 optimal resolution of eeg/meg source imaging by spatial filtering wan xiaohong 1,2 1 niche, department of qutantum science and energy engineering, tohoku university, sendai, japan, 2 niche, tohoku university, sendai, japan nowadays, electro-and magnetoencephalography (eeg/meg) is the sole invasive technique which is able to directly measure the human brain neural cortical dynamics. although we are well aware that it is impossible to accurately estimate the 3-d neural cortical activity using the 2-d eeg/meg surface potential topography, the upper limit of these techniques is not well described. during the past decades, various inverse approaches based on different criteria have been proposed, from the single dipole or multiple dipoles to the distributed current dipoles. however, it is difficult to systematically evaluate their efficiencies due to the different criteria and regularizations adopted in these methods. in this paper, we ask the question whether there exists an optimal approach based on a systematical criterion. this motivation firstly seems to be conflicted with the primary knowledge that there is no unique solution for the bioelectromagnetic inverse problem. essentially, here we are trying to find an optimal inverse solution that is closest to the real current distribution. ps3a-c192 sensitivity of serotonin synthesis to synthesis inhibitor gtp cyclohydrolase i in senescence-accelerated mouse-prone inbred strain (samp8) nobuyuki karasawa 1 , kazuko watanabe 2 , keiki yamada 3 1 seijoh universitry, tokai, japan, 2 dept. physio., sch. med., gifu univ., gifu, japan, 3 dept. anat., sch. health sci., fujita health univ., toyoake, japan to study the relationship between aging and levels of monoaminergic neurons, 2,4-diamino-6-hydroxypyrimidine (dahp), an inhibitor of monoamine synthesis, was intraperitoneally administered to senescence-accelerated mouse-prone (samp8) mice. time course of immunoreactive intensity for serotonergic (5-ht) neurons in the dorsal raphe nucleus, which were stained using laboratory-raised serotonin-specific antibody, was quantitatively evaluated using an image analysis system. results showed that 5-ht neruons are not highly sensitive to a synthesis inhibitor common to both catecholaminergic and 5-ht neurons. katsuya yamada 1,2,5 , yoshihiro matsumura 2 , takashi miki 3 , makoto wakui 1 ␣-smooth muscle actin + arterioles were fewer in kir6.1(−/−) barrel cortex than in wild-type one. in addition, whisker stimulation-induced increase in local cerebral blood flow was much smaller in kir6.1(−/−) barrel cortex than in wild-type one for short (2 s, 5 hz) but not long (30 s) stimulation, suggesting crtical involvement of thin arterioles in a short-time neuro ps3a-h135 learning to use sensory-tools by japanese monkeys yumiko yamazaki 1 , hiromi namba 1 support by public health research foundation (japan). acknowledgement supported by hkrgc. we wish to thank professor miyashita for valuable advice. ps2p-h147 mechanisms for processing of intellectual excitement kazuhiko yanai, hongmei dai dept. pharmacol tohoku grad. univ. sch. med., sendai, japanthe aim of this study was to investigate the role of histamine h1 receptor (h1r) in cognition in physiological and pathological conditions by using h1r mutant (h1−/−) mice. in normal condition, several behavioral studies indicated h1−/− mice show impaired object recognition and spatial memory, improved conditioned fear memory. moreover, hippocampal long-term potentiation was reduced in h1−/− mice. these results indicate h1 receptor is involved in memory process for which the frontal cortex, amygdala and hippocampus interact. in pathological condition, both h1−/− and control mice were subjected to social isolation, an animal model of schizophrenia. social isolation impaired locomotion, prepulse inhibition of startle response and water maze performance in control mice, but not in h1−/− mice. mutation of h1 receptor decreases isolation-induced hyperactivity of cortical dopaminergic neurons. these findings indicate blockage of h1r attenuates social isolation-induced behavioral changes. in conclusion, blockage of h1r impairs cognition in normal conditions, whereas h1r blocking inversely improves cognition in disease models of schizophrenia.research funds: kakenhi (17390156; 14370027) ps3a-h133 5-ht2a receptor gene polymorphism modulates activation in the human ventrolateral frontal lobe during go/no-go task michio nomura 1,2 , hirohito-m. kondo 2 , makio kashino 2,3 1 department of psychology, tokai women's university, kakamigahara, japan; 2 ntt communication science laboratories, ntt corporation, atsugi, japan; 3 shimojo implicit brain function project, erato, jst, kawaguchi, japan impulsive behavior has been suggested to be due to a dysfunction of 5-ht neurotransmission. we examined whether this 5-ht2a receptor gene polymorphism is involved in impulsive aggression by evaluating a reward-punishment go/no-go task using fmri. participants were required to learn to respond to active stimuli and inhibit their response to passive stimuli both under the reward-only (r) condition and the punishment-only (p) condition. the r condition, compared with the p condition caused right prefrontal activation mainly seen in ventrolateral regions. it has been reported that the possible involvement of the 5-ht2a receptor gene polymorphism in impulsive behavior (nomura et al., 2005) , together with the present findings, this observation indicates the involvement of 5-ht2a receptor gene polymorphisms in ventrolateral frontal lobe.research funds: shimojo implicit brain function project, erato, jst ps3a-h134 role of cortical thin arterioles in neurovascular coupling; analyses of kir6.1-containing atpsensitive potassium channel-deficient mice ps3p-g095 rem sleep deprivation increases serum ceruloplasmin level manoj jaiswal 1 , chinmay k. mukhopadhyay 2 , birendra n. mallick 1 1 school of life sciences, jawaharlal nehru university, new delhi, india; 2 special centre of molecular medicine, jawaharlal nehru university, new delhi, india rapid eye movement sleep (rems) is present across higher species and is essential for life. its loss predisposes one to several pathophysiological conditions. continuous loss of rems leads to several diseases and extreme loss may be fatal. rems loss is reported to increase metabolism and food intake though associated with hypothermia. hence, we proposed that the rems deprivation would affect acute phase response protein. in this study rats were rems deprived by platform method. free moving normal, large platform and recovery from rems loss were used as controls. blood was collected from the same rat before and after experimental as well as control periods. level of serum cruloplasmin, a positive acute phase response protein, was detected using western-blot analysis. the results showed that rems deprivation increased the serum ceruloplasmin level suggesting that the rems deprivation triggers an acute phase response at least in rats.research funds: council of scientific and industrial research, india and dst, india the indirect cytopathic effect in hiv-1 and the direct infection of hsv-1 are critical in their pathogenesis. we established murine neurosphere and evaluated with cocultivation of hiv-1 jrfl-infected macrophages or with hsv-1. the generation of primary neurospheres did not suppressed by hiv-1-infection or by hsv-1 infection at no more that moi 10. in the secondary neurospheres, cd133 + neural stem cells were intact in these infections, although beta-3-tubulin + cells were decreased in hiv-1 infection and intact in hsv-1-infection. in the differentiation assay, neun + nfp + neurons in hiv-1-infection and gfap + s100 + astrocytes in hsv-1 infection were significantly decreased. the migration capacity of the neurosphere cells was suppressed in hiv-1 infection and in hsv-1 infection. we conclude that neural stem cells in vitro are resistant to cytopathic effect by hiv-1 and hsv-1 infection and their differentiation capacities are different in these infections. our assay will be one of the significant methods in neurovirological research.research funds: kakenhi 17790579 grant-in-aid for young scientists(b) ps3p-g097 effects of attraction to favorite opposite gender on nervous, endocrine, and immune systems masahiro matsunaga 1,2 , taeko yamauchi 2 , toshihiro konagaya 2 , hideki ohira 1 1 department of psychology, nagoya university, japan; 2 department of internal medicine, aichi medical university school of medicine, japan everybody can "fall in love". thus everybody knows that attraction to favorite opposite gender invokes positive feelings and often makes us energetic. to investigate effects of this positive emotion on the biological systems, we recorded various parameters, namely mood states, heart rate, skin conductance level (scl), serum levels of several hormones, and proportions of t cells and natural killer (nk) cells in the lymphocytes simultaneously when subjects viewed the video films of their favorite opposite genders. when the subjects were evoked their attraction to favorite opposite gender, they became more vigorous and felt better. as for the biological systems, scl and the proportion of nk cells in the lymphocytes significantly increased. these results suggest the possibility that attraction to favorite opposite gender may have a role in activating nk cell-related innate immune system by means of the activation of scl-related sympathetic nervous system. hiroko ikeshima-kataoka 1 , shen jin-song 2 , saburo saito 1 , shigeki yuasa 3 1 dept. mol. immunol., inst. dna med., jikei. univ. sch. med., tokyo., japan; 2 dept. gene ther., inst. dna med., jikei univ. sch. med., tokyo, japan; 3 dept. ultrastruc. res., natl. inst. neurosci., ncnp, tokyo, japanto investigate the role of tenascin (tn)-expressing astrocytes played in the injured brain, we analyzed tn knockout (tn/ko) mouse. we have previously reported that tn is one of the essential molecules for proliferation of the primary culture of astrocytes. from injured mouse brain model with stab wound, gfap expression was down regulated sharply at earlier stages in tn/ko mouse than in the wt mouse. some of the inflammatory cytokines are known to be expressed in injured cns, and also those receptors are expressed in the primary culture of astrocytes. to evaluate immune responses in the cns, some of the inflammatory cytokine production was determined in the lesioned mouse brain compared with tn/ko and wt mouse. from rt-pcr method, tn seemed to have the possible roles for some of the cytokine prodution at the cns lesion sites. we are currently investigating the function of tenascin for the cytokine production around the lesion site. aiko hori 1 , tomoko yamamoto 1 , kiyoshi matsumura 2 , hiroshi hosokawa 1 , shigeo kobayashi 1 1 dept. of intelligence science and technology, grad. sch. of informatics, kyoto university, kyoto, japan; 2 dept. of information science and technology, osaka institute of technology, osaka, japan intracerebroventricular (i.c.v.) injection of arachidonic acid (aa) evokes fever. this response has been thought to occur simply because aa is converted to prostaglandin e 2 (pge 2 ), the final mediator of fever. however, our recent study suggested that aa might not only be the precursor of pge 2 but also induce an enzyme cyclooxygenase-2 (cox-2) that catalyses aa to form prostaglandins. we here examined in rats whether i.c.v. injection of aa induces cox-2, and whether cox-2 is involved in aa-induced fever. two hours after i.c.v. injection of aa, cox-2 was expressed in the perinuclear region of brain endothelial cells. aa-induced fever was partly suppressed with a cox-2 specific inhibitor, ns-398. these results indicate that aa itself or its metabolites induces cox-2 that accelerates the formation of pge 2 from aa, and, hence, enhances fever. mitsunari abe 1 , tatsuya mima 1 , shinichi urayama 1 , toshihiko aso 1 , nobukatsu sawamoto 2 , hidenao fukuyama 1 1 human brain research center, kyoto university graduate school of medicine, japan; 2 nano-medicine merger education unit, kyoto university, japanrepetitive transcranial magnetic stimulation (rtms) can induce lasting changes in the cortical excitability. however, its cellular mechanism remains unknown. diffusion weighted imaging (dwi) is a useful tool for measuring microscopic states of the brain tissue by probing water diffusion.we examined changes of dwi following rtms to further understand its effects. four healthy volunteers received rtms at 0.9 hz (10 min; 90% of the rest motor threshold) applied over the left primary motor area (m1). we scanned 4 sets of dwi (before, and 0 min, 10 min and 20 min after rtms) using 3-t mr scanner, and calculated apparent diffusion coefficient (adc). in 3 out of 4 subjects, the adc decreased (mean 1.72 × 10 −5 /mm 2 s −1 ) in the left m1 just after the rtms, which recovered at 20 min. it is possible that the rtms-induced change of adc might occur as the cellular response. further examination is needed for confirming this point. vahe poghosyan, andreas a. ioannides laboratory for human brain dynamics, brain science institute riken, wako-shi, japanretinotopic areas v6 and v6a in macaques occupy almost the entire extend of the anterior bank of parieto-occipital sulcus (pos). v6a located more dorsal has a larger receptive field size then v6. both areas lack a foveal magnification. we used meg to record brain activity while human subjects were viewing stimuli presented at two different eccentricities in each quadrant of visual fields. to verify the reliability of results, for each subject, the experiment was repeated on three different days. tomographic analysis of meg signal, in each subject, identified highly reproducible activations throughout visual cortex in accord with the known organization. two new areas along the pos with a similar retinotopy to that of macaques v6 and v6a were identified. in the ventral one, activations in response to each stimulus were spatially separated. the foveal magnification was much reduced compared to v1 in both areas and in the more dorsal area activations elicited by stimuli in the same quadrant could not be separated. given the above finding we suggest these areas as possible homologues of macaques v6 and v6a.ps3p-i144 measurement of magnetic evoked field of ratmeasurement of magnetic evoked field of rat using micro squid naohiro tsuyuguchi department of neurosurgery, osaka city university graduate school of medicine, japanthe study of neural activity in rodents would be enhanced by the stimulation of neuronal function in vivo. magnetoencephalography (meg) is used to study brain function in humans, but the limited resolution and sensitivity of conventional instruments have precluded the use of meg to study neuronal function in rodents. we demonstrate that micro meg developed for use with small animals, can be used to detect assess neuronal activity in conscious rodent brain. we used a micro 9-channel magnetometer consisting of a 3 × 3 matrix of superconducting quantum interference device (squid) with its integrated base of 2.5 × 2.5 mm to measure the visual evoked magnetic field (vef) and auditory evoked field (aef) of rats. we obtained the vef wave with 60-80 ms peak by the white led flashing stimulation and the aef wave with 10-20 ms peak by the tone and burst stimulation. this study demonstrate that micro meg can be used for serial assessment of neuronal function of individual, live animals with a minimal degree of invasiveness, has the potential for use in the study of brain function and plasticity. kentaroh takagaki, michael t. lippert, jian-young wu department of physiology and biophysics, georgetown university, washington, dc, usa voltage-sensitive dye (vsd) imaging is used to study visually evoked responses in rat visual cortex, in single trials without averaging. the signal is small, and a diode array with an effective dynamic range of 19 bits was used, along with a "blue dye" (rh 1691) with small heartbeat artifact. a subtraction algorithm was used to further remove heartbeat artifact in the data. with the combination of the array, the blue dye and the algorithm, we were able to visualize sensory evoked wave activity with a high signal-to-noise ratio in single trials. the signals were 0.1-0.05% of the resting fluorescence intensity. spatiotemporally, the evoked response manifested as propagating waves in the visual areas. there were large trial-to-trial variations in the propagating velocity and directions of the waves. the evoked wave apparently interacted with spontaneous waves in the cortex, and varied greatly according to anesthetic regimen. visualizing evoked waves may contribute to the understanding of cortical dynamics underlying sensory processing. masahito nemoto 1 , yoko hoshi 1 , chie sato 1 , susumu terakawa 2 1 tokyo institute of psychiatry, tokyo, japan; 2 photon medical research centre, hamamatsu university school of medicine, hamamatsu, japanwe investigated interhemispheric interactions and neurovascular coupling by simultaneous recordings of neuronal and hemodynamic signals in rats. bilateral somatosensory cortices were activated with a stimulus time lag between test stimuli (electrical pulses to contralateral hindpaw) and conditioning stimuli (to a homologous somatosensory region of the contralateral hemisphere). we measured electrophysiological signals (local field potentials and multiunit activity) and optical intrinsic signals (586 and 610 nm, indicators of cbv and oxygenation), and analyzed the dependence of the signals on the time lag. the results showed that both neuronal and hemodynamic signals were suppressed around 20-ms time lag. average and trial-by-trial correlation analyses suggested that the hemodynamic signals reflected a balance of neuronal excitation and inhibition via callosal connections. we can infer some parts of underlying neural interactions by imaging of the hemodynamic signals. ps3p-j147 multiple-site optical detection of spontaneous activity in the rat sensorimotor cortex akihiko hirota, shin-ichi ito department of physiology, shimane university school of medicine, izumo, japan multiple-site optical recording provides a powerful tool for the cerebral cortical neurophysiology, but its application has largely been restricted to reproducible, stimulus-evoked activation. we have developed the recording system with longer continuous recording capacity and larger signal-to-noise ratio to detect spontaneous activity in a single sweep. we applied this system to the sensorimotor cortex of rats anesthetized with a mixture of urethane and ␣-chloralose. the hindlimb region was exposed and stained with rh414, a voltage sensitive dye. optical records, after compensation for pulsation artifacts, contained deflections time-locked to the high amplitude transient waves, characteristic to ␣-chloralose anesthesia, recorded with a wire electrode placed in the optically sampling area. as the transient in the electrocorticogram fluctuated, the optical signal also varied. this signal was distributed over a broad region, whose latency, amplitude or shape varied systematically within the region, probably reflecting the regional differences in the transient activity. yuko tanaka, r. allen waggoner, kenichi ueno, keiji tanaka, kang cheng bsi, riken, saitama, japanobjective: in this fmri study, we attempted to identify the brain regions involved in the process completing objects with degraded image information.method: fourteen healthy subjects were studied using a 4t mri scanner while performing a matching-to-sample task with three task conditions. the main condition required the subject to judge in a 4-s trial if a trial-unique, degraded animal image matched a contour image. in the comparison condition, we reversed the order presenting intact and degraded images (id epoch).results: comparing images acquired in di epochs with those acquired in id epochs, significant activation was found in the left parietooccipital cortex spanning the cuneus (ba18), superior occipital gyrus around the parieto-occipital sulcus (ba19) and superior parietal lobule (ba7). other activated foci include the left anterior cingulated cortex, left dorsal frontal gyrus and right middle frontal gyrus.conclusion: these results indicate that the parieto-occipital cortex is critically involved in the object completion with degraded images.ps3p-j155 influence of task difficulty during meter inspection: an fmri study naoki miura 1,2 , makoto takahashi 3 , jobu watanabe 2,4 , shinya uchida 2,5 , shigeru sato 6 , kaoru horie 6 , masaharu kitamura 2 , toshio wakabayashi 2 , katsuki nakamura 1,7 , ryuta kawashima 2 1 crest, jst, kawaguchi, japan; 2 niche, tohoku univ. sendai, japan; 3 graduate school of engineering, tohoku univ. sendai, japan; 4 bme institute, waseda univ., tokyo, japan; 5 idac, tohoku univ., sendai, japan; 6 lbc research center, tohoku univ., sendai, japan; 7 department of animal models for human disease, ncnp, tokyo, japanthe purpose of the study was to analyze the cognitive process of a subject facing a human-machine interface (hmi) using fmri. we compared brain activation during meter inspection tasks with different task difficulty. during the meter inspection tasks, the subjects were instructed to inspect the three meters, and to press the button, if the subject found abnormal state. the task difficulty was devised by controlling the rate of change for the value to be displayed. in the right occipitotemporal area and the left cerebellar posterior lobule, activation during analog meter inspection was greater when the task difficulty was higher case. the results suggest that these regions are related to attention and perception of visual appearance of hmi.ps3p-j156 neural connectivity among brain areas related to language function shinya uchida 1,2,3 , naoki miura 2,4 , jobu watanabe 5 , shigeo kinomura 1 , kazunori sato 1 , yasuyuki taki 1 , kentaro inoue 1 , ryoi goto 1 , ai fukushima 2 , kaoru horie 6 , shigeru sato 6 , katsuki nakamura 3 , hiroshi fukuda 1 , ryuta kawashima 2 1 department of nuclear medicine and radiology, idac, tohoku university, sendai, japan; 2 niche, tohoku university, sendai, japan; 3 national institute of neuroscience, ncnp, kodaira, japan; 4 japan science and technology agency, kawaguchi, japan; 5 bme institute, asmew, waseda university, tokyo, japan; 6 graduate school of international cultural studies, tohoku university, sendai, japanthe present study examined the neural connectivity of languagerelated regions using functional mri (fmri) and diffusion tensor imaging tractography (dtt). twenty subjects were participated. functional region of interest (roi) in the left inferior frontal gyrus (lifg) defined by fmri during speech production task was used as a seed point for dtt. in more than 80% of subjects, tracts between the roi and the left thalamus (lth) were estimated. post hoc fmri analysis showed activation in the lth during speech production tasks. therefore, cortical connectivity between the lifg and lth may have certain functional roles in speech production. keisuke wakusawa 1,2 , motoaki sugiura 3 , yuko sassa 1,4 , hyeonjeong jeong 1,5 , kaoru horie 5 , shigeru sato 5 , hiroyuki yokoyama 2 , kazuie inuma 2 , ryuta kawashima 1 1 niche, tohoku univ., sendai, japan; 2 department of pediatrics, tohoku univ. graduate school of medicine, sendai, japan; 3 miyagi univ. of education, sendai, japan; 4 ristex, jst, tohoku univ., sendai, japan; 5 gsics, tohoku univ., sendai, japan; 6 lbc rc, tohoku univ., sendai, japanthis study examines the cortical mechanisms of comprehension of implicit social meanings such as irony and metaphor. healthy subjects judged whether the utterance in a picture such as irony, metaphor, or control expressions was situationally appropriate (s), or literally correct (l). greater activation during s than l task was analyzed to identify the activation for implicit meanings and neural responses to irony or metaphor were analyzed. the left medial prefrontal cortex showed higher activity during the s than l task. the medial orbitofrontal cortex and the right temporal pole showed responses selective to the irony; the responses in the former were observed during s task only, while the latter in both tasks. no selective response to metaphor was observed. keiichi onoda 1 , yasumasa okamoto 1 , kazuhiro shishida 1 , akiko kinoshita 1 , shigeru toki 1 , kazutaka ueda 2 , hidehisa yamashita 1 , shigeto yamawaki 1 1 department of psychiatry and neuroscience, hiroshima university, hiroshima, japan; 2 training and research center for clinical psychology, hiroshima university, higashi-hiroshima, japan anticipation of emotional events may affect perceptual and cognitive processes when the events actually happen. we studied the effects of anticipation of positive and negative affective images on neural processes estimated with meg and event-related fmri. participants were presented emotionally positive or negative images with cue stimuli. the cue stimulus indicated the emotional valence of the image which followed a few seconds later. in meg study, visual evoked field (vef) was smaller for the anticipatable negative image than the anticipatable positive image. this result suggests that when the presentation of a negative image is anticipated before the event, neural processing for the image is depressed compared to when a positive image is anticipated. furthermore, we report the difference of brain activation between anticipation of positive images and that of negative images in event-related fmri study. makoto wada 1,3,4 , kenji yoshimi 1,2 , noriyuki higo 4 , yong-ri ren 2 , hideki mochizuki 2 , yoshikuni mizuno 2 , shigeru kitazawa 1,3,4 1 dept. of physiol, juntendo univ. schl. of medicine, tokyo, japan; 2 dept. of neurol., juntendo univ. schl. of medicine, tokyo, japan; 3 crest, jst, tokyo, japan; 4 neurosci. res. inst., aist, tsukuba, japanwe developed a new method for comparing immunopositive cell densities across groups of animals and creating statistical parametric maps on standardized sections. as an example, we compared iba-1 positive glial cell densities in rats with and without unilateral injection of mpp+. immunopositive cell density map was automatically created in each animal over a coronal section in the midbrain (bregma −5.9 mm). after the map was normalized to a template section, positive cell densities of the two groups were compared in each pixel and a statistical parameter was mapped on each pixel. we were able to detect significant increases of microglias in the side of the injection not only in the substantia nigra pars compacta but also in the white matters. the new method was proven to be useful for detecting significant changes of cell densities over the entire area of immunostained sections.